Nonsteroidal anti-inflammatory drugs as scaffolds for the design of 5- lipoxygenase inhibitors

Article abstract of DOI:10.1021/jm9606150

Representative nonsteroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitors such as ibuprofen, naproxen, and indomethacin were used as orally bioavailable scaffolds to design selective 5-lipoxygenase (5- LO) inhibitors. Replacement of the NSAID c

Full text of DOI:10.1021/jm9606150

J . Med. Chem. 1997, 40, 819-824
819
Non ster oid a l An ti-In fla m m a tor y Dr u gs a s Sca ffold s for th e Design of
5-Lip oxygen a se In h ibitor s
Teodozyj Kolasa, Clint D. W. Brooks,* Karen E. Rodriques, J ames B. Summers, J oseph F. Dellaria,
Keren I. Hulkower, J ennifer Bouska, Randy L. Bell, and George W. Carter
Abbott Laboratories, Immunoscience Research, D-47K, AP10, 100 Abbott Park Road, Abbott Park, Illinois 60064
Received August 28, 1996X
Representative nonsteroidal anti-inflammatory drug (NSAID) cyclooxygenase inhibitors such
as ibuprofen, naproxen, and indomethacin were used as orally bioavailable scaffolds to design
selective 5-lipoxygenase (5-LO) inhibitors. Replacement of the NSAID carboxylic acid group
with a N-hydroxyurea group provided congeners with selective 5-LO inhibitory activity.
Sch em e 1. Synthesis of “Terminal” N-Hydroxyurea
NSAID Analogs 4a , 5a , and 6a a
In tr od u ction
Cyclooxygenase enzymes1 (COX-1 and COX-2) and
5-lipoxygenase2 (5-LO) are involved in the oxidative
metabolism of arachidonic acid resulting in important
biosynthetic products such as prostaglandins, prosta-
cyclin, thromboxane, and leukotrienes.3 Nonsteroidal
anti-inflammatory drugs (NSAIDs) provide well-estab-
lished anti-inflammatory therapy acting via inhibition
of cyclooxygenases.4 Recent studies have demonstrated
therapeutic benefit for 5-LO inhibitors in asthma.5 We
explored the possibility of designing orally bioavailable
5-LO inhibitors using NSAIDs as a structural scaffold.
Based on our previous studies of hydroxamate- and
N-hydroxyurea-containing 5-LO inhibitors,6 it seemed
reasonable to suggest that replacing the carboxylate
group of common NSAIDs with the N-hydroxyurea
group might offer 5-LO inhibitors suitable for clinical
development. Realization of the actual pharmacological
properties of these analogs required experimental evalu-
ation. This report describes the synthesis of orally
active, selective, N-hydroxyurea-containing 5-LO inhibi-
tors from NSAID scaffolds7 and their properties as
compared to the reference 5-LO inhibitor zileuton (1).6b
a
Reagents: (a) DPPA, benzene, TEA, 90 °C, 1 h, then NH(R)O-
H:HCl, TEA, H2O, 90 °C, 18 h.
studies revealed that the primary mechanism of leu-
kotriene inhibitory activity of 3 was due to interfering
with 5-LO-activating protein (FLAP) and not by direct
inhibition of 5-LO catalysis.14
Studies by others8 described the derivatization of the
carboxylate function of representative NSAIDs as simple
hydroxamates to provide dual inhibitors of both COX
and 5-LO. However, these inhibitors are more ap-
propriately classified as prodrugs due to the facile in
vivo hydrolysis of the 5-LO-inhibiting hydroxamate back
to the parent COX inhibitor carboxylate.9 Two N-
hydroxyurea analogs of indomethacin, 2a ,b, have been
reported to have in vitro activity as 5-LO inhibitors, but
no in vivo results were provided.10 Another study
converted the carboxylate funtion of representative
fenamate NSAIDs into oxadiazole, thiadiazole, and
triazole analogs resulting in compounds with dual 5-LO/
COX inhibitory activity in vitro but lacking oral in vivo
activity.11
Our investigation, described herein, had the following
objectives: replace the carboxylate function of repre-
sentative NSAIDs with the N-hydroxyurea function and
evaluate the pharmacological properties of these analogs
for 5-LO inhibitory activity.
Inducing a change in inhibitory specificity from a
cyclooxygenase inhibitor to a leukotriene biosynthesis
inhibitor was demonstrated using naproxen as a scaf-
fold.12 Replacing the 6-methoxy group with the larger
lipophilic quinolylmethoxy substituent resulted in 3,
which was first described as a 5-LO inhibitor.13 Further
Ch em istr y
Methods to synthesize the N-hydroxyurea analogs
(shown in Table 1) were devised utilizing the represen-
tative NSAIDs naproxen (4), ibuprofen (5), and in-
domethacin (6) as starting templates. The “terminal”
regioisomeric N′-hydroxyurea congeners 4a , 5a , and 6a
were prepared as shown in Scheme 1. A Curtius
rearrangement reaction of the NSAID with diphenyl
phosphorazidate15 (DPPA) provided the corresponding
NSAID isocyanate intermediate which was reacted with
* Address correspondence to this author at Abbott Laboratories,
Chemical Sciences, D-41K, R-13-4, 1401
Chicago, IL 60064.
