MXPA02008368A - Anti infective ectatm. - Google Patents

Abstract

The present invention provides compositions and methods for targeting toxic anti metabolites to inhibit the growth of antibiotic resistant microbial infections. It provides a means of taking advantage of a key disease resistance mechanism to activate these drugs locally, and to overcome the resistance phenotype of the microbes. In addition, the invention provides methods for treating a subject infected with an antibiotic resistant microorganism by administering the compounds or compositions of the invention.

Description

ECTA-T "M1 ANTI-INFECTIOUS CROSS REFERENCE TO RELATED REQUESTS This application claims priority under 35 U.S.C. § 119 (e) to US Provisional Application No. 60 / 185,479, filed on February 28, 2000, the contents of which are incorporated herein by reference in the present description. TECHNICAL FIELD The present invention relates to the field of Enzyme Catalyzed Therapeutic Activation therapy (ECTA ™) and specifically to enzyme substrates that are expressed by infectious agents and thus block the efficacy of currently available drugs. BACKGROUND Throughout this description, several publications are put in reference by the first author and the date, in parentheses, patent number or publication number. The complete bibliographic reference is given at the end of the application, immediately before the claims. The descriptions of these references are incorporated herein by reference in this description, to more fully describe the state of the art to which this application pertains. Resistance to antimicrobial agents is a recognized medical problem (Schaechter et al., 1993; Murray, 1997). The problem was recognized earlier as resistance to penicillin in Staphylococci, and is now a recognized problem for the treatment of many bacterial infections, including essentially all nosocomial bacterial infections (acquired in hospital) (Bus, 1988; Steinberg et al., 1996 Murria, 1997). Hospital infections occur in 5% of patients admitted to the hospital (approximately 2 million patients per year in the United States); they cause an estimated 20,000 deaths per year and contribute to an additional 60,000 deaths in the hospital. It is estimated that nosocomial infections add approximately 7.5 million hospital days and $ 1 trillion in health care costs each year (ilson et al., 1991). The importance of antibiotic-resistant bacteria has increased, in that many organisms, for example, Staphylococcus aureus, have developed resistance to several different antibiotics (the "multi-resistant" phenotype.) Enzymes involved in drug resistance include Penicillinases, beta-lactams, cefporporinases, and others.These enzymes inactivate antibiotics by modifying them to inactivate compounds.The resistance caused by enzymes also includes modification of the antibiotic by chloramphenicol acetyltransferases and other aminoglycoside modification enzymes (Murray). , 1997) Other mechanisms that contribute to antibiotic resistance include drug permeability mutations, the expression of transport proteins that actively expel antibiotics from target organisms, and mutations in drug targets themselves (Murray, 1997). Characteristics of the A Antibiotics Antibiotics are drugs that have cytostatic or cytotoxic effects on target organisms. The key to success for an antibiotic is the selectivity for the target of the disease, and the lack of toxicity to the host or patient. Many antibiotics are purified from cultures of microbial organisms by themselves, while others are synthetic derivatives of naturally occurring antibiotics (Wilson et al., 1991). The most useful antibiotics against infections are those that attack a specific target of the microbe. For example, ß-lactam antibiotics interfere with the synthesis of the cell wall by binding to the precursors of the cell wall. Since mammalian cells lack the cell walls of bacteria, these drugs have a tremendous margin of safety for the patient. The most common form of resistance to β-lactam antibiotics is the production of β-lactamases, which degrade the antibiotic molecule. The β-lactamases are encoded either by the plasmid or chromosomal genes. Although inactivation of antibiotics and probably the most common mechanism for drug resistance, resistance also occurs as a result of mutations in drug targets themselves. The best characterized of these are the mutations in the penicillin binding proteins (PBPs), which lead to a decrease or loss in the binding of antibiotics by these proteins and a corresponding decrease or loss in the activity of the antibiotic. The ß-lactam antibiotics include penicillin, ampicillin, carbenicillin, and cephalosporins (including cephalexin, cefaclor, cefoxitin, cefotaxime, and cefoperazone). Because resistance is very common via the production of high levels of β-lactamases, new drugs have been developed to inhibit these enzymes, thus increasing the efficacy of β-lactam antibiotics. Examples of β-lactamase inhibitors include clavulanate, timentin and sulbactam (Bush, 1988; ilson et al., 1991; Schaechter collaborators, 1993). The combination of the ß-lactam antibiotic with the ß-lactamase inhibitor has extended the useful pharmacological lifetimes of these antibiotics (Bus, 1988). Disadvantages of current antimicrobial agents Current agents have well-characterized targets or targets for action. Several examples are given below: Other antibiotics work by blocking DNA replication, cellular RNA production or by modifying multiple cell targets. { Schaechter et al., 1993). The occurrence of resistance to antibiotics is very common, and many of the mechanisms have been described (Schaechter et al., 1993, Murray, 1997). These mechanisms include overexpression of the target enzyme, the expression of an antibiotic inactivation enzyme or the mutation of the target so that it is no longer recognized by the antibiotic. Examples of these are given below: The increased resistance of bacterial infection to antibiotic treatment has been carefully documented (see, for example, Steinberg et al., 1996), and has now become a generally recognized problem (Murray, 1997). Each "new antibiotic derived from its previous generation (for example, penicillin cephalosporin) is initially successful, but then has had increased reports of resistance.The progression of ß-lactamase antibiotics is typical of the field.Each antibiotic successively is more resistant to degradation by ß-lactamase, and the organism then produces larger amounts of ß-lactamase This is especially a problem for nosocomial infections (acquired in hospital) (Wilson et al., 1991; Murray, 1997). The most common mechanism for the transmission of the drug resistance phenotype is via the plasmids, although some modulators of antibiotic resistance are located on the bacterial chromosome (Schaechter et al., 1993) .The despair of the medical community has been directed by the production of ß-lactamase inhibitors Unfortunately, although ß-lactamases have substratum specificities To overlap, they have evolved differently to have different amino acid sequences, but related. This problem is expressed by the widely varying efficiencies of each β-lactamase inhibitor for different enzymes. Vancomycin inhibits the synthesis and assembly of the second stage of the peptidoglycan polymers of the cell wall by forming the complex with its precursor of D-alanyl-D-alanine. that fits in a "cavity" in the vancomycin molecule, to prevent in this way its binding to the terminal peptidoglycan that is the target of the transglycolase and transpetidase enzymes. In addition, vancomycin may impair RNA synthesis and damage protoplasts by altering the permeability of its cytoplasmic membrane. Vancomycin-resistant enterococci (VRE) emerged as important pathogens in hospitals in the United States. S. aureus strains that were intermediate vancomycin resistant (VIRSA) were detected in the United States in 1997. VRE and VIRSA have highlighted serious problems about the continued effectiveness of vancomycin in the treatment of these infections. Vancomycin-resistant enterococci produce two new enzymes, a ligase and a dehydrogenase, with the formation of a new D-ala-D-lactate terminal depsipeptide, to the pentapeptide. This substitution allows the synthesis of the cell wall to continue in the presence of vancomycin. The new generation of antibiotics are usually more toxic than their predecessors, and they can not be administered to patients in a convenient manner. A cycle of resistance to the drug has been established, which requires a new approach to solve. Therefore, there is a need for a new generation of antibiotics that are not susceptible to the established mechanisms of drug evasion. This invention satisfies this need and also provides related advantages.

