Sulfaguanidine Hybrid with Some New Pyridine-2-One Derivatives: Design, Synthesis, and Antimicrobial Activity against Multidrug-Resistant Bacteria as Dual DNA Gyrase and DHFR Inhibitors

Herein, a series of novel hybrid sulfaguanidine moieties, bearing 2-cyanoacrylamide 2a–d, pyridine-2-one 3–10, and 2-imino-2H-chromene-3-carboxamide 11, 12 derivatives, were synthesized, and their structure confirmed by spectral data and elemental analysis. All the synthesized compounds showed moderate to good antimicrobial activity against eight pathogens. The most promising six derivatives, 2a, 2b, 2d, 3a, 8, and 11, revealed to be best in inhibiting bacterial and fungal growth, thus showing bactericidal and fungicidal activity. These derivatives exhibited moderate to potent inhibition against DNA gyrase and DHFR enzymes, with three derivatives 2d, 3a, and 2a demonstrating inhibition of DNA gyrase, with IC50 values of 18.17–23.87 µM, and of DHFR, with IC50 values of 4.33–5.54 µM; their potency is near to that of the positive controls. Further, the six derivatives exhibited immunomodulatory potential and three derivatives, 2d, 8, and 11, were selected for further study and displayed an increase in spleen and thymus weight and enhanced the activation of CD4+ and CD8+ T lymphocytes. Finally, molecular docking and some AMED studies were performed.


Introduction
The emergence and outbreak of methicillin-resistant S. aureus (MRSA), vancomycinresistant Enterococci (VRE), and penicillin-resistant S. pneumoniae (PRSP) made antibioticresistant infections a critical healthcare concern worldwide [1]. A report published by the United Kingdom government predicted that antibiotic-resistant infections will result in 10 million deaths worldwide per year by 2050 if new antimicrobial strategies are not discovered [2]. Therefore, antimicrobial resistance is a significant public health threat. Although resistance occurs naturally, the overuse and misuse of broad-spectrum antibiotics has accelerated the increase in resistance [3]. Therefore, to avoid unintentional elicitation of antibiotic resistance or disruption of microbiota, the use of a species-selective antimicrobial agent, which specifically targets and kills the disease-causing strain, has been suggested [4][5][6]. However, no new class of antibiotics has been developed for infections triggered by Gram-negative bacteria, including P. aeruginosa, in the last 40 years; in addition, Figure 1. Rational study design, illustrating the structure of the newly designed sulfa guanidine derivatives with representative examples for antimicrobial drugs that contain guanidine, pyridine, and sulfonamide moieties.

Chemistry
The synthetic strategies adopted for constructing the target molecules are illustrated in Schemes 1-3. The starting material, N-(4-(N-carbamimidoylsulfamoyl) phenyl)-2-cyano acetamide 1, was prepared by cyanoacetylation of the sulfaguanidine with ethyl cyanoacetate in refluxing DMF. Elemental analysis and spectral data were in favor of the proposed structure. The IR spectrum of 2-cyanoacetamide derivative 1 showed absorption bands at ʋ 3441, 3336, 3232, 2264, and 1693 cm −1 due to the NH2, NH, CN, and CO groups. The 1 H NMR spectrum showed a new singlet signal at δ 3.94 ppm corresponding to the methylene group, and a broad signal at δ 6.70 ppm due to four protons of the guanidine moiety. In addition, a singlet signal at δ 10.58 ppm was exchangeable with D2O for an NH Figure 1. Rational study design, illustrating the structure of the newly designed sulfa guanidine derivatives with representative examples for antimicrobial drugs that contain guanidine, pyridine, and sulfonamide moieties.

Chemistry
The synthetic strategies adopted for constructing the target molecules are illustrated in Schemes 1-3. The starting material, N-(4-(N-carbamimidoylsulfamoyl) phenyl)-2-cyano acetamide 1, was prepared by cyanoacetylation of the sulfaguanidine with ethyl cyanoacetate in refluxing DMF. Elemental analysis and spectral data were in favor of the proposed structure. The IR spectrum of 2-cyanoacetamide derivative 1 showed absorption bands at υ 3441, 3336, 3232, 2264, and 1693 cm −1 due to the NH 2 , NH, CN, and CO groups. The 1 H NMR spectrum showed a new singlet signal at δ 3.94 ppm corresponding to the methylene group, and a broad signal at δ 6.70 ppm due to four protons of the guanidine moiety. In addition, a singlet signal at δ 10.58 ppm was exchangeable with D 2 O for an NH proton, and two doublet signals in the region δ 7.65 and 7.72 ppm, with a coupling constant (J = 7.60 and 6.8 Hz) corresponding to the four aromatic protons. 13 C NMR spectra showed two specific singlet signals, one at δ 27.38 ppm related to the methylene group and the second Scheme 1. Illustrate synthesis of 2-cyanoacetamide derivative 1 that contains the sulfonyl guanidine moiety and its reaction with a different aldehyde or arylidine to synthesize the acrylamide 2a-d and pyridine-2-one 3a-d derivatives.
Scheme 2. Suggested pathway to produce 6-amino pyridine-2-one derivatives 4, 6 via Michael addition of the 2-cyanoacrylamide 2b derivative. Scheme 1. Illustrate synthesis of 2-cyanoacetamide derivative 1 that contains the sulfonyl guanidine moiety and its reaction with a different aldehyde or arylidine to synthesize the acrylamide 2a-d and pyridine-2-one 3a-d derivatives.
Antibiotics 2021, 10, x FOR PEER REVIEW 4 of 31 proton, and two doublet signals in the region δ 7.65 and 7.72 ppm, with a coupling constant (J = 7.60 and 6.8 Hz) corresponding to the four aromatic protons. 13 C NMR spectra showed two specific singlet signals, one at δ 27.38 ppm related to the methylene group and the second signal at δ 112.88 ppm for the cyano group, in addition to the presence of aromatic carbons in the region between δ 116.24 and 141.17 ppm, followed by two characteristic signals at δ 158.52 and 162.06 ppm for the C=N and C=O, respectively. Scheme 1. Illustrate synthesis of 2-cyanoacetamide derivative 1 that contains the sulfonyl guanidine moiety and its reaction with a different aldehyde or arylidine to synthesize the acrylamide 2a-d and pyridine-2-one 3a-d derivatives.

