Synthesis and biological evaluation of N-aryl-5-aryloxazol-2-amine derivatives as 5-lipoxygenase inhibitors

Article abstract of DOI:10.1248/cpb.c15-00033

We describe the synthesis and biological evaluation of N-aryl-5-aryloxazol-2-amine derivatives that are able to inhibit 5-lipoxygenase (5-LOX), a key enzyme of leukotriene synthesis, for the treatment of inflammationrelated diseases including asthma and r

Full text of DOI:10.1248/cpb.c15-00033

Vol. 63, No. 8
Chem. Pharm. Bull. 63, 573–578 (2015)
573
Regular Article
Synthesis and Biological Evaluation of N-Aryl-5-aryloxazol-2-amine
Derivatives as 5-Lipoxygenase Inhibitors
,c
Jee Hee Suh,a Eul Kgun Yum,b and Young Sik Cho*
a Pharmacological Research Center, Bio-organic Division, Korea Research Institute of Chemical Technology; P.O.
b
Box 107, Yuseong-gu, Daejeon 305–600, Korea: Department of Chemistry, Chungnam National University; Yuseong-
c
gu, Daejeon 305–764, Korea: and College of Pharmacy, Keimyung University; 1000 Sindang-dong, Dalseo-gu,
Daegu 704–701, Korea.
Received January 13, 2015; accepted May 19, 2015; advance publication released online June 3, 2015
We describe the synthesis and biological evaluation of N-aryl-5-aryloxazol-2-amine derivatives that are
able to inhibit 5-lipoxygenase (5-LOX), a key enzyme of leukotriene synthesis, for the treatment of inflam-
mation-related diseases including asthma and rheumatoid arthritis. A novel structural moiety containing
oxazole was initially identified from a chemical library using an in vitro enzymatic and cell-based assay, and
its synthesized oxazole derivatives were further examined to develop a structure–activity relationship (SAR).
SAR analysis demonstrated that a hydroxyl or amino group at the p-position on N-phenyl was essential for
the 5-LOX-inhibitory activities of the derivatives, and that other halogen and methyl group-substituted de-
rivatives affected the potency, positively or negatively. As a result, derivatives selected through first-round
screening were further optimized using a cell-based assay and an in vivo assay to develop a potent, selective
5-LOX inhibitor. A final hit exhibited an improved efficacy in arachidonic acid-induced ear edema when ap-
plied topically but not orally. Moreover, it showed the additional advantage of sustainable antiinflammatory
activity over a reference compound, zileuton. Taken together, chemical entities bearing an oxazole scaffold
could be promising as therapeutic drugs for the treatment of chronic inflammatory skin disorders.
Key wordsꢀ 5-lipoxygenase;ꢀantiinflammatoryꢀactivity;ꢀoxazol-2-amine;ꢀleukotriene
Inꢀ responseꢀ toꢀ externalꢀ stresses,ꢀ severalꢀ enzymesꢀ areꢀ in- [3,2-a]-s-triazine-5,7-dione,ꢀ whichꢀ showsꢀ anti-inflammatoryꢀ
volvedꢀinꢀtheꢀproductionꢀofꢀinflammatoryꢀchemicalꢀmediatorsꢀ activity in a carrageenan-induced edema test.13) Additionally,
from arachidonic acid (AA) that is released from the cytoplas- 4,5-diaryloxazoleꢀ hasꢀ beenꢀ designedꢀ asꢀ aꢀ novelꢀ anti-inflam-
mic membrane.1)ꢀSpecifically,ꢀprostaglandinsꢀ(PGs)ꢀandꢀleukot- matory drug, based on the docking method of interaction
rienes (LTs) are, respectively, formed from an AA substrate, betweenꢀoxaprozinꢀandꢀcyclooxygenase-2.14) In addition to the
catalyzedꢀ byꢀ twoꢀ individualꢀ enzymes,ꢀ cyclooxygenaseꢀ (COX)ꢀ anti-inflammatoryꢀ activityꢀ ofꢀ oxazoles,ꢀ theirꢀ derivativesꢀ haveꢀ
andꢀ lipoxygenaseꢀ (LOX),ꢀ atꢀ theꢀ bifurcationꢀ ofꢀ theꢀ metabolicꢀ beenꢀdocumentedꢀtoꢀhaveꢀaꢀwideꢀrangeꢀofꢀbiologicalꢀactivities,ꢀ
pathway.2,3)ꢀ Initialꢀ approachesꢀ targetingꢀ COX-2ꢀ selectivelyꢀ inꢀ such as analgesic, antibacterial, antiproliferative, and hypogly-
anꢀ effortꢀ toꢀ developꢀ anti-inflammatoryꢀ drugsꢀ provedꢀ toꢀ beꢀ cemic activity.15–18)
inappropriate due to their adverse cardiac effects.4) Alternative
Inꢀ anꢀ effortꢀ toꢀ developꢀ smallꢀ moleculesꢀ againstꢀ 5-LOX,ꢀ
waysꢀ toꢀ controlꢀ inflammatoryꢀ mediatorsꢀ haveꢀ beenꢀ pursuedꢀ chemicalsꢀ bearingꢀ oxazoleꢀ scaffoldsꢀ asꢀ aꢀ bioisostereꢀ ofꢀ thia-
forꢀ theꢀ treatmentꢀ ofꢀ inflammation-relatedꢀ diseases.ꢀ Theꢀ 5-li- zoleꢀwereꢀexploredꢀfromꢀchemicalꢀlibrariesꢀdepositedꢀatꢀKoreaꢀ
poxygenaseꢀ(5-LOX)ꢀisꢀaꢀkeyꢀenzymeꢀinꢀtheꢀfirstꢀstepꢀofꢀleu- ResearchꢀInstituteꢀofꢀChemicalꢀTechnologyꢀ(KRICT,ꢀDaejeon,ꢀ
kotriene (LT) synthesis, and its dysregulation causes various SouthꢀKorea)ꢀusingꢀanꢀenzymaticꢀscreeningꢀassayꢀasꢀdescribedꢀ
inflammatoryꢀ diseases,ꢀ suchꢀ asꢀ atopyꢀ andꢀ asthma.5,6) Among previously.19,20)ꢀAsꢀaꢀresult,ꢀweꢀidentifiedꢀN-aryl-5-aryloxazol-
the LTs, leukotriene B4 (LTB4) is a key chemoattractant for 2-amineꢀderivativesꢀthatꢀcouldꢀinhibitꢀ5-LOXꢀcatalyticꢀactivityꢀ
neutrophilsꢀ recruitment,ꢀ aꢀ hallmarkꢀ ofꢀ acuteꢀ inflammation7) potently. Accordingly, a series of N-aryl-5-aryloxazol-2-amineꢀ
and the others, termed collectively the “cysteinyl LTs,” act as derivativesꢀ wasꢀ synthesized,ꢀ andꢀ testedꢀ inꢀ anꢀ in vitroꢀ enzy-
theꢀslowlyꢀreactingꢀsubstancesꢀofꢀanaphylaxis.8)ꢀThus,ꢀ5-LOXꢀ matic assay to determine the structure–activity relationship
inhibitors that are able to block LTs production could be effec- (SAR)ꢀ ofꢀ thoseꢀ novelꢀ 5-LOXꢀ inhibitors.ꢀ Someꢀ compoundsꢀ
tiveꢀ therapeuticꢀ agentsꢀ toꢀ treatꢀ inflammatoryꢀ diseases.ꢀ Pres- withꢀ potentꢀ inhibitoryꢀ activityꢀ wereꢀ finallyꢀ confirmedꢀ inꢀ anꢀ
ently,ꢀonlyꢀzileutonꢀhasꢀbeenꢀapprovedꢀbyꢀtheꢀFoodꢀandꢀDrugꢀ ear-edema mouse model after topical or oral administration,
Administrationꢀ(FDA)ꢀasꢀaꢀdirectꢀ5-LOXꢀinhibitor.9)ꢀHowever,ꢀ andꢀtheyꢀwereꢀassessedꢀforꢀsustainedꢀefficacyꢀafterꢀtopicalꢀap-
thereꢀareꢀmanyꢀanti-inflammatoryꢀdrugsꢀbasedꢀonꢀtheꢀabroga- plication.