DESCRIPTION OF THE INVENTION Many combinations of enzyme-prodrug have been described in detail. Applications have included antiviral drugs similar to ganciclovir (Straus, 1993) and the antibody or expression directed to the gene of bacterial enzymes to treat cancer (Melton and Sher ood, 1996).; Stosor et al., 1996). The current invention redirects this technology to treat infectious disease resistant to antibiotic therapy. Thus, this invention provides prodrugs and methods for selectively inhibiting the proliferation of the antibiotic-resistant microorganism by contacting a sample containing such a microorganism with an effective amount of these prodrugs. In addition, the invention provides methods for treating an infected subject with an antibiotic-resistant microorganism by administering the compositions of the invention. The prodrugs of this invention have the general structure shown below: X, Y, Z and R 'are specifically defined throughout this application. MODES FOR CARRYING OUT THE INVENTION As used herein, certain terms may have the following defined meanings. The singular form "a", "an" and "the" includes plural references, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "comprising" is intended to mean that the compositions and methods include the elements mentioned, but do not exclude others. "Consisting essentially of" when used to define compositions and methods, shall mean that it excludes other elements of any essential meaning for the combination. Thus, a composition consisting essentially of the elements as defined herein, does not exclude minor contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consisting of" shall mean that it excludes more of the minor elements from other ingredients and the substantial steps of the method for administering the compositions of this invention. The modalities defined by each of these transition terms are within the scope of this invention. As used herein, the term "prodrug" means a precursor or derivative form of a pharmaceutically active agent or substance that is less cytotoxic to a target cell compared to the drug metabolite and is capable of being enzymatically activated or converted to the most active way A "composition" is proposed to mean a combination of active agent and another compound or composition, inert (eg, a detectable agent or label or a pharmaceutically acceptable carrier) or active, such as an adjuvant. A "pharmaceutical composition" is proposed to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. As used herein, the term "pharmaceutically acceptable carrier" comprises any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ.Co Easton (1975)). An "effective amount" is an amount sufficient to effect the beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. A "control" is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative". An antibiotic-resistant microorganism is a microorganism with the ability to decrease or inhibit the ability of the antibiotic to inhibit growth or kill the microorganism. A "β-lactam-resistant microorganism" is a microorganism with the ability to synthesize a protein that neutralizes a β-lactam antibiotic. "Inhibition of growth" of a microorganism means reducing, by contact with an agent, the rate of proliferation of such a microorganism, as compared to a control microorganism of the same species not in contact with this agent. A "subject" is a plant or a vertebrate such as a fish, a bird or a mammal, and preferably a human. Fish includes, but no. is limited to, pets and farm animals. Birds include, but are not limited to, pets, sport animals and farm animals. Mammals include, but are not limited to, murines, apes, humans, farm animals, sports animals and pets. The present invention provides methods for directing toxic antimetabolites to antibiotic-resistant microbial infections. In one embodiment, the invention provides a means to take advantage of a key resistance mechanism of the disease, for example, the overproduction of β-lactamase enzyme, to activate these drugs locally, and to overcome the resistance phenotype and inhibit the growth of microbes. The invention further provides methods for treating a subject infected with antibiotic-resistant microorganism by administering an effective amount of the compounds or compositions of the invention. In one aspect, the invention provides a prodrug compound having the structure: wherein R 'is selected from the group consisting of hydrogen, alkyl, aryl, halogenated aryl, phenol, nitr aryl, ammonium, methylamine, dimethylamine, lower alkylamin, bis (lower alkyl) amine, glycol, glycerol, sorbitol, polyethylene glycol (PEG) ), salt form (sodium, potassium, lithium), THAM (2-amino-2-hydroxymethyl-1,3-propanediol) and a pharmaceutically acceptable salt thereof; where X is absent or is selected from the group consisting of carbonyl, methylene, oxygen, nitrogen sulfur; wherein Y is selected from the group consisting of methylene, methyl alkenyl, methylene alkynyl, methyleneoxycarbonyl, vinyl and an alkynyl of 1 to 6 carbon atoms; and where Z is a toxophore. In one aspect, the toxophore Z is selected from the group consisting of 1-fluoro-l-carbonylmethyl and l-nitro-2-carbonylethyl. In another aspect, Z is selected from the group consisting of doxorubicin, bis- (2-chloroethyl) amine, mitomycin, trichlorcarban, trichlorocarbanilide, tribromo-salicylanilide, sulfamethoxazole, chloramphenicol, cycloserine, trimethoprim, chlorhexidine, hexachlorophene, 2-mercaptopyridine-N -oxide, camptothecin, apoptolidene, cis-platina, anthracycline, epothilone, halicondrine, herniasterlin, methioprim, tapsigargin and fentichlor. In a further aspect, the toxophore Z is a phenol substituted with chlorine. These include, but are not limited to, the group consisting of 5-chloro-2- (2, -dichlorophenoxy) phenol, 4-chloro-2- (2,4-dichlorophen-oxy) phenol, 3-chloro-2- ( 2, -dichlorophenoxy) phenol, 6-chloro-2- (2,4-dichlorophenoxy) phenol, 5-chloro-2- (3,4-dichlorophenoxy) phenol, 5-chloro-2- (2,5-dichlorophenoxy) phenol and 5-chloro-2- (3,5-dichloro-phenoxy) phenol. Alternatively, the toxophore Z is 2,2'-dihydroxy biphenyl ether. Even in a further embodiment, the toxophore Z is a halogenated 2-hydroxybenzophenone. In a further embodiment, Y and X in combination is a substituent having a structure selected from the group consisting of where T is selected from the group consisting of oxygen, nitrogen, sulfur and carbon. Specific embodiments of the invention, include, but are not limited to the following modifications of the structure shown above: 1. Z is absent; 2. Y is an alkynyl of 2 to 3 carbon atoms; 3. X is carbonyl or methylene; 4. X is methylene; and 5. Prodrugs that. They have the structures: Any of the compounds described herein may be combined with a carrier, for example, a pharmaceutically acceptable carrier. This invention also provides a composition comprising the prodrug compounds as described above, alone or in combination with other compounds or other agents, known or still to be discovered, and a carrier. In one embodiment, the carrier is a pharmaceutically acceptable carrier. This invention also provides an in vitro method for analyzing drugs that inhibit or kill antibiotic-resistant microorganisms, comprising the steps of contacting the drug with an antibiotic-resistant microorganism and separately contacting the antibiotic-resistant microorganism with a compound Pro-drug of this invention and compare the growth of microorganisms, in order to analyze the drugs that inhibit or kill antibiotic-resistant microorganisms. Drugs with activity to inhibit or kill antibiotic-resistant microorganisms similar to the compounds of this invention are considered therapeutically relevant for further testing and development. The method is particularly suitable for analyzing drugs that are effective against ß-lactam or vancomycin resistant microorganisms. The ß-lactam-resistant microorganism is a Gram-negative or Gram-positive bacterium. Examples of such 'include, but are not limited to, Gram-negative bacteria selected from the group consisting of Neisseria, Moraxella, Campylobacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides and Gram-positive bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis and other staphylococci negative to coagulase, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus. This invention also provides a method for inhibiting the growth of an antibiotic-resistant microorganism by contacting the microorganism with an effective amount of a prodrug compound of this invention. The contact operation can be performed in vitro or in vivo. When contacted in vitro, the method provides a means to control the growth of antibiotic-resistant microorganisms on surfaces and to be used as a disinfectant. In vivo, the method provides a positive control for animal models to test new potential drugs. Variant concentrations of the potential agent are put in contact with the sample to determine the optimum effective concentration of the agent. Thus, in one aspect, this invention relates to the discovery and use of the same agents that are selective substrates for enzymes that confer resistance to the drug to microorganisms. Also provided by this invention are kits (kits) containing the prodrugs as described herein, and instructions necessary to perform the test. Cell or tissue samples as used herein, comprise cells or tissues characterized by the presence of drug resistance, drug resistance which is the result of overexpression of an enzyme by the infectious microorganism. The cell can be a eukaryotic cell, i.e., a mammalian cell, for example, a mouse cell, a rat cell, a hamster cell or a human cell. The cell can also be a prokaryotic cell such as a bacterial cell. The cell can be continuously cultured or isolated from an infected animal or human subject.

