Salicylanilide N-monosubstituted carbamates: Synthesis and in vitro antimicrobial activity

Article abstract of DOI:10.1016/j.bmc.2016.02.004

The research of innovative antimicrobial agents represents a cutting edge topic. Hence, we synthesized and characterised novel salicylanilide N-monosubstituted carbamates. Twenty compounds were evaluated in vitro against eight bacterial strains and eight fungal species. The lowest minimum inhibitory concentrations (MICs) were found to be ≤0.49 μM. Genus Staphylococcus, including methicillin-resistant Staphylococcus aureus, and fungus Trichophyton mentagrophytes showed uniformly the highest rate of susceptibility, whilst Gram-negative bacteria and most of the fungi were less susceptible. A wide range of carbamates provided comparable or superior in vitro antimicrobial activity in comparison to established drugs. Interestingly, extended-spectrum β-lactamase producing strain of Klebsiella pneumoniae was inhibited with MICs starting from 31.25 μM. With respect to Staphylococci, 2-[(4-bromophenyl)carbamoyl]-4-chlorophenyl phenylcarbamate exhibited the lowest MIC values (≤0.98 μM). 2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl benzylcarbamate showed the widest spectrum of antifungal action. The results indicate that some salicylanilide carbamates can be considered to be promising candidates for future investigation.

Full text of DOI:10.1016/j.bmc.2016.02.004

Bioorganic & Medicinal Chemistry 24 (2016) 1322–1330
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Salicylanilide N-monosubstituted carbamates:
Synthesis and in vitro antimicrobial activity
⇑
Martin Krátk y´ , Jarmila Vinšová
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The research of innovative antimicrobial agents represents a cutting edge topic. Hence, we synthesized
and characterised novel salicylanilide N-monosubstituted carbamates. Twenty compounds were
evaluated in vitro against eight bacterial strains and eight fungal species. The lowest minimum inhibitory
Received 19 August 2015
Revised 2 February 2016
Accepted 3 February 2016
Available online 4 February 2016
concentrations (MICs) were found to be 60.49 lM. Genus Staphylococcus, including methicillin-resistant
Staphylococcus aureus, and fungus Trichophyton mentagrophytes showed uniformly the highest rate of sus-
ceptibility, whilst Gram-negative bacteria and most of the fungi were less susceptible. A wide range of
carbamates provided comparable or superior in vitro antimicrobial activity in comparison to established
drugs. Interestingly, extended-spectrum b-lactamase producing strain of Klebsiella pneumoniae was
Keywords:
Antibacterial activity
Antifungal activity
In vitro activity
Salicylanilide carbamate
Staphylococcus aureus
inhibited with MICs starting from 31.25
carbamoyl]-4-chlorophenyl phenylcarbamate exhibited the lowest MIC values (60.98
l
M. With respect to Staphylococci, 2-[(4-bromophenyl)
M).
l
2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl benzylcarbamate showed the widest spectrum of anti-
fungal action. The results indicate that some salicylanilide carbamates can be considered to be promising
candidates for future investigation.
Ó 2016 Elsevier Ltd. All rights reserved.
1
. Introduction
Salicylanilide derivatives have exhibited significant antimicro-
11,12
bial activity against various pathogenic species. Salicylanilides,
1
3
11,14,15
8
Problems related to drug-resistance have been reported for
salicylanilide benzoates, 4-substituted benzoates,
N,N-dis-
many human pathogens including methicillin-resistant Staphylo-
coccus aureus (MRSA), vancomycin-resistant enterococci, and the
family of Enterobacteriaceae that produce extended-spectrum b-
lactamases (ESBL) or genus Candida. Mycoses have become an
important public health issue because of the increasing number
ubstituted methyl/phenyl salicylanilide carbamates and N-alkyl
salicylanilide carbamates
1
6,17
were investigated as potential
antibacterial and antifungal agents. Although lipophilicity seems
to be one of the factors modulating positively the antimicrobial
activity, an escalated lipophilicity could hamper in vitro evaluation
1
,2
11,14,15
of immunocompromised patients and nosocomial infections.
due to solubility problems in testing media.
Some of these infections are difficult to treat with established
antimicrobial drugs. The search for antibacterial and antifungal
agents, especially those with innovative mechanisms of action,
should bring novel compounds to overcome the microbial resis-
tance to clinically used drugs.
In this study, we synthesized and evaluated twenty novel sali-
cylanilide N-cycloalkyl/aryl/arylalkyl carbamates as potential
antimicrobial agents. In contrast to the Zadrazilova’s paper,17
which presents only MRSA inhibition activity of our earlier antimy-
cobacterial active N-monoalkylated carbamates (Fig. 1),16 this
study includes expansion of new compounds evaluated against
panel of eight bacterial and eight fungal strains as our ongoing
research. Here presented carbamates cover varied substitution pat-
terns in both salicylanilide and carbamate parts to identify possible
structure–activity relationship. The structures of previously pub-
lished salicylanilide carbamates in comparison with novel deriva-
tives is depicted in Figure 1.
Phenolic compounds have been investigated widely as potential
antimicrobial compounds, especially those obtained from natural
sources. Some of them have displayed in vitro activity even against
2
–6
drug-resistant bacterial and fungal strains,
or they were syner-
7
gic with antimycotics. O-Aromatic carbamates have been reported
as potential antimicrobial agents including their involvement in
the prodrug design strategy.8
–10
⇑
Corresponding author. Tel.: +420 495067343; fax: +420 495067166.
E-mail address: martin.kratky@faf.cuni.cz (M. Krátk y´ ).
http://dx.doi.org/10.1016/j.bmc.2016.02.004
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
0

M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
1323
R1
R2
R2
R2
O
O
O
O
O
O
R1
R1
Cl
N
H
N
H
N
H
R3
R3
O
N
H
X
N
O
N
H
n
4
R
R1 = H, Cl
1
2
3
1
2
3
R
R
R
= H, Cl
R
R
R
= H, Cl
= H, F, Cl, Br, CF3
2
R
= H, Cl
= H, F, Cl, Br
= cyclohexyl, phenyl, benzyl,
phenethyl, adamantan-1-yl
n = 1-10
4
= R = methyl or phenyl
Ferriz et al.16
X = O, S
Zadrazilova et al.17
Krátký et al.8
current work
Figure 1. Comparison of the general formulas of antimicrobial active salicylanilide carbamates.
2
2
2
. Material and methods
.1. Chemistry
served as a convenient solvent for phenyl derivatives 2b and 3e,
respectively. The synthesis of carbamates 1b and 3b was carried
out in a mixture of MeCN and dry acetone (a ratio of 1:1) with
addition of a catalytic amount of triethylamine (20 lL).
.1.1. General methods
All of the reagents and solvents were purchased from Sigma–
2.1.2.1. 4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl cyclo-
hexylcarbamate (1a). White solid; yield 83%; mp 192.5–
Aldrich (Darmstadt, Germany) or Penta Chemicals (Prague, Czech
Republic) and used as received. The reactions and the purity of
the products were monitored by thin-layer chromatography using
a mixture with a ratio of toluene to ethyl acetate of 4:1 or a ratio of
toluene to methanol of 9:1 as the eluent. The plates were coated
with 0.2-mm Merck 60 F254 silica gel and were visualised by UV
irradiation (254 nm). The melting points were determined on a
Büchi Melting Point B-540 apparatus (BÜCHI, Flawil, Switzerland)
using open capillaries, and the reported values are uncorrected.
Elemental analysis (C, H, and N) was performed on an automatic
microanalyser CHNS-O CE instrument (FISONS EA 1110, Milano,
Italy). Infrared spectra (ATR) were recorded on a FT-IR spectrome-
194 °C. IR (ATR): 3261 (N–H), 2944, 2929, 2854, 1720 (C@O carba-
mate), 1684 (amide I), 1591, 1551, 1528 (amide II), 1490, 1478,
1400, 1316, 1266, 1221, 1104, 1090, 1013, 883, 838, 824, 734,
ꢀ
1
1
668 cm
. H NMR (300 MHz, DMSO): d 10.40 (1H, bs, amide
0
0
NH), 7.78–7.68 (3H, m, carbamate NH, H2 , H6 ), 7.65 (1H, d,
J = 2.7 Hz, H3), 7.56 (1H, dd, J = 2.7 Hz, J = 8.8 Hz, H5), 7.38 (2H, d,
0
0
J = 8.9 Hz, H3 , H5 ), 7.24 (1H, d, J = 8.7 Hz, H6), 3.25–3.13 (1H, m,
CH), 1.72–1.43 (5H, m, cyclohexyl), 1.27–0.98 (5H, m, cyclohexyl).
