EP2225207A2 - Tetrahydro-isoquinoline ppat inhibitors as antibacterial agents - Google Patents

Abstract

Disclosed are Tetrahydro-isoquinoline derivatives such as structural formula (I), pharmaceutical compositions comprising them and methods of treating bacterial infections. The disclosed compounds are inhibitors of PPAT (phosphopantetheine adenyl transferase), and are useful in the treatment and prevention of diseases caused by bacteria, particularly bacteria dependent on PPAT, for example, species such Escherichia coli, Helicobacter pylori, Staphyloccocus aureus, and the like. Description of the variables in structural formula (I) are provided herein.

Description

TETRAHYDRO-ISOOUINOLINE PPAT INHIBITORS AS ANTIBACTERIAL AGENTS

Related Application This application claims priority to U.S. Provisional Application No. 60/991,525,

Attorney Docket No. NPZ-006-1, filed November 30, 2007, entitled "TETRAHYDRO- ISOQUINOLINE PPAT INHIBITORS AS ANTIBACTERIAL AGENTS." The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.

Technical Field

The present invention relates to compositions which are PPAT inhibitors and methods and uses thereof.

Background of the Invention

In the last century, antibiotics were developed that led to significant reductions in mortality. Unfortunately, widespread use has led to the rise of antibiotic resistant bacteria, e.g., methicillin resistant Staphyloccocus aureus (MRS A), vancomycin resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumonias (PRSP). Some bacteria are resistant to a range of antibiotics, e.g., strains of Mycobacterium tuberculosis resist isoniazid, rifampin, ethambutol, streptomycin, ethionamide, kanamycin, and rifabutin. In addition to resistance, global travel has spread relatively unknown bacteria from isolated areas to new populations. Furthermore, there is the threat of bacteria as biological weapons. These bacteria may not be easily treated with existing antibiotics.

Infectious bacteria employ the coenzyme A (CoA) biosynthesis pathway, and, particularly in the penultimate step of the pathway, depend on phosphopantetheine adenyl transferase (PPAT), which transfers an adenyl moiety from adenosine triphosphate (ATP) to 4'-phosphopanthetheine, forming dephospho-CoA (dPCoA). While PPAT is present in mammalian cells, bacterial and mammalian PPAT enzymes differ substantially in primary sequence (about 18% identity) and physical properties. Thus, PPAT presents a desirable, selective target for new antibiotics.

Recent efforts have resulted in the identification of compounds that inhibit E. coli PPAT (Leslie, et al. "Antibacterial Anthranilates with a Novel Mode of Action"; Zhao, et al. "Inhibitors of Phosphopantetheine Adenylyltransferase"; Presented at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), San Diego, CA, 2002). However, these compounds are not appropriate for drug development. Furthermore, in one case, the structures are peptidic, while in the other case, representative compounds exhibited poor activity against purified PPAT. Therefore, there is a need for new antibiotics that target PPAT, whereby infections from bacteria dependent on PPAT can be treated.

Summary of the Invention

The present invention relates to certain tetrahydro-isoquinoline PPAT inhibitors as antibacterials. The disclosed compounds have antibiotic activity against bacteria, including drug-resistant bacteria. Thus, compounds that are PPAT inhibitors, methods of treatment with the disclosed PPAT inhibitors, and pharmaceutical compositions comprising the disclosed PPAT inhibitors are provided herein.

In one aspect, the invention provides a method of treating a subject for a bacterial infection, comprising administering to a subject in need of treatment for a bacterial infection an effective amount of a compound represented by structural Formula I:

I and pharmaceutically acceptable salts, solvates, hydrates, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof. The invention is useful for treating (therapeutically or prophylactically) bacterial infections, particularly infections caused by bacteria that depend on the CoA biosynthesis pathway, and more particularly, infections caused by bacteria that express the PPAT enzyme. Furthermore, it is useful against bacteria that have developed antibiotic resistance, especially multiple drug resistant strains, because it is believed to act through a different mechanism than existing, widely used antibiotics. Detailed Description of the Invention

The invention is generally related to methods, compounds, and pharmaceutical compositions for treating and preventing bacterial infections. In particular, the invention relates to substituted tetrahydro-isoquinoline derivatives that are PPAT inhibitors. In a preferred embodiment, the compound is represented by structural Formula I:

I and pharmaceutically acceptable salts, solvates, hydrates, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof; wherein X, Y, and Z are each independently, -H, OH, -OR^a, -SR^a, halogen, NH₂, NHR^a,

NR^a ₂, NR^aN₂, alkyl, aryl ring, heterocycle, or a fused ring;

R₃ is optionally substituted aryl, aralkyl, heteroaryl, heteroaralkyl, C₃-C₇ cycloaliphatic, or C₃-C₇ cycloalkyl;

L is -(CH₂)-, -(CO)-, -(CS)-, -(SO)-, or -(SO₂)-; R₄ is an alkyl, alkenyl, aryl, biaryl, heteroaryl, biheteroaryl, heteroaryl- aryl, aryl- heteroaryl, aralkyl, heteroaralkyl, C₁-Cs aliphatic, C₃-C₇ cycloalkyl, C₅-C₇ cycloaliphatic, or a 3-7 membered non-aromatic heterocyclic group; wherein R₄ can be substituted with halogen, -(C0)0R^a, -(C0)0(C0)R^a, - (CS)OR^a, -(SO)OR^a, SO₃R^a, -OSO₃R^a, -P(0R^a)₂, -(P0)(0R^a)₂, -0(P0)(0R^a)₂, -B(OR^a)₂, -(C0)NR^b ₂, -NR^c(C0)R^a, -SO₂NR^b ₂, or -NR^cS0₂R^a;

R₅ is -H, -(C0)0R^a, -(C0)0(C0)R^a, -(CS)OR^a, -(SO)OR^a, SO₃R^a, -OSO₃R^a, - P(OR^a)₂, -(PO)(OR^a)₂, -O(PO)(OR^a)₂, -B(0R^a)₂, -(C0)NR^b ₂, -NR^c(C0)R^a, -SO₂NR^b ₂, or -NR^cS0₂R^a;

R₆ is -H, -OH, halogen, or optionally substituted C₁-C₃ alkyl or alkoxy; each R^a and R^c are, independently, -H, C₁-C₅ alkyl, aryl, or aralkyl; each R^b is, independently, -H, C₁-C₅ alkyl, aryl, or aralkyl, or NR^b ₂ is a nonaromatic heterocyclic group. In one embodiment, X, Y, and Z are, independently, OR^a or a heterocycle, wherein each R^a is, independently, -H, C1-C5 alkyl, aryl, or aralkyl.

In another embodiment, R₃ in structural Formula I is an optionally substituted phenyl, pyridyl, benzo[l,3]dioxolyl, 2,3-dihydro-benzo[l,4]dioxinyl, pyrimidyl, pyrazyl, furanyl, pyrrolyl, thienyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, imidazolyl naphthyl, quinolinyl, biphenyl, benzopyrimidyl, benzopyrazyl, benzofuranyl, indolyl, benzothienyl, benzoxazolyl, benzoisooxazolyl, benzothiazolyl, benzoisothiazolyl, or benzimidazolyl group. Suitable optional substituents for the group represented by R₃ are provided herein below.