The method can be practiced in vi tro, ex vivo or in vivo. The in vivo practice of the invention in an animal such as a rat or mouse, provides a convenient animal model system that can be used prior to the clinical testing of the therapeutic agent or prodrug. In this system, a potential prodrug will be successful if the microbial load is reduced or the symptoms of the infection are improved, each one compared to an infected, untreated animal. It may also be useful to have a negative, separate, control group of cells or animals that has not been infected, which provides a basis for comparison. When practiced in vivo, the candidate prodrug is administered to the animal in effective amounts. As used herein, the term "administration" or "delivery" for in vivo or ex vivo purposes (if the target cell population is to be returned to the same (autologous) or another patient (allogeneic)) means that the subject is provided with an effective amount of the candidate prodrug, effective in reducing the bacterial load. In these cases, the agent or prodrug can be administered with a pharmaceutically acceptable carrier. The agents, prodrugs and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration according to conventional procedures, such as an active ingredient in pharmaceutical compositions. The methods of administration of pharmaceutical compositions are. well known to those of ordinary skill in the art and include, but are not limited to, administration by microinjection, intravenous? parenteral The compositions are proposed for topical, oral or local administration, as well as intravenous, subcutaneous or intramuscularly. The administration can be carried out continuously or intermittently throughout the course of the treatment. The methods of determining the most effective medium and the dose of administration are well known to those skilled in the art and will vary with the prodrug used for the therapy, the purpose of the therapy, the microorganism being treated, the severity of the infection and the subject that is treated. Individual or multiple administrations can be carried out with the level and dose pattern that is selected by the attending physician. For example, the compositions can be administered to a subject who already suffers from an antibiotic-resistant bacterial infection. In this situation, an effective "therapeutic amount" of the composition is administered to prevent it from continuing and at least partially stop the microbial growth and growth. proliferation, and improve the associated symptoms cori an infection.

However, prodrugs can be administered to subjects or individuals susceptible to, or at risk of developing an infection. In these embodiments, a "prophylactically effective amount" of the composition is administered to maintain cell viability and function at a level close to the level of preinfection. It should be understood that by preventing or inhibiting unwanted cell death in a subject or individual, the prodrug compositions and methods of this invention also provide methods to treat, prevent or ameliorate the symptoms associated with a disease characterized by unwanted infection. Such diseases include, but are not limited to, Gram-negative and Gram-positive infections, shown in the following table.

