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J . M ed. Chem. 1994,37, 3663-3667 3883

NotesSynthesis and 5-L ipoxygenase Inhibitory Activitiesof Some Novel 2-Substituted5-Benzofuran Hydroxamic AcidsKwasi A. Ohemeng,* Mary A. Appollina, Van N. Nguyen, Charles F. Schwender, Monica Singer, Michele Steber,J ustin Ansell, Dennis Argentieri, and William HagemanDiscovery Research, The R . W. ohnson Pharmaceutical Research Institute, Rar itan, New J ersey 08869Received May 23, 1994"

A series of 2-substituted benzofuran hydroxyamic acids were synthesized as rigid analogs ofsimple (benzy1oxy)phenyl hydroxamates, evaluated for their i n vitro and n vivo 5-lipoxygenaseactivity and found to be potent inhibitors of the enzyme. Substituents which enhancedlipophilicity near the 2-position of the benzofuran nucleus increased inhibitor potency butreduced oral activity. Incorporation of small polar substituents such asmethoxymethylene,hydroxymethylene, and amino (urea) on the acyl group led to more consistent oral activity.Themost potent inhibitors of this seriesi n vitro wereN- hydroxy- N- [ l - ( 2- phenyl - 5- benzof uranyl ) -ethyllfurancarboxamide ( 12) and methyl 5- [N- hydroxy- N- [ l - ( 2- (3, 4, 5- t r i methoxyphenyl ) - 5-benzof uranyl l et hyl l - 5- oxopent anoate17),both with IC50 values of 40nM,andin vivo the mostpotent compound wasN- hydr oxy- N- [ l - ( 2- phenyl - 5- benzof ur anyl ) et hyl l urea,0,with an ED50=10.3mglkg.

IntroductionLeukotrienes (LTs) are biological mediators derivedfrom arachidonic acid through the actionof the enzyme5-lipoxygenase (5-LO)l and are implicated in severalinflammatory and allergic reaction^^ ^ The non-peptidicleukotriene LTB4 is a potent chemotactic agent to anumber of pro-inflammatory leukocytes i n vi t ro andpromotes aggregation, chemokinesis, and superoxiderelease by these cell ^^ Invivo LTB4 causes leukocyteaccumulation in both animals and The

peptidoleukotrienes LTC4, LTD4, and LTE4 are knownto induce contraction of human airway smooth musclepreparation and mucus formation in human ai n~ays.~J ~In addition, it is becoming very evident that LTs areinvolved in several human disease states such asasthma, allergic rhinitis, rheumatoid arthritis, gout, andinflammatory bowel disease.lJ -13 Thus, the control ofleukotriene biosynthesis through the inhibitionof 5-li-poxygenase represents a potential method for treatingsuch diseases. Known inhibitors of the enzyme includea variety of molecules containing the hydroxamic acidfunctionality, such as compounds derived from the(phenyla1koxy)benzylamines 1-3.14J5In the search for novel 5-lipoxygenase nhibitors, wesynthesized a series of 2-substituted 5-benzofuran hy-droxamic acids as rigid analogs of compounds1-3, withthe phenylalkoxy portion incorporated into a substitutedfuran ring and fused onto the phenyl ring. Severalderivatives were synthesized to help establish thestructure-activity relationships ( SAR )of this seriesofcompounds. The synthesis and biological activities ofthese compounds are reported herein.Chemistry

The synthetic route to these compoundsis shown inScheme1. The synthesis of 26ahas been reported by@ Abstract published inAdvance ACS A bstracts, September 1,1994.

Scheme 1"Rl-CECH + a) [(C,H5)3P)]2Pd(02CCH3)2/Cu~

b) PyridindCuzOO24a-e 25

CH3

26a-e 27a-ep 3NaBH3CNIHCl

HRI28a-e

4 - 19& 22 23 20 &21(a)RI =phenyl; (b)R1 =3,4,5-trimethoxyphenyl; c) R1 =6-methoxy-2-naphthyl; d)R1=n-decyl; e)R1=n-butyl.

