Antibacterial profile against drug-resistant Staphylococcus epidermidis clinical strain and structure-activity relationship studies of 1H-pyrazolo[3,4-b]pyridine and thieno[2,3-b]pyridine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.07.035

Antibacterial resistance is a complex problem that contributes to health and economic losses worldwide. The Staphylococcus epidermidis is an important nosocomial pathogen that affects immunocompromised patients or those with indwelling devices. Currently,

Full text of DOI:10.1016/j.bmc.2008.07.035

Bioorganic & Medicinal Chemistry 16 (2008) 8196–8204
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antibacterial profile against drug-resistant Staphylococcus epidermidis
clinical strain and structure–activity relationship studies of
1H-pyrazolo[3,4-b]pyridine and thieno[2,3-b]pyridine derivatives
a
a
Bruno Leal a, Ilídio F. Afonso b, Carlos R. Rodrigues b, , Paula A. Abreu , Rafael Garrett ,
*
Luiz Carlos S. Pinheiro c, Alexandre R. Azevedo c, Julio C. Borges c, Percilene F. Vegi c, Cláudio C. C. Santos d,
Francisco C. A. da Silveira a, Lúcio M. Cabral b, Izabel C. P. P. Frugulhetti a, Alice M. R. Bernardino c,
Dilvani O. Santos a, Helena C. Castro a,
*
a Universidade Federal Fluminense, Instituto de Biologia, Departamento de Biologia Celular e Molecular, LABioMol, 24210-130 Niterói, RJ, Brazil
b Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, ModMolQSAR, 24020-150 Rio de Janeiro, RJ, Brazil
c Universidade Federal Fluminense, Instituto de Química, Departamento de Química Orgânica, Programa de Pós-Graduação em Química Orgânica, Campus do Valonguinho 24210-150,
Niterói, RJ, Brazil
d Fundação Ataulpho de Paiva, FAP, 20941-070 Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Antibacterial resistance is a complex problem that contributes to health and economic losses worldwide.
The Staphylococcus epidermidis is an important nosocomial pathogen that affects immunocompromised
patients or those with indwelling devices. Currently, there are several resistant strains including S. epide-
rmidis that became an important medical issue mainly in hospital environment. In this work, we report
the biological and theoretical evaluations of a 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylic acids series ( 1, 1a–m) and the comparison with a new isosteric ring nucleus series,
4-(arylamino)thieno[2,3-b]pyridine-5-carboxylic acids derivatives (2, 2a–m). Our results revealed the
1H-pyrazolo[3,4-b]pyridine derivatives significant antibacterial activity against a drug-resistant S. epide-
rmidis clinical strain in contrast to the thieno[2,3-b]pyridine series. The minimal inhibitory concentration
(MIC) of the most active derivatives ( 1a, 1c, 1e, and 1f) against S. epidermidis was similar to that of
oxacillin and twofold better than chloramphenicol. Interestingly, the position of the functional groups
has a great impact on the activity as observed in our structure–activity relationship (SAR) study. The
SAR of 1H-pyrazolo[3,4-b]pyridine derivatives shows that the highest inhibitory activity is observed
when the meta position is occupied by electronegative substituents. The molecular modeling analysis
of frontier molecular orbitals revealed that the LUMO density is less intense in meta than in ortho and
para positions for both series (1 and 2), whereas HOMO density is overconcentrated in 1H-pyrazol-
o[3,4-b]pyridine ring nucleus compared to the thieno[2,3-b]pyridine system. The most active derivatives
of series 1 were submitted to in silico ADMET screening, which confirmed these compounds as potential
antibacterial candidates.
Received 1 June 2008
Revised 15 July 2008
Accepted 16 July 2008
Available online 20 July 2008
Keywords:
Antimicrobial activity
Antibacterial activity
Pyrazolopyridine
MIC
Staphylococcus epidermidis
Structure–activity relationship (SAR)
In silico ADMET screening
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Staphylococcus epidermidis is among the most compromising
resistant strains.4–6 This Gram-positive bacterium is a major com-
The global threat of antimicrobial resistance is a serious matter
under review by the WHO and many countries throughout the
world.1–4 Currently, the widespread selective pressure and the effi-
cient dissemination channels for multidrug-resistant organisms
are major factors that may have contributed to the rapid emer-
gence of the resistant organisms.1–5
ponent of the normal human biota, and is recognized as an impor-
tant nosocomial pathogen affecting immunocompromised patients
or those with indwelling devices (i.e., joint prostheses, prosthetic
heart valves, and central venous catheters). The resistant strains
complicate the treatment, and extremely affect the recovery of
patients.4–6
Fused heterocyclic containing pyrazolopyridine systems have
been described associated with several biological and medicinal
activities including anxiolytic,7 antiviral,8,9 antileishmanial,10 and
anti-inflammatory11 profiles. In particular, thieno-pyridines are of
special importance due to the reported biological activities,12
* Corresponding authors. Tel.: +55 21 25626444 (C.R.R.); +55 21 26292294
(H.C.C.).
E-mail addresses: rangel@pharma.ufrj.br (C.R. Rodrigues), hcastrorangel@
vm.uff.br, hcastrorangel@yahoo.com.br (H.C. Castro).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.035

B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
8197
including antibacterial,13 anti-inflammatory,14 antiviral,15 and
antiparasitic16 profiles.
followed by ‘chlorocyclization’ with phosphorous oxychloride.
Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxyl-
ate (4) on fusion with appropriate substituted anilines generated
the required ethyl 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyr-
idine-5-carboxylates (3, 3a–m) in yields 68–64%, respectively.
Subsequent hydrolysis of 3 and 3a–m afforded the corresponding
4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acids (1, 1a–m) in high yields, 93–86%, respectively.
