Enzyme-catalyzed prodrug approaches for the histamine H3-receptor agonist (R)-α-methylhistamine

Article abstract of DOI:10.1016/S0968-0896(00)00237-6

Five novel prodrug types of the potent and selective histamine H₃-receptor agonist (R)-α-methylhistamine (1) were prepared and pharmacologically tested in vitro as well as in vivo. In particular, an amide of fatty acid, mono- and dicarbamates, an (acyloxy)alkylcarbamate, and a diphthalidyl derivative were synthesized, all of which require initial prodrug activation through an enzyme-catalyzed reaction in contrast to formerly developed azomethine prodrugs which are cleaved by chemical hydrolysis only. Further drug liberation may ensue spontaneously in a cascade to give 1. Since they have diverse stabilities the prodrugs were investigated for drug liberation in vitro under neutral, acidic, and basic conditions at different temperatures as well as with liver homogenates. In vivo investigation of prodrugs after oral administration to mice proved that the fatty amide 2, the N^α-methylcarbamate 4a, and the N^α-(1-(acetyloxy)ethylcarbamate) 5 showed moderate to high plasma levels of 1. Compound 5 displayed even more than 2.5 times the AUC for 1 than that of the reference azomethine prodrug BP2.94 in the periphery and also displayed a detectable drug level in the central nervous system. It was shown that prodrug approaches based on an initial enzyme-catalyzed liberation step are successfully applicable to different pro-moieties for improved bioavailability and prolonged half-live. These approaches may also be used for other aminergic compounds of this class to optimize pharmacokinetic behavior.

Full text of DOI:10.1016/S0968-0896(00)00237-6

Bioorganic & Medicinal Chemistry 9 (2001) 191±198
Enzyme-Catalyzed Prodrug Approaches for the Histamine
H -Receptor Agonist (R)-ꢀ-Methylhistamine
3
a,
a
b
b
Holger Stark, * Michael Krause, Agne
Á
b
s Rouleau, Monique Garbarg,
a
Jean-Charles Schwartz and Walter Schunack
a
Institut fuÈr Pharmazie, Freie UniversitaÈt Berlin, KoÈnigin-Luise-Strasse 2+4, 14195 Berlin, Germany
b
Unit e de Neurobiologie et Pharmacologie Mol e culaire (U. 109), Centre Paul Broca de l'INSERM,
ter rue d'AleÂsia, 75014 Paris, France
2
Received 30 June 2000; accepted 25 August 2000
AbstractÐFive novel prodrug types of the potent and selective histamine H -receptor agonist (R)-a-methylhistamine (1) were pre-
3
pared and pharmacologically tested in vitro as well as in vivo. In particular, an amide of fatty acid, mono- and dicarbamates, an
acyloxy)alkylcarbamate, and a diphthalidyl derivative were synthesized, all of which require initial prodrug activation through an
(
enzyme-catalyzed reaction in contrast to formerly developed azomethine prodrugs which are cleaved by chemical hydrolysis only.
Further drug liberation may ensue spontaneously in a cascade to give 1. Since they have diverse stabilities the prodrugs were investi-
gated for drug liberation in vitro under neutral, acidic, and basic conditions at di€erent temperatures as well as with liver homo-
a
genates. In vivo investigation of prodrugs after oral administration to mice proved that the fatty amide 2, the N -methylcarbamate 4a,
a
and the N -(1-(acetyloxy)ethylcarbamate) 5 showed moderate to high plasma levels of 1. Compound 5 displayed even more than 2.5
times the AUC for 1 than that of the reference azomethine prodrug BP2.94 in the periphery and also displayed a detectable drug level
in the central nervous system. It was shown that prodrug approaches based on an initial enzyme-catalyzed liberation step are suc-
cessfully applicable to di€erent pro-moieties for improved bioavailability and prolonged half-live. These approaches may also be used
for other aminergic compounds of this class to optimize pharmacokinetic behavior. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
strong antisecretory and anti-in¯ammatory properties8,9
of H -receptor agonists on sensory C-®bers and entero-
3
The classi®cation of histamine receptors by classical
pharmacology as well as by molecular biology approaches
has well de®ned three di€erent receptor subtypes named
chroman-like cells, potential clinical targets are
0,11
ulcers,1
myocardial ischemia. In addition, H -receptor agonists
bowl disorders, asthma bronchiale as well as
8
12
13
3
1
14
15
H , H , and H . Whereas in the beginning of 1950 his-
1
display sedative and potential antimigraine actions.
