WO2013152272A1

WO2013152272A1 - ΑΖΑ-β-LACTAM COMPOUNDS AND METHODS OF USING

Info

Publication number: WO2013152272A1
Application number: PCT/US2013/035402
Authority: WO
Inventors: Benjamin Cravatt; Andrea ZUHL; Dan Bachovchin; Megan MATTHEWS; Gregory C. Fu; Justin T. MOHR; Jacob M. BERLIN
Original assignee: Scripps Research Institute; Massachusetts Institute of Technology
Current assignee: Scripps Research Institute; Massachusetts Institute of Technology
Priority date: 2012-04-05
Filing date: 2013-04-05
Publication date: 2013-10-10
Anticipated expiration: 2014-10-05

Description

ΑΖΑ-β-LACTAM COMPOUNDS AND METHODS OF USING

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number MH084512 and CA132630, awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application Serial Number 61/686,441, filed April 5, 2012, the disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND

Serine hydrolases constitute about 1% of all proteins encoded by mammalian genomes and play important roles in a wide range of (patho)physiologic processes. Many serine hydrolases, however, remain unannotated with respect to their natural substrates and functions. Selective chemical inhibitors have served as valuable probes for the functional annotation of serine hydrolases and led to approved drugs for treating disorders such as obesity, diabetes, microbial infections, and Alzheimer's disease.

Efforts to develop inhibitors for serine hydrolases have uncovered specialized chemotypes. For example, structural features can be introduced into the inhibitors to tailor their selectivity for individual serine hydrolases. Despite advances in serine hydrolase inhibitor development much of the serine hydrolase class still lacks selective inhibitors that are suitable for pharmacological studies in living systems, and which are needed for assigning functions to these enzymes.

For example, the serine hydrolase ABHD10 (alpha beta hydrolase domain containing 10) is a 297 amino acid (aa) protein with a predicted MW of 33 kilodaltons (kDa). Proteomic studies have identified ABHD10 as a mitochondrial protein with a predicted leader sequence and proteolytic cleavage site at aa's 46-47. Inhibitors of ABHD10 have not yet been described, nor have any substrates or functions been discovered for the enzyme. These factors, combined with the very limited sequence homology (< 20%) that ABHD10 shares with other mammalian serine hydrolases, may indicate that the enzyme is an attractive target for chemical probe development. Development of such serine hydrolase modulators can also be challenging since such modulators typically lack the selectivity required for general use as in vivo pharmaceutically acceptable agents, for example, modulators that are selective with respect to serine hydrolase protein-phosphatase methylesterase- 1 (PME-1). Such serine hydrolase modulators may be useful for treating inflammation, metabolic disorders and the like.

SUMMARY

In various embodiments, the invention is directed to compounds and compositions which can be used as modulators of serine hydrolase ABHD10, and to methods of use employing aza- -lactam compounds as disclosed and claimed herein.

In various embodiments, the invention provides a method of modulating serine hydrolase ABHD10, comprising contacting the ABHD10 with an effective amount or concentration of compound of formula (I)

wherein

R¹ is a (C1-C6) alkyl group optionally substituted with halo;

Ar is an aryl or heteroaryl group, substituted with n independently selected R², wherein n = 0, 1, 2, or 3;

R² is (Cl-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C6)cycloalkyl, (Cl- C6)alkoxy, halo, cyano, nitro, R^aR^bN-, R^aR^bNS0₂, R^aR^bNC(=0), R^aS(0)_qNR^b, or R^aS(0)_q wherein q = 0, 1 or 2;

wherein any independently selected alkyl, alkenyl, alkynyl, cycloalkyl, or alkoxy group of R² is optionally mono- or independently multi-substituted with halo, cyano, or hydroxyl;

R^a and R^b are independently selected for each occurrence, from the group consisting of hydrogen and (Cl-C6)alkyl, optionally mono- or independently multi-substituted with halo, cyano, oxo or hydroxyl; or,

R^a and R^b, together with a nitrogen atom to which they are bonded form a 4-6 membered heterocyclic ring, optionally comprising an additional heteroatom selected from O, S(0)_q, or N; wherein the 4-6 membered heterocyclic ring is optionally substituted with one or more substituents selected from the group consisting of halo, cyano, oxo or hydroxyl;

R³ is alkyl or arylalkyl;

or a pharmaceutically acceptably salt thereof.

In various embodiments, the compound of formula (I) is the (R)-enantiomer at the carbon atom a to the azalactam carbonyl group.

In various embodiments, the modulation of ABHDIO can be selective with respect to modulation of other serine hydrolases. For example, the modulation of ABHDIO can be selective with respect to modulation of serine hydrolase PME-1.

In various embodiments, the invention provides a compound of formula (I)

wherein

R¹ is a (C1-C6) alkyl group optionally substituted with halo;

Ar is an aryl or heteroaryl group, substituted with n independently selected R², wherein n = 1, 2, or 3;

R³ is alkyl or arylalkyl;

including any stereoisomer thereof; or a pharmaceutically acceptably salt thereof;

provided that when R¹ is ethyl and R³ is methyl, then R² is not 2-methyl, 3 -methyl, 2- methoxy, 4-methoxy, or 4-chloro.

In various embodiments, the invention provides a pharmaceutically acceptable composition comprising a compound of the invention, or a compound useful for practice of a method of the invention, and a pharmaceutically acceptable excipient.

In various embodiments, the invention provides a use of a compound of the invention, or a compound useful for practice of a method of the invention for preparation of a medicament for treatment of a condition in a mammal for which modulation of serine hydrolase ABHDIO is indicated. For example, the condition can comprise pain,

inflammation, metabolic disorders, solid tumors, or obesity.

FIGURE

Figure 1 indicates competitive ABPP (activity based protein profiling) of disclosed compound ABL303 in situ. (A) Structures of ABL probes. (B and C) Gel-based ABPP of membrane (B) and soluble (C) proteomes from Neuro-2A cells treated with ABL303 (10 - 0.001 μΜ, 2 h), afforded an (D) IC₅₀ value of 21 nM for inhibition of ABHDIO and no significant inhibition of PME-1. Data are presented as mean values + SEM; n = 3/group. (E) ABPP-SILAC analysis of Neuro-2A cells treated with ABL303 (light) or ABL127 (dark) (100 nM, 2 h; heavy samples) versus DMSO (light samples) revealed selective inhibition of ABHDIO and PME-1, respectively. Data are reported as mean values + SEM of all peptides quantified for each serine hydrolase.

DETAILED DESCRIPTION

As used in the specification and the appended claims, the singular forms "a," "an" and

"the" include plural referents unless the context clearly dictates otherwise.

The term "about" as used herein, when referring to a numerical value or range, allows for a degree of variability in the value or range, for example, within 10%, or within 5% of a stated value or of a stated limit of a range.

All percent compositions are given as weight-percentages, unless otherwise stated.

As used herein, "individual" (as in the subject of the treatment) or "patient" can mean both mammals and non-mammals, but mammals, including humans, are always included. Mammals include, for example, humans; non-human primates, e.g. apes and monkeys; and non-primates, e.g. dogs, cats, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

The term "disease" or "disorder" or "malcondition" are used interchangeably, and are used to refer to diseases or conditions wherein a serine hydrolase such as ABHDIO plays a role in the biochemical mechanisms involved in the disease or malcondition or symptom(s) thereof such that a therapeutically beneficial effect can be achieved by acting on serine hydrolase such as ABHDIO. "Acting on" serine hydrolase such as ABHDIO, or

"modulating" serine hydrolase such as ABHDIO, can include binding to serine hydrolase such as ABHDIO and/or inhibiting the bioactivity of serine hydrolase such as ABHDIO and/or allosterically regulating the bioactivity of serine hydrolase such as ABHDIO in vivo.

The expression "effective amount", when used to describe therapy to an individual suffering from a disorder, refers to the amount of a compound of the invention that is effective to inhibit or otherwise act on serine hydrolase such as ABHDIO in the individual's tissues wherein serine hydrolase such as ABHDIO involved in the disorder is active, wherein such inhibition or other action occurs to an extent sufficient to produce a beneficial therapeutic effect.

"Substantially" as the term is used herein means completely or almost completely; for example, a composition that is "substantially free" of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is

"substantially pure" is there are only negligible traces of impurities present.

"Treating" or "treatment" within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder, or curing the disease or disorder. Similarly, as used herein, an "effective amount" or a

"therapeutically effective amount" of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition. In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects. Phrases such as "under conditions suitable to provide" or "under conditions sufficient to yield" or the like, in the context of methods of synthesis, as used herein refers to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within ordinary skill for an experimenter to vary, that provide a useful quantity or yield of a reaction product. It is not necessary that the desired reaction product be the only reaction product or that the starting materials be entirely consumed, provided the desired reaction product can be isolated or otherwise further used.

By "chemically feasible" is meant a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim. The structures disclosed herein, in all of their embodiments are intended to include only

"chemically feasible" structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed or claimed herein.

An "analog" of a chemical structure, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure. A related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative." When a substituent is specified to be an atom or atoms of specified identity, "or a bond", a configuration is referred to when the substituent is "a bond" that the groups that are immediately adjacent to the specified substituent are directly connected to each other in a chemically feasible bonding configuration.

All chiral, diastereomeric, racemic forms of a structure are intended, unless a particular stereochemistry or isomeric form is specifically indicated. In several instances though an individual stereoisomer is described among specifically claimed compounds, the stereochemical designation does not imply that alternate isomeric forms are less preferred, undesired, or not claimed. Compounds used in the present invention can include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions, at any degree of enrichment. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention.

As used herein, the terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.

When a group, e.g., an "alkyl" group, is referred to without any limitation on the number of atoms in the group, it is understood that the claim is definite and limited with respect the size of the alkyl group, both by definition; i.e., the size (the number of carbon atoms) possessed by a group such as an alkyl group is a finite number, less than the total number of carbon atoms in the universe and bounded by the understanding of the person of ordinary skill as to the size of the group as being reasonable for a molecular entity; and by functionality, i.e. , the size of the group such as the alkyl group is bounded by the functional properties the group bestows on a molecule containing the group such as solubility in aqueous or organic liquid media. Therefore, a claim reciting an "alkyl" or other chemical group or moiety is definite and bounded, as the number of atoms in the group cannot be infinite.

When a group is recited, wherein the group can be present in more than a single orientation within a structure resulting in more than single molecular structure, e.g., a carboxamide group C(=0)NR, it is understood that the group can be present in any possible orientation, e.g., X-C(=0)N(R)-Y or X-N(R)C(=0)-Y, unless the context clearly limits the orientation of the group within the molecular structure.

The inclusion of an isotopic form of one or more atoms in a molecule that is different from the naturally occurring isotopic distribution of the atom in nature is referred to as an "isotopically labeled form" of the molecule. All isotopic forms of atoms are included as options in the composition of any molecule, unless a specific isotopic form of an atom is indicated. For example, any hydrogen atom or set thereof in a molecule can be any of the isotopic forms of hydrogen, i.e., protium (^lH), deuterium (²H), or tritium (³H) in any combination. Similarly, any carbon atom or set thereof in a molecule can be any of the isotopic form of carbons, such as ⁿC, ¹²C, ¹³C, or ¹⁴C, or any nitrogen atom or set thereof in a molecule can be any of the isotopic forms of nitrogen, such as ¹³N, ¹⁴N, or ¹⁵N. A molecule can include any combination of isotopic forms in the component atoms making up the molecule, the isotopic form of every atom forming the molecule being independently selected. In a multi-molecular sample of a compound, not every individual molecule necessarily has the same isotopic composition. For example, a sample of a compound can include molecules containing various different isotopic compositions, such as in a tritium or ¹⁴C radiolabeled sample where only some fraction of the set of molecules making up the macroscopic sample contains a radioactive atom. It is also understood that many elements that are not artificially isotopically enriched themselves are mixtures of naturally occurring isotopic forms, such as 14 N and 15 N, 32 S and 34 S, and so forth. A molecule as recited herein is defined as including isotopic forms of all its constituent elements at each position in the molecule. As is well known in the art, isotopically labeled compounds can be prepared by the usual methods of chemical synthesis, except substituting an isotopically labeled precursor molecule. The isotopes, radiolabeled or stable, can be obtained by any method known in the art, such as generation by neutron absorption of a precursor nuclide in a nuclear reactor, by cyclotron reactions, or by isotopic separation such as by mass spectrometry. The isotopic forms are incorporated into precursors as required for use in any particular synthetic route. For example, 14 C and 3 H can be prepared using neutrons generated in a nuclear reactor.

Following nuclear transformation, 14 C and 3 H are incorporated into precursor molecules, followed by further elaboration as needed.

The term "amino protecting group" or "N-protected" as used herein refers to those groups intended to protect an amino group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used amino protecting groups are disclosed in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999). Amino protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2- chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, a- chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; alkoxy- or aryloxy- carbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3 ,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,

4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,

3,4,5-trimethoxybenzyloxycarbonyl, l-(p-biphenylyl)-l-methylethoxycarbonyl,

a,a-dimethyl-3 ,5 -dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,

methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2- trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl- 9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl,

cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Amine protecting groups also include cyclic amino protecting groups such as phthaloyl and dithiosuccinimidyl, which incorporate the amino nitrogen into a heterocycle. Typically, amino protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, Alloc, Teoc, benzyl, Fmoc, Boc and Cbz. It is well within the skill of the ordinary artisan to select and use the appropriate amino protecting group for the synthetic task at hand.

