Discovery of Hepatitis B Virus Surface Antigen Suppressor GS-8873.

Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.

Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.

A small-molecule inhibitor of hepatitis C virus infectivity.

One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.

Discovery of GS-9669, a thumb site II non-nucleoside inhibitor of NS5B for the treatment of genotype 1 chronic hepatitis C infection.

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure-activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.

Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).

Tri-substituted acylhydrazines as tertiary amide bioisosteres: HCV NS5B polymerase inhibitors.

The use of a tri-substituted acylhydrazine as an isostere of a tertiary amide was explored in a series of HCV NS5B thumb site II inhibitors. Direct replacement generated an analog with similar conformational and physicochemical properties. The series was extended to produce compounds with potent binding affinities and encouraging levels of cellular potency.

Optimization of Pharmacokinetics through Manipulation of Physicochemical Properties in a Series of HCV Inhibitors.

A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).

Borabicyclo[3.3.2]decanes and the stereoselective asymmetric synthesis of 1,3-diol stereotriads from 1,3-diborylpropenes.

The synthesis of mixed borabicyclodecane (BBD)-derived 1,3-diborylpropenes (trans-1) is described. These new bimetallic reagents are effective for the selective asymmetric allylboration first of ketones (or ketimines) and second of aldehydes (or aldimines). Formed as a thermodynamic mixture of trans regioisomers from cis-1 through a series of 1,3-borotropic shifts, only trans-1 undergoes the monoallylation of ketones. After this single addition, this process is effectively shut down after the reaction of the 10-Ph-9-BBD portion in 1. Serving as a molecular gate, the rearranged 10-TMS-9-BBD trans-allylborane intermediate 11 reacts only after an aldehyde (or aldimine) is added. This allylation fixes the last two stereogenic centers of the 2-vinyl-1,3-diol stereotriad, ultimately resulting in 16 (or 1,3-amino alcohols) in 50-72% yield (>98% ee) as single observable diastereomers. These reagents 1 uniquely function as the equivalent of 1,1-bimetallic allylic reagents, adding sequentially first to ketones and second to aldehydes.

Highly enantioselective [4 + 2] cycloaddition reactions catalyzed by a chiral N-methyl-oxazaborolidinium cation.

The reaction of lithium aryl borohydrides with salts of beta-amino alcohols provides a new route for the synthesis of oxazaborolidines. This method also leads to the first synthesis of hitherto elusive N-methyl oxazaborolidine cations, specifically the cationic proline derivative 3. Compound 3 is a strong chiral Lewis acid which is very effective for catalysis of [4 + 2]-cycloaddition reactions in good yield and with high enantioselectivity. Several diverse examples illustrate the scope of these catalytic reactions.

Nonracemic 3 degrees-carbamines from the asymmetric allylboration of N-trimethylsilyl ketimines with B-allyl-10-phenyl-9-borabicyclo[3.3.2]decanes.

The simple and efficient asymmetric synthesis of 3 degrees -carbamines 7 from N-TMS enamines (3) and either enantiomeric form of beta-allyl-10-(phenyl)-9-borabicyclo[3.3.2]decane (1) is reported. The high reactivity (<1 h, -78 degrees C) and enantioselectivity (60-98% ee) of these substrates can be attributed to the fact that the complexation of 3 with 1 facilitates its isomerization to the corresponding syn-N-TMS ketimine complex from which allylation can occur. In addition to providing the homoallylic amines 7 with predictable stereochemistry, the procedure also permits the efficient recovery of the chiral boron moiety (50-65%) as air-stable crystalline pseudoephedrine complexes 8, which are directly converted back to 1 with allylmagnesium bromide in ether (98%).

N-propargylamides via the asymmetric michael addition of B-alkynyl-10-TMS-9- borabicyclo[3.3.2]decanes to N-acylimines.

[Structure: see text] The asymmetric synthesis of N-propargylamides through Michael addition of the alkynylborane 1 to N-acylimines is reported. The N-acetylimines provide the best substrates for the process exhibiting high selectivity (56-95% ee) with predictable stereochemistry. In several cases, 5 crystallizes in essentially pure form (97-99% ee) and a single-crystal X-ray structure was also obtained for 5g (R1=R2=Me, R3=o-Cl-C6C4). The process regenerates 4 for its direct conversion back to 1 and facilitates the efficient recovery of the pseudoephedrine.

B-allyl-10-Ph-9-borabicyclo[3.3.2]decanes: strategically designed for the asymmetric allylboration of ketones.

The simple and efficient syntheses of B-allyl-10-(phenyl)-9-borabicyclo[3.3.2]decane (1) in both enantiomeric forms are reported. The remarkable enantioselectivity (81-99% ee) of these reagents in the allylboration process at -78 degrees C is only modestly diminished when the process is conducted at 0 degrees C, a phenomenon attributable to its rigid bicyclic structure. In addition to providing the homoallylic alcohols 6 efficiently (70-92%), the procedure also permits the efficient recovery of the chiral boron moiety (67-82%) as an air-stable crystalline N-methylpseudoephedrine complex 4 for the direct regeneration of 1 with allylmagnesium bromide in ether (98%). The reagent gives predictable stereochemistry, providing a strategically designed "chiral pocket" which is particularly receptive to leading methyl groups (e.g., methyl ethyl ketone, 87% ee).

Asymmetric allyl- and crotylboration with the robust, versatile, and recyclable 10-TMS-9-borabicyclo[3.3.2]decanes.

The remarkable versatility and selectivity of the 10-(trimethylsilyl)-9-borabicyclo[3.3.2]decanes (10-TMS-9-BBDs) in the allyl- and crotylboration of representative aldehydes are reported. The new reagents are prepared through air-stable crystalline pseudoephedrine borinic ester complexes of the 10-TMS-9-BBDs (4), which are available in 63% overall yield from B-MeO-9-BBN through a simple two-step procedure. These complexes 4 are directly converted to the corresponding B-allyl-10-TMS-9-BBDs (1) with allylmagnesium bromide, which either can be isolated (98%) or used in situ for the allylations. The remarkable enantioselectivity (96 to > or =99% ee) of these reagents in the rapid (<3 h), asymmetric allylboration process at -78 degrees C is only slightly diminished when it is conducted at 25 degrees C, a phenomenon attributable to its rigid bicyclic structure. In addition to providing the homoallylic alcohols 6 efficiently (68-80%), the procedure also permits the efficient recovery of 4 (68-84%) for the direct regeneration of 1. Alternatively, an oxidative workup procedure can be used for the preparation of 6. The reagent gives predictable stereochemistry and exhibits an extremely high level of reagent control in the allylboration of d-glyceraldehyde acetonide. A simple and efficient procedure has been developed for the preparation of all four geometric and enantiomeric isomers of the B-crotyl-10-TMS-9-BBDs (10) from optically pure enantiomers of B-MeO-10-TMS-9-BBD (3). These reagents 10 also add rapidly (<3 h) and efficiently to representative aldehydes at -78 degrees C, providing ready access to all four of the possible stereoisomers of the beta-methyl homoallylic alcohols 12-15 (69-92%) in high dr (> or =98:2) and ee (94-99%).