This disclosure relates to amphiphilic compounds containing a cyclobutene or cyclobutane moiety. In some embodiments, the compounds are useful for treating infection by Mycobacterium such as Mycobacterium tuberculosis. Cyclobutene containing compounds are also useful as monomers in the preparation of amphiphilic polymers.

TECHNICAL FIELD

This disclosure relates to amphiphilic compounds containing a cyclobutene or cyclobutane moiety. In some embodiments, the compounds are useful for treating infection bymycobacteriumsuch asMycobacterium tuberculosis. Cyclobutene containing compounds are also useful as monomers in the preparation of amphiphilic polymers.

BACKGROUND

Tuberculosis resulting from infection byMycobacterium tuberculosis(M. tb) poses a significant disease threat. Based on skin test reactivity, it is estimated that one-third of the world's population has been exposed, resulting annually in approximately nine million cases and 1.4 million deaths (2010 data) (see Raviglione, M. et al.Lancet2012, 379, 1902-1913). The current vaccine,M. bovisBCG, yields inconsistent protection and can interfere with diagnostic skin tests. Although numerous candidate vaccines are being tested, their overall safety and efficacy has not been established. Although a number of therapeutic agents have been developed, current treatment regimens require patients to take multiple drugs over a period of months. This, combined with significant drug side effects, commonly results in patient noncompliance leading to relapses and the emergence of drug resistance;3a high fraction of active cases involve multi-drug resistant (MDR, XDR) strains.

SUMMARY

It is believed that much of the hardiness and drug resistance of mycobacteria are due to an unusually thick lipid cell wall containing a significant proportion of mycolic acids, a unique class of C54-C63branched-chain fatty acids. A number of existing treatments for M. tb, exemplified by isoniazid and ethionamide, inhibit mycolic acid biosynthesis. Mycobacteria incorporate C16and C18fatty acids as biosynthetic feedstocks. The compounds provided herein mimic these feedstocks and the uptake of these specifically functionalized fatty acids is thought to inhibit downstream mycolic acid biosynthesis, leading to virulence-attenuating or even lethal alterations in the mycobacterial cell wall structure.

Provided herein are compounds of Formula (I):

or a pharmaceutically acceptable salt thereof,
wherein:----- represents an optionally present double bond;R1and R2are independently selected from the group consisting of: H, halo, OR5, and ═O;R3is absent or is an optionally substituted C1-C40alkyl;R4is H or an optionally substituted C1-C40alkyl; andZ is selected from the group consisting of: COR6, CO2R6, NHC(O)NR6R7, CONR6, OCOR6, OR6, SR6, NR6R7, and OPO3R6;R5is H or C1-C6alkyl;R6and R7are independently H or C1-C6alkyl;
wherein the hydrocarbon backbone formed by R4and R3—Z is amphiphilic,
with the proviso that the compound of Formula (I) is not:

In some embodiments, the compound provided herein can be used in a method for treating a mycobacterial infection in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. For example, a compound provided herein can be used to treat a mycobacterial infection such asM. tuberculosis.

Further provided herein are polymers prepared from a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof,
wherein:R3is absent or is an optionally substituted C1-C40alkyl;R4is H or an optionally substituted C1-C40alkyl; andZ is selected from the group consisting of: CORE, CO2R6, NHC(O)NR6R7, CONR6, OCOR6, OR6, SR6, NR6R7, and OPO3R6;R5is H or C1-C6alkyl;R6and R7are independently H or C1-C6alkyl;
wherein the hydrocarbon backbone formed by R4and R3—Z is amphiphilic.

For example, a compound of Formula (IV) can be prepared by reacting an unsaturated fatty acid or derivative thereof having a backbone comprising at least one carbon-carbon double bond with dihaloketene to achieve a stereospecific cycloaddition across the at least one carbon-carbon double bond, thereby yielding a cycloketone-containing fatty acid derivative comprising a cycloketone along the backbone, wherein the cycloketone comprises at least four carbon atoms, with at least two of the at least four carbon atoms being part of the backbone of the cycloketone-containing fatty acid derivative; reducing the cycloketone-containing fatty acid derivative to yield an amphiphilic cycloalkene-containing fatty acid derivative having a hydrophobic end, a hydrophilic end, and a backbone therebetween comprising a cycloalkene, wherein the cycloalkene comprises at least four carbon atoms, with at least two of the four carbon atoms being part of the backbone of the amphiphilic cycloalkene-containing fatty acid derivative.

In some embodiments, a multiplicity of compounds of Formula (IV) can be polymerized to yield a polymer comprising carbon-carbon double bonds. In some such embodiments, the polymerization can further include hydrogenating the carbon-carbon double bonds of the polymer; cleaving at least some of the carbon-carbon double bonds in the polymer via oxidation to yield monomeric products; cross-linking the amphiphilic cycloalkene-containing fatty acid derivatives via the carbon-carbon double bond in the cycloalkene; ring-opening metathesis reactions; aligning the backbones of the multiplicity of the amphiphilic cycloalkene-containing fatty acid derivatives before polymerizing the multiplicity of the amphiphilic cycloalkene-containing fatty acid derivatives; polymerizing via click-chemistry; and mixtures thereof.

DETAILED DESCRIPTION

Definitions

For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the term “about” is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term “about”, whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

The term “alkenyl” includes aliphatic groups that may or may not be substituted, as described above for alkyls, containing at least one double bond and at least two carbon atoms. For example, the term “alkenyl” includes straight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, and decenyl) and branched-chain alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has twelve or fewer carbon atoms in its backbone (e.g., C2-C12for straight chain; C3-C12for branched chain). The term C2-C12includes alkenyl groups containing 2 to 12 carbon atoms.

