In Vivo Half-Life Extension of BMP1/TLL Metalloproteinase Inhibitors Using Small-Molecule Human Serum Albumin Binders.

Reducing the required frequence of drug dosing can improve the adherence of patients to chronic treatments. Hence, drugs with longer in vivo half-lives are highly desirable. One of the most promising approaches to extend the in vivo half-life of drugs is conjugation to human serum albumin (HSA). In this work, we describe the use of AlbuBinder 1, a small-molecule noncovalent HSA binder, to extend the in vivo half-life and pharmacology of small-molecule BMP1/TLL inhibitors in humanized mice (HSA KI/KI). A series of conjugates of AlbuBinder 1 with BMP1/TLL inhibitors were prepared. In particular, conjugate c showed good solubility and a half-life extension of >20-fold versus the parent molecule in the HSA KI/KI mice, reaching half-lives of >48 h with maintained maximal inhibition of plasma BMP1/TLL. The same conjugate showed a half-life of only 3 h in the wild-type mice, suggesting that the half-life extension was principally due to specific interactions with HSA. It is envisioned that conjugation to AlbuBinder 1 should be applicable to a wide range of small molecule or peptide drugs with short half-lives. In this context, AlbuBinders represent a viable alternative to existing half-life extension technologies.

DNA-Encoded Library Technology-Based Discovery, Lead Optimization, and Prodrug Strategy toward Structurally Unique Indoleamine 2,3-Dioxygenase-1 (IDO1) Inhibitors.

We report the discovery of a novel indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor class through the affinity selection of a previously unreported indole-based DNA-encoded library (DEL). The DEL exemplar, spiro-chromane 1, had moderate IDO1 potency but high in vivo clearance. Series optimization quickly afforded a potent, low in vivo clearance lead 11. Although amorphous 11 was highly bio-available, crystalline 11 was poorly soluble and suffered disappointingly low bio-availability because of solubility-limited absorption. A prodrug approach was deployed and proved effective in discovering the highly bio-available phosphonooxymethyl 31, which rapidly converted to 11 in vivo. Obtaining crystalline 31 proved problematic, however; thus salt screening was performed in an attempt to circumvent this obstacle and successfully delivered greatly soluble and bio-available crystalline tris-salt 32. IDO1 inhibitor 32 is characterized by a low calculated human dose, best-in-class potential, and an unusual inhibition mode by binding the IDO1 heme-free (apo) form.

Development of a Selection Method for Discovering Irreversible (Covalent) Binders from a DNA-Encoded Library.

DNA-encoded libraries (DELs) have been broadly applied to identify chemical probes for target validation and lead discovery. To date, the main application of the DEL platform has been the identification of reversible ligands using multiple rounds of affinity selection. Irreversible (covalent) inhibition offers a unique mechanism of action for drug discovery research. In this study, we report a developing method of identifying irreversible (covalent) ligands from DELs. The new method was validated by using 3C protease (3CP) and on-DNA irreversible tool compounds (rupintrivir derivatives) spiked into a library at the same concentration as individual members of that library. After affinity selections against 3CP, the irreversible tool compounds were specifically enriched compared with the library members. In addition, we compared two immobilization methods and concluded that microscale columns packed with the appropriate affinity resin gave higher tool compound recovery than magnetic beads.

Design and Application of a DNA-Encoded Macrocyclic Peptide Library.

A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT).

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

Encoded library technology as a source of hits for the discovery and lead optimization of a potent and selective class of bactericidal direct inhibitors of Mycobacterium tuberculosis InhA.

Tuberculosis (TB) is one of the world's oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.

Discovery of thieno[3,2-d]pyrimidine-6-carboxamides as potent inhibitors of SIRT1, SIRT2, and SIRT3.

The sirtuins SIRT1, SIRT2, and SIRT3 are NAD(+) dependent deacetylases that are considered potential targets for metabolic, inflammatory, oncologic, and neurodegenerative disorders. Encoded library technology (ELT) was used to affinity screen a 1.2 million heterocycle enriched library of DNA encoded small molecules, which identified pan-inhibitors of SIRT1/2/3 with nanomolar potency (e.g., 11c: IC50 = 3.6, 2.7, and 4.0 nM for SIRT1, SIRT2, and SIRT3, respectively). Subsequent SAR studies to improve physiochemical properties identified the potent drug like analogues 28 and 31. Crystallographic studies of 11c, 28, and 31 bound in the SIRT3 active site revealed that the common carboxamide binds in the nicotinamide C-pocket and the aliphatic portions of the inhibitors extend through the substrate channel, explaining the observable SAR. These pan SIRT1/2/3 inhibitors, representing a novel chemotype, are significantly more potent than currently available inhibitors, which makes them valuable tools for sirtuin research.

Discovery of Clinical Candidate GSK1842799 As a Selective S1P1 Receptor Agonist (Prodrug) for Multiple Sclerosis.

