Salicylanilide carbamates: Antitubercular agents active against multidrug-resistant Mycobacterium tuberculosis strains

Article abstract of DOI:10.1016/j.bmc.2009.12.055

A series of 27 salicylanilide-based carbamates was prepared as a part of our ongoing search for new antituberculosis drugs. These compounds exhibited very good in vitro activity against Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium and, in particular, against five multidrug-resistant strains, with MIC values between 0.5-2 μmol/L. Moreover, they displayed moderate toxicity against intestinal cells with the selectivity index being up to 96. Furthermore, acid stability and a half-life of 43 h at pH 7.4 were shown. Thus, these novel salicylanilide derivatives are drug candidates which should be seriously consider for further screening.

Full text of DOI:10.1016/j.bmc.2009.12.055

Bioorganic & Medicinal Chemistry 18 (2010) 1054–1061
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Salicylanilide carbamates: Antitubercular agents active against
multidrug-resistant Mycobacterium tuberculosis strains
a
a
b,c
d
e
ˇ
´
ˇ
ˇ
Juana M. Férriz , Katerina Vávrová , Filip Kunc , Aleš Imramovsky , Jirina Stolaríková ,
a
a,
*
ˇ
Eva Vavríková , Jarmila Vinšová
a Charles University, Faculty of Pharmacy, Department of Inorganic and Organic Chemistry, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Charles University, Faculty of Medicine in Hradec Králové, Department of Medicinal Biology, Šimkova 870, 500 38 Hradec Králové, Czech Republic
c Generi Biotech, Machkova 587, 500 11 Hradec Králové, Czech Republic
d Institute of Organic Chemistry and Technology, Faculty of Chemistry and Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic
e Laboratory for TBC, Health Institute in Ostrava, 702 00 Ostrava, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2009
Revised 15 December 2009
Accepted 20 December 2009
Available online 29 December 2009
A series of 27 salicylanilide-based carbamates was prepared as a part of our ongoing search for new anti-
tuberculosis drugs. These compounds exhibited very good in vitro activity against Mycobacterium tuber-
culosis, Mycobacterium kansasii and Mycobacterium avium and, in particular, against five multidrug-
resistant strains, with MIC values between 0.5–2 lmol/L. Moreover, they displayed moderate toxicity
against intestinal cells with the selectivity index being up to 96. Furthermore, acid stability and a half-life
of 43 h at pH 7.4 were shown. Thus, these novel salicylanilide derivatives are drug candidates which
should be seriously consider for further screening.
Keywords:
Salicylanilide carbamates
Multidrug-resistant tuberculosis
In vitro antimycobacterial activity
Cytotoxicity
Ó 2009 Elsevier Ltd. All rights reserved.
Chemical hydrolysis
1. Introduction
antimycobacterial effect.15 Their activity results from multiple
mechanisms. SAL were identified as inhibitors of the two-com-
The recent worldwide emergence of drug-resistant tuberculosis
(TB) is alarming, especially the increase of multidrug-resistant
tuberculosis (MDR-TB),1 and most recently, extensively drug-resis-
tant tuberculosis (XDR-TB).2 Every year around 9 million people
develop the active and contagious pulmonary TB and 20% of them
die of their infection.3 More than four percent of all the worldwide
tuberculosis patients are resistant to at least one of the current first
line medications.4 Furthermore, every year almost 500,000 people
are infected with MDR-TB and there are estimated 40,000 new
cases of XDR-TB annually.5,6 TB in co-occurrence with the spread
of human immunodeficiency virus infection7 belong amongst the
most serious worldwide health threats. Therefore, effective new
drugs8 and strategies9 to treat the TB bacilli as well as its resistance
pattern are an urgent demanding task.
ponent regulatory systems16 of bacteria17,18 by a mechanism re-
lated to the effects on uncoupling oxidative phosphorylation. In
recent studies, they were also found to be selective inhibitors
of interleukin-12p40 production that play specific role in the ini-
tiation, expansion and control of the cellular response to TB as
well.19,20
In the recent past, a number of organic carbamates have been
found as potential antibacterial and antiviral agents.21 The carba-
mate residue present in these new molecules contributes as a core
component22 or incorporated into a known molecule, contributes
to the improvement of its pharmacodynamic and pharmacokinetic
properties.23 In particular, carbamate was successfully used to pro-
tect phenolic drugs.24
Thus, we hypothesised that masking the phenolic hydroxyl in
SAL by carbamate formation may protect the molecule against
extensive first-pass metabolism following oral administration,
broaden its activity profile and improve its physicochemical
and pharmacokinetic properties. In this context, the aim of this
article was to describe the synthesis, antimycobacterial activity
and cytotoxicity of a series of SAL carbamates as well as to eval-
uate their stability against chemical hydrolysis at various pH
values.
Salicylanilides (SAL) are an important class of aromatic com-
pounds with a wide range of pharmacological activities, such
as antibacterial,10–12 antifungal13 and anti-inflammatory,14
among others. Furthermore; several studies reported their potent
* Corresponding author.
E-mail address: vinsova@faf.cuni.cz (J. Vinšová).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.055

J. M. Férriz et al. / Bioorg. Med. Chem. 18 (2010) 1054–1061
1055
Scheme 1. Synthesis of 4-chloro-2-(R1-chlorophenylcarbamoyl)phenyl alkylcarbamates (R1 = 3-Cl, 3,4-diCl, 4-Cl, R2 = ethyl, butyl, pentyl, hexyl, heptyl, oktyl, nonyl, decyl,
undecyl). Reagents and conditions: (a) PCl3, chlorobenzene, microwave irradiation (530 W); (b) TEA, ACN, rt.
2.2. Antimycobacterial activities
2. Results and discussion
2.1. Chemistry
The prepared carbamates 4 were tested in vitro for their anti-
mycobacterial activity in the Laboratory for TBC, Health Institute in
Ostrava, against M. tuberculosis 331/88 and against some non-TB
strains such as Mycobacterium avium (330/88) and Mycobacterium
kansasii (235/80 and 6509/96), where the first line drug isoniazid
(INH) shows no activity. The anti-TB screening results of the com-
pounds 4 are summarized in Table 1.
The preparation of the carbamates is outlined in Scheme 1. The
starting SAL 3 were selected according to previous results showing
high in vitro activity against Mycobacterium tuberculosis25 They
were routinely prepared by the reaction of 5-chlorosalicylic acid
1 with the appropriate aniline 2 in chlorobenzene with PCl3.26 By
using microwave irradiation, the reaction time was shortened from
several hours to minutes.
In general, there are two factors influencing the activity of these
compounds. Firstly, themost active derivatives with minimalinhibi-
tion concentration (MIC) values from 0.5 to 2 lmol/L possess two
For the synthesis of the corresponding carbamates 4, a suspen-
sion of SAL 3 in acetonitrile (ACN) was treated with one equivalent
of triethylamine (TEA), adding then the corresponding isocyanate.
