Novel histamine H3-receptor antagonists with carbonyl-substituted 4-(3-(phenoxy)propyl)-1H-imidazole structures like ciproxifan and related compounds

Article abstract of DOI:10.1021/jm000966l

Novel histamine H₃-receptor antagonists possessing a 4-(3-(phenoxy)propyl)-1H-imidazole structure generally substituted in the para-position of the phenyl ring have been synthesized according to Mitsunobu or S(N)Ar reactions. With in vitro and

Full text of DOI:10.1021/jm000966l

J . Med. Chem. 2000, 43, 3987-3994
3987
Novel Hista m in e H3-Recep tor An ta gon ists w ith Ca r bon yl-Su bstitu ted
4-(3-(P h en oxy)p r op yl)-1H-im id a zole Str u ctu r es lik e Cip r oxifa n a n d Rela ted
Com p ou n d s†
Holger Stark,*,# Bassem Sadek,# Michael Krause,# Annette Hu¨ls,# Xavier Ligneau,‡ C. Robin Ganellin,|
J ean-Michel Arrang,§ J ean-Charles Schwartz,§ and Walter Schunack#
Institut fu¨r Pharmazie, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 2+4, 14195 Berlin, Germany; Laboratoire Bioprojet,
30 rue des Francs-Bourgeois, 75003 Paris, France; Department of Chemistry, Christopher Ingold Laboratories, University
College London, 20 Gordon Street, London WC1H 0AJ , U.K.; and Unite´ de Neurobiologie et Pharmacologie Mole´culaire
(U. 109), Centre Paul Broca de l’INSERM, 2ter rue d’Ale´sia, 75014 Paris, France
Received April 6, 2000
Novel histamine H3-receptor antagonists possessing a 4-(3-(phenoxy)propyl)-1H-imidazole
structure generally substituted in the para-position of the phenyl ring have been synthesized
according to Mitsunobu or SNAr reactions. With in vitro and in vivo screening for H3-receptor
antagonist potency, the carbonyl-substituted derivatives proved to be highly active compounds.
A number of compounds showed in vitro affinities in the subnanomolar concentration range,
and the 4-hexanoyl (10) and 4-acetyl-3-methyl (29) substituted derivatives showed in vivo
antagonist potencies of about 0.1 mg/kg after po administration. Many proxifans were also
tested for their affinities at other histamine receptor subtypes thereby demonstrating their
pronounced H3-receptor subtype selectivity. Since the cyclopropyl ketone derivative 14
(ciproxifan) had high affinity in vitro as well as high potency in vivo, it was selected for further
studies in monkeys. It showed good oral absorption and long-lasting, dose-dependent plasma
levels making it a promising compound for drug development.
Ch a r t 1. Histamine H3-Receptor Antagonists
In tr od u ction
Histamine is a well-known messenger in various
(patho)physiological conditions. Receptor-dependent ef-
fects are mediated by three different established pro-
teins: H1, H2, and H3 receptors.3,4 Whereas the H1 and
H2 receptors are located postsynaptically, the H3 recep-
tors can be located both pre- and postsynaptically.
Discovery of the H3 receptor helped to clarify the
neurotransmitter function of histamine. On H3-receptor
activation, inhibition of histamine release5 and inhibi-
tion of histamine synthesis6 have been observed on
histaminergic axon terminals. H3-Receptor antagonists
increase histaminergic neuron activity by inhibition of
this feedback mechanism. H3 Heteroreceptors on non-
histaminergic neurons in the brain as well as in the
periphery influence a portfolio of different classical
neurotransmitters and neuropeptides.7 Therefore, nu-
merous potential therapeutic targets for histamine H3-
receptor antagonists have been proposed, most of them
derived from pharmacological in vitro or in vivo studies.
Since the highest density of H3 receptors is found in
distinct areas of the central nervous system (CNS), the
treatment of neurological and psychiatric diseases
seems to be most promising, e.g., schizophrenia, atten-
tion-deficit hyperactivity disorder (ADHD), dementia,
epilepsy, or narcolepsy.8
Developments in the medicinal chemistry field of
histamine H3-receptor antagonists have yielded numer-
ous compounds that are highly active in vitro and some
of them also in vivo. Although these compounds possess
different functionalities, they all have more or less in
common an imidazole heterocycle connected by a spacer
with a polar group being optionally connected by
another spacer to a lipophilic group.8 With this modular
construction pattern it seems that one element can be
replaced by another structurally different moiety if the
rest of the molecule displays enough receptor affinity.
Classical antagonists are thioperamide and clobenpropit
(Chart 1). Newer antagonists such as UCL 1390,9 FUB
470,10 or the (un)labeled iodoproxyfan,11 which displays
partial agonism in some test models, have been de-
scribed which offer some potential benefit in affinity,
selectivity, and/or toxicity. Most recently the chiral
compound GT-233112 (Perceptin) has been described as
† Presented in part at the XVth International Symposium on
Medicinal Chemistry, Edinburgh, U.K., Sept 6, 1998,1 and WO Patent
96/29 315.2
* To whom correspondence should be addressed. Tel: +49 30 838
53928. Fax: +49 30 838 53854. E-mail: stark@schunet.pharmazie.fu-
berlin.de.
#
Freie Universita¨t Berlin.
‡ Laboratoire Bioprojet.
| University College London.
§ Centre Paul Broca de l’INSERM.
10.1021/jm000966l CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/21/2000

3988 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Stark et al.
Sch em e 1. General Procedures for Synthesis of Arylproxifansa
a
Trt, trityl; (a) Ph3P, DEAD, corresponding phenol, THF; (b) HCl, acetone; (c) (i) NaH, toluene, (ii) corresponding fluoroaromate, (iii)
HCl, THF.
Sch em e 2. Synthesis of 13a
entering phase II in clinical trials for the treatment of
ADHD.13 The outcome of this first clinical trial of H3-
receptor antagonists and the recent cloning of human14
and rodent15 histamine H3 receptors will highly influ-
ence further developments.
Since compounds such as UCL 1390 and FUB 470,
which have an imidazolylpropoxy moiety in common,
have high in vivo activity (cf. Table 1) we selected the
para-substituted 4-(3-(phenoxy)propyl)-1H-imidazole as
a pharmacophore for further elaboration. The triple
bond of the cyano or alkynyl group should be replaced
by other moieties, preferably by electron-withdrawing
groups which are expected to be nontoxic. This study is
focused on carbonyl-substituted derivatives since the
antagonist potency of these compounds was found to be
higher than that of compounds with other substituents.
