WO2015157618A1

WO2015157618A1 - Novel inhibitors of the new delhi metallo beta lactamase (ndm-1)

Info

Publication number: WO2015157618A1
Application number: PCT/US2015/025275
Authority: WO
Inventors: James C. Sacchettini; Joseph Andrew MIRE; Cory C. THURMAN; Nian W. ZHOU; Andrzej Joachimiak; Gyorgy BABNIGG; Kim YOUNGCHANG
Original assignee: The Texas A&M University System
Priority date: 2014-04-11
Filing date: 2015-04-10
Publication date: 2015-10-15

Abstract

Novel inhibitors of the New Delhi Metallo-B-lactamase (NDM-1) and variants thereof are provided. In certain embodiments, the inhibitors are thiourea derivatives. Methods of using the inhibitors for inhibition of NDM-1 and bacteria expressing NDM-1 are also provided.

Description

NOVEL INHIBITORS OF THE NEW DELHI METALLO BETA LACTAMASE

(NDM-1)

[0001] The present invention was developed using funding from the National Institutes of Health, Grant No. NIH401GM094568. The United States government has certain rights in the invention.

TECHNICAL FIELD

[0002] The present disclosure relates to compositions comprising thiourea derivatives able to inhibit metallo-B-lactamase enzymes, and NDM-1 in particular, and associated methods for their use, including their use in combination with B-lactam antibiotics.

BACKGROUND

[0003] Antibiotic resistance is a mounting public health concern, as previously treatable pathogenic bacteria evolve or acquire modes of defense against antiobiotic drugs. Of particular concern is bacterial resistance to the β-lactam antibiotics, which include penicillin derivatives (penams), cephalosporins (cephems), monobactams and carbapenems.

[0004] B-lactam antibiotics are the most broadly used antibacterials in the world due to their effectiveness at irreversibly inhibiting cell wall biosynthesis in a broad spectrum of Gram-positive and Gram-negative bacteria. The B-lactam ring which characterizes the B-lactam antibiotics interferes with synthesis of peptidoglycan, the primary component of the bacterial cell wall, preventing bacterial division. Many bacterial strains now express B-lactamase enzymes, which hydrolyze the B-lactam ring that characterizes the B-lactam antibiotics, thereby inactivating the drugs.

[0005] To circumvent the development of B-lactam antibiotic resistance by the production of B-lactamase enzymes, several B-lactam antibiotics are now co-administered with B-lactamase inhibitors. For example, amoxicillin is frequently administered with clavulinic acid, while ampicillin is often combined with sulbactam and piperacillin is often combined with tazobactam. Additionally, the carbapenem family of B-lactam antibiotics, which includes imipenem, meropenem, ertapenem and others, possesses a structure which makes them highly resistant to most B-lactamases. Carbapenems are therefore employed as the antibiotics of last resort for many bacterial infections.

[0006] Recently, however, carbapenemases, which confer resistance to the carbapenem antibiotics, have emerged as a serious threat to public health. One such enzyme of particular concern is the New Delhi Metallo-B-lactamase 1 (NDM-1), which is capable of degrading all classes of B-lactam antibiotics except for the monobactams. NDM-1 requires zinc for its enzymatic activity, and is therefore termed a metallo-B-lactamase. NDM-1, moreover, is horizontally transmissible on bacterial plasmids, and is therefore readily spread amongst bacterial strains. Since the discovery of NDM-1 in 2008, the enzyme has arisen in 32 countries in many pathogenic bacteria, including K. pneumonia, E. coli, A. baumanii, C. freundii, C. braaci, K. oxytoca, E. cloacae, P. mirabilis, P. aeruginosa, and many others. Many of these infectious species are already resistant to frontline therapeutics, making them completely resistant to B-lactam antibiotics. NDM variants contain mutations in the enzyme active site and thus exhibit slightly different resistance profiles. As for NDM-1, no clinically promising inhibitors have been identified to date.

[0007] Therefore, a need exists for inhibitors of metallo-B-lactamases, and particularly inhibitors of NDM-1 and variants thereof.

SUMMARY

[0008] The present disclosure relates generally to compositions and methods for inhibiting bacterial enzymes that degrade B-lactam antibiotics. More particularly, the present disclosure relates to compositions and methods for inhibiting metallo- B-lactamases, and for inhibiting NDM-1 and variants thereof in particular.

[0009] In certain embodiments in accordance with the disclosed subject matter, the present disclosure provides a composition comprising a metallo-B-lactamase inhibitor. In some embodiments, the composition is an NDM-1 inhibitor. In some embodiments, the compositition is an inhibitor of at least one of NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7. In some embodiments, the composition is an inhibitor of all variants of the NDM metallo-B-lactamase.

