Patent Application
20210122776
The present invention encompasses compounds and methods for inhibiting the adhesin protein FimH and treating and preventing urinary tract infections and inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis).
Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is one of the most common infectious diseases in women. The morbidity and economic impact are enormous, with over $2.5 billion spent annually on treatment. Further, recurrent infections are a significant problem despite appropriate antibiotic therapy of the index case. The high rates of recurrence, and the large numbers of women that end up in urology clinics due to their chronic recurrent UTIs highlights the need for a better understanding of the pathogenic mechanisms involved in this disease and the development of new and better therapies.
Gram-negative bacteria are the causative agents of a wide variety of acute and chronic infectious diseases. Many of these infections are initiated by a critical interaction between host ligands (frequently polysaccharide moieties) and bacterial adhesins (frequently expressed at the distal tip of polymeric pilus fibers assembled by the chaperone/usher pathway). The mannose binding FimH adhesin of type 1 pili is critical for the colonization and invasion into the bladder epithelium. After invasion, UPEC are able to rapidly multiply inside superficial umbrella cells of the bladder forming biofilm-like intracellular bacterial communities (IBCs). Upon maturation, bacteria disperse from the IBC, spread to neighboring cells, and form next generation IBCs. This is the mechanism by which UPEC rapidly amplify in numbers in the urinary tract and cause disease.
The X-ray crystal structure of FimH bound to mannose showed that mannose is bound in a negatively charged pocket on FimH. The mannose binding site is highly conserved as it is invariant in 300 fimH genes sequenced from clinical UPEC strains. Thus, FimH is the critical node of the entire UPEC pathogenic cascade.
Recurrence is a serious problem for many women. Women who present with an initial episode of acute UTI have a 25-44% chance of developing a second and a 3% chance of experiencing three episodes within six months of the initial UTI. Recurrence occurs despite appropriate antibiotic treatment and clearance of the initial infection from the urine. A large percentage of recurrent UTI are caused by the same strain of bacteria as the initial infection. One study followed 58 women and found that 68% of recurrences were caused by the same initial index strain of UPEC as determined by restriction fragment length polymorphism (RFLP) analysis. In a separate study, 50% of recurrent strains isolated from female college students appeared genotypically identical to the bacterial strain corresponding to the initial UTI. Another long-term prospective study demonstrated that the same strain of UPEC can cause a recurrent UTI up to 3 years later. The high frequency of same-strain recurrences supports the notion that a UPEC reservoir can exist in the affected individual. The inventors have shown that a quiescent intracellular reservoir (QIR) can form in the bladder tissue itself after acute infection and persist even after antibiotic therapy and urine cultures become sterile. Thus, reactivation of bacteria in QIRs may also be a contributing factor in recurrent UTIs.
Inflammatory bowel disease (IBD) mainly consists of two disorders, ulcerative colitis and Crohn's disease (CD), with a combined prevalence of ˜150-200 cases per 100,000 in Western countries. The abnormal inflammatory response observed in IBD requires interplay between host genetic factors and the intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) have previously been shown to induce gut inflammation in patients with Crohn's disease (CD). Mannosides have been shown to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. Given the key role of AIEC in the chronic intestinal inflammation of CD patients, these results suggest a potential anti-adhesive treatment with the FimH inhibitors developed.
Therefore, there is a need for effective treatments that can cure urinary tract infections and prevent the formation of quiescent intracellular reservoir that are the source of so many recurrent infections. As well as effective treatments that can cure, prevent or reduce symptoms associated with Crohn's disease.
One aspect of the present invention encompasses a compound comprising formula (I):
wherein:
Another aspect of the present invention encompasses a compound comprising Formula (II):
wherein:
Still another aspect of the present invention encompasses a compound comprising Formula (III):
wherein:
Yet still another aspect of the present invention encompasses a compound comprising Formula (IV):
wherein:
Yet still another aspect of the present invention encompasses a compound comprising Formula (V):
wherein:
Yet still another aspect of the present invention encompasses a compound comprising Formula (VI):
wherein:
The invention also encompasses a method of treating a urinary tract infection. The method comprises administering a compound of the invention to a subject in need thereof.
Further, the invention encompasses a method of preventing a urinary tract infection. The method comprises administering a compound of the invention to a subject in need thereof.
In another aspect, the invention encompasses a method of reducing the resistance of a bacterium to a bactericidal compound. The method comprises administering a compound of the invention a subject in need thereof.
In yet another aspect, the invention encompasses a method of treating inflammatory bowel disease. The method comprises administering a compound of the invention to a subject in need thereof.
In still yet another aspect, the invention encompasses a method of inhibiting FimH binding to mannose. The method comprises contacting a compound of the invention with FimH, wherein the compound binds FimH and inhibits binding to mannose.
In still yet another aspect, the invention encompasses a method of treating a catheter-associated urinary tract infection. The method comprises administering a compound of the invention to a subject in need thereof.
The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Compounds that inhibit the function of type 1 pili of bacteria have been developed. The compounds may be useful for the treatment of urinary tract infections and Crohn's Disease. Significantly, the compounds may prevent bacterial colonization and invasion of the bladder tissue to prevent infection and the establishment of reservoirs that can serve as a source of recurrent infections. The invention also encompasses methods of use of a compound of the invention.
One aspect of the invention is a compound of Formula (I):
wherein:
In one embodiment, a compound of the invention comprises Formula (I), wherein:
In another embodiment, a compound of the invention comprises Formula (I), wherein:
In an exemplary alternative of each of the foregoing embodiments, a compound comprising formula (I) is a compound comprising any of the Formulas in Table 1.
In a further exemplary alternative of each of the foregoing embodiments, a compound of the invention is Examples 1-23 and 25 from Table 1.
Another aspect of the invention is a compound of Formula (II):
wherein:
In one embodiment, a compound of the invention comprises Formula (II), wherein:
In another embodiment, a compound of the invention comprises Formula (II), wherein:
In another embodiment, a compound of the invention comprises Formula (II), wherein:
In another embodiment, a compound of the invention comprises Formula (II), wherein:
In still another embodiment, a compound of the invention comprises Formula (II), wherein:
In still another embodiment, a compound of the invention comprises Formula (II), wherein:
In still another embodiment, a compound of the invention comprises Formula (II), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (II), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (II), wherein:
In an exemplary alternative of each of the foregoing embodiments, a compound comprising formula (II) is a compound comprising any of the Formulas in Table 1.
In a further exemplary alternative of each of the foregoing embodiments, a compound of the invention is Example 1-16, 18-23 and 25 from Table 1.
Another aspect of the invention is a compound of Formula (III):
wherein:
In one embodiment, a compound of the invention comprises Formula (IV), wherein:
In another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In an exemplary alternative of each of the foregoing embodiments, a compound comprising formula (IV) is a compound comprising any of the Formulas in Table 1.
In a further exemplary alternative of each of the foregoing embodiments, a compound of the invention is Example 7-16 from Table 1.
Another aspect of the invention is a compound of Formula (IV):
wherein:
In one embodiment, a compound of the invention comprises Formula (IV), wherein:
In another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
R12 is selected from the group consisting of H, alkyl, CH2R13, CH2COR13, CH2CONHR13, CH2CONHR13R14, CH2CONH(CH2)2R14, (CH2)2NR13, (CH2)nNR13, CH2COOH, CH2CONH(CH2)2NH2, and (CH2)2N(CH3)2;
In still yet another embodiment, a compound of the invention comprises Formula (IV), wherein:
In an exemplary alternative of each of the foregoing embodiments, a compound comprising formula (IV) is a compound comprising any of the Formulas in Table 1.
In a further exemplary alternative of each of the foregoing embodiments, a compound of the invention is Example 7-16 from Table 1.
Yet another aspect of the invention is a compound of Formula (V):
wherein:
In one embodiment, a compound of the invention comprises Formula (V), wherein:
In another embodiment, a compound of the invention comprises Formula (V), wherein:
In yet another embodiment, a compound of the invention comprises Formula (V), wherein:
In another embodiment, a compound of the invention comprises Formula (V), wherein:
In still another embodiment, a compound of the invention comprises Formula (V), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (V), wherein:
In still yet another embodiment, a compound of the invention comprises Formula (V), wherein:
In an exemplary alternative of each of the foregoing embodiments, a compound comprising formula (V) is a compound comprising any of the Formulas in Table 1.
In a further exemplary alternative of each of the foregoing embodiments, a compound of the invention is Example 17 from Table 1.
Yet still another aspect of the invention is a compound of Formula (VI):
wherein:
In one embodiment, a compound of the invention comprises Formula (VI), wherein:
In another embodiment, a compound of the invention comprises Formula (VI), wherein:
In an exemplary alternative of each of the foregoing embodiments, a compound comprising formula (VI) is a compound comprising any of the Formulas in Table 1.
In a further exemplary alternative of each of the foregoing embodiments, a compound of the invention is Examples 5-6 from Table 1.
In certain embodiments, the sugar residue of the above compounds may encompass a stereoisomer of mannose. In other embodiments, the sugar residue of the above compounds may encompass any stereoisomer of mannose other than glucose. In an exemplary embodiment, the sugar residue of the above compounds is alpha D mannose.
Exemplary methods of synthesizing a compound of the invention are detailed in the Examples.
