Synthesis and antimicrobial activity of sulphamethoxazole-based ureas and imidazolidine-2,4,5-triones

Article abstract of DOI:10.1515/chempap-2015-0109

Progression of drug resistance among bacterial and fungal pathogens justifies the development of novel antimicrobial agents. Thus, a series of novel sulphamethoxazole-based ureas and imidazolidine-2,4,5-triones have been designed and synthesised. The urea

Full text of DOI:10.1515/chempap-2015-0109

Chemical Papers 69 (8) 1108–1117 (2015)
DOI: 10.1515/chempap-2015-0109
ORIGINAL PAPER
Synthesis and antimicrobial activity of sulphamethoxazole-based
ureas and imidazolidine-2,4,5-triones
a
b
b
c,d
Martin Krátký*, Jana Mandíková, František Trejtnar, Vladimír Buchta,
e
a
Jiřina Stolaříková, Jarmila Vinšová
aDepartment of Inorganic and Organic Chemistry, bDepartment of Pharmacology and Toxicology, cDepartment of Biological
and Medical Sciences, Faculty of Pharmacy, Charles University in Prague,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
dDepartment of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University in Prague,
Sokolská 581, 500 12 Hradec Králové, Czech Republic
eLaboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava,
Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic
Received 26 December 2014; Revised 1 February 2015; Accepted 7 February 2015
Progression of drug resistance among bacterial and fungal pathogens justifies the development of
novel antimicrobial agents. Thus, a series of novel sulphamethoxazole-based ureas and imidazolidine-
2,4,5-triones have been designed and synthesised. The urea derivatives were obtained by the reaction
of sulphamethoxazole and isocyanates, and their cyclisation to imidazolidine-2,4,5-triones was per-
formed via oxalyl chloride. All synthesised derivatives were evaluated in vitro to determine their
activity against gram-positive and gram-negative bacteria, fungi, Mycobacterium tuberculosis, and
atypical mycobacteria and their cytotoxicity. The growth of mycobacteria was inhibited within
the range of 4–1000 µM and M. tuberculosis was the least-susceptible strain. 4-(3-Heptylureido)-
N-(5-methylisoxazol-3-yl)benzenesulphonamide was identified as the most promising compound
because it exhibited the highest activity against atypical mycobacteria at minimum inhibitory
concentrations, from 4 µM, and with acceptable toxicity (selectivity indices for M. avium and
M. kansasii higher than 16 and 62.5, respectively). Gram-positive bacteria, including methicillin-
resistant Staphylococcus aureus, were inhibited at concentrations starting from 125 µM, whereas the
investigated derivatives exhibited almost no antifungal potency and activity against gram-negative
species.
ꢀ
c 2015 Institute of Chemistry, Slovak Academy of Sciences
Keywords: antimicrobial activity, antimycobacterial activity, imidazolidine-2,4,5-triones, sul-
phamethoxazole, ureas
Introduction
Mycobacterium tuberculosis (M. tuberculosis), nontu-
berculous (atypical) mycobacteria, and gram-positive
and gram-negative bacteria, including methicillin-
resistant Staphylococcus aureus (MRSA) or Entero-
coccus sp. Research studies should provide new an-
timicrobial molecules; also, the modification of known
drugs represents an effective approach in drug design.
Sulphonamides have been widely used for the ther-
The alarming progression of drug resistance among
human pathogens and the inappropriate use or misuse
of antimicrobial chemotherapeutics justifies the de-
velopment of novel antimicrobial agents. Therapeutic
difficulties and resistance-related complications have
been reported for many bacterial species, including
*Corresponding author, e-mail: martin.kratky@faf.cuni.cz
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M. Krátký et al./Chemical Papers 69 (8) 1108–1117 (2015)
1109
apy of both gram-positive and gram-negative bacte-
rial infections; moreover, these derivatives were used
for the treatment of tuberculosis (TB) in the early
(B u¨ chi, Flawil, Switzerland) with open capillaries, and
the reported values are uncorrected.
Elemental analysis was performed with an auto-
matic Fisons EA 1110 CHNS-O CE microanalyzer
(Fisons, Milano, Italy). Infrared spectra (ATR) were
recorded on a Nicolet 6700 FTIR spectrometer in the
1
950s but were later abandoned mostly for their toxi-
city and the accessibility of novel more efficient drugs.
More recently, these derivatives were considered use-
less for the treatment of TB but concomitantly useful
for the therapy of some nontuberculous mycobacte-
rial infections. Based on the findings that both drug-
susceptible and drug-resistant M. tuberculosis are sus-
ceptible in vitro to clinically achievable concentrations
of sulphamethoxazole (SMX) and its combination
with trimethoprim, this molecule has “resurrected”
as a potential antimycobacterial agent with potential
use in the treatment of multidrug-resistant TB- and
HIV-co-infected patients and infections with nontu-
berculous mycobacteria (Alsaad et al., 2013; Ameen
−
13
1
range of 400–4000 cm . NMR spectra (500 MHz for
1
H and 125 MHz for C) were measured in DMSO-
d6 or THF-d8 at ambient temperature using a Var-
ian VNMR S500 instrument (Varian, Palo Alto, CA,
1
USA) or a Varian Mercury-Vxbb 300 (300 MHz for H
and 75.5 MHz for 13C; Varian, Palo Alto, CA, USA).
Tetramethylsilane was used as the internal standard.
The calculated logP values (ClogP) were deter-
mined using the CS ChemOffice Ultra version 12.0
program (CambridgeSoft, Cambridge, MA, USA).
&
Drancourt, 2013; Huang et al., 2012; Krátký et al.,
General procedure for the synthesis of ureas II
2
012). Although SMX is active against M. tuberculo-
sis, further studies for the identification of more active
and lipid-soluble sulphonamides preferably with im-
proved pharmacokinetic and pharmacodynamic pro-
files are necessary (Huang et al., 2012).