2
3
tamine was mainly associated with allergic reactions
H ) and then, from 1972 onward, expanded to mainly
H -receptor-mediated reactions on gastric acid secre-
(
The reference agonist used most frequently is (R)-a-
methylhistamine (1). This hydrophilic compound is a
1
1
2
tion, nowadays it is clear that histamine is also a neu-
2
chiral-methylated derivative of the endogenous ligand
histamine. Despite its good pharmacodynamic proper-
ties, insucient oral absorption, poor brain penetration,
and rapid inactivation in man by histamine N-methyl-
rotransmitter. Especially the histamine H receptor has
3
been found in the central nervous system (CNS) and in
the periphery to be an important regulator of histamine
2
8
neuron activity as well as a modulator of release of other
3
transferase (E.C. 2.1.1.8) limit its clinical use. Most
histamine H -receptor agonists are 4-monosubstituted
4
neurotransmitters, e.g. dopamine, serotonin, noradrena-
5
3
6
7
line, acetylcholine, and various neuropeptides. Many
pharmacological investigations on the physiological and
pathophysiological functions of H receptors in CNS
imidazole derivatives having in a certain distance a basic
moiety which is mainly an aliphatic amino functionality.
Speci®c rigidisation by side-chain branching or ring
incorporation increases anity and selectivity in some
3
and peripheral tissues have been made possible by the
availability of potent and selective agonists, and di€erent
therapeutic applications have been suggested. Due to
1
6
cases as described among many others for immepyr,
Sch 50971,17 immepip, and imifuramine (Chart 1).
18
19
These structure elements are not restricted to agonists
only. The partial agonist impentamine also possesses a
comparable structure.20 These structural similarities
*
5
Corresponding author. Tel.: +49-30-838-53928; fax: +49-30-838-
3854; e-mail: stark@schunet.pharmazie.fu-berlin.de
0968-0896/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00237-6

1
92
H. Stark et al. / Bioorg. Med. Chem. 9 (2001) 191±198
nists.25,26 Both these di€erent approaches are promising
ways to overcome the problems with the hydrophilic
agonists.
To provide a more side-speci®c alternative to azomethine
prodrugs which merely undergo straightforward chemi-
cal hydrolysis, di€erent enzyme-dependent prodrug
approaches applicable to primary and secondary alipha-
tic aminergic compounds were developed with 1. In par-
ticular, amide, carbamate, and ester derivatives were
prepared having 1 as active agonist element, which was
chosen to allow direct comparison with formerly devel-
oped azomethine prodrugs and to use the elaborated
analytics. In all compounds modi®cation of the amino
group of 1 is likely to inhibit ®rst pass inactivation by
histamine N-methyltransferase as in the case for the
azomethine prodrugs.8
Prodrug conception is now widely accepted and applied
in drug development.27 Due to the ubiquitous presence
of unspeci®c esterases ester derivatives are most promi-
nent with prodrugs. Monoesters of steroids, antibiotics,
neuroleptics etc. are often used as prodrugs with retard
e€ects. Multiple esters like (acyloxy)alkylcarbonates in
Chart 1.
1
potentially led to comparable problems with these
ligands concerning bioavailability, blood±brain barrier
penetration, and metabolism. Azomethine prodrugs of 1
have been described to overcome these pharmacokinetic
problems having BP2.94 as parent compound currently
cefpodoxime-proxetil (Orelox ) are modern develop-
2
8
ments of prodrugs with improved pharmacokinetics.
Despite these developments and the general statement
prodrug generation has been successfully performed in
most cases on hydroxy functionalities, but only to a
much smaller extend on amine moieties. Progabide, an
azomethine derivative related to BP2.94, is an example of
an aminergic prodrug introduced to therapy, but mean-
while also withdrawn.29 To make a broad prodrug
8
,21�24
in phase II for clinical development.
Furthermore,
novel (partial) agonists have been described most
recently to display full intrinsic activity in vivo devoid of
the basic structural similarity described for former ago-
Figure 1. Proposed mechanisms of prodrug activations. Arrows: bold, mainly enzymatic process; regular, spontaneous decomposition; swinging,
bond cleavage. Compounds in brackets are unstable and were not characterized.

H. Stark et al. / Bioorg. Med. Chem. 9 (2001) 191±198
193
approach di€erent structures were developed which
should include di€erent bioactivation steps mainly
depending on amidases and esterases. Derivatization
was performed on the aliphatic amino group as well as
on the basic imidazole nucleus for some compounds.
acterization and preparation of the related diacylated
product were omitted. Dialkylation of 1 with two
equivalents of 3-bromophthalide under similar reaction
conditions resulted in low yields of 6 (Fig. 2). Attempts
for monoalkylation with one equivalent led to an unse-
parable mixture of products.
For the preparation of the (acyloxy)alkylcarbamate 5
4
Results and Discussion
-nitrophenol was reacted with a-chloroethyl chloro-
By conception carboxylic amide 2, carbamates 3,4, (acyl-
oxy)alkylcarbamate 5, and diphthalidyl derivative 6
should ®rstly undergo enzymatic hydrolysis by unspeci®c
esterases or amidases depending on their pro-moieties.
After enzymatic hydrolysis the next step of decomposi-
tion for release of 1 should take place spontaneously as
shown in Figure 1.
formate to the carbonate intermediate, which then
underwent nucleophilic substitution in acetic acid by
means of Hg(acetate) (Fig. 3). Having 4-nitrophenol
2
as an excellent leaving group the aminolysis with 1 can
be performed under mild reaction conditions.
