Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists

Article abstract of DOI:10.1021/jm9608117

The synthesis and pharmacological evaluation of 5 (L-738,167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC₅₀ value of 8 nM and was found to be >33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by >85% 24 h after the oral administration of 5 to dogs at 100 μg/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Full text of DOI:10.1021/jm9608117

J . Med. Chem. 1997, 40, 1779-1788
1779
Non -P ep tid e Glycop r otein IIb/IIIa In h ibitor s. 17. Design a n d Syn th esis of
Or a lly Active, Lon g-Actin g Non -P ep tid e F ibr in ogen Recep tor An ta gon ists
Benny C. Askew,*,† Rodney A. Bednar,‡ Bohumil Bednar,‡ David A. Claremon,† J acquelynn J . Cook,‡
Charles J . McIntyre,† Cecila A. Hunt,† Robert J . Gould,‡ Robert J . Lynch,‡ J oseph J . Lynch, J r.,‡
Stanley L. Gaul,‡ Maria T. Stranieri,‡ Gary R. Sitko,‡ Marie A. Holahan,‡ J oan D. Glass,‡ Terrence Hamill,‡
Lynn M. Gorham,§ Thomayant Prueksaritanont,§ J ohn J . Baldwin,†,| and George D. Hartman†
Merck Research Laboratories, Departments of Medicinal Chemistry, Pharmacology, and Drug Metabolism,
West Point, Pennsylvania 19486
Received November 27, 1996X
The synthesis and pharmacological evaluation of 5 (L-738,167), a potent, selective non-peptide
fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human
gel-filtered platelets with an IC50 value of 8 nM and was found to be >33000-fold less effective
at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and
vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was
inhibited by >85% 24 h after the oral administration of 5 to dogs at 100 µg/kg. The extended
pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding
to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.
In tr od u ction
displays an extremely long pharmacodynamic profile
following intravenous or oral administration in dogs.
When a blood vessel is damaged acutely by trauma
induced by clinical interventions such as angioplasty,
or more chronically by the pathophysiological processes
of atherosclerosis, platelets become activated and adhere
to the site of injury and to each other. This activation,
adherence, and aggregation may lead to the formation
of occlusive thrombi in the vessel lumen resulting in
acute thrombotic disorders.1-4 The final obligatory step
in platelet aggregation, regardless of the activating
signal, is the binding of fibrinogen to activated glyco-
protein IIb/IIIa (GPIIb/IIIa) on the surface of activated
platelets.5-7 Cyclic peptides containing the tripeptide
sequence Arg-Gly-Asp (RGD) have been demonstrated
to be potent inhibitors of platelet aggregation;8-13
therefore, small molecules and peptidomemetics that
feature elements of this tripeptide sequence have been
pursued as inhibitors of platelet aggregation.14 Design
strategies employed by several groups use rigid central
constraints to direct the vectors of the pharmacophoric
moieties: a guanidine or guanidine surrogate mimicking
the arginyl side chain and a carboxylic acid representing
the side chain carboxylic acid of aspartic acid.15-18
Researchers at these and other laboratories have
reported that incorporation of sulfonamido or carbamoyl
moieties R to the carboxylate resulted in increased in
vitro potency and improvements in the in vivo profiles
of centrally constrained analogs.16a,17,18 Recently, we
reported that incorporation of an R-n-butanesulfona-
mide moiety on the centrally constrained pyrazolopip-
erazinone 1 to give 2 (L-734,115) resulted in a 28-fold
increase in in vitro antiaggregatory potency (Table 1)
and afforded a compound that produced profound activ-
ity following oral administration to dogs and primates.17c
In this report we describe modification of the homolo-
gous pyrazolodiazepinone 3 to provide 5 (L-738,167), a
potent, selective fibrinogen receptor antagonist that
Ch em istr y
The pyrazolopiperazinone analog 1 was prepared as
illustrated in Schemes 1 and 2. The N-terminal pip-
eridine moiety utilized in all of the analogs was derived
from commercially available alcohol 6 (Scheme 1).
Following protection of 6 with Boc2O, the resulting
alcohol was converted to the iodide 7 using conditions
described by Garegg.19 Reaction of 7 with excess NaN3
in DMSO and reduction of the resulting azide afforded
the amine 8 in 98% yield. Commercially available 3,5-
pyrazoledicarboxylic acid (9) served as the key synthon
of the bicyclic central constraints. Reaction of 9 with
methanolic HCl afforded the diester 10, which was
alkylated with excess 1,2-dibromoethane or 1-bromo-3-
chloropropane to give the alkyl halides 11a and 11b.
Reaction of 8 with 11a and saponification of the result-
ing ester afforded the pyrazolopiperazinone 12b in 49%
yield. Coupling 12b with â-alanine tert-butyl ester and
deprotection of the resulting doubly protected compound
with hydrogen chloride gas in ethyl acetate afforded 1
(Scheme 2).
Since analogous preparation of the pyrazolodiazepi-
none analog 3 was problematic due to intramolecular
cyclization of the chloride 11b to a bicyclic pyrazolium
salt,16d an alternate route to 3 was developed (Scheme
3). Reaction of 11b with excess NaN3 afforded the
corresponding azide which was converted to the pyra-
zolodiazepinone 13 in 95% yield by treatment with
palladium on carbon under a hydrogen atmosphere.
Alkylation of 13 with the iodide 7, followed by saponi-
fication, afforded the acid 14b. Conversion of 14b to 3
was carried out as illustrated in Scheme 2 for the
synthesis of 1.
Compounds 2, 4, and 5 were prepared from interme-
diates 12b and 14b as illustrated in Scheme 5. The
R-sulfonamido-â-alanines 17a and 17b, prepared from
L-asparagine as depicted in Scheme 4, were coupled with
12b and 14b without carboxylate protection using the
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
§ Department of Drug Metabolism.
| Present address: Pharmacopia, Princeton, NJ 08540.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00811-4 CCC: $14.00 © 1997 American Chemical Society

1780 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Askew et al.
Sch em e 1
a
(a) Boc2O, CH2Cl2; (b) I2, Ph3P, imidazole, toluene; (c) NaN3, DMSO; (d) H2, Pd/C, MeOH.
Sch em e 2
a
(a) HCl, MeOH; (b) Br(CH2)2Br or Br(CH2)3Cl, CH3CN, K2CO3; (c) 8, CH3CN, Et3N; (d) LiOH, THF/H2O; (e) â-alanine t-Bu ester‚HCl,
EDC, Et3N, CH2Cl2; (f) HCl, EtOAc.
Sch em e 3
a
(a)NaN3, DMSO; (b) H2, Pd/C, MeOH; (c) NaH, 7, DMF; (d) LiOH, THF/H2O.
Sch em e 4
a
(a) RSO2Cl, NaOH, dioxane/H2O; (b) Br2, NaOH, H2O.
mixed anhydride method. This protocol avoids the
racemization observed when alkyl esters of 17a were
used to prepare 2.17c The resulting analogs, 18a -c,
were treated with HCl to effect removal to the Boc
protecting group and then purified by ion exchange
chromatography to afford analytically pure 2, 4, and 5.
strained pyrazolopiperidinone 1 resulted in a 28-fold
increase in potency and afforded 2, a compound that
produced profound antiaggregatory activity following
oral administration to dogs and primates.17c Similar
modification of the homologous pyrazolodiazepinone 3
afforded 4 and 5 which also inhibited ADP mediated
aggregation of human gel-filtered platelets at low na-
nomolar concentrations (Table 1). Since the platelet
concentration in the in vitro aggregation assay is
adjusted to 2 × 108 platelets/mL and platelets from
Resu lts a n d Discu ssion
As we previously reported, incorporation of an (S)-R-
n-butanesulfonamide moiety on the centrally con-

Non-Peptide Glycoprotein IIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1781
Sch em e 5
a
(a) i-BuOCOCl, N-methylmorpholine, THF; (b) 17a or 17b, THF/H2O; (c) HCl, EtOAc.