Bisangni and Royer,16 involving cyclodehydration of2-hydroxy-5-acetylbenzylphenyl ketone(30).Thisrouteto the benzofurans nucleus was found unattractive dueto the inaccessibility of 30and its analogs, which wereobtained in low yields from degradationof 1-(3-benzoyl-2- ethyl - 5- benzofurany1) ethanone29)(Scheme 2).16 Com-pounds 26a-e were therefore synthesized by reactingthe appropriately substituted acetylenes 24a-e with3- i odo- 4- hydroxyacetophenone25) n the presence ofeitherbi s(t ri phenyl phosphi ne)pal l adi umI I ) acetate andcuprous iodide in anhydrous dimethylf~rmamidel~rcuprous oxide in pyridine.18 The ketones were thenconverted to the oximes 27a-e with hydroxylamine0022-2623/94/1837-3663$04.50IO 0 1994American Chemical Society

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3664 J ournal of Medicinal Chemistry, 1994, Vol.37, No. 21Scheme216

Notes

C H 3 W - C H 3 w 6aOHEt

309

Table1. I n Vitro5-L ipoxygenase Inhibi tory A ctivities ofCompounds 1-315

~in vitro 5-LO i nhibn:no. n R IC50OcM) (95%CL )1 0 H 0.37 (0.24-0.51)152 0 CH3 0.50 (0.50-0.64)153 1 H 0.41 (0.36-0.48)15

hydrochloride and sodium hydroxide (compounds27cand27ewere reducedto the final products without fullcharacterization), followed by reduction with sodiumcyanoborohydride under acidic conditionslgto give hy-droxylamines28a-e. The hydroxamic acids4-19 and22were prepared by acylating the hydroxylamines withthe appropriate acid chlorides in the presence of tri-ethylamine. The ester 14 obtained from 28a, andacetoxyacetyl chloride was selectively hydrolyzed withlithium hydroxide in 2-propanol and water20 o give thehydroxyl derivative23. The urea analogs 20 and 21were prepared by reaction of 28a and28b respectivelywith phosgene followed by ammonium hydroxide.20Biological R esults and Discussions

Simple compounds such as 1 have been reported topossess reasonable in vitro and in vivo 5-lipoxygenaseinhibitory activity with a rather short durationof actiondue to metabolic oxidation at the benzylic position(Tablel).14J 5 Structural modifications to limit such invivo metabolism either reduced or eliminated the invitro and/or in vivo inhibitory activities associated withthe resulting compounds.15 I t was envisioned thatmetabolic inactivation of these compounds could beblocked by incorporation of the phenylalkoxy portioninto a substituted furan ring, assuming the resultingcompounds will be active. Table 2 contains the in vitroand in vivo activities of the test compounds, comparedto the clinical candidate, zileuton.21As shown in thetable, compound4 exhibited in vitro and in vivo activi-ties, but was10times less potent in vivo than zileuton.Other derivatives were therefore synthesizedto improveboth the in vitro and/or in vivo potencies. Severalfactors regarding the enzyme and its inhibitors wereconsidered in the design of derivatives of 4. In anearlier review Cashman has summarized the key struc-tural elements of the 5-lipoxygenase active site as a non-heme ferric iron, a hydrophobic domain, and a carbox-ylate binding area.22 Other studies since then havesupported some of the suggested elements in the activesite. In a study involvingQSAR of a large seriesof theso-called typeA hydroxamic acids, it was concluded thata hydrophobic binding region within the active site ofthe enzyme was a major contributor to inhibitor po-ten~y.~~urthermore other workers have reported thatstructural changes which increase hydrophobicity are

