New sulfonamide hybrids: synthesis, in vitro antimicrobial activity and docking study of some novel sulfonamide derivatives bearing carbamate/acyl-thiourea scaffolds

In this study, the novel hybrids sulfonamide carbamates were synthesized by treatment of N-substituted 4-isothiocyanatophenyl sulfonamides with ethyl carbamate in dry 1,4-dioxane at reflux temperature in the presence of triethylamine. Also, treatment of Phenylacetylisothiocyanate with sulfanilamide in refluxing acetonitrile afforded the corresponding hybrid sulfonamide acylthiourea derivatives. The anti-microbial activities of the synthesized compounds were evaluated. Ethyl ({4-[(5-methyl-1,2-oxazol-3-yl)sulfamoyl)-phenyl]carbamothioyl)carbamate and 2-Phenyl-N-((4-(N-thiazol-2-yl)sulfamoyl)-phenyl)carbamothioyl)-acetamide exhibited the best activity against tested bacteria. Molecular docking studies for the final compounds were performed using the Open Eye docking suite. Moreover, Ligand efficiency (LE) and lipophilic ligand efficiency (LLE) parameters for Ethyl ({4-[(5-methyl-1,2-oxazol-3-yl)sulfamoyl)phenyl]carbamothioyl)-carbamate and 2-Phenyl-N-((4-(N-thiazol-2yl)sulfamoyl)phenyl)carb-amothioyl)acetamide were evaluated. Quantum chemical calculations based on density functional theory (DFT) have been performed.


Introduction
The synthesis of hybrid molecules and their evaluation as diverse range of pharmacological agents and as potent drugs has been under constant escalation for the last two decades 1 .The hybrid molecules, obtained by the combination of structural features of two differently active fragments, are the most popular chemical entities to work upon for developing modified scaffolds with many improved and amazing properties in the area of biology as well as medicinal science 2 .Sulfonamides drugs are very common compounds present in literature with massive activities [3][4][5] .Some important sulfonamide derivatives are used as carbonic anhydrase inhibitors of commercial importance 6 .Over 30 drugs containing this functionality are in clinical use, including antihypertensive agent bosentan 7 , anti-bacterial 8 , antiprotozoal 9 , anti-fungal 10 , anti-inflammatory 11 , nonpeptide vasopressin receptor antagonists 12 and translation initiation inhibitors 13 .They are also effective for the treatment of urinary, intestine, and ophthalmic infections, scalds, ulcerative colitis 14 rheumatoid arthritis 15 , male erectile dysfunction as the phosphodiesterase-5 inhibitor sildenafil 16 , obesity 17 , and more recently as anticancer agents 18 .
Prodrug sulfonamides are very important in the current medicinal protocol for sulfonamide therapy.For example, sulfonyl succinyl acts as a prodrug of sulfathiazole.It used in gut infections as it ionized in the alkaline conditions of the intestine and slowly hydrolysed by enzymes in the gut.Amide group lowers the polarity of the sulfonamide and increases the hydrophobic character.It allows the drug to crosses the gut wall more easilyandmetabolised by enzymes (e.g.peptidases) in vivo to generate the primary amine.Primary amine ionizes and can form ionic interactions ionized primary amine also acts as a strong hydrogen bond (HB) 19 .
The carbamate group is a key structural motif in many approved drugs and prodrugs 20 .There is increasing use of carbamates in medicinal chemistry, and many derivatives are specifically designed to make drug target interactions through their carbamate moiety 21,22 .The carbamates emerging role in medicinal chemistry is also due to its chemical stability and its capability to increase permeability across cellular membranes.These attributes of organic carbamates have been exploited in drug design 23 .
Other uses of carbamates are well known.Particularly, the employment of carbamates in various industries as agrochemicals, in the polymer industry, and also in peptide synthesis 26,27 .Also, among the various amine protecting groups, carbamates are commonly used to enhance their chemical stability toward acids, bases, and hydrogenation 28 .
Thiourea derivatives and thiourea hybrid with other functionality are useful compounds as precursors for the synthesis of different classes of acyclic and heterocyclic compounds 29 .They have been employed as an anti-inflammatory, antimicrobial 30 , antimalarial 31 , pesticidal 32 , and anticancer agents 33,34 .The derivatives of thiourea represent one of the most promising classes of anticancer agents with a wide range of activities against various leukaemia and solid tumors [35][36][37] .Thiocarlide is a pharmacologically important thiourea drug that is used as a therapeutic agent in the treatment of tuberculosis 38 and Phenethylthiazoylthiourea (PETT) derivatives (LY73497 and trovirdine HCl) 39,40 have been discovered as potent inhibitors of HIV type 1.The development of microbial resistance to presently available antibiotics led the search for new antimicrobial agents 41 .Due to the problem of microbial resistance to antibiotics, attention is given toward biologically active components isolated from natural products, as they may offer a new source of antimicrobial activities 42 found predictive rules that help in discovery and development of small molecules as antibiotics.In this interesting result, small molecules should contain some requirements as an amine group, be an amphiphilic and rigid, and have low globularity.Such information will assist us to design our compounds.It is important to examine the something that correlates between the activity of the compounds and their structures it is the quantum chemical calculation.In general, the HOMO possesses an antibonding character between the consecutive subunits, whereas the LUMO generally shows a bonding character between the subunits.No direct correlation between HOMO or LUMO energies and antibacterial activities is highlighted.The gap in energy between the HOMO and LUMO is an important stability index 43 .Generally, the high stability indicates low chemical reactivity and small gap indicates high chemical reactivity.Softness (S) may be a property of a molecule that measures the extent of chemical reactivity.The chemical hardness (η) was related with the resistance towards the deformation of deformation cloud of chemical systems below little perturbation occurred.
A small hardness means the compound features high polarizability.Polarizability (α) measures the pliability of electrons in an exceedingly} very molecule to maneuver merely as a result of data.The softer a molecule is, the upper is its average polarizability.
In the frame of previous data [44][45][46][47][48] , we thought to design new derivatives of prodrug sulfonamides.Our strategy was designed base on linking the amino group with more labile pharmacophore than reported amide, (Figure 1).
We finally decided to install the interesting hybrid functionality from thiourea and carbamate.The thiourea was tethered with ethyl carbamate or acyl benzyl to examine the effect of the aliphatic or aromatic side chain.On the other site at sulfonamide head, we studied varieties of heterocyclic rings.Herein is verified a direct correlation between compound structures and their biological activities based on different parameters.