Ta ble 1. In Vitro Potency of Analogs 1-5 and 22
plaggin IC50
(nM)a
Kd
ED50
compd
n
R
(nM)b (nM)c
1
2
3
4
5
1
1
2
2
2
H
250
9
470
10
8
590 72
1.8 0.12
680 46
1.4 0.12
1.1
600
NHSO2(CH2)3CH3
H
NHSO2(CH2)3CH3
NHSO2Ph-4-CH3
0.08
5
22
32
a
Inhibition of aggregation of human gel-filtered platelets (GFP)
was measured by a light transmittance method at 37 °C with 2 ×
108 platelets/mL, 0.1 mg/mL human fibrinogen, and
1 mM
CaCl2.16a Aggregation was initiated by adding 10 µM ADP after
all the other components were added. The rate of aggregation in
the absence of inhibitor served as the control, and values reported
are the concentration necessary to inhibit the rate of aggregation
by 50%. At least two determinations were made for each com-
pound, and values typically varied by <20%. b Equilibrium binding
constant determined for the displacement of the fluroescent ligand
19 (L-736,622) from purified resting GPIIb/IIIa. For details, see
the In Vitro Pharmacology section of the Experimental Section.
c Measured by competition with 21 ([125I]L-692,884) for binding
to purified GPIIb/IIIa activated by coating onto yttrium silicate
Scintillation Proximity Assay Fluoromicrospheres. For details, see
the In Vitro Pharmacology section of the Experimental Section.
F igu r e 1.
values were calculated from competitive binding be-
tween compounds of interest and the 125I-labeled fi-
brinogen receptor antagonist 21 ([125I]L-692,884) to
purified GPIIb/IIIa activated by coating onto yttrium
silicate scintillation proximity assay fluoromicrospheres.
The ED50 value of 0.08 nM for 5 listed in Table 1
provides an estimate of 5’s intrinsic affinity for an
activated form of GPIIb/IIIa. The equilibrium dissocia-
tion constant for binding of 5 to resting human platelets
different donors can contain 20000-100000 receptors
per platelet, the minimum concentration of drug re-
quired to occupy 50% of the platelet receptors is 4-16
nM. Therefore, the IC50 values listed in Table 1 for
analogs 2, 4, and 5 are at the lower limit of the in vitro
aggregation assay and do not reflect their intrinsic
potency.
3
To assess the intrinsic potency of these compounds,
binding assays using the activated and unactivated
forms of purified GPIIb/IIIa29 were developed. Displace-
ment of the fluorescent fibrinogen receptor antagonist
1920 (Figure 1) from both the activated and unactivated
forms of human GPIIb/IIIa solubilized in Triton X-100
micelles was used to measure the equilibrium binding
of compounds. Table 1 lists the equilibrium dissociation
constants, KD values, for binding to the unactivated form
of GPIIb/IIIa. Equilibrium dissociation constants of 0.9
and 1.1 nM were obtained for binding of 5 to activated
and unactivated forms, respectively. However, due to
the high concentration of GPIIb/IIIa required in this
fluorescence-based assay, KD values can only be ac-
curately determined down to the low nanomolar
range.20,21 Therefore, the KD values measured for 2, 4,
and 5 likely do not reflect the intrinsic binding affinity
of these potent compounds.
was measured directly using H-labeled 522 and indi-
rectly by displacement of the fluorescent ligand 20 by
flow cytometry,23 yielding values of 0.2 and 0.11 nM,
respectively. These data indicate that 5 binds to both
forms of GPIIb/IIIa with high affinity and differs from
compound 22 (L-734,217,24 Figure 1), which has KD
values of 600 and 5 nM for binding to unactivated and
activated GPIIb/IIIa, respectively.21
The in vivo consequences of high-affinity binding to
GPIIb/IIIa on circulating platelets were evaluated by
comparing the intravenous and oral profiles of com-
pounds 2, 4, and 5 with those of 22, an orally active
compound with low affinity for resting platelets.21
Administration of 22 to dogs as a 30 µg/kg iv bolus
elicited >90% inhibition of the extent of ADP-induced
platelet aggregation between 1 and 15 min, with platelet
aggregation returning to baseline levels by 120 min
(Figure 2). Intravenous administration of compounds
2, 4, and 5 at a 3-fold lower dose (10 µg/kg), elicited peak
To extend the sensitivity of these binding assays, ED50

1782 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Askew et al.
F igu r e 3. Effect of 2, 4, 5 (0.10 mg/kg po gavage), and 22
(0.20 mg/kg po gavage) on ex vivo platelet aggregation re-
sponses to ADP (10 µM + 1 µM epinephrine) expressed as
percent inhibition over time (min) in conscious dogs. Data are
mean for n ) 4 (2, 4, and 5) and n ) 8 (22); error bars are not
shown for clarity.
F igu r e 2. Effect of 2, 4, 5 (10 µg/kg iv), and 22 (30 µg/kg iv)
on ex vivo platelet aggregation responses to ADP (10 µM + 1
µM epinephrine) expressed as percent inhibition over time
(min) in conscious dogs. Data are mean for n ) 4 (2, 4, and 5)
and n ) 8 (22); error bars are not shown for clarity.
inhibition of platelet aggregation at 1 min, with values
of 100%, 71( 11%, and 79 ( 13% observed for 2, 4, and
5, respectively. Administration of 2 resulted in complete
inhibition of platelet aggregation between 1 and 30 min,
with platelet aggregation returning to within 8% of
baseline levels by 300 min. Significant inhibition of ex
vivo platelet function was maintained throughout the
8 h protocol following administration of either 4 or 5.
In the case of compound 5, the iv pharmacodynamic
response was essentially flat, with 67 ( 21% inhibition
observed at the end of the 8 h experiment.
Differences in the pharmacodynamic profiles of com-
pounds 2, 4, 5, and 22 were also apparent following oral
administration. Aqueous solutions of each compound
(5 mL volume with sterile water as vehicle) were
administered to mongrel dogs by gastric lavage at doses
of 100 µg/kg (2, 4, and 5) or 200 µg/kg (22). At specified
time points, blood was withdrawn and the derived
platelet-rich plasma used to determine ex vivo platelet
aggregation in response to ADP and collagen. The
inhibition of ADP-mediated platelet aggregation follow-
ing oral administration of 2, 4, 5, and 22 is depicted in
Figure 3. For each compound, the inhibition of collagen
induced aggregation followed a similar time course,
however, peak levels of inhibition were typically lower
(Figure 4). Peak inhibition of ADP-induced platelet
aggregation (60 ( 12%) occurred 70 min following the
administration of 22 with platelet function returning
to baseline levels by 480 min. Compounds 2, 4, and 5
each inhibited ADP-mediated platelet aggregation by
greater than 65% at the 100 µg/kg dose. However, there
were striking differences between compounds in terms
of both the peak antiaggregatory response and the
duration of the effect. Peak inhibition of platelet
aggregation (65 ( 5%) occurred 90 min following the
administration of 2 with platelet function returning to
within 21 ( 3% of baseline by 480 min. Both of the
pyrazolodiazepinone analogs, 4 and 5, produced higher
peak levels of inhibition and the effect remained stable
through the end of the 8 h experimental protocol. For
compound 4, peak inhibition of 76 ( 8% occurred 75 min
following dosing and inhibition remained at 68 ( 9%
at 8 h. Maximal inhibition (100%) occurred 150 min
F igu r e 4. Effect of 2, 4, 5 (0.10 mg/kg po gavage), and 22
(0.20 mg/kg po gavage) on ex vivo platelet aggregation re-
sponses to collagen (10 µg/mL + 1 µM epinephrine) expressed
as percent inhibition over time (min) in conscious dogs. Data
are mean for n ) 4 (2, 4, and 5) and n ) 8 (22); error bars are
not shown for clarity.
following dosing of 5 and was maintained through the
end of the experiment. The duration of antiplatelet
activity following oral administration of crystalline 5 in
gelatin capsules was evaluated in an additional experi-
ment. Platelet aggregation was inhibited by 87 ( 7%
24 h after the administration of 5 to conscious dogs (n
) 4) at 100 µg/kg, suggesting that its pharmacodynamic
profile would allow once-a-day dosing.27
The differences in the pharmacodynamic profiles of
compounds 2, 4, 5, and 22 are likely a result of their
differing affinities for GPIIb/IIIa on resting platelets.