The term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond and two carbon atoms. For example, the term “alkynyl” includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl) and branched-chain alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has twelve or fewer carbon atoms in its backbone (e.g., C2-C12for straight chain; C3-C12for branched chain). The term C2-C12includes alkynyl groups containing 2 to 12 carbon atoms.

The term “alkoxy” is used in its conventional sense, and refers to alkyl groups linked to molecules via an oxygen atom. In some embodiments, an alkoxy has twelve or fewer carbon atoms in its backbone (e.g., a C1-C12alkoxy). For example, C1-C10, C1-C8, C1-C6, C1-C4, C1-C3, or C1-C2. Non-limiting examples of an alkoxy group include methoxy, ethoxy, propoxy, butoxy, and hexoxy.

The terms “halo” or “halogen”, used alone or in combination with other terms, refers to fluoro, chloro, bromo and iodo.

The term “haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term “Cn—Cmhaloalkyl” refers to a Cn—Cmalkyl group having n to m carbon atoms, and from at least one up to {2(n to m)+1} halogen atoms, which may either be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CCl3, CHCl2, C2Cl5and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

The term “haloalkoxy”, employed alone or in combination with other terms, refers to a group of formula —O-haloalkyl, wherein the haloalkyl group is as defined above. The term “Cn—Cmhaloalkoxy” refers to a haloalkoxy group, the haloalkyl group of which has n to m carbons. Example haloalkoxy groups include trifluoromethoxy and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

The term “amino” refers to a group of formula —NH2.

The term “carbamyl” refers to a group of formula —C(O)NH2.

The term “carbonyl”, employed alone or in combination with other terms, refers to a —C(═O)— group, which also may be written as C(O).

The term “oxo” refers to oxygen as a divalent substituent, forming a carbonyl group, or attached to a heteroatom forming a sulfoxide or sulfone group, or an N-oxide group.

The term “aromatic” refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e., having (4n+2) delocalized π (pi) electrons where n is an integer).

The term “carbocyclyl” includes a cyclic aliphatic group which may be saturated or unsaturated. For example, carbocyclyl groups include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, carbocyclyls have from 3-8 carbon atoms in their ring structure, for example, they can have 3, 4, 5 or 6 carbons in the ring structure.

In general, the term “aryl” includes groups, including 5- and 6-membered single-ring aromatic groups, such as benzene and phenyl. Furthermore, the term “aryl” includes multicyclic aryl groups, e.g., tricyclic, bicyclic, such as naphthalene and anthracene.

The term “heterocyclyl” includes non-aromatic groups, including but not limited to, 3- to 10-membered single or multiple non-aromatic rings having one to five heteroatoms, for example, oxetane, piperazine, pyrrolidine, piperidine, or homopiperazine.

The term “substituted” means that an atom or group of atoms replaces hydrogen as a “substituent” attached to another group. For aryl and heteroaryl groups, the term “substituted”, unless otherwise indicated, refers to any level of substitution, namely mono, di, tri, tetra, or penta substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In some cases, two sites of substitution may come together to form a 3-10 membered carbocyclyl or heterocyclyl ring.

Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, for example, —CH2O— is equivalent to —OCH2—. In some embodiments, one or more substituents can be a group reactive with a biologically active molecule or a detectable agent.

As used herein, chemical structures which contain one or more stereocenters depicted with dashed and bold bonds (i.e.,) are meant to indicate absolute stereochemistry of the stereocenter(s) present in the chemical structure. As used herein, bonds symbolized by a simple line do not indicate a stereo-preference. Unless otherwise indicated, chemical structures, which include one or more stereocenters, illustrated herein without indicating absolute or relative stereochemistry encompass all possible stereoisomeric forms of the compound (e.g., diastereomers, enantiomers) and mixtures thereof (e.g., racemic mixtures). Structures with a single bold or dashed line, and at least one additional simple line, encompass a single enantiomeric series of all possible diastereomers.

Also provided herein are pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the compounds provided herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the compounds provided herein can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; in some embodiments, a non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) can be used. Lists of suitable salts are found inRemington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 andJournal of Pharmaceutical Science,66, 2 (1977). Conventional methods for preparing salts are described, for example, inHandbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

The term “essentially pure” refers to chemical purity of a compound provided herein that may be substantially or essentially free of other components which normally accompany or interact with the compound prior to purification. By way of example only, a compound may be “essentially pure” when the preparation of the compound contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, an “essentially pure” compound may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. For the purposes of this document, preparations of functionalized polymers or conjugates differing only in the length of their polymer chain are considered to be essentially pure. An essentially pure compound may be obtained using chromatographic purification methods.

As used herein, “amphiphilic” or “amphiphile” refers to a chemical compound possessing regions of very different properties in terms of preferred solvation or association with a liquid interface or a solid surface. The most common examples of amphiphiles possess both hydrophilic (water-loving, polar) and lipid (fat-loving, non-polar) properties. Generally, amphiphilic compounds herein have a polar region on one half or terminal portion of the compound and a non-polar region at the opposite half or terminal portion of the compound. Fatty acids, triglycerides, and derivatives thereof are examples of amphiphilic compounds. An example of another class of amphiphiles are molecules containing either a hydrophobic or hydrophiic region on one half or terminal portion of the molecule and a thiol or similar functional group possessing high affinity for a metal on the other half or terminus.

“Fatty acid derivatives” as used herein refer to compounds having a fatty acid hydrocarbon backbone, but which may be optionally substituted as provided herein. In addition, a fatty acid derivative may have the carboxylic acid functional group replaced with another reactive group, for example, a hydroxyl group, an ester group, an aldehyde group, a carboxyl group, a sulfhydryl group, an amine group, an amide group, a carbamide group, or a phosphate group.

A “therapeutically effective amount” of a compound with respect to the subject method of treatment, refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a patient, e.g., a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.