More preferably, R₃ in structural Formula I is represented by one of structural formulas R3-i to R3-v:

1 )W

R3-i R3-ii R3-iii

R3-iv R3-v

In structural formulas R3-i to R3-v, Y is -N-, -CH-, or -CRIl-; Z is-NR^z-, -S-, or -O-, wherein R^z is -H or C₁-C₃ alkyl, more preferably -H or methyl, or most preferably - H; the variable w is 0, 1, 2, or 3; each RIl is independently, halogen, -CN, -NO₂, -CF₃, - OCF₃, -OR¹, -(CO)R¹, -(CO)OR¹, -0(CO)R¹, -(CO)O(CO)R¹, -(CS)OR¹, -(SO)OR¹, - SO₃R¹, -C0NR^m ₂, -0(C0)NR^m ₂, -NRⁿ(C0)NR^m ₂, -NR¹XCO)OR¹ , -NR¹¹COR¹, - (SO₂)NR^m ₂) -NR¹¹SO₂R¹, or optionally substituted aryl, aralkyl, or C₁-C₅ alkyl. In the preceding, u is O to 5, each R¹ and Rⁿ are independently, -H, aryl, or aralkyl, C₁-C₅ alkyl, or C₁-C₅ haloalkyl, and each R^m is independently -H, aryl, aralkyl, or C₁-C₅ alkyl, or NR^m ₂ is a nonaromatic heterocyclic group.

Even more preferably, R₃ in structural Formula I is represented by one of structural formulas R3-i' to R3-v':

R3-r R3-H¹ R3-ϊϊi"

R3-iv" R3-V¹

In structural formulas R3-i' to R3-v', w is 0, 1, 2, or 3 and each RIl is independently -OH, -NO₂, -F, -Cl, -Br, C1-C4 alkyl, C1-C4 alkoxy, -CF₃, Or -OCF₃.

Still more preferably, R₃ is represented by one of structural formulas R3a to R3r:

OMe

R3^C R3« R3» R3'

R3= R3" R3' R3i

RS" RS¹ R3™ K3"

R3° RZ" R3' Most preferably, R₃ is represented by structural Formula R3^e.

R₄ in structural Formula I is optionally further substituted as described below in the section describing suitable substituents for aryl, heteroaryl, aliphatic, and cycloalkyl groups. More preferably, R₄ is a substituted phenyl, pyridyl, pyrimidyl, pyrazyl, naphthyl, biphenyl, phenyl-pyridyl, quinolinyl, benzopyrimidyl, benzopyrazyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, or a C₂- C_δ alkenyl group.

More preferably, R₄ is represented by one of structural formulas R4-i to R4-vii:

R4-i (RIO)_1n R4-ii (RIO)_n, R4-vi R4-iii R4-iv R4-v R4-vii In structural formulas R4-i to R4-vii, each m is independently 0, 1, 2, or 3 and X is -N-, -CH-, or -CRlO-; Ring B is C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl; Rings C and D are each, independently, aryl or heteroaryl; R8 is -OR^q or -NR^r ₂; R9 is -H, aryl, aralkyl, or C₁-C₆ aliphatic, each RlO is independently halogen, -CN, -NO₂, -CF₃, -OCF₃, -OR¹, -(CO)R¹, -(CO)OR¹, -0(CO)R¹, -(CO)O(CO)R¹, -(CS)R¹, -(SO)OR¹, -SO₃R¹, - CONR₂, -0(CO)NR₂, -NR^k(C0)NR^J ₂, -NR^k(C0)0R\ -NR^kCOR\ -(SO₂)NR₂, - NR^kS0₂R\ -(CH₂)_tNR^J ₂, or optionally substituted aryl, aralkyl or C₁-C₅ alkyl. The variable t is O to 5 and each R¹ and R^k is independently -H, aryl, aralkyl, C₁-C₅ alkyl, or C₁-C₅ haloalkyl; each R^J and R^r is independently -H, aryl, aralkyl, or C₁-C₅ alkyl, or each NR^J ₂ and NR^r ₂ are independently, a nonaromatic heterocyclic group. R^q is -H or optionally substituted aryl, aroyl, aralkyl, aralkanoyl, C₁-C₅ alkyl, or C₁-C₅ alkanoyl.

Even more preferably, R₄ is represented by one of structural formulas R4-i' to

R4-vii':

R4-IV¹ R4-V¹ R4-VΪ¹

In structural formulas R4-i' to R4-vii', each m is independently 0, 1, 2, or 3; R8 is -OH, C₁-C₅ alkoxy, or C₁-C₅ alkanoyloxy; R9 is -H or C₁-C₆ aliphatic; and each RlO is independently -OH, -NO₂, -F, -Cl, -Br, C₁-C₄ alkyl, C₁-C₄ alkoxy, -CF₃, or -OCF₃.

Still more preferably, R₄ is represented by one of structural formulas R4^a to R4^q:

R4^a K4" R4" R4' R49 R4"

^(CO)R8 (CO)R8 (CO)R8 (CO)R8

R4' R4I R4^k R4>

R4^m R4" R4° R4P

Most preferably, R₄ is represented by structural formula R4^a, R4^h, or R4^q. In R4^a to R4^q R8 is -NR^y ₂, -OH, C₁-C₅ alkoxy, or C₁-C₅ alkanoyloxy, wherein each R^y is independently -H or C₁-C₃ alkyl. Even more preferably, R8 is -OH or C₁-C₄ alkoxy, or still more preferably, -OH, -OCH₃, or -OCH₂CH₃. Most preferably, R8 is OCH₃ or -OCH₂CH₃.

In preferred embodiments, in structural Formula I, R8 is -OH.

In structural Formula I, R₃ is represented by one of structural formulas R3-i to R3-v or R₄ is represented by one of structural formulas R4-i to R4-vii. More preferably, R₃ is represented by one of structural formulas R3-i to R3-v and R₄ is represented by one of structural formulas R4-i to R4-vii. In still another embodiment, in structural Formula I, R₃ is represented by one of structural formulas R3-i' to R3-v' or R₄ is represented by one of structural formulas R4-i' to R4-vii'. More preferably, R₃ is represented by one of structural formulas R3-i' to R3-v' and R₄ is represented by one of structural formulas R4- i' to R4-vii'. In another preferred embodiment, for structural Formula I, R₃ is represented by one of structural formulas R3^a to R3^r, or R₄ is represented by one of structural formulas R4^a to R4^q. Preferably, R₃ is represented by one of structural formulas R3^a to R3^r, and R₄ is represented by one of structural formulas R4^a to R4^q. More preferably, R₃ is represented by structural formula R3^e, or R₄ is represented by structural formula R4^a, R4^h, or R4^q. Even more preferably, R₃ is represented by structural formula R3^e, and R₄ is represented by structural formula R4^a, R4^h, or R4^q.

In preferred embodiments X, Y and Z are each, independently, hydrogen, C₁-C₄ alkoxy, or together form a [l,3]-dioxolo fused ring. Most preferably X, Y and Z are each independently a hydrogen, methoxy or a

[l,3]-dioxolo fused ring.

In preferred embodiments L is -C(O)-.

In preferred embodiments R₄ is a phenyl ring independently substituted one or more times with halogen, CO₂H, C₁-C₄ alkyl, C₁-C₄ alkoxy or CH₂CO₂H. In preferred embodiments R₃ is a phenyl ring substituted one or more times with halogen.

In preferred embodiments R₅ and R₆ are each hydrogen.

In an exemplary embodiment, the compound of formula I is of the formula 5:

wherein X, Y, Z and R₃ are as previously defined and R' and R" are each independently a hydrogen, halogen, hydroxyl, alkyl, nitrile, carboxylic acid or heterocycle.

