Article novel sulfamethoxazole ureas & oxalamide as potential antimycobacterial agents

Article abstract of DOI:10.3390/molecules22040535

Infections caused by Mycobacterium tuberculosis (Mtb.) and nontuberculous mycobacteria (NTM) are considered to be a global health problem; current therapeutic options are limited. Sulfonamides have exhibited a wide range of biological activities including those against mycobacteria. Based on the activity of 4-(3-heptylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide against NTM, we designed a series of homologous sulfamethoxazole-based n-Alkyl ureas (C1-C12), as well as several related ureas and an oxalamide. Fifteen ureas and one oxalamide were synthesized by five synthetic procedures and characterized. They were screened for their activity against Mtb. and three NTM strains (M. avium, M. kansasii). All of them share antimycobacterial properties with minimum inhibitory concentration (MIC) values starting from 2 μM. The highest activity showed 4,40-[carbonylbis(azanediyl)]bis[N-(5-methylisoxazol-3-yl)benzenesulfonamide] with MIC of 2-62.5 μM(i.e., 1.07-33.28 μg/mL). Among n-Alkyl ureas, methyl group is optimal for the inhibition of both Mtb. and NTM. Generally, longer alkyls led to increased MIC values, heptyl being an exception for NTM. Some of the novel derivatives are superior to parent sulfamethoxazole. Several urea and oxalamide derivatives are promising antimycobacterial agents with low micromolar MIC values.

Full text of DOI:10.3390/molecules22040535

molecules
Article
Novel Sulfamethoxazole Ureas and Oxalamide as
Potential Antimycobacterial Agents
Martin Krátký 1,*, Jirˇina Stolarˇíková 2 and Jarmila Vinšová 1
1
Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles
University, Heyrovského 1203, 50005 Hradec Králové, Czech Republic; vinsova@faf.cuni.cz
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava,
2
Partyzánské náme˘stí 7, 70200 Ostrava, Czech Republic; Jirina.Stolarikova@zu.cz
*
Correspondence: martin.kratky@faf.cuni.cz; Tel.: +420-495-067-343; Fax: +420-495-067-166
Academic Editor: Claudiu T. Supuran
Received: 2 March 2017; Accepted: 24 March 2017; Published: 28 March 2017
Abstract: Infections caused by Mycobacterium tuberculosis (Mtb.) and nontuberculous mycobacteria
(NTM) are considered to be a global health problem; current therapeutic options are limited.
Sulfonamides have exhibited a wide range of biological activities including those against mycobacteria.
Based on the activity of 4-(3-heptylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide against
NTM, we designed a series of homologous sulfamethoxazole-based n-alkyl ureas (C1–C12), as well
as several related ureas and an oxalamide. Fifteen ureas and one oxalamide were synthesized by
five synthetic procedures and characterized. They were screened for their activity against Mtb.
and three NTM strains (M. avium, M. kansasii). All of them share antimycobacterial properties
with minimum inhibitory concentration (MIC) values starting from 2
showed 4,4 -[carbonylbis(azanediyl)]bis[N-(5-methylisoxazol-3-yl)benzenesulfonamide] with MIC of
µ
M. The highest activity
0
2–62.5 µM (i.e., 1.07–33.28 µg/mL). Among n-alkyl ureas, methyl group is optimal for the inhibition of
both Mtb. and NTM. Generally, longer alkyls led to increased MIC values, heptyl being an exception
for NTM. Some of the novel derivatives are superior to parent sulfamethoxazole. Several urea and
oxalamide derivatives are promising antimycobacterial agents with low micromolar MIC values.
Keywords: antimycobacterial activity; in vitro activity; oxalamide; sulfamethoxazole; sulfonamides;
tuberculosis; ureas
1. Introduction
The progression of resistance to clinically used drugs among human pathogenic bacteria justifies
the development of new antimicrobial agents effective against, e.g., Mycobacterium tuberculosis
(Mtb.) nontuberculous (atypical) mycobacteria (NTM), methicillin-resistant Staphylococcus aureus,
and polyresistant Gram-negative species [1].
The modification of known drugs represents an established and effective approach in drug design.
Sulfonamides have been widely used for the treatment of bacterial and protozoan infections. More
recently, sulfonamides were recommended as a part of multidrug regimens for some nontuberculous
mycobacterioses [2], but not for tuberculosis. Later, e.g., Ameen and Drancourt [3,4] demonstrated
that mycobacterial strains (Mtb., Mycobacterium avium complex) are susceptible to clinically-achievable
concentrations of sulfonamides (sulfadiazine, sulfamethoxazole, including its combination with
trimethoprim) and the investigation of sulfonamide derivatives as potential antimycobacterial agents
has been initiated [1,5].
Sulfonamides are well-known inhibitors of dihydropteroate synthases involved in the folate
pathway [6], β-carbonic anhydrases [7,8], acetohydroxyacid synthase [9] or β-ketoacyl synthases [10].
Obviously, their mechanism of action against mycobacteria seems to be multiple.
Molecules 2017, 22, 535; doi:10.3390/molecules22040535
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Recently, urea derivatives obtained from sulfamethoxazole (SMX)
inhibitors of human carbonic anhydrases [11], anticancer [12
1
have been proposed as potential
,
13], and antimicrobial agents [14,15].
Importantly, we reported the synthesis and antimicrobial activity of nine sulfamethoxazole-based ureas
and corresponding cyclic analogues, imidazolidine-2,4,5-triones. They were active especially against
NTM, followed by Mtb. and Gram-positive bacteria. Gram-negative species and fungi showed almost
a complete resistance. 4-(3-Heptylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (Scheme 1;
2g) was found to be the most effective derivative in vitro with minimum inhibitory concentration
(MIC) values against NTM from 4
antimycobacterial agents [16–19].