As used herein, the term “treating” or “treatment” includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a patient's condition.

Antimicrobial Agents

Provided herein are amphiphilic compounds containing a cyclobutane or cyclobutene moiety. For example, amphiphilic acids, alcohols, esters, thiols, amides, or related groups incorporating within their backbone a four-membered ring carbocycle (cyclobutane, cyclobutene, cyclobutanone, or substituted derivatives).

A compound provided herein can include a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,
wherein:----- represents an optionally present double bond;R1and R2are independently selected from the group consisting of: H, halo, OR5, and ═O;R3is absent or is an optionally substituted C1-C40alkyl;R4is H or an optionally substituted C1-C40alkyl; andZ is selected from the group consisting of: COR6, CO2R6, NHC(O)NR6R7, CONR6, OCOR6, OR6, SR6, NR6R7, and OPO3R6;R5is H or C1-C6alkyl;R6and R7are independently H or C1-C6alkyl;wherein the hydrocarbon backbone formed by R4and R3—Z is amphiphilic

In some embodiments, the compound of Formula (I) is not:

In some embodiments, R1and R2are independently selected from the group consisting of: H, halo, OH and ═O.

In some embodiments, the substituents can be selected from the group consisting of an alkyl, a halogen, a hydroxyl, an alkoxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a suithydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a carbocyclyl, a heterocyclyl, an aralkyl, a heteroaralkyl, or an aromatic or heteroaromatic moiety.

In some embodiments, Z is CO2R6.

A compound provided herein can be selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, wherein R3, R4and Z are as described previously.

In some embodiments, a compound provided herein is a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4and Z are as described previously. In some embodiments, a compound provided herein is a compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4and Z are as described previously.

Non-limiting examples of a compound provided herein includes:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound provided herein can be selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
Pharmaceutical Compositions and Dosage Forms

When employed as pharmaceuticals, the compounds provided herein can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

In preparing a formulation, an active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If an active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If an active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

The compounds provided herein may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds provided herein can be prepared by processes known in the art, e.g., see International App. No. WO 2002/000196.

The liquid forms in which the compounds and compositions provided herein can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Topical formulations can contain one or more conventional carriers. In some embodiments, ointments can contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and the like. Carrier compositions of creams can be based on water in combination with glycerol and one or more other components, e.g. glycerinemonostearate, PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be formulated using isopropyl alcohol and water, suitably in combination with other components such as, for example, glycerol, hydroxyethyl cellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 wt % of the compound provided herein. The topical formulations can be suitably packaged in tubes of, for example, 100 g which are optionally associated with instructions for the treatment of the select indication.

In some embodiments, the compositions provided herein contain from about 5 to about 50 mg of the active ingredient. One having ordinary skill in the art will appreciate that this embodies compositions containing about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 mg of the active ingredient.

In some embodiments, the compositions provided herein contain from about 50 to about 500 mg of the active ingredient. One having ordinary skill in the art will appreciate that this embodies compositions containing about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 350 to about 400, or about 450 to about 500 mg of the active ingredient.

In some embodiments, the compositions provided herein contain from about 500 to about 1000 mg of the active ingredient. One having ordinary skill in the art will appreciate that this embodies compositions containing about 500 to about 550, about 550 to about 600, about 600 to about 650, about 650 to about 700, about 700 to about 750, about 750 to about 800, about 800 to about 850, about 850 to about 900, about 900 to about 950, or about 950 to about 1000 mg of the active ingredient.

Similar dosages may be used of the compounds described herein in the methods and uses provided herein.

Methods of Use

The compounds provided herein can be used as antibacterial agents, in particular, antimycobacterial agents. In some embodiments, the bacterial infection is a Gram-positive organism that either uptakes mycolic acid, uses mycolic acid in a biosynthetic process, synthesizes mycolic acid, or requires mycolic acid as a nutritional supplement. For example, the compounds provided herein can used to treat a patient infected with amycobacterium. In some embodiments, the compounds provided herein can inhibit the growth of amycobacterium(e.g.,Mycobacterium tuberculosis). Accordingly, provided herein is a method for treating a mycobacterial infection in a mammal comprising administering a therapeutically effective amount of a compound provided herein.

Without being bound by any theory, the compounds provided herein are thought to function through selective inhibition or activation of biological processes that would normally process fatty acids, esters, alcohols. In some embodiments, the compounds may function as competitive or irreversible inhibitors based upon their inclusion of, for example, a strained four-membered ring moiety. By way of example, certain mycolic acids are found in the cell wall ofM. tuberculosis; in particular, α-mycolic acid, which incorporates two cyclopropane rings. The presence of cyclopropanes are thought to contribute to the structural integrity of the cell wall and protect the vacillus from oxidative stress inside macrophages. Mycolic acids are assembled in mycobacteria from shorter-chain fatty acid feedstocks. The compounds provided herein can be taken up into the bacteria my mimicking these feed stocks and can, for example, disrupt mycolic acid biosynthesis through the presence of their strained four-membered ring moieties.

For example, each of the groups include the following bacteria as members:

In some embodiments, themycobacteriumis a member of MTBC. For example,M. tuberculosis, the major cause of human tuberculosis,M. bovis, M. bovisBCG,M. avium. In some embodiments, themycobacteriumisM. tuberculosis. In some embodiments, themycobacteriumisM. bovis. In some embodiments, themycobacteriumisM. aviumsubspecies paratuberculosis.