Several sulphonamide-based derivatives have re-
cently been designed and evaluated as potential an-
timycobacterial agents. Some of them show activity
against various bacterial strains (Agertt et al., 2013;
Desai et al., 2011; Krátký et al., 2012; North et al.,
Sulphamethoxazole (SMX, I) (2 mmol) was dis-
solved in dry acetonitrile (MeCN, 6 mL) and appro-
priate isocyanate (2.1 mmol) was added in one por-
tion. The solution was heated under reflux for 3.5 h
and then stirred at ambient temperature for 8 h. The
resulting crystals were filtered, washed with a small
amount of MeCN and dried. The product was recrys-
tallised from ethyl acetate (EtOAc) if necessary.
2
013; Sukdolak et al., 2005; Thomas et al., 2011).
General procedure for the synthesis
Sulphonamides generally act as structural analogues
of 4-aminobenzoic acid and therefore competitively
inhibit dihydropteroate synthase thereby disrupting
folate biosynthesis. More recently, these drugs have
been found to inhibit various β-carbonic anhydrases,
including those from Mycobacterium (Maresca et al.,
of imidazolidine-2,4,5-triones III
Oxalyl chloride (1.2 equivalent) was added in one
portion to sulphonamide-based urea (II, 0.75 mmol)
dissolved or suspended in dry THF (4 mL) and the
mixture was heated under reflux for 2 h. After cool-
ing, the crystallisation was initiated by an addition of
hexane. The resulting crystals were filtered, washed
with a small amount of dichloromethane and recrys-
tallised from EtOAc if necessary.
2
013; Minakuchi et al., 2009; Yun et al., 2012).
Based on these findings and on the fact that a
wide range of urea derivatives were found to share
antimycobacterial activity in vitro (Brown et al.,
2
011; North et al., 2013), sulphamethoxazole-based
ureas and their cyclic homologues have been designed,
synthesised, and evaluated as potential antimicrobial
agents.
Biological activity testing
The in vitro antimycobacterial activity of sulphon-
amide derivatives against M. tuberculosis 331/88
−
3
Experimental
(H37Rv; dilution of this strain was 10 ), M. avium
−
5
3
30/88 (dilution of 10 ), and two strains of M. kansa-
−
4
General
sii, namely 235/80 (dilution of 10 ) and the clinically
−
5
isolated strain 6509/96 (dilution of 10 ) was evalu-
ated using a previously described method (Krátký et
al., 2013). The following concentrations (in M) were
used: 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1. MIC
is the lowest concentration at which complete inhibi-
tion of mycobacterial growth was observed. Isoniazid
(INH) and SMX were chosen as the reference com-
pounds. The tested compounds were prepared in qua-
druplicates and the determination was repeated twice.
The in vitro antibacterial activity of II and
III was assayed against eight gram-positive and
All reagents and solvents were purchased from
Sigma–Aldrich (Darmstadt, Germany) or Penta
Chemicals (Prague, Czech Republic) and were used
as received. The reactions and purity of the products
were monitored by thin-layer chromatography using
ethyl acetate/hexane (ϕr = 2 : 1) as the eluent; the
plates were coated with 0.2 mm Merck 60 F254 sil-
ica gel (Darmstadt, Germany) and were visualised by
UV irradiation (254 nm). The melting points were de-
termined using a B u¨ chi melting point B-540 apparatus
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M. Krátký et al./Chemical Papers 69 (8) 1108–1117 (2015)
Fig. 1. Synthesis of ureas II and imidazolidine-2,4,5-triones III. Reaction conditions: i) isocyanate (R—N—C—O; for R, see
Table 1), MeCN, reflux, 3.5 h, ambient temperature, 8 h; ii) oxalyl chloride, THF, reflux, 2 h.
gram-negative strains: Staphylococcus aureus CCM
516/08, methicillin-resistant Staphylococcus aureus
H 5996/08 (MRSA), Staphylococcus epidermidis H
966/08, Enterococcus 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.
cluded: determination of 100 % viability and 0 % via-
bility (cells were treated with a 10 % DMSO solution),
no cell control, control for the determination of pos-
sible interactions of the tested compounds with the
reagents, control of the incubation medium settings,
and control of the DMSO toxicity. The results are ex-
pressed as the inhibitory concentration reducing the
cell viability to 50 % of the maximal (control) viabil-
ity (IC50). IC50 for each of the tested substances was
calculated using the GraphPad Prism software (ver-
sion 5.02; GraphPad Software, San Diego, CA, USA)
and Microsoft Excel 2010. This method is described
in detail in a previous report (Krátký et al., 2013).
4
6
The microdilution broth method with Mueller-
Hinton broth was used. The tested compounds were
dissolved in DMSO to final concentrations in the range
of 500–0.49 M. Bacitracin (BAC) and I were used
as the comparative drugs. The minimum inhibitory
concentrations were assayed as 95 % (IC95) or higher
reduction of growth compared with the control. The
measurement was repeated twice. The method used
for this determination was previously described by
Krátký et al. (2013).
The in vitro antifungal activity of II and III was
evaluated against four Candida strains (Candida al-
bicans ATCC 44859, Candida tropicalis 156, Can-
dida krusei E28, Candida glabrata 20/I), Trichosporon
asahii 1188 and three filamentous fungi (Aspergillus
fumigatus 231, Absidia corymbifera 272, Trichophyton
mentagrophytes 445). The microdilution broth method
was used in RPMI 1640 with glutamine. Flucona-
zole (FLU) was used as the reference drug. MICs
were assayed as 80 % (IC80) or higher reduction of
growth compared with the control; for filamentous
fungi, MICs are expressed as the IC50 values. The MIC
determination was repeated twice using a method pre-
viously described by Krátký and Vinšová (2012).
Cytotoxicity of II and III in the human hepatocel-
lular liver carcinoma cell line HepG2 (passage 33–39;
ECACC, Salisbury, UK) was determined using a stan-
dard colorimetric method that involves measuring the
reduction of a tetrazolium salt (CellTiter(R) 96 AQue-
ous One Solution Assay, Promega G3580, Madison,
WI, USA).
