Orally active fibrinogen receptor antagonists. 2. Amidoximes as prodrugs of amidines

Article abstract of DOI:10.1021/jm9509298

The potent and selective GP IIb-IIIa antagonist lamifiban (1, Ro 44- 9883) is currently in clinical development as an injectable antithrombotic agent for treating and preventing acute coronary syndromes. However, for secondary prevention of thrombotic occlusions, orally active inhibitors are needed. By means of a prodrug strategy, the modest oral absorption of 1 in mice was improved by a factor of 9. In addition, these studies demonstrated that an amidoxime group can serve as a prodrug functionality for an amidino group. Application of this principle to the structurally related amidino carboxylate 13 led to the amidoxime ester 18 which was absorbed approximately 20 times better, after oral administration to mice, than 13. Due to the modification of the amidino group as well as of the carboxylate group, 18 completely lost its ability to interact with purified platelet GP IIb-IIIa. After oral administration of 18 to rats, dogs, and rhesus monkeys, the bioavailability of the active derivative 13 was 26 ± 5, 25 ± 6, and 33 ± 6%, respectively, and the elimination half-life was 4.1 ± 1.7, 11.4 ± 1.1, and 5.1 ± 1.4 h, respectively. On the basis of these properties, the orally active 18 (Ro 48-3657), a double prodrug of the potent and selective non- peptide GP IIb-IIIa antagonist 13 (Ro 44-3888), was selected as clinical candidate for evaluation as a prophylactic agent in patients at high risk for arterial thrombosis.

Full text of DOI:10.1021/jm9509298

J . Med. Chem. 1996, 39, 3139-3147
3139
Or a lly Active F ibr in ogen Recep tor An ta gon ists. 2. Am id oxim es a s P r od r u gs of
Am id in es†
Thomas Weller,*,§ Leo Alig,§ Maureen Beresini,‡ Brent Blackburn,‡ Stuart Bunting,‡ Paul Hadva´ry,§
Marianne Hu¨rzeler Mu¨ller,§ Dietmar Knopp,§ Bernard Levet-Trafit,§ M. Terry Lipari,‡ Nishit B. Modi,‡
Marcel Mu¨ller,§ Canio J . Refino,‡ Monique Schmitt,§ Peter Scho¨nholzer,§ Sabine Weiss,§ and Beat Steiner§
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland, and Genentech Inc.,
460 Point San Bruno Boulevard, South San Francisco, California 94080
Received December 20, 1995X
The potent and selective GP IIb-IIIa antagonist lamifiban (1, Ro 44-9883) is currently in clinical
development as an injectable antithrombotic agent for treating and preventing acute coronary
syndromes. However, for secondary prevention of thrombotic occlusions, orally active inhibitors
are needed. By means of a prodrug strategy, the modest oral absorption of 1 in mice was
improved by a factor of 9. In addition, these studies demonstrated that an amidoxime group
can serve as a prodrug functionality for an amidino group. Application of this principle to the
structurally related amidino carboxylate 13 led to the amidoxime ester 18 which was absorbed
approximately 20 times better, after oral administration to mice, than 13. Due to the
modification of the amidino group as well as of the carboxylate group, 18 completely lost its
ability to interact with purified platelet GP IIb-IIIa. After oral administration of 18 to rats,
dogs, and rhesus monkeys, the bioavailability of the active derivative 13 was 26 ( 5, 25 ( 6,
and 33 ( 6%, respectively, and the elimination half-life was 4.1 ( 1.7, 11.4 ( 1.1, and 5.1( 1.4
h, respectively. On the basis of these properties, the orally active 18 (Ro 48-3657), a double
prodrug of the potent and selective non-peptide GP IIb-IIIa antagonist 13 (Ro 44-3888), was
selected as clinical candidate for evaluation as a prophylactic agent in patients at high risk for
arterial thrombosis.
In tr od u ction
fragment of the anti-GP IIb-IIIa monoclonal antibody
7E3 (c7E3) in high-risk angioplasty patients in a phase
III clinical trial.18,19
Human blood platelets play an important role not only
in normal hemostasis but also in arterial thrombosis,
particularly under conditions of high shear stress typical
of narrowed atherosclerotic arteries.1-3 The deposition
of platelets on thrombogenic surfaces such as ruptured
atherosclerotic plaques followed by the formation of
occlusive platelet rich aggregates are crucial events
leading to disorders such as unstable angina, myocardial
infarction, transient ischemic attacks, and stroke.4,5 The
interaction of tissue-bound von Willebrand factor (vWF)
with glycoprotein (GP) Ib-IX on the platelet surface not
only is responsible for the adhesion of platelets to
exposed subendothelium but also triggers the generation
of activation signals.6-8 As a consequence, the glyco-
protein IIb-IIIa complex (GP IIb-IIIa) becomes compe-
tent to bind adhesive proteins such as fibrinogen, vWF,
or fibronectin present in plasma. In this way, the
platelets are cross-linked and thrombus growth is
initiated.9 Due to the release and formation of a variety
of platelet activators (e.g., ADP, thrombin, collagen,
serotonin, thromboxane A2, epinephrine), additional
platelets are activated and recruited to the growing
thrombus.10-13 The efficacy of existing antiplatelet
agents, such as aspirin which inhibits only one agonist
pathway, is limited.14-16 It is expected that the higher
intrinsic potency of GP IIb-IIIa antagonists compared
to aspirin will translate into better clinical efficacy in
preventing arterial thrombus formation.14,17 Indeed,
this has been demonstrated with the chimeric Fab
In the meantime, a large number of potent and
selective GP IIb-IIIa antagonists, including snake venom
polypeptides and linear and cyclic peptides, as well as
non-peptide inhibitors have been identified.20-32 Most
of these agents seem to mimic the Arg-Gly-Asp (RGD)
recognition sequence present in various ligands of
receptors of the integrin family.33 The usefulness of
several of these low molecular weight GP IIb-IIIa
antagonists, e.g., integrelin,34,35 MK-383,36 or lamifiban
(Ro 44-9883, 1),37 as intravenous treatments of acute
thrombosis is currently being assessed in clinical trials.