[0010] In further embodiments, the present disclosure provides a method for inhibiting a metallo-B-lactamase enzyme in a patient in need thereof. In some embodiments, the metallo-B-lactamase enzyme is NDM-1. In some embodiments, the metallo-B-lactamase enzyme is NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, or NDM-7.

[0011] In additional embodiments, the present disclosure provides methods for treating bacterial infections in which the pathogenic bacteria express a metallo-B- lactamase enzyme. The methods comprise administering to a patient infected with a pathogenic bacteria expressing a metallo-B-lactamase enzyme a metallo-B-lactamase inhibitor. In some embodiments, the metallo-B-lactamase enzyme is an NDM metallo-B- lactamase. The NDM metallo-B-lactamase enzyme can be, for example, one or more of NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7. In further embodiments, the metallo-B-lactamase inhibitor is an NDM metallo-B-lactamase inhibitor. The NDM metallo-B-lactamase enzyme inhibitor can be, for example, an inhibitor of one or more of NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7.

[0012] In still further embodiments, the present disclosure provides methods for treating bacterial infections in which the pathogenic bacteria express a metallo-B- lactamase enzyme. The methods comprise administering to a patient infected with a pathogenic bacteria expressing a metallo-B-lactamase enzyme a metallo-B-lactamase inhibitor and a B-lactam antibiotic. The NDM metallo-B-lactamase enzyme can be, for example, one or more of NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7. For example, in some embodiments, the metallo-B-lactamase enzyme is NDM-1. The NDM metallo-B- lactamase enzyme inhibitor can be, for example, an inhibitor of one or more of NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7. For example, in some embodiments, the metallo-B-lactamase inhibitor is an NDM-1 inhibitor.

[0013] The features and advantages of the present invention will be apparent to those skilled in the art. While numerous changes may be made by those skilled in the art, such changes are within the spirit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] A more complete and thorough understanding of the present embodiments and advantages thereof may be acquired by referring to the following description taken in conjunction with the accompanying drawings.

[0015] Fig. 1A is a stereographic representation of the crystal structure of the NDM-1 active site in complex with NDM-1 inhibitor T0512-7750.

[0016] Fig. IB is a three-dimensional representation of the crystal structure of the NDM-1 active site in complex with B-lactam antibiotic Penicillin. [0017] Fig. 1C is a three-dimensional representation of the crystal structure of the NDM-1 active site in complex with NDM-1 inhibitor T0512-7750.

[0018] Fig. 1C is a three-dimensional representation of the crystal structure of the NDM-1 active site in complex with NDM-1 inhibitor NZ-205.

[0019] Fig. 2 is a time series plot of experimentally observed mouse plasma concentrations of NDM-1 inhibitors NZ-218, NZ-225, and T0512-7750 at 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after oral administration of 50 mg/kg of one of the inhibitors.

DESCRIPTION

[0020] The present disclosure generally relates to compositions and methods for inhibiting B-lactam antibiotic-degrading enzymes expressed by pathogenic bacteria. More particularly, the present disclosure relates to compositions and methods for inhibiting metallo-B-lactamase enzymes, including, without limitation, NDM metallo-B-lactamase enzymes, such as NDM-1 and variants thereof.

[0021] In certain embodiments, the present disclosure relates to compositions and methods for NDM metallo-B-lactamase inhibition comprising thiourea derivatives. In certain embodiments, the thiourea derivative for inhibition of an NDM metallo-B-lactamase is represented by the compound of Formula I below,

(Formula I)

wherein Ri and R₂ are independently selected from Ci-C₆-alkyl, Ci-C₆-alkenyl, Ci-C₆- alkynyl, Ci-C₆-cycloalkyl, aryl, heteroaryl and heterocycloalkyl groups, further wherein Rl and R2 are additionally substituted with from zero to four substituents chosen independently chosen independently from halogen, hydroxy, Ci-C₆-alkoxy-alkyl, -CN, nitro, -S- Ci-C₆- alkyl, amino, Ci-C₆-alkylamino, di-(Ci-C3-alkyl)amino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, Ci-C₆-alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy moieties.

[0022] In certain embodiments, the thiourea derivative for inhibition of an NDM metallo- B-lactamase is represented by the compound of Formula II below

(Formula II)

wherein R₃ and R4 are independently selected from, for example, halogen, hydroxy, Ci-C₆- alkoxy-alkyl, -CN, nitro, -S- Ci-C₆-alkyl, amino, Ci-C₆-alkylamino, di-( Ci-C₃-alkyl)amino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, Ci-C₆-alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy moieties.