The amplification of genes associated with microbial resistance can be detected and verified by a modified polymerase chain reaction (PCR) as described in U.S. Patent No. 5,085,983. Alternative analyzes include analysis of enzyme activity (Miller, 1992; Spector et al., 1997) and via the polymerase chain reaction (Spector et al., 1997).; Maher et al., 1995). The method is particularly suitable for analyzing drugs that are effective against ß-lactam or vancomycin resistant microorganisms. The ß-lactam-resistant microorganism is a Gram-positive or Gram-negative bacterium. Examples of such include, but are not limited to, Gram-negative bacteria selected from the group consisting of Neisseria, Moraxella, Campylobacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides and Gram-positive bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis and other staphylococci negative to coagulase, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus. Additionally, the invention is effective against organisms resistant to vancomycin. The compounds of the invention kill bacteria with a different mechanism of action. These prodrug compounds were designed to have double modes of function. They can kill bacteria by forming bactericide in ß-lactamase-producing strains. They may also have cidal activity against non-β-lactamase strains by the mechanism of inhibiting cell wall biosynthesis. These compounds have increased activity against the ß-lactamase-producing strains, so they also have potency against strains lacking ß-lactamase. When these compounds are treated with bacterial strains lacking ß-lactamase, they are expected to inhibit the penicillin binding protein (PBP), similar to conventional ß-lactam antibiotics. Meanwhile, the molar equality of bactericide is formed, thus producing the bactericidal activity. Therefore, for negative β-lactamase infections, the compounds of the present invention exert their antibacterial activity by the formation of bactericidal agents and also by the inhibition of PBP. In addition, by this invention, a method for treating a subject infected with an antibiotic-resistant microorganism is provided by providing the subject with an effective amount of a compound of this invention. The compound can be supplied as it is, or as a composition comprising a pharmaceutically acceptable carrier. As used herein, the subject includes, but is not limited to, plants and vertebrates such as fish, mammals or birds, as defined in the foregoing. The method is particularly suitable for analyzing or testing drugs that are effective against ß-lactam or vancomycin-resistant microorganisms. The ß-lactam-resistant microorganism is a Gram-negative or Gram-positive bacterium. Examples of such include, but are not limited to, the Gram-negative bacteria selected from the group consisting of Neisseria, Moraxella, Campylobacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides and the Gram-positive bacteria selected from the group consisting of Staphylococcus. aureus, Staphylococcus epidermis and other staphylococci negative to coagulase, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus. When an effective amount of a compound composition of this invention is administered to a subject, such as a plant, an animal or human patient, an infection can be treated or prevented. The prodrug compounds of this invention are also useful for the manufacture of a medicament for treating antibiotic-resistant microbial infections, for example, ß-lactam or vancomycin-resistant microorganisms. The ß-lactam-resistant microorganism can be a Gram-negative or Gram-positive bacteria. Examples of such include, but are not limited to, Gram-negative bacteria selected from the group consisting of Neisseria, Moraxella, Campylobacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides and Gram-positive bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis and other staphylococci negative to coagulase, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus. In addition, the invention provides a method for selecting antibiotic sensitivity, for example, β-lactam activity, since a probable mechanism by which organisms gain resistance to prodrugs is via the loss of β-lactam activity. lactamase, which makes bacteria sensitive to ß-lactam antibiotics once again. Thus, this invention provides a method for reversing resistance to the antibiotic in a microorganism by selecting the loss of the activity of the resistance enzyme. The method requires contacting the microorganism with a prodrug of this invention, in order to kill the microorganisms that express this enzyme. Using β-lactam as an example only, organisms that have lost the β-lactamase enzyme will survive. These surviving organisms are now selected for sensitivity to the original antibiotic and can be killed effectively by contacting them with this antibiotic. Thus, the invention also provides a combination therapy for the treatment of microbial infections, wherein the microorganism is a layer of developing antibiotic resistance as defined below. Combination therapy requires first treatment with the β-lactam antibiotic, then treating with a β-lactam prodrug as defined herein, then finally treating with the original β-lactam antibiotic. Also disclosed is a method for reversing resistance to the antibiotic in a microorganism by contacting the microorganism with an effective amount of the prodrug of this invention. Unlike previous work (Melton &; Sherwood, 1996), the prodrugs of the present invention need not be combined with a targeting agent. Thus, the prodrugs can be directly used, topically systemically. This invention also provides a method for selectively inhibiting the proliferation of an antibiotic-resistant microorganism by contacting the microorganism with an effective amount of a prodrug of this invention. As mentioned above, the action of contacting can be performed in vitro against samples of cells in culture or in samples, ex vivo, or in vivo in an animal system. The methods of this invention can also be practiced ex vivo using a method modification described in U.S. Patent No. 5,399,346. The prodrugs of this invention are useful for inhibiting the proliferation of a microorganism that is resistant to a β-lactam antibiotic, for example, penicillin or cephalosporin. Additionally, the prodrugs of this invention are useful for inhibiting the proliferation of a microorganism that is resistant to vancomycin. The β-lactamases can be found either extracellularly or within the periplasmic space of the microorganism. The genetic information for the β-lactamase synthesis can either be carried on a plasmid or it can occur within the bacterial chromosome; any of these can result in the production of enzymes that lead to resistance to the common ß-lactam antibiotics. Plasmid-mediated β-lactamases are especially misleading because of the ease with which these extrachromosomal elements can be transferred from one bacterial strain to another. Some β-lactamases, initially encoded for a plasmid, may have this genetic information eventually incorporated into the chromosome as a permanent addition to cellular deoxyribonucleic acid. N is unusual for bacteria to carry multiple plasmids, which code for multiple enzymes of antibiotic modification. It is also possible that multiple resistance factors can be carried out on a plasmid sol. Thus, it is becoming common for bacteria to appear with resistance to two or three classes of antibiotics. One of the most worrying aspects of the production of chromosomal β-lactamase is the ease of inducibility of these enzymes, resulting in high concentrations of β-lactamase. The best known inducers are β-lactam antibiotics, frequently those that are subsequently hydrolyzed by the induced enzyme. In some cases, a stably repressed mutant can be selected, with the total β-lactamase content representing as much as 4% of the total protein in the bacterial cell. One of the purposes of this invention is to provide prodrugs that can be activated by any ß-lactamase, to avoid in this way the problem of selecting the appropriate ß-lactamase inhibitor. Because the ß-lactam adduct of the prodrug will be broadly activated by the β-lactamases of many bacterial species (see, eg, Vrudhula et al., 1995), a single prodrug will find utility in treating many different kinds of infections, previously resistant to treatment due to high levels of β-lactamase production through the target organism. This procedure avoids the problem of mutation resistance found with β-lactamase inhibitors (Bush, 1988). This procedure is also useful because resistance to these prodrugs is likely to occur via the loss of β-lactamase activity. This will result in the bacterium regaining sensitivity to penicillins. Therefore, this invention also claims a method for causing ß-lactam antibiotic resistant organisms to be sensitive to ß-lactam antibiotics by contacting the organism with an effective amount of a prodrug of this invention or an agent identified by the test described above. Another limitation of some potent antibiotics, currently available, is its lack of specificity. Examples include doxorubicin, isolated from Streptomyces. One of the greatest challenges in drug discovery and development is the efficient targeting of the drug to a disease mechanism, with no effect on non-diseased or host organs. Because many of the antibiotics that have been discovered to date do not have good discrimination between bacterial targets and hosts, they have not been used as an anti-infective agent. Some of these compounds, however, have been employed to treat other diseases, such as cancer. This invention provides a means of targeting these toxic compounds (in the form of the prodrug) to infectious organisms with minimal exposure of the host to the toxin. Also relevant to this invention is the considerable prior art in which prodrug constructs of these antibiotics have been designed, in which they are activated by specific bacterial enzymes, such as β-lactamase. In this technology, known as prodrug therapy directed to the antibody (ADEPT) or prodrug therapy directed to the gene (GDEPT), a bacterial enzyme is localized to a tumor via an agent directed to the specific target, such as an antibody (Melton &Sherwood, 1996). The prodrug is then administered to the patient and is preferentially activated at the tumor site (where the enzyme has been localized via conjugation to the antibody). This provides a location for the antibiotic. antitumor, allowing higher concentrations of the active drug at the tumor site less systemic exposure to the active drug and its toxic activities. Several prodrugs have been prepared that are extensively activated by β-lactamases. These include β-lactam derivatives of doxorubicin (Vrudhula collaborators, 1995), paclitaxel (Rodríguez et al., 1995), mustard gases (Kerr et al., 1995), vinca pervinal alkaloids (Meyer et al., 1992) and mitomycin (Vrudhula et al. collaborators, 1995). These compounds have been shown to be activated by a broad spectrum of β-lactamases from different bacterial species (Vrudhula et al., 1995). The effectiveness of these drugs is dependent on the appropriate localization of the activation enzyme via the antibody that binds to the tumor, or by preferential expression of the activation enzyme in tumor cells. The authors of the publications cited above do not describe the utility of prodrugs as anti-infective agents. This invention does not require such localization by the antibody or other methods, since only the infecting organism is expressing the activation enzyme. The purpose of the present invention is to take advantage of this prodrug technology to treat infectious disease, not cancer. Similar prodrugs, activated by β-lactamase or other microbial enzymes normally involved in resistance to antibiotic therapy, whether expressed only by the infectious agent and / or overexpressed by the infectious agent, will be used to activate drug prodrug versions normally toxic to the host, specifically to treat infections. This "biochemical targeting" technology overcomes the lack of specificity of action of the drugs described above, by having the activated forms created at high concentrations only within or at the sites of the infectious disease. This is a novel procedure and makes possible the use of previously too toxic drugs to be used against infectious disease. Previously, Mobashery and colleagues (Mobashery and Johnson, 1986) described a polypeptide antibiotic that appeared to be activated by β-lactamase. His work failed to address several important points: (1) The activity was dependent not only on the expression of β-lactamase, but also on the transport of the peptide in the bacterial cell by peptide permeases and subsequent intracellular activation by other enzymes cell phones; (2) The peptide used was not active in an "enriched" medium (Boisvert et al., 1986). The first means of limitation that allows the entry of the drug, and the subsequent efficacy, was limited by its ability to enter the cell. Second, the peptide had no activity in the enriched media, which is likely to be the situation found in any in vivo application. The authors do not anticipate making possible the use of more toxic antibiotics (such as mitomycin or doxorubicin) as a result of this "target-directed" procedure. Similarly, groups that work on ADEPT, while recognizing the value in the application of prodrugs of β-lactam to treat cancer, do not recognize its use in infectious disease. In vivo administration can be effected in one dose, continuously or intermittently throughout the course of treatment. The methods for determining the most effective medium and dosage of administration are well known to those skilled in the art and will vary with the composition used for therapy., the purpose of the therapy, the target cell that is treated and the subject being treated. Individual or multiple administrations can be carried out at the dose level and pattern that is selected by the attending physician. Dosage formulations and suitable methods of administration of the agents can be found in the following. The pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and can take the form of tablets, pills, granules, capsules, pills, ampules, suppositories or aerosol form. It can also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions may also contain other pharmaceutically active compounds or a plurality of compounds of the invention. More particularly, an agent of the present invention also referred to herein as the active ingredient, can be administered for therapy by any suitable route including the oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal route , parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease that is treated. Ideally, the agent should be administered to achieve peak concentrations of the active compound at sites of the disease. This can be achieved, for example, by intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule, or syrup containing the active ingredient. Desirable agent levels in the blood can be. maintained by continuous infusion to provide a therapeutic amount of the active ingredient within the diseased tissue. The use of operative combinations is contemplated to provide therapeutic combinations that require a lower total dosage of each component agent that may be required when the individual therapeutic compound or drug is used alone, to thereby reduce the adverse effects. While it is possible for the agent to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other agents. therapeutic Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and not harmful to the patient. The formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods well known in the pharmacy art. Such methods include the step of carrying. in association the active ingredient with the carrier that constitutes one or more additional ingredients.