tion of the ligand–receptor interaction or indirect interference
inꢀtheꢀprocessꢀofꢀ5-LOXꢀactivation.10–12)
Results and Discussion
Recently,ꢀ thereꢀ hasꢀ beenꢀ specialꢀ interestꢀ onꢀ thiazole,ꢀ imid-
Chemistry N-Aryl-5-aryloxazol-2-amineꢀ derivativesꢀ wereꢀ
azole,ꢀ andꢀ oxazoleꢀ moietiesꢀ dueꢀ toꢀ theirꢀ rolesꢀ asꢀ pharmaco- preparedꢀ byꢀ cyclizationꢀ ofꢀ isothiocyanateꢀ (1)ꢀ withꢀ α-azido-
phores in various biologically active natural products or poten- acetophenones (2) in the presence of triphenyl phosphine in
tialꢀmedicinalꢀagents.ꢀItꢀhasꢀbeenꢀalreadyꢀreportedꢀthatꢀoxazoleꢀ refluxingꢀdioxane,ꢀasꢀshownꢀinꢀChartꢀ1.ꢀTheꢀcompoundꢀ 3–24
derivativesꢀhaveꢀanti-inflammatoryꢀactivity,ꢀandꢀleadingꢀeven- wereꢀ synthesizedꢀ withꢀ substituentꢀ variationsꢀ atꢀ theꢀ phenylꢀ
tuallyꢀ toꢀ theꢀ synthesisꢀ ofꢀ theꢀ novelꢀ bicyclicꢀ systemꢀ oxazoleꢀ groups.
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀyscho123@kmu.ac.kr
© 2015 The Pharmaceutical Society of Japan

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Vol. 63, No. 8 (2015)
(a)ꢀTriphenylꢀphosphine,ꢀdioxane,ꢀreflux,ꢀ40–82%.
Chart 1
Tableꢀ 1.ꢀ SARꢀDataꢀonꢀN-Aryl-5-aryloxazol-2-amineꢀDerivativesꢀforꢀ5-LOXꢀInhibitors
In vitro assaya)
Compounds
R1
R2
X
b)
%ꢀinhibitionꢀatꢀ1ꢀµM
IC50 (nM)
Zileuton
3
78
88
76
91
89
97
97
82
82
85
91
79
94
100
50
58
47
49
84
89
51
2
180
92
4-OH
H
4-Cl
H
4
4-OH
H
120
140
84
5
4-OH
3,4-(Cl)2
4-OCH3
4-F
H
6
4-OH
H
7
4-OH
H
330
172
120
65
8
4-OH
2,4-(F)2
4-F
H
9
4-OH
3,5-(CH3)2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4-OH
4-F
3,5-(Cl)2
4-NH2
4-NH2
4-NH2
4-NH2
4-NH2
4-NH2
3-NH2
4-NHAc
4-NHAc
4-OAc3
4-OAc
4-OAc
3-CO2H
3-CO2H
H
H
154
105
220
95
4-Cl
H
3,4-(Cl)2
4-F
H
H
2,4-(F)2
4-F
H
110
790
690
1250
907
180
190
560
ND
NDc)
3,5-(Cl)2
4-Cl
H
H
H
H
H
H
H
H
4-Cl
4-F
4-F
2,4-(F)2
3,4-(Cl)2
3-NO2
3-NH2
4
a)ꢀ5-LOXꢀenzymaticꢀactivitiesꢀwereꢀexaminedꢀinꢀtheꢀreactionꢀmixturesꢀincludingꢀchemicalꢀderivativesꢀatꢀ1ꢀµM,ꢀandꢀcomparedꢀwithꢀtheꢀvehicleꢀcontrol.ꢀb) IC50ꢀvaluesꢀwereꢀ
calculatedꢀbyꢀfittingꢀtheꢀdose–responseꢀcurvesꢀforꢀ%ꢀinhibitionꢀusingꢀtheꢀGraFitꢀsoftware.ꢀc)ꢀND,ꢀnotꢀdetermined.
Biological Activityꢀ ꢀTheꢀ synthesizedꢀ N-aryl-5-aryloxazol- hydroxyphenyl)-5-(4-fluorophenyl)ꢀoxazol-2-amineꢀ derivativesꢀ
2-amineꢀ derivativesꢀ wereꢀ preliminarilyꢀ testedꢀ withꢀ anꢀ in increasedꢀ inhibitoryꢀ activityꢀ significantlyꢀ whenꢀ comparedꢀ
vitroꢀenzymaticꢀassay.ꢀTheꢀSARꢀresultsꢀofꢀtheꢀderivativesꢀareꢀ withꢀ correspondingꢀ unsubstitutedꢀ derivativesꢀ (compoundsꢀ 7,
summarizedꢀinꢀTableꢀ1.ꢀOurꢀpreviousꢀSARꢀstudiesꢀonꢀN-aryl- 9, 10). Second, the inhibitory potencies of derivatives bearing
4-aryl-1,3-thiazol-2-amineꢀderivativesꢀshowedꢀthatꢀeitherꢀ–OHꢀ 4-amino-N-phenyl moiety (compounds 11–15)ꢀ wereꢀ furtherꢀ
or –NH2 group at the 4-position of N-phenylꢀ groupꢀ wasꢀ es- examined for varying R2 substituents. In fact, the inhibitory
sentialꢀforꢀinhibitoryꢀactivityꢀagainstꢀ5-LOX.21) In that article, activitiesꢀ ofꢀ theꢀ seriesꢀ ofꢀ 5-phenylꢀ substituentsꢀ wereꢀ notꢀ sig-
weꢀ firstꢀ presentedꢀ 4-aryl-N-(4-hydroxyphenyl)ꢀthiazol-2-amineꢀ nificantlyꢀaltered.ꢀHowever,ꢀtheꢀreplacementꢀofꢀ4-aminophenylꢀ
derivativesꢀ asꢀ aꢀ specificꢀ inhibitorꢀ ofꢀ 5-LOX,ꢀ subsequentlyꢀ withꢀ3-aminophenylꢀnegativelyꢀaffectedꢀitsꢀinherentꢀinhibitoryꢀ
leadingꢀ toꢀ theꢀ considerableꢀ suppressionꢀ ofꢀ inflammatoryꢀ re- activity, leading to a 6-fold increase in IC50ꢀ comparedꢀ withꢀ
sponsesꢀwhenꢀgivenꢀtopically,ꢀbutꢀnotꢀorally.ꢀLikewise,ꢀaꢀseriesꢀ 4-aminophenyl derivatives (compound 17). Additionally, the
of N-(4-hydroxyphenyl)ꢀoxazol-2-amineꢀ derivativesꢀ bearingꢀ placement of NHAc at position R1 decreased severely the abil-
5-phenylꢀ substituentsꢀ showedꢀ goodꢀ inhibitoryꢀ effectsꢀ againstꢀ ityꢀ toꢀ inhibitꢀ 5-LOXꢀ (compoundsꢀ 18, 19).ꢀ However,ꢀ aꢀ 4-OAcꢀ
5-LOXꢀ(compoundsꢀ3–8), indicating that electronic effects of group attached to position R1 did not affect the inhibitory
5-phenylꢀ substituentsꢀ didꢀ notꢀ significantlyꢀ affectꢀ inhibitoryꢀ activities so much (compounds 20–22), although compound
potenciesꢀofꢀoxazoleꢀscaffolds.