accompanied by an increase in potency.24 In addition,compounds containing groups capableof chelation of theferric ion have been shown to be successful inhibitorsof the enzyme.lrZ5 Due to the pivotal role of thehydroxamic acid group in this type of inhibitors, thisportion was conserved in our series and modificationsconcentrated at the positions bearing R1 and R2 tochange the properties of the moleculesto modify theirinteraction with the two remaining portions of the activesite.(a)Substituents at Position 2 (RI ). Four deriva-tives, 5-8 , bearing substituents with different lipophilicand electronic properties at position 2of the benzofuranring were prepared. Replacementof the phenyl groupwith hydrophobic groups with lower electron densitysuch as n-butyl 5 led to a 10-fold increase in vitropotency. Compound6with the larger n-decyl group wasless potent than 5 , suggesting that though hydrophobic-ity may be important in this portion of the inhibitors,there is a limiting size contribution. Replacementofthephenyl group with 3,4,54rimethoxyphenyl (7) and6-methoxynaphthyl(8) did not affect the potencyto anygreat extent, although compound8 was slightly morepotent than 4.(b) Acyl Substituents (Rz). Modifications madeincluded replacing the methyl moiety with groups withdifferent lipophilic and electronic properties such asphenyl, 9, substituted phenyl, 10 and 11, and hetero-cycles, 12 and 13. Replacementof the methyl with amore lipophilic group such as phenyl reduced thepotency almost10times while the 3,4-dimethoxyphenylanalog, 10, mproved the potency. Though the lipophi-licity of 11 is the same as 10, the presence of the 2,6-dimethoxy substitution may affect the conformation ofthe hydroxamic acid functionality, which in turn re-duced the potency. The best replacement for the methylgroup within this series isthe furan analog, 12,whichis more polar than either benzene, 9, or thiophene, 13.In general this portion of the active site appears totolerate polar substituents better, which is in agreementto someofthe reported hydroxamic acid inhibit01-s.~~epreviously reported substantial increases in in vitropotency by the introduction of various esters on the acylgroupofother hydroxamate 5-lipoxygenase nhibitors.20A similar approach within the two phenyl series re-sulted in very potent compounds, with the best com-pounds,15 and 16,possessing two and three methyleneunits between the carbonyls of the ester and thehydroxamate groups. The ester functionality could beinvolved in a hydrophilic type interaction with thecarboxylate binding area. Maintaining the methylbutyrate functionality on the acyl unit and replacing the2-position phenyl group with 3,4,5-trimethoxyphenyl(171,n-butyl (181, and n-decyl (19)did not affect thepotency of the resulting compounds. However, com-pound19was the least active among the three, probablydue to the excessive length of the molecule. Thissupports the importance of the distance between thehydrophobic and ionic binding sites.Though several of the initial modificationsof the leadcompound gave very potent compounds, in vitro, mostof the analogs had low oral activity, probably due totheir higher overall CLogP values. In an attempt toreduce the lipophilicity and improve the oral activity,derivatives20-23 containing small polar groups on the

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NotesTable 2. In Vitro and inVivo5-Lipoxygenase Inhibitory Activitiesof Compounds 4-23 Compared to Zileuton

Journal of Medicinal Chemistry, 1994,Vol. 37, No. 21 3665

i n vivo 5-LO (LTC4) nhibnb% inhibn at 30 mg/kgPOnvitro 5-LO (5-HETE) nhibn."no. Ri Rz ICs0@M) (95% CL) f SE [ED60 (mgkgPO) (95%CL )4 phenyl CH3 0.49 (0.37-0.65) - [22.1 (15.2-42.O)l5 C4H9 CH3 0.05 (0.02-0.09) 41.4f 10.66 CioHzi CH3 0.41 (0.15-0.77) 19.5f 12.07 3,4,54rimethoxyphenyl CH3 0.31 (0.18-0.35) 42.1f 8.608 6-methoxy-2-naphthyl CH3 0.12 (0.08-0.16) 25.6 f 7.409 phenyl phenyl 4.83 (3.07-7.40) 46.2 f 4.9010 phenyl 3,4-dimethoxyphenyl 0.20 (0.08-0.42) NS11 phenyl 2,6-dimethoxyphenyl 3.00 (2.06-5.55) NS12 phenyl furanyl 0.04 (0.01-0.06) 32.9f 10.213 phenyl thiophenyl 0.25 (0.19-0.35) 29.1f 12.414 phenyl CHzOAc 0.16 (0.08-0.27) 18.9f 13.015 phenyl CHzCHzCOzEt 0.05 (0.01-0.09) NS16 phenyl (CHzhCOzMe 0.06 (0.02-0.11) NS17 3,4,54rimethoxyphenyl (CHz)3COzMe 0.04 (0.02-0.06) 42.1f 8.6018 C4Hg (CHz)3COzMe 0.05 (0.04-0.07) 28.6 f 12.419 CioHzi (CHd3COzMe 0.15 (0.09-0.24) 37.3f 7.6020 phenyl NHz 0.41 (0.30-0.61) - r10.3 (5.30-15.0)]21 3,4,5-trimethoxyphenyl NH2 0.05 (0.02-0.11) - L17.5 (9.10-40.2)]