In another exemplary embodiment, the compound of formula I is of the formula 6:

wherein R₁₂, R₁₃, R₁₄ and R₁₅ are each, independently, H, Ci-_όalkyi, Ci-_όalkoxyl, or R₁₃ and R₁₄ may be linked to form:

(\)n I oi wherein n is 0, 1 or 2; R_1S is pyridine or phenyl optionally substituted with one or more halogens; and R₁₆ is pyridine or phenyl optionally, independently, substituted with one or more times with halogen, CO₂H, Ci-_όalkyi, cyano, tetrazoyl or hydroxyl.

In other embodiments the compound, the compound of the method, and the compound of the pharmaceutical composition are each represented the individual compounds provided in Table 1 :

Table 1: PPAT Inhibitors of Formula 1

10

In a particular embodiment, the compounds of Table 1 can be used to treat a bacterial infection in a subject in need thereof.

A "subject" includes mammals, e.g., humans, companion animals (e.g., dogs, cats, birds, aquarium fish, reptiles, and the like), farm animals (e.g., cows, sheep, pigs, horses, fowl, farm-raised fish and the like) and laboratory animals (e.g., rats, mice, guinea pigs, birds, aquarium fish, reptiles, and the like). Alternatively, the subject is a warm-blooded animal. More preferably, the subject is a mammal. Most preferably, the subject is human.

A subject in need of treatment has a bacterial infection (or has been exposed to an infectious environment where bacteria are present, e.g., in a hospital) the symptoms of which may be alleviated by administering an effective amount of the disclosed bicyclic derivatives. For example, a subject in need of treatment can have an infection for which the disclosed bicyclic derivatives can be administered as a treatment. In another example, a subject in need of treatment can have an open wound or burn injury, or can have a compromised immune system, for which the disclosed PPAT inhibitors can be administered as a prophylactic. Thus, a subject can be treated therapeutically or prophylactically. More preferably, a subject is treated therapeutically.

Typically, the subject is treated for a bacterial infection caused by a bacteria of a genus selected from Allochromatium, Acinetobacter, Bacillus, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Citrobacter, Escherichia, Enterobacter,

Enterococcus, Francisella, Haemophilus, Helicobacter, Klebsiella, Listeria, Moraxella, Mycobacterium, Neisseria, Proteus, Pseudomonas, Salmonella, erratia, Shigella, Stenotrophomonas, Staphyloccocus, Streptococcus, Synechococcus, Vibrio, and Yersina.

More preferably, the subject is treated for a bacterial infection from Allochromatium vinosum, Acinetobacter baumanii, Bacillus anthracis, Campylobacter jejuni, Chlamydia trachomatis, Chlamydia pneumoniae, Clostridium spp., Citrobacter spp., Escherichia coli, Enterobacter spp., Enterococcusfaecalis., Enterococcus faecium, Francisella tularensis, Haemophilus influenzas, Helicobacterpylori, Klebsiella spp., Listeria moiwcy to genes, Moraxella catarrhalis, Mycobacterium tuberculosis, Neisseria meningitidis, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella spp., Serratia spp., Shigella spp., Stenotrophomonas maltophilia, Staphyloccocus aureus, Staphyloccocus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Yersinapestis, and Yersina enterocolitica, and the like. Preferably, the subject is treated for a bacterial infection caused by a bacterium that expresses a PPAT protein. As used herein, a PPAT protein is a phosphopantetheine adenytransferase enzyme, i.e., systematic name ATP:pantetheine-4'-phosphate adenylyltransferase, IUBMB systematic classification EC 2.7.7.3, (see International Union of Biochemistry and Molecular Biology, www.chem.qmul.ac.uk/iubmb/). In one embodiment, a subject is also concurrently treated for a fungal infection, for example, a fungal infection caused by a pathogenic dermatophyte, e.g., a species of the genera Trichophyton, Tinea, Microspormn, Epidermophyton and the like; or a pathogenic filamentous fungus, e.g., a species of genera such as Aspergillus, Histoplasma, Cryptococcus, Microspormn, and the like; or a pathogenic non- filamentous fungus, e.g., a yeast, for example, a species of the genera Candida,

Malassezia, Trichosporon, Rhodotorula, Torulopsis, Blastomyces, Paracoccidioides, Coccidioides, and the like. Preferably, the subject is concurrently treated for a fungal infection resulting from a species of the genera Aspergillus or Trichophyton. Species of Trichophyton include, for example, T. mentagrophytes, T. rubrum, T. schoenleinii, T. tonsurans, T. verrucosum, and T. violaceum. Species of Aspergillus include, for example, A. fumigatus, A.flavus, A. niger, A. amstelodami, A. candidus, A. carneus, A. nidulans, A oryzae, A. restrictus, A. sydowi, A. terreus, A. ustus, A. versicolor, A. caesiellus, A. clavatus, A. avenaceus, and A. deflectus. More preferably, the subject is concurrently treated therapeutically for a fungal infection caused by a species of the genus Aspergillus selected from A. fumigatus, A. flavits, A. niger, A. canstelodami, A. candidus, A. carneus, A. nidulans, A oryzae, A. restrictus, A. sydowi, A. terreus, A. ustus, A. versicolor, A. caesiellus, A. clavatus, A. avenaceus, and. A. deflectus. Even more preferably the subject is concurrently treated therapeutically for a fungal infection caused by Aspergillus fumigatus or A spergillus niger, and most preferably, Aspergillus fumigatus.

An "effective amount" of a compound of the disclosed invention is the quantity that, when administered to a subject in need of treatment, improves the prognosis of the subject, e.g. , delays the onset of and/or reduces the severity of one or more of the subject's symptoms associated with a bacterial infection. The amount of the disclosed compound to be administered to a subject will depend on the particular disease, the mode of administration, co-administered compounds, if any, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Effective amounts of the disclosed compounds typically range between about 0.01 mg/kg per day and about 100 mg/kg per day, and preferably between 0.1 mg/kg per day and about 10 mg/kg/day. Techniques for administration of the disclosed compounds of the invention can be found in Remington: the Science and Practice of Pharmacy, 19^th edition, Mack Publishing Co., Easton, PA (1995), the entire teachings of which are incorporated herein by reference.

A "pharmaceutically acceptable salt" of the disclosed compound is a product of the disclosed compound that contains an ionic bond, and is typically produced by reacting the disclosed compound with either an acid or a base, suitable for administering to a subject.

For example, an acid salt of a compound containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like. Other examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g. (+)-tartrates, (-)-tartrates or mixtures thereof including racemic mixtures), succinates, benzoates and salts with amino acids such as glutamic acid.

Salts of compounds containing a carboxylic acid or other acidic functional groups can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N, N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, N-benzyl- 3-phenethylamine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N- methylglucamine, collidine, quinine, quinoline, and basic amino acid such as lysine and arginine.

Certain compounds and their salts may also exist in the form of solvates, for example hydrates, and the present invention includes each solvate and mixtures thereof. As used herein, a "pharmaceutical composition" is a formulation containing the disclosed compounds in a form suitable for administration to a subject. The pharmaceutical composition can be in bulk or in unit dosage form. The unit dosage form can be in any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (i.e., a formulation of the disclosed compound or salts thereof) in a unit dose of composition is an effective amount and may be varied according to the particular treatment involved. It is appreciated that it may be necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including topical, oral, pulmonary, rectal, vaginal, parenternal, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal and intranasal. The compounds described herein, and the pharmaceutically acceptable salts thereof, can be used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein. Techniques for formulation and administration of the disclosed compounds of the Invention can be found in Remington: the Science and Practice of Pharmacy, above.