However, for the chronic treatment of patients with
recurrent vascular events, compounds with a sufficiently
high bioavailability and a reasonably long half-life in
plasma after oral administration are needed. The
amidinodicarboxylate 238 is an early example of a
potent, selective non-peptide GP IIb-IIIa antagonist that
exhibits oral activity in dogs.39 However, the absolute
† Abbreviations: ADP, adenosine 5′-diphosphate; AUC, area under
the concentration-time curve; EtOEt, diethyl ether; FG, fibrinogen;
GP, glycoprotein; h-PRP, human platelet rich plasma; iv, intravenous;
po, per os; THF, tetrahydrofuran.
* Corresponding author. E-mail: thomas.weller@roche.com@internet.
‡ Genentech Inc.
§ F. Hoffmann-La Roche Ltd.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0022-2623(95)00929-0 CCC: $12.00 © 1996 American Chemical Society

3140 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Sch em e 1. Preparation of Prodrug Derivatives of 1a
Weller et al.
Sch em e 2. Preparation of Prodrug Derivatives of 2a
a
(a) 2-Propanol, H2SO4; (b) EtOCOCl, NaOH, CH2Cl2; (c)
NH2OH‚HCl, Na, MeOH.
Sch em e 3. Preparation of Prodrug Derivatives of 13a
a
(a) EtOH, H2SO4; (b) EtOCOCl, NaOH, CH2Cl2; (c) 2-propanol,
H2SO4; (d) Ac2O, K2CO3; (e) 4-NH2C(NH)C6H4COCl, pyridine; (f)
HCOOH; (g) 4-NCC6H4COCl, NaHCO3, CH2Cl2; (h) NH2OH‚HCl,
Na, MeOH.
bioavailability of 2 is not high enough for oral use in
humans. Recent reports describing orally active GP IIb-
IIIa antagonists40-51 have prompted us to disclose our
prodrug approach to increase the oral bioavailability of
1, 2, and related compounds. One of these is the orally
active alanine derivative 18 (Ro 48-3657) which has
been selected as a clinical candidate. The results
demonstrate that an amidoxime group can serve as a
prodrug functionality for an amidino group.
a
(a) EtOH, H2SO4; (b) n-BuOCOCl, NaOH, CH2Cl2; (c)
4-NCC6H4COCl, NaHCO3, CH2Cl2; (d) NH2OH‚HCl, Na, CH3OH;
(e) HCOOH; (f) 1-iodoethyl isopropyl carbonate, dicyclohexylamine,
DMF.
Ch em istr y
The target compounds indicated in Tables 1-3 were
synthesized as outlined in Schemes 1-3. Starting with
the amidino carboxylate 138 (Scheme 1), straightforward
esterification and ethoxycarbonylation52,53 protocols were
used for the preparation of 3-5. In analogy to the X-ray
structure of 2-amino-N-benzoylbenzamidine,54 all acyl-
ated benzamidines described in this report are shown
in the Z-(acylimino)amine form rather than in the
tautomeric (acylamino)imine form. The tyrosine deriva-
tive 6, obtained by coupling of N-Z-Tyr-OH with tert-
butyl (4-piperidinyloxy)acetate followed by catalytic
hydrogenation as described earlier,38 was acylated with
4-amidinobenzoyl chloride and subsequently with ethoxy-
carbonyl chloride. Before deprotection to 7 with formic
acid, the side product 7a was removed by chromatog-
raphy. Acylation of the same tyrosine derivative 6 with
4-cyanobenzoyl chloride followed by treatment with
hydroxylamine hydrochloride in the presence of sodium
methoxide55,56 and transesterification yielded the ami-
doxime derivative 8. On the basis of the X-ray structure
determined for 18 (see below) as well as for pyridine
2-amidoxime,57 all amidoximes discussed in this report
are shown in the Z-form.
F igu r e 1. Stereorepresentation of the molecular structure of
18 (Ro 48-3657) as derived from the X-ray analysis.
The amidino carboxylate 1338 and the alanine deriva-
tive 1638 (Scheme 3) were converted to the correspond-
ing prodrugs 14 and 15 as well as 17 and 18, respec-
tively, using the same method as outlined above. As
already mentioned, the X-ray structure of 18 (Figure
1) shows that the amidoxime function is Z-configurated
around the C-N double bond. In addition, in the
crystalline state the oxyacetic acid side chain prefers
an axial position at the piperidine ring. Finally, alkyl-
ation of the amidoxime acid 17 with 1-iodoethyl isopro-
pyl carbonate58 yielded the ester 19 as a mixture of
diastereoisomers.
Using the amidinodicarboxylate 238 and the nitrile
derivative 1138 (Scheme 2), similar standard transfor-
mations as described above furnished the target com-
pounds 9, 10, and 12.

Orally Active Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3141
Ta ble 1. Prodrug Derivatives of 1
Resu lts a n d Discu ssion
Assa y System s. For assessing the in vitro activity
of the compounds, inhibition of platelet aggregation in
human platelet rich plasma (h-PRP) induced by ADP
as well as inhibition of fibrinogen binding to immobilized
GP IIb-IIIa (integrin RIIbâ3) was used as described
earlier.38 The oral activity of the compounds was
initially determined in mice.
