Dibenzoxepinone hydroxylamines and hydroxamic acids: Dual inhibitors of cyclooxygenase and 5-lipoxygenase with potent topical antiinflammatory activity

Article abstract of DOI:10.1021/jm950563z

Hydroxylamine and hydroxamic acid derivatives of a known nonsteroidal antiinflammatory dibenzoxepine series display both cyclooxygenase (CO) and 5- lipoxygenase (5-LO) inhibitory properties. Many of these new dual CO/5-LO inhibitors also exhibit potent topical antiinflammatory activity in the arachidonic acid-induced murine ear edema model. On the basis of their promising profile of in vitro and in vivo activities, hydroxamic acids 24h, 3-(6,11-dihydro-11-oxodibenz[b,e]oxepin-2-yl)-N-hydroxy-N-methylpropanamide (HP 977), and 25, 3-(6,11-dihydrodibenz[b,e]oxepin-2-yl)-N-hydroxy-N- methylpropanamide (P10294), were selected as developmental candidates for the topical treatment of inflammatory skin disorders.

Full text of DOI:10.1021/jm950563z

246
J . Med. Chem. 1996, 39, 246-252
Diben zoxep in on e Hyd r oxyla m in es a n d Hyd r oxa m ic Acid s: Du a l In h ibitor s of
Cyclooxygen a se a n d 5-Lip oxygen a se w ith P oten t Top ica l An tiin fla m m a tor y
Activity
R. Richard L. Hamer, J ohn J . Tegeler,* Ellen S. Kurtz, Richard C. Allen, Steven C. Bailey, Mary Ellen Elliott,
Luther Hellyer, Grover C. Helsley, Penelope Przekop, Brian S. Freed, J ohn White, and Lawrence L. Martin
Hoechst-Roussel Pharmaceuticals Inc., Somerville, New J ersey 08876
Received J uly 28, 1995X
Hydroxylamine and hydroxamic acid derivatives of a known nonsteroidal antiinflammatory
dibenzoxepine series display both cyclooxygenase (CO) and 5-lipoxygenase (5-LO) inhibitory
properties. Many of these new dual CO/5-LO inhibitors also exhibit potent topical antiinflam-
matory activity in the arachidonic acid-induced murine ear edema model. On the basis of their
promising profile of in vitro and in vivo activities, hydroxamic acids 24h , 3-(6,11-dihydro-11-
oxodibenz[b,e]oxepin-2-yl)-N-hydroxy-N-methylpropanamide (HP 977), and 25, 3-(6,11-dihy-
drodibenz[b,e]oxepin-2-yl)-N-hydroxy-N-methylpropanamide (P10294), were selected as devel-
opmental candidates for the topical treatment of inflammatory skin disorders.
In tr od u ction
Eicosanoids have been implicated as mediators in
numerous inflammatory processes, and the role of these
20-carbon unsaturated fatty acids in the pathogenesis
of inflammatory skin disorders has received consider-
able attention in recent dermatological research. Cu-
taneous inflammatory disorders, such as atopic derma-
titis, eczema, and psoriasis,1 display elevated epidermal
levels of eicosanoids.2 Elevations in both prostaglan-
dins3 (PGE2/PGF2R) and leukotrienes4,5 (LTB4) have
been reported in psoriatic skin. Topical corticosteroids
F igu r e 1. Reference antiinflammatory agents.
impart potent antiinflammatory effects in a variety of
skin disorders, albeit with significant side effect liability
especially on chronic administration. Topical steroids
Herein are described the synthesis and SAR of this
new series of N-hydroxy-(6,11-dihydro-11-oxodibenz[b,e]-
oxepin-2-yl)alkanamines and -alkanamides and related
have been shown to attenuate arachidonate and LTB4
reverse amide derivatives as topically effective dual CO/
release6 and inhibit phospholipase A2.7 Classical non-
5-LO inhibitors. Compounds 24h and 25 were selected
for toxicological assessment.
steroidal antiinflammatory (NSAID) agents, such as
ibuprofen, have been found to impart physiological
action primarily via inhibition of the cyclooxygenase
(CO) pathway. The potent CO inhibitor indomethacin,
however, was found to exacerbate rather than amelio-
rate psoriasis, implying the importance of the lipoxy-
genase pathways.8 With this in mind, we chose to
explore the concurrent blockade of the two major
metabolic pathways of the cascade, i.e., dual inhibition
of the CO and 5-lipoxygenase (5-LO) enzymes, as a
potentially viable approach to antipsoriatic therapy.
Corey’s early discovery that arachidonate hydroxamic
acid inhibits 5-LO in vitro9 sparked a flurry of medici-
nal research toward specific 5-LO inhibitors with anti-
inflammatory activity.10 We and others11 saw the
potential of generating dual CO/5-LO inhibitors by
incorporating a hydroxamate moiety into a known CO
inhibitory NSAID nucleus such as isoxepac12 (Figure 1).
In addition, since aralkylhydroxylamines such as QA-
208,199 have been reported to be potent lipoxygenase
inhibitors,13 we also replaced the carboxylate with
substituted hydroxylamines to further explore struc-
ture-activity relationships (SAR). Other published
reports14 led us to explore several reverse hydroxamate
derivatives.
Ch em istr y
The synthetic sequence utilized in the preparation of
the required dibenzoxepine intermediates is outlined in
Scheme 1. The construction of the dibenzoxepine
nucleus utilized methodology developed for the related
thieno[3,2-c][1]benzoxepinone alkanols.15 R-(Bromo-
methyl)benzoates 1a ,b were condensed with (4-hydrox-
yphenyl)propanol (2), 4-salicylaldehyde (3), or ethyl (4-
hydroxyphenyl)alkanoates 4 with K2CO3 in 2-butanone
to afford ethyl 2-[(aryloxy)methyl]benzoates which were
directly saponified to the corresponding acids 5 and 6
and diacid 7. Cyclodehydration of 5, 6, or 7 with
trifluoroacetic anhydride provided dibenz[b,e]oxepin-2-
alkanol 8a , 2-carboxaldehyde 9, and 2-alkanoic acids
10a -d . Reduction of 2-acetic acid 10a with borane-
THF provided a separable mixture of 2-ethanol 8b and
desoxo-2-ethanol 11. Zinc in refluxing acetic acid
selectively reduced dibenzoxepinonepropanoic acid 10c
to the dibenzoxepinepropanoic acid 12.
