Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Development of Orally Bioavailable Peptides Targeting an Intracellular Protein: From a Hit to a Clinical KRAS Inhibitor.

Cyclic peptides as a therapeutic modality are attracting a lot of attention due to their potential for oral absorption and accessibility to intracellular tough targets. Here, starting with a drug-like hit discovered using an mRNA display library, we describe a chemical optimization that led to the orally available clinical compound known as LUNA18, an 11-mer cyclic peptide inhibitor for the intracellular tough target RAS. The key findings are as follows: (i) two peptide side chains were identified that each increase RAS affinity over 10-fold; (ii) physico-chemical properties (PCP) including Clog P can be adjusted by side-chain modification to increase membrane permeability; (iii) restriction of cyclic peptide conformation works effectively to adjust PCP and improve bio-activity; (iv) cellular efficacy was observed in peptides with a permeability of around 0.4 × 10-6 cm/s or more in a Caco-2 permeability assay; and (v) while keeping the cyclic peptide's main-chain conformation, we found one example where the RAS protein structure was changed dramatically through induced-fit to our peptide side chain. This study demonstrates how the chemical optimization of bio-active peptides can be achieved without scaffold hopping, much like the processes for small molecule drug discovery that are guided by Lipinski's rule of five. Our approach provides a versatile new strategy for generating peptide drugs starting from drug-like hits.

Broadly Applicable and Comprehensive Synthetic Method for N-Alkyl-Rich Drug-like Cyclic Peptides.

We report a versatile and durable method for synthesizing highly N-alkylated drug-like cyclic peptides. This is the first reported method for synthesizing such peptides in parallel with a high success rate and acceptable purity that does not require optimizations for a particular sequence. We set up each reaction condition by overcoming the following issues: (1) diketopiperazine (DKP) formation, (2) insufficient peptide bond formation due to the steric hindrance of the N-alkylated amino acid, and (3) instability of highly N-alkylated peptides under acidic conditions. Using this newly established method, we successfully synthesized thousands of cyclic peptides to explore the scope of this modality in drug discovery. We here demonstrate the syntheses of a hundred representative examples, including our first clinical N-alkyl-rich cyclic peptide (LUNA18) that inhibits an intracellular tough target (RAS), in 31% total yield and 97% purity on average after 23 or 24 reaction steps.

Optimizing the Physicochemical Properties of Raf/MEK Inhibitors by Nitrogen Scanning.

Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and 13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic stability. The impact after nitrogen substitution on interactions between a derivative and its on- and off-target proteins (Raf/MEK, CYPs, and hERG channel) was also detected, most of them contributing to weaker interactions. After identifying the positions that kept inhibitory activity on HCT116 cell growth and Raf/MEK, compound 1 (CH5126766/RO5126766) was selected as a clinical compound. A phase I clinical trial is ongoing for solid cancers.

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors.

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC50=8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED50=4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment.

Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity.

Tumors with mutant RAS are often dependent on extracellular signal-regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS.

Fluorine Scanning by Nonselective Fluorination: Enhancing Raf/MEK Inhibition while Keeping Physicochemical Properties.

A facile methodology effective in obtaining a set of compounds monofluorinated at various positions (fluorine scan) by chemical synthesis is reported. Direct and nonselective fluorination reactions of our lead compound 1a and key intermediate 2a worked efficiently to afford a total of six monofluorinated derivatives. All of the derivatives kept their physicochemical properties compared with the lead 1a and one of them had enhanced Raf/MEK inhibitory activity. Keeping physicochemical properties could be considered a benefit of monofluorinated derivatives compared with chlorinated derivatives, iodinated derivatives, methylated derivatives, etc. This key finding led to the identification of compound 14d, which had potent tumor growth inhibition in a xenograft model, excellent PK profiles in three animal species, and no critical toxicity.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Design and synthesis of peptidomimetic factor VIIa inhibitors.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other serine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound 2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expectations, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

Structural Variation in Manganase Complexes: Synthesis and Characterization of Manganese Complexes from Carboxylate-containing Chelating Ligands.

Three manganese(II) complexes, [Mn(II)(2)L(1)(2)(H(2)O)(4)](ClO(4))(2).H(2)O (1, L(1)H = (bis(2-pyridylmethyl)amino)acetic acid), [Mn(II)(2)L(2)(2)(H(2)O)(2)](BPh(4))(2).2EtOH.2H(2)O (2, L(2)H = 3-(bis(2-pyridylmethyl)amino)propionic acid), {[Mn(II)(2)L(2)(2)(H(2)O)(MeCN)](BPh(4))(2).2MeCN}(infinity) (3), and a manganese(IV) complex [Mn(IV)(2)O(2)L(2)(2)](ClO(4))(2).4H(2)O (4) were synthesized and characterized by X-ray crystallography. The compound 1 was a dinuclear Mn(II)(2) complex which crystallized in the monoclinic crystal system, space group P2(1)/n, with Z = 4, a = 12.19(1) Å, b = 14.623(8) Å, c = 21.72(1) Å, beta = 96.29(6) degrees, V = 3849(4) Å(3). The complex cation had an approximate C(2) symmtery. The two manganeses were both seven-coordinate and doubly bridged by one oxygen atom of carboxylate groups in &mgr;(2),eta(1)-mode. The compound 2 was also a dinuclear Mn(II)(2) complex which crystallized in the monoclinic crystal system, space group P2(1)/n, with Z = 2, a = 16.760(2) Å, b = 9.643(2) Å, c = 23.533(2) Å, beta = 92.984(8) degrees, V = 3798.4(7) Å(3). The complex cation of 2 also had two seven-coordinate manganese ions, but unlike 1 the nonbridging carboxylate oxygens weakly coordinate to the manganese ions. The compound 3 crystallized in the orthorhombic crystal system, space group P2(1)2(1)2(1), with Z = 4, a = 27.888(3) Å, b = 29.054(2) Å, c = 9.428(2) Å, V = 7638(2) Å(3). The cationic portion of 3 consisted of infinite chains of Mn(II) (two Mn(II) ions per an asymmetric unit) bridged by carboxylates in bidentate syn/anti mode. The compound 4 was a dinuclear bis(&mgr;-oxo) Mn(IV)(2) complex which crystallized in the trigonal crystal system, space group R&thremacr;, with Z = 8, a = 23.962(4) Å, c = 17.190(3) Å, V = 8547(3) Å(3). All these structures are made up from a common fragment "L(n)()Mn" assembling in various topologies. Variable-temperature magnetic susceptibility measurements revealed that the Mn(II) ions in 1-3 were weakly antiferromagnetically coupled (J = -0.631(6), -0.655(5), and -0.20(1) cm(-)(1) for 1-3), and that the Mn(IV) ions in 4 were strongly antiferromagnetically coupled (J = -97.5(5) cm(-)(1)). The cyclic voltammogram of 4 showed two reduction waves with E(1/2) values of -0.52 and 0.28 V (vs ferrocene). These E(1/2) values are more negative by 0.1 V than those of the closely related complex [Mn(III)Mn(IV)O(2)L(1)(2)](ClO(4)).