Design, Synthesis, and Antimicrobial Evaluation of a New Series of N-Sulfonamide 2-Pyridones as Dual Inhibitors of DHPS and DHFR Enzymes.

Sulfonamides and trimethoprim (TMP) drugs are normally used to inhibit the action of dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes, respectively. In this work, a new series of N-sulfonamide 2-pyridone derivatives that combine the inhibitory activities of DHPS and DHFR into one molecule were synthesized and evaluated for its in vitro antimicrobial activity and the ability to inhibit the action of both enzymes simultaneously. The synthesis was carried out via the reaction of novel benzothiazol sulfonylhydrazide with ketene dithioacetal derivatives, and the structures of the resultant compounds were confirmed using spectral and elemental techniques. Among the synthesized compounds, five compounds 3b, 5a, 5b, 11a, and 11b were found to possess significant antimicrobial activities against tested bacterial and fungi strains. The compounds were also examined for their cytotoxicity on HFB4 human dermal fibroblast cell line using MTT assay. The in vitro enzyme assay study of these compounds against DHPS and DHFR enzymes showed that compound 11a was the most potent inhibitor against both enzymes with IC50 values of 2.76 and 0.20 μg/mL, respectively. Docking studies showed that this compound has occupied both the p-aminobenzoic acid and pterin binding pockets of DHPS as well as the pterin binding pocket of DHFR. The results of these investigations confirmed that compound 11a is the most potent dual DHPS/DHFR inhibitor.

Introduction

Generally, sulfonamide drugs are well-known bacteriostatic antibiotics exhibiting significant activities against different bacterial strains.[1] Additionally, sulfonamides showed diverse pharmacological actions such as antidiabetic,[2] anticancer,[3,4] and antiviral potencies.[5] They are known to compete with p-aminobenzoic acid (pABA) in binding to the active site of dihydropteroate synthase (DHPS) enzyme as a result of their comparable structures and thus inhibit the action of the enzyme. DHPS is involved in the folate synthesis through the catalysis of the reaction of 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate with pABA to afford the folate intermediate 7,8-dihydropteroate (DHF).[6] Thereafter, DHF is transformed to tetrahydrofolic acid (THF) through the action of the dihydrofolate reductase (DHFR) enzyme. Inhibiting the action of either DHPS or DHFR enzymes would eventually prevent the biosynthesis of THF required for the synthesis of purines and pyrimidine and would eventually lead to the death of the bacterial cell itself.[7] It is well-documented that many bacterial and fungi strains have developed a considerable resistance against several commercial drugs bearing a sulfonamide group.[8,9] Clinically speaking, the presence of the pABA/sulfonamide binding site close to the protein surface through flexible protein loops that are amenable and tolerant to mutations was the main drive toward this bacterial resistance. Inasmuch, several research works have focused on DHFR as a viable target for enzyme inhibition through the action of different antimicrobial and anticancer agents because the enzyme is essential for both purine and thymidylate syntheses as well as the folate metabolism in cell proliferation. In the case of bacterial infections, both trimethoprim (TMP) and pyrimethamine drugs compete with the pteridine moiety of DHF in binding with the DHFR enzyme and resulting in enzyme inhibition. This is because both drugs possess a pyrimidine ring with two amino groups that are responsible for binding to the amino acids within the pterin pocket.[10,11] Combination of DHPS and DHFR inhibitors has proven to increase the therapeutic efficacy.[12] For example, it is known that sulfonamides have very little antiprotozoal action when used alone. However, combination of sulfonamides with TMP created synergistic effects. To elaborate more, the drugs separately have been shown to be bacteriostatic, while a combination of both is bactericidal. However, this combination is usually administered in the case of complicated urinary tract infections and Pneumocystis carinii infections in immune-compromised patients. Interestingly, the treatment is available orally for community acquired methicillin resistant Staphylococcus aureus (MRSA).[13]

Furthermore, 2-pyridone derivatives are interesting heterocyclic scaffolds because of their various pharmacological properties.[14] Many medications based on 2-pyridone skeletons are used in the treatment of congestive heart failure[15] and bacterial infections caused by negative gram bacteria.[16,17] Extensive studies have been conducted to develop new forms of antibacterial,[18] antivirals,[19] antifungals,[20] antitumor,[21] and anti-inflammatory[22] agents that are based on 2-pyridone structures. Interestingly, 2-pyridone ring incorporating the sulfonamide moiety showed significant impact on the inhibition of the cellar wall of different types of bacteria, as was recently demonstrated.[23,24] Moreover, N-sulfonamide 2-pyridone derivatives have exhibited good antimicrobial activity against a variety of antimicrobial strains.[25,26]

Development of drug resistance by different bacterial strains against both sulfonamide drugs and TMP[27] has encouraged us to design new compounds containing the sulfonamide group as DHPS inhibitors that are bonded to the six-membered ring such as pyridine substituted with the amino/hydroxyl group at the 6-position to inhibit DHFR, as shown in Figure . Based on our experience in the synthesis of novel compounds containing benzothiazoles, pyridines, and sulfonamide moieties,[28−33] new compounds of N-sulfonamide 2-pyridones incorporating the benzothiazole moiety were envisaged and synthesized via the reaction of novel benzothiazol sulfonylhydrazide with ketene dithioacetal derivatives. To evaluate the antimicrobial potency of the new compounds, antimicrobial assessment, minimum inhibitory concentration (MIC), cytotoxicity, and enzyme inhibition of DHPS and DHFR as well as docking study of the most potent compounds were carried out.

Figure 1

Design strategy and general structures of newly synthesized antimicrobial DHPS and DHFR inhibitors.

Results and Discussions

Chemistry

Various derivatives of N-sulfonamide 2-pyridones incorporating the benzothiazole moiety were synthesized starting with novel N′-(2-(benzo[d]thiazol-2-yl)acetyl)arylsulfonohydrazide compounds 3a,b. The latter compounds were produced via the reaction of 2-(benzo[d]thiazol-2-yl)acetohydrazide 1 with arylsulfonyl chloride 2 in pyridine at room temperature, as shown in Scheme . To confirm the proposed structure of the newly synthesized sulfonyl hydrazide compound 3b, an X-ray single crystal structure was ascertained, as shown in Figure .[34]

Scheme 1

Synthesis of N-(3-(Benzo[d]thiazol-2-yl)-5-cyano-4-(methylthio)-2-oxopyridin-1(2H)-yl)arylsulfonamide

Figure 2

X-ray single crystal structure of the title compound 3b “Reproduced with permission of the International Union of Crystallography under the open-access licence”.[34]

The reaction of N′-(2-(benzo[d]thiazol-2-yl)acetyl)arylsulfonohydrazides 3a,b with ketene dithioacetals was exploited further to provide a general approach for the synthesis of 4-methylthio 2-pyridones 5a–d and 9a–f. We studied the reactivity of the CH2 group of compounds 3a,b toward different derivatives of S,S ketene dithioacetals such as 2-(bis(methylthio)methylene)malononitrile 4a, ethyl 2-cyano-3,3-bis(methylthio)acrylate 4b, and 2-cyano-3,3-bis(methylthio)-N-arylacrylamide 6a–c compounds, as shown in Schemes and 2.

Scheme 2

Synthesis of 2-Amino-5-(benzo[d]thiazol-2-yl)-4-(methylthio)-6-oxo-N-aryl-1-(arylsulfonamido)-1,6-dihydropyridine-3-carboxamide

Reacting N′-(2-(benzo[d]thiazol-2-yl)acetyl)arylsulfonohydrazides 3a,b with either 2-(bis(methylthio)methylene)malononitrile 4a or ethyl 2-cyano-3,3-bis(methylthio)acrylate 4b in dry dimethylformamide (DMF) containing pulverized potassium hydroxide produced compounds 5a,b and 5c,d, respectively, in good yield, Scheme .

The structures of N-arylsulfonamide 2-pyridones 5a–d were assigned based on their spectral data (FT-IR, 1H NMR, and 13C NMR) and elemental analyses. The IR spectra clearly revealed that both 5a and 5b compounds had NH2 groups as evident by the absorption bands at ν 3410–3296 cm–1 range, while those of compounds 5c and 5d showed a broad band at ν 3427 and 3431 cm–1 range, respectively, corresponding to the OH groups. According to 1H NMR of 5a–d, the appearance of a singlet signal at a range of δ 2.31–2.59 ppm confirmed the presence of SCH3 protons. Additionally, in the case of compounds 5a and 5b, a broad signal at δ 7.50 and 7.63 ppm, respectively, confirmed the presence of NH2 groups, while in the case of compounds 5c and 5d, a singlet signal at δ 9.45 and 9.24 ppm, respectively, confirmed the presence of the OH group. Moreover, 1H NMR of compounds 5a–d showed four distinctive signals corresponding to the four protons of benzothiazole ring. These signals are two triplet signals at the range of δ 7.32–7.68 ppm and two doublet signals at the range of δ 7.90–8.09 ppm with each signal representing a single proton. Furthermore, 13C NMR of 5a showed a signal at δ 25 ppm for the SCH3 group and a signal at δ 117 ppm for the CN group.