1
3
C NMR (75 MHz, DMSO): d 163.23, 152.89, 147.43, 138.17,
132.00, 131.02, 129.04, 128.70, 127.44, 125.40, 122.43, 121.41,
20 2 2 3
50.06, 32.48, 25.27, 24.65. Anal. Calcd for C H20Cl N O
ꢀ1
ter (Nicolet 6700 FT-IR) in the range of 400–4000 cm . The NMR
spectra were measured in DMSO at ambient temperature using a
(407.29): C, 58.98; H, 4.95; N, 6.88. Found: C, 59.03; H, 4.84; N,
7.00.
1
Varian V NMR S500 instrument (500 MHz for H and 125 MHz
1
3
for C; Varian Comp., Palo Alto, CA, USA) or a Varian Mercury-
2.1.2.2. 4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl phenyl-
1
13
Vxbb 300 (300 MHz for H and 75.5 MHz for C; Varian, Inc., Palo
Alto, CA, USA). The chemical shifts, d, are given in ppm with respect
to tetramethylsilane, which was used as an internal standard. The
coupling constants (J) are reported in Hz.
carbamate (1b).
White solid; yield 50%; mp 210–212 °C. IR
(ATR): 3314 (N–H), 3268 (N–H), 3072, 1722 (C@O carbamate),
1650 (amide I), 1595, 1554, 1524 (amide II), 1491, 1447, 1406,
1315, 1233, 1215, 1200, 1179, 1103, 1006, 828, 754, 727, 697,
ꢀ1
1
The calculated logP values (ClogP), which are the logarithms of
the partition coefficients for octan-1-ol/water, were determined
using the CS ChemOffice Ultra program (version 13.0, Cambridge-
Soft, Cambridge, MA, USA).
670 cm
. H NMR (300 MHz, DMSO): d 10.58 (1H, bs, amide
NH), 10.25 (1H, s, carbamate NH), 7.76 (1H, d, J = 2.7 Hz, H3),
0
0
7.70 (2H, d, J = 8.8 Hz, H2 , H6 ), 7.64 (1H, dd, J = 2.7 Hz, J = 8.8 Hz,
0
0
00
00
H5), 7.47–7.34 (5H, m, H6, H3 , H5 , H2 , H6 ), 7.27 (2H, t,
0
0
00
00
13
J = 7.9 Hz, H3 , H5 ), 7.01 (1H, t, J = 7.4 Hz, H4 ).
C NMR
2
.1.2. Synthesis
(75 MHz, DMSO): d 163.16, 151.15, 146.91, 138.02, 131.83,
The parent salicylanilides SAL were synthesised according to
our previously described microwave-assisted synthetic method.
131.27, 129.68, 129.02, 128.86, 128.78, 128.66, 127.59, 125.86,
122.43, 121.57, 118.57. Anal. Calcd for C20H14Cl N O (401.24): C,
2 2 3
1
1
For the synthesis of the carbamates 1–4, we used slightly mod-
ified method according to Férriz et al.16 An equivalent of appropri-
ate salicylanilide (1 mmol) was suspended under vigorous stirring
in dry acetonitrile (MeCN; 8 mL), and then 1 mmol of tertiary
amine (triethylamine or N,N-diisopropylethylamine) was added
in one portion. The mixture was stirred for 5 min to allow complete
dissolution of the reaction mixture. Then, appropriate isocyanate
59.87; H, 3.52; N, 6.98. Found: C, 59.63; H, 3.70; N, 7.10.
2.1.2.3. 4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl benzyl-
carbamate (1c).
White solid; yield 93%; mp 187–188.5 °C. IR
(ATR): 3338 (N–H), 3282 (N–H), 3032, 1720 (C@O carbamate),
1653 (amide I), 1595, 1533, 1520 (amide II), 1493, 1476, 1400,
ꢀ1 1
1317, 1287, 1261, 1219, 1098, 1015, 921, 895, 833 cm . H NMR
(
1
1.1 of equivalents) was added in two portions, portion after
5 min, and the mixture was stirred at room temperature for addi-
(300 MHz, DMSO): d 10.50 (1H, bs, amide NH), 8.35 (1H, t,
0
0
J = 6.1 Hz, carbamate NH), 7.73 (2H, d, J = 8.8 Hz, H2 , H6 ), 7.69
(1H, d, J = 2.7 Hz, H3), 7.59 (1H, dd, J = 2.7 Hz, J = 8.7 Hz, H5), 7.40
tional 1–2 h (cyclohexyl, phenyl, benzyl and phenethyl iso-
cyanates) or 12 h (1-adamantyl isocyanate). The reaction was
monitored using TLC. Then, the resulting precipitate was collected
by filtration, washed with a small volume of cold methanol and
dried. If necessary, products were recrystallised from ethyl acetate.
The syntheses of carbamates from phenyl isocyanate and 1-ada-
mantyl isocyanate were performed under nitrogen atmosphere
and various solvents were tried. Dry acetone or tetrahydrofuran
0
0
(2H, d, J = 8.8 Hz, H3 , H5 ), 7.29 (1H, d, J = 8.7 Hz, H6), 7.25–7.18
0
0
00
00
00
00
13
(5H, m, H2 , H3 , H4 , H5 , H6 ), 4.21 (2H, d, J = 6.1 Hz, CH
2
).
C
NMR (75 MHz, DMSO): d 163.24, 154.17, 147.34, 139.18, 138.17,
132.08, 131.11, 129.31, 128.74, 128.55, 128.33, 127.48, 126.97,
126.95, 125.53, 121.48, 44.04. Anal. Calcd for C21H16Cl N O
2 2 3
(415.27): C, 60.74; H, 3.88; N, 6.75. Found: C, 60.61; H, 3.79; N,
7.00.

1
324
M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
746, 729, 698 cm 1. 1H NMR (300 MHz, DMSO): d 10.50 (1H, bs,
ꢀ
2
.1.2.4. 4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl phenethyl-
carbamate (1d). White solid; yield 95%; mp 159–161 °C. IR
amide NH), 8.36 (1H, t, J = 6.1 Hz, carbamate NH), 7.71–7.76 (3H,
0
0
(ATR): 3330 (N–H), 3275 (N–H), 3029, 2945, 1714 (C@O carbamate),
m, H3, H2 , H6 ), 7.59 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.56–7.50
0
0
1
1
6
7
655 (amide I), 1593, 1536, 1515 (amide II), 1493, 1475, 1397, 1316,
(2H, m, H3 , H5 ), 7.29 (1H, d, J = 8.7 Hz, H6), 7.25–7.17 (5H, m,
0
0
00
00
00
00
13
288, 1260, 1220, 1101, 1015, 957, 923, 863, 821, 775, 735, 698,
2
H2 , H3 , H4 , H5 , H6 ), 4.21 (2H, d, J = 6.1 Hz, CH ). C NMR
ꢀ1 1
69 cm . H NMR (300 MHz, DMSO): d 10.46 (1H, bs, amide NH),
(75 MHz, DMSO): d 163.25, 154.17, 147.33, 139.17, 138.58,
132.08, 131.12, 129.31, 128.54, 128.34, 126.98, 126.95, 125.53,
121.86, 115.55, 44.05. Anal. Calcd for C21H16BrClN O (459.72): C,
2 3
54.86; H, 3.51; N, 6.09. Found: C, 54.90; H, 3.39; N, 6.16.
0
.91 (1H, t, J = 5.7 Hz, carbamate NH), 7.74 (2H, d, J = 8.8 Hz, H2 ,
0
H6 ), 7.67 (1H, d, J = 2.6 Hz, H3), 7.57 (1H, dd, J = 2.6 Hz, J = 8.6 Hz,
H5), 7.39 (2H, d, J = 8.8 Hz, H3 , H5 ), 7.27–7.10 (6H, m, H6, H2 , H3 ,
0
0
00
00
00
00
00
H4 , H5 , H6 ), 3.19 (2H, dt, J = 6.2 Hz, J = 7.9 Hz, N-CH
2
), 2.67 (2H, t,
). C NMR (75 MHz, DMSO): d 163.21, 153.68, 147.26,
39.29, 138.16, 131.99, 130.97, 129.15, 128.83, 128.75, 128.45,
27.50, 126.25, 125.48, 121.48, 42.28, 35.26. Anal. Calcd for C22
(429.30): C, 61.55; H, 4.23; N, 6.53. Found: C, 61.65; H, 4.31; N,
13
J = 7.3 Hz, CH
1
1
2
2.1.2.9. 2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl phenethyl-
carbamate (2d).
