WO1998046566A1

WO1998046566A1 - IMPROVED β-LACTAM ANTIBIOTICS

Info

Publication number: WO1998046566A1
Application number: PCT/US1998/007690
Authority: WO
Inventors: In-Seop Cho; Scott J. Hecker; Tomasz W. Glinka; Ving J. Lee; Zhijia J. Zhang
Original assignee: Microcide Pharmaceuticals, Inc.
Priority date: 1997-04-17
Filing date: 1998-04-15
Publication date: 1998-10-22
Also published as: AU7126198A

Abstract

The present invention relates to novel β-lactam compounds, their pharmacologically acceptable salts and prodrugs, which exhibit antibiotic activity against a broad spectrum of organisms, especially bacteria which are resistant to conventional β-lactam antibiotics; e.g., methicillin-resistant Staphylococcal and ampicillin-resistant Enterococcal bacteria.

Description

DESCRIPTION

Improved /7-Lactam Antibiotics

Introduction

The present invention relates generally to organic chemistry, pharmaceutical chemistry, biochemistry, biology and medicine. More particularly, the present invention relates to novel ?-lactam compounds and their pharmacologically acceptable salts and prodrugs. Furthermore, the use of the claimed compounds, their salts, prodrugs and pharmacological compositions containing them, as antibiotics with activity against a broad spectrum of organisms, especially organisms which are resistant to conventional β- lactam antibiotics is disclosed.

Background of the Invention Over the past three decades a large variety of antibiotics has become available for clinical use. One class of antibiotics which has seen remarkable growth are the ?-lactams, over 70 of which have entered clinical use for the treatment of bacterial infections in mammals since 1965. For example, ?-lactams claimed to have antibacterial activity are described in U.S. Patent 3,992,377 and U.S. Patent 4,256,739.

Unfortunately, the wide-spread and indiscriminant use of these antibiotics has led to a rapid increase in the number of bacterial strains which are resistant to them. This resistance has emerged among clinically important microorganisms which is limiting the utility of the presently available ?-lactam antibiotics. In particular, resistant strains of Salmonella , S . pneumonix , Enterobacteria cex , and Pseudomonas have emerged which threaten to undo many of the strides made in reducing mortality and morbidity from bacterial infections.

The mode of action of ?-lactams as bacteriocides involves the inhibition of bacterial peptidoglycan biosyn- thesis. Bacterial resistance to ?-lactams occurs via three major pathways: a) the development of -lactamases capable of inactivating the ?-lactam ring; b) decreased ?-lactam penetration into the bacteria due to changes in bacterial cell wall composition; and c) poor binding to penicillin- binding proteins (PBPs) .

Pathway c) is particularly important in that the binding of ?-lactams to PBPs is essential for inhibiting bacterial cell-wall biosynthesis. For example, certain Gram-positive bacteria, namely methicillin-resistant Staphylococcus aureus ("MRSA") and enterococci are highly resistant to ?-lactam antibiotics and that resistance has been shown to be due to the presence of high levels of an unusual PBP, PBP2a, which is insensitive, or binds poorly, to /?-lactam antibiotics. Thus, as expected, the activity of ?-lactam antibiotics against PBP2a-containing organisms has been shown to correlate well with their binding affinity to PBP2a. Currently, the glycopeptides vancomycin and teicoplanin are primarily used for MRSA bacteremia. The quinolone antibacterials and some carbapenems, such as imipenem, have been reported to be active against a few MRSA strains, but their use is already being restricted due to emerging resistant MRSA strains.

Other experimental compounds which may possess utility as anti-MRSA or anti-enterococcal bactericides include the glycylcyclines (see, e.g., P.-E. Sum et al., J. Med. Che . , 37, (1994)), FK-037 (see, e.g., H. Ohki et al., J Antibiotics, 46:359-361 (1993)), RP-59,500 (see, e.g., S.K. Spangler et al., Antimicro Agents Chemother., 36:856-9 (1992)), the everninomycin complex (see, e.g., .E. Sanders et al., Antimicro. Agents Chemother., 6: 232-8 (1974)), the 2- (biaryl) carbapenems (see, e.g., U.S. Patent No. 5,025,006), 3- (benzothiazolylthio) cephems (see, e.g., EP Application No. 527686), 3- (thiazolylthio) carbacephems (see, e.g,_, R.J. Ternansky et al., J. Med. Chem. , 36:1971 (1993) and U.S. Patent No. 5,077,287) and arbekacin (S. Kondo, et al. J. Antibiotics 46:531 (1993). Recent advances in compounds, compositions and methods useful for treating infections in mammals arising from β- lacta antibiotic resistant bacteria are described in commonly owned International Application No. PCT/US95/03976 and U.S. Patent Application Serial Nos. 08/222,262, filed April 1, 1994; 08/369,798, filed January 6, 1995; 08/413,713, 08/413,714, 08/415,065, 08/413,712, 08/415,064 and 08/415,069, all of which were filed on March 29, 1995; 08/455,969, filed May 31, 1995; and 08/457,763, filed June 1, 1995, all of which are incorporated herein by reference in their entirety, including any drawings.

Summary of the Invention

The present invention relates generally to novel β- lactam compounds. In particular, it relates to antibiotic ?-lactams. In its most preferred embodiments, this inven- tions relates to ?-lactams which have biocidal activity against microorganisms resistant to conventional /?-lactam antibiotics, especially methicillin and/or ampicillin resistant disease-causing bacteria. The invention also relates to the preparation and use of pharmacological compositions of the disclosed compounds, their pharmacologically acceptable salts and prodrugs in the treatment of infections produced by disease-causing microorganisms. The compounds of this invention preferably have a minimum inhibitory concentration (MIC) that is less that

50%, more preferably less than 10%, and most preferably less than 1% of the MIC of Cefotaxime against a /?-lactam resistant organism, preferably a ?-lactam resistant

Staphylococcal or Enterococcal organism, more preferably a methicillin-resistant Staphylococcal or ampicillin-resistant

Enterococcal organism. Other preferred compounds are able to prevent or reduce mortality in mice infected with the β- lactam resistant organism to a greater extent that vancomycin or Cefotaxime.