The design of the 4-(arylamino)thieno[2,3-b]pyridine-5-carbox-
ylic acids (2, 2a–m) was based in the substitution of the 1H-pyraz-
olo[3,4-b]pyridine ring by the thieno[2,3-b]pyridine, an isosteric
ring nucleus, in the 1 series (Fig. 1). The synthesis of this new series
was performed with yield of 65–75% by using a modified process
reported elsewhere (Scheme 1).15,16 The synthesis of 4-(arylami-
Although the importance of ring systems in the drug discovery
process is recognized, the current bioactive molecules described in
literature include a limited number of unique ring types. In this
work, we have employed a bioisosteric design to get additional in-
sights into the importance of different ring systems on the antibac-
terial profile. Literature points this approach for not only getting
molecules with better bioavailability/selectivity and easier to syn-
thesize but also replacing patented structural features.17,18
Herein we compared 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acids ( 1, 1a–m) derivatives with a new
isosteric ring nucleus series, the 4-(arylamino)thieno[2,3-b]pyri-
dine-5-carboxylic acids (2, 2a–m) derivatives. Thus, the present
study reports the synthesis, the antibacterial profile against a
drug-resistantS. epidermidis clinical strain, and the structure–activ-
ity relationship (SAR) evaluation of both series (1–2). In the SAR
studies, the biological properties of these molecules were com-
pared with several theoretical parameters such as dipole moment,
EHomo, ELumo, clogP, and molecular electrostatic potential map
(MEP) calculated using a molecular modeling approach. In addition
the distribution of HOMO and LUMO over the ring core was ana-
lyzed as this feature may be related to the interaction of the mol-
ecule with the target receptor. Since the compounds are considered
for oral delivery, they were also submitted to the analysis of Lipin-
ski Rule of Five.19 Finally the compounds’ toxicity was experimen-
tally and theoretically evaluated to determine their potential as
safe leading compounds.
no)thieno[2,3-b]pyridine-5-carboxylic acids (2,
2a–m) was
outlined in Scheme 1. The derivative 4-chlorothieno[2,3-b]pyri-
dine-5-carbonitrile (5) was prepared from 2-aminothiophene
through condensation with ethyl (ethoxymethylene)cyanoacetate
to produce ethyl
a-cyano-b-(N-2-thienylammonium)acrylate
yielding 88%. The cyclization of acrylate was carried out by reflux-
ing it in dowtherm (250 °C) for 40 min and affording the
4-hydroxythieno[2,3-b]pyridine-5-carbonitrile with 78% yield.
The compound 4-hydroxythieno[2,3-b]pyridine-5-carbonitrile
was easily chlorinated in refluxing phosphorous oxychloride,
affording 4-chlorothieno[2,3-b]pyridine-5-carbonitrile (5) with
76% yield. For producing the derivatives 4-(arylamino)thieno[2,3-
b]pyridine-5-carbonitrile (6, 6a–m), an equimolar mixture of
appropriate substituted anilines was heated, without solvents, for
4 h at 140 °C producing the compounds, (6, 6a–m), with yields of
93–86%.15,16
A solution of 4-(arylamino)thieno[2,3-b]pyridine-5-carbonitr-
iles (6, 6a–m) in 6 N HCl was heated for 24 h. On cooling, the mix-
ture was alkalinized with 10% NaOH solution, and the precipitates
was filtered and recrystallized from a mixture of ethanol and
water.21
2. Results and discussion
2.1. Chemistry
2.1.1. Chemistry of 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acids (1, 1a–m) and 4-(arylamino)thi-
eno[2,3-b]pyridine-5-carboxylic acids ( 2, 2 a–m) derivatives
The synthesis of 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acids (1, 1a–m) derivatives was performed
in high yields (86–93%) by using a modified process reported else-
where.8–10,20
The synthesis of 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acids (1, 1a–m) was achieved with an
efficient synthetic route (Scheme 1). Ethyl 4-chloro-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (4) was available in our lab-
oratory, and could be easily prepared8–10,20 from 5-aminopyrazoles
through condensation with diethyl ethoxymethylenemalonate
2.2. Biological evaluation (microbiology and cytotoxicity)
2.2.1. Antibacterial susceptibility tests (ASTs)
The initial screening was performed using 5 mg/mL of the (1a–
m) and thieno[2,3-b]pyridine (2a–m) derivatives in the ASTs as de-
scribed in Section 4. Although both series included unsubstituted
(1 and 2), meta (1a–f and 2a–f), and para-substituted (1g–m and
2g–m) derivatives, the antibacterial analysis against the drug-
resistant S. epidermidis clinical strain showed only meta-substi-
tuted 1H-pyrazolo[3,4-b]pyridine derivatives (1a–f and 1l) with
R
R
O
Cl
N
O
NH
N
NH
N
O
OEt
OEt
OH
(i)
N
N
(ii)
N
N
N
N
(4)
(3, 3a-m)
(1, 1a-m)
93-86%
R
R
Cl
N
O
NH
O
NH
N
CN
CN
(iii)
(i)
OH
S
(5)
S
N
S
(6, 6a-m)
(2, 2a-m)
75-65%
Scheme 1. Reagents and conditions: (i) anilines, 140 °C, 4 h; (ii) NaOH 20%, reflux, 3 h; (iii) HCl 6 N, reflux, 24 h.

8198
B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
Lipinski rule
Halo
(mm) (μg/mL) (%)
MIC CCa
Dipole
(debye)
Comp
R
EHOMO ELUMO
PSA
(Ų)
cLogP HBA HBD MW
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1l
1m
2
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
0
ND
16
64
16
ND
16
91 -5.82 -1.50
100 -5.78 -1.47
100 -5.78 -1.45
100 -5.96 -1.67
100 -6.17 -2.43
100 -5.94 -1.70
100 -5.97 -1.68
100 -5.69 -1.29
100 -5.76 -1.32
100 -5.87 -1.47
88 -6.26 -2.41
100 -5.86 -1.58
92 -5.91 -1.77
100 -5.78 -1.56
100 -5.69 -1.60
100 -5.74 -1.52
100 -5.88 -1.59
100 -6.21 -2.33
100 -5.84 -1.55
100 -5.89 -1.60
100 -5.54 -1.29
100 -5.65 -1.35
100 -5.85 -1.58
100 -6.34 -2.35
100 -5.79 -1.46
100 -5.86 -1.60
1.33
2.45
1.73
1.20
4.51
1.18
1.27
2.50
2.23
1.73
5.52
0.72
1.22
2.89
1.82
3.12
2.93
4.45
2.79
2.93
3.83
3.55
1.36
4.04
1.93
1.32
3.28
3.15
3.76
3.83
3.31
3.43
4.11
3.15
3.76
3.83
3.31
3.43
4.11
2.97
2.85
3.46
3.53
3.01
3.13
3.80
2.85
3.46
3.53
3.01
3.13
3.80
5
6
5
5
8
5
5
6
5
5
8
5
5
4
5
4
4
7
4
4
5
4
4
7
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
330.35 58.49
360.37 65.59
344.37 58.49
364.79 58.48
375.34 97.18
348.34 59.65
409.24 58.43
360.37 65.73
344.37 58.53
364.79 58.18
375.34 97.24
348.34 58.47
409.24 59.39
270.31 46.27
300.34 53.20
284.34 46.28
304.76 46.14
315.31 84.15
288.30 46.20
349.21 46.20
300.34 53.29
284.34 46.33
304.76 46.22
315.31 84.12
288.30 46.32
349.21 46.23
m-OCH3 15
m-CH3
m-Cl
m-NO2
m-F
m-Br
p-OCH3
p-CH3
p-Cl
16
17
12
20
17
0
0
0
0
0
10
0
0
0
0
0
0
0
0
16
NDb
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
p-NO2
p-F
p-Br
H
m-OCH3
m-CH3
m-Cl
m-NO2
m-F
m-Br
p-OCH3
p-CH3
p-Cl
p-NO2
p-F
0
0
0
0
2j
2l
2m
p-Br
0
aCytotoxicity (%) represented by cellular viability at 700μM of the compounds
bNot detected.
