Bacterial carbonic anhydrase inhibitors with improved pharmacokinetics and efficacy

Abstract

The present application generally relates to compounds which are inhibitors of Neisseria gonorrhoeae. Compounds, pharmaceutical compositions and use thereof are within the scope of this disclosure.

Description

Bacterial Carbonic Anhydrase Inhibitors with Improved Pharmacokinetics and Efficacy Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 669,877, which was filed July 11, 2024, and is hereby incorporated by reference in its entirety. Government Support Clause

[0002] This invention was made with government support under AI153264 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0003] The present disclosure generally relates to therapeutics for the treatment of N. gonorrhoeae infection. Compounds, pharmaceutical formulations and methods of use and treatment are disclosed. Background

[0004] Drug-resistant gonorrhea is caused by the bacterial pathogen Neisseria gonorrhoeae, for which there is no recommended oral treatment. We have demonstrated that the FDA-approved human carbonic anhydrase inhibitor ethoxzolamide potently inhibits N. gonorrhoeae; however,is not effective at reducing N. gonorrhoeae bioburden in a mouse model. Thus, we sought to optimize the pharmacokinetic properties of the ethoxzolamide scaffold. These efforts resulted in analogs with improved activity against N. gonorrhoeae, increased metabolic stability in mouse liver microsomes, and improved Caco-2 permeability compared to ethoxzolamide. Improvement in these properties resulted in increased plasma exposure in vivo after oral dosing. Top compounds were investigated for in vivo efficacy in a vaginal mouse model of gonococcal genital tract infection, and they significantly decreased the gonococcal burden compared to vehicle and ethoxzolamide controls. Altogether, results from this study provide evidence that ethoxzolamide-based compounds have the potential to be effective oral therapeutics against gonococcal infection.

[0005] Gonorrhea, the venereal disease caused by the Gram-negative pathogen Neisseria gonorrhoeae, is a serious public health threat that is estimated to infect more than 80 million people worldwide every year.1–3Gonococcal infection often leads to inflammation of the cervix in women and of the urethra in men, causing irritation and pain. If left untreated, gonorrhea can spread throughout the body to the uterus, fallopian tubes, and bloodstream and can cause debilitating health complications including pelvic inflammatory disease, ectopic pregnancy, infertility, and skin / joint pain.1,3,4According to the Centers for Disease Control and Prevention (CDC), the incidence rates for gonococcal infection in the United States have increased 117.4% among men and 49.6% among women over the past decade, with 648,056 cases being reported in 2022 in the U.S. alone.5This steady increase in infections is especially threatening because N. gonorrhoeae has a long history of acquiring resistance to antibiotics. Since the 1940s, a multitude of drugs including penicillin, tetracycline, quinolines, and third- generation cephalosporins have become ineffective against the N. gonorrhoeae.6–8This pattern of drugresistance in gonorrhea is still prevalent today, as approximately 50% of infections in the U.S. are resistant or have increased minimum inhibitory concentrations (MICs) against at least one antibiotic.5In 2012, the CDC announced azithromycin and ceftriaxone to be used as a combination therapy, yet in 2020 the CDC updated treatment guidelines due to increasing resistance to azithromycin, removing it from the treatment regimen.9The current recommended treatment is now a single intramuscular dose of ceftriaxone.9However, ceftriaxone resistance is increasing globally with the emergence of the penA60 allele,10–12leading some to suggest there may be a future with untreatable gonorrhea.7The critical need for orally active anti-gonococcal agents has spurred pharma to invest in development of new antibiotics, as evidenced by the recent successes of gepotidacin and zoliflodacin; two novel topoisomerase inhibitors that have shown efficacy in uncomplicated urogenital gonorrhea in Phase II and Phase III clinical trials, respectively.13,14Even with these advancements, there is still an urgent need to fill the pipeline with new orally active anti-gonococcal therapeutics.

[0006] To address this need, our strategy has focused on developing anti-N. gonorrhoeae agents targeting the N. gonorrhoeae carbonic anhydrases (NgCA). Carbonic anhydrases are ubiquitous metalloenzymes that catalyze the reaction between carbon dioxide and water to produce bicarbonate and a proton, thus playing an important role in physiological processes including intracellular pH homeostasis and the biosynthesis of building blocks used in the tricarboxylic acid cycle.15–17There are eight families of CAs that are genetically distinct and vary in their metal ions and tertiary / quaternary structures.18–23N. gonorrhoeae encodes for three of these classes (α-, β-, and γ-CAs15,24,25) while humans have sixteen isoforms of only α-CAs.26The α- NgCA isoform is the most well-studied of the NgCAs. First cloned in 1997,27α-NgCA has since been identified as an essential enzyme24and has been characterized both enzymatically25andstructurally.28,29Less is known about the β- and γ-NgCAs, though a recentstudy has shown thatN. gonorrhoeae growth without the use of supplemental CO2is dependent on a single-residuesubstitution in β-NgCA.15Two FDA-approved human carbonic anhydrase (hCA) inhibitors, acetazolamide (AZM) and ethoxzolamide (EZM) (FIG.1), were originally investigated as NgCA inhibitors in 1967.30Traditionally used as diuretics and to treat glaucoma,31–34AZM and EZM were shown to reduce bacterial growth and have activity against NgCAs.30Recently, our group has focused on repurposing the AZM scaffold, reporting analogs with improved antimicrobial properties against N. gonorrhoeae.35This led our team to evaluate AZM for efficacy in a vaginal mouse model for N. gonorrhoeae infection in which it demonstrated the ability to reduce N. gonorrhoeae bioburden.36During our efforts we observed that EZM is more potent than AZM against N. gonorrhoeae and that this increased anti-gonococcal potency might be partially attributed to EZM’s greater molecule accumulation inside the bacterial cells compared to AZM.37We evaluated EZM in the same vaginal mouse model for N. gonorrhoeae infection and surprisingly observed no efficacy compared to vehicle-treated control mice (unpublished data). These results motivated our team to investigate the possible reasons for the lack of EZM efficacy in vivo and how properties of EZM may be modified to yield oral efficacy in the mouse model. Brief Summary

[0007] The present disclosure provides for compounds having the general formula (I)

[0009] where X is one or more substituents selected from halo and O-R, wherein R is H, CH3, C1-C4alkyl substituted with one or more deuterium atoms, or C1-C4alkyl substituted with one or more halo atoms and n is 0-3. In some embodiments n is 1.

[0010] In certain embodiments, compound of claim 1 selected from the group consisting of: ,,some .anti-organism activity. In some embodiments, the anti- organism activity is against Neisseria gonorrhoeae.

[0014] The present disclosure provides for a pharmaceutical composition comprising a compound, or salt thereof, selected from the group consisting of one or more of the aforementioned compositions and a pharmaceutically acceptable carrier, excipient, or diluent.

[0015] In some embodiments, the pharmaceutical composition has anti-organism activity. In certain embodiments, the organism is Neisseria gonorrhoeae.

[0016] Other aspects of the disclosure concern methods of inhibiting a pathogen comprising contacting a target pathogen with a compound a pharmaceutical composition described herein. In some embodiments, the pathogen is Neisseria gonorrhoeae.

[0017] In certain embodiments, the method of inhibiting Neisseria gonorrhoeae comprises administering a pharmaceutical composition described herein to a patent in need thereof. In some embodiments, the pharmaceutical composition comprises: .pharmaceutical composition is administered orally. Brief Description of the Drawings

[0020] A better understanding of the present disclosure will be obtained upon reference to the following description in conjunction with the accompanying drawings.

[0021] FIG.1 presents chemical structures and MICs against N. gonorrhoeae FA1090 strain for AZM and EZM and a proposed metabolic route for EZM to form the primary metabolite 1 and the glucuronide EZM-Glu.

[0022] FIG.2 presents total molecule concentration in plasma (Cp) and unbound molecule concentration in plasma (Cp,u) for EZM (A)and 1 (B) in mice (n = 3) after 10 mg / kg p.o. dose plotted over time. Cp,u calculated using mouse plasma protein binding for EZM and 1 is presented in Table 1. MICs for each molecule is shown by dotted black line. Error bars represent standard deviation (S.D.) for each time point.

[0023] FIG.3 shows sSites of metabolism predicted by P450 Sites of Metabolism Tool (Schrödinger, LLC). Highlighted carbons represent potential sites of CYP oxidation.

[0024] FIG.4 illustrates in vitro mouse liver microsome stability (FIG.4A) and Caco-2 permeability (Papp) (FIG.4B) for EZM and analogs. A = apical and B = basolateral sides of the Caco-2 monolayer.

[0025] FIG.5 shows total molecule concentration in plasma (Cp) and unbound molecule concentration in plasma (Cp,u) for 2 (A), 7 (B), and 9 (C) in mice (n = 3 for each timepoint) after 10 mg / kg p.o. dose plotted over time. Cp,u was calculated using mouse plasma protein binding for each molecule presented in Table 5. MICs for each molecule versus N. gonorrhoeae FA1090 shown by dotted black line. Error bars represent standard deviation (S.D.).

[0026] FIG.6 illustrates cell viability for EZM and lead analogs 2 and 9 tested at 32 μg / mL, 64 μg / mL, and 128 μg / mL against Vero cells (top) and ME-180 cells (bottom) measured via the MTS assay. Results are compared to DMSO (negative control); absorbance values are calculated as an average of three replicates and standard deviation values as represented by error bars. Data were analyzed via a two-way ANOVA with post hoc Dunnett’s test for multiple comparisons.

[0027] FIG.7 presents time-kill kinetics of 2, 9, and EZM tested in triplicate at 5× MIC against N. gonorrhoeae FA1090. DMSO (vehicle) served as a negative control; azithromycin (AZI) served as positive control antibiotic. Error bars represent standard deviation values for each test agent studied. The data were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons. An asterisk (*) indicates statistically significant difference (P<0.05) between treatment with drugs / compounds compared to DMSO treatment.

[0028] FIG.8 presents Log10CFU / mL of N. gonorrhoeae (average ± standard deviation) after treatment with a either ethoxzolamide (EZM), 2 or 9 (15 mg / kg, p.o., b.i.d.) orally for three consecutive days. CEF (15 mg / kg i.p. single dose) was included as positive control while the vehicle served as a negative control. The data of N. gonorrhoeae CFU counts in the vaginalsamples were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons (P<0.05). Detailed Description

[0029] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

[0030] For an initial insight about the possible reason for the differences in in vivo activity, we found that both molecules differ in their pharmacokinetic profiles. After an oral dose, AZM is excreted unmetabolized through renal clearance.38–41Alternatively, EZM also undergoes renal clearance after oral dose but only approximately 40% is isolated in the urine unmetabolized.42Further studies in rabbits have identified that EZM is subjected to both Phase I metabolism, undergoing oxidative O-dealkylation of the ethoxy substituent to form the phenol primary metabolite (1, FIG.1), and Phase II metabolism, in which the newly unmasked phenol is converted into a glucuronide conjugate (EZM-Glu).34Glucuronide-conjugated molecules often result in improved water solubility and drug clearance,43and drugs are often times no longer active against their targets once glucuronidated.44

[0031] Additionally, through previous structure-activity relationship (SAR) data, we have observed that the addition of polar functional groups eliminates the anti-gonococcal potency of the AZM scaffold.35We therefore hypothesize that the lack of in vivo efficacy for EZM is due to the metabolic modification, glucuronidation, and clearance of the compound. We sought to address EZM’s lack of efficacy by generating derivatives with increased metabolic stability thatmay improve the in vivo pharmacokinetic profiles leading to orally efficacious anti-gonococcal agents in the mouse model.

[0032] The main aim of this report is to optimize the pharmacokinetic properties of the EZM scaffold. We established SAR analysis for the scaffold and synthesized analogs were initially screened against three N. gonorrhoeae strains and CA inhibition was evaluated. The anti- gonococcal activity of prioritized EZM analogs was investigated against a panel of multidrug- resistant N. gonorrhoeae strains. In vitro ADME, cytotoxicity, killing kinetics, frequency of mutation and in vivo pharmacokinetics were assessed for the prioritized analogs. Finally, the in vivo efficacy of analogs was evaluated in the mouse model for gonococcal genital tract infection.

[0033] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[0034] The term “r.t.” indicates room temperature.

[0035] In the present disclosure the term “about” can allow for a degree of variability in a value or range, for example, within 20%, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. In the present disclosure, the term “substantially” can allow for a degree of variability in a value or range, for example, within 80%, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.

[0036] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description onlyand not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0037] The present disclosure provides for compound having the formula (I)substituents selected from halo and O-R, wherein R is H, CH3, C1-C4alkyl substituted with one or more deuterium atoms, or C1-C4alkyl substituted with one or more halo atoms and n is 0-3. In some embodiments n is 1.

[0040] The present disclosure further provides for the compound of claim 1 wherein the ethoxy is a C1 –C4optionally substituted by deuterium.

[0041] The present disclosure provides for compounds with enhanced anti- Neisseria gonorrhoeae activity. In another aspect of the disclosure provides for a compound that exhibits Neisseria gonorrhoeae inhibition activity.

[0042] Thus, the present disclosure provides for a pharmaceutical composition comprising a compound as described. It is further provided for that a pharmaceutical composition comprising a compound as described, formulated with a pharmaceutically acceptable diluent, carrier, excipient or salt thereof.

[0043] Also provided for is a method for treating a patient having Neisseria gonorrhoeae infection, and / or suffering from symptoms of Neisseria gonorrhoeae infection compromising the step of administering a therapeutically effective amount of a compound as described, to a patient in need thereof.

[0044] It is further provided for, a method for treating a patient in need thereof, a therapeutically effective amount of a compound having anti- Neisseria gonorrhoeae activity as described. It is further provided for, a method for treating a patient in need thereof, comprising administering a therapeutically effective amount of a compound as above, to a patient in need thereof, the compound having anti- Neisseria gonorrhoeae activity.

[0045] The present disclosure relates generally to a method to treat a patient with malaria. In particular, symptoms and pathologies due to Neisseria gonorrhoeae infection. The present disclosure provides for a method for treating a patient of symptoms of Neisseria gonorrhoeae infection compromising the step of administering a therapeutically effective amount of a compound selected by the disclosed methods, to the patient in need thereof.

[0046] The term “substituted” as used herein refers to a functional group in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, andsulfonamide groups; a nitrogen atom in groups such as amines, azides, hydroxylamines, cyano, nitro groups, N-oxides, hydrazides, and enamines; and other heteroatoms in various other groups.

[0047] The term “alkyl” as used herein refers to substituted or unsubstituted straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (C1-C8), or, in some embodiments, from 1 to 6 carbon atoms (C1-C6). Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0048] The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain and branched divalent alkenyl and cycloalkenyl groups having from 2 to 20 carbon atoms(C2-C20), 2 to 12 carbons (C2-C12), 2 to 8 carbon atoms (C2-C8) or, in some embodiments, from 2 to 4 carbon atoms (C2-C4) and at least one carbon-carbon double bond. Examples of straight chain alkenyl groups include those with from 2 to 8 carbon atoms such as -CH=CH-, -CH=CHCH2-, and the like. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and the like.

[0049] An alkynyl group is the fragment, containing an open point of attachment on a carbon atom that would form if a hydrogen atom bonded to a triply bonded carbon is removed from the moleculeof an alkyne. The term “hydroxyalkyl” as used herein refers to alkyl groups as defined herein substituted with at least one hydroxyl (-OH) group.

[0050] The term “cycloalkyl” as used herein refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. In some embodiments, cycloalkyl groups can have 3 to 6 carbon atoms (C3-C6). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like.

[0051] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a “formyl” group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atoms bonded to the carbonyl group. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0052] The term “aryl” as used herein refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons (C6-C14) or from 6 to 10 carbon atoms (C6-C10) in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein.

[0053] The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0054] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within themeaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0055] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0056] The term “amino group” as used herein refers to a substituent of the form -NH2, -NHR, -NR2, -NR3+, wherein each R is independently selected, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0057] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0058] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH3)2and the like.

[0059] The term “optionally substituted,” or “optional substituents,” as used herein, means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different. When using the terms “independently,” “independently are,” and “independently selected from” mean that the groups in question may be the same or different. Certain of the herein defined terms may occur more than once in the structure, and upon such occurrence each term shall be defined independently of the other.

[0060] The compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.

[0061] Similarly, the compounds described herein may include geometric centers, such as cis, trans, E, and Z double bonds. It is to be understood that in another embodiment, the invention described herein is not limited to any particular geometric isomer requirement, and that the compounds, compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. It is also to be understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other double bonds.

[0062] As used herein, the term “salts” and “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.

[0063] Pharmaceutically acceptable salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference.

[0064] The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense ofbeing compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0065] As used herein, the term “administering” includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions described herein may be administered in unit dosage forms and / or formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0066] Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like. Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.

[0067] Illustrative means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.

[0068] The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

[0069] It is to be understood that in the methods described herein, the individual components of a co-administration, or combination can be administered by any suitable means, contemporaneously, simultaneously, sequentially, separately or in a single pharmaceutical formulation. Where the co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may bethe same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

[0070] The term “therapeutically effective amount” as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.

