The co-crystal X-ray structures of two isomeric α-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors' electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional "in-action" depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

The first total synthesis of chloropeptin II (1, complestatin) is disclosed. Key elements of the approach include the use of an intramolecular Larock indole synthesis for the initial macrocyclization, adopting conditions that permit utilization of a 2-bromoaniline, incorporating a terminal alkyne substituent (−SiEt3) that sterically dictates the indole cyclization regioselectivity, and benefiting from an aniline protecting group (−Ac) that enhances the atropdiastereoselectivity and diminishes the strained indole reactivity toward subsequent electrophilic reagents. Not only did this key reaction provide the fully functionalized right-hand ring system of 1 in superb conversion (89%) and good atropdiastereoselectivity (4:1 R: S), but it also represents the first reported example of what will prove to be a useful Larock macrocyclization strategy. Subsequent introduction of the left-hand ring system enlisting an aromatic nucleophilic substitution reaction for macrocyclization with biaryl ether formation completed the assemblage of the core bicyclic structure of 1. Intrinsic in the design of the approach and by virtue of the single-step acid-catalyzed conversion of chloropeptin II (1) to chloropeptin I (2), the route also provides a total synthesis of 2.

The synthesis and evaluation of a refined series of α-ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles > 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes.

A series of alpha-ketooxazoles containing conformational constraints in the flexible C2 acyl side chain of 2 (OL-135) and representative oxazole C5 substituents were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH). Exceptionally potent and selective FAAH inhibitors emerged from the series (e.g., 6, Ki = 200 and 260 pM for rat and rhFAAH). With simple and small C5 oxazole substituents, each series bearing a biphenylethyl, phenoxyphenethyl, or (phenoxymethyl)phenethyl C2 side chain was found to follow a well-defined linear relationship between -log Ki and Hammett sigmap of a magnitude (rho = 2.7-3.0) that indicates that the substituent electronic effect dominates, confirming its fundamental importance to the series and further establishing its predictive value. Just as significantly, the nature of the C5 oxazole substituent substantially impacts the selectivity of the inhibitors whereas the effect of the C2 acyl chain was more subtle but still significant even in the small series examined. Combination of these independent features, which display generalized trends across a range of inhibitor series, simultaneously improves FAAH potency and selectivity and can provide exquisitely selective and potent FAAH inhibitors.

Three cocrystal X-ray structures of the α-ketoheterocycle inhibitors 3–5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the α-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure–activity relationships are discussed providing important insights for future design.

Full details of the initial development and continued examination of a powerful intramolecular palladium(0)-mediated indole annulation for macrocyclization closure of the strained 16-membered biaryl ring system found in complestatin (1, chloropeptin II) and the definition of factors impacting its intrinsic atropodiastereoselectivity are described. Its examination and use in an alternative, second generation total synthesis of complestatin are detailed in which the order of the macrocyclization reactions was reversed from our first generation total synthesis. In this approach and with the ABCD biaryl ether ring system in place, the key Larock cyclization was conducted with substrate 36, containing four phenols, five secondary amides, one carbamate, and four labile aryl chlorides, and provided the product 37 (56%) exclusively as a single atropisomer (>20:1, detection limits) possessing the natural (R)-configuration. In this instance, the complexity of the substrate and the reverse macrocyclization order did not diminish the atropodiastereoselectivity, rather it provided an improvement over the 4:1 selectivity that was observed with the analogous substrate used to provide the isolated DEF ring system in our first generation approach. Just as significant, the atroposelectivity represents a complete reversal of the diasteroselectivity observed with analogous macrocyclizations conducted using a Suzuki biaryl coupling.

A series of C4 substituted α-ketooxazoles were examined as inhibitors of the serine hydrolase fatty acid amide hydrolase in efforts that further define and generalize a fundamental substituent effect on enzyme inhibitory potency. Thus, a plot of the Hammett σ m versus -log K i provided a linear correlation (R 2 = 0.90) with a slope of 3.37 (ρ = 3.37) that is of a magnitude that indicates the electronwithdrawing character of the substituent dominates its effects (a one unit change in σ m provides a >1000-fold change in K i ).Fatty acid amide hydrolase (FAAH) 1,2 is the enzyme that serves to hydrolyze endogenous lipid amides 3,4 including anandamide (1a) 5 and oleamide (1b), 6 Figure 1. Its distribution is consistent with its role in degrading and regulating such signaling fatty acid amides at their sites of action. 3 Although it is a member of the amidase signature family of serine hydrolases, for which there a number of prokaryotic enzymes, it is currently the only characterized mammalian enzyme bearing the family's unusual Ser-Ser-Lys catalytic triad. 7,8 Due to the therapeutic potential of inhibiting FAAH 9 especially for the treatment of pain, 10 inflammation, 11 or sleep disorders, 12 there has been increasing interest in the development of selective and potent inhibitors of the enzyme. 9 Early studies shortly following the initial discovery and characterization of FAAH led to the demonstration that the endogenous sleepinducing molecule 2-octyl α-bromoacetoacetate is an effective FAAH inhibitor, 13 the disclosure of a series of nonselective, reversible inhibitors bearing an electrophilic ketone (e.g., trifluoromethyl ketone-based inhibitors), 14,15 and the reports of a set of irreversible inhibitors 16 (e.g., fluorophosphonates and sulfonyl fluorides). To date, two classes of inhibitors have been disclosed that provide opportunities for the development of inhibitors with therapeutic potential. One class is the reactive aryl carbamates and ureas 17-24 that irreversibly acylate a FAAH active site serine. 25 A second class is the α-ketoheterocycle-based inhibitors 26-29 that bind to FAAH via reversible hemiketal formation with an active site serine. Many of these latter competitive inhibitors are not only potent and extraordinarily selective for FAAH versus other mammalian serine hydrolases, but members of this class have been shown to be efficacious analgesics in vivo. 28In the course of these latter studies, we disclosed a fundamental substituent effect in which a well-defined correlation between the electronic character of a para substituent (Hammett σ p ) and the inhibitor potency (−log K i ) was observed. 27 Thus, the inhibitor potency was found to *Corresponding author: email boger@scripps.edu. Whereas the former para substituents are directly conjugated with the electrophilic carbonyl, the meta substituents would exert their effects through their inductive electron-withdrawing properties. Moreover and although intuitive expectations might suggest that such a nonconjugated substituent effect might...

Two cocrystal X-ray structures of the exceptionally potent α-ketoheterocycle inhibitor 1 (K i = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same "in action" state with the three catalytic residues Ser241-Ser217-Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of α-ketoheterocycle-based inhibitors bound to FAAH recently disclosed.boger@scripps.edu, stevens@scripps.edu. Supporting Information Available: Complete refs 17a, 22c, 25c , and 31e . This material is available free of charge via the Internet at