Compounds 2, 4, and 5 bound tightly to isolated,
unactivated GPIIb/IIIa with KD values of 1.8, 1.4, and
1.1 nM, respectively, while 22 had modest affinity with
KD ) 600 nM. Since 22 has modest affinity for GPIIb/
IIIa on resting platelets, its pharmacodynamic profile
is determined largely by the rate of clearance of circu-
lating drug.25 However, because 5 binds to resting
platelets with high affinity, the pharmacodynamic
profile of this compound is governed by the rate of
clearance of the platelet bound drug rather then the
3
clearance of free drug from plasma.26b When H-5 was

Non-Peptide Glycoprotein IIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1783
Ta ble 2. Inhibition of Platelet Aggregation and HUVECa
Attachment to Fibrinogen (FG), Vitronectin (VN), and
Fibrinogen (FN) by 5
chromatography, and silica gel plates (E. Merck) were used
for analytical thin-layer chromatography. All NMR spectra
were recorded on a Varian XL-300 spectrometer, and chemical
shifts are reported in parts per million relative to internal
tetramethylsilane. Melting points were determined on a
Thomas-Hoover apparatus and are uncorrected. Enantiomeric
purities were determined by HPLC using the following condi-
tions: chiral AGP column (4.0 × 100 mm); flow rate 1.00 mL/
min; 100% pH ) 3.0, 20 mM NaH2PO4 buffer; UV detection
at 255 nm. Under these conditions baseline separation
between 5 and its (R) enantiomer was obtained (retention time
for 5 ) 6.71 min; (R) enantiomer ) 10.72 min) and a detection
limit of 0.1% of the (R) isomer was established. The enanti-
omer of 5 was prepared as described for 5 with the amino acid
portion being derived from D-asparagine.
2-(N-Boc-p ip er id in -4-yl)eth yl Iod id e (7). A solution of
4-piperidineethanol (6) (13.0 g, 100 mmol) was dissolved in
CH2Cl2 (250 mL), cooled to 0 °C, and treated with di-tert-butyl
dicarbonate (22.19 g, 100 mmol). The solution was warmed
to room temperature and stirred for 4 h. The solution was
then washed with water, 10% KHSO4, and brine, dried (Na2-
SO4), filtered, and concentrated to give 2-(N-Boc-piperidin-4-
yl)ethanol (21.4 g, 98%) as a colorless oil: TLC Rf ) 0.25 (40%
ethyl acetate/hexanes); 1H NMR (300 MHz, CDCl3) δ 4.10 (bd,
2H), 3.70 (t, 2H), 2.7 (t, 2H), 1.8-1.6 (m, 5H), 1.48 (s, 9H), 1.1
(m, 2H).
A solution of the above alcohol (10.42 g, 48 mmol) in 200
mL of toluene was treated with imidazole (4.66 g, 68.0 mmol),
triphenylphosphine (15.24 g, 50 mmol), and iodine (12.2 g, 48
mmol). The resulting mixture was heated at 80 °C for 2.5 h,
then filtered, and concentrated. Flash chromatography of the
residue on silica gel (10% ethyl acetate/hexanes) afforded 7
(15.8 g, 97%) as a colorless oil: TLC Rf ) 0.45 (40% ethyl
acetate/hexanes); 1H NMR (300 MHz, CDCl3) δ 4.10 (bd, 2H),
3.19 (t, 2H), 2.65 (t, 2H), 1.8-1.6 (m, 5H), 1.45 (s, 9H), 1.1 (m,
2H).
2-(N-Boc-p ip er id in -4-yl)et h yla m in e (8). A solution of
iodide 7 (10.5 g, 31.0 mmol) in DMSO (100 mL) was treated
with NaN3 (4.0 g, 62.0 mmol) and stirred at room temperature
for 18 h. The reaction mixture was diluted with ethyl acetate
(200 mL), washed with H2O (2 × 100 mL) and brine (100 mL),
then dried over Na2SO4, filtered, and concentrated to give the
azide in quantitative yield: TLC Rf ) 0.48 (40% ethyl acetate/
hexanes); 1H NMR (300 MHz, CDCl3) δ 4.10 (bd, 2H), 3.21 (t,
2H), 2.65 (t, 2H), 1.8-1.6 (m, 5H), 1.45 (s, 9H), 1.1 (m, 2H). A
solution of the above azide (7.40 g, 31.0 mmol) in methanol
was treated with 10% Pd on C (1.2 g) and the mixture stirred
under a H2-filled balloon for 12 h. The catalyst was removed
by filtration through Celite and the filtrate concentrated to
afford 8 in quantative yield (6.65 g, 31.0 mmol): TLC Rf )
0.36 (10% methanol/ethyl acetate); 1H NMR (300 MHz, CDCl3)
δ 4.10 (bd, 2H), 3.26 (t, 2H), 2.75 (bs, 2H), 2.64(t, 2H), 1.8-
1.6 (m, 5H), 1.47 (s, 9H), 1.23 (m, 2H).
HUVEC IC50 (nM)
plaggin
IC50 (nM)
FG
VN
FN
8
>3 × 105
>3 × 105
>3 × 105
a
HUVEC-human umbilical vein endothelial cells. Experimen-
tal details of this assay have been previously reported.16a,28
administered iv at doses ranging from 3 to 250 µg/kg,
the pharmacokinetic parameters were dose-dependent;
both the clearance and volume of distribution increased
markedly with increasing doses.26b Measurement of the
concentration of 5 in whole blood (WB) and platelet-rich
(PRP) and platelet-poor plasma (PPP) following iv
administration at several doses indicated that 5 bound
tightly to circulating platelets. Following administra-
tion of 5 doses exceeding the platelet binding capacity
(70 nM in canine whole blood), excess free drug was
rapidly eliminated, with a half-life (t1/2) of approximately
0.5 h. However, the platelet bound 5 was slowly
eliminated from the PRP compartment and the terminal
half-life (t1/2) was independent of the dose given, with a
mean value of 96 h. Full details of the pharmacokinet-
ics of 5 are reported elsewhere.26b
Intrinsic potency differences between 2, 4, and 5 are
likely masked since the fluorescence-based assay can
only accurately determine KD values down to the low
nanomolar range.20,21 The greater potency of 4 com-
pared 2 is likley a consequence of the larger ring of 4.
The added flexibility may allow 4 to adopt conformations
that maximize the interactions between GPIIb/IIIa, its
charged termini, and potency-enhancing R-sulfonamide
moiety. The apparent increase in potency of 5 relative
to 4 is in agreement with results from an earlier, less
potent series of centrally constrained R-sulfonamides in
which arenesulfonamide analogs were generally 4-10-
fold more potent than alkanesulfonamides.20
Since the platelet fibrinogen receptor is a member of
the integrin superfamily of receptors, specificity for
GPIIb/IIIa over other RGD-binding integrins is believed
to be an important requirement for safety in an oral
antiplatelet agent. The specificity of 5 was evaluated
by determining IC50 values for the inhibition of human
umbilical vein endothelial cell (HUVEC) attachment to
fibrinogen-, vitronectin-, and fibronectin-coated sur-
faces.28 The results summarized in Table 2 demonstrate
that 5 is >33000-fold less effective at inhibiting cell
attachment than at inhibiting platelet aggregation.
In summary, we have reported the discovery of 5 (L-
738,167), a potent, selective fibrinogen receptor antago-
nist that displays an extended pharmacodynamic profile
following intravenous and oral administration in dogs.
Platelet aggregation was inhibited by 87 ( 7% 24 h after
the administration of 5 to conscious dogs at 100 µg/kg.
The unprecedented pharmacodymanic profile exhibited
by 5 appears to a consequence of its tight binding to
circulating platelets and may enable clinically useful
levels of inhibition of platelet aggregation to be main-
tained with small amounts of compound given once a
day.
Dim eth yl P yr a zole-3,5-d ica r boxyla te (10). A solution
of pyrazole-3,5-dicarboxylic acid (9) (75 g, 431 mmol) in 1 L of
anhydrous methanol was placed in a 2-L three-neck flask
equipped with a gas inlet, vertical condenser, and CaCl2 drying
tube. A stream of anhydrous HCl gas was rapidly passed
through the solution causing it to reflux vigorously. The HCl
addition was continued at this rate for 30 min after which the
solution was heated at reflux for 2 h and cooled and the solvent
removed at reduced pressure. The resulting white solid was
treated with 600 mL of saturated NaHCO3 and extracted into
CH2Cl2 (3 × 500 mL). The pooled extracts were dried (Na2-
SO4), filtered, and concentrated at reduced pressure. The
resulting white solid was recrystallized from methanol with
the addition of anhydrous ether to give 10 (75.0 g, 95%) as a
1
white solid: TLC Rf ) 0.45 (60% ethyl acetate/hexanes); H
NMR (CDCl3) δ 7.38 (s, 1H), 7.18 (br s, 1H), 3.98 (s, 3H), 3.93
(s, 3H).