22 bearing 3,4-dichloro at R2 position exhibited a higher IC50
The introduction of dimethyl or dichloro group into N-(4- value than 20 and 21.ꢀ Theꢀ 4-OAcꢀ substituentsꢀ atꢀ R1 position

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575
wereꢀstillꢀpotent,ꢀunlikeꢀtheꢀcounterpartꢀofꢀthiazoleꢀscaffoldꢀasꢀ enedꢀ anti-inflammatoryꢀ potency,ꢀ comparedꢀ withꢀ theꢀ methylꢀ
describedꢀabove.ꢀMoreover,ꢀplacingꢀaꢀ3-CO2H group at the R1 group-bearing derivatives (compounds 9, 10), indicating that
positionꢀ abrogatedꢀ completelyꢀ theꢀ 5-LOXꢀ inhibitoryꢀ potencyꢀ theꢀintroductionꢀofꢀtheꢀhalogenꢀgroupꢀmayꢀinterfereꢀwithꢀcellꢀ
(compounds 23, 24). Accordingly, substituent variations of R1 membrane permeability or may facilitate cellular metabolism
bonded to N-phenylꢀwereꢀimportantꢀforꢀconferringꢀ5-LOXꢀin- of those compounds although its underlying mode of action
hibitoryꢀactivityꢀonꢀtheꢀskeleton,ꢀwhileꢀvariousꢀsubstituentsꢀatꢀ remains to be determined.
R2ꢀorꢀXꢀmayꢀaffectꢀtheꢀinhibitoryꢀactivity.
Subsequently,ꢀ in vivoꢀ efficacyꢀ studiesꢀ demonstratedꢀ thatꢀ
Someꢀ derivativesꢀ drawnꢀ fromꢀ theꢀ first-roundꢀ in vitro someꢀ compoundsꢀ underꢀ investigationꢀ showedꢀ suppressiveꢀ
screeningꢀ wereꢀ furtherꢀ examinedꢀ toꢀ seeꢀ whetherꢀ suchꢀ com- effectsꢀ onꢀ AA-inducedꢀ edemaꢀ whenꢀ administeredꢀ topically,ꢀ
pounds could prevent cells from generating leukotriene B4 butꢀ notꢀ orally.ꢀ Toꢀ induceꢀ inflammatoryꢀ responses,ꢀ AAꢀ (2ꢀmgꢀ
(LTB4) after stimulation of rat basophilic leukemia-1 (RBL-1) in 20µLꢀacetone)ꢀwasꢀpaintedꢀtoꢀtheꢀinnerꢀsurfaceꢀofꢀtheꢀrightꢀ
cellsꢀ withꢀ A23187,ꢀ aꢀ calciumꢀ ionophoreꢀ (Tableꢀ 2).ꢀ Theꢀ cell- earꢀofꢀaꢀBALB/cꢀmouse.ꢀTheꢀadministrationꢀschemeꢀwasꢀper-
basedꢀassaysꢀwereꢀcarriedꢀoutꢀaccordingꢀtoꢀmethodsꢀreportedꢀ formedꢀ inꢀ theꢀ sameꢀ wayꢀ asꢀ describedꢀ before.23) As an exam-
previously.22)
ple, oral administration of compound 9ꢀtoꢀmiceꢀexertedꢀlowerꢀ
Cell-basedꢀ assayꢀ showedꢀ thatꢀ someꢀ selectedꢀ derivativesꢀ anti-inflammatoryꢀactivityꢀthanꢀthatꢀofꢀzileutonꢀ(Tableꢀ3),ꢀsug-
(compounds 7, 9–11)ꢀ wereꢀ comparableꢀ orꢀ superiorꢀ toꢀ zileu- gestingꢀ thatꢀ derivativesꢀ bearingꢀ oxazoleꢀ asꢀ aꢀ pharmacophoreꢀ
ton by criteria of IC50s,ꢀ demonstratingꢀ thatꢀ modificationsꢀ ofꢀ moietyꢀ haveꢀ poorꢀ bioavailabilityꢀ asꢀ thoseꢀ bearingꢀ thiazoleꢀ inꢀ
the R2 side group suppressed effectively the production of rats.23)ꢀ Despiteꢀ potentꢀ in vitroꢀ inhibitoryꢀ profilesꢀ ofꢀ selectedꢀ
LTB4ꢀ inꢀ A23187-stimulatedꢀ cells.ꢀ Inꢀ contrastꢀ withꢀ in vitro compoundsꢀ thatꢀ wereꢀ comparableꢀ toꢀ zileuton,ꢀ theirꢀ relativelyꢀ
enzymaticꢀ results,ꢀ theꢀ cell-basedꢀ assayꢀ showedꢀ thatꢀ N-(4- lowꢀ in vivoꢀ efficaciesꢀ withꢀ oralꢀ administrationꢀ mayꢀ beꢀ ex-
hydroxyphenyl)ꢀoxazol-2-amineꢀderivativeꢀwasꢀlessꢀpotentꢀthanꢀ plained partly by their poor bioavailability. In contrast, topical
N-(4-aminophenyl)ꢀoxazol-2-amineꢀ derivativesꢀ counterpartꢀ application of these same derivatives in mice improved the in-
withꢀrespectꢀtoꢀinterferingꢀwithꢀLTB4ꢀproductionꢀ(compoundsꢀ flammatoryꢀconditionsꢀconsiderably,ꢀsuchꢀasꢀearꢀswellingꢀandꢀ
3, 11;ꢀ dataꢀ notꢀ shown).ꢀ Intriguingly,ꢀ cellularꢀ efficaciesꢀ ofꢀ myeloperoxidaseꢀ (MPO)ꢀ activityꢀ followingꢀ AAꢀ paintingꢀ ontoꢀ
chemicalꢀ derivativesꢀ wereꢀ examinedꢀ withꢀ substituentꢀ groupsꢀ the ear (Table 3). In particular, compound 9 had more potent
varied at R2, reasoning that compounds’ cellular uptake and anti-inflammatoryꢀ activityꢀ thanꢀ anyꢀ otherꢀ compoundsꢀ whenꢀ
intracellular metabolism might be of primary concern in givenꢀ topically.ꢀ Similarly,ꢀ asꢀ shownꢀ previouslyꢀ inꢀ N-aryl-
keeping the in vitro potency in a cellular format. In effect, 4-aryl-1,3-thiazol-2-amineꢀ derivatives,ꢀ compoundꢀ 9ꢀ showedꢀ aꢀ
addition of a methyl group to the aminophenyl or hydroxy- moreꢀsustainableꢀeffectꢀagainstꢀAA-inducedꢀinflammationꢀthanꢀ
phenyl moiety enhanced the inhibitory activity of the parent zileuton.ꢀ Inꢀ fact,ꢀ theꢀ anti-inflammatoryꢀ activityꢀ ofꢀ compoundꢀ
compoundꢀ whereasꢀ substitutionꢀ withꢀ aꢀ halogenꢀ groupꢀ less- 9ꢀ wasꢀ stillꢀ substantialꢀ (approximatelyꢀ 50%ꢀ inhibition)ꢀ atꢀ 2ꢀdꢀ
afterꢀ topicalꢀ treatment,ꢀ whereasꢀ theꢀ effectsꢀ ofꢀ zileutonꢀ wereꢀ
significantlyꢀreducedꢀevenꢀatꢀ1ꢀdꢀofꢀtreatmentꢀ(Fig.ꢀ1).ꢀItꢀisꢀsug-
gested that compound 9 keeps staying longer at the painting
Tableꢀ 2.ꢀ InhibitoryꢀActivitiesꢀofꢀPotentꢀDerivativesꢀSelectedꢀthroughꢀtheꢀ
First-Roundꢀ Screeningꢀ againstꢀ LTB4ꢀ Productionꢀ inꢀ A23187-Stimulatedꢀ
spotꢀorꢀwasꢀmetabolicallyꢀstable,ꢀcomparedꢀwithꢀzileuton.
