Anti-TNF Thioester Glucocorticoid Antibody-Drug Conjugate Fully Inhibits Inflammation with Minimal Effect on Systemic Corticosterone Levels in a Mouse Arthritis Model.

We describe the discovery of a thioester-containing glucocorticoid receptor modulator (GRM) payload and the corresponding antibody-drug conjugate (ADC). Payload 6 was designed for rapid hepatic inactivation to minimize systemic exposure of nonconjugated GRM. Mouse PK indicated that 6 is cleared 10-fold more rapidly than a first-generation GRM payload, resulting in 10-fold lower exposure and 3-fold decrease in Cmax. The anti-mTNF conjugate ADC5 fully inhibited inflammation in mouse contact hypersensitivity with minimal effects on corticosterone, a biomarker for systemic GRM effects, at doses up to and including 100 mg/kg. Concomitant inhibition of P1NP suggests potential delivery to cells involved in the remodeling of bone, which may be a consequence of TNF-targeting or bystander payload effects. Furthermore, ADC5 fully suppressed inflammation in collagen-induced arthritis mouse model after one 10 mg/kg dose for 21 days. The properties of the anti-hTNF conjugate were suitable for liquid formulation and may enable subcutaneous dosing.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors.

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-ß-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).

Synthesis and evaluation of 2'-dihalo ribonucleotide prodrugs with activity against hepatitis C virus.

Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2'-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2'-deoxy-2'-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver. AIBEE prodrug 18c was compared with sofosbuvir (1) by co-dosing both compounds by oral administration in dog (5 mg/kg each) and measuring liver concentrations of the active triphosphate metabolite at both 4 and 24 h post dosing. In this study, 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir (1) at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.

[The study of risk factors of nasal septal perforation in rats].

OBJECTIVE: To study the risk factors and interaction of nasal septal perforation (NSP) in rats. METHOD: Animals (n=120) that underwent unilateral nasal obstruction using Merocel nasal packing or gelfoam with/without standard staphylococcus aureus inoculation were observed for the formation of NSP at 2, 3, 5, and 7 days after operation by endoscope system. Following sacrifice at 7 days, the obtained nasal secretions were prepared for bacterial culture. Experimental interventions were compared with normal controls (n=10). RESULT: Perforation of nasal septum was observed in 80% of the animals accepted nasal obstruction using Merocel nasal packing with standard staphylococcus aureus inoculation in 3 days (P < 0.01), while in 70% of those using abacterial Merocel nasal packing in 5 days (P < 0.05) and no significant difference than that of before (P > 0.05). There was a weak region in anteroinferior nasal septum in rats, which the almost NSPs located in. The position of NSP does not overlap Merocel. CONCLUSION: The interaction of risk factors contributes to NSP. The occurrence of NSP mainly depends on the construction of nasal septum, while dysaemia is also necessary. Obstruction of nasal drainage and infection promote the development of NSP.

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists.

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X(7) antagonists. These compounds were assayed for activity at both the human and rat P2X(7) receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X(7) receptors. Compounds 12 and 38 displayed hP2X(7)pIC(50)s>7.8 with less than 2-fold difference in potency at the rP2X(7).

Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

Synthesis and SAR of indazole-pyridine based protein kinase B/Akt inhibitors.

A series of heteroaryl-pyridine containing inhibitors of Akt are reported. The synthesis and structure-activity relationships are discussed, leading to the discovery of a indazole-pyridine analogue (K(i)=0.16 nM). These compounds bind in the ATP binding site, are potent, ATP competitive, and reversible inhibitors of Akt activity. No selectivity amongst the Akt isoforms is observed for this analogue, but there is good selectivity against an panel of other kinases. It is least selective for other members of the AGC family of kinases but is nonetheless 40-fold selective for Akt over PKA. The compound shows cellular activity and significantly slows tumor growth in vivo.

Identification of a novel 3,5-disubstituted pyridine as a potent, selective, and orally active inhibitor of Akt1 kinase.