30
Pharmacology
Alkyl carbamates are supposed to be more stable than
In vitro investigations. All prodrugs were investigated
for their ability to release the active drug 1 following
incubation under acidic conditions and in presence of
liver homogenates, since the latters are presumed to
mimic to a certain extent conditions under which drug
liberation presumably occurs in vivo. Additional experi-
ments were performed at increased temperature or other
3
0
aryl carbamates. This was the case with the carbamate
derivatives in this series (cf. 4a,b versus 4c). In related
carbamate series with an ``inverse'' functionality in his-
tamine H -receptor antagonists possessing longer alkyl
3
or aromatic substituents, these compounds were found
3
1
to be stable under in vivo conditions. Therefore, only
methyl and ethyl derivatives were prepared since these
moieties are supposed to be easily cleaved in vivo by
enzymes. Another approach exploiting side-branched
alkyl carbamates like t-butoxycarbonyl moieties, com-
monly used as protective groups in peptide chemistry,
was unsuccessful with histamine presumably due to
early decomposition in the acidic medium of the sto-
mach (results not shown). (Acyloxy)alkylcarbamates are
presumably less stable than corresponding carbamates
due to an additional obliged-fracture-structure in the
3
0
acylated hemiacetal. Recently this prodrug conception
was successfully applied to guanidine derivatives, too.32
Phthalidyl derivatives possess a potential liberation cas-
cade related to the (acyloxy)alkylcarbamates without
having a carbamate moiety. Ester hydrolysis liberates the
hemiaminal structure thereupon undergoing unpromp-
ted hydrolysis to amine and aldehyde (Fig. 1). The imi-
dazole nucleus of the thyreotropine-releasing hormone
Figure 2. Synthesis of amide (2), carbamate (3, 4), and phthalidyl
prodrugs (6). (a) Et
temp, 2±3 h. (b) Et
3
N, EtOH or MeOH, EtOAc or MeCN, ambient
ꢀ
3
N, MeCN, 65 C, 7 h.
(
leading to reduced metabolisation and prolonged half-
TRH) was derived recently by an analogous reaction
3
3
live.
Chemistry
Five di€erent classes of potential prodrugs have been
prepared in this study. Acylation of 1 with equimolar
amounts of activated carbonyl compound, i.e. palmitoyl
chloride, alkyl chloroformate, or aryl chloroformate,
a
mainly led to N -monoacylated products due to higher
nucleophilicity of the primary amine compared to the
a
t
imidazole nucleus (Fig. 2). N ,N -Diacylation took place
only to a minor extend, and puri®cation from these by-
products was easily done by salt formation. The com-
parable reaction with two equivalents led to the desired
a
t
N ,N -dicarbamates 3a, 3b (Fig. 2). (4-Nitrophenoxy)-
carbonyl prodrug 4c decomposed on isolation by stan-
dard methods, so that, due to high instability and
therefore expected missing prodrug properties, char-
Figure 3. Synthesis of 1-(acetyloxy)ethylcarbamate prodrug 5. (a) Pyri-
ꢀ
dine, CHCl
temp, 18h. (c) 1, HMPT, ambient temp, 18 h.
3
, 0 C!ambient temp, 18 h. (b) Hg(AcO)
2
, AcOH, ambient

1
94
H. Stark et al. / Bioorg. Med. Chem. 9 (2001) 191±198
Table 1. In vitro hydrolysis [%] of prodrugs under various experimental conditions
Conditions
Time (min)
0
BP 2.94a
2
3a
3b
4a
4b
5
6
ꢀ
Neutral medium, 20 C
6.0
1.4
1.7
1.8
1.7
1.9
13.6
12.4
9.8
12.1
10.8
9.5
4.8
4.8
4.8
4.8
4.8
0.5
0.5
0.7
0.5
0.5
1
3
6
5
0
0
6.0
6.5
15
11.5
13.1
1
20
10.0
ꢀ
Acidic medium, 20 C
0
3.5
4.0
5.6
8.7
1.7
1.6
1.9
1.8
2.0
0.6
0.7
0.8
0.7
0.7
0.7
0.8
0.9
0.7
0.8
11.0
13.1
11.7
15.0
14.9
11.3
11.3
12.8
11.2
12.3
4.6
4.6
5.7
8.2
8.6
0.8
0.5
0.8
1.5
0.8
1
3
6
5
0
0
1
20
14.1
ꢀ
Acidic medium, 95 C
15
87
94
100
2.5
2.9
4.1
0.6
0.6
0.9
0.6
0.6
5.3
14.2
16.5
17.9
12.3
11.8
16.0
85
100
100
1.1
0.8
1.8
3
6
0
0
ꢀ
Alkaline medium, 20 C
0
5
0
0
1.9
1.8
2.3
2.1
43.8
47
47
47.5
0.9
1.2
1.1
1.0
1
3
6
ꢀ
Liver homogenate, 37 C
0
5
0
0
3.7
6.7
10.4
19.5
27.3
1.6
2.1
2.4
2.0
2.2
1.0
0.6
0.6
0.6
0.6
1.0
0.6
0.8
0.7
0.9
13.1
10.4
8.9
6.2
11.3
12.5
11.5
6.5
8.6
7.0
23.4
67.3
70.8
73.8
76.3
0.6
0.3
0.4
0.6
0.6
1
3
6
1
20
aRef. 8.
incubation media to assess the stability of prodrugs.
