Discovery of fused tricyclic core containing HCV NS5A inhibitors with pan-genotype activity.

HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat.

Optimization of potency and pharmacokinetics of tricyclic indole derived inhibitors of HCV NS5B polymerase. Identification of ester prodrugs with improved oral pharmacokinetics.

HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current efforts are directed towards identification of novel direct acting antiviral targeting different mechanism of actions which could become part of all oral therapies. We recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase that bound to the enzyme close to the active site. In this manuscript we describe further optimization of potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a dimethyl ethyl amino ester prodrug.

Discovery of an irreversible HCV NS5B polymerase inhibitor.

The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 µM), highly selective, and safe in in vitro and in vivo assays.

Discovery of novel tricyclic indole derived inhibitors of HCV NS5B RNA dependent RNA polymerase.

The characterization of HCV genome has identified various vital functional proteins involved in the life cycle of hepatitis C virus. This has resulted in many novel enzymatic targets that are potential for development of therapeutic agents. The HCV RNA dependent RNA polymerase (HCV NS5B) is one such essential enzyme for HCV replication that has been well characterized and studied by various groups to develop novel therapies for hepatitis C. In this paper, we describe our efforts towards the identification and structure-activity relationship (SAR) of novel tricyclic indole derivatives that bind close to the palm site of the NS5B polymerase. X-ray crystal structure of an inhibitor bound to the polymerase is also described.

Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during embryonic stem cell differentiation.

BACKGROUND: Enhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27. RESULTS: Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. CONCLUSIONS: Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

Discovery and Characterization of ZL-2201, a Potent, Highly Selective, and Orally Bioavailable Small-molecule DNA-PK Inhibitor.

DNA-dependent protein kinase (DNA-PK), a driver of the non-homologous end-joining (NHEJ) DNA damage response pathway, plays an instrumental role in repairing double-strand breaks (DSB) induced by DNA-damaging poisons. We evaluate ZL-2201, an orally bioavailable, highly potent, and selective pharmacologic inhibitor of DNA-PK activity, for the treatment of human cancerous malignancies. ZL-2201 demonstrated greater selectivity for DNA-PK and effectively inhibited DNA-PK autophosphorylation in a concentration- and time-dependent manner. Initial data suggested a potential correlation between ataxia-telangiectasia mutated (ATM) deficiency and ZL-2201 sensitivity. More so, ZL-2201 showed strong synergy with topoisomerase II inhibitors independent of ATM status in vitro. In vivo oral administration of ZL-2201 demonstrated dose-dependent antitumor activity in the NCI-H1703 xenograft model and significantly enhanced the activity of approved DNA-damaging agents in A549 and FaDu models. From a phosphoproteomic mass spectrometry screen, we identified and validated that ZL-2201 and PRKDC siRNA decreased Ser108 phosphorylation of MCM2, a key DNA replication factor. Collectively, we have characterized a potent and selective DNA-PK inhibitor with promising monotherapy and combinatory therapeutic potential with approved DNA-damaging agents. More importantly, we identified phospho-MCM2 (Ser108) as a potential proximal biomarker of DNA-PK inhibition that warrants further preclinical and clinical evaluation. Significance: ZL-2201, a potent and selective DNA-PK inhibitor, can target tumor models in combination with DNA DSB-inducing agents such as radiation or doxorubicin, with potential to improve recurrent therapies in the clinic.

Differential susceptibility to axonopathy in necrotic and non-necrotic perinatal white matter injury.

BACKGROUND AND PURPOSE: White matter injury (WMI) is the leading cause of brain injury in preterm survivors and results in myelination failure. Although axonal degeneration occurs in necrotic lesions, the role of axonopathy in myelination failure remains controversial for diffuse non-necrotic WMI, which is currently the major form of WMI. We determined the burden of axonopathy in diffuse lesions. METHODS: We analyzed WMI in a preterm fetal sheep model of global cerebral ischemia that replicates the relative burden of necrotic and non-necrotic human WMI. WMI was analyzed at 1 or 2 weeks after ischemia and identified by ex vivo high-field (11.7 Tesla) magnetic resonance imaging of fixed brain tissue. Axonal integrity was analyzed by immunohistochemical detection of axon injury markers and by transmission electron microscopy to quantify axon loss and degeneration in magnetic resonance imaging-defined lesions. RESULTS: Axonal degeneration, defined by staining for neurofilament protein and ß-amyloid precursor protein, was restricted to discrete necrotic foci with robust microglial activation. Unexpectedly, axonal degeneration was not visualized in the major form of WMI, which comprised large non-necrotic lesions with diffuse reactive astrogliosis. In these major lesions, quantitative electron microscopy studies confirmed no significant differences in the density of intact and degenerating axons or in the distribution of axon diameters relative to controls. CONCLUSIONS: The mechanism of myelination failure differs significantly in perinatal WMI dependent on the burden of necrosis. Axonopathy is associated with focal necrotic injury but not with primary diffuse non-necrotic lesions, which supports that intact axons in the primary lesions are potential targets for myelination.