Scheme 3.
Synthesis of the new pyridine and chromene-3-carboxamide derivatives containing the sulfaguanidine moiety.
Knoevenagel condensation of the 2-cyanoacetamide derivative 1, with aromatic aldehydes in the ethanolic piperidine under reflux conditions, furnished the corresponding acrylamide derivatives 2a-d in good yield. The structure of acrylamide derivatives 2a-d was an assignment based on their elemental and spectral data. The infrared spectrum of compound 2b showed characteristic bands at ʋ 3360, 3309, 3206, 2222, and 1693 cm −1 assigned for the NH2, NH, CN, and C=O groups. The 1 H NMR spectrum (DMSO-d6) of these compounds revealed two singlet signals at δ 3.89 and 8.25 ppm attributed to OCH3 and the methylinic proton (CH=). Furthermore, the four guanidine protons that appear as a broad signal at δ 6.74 ppm and four doublet signals between 7.19 and 8.04 ppm are attributed to eight aromatic protons, with a coupling constant (J) from 8.0 to 8.8 Hz. Its 13 C NMR spectra afforded a new characteristic signal at δ 56.16 for the methoxy group and signals at δ 115.00, 158.56, and 163.35 ppm due to the CN, C=NH, and C=O carbons, respectively, beside signals between δ 115.42 and 151.36 ppm for the aromatic carbons.
Ternary condensation of cyanoacetamide derivative 1, aromatic aldehyde, and malononitrile in an equimolar molar ratio (1:1:1) in basic ethanol (ethanol containing three drops of piperidine) afforded the 2-pyridone derivatives 3a-d. The structure of the newly designed 2-oxopyridine derivatives 3a-d was elucidated based on its analytical and spectral data. The structure of 2-oxopyridine derivatives 3a-d was chemically confirmed by treating 2-cyanoacrylamide 2a-d with malononitrile under reflux conditions and in the presence of piperidine as a catalyst (Scheme 1). Furthermore, the reaction of acrylamide derivative 2b with 2-cyano-N-cyclohexyl acetamide in ethanolic piperidine afforded creating a product where its structure should be either 4 or 5. The structure of the product was assigned as the pyrid-2-one derivative 4 rather than 2-amino-6-oxo-pyridin-3-carboxamide derivative 5, where the formation of compound 4 is assumed to take place by an initial Michael addition of the active methylene to the double bond followed by cyclization through the addition of an NH of aliphatic acetamide to the cyano group. Finally, proton shifts occur to yield 2-aminopyridin-6-one derivative 4. Knoevenagel condensation of the 2-cyanoacetamide derivative 1, with aromatic aldehydes in the ethanolic piperidine under reflux conditions, furnished the corresponding acrylamide derivatives 2a-d in good yield. The structure of acrylamide derivatives 2a-d was an assignment based on their elemental and spectral data. The infrared spectrum of compound 2b showed characteristic bands at υ 3360, 3309, 3206, 2222, and 1693 cm −1 assigned for the NH 2 , NH, CN, and C=O groups. The 1 H NMR spectrum (DMSO-d 6 ) of these compounds revealed two singlet signals at δ 3.89 and 8.25 ppm attributed to OCH 3 and the methylinic proton (CH=). Furthermore, the four guanidine protons that appear as a broad signal at δ 6.74 ppm and four doublet signals between 7.19 and 8.04 ppm are attributed to eight aromatic protons, with a coupling constant (J) from 8.0 to 8.8 Hz. Its 13 C NMR spectra afforded a new characteristic signal at δ 56.16 for the methoxy group and signals at δ 115.00, 158.56, and 163.35 ppm due to the CN, C=NH, and C=O carbons, respectively, beside signals between δ 115.42 and 151.36 ppm for the aromatic carbons.
Ternary condensation of cyanoacetamide derivative 1, aromatic aldehyde, and malononitrile in an equimolar molar ratio (1:1:1) in basic ethanol (ethanol containing three drops of piperidine) afforded the 2-pyridone derivatives 3a-d. The structure of the newly designed 2-oxopyridine derivatives 3a-d was elucidated based on its analytical and spectral data. The structure of 2-oxopyridine derivatives 3a-d was chemically confirmed by treating 2-cyanoacrylamide 2a-d with malononitrile under reflux conditions and in the presence of piperidine as a catalyst (Scheme 1). Furthermore, the reaction of acrylamide derivative 2b with 2-cyano-N-cyclohexyl acetamide in ethanolic piperidine afforded creating a product where its structure should be either 4 or 5. The structure of the product was assigned as the pyrid-2-one derivative 4 rather than 2-amino-6-oxo-pyridin-3-carboxamide derivative 5, where the formation of compound 4 is assumed to take place by an initial Michael addition of the active methylene to the double bond followed by cyclization through the addition of an NH of aliphatic acetamide to the cyano group. Finally, proton shifts occur to yield 2-aminopyridin-6-one derivative 4. Similarly, 2-amino-N-(4-(carbamimidoylsulfamoyl) phenyl)-5-cyano-4-(4-methoxy phenyl)-6-oxo-1,6-dihydropyridine-3-carboxamide 6 was prepared through the reaction of compound 2b with cyanoacetamide in refluxing ethanol containing piperidine as catalyst (Scheme 2). The assignment of the structure of these derivatives was based on analytical and spectroscopic data. Thus, its IR spectrum of compound 6 displayed absorption bands at υ 3441, 3336, and 3232 cm −1 assignable to the NH 2 and NH groups in addition to two bands at υ 2214 and 1689 cm −1 related to CN and carbonyl groups, respectively. Further, the 1 H NMR spectrum of 6-aminopyridin-2-one derivative 6 was characterized by the existence of a methoxy group as a singlet signal at δ 3.89 in addition to the guanidine protons (NH 2 + 2NH) at 6.71 ppm, as well as eight aromatic protons ranging between δ 7.20 and 8.50 ppm and two NH groups at δ 8.25 and 10.57 ppm. While, the 1 H NMR spectrum of pyridine-2-one derivative 4 exhibited a three new singlet signals at δ 1.25, 2.75, and δ 2.90 ppm, attributed for five CH 2 groups of the hexyl moiety. In the 13 C NMR spectra of 6-aminopyridin-2-one derivative 4, we observed new signals at δ 29.50, 31.25, and 36.28 ppm for the hexyl moiety, besides methoxy, CH-pyridine and two carbonyl groups, at δ 56.17, 62.59, 162.84, and 163.37 ppm, respectively.
The reactivity of cyanoacetamide derivative 1 towards some active methylene reagents was investigated. Thus, N-carbamimidoyl-4-(4,6-diamino-3-cyano-2-oxopyridin-1(2H)-yl) benzene-sulfonamide (7) was prepared by treating of cyanoacetamide derivative 1 with malononitrile in a molar ratio of 1:1. The structure of diamino-2-oxopyridine derivative 7 was confirmed based on its elemental analysis and spectral data. The formation of diamino pyridine derivative 7 is assumed to proceed via the Michael addition of cyanoacetamide derivative 1 to the cyano group of malononitrile to form the acyclic Michael adduct, followed by in situ cyclization to the pyridin-2-one skeleton. The IR spectra of pyridine derivative 7 revealed an abroad absorption band at υ 3410, 3332, and 3224 cm −1 besides bands at υ 2214 and 1651 cm −1 for the amino and imino (NH 2 and NH), CN, and carbonyl groups, respectively. At the same time, 1 H NMR spectra demonstrated three singlet signals at δ 4.13, 7.36, and 7.53 ppm, related to the CH-of pyridine and two amino groups at position four and six in the pyridine ring as well as both aromatic and guanidine protons. 13 C NMR spectra also displayed signals at δ 71. 37, 89.32, 114.98, 159.36, 170.80, and 162.02 ppm for CH-pyridine, the carbon of pyridine attached to the nitrile group, cyano, and the carbon-holding amino and carbonyl group, respectively. Furthermore, cyclo-condensation of cyanoacetamide derivative 1 with acetylacetone furnished N-carbamimidoyl-4-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-yl) benzenesulfonamide 8 via intramolecular hetero cyclization by loss of water molecule. Analytical and spectroscopic data can provide a reaction product. The IR spectrum showed the appearance of absorption bands υ 3464, 3425, 3194, 2218, and 1651 cm −1 , corresponding to the NH 2 , NH, CN, and CO groups, respectively. Its 1 H NMR spectrum exhibited two additional singlet signals at δ 1.99 and 2.41 ppm, assignable to the protons of two methyl groups in the pyridine ring and a singlet signal at δ 6.51 ppm attributed to pyridine-H 5 ; also, its 13 C NMR afforded two additional signals at δ 21.16, 21.98 ppm due to two methyl carbons. Condensation of 4,6-diethyl-2-oxopyridine derivative 8 with aromatic aldehydes, such as p-chlorobenzaldehyde and p-anisaldehyde, afforded the corresponding styryl derivatives 9a, b based on the elemental and spectral analyses. The 1 H NMR spectrum of compound 9b revealed the absence of a methyl signal at δ 1.99 ppm and the presence of five significant signals at δ 2.41, 3.84, 7.12, 7.26, and 6.49 ppm due to the methyl and methoxy protons and two singlet signals for the styryl moiety (2 CH=CH), as well as pyridine-H 5 . Furthermore, its 13  A Gewald reaction of the activated methyl pyridone derivative 8 with elemental sulfur in EtOH/DMF, containing triethylamine as a basic catalyst, led to the formation of a product that was formulated as 4-(3-amino-6-methyl-4-oxothieno [3,4-c] pyridin-5(4H)yl)-N-carbamimidoylbenzenesulfon-amide 10. The structure of the prepared compound was elucidated based on elemental analysis and spectral data. IR spectrum of thieno [3,4-c] Antibiotics 2021, 10, 162 7 of 31 pyridine derivative 10 displayed bands at υ 3433, 3286, 3232, and 1668 cm −1 , related to the (NH 2 + NH) and carbonyl groups, beside the absent band for the cyano group. The 1 H NMR spectrum of compound 10 was characterized by the existence of a singlet signal for thiophene-H at δ 7.96 ppm in addition to pyridine-H and a methyl group at δ 6.50 and 2.41 ppm, respectively. Its 13 C NMR showed signals at δ 21.15, 160.47, 160.90, and 162.83 ppm due to the methyl, carbon of imino (C=NH), carbonyl, and carbon attached to the amino and sulfur in the thiophene ring.
Furthermore, condensation of cyanoacetanilide derivative 1 with salicylaldehyde using ammonium acetate as a catalyst gave N-(4-(N-carbamimidoylsulfamoyl) phenyl)-2-imino-2H-chromene-3-carboxamide 11 on the bases of elemental analyses and spectral data. The resulting chromene derivative 11 has latent functional constituents, which have the potential for further chemical transformations that give new routes for the preparation of substituted, polycondensed chromene derivatives. The reaction of chromene 11 with malononitrile in refluxing DMF/ethanol containing a catalytic amount of piperidine furnishes the novel chromeno [3,4-c] pyridine derivative 12 in a good yield. The molecular structure of 12 was established through analytical and spectral data. Its infrared spectrum showed absorption bands at υ 3441, 3348, 3238, 3182, 2202, and 1662 cm −1 due to the NH 2 , NH, CN, and C=O functional groups, respectively; also, its 1 H NMR and 13 C NMR agree with the proposed structure (Scheme 3). The 1 H NMR spectra of chromeno [3,4-c] pyridine derivative 12 exhibited the disappearance of two singlet signals for the CH-4 of chromene and the NH of amide, which appears as downfield of the chromene-3-carboxamide derivatives 11. Besides, the appearance of new signals related to the amine group with guanidine protons was confirmed by increasing the integration of protons at δ 6.74 ppm to six instead of four.