Per experiment, a given compound was orally admin-
istered to two mice. The first animal was sacrificed
after 1 h and the second after 3 h, and plasma was
prepared. Since GP IIb-IIIa antagonists are generally
very species selective, the inhibitory potency of the mice
plasma was assessed in a hybrid bioassay. The mouse
plasma was mixed with h-PRP, and the extent of ADP-
induced platelet aggregation was determined. From the
IC50 value (µL of plasma/400 µL), the oral dose (ID50)
was calculated that would result in 50% inhibition of
human platelet aggregation. The ID50 values presented
in Tables 1-3 were calculated from experiments, where
about 100 mg/kg of a given compound was orally
administered. The ID50 values determined from the ‘1
h mice’ and the ‘3h mice’ were pooled since no significant
differences between the two groups were detected
among the compounds presented in Tables 1-3. For
selected compounds with low ID50 values, i.e., with good
oral activity in mice, we investigated whether the ID50
was dependent on the dose administered. Compounds
15, 18, and 19 were, in addition to 100 mg/kg, also dosed
at 10 and 1 mg/kg. For all three compounds the ID50
values derived from the 1 mg/kg experiments were only
about 1.5-fold lower than those determined from the 100
or 10 mg/kg experiments. For example, compound 18
showed an ID50 of 0.25 ( 0.04 and 0.17 ( 0.02 mg/kg
for the experiments dosed at 100 and 1 mg/kg, respec-
tively. Thus, the ID50 was largely independent of the
dose administered.
inhibtn of oral activity in
inhibtn of
platelet
mice ID50
(mg/kg)c
mean (
SEM (n)
FG binding to aggregation
GP IIb-IIIa (h-PRP/ADP)
R1
R2
R3
IC50 (nM)a
IC50 (nM)b
1 H
3 H
H
H
H
Et
Et
1.4
51
>8000
>10 000
90
33
63
8.5 ( 0.8 (12)
6.9 ( 1.1 (4)
1.4 ( 0.4 (4)
0.9 ( 0.3 (4)
4 EtOCO H
5 EtOCO CH3CO i-Pr
7 EtOCO H
8 OH
39 800
>100 000
1950 10.7 ( 2.8 (2)
>100 000
H
Et
H
>10 000
2.6 ( 0.7 (5)
a
IC50 values were calculated with
a
log linear regression
analysis. The mean value of at least two independent experiments
is shown. In examples with more than five independent experi-
ments, the SD was always lower than (25% of the mean.
b
Inhibition of ADP-induced platelet aggregation was determined
in h-PRP of at least two donors. In examples with more than five
determinations, the SD was always lower than (20% of the mean.
c The indicated ID50 values represent means ( SEM. Each ID50
value was calculated as described in the Experimental Section.
The ID50 values indicate the oral dose (mg/kg) administered to
mice that, when their plasma is tested in the hybrid bioassay,
results in 50% inhibition of human platelet aggregation.
Ta ble 2. Prodrug Derivatives of 2
oral
P r od r u g Der iva tives of 1 a n d 2. As shown by the
ID50 values given in Tables 1 and 2, the potent fibrino-
gen receptor antagonists 1 and 2 exhibited similar oral
activities in mice. In a rat experiment, the absolute
bioavailability F (F (in %) ) [AUCpo]/[AUCiv] × [doseiv]/
[dosepo] × 100) of 1 was estimated to be about 1%
(results not shown). To improve the oral bioavailability
of compounds such as 1 or 2, several strategies might
be pursued as follows: (i) synthesis of structurally
related novel scaffolds, (ii) replacement of polar and/or
charged functional groups (e.g., amidino and/or carbox-
ylate function), or (iii) modification of polar and/or
charged functional groups by means of a prodrug
strategy. This approach would also protect the gas-
trointestinal tract from exposure to high concentrations
of a potent platelet inhibitor, provided the prodrug is
not a competent GP IIb-IIIa antagonist. Application of
a prodrug strategy to 1 seemed especially attractive
since this compound is already in clinial trials for
intravenous administration.37 Esterification of the car-
boxylate group in 1 did not yield a substantial increase
in oral activity in mice (3, Table 1). Only the simulta-
neous masking of the amidino function by an ethoxy-
carbonyl group to give the uncharged derivative 4
inhibtn
of platelet
FG binding to aggregation
activity in
mice ID50
(mg/kg)c
mean (
inhibtn of
GP IIb-IIIa
(h-PRP/ADP)
R1
R2
IC50 (nM)a
IC50 (nM)b
SEM (n)
2
9
H
H
H
i-Pr
0.4
900
>10 000
>10 000
51
345
>100 000
>100 000
11.2 ( 1.8 (4)
11.0 ( 2.1 (4)
1.4 ( 0.4 (5)
0.7 ( 0.2 (6)
10 EtOCO i-Pr
12 OH CH3
a-c See corresponding footnotes in Table 1.
phenolic hydroxyl group of 4 to give the triple prodrug
5 which is devoid of any in vitro activity. Interestingly,
modification of the amidino group alone, as demon-
strated in 7, did not result in a significant increase in
oral activity in mice.
Although completely inactive in inhibiting FG binding
to GP IIb-IIIa as well as in inhibiting platelet aggrega-
tion, the amidoxime ethyl ester 8, which might be
considered as intermediate in the synthesis of com-
pounds like 3,40 exhibited oral activity in the mouse
assay. Obviously, the double prodrug 8 was absorbed
and converted to an active compound in vivo, most
probably to the amidino acid 1. The metabolic reduction
of benzamidoxime derivatives to benzamidines, in vivo
as well as in vitro, is well documented.59-62 The pKa
values of benzamidoxime,63 i.e., pKa1 approximately 13
(deprotonation) and pKa2 approximately 5 (protonation),
resulted in the desired increase of oral activity.
A
similar observation was reported for the GP IIb-IIIa
antagonist BIBU 52 and its double prodrug BIBU 104.44
Further improvement was possible by acetylation of the

3142 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Ta ble 3. Prodrug Derivatives of 13
Weller et al.
oral
inhibtn of
FG binding of platelet
to aggregation
inhibtn
activity in
mice ID50
(mg/kg)c
mean (
GP IIb-IIIa (h-PRP/ADP)
R1
R2
IC50 (nM)a IC50 (nM)b
SEM (n)
13 H
14 H
15 n-BuOCO Et
17 OH
18 OH
H
Et
1.9
163
38 5.2 ( 1.5 (10)
500 1.8 ( 0.4 (6)
>100 000 0.30 ( 0.05 (6)
13 800 5.1 ( 1.6 (6)
>100 000 0.25 ( 0.04 (6)
>10 000
H
Et
594
>10 000
OH
19
>10 000
>60 000 0.34 ( 0.10 (5)
a-c See corresponding footnotes in Table 1.