Asꢀ withꢀ theꢀ poorꢀ bioavailabilityꢀ ofꢀ 4-[4-(4-fluoro-phenyl)-
thiazol-2-ylamino]-2,6-dimethyl-phenolꢀ (KR-33749)ꢀ (2%ꢀ bio-
availabilityꢀ inꢀ rat),ꢀ smallꢀ moleculesꢀ withꢀ aromaticꢀ aminoꢀ andꢀ
hydroxylꢀ groupsꢀ areꢀ subjectꢀ toꢀ extensiveꢀ metabolismꢀ inꢀ theꢀ
liver.23–25)ꢀ Forꢀ example,ꢀ oneꢀ ofꢀ theꢀ non-specificꢀ LOXꢀ inhibi-
tors,ꢀ nordihydroguaiareticꢀ acidꢀ (NDGA),ꢀ hasꢀ beenꢀ reportedꢀ
to display only topical activity in reducing both AA-induced
edemaꢀ andꢀ infiltrationꢀ ofꢀ inflammatoryꢀ cellsꢀ intoꢀ inflamedꢀ
areas.26)ꢀThus,ꢀanti-inflammatoryꢀcompoundsꢀdesignedꢀforꢀpo-
tentꢀtopicalꢀapplicationꢀcanꢀbeꢀfurtherꢀtestedꢀinꢀtheꢀinflamma-
tion-related skin lesions, such as atopic dermatitis and other
chronicꢀinflammatoryꢀdiseases.
RBL Cells
Compounds
a)
%ꢀinhibitionꢀatꢀ1ꢀµM
IC50 (nM)
Zileuton
77
31
90
97
70
85
430
ND
279
104
352
384
3
7
9
10
11
a) Cell-based assay to determine LTB4 amounts released in RBL-1 cells stimu-
latedꢀ withꢀ A23187.ꢀ Cellsꢀ wereꢀ preloadedꢀ withꢀ selectedꢀ compoundsꢀ priorꢀ toꢀ A23187ꢀ
stimulationꢀtoꢀcompareꢀtheirꢀinhibitoryꢀpotencies.ꢀLTB4ꢀlevels,ꢀindicativeꢀofꢀ5-LOXꢀ
activity,ꢀ wereꢀ measuredꢀ quantitativelyꢀ accordingꢀ toꢀ theꢀ protocolꢀ providedꢀ byꢀ theꢀ
manufacturer.
Table 3. Comparative in VivoꢀEfficacyꢀofꢀSelectedꢀCompoundsꢀagainstꢀAA-InducedꢀEdemaꢀvia p.o. or Topical Route
Administration routesa)
Compounds
Oralꢀadministrationꢀ(%ꢀinhibition)
Topicalꢀadministrationꢀ(%ꢀinhibition)
Ear thickness Myeloperoxidase
43 63
39
49
58 10
Ear thickness
75 12
18
Myeloperoxidase
Zileuton
82 20
12 10
7
8
3
7
9
6
6
58 13
66 12
80 10
30 10
44 16
20
34
5
8
7
a)ꢀForꢀp.o.ꢀadministration,ꢀcompoundsꢀandꢀzileutonꢀ(50ꢀmpk)ꢀwereꢀgivenꢀtoꢀtheꢀmiceꢀ30ꢀminꢀbeforeꢀAAꢀpaintingꢀ(2ꢀmgꢀinꢀ20ꢀµL acetone) on the right ear. Also, test com-
poundsꢀwereꢀappliedꢀtopicallyꢀatꢀaꢀdoseꢀofꢀ500ꢀµg.ꢀAtꢀ1ꢀhꢀafterꢀAAꢀapplication,ꢀtheꢀearꢀthicknessꢀofꢀtheꢀinflamedꢀrightꢀearꢀwasꢀmeasured,ꢀandꢀthenꢀtheꢀinflamedꢀearsꢀwereꢀtakenꢀ
forꢀMPOꢀactivityꢀmeasurement.ꢀEfficacyꢀofꢀtheꢀdrugꢀwasꢀevaluatedꢀbyꢀrelativeꢀsuppressionꢀofꢀearꢀthicknessꢀandꢀMPOꢀactivityꢀinꢀinflamedꢀearsꢀofꢀtreatedꢀgroupꢀvs. vehicle group.

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Vol. 63, No. 8 (2015)
Fig.ꢀ 1.ꢀ TheꢀSustainableꢀAnti-inflammatoryꢀPotencyꢀofꢀCompoundꢀ9 in an AA-Induced Ear Edema Model
To determine the sustainable action of compound 9, test compounds (500µg)ꢀwereꢀappliedꢀandꢀforꢀ48,ꢀ24,ꢀorꢀ0.5ꢀh.ꢀFollowingꢀtheꢀindicatedꢀincubationꢀtimes,ꢀearsꢀwereꢀ
stimulatedꢀwithꢀAAꢀandꢀthenꢀinflammatoryꢀindicators,ꢀsuchꢀasꢀearꢀthicknessꢀandꢀMPOꢀactivity,ꢀwereꢀmeasuredꢀ1ꢀhꢀlater.ꢀDurationꢀofꢀtestꢀandꢀreferenceꢀcompoundꢀwasꢀ
monitoredꢀbyꢀ%ꢀanti-inflammatoryꢀactivityꢀwithꢀpretreatmentꢀtimes.