For oral administration, the disclosed compounds or salts thereof can be combined with a suitable solid or liquid carrier or diluent to form capsules, tablets, pills, powders, syrups, solutions, suspensions and the like.

The tablets, pills, capsules, and the like contain from about 1 to about 99 weight percent of the active ingredient and a binder such as gum tragacanth, acacias, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch or alginic acid; a lubricant such as magnesium stearate; and/or a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor, and the like.

For parental administration of the disclosed compounds, or salts, solvates, or hydrates thereof, can be combined with sterile aqueous or organic media to form injectable solutions or suspensions. For example, solutions in sesame or peanut oil, aqueous propylene glycol and the like can be used, as well as aqueous solutions of water-soluble pharmaceutically- acceptable salts of the compounds. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

In addition to the formulations previously described, the compounds may also be formulated as a depot preparation. Suitable formulations of this type include biocompatible and biodegradable polymeric hydrogel formulations using crosslinked or water insoluble polysaccharide formulations, polymerizable polyethylene oxide formulations, impregnated membranes, and the like. Such long acting formulations may be administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection or a transdermal patch. Preferably, they are implanted in, or applied to, the microenvironment of an affected organ or tissue, for example, a membrane impregnated with the disclosed compound can be applied to an open wound or burn injury. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials, for example, as an emulsion in acceptable oil, or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

For topical administration, suitable formulations may include biocompatible oil, wax, gel, powder, polymer, or other liquid or solid carriers. Such formulations may be administered by applying directly to affected tissues, for example, a liquid formulation to treat infection of conjunctival tissue can be administered dropwise to the subject's eye, a cream formulation can be administer to a wound site, or a bandage may be impregnated with a formulation, and the like.

For rectal administration, suitable pharmaceutical compositions are, for example, topical preparations, suppositories or enemas. For vaginal administration, suitable pharmaceutical compositions are, for example, topical preparations, pessaries, tampons, creams, gels, pastes, foams or sprays. In addition, the compounds may also be formulated to deliver the active agent by pulmonary administration, e.g., administration of an aerosol formulation containing the active agent from, for example, a manual pump spray, nebulizer or pressurized metered- dose inhaler. Suitable formulations of this type can also include other agents, such as antistatic agents, to maintain the disclosed compounds as effective aerosols.

The term "pulmonary" as used herein refers to any part, tissue or organ whose primary function is gas exchange with the external environment, i.e., O₂/CO₂ exchange, within a patient. "Pulmonary" typically refers to the tissues of the respiratory tract. Thus, the phrase "pulmonary administration" refers to administering the formulations described herein to any part, tissue or organ whose primary function is gas exchange with the external environment (e.g., mouth, nose, pharynx, oropharynx, laryngopharynx, larynx, trachea, carina, bronchi, bronchioles, alveoli). For purposes of the present invention, "pulmonary" is also meant to include a tissue or cavity that is contingent to the respiratory tract, in particular, the sinuses.

A drug delivery device for delivering aerosols comprises a suitable aerosol canister with a metering valve containing a pharmaceutical aerosol formulation as described and an actuator housing adapted to hold the canister and allow for drug delivery. The canister in the drug delivery device has a head space representing greater than about 15% of the total volume of the canister. Often, the polymer intended for pulmonary administration is dissolved, suspended or emulsified in a mixture of a solvent, surfactant and propellant. The mixture is maintained under pressure in a canister that has been sealed with a metering valve.

For nasal administration, either a solid or a liquid carrier can be used. The solid carrier includes a coarse powder having particle size in the range of, for example, from about 20 to about 500 microns and such formulation is administered by rapid inhalation through the nasal passages. Where the liquid carrier is used, the formulation may be administered as a nasal spray or drops and may include oil or aqueous solutions of the active ingredients.

In addition to the formulations described above, a formulation can optionally include, or be co-administered with one or more additional drugs, e.g., other antibiotics, antiinflammatories, antirungals, antivirals, immunomodulators, antiprotozoals, steroids, decongestants, bronchodialators, and the like. For example, the disclosed compound can be co-administered with drugs such as such as ibuprofen, prednisone (corticosteroid) pentoxifylline, Amphotericin B, Fluconazole, Ketoconazol, Itraconazol, penicillin, ampicillin, amoxicillin, and the like. The formulation may also contain preserving agents, solubilizing agents, chemical buffers, surfactants, emulsifiers, colorants, odorants and sweeteners.

The term "derivative," e.g., in the term "tetrahydro-isoquinoline derivatives," refers to compounds that have a common core structure, and are substituted with various groups as described herein. For example, all of the compounds represented by structural Formula I are tetrahydro-isoquinoline derivatives, and have structural Formula I as a common core.

In the structural formulas depicted herein, a dashed line indicates a bond by which the depicted or moiety or group is connected to the remainder of the molecule. For example, the dashed line in R4-i indicates the bond that connects the depicted group to another structural formula. A dashed or solid line across a bond in a ring, for example, the solid line from RIl in R4-i, indicates that the represented bond can be connected to any substitutable atom in the ring. A zig-zag line indicates either cis or trans arrangement of the respective substituents with respect to the bond represented by the dashed line.

The term "aryl" group refers to carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl. The term "heteroaryl" group refers to hetero aromatic groups such as imidazolyl, isoimidazolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, 1,2,3- triazolyl, 1 ,2,4-triazolyl, and tetrazolyl. As used herein, a "heteroaryl" group is a 5 membered carbocyclic ring containing at least one N, S, or O atom and two double bonds, or a 6 membered carbocyclic ring containing at least one N, S, or O atom and three double bonds.

The term "nonaromatic heterocyclic" refers to non-aromatic ring systems typically having four to eight members, preferably five to six, in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom such as N, O, or S. Examples of non-aromatic heterocyclic rings include 3-tetrahydrofuranyl, 2- tetrahydropyranyl, 3-tetrahydropyranyl, 4-tetrahydropyranyl, [l,3]-dioxalanyl, [1,3]- dithiolanyl, [l,3]-dioxanyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2- morpholinyl, 3-morpholinyl, 4-raorpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, A- thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrorolidinyl, 1-piperazinyl, 2- piperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, 5- diazolonyl, N-substituted diazolonyl, and 1-pthalimidinyl.

The disclosed compounds can contain one or more chiral centers. The presence of chiral centers in a molecule gives rise to stereoisomers. For example, a pair of optical isomers, referred to as "enantiomers", exist for every chiral center in a molecule. A pair of diastereomers exists for every chiral center in a compound having two or more chiral centers. Where the structural formulas do not explicitly depict stereochemistry, for example in structural Formula I, it is to be understood that these formulas encompass enantiomers free from the corresponding optical isomer, racemic mixtures, mixtures enriched in one enantiomer relative to its corresponding optical isomer, a diastereomer free of other diastereomers, a pair of diastereomers free from other diasteromeric pairs, mixtures of diasteromers, mixtures of diasteromeric pairs, mixtures of diasteromers in which one diastereomer is enriched relative to the other diastereomer(s) and mixtures of diasteromeric pairs in which one diastereomeric pair is enriched relative to the other diastereomeric pair(s).

The term "alkyl" used alone or as part of a larger moiety (e.g., aralkyl, alkoxy, alkylamino, alkylaminocarbonyl, haloalkyl), is a straight or branched non-aromatic hydrocarbon which is completely saturated. Typically, a straight or branched alkyl group has from 1 to about 10 carbon atoms, preferably from 1 to about 5 if not otherwise specified. Examples of suitable straight or branched alkyl groups include methyl, ethyl, n-propyl, 2-propyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl. A C₁- C_1O straight or branched alkyl group or a C₃-Cs cyclic alkyl group can also be referred to as a "lower alkyl" group. An "alkoxy" group refers to an alkyl group that is connected through an intervening oxygen atom, e.g., methoxy, ethoxy, 2-propyloxy, tert-butoxy, 2- butyloxy, 3-pentyloxy, and the like.

