The role of phosphodiesterase 4B in IL-8/LTB4-induced human neutrophil chemotaxis evaluated with a phosphodiesterase 4B inhibitor.

PDE4B was previously shown to be a dominant PDE4 subtype of neutrophils. However, its physiological role in the neutrophil function has not been evaluated. In this study, the inhibitory effects of a phosphodiesterase 4B (PDE4B)- selective inhibitor (compound A) and subtype non-selective PDE4 inhibitors (roflumilast and cilomilast) were evaluated in human peripheral blood cells. Compound A, roflumilast and cilomilast in a similar manner inhibited TNF-α production by LPS-stimulated human mononuclear cells. However, the inhibitory effect of compound A on IL-8 or LTB4-induced chemotactic response of neutrophils was modest even at the highest concentration (10 µmol L(-1)), whereas roflumilast and cilomilast inhibited IL-8 or LTB4-induced neutrophil chemotaxis. Our results suggest that PDE4B does not play an important role during the chemotactic response of human neutrophils.

Identification of the 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives as highly selective PDE4B inhibitors.

A PDE4B subtype selective inhibitor is expected to have a wider therapeutic window than non-selective PDE4 inhibitors. In this Letter, two series of 7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives and 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives were evaluated for their PDE4B subtype selectivity using human PDE4B2 and PDE4D2 full length enzymes. To improve their PDE4B selectivity over PDE4D, we optimized the substituents on the pyrimidine ring and the side chain phenyl ring, resulting in several derivatives with more than 100-fold selectivity for PDE4B. Consequently, we identified 2-(3-chloro-4-methoxy-phenyl)-5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative 54 as a highly selective PDE4B inhibitor, which had potent hPDE4B inhibitory activity with an IC50 value of 3.0 nM and 433-fold PDE4B selectivity over PDE4D.

Evaluation of the therapeutic index of a novel phosphodiesterase 4B-selective inhibitor over phosphodiesterase 4D in mice.

Phosphodiesterase 4 (PDE4) inhibitors have been developed for the treatment of pulmonary inflammatory diseases, but their clinical use was dose-limited by mainly gastric adverse effects. Recent studies suggested PDE4B-selective inhibitors over PDE4D are supposed to display a wider therapeutic index than subtype non-selective PDE4 inhibitors such as roflumilast. Compound A was identified as an orally active PDE4B-selective inhibitor over PDE4D both in humans (80-fold selective) and mice (29-fold selective). In this study, the therapeutic effects of compound A and roflumilast were evaluated on lipopolysaccaride (LPS) injection-induced plasma TNF-α elevation and on LPS inhalation-induced pulmonary neutrophilia in mice. The inhibitory effect on gastric emptying in mice was evaluated as a gastric adverse effect. The therapeutic index for TNF-α production (TI(TNF) = ID50 in gastric emptying / ID50 in LPS injection-induced plasma TNF-α elevation) of compound A was larger than roflumilast (9.0 and 0.2, respectively), whereas the therapeutic index for pulmonary neutrophilia (TI(Neu) = ID50 in gastric emptying / ID50 in LPS inhalation-induced pulmonary neutrophilia) of compound A was comparable to roflumilast (1.0 and 0.5, respectively). In conclusion, the TI(Neu) of compound A was not superior compared to that of roflumilast in spite of its high selectivity for PDE4B over PDE4D in mice.

Synthesis and biological evaluation of 5-carbamoyl-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

5-Carbamoyl-2-phenylpyrimidine derivative 2 has been identified as a phosphodiesterase 4 (PDE4) inhibitor with moderate PDE4B inhibitory activity (IC50=200 nM). Modification of the carboxylic acid moiety of 2 gave N-neopentylacetamide derivative 10f, which had high in vitro PDE4B inhibitory activity (IC50=8.3 nM) and in vivo efficacy against lipopolysaccharide (LPS)-induced pulmonary neutrophilia in mice (ID50=16 mg/kg, ip). Furthermore, based on the X-ray crystallography of 10f bound to the human PDE4B catalytic domain, we designed 7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one derivative 39 which has a fused bicyclic lactam scaffold. Compound 39 exhibited excellent inhibitory activity against LPS-induced tumor necrosis factor alpha (TNF-α) production in mouse splenocytes (IC50=0.21 nM) and in vivo anti-inflammatory activity against LPS-induced pulmonary neutrophilia in mice (41% inhibition at a dose of 1.0 mg/kg, i.t.).

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC50=150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50=25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC50=7.5 nM) and TNF-α production in mouse splenocytes (IC50=9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50=18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand-enzyme complex.

Design, synthesis and SAR of novel ethylenediamine and phenylenediamine derivatives as factor Xa inhibitors.