Results and discussion
SMX-based ureas II were prepared from sulphon-
amide I and commercially available isocyanates (with
different substituents: aryl, alkyl, cycloalkyl, ary-
lalkyl) (Fig. 1) by heating under reflux for 3.5 h in dry
acetonitrile in satisfactory yields (72–95 %) except for
the adamantan-1-yl urea IIe, where the yield was only
40 %. However, this yield is significantly higher than
16 % obtained by the synthetic procedure reported
previously by North et al. (2013).
The synthesised ureas II were used for cyclisation
to form imidazolidine-2,4,5-triones III in the next step
(Fig. 1) applying oxalyl chloride (Pejchal et al., 2011)
in dry tetrahydrofuran. The yields were in the range of
47–95 %; the adamantyl derivative IIIe provided the
lowest rate of conversion. Characterisation and spec-
tral data of compounds II and III are summarised in
Tables 1 and 2.
Starting SMX (I), ureas II and imidazolidine-
2,4,5-triones III were evaluated for their in vitro
antimycobacterial activity against M. tuberculosis
331/88 (H37Rv), M. avium 330/88 and two strains
of M. kansasii, namely 235/80 and the clinical isolate
6509/96. The first-line antimycobacterial drug isoni-
azid was employed as the reference compound.
SMX and its derivatives, II and III, showed an-
timycobacterial activity with MICs of 4 M or higher
(Table 3); none of the compounds was completely inac-
tive. M. tuberculosis showed the lowest susceptibility
to the novel molecules (MICs ≥ 125 M; the best re-
sult was obtained with 4-(trifluoromethyl)phenyl urea
IIh), whereas M. kansasii, with MICs ≥ 4 M, was
The cells were routinely cultured in Eagle’s mini-
mum essential media supplemented with 10 % of foetal
bovine serum, 1 % of L-glutamine solution, and a
non-essential amino acid solution. The investigated
compounds were dissolved in a very small amount of
DMSO, and a small volume of this solution was added
to the cell culture. The tested compounds were pre-
pared in triplicates at concentrations in the range of
0
.1–2000 M. The following types of controls were in-
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Table 1. Characterisation data of newly prepared compounds
1111
wi(calc.)/%
wi(found)/%
Yield
M.p.
Compound
R
Formula
Mr
◦
C
H
N
%
73
90
91
76
40
92
72
85
84
74
87
95
83
47
80
73
86
87
C
IIa
IIb
cyclohexyl
phenyl
C17H22N4O4S
C17H16N4O4S
C18H18N4O4S
C19H20N4O4S
C21H26N4O4S
C14H18N4O4S
C18H26N4O4S
378.45
372.40
386.42
400.45
430.52
338.38
394.49
53.95
5.86
5.88
14.80
14.89
210–212.5
217–218.5
225.5–228
239–241
5
3.79
54.83
4.92
55.95
5.87
56.99
6.88
58.59
8.63
49.69
9.74
54.80
4.67
49.09
9.16
58.86
9.02
52.77
3.00
53.52
3.48
54.54
4.65
55.50
5.37
57.01
6.91
48.97
9.11
53.56
3.44
48.59
9.33
57.25
7.42
4.33
4.51
15.04
14.99
5
IIc
benzyl
4.70
5.00
14.50
14.64
5
IId
phenethyl
adamantan-1-yl
propyl
5.03
4.90
13.99
14.12
5
IIe
6.09
6.00
13.01
13.20
224.5–226
235–237
5
IIf
5.36
5.21
15.56
15.69
4
IIg
heptyl
6.64
6.80
14.20
14.00
219–220.5
246–247
5
IIh
4-CF3-phenyl
4-tert-butylphenyl
cyclohexyl
phenyl
C18H15F3N4O4S 440.40
3.43
3.25
12.72
12.83
4
IIi
C21H24N4O4S
C19H20N4O6S
C19H14N4O6S
C20H16N4O6S
C21H18N4O6S
C23H24N4O6S
C16H16N4O6S
C20H24N4O6S
428.50
432.45
426.40
440.43
454.46
484.52
392.39
448.49
5.65
5.54
13.07
12.96
221.5–224
200.5–203
236–237.5
208.5–210.5
206–207
5
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
IIIh
IIIi
4.66
4.59
12.96
12.86
5
3.31
3.47
13.14
12.99
5
benzyl
3.66
3.70
12.72
12.73
5
phenethyl
adamantan-1-yl
propyl
3.99
3.86
12.33
12.19
5
4.99
5.12
11.56
11.70
206.5–208.5
198–200.5
147.5–148.5
216–218.5
223–226
5
4.11
4.02
14.28
14.41
4
heptyl
5.39
5.47
12.49
12.60
5
4-CF3-phenyl
4-tert-butylphenyl
C20H13F3N4O6S 494.40
C23H22N4O6S 482.51
2.65
2.51
11.33
11.36
4
4.60
4.74
11.61
11.58
5
shown to be the most susceptible species. M. avium
was inhibited at concentrations of at least 32 M. All
nontuberculous mycobacteria were suppressed most
effectively by 4-(3-heptylureido)-N-(5-methylisoxazol-
II or imidazolidine-2,4,5-triones III exceeded signifi-
cantly the activity of the parent drug SMX against the
studied mycobacterial strains, and only heptylurea IIg
showed equal or slightly higher activity against non-
tuberculous strains.
3
-yl)benzenesulphonamide (IIg) with MICs of 4–16
M for M. kansasii and 32–62.5 for M. avium. Propy-
3
The investigation on the influence of N -substitu-
lurea IIf was inactive against M. tuberculosis and the
adamantyl moiety containing imidazolidine IIIe was
inactive against M. avium.
INH is much more active against M. tuberculo-
sis than the SMX-based derivatives; two derivatives
tion on the activity for M. tuberculosis revealed that
the highest activity is conferred by a longer alkyl (IIg
vs. IIf ) and 4-substituted phenyl (IIi and especially
IIh) vs. unsubstituted phenyl IIb. Hydrogenation of
the phenyl ring (IIa vs. IIb) and the insertion of a
methylene group between nitrogen and phenyl (IIb vs.