The influence of the acyl group in the para-position of
the phenyl ring, the influence of the position of the
carbonyl group in the para-substituents, and the effects
of additional substituents in the phenyl ring were
investigated. Synthesis and pharmacological H3-recep-
tor antagonist screening of the compounds are described
to provide information for structure-activity relation-
ships (SARs). Additional pharmacological screening for
histamine receptor subtype selectivity, histamine-N-
methyltransferase inhibitory activity, and in vivo kinet-
ics was also performed for selected compounds.
a
Trt, trityl; (a) EtOH, HCl, acetone, reflux, 45 min or HCOOH
(90%), ambient temperature, 24 h.
obtained were readily separated by standard chromato-
graphic procedures. The phenol for the synthesis of 24
was prepared by aldol reaction with 4-hydroxybenzal-
dehyde and acetone under standard conditions. In cases
where the corresponding methoxy derivative was com-
mercially available (14, 36, 37) the requisite phenol was
obtained most conveniently by ether cleavage using
BBr3‚etherate in dichloromethane at low temperature
according to known methods.10
Another method for ether formation is a nucleophilic
substitution (SNAr) on fluoroaromatics containing ad-
ditional electron-withdrawing substituents. This reac-
tion was performed with the sodium salt of the synthon
as nucleophile and corresponding fluorophenyl deriva-
tives, followed by deprotection for compounds 19, 26,
and 32-35 (Scheme 1). The yields vary to a large extent
depending on the aryl substituents, but this is an
expedient way to obtain the final compounds from
commercially available starting materials.
Ch em istr y
The benzyl alcohol 5 was obtained by reduction of 4
with complex hydrides (e.g., LiAlH4). The carboxylic acid
21 was prepared by acidic ester hydrolysis of 20. As
shown in Scheme 2 compound 13 was obtained from the
corresponding alkynyl-substituted phenyl ether which
was prepared by Heck reaction of (4-iodophenoxy)-
methoxymethane (Mom-protected 4-iodophenol) with
3,3-dimethylbutyne, phenol deprotection with sodium
ethanthiolate, and following Mitsunobu reaction with
the key synthon, 3-(1-trityl-1H-imidazol-4-yl)propanol.
The tritylated alkynyl precursor molecule obtained was
prepared according to a method described before for
related tert-butoxycarbonyl (Boc)-protected imidazole
derivatives for the synthesis of alkynylphenyl deriva-
tives.10 Deprotection under acidic conditions led to an
addition of water to the C-C triple bond and formation
of the tautomeric carbonyl isomer 13 (Scheme 2). Taking
corresponding trimethylsilyl- or alkyl-substituted alky-
nyl precursor molecules for the Heck reaction and
performing the analogous reaction steps, compounds 6
or 7 and 8, respectively, were also prepared by this
The key intermediate for all the compounds was the
trityl-protected 3-(1H-imidazol-4-yl)propanol which was
conveniently prepared from commercially available uro-
canic acid.11a Ether formation was effected by a Mit-
sunobu type synthesis16 with the corresponding phenol
derivative as described before (Scheme 1).9 Deprotection
under acidic conditions resulted in the final products
(1-4, 6-12, 14-18, 20, 22-24, 27-31, 38, 39, and with
modifications for 13 and 25). The mild conditions using
the Mitsunobu protocol were useful for almost all
carbonyl-substituted phenol derivatives which tend to
undergo unwanted side reactions under different reac-
tion conditions. Alternative methods such as the Will-
iamson synthesis with 4-(3-chloropropyl)-1H-imidazole17
were not successful due to intramolecular cyclization
and partial polymerization under basic conditions. The
corresponding carbonyl-substituted phenol derivatives
were prepared by Friedel-Crafts alkylation of phenol
in nitrobenzene at ambient or slightly increased tem-
perature using an excess of AlCl3. Mixtures of isomeric
ortho- and para-substituted phenol derivatives when

Novel Histamine H3-Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3989
Sch em e 3. Synthesis of 25a
affinity compared to the rather low number of other
substituents previously tested (F, CF3, CtC-R with R
) CH3, C3H7, C(CH3)3, Si(CH3)3)9,10 and in this series.
Moreover, steric factors seem to be more important than
electronic properties. Larger alkyl substituents on the
carbonyl moiety slightly decreased affinity (e.g., 6, 7 f
8 f 9, 10 f 11; 12 f 13; 14 f 15 f 16). Compounds
possessing moieties with additional binding properties
distant to the carbonyl group, e.g., 18-21, showed
obviously decreased affinity. The steric factor is also
important within the series of polysubstituted deriva-
tives (Table 2). Two methyl groups in the neighborhood
(ortho-related) of the acetyl group strongly decreased
affinity, whereas the second methyl group in another
position (meta-related) exhibited a less pronounced
effect (30 f 31). Affinity was also decreased when other
groups adjacent to the acetyl group could influence its
orientation (32, 33). In this series small lipophilic
substituents on the carbonyl group seem to be optimal
since the acetyl (6), propionyl (7), cyclopropylcarbonyl
(14), and cyclobutylcarbonyl (15) derivatives displayed
subnanomolar affinities.
a
Trt, trityl; (a) HCtCMgBr, THF, reflux, 1 h; (b) EtOH, HCl,
acetone, reflux, 45 min.
In Vivo Scr een in g in Mice CNS. The novel com-
pounds were also screened for their effect at histamine
H3 receptors in vivo on mice brain cortex after po
administration measuring the increase in Nτ-methyl-
histamine level, the main histamine metabolite.19 All
those compounds where potency could be detected in
vivo had antagonist properties at H3 receptors. Surpris-
ingly, the simple phenoxy derivative 1 as well as the
derivatives with hydroxy groups (2, 5, 21) did not show
any in vivo potency. Compared to their relative high in
vitro affinity, pharmacokinetic properties seem to be one
of the most probable reasons for the in vivo failure of
these compounds as well as of 4, 18-25, 27, 30, and
33.
method, but total yields were lower as compared to the
Mitsunobu protocol mentioned before (results not shown).
