Preventive and Therapeutic Efficacy of ONO-4641, a Selective Agonist for Sphingosine 1-Phosphate Receptor 1 and 5, in Preclinical Models of Type 1 Diabetes Mellitus.

ONO-4641, 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid (ceralifimod), is a second-generation sphingosine 1-phosphate receptor agonist selective for sphingosine 1-phosphate receptors 1 and 5, and has clinical effects in multiple sclerosis. The objective of the present study was to explore other potential indications for ONO-4641 based on its immunomodulatory effects. ONO-4641 was tested in non-obese diabetic (NOD) mice, an animal model of spontaneous type 1 diabetes mellitus, an autoimmune disease with unmet medical needs. ONO-4641 at a dose of 0.1 mg/kg prevented the onset of diabetes mellitus in NOD mice. Furthermore, ONO-4641 at doses of 0.03 and 0.1 mg/kg decreased diabetic prevalence in NOD mice after the onset of diabetes mellitus in a dose-dependent manner. Histopathological analysis demonstrated that insulin-positive areas in the islets of mice administered 0.03 and 0.1 mg/kg ONO-4641 showed a tendency of high values although they were not significantly different from the Control group, which was treated with vehicle. These observations suggest ONO-4641 may delay the onset and progression of type 1 diabetes mellitus.

Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.

Structure-activity relationship studies of sphingosine-1-phosphate receptor agonists with N-cinnamyl-ß-alanine moiety.

Structure-activity relationship of sphingosine-1-phosphate receptor agonist was examined. In terms of reducing the flexibility of molecule, hit compound 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Novel S1P agonists with cinnamyl scaffold or 1,2,5,6-tetrahydropyridine scaffold were identified.

Discovery of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P(1) receptor agonist with high selectivity against S1P(3) and enhanced efficacy in lowering peripheral lymphocyte counts in mice.

Spirodiketopiperazine-based CCR5 antagonists: Improvement of their pharmacokinetic profiles.

Spirodiketopiperazine-based CCR5 antagonists, showing improved pharmacokinetic profiles without reduction in antagonist activity, were designed and synthesized. We also demonstrate the anti-HIV activity of a representative compound 12, as measured in a p24 assay.

Discovery of orally available spirodiketopiperazine-based CCR5 antagonists.

Using the previously reported novel spirodiketopiperazine scaffold, the design and synthesis of orally available CCR5 antagonists was undertaken. Compounds possessing a carboxylic acid function in the appropriate position showed improved oral exposure (AUC) relative to the initial chemical leads without reduction in the antagonist activity. The optimized compound 40 was found to show potent anti-HIV activity. Full details of structure-activity relationship (SAR) study are presented.

Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine-1-Phosphate (S1P) Receptor Agonist Ceralifimod (ONO-4641). A S1P1 and S1P5 Selective Agonist for the Treatment of Autoimmune Diseases.

The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, is described. While it has been revealed that the modulation of the S1P1 receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P3 receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P1 over S1P3 and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing).

Design, synthesis, and biological evaluation of the combinatorial library with a new spirodiketopiperazine scaffold. Discovery of novel potent and selective low-molecular-weight CCR5 antagonists.

We previously reported the discovery of several spirodiketopiperazine derivatives as potent CCR5 antagonists with anti-HIV activity. Herein, we describe in detail the identification of these lead compounds using a combinatorial chemistry approach. A novel spirodiketopiperazine scaffold was designed on the basis of the concept of the privileged structure of G-protein-coupled receptors (GPCRs). This new framework was obtained in acceptable yield with high purity from the readily prepared isonitrile resin through the Ugi reaction, sequential transformations, and cyclative cleavage. By measuring the inhibitory activity of each compound in the initial library against the intracellular calcium mobilization stimulated by MIP-1alpha, several compounds were found to show modest but selective CCR5 antagonistic activity. After the rapid evaluation of these hit compounds, several single-digit nanomolar, low-molecular-weight CCR5 antagonists that can potently block the infectivity and replication of laboratory and clinical strains of HIV as well as those of highly drug-resistant HIV variants with minimal cytotoxicity have been identified.

Discovery of ONO-7300243 from a Novel Class of Lysophosphatidic Acid Receptor 1 Antagonists: From Hit to Lead.

Lysophosphatidic acid (LPA) evokes various physiological responses through a series of G protein-coupled receptors known as LPA1-6. A high throughput screen against LPA1 gave compound 7a as a hit. The subsequent optimization of 7a led to ONO-7300243 (17a) as a novel, potent LPA1 antagonist, which showed good efficacy in vivo. The oral dosing of 17a at 30 mg/kg led to reduced intraurethral pressure in rats. Notably, this compound was equal in potency to the α1 adrenoceptor antagonist tamsulosin, which is used in clinical practice to treat dysuria with benign prostatic hyperplasia (BPH). In contrast to tamsulosin, compound 17a had no impact on the mean blood pressure at this dose. These results suggest that LPA1 antagonists could be used to treat BPH without affecting the blood pressure. Herein, we report the hit-to-lead optimization of a unique series of LPA1 antagonists and their in vivo efficacy.

Palladium(0)-Catalyzed Isomerization Reactions of Aziridines Bearing an alpha,beta-Unsaturated Ester Group: A Thermodynamic Preference for Chiral Alkyl (2E)-4,5-cis-4,5-Epimino-N-(alkyl- or arylsulfonyl) 2-Enoates over the Other Three Stereoisomers.