IIc) did not affect the MIC values, but the derivative
with a two-carbon-long linker showed enhanced ac-
tivity (IIb vs. IId). Similar results were obtained for
M. avium with the exception of IIh, which is not signif-
icantly more active than IIb. A longer alkyl (IIg) and
4-substituted phenyl (IIh, IIi) were also the best sub-
(SMX and heptylurea IIg) exhibited activity against
the M. kansasii isolate 6509/96 comparable to that
of INH. In contrast, all imidazolidine-2,4,5-triones III
and five ureas (IId, IIe, IIg, IIh, IIi) showed higher ac-
tivity against M. kansasii 235/80 than INH, and eight
compounds (IId, IIg, IIh, IIIa, IIId, IIIf, IIIg, IIIh) ex-
hibited lower MICs for M. avium. None of the ureas
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112
M. Krátký et al./Chemical Papers 69 (8) 1108–1117 (2015)
Table 2. Spectral data of newly prepared compounds
Compound
Spectral data
IR, ν˜ /cm 1: 3356 (N—H), 1677 (C—O)
−
IIa
IIb
IIc
IId
IIf
1
1
H NMR (300 MHz, DMSO-d6), δ: 11.20 (1H, s, SO2NH), 8.79 (1H, s, N H ureido), 7.67 (2H, d, J = 8.9 Hz, H2,
H6), 7.52 (2H, d, J = 8.9 Hz, H3, H5), 6.26 (1H, d, J = 7.8 Hz, N H ureido), 6.10 (1H, s, isoxazole), 3.51–3.38 (1H,
m, H1 ), 2.28 (3H, s, CH3), 1.82–1.46 (5H, m, H2 , H3 , H4 , H5 , H6 ), 1.36–1.07 (5H, m, H2 , H3 , H4 , H5 , H6 )
C NMR (75.5 MHz, DMSO-d6), δ: 170.58, 158.12, 154.24, 145.50, 130.97, 128.53, 117.31, 95.77, 48.14, 33.17, 25.62,
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
13
2
4.72, 12.49
IR, ν˜ /cm 1: 3401 (N—H), 1696 (C—O)
−
1
1
3
H NMR (500 MHz, DMSO-d6), δ: 11.26 (1H, s, SO2NH), 9.14 (1H, s, N H ureido), 8.80 (1H, s, N H ureido), 7.75
ꢀ ꢀ
2H, d, J = 8.8 Hz, H2, H6), 7.63 (2H, d, J = 8.9 Hz, H3, H5), 7.45 (2H, d, J = 7.7 Hz, H2 , H6 ), 7.28 (2H, t, J =
(
ꢀ ꢀ ꢀ
.9 Hz, H3 , H5 ), 6.99 (1H, t, J = 7.4 Hz, H4 ), 6.12 (1H, s, isoxazole), 2.29 (3H, s, CH3)
7
13
C NMR (125 MHz, DMSO-d6), δ: 170.71, 158.15, 152.64, 144.80, 139.68, 132.04, 129.35, 128.66, 122.84, 118.99,
18.19, 95.89, 12.57
1
IR, ν˜ /cm 1: 3367 (N—H), 1671 (C—O)
−
1
1
H NMR (300 MHz, DMSO-d6), δ: 11.22 (1H, s, SO2NH), 9.09 (1H, s, N H ureido), 7.74–7.66 (2H, m, H2, H6),
.61–7.54 (2H, m, H3, H5), 7.36–7.18 (5H, m, H2 , H3 , H4 , H5 , H6 ), 6.82 (1H, t, J = 5.9 Hz, N H ureido), 6.11
1H, s, isoxazole), 4.29 (2H, d, J = 5.8 Hz, CH2), 2.28 (3H, s, CH3)
3C NMR (75.5 MHz, DMSO-d6), δ: 170.61, 158.09, 155.13, 145.44, 140.39, 131.15, 128.76, 128.54, 127.55, 127.23,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
7
(
1
1
17.55, 95.77, 43.18, 12.49
IR, ν˜ /cm 1: 3371 (N—H), 1657 (C—O)
−
1
1
H NMR (300 MHz, DMSO-d6), δ: 11.21 (1H, s, SO2NH), 9.00 (1H, s, N H ureido), 7.72–7.66 (2H, m, H2, H6),
.58–7.52 (2H, m, H3, H5), 7.33–7.16 (5H, m, H2 , H3 , H4 , H5 , H6 ), 6.31 (1H, t, J = 5.7 Hz, N H ureido), 6.11
1H, s, isoxazole), 3.34 (2H, q, J = 6.6 Hz, CH2NH) 2.74 (2H, t, J = 7.1 Hz, PhCH2), 2.28 (3H, s, CH3)
C NMR (75.5 MHz, DMSO-d6), δ: 170.36, 157.86, 154.77, 145.24, 139.58, 130.79, 128.86, 128.58, 128.29, 126.31,
17.17, 95.53, 40.81, 35.81, 12.25
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
7
(
1
3
1
IR, ν˜ /cm 1: 3368 (N—H), 1668 (C—O)
−
1
1
H NMR (300 MHz, DMSO-d6), δ: 11.20 (1H, s, SO2NH), 8.92 (1H, s, N H ureido), 7.71–7.65 (2H, m, H2, H6),
.58–7.51 (2H, m, H3, H5), 6.33 (1H, t, J = 5.7 Hz, N H ureido), 6.10 (1H, s, isoxazole), 3.03 (2H, q, J = 6.7 Hz,
3
7
CH2NH), 2.28 (3H, s, isoxazole-CH3), 1.42 (2H, sext, J = 7.3 Hz, CH2), 0.85 (3H, t, J = 7.4 Hz, CH2CH3)
1
3
C NMR (75.5 MHz, DMSO-d6), δ: 170.35, 157.86, 154.83, 145.33, 130.70, 128.27, 117.13, 95.53, 41.05, 23.02, 12.24,