Another indirect way of preparation was applied for
compound 25. The tritylated precursor of the benzal-
dehyde compound 4 was isolated and subjected to a
Grignard reaction with ethynylmagnesium bromide.
This propargyl alcohol intermediate rearranged into the
cinnamaldehyde derivative 25 in the course of acid-
catalyzed detritylation (Scheme 3). This isomerization
is known as Meyer-Schuster rearrangement and de-
scribes the acid catalyzed 1,3-rearrangement of second-
ary or tertiary alcohols with an ethynyl functionality
leading to R,â-unsaturated carbonyl derivatives via the
corresponding ketenol intermediate (Scheme 3).18 By
this rearrangement terminal alkynes result in corre-
sponding aldehydes as shown with 25. It should also
be possible to get this compound directly by reacting
4-hydroxycinnamaldehyde with the key intermediate
followed by deprotection. But the tritylated propargyl
intermediate was available to us from previous studies
for the design of histamine H3-receptor antagonists,10
and therefore, this was the most convenient route for
the preparation of 25.
Our present data do not show direct correlation
between the potencies of compounds as antagonists in
vitro and in triggering histamine neuron activation in
vivo. Very recently it has also been shown that not all
histamine H3-receptor antagonists influence the Nτ-
methylhistamine CNS level to the same extent although
they are reaching brain areas.20 Therefore, it can be
concluded that despite the direct antagonist effect on
H3 receptors other factors that influence the Nτ-meth-
ylhistamine level cannot be excluded. This complex topic
needs further investigation because different G protein
coupling, different pharmacology of somatodendritic vs
presynaptic H3 receptors, receptor cross-reactivity, in-
fluence on enzymes of histamine metabolism, etc., may
be reasons for these apparent discrepancies.
P h a r m a cologica l Resu lts a n d Discu ssion
In Vitr o Assa y on Syn a p tosom es of Ra t Cer ebr a l
Cor tex. The novel compounds were tested for their
effect at histamine H3 receptors in vitro on synapto-
somes of rat cerebral cortex for the release of [3H]-
histamine (Tables 1 and 2).19 All compounds had
antagonist properties at H3 receptors in vitro.
Most compounds displayed antagonist potencies in the
nanomolar or even subnanomolar concentration range.
It can be clearly seen that the electronic properties of
the para-substituents do not influence affinity to a large
extent because compounds possessing electron-donating
substituents (2, 3, 5, 22, 23, 38, 39) showed comparable
potency to those with electron-withdrawing substituents
(e.g., 4, 6, 24-26, 36, 37). The same is true for
polysubstituted derivatives (e.g., 6 f 28 f 29, 31 f
32; Table 2). Nevertheless, it should be stated that most
acyl derivatives possess a tendency for higher in vitro
The statement concerning the electronic and steric
effect for the in vitro affinity also applies for the in vivo
potency. Different “proxifans”, which are compounds
possessing a 3-(1H-imidazol-4-yl)propoxy moiety, showed
strikingly high in vivo potency. Antagonists 3, 6-11,
13-17, 28, 29, 31, 32, and 34 were active in a dose
range below 1 mg/kg after peroral administration and
compounds 10 and 29 even below 0.1 mg/kg. For the
different n-alkyl chains on the carbonyl group (6-21)
increasing chain length slightly increased potency reach-
ing an optimum with the hexanoyl moiety (10). Its
isomeric compound 13 was significantly less effective
suggesting that in addition to the presumed benefit of

3990 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Stark et al.
Ta ble 1. Structures, Physical Data, and Pharmacological Screening Results of Para-Substituted 4-(3-(Phenoxy)propyl)-1H-imidazoles
for Histamine H3-Receptor Antagonist Potency in Vitro and in Vivo in Rodents
b
yield
(%)
mp
(°C)
Kia (nM)
xj ( sxj
ED50 (mg/kg)
no.
R
formula
Mr
xj ( sxj
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
OH
OCH3
CHO
C12H14N2O‚0.95C2H2O4
278.8
334.3
35
30
85
85
50
80
80
75
85
75
85
85
70
55
80
80
65
20
10
55
80
75
70
45
35
25
60
163-165
138
120-121
120
134
118
136
87
104-105
113-115
116-118
95
55 ( 11
18 ( 4
>10
C
12H14N2O2‚C4H4O4