Antimicrobial Activity with Structure Activity Relationship (SAR) Study
The newly designed and modified sulfaguanidine derivatives 2a-d, 3a-d, 4, 6, 7, 8, 9a, b, 10, 11, and 12 were screened in vitro for their antimicrobial activity against six bacterial strains and two fungal pathogens. The antimicrobial activity results are described in Table 1, which were obtained by measuring the inhibition zone (mm) with the conventional paper disk diffusion method according to reported methods [61][62][63]. Tetracycline and amphotericin B were used as the positive controls, with a significantly sized inhibition zone against all the tested pathogens.
Furthermore, the reaction of acrylamide derivative 2a-d with malononitrile causes cyclization to produce 3,5-dicyano pyridine-2-one derivatives 3a-d. It is interesting to note that the 3,5-dicyano pyridine-2-one with 4-chlorophenyl in position four of pyridine derivatives 3a showed broad and higher activity than 3-(4-chlorophenyl) acrylamide derivative 2a and higher than any 3,5-dicyano pyridine-2-one derivatives 3b-d, with an inhibition zone (IZ) between 18 ± 0.16 and 31 ± 0.40 mm. Similarly, pyridine-3carboxamide derivatives 4 and 6 showed moderate activity. However, the presence of an N-cyclohexyl group in position one increased the activity from 23 ± 0.22 to 25 ± 0.35 mm for the Gram-positive strains and from 23 ± 0.74 to 27 ± 0.30 mm for the Gram-negative strains. Besides, N-hydropyridine-3-carboxamide derivative 6 demonstrated an IZ from 16 ± 0.54 to 18 ± 0.12 mm and 12 ± 0.61 to 15 ± 0.96 mm respectively. N-cyclohexyl pyridine-3-carboxamide derivative 4 exhibited no activity to the S. typhi strain, while pyridine-3-carboxamide derivative 6 showed no growth against E. faecalis and P. aeruginosa. Our study extended to study the different substituents' effect in positions four and six on pyridine-2-one derivatives 7 and 8 as the methyl and amino groups. From the obtained data, it is clear that 4,6-dimethyl pyridine-2-one derivative 8 advertised higher activity than diamino derivative 7, with an inhibition zone (IZ) from 23 ± 0.14 to 27 ± 0.50 mm compared to tetracycline (25 ± 0.22 to 20 ± 0.5 mm). The reaction of the methyl group at position four of 4,6-dimethyl pyridine-2-one derivative 8 to form 4-(arylstyrenyl) pyridine-2-one derivative 9a, b, or the condensed pyridine structure as theino [3,4-c] pyridine derivative 10, did not lead to an improvement in antimicrobial activity but displayed moderate activity and less than the parent structure 8. Replacement of the pyridine nucleus with the pyrane moiety in 2-imino chromen-3-carboamide derivative 11 displayed a significant antibacterial activity with an IZ of 28 ± 0.11, 24 ± 0.29, and 29 ±0.54 mm and 25 ± 0.43, 24 ± 0.13, and 18 ± 0.36 mm for the Gram-positive bacteria. Besides, for the Gram-negative bacterial strains used in this study, introducing the pyridine nucleus into the chromene derivative led to decreased activity, as observed in the activity of the chromeno[3,4-c] pyridine derivative 12.
For the antifungal activity determined against yeast strains C. albicans and F. oxysporum, most of the synthetized compounds showed moderate to good activity, with six derivatives (3b, 3d, 4, 7, 9a, and 12) that had no activity against F. oxysporum. Furthermore, six derivatives (2a, 2d, 3a, 3c, 8, and 11) were the most active compounds and displayed a higher or equipotent inhibition zone that ranged between 22 ± 0.21 and 27 ± 0.5 mm for C. albicans, and exhibited an IZ from 18 ± 0.45 to 22 ± 0.11 mm for F. oxysporum in comparison to the amphotericin B as a positive control (22 ± 0.2 and 18 ± 0.32 mm) against the two fungal strain respectively.
Finally, regarding the most promising activity, derivatives 2a, 2b, 2d, 3a, 8, and 11 were revealed to be the best in inhibition of the growth of bacteria, with low MIC and MBC values. These values showed that the sulfonamide derivatives exhibited bactericidal activity depending on the MBC/MIC ratio ranging between 1 and 2. Furthermore, it seems that the tested derivatives are the most potent against Gram-positive strains, and the order of antibacterial activity is as follows: 2d > 3a > 8 > 11 > 2b > 2a.