Small amount of 2-bromoacetophenone (1.00g, 5.02mmol)
Conclusion
Weꢀ identifiedꢀ anꢀ oxazole-bearingꢀ skeletonꢀ withꢀ anti- wasꢀ firstꢀ dissolvedꢀ inꢀ 15ꢀmLꢀ ofꢀ acetone,ꢀ andꢀ 0.39ꢀgꢀ
inflammatoryꢀ activityꢀ targetingꢀ 5-LOX.ꢀ Aꢀ fewꢀ compoundsꢀ (6.03ꢀmmol)ꢀ ofꢀ sodiumꢀ azideꢀ wasꢀ added,ꢀ andꢀ thenꢀ keptꢀ atꢀ
(compounds 3, 7, 9)ꢀ wereꢀ moreꢀ effectiveꢀ againstꢀ AA-inducedꢀ roomꢀ temperatureꢀ forꢀ 2ꢀh.ꢀ Afterꢀ theꢀ reactionꢀ wasꢀ completed,ꢀ
earꢀedemaꢀthanꢀzileutonꢀwhenꢀappliedꢀtopically,ꢀbutꢀnotꢀorally.ꢀ theꢀreactionꢀmixtureꢀwasꢀdilutedꢀwithꢀ20ꢀmLꢀofꢀethylꢀacetate,ꢀ
Judged from in vivoꢀtopicalꢀefficacyꢀdataꢀofꢀcompoundꢀ9ꢀwithꢀ washedꢀwithꢀwater,ꢀdriedꢀoverꢀanhydrousꢀmagnesiumꢀsulfate,ꢀ
itsꢀ sustainableꢀ activity,ꢀ itꢀ mayꢀ beꢀ applicableꢀ toꢀ aꢀ wideꢀ rangeꢀ andꢀ finallyꢀ concentratedꢀ underꢀ aꢀ reducedꢀ pressure.ꢀ Theꢀ
ofꢀskinꢀmodelsꢀassociatedꢀwithꢀ5-LOXꢀinꢀtheꢀfuture.ꢀToꢀdefineꢀ residuesꢀwereꢀpurifiedꢀusingꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ
more comprehensively molecular mechanism of a compound (hexaneꢀ:ꢀethylꢀ acetate=10ꢀ:ꢀ1)ꢀ toꢀ obtainꢀ theꢀ 2-azido-1-
9, a controlled pilot study for clarifying redox or non-redox phenylethan-1-oneꢀ (0.66ꢀg,ꢀ 82%).ꢀ 1H-NMRꢀ (300ꢀMHz,ꢀ CDCl3)
inhibitoryꢀ typesꢀ willꢀ beꢀ carriedꢀ outꢀ andꢀ itsꢀ selectivityꢀ forꢀ δ:ꢀ 4.57ꢀ (s,ꢀ 2H),ꢀ 7.50ꢀ (dd,ꢀ J=7.5ꢀHz,ꢀ 7.4ꢀHz,ꢀ 2H),ꢀ 7.63ꢀ (dd,ꢀ
5-LOXꢀoverꢀotherꢀLOXꢀisozymesꢀwillꢀbeꢀfurtherꢀexamined.
J=7.4ꢀHz,ꢀ7.4ꢀHz,ꢀ1H),ꢀ7.91ꢀ(d,ꢀJ=7.5ꢀHz,ꢀ2H).
(b)ꢀ Synthesisꢀofꢀ4-((5-Phenyloxazol-2-yl)amino)phenolꢀ(3)
ꢀTheꢀresultingꢀcompoundꢀ2-azido-1-phenylethan-1-oneꢀ(0.31ꢀg,ꢀ
Experimental
General Information All the commercially available 1.91ꢀmmol)ꢀ wasꢀ dissolvedꢀ inꢀ 10ꢀmLꢀ ofꢀ 1,4-dioxane.ꢀ Theꢀ tri-
chemicalsꢀ wereꢀ obtainedꢀ fromꢀ Aldrich,ꢀ Sigma,ꢀ Fluka,ꢀ andꢀ phenyl phosphine (0.55g, 2.09mmol) and 4-hydroxy phenyl
JunseiꢀChemicalꢀCompanyꢀandꢀusedꢀgenerallyꢀwithoutꢀfurtherꢀ isothiocyanateꢀ (0.26ꢀg,ꢀ 1.74ꢀmmol)ꢀ wereꢀ addedꢀ atꢀ roomꢀ tem-
purification.ꢀSolventsꢀincludingꢀethylꢀacetateꢀandꢀn-hexane for perature,ꢀandꢀtheꢀmixtureꢀwasꢀrefluxedꢀatꢀ80°Cꢀforꢀ1ꢀh.ꢀAfterꢀ
columnꢀchromatographyꢀwereꢀofꢀtechnicalꢀgradeꢀandꢀdistilledꢀ theꢀreactionꢀwasꢀcompleted,ꢀtheꢀreactionꢀmixtureꢀwasꢀdilutedꢀ
before use.
withꢀ10ꢀmLꢀofꢀethylꢀacetate,ꢀwashedꢀwithꢀwater,ꢀdriedꢀoverꢀan-
The 1H-NMRꢀ spectraꢀ (300ꢀMHzꢀ H)ꢀ wereꢀ recordedꢀ onꢀ hydrous magnesium sulfate, and concentrated under a reduced
BrukerꢀFourierꢀTransformꢀAG-300ꢀandꢀVarianꢀFourierꢀTrans- pressure.ꢀ Theꢀ residuesꢀ wereꢀ purifiedꢀ usingꢀ silicaꢀ gelꢀ columnꢀ
form-300 instrument in chroroform-d3ꢀ (CDCl3) or dimethyl chromatographyꢀ (hexaneꢀ:ꢀethylꢀ acetate=2ꢀ:ꢀ1)ꢀ toꢀ obtainꢀ theꢀ
sulfoxide-d6ꢀ (DMSO-d6).ꢀ Chemicalꢀ shiftsꢀ wereꢀ reportedꢀ inꢀ δ finalꢀproductꢀ 3ꢀ(0.25ꢀg,ꢀ56%).ꢀ1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6)
units (ppm), using tetramethylsilane (TMS) as internal stan- δ:ꢀ6.71ꢀ(d,ꢀJ=9.0ꢀHz,ꢀ2H),ꢀ7.25ꢀ(dd,ꢀJ=7.5ꢀHz,ꢀ7.5ꢀHz,ꢀ1H),ꢀ7.40ꢀ
dard.ꢀTheꢀfollowingꢀabbreviationsꢀwereꢀusedꢀtoꢀdescribeꢀpeakꢀ (m, 5H), 7.55 (d, J=7.5ꢀHz,ꢀ2H),ꢀ9.03ꢀ(s,ꢀ1H),ꢀ9.86ꢀ(s,ꢀ1H);ꢀMSꢀ
patternsꢀ whenꢀ appropriate:ꢀ s,ꢀ singlet;ꢀ d,ꢀ doublet;ꢀ t,ꢀ triplet;ꢀ (EI) m/z 252 [M]+.
1
q,ꢀ quartet;ꢀ m,ꢀ multiplet.ꢀ Couplingꢀ constantꢀ (J)ꢀ wasꢀ reportedꢀ
inꢀ Hertzꢀ unitꢀ (Hz).ꢀ Massꢀ spectraꢀ wereꢀ recordedꢀ onꢀ aꢀ Varianꢀ according to the general procedures described above by em-
1200ꢀLꢀGC/MSꢀusingꢀelectronꢀimpactꢀ(EI)ꢀmethod. ployingꢀ theꢀ appropriateꢀ 2-azido-1-phenylethan-1-oneꢀ andꢀ phe-
Allꢀ reactionsꢀ wereꢀ monitoredꢀ usingꢀ thinꢀ layerꢀ chromatog- nyl isothiocyanate.
Theꢀ followingꢀ oxazole-bearingꢀ derivativesꢀ wereꢀ preparedꢀ
raphy (TLC) on silica gel glass plates (E.Merck, TLC silica
4-((5-(4-Chlorophenyl)oxazol-2-yl)amino)phenol (4)
gelꢀ 60F254).ꢀ Visualizationꢀ onꢀ TLCꢀ wasꢀ achievedꢀ byꢀ UVꢀ lightꢀ 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ6.64ꢀ(d,ꢀJ=8.9ꢀHz,ꢀ2H),ꢀ7.32ꢀ
(254nm), typical TLC indicating solution (p-anisaldehyde), (d, J=8.9ꢀHz,ꢀ 2H),ꢀ 7.38ꢀ (s,ꢀ 1H),ꢀ 7.39ꢀ (d,ꢀ J=8.6ꢀHz,ꢀ 2H),ꢀ 7.48ꢀ
iodineꢀandꢀpolymolybdicꢀacidꢀindicator.ꢀFlashꢀcolumnꢀchroma- (d, J=8.6ꢀHz,ꢀ2H),ꢀ8.98ꢀ(s,ꢀ1H),ꢀ9.90ꢀ(s,ꢀ1H);ꢀMSꢀ(EI)ꢀm/z 287
tographyꢀwasꢀcarriedꢀoutꢀusingꢀMerckꢀ230-400ꢀmeshꢀASTM.