Figure 1. Comparison of the antibacterial profile against a drug-resistant Staphylococcus epidermidis clinical strain (antibacterial susceptibility test—halo, minimal inhibitory
concentration—MIC), cytotoxicity on peripheral blood mononuclear cells (CC), and theoretical parameters (EHOMO, ELUMO, dipole moment, and Lipinski Rule of Five) of the
series of 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acids (1, 1a–m) and 4-(arylamino)thieno[2,3-b]pyridine-5-carboxylic acids (2, 2 a–l).
an active biological profile (halo = 10–20 mm) (Fig. 1). No antibac-
terial activity was observed when using other drug-resistant
Gram-positive and Gram-negative clinical bacteria (not shown),
which suggested a specific mechanism against S. epidermidis. Over-
all these screening tests showed the potential of 1H-pyrazolo[3,4-
b]pyridine system as an antibacterial lead structure against the
drug-resistant S. epidermidis clinical strain in contrast to the thie-
no[2,3-b]pyridine system. In addition the meta position in the
1H-pyrazolo[3,4-b]pyridine series was also pointed as an impor-
tant structural feature to this specific biological profile.
2.2.2. Minimal inhibitory concentration (MIC) assays
The MIC assays of the active compounds (1a–e and 1f) showed
all with an antibacterial profile (MIC = 16
antibiotics current on the market (MIC = 1–40
1b that presents a methyl group at meta position (MIC = 64
l
g/mL) in the range of
g/mL), except for
g/
l
l
mL) (Figs. 1 and 2). The comparison showed that these compounds
are similar to oxacillin and are better than chloramphenicol, both
clinical antibiotics are currently in use (Fig. 2). These results rein-
forced the potential of these derivatives against the drug-resistant
S. epidermidis clinical strain.

B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
8199
A
B
C
Toxicity effects
Drug
M
T
I
R
1a
medium low
low
low
low high
low low
low low
1b
low
low
1c
1d
medium medium low low
1e
low
low
low
low
high
low
low
low
low
low
low medium
low low
low low
low low
1f
oxacillin
penicillin G
chloramphenicol
ampicillin
high low high
low low low
linezolid
high
high low low
Figure 2. Comparison of the most active 1H-pyrazolo[3,4-b]pyridine derivatives (1a, 1b, 1c, 1e, and 1f) with the clinical antibiotics currently in use. (A) Minimal Inhibitory
concentration (MIC), (B) drugscore, and (C) in silico toxicity risks. M, mutagenic; T, tumorigenic; I, irritant; R, reprodutive.
2.2.3. Effects on the bacterial growth
2.2.4. Cytotoxicity assays
Since all active compounds are meta substituted, this infers that
not only the 1H-pyrazolo[3,4-b]pyridine but also the substituent
position in the phenyl ring is biologically relevant. Then to analyze
the importance of the substituent position in these derivatives we
synthesized an ortho-F substituted 1H-pyrazolo[3,4-b]pyridine
derivative (1n) to compare with meta-F (1e) and para-F (1l) deriv-
atives on drug-resistant S. epidermidis clinical strain growth assays
(Fig. 3). The substituent (F) was selected due to its small size and
low electronic profile. Interestingly, our antibacterial data con-
firmed the importance of the restricted position in the ring for this
biological activity since only meta-F (1e) showed an antibacterial
profile in contrast to both para-F (1l) and ortho-F (1n) substituted
molecules that were inactive (Fig. 3).
To verify the cytotoxicity profile of the active compounds,
we analyzed their profile against peripheral blood mononuclear
cells (PBMCs). Interestingly our experimental results pointed 1a,
1c, 1e, and 1f as low risk compounds as the concentration used
was at least 15 times higher than the MIC with no toxicity on
PBMCs at 700 lM (Fig. 1). These results were similar to that ob-
served for antibiotics currently in use in the market (oxacillin,
chloramphenicol, ampicillin, and vancomycin), which kept
100% of PBMCs viability at 700 lM (not shown). In addition
these data were congruent with previous results obtained with
other 1H-pyrazolo[3,4-b]pyridine derivatives,8–10 which rein-
forced 1H-pyrazolo[3,4-b]pyridine nucleus as a low cytotoxic
structure.
Figure 3. Comparison of para (p-F), ortho (o-F), and meta (m-F) fluoride substituted 1H-pyrazolo[3,4-b]pyridine derivatives. (A) Effects on Staphylococcus epidermidis growth
(p < 0.05) of (para-F, ortho-F) substituted derivatives, tested at 5 mg/mL and meta-F tested at 16
lg/mL (1ꢀ MIC) and 160 ll/mL (10ꢀ MIC). (B) LUMO density encoded onto an
isosurface of the 0.002 e/au3 revealing the different degree of LUMO density distribution marked in the white box. (C) Structural alignment of all 1H-pyrazolo[3,4-b]pyridine
derivatives showing a green zone allowed by the meta position, whereas the red zone is the prohibited zone represented by the para and ortho positions in the benzene ring.

8200
B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
2.3. Molecular modeling and SAR studies of 1H-pyrazolo[3,4-
b]pyridine and thieno[2,3-b]pyridine systems
b]pyridine nucleus compared to thieno[2,3-b]pyridine system
(Fig. 4). This feature pointed out this region as feasible for main-
taining p–p stacking interactions with the target.
In this work, the structural and electronic properties of the
compounds were evaluated to identify their role in modulating
the 1H-pyrazolo[3,4-b]pyridine and thieno[2,3-b]pyridine biologi-
cal profile. Initially we calculated HOMO and LUMO energy, dipole
moment, and clogP of the derivatives that showed no clear or di-
rect correlation with antibacterial activity (Fig. 1). These data
pointed out that these parameters may not be used for establishing
a feasible relationship between these compounds structures and
the biological activity observed herein.