8
tested concerning the presumed enzyme-catalyzed drug
liberation. The reason for these disappointing in vitro
results are not clear. Further investigations are necessary
to clarify the stability of these compounds, especially in
liver homogenate.
Data obtained from BP2.94 are shown in Tables 1±3
for comparison.
Little liberation of 1 could be found for any compound
under incubation in neutral medium and enzyme-free
conditions; in presence of the liver extract liberation
kinetic was found for compound 5 only (Tables 1 and 2).
Slight time-dependent prodrug decomposition could be
observed for 2, 3a, 3b, 4b, and 6 under drastic acidic
In vivo investigations. Whatever the in vitro results, all
compounds were investigated in vivo after oral applica-
tion with regard to prodrug activation measured by
released 1 in plasma as well as CNS (Table 3).
ꢀ
conditions at 95 C. Under these conditions, compound
a
t
a
5 was totally hydrolyzed to 1 within 30min. The in vitro
results strongly supported the hypothesis of bioactivation
of 5, but gave disappointing results for the other prodrugs
As expected the N ,N -dicarbamates 3a,b, the N -
a
t
monoethylcarbamate 4b, and the N ,N -diphthalidyl
derivative 6 did not show any drug liberation neither in
plasma nor in the CNS. The N -monomethoxycarbonyl
derivative 4a showed a slight drug level in vivo, but the
total detectable amount of 9 ng h mL is presumably too
small to be considered as pharmacologically relevant.
a
�
1
Table 2. In vitro hydrolysis [% h 1] of prodrugs under various
�
experimental conditions
Conditions
BP2.94a
2
3a 3b 4a 4b
5
6
Surprisingly, the palmityl amide derivative 2 showed
good prodrug properties. Although the maximal con-
centration is much lower than that of the azomethine
BP2.94 the total amount (AUC) is comparable to that
of BP2.94 due to prolonged drug release (Fig. 4). This
result shows that the in vitro models were not able to
ꢀ
Neutral medium, 20 C
ꢀ
4.5 0.3
5.3 0.2 0.1 0.1 2.0 0.5
100
0
0.1
0
2
0
0
1
Acidic medium, 20 C
ꢀ
Acidic medium, 95 C
Alkaline medium, 20 C
2.4 0.3 4.6 6.9 4.7 100
0.2
11.8 0.3
ꢀ
1.9 0.1
26.5
ꢀ
Liver homogenate, 37 C
0
0
0
0
0
aRef. 8.
�
1
Table 3. In vivo data of 1 after p.o. administration of 24 mmol kg of prodrug to mice
Tissue
BP2.94a
2
3a
3b
4a
4b
5
6
�
n.d.b
n.d.
n.d.
n.d.
n.d.
Plasma
c
max [ng mL 1
]
115Æ9
30
186
n.d.
n.d.
73Æ8
30
169
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
18Æ3
30
9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
308Æ17
30
482
n.d.
n.d.
n.d.
n.d.
n.d.
t
AUC [ng h mL
max [min]
c
� 1
]
�
1
Cerebral cortex
c
max [ng mL
]
9.4Æ2.4
AUC [ng h mL 1]
�
15.9
a
Ref. 8..
n.d., not detectable.
AUC, area under the curve.
b
c

H. Stark et al. / Bioorg. Med. Chem. 9 (2001) 191±198
195
predict all in vivo actions. It has also to be considered
that 1 was undetectable in the CNS raising hopes that
all pharmacological results obtained by this prodrug in
derivative of palmitic acid 2, the mono- or dicarbamates
(3±4), and the diphthalidyl derivative 6 were devoid of
any hydrolysis kinetic. Despite this stability in vitro the
monomethylcarbamate 4a and the amide 2 showed
moderate to promising prodrug properties in vivo after
oral application to mice. The palmityl derivative 2 pos-
sessed AUC levels comparable to those of the parent
azomethine prodrug BP2.94. The most promising com-
pound was 5, which is supposed to release 1 by a cas-
cade reaction with an initial enzyme-induced hydrolysis.
Maximum concentration and the AUC obtained were
more than 2.5 times higher than that of the azomethine
BP2.94 with an equimolar dose tested. Drug levels could
even be detected in CNS. Amides and (acyloxy)alkyl-
carbamates of the type described are promising pro-
drugs of 1 and are also interesting leads for further
development of aminergic compounds with supposed
peripheral activity.
future will be mediated by peripheral H receptors only.
3
Furthermore, amide derivatives of fatty acids are a new
lead for further prodrug development with aminergic
compounds like 1. One might speculate that these com-
pounds are substrates for amidases after previous bio-
transformation, e.g. o-oxidation, what would explain
their failure in short time in vitro tests.