The terms "optionally halogenated alkyl", and "optionally halogenated alkoxy", as used herein, includes the respective group substituted with one or more of -F, -Cl, -Br, or -I.

The terms "alkanoyl", "aroyl", and the like, as used herein, indicates the respective group connected through an intervening carbonyl, for example, - (CO)CH₂CH₃, benzoyl, and the like. The terms "alkanoyloxy", "aroyloxy", and the like, as used herein, indicates the respective group connected through an intervening carboxylate, for example, -0(CO)CH₂CH₃, -0(CO)C₆H₅, and the like.

The term "cycloalkyl group" is a cyclic alkyl group that has from 3 to about 10 carbon atoms, preferably from 3 to about 7. Examples of suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. A "cycloalkoxy" group refers to a cycloalkyl group that is connected through an intervening oxygen atom, e.g., cyclopentyloxy, cyclohexyloxy, and the like.

The term "aliphatic" includes branched and linear alkyl groups that contain one or more units of carbon-carbon unsaturation, i.e., carbon-carbon double or triple bonds. A cycloaliphatic group is a cyclic aliphatic group, for example, cyclohexenyl or cyclopentenyl.

The terms "aralkyl", "heteroaralkyl", "cycloalkylalkyl" "cycloaliphaticalkyl", and "nonaromatic heterocycloalkyl" refer to aryl, heteroaryl, cycloalkyl, cycloaliphatic, and nonaromatic heterocyclic groups, respectively, that are connected through an alkyl chain, e.g., benzyl, -CH₂CH₂-pyridine, (3-cyclohexyl)propyl, and the like.

The terms biaryl, biheteroaryl, aryl-heteroaryl and heteroaryl-aryl, as used herein, indicate two aryl groups connected by a single covalent bond, two heteroaryl groups connected by a single covalent bond, an aryl and heteroaryl group connected by single covalent bond, and a heteroaryl and aryl group connected by a single covalent bond, respectively. Examples of biaryl, biheteroaryl, heteroaryl-aryl and aryl-heteroaryl groups include biphenyl, bipyridyl, pyrimidyl-phenyl, and phenyl-pyridyl, respectively. When a biaryl, biheteroaryl, heteroaryl-aryl or aryl-heteroaryl group is a substituent, as in the definition of R4 for structural Formula I, the first recited group is bonded to the remainder of the molecule, i.e., "L" in structural Formula I. For example, when R4 in structural Formula I is a phenyl-pyridyl group, the phenyl of the phenyl-pyridyl group is bonded to L.

An "acyclic" group is a substituent that does not contain a ring. A "monocyclic" group contains only a single ring, for example, a phenyl ring that is not fused to another ring. A "polycyclic" group is a group that contains multiple fused rings, for example, naphthyl.

A "substitutable atom" is any atom such as nitrogen or carbon that is bonded through a single covalent bond to a hydrogen atom, wherein the hydrogen atom can be replaced with another group. A "substitutable ring atom" in an aromatic ring is any ring atom, e.g., a carbon or nitrogen, which is bonded by a single covalent bond to a hydrogen atom, wherein the hydrogen atom can be replaced with another group. Suitable substituents are those that do not substantially interfere with the pharmaceutical activity of the disclosed compound. A compound or group can have one or more substituents, which can be identical or different. Examples of suitable substituents for a substitutable carbon atom in an alkyl, aliphatic, cycloalkyl, cycloaliphatic, non-aromatic heterocyclic, aryl, or heteroaryl group include -OH, halogen (-Br, -Cl, -I and -F), -R, -OR, -CH₂R, - CH₂CH₂R, -OCH₂R, -CH₂OR, - CH₂CH₂OR, -CH₂OC(O)R, -0-COR, -COR, -SR, -SCH₂R, - CH₂SR, -SOR, -SO₂R, - CN, -NO₂, -COOH, -SO₃H, -NH₂, -NHR, -N(R)₂, -COOR, -CH₂COOR, -

CH₂CH₂COOR, -CHO, -CONH₂, -CONHR, -CON(R)₂, -NHCOR, -NRCOR, - NHCONH₂, -NHCONRH, -NHCON(R)₂, -NRCONH₂, -NRCONRH, -NRCON(R)₂, - C(=NH)-NH₂, -C(=NH)-NHR, -C(=NH)-N(R)₂, -CC=NR)-NH₂, -C(=NR)-NHR, - C(=NR)-N(R)₂, -NH-C(=NH)-NH₂, -NH-C(=NH)-NHR, -NH-C(=NH)-N(R)₂, -NH- C(=NR)-NH₂, -NH-C(=NR)-NHR, -NH-C(=NR)-N(R)₂, -NRH-C(=NH)-NH₂, -NR- C(=NH)-NHR, -NR-C(=NH)-N(R)₂, -NR-C(=NR)-NH₂, -NR-C(=NR)-NHR, -NR- C(=NR)-N(R)₂, -SO₂NH₂, -SO₂NHR, -SO₂NR₂, -SH, -SO_kR (k is 0, 1 or 2) and -NH- C(=NH)-NH₂. Each R is independently an alkyl, cycloalkyl, benzyl, aromatic, heteroaromatic, or iV-anilinyl group that is optionally substituted. Preferably, R is unsubstituted. In addition, -N(R)₂, taken together, can also form a substituted or unsubstituted heterocyclic group, such as pyrrolidinyl, piperidinyl, morpholinyl and thiomorpholinyl. Examples of substituents on group represented by R include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl.

Suitable substituents on the nitrogen of a heterocyclic group or heteroaromatic group include -R', -N(R')₂, -C(O)R', -CO₂R, -C(O)C(O)R', -C(O)CH₂ C(O)R', -SO₂R', - SO₂N(R')₂, -C(=S)N(R')₂, -C(=NH)-N(R')₂, and -NR'S0₂R', wherein R' is hydrogen, an alkyl, alkoxy, cycloalkyl, cycloalkoxy, phenyl, phenoxy, benzyl, benzyloxy, heteroaromatic, or heterocyclic group that is optionally substituted. Examples of substituents on the groups represented by R' include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl. Preferably, R' is unsubstituted.

Advantageously, the present invention also provides kits for use by a consumer for treating disease. The kits comprise a) a pharmaceutical composition comprising an antibiotic and a pharmaceutically acceptable carrier, vehicle or diluent; and, optionally, b) instructions describing a method of using the pharmaceutical composition for treating the specific disease. The instructions may also indicate that the kit is for treating disease while substantially reducing the concomitant liability of adverse effects associated with antibiotic administration.

A "kit" as used in the instant application includes a container for containing the separate unit dosage forms such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art which is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a "refill" of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle which is in turn contained within a box.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It may be desirable to provide a written memory aid, where the written memory aid is of the type containing information and/or instructions for the physician, pharmacist or subject, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested or a card which contains the same type of information. Another example of such a memory aid is a calendar printed on the card e.g., as follows "First Week, Monday, Tuesday," . . . etc. . . . "Second Week, Monday, Tuesday, . . . " etc. Other variations of memory aids will be readily apparent. A "daily dose" can be a single tablet or capsule or several tablets or capsules to be taken on a given day.