1. The Compounds

A. General Structural Features.

In general, the compounds of this invention comprise an optionally substituted 4-membered ?-lactam ring fused through its nitrogen atom and the methylene group adjacent to the nitrogen to a five- or six-membered heteroalicyclic group. The five- of six-membered heteroalicyclic group is substituted with an optionally-substituted carboxylic acid group and with a sulfur atom. The sulfur atom is then bonded to a five- or six-membered heteroaromatic group (Q, below) . The five- or six-membered heteroaromatic group is both optionally and mandatorily substituted. That is, the five- or six membered heteroaromatic group must be substituted with one or two groups comprising, first, an optionally present, optionally-substituted mono- or polymethylene spacer group (alki) which in turn is bonded to a lipophilicity-enhancing group (R⁹⁹) . If the spacer group is not present, the lipophilicity-enhancing group R⁹⁹ is bonded directly to the heteroaromatic ring. The lipohili- city-enhancing group is bonded to another independent optionally-substituted mono- or polymethylene spacer group (alk₂) which, finally, is bonded to an end group (R¹²) which is either formally positively charged or is capable of attaining a positive charge at normal biological pHs . Any positions remaining open on the heteroaryl groups may be optionally substituted with a wide variety of substituents, described below. Schematically, the compounds of this invention can be visualized as follows:

(?-Lactam) (carboxyheteroalicylic) -sulfur-Q- (alki) _P-R⁹⁹-

(alk₂)_q-R¹²

Thus, in one aspect, the present invention relates to a compound selected from the group comprising:

and a pharmaceutically acceptable salt and/or prodrug thereof,

wherein: X is selected from the group including CH₂, oxygen, sulfur, SO and S0₂;

T is selected from the group including CH₂ and oxygen;

U is selected from the group including CH₂, sulfur, oxygen and

-CH (alkyl) ;

Y is selected from the group including H, -OCH₃, and -NHCHO;

R* is selected from the group including -CH(OH)CH₃, C(OH) (CH₃)₂,

-CHFCH₃, and -CH=CH₂;

R** is selected from the group including H, -CH₃, and -CH₂CH₃,

R¹ is selected from the group including -NHC(=0)ZR³; -NR⁴R⁵ and

wherein:

Z is selected from the group including -CH₂(X*)_m-,

-C(=NOR⁶)-, -CH(OR⁷)-, -C(=CHC0₂R⁸)- and -CH(NR⁹R¹⁰) wherein: X* is selected from the group including oxygen and sulfur,

m is 0 or 1;

R³ is selected from the group including cyano, alkyl, aryl, heteroaryl, heteroaralkyl and-(CH₂)_nW wherein: n is 1 - 6, inclusive; and,

is selected from the group including amino, amidino (C- or N-linked) , guanidino, and isothi- ourido;

R^4-7 are each independently selected from the group including hydrogen, alkyl, aryl and acyl; R⁸ is selected from the group including hydrogen, alkyl and aryl;

R⁹ and R¹⁰ are each independently selected from the group consisting of hydrogen, alkyl, acyl, and heterocyclecarbonyl ;

R² is selected from a group including hydrogen, alkyl, alkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, and trialkylsilyl or R² is not present at all and the C0₂ group to which it would be attached bears a negative charge;

Q is a heteroaryl group selected from the group including:

and

wherein:

A, B, D and E are selected from the group including carbon, nitrogen and sulfur and the specific juxtaposition of groups A, B, D, and E is limited to examples of heterocyclic groups known in the chemistry arts; and,

G, H, J, L and M are carbon, nitrogen or ⁺NRⁿ -(quaternary ammonium heterocycle) and the specific juxtaposition of groups G, H, J, L and M is limited to examples of heterocyclic groups known in the chemistry arts;

R¹¹ is selected from the group including hydrogen, halogen, alkyl, alkoxy, hydroxyl, amino, cyano, hydroxyalkyl, carboxamidoalkyl, optionally substituted aminoalkyl or quaternary ammonium alkyl and quaternary heteroaryliumalkyl; and,

R is - [ alkι ] _p [R 9³9³ -] q [ alk₂ ] _rR ,12

wherein alki and alk₂ are each independently selected from the group including optionally substituted methylene groups -(CR'R")-;

wherein

R' and R" are independently selected from the group including alkyl and aryl;

p is 0, 1 or 2;

R⁹⁹ is selected from the group including sulfur, sulfinyl, sulfonyl, -N (alkyl)-, oxygen, -C=C- (cis or trans) and -C≡C-;

q is 0 or 1;

r is 0, 1, 2 or 3; and,

R¹² is selected from the group including NR¹³R¹⁴ ,

wherein :

R¹³-R¹⁶ are independently selected from the group including hydrogen, hydroxy, amino, amidino, alkyl, cycloalkyl, acyl, aminoacyl and phosphoryl and, taken together, R¹³ and R¹⁴ or R¹⁵ and R¹⁶ may form a 5- or 6-membered nitrogen-containing heteroaryl or heteroalicyclic ring;

R .17 is selected from the group including hydrogen and alkyl; and,

alk₂ and R¹² taken together may form an optionally substituted 5- or 6-membered heteroalicyclic group.

Specific examples of heteroaryl groups known in the chemistry arts include the following:

As used herein, the term "alkyl" refers to a branched or unbranched hydrocarbon chain containing between one and eight, inclusive, preferably between one and four, inclusive, carbon atoms, such as, for example and without limitation, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, and 2-methylpentyl. The alkyl moiety may be optionally substituted with one or more functional groups including, for example and without limitation, hydroxyl, alkoxy, aryloxy, halo, mercapto, alkylthio, arylthio, cyano, aryl, heteroaryl, hetero- alicyclyl, carboxyl, alkoxycarbonyl, alkenyl, alkynyl, nitro, amino, amido, isothioureido, amidino, guanidino, and the like. Thus, for example and without limitation, the following are "alkyl" groups formed pursuant to this definition: trifluoromethyl, 3-hydroxyhexyl, 2-carboxy- propyl, 2-fluoroethyl, carboxymethyl, 4-cyanobutyl, 2- guanidinoethyl, 3-N,N' -dimethylisothiouroniumpropyl, and the like.

