N-(1,3,4-Oxadiazol-2-yl)Benzamides as Antibacterial Agents against Neisseria gonorrhoeae

The Centers for Disease Control and Prevention (CDC) recognizes Neisseria gonorrhoeae as an urgent-threat Gram-negative bacterial pathogen. Additionally, resistance to frontline treatment (dual therapy with azithromycin and ceftriaxone) has led to the emergence of multidrug-resistant N. gonorrhoeae, which has caused a global health crisis. The drug pipeline for N. gonorrhoeae has been severely lacking as new antibacterial agents have not been approved by the FDA in the last twenty years. Thus, there is a need for new chemical entities active against drug-resistant N. gonorrhoeae. Trifluoromethylsulfonyl (SO2CF3), trifluoromethylthio (SCF3), and pentafluorosulfanyl (SF5) containing N-(1,3,4-oxadiazol-2-yl)benzamides are novel compounds with potent activities against Gram-positive bacterial pathogens. Here, we report the discovery of new N-(1,3,4-oxadiazol-2-yl)benzamides (HSGN-237 and -238) with highly potent activity against N. gonorrhoeae. Additionally, these new compounds were shown to have activity against clinically important Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and Listeria monocytogenes (minimum inhibitory concentrations (MICs) as low as 0.25 µg/mL). Both compounds were highly tolerable to human cell lines. Moreover, HSGN-238 showed an outstanding ability to permeate across the gastrointestinal tract, indicating it would have a high systemic absorption if used as an anti-gonococcal therapeutic.


Introduction
Drug-resistant bacterial infections have become a serious global threat. Neisseria gonorrhoeae is a Gram-negative bacterial pathogen which causes gonorrhea, a sexually transmitted infection (STI) [1]. N. gonorrhoeae infects a variety of mucosal surfaces (i.e., the urethra, endocervix, pharynx, and rectum) [2] and, if left untreated, can cause drastic complications, such as pelvic inflammatory disease, ectopic pregnancy, and increased susceptibility to HIV infections [3]. The Centers for Disease Control and Prevention (CDC) considers N. gonorrhoeae an urgent threat, as it accounts for 550,000 infections per year and $133.4 million dollars in medical costs in the United States alone [4]. Globally, N. gonorrhoeae is also devastating. The World Health Organization (WHO) listed N. gonorrhoeae as a priority 2 (high) pathogen, as it is has caused 87 million new cases as well as an estimated total treatment cost of $5 billion dollars [5][6][7][8].
Efforts to develop novel antibiotics against urgent threat pathogens, especially N. gonorrhoeae, have intensified [9]. For instance, N. gonorrhoeae is now resistant to former frontline therapies such as penicillin, fluoroquinolones, and cefixime, which are now deemed ineffective as treatment options [10]. This increased resistance rate prompted a global health scare, leading the CDC to recommend treating N. gonorrhoeae with dual therapy involving ceftriaxone and azithromycin [1,11]. Yet, resistance to this dual therapy has been reported, leading to the rise of multidrug-resistant N. gonorrhoeae (commonly referred to as super gonorrhea) [2]. To make matters worse, no new classes of antibiotics to treat N. gonorrhoeae have been FDA-approved over the last two decades, warranting the public health concern that once easily treated gonorrhea infections will soon become deadly [12][13][14]. Therefore, the rise in multidrug-resistant N. gonorrhoeae infections necessitates intense research efforts to identify and develop new antibiotics.