Synthetic Procedure for the Preparation of N-Aryl-5-ar-
yloxazol-2-amine Derivatives
[M]+.
4-((5-(3,4-Dichlorophenyl)oxazol-2-yl)amino)phenol (5)
1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ6.73ꢀ(d,ꢀJ=8.7ꢀHz,ꢀ2H),ꢀ7.40ꢀ
(d, J=8.7ꢀHz,ꢀ 2H),ꢀ 7.50ꢀ (d,ꢀ J=8.4ꢀHz,ꢀ 1H),ꢀ 7.52ꢀ (s,ꢀ 1H),ꢀ 7.67ꢀ
(a)ꢀ Preparationꢀofꢀ2-Azido-1-phenylethan-1-one

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
577
(d, J=8.4ꢀHz,ꢀ1H),ꢀ7.78ꢀ(s,ꢀ1H),ꢀ9.11ꢀ(s,ꢀ1H),ꢀ10.04ꢀ(s,ꢀ1H);ꢀMSꢀ
(EI) m/z 321 [M]+.
4-((5-(4-Fluorophenyl)oxazol-2-yl)amino)phenyl Acetate
(20) 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ2.09ꢀ(s,ꢀ3H),ꢀ7.07ꢀ(d,ꢀ
4-((5-(4-Methoxyphenyl)oxazol-2-yl)amino)phenol (6) J=8.7ꢀHz,ꢀ2H),ꢀ7.29ꢀ(m,ꢀ2H),ꢀ7.43ꢀ(s,ꢀ1H),ꢀ7.60–7.70ꢀ(m,ꢀ4H),ꢀ
1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 3.78ꢀ (s,ꢀ 3H),ꢀ 6.70ꢀ (d,ꢀ 10.38 (s, 1H); MS (EI) m/z 312 [M]+.
J=8.8ꢀHz,ꢀ 2H),ꢀ 6.98ꢀ (d,ꢀ J=8.8ꢀHz,ꢀ 2H),ꢀ 7.22ꢀ (s,ꢀ 1H),ꢀ 7.39ꢀ (d,ꢀ
J=8.7ꢀHz,ꢀ 1H),ꢀ 7.47ꢀ (d,ꢀ J=8.7ꢀHz,ꢀ 1H),ꢀ 9.01ꢀ (s,ꢀ 1H),ꢀ 9.83ꢀ (s,ꢀ tate (21) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 2.26ꢀ (s,ꢀ 3H),ꢀ
1H); MS (EI) m/z 282 [M]+.
7.09 (s, 2H), 7.21–7.31 (m, 2H), 7.44 (m, 1H), 7.61–7.67 (m,
4-((5-(2,4-Difluorophenyl)oxazol-2-yl)amino)phenyl Ace-
4-((5-(4-Fluorophenyl)oxazol-2-yl)amino)phenol (7) 3H), 10.49 (s, 1H); MS (EI) m/z 330 [M]+.
1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6,) δ:ꢀ 6.72ꢀ (d,ꢀ J=8.9ꢀHz,ꢀ 2H),ꢀ
4-((5-(3,4-Dichlorophenyl)oxazol-2-yl)amino)phenyl Ace-
7.05–7.42 (m, 5H), 7.56–7.61 (m, 2H), 9.05 (s, 1H), 9.91 (s, tate (22) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 2.25ꢀ (s,ꢀ 3H),ꢀ
1H); MS (EI) m/z 270 [M]+.
7.09 (d, J=8.7ꢀHz,ꢀ 2H),ꢀ 7.53ꢀ (d,ꢀ J=8.4ꢀHz,ꢀ 1H),ꢀ 7.63–7.71ꢀ (m,ꢀ
4-((5-(2,4-Difluorophenyl)oxazol-2-yl)amino)phenol
(8) 4H), 7.83 (s, 1H), 10.49 (s, 1H); MS (EI) m/z 363 [M]+.
1H-NMRꢀ (300ꢀMHzꢀ DMSO-d6) δ:ꢀ 6.73ꢀ (d,ꢀ J=9.0ꢀHz,ꢀ 2H),ꢀ
3-((5-(3-Nitrophenyl)oxazol-2-yl)amino)benzoic Acid (23)
7.22–7.24 (m, 2H), 7.37–7.43 (m, 3H), 7.60 (m, 1H), 9.08 (s, 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ4.35ꢀ(br,ꢀ2H),ꢀ7.25ꢀ(m,ꢀ1H),ꢀ
1H), 10.03 (s, 1H); MS (EI) m/z 288 [M]+.
7.36 (d, J=7.5ꢀHz,ꢀ 1H),ꢀ 7.55ꢀ (m,ꢀ 1H),ꢀ 7.62ꢀ (s,ꢀ 1H),ꢀ 7.64ꢀ (m,ꢀ
4-((5-(4-Fluorophenyl)oxazol-2-yl)amino)-3,5-dimethyl- 1H), 7.84 (d, J=7.8ꢀHz,ꢀ 1H),ꢀ 7.91ꢀ (d,ꢀ J=9.6ꢀHz,ꢀ 1H),ꢀ 8.13ꢀ (s,ꢀ
phenol (9) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 2.21ꢀ (s,ꢀ 6H),ꢀ 1H), 8.18 (s, 1H), 10.50 (s, 1H); MS (EI) m/z 325 [M]+.
7.28 (m, 2H), 7.44 (s, 1H), 7.58–7.65 (m, 4H), 9.68 (s, 1H),
3-((5-(3-Aminophenyl)oxazol-2-yl)amino)benzoic
Acid
10.41 (s, 1H); MS (EI) m/z 298 [M]+.
(24) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 4.35ꢀ (br,ꢀ 2H),ꢀ 7.25ꢀ
4-((5-(4-Fluorophenyl)oxazol-2-yl)amino)-3,5-dichloro- (m, 1H), 7.36 (d, J=7.8ꢀHz,ꢀ 1H),ꢀ 7.55ꢀ (m,ꢀ 1H),ꢀ 7.62ꢀ (s,ꢀ 1H),ꢀ
phenol (10) 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ7.28ꢀ(m,ꢀ2H),ꢀ 7.63 (m, 1H), 7.84 (d, J=7.8ꢀHz,ꢀ 1H),ꢀ 7.91ꢀ (d,ꢀ J=9.6ꢀHz,ꢀ 1H),ꢀ
7.44 (s, 1H), 7.58–7.65 (m, 4H), 9.68 (s, 1H), 10.41 (s, 1H); MS 8.13 (s, 1H), 8.18 (s, 1H), 10.50 (s, 1H); MS (EI) m/z 295 [M]+.
(EI) m/z 339 [M]+.