The term "alkenyl" refers to an alkyl group as defined above having at least one double bond in the one to eight or one to four carbon atom branched or unbranched hydrocarbon chain; e.g., and without limitation, allyl, 3-hydroxy-2- buten-1-yl, l-methyl-2-propen-l-yl and the like.

The term "alkynyl" refers to an alkyl group as defined above having at least one triple bond in the one to eight or one to four carbon atom branched or unbranched hydrocarbon chain; e.g., and without limitation, acetylenyl, propynyl, 3-methyl-lbutynyl, 4-methyl-2-pentynyl and the like.

The term "aryl", refers to an all-carbon monocyclic or fused-ring group (i.e., rings which share adjacent pairs of carbon atoms) having a completely conjugated pi-electron system. < Examples, without limitation, of aryl groups are phenyl, naphthyl, indenyl, anthracenyl and the like. The aryl group may be substituted or unsubstituted. When substituted, the substituted group (s) is preferably one or more selected from the group including, without limitation, alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, cyanoamido, heteroaryl, heteroalicyclyl, carbonyl, carboxyl, alkoxy- carbonyl, nitro, amino, amido, and the like, to form aryl groups such as, without limitation, biphenyl, iodobiphenyl, methoxybiphenyl, anthryl, bromophenyl, iodophenyl, chloro- phenyl, hydroxyphenyl, methoxyphenyl, formylphenyl, acetyl- phenyl, trifluoromethylthiophenyl, trifluoromethoxyphenyl, alkylthiophenyl, trialkylammoniumphenyl, amidophenyl, thia- zolylphenyl, oxazolylphenyl, imidazolylphenyl, imidazolyl- methylphenyl, cyanophenyl, pyridylphenyl, pyrrolylphenyl, pyrazolylphenyl, triazolylphenyl, tetrazolylphenyl and the like. The term "heteroaryl" (sometimes abbreviated "HetAr" herein) refers to a monocyclic or fused-ring (i.e., rings which share two adjacent atoms) group having one or more atoms independently selected from the groups including nitrogen, oxygen and sulfur in the ring(s) and, furthermore, having a completely conjugated pi-electron system. Examples of heteroaryl groups include, without limitation, furanyl, thienyl, imidazolyl, indolyl, pyridinyl, thiadiazolyl, thiazolyl, piperazinyl, dibenzfuranyl, dibenzthienyl . The heteroaryl group may be substituted or unsubstituted. When substituted, the substituted group (s) is preferably selected from a group including, without limitation, alkyl, aryl, hydroxyl, alkoxy, aryloxy, halo, mercapto, thioalkoxy, thioaryloxy, cyano, cyanoamido, heteroaryl, heteroalicyclyl, carboxyl, carbonyl, alkoxycarbonyl, nitro, amino, amido, and the like, to form rings such as, for example and without limitation, 2-aminothiazol-4-yl, 2,3-dioxo- piperazinyl, 4- alkylpiperazinyl, 2-iodo-3-dibenzfuranyl, 3-hydroxy-4- dibenzthienyl and the like. The term "heteroalicyclic" refers to a monocyclic or fused-ring (i.e., rings which share two adjacent atoms) group having one or more atoms independently selected from the groups including nitrogen, oxygen and sulfur in the ring(s) wherein the ring system does not contain a fully conjugated pi-electron system.

The term "carboxyheteroalicylic" refers to a heteroalicyclic group substituted with a carboxylic acid or carboxylic acid ester group as defined herein. The term "alkoxy" refers to an -0-alkyl group, alkyl being previously defined.

The term "aryloxy" refers to an -0-aryl group, aryl being previously defined.

The term "thioalkoxy" refers to an -S-alkyl group, alkyl being previously defined.

The term "thioaryloxy" refers to an -S-aryl group, aryl being previously defined.

The term "acyl" refers to an -C(=0) -alkyl group, alkyl being previously defined. The term "aralkyl" refers to an aryl-alkyl- group, aryl and alkyl being previously defined.

The term "heteroaralkyl" refers to a HetAr-alkyl- group, HetAr and alkyl being previously defined herein.

The term "trialkylsilyl" refers to an -Si (alkyl) ₃ group, wherein each alkyl may be the same or an independently selected alkyl group as defined herein.

The term "trialkylammonium" refers to an -⁺N (alkyl) ₃, group wherein each alkyl may be the same or an independently selected alkyl as defined herein. The term "amino" denotes the group -NRR' , where R and R' are independently selected from the group including hydrogen, alkyl, aryl, heteroaryl, alicyclyl and heteroalicyclyl as each term is defined herein. The term "carboxylic acid" refers to a -C(=0)-OH group. The term "carboxylic acid ester" refers to a -C(=0)-OR where R is alkyl or aryl as defined herein.

The terms "amido" and "carboxamido" refer to a -C(=0)NRR' group, where R and R' are as previously defined herein.

The term "halo" refers to fluorine, chlorine, bromine and iodine.

The term "cyanamido" refers to the -NR-CDN group where R is as previously defined herein.

The terms "lipophilicity enhancing" or "lipophilic" group refers to a group which increases the propensity for the molecule containing such a group to partition preferentially into an oily or fatty milieu over an aqueous one. Classically, the measure of lipophilicity is termed the "octane/water partition coefficient", which is well- known to those of ordinary skill in the art.