N Sheridan Rd, North
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00615-2 CCC: $14.00 © 1997 American Chemical Society

820 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
Sch em e 2. Synthesis of “Internal” N-Hydroxyurea
NSAID Analogs 4d and 5d a
Resu lts a n d Discu ssion
Direct evaluation of inhibition of 5-LO catalysis was
conducted using a lysate from sonicated rat basophilic
leukemia cells and measuring 5-HETE product forma-
tion. Additional biochemical characterization was con-
ducted in a human whole blood assay where leukotriene
biosynthesis was stimulated by calcium ionophore
(A23187). This assay provided a measurement of a
compound’s ability to inhibit leukotriene inhibition in
the complex medium of whole blood with potentially
interfering components such as binding to plasma
proteins. Separate recombinant human COX-1 and
COX-2 assays were also used to evaluate inhibitory
activity. The selective 5-LO inhibitor zileuton (1) was
used as reference standard. The results of the biological
evaluation of the compounds of this study are sum-
marized in Table 1 and described as follows. No
significant inhibitory activity was observed for 1 in
either the COX-1 or COX-2 assay at 100 and 10 µM
concentrations, respectively. The NSAID examples used
in this study, naproxen (4), ibuprofen (5), and in-
domethacin (6), demonstrated inhibitory activity in both
COX assays. Naproxen had similar activity against
both COX-1 and COX-2 enzymes (IC50s of 3.2 and 2.5
µM, respectively), whereas ibuprofen was approximately
100-fold more potent for COX-2 (IC50 ) 0.1 µM) than
for COX-1 (IC50 ) 11 µM), and indomethacin was about
50-fold more potent for COX-1 (IC50 ) 0.012 µM) than
for COX-2 (IC50 ) 0.56 µM). As expected, the NSAID
examples 4-6 did not provide significant inhibition of
5-LO in the broken cell or human whole blood assays
at concentrations up to 100 µM.
a
Reagents: (a) DPPA, benzene, TEA, 90 °C, 1 h, then tert-butyl
alcohol, 90 °C, 1 h, cool to rt, 10% aqueous HCl; (b) 4 N HCl,
dioxane, rt, 1 h; (c) p-anisaldehyde, Na2CO3, methanol, rt, 18 h;
(d) 3-chloroperbenzoic acid, dichloromethane, -20 °C f rt, 8 h;
(e) NH2OH:HCl, methanol, rt, 18 h; (f) TMS-NdCdO, THF, rt,
0.5 h.
Sch em e 3. Synthesis of the Homologated “Internal”
N-Hydroxyurea Indomethacin Analog 7a
a
Reagents: (a) BH3:THF, THF, 5 °C, 18 h; (b) PPh3, N,O-
bis(tert-butoxycarbonyl)hydroxylamine, THF, diisopropyl azodi-
carboxylate; (c) trifluoroacetic acid, dichloromethane, 0 °C, 20 min;
(d) TMS-NdCdO, THF, rt, 12 h.
the requisite hydroxylamine under standard conditions
to afford the target compounds.
The modification of naproxen (4) to exchange the
carboxylate for a hydroxyurea function provided con-
geners with 5-LO inhibitory activity. The internal
N-hydroxyurea 4d was a more potent inhibitor of 5-LO
in both the broken cell and whole blood assays (IC50s of
0.4 and 1.6 µM, respectively) than the terminal regio-
isomer 4a (IC50s of 4.8 and 7.1 µM, respectively). These
N-hydroxurea congeners 4a ,d exhibited no inhibitory
activity against both COX enzyme assays at concentra-
tions up to 30 µM.
Effecting this pharmacophore change in ibuprofen (5)
resulted in 5-LO inhibitors with a similar trend. The
internal N-hydroxurea congener 5d was more potent
than the terminal regioisomer 5a (Table 1). Both of
these hydroxyurea compounds retained some weak
COX-1 inhibitory activity at 30 µM but demonstrated
no inhibition of COX-2 activity at that concentration.
The indomethacin-derived terminal N-hydroxyurea
6a was a potent 5-LO inhibitor with IC50s of 0.2 and
1.0 µM in the broken cell and whole blood assays,
respectively. This congener also had weak activity
against both COX enzymes with 64% and 88% inhibition
at 30 µM, respectively. The indomethacin-derived
internal N-hydroxyureas 2a ,b were previously re-
ported10 to inhibit 5-LO in a similar broken cell assay
with IC50s of 0.34 and 1.4 µM, respectively. The
homologous internal N-hydroxyurea 7 was prepared and
evaluated. This compound was an effective 5-LO in-
hibitor in the broken cell assay (IC50 ) 0.4 µM) but more
than 10-fold less potent in whole blood (IC50 ) 6.1 µM).