The N-hydroxydibenz[b,e]oxepin-2-alkanamines were
prepared as shown in Scheme 2. Conversion of alkanols
8a ,b and 11 to their methanesulfonates 13a ,b and 14
followed by nucleophilic displacement with secondary
hydroxylamines yielded the desired aralkylhydroxyl-
amines 15a -d and the desoxo analog 17, respectively.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0246$12.00/0 © 1996 American Chemical Society

Topically Active Dual Inhibitors of CO and 5-LO
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 247
Sch em e 1. Dibenzoxepin Intermediatesa
compound 20. Boc-deprotection and acetylation pro-
vided N-(benzyloxy)amine 21 and N-(benzyloxy)aceta-
mide 22 successively. Ammonium formate hydrogenol-
ysis of 22 resulted in (arylethyl)-N-hydroxyacetamide
23b.
Physiochemical data for (hydroxylamino)alkyl com-
pounds 15a -d , 16, and 17 and for the reversed N-
hydroxyamides 23a ,b are summarized in Table 1.
Treatment of the acid chlorides of compounds 10a -d
and 12 with various hydroxylamines yielded hydroxamic
acids 24a -m and 25, respectively. Physiochemical data
for hydroxamic acids 24a -m and 25 are summarized
in Table 2.
Sch em e 3. Hydroxamic Acids
a
(a) K2CO3, 2-butanone; (b) KOH, aqueous EtOH; (c) trifluo-
Biologica l Resu lts a n d Discu ssion
roacetic anhydride, CH2Cl2; (d) borane-THF; (e) Zn, HOAc.
Biological assay results for (hydroxylamino)alkyl
compounds 15a -d , 16, and 17 and for the reversed
N-hydroxyamides 23a ,b are summarized in Table 3.
Table 4 contains similar data for hydroxamic acids
24a -m and 25. The 5-LO inhibitory activity of these
compounds was assessed by measurement of 5-HETE
levels utilizing the method of Cochran and Finch-
Arietta17 with RBL-1 cell-free supernatant prepared
according to the method of J akschik and Kuo.18 The
CO inhibitory potency was evaluated by attenuation of
PGE2 levels in cultured 3T3 fibroblasts by the method
of Lindgren.19 Topical antiinflammatory activity was
determined via the arachidonic acid-induced mouse ear
edema model of Young20 using a 3:7 propylene glycol:
ethanol vehicle. This vehicle was utilized due to its
reduced volatility, improved reproducibility, and rel-
evancy to known topical vehicles versus the reported
vehicle acetone.
Sch em e 2. Hydroxylamine Derivativesa
The (hydroxylamino)alkyl compounds 15a -d , 16, and
17 and the reversed N-hydroxyamides 23a ,b in Table
3 all displayed potent in vitro 5-LO and CO inhibition
and in vivo topical antiinflammatory activity. Primary
differences occurred in their relative CO inhibitory
potencies. Methyl- and ethylhydroxylamines 15a ,b
were nearly 1 order of magnitude more potent than the
corresponding isopropylhydroxylamine 15c. Reduction
of the diaryl ketone unit of 15b to the diarylmethanol
16 had no effect on in vitro potency; however, further
reduction to the diarylmethane 17 caused a 5-fold
reduction in CO inhibitory activity. Extending the
methylene linker in N-acetyl derivative 23a to the two-
carbon homolog 23b had no effect on in vitro activity
but produced a modest improvement in topical activity.
A more diverse SAR was observed with hydroxamic
acids 24a -m and 25 in Table 4. Among the aryl
acetamides 24a -e, N-alkylation appears important for
both 5-LO inhibition and topical antiinflammatory
activity. As seen in other studies, unsubstituted 24a
was almost 10-fold weaker against 5-LO21 and nearly
devoid of in vivo activity in the arachidonic acid ear
edema model. Alkyl analogs 24b-e all have micromo-
lar 5-LO potency. Increasing steric bulk in N-cyclohexyl
a
(a) CH3SO3Cl, Et3N; (b) R3NHOH-HCl, KOtBu; (c) NaBH4;
(d) NH2OH-HCl, pyridine; (e) borane-pyridine; (f) Boc-NHOBn,
NaH, DMF; (g) AcCl, Et3N; (h) HCl(g), EtOAc; (i) LiOH; (j) 5%
Pd/C, HCO2NH4.
Borohydride reduction of ethylhydroxylamine 15b pro-
vided the diarylmethanol analog 16.
The one- and two-carbon-linked reverse N-acetyl-N-
hydroxydibenz[b,e]oxepin-2-alkanamines 23a ,b were
prepared as illustrated in Scheme 2. Conversion of
aldehyde 9 to its oxime followed by borane-pyridine
reduction yielded hydroxylamine 18. N,O-Diacetylated
derivative 19 was subjected to selective hydrolysis with
LiOH14 to provide (arylmethyl)-N-hydroxyacetamide
23a .
An alternate route was used in the preparation of the
two-carbon-linked derivative due to difficulties in the
preparation of the corresponding acetaldehyde needed
for the above methodology. Methanesulfonate 13a was
condensed with N-Boc-O-benzylhydroxylamine16 to give

248 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Ta ble 1. Physicochemical Data of (Hydroxylamino)alkyl Analogsa
Hamer et al.
starting
material
recrystn
solventd
no.
n
X
R3
CH3
C2H5
CH(CH3)2
CH3
C2H5
C2H5
C(O)CH3
C(O)CH3
method
mpb (°C)
yieldc (%)
formula
anal.e
15a
15b
15c
15d
16
17
23a
23b
2
2
2
3
2
2
1
2
O
O
O
O
H, OH
H, H
O
8b
8b
8b
8a
15b
11
A
A
A
A
B
A
C
D
101-102
86-88f
19
42
32
52
65
23
52
71
A
C17H17NO3
C18H19NO3
C19H21NO3
C18H19NO3
C18H21NO3
C18H21NO2
C17H15NO4
C18H17NO4
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
122-124
113-114.5
116-118
102-103
129-130
113-115
B
B
C
C/D
E/F
E/F
19
22
O
a
b
All compounds exhibited IR, 1H NMR, and MS spectra consistent with the assigned structure. Melting points are uncorrected. c Yields
d
were calculated from the indicated starting material unless otherwise noted and are not optimized. A ) methanol; B ) acetonitrile; C
) ethyl ether; D ) cyclohexane; E ) ethyl acetate; F ) hexane; G ) ethanol; H ) acetone. e Analytical results are within (0.4% of
theoretical values unless otherwise noted. f Chromatographed, unrecrystallized.