>10
C13H16N2O2‚C4H4O4‚0.25H2O 352.9
13H14N2O2‚C4H4O4 346.3
C13H16N2O2‚C4H4O4‚0.25H2O 352.9
2.2 ( 0.4
12 ( 3
0.20 ( 0.08
∼30
C
CH2OH
8.9 ( 1.5
0.80 ( 0.16
0.74 ( 0.07
1.9 ( 0.4
2.6 ( 0.7
2.5 ( 0.3
7.0 ( 1.3
4.8 ( 0.6
27 ( 10
>10
CO-CH3
C14H16N2O2‚C4H4O4
C15H18N2O2‚C4H4O4
C16H20N2O2‚C4H4O4‚H2O
C17H22N2O2‚C4H4O4
C18H24N2O2‚C4H4O4
360.4
374.4
406.4
402.4
416.5
0.24 ( 0.06
0.27 ( 0.09
0.17 ( 0.05
0.15 ( 0.04
0.098 ( 0.029
0.15 ( 0.05
3.9 ( 1.9
0.85 ( 0.14
0.14 ( 0.03c
0.19 ( 0.09
0.85 ( 0.33
0.46 ( 0.11
>10
CO-C2H5
CO-C3H7
CO-C4H9
CO-C5H11
CO-C6H13
CO-CH(CH3)2
CO-CH2-C(CH3)3
CO-cyclopropyl
CO-cyclobutyl
CO-cyclopentyl
CO-CH2-Ph
CO-(CH2)3-piperidino
C19H26N2O2‚C4H4O4‚0.25H2O 435.0
C16H20N2O2‚C4H4O4‚0.5H2O
C18H24N2O2‚C4H4O4
397.4
416.5
386.4
139
C
16H18N2O2‚C4H4O4
118-120 0.49 ( 0.09c
C17H20N2O2‚C4H4O4‚0.25H2O 404.9
18H22N2O2‚C4H4O4‚0.25H2O 419.0
C20H20N2O2‚C2H2O4‚0.75H2O 423.9
130
141
174-176
190-192
70
oil
97
89
104
0.89 ( 0.18
7.2 ( 1.1
5.9 ( 2.4
ndd
410 ( 139
122 ( 17
49 ( 10
53 ( 11
91 ( 20
20 ( 5
C
C21H29N3O2‚2C2H2O4
535.5
782.8
478.5f
388.8
383.4
397.4
395.4
381.4
396.4
337.9
CO-(CH2)3-pyridoindole C30H29FN4O2‚2C4H4O4‚3H2O
>10
CO-(CH2)3-COOC2H5
CO-(CH2)3-COOH
CH2-CO-CH3
(CH2)2-CO-CH3
(CH)2-CO-CH3
(CH)2-CHO
C19H24N2O4‚C4H4O4‚H2O
C17H20N2O4‚HCl‚2H2O
>10
>10
C15H18N2O2‚C4H4O4‚0.5H2O
C16H20N2O2‚C4H4O4‚0.5H2O
C16H18N2O2‚C4H4O4‚0.5H2O
C15H16N2O2‚C4H4O4‚0.5H2Og
C13H16N2O3S‚C4H4O4
>10
>10
123
119-121
135
>10
25
26
27
32 ( 7
40 ( 10
nd
∼30
SO2-CH3
8.8 ( 2.2
∼30
cyclopentyl
C
17H22N2O‚0.75C2H2O4
224-225
UCL 1390
FUB 470
thioperamide
clobenpropit
(R ) C≡N)h
12 ( 3
0.54 ( 0.23
0.12 ( 0.07
1.0 ( 0.5c
26 ( 7c
(R ) C≡CH) j
2.3 ( 0.8
4 ( 1c
0.6 ( 0.1c
a
b
Functional H3-receptor assay in vitro on synaptosomes of rat cerebral cortex.19 Central H3-receptor screening in vivo after oral
administration to mice.19 c Ref 21a and literature therein. nd, not determined. e Pyridoindol, 8-fluoro-2,3,4,5-tetrahydro-1H-pyrido[4,3-
d
b]indol-2-yl. f Elemental analyses not performed. C,H; N: calcd, 7.35; found, 8.02. Ref 9. Ref 10.
g
h
j
Ta ble 2. Structures, Physical Data, and Pharmacological Screening Results of Polysubstituted 4-(3-(Phenoxy)propyl)-1H-imidazoles
for Histamine H3-Receptor Antagonist Potency in Vitro and in Vivo in Rodents
b
yield
(%)
mp
(°C)
Kia (nM)
xj ( sxj
ED50 (mg/kg)
no. R1
R2
R3
R4
formula
Mr
xj ( sxj
28
29
30
31
32
33
34
35
36
37
38
39
CO-CH3
F
H
H
CH3
H
H
H
H
H
H
H
H
H
C14H15FN2O2‚C4H4O4‚0.25H2O 382.9
80
80
85
80
55
60
35
60
40
75
40
20
114
109
92
126
117
124
119-121
151-152
147
1.1 ( 0.2
1.2 ( 0.3
90 ( 19
4.8 ( 0.8
17 ( 5
325 ( 164
57 ( 11
ndc
0.15 ( 0.06
0.08 ( 0.02
>10
0.39 ( 0.10
0.64 ( 0.14
>10
0.9 ( 0.2
5.5 ( 2.8
1.5 ( 0.5
7 ( 4
1.3 ( 0.2
3.3 ( 1.3
CO-CH3
CH3
CH3
H
CF3
OCH3
F
C15H18N2O2‚C4H4O4
374.4
397.4
387.9
CO-CH3
C16H20N2O2‚C4H4O4‚0.5H2O
CO-CH3
CH3 C15H18N2O2‚C4H4O4‚0.25H2O
CO-CH3
H
H
H
C15H15F3N2O2‚C2H2O4‚0.5H2O 411.3
C15H18N2O3‚C4H4O4‚0.5H2O 399.4
C15H17FN2O2‚C4H4O4‚0.25H2O 396.9
CO-CH3
CO-C2H5
CO-cyclopropyl
H
NO2 C16H17N3O4‚C4H4O4
431.4
390.4
395.4
314.3
332.9
-CO-CH2-CH2-
-CO-CH2-CH2-CH2-
-CH2-CH2-CH2-
H
H
H
H
C15H16N2O2‚C4H4O4‚H2O
C16H18N2O2‚C4H4O4‚0.5H2O
C15H18N2O‚0.8C2H2O4
17( 4
H
H
H
102
188-190
171-173
13 ( 3
38 ( 6
-CH2-CH2-CH2-CH2-
C16H20N2O‚0.85C2H2O4
45 ( 14
a
b
Functional H3-receptor assay in vitro on synaptosomes of rat cerebral cortex.19 Central H3-receptor screening in vivo after oral
administration to mice.19 c nd, not determined.
increased lipophilicity, steric factors have to be consid-
ered. The steric demands of this alkyl substituent were
impressively shown by the isopropyl derivative 12
compared to its cyclized analogue 14. In particular, the

Novel Histamine H3-Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3991
Ta ble 3. Activity of Selected Compounds at Histamine
Receptor Subtypes
b
c
d
no.
H3 pKia
H3 pKb
H2 pKb
H1 pKb
3
4
5
6
7
8.7
7.9
8.1
9.1
9.1
8.7
8.6
8.6
8.3
9.3
9.1
8.1
7.3
7.1
8.9
8.9
7.8
7.4
6.3
6.7
7.8
8.0
8.1
8.4
8.4
7.1
8.4
8.5
7.9
6.2
6.9
7.9
7.9
4.3
4.0
4.1e
4.6
4.9
5.4
4.7
5.3
4.9
5.4
5.8
4.2e
5.6
4.8
5.1
5.3
4.9
4.6
<4.0
5.0
8
9
10
12
14f
15
16
22
23
28
29
32
F igu r e 1. Kinetics of ciproxifan (14) in monkeys (n ) 6-11)
after single oral administration.