Minimal Inhibitory/Fungicidal Concentrations (MIC) and (MFC)
Similarly, the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were determined against a panel of fungal strains in Table 3 and expressed in µM. Among the promising derivatives, only two derivatives, 3-(3-hydroxy-4methoxyphenyl) acrylamide derivative 2d and 3,5-dicyanopyridin-2-one derivative 3a, appeared to be sensitive against both C. albicans and F. oxysporum, while the other four derivatives, 2a, 2b, 8, and 11, were the most resistant. Finally, all the promising derivatives indicated moderate to good antifungal activity, especially 2-cyano acrylamide derivative 2d and another 3,5-dicyanopyridin-2-one derivative 3a, which appeared to be the two most active derivatives. Besides, all the newly designed compounds showed MFC/MIC ratios between 1 and 2, proving that these derivatives have fungicidal activity.
Finally, the most promising derivatives exhibited good activity against the MDRB strains used in this study, and according to the Clinical and Laboratory Standards Institute (CLSI) standards [66,67], these derivatives shown bactericidal potential. derivative from 2-cyanoacrylamide 2a. Alternatively, an electron-withdrawing group, such as the chloro group in the phenyl ring and cyano in 6-amino-3,5-dicyanopyridin-2one derivative 3a, enhanced the antibacterial activity, which was still less than acrylamide derivative 2d but higher than dimethylpyridine-2-one 8 and chromene-3-carboxamide derivative 11. Finally, the most promising derivatives exhibited good activity against the MDRB strains used in this study, and according to the Clinical and Laboratory Standards Institute (CLSI) standards [66,67], these derivatives shown bactericidal potential. creasing electron contributing groups (OH, OMe) in the aryl group rather than another derivative from 2-cyanoacrylamide 2a. Alternatively, an electron-withdrawing group, such as the chloro group in the phenyl ring and cyano in 6-amino-3,5-dicyanopyridin-2one derivative 3a, enhanced the antibacterial activity, which was still less than acrylamide derivative 2d but higher than dimethylpyridine-2-one 8 and chromene-3-carboxamide derivative 11. Finally, the most promising derivatives exhibited good activity against the MDRB strains used in this study, and according to the Clinical and Laboratory Standards Institute (CLSI) standards [66,67], these derivatives shown bactericidal potential.  N-carbamimidoylsulfamoyl derivatives  2a, 2b, 2d, 3a, 8, and 11 were evaluated to explore the antibacterial mechanism of hybrids series for their inhibitory activity against in vitro S. aureus DNA gyrase and E. coli DHFR   N-carbamimidoylsulfamoyl derivatives  2a, 2b, 2d, 3a, 8, and 11 were evaluated to explore the antibacterial mechanism of hybrids series for their inhibitory activity against in vitro S.  N-carbamimidoylsulfamoyl derivatives  2a, 2b, 2d, 3a, 8, and 11 were evaluated to explore the antibacterial mechanism of hybrids series for their inhibitory activity against in vitro S. aureus DNA gyrase and E. coli DHFR   N-carbamimidoylsulfamoyl derivatives  2a, 2b, 2d, 3a, 8, and 11 were evaluated to explore the antibacterial mechanism of hybrids series for their inhibitory activity against in vitro S. aureus DNA gyrase and E. coli DHFR   38, 9.38, and 8.32 µM), respectively. This activity might due to increasing electron contributing groups (OH, OMe) in the aryl group rather than another derivative from 2-cyanoacrylamide 2a. Alternatively, an electron-withdrawing group, such as the chloro group in the phenyl ring and cyano in 6-amino-3,5-dicyanopyridin-2-one derivative 3a, enhanced the antibacterial activity, which was still less than acrylamide derivative 2d but higher than dimethylpyridine-2-one 8 and chromene-3-carboxamide derivative 11.
Finally, the most promising derivatives exhibited good activity against the MDRB strains used in this study, and according to the Clinical and Laboratory Standards Institute (CLSI) standards [66,67], these derivatives shown bactericidal potential.