Significant inhibition of COX-2 but not COX-1 was
observed for 7 at 10 µM. Thus homologation of the
3-indolyl substituent appears to shift COX-1/COX-2
The “internal” regioisomeric N-hydroxyurea conge-
ners 4d and 5d , with the hydroxy substituent on the
same nitrogen atom as the NSAID template, were
prepared as shown in Scheme 2. The Curtius reaction
provided the corresponding NSAID amines 4b and 5b
which were converted to the corresponding NSAID
hydroxylamines 4c and 5c by the method of Grundke.16
This method involved the formation of an imine with
p-anisaldehyde followed by oxidation to an oxaziridine.
The oxaziridine intermediate was cleaved with
hydroxylamine to provide the desired NSAID
hydroxylamine and p-anisaldehyde oxime which were
separated by standard workup conditions. The result-
ing NSAID hydroxylamine was then converted to the
target N-hydroxyureas 4d and 5d by treatment with the
trimethylsilyl isocyanate.
The homologous internal N-hydroxyurea indometha-
cin analog 7 was prepared as shown in Scheme 3. The
hydroxy analog 6b was subjected to a Mitsunobu
reaction with N,O-bis(tert-butoxycarbonyl)hydroxyl-
amine17 followed by hydrolysis with TFA to provide the
hydroxylamine derivative 6d which was converted to 7
by treatment with trimethylsilyl isocyanate.

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 821
Ta ble 1. Inhibitory Activity of Compounds in 5-Lipoxygenase and Cyclooxygenase Assays
5-LO broken cella
IC50 (µM) or % I at µM
5-LO HWBLb IC50
(µM) or % I at µM
COX-1c IC50 (µM)
or % I at µM
COX-2d IC50 (µM)
or % I at µM
compound
formula
1 (zileuton)
4 (naproxen)
4a
4d
5 (ibuprofen)
5a
5d
6 (indomethacin)
6a
7
C11H12O2S
0.5 (0.4-0.6)
0.76 (0.44-1.5)
0% at 100 µM
3.2 (2.2-4.6)
0% at 30 µM
0% at 30 µM
11.1 (6.4-20)
51% at 30 µM
37% at 30 µM
0.012 (0.01-0.02)
64% at 30 µM
9% at 10 µM
0% at 10 µM
2.5 (1.0-6.0)
2% at 30 µM
0% at 30 µM
0.10 (0.06-0.16)
0% at 30 µM
0% at 30 µM
0.56 (0.27-0.86)
88% at 30 µM
48% at 10 µM
C
C
C
C
C
C
C
C
C
14H14O3
17% at 100 µM
4.8 (3.9-6.1)
14H16N2O3
14H16N2O3
12H16O2
13H20N2O2
13H20N2O2
17H16NO4Cl
20H20ClN3O4
20H20ClN3O4
7.1 (6.5-7.7)
1.6 (0.5-2.0)
17% at 100 µM
23 (19-36)
2.5 (2.2-2.8)
7% at 100 µM
1.0 (0.86-1.4)
6.1 (5.7-6.4)
0.4 (0.4-0.5)
14% at 100 µM
2.9 (2.4-3.6)
0.6 (0.53-0.77)
12% at 100 µM
0.2 (0.17-0.24)
0.4 (0.38-0.43)
a
A 20000g supernatant from sonicated rat basophilic leukemia (RBL) cells expressing 5-LO activity. Assays run in duplicate; 95%
b
confidence limits for IC50 values are shown in parentheses. Human whole blood stimulated with calcium ionophore (A23187) and LTB4
measured by enzyme immunoassay. Assays run in duplicate; 95% confidence limits for IC50 values are shown in parentheses. c Sf9 insect
cells expressing recombinant human prostaglandin H synthase-1 (COX-1), arachidonic acid initiation, and PGE2 measured by enzyme
d
immunoassay. Assays run in duplicate; 95% confidence limits for IC50 values are shown in parentheses. Sf9 insect cells expressing
recombinant human prostaglandin H synthase-2 (COX-2), arachidonic acid initiation, and PGE2 measured by enzyme immunoassay.
Assays run in duplicate; 95% confidence limits for IC50 values are shown in parentheses.
