A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058.

The difluoromethyl-allo-threonyl hydroxamate-based compound LPC-058 is a potent inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) in Gram-negative bacteria. A scalable synthesis of this compound is described. The key step in the synthetic sequence is a transition metal/base-catalyzed aldol reaction of methyl isocyanoacetate and difluoroacetone, giving rise to 4-(methoxycarbonyl)-5,5-disubstituted 2-oxazoline. A simple NMR-based determination of enantiomeric purity is also described.

Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling.

The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28(192)A, F3.36(200)A, W5.43(279)A, W6.48(356)A, and F3.25(189)A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28(192) that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569's ability to increase equilibrium binding of CP55,940. This site also explains ORG27569's ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63(176). 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

The importance of hydrogen bonding and aromatic stacking to the affinity and efficacy of cannabinoid receptor CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528).

Despite the therapeutic promise of the subnanomolar affinity cannabinoid CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528, 1), little is known about its binding site interactions and no primary interaction site for 1 at CB2 has been identified. We report here the results of Glide docking studies in our cannabinoid CB2 inactive state model that were then tested via compound synthesis, binding, and functional assays. Our results show that the amide functional group of 1 is critical to its CB2 affinity and efficacy and that aromatic stacking interactions in the TMH5/6 aromatic cluster of CB2 are also important. Molecular modifications that increased the positive electrostatic potential in the region between the fenchyl and aromatic rings led to more efficacious compounds. This result is consistent with the EC-3 loop negatively charged amino acid, D275 (identified via Glide docking studies) acting as the primary interaction site for 1 and its analogues.

Conformationally constrained farnesoid X receptor (FXR) agonists: alternative replacements of the stilbene.

To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.

Conformationally constrained farnesoid X receptor (FXR) agonists: heteroaryl replacements of the naphthalene.

To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.

FXR agonist activity of conformationally constrained analogs of GW 4064.

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands.

A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ERalpha and ERbeta binding affinity ranging from micromolar to low nanomolar.

Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064.

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.

High-throughput manual parallel synthesis using SynPhase crowns and lanterns.

The high-throughput manual solid-phase parallel synthesis of libraries comprising thousands of discrete samples using pellicular supports (i.e. SynPhase crowns and lanterns) and a suite of novel tools and techniques is described. Key aspects of this approach include the combination of a split-split-split synthesis strategy with spatial encoding to differentiate thousands of crowns, the rapid washing and filtration of up to 48 reaction vessels in parallel, the application of an inexpensive and environmentally friendly technique to remove trifluoroacetic acid from sixteen 96-well plates in parallel, and a high-throughput method for removing cleaved crowns from reusable pin racks. Tens of thousands of discrete samples have been produced in-house using this conceptually and operationally straightforward strategy.

2-(Anilinomethyl)imidazolines as alpha1 adrenergic receptor agonists: alpha1a subtype selective 2'-heteroaryl compounds.

The structure-activity relationship of 2'-pyrrole, pyrazole and triazole substituted 2-(anilinomethyl)imidazolines as alpha(1) adrenergic agonists was investigated. The size and orientation of substituents, as well as the position of the heteroatoms, were found to have a profound effect on the potency and selectivity of the molecules. Potent alpha(1A) subtype selective agonists have been identified.

Alpha(1)-adrenoceptor activation: a comparison of 4-(anilinomethyl)imidazoles and 4-(phenoxymethyl)imidazoles to related 2-imidazolines.

Literature reports suggest that disruption of an interhelical salt bridge is critical for alpha(1)-adrenoceptor activation, and the basic amine found in adrenergic receptor ligands is responsible for the disruption. Novel 4-(anilinomethyl)imidazoles and 4-(phenoxymethyl)imidazoles are agonists of the cloned human alpha(1)-adrenoceptors in vitro, and potent, selective alpha(1A)-adrenoceptor agonists have been identified in this series. These imidazoles demonstrate similar potencies and alpha(1)-subtype selectivities as the corresponding 2-substituted imidazolines. The extremely close SAR suggests that, in spite of the large difference in basicity, these imidazoles and imidazolines may establish the same interactions to activate alpha(1)-adrenoceptors.

2-(Anilinomethyl)imidazolines as alpha1 adrenergic receptor agonists: the discovery of alpha1a subtype selective 2'-alkylsulfonyl-substituted analogues.

A series of 2'-alkylthio-2-(anilinomethyl)imidazolines were prepared to examine the effect of the alkyl group size, sulfur oxidation state, and phenyl ring substitution on ligand binding and agonism of alpha-adrenergic receptor subtypes alpha1a, alpha1b, alpha1d, alpha2a, and alpha2c. Binding at all receptor subtypes decreased for compounds in the sulfone oxidation state as compared to their sulfide analogues. While sulfides were generally potent, nonselective agonists, sulfones exhibited alpha1a subtype selectivity in a cell-based functional assay. Sulfone (32) was 250-7000-fold selective for alpha1a vs all other subtypes.

2-(anilinomethyl)imidazolines as alpha1A adrenergic receptor agonists: 2'-heteroaryl and 2'-oxime ether series.

A series of 2'-heteroaryl and 2'-oxime anilinomethylimidazolines was prepared and evaluated in in vitro functional assays for cloned human alpha1A, alpha1B, and alpha1D receptor subtypes. Potent and selective alpha1A agonists have been identified in these series.

alpha(1)-Adrenoceptor agonists: the identification of novel alpha(1A )subtype selective 2'-heteroaryl-2-(phenoxymethyl)imidazolines.

Novel 2'-heteroaryl-2-(phenoxymethyl)imidazolines have been identified as potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The nature of the 2'-heteroaryl group can have significant effects on the potency, efficacy, and subtype selectivity in this series. alpha(1A) Subtype selective agonists have been identified.