[0071] Depending upon the route of administration, a wide range of permissible dosages are contemplated herein, including doses falling in the range from about 1 μg / kg to about 1 g / kg. The dosages may be single or divided, and may administered according to a wide variety ofprotocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like. In each of these cases it is understood that the therapeutically effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.

[0072] In addition to the illustrative dosages and dosing protocols described herein, it is to be understood that an effective amount of any one or a mixture of the compounds described herein can be determined by the attending diagnostician or physician by the use of known techniques and / or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0073] The term “patient” includes human and non-human animals such as companion animals (dogs and cats and the like) and livestock animals. Livestock animals are animals raised for food production. The patient to be treated is preferably a mammal, in particular a human being.

[0074] The present disclosure may be better understood in light of the following non-limiting compound examples and method examples.EXAMPLES In vivo pharmacokinetics of EZM and its primary metabolite.

[0075] While the background literature suggests that EZM may be metabolized and cleared rapidly, the data is provided in humans and rabbits but not in mice. Thus, to investigate the first part of our hypothesis that the lack of EZM’s efficacy in the in vivo mouse model for gonococcal infection is attributed to its pharmacokinetic profile, we carried out a study to compare plasma concentration of both EZM and the primary metabolite 1 after oral dosing. BALB / c mice were administered 10 mg / kg EZM or the primary metabolite 1 via oral gavage. Then, blood samples were drawn at eight timepoints and plasma concentration of each molecule was plotted versus time (FIG.2) and the data was used to calculate pharmacokinetic metrics for each molecule (Table 1). Mice were sampled out to 24 hours; however, each molecule was below the limit of detection in the plasma after 4 – 8 hours. EZM and 1 both displayed high clearance rates at 649 ± 107 mL / min / kg and 315 ± 4 mL / min / kg, respectively. Additionally, the molecules displayed relatively low maximum plasma concentrations (Cp,max) and areas under the curve (AUCp,0- last) that were paired with high volumes of distribution at the terminal phase of elimination (Vz). When taken into context of unbound molecule in plasma the unbound Cmax (Cp,u,max) for EZM and 1 were 77 ± 10 ng / mL and 334 ± 175 ng / mL, respectively. Doing the same for unbound AUC (AUCp,u,0-last) for EZM and 1 provides values of 63 ± 12 h*ng / mL and 195 ± 60 h*ng / mL. The MIC values for each molecule are overlaid on the plasma concentration curves to offer additional context to plasma levels with respect to MIC, although we have yet to investigate the PK / PD drivers for the scaffold. Moreover, CA inhibitors are known to partition into the red blood cell portion of the blood over the plasma due to expression of endogenous hCAs45,46, mainly hCA I and hCA II. We therefore evaluated the blood-to-plasma ratio (Rb) forthe molecules and showed that EZM has an Rb value of 8.0 ± 0.8 into mouse erythrocytes, while the Rb value for 1 is much greater at 124 ± 7. These pharmacokinetic parameters are similar to those observed in rabbits and are not problematic if the goal is to target hCAs, as both EZM and 1 maintain sufficient potency versus the hCAs.34The molecules also distribute to various other tissues such as the eye and kidneys, where the hCA targets reside for the treatment of glaucoma or use as a diuretic.34Conversely, these overall pharmacokinetic profiles for EZM and 1 may not be ideal for distributing enough molecule efficiently to the site of gonococcal infection to have a sustained antibacterial effect. Drawing a correlation from the EZM data in rabbits in which 40% is excreted unchanged in the urine, it is feasible to believe a major metabolite may be the glucuronide that we hypothesize is inactive versus N. gonorrhoeae. Design of analogs to limit or block metabolism could result in increased active agent in the plasma, thereby increasing the anti-gonococcal activity in vivo. Further tests on the glucuronide would be needed to confirm this hypothesis, however, we were unable to obtain this metabolite synthetically. FIG.2. Design Rationale and Synthesis of Analogs

[0076] EZM and metabolite 1 displayed potent anti-gonococcal activity, but the pharmacokinetic profiles indicated the molecules may be cleared too rapidly or distributed into other tissues making less molecule access the site of infection in the N. gonorrhoeae mouse model. Therefore, our first goal was to design analogs to reduce or prevent in vivo metabolism of the scaffold while still maintaining antibacterial activity. To provide a prediction of potential metabolic hotspots mediated through cytochrome P450 (CYP) oxidation, analogs were submitted to the P450 Sites of Metabolism prediction tool in Maestro (version 2022-2, Schrödinger, LLC) to assess potential sites of metabolic liability from CYP isoforms 2C9, 2D6,and 3A4. Atoms in each molecule are scored based on a combination of intrinsic reactivity and accessibility to the heme iron centers of the P450 enzymes, calculated via induced-fit docking. The prediction tool highlighted the methylene carbon in the ethoxy group of EZM as a likely site of CYP oxidation (FIG.3), which would hydroxylate the carbon next to the ether oxygen, creating a hemiacetal intermediate that is then hydrolyzed to yield the free hydroxyl primary metabolite. This aligns with the O-dealkylated metabolite 1 that has previously been observed for EZM. Additionally, in preparation to test an analog with the ethoxy group removed altogether, we assessed an unsubstituted benzothiazole analog (2, FIG.3) for potential CYP oxidation. The predictor tool indicated that there is possibility for oxidation at either the 5- or 6- position of the benzothiazole heterocycle, which would yield the same or similar primary metabolite 1 and would conceivably continue through Phase II glucuronidation and subsequent clearance. Therefore, our analog design also focused on modification of the phenyl portion to alleviate the potential for oxidation at these positions.

[0077] The mechanism of CYP oxidation relies on hydrogen abstraction from the carbon by the heme Fe3+.47A common strategy to modulate CYP metabolism is to replace the vulnerable C-H bond with an isosteric atom such as deuterium or fluorine. With respect to deuterium substitution, there are well-established kinetic isotope effects for CYP activity on molecules containing a deuterium in place of a hydrogen as CYPs demonstrate slower rates of oxidation on deuterium containing molecules compared to hydrogen.47–50In the case of fluorine, the atom is often exchanged with metabolically labile hydrogens to protect against CYP oxidation due to the relatively similar size of hydrogen and fluorine and the C-F bond being stronger than the C-H bond.51–54Thus, we incorporated both strategies in the design of new EZM-based analogs.Synthesis of analogs. Synthesis of the primary metabolite 1 was accomplished by dealkylation of EZM using aluminum trichloride in a single step as previously reported (Scheme 1). (Carta, F.; Di Cesare Mannelli, L.; Pinard, M.; Ghelardini, C.; Scozzafava, A.; McKenna, R.; Supuran, C. T. A Class of Sulfonamide Carbonic Anhydrase Inhibitors with Neuropathic Pain Modulating Effects. Bioorg Med Chem 2015, 23 (8), 1828–1840, incorporated herein by reference.)55The benzothiazole analog 2 was purchased as a dry powder (1 Click Chemistry Inc., catalog no. MFCD11870742). The remaining analogs in the study (6-11) were synthesized in three steps using previously reported synthetic routes (Schoenwald, R. D.; Eller, M. G.; Dixson, J. A.; Barfknecht, C. F. Topical Carbonic Anhydrase Inhibitors. J Med Chem 1984, 27 (6), 810–812; Petrova, E.; Rasina, D.; Jirgensons, A. N-Sulfonylcarboxamide as an Oxidizing Directing Group for Ruthenium- Catalyzed C–H Activation / Annulation. European J Org Chem 2017, 2017 (13), 1773–1779; KORMAN, J. Carbonic Anhydrase Inhibitors. I. Benzothiazole Derivatives. J Org Chem 1958, 23 (11), 1768–1771; Handte, R.; Willms, L.; Blume, E. Process for the Preparation of 2-Mercaptobenzothiazoles.4,431,813, 1984; Breiten, B.; Lockett, M. R.; Sherman, W.; Fujita, S.; Al-Sayah, M.; Lange, H.; Bowers, C. M.; Heroux, A.; Krilov, G.; Whitesides, G. M. Water Networks Contribute to Enthalpy / Entropy Compensation in Protein–Ligand Binding. J Am Chem Soc 2013, 135 (41), 15579–15584, each incorporated herein by reference.).56–60The synthesis starts from substituted 2-chloro anilines 3a-f (Scheme 2), which were commercially available reagents except for 3a. The intermediate 3a was synthesized by adapting a previously reported procedure (Esteve-Turrillas, F. A.; Agulló, C.; Mercader, J. V; Abad-Somovilla, A.; Abad-Fuentes, A. Rationally Designed Haptens for Highly Sensitive Monoclonal Antibody-Based Immunoanalysis of Fenhexamid. Analyst 2018, 143 (17),4057–4066, incorporated herein by reference.)61to convert 4-amino-3-chlorophenol to thesubsequent amide intermediate 12 (Scheme 3). This was followed by coupling 12 with d5-iodoethane to afford intermediate 13 and subsequent amide deprotection to yield 3a. The substituted reagents 3a-f were converted to benzothiazole-2-thiol intermediates 4a-f via cycloaddition with potassium ethyl xanthate.59,60The thiol was then treated with NaOH, ammonium hydroxide, and sodium hypochlorite to produce the sulfenamide intermediates 5a-f.56,58,60The sulfenamides were oxidized to their corresponding sulfonamides usingKMnO4inH2O:acetone (1:1)(Petrova, E.; Rasina, D.; Jirgensons, A. N-Sulfonylcarboxamide as an OxidizingDirecting Group for Ruthenium-Catalyzed C–H Activation / Annulation. European J Org Chem 2017, 2017 (13), 1773–1779; Korman J. Carbonic Anhydrase Inhibitors. I. Benzothiazole Derivatives. J OrgChem 1958, 23 (11), 1768–1771,each incorporated herein by reference.)57,58 to yield benzothiazolesulfonamides 6-11 as the final products.

[0078] Reference Compound Ethoxzolamide (EZM)

[0082] Analog Compound 2 (CAI0094)

[0092] Analog Compound 10 (CAI0128)General Synthesis Methods

[0096] Chemistry: General Methods. H and13C NMR spectra were recorded on either a Bruker DRX500 (operating at 500 and 126 MHz) or Avance-III-800 spectrometer (operating at 800 and 200 MHz) in DMSO-d6 with or without the internal standard of TMS at 0.05% v / v. Chemical shifts (δ) are reported as parts per million (ppm), and the coupling constants are reported as s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet. The purity of all final compounds was >95% as assessed by HPLC using an Agilent 1200 series chromatograph. The chromatographic method utilized a ThermoScientific Hypersil GOLD C-18. UV detection wavelength = 220 / 254 nm; flow-rate = 1.0 mL / min; mobile phases of (1) acetonitrile / water with 0.1% formic acid. An Advion CMS-L compact mass spectrometer with an APCI source and an atmospheric solids analysis probe (ASAP) was used for mass spectrometry analysis. Most final compounds were primarily prepared according to General Procedures 1-4, detailed below. EZM (Sigma-Aldrich, catalog no.333328) and 2 (1 ClickChemistry Inc., catalog no. MFCD11870742) were purchased from commercial vendors, then fully characterized to confirm identity and purity prior to biological evaluation. Chemistry

[0097] 6-Ethoxybenzo[d]thiazole-2-sulfonamide (Ethoxzolamide, EZM). EZM was purchased from a commercial vendor (Sigma Aldrich, catalog no.333328) and characterized prior to biological evaluation.1H NMR (500 MHz, DMSO) δ 8.22 (s, 2H), 7.99 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 2.5 Hz, 1H), 7.20 (dd, J = 9.0, 2.4 Hz, 1H), 4.09 (q, J = 6.8 Hz, 2H), 1.34 (t, J = 6.9 Hz, 3H).13C NMR (126 MHz, DMSO) δ 166.5, 158.3, 146.3, 137.8, 125.2, 118.1, 105.6, 64.2, 14.8. APCI-MS: m / z 259.0 [M+H]. HPLC retention time: 6.127 min HPLC purity: 96.7%.

[0098] 6-Hydroxybenzo[d]thiazole-2-sulfonamide (1). To an argon-flushed vial was added EZM (0.400 g, 1.55 mmol, 1.0 equiv) in anhydrous DCM (20 mL), followed by the addition of aluminum trichloride (0.723 g, 5.42 mmol, 3.5 equiv) portion-wise at 0 °C. The reaction was stirred at 0 ˚C for 10 min and then allowed to warm to room temperature overnight. The reaction mixture was then cooled to 0 °C and quenched with 1.0 M HCl. The white solid was collected by filtration, washed with H2O, and dried under vacuo to yield the product (0.322 g, 1.40 mmol, 90%) as an off-white solid.1H NMR (500 MHz, DMSO) δ 10.23 (s, 1H), 8.17 (s, 2H), 7.92 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 9.0, 2.4 Hz, 1H).13C NMR (126 MHz, DMSO) δ 165.5, 157.5, 145.5, 137.8, 125.3, 117.9, 107.2. APCI-MS: m / z 230.9 [M+H]. HPLC retention time: 5.99 min. HPLC purity: 96.4%.

[0099] 1,3-Benzo[d]thiazole-2-sulfonamide (2).1,3-benzothiazole-2-sulfonamide was purchased from a commercial vendor (1 Click Chemistry Inc., catalog no. MFCD11870742) and characterized prior to biological evaluation.1H NMR (500 MHz, DMSO) δ 8.33 (s, 2H), 8.23 (d,J = 8.0 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.67 – 7.56 (m, 2H).13C NMR (126 MHz, DMSO) δ 169.7, 152.1, 135.9, 127.9, 127.8, 124.5, 123.5. APCI-MS: m / z 215.0 [M+H]. HPLC retention time: 5.527 min HPLC purity: 96.4%. General Procedure for Compounds 6-11, described in detail for 6.

[0100] 6-(Ethoxy-d5)benzo[d]thiazole-2-thiol (4a). To an argon-flushed vial was added potassium ethyl xanthate (0.438 g, 2.72 mmol, 3.0 equiv) in anhydrous DMF and the solution was warmed to 150 ˚C. A solution of 3a (0.160 g, 0.906 mmol, 1.0 equiv) dissolved in anhydrous DMF was added to the stirring solution dropwise over an hour, then was left to stir at reflux for 18 h. The reaction mixture was allowed to cool to 80 ˚C and stirred into ice- water, then acidified with concentrated HCl. The resulting precipitate was collected by filtration to afford the product as an off-white solid (0.095 g, 0.440 mmol, 48%).1H NMR (500 MHz, DMSO) δ 13.58 (s, 1H), 7.29 (d, J = 2.7 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 6.94 (dd, J = 8.9, 2.6 Hz, 1H). APCI-MS: m / z 216.9, [M+H]+.

[0101] 6-(Ethoxy-d5)benzo[d]thiazol-2-yl)thiohydroxylamine (5a). Ammonium hydroxide solution (28% w / v, 1.9 mL, 13 mmol, 30 equiv) was stirred in a vial at 0 ˚C. Sodium hypochlorite solution (11% w / v, 0.40 mL, 0.70 mmol, 1.6 equiv) and a solution of 4a (0.095 g, 0.44 mmol, 1.0 equiv) in 2.0 M NaOH (0.61 mL) were simultaneously added to the vial dropwise at 0˚C over 1 h. Stirring was then allowed for an additional 15 minutes. The mixture was filtered and the solid was rinsed with copious amounts of water to rid any remaining NH4. The light brown solid was collected as the product (0.080 g, 0.35 mmol, 79%).1H NMR (500 MHz, DMSO) δ 7.62 – 7.39 (m, 2H), 6.94 (dd, J = 8.8, 2.7 Hz, 1H), 4.86 (s, 2H) APCI-MS: 231.9, [M+H]+.

[0102] 6-(Ethoxy-d5)benzo[d]thiazole-2-sulfonamide (6). To a stirring mixture of 5a (0.080 g, 0.35 mmol, 1.0 equiv) in a solution of 1:1 acetone:water (4 mL) was added a solution of potassium permanganate (0.082 g, 0.52 mmol, 1.5 equiv) dissolved in water (6 mL) portion-wise over 30 min. The reaction mixture was stirred for another hour, treated with charcoal (0.2 g) and 10 M NaOH (0.5 mL), then stirred for another 30 min. The mixture was filtered through celite, and the resulting filtrate was acidified with HCl. The product was extracted with ethyl acetate three times and the combined organic layers were washed with water, dried over Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography (20-50% EtOAc:hexanes) to afford the product (0.035 g, 0.13 mmol, 38%) was isolated as a white solid. 1H NMR (500 MHz, DMSO) δ 8.22 (s, 2H), 8.00 (d, J=9.0 Hz, 1H), 7.75 (d, J=2.7 Hz, 1H), 7.20 (dd, J=9.0, 2.6 Hz, 1H).13C NMR (126 MHz, DMSO) δ 166.9, 158.7, 146.7, 138.2, 125.6, 118.5, 106.0. APCI-MS: m / z 264.6 [M+H]+. HPLC retention time: 6.541 min HPLC purity: 96.7%. Compounds 4b-4f.