The term "hydroxyl protecting group" or "O-protected" as used herein refers to those groups intended to protect an OH group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used hydroxyl protecting groups are disclosed in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999). Hydroxyl protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t- butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,

o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; acyloxy groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4- dimethoxybenzyloxycarbonyl, 3 ,5 -dimethoxybenzyloxycarbonyl, 2,4- dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5- dimethoxybenzyloxycarbonyl, 3 ,4,5 -trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)- 1 - methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl,

benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2- trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4- nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. It is well within the skill of the ordinary artisan to select and use the appropriate hydroxyl protecting group for the synthetic task at hand.

In general, "substituted" refers to an organic group as defined herein in which one or more bonds to a hydrogen atom contained therein are replaced by one or more bonds to a non-hydrogen atom such as, but not limited to, a halogen (i.e., F, CI, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents J that can be bonded to a substituted carbon (or other) atom include F, CI, Br, I, OR', OC(0)N(R')₂, CN, NO, N0₂, ON0₂, azido, CF₃, OCF₃, R, O (oxo), S (thiono), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR, S0₂R, S0₂N(R)₂, S0₃R, C(0)R, C(0)C(0)R, C(0)CH₂C(0)R, C(S)R, C(0)OR, OC(0)R, C(0)N(R)₂,

OC(0)N(R)₂, C(S)N(R)₂, (CH₂)₀-₂N(R)C(O)R, (CH₂)₀-₂N(R)N(R)₂, N(R)N(R)C(0)R, N(R)N(R)C(0)OR, N(R)N(R)CON(R)₂, N(R)S0₂R, N(R)S0₂N(R)₂, N(R)C(0)OR, N(R)C(0)R, N(R)C(S)R, N(R')C(0)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(=NH)N(R)₂, C(0)N(OR)R, or C(=NOR)R wherein R' can be hydrogen or a carbon- based moiety, and wherein the carbon-based moiety can itself be further substituted; for example, wherein R' can be hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl or R' can be independently mono- or multi- substituted with J; or wherein two R' groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl, which can be mono- or independently multi- sub stituted with J .

In various embodiments, J can be halo, nitro, cyano, OR, NR₂, or R, or is C(0)OR, C(0)NR₂, OC(0)OR, OC(0)NR₂, N(R)C(0)OR, N(R)C(0)NR₂ or thio/thiono analogs thereof. By "thio/thiono analogs thereof, with respect to a group containing an O, is meant that any or all O atoms in the group can be replaced by an S atom; e.g., for group C(0)OR, a "thio/thiono analog thereof includes C(S)OR, C(0)SR, and C(S)SR; e.g., for group

OC(0)NR₂, a "thio/thiono analog thereof includes SC(0)NR₂, OC(S)NR₂, and SC(S)NR₂; and so forth.

In various embodiments, J is any of halo, (Cl-C6)alkyl, (Cl-C6)alkoxy, (Cl- C6)haloalkyl, hydroxy(Cl-C6)alkyl, alkoxy(Cl-C6)alkyl, (Cl-C6)alkanoyl, (Cl- C6)alkanoyloxy, cyano, nitro, azido, R₂N, R₂NC(0), R₂NC(0)0, R₂NC(0)NR, (Cl- C6)alkenyl, (Cl-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl, (C6- C10)aryl(Cl-C6)alkyl, (C6-C10)aryl(Cl-C6)alkoxy, (C6-C10)aryloxy(Cl-C6)alkyl, (C6- C10)aryloxy(Cl-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to 9- membered)heterocyclyl(Cl-C6)alkyl, (3- to 9-membered)heterocyclyl(Cl-C6)alkoxy, (5- to 10-membered)heteroaryl, (5- to 10-membered)heteroaryl(Cl-C6)alkyl, (5- to 10- membered)heteroaryl(Cl-C6)alkoxy, or (5- to 10-membered)heteroaroyl. For example, R independently at each occurrence can be H, (Cl-C6)alkyl, or (C6-C10)aryl, wherein any alkyl or aryl group is substituted with 0-3 J.

When a substituent is monovalent, such as, for example, F or CI, it is bonded to the atom it is substituting by a single bond. When a substituent is more than monovalent, such as

0, which is divalent, it can be bonded to the atom it is substituting by more than one bond,

1. e., a divalent substituent is bonded by a double bond; for example, a C substituted with O forms a carbonyl group, C=0, which can also be written as "CO", "C(O)", or "C(=0)", wherein the C and the O are double bonded. When a carbon atom is substituted with a double-bonded oxygen (=0) group, the oxygen substituent is termed an "oxo" group. When a divalent substituent such as NR is double-bonded to a carbon atom, the resulting C(=NR) group is termed an "imino" group. When a divalent substituent such as S is double-bonded to a carbon atom, the results C(=S) group is termed a "thiocarbonyl" or "thiono" group.

Alternatively, a divalent substituent such as O or S can be connected by two single bonds to two different carbon atoms. For example, O, a divalent substituent, can be bonded to each of two adjacent carbon atoms to provide an epoxide group, or the O can form a bridging ether group, termed an "oxy" group, between adjacent or non-adjacent carbon atoms, for example bridging the 1,4-carbons of a cyclohexyl group to form a [2.2.1]-oxabicyclo system. Further, any substituent can be bonded to a carbon or other atom by a linker, such as (CH₂)_n or (CR'₂)_n wherein n is 1, 2, 3, or more, and each R' is independently selected.

Another divalent substituent is an alkylidene carbon, represented as C= and signifying that the carbon atom so indicated, which also bears two additional groups, is double bonded to a third group. For example, (C1¾)₂C= indicates an isopropylidene group bonded to another carbon or nitrogen atom.

C(O) and S(0)₂ groups can also be bound to one or two heteroatoms, such as nitrogen or oxygen, rather than to a carbon atom. For example, when a C(O) group is bound to one carbon and one nitrogen atom, the resulting group is called an "amide" or "carboxamide." When a C(O) group is bound to two nitrogen atoms, the functional group is termed a "urea." When a C(O) is bonded to one oxygen and one nitrogen atom, the resulting group is termed a "carbamate" or "urethane." When a S(0)₂ group is bound to one carbon and one nitrogen atom, the resulting unit is termed a "sulfonamide." When a S(0)₂ group is bound to two nitrogen atoms, the resulting unit is termed a "sulfamide." Substituted alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups as well as other substituted groups also include groups in which one or more bonds to a hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a carbon atom, or to a heteroatom such as, but not limited to, oxygen in carbonyl (oxo), carboxyl, ester, amide, imide, urethane, and urea groups; and nitrogen in imines, hydroxyimines, oximes, hydrazones, amidines, guanidines, and nitriles.

Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and fused ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups can also be substituted with alkyl, alkenyl, and alkynyl groups as defined herein.

By a "ring system" as the term is used herein is meant a moiety comprising one, two, three or more rings, which can be substituted with non-ring groups or with other ring systems, or both, which can be fully saturated, partially unsaturated, fully unsaturated, or aromatic, and when the ring system includes more than a single ring, the rings can be fused, bridging, or spirocyclic.

By "spirocyclic" is meant the class of structures wherein two rings are fused at a single tetrahedral carbon atom, as is well known in the art.

As to any of the groups described herein, which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this disclosed subject matter include all stereochemical isomers arising from the substitution of these compounds.

Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed above, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. Exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as Ci_₆alkyl, Ci-₄alkyl, and Ci_₃alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-l -butyl, 3-methyl-2-butyl, 2-methyl-l-pentyl, 3-methyl-l-pentyl, 4- methyl-l-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l- butyl, 3, 3 -dimethyl- 1 -butyl, 2-ethyl- 1 -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.

Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term

"cycloalkenyl" alone or in combination denotes a cyclic alkenyl group.

The terms "carbocyclic," "carbocyclyl, " and "carbocycle" denote a ring structure wherein the atoms of the ring are carbon, such as a cycloalkyl group or an aryl group. In some embodiments, the carbocycle has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms is 4, 5, 6, or 7. Unless specifically indicated to the contrary, the carbocyclic ring can be substituted with as many as N-1 substituents wherein N is the size of the carbocyclic ring with, for example, alkyl, alkenyl, alkynyl, amino, aryl, hydroxy, cyano, carboxy, heteroaryl, heterocyclyl, nitro, thio, alkoxy, and halogen groups, or other groups as are listed above. A carbocyclyl ring can be a cycloalkyl ring, a cycloalkenyl ring, or an aryl ring. A carbocyclyl can be monocyclic or polycyclic, and if polycyclic each ring can be independently be a cycloalkyl ring, a cycloalkenyl ring, or an aryl ring.

(Cycloalkyl)alkyl groups, also denoted cycloalkylalkyl, are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkyl group as defined above.

Alkenyl groups include straight and branched chain and cyclic alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to about 20 carbon atoms, and typically from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=CH(CH₃), -CH=C(CH₃)₂, -C(CH₃)=CH₂, -C(CH₃)=CH(CH₃), -C(CH₂CH₃)=CH₂, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others. Exemplary alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as C₂_₆alkenyl, and C₃_₄alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.

Cycloalkenyl groups include cycloalkyl groups having at least one double bond between 2 carbons. Thus for example, cycloalkenyl groups include but are not limited to cyclohexenyl, cyclopentenyl, and cyclohexadienyl groups. Cycloalkenyl groups can have from 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like, provided they include at least one double bond within a ring. Cycloalkenyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above.

(Cycloalkenyl)alkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above.

Alkynyl groups include straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to about 20 carbon atoms, and typically from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to -C≡CH, -C≡C(CH₃), -

C≡C(CH₂CH₃), -CH₂C≡CH, -CH₂C≡C(CH₃), and -CH₂C≡C(CH₂CH₃) among others.

The term "heteroalkyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group.

Examples include: -0-CH₂-CH₂-CH₃, -CH₂-CH₂CH₂-OH, -CH₂-CH₂-NH-CH₃, -CH₂-S-CH₂-CH₃, -CH₂CH₂-S(=0)-CH₃, and -CH₂CH₂-0-CH₂CH₂-0-CH₃. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃, or

-CH₂-CH₂-S-S-CH₃.

A "cycloheteroalkyl" ring is a cycloalkyl ring containing at least one heteroatom. A cycloheteroalkyl ring can also be termed a "heterocyclyl, " described below.

The term "heteroalkenyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain monounsaturated or di-unsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Up to two heteroatoms may be placed consecutively. Examples

include -CH=CH-0-CH₃, -CH=CH-CH₂-OH, -CH₂-CH=N-OCH₃, -CH=CH-N(CH₃)-CH₃, -C H₂-CH=CH-CH₂-SH, and and -CH=CH-0-CH₂CH₂-0-CH₃.

Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined above. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6- substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl group are alkenyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.

Heterocyclyl groups or the term "heterocyclyl" includes aromatic and non-aromatic ring compounds containing 3 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Thus a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those comprising fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed above. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl,

dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed above. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed above.

Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5 -imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl, l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5 -pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5 -pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),

6- (2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1 -benzimidazolyl,

2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8 -benzimidazolyl), benzoxazolyl (1 -benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5 -benzothiazolyl, 6-benzothiazolyl,

7 - benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-l-yl, 5H-dibenz[b,f]azepine-2-yl,

5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,1 l-dihydro-5H-dibenz[b,f]azepine (10,1 l-dihydro-5H-dibenz[b,f]azepine-l-yl,

10,l l-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,l l-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,l l-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,l l-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group as defined above is replaced with a bond to a heterocyclyl group as defined above. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

Heteroarylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above.

The term "alkoxy" refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci_₆alkoxy, and C_2- ₆alkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, and the like.

An alkoxy group can include one to about 12-20 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structures are substituted therewith.

The term "cycloalkoxy" as used herein refers to a cycloalkyl group attached to oxygen (cycloalkyl-O-). Examples of cyclic alkoxy include but are not limited to

cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Exemplary cycloalkoxy groups include, but are not limited to, cycloalkoxy groups of 3-6 carbon atoms, referred to herein as C3_₆cycloalkoxy groups. Exemplary cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclohexyloxy, and the like. The terms "halo" or "halogen" or "halide" by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.

A "haloalkyl" group includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1 ,2-dichloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.

A "haloalkoxy" group includes mono-halo alkoxy groups, poly-halo alkoxy groups wherein all halo atoms can be the same or different, and per-halo alkoxy groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkoxy include trifluoromethoxy, 1,1-dichloroethoxy, 1 ,2-dichloroethoxy, l,3-dibromo-3,3- difluoropropoxy, perfluorobutoxy, and the like.

The term "(C_x-C_y)perfluoroalkyl," wherein x < y, means an alkyl group with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein all hydrogen atoms are replaced by fluorine atoms. Preferred is -(Ci-C6)perfluoroalkyl, more preferred is -(Ci-C3)perfluoroalkyl, most preferred is -CF₃.