To expand on the above findings, reactions involving alternative derivatives of S,S ketene dithioacetal compounds, namely, N-substituted bis(methylthiomethylene)(cyano)acetamide derivatives 6a–c, were investigated. The reactions of 6a–c with compounds 3a,b in dry 1,4-dioxane containing a catalytic amount of potassium hydroxide afforded N-(4-methylthio-6-oxopyridin-1-yl)arylsulfonamides 9a–f, whose structures were confirmed by analytical and spectral data, as shown in Scheme . IR spectra of compounds 9a–f showed a characteristic broad band at the range of ν 3413–3268 cm–1 corresponding to NH2 and NH groups. The 1H NMR spectrum of 9e, as an example, revealed the presence of both NH2 group as a broad signal at δ 7.50 ppm and NHSO2 and CONH groups as two singlet signals at δ 10.35 and 11.46 ppm, respectively. The 13C NMR spectrum of 9f confirmed the presence of CH3 and SCH3 at δ 19.5 and 21.4 ppm, respectively, and the OCH3 group at δ 55.1 ppm.

A possible mechanism for the formation of compounds 5a–d and 9a–f starts with the Michael addition of the active methylene carbon atom of compounds 3a,b to the double bond of the S,S ketene dithioacetal compounds 4a,b and 6a–c, followed by the elimination of CH3SH and subsequent intramolecular cyclization resulting from the addition of a NH2 proton to either the cyano group to form compounds 5a,b and 9a–f or the carbonyl group to form compounds 5c and 5d, as shown in Scheme .

Finally, upon heating N-arylsulfonamide 2-pyridones 5a,b with hydrazine hydrate in a mixture of methanol/DMF (50:50), 1H-pyrazolo[4,3-c]pyrid-2-ones 11a,b were successfully synthesized. Elemental analysis and spectral data confirmed the proposed structures of 11a,b, as shown in Scheme . The disappearance of the CN signal in both IR and 13C NMR spectra and the singlet signal of SCH3 at 1H NMR as well as the presence of the additional NH2 group at δ 5.57 confirmed the transformation of pyrido-2-ones 5a,b to pyrazolopyrid-2-ones 11a,b. Plausibly, the reaction proceeded via the hydrazinolysis of 5a,b through the removal of the methylthio group and a subsequent cyclization through the NH2 group attack on the cyano group in pyrid-2-one ring to finally afford pyrazolopyrid-2-ones 11a,b.

Scheme 3

Synthesis of N-(3,4-Diamino-7-(benzo[d]thiazol-2-yl)-6-oxo-1H-pyrazolo[4,3-c]pyridin-5(6H)-yl)arylsulfonamide

Biological Evaluation

Antimicrobial

The newly synthesized compounds were examined for their in vitro antibacterial potency against three Gram −ve bacteria, namely, Klebsiella pneumonia, Pseudomonas aeruginosa, and Escherichia coli, as well as two Gram +ve bacteria, namely, Streptococcus mutans and Staphylococcus aureus. They were also evaluated for their antifungal potency against Candida albicans fungal strain. The agar-diffusion method was used to determine the preliminary antibacterial and antifungal potencies. Standard drugs such as Ampicillin, Gentamicin, and Nystatin were also used against Gram +ve bacterial, Gram −ve bacterial, and fungal strains, respectively. Sulfonamide antibiotics such as sulfadiazine (SD) have also been tested and compared for its antibacterial efficacy versus that of the newly synthesized compounds. The antimicrobial results were reported as the average diameter of inhibition zones of the microbial growth around the disks in mm ± SD, as summarized in Table . The MIC measurements were also determined for the most potent compounds 3b, 5a, 5b, 11a, and 11b using twofold serial dilution method, and the results are also summarized in Table .

Table 1

Antibacterial Inhibition Zone in mm ± Standard Deviation of Synthesized Compounds

	diameter of the inhibition zone (mm)
	Gram (−ve) bacteria			Gram (+ve) bacteria		fungi
compd no.	E. coli (ATCC: 3008)	K. pneumonia (ATCC: 4415)	P. aeruginosa (ATCC: 27853)	S. aureus (ATCC: 6538)	S. mutans (ATCC: 25175)	C. albicans (ATCC: 10231)
3a	−a	20.0 ± 3.0	−	−	−	−
3b	17.3 ± 2.5	32.0 ± 2.0	−	17.6 ± 2.5	10.6 ± 0.5	−
5a	26.0 ± 0.5	20.6 ± 1.0	−	−	17.6 ± 1.5	21.0 ± 1.0
5b	21.3 ± 3.0	25.6 ± 1.1	23.3 ± 0.5	30.3 ± 0.5	18.6 ± 1.0	−
5c	23.3 ± 1.5	27.3 ± 1.5	−	17.6 ± 1.5	−	−
5d	−	18.2 ± 0.5	−	17.5 ± 2.0	−	−
9a	−	−	−	31 ± 1.0	−	−
9b	−	−	−	27.1 ± 0.2	−	−
9c	14.8 ± 0.2	29.2 ± 0.3	−	−	20.2 ± 1.0	−
9d	−	−	−	26.8 ± 1.5	8.8 ± 1.0	28.0 ± 0.7
9e	−	−	−	19.6 ± 1.0	−	−
9f	−	29.6 ± 1.5	−	−	−	−
11a	26.0 ± 0.5	15.2 ± 0.5	−	35.2 ± 1.5	22.3 ± 0.5	21.0 ± 1.7
11b	26.0 ± 1.0	17.5 ± 0.5	−	25.2 ± 1.0	21.0 ± 1.0	−
Gentamicin	25.0 ± 0.1	32.0 ± 0.1	35.0 ± 0.1	NT	NT	NT
Ampicillin	NTb	NT	NT	32.0 ± 0.5	22.0 ± 0.5	NT
Nystatin	NT	NT	NT	NT	NT	28.0 ± 0.1
SD	19.8 ± 0.4	20.4 ± 0.9	24.0 ± 1.4	31.2 ± 2.1	16.3 ± 1.1	NA

No activity.

Not tested.

Table 2

MIC of the Most Active Compounds 3b, 5a, 5b, 11a, and 11b

	The MIC (μg/mL)
organism	3b	5a	5b	11a	11b	SD	standard
							Gentamicin
E. coli (ATCC: 3008)	250	62.5	125	31.25	31.25	250	31.25
K. pneumonia (ATCC: 4415)	1000	1000	1000	125	125	250	62.5
P. aeruginosa (ATCC: 27853)	NT	NT	1000	NT	NT	250	125
							Ampicillin
S. aureus (ATCC: 6538)	1000	NT	250	31.25	500	500	62.5
S. mutans (ATCC: 25175)	1000	1000	1000	1000	500	125	62.5
							Nystatin
C. albicans (ATCC: 10231)	NT	62.5	NT	125	NT	NT	31.25

As shown in Table , all the synthesized compounds showed no apparent activity against P. aeruginosa except for compound 5b. It is equally clear that the antibacterial activity of sulfonyl hydrazides 3a showed some activities against a particular bacterial strain, K. pneumonia, while 3b showed similar inhibition zone against K. pneumonia when compared to Gentamicin (inhibition zone 32.0 ± 2.0 mm, MIC, 250 μg/mL) while showing lower activities than those of the standard drugs against E. coli, S. aureus, and S. mutans.

It was also noticed that arylsulfonylamino-2-pyridone derivatives 5a–d (Scheme ) displayed considerable antibacterial and fungal activities. In some instances, these compounds exhibited similar or slightly lower activities than those of the corresponding standard drugs. In this series, it was clear that compound 5a showed activities slightly higher than Gentamicin and, therefore, was marked as the most potent against E. coli (inhibition zone 26.0 ± 0.5 mm, MIC, 62.5 μg/mL), while compound 5b was marked as the most potent agent against S. aureus (inhibition zone 30.3 ± 0.5 mm, MIC, 250 μg/mL). Additionally, compound 5c revealed strong activity against K. pneumonia (inhibition zone 27.3 ± 1.5 mm) in comparison to that of other derivatives in this series. Interestingly, compound 5a was shown to be the only compound in this series that exhibited some antifungal activity against C. albicans (inhibition zone 21.0 ± 1.0 mm, MIC, 62.5 μg/mL).