The molecular electrostatic potential map (MEP) energy isosur-
faces is an alternative approach for understanding the electrostatic
contribution on binding of receptor and drugs.9 The MEP analysis
of both series revealed a higher electronic density over 1H-pyraz-
olo[3,4-b]pyridine nucleus than that observed in the thieno-pyri-
dine bioisosteric ring (Fig. 4). The SAR studies of 1 series MEPs
suggested that the electron density concentrated in the substituted
phenyl ring apparently rules the activity profile of these systems
(i.e., 1b) (Fig. 4). Meanwhile derivatives 1a, 1c, 1e, and 1f contain-
ing electronegative atoms as substituents (methoxy, chlorine, fluo-
rine, and bromine, respectively) showed better biological results
than 1b, which inferred that the methyl low electronic contribu-
tion in 1b may avoid important interactions with the receptor
compared with the other active compounds (Fig. 4). Importantly,
the electron withdrawing meta nitro substituent caused a signifi-
cant modification on the MEP profile at the substituted phenyl ring
(Fig. 4). The nitro group is a strongly deactivating group, and also
led to a shift of the LUMO to the same region compared to the other
derivatives (Fig. 4), which apparently compromised the activity de-
spite its meta location.
The evaluation of the electron density/LUMO encoded onto an
isosurface of 0.002 e/au3 showed the thieno[2,3-b]pyridine system
with an intense blue color (high orbital density) over the carboxyl-
ate group, different from 1H-pyrazolo[3,4-b]pyridine system (Fig.
4). Our results also showed differences on LUMO density among
the active derivative 1e (meta-F) that presented the lowest density,
and the inactive derivatives 1l (para-F) and 1n (ortho-F) (Fig. 3).
The comparison of the structural alignment of the most active
derivatives (1a–f) with other substituted compounds of 1 series
suggested a steric hindrance when these substituents are at para
and ortho positions of the phenyl ring, probably creating a prohib-
itive (red) zone for the antibacterial profile (Fig. 3). In fact the con-
formational analysis of the meta-substituted derivatives revealed
one local energy minima where the best conformer of each meta
compound was 10 kcal/mol more stable when the substituent
was near from the pyrazole ring (not shown). This is probably
caused by the hydrogen bond formed between the NH (donor) of
the 1H-pyrazolo[3,4-b]pyridine system and the oxygen atom
(acceptor) of the carboxylate group in this specific conformation,
noticed only when the structure presents a meta substitutent
(not shown). Thus, the more stable meta conformation probably al-
lows the correct orientation of the substituent in the active deriv-
atives for interacting in a perfect match with the S. epidermidis
target.
The analysis of some structural properties (molecular weight,
number of hydrogen bond acceptors—nHBA, number of hydrogen
bond donors—nHBD, and topological surface area—tpsa) of the
two series revealed that the 1H-pyrazolo[3,4-b]pyridine system
presented higher values of lipophilicity, weight, nHBA, and tpsa
than thieno[2,3-b]pyridine compounds (Fig. 1). As only the 1H-pyr-
azolo[3,4-b]pyridine compounds presented an antibacterial profile,
it seems that these structural and electronic features may be
important for the permeation across membranes and for the
Our theoretical study using HOMO depicted as a map of the
orbital coefficient onto the van der Waals surface of the com-
pounds (red = low to blue = high HOMO density) showed a higher
electron density significantly concentrated over 1H-pyrazolo[3,4-
Figure 4. Electronic properties of 1H-pyrazolo[3,4-b]pyridine (1–1l) and thieno[2,3-b]pyridine (2–2l) derivatives. (A) Molecular electrostatic potential energy isosurfaces
(MEP) superimposed onto total electrons density of 0.002e/au3. The color code is in the range of ꢁ25 (deepest red) to +55 kcal/mol (deepest blue). (B) HOMO, and (C) LUMO
densities encoded onto a van der Waals surface (isodensity 0.002 e/au3), the orbital absolute density coefficient was mapped from deepest red (0.00) to deepest blue (0.01).

B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
8201
interaction through interatomic contacts and interface comple-
mentarity with the S. epidermidis target.
In this work, we submitted the active compounds to the analy-
sis of Lipinski Rule of Five that indicates if a chemical compound
could be an orally active drug in humans. The rule states that most
by electronegative substituents and (b) the 1H-pyrazolo[3,4-b]pyr-
idine derivatives present higher HOMO electron density. The active
compounds also showed low experimental and theoretical toxicity,
and were theoretically feasible as orally active drugs in humans as
they fulfilled the Lipinski Rule of Five. These new lead compounds
and the analyses of their molecular properties may be useful for
designing new and more efficient antibacterial drugs for fighting
the emergence of new resistant bacterial strains, an important is-
sue for public health that still lies ahead.
‘drug-like’ molecules have clogP
6
5, molecular weight
(MW) 6 500, PSA 6 140, and number of hydrogen bond acceptors
610 and donors 65. Molecules violating more than one of these
rules may have problems with bioavailability (Lipinski, 2001).
Our results showed that all active compounds (1a, 1b, 1c, 1e, and
1f) fulfilled this rule (molecular weight = 344.37–409.24,
clogP = 3.15–4.11, nHBA = 6–7, and nHBD = 2) (Fig. 1), similarly
to commercial drugs (i.e. chloramphenicol, oxacillin, ampicillin,
penicillin G, and linezolid) (not shown).
4. Experimental
4.1. Chemistry
All reagents and solvents used were of analytical grade. The 1H
Nuclear Magnetic Resonance (NMR) spectra were obtained on a
300 MHz, Varian Unity Plus instrument using tetramethylsilane
as internal standard. The chemical shifts (d) are reported in ppm
and the coupling constants (J) in Hertz. Fourier transform infrared
(FT-IR) spectra were recorded in a Perkin-Elmer Spectrum One FT-
IR. The solid samples were determined in potassium bromide (KBr)
pellets. Melting points (mp) were determined with a Fisher–Johns
apparatus. TLC was carried out using silica gel F-254 Glass Plate
(20 ꢀ 20 cm). All reagents and solvents used were of analytical
grade. The EI-MS spectra were recorded using a Finingan MAT
711 A instrument. The ionization energy was 70 eV with the source
200 °C and an accelerative voltage of 8 kV. Samples were intro-
duced by the standard direct insertion probe. High-resolution data
were obtained with the instrument using 10,000 resolution.