The term "(C_x-C_y)perfluoroalkylene," wherein x < y, means an alkyl group with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein all hydrogen atoms are replaced by fluorine atoms. Preferred is -(Ci-C6)perfluoroalkylene, more preferred is -(Ci-C₃)perfluoroalkylene, most preferred is -CF₂-.

The terms "aryloxy" and "arylalkoxy" refer to, respectively, an aryl group bonded to an oxygen atom and an aralkyl group bonded to the oxygen atom at the alkyl moiety.

Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy.

An "acyl" group as the term is used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a "formyl" group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-20 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3-carbonyl) group is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group.

The term "amine" includes primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH₂, for example, alkylamines, arylamines, alkylarylamines; R₂NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R₃N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

An "amino" group is a substituent of the form -NH₂, -NHR, -NR₂, -NR₃ ^"1", wherein each R is independently selected, and protonated forms of each, except for -NR₃ ^"1", which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary or quaternary amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group.

An "ammonium" ion includes the unsubstituted ammonium ion NH₄ ^"1", but unless otherwise specified, it also includes any protonated or quaternarized forms of amines. Thus, trimethylammonium hydrochloride and tetramethylammonium chloride are both ammonium ions, and amines, within the meaning herein.

The term "amide" (or "amido") includes C- and N-amide groups, i.e., -C(0)NR₂, and

-NRC(0)R groups, respectively. Amide groups therefore include but are not limited to primary carboxamide groups (-C(0)NH₂) and formamide groups (-NHC(O)H). A

"carboxamido" group is a group of the formula C(0)NR₂, wherein R can be H, alkyl, aryl, etc.

The term "azido" refers to an N3 group. An "azide" can be an organic azide or can be a salt of the azide (N₃ ^~) anion. The term "nitro" refers to an N0₂ group bonded to an organic moiety. The term "nitroso" refers to an NO group bonded to an organic moiety. The term nitrate refers to an ON0₂ group bonded to an organic moiety or to a salt of the nitrate (NO₃ ) anion.

The term "urethane" ("carbamoyl" or "carbamyl") includes N- and O-urethane groups, i.e., -NRC(0)OR and -OC(0)NR₂ groups, respectively. The term "sulfonamide" (or "sulfonamido") includes S- and N-sulfonamide groups, i.e., -SO₂NR₂ and -NRSO₂R groups, respectively. Sulfonamide groups therefore include but are not limited to sulfamoyl groups (-SO₂NH₂). An organosulfur structure represented by the formula -S(0)(NR)- is understood to refer to a sulfoximine, wherein both the oxygen and the nitrogen atoms are bonded to the sulfur atom, which is also bonded to two carbon atoms.

The term "amidine" or "amidino" includes groups of the formula -C(NR)NR₂.

Typically, an amidino group is -C(NH)NH₂.

The term "guanidine" or "guanidino" includes groups of the formula -NRC(NR)NR₂. Typically, a guanidino group is -NHC(NH)NH₂.

Standard abbreviations for chemical groups such as are well known in the art are used; e.g., Me = methyl, Et = ethyl, i-Pr = isopropyl, Bu = butyl, t-Bu = tert-butyl, Ph = phenyl, Bn = benzyl, Ac = acetyl, Bz = benzoyl, and the like.

A "salt" as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion. For example, acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as NH₄ ^" or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium, or other cations such as trimethylsulfonium, and the like. A "pharmaceutically acceptable" or "pharmacologically acceptable" salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt. A "zwitterion" is an internal salt such as can be formed in a molecule that has at least two ionizable groups, one forming an anion and the other a cation, which serve to balance each other. For example, amino acids such as glycine can exist in a zwitterionic form. A

"zwitterion" is a salt within the meaning herein. The compounds of the present invention may take the form of salts. The term "salts" embraces addition salts of free acids or free bases which are compounds of the invention.

Salts can be "pharmaceutically-acceptable salts. " The term

"pharmaceutically-acceptable salts" refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds of the invention. "Pharmaceutically or

pharmacologically acceptable" include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.

Suitable pharmaceutically- acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,

benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

A "hydrate" is a compound that exists in a composition with water molecules. The composition can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a "hydrate" refers to a solid form, i.e., a compound in water solution, while it may be hydrated, is not a hydrate as the term is used herein.

A "solvate" is a similar composition except that a solvent other that water replaces the water. For example, methanol or ethanol can form an "alcoholate", which can again be stoichiometric or non-stoichiometric. As the term is used herein a "solvate" refers to a solid form, i.e., a compound in solution in a solvent, while it may be solvated, is not a solvate as the term is used herein.

A "prodrug" as is well known in the art is a substance that can be administered to a patient where the substance is converted in vivo by the action of biochemicals within the patients body, such as enzymes, to the active pharmaceutical ingredient. Examples of prodrugs include esters of carboxylic acid groups, which can be hydrolyzed by endogenous esterases as are found in the bloodstream of humans and other mammals. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). For example, if a compound of the invention or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (Ci_8)alkyl, (C2-i2)alkylcarbonyloxymethyl, l-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1 -methyl- l-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1- (alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1 -methyl- 1- (alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, l-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(Ci_ 2)alkylamino(C2-3)alkyl (such as β-dimethylaminoethyl), carbamoyl-(Ci_2)alkyl, N,N-di(Ci_ 2)alkylcarbamoyl-(Ci-2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-3)alkyl.

Similarly, if a compound of the invention, or a compound useful in practice of a method of the invention, contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (Ci_

6)alkylcarbonyloxymethyl, l-((Ci_6)alkylcarbonyloxy)ethyl, 1 -methyl- l-((Ci_

6)alkylcarbonyloxy)ethyl (Ci_6)alkoxycarbonyloxymethyl, N-(Ci_

6)alkoxycarbonylaminomethyl, succinoyl, (Ci_6)alkylcarbonyl, a-amino(Ci_4)alkylcarbonyl, arylalkylcarbonyl and a-aminoalkylcarbonyl, or a-aminoalkylcarbonyl-a-aminoalkylcarbonyl, where each a-aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(0)(OH)2, -P(0)(0(Ci_6)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).

Alternatively or additionally, if a compound of the invention, or a compound useful in practice of a method of the invention, incorporates an amine functional group, a prodrug can be formed, for example, by creation of an amide or carbamate, an N-alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine. In addition, a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can metabolically cleaved to generate a bioactive primary or secondary amine. For examples, see Simplicio, et al, Molecules 2008, 13, 519 and references therein.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Moreover, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any combination of individual members or subgroups of members of Markush groups. Thus, for example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, and Y is described as selected from the group consisting of methyl, ethyl, and propyl, claims for X being bromine and Y being methyl are fully described.

If a value of a variable that is necessarily an integer, e.g., the number of carbon atoms in an alkyl group or the number of substituents on a ring, is described as a range, e.g., 0-4, what is meant is that the value can be any integer between 0 and 4 inclusive, i.e., 0, 1, 2, 3, or 4.

In various embodiments, the compound or set of compounds, such as are used in the inventive methods, can be any one of any of the combinations and/or sub-combinations of the above-listed embodiments.

In various embodiments, a compound as shown in any of the Examples, or among the exemplary compounds, is provided. Provisos may apply to any of the disclosed categories or embodiments wherein any one or more of the other above disclosed embodiments or species may be excluded from such categories or embodiments.

The present invention further embraces isolated compounds of the invention. The expression "isolated compound" refers to a preparation of a compound of the invention, or a mixture of compounds the invention, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds. "Isolated" does not mean that the preparation is technically pure

(homogeneous), but it is sufficiently pure to compound in a form in which it can be used therapeutically. Preferably an "isolated compound" refers to a preparation of a compound of the invention or a mixture of compounds of the invention, which contains the named compound or mixture of compounds of the invention in an amount of at least 10 percent by weight of the total weight. Preferably the preparation contains the named compound or mixture of compounds in an amount of at least 50 percent by weight of the total weight; more preferably at least 80 percent by weight of the total weight; and most preferably at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation.

The compounds of the invention and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystallization or chromatography, including flash column chromatography, or HPLC.

Isomerism and Tautomerism in Compounds of the Invention

Tautomerism

Within the present invention it is to be understood that a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms. However, it is also to be understood that the invention encompasses any tautomeric form, and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been convenient to show graphically herein. For example, tautomerism may be exhibited by a pyrazolyl group bonded as indicated by the wavy line. While both substituents would be termed a 4-pyrazolyl group, it is evident that a different nitrogen atom bears the hydrogen atom in each structure.

Such tautomerism can also occur with substituted pyrazoles such as 3 -methyl, 5- methyl, or 3,5-dimethylpyrazoles, and the like. Another example of tautomerism is amido- imido (lactam-lactim when cyclic) tautomerism, such as is seen in heterocyclic compounds bearing a ring oxygen atom adjacent to a ring nitrogen atom. For example, the equilibrium:

is an example of tautomerism. Accordingly, a structure depicted herein as one tautomer is intended to also include the other tautomer.

Optical Isomerism

It will be understood that when compounds of the present invention contain one or more chiral centers, the compounds may exist in, and may be isolated as single and substantially pure enantiomeric or diastereomeric forms or as racemic mixtures. The present invention therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of the compounds of the invention.

The compounds of the invention, or compounds used in practicing methods of the invention, may contain one or more chiral centers and, therefore, exist as stereoisomers. The term "stereoisomers" when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols "(+)," "(-)," "R" or "S," depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated "(±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.

The compounds of the invention, or compounds used in practicing methods of the invention, may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. The symbol =. denotes a bond that may be a single, double or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration wherein the terms "Z" and "£"' are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the "£"' and "Z" isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as "cis" or "trans," where "cis" represents substituents on the same side of the double bond and "trans" represents substituents on opposite sides of the double bond.

Compounds of the invention, or compounds used in practicing methods of the invention, may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the "Z" or "E" configuration wherein the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both "Z" and "E" isomers. Substituents around a carbocyclic or heterocyclic rings may also be referred to as "cis" or "trans", where the term "cis" represents substituents on the same side of the plane of the ring and the term "trans" represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated "cis/trans."

Individual enantiomers and diastereomers of contemplated compounds can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective syntheses, a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art. Stereoselective syntheses encompass both enantio- and

diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley- VCH: Weinheim, 2009.

The isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called "enantiomers." Single enantiomers of a pure compound are optically active, i. e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system. The priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated as having an (R) absolute configuration, and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated as having an (5) absolute configuration. In the example in the Scheme below, the Cahn-Ingold-Prelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.

(R) configuration (5) configuration

A carbon atom bearing the A-D atoms as shown above is known as a "chiral" carbon atom, and the position of such a carbon atom in a molecule is termed a "chiral center."

The present invention is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof.

Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.

"Isolated optical isomer" or "isolated enantiomer" means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula.

Preferably, the isolated isomer is at least about 80%, more preferably at least 90%

enantiomerically pure, even more preferably at least 98% enantiomerically pure, most preferably at least about 99% enantiomerically pure, by weight. By "enantiomeric purity" is meant the percent of the predominant enantiomer in an enantiomeric mixture of optical isomers of a compound. A pure single enantiomer has an enantiomeric purity of 100%.

Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the invention, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of

DAICEL^® CHIRALPAK^® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.

Another well-known method of obtaining separate and substantially pure optical isomers is classic resolution, whereby a chiral racemic compound containing an ionized functional group, such as a protonated amine or carboxylate group, forms diastereomeric salts with an oppositely ionized chiral nonracemic additive. The resultant diastereomeric salt forms can then be separated by standard physical means, such as differential solubility, and then the chiral nonracemic additive may be either removed or exchanged with an alternate counter ion by standard chemical means, or alternatively the diastereomeric salt form may retained as a salt to be used as a therapeutic agent or as a precursor to a therapeutic agent.

Another aspect of an embodiment of the invention provides compositions of the compounds of the invention, alone or in combination with another medicament. As set forth herein, compounds of the invention include stereoisomers, tautomers, solvates, prodrugs, pharmaceutically acceptable salts and mixtures thereof. Compositions containing a compound of the invention can be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy, 19th Ed., 1995, or later versions thereof, incorporated by reference herein. The compositions can appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.

Typical compositions include a compound of the invention and a pharmaceutically acceptable excipient which can be a carrier or a diluent. For example, the active compound will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which can be in the form of an ampoule, capsule, sachet, paper, or other container. When the active compound is mixed with a carrier, or when the carrier serves as a diluent, it can be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound. The active compound can be adsorbed on a granular solid carrier, for example contained in a sachet. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and

polyvinylpyrrolidone. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The formulations can be mixed with auxiliary agents which do not deleteriously react with the active compounds. Such additives can include wetting agents, emulsifying and suspending agents, salt for influencing osmotic pressure, buffers and/or coloring substances preserving agents, sweetening agents or flavoring agents. The compositions can also be sterilized if desired.

The route of administration can be any route which effectively transports the active compound of the invention to the appropriate or desired site of action, such as oral, nasal, pulmonary, buccal, subdermal, intradermal, transdermal or parenteral, e.g., rectal, depot, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment, the oral route being preferred.