II. Novel HCV NS5B polymerase inhibitors: discovery of indole C2 acyl sulfonamides.

Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase (NS5B IC(50)=0.053µM, replicon EC(50)=4.8µM), is described. Initial screening identified an acyl sulfonamide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in identification of acyl sufonamide analog 7q (NS5B IC(50)=0.039µM, replicon EC(50)=0.011µM) with >100-fold improved replicon activity.

I. Novel HCV NS5B polymerase inhibitors: discovery of indole 2-carboxylic acids with C3-heterocycles.

SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 µM, replicon EC(50)>100 µM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 µM, replicon EC(50)=1.4 µM) and 7r (NS5B IC(50)=0.017 µM, replicon EC(50)=0.3 µM) with improved enzyme and replicon activity.

P4 capped amides and lactams as HCV NS3 protease inhibitors with improved potency and DMPK profile.

SAR studies on the extension of P3 unit of Boceprevir (1, SCH 503034) with amides and lactams and their synthesis is described. Extensive SAR studies resulted in the identification of 36 bearing 4, 4-dimethyl lactam as the new P4 cap unit with improved potency (K(i)( *)=15nM, EC 90=70nM) and pharmacokinetic properties (Rat AUC (PO)=3.52microMh) compared to 1.

The introduction of P4 substituted 1-methylcyclohexyl groups into Boceprevir: a change in direction in the search for a second generation HCV NS3 protease inhibitor.

In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.

Challenges in modern drug discovery: a case study of boceprevir, an HCV protease inhibitor for the treatment of hepatitis C virus infection.

More than 170 million people worldwide are affected by the hepatitis C virus (HCV). The disease has been described as a "silent epidemic" and "a serious global health crisis". HCV infection is a leading cause of chronic liver disease such as cirrhosis, carcinoma, or liver failure. The current pegylated interferon and ribavirin combination therapy is effective in only 50% of patients. Its moderate efficacy and apparent side effects underscore the need for safer and more effective treatments. The nonstructural NS3 protease of the virus plays a vital role in the replication of the HCV virus. The development of small molecule inhibitors of NS3 protease as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, it is a daunting task. The protease has a shallow and solvent-exposed substrate binding region, and the inhibitor binding energy is mainly derived from weak lipophilic and electrostatic interactions. Moreover, lack of a robust in vitro cell culture system and the absence of a convenient small animal model have hampered the assessment of both in vitro and in vivo efficacy of any antiviral compounds. Despite the tremendous challenges, with access to a recently developed cell-based replicon system, major progress has been made toward a more effective small molecule HCV drug. In our HCV program, facing no leads from our screening effort, a structure-based drug design approach was carried out. An alpha-ketoamide-type electrofile was designed to trap the serine hydroxyl of the protease. Early ketoamide inhibitors mimicked the structures of the peptide substrates. With the aid of X-ray structures, we successfully truncated the undecapeptide lead that had a molecular weight of 1265 Da stepwise to a tripeptide with a molecular weight of 500 Da. In an attempt to depeptidize the inhibitors, various strategies such as hydrazine urea replacement of amide bonds and P2 to P4 and P1 to P3 macrocyclizations were examined. Further optimization of the tripeptide inhibitors led to the identification of the best moieties for each site: primary ketoamide at P', cyclobutylalanine at P1, gem-dimethylcyclopropylproline at P2, tert-leucine at P3, and tert-butyl urea as capping agent. The combination of these led to the discovery of compound 8 (SCH 503034, boceprevir), our clinical candidate. It is a potent inhibitor in both enzyme assay (Ki* = 14 nM) and cell-based replicon assay (EC 90 = 0.35 microM). It is highly selective (2200x) against human neutrophil elastase (HNE). Boceprevir is well tolerated in humans and demonstrated antiviral activity in phase I clinical trials. It is currently in phase II trials. This Account details the complexity and challenges encountered in the drug discovery process.

Novel potent inhibitors of hepatitis C virus (HCV) NS3 protease with cyclic sulfonyl P3 cappings.

Extensive SAR studies of the P3 capping group led to the discovery of a series of potent inhibitors with sultam and cyclic sulfonyl urea moieties as the P3 capping. The bicyclic thiophene-sultam or phenyl-sultam cappings were selected for further SAR development. Modification at the P3 side chain determined that the tert-butyl group was the best choice at that position. Optimization of P1 residue significantly improved potency and selectivity. The combination of optimal moieties at all positions led to the discovery of compound 33. This compound had the best overall profile in potency and PK profile: excellent K(i)(*) of 5.3 nM and activity in replicon (EC(90)) of 80 nM, extremely high selectivity of 6100, and a good rat PO AUC of 1.43 microMh.

Potent and selective small molecule NS3 serine protease inhibitors of Hepatitis C virus with dichlorocyclopropylproline as P2 residue.