Ta ble 2. Physicochemical Data of Hydroxamic Acid Analogsa
starting
material
yieldc,g
(%)
recrystn
solventd
no.
X
R
R6
R7
mpb (°C)
formula
anal.e
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
24k
24l
24m
25
O
O
O
O
O
O
O
O
O
O
O
O
O
CH2
CH2
CH2
CH2
CH2
CH(CH3)
CH(CH3)
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
H
CH3
H
H
H
H
H
H
H
H
H
H
H
CH3
H
10a
10a
10a
10a
10a
10b
10b
10c
10c
10c
10c
10c
10d
12
152-155
118-120
119-121
144-145
130-131
34
17
49
40
51
68
30
64
47
29
31
60
55
50
G
H
H
H
C16H13NO4
C17H15NO4
C18H17NO4
C19H19NO4
C22H23NO4
C18H17NO4
C19H19NO4
C18H17NO4
C19H19NO4
C20H21NO4
C23H25NO4
C19H19NO4
C20H21NO4
C18H19NO3
H,N,Ch
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
H,N,Ci
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C2H5
iC3H7
cC6H13
CH3
C2H5
CH3
C2H5
iC3H7
cC6H13
CH3
H
oil
oil
E
E/F
H/D
E/F
E
101-103
120-121
117-118
146-147
112-114
192-194
120-122
CH2C(CH3)2
(CH2)2
CH3
CH3
E/A
E
H, H
H
g
h
i
a-e See Table 1. Method E. Anal. (C16H13NO4) H, N; C: calcd, 67.84; found, 67.11. Anal. (C19H19NO4) H, N; C: calcd, 70.14; found,
69.64.
24e significantly decreased topical effectiveness relative
oxidant nordihydroguaiaretic acid (NDGA)21 and more
than 25-fold more potent than the topical dual 5-LO/
CO inhibitor BMY-30094.22 To address cell penetration
concerns, compound 24h was also evaluated in an
A-23187-stimulated HL-60 whole cell 5-LO assay and
found to possess similar potency to that seen in the cell-
free assay. Compounds 24h and 25 are mild CO
inhibitors being slightly weaker than ibuprofen and
BMY-30094 but 30-50-fold weaker than indomethacin.
Compounds 24h and 25 displayed potent topical anti-
inflammatory activity in the arachidonic acid ear edema
model relative to topical agents NDGA and BMY-30094.
Isoxepac was inactive in both the in vitro 5-LO and in
vivo ear edema assays. On the basis of this profile,
compounds 24h and 25 were selected for more in depth
biological evaluation (to be reported elsewhere) and
toxicological assessment.
to N-methyl-, -ethyl-, and -isopropylacetamides 24b-
d . R-Methylation (24f,g) also diminished in vivo po-
tency. Lengthening the acetamide chain of N-methyl
24b by a methylene unit to 24h produced a marked 120-
fold enhancement in 5-LO inhibitory potency. The
related N-alkylpropanamides 24i-k displayed a more
modest 2-4 fold improvement in 5-LO potency. O-
Methylation of the hydroxamide moiety (24l) negated
5-LO activity as has been previously observed.21 The
SAR trend for ear edema activity of 24i-k followed that
seen with the corresponding acetamides. R,R-Dimethy-
lation (24m ) caused a 10-fold reduction in 5-LO potency
versus 24h and surprisingly produced a proinflamma-
tory response in the arachidonic acid ear edema assay.
Finally, diarylmethane analog 25 retained the potent
antiinflammatory profile of 24h .
In vivo dose-ranging studies led to the selection of
N-methylhydroxamic acids 24h and 25 as the most
promising candidates for further study. The bioprofile
of these compounds relative to standards is highlighted
in Table 5. Compounds 24h and 25 are among the most
potent 5-LO inhibitors known. They are nearly 10-fold
more potent at inhibiting cell-free 5-LO than the anti-
Exp er im en ta l Section
Ch em istr y. Melting points were obtained on a Thomas
Hoover capillary melting point apparatus and are uncorrected.
1H NMR spectra were recorded on a Varian XL-200 spectrom-
eter using tetramethylsilane as an internal standard. IR

Topically Active Dual Inhibitors of CO and 5-LO
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 249
Ta ble 3. Biological Data of (Hydroxylamino)alkyl Analogs 15-17 and Reversed Hydroxyamides 23a ,ba
IC50 (µM)
c
no.
n
X
R3
CH3
C2H5
CH(CH3)2
CH3
C2H5
C2H5
C(O)CH3
C(O)CH3
5-HETEb
PGE2
AAEEd change (%)
15a
15b
15c
15d
16
17
23a
23b
2
2
2
3
2
2
1
2
O
O
O
O
0.24 (0.21-0.26)
0.48 (0.44-0.52)
0.13 (0.09-0.13)
0.62 (0.58-0.63)
0.45 (0.41-0.49)
0.32 (0.32-0.34)
0.21 (0.12-0.34)
0.19 (0.18-0.20)
0.08 (0.02-0.75)
0.19 (0.06-1.28)
(-47%)*
0.24 (0.21-0.28)
0.12 (0.01-1.95)
0.46 (0.14-1.05)
0.75 (0.15-10.82)
(-53%)**
-46**
-50**
-47**
-53**
-47**
-50**
-47**
-61**
H, OH
H, H
O
O
a
b
Where indicated, significance was determined by the Students T-test: * indicates p < 0.05; ** indicates p < 0.01. Inhibition of
5-HETE levels in RBL-1 cell-free supernatant. Number in parentheses represents 95% confidence range or percent change at 50 µM.
c Inhibition of PGE2 levels in murine 3T3 fibroblast culture. Number in parentheses represents 95% confidence range or percent change
d
at 1.0 µM. Percent change in arachidonic acid-induced mouse ear edema versus vehicle (70:30 ethanol:propylene glycol) control at 1.0
mg/ear.
Ta ble 4. Biological Data of Hydroxamic Acid Analogsa
IC50 (µM)
R6
R7
5-HETEb
PGE2
AAEEd change (%)
c
no.
