New antituberculotics originated from salicylanilides with promising in vitro activity against atypical mycobacterial strains

Article abstract of DOI:10.1016/j.bmc.2009.04.008

A new series of 30 N-protected amino acid esters were prepared as a part of ongoing search for new anti-tuberculosis active salicylanilides. The esters possess high in vitro activity against Mycobacterium tuberculosis, Mycobacterium avium, and two strains of Mycobacterium kansasii, where one is an isolate from the patient, with MIC in the range 1-32 μmol/L for all tested strains. The prepared esters can be considered as prodrugs with better bio-availability and as more efficient transport forms through the mycobacterial cell membranes due to the higher lipophilicity. The experimental and calculated lipophilicity, stability, antituberculotic activity, cytotoxicity as well as the quantitative structure-activity relationships (QSARs) explored by the Intelligent Problem Solver (IPS) in Trajan Neural Network Simulator 6.0 are presented.

Full text of DOI:10.1016/j.bmc.2009.04.008

Bioorganic & Medicinal Chemistry 17 (2009) 3572–3579
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New antituberculotics originated from salicylanilides with promising
in vitro activity against atypical mycobacterial strains
a,
b,
b
b
c,d
*
*
´
ˇ
Aleš Imramovsky , Jarmila Vinšová , Juana Monreal Férriz , Rafael Dolezal , Josef Jampílek
,
Jarmila Kaustová e, Filip Kunc f,g
a Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, nam. Cs. Legii 565, 532 10 Pardubice, Czech Republic
b Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
c Zentiva k.s., U kabelovny 130, 102 37 Prague, Czech Republic
d Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
e National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Public Health, Partyzanske namesti 7, 702 00 Ostrava, Czech Republic
f Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic
g Generi-Biotech s.r.o. Machkova 587, 500 11 Hradec Kralove, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 30 N-protected amino acid esters were prepared as a part of ongoing search for new anti-
tuberculosis active salicylanilides. The esters possess high in vitro activity against Mycobacterium tuber-
culosis, Mycobacterium avium, and two strains of Mycobacterium kansasii, where one is an isolate from the
Received 2 March 2009
Revised 3 April 2009
Accepted 6 April 2009
Available online 11 April 2009
patient, with MIC in the range 1–32 lmol/L for all tested strains. The prepared esters can be considered as
prodrugs with better bio-availability and as more efficient transport forms through the mycobacterial cell
membranes due to the higher lipophilicity. The experimental and calculated lipophilicity, stability, anti-
tuberculotic activity, cytotoxicity as well as the quantitative structure–activity relationships (QSARs)
explored by the Intelligent Problem Solver (IPS) in Trajan Neural Network Simulator 6.0 are presented.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Salicylanilides
Amino acid esterification
Lipophilicity
In vitro antimycobacterial activity
Cytotoxicity, hydrolytic stability, QSAR
Artificial neural networks
1. Introduction
spect to these MDR strains. Particularly in countries of Africa,
tuberculosis incidence has been increasing, primarily as a result
Tuberculosis (TB) was one of the first identified infectious dis-
eases. Nevertheless, it has become the most deadly infectious dis-
ease on the planet killing nearly 2 million people per year. It is
estimated that about one-third of the world’s population (2 billion
people) is infected with Mycobacterium tuberculosis (MTB) and
10% of them will progress to the active disease. The highest inci-
dence and burden of disease is observed in India, China, Indonesia,
Nigeria, and Bangladesh with the rates ranging from 293 to 102
new cases per 100,000 people of country’s population per year.1
Excessive use of antibiotics is generally accepted to be the main
reason for increased antibiotic resistance among bacteria.2,3 Resis-
tance to antimicrobial agents is an unavoidable side effect of their
use and goes hand in hand with an inexorable drive of bacterial
evolution. The situation in prevention and control of tuberculosis
is further fuelled by the deadly rise of multidrug-resistant TB
(MDR-TB).4 Currently available antibiotics are ineffectual with re-
of the HIV/AIDS epidemic. This co-infection (HIV/AIDS–TB) is one
of the major problems in the treatment and control of TB.5
Over the past 50 years, no new drug classes have been intro-
duced to the treatment of tuberculosis.1 Currently, patients require
between 6 and 9 months of treatment. This long period leads to the
lack of compliance, which in turn can be responsible for the relapse
and emergence of MDR-TB strains.6 Hence, there is an urgent need
to develop new, potent, and fast acting anti-tuberculosis drugs
with low toxicity profiles that can be used in conjunction with
drugs used for HIV infection treatment and are active against both
actively growing and latent infections.
Salicylanilides (2-hydroxy-N-phenylbenzamides) have been the
subject of intensive interest in medicinal chemistry, due to a wide
variety of their biological activities, particularly antimycobacterial
and antifungal activities.7–10 They exhibit high effect against M.
tuberculosis and isoniazid resistant strains Mycobacterium avium
and Mycobacterium kansasii.11 Even if their mechanism of action
is still under investigation, they serve as inhibitors of protein ki-
nase epidermal growth factor receptor (EGFR PTK).12 They are gen-
erally designed to compete with ATP for binding in catalytic
* Corresponding authors. Tel.: +420 466038007; fax: +420 466038004.
E-mail addresses: Ales.Imramovsky@upce.cz (A. Imramovsky´), Jarmila.Vinsova@
faf.cuni.cz (J. Vinšová).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.008

´
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A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
domain of tyrosin kinase.13,14 The latest studies specified them also
as selective inhibitors of interleukin-12p40 production that plays a
specific role in initiation, expansion, and control of cellular re-
sponse to tuberculosis.15,16
various lipophilicity values of individual positional isomers, that is,
the same log P/Clog P data were calculated for series 4-Cl (3a–3x)
and series 5-Cl (3y–3dd) as well as for C(3)/C(4) substitution in phe-
nylcarbamoyl moiety, that is, compounds 3a–3g, 3h–3l.
Experimentally determined values of log k are in accordance
with the calculated outcomes for both groups (series 4-Cl and ser-
ies 5-Cl) of compounds 3. Lipophilicity factor log k as well as calcu-
lated log P (ACD/log P) and log P/Clog P values (CHEMDRAW) increase
in dependence on amino acid type (glycine, alanine, valine, and
phenylalanine), that is, lipophilicity of the compounds substituted
in R3 increases: H < CH3 < CH(CH3)2 < CH2C6H5. The compounds
substituted by chlorine in C(5) position of benzene ring showed
higher lipophilicity according to program ACD/log P than the com-
pounds with C(4) chlorine substitution, but experimental log k for
C(4)-series is higher than that for C(5)-series. As expected, lipophil-
icity of the compounds substituted in the anilide part of the mole-
cule increases: Cl < Br < diCl. Compounds 3a–3g substituted by
chlorine in C0ð3Þ position showed higher lipophilicity according to
program ACD/log P than compounds 3h–3l with C0ð4Þ chlorine sub-
stitution, but experimental log k for C0ð4Þ chlorine substitution is
higher. The results are shown in Table 1.
2. Chemistry
Modifications of salicylanilides 1 should bring prodrugs with
improved physico-chemical properties important for passing bet-
ter through the cell membrane and lower toxicity. Previously stud-
ied salicylanilide acetates exhibited promising antimycobacterial
activity against M. tuberculosis and some atypical mycobacterial
strains M. avium and M. kansasii, but their toxicity did not decrease
significantly in comparison with salicylanilides.17 Therefore a new
prodrug form of amino acid esters and salicylanilides was pro-
posed. Desired compounds will have higher lipophilicity than
starting salicylanilides. Several perspective models of salicylanilide
amino acid esters with high antifungal activity confirm our sugges-
tions. The respective results were published recently.10
On the basis of the known results, the series of the above-men-
tioned salicylanilide derivatives was synthesized in two steps. Ani-
lines and salicylic acids were reacted under microwave heating,
using PCl3 in chlorobenzene, to form starting salicylanilides 1. In
the second step, N-benzyloxycarbonyl amino acids (N-Cbz-AA) 2
were esterified with salicylanilides 1 by using N,N0-dicyclohexylcar-
bodiimide activation as an optimal method.18 Lipophilic N-Cbz-AA
such as glycin, R/S-alanine, R/S-valine, and R/S-phenylalanine were
used for esterification. As expected, in most cases the reaction gave
Log k data specify lipophilicity within this series of the dis-
cussed compounds and all the chiral compounds were measured
several times giving the same results. The determined differences
in log k parameters for individual R/S-enantiomers cannot be ex-
plained on the basis of the results presented here.
5. Cytotoxicity assay
the desired esters 3 in high yields. When Cbz-Gly and Cbz-L-Ala were
esterified with salicylanilide that did not have halogen in position
C0ð3Þ, the major isolated product was unambiguously identified by
MS and gHMBC 2D NMR experiments as benzoxazepine-2,5-
dione.19 The general synthetic pathway is shown in Scheme 1.
Cytotoxicity assessment made on human intestinal cell line
HCT-8 (ECACC, UK) showed medium cytotoxicity for all measured
compounds, in comparison to the standard INH. The selectivity in-
dex (SI) is defined as the ratio of the measured EC50 (mammalian
cell toxicity) to the MIC. All observed compounds have SI value
higher than 10, it means the compounds may be considered suit-
able for further screening21 (Table 2).
