Novel Sulfamethoxazole Ureas and Oxalamide as Potential Antimycobacterial Agents

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 4,4′-[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.


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].

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 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 3 was obtained by treatment of SMX 1 with oxalyl chloride (Scheme 5) in stoichiometric amounts.
All of the compounds (Table 1) were characterized by 1 H-, 13 C-NMR (nuclear magnetic resonance), and infrared (IR) spectra and melting points. Their purity was checked by thin-layer chromatography (TLC) and elemental analysis.
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 1 connected via a carbonyl Scheme 1. Design of novel antimycobacterial sulfamethoxazole ureas based on 3-heptylurea derivative (n = 0-11; R 1 = isopropyl, cyclopropyl, 4-[N-(5-methylisoxazol-3-yl)sulfamoyl]phenyl; R 2 = H, isopropyl).

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 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 3 was obtained by treatment of SMX 1 with oxalyl chloride (Scheme 5) in stoichiometric amounts.
All of the compounds (Table 1) were characterized by 1 H-, 13 C-NMR (nuclear magnetic resonance), and infrared (IR) spectra and melting points. Their purity was checked by thin-layer chromatography (TLC) and elemental analysis.
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 1 connected via a carbonyl bridge or as simplified imidazolidine-2,4,5-trione [1] that has arisen from the removal of a -CO-CObridge. Oxalamide 3 was intended as another "open analogue" of imidazolidine-2,4,5-trione designed by the removal of the carbonyl bridge.

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., H 37 Rv) 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.
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 [20,21]. Mycolic acids, essential components 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 were obtained by symmetrically disubstituted N 1 ,N 2 -oxalamide 3; interestingly, this molecule was inactive against M. avium. On the other hand, N 3 ,N 3 -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 µM, i.e., 4.97-9.93 µg/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 µM). The mildest activity is connected with an intermediate length of alkyl (five-, six-, and seven-membered chains, 2e-2g, 250-500 µM). Based on decylurea 2j (MIC = 125 µM), 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 [1] seems to be an anomaly. The superior activity against NTM displayed methyl (2a, 8-125 µM, 2.48-38.75 µg/mL) and especially heptyl (2g, 4-62.5 µM, i.e., 1.58-24.66 µg/mL). For M. avium, concomitantly with the increasing length of the chain 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. N 3 -Substituted ureas by from C 2 to C 6 (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 3 as well as previously reported heptylurea 2g [1] were found to be the most successful modifications of the sulfa drug 1.
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].

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 Nicolet 6700 FTIR spectrometer (ThermoFisher Scientific, Waltham, MA, USA) in the range of 400 to 4000 cm −1 . The NMR spectra were measured in DMSO-d 6 at ambient temperature on a Varian VNMR S500 instrument (500 MHz for 1 H and 125 MHz for 13 C; Varian Comp., Palo Alto, CA, USA) or a Varian Mercury-Vxbb 300 (300 MHz for 1 H and 75.5 MHz for 13 C; Varian Comp., Palo Alto, CA, USA). The chemical shifts, δ, are given in ppm, with respect to tetramethylsilane as an internal standard. 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).

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,Ndiisopropylethylamine (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
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.

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.

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.