According to the antimicrobial results, compounds 9a–e showed some activities against Gram +ve bacterial strains, while the two compounds 9c and 9f showed some activities against Gram −ve bacterial strains, and only one compound, 9d, showed equal potency against C. albicans fungal strain (inhibition zone 28.0 ± 0.7 mm) as compared to that of the standard drug Nystatin (inhibition zone 28.0 ± 0.1 mm). As shown in Table , both compounds 9c and 9f revealed slightly lower activities than Gentamicin against K. pneumonia (inhibition zone 29 mm). Furthermore, compound 9a was the most active compound in this series against S. aureus (inhibition zone 31.0 ± 1.0 mm). Only one compound 9c in this series revealed slightly lower activity than Ampicillin against S. mutans (inhibition zone 20.2 ± 1.0 mm).

Interestingly, transformation of pyrid-2-ones 5a,b to 1H-pyrazolo[4,3-c]pyrid-2-ones 11a,b, as shown in Scheme , significantly improved its antibacterial activities to a greater extent. The main observation was the effect of compound 11a on the three bacterial strains, namely, S. aureus, E. coli, and S. mutans. Quite remarkably, compound 11a showed a higher antimicrobial activity against S. aureus (inhibition zone 35.2 ± 1.5 mm, MIC, 31.25 μg/mL) than that of Ampicillin (inhibition zone 32.0 ± 0.5 mm, MIC, 62.5 μg/mL) with much lower MIC value. Moreover, compound 11a revealed higher activity (inhibition zone 26.0 ± 0.5 mm, MIC, 31.25 μg/mL) as compared to that of Gentamicin (inhibition zone 25.0 ± 0.1 mm, MIC, 31.25 μg/mL) against E. coli. On comparing the antifungal effect of the standard drug, Nystatin (inhibition zone 28.0 ± 0.1 mm, MIC, 31.25 μg/mL), with that of compound 11a, the latter showed good activity against C. albicans (inhibition zone 21.0 ± 1.7 mm, MIC, 125 μg/mL). Furthermore, compound 11b possessed higher potency (inhibition zone 26.0 ± 1 mm, MIC, 31.25 μg/mL) than Gentamicin (inhibition zone 25.0 ± 0.1 mm, MIC, 31.25 μg/mL) against E. coli and a comparable activity (inhibition zone 21.0 ± 1.0 mm, MIC, 500 μg/mL) to that of Ampicillin (inhibition zone 22.0 ± 0.5 mm, MIC, 62.5 μg/mL) against S. mutans.

Additionally, the antibacterial efficacy of the synthesized compounds as detailed above was also compared to that of the well-known standard drug, SD. It was noticed that some of the tested compounds showed superior activities to the standard drug against different bacterial strains. Both thiomethylpyridon-2-ones 5a–c and 1H-pyrazolopyrid-2-ones, 11a and 11b, exhibited higher potency than that of SD (inhibition zone rang 19.8 ± 0.4 mm, MIC, 250 μg/mL) against E. coli. Sulfonyl hydrazides 3a and 3b and thiomethylpyridon-2-ones 5a–c exhibited excellent activity against K. pneumonia as compared to SD (inhibition zone 20.4 ± 0.9 mm, MIC, 250 μg/mL). Remarkably, unlike all other compounds in the series, 1H-pyrazolopyrid-2-one, compound 11a showed higher potency against S. aureus than SD (inhibition zone 31.2 ± 2.1 mm, MIC, 500 μg/mL), which demonstrates the remarkable overall potencies of this compound against various bacterial strains.

Although SD had no activity against C. albicans, some of newly synthesized compounds such as compounds 5a, 9d, and 11a revealed some potency (inhibition zone rang 21–28 mm).

Structural–Activity Relationship

The results of the antimicrobial examination demonstrated the following assumptions about the structural–activity relationship (SAR). The physicochemical properties evaluated in terms of the partition coefficient, log P, of the synthesized compounds were performed using Molinspiration, Table . Compounds with log P values ranging from 0 to 5 are considered to be a good oral bioavailability.[35] The results indicated that all synthesized compounds are having log P values less than 5. In the case of sulfonyl hydrazides 3a and 3b, it was observed that compound 3b, which have a methyl group at the para-position of the benzene sulfonamide moiety, is more active than 3a against the tested bacterial strains. It is also noticed that the lipophilic character of compound 3b, log P of 2.50, has increased as a result of the presence of a methyl group at the para position. The thiomethylpyridon-2-one derivatives, 5a–d, showed a strong antimicrobial activity. In the case of Y = NH2, Scheme , the substituted benzene sulfonamide moiety with the methyl group at the para-position caused 5b to be more active than that with an unsaturated benzene sulfonamide, 5a, against the tested microbial strains except for E. coli and C. albicans. However, in the case of Y = OH, the test revealed greater activity for 5c versus that of 5d against the Gram −ve bacteria such as K. pneumonia and E. coli. An evidence of how influential the Y group in this series of compounds is on its antimicrobial activity.

Table 3

Calculated Lipophilic Character log P of the Synthesized Compounds

compd no.	Y	Ar	Ar1	log Pa
3a	−	Ph	−	2.06
3b	−	4-Me-Ph	−	2.50
5a	NH2	Ph	−	3.27
5b	NH2	4-Me-Ph	−	3.72
5c	OH	Ph	−	3.57
5d	OH	4-Me-Ph	−	4.02
9a	NH2	Ph	Ph	4.41
9b	NH2	4-Me-Ph	Ph	4.86
9c	NH2	Ph	4-Me-Ph	4.86
9d	NH2	4-Me-Ph	4-Me-Ph	5.30
9e	NH2	Ph	4-MeO-Ph	4.46
9f	NH2	4-Me-Ph	4-MeO-Ph	4.91
11a	NH2	Ph	−	2.43
11b	NH2	4-Me-Ph	−	2.87
SD				–0.04

Calculations were performed using Molinspiration online property calculation toolkit (http://www.molinspiration.com). log P: logarithm of compound partition coefficient between n-octanol and water.

Interestingly, structure manipulation in terms of various substituents on both the benzene ring of the sulfonamide moiety and that of the anilide group, thiomethylpyridon-2-one derivatives 9a–f, led to a corresponding variation in their antimicrobial activities. To relate the impact of the molecular structure on the antimicrobial activities of this series of derivatives, substituents on one moiety were varied while keeping the other moiety intact and vice versa. In the case of no substituents on the benzene ring of the anilide group, compounds 9a,b, the antimicrobial effectiveness revealed that compound 9a, unsubstituted benzene sulfonamide derivative, had a higher activity against S. aureus than that of compound 9b, having a methyl group at the para-position of the benzene sulfonamide moiety. Similar antimicrobial activities were also observed for compounds 9c,d with a methyl group at the benzene ring of the anilide group. In the case of a methoxy group on the benzene ring of the anilide group, compounds 9e,f, the antimicrobial activities revealed that compound 9e, unsubstituted benzene sulfonamide derivative, had no activity against K. pneumonia, while compound 9f with a methyl group at the para-position of the benzene sulfonamide moiety had strong activity. Interestingly, the opposite behavior was observed when testing the two derivatives against S. aureus.

It is interesting here to note that 1H-pyrazolo[4,3-c]pyrid-2-ones 11a and 11b showed the strongest antimicrobial activity among all tested compounds and a good lipophilic character confirmed by log P values of 2.43 and 2.87, respectively, attributed to the fusion of the bioactive pyrazole moiety with the 2-pyridinone ring. It was also observed that compound 11a with an unsubstituted benzene sulfonamide moiety had a higher potency than compound 11b having a methyl group at the para-position of the benzene sulfonamide moiety against all tested bacterial strains except for K. pneumonia. Surprisingly, compound 11a also showed good antifungal activity against C. albicans, while compound 11b showed no potency.

In general, the presence of a methyl group at the para position of the benzene sulfonamide moiety has depressed the antimicrobial activities of N-arylsulfonamide 2-pyridones as compared to that of unsubstituted benzene sulfonamide moiety.

According to the aforementioned results of antimicrobial inhibition zones, MIC and log P, most of the newly synthesized compounds and in particular compounds 3b, 5a, 5b, 11a, and 11b exhibited notable potency as antimicrobial agents, which prompted us to further study their cytotoxicity and inhibition activities against both DHPS and DHFR enzymes.

Cytotoxicity Assessment

To study the inhibition activities of compounds exhibiting the highest activities namely, compounds 3b, 5a, 5b, 11a, and 11b, in vitro cytotoxicity was evaluated against HFB4 human dermal fibroblast cell line using MTT assay with SD as a reference drug. Plotting the cell viability percent against the concentration of test compounds was done to determine the human dermal cell line viability toward the specified compounds, Figure . Values of the IC50 of the examined compounds are summarized in Table .

Figure 3

Percentage of cell viability determined for 3b, 5a, 5b, 11a, 11b, and SD using the MTT assay.