Another specific embodiment of a kit is a dispenser designed to dispense the daily doses one at a time. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter, which indicates the number of daily doses that, has been dispensed. Another example of such a memory-aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

EXEMPLIFICATION

Example 1: Synthesis of PPAT inhibitors of structural Formula I:

R' and R" are each independently hydrogen, halogen, hydroxy, alkyl, nitrile, carboxylic acid or a heterocycle

A mixture of amine 1 and aldehyde 2 in trifluoroacetic acid is heated to reflux under N₂ for two days. The mixture is allowed to cool to room temperature and most of TFA is removed in-vacuo. The residue is cooled to 0^QC and is treated with CH₂Cl₂ and 1 N NaOH. The organic layer is separated and the aqueous layer is extracted with CH₂Cl₂. The combined organic layer is dried with Na₂SO₄ and the solvent is removed in-vacuo. The product 3 is further purified by column chromatography on silica gel. A solution of compound 3 and anhydride 4 in DMSO is heated at 65^QC (bath temperature) under N₂ for 5 hours. The mixture is allowed to cool to room temperature and is treated with a 1:1 etheπethyl acetate mixture. This is washed with brine solution followed by water and then dried with Na₂SO₄. The solvent is removed in vacuo and the residue is purified by column chromatography on silica gel to give compound 5. Example 2: Synthesis of PPAT Inhibitor Compound 15

NH

HX

£ H₁C

O O CO₂H

O N

H₃CO-, \ F

Cl O , ^F _{c |}

H₃C = 4 5

A mixture of amine 1 (500 mg, 2.76 mM) and aldehyde 2 (398 mg, 2.80 mM) in trifluoroacetic acid (15 mL) was heated to reflux under N₂ for two days. The mixture was allowed to cool to room temperature and most of the triflouroacetic acid was removed in vacuo. The residue was cooled to 0⁰C and was treated with CH₂Cl₂ (30 mL) and IN NaOH (10 mL). The organic layer was separated and the aqueous layer was extracted with CH₂Cl₂ (2X 2OmL, IX 1OmL). The combined organic layer was dried with Na₂SO₄ and the solvent was removed in vacuo. The dark oily residue was purified by column chromatography on silica gel (ethyl acetate-ethyl acetate/methanol (97:3) to give compound 3 as waxy solid (296 mg, 35 %). ¹H NMR (DMSO-d₆): 2.7 (4 H, Ar- CH₂CH₂-NH), 3.49 and 3.76 (3 H each, two methyl groups), 5.37 (s, 1 H, ArAr'-CH- NH), 6.32 and 6.38 (both s, 1 H each, two aromatic protons next to methoxy). 6.45, 6.96 and 7.24 (IH each, rest of aromatic protons). A solution of compound 3 (102 mg, 0.33 mM) and anhydride 4 (105 mg, 0.48 mM) in DMSO (10 mL) was heated at 65°C (bath temperature) under N₂ for 5 hours. The mixture was allowed to cool to room temperature and was treated with 250 mL (ether/ethylacetate: 1/1). The mixture was washed with brine solution (2X 5OmL) followed by water (3X 50 mL) and then dried with Na₂SO₄. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (ethyl acetate/ hexane: ¹A to 1/3) to give compoud 5. This was recrystallized from chloroform/hexane and dried in vacuum oven at 65°C overnight to give 5 as a white powder (110 mg, 60 %). ¹H NMR (dmso-d₆): was not well defined due to rotomers. MS: m+1 = 522 Example 3: Bacteria are dependent on PPAT, a general target for antibiotics

The gene for PPAT, named coaD (alternatively, kdtE), has been identified: see Geerlof, et al., "Purification and characterization of Phosphopantetheine Adenylyltransferase from E. CoIi" J. Biol. Chem., 1999, 274(38), pp. 27105-11, the entire teachings of which are incorporated herein by reference. The gene sequence has been searched in a range of bacteria and in mammals using BLAST® (Basic Local Alignment Search Tool, available online at http://www.ncbi.nkri.nih.gov/BLAST/). The results are provided in Table 2.

Table 2: Conservation of PPAT gene (coaD) among range of bacterial species

PPAT is seen to be highly conserved across a range of bacterial pathogens. Thus, PPAT is a general target for antibiotics. Furthermore, although PPAT is present in mammalian cells, the mammalian sequence is sufficiently different to indicate that the disclosed PPAT inhibitors can be selective for bacterial PPAT.

The gene for PPAT, coaD, is disrupted from a range of bacteria by allelic exchange; see, for example, Geerlof, et al, above, and Freiberg, et al. 2001. "Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria" J MoI Microbiol Biotechnol 2001, 3, pp 483-9, the entire teachings of which are incorporated herein by reference. The survival of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Streptococcus pneumoniae in complex growth media is studied. The inability of the modified bacteria to survive without the coaD gene indicates that PPAT is necessary for bacterial survival and is thus a potential antibiotic target.

An additional experiment can test the survival of Escherichia coli in media containing exogenous dePhospho-CoA and/or CoA. Mammalian, including human cells, can make CoA from pantothenate (vitamin B₅) scavenged from the environment. Thus, it is possible that in a human subject, human cells/tissues could supply CoA to a bacterium that is unable to synthesize CoA. The inability of modified Escherichia coli to survive in media containing exogenous dePhospho- CoA and/or CoA further indicates that PPAT can be an antibacterial target.

Example 4: Kinetic assay of PPAT inhibition

The IC₅₀ (Inhibition Concentration at 50 percent) values for the disclosed compounds against PPAT are determined with various concentrations of the compounds in a range of 0.003 ~ 200μg/ml. Compounds with IC₅₀ values >200 can have a measurable IC₅₀ using a different assay method.

These inhibition assays are performed in 96-well assay plates, using a similar method to the screening assay above. The reaction buffer is 2OmM Hepes (pH 7.5), 10OmM NaCl, ImM MgCl₂, 0.5mM DTT, 0.006% Brij 35, 10% Glycerol, 25μM PPT, 0.5mM ATP, 0.2 Unit of pyrophosphatase, 200ng of PPAT in a total volume of lOOμl. The reaction is performed for 2 minutes, and then stopped with 150ml Malachite Green reagent. Absorption at 650nm is measured after 10 minutes of color development. The IC₅o's are determined with fitting data to the four-parameter method using XLfit (ID Business Solutions Inc., Cambridge, MA). The IC₅₀ value is derived from the curve as the compound concentration that gives 50% inhibition of the enzymatic reaction. The IC₅₀ data for compound of the invention is provided in Table 3.

Table 3: IC₅₀ data for PPAT inhibitors of formula 1

In order to perform the IC₅₀ assays, purified PPAT is needed. The E. coli PPAT gene is cloned into the pET28a expression vector (Novagen, Inc., Madison, WT) and expressed in E. coli BL21(DE3) cells. A chromatographic purification procedure employs Q-sepharose, gel filtration, and MonoQ chromatography, as follows. The methods are described in detailed in Geerlof, et al, above.

