Stereodivergent Synthesis of Biologically Active Spironucleoside Scaffolds via Catalytic Cyclopropanation of 4-exo-Methylene Furanosides.

Cyclopropane fusion of the only rotatable carbon-carbon bond in furanosyl nucleosides (i.e., exocyclic 4'-5') is a powerful design strategy to arrive at conformationally constrained analogues. Herein, we report a direct stereodivergent route toward the synthesis of the four possible configurations of 4-spirocyclopropane furanoses, which have been transformed into the corresponding 4'-spirocyclic adenosine analogues. The latter showed differential inhibition of the protein methyltransferase PRMT5-MEP50 complex, with one analogue inhibiting more effectively than adenosine itself, demonstrating the utility of rationally probing 4'-5' side chain orientations.

Regio- and Stereoselective Synthesis of C-4' Spirocyclobutyl Ribofuranose Scaffolds and Their Use as Biologically Active Nucleoside Analogues.

Novel C-4',C-5' cyclobutane-fused spirocyclic ribonucleoside analogues were prepared. Thermal [2 + 2] cycloaddition between dichloroketene and readily derived 4'-exo-methylene furanoses afforded a first entry to the required constrained ribofuranoses, relying on a carbonyl transposition sequence. Alternatively, an unusual stereoselective ionic [2 + 2] cycloaddition using methyl propiolate promoted by methylaluminoxane gave a complementary, more direct approach to such ribofuranoses. Further conversion to the constrained adenosine analogues revealed promising structure-dependent inhibition of the protein methyltransferase PRMT5:MEP50 complex in the (sub)micromolar range.

Discovery and Pharmacological Characterization of JNJ-64619178, a Novel Small-Molecule Inhibitor of PRMT5 with Potent Antitumor Activity.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.

A Chemical Probe for the Methyl Transferase PRMT5 with a Novel Binding Mode.

Protein arginine methyltransferase 5 (PRMT5) is an enzyme that can symmetrically dimethylate arginine residues in histones and nonhistone proteins by using S-adenosyl methionine (SAM) as the methyl donating cofactor. We have designed a library of SAM analogues and discovered potent, cell-active, and selective spiro diamines as inhibitors of the enzymatic function of PRMT5. Crystallographic studies confirmed a very interesting binding mode, involving protein flexibility, where both the cofactor pocket and part of substrate binding site are occupied by these inhibitors.

Synthesis and Reactivity of Spirocarbocycles as Scaffolds for Nucleoside Analogues.

A novel class of substituted spiro[3.4]octanes can be accessed via a [2 + 2]-cycloaddition of dichloroketene on a readily prepared exo-methylene cyclopentane building block. This reaction sequence was found to be robust on a multigram scale and afforded a central spirocyclobutanone scaffold for carbocyclic nucleosides. The reactivity of this constrained building block was evaluated and compared to the corresponding 4'-spirocyclic furanose analogues. Density functional theory calculations were performed to support the observed selectivity in the carbonyl reduction of spirocyclobutanone building blocks. Starting from novel spirocyclic intermediates, we exemplified the preparation of an undescribed class of carbocyclic nucleoside analogues and provided a proof of concept for application as inhibitors for the protein methyltransferase target PRMT5.

Preparation of 4'-Spirocyclobutyl Nucleoside Analogues as Novel and Versatile Adenosine Scaffolds.

Despite the large variety of modified nucleosides that have been reported, the preparation of constrained 4'-spirocyclic adenosine analogues has received very little attention. We discovered that the [2+2]-cycloaddition of dichloroketene on readily available 4'-exo-methylene furanose sugars efficiently results in the diastereoselective formation of novel 4'-spirocyclobutanones. The reaction mechanism was investigated via density functional theory (DFT) and found to proceed either via a non-synchronous or stepwise reaction sequence, controlled by the stereochemistry at the 3'-position of the sugar substrate. The obtained dichlorocyclobutanones were converted into nucleoside analogues, providing access to a novel class of chiral 4'-spirocyclobutyl adenosine mimetics in eight steps from commercially available sugars. Assessment of the biological activity of designed 4'-spirocyclic adenosine analogues identified potent inhibitors for protein methyltransferase target PRMT5.

Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS.

Synthesis of 3-amino-4-fluoropyrazoles.

Fluorinated pyrazoles bearing additional functional groups that allow further functionalization are of considerable interest as building blocks in medicinal chemistry. The developed synthetic strategy for new 3-amino-4-fluoropyrazoles consists of a monofluorination of ß-methylthio-ß-enaminoketones using 1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor) toward the corresponding monofluorinated enaminoketones, followed by condensation with different hydrazines.

Characterization of 2-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-5-thiazolemethanol (JNJ-1930942), a novel positive allosteric modulator of the {alpha}7 nicotinic acetylcholine receptor.

The α(7) nicotinic acetylcholine receptor (nAChR) is a potential therapeutic target for the treatment of cognitive deficits associated with schizophrenia, Alzheimer's disease, Parkinson's disease, and attention-deficit/hyperactivity disorder. Activation of α(7) nAChRs improved sensory gating and cognitive function in animal models and in early clinical trials. Here we describe the novel highly selective α(7) nAChR positive allosteric modulator, 2-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-5-thiazolemethanol (JNJ-1930942). This compound enhances the choline-evoked rise in intracellular Ca(2+) levels in the GH4C1 cell line expressing the cloned human α(7) nAChR. JNJ-1930942 does not act on α4ß2, α3ß4 nAChRs or on the related 5-HT3A channel. Electrophysiological assessment in the GH4C1 cell line shows that JNJ-1930942 increases the peak and net charge response to choline, acetylcholine, and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (PNU-282987). The potentiation is obtained mainly by affecting the receptor desensitization characteristics, leaving activation and deactivation kinetics as well as recovery from desensitization relatively unchanged. Choline efficacy is increased over its full concentration response range, and choline potency is increased more than 10-fold. The potentiating effect is α(7) channel-dependent, because it is blocked by the α(7) antagonist methyllycaconitine. Moreover, in hippocampal slices, JNJ-1930942 enhances neurotransmission at hippocampal dentate gyrus synapses and facilitates the induction of long-term potentiation of electrically evoked synaptic responses in the dentate gyrus. In vivo, JNJ-1930942 reverses a genetically based auditory gating deficit in DBA/2 mice. JNJ-1930942 will be a useful tool to study the therapeutic potential of α(7) nAChR potentiation in central nervous system disorders in which a deficit in α(7) nAChR neurotransmission is hypothesized to be involved.

Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.

The development of HIV integrase (IN) strand transfer inhibitors (INSTIs) and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) intasome with raltegravir and elvitegravir, establishing the general INSTI binding mode. We now present an expanded set of cocrystal structures containing PFV intasomes complexed with first- and second-generation INSTIs at resolutions of up to 2.5 Å. Importantly, the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the INSTI-bound intasome active site. We show that like the Q148H/G140S and N155H HIV-1 IN variants, the analogous S217H and N224H PFV INs display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in INSTI-free and -bound forms revealed that the amino acid substitutions necessitate considerable conformational rearrangements within the IN active site to accommodate an INSTI, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an interaction between HIV-1 IN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/A mutations in drug-resistant viral strains.

Synthesis of 5-amino- and 5-hydroxy-3,3-difluoropiperidines.

Synthetic routes toward new 5-amino- and 5-hydroxy-3,3-difluoropiperidines, which are of high interest as building blocks in medicinal chemistry, are described. The key step involves the N-halosuccinimide-induced cyclization of 2,2-difluoro-4-pentenylamines toward 5-halo-3,3-difluoropiperidines, which were used to synthesize 5-amino-3,3-difluoropiperidine. In a second strategy, iodolactonization of 2,2-difluoro-4-pentenoic acid gave the corresponding γ-lactone, which was transformed into 5-hydroxy-3,3-difluoropiperidine.

Synthesis of aminomethylated 4-fluoropiperidines and 3-fluoropyrrolidines.

A short and efficient synthesis of 4-aminomethyl-4-fluoropiperidines and 3-aminomethyl-3-fluoropyrrolidines is described. These fluorinated azaheterocycles are of specific interest as bifunctional building blocks for fluorinated pharmaceutical compounds. The key step of the synthetic pathway involves the regioselective bromofluorination of N-Boc-4-methylenepiperidine and 3-methylenepyrrolidine using Et(3)N.3HF and NBS.