F igu r e 2. Plasma levels of 13 upon iv and po administration
of 13 and 18, respectively, and plasma levels of 17 upon po
administration of 18 versus time as determined by HPLC in
dogs. Data are mean ( SD.
indicate that 8 remains uncharged over a wide pH
range. This property which also characterizes com-
pounds 4 and 5 might enhance oral absorption.
Ta ble 4. Pharmacokinetic Parameters of 13 after Oral
Administration of 18
On the basis of the results shown in Table 1, the
prodrug concept was then applied to the amidino
dicarboxylate 2 (Table 2). Again, only the modification
of the amidino group and the carboxylate groups to give
10 or 12 was successful, while after esterification of 2
to 9 no improvement of the oral activity in mice was
observed. Contrary to the derivatives of 1 (Table 1), the
amidoxime dimethyl ester 12 is superior to the other
derivatives shown in Table 2 with respect to the oral
activity in mice.
species (n)
rat (3)
dog (4)
rhesus monkey (5)
dose (mg/kg)
t
1/2â (h)a,b
F (%)a,c
4
2
1
4.1 ( 1.7
26 ( 5
25 ( 6
33 ( 6
11.4 ( 1.1
5.1 ( 1.4
a
Data are mean ( SD. b The apparent elimination half-life (t1/2â
)
was determined from the linear portion of the log plasma
concentration-time profile. c The bioavailability (F) of 13 following
po administration of 18 was calculated accounting for the differ-
ences in molecular weights and doses.
P r od r u g Der iva tives of 13. We then focused our
attention on prodrugs of the alanine derivative 13 (Table
3), a highly potent and selective FG receptor antagonist
structurally related to 1 but with a lower molecular
weight.38 According to the ID50 (Table 3) 13 showed
better oral activity as compared to 1. Following the
trend already discussed above, the ethyl ester 14
exhibited a 3 times higher oral activity than 13 in the
mouse model. A further 6-7-fold gain in oral activity
was again observed with the (n-butoxycarbonyl)amidino
derivative 15 as well as with the amidoxime ethyl ester
18, while the amidoxime acid 17 showed about the same
activity after oral administration as the amidino acid
13. Replacement of the ethyl ester in 18 by an [(iso-
propoxycarbonyl)oxy]ethyl ester to give 19 (as a mixture
of diastereoisomers) did not lead to a further improve-
ment of oral activity in mice. Therefore, this compound
was not further investigated. Since the bioavailability
of 13 after oral administration of 15 to rats (F ) 9.8%,
n ) 2), dogs (F ) 10.1%, n ) 3), and rhesus monkeys (F
) 4.0%, n ) 2) was clearly lower as compared to 18,
the pharmacokinetics of the latter compound were
characterized in more detail. The results are sum-
marized in Table 4.
(rat, dog, and selected monkey samples; detection limit
2.5 ng/mL) or by a solid phase FG binding assay
(monkey samples; detection limit 0.5 ng/mL).
After oral administration of 18 in solution at a dose
of 4 mg/kg to male rats (n ) 3), the active drug 13 as
well as the amidoxime acid 17 were detected in plasma.
While 13 showed a relatively long elimination half-life
(Table 4), 17 disappeared more rapidly (t1/2â ) 0.35 (
0.025 h, mean ( SD). It was gratifying to see that 18
was absorbed and metabolized to the active drug 13,
suggesting that the antiplatelet activity seen in the
mouse assay of 18 was due to in vivo metabolism to 13.
The metabolic consequence of in vivo production of the
amidoxime acid 17 remains to be elucidated. Despite
the two metabolic steps involved, the interindividual
variability of the plasma concentrations of 13 was low
(in eight rats dosed at 5 mg/kg the coefficient of
variation, CV, of AUC(13) was e10%). Compared to the
tyrosine derivative 1, a more than 25-fold increase of
the bioavailability in rats was achieved.
When compound 18 was given orally to fasted male
beagle dogs as 25 mg tablets, a pharmacokinetic pattern
similar to that described above for rats was observed.
As depicted in Figure 2, peak plasma levels of 17 were
significantly higher and reached earlier than peak
plasma levels of 13. Again, the half-life of 13 was
significantly longer (Table 4) than that of 17 (t1/2â ) 0.96
( 0.07 h, mean ( SD).
Oral bioavailability F was calculated by comparing
the individual AUCs of 13 after oral administration of
the double prodrug 18 with the mean value after
intravenous administration of the active drug 13, i.e.,
F (in %) ) [AUC(18)po]/[AUC (13)iv] × [dose(13)iv]/[dose-
(18)po] × 100, where the oral dose was corrected by the
corresponding molecular weights of 13 and 18. The
plasma concentrations were determined either by HPLC
Comparison of the AUCs in rhesus monkeys after
intravenous or oral administration of 13 or 18, respec-
tively, is shown in Figure 3. After administration of 18

Orally Active Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3143
IIIa antagonist 13 (Ro 44-3888), was selected as a
clinical candidate for evaluation as a prophylactic agent
in patients at high risk for arterial thrombosis.
Exp er im en ta l Section
Ch em istr y. Reagent grade solvents were used without
further purification. Evaporation implies the removal of
solvent by use of a Bu¨chi rotary evaporator at 40-50 °C in
vacuo. All organic extracts were dried over Na2SO4 or MgSO4.
Normal phase silica gel used for flash chromatography (FC)
was Kieselgel-60 (230-400 mesh); reverse phase silica gel used
was Lichroprep RP 18 (40-63 µm); both were supplied by E.
Merck, A.G., Darmstadt, Germany. TLC plates coated with
silica gel 60 F254 (Merck) were used; detection by UV light (254
nm) or I2 vapor was applied. Melting points were determined
with a Bu¨chi 510 or Electrothermal 9100 apparatus and are
uncorrected. Proton NMR spectra (NMR) were recorded on a
Bruker AC 250 spectrometer; δ values in ppm relative to
tetramethylsilane are given. IR spectra (IR) were recorded
with the compound (neat) on a sodium chloride disk or as KBr
pellets using a Nicolet 7199-FT-IR spectrometer. Optical
rotations, [R]20D, were determined with a Perkin Elmer 241
polarimeter, c in g/100 mL. Mass spectra (MS) were recorded
with an instrument of type API III from Perkin Elmer Sciex
using the pneumatically assisted electrospray technique.