In some embodiments, one or more compounds provided herein are administered with another antibiotic useful for treating mycobacterial infections. These additional antibiotics may include: ethambutol, isoniazid, pyrazinamide, members of the rifamycin class (including, for example, rifampicin), aminoglycosides (for example, amikacin or kanamycin), polypeptides (for example, capreomycin, viomycin, or enviomycin), fluoroquinolones (for example ciprofloxacin levofloxacin, or moxifloxacin), ethionamide, prothionamide, cycloserine, any other antibiotic employed as part of a therapeutic treatment for mycocaterial infections, and any combination of the antibiotics listed above. The additional antibiotic can be administered before, after, or simultaneously with a compound as provided herein.

Certain of the compounds provided herein are can be used as monomers to produce amphiphilic polymers. Such reactions can prepare a variety of polymers such as those shown in Scheme 1.

Compounds of Formula (IV) can be used to prepare polymers using any number of known polymerization reactions. A compound of Formula (IV) includes:

or a pharmaceutically acceptable salt thereof,
wherein:R3is absent or is an optionally substituted C1-C40alkyl;R4is H or an optionally substituted C1-C40alkyl; andZ is selected from the group consisting of: CORE, CO2R6, NHC(O)NR6R7, CONR6, OCOR6, OR6, SR6, NR6R7, and OPO3R6;R5is H or C1-C6alkyl;R6and R7are independently H or C1-C6alkyl;
wherein the hydrocarbon backbone formed by R4and R3—Z is amphiphilic.

In some embodiments, the compound of Formula (I) is not:

In some embodiments, the substituents can be selected from the group consisting of an alkyl, a halogen, a hydroxyl, an alkoxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a carbocyclyl, a heterocyclyl, an aralkyl, a heteroaralkyl, or an aromatic or heteroaromatic moiety.

In some embodiments, the hydrocarbon backbone formed by R3and R4is a fatty acid or a derivative thereof which contains a cyclobutene moiety. In some embodiments, R3and R4together with the cyclobutene moiety form a hydrocarbon chain with a reactive group at one terminus (e.g., carboxylic acid or a Z moiety as defined above). In some embodiments, the hydrocarbon chain ranges from 10 to 30 carbon atoms and may be substituted or unsubstituted. Non-limiting examples include myristoleic acid, pamitoleic acid, sapienic acid, oleic acid, linoleic acid, linolenic acid, arachadonic acid, elaidic acid, and vaccenic acid.

In some embodiments, Z is CO2R6.

As noted above, any known method of polymerization for alkenes may be used with the monomers described herein. For example, in the presence of selected transition metal catalysts, a monomer as provided herein can undergo metathesis with other alkenes. In the absence of a thermodynamic driving force, these exchange reactions typically produce mixtures of the starting and product alkenes. For the monomers provided herein, however, the metathesis reaction is driven forward by relief of strain to result in rapid and typically irreversible ring-opening metathesis polymerization (ROMP). As shown in Scheme 2 for a ROMP process with a generic cycloalkane.

With a monomer as provided herein, an example reaction is as shown in Scheme 3.

In some embodiments, one or more monomers as provided herein can be reacted with other alkenes, either monomeric alkenes or alkene-containing polymers, through cross metathesis (CM) wherein two alkenes can undergo transalkylidenation under release of ethane in the presence of a ruthenium carbenoid (e.g., Grubbs' Catalyst). In the absence of a thermodynamic driving force, these exchange reactions typically produce mixtures of the starting and product alkenes. For the cyclobutene-containing monomers provided herein, however, the cross metathesis reaction is driven forward by relief of strain. Two exemplary reactions with monomeric alkenes are shown in Scheme 4.

For example, a polymer can be prepared as follows. Under an inert atmosphere, a solution of catalyst (Grubbs' I or II) in THF was added to a vigorously stirred solution of 0.2 M monomer in THF. The reaction mixture is then stirred for about 30 min at room temperature after which the reaction is terminated by addition of a small amount of ethyl vinyl ether. The mixture is allowed to stir at room temperature for 30 min. and poured into methanol to precipitate the polymer. The polymer is then resolubilized in dry THF and the solutions dried with typical agents (magnesium sulfate, sodium sulfate) before further purification reprecipitation using dry methanol (2×). The polymer can be analyzed by gel permeation chromatography (GPC) in THF or by proton1H NMR as a solution in CDCl3.

In some embodiments, radical polymerization methods may be used to prepare a polymer as provided herein. Free radical polymerization is a method of polymerization by which a polymer forms by the successive addition of free radical building blocks. Free radicals can be formed via a number of different mechanisms usually involving separate initiator molecules. Following its generation, the initiating free radical adds (nonradical) monomer units, thereby growing the polymer chain. Any reasonable initiator may be used in these polymerization reactions. An example of such a reaction is shown in Scheme 5.

The cyclobutene compounds provided herein are also useful in bioorthogonal “click” chemistries. Bioorthogonal reactions for coupling materials in the presence of complex biological milieu are of great interest in biology and medicine. Such reactions have become key components in a variety of applications including protein engineering, immunoassay development, and cell surface modification. (Link J A et al., 2003, Curr Opin Biotechnol14:603-609; Wang Q et al., 2003,J Am Chem Soc12:3192-3193; Dimandis E P et al., 1991,Clin Chem37:625-636; Baskin J M et al., 2007, Proc Natl Acad Sc. USA104:16793-16797; Link J A et al., 2003,J Am Chem Soc125:11164-11165). Presently, a few types of bioorthogonal reactions have been reported, the most popular being the Staudinger ligation and the [3+2] cycloaddition “click” reaction between azides and alkynes. (Prescher J A et al., 2004,Nature430(7002):873-877; Rostovtsev V V et al., 2002,Angew Chem Int Ed41(14):2596-2599).