A compound of the invention may also be an intermediate in the synthesis of a compound of formula (I)-(IV). For instance, in one embodiment, a compound of the invention may be an ester intermediate in the synthesis of a compound of formula (I)-(IV). In another embodiment, a compound of the invention may be a boronate ester of a mannoside or a boronic acid ester of a mannoside. In still another embodiment, a compound of the invention may be a compound illustrated in Schemes I-XII in the Examples below.
A compound of the invention may also comprise an imaging agent, such as a fluorescent moiety. In an embodiment, the imaging agent is bound to the sugar portion of a compound of the invention, either directly, or via a linker.
Compounds of the invention may block the function of FimH of the type 1 pili of pathogenic bacteria and prevent bacterial adherence and invasion and thus prevent bacterial amplification in the IBC and subsequent spreading and repeated rounds of amplification via new generation IBCs.
FimH functional assays used to measure activity of the compounds are known to individuals skilled in the art. Non-limiting examples of functional assays include hemmagglutination titer using guinea pig red blood cells, affinity of binding to FimH, and the ability of the compounds to prevent biofilm formation.
In some embodiments, activity of the compound is measured using hemmagglutination titer of guinea pig red blood cells. Hemagglutination of guinea pig red blood cells by type1 piliated UPEC is dependent upon FimH mannose binding ability and serial dilutions allow a quantitative analysis. Hemagglutination titer may generally be defined as the amount of compound required for decreasing hemagglutination by 75%. In some embodiments, the hemmagglutination titer of the compound of the invention may be less than about 5, 4, 3, 2, or 1 μM. In a preferred alternative of the embodiments, the hemmagglutination titer of the compound of the invention may be less than about 1, 0.5, 0.4, 0.3, 0.2, or 0.1 μM. In another preferred alternative of the embodiments, the hemmagglutiantion titer of the compound of the invention may be less than about 0.1, 0.05, 0.04, 0.03, 0.02, 0.01 μM. In yet another preferred alternative of the embodiments, the hemmagglutination titer of the compound of the invention may less than about 0.01 μM.
In yet other embodiments, activity of the compound may be measured using the ability of the compound to prevent or disrupt biofilm formation. In general, titration curves measuring the ability of a compound inhibit biofilm formation may be performed to determine the IC50. In some embodiments, the IC50 of the compound may be less than about 700, 600, 500, 400, 300, 200 or 100 μM. In other embodiments, the IC50 of the compound may be less than about 500, 400, 300, 200, 100, 50, 40, 30, 20 10, 9, 8, 7, 6, or 5 μM. In preferred embodiments, the IC50 of the compound may be less than about 20 μM. In other preferred embodiments, the IC50 of the compound may be less than about 9 μM.
Another aspect of the present invention encompasses a combination of a compound of the invention (described in Section I above) with one or more bactericidal compounds. In some embodiments, a compound of the invention may comprise a combination with 1, 2, 3, 4, or 5 bactericidal compounds. In one embodiment, the bactericidal compound is an antibiotic. Suitable antibiotics are known in the art, and may include Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Geldanamycin, Herbimycin, Carbacephem, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cephalosporins, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spectinomycin, Aztreonam, Furazolidone, Nitrofurantoin, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole (SMZ), Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim (TMP), Trimethoprim-Sulfamethoxazole (such as Bactrim, Septra), Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Linezolid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Rifaximin, Thiamphenicol, or Tinidazole. In an exemplary embodiment, the antibiotic is TMP, SMZ, or a combination thereof.
Yet another aspect of the invention encompasses a pharmaceutical composition. A compound of the invention described in Section I above may exist in tautomeric, geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-geometric isomers, E- and Z-geometric isomers, R- and S-enantiomers, diastereomers, d-isomers, I-isomers, the racemic mixtures thereof and other mixtures thereof. Pharmaceutically acceptable salts of such tautomeric, geometric or stereoisomeric forms are also included within the invention. The terms “cis” and “trans”, as used herein, denote a form of geometric isomerism in which two carbon atoms connected by a double bond will each have a hydrogen atom on the same side of the double bond (“cis”) or on opposite sides of the double bond (“trans”). Some of the compounds described contain alkenyl groups, and are meant to include both cis and trans or “E” and “Z” geometric forms. Furthermore, some of the compounds described contain one or more stereocenters and are meant to include R, S, and mixtures of R and S forms for each stereocenter present.
In a further embodiment, the inhibitors of the present invention may be in the form of free bases or pharmaceutically acceptable acid addition salts thereof. The term “pharmaceutically-acceptable salts” are salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt may vary, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of compounds for use in the present methods may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, stearic, algenic, algenic, hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts of compounds of use in the present methods include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N, N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine- (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid or base with any of the compounds of the invention.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injectables. Dimethyl acetamide, surfactants including ionic and non-ionic detergents, and polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound is ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the compound can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, or magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings.
For therapeutic purposes, formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. For instance, a compound of the invention may be administered with a carrier. Non-limiting examples of such a carrier include protein carriers and lipid carriers.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
The amount of the compound of the invention that may be combined with the carrier materials to produce a single dosage of the composition will vary depending upon the subject and the particular mode of administration. Those skilled in the art will appreciate that dosages may also be determined with guidance from Goodman & Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman & Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.
A compound of the invention may also be formulated as a prodrug. Such a prodrug formulation may increase the bioavailability of a compound of the invention. In one embodiment, the sugar portion of a compound of the invention may encompass a prodrug. In another embodiment R3 may comprise a prodrug. Non-limiting examples of a compound of the invention formulated as a prodrug include the compounds below:
Compounds of the invention may be used in methods of treating a bacterial infection and methods of reducing resistance to a bactericidal compound in a bacterium.
One embodiment of the invention encompasses a method for treating bacterial infections. Or, more specifically, the invention encompasses a method for treating a urinary tract infection. As used herein, “treating” refers to preventing infection in a subject not currently infected, and reducing or eliminating infection in a subject that is currently infected. As such, the invention also encompasses a method for preventing UTI. Generally, such a method comprises administering a pharmaceutical composition comprising a compound of the invention to a subject. As used herein, “subject” includes any mammal prone to urinary tract infections by E. coli. In one embodiment, a subject is prone to recurring UTIs. In some embodiments, a subject may not have clinical symptoms of a UTI. In such embodiments, the subject may have a latent infection. In other embodiments, a subject may have clinical symptoms of a UTI.
In some embodiments, a compound of the invention may be administered to a subject in combination with a bactericidal compound as described in Section II above. When administered in a combination, a compound of the invention may be administered before, simultaneously, or after administration of a bactericidal compound. When administered before or after a bactericidal compound, the time between administration of a compound of the invention and a bactericidal compound may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 min. In another embodiment, the time between administration of a compound of the invention and a bactericidal compound may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours.
A compound or pharmaceutical composition of the invention may be administered by several different means that will deliver a therapeutically effective dose. Such compositions may be administered orally, parenterally, by inhalation spray, rectally, intradermally, intracisternally, intraperitoneally, transdermally, bucally, as an oral or nasal spray, topically (i.e. powders, ointments or drops), or via a urinary catheter in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection, or infusion techniques. In an exemplary embodiment, the pharmaceutical composition will be administered in an oral dosage form. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).
The amount of a compound of the invention that constitutes an “effective amount” can and will vary. The amount will depend upon a variety of factors, including whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, and weight. Those skilled in the art will appreciate that dosages may also be determined with guidance from Goodman & Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman & Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.
In order to selectively control the release of an inhibitor to a particular region of the gastrointestinal tract for release, the pharmaceutical compositions of the invention may be manufactured into one or several dosage forms for the controlled, sustained or timed release of one or more of the ingredients. In this context, typically one or more of the ingredients forming the pharmaceutical composition is microencapsulated or dry coated prior to being formulated into one of the above forms. By varying the amount and type of coating and its thickness, the timing and location of release of a given ingredient or several ingredients (in either the same dosage form, such as a multi-layered capsule, or different dosage forms) may be varied.
In an exemplary embodiment, the coating may be an enteric coating. The enteric coating generally will provide for controlled release of the ingredient, such that drug release can be accomplished at some generally predictable location in the lower intestinal tract below the point at which drug release would occur without the enteric coating. In certain embodiments, multiple enteric coatings may be utilized. Multiple enteric coatings, in certain embodiments, may be selected to release the ingredient or combination of ingredients at various regions in the lower gastrointestinal tract and at various times.
As will be appreciated by a skilled artisan, the encapsulation or coating method can and will vary depending upon the ingredients used to form the pharmaceutical composition and coating, and the desired physical characteristics of the microcapsules themselves. Additionally, more than one encapsulation method may be employed so as to create a multi-layered microcapsule, or the same encapsulation method may be employed sequentially so as to create a multi-layered microcapsule. Suitable methods of microencapsulation may include spray drying, spinning disk encapsulation (also known as rotational suspension separation encapsulation), supercritical fluid encapsulation, air suspension microencapsulation, fluidized bed encapsulation, spray cooling/chilling (including matrix encapsulation), extrusion encapsulation, centrifugal extrusion, coacervation, alginate beads, liposome encapsulation, inclusion encapsulation, colloidosome encapsulation, sol-gel microencapsulation, and other methods of microencapsulation known in the art. Detailed information concerning materials, equipment and processes for preparing coated dosage forms may be found in Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. (Media, Pa.: Williams & Wilkins, 1995).
