HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 µM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 µM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

A New Inhibitor of ADAM17 Composed of a Zinc-Binding Dithiol Moiety and a Specificity Pocket-Binding Appendage.

A disintegrin and metalloproteinase 17 (ADAM17) is a zinc-dependent enzyme that catalyzes the cleavage of the extracellular domains of various transmembrane proteins. ADAM17 is regarded as a promising drug target for the suppression of various diseases, including cancer metastasis. We synthesized a new ADAM17 inhibitor, SN-4, composed of a zinc-binding dithiol moiety and an appendage that specifically binds to a pocket of ADAM17. We show that SN-4 inhibits the ability of ADAM17 to cleave tumor necrosis factor α (TNF-α) in vitro. This activity was reduced by the addition of zinc, indicating the importance of the zinc chelating dithiol moiety. Inhibition of TNF-α cleavage by SN-4 in cells was also observed, and with an IC50 of 3.22 µM, SN-4 showed slightly higher activity than the well-studied ADAM17 inhibitor marimastat. Furthermore, SN-4 was shown to inhibit cleavage of CD44 by ADAM17, but not by ADAM10, and to suppress cell invasion. Molecular docking showed good fitting of the specificity pocket-binding group and one SH of SN-4 and hinted at possible means of structural optimization. This study provides clues for the development of potent and selective ADAM17 inhibitors.

Structural analysis of TIFA: Insight into TIFA-dependent signal transduction in innate immunity.

TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-ß-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA-TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-κB activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA-TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants-Thr9 mutated to either Asp or Glu to mimic the phosphorylation state-to obtain the structural information for oligomer formation of the TIFA-TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA-TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA-TRAF6 signalling complex.

Antiproliferative S-Trityl-l-Cysteine -Derived Compounds as SIRT2 Inhibitors: Repurposing and Solubility Enhancement.

S-trityl-l-cysteine (STLC) is a well-recognized lead compound known for its anticancer activity owing to its potent inhibitory effect on human mitotic kinesin Eg5. STLC contains two free terminal amino and carboxyl groups that play pivotal roles in binding to the Eg5 pocket. On the other hand, such a zwitterion structure complicates the clinical development of STLC because of the solubility issues. Masking either of these radicals reduces or abolishes STLC activity against Eg5. We recently identified and characterized a new class of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of sirtuin protein (SIRT2) inhibitors that can be utilized as cytotoxic agents based on an S-trityl-l-histidine scaffold. Herein, we propose new STLC-derived compounds that possess pronounced SIRT2 inhibition effects. These derivatives contain modified amino and carboxyl groups, which conferred STLC with SIRT2 bioactivity, representing an explicit repurposing approach. Compounds STC4 and STC11 exhibited half maximal inhibitory concentration values of 10.8 ± 1.9 and 9.5 ± 1.2 µM, respectively, against SIRT2. Additionally, introduction of the derivatizations in this study addressed the solubility limitations of free STLC, presumably due to interruption of the zwitterion structure. Therefore, we could obtain drug-like STLC derivatives that work by a new mechanism of action. The new derivatives were designed, synthesized, and their structure was confirmed using different spectroscopic approaches. In vitro and cellular bioassays with various cancer cell lines and in silico molecular docking and solubility calculations of the synthesized compounds demonstrated that they warrant attention for further refinement of their bioactivity.

Novel p97/VCP inhibitor induces endoplasmic reticulum stress and apoptosis in both bortezomib-sensitive and -resistant multiple myeloma cells.

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.

Minimum structural requirements for inhibitors of the zinc finger protein TRAF6.

Zinc fingers have rarely been regarded as drug targets. On the contrary, the zinc-binding site of enzymes has often been considered a target of inhibitors. We previously developed a dithiol compound called SN-1 that binds to the zinc finger protein tumor necrosis factor receptor-associated factor 6 (TRAF6) and suppresses downstream nuclear factor-κB (NF-κB) signaling. To determine the minimal structure requirements of TRAF6 inhibitors based on SN-1, NF-κB inhibitory activity and cytotoxicity of its derivatives including new compounds were examined. SN-2, an oxidative type of prodrug of SN-1 with 2-nitrophenylthio groups via disulfide, has the minimum structure for an inhibitor of TRAF6, as seen with cellular experiments. The importance of two side chains with a thiol group was shown with molecular modelling. This study may lead to development of selective TRAF6 inhibitors in the near future.

Neuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal CA1 damage after transient cerebral ischemia.