3. Antimycobacterial activity of the prepared compounds
All the prepared N-Cbz-amino acid esters 3 were tested in vitro
for their antimycobacterial activity in the National Reference Lab-
oratory for Mycobacterium kansasii, against M. tuberculosis (M. tbc.)
331/88 and moreover for some non-tuberculous strains such as M.
avium (330/88) and M. kansasii (235/80 and 6509/96). The anti-TB
screening results of compounds 3 are summarized in Table 1.
6. Study of hydrolytic stability of the most active compounds
against M. avium and M. kansasii (3n, 3o, 3q, 3bb, 3cc, 3dd)
The studied derivatives were designed as a new prodrug form of
salicylanilides with prolonged liberation. To simulate blood or ser-
um and afflicted tissue, the stability in two types of phosphate buf-
fer at pH 7.4 (7 ꢀ 10ꢁ2 M, 37 °C) and pH 5.5 (7 ꢀ 10ꢁ2 M, 37 °C)
was measured, using UV/vis spectroscopy.22 During the hydrolysis
we observed an absorbance decrease at 224 nm. This absorption
band decrease corresponds to the rate of hydrolyses. From experi-
mental data measured at 224 nm, the half-times (t1/2) and rate
constants were calculated. The results are summarized in Table 2.
Experimental results for both types of hydrolysis are compara-
ble, acidic hydrolysis is slightly faster. The influence of amino acid
on the rate of hydrolysis requires further experiments. The hydro-
4. Lipophilicity properties of the prepared compounds
Hydrophobicities (log P/Clog P values) of the studied com-
pounds 3 were calculated using two commercially available pro-
grams (CHEMDRAW Ultra 10.0 and ACD/log P) and measured by
means of RP-HPLC determination of capacity factors k with subse-
quent calculation of log k.20
Both used programs do not resolve lipophilicity parameters
within the series of enantiomers. Program CHEMDRAW did not resolve
O
R2
R3
O
N
R1
2
O
R2
a
H
+
1
O
HOOC
N
O
N
R1
H
H
H
N
O
O
OH
R3
O
1
2
3
R1 = 4-Cl; 5-Cl
R2 = 4-Cl; 3-Cl
R3 = H; Me; i-Pro; -CH2-Ph
Scheme 1. Synthesis of investigated salicylanilide derivatives. Reagents and condition: (a) DCC, DMF, ꢁ10 °C.

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A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
3574
Table 1
Evaluation of Cbz-amino acid esters 3
Compd
R1
R2
R3
MIC (l
M)
Lipophilicity factor
M. tbc 331/88
M. avium 330/88
M. kansasii 235/80
M. kansasii 6509/96
log k
log P
log P/Clog P
ACD/log P
ChemOffice
14d
21d
14d
21d
7d
14d
21d
7d
14d
21d
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
4-Br
4-Br
4-Br
H
4
4
8
4
8
16
4
4
4
4
4
4
2
4
2
4
2
1
2
2
2
2
2
2
4
4
4
4
4
4
0.5
8
8
8
4
8
16
4
4
4
4
4
4
4
4
4
8
4
2
4
4
4
4
2
2
8
8
8
4
4
4
0.5
16
32
32
16
32
32
16
16
16
16
16
16
16
8
32
62.5
32
16
32
62.5
32
16
16
16
16
16
16
16
16
16
16
32
32
32
32
16
32
32
16
16
16
16
8
16
16
16
8
8
32
8
8
8
8
8
8
4
4
4
4
2
8
4
8
4
4
4
4
4
8
4
4
4
4
>250
16
16
16
16
16
32
16
16
16
8
16
8
8
8
8
8
4
8
4
8
4
4
4
16
16
16
16
16
32
16
16
16
16
16
8
8
8
8
8
4
16
8
16
8
8
8
8
8
8
8
32
8
8
8
8
8
8
8
8
8
16
8
8
8
4
8
8
8
8
8
4
4
4
4
4
4
16
16
16
16
16
32
16
8
8
8
16
8
16
8
8
16
8
8
16
8
8
8
8
8
16
16
16
16
16
32
16
16
16
16
16
16
16
8
8
16
8
8
16
8
0.6119
0.6995
0.6990
0.8972
0.8954
1.0465
1.0476
0.7294
0.9417
0.9421
1.0971
1.0678
0.7527
0.9803
0.9805
1.1258
1.1267
0.8639
0.9408
0.9380
1.1567
1.1559
1.3056
1.3030
0.6337
0.6334
0.8804
0.8816
1.0304
1.0315
5.04 0.48
5.39 0.48
5.39 0.48
6.27 0.48
6.27 0.48
7.32 0.49
7.32 0.49
5.35 0.48
6.23 0.48
6.23 0.48
7.03 0.58
7.28 0.40
5.53 0.54
6.41 0.54
6.41 0.54
7.46 0.55
7.46 0.55
5.91 0.50
6.26 0.50
6.26 0.50
7.14 0.50
7.14 0.50
8.19 0.51
8.19 0.51
5.63 0.48
5.63 0.48
5.61 0.48
5.61 0.48
7.56 0.49
7.56 0.49
4.71/5.14625
5.20/5.45525
5.20/5.45525
6.09/6.38325
6.09/6.38325
6.88/6.87325
6.88/6.87325
5.20/5.45525
6.09/6.38325
6.09/6.38325
6.88/6.87325
6.88/7.19557
5.47/5.60525
6.36/6.53325
6.36/6.53325
7.15/7.02325
7.15/7.02325
5.27/5.7738
5.76/6.0828
5.76/6.0828
6.65/7.0108
6.65/7.0108
7.44/7.5008
7.44/7.5008
5.20/5.45525
5.20/5.45525
6.09/6.38325
6.09/6.38325
6.88/6.87325
6.88/6.87325
(S)-CH3
(R)-CH3
(S)-CH–(CH3)2
(R)-CH–(CH3)2
(S)-CH2-phenyl
(R)-CH2-phenyl
(R)-CH3
(S)-CH–(CH3)2
(R)-CH–(CH3)2
(S)-CH2-phenyl
(R)-CH2-phenyl
(R)-CH3
(S)-CH–(CH3)2
(R)-CH–(CH3)2
(S)-CH2-phenyl
(R)-CH2-phenyl
H
16
16
8
4-Br
4,3-diCl
4,3-diCl
4,3-diCl
4,3-diCl
4,3-diCl
4,3-diCl
4,3-diCl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
16
16
16
16
16
16
16
16
16
16
8
3s
3t
(S)-CH3
(R)-CH3
3u
3v
3w
3x
3y
3z
3aa
3bb
3cc
3dd
INH
(S)-CH–(CH3)2
(R)-CH–(CH3)2
(S)-CH2-phenyl
(R)-CH2-phenyl
(S)-CH3
8
16
8
8
8
8
8
8
8
8
8
4
8
8
8
8
8
8
>250
8
8
8
8
8
8
8
8
16
16
16
8
8
8
(R)-CH3
(S)-CH–(CH3)2
(R)-CH–(CH3)2
(S)-CH2-phenyl
(R)-CH2-phenyl
8
8
>250
4-Cl
8
>250
>250
8
Table 2
Rate constants and half-times of the most active compounds in phosphate buffer solution pH 7.4 and pH 5.5
Compd
Rate constant kobs (sꢁ1) pH 7.4
t1/2 (s) for pH 7.4
Rate constant kobs (sꢁ1) pH 5.5
t1/2 (s) for pH 5.5
EC50
(lM)
SI for M. tbc
3n
3o
3q
3bb
3cc
3dd
7.79 ꢀ 10ꢁ4
7.70 ꢀ 10ꢁ4
3.37 ꢀ 10ꢁ4
5.65 ꢀ 10ꢁ4
4.56 ꢀ 10ꢁ4
5.24 ꢀ 10ꢁ4
889.94
897.47
2057.7
1226.10
1520.80
1323.30
8.06 ꢀ 10ꢁ4
7.81 ꢀ 10ꢁ4
3.56 ꢀ 10ꢁ4
5.69 ꢀ 10ꢁ4
5.29 ꢀ 10ꢁ4
4.35 ꢀ 10ꢁ4
860.1
887.25
1948.5
1218.1
1311.5
1074.0
121.8
121.3
35.5
106.7
82.94
104.2
16.74
33.35
10.61
13.75
20.73
26.05
lysis half-time in the range of 14–34 min should be sufficient for
the release of active molecules at the site of action.
found as the most important statistically for the activity against
M. tuberculosis 331/88, Free-Wilson descriptor of R2 = 4-Cl, for
the activity against M. avium 330/88, Free-Wilson descriptor
of R2 = 4-Br, for the activity against M. kansasii 235/80, and
Free-Wilson descriptor of R2 = 3-Cl, for the activity against M.
kansasii 6509/96. Substituents in R2 of N-Cbz-amino acid esters
3 seem to have a greater influence on the activity than those in
R1 or R3. Log k of N-Cbz-amino acid esters 3 appears to be of
higher importance only for the activity against M. kansasii
235/80.
7. Quantitative structure–activity relationships
7.1. QSARs analyses
For 30 N-Cbz-amino acid esters 3 QSAR analyses in Trajan 6.023
were carried out. Relationships between the antimycobacterial
activities against M. tuberculosis 331/88; M. avium 330/88; M. kans-
asii 235/80; M. kansasii 6509/96 (i.e., log MIC) after 21 days of incu-
bation and 17 descriptors of the compounds were sought. Free-
Wilson binary presence/absence descriptors (of substituents in
R1, R2, and R3), experimentally determined log k, Hammett substi-
Description and details of used QSAR method, characterization
of artificial neural networks, and the assessed results are provided
in the Supplementary data.
tuent
r
constants (for substituents in R1 and R2),24 and Taft induc-
8. SAR and QSAR
tive r* constants (for substituents in R3)25 were used as
descriptors. These descriptors are able to express lipophilic, elec-
tronic, and steric properties of the structures studied and can be
easily attainable from the formula or from the literature.