µM [1]. Moreover, ureas are largely investigated as potential
This unique activity of the 3-heptylureido derivative within the series inspired us to synthesize
and evaluate a homologous series of sulfamethoxazole-based monoalkylureas (C1–C12) to determine
possible relationships between the length of the alkyl and antimycobacterial activity. Additionally,
we synthesized several other ureas (Scheme 1) and one analogue of 2,4,5-trioxoimidazolidines,
oxalamide, as potential antimycobacterial agents.
Scheme 1. Design of novel antimycobacterial sulfamethoxazole ureas based on 3-heptylurea derivative
(n = 0–11; R1 = isopropyl, cyclopropyl, 4-[N-(5-methylisoxazol-3-yl)sulfamoyl]phenyl; R2 = H, isopropyl).
2. Results and Discussion
2.1. Chemistry
Sulfamethoxazole-based ureas
2
were obtained from parent sulfonamide
1 and isocyanates,
predominantly (reflux, 3 h; compounds 2b
–
2l). Method A involved commercially available isocyanates;
decyl isocyanate was generated in situ from decylamine using triphosgene (Method B; Scheme 2).
In general, it provided satisfactory yields of 71–85%. Methylurea 2a was synthesized by treating of
1
with 2,5-dioxopyrrolidin-1-yl methylcarbamate (N-succinimidyl N-methylcarbamate) as a methyl
isocyanate substitute in the presence of Hunig’s base (Method C; yield of 89%, Scheme 3). The last
0
approach (Method D) consisted of a one-pot reaction of
1
with 1,1 -carbonyldiimidazole (CDI) to form
the (1H-imidazole-1-yl)urea intermediate that undergoes aminolysis by an excess of an amine added
into the reaction mixture (Scheme 4). In this case, yields are varying (58–95%). Method D was favorable
for a secondary amine (diisopropylamine) and for primary amines of which isocyanates were not
available for us (cyclopropylamine for 2m, SMX itself for the synthesis of “bis-SMX-urea” 2n).
Oxalamide derivative
stoichiometric amounts.
3 was obtained by treatment of SMX 1 with oxalyl chloride (Scheme 5) in
All of the compounds (Table 1) were characterized by 1H-, 13C-NMR (nuclear magnetic resonance),
and infrared (IR) spectra and melting points. Their purity was checked by thin-layer chromatography
(TLC) and elemental analysis.

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Based on the previously reported activity of isolated propyl- and heptyl-ureas 2c and 2g [1],
we completed a homologous series of unbranched alkyl chains from methyl to dodecyl to reveal the
dependence of antimycobacterial activity on the alkyl length. To investigate the possible influence of
a cycloalkyl and a branched alkyl, cyclopropyl derivative 2m and isopropyl derivative 2o were also
synthesized. Urea 2n may be considered as a “double molecule” of SMX
bridge or as simplified imidazolidine-2,4,5-trione [ ] that has arisen from the removal of a –CO–CO–
bridge. Oxalamide was intended as another “open analogue” of imidazolidine-2,4,5-trione designed
by the removal of the carbonyl bridge.
1 connected via a carbonyl
1
3
Scheme 2. Synthesis of the ureas 2b–2l (Methods A and B; Et3N = triethylamine; DCM = dichloromethane;
MeCN = acetonitrile; R = C2 to C12 n-alkyl).
Scheme 3. Synthesis of the methyl derivative 2a (Method C; DIPEA = N,N-diisopropylethylamine).
Scheme 4. Synthesis of the urea derivatives 2m
R1 = cyclopropyl, isopropyl, 4-[N-(5-methylisoxazol-3-yl)sulfamoyl]phenyl; R2 = H, isopropyl).
–
2o (Method D; CDI = 1,10-carbonyldiimidazole;
Scheme 5. Synthesis of the oxalamide 3.

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Table 1. Antimycobacterial activity of sulfamethoxazole-based ureas 2 and oxalamides 3.
MIC (µM)
Mycobacterium
tuberculosis 331/88
Mycobacterium
avium 330/88
Mycobacterium kansasii
Mycobacterium kansasii
ClogP
R
Code
235/80
6509/96
14 d
21 d
14 d
21 d
7 d
14 d
21 d
7 d
14 d
21 d
2a
2b
2c
2d
2e
2f
2g
2h
2i
Methyl
Ethyl
16
62.5
>1000
62.5
250
32
62.5
>1000
62.5
500
250 *
500
125 *
125 *
125
62.5
1000
1000
1000
500
250
32
125 *
125 *
125
125 *
125 *
125
125
1000
1000
1000
1000
250
62.5
125 *
125 *
125
32
125
500
125
250
250
4
125 *
125 *
125
125 *
125 *
4
62.5
250
1000
250
250
250
8
125 *
125 *
125
125 *
125 *
4
125
250
1000
250
500
500
16
125 *
125 *
125
125 *
125 *
8
8
16
500
500
500
500
250
4
125 *
125 *
125
125 *
125 *
16
32
500
500
500
500
250
8
125 *
125 *
125
125 *
125 *
16
0.48
0.82
1.31
1.72
2.14
2.56
2.97
3.39
3.81
4.23
4.64
5.06
0.78
250
250
250
250
125
4
125 *
125 *
125
125 *
125 *
8
Propyl a
Butyl
Pentyl
Hexyl
250
250
Heptyl a
Octyl
125 *
125 *
125
125 *
125 *
2
Nonyl
Decyl
Undecyl
Dodecyl
Cyclopropyl
2j
2k
2l
125 *
125 *
4
125 *
125 *
125
2m
2n
2o
-
-
2
125
2
250
32
>1000
62.5
>1000
2
250
2
250
2
250
2
500
4
500
4
500
1.78
2.03

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Table 1. Cont.