A bacterium may be contacted with a compound of the invention in vivo, in vitro, in situ, or ex vivo. In some embodiments, a bacterium may be directly contacted with the compound of the invention. In other embodiments, an intracellular bacterium may be contacted with a compound of the invention. Suitable cells comprising one or more bacteria may be grown, sub-cultured, stored and manipulated using standard techniques known to individuals skilled in the art. Cell culture and microbiological techniques for growing, culturing, storing, and manipulating cells comprising one or more bacteria are commonly known in the art.
Another method of the invention comprises reducing the resistance of a bacterium to a bactericidal compound. Such a method comprises contacting a bacterium resistant to a bactericidal compound with a compound of the invention. For instance, a subject infected with a bacterium resistant to a bactericidal compound may be administered a compound of the invention, as described in Section IV(a) above. In an exemplary embodiment, a method comprises contacting a bacterium resistant to an antibiotic with a compound of the invention. In a further exemplary embodiment, a method comprises contacting a bacterium resistant to TMP or SMZ with a compound of the invention.
Methods of measuring resistance of a bacterium to an antibiotic are known in the art. For more details, see the examples.
In a further embodiment, a method of the invention encompasses a method for treating catheter-associated urinary tract infections. As used herein, “treating” refers to preventing infection in a subject not currently infected, and reducing or eliminating infection in a subject that is currently infected. Generally, such a method comprises administering a pharmaceutical composition comprising a compound of the invention to a subject. For this embodiment, “subject” refers to any mammal with an indwelling urinary catheter. In one embodiment, a subject with a urinary catheter is prone to recurring UTIs. In some embodiments, a subject with a urinary catheter may not have clinical symptoms of a UTI. In such embodiments, the subject may have a latent infection. In other embodiments, a subject with a urinary catheter may have clinical symptoms of a UTI.
In some embodiments, a compound of the invention may be administered to a subject in combination with a bactericidal compound as described in Section II and Section IV(a) above.
In a further embodiment, a method of the invention encompasses a method for treating inflammatory bowel disease. Inflammatory bowel disease (IBD) involves chronic inflammation of all or part of the digestive tract. IBD may include ulcerative colitits, Crohn's disease, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's disease and indeterminate colitis. As used herein, “treating” refers to reducing symptoms associated with inflammatory bowel disease. Alternatively, a method of the invention encompasses a method for reducing symptoms associated with inflammatory bowel disease. Symptoms may include ulcers, reduced appetite, rectal bleeding, rectal pain, a feeling of urgency or frequent, small bowel movements, bloody diarrhea, abdominal cramps and pain, inability to move the bowels in spite of the urge to do so (tenesmus), pain on the left side, unintended weight loss, fatigue, significant weight loss, profuse diarrhea, dehydration, shock, fever, fatigue, arthritis, eye inflammation, skin disorders, and inflammation of the liver or bile ducts.
Generally, such a method comprises administering a pharmaceutical composition comprising a compound of the invention to a subject. For this embodiment, “subject” refers to any mammal with inflammatory bowel disease.
An additional aspect of the present invention encompasses coatings comprising a compound of the invention. Such a coating may be used on a medical device to prevent bacterial adherence or infection of the host. Suitable means of coating medical devices are known in the art. In one embodiment, a catheter may be coated with a compound of the invention. In another embodiment, a urinary catheter may be coated with a compound of the invention.
An alternative aspect of the present invention encompasses a nutritional supplement that comprises a compound of the invention. Such a supplement may be used to treat a bacterial infection as described in section IV above.
The term “acyl,” as used herein alone or as part of another group, denotes the moiety formed by removal of the hydroxyl group from the group —COOH of an organic carboxylic acid, e.g., RC(O)—, wherein R is R′, R1O—, R′R2 N—, or R1S—, R1 is hydrocarbyl, heterosubstituted hydrocarbyl, or heterocyclo and R2 is hydrogen, hydrocarbyl or substituted hydrocarbyl.
The term “acyloxy,” as used herein alone or as part of another group, denotes an acyl group as described above bonded through an oxygen linkage (—O—), e.g., RC(O)O— wherein R is as defined in connection with the term “acyl.”
Unless otherwise indicated, the alkyl groups described herein are preferably lower alkyl containing from one to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain or cyclic, also known as a cycloalkyl, and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.
Unless otherwise indicated, the alkenyl groups described herein are preferably lower alkenyl containing from two to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain or cyclic and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.
Unless otherwise indicated, the alkynyl groups described herein are preferably lower alkynyl containing from two to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.
The terms “aryl” or “ar” as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic groups containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl. Phenyl and substituted phenyl are the more preferred aryl.
As used herein, the term “functional group” includes a group of atoms within a molecule that is responsible for certain properties of the molecule and/or reactions in which it takes part. Non-limiting examples of functional groups include, alkyl, carboxyl, hydroxyl, amino, sulfonate, phosphate, phosphonate, thiol, alkyne, azide, halogen, and the like.
The terms “halogen” or “halo” as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.
The terms “heterocyclo” or “heterocyclic” as used herein alone or as part of another group denote optionally substituted, fully saturated or unsaturated, monocyclic or bicyclic, aromatic or nonaromatic groups having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring. The heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to the remainder of the molecule through a carbon or heteroatom. Exemplary heterocyclo include heteroaromatics such as furyl, thienyl, pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like. Exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, thiol, ketals, acetals, esters and ethers.
The term “heteroaromatic” as used herein alone or as part of another group denote optionally substituted aromatic groups having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring. The heteroaromatic group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to the remainder of the molecule through a carbon or heteroatom. Exemplary heteroaromatics include furyl, thienyl, pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like. Exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, thiol, ketals, acetals, esters and ethers.
The terms “hydrocarbon” and “hydrocarbyl” as used herein describe organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, and aryl moieties. These moieties also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwise indicated, these moieties preferably comprise 1 to 20 carbon atoms.
The “substituted hydrocarbyl” moieties described herein are hydrocarbyl moieties which are substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted (i.e. replaced) with a hetero atom such as nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen atom. These moieties may include halogen, carbocycle, aryl, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protected hydroxy, keto, acyl, acyloxy, nitro, amino, amido, nitro, cyano, thiol, ketals, acetals, esters and ethers.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
Certain compounds may exist as mixtures of isomers in equilibrium as described for isoquinolone isomer A in the scheme below which is in equilibrium with the hydroxyquinoline isomer B:
Therefore, it is understood that compounds containing isoquinolones may exist in the hydroxyisoquinoline form and the synthesis of analogs thereof may lead to the production of either one isomer A1 or B1 exclusively or a mixture. It is not always possible to confirm the identity of each individual isomer (e.g. A1 or B1). Thus, all possible isomers are claimed as the final product in examples which contain the isoquinolone ring.
Starting materials, reagents, and solvents were purchased from commercial vendors unless otherwise noted. In general anhydrous solvents are used for carrying out all reactions. 1H NMR spectra were measured on a Varian 400 MHz NMR instrument equipped with an auto sampler. The chemical shifts were reported as δ ppm relative to TMS using residual solvent peak as the reference unless otherwise noted. The following abbreviations were used to express the peak multiplicities: s=singlet; d=doublet; t=triplet; q=quartet; m=multiplet; br=broad. High-performance liquid chromatography (HPLC) was carried out on GILSON GX-281 using Waters C18 5 μM, 4.6*50 mm and Waters Prep C18 5 μM, 19*150 mm reverse phase columns, eluted with a gradient system of 5:95 to 95:5 acetonitrile:water with a buffer consisting of 0.05-0.1% TFA. Mass spectroscopy (MS) was performed on HPLC/MSD using a gradient system of 5:95 to 95:5 acetonitrile:water with a buffer consisting of 0.05-0.1% TFA on a C18 or C8 reversed phased column and electrospray ionization (ESI) for detection. All reactions were monitored by thin layer chromatography (TLC) carried out on Merck silica gel plates (0.25 mm thick, 60F254), visualized by using UV (254 nm) or dyes such as KMnO4, p-Anisaldehyde and CAM (Hannesian's Stain). Silica gel chromatography was carried out on a Teledyne ISCO CombiFlash purification system using pre-packed silica gel columns (12 g-330 g sizes). All compounds used for biological assays are greater than 95% purity based on NMR and HPLC by absorbance at 220 nm and 254 nm wavelengths.