Materials and methods
All analyses were done at the Microanalytical Center, Cairo University, Cairo (Egypt).Melting points (uncorrected) were determined in open capillaries on a Gallenkamp melting point apparatus (Sanyo Gallenkamp, Southborough, UK).IR spectra (KBr discs) were recorded using Schimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan), NMR Spectra were recorded on a Bruker spectrophotometer (Bruker, Karlsruhe, Germany). 1 H spectrum was run at 300 MHz, and the 13 C spectrum was run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d6).Chemical shifts (δ) are reported relative to MS as an internal standard.Mass spectral data were given by a GCMS-QP1000 EX-spectrometer (Shimadzu, Kyoto, Japan) at 70 eV.Elemental analyses were done on a model 2400 CHNSO instrument (Perkin Elmer, Waltham, MA, USA).All reagents used were of the Analytical grade.

General Procedure for Synthesis of carbamates 3a-e Method A:
A mixture of ethyl carbamate (0.01 mol) and isothiocyanate2 (0.01 mol) and triethyl amine (0.01 mol) in 1,4-dioxane (10 mL) was refluxed for 3 h.The reaction mixture was left to cool at room temperature.The solid product, so formed, was collected by filtration and recrystallized from an appropriate solvent.Method B: A mixture of 1 (0.01 mol), ethoxycarbonylisothiocyanate (0.01 mol) [prepared by adding ammonium thiocyanate (0.01 mol) to a solution of ethyl chloroformate (0.01 mol) in 1,4dioxane (10 mL) and heat for 1/2 h followed by isolation of the byproduct, ammonium chloride] and triethylamine (0.01 mol) was refluxed for 1 h.The resulting solid product was collected by filtration and recrystallized to give 3.

General procedure for the synthesis of acylthioureas 7a-e
A solution of phenylacetyl chloride 5 (0.01 mol) and ammonium thiocyanate (0.01 mol) in acetonitrile (10 mL) was heated under reflux for 10-20 min.After the reaction mixture was cooled to room temperature and the formed precipitate (NH4Cl) was filtered off.To the freshly prepared solution of Phenylacetylisothiocyanate 6, sulfanilamide 1 (0.01 mol) was added, and the mixture was refluxed for 2 h.Upon completion of reaction (checked by TLC), the resulting precipitate was collected by filtration and recrystallized to give the product 7.