Synthesis of 4-substituted 3,3-difluoropiperidines.

Synthetic strategies toward 4-substituted 3,3-difluoropiperidines were evaluated. 4-Alkoxymethyl- and 4-aryloxymethyl-3,3-difluoropiperidines were synthesized via 1,4-addition of ethyl bromodifluoroacetate to 3-substituted acrylonitriles in the presence of copper powder, followed by borane reduction of the cyano substituent, lactamization, and reduction of the lactam. This method was applied to establish the synthesis of N-protected 3,3-difluoroisonipecotic acid, a fluorinated gamma-amino acid. 4-Benzyloxy-3,3-difluoropiperidine was prepared using an analogous methodology and was converted to N-protected 3,3-difluoro-4,4-dihydroxypiperidine, a compound with high potential as a building block in medicinal chemistry.

Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes.

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

Respiratory syncytial virus fusion inhibitors. Part 7: structure-activity relationships associated with a series of isatin oximes that demonstrate antiviral activity in vivo.

A series of bezimidazole-isatin oximes were prepared and profiled as inhibitors of respiratory syncytial virus (RSV) replication in cell culture. Structure-activity relationship studies were directed toward optimization of antiviral activity, cell permeability and metabolic stability in human liver micorosomes (HLM). Parallel combinatorial synthetic chemistry was employed to functionalize isatin oximes via O-alkylation which quickly identified a subset of small, lipophilic substituents that established good potency for the series. Further optimization of the isatin oxime derivatives focused on introduction of nitrogen atoms to the isatin phenyl ring to provide a series of aza-isatin oximes with significantly improved PK properties. Several aza-isatin oximes analogs displayed targeted metabolic stability in HLM and permeability across a confluent monolayer of CaCo-2 cells. These studies identified several compounds, including 18i, 18j and 18n that demonstrated antiviral activity in the BALB/c mouse model of RSV infection following oral dosing.

Synthesis of 1-Boc-3-fluoroazetidine-3-carboxylic acid.

Synthetic strategies toward 3-fluoroazetidine-3-carboxylic acid, a new cyclic fluorinated beta-amino acid with high potential as building block in medicinal chemistry, were evaluated. The successful pathway includes the bromofluorination of N-(diphenylmethylidene)-2-(4-methoxyphenoxymethyl)-2-propenylamine, yielding 1-diphenylmethyl-3-hydroxymethyl-3-fluoroazetidine after reduction of the imino bond, ring closure, and removal of the 4-methoxybenzyl group. Changing the N-protecting group to a Boc-group allows further oxidation to 1-Boc-3-fluoroazetidine-3-carboxylic acid, a new fluorinated heterocyclic amino acid.

New synthesis of 3-fluoropyrroles.

5-Alkoxymethyl-2-aryl-3-fluoro-1H-pyrroles and 2-aryl-3-fluoro-1H-pyrrole-5-carbaldehydes were efficiently prepared from the corresponding 2-aryl-5-(bromomethyl)-1-pyrrolines via electrophilic alpha,alpha-difluorination of the imino bond, using Selectfluor (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bistetrafluoroborate) and subsequent aromatization by dehydrofluorination. This methodology provides a new and easy entry toward various new 3-fluorinated pyrroles.

New entries toward 3,3-difluoropiperidines.

Difluoropiperidines attract considerable interest from organic and medicinal chemists, but their synthesis is often problematic. This paper describes a new synthetic pathway toward valuable 3,3-difluoropiperidines starting from suitable delta-chloro-alpha,alpha-difluoroimines. The latter imines can be synthesized via electrophilic fluorination of the corresponding delta-chloroimines using NFSI (N-fluorodibenzenesulfonimide) in acetonitrile. After hydride reduction of the imino bond and subsequent intramolecular substitution of the chloride atom, new 3,3-difluoropiperidines were obtained in good yields. In addition, this methodology was applied to establish the first synthesis of N-protected 3,3-difluoropipecolic acid, a new fluorinated amino acid.