To a round-bottomed flask equipped with a reflux condenser and N2 line was added [(2R,3R,4S,5R,6S)-4,5-diacetoxy-6-(acetoxymethyl)-2-(4-bromo-2-methyl-phenoxy)tetrahydropyran-3-yl] acetate (0.52 g, 1.0 mmol), (3-methoxycarbonylphenyl)boronic acid (0.22 g, 1.2 mmol), Cs2CO3 (0.98 g, 3 mmol) and Pd(Ph3)4 (0.12 g, 0.1 mmol) followed by 5:1 mixture of 1,4-dioxane/water (30 mL). The reaction flask was placed under high vacuum and then repressurized with N2 repeated 3 times. The reaction was heated to 80° C. under a N2 atmosphere for 1 h. The solvent was removed in vacuo and the residue was dissolved in CHCl3 and filtered. The filtrate was purified by silica gel chromatography (ISCO MPLC, MeOH/CH2Cl2, 0-10% gradient). Pure fractions as determined by TLC and LCMS were combined and then concentrated in vacuo. The residue was dissolved in MeOH (10 mL) and then charged with 0.002 M NaOMe/MeOH (5 mL). After the reaction was complete determined by LCMS, DOWEX 50WX4-100 ion exchange resin was added. After 15 minutes, the resin was filtered, washed with MeOH and then the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (0-25% MeOH/CH2Cl2) to yield the title compound (0.222 g, 55%) as a white solid. LCMS (ESI, M+Na+=427.3),
To a solution of methyl 3-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoate (0.222 g, 0.55 mmol) in MeOH (70 mL) was added 0.2 M NaOH (30 mL). The reaction was stirred overnight at RT. DOWEX 50WX4-100 ion exchange resin was added. After 15 minutes, the resin was filtered, washed with MeOH and then the filtrate was concentrated in vacuo to yield the title compound (0.2025 g, 94%) as a white solid. LCMS (ESI, M+Na+=413.3); 1H NMR δ ppm (d3-MeOD; 2.31 (s, 3H) 3.61 (ddd, J=9.78, 5.09, 2.74 Hz, 1H) 3.69-3.84 (m, 3H) 3.97 (dd, J=9.39, 3.52 Hz, 1H) 4.08 (dd, J=3.33, 1.76 Hz, 1H) 5.56 (d, J=1.96 Hz, 1H) 7.31 (d, J=8.22 Hz, 1H) 7.39-7.48 (m, 2H) 7.51 (t, J=7.83 Hz, 1H) 7.76-7.84 (m, 1H) 7.95 (dt, J=7.83, 1.37 Hz, 1H) 8.21 (t, J=1.76 Hz, 1H)).
To a stirred solution of 3-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzoic acid (0.039 g, 0.1 mmol) and HATU (0.046 g, 0.12 mmol) in DMF (5 mL) under a N2 atmosphere and cooled to 0° C. was added 4-aminopyridine (0.011 g, 0.12 mmol), and DIPEA (0.054 mL, 0.3 mmol). The reaction was allowed to warm to RT and then stirred overnight. The solvent was removed in vacuo and the residue purified by reversed phase HPLC (5-85% acetonitrile/water/0.05% TFA). Pure fractions were combined and lyophilized to give the title compound as a white powder (0.047 g, 100%). LCMS (ESI, M+H+=467.3); 1H NMR δ ppm (d3-MeOD; 2.34 (s, 3H) 3.60 (ddd, J=9.78, 5.28, 2.54 Hz, 1H) 3.68-3.85 (m, 3H) 3.98 (dd, J=9.59, 3.33 Hz, 1H) 4.09 (dd, J=3.33, 1.76 Hz, 1H) 5.58 (d, J=1.57 Hz, 1H) 7.34 (d, J=8.61 Hz, 1H) 7.44-7.58 (m, 2H) 7.63 (t, J=7.63 Hz, 1H) 7.84-8.00 (m, 2H) 8.19-8.27 (m, 1H) 8.37-8.45 (m, 2H) 8.62-8.72 (m, 2H)).
Synthesized in a similar manner to Example 3 using 3-aminopyridine to give (0.043 g, 94%). LCMS (ESI, M+H+=467.3); 1H NMR δ ppm (d3-MeOD; 2.33 (s, 3H) 3.61 (m, 1H) 3.76 (m, 3H) 3.97 (d, J=9.39 Hz, 1H) 4.08 (m, 1H) 5.57 (d, 1H) 7.33 (d, J=6.26 Hz, 1H) 7.43-7.56 (m, 2H) 7.61 (m, 1H) 7.85 (m, 1H) 7.94 (m, 2H) 8.22 (m, 1H) 8.55 (m, 1H) 8.66 (d, J=6.26 Hz, 1H) 9.47 (m, 1H)).
Synthesized in a similar manner to Example 1 using 2-methyl-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-1,3,4-oxadiazole (purchased from Boron Molecular). LCMS (ESI, M+H+=429.3); 1H NMR δ ppm (d3-MeOD; 2.28 (s, 3H) 2.63 (s, 3H) 3.53-3.65 (m, 1H) 3.70-3.88 (m, 3H) 3.98 (dd, J=9.59, 3.33 Hz, 1H) 4.09 (dd, J=3.13, 1.96 Hz, 1H) 5.57 (d, J=1.17 Hz, 1H) 7.24 (d, J=8.22 Hz, 1H) 7.38-7.45 (m, 2H) 7.48 (s, 1H) 7.69 (m, J=8.61 Hz, 1.5H) 8.02 (m, J=8.22 Hz, 1.5H)).
Synthesized in a similar manner to Example 1 using [3-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl]boronic acid (purchased from Apollo Scientific). LCMS (ESI, M+H+=429.3); 1H NMR δ ppm (d3-MeOD; 2.08 (s, 1.5H) 2.32 (s, 1.5H) 2.33 (s, 1.5H) 2.65 (s, 1.5H) 3.55-3.65 (m, 1H) 3.69-3.83 (m, 3H) 3.98 (dt, J=9.49, 2.69 Hz, 1H) 4.06-4.12 (m, 1H) 5.54-5.60 (m, 1H) 7.27-7.38 (m, 1H) 7.44-7.65 (m, 3H) 7.75-7.98 (m, 2H) 8.07-8.25 (m, 1H)).
Synthesized in a similar manner to Example 1 using [4,5-diacetoxy-6-(acetoxymethyl)-2-[2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]tetrahydropyran-3-yl] acetate (Han et. al., J. Med. Chem. 2012, 55, 3945-3959) and 7-bromo-2H-isoquinolin-1-one (purchased from AstaTech). LCMS (ESI, M+H+=414.3); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.57-3.67 (m, 1H) 3.70-3.85 (m, 3H) 3.94-4.02 (m, 1H) 4.05-4.13 (m, 1H) 5.57 (d, J=1.57 Hz, 1H) 6.70 (d, J=7.00 Hz, 1H) 7.17 (d, J=7.04 Hz, 1H) 7.33 (d, J=8.22 Hz, 1H) 7.54 (s, 2H) 7.70 (d, J=8.61 Hz, 1H) 7.89-8.04 (m, 1H) 8.49 (d, J=1.96 Hz, 1H)).
To a solution of [4,5-diacetoxy-6-(acetoxymethyl)-2-[2-methyl-4-(1-oxo-2H-isoquinolin-7-yl)phenoxy]tetrahydropyran-3-yl] acetate (0.116 g, 0.2 mmol) in DMF (5 mL) cooled to 0° C. under a N2 atmosphere was slowly added NaH (0.024 g, 0.6 mmol, 60% dispersion in mineral oil). After 10 min, methyl 2-bromoacetate (0.018 mL, 0.19 mmol) was added and the reaction was stirred for 1 h at 0° C. under a N2 atmosphere. The solvent was removed under high vacuum and the residue was dissolved in MeOH (5 mL) followed by the addition of 0.02 M NaOMe/MeOH (3 mL) and the reaction was stirred overnight at RT. DOWEX 50WX4-100 ion exchange resin was added. After 15 minutes, the resin was filtered, washed with MeOH and then the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (0-20% MeOH/CH2Cl2) to give the title product (0.0558 g, 57%) as a white solid. LCMS (ESI, M+H+=486.3); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.61 (ddd, J=9.68, 4.99, 2.54 Hz, 1H) 3.68-3.85 (m, 3H) 3.78 (s, 3H) 3.98 (dd, J=9.59, 3.33 Hz, 1H) 4.04-4.14 (m, 1H) 4.82 (s, 2H) 5.53-5.62 (m, 1H) 6.72 (d, J=7.04 Hz, 1H) 7.32 (dd, J=7.83, 3.91 Hz, 2H) 7.44-7.58 (m, 2H) 7.70 (d, J=8.22 Hz, 1H) 7.97 (dd, J=8.22, 1.96 Hz, 1H) 8.43-8.49 (m, 1H)).
Following a similar procedure to Example 2 using methyl 2-[7-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6 (hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]-1-oxo-2-isoquinolyl]acetate (0.050 g, 0.1 mmol) the title product was obtained as a white solid (0.045 g, 96%). LCMS (ESI, M+H+=472.3); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.61 (ddd, J=9.59, 5.28, 2.35 Hz, 1H) 3.67-3.86 (m, 3H) 3.98 (dd, J=9.59, 3.33 Hz, 1H) 4.08 (dd, J=3.33, 1.76 Hz, 1H) 4.79 (s, 2H) 5.50-5.62 (m, 1H) 6.72 (d, J=7.43 Hz, 1H) 7.32 (dd, J=8.02, 2.93 Hz, 2H) 7.44-7.59 (m, 2H) 7.70 (d, J=8.22 Hz, 1H) 7.97 (dd, J=8.22, 1.96 Hz, 1H) 8.44-8.55 (m, 1H)).