This reaction was performed at room temperature due to thermal
instability of the products. The prepared carbamates 4a–zz belong
into three series: those having chlorine at position 4 of salicylic
part, and at positions 3 and 4, respectively, of the anilide ring
(see Table 1). The yields of the synthesized compounds varied in
the interval of 35–80%. The carbamates 4 were characterised by
means of infrared, NMR spectroscopy and elemental analyses.
chlorines at positions 3 and 4 of the aniline moiety (4j–4r). Such
presence of an electron acceptor substituent in the aniline moiety
was previously found to play an important role in the activity of
SAL.25 Focusing our attention on the alkyl chain, the compounds
with 5, 6 or 7 carbons (4d, 4l, 4m, 4n and 4u) showed the highest
activity with MIC of 0.5–1 lmol/L. This suggests that the lipophilic-
ityofthecarbamatesplaysanimportantroleintheirbiologicalactiv-
ity. The Log P values of these compounds (Table 1), that is, the
logarithm of the partition coefficient for n-octanol/water, calculated
Table 1
Antimycobacterial activity and calculated lipophilicity of SAL alkylcarbamates 4
R1
R2
MIC (
l
mol/L)
M. kansasii 235/80
log P/C log Pa
M. tuberculosis 331/88
M. avium 330/88
M. kansasii 6509/96
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
Ethyl
Butyl
2
2
2
0.5
1
2
1
1
2
0.5
0.5
0.5
0.5
0.5
0.5
2
2
2
1
2
2
4
4
1
2
2
2
2
2
1
1
0.5
0.5
1
1
2
2
2
2
2
0.5
2
2
2
2
8
8
8
8
8
4
2
8
8
16
8
8
4
4
4
8
8
16
8
4
8
4
8
8
8
8
8
16
8
8
8
4
4
8
8
32
16
16
8
8
8
8
8
16
8
8
8
16
8
4
4
8
4
8
8
2
2
2
2
2
2
8
4
4
4
4
2
4
8
8
4
8
16
8
4
8
8
4
8
8
4
2
2
2
2
4
8
4
4
8
4
4
4
8
8
4
8
8
8
4
8
4
4
8
8
2
2
2
1
2
2
4
2
4
4
2
4
2
4
4
4
8
8
4
8
16
8
4
8
8
4
8
8
4
4
4
2
4
4
4
4
4
8
4
4
4
4
4
4
8
8
4
3.79/4.19
4.69/5.25
5.11/5.78
5.53/6.31
5.94/6.84
6.36/7.37
6.78/7.89
7.19/8.42
7.61/8.95
4.35/4.82
5.25/5.88
5.67/6.41
6.08/6.93
6.50/6.46
6.92/7.99
7.34/8.52
7.77/9.05
8.17/9.58
3.79/4.19
4.69/5.25
5.11/5.78
5.53/6.31
5.94/6.84
6.36/7.37
6.78/7.89
7.19/8.42
7.61/8.95
—
Pentyl
Hexyl
Heptyl
Octyl
Nonyl
Decyl
Undecyl
Ethyl
3-Cl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diCl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
Nonyl
Decyl
Undecyl
Ethyl
4s
4t
Butyl
4u
4v
4w
4x
4y
4z
4zz
INH
Pentyl
Hexyl
Heptyl
Octyl
Nonyl
Decyl
Undecyl
—
0.5
1
2
2
2
2
2
0.5
8
8
8
8
16
8
8
2
2
0.5
8
8
>250
8
8
>250
8
>250
—
>250
a
The Log P values were calculated using the program CS ChemOffice Ultra ver. 9.0 (CambridgeSoft, Cambridge, MA, USA).

1056
J. M. Férriz et al. / Bioorg. Med. Chem. 18 (2010) 1054–1061
Table 2
Activity of selected carbamates against MDR-TB strains
MIC (
M. tuberculosis
9449/06b
14 d 21 d
l
mol/L)
M. tuberculosis
2092/05c
14 d 21 d
EC50
(
l
mol /L) SI*
SI*
M. tuberculosis
M. tuberculosis
M. tuberculosis
M. tuberculosis
For MTB For MDR-TB
331/88
7357/98a
Praha 1d
Praha 128e
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
4d
4j
4k
4l
4m
4n
4o
4u
0.5
1
1
1
1
0.5
0.5
1
1
0.5
0.5
1
1
1
1
0.5
1
0.5
1
1
0.5
1
0.5
0.5
0.5
1
1
1
2
0.5
1
0.5
0.5
1
1
1
16
1
2
1
1
1
1
1
2
2
16
1
2
0.5
1
0.5
0.5
1
1
1
16
14.9
31.0
38.0
40.0
27.9
43.4
48.5
17.4
>100
14.9
31.0
38.0
80.0
55.8
43.4
48.5
34.8
>200
7.4–14.9
31–62
38–76
40–80
28–56
43.4–86.8
24–97
8.7–17.4
>6.25
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
1
0.5
1
16
2
16
1
16
1
16
INH 0.5
16
16
16
a
b
c
Resistant to INH, RMP, ETA, STM, OFX and ansamycin.
Resistant to INH, STM, RMP and ansamycin.
Resistant to INH, RMP, ETA, STM, OFX and ansamycin.
Resistant to INH, RMP, ETA, STM, CFZ and ansamycin.
d
e
Resistant to INH, RMP, ETA, STM, GTM, CFZ, ansamycin and AK.
*
SI = EC50/MIC.
using the program CS ChemOffice Ultra ver. 9.0 (CambridgeSoft,
Cambridge, MA, USA), are from 5 to 6.4, which does not correlate
with Lipinski rule of five. However, we can speculate that due to high
lipophilicity, the molecule presents higher membrane permeability,
making more effective the delivery of the parent drug. This hypoth-
esis is supported by the results of our previous research,26 where we
presented a series of 30 N-protected amino acid esters of salicylani-
lides.27 In this study, the most active derivatives resulted to be those
with log P values of approximately 6.
Although higher lipophilicity may be the reason for the increase in
the activities of the carbamates 4 against M. tuberculosis, kansasii and
avium compared to the starting SAL,25 it cannot explain their higher
potencycompared to the correspondingN-protectedamino acidester
derivatives, whose lipophilicities are comparable.27 For example,
compound 4mwas 16times morepotent that the parentSAL and four
times more active that the most active corresponding N-protected
amino acid ester derivative against M. tuberculosis (331/88).
Nevertheless, all the studied compounds displayed the selectivity
index (SI) value, defined as a ratio of EC50 to MIC, higher than 10
for both TB and MDR-TB strains (Table 2), what means that they
should be seriously considered for further screening.33
2.4. Stability of compound 4m
The chemical hydrolysis of compound 4m was studied in aque-
ous buffer solutions at pH from 3 to 8 at 37 °C. The decomposition
of 4m and the release of the parent SAL were monitored by HPLC
for 48 h. The carbamate bond was stable at acidic pH values but
decomposed in alkaline environment (Table 3, Fig. 1).