In Vitro S. aureus DNA Gyrase and E. coli DHFR Enzymatic Assay
The most promising derivatives containing N-carbamimidoylsulfamoyl derivatives 2a, 2b, 2d, 3a, 8, and 11 were evaluated to explore the antibacterial mechanism of hybrids series for their inhibitory activity against in vitro S. aureus DNA gyrase and E. coli DHFR (dihydrofolate reductase) assays, with ciprofloxacin and trimethoprim as the positive control, respectively.
As described in Table 6 and Figure 2, in vitro S. aureus DNA gyrase inhibitory capacity expressed by (IC 50 µM) was firstly assessed for the most promising guanidine derivatives and displayed moderate to potent inhibitory activity, with IC 50 values ranging between 18.17± 1.18 and 39.41 ± 1.15 µM as compared to the positive control ciprofloxacin (IC 50 = 26.32 ± 1.76 µM). Compound 2d, having an acrylamide derivative containing in position three aryl groups as 3-hydroxy-4-methoxyphenyl, has shown excellent inhibitory activity and found to be the most promising derivative (IC 50 = 18.17 ± 1.18 µM), while exchanging this aryl with other aryl groups as 4-chlorophenyl or 4-methoxyphenyl in 2a and 2b showed less activity (IC 50 = 23.87± 1.22 and 29.14 ± 1.93 µM). Notably, 6-amino-4-(4-chlorophenyl)-3,5-dicyanopyridin-2-one derivative 3a discovered the second inhibitory potency derivatives (IC 50 = 21.97± 1.35 µM), with a nearly 1.19-fold higher value than for ciprofloxacin. Furthermore, 4,6-dimethyl pyridine-2-one (containing a methyl group in position four instead of an aryl as well as a methyl group rather than the amino group in position six) derivative 8 and 2-imino-2H-chromene-3-carboxamide (containing a chromone moiety instead of a pyridine nucleus) derivative 11 exhibited the least inhibition towards S. aureus DNA gyrase in comparison to the other tested derivatives.  Finally, it can conclude that sulfaguanidine's hybridization enhances the antimicrobial activity (MICs and MBCs values), with a lower micromole, especially in hybrids with some acrylamide or pyridin-2-one derivatives and those having a specific substituent, as shown in both 2d and 3a. Moreover, the most promising derivatives with the guanidinesulfonyl moiety as a sidechain core indicated that they could inhibit bacterial growth through different mechanisms. Significantly, the antimicrobial activity depended on the nature of the design and the hybrid's core, and the aromatic moiety.