3
H
4
4
125 *
125 *
8
8
8
8
8
8
1.23
SMX (1) a
INH
32
1
32
1
32
>250
62.5
>250
8
16
>250
16
>250
4
8
4
8
4
8
1.5
−0.64
>250
INH: isoniazid; SMX = sulfamethoxazole; ClogP = calculated logP values. Minimum inhibitory concentration (MIC) values were determined after incubation for 14 and 21 days, for
M. kansasii additionally for seven days. The best MIC values for each strain are shown in bold. Compounds and data a were taken from [
strain was observed; at duplex concentration, there was a precipitate present and/or a turbidity; therefore it was not possible to determine exact MIC value.
1]. * At presented concentration, the growth of the

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2.2. Antimycobacterial Activity
All derivatives
2
and
3
together with parent SMX
1
and isoniazid (INH) were evaluated for their
in vitro antimycobacterial activity against Mycobacterium tuberculosis CNCTC 331/88 (i.e., H37Rv) and
three strains of NTM, namely Mycobacterium avium CNCTC 330/88, Mycobacterium kansasii CNCTC
235/80 and M. kansasii 6509/96 (a clinically isolated strain). The minimal inhibitory concentration
(MIC) is the lowest concentration of the substance that prevents the growth of Mycobacterium
species completely.
An escalated lipophilicity of higher homologues abolished their antimycobacterial evaluation.
Octyl derivative 2h (ClogP = 3.39), nonyl 2i, undecyl 2k, and dodecyl 2l ureas were soluble in the
testing medium only up to the concentration of 125 µM. The growth of mycobacteria was observed
at this concentration but, at higher concentrations, there was a precipitate present and/or turbidity.
That is why it was not possible to determine MIC values.
In general, all soluble ureas (2a
from 2 M. In this series, M. avium showed the highest rate of resistance among mycobacterial strains.
In contrast, the susceptibility of Mtb. and M. kansasii was well marked. MIC values for M. avium were
within the range of 32–1000 M; N,N-diisopropylurea 2o was inactive. Heptylurea 2g ], methylurea
2a, and “bis-SMX-urea” 2n produced an activity comparable to SMX ; neither of the derivatives and
were superior to the parent drug. Three derivatives (cyclopropylurea 2m, “bis-SMX-urea” 2n, and
–2g, 2j, 2m–2o) and oxalamide 3 exhibited MIC values starting
µ
µ
[1
1
2
3
oxalamide
3
) exceeded the in vitro activity of SMX against Mtb. up to sixteen times (2m and 2n; MIC
g/mL and 1.07 g/mL, respectively) and methylurea 2a showed an equal
inhibition. Additionally, ethyl 2b, butyl 2d and decyl 2j ureas were superior to original heptylurea 2g
Both strains of M. kansasii share a similar susceptibility of 2–1000 M, only methylurea 2a exhibited
moderately lower MIC values for the clinical isolate. The most active derivatives, 2m 2n, and , share
an identical or a slightly superior activity than both parent SMX 1 and heptylurea 2g.
of 2–4
µM, i.e., 0.67–1.35
µ
µ
.
µ
,
3
Focusing on structure-activity relationship, the investigation of 3-ureido substitution identified
4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl (2n) as the most convenient substituent. Obviously,
the link of two SMX molecules to one molecular entity via a carbonyl bridge led to an advantageous
augmentation of the antimycobacterial activity; this “double” sulfonamide is the most active within
the series, being comparable (M. avium, M. kansasii 6509/96) or considerably superior to the parent
drug. The introduction of 3-cyclopropyl moiety also produced highly active urea 2m. Evidently, the
cyclization of n-propyl to cyclopropyl increased the activity sharply (2c vs. 2m). The cyclopropylamino
fragment is frequent in the molecules of many (fluoro)quinolones, known antimycobacterial agents,
or in antimycobacterials in preclinical development [20
of the mycobacterial cell wall, also contain a cyclopropane motif [22]; thus, interference with these
biomolecules may contribute to excellent antimycobacterial action. Interestingly, a review of Talele [23
emphasized the unique role of cyclopropyl in the structures of drug candidates. Similar MIC values
,21]. Mycolic acids, essential components
]
were obtained by symmetrically disubstituted N1,N2-oxalamide
3; interestingly, this molecule was
inactive against M. avium. On the other hand, N3,N3-disubstitution did not improve antimycobacterial
potency. Diisopropylurea 2o was ineffective against M. avium and its activity against remaining strains
was moderate.
Considering homologous n-alkyls, we can distinguish a clear trend for the tuberculous strain
331/88. The best activity against Mtb. is conferred by the lowest alkyl methyl (2a, 16–32
4.97–9.93 g/mL) and MIC values are increasing with the length of the alkyl chain (moderate activity
of ethyl 2b and butyl 2d ureas of 62.5 M). The mildest activity is connected with an intermediate
length of alkyl (five-, six-, and seven-membered chains, 2e 2g, 250–500 M). Based on decylurea 2j
(MIC = 125 M), it may be expected that the antitubercular properties are improved with a further
prolongation of alkyl chain, but limited solubility of such compounds in the testing medium interferes
with the evaluation. Previously described propylurea 2c ] seems to be an anomaly. The superior
activity against NTM displayed methyl (2a, 8–125 M, 2.48–38.75 g/mL) and especially heptyl (2g
4–62.5 M, i.e., 1.58–24.66 g/mL). For M. avium, concomitantly with the increasing length of the chain
µM, i.e.,
µ
µ
–
µ
µ
[
1
µ
µ
,
µ
µ

Molecules 2017, 22, 535
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from methyl, the activity is decreased dramatically (2a vs. 2b
–2e) but after pentyl (2e), it is enhanced
again to urea 2g. Decyl derivative 2j exhibited a moderate MIC of 125 µM. The drop in the activity
caused by the lengthening of the alkyl was not too sharp for both M. kansasii strains. N3-Substituted
ureas by from C2 to C6 (2b–2f) share a comparable activity.