White solid; yield 56%; mp 164–166 °C. IR
H18Cl
2
(ATR): 3337 (N–H), 3281 (N–H), 3029, 2945, 1716 (C@O carbamate),
1655 (amide I), 1589, 1535, 1513 (amide II), 1490, 1476, 1393, 1315,
1288, 1258, 1215, 1103, 1071, 1012, 958, 922, 836, 817, 774, 735,
2 3
N O
6.45.
ꢀ1 1
6
98, 664 cm . H NMR (300 MHz, DMSO): d 10.46 (1H, bs, amide
2
.1.2.5. 4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl adam-
White solid; yield 69%; mp 234–
36.5 °C. IR (ATR): 3299 (N–H), 2927, 2907, 2855, 1716 (C@O
carbamate), 1663 (amide I), 1589, 1534, 1519 (amide II), 1489, 1476,
NH), 7.91 (1H, t, J = 5.7 Hz, carbamate NH), 7.74–7.63 (3H, m, H3,
0
0
antan-1-ylcarbamate (1e).
2
H2 , H6 ), 7.57 (1H, dd, J = 2.7 Hz, J = 8.8 Hz, H5), 7.54–7.47 (2H, m,
0
0
00
00
00
00
00
H3 , H5 ), 7.27–7.09 (6H, m, H6, H2 , H3 , H4 , H5 , H6 ), 3.19 (2H,
13
2 2
dt, J = 6.2 Hz, J = 7.9 Hz, N-CH ), 2.67 (2H, t, J = 7.3 Hz, CH ). C NMR
1
9
397, 1359, 1310, 1297, 1283, 1252, 1214, 1108, 1087, 1023, 1011,
(75 MHz, DMSO): d 163.25, 153.70, 147.27, 139.30, 138.59, 132.01,
131.67, 131.00, 129.15, 128.85, 128.47, 126.27, 125.48, 121.86,
ꢀ1 1
20, 882, 823, 756, 674 cm . H NMR (300 MHz, DMSO): d 10.34
0
0
(
1H, bs, amide NH), 7.72 (2H, d, J = 8.4 Hz, H2 , H6 ), 7.65 (1H, d,
2 3
115.57, 42.28, 35.27. Anal. Calcd for C22H18BrClN O (473.75): C,
J = 2.6 Hz, H3), 7.59–7.52 (2H, m, carbamate NH, H5), 7.38 (2H, d,
55.78; H, 3.83; N, 5.91. Found: C, 55.64; H, 3.88; N, 6.03.
0
0
J = 8.4 Hz, H3 , H5 ), 7.20 (1H, d, J = 8.9 Hz, H6), 1.98–1.89 (3H, m,
1
3
CH), 1.84–1.74 (6H, m, C-CH
2
), 1.63–1.47 (6H, m, CH-CH
2
).
C
2.1.2.10.
2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl
White solid; yield 63%; mp
NMR (75 MHz, DMSO): d 165.12, 157.47, 147.33, 138.18, 137.40,
adamantan-1-ylcarbamate (2e).
1
4
33.12, 128.85, 128.60, 127.90, 122.34, 120.01, 119.45, 55.94,
4.63, 35.20, 29.26. Anal. Calcd for C24 (459.36):
212–213.5 °C. IR (ATR): 3295 (N–H), 2924, 2855, 1715 (C@O carba-
mate), 1665 (amide I), 1585, 1534, 1518 (amide II), 1486, 1476,
1393, 1359, 1310, 1298, 1283, 1252, 1215, 1107, 1066, 1022,
H
2 2 3
24Cl N O
C, 62.75; H, 5.27; N, 6.10. Found: C, 62.74; H, 5.50; N, 6.22.
ꢀ1 1
1
007, 978, 921, 882, 837, 821, 756, 669 cm . H NMR (300 MHz,
0
2
.1.2.6. 2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl cyclo-
hexylcarbamate (2a). White solid; yield 83%; mp 196.5–
98.5 °C. IR (ATR): 3262 (N–H), 2941, 2929, 2854, 1710 (C@O
carbamate), 1655 (amide I), 1587, 1551, 1527 (amide II), 1487, 1477,
DMSO): d 10.34 (1H, bs, amide NH), 7.71–7.62 (3H, m, H3, H2 ,
0
0
0
H6 ), 7.60–7.46 (4H, m, carbamate NH, H5, H3 , H5 ), 7.20 (1H, d,
J = 8.7 Hz, H6), 1.97–1.89 (3H, m, CH), 1.85–1.74 (6H, m, C-CH ),
). C NMR (75 MHz, DMSO): d 163.27,
1
2
1
3
1.62–1.47 (6H, m, CH-CH
2
1
7
394, 1315, 1278, 1265, 1219, 1105, 1068, 1009, 882, 838, 820,
82, 734, 700, 668 cm . H NMR (300 MHz, DMSO): d 10.40 (1H,
151.41, 143.44, 138.61, 132.08, 131.56, 131.03, 128.91, 128.44,
125.37, 121.89, 115.48, 50.47, 40.97, 36.04, 28.92. Anal. Calcd for
ꢀ1 1
bs, amide NH), 7.75 (1H, d, J = 8.0 Hz, carbamate NH), 7.71–7.46
24 2 3
C H24BrClN O (503.82): C, 57.21; H, 4.80; N, 5.56. Found: C,
57.40; H, 4.90; N, 5.38.
0
0
0
0
(
3
6H, m, H3, H5, H2 , H3 , H5 , H6 ), 7.24 (1H, d, J = 8.8 Hz, H6),
.26–3.12 (1H, m, CH), 1.72–1.43 (5H, m, cyclohexyl), 1.27–0.98
1
3
(
5H, m, cyclohexyl). C NMR (75 MHz, DMSO): d 163.21, 152.86,
2.1.2.11. 4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl cyclo-
1
1
47.41, 138.56, 131.97, 131.74, 131.00, 129.01, 128.46, 125.37,
21.78, 115.48, 50.04, 32.45, 25.24, 24.62. Anal. Calcd for C20
(451.74): C, 53.18; H, 4.46; N, 6.20. Found: C, 52.97; H,
.43; N, 6.25.
hexylcarbamate (3a).
White solid; yield 81%; mp 216–
H
20
218.5 °C. IR (ATR): 3321 (N–H), 3276 (N–H), 2937, 2855, 1703
(C@O carbamate), 1650 (amide I), 1570, 1531, 1512 (amide II),
1477, 1409, 1317, 1281, 1246, 1215, 1100, 1020, 893, 831, 802,
2 3
BrClN O
4
ꢀ
1 1
7
59, 691, 667, 652 cm . H NMR (500 MHz, DMSO): d 10.31 (1H,
0
0
2
.1.2.7. 2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl phenyl-
White solid; yield 55%; mp 221–223.5 °C. IR
ATR): 3304 (N–H), 3272 (N–H), 1716 (C@O carbamate), 1652
amide I), 1604, 1536 (amide II), 1516, 1489, 1476, 1445, 1394,
bs, amide NH), 7.78–7.62 (4H, m, carbamate NH, H3, H2 , H6 ),
0
carbamate (2b).
(
(
7.56 (1H, dd, J = 2.7 Hz, J = 8.5 Hz, H5), 7.27–7.12 (3H, m, H6, H3 ,
0
H5 ), 3.27–3.14 (1H, m, CH), 1.81–1.43 (5H, m, cyclohexyl), 1.30–
1
3
0.95 (5H, m, cyclohexyl). C NMR (125 MHz, DMSO): d 163.27,
158.40 (d, J = 240.3 Hz), 152.91, 147.42, 135.58 (d, J = 2.5 Hz),
132.11, 130.92, 129.04, 128.48, 125.43, 121.69 (d, J = 8.1 Hz),
115.36 (d, J = 22.3 Hz), 50.06, 33.54, 32.49, 24.65. Anal. Calcd for
C H20ClFN O (390.84): C, 61.46; H, 5.16; N, 7.17. Found: C,
20 2 3
61.55; H, 4.99; N, 7.20.