22 phenyl CHzOMe 0.16 (0.03-0.33) - [18.61(9.80-27.6)123 phenyl CHzOH 0.70 (0.61-0.79) - 27.5 (19.2-59.8)lZileuton 0.37 (0.24-0.54) - L2.71 (1.39-6.93)]a IC50with 95% confidence limitsz7 n parentheses for the invitro inhibitionof 5-lipoxygenase (5-HETE) rom 9OOOgsupernatantofRBL broken cell assay (see Methods). ED60with 95% confidence limitsz7 n parentheses or mean percent inhibition values+SEM forinhibitionof 5-lipoxygenase (LTC4) in the mouse zymosan peritonitis assay (see methods). NS=no significant activity at 30 mgkg.

acyl unit were prepared. One substituent which hasbeen an effective replacement for the acyl unit ininhibiting the enzyme is urea.26 This was foundto beeffective within this series also. Conversion of com-pounds4 and7 to the urea analogs20and21 improvedthe in vivo potencies for both compounds and alsoimproved the in vitro potency for 4 while maintainingthatof7. Compound20was foundtobe the most orallypotent inhibitor within the series. An important findingwithin this series is that the methoxymethylene and thehydroxylmethylene groups (22 and 23) also serve asefficient bioisosteric replacements for the acyl methylgroupof the acetyl hydroxamic acids, while maintainingboth the i n vitro and in vivo potencies.In summary, a novel series of 2-substituted benzo-furan hydroxamic acids were shownto be potent inhibi-tors of the enzyme, 5-lipoxygenase. The more rigidbenzofurans were equipotentto the simple (benzyloxy)-phenyl derivatives,1- 3. In addition, we have furtherdemonstrated the limiting but important hydrophobicbinding region of the enzyme. In the region adjacentto the hydroxamate binding site, a hydrophilic area wasfound which can be used to alter the physicochemicalparameters and thus the pharmacodynamics of theinhibitor molecules.Experimental Section

Melting points were determined onaMeltemp I1 apparatusand are uncorrected. Elemental analyses (within 0.4% of thetheoretical values unless otherwise indicated) and the massspectral data (chemical ionization technique) were performedby the analytical groupat theR. W. Johnson PharmaceuticalResearch Institute. All IH NMR spectra were recorded on aGE-300 spectrometer, and values are reported in ppm fromMersi.General Procedure for the Preparation of 1-(2-Sub-stituted5-benzofurany1) ethanones26a-e. The following

procedures for the preparation of 1-(2-pheny1-5-benzo-furany1)ethanone 26a)are representative. MethodA. Toastirred suspensionof anhydrous sodium acetate(6.65g,81.1"01) in DMF (75 mL) was added phenylacetylene (6.63 g,64.9 mmol),4-hydr oxy- 3- i odoacet ophenone8.50g, 32.4 mmol),bi s(t ri phenylphosphi ne)pal l adi umI I ) acetate (487 mg, 0.65mmol), and copper(1) iodide (246 mg, 1.30 mmol). The mixturewas then heatedat65-70 "C for 4 h under nitrogen and cooledto 25 "C, and HzO (600mL) was added. The resulting tansolid was filtered, air-dried, packed on silica gel column, andeluted with EtOAcihexane(15) o give 6.68 g (87%) of a tansolid, mp 156-159 (lit.16 mp 160 "C).MethodB. To a suspensionofcuprous oxide (179 mg, 1.25mmol) in pyridine(8mL) was added phenylacetylene (213 mg,2.09 mmol) and 4- hydr oxy- 34odoacet ophenone524 mg, 2.00mmol), and the mixture was refluxed for 5 h under nitrogen.The mixture was then filtered, HzO (50mL) was added, andthe resulting solid was filtered, air-dried, packed on silica gelcolumn, and purified as before to give 0.37 g (78%) of theproduct.General Procedure for the Preparation of 1-(2-Sub-stituted 5-benzofurany1)ethanoneOximes 27a-e. Thefollowing procedure for the preparation of 1-(2-phenyl-5-benzofurany1)ethanoneoxime (27a)s representative. Toa mixture of 26a (3.93 g, 16.6 mmol) and hydroxylaminehydrochloride (4.05 g,58.3mmol) n EtOH (50 mL) was addedpowdered NaOH (5.90 g, 0.15 mol) in small portions. Afteraddition, the mixture was stirredat25 "C for 30 min, refluxedfor 10 min, cooled to 25 "C, and poured intoamixture of 12 NHCl(25 mL) and HzO (100 mL). The resulting precipitate wasfiltered and recrystallized from aqueous EtOH to give 3.00g(72%) of a tan solid: mp 207-209 "C;M S (CI, CHI) MH+at254; 'H NM R (DMSO-&) 6 2.23 (s,3H, CH3), 7.47 (m, 4H, 3H,and 3', 4, nd 5' phenyl Hs), 7.61 (m, 2H, 2 and 6' phenylHs), 7.92 (m, 3H, 4,6, and 7Hs)5.85, 11.15 s, lH, OH). Anal.(Ci6Hi3NiOz4.5HzO) C, H,N.General Procedure for the Preparation of N-Hydroxy-l-(a-substituted5-benzof urany1)et hanamnes. The fol-lowing procedure for the preparation of N-hydroxy-l-(2-phenyl - 5- benzof urany1) ethanamne%a)srepresentative.To a solution of 27a (1.76 g, 6.76 mmol), NaBH3CN (0.88g,