X
R
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
24k
24l
24m
25
O
O
O
O
O
O
O
O
O
O
O
O
O
CH2
CH2
CH2
CH2
CH2
CH(CH3)
CH(CH3)
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
H
CH3
H
H
H
H
H
H
H
H
H
H
H
CH3
H
9.0 (8.35-9.22)
1.19 (1.14-1.25)
0.58 (0.48-0.60)
1.23 (1.12-1.36)
0.67 (0.60-0.74)
1.3 (1.26-1.53)
(-94%)**
0.01 (0.006-0.022)
0.15 (0.14-0.15)
0.58 (0.52-0.62)
2.0 (1.75-2.12)
>50
0.66 (0.52-0.85)
0.02 (0.019-0.029)
(-86%)**
>1.0
-19
-63**
-52**
-52**
-29
C2H5
iC3H7
cC6H13
CH3
C2H5
CH3
C2H5
iC3H7
cC6H13
CH3
(-47%)**
>1.0
>1.0
0.21 (0.06-0.40)
(-62%)**
0.53 (0.18-1.28)
(-73%)**
(-45%)**
>1.0
-44
-38*
-66**
-55**
-63**
-14
NT
NT
(-60%)**
-41**
+29
-67**
CH2C(CH3)2
(CH2)2
CH3
CH3
H, H
H
a-d
See Table 3.
Ta ble 5. Comparative Biological Profiles of 24h and 25 vs Standardsa
IC50 (µM)
5-HETEb
PGE2
AAEEd ED50 (mg/ear)
c
compd
24h
25
0.01 (0.006-0.022)e
0.02 (0.019-0.029)
0.12 (0.09-0.16)
0.48 (0.42-0.55)
0.53 (0.18-1.28)
(-60%)**
0.69 (0.60-0.80)
0.70 (0.44-0.93)
3.10 (2.52-4.11)
2.56 (2.12-3.53)
1.63 (1.22-2.38)
(-11%)
NDGAf
BMY-30094
indomethacin
ibuprofen
isoxepac
0.11 (0.02-0.48)
0.02 (0.008-0.05)
0.22 (0.21-0.24)
(-1.5%)
(+55%)
d
a-c See Table 3. Effective dose to produce a 50% reduction in arachidonic acid-induced mouse ear edema versus vehicle (70:30 ethanol:
propylene glycol) control. Number in parentheses represents 95% confidence range or percent change at 1.0 mg/ear. e IC50 (HL-60 whole
cell 5-LO): 0.014 µM (0.0128-0.0161). Inhibition of 5-HETE levels in A-23187-stimulated HL-60 cell culture. Number in parentheses
represents 95% confidence range. f NDGA ) nordihydroguaiaretic acid.
spectra were recorded on a Perkin Elmer 841 or 1420 spec-
trometer. Mass spectra (MS) were generated with a Finnigan
4500 GC-MS instrument equipped with an INCOS data
system. Elemental analysis (C,H,N) results are within (0.4%
of theoretical values unless indicated otherwise. Microanaly-
ses were performed by Oneida Research Services, Inc., Whites-
boro, NY. Reactions were routinely performed under N2
atmosphere unless otherwise indicated.
g, 0.38 mol) in 2-butanone (500 mL) was added dropwise to a
stirred suspension of 3-(4-hydroxyphenyl)propanol (2; 57.9 g,
0.38 mol), K2CO3 (157 g, 1.14 mol), and a catalytic amount of
KI in 2-butanone (500 mL). The resulting mixture was
warmed to reflux and maintained there for 24 h. After the
mixture had cooled to room temperature, filtration and
concentration gave an oil. A solution of the crude product in
CH2Cl2 (500 mL) was washed with 10% aqueous NaOH (1500
mL) and water, dried (Na2SO4), and concentrated. The residue
was chromatographed via HPLC (Waters Associates Prep LC/
2-[[4-(3-Hyd r oxyp r op yl)p h en oxy]m eth yl]ben zoic Acid
(5). A solution of methyl 2-(bromomethyl)benzoate (1b; 87.6

250 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Hamer et al.
System 500, silica gel, 60% hexane/EtOAc) to give 68.7 g (60%)
of methyl 2-[[4-(3-hydroxypropyl)phenoxy]methyl]benzoate as
a solid: mp 50.5-52 °C; 1H NMR (CDCl3) δ 1.70 (s, 1H), 1.87
(m, 2H), 2.65 (t, 2H, J ) 6.6 Hz), 3.66 (t, 2H, J ) 8.0 Hz), 3.9
(s,3H), 5.47 (s, 2H), 6.91 (d, 2H, J ) 8.8 Hz), 7.10 (d, 2H, J )
8.6 Hz), 7.40 (t, 1H, J ) 6.4 Hz), 7.58 (t, 1H, J ) 7.4 Hz), 7.75
(d, 1H, J ) 7.4 Hz), 8.0 (d, 1H, J ) 7.8 Hz); EIMS 300 (M•+).
Anal. (C18H20O4) C, H.
lamine hydrochloride (3.95 g, 0.04 mol) was added to a solution
of potassium tert-butoxide (4.48 g, 0.04 mol) in absolute EtOH
(500 mL). 2-(6,11-Dihydro-11-oxodibenz[b,e]oxepin-2-yl)etha-
nol methanesulfonate23 (13b; 3.32 g, 0.01 mol) was added to
the resulting suspension, and the slurry was warmed to reflux
and maintained there for 4.5 h. The cooled mixture was
diluted with water (500 mL) and concentrated. The resulting
aqueous suspension was diluted with CH2Cl2 (500 mL) and
washed with water. The organic extract was dried (Na2SO4)
and concentrated. The residue was chromatographed via
HPLC (Waters Associates Prep LC/System 500, silica gel, 20%
hexane/EtOAc) to give 1.24 g (42%) of 15b as a solid: mp 86-
88 °C; 1H NMR (CDCl3) δ 1.16 (t, 3H, J ) 7.0 Hz), 2.77 (q, 2H,
J ) 7.0 Hz), 2.93 (s, 4H), 5.16 (s, 2H), 6.60 (br s, 1H), 6.98 (d,
1H, J ) 8.4 Hz), 7.35-7.55 (m, 4H), 7.9 (d, 1H, J ) 7.4 Hz),
8.1 (s, 1H); EIMS 297 (M•+). Anal. (C18H19NO3) C, H, N.
The properties of 15a ,c,d and 17, prepared in a similar man-
ner from 8a ,b26 and 11, respectively, are included in Table 1.