Following a similar procedure to Example 3 using 2-[7-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]-1-oxo-2-isoquinolyl]acetic acid (0.024 g, 0.05 mmol) and 3-aminopyridine the title compound was obtained (22 mg, 81%) as a white solid. LCMS (ESI, M+H+=548.4); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.55-3.66 (m, 1H) 3.66-3.85 (m, 3H) 3.97 (dd, J=9.59, 3.33 Hz, 1H) 4.08 (dd, J=3.13, 1.96 Hz, 1H) 4.96 (s, 2H) 5.46-5.63 (m, 1H) 6.77 (d, J=7.04 Hz, 1H) 7.27-7.44 (m, 2H) 7.46-7.58 (m, 2H) 7.73 (d, J=8.22 Hz, 1H) 7.88 (dd, J=8.61, 5.48 Hz, 1H) 8.00 (dd, J=8.22, 1.96 Hz, 1H) 8.42 (dd, J=8.61, 1.17 Hz, 1H) 8.49 (s, 2H) 9.18-9.29 (m, 1H)).
Following a similar procedure to Example 10 using 4-aminopyridine the title compound was obtained (13.3 mg, 52%). LCMS (ESI, M+H+=548.4); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.60 (ddd, J=9.78, 5.09, 2.74 Hz, 1H) 3.67-3.84 (m, 3H) 3.97 (dd, J=9.59, 3.33 Hz, 1H) 4.08 (dd, J=3.33, 1.76 Hz, 1H) 5.00 (s, 2H) 5.57 (d, J=1.57 Hz, 1H) 6.78 (d, J=7.43 Hz, 1H) 7.38 (d, J=7.43 Hz, 2H) 7.33 (d, J=8.61 Hz, 1H) 7.49-7.58 (m, 2H) 7.75 (d, J=8.22 Hz, 1H) 8.01 (dd, J=8.41, 2.15 Hz, 1H) 8.18 (m, J=7.04 Hz, 2H) 8.49 (d, J=1.96 Hz, 1H) 8.64 (m, J=7.43 Hz, 2H)).
Following a similar procedure to Example 10 using 1-methylpiperazine the title compound was obtained (25.9 mg, 88%). LCMS (ESI, M+H+=554.4); 1H NMR δ ppm (d3-MeOD; 2.33 (s, 3H) 2.99 (s, 3H) 3.26 (dt, J=3.23, 1.71 Hz, 1H) 3.34-3.42 (m, 1H) 3.49 (dd, J=3.52, 1.57 Hz, 1H) 3.61 (ddd, J=9.78, 5.28, 2.54 Hz, 3H) 3.70-3.86 (m, 4H) 3.97 (dd, J=9.59, 3.33 Hz, 2H) 4.08 (dd, J=3.52, 1.96 Hz, 2H) 4.81 (s, 1H) 5.57 (d, J=1.96 Hz, 1H) 6.75 (d, J=7.43 Hz, 1H) 7.20-7.40 (m, 3H) 7.45-7.59 (m, 2H) 7.72 (d, J=8.22 Hz, 1H) 7.99 (dd, J=8.22, 1.96 Hz, 1H) 8.49 (d, J=1.96 Hz, 1H)).
Following a similar procedure to Example 10 using 1,2-diaminoethane the title compound was obtained (11.2 mg, 55%). LCMS (ESI, M+H+=514.4); 1H NMR δ ppm (d3-MeOD; 2.33 (s, 3H) 3.11 (t, J=5.67 Hz, 2H) 3.54 (t, J=5.67 Hz, 2H) 3.56-3.67 (m, 1H) 3.68-3.84 (m, 3H) 3.97 (dd, J=9.59, 3.33 Hz, 1H) 4.08 (dd, J=3.33, 1.76 Hz, 1H) 4.73 (s, 2H) 5.48-5.64 (m, 1H) 6.78 (d, J=7.43 Hz, 1H) 7.34 (d, J=7.83 Hz, 2 H) 7.46-7.59 (m, 2H) 7.74 (d, J=8.22 Hz, 1H) 8.00 (dd, J=8.22, 1.96 Hz, 1H) 8.50 (s, 1H)).
Following a similar procedure to Example 8 using [4,5-diacetoxy-6-(acetoxymethyl)-2-[2-methyl-4-(1-oxo-2H-isoquinolin-7-yl)phenoxy]tetrahydropyran-3-yl] acetate (0.1 mmol) and 2-bromo-N,N-dimethyl-ethanamine (0.1 mmol), the title compound was obtained (0.0426 g, 88%). LCMS (ESI, M+H+=485.4); 1H NMR δ ppm (d3-MeOD; 2.33 (s, 3H) 3.05 (s, 6H) 3.52-3.67 (m, 3H) 3.68-3.84 (m, 3H) 3.97 (dd, J=9.39, 3.52 Hz, 1H) 4.08 (dd, J=3.33, 1.76 Hz, 1H) 4.46 (t, J=5.87 Hz, 2H) 5.47-5.64 (m, 1H) 6.80 (d, J=7.43 Hz, 1H) 7.38 (d, J=7.43 Hz, 1H) 7.34 (d, J=8.61 Hz, 1H) 7.47-7.59 (m, 2H) 7.73 (d, J=8.61 Hz, 1H) 8.01 (dd, J=8.41, 1.76 Hz, 1H) 8.49-8.60 (m, 1H)).
Following a similar procedure to Example 14 using 4-(bromomethyl)pyridine, the title compound was obtained (0.046 g, 92%). LCMS (ESI,M+H+=505.4); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.53-3.66 (m, 1H) 3.67-3.83 (m, 3H) 3.96 (dd, J=9.59, 3.33 Hz, 1H) 4.07 (dd, J=3.13, 1.96 Hz, 1H) 5.52 (s, 2H) 5.56 (s, 1H) 6.84 (d, J=7.43 Hz, 1H) 7.33 (d, J=8.61 Hz, 1H) 7.44-7.57 (m, 3H) 7.70-7.86 (m, 3H) 8.02 (dd, J=8.22, 1.96 Hz, 1H) 8.49 (s, 1H) 8.73 (d, J=6.65 Hz, 2H)).
Following a similar procedure to Example 14 using 3-(bromomethyl)pyridine, the title compound was obtained (0.046 g, 92%). LCMS (ESI,M+H+=505.4); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 3.51-3.66 (m, 1H) 3.66-3.85 (m, 3H) 3.97 (dd, J=9.59, 3.33 Hz, 1H) 4.08 (dd, J=3.13, 1.57 Hz, 1H) 5.42 (s, 2H) 5.56 (s, 1H) 6.80 (d, J=7.43 Hz, 1H) 7.33 (d, J=8.22 Hz, 1H) 7.43-7.61 (m, 3H) 7.72 (d, J=8.22 Hz, 1H) 7.89 (dd, J=8.02, 5.67 Hz, 1H) 7.99 (dd, J=8.41, 1.76 Hz, 1H) 8.42 (d, J=8.22 Hz, 1H) 8.46-8.54 (m, 1H) 8.71 (d, J=5.48 Hz, 1H) 8.86 (s, 1H)).
Methyl 5-bromo-3-ureido-thiophene-2-carboxylate (Han et. al., J. Med. Chem. 2012, 55, 3945-3959) (0.5 g) was stirred with 40 mL of 33% methylamine in EtOH overnight at RT. The solvent was removed in vacuo and the residue was triturated in CH2Cl2. The precipitate was filtered and dried to yield the title product as a white solid (0.26 g). LCMS (ESI, M+Na+=300.1).
Synthesized in a similar manner to Example 7 using 5-bromo-N-methyl-3-ureido-thiophene-2-carboxamide to give the title compound as a white powder (19 mg). LCMS (ESI, M+H+=468.3); 1H NMR δ ppm (d3-MeOD; 2.27 (s, 3H) 2.87 (s, 3H) 3.52-3.61 (m, 1H) 3.76 (d, J=1.17 Hz, 3H) 3.91-3.99 (m, 1H) 4.03-4.09 (m, 1H) 5.56 (d, J=1.17 Hz, 1H) 7.27 (m, 1H) 7.47 (m, 2H) 8.06 (s, 1H)).
Under a N2 atmosphere, to a solution of [(2R,3R,4S,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl] acetate (1.164 g, 2 mmol) in acetonitrile (20 mL) was added BF3.OEt2 (0.05 mL, 0.4 mmol) at 0° C. The mixture was stirred at RT until completion confirmed by TLC. The solvent was removed in vacuo and the resulting residue was partitioned between dichloromethane and water. The organic layer was collected, dried with Na2SO4 and concentrated. The residue was purified by silica gel chromatography using a EtOAc/Hexane gradient to give (2R,3S,4R,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-carbonitrile (0.560 g) in 51% yield. MS (ESI): found [M+Na+], 572.2.
At −78° C., DIBAL/Hexanes (1.0 M, 0.52 mL) was added dropwise into the solution of (2R,3S,4R,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-carbonitrile (0.258 g, 0.47 mmol) in CH2Cl2 (5 mL). Then the mixture was warmed slowly to −40° C. over 1 h. 0.5 N HCl aqueous was used to quench the reaction and EtOAc was used for extraction. The organic layer was collected, dried with Na2SO4 and concentrated to give (2S,3R,4S,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-carbaldehyde (0.235 g) as crude product for the next step without further purification. Into another flask containing 5-bromo-2-iodotoluene (0.42 mL, 3.0 mmol) in ether (5 mL) was added BuLi/Hexanes (2.5 M, 1.0 mL) at −78° C. One hour later, (2S,3R,4S,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-carbaldehyde (0.235 g) was added. The mixture was warmed slowly to −20° C. over 1 h 40 min. 0.5 N HCl aqueous was used to quench the reaction and EtOAc was use for extraction. The organic layer was collected, dried with Na2SO4 and concentrated. The resulting residue was purified by silica gel chromatography with a EtOAct/Hexane gradient as eluent to give (4-bromo-2-methyl-phenyl)-[(2R,3R,4S,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]methanol (A), (0.130 g) in 38% yield. MS (ESI): found [M+Na+], 745.4.