To develop effective oral treatment for multiple sclerosis (MS), we discovered a series of alkyl-substituted biaryl amino alcohols as selective S1P1 modulators. One exemplar is (S)-2-amino-2-(5-(4-(octyloxy)-3-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)propan-1-ol (10, GSK1842799). Upon phosphorylation, the compound (10-P) showed subnanomole S1P1 agonist activity with >1000× selectivity over S1P3. The alcohol 10 demonstrated good oral bioavailability and rapid in vivo conversion to 10-P. Dosed orally at 0.1 mg/kg, 10 significantly reduced blood lymphocyte counts 6 h postdose, and at 3 mg/kg, 10 achieved efficacy equivalent to FTY720 in the mouse EAE model of MS. Further pharmacokinetic/pharmacodynamic (PK/PD) study with cynomolgus monkeys indicated that, after oral dosing of 10 at 3.8 mg/kg, the active phosphate reached plasma levels that are comparable to FTY-720 phosphate (FTY-P) revealed in human clinical pharmacokinetics studies. On the basis of the favorable in vitro ADME and in vivo PK/PD properties as well as broad toxicology evaluations, compound 10 (GSK1842799) was selected as a candidate for further clinical development.

Exploring amino acids derivatives as potent, selective, and direct agonists of sphingosine-1-phosphate receptor subtype-1.

In the quest to discover a potent and selective class of direct agonists to the sphingosine-1-phosphate receptor, we explored the carboxylate functional group as a replacement to previously reported lead phosphates. This has led to the discovery of potent and selective direct agonists with moderate to substantial in vivo lymphopenia. The previously reported selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) in the phenylamide and phenylimidazole scaffolds were crucial to obtaining selectivity for S1P receptor subtype 1 over 3.

The Discovery of VX-745: A Novel and Selective p38α Kinase Inhibitor.

The synthesis of novel, selective, orally active 2,5-disubstituted 6H-pyrimido[1,6-b]pyridazin-6-one p38α inhibitors is described. Application of structural information from enzyme-ligand complexes guided the selection of screening compounds, leading to the identification of a novel class of p38α inhibitors containing a previously unreported bicyclic heterocycle core. Advancing the SAR of this series led to the eventual discovery of 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one (VX-745). VX-745 displays excellent enzyme activity and selectivity, has a favorable pharmacokinetic profile, and demonstrates good in vivo activity in models of inflammation.

Exploration of amino alcohol derivatives as novel, potent, and highly selective sphingosine-1-phosphate receptor subtype-1 agonists.

In pursuit of a potent and highly selective sphingosine-1-phosphate receptor agonists with an improved in vivo conversion of the precursor to the active phospho-drug, we have utilized previously reported phenylamide and phenylimidazole scaffolds to identify a selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) within both pharmacophores. SEM and SEO have allowed for over 100 to 500-fold improvement in selectivity for S1P receptor subtype 1 over subtype 3. Utility of SEM and SEO and further SAR study allowed for discovery of a potent and selective preclinical candidate PPI-4955 (21b) with an excellent in vivo potency and dose responsiveness and markedly improved overall in vivo pharmacodynamic properties upon oral administration.

Synthesis and evaluation of arylalkoxy- and biarylalkoxy-phenylamide and phenylimidazoles as potent and selective sphingosine-1-phosphate receptor subtype-1 agonists.

In pursuit of potent and selective sphingosine-1-phosphate receptor agonists, we have utilized previously reported phenylamide and phenylimidazole scaffolds to explore extensive side-chain modifications to generate new molecular entities. A number of designed molecules demonstrate good selectivity and excellent in vitro and in vivo potency in both mouse and rat models. Oral administration of the lead molecule 11c (PPI-4667) demonstrated potent and dose-responsive lymphopenia.

Synthesis and evaluation of alkoxy-phenylamides and alkoxy-phenylimidazoles as potent sphingosine-1-phosphate receptor subtype-1 agonists.

In the design of potent and selective sphingosine-1-phosphate receptor agonists, we were able to identify two series of molecules based on phenylamide and phenylimidazole analogs of FTY-720. Several designed molecules in these scaffolds have demonstrated selectivity for S1P receptor subtype 1 versus 3 and excellent in vivo activity in mouse. Two molecules PPI-4621 (4b) and PPI-4691 (10a), demonstrated dose responsive lymphopenia, when administered orally.

Copper-catalyzed domino annulation approaches to the synthesis of benzoxazoles under microwave-accelerated and conventional thermal conditions.