Table 4

Anti-proliferative Activities of 3b, 5a, 5b, 11a, 11b, and SD against HFB4 Human Skin Normal Cell Line Using MTT Assay

comp.	IC50 (μg/mL)a
3b	164.76 ± 3.04
5a	167.28 ± 21.83
5b	145.73 ± 26.99
11a	233.51 ± 20.73
11b	295.14 ± 19.05
SD	242.56 ± 46.12

The data are expressed as the mean ± SD of three independent experiments.

The IC50 values shown in the table for compounds 3b, 5a, 5b, 11a, and 11b were found to be 164.76, 167.28, 145.73, 233.51, and 295.14 μg/mL, respectively. Interestingly, compounds 11a and 11b exhibited a high IC50 values, while compounds 3b, 5a and 5b showed low values toward the human dermal cell line as compared to that of SD. The results indicate that compounds 11a and 11b demonstrated a lower toxicity toward HFB4 human dermal fibroblast cell than SD did, while compounds 3b, 5a and 5b exhibited a higher toxicity.

DHPS and DHFR in Vitro Enzyme Assay

The in vitro growth inhibitory activity of the most potent compounds 3b, 5a, 5b, 11a, and 11b was studied on both DHPS and DHFR enzymes using DHPS catalytic assay and DHFR inhibitor screening kit, respectively. For the purpose of comparison, SD and TMP were used as reference drugs for DHPS and DHFR, respectively. The concentration of the tested compounds that is required to inhibit 50% of the cell population (IC50) was determined and is shown in Tables –7.

Table 5

IC50 Values of the Compounds 3b, 5a, 5b, 11a, and 11b on the DHPS and DHFR Enzymes Using SD and TMP as Reference Drugs

compd no.	IC50a (DHPS, μg/mL)	IC50a (DHFR, μg/mL)
3b	6.90 ± 0.44	0.37 ± 0.009
5a	14.65 ± 0.96	0.26 ± 0.009
5b	13.15 ± 0.74	0.30 ± 0.011
11a	2.76 ± 0.16	0.20 ± 0.007
11b	10.46 ± 0.07	5.52 ± 0.070
SD	2.05 ± 0.12	NT
TMP	NT	0.17 ± 0.005

The data are expressed as the mean ± SD of three independent experiments.

Table 7

Inhibition Activity of Compounds 3b, 5a, 5b, 11a, 11b, and TMP at Different Concentrations on the DHFR Enzyme

compd no.	10 (μg/mL), %	1 (μg/mL), %	0.1 (μg/mL), %	0.01 (μg/mL), %
3b	81.47	60.91	33.38	17.89
5a	75.89	57.27	41.84	29.97
5b	79.44	64.97	37.44	19.80
11a	71.81	66.96	44.13	29.59
11b	60.08	31.25	19.26	12.37
TMP	88.07	66.11	43.02	25.51

The resulting data were thus used to plot a dose response curves, as shown in Figures and 5. All the IC50 values of the tested compounds were compared to that of the reference drugs (p < 0.0001 for compounds 3b, 5a, 5b, and 11b and p < 0.01 for compound 11a).

Figure 4

Inhibition efficacy of the compounds toward (A) DHPS enzyme, (B) DHFR enzyme. The data are expressed as means ± SD of three separate experiments. p values were determined using GraphPad Prism Software (Student’s test). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs reference drugs.

Figure 5

Survival percentage of (A) DHFR enzyme and (B) DHPS enzyme at different concentrations of the compounds.

It is clear from the tables that the formation of 4-methylthio 2-pyridones 5b (IC50 = 13.15 μg/mL) as compared to its starting compound 3b (IC50 = 6.90 μg/mL) had resulted in a decreased potency against DHPS. However, its activities against DHFR (IC50 = 0.30 μg/mL) as compared to its starting compound 3b (IC50 = 0.37 μg/mL) had increased. Obviously, the presence of the methyl group at the para-position of the benzene sulfonamide moiety plays an important role on the IC50 values. This has caused the increase in the IC50 values of methylthio 2-pyridones 5b as compared to the unsubstituted benzene sulfonamide moiety 5a against DHFR, while the IC50 values of 5b had decreased when compared to that of the unsubstituted benzene sulfonamide moiety 5a against DHPS. It was also observed that the presence of a pyrazole moiety fused with a 2-pyridone ring has remarkably affected the potency of the compounds against DHPS. In the case of compound 11a, which contained the pyrazole moiety and an unsubstituted benzene sulfonamide moiety, the IC50 value has dropped to 2.76 μg/mL, while in the case of compound 11b which has a pyrazole moiety and a methyl group at the para-position of the benzene sulfonamide moiety, the IC50 value reached 10.46 μg/mL. The resulting data indicate that the presence of the unsubstituted benzene moiety in 11a led to an increase in the activities of the compound toward DHPS when compared to the corresponding compound with a methyl group at the para-position of the benzene sulfonamide moiety 11b. Similar observation was also found when the activities of both compounds, 11a and 11b, against DHFR enzyme was evaluated and compared. The results show clearly that compound 11a had the lowest IC50 value among all tested compounds against both DHPS and DHFR enzymes. To ascertain the potency of this compound versus that of standard drugs, its IC50 value (IC50 = 2.76 μg/mL) was compared to that of SD (IC50 = 2.05 μg/mL) against DHPS enzyme. Similarly, the IC50 value (IC50 = 0.20 μg/mL) of compound 11a was also compared to that of the TMP standard drug (IC50 = 0.17 μg/mL) against DHFR enzyme.

According to the inhibition results, DHPS was treated with the synthesized compounds and the reference drug, SD, at four different concentrations, 100, 10, 1, and 0.1 (μg/mL) to evaluate the IC50 of each one. It was noticed that compound 11a was the only compound that showed 77.28 inhibition % which is similar to the inhibition % of SD, 76.38%, at a concentration of 100 μg/mL. However, the inhibition % of SD, 26.15%, at a concentration of 0.1 μg/mL, was higher than the inhibition % of compound 11a, 21.01%. Although compound 11a showed similar inhibition to SD at a higher concentration, it showed lower inhibition than SD at a lower concentration, which led to increasing the IC50 of compound 11a. In contrary, the inhibition % of DHFR when treated by compound 11a was 71.81%, which is lower than that of TMP, 88.07%, at a concentration of 10 μg/mL and higher inhibition values, 29.59%, than that of TMP, 25.51%, at the low concentration of 0.01 μg/mL.

According to the above mentioned data, it is clearly showed that compound 11a exhibited the strongest growth inhibition among all other tested compounds 3b, 5a, 5b, and 11b against both DHPS and DHFR enzymes. More importantly, the IC50 values of compound 11a were found to be quite comparable to those of the reference drugs SD and TMP against the DHPS and DHFR enzymes, respectively. Therefore, this observed superior dual effect of compound 11a makes it potentially of high significance in the treatment of bacterial infections.

Docking Studies

Based on the results of the antimicrobial activities and enzyme assay, docking simulation was performed to better understand the interaction of the most potent compound, 11a, with the binding sites of DHPS and DHFR enzymes. Docking studies were carried out using a crystal structure of DHPS (PDB ID: 3TYE)[36] and DHFR (PDB ID: 3FRB),[10] separately obtained from the Protein Data Bank server (www.pdb.org).

First, the docking studies were validated by docking the cocrystallized ligands of DHPS and DHFR inside the active sites after their extraction from the respective receptors. The re-docking of the cocrystallized ligands (XTZ for 3TYE and TOP for 3FRB) were carried out by removing the bound ligands, XTZ and TOP, from the complex followed by its docking back into the binding sites, which yielded root mean square deviation (rmsd) values of 2.4 Å and 2.2 Å, respectively. The top ranked poses obtained from the MOE docking simulation were thus selected for the compound under study. Figure A–D shows the 2D and 3D interaction diagrams of both the cocrystallized ligand XTZ and the synthesized inhibitor, 11a, within the active site of DHPS, while Figure A–D shows the 2D and 3D interaction diagrams of both the cocrystallized ligand TOP and the synthesized inhibitor, 11a, within the active site of DHFR.

Figure 6

Binding mode of both the ligand (XTZ) and compound 11a inside DHPS active site (PDB ID: 3TYE) using MOE. (A) 2D interaction of the ligand with DHPS (B) 3D interaction of the ligand (brown) with DHPS (C) 2D interaction of 11a with DHPS (D) 3D interaction of 11a (green) with DHPS.

Figure 7

Binding mode of both the ligand (TOP) and compound 11a inside the DHFR active site (PDB ID: 3FRB) using MOE. (A) 2D interaction of the ligand with DHFR (B) 3D interaction of the ligand (brown) with DHFR (C) 2D interaction of 11a with DHFR (D) 3D interaction of 11a (green) with DHFR.