The conversion of primary amino group of sulfonamide
1 to a urea or a carboxamide led to
controversial results. In some cases, antimycobacterial properties are enhanced (cyclopropylurea 2m
for Mtb., M. kansasii 235/80; “bis-SMX-urea” 2n for M. tuberculosis and M. kansasii 235/80; oxalamide
3
for Mtb.), retained (generally methylurea 2a, heptylurea 2g in the case of NTM, urea 2n for the
clinically-isolated M. kansasii) or even diminished (a majority of derivatives against M. avium and
M. kansasii 6509/96). For several compounds, a sharply increased lipophilicity prevented the exact
determination of MIC. In general, the formation of cyclopropylurea 2m, “double” SMX urea 2n
oxalamide as well as previously reported heptylurea 2g ] were found to be the most successful
modifications of the sulfa drug 1.
The main impetus for this subsequent study was a unique activity of heptylurea 2g against NTM
within previous series of SMX-based ureas [ ]. Now, we obtained seven compounds (2a 2b 2d 2j
2m 2n, and ) that are more efficacious in vitro against Mtb. (up to 250 times). However, only two
,
3
[1
1
,
,
,
,
,
3
derivatives (methyl derivative 2a and “bis-SMX-urea” 2n) produced a comparable inhibition of M.
avium. Urea 2n is also superior for M. kansasii 235/80 and comparable in the case of the strain 6509/96.
Cyclopropropylurea 2m and amide
Drawing a comparison to INH, a first-line oral antimycobacterial drug, “bis-SMX-urea” 2n showed
an equivalent in vitro activity ( 1 dilution) against Mtb. while the growth of M. avium and both strains
of M. kansasii was suppressed more effectively by 2n. Four additional derivatives (2a 2f 2j, and 2m
were also effective against INH-resistant M. avium, evidently. Focusing on INH-resistant M. kansasii
235/80, all of the soluble derivatives displayed lower MIC values with an exception (propylurea 2c ]).
Four derivatives (2g 2m 2n ) share an activity against M. kansasii isolate 6509/96 comparable or
3 inhibited both strains of M. kansasii at similar concentration levels.
±
,
,
)
[1
,
,
, 3
superior to that on INH, followed by methylurea 2a.
The uniform and broad-spectrum activity of
2 and 3 against INH-resistant strains indicate
that these derivatives do not share any cross-resistance with this established antitubercular drug.
Additionally, NTM are usually more chemoresistant than Mtb. Consequently, cellular targets of the
ureas and oxalamide, although not elucidated, seem to be different from INH. INH is highly specific
for Mtb. and its mechanism of action includes multiple effects on various cellular structures and
molecules. After metabolic activation, it inhibits enoyl-acyl carrier protein reductase (InhA) involved
in the biosynthesis of mycolic acids [24].
3. Materials and Methods
3.1. Chemistry
3.1.1. General
All of the reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or
Penta Chemicals (Prague, Czech Republic), and they were used as received. Melting points were
determined on a Büchi Melting Point machine B-545 apparatus (BÜCHI, Flawil, Switzerland) using
open capillaries, and the reported values are uncorrected. The progress of the reactions and the purity
of the products were monitored by TLC using a mixture of ethyl acetate with n-hexane (2:1, v/v).
Plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck Millipore, Darmstadt, Germany) and
were visualized by UV irradiation (254 nm).
Elemental analysis (C, H, N) was performed on a CHNS-O CE automatic microanalyzer
instrument (FISONS EA 1110, Milano, Italy). Infrared spectra (ATR) were recorded on a Nico−le1t
6700 FTIR spectrometer (ThermoFisher Scientific, Waltham, MA, USA) in the range of 400 to 4000 cm
.
The NMR spectra were measured in DMSO-d6 at ambient temperature on a Varian VNMR S500
instrument (500 MHz for 1H and 125 MHz for 13C; Varian Comp., Palo Alto, CA, USA) or a Varian

Molecules 2017, 22, 535
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1
Mercury-Vxbb 300 (300 MHz for H and 75.5 MHz for 13C; Varian Comp., Palo Alto, CA, USA).
The chemical shifts, , are given in ppm, with respect to tetramethylsilane as an internal standard.
δ
The coupling constants (J) are reported in Hz.
The calculated logP values (ClogP), which are the logarithms of the partition coefficients
for octan-1-ol/water, were determined using the program CS ChemOffice Ultra version 15.0
(CambridgeSoft, Cambridge, MA, USA).
3.1.2. Synthesis
Synthesis of Urea Derivatives 2
Method A
Sulfamethoxazole 1 (1 mmol) was dissolved in dry acetonitrile (MeCN, 6 mL) and appropriate
isocyanate (1.1 mmol) was added in one portion. The solution was heated under reflux for 3 h and then
stirred at ambient temperature overnight. The resulting crystals were filtered, washed with a small
volume of MeCN and dried. The product was recrystallized from ethyl acetate (EtOAc) if necessary [
1].