Immunomodulatory Activity
In Vitro Intracellular Killing Activities One of the broad methods used for determining immune disorders for patients in hospitals is the NBT test to measure their immune responses. The reduction in NBT dye provides information about the phagocytic and intracellular killing functions of neutrophils, which are important for microbiocidal activity [68]. The most promising derivatives (2a, 2b, 2d, 3a, 8, and 11) were evaluated against in vitro intracellular killing activities using a nitro blue tetrazolium (NBT) reduction assay and the obtained results expressed in percentage (%): increasing the percentage led to an improvement in the killing ability of the neutrophils used as innate immunity.
As represented in Table 7, intracellular killing activities displayed potency as an immunomodulatory agent with a percentage of 82.8 ± 0.37 to 142.4 ± 0.98. Remarkably, 3-(3hydroxy-4-methoxyphenyl) acrylamide derivative 2d showed the highest and most promising immunomodulatory action by killing activities (142.4 ± 0.98 %), and the activity dramatically decrease in order of 8 > 11 > 3a > 2b > 2a.  To explain the antimicrobial activity and investigate another mechanism, the most active derivatives were assayed against in vitro E. coli DHFR, with the IC 50  Finally, it can conclude that sulfaguanidine's hybridization enhances the antimicrobial activity (MICs and MBCs values), with a lower micromole, especially in hybrids with some acrylamide or pyridin-2-one derivatives and those having a specific substituent, as shown in both 2d and 3a. Moreover, the most promising derivatives with the guanidine-sulfonyl moiety as a sidechain core indicated that they could inhibit bacterial growth through different mechanisms. Significantly, the antimicrobial activity depended on the nature of the design and the hybrid's core, and the aromatic moiety.

Immunomodulatory Activity In Vitro Intracellular Killing Activities
One of the broad methods used for determining immune disorders for patients in hospitals is the NBT test to measure their immune responses. The reduction in NBT dye provides information about the phagocytic and intracellular killing functions of neutrophils, which are important for microbiocidal activity [68]. The most promising derivatives (2a, 2b,  2d, 3a, 8, and 11) were evaluated against in vitro intracellular killing activities using a nitro blue tetrazolium (NBT) reduction assay and the obtained results expressed in percentage (%): increasing the percentage led to an improvement in the killing ability of the neutrophils used as innate immunity.

In Vivo Immunomodulatory Investigation
Depending on NBT assay, our work extended to study the effect of the most active three derivatives (2d, 8, and 11) on the in vivo immunomodulatory investigations and determining their effect on the immune organs, the thymus and spleen. Both the spleen and thymus have important antibacterial and fungal immune organ responses, and any change in weight, and indexes can reflect the level of immune regulation [47,69]. Additionally, the relative spleen and thymus indexes are important indices for nonspecific immunity and potential immunomodulatory compounds that increase the spleen and thymus weight. This increase is due to immune cells' stimulation in the spleen and thymus [70][71][72].
The effect of 3-(3-hydroxy-4-methoxyphenyl) acrylamide derivative 2d, 4,6-dimethyl pyridine-2-one derivative 8, and 2-imino-2H-chromene-3-carboxamide derivative 11 on spleen and thymus weight and other indices are presented in Table 8, compared to the normal values and vitamin C as the positive control. Our study displayed that the promising three derivatives (2d, 8, and 11) exhibited a significant increase in spleen and thymus weight (p ≤ 0.05) compared to the normal control and nearly positive control. The 2cyano-acrylamide derivative 2d demonstrated an increase in spleen and thymus weights (250 ± 0.03, 15.56 ± 0.54 mg) compared to the normal (90 ± 0.02, 19.63 ± 0.35 mg) and nearly to vitamin C, with 260 ± 0.028 and 17.2 ± 0.31 mg, as well as slightly decreased spleen and thymus indices (0.018 ± 0.00 and 0.532 ± 0.06 mg/g) in comparison to the positive control (0.019 ± 0.00 and 0.633 ± 0.06 mg/g). Although, pyridine-2-one derivative 8 and 2-imino-2H-chromene-3-carboxamide derivative 11 showed good indexed results compared to the normal and positive control. Moreover, acrylamide derivative 2d showed the highest spleen and thymus indexed values among the tested compounds. Finally, it can be concluded that the three derivatives have immunomodulatory potential. Activation of immune cells as CD4 + and CD8 + T cells is one possible mechanism of a drug's immune stimulus activity. An increase in these cells is thought to be a good indicator of an active immune response to infections [73]. So, our work extended to study the effect of the most promising derivatives (2d, 8, and 11) on T lymphocyte subsets (CD4 + and CD8 + ) from peripheral blood, and determined the relative level in model mice that analyzed by flow cytometry after the period of treating.
As represented in Table 9, there is diversity in the percentage of CD4 + and CD8 + . 2-Cyano acrylamide derivative 2d, having an aryl group with electron-rich hydroxy and methoxy groups, showed the highest percentage, 79.61 ± 0.3. While, 2-imino-2H-chromene-3-carboxamide derivative 11 exhibited the lowest values, 75. 20 ± 0.97%, compared to the positive control, with a 76.74 ± 0.7% ratio for the T lymphocyte subsets CD4 + . Similarly, the percentage of CD8 + T lymphocytes for the tested derivative ranged between 18.44 ± 0.2 and 27.05 ± 0.5. The acrylamide derivative 2d displayed the highest percentage, 27.05 ± 0.5, and pyridine-2-one derivative 8 had the lowest value, 18.44 ± 0.2%, compared to the positive control (19.62 ± 0.21%). Finally, this increase and decrease in the percentage of the two T lymphocyte subsets suggested that the newly designed compounds 2d, 8, and 11 could enhance the stimulation and activation of CD4 + and CD8 + T lymphocytes, suggesting the T lymphocytes as a possible mechanism.