In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary to form the product. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, pills or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as a liquid oil-in-water emulsion or a liquid water-in-oil emulsion. The active ingredient can also be presented as a bolus, electuary or paste. A tablet can be made by cossion or molding, optionally with one or more additional ingredients. Cossed tablets can be prepared by cossing in a suitable machine the active ingredient in a form of free fluidity such as a powder or granules, optionally mixed with a binder (eg, povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active agent or dispersant. The molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated or scored and may be formulated to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. The tablets may optionally be provided with an enteric coating to provide release in parts of the intestines other than the stomach. Formulations suitable for topical administration in the mouth include tablets cosing the active ingredient in a flavor base, usually sucrose and acacia or tragacanth; pills cosing the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouth rinses cosing the active ingredient in a suitable liquid carrier. The pharmaceutical compositions for topical administration according to the present invention can be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, a formulation may cose a patch or a dressing such as a bandage or adhesive plaster ignated with the active ingredients and optionally one or more excipients or diluents.

If desired, the aqueous phase of the cream base may include, for example, at least about 30% w / w of a polyhydric alcohol, ie, an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The . Topical formulations can desirably include a compound that increases the absorption or penetration of the agent through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogues. The oily phase of the emulsions of this invention can be constituted of known ingredients in a known manner. While this phase may simply cose an emulsifier (otherwise known as an emulsifier), it desirably coses a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier (s) with or without stabilizer (s) constitute the so-called emulsifying wax, and the wax together with the oil and / or fat constitute the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Emulsifiers and emulsifier stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The selection of oils or greases suitable for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most of the oils, which are probably used in pharmaceutical emulsion formulations, is very low. Thus, the cream should preferably be a non-greasy, non-washable and washable product, with a suitable consistency to avoid the leakage of the tubes or other containers. The branched straight chain mono- or dibasic alkyl esters such as diisoadipate, isocetyl stearate propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or Mix of branched chain esters known as Crodamol CA can be used, the last three being the preferred stere. These can be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used. Formulations suitable for topical administration to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent. Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns, which is administered in the manner in which it is administered. aspiration is taken, that is, by rapid inhalation through the nasal passage from a container of dust held close to the nose. Suitable formulations wherein the carrier is a liquid for administration such as, for example, nasal spray, nasal drops or by aerosol administration by nebulizer, include aqueous or oily solutions of the agent.

Formulations suitable for parenteral administration include sterile, isotonic, aqueous or non-aqueous injection solutions, which may contain antioxidants, regulatory solutions, bacteriostats and solutes which render the formulation isotonic with the blood of the proposed recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems that are designed to direct the compound to the blood components or to one or more organs. The formulations may be presented in sealed unit dose or multiple dose containers, for example, ampoules and flasks, and may be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid carrier, eg, water for injections. , immediately before use. The extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as mentioned hereinbefore, or an appropriate fraction thereof, of an agent. Must be . understand that in addition to the ingredients particularly mentioned in the foregoing, the formulations of this invention may include other agents conventional in the art, having considered the type of formulation in question, for example, those suitable for oral administration may include such additional agents as agents sweeteners, thickeners and flavorings. It is also proposed that the agents, compositions and methods of this invention be combined with other suitable compositions and therapies. These agents of this invention and the compounds mentioned in the foregoing and their derivatives can be used for the preparation of medicaments for use in the methods described herein. In the clinical use of pro-drug antibiotics will purposely follow well-established principles. Dosage on purpose will be similar to those already used for most other antibiotics. It is estimated that a prodrug dose will be in the range of 100 mg to 1 gm, given once every eight hours, or once a day, for one or two weeks, or until the patient is tested negative for infectious organisms. In one aspect, the invention comprises a method of treating or protecting plants from bacterial infections resistant to antibiotics, which comprises applying an effective amount of the prodrug. In order to achieve good dispersion and adhesion of the compounds when used to treat plants, it may be advantageous to formulate the compounds with components that aid dispersion and adhesion. Suitable formulations will be known to those skilled in the art. This invention also provides a method of treating or protecting plants from infection by antibiotic-resistant bacteria by applying an effective amount of the prodrug compound to the foliage, roots or the soil surrounding the plants or roots. These isolated compounds can be combined with known pesticides or insecticides. The compounds within the present invention, when used to treat or protect plants from bacterial infections resistant to antibiotics, can be formulated as wettable powders, granules and the like, or they can be microencapsulated in a suitable medium and the like. Examples of other formulations include, but are not limited to, soluble powders, wettable granules, dry flowable, waterborne fluids, wettable dispersible granules, emulsifiable concentrates and aqueous suspensions. Other suitable formulations will be known to those skilled in the art. This invention further provides a method for administering the prodrug compound to fish, in an amount effective to either prevent or treat an antibiotic-resistant bacterial infection. The compound can be administered by incorporating the compound into the food supply for the fish. Alternatively, the compound can be added to the water in which the fish live, or is contained inside. Finally, the compound can be administered to the fish as a suitable pharmaceutical preparation. Other suitable formulations will be known to those skilled in the art.