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3666 J ournal of Medicinal Chemistry, 1994, Vol.37, No. 2114.0 mmol) in MeOH (50mL), and THF (50 mL) containingmethyl orange(1mg) was added dropwise 12 N HCl until thecolor remained pink. The mixture was stirred continuouslyfor 4 h with the occasional addition of 12 N HC1to maintainthe pink color of the reaction. The reaction mixture was thenevaporated to dryness under reduced pressure, the solid wassuspended in HzO (50 mL), and enough sodium hydroxide wasadded to adjust the pH to 9 and extracted with CHzClz (3x250 mL). The combined organic extracts were dried withmagnesium sulfate and concentratedto dryness to give anoff-white solid. The solid material was packed on a silica gelcolumn and eluted with EtOAdhexane (3:l) t o give 1.70 g(99%)of awhite product: mp 164-166 "C; MS (CI, CHI) MH+at 253; lH NMR (DMSO-de)6 1.3 (d, 3H, CH3), 4.16 (q, lH,CH), 7.18 (s, lH, 3H),5.85 (br s, lH, OH), 7.28-7.5 (m, 6H,phenyl Hs and 7H), 7.9 (d, 2H, 4 and 7 Hs)(s, lH , NH). Anal.(CisHi5Ni02)C, H, N.General Procedure for the Preparation of N-hydroxy-N-[l-(2-substituted- benzof urany1)ethyl l acetatni des4-19and 22. The following procedure for the preparation ofN-hydroxy-N-[1- ( 2- pheny1- 5- benzof ur anyl ) ethyl ] acet-amide (4) s representative. To a solution of 28a(0.50g, 1.97mmol) and triethylamine (1mL) in dry THF (50 mL) wasadded acetyl chloride (0.16 g, 1.97 mmol). The reactionmixture was stirred at 25 "C for 20 min, concentrated todryness, and recrystallized from H20, THF, and a few dropsof AcOH to give 0.39 g (66%) of an off-white solid: mp 181-183 "C; MS (CI, CH4) MH' at296; lH NMR (DMSO-dd6 1.517.61(m,7H, phenyl Hs, 3 and 4 Hs), 7.95 (d, 2H, 6 and 7 Hs),9.58 (br s, lH, OH). Anal. (C18H17N103) C, H, N.General Procedure for the Preparation of N-Hydroxy-N-[l-(2-substituted - benzofurany1) ethyl l ureas20 and21. The following procedure for the preparation of N-hy-dr oxy-N- [ l - ( 2- phenyl - 5- benzof ranyl ) et hyl l ur ea20) srepresentative. Dry HC1 gas was bubbled through asolutionof 28a (0.20 g, 0.79 mmol) in THF (35 mL) for 5 min. Thesolution was added dropwise to a stirred 20% phosgene intoluene solution (4.09 mL, 7.90 mmol), stirred at 25 "C for 4h, and poured into cold aqueous 38% NH40H solution (50 mL).The resulting solution was extracted with EtOAc (3x 125 mL).The combined organic extracts were washed with H2O (100mL), dried with magnesium sulfate, and concentrated todryness. The solid obtained was packed on a silica gel columnand eluted with EtOAdhexane (1:4) to give 0.14 g (62%)of awhite product: mp 164-166 "C; MS (CI, CH4) MH+at 297;(s, 2H, NHz), 7.3-7.6 (m, 7H, phenyl Hs, 3 and 4 Hs), 7.92 (d,2H,6and 7 Hs), 9.07(s, lH, OH). Anal. (C I ~H I ~N~O~),, H,N.N,P-Dihydroxy-N-[- ( 2- phenyl - 5- benzof ranyl ) et hyl l -acetamide (23). To a solution of 14(0.43 g, 1.21 mmol) inwarm 2-propanol (50mL) and THF (10 mL) was added H2O(2 mL), and the mixture was cooledto 25"C. Solid LiOH (0.46g, 19.0 mmol) was added and the mixture stirredat25 "C for2 h. Enough 2 N HC1 was added to bring the pH to about 2,followed by H2O (50 mL). The resulting precipitate wasfiltered, and the aqueous layer was extracted with EtOAc (2x 50 mL), washed with H2O (2 x 75 mL), and dried withmagnesium sulfate. The solvent was evaporated, and theresulting solid was added to the filtered solid, packed on asilica gel column, and eluted with EtOAdhexane (2:3) to give0.18 g (47%)of an off-white product: mp 187-190 "C;MS (CI,CH4) MH+at 312; 'H NMR (DMs0-d~) 1.5 (d, 3H, CH3), 4.13(m, 2H, CHd, 4.5 (q, lH, CHI, 5.70 (q, lH, CHI, 7.28-7.62 (m,7H, phenyl Hs, 3 and 4Hs),7.90 (d, 2H, 6 and 7 Hs), 9.50(s,