1.47
1
IR, ν˜ /cm 1: 3378 (N—H), 1681 (C—O)
−
IIg
1
1
H NMR (300 MHz, DMSO-d6), δ: 11.20 (1H, s, SO2NH), 8.91 (1H, s, N H ureido), 7.71–7.65 (2H, m, H2, H6),
.57–7.51 (2H, m, H3, H5), 6.30 (1H, t, J = 5.7 Hz, N H ureido), 6.10 (1H, s, isoxazole), 3.06 (2H, q, J = 6.5 Hz,
3
7
CH2NH), 2.28 (3H, s, isoxazole-CH3), 1.40 (2H, quint, J = 6.9 Hz, CH2CH2NH), 1.31–1.20 (8H, m), 0.83 (3H, t, J
=
6.9 Hz, CH2CH3)
1
3
C NMR (75.5 MHz, DMSO-d6), δ: 170.58, 158.10, 155.04, 145.57, 130.93, 128.51, 117.36, 95.77, 39.48, 31.70, 30.01,
28.87, 26.74, 22.50, 14.39, 12.49
IR, ν˜ /cm 1: 3345 (N—H), 1698 (C—O)
−
IIh
IIi
1
3
1
H NMR (500 MHz, DMSO-d6), δ: 11.28 (1H, s, SO2NH), 9.27 (1H, s, N H ureido), 9.23 (1H, s, N H ureido),
ꢀ ꢀ ꢀ ꢀ
.79–7.75 (2H, m, H2, H6), 7.68–7.61 (6H, m, H3, H5, H2 , H3 , H5 , H6 ), 6.13 (1H, s, isoxazole), 2.29 (3H, s, CH3)
C NMR (125 MHz, DMSO-d6), δ: 170.40, 157.79, 152.15, 144.07, 143.15, 132.16, 128.33, 126.30 (q, J = 3.8 Hz),
24.66 (d, J = 271.1 Hz), 122.43 (q, J = 32.0 Hz), 118.35, 118.17, 95.56, 12.23
7
13
1
IR, ν˜ /cm 1: 3351 (N—H), 1698 (C—O)
−
1
1
3
H NMR (300 MHz, DMSO-d6), δ: 11.25 (1H, s, SO2NH), 9.09 (1H, s, N H ureido), 8.72 (1H, s, N H ureido), 7.77–
.71 (2H, m, H2, H6), 7.65–7.58 (2H, m, H3, H5), 7.39–7.27 (4H, m, H2 , H3 , H5 , H6 ), 6.12 (1H, s, isoxazole),
ꢀ
ꢀ
ꢀ
ꢀ
7
2
.29 (3H, s, isoxazole-CH3), 1.25 (9H, s, CH3 of tert-butyl)
13
C NMR (75.5 MHz, DMSO-d6), δ: 170.36, 157.80, 152.30, 144.82, 144.53, 136.68, 131.55, 128.30, 125.61, 118.49,
17.75, 95.53, 34.09, 31.40, 12.23
1
IR, ν˜ /cm 1: 1732 (C—O)
−
IIIa
IIIb
1
H NMR (300 MHz, THF-d8), δ: 10.41 (1H, s, SO2NH), 8.00–7.94 (2H, m, H2, H6), 7.77–7.73 (2H, m, H3, H5),
ꢀ ꢀ ꢀ
.25 (1H, s, isoxazole), 4.09 (1H, tt, J = 12.3 Hz, J = 3.7 Hz, H1 ), 2.30 (3H, s, CH3), 1.91–1.64 (5H, m, H2 , H3 ,
H4 , H5 , H6 ), 1.47–1.16 (5H, m, H2 , H3 , H4 , H5 , H6 )
3C NMR (75.5 MHz, THF-d8), δ: 171.19, 158.71, 156.65, 155.88, 153.32, 140.53, 136.17, 128.51, 126.31, 95.98,
6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
5
3.12, 30.30, 26.62, 25.98, 12.25
IR, ν˜ /cm 1: 1738 (C—O)
−
1
H NMR (500 MHz, THF-d8), δ: 11.61 (1H, s, SO2NH), 8.08–8.05 (2H, m, H2, H6), 7.74–7.70 (2H, m, H3, H5),
ꢀ ꢀ ꢀ ꢀ ꢀ
.57 (2H, t, J = 7.6 Hz, H3 , H5 ), 7.49 (1H, t, J = 7.6 Hz, H4 ), 7.44 (2H, d, J = 7.6 Hz, H2 , H6 ), 6.17 (1H, s,
7
isoxazole), 2.30 (3H, s, CH3)
1
3C NMR (125 MHz, THF-d8), δ: 170.74, 157.49, 156.05, 155.86, 152.15, 139.34, 134.77, 130.51, 129.46, 129.17,
128.14, 127.19, 126.79, 95.66, 12.26
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1113
Table 2. (continued)
Compound
Spectral data
IR, ν˜ /cm 1: 1734 (C—O)
−
IIIc
IIId
1
H NMR (300 MHz, THF-d8), δ: 11.60 (1H, s, SO2NH), 8.05–7.99 (2H, m, H2, H6), 7.70–7.64 (2H, m, H3, H5),
ꢀ ꢀ ꢀ ꢀ ꢀ
.45–7.26 (5H, m, H2 , H3 , H4 , H5 , H6 ), 6.16 (1H, s, isoxazole), 4.78 (2H, s, CH2), 2.29 (3H, s, CH3)
C NMR (75.5 MHz, THF-d8), δ: 170.74, 157.50, 157.08, 156.06, 153.09, 139.05, 135.27, 134.89, 128.63, 128.01,
27.86, 127.02, 95.63, 42.28, 12.27
7
13
1
IR, ν˜ /cm 1: 1736 (C—O)
−
1
H NMR (300 MHz, THF-d8), δ: 11.60 (1H, s, SO2NH), 8.06–8.00 (2H, m, H2, H6), 7.68–7.62 (2H, m, H3, H5),
ꢀ ꢀ ꢀ ꢀ ꢀ
.36–7.20 (5H, m, H2 , H3 , H4 , H5 , H6 ), 6.16 (1H, s, isoxazole), 3.79 (2H, t, J = 7.8 Hz, CH2N) 2.82 (2H, t, J
7
=
7.8 Hz, PhCH2), 2.30 (3H, s, CH3)
C NMR (75.5 MHz, THF-d8), δ: 170.72, 157.47, 156.78, 156.01, 152.84, 139.11, 137.96, 134.79, 128.83, 128.74,
1
3
128.03, 126.96, 126.80, 95.63, 40.16, 33.46, 12.25
IR, ν˜ /cm 1: 1730 (C—O)
−
IIIe
IIIf
1