Materials and Methods Processes for the Preparation of Pro Drug Compounds A process for producing the prodrugs of this invention is also provided. In general, the process requires the following steps: ECTA 7-a-bromocephalosporanic acid (2) This compound is prepared according to Rosati's procedures (North American patent No. 4,429,128, issued January 31, 1984) in an 80% yield as a whitish foam. It is used without purification in the next step. 3-Acetoxymethylcef-3-em-4-carboxylic acid (3) 7-a-bromocephalosporinic acid is debrominated with tributylphosphine in MeOH using the method of Chern et al., 1988. A yellow foam is obtained in quantitative yield. 3-Acetoxymethyl-cef-3-em-4-carboxylate of 4-nitrobenzylic acid (4) The crude cef-3-em-4-carboxylic acid obtained from the above is dissolved in dimethylformamide (DMF). Diisopropylethylamine (1 equivalent) is added dropwise under cooling with water. 4-Nitrobenzyl bromide (1 equivalent) is added in small portions. The water bath was removed and the reaction mixture is stirred at room temperature for 4 hours. The solvent and volatile components are removed in vacuo and the residue is taken up in EtOAc. The organic solution is washed with water, 0.5 N HCl and saturated NaHCO3 solution. After drying over anhydrous MgSO 4, the solvent is removed to give the crude product which is chromatographed on silica gel to give the product as a whitish foam. 3- (Iodomethyl) cef-3-em-4-carboxylate of 4-nitroben-cl (6) Bonjouklian's method, 1981, was used. To a 4-nitrobenzyl 4-cephalosporanate solution in CH 2 C 12 is added iodotrimethylsilane dropwise at 20 ° C under a nitrogen atmosphere. The reaction is stirred at room temperature for 3 hours. The reaction mixture is diluted with CH2C12 and washed with 10% Na2S203, brine and water, dried over MgSO4 and evaporated in vacuo to give the title compound as a light brown oil. 3- ((5-Chloro-2- (2,4-dichlorophenoxy) phenoxy) -triethyl) -cef-3-em-4-carboxylate of 4-n-fcrobenzyl (7) A mixture of the iodomethyl compound 6 (1 equivalent) ), triclosan (2, 4, 4'-tpchloro-2'-hydroxydiphenyl, 1 equivalent), NaHCO 3 (1.2 equivalents) in DMF is stirred at room temperature until all of the starting material 6 is consumed, as verified by thin layer chromatography. The DMF is evaporated in vacuo and the residue partitioned between EtOAc and water. The organic layer is separated and washed with brine, dried over MgSO4 and concentrated to give a crude product. Purification by column chromatography produces a white solid. 3- ((5-Chloro-2- (2,4-dichloro-binoxy) phenoxy) -methyl) -cef-3-em-4-carboxylic acid (8) The 4-nitrobenzyl ester 7 is dissolved in MeOH, a catalyst of 5% Pd / C is added and the mixture is stirred under a hydrogen atmosphere. When the reaction is complete, the catalyst is removed by filtration. The filtrate is concentrated under reduced pressure and taken up in EtOAc. The organic phase is extracted with saturated NaHCO3 twice. The combined NaHCO3 extracts are cooled to 0 ° C and acidified (pH < 2) with IN HCl. The precipitated crystals are harvested, washed with CH2C12 and dried in vacuo to give the desired product. The additional purification is achieved by recrystallization.

Compound 10 3-. { pyrid-2-yl-N-oxide) iomethyl-cef-3-em-4-carboxylato (10) A mixture of the iodomethyl compound 6 (1.0 equivalent), 2-mercaptopyridine-N-oxide (1.0 equivalent) and NaHCO 3 (1.1 equivalents) in DMF is stirred at room temperature until all of the starting material 6 is consumed, as verified by thin layer chromatography. The DMF is evaporated in vacuo and the residue is partitioned between EtOAc and water. The organic layer is separated, washed with brine, dried over MgSO4 and concentrated to give a crude product. Purification by column chromatography gives a solid. Deprotection of the p-nitrobenzyl group in the procedure described for compound 8 gave compound 10.

Compound 11 3- (N, N-bis (2-chloroethyl) carbamoyl) methyl-cef-3-em-4-carboxylate (11) A solution of the hydroxymethyl compound 3 (1.0 equivalent) and pyridine (1.0 equivalent) in anhydrous CH 2 C 12 is cooled in a bath with ice and N, -bis (2-chloroethyl) carbamoyl chloride is slowly introduced via a syringe. After thirty minutes the reaction is washed with water, brine, dried over MgSO and concentrated to give the crude product. Purification by column chromatography gives a solid. The deprotection of the p-nitrobenzyl group in the. procedure described for compound 8 provides compound 11.

I CQaHX OX Compound 12 3- (4-Amino-3-oxo-isoxazo-lidin-2-yl) methyl compound (12) A mixture of the hydroxymethyl compound 3 (1.0 equivalent), triphenylphosphine (1.0) equivalent), diethylazodicarboxylate (1.0 equivalent) and tert-butyl ester of (3-oxo-isoxazolidin-4-yl) -carbamic acid (1.0 equivalent) in anhydrous THF is stirred at room temperature for 4 hours. EtOAc is then added and the reaction mixture is washed with water, brine, dried over MgSO. Concentration and purification by column chromatography gives a solid. Deprotection of the p-nitrobenzyl group in the procedure described for compound 8 provides compound 12.