The results of in vitro evaluation showed an interesting activity
for all compounds. Cbz-Amino acid esters 3 have exhibited an
activity comparable with isoniazid (INH) as an internal standard
against M. tuberculosis. Activities against M. avium and M. kansasii
exceed the activity of INH considerably.
According to sensitivity analysis of QSAR models for N-Cbz-
amino acid esters 3, Free-Wilson descriptor of R2 = 3-Cl was

´
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A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
Compounds 3r, 3w/x, 3m, 3o, 3q, and 3s–3v exhibited the high-
est antituberculotic activity against M. tuberculosis. Based on these
observations it can be concluded that both substitutions R1 = 4-Cl
and R2 = 3,4-Cl or 4-Br are necessary for high activity while substi-
tution of R3 rather modulates lipophilicity that was found to be a
secondary parameter with the range of log k 0.94–1.16. Based on
the QSAR results the substituent in R2 was selected as the deter-
mining factor for the change of anti-TB activity against M.
tuberculosis.
Compounds 3cc/dd, 3bb, 3q, and 3n showed the highest activ-
ity against M. avium. From this fact it can be assumed that substi-
tutions of both R1 = R2 in the C(4) positions are strongly important
for high activity as well as substitution of R3 by branched substitu-
ent (benzyl or isopropyl—radical analogy).
Compounds 3q, 3u, 3w/x, and 3bb–3dd showed the highest
antimycobacterial activity against both evaluated strains of M.
kansasii. It means that both substitutions R1 = 4-Cl and R2 = 4-Br
or 3,4-Cl in C(4) positions are recommended for high activity. Fur-
thermore it can be specified that R1 = 5-Cl is a more advantageous
substitution for higher active compounds against M. kansasii 6509/
96. The substitution of R3 by branched substituent (benzyl or iso-
propyl) is also connected with the activity (radical analogy). Final-
ly, lipophilicity is another important parameter, the lipophilicity
range is 0.88–1.16, and the optimum is about 1.1. The substituents
R1 and R2 are relevant for the change of antituberculotic activity in
accordance with the results of QSAR analysis.
A very important parameter influencing the activity is stereo-
isomerism, because individual enantiomers demonstrate consider-
able difference in their antituberculosis/antimycobacterial activity,
for example, (S)-enantiomer 3p showed much lower antitubercu-
losis activity than (R)-enantiomer 3q and enantiomers 3n/o and
3u/v showed different antimycobacterial activity. The determined
differences in antituberculosis/antimycobacterial activity for indi-
vidual R/S-enantiomers cannot be explained on the basis of the re-
sults presented here. Due to these facts it can be assumed that
stereospecific bond can be probably formed between the com-
pound and an enzyme in Mycobacterium sp. with subsequent en-
zyme inhibition.
(254 nm). The products were purified by crystallization, by means
of column flash chromatography employing Silica Gel 60 (Merck).
All melting points were determined on Melting Point B-545 appa-
ratus (Büchi, Germany) and are uncorrected. Optical activities were
measured on polarimeter ADP 220 BS (Bellingham Stanley Ltd).
Infrared spectra (KBr pellets) were recorded on FT-IR spectrometer
Nicolet 6700 FT-IR in the range of 4000–400 cmꢁ1. NMR spectra
were measured in CDCl3 or DMSO-d6 solutions (if not specified
otherwise) on Varian Mercury—Vxbb 300 (300 MHz for 1H and
75.5 MHz for 13C; Varian Comp. Palo Alto, CA, USA). The chemical
shifts d are given in ppm, relating to tetramethylsilane (TMS) as
an internal standard. The coupling constants (J) are reported in
hertz. Elemental analyses (C, H, N) were performed on automatic
microanalyzer CHNS–O CE Instrument (FISONS EA 1110, Milano,
Italy).
10.1.2. Procedures and data of the prepared target compounds
10.1.2.1. General procedure for preparation of esters of Cbz-
a-
amino acids and substituted salicylanilides 3. N-Benzyloxy-
carbonyl (Cbz) protected 2-amino acid 2 (10 mmol) and substi-
tuted salicylanilide 1 (10 mmol) were dissolved in dry N,N-
dimethylformamide (DMF, 45 mL). The solution was cooled to
ꢁ10 °C and N,N0-dicyclohexylcarbodiimide (DCI, 11 mmol) was
added in three portions during 1 h. The mixture was then stirred
for 3 h at the same temperature and stored at +4 °C for 20 h. The
precipitate of N,N0-dicyclohexylurea was removed by filtration
and the solvent was evaporated in vacuo. The crude product 3
was purified by crystallization from ethyl acetate–hexane.
10.1.2.2. Data of prepared derivatives 3.
4-Chloro-2-(3-
chlorophenylcarbamoyl)phenyl 2-(benzyloxycarbonylamino)ace-
tate (3a).10
(S)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl
2-(benzyl-
oxycarbonylamino)propanoate (3b).10
(R)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)propanoate (3c).10
(S)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3d). White solid; yield 47%;
mp 149–151 °C; ½a D25
ꢂ
ꢁ30.7 (c 2.1, CHCl3), ½a D23
ꢁ86.0 (c 1.4, ethyl
ꢂ
acetate). IR (KBr pellet): 3330, 2965, 1764 (CO ester), 1712, 1667,
1592, 1532, 1481, 1424, 1310, 1199, 1104, 896, 781, 752, 700,
9. Conclusion
530 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d 8.21 (1H, br s, NH),
.
Esterification of active molecules is one of the most frequent
structural modifications that lead to better bioavailability. Lipo-
philic simple amino acids were chosen as natural molecules with
increased lipophilicity and consequently permeability through cell
membranes. The life-time of the prepared esters seems to be con-
venient for transport to the target. All 30 salicylanilide esters
showed high antimycobacterial activity (comparable with or high-
er than the standard) against all tested mycobacterial strains and
the selected six derivatives showed selectivity index higher than
ten. The present study encourages further research of the series
of compounds in order to find a new and novel antitubercular drug
that would also be active in MDR cases. Structure analyses rela-
tionship is in accordance with QSAR results.
7.82–7.72 (2H, m, H3, H20), 7.53–7.40 (2H, m, H5, H60), 7.36–7.28
(4H, m, H50, H300, H400, H500), 7.28–7.20 (2H, m, H200, H600), 7.16–
7.09 (2H, m, H40, H6), 5.28 (1H, d, J = 8.0 Hz, NH), 5.11 (1H, d,
J = 12.1 Hz, OCH2), 5.02 (1H, d, J = 12.1 Hz, OCH2), 4.39 (1H, dd,
J = 8.0 Hz, J = 5.2 Hz, CH), 2.37–2.17 (1H, m, CH), 1.03 (3H, d,
J = 6.9 Hz, CH3), 0.93 (3H, d, J = 6.9 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6) d 170.6, 162.8, 156.4, 145.6, 138.6, 135.8, 134.6, 132.2,
132.0, 130.0, 129.9, 129.8, 128.6, 128.3, 128.2, 124.9, 124.3,
120.7, 118.5, 67.4, 59.7, 30.4, 19.2, 17.6. Anal. Calcd for
C26H24Cl2N2O5 (515.40): C, 60.59; H, 4.69; N, 5.44. Found: C,
60.40; H, 4.99; N, 5.57.
(R)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3e). White solid; yield 34%;
mp 159–162 °C; ½a D26
ꢂ
18.7 (c 2.4, CHCl3). IR (KBr pellet): 3332,
2964, 1764 (CO ester), 1734, 1712, 1667, 1592, 1533, 1480, 1423,
10. Experimental
10.1. Chemistry
1310, 1202, 1104, 1038, 896, 782, 753, 586, 531 cmꢁ1 1H NMR
.
(300 MHz, DMSO-d6) d 8.21 (1H, br s, NH), 7.82–7.72 (2H, m, H3,
H20), 7.53–7.40 (2H, m, H5, H60), 7.36–7.28 (4H, m, H50, H300, H400,
H500), 7.28–7.20 (2H, m, H200, H600), 7.16–7.09 (2H, m, H6, H40),
5.28 (1H, d, J = 8.0 Hz, NH), 5.11 (1H, d, J = 12.0 Hz, OCH2), 5.02
(1H, d, J = 12.1 Hz, OCH2), 4.39 (1H, dd, J = 8.0 Hz, J = 5.2 Hz, CH),
2.37–2.17 (1H, m, CH), 1.03 (3H, d, J = 6.9 Hz, CH3), 0.93 (3H, d,
J = 7.0 Hz, CH3). 13C NMR (75 MHz, DMSO-d6) d 170.7, 162.3,
156.4, 145.6, 138.6, 135.8, 134.6, 132.2, 132.0, 130.0, 129.9,
10.1.1. Instrumentation and chemicals
The chemicals were purchased from commercial sources (Sig-
ma–Aldrich, Merck). Commercial grade reagents were used with-
out further purification. Reactions were monitored by means of
thin layer chromatography plates coated with 0.2 mm Silica Gel
60 F254 (Merck). TLC plates were visualized by UV irradiation

´
A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
3576
129.8, 128.6, 128.3, 128.2, 124.9, 124.3, 120.7, 118.5, 67.4, 59.7,
30.4, 19.2, 17.6. Anal. Calcd for C26H24Cl2N2O5 (515.40): C, 60.59;
H, 4.69; N, 5.44. Found: C, 60.59; H, 5.09; N, 5.78.