Dim eth yl 1-(2-Br om oeth yl)p yr a zole-3,5-d ica r boxyla te
(11a ). To solution of 10 (5.0 g, 27.2 mmol) in 150 mL of
anhydrous acetonitrile were added K2CO3 (5.2 g, 40.0 mmol)
and 1,2-dibromoethane (25.0 mL, 291 mmol). The resulting
mixture was heated at reflux under Ar. After 25 min, TLC
analysis (silica, 70:30 EtOAc/hexane) showed that no 10
Exp er im en ta l Section
Unless otherwise noted, starting materials were obtained
from commercial sources and used without purification. Silica
gel (E. Merck, 230-400 mesh) was used for flash column

1784 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Askew et al.
remained. The reaction suspension was cooled and filtered
and the filtrate evaporated to dryness at reduced pressure and
placed on a high vacuum line for 12 h. The resulting white
solid was recrystallized from hexane containing 10% ethyl
acetate, giving 5.61 g of a white solid (71%): 1H NMR (CDCl3)
δ 7.38 (s, 1H); 5.03 (t, J ) 8.2 Hz, 2H), 3.98 (s, 3H), 3.93 (s,
3H), 3.75 (t, J ) 8.5 Hz, 2H).
38.5 mmol) in 100 mL of DMSO was treated with NaN3 (8.83
g, 138.0 mmol), and the mixture was stirred at 25 °C for 16 h.
The reaction mixture was then diluted with 100 mL of H2O
and extracted with ethyl acetate (3 × 100 mL). The combined
organic extracts were washed with water (2 × 100 mL) and
brine (1 × 100 mL), dried over Na2SO4, and concentrated to
give dimethyl 1-(3-azidopropyl)pyrazole-3,5-dicarboxylate (9.8
g, 95%) as a colorless oil: TLC Rf ) 0.65 (50% ethyl acetate/
hexanes); 1H NMR (CDCl3) δ 7.38 (s, 1H), 4.95 (t, J ) 8.2 Hz,
2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.75 (t, J ) 8.5 Hz, 2H), 2.51
(m, 2H). A solution of the above azide (8.5 g, 32.5 mmol) in
100 mL of MeOH saturated with HCl gas was treated with
500 mg of 10% Pd on C and the mixture shaken on a Parr
hydrogenator at 45 psi for 5 h. The catalyst was removed by
filtration through Celite, and the filtrate was concentrated to
give a colorless oil. This oil was dissolved in 250 mL of MeOH,
treated with Et3N (6.70 mL, 48.8 mmol), refluxed for 1.5 h,
and then concentrated to a volume of 50 mL. The resulting
white solid was filtered to give 13 (6.53 g, 91%). mp 220-221
°C; TLC Rf ) 0.40 (10% methanol/ethyl acetate); 1H NMR (300
MHz, CDCl3) δ 7.36 (s, 1H); 6.42 (br t, 1H); 4.58 (t, J ) 8.0
Hz, 2H), 3.95 (s, 3H), 3.39 (m, 2H), 2.31 (m, 2H).
Dim eth yl 1-(3-Ch lor op r op yl)p yr a zole-3,5-d ica r boxy-
la te (11b). Dimethyl pyrazole-3,5-dicarboxylate (10) (5.0 g,
27.2 mmol) in 150 mL of anhydrous acetonitrile was treated
with K2CO3 (5.2 g, 40.0 mmol) and 1-bromo-3-chloropropane
(10.8 mL, 109 mmol). The resulting mixture was heated to
reflux under Ar. After 25 min, the reaction suspension was
cooled and filtered and the fitrate evaporated to dryness at
reduced pressure and placed on a high vacuum line for 12 h.
The resulting white solid was recrystallized from hexane
containing 10% ethyl acetate, giving 11b (5.52 g, 78%) as a
1
white solid: TLC Rf ) 0.60 (50% ethyl acetate/hexanes); H
NMR (CDCl3) δ 7.38 (s, 1H), 4.95 (t, J ) 8.2 Hz, 2H), 3.95 (s,
3H), 3.92 (s, 3H), 3.75 (t, J ) 8.5 Hz, 2H), 2.51 (m, 2H).
Meth yl [4,5,6,7-Tetr a h yd r o-4-oxo-5-[2-(N-Boc-p ip er i-
d in -4-yl)eth yl]-4H-p yr a zolo[1,5-a ][1,4]p yr a zin e-2ca r box-
yla te (12a ). A solution containing 11a (14.0g, 48.0 mmol),
N,N-diisopropylethylamine (25 mL, 144 mmol), Boc-4-(ami-
noethyl)piperdine (8) (12.0 g, 52.6 mmol), and potassium iodide
(2.39 g, 0.3 mmol) in 250 mL of CH3CN was refluxed under
N2 for 4.5 h, cooled, filtered, and evaporated at reduced
pressure. The resulting yellow residue was chromatographed
on silica gel using EtOAc as eluent to give 9.53 g of an off-
white crystaline solid (49%): 1H NMR (CDCl3) δ 7.15 (s, 1H),
4.29(t, J ) 7.0 Hz, 2H), 3.93 (br d, J ) 12 Hz, 2H), 3.76 (s,
3H), 3.61(t, J ) 5.3 Hz, 2H), 3.42 (t, J ) 7.3 Hz, 2H), 2.65 (t,
J ) 7.6 Hz, 2H), 1.55 (d, J ) 12.5 Hz, 2H), 1.38 (m, 2H), 1.33-
1.25 (m, 1H), 1.27 (s, 9H), 1.01 (m, 2H).
Meth yl 5,6,7,8-Tetr a h yd r o-4-oxo-5-[2-(N-Boc-p ip er id in -
4-yl)et h yl]-4H-p yr a zolo[1,5-a ][1,4]d ia zep in e-2-ca r b oxy-
la te (14a ). A solution of 13 (17.5 g, 83.0 mmol) in 150 mL of
DMF was treated with 60% NaH (3.6 g, 91 mmol), and the
mixture was stirred under N2 at -15 °C for 30 min. This
mixture was treated dropwise with a solution of 7 (28.3 g, 83.0
mmol) in 50 mL of DMF over 20 min. The resulting solution
was stirred for 30 min at -15 °C, then warmed to room
temperature, and stirred overnight. The DMF was removed
at reduced pressure and the residue dissolved in ethyl acetate,
filtered, and chromatographed on silica gel using ethyl acetate
as eluent to afford 14a (29.8 g, 85.5%) as a colorless glass:
1
TLC Rf ) 0.50 (ethyl acetate); H NMR (300 MHz, CDCl3) δ
4,5,6,7-Tet r a h yd r o-4-oxo-5-[2-(N-Boc-p ip er id in -4-yl)-
eth yl]-4H-p yr a zolo[1,5-a ][1,4]p yr a zin e-2-ca r boxylic Acid
(12b). A solution contaning LiOH (140 mg, 3.35 mmol) in 10
mL of H2O was added to a solution of the 12a (908 mg, 2.23
mmol) in 10 mL of CH3OH, and the resulting mixture was
heated to 60 °C for 2.5 h and then cooled and the CH3OH
removed at reduced pressure. The remaning aqueous phase
was acidified with 10% aqueous citric acid and extracted with
CH2Cl2 (2 × 50 mL). The pooled organic extracts were dried
over Na2SO4 and then evaporated, giving 883 mg of a white
solid (98.7%): 1H NMR (CDCl3) δ 7.43 (s, 1H), 4.48 (t, J ) 7.0
Hz, 2H), 4.01 (br d, J ) 12 Hz, 2H), 3.77 (t, J ) 5.3 Hz, 2H),
3.51 (t, J ) 7.3 Hz, 2H), 2.71 (t, J ) 8.3 Hz, 2H), 1.72 (d, J )
12.5 Hz, 2H), 1.53 (m, 2H), 1.42-1.37 (m, 1H), 1.35 (s, 9H),
1.10 (m, 2H).