B. Preferred Structural Features. With respect to R², the preferred embodiments of this invention are those in which R² is hydrogen since, in general, only those compounds in which R² is hydrogen are biologically active. However, the present invention also contemplates other R² substituents which are easily hydrolyzed under biological conditions, i.e., such groups which can be cleaved easily after injection or ingestion of a compound of the invention by an organism, especially a mammal (see, e.g., European Patent Application No. 527,686 Al to Tsushima, et al., which is fully incorporated, including any drawings, herein by reference) . The present invention further contemplates substituents R² which are effective to protect the carboxyl group from unwanted reactions during synthesis of the compounds of the invention. Many such protective groups are well-known in the art (see, e.g., Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991), which is incorporated herein by reference) . Examples of such groups include allyl, t- amyl, benzhydryl, t-butyl, t-butyldimethylsilyl, benzyl, 2- chloroallyl, 3, 3-dimethylallyl, 2, 4-dimethoxybenzyl, 3,4- dimethoxybenzyl, 4, 41-dimethoxytrityl, 4-methoxybenzyl, 2- methoxybenzyl, 4-methoxytrityl, methoxymethyl, 4- nitrobenzyl, 2-nitrobenzyl, phenacyl, 2, 2, 2-trichloroethyl, trimethylsilyl, 2- (trimethylsilyl) ethyl, and trityl as well as the trifluoro-acetate, hydrochloride, hydrobromide and sulfate salts thereof.

Other preferred embodiments of this invention are those in which, in structures A, B and C above:

R* is -CH(OH)CH3 or -C(OH) (CH3)₂

R** is hydrogen

Y is hydrogen

R¹ is -NHC(=0)ZR^J

Z is selected from the group including -CH2(X*)_m- and

-C(=NOR^D)-;

R³ is selected from the group including cyano, aryl and heteroaryl;

p is 0 or 1; q is 1 ;

r is 1 , 2 or 3 ; and

R¹² is selected from the group including -NR¹³R¹⁴,

wherein

_R ⁱ³-ⁱ⁷ _are selected from the group including hydrogen and alkyl; or,

alk₂ and R¹² combine to form an optionally substituted 5- or 6- membered heteroalicyclic group containing one nitrogen, examples of which, as preferred embodiments but without limitation, are:

Especially preferred embodiments of this invention include those in which:

R³ is a heteroaryl group on which at least one of the substitutents is an amino group;

p is 0 or 1;

q is 1;

r is 1 or 2;

R .99 is sulfur; and,

R¹² is NR¹³R¹⁴

wherein R¹³ and R¹⁴ are hydrogen.

2. Pharmacological Compositions and Therapeutic Applications A. Compositions .

While it is possible to administer a compound of this invention alone, it is often preferable to administer it in a pharmacological composition containing the compound exactly as shown in the above structures or as a pharmaceutically acceptable salt or prodrug thereof. Thus, in another aspect, the present invention provides for the administration of a pharmacological composition containing a therapeutically effective amount of one or more of the compounds described herein, or a pharmaceutically acceptable salt or prodrug thereof to an organism suffering from infection by a disease-causing microorganism.

As used herein, "organism" refers to any multicellular life form but preferably refers to mammals such as, without limitation, mice, rats, cats, dogs, horses, monkeys, pigs, goats, etc. Most preferably, the organism is a human being.

A "pharmacologically acceptable salt" refers to a salt including, but not limited to, sodium, potassium, arginine, glycine, alanine and threonine. These may be prepared in water mixed with a suitable surfactant such as hyroxypropyl- cellulose. Other salts, surfactants and methods of preparation are well known to those skilled in the art.

The term "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because,' in some situations, they may be easier to administer than the parent drug. They also may be bioavailable by oral administration whereas the parent may not be. The prodrug may also have improved solubility in a pharmacological composition over the parent. The use of a bioreactive R² group, discussed above, is an example of a prodrug. That is, as noted previously, when R² is anything but hydrogen, the compound is generally biologically inactive. However, some types of R² groups can be removed in vivo, by naturally occurring biological processes, leaving the compound in the same (biologically active) form as if it had begun with R² being hydrogen. Such a compound, with R² being a biologically removable group, is an example of a "prodrug".

As used herein, "infection" refers to the entrance, growth and multiplication, in the body of an organism, of a disease-causing microorganism, in particular, for the purposes of this invention, disease-causing bacteria and even more particularly, /?-lactam resistant disease-causing bacteria such as, for example and without limitation, MRSA and enterococci, discussed above.

As used herein, a "disease-causing microorganism" refers to a virus, fungus, bacterium, paramecium, amoeba, etc. which, when it enters the body of an organism, causes a pathological condition in that organism.

As used herein "?-lactam resistant disease-causing bacteria" refers to bacteria which, when they enter an organism, cause an infection and for which conventional β- lactam antibiotics such as, for example, methicillin or ampicillin have a minimum inhibitory concentration (MIC) greater than 32 mg/ml.

As used herein, a "therapeutically effective amount" refers to that amount of the compound being administered which will eliminate an infection or which will to some extent relieve one or more of its symptoms.

As used herein, a "pharmaceutical composition" refers to mixture of one or more of the compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

As used herein, a "physiologically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Carriers may be solids or liquids. Solid carriers include, e.g., starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, and, optionally, other therapeutic ingredients. Liquid carriers include, e.g., sterile water, polyethylene glycols, non-ionic surfactants, and edible oils such as corn, peanut and sesame oils. In addition, various adjuvants such as are commonly used in the art may be included. For example: flavoring agents, coloring agents, preservatives, and antioxidants, e.g., vitamin E, ascorbic acid, BHT and BHA. Various other considerations regarding physiologically acceptable carriers are well-known to those skilled in the art and are described in texts such as Goodman and Gilman: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press (1990) , et al. As used herein, an "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.

A pharmacological composition can assume a variety of forms. These include, for example, solid, semi-solid and liquid forms, such as tablets, pills, powders, capsules, liquid solutions or suspensions, liposomes and injectable and infusible solutions.

Depending on the particular condition being treated, pharmacological compositions can be specifically prepared for administration systemically or locally. The choice, and techniques for preparation, of pharmacological compositions may be found in Remington's Pharmaceutical Sciences, 18th ed. , Mack Publishing Co., Easton, PA (1990) as well as in numerous other publications well known to those skilled in the art.