It is well known that DHPS has two binding pockets. One is p-amino benzoic acid binding pocket (pABA-binding pocket) capable of binding a p-amino benzoic acid group. The other is pterin-binding pocket capable of binding a dihydropterin pyrophosphate group.[37] Five amino acid residues, Asn120, Asp184, Lys220, Arg254, and His256, are considered as the key amino acids for the binding to the pterin-binding pocket, while the three amino acid residues, Lys220, Ser218 and Phe189, are the essential amino acids for the binding to the pABA-binding pocket of DHPS enzyme. Benzene-sulfonamide moieties are known to occupy the pABA-binding pocket and thus preventing the key substrate (pABA) from binding to it, a common observation for all sulfa drugs.[38]

The docking results showed that the cocrystallized ligand XTZ has four hydrogen-bond acceptors with Asn120, Lys220, and Ser22, three hydrogen-bond donors with Asn120 and Asp184, and one arene–H interaction with Lye220 within the DHPS pockets. The binding energy of compound 11a was determined from the docking study to be −6.6785 kcal/mol. It was also shown from the docking patterns of compound 11a that it fits well inside both the DHPS pterin- and pABA-binding pockets. As shown in Figure , compound 11a exhibited four hydrogen-bond acceptors. One hydrogen bond is formed between the nitrogen atom of the benzothiazole ring and the NH2 group of Lys220 amino acid with a bond length of 2.95 Å. Two hydrogen bonds are located between the oxygen atom of the pyridone ring and both the NH2 and NH groups of Arg254 amino acid with bond lengths of 3.47 and 3.61 Å, respectively. The fourth one is formed between the oxygen atom of the sulfonamide group and the NH2 group of Asn27 amino acid with a bond length of 3.28 Å. According to these results, compound 11a is attached to the protein molecule not only through one hydrogen bond with Lys220 amino acid within the pABA-binding pocket but also through three hydrogen bonds with Lys220 and Arg254 amino acids within the pterin-binding pocket. The advantage of binding the compound to the pterin site is that it can get around the most prevalent method of bacterial resistance, which takes place within the pABA binding pocket.

The interaction of the cocrystallized ligand TOP with DHFR revealed that the two amino groups at the pyrimidine and benzene rings are often the sites of interaction. The benzene ring created arene–H interactions, while the two amino groups established hydrogen bond donors with Phe92, Leu5, and Asp27 amino acids. Concerning the docking study of the tested compound 11a, it showed that this compound can fit into the DHFR pocket with two hydrogen bonds donors with Phe92 and Asp27 amino acids similar to that of the cocrystallized ligand TOP, with bond lengths of 3.24 and 3.38 Å, respectively. Additionally, compound 11a exhibited the third hydrogen bond donor between NH of the pyrazole ring and Ser49 amino acid with a bond length of 3.00 Å. Figure illustrated that the arene–H interaction has occurred between the pyridone ring and Thr46 amino acid. The binding energy of compound 11a scored −7.4515 kcal/mol.

These interactions with the amino acids inside the pockets have provided a major explanation for the basis of the inhibition effect of compound 11a on both the DHPS and DHFR enzymes, which highlighted its strong potential as a valid dual inhibitor for both DHPS and DHFR enzymes.

Conclusions

In this work, novel compounds of N-sulfonamide 2-pyridones incorporating the benzothiazole moiety were produced through the reaction of newly synthesized benzothiazol sulfonylhydrazide compounds with ketene dithioacetal derivatives. Spectral and elemental analyses were both utilized to confirm the structures of the synthesized compounds. The inhibition zones, MIC and logP have all been assessed through the evaluation of the antimicrobial activities of the newly synthesized compounds. It was determined from the study that the majority of the synthesized compounds and in particular compounds 3b, 5a, 5b, 11a, and 11b exhibited a notable potency as antimicrobial agents. The cytotoxicity and inhibition activities against both DHPS and DHFR enzymes showed that compound 11a displayed the strongest growth inhibition among all tested compounds 3b, 5a, 5b, and 11b against both DHPS and DHFR enzymes with IC50 values comparable to those of the reference drugs, SD and TMP. Docking simulation of the most potent compound, 11a, showed strong interactions with the amino acids inside the binding pockets of both DHPS and DHFR enzymes, which explains its remarkable inhibition effect on both DHPS and DHFR enzymes. The superior dual effect of compound 11a makes it potentially of high significance in the treatment of bacterial infections.

Experimental Section

Melting points were determined on a digital SMP3 melting point apparatus using open capillary tubes and are uncorrected. IR spectra were recorded on an FTIR plus 460 or pyeunicam SP-1000 spectrophotometer using KBr pellets. 1H NMR (400 MHz) and 13C NMR (100 MHz) were done in the Center of Drug Discovery Research and Development at Ain Shams University, and spectra were recorded on a Bruker ADVANCE (III) model ultra shield NMR spectrometer in DMSO-d6 as a solvent using tetramethylsilane as an internal standard, and chemical shifts are reported as δ ppm units. The elemental analyses were done at the microanalytical data unit at Cairo University and performed on a vario EI III Elemental CHNS analyzer. Progress of the reactions were monitored by TLC using aluminum sheet coated with silica gel MERCK 60F 254 and was visualized using a UV lamp. The reagents and solvents were purchased in commercially available grade purity.

General Procedure for the Synthesis of 3a,b

A solution of arylsulfonyl chloride 2a,b (0.015 mol) in pyridine (10 mL) was added gradually to a stirred solution of 2-(benzo[d]thiazol-2-yl)acetohydrazide 1 (0.01 mol) in pyridine (10 mL) at 0 °C. The reaction mixture was stirred at room temperature for 3 h (TLC control). After the reaction was completed, the mixture poured onto ice water with continues stirring and neutralized with 1 N HCl solution until the pH of the solution reached to 7. The formed precipitate was filtrated off, washed with water, and recrystallized from ethanol.

N′-(2-(Benzo[d]thiazol-2-yl)acetyl)benzenesulfonohydrazide (3a)

It was obtained as a white solid; (EtOH), yield 87%, mp 208–210 °C; IR (KBr, cm–1) ν: 3441 (NH), 3083 (ArCH), 2926 (Aliph-H), 1672 (CO), 1525 (C=N), 1341, 1159 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 3.98 (s, 2H, CH2), 7.41–7.45 (m, 3H, Ar-H), 7.51 (t, J = 8 Hz, 1H, benzothiazole-H), 7.57 (t, J = 8 Hz, 1H, benzothiazole-H), 7.80–7.78 (m, 2H, Ar-H), 7.96 (d, J = 8 Hz, 1H, benzothiazole H), 8.06 (d, J = 8 Hz, 1H, benzothiazole H), 10.07 (s, 1H, NH), 10.55 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 39.0 (CH2), 122.5, 122.7, 125.5, 126.5, 128.0, 129.2, 133.4, 135.8, 139.3, 152.6, 164.6, 166.5 (Ar-C); Anal. Calcd for C15H13N3O3S2 (347.41): C %, 51.86; H %, 3.77; N %, 12.10. Found: C %, 51.88; H %, 3.73; N %, 12.14.

N′-(2-(Benzo[d]thiazol-2-yl)acetyl)-4-methylbenzenesulfonohydrazide (3b)[34]

It was obtained as colorless crystals; (EtOH), yield 85%, mp 185 °C; IR (KBr, cm–1) ν: 3427 (NH), 3164 (ArCH), 2993 (Aliph-H) 1692 (CO), 1547 (C=N), 1344, 1168 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.27 (s, 3H, CH3), 3.96 (s, 2H, CH2), 7.15 (d, J = 8 Hz, 2H, Ar-H), 7.44 (t, J = 8 Hz, 1H, benzothiazole-H), 7.52 (t, J = 8 Hz, 1H, benzothiazole-H), 7.63 (d, J = 8 Hz, 2H, Ar-H), 7.97 (d, J = 8 Hz, 1H, benzothiazole-H), 8.07 (d, J = 8 Hz, 1H, benzothiazole-H), 9.95 (s, 1H, NH), 10.52 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 21.4 (CH3), 39.1 (CH2), 122.4, 122.7, 125.5, 126.5, 128.1, 129.6, 130.3, 135.8, 143.7, 152.6, 164.6, 166.3 (Ar-C); Anal. Calcd for C16H15N3O3S2 (361.44): C %, 53.17; H %, 4.18; N %, 11.63. Found: C %, 53.19; H %, 4.22; N %, 11.61.

General Procedure for the Synthesis of 5a–d

2-(Benzo[d]thiazol-2-yl)-3,3-bis(methylthio)acrylonitrile 4a (0.01 mol) or ethyl 2-cyano-3,3-bis(methylthio)acrylate 4b was added to a solution of N′-(2-(benzo[d]thiazol-2-yl)acetyl)arylsulfonohydrazides 3a,b (0.01 mol) in dry DMF (30 mL) containing pulverized potassium hydroxide (0.01 mol). The reaction mixture was refluxed with stirring for 2 h (TLC monitoring). After cooling, the reaction mixture was poured into ice-cold water and neutralized with HCl. The solid product was filtered off, washed with water, and dried. It was further purified from hot ethyl acetate: petroleum ether (1:1). The precipitated solid was crystallized from DMF.