Synthesis and Antigonococcal Activity of N-(1,3,4-oxadiazol-2-yl)benzamides
We previously reported that trifluoromethylsulfonyl (SO 2 CF 3 ), trifluoromethylthio (SCF 3 ), and pentafluorosulfanyl (SF 5 ) containing N-(1,3,4-oxadiazol-2-yl)benzamides (compounds 6, 12, and 13, respectively) were potent against a panel of drug-resistant Gram-positive bacteria [17]. We wondered if these compounds would be active against N. gonorrhoeae and discovered that compounds 6, 12, and 13 have quite potent activity against N. gonorrhoeae strain 181, with minimum inhibitory concentrations (MICs) of 0.5, 0.06, and 0.06 µg/mL, respectively (see Table 1). While all three compounds have favorable CLogP values, they also contain an unsubstituted thiophene moiety ( Figure 1) which can cause toxicity. For example, the cytochrome P450-mediated oxidation of thiophene moeities can lead to reactive metabolites such as thiophene epoxides [18], thiophene-S oxides [18,19], and sulphenic acids [20], which can react with nucleophiles such as glutathione and/or water [21]. However, since Compounds 6, 12, and 13 have shown excellent activities against N. gonorrhoeae as well as adequate CLogP values, we desired to further optimize these compounds via the synthesis of new analogs. We proceeded to use computational methods to guide our synthetic strategy. We began to substitute the benzamide ring with the trifluoromethoxy (OCF 3 ) group due to its importance in medicinal chemisty [22,23]. For instance, it was reported that the electronegativity of the OCF 3 group allows for enhanced in vivo uptake and transport in biological systems [22]. Thus, utilizing this strategy has led to the synthesis of HSGN-235, which contained a fluoro atom ortho to the OCF 3 group as well as a trifluoromethyl phenyl. Yet, HSGN-235 was found to contain a much larger CLogP value compared to previously synthesized analogs ( Figure 1). Since LogP shows a positive correlation between low aqueous solubility and compromising bioavailability (an extremely important attribute when creating antibacterial agents against N. gonorrhoeae) [24], we replaced the thiophene moiety with a substituted thiophene or phenyl group as unsubstituted thiophene could be a toxicophore, as mentioned above.
Considering that the addition of halogens to compounds has been shown to improve drug properties and metabolic stability [25][26][27][28][29], our new analogs were made up of compounds with halogen substitutions to a phenyl ring ( Figure 1).
The synthesis of these compounds started with a substituted aryl aldehyde, followed by the addition of semicarbazide and sodium acetate to give the corresponding semicarbazone. Then, using bromine and sodium acetate, the semicarbazone was converted into the subsequent aryl 1,3,4-oxadizol-2-amine. Amide coupling between the aryl 1,3,4-oxadiazol-2-amine and 4-trifluoromethoxy benzoic acid using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent gave the desired N-(1,3,4-oxadiazol-2-yl)benzamides (Scheme 1).  The synthesis of these compounds started with a substituted aryl aldehyde, followed by the addition of semicarbazide and sodium acetate to give the corresponding semicarbazone. Then, using bromine and sodium acetate, the semicarbazone was converted into the subsequent aryl 1,3,4-oxadizol-2-amine. Amide coupling between the aryl 1,3,4-oxadiazol-2-amine and 4-trifluoromethoxy benzoic acid using benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent gave the desired N-(1,3,4-oxadiazol-2-yl)benzamides (Scheme 1).  The synthesis of these compounds started with a substituted aryl aldehyde, followed by the addition of semicarbazide and sodium acetate to give the corresponding semicarbazone. Then, using bromine and sodium acetate, the semicarbazone was converted into the subsequent aryl 1,3,4-oxadizol-2-amine. Amide coupling between the aryl 1,3,4-oxadiazol-2-amine and 4-trifluoromethoxy benzoic acid using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent gave the desired N-(1,3,4-oxadiazol-2-yl)benzamides (Scheme 1).  (Table 1). Interestingly, the substitution of the 4-trifluoromethoxy phenyl group with a fluorine, as well as the substitution of the thiophene moiety with trifluoromethylphenyl (HSGN-235) only had moderate activity when tested against N. gonorrhoeae strain 181 (Table 1). Since both HSGN-237 and HSGN-238 contained aromatic rings bearing a halogen atom, we speculate that the loss of activity for HSGN-235 is due to the addition of the fluorine atom ortho to the trifluoromethoxy group (see Figure 1 and Table 1 for comparisons). Table 1. MICs (µg/mL) of the previously reported analogs (compounds 6, 12, and 13) and the new compounds (HSGN-235, -237, and -238) against N. gonorrhoeae strain 181. The experiment was repeated 3 independent times and the same MIC values were obtained. Compounds tested are in bold while control drugs are in regular script. After the initial screening against N. gonorrhoeae 181, the anti-gonococcal activity of HSGN-235, -237, and -238 was explored against a panel drug-resistant pathogenic N. gonorrhoeae strains, including one WHO reference strain (N. gonorrhoeae WHO L) which has a well-characterized antibiogram and phenotypic and genetic markers [30]. As depicted in Table 2, HSGN-237 and -238 exhibited potent activity against the tested strains, with inhibitions of their growth at concentrations ranging from 0.03 to 0.125 µg/mL. Both were superior to azithromycin and tetracycline against the tested isolates. On the other hand, HSGN 235 inhibited the growth of the tested strains at concentrations ranging from 1 to 2 µg/mL. Interestingly, the minimum bactericidal concentration (MBC) values of HSGN235, -237, and -238 were the same as or one-fold higher than their corresponding MIC values, indicating that the compounds exhibit bactericidal activity against the tested N. gonorrhoeae strains.