B. Applications. i . Routes of Administration. Routes for the administration of the compounds of this invention include oral, rectal, transdermal, vaginal, trans- mucosal, or intestinal administration; parenteral delivery, including intramuscular subcutaneous, intramedullary injections, as well an intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.

Generally, preferred routes of administration are oral or intravenous, intraperitoneal or intramuscular injection, ii. Dosage . According to this invention, a therapeutically effective amount of one or more compounds of this invention is administered to a organism suffering from a ?-lactam resistant infection, including, but not limited to, methicillin-resistant, vancomycin-resistant or ampicillin- resistant bacterial infections in an amount effective to at least partially relieve the infection. Especially important are infections resulting from y#-lactam resistant strains having ?-lactam resistance similar to strains such as S . aureus ATCC 29213, S . aureus ATCC 25913, S . aureus ATCC 31432, S. aureus col (Meth^R) (lac^") , S. aureus col (Meth^R) (lac⁺) , S . aureus ColBA, (Meth^s) (lac^") , E. faecium ATCC 35667 and E . faecalis ATCC 29212. The proper dosage will depend on the severity and course of the infection, previous therapy, the patient's health status, his or her response to the drugs, etc., all of which are well within the knowledge, expertise and judgment of the treating physician. In general, however, a suitable effective dose of a compound of this invention will be in the range of 0.1 to 1000 milligram (mg) per recipient per day, preferably in the range of 1 to 100 mg per day. The desired dosage may be administered in one dose or, preferably, in two, three, four or more subdoses administered at appropriate intervals throughout the day. These subdoses can be administered as unit dosage forms, for example, containing 5 to 1000 mg, preferably 10 to 100 mg of active ingredient per unit dosage form. Preferably, the compounds of the invention will be administered in amounts of between about 2.0 mg/kg to 250 mg/kg of patient body weight, between one and four times per day.

Once improvement of the patient's conditions has occurred, a maintenance dose may be administered if desired by the treating physician. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the response of the patient, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment can cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of the disease symptoms. iii. Prophylactic use. As well as being useful to treat patients with an ongoing infection, the compounds of this invention may be used in a prophylactic manner. That is, compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular infection. Such an amount is defined to be a "prophylactically effective amount or dose." In this use, the precise amounts again depend on the patient's state of health, weight, and the like.

3. Synthesis and Biological Evaluation

A. Synthesis of Compounds of Structures A-C. The compounds of the present invention may be readily prepared in accordance with the procedures described in the references provided in this section. It will, however, be appreciated by those skilled in the art that other synthetic pathways for forming the compounds of the invention are available and that the following is offered merely by way of example, and not limitation. Generally, the synthesis of the compounds of the present invention may be achieved using well-known methods and readily available materials (see, e.g., March; Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH Publishers, 1989); and G. I. Georg, THE ORGANIC CHEMISTRY OF ?-lactams, (VCH 1992), each of which is incorporated herein by reference) .

Thus, the preferred approach to the synthesis of these compounds is shown in Scheme I:

Scheme I

Thus, treatment of the triflate 1 with the desired optionally protected thiolate nucleophile 2, using standard methods such as those described in Farina, et al., J. Org. Chem., 54:4962 (1989) and U.S. Patent No. 4,870,168 to Baker, et al. (both of which, including any drawings, are incorporated by reference herein) , provides the 3-thio derivative 3. Subsequent deprotection (removal of Pg and, optionally, R²) using procedures well known to those skilled in the art affords the biologically active 4-carboxy-?- lactam or, if R² is left in place, a prodrug thereof.

The substituent R¹ may be any of the groups described above and are either available commercially (e.g., from Aldrich, Milwaukee, WI) or can be formed using known techniques and starting materials (see, e.g., March; Larock) . These groups can be substituted for those present on the starting material by variety of well known techniques (see, e.g., Barrett, J.C.S. Perkin I, 1629 (1979) or Chauvette, J. Org. Chem. 36:1259 (1971), both of which are incorporated herein by reference) , such as by transamination of an existing substituent for the desired substituent, or hydrolytic removal of the existing substituent followed by reaction with a suitably reactive form of the desired substituent, such as an acyl chloride. Again, the appropriate reagents and techniques will be apparent to those of skill in the art. The carboxyl group R² may be those protecting groups amenable to reductive cleavage, such as benzyl, p- or o- nitrobenzyl, 2, 2, 2-trichloroethyl, allyl, cinnamyl, benzhydryl, 2-chloroallyl and the like. Alternatively, R² may be a protecting group amenable to acidic cleavage, such as t- butyl, t-amyl, trityl, 4-methoxytrityl, 4, 4 ' -dimethoxy- trityl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, S- (trimethylsilyl) ethyl, benzyl, 4- (or 2-methoxybenzyl, 2,4- di ethoxybenzyl, 3, 4-dimethoxybenzyl, 2, 4 , 6-trimethoxy- benzyl, methoxymethyl, benzhydryl, or 3, 3-dimethylallyl. Preferred protecting groups are p-methoxybenzyl, p- nitrobenzyl, allyl and benzhydryl. Such groups may be attached to the unprotected carboxyl group of the /?-lactam starting material using known reagents and techniques, such as those described in Green and Wuts. Preferred amine protecting groups include trityl, formyl, phenoxyacetyl, trichloroacetyl, chloroacetyl, bromoacetyl, iodoacetyl, urethane-type protecting groups

[such as t-butoxycarbonyl, benzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2- chloroallylcarbonyl, allyoxycarbonyl, 2- (trimethylsilyl) ethoxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, (C₄ C₆) -cycloalkanyloxycarbonyl or 9-fluorenylmethoxycarbonyl (FMOC) . Especially preferred protecting groups are trityl, allyoxycarbonyl, benzyloxycarbonyl, phenoxyacetyl, and t- butoxycarbonyl . These may be attached and removed using standard techniques (see Green and Wuts) . The selection of the amine-protecting group to be employed will depend on the stability of the protected /?-lactam to the subsequent reaction conditions.

