Discovery of highly potent and selective orexin 1 receptor antagonists (1-SORAs) suitable for in vivo interrogation of orexin 1 receptor pharmacology.

While a correlation between blockade of the orexin 2 receptor (OX2R) with either a dual orexin receptor antagonist (DORA) or a selective orexin 2 receptor antagonist (2-SORA) and a decrease of wakefulness is well established, less is known about selective blockade of the orexin 1 receptor (OX1R). Therefore, a highly selective orexin 1 antagonist (1-SORA) with suitable properties to allow in vivo interrogation of OX1R specific pharmacology in preclinical species remains an attractive target. Herein, we describe the discovery of an optimized 1-SORA series in the piperidine ether class. Notably, a 4,4-difluoropiperidine core coupled with a 2-quinoline ether linkage provides OX1R selective compounds. The combination with an azabenzimidazole or imidazopyridine amide substituent leads to analogs 47 and 51 with >625-fold functional selectivity for OX1R over OX2R in rat. Compounds 47 and 51 possess clean off-target profiles and the required pharmacokinetic and physical properties to be useful as 1-SORA tool compounds.

Discovery of piperidine ethers as selective orexin receptor antagonists (SORAs) inspired by filorexant.

Highly selective orexin receptor antagonists (SORAs) of the orexin 2 receptor (OX2R) have become attractive targets both as potential therapeutics for insomnia as well as biological tools to help further elucidate the underlying pharmacology of the orexin signaling pathway. Herein, we describe the discovery of a novel piperidine ether 2-SORA class identified by systematic lead optimization beginning with filorexant, a dual orexin receptor antagonist (DORA) that recently completed Phase 2 clinical trials. Changes to the ether linkage and pendant heterocycle of filorexant were found to impart significant selectivity for OX2R, culminating in lead compound PE-6. PE-6 displays sub-nanomolar binding affinity and functional potency on OX2R while maintaining >1600-fold binding selectivity and >200-fold functional selectivity versus the orexin 1 receptor (OX1R). PE-6 bears a clean off-target profile, a good overall preclinical pharmacokinetic (PK) profile, and reduces wakefulness with increased NREM and REM sleep when evaluated in vivo in a rat sleep study. Importantly, subtle structural changes to the piperidine ether class impart dramatic changes in receptor selectivity. To this end, our laboratories have identified multiple piperidine ether 2-SORAs, 1-SORAs, and DORAs, providing access to a number of important biological tool compounds from a single structural class.

Structural basis for the inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase H active site-directed inhibitors.

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes as thrombin inhibitors.

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.

Purification of untagged HIV-1 reverse transcriptase by affinity chromatography.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.

Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold.

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC(50)=0.045 µM, HIV RT RNase H; 13 µM, HIV RT-polymerase; 24 µM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC(50)=0.19 µM) with a modest window with respect to cytotoxicity (CC(50)=3.3 µM).

Neonatal Suckling, Oxytocin, and Early Infant Attachment to the Mother.

The neuropeptide oxytocin (OT) promotes maternal care and social affiliation in adults but its importance in infant attachment still remains unknown. True animal models of infant attachment are extremely rare, and the sheep (in complement to non-human primates) is one of the few that provides the opportunity to investigate its neuroendocrinological basis. In the lamb, access to the udder has strong rewarding properties for the establishment of a preferential relationship with the mother. Therefore, the present study explored the possible involvement of OT through its release during close social contacts with the mother. The first experiment revealed that lambs having free access to the udder from birth onward developed, by 12 h of age, a clear preference for their mothers over another maternal ewe. Delaying access to the udder for six, four or even only 2 h starting at birth, by covering the ewe's udder, resulted in the lack of such a preference without affecting general activity. These effects persisted in most cases at 24 h but by 72 h of age a bond with the mother was clearly expressed. Experiment two showed that social interactions with the mother were followed by a release of OT in the plasma when lambs had the possibility to suckle. Non-nutritive interactions were without effects. Preliminary data on two subjects suggested that OT might also increase in the cerebrospinal fluid after suckling. Finally, in the third experiment, oral administration of a non-peptide OT receptor antagonist (L-368-899, Merck) over the first 4 h after birth led to decreased exploration of the mother's body compared to lambs receiving saline, and impaired the expression of a preference for the mother at 24 h. The effects were no longer observed at 48 h. Our findings demonstrate that both delayed access to the mother's udder and OT receptor antagonist alter the onset of mother preference in newborn lambs. This suggests that central OT facilitates the development of filial attachment through its release during suckling.