[0023] In certain embodiments, the thiourea derivative for inhibition of an NDM metallo- β-lactamase is represented by the compound of Formula III below

(Formula III) wherein R_ls R₂, and R₃ are independently selected from, for example, halogen, hydroxy, Ci- C₆-alkoxy-alkyl, -CN, nitro, -S- Ci-C₆-alkyl, amino, Ci-C₆-alkylamino, di-( Ci-C₃- alkyl)amino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, Ci-C₆- alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy moieties.

[0024] In certain embodiments, the thiourea derivatives for inhibition of an NDM metallo- β-lactamase is represented by the compound of Formula IV below

(Formula IV)

wherein R₅ is selected from one of the following structures:

[0025] The disclosed thiourea derivatives can inhibit one or more of NDM-1 , NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7, and are also referred to accordingly herein as NDM inhibitors. For example, in certain embodiments, the thiourea derivative is an NDM-1 inhibitor. Inhibitor structures and corresponding IC₅₀ values for NDM-1 inhibition for representative thiourea derivative inhibitors of the present disclosure are provided in Table 1 below for purpose of illustration.

Table I: Representative Thiourea Derivative NDM Inhibitor Compounds

Ethyl 3 - { [(2- {imidazo[2, 1 -b] [ 1 ,3 ]

thiazol- 6 -yl } ace tohydrazido)methanethioyl]

amino } benzoate

NZ-205

[0026] The thiourea derivative NDM inhibitors of the present disclosure possess a conserved thiosemicarbazide scaffold, which is significant for NDM binding. This region is indicated in the representative formula below.

Additional structure-activity relationship considerations of the disclosed thiourea derivative NDM inhibitors are described in the Examples below. The NDM metalloproteinase inhibitors of the present disclosure can be synthesized by methods known in the art and/or as described in non-limiting fashion in the Examples below.

[0027] In additional embodiments, the present disclosure relates to compositions and methods for NDM metallo-B-lactamase inhibition comprising the compounds provided in Table 2 below. As described in the Examples below, such compounds were discovered to exhibit potent inhibition of NDM-1. Accordingly, the compounds provided in Table 2 are also NDM inhibitors according to the present disclosure.

Table 2. Potent NDM-1 Inhibitor Compounds Identified by HTS

2-[(4Z)-4-{4-[(4-Chlorophenyl)

sulfanyl]benzylidene } -2, 5 -dioxo

- 1 -imidazolidinyl] -N-(2- 90 methylphenyl) acetamide

5-(4-{[(2,3-Dimethylphenyl)

carbamoyl]amino}phenyl)-N-(4- methylphenyl)- 1 ,3 ,4-thiadiazole-2- 93

carboxamide

[0028] The NDM metalloproteinase enzymes confer broad resistance to the B- lactam antibiotics. NDM-1 is encoded by the plasmid-borne blaNDM-1 gene, and has been identified in several pathogenic bacteria to date since its isolation in 2008. Bacterial species known to possess or to have the potential to acquire the blaNDM-1 gene include Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Serratia marcescens, Proteus mirabilis, Haemophilus influenza, Neisseria gonorrhoeae, Staphylococcus aureus, Klebsiella oxytoca, Citrobacter freundii, Listeria monocytogenes, Streptococcus pneumoniae, Neisseria meningitides, Enterococcus faecalis, Bacillus anthracis, Streptococcus pyogenes.

[0029] Thus, in accordance with the present disclosure, methods of treating and/or preventing infection with a bacterium expressing an NDM metalloproteinase enzyme, and methods of inhibiting a bacterium expressing an NDM metalloproteinase enzyme, are disclosed. In related aspects of the present disclosure, methods of treating and/or preventing infection with a bacterium known to or believed to express an NDM metalloproteinase enzyme, and methods of inhibiting a bacterium known to or believed to express an NDM metalloproteinase enzyme, are disclosed. In further related aspects, methods of sensitizing a bacterium expressing, known to express, or believed to express, are disclosed. As used herein, the phrase "known to express" will refer to both knowledge that such bacterium is expressing an NDM metalloproteinase, as determined by, for example, observed B-lactam antibiotic resistance, laboratory testing, such as, without limitation, immunoassay, enzyme assay, or PCT/RT-PCR techniques, and to knowledge that such bacterium is capable of acquiring the ability to express NDM. Such methods generally include administering, such as by administering a pharmaceutical composition comprising, to an organism infected with or susceptible to infection by such bacterium (e.g., a patient), one or more NDM inhibitors according to the present disclosure. In certain embodiments, the methods of the present disclosure cause or result in contacting the bacterium and/or the NDM enzyme with the one or more inhibitors. Such methods can further include administration of one or more β-lactam antibiotics to an organism infected with or susceptible to infection by such bacterium. In certain embodiments, the NDM inhibitor according to the present disclosure is an NDM-1 inhibitor. In certain embodiments, the NDM inhibitor according to the present disclosure inhibits one or more of NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, NDM-6, and NDM-7.