Antibacterial Activity of N-(1,3,4-oxadiazol-2-yl)benzamides against Other Bacterial Species
While the focus of these new N-(1,3,4-oxadiazol-2-yl)benzamides is towards N. gonorrhoeae, we proceeded to test their activity against other Gram-positive and Gram-negative pathogens. Intriguingly, HSGN-235, HSGN-237, and HSGN-238 had potent activity against the tested Gram-positive bacterial pathogens. For instance, all three compounds had potent activity against the staphylococcal strains, with MICs ranging from 0.25 to 1 µg/mL (Table 3). Furthermore, HSGN-235, HSGN-237, and HSGN-238 maintained potent activity against clinically relevant Gram-positive bacterial pathogens such as vancomycin-resistant enterococci (VRE) and Listeria monocytogenes (Table 3). Additionally, we moved to test if HSGN-235, HSGN-237, and HSGN-238 were active against other Gram-negative bacterial pathogens. These compounds were found to be inactive against E. coli BW25113. This lack of activity against Gram-negative bacteria appears to be due to HSGN-235, HSGN-237, and HSGN-238 being a substrate for efflux. This can be seen by the shift in the MICs observed for HSGN-235, HSGN-237, and HSGN- HSGN-235, HSGN-237, and HSGN-238 improves to 4, 0.25, and 0.06 µg/mL, respectively; Table 3). A similar result was observed with linezolid, an antibiotic known to be a substrate for the AcrAB-TolC efflux pump in Gram-negative bacteria, as reported in previous reports [31,32]. Interestingly, HSGN-235, HSGN-237, and HSGN-238 appeared to be bacteriostatic agents, as their MBCs were more than three-fold higher than their corresponding MICs against the tested bacterial strains (Table 3).  An increase in MIC due to antibiotics being highly protein-bound has been documented in several classes of antibiotics [33][34][35]. Therefore, we evaluated our compounds' activity against N. gonorrhoeae in the presence of different concentrations of fetal bovine serum (FBS). As presented in Table S2, the activity of HSGN-235, -237, and -238 was reduced in the presence of FBS. The addition of 1%, 5%, and 10% FBS to the media increased the MIC of HSGN-237 to 0.125, 1, and 4 µg/mL, respectively (see Table S2). Similarly, the MIC of HSGN-238 also changed to 0.25 and 1 µg/mL (still considered good potency) in the presence of 1% and 5% FBS, respectively, but stayed at 1 µg/mL with the addition of 10% FBS (Table S2). The MIC of HSGN-235 did not change in the presence of 1% FBS, but increased to 4 and 8 µg/mL in the presence of 5% and 10% FBS, respectively (Table S2).
Hydrophobic antibiotics such as antimicrobial peptides and lipopeptides have been found to show an increase in MIC upon the addition of serum to media [36,37]. We predict that the hydrophobicity of these N-(1,3,4-oxadiazol-2-yl)benzamides contributes to the rise in MIC in the presence of FBS. Future studies will attempt to develop analogs thereof with fewer protein-binding properties.