Results of elemental analysis were within 0.4% of the theoreti-
cal values unless otherwise noted.
F igu r e 3. Plasma levels of 13 upon iv and po administration
of 13 and 18, respectively, versus time as determined by
bioassay in rhesus monkeys. Data are mean ( SD.
as either a pressed tablet (n ) 2) or an enteric coated
capsule (n ) 3), no significant differences in the plasma
concentrations were seen. Therefore, the data from the
two groups were pooled (Table 4). Compared to the
experiments in dogs, the half-life of 13 was shorter while
its bioavailability was higher in this primate model of
oral activity. In addition to solid phase FG binding
assay, plasma samples from those animals dosed with
18 formulated as a pressed tablet were also analyzed
by reverse phase HPLC (Supporting Information). The
concentrations of 13 determined by these two indepen-
dent methods are highly correlatable (slope ) 0.989, R
) 0.94). Since the enantiomer of 13 is 10-fold less
potent than 13,38 these data suggest that 18, upon
absorption and metabolism to 13, is stereochemically
stable in vivo. HPLC analysis also indicated the pres-
ence of 17 having a similar plasma disposition seen in
the rats and dogs described above.
(S)-[[1-[2-[[4-(Am in oim in om et h yl)b en zoyl]a m in o]-1-
oxop r op yl]-4-p ip er id in yl]oxy]a cetic Acid Eth yl Ester
(14) (Sta n d a r d p r oced u r e A). To a solution of the amidino
acid 1338 (4.85 g, 12.9 mmol) in EtOH (50 mL) was added
concentrated sulfuric acid (3 mL). After 14 h at 23 °C, the
solvents were removed in vacuo to a volume of about 10 mL,
water was added (25 mL), and the resulting solution was
neutralized by careful addition of solid NaHCO3 in portions.
After evaporation of the solvents, the residue was passed
through silica gel RP-18 (180 g, H2O/EtOH, 0-5%). The pure
fractions were combined and the solvents evaporated, and the
residue was triturated with EtOH to give the hemisulfate salt
of 14 (4.39 g, 75%): mp 196-198 °C; [R]20 ) +36.5° (c ) 0.6,
D
EtOH); NMR (DMSO-d6) δ 1.19 and 1.20 (2 t, J ) 7 Hz, 3 H,
rotamers), 1.30 (d, J ) 5 Hz, 3 H), 1.40 (m, 2 H), 1.85 (m, 2
H), 2.92-3.45 (m, 2 H), 3.52-3.88 (m, 3 H), 4.10 (m, 2 H), 4.14
(2 s, 2 H, rotamers), 4.98 (m, 1 H), 7.88 (part of AA’BB’, 2 H),
8.10 (part of AA’BB’, 2 H), 8.92 (2 d, J ) 7 Hz, NH, rotamers),
9.94 (bs, 2 × NH2); MS m/ z (FAB) 405 (M + H)+. Anal.
(C20H28N4O5‚0.5 H2SO4) C, H, N.
Oral administration of 18 to dogs and rhesus monkeys
had no effect on complete blood cell counts. Further-
more, iv administration of 13 or po administration of
18 to rhesus monkeys had no effect on heart rate or
mean arterial pressure.
Compounds 3 and 9 were prepared from 138 and 2,38
respectively, according to standard procedure A using the
corresponding alcohols.
(Z)-(S)-[[1-[2-[[4-[Am in o[(n -b u t oxyca r b on yl)im in o]-
m eth yl]ben zoyl]a m in o]-1-oxop r op yl]-4-p ip er id in yl]oxy]-
a cetic Acid Eth yl Ester (15) (Sta n d a r d p r oced u r e B). A
solution of the hemisulfate salt of 14 (3.71 g, 8.2 mmol) in
water (100 mL) was neutralized by addition of solid NaHCO3
in portions at 5 °C. After addition of CH2Cl2 (100 mL), the
reaction mixture was treated with n-butyl chloroformate (2.05
g, 15 mmol). With vigorous stirring aqueous NaOH (0.5 N,
40 mL) was added dropwise within 1 h. After transferring
the reaction mixture to a separatory funnel, the layers were
separated. The organic extracts were washed with water and
dried and the solvents evaporated. After chromatography
(silica gel, EtOAc) 15 (2.3 g, 56%) was obtained as a colorless
Con clu sion s
In summary, by pursuing a prodrug approach we were
able to improve the oral bioavailability of potent and
selective GP IIb-IIIa antagonists such as 13 by a factor
of approximately 20. With double prodrugs like the
amidoxime ethyl ester 18, we have demonstrated that
an amidoxime group can serve as a prodrug functional-
ity for an amidino group. Although the mechanistic
details for this reduction remain to be elucidated, these
findings might be of interest in those cases where highly
basic, charged amidino or guanidino groups have to be
replaced by neutral surrogates. The similar bioavail-
ability of 13 after oral administration of 18 in rats, dogs,
and rhesus monkeys suggests the absence of a species
dependent conversion of the double prodrug to the active
moiety. In addition, a favorable elimination half-life of
13 after oral administration of 18 was observed in the
same three animal species. On the basis of these
properties, the orally active 18 (Ro 48-3657), a double
prodrug of the potent and selective non-peptide GP IIb-
foam: [R]20 ) +36.2° (c ) 1, EtOH); NMR (CDCl3) δ 0.95 (t,
D
J ) 7 Hz, 3 H), 1.30 (t, J ) 7 Hz, 3 H), 1.35-1.50 (m, 2 H),
1.44 (d, J ) 7 Hz, 3 H), 1.60-2.00 (m, 6 H), 3.29-4.00 (m, 5
H), 4.14 (bs, 2 H), 4.23 (2 q, J ) 7 Hz, 4 H), 5.06 (m, 1 H), 7.56
(m, NH), 7.89 (AA’BB’ system, 4 H), 8.17 and 9.90 (2 bs, NH2);
MS m/ z (EI) 505 (M + H)+. Anal. (C25H36N4O7‚1.25 C4H8O2)
C, H, N.