Table 3
Half-lives (t1/2) of the carbamate 4m at various pH values
pH
t1/2 (h)
The most active derivatives of this series were tested against
five clinically isolated MDR-TB strains as well, including M. tuber-
culosis 7357/98, resistant to INH, rifampicine (RMP), ethambutol
(ETA), streptomycin (STM), ofloxacin (OFX) and ansamycin, M.
tuberculosis 9449/06, resistant to INH, STM, RMP and ansamycin,
M. tuberculosis 2092/05, resistant to INH, RMP, ETA, STM, OFX
and ansamycin, M. tuberculosis Praha 1, resistant to INH, RMP,
ETA, STM, clofazimine (CFZ) and ansamycin, and M. tuberculosis
Praha 128 resistant to IHN, RMP, ETA, STM, gentamicin (GTM),
CFZ, ansamycin and amikacin (AK). All the studied compounds
exhibited high activity against the MDR-TB strains, with MIC val-
3
4
5
6
7
7.4
8
nd
nd
nd
117.5 19.5
64
43
5
8
6
2
Mean SEM, n = 3.
nd = no significant decomposition observed (p <0.05).
ues between 0.5–2 lmol/L (Table 2). These activities are compara-
100
80
60
40
20
0
ble with that presented by compounds undergoing PhaseII clinical
trials such a nitroimidazopyran PA-824,28,29 with MIC values be-
tween 0.1–0.7
diamine analogue of ETA, SQ109,30,31 with MIC values between
0.5–1.8 mol/L against strains resistant to ETA, INH and RMP.
lmol/L against mono and MDR-TB strains or the
l
2.3. Cytotoxicity of the most active compounds
4m
SAL
The in vitro mammalian cell toxicity of the most active com-
pounds 4d, 4j, 4k, 4l, 4m, 4n, 4o and 4u was assessed on human
intestinal cell line HCT-8 (ECACC, UK) using the XTT assay.32 The
cytotoxicity results presented in Table 2 are expressed as the con-
centration inhibiting 50% of the cell growth (EC50). The studied
compounds show moderate cytotoxicity in comparison to the stan-
0
6
12 18 24 30 36 42 48
time (h)
Figure 1. Hydrolysis of the carbamate 4m to the corresponding SAL in 100 mM
phosphate buffer at pH 7.4 at 37 °C.
dard INH with the EC50 values in the range of 14.9–48.5 lmol/L.

J. M. Férriz et al. / Bioorg. Med. Chem. 18 (2010) 1054–1061
1057
At physiological pH of 7.4, the carbamate 4m showed a t1/2 of
almost 2 days. Although these data describe chemical hydrolysis
only, we hypothesize that these compounds may also be stable
in plasma for sufficient time to reach its site of action, that is, the
mycobacteria. This assumption is supported by the relative stabil-
ity of the carbamate bond towards esterases.34 Moreover, in a pre-
vious study, a carbamate of a similar structure displayed a t1/2 of
15.9 and 2.7 h in a buffer and plasma, respectively.35 In addition,
due to the stability in acidic environment, these compounds may
be good candidates for oral administration. Furthermore, protec-
tion of the phenolic hydroxyl may render the compound less sus-
ceptible towards first-pass metabolism. However, the stability of
the synthesized SAL carbamates in biological environments and
their bioavailability warrants further investigation.
4.1.1.2. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl butylc-
arbamate (4b). (40%) as a white solid; mmax (KBr) 1654 (CO),
;
1723 (CO), 3279 (NH), 3343 (NH) cmꢀ1 1H NMR (THF-d8,
300 MHz): d ppm 9.58 (s, 1H, NH), 7.89 (dd, J = 2.0, 2.0 Hz, 1H,
H20), 7.65 (d, J = 2.6 Hz, 1H, H3), 7.56 (ddd, J = 8.2, 2.0, 0.9 Hz, 1H,
H40), 7.45 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.30–7.20 (m, 2H, H6, H50),
7.06 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H, H60), 3.11 (dt, J = 6.8, 5.6 Hz, 2H,
CH2), 1.49–1.24 (m, 4H, CH2), 0.85 (t, J = 7.2 Hz, 3H, CH3); 13C
NMR (THF-d8, 75 MHz): d ppm 163.9, 154.5, 148.5, 141.7, 134.9,
133.0, 131.5, 130.7, 130.6, 129.3, 125.7, 124.2, 120.2, 118.4, 41.6,
32.8, 20.6, 14.1. Anal. Calcd for C18H18Cl2N2O3 (381.25): C, 56.71;
H, 4.76; N, 7.35. Found: C, 56.83; H, 4.59; N, 7.49.
4.1.1.3. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl pentylc-
arbamate (4c). (63%) as a white solid; mmax (KBr) 1655 (CO),
1713 (CO), 3257 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d ppm
;
3. Conclusion
9.58 (s, 1H, NH), 7.89 (dd, J = 2.0, 2.0 Hz, 1H, H20),7.65 (d,
J = 2.6 Hz, 1H, H3), 7.56 (ddd, J = 7.9, 2.0, 0.9 Hz, 1H, H40), 7.45
(dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–7.29 (m, 2H, H6, H50), 7.13 (t,
J = 5.5 Hz, 1H, NH), 7.07 (ddd, J = 7.5, 2.0, 0.9 Hz, 1H, H60), 3.10
(dt, J = 6.1, 5.5 Hz, 2H, CH2), 1.51–1.40 (m, 2H, CH2), 1.31–1.23
(m, 4H, CH2), 0.86 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.8, 154.4, 148.4, 141.6, 134.8, 132.8, 131.4,
130.6, 130.5, 129.2, 125.6, 124.1, 120.1, 118.3, 41.8, 30.2, 29.6,
23.1, 14.2. Anal. Calcd for C19H20Cl2N2O3 (394.28): C, 57.73; H,
5.10; N, 7.09. Found: C, 57.52; H, 5.29; N, 7.04.
The results obtained in this study show that the derivatization
of the phenolic group of SAL in the form of carbamate appears to
be a useful approach to increase their antimycobacterial activity.
Whether this is a simple effect of an increased hydrophobicity
and therefore better permeability through the lipophilic mycobac-
terial cell wall or a direct effect of the carbamate moiety remains to
be elucidated. However, the high potencies of these newly pre-
pared compounds, particularly those against the MDR-TB strains,
together with their favorable selectivity makes these SAL-derived
carbamates promising drug candidates.