Materialsꢀ ꢀXylenol,ꢀ ironꢀ sulfate,ꢀ ATP,ꢀ hexadecyltrimethylꢀ
N1-(5-Phenyloxazol-2-yl)benzene-1,4-diamine (11) ammonium bromide (HTAB), α-phophatidyl choline (PC), and
1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 4.75ꢀ (br,ꢀ 2H),ꢀ 6.54ꢀ (d,ꢀ zileutonꢀ wereꢀ obtainedꢀ fromꢀ Sigmaꢀ (St.ꢀ Louis,ꢀ MO,ꢀ U.S.A.).ꢀ
J=8.7ꢀHz,ꢀ2H),ꢀ7.21–7.29ꢀ(m,ꢀ3H),ꢀ7.36–7.43ꢀ(m,ꢀ3H),ꢀ7.53ꢀ(d,ꢀ Arachidonicꢀ acidꢀ (AA)ꢀ andꢀ A23187ꢀ wereꢀ obtainedꢀ fromꢀ Cal-
J=8.0ꢀHz,ꢀ2H),ꢀ9.72ꢀ(s,ꢀ1H);ꢀMSꢀ(EI)ꢀm/z 251 [M]+.
biochemꢀ (Laꢀ Jolla,ꢀ CA,ꢀ U.S.A.).ꢀ Theꢀ leukotrieneꢀ B4ꢀ (LTB4)ꢀ
N1-(5-(4-Chlorophenyl)oxazol-2-yl)benzene-1,4-diamine enzymeꢀ immunoassayꢀ (EIA)ꢀ kitꢀ wasꢀ obtainedꢀ fromꢀ Amersh-
(12) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 4.76ꢀ (br,ꢀ 2H),ꢀ 6.52ꢀ amꢀ(NJ,ꢀU.S.A.).ꢀAꢀseriesꢀofꢀchemicalꢀderivativesꢀwasꢀsynthe-
(d, J=8.6ꢀHz,ꢀ2H),ꢀ7.24ꢀ(d,ꢀJ=8.6ꢀHz,ꢀ2H),ꢀ7.41ꢀ(s,ꢀ1H),ꢀ7.47ꢀ(d,ꢀ sizedꢀasꢀdescribedꢀbelow.ꢀAllꢀotherꢀreagentsꢀwereꢀofꢀanalyticalꢀ
J=9.3ꢀHz,ꢀ 2H),ꢀ 7.52ꢀ (d,ꢀ J=9.3ꢀHz,ꢀ 2H),ꢀ 9.76ꢀ (s,ꢀ 1H);ꢀ MSꢀ (EI)ꢀ grade and from Sigma.
m/z 285 [M]+.
Cell Culture and Animal Careꢀ ꢀRBL-1ꢀ cellsꢀ wereꢀ ob-
N1-(5-(3,4-Dichlorophenyl)oxazol-2-yl)benzene-1,4-di- tained from the American Type Culture Collection (Manassas,
amine (13) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 4.80ꢀ (br,ꢀ VA,ꢀ U.S.A.)ꢀ andꢀ maintainedꢀ inꢀ Dulbecco’sꢀ modifiedꢀ Eagle’sꢀ
2H), 6.54 (d, J=8.6ꢀHz,ꢀ 2H),ꢀ 7.25ꢀ (d,ꢀ J=8.6ꢀHz,ꢀ 1H),ꢀ 7.43ꢀ (d,ꢀ mediumꢀ (DMEM),ꢀ supplementedꢀ withꢀ 10%ꢀ heat-inactivatedꢀ
J=8.4ꢀHz,ꢀ 1H),ꢀ 7.56ꢀ (s,ꢀ 1H),ꢀ 7.66ꢀ (d,ꢀ J=8.4ꢀHz,ꢀ 1H),ꢀ 7.77ꢀ (s,ꢀ fetalꢀ calfꢀ serum,ꢀ glutamine,ꢀ penicillin,ꢀ andꢀ streptomycin.ꢀ Forꢀ
1H), 9.83 (s, 1H); MS (EI) m/z 320 [M]+.
in vivoꢀ efficacy,ꢀ BALB/cꢀ miceꢀ (males,ꢀ 8ꢀ weeksꢀ old)ꢀ wereꢀ
N1-(5-(4-Fluorophenyl)oxazol-2-yl)benzene-1,4-diamine bred, and cared for in a barrier system in a temperature- and
(14) 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ4.76ꢀ(br,ꢀ2H),ꢀ6.54ꢀ(d,ꢀ humidity-controlledꢀroomꢀwithꢀaꢀ12/12-hꢀlight/darkꢀcycle,ꢀandꢀ
J=8.6ꢀHz,ꢀ 2H),ꢀ 7.23-7.32ꢀ (m,ꢀ 4H),ꢀ 7.34ꢀ (s,ꢀ 1H),ꢀ 7.55–7.59ꢀ (m,ꢀ acclimatedꢀforꢀ1ꢀweekꢀbeforeꢀexperiments.ꢀAllꢀanimalꢀexperi-
2H), 9.71 (s, 1H); MS (EI) m/z 269 [M]+.
mentsꢀ wereꢀ carriedꢀ outꢀ inꢀ accordanceꢀ withꢀ theꢀ Guideꢀ forꢀ theꢀ
N1-(5-(2,4-Difluorophenyl)oxazol-2-yl)benzene-1,4-di- Careꢀ andꢀ Useꢀ ofꢀ Laboratoryꢀ Animals,ꢀ andꢀ approvedꢀ byꢀ theꢀ
amine (15) 1H-NMRꢀ(300ꢀMHz,ꢀDMSO-d6) δ:ꢀ4.78ꢀ(br,ꢀ2H),ꢀ Institutionalꢀ Animalꢀ Careꢀ andꢀ Useꢀ Committeeꢀ ofꢀ theꢀ Koreaꢀ
6.55 (d, J=8.6ꢀHz,ꢀ2H),ꢀ7.19–7.29ꢀ(m,ꢀ4H),ꢀ7.37ꢀ(m,ꢀ1H),ꢀ7.58ꢀ Researchꢀ Instituteꢀ ofꢀ Chemicalꢀ Technologyꢀ (Daejeon,ꢀ Southꢀ
(m, 1H), 9.83 (s, 1H); MS (EI) m/z 287 [M]+.
Korea).
3,5-Dichloro-N1-(5-(4-fluorophenyl)oxazol-2-yl)ben-
In Vitro 5-LOX Assayꢀ ꢀAꢀ5-LOXꢀenzymeꢀassayꢀwasꢀcar-
zene-1,4-diamine (16) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ riedꢀ outꢀ withꢀ someꢀ modificationsꢀ ofꢀ theꢀ ferricꢀ oxidationꢀ ofꢀ
5.13 (br, 2H), 7.27 (m, 2H), 7.41(s, 1H), 7.56–7.61 (m, 4H), xylenolꢀ orangeꢀ (FOX)ꢀ assay,ꢀ whichꢀ isꢀ basedꢀ onꢀ theꢀ complexꢀ
10.21 (s, 1H); MS (EI) m/z 337 [M]+.
formationꢀ ofꢀ Fe3+/xylenolꢀ orangeꢀ withꢀ absorptionꢀ atꢀ visibleꢀ
N1-(5-(4-Chlorophenyl)oxazol-2-yl)benzene-1,3-diamine wavelengthsꢀ19,ꢀ20).ꢀTheꢀenzymeꢀsourcesꢀwereꢀpreparedꢀfromꢀ
(17) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 5.07ꢀ (br,ꢀ 2H),ꢀ 6.18ꢀ insectꢀ cellꢀ (Sf21)ꢀ lysatesꢀ highlyꢀ expressingꢀ ratꢀ 5-LOXꢀ asꢀ
(d, J=7.8ꢀHz,ꢀ1H),ꢀ6.70ꢀ(d,ꢀJ=7.8ꢀHz,ꢀ1H),ꢀ6.89–6.94ꢀ(m,ꢀ2H),ꢀ described previously. Cell lysates (15µgꢀ protein)ꢀ wereꢀ prein-
7.47–7.60 (m, 5H), 10.00 (s, 1H); MS (EI) m/z 286 [M]+.