2-(6,11-Dih yd r o-11-h yd r oxyd iben z[b,e]oxep in -2-yl)-N-
eth yl-N-h yd r oxyeth yla m in e (16). Meth od B. A solution
of 15b (4.1 g, 13.7 mmol) in MeOH (100 mL) at 0 °C was
treated with sodium borohydride (1.04 g, 27.5 mmol) in two
portions and stirred for 2 h, allowing the mixture to warm to
room temperature. The mixture was concentrated, diluted
with CHCl3, and washed with saturated NaHCO3. Concentra-
tion of the organics gave a residue that was purified by flash
chromatography (silica gel, 5% MeOH/CHCl3) and recrystal-
lization (Et2O) to yield 2.7 g (65%) of 16 as white crystals: mp
116-118 °C; 1H NMR (CDCl3) δ 1.14 (t, 3H, J ) 7.2 Hz), 2.68-
2.84 (m, 7H), 4.97 (d, 1H, J ) 13.0 Hz), 5.57 (s, 1H), 5.75 (d,
1H, J ) 13.0 Hz), 6.85 (d, 1H, J ) 8.2 Hz), 7.02-7.40 (m, 6H);
CIMS 300 (MH•+). Anal. (C18H21NO3) C, H, N.
N-Acetoxy-N-[2-(6,11-dih ydr o-11-oxodiben z[b,e]oxepin -
2-yl)et h yl]a cet a m id e (19). A mixture of 6,11-dihydro-11-
oxodibenz[b,e]oxepin-2-carboxaldehyde (9; 10.8 g, 45 mmol),
hydroxylamine hydrochloride (6.4 g, 91 mmol), pyridine (75
mL), and ethanol (125 mL) was stirred at 50 °C for 3 h. After
concentrating, diluting with CHCl3, and washing with 2 N HCl
and water, the organic extract was dried (Na2SO4). Concen-
tration provided 10.7 g (93%) of 6,11-dihydro-11-oxodibenz-
[b,e]oxepin-2-carboxaldehyde oxime as an oil that was used
directly: 1H NMR (CDCl3) δ 5.25 (s, 2H), 7.10 (d, 1H, J ) 9.0
Hz), 7.40 (dd, 1H, J 1 ) 2.0 Hz, J 2 ) 7.0 Hz), 7.45-7.66 (m,
3H), 7.86 (dd, 1H, J 1 ) 3.0 Hz, J 2 ) 13.0 Hz), 7.93 (dd, 1H, J 1
) 2.0 Hz, J 2 ) 12.0 Hz), 8.20 (s, 1H), 8.33 (d, 1H, J ) 2.0 Hz).
A mixture of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-car-
boxaldehyde oxime (8.5 g, 34 mmol), borane-pyridine complex
(10.9 g, 117 mmol), and ethanol (100 mL) was stirred at 0 °C
as 4 M HCl (65 mL) was added dropwise. The resulting
mixture was stirred at room temperature for 1 h and then
made basic with saturated K2CO3 solution and concentrated
to remove ethanol. The resulting aqueous mixture was
extracted with CH2Cl2, and the organic extract was dried (Na2-
SO4). Concentration and flash chromatography provided 5.9
g (68%) of 1-(6,11-dihydro-11-oxodibenz[b,e]oxepin-2-yl)-N-
hydroxymethylamine (18) as a yellow solid which was used
directly.
A mixture of methyl 2-[[4-(3-hydroxypropyl)phenoxy]methyl]-
benzoate (59.4 g, 0.20 mol) and KOH (127.1 g, 2.27 mol) in
95% ethanol (500 mL) was warmed to reflux and maintained
there overnight. After cooling and diluting with water (900
mL), the mixture was acidified with concentrated HCl (200
mL) and partitioned with EtOAc (1 L). The organic extract
was washed with water, dried (Na2SO4), and concentrated. The
residue was recrystallized (EtOH/water) to give 49.1 g of 5 as
1
white needles: mp 139-140 °C; H NMR (DMSO-d6) δ 1.67
(m, 2H), 2.54 (t, 2H, J ) 7.7 Hz), 3.40 (t, 2H, J ) 6.6 Hz), 4.4
(br s, 1H), 5.41 (s, 2H), 6.88 (d, 2H, J ) 8.8 Hz), 7.11 (d, 2H,
J ) 8.6 Hz), 7.43 (t, 1H, J ) 7.3 Hz), 7.58 (m, 2H), 7.90 (d,
1H, J ) 8.0 Hz), 13.0 (br s, 1H); EIMS 286 (M•+). Anal.
(C17H18O4) C, H.
Following this procedure, utilizing starting materials 3 and
4a -d provided benzoic acids 6 and 7a -d , respectively.12,23,24
3-(6,11-Dih yd r o-11-oxod ib e n z[b,e]oxe p in -2-yl)p r o-
p a n ol (8a ). Trifluoroacetic anhydride (82.7 g, 0.39 mol) was
added dropwise to a suspension of 5 (46.7 g, 0.16 mol) in CH2-
Cl2 (500 mL). The resulting solution was warmed to reflux
and maintained there for 4 h. After cooling and washing with
water (200 mL), the organic extract was concentrated to give
56.6 g of a solid. A portion of the residue (54 g) was diluted
with acetone (300 mL) and 10% aqueous HCl (250 mL), and
the resulting solution was warmed to reflux and maintained
there for 24 h. After cooling and concentration to remove
acetone, the resulting mixture was brought to pH 8 with
saturated NaHCO3 and extracted with CH2Cl2 (1 L). The
organic extract was washed with water, dried (Na2SO4), and
concentrated. The residue was chromatographed via HPLC
(Waters Associates Prep LC/System 500, silica gel, 1:1 hexane:
EtOAc) to give 38.8 g (86%) of 8a as an oil: 1H NMR (DMSO-
d6) δ 1.73 (m, 2H), 2.65 (t, 2H, J ) 7.7 Hz), 3.42 (dd, 2H, J 1
)
6.4 Hz, J 2 ) 11.6 Hz), 4.45 (t, 1H, J ) 5.0 Hz), 5.27 (s, 2H),
7.03 (d, 1H, J ) 8.4 Hz), 7.4-7.7 (m, 4H), 7.80 (d, 1H, J ) 7.8
Hz), 7.90 (d, 1H, J ) 2.4 Hz); EIMS 268 (M•+). Anal.
(C17H16O3) C, H.