4.1.1.4. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl hexylc-
arbamate (4d). (68%) as a white solid; mmax (KBr) 1657 (CO),
4. Experimental
4.1. General
1716 (CO), 3262 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d ppm
;
9.57 (s, 1H, NH), 7.88 (dd, J = 2.0, 2.0 Hz, 1H, H2ꢀ), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.58–7.53 (m, 1H, H40), 7.45 (dd, J=8.7, 2.6 Hz,
1H, H5), 7.24 (m, 2H, H6, H50), 7.13 (t, J = 5.5 Hz, 1H, NH), 7.08–
7.05 (m, 1H, H60), 3.10 (dt, J = 6,18, 5,53 Hz, 2H, CH2), 1.44 (m,
2H, CH2), 1.27 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H); 13C NMR (THF-d8,
75 MHz): d ppm 163.9, 154.5, 148.5, 141.7, 135.0, 132.9, 131.5,
130.7, 130.6, 129.3, 125.7, 124.2, 120.2, 118.4, 41.9, 32.4, 30.6,
27.3, 23.4, 14.4. Anal. Calcd for C20H22Cl2N2O3 (409.31): C, 58.69;
H, 5.42; N, 6.84. Found: C, 58.22; H, 5.51; N, 7.00.
All reagents and solvents were purchased from commercial
sources (Sigma–Aldrich, Merck). Reactions were monitored by thin
layer chromatography plates coated with 0.2 mm silica gel 60 F254
(Merck). During the measurement of melting point, carbamates un-
dergo thermal degradation.36 Melting points of degradated products
were in therange of 212–248 °C andarenotpresented. Infrared spec-
tra (KBr pellets) were evaluated on FT-IR spectrometer Nicolet 6700
FT-IR in the range of 400–4000 cmꢀ1. NMR spectra were measured in
THF solutions at room temperature on a Varian Mercury-Vxbb 300
(300 MHz for 1H and 75.5 MHz for 13C; Varian Comp. Palo Alto, CA,
USA). Elemental analyses (C, H, N) were performed on an automatic
microanalyserCHNS-OCEinstrument(FisonsEA1110, Milano,Italy).
4.1.1.5. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl heptylc-
arbamate (4e). (40%) as a white solid; mmax (KBr) 1655 (CO),
;
1713 (CO), 3267 (NH), 3331 (NH) cmꢀ1 1H NMR (THF-d8,
300 MHz): d ppm 9.58 (s, 1H, NH), 7.88 (dd, J = 2.0, 2.0 Hz, 1H,
H20), 7.65 (J = 2.6 Hz, 1H, H3), 7.58–7.54 (m, 1H, H40), 7.45 (dd,
J = 8.7, 2.6 Hz, 1H, H5), 7.30–7.20 (m, 2H, H6, H50), 7.13 (t,
J = 5.3 Hz, 1H, NH), 7.07 (ddd, J = 8.1, 1.8, 1.1 Hz, 1H, H60), 3.10
(dt, J = 6.1, 5.3 Hz, 2H, CH2), 1.50–1.40 (m, 2H, CH2), 1.34–1.22
(m, 8H, CH2), 0.88 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.8, 154.4, 148.4, 141.6, 134.8, 132.8, 131.4,
130.6, 130.5, 129.2, 125.6, 124.1, 120.1, 118.3, 41.8, 32.6, 30.5,
29.8, 27.5, 23.4, 14.3. Anal. Calcd for C21H24Cl2N2O3 (423.33): C,
59.58; H, 5.71; N, 6.62. Found: C, 59.22; H, 5.43; N, 6.60.
4.1.1. General procedure for the preparation of 4-chloro-2-(R1-
chlorophenylcarbamoyl)-phenyl alkylcarbamates 4
To a suspension of the corresponding SAL 3 (1 mmol) in ACN
(7 mL) was added one equivalent of TEA (1 mmol, 101 mg). After
SAL dissolved, the appropriate isocyanate (1 mmol) was added in
four fractions during 2 h at room temperature under stirring. After
the complete addition of the isocyanate, the reaction mixture was
stirred for two more hours at room temperature. The resulting
crystals were filtered off and washed with methanol.
4.1.1.6. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl octylcar-
bamate (4f). (35%) as a white solid; mmax (KBr) 1659 (CO), 1713
4.1.1.1. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl ethylcar-
bamate (4a). (35%) as a white solid; mmax (KBr) 1660 (CO), 1715 (CO),
(CO), 3325 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d ppm 9.57
;
(s, 1H, NH), 7.88 (dd, J = 1.9, 1.9 Hz, 1H, H20), 7.65 (d, J = 2.6 Hz,
1H, H3), 7.56 (ddd, J = 8.1, 1.9, 0.9 Hz, H40), 7.45 (dd, J = 8.7,
2.6 Hz, 1H, H5), 7.28–7.20 (m, 2H, H6, H50), 7.12 (t, J = 5.4 Hz, 1H,
NH), 7.06 (ddd, J = 8.1, 1.9, 0.9 Hz, 1H, H60), 3.09 (dt, J = 6.7,
5.4 Hz 2H, CH2), 1.51–1.39 (m, 2H, CH2), 1.31–1.25 (m, 10H), 0.88
(t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8, 75 MHz): d ppm 163.9,
154.5, 148.5, 141.7, 135.0, 132.9, 131.5, 130.7, 130.6, 129.4,
125.7, 124.2, 120.2, 118.4, 41.9, 32.8, 30.7, 30.2, 30.2, 27.6, 23.5,
3279 (NH), 3343 (NH) cmꢀ1; 1H NMR (THF-d8, 300 MHz): d ppm 9.57
(s, 1H, NH), 7.88 (s, 1H, H20), 7.65 (s, 1H, H3), 7.55 (d, J = 8.0 Hz, 1H,
H40), 7.45 (d, J = 8.6 Hz, 1H, H5), 7.29–7.21 (m, 2H, H6, H50), 7.16–
6.94 (m, 2H, NH, H60), 3.21–3.05 (m, 2H, CH2), 1,10 (t, J = 7.1 Hz,
3H, CH3); 13C NMR (THF-d8, 75 MHz): d ppm 163.9, 154.4, 148.5,
141.7, 134.9, 132.9, 131.5, 130.7, 129.4, 125.7, 124.2, 120.2, 118.4,
36.7, 15.2. Anal. Calcd for C16H14Cl2N2O3 (353.20): C, 54.41; H,
4.00; N, 7.93. Found: C, 54.23; H, 3.89; N, 7.90.

1058
J. M. Férriz et al. / Bioorg. Med. Chem. 18 (2010) 1054–1061
14.4. Anal. Calcd for C22H26Cl2N2O3 (437.36): C, 60.42; H, 5.99; N,
6.41. Found: C, 59.84 H, 5.85; N, 6.38.
140.3, 132.9, 132.8, 131.7, 131.2, 130.6, 129.2, 127.0, 125.7, 121.9,
120.0, 41.6, 32.8, 20.6, 14.0. Anal. Calcd for C18H17Cl3N2O3 (415.70):
C, 52.01; H, 4.12; N, 6.74. Found: C, 52.13; H, 4.18; N, 7.01.