cubatedꢀ withꢀ variousꢀ concentrationsꢀ ofꢀ eitherꢀ someꢀ selectedꢀ
N-(4-((5-(4-Chlorophenyl)oxazol-2-yl)amino)phenyl)acet- chemicalsꢀorꢀzileutonꢀ(inꢀDMSO)ꢀinꢀ50ꢀµL of 50mM Tris–HCl
amide (18) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 2.00ꢀ (s,ꢀ 3H),ꢀ buffer (pH 7.4) containing 0.4mM CaCl2, 24µg/mL PC and
7.48-7.61 (m, 9H), 9.82 (s, 1H), 10.24 (s, 1H); MS (EI) m/z 327 20µMꢀATPꢀatꢀroomꢀtemperatureꢀforꢀ4ꢀmin.ꢀTheꢀreactionsꢀwereꢀ
[M]+.
started by the addition of 40µM AA, kept for another 4min,
N-(4-((5-(4-Fluorophenyl)oxazol-2-yl)amino)phenyl)acet- and terminated by the addition of 100µLꢀ FOXꢀ reagent:ꢀ sul-
amide (19) 1H-NMRꢀ (300ꢀMHz,ꢀ DMSO-d6) δ:ꢀ 2.02ꢀ (s,ꢀ 3H),ꢀ furic acid (25mM), xylenol orange (100µM), iron(II) sulfate
7.29 (dd, J=8.9ꢀHz,ꢀ 8.7ꢀHz,ꢀ 2H),ꢀ 7.42ꢀ (s,ꢀ 1H),ꢀ 7.49–7.64ꢀ (m,ꢀ (100 µM),ꢀ methanol–waterꢀ (9ꢀ:ꢀ1,ꢀ v/v).ꢀ Aꢀ blankꢀ assayꢀ wasꢀ doneꢀ
6H), 9.82 (s, 1H), 10.20 (s, 1H); MS (EI) m/z 311 [M]+.
withꢀnoꢀsubstrateꢀinꢀtheꢀincubation.

578
Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
LTB4 Secretion in RBL-1 Cells Activated by A23187
References
Aꢀ cell-basedꢀ assayꢀ wasꢀ carriedꢀ outꢀ asꢀ reportedꢀ previously.22)
Briefly,ꢀ RBL-1ꢀ cellsꢀ wereꢀ platedꢀ atꢀ aꢀ densityꢀ ofꢀ 4×104 cells
inꢀ 96-wellꢀ plates.ꢀ Increasingꢀ dosesꢀ ofꢀ compoundsꢀ orꢀ zileutonꢀ
dissolvedꢀinꢀDMSOꢀ(finalꢀ0.5%)ꢀwereꢀaddedꢀtoꢀplatesꢀandꢀin-
cubationꢀ forꢀ 10ꢀminꢀ atꢀ 37°Cꢀ priorꢀ toꢀ theꢀ stimulationꢀ withꢀ theꢀ
Ca2+ꢀionophoreꢀA23187ꢀ(finalꢀ10ꢀµMꢀinꢀDMSO).ꢀAfterꢀfurtherꢀ
incubationꢀforꢀ15ꢀmin,ꢀcultureꢀmediaꢀwereꢀcollectedꢀandꢀLTB4ꢀ
contentꢀ inꢀ theꢀ supernatantꢀ wasꢀ measuredꢀ usingꢀ anꢀ EIAꢀ kitꢀ
(Amersham,ꢀNJ,ꢀU.S.A.).
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Arachidonic Acid-Induced Ear Inflammation in Mice
Toꢀinduceꢀanꢀinflammatoryꢀresponse,ꢀ2ꢀmgꢀofꢀAAꢀinꢀ20ꢀµL of
acetoneꢀ wasꢀ paintedꢀ toꢀ theꢀ innerꢀ surfaceꢀ ofꢀ theꢀ rightꢀ earꢀ ofꢀ
aꢀ BALB/cꢀ mouseꢀ (8ꢀ weeksꢀ ofꢀ age,ꢀ male,ꢀ n=5–6 per group).
Some potent derivative (3, 7, 9)ꢀandꢀzileutonꢀwereꢀpaintedꢀdi-
rectly on the ears (500µg/ear) or given orally to mice (50mpk)
1h before AA treatment (2mg in 20µLꢀ acetone).ꢀ Forꢀ vehicleꢀ
control for per os (p.o.)ꢀ administration,ꢀ anꢀ equalꢀ volumeꢀ ofꢀ
0.5%ꢀ carboxymethylꢀ celluloseꢀ (CMC)ꢀ wasꢀ given.ꢀ Atꢀ 1ꢀhꢀ afterꢀ
AA stimulation, ear thickness of the treated- and untreated-
earsꢀ wasꢀ measuredꢀ withꢀ aꢀ dialꢀ thicknessꢀ gaugeꢀ (Mitutoyo,ꢀ
Tokyo, Japan). To further examine the long-lasting effect
of a potent compound against an ear-edema animal model,
mouseꢀ earsꢀ wereꢀ preloadedꢀ topicallyꢀ withꢀ eitherꢀ zileutonꢀ orꢀ
compound 9ꢀ overꢀ theꢀ indicatedꢀ timesꢀ andꢀ thenꢀ theꢀ skinꢀ wasꢀ
inflamedꢀ withꢀ AAꢀ treatment.ꢀ Theꢀ effectsꢀ ofꢀ chemicalꢀ deriva-
tivesꢀ wereꢀ calculatedꢀ asꢀ theꢀ percentꢀ differenceꢀ inꢀ earꢀ thick-
ness from the mean values of the contralateral untreated ear.
Myeloperoxidaseꢀ (MPO)ꢀ activityꢀ inꢀ theꢀ inflamedꢀ earꢀ tissueꢀ
wasꢀ determinedꢀ accordingꢀ toꢀ theꢀ methodꢀ describedꢀ before.27)
Inꢀbrief,ꢀpunchedꢀearsꢀ(2ꢀmmꢀinꢀdiameter)ꢀwereꢀchopped,ꢀandꢀ
homogenizedꢀinꢀ300ꢀµL of 50mM potassium phosphate buffer,
pHꢀ 6.0,ꢀ containingꢀ 0.5%ꢀ HTAB.ꢀ Theꢀ homogenateꢀ wasꢀ thenꢀ
centrifuged and the resulting supernatant (100µL)ꢀ wasꢀ al-
lowedꢀ toꢀ reactꢀ withꢀ 0.167ꢀmg/mLꢀ o-dianisidineꢀ andꢀ 0.0005%ꢀ
H2O2 in order to determine the absorbance at 450nm.
Statistical Analysisꢀ ꢀResultsꢀ wereꢀ expressedꢀ asꢀ means
standardꢀerrorꢀofꢀtheꢀmeanꢀ(S.E.M.)ꢀofꢀtwoꢀexperiments,ꢀeachꢀ
carriedꢀoutꢀinꢀtriplicate.ꢀStatisticalꢀsignificanceꢀwasꢀevaluatedꢀ
using Student’s t-test, and p<0.05ꢀwasꢀconsideredꢀtoꢀindicateꢀ
statisticalꢀsignificance.
Acknowledgment Thisꢀ researchꢀ wasꢀ supportedꢀ byꢀ Basicꢀ
Science Research Program through the National Research
FoundationꢀofꢀKoreaꢀ(NRF)ꢀfundedꢀbyꢀtheꢀMinistryꢀofꢀEduca-
tion (2013R1A1A2010212).
27)ꢀ BradleyꢀP.ꢀP.,ꢀPriebatꢀD.ꢀA.,ꢀChristensenꢀR.ꢀD.,ꢀRothsteinꢀG.,ꢀJ. In-
vest. Dermatol., 78, 206–209 (1982).
Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
interest.