Compounds 4 and 10 were prepared from 3 and 9, respec-
tively, according to standard procedure B using ethyl chloro-
formate.
(Z)-(S)-[[1-[2-[[4-[Am in o[(eth oxyca r bon yl)im in o]m eth -
yl]ben zoyl]a m in o]-3-(4-a cetoxyp h en yl)p r op ion yl]p ip er i-

3144 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Weller et al.
d in -4-yl]oxy]a cetic Acid Isop r op yl Ester (5). Using stan-
dard procedure A (with 2-propanol) and standard procedure
B (with ethyl chloroformate) 1 (4.68 g, 10 mmol) was converted
to (Z)-(S)-[[1-[2-[[4-[amino[(ethoxycarbonyl)imino]methyl]ben-
zoyl]amino]-3-(4-hydroxyphenyl)propionyl]piperidin-4-yl]oxy]-
acetic acid isopropyl ester (1.20 g, 21% overall) as a colorless
foam: MS m/ z (FAB) 583 (M + H)+.
To a solution of this material (291 mg, 0.5 mmol) in acetone
(10 mL) was added acetic anhydride (102 mg, 1 mmol). After
40 min at 23 °C, solid K2CO3 (173 mg, 1.25 mmol) was added.
After 1 h the volume was reduced to 2 mL and the residue
distributed between water and EtOAc. The organic extracts
were washed with water and dried and the solvents evapo-
rated. The residue was purified by chromatography (silica gel,
EtOAc) to give 5 (155 mg, 50%) as a colorless foam: NMR
(CDCl3) δ 1.24 and 1.25 (2 t, J ) 7 Hz, 6 H), 1.37 (t, J ) 7 Hz,
3 H), 1.45-1.90 (m, 4 H), 2.27 and 2.28 (2 s, 3 H, rotamers),
2.90-3.34 (m, 3 H), 3.40-3.80 (m, 4 H), 4.02 and 4.05 (2 bs, 2
H, rotamers), 4.24 (q, J ) 7 Hz, 2 H), 5.07 (m, 1 H), 5.34 (m,
1 H), 7.01 (m, 2 H), 7.17-7.32 (m, 3 H), 7.80 (m, 2 H), 7.91
(m, 2 H), 9.70 (bs, NH2); MS m/ z (FAB) 625 (M + H)+. Anal.
(C32H40N4O9‚1.2 C4H8O2) H, C: calcd, 60.52; found, 59.55. N:
calcd, 7.68; found, 8.22.
cm-1; NMR (DMSO-d6) δ 1.18 and 1.19 (2 t, J ) 7 Hz, 3 H,
rotamers), 1.20-1.80 (m, 4 H), 2.75-3.98 (m, 7 H), 4.09 (m, 2
H), 4.11 (bs, 2 H), 5.04 (m, 1 H), 5.89 (bs, NH2), 6.63 (part of
AA’BB’, 2 H), 7.07 (part of AA’BB’, 2 H), 7.73 (part of AA’BB’,
2 H), 7.83 (part of AA’BB’, 2 H), 8.74 (m, 1 H), 9.17 (bs, OH),
9.80 (bs, OH); MS m/ z (FAB) 513 (M + H)+. Anal.
(C26H32N4O7) H, C: calcd, 60.93; found, 60.52. N: calcd, 10.93;
found, 10.37.
Compound 1138 was converted to the corresponding ami-
doxime 12 according to standard procedure C.
(Z)-(S)-[[1-[2-[[4-[Am in o(h ydr oxyim in o)m eth yl]ben zoyl]-
a m in o]-1-oxop r op yl]-4-p ip er id in yl]oxy]a cetic Acid (17).
The amine 1338 (43 g, 0.15 mol) in CH2Cl2/saturated NaHCO3
(1:1, 600 mL) was acylated with 4-cyanobenzoyl chloride (26
g, 0.16 mol) as described earlier38 to give (S)-[[1-[2-[(4-cy-
anobenzoyl)amino]-1-oxopropyl]-4-piperidinyl]oxy]acetic acid
tert-butyl ester (50 g, 80%): mp 134-137 °C (from EtOEt);
[R]20D ) +39.6° (c ) 1.0, EtOH); MS m/ z (EI) 359 (M - C4H8)+.
This material (50 g, 0.12 mol) was converted to (Z)-(S)-[[1-
[2-[[4-[amino(hydroxyimino)methyl]benzoyl]amino]-1-oxopro-
pyl]-4-piperidinyl]oxy]acetic acid tert-butyl ester according to
standard procedure C (46 g, 85%): mp 193-194 °C (from
MeOH); MS m/ z (FAB) 449 (M + H)+.
(Z)-(S)-[[1-[2-[[4-[Am in o[(eth oxyca r bon yl)im in o]m eth -
yl]b en zoyl]a m in o]-3-(4-h yd r oxyp h en yl)p r op ion yl]p ip -
er id in -4-yl]oxy]a cetic Acid (7). The amine 6 was acylated
with 4-amidinobenzoyl chloride as described earlier38 to give
(S)-[[1-[2-[[4-(aminoiminomethyl)benzoyl]amino]-3-(4-hy-
droxyphenyl)propionyl]piperidin-4-yl]oxy]acetic acid tert-butyl
ester: MS m/ z (FAB) 525 (M + H)+.
This material (787 mg, 1.5 mmol) was treated with ethyl
chloroformate (216 mg, 2 mmol) according to standard proce-
dure B to give, after chromatography (silica gel, CH2Cl2/MeOH,
49:1), 7a (170 mg, 17%; mp 72-73 °C; MS m/ z (FAB) 669 (M
+ H)+) together with the desired monoacylated product (180
mg, 20%) which was dissolved in formic acid (5 mL) and heated
to 50 °C. After 1 h the solvents were removed by evaporation,
and the residue was crystallized upon addition of CH3CN to
give 7 (115 mg, 71%): mp 134-135 °C; NMR (DMSO-d6) δ
1.22 (t, J ) 7 Hz, 3 H), 1.20-1.80 (m, 4 H), 2.75-3.98 (m, 7
H), 4.07 (q, J ) 7 Hz, 2 H), 4.11 (s, 2 H), 5.08 (m, 1 H), 6.64
(part of AA’BB’, 2 H), 7.07 (part of AA’BB’, 2 H), 7.90 (part of
AA’BB’, 2 H), 8.02 (part of AA’BB’, 2 H), 8.85 (m, 1 H), 9.20
(bs, NH2, 2 OH); MS m/ z (FAB) 539 (M - H)-. Anal.