Ischemia-reperfusion injuries produce reactive oxygen species that promote the peroxide lipid oxidation process resulting in the production of an endogenic lipid peroxide, 4-hydroxy-trans-2-nonenal (4-HNE), a highly cytotoxic aldehyde that induces cell death. We synthesized a novel 4-HNE scavenger - a carnosine-hydrazide derivative, l-carnosine hydrazide (CNN) - and examined its neuroprotective effect in a model of transient ischemia. PC-12 cells were pre-incubated with various doses (0-50 mmol/L) of CNN for 30 min, followed by incubation with 4-HNE (250 µM). An MTT assay was performed 24 h later to examine cell survival. Transient ischemia was induced by bilateral common carotid artery occlusion (BCCO) in the Mongolian gerbil. Animals were assigned to sham-operated (n = 6), placebo-treated (n = 12), CNN pre-treated (20 mg/kg; n = 12), CNN post-treated (100 mg/kg; n = 11), and histidyl hydrazide (a previously known 4-HNE scavenger) post-treated (100 mg/kg; n = 7) groups. Heat shock protein 70 immunoreactivity in the hippocampal CA1 region was evaluated 24 h later, while delayed neuronal death using 4-HNE staining was evaluated 7 days later. Pre-incubation with 30 mmol/L CNN completely inhibited 4-HNE-induced cell toxicity. CNN prevented delayed neuronal death by >60% in the pre-treated group (p < 0.001) and by >40% in the post-treated group (p < 0.01). Histidyl hydrazide post-treatment elicited no protective effect. CNN pre-treatment resulted in high heat shock protein 70 and low 4-HNE immunoreactivity in CA1 pyramidal neurons. Higher 4-HNE immunoreactivity was also found in the placebo-treated animals than in the CNN pre-treated animals. Our novel compound, CNN, elicited highly effective 4-HNE scavenging activity in vitro. Furthermore, CNN administration both pre- and post-BCCO remarkably reduced delayed neuronal death in the hippocampal CA1 region via its induction of heat shock protein 70 and scavenging of 4-HNE.

Blockade of TGF-ß/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis.

BACKGROUND: Transforming growth factor-ß (TGF-ß)/Smad signaling is well known to play a critical role in the pathogenesis of systemic sclerosis (SSc). We previously developed an artificial molecule, the histidine-pyridine-histidine ligand derivative HPH-15, which may have an antifibrotic effect. The purpose of the present study was to clarify the effects of this drug in human skin fibroblasts and in a preclinical model of SSc. METHODS: The effects of HPH-15 on expression of extracellular matrix components and TGF-ß signaling in human dermal fibroblasts were analyzed. The antifibrotic properties of HPH-15 and its mechanisms were also examined in a bleomycin-induced skin fibrosis mouse model. RESULTS: HPH-15 suppressed the TGF-ß-induced phosphorylation of Smad3 and inhibited the expression of collagen I, fibronectin 1, connective tissue growth factor, and α-smooth muscle actin induced by TGF-ß in cultured human skin fibroblasts. In the bleomycin-induced skin fibrosis model, oral administration of HPH-15 protected against the development of skin fibrosis and ameliorated established skin fibrosis. Additionally, HPH-15 suppressed the phosphorylation of Smad3 in various cells, including macrophages in the bleomycin-injected skin. Further, in the treated mice, dermal infiltration of proinflammatory macrophages (CD11b+Ly6Chi) and M2 profibrotic macrophages (CD11b+CD204+ or CD11b+CD206+) was significantly decreased during the early and late stages, respectively. HPH-15 treatment resulted in decreased messenger RNA (mRNA) expression of the M2 macrophage markers arginase 1 and Ym-1 in the skin, whereas it inversely augmented expression of Friend leukemia integration 1 and Krüppel-like factor 5 mRNAs, the transcription factors that repress collagen synthesis. No apparent adverse effects of HPH-15 were found during the treatment. CONCLUSIONS: HPH-15 may inhibit skin fibrosis by inhibiting the phosphorylation of Smad3 in dermal fibroblasts and possibly in macrophages. Our results demonstrate several positive qualities of HPH-15, including oral bioavailability, a good safety profile, and therapeutic effectiveness. Thus, this TGF-ß/Smad inhibitor is a potential candidate therapeutic for SSc clinical trials.

A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group.

Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity.

Zinc-mediated binding of a low-molecular-weight stabilizer of the host anti-viral factor apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination.

Synthesis of the biotinylated anti-HIV compound BMMP and the target identification study.

BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], an inhibitor of HIV-1 replication, was linked to biotin to study the interaction with the presumed target, HIV-1 Pr55(Gag) or CA, by means of surface plasmon resonance. The synthesized Biotin-BMMP inhibited HIV-1 replication to a similar extent as BMMP alone, but did not interact with Pr55(Gag) or CA, suggesting that BMMP exerts its activity by a different mechanism.

Novel metal chelating molecules with anticancer activity. Striking effect of the imidazole substitution of the histidine-pyridine-histidine system.

Previously we have reported a metal chelating histidine-pyridine-histidine system possessing a trityl group on the histidine imidazole, namely HPH-2Trt, which induces apoptosis in human pancreatic adenocarcinoma AsPC-1 cells. Herein the influence of the imidazole substitution of HPH-2Trt was examined. Five related compounds, HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me were newly synthesized and screened for their activity against AsPC-1 and brain tumor cells U87 and U251. HPH-1Trt and HPH-2Trt were highly active among the tested HPH compounds. In vitro DNA cleavage assay showed both HPH-1Trt and HPH-2Trt completely disintegrate pUC19 DNA. The introduction of trityl group decisively potentiated the activity.

l-Histidyl-glycyl-glycyl-l-histidine. Amino-acid structuring of the bleomycin-type pentadentate metal-binding environment capable of efficient double-strand cleavage of plasmid DNA.

A tetrapeptide, l-histidyl-glycyl-glycyl-l-histidine (HGGH), was synthesized and the pUC19 plasmid DNA cleaving activity by copper(II) complex of HGGH (Cu(II)-HGGH) was investigated. Cu(II)-HGGH showed bleomycin-like DNA cleaving activity and, at 50nM, converted a supercoiled DNA efficiently to a linear DNA in the presence of 500µM H2O2/sodium ascorbate through an oxidative pathway.

Design and synthesis of lipid-coupled inositol 1,2,3,4,5,6-hexakisphosphate derivatives exhibiting high-affinity binding for the HIV-1 MA domain.

The precursor of Gag protein (Pr55(Gag)) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind d-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55(Gag), the MA domain, plays a critical role in the binding of Pr55(Gag) to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 µM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 µM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 µM), suggesting the possibility that the PIP5 derivative blocks Pr55(Gag) membrane binding by competing with PIP2 in MA-binding.

Comparison of pharmacokinetics between loxoprofen and its derivative with lower ulcerogenic activity, fluoro-loxoprofen.

  We recently reported that, compared to loxoprofen (LOX, an non-steroidal anti-inflammatory drug), the LOX derivative fluoro-loxoprofen (F-LOX) is less ulcerogenic but has similar anti-inflammatory activity. Our previous in vitro studies suggested that both LOX and F-LOX are pro-drugs, the active metabolites of which are their trans-alcohol forms. In this study, we compared the pharmacokinetics of F-LOX and LOX in rats. Overall, the pharmacokinetic characteristics of F-LOX, including the formation of metabolites in vivo and in vitro, were comparable to those of LOX. However, F-LOX disappeared from the plasma more rapidly than LOX, which could potentially explain its lower ulcerogenicity. However, we showed that F-LOX produced fewer gastric lesions than LOX, even when a higher plasma concentration of F-LOX was maintained. Similar to LOX, F-LOX was readily metabolized to its trans- and cis-alcohol forms, with a higher level of the trans-alcohol form being observed after oral or intravenous administration of the drug. The preferential formation of the trans-alcohol form was also observed after incubation of F-LOX with rat liver homogenates in vitro. These results suggest that, similar to LOX, F-LOX acts as a pro-drug and that there is a metabolic system that selectively produces its active metabolite.

Identification of a unique nsaid, fluoro-loxoprofen with gastroprotective activity.

We previously proposed that direct cytotoxicity of NSAIDs due to their membrane permeabilization activity, together with their ability to decrease gastric prostaglandin E(2), contributes to production of gastric lesions. Compared to loxoprofen (LOX), fluoro-loxoprofen (F-LOX) has much lower membrane permeabilization and gastric ulcerogenic activities but similar anti-inflammatory activity. In this study, we examined the mechanism for this low ulcerogenic activity in rats. Compared to LOX, the level of gastric mucosal cell death was lower following administration of F-LOX. However, the gastric level of prostaglandin E(2) was similar in response to treatment with the two NSAIDs. Oral pre-administration of F-LOX conferred protection against the formation of gastric lesions produced by subsequent administration of LOX and orally administered F-LOX resulted in a higher gastric pH value and mucus content. In the presence of a stimulant of gastric acid secretion, the difference in the ulcerogenic activity of F-LOX and LOX was less apparent. Furthermore, an increase in the mucus was observed in gastric cells cultured in the presence of F-LOX in a manner dependent of increase in the cellular level of cAMP. These results suggest that low ulcerogenic activity of F-LOX involves its both low direct cytotoxicity and protective effect against the development of gastric lesions. This protective effect seems to be mediated through an increase in a protective factor (mucus) and a decrease in an aggressive factor (acid).