RBL -15-L ipoxygenase nhibition. RBL l cells from theAmerican Type Culture Collection (ATCC) were grown insuspension cultures and harvested by centrifugation at 2000gfor 5 min. Washed cells at a concentration of 5 x lo7 cells/mL were suspended in NaHPOJCaClz buffer, homogenizedat0"C, and then centrifuged at9OOOgfor 50min. The 5-lipoxy-genase activity in the 9OOOg supernatant was determinedradiometrically by measuring the conversion of arachidonicacid to 5-HETE. Increasing logarithmic doses of test com-

(d, 3H, CH3), 2.02 (s, 3H, CH&(O)), 5.88 (9, lH, CHI, 7.2-

'H NMR (DMSO-ds)6 1.47 (d, 3H, CH3), 5.4 (9, lH, CH), 6.28

lH, OH). Anal. (C18H17N104G.lHzO) C, H, N.

Notespound were utilized in order to determine a dose-responsecurve for each drug. Doses were chosen such that the IC50concentration of the drug fell within the linear portion of thesigmoidal dose response curve. A mixture of 5.5pL of testcompound and 500 pL of enzyme supernatant was pre-incubated for 5min at 37 "C. Then, 10 pL of 50pCi/mL [I4C]-arachidonic acid was added to each sample followed by a20min incubation period at 37 "C. The reaction was stopped bythe addition of 1.0 mL of 2 N formic acid per sample. Theprimary 5-LO product, 5-HETE, was isolated by chloroformextraction, followed by TLC on silica gel, and detection ofradioactive emissions of product via a Bioscan imaging systemplate scanner. The inhibition of 5-LO product formation isexpressed as apercentage of the arachidonic acid convertedto 5-HETE by the control group vs the drug treatment group.IC50 values with 95% confidence limits (CL) were determinedby the method of F i nne~. ~~