H NMR (500 MHz, THF-d8), δ: 10.44 (1H, s, SO2NH), 7.97–7.94 (2H, m, H2, H6), 7.68–7.64 (2H, m, H3, H5), 6.24
1H, s, isoxazole), 2.49 (6H, m, CCH2CH) 2.30 (3H, s, CH3), 2.16–2.10 (3H, m, CH), 1.81–1.68 (6H, m, CHCH2CH)
C NMR (125 MHz, THF-d8), δ: 171.15, 158.72, 157.38, 155.37, 153.67, 140.57, 136.04, 128.41, 126.70, 95.99, 62.46,
0.83, 36.89, 30.94, 12.25
(
1
3
4
IR, ν˜ /cm 1: 1732 (C—O)
−
1
H NMR (300 MHz, DMSO-d6), δ: 11.56 (1H, s, SO2NH), 8.04–7.99 (2H, m, H2, H6), 7.68–7.62 (2H, m, H3, H5),
6.16 (1H, s, isoxazole), 3.53 (2H, t, J = 7.0 Hz, CH2N), 2.30 (3H, s, isoxazole-CH3), 1.62 (2H, sext, J = 7.3 Hz,
CH2), 0.91 (3H, t, J = 7.4 Hz, CH2CH3)
1
3C NMR (75.5 MHz, DMSO-d6), δ: 170.97, 157.77, 157.43, 156.40, 153.44, 139.29, 135.18, 128.25, 127.24, 95.89,
40.91, 21.30, 12.52, 11.59
IR, ν˜ /cm 1: 1736 (C—O)
−
IIIg
1
H NMR (500 MHz, DMSO-d6), δ: 11.58 (1H, s, SO2NH), 8.03–8.00 (2H, m, H2, H6), 7.67–7.63 (2H, m, H3, H5),
6.15 (1H, s, isoxazole), 3.55 (2H, q, J = 7.1 Hz, CH2NH), 2.30 (3H, s, isoxazole-CH3), 1.59 (2H, quint, J = 7.1 Hz,
CH2CH2NH), 1.36–1.18 (8H, m), 0.85 (3H, t, J = 6.7 Hz, CH2CH3)
1
3C NMR (125 MHz, DMSO-d6), δ: 170.70, 157.49, 157.11, 156.14, 153.14, 138.92, 134.92, 127.98, 126.95, 95.62,
38.96, 31.31, 28.41, 27.57, 26.15, 22.22, 14.06, 12.25
IR, ν˜ /cm 1: 1746 (C—O)
−
IIIh
IIIi
1
ꢀ
H NMR (500 MHz, THF-d8), δ: 10.48 (1H, s, SO2NH), 8.05–8.01 (2H, m, H2, H6), 7.87 (2H, d, J = 8.5 Hz, H3 ,
H5 ), 7.79–7.73 (4H, m, H3, H5, H2 , H6 ), 6.25 (1H, s, isoxazole), 2.30 (3H, s, CH3)
C NMR (125 MHz, THF-d8), δ: 171.26, 160.45, 158.67, 155.44, 152.28, 141.20, 135.69, 130.82 (q, J = 32.7 Hz),
28.76, 127.12 (q, J = 3.7 Hz), 126.96, 126.74, 124.93 (d, J = 272.0 Hz), 118.87, 118.55, 95.99, 12.26
ꢀ
ꢀ
ꢀ
13
1
IR, ν˜ /cm 1: 1736 (C—O)
−
1
H NMR (500 MHz, DMSO-d6), δ: 11.61 (1H, s, SO2NH), 8.06 (2H, d, J = 8.3 Hz, H2, H6), 7.71 (2H, d, J = 8.4 Hz,
H3, H5), 7.59 (2H, d, J = 8.3 Hz, H3 , H5 ), 7.36 (2H, d, J = 8.1 Hz, H2 , H6 ), 6.17 (1H, s, isoxazole), 2.30 (3H, s,
ꢀ
ꢀ
ꢀ
ꢀ
isoxazole-CH3), 1.32 (9H, s, CH3 of tert-butyl)
1
3C NMR (125 MHz, DMSO-d6), δ: 170.72, 157.48, 156.18, 155.90, 152.23, 151.77, 139.30, 134.79, 128.11, 127.88,
127.18, 126.39, 126.26, 95.65, 34.73, 31.18, 12.25
stitution patterns for the inhibition of M. kansasii. In
this case, the cyclohexyl (IIa) and benzyl (IIc) deriva-
tives showed somewhat lower activity than phenylurea
IIb, and phenethyl IId and adamantyl urea IIe exhib-
ited slightly lower MICs.
However, replacement of the 4-amino group of
SMX with an R-ureido moiety decreased predom-
inantly the antimycobacterial activity of the par-
ent compound, especially against the M. tuberculo-
sis strain, which can be explained by weaker interac-
tion with the target structure(s). Additionally, ClogP
values are not sufficiently satisfactory predictors of
antimicrobial activity. Similar results were obtained
for sulphonamide–phtalimide hybrids by Akg u¨ n et al.
The findings are not uniform. In general, cyclisation
of more efficient ureas produced derivatives with sim-
ilar (IIg or IIi for M. tuberculosis and M. avium and
IId for atypical mycobacteria) or higher (IIg or IIi
for M. kansasii, IId for M. tuberculosis, and IIh for
all strains) MIC values. In contrast, the modification
of ureas II with weaker antimycobacterial activity re-
sulted in products III with enhanced in vitro efficacy,
particularly for nontuberculous strains (IIa and IIf for
all strains and IIb and IIc for M. kansasii). Analy-
sis of the remaining compounds and strains revealed
no significant differences between imidazolidine-2,4,5-
triones and ureas.