Synthesis and biological evaluation of derivatives of 2-{2-fluoro-4-[(2-oxocyclopentyl)methyl]phenyl}propanoic acid: nonsteroidal anti-inflammatory drugs with low gastric ulcerogenic activity.

We previously reported that 2-fluoroloxoprofen has lower gastric ulcerogenic activity than loxoprofen, a nonsteroidal anti-inflammatory drug (NSAID) without selectivity for COX-2. We synthesized derivatives of 2-fluoroloxoprofen and studied their properties. Compared to 2-fluoroloxoprofen, one derivative, 11a, exhibited higher anti-inflammatory activity and an equivalent ulcerogenic effect. These results suggest that 11a could be therapeutically beneficial for use as an NSAID.

An artificial copper complex incorporating a cell-penetrating peptide inhibits nuclear factor-κB (NF-κB) activation.

Nuclear factor-κB (NF-κB) is an inducible transcription factor activated by a variety of cytokines, and promotes the transcription of genes involved in cancer, inflammation, autoimmune disease, and viral infection, among others. Because of its involvement in numerous disease processes, considerable research has focused on NF-κB as a potential drug target. We previously reported that cupric ion (Cu(2+)) blocks NF-κB activation. However, Cu(2+) is unsuitable for drug applications. The copper complex of an artificial peptide HPH-Pep (HPH-Pep-Cu(2+)) was a promising alternative, but it did not easily cross the cell membrane. We report the development of a NF-κB inhibiting Cu(2+) complex with improved cell-penetrating activity arising from the coupling of a Tat peptide to HPH-Pep-Cu(2+).

Design and synthesis of biotinylated inositol 1,3,4,5-tetrakisphosphate targeting Grp1 pleckstrin homology domain.

A bifunctional molecule containing biotin and d-myo-inositol 1,3,4,5-tetrakisphosphate was synthesized. This molecule was designed on the basis of X-ray structure of the complex of d-myo-inositol 1,3,4,5-tetrakisphosphates, Ins(1,3,4,5)P(4), and Grp1 PH (general receptor of phosphoinositides pleckstrin homology) domain for the application to the widely employed biotin-avidin techniques. The building block of inositol moiety was synthesized starting with myo-inositol and assembled with the biotin-linker moiety through a phosphate linkage. The equilibrium dissociation constant K(D) of biotinylated Ins(1,3,4,5)P(4) binding of original Grp1 PH domain was 0.14 µM in pull-down analysis, which was comparable to that of unmodified Ins(1,3,4,5)P(4). Furthermore, biotinylated Ins(1,3,4,5)P(4) had an ability to distinguish Grp1 PH domain from PLCδ(1) PH domain. Thus, biotinylated Ins(1,3,4,5)P(4) retained the binding affinity and selectivity of original Grp1 PH domain, and realized the intracellular Ins(1,3,4,5)P(4) despite a tethering at the 1-phosphate group of inositol.

Synthesis and biological evaluation of loxoprofen derivatives.

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory actions through an inhibitory effect on cyclooxygenase (COX). Two COX subtypes, COX-1 and COX-2, are responsible for the majority of COX activity at the gastrointestinal mucosa and in tissues with inflammation, respectively. We previously suggested that both gastric mucosal cell death due to the membrane permeabilization activity of NSAIDs and COX-inhibition at the gastric mucosa are involved in NSAID-induced gastric lesions. We have also reported that loxoprofen has the lowest membrane permeabilization activity among the NSAIDs we tested. In this study, we synthesized a series of loxoprofen derivatives and examined their membrane permeabilization activities and inhibitory effects on COX-1 and COX-2. Among these derivatives, 2-{4'-hydroxy-5-[(2-oxocyclopentyl)methyl]biphenyl-2-yl}propanoate 31 has a specificity for COX-2 over COX-1. Compared to loxoprofen, oral administration of 31 to rats produced fewer gastric lesions but showed an equivalent anti-inflammatory effect. These results suggest that 31 is likely to be a therapeutically beneficial and safer NSAID.