N-(6-Amino-3-(benzo[d]thiazol-2-yl)-5-cyano-4-(methylthio)-2-oxopyridin-1(2H)-yl)benzenesulfonamide (5a)

It was obtained as a greenish yellow solid; (DMF), yield 78%, mp 300–301 °C; IR (KBr, cm–1): ν 3406, 3296 (NH, NH2), 3074 (ArCH), 2921 (Aliph-H), 2208 (CN), 1626 (CO), 1592 (C=N), 1367, 1132 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.46 (s, 3H, SCH3), 7.34–7.39 (m, 4H, 3Ar-H & benzothiazole-H), 7.45 (t, J = 8 Hz, 1H, benzothiazole-H), 7.50 (br, 2H, NH2), 7.73–7.76 (m, J = 8 Hz, 2H, Ar-H), 7.93 (d, J = 8 Hz, 1H, benzothiazole H), 8.99 (d, J = 8 Hz, 1H, benzothiazole H); 13C NMR (100 MHz, DMSO-d6): 25.9 (CH3), 117.8 (CN), 73.9, 110.7, 121.9, 122.5, 124.6, 126.0, 126.6, 127.5, 128.2, 129.6, 130.3, 135.8, 152.0, 157.9, 159.02, 162.8 (Ar-C); Anal. Calcd for C20H15N5O3S3 (469.56): C %, 51.16; H %, 3.22; N %, 14.91. Found: C %, 51.13; H %, 3.20; N %, 14.89.

N-(6-Amino-3-(benzo[d]thiazol-2-yl)-5-cyano-4-(methylthio)-2-oxopyridin-1(2H)-yl)-4-methylbenzenesulfonamide (5b)

It was obtained as a greenish yellow solid; (DMF), yield 75%, mp 315–317 °C; IR (KBr, cm–1) ν: 3410, 3300 (NH, NH2), 3062 (ArCH), 2921 (Aliph-H), 2206 (CN), 1627 (CO), 1593 (C=N), 1435, 1132 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.31 (s, 3H, CH3), 2.45 (s, 3H, SCH3), 7.14 (d, J = 8 Hz, 2H, Ar-H), 7.37 (t, J = 8 Hz, 1H, benzothiazole-H), 7.46 (t, J = 8 Hz, 1H, benzothiazole-H), 7.49 (br, 2H, NH2), 7.62 (d, J = 8 Hz, 2H, Ar-H), 7.93 (d, J = 8 Hz, 1H, benzothiazole-H), 8.01 (d, J = 8 Hz, 1H, benzothiazole-H); 13C NMR (100 MHz, DMSO-d6): 19.7, 21.4 (CH3), 117.8 (CN), 73.9, 110.7, 121.9, 122.5, 124.6, 126.0, 126.6, 127.5, 128.2, 129.6, 130.3, 135.8, 152.0, 157.9, 159.0, 162.8 (Ar-C); Anal. Calcd for C21H17N5O3S3 (483.59): C %, 52.16; H %, 3.54; N %, 14.48. Found: C %, 52.12; H %, 3.51; N %, 14.46.

N-(3-(Benzo[d]thiazol-2-yl)-5-cyano-6-hydroxy-4-(methylthio)-2-oxopyridin-1(2H)-yl)benzenesulfonamide (5c)

It was obtained as an army green solid; (acetone), yield 72%, mp 303–306 °C; IR (KBr, cm–1) ν: 3427 (OH), 3265 (NH), 3078 (ArCH), 2919 (Aliph-H), 2208 (CN), 1670 (CO), 1585 (C=N), 1340, 1163 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.59 (s, 3H, SCH3), 7.45 (t, J = 8 Hz, 1H, benzothiazole-H), 7.53–7.59 (m, 3H, Ar-H), 7.66 (t, J = 8 Hz, 1H, benzothiazole-H), 7.91 (d, J = 8 Hz, 2H, Ar-H), 8.07–8.09 (m, 2H, benzothiazole-H), 10.42 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 20.0 (CH3), 118.2 (CN), 101.0, 118.7, 122.5, 125.7, 127.5, 127.7, 127.8, 129.2, 129.6, 133.2, 141.7, 157.2, 159.9, 161.6, 165.9, 167.8 (Ar-C); Anal. Calcd for C20H14N4O4S3 (470.54): C %, 51.05; H %, 3.00; N %, 11.91. Found: C %, 51.02; H %, 3.03; N %,11.95.

N-(3-(Benzo[d]thiazol-2-yl)-5-cyano-6-hydroxy-4-(methylthio)-2-oxopyridin-1(2H)-yl)-4-methylbenzenesulfonamide (5d)

It was obtained as an army green solid; (acetone), yield 70%, mp 277–278 °C; IR (KBr, cm–1) ν: 3431 (OH), 3295, (NH), 3071 (ArCH), 2923 (Aliph-H), 2210 (CN), 1671 (CO), 1589 (C=N), 1342, 1161 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.39 (s, 3H, CH3), 2.56 (s, 3H, SCH3), 7.36 (d, J = 8 Hz, 2H, Ar-H), 7.42 (t, J = 8 Hz, 1H, benzothiazole-H), 7.53 (t, J = 8 Hz, 1H, benzothiazole-H), 7.78 (d, J = 8 Hz, 2H, Ar-H), 8.03–8.07 (m, 2H, benzothiazole-H), 9.23 (br, 1H, OH), 10.22 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 19.7, 21.5 (SCH3), 118.2 (CN), 101.0, 118.8, 120.6, 122.2, 124.9, 127.9, 129.3, 130.3, 134.3, 145.0, 157.2, 160.0, 161.6, 165.9, 167.8 (Ar-C); Anal. calcd for C21H16N4O4S3 (484.57): C %, 52.05; H %, 3.33; N %, 11.56. Found: C %, 52.01; H %, 3.34; N %, 11.60.

General Procedure for the Synthesis of 9a–f

N-Substituted bis(methylthiomethylene) (cyano)acetamide derivatives 6a–c (0.01 mol) were added to N′-(2-(benzo[d]thiazol-2-yl)acetyl)arylsulfonohydrazides 3a,b in dry dioxane (30 mL) containing pulverized potassium hydroxide (0.01 mol). The reaction mixture was refluxed until completion (TLC monitoring, 6–8 h). After cooling, the mixture was poured into ice water. The resulting solid was filtrated off, washed with water, and recrystallized from the appropriate solvent.

2-Amino-5-(benzo[d]thiazol-2-yl)-4-(methylthio)-6-oxo-N-phenyl-1-(phenylsulfonamido)-1,6-dihydropyridine-3-carboxamide (9a)

It was obtained as a greenish yellow solid; (acetone), yield 71%, mp 240–242 °C; IR (KBr, cm–1) ν: 3424, 3315 (NH, NH2), 3059 (ArCH), 2922 (Aliph-H), 1634 (CO), 1594 (C=N), 1321, 1162 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.24 (s, 3H, SCH3), 7.11 (t, J = 7.2 Hz, 1H, benzothiazole-H), 7.32–7.55 (m, 9H, 2NH2, 6Ar-H & benzothiazole-H), 7.76 (d, J = 7.2 Hz, 2H, Ar-H), 7.81 (d, J = 7.2 Hz, 2H, Ar-H), 7.88 (d, J = 8 Hz, 1H, benzothiazole-H), 7.97 (d, J = 8 Hz, 1H, benzothiazole-H); 10.40 (s, 1H, NH); Anal. Calcd for C26H21N5O4S3 (563.67): C %, 55.40; H %, 3.76; N %, 12.42. Found: C %, 55.45; H %, 3.78; N %, 12.46.

2-Amino-5-(benzo[d]thiazol-2-yl)-1-(4-methylphenylsulfonamido)-4-(methylthio)-6-oxo-N-phenyl-1,6-dihydropyridine-3-carboxamide (9b)

It was obtained as a greenish yellow solid; (acetone), yield 68%, mp 206–209 °C; IR (KBr, cm–1) ν: 3431, 3270 (NH, NH2), 3074 (ArCH), 2921 (Aliph-H), 1633 (CO), 1562 (C=N), 1367, 1175 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.24 (s, 3H, CH3), 2.34 (s, 3H, SCH3), 7.12 (t, J = 7.2 Hz, 1H, benzothiazole-H), 7.24 (t, J = 7.2 Hz, 1H, benzothiazole-H), 7.31–7.47 (m, 7H, NH2, 5Ar-H), 7.64 (d, J = 7.6 Hz, 2H, Ar-H), 7.69 (d, J = 7.6 Hz, 2H, Ar-H), 7.99 (d, J = 8 Hz, 1H, benzothiazole-H), 7.97 (d, J = 8 Hz, 1H, benzothiazole-H); 10.45 (s, 1H, NH), 11.21 (s, 1H, NH); Anal. Calcd for C27H23N5O4S3 (577.70): C %, 56.13; H %, 4.01; N %, 12.12. Found: C %, 56.19; H %, 4.05; N %, 12.18.