Currently there are many approaches that assess a compound
drug-likeness based on topological descriptors, fingerprints of
molecular drug-likeness structure keys or other properties such
as clogP and molecular weight.22 In the Osiris program (http://
www.organic-chemistry.org/prog/peo) the occurrence frequency
of each fragment is determined within the collection created by
shreddering 3300 traded drugs as well as 15,000 commercially
available chemicals (Fluka) yielding a complete list of all available
fragments. In this case, positive values point out that the molecule
contains predominantly the better fragments, which are frequently
present in commercial drugs but not in the non-drug-like collec-
tion of Fluka compounds. In this work, we used the Osiris program
for calculating the fragment based drug-likeness of the active com-
pounds also comparing them with penicillin G, chloramphenicol,
oxacillin, ampicillin, and linezolid (Fig. 4).
Interestingly, the derivatives 1a, 1b, 1c, 1e, and 1f presented
better drug-likeness values (from ꢁ3.68 to ꢁ0.12) than chloram-
phenicol (ꢁ4.61) and linezolid (ꢁ4.08) (not shown). In this study
we also verified the drugscore, which combines drug-likeness,
clogP, logS, molecular weight, and toxicity risks in one value and
this may be used to judge the compound’s overall potential to
qualify for a drug.23 Our theoretical data showed that 1a–f deriva-
tives presented values once again higher than chloramphenicol
and linezolid (Fig. 2).
4.1.1. General procedure for synthesis of the 4-(arylamino)-1-
phenyl-1H-pyrazolo[3,4-b]pyridines (1, 1a–m)
A mixture of the derivatives (3, 3a–m) (2 mmol), 10 mL of 20%
sodium hydroxide solution, and 10 mL of ethanol was heated un-
der reflux for 1–3 h. On cooling the mixture was acidified with di-
luted hydrochloric acid (1:3), and the precipitated acids were
filtered and recrystallized from a mixture of DMF and water.8–10,20
Drug toxicity is a factor of great importance for a potential com-
mercial drug, since a significant number of drugs are disapproved
in clinical trials based on their high toxicity profile. Herein, we
used the Osiris program (Fig. 2) to predict the overall toxicity of
the most active derivatives as it may point to the presence of some
fragments generally responsible for the irritant, mutagenic, tumor-
igenic, or reproductive effects in these molecules. Interestingly,
most of the active derivatives presented a low in silico toxicity risk
profile, similar to oxacillin, ampicillin, and penicillin G, and even
lower than that observed for chloramphenicol and linezolid (Fig.
2). These theoretical data reinforced the cytotoxicity experimental
data described in this work pointing these compounds as lead
compounds with low cytotoxicity.
4.1.1.1. 4-Phenylamino-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylic acid. Yield: 86%, mp 229 °C; IR (KBr, cmꢁ1): (
m
OH
3435–2598;
m
CAO 1654); 1H NMR (300 MHz, DMSO-d6, TMS, J
in Hz, d in ppm) 7.01 (s, H3, 1H), 9.02 (s, H6, 1H), 8.23 (d, 8.1, H-
20,60, 2H), 7.86–7.45 (m, 8H), 10.78 (s, N-H); EI (70 eV) m/z (%):
M+ꢀ 330.3127 (100).
4.1.1.2. 4-(30-Methoxyphenylamino)-l-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1a). Yield: 90%, mp 237 °C; IR (KBr,
cmꢁ1): ( CAO 1654); 1H NMR (300 MHz, DMSO-
m OH 3330–2590; m
d6, TMS, J in Hz, d in ppm) 6.91 (s, H3, 1H), 9.05 (s, H6, 1H), 8.27 (d,
7.5, H-20,60, 2H), 7.69 (dd, 7.5, H-30,50, 2H), 7.51 (dd, 7.5, H40, 1H),
7.66–7.61 (m, 4H), 10.88 (s, N-H), 3.95 (s, Ar-OCH3); EI (70 eV)
m/z (%): M+ꢀ 360.1237 (100).
3. Conclusion
4.1.1.3.
b]pyridine-5-carboxylic acid (1b). Yield: 90%, mp 237 °C; IR
(KBr, cmꢁ1): ( C@O 1650); 1H NMR (300 MHz,
OH 3482–2592;
DMSO-d6, TMS, J in Hz, d in ppm) 6.71 (s, H3, 1H), 8.95 (s, H6,
1H), 8.16 (d, 7.5, H-20,60, 2H), 7.60 (dd, 7.5, H-30,50, 2H), 7.52 (dd,
7.5, H40, 1H), 7.44–7.32 (m, 4H), 10.81 (s, N-H), 2.21 (s, Ar-CH3);
EI (70 eV) m/z (%): M+ꢀ 344.1317 (95).
4-(30-Methylphenylamino)-l-phenyl-1H-pyrazolo[3,4-
In this work, we synthesized 26 4-(arylamino)-1-phenyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxylic acids ( 1, 1a–m) and
4-(arylamino)thieno[2,3-b]pyridine-5-carboxylic acids (2, 2a–m)
derivatives to evaluate their antibacterial profile and to correlate
the biological results with some molecular properties. The study
revealed new four lead 1H-pyrazolo[3,4-b]pyridine compounds
with significant antibacterial activity on a drug-resistant S. epide-
rmidis clinical strain, similar to oxacillin, and are twofold better
than chloramphenicol. Interestingly, the 1H-pyrazolo[3,4-b]pyri-
dine system and the position of functional groups have a great im-
pact on the activity as observed in the SAR study. The highest
inhibitory activity is noticed when (a) the meta position is occupied
m
m
4.1.1.4. 4-(30-Chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1c). Yield: 93%, mp 236 °C; IR
(KBr, cmꢁ1): ( CAO 1649); 1H NMR (300 MHz,
m OH 3488–2766; m
DMSO-d6, TMS, J in Hz, d in ppm) 7.01 (s, H3, 1H), 9.07 (s, H6,
1H), 8.27 (d, 7.5, H-20,60, 2H), 7.74–7.62 (m, 5H), 7.52 (dd, 7.5,

8202
B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
H40, 1H), 7.79 (s, H200, 1H), 10.92 (s, N-H); EI (70 eV) m/z (%): M+ꢀ
364.0792 (100).
H-30,50, 2H), 7.52 (dd, 7.5, H40, 1H), 7.71 (d, 8,1, H200, 1H),
7.70 (dd, 8.1, H300, 1H), 7.70 (dd, 8.1, H500, 1H), 7.71 (d, 8.1,
H600, 1H), 10.90 (s, N-H); EI (70 eV) m/z (%): M+ꢀ 348.0960
(100).