Bioorthogonal “click” chemistries are widely used in chemical biology for a myriad of applications such as activity based protein profiling, crosslinking of proteins, monitoring cell proliferation, generation of novel enzyme inhibitors, monitoring the synthesis of newly formed proteins, protein target identification, and studying glycan processing. Perhaps the most fascinating applications involve using these bioorthogonal chemistries to assemble molecules in the presence of living systems such as live cells or even whole organisms (Baskin et al., 2007, Proc Natl Acad Sci USA,104, 16793-7; Laughlin et al., 2008,Science,320, 664-7; Prescher and Bertozzi, 2005,Nat Chem Biol,1, 13-21; Neef and Schultz, 2009,Angew Chem Int Ed Engl,48, 1498-500; Ning et al., 2008,Angewandte Chemie-International Edition,47, 2253-2255). These latter applications require that the chemistry be non-toxic and possess kinetics that allow fast reaction to occur with micromolar concentrations of reagents in a time span of minutes to hours.

Bioconjugation methods using inverse electron demand Diels-Alder cycloadditions between tetrazines and highly strained dienophiles such as norbomene and trans-cyclooctene are known in the literature, however the tetrazine used has limited stability to aqueous media. (Blackman et al., 2008,J Am Chem Soc,130, 13518-9; Devaraj et al., 2009,Angew Chem Int Ed Engl,48, 7013-6; Devaraj et al., 2008,Bioconjug Chem,19, 2297-9; Pipkom et al., 2009,J Pept Sci,15, 235-41). To improve upon the stability of the tetrazine, a novel asymmetric tetrazine was employed that demonstrated superior stability in water and serum and can react with trans-cyclooctene at rates of approximately 103M−1sec−1at 37° C. (Devaraj et al., 2009, Angew Chem Int Ed Engl, 48, 7013-6). This extremely fast rate constant allows for the labeling of extracellular targets at low nanomolar concentrations of tetrazine labeling agent, concentrations that are sufficiently low to allow for real-time imaging of probe accummulation.

In some embodiments, the ligand, e.g., an antibody, small molecule or other biomolecule, can be physically attached to the dienophile. In some embodiments, the ligand carries a functional group such as an amine, alcohol, carboxylic acid or ester, or other group of atoms on the ligand that can undergo a chemical reaction allowing attachment to the dienophile. Alternatively or in addition, the dienophile or heterodienophile (e.g., a cyclobutene compound as provided herein) possesses a reactive functional group for attachment to the ligand (e.g., Z). Thus, the reactive functional group on the ligand and/or dienophile undergoes a chemical reaction to form a link between the two.

In some embodiments, the diene can be a substituted tetrazine or other heteroaromatic ring system with at least two nitrogens adjacent to each other and which is a highly reactive participant in the inverse electron demand Diels-Alder reaction. The diene is linked to the payload (which can be, e.g., a therapeutic agent, fluorescence dye, or other detectable agent). In these embodiments, the diene possesses a reactive group such as an amine, alcohol, carboxylic acid or ester, or other group that can undergo a chemical reaction with the reactive moiety on the payload to form a link between the two.

Dienes useful in the present disclosure include but are not limited to aromatic ring systems that contain two adjacent nitrogen atoms, for example, tetrazines, pyridazines, substituted or unsubstituted 1,2-diazines. Other 1,2-diazines can include 1,2-diazines annelated to a second n-electron-deficient aromatic ring such as pyrido[3,4-d]pyridazines, pyridazino[4,5-d]pyridazines, and 1,2,4-triazines. Pyridazines can also be fused with a five-membered heterocycle such as imidazo[4,5-d]pyridazines and 1,2,3-triazolo[4,5-d]pyridazines. In some preferred embodiments, the diene is an asymmetrical tetrazine as described herein, e.g., 3-(p-Benzylamino)-1,2,4,5-tetrazine:

Two non-limiting examples of such a “click” chemistry reaction is shown in Scheme 6.

Accordingly, provided herein is an amphiphilic polymer formed by polymerizing a compound as provided herein (e.g., a compound of Formula (IV) or an amphiphilic cycloalkene-containing fatty acid derivative), each having a hydrophobic end, a hydrophilic end, and a backbone there between comprising a cycloalkene, wherein the cycloalkene comprises at least four carbon atoms, with at least two of the four carbon atoms being part of the backbone of the amphiphilic cycloalkene-containing fatty acid derivatives. In some embodiments, the polymer comprises amphiphilic fatty acid derivatives cross-linked via an unsaturated hydrocarbon chain formed by cleavage of the cycloalkene. In some embodiments, the polymer is ordered in two or three dimensions.

Further provided herein is a polymer comprising a repeating unit having the structure
═(═C—CR1—CR2—C═)═,
where:R1is hydrophobic and comprises a substituted or unsubstituted alkyl group having at least one carbon atom or a substituted or unsubstituted alkenyl group having at least two carbon atoms, andR2is hydrophilic and comprises a substituted alkyl group having at least one carbon atom or a substituted alkenyl group having at least two carbon atoms.

In some embodiments, the substituted alkyl group or substituted alkenyl group of R2comprises a hydroxyl group, an ester group, an aldehyde group, a carboxyl group, a sulfhydryl group, an amine group, an amide group, a carbamide group, or a phosphate group.

The polymers provided herein may be used in a number of applications. For example, the polymers may undergo further manipulation such as cleavage, stabilization, and/or cross-linking. For example, the carbon-carbon double bonds introduced through certain polymerization reactions (e.g., ROMP polymerization) can be cleaved by oxone or a similar oxidant, thus providing a means of breaking the polymer back to monomeric components. In some cases, the carbon-carbon double bonds which provide the cross-linking element can be saturated (hydrogenated) to form highly stable alkane linkages, making the polymer much less susceptible to oxidative cleavage. In some embodiments, the carbon-carbon double bonds which provide the cross-linking element can be cross-linked by the same methods used for latex of butadiene rubbers, allowing the incorporation of a two-dimensional crosslink within a coating or layer.