4.1.1.7. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl nonylc-
arbamate (4g). (54%) as a white solid; mmax (KBr) 1656 (CO),
4.1.1.12. 4-Chloro-2-(3,4dichlorophenylcarbamoyl)-phenyl pen-
1716 (CO), 3259 (NH), 3323 (NH) cmꢀ1
;
1H NMR (THF-d8,
tylcarbamate (4l). (75%) as a white solid; mmax (KBr) 1659 (CO),
300 MHz): d ppm 9.56 (s, 1H, NH), 7.88 (dd, J = 1.9, 1.9 Hz, 1H,
H20), 7.65 (d, J = 2.6 Hz, 1H, H3), 7.59–7.53 (m, 1H, H40), 7.45 (dd,
J = 8.7, 2.6 Hz, 1H, H5), 7.24 (m, 2H, H6, H50), 7.12 (t, J = 5.2 Hz,
1H, NH), 7.06 (ddd, J = 8.0, 1.9, 1.0 Hz, 1H, H60), 3.10 (dt, J = 6.6,
5.2 Hz, 2H, CH2), 1.31–1.24 (m, 12H, CH2), 0.89 (t, J = 6.7 Hz, 3H,
CH3); 13C NMR (THF-d8, 75 MHz): d ppm 163.9, 154.5, 148.5,
141.7, 135.0, 133.0, 131.5, 130.7, 130.6, 129.4, 125.7, 124.2,
120.2, 118.4, 41.9, 32.8, 30.7, 30.6, 30.5, 30.3, 27.6, 23.6, 14.4. Anal.
Calcd for C23H28Cl2N2O3 (451.39): C, 61.20; H, 6.25; N, 6.21. Found:
C, 61.28; H, 5.85; N, 6.18.
1715 (CO), 3328 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d ppm
;
9.67 (s, 1H, NH), 8.03 (d, J = 2.4 Hz, 1H, H20), 7.65 (d, J = 2.7 Hz, 1H,
H3), 7.57 (dd, J = 8.7, 2.4 Hz, 1H, H60), 7.49–7.41 (m, 2H, H5, H50),
7.21 (d, J = 8.8 Hz, 1H, H6), 7.11 (t, J = 5.4 Hz, 1H, H50), 3.09 (dt,
J = 6.7, 5.4, 2H, CH2), 1.50–1.39 (m, 2H, CH2), 1.32–1.21 (m, 4H,
CH2), 0.86 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (THF, 75 MHz): d ppm
164.1, 154.5, 148.6, 140.2, 132.9, 132.7, 131.6, 131.2, 130.6, 129.2,
127.0, 125.7, 121.9, 120.0, 41.9, 30.3, 29.7, 23.2, 14.3. Anal. Calcd
for C19H19Cl3N2O3 (429.72): C, 53.10; H, 4.46; N, 6.52. Found: C,
53.01; H, 4.23; N, 6.72.
4.1.1.8. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl decylc-
4.1.1.13. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl hex-
arbamate (4h). (63%) as a white solid; mmax (KBr) 1660 (CO),
ylcarbamate (4m). (74%) as a white solid; mmax (KBr) 1664 (CO),
1713 (CO), 3270 (NH), 3329 (NH) cmꢀ1
;
1H NMR (THF-d8,
1724 (CO), 3286 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d ppm
;
300 MHz): d ppm 9.57 (s, 1H, NH), 7.88 (dd, J = 2.0, 2.0 Hz, 1H,
H20), 7.65 (d, J = 2.6 Hz, H3), 7.58–7.53 (m, 1H, H40), 7.45 (dd,
J = 8.7, 2.6 Hz, 1H, H5), 7.28–7.20 (m, 2H, H6, H50), 7.12 (t,
J = 5.4 Hz, 1H, NH), 7.06 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H, H60), 3.09
(dt, J = 6.8, 5.4 Hz, 2H, CH2), 1.49–1.39 (m, 2H, CH2), 1.33–1.23
(m, 14H, CH2), 0.89 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.9, 154.5, 148.5, 141.7, 134.9, 132.9, 131.5,
130.7, 129.3, 125.7, 124.2, 120.2, 118.4, 41.9, 32.9, 30.7, 30.6,
30.5, 30.3, 30.3, 27.6, 23.6, 14.4. Anal. Calcd for C24H30Cl2N2O3
(465.41): C, 61.94; H, 6.50; N, 6.02. Found: C, 61.88; H, 6.75; N,
6.05.
9.67 (s, 1H, NH), 8.03 (d, J = 2.4 Hz, 1H, H20), 7.65 (d, J = 2.6 Hz, 1H,
H3), 7.57 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.48–7.41 (m, 2H, H5, H50),
7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.5 Hz, 1H, NH), 3.09 (dt,
J = 6.9, 5.5 Hz, 2H, CH2), 1.49–1.38 (m, 2H, CH2), 1.33–1.21 (m, 6H,
CH2), 0.87 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8, 75 MHz): d
ppm 164.1, 154.5, 148.6, 140.3, 132.9, 132.8, 131.7, 131.2, 130.6,
129.3, 127.0, 125.7, 121.9, 120.0, 41.9, 32.5, 30.6, 27.3, 23.5, 14.4.
Anal. Calcd for C20H21Cl3N2O3 (443.75): C, 54.13; H, 4.77; N, 6.31.
Found: C, 53.92; H, 4.55; N, 6.42.
4.1.1.14. 4-Chloro-2-(3,4dichlorophenylcarbamoyl)-phenyl hep-
tylcarbamate (4n). (53%) as a white solid; mmax (KBr) 1659 (CO),
4.1.1.9. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl undecyl-
1714 (CO), 3268 (NH), 3330 (NH) cmꢀ1 1H NMR (THF-d8,
;
carbamate (4i). (82%) as a white solid;
m
max (KBr) 1659 (CO), 1714
300 MHz): d ppm 9.67 (s, 1H, NH), 8.03 (d, J = 2.4 Hz, 1H, H20),
7.65 (d, J = 2.6 Hz, 1H, H3), 7.58 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.48–
7.41 (m, 2H, H5, H50), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t,
J = 5.6 Hz, 1H, NH), 3.09 (dt, J = 6.9, 5.6, 2H, CH2), 1.49–1.38 (m,
2H, CH2), 1.33–1.25 (m, 8H, CH2), 0.88 (t, J = 6.9 Hz, 3H, CH3); 13C
NMR (THF-d8, 75 MHz): d ppm 164.1, 154.5, 148.6, 140.3, 132.9,
132.8, 131.7, 131.2, 130.6, 129.3, 127.0, 125.7, 121.9, 120.0, 41.9,
32.7, 30.7, 29.9, 27.6, 23.5, 14.4. Anal. Calcd for C21H23Cl3N2O3
(457.78): C, 55.10; H, 5.06; N, 6.12. Found: C, 54.97; H, 4.88; N, 6.03.