4.3
4.5
5.0e
4.8
3.9
4.1
4.7e
4.8
cyclopropyl derivative 14 is more than 25 times more
potent than the isopropyl derivative 12. Even the
homologous cycloalkyl derivatives (15, 16) are more
potent than 12. Shifting of the carbonyl moiety away
from the phenyl ring (22-25), its exchange to sulfone
(26), or its omission (27) led to strong decreases in
potencies. The carboxylic acid 21 was synthesized for
the preparation of antibodies for the development of a
specific radioimmunoassay for the main compounds of
this series (Ligneau et al., unpublished results).
a
Functional H3-receptor assay on synaptosomes of rat cerebral
cortex.19 Functional H3-receptor assay on guinea pig ileum.22
b
c Functional H2-receptor test on guinea pig atrium.23 Functional
d
H1-receptor test on guinea pig ileum.23 e Ref 21b. f Ref 21a.
system (-log Ki ) 5.6, using a test system based on
human placental microsomes) (Hartmann et al., unpub-
lished results).
Scr een in g of Selected Com p ou n d s a t F u n ction a l
Hista m in e Recep tor Mod els on Gu in ea P ig Tis-
su es. In addition to the extensive selectivity studies on
ciproxifan,21 selected compounds of this series were also
tested on different functional models on isolated organs
of guinea pigs for their histamine H3-, H2-, and/or H1-
receptor activities.22,23 All compounds tested showed low
activity at H1 or H2 receptors proving their selectivity
(Table 3). For a number of compounds, the differences
between the two functional H3-receptor test systems
were striking being sometimes more than 1.5 orders of
magnitude (4). These differences are also observed in
comparable assays even in the same species with
compounds of other classes. However, the reason for this
is not clarified to date.24
Additional substitution of the phenyl ring was ac-
cepted when the substituents did not influence too much
the little steric demands of the carbonyl moiety. One
methoxy group (33) or two methyl groups (30) in the
ortho-position relative to the carbonyl group led to
inactive compounds, whereas a nitro group in the
corresponding meta-position (35) only caused a decrease.
Bicyclic compounds (36-39) gave no improvements.
Additional substitution was tolerated with one fluoro
(28, 34), methyl (29, 31), or trifluoromethyl (32) group
since it led to retention of activity. Although compounds
28 and 29 showed an improvement in potency compared
to 6, this cannot be taken as a general rule (cf. 7 f 34).
Compounds 10, 14, and 29 were the most active
antagonists in vivo in this series and are also among
the most active ones known so far to our knowledge.
The potencies of these compounds were not statistically
significantly different from each other. Compound 14,
named ciproxifan, was selected for further studies
because it showed extremely high activity in vitro as
well as in vivo. With these studies ciproxifan appears
to be an orally bioavailable, extremely potent, and
selective histamine H3-receptor antagonist whose vig-
ilance- and attention-promoting effects are promising
for therapeutic applications in aging or degenerative
disorders.21 Oral bioavailability and detectable plasma
levels for more than 6 h with different doses of ciproxi-
fan are shown by the preclinical kinetic data in monkeys
in Figure 1 although rapid formation of an active
metabolite cannot be excluded by the method of deter-
mination. The areas under the curves (AUC) were 6.2,
26.6, and 61.6 µM/h for the 3, 10, and 30 mg/kg po doses,
respectively. Furthermore, ciproxifan showed low in-
hibitory effect on the histamine-metabolizing enzyme,
histamine-N-methyltransferase (E.C. 2.1.1.8) (IC50 ) 9.0
( 1.2 µM), indicating that this enzyme is not influenced
in therapeutic concentrations. Ciproxifan also showed
low inhibitory affinity for the cytochrome P450 enzyme
Con clu sion s
Compounds of the proxifan class possessing a para-
substituted 4-(3-(phenoxy)propyl)-1H-imidazole moiety
were prepared by different methods and screened for
their histamine H3-receptor potency in different assays.
The hexanoyl derivative (10), cyclopropylcarbonyl de-
rivative (14, ciproxifan), and the acetyl derivative with
an additional methyl substituent (29) were extremely
potent in the in vivo assay displaying ED50 values in
the low 0.1 mg/kg dosage range on oral administration.
The H3-receptor selectivity for many compounds of this
class was also proved by testing activity at other
histamine receptor subtypes. Preclinical kinetic data of
ciproxifan (14) in primates showed oral bioavailability
and plasma levels lasting for 6 h or more, which makes
this compound attractive for further development.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
determined on an Electrothermal IA 9000 digital or a Bu¨chi
512 apparatus and are uncorrected. For all compounds 1H
NMR spectra were recorded on a Bruker AC 300 (300 MHz)
spectrometer. Chemical shifts are expressed in ppm downfield
from internal TMS as reference. 1H NMR data are reported

3992 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Stark et al.
in the following order: multiplicity (s, singlet; d, doublet; dd,
double doublet; t, triplet; m, multiplet; Im, imidazole; Mal,
maleic acid), number of protons, and approximate coupling
constants in hertz (Hz). Mass spectra were obtained on an EI-
MS Finnigan MAT CH7A (170 °C, 70 eV) or a Finnagan MAT
CH5DF (FAB+/-; Xe, Me2SO/glycerol). Elemental analyses (C,
H, N) for the compounds were measured on Perkin-Elmer 240
B or Perkin-Elmer 240 C instruments and were within (0.4%
of the theoretical values, unless otherwise stated. Preparative,
centrifugally accelerated rotatory chromatography was per-
formed using a Chromatotron 7924T (Harrison Research) and
glass rotors with 4-mm layers of silica gel 60 PF254 containing
gypsum (Merck). Column chromatography was carried out
using silica gel 63-200 µm (Macherey, Nagel & Co.). TLC was
performed on silica gel PF254 plates (Merck); the spots were
visualized with fast blue salt BB. Abbreviations for solvents
are the following: Et2O, diethyl ether; EtOH, ethanol; EtOAc,
ethyl acetate; MeOH, methanol; THF, tetrahydrofuran. Syn-
thesis procedures and spectral data are shown only for parent
compounds (5, 6, 13, 21, 25, 26) that were obtained by different
reactions or methods and for the most active ones (10, 14, 29).
4-(3-(1H-Im id a zol-4-yl)p r op yloxy)p h en ylm eth a n ol (5).