The polymers provided herein can form two-dimensional or three-dimensional ordered polymers or coatings. For example, see Scheme 7.

Examples of applications of such polymers or coatings include:1) Polymerization of monolayer or multilayer films derived from Langmuir-Blodgett techniques (either directly on a Langmuir trough or after transfer of a monolayer or multi layers to a substrate by dipping).2) Polymerization of monolayer or multilayer films derived from polar interactions between the amphiphile and inorganic or organometallic colloids such as quantum dots.3) Polymerization within micelles, bilayers, liquid crystals, and liposomes. These aggregates may be composed solely of the amphiphilic cyclobutenes or else mixtures of the amphiphilic cyclobutenes with other amphiphilic components.4) Polymerization of monolayer or multilayer films of the amphiphilic cyclobutenes onto a chromatographic support such as silica to create a remarkably stable reverse-phase chromatographic layer capable of displaying any desired functional group that can be incorporated into the cyclobutene precursors. By way of comparison, commercial reverse phase chromatography columns are prepared by chemical bonding of C8 or C18 alkylsilanes to a silica surface via siloxane bonds. The resulting siloxanes are only stable within a fairly modest pH range. If a reverse-phase chromatography column was instead prepared using a cyclobutene-substituted monomer (for example, with a terminal siloxane grafted onto or in place of the carboxylic acid), treatment with a ROMP catalyst could provide a new chromatographic support maintaining the same fundamental features of existing supports yet possessing far greater hydrolytic stability.5) The ability to prepare cyclobutenes having a close structural analogy to natural fatty acids, combined with the facility of achieving cross-metathesis of the cyclobutenes with other alkenes, will enable the use of the cyclobutene fatty acid as a means of stabilizing or solubilizing proteins or chemical reagents within biological membranes. A targeted protein or agent would be functionalized with one or more alkene units (in the form of allyl ethers, allyl esters, or allyl amides) and then subjected with cross-metathesis with a cyclobutene fatty acid to create a protein “decorated” with fatty acids, fatty acid esters, phospholipids, or triglycerides.6) Polymerization or cross-metathesis of cyclobutene-containing fatty acids, or their conjugates with proteins, after absorption within the skin, hair, or nail, would result in a new class of remarkably durable cosmetic and therapeutic agents. The ability to polymerize the absorbed fatty acid under conditions that selectively target the cyclobutene would create strong and biomimetic coatings, with potential applications for cosmetics, skin protective or tanning agents, hair thickeners, and wound coatings.

Synthesis

The compounds provided herein, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

Preparation of the compounds provided herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, inProtecting Group Chemistry,1stEd., Oxford University Press, 2000;March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,5thEd., Wiley-Interscience Publication, 2001; and Peturssion, S. et al., “Protecting Groups in Carbohydrate Chemistry,” J Chem. Educ.,74(11), 1297 (1997).

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved Compound Specific Method Optimization” K. F. Blom, et al.,J. Combi. Chem.6(6), 874 (2004) and normal phase silica chromatography.

In some embodiments, the compounds provided herein can be prepared as described in the Examples provided herein and as illustrated in Scheme 8:

As shown above, the preparation of cyclobutene compounds as provided herein (e.g., 1) can occur using the following procedures. The benzyl ester of oleic acid undergoes cycloaddition with dichloroketene to afford a mixture of regioisomeric dichlorocyclobutanones, each predominantly as the cis stereoisomer. The dichlorocyclobutanones possessed limited stability and are directly reduced with sodium borohydride to furnish a mixture of regioisomeric 2,2-dichloro-1-cyclobutanols. Conversion to the corresponding methanesulfonate (mesylate) esters is followed by reaction with sodium in ammonia, resulting in simultaneous deprotection of the benzyl ester and fragmentation/reduction of the β-chloromethanesulfonate, generating cyclobutene 1 in quantities of up to several hundred milligrams. Hydrogenation of the compound can provide the corresponding cyclobutane (e.g., 2). A cyclobutanone (e.g., 3) and cyclobutanol (e.g., 4) analogs can be prepared through a variation of the above in which the initial dichlorocyclobutanone is dehalogenated, for example, with excess zinc in acetic acid. A mixture of regioisomeric monochlorocyclobutanones (e.g., 5) can be prepared by partial reduction of the dichlorocyclobutanone with 1.1 equiv of Zn or Zn(Cu) in acetic acid. For the preparation of cyclobutanone, cyclobutanol and monochlorocyclobutanones, the final step can include removal of the benzyl ester by Pd-mediated hydrogenolysis.

Compound having shorter chains (e.g., 8-11) can be prepared analogously from 9-decenoic acid, furnishing comparably functionalized substrates with lower log P. The trans-stereoisomers of cyclobutenes (e.g., 2) and monochlorocyclobutanones (e.g., 7) can be prepared using a similar route based upon elaidic acid (Scheme 9). In some cases, the cycloaddition step can be conducted only in the presence of activated Zn or Zn/Cu.

Accordingly, provided herein is a method comprising:reacting an unsaturated fatty acid or derivative thereof having a backbone comprising at least one carbon-carbon double bond with dihaloketene (e.g., dichloroketene or dibromoketene) to achieve a stereospecific cycloaddition across the at least one carbon-carbon double bond, thereby yielding a cycloketone-containing fatty acid derivative comprising a cycloketone along the backbone, wherein the cycloketone comprises at least four carbon atoms, with at least two of the at least four carbon atoms being part of the backbone of the cycloketone-containing fatty acid derivative;reducing the cycloketone-containing fatty acid derivative to yield an amphiphilic cycloalkene-containing fatty acid derivative having a hydrophobic end, a hydrophilic end, and a backbone therebetween comprising a cycloalkene, wherein the cycloalkene comprises at least four carbon atoms, with at least two of the four carbon atoms being part of the backbone of the amphiphilic cycloalkene-containing fatty acid derivative.