If a solid carrier is used for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. If a liquid carrier is used, the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

Injectable dosage forms generally include aqueous suspensions or oil suspensions which can be prepared using a suitable dispersant or wetting agent and a suspending agent Injectable forms can be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer' s solution, or an isotonic aqueous saline solution. Alternatively, sterile oils can be employed as solvents or suspending agents. Preferably, the oil or fatty acid is non- volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the formulation can also be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations can optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The compounds can be formulated for parenteral administration by injection such as by bolus injection or continuous infusion. A unit dosage form for injection can be in ampoules or in multi-dose containers.

The formulations of the invention can be designed to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. Thus, the formulations can also be formulated for controlled release or for slow release.

Compositions contemplated by the present invention can include, for example, micelles or liposomes, or some other encapsulated form, or can be administered in an extended release form to provide a prolonged storage and/or delivery effect. Therefore, the formulations can be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections. Such implants can employ known inert materials such as silicones and biodegradable polymers, e.g., polylactide-polyglycolide. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). For nasal administration, the preparation can contain a compound of the invention, dissolved or suspended in a liquid carrier, preferably an aqueous carrier, for aerosol application. The carrier can contain additives such as solubilizing agents, e.g., propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens.

For parenteral application, particularly suitable are injectable solutions or

suspensions, preferably aqueous solutions with the active compound dissolved in

polyhydroxylated castor oil.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrier or binder or the like are particularly suitable for oral application. Preferable carriers for tablets, dragees, or capsules include lactose, corn starch, and/or potato starch. A syrup or elixir can be used in cases where a sweetened vehicle can be employed.

A typical tablet that can be prepared by conventional tableting techniques can contain: Core:

Active compound (as free compound or salt thereof) 250 mg

Colloidal silicon dioxide (Aerosil®) 1.5 mg

Cellulose, microcryst. (Avicel®) 70 mg

Modified cellulose gum (Ac-Di-Sol®) 7.5 mg

Magnesium stearate Ad.

Coating:

HPMC approx. 9 mg

*Mywacett 9-40 T approx. 0.9 mg *Acylated monoglyceride used as plasticizer for film coating.

A typical capsule for oral administration contains compounds of the invention (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture is passed through a 60 mesh sieve and packed into a No. 1 gelatin capsule. A typical injectable preparation is produced by aseptically placing 250 mg of compounds of the invention into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 mL of sterile physiological saline, to produce an injectable preparation.

The compounds of the invention can be administered to a mammal, especially a human in need of such treatment, prevention, elimination, alleviation or amelioration of a malcondition. Such mammals include also animals, both domestic animals, e.g. household pets, farm animals, and non-domestic animals such as wildlife.

The compounds of the invention are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from about 0.05 to about 5000 mg, preferably from about 1 to about 2000 mg, and more preferably between about 2 and about 2000 mg per day can be used. A typical dosage is about 10 mg to about 1000 mg per day. In choosing a regimen for patients it can frequently be necessary to begin with a higher dosage and when the condition is under control to reduce the dosage. The exact dosage will depend upon the activity of the compound, mode of administration, on the therapy desired, form in which administered, the subject to be treated and the body weight of the subject to be treated, and the preference and experience of the physician or veterinarian in charge.

Generally, the compounds of the invention are dispensed in unit dosage form including from about 0.05 mg to about 1000 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.

Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration include from about 125 μg to about 1250 mg, preferably from about 250 μg to about 500 mg, and more preferably from about 2.5 mg to about 250 mg, of the compounds admixed with a pharmaceutically acceptable carrier or diluent.

Dosage forms can be administered daily, or more than once a day, such as twice or thrice daily. Alternatively dosage forms can be administered less frequently than daily, such as every other day, or weekly, if found to be advisable by a prescribing physician.

Aza- -Lactam Compounds

In various embodiments, the invention is directed to compounds and compositions which cab be used as modulators of serine hydrolase ABHD10.

wherein

R¹ is a (C1-C6) alkyl group optionally substituted with halo;

R³ is alkyl or arylalkyl;

or a pharmaceutically acceptably salt thereof.

In various embodiments, the compound of formula (I) is the (R)-enantiomer at the carbon atom a to the azalactam carbonyl group. As shown in Table 1, below, the (R)- enantiomer can exhibit much more effective modulation of ABHDIO than does the (S)- enantiomer.

For example, the invention can provide a method of modulating serine hydrolase

ABHDIO with a compound of formula (I) wherein R¹ is ethyl, or wherein R² is methyl, trifluoromethyl, fluoro, methoxy, or trifluoromethoxy.

In various embodiments, R³ is isopropyl. More specifically, the compound of formula (I) can be any one of

or a pharmaceutically acceptable salt thereof.

For example, the invention provides a method of modulating serine hydrolase

ABHDIO wherein the ABHDIO is disposed in a living cell, and contacting comprises contacting the cell with the effective amount or concentration of the compound of formula (I), or a pharmaceutically acceptable salt thereof. More specifically, the ABHDIO can be disposed in the living tissue of a patient suffering from a condition wherein modulation of ABHDIO is indicated, and contacting comprises administering an effective dose of the compound of formula (I) to the patient. More specifically, the condition can comprise pain, inflammation, metabolic disorders, solid tumors, or obesity.

In various embodiments, the invention provides a method of modulating serine hydrolase ABHDIO wherein modulation of ABHDIO is selective with respect to modulation of serine hydrolase PME-1. This selectivity for modulation of serine hydrolase ABHDIO with respect to modulation of other serine hydrolases such as PME-1 can avoid unwanted side-effects in using the methods disclosed and claimed herein, administering an aza- -lactam compound to a patient afflicted with a condition for which modulation of ABHDIO is indicated, such as pain, inflammation, metabolic disorders, solid tumors, or obesity.

In various embodim nd of formula (I)

wherein

R¹ is a (C1-C6) alkyl group optionally substituted with halo;

Ar is an aryl or heteroaryl group, substituted with n independently selected R², wherein n = 1, 2, or 3; R² is (Cl-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C6)cycloalkyl, (Cl- C6)alkoxy, halo, cyano, nitro, R^aR^bN-, R^aR^bNS0₂, R^aR^bNC(=0), R^aS(0)_qNR^b, or R^aS(0)_q wherein q = 0, 1 or 2;

R³ is alkyl or arylalkyl;

including any stereoisomer thereof;

or a pharmaceutically acceptably salt thereof;

provided that when R¹ is ethyl and R³ is methyl, then R² is not 2-methyl, 3-methyl, 2- methoxy, 4-methoxy, or 4-chloro.

In various embodiments the invention provides a compound of formula (I) wherein the compound is the (R)-enantiomer thereof. As shown in Table 1, below, the (R)- enantiomer can exhibit much more effective modulation of ABHD10 than does the (S)- enantiomer.

For example, for a compound of the invention of formula (I), R¹ can be ethyl, and R² can be methyl, trifluoromethyl, fluoro, methoxy, or trifluoromethoxy. More specifically, R² can be 3-methyl, 3 -trifluoromethyl, 3-fluoro, 3-methoxy, 3-trifluoromethoxy, 4-methyl, 4- trifluoromethyl, 4-fluoro, 4-methoxy, or 4-trifluoromethoxy. For example, R³ can be isopropyl.

For example, the compound of formula (I) can be any one of

or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides a pharmaceutically acceptable composition comprising a compound of the invention, or a compound useful for practice of a method of the invention, and a pharmaceutically acceptable excipient. This disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) rectal, vaginal, or aerosol administration, although the most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used. For example, disclosed compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration.

Exemplary pharmaceutical compositions may be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which includes one or more of a disclosed compound, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease.

For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a disclosed compound or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation

compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.

Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non- irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.

Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Compositions and compounds disclosed herein may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Pharmaceutical compositions suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacterio stats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants

Also contemplated are enteral pharmaceutical formulations including a disclosed compound and an enteric material; and a pharmaceutically acceptable carrier or excipient thereof. Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, and that are predominantly soluble in intestinal fluids at specific pHs. The small intestine is the part of the gastrointestinal tract (gut) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum. The pH of the duodenum is about 5.5, the pH of the jejunum is about 6.5 and the pH of the distal ileum is about 7.5.

Accordingly, enteric materials are not soluble, for example, until a pH of about 5.0, of about 5.2, of about 5.4, of about 5.6, of about 5.8, of about 6.0, of about 6.2, of about 6.4, of about 6.6, of about 6.8, of about 7.0, of about 7.2, of about 7.4, of about 7.6, of about 7.8, of about 8.0, of about 8.2, of about 8.4, of about 8.6, of about 8.8, of about 9.0, of about 9.2, of about 9.4, of about 9.6, of about 9.8, or of about 10.0. Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose acetate butyrate, cellulose acetate propionate, copolymer of methylmethacrylic acid and methyl methacrylate, copolymer of methyl acrylate, methylmethacrylate and methacrylic acid, copolymer of methylvinyl ether and maleic anhydride (Gantrez ES series), ethyl methyacrylate-methylmethacrylate- chlorotrimethylammonium ethyl acrylate copolymer, natural resins such as zein, shellac and copal collophorium, and several commercially available enteric dispersion systems (e. g. , EudragitL30D55, Eudragit FS30D, Eudragit L100, Eudragit S100, Kollicoat EMM30D, Estacryl 30D, Coateric, and Aquateric). The solubility of each of the above materials is either known or is readily determinable in vitro. The foregoing is a list of possible materials, but one of skill in the art with the benefit of the disclosure would recognize that it is not comprehensive and that there are other enteric materials that would meet the objectives of the present disclosure

In various embodiments, the invention provides a use of a compound of the invention, or a compound useful for practice of a method of the invention for preparation of a medicament for treatment of a condition in a mammal for which modulation of serine hydrolase ABHD10 is indicated. For example, the condition can comprise pain,

inflammation, metabolic disorders, solid tumors, or obesity.

In various embodiments, the invention provides methods that comprise exposing said enzyme to a compound described herein. In some embodiments, the compound utilized by one or more of the foregoing methods is one of the generic, subgeneric, or specific compounds described herein, such as a compound of Formula I. The ability of compounds described herein to modulate or inhibit ABHD10 can be evaluated by procedures known in the art and/or described herein. Another aspect of this disclosure provides methods of treating a disease associated with expression or activity of ABHD10 in a patient. For example, provided herein are compounds that may be selective in inhibiting ABHD10 as compared to inhibition of other serine hydrolases e.g., PME-1 , e.g. 10, 100, 1000 or more fold inhibition of ABHD10 over PME-1.

Also contemplated herein are methods of treating and/or preventing in a patient in need thereof a disorder such as one or more of acute or chronic pain, obesity, metabolic disorders (such as syndrome X), vomiting or nausea, eating disorders such as anorexia and/or bulimia; dislipidaemia, neuropathy such as diabetic neuropathy, pellagric neuropathy, alcoholic neuropathy, Beriberi neuropathy, burning feet syndrome, neurodegenerative disorders such as multiple sclerosis, Parkinson's disease, Huntington's chorea, Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy, sleep disorders, cardiovascular diseases, hypertension, dyslipidemia, atherosclerosis, osteoporosis, osteoarthritis, emesis, epilepsy, mental disorders such as schizophrenia and depression, glaucoma, cachexia, insomnia, traumatic brain injury, spinal cord injury, seizures, excitotoxin exposure, ischemia, AIDS wasting syndrome, renal ischaemia, cancers (e.g., solid tumor cancers such as breast, lung, head and neck, ovarian, sarcoma, melanoma, and/or prostate cancer); cancers such as melanoma, metastatic tumors, kidney or bladder cancers, brain, gastrointestinal cancers (e.g., colon cancer), leukemia or blood cancers (e.g. myeloid, lymphoid or monocytic cancers), inflammatory disorders (e.g. bladder inflammation), including inflammatory pain, and/or psychological disorders including anxiety disorders (e.g., panic disorder, acute stress disorder, post-traumatic stress disorder, substance-induced anxiety disorders, obsessive-compulsive disorder, agoraphobia, specific phobia, social phobia. Contemplated methods include administering a pharmaceutically effective amount of a disclosed compound.

For example, provide herein is a method for treating chronic pain such as

inflammatory pain, visceral pain, post operative pain, pain related to migraine, osteoarthritis, or rheumatoid arthritis, back pain, lower back pain, joint pain, abdominal pain, chest pain, labor, musculoskeletal diseases, skin diseases, toothache, pyresis, burn, sunburn, snake bite, venomous snake bite, spider bite, insect sting, neurogenic bladder, interstitial cystitis, urinary tract infection, rhinitis, contact dermatitis/hypersensitivity, itch, eczema, pharyngitis, mucositis, enteritis, irritable bowel syndrome, cholecystitis, pancreatitis, postmastectomy pain syndrome, menstrual pain, endometriosis, pain, pain due to physical trauma, headache, sinus headache, tension headache, or arachnoiditis.

For example, contemplated herein are methods for treating neuropathic pain (e.g., neuropathic low back pain, complex regional pain syndrome, post trigeminal neuralgia, causalgia, toxic neuropathy, reflex sympathetic dystrophy, diabetic neuropathy, chronic neuropathy caused by chemotherapeutic agents) in a patient in need thereof, comprising administering a pharmaceutically effective amount of a disclosed compound.