(CO), 3270 (NH), 3330 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.57 (s, 1H, NH), 7.88 (dd, J = 2.0 Hz, 1H, H20), 7.65 (d,
J = 2.6 Hz, H3), 7.58–7.53 (m, 1H, H40), 7.45 (dd, J = 8.7, 2.6 Hz,
1H, H5), 7.28–7.20 (m, 2H, H6, H50), 7.12 (t, J = 5.5 Hz, 1H, NH),
7.06 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H, H60), 3.09 (dt, J = 6.81, 5.49 Hz,
2H, CH2), 1.50–1.40 (m, 2H, CH2), 1.34–1.23 (m, 16H, CH2), 0.89
(t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8, 75 MHz): d ppm 163.9,
154.5, 148.5, 141.7, 135.0, 133.0, 131.5, 130.7, 130.6, 129.4,
125.7, 124.2, 120.2, 118.4, 42.0, 32.9, 30.7, 30.6, 30.6, 30.5, 30.3,
30.3, 27.6, 23.6, 14.4. Anal. Calcd for C25H32Cl2N2O3 (479.44): C,
62.63; H, 6.73; N, 5.84. Found: C, 62.40; H, 6.76; N, 6.00.
4.1.1.15. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl octyl-
carbamate (4o). (39%) as a white solid; mmax (KBr) 1659 (CO), 1713
(CO), 3266 (NH), 3330 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
4.1.1.10. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl eth-
ppm 9.66 (s, 1H, NH), 8.03 (d, J = 2.4 Hz, 1H, H20), 7.65 (d, J = 2.6 Hz,
1H, H3), 7,57 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.48–7.40 (m, 2H, H5,
H50), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.12 (t, J = 5.5 Hz, 1H, NH), 3.09 (dt,
J = 6.5, 5.5 Hz, 2H, CH2), 1.49–1.38 (m, 2H, CH2), 1.33–1.25 (m, 10H,
CH2), 0.89 (t, J = 6.5 Hz, 3H, CH3); 13C NMR (THF-d8, 75 MHz): d
ppm 164.1, 154.5, 148.6, 140.3, 132.9, 132.8, 131.7, 131.2, 130.7,
129.3, 127.0, 125.7, 121.9, 120.0, 41.9, 32.8, 30.7, 30.2, 30.2, 27.6,
23.5, 14.5. Anal. Calcd for C22H25Cl3N2O3 (471.80): C, 56.01; H, 5.34;
N, 5.94. Found: C, 56.12; H, 5.20; N, 6.05.
ylcarbamate (4j). (64%) as a white solid;
mmax (KBr) 1661 (CO), 1716
(CO), 3266 (NH), 3336 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.68 (s, 1H, NH), 8.04 (d, J = 2.4 Hz, 1H, H20),7.66 (d,
J = 2.6 Hz, 1H, H3), 7.58 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.49–7.42 (m,
2H, H5, H50), 7.23 (d, J = 8.7 Hz, 1H, H6), 7.12 (t, J = 4.9 Hz, 1H,
NH), 3.18–3.08 (m, 2H, CH2), 1.07 (t, J = 7.2 Hz, 3H, CH3); 13C NMR
(THF-d8, 75 MHz): d ppm 164.1, 154.4, 148.6, 140.2, 132.9, 132.7,
131.7, 131.2, 130.6, 129.3, 127.0, 125.7, 121.9, 120.0, 36.7, 15.3.
Anal. Calcd for C16H13Cl3N2O3 (387.00): C, 49.57; H, 3.38; N, 7.23.
Found: C, 49.47; H, 3.25; N, 7.03.
4.1.1.16. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl non-
ylcarbamate (4p). (58%) as a white solid;
mmax (KBr) 1659 (CO), 1715
4.1.1.11. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl butyl-
(CO), 3266 (NH), 3329 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
carbamate (4k). (75%) as a white solid; mmax (KBr) 1656 (CO), 1720
ppm 9.67 (s, 1H, NH), 8.02 (d, J = 2.4 Hz, 1H, H20), 7.65 (d,
J = 2.5 Hz, 1H, H3), 7.58 (dd, J = 8.6, 2.4 Hz, 1H, H60), 7.49–7.41 (m,
2H, H5, H50), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.4 Hz, 1H, NH),
3.08 (dt, J = 6.0, 5.4 Hz, 2H, CH2), 1.48–1.37 (m, 2H, CH2), 1.36–1.20
(m, 12H, CH2), 0.89 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 164.1, 154.5, 148.6, 140.3, 132.9, 132.8, 131.7,
131.7, 130.6, 129.3, 127.0, 125.7, 121.9, 120.0, 41.9, 32.8, 30.7,
(CO), 3266 (NH), 3338 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.68 (s, 1H, NH), 8.04 (d, J = 2.4 Hz, 1H, H20), 7.65 (d, J = 2.6 Hz,
1H, H3), 7.58 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.48–7.42 (m, 2H, H5,
H50), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.4 Hz, 1H, H50), 3.10 (dt,
J = 6.7, 5.4 Hz, 2H, CH2), 1.49–1.23 (m, 4H, CH2), 1.07 (t, J = 7.2 Hz,
3H, CH3); 13C NMR (THF-d8, 75 MHz): d ppm 164.1, 154.5, 148.6,

J. M. Férriz et al. / Bioorg. Med. Chem. 18 (2010) 1054–1061
1059
30.5, 30.3, 27.6, 25.8, 23.6, 14.5. Anal. Calcd for C23H27Cl3N2O3
(485.83): C, 56.86; H, 5.60; N, 5.77. Found: C, 56.58; H, 5.49; N, 6.00.
4.1.1.22. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl hexylc-
arbamate (4v). (36%) as a white solid; mmax (KBr) 1657 (CO), 1720
(CO), 3285 (NH), 3332 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
4.1.1.17. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl decyl-
ppm 9.53 (s, 1H, NH), 7.73–7.68 (m, 2H, H30, H50), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–7.26 (m,
2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.5 Hz, 1H,
NH), 3.09 (dt, J = 6.8, 5.5 Hz, 2H, CH2), 1.49–1.38 (m, 2H, CH2),
1.33–1.20 (m, 6H), 0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.8, 154.6, 148.5, 139.2, 133.1, 131.4, 130.6,
129.4, 129.4, 129.0, 125.7, 121.7, 41.9, 32.5, 30.6, 27.3, 23.5, 14.4.
Anal. Calcd for C20H22Cl2N2O3 (409.31): C, 58.69; H, 5.42; N, 6.84.