In some embodiments, the method further comprises polymerizing a multiplicity of the amphiphilic cycloalkene-containing fatty acid derivatives to yield a polymer comprising carbon-carbon double bonds. In some such embodiments, polymerization can include cross-linking the amphiphilic cycloalkene-containing fatty acid derivatives via the carbon-carbon double bond in the cycloalkene. In some embodiments, polymerizing the multiplicity of the amphiphilic cycloalkene-containing fatty acid derivatives comprises ring-opening metathesis reactions. In some embodiments, polymerizing the multiplicity of the amphiphilic cycloalkane-containing fatty acid derivatives comprises click-chemistry. For example, the amphiphilic cycloalkane-containing fatty acid derivative can be reacted with a substituted or unsubstituted tetrazine.

In some embodiments, the method further comprises hydrogenating the carbon-carbon double bonds of the polymer. In some embodiments, the method further comprises cleaving at least some of the carbon-carbon double bonds in the polymer via oxidation to yield monomeric products.

The backbones of the multiplicity of the amphiphilic cycloalkene-containing fatty acid derivatives can be aligned before polymerizing the multiplicity of the amphiphilic cycloalkene-containing fatty acid derivatives.

EXAMPLES

General Procedures

All reagents and solvents were used as purchased except for pyridine and CH2Cl2(distilled from CaH2and kept under N2) and THF (distilled from Na/benzophenone under N2). Thin-layer chromatography (TLC) was performed on 0.25 mm hard-layer silica G plates; developed plates were visualized with a hand-held UV lamp or by staining: 1% Ce(SO4)2and 10% (NH4)2MoO4in 10% aq. H2SO4(general stain, after heating); 1% aq. KMnO4(for unsaturated compounds); 3% vanillin in 3% H2SO4in EtOH (general stain, after heating). Unless otherwise noted, NMR (spectra were recorded at 400 MHz (1H) or 100 MHz (13C) in CDCl3; peaks are reported as: chemical shift (multiplicity, J couplings in Hz, number of protons); “app” and “br” refer to apparent and broad signals, respectively. IR spectra were recorded as neat films (ZnSe, ATR mode) with selected absorbances reported in wavenumbers (cm−1). Flash chromatography was performed on 32-60 μm silica gel. Preparative HPLC was performed on a 21×250 mm normal phase Si (8 micron) column at 10 mL/min of the indicated solvent unless otherwise noted. Analytical purity of compounds was checked using an analytical column (250 mm×4.6 mm; Microsorb) at 1 mL/min of 20% EtOAc/Hexane; detection was accomplished using a differential refractometer interfaced with a data module. All compounds tested for biological activity showed >97% purity by HPLC analysis except for 5 and 7. Melting points are uncorrected. Mass spectral analyses were carried out by HRFAB on 3-nitrobenzyl alcohol (3-NBA) or glycerol matrices at the Nebraska Center for Mass Spectrometry.

Example 1—Preparation of Compounds

Small quantities of individual isomers could be separated for analysis by HPLC using 10% EtOAc/Hex. The stereochemical assignments were confirmed by 2D-NMR experiments and by preparation of individual methanesulfonates from individual samples of alchols described in the previous step.

Example 2—Determination of Inhibitor Aqueous Stability and Solubility

Although cyclobutenes are known to undergo thermally induced electrocyclic ring-opening, assessment of the thermal stability of cyclobutene using differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) found no decomposition occurred at temperatures below 150° C. (FIG. 1).

One-dimensional (1D)1H-NMR spectroscopy was used to verify the chemical purity, aqueous stability, and concentration dependent micelle formation of eleven fatty acid analogs. Each compound was dissolved in deuterated dimethyl sulfoxide (DMSO-d6) to obtain a stock concentration of 20 mM. Four different concentrations were prepared from the stock solutions for NMR analysis: 1.00 mM, 750 μM, 500 μM, 100 μM. All NMR samples consisted of 600 μL of a deuterated 50 mM potassium phosphate buffer at pH 7.2 with 50 μM of 3 (trimethylsilyl) propionic-2,2,3,3-d4acid sodium salt (TMSP). Each 600 μL NMR sample contains 30 μL (5%) of DMSO-d6and was transferred to a 5 mm NMR tube for analysis.

A Bruker Avance DRX 500-MHz spectrometer equipped with a 5 mm triple-resonance cryoprobe (1H,13C,15N) with a z-axis gradient was utilized for all 1D1H NMR experiments. Acquisition of NMR spectra was automated using a BACS-120 sample changer and Icon NMR software. All spectra were acquired at 298.15 K with 16 dummy scans, 64 scans, 32K data points, a spectral width of 5482.46 Hz, and a relaxation delay of 1.5 s. The NMR spectra were processed and analyzed using ACD/1D NMR Manager (Advanced Chemistry Development). The resulting 1D1H NMR spectra were visually inspected for evidence of micelle formation (peak broadening), or chemical instability/impurities (additional peaks).

As exemplified inFIG. 2, the results demonstrate the analogs to be stable in aqueous buffer. The analogs based upon an octadecanoic acid (1, 2, 3, 4, 5, 6, and 7) scaffold aggregate at concentrations as low as 100 μM. The analogs based upon a decanoic acid backbone (8, 9, 10, and 11) do not form micelles or other aggregates at concentrations below 1 mM, the measured MICs (vida infra).

Example 3—Measurement of Nonspecific Cytotoxicity

RAW 264.7 macrophages were incubated with 0-100 μM of oleic acid or the C18 cyclobutene fatty acid 1, each delivered as bovine serum albumin complexes. After 24 h, cell viability was relative to untreated controls was assessed using an IN CTYOTOX-CVDE Crystal violet Dye Elution Kit.