3-[[[4-Oxo-5-(2-p ip er id in -4-yl-eth yl)-4,5,6,7-tetr a h yd r o-
p yr a zolo[1,5-a ]p yr a zin -2-yl]ca r b on yl]a m in o]p r op ion ic
Acid Hyd r och lor id e (1). A solution containing 12b (79 mg,
0.20 mmol), â-alanine tert-butyl ester hydrochloride (40 mg,
0.22 mmol), and EDC (41.5 mg. 0.22 mmol) in CH2Cl2 (20 mL)
was treated with Et3N (30.8 µL, 0.22 mmol) and stirred at room
temperature for 13.5 h. the resulting solution was washed
successively with 10% citric acid, H2O, saturated NaHCO3, and
brine (10 mL each), dried over Na2SO4, and evaporated to
afford the doubly protected intermediate as a glass (88.5 mg,
98%): 1H NMR (CDCl3) δ 7.38 (t, 1H), 7.31 (s, 1H), 4.48 (t, J
) 7.0 Hz, 2H), 4.01 (br d, J ) 12 Hz, 2H), 3.77 (t, J ) 5.3 Hz,
2H), 3.73 (t, 2H), 3.58 (t, J ) 7.3 Hz, 2H), 2.71 (m, 4H), 1.72
(d, J ) 12.5 Hz, 2H), 1.53 (m, 2H), 1.42-1.37 (m, 1H), 1.35 (s,
9H), 1.28 (s, 9H), 1.10 (m, 2H). The above ester was dissolved
in EtOAc (10 mL) cooled to 0 °C and treated with anhydrous
HCl gas for 15 min. After being stirred at 0 °C for 1 h, the
resulting suspension was diluted with anhydrous ether (25
mL), filtered, and dried under vacuum to afford 1 as a
hydrochloride salt: 1H NMR (DMSO-d6) δ 8.60 (br s, 1H), 8.38
(br s, 1H), 8.18 (t, 1H), 7.00 (s, 1H), 4.40 (t, J ) 7.0 Hz, 2H),
3.78 (t, J ) 5.3 Hz, 2H), 3.5-3.1 (m, 6H), 2.81 (m, 2H), 1.72
(d, J ) 12.5 Hz, 2H), 1.53 (m, 2H), 1.37 (m, 1H), 1.10 (m, 2H);
FAB MS (MH+ ) 364). Anal. (C17H25N5O4‚HCl‚0.6H2O) C,
H, N.
7.24 (s, 1H), 4.50 (t, J ) 7.0 Hz, 2H), 3.93 (br d, J ) 12 Hz,
2H), 3.94 (s, 3H), 3.61 (t, J ) 5.3 Hz, 2H), 3.42 (t, J ) 7.3 Hz,
2H), 2.7 (br t, J ) 6.3 Hz, 2H), 2.3 (m, 2H), 1.55 (d, J ) 12.5
Hz, 2H), 1.38 (m, 2H), 1.33-1.25 (m, 1H), 1.27 (s, 9H), 1.01
(m, 2H).
5,6,7,8-Tet r a h yd r o-4-oxo-5-[2-(N-Boc-p ip er id in -4-yl)-
eth yl]-4H-pyr azolo[1,5-a ][1,4]diazepin e-2-car boxylic Acid
(14b). A solution of 14a (16.6 g, 39.5 mmol) in 100 mL of THF
was treated with 1 N LiOH (43.5 mL, 43.0 mmol). The
resulting solution was stirred at room temperature for 18 h,
and then THF was removed at reduced pressure. The remain-
ing aqueous phase was acidified with 10% KHSO4, and the
resulting white solid was filtered, washed with H2O, and dried
to give 14b (16.0 g, 99%): TLC Rf ) 0.30 (CHCl3/CH3OH/NH4-
OH, 94:5:1); 1H NMR (300 MHz, CDCl3) δ 7.29 (s, 1H), 4.52 (t,
J ) 7.0 Hz, 2H), 4.12 (br d, J ) 12 Hz, 2H), 3.94 (s, 3H), 3.61
(t, J ) 5.23 Hz, 2H), 3.42 (t, J ) 7.3 Hz, 2H), 2.7 (br t, J ) 6.3
Hz, 2H), 2.3 (m, 2H), 1.55 (d, J ) 12.5 Hz, 2H), 1.38 (m, 2H),
1.33-1.25 (m, 1H), 1.27 (s, 9H), 1.01 (m, 2H).
3-[[4-Oxo-5-(2-p ip er id in -4-yl-eth yl)-5,6,7,8-tetr a h yd r o-
p yr a zolo[1,5-a ]d ia zep in -2-yl]a m in o]p r op ion ic Acid Hy-
d r och lor id e (3). Prepared from 14b in a manner similar to
that described above for the preparation of 1: 1H NMR
(DMSO-d6) δ 8.80 (br s, 2H), 8.18 (t, 1H), 6.98 (s, 1H), 4.45 (t,
J ) 7.0 Hz, 2H), 3.52 (t, J ) 5.3 Hz, 2H), 3.5-3.41 (m, 2H),
3.25 (d, J ) 12.7 Hz, 2H), 2.84 (m, 2H), 2.20 (t, 2H), 2.21 (m,
2H), 1.85 (d, J ) 12.5 Hz, 2H), 1.53 (m, 3H), 1.38 (m, 2H).
Anal. (C18H27N5O4‚HCl‚0.65H2O) C, H, N.
N-(n -Bu tylsu lfon yl)-L-a sp a r a gin e (16a ). A solution con-
taining L-asparagine (6.45g, 48.9 mmol) and NaOH (2.0 g, 50.0
mmol) in 100 mL of 50% aqueous dioxane was cooled to 0 °C
in an ice bath. To this rapidly stirred mixture were added
alternately a solution of NaOH (2.2 g, 55.0 mmol) in 50 mL of
water and neat n-butanesulfonyl chloride (7.0 mL, 53.9 mmol)
over a period of 30 min. After the mixture was mixed at 0 °C
for 30 min, HPLC analysis showed the reaction to be ap-
proximately 60% complete. Neither longer reaction times nor
the use of additional butanesulfonyl chloride resulted in
increased product formation. The reaction solution was
concentrated to a volume of 50 mL at reduced pressure, and
the aqueous residue was cooled, acidified with concentrated
Meth yl 5,6,7,8-Tetr a h yd r o-4-oxo-4-H-p yr a zolo[1,5-a ]-
[1,4]d ia zep in e-2-ca r boxyla te (13). A solution of 11b (10 g,

Non-Peptide Glycoprotein IIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1785
HCl, and extracted into ethyl acetate (3 × 100 mL). The
organic extracts were dried over Na2SO4 and concentrated to
a volume of approximately 50 mL, anhydrous ether (50 mL)
was added, and the resulting white precipitate was isolated
by vacuum filtration to give 16a (5.31 g, 43.1%): mp 154-
155 °C; 1H NMR (DMSO-d6) δ 11.9 (br s, 1H), 7.58 (d, J ) 8.6
Hz, 1H), 7.46 (br s, 1H), 7.01 (br s, 1H), 4.14 (m, 1H), 3.00 (t,
J ) 7.8 Hz, 2H), 2.62-2.38 (m, 2H), 1.65 (m, 2H), 0.88 (t, J )
7.3 Hz, 3H).
N-p-Tolu en esu lfon yl-L-Asp a r a gin e (16b). L-Asparagine
(10.0g, 75.7 mmol) was placed in a 500 mL round bottom flask
equipped with a magnetic stir bar and an addition funnel.
NaOH (1 N, 170 mL, 166.5 mmol) was added along with 50
mL of dioxane. p-Toluenesulfonyl chloride (15.88 g, 83.27
mmol) dissolved in dioxane (50 mL) was added to the reaction
mixture with vigorous stirring. The reaction mixture was then
stirred for 2 h at room temperature, cooled to 0 °C, and
acidified to pH 2-3 with hydrochloric acid (concentrated). The
product 16b formed as a white crystalline solid (20.3 g, 94%)
that was collected by filtration: TLC Rf 0.55 (10:0.1 CH3OH:
NH4OH); 1H NMR (DMSO-d6) δ 7.91 (d, J ) 8.79 Hz, 1H), 7.64
(d, J ) 8.06 Hz, 2H), 7.32 (s, d (overlapping), J ) 8.06 Hz,
3H), 6.87 (s, br, 1H), 4.03 (m, 1H), 2.49 (m, 1H), 2.43 (d, d, J
) 7.08, 15.38 Hz, 1H), 2.35 (s, 3H), 2.21 (dd, J ) 6.11, 15.38
Hz, 1H).
2(S)-[(n -Bu tylsu lfon yl)a m in o]-â-a la n in e (17a ). A solu-
tion containing NaOH (6.04 g, 151 mmol) in 50 mL of H2O
was cooled to 0 °C, and bromine (1.40 mL, 26.9 mmol) was
added. The resulting solution was stirred at 0 °C for 5 min.