(C27H32N4O8) H, N, C: calcd, 59.99; found, 59.27.
(Z)-(S)-[[1-[2-[[4-[Am in o(h ydr oxyim in o)m eth yl]ben zoyl]-
am in o]-3-(4-h ydr oxyph en yl)pr opion yl]piper idin -4-yl]oxy]-
a cetic Acid Eth yl Ester (8). The amine 6 (3.65 g, 9.64 mmol)
in CH2Cl2/saturated NaHCO3 (1:1, 140 mL) was acylated with
4-cyanobenzoyl chloride (1.75 g, 10.6 mmol) as described
earlier38 to give (S)-[[1-[2-[(4-cyanobenzoyl)amino]-3-(4-hy-
droxyphenyl)propionyl]piperidin-4-yl]oxy]acetic acid tert-butyl
ester as a colorless foam (3.10 g, 63%): IR (KBr) 2231, 1746,
1628 cm-1; MS m/ z (FAB) 508 (M + H)+.
Sta n d a r d p r oced u r e C: To a solution of Na (124 mg, 5.4
mmol) in MeOH (100mL) were successively added NH2OH‚
HCl (392 mg, 5.63 mmol) and (S)-[[1-[2-[(4-cyanobenzoyl)-
amino]-3-(4-hydroxyphenyl)propionyl]piperidin-4-yl]oxy]ace-
tic acid tert-butyl ester (1.90 g, 3.75 mmol). After 14 h at 23
°C, the solvents were evaporated, and the residue was purified
by chromatography (silica gel, CH2Cl2/CH3OH, 15:1) to give
(Z)-(S)-[[1-[2-[[4-[amino(hydroxyimino)methyl]benzoyl]amino]-
3-(4-hydroxyphenyl)propionyl]piperidin-4-yl]oxy]acetic acid tert-
butyl ester as a colorless foam (930 mg, 46%): MS m/ z (FAB)
541 (M + H)+.
This material (46 g, 0.10 mol) in formic acid (150 mL) was
heated to 80 °C. After 3 h the solvents were evaporated, and
the residue was crystallized upon addition of EtOAc (150 mL).
The resulting suspension was filtered; the filter cake was
washed with EtOAc (50 mL) and dried in vacuo to give 17 (34.5
g, 86%): mp 176-178 °C; [R]20D ) +44.2° (c ) 1.0, H2O); NMR
(DMSO-d6) δ 1.27 (bd, J ) 5 Hz, 3 H), 1.43 (m, 2 H), 1.85 (m,
2 H), 2.95-3.45 (m, 3 H), 3.55-3.80 (m, 2 H), 4.04 (2 s, 2 H,
rotamers), 4.96 (m, 1 H), 5.90 (bs, NH2), 7.75 (part of AA’BB’,
2 H), 7.88 (part of AA’BB’, 2 H), 8.62 (m, NH, rotamers), 9.79
(bs, N-OH), 12.59 (bs, COOH); MS m/ z (FAB) 391 (M - H)-.
Anal. (C18H24N4O6) C, H, N.
(Z)-(S)-[[1-[2-[[4-[Am in o(h ydr oxyim in o)m eth yl)ben zoyl]-
a m in o]-1-oxop r op yl]-4-p ip er id in yl]oxy]a cetic Acid Eth yl
Ester (18). Concentrated sulfuric acid (15 mL) was added
with ice cooling to a suspension of 17 (19.6 g, 50 mmol) in
EtOH (300 mL). After removal of the ice bath, the solution
was stirred for 14 h at 23 °C. Subsequently, the solvents were
evaporated, and the residue was dissolved in water (300 mL).
At ice bath temperature the solution was neutralized upon
addition of an aqueous solution (28%) of sodium hydroxide.
The precipitates were collected by filtration, washed with
water, and recrystallized from EtOH to give 18 (11.7 g, 56%):
mp 201-203 °C; [R]20D ) +75.3o (c ) 0.9, AcOH); NMR (DMSO-
d6) δ 1.19 (2 t, J ) 7 Hz, 3 H, rotamers), 1.29 (bd, J ) 6 Hz,
3 H, rotamers), 1.42 (m, 2 H), 1.82 (m, 2 H), 2.90-3.33 (m, 2
H), 3.52-4.04 (m, 3 H), 4.09 (m, 4 H), 4.94 (m, 1 H), 5.90 (bs,
NH2), 7.75 (part of AA’BB’, 2 H), 7.88 (part of AA’BB’, 2 H),
8.68 (m, NH, rotamers), 9.80 (bs, N-OH); MS m/ z (FAB) 421
(M + H)+. Anal. (C20H28N4O6) C, H, N.
X-r a y Cr ysta llogr a p h ic Str u ctu r e An a lysis of 18. From
EtOH, crystals of 18 (C20H28N4O6, formula weight 420.46)
formed in the monoclinic space group P21. A representative
crystal was surveyed, and a total of 1634 reflections with 2θ
less than 113.5° were measured at 173 K on a Siemens P4/
RA, M18XHF diffractometer using monochromatic copper
radiation. The structure was solved by direct methods using
the SHELXS-86 package; refinement was facilitated by the
SHELXL-9364 system. Molecules/unit cell Z ) 2; cell dimen-
sions a ) 11.390(4) Å, b ) 5.386(2) Å, c ) 16.793(7) Å, â )
90.04(3)°; calculated density 1.355 g cm-3. Full-matrix least-
squares refinement was conducted with anisotropic temper-
ature factors for all atoms except hydrogen which were
included at calculated positions with isotropic temperature
factors. The final R-factor of 0.066 was obtained for 1542
observed reflections (I > 2σ(I) ); largest difference peak and
hole were 0.61 and -0.35 eÅ-3, respectively. Coordinates,
anisotropic temperature factors, bond lengths, and angles are
available as Supporting Information and will be deposited at
the Cambridge Crystallographic Data Centre.