4.1.1.5. 4-(30-Nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1d). Yield: 90%, mp 255 °C, IR
(KBr, cmꢁ1): (
m
OH 3401–2603;
m
CAO l674); 1H NMR (300 MHz,
4.1.1.13. 4-(40-Bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1m). Yield: 90%, mp 265 °C; IR
DMSO-d6, TMS, J in Hz, d in ppm) 7.12 (s, H3, 1H), 9.04 (s, H6,
1H), 8.22 (d, 7.5, H-20,60, 2H), 7.65 (dd, 7.5, H-30,50, 2H), 7.46 (dd,
7.5, H40, 1H), 8.42 (s, H200, 1H), 8.35 (d, 8.7, H400, 1H), 7.88 (d, 8.7,
H500, 1H), 8.03 (d, 8.7, H600, 1H), 10.99 (s, N-H); EI (70 eV) m/z
(%): M+ꢀ 375.0968 (100).
(KBr, cmꢁ1): ( CAO 1651); 1H NMR (300 MHz,
m OH 3431–2580; m
DMSO-d6, TMS, J in Hz, d in ppm) 6.87 (s, H3, 1H), 8.95 (s, H6,
1H), 8.16 (d, 7.5, H-20,60, 2H), 7.59 (dd, 7.5, H-30,50, 2H), 7.40 (dd,
7.5, H40, 2H), 7.50 (d, 8.7, H200, 1H), 7.78 (d, 8.7, H300, 1H), 7.78 (d,
8.7, H500, 1H), 7.50 (d, 8.7, H600, 1H), 13.00 (s, CO2H), 10.73 (s, N-
H); EI (70 eV) m/z (%): M+ꢀ 410.0073 (100).
4.1.1.6. 4-(30-Fluorophenylamino)-l-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1e). Yield: 93%, mp 258 °C; IR (KBr,
cmꢁ1): (
m
OH 3412–2586;
m
CAO 1675); 1H NMR (300 MHz, DMSO-
4.1.2. General procedure for synthesis of new 4-(arylamino)thi-
eno[2,3-b]pyridine-5-carboxylic acids (2b, 2c, 2g–m)
(a) Acid hydrolysis. A solution of 4-(arylamino)thieno[2,3-b]pyr-
idine-5-carbonitriles (6b, 6c, 6g–m) (2 mmol) in 6 N HCl (5 mL)
was heated under reflux for 24 h. On cooling, the mixture was alka-
linized with 10% aq NaOH solution, and the precipitated was fil-
tered and recrystallized from a mixture of ethanol and water.
The compounds (2b, 2c, 2g–m) were isolated in good yields (72–
65%).21
(b) Alkaline hydrolysis. A solution of 4-(arylamino)thieno[2,3-
b]pyridine-5-carbonitriles (6b, 6c, 6g–m) (2 mmol), 2 g of potas-
sium hydroxide pellets, and 4 mL of ethyleneglycol was heated
under reflux for 24 h. On cooling the mixture was acidified with
diluted HCl (1:3), and the precipitate was filtered and recrystal-
lized from a mixture of ethanol and water. The derivatives (2b,
2c, 2g–m) were obtained with lower yield.
d6, TMS, J in Hz, d in ppm) 7.04 (s, H3, 1H), 9.08 (s, H6, 1H), 8.28
(d,7.5, H-20,60, 2H), 7.79–7.54 (m, 6H), 7.49 (dd,7.5, H40, 1H),
10.98 (s, N-H); EI (70 eV) m/z (%): M+ꢀ 348.1013 (100).
4.1.1.7. 4-(30-Bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1f). Yield: 90%, mp 246 °C; IR (KBr,
cmꢁ1): ( CAO 1650); 1H NMR (300 MHz, DMSO-
m OH 3490–2580; m
d6, TMS, J in Hz, d in ppm) 6.65 (s, H3, 1H), 8.72 (s, H6, 1H), 7.93 (d,
7.5, H-20,60, 2H), 7.50–7.31 (m, 5H), 7.17 (dd, 7.5, H40, 1H), 7.57 (s,
H200, 1H), 12.90 (s, CO2H), 10.57 (s, N-H); EI (70 eV) m/z (%): M+ꢀ
410.0042 (100).
4.1.1.8. 4-(40-Methoxyphenylamino)-l-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1g). Yield: 91%, mp 261 °C; IR (KBr,
m OH 3430–2588; m
cmꢁ1): ( C = O 1654); 1H NMR (300 MHz,
DMSO-d6, TMS, J in Hz, d in ppm) 6.70 (s, H3, 1H), 9.03 (s, H6,
1H), 8.26 (d, 7.5, H-20,60, 2H), 7.69 (dd, 7.5, H-30,50, 2H), 7.51 (dd,
7.5, H40, 1H), 7.28 (d, 8.7, H200, 1H), 7.56 (s, H300, 1H), 7.56 (d, 8.7,
H500, 1H), 7.28 (d, 8.7, H600, 1H), 10.77 (s, N-H), 4.02 (s, Ar-OCH3);
EI (70 eV) m/z (%): M+ꢀ 360.1245 (100).
4.1.2.1. 4-(40-Methoxyphenylamino)thieno[2,3-b]pyridine-5-
carboxylic acid21 (2b). Yield: 70%, mp >300 °C; IR (KBr, cmꢁ1):
(
TMS, J in Hz, d in ppm) 7.59 (d, 6.0, H2, 1H); 6.38 (d, 6.0,
H3, 1H); 9.23 (s, H6, 1H); 7.39 (d, 8.7, 2H); 7.28 (d, 9.0, 2H); 3.42
(s, Ar-OCH3); 10.45 (s, CO2H); EI (70 eV) m/z (%): M+ꢀ 301.0951
(100).
m OH 3200–2800, m
CAO 1672); 1H NMR (300 MHz, DMSO-d6,
4.1.1.9. 4-(40-Methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1h). Yield: 92%, mp 258 °C; IR
(KBr, cmꢁ1): ( CAO 1654); 1H NMR (300 MHz,
m OH 3430–2603; m
DMSO-d6, TMS, J in Hz, d in ppm) 6.67 (s, H3, 1H), 8.95 (s, H6,
1H), 8.18 (d, 7.5, H-20,60, 2H), 7.61–7.40 (m, 7H), 10.73 (s, N-H),
2.47 (s, Ar-CH3); EI (70 eV) m/z (%): M+ꢀ 344.1336 (95).