Next, a cooled solution of 16a (5.23 g, 20.7 mmol) and NaOH
(1.66 g, 41.4 mmol) in 15 mL of H2O was added at once and
mixture stirred at 0 °C for 5 min and then heated to 80 °C for
15 min. The solution was then cooled to 0 °C, neutralized with
12 N HCl (9.5 mL), and stirred until gas evolution ceased.
None of the amino acid 12 crystallized on standing, so the
solution was then made basic by the addition of 2 N NaOH
and 20 mL of THF was added along with di-tert-butyl dicar-
bonate (9.0 g, 41.4 mmol). After the solution was stirred
overnight at 25 °C the THF was removed at reduced pressure
and the basic aqueous phase extracted ethyl acetate (2 × 50
mL). The aqueous phase was then made acidic with 10%
KHSO4 and extracted with ethyl acetate (3 × 100 mL). The
pooled acidic extracts were dried over Na2SO4, filtered, and
evaporated, giving a white solid (5.64 g, 84%): mp, 111-112
°C; 1H NMR (DMSO-d6) δ 12.1 (br s, 1H), 7.44 (d, J ) 9.0 Hz,
1H); 6.89 (m, 1H), 4.91 (m, 1H), 3.3 -3.05 (m, 2H), 2.93 (t, J
) 7.7 Hz, 2H), 1.65 (m, 2H), 1.40 (m, 2H), 1.33 (s, 9H), 0.86 (t,
J ) 7.3 Hz, 3H). A solution of this Boc sulfonamide (3.83 g,
11.8 mmol) in 200 mL of ethyl acetate was cooled to 0 °C and
treated with HCl gas for 5 min. The solution was then warmed
to 25 °C and stirred for 30 min, then concentrated at reduced
pressure to 50% of its volume, and diluted with 100 mL of
ether. The resulting white solid was collected by vacuum
filtration, giving 17a (2.70 g, 92%): mp, 122-125 °C; 1H NMR
(DMSO-d6) δ 8.20 (br s, 2H), 7.78 (d, J ) 9.0 Hz, 1H); 4.21 (m,
1H), 3.2-3.05 (m, 3H), 2.93 (m, 1H), 1.65 (m, 2H), 1.40 (m,
2H), 0.87 (t, J ) 7.3 Hz, 3 H).
2-(S)-(p-Tolu en esu lfon yla m in o)-â-a la n in e (17b). Bro-
mine (5.03 mL, 97.5 mmol) was added to a chilled solution
(-15 °C) of sodium hydroxide (21 g, 525 mmol) in water (300
mL) at such a rate as to keep the temperature below 0 °C.
The solution was mixed for 10 min, and then a cold (0 °C)
solution of N-p-toluenesulfonyl-L-asparagine (16b) (21.5 g,
75.0mmol) in 40 mL of 10% NaOH was added in a single
portion to the sodium hypobromite solution. This mixture was
stirred with cooling for 20 min, placed in an oil bath, and
heated at 80-90 °C for 40 min. The solution was then cooled
in an ice bath and adjusted to pH 7 by adding hydrochloric
acid (concentrated) dropwise. The resulting white solid was
isolated by vacuum filtration and then dried in a vacuum oven
to afford 17b (14.2 g, 73.6%): TLC Rf ) 0.40 (10:0.1 CH3OH:
NH4OH); 1H NMR (DMSO-d6) δ 8.2-7.2 (br, 2H, (NH,COOH)),
7.70 (d, J ) 8.18 Hz, 2H), 7.38 (d, J ) 8.18 Hz, 2H), 3.7-3.0
(br, 2H, (NH2)), 3.12 (q, J ) 4.76 Hz, 1H), 2.99 (d, d, J ) 4.64,
11.96 Hz, 1H), 2.79 (d, d, J ) 9.52, 11.96 Hz, 1H). 2.36 (s, 3H).
2(S)-[(n -Bu tylsu lfon yl)a m in o]-3-[[[4,5,6,7-tetr a h yd r o-
4-oxo-5-[2-(N-Boc-p ip er id in -4-yl)eth yl]-4H-p yr a zolo[1,5-
a ][1,4]pyr azin -2-yl]car bon yl]am in o]pr opion ic Acid (18a).
Isobutyl chloroformate (1.75 mL, 13.35 mmol) was added to a
cooled solution (0 °C) containing 12a (4.98 g, 12.72 mmol) and
N-methylmorpholine (1.53 mL, 14.00 mmol) in 100 mL of THF.
This mixture was stirred under an atmosphere of dry nitrogen.
After the reaction continued for 1 h, HPLC analysis of an
aliquot indicated that the reaction was >90% complete. The
N-methylmorpholine hydrochloride was removed by filtration
and the filtrate poured into a solution containing 17a hydro-
chloride (4.30 g, 16.54 mmol), N,N-diisopropylethylamine (4.27
mL, 33.10 mmol), THF (60 mL), and H2O (20 mL). HPLC
analysis indicated that the mixed anhydride was consumed
within 15 min with an 83% conversion to product the remain-
der being hydrolyzed to starting acid. The THF was then
removed from the reaction solution at reduced pressure and
the remaining aqueous portion acidified with saturated NaH-
SO4 and extracted with ethyl acetate (3 × 200 mL). Pooled
extracts were dried over Na2SO4, filtered, and concentrated,
giving a red oil from which a white solid formed on standing.
Solid 18a was collected by vacuum filtration (4.45 g, 58.8%);
additional material was obtained from the filtrate in a second
crop (total yield ) 65.6%): 1H NMR (DMSO-d6) δ 8.31 (t, J )
6 Hz, 1H), 7.62 (d, J ) 8.5 Hz, 1H), 7.01 (s, 1H), 4.43 (t, J )
6.6 Hz, 2H), 4.11 (m, 1H), 3.92 (d, J ) 12 Hz, 2H), 3.80 (t, J )
6.6 Hz, 2H), 3.51 (t, J ) 7.3 Hz, 2H), 3.65 (m, 2H), 3.51 (t, J
) 6.8 Hz, 2H), 2.96 (t, J ) 7.2 Hz, 2H), 1.70 (d, J ) 11 Hz,
2H), 1.53 (m, 2H), 1.60-1.49 (overlaping m, 5H), 1.40 (s, 9H),
1.28 (q, J ) 7.1 Hz, 2H), 1.05 (m, 2H), 0.79 (t, J ) 7.1 Hz,
3H).
2(S)-[(n -Bu tylsu lfon yl)a m in o]-3-[[[4,5,6,7-tetr a h yd r o-
4-oxo-5-(2-p ip er id in -4-ylet h yl)-4H -p yr a zolo[1,5-a ][1,4]-
d ia zep in -2-yl]ca r bon yl]a m in o]p r op ion ic Acid (2). A so-
lution contaning 18a (278 mg, 0.437 mmol) in 30 mL of ethyl
acetate was cooled to 0 °C and HCl gas bubbled through for 3
min. The reaction mixture was warmed to room temperature,
stirred for 30 min, and then taken to dryness on a rotary
evaporator. The resulting hydrochloride salt was subjected
to ion exchange chromatography using Dowex 50 × 8-200 resin
with ammonium hydroxide:acetonitrile:water (50:25:25) as
eluent. Fractions containing 2 were combined and then
concentrated at high vacuum, and the resulting white foam
was dried for 8 h under high vacuum and then recrystallized
from water to afford 2 (223 mg, 95.3%) as an analytically pure
1
crystaline solid: mp 283 °C dec; H NMR (DMSO-d6) δ 8.95
(br s, 1H), 8.33 (t, J ) 5.7 Hz, 1H), 7.64 (d, J ) 9 Hz, 1H),
7.02 (s, 1H), 4.35 (t, J ) 5.1 Hz, 2H), 4.10 (m, 1H), 3.81 (t, J
) 5.2 Hz, 2H), 3.6-3.4 (m, 4H), 3.21 (d, J ) 10.5 Hz, 2H),
2.95 (t, J ) 7.8 Hz, 2H), 2.81 (br m, 2H), 1.96 (d, J ) 11 Hz,
2H), 1.62-1.2 (overlaping multiplets, 9H), 0.80 (t, J ) 7.3 Hz,
3H); FAB-MS m/e 499 (M + H)+; enantiomeric excess >99.8%
by HPLC. Anal. (C21H34N6O6S‚3.0H2O) C, H, N.