Starting from a pentapeptide Hepatitis C virus NS3 protease inhibitor, a number of alpha-ketoamide inhibitors based on novel dichlorocyclopropylproline P2 core were synthesized and investigated for their HCV NS3 serine protease activity. The key intermediate 3,4-dichlorocyclopropylproline was obtained through a dichloro carbene insertion to 3,4-dehydroproline. The size of the molecules was reduced significantly through a series of truncations of the initial pentapeptide. By varying P1 side chain in length and size, potency and selectivity were improved. A variety of aliphatic carbamate and urea capping groups were examined. In general, compounds with urea cappings were more potent and selective than their carbamate counterparts. The most potent compound was a tert-butyl urea analog. Variations at P3 position were also investigated. Among the three residues incorporated, tert-leucine was clearly superior, leading to compounds that had excellent enzyme potency and selectivity. The most potent compound achieved cell-based replicon assay EC50 of 40 nM. The most promising compound of all had excellent potency in both enzyme (Ki* = 9 nM) and replicon assays (EC50 = 100 nM). Its bioavailabilities were above 10% in all three animal species (rats, monkeys, and dogs). It has provided a lead for future investigations.

GNE-781, A Highly Advanced Potent and Selective Bromodomain Inhibitor of Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP).

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.

Potent 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-based macrocyclic inhibitors of hepatitis C virus NS3 protease.

The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are alpha-ketoamide-type HCV serine protease inhibitors with modest potency. We envisioned that the 1,2,3,4-tetrahydroisoquinoline-3-carboxylamide (Tic) moiety could be cyclized to the P3 capping group. The resulting macrocycle could enhance the binding through its extra contact with the Ala156 methyl group. Macrocyclization could also provide a less peptidic HCV inhibitor. Synthesis started from dipeptide 5, which was obtained via a coupling of two amino acid derivatives. The N-terminal was capped as hept-6-enoylamide to give 6. Hydroboration of the double bond afforded alcohol 7, the precursor to the macrocycle 8. The macrocyclization was achieved under Mitsunobu conditions (PPh(3), ADDP). The macrocyclic acid 9 was then combined with appropriate right-hand fragments 12, 14, or 16, which was prepared from common intermediate 11. Finally, oxidation of alpha-hydroxyamide provided target molecule alpha-ketoamides 17, 18, and 21. The C-terminal esters were then elaborated to carboxylic acids 19 and 20, and amides 20 and 23. The inhibitors 17-23 were tested in HCV NS3 protease continuous assay. Tripeptide 17 was more potent than the larger acyclic tetrapeptide 1. The tetrapeptides 18-20 were as active as 17. Most significantly, the pentapeptides (21-23) were much better inhibitors (K(i) = 0.015-0.26 microM). The carboxylic acid (22) and amide (23) were 57-80 times more potent than the acyclic analogue 2. The X-ray crystal structure of compound 23 bound to the protease revealed that the macrocycle adopted a donutlike conformation and had close contact with the Ala156 methyl group. The ketone carbonyl formed a reversible covalent bond with Ser139. The n-propyl of P1 novaline and the aromatic ring of P2' phenylglycine formed a C-shaped clamp around the Lys136 side chain.

Novel potent hepatitis C virus NS3 serine protease inhibitors derived from proline-based macrocycles.

The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

Design, synthesis, and biological activity of m-tyrosine-based 16- and 17-membered macrocyclic inhibitors of hepatitis C virus NS3 serine protease.

The limited efficacy and considerable side effects of currently available therapies for the treatment of hepatitis C virus (HCV) infection have prompted significant efforts toward the development of safe and effective new therapeutics. The pentapeptide alpha-ketoamides of type 1 were weak HCV inhibitors with a binding constant, Ki, above 5 microM. We envisioned that cyclization of a P2 phenyl side chain to a P3 capping group could enhance binding through an interaction of the resulting macrocycle with the methyl group of Ala156 on the enzyme backbone. The macrocyclic dipeptide moiety would also decrease the peptidic nature of the inhibitors. The synthesis of macrocyclic HCV inhibitors started from m-tyrosine methyl ester. Two consecutive couplings, first, with Boc-cyclohexylglycine and, then, with hept-6-enoic acid, provided compound 6. The alkene was converted to an alcohol via hydroboration. The key macrocyclization of phenol alcohol 7 was achieved through a Mitsunobu reaction. Both 16- and 17-membered macrocycles (8 and 21) were prepared. After hydrolysis, the macrocyclic acids (15 and 22) were coupled to the right-hand tripeptide (14) to afford alpha-hydroxyamides, which upon Dess-Martin periodinane oxidation furnished the desired alpha-ketoamides. Esters, acids, and amides were incorporated at the C-terminal of these peptides. These inhibitors were tested in an HCV protease continuous assay. The binding constants (Ki) indicated that the 16-membered macrocyclic inhibitors (23 and 24) were less potent than the 17-membered analogues (16-19). It was also evident that C-terminal acids (i.e., 17) and amides (18 and 19) (Ki range: 0.16-0.31 microM) were much better inhibitors than tert-butyl esters (16 and 23). The X-ray crystal structure of compound 17 bound to the enzyme revealed that the macrocycle formed a "donut"-shaped ring around the methyl group of Ala156. P2' phenyl and P1 propyl groups wrapped around the Lys136 side chain, forming a "C"-shaped clamp. The 17-membered macrocyclic inhibitors 17-19 were significantly more potent than the acyclic pentapeptide 1.