F igu r e 2. Mean plasma concentration of 4d determined at
1, 2, 4, and 8 h intervals from two rats dosed by oral gavage
F igu r e 1. Inhibition of LTE4 in a rat anaphylaxis model by
4d . Rats were dosed by oral gavage with 4d , 1 h prior to
with 200 µmol/kg 4d . Compound plasma concentrations were
antigen challenge. Data presented are the mean values of 8
determined by HPLC.
rats/dose group. Error bars are SEM.
modification to the corresponding N-hydroxyureas. Each
new N-hydroxyurea congener had individual character-
inhibitory selectivity to favor COX-2. A recent report
of indomethacincarboxylate homologs found a shift to
istics which imparted differences in the amount of
COX-2 inhibition selectivity.18
cyclooxygenase inhibitory activity retained and oral
The naproxen congener 4d appeared promising as a
selective 5-LO inhibitor with an in vitro activity profile
similar to that of zileuton (1). The oral activity in vivo
pharmacology of 4d was examined in a rat anaphylaxis
bioavailability observed in the rat. The demonstration
of the conversion of the widely used NSAID naproxen
into a selective, orally active 5-LO inhibitor 4d with
comparable biochemical activity to zileuton (1) by a
model of leukotriene formation in the peritoneal cav-
pharmacophore modification reaffirms the preferential
ity.19 This model was instrumental for the pharmaco-
affinity of the N-hydroxyurea function for the 5-LO
logical optimization of 5-LO inhibitors leading to the
enzyme.
selection of zileuton (1) for clinical investigation.6c The
dose-response curve of 4d in this rat model of leukot-
riene formation is shown in Figure 1. Compound 4d
had an oral ED50 of 5 mg/kg, which was comparable to
that found for 1 (4.4 mg/kg).
Exp er im en ta l Section
Ch em istr y. Gen er a l. Melting points were determined in
open glass capillaries and are uncorrected. 1H NMR chemical
shifts are reported in parts per million (ppm, δ) relative to
The results of a preliminary pharmacokinetic evalu-
ation of 4d in rats dosed orally with 200 µmol/kg are
shown in Figure 2. The maximum mean drug concen-
tration from two rats was 39 µM at 4 h postdose.
Similar studies in the rat with the terminal N-
hydroxyurea 4a demonstrated substantially lower plasma
levels (Cmax ) 5 µM at 1 h) after a 200 µmol/kg oral dose.
Both ibuprofen N-hydroxyureas 5a ,d gave undetectable
drug plasma concentrations 1-8 h after a 200 µmol/kg
oral dose in the rat suggesting rapid metabolism or
elimination.
tetramethylsilane as an internal standard. Elemental analy-
ses (C, H, N) were performed at Abbott Laboratories or
Robertson Microlit Laboratories, Inc., Madison, N.J . Silica gel
60 (E. Merck; 230-400 mesh) was used for preparative column
chromatography. THF was freshly distilled from sodium
benzophenone ketyl. Other solvents were HPLC grade. Re-
agents were obtained commercially and used without further
purification. Chemical yields reported are unoptimized specific
examples of one prepartion. Analytical TLC was conducted
with E. Merck F254 commercial plates to follow the course of
reactions.
N ′-H y d r o x y -N -[1-(6-m e t h o x y n a p h t h a l-2-y l)e t h y l]-
u r ea (4a ): prepared according to the procedure for 6a
substituting naproxen for indomethacin and neat O-(trimeth-
ylsilyl)hydroxylamine for the solution of N-methylhydroxyl-
amine HCl and TEA in water (40% yield); recrystallized from
EtOAc/hexane; mp 174.5-175.5 °C; 1H NMR (300 MHz,
Con clu sion
Representative NSAIDs were used as a scaffold to
create 5-lipoxygenase inhibitors by pharmacophore

822 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
DMSO-d6) δ 1.47 (d, 3H, J ) 7.5 Hz), 3.86 (s, 3H), 4.97 (m,
1H), 7.02 (d, 1H, J ) 9 Hz), 7.14 (dd, 1H, J ) 2.5, 9 Hz), 7.28
(d, 1H, J ) 3 Hz), 7.50 (dd, 1H, J ) 1.5 Hz, 9 Hz), 7.75 (m,
3H), 8.37 (d, 1H, J ) 0.5 Hz), 8.64 (d, 1H, J ) 1 Hz); MS 261
(M + H)+. Anal. (C14H16N2O3) C, H, N.
N-H yd r oxy-N-[1-(6-m et h oxyn a p h t h a len -2-yl)et h yl]-
u r ea (4d ): prepared according to the procedure for 5d sub-
stituting naproxen for ibuprofen (5% overall yield); recrystal-
lized from EtOAc/hexane; mp 172.5-173.0 °C; 1H NMR δ 1.49
(d, 3H, J ) 6.5 Hz), 3.86 (s, 3H), 5.43 (q, 1H, J ) 6.5 Hz), 6.31
(bs, 2H), 7.13 (dd, 1H, J ) 2.5, 9 Hz), 7.27 (d, 1H, J ) 2.5 Hz),
7.47 (dd, 1H, J ) 1.5, 9 Hz), 7.72-7.81 (m, 3H), 9.07 (s, 1H);
MS 261 (M + H)+. Anal. (C14H16N2O3) C, H, N.