In certain embodiments, a disclosed compound utilized by one or more of the foregoing methods is one of the generic, subgeneric, or specific compounds described herein, such as a compound of Formula (I). Disclosed compounds may be administered to patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician. For treating clinical conditions and diseases noted above, a contemplated compound disclosed herein may be administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Parenteral administration may include subcutaneous injections, intravenous or intramuscular injections or infusion techniques.

Examples

The compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated. The starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials. All commercially available chemicals were obtained from Aldrich, Alfa Aesare, Wako, Acros, Fisher, Fluka, Maybridge or the like and were used without further purification, except where noted. Dry solvents are obtained, for example, by passing these through activated alumina columns.

Nucleophilic catalyst PPY* was prepared by literature methods. See, for example, Cook AH, Jones OG J Chem Soc. 1941 :184-187; Fu GC Acc Chem Res. 2004; 37:542-547; Lee EC, McCauley KM, Fu GC Angew Chem, Int Ed. 2007; 46:977-979; Dai X, Nakai T, Romero JAC, Fu GC Angew Chem, Int Ed. 2007; 46:4367-4369; Lee EC, Hodous BL, Bergin E, Shih C, Fu GC. J Am Chem Soc. 2005; 127:11586-11587; Hodous BL, Fu GC. J Am Chem Soc. 2002; 124:1578-1579; Wilson JE, Fu GC. Angew Chem, Int Ed. 2004, 43:6358-6360. p-Tolylacetic acid (Alfa Aesar), iodoethane (Aldrich), w-butyllithium solution (2.5 M in hexane, Aldrich), thionyl chloride (Aldrich), anhydrous CH₂CI₂ (Aldrich), N,N-dimethylethylamine (Aldrich or Alfa Aesar), dimethylazodicarboxylate (Wako), diisopropylazodicarboxylate (Aldrich), and dibenzylazodicarboxylate (Alfa Aesar) were purchased and used as received. Anhydrous tetrahydrofuran was dried by passage through a column of activated alumina under an argon atmosphere or purchased (Aldrich) and used as received. Reagent grade solvents for extractions and workups were purchased from Aldrich or VWR and used as received. HPLC grade solvents were purchased from Aldrich.

Unless otherwise stated, reactions were performed in oven-dried glassware under a nitrogen atmosphere using dry, deoxygenated solvents. Thin-layer chromatography (TLC) was performed using E. Merck silica gel 60 F254 precoated plates (0.25 mm) purchased from Silicycle and visualized by UV fluorescence quenching. Zeochem ZeoPrep 60 Eco silica gel (40-63 μιη particle size) was used for flash chromatography. Automated silica gel chromatography was performed with a Biotage Isolera Four using SNAP cartridges with UV visualization at 210 and 230 nm. Analytical GC analyses were carried out with an Agilent

6890 GC using a J & W Scientific HP-5 column. Analytical HPLC analyses were carried out with an Agilent 1100 series system using Daicel CHIRALPAK® columns (internal diameter 4.6 mm, column length 250 mm, particle size 5 or 3 μ) with visualization at 210, 230, and 254 nm. Preparative HPLC separations were carried out with a Gilson PLC 2020 using Daicel CHIRALPAK® columns (internal diameter 20.0 mm, column length 250 mm, particle size 5 μ) with visualization at 210 and 230 nm.

lU NMR and ¹³C NMR data were collected with a Bruker Avance 400 spectrometer (at 400 and 100 MHz, respectively) or a Varian Inova 500 spectrometer (at 500 and 125 MHz, respectively) at ambient temperature using the solvent residual peak as an internal standard (CHCI₃ at 7.27 ppm and CDC1₃ at 77 ppm). ¹⁹F NMR data were collected with a Varian

Mercury 300 spectrometer (at 282 MHz) at ambient temperature using external CF₃CO₂H in CDCI₃ as a standard (-76.53 ppm). IR spectra were recorded on a Perkin-Elmer 2000 FT-IR spectrometer and are reported in frequency of absorption (cm⁴). Optical rotations were measured with a Jasco P-1010 polarimeter at 589 nm. High resolution mass spectra (HRMS) were recorded on an Agilent LC/MSD TOF mass spectrometer by electrospray ionization time of flight reflectron experiments

Chemical shifts are typically recorded in ppm relative to tetramethylsilane (TMS) with multiplicities given as s (singlet), bs (broad singlet), d (doublet), t (triplet), dt (doublet of triplets), q (quadruplet), qd (quadruplet of doublets), m (multiplet). Example A: Procedure for Preparation of Ketenes

SI-3 SI-4 Ethyl p-tolyl ketene (SI-4)

To a 0 °C solution of p-tolylacetic acid (SI-1, 10.00 g, 66.59 mmol, 1.0 equiv) in THF (100 mL) was added a soln of w-BuLi (59.67 mL, 2.5 M in hexanes, 149.16 mmol, 2.24 equiv) dropwise over 20 min. Some bubbling was observed, and a precipitate formed during the addition. When the base addition was complete, the resulting orange-brown slurry was stirred for 2 h. Neat iodoethane (6.42 mL, 79.91 mmol, 1.2 equiv) was added dropwise over 5 min. The mixture was warmed gradually to ambient temperature and stirred overnight. The reaction was quenched by addition of H₂0 (4 mL) and the volatiles were removed by rotary evaporation. The resulting paste was dissolved with Et₂0 (50 mL) and H₂0 (15 mL). The aq phase was brought to pH 1 by dropwise addition of coned HC1. The phases were separated and the aq phase was extracted with Et₂0 (3 x 20 mL). The organic phases were combined, washed with brine (1 x 7 mL), and dried over MgS0₄. After filtration and concentration the crude acid SI-2 was obtained as an off-white solid and used directly in the next step. ^lU NMR (500 MHz, CDC1₃) δ 7.20 (d, / = 8.0 Hz, 2H), 7.14 (d, / = 8.0 Hz, 2H), 3.42 (app. t, / = 7.7 Hz, 1H), 2.33 (s, 3H), 2.09 (ddq, / = 14.9, 7.4, 7.4 Hz, 1H), 1.79 (ddq, J = 14.9, 7.5, 7.5 Hz, 1H), 0.90 (app. t, / = 7.4 Hz, 3H)

The crude butanoic acid from above was dissolved in CH₂C1₂ (10 mL) and the flask was immersed in a room temperature water bath. Neat SOCl₂ (14.49 mL, 199.77 mmol, 3.0 equiv) was added dropwise via syringe over 10 min. Some gas and heat evolution was observed. The homogenous solution was stirred for 15 h. The mixture was concentrated to an oil by rotary evaporation. The residue was vacuum distilled through a short path distillation head to yield the acid chloride SI-3 as a colorless oil (10.42 g, 80% yield for 2 steps), bp 59-60 °C (400 mTorr), 80 °C oil bath temperature (lit. 118-120 °C (12 Torr)) 1H NMR (500 MHz, C₆D₆) δ 6.93 (d, / = 8.2 Hz, 2H), 6.88 (d, / = 8.4 Hz, 2H), 3.49 (app. t, J = 7.4 Hz, 1H), 2.03 (s, 3H), 1.89 (ddq, / = 14.7, 7.4, 7.4 Hz, 1H), 1.55 (ddq, / = 14.9, 7.5, 7.5 Hz, 1H), 0.60 (app. t, / = 7.4 Hz, 3H)

To a 0 °C soln of the distilled acid chloride (10.42 g, 53.00 mmol, 1.0 equiv) in THF (66 mL) was added N,N-dimethylethylamine (28.7 mL, 265.01 mmol, 5.0 equiv) over 10 min. A white precipitate formed immediately and the liquid phase became bright yellow. The mixture was stirred at 0 °C for 16 h, and then warmed to ambient temperature. The solids were removed by filtration under an atmosphere of dry N₂ using a flip-frit apparatus with a medium porosity sintered glass frit. The solids were washed with a small amount of dry Et₂0. The yellow-orange filtrate solution was concentrated by rotary evaporation at ambient temperature. The residue was immediately distilled through a short path distillation head to yield ethyl p-tolyl ketene as an orange liquid (bp 44 °C (235 mTorr), 75 °C oil bath temp). The distillate was immediately transferred to a nitrogen-atmosphere glovebox where the mass was measured (4.6313 g, 54.5% yield). The ketene SI-4 was divided into small vials that were sealed with Teflon- lined caps and tape to exclude air and moisture. The neat ketene was stored outside the glovebox in a -20 °C freezer and handled exclusively in the glovebox. ^XH NMR (500 MHz, CDC1₃) δ 7.13 (d, / = 8.2 Hz, 2H), 6.93 (d, / = 8.0 Hz, 2H), 2.42 (q, / = 7.4 Hz, 2H), 2.31 (s, 3H), 1.21 (t, / = 7.4 Hz, 3H)

SI-6 Following the general procedure, acid SI-5 (3.75 g, 25.00 mmol) was alkylated with iodomethane to yield crude acid SI-6, which was used directly in the next step without purification. ^lU NMR (400 MHz, CDC1₃) δ 10.87 (br s, 1H), 7.33-7.05 (comp. m, 4H), 3.87- 3.66 (m, 1H), 2.41 (s, 3H), 1.57 (d, / = 7.2 Hz, 3H);

1³C NMR (100 MHz, CDC1₃) δ 180.8, 130.7, 138.2, 128.5, 128.2, 128.1 , 124.5, 45.3, 21.3, 18.0;IR (neat film, NaCl) 3022, 2977, 2935, 1707, 1607, 1459, 1413, 1245, 1217, 939, 905,

Following the general procedure with crude acid SI-6, the acid chloride SI-7 was obtained as a colorless liquid after distillation (bp 44-45 °C at 115 mTorr) and used immediately in the next step. ^XH NMR (400 MHz, CDC1₃) δ 7.29 (app. t, / = 7.6 Hz, 1H), 7.17 (d, / = 7.6 Hz, 1H), 7.13-7.09 (comp. m, 2H), 4.11 (q, / = 7.1 Hz, 1H), 2.39 (s, 3H), 1.60 (d, / = 7.1 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 175.6, 138.8, 137.4, 128.9, 128.6, 124.9, 57.4, 21.4, 18.7;IR (neat film, NaCl) 2984, 2937, 2874, 1810, 1782, 1708, 1608, 1490, 1456, 1089, 1032, 928, 871, 789, 719 cnT¹

SI-8

Following the general procedure with acid chloride SI-7, ketene SI-8(1.49 g, 41% yield, 3 steps) was obtained as a yellow liquid after distillation (bp 24 °C at 180 mTorr).

XH NMR (400 MHz, CDC1₃) δ 7.25 (app. t, / = 8.1 Hz, 1H), 6.93 (d, / = 7.9 Hz, 1H), 6.89- 6.85 (comp. m, 2H), 2.37 (s, 3H), 2.03 (s, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 205.8, 138.5, 133.2, 128.8, 125.0, 124.3, 120.7, 33.6, 26.5, 21.5, 8.6;IR (neat film, NaCl) 3029, 2950, 2921, 2099, 1750, 1606, 1490, 1456, 1378, 1276,1256, 1191, 1145, 1094, 1072, 785, 693 cnT¹

Following the general procedure, acid SI-9 (5.00 g, 33.29 mmol) was alkylated with iodoethane to yield crude acid SI-10, which was used directly in the next step without purification. ^XH NMR (400 MHz, CDC1₃) δ 11.06 (br s, 1H), 7.34 (app. t, 7 = 7.5 Hz, 1H), 7.29-7.23 (comp. m, 2H), 7.20 (d, / = 7.5 Hz, 1H), 3.56 (app. t, / = 7.7 Hz, 1H), 2.46 (s, 3H), 2.31-2.17 (m, 1H), 2.00-1.87 (m, 1H), 1.04 (app. t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 180.0, 138.3, 138.1, 128.7, 128.4, 128.0, 125.0, 53.2, 26.7, 26.2, 21.2, 12.0;

IR (neat film, NaCl) 3029, 2966, 2928, 2876, 1705, 1607, 1460, 1413, 1284, 1213, 930, 782, 717 cm^"1

si-11

Following the general procedure with crude acid SI-10, acid chloride SI-11 (3.79 g,

58% yield, 2 steps) was obtained as a colorless liquid after distillation (bp 64-69 °C at 225 mTorr). ^XH NMR (400 MHz, CDC1₃) δ 7.28 (app. t, / = 7.9 Hz, 1H), 7.16 (d, / = 7.7 Hz, 1H), 7.11-7.07 (comp. m, 2H), 3.86 (app. t, 7 = 7.5 Hz, 1H), 2.38 (s, 3H), 2.22 (ddq, / = 14.2, 7.3, 7.3 Hz, 1H), 1.86 (ddq, / = 14.8, 7.5, 7.5 Hz, 1H), 0.95 (app. t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCI3) δ 175.0, 138.8, 135.8, 129.0, 129.0, 128.9, 125.4, 65.2, 26.6, 21.4,

11.8;IR (neat film, NaCl) 2970, 2935, 2877, 1800, 1706, 1608, 1490, 1457, 1114, 1045, 995, 971, 818, 785, 752, 711 cnT¹