Compound 4 (free base, 2 mmol, 700 mg) in 20 mL of freshly
distilled THF was slowly added to a suspension of LiAlH4 (1
mmol, 40 mg) in 20 mL of freshly distilled THF. After heating
for 2 h 5 mL of 2 N NaOH was added dropwise at ambient
temperature. The organic phase was separated, washed with
brine, dried (Na2SO4) and purified by column chromatography
(eluent: CH2Cl2/MeOH (96/4), ammonia atmosphere). The
colorless oil obtained was crystallized as hydrogen maleate in
EtOH/Et2O: 1H NMR (Me2SO-d6) δ 8.87 (s, 1H, Im-2-H), 7.41
(s, 1H, Im-5-H), 7.22 (d, J ) 8.5 Hz, 2H, Ph-2-H, Ph-6-H), 6.86
(d, J ) 8.6 Hz, 2H, Ph-3-H, Ph-5-H), 6.04 (s, 2H, Mal), 5.00 (s,
1H, OH), 4.41 (m, 2H, CH2-OH), 3.98 (t, J ) 6.2 Hz, 2H, CH2-
O), 2.79 (t, J ) 7.6 Hz, 2H, Im-CH2), 2.06 (m, 2H, Im-CH2-
CH2); MS m/z 232 ([M•+], 4), 109 (100), 96 (22), 82 (71), 81
(39), 72 (11).
1-(4-(3-(1H -Im id a zol-4-yl)p r op yloxy)p h e n yl)-3,3-d i-
m eth ylbu ta n on e (13). 3-(1-Triphenylmethyl-1H-imidazol-4-
yl)propanol11a (3.7 mmol, 1.36 g) and 4-(3,3-dimethylbutynyl)-
phenol10 (3.7 mmol, 0.65 g) were reacted analogous to general
procedure A. The trityl-protected intermediate was hydrolyzed
either by heating in 5 mL of EtOH, 5 mL of acetone, and 30
mL of 2 N HCl for 45 min or by stirring in 20 mL of formic
acid for 24 h at ambient temperature. After extraction with
Et2O, alkalization with NH3, subsequent extraction with Et2O
and following rotatory chromatographic purification of the
organic extracts (eluent: CH2Cl2/MeOH (gradient from 95/5
to 90/10), ammonia atmosphere) the colorless oil was crystal-
lized as hydrogen maleate in EtOH/Et2O to give the product
in the same yield for both methods of hydrolysis: 1H NMR
(Me2SO-d6) δ 8.88 (s, 1H, Im-2-H), 7.93 (d, J ) 8.8 Hz, 2H,
Ph-2-H, Ph-6-H), 7.43 (s, 1H, Im-5-H), 6.99 (d, J ) 8.8 Hz,
2H, Ph-3-H. Ph-5-H), 6.05 (s, 2H, Mal), 4.11 (t, J ) 6.1 Hz,
2H, CH2-O), 2.82 (m, 4H, Im-CH2, CO-CH2), 2.09 (m, 2H, Im-
CH2-CH2), 0.99 (s, 9H, C(CH3)3); MS m/z 300 ([M•+], 2), 229
(7), 109 (100), 96 (36), 82 (54).
Gen er a l P r oced u r e A for Mit su n ob u Typ e E t h er
F or m a tion . Triphenylphosphine (6 mmol, 1.57 g) was dis-
solved in 15 mL of freshly distilled THF under Ar atmosphere
together with 3-(1-triphenylmethyl-1H-imidazol-4-yl)propanol11a
(5 mmol, 1.84 g) and 6 mmol of the phenol derivative and
cooled in an ice bath. Then, diethyl azodicarboxylate (DEAD)
(6 mmol, 0.95 mL) was slowly added followed by additional
stirring for 12-72 h at ambient temperature. After removal
of the solvent in vacuo purification was performed by column
chromatography (eluent: EtOAc). The oil obtained was detri-
tylated by heating for reflux in 20 mL of 2 N HCl and 20 mL
of acetone (except for compound 20 in 30 mL of formic acid
only) for 30-90 min. Acetone was removed under reduced
pressure and the residue was extracted with Et2O. The
aqueous phase was neutralized with potassium carbonate and
extracted with CH2Cl2 (3 × 20 mL). The combined organic
extracts were dried (Na2SO4), concentrated under reduced
pressure and purified by rotatory chromatography (eluent:
CH2Cl2/MeOH (gradient from 99.5/0.5 to 90/10), ammonia
atmosphere). The product obtained was crystallized as hydro-
gen maleate or hydrogen oxalate in EtOH/Et2O.
5-(4-(3-(1H-Im id a zol-4-yl)p r op yloxy)p h en yl)-5-oxova le-
r ia n ic Acid (21). Compound 20 (∼0.5 mmol, 200 mg) was
dissolved in 1.6 mL of acetone, 0.48 mL of 6 N HCl, and 1.12
mL of water and stirred for 6 h at ambient temperature. Then
10 mL of acetone was added and carefully concentrated under
reduced pressure. Compound 21 slowly crystallized in form of
its hydrochloride salt on standing: 1H NMR (Me2SO-d6) δ 8.84
(s, 1H, Im-2-H), 7.94 (d, J ) 8.7 Hz, 2H, Ph-2-H, Ph-6-H), 7.39
(s, 1H, Im-5-H), 7.02 (d, J ) 8.7 Hz, 2H, Ph-3-H, Ph-5-H), 4.11
(t, J ) 6.0 Hz, 2H, CH2-O), 2.99 (t, J ) 7.2 Hz, 2H, CO-CH2),
2.84 (t, J ) 7.5 Hz, 2H, Im-CH2), 3.32 (t, J ) 7.3 Hz, 2H, CH2-
COO), 2.11 (m, 2H, Im-CH2-CH2), 1.83 (m, 2H, CO-CH2-CH2);
MS m/z (FAB-) 351 ([M + Cl], 25), 315 ([M - H]-, 35), 207
(20).
1-(4-(3-(1H -I m id a zo l-4-y l)p r o p y lo x y )p h e n y l)e t h a -
n on e (6): 1H NMR (Me2SO-d6) δ 8.89 (s, 1H, Im-2-H), 7.93 (d,
J ) 8.8 Hz, 2H, Ph-2-H, Ph-6-H), 7.43 (s, 1H, Im-5-H), 7.02
(d, J ) 8.8 Hz, 2H, Ph-3-H, Ph-5-H), 6.05 (s, 2H, Mal), 4.11 (t,
J ) 6.2 Hz, 2H, CH2-O), 2.80 (t, J ) 7.5 Hz, 2H, Im-CH2), 2.50
(s, 3H, CH3), 2.09 (m, 2H, Im-CH2-CH2); MS m/z 244 ([M•+],
2), 121 (10), 109 (87), 96 (51), 82 (100), 81 (74), 54 (12).