[0103] 6-Methoxybenzo[d]thiazole-2-thiol (4b). Prepared according to the synthesis of 4a in General Procedure 1 using potassium ethyl xanthate (1.54 g, 9.52 mmol, 3.0 equiv) and 2-chloro- 4-methoxyaniline (0.434 mL, 3.17 mmol, 1 equiv). Isolated as a light brown product (0.326 g, 1.65 mmol, 52%).1H NMR (500 MHz, DMSO) δ 13.60 (s, 1H), 7.31 (d, J = 2.5 Hz, 1H), 7.18 (d, J = 8.9 Hz, 1H), 6.95 (dd, J = 8.9, 2.6 Hz, 1H), 3.72 (s, 3H). APCI-MS: m / z 198.0 [M+H]+.

[0104] 6-(Trifluoromethoxy)benzo[d]thiazole-2-thiol (4c). Prepared according to the synthesis of 4a in General Procedure 1 using potassium ethyl xanthate (0.915 g, 5.67 mmol, 3.0 equiv) and 2- chloro-4- (trifluoromethoxy)aniline (0.262 mL, 1.89 mmol, 1.0 equiv). Isolated as an off-whitesolid (0.315 g, 1.25 mmol, 0 MHz, DMSO) δ 13.90 (s, 1H), 7.83 (d, J = 2.4 Hz, 1H), 7.41 – 7.34 (m, 1H), 7.34 – 7.28 (m, 1H). APCI-MS: m / z 252.4, [M+H]+.

[0105] 6-Fluorobenzo[d]thiazole-2-thiol (4d). Prepared according to the synthesis of 4a in General Procedure 1 using potassium ethyl xanthate (1.66 g, 10.3 mmol, 3.0 equiv) and 2-chloro- 4-fluoroaniline (0.500 g, 3.44 mmol, 1.0 equiv). Isolated as a light orange solid (0.432 mg, 2.33 mmol, 68%).1H NMR (500 MHz, DMSO) δ 13.83 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.38 – 7.14 (m, 2H). APCI-MS: m / z 185.9, [M+H]+.

[0106] 5-Fluorobenzo[d]thiazole-2-thiol (4e). Prepared according to the synthesis of 4a in General Procedure 1 using potassium ethyl xanthate (1.66 g, 10.3 mmol, 3.0 equiv) and 2-chloro- 5-fluoroaniline (0.500 g, 3.44 mmol, 1.0 equiv). Isolated as a light orange solid (0.454 g, 2.45 mmol, 71%).1H NMR (500 MHz, DMSO) δ 13.85 (s, 1H), 7.73(dd, J = 8.8, 5.2 Hz, 1H), 7.19 (td, J = 9.1, 7.5, 2.5 Hz, 1H), 7.08 (dd, J = 9.1, 2.4 Hz, 1H). APCI-MS: m / z 185.9, [M+H]+.

[0107] 5,6-Difluorobenzo[d]thiazole-2-thiol (4f). Prepared according to the synthesis of 4a in General Procedure 1 using potassium ethyl xanthate (1.48 g, 9.17 mmol, 3.0 equiv) and 2-chloro- 4,5-fluoroaniline (0.33 mL, 3.06 mmol, 1.0 equiv). Isolated as a light brown solid (0.429 g, 2.11 mmol, 69%).1H NMR (500 MHz, DMSO) δ 13.87 (s, 1H), 7.87 (dd, J = 10.1, 7.3 Hz, 1H), 7.27 (dd, J = 9.9, 6.6 Hz, 1H). APCI-MS: m / z 203.9, [M+H]+. Compounds 5b-5f.

[0108] S-(6-Methoxybenzo[d]thiazol-2-yl)thiohydroxylamine (5b). Prepared according to the synthesis of 5a in General Procedure 1 using ammonium hydroxide (28% w / v, 6.50 mL, 45.6 mmol, 30 equiv), sodium hypochlorite (11% w / v 1.40 mL, 2.43 mmol, 1.6 equiv), and 4b (0.300 g, 1.52 mmol, 1.0 equiv) in 2.0 M NaOH (2.13 mL). Product was isolated as an off-white solidproduct (0.225 g, 1.06 mmol, 70%).1H NMR (500 MHz, DMSO) δ 7.61 – 7.54 (m, 2H), 6.96 (dd, J = 9.0, 2.5 Hz, 1H), 4.87 (s, 2H), 3.76 (s, 3H). APCI-MS: 212.5 [M+H]+.

[0109] S-(6-Trifluoromethoxy)benzo[d]thiazol-2-yl)thiohydroxylamine (5c). Prepared according to the synthesis of 5a in General Procedure 1 using ammonium hydroxide (28% w / v, 8.55 mL, 59.7 mmol, 30 equiv), sodium hypochlorite (11% w / v, 1.76 mL, 3.184 mmol, 1.6 equiv), and 4c (0.500 g, 1.99 mmol, 1.0 equiv) in 2.0 M NaOH (2.8 mL). Product was collected as an off-white solid (0.394 g, 1.48 mmol, 74%.1H NMR (500 MHz, DMSO) δ 8.11 (d, J = 2.7 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.36 (dd, J = 9.0, 2.8 Hz, 1H), 5.01 (s, 2H). APCI-MS: m / z 267.0 [M+H]+.

[0110] S-(6-Fluorobenzo[d]thiazol-2-yl)thiohydroxylamine (5d). Prepared according to the synthesis of 5a in General Procedure 1 using ammonium hydroxide (28% w / v, 5.70 mL, 40.5 mmol, 20 equiv), sodium hypochlorite (1.46 g, 1.20 mL, 11% w / v, 2.16 mmol, 1.6 equiv), and 4d (0.250 g, 1.35 mmol, 1.0 equiv) in 2.0 M NaOH (2.9 mL). Product was collected as an off- white solid (0.18 mg, 0.899 mmol, 67%) 1H NMR (500 MHz, DMSO) δ 7.98 – 7.81 (m, 1H), 7.69 (td, J = 8.6, 4.9 Hz, 1H), 7.23 (dd, J = 12.7, 5.9 Hz, 1H), 4.95 (s, J = 7.3 Hz, 2H). APCI- MS: m / z 201.0, [M+H]+.

[0111] S-(5-Fluorobenzo[d]thiazol-2-yl)thiohydroxylamine (5e). Prepared according to the synthesis of 5a in General Procedure 1 using ammonium hydroxide (28% w / v, 3.45 mL, 24.3 mmol, 30 equiv), sodium hypochlorite (11% w / v, 0.73 mL, 1.3 mmol, 1.6 equiv), and 4e (0.150 g, 0.810 mmol, 1.0 equiv) in 2.0 M NaOH (1.74 mL). Product was collected as an off-white solid (0.112 g, 0.559 mmol, 69%).1H NMR (500 MHz, DMSO) δ 8.03 (dd, J = 8.8, 5.4 Hz, 1H), 7.57 (dd, J = 10.1, 2.6 Hz, 1H), 7.18 (td, J = 9.1, 2.7 Hz, 1H), 5.00 (s, 2H). APCI-MS: m / z 201.0, [M+H]+.

[0112] S-(5,6-Difluorobenzo[d]thiazol-2-yl)thiohydroxylamine (5f). Prepared according to the synthesis of 5a in General Procedure 1 using ammonium hydroxide (28% w / v, 5.20 mL, 36.9 mmol, 30 equiv), sodium hypochlorite (11% w / v, 1.10 mL, 1.97 mmol, 1.6 equiv), and 4f (0.250 g, 1.23 mmol, 1.0 equiv) in 2.0 M NaOH (2.07 mL). Product was collected as an off-white solid (0.189 g, 0.866 mmol, 70%).1H NMR (500 MHz, DMSO) δ 8.29 – 8.07 (m, 1H), 7.94 – 7.73 (m, 1H), 5.03 (s, 2H). APCI-MS: m / z 219.0, [M+H]+. Compounds 7-11

[0113] 6-Methoxybenzo[d]thiazole-2-sulfonamide (7). Prepared according to the synthesis of 6 in General Procedure 1 using 5b (0.100 g, 0.471 mmol, 1.0 equiv) in a solution of acetone (2 mL) and water (2 mL) and potassium permanganate (0.112 g, 0.707 mmol, 1.5 equiv) dissolved in water (6 mL). The crude product was purified by column chromatography (20-50% EtOAc:Hexanes) to afford the product (0.029 g, 0.12 mmol, 25%) as a white solid.1H NMR (500 MHz, DMSO) δ 8.23 (s, 2H), 8.01 (d, J = 9.1 Hz, 1H), 7.78 (d, J = 2.7 Hz, 1H), 7.22 (dd, J = 9.1, 2.7 Hz, 1H), 3.83 (s, 3H).13C NMR (126 MHz, DMSO) δ 166.6, 159.0, 146.4, 137.8, 125.2, 117.8, 105.1, 56.1. APCI-MS: m / z 245.5 [M+H]+. HPLC retention time: 6.226 min HPLC purity: 97.0%.

[0114] 6-(Trifluoromethoxy)benzo[d]thiazole-2-sulfonamide (8). Prepared according to the synthesis of 6 in General Procedure 1 using 5c (0.100 g, 0.751 mmol, 1.0 equiv) in a solution of acetone (2 mL) and water (2 mL) and potassium permanganate (0.089 g, 0.56 mmol, 1.5 equiv) The final product (0.024 g, 0.08 mmol, 21%) was isolated as a white solid.1H NMR (500 MHz, DMSO) δ 8.41 (s, 2H), 8.38 (s, 1H), 8.26 (dd, J=9.0, 3.1 Hz, 1H),

[0115] 7.76 – 7.43 (m, 1H).13C NMR (126 MHz, DMSO) δ 171.7, 151.0, 147.2, 137.3, 126.3, 121.9, 120.5 (q, J = 257.4 Hz), 116.3. APCI-MS: m / z 299.5 [M+H]+. HPLC retention time: 6.392 min HPLC purity: 96.0%.

[0116] 6-Fluorobenzo[d]thiazole-2-sulfonamide (9). Prepared according to the synthesis of 6 in General Procedure 1 using 5d (0.300 g, 1.50 mmol, 1.0 equiv) in a solution of acetone (2 mL) and water (2 mL) and potassium permanganate (0.355 g, 2.25 mmol, 1.5 equiv) dissolved in water (6 mL). The product (0.080 g, 0.34 mmol, 23%) was isolated as white solid.1H NMR (500 MHz, DMSO) δ 8.35 (s, 2H), 8.18 (ddd, J = 9.1, 4.9, 1.5 Hz, 1H), 8.13 (dt, J = 8.7, 2.1 Hz, 1H), 7.52 (td, J = 9.1, 2.6 Hz, 1H).13C NMR (126 MHz, CDCl3) δ 169.8, 161.0 (d, J = 246.0 Hz), 148.9, 137.4 (d, J = 12.4 Hz), 126.2 (d, J = 9.7 Hz), 116.9 (d, J = 25.2 Hz), 109.7 (d, J = 28.0 Hz). APCI-MS: m / z 233.2 [M+H]+. HPLC retention time: 6.254 min HPLC purity: 97.6%.

[0117] 5-Fluorobenzo[d]thiazole-2-sulfonamide (10). Prepared according to the synthesis of 6 in General Procedure 1 using 5e (0.100 g, 0.499 mmol, 1.0 equiv) in a solution of acetone (2 mL) and water (2 mL) and potassium permanganate (0.118 g, 0.749 mmol, 1.5 equiv) dissolved in water (6 mL). The product (0.034 mg, 0.15 mmol, 29%) was isolated as a white solid.1H NMR (500 MHz, DMSO) δ 8.35 (s, 2H), 8.29 (dd, J = 8.9, 5.4 Hz, 1H), 8.02 (dd, J = 9.7, 2.7 Hz, 1H), 7.53 (td, J = 9.2, 2.6 Hz, 1H).13C NMR (126 MHz, DMSO) δ 172.4, 162.1 (d, J = 243.1 Hz), 153.1 (d, J = 12.5 Hz), 132.0, 125.3 (d, J = 10.1 Hz), 116.8 (d, J = 25.3 Hz), 110.5 (d, J = 23.8 Hz). APCI-MS: m / z 233.4 [M+H]+. HPLC retention time: 6.257 min HPLC purity: 99.2%.

[0118] 5,6-Difluorobenzo[d]thiazole-2-sulfonamide (11). Prepared according to the synthesis of 6 in General Procedure 1 using 5f (0.100 g, 0.458 mmol, 1.0 equiv) in a solution of acetone (2 mL) and water (2 mL) and potassium permanganate (109 mg, 0.687 mmol, 1.5 equiv) dissolved in water (6 mL). The product (0.036 mg, 0.14 mmol, 31%) was isolated as a white solid.1HNMR (500 MHz, DMSO) δ 8.44-8.36 (m, 3H), 8.32 (ddd, J=11.4, 7.3, 4.1 Hz, 1H).13C NMR (126 MHz, DMSO) δ 171.9, 148.9 (dd, J = 248.8, 15.0 Hz), 150.5 (dd, J = 246.5, 15.1 Hz), 148.6 (d, J = 10.4 Hz), 132.31 (d, J = 9.7 Hz), 112.6 (d, J = 19.6 Hz), 111.6 (d, J = 22.9 Hz). APCI-MS: m / z 251.5 [M+H]+. HPLC retention time: 6.502 min HPLC purity: 98.1%. Synthesis for intermediate 3a.

[0119] N-(2-Chloro-4-hydroxyphenyl)acetamide (12). To a stirring mixture of 4-amino-3- chlorophenol (1.00 g, 6.97 mmol, 1.0 equiv) in THF (10 mL) and water (5.0 mL) was added dropwise acetic anhydride (0.724 mL, 7.66 mmol, 1.1 equiv) at room temperature. The reaction was allowed to stir for 1.5 hours, then was poured into water and extracted with ether three times. The combined organic layers were washed with NaHCO3, then brine, then dried over Na2SO4. The organic layer was then concentrated in vacuo to afford the off-white product (0.995 g, 5.36 mmol, 77%).1H NMR (500 MHz, DMSO) δ 9.78 (s, 1H), 9.29 (s, 1H), 7.25 (d, J = 8.8 Hz, 1H), 6.80 (d, J= 2.8 Hz, 1H), 6.67 (dd, J = 8.7, 2.9 Hz, 1H), 1.97 (d, J = 2.9 Hz, 3H). APCI-MS: m / z 186.0 [M+H]+.

[0120] N-(2-Chloro-4-(ethoxy-d5)phenyl)acetamide (13). To an argon-flushed round bottom flask containing N-(2- chloro-4-hydroxyphenyl)acetamide (0.400 g, 2.16 mmol, 1.0 equiv) in anhydrous acetone (30 mL) was added potassium carbonate (1.49 g, 10.8 mmol, 5.0 equiv), then 1-iodoethane-1,1,2,2,2-d5 (Cambridge Isotope Laboratories catalog # DLM-272-5, 0.694 g, 4.31 mmol, 2.0 equiv). The reaction was stirred at reflux for 1 h, then allowed to cool to room temperature. The solution was filtered through Celite, washing with acetone, and the filtrate was concentrated in vacuo to afford the off-white product (0.353 g, 1.61 mmol, 75%).1H NMR (500MHz, DMSO) δ 9.38 (s, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.00 (d, J = 2.9 Hz, 1H), 6.84 (dd, J, 8.7, 2.9 Hz, 1H), 2.00 (s, 3H). APCI-MS: m / z 219.0, [M+H]+.

[0121] 2-Chloro-4-(ethoxy-d5)aniline (3a). To a stirring solution of N-(2-chloro-4-(ethoxy- d5)phenyl)acetamide (0.20 g, 0.92 mmol, 1.0 equiv) in anhydrous methanol was added HCl in dioxane (4 M, 2.4 mL, 10.5 equiv). the reaction was refluxed under argon for 2.5 hours, then allowed to cool to r.t. The solution was poured into water and basified to pH 9-10 using 3 M NaOH, then extracted with ether. The combined organic layers were washed with brine and dried over anhydrous Na2SO4. The solution was concentrated in vacuo to afford the product (0.14 g, 0.79 mmol, 87%).1H NMR (500 MHz, DMSO) δ 6.77 (d, J = 2.8 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 6.64 (dd, J = 8.7, 2.8 Hz, 1H), 4.79 (s, 2H). APCI-MS: m / z 177.0, [M+H]+. Biological Evaluation

[0122] Bacterial strains, Media, and Chemicals. N. gonorrhoeae strains used in the study were obtained from the American Type Culture Collection (ATCC) and the CDC.

[0123] Carbonic Anhydrase CO2Hydration Catalytic Assay and Ki Determination. The assay was performed as previously described (Hewitt, C. S.; Abutaleb, N. S.; Elhassanny, A. E. M.; Nocentini, A.; Cao, X.; Amos, D. P.; Youse, M. S.; Holly, K. J.; Marapaka, A. K.; An, W.; Kaur, J.; Krabill, A. D.; Elkashif, A.; Elgammal, Y.; Graboski, A. L.; Supuran, C. T.; Seleem, M. N.; Flaherty, D. P. Structure-Activity Relationship Studies of Acetazolamide-Based Carbonic Anhydrase Inhibitors with Activity against Neisseria Gonorrhoeae. ACS Infect Dis 2021, 7 (7), 1969–1984 and Abutaleb, N. S.; Elhassanny, A. E. M.; Nocentini, A.; Hewitt, C. S.; Elkashif, A.; Cooper, B. R.; Supuran, C. T.; Seleem, M. N.; Flaherty, D. P. Repurposing FDA-Approved Sulphonamide Carbonic Anhydrase Inhibitors for Treatment of Neisseria Gonorrhoeae. J Enzyme Inhib Med Chem 2022, 37 (1), 51–61, each incorporated herein by reference.).35,37Recombinant N. gonorrhoeae CAs were produced as previouslydescribed.35Recombinant human CAs I and II were purchased from Millipore Sigma (hCAI Catalog # C4396-5MG; hCAII Catalog # C6624-500UG). Ki values were calculated by inputting IC50 values into the Cheng-Prusoff equation for Ki from catalytic inhibition constants (Yung- Chi, C.; Prusoff, W. H. Relationship between the Inhibition Constant (KI) and the Concentration of Inhibitor Which Causes 50 per Cent Inhibition (I50) of an Enzymatic Reaction. Biochem Pharmacol 1973, 22 (23), 3099–3108, incorporated herein by reference.).109Antibacterial Activity of Analogs against N. gonorrhoeae.