SI-12

Following the general procedure, acid chloride SI-11 (3.70 g, 18.81 mmol), yielded ketene SI-12 (1.01 g, 33% yield) as a yellow liquid after distillation (bp 65-69 °C at 550 mTorr). *H NMR (400 MHz, CDC1₃) δ 7.23 (app. t, J = 8.0 Hz, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.89-6.85 (comp. m, 2H), 2.46 (q, / = 7.4 Hz, 2H), 2.36 (s, 3H), 1.26 (t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 205.5, 138.6, 132.6, 128.8, 125.0, 124.7, 121.1, 41.7, 21.5, 17.0, 12.9;IR (neat film, NaCl) 3031, 2969, 2932, 2877, 2094, 1603, 1582, 1491, 1458, 1259, 775, 692 cm^"1

Following the general procedure, acid SI-13 (5.00 g, 30.09 mmol) was alkylated with iodoethane to yield crude acid SI-14, which was used directly in the next step without purification. ^lU NMR (400 MHz, CDC1₃) δ 10.29 (br s, 1H), 7.27 (app. t, J = 7.7 Hz, 1H),

6.98-6.90 (comp. m, 2H), 6.85 (dd, / = 8.3, 2.5 Hz, 1H), 3.82 (s, 3H), 3.47 (app. t, / = 7.7 Hz, 1H), 2.13 (ddq, / = 13.9, 7.4, 7.4 Hz, 1H), 1.84 (ddq, / = 14.0, 7.9, 7.4 Hz, 1H), 0.95 (app. t, 7 = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 179.7, 159.6, 139.9, 129.5, 120.4, 113.7, 112.6, 55.0, 53.2, 26.2, 12.0; IR (neat film, NaCl) 3065, 2965, 2837, 1706, 1600, 1586, 1491, 1456, 1261, 1152, 1050, 934, 781 cm

SI-15

Following the general procedure with crude acid SI-14, acid chloride SI-15 (3.56 g, 56% yield, 2 steps) was obtained as a colorless liquid after distillation (bp 85-90 °C at 165 mTorr). ^lU NMR (400 MHz, CDC1₃) δ 7.31 (app. t, / = 8.1 Hz, 1H), 6.91-6.86 (comp. m, 2H), 6.83 (app. t, / = 2.1 Hz, 1H), 3.87 (app. t, / = 7.5 Hz, 1H), 3.83 (s, 3H), 2.22 (ddq, / = 14.1 , 7.3, 7.3 Hz, 1H), 1.86 (ddq, / = 13.9, 7.5, 7.5 Hz, 1H), 0.95 (app. t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 174.8, 160.0, 137.3, 130.0, 120.7, 114.2, 113.4, 65.1 , 55.2, 26.5, 11.7;IR (neat film, NaCl) 2969, 2938, 1798, 1707, 1601, 1586, 1493, 1456, 1437, 1262,

877, 817, 785, 757, 712 cm^"1

SI-16

Following the general procedure with acid chloride SI-15 (3.50 g (16.46 mmol), ketene SI-16 (1.23 g, 42% yield) was obtained as a yellow liquid after distillation (bp 90-

95 °C at 550 mTorr). ^lU NMR (400 MHz, CDC1₃) δ 7.24 (app. t, / = 8.0 Hz, 1H), 6.68-6.63 (comp. m, 2H), 6.60 (t, / = 2.0 Hz, 1H), 3.82 (s, 3H), 2.44 (q, / = 7.4 Hz, 2H), 1.25 (t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 204.9, 160.2, 134.4, 129.8, 116.6, 109.8, 109.2, 55.1 , 42.0, 16.9, 12.8;IR (neat film, NaCl) 2967, 2936, 2097, 1797, 1603, 1579, 1493, 1458, 1436, 1292, 1273, 1212, 1170, 1051, 858, 834, 769, 688 cm^"1

Following the general procedure, SI-17 (5.00 g, 32.44 mmol) was alkylated with iodoethane to yield the crude acid SI-18, which was used directly in the next step without purification. ^lU NMR (400 MHz, CDC1₃) δ 9.86 (br s, 1H), 7.32-7.24 (m, 1H), 7.12-7.04 (comp. m, 2H), 6.97 (app. ddt, / = 8.5, 2.6, 0.9 Hz, 1H), 3.47 (app. t, / = 7.7 Hz, 1H), 2.17- 2.04 (m, 1H), 1.89-1.75 (m, 1H), 0.92 (app. t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 178.9, 162.8 (d, /_C-F = 246.0 Hz), 140.9 (d, /_C-F = 7.3 Hz), 129.9 (d, /_C-F = 8.3 Hz), 123.8 (d, Jc-F = 2.9 Hz), 115.0 (d, /_C-F = 21.9 Hz), 114.2 (d, /_C-F = 21.1 Hz), 53.0 (d, /_C-F = 1.3 Hz), 26.3, 11.9; ¹⁹F NMR (282 MHz, CDC1₃) δ -115.8 (app. d, J =7.8 Hz); IR (neat film, NaCl)

, 1707, 1614, 1591, 1490, 1414, 1256, 1231, 1144, 942, 784 cm^"1

SI-19

Following the general procedure with crude SI-18, acid chloride SI-19 (5.2554 g, 81% yield, 2 steps) was obtained as a colorless liquid after distillation (bp 42-44 °C at 380 mTorr). ^lU NMR (400 MHz, CDC1₃) δ 7.41-7.33 (m, 1H), 7.12-6.99 (comp. m, 3H), 3.90 (app. t, / = 7.4 Hz, 1H), 2.30-2.16 (m, 1H), 1.95-1.81 (m, 1H), 0.95 (app. t, / = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 174.5, 163.0 (d, J_C-F = 247.1 Hz), 138.1 (d, J_C-F = 7.3 Hz), 130.5 (d, Jc-F = 8.2 Hz), 124.2 (d, /_C-F = 3.0 Hz), 115.4 (d, /_C-F = 22.1 Hz), 115.3 (d, /_C-F = 21.0 Hz), 64.7, 26.5, 11.6; ¹⁹F NMR (282 MHz, CDC1₃) δ -115.8 (app. q, J =8.7 Hz);

Cl) 2970, 1707, 1591, 1490, 1450, 1255, 1227, 943, 784 cm^"1;

SI-20

Following the general procedure with 5.2554 g (26.19 mmol) of acid chloride SI-19, ketene SI-20 (2.0222 g, 47% yield) was obtained as a yellow liquid after distillation (bp 30 °C at 440 mTorr). ^lU NMR (400 MHz, CDC1₃) δ 7.28 (app. q, / = 7.8 Hz, 1H), 6.85-6.73 (comp. m, 3H), 2.44 (q, / = 7.4 Hz, 2H), 1.26 (t, / = 7.4 Hz, 3H); ¹³C NMR (125 MHz, CDCI3) δ 203.8, 163.5 (d, /_C-F = 245.1 Hz), 135.6 (d, /_C-F = 8.7 Hz), 130.2 (d, /_C-F = 8.9 Hz), 119.6 (d, Jc-F = 2.6 Hz), 110.8 (d, /_C-F = 21.1 Hz), 110.6 (d, /_C-F = 23.3 Hz), 42.1 (d, /_C-F = 2.5 Hz), 16.9, 12.7; ¹⁹F NMR (282 MHz, CDC1₃) δ -115.8 (app. q, J =9.4 Hz);IR (neat film, NaCl) 2972, 2936, 2879, 2101, 1799, 1613, 1585, 1490, 1444, 1258, 1183, 1161, 964, 857, 776, 683 cm^"1 Example B General Procedures for Cycloadditions

(+)-Diisopropyl 3-ethyl-4-oxo-3-(p-tolyl)-l ,2-diazetidine- 1 ,2-dicarboxylate (ABL303).

In a nitrogen- atmosphere glovebox, a solution of ethyl p-tolyl ketene (SI-4, 160.2 mg, 1.00 mmol, 1.0 equiv) in CH₂CI₂ (59 mL) was prepared in a 200 mL round bottom flask. A solution of diisopropylazodicarboxylate (202.2 mg, 1.00 mmol, 1.0 equiv) in CH₂CI₂ (4 mL) was added and the vial containing the azo-compound was rinsed with additional CH₂CI₂ (3 x 3 mL). The flask was sealed with a rubber septum and taped. In a separate vial, a solution of (+)-PPY* (18.8 mg, 0.05 mmol, 0.05 equiv) in CH2CI2 (2 mL) was prepared and the vial was closed with a septum cap. The flask and vial were removed from the glovebox and the flask containing the yellow-orange ketene/azodicarboxylate solution was cooled to -30 °C in a dry ice/CHCl₃ bath. The dark purple catalyst solution was added via syringe in one portion leading to an immediate color change to green. The mixture was stirred overnight, warming gradually to ambient temperature. After 14 h, the mixture was concentrated under vacuum to an oil. The residue was purified by automated silica gel chromatography using a Biotage Isolera Four (25 g SNAP S1O₂ cartridge, linear gradient from 10-100% Et₂0 in hexanes) to provide (+)-ABL303 as a colorless oil (241.2 mg, 67% yield).

Separation of enantiomers was achieved by semi-preparative HPLC using a Gilson PLC 2020 and a Chiralpak IB column (20 x 250 mm) using 2% -PrOH in hexanes as eluent (isocratic 17.0 mL/min flow rate, retention times: 6.6 min, 8.4 min). Yield of fast-eluting enantiomer (ABL303): 84.9 mg; yield of slow-eluting enantiomer (ent-ABL303): 97.3 mg. Both isolates from the chromatographic resolution were >99% ee by analytical HPLC (4.6 x 250 mm Chiralpak IB-3 column, 2% -PrOH in hexanes eluent, isocratic 1 mL/min flow rate, retention times: 8.834 min, 11.724 min or 4.6 x 250 mm Chiralpak OD-H column, 2% i- PrOH in hexanes eluent, isocratic 1 mL/min flow rate, retention times: 9.295 min, 14.389 min). Gas chromatographic analysis (J & W Scientific HP-5 column, 100-310 °C ramp) of the resolved enantiomers found a sing le nonsolvent component. ^lU NMR (500 MHz, CDC1₃) δ 7.43 (d, / = 8.1 Hz, 2H), 7.20 (d, J = 1.9 Hz, 2H), 5.09 (septet, / = 6.2 Hz, 1H), 4.96 (septet, 7 = 5.9 Hz, 1H), 2.40 (dq, / = 15.1 , 7.7 Hz, 1H), 2.35 (s, 3H), 2.25 (dq, / = 14.6, 7.2 Hz, 1H), 1.36 (d, 7 = 6.2 Hz, 3H), 1.35 (d, 7 = 6.2 Hz, 3H), 1.27 (d, 7 = 6.3 Hz, 3H), 1.16-1.07 (br s, 3H), 1.08 (app t, / = 7.3 Hz, 3H); ¹³C NMR (125 MHz, CDC1₃) δ 165.2, 157.3, 147.8, 139.0, 132.2, 129.5, 126.4, 90.3, 72.8, 71.6, 28.4, 22.0, 21.9, 21.3, 8.7;IR (neat film, NaCl) 2983, 2940, 2884, 1835, 1767, 1739, 1467, 1376, 1358, 1301, 1267, 1233, 1180, 1146, 1102, 1051 , 920 cm ; LCMS (ES+) mlz: cacld for C19H27N2O5 [M + H]⁺ 363.2, found: 363.1 ;

HRMS m/r. calcd for C19H26N2O5 [M + H]⁺ 363.1914, found: 363.1919;

Optical rotation of fast-eluting enantiomer (ABL303): [a]^{23 9} _D -7.2° (c 1.27, CH₂C1₂, >99 ee). Optical rotation of slow-eluting enantiomer (ent-ABL303): [a]^{23 8} _D +7.0° (c 1.17,

Cycloaddition Example 1

(-)-Diisopropyl 3-ethyl-4-oxo-3-(p-tolyl)- 1 ,2-diazetidine- 1 ,2-dicarboxylate (ABL303).

In a nitrogen- atmosphere glovebox, a solution of ethyl p-tolyl ketene (SI-4, 320.4 mg,

2.00 mmol, 1.0 equiv) in CH2CI2 (110 mL) was prepared in a 300 mL round bottom flask. A solution of diisopropylazodicarboxylate (404.4 mg, 2.00 mmol, 1.0 equiv) in CH2CI2 (4 mL) was added and the vial containing the azo-compound was rinsed with additional CH2CI2 (1 x 3 mL). The flask was sealed with a rubber septum and taped. In a separate vial, a solution of (-)-PPY* (37.6 mg, 0.10 mmol, 0.05 equiv) in CH2CI2 (3 mL) was prepared and the vial was closed with a septum cap. The flask and vial were removed from the glovebox and the flask containing the yellow-orange ketene/azodicarboxylate solution was cooled to -20 °C in an immersion cooling bath. The dark purple catalyst solution was added via syringe in one portion leading to an immediate color change to dark green. The mixture was stirred 18 h at -20 °C, during which time the green color subsided and the solution was again dark purple in color. The mixture was removed from the cooling bath and then concentrated to an oil by rotary evaporation. The residue was purified by automated silica gel chromatography using Biotage Isolera Four (25 g SNAP Si0₂ cartridge, linear gradient from 5-100% Et₂0 in hexanes) to provide ABL303 as a colorless oil (362.2 mg, 50% yield). Analytical chiral HPLC found 56% ee (4.6 x 250 mm Chiralpak IB-3 column, 2% i^'-PrOH in hexanes eluent, isocratic 1 mL/min flow rate, retention times: 8.834 min (major), 11.724 min (minor) or 4.6 250 mm Chiralpak OD-H column, 2% i^'-PrOH in hexanes eluent, isocratic 1 mL/min flow rate, retention times: 8.714 min (major), 11.509 min (minor)).