The most promising results are obtained from the Na-
(
1-(acetyloxy)ethylcarbamate) 5. By oral administration
�
1
of this prodrug in a dose of 24 mmol kg high levels of 1
were readily detectable in plasma and were sustained for
more than 6 h (Fig. 4). The AUC is about 2.5 times
higher than that obtained with BP2.94 proving a high
absorption rate. For a related prodrug, cefpodoxime-
proxetil, an enzymatic cleavage by esterases is discussed
to take place already during the passage of the gastro-
2
8,34
intestinal mucosa.
A comparable mechanism for 5
Experimental
Chemistry
could explain the high plasma level of 1. Reasonable
drug concentrations are also found in the CNS, and its
AUC is even higher than that obtained with compound
4
1
a in plasma (Table 3). The detectable concentration of
in CNS could be related to the extremely high plasma
General procedures. Melting points were determined on
È
an Electrothermal IA 9000 digital or a Buchi 512 appa-
1
levels. The relation of peripheral to central AUCs
supports the hypothesis that prodrugs of the
ratus and are uncorrected. For all compounds H NMR
spectra were recorded on a Bruker DPX 400 Avance
(400 MHz) spectrometer. Chemical shifts are expressed
(
acyloxy)alkylcarbamate type are acting more strongly
1
at peripheral than at central targets. This type of pro-
drugs is the most promising approach in this series
which does not seem to be restricted to the primary ali-
phatic amine group of 1, but can also be applied to
other compounds containing primary or secondary
amine functionalities.
in ppm down®eld from internal TMS as reference. H
NMR data are reported in the following order: multi-
plicity (br, broad; s, singlet; d, doublet; dd, double doub-
let; t, triplet; q, quintett; m, multiplet; *, exchangeable by
D O; ** exchangeable by F CCOOD; Im, imidazolyl;
2
3
Mal, maleic acid; Me, methyl; Ph, phenyl), number of
protons, and approximate coupling constants in hertz
(
Hz). Mass spectra were obtained on a Finnigan MAT
CH7A (EI±MS) and a Finnigan MAT CH5DF (FAB±
MS). Optical rotation was determined on a Perkin±Elmer
241 MC. Elemental analyses (C, H, N) for all compounds
were measured on Perkin±Elmer 240 B or Perkin±Elmer
240 C instruments and were within Æ0.4% of theoretical
values. Column chromatography was carried out using
silica gel 63±200 mm (Machery & Nagel). Preparative,
centrifugally accelerated, rotatory chromatography was
performed using a Chromatotron 7924T (Harrison
Research) and glass rotors with 4 mm layers of silica gel
Conclusions
Five di€erent types of prodrugs for the histamine H3-
receptor agonist (R)-a-methylhistamine (1) have been
described as its N -position or its N ,N -position were
derivatized simultaneously under mild reaction condi-
tions. In vitro testing under di€erent conditions for the
1-(acetyloxy)ethylcarbamate 5 showed marked lability
under enzymatic environment, whereas the amide
a
a
t
60 PF254 containing gypsum (Merck). For analytical data
on mp. and elemental analyses see Table 4.
Synthesis of prodrugs
Amide of fatty acid
(
R)-(� )-N-(1-(1H-Imidazol-4-yl)-2-propyl)hexadecanoic
amide (2). A solution of palmitoyl chloride (1.6 mmol,
.44 g) in 20 mL of MeCN was slowly added to 1
1.6 mmol, 0.2 g) and Et N (1.6 mmol, 0.16 g) in 20 mL
0
(
3
of EtOH and 5 mL of MeCN. After stirring for 1 h at
ambient temp the reaction mixture was concentrated to
1
was diluted with CHCl , washed (K CO , H O), dried
(MgSO ), and evaporated to dryness. Compound 2 was
4
0 mL under reduced pressure. The mixture obtained
Figure 4. Plasma level of released 1 after p.o. administration of 2 ( ),
�
3
2
3
2
1
4
a ( ), and 5 ( ) to mice in a dose of 24 mmol kg
.

1
96
H. Stark et al. / Bioorg. Med. Chem. 9 (2001) 191±198
Table 4. Analytical data of prodrugs 2±6
Calculated
Found
H
ꢀ
No.
Formula
MW
Mp [ C]
C
H
N
C
N
.
O C
2
C
C
C
C
C
C
C
22
10
12
H
H
H
41
15
19
N
N
N
3
3
3
4
H
4
O
4
479.7
241.3
269.3
299.3
313.3
380.4
393.9
119
79
63
104
106
86±88
163
65.1
49.8
53.5
48.2
49.8
47.4
67.1
9.46
6.27
7.11
5.73
6.11
5.83
4.99
8.76
17.4
64.9
49.8
53.4
47.8
49.8
47.7
67.1
9.39
6.33
7.31
5.77
6.21
5.68
4.84
8.84
17.5
3
3
4
4
5
6
a
b
a
b
O
O
4
4
2.