As illustrated inFIG. 4, the cyclobutene analogue 1 showed little toxicity at 50 μM and only modest toxicity at 100 μM; most naturally occurring fatty acids, represented here by oleate, display some toxicity this cell line at concentrations ≥200 μM (Russell, D. G. et al.,Science2010, 328: 852-856; and McShane, H.,Trans. R. Soc. B2011, 366, 2782-2789). The low toxicity against a mammalian cell line supports the potential investigations into the modified fatty acids as potential antimicrobials.

Example 4—Bacterial Strains and Culture Conditions

Bacterial strains used in this study areM. smeg(mc2155) and two strains of M. tb (CDC 1551 and H37Rv). Bacterial cells were grown with shaking at 37° C. in complete Middlebrook 7H9 broth supplemented with 0.05% v/v Tween 80 to an OD600 of 0.6-1.2. For MIC determinations, cells were inoculated into a modified previously reported minimal medium (Chacon et al 2002 REF) with components and final concentrations as follows: 22 mM dibasic potassium phosphate, 16 mM monobasic potassium phosphate, 2.8×10-5 mM ferric chloride, 8.7×10-3 mM zinc sulfate, 8.4×10-4 mM cobalt(II) chloride, 1.0×10-2 mM manganese chloride, 6.8×10-2 mM calcium chloride, 2.4 mM magnesium sulfate, 5.0 mM ammonium chloride, 25 mM glycerol, and 0.02% v/v Tyloxapol. The use of minimal media was dictated by preliminary experiments indicating that standard complete Middlebrook 7H9 media components may interfere with the MIC assay for some of these lipid analog compounds.

Example 5—Druz Susceptibility Assays

For MIC testing, each fatty acid analog compound was suspended in 100% DMSO-d6at either 100 mM (M. smeg) or 100 mg/ml (M. smegand M. tb). An equivalent concentration DMSO-d6was tested separately. The concentration gradients, expressed in μg/ml (compound) and % DMSO-d6, were generated using a power-2 series and varied between experiments. MICs were determined by a 96-well microplate twofold dilution method. Bacteria were harvested, washed 2× with minimal media, and inoculated to an initial concentration of approximately 1.0×105 colony forming units (CFU) per well. The initial inoculum was plated to verify retrospectively the desired CFU/ml for each strain. Plates were incubated at 37° C. for 3-4 day (M. smeg) or 5-7 weeks (M. tb). MIC values were determined by the consistent results of three biological and three technical replicates. The MIC was defined by taking the mode of three independent cultures where the MIC did not differ by more than one doubling dilution, discarding any results that are two doubling dilutions away from the mode.

MICs were initially determined againstM. smeg(mc2155), a non-pathogenic mycobacteria used as a model for M. tb to analyze processes that are likely to be conserved in the genus, and against two M. tb strains (CDC1551 and H37Rv). DSC, a clinically used TB drug, was employed as a control since MIC determinations for this compound have been standardized numerous times under different conditions in our laboratories. The analogs were added to wells as solutions in DMSO-d6; the deuterated solvent was chosen for convenience to enable consistency with planned NMR-based metabolomics studies. Importantly, the DMSO-d6had no apparent effect on the growth ofM. smeg, and contamination was not evident in the compound stock control wells or the media control wells. For M. tb, the combined analysis of 12 replicates for each of three independent cultures indicated no significant inhibition by DMSO-d6for either M. tb strain. Results for the fatty acid analogs are shown in Table 2 andFIG. 3.M. smegwas in general more resistant than M. tb to these analogs and DCS. The best MIC values againstM. smegwere observed for 2, the C18-cyclobutanone (3), 8, and the C10-cyclobutanol 11, and were similar to the MIC for DCS (313 μM). Notably, four of the eleven molecules have MIC values <100 μM for both M. tb strains: these include the C18cyclobutane (2), the C10cyclobutene (8), the C10cyclobutane (9), and the C18cyclobutanol (4). These MIC values were lower than that measured (122 μM) for the second line antituberculosis drug D-cycloserine (DCS). Four other analogs yielded lower MICs compared with DCS in M. tb H37Rv; three others yielded higher MIC values than DCS.

The MIC results demonstrate promising levels of inhibition with a variety of four-membered ring carbocycles (cyclobutanol, cyclobutanone, cyclobutene, and cyclobutane). The measured MICs vary more than a hundred fold against M. tb H37Rv and more than thirty fold against strain CDC1551; a much smaller range is observed againstM. smeg. Moreover, the nature of the functionality clearly has an impact on activity. The different influences of functionality within the C18 and C10 series are also notable; for example, the short-chain cyclobutanone (10) and cyclobutanol (11) are less potent toward mycobacteria than the corresponding long-chain analogs (3 and 4), while the opposite trend is observed with the cyclobutenes (8 vs. 1 or 6) or cyclobutanes (9 vs. 2 or 7). The cis/trans configuration of the four-membered carbocycle (monochlorocyclobutanones 5 vs. 7) appears to have a substantial influence on MICs for M. tb. (H37Rv); however, no different is observed for cyclobutenes 1 and 6.

Most of our analogs (8 out of 11) show lower MIC values in M. tb thanM. smeg. The five to ten fold differences between MICs forM. smegand M. tb with some analogs (2, 4, 6, 8 and 9) may result from a mechanism of action specific to M. tb. Isoniazid, for example, demonstrates 100-fold more potent inhibition of M. tb compared withM. smegand possesses no toxicity toward other mycobacteria and prokaryotic pathogens. The influence of the analogs may be specific to a metabolic pathway only found in M. tb such as at the final elongation step. It is also possible that our four-membered ring analogs are converted into analogs of mycolic acids and that the physical properties of these unnatural cell wall constituents results in cell death.

Other Embodiments