J = 2.5 Hz, H5), 7.36–7.21 (10H, m, H30, H50, Ar-phenyl), 7.19–7.11
(2H, m, Ar-phenyl), 6.93 (1H, d, J = 8.7 Hz, H6), 5.25 (1H, d,
J = 7.2 Hz, NH), 5.04 (1H, d, J = 12.0 Hz, OCH2), 4.97 (1H, d,
J = 12.3 Hz, OCH2), 4.75 (1H, q, J = 7.2 Hz, NCH), 3.22 (1H, dd,
J = 13.5 Hz, J = 6.4 Hz, CH2), 3.10 (1H, dd, J = 13.5 Hz, J = 7.4 Hz,
CH2). 13C NMR (75 MHz, CDCl3) d 170.1, 162.1, 155.9, 145.7,
135.9, 13,5.8, 134.9, 132.3, 132.1, 130.0, 129.5, 129.2, 129.0,
128.9, 128.6, 128.4, 128.3, 128.1, 127.6, 124.3, 121.9, 67.4, 55.4,
37.4. Anal. Calcd for C30H24Cl2N2O5 (563.43): C, 63.95; H, 4.29; N,
4.97. Found: C, 64.285; H, 4.57; N, 5.13.
(S)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-phenylpropanoate (3f). White solid; yield 33%;
mp 157–159 °C; ½a D26
ꢁ12.2 (c 1.8, CHCl3). IR (KBr pellet): 3326,
ꢂ
2929, 2851, 1761 (CO ester), 1705, 1658, 1627, 1594, 1538, 1483,
1424, 1311, 1262, 1200, 1161, 1106, 1082, 1054, 1029, 779, 698,
682 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.25 (1H, br s, NH), 7.80
.
(1H, d, J = 2.1 Hz, H3), 7.73 (1H, m, H20), 7.49–7.46 (1H, m, H40),
7.41 (1H, dd, J = 8.5 Hz, J = 2.1 Hz, H5), 7.32–7.27 (10H, m, H60,
H50, Ar-phenyl), 7.22–7.16 (2H, m, Ar-phenyl), 6.93 (1H, d,
J = 8.5 Hz, H6), 5.27 (1H, d, J = 7.2 Hz, NH), 5.06 (1H, d,
J = 12.3 Hz, OCH2), 4.99 (1H, d, J = 12.3 Hz, OCH2), 4.75 (1H, q,
J = 6.9 Hz, NCH), 3.24 (1H, dd, J = 13.9 Hz, J = 6.3 Hz, CH2), 3.10
(1H, dd, J = 13.9 Hz, J = 7.5 Hz, CH2). 13C NMR (75 MHz, CDCl3) d
170.2, 162.2, 155.9, 145.7, 138.6, 135.7, 134.9, 134.5, 132.2,
132.1, 130.0, 129.9, 129.4, 129.2, 128.9, 128.5, 128.3, 128.1,
127.5, 124.9, 124.3, 120.8, 118.7, 67.4, 55.4, 37.3. Anal. Calcd for
C30H24Cl2N2O5 (563.43): C, 63.95; H, 4.29; N, 4.97. Found: C,
64.35; H, 4.50; N, 5.32.
(R)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl 2-(benzyloxy-
carbonylamino)propanoate (3m). White solid; yield 3%; mp 190–
192 °C; ½a 2D5
ꢂ
38.1 (c 1.8, CHCl3). IR (KBr pellet): 3313, 2935, 1767
(CO ester), 1697, 1657, 1533, 1489, 1455, 1403, 1314, 1262,
1196, 1166, 1102, 1071, 1010, 882, 822, 736, 696, 506 cmꢁ1 1H
.
NMR (300 MHz, CDCl3) d 8.15 (1H, br s, NH), 7.76 (1H, m, H3),
7.52–7.33 (10H, m, H5, H20, H60, H30, H50, H200, H300, H400, H500,
H600), 7.11 (1H, d, J = 8.7 Hz, H6), 5.31 (1H, d, J = 7.2 Hz, NH), 5.11
(1H, d, J = 12.3 Hz, OCH2), 5.03 (1H, d, J = 12.3 Hz, OCH2), 4.53
(1H, m, NCH), 1.48 (3H, d, J = 7.2 Hz, CH3). 13C NMR (75 MHz,
CDCl3) d 171.4, 162.2, 155.8, 145.8, 136.5, 135.6, 132.3, 132.1,
132.0, 129.7, 128.6, 128.3, 128.1, 124.5, 122.0, 117.6, 67.3, 50.1,
33.8, 17.5. Anal. Calcd for C24H20BrClN2O5 (531.78): C, 54.21; H,
3.79; N, 5.27. Found: C, 54.28; H, 3.92; N, 5.42.
(R)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-phenylpropanoate (3g). White solid; yield 48%;
mp 161–163 °C; ½a D26
ꢂ
19.1 (c 2.9, CHCl3). IR (KBr pellet): 3299,
3064, 3033, 1761 (CO ester), 1705, 1658, 1594, 1538, 1483, 1424,
1306, 1262, 1200, 1161, 1148, 1106, 1081, 1054, 883, 779, 698,
(S)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3n). White solid; yield 48%;
682 cmꢁ1
.
1H NMR (300 MHz, CDCl3) d 8.20 (1H, br s, NH), 7.76
mp 144–146 °C; ½a D26
ꢁ24.3 (c 2.2, CHCl3). IR (KBr pellet): 3308,
ꢂ
(1H, d, J = 2.4 Hz, H3), 7.73 (1H, m, H20), 7.48–7.45 (1H, m, H40),
7.41 (1H, dd, J = 8.7 Hz, J = 2.4 Hz, H5), 7.33–7.28 (10H, m, H60,
H50, Ar-phenyl), 7.23–7.10 (2H, m, Ar-phenyl), 6.93 (1H, d,
J = 8.7 Hz, H6), 5.25 (1H, d, J = 7.2 Hz, NH), 5.06 (1H, d,
J = 12.0 Hz, OCH2), 4.99 (1H, d, J = 12.3 Hz, OCH2), 4.75 (1H, q,
J = 6.6 Hz, NCH), 3.24 (1H, dd, J = 14.0 Hz, J = 6.2 Hz, CH2), 3.10
(1H, dd, J = 14.0 Hz, J = 7.7 Hz, CH2). 13C NMR (75 MHz, CDCl3) d
170.2, 162.2, 155.9, 145.7, 138.6, 135.7, 134.9, 134.5, 132.3,
132.1, 130.0, 129.9, 129.4, 129.1, 128.9, 128.5, 128.3, 128.1,
127.5, 125.0, 124.3, 120.8, 118.7, 67.4, 55.4, 37.3. Anal. Calcd for
C30H24Cl2N2O5 (563.43): C, 63.95; H, 4.29; N, 4.97. Found: C,
64.15; H, 4.60; N, 5.09.
2960, 1748 (CO ester), 1707, 1668, 1531, 1489, 1394, 1315, 1252,
1189, 1105, 1069, 1008, 815, 744, 695 cmꢁ1 1H NMR (300 MHz,
.
DMSO-d6) d 8.17 (1H, br s, NH), 7.82–7.77 (1H, m, H3), 7.54–7.28
(10H, m, H5, H20, H30, H50, H60, H200, H300, H400, H500, H600), 7.12
(1H, d, J = 8.8 Hz, H6), 5.27 (1H, d, J = 8.2 Hz, NH), 5.10 (1H, d,
J = 12.4 Hz, OCH2), 5.01 (1H, d, J = 12.4 Hz, OCH2), 4.39 (1H, dd,
J = 8.2 Hz, J = 5.5 Hz, CH), 2.35–2.18 (1H, m, CH), 1.03 (3H, d,
J = 6.9 Hz, CH3), 0.93 (3H, d, J = 6.9 Hz, CH3). 13C NMR (75 MHz,
DMSO-d6) d 170.5, 162.2, 156.4, 145.6, 136.5, 135.8, 132.2, 132.0,
131.9, 130.0, 129.8, 128.6, 128.4, 128.1, 124.3, 122.1, 117.6, 67.4,
59.7, 30.5, 19.2, 17.6. Anal. Calcd for C26H24BrClN2O5 (559.83): C,
55.78; H, 4.32; N, 5.00. Found: C, 56.065, H, 4.565; N, 4.97.
(R)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)propanoate (3h). White solid; yield 12%; mp 137–
(R)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3o). White solid; yield 32%;
139 °C; ½a 2D5
ꢂ
37.0 (c 2.3, CHCl3). IR (KBr pellet): 3307, 1768 (CO es-
mp 147–150 °C; ½a D26
ꢂ
16.4 (c 2.1, CHCl3). IR (KBr pellet): 3331,
2964, 1763 (CO ester), 1705, 1669, 1601, 1533, 1489, 1394, 1315,
ter), 1698, 1657, 1538, 1533, 1493, 1455, 1404, 1315, 1262, 1196,
1101, 1065, 825, 736, 697, 508 cmꢁ1
.