Method B
Triphosgene (0.4 mmol) was dissolved in anhydrous dichloromethane (DCM; 5 mL) under argon
atmosphere and decylamine (1.01 mmol) in dry DCM (3 mL) was added dropwise. This mixture was
stirred for 30 min at room temperature and then treated with triethylamine (2.1 mmol) in dry DCM
(3 mL). After 30 min, a solution of sulfamethoxazole 1 (1.01 mmol) in 5 mL of dry MeCN was added.
The reaction mixture was stirred for 12 h at room temperature. Then 1 mL of glacial acetic acid was
added and the reaction mixture was stirred for additional 2 h. The mixture was evaporated to dryness.
After addition of 10 mL of MeCN, the suspension was let to stay overnight. The resulting crystals were
filtered, washed with a small volume of diethylether and dried to obtain the final product.
Method C
Sulfamethoxazole
1 (1 mmol) was dissolved in dry acetonitrile (5 mL), then N,N-
diisopropylethylamine (2 mmol) followed by 2,5-dioxopyrrolidin-1-yl methylcarbamate (1.5 mmol)
was added. The solution was stirred for 24 h. The resulting crystals were filtered, washed with a small
volume of MeCN and dried. The product was recrystallized from EtOAc if necessary.
Method D
0
Sulfamethoxazole
1
(1 mmol) was dissolved in dry acetonitrile (6 mL) and 1,1 -carbonyldiimidazole
(1.2 mmol) was added. The mixture was refluxed for 12 h and then treated with amine (1.2 mmol; 3
h refluxing, then overnight at room temperature). The resulting crystals were filtered, washed with
a small amount of MeCN and dried to obtain the final product.
N-(5-Methylisoxazol-3-yl)-4-(3-methylureido)benzenesulfonamide (2a) [25]. White solid; yield 89% (Method
1
C); m.p. 242.5–244 ◦C. IR: 3375 (N–H), 1662 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ
11.22 (1H, s,
SO2NH), 9.04 (1H, s, N1–H ureido), 7.70–7.66 (2H, m, H2, H6), 7.57–7.54 (2H, m, H3, H5), 6.20 (1H, q,
J = 4.5 Hz, N3-H ureido), 6.11 (1H, d, J = 1.0 Hz, isoxazole), 2.63 (3H, d, J = 4.6 Hz, CH3), 2.28 (3H, d,
J = 1.0 Hz, CH3 isoxazole). 13C-NMR (126 MHz, DMSO):
δ 170.36, 157.88, 155.44, 145.38, 130.70, 128.25,
117.16, 95.53, 26.44, 12.26. Anal. Calcd. for C12H14N4O4S (310.33): C, 46.45; H, 4.55; N, 18.05. Found: C,
46.54; H, 4.88; N, 17.99.
4-(3-Ethylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2b). White solid; yield 80% (Method A);
1
m.p. 240–241.5 ◦C. IR: 3374 (N–H), 1684 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.19 (1H, s,
SO2NH), 8.93 (1H, s, N1–H ureido), 7.70–7.66 (2H, m, H2, H6), 7.57–7.53 (2H, m, H3, H5), 6.29 (1H,
t, J = 5.6 Hz, N3–H ureido), 6.10 (1H, d, J = 0.9 Hz, isoxazole), 3.09 (2H, td, J = 7.2 Hz, J = 5.6 Hz,

Molecules 2017, 22, 535
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CH2), 2.28 (3H, d, J = 1.0 Hz, CH3 isoxazole), 1.03 (3H, t, J = 7.1 Hz, CH3 alkyl). 13C-NMR (126 MHz,
DMSO): 170.32, 157.84, 154.72, 145.32, 130.72, 128.24, 117.15, 95.52, 34.19, 15.45, 12.22. Anal. Calcd.
δ
for C13H16N4O4S (324.36): C, 48.14; H, 4.97; N, 17.27. Found: C, 48.29; H, 4.87; N, 17.44.
N-(5-Methylisoxazol-3-yl)-4-(3-propylureido)benzenesulfonamide (2c). Compound 2c was synthesized
following a procedure described by Krátký et al. [1].
4-(3-Butylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2d). White solid; yield 73% (Method A);
◦
m.p. 216–218 C. IR: 3369 (N–H), 1683 (C=O) cm−1 1H-NMR (500 MHz, DMSO):
. δ 11.19 (1H, s,
SO2NH), 8.90 (1H, s, N1–H ureido), 7.70–7.66 (2H, m, H2, H6), 7.56–7.51 (2H, m, H3, H5), 6.30 (1H, t,
J = 5.7 Hz, N3–H ureido), 6.10 (1H, d, J = 1.0 Hz, isoxazole), 3.07 (2H, td, J = 6.9 Hz, J = 5.6 Hz, C1H2),
2.28 (3H, d, J = 1.0 Hz, CH3 isoxazole), 1.43–1.36 (2H, m, C2H2), 1.33–1.24 (2H, m, C3H2), 0.87 (3H, t,
J = 7.3 Hz, CH3 alkyl). 13C-NMR (126 MHz, DMSO):
δ 170.31, 157.85, 154.79, 145.31, 130.71, 128.24,
117.11, 95.52, 38.89, 31.86, 19.65, 13.83, 12.22. Anal. Calcd. for C15H20N4O4S (352.41): C, 51.12; H, 5.72;
N, 15.90. Found: C, 51.29; H, 5.90; N, 15.74.