Prediction of Some Physicochemical, Pharmacokinetic, and Toxicity Properties
Physicochemical properties are a widely recognized tool for designing bioavailable drugs and many efforts to assess drug "developability" depend on the calculated and measured physicochemical parameters [74]. As described in Table 10, some of the molecular properties for the most promising derivatives, 2a, 2b, 2d, 3a, 8, and 11, were calculated using the Swiss ADME tool (http://swissadme.ch/index.php (accessed on 21 October 2020)), as described in previously reported methods [59], to evaluate the drug-likeness properties. All the most active derivatives were found to have a number of rotatable bonds, a hydrogen bond acceptor and donor, as well as molecular weight and lipophilicity properties (MLogP), with the criteria limitation of Lipinski's rule without any violation. These derivatives are suggested to have oral bioavailability properties. Similarly, three pharmacokinetic items were selected to be determined in our study, which are known as the blood-brain barrier (BBB) permeation, gastrointestinal absorption (GI), and permeability glycoprotein (P-gp). The designed compounds exhibited lower gastrointestinal absorption (GI) without blood-brain barrier (BBB) permeation and a non-substrate of the permeability glycoprotein (P-gp). Furthermore, toxicity prediction is very important in the drug design process, because it makes it safer, more timely, cost less money, and reduces the number of animals for experimental purposes [75]. In our study, the most active six derivatives, 2a, 2b, 2d,  3a, 8, and 11, were evaluated against some toxicity items, such as the AMES toxicity, carcinogens, and acute oral toxicity category, using the admetSAR online program (http: //lmmd.ecust.edu.cn/admetsar1/predict/ (accessed on 21 October 2020)). As represented in Table 11, the toxicity web tool admetSAR-predicted results, the promising derivatives revealed non-carcinogens and non-AMES toxicity, and all derivatives belonging to category III in acute oral toxicity observed LD 50 values greater than 500 mg/kg but less than 5000 mg/kg; also, trimethoprim showed an acute oral toxicity category IV that displayed LD 50 values greater than 5000 mg/kg. This web tool classified acute oral toxicity, depending on the LD 50 values, into four categories based on the criterion of the US EPA. Further, ciprofloxacin demonstrated AMES toxicity, and both the standard drugs exhibited noncarcinogenic chemicals. Table 11. In silico some pharmacokinetic properties and toxicity prediction of the most promising sulfaguanidine derivatives as well as standard drugs.

Molecular Docking Studies
According to the antimicrobial results, the most active compounds (2a, 2b, 2d, 3a, 8, and 11) were selected for molecular modeling simulation into DNA gyrase (PDB: 2XCT) and DHFR (PDB: IDLS) to investigate and understand the binding mode. As shown in Table 12, all docked compounds fitted well in the binding cavity of both the DNA gyrase and DHFR active sites and exhibited interaction, especially hydrogen bonding, arene-arene interactions, and arene-cation interactions. The molecular docking study was performed using Molecular Operating Environment software 10.2008 (MOE), Chemical Computing Group Inc., Montreal, Quebec, Canada.
As described in Table 12, the docking results observed a binding energy nearly or equal to the standard drugs. Firstly, analysis of the docking score energy (S) of the promising compounds inside the active site of DNA gyrase (PDB: 2XCT) exhibited that these derivatives had a comparable docking score energy, ranging between −15.98 and 21.67 Kcal/mol compared with ciprofloxacin (−9.87 Kcal/mol). The ciprofloxacin formed one hydrogen bond donor between the Thr580 and NH of the piperazine addition to the hydrogen bond acceptor between His1081 and oxygen of the carboxylate, with a bond length and strength of 2.6 • A (46%) and 2.3 • A (37%), respectively. The docking score of the most promising derivatives can be arranged in order 2d > 3a > 2a > 8 > 2b > 11 (see all figures of the docking study in the Supplementary Material).
For the guanidine derivative that has 3-(3-hydroxy-4-methoxyphenyl) acrylamide derivative 2d, the lowest docking score energy was observed (S = −21.67 Kcal/mol), and considered the best compound in the active binding site of S. aureus DNA gyrase. This compound can form four side-chain hydrogen bond acceptors, or between His1079, Lys1043, Ser1173, and Gln1267 with the oxygen of sulfonyl, oxygen of acetamide, the phenolic hydroxy group of aldehyde and oxygen of methoxy group, respectively, with a bond length ranging between 2.5 and 2.8 • A and a strength varying from 24 to 66%; also, the phenyl of the aldehyde derivative formed an arene-cation with Arg1092 (Figure 3a). Phe34 with pyrazine ring --* STD means standard; for DNA gyrase it is ciprofloxacin and for DHFR it is methotrexate, (-) arene-cation interaction, (--) arene-arene interaction, and docking score energy represented as S = (Kcal/mol). --* STD means standard; for DNA gyrase it is ciprofloxacin and for DHFR it is methotrexate, (-) arene-cation interaction, (--) arene-arene interaction, and docking score energy represented as S = (Kcal/mol).

Chemistry
Uncorrected melting points (MPs) of all the newly designed compounds were determined and recorded on a digital Gallen Kamp MFB-595 instrument using open capillaries and the recorded MPs were reported as such. A SHIMAZDU IR AFFINITY-I FTIR spectrophotometer was used for recording the IR spectra within the range of 400-4000 cm −1 . A Bruker 400 MHz spectrometer was used to assessment of the 1 H and 13 C signals in the Finally, the docking results displayed that these new hybrid derivatives 2a, 2b, 2d, 3a, 8, and 11 fitted well and interacted with the residues inside the active cavities in both DNA gyrase (PDB: 2XCT) and DHFR (PDB: IDLS), suggesting that they could potential be one of the antimicrobial agents (all the figures are in the Supplementary Material).