Compounds I–III were investigated in vitro against
four gram-positive bacterial strains, namely Staphy-
lococcus aureus, methicillin-resistant Staphylococcus
aureus (MRSA), Staphylococcus epidermidis, Entero-
coccus sp., and four gram-negative strains, namely Es-
(
2012).
Another goal of this study was to describe the in-
fluence of the cyclisation of ureas II to imidazolidine-
,4,5-triones III on their antimycobacterial activity.
2
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114
M. Krátký et al./Chemical Papers 69 (8) 1108–1117 (2015)
Table 3. Antimycobacterial activity of SMX, INH, ureas II and imidazolidine-2,4,5-triones III
MIC/ Ma
Compound
R
M. tuberculosis 331/88 M. avium 330/88
M. kansasii 235/80
M. kansasii 6509/96
ClogP
14 d
21 d
14 d
21 d
7 d
14 d
21 d
7 d 14 d 21 d
IIa
IIIa
IIb
IIIb
IIc
IIIc
IId
IIId
IIe
IIIe
IIf
IIIf
IIg
IIIg
IIh
IIIh
IIi
1000
500
1000
1000
1000
500
250
500
500
500
> 1000
250
250
250
125
500
250
250
32
1000
500
1000
250
500
1000
1000
500
250
250
1000
> 500
1000
250
1000
250
500
32
1000
62.5
500
250
1000
250
125
125
250
250
1000
62.5
8
62.5
62.5
125
62.5
125
16
1000
125
500
500
1000
500
125
125
250
250
1000
62.5
16
125
125
125
62.5
250
16
250
32
250
500 1000
62.5 62.5
2.03
2.01
2.14
2.13
2.21
2.20
2.49
2.48
2.31
2.29
1.31
1.29
2.97
2.96
3.07
3.05
3.85
3.83
0.86
–
cyclohexyl
phenyl
1000
1000
1000
> 500
250
1000
500
> 500
> 1000
250
500
250
125
500
250
500
1000
1000
1000
> 500
500
250
250
500
125
125
125
125
125
500
32
4
32
16
62.5
32
500
500
250
62.5 125
250
62.5 125
125
125
125
250
250
32
500 1000
benzyl
250
250
250
250
500
500
125
8
250
250
250
500
500
62.5
4
phenethyl
adamantan-1-yl
propyl
500
1000
> 500
1000
250
62.5
125
125
250
500
500
32
4
heptyl
62.5
62.5
250
500
500
62.5 125
16 32
62.5 125
16
125
4
250
62.5
250
32
500
4
4
-CF3-phenyl
32
250
4
4
-tert-butylphenyl
IIIi
SMX
INH
62.5
8
–
–
32
1
32
> 250
62.5
0.5
> 250 > 250 > 250 > 250
4
8
8
a) One or two of the best MIC value(s) for each strain are shown in bold.
cherichia coli, Klebsiella pneumoniae, ESBL-positive
Klebsiella pneumoniae and Pseudomonas aeruginosa.
Bacitracin (BAC) was used as the comparative drug
500 M, and its cyclic analogue IIIg affected the
growth of Candida glabrata and C. krusei with MICs
of 250 M and 500 M, respectively.
(
Table 4).
None of the derivatives inhibited the growth of P.
aeruginosa and K. pneumoniae at concentrations of
00 M or lower, whereas E. coli was inhibited by
Cytotoxicity of the tested compounds I–III was
measured using the standard hepatic cell line HepG2
(Table 5). The used CellTiter 96 assay was based
on the reduction of tetrazolium dye in living cells
to formazan. The cytotoxicity is expressed as IC50,
i.e., the concentration reducing the viability of the
cells to 50 % of the maximal viability. It was not
possible to determine the exact IC50 value for three
derivatives (IIf, IIIf, IIg) due to their limited solu-
bility in the cell culture medium used in this experi-
ment.
The experiments showed that the studied com-
pounds can be divided into three groups. The first
group includes compounds IIe, IIh, IIi, IIIh, and IIIg,
which exhibit comparatively higher toxicity in HepG2
cells (IC50 in the range of 103.3–237.9 M). The sec-
ond group consists of IIIa–IIId, IIIi, IIIe, IIb, and
IId, which have moderate toxicity (348.3–910.6 M),
and the third group includes molecules with low
in vitro cytotoxicity (I, IIa, IIc, IIf, IIg, IIIf with
IC50 > 1000 M). Thus, the enhanced cytotoxicity
of SMX derivatives is observed in the presence of 4-
substituted phenyl (IIh, IIIh, IIi, IIIi) and adamantyl
(IIe, IIIe) moieties, whereas the cyclohexyl-, benzyl-,
phenethyl-, and alkyl-containing ureas showed com-
paratively milder IC50 values.
5
two ureas (IIf and IIi) at this concentration. Gram-
positive cocci were more susceptible, i.e., they were in-
hibited by some derivatives with MICs starting from
1
25 M. S. aureus exhibited the highest susceptibil-
ity with no marked difference between methicillin-
susceptible and MRSA strains.
Five ureas (IIa–IIe) and three imidazolidine-2,4,5-
triones (IIIb, IIIc, IIIg) did not exhibit any antibacte-
rial activity at the investigated concentrations. In con-
trast, IIg showed the highest in vitro potency (MICs
against S. aureus of 125–250 M), which is compa-
rable or slightly superior to that of the parent SMX.
However, none of II and III was proved to be supe-
rior to BAC or to produce low micromolar MICs. In
most cases, imidazolidine-2,4,5-triones III showed bet-
ter antibacterial properties than the parent ureas II
(IIa vs. IIIa, IId vs. IIId, IIf vs. IIIf, and IIi vs. IIi),
while two ureas were superior to III (IIg vs. IIIg and
IIh vs. IIIh), indicating that neither of the two ureido
hydrogens is essential for this action.