Following this procedure, utilizing starting materials 6 and
7a -d provided dibenzoxepinones
9 and 10a -d , respec-
tively.12,23,24,25
2-(6,11-Dih ydr odiben z[b,e]oxepin -2-yl)eth an ol (11). The
preparation of 2-(6,11-dihydro-11-oxodibenz[b,e]oxepin-2-yl)-
ethanol26 (8b) was reported via reduction of known 2-(6,11-
dihydro-11-oxodibenz[b,e]oxepin-2-yl)acetic acid12 (10a ) with
borane-THF. Extending the reaction time at room temper-
ature from 16 to 72 h, starting with 10a (100 g, 0.37 mol) and
1.0 M borane-THF (0.37 mol), followed by flash chromatog-
raphy (silica gel, 5:3 hexane:EtOAc) resulted in isolation of
both 8b (55.6 g, 59%) and 11 (14.8 g, 17%). Compound 11, an
oil, was used directly: 1H NMR (CDCl3) δ 2.74 (t, 2H, J ) 6.7
Hz), 2.90 (br s, 1H), 3.80 (t, 2H, J ) 6.7 Hz), 4.31 (s, 2H), 5.28
(s, 2H), 6.80 (d, 1H, J ) 8.0 Hz), 6.88-7.08 (m, 2H), 7.10-
7.33 (m, 4H).
3-(6,11-Dih yd r od iben z[b,e]oxep in -2-yl)p r op a n oic Acid
(12). Zinc dust (18.0 g, 275 mmol) was added portionwise to
a solution of 10c (6.0 g, 21 mmol) in glacial acetic acid (120
mL). The resulting slurry was warmed to reflux and main-
tained there for 2 h. Filtration and concentration gave a
residue that was taken up in EtOAc and washed with 2 N HCl.
The organic extract was dried and concentrated and the
resulting solid recrystallized (EtOAc) to give 4.3 g (76%) of 12
as white crystals: mp 181-183 °C; 1H NMR (DMSO-d6) δ
2.40-2.80 (m, 4H), 4.21 (s, 2H), 5.27 (s, 2H), 6.63 (d, 1H, J )
8.2 Hz), 6.89-6.98 (m, 2H), 7.22-7.34 (m, 4H), 12.10 (br s,
1H); EIMS 268 (M•+). Anal. (C17H16O3) H; C: calcd, 76.10;
found, 75.59.
Acetyl chloride (4.0 g, 50 mmol) was added dropwise to a
solution of 18 (5.9 g, 23 mmol) and Et3N (7.0 g, 69 mmol) in
CH2Cl2 at 0 °C. After 1 h at 0 °C, the reaction was quenched
into 2 N HCl (100 mL) and the separated aqueous phase was
extracted with CH2Cl2. The organic extracts were dried (Na2-
SO4) and concentrated. The residue was purified by flash
chromatography (silica gel, 2:1 hexane:EtOAc) and recrystal-
lization (Et2O/hexane) to yield 2.8 g (36%) of 19 as off-white
crystals: mp 107-109 °C; 1H NMR (CDCl3) δ 2.10 (s, 3H), 2.15
(s, 3H), 4.85 (s, 2H), 5.20 (s, 2H), 7.05 (d, 1H, J ) 8.4 Hz),
7.35-7.60 (m, 4H), 7.9 (d, 1H, J ) 7.4 Hz), 8.1 (s, 1H); CIMS
340 (MH•+). Anal. (C19H17NO5) C, H, N.
N-[(6,11-Dih ydr o-11-oxodiben z[b,e]oxepin -2-yl)m eth yl]-
N-h yd r oxya ceta m id e (23a ). Meth od C. A solution of
lithium hydroxide monohydrate (2.1 g, 50 mmol) in water (50
mL) was added dropwise to a solution of 19 (2.4 g, 7.1 mmol)
in 2-propanol (120 mL) at room temperature. The resulting
mixture was stirred for 1 h and concentrated. The residue
was diluted with 2 N HCl and extracted with Et2O. The
2-(6,11-Dih yd r o-11-oxod iben z[b,e]oxep in -2-yl)-N-eth yl-
N-h yd r oxyeth yla m in e (15b). Meth od A. N-Ethylhydroxy-

Topically Active Dual Inhibitors of CO and 5-LO
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 251
organic extracts were dried (Na2SO4) and concentrated. The
residue was purified by flash chromatography (silica gel, 2:1
hexane:EtOAc) and recrystallization (EtOAc/hexane) to yield
1.1 g (52%) of 23a as white crystals: mp 129-130 °C; 1H NMR
(CDCl3) δ 2.10 (s, 3H), 4.85 (s, 2H), 5.10 (br s, 3H), 7.0 (br s,
1H), 7.35-7.90 (m, 4H), 8.05 (s, 1H), 8.35 (br s, 1H); EIMS
297 (M•+). Anal. (C17H15NO4) C, H, N.
N-[2-(6,11-Dih ydr o-11-oxodiben z[b,e]oxepin -2-yl)eth yl]-
N-(p h en ylm eth oxy)a ceta m id e (22). A solution of tert-butyl
N-(benzyloxy)carbamate (26.8 g, 0.12 mol) in anhydrous DMF
(100 mL) was added dropwise to a slurry of sodium hydride
(3.16 g, 0.13 mol) in DMF (50 mL). The resulting solution was
added to a solution of 2-(6,11-dihydro-11-oxodibenz[b,e]oxepin-
2-yl)ethanol methanesulfonate23 (13b ) (40 g, 0.12 mol) in
DMF (150 mL) and warmed at 100 °C overnight. After the
mixture was diluted with water, extraction (EtOAc), drying,
concentration, and preparative HPLC (silica gel, 2:1 hexane:
EtOAc) provided 30 g (54%) of N-[2-(6,11-dihydro-11-oxod-
ibenz[b,e]oxepin-2-yl)ethyl]-N-(phenylmethoxy)-1,1-(dimet-
hylethyl)carbamate (20) as an oil: 1H NMR (CDCl3) δ 1.43
(s, 9H), 2.90 (t, 2H, J ) 8.0Hz), 3.64 (t, 2H, J ) 8.0 Hz), 4.83
(s, 2H), 5.17 (s, 2H), 6.96 (d, 1H, J ) 8.0 Hz), 7.15-7.60 (m,
9H), 7.90 (dd, 1H, J 1 ) 2.0 Hz, J 2 ) 6.7 Hz), 8.07 (d, 2H, J )
3.0 Hz).
A solution of 20 (26.0g, 57 mmol) in anhydrous EtOAc (200
mL) was treated with saturated ethereal HCl (90 mL) and
stirred for 10 min. The resulting precipitate was collected and
dried to give 10.6 g (52%) of 2-(6,11-dihydro-11-oxodibenz[b,e]-
oxepin-2-yl)-N-(phenylmethoxy)ethylamine hydrochloride (21)-
as a powder that was used directly.