[0124] The MICs of EZM and analogs were determined using a broth microdilution assay, as previously described. (Hewitt, C. S.; Abutaleb, N. S.; Elhassanny, A. E. M.; Nocentini, A.; Cao, X.; Amos, D. P.; Youse, M. S.; Holly, K. J.; Marapaka, A. K.; An, W.; Kaur, J.; Krabill, A. D.; Elkashif, A.; Elgammal, Y.; Abutaleb, N. S.; Elhassanny, A. E. M.; Nocentini, A.; Hewitt, C. S.; Elkashif, A.; Cooper, B. R.; Supuran, C. T.; Seleem, M. N.; Flaherty, D. P. Repurposing FDA- Approved Sulphonamide Carbonic Anhydrase Inhibitors for Treatment of Neisseria Gonor- rhoeae. J Enzyme Inhib Med Chem 2022, 37 (1), 51–61; Graboski, A. L.; Supuran, C. T.; Seleem, M. N.; Flaherty, D. P. Structure-Activity Relationship Studies of Acetazolamide-Based Carbonic Anhydrase Inhibitors with Activity against Neisseria Gonorrhoeae. ACS In-fect Dis 2021, 7 (7), 1969–1984, Giovannuzzi, S.; Abutaleb, N. S.; Hewitt, C. S.; Carta, F.; Nocentini, A.; Seleem, M. N.; Flaherty, D. P.; Supuran, C. T. Dithiocarbamates Effectively Inhibit the α- Carbonic Anhydrase from Neisseria Gonorrhoeae. J Enzyme Inhib Med Chem 2022, 37 (1), 1–8; Naclerio, G. A.; Abutaleb, N. S.; Alhashimi, M.; Seleem, M. N.; Sintim, H. O. N-(1,3,4- Oxadiazol-2- Yl)Benzamides as Antibacterial Agents against Neisseria Gonor-rhoeae. International Journal of Molecular Sciences.2021, each incorporated herein by ref- erence.)35,37,110,111N. gonorrhoeae strains were grown overnight on chocolate II agar plates. Abacterial solution equivalent to a 1.0 McFarland standard was prepared and diluted in brucella broth supplemented with yeast extract, dextrose, proteose-peptone, NAD, pyrodixal, hematin, and IsoVitaleX to reach a bacterial count of about 1×106 CFU / mL. Test agents were serially diluted along the plates. Media containing bacteria (without test agents) were included in the assays as a growth control. Plates were then incubated aerobically and in the presence of 5% CO2 for 24 h at 37 ˚C prior to recording the MIC, as observed visually. Aqueous Solubility.

[0125] The solubility was assessed as described by Kerns et al (Kerns, H. E.; Di, L.; Carter, T. G. In Vitro Solubility Assays in Drug Discovery. Current Drug Metabolism.2008, pp 879–885, incorporated herein by reference.112A 20 mM DMSO stock solution of each compound was diluted to 200 µM in PBS (pH 7.4) and was equilibrated on an end-to-end rotator at 25 ºC for 24 hours. Solutions were then centrifuged, filtered, and subjected to HPLC analysis. Sample concentrations were determined in triplicate by measuring HPLC UV peak area and were compared to standard curves. Log D calculations.

[0126] The method and quantification were performed as previously described (Abutaleb, N. S.; Shrinidhi, A.; Bandara, A. B.; Seleem, M. N.; Flaherty, D. P. Evaluation of 1,3,4- Thiadiazole Carbonic Anhydrase Inhibitors for Gut Decolonization of Vancomycin-Resistant Enterococci. ACS Med Chem Lett 2023, 14 (4), 487–492, incorporated herein by reference.).113Briefly, 10 µL solutions of each compound were pipetted into a pre-determined volume (0.5, 0.66, or 0.8 mL) of pre-saturated PBS (pH 7.4) and mixed well for 5 min. Solutions were centrifuged for 5 min, then pre- saturated 1-octanol (0.5, 0.33, or 0.2 mL) was added to yield a 1.0 mL solution of 1:1, 2:1, or4:1 PBS:buffer. Solutions were equilibrated on an end- to-end rotator for 2 h, then centrifuged for 10 min.200 µL aliquots of each organic phase and aqueous phase were subjected to HPLC analysis, using standard curves for quantitation. LogD was calculated by the equation ^^^^^^^^ =

[0127] ^^^^^^ ["#$%&'( *%+,#].

[0128] [. / 0,102 *%+,#] Caco-2 Permeability Assay.

[0129] Assays and data analysis were performed by Eurofins Panlabs (MO, USA), according to the procedure by Hidalgo et al. (Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Characterization of the Human Colon Carcinoma Cell Line (Caco-2) as a Model System for Intestinal Epithelial Permeability. Gastroenterology 1989, 96 (3), 736–749, incorporated herein by reference.)114Mouse Liver Microsome Stability.

[0130] Assays and data analysis were performed by Eurofins Panlabs (MO, USA) according to the procedure by Obach et al. (Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C.; Macintyre, F.; Rance, D. J.; Wastall, P. The Prediction of Human Pharmacokinetic Parameters from Preclinical and In Vitro Metabolism Data. Journal of Pharmacology and Experimental Therapeutics 1997, 283 (1), 46–58, incorporated herein by reference.)115Plasma Protein Binding.

[0131] Assays and data analysis were performed by Eurofins Panlabs (MO, USA), according to the procedure by Banker et al. (Banker, M. J.; Clark, T. H.; Williams, J. A. Development and Validation of a 96-Well Equilibrium Dialysis Apparatus for Measuring Plasma Protein Binding. J Pharm Sci 2003, 92 (5), 967–974, incorporated herein by reference.)116Blood Partitioning.

[0132] Assays and data analysis were performed by Eurofins Panlabs (MO, USA) according to the procedure by Yu et al. (Yu, S.; Li, S.; Yang, H.; Lee, F.; Wu, J.-T.; Qian, M. G. A Novel Liquid Chromatography / Tandem Mass Spectrometry Based Depletion Method for Measuring Red Blood Cell Partitioning of Pharmaceutical Compounds in Drug Discovery. Rapid Communications in Mass Spectrometry 2005, 19 (2), 250–254, incorporated herein by reference.)117In Vivo Pharmacokinetics.

[0133] Assays and data analysis were performed by Eurofins Panlabs (Taiwan). All aspects of the work were performed in general accordance with the Guide for the Care and Use of Laboratory Animals: Eighth Edition in an AAALAC-accredited laboratory animal facility. The animal care and use protocol was reviewed and approved by the Institutional Animal Cares and Use Committee at Pharmacology Discovery Services Taiwan, Ltd. BALB / c mice (n = 3 per time point per molecule tested) were dosed with test compound at 10 mg / kg by oral gavage (PO) in vehicle (10% dimethylacetamide / 20% propylene glycol / 40% polyethylene glycol 400 / 30% phosphate buffered saline). Blood aliquots were collected at 8 timepoints (0.167, 0.5, 1, 2, 4, 6, 8, and 24 h) after PO administration from each mouse. The first seven timepoint blood aliquots were collected via facial vein (0.05 mL) and the final timepoint was collected via cardiac puncture (0.3 mL). Samples were then centrifuged at 2,500 x g for 15 minutes at 4 ºC, within 1 hour of collection. The plasma samples were processed using acetonitrile precipitation and analyzed by LC-MS / MS.

[0134] The exposure levels (ng / mL) of all molecules in plasma samples determined by LC- MS / MS. Plots of plasma concentrations (mean ± SD) vs. time for molecule were constructed.The fundamental PK parameters (t1 / 2, Tmax, Cmax, AUClast, AUCInf, AUCExtr MRT, Vz and CL) after PO administration were obtained from the NCA of the plasma data using WinNonlin (best-fit mode). In vitro Cytotoxicity. Compounds were assayed at concentrations of 32, 64, and 128 µg / mL against money fibroblast (Vero) cells and human endocervical (ME-180) cells, as described elsewhere (Almolhim, H.; Elhassanny, A. E. M.; Abutaleb, N. S.; Abdelsattar, A. S.; Seleem, M. N.; Carlier, P. R. Substituted Salicylic Acid Analogs Offer Improved Potency against Multidrug-Resistant Neisseria Gonorrhoeae and Good Selectivity against Commensal Vaginal Bacteria. Sci Rep 2023, 13 (1), 14468; (119) Hagras, M.; Abuelkhir, A. A.; Abutaleb, N. S.; Helal, A. M.; Fawzy, I. M.; Hegazy, M.; Seleem, M. N.; Mayhoub, A. S. Novel Phenylthiazoles with a Tert-Butyl Moiety: Promising Antimicrobial Activity against Multidrug-Resistant Pathogens with Enhanced ADME Properties. RSC Adv 2024, 14 (2), 1513–1526; (120) Wagdy, R. A.; Abutaleb, N. S.; Fathalla, R. K.; Elgammal, Y.; Weck, S.; Pal, R.; Fischer, P. D.; Ducho, C.; Abadi, A. H.; N Seleem, M.; Engel, M.; Abdel-Halim, M. Discovery of 1,2-Diaryl-3- Oxopyrazolidin-4-Carboxamides as a New Class of MurA Enzyme Inhibitors and Characterization of Their Antibacterial Activity. Eur J Med Chem 2023, 261, 115789; (121) Dokla, E. M. E.; Abutaleb, N. S.; Milik, S. N.; Kandil, E. A. E. A.; Qassem, O. M.; Elgammal, Y.; Nasr, M.; McPhillie, M. J.; Abouzid, K. A. M.; Seleem, M. N.; Imming, P.; Adel, M. SAR Investigation and Optimization of Benzimidazole-Based Derivatives as Antimicrobial Agents against Gram-Negative Bacteria. Eur J Med Chem 2023, 247, 115040, each incorporated herein by reference.).118–121Briefly, cells incubated with the compounds (in triplicate) in a 96-well plate for 24 hours at 37 ˚C. Control cells received DMSO (the solvent of the compounds) at a concentration equal to that in drug-treated wells. The assay reagent MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was subsequently added and the plates were incubated for up to four hours. Absorbance readings (at OD490) were recorded using a kinetic microplate reader (Synergy H1, BioTek, USA). The quantity of viable cells after treatment of each compound was expressed as a percentage of the viability of the DMSO-treated control cells. The data were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons. Anti-bacterial killing kinetics.

[0135] A standard time-kill kinetics assay was performed against N. gonorrhoeae FA1090 as described previously. (Abutaleb, N. S.; Seleem, M. N. Antivirulence Activity of Auranofin against Vancomycin-Resistant Enterococci: In Vitro and in Vivo Studies. Int J Antimicrob Agents 2020, 55 (3), 105828; S., A. N.; N., S. M. Repurposing the Antiamoebic Drug Diiodohydroxyquinoline for Treatment of Clostridioides Difficile Infections. Antimicrob Agents Chemother 2020, 64 (6), 10.1128 / aac.02115-19; Elsebaei, M. M.; Abutaleb, N. S.; Mahgoub, A. A.; Li, D.; Hagras, M.; Mohammad, H.; Seleem, M. N.; Mayhoub, A. S. Phenylthiazoles with Nitrogenous Side Chain: An Approach to Overcome Molecular Obesity. Eur J Med Chem 2019, 182, 111593; Shao, X.; AbdelKhalek, A.; Abutaleb, N. S.; Velagapudi, U. K.; Yoganathan, S.; Seleem, M. N.; Talele, T. T. Chemical Space Exploration around Thieno[3,2-d]Pyrimidin-4(3H)- One Scaffold Led to a Novel Class of Highly Active Clostridium Difficile Inhibitors. J Med Chem 2019, 62 (21), 9772– 9791, each incorporated herein by reference.)122–125N. gonorrhoeae was grown in GC broth to logarithmic phase, then diluted to reach an initial inoculum of ~ 106 CFU / mL. EZM, 2, 9, and azithromycin were added at 5× MIC in triplicate and incubated in ambient air at 37°C for 24 hours. Bacteria exposed to DMSO served as a control. An aliquot from each sample was collected at each timepoint and subsequently serially diluted and platedonto chocolate II agar plates. Plates were incubated at 37°C for 24 h before viable CFU / mL was determined. Frequency of mutations.

[0136] N. gonorrhoeae FA1090 was tested against EZM, 2, 9, azithromycin, ceftriaxone, and rifampicin for a single-step mutation assay as previously described. (M., B. M.; L., W. S.; L., C. K.; E., J. A.; Weirui, C.; E., L. R.; A., K. S.; L., S. D.; L., B. T. Aminomethyl Spectinomycins as Therapeutics for Drug-Resistant Gonorrhea and Chlamydia Coinfections. Antimicrob Agents Chemother 2018, 62 (5), 10.1128 / aac.00325-18; Naclerio, G. A.; Abutaleb, N. S.; Li, D.; Seleem, M. N.; Sintim, H. O. Ultrapotent Inhibitor of Clostridioides Difficile Growth, Which Suppresses Recurrence In Vivo. J Med Chem 2020, 63 (20), 11934–11944; Thangamani, S.; Mohammad, H.; Abushahba, M. F. N.; Sobreira, T. J. P.; Hedrick, V. E.; Paul, L. N.; Seleem, M. N. Antibacterial Activity and Mechanism of Action of Auranofin against Multi-Drug Resistant Bacterial Pathogens. Sci Rep 2016, 6 (1), 22571; each incorporated herein by reference.)126–128Plates were prepared by mixing drugs with GC agar supplemented with IsoVitaleX at the concentrations of 4× and 8× MIC, then were dried at room temperature. A bacterial suspension (~109 CFU / mL) was prepared and applied to the plates, which were incubated at 37 ˚C for 48 hours before determining the bacterial CFU. In vivo efficacy in a mouse model of N. gonorrhoeae infection.

[0137] Animal housing and experiments in this study were reviewed, approved, and performed under the guidelines of the Virginia Polytechnic Institute and State University Animal Care and Use Committee and carried out in strict accordance with the recommendations in the Guide forthe Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in individually ventilated cages (5 per cage) with free access to food and water. All mice were acclimatized for seven days before the start of the experiment. The mouse model of N. gonorrhoeae genital tract infection was performed as described previously (Schmitt, D. M.; Connolly, K. L.; Jerse, A. E.; Detrick, M. S.; Horzempa, J. Antibacterial Activity of Resazurin- Based Compounds against Neisseria Gonorrhoeae in Vitro and in Vivo. Int J Antimicrob Agents 2016, 48 (4), 367–372; (36) Abutaleb, N. S.; Elhassanny, A. E. M.; Seleem, M. N. In Vivo Efficacy of Acetazolamide in a Mouse Model of Neisseria Gonorrhoeae Infection. Microb Pathog 2022, 164, 105454; (62); Song, W.; Condron, S.; Mocca, B. T.; Veit, S. J.; Hill, D.; Abbas, A.; Jerse, A. E. Local and Humoral Immune Responses against Primary and Repeat Neisseria Gonorrhoeae Genital Tract Infections of 17beta-Estradiol-Treated Mice. Vaccine 2008, 26 (45), 5741–5751; (68); Kikiowo, B.; Bandara, A. B.; Abutaleb, N. S.; Seleem, M. N. Colonization Efficiency of Multidrug- Resistant Neisseria Gonorrhoeae in a Female Mouse Model. Pathog Dis 2023, 81, ftad030; Raterman, E. L.; Jerse, A. E. Female Mouse Model of Neisseria Gonorrhoeae Infection. In Neisseria gonorrhoeae: Methods and Protocols; Christodoulides, M., Ed.; Springer New York: New York, NY, 2019; pp 413–429; Elhassanny, A. E. M.; Abutaleb, N. S.; Seleem, M. N. Auranofin Exerts Antibacterial Activity against Neisseria Gonorrhoeae in a Female Mouse Model of Genital Tract Infection. PLoS One 2022, 17 (4), e0266764, each incorporated herein by reference.).2,36,62,68,129,130

[0138] Estradiol implantation. On Day -2, ovariectomized 8-week-old female BALB / c mice were implanted with a 5- mg, 21-day controlled-release estradiol pellet subcutaneously, using precision trocars. Afterwards, a drop of tissue adhesive was applied, and the skin was pressed to close the wound.