1.2 mL, 7.46 mmol). The mixture was stirred for 0.5 h, diluted
with saturated aqueous NH4Cl (10 mL), and extracted with
EtOAc (3 × 10 mL). The combined organic extract was washed
with brine (1 × 10 mL), dried (MgSO4), and concentrated in
vacuo, and the residue was chromatographed (silica gel, ether:
MeOH, 95:5) and crystallized from EtOAc/hexanes to afford
5d (0.40 g, 3.5% overall yield): mp 145-146 °C; 1H NMR (300
MHz, DMSO-d6) δ 0.86 (d, 6H, J ) 6.5 Hz), 1.39 (d, 3H, J )
6.5 Hz), 1.81 (m, 1H), 2.41 (d, 2H, J ) 6.5 Hz), 5.27 (m, 1H),
6.28 (bs, 2H), 7.07 (m, 2H), 7.24 (m, 2H), 9.02 (s, 1H); MS 237
(M + H)+. Anal. (C13H20N2O2) C, H, N.
N′-Hyd r oxy-N′-m eth yl-N-[(1-(4-ch lor oben zoyl)-5-m eth -
oxy-2-m eth yl-3-in d olyl)m eth yl]u r ea (6a ). To a solution of
indomethacin (3.0 g, 8.4 mmol) in benzene (40 mL) was added
triethylamine (TEA; 0.85 g, 8.4 mmol) followed by DPPA (2.31
g, 8.4 mmol), and the mixture was heated at 90 °C for 1 h. A
solution of N-methylhydroxylamine HCl (1.40 g, 16.8 mmol)
and TEA (1.70 g, 16.8 mmol) in H2O (3 mL) was added, and
the reaction mixture was stirred at 90 °C for 18 h. The
reaction mixture was cooled to room temperature and diluted
with aqueous saturated NH4Cl (40 mL), the layers were
separated, and the aqueous portion was extracted with ethyl
acetate (2 × 40 mL). The combined organic extracts were dried
(MgSO4), concentrated in vacuo, and chromatographed (silica
gel, CH2Cl2:MeOH, 98.5:1.5) to afford a solid which was
crystallized from ethyl acetate/hexanes to afford 6a (1.32 g,
39%): mp 179-180 °C; 1H NMR (300 MHz, DMSO-d6) δ 2.30
(s, 3H), 2.95 (s, 3H), 3.76 (s, 3H), 4.31 (bd, 2H, J ) 6 Hz), 6.70
(dd, 1H, J ) 3, 9 Hz), 6.92 (1H, d, J ) 9 Hz), 7.37 (m, 2H),
N ′-H yd r oxy-N -[1-(4-(2-m e t h ylp r op yl)p h e n yl)e t h yl]-
u r ea (5a ): prepared according to the procedure for 6a substi-
tuting ibuprofen for indomethacin and neat O-(trimethylsilyl)-
hydroxylamine for the solution of N-methylhydroxylamine HCl
and TEA in water (40% yield); recrystallized from EtOAc/
hexane; mp 131.5-132.0 °C; 1H NMR (300 MHz, DMSO-d6) δ
0.85 (d, 6H, J ) 7 Hz), 1.37 (d, 3H, J ) 7.5 Hz), 1.80 (septet,
1H), 2.41 (d, 2H, J ) 7.5 Hz), 4.81 (m, 1H), 6.89 (m, 1H), 7.08
(m, 2H), 7.24 (m, 2H), 8.34 (d, 1H, J ) 1 Hz), 8.61 (d, 1H, J )
1.5 Hz); MS 237 (M + H)+. Anal. (C13H20N2O2) C, H, N.
N -H yd r oxy-N -[1-(4-(2-m e t h ylp r op yl)p h e n yl)e t h yl]-
u r ea (5d ). To a solution of ibuprofen (10.0 g, 49 mmol) in
benzene (100 mL) was added TEA (6.8 mL, 49 mmol) followed
by DPPA (10.6 mL, 49 mmol), and the mixture was heated at
reflux for 1 h. tert-Butyl alcohol (9.1 mL, 97 mmol) was added,
and the mixture was refluxed for 1 h. The mixture was cooled
to room temperature, 10% aq HCl (80 mL) was added, the
layers were separated, and the aqueous portion was extracted
with ethyl acetate, washed with aqueous saturated NaHCO3,
and brine, dried (MgSO4), and concentrated in vacuo to afford
the crude urethane (10.4 g) which was used directly in the
following reaction.
The crude urethane (10 g) in 4 N HCl/dioxane (20 mL) was
stirred for 1 h and concentrated in vacuo, and the residue was
taken up in ether and agitated. The resulting white solid was
collected by filtration and washed with ether to afford 1-[4-
(2-methylpropyl)phenyl]ethylamine HCl (5b) (3.3 g, 41%): 1H
NMR (300 MHz, DMSO-d6) δ 0.86 (d, 6H, J ) 6.5 Hz), 1.51 (d,
3H, J ) 6.5 Hz), 1.83 (septet, 1H), 2.46 (d, 2H, J ) 6.5 Hz),
4.33 (bm, 1H), 7.20 (m, 2H), 7.43 (m, 2H), 8.52 (bs, 2H).