Similarly, the thiolate nucleophile 2 may be formed using known procedures and commercially available starting materials. The following references, which are incorporated, including any drawings, by reference herein, provide experimental details for accomplishing the synthesis shown in Scheme I for compounds of types A, B and C:

1. 6-hydroxyethyl-2- ( (heteroaryl) thio) carbapenems (Structure A, U = CH₂)

These compounds may be synthesized using the techniques described in: (1) D.G. Melillo, I. Shinkai, T. Liu, K. Ryan and M. Sletzinger, Tetrahedron Letters, 2783 (1980); (2) R.I. Ponsford and R. Southgate, J.C.S., Chem. Commun., 846 (1979); (3) D.H. Shih, F. Baker, L. Cama and B.G. Christensen, Heterocycles, 21, 29 (1984); (4) J.H. Bateson, P.M. Roberts, T.C. Smale and R. Southgate, J.C.S., Chem. Commun . , 185 (1980); (5) European Patent Application 10,312; (6) U.S. Patent 4,153,714; (7) C.-H. Cho and J.-H. Cho, J^

Antibiotics, 47, 126 (1994); (8) M. Sunagawa, H. Matsumura,

T. Inoue, H. Yamaga and M. Fukasawa, J. Antibiotics, 45, 971

(1992); (9) M.J. Basker, D.F. Corbett, S. Coulton and R.

Southgate, J. Antibiotics, 43, 847 (1990); (10) A. Yoshida, Y. Tajima, N. Takeda and S. Oida, Tetrahedron Letters, 25, 2793 (1984); (11) J. Fetter, K. Le pert, M. Kajtar-Peredy, G. Bujtas, and G. Simig, J. Chem. Res. (S) , 28 (1987); J. Chem. Res. (M) , 349 (1987); and (12) T. Shibata and Y. Sugimura, J. Antibiotics, 42, 374 (1989) .

2. 6-hydroxyethyl-2- ( (heteroaryl) thio) penems (Structure A, U - S)

These compounds may be synthesized using the techniques described in (1) S. Oida, A. Yoshida, T. Hayashi, N. Takeda and E. Ohki, Chem. Pharm. Bull., 28, 3232 (1980); (2) I. Ernest, A.J. Main and R.B. Woodward, Helv. Chim. Acta., 64, 1303 (1981); (3) A. Alfonso, F. Hon, J. Weinstein and A.K. Ganguly, J. Am. Chem. Soc, 104, 6138 (1982); (4) A. Yoshida, T. Hayashi, N. Takeda, S. Oida and E. Ohki, Chem. Pharm. Bull., 31, 768 (1983); (5) U.S. Patents 4,782,145 and 4,782,146; (6) M. Sunagawa, H. Matsumura, T. Inoue and M. Fukasawa, J. Antibiotics, 45, 500 (1992); and (7) D. Phillips and B.T. O'Neill, Tetrahedron Letters, 31, 3291 (1990) .

3. 7-acylamido-3- ( (heteroaryl) thio) carbacephems (Structure B, T = CH₂)

These compounds may be synthesized using the techniques described in (1) R.J. Ternansky, S.E. Draheim, A.J. Pike, F.W. Bell, S . J. West, C.L. Jordan, CY. Ernie Wu, D.A. Preston, W. Alborn, Jr., J.S. Kasher and B.L. Hawkins, J^_ Med. Chem., 36, 1971 (1993); and (2) M. Hatanaka and T. Ishimaru, Tetrahedron Letters, 24, 4837 (1983) .

4. 7-acylamido-3- ( (heteroaryl) thio) oxacephems (Structure B, T = 0) These compounds may be synthesized using the techniques described in (1) Y. Hamashima, S. Yamamoto, T. Kubota, K. Tokura, K. Ishikura, K. Minami, F. Matsubara, M. Yamaguchi, I. Kikkawa, and W. Nagata, Tetrahedron Letters, 4947 (1979) .

5. 7-acylamido-3- ( (heteroaryl) thio) isocephems (Structure C, X = S)

These compounds may be synthesized using the techniques described in (1) J. Aszodi, A. Bonnet, J.-F. Chantot, G. Costerousse and G. Teutsch in "Recent Advances in the Chemistry of b-Lactam Antibiotics", ed. P.H. Bentley and R. Southgate, Specialist Publication No. 70, Royal Society Chemistry; London ( 1989) -Chapter 23; and (2) H. Tsubouchi, K. Tsuji, K. Yasumura, N. Tada, S. Nishitani, J. Minamikawa and H. Ishikawa, Tetrahedron: Asymmetry, 5, 441 (1994) .

6. 7-acylamido-3- ( (heteroaryl) thio) isoxacephems (Structure C, X = 0)

These compounds may be synthesized using the techniques described in S.W. McCombie, W.A. Metz and A. Alfonso, Tetrahedron Letters, 27, 305 (1986) .

B. Biological Evaluation.

It will be appreciated that, in any given series of compounds, a spectrum of biological activity will be afforded. In its most preferred embodiment, this invention relates to novel ?-lactam antibiotics demonstrating activity superior to conventional ?-lactams such as methicillin, ampicillin and vancomycin against bacterial infections. The following assays are employed to select those compounds demonstrating the optimal degree of the desired activity. i. In Vitro.

The compounds of the invention can be evaluated against ?-lactam resistant (for instance, but not limited to, methicillin-resistant , vancomycin-resistant and/or ampicillin-resistant) bacteria strains by determining the minimum inhibitory concentration (MIC, μg/ml) of each compound with respect to each strain. The MIC, the lowest concentration of antibiotic which inhibits growth of the test organism, is determined by the agar dilution method.

