5-Lipoxygenase inhibition by N-hydroxycarbamates in dual-function compounds

Article abstract of DOI:10.1016/j.bmcl.2004.12.023

A series of N-hydroxycarbamates containing a histaminergic H₁ receptor antagonist pharmacophore was synthesized. In vitro assays determined the compounds had both histaminergic binding and 5-lipoxygenase inhibiting activities comparable to the

Full text of DOI:10.1016/j.bmcl.2004.12.023

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1083–1085
5-Lipoxygenase inhibition by N-hydroxycarbamates in
dual-function compounds
Timothy A. Lewis,a,* Lynn Bayless,a Alan J. DiPesa,a Joseph B. Eckman,a Michel Gillard,b
Lyn Libertine,a Ralph T. Scannell,a Donna M. Wypija and Michelle A. Younga
aUCB Research, 840 Memorial Dr., Cambridge, MA 02139, USA
bInVitro Pharmacology, UCB Pharma SA, Chemindu Foriest, B-1420 Braine l’Alleud, Belgium
Received 9 October 2004; revised 6 December 2004; accepted 9 December 2004
Available online 12 January 2005
Abstract—A series of N-hydroxycarbamates containing a histaminergic H1 receptor antagonist pharmacophore was synthesized. In
vitro assays determined the compounds had both histaminergic binding and 5-lipoxygenase inhibiting activities comparable to the
corresponding N-hydroxyurea analog. Animal models demonstrated antihistaminergic and the 5-lipopxygenase inhibitory activity,
with the N-hydroxyurea analog having a better overall profile.
Ó 2004 Elsevier Ltd. All rights reserved.
Histamine plays a role in the pathophysiology of asthma
alongside its key role in allergic response. Clinical stud-
ies have demonstrated that asthmatics treated simulta-
neously with an H1 receptor antagonist and a LTD4
receptor antagonist experienced less airway obstruction
than those patients treated with either drug alone.1 A
similar combination proved to be as efficacious as a cor-
ticosteroid in treating the airway symptoms of allergic
rhinitis and asthma.2 Inhibition of 5-lipoxygenase (5-
LO) is potentially a more efficacious treatment than
antagonism of leukotriene receptors, as biosynthesis of
all the pro-inflammatory leukotrienes would be
diminished.
O
F
N
O
N
N
NH2
OH
1
F
Synthesis of 1 began by reacting 4-iodophenol with 1,4-
dibromobutane to give 2 (Scheme 1). Palladium cata-
lyzed coupling of 2 with 3-butyn-1-ol gave 3. N-Alkyl-
ation of 1-bis-(4-fluorophenyl)methyl piperazine with 3
gave alcohol 4, which was converted to N-hydroxyurea
1 using a literature procedure.5 Synthesis of the carba-
mate analogs began with alcohol 4, which was converted
into hydroxylamine 5 via mesylation and displacement
with aqueous hydroxylamine. Treatment of 5 with ethyl
chloroformate gave the desired N-hydroxycarbamate 6.
However, treatment of 5 with isopropyl chloroformate
gave predominately the undesired N,O-bis-acylated
product 7 in 43% yield. Treatment of 7 with KOH/
i-PrOH hydrolyzed only the carbonate group to afford
N-hydroxycarbamate 8. This two step procedure, alkyl
chloroformate addition to 5 making the diacyl product
followed by basic hydrolysis, also proved more conve-
nient for the preparation of the methyl, isobutyl, and
benzyl N-hydroxycarbamates 9–11.6
Our work has focused on dual-function antihistaminer-
gic/5-LO inhibiting compounds such as the N-hydroxy-
urea UCB 62045 (1), which demonstrates both activities
in vitro and in vivo.3 UCB 62045 is effective in reducing
ovalbumin-induced bronchoconstriction in the guinea
pig bronchoconstriction model with oral dosing, and
has been investigated as a treatment for asthma and
allergic rhinitis. Reports on the 5-LO inhibitory effect
of N-hydroxycarbamates4 supported investigating this
moiety in similar dual-function molecules.
Keywords: 5-LO inhibitor; N-Hydroxycarbamate; Antihistamine.
*
Human H1 receptor binding was performed using CHO-
K1 cells expressing the recombinant H1 receptor7 (Table
Corresponding author. Tel.: +1 508 460 5099; e-mail: timlewis1201@
comcast.net
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.023