2-Amino-5-(benzo[d]thiazol-2-yl)-4-(methylthio)-6-oxo-1-(phenylsulfonamido)-N-(p-tolyl)-1,6-dihydropyridine-3-carboxamide (9c)

It was obtained as a beige solid; (acetone), yield 74%, mp 240–242 °C; 3429, 3268 (NH, NH2), 3076 (ArCH), 2962 (Aliph-H), 1657 (CO), 1387, 1168 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.31 (s, 3H, CH3), 2.50 (s, 3H, SCH3), 7.21 (d, J = 7.6 Hz, 2H, Ar-H), 7.51 (d, J = 7.6 Hz, 2H, Ar-H), 7.59 (t, J = 7.6 Hz, 1H, benzothiazole H), 7.73–7.76 (m, 3H, Ar-H), 7.74 (t, J = 7.6 Hz, 1H, benzothiazole H), 8.01 (d, J = 8 Hz, 2H, Ar-H), 8.20 (d, J = 8 Hz, 1H, benzothiazole-H), 8.46 (d, J = 8 Hz, 1H, benzothiazole-H), 9.28 (br, 2H, NH2), 11.17 (s, 1H, NH); Anal. Calcd for C27H23N5O4S3 (577.70): C %, 56.13; H %, 4.01; N %, 12.12. Found: C %, 56.15; H %, 4.02; N %, 12.13.

2-Amino-5-(benzo[d]thiazol-2-yl)-1-(4-methylphenylsulfonamido)-4-(methylthio)-6-oxo-N-(p-tolyl)-1,6-dihydropyridine-3-carboxamide (9d)

It was obtained as a greenish yellow solid; (acetone), yield 72%, mp 209–210 °C; IR (KBr, cm–1) ν: 3420, 3311 (NH, NH2), 3059 (ArCH), 2919 (Aliph-H), 1642 (CO), 1590 (C=N), 1344, 1160 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.23 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.36 (s, 3H, SCH3), 7.18 (d, J = 7.6 Hz, 2H, Ar-H), 7.36–7.38 (m, 3H, 2Ar-H & benzothiazole-H), 7.43–7.47 (m, 3H, 2Ar-H & benzothiazole-H), 7.63 (d, J = 7.6 Hz, 2H, Ar-H), 7.69 (d, J = 7.6 Hz, 2H, Ar-H), 7.90 (d, J = 8 Hz, 1H, benzothiazole-H), 8.00 (d, J = 8 Hz, 1H, benzothiazole-H), 10.36 (s, 1H, NH), 11.32 (s, 1H, NH); Anal. Calcd for C28H25N5O4S3 (591.72): C %, 56.83; H %, 4.26; N %, 11.84. Found: C %, 56.84; H %, 4.27; N %, 11.81.

2-Amino-5-(benzo[d]thiazol-2-yl)-N-(4-methoxyphenyl)-4-(methylthio)-6-oxo-1-(phenylsulfonamido)-1,6-dihydropyridine-3-carboxamide (9e)

It was obtained as a beige solid; (dioxane), yield 77%, mp 249–250 °C; IR (KBr, cm–1) ν: 3429, 3316 (NH, NH2), 3057 (ArCH), 2920 (Aliph-H), 1656 (CO), 1596 (C=N), 1353, 1163 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.25 (s, 3H, SCH3), 3.76 (s, 3H, OCH3), 6.96 (d, J = 8 Hz, 2H, Ar-H), 7.35 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.45 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.50 (br, 2H, NH2), 7.55–7.60 (m, 2H, Ar-H), 7.67–7.70 (m, 3H, Ar-H), 7.82 (d, J = 8 Hz, 2H, Ar-H), 7.90 (d, J = 8 Hz, 1H, benzothiazole-H), 7.98 (d, J = 8 Hz, 1H, benzothiazole-H), 10.35 (s, 1H, NH), 11.46 (s, 1H, NH); Anal. Calcd for C27H23N5O5S3 (593.70): C %, 54.62; H %, 3.90; N %, 11.80. Found: C %, 54.66; H %, 3.92; N %, 11.72.

2-Amino-5-(benzo[d]thiazol-2-yl)-N-(4-methoxyphenyl)-1-(4-methylphenylsulfonamido)-4-(methylthio)-6-oxo-1,6-dihydropyridine-3-carboxamide (9f)

It was obtained as a greenish yellow solid; (MeOH), yield 76%, mp 250–252 °C; IR (KBr, cm–1) ν: 3413, 3399 (NH, NH2), 3054 (ArCH), 2921 (Aliph-H), 1630 (CO), 1567 (C=N), 1466, 1172 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.22 (s, 3H, CH3), 2.33 (s, 3H, SCH3), 3.76 (s, 3H, OCH3), 6.94 (d, J = 8 Hz, 2H, Ar-H), 7.13–7.18 (m, 4H, 2Ar-H & NH2), 7.31 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.41 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.67–7.70 (m, 4H, Ar-H), 7.86 (d, J = 8 Hz, 1H, benzothiazole-H), 7.97 (d, J = 8 Hz, 1H, benzothiazole-H), 10.15 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ 19.6 (CH3), 21.4 (SCH3), 55.1 (OCH3), 97.6, 108.6, 114.3, 121.4, 121.6, 121.7, 124.1, 125.6, 125.8, 128.4, 133.1, 135.5, 139.0, 150.8, 151.9, 154.0, 155.8, 159.3, 164.2, 166.0 (Ar-C); Anal. Calcd for C28H25N5O5S3 (607.72): C %, 57.34; H %, 4.15; N %, 11.52; S %, 15.83. Found: C %, 57.32; H %, 4.13; N %, 11.50; S %, 15.84.

General Procedure for the Synthesis of 11a,b

A mixture of 5a,b (0.01 mol) and hydrazine hydrate (0.02 mol) in a mixture of methanol/DMF (50:50 vol %) containing three drops of piperidine was heated under reflux for 2 h (TLC monitoring). The mixture was then concentrated under reduced pressure to afford a solid residue that was triturated with ethanol, filtered off, and washed with ethanol. The solid product was recrystallized from methanol.

N-(3,4-Diamino-7-(benzo[d]thiazol-2-yl)-6-oxo-1H-pyrazolo[4,3-c]pyridin-5(6H)-yl)benzenesulfonamide (11a)

It was obtained as a pale yellow solid; (MeOH), yield 68%, mp 309–310 °C; IR (KBr, cm–1) ν: 3396, 3297 (NH, NH2), 3065 (ArCH), 1661 (CO), 1603 (C=N), 1321, 1137 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 5.58 (br, 2H, NH2), 7.13 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.33 (t, J = 7.6 Hz, 1H, benzothiazole-H), 7.33–7.39 (m, 3H, Ar-H), 7.56 (br, 2H, NH2), 7.80–7.86 (m, 4H, 2Ar-H & 2benzothiazole-H), 10.86 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ 86.3, 119.9, 121.4, 121.9, 125.4, 126.6, 128.0, 129.7, 133.2, 144.1, 148.5, 150.8, 151.7, 153.2, 159.6, 162.8, 165.0 (Ar-C); Anal. Calcd. for C19H15N7O3S2 (453.50): C %, 50.32; H %, 3.33; N %, 21.62. Found: C %, 50.33; H %, 3.32; N %, 21.63.

N-(3,4-Diamino-7-(benzo[d]thiazol-2-yl)-6-oxo-1H-pyrazolo[4,3-c]pyridin-5(6H)-yl)-4-methylbenzenesulfonamide (11b)

It was obtained as a pale yellow solid; (MeOH), yield 62%, mp 310–312 °C; IR (KBr, cm–1) ν: 3395, 3330 (NH, NH2), 3063 (ArCH), 2922 (Aliph-H), 1644 (CO), 1605 (C=N), 1321, 1131 (O=S=O); 1H NMR (400 MHz, DMSO-d6): δ 2.35 (s, 3H, CH3), 5.59 (br, 2H, NH2), 7.14 (t, J = 8 Hz, 1H, benzothiazole-H), 7.19 (d, J = 8 Hz, 2H, Ar-H), 7.33 (t, J = 8 Hz, 1H, benzothiazole-H), 7.51 (br, 2H, NH2), 7.70 (d, J = 8 Hz, 2H, Ar-H), 7.82 (d, J = 8 Hz, 1H, benzothiazole-H), 7.85 (d, J = 8 Hz, 1H, benzothiazole-H), 11.37 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ 21.6 (CH3), 81.4, 120.1, 121.5, 123.2, 126.1, 126.2, 129.9, 135.0, 144.4, 151.4, 152.0, 156.0, 162.0, 164.3 (Ar-C); Anal. Calcd for C20H17N7O3S2 (467.52): C %, 51.38; H %, 3.67; N %, 20.97. Found: C %, 51.39; H %, 3.67; N %, 20.99.