To a solution of amine (3.3 g, 15.5 mmol) in MeOH (15 mL)
were added p-anisaldehyde (1.9 mL, 15.5 mmol) and Na2CO3
(2.50 g, 23.2 mmol) and the reaction mixture was stirred for
18 h. The mixture was filtered through Celite, and the
filtrated was concentrated in vacuo to afford crude N-[1-(4-
(2-methylpropyl)phenyl)ethyl]benzaldehyde imine (6.4 g) which
was used directly in the next reaction.
7.66 (m, 4H), 9.33 (s, 1H); MS 402 (M + H)+. Anal. (C20H20
ClN3O4) C, H, N.
-
N-[2-(1-(4-Ch lor oben zoyl)-5-m eth oxy-2-m eth ylin d ol-3-
yl)-1-eth yl]-N-h yd r oxyu r ea (7). To a solution of indometha-
cin (7.14 g, 20 mmol) in THF (100 mL) at -20 °C was added
dropwise BH3‚THF (1 M solution in THF, 22 mL, 22 mmol),
and the resulting mixture was kept at 5 °C for 18 h. Acetic
acid (1 mL) was added to decompose any excess BH3, and the
mixture was concentrated in vacuo. To the residue was added
20% aqueous NaHCO3 to pH 8; the product was extracted with
EtOAc, washed with brine, dried (MgSO4), and concentrated
in vacuo to afford 2-[1-(4-chlorobenzoyl)-5-methoxy-2-meth-
ylindol-3-yl]ethan-1-ol (6.75 g, 98%).
To a solution of the alcohol intermediate (343 mg, 1 mmol),
Ph3P (262 mg, 1 mmol), and N,O-bis(tert-butoxycarbonyl)-
hydroxylamine (233 mg, 1 mmol) in THF (30 mL) was added
dropwise a solution of diisopropyl azodicarboxylate (DIAD; 0.2
mL, 1 mmol) in THF (5 mL), and the resulting mixture was
stirred at room temperature for 3 h. The mixture was
concentrated in vacuo, and the residue was chromatographed
(silica gel, CH2Cl2:EtOAc, 19:1) to afford the desired bis(BOC)-
indolyl intermediate as an oil (540 mg, 97%) which was used
directly in the next reaction.
To a solution of the crude imine (4.58 g) in CH2Cl2 (12 mL)
at -20 °C was added dropwise a solution of 85% m-chloro-
peroxybenzoic acid (2.70 g, 15.51 mmol) in CH2Cl2 (40 mL).
The reaction mixture was allowed to warm to room temper-
ature and stirred for 8 h, saturated aqueous NaHCO3 (50 mL)
was added, the layers were separated, and the aqueous portion
was extracted with EtOAc, washed with brine, dried (MgSO4),
and concentrated in vacuo to afford the crude oxaziridine 5c
(6.5 g) which was used directly in the next reaction.
A solution of the bis(BOC)indolyl intermediate in CH2
Cl2
(10 mL) was treated with trifluoroacetic acid (TFA; 5 mL) for
20 min at 0 °C. The mixture was concentrated in vacuo; the
residue was neutralized with aqueous saturated NaHCO3
,
extracted with Et2O, washed with water and brine, dried
(MgSO4), and concentrated in vacuo to provide N-[2-(1-(4-
ch lor oben zoyl)-5-m et h oxy-2-m et h ylin dol-3-yl)-1-et h yl]-
hydroxylamine (350 mg, 98%).
To a solution of the crude oxaziridine (4.8 g) in MeOH (50
mL) was added hydroxylamine HCl (2.70 g, 38.6 mmol), and
the reaction mixture was stirred for 18 h. The mixture was
concentrated in vacuo, the residue was taken up in H2O (50
mL), and the solids were removed by filtration. The aqueous
filtrate was washed with Et2O (2 × 50 mL) and EtOAc (2 ×
50 mL) to remove any residual p-anisaldehyde oxime. The
aqueous portion was neutralized with excess solid NaHCO3,
extracted with EtOAc, washed with brine, dried (MgSO4), and
concentrated in vacuo, and the residue was chromatographed
(silica gel, ether:hexanes, 1:1) to afford the desired hydroxyl-
A solution of the hydroxylamine from above and trimeth-
ylsilyl isocyanate (0.5 mL, 4 mmol) in THF (15 mL) was stirred
at room temperature for 12 h and then concentrated in vacuo.