To determine the MIC for bacterial isolates, the test compound is incorporated in a series of two-fold dilutions into liquefied Mueller-Hinton agar. Upon solidification, a number of different bacterial strains are spot inoculated with a replicating device onto the agar surface. After overnight incubation, the MIC breakpoint is determined as the lowest drug concentration that completely inhibited growth, disregarding a single colony or a faint haze. The procedures used in these studies have been standardized by the National Committee for Clinical Laboratory Standards (NCCLS) , as per the NCCLS publication entitled METHODS FOR DILUTION ANTIMICROBIAL SUSCEPTIBILITY TESTS (1991), which is incorporated herein by reference.

Aliquots of antimicrobial agents are prepared in phosphate buffered saline (PBS) at pH 7.2. Tween 20 or DMSO is used as a solubilizing vehicle as needed. Standard methods of vortexing, sonicating and gentle heat are used to facilitate solubilizing the test agent. Typically, the concentration of the stock solution is 10X that of the highest drug concentration tested. A 1.28 mg/mL stock solution is used with a subsequent highest working concentration of 128 μg/mL. Serial two-fold dilutions are done through > 0.25 μg/mL. Each drug level is tested in duplicate. Two-fold drug dilutions are done in sterile 50 mL tubes with a final drug volume of 5 mL. Upon the addition of 45 mL of molten agar, a 10-fold dilution results. Two, 25 mL. plates are then poured into 15x150 mm square Petri plates with grids and allowed to harden. A control plate with a reference drug, either cefotaxime, vancomycin or imipenem, is used as the positive growth control. Stock concentrations of reference antibiotics are prepared and frozen at -80° C. Upon preparation, the control plates are sealed and stored in the refrigerator for up to 1 week prior to use; however, imipenam control plates have to be prepared just prior to use. All test plates are used within 24 hours of preparation. Satisfactory results are obtained where the inoculum contained about 10⁴ colony forming units (cfu) + 0.5 logs. Starting with pure cultures of the test isolates on agar plates, a few isolated colonies are transferred to a tube of nutrient broth and allowed to grow 4-6 hours at 35-36° C to reach log-phase growth. Dropwise addition of the broth culture to PBS is done to match a 0.5 McFarland turbidity standard equal to 10⁸ cfu/mL. This is further diluted tenfold in PBS to reach a working inoculum concentration of 10⁷ cfu/mL. When 1 μL of the working inoculum is applied to the agar surface a concentration of about 10⁴ CfU per spot is obtained.

Disposable sterile 1 μL loops are used to inoculate test plates, with each isolate in a designated grid on the agar plate. An alternate method of inoculation involves the use of a replica plater, a device with 48 steel pins allowing the simultaneous inoculation of multiple isolates. After the spots have dried, the plates are incubated at 35- 36° C for 16-20 hours. Endpoints are assessed as the minimum inhibitory concentration (MIC) of antimicrobial agent. ii. In Vivo. Compounds with superior activity in vitro when compared to reference antibiotics, are further evaluated in a murine model for lethal bacteremic peritonitis. Groups of 5 female Swiss-Webster mice (Simonsen, Gilroy, CA) each are challenged by the intraperitoneal (IP) route with tenfold increments of a bacterial inoculum. This permits calculation of the mean lethal dose (LD₅₀) and the LDioo. For preliminary evaluation of a new antibiotic, mice are challenged IP with an LDioo titer of bacteria. In two equal doses administered at the time of bacterial challenge and 2 hours later, groups of 10 mice each are treated subcutaneously with two-fold increments of the test drug and an antibiotic of known efficacy in mice and humans (i.e., positive control) . Mice are observed for 72h. Those alive at 72h are considered long term survivors. The total drug dose in mg/kg that protects 50% of mice in a group from death is termed the mean protective dose (PD₅₀) . PD₅₀s are similarly determined for several pathogens. The quanti- tative endpoints for the new drug are then compared with those obtained with reference antibiotics.

Six ten-fold dilutions of inoculum suspended in 0.5 mL of sterilized 7% hog gastric mucin (Sigma) are injected IP in groups of 5 mice each. A control group of 5 mice receive mucin alone. Mice are observed for 72h. Those alive at 72h are considered long term survivors. The mean lethal dose

(LD₅o) and 100% lethal dose (LDioo) are determined by the probit test. For antibiotic efficacy studies, mice are challenged IP with bacterial titers that will afford an LDioo for the test strain. In two equal doses administered at the time of bacterial challenge and 2 hours later, groups of 10 mice each are treated by the subcutaneous route (SC) with twofold increments of the test antibiotic; another group is treated similarly with a reference antibiotic of known efficacy in animals and man. Drug doses can range from 0.01 to 512 mg/kg. If the drug is poorly soluble, Tween 20 or propylene glycol will be employed to solubilize the drug. Animals are observed for 72h. The 50% protective dose (PD₅₀) is calculated in mg/kg by the probit method. The PD₅₀, is the same as the 50% effective dose (ED₅o) and the 50% curative dose (CD₅₀) . Samples of blood from the hearts of all animals that die and from half the mice that survive are cultured on brain-heart infusion agar. Animals that received a protective dosage of the test drug will be alive at 72h, although they may appear moderately ill to very ill during the observation period. Infected, placebo-treated control mice and those receiving non-effective i.e. lower dosages of the test drug will demonstrate a high rate of mortality. Most of these mice will die within 6 to 48h.

Those alive at 72h will be considered long term survivors.

Thus, it will be appreciated that the compounds, methods and compositions of the invention are effective against various /?-lactam resistant strains of bacteria which pose an increasing health risk to society.

Although certain embodiments and examples have been used to describe the present invention, it will be apparent to those skilled in the art that changes to the embodiments and examples shown may be made without departing from the scope or spirit of the invention.

Those references not previously incorporated herein by reference, including both patent and non-patent references, are expressly incorporated herein by reference for all purposes .

Other embodiments are within the following claims.