Antimicrobial Activity

Antimicrobial assessment and the MIC performed at the Microbiology Unit in Biochemistry Central Lab, Faculty of Science, Cairo University, Cairo, Egypt. Chemical compounds were individually tested against a panel of Gram positive and Gram-negative bacterial pathogens and the fungi utilizing agar well diffusion method.[39] The compounds were tested at a concentration of 15 mg/mL against both bacterial and fungal strains. Microbial suspension was prepared in sterilized saline equivalent to McFarland 0.5 standard solution (1.5 × 105 cfu mL–1), and its turbidity was adjusted to the optical density (OD) = 0.13 using a spectrophotometer at 625 nm. Optimally, within 15 min after adjusting the turbidity of the inoculum suspension, a sterile cotton swab was dipped into the adjusted suspension, was flooded on the dried agar surface and then allowed to dry for 15 min with lid in place. Wells of 6 mm diameter was made in the solidified media with the help of sterile borer. The solution of the tested compound (100 μL) was added to each well with the help of micropipette. The plates were incubated at 37 °C. The zone of inhibition (mm) was measured after 24 h incubation at 30 °C in the case of bacterial plates and 48 h in the case fugal plates. This experiment was carried out in triplicate, diameters of the inhibition zones were measured in millimeters, and the results were recorded for each tested compound as % inhibition ± SD.

MIC Measurement

Stock solutions of the tested compounds, Ampicillin, Gentamicin, and Nystatin were prepared in DMSO at a concentration of 1000 μg/mL followed by serial twofold dilution at concentrations of (500, 250,125, 62.5, and 31.25 μg/mL). Each concentration was mixed with sterile nutrient agar (Sigma-Aldrich, USA) in a sterile plate followed by the inoculation of a defined microbial inoculum onto the agar plate surface. The plates were incubated at 37 °C in a humid chamber, and MIC endpoints were read after 24 h. The MIC end point is recorded as the lowest concentration of the antimicrobial agent that completely inhibits growth under suitable incubation conditions.

Cytotoxicity Effect

The toxicity effect tests were performed at the Tissue Culture Unit, the Egyptian Company for Production of Vaccines, Sera and Drugs (VACSERA), Giza, Egypt. The toxicity effect on human dermal cell line (HFB4) was studied for the most active compounds by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Potential cytotoxicity of the compounds was tested as follows: cells were plated in 96 multi-well plates (104 cells/well) for 24 h before treatment with the compound(s) to allow attachment to the wall of the plate. Different concentrations of the compounds (0, 6.25, 12.5, 25, 50, and 100 μg/mL) added to the cell monolayer triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the compound(s) for 48 h at 37 °C. Subsequently, an amount of 20 μL of MTT solution was added to the wells and incubated for 4 h. DMSO (200 μL) was added to each wall for dissolving the formed dark blue formazan crystals. Their OD of visible cells was detected using a spectrophotometer. In order to determine the cell viability for the tested compounds, OD of each tested compound was compared with OD of untreated control.

Enzyme Assay

Enzyme assay experiments were carried out at the Tissue Culture Unit, the Egyptian Company for Production of Vaccines, Sera and Drugs (VACSERA), Giza, Egypt.

DHPS Assay

DHPS activity was determined by bacterial-DHPS catalytic assay. The reaction mixture consisted of 5 mM magnesium chloride (MgCl2), 40 mM Tris-HCl (pH 8.2), 1 μM radiolabeled p-amino benzoic acid [3H]pABA, 10 mM dithiothreitol (DTT), and 10 μM 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (H2PtCH2OPP) in a total volume of 100 μL. Stock solutions of the tested compounds were prepared by dissolving each tested compounds in DMSO, 100 μg/mL. The reaction was initiated via adding 100 μL of DHPS-containing lysates (containing 0.5 mU of enzyme) at 37 °C. Reaction mixtures without lysate or H2PtCH2OPP served as blanks. After 30 min incubation, the reactions were terminated by placing the reaction tubes on ice, and then, 100 μL of the reaction volume was spotted onto 3 × 30 cm strips of 3MM chromatography paper (Whatman Laboratory Products Inc.). The strips were developed in a descending chromatography tank utilizing 0.1 M KH2PO4, pH 7.0, as an elution buffer. Under such conditions, unreacted [3H]PABA migrates with the solvent front and radiolabeled product, 7,8-dihydropteroate (DHF, H2Pte), remains at the origin. The origins that contain the labeled products of the reaction were cut from the strips and placed in scintillation vials. The radioactive activity of the labeled products was measured by counting the resultant photon emission in a liquid scintillation counter (Packard Tri-Carb; Packard Instrument Co. Inc., Downers Grove) 24 h after the addition of 9.5 mL of counting cocktail (3A70b; Research Products International Corp., Mt. Prospect). Results were expressed as the mean and standard deviation of triplicate samples in picomoles of product formed per milligram of total protein.

DHFR Assay

DHFR inhibitor screening kit (BioVision, Catalog #K247-100) was used for this assay. The assay for the inhibitory effect of target compounds against DHFR enzyme was applied as indicated in the DHFR assay kit. DHFR provided with the kit is human DHFR recombinant expressed in E. coli. Stock solutions of the tested compounds with different concentrations were prepared by dissolving each tested compounds to 100× in DMSO. Different concentrations (2 μL) of the tested compounds were added into wells of 96-well clear plate. Stock solutions of the DHFR were prepared by adding 2 μL of DHFR to 798 μL of supplied DHFR assay buffer. An amount of 98 μL of diluted DHFR was added into the wells containing the tested samples and enzyme control. An amount of 40 μL of diluted NADPH (10 μL of NADPH in 390 μL DHFR assay buffer) was also added to the aforementioned wells and then mixed well by using vortex. After incubation at room temperature for 10–15 min without exposing to light, 60 μL of diluted DHFR substrate (40 μL of DHFR stock substrate in 560 μL DHFR assay buffer) was added to each well in 96-well plate that contains the test samples and enzyme control and mixed well. Consequently, the total volume is 200 μL. Absorbance was immediately measured at 340 nm in kinetic mode for 10–20 min at room temperature using ELISA reader. The relationship between absorbance with time was plotted, and the slope for all test inhibitor samples was calculated. The percentages of both of relative inhibition and relative activity were determined using eqs and 2, respectively.where, S is sample screening and EC is enzyme control.

Statistical Analysis

The experiments was repeated three times, and all data were expressed as mean ± SD. Significant difference was tested via applying Student’s test between two groups to the data. Such statistical analyses was carried out using GraphPad Prism Software version 6 (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Molecular Docking

The molecular studies were carried out using Molecular Operating Environment (MOE 2014) All the minimizations were performed with MOE until a rmsd gradient of 0.01 kcal/mol Å with MMFF94X force field, and the partial charges were automatically calculated. Docking simulations were performed using the crystal structure of DHPS enzyme (PDB ID: 3TYE) in complex to XTZ and the crystal structure of DHFR enzyme (PDB ID: 3FRB) in complex to TOP which was obtained from Protein Data Bank. Enzyme structures were checked for missing atoms, bonds, and contacts. Water molecules and bound ligands were removed. Protonate 3D application of MOE was used to add the missing hydrogens and properly assign the ionization states. The ligand molecules were constructed using the builder molecule and were energy minimized. The active site was generated using the MOE-Alpha site finder. Dummy atoms were created from the obtained alpha spheres. Ligands were docked within the active sites using the MOE-Dock. The generated poses were energy minimized using the MMFF94x force field. Finally, the optimized poses were ranked using the GBVI/WSA DG free-energy estimates. Docking poses were visually inspected, and interactions with binding pocket residues were analyzed.

Table 6

Inhibition Activity of Compounds 3b, 5a, 5b, 11a, 11b, and SD at Different Concentrations on the DHPS Enzyme

compd no.	100 (μg/mL), %	10 (μg/mL), %	1 (μg/mL), %	0.1 (μg/mL), %
3b	74.41	53.94	34.16	7.63
5a	66.66	49.27	20.74	12.24
5b	66.59	52.91	21.81	5.87
11a	77.28	63.44	42.71	21.01
11b	71.96	48.82	25.80	9.94
SD	76.38	67.79	42.76	26.15