Under nitrogen atmosphere, the mixture of A (0.130 g, 0.18 mmol), N-methyl-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzamide (0.071 g, 0.27 mmol), cesium carbonate (0.176 g, 0.54 mmol) and tetrakis(triphenylphosphine)palladium (0.021 g, 0.018 mmol) in dioxane/water (5 mL/1 mL) was heated at 80° C. with stirring for 1 h. The solvent was removed and the resulting residue was purified by silica gel chromatography to give 3-[4-[(R)-hydroxy-[(2R,3R,4S,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]methyl]-3-methyl-phenyl]-N-methyl-benzamide (B), (0.046 g) and 3-[4-[(S)-hydroxy-[(2R,3R,4S,5R,6S)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]methyl]-3-methyl-phenyl]-N-methyl-benzamide (C), (0.055 g). MS (ESI): found [M+Na+], 800.6.
A mixture of intermediate B (0.046 g, 0.059 mmol) and Pd/C (10 wt %) (0.050 g, 0.024 mmol) in MeOH (5 mL) was stirred under H2 atmosphere overnight. Pd/C was filtered off and the filtrate was concentrated in vacuo. The resulting residue was purified by purified by HPLC (C18, 15150 mm column; eluent: acetonitrile/water (0.05% TFA) to give Example 18A (0.020 g) in 81% yield. Example 19 was also isolated as a product (0.0030 g). Following the same procedure for Intermediate B, Intermediate C was converted to Example 18B and 19 in the same fashion.
LCMS (ESI, M+Na+=440.3); 1H NMR δ ppm (d3-MeOD; 2.51 (s, 3H) 2.95 (s, 3H) 3.57-3.78 (m, 4H) 4.00-4.07 (m, 1H) 4.10 (dd, J=6.85, 2.54 Hz, 1H) 4.25 (t, J=2.93 Hz, 1H) 5.24 (d, J=6.65 Hz, 1H) 7.45-7.57 (m, 3H) 7.62 (d, J=8.22 Hz, 1H) 7.71-7.83 (m, 2H) 8.07 (t, J=1.56 Hz, 1H)).
LCMS (ESI, M+Na+=440.3); 1H NMR δ ppm (d3-MeOD; 2.51 (s, 3H) 2.95 (s, 3H) 3.56 (dd, J=1.00 Hz, 1H) 3.67 (m, 1H) 3.70-3.82 (m, 3H) 3.91 (m, 1H) 4.10 (dd, J=9.00, 1.96 Hz, 1H) 5.28 (d, J=8.61 Hz, 1H) 7.34-7.63 (m, 4H) 7.69-7.90 (m, 2H) 8.07 (s, 1H)).
*Note: the assignment of the R stereochemistry for 18A and S stereochemistry for 188 is only arbitrary and tentatively assigned by but not confirmed.
LCMS (ESI, M+H+=402.3); 1H NMR δ ppm (d3-MeOD; 2.44 (s, 3H) 2.95 (s, 3H) 3.04 (d, J=7.43 Hz, 2H) 3.69 (m, 3H) 3.83 (m, 2H) 3.86-3.92 (m, 1H) 4.04-4.21 (m, 1H) 7.31 (d, J=7.83 Hz, 1H) 7.42-7.47 (m, 1H) 7.50 (m, 2H) 7.75 (m, 2H) 8.05 (s, 1H)).
N-methyl-3-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide (Han et. al., J. Med. Chem. 2012, 55, 3945-3959), (0.072 g, 0.178 mmol) was dissolved in anhydrous pyridine (1 mL) and acetic anhydride (1 mL). The solvent was removed in vacuo and the residue purified by reversed phase HPLC (5-95% acetonitrile/water/0.05% TFA). Pure fractions were combined and lyophilized to give the title compound as a white powder (0.063 g). LCMS (ESI, M+Na+=594.3); 1H NMR δ ppm (d6-DMSO; 1.94 (s, 3H) 2.00 (s, 3H) 2.05 (s, 3H) 2.16 (s, 3H) 2.32 (s, 3H) 2.81 (d, J=4.30 Hz, 3H) 3.93-4.11 (m, 2H) 4.19 (dd, J=12.13, 5.09 Hz, 1H) 5.22 (t, J=9.98 Hz, 1H) 5.33-5.45 (m, 2H) 5.80 (s, 1H) 7.23 (d, J=8.61 Hz, 1H) 7.46-7.65 (m, 3H) 7.77 (d, J=7.83 Hz, 2H) 8.07 (s, 1H) 8.54 (d, J=4.30 Hz, 1H)).
N-methyl-3-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide (Han et. al., J. Med. Chem. 2012, 55, 3945-3959), (0.20 g, 0.5 mmol) was dissolved in trimethyl phosphate (5 mL) and water (9 uL, 0.5 mmol). The reaction was cooled to 0° C. and then phosphoryl trichloride (142 uL, 1.5 mmol) was slowly added and then stirred for 3 h at 0° C. The reaction was neutralized by adding crushed ice and then conc. ammonia. The solvent was removed in vacuo and the residue purified by reversed phase HPLC (5-95% acetonitrile/water/0.05% TFA). Pure fractions were combined and lyophilized to give the title compound as a white powder (0.070 g). LCMS (ESI, M+H+=484.3); 1H NMR δ ppm (d6-DMSO; 2.26 (s, 3H) 2.81 (d, J=4.70 Hz, 3H) 3.42-3.68 (m, 3H) 3.75 (dd, J=9.00, 3.13 Hz, 1H) 3.86-3.97 (m, 2H) 4.03 (dd, J=9.78, 5.87 Hz, 1H) 5.45 (d, J=1.96 Hz, 1H) 7.24 (d, J=8.61 Hz, 1H) 7.43-7.60 (m, 3H) 7.76 (dd, J=7.43, 1.57 Hz, 2H) 8.06 (s, 1H) 8.56 (d, J=4.30 Hz, 1H)).
At 0° C. TMSCI (0.35 mL, 2.75 mmol) was added slowly into the solution of N-methyl-3-[3-methyl-4-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]benzamide (Han et. al., J. Med. Chem. 2012, 55, 3945-3959), (0.202 g, 0.5 mmol) and Et3N (0.38 mL, 2.75 mmol) in DMF (2 mL). The mixture was stirred at RT for 3.5 h, then partitioned between EtOAc and water. The organic layer was collected, dried with Na2SO4 and concentrated. To the resulting residue, acetone (1 mL) and MeOH (1.5 mL) was added. Then the mixture was cooled at 0° C. while AcOH (0.055 mL, 0.96 mmol) was added. The mixture was stirred at RT for 9 h, then NaHCO3 (0.16 g, 1.9 mmol) was added. The solvents were removed. The resulting residue was purified by silica gel chromatography with a EtOAc/Hexanes gradient as eluent to give 3-[4-[(2R,3R,4S,5R,6S)-6-(hydroxymethyl)-3,4,5-tris(trimethylsilyloxy)tetrahydropyran-2-yl]oxy-3-methyl-phenyl]-N-methyl-benzamide (D), (0.190 g) in 61% yield. Into the mixture of N,N′-dimethylglycine hydrochloride (0.0154, 0.11 mmol), DMAP (0.0024 g, 0.02 mmol), 1Pr2NEt (0.035 mL, 0.2 mmol) and intermediate D (0.062 g, 0.1 mmol) in dichloromethane (2 mL) was added N,N′-diisopropylcarbodiimide (0.02 mL, 0.13 mmol). The mixture was stirred overnight at RT. The solvent was removed and the resulting residue was dissolved in acetonitrile (3 mL). Then trifluoroacetic acid (0.08 mL) was added at 0° C. The mixture was stirred for 2 h at 0° C. The solvent was removed and the resulting residue was purified by HPLC (C18, 15*150 mm column; eluent: acetonitrile/water (0.05% TFA) to give the title compound (0.015 g) in 31% yield. LCMS (ESI, M+H+=489.4); 1H NMR δ ppm (d3-MeOD; 2.32 (s, 3H) 2.89 (s, 6H) 2.95 (s, 3H) 3.71-3.85 (m, 2H) 3.94-4.00 (m, 1H) 4.06 (d, J=5.48 Hz, 2H) 4.11 (t, J=2.54 Hz, 1H) 4.42 (m, 1H) 4.61 (dd, J=11.74, 1.56 Hz, 1H) 5.57 (d, J=1.57 Hz, 1H) 7.23 (d, J=8.61 Hz, 1H) 7.34-7.61 (m, 3H) 7.66-7.88 (m, 2H) 7.99-8.17 (m, 1H)).