N-(5-Methylisoxazol-3-yl)-4-(3-pentylureido)benzenesulfonamide (2e). White solid; yield 71% (Method A);
1
m.p. 213.5–214.5 ◦C. IR: 3370 (N–H), 1682 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ
11.19 (1H, s,
SO2NH), 8.90 (1H, s, N1–H ureido), 7.70–7.66 (2H, m, H2, H6), 7.56–7.52 (2H, m, H3, H5), 6.30 (1H, t,
J = 5.7 Hz, N3-H ureido), 6.10 (1H, d, J = 1.0 Hz, isoxazole), 3.06 (2H, td, J = 6.9 Hz, J = 5.6 Hz, C1H2),
2.28 (3H, d, J = 0.9 Hz, CH3 isoxazole), 1.45–1.37 (2H, m, C2H2), 1.32–1.20 (4H, m, C3H2, C4H2), 0.86
(3H, t, J = 7.0 Hz, CH3 alkyl). 13C-NMR (126 MHz, DMSO):
δ 170.31, 157.83, 154.77, 145.30, 130.69,
128.24, 117.10, 95.51, 39.19, 29.41, 28.71, 22.02, 14.10, 12.21. Anal. Calcd. for C16H22N4O4S (366.44): C,
52.44; H, 6.05; N, 15.29. Found: C, 52.32; H, 5.92; N, 14.99.
4-(3-Hexylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2f). White solid; yield 82% (Method A);
1
m.p. 218.5–220 ◦C. IR: 3373 (N–H), 1683 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.08 (1H, s,
SO2NH), 8.80 (1H, s, N1–H ureido), 7.59–7.56 (2H, m, H2, H6), 7.46–7.42 (2H, m, H3, H5), 6.20 (1H,
t, J = 5.7 Hz, N3–H ureido), 6.00 (1H, d, J = 0.9 Hz, isoxazole), 2.96 (2H, td, J = 6.9 Hz, J = 5.6 Hz,
C1H2), 2.18 (3H, d, J = 0.9 Hz, CH3 isoxazole), 1.35–1.26 (2H, m, C2H2), 1.32–1.20 (6H, m, C3H2, C4H2,
C5H2), 0.74 (3H, t, J = 7.1 Hz, CH3 alkyl). 13C-NMR (126 MHz, DMSO):
δ 170.30, 157.83, 154.77, 145.30,
130.70, 128.23, 117.10, 95.51, 39.19, 31.16, 29.70, 26.17, 22.24, 14.08, 12.21. Anal. Calcd. for C17H24N4O4S
(380.46): C, 53.67; H, 6.36; N, 14.73. Found: C, 54.00; H, 6.58; N, 14.86.
4-(3-Heptylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2g). Compound 2g was synthesized
following a procedure described by Krátký et al. [1].
N-(5-Methylisoxazol-3-yl)-4-(3-octylureido)benzenesulfonamide (2h). White solid; yield 83% (Method A);
◦
m.p. 214–216 C. IR: 3368 (N–H), 1677 (C=O) cm−1 1H-NMR (500 MHz, DMSO):
. δ 11.18 (1H, s,
SO2NH), 8.89 (1H, s, N1–H ureido), 7.69–7.65 (2H, m, H2, H6), 7.56–7.52 (2H, m, H3, H5), 6.30 (1H, t,
J = 5.7 Hz, N3-H ureido), 6.10 (1H, d, J = 1.1 Hz, isoxazole), 3.06 (2H, td, J = 6.8 Hz, J = 5.6 Hz, C1H2),
2.28 (3H, d, J = 1.0 Hz, CH3 isoxazole), 1.44–1.36 (2H, m, C2H2), 1.32–1.20 (10H, m, C3H2, C4H2, C5H2,
C6H2, C7H2), 0.84 (3H, t, J = 6.9 Hz, CH3 alkyl). 13C-NMR (126 MHz, DMSO):
δ 170.28, 157.85, 154.76,
145.28, 130.71, 128.22, 117.09, 95.50, 39.20, 31.40, 29.72, 28.88, 28.84, 26.50, 22.24, 14.11, 12.21. Anal.
Calcd. for C19H28N4O4S (408.52): C, 55.86; H, 6.91; N, 13.71. Found: C, 56.02; H, 6.92; N, 14.02.
N-(5-Methylisoxazol-3-yl)-4-(3-nonylureido)benzenesulfonamide (2i). White solid; yield 84% (Method A);
1
m.p. 211–212.5 ◦C. IR: 3382 (N–H), 1679 (C=O) cm−1. H-NMR (300 MHz, DMSO):
δ 11.20 (1H, s,
SO2NH), 8.91 (1H, s, N1–H ureido), 7.70–7.65 (2H, m, H2, H6), 7.57–7.51 (2H, m, H3, H5), 6.30 (1H, t,
J = 5.7 Hz, N3–H ureido), 6.10 (1H, d, J = 0.9 Hz, isoxazole), 3.05 (2H, q, J = 6.5 Hz, C1H2), 2.28 (3H,
d, J = 0.9 Hz, CH3 isoxazole), 1.44–1.36 (2H, m, C2H2), 1.30–1.18 (12H, m, C3H2, C4H2, C5H2, C6H2,
C7H2, C8H2), 0.84 (3H, t, J = 6.9 Hz, CH3 alkyl). 13C-NMR (75 MHz, DMSO):
δ 170.35, 157.88, 154.81,
145.35, 130.70, 128.28, 117.13, 95.53, 38.88, 31.49, 29.77, 29.20, 28.97, 28.86, 26.53, 22.30, 14.15, 12.24.