[0139] Antibiotic administration. Antibiotics were administered to increase the susceptibility to N. gonorrhoeae as described. (Raterman, E. L.; Jerse, A. E. Female Mouse Model of Neisseria Gonorrhoeae Infection. In Neisseria gonorrhoeae: Methods and Protocols; Christodoulides, M., Ed.; Springer New York: New York, NY, 2019; pp 413–429, incorporated herein by reference.)129Mice were injected intraperitonially with 0.6 mg of vancomycin and 1.2 mg of streptomycin on Days −2 through +1. The drinking water was replaced on Day −2 with sterilized water containing 0.4 g / L trimethoprim. Trimethoprim water was renewed every other day throughout the duration of the experiment. Streptomycin sulfate (2.4 g / L) was added to the trimethoprim water starting on Day +2 until the end of the experiment.

[0140] N. gonorrhoeae infection and treatment. Two days after pellet implantation (Day 0), the vagina of each mouse was rinsed with 50 mM HEPES (pH = 7.4), and mice were inoculated intravaginally with 1.94 × 106 CFU / mouse of N. gonorrhoeae FA1090 (each mouse was infected with 15 µL from the bacterial inoculum). Two days post- infection (Day +2), mice were randomly allocated into groups (n = 6). Mice were treated with test agents at the dose of 15 mg / kg twice daily for three consecutive days via oral gavage. As controls, mice were administered either the vehicle (10% dimethylacetamide: 20% propylene glycol: 40% polyethylene glycol 400:30% PBS), orally twice daily for three consecutive days or a single intraperitoneal dose of CEF (15 mg / kg in water).

[0141] Sample collection and counting of N. gonorrhoeae. Vaginal swabs were collected daily by gently inserting a moistened Dacron swab into the vagina of anaesthetized mice and suspending the swab in 0.1 mL of GC broth containing 0.05% saponin. Samples were serially diluted and plated on GC agar supplemented with vancomycin, colistin, nystatin and trimethoprim. A small portion of the sample was also cultured on heart infusion agar to monitorthe presence of potentially inhibitory commensal flora. Gram staining was performed to further identify the grown commensals (if any). If any mice were found to be colonized with enteric Gram-negative rods that may contribute to the clearance of N. gonorrhoeae, these mice were excluded from the experiment and the data analysis. Mice were humanely euthanized via carbon dioxide asphyxiation after the end of the experiment.

[0142] Statistical analysis. Data was analyzed using GraphPad Prism version 9.0 for Windows (GraphPad Software, La Jolla, CA. The data of N. gonorrhoeae CFU counts in the vaginal samples were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons (P<0.05).benzo[d]thiazole-2-sulfonamide (CAI0094):

[0143] 1,3-benzothiazole-2-sulfonamide was purchased from a commercial vendor (1 Click Chemistry Inc., catalog no. MFCD11870742) and characterized prior to biological evaluation.1H NMR (500 MHz, DMSO) δ 8.33 (s, 2H), 8.23 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.67 – 7.56 (m, 2H).13C NMR (126 MHz, DMSO) δ 169.74, 152.11, 135.98, 127.92, 127.82, 124.55, 123.55. APCI-MS: m / z 215.0 [M+H]. HPLC retention time: 5.527 min HPLC purity: 96.4%.6-hydroxybenzo[d]thiazole-2-sulfonamide (CAI0098):

[0144] To an argon-flushed vial was added EZM (400 mg, 1 Eq, 1.55 mmol) in anhydrous DCM (20 mL), followed by the addition of aluminum trichloride (723 mg, 3.5 Eq, 5.42 mmol) portion- wise at 0 °C. The reaction was stirred at 0 ˚C for 10 min and then allowed to warm to room temperature overnight. The reaction mixture was then cooled to 0 °C and quenched with 1.0 M HCl. The white solid was collected by filtration, washed with H2O, and dried MHz, DMSO) δ 10.23 (s, 1H), 8.17 (s, 2H), 7.92 (d, J = 8.9 Hz, 1H), 7.46 (d, J=2.4Hz,1H), 7.07 (dd, J = 9.0, 2.4 Hz, 1H).13C NMR (126 MHz, DMSO) δ 165.52, 157.53, 145.51, 137.86, 125.34, 117.93, 107.25. APCI-MS: m / z 230.9 [M+H]. HPLC retention time: 5.99 min. HPLC purity: 96.4% under vacuo to yield the product (322 mg, 1.40 mmol, 90%) as an off-white solid.1H NMR (500 MHz, DMSO) δ 10.23 (s, 1H), 8.17 (s, 2H), 7.92 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 9.0, 2.4 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 165.52, 157.53, 145.51, 137.86, 125.34, 117.93, 107.25. APCI-MS: m / z 230.9 [M+H]. HPLC retention time: 5.99 min. HPLC purity: 96.4%.6-(ethoxy-d5)benzo[d]thiazole-2-sulfonamide (CAI0112):

[0145] Step 1: To a stirring mixture of 4-amino-3-chlorophenol (1.00 g, 1 Eq, 6.97 mmol) in THF (10 mL) and water (5.0 mL) was added dropwise acetic anhydride (782 mg, 724 µL, 1.1 Eq, 7.66 mmol) at room temperature. The reaction was allowed to stir for 1.5 hours, then was poured into water and extracted with ether three times. The combined organic layers were washed with NaHCO3, then brine, then dried over Na2SO4. The organic layer was then concentrated in vacuo to afford the off-white product (995 mg, 5.36 mmol, 77%).

[0146] Step 2: To an argon-flushed round bottom flask containing N-(2-chloro-4- hydroxyphenyl)acetamide (400 mg, 1 Eq, 2.16 mmol) in anhydrous acetone (30 mL) was added potassium carbonate (1.49 g, 5 Eq, 10.8 mmol), then 1-iodoethane-1,1,2,2,2-d5 (694 mg, 2 Eq, 4.31 mmol). The reaction was stirred at reflux for 1 h, then allowed to cool to room temperature. The solution was filtered through Celite, washing with acetone, and the filtrate was concentrated in vacuo to afford the off-white product (353 mg, 1.61 mmol, 75%).

[0147] Step 3: To a stirring solution of N-(2-chloro-4-(ethoxy-d5)phenyl)acetamide (200 mg, 1.0 Eq, 915 µmol) in anhydrous methanol was added HCl in dioxane (4 M, 2.4 mL). the reaction was refluxed under argon for 2.5 hours, then allowed to cool to r.t. The solution was poured into water and basified to pH 9-10 using 3 M NaOH, then extracted with ether. The combined organiclayers were washed with brine and dried over anhydrous Na2SO4. The solution was concentrated in vacuo to afford the product.

[0148] Step 4: using potassium ethyl xanthate (438 mg, 3 Eq, 2.72 mmol) and 2-chloro-4- (ethoxy-d5)aniline (160 mg, 1 Eq, 906 µmol). Isolated as an off-white solid (95 mg, 0.44 mmol, 48%).

[0149] Step 5: ammonium hydroxide (1.6 g, 1.9 mL, 28% Wt, 30 Eq, 13 mmol), sodium hypochlorite (0.48 g, 0.40 mL, 11% Wt, 1.6 Eq, 0.70 mmol), and 6-(ethoxy-d5)benzo[d]thiazole- 2-thiol (95 mg, 1 Eq, 0.44 mmol) in 2.0 M NaOH (0.61 mL). Product was collected as a light brown solid (80 mg, 0.35 mmol, 79%).

[0150] Step 6: S-(6-(ethoxy-d5)benzo[d]thiazol-2-yl)thiohydroxylamine (80 mg, 1 Eq, 0.35 mmol) and potassium permanganate (82 mg, 1.5 Eq, 0.52 mmol). The product (35 mg, 0.13 mmol, 38%) was isolated as a white solid.1H NMR (500 MHz, DMSO) δ 8.22 (s, 2H), 8.00 (d, J = 9.1 Hz, 1H), 7.75 (d, J = 2.7 Hz, 1H), 7.24 – 7.17 (m, 1H).13C NMR (126 MHz, DMSO) δ 166.98, 158.77, 146.72, 138.28, 125.65, 118.55, 106.07. APCI-MS: m / z 263.7 [M+H]. HPLC retention time: 6.541 min HPLC purity: 96.7%. 6-(trifluoromethoxy)benzo[d]thiazole-2-sulfonamide (CAI0113):

[0151] Step 1: To an argon-flushed round-bottom flask was added potassium ethyl xanthate (915 mg, 3 Eq, 5.67 mmol) in anhydrous DMF and the solution was warmed to 150 ˚ C. A solution of 2-chloro-4-(trifluoromethoxy)aniline (400 mg, 262 µL, 1 Eq, 1.89 mmol) dissolved in anhydrous DMF was added to the stirring solution dropwise over an hour. The reaction mixture was then stirred at reflux for 12 hours overnight. The reaction was allowed to cool to 80 ˚ C and stirred into ice-water. The solution was then acidified with concentrated HCl and the resultingprecipitate was collected by filtration to afford the product as an off-white solid (315 mg, 1.25 mmol, 66%).

[0152] Step 2: Ammonium hydroxide (498.2 mg, 0.57 mL, 28% Wt, 2 Eq, 3.980 mmol) was stirred in a vial at 0 ˚ C. A solution of sodium hypochlorite (2.694 g, 2.2 mL, 11% Wt, 2 Eq, 3.980 mmol), and a solution of 6-(trifluoromethoxy)benzo[d]thiazole-2-thiol (500.0 mg, 1 Eq, 1.990 mmol) in 2.0 M NaOH (2.8 mL) were simultaneously added to the vial dropwise at 0 ˚ C over 1 h. Stirring was then allowed for an additional 15 minutes. The mixture was filtered and the solid was rinsed with copious amounts of water to rid any remaining NH4. Product was collected as an off-white solid (394 mg, 1.48 mmol, 74%).

[0153] Step 3: To a stirring mixture of S-(6-(trifluoromethoxy)benzo[d]thiazol-2- yl)thiohydroxylamine (100 mg, 1 Eq, 376 µmol) and potassium carbonate (182 mg, 3.5 Eq, 1.31 mmol) in ethanol (7.5 mL) and water (2.5 mL) was added 3-chlorobenzoperoxoic acid (194 mg, 3 Eq, 1.13 mmol) in ethanol (7.5 mL) over a ten minute period. The reaction was allowed to stir for two hours. The reaction was quenched with sodium thiosulfate (0.1 g) and concentrated in vacuo. The mixture was then taken up in water and acidified with conc. HCl. The solution was extracted three times with ethyl acetate and the combined organic layers were washed with water once, saturated sodium bicarbonate solution twice, then dried over Na2SO4and concentrated in vacuo. The crude product was purified by column chromatography (20-50% EtOAc:Hexanes) to afford the product as a white solid (14 mg, 47 µmol, 12%).1H NMR (500 MHz, DMSO) δ 8.41 (s, 2H), 8.39 – 8.36 (m, 1H), 8.26 (dd, J = 9.0, 3.1 Hz, 1H), 7.67 – 7.62 (m, 1H).13C NMR (126 MHz, DMSO) δ 171.61, 150.87, 147.07, 137.23, 126.17, 121.86, 116.26. APCI-MS: m / z 299.6 [M+H]. HPLC retention time: 6.392 min HPLC purity: 96.0%6-methoxybenzo[d]thiazole-2-sulfonamide (CAI0114s1):

[0154] Step 1: To an argon-flushed round-bottom flask was added potassium ethyl xanthate (1.54 g, 3 Eq, 9.52 mmol) in anhydrous DMF and the solution was warmed to 150 ˚ C. A solution of 2-chloro-4-methoxyaniline (500 mg, 434 µL, 1 Eq, 3.17 mmol) dissolved in anhydrous DMF was added to the stirring solution dropwise over an hour. The reaction mixture was then stirred at reflux for 12 hours overnight. The reaction was allowed to cool to 80 ˚ C and stirred into ice-water. The solution was then acidified with concentrated HCl and the resulting precipitate was collected by filtration to afford the product (326 mg, 1.65 mmol, 52.1%) as a light brown solid.

[0155] Step 2. Ammonium hydroxide (5.71 g, 6.5 mL, 28% Wt, 30 Eq, 45.6 mmol) was stirred in a vial at 0 ˚C. A solution of sodium hypochlorite (1.65 g, 1.4 mL, 11% Wt, 1.6 Eq, 2.43 mmol) and a solution of 6-methoxybenzo[d]thiazole-2-thiol (300 mg, 1 Eq, 1.52 mmol) in 2.0 M NaOH (2.13 mL) were simultaneously added to the vial dropwise at 0 ˚ C over 1 h. Stirring was then allowed for an additional 15 minutes. The mixture was filtered and the solid was rinsed with copious amounts of water to rid any remaining NH4. The off-white solid was collected as the product (225 mg, 1.06 mmol, 70%).

[0156] Step 3. To a stirring mixture of S-(6-methoxybenzo[d]thiazol-2-yl)thiohydroxylamine (100 mg, 1 Eq, 471 µmol) in a solution of acetone (2 mL) and water (2 mL) was added a solution of potassium permanganate (112 mg, 41 µL, 1.5 Eq, 707 µmol) in water (4 mL) portion-wise over 30 min. The reaction mixture was stirred for another hour, then treated with charcoal (200 mg) and 10 M NaOH (0.5 mL), then stirred for another 30 min. The mixture was filtered through celite, and the resulting filtrate was acidified with HCl. The product was extracted with ethyl acetate three times and the combined organic layers were washed with water, dried overNa2SO4, and concentrated in vacuo. The crude product was purified by column chromatography (20-50% EtOAc:Hexanes) to afford the product (29 mg, 0.12 mmol, 25%) as a white solid.1H NMR (500 MHz, DMSO) δ 8.23 (s, 2H), 8.01 (d, J = 9.1 Hz, 1H), 7.78 (d, J = 2.7 Hz,7.22 (dd, J = 9.1, 2.7 Hz, 1H), 3.83 (s, 3H).13C NMR (126 MHz, DMSO) δ 166.66, 159.07, 146.42, 137.86, 125.22, 117.89, 105.16, 56.19. APCI-MS: m / z 244.9 [M+H] HPLC retention time: 6.226 min HPLC purity: 97.0%. 6-fluorobenzo[d]thiazole-2-sulfonamide (CAI0124):

[0157] Step 1: To an argon-flushed round-bottom flask was added potassium ethyl xanthate (1.66 g, 3 Eq, 10.3 mmol) in anhydrous DMF and the solution was warmed to 150 ˚ C. A solution of 2-chloro-4-fluoroaniline (500 mg, 1 Eq, 3.44 mmol) dissolved in anhydrous DMF was added to the stirring solution dropwise over an hour. The reaction mixture was then stirred at reflux for 12 hours overnight. The reaction was allowed to cool to 80 ˚ C and stirred into ice- water. The solution was then acidified with concentrated HCl and the resulting precipitate was collected by filtration to afford the product as a light orange solid (432 mg, 2.33 mmol, 68%).

[0158] Step 2: Ammonium hydroxide (3.38 g, 3.8 mL, 28% Wt, 20 Eq, 27.0 mmol) was stirred in a vial at 0 ˚ C. A solution of sodium hypochlorite (1.46 g, 1.2 mL, 11% Wt, 1.6 Eq, 2.16 mmol and a solution of 6-fluorobenzo[d]thiazole-2-thiol (250 mg, 1 Eq, 1.35 mmol) in 2.0 M NaOH (2.9 mL) were simultaneously added to the vial dropwise at 0 ˚ C over 1 h. Stirring was then allowed for an additional 15 minutes. The mixture was filtered and the solid was rinsed with copious amounts of water to rid any remaining NH4. Product was collected as an off-white solid (180 mg, 0.899 mmol, 67%).

[0159] Step 3: To a stirring mixture of S-(6-fluorobenzo[d]thiazol-2-yl)thiohydroxylamine (160 mg, 1 Eq, 799 µmol) and potassium carbonate (361 mg, 3.27 Eq, 2.61 mmol), in ethanol (7.5 mL) and water (2.5 mL) was added 3-chlorobenzoperoxoic acid (414 mg, 3 Eq, 2.40 mmol) in ethanol over a ten minute period. The reaction was allowed to stir for two hours. The reaction was quenched with sodium thiosulfate (0.1 g) and concentrated in vacuo. The mixture was then taken up in water and acidified with conc HCl. The solution was extracted three times with ethyl acetate and the combined organic layers were washed with water once, saturated sodium bicarbonate twice, then dried over Na2SO4and concentrated in vacuo to afford the product (39 mg, 0.17 mmol, 21%) as a white solid.1H NMR (500 MHz, DMSO) δ 8.35 (s, 2H), 8.18 (ddd, J = 9.1, 4.9, 1.5 Hz, 1H), 8.13 (dt, J = 8.7, 2.1 Hz, 1H), 7.52 (td, J = 9.1, 2.6 Hz, 1H).13C NMR (126 MHz, DMSO) δ 169.82, 161.04 (d, J = 245.8 Hz), 148.98, 137.40 (d, J = 11.9 Hz), 126.26 (d, J = 9.7 Hz), 116.90 (d, J = 25.5 Hz), 109.76 (d, J = 27.7 Hz). APCI-MS: m / z 232.9 [M+H]. HPLC retention time: 6.254 min HPLC purity: 97.6%. 5,6-difluorobenzo[d]thiazole-2-sulfonamide (CAI0127):

[0160] Step 1: To an argon-flushed round-bottom flask was added potassium ethyl xanthate (1.48 g, 3 Eq, 9.17 mmol) in anhydrous DMF and the solution was warmed to 150 ˚ C. A solution of 2-chloro-4,5-fluoroaniline (500 mg, 0.33 mL, 1 Eq, 3.06 mmol) dissolved in anhydrous DMF was added to the stirring solution dropwise over an hour. The reaction mixture was then stirred at reflux for 12 hours overnight. The reaction was allowed to cool to 80 ˚ C and stirred into ice-water. The solution was then acidified with concentrated HCl and the resulting precipitate was collected by filtration to afford the product as a light brown solid (429 mg, 2.11 mmol, 69%).