1H NMR δ ppm (d3-MeOD
The inventors set out to develop and optimize orally active mannoside small-molecule antagonists of FimH bacterial adhesion for treatment and prevention of recurring urinary tract infection (UTI). The endpoint desired to determine orally active compounds was drug unchanged in the urine and/or bladder. First, the inventors rationally designed biaryl mannosides with potency and desirable properties. To do this, structure activity relationships (SAR) of substituents was determined. Ortho substitution on the biaryl ring was evaluated for FimH activity. Solubility, LogD and pKa was improved with heterocycles. It was further discovered that replacements to the glycosidic bond could improve metabolic stability and bioavailability. Alternate linkers of mannose to the biaryl ring were identified. N-, S- and C-mannosides were synthesized. Murine animal models of both acute and chronic UTI were used to further evaluate compound efficacy.
The inventors have developed compounds with a 2000-fold increase in cellular potency by X-ray structure-based design. Mannosides show good oral compound exposure for 6h at 100 mg/kg dose and prophylactically prevent IBC formation of UTI89 bacteria in vivo. Some metabolism/hydrolysis products (phenol) detected in the urine. Importantly, mannosides reverse antibiotic TMP-SMZ resistant strains of UTI in vivo. Ongoing optimization for decreased CI, increase t1/2, Vdss (tissue exposure), and improved bioavailability by compound PK screening in plasma and urine. Also, ongoing efficacy model development for demonstrating antibacterial effects post-infection as monotherapy and in combination with antibiotics. Further, the inventors are optimizing prodrugs and non-sugar mannoside mimetics.
The efficacy of in vivo mannoside treatment was evaluated after orally dosing animals with 50 mg/kg of mannosides ZFH-4269 (
Mannoside compounds FIM-4269, FIM-5240, FIM-5254, FIM-1CJ82 and FIM-1CJ66 (
Based on these results, oral PK studies were performed in mice. Compounds ZFH-4269 (
The efficacy of in vivo mannoside treatment was evaluated after orally dosing animals with 25 mg/kg of mannosides ZFH269, Prodrug FIM-4269, ZFH-5254, and ZFH-5240 in 10% cyclodextrin or 10% cyclodextrin 30 min prior to infecting with UTI89. At 6 hours post-infection (hpi) the bladders were removed and total bacterial CFUs were quantitated. In all of the mannoside-treated cohorts, there was a drop in bacterial counts demonstrating the efficacy of these mannosides in reducing overall colonization of the bladder (
Clinically, it has been presumed that UPEC infection consists of a relatively simple extracellular colonization of the luminal surface after inoculation of fecal flora into the bladder via the urethra. In contrast, using a murine model of UPEC infection of the UT, the inventors have detailed an unexpectedly complex UPEC pathogenesis cycle that involves both intracellular and extracellular niches. Using genetic, biochemical and cell biological approaches together with a variety of imaging techniques including transmission, quick freeze-deep etch and scanning electron microscopy, as well as confocal and time lapse video microscopy, the inventors discovered that UPEC invade bladder facet cells via a FimH-dependent mechanism (see below). After invasion, cytoplasmic intracellular bacterial communities (IBCs) are formed. Rapid replication of the initial invading bacteria results in the formation of an early IBC of loosely-packed rod-shaped bacteria. The bacteria continue to replicate and progress to form a large densely packed mid-stage IBC of morphologically coccoid bacteria, with biofilm-like characteristics including positive periodic acid-Schiff (PAS) staining and differential gene expression throughout the community. After the IBC matures, bacteria detach from the biomass, often become filamentous, and spread to neighboring cells forming new generation IBCs. Thus, the IBC pathway facilitates massive expansion of the invading bacteria in a niche protected from host defenses. Translational studies have shown that the majority of UPEC isolates form IBCs when introduced into the murine bladder and that IBCs and filamentous bacteria occur in the urine of human UTI patients. Population dynamic studies conducted by the inventors using ex vivo gentamicin protection assays demonstrated that ˜104 UPEC of an initial 107 inoculum invaded the bladder tissue within 15 minutes after infection and that one percent of the invaded bacteria went on to form IBCs, resulting in an average of 100 IBCs per infected mouse bladder. If this is extrapolated to the human situation, innate defenses in the bladder most likely prevent the majority of bacterial inoculation events into the bladder from leading to disease. However, the ramifications of the IBC cascade are striking. Invasion of a single infecting bacterium can lead to rapid expansion of the infection via IBC formation, replicating within hours to 104 bacteria and even higher numbers followed by dispersal of the bacteria from the biomass and spreading to neighboring cells to reinitiate the IBC cascade. This process allows the bacteria to gain a critical foothold. Bacterial descendents of the acute IBC cascade have been shown using a murine model, to be able to form a quiescent intracellular reservoir (QIR) that can persist, protected from antibiotics and seemingly undetected by the host immune system even after the acute infection is resolved and bacteria are no longer detectable in the urine. Bacteria in the QIR can later seed a recurrent infection, manifested by IBC formation, bacteruria and inflammation.
There are several key implications from understanding UPEC pathogenesis. Mannosides and pilicides that block FimH function will prevent bacterial adherence and invasion and thus prevent bacterial amplification in the IBC and subsequent spreading and repeated rounds of amplification via new generation IBCs. These compounds will have potent therapeutic activity by preventing bacterial expansion which may also have the consequence of eliminating or significantly reducing the QIR thus reducing predisposition to recurrent infection.
Type 1 pili/FimH are Critical for UPEC Pathogenesis in the UT.
Type 1 pili are essential cystitis virulence determinants. Using scanning and high-resolution EM and the mouse cystitis model developed by the inventors, it was shown that adhesive type 1 piliated bacteria are able to bind and invade host superficial umbrella cells, while UPEC lacking type 1 pili are not. Colonization and invasion of the bladder epithelium is dependent on the FimH adhesion located at the distal end of the pilus that binds mannose residues on bladder epithelial cells. High-resolution freeze-dry/deep-etch EM revealed that FimH interacts directly with receptors on the luminal surface of the bladder (
Adhesive type 1 pili are prototypic structures of a family of adhesive fibers produced by diverse Gram-negative bacteria via the chaperone/usher assembly pathway. Using biochemistry, mutational studies, nuclear magnetic resonance, and x-ray crystallography, the molecular basis of pili assembled by the chaperone/usher pathway in gram-negative bacteria, including type 1 pili of UPEC, were delineated (
FimH is a two domain protein, with a receptor binding domain linked to a typical pilin domain that joins the adhesin to the pilus fiber. The structure of the complex of the FimC chaperone bound to FimH (which was bound to D-mannopyranoside) was determined to 2.8 Å resolution. The mannose binding site of FimH is a deep negatively charged pocket at the tip of its receptor-binding domain. The FimH pocket engages in extensive hydrogen bonding to mannose (
The FimH-mannose interaction was further investigated in an effort to develop potential ligand-based antagonists of UTIs. The chitobiose unit on oligomannose was found to bridge various mannose derivatives to the asparagine in the Asn-X-Ser/Thr motif of FimH resulting in higher affinity binding. Crystallization of FimH in complex with oligomannose-3 revealed the mechanism of this higher affinity binding. The non-reducing Man4 anchors into the mannose-binding pocket while the GlcNAc folds over Thr51 allowing specific interactions with a hydrophobic tyrosine gate. Heptyl mannoside mimics the GlcNAc tail of oligomannose-3 and extends it further to increase interactions outside the binding pocket resulting in high affinity binding (Kd=5 nM). Based on the high affinity of heptyl mannose for FimH, the ability of heptyl mannose to reduce bacterial infection in our mouse model of UTI was tested. First, biofilm formation as a surrogate for IBCs formed in the bladder was evaluated. Heptyl mannose at 1 mM inhibited UPEC biofilm formation in vitro, suggesting that the mannose binding properties of the FimH adhesin is required for biofilm formation. Thus, UPEC strain UTI89 was incubated with heptyl mannose prior to inoculation into the bladders of mice. This resulted in a significant attenuation of virulence at 6 hours post-infection at 5 mM heptyl mannose. The ability of these compounds to significantly attenuate virulence establishes mannosides as a potential treatment for UTI. Therefore, more potent mannosides that mimic the natural receptor for FimH but with increased affinity and avidity in order to ultimately block bacterial colonization, invasion, IBC formation and disease were developed as described below.
The first-line treatment of choice for UTI has traditionally been a 3-day course of TMP-SMZ. Women suffering from chronic/recurrent UTIs are often given TMP-SMZ prophylactically to prevent recurrence. However, resistance to this TMP-SMZ regimen is rapidly expanding. It was hypothesized that by preventing bacterial invasion into the bladder tissue, a FimH inhibitor may result in anti-virulence synergism with TMP-SMZ and may curtail or circumvent the problem of TMP-SMZ resistance. This theory was evaluated in a preclinical animal model where mice given TMP-SMZ for 3 days were infected with either UTI89 or the TMP-SMZR strain, PBC-1. Mice were IP treated with 6 30 min prior to inoculation with bacteria and compared to a control group of untreated animals. After inoculation with UTI89 or PBC-1, bacterial CFUs were quantified at 6 hpi. As expected, treatment with TMP-SMZ alone resulted in a significant drop in bacterial load in the UTI89-infected mice but had no effect on PBC-1, since it is resistant to TMP-SMZ. Upon treatment with 6 alone there was a significant drop in bacterial load of both strains in the bladder. In the dual treatment group there was also a significant drop in bacterial CFUs compared to mannoside alone or TMP-SMZ alone for both strains which was most pronounced for PBC-1 (
Having established that FimH is required for UPEC virulence in implanted bladders, we investigated this as a potential therapeutic target for CAUTI using small molecules inhibitors designed to interfere with FimH binding to mannosylated residues. This family of small molecules, called mannosides, has recently been shown to prevent acute and chronic UPEC infections and potentiated the effectiveness of antibiotics in combinatorial treatment.