4.1.2.2. 4-(40-Nitrophenylamino)thieno[2,3-b]pyridine-5-car-
boxylic acid (2c). Yield: 65%, mp >300 °C; IR (KBr, cmꢁ1): (
m
OH
3200–2800,
m
C = O 1672); 1H NMR (300 MHz, DMSO-d6, TMS, J
in Hz, d in ppm) 7.02 (d, 6.0, H2, 1H); 6.73 (d, 6.0, H3, 1H); 9.02
(s, H6, 1H); 8.28–7.32 (m, 4H); 9.06 (s, N-H); EI (70 eV) m/z (%):
M+ꢀ 316.0283 (100).
4.1.1.10. 4-(40-Chlorophenylamino)-l-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1i). Yield: 89%, mp 268 °C; IR
(KBr, cmꢁ1): ( CAO 1654); 1H NMR (300 MHz,
m OH 3430–2603; m
DMSO-d6, TMS, J in Hz, d in ppm) 6.87 (s, H3, 1H), 8.97 (s, H6,
1H), 8.17 (d, 7.5, H-20,60, 2H), 7.61 (dd, 7.5, H-30,50, 2H), 7.42 (dd,
7.5, H40, 1H), 7.58 (d, 8.7, H200, 1H), 7.67 (d, 8.7, H300, 1H), 7.67 (d,
8.7, H500, 1H), 7.58 (d, 8.7, H600, 1H), 10.77 (s, N-H); EI (70 eV) m/z
(%): M+ꢀ 364.0782 (100).
4.1.2.3. 4-(30-Bromophenylamino)thieno[2,3-b]pyridine-5-car-
boxylic acid (2g). Yield: 71%, mp >300 °C; IR (KBr, cmꢁ1): (
m
OH
3200–2800,
m
CAO 1670); 1H NMR (300 MHz, DMSO-d6, TMS, J
in Hz, d in ppm) 7.58 (d, 6.0, H2, 1H), 6.59 (d, 6.0, H3, 1H), 9.18
(s, H6, 1H), 7.49–7.09 (m, 4H); EI (70 eV) m/z (%): M+ꢀ 350.8931
(100).
4.1.1.11.
b]pyridine-5-carboxylic acid (1j). Yield: 93%, mp 256 °C; IR
(KBr, cmꢁ1): ( CAO 1678); 1H NMR (300 MHz,
OH 3409–2603;
4-(40-Nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-
4.1.2.4. 4-(30-Chlorophenylamino)thieno[2,3-b]pyridine-5-car-
m
m
boxylic acid (2h). Yield: 72%, mp >300 °C; IR (KBr, cmꢁ1): (
3200–2800, m
m
OH
DMSO-d6, TMS, J in Hz, d in ppm) 7.16 (s, H3, 1H), 9.06 (s, H6,
1H), 8.23 (d, 7.5, H-20,60, 2H), 7.65 (dd, 7.5, H-30,50, 2H), 7.46 (dd,
7.5, H40, 1H), 7.74 (d, 8.7, H200, 1H), 8.40 (d, 8.7, H300, 1H), 8.40 (d,
8.7, H500, 1H), 7.74 (d, 8.7, H600,1H), 10.98 (s, N-H); EI (70 eV) m/z
(%): M+ꢀ 375.0957 (100).
CAO 1679); 1H NMR (300 MHz, DMSO-d6, TMS, J in
Hz, d in ppm) 7.58 (d, 6.0, H2, 1H); 6.55 (d, 6.0, H3, 1H); 8.96 (s,
H6, 1H); 7.52–7.22 (m, 4H); 10.45 (s, CO2H); EI (70 eV) m/z (%):
M+ꢀ 305.0090 (100).
4.1.2.5. 4-(30-Fluorophenylamino)thieno[2,3-b]pyridine-5-car-
4.1.1.12. 4-(40-Fluorophenylamino)-l-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid (1l). Yield: 93%, mp 232 °C; IR
boxylic acid (2i). Yield: 67%, mp >300 °C; IR (KBr, cmꢁ1): (
m
OH
3200–2800,
m
CAO 1670); 1H NMR (300 MHz, DMSO-d6, TMS, J in
(KBr, cmꢁ1):
(m
OH 3487–2586;
m
CAO 1649); 1H NMR
Hz, d in ppm) 7.81 (d, 6.0, H2, 1H), 6.33 (d, 6.0, H3, 1H), 9.19 (s,
H6, 1H), 7.52–6.80 (m, 4H), 10.79; (s, CO2H); EI (70 eV) m/z (%):
M+ꢀ 289.0360 (100).
(300 MHz, DMSO-d6, TMS, in Hz,
J
d
in ppm) 6.82 (s, H3,
1H), 9.06 (s, H6, 1H), 8.27 (d, 7.5, H-20,60, 2H), 7.57 (dd, 7.5,

B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
8203
4.1.2.6.
boxylic acid (2j). Yield: 68%, mp >300 °C; IR (KBr, cmꢁ1): (
3200–2800,
C = O 1684); 1H NMR (300 MHz, DMSO-d6, TMS, J
4-(30-Nitrophenylamino)thieno[2,3-b]pyridine-5-car-
OH
m
at least in duplicate in four separate experiments, and a reference
m
antibiotic (vancomycin) was used as a positive control (MIC = 2
mL).
lg/
in Hz, d in ppm) 7.71 (d, 6.0, H2, 1H), 6.73 (d, 6.0, H3, 1H), 9.02
(s, H6, 1H), 8.14–7.69 (m, 4H), 10.45 (s, CO2H), EI (70 eV) m/z
(%): M+ꢀ 316.0294 (100).
4.2.4. Effects on the bacterial growth
The importance of the meta, para, and ortho positions in the
phenyl ring to the antibacterial profile was determined by testing
the fluoride (meta-F, para-F, and ortho-F) substituted derivatives ef-
fects on drug-resistant S. epidermidis clinical strain culture growth
monitored during 6 h at 560 nm as described previously24 Briefly,
cultures in the logarithimic growth phase were incubated with the
fluoride compounds at 1 and 10ꢀ MIC and 5 mg/mL. The values of
optical density were presented as growth (%) compared to the control.
4.1.2.7. 4-(30-Methoxyphenylamino)thieno[2,3-b]pyridine-5-car-
boxylic acid (2l). Yield: 72%, mp >300 °C; IR (KBr, cmꢁ1): (
m OH
3200–2800,
m
CAO 1671); 1H NMR (300 MHz, DMSO-d6, TMS, J in
Hz, d in ppm) 7.52 (d, 6.0, H2, 1H), 6.38 (d, 6.0, H3, 1H), 9.17 (s,
H6, 1H), 7.49–7.23 (m, 4H), 3.27 (s, Ar-OCH3), 10.62 (s, CO2H), EI
(70 eV) m/z (%): M+ꢀ 301.0389 (100).