Mouse Zymosan Peritonitis Model. Male mice (CD-11,18-25 g, were dosed orally with test compound suspended inpolyethylene glycol 200. One hour later, the animals wereinjected (ip) with 3 mgof zymosan-A suspended in 0.5mL of0.9% sterile saline. Fifteen minutes after receiving zymosan,the mice were sacrificed by COz inhalation. The abdomenswere injected with 2 mL of a 10 pM indomethacin solution.Subsequentto massaging the abdominal area, the skin wasremoved and the abdominal wall was opened. A 0.2 mLaliquot of peritoneal fluid was withdrawn and added to1mLof cold 95% ethanol. The solutions were incubated in an icebath (minimum of 30 min) and then centrifuged at 28000gfor15min at 4 "C. Supernatant fractions were decanted andevaporated under astream of nitrogen at room temperature.The samples were capped and stored at -70 "C until assayed.Radioimmunoassays (RIAs) for LTC4 were performed ona1:20dilution oforiginal samples using [3H]RIAkits from AdvancedMagnetics, Inc., according to kit instructions. ED50 values(that dose calculated to causea508 reduction in the immu-noreactive LTC4 with 95% confidence limits of (CL)) werecalculated from the percentage of inhibition determined foreach animal at the doses tested and then fitted to a straightline by a log-linear regression analysis according to themethod of F i nne~.~~

ReferencesMusser, J . H.; K reft, A. F. 5-L ipoxygenase: Properties, Phar-macology and the Quino1inyKbridged)aryl Class of Inhibitors.J .M ed. Chem. 1992, 35, 2501- 2524.Samuelsson, B. Leukotrienes: Mediators of Immediate Hyper-sensitivity Reactions and Inflammation. Science 1983, 220,568-575.Lewis, R. A.; Austen, K . F.M ediationofLocal Homeostasis andInflammation by Leukotrienes and Other M ast Cell-DependentCompounds. Nature 1981, 293, 03- 108.Ford-Hutchinson, A. W.; Bray, M. A.; Doig, M . V.; Shipley, M.E.; Smith, M. J .H. L eukotriene B, a Potent Chemokinetic andAggregating Substance Released from PolymorphonuclearLeukocytes. Nature 1980, 286, 64-265.Palmer, R. M. J .;Stepney, R. J .;Higgs, G. A.; Eakins, K. E.Chemokinetic Activity of Arachidonic Acid Lipoxygenase Prod-ucts on Leukocytes of Dif ferent Species. Prostaglandins 1980,20, 411-418.Bray, M. A.; C unnigham, F. M.; Ford-Hutchinson, A. W.; Smith,M. J .H. LeukotrieneB4: A M ediator of Vascular Permeabili ty.Br. J . Pharmacol. 1981, 72, 483-486.Wedmore, C. V.; Williams, T. J .Control ofVascular Permeabili tyby Polymorphonuclear Leukocytes in I nflammation. Nature1981, 289,646-650.Bray, M. A.; F ord-Hutchinson, A. W.; Smith, M. J .H. Leuko-triene B4: An Inflammatory M ediator I n V ivo. P rostaglandins1981, 22, 213-222.Hanna, C. J . ; Bach, M. I C; Pare,P.D.; Schellenberg, R. R. Slow-reactingSubstances( 1eukot r i enes)Contract Human A irway andPulmonary Vascular Smooth MuscleI n Vitro. Nature 1981,290,343-344.Marom,Z. ; helhamer, J .H.; B ach,M. I C;Morton, D.R.; K aliner,M. Slow-reacting Substances, L eukotrienes Cq and D4, Increasethe Release of Mucus from Human Airways in vitro. A m. Rev.Respir. D s. 1982, 126, 449-451.Ford-Hutchinson,A. W. Leukotrienes: Their Formation and Roleas Inflammatory Mediators. F ed. Proc. 1985,44, 25-29.

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Notes J ournal of M edicinal Chemistry, 1994, Vol.37,No. 21 3667(12) Weinblatt, M.; Kremer, J .;Helfgott, S.; Coblyn, J .;M aier, A.;Sperling, R.; Petrill o, G.; Kesterson, J .;Dube, L.; Henson, B.;Teoh, N.; Rubin, P.A 5-Lipoxygenase Inhibitor in RheumatoidA rthritis (RA). Arthritis Rheum. 1990,33,D111.(13) Stenson, W. F.; L auritsen, I C; L aursen, L. S.; Rasak-Madsen,J .; acobsen,0.;Naesdal, J .;Cort, D.; Goebell, H.; Pesker, B.;Hanauer, S.;Swanson, L .; Dube, L.; Rubin, P. A Cl inical Trialof Zileuton, A Specific nhibitor of 5-L ipoxygenase, in UlcerativeColitis. Gastroenterology 1991, 100, A253.(14) Summers, J . B.; Gunn, B. P.; Martin, G. J .;Mazdiyasni, H .;Stewart,A. 0.;Y oung, P.R.; Goetze, A. M.; Bouska, J .B.; Dyer,R. D.; Brooks, D. W.; C arter, G. W. Orally Active HydroxamicAcid Inhibitors Of Leukotriene Biosynthesis. J .M ed. Chem.