Found: C, 58.74; H, 5.67; N, 6.75.
carbamate (4q). (70%) as a white solid; mmax (KBr) 1660 (CO), 1715
(CO), 3266 (NH), 3329 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.66 (s, 1H, NH), 8.02 (d, J = 2.4 Hz, 1H, H20), 7.65 (d, J = 2.6 Hz,
1H, H3), 7.58 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.48–7.41 (m, 2H, H5,
H50), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.6 Hz, 1H, NH), 3.09 (dt,
J = 6.8, 5.6 Hz, 2H, CH2), 1.50–1.39 (m, 2H, CH2), 1.36–1.20 (m, 14H,
CH2), 0.89 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8, 75 MHz): d ppm
164.1, 154.5, 148.6, 140.3, 132.9, 132.7, 131.7, 131.2, 130.6, 129.3,
128.1, 127.0, 125.7, 121.9, 41.9, 32.9, 30.7, 30.6, 30.6, 30.4, 30.3,
27.6, 23.6, 14.5. Anal. Calcd for C24H29Cl3N2O3 (499.86): C, 57.67; H,
5.85; N, 5.60. Found: C, 57.45; H, 5.73; N, 6.01.
4.1.1.23. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl heptyl-
carbamate (4w). (67%) as a white solid;
mmax (KBr) 1654 (CO), 1712
(CO), 3274 (NH), 3332 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
4.1.1.18. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)-phenyl un-
ppm 9.52 (s, 1H, NH), 7.73–7.68 (m, 2H, H30, H50), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–7.26 (m,
2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.5 Hz, 1H,
NH), 3.09 (dt, J = 6.8, 5.5 Hz, 2H, CH2), 1.49–1.38 (m, 2H, CH2),
1.34–1.22 (m, 8H), 0.89 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.7, 154.6, 148.5, 139.2, 133.1, 131.4, 129.6,
129.4, 129.3, 129.1, 125.7, 121.7, 41.9, 32.8, 30.7, 30.0, 27.6, 23.5,
14.4. Anal. Calcd for C21H24Cl2N2O3 (423.33): C, 59.58; H, 5.71; N,
6.84. Found: C, 59.28; H, 5.54; N, 6.93.
decylcarbamate (4r). (78%) as a white solid;
m
;
max (KBr) 1659 (CO),
1714 (CO), 3266 (NH), 3328 (NH) cmꢀ1
1H NMR (THF-d8,
300 MHz): d ppm 9.66 (s, 1H, NH), 8.03 (d, J = 2.4 Hz, 1H, H20),
7.65 (d, J = 2.6 Hz, 1H, H3), 7.58 (dd, J = 8.8, 2.4 Hz, 1H, H60), 7.51–
7.40 (m, 2H, H5, H50), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t,
J = 5.5 Hz, 1H, NH), 3.09 (dt, J = 6.6, 5.5 Hz, 2H, CH2), 1.48–1.40
(m, 2H, CH2), 1.36–1.20 (m, 14H, CH2), 0.89 (t, J = 6.6 Hz, 3H,
CH3); 13C NMR (THF-d8, 75 MHz): d ppm 164.2, 154.5, 148.6,
140.2, 132.9, 132.8, 131.7, 131.2, 130.6, 129.3, 127.0, 125.7, 121.9,
120.0, 41.9, 32.9, 30.7, 30.6, 30.6, 30.5, 30.5, 30.3, 27.6, 23.6, 14.4.
Anal. Calcd for C25H31Cl3N2O3 (513.88): C, 58.43; H, 6.08; N, 5.45.
Found: C, 58.66; H, 5.94; N, 5.89.
4.1.1.24. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl octylc-
arbamate (4x). (46%) as a white solid; mmax (KBr) 1655 (CO),
;
1716 (CO), 3276 (NH), 3334 (NH) cmꢀ1 1H NMR (THF-d8,
300 MHz): d ppm 9.53 (s, 1H, NH), 7.74–7.68 (m, 2H, H30, H50),
7.65 (d, J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–
7.25 (m, 2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.11 (t,
J = 5.4 Hz, 1H, NH), 3.09 (dt, J = 6.8, 5.4 Hz, 2H, CH2), 1.51–1.39
(m, 2H, CH2), 1.35–1.19 (m, 10H), 0.89 (t, J = 6.5 Hz, 3H, CH3); 13C
NMR (THF-d8, 75 MHz): d ppm 163.7, 154.5, 148.5, 139.2, 133.1,
131.4, 130.6, 129.4, 129.4, 129.0, 125.7, 121.7, 41.9, 32.8, 30.7,
30.2, 30.2, 27.6, 23.5, 14.5. Anal. Calcd for C22H26Cl2N2O3
(437.36): C, 60.42; H, 5.99; N, 6.41. Found: C, 60.34; H, 5.88; N,
6.27.
4.1.1.19. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl ethylc-
arbamate (4s). (46%) as a white solid; mmax (KBr) 1654 (CO), 1714
(CO), 3273 (NH), 3335 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.52 (s, 1H, NH), 7.74–7.68 (m, 2H, H30, H50), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.32–7.26 (m,
2H, H20, H60), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.2 Hz, 1H,
NH), 3.18–3.07 (m, 2H, CH2), 1.06 (t, J = 7.2 Hz, 3H, CH3); 13C
NMR (THF-d8, 75 MHz): d ppm 163.7, 154.4, 148.5, 139.2, 133.0,
131.4, 130.6, 129.6, 129.4, 129.0, 125.7, 121.7, 36.7, 15.2. Anal.
Calcd for C16H14Cl2N2O3 (353.20): C, 54.41; H, 4.00; N, 7.93. Found:
C, 54.20; H, 3.79; N, 7.56.
4.1.1.25. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl nonylc-
arbamate (4y). (71%) as a white solid; mmax (KBr) 1657 (CO), 1712
4.1.1.20. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl butylc-
(CO), 3282 (NH), 3342 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
arbamate (4t). (48%) as a white solid; mmax (KBr) 1656 (CO), 1714
ppm 9.52 (s, 1H, NH), 7.73–7.68 (m, 2H, H30, H50), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–7.25 (m,
2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.10 (t, J = 5.2 Hz, 1H,
NH), 3.09 (dt, J = 6.7, 5.2 Hz, 2H, CH2), 1.49–1.39 (m, 2H, CH2),
1.34–1.20 (m, 12H), 0.89 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-
d8, 75 MHz): d ppm 163.8, 154.6, 148.5, 139.2, 133.1, 131.4,
130.6, 129.6, 129.4, 129.0, 125.7, 121.7, 42.0, 30.7, 30.5, 30.3,
30.3, 23.6, 14.5. Anal. Calcd for C23H28Cl2N2O3 (451.39): C, 61.20;
H, 6.25; N, 6.21. Found: C, 61.07; H, 6.22; N, 6.35.