Chemistry
Uncorrected melting points (MPs) of all the newly designed compounds were determined and recorded on a digital Gallen Kamp MFB-595 instrument using open capillaries and the recorded MPs were reported as such. A SHIMAZDU IR AFFINITY-I FTIR spectrophotometer was used for recording the IR spectra within the range of 400-4000 cm −1 . A Bruker 400 MHz spectrometer was used to assessment of the 1 H and 13 C signals in the NMR spectra relative to (Me) 4 Si as an internal standard. Chemical shift values were reported in ppm units. The data were presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, and app = apparent), coupling constant(s) in Hertz (Hz), and integration. Mass spectra were recorded on a Thermo Scientific ISQLT mass spectrometer at the Regional Center for Mycology and Biotechnology, Al-Azhar University. Elemental analyses were carried out at Micro Analytical Unit, Cairo University, Cairo, Egypt. The progress of the reaction was monitored by TLC using UV detection. The information of the chemical used in this study was listed and provided in a Supplementary Material file. 2-Amino-N-(4-(carbamimidoylsulfamoyl) phenyl)-5-cyano-4-(4-methoxyphenyl)-6-oxo-1,6-dihydrop yridine-3-carboxamide (6) A mixture of 2-cyanoacrylamide derivative 2b (3.9 g, 0.01 mol) and cyanoacetamide (0.84 g, 0.01 mol) in DMF/ethanol (1:1), including three drops of piperidine, was refluxed for 5 h, then cooled and poured onto ice. The solid product formed was collected by filtration, dried, and recrystallized from ethanol. N-Carbamimidoyl-4-(4,6-diamino-3-cyano-2-oxopyridin-1(2H)-yl) benzenesulfonamide (7) A mixture of 2-cyanoacetamide derivative 1 (2.81 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) in DMF/ethanol (1:1) in the presence of three drops of piperidine was refluxed for 6 h, cooled, and poured onto ice. The solid product obtained was collected by filtration, dried and recrystallized from DMF.  A mixture of 4,6-diamino-3-cyano-2-oxo-pyridine derivative 8 (3.45 g, 0.01 mol), pchloro-benzaldehyde or p-anisaldehyde (0.01mol) in DMF/Ethanol (1:1) in the presence of three drops of piperidine was heated under reflux for 3 h, cooled, and poured onto ice. The solid product obtained was collected by filtration, dried and recrystallized from ethanol.

Determination of Inhibition Zones (mm)
The antimicrobial activity performed at the bacteriology laboratory, Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt. The inhibition zone (IZ) of the tested derivatives expressed as the diameter (mm) according to the agar plate diffusion method [36,40,61,76].

Determination the Minimal Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
According to Clinical and Laboratory Standards Institute (CLSI) guidelines, the minimal inhibition concentration (MIC) of the most promising derivatives, depending on preliminary screening of 2a, 2b, 2d, 3a, 8, and 11, were dissolved and loaded on paper disks with different concentrations according to reported methods [61,67]. The experiment was repeating for three-times. The MBC assay was determined by plating 10 µL of culture volume from the MIC assay onto TSB agar plates and colony formation was examined after 24 h at 37 • C and according to the reported method [65]. MBC is defined as the lowest compound concentration resulting in a ≥ 3-log reduction in the number of colony-forming units (CFU) [77].

Determination DNA Gyrase and DHFR Inhibitory Assay
The in-vitro enzymic assay for both S. aureus DNA gyrase and E.coli dihydrofolate reductase (DHFR) enzymes for the most promising derivatives with ciprofloxacin and trimethoprim as positive control was carried out in the confirmatory diagnostic unit, Vacsera, Egypt, according to the previously reported procedures and represented as IC 50 values [62,78,79].

Determination Immunomodulatory Activity
In vitro intracellular killing activities for the most promising six derivatives 2a, 2b, 2d, 3a, 8, and 11 were performed according to reported methods [65,68,80]. While in vivo, the immunomodulatory aspects involved (relative immune organs weight and indexes as well as determining the T lymphocytes subsets (CD4 + and CD8 + )) for the most active three derivatives, 2d, 8, and 11, were evaluated according to reported methods [47,69].

Molecular Docking Study
Docking simulations were performed using Molecular Operating Environment (MOE) software version 2008. 10. The most active compounds, 2a, 2b, 2d, 3a, 8, and 11, were drawn by chem draw 2014, and then exported to MOE. Energy minimization using the MMFF94x force field was calculated for each molecule. The crystal structure of both dihydrofolate reductase enzyme (DHFR), which co-crystallized with methotrexate (MTX) and S. aureus DNA gyrase in complex with ciprofloxacin, were downloaded from the protein data bank (PDB ID: 1DLS, 2XCT) [81,82]. For DNA gyrase, the protein structure prepared by selecting the G chain only for molecular docking and other repeated chain was deleted as well as the water molecules and the trigonal matcher placement used for this study. Further, DHFR (1DLS) that contains only one chain and the alpha trigonal matcher placement methods were selected for the previous protein and the London dG scoring function was used in the docking protocol. The docking process methodology was first validated by redocking the original ligand that co-crystalized inside the active site, with deviation (RMSD) values of 1.12 and 2.6 Å for DNA gyrase and DHFR, respectively. The docking of new compounds was performed according to standard protocols and previously reported works [62,65].

Ethics Statement for Both Animal Models and for Using Volunteer Blood Cells
All experimental protocols were approved and performed in compliance with the guide for the care and use of laboratory animals, published by the National Institutes of Health (USA), and performed according to Institutional guidelines. In addition, all volunteers gave their consent by signing a consent form before they participated.

Conclusions
In this study, eighteen compounds based on a sulfaguanidine moiety hybrid with acetamide, acrylamide, 2-oxopyridine, and chromene-3-carboxamide derivatives were synthetized and evaluated against several bacterial and fungal strains. ; therefore, these derivatives exhibited bactericidal potential. Additionally, the most promising 2a, 2b, 2d, 3a, 8, and 11 derivatives showed good interaction with DNA gyrase and DHFR enzymes with IC 50 values ranging between 18.17 and 39.41 µM and 4.33 and 17.64 µM, respectively, as compared to ciprofloxacin (26.32 ± 1.76 µM) and trimethoprim (5.16 ± 0.12 µM). Among them, three derivatives, 2d, 3a and 2a, demonstrated IC 50 values as potent, or nearly so, as the positive controls for both enzymes. The in vitro intracellular killing activities represented potency as an immunomodulatory agent with a percentage from 82.8 ± 0.37 to 142.4 ± 0.98. Similarly, the most active three derivative, 2d, 8, and 11, exhibited a significant increase in spleen and thymus weight (p ≤ 0.05) and enhance the stimulation of the CD4 + and CD8 + T lymphocytes. Finally, it is interesting to mention that the molecular docking study matches with the experimental results. Besides, these derivatives obey Lipinski's rule and revealed non-carcinogens, non-AMES toxicity, and all derivatives belong to category III regarding acute oral toxicity.