A mixture of 21 (10.6 g, 30 mmol) and triethylamine (5.3 g,
52 mmol) in THF (150 mL) was treated with acetyl chloride
(2.4 g, 30 mmol) and stirred for 2 h. The resulting mixture
was filtered, concentrated, and flashed chromatographed (silica
gel, 2:1 hexane:EtOAc) to yield 4.1 g (34%) 22 as a semisolid
that was used directly: 1H NMR (CDCl3) δ 2.06 (s, 3H), 2.93
(t, 2H, J ) 7.5 Hz), 3.84 (t, 2H, J ) 7.5 Hz), 4.80 (s, 2H), 5.17
(s, 2H), 6.97 (d, 1H, J ) 8.0 Hz), 7.20-7.63 (m, 9H), 7.88 (dd,
1H, J 1 ) 2.0 Hz, J 2 ) 6.7 Hz), 8.10 (d, 2H, J ) 3.0 Hz).
The properties of 24a -g,i-m , and 25, prepared in a similar
manner from 10a -d 12,23,24 and 12, respectively, are included
in Table 2. Representative 1H NMR data are given below.
24a : 1H NMR (DMSO-d6) δ 3.35 (s, 2H), 5.30 (s, 2H), 7.05
(d, 1H, J ) 8.4 Hz), 7.40-7.70 (m, 4H), 7.78 (d, 1H, J ) 7.4
Hz), 7.92 (d, 1H, J ) 2.4 Hz), 8.90 (br s, 1H), 10.50 (br s, 1H).
24e: 1H NMR (DMSO-d6) δ 1.00-1.80 (m, 10H), 3.75 (s, 2H),
4.15 (br s, 1H), 5.30 (s, 2H), 7.04 (d, 1H, J ) 8.4 Hz), 7.40-
7.80 (m, 5H), 7.92 (d, 1H, J ) 2.4 Hz), 9.50 (br s, 1H).
24g: 1H NMR (DMSO-d6) δ 1.05 (t, 3H, J ) 7.0 Hz), 1.30
(t, 3H, J ) 7.0 Hz), 3.40-3.60 (m, 2H), 4.20-4.40 (m, 1H),
5.30 (s, 2H), 7.05 (d, 1H, J ) 8.4 Hz), 7.50-7.70 (m, 4H), 7.78
(d, 1H, J ) 7.6 Hz), 7.98 (d, 1H, J ) 2.4 Hz), 9.70 (br s, 1H).
24j: 1H NMR (DMSO-d6) δ 1.05 (t, 6H, J ) 6.6 Hz), 2.15 (t,
2H, J ) 7.0 Hz), 2.84 (t, 2H, J ) 2.0 Hz), 4.40-4.60 (m, 1H),
5.30 (s, 2H), 7.05 (d, 1H, J ) 8.4 Hz), 7.45-7.70 (m, 4H), 7.78
(d, 1H, J ) 7.6 Hz), 7.92 (d, 1H, J ) 2.4 Hz), 9.25 (br s, 1H).
24l: 1H NMR (CDCl3) δ 2.75 (t, 2H, J ) 7.0 Hz), 3.00 (t,
2H, J ) 2.0 Hz), 3.15 (s, 3H), 3.65 (s, 3H), 5.15 (s, 2H), 7.00
(d, 1H, J ) 8.4 Hz), 7.35-7.60 (m, 4H), 7.92 (d, 1H, J ) 7.4
Hz), 8.10 (s, 1H).
24m : 1H NMR (DMSO-d6) δ 1.15 (s, 6H), 3.00 (s, 2H), 3.15
(s, 3H), 5.28 (s, 2H), 7.00 (d, 1H, J ) 8.4 Hz), 7.30-7.80 (m,
5H), 7.90 (s, 1H), 9.95 (s, 1H).
25: 1H NMR (DMSO-d6) δ 2.55-2.75 (m, 4H), 3.10 (s, 3H),
4.21 (s, 2H), 5.27 (s, 2H), 6.65 (d, 1H, J ) 8.4 Hz), 6.90-7.00
(m, 2H), 7.20-7.35 (m, 4H), 9.75 (br s, 1H).
Biologica l Meth od s. In Vitr o Cell-F r ee RBL-1 5-Li-
p oxygen a se Assa y. 5-LO inhibition was determined by
measurement of the inhibition of (S)-5-hydroxy-6-trans-8,11,-
14-cis-eicosatetraenoic acid (5-HETE) levels in a modification
of the method reported by Cochran and Finch-Arietta.17 Test
compounds, dissolved in dimethyl sulfoxide (DMSO), were
diluted to various concentrations maintaining a final DMSO
concentration of 1%. RBL-1 cell-free supernatants were
prepared by a modification of the method reported by J akschik
and Kuo.18 The supernatants were frozen with liquid N2 and
stored at -80 °C in 200 µL aliquots. The supernatants were
quick-thawed and kept on ice until assay. Duplicate reaction
tubes (10 × 75 mm) containing 35 mM NaH2PO4 (pH 7.0), 5
µM ATP, 50 µM CaCl2, test compound or standard, 0.1%
DMSO, and 10 µM arachidonic acid were prepared and
equilibrated at 37 °C. The reaction was initiated by addition
of supernatant protein (150 µg) to each assay tube. The
reaction was terminated after 10 min by the addition of 10 µL
of citric acid solution (0.3 M) and the mixture diluted with
BGGE (pH 8.5) buffer. The diluted sample 5-HETE levels
were quantitated by radioimmunoassay (Advanced Magnetics,
Bulletin 6010). The samples were incubated for 2 h at 25 °C,
and dextran-coated charcoal was used to absorb the unbound
ligand. Nonspecific binding (without antiserum), total binding
(with nonsample media), and standard curve samples were run
concurrently with test samples. Antibody-bound 5-HETE was
quantitated by scintillation counting. 5-HETE (ng/10 min at
37 °C) was quantitated from the standard curve. The mean
5-HETE levels were determined, and the percent change from
control values was calculated.