[0030] Without limitation to any particular theory, it is believed that the inhibitors irreversibly bind to the NDM-1 enzyme, preventing the enzyme from hydro lyzing the β-lactam ring of β-lactam antibiotics. Accordingly, any β-lactam antibiotic will be suitable for administration with any of the thiourea derivatives recited above. The NDM metalloproteinase inhibitors of the present disclosure can additionally or alternatively be administered with one or more β-lactamase inhibitors. Suitable β-lactamase inhibitors include, without limitation, clavulanic acid, tazobactam, and sulbactam.

[0031] As used herein, the phrase "administration with" will generally include simultaneous coadministration (such as by a single pharmaceutical composition (e.g., formulation) or simultaneous adminstration, by one or more routes, of two or more compositions), contemporaneous administration, such as overlapping administration schedules, and non-overlapping administration, provided that administration of the two or more administered compounds and/or compositions (e.g., inhibitors and/or β-lactam antibiotics) is provided to a single organism, such as a patient. For avoidance of doubt, the terms "adminstration," "administering," "administration to," and the like, include self- administration, dispensation, and supply.

[0032] For example, in certain non-limiting embodiments according to the disclosed subject matter, one or more of the NDM inhibitors of the present disclosure are simultaneously administered with a β-lactam antibiotic. In further embodiments, the inhibitors are administered prior to and concomitantly with β-lactam antibiotic administration. In still further embodiments, the inhibitors are administered prior to, concomitantly with, and after β-lactam antibiotic administration. Likewise, in additional or alternative nonlimiting embodiments, two or more of the NDM metalloproteinase inhibitors of the present disclosure can be administered simultaneously or in sequence, particularly during the course of antibiotic treatment. The administration of two or more NDM metalloproteinase inhibitors may help prevent the development of resistance to one or more of the inhibitors.

[0033] The NDM metalloproteinase inhibitors of the present disclosure are suitably administered with a β-lactam antibiotic. Suitable B-lactam antibiotics include, without limitation, the penams (penicillins), including Amoxicillin, Ampicillin, Epicillin, Carbenicillin, Ticarcillin, Temocillin, Azlocillin, Piperacillin, Mezlocillin, Mecillinam, Sulbenicillin, Benzylpenicillin, Clometocillin, Benzathine benzylpenicillin, Procaine benzylpenicillin, Azidocillin, Penamecillin, Phenoxymethylpenicillin, Propicillin, Benzathine phenoxymethylpenicillin, Pheneticillin, Cloxacillin, Oxacillin, Meticillin, Nafcillin; the penems, including Faropenem, the Carbapenems, including Biapenem, Ertapenem, Doripenem, Imipenem, Meropenem, Panipenem, Tebipenem; the cephalosporins, including Cefazolin, Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefminox, Cefonicid, Ceforanide, Cefotiam, Cefprozil, Cefbuperazone, Cefuroxime, Cefuzonam, Cephamycin, Carbacephem, Cefixime, Ceftriaxone, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime, Cefsulodin, Cefteram, Ceftibuten, Ceftiolene, Ceftizoxime, Moxalactam, Flomoxef, Cefepime, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Ceftaroline fosamil, Ceftolozane; and the monobactams, including Aztreonam, Tigemonam, Carumonam, and Nocardicin A.

[0034] The NDM inhibitors can be administered in an amount and for a time sufficient to inhibit a metallo-B-lactamase enzyme, or to improve the ability of a B-lactam antibiotic to kill a pathogen. Such amounts and times can be readily determined by those having ordinary skill in the art with the benefit of this disclosure, including the pharmacokinetic data disclosed herein. In some embodiments, the NDM inhibitors can be administered during an entire course of antibiotic treatment. Frequency of administration may be such that a selected minimum amount of NDM inhibitor remains biologically available at all times during the course of administration.

[0035] In additional embodiments in accordance with the disclosed subject matter, the one or more NDM inhibitors is administered orally (i.e. by enteral administration). Such oral administration can be by solid pill, liquid capsule, drink, nutritional supplement, meal, or any other means of oral administration. In alternative embodiments, the NDM inhibitors are administered by parenteral administration, including, without limitation, intravenous infusion.

[0036] Patients can include mammals, such as humans, pets, such as dogs and cats, and livestock, such as cattle, sheep, horses, and pigs. Patients can also include birds, such as chickens, turkeys, ducks, geese, quail and other poultry. Patients can further include any animal from which a bacterium susceptible to a β-lactam antibiotic may be eliminated. Patients can be infected with a bacteria that is resistant to a β-lactam antibiotic due to the presence of a bacterial metallo-B-lactamase enzyme, such as NDM-1. The NDM inhibitor(s) can be used to restore full or partial susceptibility of the bacteria to the β-lactam antibiotic. In instances where only partial susceptibility is restored or expected to be restored, the β-lactam antibiotic may be administered in higher doses or more frequently than normally used with non-resistant bacteria.