N-(1,3,4-Oxadiazol-2-yl)benzamides Are Highly Tolerable to Human Cell Lines
Prokaryotic cell selectivity is highly important for an antibiotic candidate. Therefore, since HSGN-237 and -238 were found to be the most potent analogs against N. gonorrhoeae, they were assessed for toxicity to mammalian cells over 24-and 48-h periods (Figure 2A,B). Both compounds showed excellent safety profiles against human colorectal cells (Caco-2). For instance, HSGN-237 was non-toxic at concentrations higher than 64 µg/mL, which is 512-times higher than the compound's corresponding MIC values against N. gonorrhoeae (Figure 2A,B). Additionally, HSGN-238 was non-toxic at concentrations of up to 16 µg/mL which is 128 times higher than the compound's corresponding MIC values against N. gonorrhoeae (Figure 2A,B). activity against N. gonorrhoeae in the presence of different concentrations of fetal bovine serum (FBS). As presented in Table S2, the activity of HSGN-235, -237, and -238 was reduced in the presence of FBS. The addition of 1%, 5%, and 10% FBS to the media increased the MIC of HSGN-237 to 0.125, 1, and 4 µg/mL, respectively (see Table S2). Similarly, the MIC of HSGN-238 also changed to 0.25 and 1 µg/mL (still considered good potency) in the presence of 1% and 5% FBS, respectively, but stayed at 1 µg/mL with the addition of 10% FBS (Table S2). The MIC of HSGN-235 did not change in the presence of 1% FBS, but increased to 4 and 8 µg/mL in the presence of 5% and 10% FBS, respectively (Table S2). Hydrophobic antibiotics such as antimicrobial peptides and lipopeptides have been found to show an increase in MIC upon the addition of serum to media [36,37]. We predict that the hydrophobicity of these N-(1,3,4-oxadiazol-2-yl)benzamides contributes to the rise in MIC in the presence of FBS. Future studies will attempt to develop analogs thereof with fewer protein-binding properties.

N-(1,3,4-oxadiazol-2-yl)benzamides are highly tolerable to human cell lines:
Prokaryotic cell selectivity is highly important for an antibiotic candidate. Therefore, since HSGN-237 and -238 were found to be the most potent analogs against N. gonorrhoeae, they were assessed for toxicity to mammalian cells over 24-and 48-hour periods (Figure 2A ,B). Both compounds showed excellent safety profiles against human colorectal cells (Caco-2) . For instance, HSGN-237 was non-toxic at concentrations higher than 64 µg/mL, which is 512-times higher than the compound's corresponding MIC values against N. gonorrhoeae (Figure 2A,B). Additionally, HSGN-238 was non-toxic at concentrations of up to 16 µg/mL which is 128 times higher than the compound's corresponding MIC values against N. gonorrhoeae (Figure 2A,B).

HSGN-238 Demonstrates High Intestinal Permeability:
Oral bioavailability is a highly important consideration when developing bioactive molecules as therapeutic agents [38]. A critical factor of oral bioavailability is human intestinal absorption. The Caco-2 bidirectional permeability assay is the most widely used in vitro model for predicting if a bioactive molecule can have adequate systemic absorption [39,40]. Thus, we selected HSGN-238 to act as a model to analyze the drug-like properties of newly synthesized N-(1,3,4-oxadiazol-2-yl)benzamides. The assay demonstrated that HSGN-238 showed an outstanding ability to permeate across Caco-2 bilayers (apparent permeability, Papp = 82.3 × 10 −6 cm s −1 from the apical to basolateral and Papp = 32.9 × 10 −6 cm s −1 from the basolateral to apical; see Table 4). This permeability is comparable to propranolol (Papp = 37.2 × 10 −6 cm s −1 from the apical to basolateral and Papp = 22.7 × 10 −6 cm s −1 from the basolateral to apical (Table 4)), a drug that is known to have a high

HSGN-238 Demonstrates High Intestinal Permeability:
Oral bioavailability is a highly important consideration when developing bioactive molecules as therapeutic agents [38]. A critical factor of oral bioavailability is human intestinal absorption. The Caco-2 bidirectional permeability assay is the most widely used in vitro model for predicting if a bioactive molecule can have adequate systemic absorption [39,40]. Thus, we selected HSGN-238 to act as a model to analyze the drug-like properties of newly synthesized N-(1,3,4-oxadiazol-2-yl)benzamides. The assay demonstrated that HSGN-238 showed an outstanding ability to permeate across Caco-2 bilayers (apparent permeability, P app = 82.3 × 10 −6 cm s −1 from the apical to basolateral and P app = 32.9 × 10 −6 cm s −1 from the basolateral to apical; see Table 4). This permeability is comparable to propranolol (P app = 37.2 × 10 −6 cm s −1 from the apical to basolateral and P app = 22.7 × 10 −6 cm s −1 from the basolateral to apical (Table 4)), a drug that is known to have a high permeability across Caco-2 bilayers. Ranitidine was used as a low-permeability control, as its P app = 0.5 × 10 −6 cm s −1 from the apical to basolateral and P app = 1.3 × 10 −6 cm s −1 from the basolateral to apical. Therefore, the Caco-2 permeability results indicate that HSGN-238 has a high potential to be strongly absorbed after being administered orally.