Claims

1. A compound selected from the group consisting of the following structures:

or a physiologically acceptable salt or prodrug thereof,

wherein

X is selected from the group consisting of CH₂, oxygen, sulfur, SO and S0₂;

T is selected from the group consisting of CH₂ and oxygen; U is selected from the group consisting of CH₂, sulfur, oxygen and -CH (alkyl), where alkyl is selected from the group comprising Ci to C alkyl;

Y is selected from the group consisting of hydrogen, -OCH₃ and -NHCHO;

R* is selected from the group consisting of -CH(OH)CH₃, -C(OH) (CH₃)₂, -CHFCH₃ and -CH=CH₂;

R** is selected from the group consisting of hydrogen, CH₃ and -CH₂CH₃;

R¹ is selected from the group consisting of -NHC(=0)ZR³, NR⁴R⁵ and

wherein

Z is selected from the group consisting of -CH₂(X*)_m-, -C(=NOR⁶)-, -CH(OR⁷)-, =C(CHC0₂R⁸)- and -CH (NR⁹R¹⁰) -;

wherein

X* is selected from the group consisting of oxygen and sulfur,

m is 0 or 1; R³ is selected from the group consisting of cyano, alkyl, aryl, heteroaryl, heteroaralkyl and (CH₂)_nW

wherein

n is 1 to 6; and

W is selected from the group consisting of amino, amidino, (C- or N- linked) , guanidino, and isothiourido, optionally substituted by alkyl, aryl, hydroxy or amino;

R^4"7 are independently selected from the group consisting of hydrogen, alkyl, aryl and acyl;

R⁸ is selected from the group consisting of hydrogen, alkyl and aryl;

R⁹ and R are independently selected from the group consisting of hydrogen, alkyl, acyl, and heterocyclecarbonyl ;

R² is selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, and trialkylsilyl or R² is not present at all and the C0₂ group to which it would be attached bears a negative charge;

Q is a heteroaryl group selected from the group consisting of:

and

wherein

A, B, D and E are selected from the group consisting of carbon, nitrogen and sulfur and the specific juxtaposition of groups A, B, D, and E is limited to examples of heterocyclic groups known in the chemistry arts;

G, H, J, L and M are selected from the group consisting of carbon, nitrogen or -⁺NR^╬╣:L (quaternary ammonium heterocycle) and the specific juxtaposition of groups G, H, J, L and M is limited to examples of heterocyclic groups known in the chemistry arts;

R 11 is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, hydroxyl, amino, cyano, hydroxyalkyl, carboxamidoalkyl, optionally substituted aminoalkyl or quaternary ammonium alkyl and quaternary heteroaryliumalkyl ;

R*** is - [alkil p ER 9H9. q talkzl rR > 12

wherein alki and alk₂ are each independently selected from the group consisting of optionally substituted methylene groups -(CR'R")-

wherein

R' and R" are independently selected from the group consisting of alkyl and aryl;

p is 0, 1 or 2;

R⁹⁹ is selected from the group consisting of sulfur, sulfinyl, sulfonyl, -N (alkyl)-, oxygen, -C=C- (cis or trans) and -CΓëíC-;

q is 0 or 1;

r is 0, 1, 2 or 3; and,

R¹² is selected from the group consisting of

NR¹³R¹⁴ ,

O _NR15_R1╬▓ Xm'v _ \ 2i3 _┬½ ^R", , a _nd ⁺ jN|0R┬½R^K wherein _R ⁱ³__R ⁱ⁶ _are independently selected from the group consisting of hydrogen, hydroxy, amino, amidino, alkyl, cycloalkyl, acyl, aminoacyl and phosphoryl and, taken together, R¹³ and R¹⁴ or R¹⁵ and R¹⁶ may form a 5- or 6-membered nitrogen-containing heteroaryl or heteroalicyclic ring;

R¹⁷ is selected from the group consisting of hydrogen and alkyl; and,

2. The compound, salt or prodrug of claim 1 wherein R* is -CH(OH)CH₃.

3. The compound, salt or prodrug of claim 1 wherein R¹ is -NH(C=0)ZR³.

4. The compound, salt or prodrug of claim 3 wherein Z is -C(NOR⁶)-,

The compound, salt or prodrug of claim 4 wherein R⁶ is hydrogen.

The compound, salt or prodrug of claim 5 wherein R³ is a heteroaryl group.

7. The compound, salt or prodrug of claim 6 wherein said heteroaryl group is an amino-substituted heteroaryl group.

8. The compound, salt or prodrug of claim 5 wherein R³ is selected from the group consisting of 2-aminothiazol-4- yl, 2-amino-5-chlorothiazol-4-yl and 2-aminothiadiazol-4-yl.

9. The compound, salt or prodrug of claim 8 wherein R⁹⁹ is sulfur.

10. The compound, salt or prodrug of claim 9 wherein R¹² is -NR¹³R¹⁴.

11. The compound, salt or prodrug of claim 10 wherein R¹³ and R¹⁴ are hydrogen.

12. A method for administering to an organism suffering from a ?-lactam antibiotic resistant bacterial infection, a therapeutically effective amount of said compound, salt or prodrug of claim 1.

13. The method of claim 12 wherein said organism is a mammal .

14. The method of claim 13 wherein said mammal is a human.

15. The method of claim 12 wherein said ?-lactam antibiotic resistant bacterial infection comprises a ╬▓- lactam antibiotic resistant Staphylococcal or Enterococcal bacteria.

16. The method of claim 15 wherein said ?-lactam antibiotic resistant Staphylococcal or Enterococcal bacteria comprise methicillin-resistant Staphylococcal or ampicillin- resistant Enterococcal bacteria.

17. A pharmaceutical composition of said compound, salt or prodrug of claim 1.

18. A method for administering said pharmaceutical composition of claim 17 to an organism suffering from a ╬▓~ lactam antibiotic resistant bacterial infection.

19. The method of claim 18 wherein said ?-lactam antibiotic resistant bacterial infection comprises ^" a ╬▓- lactam antibiotic resistant Staphylococcal or Enterococcal bacteria.

20. The method of claim 19 wherein said ?-lactam antibiotic resistant Staphylococcal or Enterococcal bacteria comprise methicillin-resistant Staphylococcal or ampicillin- resistant Enterococcal bacteria.