1H NMR (300 MHz, CDCl3) d
1253, 1199, 1105, 1070, 1008, 827, 696, 507 cmꢁ1 1H NMR
.
8.13 (1H, br s, NH), 7.77 (1H, m, H3), 7.56–7.28 (10H, m, H20,
H60, H4, H30, H50, H200, H300, H400, H500, H600), 7.11 (1H, d, J
= 8.7 Hz, H6), 5.30 (1H, d, J = 6.9 Hz, NH), 5.11 (1H, d, J = 12.0 Hz,
OCH2), 5.04 (1H, d, J = 12.0 Hz, OCH2), 4.54 (1H, m, NCH), 1.48
(3H, d, J = 7.2 Hz, CH3). 13C NMR (75 MHz, CDCl3) d 171.4, 162.2,
155.8, 145.8, 136.0, 135.9, 132.3, 132.1, 130.0, 129.9, 129.7,
129.0, 128.6, 128.3, 128.1, 124.5, 121.7, 67.3, 50.1, 17.5. Anal. Calcd
for C24H20Cl2N2O5 (487.33): C, 59.19; H, 4.14; N, 5.75. Found: C,
59.02; H, 4.43; N, 5.85.
(300 MHz, CDCl3) d 8.18 (1H, br s, NH), 7.79 (1H, m, H3), 7.52–
7.32 (10H, m, H5, H20, H60, H30, H50, H200, H300, H400, H500, H600),
7.12 (1H, d, J = 8.7 Hz, H6), 5.28 (1H, d, J = 8.4 Hz, NH), 5.10 (1H,
d, J = 12.3 Hz, OCH2), 5.00 (1H, d, J = 12.0 Hz, OCH2), 4.39 (1H, dd,
J = 4.8 Hz, J = 1.4 Hz, NCH), 2.30–2.23 (1H, m, CH), 1.03 (3H, d,
J = 6.9 Hz, CH3), 0.93 (3H, d, J = 6.9 Hz, CH3). 13C NMR (75 MHz,
CDCl3) d 170.5, 162.2, 156.4, 145.6, 136.5, 135.8, 132.2, 132.0,
131.9, 130.0, 129.8, 128.6, 128.4, 128.1, 124.3, 122.1, 117.6, 67.4,
59.7, 30.5, 19.2, 17.6. Anal. Calcd for C26H24BrClN2O5 (559.84): C,
55.78; H, 4.32; N, 5.00. Found: C, 55.79; H, 4.61; N, 5.11.
(S)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl
2-(benzyl-
oxycarbonylamino)-3-methylbutanoate (3i).10
(S)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl 2-(benzyloxy-
carbonylamino)-3-phenylpropanoate (3p). White solid; yield 32%;
(R)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)-3-methylbutanoate (3j).10
mp 168–170 °C; ½a D26
ꢁ15.2 (c 1.65, CHCl3). IR (KBr pellet): 3312,
ꢂ
(S)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl
2-(benzyl-
1761 (CO ester), 1704, 1659, 1593, 1533, 1489, 1456, 1402, 1313,
oxycarbonylamino)-3-phenylpropanoate (3k).10,19
1261, 1199, 1101, 1050, 1010, 820, 750, 698, 504 cmꢁ1 1H NMR
.
(R)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl2-(benzyloxy-
(300 MHz, CDCl3) d 8.15 (1H, br s, NH), 7.80 (1H, d, J = 2.4 Hz,
H3), 7.49 (2H, m, H20, H60), 7.50–7.31 (11H, m, H5, H30, H50, Ar-phe-
nyl), 7.16 (2H, m, Ar-phenyl), 6.92 (1H, d, J = 8.7 Hz, H6), 5.25 (1H,
d, J = 6.9 Hz, NH), 5.04 (1H, d, J = 12.3 Hz, OCH2), 4.96 (1H, d,
J = 12.3 Hz, OCH2), 4.75 (1H, q, J = 6.9 Hz, NCH), 3.23 (1H, dd,
J = 14.0 Hz, J = 6.2 Hz, CH2), 3.10 (1H, dd, J = 14.0 Hz, J = 7.5 Hz,
CH2). 13C NMR (75 MHz, CDCl3) d 170.1, 162.1, 155.9, 145.7,
carbonylamino)-3-phenylpropanoate (3l). White solid; yield 46%;
mp 163–166 °C; ½a D25
ꢂ
18.4 (c 3.5, CHCl3). IR (KBr pellet): 3226,
2922, 2851, 1762 (CO ester), 1706, 1663, 1597, 1534, 1493, 1403,
1313, 1260, 1198, 1147, 1101, 826, 751, 699 cmꢁ1 1H NMR
.
(300 MHz, CDCl3) d 8.14 (1H, br s, NH), 7.81 (1H, d, J = 2.5 Hz,
H3), 7.55–7.53 (2H, m, H20, H60), 7.40 (1H, dd, J = 8.5 Hz,

´
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A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
136.5, 135.7, 134.9, 132.3, 132.1, 131.9, 130.0, 129.4, 129.2, 128.9,
128.6, 128.3, 128.1, 127.6, 124.3, 122.3, 117.6, 67.4, 55.3, 37.4.
Anal. Calcd for C30H24BrClN2O5 (607.88): C, 59.28; H, 3.98; N,
4.61. Found: C, 59.23; H, 4.00; N, 4.60.
(R)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)-3-methylbutanoate (3v). White solid; yield
15%; mp 150–152 °C; ½a D26
ꢂ
19.5 (c 2.7, CHCl3). IR (KBr pellet):
3324, 2967, 1767 (CO ester), 1704, 1670, 1588, 1525, 1477, 1382,
(R)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl 2-(benzyloxy-
carbonylamino)-3-phenylpropanoate (3q). White solid; yield 37%;
1308, 1202, 1104, 1028, 821, 698 cmꢁ1 1H NMR (300 MHz,
.
DMSO-d6) d 8.32 (1H, br s, NH), 7.88 (1H, d, J = 2.0 Hz, H3), 7.81
(1H, d, J = 2.2 Hz, H20), 7.52–7.41 (2H, m, H5, H50), 7.38–7.29 (6H,
m, H60, H200, H300, H400, H500, H600), 7.13 (1H, d, J = 8.5 Hz, H6), 5.27
(1H, d, J = 7.7 Hz, NH), 5.12 (1H, d, J = 12.2 Hz, OCH2), 5.02 (1H, d,
J = 12.2 Hz, OCH2), 4.35 (1H, dd, J = 7.7 Hz, J = 5.5 Hz, CH), 2.36–
2.19 (1H, m, CH), 1.06 (3H, d, J = 6.9 Hz, CH3), 0.97 (3H, d,
J = 6.9 Hz, CH3). 13C NMR (75 MHz, DMSO-d6) d 170.5, 162.3,
156.5, 145.6, 137.0, 135.7, 134.4, 132.6, 132.3, 132.2, 130.4,
130.2, 129.3, 128.6, 128.4, 128.1, 124.3, 122.5, 120.0, 67.5, 59.9,
30.4, 19.2, 17.8. Anal. Calcd for C26H23Cl3N2O5 (549.83): C, 56.80;
H, 4.22; N, 5.09. Found: C, 57.20; H, 4.22; N, 5.12.
mp 169–171 °C; ½a D26
ꢂ
17.8 (c 0.9, CHCl3). IR (KBr pellet): 3309,
1762 (CO ester), 1704, 1659, 1533, 1489, 1455, 1401, 1313, 1261,
1198, 1140, 1104, 1029, 1010, 822, 750, 698 cmꢁ1 1H NMR
.
(300 MHz, CDCl3) d 8.19 (1H, br s, NH), 7.79 (1H, d, J = 2.4 Hz,
H3), 7.48 (2H, m, H20, H60), 7.50–7.28 (11H, m, H5, H30, H50, Ar-phe-
nyl), 7.16 (2H, m, Ar-phenyl), 6.92 (1H, d, J = 8.7 Hz, H6), 5.27 (1H,
d, J = 7.2 Hz, NH), 5.04 (1H, d, J = 12.3 Hz, OCH2), 4.96 (1H, d,
J = 12.3 Hz, OCH2), 4.75 (1H, q, J = 7.2 Hz, NCH), 3.23 (1H, dd,
J = 14.0 Hz, J = 6.2 Hz, CH2), 3.10 (1H, dd, J = 14.0 Hz, J = 7.2 Hz,
CH2). 13C NMR (75 MHz, CDCl3) d 170.1, 162.1, 155.9, 145.7,
136.5, 135.7, 134.9, 132.2, 132.1, 131.9, 130.0, 129.5, 129.2,
128.9, 128.6, 128.3, 128.1, 127.5, 124.3, 122.3, 117.6, 67.4, 55.3,
37.4. Anal. Calcd for C30H24BrClN2O5 (607.88): C, 59.28; H, 3.98;
N, 4.61. Found: C, 59.08; H, 4.28; N, 4.70.
(S)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)-3-phenylpropanoate (3w). White solid; yield
27%; mp 169–172 °C; ½a D26
ꢁ10.1 (c 2.1, CHCl3). IR (KBr pellet):
ꢂ
3308, 1761 (CO ester), 1703, 1658, 1592, 1477, 1456, 1401, 1378,
1306, 1262, 1200, 1139, 1104, 1053, 1029, 1053, 1029, 883, 813,
4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)acetate (3r).10
698 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.33 (1H, br s, NH), 7.84
.