1
7
318, 1228, 1205, 1101, 1070, 1016, 1001, 837, 818, 758, 743,
ꢀ1 1
18, 690, 665 cm
. H NMR (300 MHz, DMSO): d 10.58 (1H, bs,
amide NH), 10.26 (1H, s, carbamate NH), 7.76 (1H, d, J = 2.6 Hz,
H3), 7.68–7.61 (3H, m, H5, H2 , H6 ), 7.52–7.37 (5H, m, H6, H3 ,
H5 , H2 , H6 ), 7.27 (2H, t, J = 7.9 Hz, H3 , H5 ), 7.02 (1H, t,
J = 7.4 Hz, H4 ). C NMR (75 MHz, DMSO): d 163.17, 151.14,
0
0
0
0
00
00
00
00
00
13
1
1
C
46.91, 138.44, 131.82, 131.77, 131.68, 131.28, 129.68, 129.01,
28.96, 128.66, 125.85, 122.77, 121.94, 115.68. Anal. Calcd for
2.1.2.12. 4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl phenyl-
carbamate (3b).
White solid; yield 79%; mp 204–206.5 °C. IR
20
H
14BrClN
2
O
3
(445.69): C, 53.90; H, 3.17; N, 6.29. Found:
(ATR): 3330 (N–H), 3082, 1748 (C@O carbamate), 1641 (amide I),
C, 53.97; H, 3.34; N, 6.33.
1599, 1538, 1509 (amide II), 1476, 1442, 1415, 1316, 1238, 1210,
ꢀ1 1
1192, 1179, 1106, 1001, 994, 834, 822, 772, 759, 691 cm . H NMR
2
.1.2.8. 2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl benzyl-
carbamate (2c). White solid; yield 65%; mp 179.5–181.5 °C.
IR (ATR): 3338 (N–H), 3278 (N–H), 3066, 1713 (C@O carbamate),
(300 MHz, DMSO): d 10.49 (1H, bs, amide NH), 10.25 (1H, s, carbamate
0
0
NH), 7.75 (1H, d, J = 2.6 Hz, H3), 7.71–7.66 (2H, m, H2 , H6 ), 7.63 (1H,
0
0
dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.47–7.42 (2H, m, H3 , H5 ), 7.39 (1H, d,
0
0
00
1
1
655 (amide I), 1591, 1534, 1511 (amide II), 1489, 1478, 1394,
314, 1290, 1264, 1216, 1102, 1072, 1025, 1013, 921, 890, 823,
J = 8.7 Hz, H6), 7.28 (2H, t, J = 7.9 Hz, H2 , H6 ), 7.28 (2H, t, J = 7.9 Hz,
00
00
00 13
H3 , H5 ), 7.02 (1H, t, J = 7.5 Hz, H4 ). C NMR (75 MHz, DMSO): d

M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
1325
1
62.97, 158.49 (d, J = 240.3 Hz), 151.15, 146.90, 135.41 (d, J = 2.6 Hz),
2.1.2.17. 2-(Phenylcarbamoyl)phenyl benzylcarbamate (4c)
White solid; yield 92%; mp 154.5–155.5 °C. IR (ATR): 3350 (N–H),
3292 (N–H), 3058, 2924, 1708 (C@O carbamate), 1651 (amide I),
1598, 1533 (amide II), 1518, 1498, 1486, 1441, 1326, 1273, 1220,
131.95, 131.13, 129.66, 128.98, 128.93, 128.61, 125.83, 121.88 (d,
J = 7.9 Hz), 118.34, 115.42 (d, J = 22.3 Hz). Anal. Calcd for C20
O
H14ClFN
2
3
(384.79): C, 62.43; H, 3.67; N, 7.28. Found: C, 62.29; H, 3.64; N, 7.29.
ꢀ1 1
1
087, 1025, 921, 899, 873, 763, 749, 698, 691, 669 cm . H NMR
2
.1.2.13. 4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl ben-
zylcarbamate (3c). White solid; yield 82%; mp 227–
29.5 °C. IR (ATR): 3315 (N–H), 3233 (N–H), 3033, 1711 (C@O car-
bamate), 1652 (amide I), 1627, 1572, 1537, 1511 (amide II), 1471,
(300 MHz, DMSO): d 10.24 (1H, bs, amide NH), 8.31 (1H, t,
0
0
J = 6.1 Hz, carbamate NH), 7.75 (2H, d, J = 7.9 Hz, H2 , H6 ), 7.63
(1H, dd, J = 1.8 Hz, J = 7.6 Hz, H3), 7.53 (1H, td, J = 1.7 Hz,
2
0
0
00
00
00
J = 7.8 Hz, H4), 7.39–7.16 (9H, m, H5, H6, H3 , H5 , H2 , H3 , H4 ,
0
0
00
0
1
7
454, 1415, 1315, 1251, 1215, 1110, 1096, 1030, 827, 816, 788,
51, 730, 697, 669 cm . H NMR (500 MHz, DMSO): d 10.41 (1H,
2
H5 , H6 ), 7.09 (1H, t, J = 7.4 Hz, H4 ), 4.23 (2H, d, J = 6.1 Hz, CH ).
C NMR (75 MHz, DMSO): d 164.57, 154.55, 148.47, 139.46,
ꢀ1
1
13
bs, amide NH), 8.35 (1H, t, J = 6.2 Hz, carbamate NH), 7.74–7.70
139.32, 131.29, 130.83, 128.98, 128.79, 128.33, 126.99, 126.89,
125.33, 123.67, 123.58, 119.94, 44.04. Anal. Calcd for C21H N O
18 2 3
0
0
(
2H, m, H2 , H6 ), 7.68 (1H, d, J = 2.6 Hz, H3), 7.58 (1H, dd,
J = 2.7 Hz, J = 8.6 Hz, H5), 7.29 (1H, d, J = 8.7 Hz, H6), 7.26–7.15
(346.38): C, 72.82; H, 5.24; N, 8.09. Found: C, 72.88; H, 5.14; N,
8.21.
0
0
00
00
00
00
00
(
7H, m, H3 , H5 , H2 , H3 , H4 , H5 , H6 ), 4.22 (2H, d, J = 6.1 Hz,
1
3
2
CH ).
C NMR (125 MHz, DMSO): d 163.03, 158.45 (d,
J = 240.0 Hz), 154.18, 147.33, 139.18, 135.57 (d, J = 2.6 Hz),
2.1.2.18.
(4d).
2-(Phenylcarbamoyl)phenyl
White solid; yield 72%; mp 154.5–157 °C. IR (ATR):
phenethylcarbamate
1
1
32.18, 130.98, 129.29, 128.51, 128.32, 126.98, 126.94, 125.54,
21.76 (d, J = 7.8 Hz), 115.38 (d, J = 22.1 Hz), 44.05. Anal. Calcd
3352 (N–H), 3316 (N–H), 3026, 1710 (C@O carbamate), 1655
for C21
H
16ClFN
2
O
3
(398.81): C, 63.24; H, 4.04; N, 7.02. Found: C,
(amide I), 1599, 1533 (amide II), 1517, 1499, 1485, 1440, 1325,
1265, 1241, 1217, 1074, 946, 892, 794, 778, 753, 698, 668 cm .
ꢀ1
6
2.99; H, 4.30; N, 7.11.
1
H NMR (300 MHz, DMSO): d 10.18 (1H, bs, amide NH), 7.85 (1H,
0
0
2
.1.2.14.
4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl
White solid; yield 67%; mp 231–
32.5 °C. IR (ATR): 3340 (N–H), 3258 (N–H), 3066, 1714 (C@O car-
bamate), 1658 (amide I), 1538, 1511 (amide II), 1483, 1464, 1414,
t, J = 5.7 Hz, carbamate NH), 7.73 (2H, d, J = 7.9 Hz, H2 , H6 ), 7.61
(1H, dd, J = 1.9 Hz, J = 7.6 Hz, H3), 7.50 (1H, td, J = 1.7 Hz,
J = 7.8 Hz, H4), 7.37–7.13 (9H, m, H5, H6, H3 , H5 , H2 , H3 , H4 ,
H5 , H6 ), 7.07 (1H, t, J = 7.3 Hz, H4 ), 3.19 (2H, dt, J = 6.2 Hz,
2 2
J = 7.9 Hz, N-CH ), 2.68 (2H, t, J = 7.2 Hz, CH ). C NMR (75 MHz,
phenethylcarbamate (3d).