Each cell pellet is suspended in a 4 fold- volume of lysis buffer (5OmM KH₂PO₄ pH 8.0, 10OmM NaCl, 2mM EGTA, and 10% glycerol. Cells are broken by passage through a Microfluidics cell disrupter 4 times, and the cell lysate is centrifuged at 3,000 g for 20 minutes. The supernatant is then applied to a pre-equilibrated Q-sepharose column (1OmM Tris-HCl pH 8.0, 0.ImM EGTA, ImM PMSF, 10OmM NaCl, 10% glycerol, 0.1% p-mercaptoethanol, and 0.02% Brij 35). PPAT is eluted with NaCl gradient (0.1-lM) in the equilibrium buffer. The major peak fractions are pooled and concentrated, then applied to a Sephacryl S200 HR column (1OmM Tris-HCl pH 7.5, 15OmM NaCl, 0.ImM EGTA, 0.ImM PMSF, 10% glycerol, 0.1% β-mercaptoethanol, and 0.02% Brij 35). PPAT is eluted with the same buffer. The major peak fractions are pooled and loaded on a pre-equilibrated MonoQ column (1OmM Tris-HCl, pH 7.0, 0.ImM EGTA, 0.ImM PMSF, 10% glycerol, 0.1% β-mercaptoethanol, and 0.02% Brij 35). PPAT is eluted with a gradient of NaCl from 10OmM up to 100OmM. The peak fractions are pooled and dialyzed in the storing buffer (1OmM MOPS pH7.0,150mM NaCl, 0.ImM EGTA, 50% glycerol, 0.02% Brij 35), then stored at -20⁰C.

Example 5: Measuring disclosed PPAT inhibitors' antibiotic activity against drug- resistant bacteria

Potency, spectrum, target specificity and serum effect is evaluated by measuring the MIC (Minimum Inhibitory Concentration). This is the lowest concentration, in μg/mL, in a series of 2-fold dilutions of the compound that completely inhibits growth, for a panel of pathogenic bacteria. The strains comprising the bacterial panel are either obtained from American Type Culture Collection (ATCC, Manassas, VA), or genetically engineered to express varying levels of PPAT. The ATCC strains include the following: Escherichia coli (ATCC 35218), Staphylococcus aureus (ATCC 700699), and Enterococcusfaeciim (ATCC 700221). Other strains include Staphylococcus aureus RN4220, Escherichia coli WO-0159, Escherichia coli WO-0153, and Bacillus subtilis BD 170 with endogenous PPAT disrupted and complemented with PPAT under the regulation of inducible promoter, P_space-

The MIC assays are performed essentially as described in the NCCLS recommendations, the entire teachings of which are incorporated herein by reference (National Center for Clinical Laboratory Standards, 1997, (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria the Grow Aerobically), 4th ed.; approved standard. NCCLS document M7-A4. NCCLS, Wayne, PA.), with the following exceptions: both Tryptic Soy broth, and Mueller Hinton broth with and without the presence of serum are used as the growth medium. The concentration range tested is from 200 to 0.39 mcg/ml. Concentrations of 50-fold the desired final concentration are made by 2-fold serial dilutions in 96- well microtiter plates, after which 2 μL are transferred to the assay plates. Cells are grown up in the appropriate culture media and diluted back to final OD_60O of 0.001, after which 98 μL is inoculated into the assay plates. The final volume in each assay well is 100 μL. After an overnight incubation at 37°C, the assay plates are read. The MIC is determined as the minimal concentration that results in >80% inhibition of growth.

GOVERNMENT SUPPORT This invention was made with government support from the National Institutes of Health. The government has certain rights in the invention.

Claims

1. A compound represented by structural Formula I:

I and pharmaceutically acceptable salts, solvates, hydrates, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein:

X, Y, and Z are each independently -H, OH, -OR, -SR, halogen, NH₂, NHR^a, NR^a ₂, NR^aN₂, alkyl, aryl ring, heterocycle, or a fused ring;

R₃ is -H, optionally substituted aryl, aralkyl, heteroaryl, heteroaralkyl, C₃-C₇ cycloaliphatic, C₃-C₇ cycloalkyl, or C₁-C₅ alkyl; L is -(CH₂)-, -(CO)-, -(CS)-, -(SO)-, or -(SO₂ )-;

R₄ is an aryl, biaryl, heteroaryl, biheteroaryl, heteroaryl-aryl, aryl-heteroaryl, aralkyl, heteroaralkyl, C₁-Cs aliphatic, C₃-C₇ cycloalkyl, C₅-C₇ cycloaliphatic, or a 3-7 membered non-aromatic heterocyclic group, wherein the group represented by R₄ is substituted with halogen, -(C0)0R^a, -(C0)0(C0)R^a, -(CS)OR^a, -(SO)OR^a, SO₃R^a, - OSO₃R^a, -P(OR^a)₂, -(PO)(OR^a)₂, -O(PO)(OR^a)₂, -B(0R^a)₂, -(C0)NR^b ₂, -NR^c(C0)R^a, - SO₂NR^b ₂, or -NR^cS0₂R^a;

R₅ is -H, -(C0)0R^a, -(C0)0(C0)R^a, -(CS)OR^a, -(SO)OR^a, SO₃R^a, -OSO₃R^a, - P(OR^a)₂, -(PO)(OR^a)₂, -O(PO)(OR^a)₂, -B(0R^a)₂, -(C0)NR^b ₂, -NR^c(C0)R^a, -SO₂NR^b ₂, or -NR^cS0₂R^a; R₆ is -H, -OH, halogen, or optionally substituted C₁-C₃ alkyl or alkoxy; and each R^a and R^c is independently -H, C₁-C₅ alkyl, aryl, or aralkyl; and each R^b is independently -H, C₁-C₅ alkyl, aryl, or aralkyl, or NR^b ₂ is a nonaromatic heterocyclic group.

2. The compound of claim 1 where X, Y, and Z are OR^a or a heterocycle; wherein R^a is independently -H, C₁-C₅ alkyl, aryl, or aralkyl.

3. The compound of claim 2 wherein R₃ is an optionally substituted phenyl, pyridyl, benzo[l,3]dioxolyl, 2,3-dihydro-benzo[l,4]dioxine, pyrimidyl, pyrazyl, furanyl, pyrrolyl, thienyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, imidazolyl, naphthyl, quinolinyl, biphenyl, benzopyrimidyl, benzopyrazyl, benzofuranyl, indolyl, benzothienyl, benzoxazolyl, benzoisooxazolyl, benzothiazolyl, benzoisothiazolyl, or benzimidazolyl group.

4. The compound of claim 3 wherein R₄ is a substituted phenyl, pyridyl, pyrimidyl, pyrazyl, naphthyl, biphenyl, phenyl-pyridyl, bipyridyl, quinolinyl, benzopyrimidyl, benzopyrazyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, or C₂-Cs alkenyl group.

5. The compound of claim 4 wherein R₄ is represented by one of structural formulas R4-i to R4-vii:

R4-Ϊ (R10)_m R4-ii (R10)_m R₄-Vi

R4-iii R4-iv R4-v R4-vii

wherein: each m is independently 0, 1, 2, or 3;

X is -N-, -CH-, or -CR10-; Ring B is C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl; Rings C and D are each, independently, aryl or heteroaryl;

R8 is -OR^q or -NR^r ₂;

R9 is -H, aryl, aralkyl, or C₁-C₆ aliphatic; each RlO is independently halogen, -CN, -NO₂, -CF₃, -OCF₃, -OR¹, -(CO)R¹, - (CO)OR¹, -0(CO)R¹, -(CO)O(CO)R¹, -(CS)OR¹, -(SO)OR¹, -SO₃R¹, -CONR₂, - O(CO)NR^J2, -NR^k(CO)NR^J ₂, -NR^k(CO)OR\ -NR^kCOR\ -(SO₂)NR^J ₂, -NR^kSO₂R\ - (CH₂)_tNR^J ₂, or optionally substituted aryl, aralkyl or C₁-C₅ alkyl; each R¹ and R^k are independently, -H, aryl, aralkyl, C₁-C₅ alkyl, or C₁-C₅ haloalkyl; each R^J and R^r are independently, -H, aryl, aralkyl, or C₁-C₅ alkyl, or each NR^J ₂ and NR^r ₂ are independently, a nonaromatic heterocyclic group;

R^q is -H or optionally substituted aryl, aroyl, aralkyl, aralkanoyl, C₁-C₅ alkyl, or C₁-C₅ alkanoyl; and t is 0 to 5.