Docking studies
Crystallographic structure for dihydropteroate synthase (DHPS) (ID: 3TZF) 57 contains the standard sulfamethoxazole co-crystalized inside the receptor was chosen for docking.A library of our designed sulfonamides linked thiourea-carbamate including standard drugs was designed, and energy minimized using MMFF94 force field calculations the catalytic domain of DHPS which was obtained from protein data bank (PDB code 3TZF)) and was prepared for docking using Open Eye 58-60 software.Omega application was used to prepare conformers for docking, and FRED application was selected to generate a physical property (ΔG) reflecting the predicted energy profile of the ligand-receptor complex

Quantum chemical calculations
The Quantum chemical calculations were performed using B3LYP that includes a mixture of Hartree-Fock with DFT exchange terms associated with the gradient-corrected exchange-correlation functional of Lee, Yang and Parr (LYP).It has fewer convergence problems than those found in the pure DFT methods.Thus, B3LYP has been used in this paper to carry out quantum calculations.Full geometry optimizations of all additives were carried out with the standard B3LYP/6-311G++ (d, p) basis set using Gaussian 09.

Chemistry
Isothiocyanates are useful and widely used building blocks in the synthesis of nitrogen, sulfur and oxygen heterocyclic and organometallic compounds of academic, pharmaceutical and industrial interest 50 .The high electrophilicity and nucleophilicity related to the carbon and sulfur atoms, severally, of the isothiocyanates and their extended π electron system, create them distinctive precursors of an outsized type of target molecules.Consequently, many classes of five and six-membered nitrogen and sulfur heterocyclic, either carrying various substituents or fused with benzo or non-benzo nuclei to interesting poly heterocyclic, have been synthesized from isothiocyanates which are undoubtedly a landmark in organ sulfur chemistry 51 .The intermediate, Nsubstituted 4-isothiocyanatophenyl sulfonamides 2 used for the preparation of target compounds have been synthesized in high yield via thiophosgenation of sulfonamides 1 at room temperature in the presence of dilute hydrochloric acid, according to literature procedure 52 (Scheme 1).The synthetic route designed for the carbamates 3a-e containing sulfonamide moiety is outlined in (Scheme 2).
The treatment of N-substituted 4isothiocyanatophenyl sulfonamides 2a-e with ethyl carbamate in dry 1,4-dioxane at reflux temperature in the presence of triethylamine afforded the novel hybrid carbamates 3a-e.
The structure of all newly synthesized carbamate derivatives 3a-e was determined by analytical and spectroscopic analysis, which were consistent with the proposed structure.Carbamates 3a-e were confirmed by the absence of characteristic infrared absorption peak at 2000-2200 cm −1 (N=C=S group).Also, the infrared spectra of the isolated compounds 3a-e contained absorption bands 3349-3150 cm -1 due to stretching vibrations of the NH group and at 1736-1711 cm -1 for the carbonyl group in addition to the presence of sulfamoyl and thiocarbonyl groups.For example, the 1 H NMR spectrum of compound 3a (DMSO-d6) revealed a triplet signal at δH 1.27 ppm and a quartet at δH 4.17 ppm assigned to the ethyl group and a broad singlet at δH 7.41 ppm assigned for the NH2 and two doublet signals at δH 7.42-7.83ppm assigned to the aromatic protons.Also, in the 1 H NMR spectrums; two N-H protons at δH 11.32 and 11.68 ppm appeared as singlet signals.These are the most deshielded signals because of intramolecular hydrogen bonding that results in shifting the signals towards higher ppm value, thus justify the presence of thio core in thiourea 53 .In the 13 C-NMR spectral data of compounds 3a, c, e the thiocarbonyl group of thiourea moiety was also observed at around δC 169-179.38 ppm and carbonyl carbon appeared at around δC 153.4-153.9ppm.Moreover, the mass spectral data of compounds 3b-d were fully consistent with the assigned structures.The molecular ion peaks of synthesized compounds 3b, 3c and 3d were observed at m/z 345(3.38%),386(2.06%)and 384(46%) which compatible with its molecular weights, respectively.The molecular ion peak of compound 3c underwent fragmentation to produce a peak at m/z 297 (44%), corresponding to the molecular ion of N-(2-thiazolyl)-4-isothiocyanato phenyl sulfonamides.It underwent further loss of SO2, phenyl isothiocyanate and 2thiazolyl sulfamoyl yielded peaks at m/z 233(58.45%),163(1.58%)and 134 (100%: base peak), respectively.

Scheme 1. synthesis of p-substituted sulfamoylphenylisothiocyanate 2a-e Scheme 2. synthesis of carbamates 3a-e containing sulfonamide moiety
The formation of carbamate 3 can be explained by the reaction pathway depicted in (Scheme 3).The formation of 3 is assumed to proceed via the nucleophilic attack of the amino group of the ethyl carbamate on thiocarbonyl group of isothiocyanate to form the intermediate (A), followed by deprotonation and protonation to yield the carbamate skeleton 45 .A further evidence for the formation of carbamate 3 was obtained by an independent synthesis of compound 3a via treatment of sulfanilamide 1 with ethoxycarbonylisothiocyanate 4 in 1,4-dioxane in the presence of a catalytic amount of triethylamine to yield a product identical in all respect (mp.TLC and spectra) with that obtained previously from reaction of 2a with ethyl carbamate (Scheme 2).The physical properties, spectral information, and mass analysis of all the synthesized compounds 3a-e are illustrated in the experimental section.