(S)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)propanoate (3s). White solid; yield 13%; mp
(1H, d, J = 1.9 Hz, H3), 7.80 (1H, d, J = 2.1 Hz, H20), 7.46 (1H, dd,
J = 8.7 Hz, J = 2.0 Hz, H5), 7.42 (1H, dd, J = 8.7 Hz, J = 2.4 Hz, H60),
7.34–7.27 (9H, m, H50, Ar-phenyl), 7.18 (2H, m, Ar-phenyl), 6.90
(1H, d, J = 8.7 Hz, H6), 5.27 (1H, d, J = 6.9 Hz, NH), 5.06 (1H, d,
J = 12.3 Hz, OCH2), 4.99 (1H, d, J = 12.3 Hz, OCH2), 4.73 (1H, q,
J = 7.2 Hz, NCH), 3.23 (1H, dd, J = 13.8 Hz, J = 6.3 Hz, CH2), 3.10
(1H, dd, J = 13.8 Hz, J = 7.5 Hz, CH2). 13C NMR (75 MHz, CDCl3) d
170.2, 162.1, 156.0, 145.7, 136.9, 135.6, 134.7, 132.6, 132.3,
132.2, 130.4, 130.2, 129.8, 129.6, 129.1, 129.0, 128.6, 128.4,
128.1, 127.6, 124.4, 122.6, 120.2, 67.5, 55.4, 37.3. Anal. Calcd for
C30H23Cl3N2O5 (597.87): C, 60.27; H, 3.88; N, 4.69. Found: C,
60.59; H, 4.03; N, 4.82.
136–138 °C; ½a 2D6
ꢁ36.1 (c 1.7, CHCl3). IR (KBr pellet): 3305, 1768
ꢂ
(CO ester), 1700, 1659, 1589, 1525, 1477, 1454, 1381, 1307, 1257,
1199, 1103, 1065, 1028, 881, 818, 736, 697 cmꢁ1 1H NMR
(300 MHz, CDCl3) d 8.32 (1H, br s, NH), 7.86–7.75 (2H, m, H3, H20),
7.48–7.44 (2H, m, H5, H60), 7.35–7.33 (6H, m, H50, H200, H300, H400,
H500, H600), 7.10 (1H, d, J = 8.7 Hz, H6), 5.32 (1H, d, J = 6.9 Hz, NH),
5.12 (1H, d, J = 12.0 Hz, OCH2), 5.02 (1H, d, J = 12.0 Hz, OCH2), 4.51
(1H, m, NCH), 1.49 (3H, d, J = 7.2 Hz, CH3). 13C NMR (75 MHz, CDCl3)
d 171.4, 162.3, 155.9, 145.8, 136.9, 135.8, 132.7, 132.2, 131.0, 130.4,
129.9, 129.2, 128.6, 128.3, 128.1, 127.0, 124.4, 122.4, 119.9, 67.4,
50.1, 17.3. Anal. Calcd for C24H19Cl3N2O5 (521.78): C, 55.25; H,
3.67; N, 5.37. Found: C, 55.15; H, 3.75; N, 5.44.
(R)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)-3-phenylpropanoate (3x). White solid; yield
(R)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-(benzyl-
oxycarbonylamino)propanoate (3t). White solid; yield 4%; mp
10%; mp 167–170 °C; ½a D26
ꢂ
35.9 (c 1.6, CHCl3). IR (KBr pellet):
3320, 2929, 2851, 1761 (CO ester), 1703, 1658, 1628, 1588, 1525,
1477, 1378, 1307, 1262, 1200, 1104, 1052, 1029, 814, 749,
134–136 °C; ½a 2D5
ꢂ
34.5 (c 2.0, CHCl3). IR (KBr pellet): 3305, 1768,
1699, 1659, 1589, 1526, 1477, 1455, 1381, 1300, 1261, 1200,
698 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.32 (1H, br s, NH), 7.85
.
1103, 1067, 1029, 881, 819, 736, 697 cmꢁ1
.
1H NMR (300 MHz,
(1H, d, J = 2.1 Hz, H3), 7.80 (1H, d, J = 2.5 Hz, H20), 7.47 (1H, dd,
J = 8.7 Hz, J = 2.1 Hz, H5), 7.42 (1H, dd, J = 8.7 Hz, J = 2.4 Hz, H60),
7.34–7.27 (9H, m, H50, Ar-phenyl), 7.18 (2H, m, Ar-phenyl), 6.91
(1H, d, J = 8.7 Hz, H6), 5.26 (1H, d, J = 6.6 Hz, NH), 5.06 (1H, d,
J = 12.3 Hz, OCH2), 5.00 (1H, d, J = 12.3 Hz, OCH2), 4.73 (1H, q,
J = 7.2 Hz, NCH), 3.23 (1H, dd, J = 13.8 Hz, J = 6.3 Hz, CH2), 3.10
(1H, dd, J = 13.8 Hz, J = 7.7 Hz, CH2). 13C NMR (75 MHz, CDCl3) d
170.2, 162.1, 156.0, 145.7, 137.0, 135.7, 134.8, 132.6, 132.3,
132.2, 130.4, 130.3, 129.9, 129.6, 129.2, 129.0, 128.6, 128.4,
128.1, 127.6, 124.3, 122.6, 120.2, 67.4, 55.4, 37.3. Anal. Calcd for
C30H23Cl3N2O5 (597.87): C, 60.27; H, 3.88; N, 4.69. Found: C,
60.65; H, 4.35; N, 5.01.
CDCl3) d 8.30 (1H, br s, NH), 7.87–7.77 (2H, m, H3, H20), 7.48–
7.32 (8H, m, H5, H50, H60, H200, H300, H400, H500, H600), 7.10 (1H, d,
J = 8.7 Hz, H6), 5.30 (1H, d, J = 6.6 Hz, NH), 5.11 (1H, d,
J = 12.0 Hz, OCH2), 5.02 (1H, d, J = 12.3 Hz, OCH2), 4.52 (1H, m,
NCH), 1.50 (3H, d, J = 7.2 Hz, CH3). 13C NMR (75 MHz, CDCl3) d
171.4, 162.3, 156.0, 145.8, 137.0, 135.8, 132.3, 130.4, 130.0,
129.7, 129.2, 128.6, 128.4, 128.1, 127.0, 126.7, 124.4, 122.4,
119.9, 67.4, 50.1, 17.3. Anal. Calcd for C24H19Cl3N2O5 (521.78): C,
55.25; H, 3.67; N, 5.37. Found: C, 55.575; H, 3.815; N, 5.52.
(S)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3u). White solid; yield 33%;
mp 144–147 °C; ½a D26
ꢂ
ꢁ20.9 (c 1.5, CHCl3). IR (KBr pellet): 3324,
(S)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)propanoate (3y). White solid; yield 57%; mp 157–
2966, 1763 (CO ester), 1706, 1669, 1587, 1523, 1477, 1382, 1308,
1200, 1104, 1027, 820, 745, 698, 578 cmꢁ1
.
1H NMR (300 MHz,
160 °C; ½a 2D6
ꢁ39.0 (c 1.7, CHCl3). IR (KBr pellet): 3325, 1769 (CO
ꢂ
DMSO-d6) d 8.32 (1H, br s, NH), 7.88 (1H, d, J = 2.0 Hz, H3), 7.81
(1H, d, J = 2.2 Hz, H20), 7.52–7.41 (2H, m, H5, H50), 7.38–7.29 (6H,
m, H60, H200, H300, H400, H500, H6), 7.13 (1H, d, J = 8.5 Hz, H6), 5.27
(1H, d, J = 7.7 Hz, NH), 5.12 (1H, d, J = 12.2 Hz, OCH2), 5.02 (1H, d,
J = 12.2 Hz, OCH2), 4.35 (1H, dd, J = 7.7 Hz, J = 5.5 Hz, CH), 2.36–
2.19 (1H, m, CH), 1.06 (3H, d, J = 6.9 Hz, CH3), 0.97 (3H, d,
J = 6.9 Hz, CH3). 13C NMR (75 MHz, DMSO-d6) d 170.5, 162.3,
156.5, 145.6, 137.0, 135.7, 134.4, 132.6, 132.3, 132.2, 130.4,
130.2, 129.3, 128.6, 128.4, 128.1, 124.3, 122.5, 120.0, 67.5, 59.9,
30.4, 19.2, 17.8. Anal. Calcd for C26H23Cl3N2O5 (549.83): C, 56.80;
H, 4.22; N, 5.09. Found: C, 56.74; H, 4.56; N, 5.14.
ester), 1691, 1663, 1595, 1537, 1493, 1454, 1399, 1313, 1253,
1165, 1095, 1070, 1015, 910, 824, 738, 696, 508 cmꢁ1 1H NMR
.
(300 MHz, CDCl3) d 8.19 (1H, br s, NH), 7.74 (1H, d, J = 8.4 Hz,
H3), 7.65 (2H, m, H20, H60), 7.32–7.29 (8H, m, H4, H30, H50, H200,
H300, H400, H500, H600), 7.20 (1H, m, H6), 5.32 (1H, d, J = 6.9 Hz, NH),
5.12 (1H, d, J = 12.3 Hz, OCH2), 5.04 (1H, d, J = 12.0 Hz, OCH2),
4.53 (1H, m, NCH), 1.48 (3H, d, J = 7.2 Hz, CH3). 13C NMR
(75 MHz, CDCl3) d 171.1, 162.7, 155.8, 147.8, 137.7, 136.1, 135.8,
131.0, 129.9, 129.0, 128.6, 128.3, 128.1, 127.0, 126.7, 123.4,
121.8, 37.3, 50.1, 17.4. Anal. Calcd for C24H20Cl2N2O5 (487.33): C,
59.15; H, 4.14; N, 5.75. Found: C, 59.23; H, 4.235; N, 5.78.