1084
T. A. Lewis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1083–1085
O
c
Br
b
O
a
Br
HO
I
I
OH
3
2
F
O
N
N
d
1
OH
4
F
e
F
F
O
O
N
N
f or g
N
N
O
OR
N
OR'
NHOH
F
F
5
6 (R = Et, R' = H)
7 (R = iPr, R' = COiPr)
8 (R = iPr, R' = H)
9 (R = Me, R' = H)
10 (R = iBu, R' = H)
11 (R = CH Ph, R' = H)
2
Scheme 1. Reagents and conditions: (a) 1,4-dibromobutane, K2CO3/DMF (83%); (b) 3-butyn-1-ol, (Ph3P)2PdCl2, CuI, Et3N/CH2Cl2 (76%); (c) 1-
[bis-(4-fluorophenyl)methyl]piperazine, K2CO3/DMF (77%); (d) (1) PPh3, DIAD, PhOCO2NHCOOPh/THF; (2) NH3/MeOH (25%); (e) (1)
MeSO2Cl, Et3N/CH2Cl2; (2) NH2OH (aq), THF/MeOH (39%); (f) for 6: EtOCOCl, Et3N/CH2Cl2 (39%); (g) (1) ROCOCl, Et3N/CH2Cl2; (2) KOH/
ROH (8—21%, 9—23%, 10—32%, 11—15%, two steps).
1). Hydroxyurea 1 displayed the best binding,8 better
than the reference standard, cetirizine. In the carbamate
series, the compounds with the smaller alkoxy groups
(OR) displayed better H1 binding, the methyl carbamate
9 binding better than cetirizine, the ethyl carbamate 6
binding equally, and all others binding with lower affin-
ity. The overall variation in H1 binding was not pro-
nounced throughout the entire series.
mation10 (Table 1). All the compounds tested were more
potent than zileuton, the reference standard, with little
variation occurring between the carbamate analogs.
O
HO
O
O
Cl
N
OH
N
NH2
N
S
Inhibition of 5-LO was tested with human recombinant
5-LO (HR),9 and a human whole blood (HWB) assay,
which monitors calcium ionophore-induced LTB4 for-
cetirizine
zileuton
N-Hydroxyurea 1 and N-hydroxycarbamates 6 and 8
were tested in animal models with oral dosing (2 mg/
kg). While methyl carbamate 9 was more potent in the
H1 and 5-LO (HR) assays, analogs 6 and 8 were ex-
pected to have better metabolic stability. Compounds
6 and 8 are both orally active antihistamines with 5-
LO inhibitory activity for up to 6 h.
Table 1. In vitro activities of dual-function compoundsa
Compound H1 binding 5-LO inhibition 5-LO inhibition
(Ki nM)b
(HR, IC50, nM)c (HWB, IC50, nM)d
1
3.63 0.52 193 76
5.89 0.49 365 55
9.55 0.62 335 115
5.01 0.59 170 60
7.59 0.66 420 30
17.8 0.60 350 20
89 34
72 40
102 69
90 11
176 116
150 56
—
6
8
9
10
The H1 antagonist activity was determined using the
Konzett–Ro¨ssler protocol, which monitors histamine-
induced bronchoconstriction (Fig. 1).11 Antihistaminer-
gic activity profiles of the three compounds were differ-
ent; hydroxyurea 1, after a slow onset, displayed nearly
complete inhibition of bronchoconstriction at 3 and 6 h.
Ethyl carbamate 6 had a faster onset and increasing
activity with time, while isopropyl carbamate 8 had slow
onset and the least activity at 3 and 6 h. None of the
11
Cetirizine
Zileuton
5.89 0.81
—
—
518 146b
873 391b,e
a Mean SD.
b n P 6.
c n P 2.
d n P 3.
e Lit. value: 900.10

T. A. Lewis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1083–1085
1085
fully determine the therapeutic potential of N-
hydroxycarbamates.13
100
80
Supplementary data
60
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2004.12.023.
40
20
0
References and notes
0
2
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Time (h)
1
6
8
Cetirizine
Figure 1. The effect of dual-function compounds on histamine-induced
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0.5 mg/kg, po, n = 3 animals/timepoint).
3. Lewis, T. A.; Bayless, L.; Eckman, J. B.; Ellis, J. L.;
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dual-function compounds are as active as cetirizine,
which displayed nearly complete inhibition of hista-
mine-induced bronchoconstriction at all time points
with a lower dose (0.5 mg/kg).
The ex vivo 5-LO assay monitors the inhibition of LTB4
production after calcium ionophore-induced stimula-
tion12 (Fig. 2). Zileuton was tested alongside the dual-
function compounds as a reference. Inhibition of 5-LO
activity by the three dual-function molecules was de-
tected up to six hours after challenge. N-Hydroxyurea
1 has better activity than the two N-hydroxycarbamates
tested, and the activity increases with time. The N-
hydroxycarbamate activities are fairly constant in this
assay, or decrease with time.
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5020.
In conclusion, in a dual-function antihistaminergic sys-
tem, N-hydroxycarbamates demonstrate 5-LO inhibi-
tory activity, both in vitro and in vivo with oral
dosing. However, the ex vivo 5-LO activities of the N-
hydroxycarbamates are lower at 1, 3, and 6 h than the
analogous N-hydroxyurea. Further work is required to
6. All compounds tested were characterized by 1H NMR,
mass spectral, and IRanalysis; purity was >95% as
determined by HPLC analysis with UV detection at
254 nm and tandem mass spectral detection. Recrystalli-
zation from EtOAc gave analytically pure material as
determined by combustion analysis (C, H, N 0.4% of
calculated values).
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and in Refs. 3b,c is due to testing at two different
laboratories using different cell lines with different batches
of 1.
100
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60
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Time (h)
1
6
8
12. Spaethe, S. M.; Snyder, D. W.; Pechous, P. A.; Clarke, T.;
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13. Experimental procedures for the synthesis of all interme-
diates and final compounds, plus the biological experi-
mental procedures can be found in the Supplementary
material.
Zileuton
Figure 2. The effect of dual-function compounds and of zileuton on
the inhibition of A-23187-stimulated LTB4 production in guinea pigs
(for 1, 6, 8, 2 mg/kg, po; zileuton, 5 mg/kg, po, n = 3 animal/
timepoint).