[0161] . Step 2: Ammonium hydroxide (4.62 g, 5.2 mL, 28% Wt, 30 Eq, 36.9 mmol) was stirred in a vial at 0 ˚ C. A solution of sodium hypochlorite (1.33 g, 1.1 mL, 11% Wt, 1.6 Eq, 1.97 mmol), and a solution of 5,6-difluorobenzo[d]thiazole-2-thiol (250 mg, 1 Eq, 1.23 mmol) in 2.0 M NaOH (2.07 mL) were simultaneously added to the vial dropwise at 0 ˚ C over 1 h. Stirring was then allowed for an additional 15 minutes. The mixture was filtered and the solid was rinsed with copious amounts of water to rid any remaining NH4. Product was collected as an off-white solid (189 mg, 0.866 mmol, 70%)

[0162] Step 3: To a stirring mixture of S-(5,6-difluorobenzo[d]thiazol-2-yl)thiohydroxylamine (100 mg, 1 Eq, 458 µmol) in a solution of acetone (2 mL) and water (2 mL) was added a solution of potassium permanganate (109 mg, 1.5 Eq, 687 µmol) in water (4 mL) portion-wise over 30 min. The reaction mixture was stirred for another hour, then treated with charcoal (200 mg) and 10 M NaOH (0.5 mL), then stirred for another 30 min. The mixture was filtered through celite, and the resulting filtrate was acidified with HCl. The product was extracted with ethyl acetate three times and the combined organic layers were washed with water, dried over Na2SO4, and concentrated in vacuo. The reaction mixture was stirred for another hour, then treated with charcoal (200 mg) and 10 M NaOH (0.5 mL), then stirred for another 30 min. The mixture was filtered through celite, and the resulting filtrate was acidified with HCl. The product was extracted with ethyl acetate three times and the combined organic layers were washed with water, dried over Na2SO4, and concentrated in vacuo. The product (36 mg, 0.14 mmol, 31%) was isolated as a white solid.1H NMR (500 MHz, DMSO) δ 8.35 (s, 2H), 8.29 (dd, J = 8.9, 5.4 Hz, 1H), 8.06 – 7.96 (m, 1H), 7.53 (td, J = 9.0, 2.5 Hz, 1H).13C NMR (126 MHz, DMSO) δ 171.77, 112.49 (d, J = 19.5 Hz), 111.53 (d, J = 23.0 Hz). APCI-MS: m / z 251.0 [M+H]. HPLC retention time: 6.502 min HPLC purity: 98.1%.5-difluorobenzo[d]thiazole-2-sulfonamide (CAI0128):

[0163] Step 1: To an argon-flushed round-bottom flask was added potassium ethyl xanthate (1.66 g, 3 Eq, 10.3 mmol) in anhydrous DMF and the solution was warmed to 150 ˚ C. A solution of 2-chloro-5-fluoroaniline (500 mg, 1 Eq, 3.44 mmol) dissolved in anhydrous DMF was added to the stirring solution dropwise over an hour. The reaction mixture was then stirred at reflux for 12 hours overnight. The reaction was allowed to cool to 80 ˚ C and stirred into ice- water. The solution was then acidified with concentrated HCl and the resulting precipitate was collected by filtration to afford the product as a light orange solid (454 mg, 2.45 mmol, 71%).

[0164] Step 2: Ammonium hydroxide (2.03 g, 2.3 mL, 28% Wt, 20 Eq, 16.2 mmol) was stirred in a vial at 0 ˚ C. A solution of sodium hypochlorite (877 mg, 0.73 mL, 11% Wt, 1.6 Eq, 1.30 mmol), and a solution of 5-fluorobenzo[d]thiazole-2-thiol (150 mg, 1 Eq, 810 µmol) in 2.0 M NaOH (1.74 mL) were simultaneously added to the vial dropwise at 0 ˚ C over 1 h. Stirring was then allowed for an additional 15 minutes. The mixture was filtered and the solid was rinsed with copious amounts of water to rid any remaining NH4. Product was collected as an off-white solid (112 mg, 0.559 mmol, 69%).

[0165] Step 3: To a stirring mixture of S-(5-fluorobenzo[d]thiazol-2-yl)thiohydroxylamine (100 mg, 1 Eq, 499 µmol) in a solution of acetone (2 mL) and water (2 mL) was added a solution of potassium permanganate (118 mg, 1.5 Eq, 749 µmol) in water (4 mL) portion-wise over 30 min. The reaction mixture was stirred for another hour, then treated with charcoal (200 mg) and 10 M NaOH (0.5 mL), then stirred for another 30 min. The mixture was filtered through celite, and the resulting filtrate was acidified with HCl. The product was extracted with ethyl acetate three times and the combined organic layers were washed with water, dried over Na2SO4, and concentratedin vacuo. The crude product was purified by column chromatography (20-50% EtOAc:Hexanes) to afford the product (34 mg, 0.15 mmol, 29%) as a white solid.1H NMR (500 MHz, DMSO) δ 8.35 (s, 2H), 8.29 (dd, J = 8.9, 5.4 Hz, 1H), 8.02 (dd, J = 9.7, 2.7 Hz, 1H), 7.53 (td, J = 9.2, 2.6 Hz, 1H).13C NMR (126 MHz, DMSO) δ 172.33, 131.94, 125.20, 116.73 (d, J = 24.7 Hz), 110.41 (d, J = 24.2 Hz). APCI-MS: m / z 232.9 [M+H] HPLC retention time: 6.257 min HPLC purity: 99.2%. Testing of Analog Compound

[0166] Following the methods of Gao, S. et al. (Development and validation of an UPLC- MS / MS method for the quantification of ethoxzolamide in blood, brain tissue, and bioequivalent buffers: applications to absorption, brain distribution, and pharmacokinetic studies. J. Chromatogr. B, Anal. Technol. Biomed. life Sci.986–987, 54–59 (2015)) and Maren, T. H. (Carbonic anhydrase: chemistry, physiology, and inhibition. Physiol. Rev.47, 595–781 (1967)), the analog compounds were tested for activity. In vitro anti-gonococcal activity

[0167] Analogs were evaluated for antibacterial activity by measuring MICs (Table 2) against two CDC N. gonorrhoeae clinical isolates and N. gonorrhoeae FA1090 strain that has previously been used in the in vivo mouse models of gonococcal genital tract infections. (Abutaleb, N. S.; Elhassanny, A. E. M.; Seleem, M. N. In Vivo Efficacy of Acetazolamide in a Mouse Model of Neisseria Gonorrhoeae Infection. Microb Pathog 2022, 164, 105454; (62); Song, W.; Condron, S.; Mocca, B. T.; Veit, S. J.; Hill, D.; Abbas, A.; Jerse, A. E. Local and Humoral Immune Responses against Primary and Repeat Neisseria Gonorrhoeae Genital Tract Infections of17beta-Estradiol-Treated Mice. Vaccine 2008, 26 (45), 5741–5751; Jerse, A. E.; Wu, H.; Packiam, M.; Vonck, R. A.; Begum, A. A.; Garvin, L. E. Estradiol-Treated Female Mice as Surrogate Hosts for Neisseria Gonorrhoeae Genital Tract Infections. Front Microbiol 2011, 2, 107; E., J. A. Experimental Gonococcal Genital Tract Infection and Opacity Protein Expression in Estradiol-Treated Mice. Infect Immun 1999, 67 (11), 5699–5708, each incorporated herein by reference.)36,62–64EZM exhibited MIC values ranging from 232 – 484 nM (0.06 – 0.125 µg / mL), while MICs for the synthesized analogs ranged from 65 nM – 4µM (0.015 – 1 µg / mL). Several analogs exhibited improved antimicrobial activity compared to EZM. The primary metabolite 1 maintained antibacterial activity with MICs ranging from 65 – 543 nM (0.015 – 0.125 µg / mL). The most potent analog across all four isolates was 2 with MICs ranging from 70 – 280 nM (0.015 – 0.06 µg / mL). The 6-d5-ethoxy (6) and methoxy (7) analogs were equipotent against the N. gonorrhoeae strains compared to EZM. Swapping the methoxy from 7 for trifluoromethoxy in 8 resulted in reduced potency against all four strains with activity versus FA1090 dropping by 16-fold. Out of the three fluorinated benzothiazole derivatives, the 6-F analog 9 performed the best across the four strains with MICs ranging from 129 – 538 nM (0.03 – 0.125 µg / mL). This was followed the 5-F analog 10 that maintained 129 nM activity versus FA1090 but was less active toward strains CDC 178 and CDC 181 compared to 9. Finally, the 5,6-di-F analog 11 was the weakest in terms of anti- gonococcal potency of the entire analog set with MICs ranging from 2 to 4µM (0.5 to 1 µg / mL). Analogs were also tested against N. gonorrhoeae strains in 5% CO2 conditions to validate on-target activity versus the intracellular carbonic anhydrases. CO2is a substrate of carbonic anhydrases, and at elevated levels of the gas may outcompete the molecule resulting in reduced susceptibility to CA inhibitors.30,65Alternatively, it is known that CO2and water can spontaneously react to produce bicarbonate in solution and that at higher CO2 levels,enough bicarbonate is produced from this process to mitigate the need for the CA activity.66In either scenario, if a molecule’s antibacterial effect is mediated via CA inhibition then the bacteria will exhibit reduced susceptibility to the molecules at 5% CO2. As expected, EZM and its analogs were shown to no longer be effective at the highest concentration tested (64 µg / mL) in 5% CO2conditions (Table 2), suggesting the antibacterial response is mediated, at least in part, by CA inhibition. Importantly, controls azithromycin (AZI) and ceftriaxone (CEF), which each have different mechanisms of action for antibacterial activity other than CA inhibition, were tested in both ambient air and elevated CO2conditions and did not display differential susceptibility between the two conditions as previously reported,35suggesting no global change to the cells in presence of 5% CO2that broadly reduced potency of multiple classes of antibiotics. These data suggest the CAs could be the intracellular targets of the EZM-based analogs.

[0168] Two of the most potent analogs 2 and 9, along with EZM, AZI, and CEF, were tested against a broader panel of multidrug-resistant N. gonorrhoeae clinical isolates (Table 3). Compounds 2 and 9 demonstrated potent activity against all the tested strains, with MICs ranging from 0.008 to 0.06 µg / mL. The analogs were capable of inhibiting the growth of 50% and 90% of the tested isolates (MIC50 and MIC90 values, respectively) at concentrations of 0.03 and 0.06 µg / mL, respectively. These values were 2- to 4-fold lower than the MIC50 / MIC90 value of EZM (0.125 µg / mL). Interestingly, the MIC values of 2 and 9 were comparable to that of the standard of care CEF. CEF inhibited 90% of the tested isolates at 0.125 µg / mL, though it inhibited the growth of some strains at concentrations lower that 0.001 µg / mL. The EZM-based analogs were 16-fold more potent than CEF against WHO-X, a strain previously identified to have high-level resistance against CEF.67,68AZI was less potent against the panel, with MIC50and MIC90 values of 0.5 and 1, respectively. As expected, AZI had elevated MICs against CDC 181, WHO-P, and WHO-U strains, which have previously been shown to exhibit AZI resistance67,68, while EZM, 2, and 9 were potent against these strains. In vitro carbonic anhydrase inhibition

[0169] Improvement of pharmacokinetic properties while maintaining antimicrobial activity was the primary goal for this study and the driver for our decision process to improve the outcome in the N. gonorrhoeae mouse model. However, in addition to antimicrobial testing, analogs were assessed for their activity against both the α- and β- NgCA. Additionally, with respect to potential adverse effects that may arise from translation to in vivo studies, the molecules were also evaluated against five human carbonic anhydrases. The human isoforms hCAI and hCAII were selected because these are the predominant isoforms observed in human erythrocytes and gastrointestinal tract16and their binding may affect in vivo drug distribution and plasma concentration. Isoforms hCAIV, hCAXII, and hCAXIV were selected because they areexpressed in the kidney, along with hCAII, and are linked to thediuretic effects of CAinhibitors.69–71The inhibition constants (Ki) were collected by measuring the CO2hydration activity of all CAs with dose-response with inhibitor as previously described (Marapaka, A. K.; Nocentini, A.; Youse, M. S.; An, W.; Holly, K. J.; Das, C.; Yadav, R.; Seleem, M. N.; Supuran, C. T.; Flaherty, D. P. Structural Characterization of Thiadiazolesulfonamide Inhibitors Bound to Neisseria Gonorrhoeae Α-Carbonic Anhydrase; Hewitt, C. S.; Abutaleb, N. S.; Elhassanny, A. E. M.; Nocentini, A.; Cao, X.; Amos, D. P.; Youse, M. S.; Holly, K. J.; Marapaka, A. K.; An, W.; Kaur, J.; Krabill, A. D.; Elkashif, A.; Elgammal, Y.; Graboski, A. L.; Supuran, C. T.; Seleem, M. N.; Flaherty, D. P. Structure-Activity Relationship Studies of Acetazolamide-BasedCarbonic Anhydrase Inhibitors with Activity against Neisseria Gonorrhoeae. ACS Infect Dis 2021, 7 (7), 1969–1984 and Abutaleb, N. S.; Elhassanny, A. E. M.; Nocentini, A.; Hewitt, C. S.; Elkashif, A.; Cooper, B. R.; Supuran, C. T.; Seleem, M. N.; Flaherty, D. P. Repurposing FDA-Approved Sulphonamide Carbonic Anhydrase Inhibitors for Treatment of Neisseria Gonorrhoeae. J Enzyme Inhib Med Chem 2022, 37 (1), 51–61; Nocentini, A.; Hewitt, C. S.; Mastrolorenzo, M. D.; Flaherty, D. P.; Supuran, C. T. Anion Inhibition Studies of the α- Carbonic Anhydrases from Neisseria Gonorrhoeae. J Enzyme Inhib Med Chem 2021, 36 (1), 1061–1066; Giovannuzzi, S.; Marapaka, A. K.; Abutaleb, N. S.; Carta, F.; Liang, H.-W.; Nocentini, A.; Pisano, L.; Seleem, M. N.; Flaherty, D. P.; Supuran, C. T. Inhibition of Pathogenic Bacterial Carbonic Anhydrases by Monothiocarbamates. J Enzyme Inhib Med Chem 2023, 38 (1), each incorporated herein by reference.).29,35,37,72,73

[0170] In general, the molecules were more potent against the human isoforms over the bacterial CAs, and the potency trends observed across the analogs against α-NgCA were similar for the human isoforms (Table 4). This is somewhat expected as the human isoforms also belong to the α -CA class of enzymes. Most analogs displayed modest reduction of bacterial CA activity compared to EZM except for the deuterated ethoxy analog 6, which was equipotent demonstrating little-to-no change in activity upon swapping hydrogens for deuterium. Theprimary metabolite phenol 1 (α-NgCA Ki= 131 nM; β-NgCA Ki= 517 nM) and thebenzothiazole derivative 2 (α-NgCA Ki= 270 nM; β-NgCA Ki= 794 nM) each displayed astepwise decrease in potency versus both NgCAsdemonstrating the value of the ethoxy andphenol functionalities toward binding to the NgCAs. The same wasobserved against hCAI andhCAII. Restoring a methoxy (7,α-NgCA Ki= 125 nM; β-NgCA Ki= 558 nM) andtrifluoromethoxy (8,α-NgCA Ki= 105 nM; β-NgCA Ki= 605 nM) groups at the 6-positionimproved potencyagainst α-NgCA, back in line with EZM and 1 further supporting a role inthe O-alkylation for binding between this CA isoform. However, the improvement of activity was less pronounced against the β-NgCA and did not reach the same potency as observed withEZM. Addition of one or two fluorine atoms to the benzothiazole asdepicted by analogs 9 (6-F;α-NgCA Ki= 222 nM; β-NgCA Ki= 695 nM), 10 (5-F;α-NgCA Ki= 249 nM; β-NgCAKi= 701 nM), 11 (5,6-di-F; α-NgCA Ki= 169 nM; 3-NgCA Ki= 674 nM) all similar, or slightlyimproved,in activity against both NgCAs compared to the benzothiazole analog 2 but were lessactive than both EZM and 1. Overall, the SAR for changes to bacterial CA inhibition did not trend with the SAR observed for antibacterial potency. A few possible explanations for this will be addressed in the discussion section.