´
A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
3578
(R)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
1258, 1193, 1143, 1093, 1079, 1056, 908, 826, 743, 699, 508 cm
ꢁ1
carbonylamino)propanoate (3z). White solid; yield 21%; mp 155–
.
1H NMR (300 MHz, DMSO-d6) d 8.31 (1H, br s, NH), 10.59 (1H,
157 °C; ½a 2D6
ꢂ
42.1 (c 1.65, CHCl3), ½a D23
ꢂ
52.3 (c 1.1, ethyl acetate).
IR (KBr pellet): 3325, 1769 (CO ester), 1691, 1663, 1595, 1538,
1494, 1454, 1399, 1313, 1267, 1166, 1095, 1071, 824, 738, 696,
d, J = 7.8 Hz, H3), 7.73 (4H, m, H6, H4, H20, H60), 7.53 (2H, m, H30,
H50), 7.39–7.22 (10H, m, Ar-phenyl), 4.95 (1H, d, J = 10.5 Hz, NH),
4.94 (2H, m, OCH2), 4.50–4.43 (1H, m, NCH), 3.20 (1H, dd,
J = 13.8 Hz, J = 4.5 Hz, CH2), 2.89 (1H, dd, J = 13.8 Hz, J = 10.8 Hz,
CH2). 13C NMR (75 MHz, DMSO-d6) d 170.2, 163.3, 156.2, 148.4,
138.1, 137.4, 137.0, 135.5, 129.3, 129.0, 128.8, 128.7, 128.5,
128.2, 128.0, 127.8, 127.6, 126.8, 126.6, 123.3, 122.3, 65.7, 55.8,
36.1. Anal. Calcd for C30H24Cl2N2O5 (563.43): C, 63.95; H, 4.29; N,
4.97. Found: C, 64.15; H, 4.40; N, 5.00.
507 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d 8.23 (1H, br s, NH),
.
7.73 (1H, d, J = 8.2 Hz, H3), 7.61–7.49 (2H, m, H20, H60), 7.38–7.17
(9H, m, H4, H6, H30, H50, H200, H300, H400, H500, H600), 5.36 (1H, d,
J = 6.7 Hz, NH), 5.12 (1H, d, J = 12.1 Hz, OCH2), 5.03 (1H, d,
J = 12.1 Hz, OCH2), 4.61–4.44 (1H, m, CH), 1.48 (3H, d, J = 7.2 Hz,
CH3). 13C NMR (75 MHz, DMSO-d6) d 171.1, 162.7, 155.9, 147.8,
137.7, 136.1, 135.8, 130.9, 129.8, 129.0, 128.6, 128.3, 128.1,
127.0, 126.7, 123.4, 121.8, 67.3, 50.1, 17.4. Anal. Calcd for
C24H20Cl2N2O5 (487.33): C, 59.15; H, 4.14; N, 5.75. Found: C,
59.42; H, 4.45; N, 5.89.
10.2. HPLC determination of capacity factor k/calculated log k
The HPLC separation module Waters Alliance 2695 XE and
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, USA) were used. The chromatographic column SymmetryÒ
(S)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3aa). White solid; yield 16%;
mp 149–151 °C; ½a D26
ꢂ
ꢁ25.5 (c 2.7, CHCl3), ½a D23
ꢂ
ꢁ37.5 (c 1.5, ethyl
C18
5
l
m, 4.6 ꢀ 250 mm, Part No. WAT054275, (Waters Corp., Mil-
acetate). IR (KBr pellet): 3303, 2968, 1758 (CO ester), 1684, 1684,
1664, 1603, 1534, 1493, 1400, 1350, 1313, 1250, 1196, 1128,
ford, MA, USA) was used. The HPLC separation process was moni-
tored by Millennium32Ò Chromatography Manager Software,
Waters 2004 (Waters Corp., Milford, MA, USA). The mixture of
MeOH p.a. (70.0%) and H2O-HPLC—Mili-Q Grade (30.0%) was used
as a mobile phase. The total flow of the column was 1.0 mL/min,
1092, 1073, 1045, 820, 698, 508 cmꢁ1 1H NMR (300 MHz, CDCl3)
.
d 8.23 (1H, br s, NH), 7.78 (1H, d, J = 8.4 Hz, H3), 7.58–7.55 (2H,
m, H20, H60), 7.33–7.25 (8H, m, H4, H30, H50, H200, H300, H400, H500,
H600), 7.21 (1H, m H6), 5.29 (1H, d, J = 8.1 Hz, NH), 5.10 (1H, d,
J = 12.3 Hz, OCH2), 5.01 (1H, d, J = 12.0 Hz, OCH2), 4.38 (1H, dd,
J = 5.4 Hz, J = 2.7 Hz, NCH), 2.26 (1H, m, CH), 1.03 (3H, d,
J = 6.9 Hz, CH3), 0.93 (3H, d, J = 6.9 Hz, CH3). 13C NMR (75 MHz,
CDCl3) d 170.3, 162.6, 156.4, 147.6, 137.6, 136.1, 135.8, 131.3,
129.8, 128.9, 128.6, 128.4, 128.1, 127.0, 126.8, 123.3, 121.9, 67.4,
59.7, 30.5, 19.2, 17.6. Anal. Calcd for C26H24Cl2N2O5 (515.40): C,
60.59; H, 4.69; N, 5.44. Found: C, 60.375; H, 4.97; N, 5.47.
injection 30 lL, column temperature 45 °C and sample tempera-
ture 10 °C. The detection wavelength 210 nm was chosen. The KI
methanolic solution was used for dead time (tD) determination.
Retention times (tR) were measured in minutes.20
The capacity factors k were calculated using Millennium32Ò
Chromatography Manager Software according to formula
k = (tR ꢁ tD)/tD, where tR is the retention time of the solute, whereas
tD denotes the dead time obtained via an unretained analyte. Log k,
calculated from the capacity factor k, is used as the lipophilicity in-
dex converted to log P scale. The log k values of individual com-
pounds are shown in Table 1.
(R)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-methylbutanoate (3bb). White solid; yield 42%;
mp 148–150 °C; ½a D26
ꢂ
33.0 (c 1.3, CHCl3). IR (KBr pellet): 3303,
2968, 1758 (CO ester), 1684, 1664, 1603, 1523, 1534, 1493, 1399,
1350, 1314, 1250, 1196, 1175, 1127, 1092, 1073, 1045, 1014,
10.3. Lipophilicity calculations
827, 698, 659, 508 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.22 (1H,
.
br s, NH), 7.78 (1H, d, J = 8.1 Hz, H3), 7.33–7.25 (10H, m, H4,H20,
H60, H30, H50, H200, H300, H400, H500, H600), 7.21 (1H, m, H6), 5.29
(1H, d, J = 8.1 Hz, NH), 5.10 (1H, d, J = 12.0 Hz, OCH2), 5.01 (1H, d,
J = 12.0 Hz, OCH2), 4.38 (1H, dd, J = 5.4 Hz, J = 3.0 Hz, NCH), 2.29–
2.23 (1H, m, CH), 1.04 (3H, d, J = 6.9 Hz, CH3), 0.94 (3H, d,
J = 6.9 Hz, CH3). 13C NMR (75 MHz, CDCl3) d 170.3, 162.6, 156.4,
147.6, 137.6, 136.1, 135.8, 131.3, 129.8, 128.9, 128.6, 128.4,
128.1, 127.0, 126.8, 123.3, 121.9, 67.4, 59.7, 30.5, 19.2, 17.6. Anal.
Calcd for C26H24Cl2N2O5 (515.40): C, 60.59; H, 4.69; N, 5.44. Found:
C, 60.67; H, 5.00; N, 5.43.
Log P, that is, logarithm of the partition coefficient for n-octanol/
water, was calculated using programsCS ChemOffice, CHEMDRAW Ultra
ver. 10.0 (CambridgeSoft, Cambridge, MA, USA), and ACD/log P ver.
1.0 (Advanced Chemistry Development Inc., Toronto, Canada).
Clog P values (the logarithm of n-octanol/water partition coefficient
based on established chemical interactions) were generated
by means of CS ChemOffice Ultra ver. 10.0 (CambridgeSoft,
Cambridge, MA, USA) software. The results are shown in Table 1.
10.4. Antimycobacterial evaluation
(S)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-phenylpropanoate (3cc). White solid; yield 42%;
The in vitro antimycobacterial activity of all prepared com-
mp 185–187 °C; ½a D25
ꢂ
ꢁ14.8 (c 1.8, CHCl3). IR (KBr pellet): 3327,
pounds was evaluated against M. tuberculosis CNCTC My 331/88
1764 (CO ester), 1705, 1658, 1533, 1494, 1455, 1401, 1316, 1258,
(dilution of the strain was 10ꢁ3
l
mol/L), M. kansasii CNCTC My
235/80 (dilution of the strain was 10ꢁ4
mol/L), M. kansasii
6509/96 (dilution of the strain was 10ꢁ4
mol/L), and M. avium
CNCTC My 330/88 (dilution of the strain was 10ꢁ5
mol/L) in the
1143, 1093, 1080, 1056, 826, 743, 699, 508 cmꢁ1
.