(CO), 3286 (NH), 3335 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.54 (s, 1H, NH), 7.74–7.68 (m, 2H, H30, H50), 7.64 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–7.26 (m,
2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.10 (t, J = 5.4 Hz, 1H, NH),
3.10 (dt, J = 6.7, 5.4 Hz, 2H, CH2), 1.48–1.23 (m, 4H, CH2), 0.85 (t,
J = 7.2 Hz, 3H, CH3); 13C NMR (THF-d8, 75 MHz): d ppm 163.8,
154.6, 148.5, 139.2, 133.1, 131.4, 130.6, 129.6, 129.4, 129.0, 125.7,
121.7, 41.6, 32.8, 20.6, 14.1. Anal. Calcd for C18H18Cl2N2O3
(381.25): C, 56.71; H, 4.76; N, 7.35. Found: C, 56.42; H, 4.83; N, 7.22.
4.1.1.26. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl decylc-
4.1.1.21. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl pentyl-
arbamate (4z). (51%) as a white solid; mmax (KBr) 1656 (CO), 1713
carbamate (4u). (63%) as a white solid;
m
max (KBr) 1657 (CO), 1717
(CO), 3278 (NH), 3330 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
(CO), 3282 (NH), 3336 (NH) cmꢀ1 1H NMR (THF-d8, 300 MHz): d
;
ppm 9.53 (s, 1H, NH), 7.74–7.68 (m, 2H, H30, H50), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.32–7.25 (m,
2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.5 Hz, 1H,
NH), 3.09 (dt, J = 6.7, 5.5 Hz, 2H, CH2), 1.48–1.39 (m, 2H, CH2),
1.34–1.20 (m, 14H), 0.89 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-
d8, 75 MHz): d ppm 163.8, 154.6, 148.5, 139.2, 133.1, 131.4,
130.6, 129.4, 129.4, 129.0, 125.7, 121.7, 41.9, 32.9, 30.7, 30.6,
30.5, 30.3, 30.3, 27.6, 23.6, 14.5. Anal. Calcd for C24H30Cl2N2O3
(465.41): C, 61.94; H, 6.50; N, 6.02. Found: C, 61.87; H, 6.74; N,
5.87.
ppm 9.53 (s, 1H, NH), 7.74–7.68 (m, 2H, H30, H50), 7.65 (d,
J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.32–7.26 (m,
2H, H20, H60), 7.21 (d, J = 8.7 Hz, 1H, H6), 7.10 (t, J = 5.6 Hz, 1H,
NH), 3.09 (dt, J = 6.8, 5.6 Hz, 2H, CH2), 1.51–1.40 (m, 2H, CH2),
1.32–1.23 (m, 4H), 0.87 (t, J = 6.7 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.7, 154.5, 148.5, 139.2, 133.1, 131.4, 130.6,
129.4, 129.4, 129.0, 125.7, 121.7, 41.9, 30.3, 29.8, 23.2, 14.3. Anal.
Calcd for C19H20Cl2N2O3 (395.28): C, 57.73; H, 5.10; N, 7.09. Found:
C, 57.55; H, 4.89; N, 7.15.

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J. M. Férriz et al. / Bioorg. Med. Chem. 18 (2010) 1054–1061
4.1.1.27. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl unde-
The reactions were stirred at 37 °C and at predetermined time
intervals, 300 l of each solution was withdrawn and analyzed
for 4m and the hydrolysis product. 20 l of the sample was in-
jected into a LiChroCART 125-4 column (LichroSpher RP-18e,
m; Merck, Darmstadt, Germany) equipped with a LiChroCART
4-4 precolumn with the same sorbent. Acetonitrile/20 mM phos-
phate buffer at pH 6.5 (7:3 v/v) was used as the mobile phase at
a flow rate of 2 mL/min and was monitored at 263 nm. The reten-
tion of 4m and the hydrolysis product was 4.0 and 1.6 min, respec-
tively. The calibration curves of 4m and the hydrolysis product
were linear (p <0.0001, R2 = 0.9997 and p <0.0001, R2 = 0.996,
cylcarbamate (4zz). (80%) as a white solid; mmax (KBr) 1656 (CO),
l
1713 (CO), 3278 (NH), 3338 (NH) cmꢀ1
;
1H NMR (THF-d8,
l
300 MHz): d ppm 9.52 (s, 1H, NH), 7.74–7.68 (m, 2H, H30, H50), 7.65
(d, J = 2.6 Hz, 1H, H3), 7.44 (dd, J = 8.7, 2.6 Hz, 1H, H5), 7.31–7.26
(m, 2H, H20, H60), 7.20 (d, J = 8.7 Hz, 1H, H6), 7.11 (t, J = 5.6 Hz, 1H,
NH), 3.09 (dt, J = 6.8, 5.6 Hz, 2H, CH2), 1.48–1.38 (m, 2H, CH2),
1.36–1.22 (m, 16H), 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (THF-d8,
75 MHz): d ppm 163.8, 154.6, 148.5, 139.2, 133.1, 131.4, 130.6,
129.6, 129.4, 129.0, 125.7, 121.7, 41.9, 32.9, 30.7, 30.6, 30.6, 30.5,
30.3, 30.3, 27.6, 23.6, 14.4. Anal. Calcd for C25H32Cl2N2O3 (479.44):
C, 62.63; H, 6.73; N, 5.84. Found: C, 62.75; H, 6.57; N, 6.58.
5
l
respectively) in the range of concentrations of 0.5–100 lg/ml.
The accuracy ranged from 93.13% to 108.55% and relative standard
deviation was less than 6.0%. Half-lives for the hydrolysis of the
carbamates were calculated from the linear slopes of plots of the
logarithm of remaining carbamate against time.
4.2. Materials and methods
4.2.1. In vitro antimycobacterial assay
In vitro antimycobacterial activity of the synthesized com-
pounds was evaluated against Mycobacterium tuberculosis My
331/88, M. avium My 330/88, M. kansasii My 235/80 and M. kansasii
6509/96. The most active compounds 4d, 4j, 4k, 4l, 4m, 4n, 4o, 4u
were tested as well against five MDR-TB strains: 7357/98, 9449/06,
2092/05, Praha 1 and Praha 128. For this purpose, the micro-
method37 for the determination of the minimum inhibitory con-
centration (MIC) was used. All the strains were obtained from
the Czech National Collection of Type Cultures (CNCTC) with the
exception of M. kansasii 6509/96 and the five MDR-TB strains
which were clinically isolated. The antimycobacterial activities of
the compounds were determined in a Šula semisynthetic medium
(SEVAC, Prague). The compounds were added to the medium in
DMSO solutions. The following concentrations were used: 1000,
Acknowledgment
This work was financially supported by the Research project
MSM 0021620822, GAUK 76807/2007 and IGA NS 10367-3.
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