[0171] As mentioned, the SAR for α-NgCA activity generally correlated with changes to hCA activity. Inhibition of hCAII and α-NgCA correlated the closest when comparing the bacterial CA to the hCAs (Table 4). This is to be expected, as hCAII and α-NgCA are the most similar within their active sites.28,29The two enzymes maintain the same amino acid residues involved in binding to CA inhibitors: Gln90, Thr199, and Thr200 of hCAII, and Gln92, Thr177, and Thr178 in α-NgCA. Alternatively, the hCAI isoform has a histidine (His200) in place of Thr200 from hCAII. This alters the binding interactions with the CA inhibitors and may be a reason for some observed differences in the SAR trends between α-NgCA and hCAI. The analogs showed comparable changes in potency against the three kidney hCA isoforms tested as well with a few exceptions. Interestingly, as compared to EZM, the dealkylated phenol derivative 1 was approximately 4- to 18-fold less potent against hCAI, hCAII, and hCAXIV but was 2-to-5-fold more active against the kidney isoforms hCAIV and hCAXII suggesting a role in binding for the unmasked phenol against these enzymes. This is also notable as it was previously speculatedthat EZM can provide the desired biological effect despite being O-dealkylated because the primary metabolite 1 still maintained activity versus the relevant hCAs in the human body.34Themolecules that displayed the largestdecrease in activity across all five hCAs were 2 and 9.Analog 2 demonstrated hCA Kivalues in the range 59 –423 nM, which represents 4.5- to 23-fold decrease in activity against the hCAs compared to EZM. Similarly,analog 9 demonstratedhCA Kivalues range of 62 – 306 nM, representing 2- to 25-fold decrease in activitycomparedto EZM. These two analogs were among the most potent against N. gonorrhoeae FA1090 with MICs of 70 and 129 nM, respectively. While it would be desirable for analogs to be more potent against the pathogen than against the hCAs, the fact that they are at least comparable in these values represents an improvement over EZM and the potential to have reduced off-target diuretic effects that could be undesired in vivo. In vitro ADME Evaluation of EZM and analogs

[0172] As the goal is to develop molecules that will demonstrate oral efficacy in the mouse model of N. gonorrhoeae infection, evaluating parameters such as solubility, Caco-2 permeability, and liver microsome stability will provide data to select analogs to advance to in vivo pharmacokinetic evaluation. EZM and analogs were examined for in vitro ADME properties to understand how the compounds will potentially fare in vivo (Table 5). Both aqueous solubility in phosphate-buffered saline (PBS) at pH 7.4 (1% v / v DMSO) and logD7.4 values were collected for all analogs. Compounds were soluble in PBS (1% v / v DMSO) at pH 7.4 in a range from 107 µM to 196 µM, well over the 10 µM threshold commonly used as a benchmark for drug candidates to be considered for preclinical testing.74 These data suggest solubility of the analogs will likely not be a limiting factor for advancement. The LogD7.4 wasmeasured via the shake-flask method in PBS / 1-octanol and all analogs maintained LogD7.4 values between 0.81 and 2.77 indicating they remain in the drug-like chemical space.75–77Comparing the analogs’ logD7.4 values reveals that their variable functional groups follow the general trends of lipophilicity found in the literature. The OCF3 is reported to contribute the most to lipophilicity, followed by OEt, then F and OMe, and lastly OH, which significantly decreases lipophilicity.77Likewise, OCF3 analog 8 had the highest logD7.4 value, followed by EZM and 6. The di-fluorinated analog 11 had a slightly higher logD7.4 than the monofluorinated analogs 9 and 10, though the methoxy analog 7 was approximately equally lipophilic to 11. As expected, 1 was the least lipophilic due to its hydroxyl group.

[0173] Since the analogs were designed to modulate EZM metabolic liability, all compounds were assessed for their stability in mouse liver microsomes (MLM) (Table 5 and FIG.4). As a baseline, EZM was evaluated and exhibited a decline over 60 minutes with a half-life (t1 / 2) of 23 minutes and 19% of molecule remained after 60 minutes. This resulted in intrinsic clearance (Clint) value of 297.1 µL / min / mg. The remaining molecules all demonstrated improved metabolic stability compared to EZM exhibiting >93% molecule remaining at the 60- minute timepoint except for analog 6. Interestingly, while the stability of deuterated analog 6 was improved compared to EZM, the molecule did not block metabolic activity in MLM completely. This is likely attributed to the kinetic isotope effect previously described with the deuterium reacting slower than hydrogen, but not unreactive, with CYPs in the enzyme atom extraction step.48,50Surprisingly, the methoxy analog 7 was metabolically stable despite differing from EZM by a single methylene. Evaluation of 7 in the P450 Sites of Metabolism tool suggested the CH3would be a site susceptible for CYP oxidation. However, the experimental data in the MLM suggest the methoxy is stable to the CYPs present. At this point, we chose to deprioritize threeanalogs from further evaluation. The deuterated analog 6 was deprioritized due to lower metabolic stability compared to the remaining analogs and both analogs 8 and 11 were deprioritized because these were the least active against the N. gonorrhoeae FA1090 strain used in the mouse model for infection. The remainder of molecules advanced for further testing.

[0174] Since the analogs were designed to modulate EZM metabolic liability, all compounds were assessed for their stability in mouse liver microsomes (MLM) (Table 5 and FIG.4). As a baseline, EZM was evaluated and exhibited a decline over 60 minutes with a half-life (t1 / 2) of 23 minutes and 19% of molecule remained after 60 minutes. This resulted in intrinsic clearance (Clint) value of 297.1 µL / min / mg. The remaining molecules all demonstrated improved metabolic stability compared to EZM exhibiting >93% molecule remaining at the 60- minute timepoint except for analog 6. Interestingly, while the stability of deuterated analog 6 was improved compared to EZM, the molecule did not block metabolic activity in MLM completely. This is likely attributed to the kinetic isotope effect previously described with the deuterium reacting slower than hydrogen, but not unreactive, with CYPs in the enzyme atom extraction step.48,50Surprisingly, the methoxy analog 7 was metabolically stable despite differing from EZM by a single methylene. Evaluation of 7 in the P450 Sites of Metabolism tool suggested the CH3 would be a site susceptible for CYP oxidation. However, the experimental data in the MLM suggest the methoxy is stable to the CYPs present. At this point, we chose to deprioritize three analogs from further evaluation. The deuterated analog 6 was deprioritized due to lower metabolic stability compared to the remaining analogs and both analogs 8 and 11 were deprioritized because these were the least active against the N. gonorrhoeae FA1090 strain used in the mouse model for infection. The remainder of molecules advanced for further testing.

[0175] To further assess our analogs in the context of orally bioavailable and efficacious agents, the analogs were next evaluated for permeability across human colon carcinoma (Caco-2) cell monolayer. The Caco-2 system is a surrogate to estimate molecule absorption across the gastrointestinal tract that can inform for oral bioavailability.78Molecules were evaluated for apparent permeability (Papp) in bidirectional mode, testing both apical to basolateral (A—>B) and basolateral to apical (B—>A) (Table 5 and FIG.4). The donor and receiver chambers were maintained at pH 6.5 and 7.4, respectively, to reflect the pH gradient between the intestinal lumen and blood. EZM had previously been reported to have Papp of 15.9 x 10-6 cm / s (A—>B) and 14.9 x 10-6 cm / s (B—>A) with pH 7.4 in both chambers.79These values would classify EZM at the low end of the ‘highly permeable” classification according to the Food and Drug Administration (FDA).78 Analog 1 was comparable to EZM with Papp values of 14.3 x 10-6 cm / s (A—>B) and 12.3 x 10-6 cm / s (B—>A). The remaining analogs all exhibited a significant increase in Papp,A—>B compared to EZM with values ranging from 31.0 to 67.8 x 10-6 cm / s. The molecules also appeared to display reduced relative Papp,B—>A rates that resulted in efflux ratios (Papp,B—>A / Papp,A—>B) in the range of 0.29 to 0.43. Within the cohort, analog 2 had the highest permeability rate (Papp,A—>B = 67.8 x 10-6 cm / s), followed closely by 7 and 9 with Papp,A—>B = 54.7 and 54.4 x 10-6 cm / s, respectively. These values would place the analogs at the upper end of the “highly permeable” classification with predicted 100% gastrointestinal tract absorption.78 Analog 10 still displayed high Caco-2 permeability (Papp A—>B = 31.0 x 10-6 cm / s) but was the lowest of the four molecules tested. These data suggest the analogs should have good intestinal absorption that could lead to adequate oral bioavailability for testing in in vivo assays.

[0176] Analogs were also tested for in vitro protein binding in mouse plasma and blood-to- plasma ratio (Rb) as these metrics could affect molecule distribution to site of action in vivo (Table 5). EZM was reported to be highly protein bound in human plasma (96%) and rat plasma (94%).30 The mouse plasma protein binding for EZM was determined in this study to be 74% protein bound, somewhat lower than in rat and human, but consistent with trends that indicate molecules on average exhibit lower binding to mouse plasma proteins compared to human.80 Analogs 7, 9, and 10 were comparable to EZM ranging from 72% to 77% bound while 1 and 2 displayed reduced mouse plasma protein binding with 53% and 56%, respectively.

[0177] In the Rb studies, EZM had the lowest Rb at 8:1 or 89% in the red blood cells (RBCs) over the plasma portion, though EZM’s phase I metabolite 1 is the most highly sequestered in RBCs at a ratio of 124:1. Compound 2 partitioned into RBCs 1.7-fold more than EZM for an Rb of 14:1 or 93%, while the Rb for compounds 7 and 9 were more than double that of 2 at 39:1 (98%) and 33:1 (97%). Overall, the results from these in vitro studies revealed that our analogs have favorable properties for drug leads, encouraging us to further investigate our top compounds in vivo. Mouse In vivo Pharmacokinetics Studies

[0178] Based on the MICs versus N. gonorrhoeae FA1090, in vitro ADME and hCA potencies, three lead compounds 2, 7, and 9 were selected to be assessed for in vivo pharmacokinetic evaluation in mice and compared to EZM and its metabolite 1. Female BALB / c mice (n = 3 for each timepoint) were treated with test molecules at a dose of 10 mg / kg by oral gavage and plasma concentrations were determined at eight time points (0.167, 0.5, 1, 2, 4, 6, 8, and 24 h) over 24 hours (Table 6, FIG.5). All analogs had peak plasma concentrations at the 10- minutetimepoint. Of the three new analogs, 9 demonstrated the greatest Cp,max at 3136 ± 673 ng / mL, followed by 2 (2585 ± 680 ng / mL) and 7 (1916 ± 295 ng / mL), all considerable improvements over EZM. When adjusted for the unbound fraction Cp,u,max, analog 2 had the best value (1137 ± 299 ng / mL) by virtue of a lower mouse PPB at 56% compared to 76% and 77% for 7 and 9, respectively. Comparing the areas under the curve at the 24 h timepoint (AUCp,0—>24) for plasma concentration of the three leads revealed analog 9 (4356 ± 1601 h*ng / mL) outperformed both 2 and 7, which had values of 1526 ± 243 and 792 ± 62 h*ng / mL, respectively. When adjusted for the fraction unbound portion in the plasma (AUCp,u,0—>24), analog 9 still maintained an edge over 2, unlike what was observed for the Cp,u,max values, despite the higher mouse PPB. Taken together, these data result in the 2.5- fold lower plasma clearance (CLp) for 9 compared to 2 with values of 41 ± 18 and 108 ± 19 mL / min / kg, respectively. It is reasonable to assume 2 and 9 are cleared by the kidney similar to EZM, thus, this difference in clearance between these two analogs is possibly a result of 9 being more protein bound than 2 as any drug that is not bound to plasma protein will be filtered out by the kidney.81Further comparing our leads revealed that the Vz in mice for EZM and 1 were more than 3-fold higher than the Vz values of 2, 7, and 9, although the Vz values for the new analogs still suggest high distribution to other tissues. Collectively, the in vivo pharmacokinetic data for the new analogs compared to EZM and 1 indicates the molecules possess improved metrics and may exhibit efficacy in the mouse model for N. gonorrhoeae infection. Thus, we prioritized analogs 2 and 9 based on the overall combined profiles of anti-gonococcal activity versus N. gonorrhoeae strains, in vitro ADME, and in vivo pharmacokinetics of the molecules, particularly the higher Cmax and AUC values combined with lower CL values.In vitro cytotoxicity against mammalian cell lines

[0179] After the pharmacokinetics experiments revealed our molecules to have improved properties, we were motivated to further explore the potential for translation to in vivo efficacy of our lead analogs. For this, we prioritized leads 2 and 9 based on the pharmacokinetics metrics and compared them to EZM for cytotoxicity in relevant cell lines using an MTS assay (FIG.6). EZM is known to undergo renal clearance and the kidneys are the site of action for the diuretic effect of carbonic anhydrase inhibitors, therefore, we chose to assess toxicity in monkey kidney fibroblast (Vero) cells to determine how our compounds would affect mammalian kidney cells. All three compounds tested were well-tolerated by the cells and were non-toxic up to 128 µg / mL in Vero cells, showing 100% viability relative to the negative control (DMSO). Cytotoxicity in human endocervical (ME-180) cells was also assessed due to N. gonorrhoeae primarily infecting the cervix. At 32 and 64 µg / mL, 2, 9, and EZM were non-toxic; however, at 128 µg / mL 2 and 9 resulted in less than 20% cell viability while EZM showed slight toxicity at this concentration. Despite this drastic increase in toxicity at 128 µg / mL, this concentration is greater than 1000-fold higher than the MIC values for 2 and 9 and the 100% cell viability at 64 µg / mL still demonstrates that the analogs are highly tolerable. Anti-bacterial Killing Kinetics

[0180] EZM had previously been evaluated for its killing kinetics to better understand its antimicrobial properties in comparison to AZM.37 We therefore sought to measure the killing kinetics of our lead analogs to compare against EZM. The time-kill assay revealed 2 and 9 to have the same bacteriostatic effect as EZM (FIG.7) when dosed at 5× MIC. However, the compounds did reduce N. gonorrhoeae burden after 24 h by approximately 1.0-log10 units andhad a significantly reduced N. gonorrhoeae burden compared to DMSO-treated culture (negative control) at 24 h by 4-log10 units. AZI was used as the positive control and reduced N. gonorrhoeae burden below the limit of detection within 6 h, consistent with previous reports.37,85Frequency of spontaneous mutation

[0181] With the ever-increasing rise of antibiotic resistance in N. gonorrhoeae and a possible future of untreatable gonococcal infections on the horizon, it was important to assess the likelihood of N. gonorrhoeae developing a resistance to our molecules. Using a single step mutation assay, N. gonorrhoeae FA1090 was dosed with compounds at 4× and 8× MIC and investigated for spontaneous mutation frequencies (Table 7). There was no evidence of resistant mutants when exposed to both concentrations of EZM, 2, and 9, indicating that the frequency of mutation is less than 1.5 × 10-9. AZI and CEF yielded low mutation frequencies as well (< 1.5 × 10-9), while rifampicin, used as a positive control, was shown to have a higher mutation frequency (3 – 4.5 × 10-7), in line with previous reports.37,86,87In vivo mouse model for gonococcal infection

[0182] Based on the in vitro antimicrobial activity, the in vitro ADME, and the in vivo pharmacokinetic evaluation, our studies suggest that our lead compounds have the potential to demonstrate in vivo efficacy in the mouse model for N. gonorrhoeae infection. For the assay, female ovariectomized BALB / c mice were infected intravaginally with N. gonorrhoeae FA1090, then treated beginning at two days post-infection with either EZM, 2, or 9 at 15 mg / kg by oral gavage (p.o.) twice daily (b.i.d.) for three consecutive days. In our prior experience with EZM dosing in mice we had observed mice did not tolerate a 50 mg / kg / day oral dose of EZM for threeconsecutive days but were able to tolerate approximately 30 mg / kg dose (unpublished results). Based on the plasma concentrations for analogs 2 and 9 showing the unbound fractions dropped below the MIC at 4 hours we opted for a twice a day dosing schedule. Without knowing the PK / PD drivers for this scaffold at this stage of the project, we used this data to at least provide an estimate of dose size and regimen, with the caveat that molecule distribution to other tissues or unmetabolized molecule in the urine likely also have an impact of efficacy. We sought to have a direct comparator cohort with EZM for the analogs 2 and 9, this led us to select a 15 mg / kg dose p.o. and b.i.d. to provide the adequate exposure and increase the likelihood that mice would tolerate EZM in the cohort. CEF was used as a positive control and vehicle was the negative control. CEF, as previously reported, could clear gonococcal infection after a single I.P. dose.2,36After three days, 9 significantly reduced gonococcal burden in infected mice by 90% (1.0-log10 reduction) compared to the vehicle, while 2 reduced gonococcal burden by 95% (1.3-log10 reduction) (FIG.8). Remarkably, treatment with analog 2 has resulted in the decrease in N. gonorrhoeae bioburden up to 3-log10 reduction in 50% of the mice (three mice), indicating the possibility of clearance of the infection if treatment was extended. Contrarily, mice treated with EZM did not show significant reduction in the bacterial load and the count remained similar to that of the vehicle-treated mice (negative control). The CFU / mL counts for vehicle-treated mice were sustained for the duration of the study in the 105 range, providing evidence that the significant reduction in N. gonorrhoeae observed in mice treated with 2 and 9 could be attributed to the molecule treatment. It was also observed during the experiment that mice treated with EZM displayed adverse side effects such as hypothermia, lethargy, and dehydration while animals treated with analogs 2 and 9 did not exhibit these side effects. As mentioned, this was previously observed in a separate study as mentioned above when dosing mice with 50 mg / kgEZM (unpublished results). Further investigation is necessary to determine cause of the differential side effect profile, however, at the current dosing regimen analogs 2 and 9 appear to be well-tolerated.