1-(4-(3-(1H -I m id a zo l-4-y l)p r o p y lo x y )p h e n y l)h e x -
a n on e (10): 1H NMR (Me2SO-d6) δ 8.89 (s, 1H, Im-2-H), 7.92
(d, J ) 8.8 Hz, 2H, Ph-2-H, Ph-6-H), 7.43 (s, 1H, Im-5-H), 7.01
(d, J ) 8.8 Hz, 2H, Ph-3-H, Ph-5-H), 6.05 (s, 2H, Mal), 4.12 (t,
J ) 6.1 Hz, 2H, CH2-O), 2.93 (m, 2H, CO-CH2), 2.80 (t, J )
7.5 Hz, 2H, Im-CH2), 2.09 (m, 2H, Im-CH2-CH2), 1.59-1.28
(m, 6H, CO-CH2-(CH2)3), 0.86 (m, 3H, CH3); MS m/z 300 ([M•+],
<1), 121 (15), 109 (100), 96 (201), 82 (64), 41 (12).
Cyclop r op yl 4-(3-(1H-im id a zol-4-yl)p r op yloxy)p h en yl
m eth a n on e (14): 1H NMR (Me2SO-d6) δ 8.50 (s, 1H, Im-2-
H), 8.03 (d, J ) 8.8 Hz, 2H, Ph-2-H, Ph-6-H), 7.25 (s, 1H, Im-
5-H), 7.05 (d, J ) 8.8 Hz, 2H, Ph-3-H, Ph-5-H), 6.03 (s, 2H,
Mal), 4.12 (t, J ) 6.2 Hz, 2H, CH2-O), 2.85 (m, 1H, CO-CH),
2.77 (t, J ) 7.5 Hz, 2H, Im-CH2), 2.08 (m, 2H, Im-CH2-CH2),
0.98 (d, J ) 6.1 Hz, 4H, cyclopropyl (CHH)2); MS m/z 541 ([2M
+ H]+, 1), 333 ([M + Cu]+, 11), 271 ([M + H]+, 70), 203 (2),
121 (8), 109 (100), 95 (13), 82 (44), 81 (34), 59 (14).
(E)-3-(4-(3-(1H-Im id a zol-4-yl)p r op yloxy)p h en yl)p r op e-
n a l (25). 3-(1-Triphenylmethyl-1H-imidazol-4-yl)propanol11a (5
mmol, 1.84 g) and 4-hydroxybenzaldehyde (5 mmol, 0.61 g)
were reacted according to general procedure A to result in the
precursor for compound 4. Ethynylmagnesium bromide (0.5
M solution in THF, 5 mmol, 10 mL) was added to the trityl-
protected intermediate in 25 mL of freshly distilled THF and
heated under reflux for 1 h. The mixture was evaporated,
hydrolyzed in EtOH, acetone and 2 N HCl, and extracted as
described before. The dried organic extracts were crystallized
and recrystallized as hydrogen maleate in EtOH/Et2O: 1H
NMR (Me2SO-d6) δ 9.62 (d, J ) 7.8 Hz, 1H, CHO), 8.94 (s,
1H, Im-2-H), 7.70 (m, 3H, Ph-CH)CH, Ph-2-H, Ph-6-H), 7.46
(s, 1H, Im-5-H), 7.02 (d, J ) 8.7 Hz, 2H, Ph-3-H, Ph-5-H), 6.74
(dd, J H,H(E) ) 15.9 Hz, J H,CHO ) 7.8 Hz, 1H, CH-CHO), 6.07
(s, 2H, Mal), 4.10 (t, J ) 6.1 Hz, 2H, CH2-O), 2.82 (t, J ) 7.5
Hz, 2H, Im-CH2), 2.08 (m, 2H, Im-CH2-CH2); MS m/z 256
([M•+], 5), 240 (1), 228 (3), 109 (100), 82 (30).
Gen er a l P r oced u r e B for E t h er F or m a t ion b y SNAr
Rea ction . 3-(1-Triphenylmethyl-1H-imidazol-4-yl)propanol11a
(5 mmol, 1.84 g) was stirrred at 60 °C for 2 h with NaH (6
mmol, 240 mg of 60% in paraffin oil) under Ar in 20 mL of
toluene. Then, the corresponding fluoroaromate (6 mmol) was
added at ambient temperature in 5 mL of toluene, the mixture
3
3
1-(4-(3-(1H-Im id a zol-4-yl)p r op yloxy)-2-m eth ylp h en yl)-
1
eth a n on e (29): H NMR (Me2SO-d6) δ 8.82 (s, 1H, Im-2-H),
7.82 (d, J ) 8.6 Hz, 1H, Ph-6-H), 7.38 (s, 1H, Im-5-H), 6.83 (s,
1H, Ph-3-H), 6.81 (d, J ) 8.6 Hz, 1H, Ph-5-H), 6.02 (s, 2H,
Mal), 4.06 (t, J ) 6.2 Hz, 2H, CH2-O), 2.77 (t, J ) 7.5 Hz, 2H,
Im-CH2), 2.43 (s, 6H, 2CH3), 2.06 (m, 2H, Im-CH2-CH2); MS
m/z 258 ([M•+], 8), 149 (11), 109 (100), 96 (34), 82 (68), 43 (10).

Novel Histamine H3-Receptor Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3993
warmed slowly to 70 °C and stirred for 2-8 h (TLC control).
In some cases, yields could be increased by repeating the
procedure by addition of NaH and fluoroaromate in 2 mmol
amounts, but reaction conditions were not optimized. The
mixture was evaporated, water was carefully added, and then
it was heated to reflux in 35 mL of 2 N HCl and 20 mL of
THF for 1.5 h. The organic solvent was removed under reduced
pressure, and the residue extracted with Et2O (3 × 20 mL).
The aqueous solution was basified with K2CO3 and extracted
with CH2Cl2 (3 × 20 mL). The combined organic extracts were
evaporated and subjected to rotatory chromatography (elu-
ent: CH2Cl2/MeOH (gradient from 99.5/0.5 to 90/10), ammonia
atmosphere). The resulting product was converted into a salt
(cf. Tables 1 and 2) and recrystallized from EtOH/Et2O.
gation (100g, 10 min, 4 °C) in the serum using a radioreceptor
assay derived from the [3H](R)-R-methylhistamine binding
assay described before with mice.19,21a Values given represent
the mean with SEM of an experiment with 6-11 monkeys for
each dosage.