Antifungal properties of SMX derivatives were
evaluated in vitro against eight species. However, the
investigated sulphonamides showed almost no anti-
fungal activity, except for 3-heptyl derivatives IIg
and IIIg. Urea IIg inhibited Absidia corymbifera at
In general, although the newly synthesised deriva-
tives did not exhibit significant cytotoxicity, all modifi-
cations of the parent SMX (IC50 = 2622 M) increased
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Table 4. Antibacterial activity of SMX, ureas II and imidazolidine-2,4,5-triones III
MIC/ Ma
1115
Compound
R
S. aureus
4 h 48 h
MRSA
24 h
S. epidermidis Enterococcus sp.
E. coli
24 h 48 h
2
48 h
24 h
48 h
24 h
48 h
IIa
IIIa
IIb
IIIb
IIc
IIIc
IId
IIId
IIe
IIIe
IIf
IIIf
IIg
IIIg
IIh
IIIh
IIi
> 500
500
> 500
500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
500
> 125
500
> 500
500
250
> 500
125
500
500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 125
500
> 500
> 500
250
> 500
250
> 500
> 500
> 500
> 500
> 500
> 500
> 500
500
> 125
500
> 500
500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 125
> 500
> 500
> 500
250
> 500
> 500
> 500
> 500
> 500
> 500
> 500
500
> 125
500
> 500
500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 125
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 125
> 500
500 > 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 125
> 500
cyclohexyl
phenyl
> 500
> 500
> 500
> 500
> 500
500
> 125
500
> 500
500
125
> 500
125
500
250
> 500
> 500
> 500
> 500
> 500
> 500
> 125
500
> 500
> 500
250
> 500
500
benzyl
phenethyl
adamantan-1-yl
propyl
> 500
> 500
> 500
> 500
> 500
125
500
heptyl
> 500
> 500
500
> 500
250
> 500
> 500
> 500
> 500
500
> 500
> 500
500
> 500
250
4
-CF3-phenyl
500
500
500
500
500
> 500
500
500 > 500
> 500
> 500
4
-tert-butylphenyl
IIIi
SMX
250
250
250
62.5
> 500
> 500
–
250
250
500
> 500
BAC
–
15.62
31.25
15.62
31.25
15.62
31.25
31.25
31.25 > 500
> 500
a) One or two of the best MIC value(s) for each strain are shown in bold.
Table 5. Cytotoxicity and selectivity indices of SMX, ureas II and imidazolidine-2,4,5-triones III
Selectivity indexa
Compound
R
IC50 HepG2/ M
M. tuberculosis
M. avium
M. kansasii
Staphylococci
IIa
IIIa
IIb
IIIb
IIc
IIIc
IId
IIId
IIe
IIIe
IIf
IIIf
IIg
IIIg
IIh
IIIh
IIi
1129.0
375.2
499.5
667.0
1263.0
486.8
910.6
867.2
114.5
352.7
> 1500
> 1000
> 1000
237.9
176.4
181.3
103.3
348.3
2622.0
1.13
0.75
0.50
0.67
1.26
1.13
1.50
0.50–1.00
0.67
1.13–4.52
3.00–11.73
1.00–2.00
1.33–10.67
1.26–5.05
0.97–7.79
3.64–7.28
3.47–6.94
0.46–0.92
0.71–2.82
> 1.50–4.00
> 8.00–31.25
< 2.26
≤ 0.75
< 1.00
< 1.33
< 2.53
< 0.97
< 1.82
≤ 1.73
< 0.92
≤ 0.71
nd
cyclohexyl
phenyl
1.26
benzyl
≤ 0.97
≤ 0.97
1.82–3.64
1.73–3.47
0.11
> 0.71
> 1.50
> 4.00
3.64
phenethyl
adamantan-1-yl
propyl
0.87–1.73
0.23
≤ 0.71
nd
> 4.00
> 2.00–4.00
0.95
1.41
0.36
0.41
0.70–1.39
81.94
nd
> 16.00–31.25 > 62.50–250.00 > 4.00–8.00
heptyl
1.90–3.81
1.41–2.82
0.73
0.21
0.70
1.90–7.43
1.41–11.03
0.73–2.90
1.65–3.23
0.70–5.57
< 0.48
≤ 1.41
4
-CF3-phenyl
≤ 0.36
≤ 0.41
4
-tert-butylphenyl
IIIi
SMX
0.70–1.39
5.24–41.95
–
41.95–81.94
163.88–655.50
a) The best selectivity index value (SI = IC50/MIC) for each strain is shown in bold; nd = not determined (the IC50 and MIC
values are not exactly known).
the cytotoxicity by up to one order of magnitude for
the most toxic compounds.
The selectivity indices (SIs) of all sulphonamides
for M. tuberculosis, atypical mycobacteria and S. au-
reus were calculated. SI is defined as the ratio of IC50
to MIC, and values higher than 10 indicate rather ac-
ceptable toxicity (based on the analogy of the thera-
peutic index). In some cases it was not possible to de-
termine exact SI values due to the limited solubility of
the compounds in the medium used for the cytotoxic-
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M. Krátký et al./Chemical Papers 69 (8) 1108–1117 (2015)
ity assays (IIf, IIIf, IIg) or the anti(myco)bacterial as-
says. The analysis of M. tuberculosis revealed that the
propyl and heptyl derivatives IIIf and IIg exhibit the
best profiles, but their values are low. Similar results
were found for Staphylococci. The unsatisfactory SIs
are a consequence of low antibacterial activity; hep-
tylurea IIg was found to be favourable with SI values
higher than 4 or 8, but it was not possible to deter-
mine the exact value due to its limited solubility in
the testing medium. IIg also exhibited the most con-
venient toxicity properties for atypical mycobacterial
strains, i.e., its SI values for M. avium and for both
strains of M. kansasii ranged from 16 to 31.25 and
from 62.5 to 250, respectively.
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