1988,31, -5.(15) Summers, J . B.; Gunn, B. P.; M artin, J . G.; M artin, M. B.;Mazdivasni. H.: Stewart. A. 0. :Y oung, P. R.: Bouska, J . B.:Goetz,"A. M.'; Dyer, R. D.; Brooks,'D. W.;-darter,G.W. Str&ure-Activity Analysis of aCl ass of Orally Active Hydroxamic AcidInhibi tors of L eukotriene Biosynthesis. J .M ed. Chem. 1988,31, 1960-1964.(16) Bisagni, E.; Royer, R. Recherches sur le Benzofuranne. V.Structure des Dicetone Obtenues par Acylation des Ethyl-2-Acyl-3 Benzofurannes. (Study of benzofuran. V. Structuke ofthe diketones obtained from the A cylation of 2-ethyl-3-acy-benzofurans.) Bull. SOC. him. 1960, 1968-1976.(17) A rcadi, A.; Marinelli, F.;Cacchi, S.Palladium-Catalyzed Reac-tion of 2-Hydroxyaryl and Hydroxyheteroaryl Halides with1-Alkynes: An Improved Route to the Benzoblfuran RingSystem. Synthesis 1986, 749-751.(18) Doad. G. J . S.: BarltroD. J . A.: Pettv. C. M.: Owen. T. C. AV ersatile and Convenie'nt Synthesi sof Benzofurans. Tetra-hedron L ett. 1989, 1597-1598.

(19) Borch, R. F.; Bernstein, M . D.; Durst, H. D. The Cyanohydri-doborate Anion as a Selective Reducing Agent. J .Am . Chem.SOC. 971,93, 2897-2904.(20) Ohemeng, K. A.; Nguyen, V. N.; Schwender, C. F.;Singer, M.;Steber, M.; Ansell, J .;and Hageman, W. Novel Bishydroxami-cAcidsa55-L ipoxygenase Inhibi tors. Bioorg. M ed. Chem.1994,(21) Carter, G. W.; Young, P. R.; Albert, D. H.; B ouska, J .;Dyer, R.;Bell, R. L.; Summers, J . B.; Brooks, D. W. 5-LipoxygenaseInhibitory A ctivity of Zileuton. J .Pharmac. Exp. Ther. 1991,256,929-937.(22) Cashman, J . R. Leukotriene Biosynthesis Inhibitors. Pharm.Res. 1986,2, 253-261.(23) Summers, J . B.; K im, K. H.; M azdiyasni, H.; Holms, J . H.;Ratajczyk, J . D.; Stewart, A. 0.;Dyer, R. D.; Carter, G. W.Hydroxamic Acid Inhibitors of 5-L ipoxygenase: QuantitativeStructure - Activity Relationships. J .M ed. Chem. 1990,33,(24) For example: Hammond, M. L .; Kopka, I. E.; Zambias, R. A.;Caldwell, C. G.; Boger, J .; Baker, F.; Bach, T.; Luell, S.;MacIntyre, D. E. 2, 3- D hydro- 5- benzofuranol ssAntioxidant-Based I nhibitors of Leukotriene Biosynthesis. J .M ed. Chem.1989,32, 1006-1020.(25) McMillan, R. M.; Walker, E. R. H. Designing TherapeuticallyEffective 5-L ipoxygenase Inhibitors. Trends Pharmacol. Sci.1992,13,323-330.(26) Garland, L. G.; Salmon, J . A. Hydroxamic Acids and Hydroxy-ureas as Inhibitors of A rachidonate 5-L ipoxygenase. Dr ugsF uture 1991, 16, 547.(27) Finney, D. J . n Statistical Method in Biological Assay, 3rd ed.;Charles Griffin and Co. Ltd: London, 1978; pp 39-67.

2, 187-193.

992-998.