Based on lead compounds 2 and 3 a series of 3,5-disubstituted pyridines have been designed and evaluated for inhibition of AKT/PKB. Modifications at the 3 position of the pyridine ring led to a number of potent compounds with improved physical properties, resulting in the identification of 11g as a promising, orally active Akt inhibitor. The synthesis, structure-activity relationship studies, and pharmacokinetic data are presented in this paper.

Discovery of trans-3,4'-bispyridinylethylenes as potent and novel inhibitors of protein kinase B (PKB/Akt) for the treatment of cancer: Synthesis and biological evaluation.

A novel series of Akt/PKB inhibitors derived from a screening lead (1) has been prepared. The novel trans-3,4'-bispyridinylethylenes described herein are potent inhibitors of Akt/PKB with IC(50) values in the low double-digit nanomolar range against Akt1. Compound 2q shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to modest selectivity against closely related kinases in the AGC and CMGC families. The cellular activities including inhibition of cell growth and phosphorylation of downstream target GSK3 are also described. The X-ray structure of compound 2q complexed with PKA in the ATP binding site was determined.

Synthesis and structure-activity relationship of 3,4'-bispyridinylethylenes: discovery of a potent 3-isoquinolinylpyridine inhibitor of protein kinase B (PKB/Akt) for the treatment of cancer.

Structure-based design and synthesis of the 3,4'-bispyridinylethylene series led to the discovery of 3-isoquinolinylpyridine 13a as a potent PKB/Akt inhibitor with an IC(50) of 1.3nM against Akt1. Compound 13a shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to marginal selectivity against closely related kinases in the AGC and CMGC families. Moreover, 13a demonstrates potent cellular activity comparable to staurosporine, with IC(50) values of 0.42 and 0.59microM against MiaPaCa-2 and the Akt1 overexpressing FL5.12-Akt1, respectively. Inhibition of phosphorylation of the Akt downstream target GSK3 was also observed in FL5.12-Akt1 cells with an EC(50) of 1.5microM. The X-ray structures of 12 and 13a in complex with PKA in the ATP-binding site were determined.

Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo.

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (K(i) = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses approximately 2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.

Benzimidazolones and indoles as non-thiol farnesyltransferase inhibitors based on tipifarnib scaffold: synthesis and activity.

A series of analogs of tipifarnib (1) has been synthesized as inhibitors of FTase by substituting the benzimidazolones and indoles for the 2-quinolone of tipifarnib. The novel benzimidazolones are potent and selective FTase inhibitors (FTIs) with IC(50) values of the best compounds close to that of tipifarnib. The current series demonstrate good cellular activity as measured in their inhibiting the Ras processing in NIH-3T3 cells, with compounds 2c and 2f displaying EC(50) values of 18 and 22nM, respectively.

Design, synthesis, and activity of 4-quinolone and pyridone compounds as nonthiol-containing farnesyltransferase inhibitors.

As a part of our efforts to identify potent inhibitors of farnesyltransferase (FTase), modification of the structure of tipifarnib through structure-based design was undertaken by replacing the 2-quinolones with 4-quinolones and pyridones, and subsequent relocation of the D-ring to the N-methyl group on the imidazole ring. This study has yielded a novel series of potent and selective FTase inhibitors. The X-ray structure of tipifarnib (1) in complex with FTase was described.

Synthesis and activity of 1-aryl-1'-imidazolyl methyl ethers as non-thiol farnesyltransferase inhibitors.

A series of imidazole-containing methyl ethers (4-5) have been designed and synthesized as potent and selective farnesyltransferase inhibitors (FTIs) by transposition of the D-ring to the methyl group on the imidazole of the previously reported FTIs 3. Several compounds such as 4h and 5b demonstrate superior enzymatic activity to the current benchmark compound tipifarnib (1) with IC(50) values in the lower subnanomolar range, while maintaining excellent cellular activity comparable to tipifarnib. The compounds are characterized as being simple, easier to make, and possess no chiral center involved.

Discovery of potent imidazole and cyanophenyl containing farnesyltransferase inhibitors with improved oral bioavailability.

A pyridyl moiety was introduced into a previously developed series of farnesyltransferase inhibitors containing imidazole and cyanophenyl (such as 4), resulting in potent inhibitors with improved pharmacokinetics.