The residue was chromatographed (silica gel, CH2Cl2:EtOH,
9:1) to afford 7 (225 mg, 56%): recrystallized from EtOAC/
hexane; mp 172-173 °C; 1H NMR (300 MHz, DMSO-d6) δ 2.18
(s, 3H), 2.86 (m, 2H), 3.52 (m, 2H), 3.78 (s, 3H), 6.34 (s, 2H),
6.72 (dd, J ) 9, 3 Hz, 1H), 7.00 (d, J ) 9 Hz, 1H), 7.10 (d, J )
3 Hz, 1H), 7.66 (m, 4H), 9.39 (s, 1H); MS (DCI/NH3) m/ z 402
(M + H)+, 419 (M + NH4)+. Anal. (C20H20ClN3O4) C, H, N.
Biologica l Meth od s. Percent inhibition was computed by
comparing individual values in treatment groups to the mean
value of the control group. Statistical significance was deter-
mined using one-way analysis of variance and Tukeys multiple
comparison procedure. Linear regression was used to estimate
IC50 and ED50 values.
1
amine (0.72 g, 24%): mp 57 °C; H NMR (300 MHz, DMSO-
d6) δ 0.86 (d, 6H, J ) 6.5 Hz), 1.19 (d, 3H, J ) 6.5 Hz), 1.80
(septet, 1H), 2.41 (d, 2H, J ) 6.5 Hz), 3.88 (q, 1H, J ) 6.5 Hz),
5.73 (bs, 1H), 7.07 (m, 2H), 7.14 (s, 1H), 7.23 (m, 2H).
To a solution of the hydroxylamine (0.72 g, 3.73 mmol) in 5
mL of THF was added trimethylsilyl isocyanate (85% purity,

Notes
5-LO Br ok en Cell Assa y. Adherent rat basophilic leuke-
mia (RBL-1) cell (2H3 subline) lysate was centrifuged at
20000g for 20 min and the supernatant containing 5-LO
activity stored frozen until used. Compounds were evaluated
for 5-lipoxygenase inhibitory activity according to the method
described by Carter et al.6d Data are from duplicate incuba-
tions.
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 823
porting departments at Abbott Laboratories for techni-
cal assistance.
Refer en ces
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C. D.; Smith, W. L. Differential inhibition of human prostag-
landin endoperoxide H synthases-1 and -2 by nonsteroidal anti-
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5-LO Hu m a n Wh ole Blood Assa y (HWBL). Aliquots of
heparinized (20 USP units/mL) human blood (0.3 mL) were
preincubated with drug or vehicle for 15 min at 37 °C, and
ecosanoid biosynthesis was initiated by adding calcium iono-
phore A23187 according to the method described by Carter et
al.6d The amount of LTB4 in aliquots of the extracts was
analyzed by enzyme immunoassay (EIA). Similarly, cyclooxy-
genase activity was determined by analysis of plasma samples
for thromboxane B2 by EIA. 12- and 15-HETE were analyzed
using commercially available radioimmunoassay (RIA) kits.
All results are means of at least duplicate and in most cases
triplicate determinations.
(2) Steinhilber, D. 5-Lipoxygenase: enzyme expression and regula-
tion of activity. Pharm. Helv. Acta 1994, 69, 3-14.
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hypersensitivity reactions and inflammation. Science 1983, 220,
568-575. (b) Lewis, R. A.; Austen, K. F.; Soberman, R. J .
Leukotrienes and other products of the 5-lipoxygenase pathway.
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Siegel, S. C.; Tinkelman, D.; Murray, J . J .; Busse, W.; Segal, A.
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1066. (b) Israel, E.; Fischer, A. R.; Rosenberg, M. W.; Lilly, C.
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(6) (a) Summers, J . B.; Gunn, B. P.; Martin, J . G.; Martin, M. B.;
Mazdiyasni, H.; Stewart, A. O.; Young, P. R.; Bouska, J . B.;
Goetze, A. M.; Dyer, R. D.; Brooks, D. W.; Carter, G. W.
Structure-activity analysis of a class of orally active hydroxamic
acid inhibitors of leukotriene biosynthesis. J . Med. Chem. 1988,
31, 1960-1964. (b) Carter, G. W.; Young, P. R.; Albert, D. H.;
Bouska, J .; Dyer, R.; Bell, R. L.; Summers, J . B.; Brooks, D. W.
5-Lipoxygenase inhibitory activity of zileuton. J . Pharmacol.
Exp. Ther. 1991, 256, 929-937. (c) Brooks, D. W.; Carter, G. W.
The discovery of zileuton. In The Search for Anti-Inflammatory
Drugs; Merluzzi, V. J ., Adams, J ., Eds.; Birkhauser: Boston,
1995; Chapter 5, pp 129-160. (d) Brooks, D. W.; Summers, J .