1H NMR
l
(300 MHz, CDCl3) d 10.35 (1H, br s, NH), 8.02 (1H, d, J = 7.8 Hz, H3),
7.61 (4H, m, H6, H4, H20, H60), 7.53 (2H, m, H30, H50), 7.32–7.21
(10H, m, Ar-phenyl), 5.17 (1H, d, J = 11.4 Hz, NH), 4.92 (1H, d,
J = 12.3 Hz, OCH2), 4.86 (1H, d, J = 12.3 Hz, OCH2), 4.47 (1H, q,
J = 5.1 Hz, NCH), 3.23 (1H, dd, J = 13.9 Hz, J = 4.5 Hz, CH2), 2.89 (1H,
dd, J = 13.9 Hz, J = 10.5 Hz, CH2). 13C NMR (75 MHz, CDCl3) d 170.2,
163.3, 156.0, 148.2, 138.1, 137.3, 137.0, 135.4, 129.2, 129.0, 128.8,
128.6, 128.4, 128.2, 128.0, 127.9, 127.7, 126.8, 126,6, 123.4, 122.3,
65.7, 55.8, 36.1. Anal. Calcd for C30H24Cl2N2O5 (563.43): C, 63.95;
H, 4.29; N, 4.97. Found: C, 64.30; H, 4.65; N, 5.35.
l
l
National Reference Laboratory for M. kansasii, Regional Institute
of Hygiene, Ostrava, Czech Republic. All strains were obtained
from the Czech National Collection of Type Cultures (CNCTC),
except M. kansasii 6509/96, which was clinically isolated. Antimy-
cobacterial activities were determined in the Sula semisynthetic
medium (SEVAC, Prague, Czech Republic). The tested compounds
were added to the medium as dimethyl sulfoxide solutions. The
following concentrations were used: 250, 125, 62, 32, 16, 8, 4, 2,
(R)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-(benzyloxy-
carbonylamino)-3-phenylpropanoate (3dd). White solid; yield 44%;
and 1
l
mol/L. The MIC values were determined after incubation
at 37 °C for 7, 14, and 21 days. MIC (
substance concentration at which the inhibition of growth of
mycobacteria occurred, see Table 1.
l
mol/L) was the lowest
mp 181–184 °C; ½a D26
ꢂ
18.2 (c 2.7, CHCl3). IR (KBr pellet): 3328,
1763 (CO ester), 1705, 1657, 1597, 1534, 1493, 1455, 1401, 1316,

´
3579
A. Imramovsky et al. / Bioorg. Med. Chem. 17 (2009) 3572–3579
10.5. Study of hydrolytic behavior
Acknowledgments
Hydrolysis was performed on a Hewlett Packard UV/vis 8435
Diode Array apparatus in 10 mm closable quartz cell. The temper-
ature during all experiments was 37 °C. The compounds were
This work was financially supported by the GAUK 76807/2007
and by the Ministry of Education, Youth and Sports of the Czech
Republic (MSM 002 162 7501 and MSM 002 162 0822).
tested in methanolic solution (10 or 20 lL), which was added into
the tempered solution of phosphate buffer (2 mL) with defined pH
(7.4 or 5.5). Rate constants kobs (sꢁ1) (pseudo-first-order) were cal-
culated from the experimental data using the equation: kobst = ln -
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.04.008.
D
A + const., where
D
A = (A1 ꢁ At) or (At ꢁ A1).
10.6. Cytotoxic assay
References and notes
1. Laughon, E. L. Curr. Top. Med. Chem. 2007, 7, 463.
2. Levy, S. B. Adv. Drug Delivery Rev. 2005, 57, 1446.
3. Poole, K. Curr. Opin. Microbiol. 2001, 4, 500.
The cytotoxic effect of the compounds was tested by XTT assay on
human intestinal cell line HCT-8 (ECACC, UK). The cells were grown
in EMEM supplemented by 5% fetal bovine serum at 37 °C in a
humidified atmosphere of 5% CO2. For the experiments, the cells
were harvested with trypsin, resuspended in a fresh medium to a fi-
nal concentration of 2 ꢀ 105 cells/mL, and seeded in aliquots
4. Espinal, M. A. Tuberculosis 2003, 83, 44.
5. Swaminathan, S.; Narendran, G. J. Biosci. 2008, 33, 527.
6. Matsumoto, M.; Hashizume, H.; Tsubouchi, H.; Sasaki, H.; Itotani, M.; Kuroda,
H.; Tomishige, T.; Kawasaki, M.; Komatsu, M. Curr. Top. Med. Chem. 2007, 7, 499.
7. Vinšová, J.; Imramovsky´, A. Cesk. Slov. Farm. 2004, 53, 294.
8. De La Fluente, R.; Sonawane, N. D.; Arumainayagam, D.; Verman, A. S. British J.
Pharmacol. 2006, 149, 551.
(100 lL) onto 96-well tissue culture plates (TPP AG, Switzerland).
The medium was removed after 24 h of cell incubation and replaced
by EMEM culture containing the tested compounds dissolved in
DMSO. In control wells, the cells were incubated in a medium con-
taining DMSO without the tested compound (positive control for cell
viability) and in the medium containing 20% DMSO (positive control
for cytotoxic effect). The ability of the compounds to inhibit cellular
9. Dahlgren, M. K.; Kauppi, A. M.; Olsson, I. M.; Linusson, A.; Elofs, M. J. Med. Chem.
2007, 50, 6177.
´
10. Imramovsky, A.; Vinšová, J.; Férriz, J. M.; Buchta, V.; Jampílek, J. Bioorg. Med.
Chem. Lett. 2009, 19, 348.
11. Vinšová, J.; Imramovsky´, A.; Buchta, V.; Cecková, M.; Dolezal, M.; Štaud, F.;
Jampílek, J.; Kaustová, J. Molecules 2007, 11, 1.
ˇ
ˇ
ˇ
12. Kamatb, S.; Buolamwini, J. K. Med. Res. Rev. 2006, 26, 569.
13. Liechti, C.; Sequin, U.; Bold, G.; Furet, P.; Meyer, T.; Traxler, P. Eur. J. Med. Chem.
2004, 39, 11.
14. Guo, L.; Wang, Q. L.; Jiang, Q. Q.; Jiang, J. J.; Jiang, Y. B. J. Org. Chem. 2007, 72,
9947.
15. Brown, M. E.; Fitzner, J. N.; Stevens, T.; Chin, W.; Wright, C. D.; Boyce, J. P.
Bioorg. Med. Chem. 2008, 16, 8760.
16. Boyce, J. P.; Brown, M. E.; Chin, W.; Fitzner, J. N.; Paxton, R. J.; Shen, M.; Stevens,
growth was determined after 72 h by adding XTT solution (20 lL,
1 mg/mL, F. Hoffmann-La Roche Ltd, Switzerland) to each well. After
incubation for 4 h absorbance was read at 450 nm using multiplate
spectrometer LM 01 A (Beckmann Coulter Inc., USA). Each concen-
tration of the compounds was tested in triplicates.
T.; Wolfson, F.; Wright, C. D. Bioconjugate Chem. 2008, 19, 1775.
ˇ
ˇ
ˇ
17. Vinšová, J.; Imramovsky´, A.; Buchta, V.; Cecková, M.; Dolezal, M.; Štaud, F.;
10.7. QSAR analysis
Jampílek, J.; Kaustová, J. Molecules 2007, 11, 1.
18. Bodansky, M. Principle of Peptide Synthesis, 2nd ed.; Springer: Berlin, 1993.
ˇ
19. Imramovsky´, A.; Vinšová, J.; Férriz, J. M.; Kuneš, J.; Pour, M.; Dolezal, M.
Quantitative structure analyses relationships between antitu-
berculotic activity (log MIC) against M. tuberculosis (M. tbc.) 331/
88, M. avium (330/88), M. kansasii (235/80, and clinically isolated
6509/96) after 21 days of incubation and Free-Wilson binary
descriptors, experimentally determined log k, Hammett polar con-
Tetrahedron Lett. 2006, 47, 5007.
ˇ
20. Dolezal, M.; Pálek, L.; Vinšová, J.; Buchta, V.; Jampílek, J.; Králová, K. Molecules
2006, 11, 242.
21. http://www.taacf.org/Process-text.htm
ˇ
22. Sedlák, M.; Pravda, M.; Štaud, F.; Kubicová, L.; Tycova, K.; Ventura, K. Bioorg.
Med. Chem. 2007, 15, 4069.
stants r
, and Taft inductive r* were investigated using the Intelli-
23. Trajan Neural Network Simulator 6.0D; Trajan Software Ltd, Lincs, United
Kingdom; http://www.trajan-software.demon.co.uk/.
24. Hansch, C.; Leo, A. J. Substituent Constants for Correlation Analysis in Chemistry
and Biology; John Wiley & Sons: New York, 1979.
25. Taft, R. W. Steric Effects in Organic Chemistry; J. Wiley & Sons: New York,
1962.
gent Problem Solver (IPS) in Trajan 6.0 and this method is
completely described in Supplementary data. The IPS automati-
cally generates and examines different Artificial Neural Networks
(ANNs) and observes their statistical performance.