[0037] In accordance with the disclosed subject matter, a pharmaceutical composition can be formed comprising NDM inhibitor as disclosed herein. The pharmaceutical composition can contain other pharmaceutically acceptable components, such as salts or other pharmaceutically acceptable variations of the NDM inhibitor that retain its biological function. The composition can contain additional compositions to stabilize or preserve the inhibitor. The composition may further contain compositions to increase uptake, particularly enteral uptake, or bioavailability of the inhibitor. Furthermore, the composition can comprise other therapeutically active agents to be co-administered with the inhibitor, such as an additional NDM inhibitor as disclosed, a β-lactam antibiotic, or an additional β- lactamase inhibitor.

[0038] The present disclosure is based in part on the observation that certain thiourea derivatives function as potent inhibitors of NDM metalloproteinases. As described in the Examples below, the thiourea derivatives were identified by screening the NDM-1 enzyme against a diverse chemical library of small molecules. Using the crystal structure of NDM-1 (described in Structure of Apo- and Monometalated Forms of NDM-1, A Highly Potent Carbapenem-Hydrolyzing Metallo-fi-Lactamase, PLOS One, September 2011, hereby incorporated by reference in its entirety), structure-activity relationships between the NDM-1 enzyme and thiourea derivatives were determined. By resolving the crystal structure of the NDM-1 enzyme in complex with the thiourea derivative inhibitors, and subsequently analyzing the structure-activity relationship of the thiourea derivative-NDM-1 complex scaffold, specific chemical modifications to the thiourea derivatives associated with enhanced potency of inhibition of the NDM-1 enzyme were determined. Moreover, the thiourea derivatives have been determined to be non-cytotoxic to a human cell line. The crystal structures of NDM-2 through NDM-7 were also solved to better than 1.5 A resolution, and the substrate specificity of purified recombinant NDM-2-7 was determined using a panel of different β-lactam antibiotics. Inhibition of the NDM-2 - NDM-7 metalloproteinases was also observed with the thiourea derivatives tested.

[0039] These and other aspects of the disclosure are discussed in detail below in the non-limiting Examples that follow.

EXAMPLES

[0040] The following examples are provided to further illustrate certain aspects and/or embodiments of the disclosure. They are not intended to disclose or describe each and every aspect of the disclosure in complete detail and should be not be so interpreted.

Identification of Candidate NDM Inhibitors

[0041] A 50,000-compound high-diversity library was screened using an enzyme assay to identify putative inhibitors of NDM-1. The library was selected from over 3 million commercially available compounds compiled from multiple vendors' catalogs. The library was designed with drug discovery in mind, incorporating Lipinski's Rules, such as MW < 550 Da and logP < 5. Furthermore, potentially reactive chemotypes such as disulfides and acyl halides were removed. To maximize chemical diversity and avoid redundant scaffolds, a unique subset was selected using an algorithm to first filter the 3 million commercial compounds for drug-like properties, then to cluster, and then to select most representative compounds, such that no pair of compounds has a Tanimoto coefficient (degree of chemical similarity) > 0.7.

[0042] The NDM-1 enzyme assay was screened in 384-well plate format using nitrocefin as the substrate. Nitrocefm is a chromogenic beta-lactam, whose turnover is measured by an increase in absorbance at 486 nm. The primary screen was done at a compound concentration of 10 μΜ. 344 initial candidate compounds were identified based on reduction of enzyme activity by more than 4 standard deviations below the mean. These compounds were selected on a single 384-well hit plate for further studies. Twelve of the 344 compounds were eliminated based on observed cytotoxicity to human cells. Using a threshold inhibition of >20% at 10 μΜ yielded 138 candidates from the screen.

[0043] Differential scanning fluorimetry (DSF) was then employed as a secondary screen to eliminate chelator compounds. DSF is an assay of direct protein binding typically use for fragment screening which employs a hydrophobic fluorescent probe to monitor the folded state of a protein as a function of temperature, and can thus detect a shift in the melting temperature (T_M) of protein due to the binding of a ligand, with the T_M calculated at the inflection point of the melting curve. To remove general chelators, as well as protein destabilizers and aggregators, DSF was performed on the remaining 138 candidate compounds. With EDTA chelator as positive control, a decrease in 4°C in T_m was observed due to the loss of the two zinc ions in the active site, resulting in a less stable enzyme, whereas an increase of 4°C was observed with incubation of NDM-1 with potent inhibitor candidate compounds relative to DMSO negative control (Figure 2).