0
0
00
00
00
2
0
0
00
0
1
3
1
8
1
7
313, 1286, 1268, 1213, 1157, 1115, 1098, 1022, 968, 902, 833,
24, 786, 749, 699, 682, 661 cm . H NMR (500 MHz, DMSO): d
0.37 (1H, bs, amide NH), 7.90 (1H, t, J = 5.7 Hz, carbamate NH),
ꢀ1 1
DMSO): d 164.52, 154.04, 148.39, 139.42, 139.37, 131.16, 130.69,
128.93, 128.85, 128.78, 128.45, 126.23, 125.18, 123.68, 123.52,
119.92, 42.30, 35.33. Anal. Calcd for C22H N O (360.41): C,
20 2 3
73.32; H, 5.59; N, 7.77. Found: C, 73.33; H, 5.49; N, 8.00.
0
0
.74–7.70 (2H, m, H2 , H6 ), 7.66 (1H, d, J = 2.6 Hz, H3), 7.56 (1H,
0
0
00
dd, J = 2.7 Hz, J = 8.7 Hz, H5), 7.26–7.11 (8H, m, H6, H3 , H5 , H2 ,
0
0
00
00
00
H3 , H4 , H5 , H6 ), 3.20 (2H, dt, J = 5.9 Hz, J = 7.7 Hz, N-CH
2
),
). C NMR (125 MHz, DMSO): d 163.00,
58.46 (d, J = 240.3 Hz), 153.69, 147.25, 139.29, 135.55 (d,
13
2
1
.68 (2H, t, J = 7.3 Hz, CH
2
2.1.2.19. 2-(Phenylcarbamoyl)phenyl adamantan-1-ylcarba-
mate (4e).
White solid; yield 89%; mp 172–174.5 °C. IR
J = 2.5 Hz), 132.07, 130.86, 129.13, 128.83, 128.44, 128.38,
(ATR): 3272 (N–H), 3065, 2908, 2859, 1712 (C@O carbamate),
1657 (amide I), 1598, 1551, 1537 (amide II), 1499, 1484, 1444,
1327, 1298, 1279, 1212, 1088, 1016, 921, 775, 752, 711, 689,
1
4
4
26.24, 125.47, 121.76 (d, J = 7.9 Hz), 115.39 (d, J = 22.2 Hz),
2.29, 35.27. Anal. Calcd for C22 18ClFN (412.84): C, 64.00; H,
H
2 3
O
ꢀ1
1
.39; N, 6.79. Found: C, 64.13; H, 4.30; N, 6.77.
671 cm
.
H NMR (300 MHz, DMSO): d 10.00 (1H, bs, amide
0
0
NH), 7.71 (2H, d, J = 8.0 Hz, H2 , H6 ), 7.61 (1H, dd, J = 1.8 Hz,
J = 7.5 Hz, H3), 7.52–7.46 (2H, m, carbamate NH, H4), 7.35–7.27
(3H, m, H5, H3 , H5 ), 7.16 (1H, d, J = 8.1 Hz, H6), 7.06 (1H, t,
J = 7.3 Hz, H4 ), 1.96–1.90 (3H, m, CH), 1.86–1.76 (6H, m, C-CH
1.61–1.48 (6H, m, CH-CH
2
.1.2.15. 2-(Phenylcarbamoyl)phenyl cyclohexylcarbamate (4a).
0
0
White solid; yield 80%; mp 154–155 °C. IR (ATR): 3322 (N–H), 3262
N–H), 3057, 2932, 2852, 1704 (C@O carbamate), 1655 (amide I),
0
(
2
),
1
3
1
1
597, 1541 (amide II), 1499, 1454, 1438, 1329, 1315, 1275, 1232,
2
). C NMR (75 MHz, DMSO): d 164.39,
ꢀ1
1
215, 1096, 1019, 895, 779, 745, 705, 688, 667 cm
.
H NMR
151.77, 148.39, 139.36, 131.26, 130.63, 129.00, 128.66, 125.01,
123.63, 123.47, 119.97, 50.38, 41.01, 36.04, 28.92. Anal. Calcd for
C H N O (390.47): C, 73.82; H, 6.71; N, 7.17. Found: C, 73.70;
24 26 2 3
(
300 MHz, DMSO): 10.10 (1H, bs, amide NH), 7.74–7.66 (3H, m,
0
0
H3, H2 , H6 ), 7.63–7.46 (2H, m, carbamate NH, H4), 7.35–7.26
0
0
(
3H, m, H5, H3 , H5 ), 7.19 (1H, d, J = 8.0 Hz, H6), 7.06 (1H, t,
H, 6.90; N, 6.98.
0
J = 7.3 Hz, H4 ), 3.28–3.14 (1H, m, CH), 1.80–1.42 (5H, m, cyclo-
hexyl), 1.27–0.96 (5H, m, cyclohexyl). 13C NMR (75 MHz, DMSO):
d 164.48, 153.25, 148.54, 139.41, 131.24, 130.68, 128.98, 128.73,
2.2. Biology
1
25.09, 123.64, 123.47, 119.87, 50.00, 32.55, 25.29, 24.69. Anal.
Calcd for C20 (338.40): C, 70.99; H, 6.55; N, 8.28. Found:
C, 71.06; H, 6.80; N, 8.37.
2.2.1. In vitro antibacterial evaluation14
The in vitro antibacterial activities were assayed against eight
Gram-positive and Gram-negative strains: Staphylococcus aureus
H
22
N
2
O
3
CCM 4516/08, methicillin-resistant Staphylococcus aureus
H
2
.1.2.16. 2-(Phenylcarbamoyl)phenyl phenylcarbamate (4b).
White solid; yield 74%; mp 160.5–163 °C. IR (ATR): 3349 (N–H),
313 (N–H), 1720 (C@O carbamate), 1655 (amide I), 1598, 1530
amide II), 1498, 1482, 1443, 1323, 1219, 1195, 1098, 1011, 919,
5996/08 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococ-
cus sp. J 14365/08; Escherichia coli CCM 4517, Klebsiella pneumoniae
D 11750/08, ESBL-positive Klebsiella pneumoniae J 14368/08, and
Pseudomonas aeruginosa CCM 1961.
The microdilution broth method modified according to stan-
dard M07-A07 in Mueller-Hinton broth (HiMedia Laboratories,
Mumbai, India) adjusted to pH 7.4 (±0.2) was used. The tested
compounds were dissolved in DMSO to final concentrations
3
(
ꢀ1 1
7
93, 751, 705, 689, 671 cm . H NMR (300 MHz, DMSO): d 10.33
(
1H, bs, amide NH), 10.21 (1H, s, carbamate NH), 7.76–7.64 (3H,
0
0
m, H3, H2 , H6 ), 7.61–7.53 (1H, m, H4), 7.50–7.23 (8H, m, H5,
H3 , H5 , H2 , H3 , H4 , H5 , H6 ), 7.09–6.95 (2H, m, H6, H4 ).
0
0
00
00
00
00
00
0
13
C
NMR (75 MHz, DMSO): d 164.53, 151.49, 148.05, 139.32, 131.41,
ranging from 500 to 0.49 lM. Benzylpenicillin (penicillin G; PNC)
1
1
4
30.65, 128.98, 128.92, 128.79, 125.67, 123.86, 123.78, 121.15,
20.02, 118.35. Anal. Calcd for C20 (332.35): C, 72.28; H,
.85; N, 8.43. Found: C, 72.06; H, 4.99; N, 8.31.
and bacitracin (BAC) were used as comparative drugs. A bacterial
16 2 3
H N O
inoculum in sterile water was prepared to reach 0.5 on the
8
McFarland scale (1.5 ꢁ 10 CFU/mL). The minimum inhibitory

1
326
M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
concentrations were assayed as a reduction in growth of at least
0% (IC90) compared with the control. The results were analysed
both visually and spectrophotometrically at 540 nm. The MIC val-
ues were determined after 24 and 48 h of incubation in the dark at
side products 5b and 5e. These unwanted by-products can be the
result of the presence of water in the reaction mixture and the
extended reaction time promotes this reaction, especially for 1-
adamantyl isocyanate. The formation of ureas via carbamic acid
unstable intermediate is depicted in Scheme 2. Thus, strictly anhy-
drous conditions had a beneficial effect on the suppression of this
side reaction. The change of the solvent can also improve yields of
carbamates 1–4 instead of ureas 5. However, isocyanates are con-
verted to symmetric 1,3-disubstituted ureas directly in the pres-
9
3
5 °C (±0.1) in a humid atmosphere.