15.6
14.0
13.4
11.0
10.7
15.8
13.7
13.6
10.8
10.7
8
9
H
13
N
3
O C
4
H
H
4
O
O
4
H
15
N
3
O C
2.
4
4
4
11
H
17
N
N
3
O C
4.
4
H
4
O
4.
0.5H
2
O
22
H
19
3
O
4.
0.25H
2
O
crystallized as salt of maleic acid in EtOH/Et O (yield:
2
ing to 3a with methyl chloroformate (2 mmol, 0.19 g).
Compound 4a was crystallized as salt of maleic acid in
20
1
0
.5 g, 65%). ‰ꢀŠ =� 53.8 (c=1 g/100 mL in MeOH); H
D
20
NMR ([d ]DMSO) d 8.86 (s, 1H, Im-2-H), 7.72 (d**,
EtOH/Et O (yield: 0.4 g, 66%). ‰ꢀŠ =+9.2 (c=1 g/
6
2
D
1
J=8.2 Hz, 1H, NH), 7.32 (s, 1H, Im-5-H), 6.04 (s, 2H,
Mal), 4.06 (m, 1H, CH), 2.72 (d, J=6.8 Hz, 2H, Im-
CH ), 1.99 (t, J=7.3 Hz, 2H, CO-CH ), 1.41 (m, 2H,
100 mL in MeOH); H NMR ([d ]DMSO) d 8.90 (s, 1H,
6
Im-2-H), 7.36 (s, 1H, Im-5-H), 7.16 (d**, J=8.1 Hz, 1H,
NH-CO), 6.06 (s, 2H, Mal), 3.81 (m, 1H, CH), 3.49 (s,
2
2
CO-CH -CH ), 1.23 (m, 24H, alkyl-H), 1.05 (d, J=
2
3H, O-Me), 2.73 (d, J=6.7 Hz, 2H, Im-CH ), 1.07 (d,
2
2
6
.6 Hz, 3H, CH-Me), 0.85 (t, J=7.0 Hz, 3H, CH -Me);
2
J=6.6 Hz, 3H, CH-Me); MS (70 eV), m/z (%) 183 (1)
+
+
MS (70 eV), m/z (%) 363 (2) [M ], 256 (3), 167 (6), 108
89), 82 (100).
[M ], 140 (12), 101 (16), 86 (100), 82 (16).
(
(
R)-(+)-N-(1-(1H-Imidazol-4-yl)-2-propyl)carbamate
ethylester (4b). The synthesis was performed according
to 3b with ethyl chloroformate (2 mmol, 0.19 g). Com-
pound 4b was crystallized as salt of maleic acid in
Dicarbamates
R)-(+)-N-(1-(1-(Methoxycarbonyl)-1H-imidazol-4-yl)-
-propyl)carbamate methylester (3a). A solution of
(
2
20
EtOH/Et O (yield: 0.4 g, 63%). ‰ꢀŠ =+7.4 (c=1 g/
2
D
1
methyl chloroformate (4 mmol, 0.38 g) in 10 mL of ethyl
acetate was slowly added to 1 (2 mmol, 0.25 g) and Et N
100 mL in MeOH); H NMR ([d ]DMSO) d 8.91 (s, 1H,
6
Im-2-H), 7.35 (s, 1H, Im-5-H), 7.12 (d**, J=8.2 Hz,
NH-CO), 6.05 (s, 2H, Mal), 3.91 (q, J=7.0 Hz, 2H, O-
CH ), 3.81 (m, 1H, CH), 2.72 (d, J=6.7 Hz, 2H, Im-
2
3
(
acetate. After stirring for 3 h at ambient temp Et N hy-
4 mmol, 0.4 g) in 5 mL of MeOH and 10 mL of ethyl
.
3
drochloride was ®ltrated and the residue evaporated
under reduced pressure. The oily compound 3a was
CH ), 1.11 (t, J=7.0 Hz, 3H, O-CH -Me), 1.06 (d, J=
2
6.6 Hz, 3H, CH-Me); MS (70 eV), m/z (%) 197 (1) [M ],
154 (84), 109 (13), 86 (32), 81 (100).
2
+
crystallized as salt of maleic acid in EtOH/Et O at
2
ꢀ
�
5 C and was recrystallized in Et O (yield: 0.2 g, 41%).
2
20
1
‰
ꢀŠ =+2.3 (c=1 g/100 mL in MeOH); H NMR
D
(
Acetyloxy)alkylcarbamate
(
[d ]DMSO) d 8.17 (d, J=1.0 Hz, 1H, Im-2-H), 7.31 (s,
6
1H, Im-5-H), 7.07 (d**, J=8.0 Hz, 1H, NH), 3.95 (s,
3H, Im-COO-Me), 3.75 (m, 1H, CH), 3.49 (s, 3H, NH-
(1-Chloroethyl)-(4-nitrophenyl)carbonate.30 a-Chloroethyl
chloroformate (35 mmol, 5 g) was added slowly at 0 C
ꢀ
to 4-nitrophenol (35 mmol, 4.87 g) and pyridine
COO-Me), 2.62 (d, J=6.8 Hz, 2H, Im-CH ), 1.02 (d,
2
J=6.6 Hz, 3H, CH-Me); MS (70 eV), m/z (%) 241 (1)
+
(35 mmol, 2,77 g) in 100 mL of CHCl . After 30 min at
3
0 C the mixture was stirred at ambient temp for 18 h.
+
ꢀ
Washing of the mixture (H O, 0.5% NaOH, H O),
[
M ], 210 (4) [M � MeO], 166 (16), 140 (100), 102 (17),
81 (45).