2(S)-[(n -Bu tylsu lfon yl)a m in o]-3-[[[5,6,7,8-tetr a h yd r o-
4-oxo-5-(2-p ip er id in -4-ylet h yl)-4H -p yr a zolo[1,5-a ][1,4]-
d ia zep in -2-yl]ca r bon yl]a m in o]p r op ion ic Acid (4). Pre-
pared from 14b and 17a in a manner similar to that described
above for the preparation of 2: 1H NMR (DMSO-d6) δ 8.95 (br
s, 1H), 8.33 (t, J ) 5.7 Hz, 1H), 7.64 (d, J ) 9 Hz, 1H), 7.02 (s,
1H), 4.35 (t, J ) 5.1 Hz, 2H), 4.10 (m, 1H), 3.81 (t, J ) 5.2 Hz,
2H), 3.6-3.4 (m, 4H), 3.21 (d, J ) 10.5 Hz, 2H), 2.95 (t, J )
7.8 Hz, 2H), 2.81 (br m, 2H), 1.96 (d, J ) 11 Hz, 2H), 1.62-
1.2 (overlaping multiplets, 9H), 0.80 (t, J ) 7.3 Hz, 2H); FAB-
MS (MH+
(C22H36N6O6S‚2.5H2O) C, H, N.
) 513); enantiomeric excess ) 99.8%. Anal.
2-(S)-(p-Tolu en esu lfon yla m in o)-3-[[[5,6,7,8-tetr a h yd r o-
4-oxo-5-[2-(N-Boc-p ip er id in -4-yl)eth yl]-4H-p yr a zolo[1,5-
a ][1,4]diazepin -2-yl]car bon yl]am in o]pr opion ic acid (18c).
A solution of 14b (5.0 g, 12.3 mmol) in THF (150 mL) was
cooled to 0 °C, and N-methylmorpholine (2.11 mL, 19.2 mmol)
was added via syringe. After being mixed for 20 min, isobutyl
chloroformate (2.38 mL, 18.2 mmol) was added dropwise via
syringe. After 0.5 h, HPLC analysis indicated that 14b had
been >95% consumed. The amine 17b (7.00 g, 27.1 mmol),
THF (125 mL), and N,N-diisopropylethylamine (4.71 mL, 27.1
mmol) were combined in a 500 mL round bottom flask with a
magnetic stir bar. Water was added in small portions until a

1786 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Askew et al.
clear solution resulted. The resulting solution was cooled in
an ice bath. The mixed anhydride suspension was added in a
single portion to the solution of 17b with vigorous mixing.
After 20 min, HPLC analysis of the crude reaction solution
indicated that the coupled product had formed in 86% yield.
At this time there was no evidence from HPLC of any mixed
anhydride remaining in the reaction solution. The reaction
solution was concentrated to remove THF. The remaining
aqueous material was acidified with 10% KHSO4. The result-
ing percipitate was filtered to give a white solid. This material
was subjected to flash column chromatography using silica
(EM Science, 230-400 mesh, 10 × 20 cm). The column was
eluted with methylene chloride:methanol:ammonium hydrox-
ide (98:2:0.2, 95:5:0.5, 90:10:1, and then 85:15:1.5) to give pure
18c (6.2 g, 78%) as a white solid: TLC Rf ) 0.30 (85:15:1.5
abbreviated GP IIb/IIIa/SPA. The binding of the 125I-labeled
RGD-containing heptapetide 21 ([125I]L-692,884) (New En-
gland Nuclear, NEX-330) to the yttrium silicate containing IIb/
IIIa/SPA is detectable, without the necessity of separation of
bound from free, in a Top Count Scintillation Counter. The
ED50 for a nonradiolabeled compound was determined by
competition with the binding of 125I-containing 21 to GP IIb/
IIIa/SPA at pH 7.5 (20 mM HEPES, 0.15 M NaCl, room
temperature) with ∼0.3 nM of IIb/IIIa/SPA, ∼0.3 nM of 21,
and a wide range of concentrations of the competing nonra-
diolabeled compounds. After equilibration, the bound CPM
was measured and the ED50 value determined by a nonlinear
least-squares fit of CPM ) (Bmax - Bmin)/(1 + (I/ED50)B + Bmin
,
where I is the concentration of the test compound, B is the
Hill slope, Bmax is the maximum binding observed without the
test compound, and Bmin is the nonspecific binding signal. The
average standard error of mean for ED50 determinations was
(20%.
1
CH2Cl2:CH3OH:NH4OH); H NMR (DMSO-d6) δ 8.23 (q, J )
3.40 Hz, 1H), 7.64 (d, J ) 8.20 Hz, 2H), 7.32 (d, J ) 8.20 Hz),
7.2-7.0 (br, 1H), 6.86 (s, 1H), 4.36 (t, J ) 6.70 Hz, 2H), 3.89
(d, br, J ) 12.21 Hz, 2H), 3.59 (m, 1H), 3.47 (t, J ) 7.08 Hz,
2H), 3.5-3.1 (m, br, 5H), 2.8-2.6 (br, 2H), 2.33 (s, 3H), 2.17
(t, J ) 6.47 Hz, 2H), 1.66 (d, br, J ) 11.97 Hz, 2H), 1.55-1.45
(m, br, 3H), 1.37 (s, 9H), 1.1-0.9 (m, br, 2H).
Equ ilibr iu m Dissocia tion Con sta n t (KD) for [3H]-5 to
P la telets. Human gel filtered platelets (10 mL of 2 × 106
platelets/mL) were incubated at room temperature in a platelet
buffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.0 mM
CaCl2, 3.3 mM NaH2PO4, 3.8 mM HEPES, containing 1%
glucose and 1% BSA at pH 7.4) with 14 concentrations of [3H]-
5 (0.02-10 nM; radiolabel diluted 10-fold with unlabeled 5)
in the presence or absence of 10 µM unlabeled 5 for 3 h to
attain equilibrium. Platelets were then pelleted by centrifuga-
2(S)-(p-Tolu en esu lfon yla m in o)-3-[[[5,6,7,8-tetr a h yd r o-
4-oxo-5-(2-p ip er id in -4-ylet h yl)-4H -p yr a zolo[1,5-a ][1,4]-
d ia zep in -2-yl]ca r bon yl]a m in o]p r op ion ic Acid (5). The
above Boc-protected material 18c (7.42 g, 11.48 mmol) was
placed in a 1 L round bottom flask equipped with a magnetic
stir bar. Methylene chloride (300 mL) was added, and the
reaction mixture was cooled to 0-5 °C. Hydrogen chloride was
bubbled through the suspension with stirring. After 30 min
the contents of the reaction flask were concentrated, and the
resulting hydrochloride of 5 was collected by filtration. This
hydrochloride salt was subjected to ion exchange chromatog-
raphy using Dowex 50 × 8-200 ion exchange resin (110 g, 4.11
mequiv/g) using ammonium hydroxide:acetonitrile:water (50:
25:25) as eluent. Fractions containing 5 were combined and
then concentrated at high vacuum, and the resulting white
foam was dried for 8 h under high vacuum and then recrystal-
lized from water to afford 5 (7.43 g, 77.6%) as a white solid:
mp 187 °C; TLC Rf 0.55 (10:0.1 CH3OH:NH4OH); 1H NMR
(DMSO-d6) δ 9.0-8.5 (br, 1H), 8.17 (m, 1H), 7.67 (d, J ) 8.18
Hz, 2H), 7.32 (d, J ) 8.18 Hz, 2H), 6.89 (s, 1H), 4.38 (t, J )
6.84 Hz, 2H), 3.75-3.65 (m, br, 1H), 3.46 (t, br, 2H), 3.5-3.1
(m, br, 8H, H2O), 2.77 (t, br, J ) 11.36, 2H), 2.35 (s, 3H), 2.17
(t, J ) 6.47 Hz, 2H), 1.80 (d, br, J ) 12.7 Hz, 2H), 1.53-1.42
(m, br, 3H), 1.33-1.24 (m, br, 2H); FAB-MS (MH ) 547+);
enantiomeric excess ) 99.8%. Anal. (C25H34N6O6S‚3.0H2O C,
H, N.
In Vitr o P h a r m a cology. P u r ifica tion of Hu m a n GP
IIb/IIIa . GPIIb/IIIa was purified from outdated human
platelets by passing platelet lysates sequentially over con-
canavalin A affinity, Sepharose 4B-hexyl-RGDS affinity and
Sephacryl S-300HR size exclusion columns by a modification
of the method of Kouns et al.29 GPIIb/IIIa fractions which
bound to the RGDS-affinity column bound fibrinogen (acti-
vated form), while the fractions purified on Sephacryl S-300HR
did not (unactivated form).