Palladium(0)-catalyzed reactions of five sets of four stereoisomeric 4,5-epimino-N-(methanesulfonyl) or -N-(arylsulfonyl) 2-enoates reveal that 4,5-cis-(2E)-isomers are thermodynamically more stable than other isomers, in accord with calculations. A highly stereoselective synthesis of (E)-alkene dipeptide isosteres having the desired stereochemistries from unwanted stereoisomeric 4,5-epimino-N-(arylsulfonyl) 2-enoates is also presented.

Suppressive effects of a novel CC chemokine receptor 4 antagonist on Th2 cell trafficking in ligand- and antigen-induced mouse models.

CC chemokine receptor 4 (CCR4) has been implicated as a preferential marker for T helper type 2 (Th2) cells, and is believed to be involved in the pathology of allergic diseases by controlling Th2 cell trafficking into inflamed tissues. The objective of the study was to characterize the pharmacological properties of E0001-163, a novel CCR4 antagonist. E0001-163 was tested in both in vitro chemotaxis assays as well as in vivo mouse models of CCR4 ligand-induced air pouch and antigen-induced airway inflammation by utilizing in vitro-polarized Th2 cells. In vitro, E0001-163 inhibited migratory response of human Th2-polarized cells to CCL22, a CCR4 ligand, with an IC50 value of 11.9 nM. E0001-163 significantly suppressed CCL22-induced Th2 cell trafficking into mouse air pouch in a dose-dependent manner at doses of 3 and 10mg/kg, suggesting that E0001-163 has an inhibitory effect on CCR4-mediated T cell trafficking in vivo. In addition, E0001-163 partially decreased Th2 cell trafficking and the level of IL-4 in the lungs in Th2-tansferred and ovalbumin (OVA)-challenged mice. T cell trafficking involves multiple chemokine receptors both in acute and chronic phases, and our findings suggest that CCR4, together with other chemokine receptors, may be involved in Th2 cell trafficking under disease conditions.

Spirodiketopiperazine-based CCR5 antagonists: Lead optimization from biologically active metabolite.

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.

Discovery of a new chemical lead for a matrix metalloproteinase inhibitor.

A series of N-benzoyl gamma-aminobutyric hydroxamic acids were synthesized and evaluated as matrix metalloproteinase inhibitors. First, we focused on chemical modification of the N-benzoyl residue. Introduction of electron-rich para-substituents was effective to increase the inhibitory activity. Especially, some of the analogs with relatively more planar N-acyl residues, such as 10 and 11, demonstrated more potent activity. Second, chemical modification of the gamma-aminobutyric hydroxamic acid moiety was carried out to optimize the three-dimensional arrangement of the two pharmacophores (hydroxamic acid and N-acyl residues). Among the tested, the gamma-aminobutyric hydroxamic acid moiety was found to be the best spacer for connecting the above-mentioned two pharmacophores. Synthesis and structure-activity relationships are discussed.

Design and synthesis of novel metalloproteinase inhibitors.

A series of N-benzoyl 4-aminobutyric acid hydroxamate analogs were synthesized and evaluated as matrix metalloproteinase inhibitors. Synthetic work was focused on the chemical modification of the 4-aminobutyric acid part using easily available starting materials. As such, chemical modification was carried out using commercially available starting materials such as 4-aminobutyric acid, (+)- and (-)-malic acid, and D- and L-glutamic acid derivatives. Among the compounds tested, N-[4-(benzofuran-2-yl)benzoyl] 4-amino-4S-hydroxymethylbutyric acid hydroxamates derived from L-glutamic acid demonstrated more potent inhibitory activity against MMP-2 and MMP-9 compared with the corresponding 2S-hydroxy analogs or 3S-hydroxy analogs, respectively, which were derived from (-)-malic acid. Structure-activity relationship study is presented.

Novel matrix metalloproteinase inhibitors: generation of lead compounds by the in silico fragment-based approach.

Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico technique. In order to identify the small fragment interacting with residues in the S1' pocket of MMP-1 through hydrogen bonds, we performed in silico screening using the LUDI program. As a result, acetyl-L-alanyl-(N-methyl)amide (Ac-L-Ala-NHMe) was selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the L-glutamic acid derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.

Novel and potent anti-malarial agents.

Readily accessible, novel, and potent anti-malarial compounds have been developed. Optimization of the initial lead structure resulted in derivatives with IC50 values from 7 to 35 nM against chloroquine-sensitive and 70-350 nM against chloroquine-resistant strains of Plasmodium falciparum.

Certification of the critical importance of L-3-(2-naphthyl)alanine at position 3 of a specific CXCR4 inhibitor, T140, leads to an exploratory performance of its downsizing study.

We have previously found that a 14-amino acid residue-peptide, T140, inhibits infection of target cells by T cell line-tropic HIV-1 (X4-HIV-1) through its specific binding to a chemokine receptor, CXCR4. Here, the importance of an L-3-(2-naphthyl)alanine (Nal) residue at position 3 in T140 for high anti-HIV activity and inhibitory activity against Ca(2+) mobilization induced by stromal cell-derived factor (SDF)-1alpha-stimulation through CXCR4 has initially been shown by the synthesis and biological evaluation of several analogues, where Nal(3) is substituted by diverse aromatic amino acids. Next, the order of the N-terminal 3 residues (Arg(1)-Arg(2)-Nal(3)) has been proved to be important from the structure--activity relationship (SAR) study shuffling these residues. Based on these results, we have found 10-residue peptides possessing modest anti-HIV activity by systematic antiviral evaluation of a series of synthetic, shortened analogues of T140.