ABL248

Following the general procedure with ketene SI-8 (100.0 mg, 0.68 mmol) and diisopropylazodicarboxylate, ABL248 (96.3 mg, 41% yield) was isolated as a thick colorless oil after Biotage purification. ^lU NMR (400 MHz, CDC1₃) δ 7.37-7.33 (comp. m, 2H), 7.27 (app. t, / = 7.7 Hz, 1H), 7.15 (d, / = 7.5 Hz, 1H), 5.08 (app. septet, / = 6.2 Hz, 1H), 5.00 (app. septet, /= 6.2 Hz, 1H), 2.35 (s, 3H), 1.91 (s, 3H), 1.35 (d, / = 6.3 Hz, 3H), 1.33 (d, / = 6.3 Hz, 3H), 1.27 (d, / = 6.3 Hz, 3H), 1.17 (d, / = 6.1 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 165.1, 157.3, 147.7, 138.5, 135.6, 129.6, 128.6, 126.3, 122.8, 85.5, 72.6, 71.6, 30.3, 21.8, 21.71, 21.69, 21.6, 21.4; IR (neat film, NaCl) 2984, 2938, 1835, 1767, 1738, 1468, 1377, 1358, 1299, 1254, 1182, 1101, 1040, 965, 938, 915, 830, 790, 752, 721 cm^"1;

HRMS m/r. calcd for Ci₈H₂₄N₂0₅ [M + H]⁺ 349.1758, found: 349.1762

HPLC analysis found 84% ee (4.6 x 250 mm Chiralpak IC column, 5% i^'-PrOH in hexanes eluent, isocratic 1.0 mL/min flow rate, retention times: 16.9 min (major), 20.0 min (minor)). Separation of enantiomers was achieved by semipreparative HPLC (20 x 250 mm Chiralpak IC column, 5% i^'-PrOH in hexanes eluent, isocratic 20.0 mL/min flow rate) provided samples of both ABL248 (major component, retention time: 11.2 min) and ent-ABL248 (minor component, retention time: 13.7 min) with >99% ee. Yield of fast-eluting enantiomer

(ABL248): 60.9 mg; yield of slow-eluting enantiomer (ent- ABL248): 2.3 mg.

Optical rotation of major enantiomer: [a]^{25 5} _D +2.4° (c 2.84, CH₂C1₂, >99% ee) Cycloaddition Example 3

ABL223

Following the general procedure with ketene SI-20 (100.0 mg, 0.61 mmol) and diisopropylazodicarboxylate, ABL223 (71.1 mg, 32% yield) was isolated as a thick colorless oil after Biotage purification.

^XH NMR (400 MHz, CDC1₃) δ 7.40-7.31 (comp. m, 2H), 7.31-7.28 (m, 1H), 7.09-7.06 (m, 1H), 5.10 (app. septet, / = 6.3 Hz, 1H), 5.04 (app. septet, / = 6.3 Hz, 1H), 2.39 (dq, / = 14.7, 7.3 Hz, 1H), 2.24 (dq, / = 14.9, 7.5 Hz, 1H), 1.37 (d, / = 6.3 Hz, 3H), 1.35 (d, / = 6.3 Hz, 3H), 1.31 (d, / = 6.3 Hz, 3H), 1.24 (d, / = 6.2 Hz, 3H), 1.08 (app. t, / = 7.4 Hz, 3H);

¹³C NMR (100 MHz, CDC1₃) δ 164.2, 162.9 (d, J_C-F = 247.2 Hz), 156.8, 147.5, 137.8 (d, J_C-F = 7.0 Hz), 130.3 (d, J_C-F = 8.2 Hz), 121.7 (d, J_C-F = 3.0 Hz), 115.8 (d, J_C-F = 21.1 Hz), 113.6 (d, Jc-F = 23.4 Hz), 89.3, 72.8, 71.9, 28.7, 21.8, 21.70, 21.68, 21.65, 8.5 ;

1⁹F NMR (282 MHz, CDC1₃) δ -114.6; IR (neat film, NaCl) 2984, 2942, 1836, 1767, 1738, 1614, 1590, 1489, 1446, 1359, 1299, 1251, 1182, 1102, 1053, 944, 791 cnT¹;

HRMS m/r. calcd for C18H23FN2O5 [M + H]⁺ 367.1664, found: 367.1664;

HPLC analysis found 38% ee (4.6 x 250 mm Chiralpak IC column, 3% i^'-PrOH in hexanes eluent, isocratic 1.0 mL/min flow rate, retention times: 12.3 min (major), 18.4 min (minor)). Separation of enantiomers was achieved by semipreparative HPLC (20 x 250 mm Chiralpak IC column, 3% i^'-PrOH in hexanes eluent, isocratic 20.0 mL/min flow rate) provided samples of both ABL223 (major component, retention time: 10.4 min) and ent-ABL223 (minor component, retention time: 14.3 min) with >99% ee. Yield of fast-eluting enantiomer

(ABL223): 36.7 mg; yield of slow-eluting enantiomer (ent-ABL223): 15.3 mg.

Optical rotation of major enantiomer: [a]^{25 6} _D -17.3° (c 1.83, CH₂C1₂, >99% ee) Cycloaddition Example 4

ABL243

Following the general procedure with ketene SI-12 (100.0 mg, 0.62 mmol) and diisopropylazodicarboxylate, ABL243 (64.9 mg, 29% yield) was isolated as a thick colorless oil after Biotage purification. ^lU NMR (400 MHz, CDC1₃) δ 7.38-7.33 (comp. m, 2H), 7.28 (app. t, / = 7.7 Hz, 1H), 7.17 (d, / = 7.4 Hz, 1H), 5.10 (app. septet, / = 6.3 Hz, 1H), 4.99 (app. septet, /= 6.2 Hz, 1H), 2.42 (dq, / = 14.6, 7.3 Hz, 1H), 2.37 (s, 3H), 2.26 (dq, /= 14.7, 7.3 Hz, 1H), 1.37 (d, / = 6.1 Hz, 3H), 1.36 (d, / = 6.1 Hz, 3H), 1.29 (d, / = 6.3 Hz, 3H), 1.15 (br s, 3H), 1.09 (app. t, / = 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 164.9, 157.1, 147.6,

138.4, 135.0, 129.6, 128.5, 126.8, 123.3, 90.2, 72.6, 71.5, 28.4, 21.8, 21.73, 21.72, 21.5, 21.4, 8.6;

IR (neat film, NaCl) 2983, 2940, 1836, 1767, 1741, 1467, 1376, 1358, 1302, 1249, 1181,

1146, 1102, 1052, 941, 790 cm^-1;

HRMS m/z: calcd for C19H2₆N2O5 [M + H]⁺ 363.1914, found: 363.1918;HPLC analysis found

52% ee (4.6 x 250 mm Chiralpak IC column, 3% i^'-PrOH in hexanes eluent, isocratic 1.0 mL/min flow rate, retention times: 17.3 min (major), 24.3 min (minor)).

Separation of enantiomers was achieved by semipreparative HPLC (20 x 250 mm Chiralpak

IC column, 3% i^'-PrOH in hexanes eluent, isocratic 20.0 mL/min flow rate) provided samples of both ABL243 (major component, retention time: 13.4 min) and ent-ABL243 (minor component, retention time: 16.3 min) with >99% ee. Yield of fast-eluting enantiomer

(ABL243): 23.9 mg; yield of slow-eluting enantiomer (ent-ABL243): 6.1 mg.

Optical rotation of major enantiomer: [a]^{25 8} _D -8.8° (c 1.59, CH₂C1₂, >99% ee.

Cycloaddition Example 5

Following the general procedure with ketene SI-16 (100.0 mg, 0.57 mmol) and diisopropylazodicarboxylate, ABL179 (60.4 mg, 28% yield) was isolated as a thick colorless oil after Biotage purification. ^lU NMR (400 MHz, CDC1₃) δ 7.31 (app. t, 7 = 8.0 Hz, 1H), 7.15 (d, 7 = 7.8 Hz, 1H), 7.12 (app. t, 7 = 2.1 Hz, 1H), 6.90 (dd, 7 = 8.3, 1.9 Hz, 1H), 5.10 (app. septet, 7 = 6.3 Hz, 1H), 5.02 (app. septet, 7 = 6.3 Hz, 1H), 3.82 (s, 3H), 2.40 (dq, 7 = 14.7, 7.3 Hz, 1H), 2.26 (dq, 7 = 14.8, 7.4 Hz, 1H), 1.37 (d, 7 = 6.0 Hz, 3H), 1.36 (d, 7 = 6.1 Hz, 3H), 1.30 (d, 7 = 6.3 Hz, 3H), 1.20 (d, 7 = 6.1 Hz, 3H), 1.09 (app. t, 7 = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 164.7, 159.7, 157.0, 147.6, 136.7, 129.7, 118.3, 114.5, 111.8, 90.0, 72.7, 71.6, 55.3, 28.6, 21.8, 21.74, 21.72, 21.6, 8.6;IR (neat film, NaCl) 2983, 2940, 2885, 2839, 1835, 1767, 1739, 1603, 1585, 1467, 1358, 1294, 1256, 1212, 1181, 1102, 1050, 942, 788 cm^"1;

HRMS m/r. calcd for Ci₉H₂₆N₂0₆ [M + H]⁺ 379.1864, found: 379.1861;

HPLC analysis found 45% ee (4.6 x 250 mm Chiralpak IC column, 5% i^'-PrOH in hexanes eluent, isocratic 1.0 mL/min flow rate, retention times: 17.7 min (major), 24.5 min (minor)). Separation of enantiomers was achieved by semipreparative HPLC (20 x 250 mm Chiralpak IC column, 5% i^'-PrOH in hexanes eluent, isocratic 20.0 mL/min flow rate) provided samples of both ABL179 (major component, retention time: 13.5 min) and ent-ABL179 (minor component, retention time: 18.8 min) with >99% ee. Yield of fast-eluting enantiomer

(ABL179): 26.5 mg; yield of slow-eluting enantiomer (ent-ABL179): 7.6 mg.

Optical rotation of major enantiomer: [a]^{25 8} _D -17.7° (c 1.77, CH₂C1₂, >99% ee)

Cycloaddition Example 6

ABL245

Following the general procedure with ketene SI-12 (100.0 mg, 0.62 mmol) and dibenzylazodicarboxylate, ABL245 (92.9 mg, 33% yield) was isolated as a thick colorless oil after Biotage purification. ^XH NMR (400 MHz, CDC1₃) δ 7.43-7.22 (comp. m, 14H), 7.15 (d, 7 = 7.4 Hz, 1H), 5.34 (d, 7 = 12.3 Hz, 1H), 5.30 (d, 7 = 12.4 Hz, 1H), 5.23 (d, 7= 12.0 Hz, 1H), 5.13 (d, 7 = 11.6 Hz, 1H), 2.35 (dq, 7 = 14.4, 7.0 Hz, 1H), 2.33 (s, 3H), 2.21 (dq, 7 = 14.8, 7.4 Hz, 1H), 0.96 (app. t, 7 = 7.3 Hz, 3H); ¹³C NMR (125 MHz, CDC1₃) δ 164.6, 148.0, 138.5, 134.7, 134.4, 129.7, 128.61, 128.59, 128.57, 128.5, 128.2, 126.6, 123.1, 91.1, 69.2, 69.0, 28.6, 21.4, 8.5; IR (neat film, NaCl) 2976, 1837, 1769, 1741, 1499, 1455, 1382, 1302, 1246, 1190, 1054, 752 cm^"1;

HRMS m/z: calcd for C27H2₆N2O5 [M + H]⁺ 459.1914, found: 459.1910;HPLC analysis found 82% ee (4.6 x 250 mm Chiralpak IB-3 column, 5% i^'-PrOH in hexanes eluent, isocratic 0.8 mL/min flow rate, retention times: 13.5 min (major), 16.6 min (minor)).

Separation of enantiomers was achieved by semipreparative HPLC (20 x 250 mm Chiralpak IB column, 3% i^'-PrOH in hexanes eluent, isocratic 20.0 mL/min flow rate) provided samples of both ABL245 (major component, retention time: 9.7 min) and ent-ABL245 (minor component, retention time: 13.0 min) with >99% ee. Yield of fast-eluting enantiomer

(ABL245): 63.3 mg; yield of slow-eluting enantiomer (ent-ABL245): 6.1 mg.

Optical rotation of major enantiomer: [a]^{25 7} _D -6.4° (c 3.17, CH₂C1₂, >99% ee)

Example 7 Serine Hydrolase Activity

Materials.

FP-Biotin and FP-Rh were synthesized as described previously. Chemical reagents were obtained from Sigma- Aldrich or ThermoFisher unless otherwise indicated. Cell culture media and supplements were obtained from CellGro and Omega Scientific.

Preparation of Mouse Tissue Proteomes.