Scheme 3. the proposed mechanism for the formation of carbamates 3a-e Scheme 4. synthesis of acetamides7a-e containing sulfonamide moiety
The chemistry of acyl isothiocyanates is very rich and diverse and has been employed in the synthesis of some biologically important organic compounds 54 .In recent years,1-(acyl/aroyl)-3-(substituted)thioureas have been seen increasing importance in organic chemistry with privileged structures.They have been the subject of extensive study in coordination chemistry, and are also known to play a promising role in the fields of material sciences, molecular electronics, molecular recognition, agriculture, biological activities and pharmaceuticals 55 .The structures of acylthiourea derivatives 7a-e were established by their elemental analysis and spectral data.The infrared spectra of compounds 7 indicated the characteristic absorption bands at 1690-1713 cm -1 for the C=O group in addition to the presence of NH, C=S and SO2 groups.The 1 HNMR spectral data were also consistent with the assigned structures.For example, the 1 H NMR spectrum of compound 7c (DMSO-d6) showed a singlet signal at δH 2.28 ppm assigned to the methyl protons, a singlet signal at δH = 3.81 ppm assigned to the methylene protons, a singlet signal at δH 6.14 ppm due to proton of isoxazole ring in addition to the presence of aromatic protons.Also, in 1 H NMR the characteristic broad signals at δH 11.43 and 12.57 ppm for protons of N1 and N3, respectively.In the mass spectrum of compound 7a, 7c and 7d molecular ion peaks were observed at m/z 349 (38.68%), m/z 430 (0.5%) and m/z 444 (1%) which compatible with its molecular weights.
A plausible mechanism for the synthesis of acyl thioureas 7a-e is depicted in (Scheme 5).

Antimicrobial activity
The in vitro antimicrobial activities of the synthesized compounds were evaluated for four Gram-positive bacteria viz.Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Bacillus subtillis, Streptococcus pyogenes, and three Gram-negative organisms viz.Escherichia coli, Proteus vulgaris, Erwiniacarotovora, as well as one fungi viz.Candida Albicans by agar well diffusion method 56 .Ampicillin and penicillin were used as standards.The antibacterial and antifungal data were depicted in Table 1.Compounds 7b and 3d exhibited appreciable broad spectrum (87.5%) against both Gram-positive bacteria and Gram-negative bacteria (Figure 3), followed by 2e (37.5%, each) then 7a, 7c, and 3e (25%).The pathogen S. aureus was the most sensitive bacteria in case of 7a (1.4 mm diameter of inhibition zone), whereas, Proteus vulgaris was the most sensitive in case of 3d (1.3 mm diameter).Compounds 7b and 3d were selected for determination of MIC as the highest for inhibition the growth of Bacillus subtilis.The minimum inhibitory concentration of 7b and 3d on Bacillus subtilis were 70 and 80 μ/ml, respectively, (Figure 2).Table 1.The antibacterial and antifungal data of prepared compounds.

Docking Study
Sulfonamides drugs are reported to inhibit bacterial dihydropteroate synthase (DHPS) which has an important role in the biosynthesis of the tetrahydrofolate cofactor which is crucial to pyrimidine and DNA biosynthesis.
Crystallographic structure for dihydropteroate synthase (DHPS) (ID: 3TZF) 57 illustrated that the active site region contains the standard sulfamethoxazole co-crystalized inside the receptor with the formation of hydrogen bonding (HB) with amino acids Lys 221 AA, Ser 222 AA, and Ther 62 AA through its sulfonamide and para-amino group.Also, benzene ring form π-π bonding interactions with amino acids Arg 63, Pro 64 57 .The docking of our target compounds with (DHPS) represents consensuses scores and binding modes correlated with their biological activity as antimicrobial.A library of our designed sulfonamides linked thiourea-carbamate including standard drugs was designed.The catalytic domain of DHPS which was obtained from protein data bank (PDB code 3TZF)) was prepared for docking using Open Eye 58-60 software.To validate our docking, the study commenced with docking with standard sulfamethoxazole and another standard which showed docking pose and a mode similar to cocrystalized pose 57 .The active compound 3d (sulfamethoxazole linked thiourea-ethyl carbamate) lays in the active sit with the formation of four HBs; two HBs with Ser 222 AA through both sulfonamide and nitrogen of isoxazole ring (acceptor).Also forms HB with Arg 225 AA through the thiourea part (acceptor).The carbonyl of ethyl carbamate group interacts with Thr 62 though the formation of HB (acceptor), (Figure 3).