Compound 13 3- [(4-Amino-benzenesul-fonyl) - (5-methyl-isoxazol-3-yl) -a] n-methyl] cef-3-em-4-carboxylate A mixture of the hydroxymethyl compound 3 (1.0 equivalent), triphenylphosphine (1.0 equivalent), diethylazodicarboxylate (1.0 equivalent) and sulfamethoxazole (1.0 equivalent) in anhydrous THF is stirred at room temperature for 1 hour. EtOAc is then added and the reaction mixture is washed with water, brine, dried over MgSO4. Concentration and purification by column chromatography gives a solid. Deprotection of the p-nitrobenzyl group in the procedure described for compound 8 gives compound 13.

Compound 14 Ce -3-em-4-carboxylate 3- (initomicincarbamoyl-oxy) methyl (14) A solution of the hydroxymethyl compound 3 (1.0 equivalent), 2,6-lutidine (1.0 equivalent) in anhydrous THF is cooled in a bath with ice and 4-nitrophenylchloroformate is added slowly. After 30 minutes, a solution of N-BOC mitomycin C in anhydrous THF is added under an argon atmosphere. The ice bath is then removed and the stirring is continued at room temperature until completion of the reaction, as verified by thin layer chromatography. The reaction is then washed with water, brine and dried over MgSO4. Concentration and purification by column chromatography gives a solid. Deprotection of the p-nitrobenzyl group in the procedure described for compound 8 gives compound 14.

In Vitro Analysis The susceptibility test is made by using the NCCLS method (National Committee for Clinical Laboratory Standards) to determine the MICs of the antimicrobial compounds, modified for the high performance test. MIC is defined as the lowest concentration at which bacterial growth (equivalent to visible growth) was inhibited after 16 to 18 hours of incubation at the appropriate temperature required for bacterial growth. All extracts of tested compounds are prepared either in water or in DMSO, depending on the solubility. At the highest concentration tested, the DMSO content should not exceed 0.5%. In summary, 20 serial 2-fold dilutions of the highest concentration test compounds are made in a 384-cavity microtiter plate. Each cavity is inoculated with test bacteria in broth at a final concentration of approximately 1-1.5 x 106 cells / ml. Bacterial growth is determined by the increase in optical density at 600 nm using a microplate reader (Tecan SpectraFluor Plus).

Toxicity Analysis The toxicity of the ECTA compound is determined by intravenously injecting groups of ICR-CD1 male mice (weighing approximately 22 to 25 grams) with various concentrations of the prodrug compound. The vehicle of the compound is used as a control. Animals are observed twice a day within 14 days after inoculation and death is registered. BAT is determined (maximum tolerated dose).

In Vivo Analysis The in vivo efficacy of the compound is evaluated by intraperitoneally inoculating ECR-CD1 mice with 0.5 ml of bacteria at 100 times of MLD. Mucin is used as the control. An individual or multiple administration of the pro-drug compound (either intravenously, subcutaneously, intramuscularly or orally) is administered after inoculation. The vehicle of the compound is used as the control. Animals are observed twice a day within 14 days after inoculation and death is registered. The ED50 (average effective dose) of the compounds are determined. It will be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and the following examples are intended to illustrate and not to limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

REFERENCES Anderson et al., North American Patent No. ,399,346, issued March 21, 1995. Boisvert et al. (1986) J. Bíol. Chem. 261: 7871-7878.

Bonjouklian (1981) Tetrahedron Lett. 22: 3915-3918. Bush (1988) Clinical Microbial Rev. 1: 109-123. Chern et al. (1988) Heterocycles 27: 1349-1351. Kerr D.E. et al. (1995) Cancer Research 55: 3558-3563. Maher et al. (1995) Mol. Cell Probes 9: 265-276. Martin, REMINGTON 'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

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Miller, J.H. A Short Course In Bacterial Genetics: A Laboratory Manual And Handbook For E. Coli And Related Bacteria. Cold Spring Harbor Press (1992). Mobashery et al (1986) J. Biol. Chem. 261 (17): 7879-7887. Murray (1997) Antibiotic Resistance, Adv. Int. Med. 42: 339-367. Physicians Desk Reference, 50 ^ Edition (1996) Publ. Medical Economics Co., Montvale, NJ. Rodrigues et al. (1995) Chemistry And Biology 2: 223-227. Rosati, U.S. Patent No. 4,429,128, issued January 31, 1984. Scanlon, U.S. Patent No. 5,085,983, issued February 4, 1992. Schaechter et al. , Mechanisms of microbial disease (2nd Ed). Publ. Williams & Wilkins. pp.973 (1993). Steinberg J.P. and collaborators (1996) Clinical Infectious Diseases 23: 255-259. Stosor V. et al. (1996) Infect. Med. 13: 487-488 (1996), Opit. Pp. 493-498 (1996). Straus, S.E., Strategies To Combat Viral Infections, In Mechanisms Of Mícrobial Disease (2nd Ed.) Ed. T.S Satterfield, Publ. Williams & Williams, Baltimore, Md pp. 537-550,973 (1993). Vrudhula et al. (1995) J. Med. Chem. 38: 1380-1385.

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Claims (57)