2
.2.2. In vitro antifungal evaluation11
The antifungal properties were evaluated in vitro against four
Candida strains (Candida albicans ATCC 44859, Candida tropicalis
56, Candida krusei E28, and Candida glabrata 20/I), Trichosporon
asahii 1188 and three filamentous fungi (Aspergillus fumigatus
1
8
1
ence of triethylamine or other tertiary bases. This unintentional
reaction can be circumvent sufficiently by the difference choice
of solvent (dry acetone, toluene, tetrahydrofuran or mixture of sol-
2
4
31, Absidia corymbifera 272, and Trichophyton mentagrophytes
45).
3
vents), replacement of Et N by DIPEA and/or by addition of only a
The microdilution broth method was used according to the CLSI
catalytic amount of triethylamine.
M27-A3 and M38-A2 guidelines in RPMI 1640 with glutamine
KlinLab, Prague, Czech Republic) buffered to pH 7.0 with
.165 mol of 3-morpholino-propane-1-sulfonic acid (Sigma–
Compounds were characterised by melting points, IR and NMR
spectra; their purity was checked by thin-layer chromatography
and elemental analysis. In the IR spectra of carbamates, sharp
(
0
ꢀ1
Aldrich, Darmstadt, Germany). DMSO served as a diluent for all
and strong bands appear at around 1703–1748 cm (carbamic
of the compounds. In yeast, the final size of the inoculum was
C@O) and 1641–1684 (amide I). Amide II bands were observed at
3
ꢀ1
5
ꢁ 10 ± 0.2 CFU/mL, and in the case of the moulds, the final size
around 1509–1541 cm . Most of the derivatives displayed also
4
ꢀ1
of the inoculum was 0.5–5 ꢁ 10 CFU/mL. Fluconazole (FLU) and
amphotericin B (AMB) were used as reference drugs. The MIC val-
ues for yeasts and filamentous fungi were assayed as a reduction of
growth of at least 80% (IC80) or of at least 50% (IC50) compared with
the control, respectively. The results were analysed visually and
spectrophotometrically at 540 nm. The MIC values were deter-
mined after 24 and 48 h of incubation in the dark at 35 °C (±0.1)
in a humid atmosphere, but for T. mentagrophytes, the final MIC
values were determined after 72 and 120 h of incubation.
two visible N–H stretch bands (3261–3352 cm ); in some cases,
only one N–H band was well-marked.
Twenty novel carbamates 1–4 are summarised in Table 1. In
contrast to previously reported salicylanilide carbamates,8 here
presented carbamates 1–4 have substituted both aromatic rings
of salicylanilide core by various and/or different substituents not
,17
1
6
prepared before. Férriz et al.
2
reported N-alkyl (C –C11)
carbamates of salicylanilides synthesized from 5-chlorosalicylic
acid and 3-chloro-, 4-chloro- or 3,4-dichloro-anilines. Previously,
we reported three types of salicylanilide N,N-disubstituted carba-
mates (dimethyl, methyl(phenyl) and diphenyl carbamates) and
3
3
. Results and discussion
8
N,N-dimethylthiocarbamates. In this study, ‘salicylic part’ of
compounds 1–4 was synthesized from 5-chlorosalicylic or salicylic
acids, ‘anilide part’ from unsubstituted aniline, 4-Cl/Br/F-anilines
and N-cycloalkyls (derivatives a, e), N-phenyl b and N-phenylalkyl
.1. Chemistry
The synthetic plan is depicted in a Scheme 1. Parent salicy-
(c, d) carbamates are involved.
lanilides SAL were synthesised using microwave irradiation via a
previously described method.11
3
3
.2. Biology
Salicylanilide carbamates 1–4 were synthesised by a reaction of
salicylanilides with isocyanates in the presence of 1 equiv of tri-
ethylamine or Hünig’s base (N,N-diisopropylethylamine, DIPEA)
at rt. Acetonitrile (MeCN) was used as the solvent for most of the
products. In this case, the average reaction time was 12 h for 1-
adamantyl isocyanate and 2 hours for remaining isocyanates. In
general, yields ranged from 50% to 95%.
When phenyl isocyanate and 1-adamantyl isocyanate served as
starting material, the formation of desired carbamates 1–4 was
diminished or even abolished completely due to formation of urea
.2.1. Antibacterial activity
Salicylanilide carbamates 1–4 were evaluated in vitro for their
antibacterial activity against four Gram-positive strains: Staphylo-
coccus aureus, methicillin-resistant Staphylococcus aureus (MRSA),
Staphylococcus epidermidis, Enterococcus sp., and four Gram-nega-
tive strains: Escherichia coli, Klebsiella pneumoniae, ESBL-positive
Klebsiella pneumoniae (KP-E) and Pseudomonas aeruginosa (Table 1).
Benzylpenicillin (G-penicillin; PNC) and bacitracin (BAC) were
used as the reference drugs for the comparison. Solubility
problems in the testing medium did not allow evaluation of one
adamantane derivative (4e).
R2
In general, Gram-positive bacteria showed higher susceptibility
O
6
R1
COOH
H
2
N
R1
5
4
1
to investigated compounds (MICs starting from 0.49
Gram-negative species (MICs P 31.25 M). None of the derivatives
inhibited the growth of P. aeruginosa at a concentration of 125 lM
lM) than
PCl
3
N
H
+
l
OH
R2
MW, PhCl
2
OH
3
or lower. All carbamates 1–4 were active at least against three bac-
terial strains. Dihalogenated derivatives 1–3 showed unequivocally
improved antibacterial activity than carbamates 4 derived from
unsubstituted salicylanilide (MICs P 125 lM).
Despite the presence of drug-resistance, the genus Staphylococ-
cus (S. aureus, S. epidermidis) was the most susceptible. They were
SAL
R2
R2
O
O
2
6
3
3
R1
5
4
1
2
R1
4
5
N
H
N
H
O
base
+
C
R3
N
OH
SAL
various
solvents
O
1
6
R3
O
N
H
inhibited by all of the carbamates 1–4 with MICs 0.49–500
lM. 2-
[
(4-Bromophenyl)carbamoyl]-4-chlorophenyl phenylcarbamate 2b
1-4
showed the best MIC values (60.49–0.98 lM). When focusing on
Scheme 1. Synthesis of salicylanilides SAL and carbamates 1–4. [R = H, Cl; R2 = H,
1
established antibiotics, five derivatives (2a–e) were in vitro
comparable to PNC and among halogenated carbamates, only the
compound 1b expressed lower activity than BAC. All of the halogen
3
Cl, Br, F;
R
= cyclohexyl, phenyl, benzyl, phenethyl, adamantan-1-yl; MW:
microwave irradiation (530 W, 600 rpm, 22 min); PhCl: chlorobenzene].

M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
1327
O
C
O
O
C
R3
R3
R3 NH2
O
R3
R3
R3
+
H O
+
C
2
N
HO
N
H
N
N
H
N
H
-
CO2
5
Scheme 2. Pathway of side-product urea 5 formation in the presence of traces of water (R3 = phenyl, adamantan-1-yl).