4.1.2.8. 4-(30-Methylphenylamino)thieno[2,3-b]pyridine-5-car-
4.2.5. Cytotoxicity assays
boxylic acid (2m). Yield: 72%, mp >300 °C; IR (KBr, cmꢁ1): (
m
OH
Peripheral blood mononuclear cells (PBMCs) from healthy hu-
man donors were obtained by density gradient centrifugation(Hyst-
opaque, SigmaChem. Co, St. Louis, MO)from buffy coatpreparations.
Cells were resuspended in RPMI 1640 supplemented with 10% heat-
inactivated fetal bovine serum (FBS, Hyclone, Logan, UT), penicillin
3200–2800,
m
CAO 1671); 1H NMR (300 MHz, DMSO-d6, TMS, J in
Hz, d in ppm) 7.55 (d, 6.0, H2, 1H), 6.41 (d, 6.0, H3, 1H), 9.00 (s,
H6, 1H), 7.47–7.18 (m, 4H), 2.44 (s, Ar-CH3), 10.69 (s, CO2H), EI
(70 eV) m/z (%): M+ꢀ 285.0376 (100).
(100 U/mL), streptomycin (100
10 mM Hepes, stimulated with 5
l
g/mL), 2 mM glutamine, and
g/mL of phytohemagglutinin
4.2. Antibacterial assays
l
(PHA, Sigma) during 2–3 days, and further maintained in culture
medium containing 5 U/mL of recombinant human interleukin-2
(Sigma). After these cells were treated with several concentrations
of the compounds for 3–5 days incubated in 96-multiwell plates
on the conditions of 5% CO2, at 37 °C cell viability was determined
4.2.1. Bacteria
The nine drug-resistant Gram-positive (Enterococcus faecalis
and Staphylococcus epidermidis) and Gram-negative (Escherichia
coli, Serratia marcencens, Proteus mirabilis, Pseudomonas aeruginosa,
Enterobacter cloacae, Acinetobacter calcoaceticus, and Klebsiella
pneumoniae) clinical bacteria were isolated from patients of the
Hospital Antônio Pedro from Fluminense Federal University, and
were grown at 37 °C as described elsewhere.24 All other reagents
were from Sigma (St. Louis, MO). After isolation, the bacterial
strains were kept frozen in 10% milk sterilized solution containing
10% glycerin until used in the antibacterial susceptibility tests
(AST) and minimal inhibitory concentration (MIC) assays.
colorimetrically by the XTT method25 In brief, 50
ll of XTT was
added to wells, and the optical density (OD) was measured at
450 nm, 2 h later. All experiments were performed in duplicate at
least three times of each derivative (700
phenicol, ampicillin, vancomycin are clinical antibiotics and were
used as standard (700 M) in the assays.
lM). Oxacillin, chloram-
l
4.2.6. Molecular modeling and SAR studies
The molecular modeling study was performed using SPAR-
TAN’06 (Wavefunction Inc. Irvine, CA, 2006) and Osiris programs
(http://www.organic-chemistry.org/prog/peo/druglikeness.html) as
described elsewhere.9 The conformation analysis was obtained
through AM1 and angles of rotation in the range of 30/30°. Single
Point Calculation in DFT/B3LYP was performed with database
6.31G*. Molecular electrostatic potential maps (MEPs), HOMO,
and LUMO eigenvalues and orbital coefficients, and the molecular
dipole moments were calculated. In this work, we also studied
the drug-likeness and the drugscore of the compounds, which is
based on topological descriptors, fingerprints of molecular drug-
likeness, structural keys or other properties as clogP and molecular
weights. In the case of Osiris Property Explorer (http://www.organ-
ic-chemistry.org/), the occurrence frequency of each fragment is
determined within the collection of traded drugs and within the
supposedly non-drug-like collection of Fluka compounds.
Since the compounds are considered for oral delivery, they were
also submitted to the analysis of Lipinski Rule of Five,19 which eval-
uate some properties of a compound that would make it a likely
orally active drug in humans. These structural parameters were per-
formed using Molispiration program (http://www.molinspiration.
com/cgi-bin/properties).
4.2.2. Antibacterial susceptibility test (AST)
The assays were performed according to the National Commit-
tee for Clinical Laboratory Standards (NCCLS), in Müeller–Hinton
medium as described elsewhere24 Briefly, the strains were grown
at 37 °C in Müeller–Hinton medium, and 1 lL of the stock solution
(5 mg/mL) of each derivative in dimethyl sulfoxide (DMSO) was
placed in Whatman disks (5 mm diameter). The disks were put
on exponentially growing plated cultures with appropriate dilution
to 1.0 ꢀ 107 colony forming unit (CFU/mL), which were then incu-
bated for 24 h at 37 °C. The inocula used in growth method were
those where turbidity was equal to 0.5 McFarland Standard. The
results were verified by measuring the inhibitory zones surround-
ing the disk. Ciprofloxacin and vancomycin were used as positive
controls, and the halo >15 mm was considered the minimum value
for positive antibacterial activity as it generally leads to a minimal
inhibitory concentration (MIC) near that observed for the newest
antibiotics which are currently present in the market (MIC = 1–
40 lg/mL) using these assays. Vancomycin and ciprofloxacin pre-
sented halos ꢂ15–17 and 23–25 mm, respectively, in the strains
tested herein (p < 0.005).
4.2.3. Minimal inhibitory concentration assays (MIC)
MIC was determined only for active compounds on the AST by
using the macro-dilution broth method. All MIC were performed
in triplicate as described previously.24 Briefly, after 5 h of the bac-
terial growth, the culture was diluted to obtain 1.0 ꢀ 105 colony
forming unit (CFU/mL). Then each compound was added to reach
a final concentration from 0.5 to 1024 lg/mL, and was incubated
at 37 °C for 24 h. MIC was defined as the lowest compound concen-
Acknowledgments
We thank the Fundação de Amparo à Pesquisa do Estado do Rio
de Janeiro (FAPERJ) fellowships of Castro H.C.C., Bernardino
A.M.R.B., Borges J.C., and Pinheiro L.C.S.; Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Coordenação
de Aperfeiçoamento de Pessoal Docente (CAPES), and Universidade
Federal Fluminense (PROPP-UFF) for the financial support.
tration preventing visible bacterial growth. All strains were tested

8204
B. Leal et al. / Bioorg. Med. Chem. 16 (2008) 8196–8204
13. Bompart, J.; Giral, L.; Malicorne, G.; Puygrenier, M. Eur. J. Med. Chem. 1987, 22,
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