[0183] This study sought to modify the human CA inhibitor, EZM, by designing analogs with improved metabolic stability and in vivo pharmacokinetic metrics to translate toward orally efficacious leads for the treatment of N. gonorrhoeae infections. Previous studies have shown that the human CA inhibitor, AZM, demonstrated efficacy in reducing N. gonorrhoeae burden in an in vivo mouse model of gonococcal infection.36 During this study, we observed that EZM was not effective in vivo despite being 16-fold more potent in vitro against multiple N. gonorrhoeae isolates.37 In the current study, we hypothesized the difference in efficacy between AZM and EZM may be attributed to the differences in metabolic fate between the two molecules. Therefore, the analogs were designed to modulate the metabolism of EZM and subsequently improve the in vivo pharmacokinetics and, in turn, oral efficacy against N. gonorrhoeae in a mouse model.

[0184] It was noted in the in vitro CA inhibition section that MIC data did not correlate with inhibition of α-NgCA and / or β-NgCA inhibition. This may be explained in a few ways. First, the polypharmacology of inhibiting two enzymes likely complicates the correlation to whole cell data, especially when there is little data on the degree to which each enzyme is expressed and / or essential. Evidence suggesting N. gonorrhoeae encodes a γ-CA isoform further complicates this matter, as it is unknown if γ-NgCA is expressed and whether our analogs would inhibit this enzyme as well. Second, as reported by Rubin et al.,15 many N. gonorrhoeae strains possess a hypomorphic Glu19Gly single-point mutation to the β-NgCA. The Gly19 mutation reduces, but does not kill, the catalytic activity of β-NgCA and confers the strains’ dependence on CO2 togrow. This suggests that reduction of the catalytic activity of β-NgCA, but not full inhibition, may be sufficient to affect N. gonorrhoeae growth in non-CO2growth conditions. Thus, there may be a level of β-NgCA inhibition that is sufficient to sustain antimicrobial activity and the Ki values for the analogs still meet this threshold. A third reason for potential non-correlation of MIC and NgCA Ki values could be differences in permeability among analogs which may affect access to the intracellular target(s). The true answer may be any one reason or likely a combination. All three scenarios require additional investigation that are outside the scope of the current study. Regardless, the MIC potency was the main driver for selection of lead molecules to advance.

[0185] Oral bioavailability is dictated by several factors including aqueous solubility, Phase I metabolism, permeability, and susceptibility to efflux.88Toward this goal, we designed the analogs of the EZM core for predicted improvement in metabolic stability. All analogs were evaluated for antibacterial activity versus N. gonorrhoeae strains, in vitro bacterial and human CA inhibition, and MLM stability to down-select potential leads to transition to in vivo pharmacokinetics and ultimately in vivo efficacy. These studies revealed that most molecules maintained or improved the anti-gonococcal activity compared to EZM and all molecules demonstrated greater liver microsome stability over the 1-hour incubation. The four analogs that demonstrated combined improvements in potency and liver microsome stability, plus the primary metabolite 1, were assessed for bidirectional permeability in the Caco-2 model. These molecules showed significantly greater Papp values than both EZM or 1 with an increased rate in the A->B direction over the B—>A direction. EZM displayed a Papp,A—>B value of 15.9 × 10-6 cm / s, which is considered highly Caco-2 permeable and is predicted to have at least 90% intestinal absorption.78 Analogs 2, 7, and 9 possess Papp,A —>B values > 40 × 10-6 cm / s and aretherefore predicted to have around 100% intestinal absorption.78 The efflux ratios for EZM and 1 were close to 1, indicating these molecules are likely to traverse the Caco-2 monolayer via passive diffusion or via active transport where the net flux is equal to transport in each direction.89 Interestingly, the new analogs demonstrated efflux ratios of 0.29 – 0.43, indicating greater than 2-to-1 permeability rates in the A—>B direction over the B—>A. This may be a result of increased active uptake in the A—>B direction, though further investigation is necessary to determine flux rate and the potential transporters that would result in increased uptake for these analogs though they are structurally similar to EZM.

[0186] Three analogs (2, 7, and 9) were selected for evaluation for plasma concentration in mice after oral administration. All three exhibited improved pharmacokinetic parameters after oral administration with > 6-fold in Cmax and >3-fold improvement in AUC compared to EZM. There is likely a combination of variables that contribute to the increase in plasma exposure such as improvements to both intestinal absorption and first-pass hepatic metabolism already discussed above. Additionally, EZM is known to undergo renal clearance and it is feasible that the leads also go through the same excretion pathway. If this is the case, there is also a possibility of differences in the rate of renal excretion among the analogs that could be a contributing factor to the prolonged plasma exposure for 2, 7, and 9 compared to EZM. Moreover, as mentioned previously, CA inhibitors have a propensity to partition into erythrocytes and bind to hCAI and II.45,46This is indeed the case for the leads reported here as evidenced by the Rb data that showsinto the erythrocytes at high percentages. Thus, measuring the plasma concentration underrepresents the total molecule present in the blood stream84and the levels of leads 2, 7, and 9 would likely be much greater than EZM if whole blood was the medium analyzed. Further studies to determine the role of blood partitioning and assessing invivo pharmacokinetics on whole blood would provide more insight into the properties of the molecule being cleared from each medium.84,45,46

[0187] A final pharmacokinetic metric that was different between EZM and the three analogs was the volume of distribution at terminal phase of elimination (VZ). The VZ value for EZM was 144 ± 58 L / kg, indicating the molecule is heavily distributed to tissues over the plasma.92 The three lead analogs had reduced VZ values ranging from 30 – 40 L / kg, which still indicates high tissue distribution, but to a lesser degree than EZM. Although the molecules are quite similar in terms of chemical structure, there are multiple factors that could explain the differences in VZ. First, modulation of lipophilicity influences volume of distribution, with increased logD7.4 correlating with greater volume of distribution.92EZM was the most lipophilic of the four molecules discussed, so this metric alone could explain the observed higher VZ. A second factor to consider is the broad CA inhibitory activity for EZM. Mammals have sixteen CA isoforms ubiquitously expressed in all tissues,16 and generally mouse and human isoforms are highly similar.16,93,94Therefore, one would not expect large differences in inhibitor potency for mouse versus human CAs. EZM is broadly potent against twelve isoforms that have been recombinantly expressed and purified, with Ki values < 100 nM against all twelve.16Thus, it is reasonable to suggest that EZM may readily distribute into tissues and is then sequestered by virtue of binding to the ubiquitously expressed CA isoforms, similar to what is observed in red blood cells via potent binding to hCA I and II.95,96 Analogs 2, 7, and 9 exhibited reduced hCA binding, which may also translate to murine CAs and could therefore result in reduced sequestration in tissues. Thus, differences in logD7.4 and broad CA inhibition could both contribute to the lower VZ observed for analogs 2, 7, and 9.

[0188] Based on the combined data, analogs 2 and 9 were nominated to be carried forward into the mouse model for N. gonorrhoeae infection. Each molecule demonstrated the ability to reduce N. gonorrhoeae bioburden by at least 1.0-log10 CFU compared to both vehicle and EZM-treated control groups when dosed at 15 mg / kg p.o. b.i.d. for three consecutive days. These results are promising and suggest the initial hypothesis— that modulating pharmacokinetic parameters of EZM to improve metabolic stability and exposure will improve in vivo efficacy— was true. Additionally, the results for the 2 and 9 displayed the comparable or slightly better in vivo efficacy compared to a prior study with AZM, in which the molecule was dosed at 50 mg / kg and resulted in 1-log10 CFU reduction of gonococcal burden in mice at day three.97

[0189] While the results are promising, there are still questions that remain to be addressed that may be able to improve the efficacy. It remains to be investigated how the molecules are distributed in tissues at the vaginal site of infection and how they are cleared. One could argue that drugs that have lower volumes of distribution and that undergo renal clearance with a predominantly unmetabolized form would be more suited for treatment of gonococcal infections in the urogenital tissues. In fact, the current drug of choice to treat gonorrhea, CEF, represents this profile. Depending on the study, after intramuscular or intravenous injection CEF typically has a volume of distribution ranging from 4.5 – 13.3 L,98,99with one study in critically ill patients showing higher distribution at 20 L,100 and has renal clearance with up to 40% of the accumulated dose in the urine unchanged at 48 hours.98,100The volumes of distribution suggest some distribution to the tissues, but the accumulated dose in the urine coupled with the potent bactericidal activity of CEF make it effective as a single-dose intramuscular injection. These attributes may be partially responsible for the efficacy observed for AZM as well. Even though the MIC for AZM against N. gonorrhoeae FA1090 is 8-fold less potent than EZM, the moleculedemonstrated modest reduction of N. gonorrhoeae bioburden in the same model with 50 mg / kg oral dose over 3 days while EZM did not at the same dosing regimen. This is may due to the pharmacokinetic properties of AZM, such as a lower volume of distribution (0.2 – 0.4 L / kg)38,101and clearance by the kidney in which 74% of the unmetabolized form is excreted in the urine within the first 4 hours.102–104Thus, despite having a higher MIC, the molecule may better concentrate at the site of infection. Future research will evaluate the tissue distribution of the EZM analogs, clearance mechanisms including concentration in urine, and pharmacokinetics in whole blood, all toward optimization of dosing to improve efficacy. Additional analog design may improve the pharmacokinetic profile to further reduce the tissue distribution while maintaining potency against the pathogen.

[0190] We have shown a cohort of EZM-based CA inhibitors that exhibited potent anti- gonococcal activity in vitro and in vivo. Our compounds exhibited sustained or improved antimicrobial activity against N. gonorrhoeae with analog 2 exhibiting an MIC value as low as 0.015 µg / mL, a 4-fold improved potency compared to EZM. The analogs were also shown to inhibit their presumed intracellular target NgCA, exhibiting Kis ranging from 96.8 to 249.3 nM against the α isoform and from 425.3 to 794.3 nM against the β isoform. Importantly, seven of the eight analogs had decreased potency against the human CAs compared to EZM, which may result in reduced off-target binding in vivo. Compounds were designed with the intent of evading in vivo metabolism, as we hypothesized that EZM’s inefficacy in N. gonorrhoeae-infected mice could be attributed to its metabolic fate. Lead compounds 2, 7, and 9 exhibited lower efflux ratios, reduced plasma protein binding, and had lower volumes of distribution and clearance in vivo. Remarkably, the mono-fluorinated analog 9 extended the in vivo half-life to 10 hours, a 4- fold increase compared to that of EZM. Additionally, these analogs exhibited low cytotoxicityagainst mammalian cell lines and resulted in low spontaneous mutation frequencies against N. gonorrhoeae, further emphasizing their potential to be anti-gonococcal agents. A proof-of- concept study demonstrated oral dosing of 2 and 9 in infected mice reduced the gonococcal burden by 90% and 95%, respectively, compared to EZM which had no effect compared to vehicle.

[0191] The current landscape of N. gonorrhoeae treatment is limited to single intramuscular dose of ceftriaxone as the first-line treatment for which resistance is already growing. Newer treatments are in the pipeline, including Gepotidacin as an oral option, however, there is still a need to develop alternative orally efficacious therapeutics for N. gonorrhoeae. The molecules reported herein are a step toward filling this gap demonstrating both oral efficacy with a novel mechanism of action. While improving the ADME properties of EZM to result in an in vivo therapeutic to treat gonococcal infection is a significant development as N. gonorrhoeae, work remains to be done on this scaffold. One of the limitations of the study is a lack of understanding for the tissue distribution of the leads. Future work will evaluate distribution of leads to tissues and excretion via the kidney in the urine. A second limitation includes little knowledge of the PK / PD drivers for in vivo efficacy. Once this is established a dosing regimen can be optimized for greater efficacy. Regardless, the studies reported herein as proof-of-concept that the EZM class of bacterial carbonic anhydrase inhibitors can be designed into orally efficacious agents is an important first step in these directions. TABLESTable 8 t1 / 2 (hrs) Cmax MRT Vz CL AUClast AUCInf AUC / D Compound mg)

[0192] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.

[0193] While the disclosure has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0194] It is intended that the scope of the present methods and apparatuses be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims. Enumerated Embodiments (EE)

[0195] The following list of enumerated embodiments presents claims with multiply dependent claims depending from multiply dependent claims for presentation in those jurisdictions where such dependencies are allowed as well as additional claims, which may be presented during the examination of the application or any divisional or continuation thereof.

[0196] EE 1. A compound having the general formula (I)

[0198] where X is one or more substituents selected from halo and O-R, wherein R is H, CH3, C1-C4alkyl substituted with one or more deuterium atoms, or C1-C4alkyl substituted with one or more halo atoms and n is 0-3.

[0199] EE 2. The compound of EE 1, wherein n is 1 or 2.

[0200] EE 3. The compound of EE 1, wherein n is 1.

[0201] EE 4. The compound of any one of EE 1 to EE 3, wherein the compound has one X selected from H, OH, CH3, CF3, F, and C2D5.

[0202] EE 5. The compound of any one of EE 1 to EE 3, wherein the compound has two X groups that are each F.

[0203] EE 6. The compound of EE 1 selected from the group consisting of: ,,.of any one of EE 1 to EE 7, wherein the compound has anti- organism activity.

[0210] EE 9. The compound of EE 8, wherein the organism is Neisseria gonorrhoeae.

[0211] EE 10. A pharmaceutical composition comprising a compound of EE 1, or salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0212] EE 11. The pharmaceutical composition of EE 10, wherein n is 1 or 2.

[0213] EE 12. The pharmaceutical composition of EE 10, wherein n is 1.

[0214] EE 13. The pharmaceutical composition of any one of EE 10 to EE 12, wherein the compound has one X selected from H, OH, CH3, CF3, F, and C2D5.

[0215] EE 14. The pharmaceutical composition of any one of EE 10 to EE 12, wherein the compound has two X groups that are each F.

[0216] EE 15. The pharmaceutical composition of EE 10, comprising a compound is of the structure:

[0220] EE 16. The pharmaceutical composition of EE 10, comprising a compound is of the structure: .composition of any one of EE 10 to EE 16, wherein the compound has anti-organism activity.

[0223] EE 18. The pharmaceutical composition of EE 17, wherein the organism is Neisseria gonorrhoeae.

[0224] EE 19. A method of inhibiting a pathogen comprising contacting a target pathogen with a compound of any one of EE 1 to EE 9 or a pharmaceutical composition of any one of EE 10 to EE 18.

[0225] EE 20. The method of EE 19, where the pathogen is Neisseria gonorrhoeae.

[0226] EE 21. The method of EE 19 comprising contacting the target pathogen with a pharmaceutical composition of any one of EE 10 to EE 18.

[0227] EE 22. The method of EE 19, wherein the compound of EE 1 or the pharmaceutical composition of EE 10 comprises at least one F substituent.

[0228] EE 23. The method of EE 19, wherein the compound of EE 1 or the pharmaceutical composition of EE 10 comprises: .EE 20 wherein the compound of EE 1 or the pharmaceutical composition of EE 10 comprises: .EE 20, wherein inhibiting Neisseria gonorrhoeae comprises administering the pharmaceutical composition of claim 6 comprising:

[0234] EE 26. The method of EE 25, wherein the pharmaceutical composition is administered orally.

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Claims

WE CLAIM:

1. A compound having the general formula (I)one or more substituents selected from halo and O-R, wherein R is H, CH3, C1-C4 alkyl substituted with one or more deuterium atoms, or C1-C4 alkyl substituted with one or more halo atoms and n is 0-3.

2. The compound of claim 1, wherein n is 1.

3. The compound of claim 1 selected from the group consisting of: ,,4..

5. 1, wherein the compound has anti-organism activity.

6. The compound of claim 5, wherein the organism is Neisseria gonorrhoeae.

7. A pharmaceutical composition comprising a compound of claim 1, or salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

8. The pharmaceutical composition of claim 7, wherein n is 1.

9. The pharmaceutical composition of claim 7, comprising a compound is of the structure:

10. The pharmaceutical composition of claim 7, comprising a compound is of the structure: .of claim 7, wherein the compound has anti-organism activity.

12. The pharmaceutical composition of claim 11, wherein the organism is Neisseria gonorrhoeae.

13. A method of inhibiting a pathogen comprising contacting a target pathogen with a compound of claim 1 or a pharmaceutical composition of claim 7.

14. The method of claim 13, where the pathogen is Neisseria gonorrhoeae.

15. The method of claim 13 comprising contacting the target pathogen with a pharmaceutical composition of claim 7.

16. The method of claim 13, wherein the compound of claim 1 or the pharmaceutical composition of claim 7 comprises at least one F substituent.

17. The method of claim 13, wherein the compound of claim 1 or the pharmaceutical composition of claim 6 comprises: .14, wherein the compound of claim 1 or the pharmaceutical composition of claim 6 comprises: .wherein inhibiting Neisseria gonorrhoeae comprises administering the pharmaceutical composition of claim 6 comprising: a patent in need thereof. 20.wherein the pharmaceutical composition is administered orally.

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