B.; Stewart, A. O.; Bell, R. L.; Bouska, J .; Lanni, C.; Rubin, P.;
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Fuhrer, W., Giger, R., Eds.; Verlag: Basel, 1993; Chaper 9, pp
119-134. (e) Brooks, C. D. W.; Stewart, A. O.; Basha, A.; Bhatia,
P.; Ratajczyk, J . D.; Martin, J . G.; Craig, R. A.; Kolasa, T.;
Bouska, J . B.; Lanni, C.; Harris, R. R.; Carter, G. W.; Bell, R. L.
(R)-(+)-N-[3-[5(4-Fluorophenyl)methyl]-2-thienyl]-1-methyl-2-
propynyl]-N-hydroxyurea (ABT-761), a second-generation 5-li-
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Patent 5,220,059.
Ra t P er iton ea l An a p h yla xis Mod el. This in vivo leu-
kotriene assay was conducted as described by Young et al.19
Male, Sprague-Dawley-derived rats were passively sensitized
by intraperitoneal (ip) injection of rabbit antisera (5 mL) to
bovine serum albumin (anti-BSA) in phosphate-buffered saline
(PBS), pH 7.1. Three hours after this sensitization, the rats
(8 animals/group) were injected ip with 4 mg of BSA (fraction
V, ICN Immunobiologicals, Lisle, IL) in 5 mL of PBS. Test
drug was administered by oral gavage 1 h prior to antigen
challenge unless stated otherwise. The rats were euthanized
with CO2 15 min after challenge. The peritoneal cavity was
opened, and the fluid contents were collected with a plastic
trocar and disposable pipets. The cavities were rinsed with 5
mL of cold PBS containing 0.1% gelatin, 0.1% sodium azide,
and 10 mM disodium ethylenediaminetetraacetic acid (EDTA).
The fluids were transferred to 20 mL of ice cold methanol,
allowed to stand for 20 min, vortexed, and then centrifuged
at 1000g for 15 min. Volumes were measured, and the
samples were stored at -80 °C until analyzed for leukotrienes
by enzyme immunoassay (EIA reagents for LTE4, LTB4, and
thromboxane, Cayman Chemical Co., Ann Arbor, MI; LTB4
antibody from Advance Magnetics, Cambridge, MA; LTE4
tracer prepared at Abbott).
Recom bin a n t Hu m a n P GHS-1 a n d P GHS-2 Assa ys.
Compound (3.3% DMSO final concentration) was preincubated
for 60 min with aliquots of CHAPS (3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate)-solubilized microsomes
prepared from baculovirus-infected Sf9 insect cells expressing
recombinant human prostaglandin H synthase-1 or -2 in a
reaction mixture containing hematin and phenol cofactors.
Arachidonic acid (10 µM final concentration; Nu-Chek Prep
Inc., Elysian, MN) was added to start the reactions. Following
an incubation time of 2.5 min at 25 °C, the reactions were
quenched with HCl and neutralized with NaOH. PGE2
production by the reaction mixtures was measured by EIA
(Cayman Chemical Co., Ann Arbor, MI).
(8) Flynn, D. L.; Capiris, T.; Cetenko, W. J .; Connor, D. T.; Dyer,
R. D.; Kostlan, C. R.; Nies, D. E.; Schrier, D. J .; Sircar, J . C.
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Deter m in a tion of Dr u g P la sm a Con cen tr a tion s. Com-
pounds for oral administration were suspended in 0.2% HPMC
with a Potter-Elvehjem homogenizer equipped with a Teflon-
coated pestle and administered orally to cynomolgus monkeys.
Blood samples were collected at various times following
compound administration and centrifuged, and the plasma was
removed and stored frozen until assayed. Plasma samples
were thawed, 2 vol of methanol added, and precipitated plasma
proteins removed by centrifugation. Supernatants were in-
jected directly onto a C18 reversed phase column (Adsorbo-
sphere HL 7 µm column) and chromatographed using a mobile
phase composed of 55% acetonitrile containing 10 mM aceto-
hydroxamic acid and 8 mM triethylamine acetate, pH 6.5, at
a flow rate of 1 mL/min. Compound peaks were quantitated
by UV absorbance at 260 nm using an external calibration
curve. Data presented are means from at least three monkeys.
(9) Summers, J . B.; Gunn, B. P.; Mazdiyasni, H.; Goetze, A. M.;
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Wright, C. D. J . Med. Chem. 1993, 36, 1802-1810.
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(13) Grimes, D.; Sturm, R. J .; Marinari, L. R.; Carlson, R. P.;
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Ack n ow led gm en t. We thank the members of the
Leukotriene Biosynthesis Regulators Project and sup-

824 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
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may infer that the resulting N-hydroxyurea has the same
absolute configuration as the starting NSAID.
(18) Black, W. C.; Bayly, C.; Belley, M.; Chan, C.-C.; Charleson, S.;
Denis, D.; Gauthier, J . Y.; Gordon, G. R.; Guay, D.; Kargman,
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Synthesis 1987, 275-276.
a versatile Gabriel reagent.
J M9606150