Table 1
Antibacterial activity of carbamates 1–4
R2
O
O
R1
N
H
R3
O
N
H
Code
R1
R2
R3
MIC/IC95
[lM]
SA
MRSA
SE
EF
EC
KP
KP-E
48 h
2
4 h
48 h
24 h
48 h
24 h
24 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
a
b
c
d
e
a
b
c
d
e
a
b
c
d
e
a
b
c
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
F
F
F
F
F
H
H
H
H
H
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
1.95
31.25
3.9
3.9
3.9
60.49
60.49
60.49
60.49
0.98
15.62
7.81
15.62
7.81
15.62
250
7.81
31.25
15.62
15.62
15.62
0.98
0.98
0.98
1.95
1.95
15.62
7.81
15.62
15.62
15.62
250
3.9
15.62
31.25
15.62
15.62
31.25
1.95
0.98
1.95
1.95
3.9
31.25
7.81
31.25
62.5
62.5
500
0.98
31.25
3.9
3.9
3.9
60.49
60.49
60.49
60.49
0.98
7.81
7.81
15.62
15.62
250
0.98
31.25
3.9
15.62
3.9
0.98
0.98
0.98
0.98
1.95
7.81
7.81
15.62
15.62
250
>125
500
>125
500
250
500
>125
500
>125
500
250
500
>125
500
>125
125
>125
500
>125
500
>125
500
125
>125
500
125
>125
62.5
62.5
31.25
62.5
>500
>500
>500
>500
31.25
62.5
250
>500
>500
>500
>500
>500
>500
>500
NT
>125
62.5
125
31.25
62.5
>500
>500
>500
>500
125
31.25
7.81
3.9
500
500
3.9
>250
>500
>500
>500
>500
>250
>125
>500
>500
>500
>500
>500
>500
>500
>500
NT
>250
>500
>500
>500
>500
>250
>125
>500
>500
>500
>500
>500
>500
>500
>500
NT
>250
>500
>500
>500
>500
>250
>125
250
>500
>500
>500
>500
>500
>500
>500
NT
>250
>500
>500
>500
>500
>250
>125
250
>500
>500
>500
>500
>500
>500
>500
NT
60.49
60.49
60.49
0.98
1.95
31.25
7.81
15.62
15.62
62.5
500
500
500
Benzyl
>500
>500
>500
>125
250
>500
>500
125
>500
>500
>500
>125
250
>500
>500
125
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
62.5
250
Benzyl
>500
>500
>500
>500
>500
>500
>500
NT
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
500
500
500
125
500
500
500
125
500
500
H
H
H
H
500
250
125
NT
500
250
125
NT
500
500
125
NT
500
500
125
NT
>500
>500
>500
NT
>500
>500
>500
NT
d
e
NT
NT
PNC
BAC
0.98
7.81
0.98
15.62
62.5
15.62
125
15.62
250
15.62
250
31.25
7.81
15.62
15.62
62.5
125
>500
125
>500
500
>500
500
>500
>500
>500
>500
>500
SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus epidermidis H 6966/08; EF: Enterococcus sp. J 14365/
8. Escherichia coli CCM 4517, Klebsiella pneumoniae D 11750/08; ESBL-positive Klebsiella pneumoniae J 14368/08. PNC: benzylpenicillin; BAC: bacitracin.
0
The best values for each strain are provided in bold.
derivatives 1–3 were superior to benzylpenicillin against MRSA
and seventeen of them exceeded activity of bacitracin (with excep-
tions of 1b, 3a, and 3e). For S. epidermidis, one carbamate (3e) was
comparable to PNC but remaining halogenated molecules and one
non-halogenated 4d displayed higher in vitro efficacy. Addition-
ally, most of the carbamates were also superior to BAC (Fig. 2).
Derivatives of N-(4-bromophenyl)-5-chloro-2-hydroxybenza-
mide (i.e. carbamates 2) showed improved activity when compared
to other substitution patterns, followed by 5-chloro-N-(4-chloro-
phenyl)-2-hydroxybenzamide-based compounds 1. Focusing on
N-substitution of carbamates, the compounds were ordered based
on decreasing activity against Staphylococci as follows: cyclohexyl
a P benzyl c > phenethyl d > phenyl b > adamantan-1-yl e.
showed higher susceptibility than ESBL-negative K. pneumoniae
strain (affected by only four compounds with MICs starting from
125 lM; 1b–1d, 3b). The evidence that some salicylanilide carba-
mates are able to inhibit Klebsiella pneumoniae including the
ESBL-producing strain is unique, whereas previously reported sal-
icylanilide O-derivatives avoided any considerable activity (e.g.,
Refs. 8,13–15). Derivatives 1 synthesized from 5-chloro-N-(4-
chlorophenyl)-2-hydroxybenzamide and N-phenyl carbamates b
seems to be optimal choice for design of potential agents against
Gram-negative bacteria. Nevertheless, their MIC values are com-
paratively higher than those determined for Staphylococcus spp.
Previously, various salicylanilide esters and carbamates have
8
,11,13–15,17
been revealed as potential antibacterial agents.
The
The activity against Enterococcus sp. was lower and not uniform.
increased lipophilicity in comparison to parent salicylanilides
should be beneficial for better crossing through biomembranes or
absorption. However, the sharply increased lipophilicity (Table 2)
and corresponding low solubility in the testing media hampered
in vitro evaluation of a wide range of 4-(trifluoromethyl)ben-
Nine carbamates showed MICs within the range of 125–500
1b, 1d, 1e, 2a, 2b, 3b, 3d, 3e, 4a), however, all of them were less
active than both BAC and PNC.
Among Gram-negative strains, E. coli was susceptible to only
two molecules, 1b and especially 1d (MIC values 125–500 M).
The later carbamate was also the most active compound against
ESBL-positive K. pneumoniae with MIC of 31.25 M. In summary,
seven carbamates (1b–1e, 2e, 3a, 3b) affected the growth of KP-E
with MICs within the range of 31.25–250 M. Interestingly, KP-E
lM
(
1
4
15
l
zoates and other highly lipophilic 4-substituted benzoates,
whereas no problems with the solubility of benzoic acid esters
13
1
7
l
and N-alkyl carbamates
were reported. N,N-Disubstituted
carbamates showed sufficient solubility for biological evaluation,
even derivatives with an escalated lipophilicity (N,N-diphenyl
l

1
328
M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
250 250
70
60
50
40
30
20
10
0
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e PNC BAC
SA MRSA SE
Figure 2. Antibacterial activity: comparison of dihalogenated carbamates 1–3 with control drugs against three Gram-positive strains after 24 h incubation (SA: S. aureus,
MRSA: methicillin-resistant S. aureus, SE: S. epidermidis, PNC: benzylpenicillin, BAC: bacitracin).
carbamates; Table 2). Presented compounds belong to N-monosub-
stituted derivatives and ‘carbamic’ hydrogen confers more
hydrophilic properties when compared to other salicylanilide
O-derivatives. For example, ClogP value of N-phenyl carbamate
identified against Staphylococcus epidermidis and Klebsiella pneu-
moniae including an ESBL-producing strain.
A possible additional mechanism of action was proposed for N-
alkyl carbamates with a longer alkyl chain. The long, unbranched
hydrophobic tail can cause membrane damage as non-ionic surfac-
tants. The disruption of biomembranes is followed by leakage of
the intracellular content and facilitation of the entry of active
agents in the cells where it can interact with specific target(s).17
However, this ‘detergent-like’ mechanism of action seems to be
an unlikely for carbamates 1–4 due to their molecular structures.
The antibacterial activity is also independent of the presence 4-
chlorophenyl group as postulated by Ref. 17, moreover, its replace-
ment by 4-bromophenyl decreased MIC values substantially.
1
b is lower than those calculated for corresponding benzoate
(
5.11 vs 5.44, respectively; Table 2) and concomitantly, it is suffi-
ciently increased than in the case of parent salicylanilide (3.57).
Presented salicylanilide N-monosubstituted carbamates 1–4
exhibited more potent antibacterial action than N,N-disubstituted
8
carbamates, that is, they offer substantially lower MIC values
(60.49 lM vs 62.5 lM for the best derivatives, respectively) and
also more uniform activity. Among N,N-disubstituted carbamates,
only two derivatives from total fifteen were able to stop the growth
of bacteria. Drawing a comparison between N-monoalkyl carba-
1
7
mates and derivatives 1–4, the most active compounds provide
similar low micromolar or submicromolar MIC values for Staphylo-
coccus aureus despite the presence of methicillin-resistance.
Among carbamates synthesized from 5-chloro-N-(4-chlorophe-
3.2.2. Antifungal activity
The antifungal properties of carbamates 1–4 were evaluated
in vitro against eight species: Candida albicans, C. tropicalis, C. kru-
sei, C. glabrata, Trichosporon asahii, Aspergillus fumigatus, Absidia
corymbifera and Trichophyton mentagrophytes (Table 3). Flucona-
zole (FLU) and amphotericin B (AMB) were involved as reference
drugs. As in the case of antibacterial tests, it was not possible to
evaluate adamantane derivative 4e due to limited solubility.