Two domino annulation approaches for benzoxazole synthesis have been developed. In the first approach, copper-catalyzed intermolecular cross-coupling of 1,2-dihaloarenes with primary amides initially forms the Ar-N bond of the benzoxazole ring, followed by copper-catalyzed intramolecular cyclization to form the Ar-O bond. Benzoxazoles were formed in good yields for the reaction of 1,2-dibromobenzene, but the reaction was not regioselective for the reaction of 3,4-dibromotoluene. Furthermore, the method is limited by the availability of 1,2-dihaloarenes. As a result of these limitations, an alternative more versatile one-pot domino annulation strategy was developed involving reaction of 2-bromoanilines with acyl chlorides in the presence of Cs2CO3, catalytic CuI, and the non-acylatable ligand 1,10-phenanthroline. Under these conditions initial acylation of the aniline is followed by copper-catalyzed intramolecular cyclization of the resultant 2-haloanilide to form the Ar-O bond of the benzoxazole ring. Optimized conditions using microwave irradiation achieved much shorter reaction times than conventional heating (i.e., 210 degrees C for 15 min versus 95 degrees C for 24 h) and were applied to the synthesis of a small library of benzoxazoles. These copper-catalyzed approaches complement existing strategies for benzoxazole synthesis, which typically utilize 2-aminopheonls as precursors.

Peptide-heterocycle hybrid molecules: solid-phase-supported synthesis of substituted N-terminal 5-aminotetrazole peptides via electrocyclization of peptidic imidoylazides.

A method for the synthesis of polypeptides modified with a tetrazole ring at the N-terminus is described. Reaction of the N-terminal amino group of solid-supported peptides with arylisothiocyanates generates thiourea intermediates, which upon treatment with Mukaiyama's reagent (2-chloro-1-methylpyridinium iodide) generate electrophilic carbodiimide functionality. Trapping by the azide anion and electrocyclization of the intermediate imidoylazide generates an aryl-substituted 5-aminotetrazole at the N-terminus of the peptide. To prevent competitive cyclization of a neighboring amide N-H into the carbodiimide, there should not be a free N-H at the [X-1] position relative to the activated carbodiimide. Protection of the N-H group at this position or incorporation of a secondary amino acid is thus required for optimal tetrazole formation. Cleavage from the resin releases the hybrid molecules incorporating a 5-aminotetrazole ring conjugated onto a peptidic fragment.

Peptide-heterocycle hybrid molecules: solid-phase synthesis of a 400-member library of N-terminal 2-iminohydantoin peptides.

A method for the heterocyclic modification of the N-terminus of a peptide is described. Reaction of the N-terminal amino group of solid-supported peptides with arylisothiocyanates generates a thiourea intermediate, as in the first step of Edman degradation. Treatment of the resin-supported peptide-thioureas with Mukaiyama's reagent (2-chloro-1-methylpyridinium iodide) generates an electrophilic carbodiimide functionality, which undergoes rapid intramolecular trapping by the adjacent amide group to give a 2-iminohydantoin ring at the N-terminus of the peptide. The dehydrothiolation step in the presence of Mukaiyama's reagent prevents Edman degradation from occurring, in essence leading to a "diversion" of the Edman degradation. Cleavage from the resin then releases the hybrid molecules incorporating a 2-iminohydantoin ring conjugated onto a peptidic fragment. A 400-member library of the iminohydantoin-peptide hybrids was synthesized using this approach, starting from a chlorotrityl resin-supported tripeptides.

Parallel synthesis of a library of benzoxazoles and benzothiazoles using ligand-accelerated copper-catalyzed cyclizations of ortho-halobenzanilides.

A general method for the formation of benzoxazoles via a copper-catalyzed cyclization of ortho-haloanilides is reported. This approach complements the more commonly used strategies for benzoxazole formation which require 2-aminophenols as substrates. The reaction involves an intramolecular C-O cross-coupling of the ortho-haloanilides and is believed to proceed via an oxidative insertion/reductive elimination pathway through a Cu(I)/Cu(III) manifold. The reaction is also applicable to the formation of benzothiazoles. A variety of ligands including 1,10-phenanthroline and N,N'-dimethylethylenediamine were shown to provide ligand acceleration/stabilization in the reaction. Optimal conditions for cyclization used a catalyst combination of CuI and 1,10-phenanthroline (10 mol %). The method was amenable to a parallel-synthesis approach, as demonstrated by the synthesis of a library of benzoxazoles and benzothiazoles substituted at various positions in the ring. Most examples utilized the cyclization of ortho-bromoanilides, but ortho-iodoanilides and ortho-chloroanilides also undergo a reaction under these conditions. The rate of reaction of the ortho-haloanilides follows the order I > Br > Cl, consistent with oxidative addition being the rate-determining step.

Copper- and palladium-catalyzed intramolecular C-S bond formation: a convenient synthesis of 2-aminobenzothiazoles.

Copper- and palladium-catalyzed intramolecular C-S bond formation by cross-coupling between an aryl halide and thiourea functionality is demonstrated for the synthesis of 2-aminobenzothiazoles, wherein the Cu-catalyzed protocol is generally superior and more cost effective than the Pd-catalyzed protocol; the Cu-catalyzed reaction also further expands recent studies exploring the utility of Cu salts as replacements for Pd in carbon-heteroatom bond-forming reactions. A one-pot variant combining the synthesis of the thiourea and the cyclization was also demonstrated.