Anal. Calcd. for C20H30N4O4S (422.54): C, 56.85; H, 7.16; N, 13.26. Found: C, 56.97; H, 6.95; N, 13.40.

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4-(3-Decylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2j). White solid; yield 85% (Method B);
1
m.p. 212–213.5 ◦C. IR: 3377 (N–H), 1682 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ 11.19 (1H, s,
SO2NH), 8.90 (1H, s, N1–H ureido), 7.70–7.66 (2H, m, H2, H6), 7.56–7.52 (2H, m, H3, H5), 6.30 (1H, t,
J = 5.7 Hz, N3–H ureido), 6.10 (1H, d, J = 1.0 Hz, isoxazole), 3.06 (2H, td, J = 6.9 Hz, J = 5.6 Hz, C1H2),
2.28 (3H, d, J = 0.8 Hz, CH3 isoxazole), 1.44–1.37 (2H, m, C2H2), 1.29–1.17 (14H, m, C3H2, C4H2, C5H2,
C6H2, C7H2, C8H2, C9H2), 0.84 (3H, t, J = 7.0 Hz, CH3 alkyl). 13C-NMR (126 MHz, DMSO):
δ 170.30,
157.85, 154.79, 145.31, 130.73, 128.24, 117.11, 95.51, 39.21, 31.47, 29.73, 29.20, 29.14, 28.93, 28.87, 26.50,
22.27, 14.12, 12.22. Anal. Calcd. for C21H32N4O4S (436.57): C, 57.77; H, 7.39; N, 12.83. Found: C, 55.65;
H, 7.52; N, 12.84.
N-(5-Methylisoxazol-3-yl)-4-(3-undecylureido)benzenesulfonamide (2k). White solid; yield 79% (Method
1
A); m.p. 218.5–220 ◦C. IR: 3374 (N–H), 1680 (C=O) cm−1. H-NMR (300 MHz, DMSO):
δ
11.20 (1H, s,
SO2NH), 8.90 (1H, s, N1–H ureido), 7.70–7.65 (2H, m, H2, H6), 7.56–7.51 (2H, m, H3, H5), 6.30 (1H, t,
J = 5.6 Hz, N3–H ureido), 6.10 (1H, d, J = 1.0 Hz, isoxazole), 3.05 (2H, q, J = 6.5 Hz, C1H2), 2.28 (3H,
d, J = 0.9 Hz, CH3 isoxazole), 1.45–1.35 (2H, m, C2H2), 1.29–1.18 (16H, m, C3H2, C4H2, C5H2, C6H2,
C7H2, C8H2, C9H2, C10H2), 0.84 (3H, t, J = 6.7 Hz, CH3 alkyl). 13C-NMR (75 MHz, DMSO):
δ 170.31,
157.85, 154.77, 145.31, 130.67, 128.25, 117.09, 95.51, 39.21, 31.48, 29.74, 29.22, 29.20, 29.18, 28.94, 28.90,
26.51, 22.28, 14.14, 12.23. Anal. Calcd. for C22H34N4O4S (450.59): C, 58.64; H, 7.61; N, 12.43. Found: C,
58.45; H, 7.50; N, 12.51.
4-(3-Dodecylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2l). White solid; yield 82% (Method
1
A); m.p. 211.5–213 ◦C. IR: 3377 (N–H), 1683 (C=O) cm−1. H-NMR (500 MHz, DMSO):
δ
11.18 (1H, s,
SO2NH), 8.89 (1H, s, N1–H ureido), 7.69–7.65 (2H, m, H2, H6), 7.56–7.52 (2H, m, H3, H5), 6.29 (1H,
t, J = 5.7 Hz, N3–H ureido), 6.10 (1H, s, isoxazole), 3.05 (2H, q, J = 6.6 Hz, C1H2), 2.28 (3H, s, CH3
isoxazole), 1.44–1.36 (2H, m, C2H2), 1.30–1.15 (18H, m, C3H2, C4H2, C5H2, C6H2, C7H2, C8H2, C9H2,
C10H2, C11H2), 0.83 (3H, t, J = 6.9 Hz, CH3 alkyl). 13C-NMR (126 MHz, DMSO):
δ 170.27, 157.82, 154.75,
145.29, 130.69, 128.22, 117.08, 95.49, 39.20, 31.46, 29.72, 29.21, 29.18, 29.17, 29.16, 28.91, 28.87, 26.49,
22.26, 14.10, 12.20. Anal. Calcd. for C23H36N4O4S (464.62): C, 59.46; H, 7.81; N, 12.06. Found: C, 59.58;
H, 7.63; N, 11.91.
4-(3-Cyclopropylureido)-N-(5-◦methylisoxazol-3-yl)benzenesulfonamide (2m). White solid; yield 95%
1
(Method D); m.p. 144–146 C. IR: 3352 (N–H), 1662 (C=O). H-NMR (500 MHz, DMSO):
δ 11.19
(1H, s, SO2NH), 9.05 (1H, s, N1–H ureido), 7.66–7.59 (2H, m, H2, H6), 7.50–7.46 (2H, m, H3, H5), 6.28
(1H, d, J = 5.7 Hz, N3–H ureido), 6.10 (1H, d, J = 1.0 Hz, isoxazole), 2.53–2.44 (1H, m, CH), 2.26 (3H,
d, J = 0.9 Hz, CH3 isoxazole), 0.65–0.55 (2H, m, CH2CH2), 0.43–0.33 (2H, m, CH2CH2). 13C-NMR
(126 MHz, DMSO):
δ 167.31, 163.80, 152.30, 141.71, 138.17, 127.31, 117.61, 96.55, 22.79, 12.34, 4.36. Anal.