Mouse brains were Dounce-homogenized on ice in PBS (pH7.5) followed by a low- speed spin (1,400 x g, 5 min) to remove debris. After sonication, the supernatant was then centrifuged (100,000 x g, 45 min) to provide the cytosolic fraction in the supernatant and the membrane fraction as a pellet. The pellet was washed and resuspended in PBS by sonication. Protein concentrations were determined using a protein assay kit (Bio-Rad). Samples were stored at -80 °C until use.

Recombinant expression of mABHDIO in COS-7 cells.

Full length cDNA (Open Biosystems, Clone ID 6820515) was used to subclone mouse ABHD10 into the pcDNA3.1+ vector (Invitrogen). COS-7 cells were grown in DMEM media supplemented with 10% fetal bovine serum and 2 mM L-glutamine in a humidified 5% CO₂ incubator at 37 °C to ~ 50% confluence. Cells were transiently transfected using the Fugene 6 reagent (Roche Applied Science) following the

manufacturer's protocols. After 48 h, cells were washed 2x with PBS (pH 7.5) and scraped into cold PBS. Cell pellets were isolated by centrifugation (1,400 x g, 3 min), resuspended in PBS, sonicated, and used as whole cell proteomes. Samples were stored at -80 °C until use. Cell Culture and Preparation ofNeuro2A Proteomes.

Neuro2A murine neuroblastoma cells were grown in DMEM media supplemented with 10% fetal bovine serum and 2 mM L-glutamine in a humidified 5% C0₂ incubator at 37 °C. For in vitro experiments, cells were grown to 90-100% confluence, washed 2x with PBS (pH 7.5) and scraped into cold PBS. Cell pellets were isolated by centrifugation (1,400 x g, 3 min), resuspended in PBS, sonicated, separated into membrane and soluble fractions as described for mouse tissue proteomes, and stored at -80 °C until use.

Competitive ABPP Assays in Proteomes.

For in vitro experiments, proteomes were diluted to 1 mg/mL in PBS (pH 7.5, 50 μL· total reaction volume) and incubated with compound at the indicated concentrations (1 μL· oί a 50x stock in DMSO) for 30 min at 37 °C, followed by labeling with 1 μΜ FP-Rh (1 μΐ. of a 50x stock in DMSO) for 30 min at 25 °C. Reactions were quenched with 4x SDS-PAGE loading buffer, boiled for 5 min at 90 °C, separated by SDS-PAGE and visualized by in-gel fluorescence scanning (Hiatchi FMBio He, MiraBio). For in situ experiments, cells were treated with compound at the indicated concentrations (15 μΕ of a 200x DMSO stock) in a 6 cm dish (3 mL total media volume). Cells were harvested and separated into membrane and soluble fractions as described for mouse tissue proteomes. Total protein concentrations of each fraction were adjusted to 1 mg/mL in PBS (50 μΕ total reaction volume), labeled with FP-Rh and analyzed as described above. The percentage activity remaining was quantified by measuring the integrated optical density of the individual serine hydrolase band relative to a DMSO-only (no compound) control using ImageJ software. IC5₀ values for inhibition were determined from dose-response curves from three replicates at each inhibitor concentration fitted using Prism software (GraphPad).

Competitive ABPP-MudPIT in Mouse Tissue Proteomes.

Mouse brain membrane proteome (1 mg/mL in PBS) was treated with 2 μΜ ABL117

(2 μΐ, of a lOOOx DMSO stock) or DMSO (2 μ_ϋ) for 30 min at 25 °C, followed by 5 μΜ FP- biotin for 2 h at 25 °C (1 mL total reaction volume). The proteomes were then solubilized with 1% Triton X-100 and rotated at 4 °C for 1 h, desalted over PD-10 desalting columns (GE Healthcare), and FP-labeled proteins were enriched with streptavidin beads as previously described. The beads were washed with 1 % SDS in PBS, 6M urea, and PBS, then resuspended in 8 M urea in 25 mM ammonium bicarbonate, reduced with 10 mM TCEP for 30 min at 25 °C, and alkylated with 12 mM iodoacetamide for 30 min at 25 °C in the dark. On-bead digestions were performed for 12 h at 37 °C with sequence-grade modified trypsin (Promega; 2 μg) in 2M urea in the presence of 2 mM CaCl₂. Peptide samples were acidified to a final concentration of 5 % (v/v) formic acid, pressure-loaded on to a biphasic (strong cation exchange/reversed phase) capillary column. MudPIT analysis of eluted peptides was carried out as previously described on a coupled Agilent 1100 LC-ThermoFinnigan LTQ-MS instrument. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.7; publicly available at the website fields.scripps.edu/downloads.php) MS2 spectra data were searched using the SEQUEST algorithm (Version 3.0) against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. SEQUEST searches allowed for static modification of cysteine residues (+57.02146 due to alkylation) and methionine oxidation (+15.9949). The resulting MS2 spectra matches were assembled into protein identifications and filtered using

DTASelect (version 2.0) using the— trypstat options (applies different statistical models for the analysis of peptide digestion state). Peptides with cross-correlation scores greater than 1.8 (+1), 2.5 (+2), 3.5 (+3) and delta CN scores greater than 0.08 were included in the spectral counting analysis. Only proteins for which 10 or more spectral counts were identified in the DMSO-treated samples were considered for comparative analysis.

Isotopic Competitive ABPP-MudPIT in Cells.

Neuro2A and BW5147-derived murine T-cells were initially grown for 10 passages in light/heavy SILAC DMEM (Neuro2A) or RPMI (T-cells) supplemented with 10% dialyzed FCS and 2 mM L-glutamine. Light media was supplemented with 100 μg/mL L-arginine and 100 μg/mL L-lysine. Heavy media was supplemented with 100 μg/mL [¹³C6¹⁵N₄]-L-Arginine and 100 μg/mL [¹³C₆ ¹⁵N₂]-L-Lysine.

Neuro2A:

Heavy Neuro2A cells were treated with either ABL303 or ABL127 (50 uL of a 200x stock in DMSO) and light cells were treated with DMSO (50 uL) for 2 h at 37 °C in 10 cm dishes (10 mL total media volume). Cells were washed 2x with PBS, harvested, and homogenized by sonication in PBS. The soluble and membrane fractions were isolated by centrifugation (100,000 x g, 45 min) and the protein concentration was adjusted to 2 mg/mL with PBS in each fraction. The light and heavy proteomes were labeled with 10 μΜ of FP- biotin (500 \L total reaction volume) for 2 h at 25 °C.

BW5147 -derived T-cells:

Heavy BW5147 -derived T-cells were treated with either ABL303 or ABL127 (250 μΐ, of a 200x stock in DMSO) and light cells were treated with DMSO (250 pL) for 2 h at 37 °C in T-150 flasks (50 mL total media volume). Cells were harvested by centrifugation (1,400 x g, 3 min), resuspended in PBS, sonicated, and separated into membrane and soluble fractions. The protein concentration was adjusted to 1 mg/mL with PBS in each fraction. The light and heavy proteomes were labeled with 5 μΜ of FP-biotin (1 mL total reaction volume) for 2 h at 25 °C.

After incubation, light and heavy proteomes were mixed in 1 : 1 ratio, and the membrane proteomes were additionally solubilized with 1% TritonX-100. The proteomes were desalted over PD-10 desalting columns (GE Healthcare) and FP-labeled proteins were enriched with streptavidin beads (Sigma) as previously described. The beads were washed with 1% SDS in PBS (lx), 6M urea (lx), and PBS (2x), then resuspended in 6 M urea, reduced with 5 mM TCEP for 20 min at 25 °C, and alkylated with 10 mM iodoacetamide for 30 min at 25 °C in the dark. On-bead digestions were performed for 12 h at 37°C with sequence-grade modified trypsin (Promega; 2 μg) in 2M urea in the presence of 2 mM CaCl₂. Peptide samples were acidified to a final concentration of 5 % (v/v) formic acid, pressure- loaded on to a biphasic (strong cation exchange/reversed phase) capillary column.

Digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography (2D-LC) separation in combination with tandem mass spectrometry as previously described using an Agilent 1200-series quaternary pump and Thermo Scientific LTQ-Orbitrap Velos ion trap mass spectrometer. Peptides were eluted in a 5 -step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (20 s, repeat of 1). Specifically, one full MS (MSI) scan (400-1800 m/z) was followed by 30 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor and searched using the ProLuCID algorithm (publicly available at the website fields.scripps.edu/doadload.php against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false- discovery rates. Pro Lucid searches allowed for static modification of cysteine residues

(+57.02146 due to alkylation), methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, 8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the— modstat, -mass, and -trypstat options (applies different statistical models for the analysis of high resolution masses, peptide digestion state, and methionine oxidation state respectively). Ratios of Heavy/Light peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were >3 standard deviations from the median peptide value. Compounds demonstrated activity in the assays of this Example as indicated in the following table (Table 1).

Table 1. In vitro serine hydrolase profiles.

ICso (nM)

5

Compound R ^f R² R³ A8HD10 ABHDS PRE P P E-1

ABU 17 Et 3- e Me 86% (B) 210 1 ,900 ¾eoo 250

ABL143 Et H Et 80% (R) 750 1300 3, 100 910

AS LS I Et H e 83% (R) 1 500 10,000 ^•.- 10,000 -

ABL1 e 2-Me e 89% (ft) 1 ,800 9 500 >10_;000

ABL243 Et 3~Me ipr 90% ( S3 1 ,300 810

ABL245 Et 3 Me Bn ee% (R) 410 180 260 220

ABL24S Me 31,1« ^sPr 99% ( ) 120 1 _f80O 7,700

ABL223 Et 3-F *Pr 95% (R) 40 2,400 2,200 -

ABL179 Et 3-GMe ^sPr 99% (R) 220 2, 100 1 200

ABL3G3 Et 4-Me ^sPr 99% (R) 30 3,000 4,500 1 ,300 estt-ABL303 Et 4- e ^sPr 99% (S) >10,000 > 10,000 >io,ooo

All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of modulating serine hydrolase ABHDIO, comprising contacting the ABHDIO with an effective amount or concentration of compound of formula (I)

wherein

R is a (C1-C6) alkyl group optionally substituted with halo;

R^a and R^b are independently selected for each occurrence, from the group consisting of hydrogen and (Cl-C6)alkyl, optionally mono- or independently multi- substituted with halo, cyano, oxo or hydroxyl; or,

R³ is alkyl or arylalkyl;

or a pharmaceutically acceptably salt thereof.

2. The method of claim 1, wherein the compound of formula (I) is the (R)- enantiomer thereof.

3. The method of claim 1, wherein R¹ is ethyl.

4. The method of claim 1, wherein R² is methyl, trifluoromethyl, fluoro, methoxy, or trifluoromethoxy.

5. The method of claim 1, wherein R³ is isopropyl.

The method of claim 1 , wherein the compound of formula (I) is any one of

or a pharmaceutically acceptable salt thereof.

7. The method of claim 1, wherein the ABHDIO is disposed in a living cell, and contacting comprises contacting the cell with the effective amount or concentration of the compound of formula (I), or a pharmaceutically acceptable salt thereof.

8. The method of claim 1, wherein the ABHDIO is disposed in the living tissue of a patient suffering from a condition wherein modulation of ABHDIO is indicated, and contacting comprises administering an effective dose of the compound of formula (I) to the patient.

9. The method of claim 8, wherein the condition comprises pain, inflammation, metabolic disorders, solid tumors, or obesity.

10. The method of claim 1 wherein modulation of ABHD10 is selective with respect to modulation of serine hydrolase PME-1.

11. A compound of formula (I)

wherein

R¹ is a (C1-C6) alkyl group optionally substituted with halo;

Ar is an aryl or heteroaryl group, substituted with n independently selected R², wherein n = 1 , 2, or 3 ;

R³ is alkyl or arylalkyl;

including any stereoisomer thereof;

or a pharmaceutically acceptably salt thereof; provided that when R¹ is ethyl and R³ is methyl, then R² is not 2-methyl, 3- methyl, 2-methoxy, 4-methoxy, or 4-chloro.

12. The compound of claim 11, wherein the compound of formula (I) is the (R)- enantiomer thereof.

13. The compound of claim 11, wherein R¹ is ethyl.

14. The compound of claim 11, wherein R² is methyl, trifluoromethyl, fluoro, methoxy, or trifluoromethoxy.

15. The compound of claim 11, wherein R² is 3 -methyl, 3 -trifluoromethyl, 3- fluoro, 3-methoxy, 3-trifluoromethoxy, 4-methyl, 4-trifluoromethyl, 4-fluoro, 4- methoxy, or 4-trifluoromethoxy.

16. The compound of claim 11, wherein R³ is isopropyl.

17. The compound of claim 11, wherein the compound of formula (I) is any one of

or a pharmaceutically acceptable salt thereof.

18. A pharmaceutically acceptable composition comprising a compound of any one of claims 11-17, and a pharmaceutically acceptable excipient.

19. Use of a compound of any one of claims 11-17 for preparation of a medicament for treatment of a condition in a mammal for which modulation of serine hydrolase ABHD10 is indicated.

20. The use of claim 19 wherein the condition comprises pain, inflammation, metabolic disorders, solid tumors, or obesity.