[0044] 78 candidate inhibitor compounds passed both the primary and secondary screens. These remaining compounds were manually clustered based on chemical scaffold and chemotype similarity, and compounds with potentially reactive groups were eliminated.

[0045] Thiourea derivatives, including diaryl-thiosemicarbazides, were identified in the described screening as a chemical series exhibiting potent NDM-1 inhibition. Dose-response assays confirmed the potency of these candidates, with the most potent candidate (T0512-7750) exhibiting an IC₅₀ of 0.6 μΜ. The most potent first-generation inhibitors identified were T5359810, T0512-7750, and NZ-205, shown in Table 1 above. The most potent of the remaining compounds identified are provided in Table 2 above.

Structure-Activity Relationship Analysis of Diaryl-Thiosemicarbazide Thiourea Derivatives

[0046] The crystal structure of the NDM-1 enzyme was defined previously to determine the mechanism of β-lactam antibiotic degradation. The crystal structure of NDM-1 illustrates that two Zn²⁺ ions in the active site cooperate to cleave the lactam/penem ring. Unlike other β -lactamases (classes A, C, and D), which use an active-site Ser as a nucleophile to attack and hydro lyze substrates, NDM-1 is a class B β-lactamase, which utilizes two divalent cations in the active site. The crystal structures of the apo-form of the enzyme, as well as complexes with substrates like ampicillin, have been determined to high resolution. The enzyme has a relatively open active site, explaining its broad substrate specificity. As shown in Fig. 4A, NDM-1 binds two Zn²⁺ ions, spaced about 5 A apart, labeled as site Znl (higher-affinity, coordinated by 3 His) and site Zn2 (Kd=2uM, coordinated by Asp90, Cysl68, and His210). The Zn²⁺ in the higher-affinity Znl site activates a water for nucleophilic attack on the β-lactam ring, while the second Zn²⁺ is believed to stabilize the polar intermediate in ring cleavage. The crystal structure of NDM-1 bound with penicillin is shown in Fig. 4B.

[0047] The crystal structure of the NDM-1 enzyme in complex with T0512- 7750 was solved to investigate the structure-activity relationship (SAR) of the thiourea derivatives. It was determined that the sulphur residue of T0512-7750 binds Zn²⁺ (ZN1) and the phenyl ring displaces the other active site Zn²⁺ (ZN2) accompanied by reorientation of the groups that were originally chelating the Zn. As depicted stereographically in Fig. 4A and three-dimensionally Fig. 4C, the sulfur atom forms a direct interaction (1.93 A°) with the Znl atom. Significantly, it was observed that the large pendant aromatic groups of the diaryl- thiosemicarbazide thiourea derivatives pack into adjacent hydrophobic pockets in the active site, and thus optimization of these interactions was identified as a strategy to improve potency.

High-Potency Second Generation NDM Inhibitors

[0048] Further SAR analysis was conducted on thiosemicarbazide analogs synthesized to contain modifications to the benzothiazole domain of the most potent first- generation thiourea derivative NDM-1 inhibitor. Initial efforts focused on determining activity of derivatives having alternatives to the phenyl ring with the para-difluoromethoxy substituent in T0512-7750. While replacement with a carboyxlate or hydroxamate resulted in loss of activity, replacement with the ethyl ester (NZ-205) increased activity by 4-fold to 150 nM. Subsequent improvements yielded a compound with an IC₅₀ of 35 nM (NZ-218) as measured with 100 μΜ Zn²⁺ in the assay condition. Binding of high-potency NDM-1 inhibitor NZ-205 with the NDM-1 active site is depicted in Fig. 4D. Representative high- potency second generation inhibitors are shown in Table 1 above.

[0049] Titration of enzyme inhibition activity with zinc concentration showed that, for all compounds in the series, a decrease of zinc resulted in a concomitant decrease in IC₅₀. However, even at 25 μΜ Zn, the observed K_m of the inhibitors was an order of magnitude greater than that of the lower affinity second Zn²⁺ site. Given that zinc is a trace element, the free zinc concentration in vivo is orders of magnitude less than those used in enzyme assay conditions. Thus, zinc is not believed to reduce in vivo activity of the disclosed inhibitors.

Thiourea Derivative NDM-1 Inhibitor Synthesis

[0050] The diaryl-thiosemicarbazide derivatives were synthesized according to the following scheme:

2 3 4

The starting material, an aromatic amine (1), was reacted with thiophosgene in the presence of a base, such as triethylamine, to produce an intermediate isothiocyanate (2). Treatment of this isothiocyanate (2) with hydrazine gave the corresponding thiosemicarbazide (3). The acyl-thiosemicarbazide product (4) was obtained by condensation of thiosemicarbazide (3) with acid in the presence of a coupling reagent. All analogs were purified to >95% purity, based on LC-MS and NMR.