We previously reported on a series of cyclohexanediamine derivatives as highly potent factor Xa inhibitors. Herein, we describe the modification of the spacer moiety to discover an alternative scaffold. Ethylenediamine derivatives possessing a substituent at the C1 position in S configuration and phenylenediamine derivatives possessing a substituent at the C5 position demonstrated moderate to strong anti-fXa activity. Further SAR studies led to the identification of compound 30 h which showed both good in vitro activity (fXa IC(50) = 2.2 nM, PTCT2 = 3.9 µM) and in vivo antithrombotic efficacy.

Design, synthesis, and SAR of cis-1,2-diaminocyclohexane derivatives as potent factor Xa inhibitors. Part I: exploration of 5-6 fused rings as alternative S1 moieties.

A series of cis-1,2-diaminocyclohexane derivatives were synthesized with the aim of optimizing previously disclosed factor Xa (fXa) inhibitors. The exploration of 5-6 fused rings as alternative S1 moieties resulted in two compounds which demonstrated improved solubility and reduced food effect compared to the clinical candidate, compound A. Herein, we describe the synthesis and structure-activity relationship (SAR), together with the physicochemical properties and pharmacokinetic (PK) profiles of some prospective compounds.

Design, synthesis, and SAR of cis-1,2-diaminocyclohexane derivatives as potent factor Xa inhibitors. Part II: exploration of 6-6 fused rings as alternative S1 moieties.

A series of cis-1,2-diaminocyclohexane derivatives possessing a 6-6 fused ring for the S1 moiety were synthesized as novel factor Xa (fXa) inhibitors. The synthesis, structure-activity relationship (SAR), and physicochemical properties are reported herein, together with the discovery of compound 45c, which has potent anti-fXa activity, good physicochemical properties and pharmacokinetic (PK) profiles, including a reduced negative food effect.

Discovery of N-[(1R,2S,5S)-2-{[(5-chloroindol-2-yl)carbonyl]amino}-5-(dimethylcarbamoyl)cyclohexyl]-5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxamide hydrochloride: a novel, potent and orally active direct inhibitor of factor Xa.

In the early 1990's, we reported on the low-molecular selective fXa inhibitor DX-9065a having two amidino groups. However, it had poor oral bioavailability due to its strong basic amidino groups. To obtain fXa inhibitors with improved oral bioavailability, we investigated various non-amidino fXa inhibitors and finally discovered cis-1,2-diaminocyclohexane derivative 4c to have potent fXa inhibition, promising anticoagulant activity, and good oral bioavailability, compared with amidino compound DX-9065a. In addition, we will discuss the influence of the third substituent on the cyclohexane ring on anti-fXa activity, anticoagulant activity, PK profile, and lipophilicity.

Stereoselective synthesis and biological evaluation of 3,4-diaminocyclohexanecarboxylic acid derivatives as factor Xa inhibitors.

There have been few reports on synthetic methods for cis-1,2-diaminocyclohexane bearing a third ring substituent. Starting from 3-cyclohexenecarboxylic acid, we developed efficient methods for synthesizing the 3,4-diaminocyclohexanecarboxylic acid derivatives 2-5. We also evaluated their anti-Xa and anticoagulant activities. Among the compounds, acid 2a and amide 2b exhibited the most potent in vitro anti-fXa activity, indicating that the position and stereochemistry of a polar functional group on the cyclohexane ring greatly affected the in vitro anti-fXa activity.

Cycloalkanediamine derivatives as novel blood coagulation factor Xa inhibitors.

This paper describes the synthesis of orally available potent fXa inhibitors 2 and 3 by modification of the piperazine part of lead compound 1. Carbonyl derivative 3 showed potent fXa activity but not sulfonyl derivative 2. Among the compounds synthesized, cyclohexane derivatives 3g and 3h and cycloheptane derivative 3j had potent anticoagulant activity as well as anti-fXa activity. Synthetic study of the optical isomers of 3g demonstrated that (-)-3g had more potent activity.

A total synthesis of xestodecalactone a and proof of its absolute stereochemistry: interesting observations on dienophilic control with 1,3-disubstituted nonequivalent allenes.

A concise total synthesis of xestodecalactone A, utilizing a Diels-Alder strategy is described. The focal Diels-Alder reaction relied on an "ynoate" dienophile to rapidly assemble the required resorcylinic acid scaffold. During this study, Diels-Alder cycloaddition reactions involving 1,3-disubstituted nonequivalent allene dienophiles were studied, and some surprising results were encountered.

Design, synthesis, and biological activity of novel factor Xa inhibitors: improving metabolic stability by S1 and S4 ligand modification.