1. CLAIMS 1. A prodrug compound that has the structure: characterized in that R 'is selected from the group consisting of hydrogen, alkyl, aryl, halogenated aryl, phenol, nitro aryl, ammonium, methylamine, dimethylamine, * lower alkylamine, bis (lower alkyl) amine, glycol, glycerol, sorbitol, polyethylene glycol ( PEG), salt form (sodium, potassium, lithium), THAM (2-amino-2-hydroxymethyl-l, 3-propanediol) and pharmaceutically acceptable salts thereof; r wherein X is absent or selected from the group consisting of carbonyl, methylene, oxygen, sulfur and nitrogen; wherein Y is selected from the group consisting of methylene, methyl alkenyl, methylene alkynyl, methyleneoxycarbonyl, vinyl and an alkynyl of 1 to 6 carbon atoms; and where Z is a toxophore.
2. 2. The compound according to claim 1 ,. characterized in that the toxophore Z is selected from the group consisting of 1-fluoro-l-carbonylmethyl and l-nitro-2-carbonylethyl.
3. 3. The compound according to claim 1, characterized in that the toxophore Z is selected from the group consisting of doxorubicin, bis- (2-chloroethyl) amine, mitomycin, trichlorcarban, triclorscarbanilide, tribromosalicylanilide, sulfamethoxazole, chloramphenicol, cycloserine, trimethoprim, chlorhexidine, hexachlorophene, 2-mercaptopyridine-N-oxide, camptothecin, apoptolidene, cisplatin, anthracycline, epothilone, halicondrine, hemiaster-lina, methioprim, tapsigargin and fentichlor.
4. 4. The compound according to claim 1, characterized in that the Z-toxophore is a phenol substituted with chlorine.
5. The compound according to claim 4, characterized in that the phenol substituted with chlorine is selected from the group consisting of 5-chloro-2- (2,4-dichlorophenoxy) phenol, 4-chloro-2- (2, 4) -dichlorophenoxy) phenol, 3-chloro-2- (2, -dichlorophenoxy) phenol, 6-chloro-2- (2, -di-chlorophenoxy) phenol, 5-chloro-2- (3, -dichlorophenoxy) phenol, -chloro-2- (2, 5-dichlorophenoxy) phenol and 5-chloro-2- (3,5-dichloro-phenoxy) phenol.
6. 6. The compound according to claim 1, characterized in that the Z-toxophore is 2,2'-dihydroxy biphenyl ether.
7. 7. The compound according to claim 1, characterized in that the Z toxophore is a halogenated 2-hydroxybenzophenone.
8. 8. The compound according to claim 1, characterized in that Y and X in combination is a substituent having a structure selected from the group consisting of wherein T is selected from the group consisting of oxygen, nitrogen, sulfur and carbon.
9. 9. The compound according to any of claims 1 to 8, characterized in that Z is absent.
10. 10. The compound according to any of claims 1 to 8, characterized in that Y is an alkynyl of 2 to 3 carbon atoms.
11. 11. The compound according to claim 1, characterized in that X is carbonyl or methylene.
12. 12. The compound according to claim 1, characterized in that X is methylene.
13. 13. A prodrug compound, characterized in that it has the structure:
14. 14. A prodrug compound, characterized in that it has the structure:
15. 15. A prodrug compound, characterized in that it has the structure:
16. 16. A prodrug compound, characterized in that it has the structure:
17. 17. A prodrug compound, characterized in that it has the structure:
18. 18. A composition, characterized in that it comprises the compound of any of claims 1 to 8 or 11-17 and a carrier.
19. 19. A composition, characterized in that it comprises • the compound of claim 9 and a carrier.
20. 20. A composition, characterized in that it comprises the compound of claim 10 and a carrier.
21. 21. The. composition according to claim 18, characterized in that the carrier is a pharmaceutically acceptable carrier.
22. 22. The composition according to claim 19, characterized in that the carrier is a pharmaceutically acceptable carrier.
23. 23. The composition according to claim 20, characterized in that the carrier is a pharmaceutically acceptable carrier. *
24. 24. An in vitro method to analyze drugs that inhibit or kill antibiotic-resistant microorganisms, characterized in that it comprises the steps of: a) contacting the drug with an antibiotic-resistant microorganism and contacting separately the • antibiotic-resistant microorganism with a The compound of claim 1; and b) to compare the growth of the microorganisms, in order to analyze the drugs that inhibit or kill antibiotic resistant microorganisms. 20
25. 25. The method according to the claim 24, characterized in that the antibiotic resistant microorganism is a β-lactam resistant microorganism.
26. 26. The method according to claim 24, characterized in that the microorganism resistant to The antibiotic is a microorganism resistant to vancomycin.
27. 27. The method according to claim 25, characterized in that the ß-lactam-resistant microorganism is a Gram-negative bacterium.
28. The method according to claim 27, characterized in that the Gram-negative bacteria are selected from the group consisting of Neisseria, Moraxella, Campylobacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides.
29. 29. The method according to claim 25, characterized in that the β-lactam resistant microorganism is a Gram-positive bacterium.
30. 30. The method according to claim 29, characterized in that the Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis, Coagulase negative Staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus.
31. 31. A method for inhibiting the growth of an antibiotic-resistant microorganism, characterized in that it comprises contacting the microorganism with an effective amount of the compound of claim 1.
32. 32. The method according to claim 31, characterized in that the resistant microorganism The antibiotic is a microorganism resistant to ß-lactam.
33. 33. The method according to claim 31, characterized in that the antibiotic-resistant microorganism is a vancomycin-resistant microorganism,
34. 34. The method according to claim 1. 32, characterized in that the microorganism resistant to β-lactam is a Gram-negative bacterium.
35. 35. The method according to claim 34, characterized in that the Gram-negative bacteria are selected from the group consisting of Neisseria, Moraxella, Campylbbacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides.
36. 36. The method according to claim 32, characterized in that the ß-lactam-resistant microorganism is a Gram-positive bacterium.
37. 37. The method according to claim 36, characterized in that the Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus.
38. 38. A method for treating a subject infected with an antibiotic-resistant microorganism, characterized in that it comprises supplying the subject with an effective amount of the compound of claim 1.
39. 39. The method according to claim 38, characterized in that the antibiotic-resistant microorganism is a microorganism resistant to ß-lactam.
40. 40. The method of compliance with the claim 38, characterized in that the antibiotic-resistant microorganism is a vancomycin-resistant microorganism.
41. 41. The method according to the claim 39, characterized in that the ß-lactam-resistant microorganism is a Gram-negative bacterium.
42. 42. The method according to claim 41, characterized in that the Gram-negative bacteria are selected from the group consisting of Neisseria, Moraxella, Campylobacter, Enterobacteriaceae, Pseudomonas, Acinetobacter, Haemophilus and Bacteroides.
43. 43. The method according to claim 39, characterized in that the β-lactam-resistant microorganism is a Gram-positive bacterium.
44. 44. The method according to claim 43, characterized in that the Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis, Coagulase negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus.
45. 45. The method according to claim 38, characterized in that the subject is a plant.
46. 46. The method according to claim 38, characterized in that the subject is a vertebrate.
47. 47. The method according to claim 46, characterized in that the vertebrate is a fish, mammal or bird.
48. 48. Use of a compound according to any of claims 1 to 8 or 11 to 17, characterized in that it is for the manufacture of a medicament for treating antibiotic-resistant infections.
49. 49. Use of a compound according to claim 10, characterized in that it is for the manufacture of a medicament for treating antibiotic-resistant infections.
50. 50. Use of a compound according to claim 11, characterized in that it is for the manufacture of a medicament for treating antibiotic-resistant infections.
51. 51. The use according to claim 48, characterized in that the antibiotic-resistant microorganism is a β-lactam or vancomycin-resistant microorganism.
52. 52. The use according to claim 49, characterized in that the antibiotic-resistant microorganism is a β-lactam or vancomycin-resistant microorganism.
53. 53. The use according to claim 50, characterized in that the antibiotic-resistant microorganism is a ß-lactam or vancomycin-resistant microorganism.
54. 54. A method for selecting antibiotic sensitivity to reverse antibiotic resistance in a microorganism that expresses an enzyme that confers antibiotic resistance to the microorganism, characterized in that it comprises contacting the microorganism with a compound of claim 1, to kill this way the microorganisms that express this enzyme.
55. 55. The method of compliance with the claim 54, characterized in that the enzyme is β-lactamase.
56. 56. A method for inhibiting the growth or killing a microorganism, characterized in that it comprises contacting the microorganism first with an antibiotic and subsequently contacting the microorganisms with a compound of claim 1.
57. 57. A method for reversing the resistance to antibiotic in a microorganism, characterized in that it comprises contacting the microorganism with an effective amount of a compound of claim 1.