Whole-Cell Activity, In Vitro Cytotoxicity, and In Vivo Toxicity and Pharmacokinetics

[0051] To evaluate whole-cell activity of the inhibitors, an experimental carbapenem-resistant strain of E. coli was generated by cloning the NDM-1 gene into plasmid pBAD 18 and transforming E. coli K12 with the plasmid. The wild type K12 E. coli strain showed an IC₅₀ for faropenem of 1 μ^ιηΐ. The NDM-1 expressing recombinant K12 strain was completely resistant to faropenem at the highest concentrations tested, 50 μ^ιηΐ (without induction by arabinose). However, addition of 6.25 μΜ NZ-205 dramatically reduced the MIC of faropenem to 0.7 μ^ιηΐ, similar to the sensitivity of the parental strain of E. coli. NZ-205 alone caused no detectable growth inhibition in E. coli K12 up to 800 uM.

[0052] The most potent second-generation inhibitors (NZ-218, NZ-205, and T0512-7750) were evaluated in short term cytotoxicity assays on human dermal fibroblasts (HDF). Cells were plated in media into 384-well plates and allowed several hours to attach prior to compound addition. Cells were cultured for 48 hours with compounds at which point 40 ng/ml resazurin blue was added. After 16-24 hours fluorescent signal was read on a plate reader. Percent growth inhibition was determined by comparing the growth of cells in the presence of compound to the growth with no compound. All DATS compounds were found to be non-toxic up to the highest concentration tested, 40 μΜ.

[0053] Preliminary mouse toxicity testing was conducted using a single oral dose of 50 mg/kg T0512-7750 formulated in oil (w/ 10 % DMSO) given to two mice. Behavior was monitored closely for eight hours following administration. Only slight and temporary subdued behavior patterns were observed, which onset 10 minutes after dosing and subsided within 30 minutes. After 24 hours, the mice appeared to be behaving normally and there were no overt toxic effects. Plasma was collected at 8 hours after administration and upon sacrifice 24 hours after administration. Drug concentrations were determined by mass spectrometry.

[0054] A similar study was conducted using T0512-7750, NZ-218, and NZ- 225. All 3 compounds were similarly dosed (50 mg/kg). Abnormal behavior patterns were not observed with NZ-218 and NZ-225. Plasma samples were collected from two treated mice 30 minutes, 1 hour, 2 hours and 4 hours after administration. Drug concentrations were determined by mass spectrometry.

[0055] Observed inhibitor concentrations are plotted in Fig. 2. For T0512-7750, observed AUC was 1.94 μg.hr.ml^"1; T_1/2 was 1 hr; and Cmax was 0.23 μg/ml. For NZ-218, observed AUC was 0.23 μg.hr.ml-l; T_1/2 was 0.5 hr; and Cmax was 0.14 μg/ml. For NZ-225, observed AUC= 0.62 μg.hr.ml-l; T_1/2= 0.5 hr; Cmax= 0.17 μg/ml. Thus exposure above the IC₅₀ was observed over several hours with the compounds and dosing described. Based on these preliminary studies, an approximate human equivalent dose can be between about 1 mg/kg and about 100 mg/kg, or about 1 mg/kg and about 20 mg/kg, or about 1 mg/kg and about 10 mg/kg.

* * *

[0056] While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.

Claims

CLAIMS What is claimed is:

1. A compound for inhibiting β-lactam antibiotic degradation by a metallo-B-lactamase enzyme, the compound represented by the following formula:

wherein Ri and R₂ are independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl groups, further wherein RI and R2 are additionally substituted with from zero to four substituents chosen independently from halogen, hydroxy, alkoxy-alkyl, -CN, nitro, -S-alkyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy moieties.

2. A compound for inhibiting B-lactam antibiotic degradation by a metallo-B-lactamase enzyme, the compound represented by the following formula:

wherein R₃ and R4 are independently selected from, for example, halogen, hydroxy, Ci-C₆- alkoxy-alkyl, -CN, nitro, -S- Ci-C₆-alkyl, amino, Ci-C6-alkylamino, di-( Ci-C₃-alkyl)amino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, Ci-C6-alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy moieties.

3. A compound for inhibiting β-lactam antibiotic degradation by a metallo-B-lactamase enzyme, the compound represented by the following formula:

wherein R_{l s} R₂, and R₃ are independently selected from halogen, hydroxy, Ci-C₆-alkoxy- alkyl, -CN, nitro, -S- Ci-C₆-alkyl, amino, Ci-C₆-alkylamino, di-( Ci-C₃-alkyl)amino, dialkylaminoalkyl, carboxy, carboalkoxy, acyl, carboxamido, Ci-C₆-alkylsulfoxide, acylamino, phenyl, benzyl, phenoxy and benzyloxy moieties.