To investigate the potential therapeutic effects of mannosides on CAUTI, we first assessed the inhibitory effects of methyl-α-D-mannopyranoside (methyl mannose), on UTI89 biofilm formation in urine under flow. Similar to the deletion of fimH, UTI89 biofilms grown in presence of 1% methyl mannose had significantly reduced biomass (p=0.0022) and biofilm-adherent cells (p=0.0012), compared to untreated controls. Since methyl mannose is a FimH antagonist, these data confirm the critical role of type 1 pili to biofilm formation in urine as was previously described for biofilms formed in LB media.
The effects of mannoside treatment were then assessed in vivo by using IBC formation as well as implant and urinary tract colonization as benchmarks of disease progression. Mice were treated intraperitoneally (i.p.) with saline or 5 mg/kg of mannoside 6, which is more potent than methyl mannose in vitro and in vivo, in PBS 30 min prior to urinary implantation. Catheter implantation was immediately followed by transurethral inoculation of UTI89. IBC formation and bacterial colonization were assayed by LacZ staining and CFU enumeration of implants, bladders, and kidneys at 6 hpi and 24 hpi, respectively. Mannoside treatment further reduced IBC formation (p=0.0051) and bladder colonization (p=0.0114) in implanted animals at 6 hpi, suggesting that this treatment prevents intracellular infection. While eliminated from their intracellular niche, data further indicated that UPEC were able to persist in the extracellular milieu where they can colonize the surface of the implants to relatively similar levels as saline-treated animals (p=0.0547). No statistical difference was observed in kidney colonization in the presence or absence of mannosides. By 24 hpi, a time point at which the mannosides have been eliminated from the bladder, similar bacterial loads were recovered from implants, bladders, and kidneys in implanted animals in the presence or absence of mannoside treatment.
In order to examine whether mannosides could prevent establishment of CAUTI when used in combination with antibiotics, animals were treated with 54 and 270 μg/ml of TMP-SMZ, respectively, in their drinking water for three days and then treated with saline or mannoside (5 mg/kg) i.p. 30 min prior to implantation and bacterial inoculation. At 6 hpi, UPEC colonized the implants and bladders at significantly lower levels in animals that only received antibiotics compared to those who received water or were only administered mannoside. Interestingly, mannoside treatment in addition to TMP-SMZ further decreased UPEC colonization of implants, bladders, and kidneys compared to treatment with antibiotic alone (p<0.0005 in all cases). Furthermore, treatment with mannosides alone did not reduce bacterial titers from a 24h old UPEC infection and in combination with TMP-SMZ showed no additive effects on established UPEC CAUTI 24 hpi (data not shown). Together, these findings indicate that virulence-targeted therapies in combination with established antibiotic treatment can help prevent or delay the onset of CAUTI and that further research is warranted for enhancing mannosides potential as therapeutics against CAUTIs.
Biofilm Assay. UTI89 was grown in LB broth in wells of PVC microtiter plates at 23° C. in the presence of individual mannosides at varying concentrations. After 48 h of growth, wells were rinsed with water and stained with crystal violet for quantification as described. For biofilm disruption activity in PVC plates, UTI89 was grown in LB broth in wells of PVC microtiter plates at 23° C. After 24 h of growth, mannoside was added and biofilms were grown for an additional 16h. Wells were then rinsed, stained with crystal violet and quantified. For biofilm disruption activity on PVC coverslips, UTI89 was grown in LB broth in 50 mL conicals containing PBC coverslips at 23° C. After 24 h of growth, 0.3 μM ZFH-2056 was added and biofilm was grown for an additional 16 h. Coverslips were then rinsed, fixed with 2% paraformaldehyde (v/v), stained with SYTO9 (1:1000 in PBS; Molecular Probes) and observed with a Zeiss LSM410 confocal laser scanning microscope under a 63X objective.
Animal infections. Bacteria were grown under type 1 pili-inducing conditions (2×24 h at 37° C. statically in LB). The bacteria were harvested and resuspended to an OD600 of 0.5 in PBS. Eight-week-old C3H/HeN (Harlan) female mice were anesthetized by inhalation of isoflurane and infected via transurethral catheterization with 50 pl of the bacterial suspension, resulting in 1-2×107 inoculum. At 6 hpi, mice were sacrificed by cervical dislocation under anesthesia and the bladders were immediately harvested and processed as described below. All animal studies using mice were approved by the Animal Studies Committee of Washington University (Animal Protocol Number 20100002).
Pharamacokinetic analysis. For intraperitoneal dosing, 50 μl of a 2 mg/ml (5 mg/kg) or 4 mg/ml (10 mg/kg) solution of ZFH-2056 in PBS was injected into the peritoneal cavity of the mouse. For oral dosing, 100 μl of a 20 mg/ml (100 mg/kg) solution of ZFH-2056 in 8% DMSO was inoculated with a gavage needle into the mouse stomach. Urine was collected at 30 min, 1, 2, 3, 4, 6, and 8 h post-treatment. An equal volume of 10 μM internal standard (ZFH-2050) was added to the urine. Mannosides were extracted from the urine by loading on C18 columns (100 mg, Waters), washing with 30% methanol, and eluting with 60% methanol. Vacuum-concentrated eluates were analyzed using liquid chromatography-mass spectrometry system 30 with a lower heated capillary temperature of 190° C. and a gradient as follows: Solvent B (80% acetonitrile in 0.1% formic acid) was held constant at 5% for 5 minutes, increased to 44% B by 45 minutes, and then to a 95% B by 65 minutes. SRM mode quantification was performed with collision gas energy of 30% for the following MS/MS transitions (precursor m/z/product m/z): compound ZFH-2056, 447/285; compound ZFH-2050, 390/228. Absolute quantification was achieved by comparison to a calibration curve.
Bladder tissue bacterial titer determination. Mannoside ZFH-2056 was administered either IP (5 mg/kg) or orally (100 mg/kg) 30 min prior to inoculation with UTI89. To enumerate the bacteria present, mice were sacrificed at 6 hpi and bladders were aseptically removed and homogenized in 1 ml PBS, serially diluted and plated onto LB agar plates. CFU was enumerated after 16 h of growth at 37° C.
Gentamicin protection assay. To enumerate bacteria present in the intracellular versus extracellular compartments, bladders were aseptically harvested at 6 hpi. The bladders were then bisected twice and washed three times in 500 μl of PBS each. The wash fractions were pooled, lightly spun at 500 rpm for 5 min to pellet exfoliated bladder cells, serially diluted, and plated onto LB agar to obtain the luminal fraction. The bladders were treated with 100 μg of gentamicin/mi for 90 min at 37° C. After treatment, the bladders were washed twice with PBS to eliminate residual gentamicin, homogenized in 1 ml of PBS, serially diluted, and plated onto LB agar to enumberate the CFUs in the intracellular fraction.
Antibiotic treatment. Mice were given TMP-SMZ in the drinking water at a concentration of 54 μg/ml and 270 μg/ml, respectively. Water was changed daily for 3 days prior to inoculation with UTI89. Mice remained on TMP-SMZ during the infection. To determine TMP-SMZ concentration in the urine, urine was collected after 3 days of TMP-SMZ treatment and quantified by LC-MS following addition of sulfisoxazole as an internal standard.
Growth curve. An overnight culture of PBC-1 was diluted 1:1000 in LB in the absence or presence of TMP-SMZ and/or mannoside ZFH-2056. The highest concentration of TMP-SMZ used was 512 μg/ml and 2560 μg/ml, respectively. Two-fold dilutions of TMP-SMZ were performed. Mannoside ZFH-2056 was added at 100 μM. Growth curves were performed in a 96-well plate at 37° C. with A600 readings taken every 30 min for 8 h.
Hemagglutination assay. PBC-1 was grown statically in LB in the absence or presence of TMP-SMZ for 2×24 h at 37° C. The highest concentration of TMP-SMZ used was 256 μg/ml and 1280 μg/ml, respectively. Two-fold dilutions of TMP-SMZ were performed. Hemagglutination assays for mannose-sensitive agglutination of guinea pig red blood cells were performed as previously described.
Statistical analysis. Observed differences in bacterial titers and IBC numbers were analyzed for significance using the nonparametric Mann-Whitney U test (Prizm; GraphPad Software).
This application is a continuation of U.S. application Ser. No. 15/915,490, filed Mar. 8, 2018, which is a continuation of U.S. application Ser. No. 14/894,927, filed Nov. 30, 2015, now U.S. Pat. No. 9,957,289, issued May 1, 2018, which claims the benefit of PCT International Patent Application No. PCT/US2014/040355, filed May 30, 2014, which claims the priority of U.S. provisional application No. 61/828,954, filed May 30, 2013, each of the disclosures of which are hereby incorporated by reference in its entirety.
This invention was made with government support under RO1AI029549, DJ086378, P50DK064540 and RO1BK051406-12 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61828954 | May 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15915490 | Mar 2018 | US |
| Child | 17135256 | US | |
| Parent | 14894927 | Nov 2015 | US |
| Child | 15915490 | US |