2
2
drying (Na SO ), and evaporation to dryness resulted in
2
4
ꢀ
(R)-(+)-N-(1-(1-(Ethoxycarbonyl)-1H-imidazol-4-yl)-2-
an oil, which slowly crystallized at � 5 C. Recrystalli-
zation in ethyl acetate/petrol ether gave 81% yield (7 g),
which can be used directly for further reactions. Mp.
70±71 C; H NMR ([d ]DMSO) d 8.35 (dd, J=9.0 Hz,
J=2.8 Hz, 2H, Ph-3-H, Ph-5-H), 7.62 (dd, J=9.0 Hz,
J=2.8 Hz, 2H, Ph-2-H, Ph-6-H), 6.61 (q, J=5.8 Hz,
propyl)carbamate ethylester (3b). The synthesis was
made according to 3a with ethyl chloroformate and
EtOH instead of MeOH. Crystallization was performed
ꢀ
1
3
6
2
D
0
4
4
3
in Et O (yield: 0.24 g, 44%). ‰ꢀŠ =+1.8 (c=1 g/
2
1
1
00 mL in MeOH); H NMR ([d ]DMSO) d 8.16 (d,
6
J=1.0 Hz, 1H, Im-2-H), 7.29 (s, 1H, Im-5-H), 7.02 (d**,
J=8.1 Hz, NH), 4.39 (q, J=7.1 Hz, 2H, Im-COO-CH2),
1H, CH), 1.86 (d, J=5.7 Hz, 3H, Me); MS (70 eV), m/z
+
+
(%) 245 (1) [M ], 210 (8) [M � Cl], 166 (14), 139 (99),
122 (19), 109 (28), 93 (13), 81 (11), 76 (11); (C H
8
3.94 (q, J=7.1 Hz, 2H, NH-COO-CH ), 3.77 (m, 1H,
CH), 2.62 (d, J=6.4 Hz, 2H, Im-CH ), 1.33 (t, J=
2
9
ClNO5).
2
7.1 Hz, 3H, Im-COO-CH -Me), 1.13 (t, J=7.1 Hz, 3H,
NH-COO-CH -Me), 1.02 (d, J=6.5 Hz, 3H, CH-Me);
2
(1-(Acetoxy)ethyl)-(4-nitrophenyl)carbonate.30 The pro-
duct obtained above (28 mmol, 6.87g) in 50mL of acetic
acid was mixed with Hg(H CCOO) (34 mmol, 10.83 g)
2
+
MS (70 eV), m/z (%) 269 (2) [M ], 224 (5), 180 (17), 154
(
100), 116 (11), 81 (77).
3
2
and stirred for 18 h at ambient temp. After evaporation
to dryness the residue was dissolved in Et O, ®ltrated,
2
Monocarbamates
R)-(+)-N-(1-(1H-Imidazol-4-yl)-2-propyl)carbamate
methylester (4a). The synthesis was performed accord-
washed (H O, NaHCO , H O), dried (Na SO ), and
2
3
2
2
4
(
once more evaporated to dryness. The oil obtained
was puri®ed by column chromatography (eluent:

H. Stark et al. / Bioorg. Med. Chem. 9 (2001) 191±198
197
1
ꢀ
ꢀ
CH Cl :petrol ether; 1:1) (yield: 5.5 g, 73%); H NMR
2
raised in rabbits for 1 h at 37 C and left at 4 C over-
night after transfer in swine anti-rabbit IgG-coated 96-
well plates and addition of an [1 I]iodinated tracer. The
radioactivity bound to the wells was then counted in a
g-spectrometer with an eciency of 82%. The percen-
tage ratio of level of 1 measured in the sample and that
corresponding to complete hydrolysis of prodrug was
calculated at various times. A blank value correspond-
ing to a level of 1 at zero time incubation (and repre-
senting hydrolysis occurring during the RIA) was
determined and subtracted. The hydrolysis rate of pro-
drugs was then expressed as percent compound hydro-
lyzed per h.
2
(
CDCl ) d 8.29 (d, J=9.1 Hz, 2H, Ph-3-H, Ph-5-H),
.41 (d, J=9.1 Hz, 2H, Ph-2-H, Ph-6-H), 6.84 (q,
J=5.5 Hz, 1H, CH), 2.14 (s, 3H, CO-Me), 1.62 (d,
J=5.4 Hz, 3H, CH-Me); MS (70 eV), m/z (%) 268 (0.3)
M � H], 210 (2) [M � AcO], 181 (1), 166 (4), 139 (2),
3
25
7
+
+
[
1
22 (5), 87 (33), 43 (100); (C H NO ).