In Vitr o Mu r in e 3T3 F ibr obla st Cyclooxygen a se As-
sa y. CO inhibition was determined by measurement of the
inhibition of prostaglandin E2 (PGE2) levels in a modification
of the method reported by Lindgren.19 Test compounds,
dissolved in absolute ethanol, were diluted to various concen-
trations in Dulbecco’s modified Eagle’s medium (DMEM)
where ethanol concentrations were <0.1%. 3T3 mouse fibro-
blasts (CCL 92-3T3; American type culture collection) were
seeded in 24 mm tissue culture wells (2 × 10-4 cells/well) and
cultured for 24 h. Media were removed by aspiration and
replaced with complete DMEM (1 mL/well) containing test
compound, standard, or vehicle (4 wells/treatment). The cells
were incubated for 24 h, media removed, and the media PGE2
levels quantitated by radioimmunoassay (Advanced Magnetics,
Bulletin 6001). The samples were incubated for 2 h at 25 °C,
and charcoal was used to absorb the unbound ligand. Non-
specific binding (without antiserum), total binding (with
nonsample media), and standard curve samples were run
concurrently with test samples. The duplicate determinations
N-[2-(6,11-Dih ydr o-11-oxodiben z[b,e]oxepin -2-yl)eth yl]-
N-h yd r oxya ceta m id e (23b). Meth od D. A solution of 22
(4.0 g, 10 mmol) in EtOH (50 mL) was treated with ammonium
formate (3.2 g, 51 mmol) and 5% Pd/C (0.7 g). The mixture
was stirred at room temperature for 1.5 h, filtered through a
pad of Celite, and concentrated. The residue was purified
by flash chromatography (silica gel, 2:1 hexane:EtOAc) and
recrystallization (EtOAc/hexane) to yield 1.3 g (42%) of 23b
1
as white crystals: mp 113-115 °C; H NMR (CDCl3) δ 2.10
(s, 3H), 3.05 (m, 2H), 3.85 (m, 2H), 5.15 (s, 2H), 7.0 (d, 1H, J
) 8.2 Hz), 7.25-7.60 (m, 4H), 9.95 (d, 1H, J ) 8.0 Hz), 8.05
(s, 1H), 8.6 (br s, 1H); EIMS 311 (M•+). Anal. (C18H17NO4) C,
H, N.
3-(6,11-Dih yd r o-11-oxod ib en z[b,e]oxep in -2-yl)-N-h y-
d r oxy-N-m eth ylp r op a n a m id e (24h ). Meth od E. A slurry
of 3-(6,11-dihydro-11-oxodibenz[b,e]oxepin-2-yl)propanoic acid24
(10c; 9.9 g, 35 mmol) in CH2Cl2 (100 mL) containing DMF (0.3
mL) was treated dropwise with oxalyl chloride (6.1 mL, 70
mmol) at 0 °C. The resulting mixture was warmed to room
temperature, stirred for 2 h, and evaporated to give 10.6 g of
crude 3-(6,11-dihydro-11-oxodibenz[b,e]oxepin-2-yl)propanoyl
chloride.
A solution of the acid chloride (5.3 g, 17.5 mmol) in THF
(50 mL) was added dropwise to a mixture of N-methylhydroxy-
lamine hydrochloride (2.2 g, 26.3 mmol) and triethylamine (9.7
mL, 70 mmol) in THF:H2O (4:1, 125 mL) at 0 °C. After the
mixture had stirred for 4 h at room temperature, EtOAc (600
mL) was added and the organics were washed successively
with dilute HCl, water, and dilute NaHCO3 and dried.
Evaporation gave a foam which solidified on trituration with
Et2O. Recrystallization from ethyl acetate gave 3.5 g (64%)
of 24h as off-white crystals: mp 101-103 °C; 1H NMR (DMSO-
d6) δ 2.67 (t, 2H, J ) 7.4 Hz), 2.84 (t, 2H, J ) 7.4 Hz), 3.10 (s,
3H), 5.27 (s, 2H), 6.63 (d, 1H, J ) 8.2 Hz), 7.44-7.70 (m, 4H),
7.78 (d, 1H, J ) 8.0 Hz), 7.92 (d, 1H, J ) 2.4 Hz), 9.6 (br s,
1H); EIMS 311 (M•+). Anal. (C18H17NO4) C, H, N.

252 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Hamer et al.
(10) Summers, J . B.; Kim, K. H.; Mazdiyashi, H.; Holms, J . H.;
Ratajczyk, J . D.; Stewart, A. O.; Dyer, R. D.; Carter, G. W.
Hydroxamic Acid Inhibitors of 5-Lipoxygenase: Quantitative
Structure Activity Relationships. J . Med. Chem. 1990, 33, 992-
998 and references cited therein.
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Nonsteroidal Antiinflammatory Drug Hydroxamic Acids. Dual
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(13) Schnyder, J .; Hunziker, T.; Strasser, M.; Richardson, B.; Krebs,
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Dibenz[b,e]oxepinalkanoic Acids as Nonsteroidal Antiinflam-
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of PGE2 (pg/well) in each sample was quantitated from the
standard curve. The mean PGE2 level was determined for
each treatment group and the percent change from control
values quantified.
In Vivo Ar a ch id on ic Acid -In d u ced Mou se Ea r Ed em a
Assa y. Ear edema determinations were performed by a
modification of the method reported by Young.20 Arachidonic
acid (5,8,11,14-eicosatetraenoic acid; Sigma Chemical Co.) was
dissolved in 3:7 propylene glycol:ethanol at a final concentra-
tion of 4 mg/20 µL, and the test compound was dissolved in
3:7 propylene glycol:ethanol at a concentration of 1 mg/20 µL.
Male CFW (Swiss Webster, Charles River) mice (N ) 6)
received vehicle alone or test compound in vehicle on the right
ear (10 µL/outer ear and 10 µL/inner ear). After 30 min, the
right ear of all groups received arachidonic acid (4 mg/20 µL)
and the left ear received vehicle alone (20 µL/ear). After an
additional 1 h, mice were sacrificed by cervical dislocation and
an ear punch was taken from each ear with a 4 mm trephine.
The punch biopsies were weighed on an analytical balance.
The difference in right and left ear punch weights was
determined, and the mean difference in ear weights was
calculated for each group. The percent change in mean ear
weights from the vehicle control group was quantitated for
each compound treatment group.
Ack n ow led gm en t. We are grateful to Marc N.
Agnew, Dana L. Hallberg, and Anastasia Linville for
spectral data.
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