Design and synthesis of substituted 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides, novel HIV-1 integrase inhibitors.

A series of 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides was synthesized and tested for their inhibition of HIV-1 integrase catalytic activity and HIV-1 replication in cells. Structure-activity studies around lead compound 5 indicated that a coplanar relationship of metal-binding heteroatoms provides optimal binding to the integrase active site. Identification of potency-enhancing substituents and adjustments in lipophilicity provided 17b which inhibits integrase-catalyzed strand transfer with an IC(50) value of 74 nM and inhibits HIV-1 replication in cell culture in the presence of 50% normal human serum with an IC(95) value of 63 nM.

10-Hydroxy-7,8-dihydropyrazino[1',2':1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones: potent, orally bioavailable HIV-1 integrase strand-transfer inhibitors with activity against integrase mutants.

A series of 10-hydroxy-7,8-dihydropyrazino[1',2':1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones was synthesized and tested for their inhibition of HIV-1 replication in cell culture. Structure-activity studies indicated that high antiviral potency against wild-type virus as well as viruses containing integrase mutations that confer resistance to three different structural classes of integrase inhibitors could be achieved by incorporation of small aliphatic groups at certain positions on the core template. An optimal compound from this study, 16, inhibits integrase strand-transfer activity with an IC(50) value of 10 nM, inhibits HIV-1 replication in cell culture with an IC(95) value of 35 nM in the presence of 50% normal human serum, and displays modest pharmacokinetic properties in rats (i.v. t(1/2)=5.3 h, F=17%).

Design and Synthesis of Piperazine Sulfonamide Cores Leading to Highly Potent HIV-1 Protease Inhibitors.

Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718.

Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors.

In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.

A nonpeptide oxytocin receptor antagonist radioligand highly selective for human receptors.

A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity with [35S]. In binding studies, this compound exhibits sub-nanomolar affinity and a high degree of selectivity (900-1800-fold) for human oxytocin receptors compared to human vasopressin receptors. This compound appears suitable for studying the pharmacology of oxytocin receptors in human and nonhuman primate tissues, for which there is currently a paucity of highly selective tools. It may also be useful as a nonlabeled competitor or as a radioligand in autoradiographic studies of oxytocin receptor localization in these tissues.

Dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors.

A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC(95) of 0.31 microM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.

Tetrazole thioacetanilides: potent non-nucleoside inhibitors of WT HIV reverse transcriptase and its K103N mutant.

A series of aryltetrazolylacetanilides was synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors on wild-type virus and on the clinically relevant K103N mutant strain. Extensive SAR investigation led to potent compounds, with nanomolar activity on K103N, and orally bioavailable in rats.

Discovery of potent, selective 4-fluoroproline-based thrombin inhibitors with improved metabolic stability.

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4R)- and (4S)-4-fluoroproline was rationalized by analyzing inhibitor-enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2xAPTT=190 nM), excellent selectivity versus the digestive enzyme trypsin (K(i)=3300 nM), and excellent oral bioavailability in dogs with moderate clearance (F=100%, CL=12 mL/min/kg).

Development of an oxazolopyridine series of dual thrombin/factor Xa inhibitors via structure-guided lead optimization.

Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.

Low molecular weight thrombin inhibitors with excellent potency, metabolic stability, and oral bioavailability.

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.