Ligand efficiency (LE) scores:
ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties are important in drug discovery and were calculated, with special emphasis on the lipophilicity requirements.LE is used in fragment-based drug discovery 62 to lead compounds with optimal combinations of physicochemical properties and pharmacological properties.LE is used to estimate the efficiency of compounds and determine binding affinity about the number of heavy atoms in a molecule.This provides a way to compare the affinity of molecules corrected for their size. 62E describes how efficient a ligand exploits its lipophilicity.If lipophilicity is too high, the likelihood of a compound to bind to multiple targets increases.Moreover, affinity is often optimized through the introduction of lipophilic groups, as these contribute favorably to the hydrophobic effect without the need for specific interactions with the target.These contrast with polar groups, which need to establish very good interactions with the target to compensate for the desolvation penalty.For this reason, polar groups are often used to improve solubility rather than affinity.Compounds 3d exhibited LE similar to 7b but it has LLE (2.6) higher than 7b (1.86), Table 2.This results emphasize the hypothesis of ethyl carbamate is better than benzyl carbamate.

Quantum chemical calculations
The ultimate goal of the structure-activity relationship (SAR) studies is to correlate the biological activity of a series of compounds with their structures.Unconstrained geometry optimizations of prepared compounds (3b,d,e and 7a-e) were carried out at gradient corrected DFT using Becke's three parameters hybrid method and the Lee-Yang -Parr correlation functional (B3LYP) 63 combined with 6-31G(d) 64 basis set using Gaussian 09 in both gas and solvent (DMSO) media.Figure 6 shows HOMO and LUMO frontier orbital's obtained for the studied compounds.It is important to examine the HOMO and LUMO for these compounds because the relative ordering of occupied and virtual orbital provides a reasonable qualitative indication of electronic properties.Molecular electrostatic potential mapping is very useful in the investigation of the physicochemical properties of the studied compounds.Different values of the electrostatic potential at the surface are represented by different colors: red represents regions of most electro negative potential, blue represents regions of most positive electrostatic potential and green represents regions of zero potential.Herein, the three MESPs are very similar and revealed that the high electronic density suitable for the electrophilic attack is located on sulfonyl oxygen atoms in the red region.
The quantum chemical calculation of the prepared compound (3b, d, e and 7a-e) was tabulated in Table 3.These data proved the ΔHE is less value in compound 3d and 7b that indicate that the two compounds are more active than another compounds.As soon as the softness value showed the high value in the same compound 3d and 7b that indicate that the two compounds highly active.From the foregoing, we find that quantum calculated data is fully compatible with biological activities.The theoretical study implies that gap, softness and hardness tend to be the best chemical descriptors to identify compounds presenting an interesting antibacterial activity.The reaction of inhibition in question seems to be mainly directed by hard-hard interactions, for example, the transfer of a proton to a hard base.In that case, the reactions are mainly controlled by electrostatic relationships as modelled by Mulliken charges that can be also considered as important descriptors.

Figure 1 :
Figure 1: a) reported drugs contain sulfonamide prodrug as antimicrobial; b) general formula of our designed compounds.

Phenylacetylisothiocyanate 6
was prepared in situ by reaction of Phenylacetyl chloride 5 with an equimolar quantity of ammonium thiocyanate in dry acetonitrile.Treatment of Phenylacetylisothiocyanate 6 with sulfanilamide 1 in refluxing acetonitrile afforded the corresponding acylthiourea derivatives 7a-e, (Scheme 4).

Scheme 5 .Figure 2 .
Scheme 5.The proposed mechanism for the synthesis of acyl thioureas 7a-e

Figure 3 .
Figure 3.A visual representation for 3d docked with PDB: 3TZF showing three HBs interaction towards the binding site

Figure 4 .
Figure 4.A visual representation for 7c docked with PDB: 3TZF showing Two HBs interaction towards the binding site.Judging from the docking study, both analogues 3d and 7c have different docking mode and pose.The R (ethyl or benzyl) group from carbamate part has a great effect, and this explains the difference in activity.Compound 7b docked with the receptor with formations of different hydrogen bonding; with Ser

Table 2 .
Summary of ligand efficiency scores to be considered during fragment-based drug Discovery (FBDD)

Table 3 .
The quantum chemical calculation of the prepared compounds.