4. A compound for inhibiting B-lactam antibiotic degradation by a metallo-B-lactamase enzyme, the compound represented by the following formula:

wherein R₅ is selected from one of the following structures:

5. A compound for inhibiting B-lactam antibiotic degradation by a metallo-B-lactamase enzyme, the compound selected from the group consisting of:

6. A compound for inhibiting B-lactam antibiotic degradation by a metallo-B-lactamase enzyme, the compound selected from the group consisting of 2-{5-Amino-4-[3-(4- methylphenyl)- 1 ,2,4-oxadiazol-5-yl]- 1H- 1 ,2,3-triazol-l -yl} -N-(3-chloro-4-methylphenyl) acetamide, 3-Benzyl-6-phenyl-2-thioxo- 1,2,3, 5-tetrahydro-4H-pyrrolo[3,2-d]pyrimidin-4- one, l-[(4-chloro-2,5-dihydroxyphenyl)sulfanyl]-N'-[(lE)-(4-fluorophenyl)methylidene] formohydrazide, 2-[(4Z)-4- {4-[(4-Chlorophenyl)sulfanyl]benzylidene} -2,5-dioxo- 1 - imidazolidinyl]-N-(2-methylphenyl)acetamide, and 5-(4- {[(2,3-Dimethylphenyl)carbamoyl] amino}phenyl)-N-(4-methylphenyl)-l,3,4-thiadiazole-2-carboxamide .

7. A compound according to any of claims 1-6, wherein the metallo-B-lactamase enzyme is a New Delhi Metallo-B-lactamase.

8. A compound according to any of claims 1-7, wherein the metallo-B-lactamase enzyme is NDM-1.

9. A method of inhibiting a Metallo-B-lactamase enzyme in a patient comprising administering to the patient, in an amount effective to inhibit the enzyme, a compound according to any of claims 1-8.

10. A method of inhibiting a bacterium expressing a Metallo-B-lactamase comprising administering to a patient infected with the bacterium a compound according to any of claims 1-8 in an amount effective to inhibit the Metallo-B-lactamase.

11. A method of sensitizing a bacterium expressing a Metallo-B-lactamase to a B-lactam antibiotic comprising administering to a patient infected with the bacterium a compound according to any of claims 1-8 in an amount effective to inhibit the Metallo-B-lactamase.

12. A method according to claims 10-11, wherein the bacterium belongs to a species selected from the group consisting of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Serratia marcescens, Proteus mirabilis, Haemophilus influenza, Neisseria gonorrhoeae, Staphylococcus aureus, Klebsiella oxytoca, Citrobacter freundii, Listeria monocytogenes, Streptococcus pneumoniae, Neisseria meningitides, Enterococcus faecalis, Bacillus anthracis, and Streptococcus pyogenes.

13. A method according to any of claims 9-11, wherein the amount effective to inhibit the Metallo-B-lactamase temporarily results in a concentration of the compound in the patient greater than the half-maximal inhibitory concentration of the compound.

14. A method according to any of claims 9-12, wherein the New Delhi Metallo-B- lactamase is NDM-1.

15. A method according to any of claims 9-13, further comprising administering to the patient a therapeutically effective amount of a B-lactam antibiotic optionally selected from the group consisting of Amoxicillin, Ampicillin, Epicillin, Carbenicillin, Ticarcillin, Temocillin, Azlocillin, Piperacillin, Mezlocillin, Mecillinam, Sulbenicillin, Benzylpenicillin, Clometocillin, Benzathine benzylpenicillin, Procaine benzylpenicillin, Azidocillin, Penamecillin, Phenoxymethylpenicillin, Propicillin, Benzathine phenoxymethylpenicillin, Pheneticillin, Cloxacillin, Oxacillin, Meticillin, Nafcillin; Faropenem, Biapenem, Ertapenem, Doripenem, Imipenem, Meropenem, Panipenem, Tebipenem; the cephalosporins, including Cefazolin, Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefminox, Cefonicid, Ceforanide, Cefotiam, Cefprozil, Cefbuperazone, Cefuroxime, Cefuzonam, Cephamycin, Carbacephem, Cefixime, Ceftriaxone, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime, Cefsulodin, Cefteram, Ceftibuten, Ceftiolene, Ceftizoxime, Moxalactam, Flomoxef, Cefepime, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Ceftaroline fosamil, Ceftolozane; Aztreonam, Tigemonam, Carumonam, and Nocardicin A.