F lu or esecen ce-Ba sed Assa y of Affin ity tow a r d Tr iton
X-100 Solu blized GP IIb/IIIa . Competitive fluorescent
displacement binding measurements were done with unacti-
vated and activated forms of GP IIb/IIIa (typically 600 nM)
solubilized in Triton X-100 buffer (0.1% Triton X-100, 20 mM
Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4)
and 1 µM of 19 (L-736,622) at room temperature. Changes in
the fluorescence of the solution upon addition of nonfluorescent
fibrinogen receptor antagonists were recorded and, after
correction for the fluorescence of GPIIb/IIIa and subtraction
of the fluorescence of L-736,622 in the buffer, were plotted as
percent of fluorescence of the initial solution (nonfluorescent
ligand present). The values of KD were calculated from
displacement measurement using the KD value of L-736,622
of 3.7 nM obtained in stopped-flow measurements.17,20
Scin tilla tion P r oxim ity Ba sed Assa y for Affin ity of a n
Act iva t ed F or m of GP IIb /IIIa . Purified GPIIb/IIIa was
activated by coating onto yttrium silicate Scintillation Proxim-
ity Assay Fluoromicrospheres (Amersham RPN 143) and is
tion (5000g for 10 min in
a fixed angle rotor) and the
supernatant decanted. Following an additional centrifugation
(5000g for 5 min) the remaining supernatant was pipetted off,
and the platelet pellet was resuspendended in 60 µL of 10 µM
unlabeled 5 and allowed to stand overnight to dissociate all
bound radiolabel. An aliquot (50 µL) was counted in 4.5 mL
of Readysafe scintillation cocktail and counted on a â counter.
The specific binding (CPMBound) was determined from the
difference in bound counts in the absence and presence of a
large excess of unlabeled 5. The concentration of total [3H]-5
required to produce half-maximal binding (K1/2) was obtained
by a nonlinear least-squares fit of the binding isotherm to
CPMBound ) Bmax/(1 + K1/2/[[3H]-5] where Bmax is the maximum
amount of specific binding. The concentration of GPIIb/IIIa
receptors were determined from the value of Bmax and the
measured specific radioactivity of [3H]-5. The concentration
of free [3H]-5 required to produce half-maximal binding (KD)
was calculated from the observed K1/2 after correction by
subtraction of 50.
In Vivo P h a r m a cology. All animals were cared for under
the standards of the NIH Guide for the Care and Use of
Laboratory Animals. All studies were reviewed by an instu-
tional aminal care and use committiee (MRL-20P).
Sin gle Dose In tr a ven ou s Ad m in istr a tion of Com -
p ou n d s to Con sciou s Dogs. Conscious purpose-bred, labo-
ratory-raised dogs of each sex were administered compounds
intravenously in sterile saline. During these studies, dogs
rested comfortably in nylon slings. At specified time points,
blood samples (6 mL) were drawn from either the saphenous
or cephalic veins (0.38% sodium citrate, final concentration).
Whole blood platelet counts were determined (1 mL), and the
remainder of the sample was used for the preparation of PRP
for ex vivo platelet aggregation as described below. For each
treatment group, blood samples were obtained before com-
pound administration (baseline) and at 1, 5, 15, 30, 45, 60,
75, 90,120, 180, 240, 300, 360, 420, and 480 min after dosing.
Measu r em en t of ex Vivo P latelet Aggr egation . Platelet-
rich plasma (PRP) was prepared by centrifugation of whole
blood at 150g for 5 min, and the platelet count was adjusted
to 2 × 108 platelets/mL with time matched platelet-poor
plasma (PPP). PRP (300 µL, 2 × 108 platelets/mL) was
incubated at 37 °C for 3 min prior to the addition of agonists.
Platelet aggregation was measured by the change in light
transmittance (PPP represents 100%) under stirring conditions
(1100 rpm) at 37 °C in a Biodata Platelet Aggregation Profiler,
Model PAP-4 and was initiated by the addition of 10 µM ADP
+ 1 µM epinephrine or 10 µg/mL collagen + 1 µM epinephrine.
Epinephrine was used to enhance the aggregation response
of canine platelets to the other agonists. The extent of

Non-Peptide Glycoprotein IIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1787
19, 461-476. (b) Austel, V.; Himmelsbach, F.; Muller, T. Non-
peptide Fibrinogen Receptor Antagonists. Drugs Future 1994,
19, 461-476. (c) Cook, N. S.; Kottirisch, G. Zerwes, H.-G. Platelet
Glycoprotein IIb/IIIa Antagonists. Drugs Future 1994, 19,
135.159.
aggregation is reported as the peak percent of aggregation
achieved based on a maximum of 100%, and the maximum
slope represents the maximum sustained rate of the aggre-
gatory response. The effect of a compound on the extent and
rate of platelet aggregation is expressed as the percent
inhibition using the baseline, pretreatment response as 100%.
(15) (a) Ku, T. W.; Ali, F. E.; Barton, L. S.; Bean, J . W.; Bondinell,
W. E.; Burges, J . L. Callahan, J . F.; Calco, R. R.; Chen. L.;
Eggleston, D. S.; Gleason, J . G.; Huffman, W. F.; Hwang, S. M.;
J akas, D. R.; Karash, C. B.; Keenan, R. M.; Kopple, K. D.; Miller
W. H.; Newlander, K. A.; Nichols, A.; Parker, M. F.; Peishoff, C.
E.; Samanen, J . M. ; Uzinskas, I.; Venslavsky, J . W. Direct
Design of a Potent Non-peptide Fibrinogen Receptor Antagonist
Based on the Strucutre and Conformation of a Highly Con-
strained Cyclic RGD Peptide. J . Am. Chem. Soc. 1993, 115,
8861-8862. (b) McDowell, R. S.; Gadek, T. R.; Barker, P. L.;
Burdick, D. J .; Chan, K. S.; Quan, C. L.; Skelton, N.; Struble,
M.; Thorsett, E. D.; Tischler, M.; Tom. J . Y.; Webb, T. R.;
Burnier, J . P. From peptide to non-peptide. 1. The Elucidation
of a Bioactive Conformation of the Arginine-Glycine-Aspartic
Acid Recognition Sequence. J . Am. Chem. Soc. 1994, 116, 5069-
5076. (c) McDowell, R. S.; Blackburn, B. K.; Gadek, T. R.; McGee,
L. R.; Rawson, T.; Reynolds, M. E.; Robarge, K. D. Sommers, T.
C.; Thorsett, E. D.; Tischler, M.; Webb, T. R.;Venuti, M. C. From
Peptide to Non-Peptide. 2. The De Novo Design of Potent, Non-
Peptidal Inhibitors of Platelet Aggregation Based on a Benzo-
diazepinedione Scaffold. J . Am. Chem. Soc. 1994, 116, 5077-
5083. (d) Ku, T. W.; Miller W. H.; Bondinell, W. E.; Erhard, K.
F.; Keenan, R. M.; Nichols, A.; Peishoff, C. E.; Samanen, J . M.;
Wong, A. S.; Huffman, W. F.; Potent Non-Peptide Fibrinogen
Receptor Antagonists Which Present an Alternate Pharmacoph-
ore. J . Med. Chem. 1995, 38, 9-12. (e) Stilz, H. U.; J ablonka,
B.; J ust, M.; Knolle, J .; Paulus, E. F.; Zoller, G. Discovery of an
Orally Active Non-Peptide Fibrinogen Receptor Antagonist. J .
Med. Chem. 1995, 39, 2118-2122. (f) Weller, T.; Alig, L. Beresini,
M.; Blackburn, B.; Bunting, S.; Hadva´ry, P.; Mu¨ller, H. M.;
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Sin gle Dose Or a l Ad m in istr a tion of Com p ou n d s to
Con sciou s Dogs by Ga str ic La va ge or Ca p su le. Conscious
purpose-bred, laboratory-raised dogs of each sex were admin-
istered compounds orally by gastric lavage in aqueous solution
(5 mL volume with sterile water as vehicle) or orally in gelatin
capsules. During these studies, dogs rested comfortably in
nylon slings. At specified time points, blood samples (6 mL)
were drawn from either the saphenous or cephalic veins (0.38%
sodium citrate, final concentration). Whole blood platelet
counts were determined (1 mL), and the remainder of the
sample was used for the preparation of PRP for ex vivo platelet
aggregation as described above. For each treatment group,
blood samples were obtained before compound administration
(baseline) and at 20, 40, 70, 90, 150, 200, 250, 300, 350, and
480 min after compound administration.
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a
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J M9608117