6. The compound of claim 5 wherein R₄ is represented by one of structural formulas R4-i to R4-vii:

R4-Ϊ (R10)_m R4-ii (R10)m R4-vi R4-iii R4-iv R4-V R4-vii wherein: each m is independently 0, 1, 2, or 3,;

X is -N-, -CH-, or -CRlO-;

Ring B is C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl; Rings C and D are each, independently, aryl or heteroaryl;

R8 is-OR^qor -NR^r ₂; R9 is -H, aryl, aralkyl, or C₁-C₆ aliphatic; each RlO is independently halogen, -CN, -NO₂, -CF₃, -OCF₃, -OR¹, -(CO)R¹, (CO)OR¹, -0(CO)R¹, -(CO)O(CO)R¹, -(CS)OR¹, -(SO)OR¹, -SO₃R¹, -CONR₂, - 0(C0)NR^J ₂, -NR^k(C0)NR^J ₂, -NR^k(C0)0R\ -NR^kCOR\ -(SO₂)NR^J ₂, -NR^kS0₂R\ - (CH₂)_tNR^J ₂, or optionally substituted aryl, aralkyl or C₁-C₅ alkyl; each R¹ and R^k are, independently, -H, aryl, aralkyl, C₁-C₅ alkyl, or C₁-C₅ haloalkyl; each R^J and R^r are, independently, -H, aryl, aralkyl, or C₁-C₅ alkyl, or each NR^J ₂ and NR^r ₂ are, independently, a nonaromatic heterocyclic group; R^q is -H or optionally substituted aryl, aroyl, aralkyl, aralkanoyl, C₁-C₅ alkyl, or

C₁-C₅ alkanoyl; and t is 0 to 5.

7. The compound of claim 6 wherein R₄ is represented by one of structural formulas R4-i' to R4-vii':

R4-ΪV R4-V¹ R4-VΪ¹ wherein: each m is independently 0, 1, 2, or 3;

R8 is -OH, C₁-C₅ alkoxy, or C₁-C₅ alkanoyloxy; R9 is -H or C₁-C₆ aliphatic; and each RlO is independently -OH, -NO₂, -F, -Cl, -Br, C₁-C₄ alkyl, C₁-C₄ alkoxy, - CF₃, Or -OCF₃.

8. The compound of claim 7 wherein, R₄ is represented by one of structural formulas R4^a to R4^q:

R4* R4^b R4= R4^d R4" R4^f R49 R4^h

(CO)RS i ^(CO)R8 1 ^(CO)RS (CO)R8

R4¹ R4I R4^k R4¹

R4^m R4ⁿ R4° R4P

R4i

(CO)R8

9. The compound of claim 8 wherein R₃ is represented by one of structural formulas R3-i to R3-v:

(R1

R3-i R3-ii R3-iii

R3-iv R3-v wherein

Y is -N-, -CH-, or -CRIl-;

Z is-NR^z-, -S-, or -O-, wherein R^z is -H or C₁-C₃ alkyl; the variable w is 0, 1, 2, or 3; each RIl are, independently, halogen, -CN, -NO₂, -CF₃, -OCF₃, -OR¹, -(CO)R¹, -(CO)OR¹, -0(CO)R¹, -(CO)O(CO)R¹, -(CS)OR¹, -(SO)OR¹, -SO₃R¹, -C0NR^m ₂, - 0(C0)NR^m ₂, -NRⁿ(CO)NR^m ₂, -NR¹¹CCO)OR¹, -NR¹¹COR¹, -(SO₂)NR^m ₂) -NR¹¹SO₂R¹, - (CH^_uNR¹^ or optionally substituted aryl, aralkyl, or C₁-C₅ alkyl; u is O to 5, each R¹ and Rⁿ are, independently, -H, aryl, or aralkyl, C₁-C₅ alkyl, or

C₁-C₅ haloalkyl; and each R^m is independently -H, aryl, aralkyl, or C₁-C₅ alkyl, or NR^m ₂ is a nonaromatic heterocyclic group.

10. The compound of claim 9 wherein R₃ is represented by one of structural formulas R3-i' to R3-v':

R3-Ϊ¹ R3-H¹ RZ-UV

R3-ΪV R3-V' wherein: w is O, 1, 2, or 3,; and each Rl 1 is independently -OH, -NO₂, -F, -Cl, -Br, C₁-C₄ alkyl, Ci-C₄ alkoxy, -

CF₃, or -OCF₃.

11. The compound of claim 10, wherein R₃ is represented by one of structural formulas R3a to R3r: R3° R3" R3° R3'

R3o R3^h R3¹ R3I

R3'¹ RS¹ R3™ R3"

R3° R3P R3i R3^r .

12. The compound of claim 11 wherein:

R3 is represented by one of structural formulas R3^a to R3^r; and R4 is represented by one of structural formulas R4^a to R4^q.

13. The compound of claim 12 wherein: R3 is represented by structural R3^e; and

R4 is represented by one of structural formulas R4^a, R4^h, or R4^q.

14. The compound of claim 13, wherein X, Y and Z are each, independently, hydrogen, C₁-C₄ alkoxy; or a [l,3]-dioxolo fused ring.

15. The compound of claim 14, wherein L is -C(O)-.

16. The compound of claim 15, wherein R₄ is a phenyl ring independently substituted one or more times with halogen, CO₂H, C₁-C₄ alkyl, C₁-C₄ alkoxy or CH₂CO₂H.

17. The compound of claim 16, wherein R₃ is a phenyl ring substituted one or more times with halogen.

18. The compound of claim 17, wherein R₅ and R₆ are each hydrogen.

19. The compound of claim 1, wherein the compound of Formula I is selected from the group consisting of the individual compounds provided in Table 1.

20. The compound of claim 1, wherein the compound is of the formula 6:

6 wherein R₁₂, R₁₃, R₁₄ and R₁₅ are each, independently, H, Q-_όalkyl, Ci-_όalkoxyl, or R₁₃ and R₁₄ may be linked to form:

(\)n I oi wherein n is 0, 1 or 2; R_1S is pyridine or phenyl optionally substituted with one or more halogens; and R₁₆ is pyridine or phenyl optionally, independently, substituted with one or more times with halogen, CO₂H, Q-_όalkyl, cyano, tetrazoyl or hydroxyl.

21. A method of treating a subject for a bacterial infection, comprising administering to a subject in need of treatment for a bacterial infection an effective amount of a compound of claim 1.

22. The method of claim 21, wherein the subject is a human.

23. The method of claim 21, wherein the infection is caused by a bacterium that expresses phosphopantetheine adenylytransferase.

24. The method of claim 21, wherein the infection is caused by a bacteria of a genus selected from Acinetobacter, Bacillus, Campylobacter, Chlamydia, Chlamydophila,

Clostridium, Citrobacter, Escherichia, Enterobacter, Enterococcus, Francisella, Haemophilus, Helicobacter, Klebsiella, Listeria, Moraxella, Mycobacterium, Neisseria, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, Staphyloccocus, Streptococcus, and Yersina.

25. A pharmaceutical composition comprising a compound of claim 1.