Chemistry
General considerations: All reagents and solvents were purchased from commercial sources. The 1 H, 13 C, and 19 F NMR spectra were acquired in DMSO-d 6 as solvent using a 500 MHz spectrometer with Me 4 Si as an internal standard. Chemical shifts are reported in parts per million (δ) and were calibrated using residual undeuterated solvent as an internal reference. Data for 1 H NMR spectra are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, or combinations thereof. High resolution mass spectra (HRMS) were obtained using electron spray ionization (ESI) technique and as TOF mass analyzer. New compounds were characterized by 1 H NMR, 13 C NMR, 19 F NMR, and HRMS data.

Bacterial Strains, Media, Reagents and Cell Lines
Neisseria gonorrhoeae clinical isolates (Table S1)

Determination of the MICs of Compounds and Control Drugs against N. gonorrhoeae Strains
The MICs of the tested compounds and control drugs; azithromycin, and tetracycline were determined using the broth microdilution as described previously [42][43][44]. Briefly, bacteria were grown overnight on chocolate agar II at 37 • C in the presence of 5% CO 2 . Afterwards, a bacterial suspension equivalent to 1.0 McFarland standard was prepared and diluted in brucella broth supplemented with yeast extract, neopeptone, hematin, pyridoxal, and NAD. Test agents were added in the first row of the 96-well plates and serially diluted along the plates. Plates were then incubated at 37 • C in the presence of 5% CO 2 for 24 h. The MICs reported in Table 1 are the minimum concentrations of the compounds and control drugs that could completely inhibit the visual growth of bacteria. The minimum bactericidal concentration (MBC) of these drugs was tested by plating 4 µL from wells with no growth onto chocolate agar II plates. Plates were then incubated at 37 • C in the presence of 5% CO 2 for 24 h. The MBC was categorized as the lowest concentration that reduced bacterial growth by 99.9% [45][46][47].

Determination of the MICs and MBCs of Compounds and Control Drugs against Clinically Important Gram-Positive and Gram-Negative Bacteria
The minimum inhibitory concentrations (MICs) of the tested compounds and control drugs, linezolid, vancomycin, and gentamicin, were determined using the broth microdilution method according to the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI) [48] against clinically relevant bacterial (Staphylococcus aureus, MRSA, Escherichia coli, Enterococcus faecalis and Enterococcus faecium strains. S. aureus, MRSA, E. coli, Enterococcus faecalis and Enterococcus faecium were grown aerobically overnight on tryptone soy agar (TSA) plates at 37 • C. Afterwards, a bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted in cation-adjusted Mueller-Hinton broth (CAMHB) (for S. aureus, MRSA, and E. coli) to achieve a bacterial concentration of about 5 × 10 5 CFU/mL. Enterococcus faecalis and Enterococcus faecium 0.5 McFarland standard solution was diluted in tryptone soy broth (TSB) to achieve a bacterial concentration of about 5 × 10 5 CFU/mL. Compounds and control drugs were added in the first row of 96-well plates and serially diluted with the corresponding media containing bacteria. Plates were then incubated as previously described. MICs reported in Table 2 are the minimum concentration of the compounds and control drugs that could completely inhibit the visual growth of bacteria. The minimum bactericidal concentration (MBC) was tested by spotting 4 µL from wells with no growth onto TSA plates. Plates were incubated at 37 • C for at least 18 h before recording the MBC. The MBC was categorized as the lowest concentration that reduced bacterial growth by 99.9% [45][46][47].

Caco-2 Permeability Assay
Assay and data analysis were performed by Eurofins Panlabs (MO, USA) according to a previously reported protocol [51,52]. The apparent permeability coefficient (P app ) of the tested agents was calculated using the equation below: where V R is the volume of the receiver chamber. C R,end is the concentration of the test compound in the receiver chamber at the end time point, ∆t is the incubation time, and A is the surface area of the cell monolayer. C D,mid is the calculated mid-point concentration of the test compound in the donor side, which is the mean value of the donor concentration at 0 minutes and the donor concentration at the end time point. C R,mid is the mid-point concentration of the test compound in the receiver side, which is one half of the receiver concentration at the end time point. Concentrations of the test compound were expressed as peak areas of the test compound.