Hista m in e H3-Recep tor An ta gon ist Activity on Gu in ea
P ig Ileu m . For selected compounds H3-receptor activity was
measured by concentration-dependent inhibition of electrically
evoked twitches of isolated guinea pig ileum segments induced
by (R)-R-methylhistamine in the presence of the antagonist
according to Ligneau et al.22 In brief, longitudinal muscle strips
were prepared from the small intestine, 20-50 cm proximal
to the ileocecal valve. The muscle strips were mounted between
two platinum electrodes (4 mm apart) in 20 mL of Krebs buffer,
containing 1 µM of mepyramine, connected to an isometric
transducer, continuously gassed with oxygen containing 5%
CO2 at 37 °C. After equilibration of the muscle segments for 1
h with washing every 10 min, they were stimulated continu-
ously with rectangular pulses of 15 V and 0.5 ms at a frequence
of 0.1 Hz. After 30 min of stimulation, a cumulative dose-
response curve was recorded. Subsequently the preparations
were washed three times every 10 min without stimulation.
The antagonist was incubated 20-30 min before redetermina-
tion of the dose-response curve of (R)-R-methylhistamine.
In Vitr o Scr een in g a t Oth er Hista m in e Recep tor s.
Selected compounds were screened for histamine H2-receptor
activity on the isolated spontaneously beating guinea pig right
atrium as well as for H1-receptor activity on the isolated guinea
pig ileum by standard methods described by Hirschfeld et al.23
Each pharmacological test was performed at least in triplicate,
but the exact type of interaction had not been determined in
each case. The given values represent the mean.
4-(3-(1H-Im id a zol-4-yl)p r op yloxy)p h en yl m eth yl su l-
1
fon e (26): H NMR (Me2SO-d6) δ 8.83 (s, 1H, Im-2-H), 7.98
(d, J ) 8.6 Hz, 2H, Ph-2-H, Ph-6-H), 7.39 (s, 1H, Im-5-H), 7.13
(d, J ) 8.6 Hz, 2H, Ph-3-H, Ph-5-H), 6.03 (s, 2H, Mal), 4.09 (t,
J ) 6.0 Hz, 2H, CH2-O), 3.13 (s, 3H, SO2-CH3), 2.79 (t, J ) 7.5
Hz, 2H, Im-CH2), 2.09 (m, 2H, Im-CH2-CH2); MS m/z 288
([M•+], 3), 109 (32), 96 (35), 81 (100), 41 (27).
P h a r m a cology. Gen er a l Meth od s. Hista m in e H3-Re-
cep tor Assa y on Syn a p tosom es of Ra t Cer ebr a l Cor tex.
Compounds were tested for their H3-receptor antagonist
activity in an assay with K+-evoked depolarization-induced
release of [3H]histamine from rat synaptosomes according to
Garbarg et al.19 A synaptosomal fraction from rat cerebral
cortex prepared according to the method of Whittaker25 was
preincubated for 30 min with L-[3H]histidine (0.4 µM) at 37
°C in a modified Krebs-Ringer solution. The synaptosomes
were washed extensively, resuspended in fresh 2 mM K+
Krebs-Ringer’s medium, and incubated for 2 min with 2 or 30
mM K+ (final concentration). Drugs and 1 µM histamine were
added 5 min before the depolarization stimulus. Incubations
were stopped by rapid centrifugation, and [3H]histamine levels
were determined after purification by liquid scintillation
spectrometry. Ki values were determined according to the
Cheng-Prusoff equation.26 The data presented are given as
mean values with standard error of the mean (SEM) for a
minimum of three separate determinations each.
Ack n ow led gm en t. The authors gratefully thank N.
Pelloux-Le´on, S. Athmani, and T. S. J ensen for the
preparation of some compounds. This work was sup-
ported by the Biomedical & Health Research Pro-
gramme (BIOMED) of the European Union and the
Fonds der Chemischen Industrie, Verband der Chemi-
schen Industrie, Frankfurt/Main, Germany.
Hista m in e H3-Recep tor An ta gon ist P oten cy in Vivo in
Mice. In vivo testing was performed after peroral administra-
tion to Swiss mice as described by Garbarg et al.19 Brain
histamine turnover was assessed by measuring the level of
the main metabolite of histamine, Nτ-methylhistamine. Mice
were fasted for 24 h before treatment. Animals were decapi-
tated 90 min after treatment, and the cerebral cortex was
prepared out. The cortex was homogenized in 10 vol of ice-
cold perchloric acid (0.4 M). The Nτ-methylhistamine level was
measured by radioimmunoassay.27 By treatment with 10 mg/
kg of thioperamide the maximal Nτ-methylhistamine level was
obtained and related to the level reached with the adminis-
tered drug, and the ED50 value was calculated as mean values
with SEM.28
Hista m in e-N-m eth yltr a n sfer a se Activity. Histamine-N-
methyltransferase (HMT) activity was quantified by measur-
ing the conversion of histamine into [3H]Nτ-methylhistamine
by using S-[3H]adenosylmethionine (SAM) as a [3H]methyl
donor. HMT was purified from rat kidney according to the
method of Bowsher et al.29 with slight modifications.30 Hista-
mine (1 µM final concentration) was incubated with HMT and
SAM (20 µM final concentration) alone or together with
different concentrations of drugs for 20 min at 37 °C. The
reaction was stopped by addition of perchloric acid (0.4 N final
concentration) followed by sodium hydroxide (1 N final con-
centration). [3H]Nτ-Methylhistamine was extracted into tolu-
ene-isoamyl alcohol (3:2) and quantified by liquid scintillation
spectrometry.19 The IC50 value for each drug tested was
calculated from the inhibition curve of the Nτ-methylhistamine
formation obtained with increasing drug concentrations.
Refer en ces
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Ciproxifan, a Novel Potent Histamine H3-Receptor Antagonist.
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Kin etic Stu d y w ith Cip r oxifa n (14) in Mon k eys. Mon-
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the concentration of ciproxifan was determined after centrifu-

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