Serine protease factor xa (fXa) inhibitor 1 showed good ex vivo anti-fXa activity upon oral administration in rats. However, it has been revealed that 1 had low metabolic stability against human liver microsomes. To improve the metabolic stability, we attempted to modify the S1 and S4 ligands of 1. These modifications resulted in compound 34b, which exhibited selective anti-fXa activity and excellent anti-coagulation activity.

Design, synthesis, and biological activity of non-basic compounds as factor Xa inhibitors: SAR study of S1 and aryl binding sites.

Compound 7 was identified as the active metabolite of 6 by HPLC and mass spectral analysis. Modification of lead compound 7 by transformation of its N-oxide 6-6 biaryl ring system and fused aromatics produced a series of non-basic fXa inhibitors with excellent potency in anti-fXa and anticoagulant assays. The optimized compounds 73b and 75b showed sub to one digit micromolar anticoagulant activity (PTCT2). Particularly, anti-fXa activity was detected in plasma of rats orally administered with 1mg/kg of compound 75b.

Synthesis and conformational analysis of a non-amidine factor Xa inhibitor that incorporates 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine as S4 binding element.

Our exploratory study was based on the concept that a non-amidine factor Xa (fXa) inhibitor is suitable for an orally available anticoagulant. We synthesized and evaluated a series of N-(6-chloronaphthalen-2-yl)sulfonylpiperazine derivatives incorporating various fused-bicyclic rings containing an aliphatic amine expected to be S4 binding element. Among this series, 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine type 61 displayed orally potent anti-fXa activity and evident prolongation of prothrombin time (PT) with the moderate bioavailability in rats. The X-ray crystal analysis afforded an obvious binding mode that 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine and 6-chloronaphthalene respectively bound to S4 and S1 subsites. In this X-ray study, we discovered a novel intramolecular S-O close contact. Ab initio energy calculations of model compounds deduced that conformers with the most close S-O proximity were most stable. The Mulliken population analysis proposed that this energy profile was caused by both of electrostatic S-O affinity and N-O repulsion. The results of these calculations and X-ray analysis suggested a possibility that the restricted conformation effected the affinity to S4 subsite of fXa.

Design, synthesis, and biological activity of non-amidine factor Xa inhibitors containing pyridine N-oxide and 2-carbamoylthiazole units.

Based on the both of results for X-ray studies of tetrahydrothiazolopyridine derivative 1c and FXV673, we synthesized a series of thiazol-5-ylpyridine derivatives containing pyridine N-oxide and 2-carbamoylthiazole units to optimize the S4 binding element. N-Oxidation of thiazol-5-ylpyridine increased the anti-fXa activity more than 10-fold independent on the position of N-oxide. The 4-pyridine N-oxide derivatives 3a and 3d excelled over the tetrahydrothiazolopyridine 1b in potency. 2-Methylpyridine N-oxide 3d exhibited 49-fold selectivity over thrombin. Our modeling study proposed a binding mode that the pyridine N-oxide ring of 3a stuck into the "cation hole" , and the oxide anion of 3a occupied in the almost same space to that of FXV673. From observations of the SAR and modeling studies, we suggested the possibilities that the formation of hydrogen bond with the oxide anion in the "cation hole" and the affinity of cationic pyridine ring to S4 subsite were responsible for increase in anti-fXa activity.

New highly active taxoids from 9beta-dihydrobaccatin-9,10-acetals. Part 3.

We synthesized novel water-soluble and orally active taxane analogues, 7-deoxy-9beta-dihydro-9,10-O-acetal taxanes. Cytotoxicities of the synthetic compounds were greater than those of paclitaxel and docetaxel, especially against resistant cancer cell lines expressing P-glycoprotein. In addition, some compounds showed potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration.

A new method for synthesis of 7-deoxytaxane analogues by hydrogenation of delta(6,7)-taxane derivatives.

A new method for the synthesis of 7-deoxytaxane analogues has been established through hydrogenation of Delta(6,7)-taxane derivatives. Among several catalysts examined, Pd-C was found to be a most effective catalyst for the preparation of target compound.

New highly active taxoids from 9beta-dihydrobaccatin-9,10-acetals. Part 2.

To investigate structure-activity relationships of the 9,10-acetal-9beta-dihydro taxoids, we modified the 7-hydroxyl groups of the 9,10-acetonide-3'-(4-pyridyl) analogue to deoxy, methoxy, alpha-F, and 7beta,8beta-methano group. As a result of this study, we found that the 7-deoxy analogue was the strongest among these analogues. In addition, we found that the 7-deoxy-3'-(4-pyridyl) and 7-deoxy-3'-(2-pyridyl) analogues showed stronger activity against cell lines expressing P-glycoprotein than the corresponding 3'-phenyl analogue.