Among all strains, only Aspergillus fumigatus showed complete
resistance to investigated compounds 1–4 and two carbamates
(3e, 4a) avoided any antifungal action. On the other side, 2-[(4-bro-
mophenyl)carbamoyl]-4-chlorophenyl benzylcarbamate 2c was
identified as an agent with a broad-spectrum in vitro antifungal
activity exhibiting the lowest or second-lowest reached MIC values
for seven fungal species.
nyl)-2-hydroxybenzamide involved in both papers, N-alkyl (C
2
,
C
4
–C , C11) derivatives showed mostly indistinguishable or slightly
9
lower MIC values than if carbamic nitrogen is substituted by
cycloalkyl/aryl/arylalkyl. In contrast to Zadrazilova et al.1 , novel
salicylanilide carbamates 1–4 were evaluated against a broader
spectrum of bacteria represented by eight Gram-positive and
Gram-negative strains. Additional actimicrobial action was
7
Table 2
Calculated lipophilicities (ClogP) of selected salicylanilide derivatives
Candida albicans was inhibited by four derivatives (2c, 3c, 3d,
4c) with MICs from 62.5 lM. None of them achieved the activity
Cl
O
Cl
of fluconazole and amphotericin B. Three carbamates (1e, 2c, 4c)
are able to stop the growth of C. tropicalis with MIC values of 125
to 250 lM, thus being superior to FLU. C. krusei and C. glabrata
showed a comparatively higher susceptibility. The first strain was
N
H
O
R
affected by ten carbamates (1a–c, 1e, 2b, 2c, 3c, 3d, 4c, 4d; MICs
Group of derivatives
Parent salicylanilides
R
ClogP
of 62.5 to 250
lM) and the second strain by eight derivatives
H
3.57
5.44
6.36
5.35
7.01
5.53
5.11
(2b–d, 3a, 3c, 3d, 4c, 4d) at a concentration of 31.25
lM (2c) and
13
Benzoates
Benzoyl
-(Trifluoromethyl)benzoates1
1,14
higher. With respect to in vitro evaluation results of two estab-
lished drugs, carbamates are comparable or superior to FLU and
significantly less active than AMB.
Among non-Candida species, Absidia corymbifera was inhibited
by five derivatives (1a, 2c, 2d, 3a, 3c), but only 3c showed identical
4
4-(Trifluoromethyl)benzoyl
Methyl(phenyl)carbamoyl
Diphenylcarbamoyl
Hexylcarbamoyl
Phenylcarbamoyl 1d
8
N,N-Disubstituted carbamates
N-Alkyl carbamates16,17
N-Monosubstituted carbamates

M. Krátk y´ , J. Vinšová / Bioorg. Med. Chem. 24 (2016) 1322–1330
1329
Table 3
Antifungal activity of carbamates 1–4
Code
MIC/IC80/IC50
CG
[lM]
CA
CT
CK
TA
AC
TM
24 h
48 h
24 h
48 h
24 h
24 h
24 h
48 h
24 h
48 h
24 h
48 h
72 h
120 h
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
a
b
c
d
e
a
b
c
d
e
a
b
c
d
e
a
b
c
>125
>125
>125
>500
>250
>125
>500
62.5
>125
>125
>125
>250
250
>125
>125
>125
>500
>250
>125
>500
62.5
>125
>125
>125
>250
>250
>500
>125
>500
>500
125
>125
>125
>125
>500
250
>125
>500
125
>125
>125
>125
>250
>250
>500
>125
>500
>500
125
>125
>125
>125
>500
250
>125
>500
125
>125
>125
>125
>250
>250
>500
>125
>500
>500
125
125
125
>125
>125
>125
>500
>250
>125
125
31.25
125
>125
125
>250
125
125
>125
>500
>500
250
>125
>125
>125
>500
>250
>125
125
125
125
125
125
7.81
1.95
1.95
1.95
3.9
1.95
3.9
3.9
7.81
1.95
1.95
1.95
3.9
1.95
3.9
3.9
62.5
62.5
>500
125
>125
250
62.5
>125
>125
>125
>250
250
62.5
62.5
>500
125
>125
250
62.5
>125
>125
>125
>250
>250
>500
>125
>500
>500
125
62.5
15.62
>500
31.25
62.5
125
15.62
62.5
>125
62.5
>250
125
62.5
15.62
>500
31.25
62.5
125
15.62
62.5
>125
>125
>250
>250
>500
>125
>500
>500
125
>125
>125
>500
>250
>125
>500
7.81
125
>125
125
>250
250
>500
>125
>500
>500
>500
>500
NT
>125
>125
>500
>250
>125
>500
7.81
125
>125
>125
>250
>250
>500
>125
>500
>500
>500
>500
NT
31.25
125
3.9
3.9
>125
>125
>250
>250
>500
>125
>500
>500
250
7.81
1.95
3.9
7.81
1.95
15.62
250
>500
>125
>500
125
15.62
62.5
NT
250
>500
>125
>500
125
15.62
62.5
NT
250
125
125
>125
>500
>500
125
>500
NT
>125
>500
>500
125
125
NT
>125
>500
>500
125
>500
NT
d
e
>500
NT
>500
NT
>500
NT
125
NT
250
NT
250
NT
>500
NT
FLU
AMB
0.24
0.12
0.24
0.49
>500
1.95
>500
1.95
125
1.95
250
1.95
31.25
0.98
500
1.95
250
0.49
500
0.98
>500
7.81
>500
7.81
7.81
1.95
125
1.95
CA: Candida albicans ATCC 44859; CT: Candida tropicalis; CK: Candida krusei E28; CG: Candida glabrata 20/I; TA: Trichosporon asahii 1188; AC: Absidia corymbifera 272; TM:
Trichophyton mentagrophytes 445. FLU: fluconazole; AMB: amphotericin B.
The best values for each strain are provided in bold.
MIC as AMB (7.81
d, 4c) abolished the growth of Trichosporon asahii at lower con-
centrations (P15.62 M for 1c and 2c) than FLU (250–500 M).
Trichophyton mentagrophytes showed the highest rate of suscepti-
bility from all of the investigated fungi. MIC values of sixteen car-
bamates (1a–3d, 4b–d) ranged of 1.95–250 lM. Most of the
compounds were superior to FLU. Interestingly, under our condi-
tions, five derivatives (1b–d, 2a, 3a) exhibited an identical mini-
l
M). Twelve carbamates (1a–c, 1e, 2a–d, 3a, 3c,
from salicylanilides and isocyanates in the presence of ter-
tiary base. The carbamoylation of salicylanilide phenol group led
to derivatives with significant antimicrobial properties and
sufficient solubility for biological evaluation. Dihalogenated
salicylanilide carbamates exhibit potent uniform antibacterial
activity against Gram-positive bacteria including MRSA and a
fungal strain of T. mentagrophytes with low micromolar or even
submicromolar MICs for the majority of the derivatives. Their
moderate activity against ESBL-producing strain of K. pneumoniae
is also noteworthy.
3
l
l
mum inhibitory concentration as amphotericin B,
efficient antimycotic drug.
a highly
The analysis of the results revealed some relationships
between substitution pattern(s) and biological response. In
general, O-carbamoylation of both N-(4-bromophenyl)-5-chloro-
In general, derivatives of bromosalicylanilide were superior to
other groups of halogenated as well as non-halogenated salicy-
lanilide derivatives. N-Monosubstituted carbamates exhibited
higher and more uniform in vitro antimicrobial activity than sali-
cylanilide N,N-disubstituted carbamates. Based on these results,
reported salicylanilide derivatives seem to be a promising starting
compounds for further research and development of novel antimi-
crobial agents.
2
-hydroxybenzamide and especially 5-chloro-N-(4-chlorophe-
nyl)-2-hydroxybenzamide led to the carbamates 2 and 1 with
enhanced in vitro antifungal potency. Unequivocally, N-benzyl car-
bamates exhibited the lowest MIC values in comparison to other N-
substituents. In most cases, longer bridge connecting nitrogen with
phenyl ring (phenethyl derivatives d) or its removal to form phenyl
carbamates b led to the decrease of activity. The effect of the phe-
nyl ring hydrogenation (a vs b) is ambiguous.
Acknowledgements
8
Previously reported salicylanilide N,N-dialkyl(thio)carbamates
This work was financially supported by IGA NT 13346 (2012).
We want to thank to Ida Dufková for the excellent performance
of antibiotic susceptibility tests, the staff of the Department of
Inorganic and Organic Chemistry, Faculty of Pharmacy, for the
technical assistance and J. Urbanová, M.A., for the language help
provided.
showed a narrow-spectrum only against T. mentagrophytes. This
strain was inhibited solely by six N,N-dimethyl (thio)carbamates
from total twenty with MIC values within the range of 1.95–
5
00 lM with superiority of the thiocarbamates. The introduction
of bulkier substituents (one or two phenyls) led to the complete
loss of the antifungal properties. Here presented carbamates 1–4
exhibited more uniform activity and the most active derivatives
References and notes
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2
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