Calcd. for C14H16N4O4S (336.37): C, 49.99; H, 4.79; N, 16.66. Found: C, 50.14; H, 4.90; N, 16.58.
4,40-[Carbonylbis(azanediyl)]bis[N-(5-methylisoxazol-3-yl)benzenesulfonamide] (2n) [26]. White solid; yield
◦
58% (Method D); m.p. 262–263.5 C. IR: 3333 (N–H), 1659 (C=O). 1H-NMR (500 MHz, DMSO):
δ
11.28
(2H, s, SO2NH), 9.30 (2H, s, NH ureido), 7.79–7.75 (4H, m, H2, H6), 7.66–7.62 (4H, m, H3, H5), 6.12 (2H,
d, J = 1.1 Hz, isoxazole), 2.29 (6H, d, J = 1.0 Hz, CH3). 13C-NMR (126 MHz, DMSO):
170.39, 157.75,
δ
152.01, 143.92, 132.24, 128.31, 118.19, 95.54, 12.22. Anal. Calcd. for C21H20N6O7S2 (532.55): C, 47.36; H,
3.79; N, 15.78. Found: C, 47.48; H, 3.91; N, 15.66.
4-(3,3-Diisopropylureido)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (2o). White solid; yield 62%
(Method D); m.p. 234.5–236.5 ◦C. IR: 3361 (N–H), 1613 (C=O). 1H-NMR (300 MHz, DMSO):
δ 11.20
(1H, s, SO2NH), 9.25 (1H, s, NH ureido), 7.68–7.61 (2H, m, H2, H6), 7.54–7.48 (2H, m, H3, H5), 5.92
(1H, d, J = 1.1 Hz, isoxazole), 3.34 (2H, sep, J = 6.4 Hz, CH), 2.18 (3H, d, J = 0.9 Hz, CH3 isoxazole), 1.20
(12H, d, J = 6.4 Hz, isopropyl CH3). 13C-NMR (75 MHz, DMSO):
δ 167.94, 162.68, 152.29, 142.19, 137.10,
127.52, 117.74, 96.42, 46.46, 19.04, 12.37. Anal. Calcd. for C17H24N4O4S (380.46): C, 53.67; H, 6.36; N,
14.73. Found: C, 53.43; H, 6.54; N, 15.00.

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Synthesis of Oxalamide 3
Sulfamethoxazole
1
(1 mmol) was dissolved in MeCN (6 mL) and oxalyl chloride (0.5 mmol) was
added in one portion. The mixture was stirred for 2 h at room temperature. The resulting crystals
were filtered, washed with a small volume of MeCN and dried. The product was recrystallized from
tetrahydrofuran/hexane.
N1,N2-Bis{4-[N-(5-methylisoxazol-3-yl)sulfamoyl]phenyl}oxalamide (
(decomp.). IR: 3344 (N–H), 1690 (C=O). 1H-NMR (300 MHz, DMSO):
s, SO2NH), 8.07–8.01 (4H, m, H2, H6), 7.89–7.82 (4H, m, H3, H5), 6.13 (2H, d, J = 1.0 Hz, isoxazole), 2.28
(6H, d, J = 1.1 Hz, CH3). 13C-NMR (75 MHz, DMSO):
170.54, 158.88, 157.69, 142.04, 134.93, 128.08,
3
). White solid; yield 84%; m.p. > 300 ◦C
δ
11.42 (2H, s, CONH), 11.26 (2H,
δ
120.82, 95.61, 12.28. Anal. Calcd. for C22H20N6O8S2 (560.56): C, 47.14; H, 3.60; N, 14.99. Found: C,
47.40; H, 3.51; N, 15.11.
3.2. Antimycobacterial Activity
The antimycobacterial activity against Mycobacterium tuberculosis 331/88 (H37Rv; dilution
of this strain was 10−3), Mycobacterium avium 330/88 (dilution of 10−5), and two strains of
Mycobacterium kansasii, namely 235/80 (dilution of 10−4) and the clinically-isolated strain 6509/96
(dilution of 10−5), was evaluated using a previously described method [27]. The following
concentrations were used: 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, and 1 µM. MIC is the lowest
concentration at which complete inhibition of mycobacterial growth was observed. The MICs were
determined after incubation at 37 ◦C for 14 and 21 days and, for M. kansasii, additionally for 7 days.
INH and parent sulfamethoxazole
1 were chosen as the reference drugs. The experiments were
prepared in quadruplicates and the determination was repeated twice.
4. Conclusions
In this work, fifteen sulfamethoxazole-based ureas and one oxalamide were synthesized
using various methods, characterized, and evaluated for their antimycobacterial activity. All of
these compounds inhibited the growth of both M. tuberculosis and nontuberculous mycobacteria
in vitro with MIC values starting from 2 µM. Several derivatives exhibited an antimycobacterial
activity comparable or superior to sulfamethoxazole and isoniazid, thus, being potentially
promising for further investigation. Regarding structure-activity relationships, methyl, cyclopropyl
and 4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl are favored as the 3-ureido substituents of
sulfamethoxazole-based urea.
Acknowledgments: This work was supported by the Czech Science Foundation project No. 17-27514Y. We would
like to thank to Jaroslava Urbanová, M.A., for the language help provided and to the staff of the Department of
Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, for the technical assistance.
Author Contributions: Martin Krátký designed experiments and synthesized all the presented compounds.
Jirˇina Stolarˇíková conducted the antimycobacterial activity determination. Martin Krátký and Jarmila Vinšová
wrote the article, and made the discussion and conclusions.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2a–2o and 3 are available from the authors.
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