A solution of this material (930 mg, 1.72 mmol) in formic
acid (20 mL) was stirred at 23 °C. After 5 h the solvents were
evaporated; the residue was washed with water, evaporated,
and triturated with EtOEt to give (Z)-(S)-[[1-[2-[[4-[amino-
(hydroxyimino)methyl]benzoyl]amino]-3-(4-hydroxyphenyl)pro-
pionyl]piperidin-4-yl]oxy]acetic acid as an amorphous solid
(800 mg, 96%): MS m/ z (FAB) 485 (M + H)+.
This material (350 mg, 0.72 mmol) was esterified in EtOH
(50 mL) in the presence of concentrated sulfuric acid according
to standard procedure A to give, after trituration with EtOEt,
8 (120 mg, 33%): mp 107-110 °C; IR (KBr) 3369, 1746, 1627
(Z)-(S)-[[1-[2-[[4-[Am in o(h ydr oxyim in o)m eth yl]ben zoyl]-
a m in o]-1-oxop r op yl]-4-p ip er id in yl]oxy]a cetic Acid (R/S)-
1-[(Isop r op oxyca r bon yl)oxy]eth yl Ester (19). To a solu-

Orally Active Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3145
tion of 17 (780 mg, 2 mmol) in N,N-dimethylformamide (20
mL) were added at 0 °C dicyclohexylamine (400 mg, 2.2 mmol)
and 1-iodoethyl isopropyl carbonate58 (620 mg, 2.4 mmol). After
14 h at 23 °C, the solvents were evaporated in vacuo at 60 °C.
The residue was dissolved in EtOAc, and the resulting solution
was washed twice with water, dried, and evaporated. After
chromatography (silica gel, EtOAc) and crystallization (EtOEt),
19 was obtained as colorless powder (220 mg, 21%): mp 113
°C dec; NMR (CDCl3) δ 1.32 (d, J ) 7 Hz, 6 H), 1.47 (d, J ) 6
Hz, 3 H), 1.54 (2 d, J ) 6 Hz, 3 H, diastereomers), 1.58-1.98
(m, 4 H), 3.35-4.05 (m, 5 H), 4.15 and 4.16 (2 s, 2 H), 4.86 (m,
3 H), 5.26 (m, 1 H), 6.82 (2 q, J ) 6 Hz, 1 H, diastereomers),
7.38 (part of AA’BB’, 2 H), 7.61 (part of AA’BB’, 2 H), 8.42 (m,
1 H), 8.98 (bs, 1 H); MS m/ z (FAB) 523 (M + H)+. Anal.
(C24H34N4O9) C, H, N.
plasma was transferred to fresh tubes and stored at -70 °C.
Prior to assay, plasma samples were thawed and filtered
through Microcon-10 microconcentrators (Amicon Inc., Beverly,
MA). The protein poor filtrate was analyzed for anti-GP IIb-
IIIa activity using the solid phase fibrinogen binding assay
as described earlier.38 The compound 13 (Ro 44-3888) was
used to generate the assay standard curve, and test results
are expressed in ng/mL of 13 (Ro 44-3888). The lower limit of
detection in this assay system was approximately 0.5 ng/mL.
The extraction procedure was validated by recovery studies
from spiked whole blood. The half-life of 13 was determined
using TopFit.65 The half-life was estimated using the last four
data points, and the bioavailability of 13 following po admin-
istration of 18 was calculated accounting for the differences
in the molecular weights and doses.
P h a r m a cology. In Vitr o Assa y System s. The inhibition
of platelet aggregation in h-PRP induced by ADP as well as
the inhibition of fibrinogen binding to immobilized GP IIb-
IIIa (integrin RIIbâ3) was used as described earlier.38
Ack n ow led gm en t. We would like to express our
thanks to the following talented and enthusiastic
people: Anita Allemann, J ulie Badillo, Markus Haas,
Marie-Paule Imhoff, Anh Le, Martine Singer, Sabine
Sokoll, Corinne Stucki, Sonja Villard, and Gabriella
Widmer-Pfenninger for skillful technical assistance;
Andre´ Bubendorf for the IR spectra; J osiane Kohler for
optical rotations; Wolf Arnold for the NMR spectra;
Walter Meister for the mass spectra; and Stephan
Mu¨ller for the microanalyses.
Assessm en t of Or a l Activity in Mice. Mice were used
for the first assessment of the oral activity of each compound.
Per experiment, about 100 mg/kg of a given compound,
suspended in 10% gelatin (particle size < 1 µm), was orally
administered to two mice. The first animal was sacrificed after
1 h and the second after 3 h to prepare plasma. Since GP
IIb-IIIa antagonists are generally very species selective, ex vivo
aggregation in mouse PRP could not be used to determine the
amount of the active constituent(s) in the plasma. The
inhibitory potency of these mice plasma samples was therefore
assessed in a hybrid bioassay. Serial dilutions of each mouse
plasma (100 µL) were mixed with human PRP (300 µL), and
platelet aggregation was induced by ADP (10 µM final con-
centration). The volume of plasma required to inhibit 50% of
aggregation was determined (IC50). From the IC50 value (µL
of plasma/400 µL), the dilution factor for each mouse plasma
was determined. The orally administered dose (mg/kg) was
then divided by this dilution factor to calculate the oral dose
(ID50) that results in 50% inhibition of ADP-induced aggrega-
tion in human PRP.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and characterization data (NMR spectra, mass spectra,
IR spectra, melting points, and microanalytical) for all com-
pounds contained in Schemes 1-3 as well as in Tables 1-3
which do not appear in the Experimental Section and crystal-
lographic data (coordinates, anisotropic temperature factors,
bond lengths, and angles) of 18 (6 pages). Ordering informa-
tion is given on any current masthead page.
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