7
11
11
(
1
(
2R)-(+)-N-(1-(1H-Imidazol-4-yl)-2-propyl)carbamate
-(acetyloxy)ethylester (5). The product obtained above
1.6 mmol, 0.43 g) and 1 (1.6 mmol, 0.2 g) were dissolved
in 30 mL of HMPT and stirred for 18 h at ambient
temp. After addition of 70 mL of H O the mixture was
2
extracted with ethyl acetate. After evaporation to dry-
ness of the combined organic extracts the resulting oil
was puri®ed by preparative rotatory chromatography
Determination of 1 and prodrug levels in plasma and cer-
8
ebral cortex of mice treated with the varius prodrugs.
Male Swiss mice (20±25 g; I€a-Credo, France) that
were given food and water ad libitum received an oral
(
pound 5 was crystallized as salt of maleic acid in EtOH/
gradient eluent: CH Cl :MeOH, 95±85:5±15). Com-
2
2
2
D
0
� 1
Et O (yield: 0.2 g, 32%). ‰ꢀŠ =+28.0 (c=1 g/100 mL
dose of 24 mmol kg of prodrug in 1% methyl cellulose
2
1
in MeOH); H NMR ([d ]DMSO) d 8.97 (s, 1H, Im-2-
and were sacri®ced by decapitation 0.5, 1, 3, or 6 h later.
Controls received the vehicle only. For determination of
1 and prodrug levels in plasma and in cerebral cortex,
blood was collected after decapitation and centrifuged
(15,000 g for 1 min), and the cerebral cortex was dis-
sected out rapidly and homogenized in 10 vol (w/v) of
6
H), 7.51 (d**, J=8.3 Hz, 1H, NH-CO), 7.34 (s, 1H, Im-
5-H), 6.59 (q, J=5.4 Hz, O-CH), 6.12 (s, 2H, Mal), 3.83
(
m, 1H, CH -CH-Me), 2.74 (d, J=6.7 Hz, CH ), 1.99 (s,
2
2
3H, CO-Me), 1.36 (d, J=5.4 Hz, O-CH-Me), 1.08 (d,
J=6.6 Hz, CH -CH-Me); FAB -MS (Xe, DMSO/Gly-
+
2
+
2
+
cerin), m/z (%) 511 (1) [M +H], 256 (66) [M +H],
152 (100), 126 (15), 109 (31), 82 (24).
ice-cold 0.4 N HClO . Plasma (brought to a ®nal con-
4
centration of 0.4 N HClO ) and cerebral extracts were
4
then centrifuged, and the clear supernatant was used for
ꢀ
the RIA immediately or stored at � 20 C. Compound 1
ꢀ
ꢁ
N ,N -Diphthalidyl derivate
Æ)-(2R)-3-(N-(1-(1-(3-Phthalidyl)-1H-imidazol-4-yl)-2-
propyl)amino)phthalide (6). 3-Bromophthalide (3.5 mmol,
.21g) was added to a solution of 1 (1.7 mmol, 0.21 g)
was derivatized and radioimmunoassayed. The plasma
of nontreated mice was also assayed in order to estimate
the interference of plasma in the RIA for 1. The deter-
minations of 1 for treated mice were then corrected
accordingly. Each determination was perfomed at least
in three di€erent animals.
(
0
and Et N (3.5 mmol, 0.35 g) in 40 mL of MeCN. After
3
stirring for 7 h at 65 C the mixture was evaporated to
ꢀ
dryness. The oily residue was puri®ed by preparative
rotatory chromatography (eluent: CH Cl :MeOH, 95:5).
The product 6 was crystallized in Et O (yield: 0.135 g,
2
2
Acknowledgements
2
1
2
0%). H NMR ([d ]DMSO) d 7.98±7.38 (m, 10H, 8 Ph-
6
H, Im-2-H, Im-5-H), 6.70 (s, 1H, Im-CH), 6.50 (m*, 1H,
NH), 5.98 (m, 1H, NH-CH), 4.37 (m, 1H, CH CH),
We greatly acknowledge the gift of (R)-a-methylhistamine
kindly provided by Dr. J.-M. Lecomte (Bioprojet Pharma,
Paris, France). This work was supported by the Biomedi-
cal & Health Research Programme (BIOMED) of the
European Union and the Fonds der Chemischen
Industrie, Verband der Chemischen Industrie, Frank-
furt/Main, Germany.
2-
2
.91 (m, 2H, CH ), 1.27 (d, J=6.7 Hz, 3H, Me); MS
2
+
+
(
(
(
70 eV), m/z (%) 389 (2) [M ], 371 (5) [M � H O], 239
2
45), 214 (25), 176 (14), 159 (88), 149 (18), 133 (100), 121
16), 108 (60), 81 (29).
Pharmacology
Determination of prodrug hydrolysis rates in vitro. A
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1