Non-peptide GPIIb/IIIa inhibitors. 20. Centrally constrained thienothiophene α-sulfonamides are potent, long acting in vivo inhibitors of platelet aggregation

Article abstract of DOI:10.1021/jm980722p

The synthesis and pharmacology of 4, a potent thienothiophene non- peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC₅₀ of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion- controlled process (k(on) = 3.3 x 10⁶ M^-1 s^-1) and that 4 binds to dog and human platelets with comparable affinity (K(d) = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 mg/kg, and an oral dose of 50-90 mg/kg followed by low daily doses of 10 mg/kg was sufficient to maintain ~80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 ± 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.

Full text of DOI:10.1021/jm980722p

J . Med. Chem. 1999, 42, 2409-2421
2409
Non -P ep tid e GP IIb/IIIa In h ibitor s. 20. Cen tr a lly Con str a in ed Th ien oth iop h en e
r-Su lfon a m id es Ar e P oten t, Lon g Actin g in Vivo In h ibitor s of P la telet
Aggr ega tion
Melissa S. Egbertson,* J acquelynn J . Cook,* Bohumil Bednar,* J ohn D. Prugh,* Rodney A. Bednar,
Stanley L. Gaul, Robert J . Gould, George D. Hartman, Carl F. Homnick, Marie A. Holahan, Laura A. Libby,
J oseph J . Lynch, J r., Robert J . Lynch, Gary R. Sitko, Maria T. Stranieri, and Laura M. Vassallo
Departments of Medicinal Chemistry and Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486
Received December 21, 1998
The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor
antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets
with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/
IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies
revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon ) 3.3 ×
106 M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd ) 0.04
and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by
an iv dose of 5 mg/kg, and an oral dose of 50-90 mg/kg followed by low daily doses of 10 mg/kg
was sufficient to maintain ∼80% inhibition of ex vivo platelet aggregation over several days.
Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 ( 7% resulted in a
moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an
approximately 10-min bleeding time. Additional doses were required to increase the bleeding
time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful
and safe separation of efficacy and bleeding time.
In tr od u ction
The binding of fibrinogen to platelet glycoprotein
GPIIb/IIIa (fibrinogen receptor) represents a final path-
way in both hemostasis and pathologically critical
thrombosis,1 and can be inhibited by proteins or pep-
tides containing an RGD tripeptide or its synthetic
mimetics.2,3 We have found two key structural modifica-
tions of some RGD mimics that have led to improved
potency. First, R-carbon substitution of a tyrosine-like
lead compound with sulfonamides improved potency
from the micromolar to the low-nanomolar level. The
potent n-butylsulfonamide intravenous therapeutic Ag-
grastat (1) (Figure 1) demonstrates the potency-enhanc-
ing affect of the R-sulfonamide moiety in this series.4,5
Second, we proposed that potent inhibitors could be
prepared by incorporation of an element of geometric
(structural) constraint at the center of the molecule to
direct the vectors of the N- and C-terminal chains. In
F igu r e 1. Small molecule inhibitors of GPIIb/IIIa.
practice, an isoindolinone moiety proved to be an
effective “central constraint”, and the nanomolar inhibi-
tor 2 was found to be orally active.6,7 Recently we have
outlined how the combination of these two key elements
of structure led to potent, orally active GPIIb/IIIa
inhibitors such as the R-pyridylsulfonamide isoindoli-
none 3.8 Compound 3 has a binding affinity for isolated
receptors9 similar to that of 1 and greater than that of
2 and demonstrates greatly improved oral activity in
dogs when compared to 1 and 2. The success of the
isoindolinone central constraint prompted us to screen
other types of bicyclic central constraints for potency
and optimal oral profile. This paper describes the
substitution of the isoindolinone central constraint with
a thienothiophene central constraint, the additional
improvements in potency associated with that substitu-
tion,10 and how this approach led to the identification
of 4 as an oral fibrinogen receptor antagonist suitable
for once-a-day dosing in dogs.
Ch em istr y
We chose to combine the N-terminal and central
portions of analogues first, so as to allow for convenient
derivatization at the C-terminus. Construction of the
substituted thienothiophenes 9 and 11 proceeded from
initial alkylation of the [2,3-b]- and [3,2-b]thienothiophene
central constraints with iodide 5. An example of our
initial synthetic approach is outlined in Scheme 1.
10.1021/jm980722p CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/15/1999

2410 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Egbertson et al.
Sch em e 1a
Sch em e 3a
a
(a) THF/n-butyllithium/5/-78 °C (45%); (b) TFA/acetone/5 min
(93%); (c) NaClO2/tert-butanol/NaH2PO4/H2O/2-methyl-2-butene
(83%).
Sch em e 2a
(a) 1 M BH3/THF, 0 °C; (b) PCC/CH2Cl2 (69% for two steps); (c)
n-BuLi, 78 °C, 2-(dioxolanyl)thieno[2,3-b]thiophene11 (77%); (d) 1
N KHSO4 (98%); (e) 2-methyl-2-butene, NaClO2 (76%); (f) Et3SiH/
TFA; (g) BOC2O/TEA/THF/H2O.
no group, deprotection of the 2-amino group, sulfonyla-
tion, and deprotection of the 3- amino group (Scheme
4).
a
(a) 10:1 THF/HMPA, n-butyllithium/5/-78 °C (80%).
N-BOC-piperidinylethyl iodide (5) was prepared from
N-BOC-piperidinylethyl alcohol5 using standard condi-
tions. The lithio anion of the [3,2-b]thienothiophene
acetal 611 or its [2,3-b] analogue was generated at -78
°C with n-butyllithium, and the alkylation proceeded
in 45% yield. The acetal 7 was then converted to the
acid 9 in high yield through deprotection with TFA
followed by oxidation with sodium hypochlorite (77% for
two steps). A [2,3-b]thienothiophene analogue of 9
possessing a three-carbon linker between the piperidine
and thienothiophene moieties was prepared in a manner
similar to that used for 9, using the appropriate N-BOC-
piperidinylpropyl iodide5 (compound 10, not shown).
While this sequence worked well, we sought a way to
reduce the number of transformations required. In the
[2,3-b] series, direct alkylation of the appropriate [2,3-
b] carboxylic acid 11 made further adjustment of the
fragment’s oxidation state unnecessary (Scheme 2). The
solubility and reactivity of the thienothiophene acid
mono- and dianions proved to be problematic but could
be improved by using HMPA as a cosolvent in the
reaction. In the absence of HMPA, greater than 50% of
the alkylation took place at the kinetically more reactive
3-position of the thienothiophene. In the presence of
HMPA, lithiation ortho to the carboxylate was dis-
rupted, the desired 5-anion predominated, and 12 was
obtained in 80% yield.
An analogue possessing only one methylene unit
between the piperidine and thienothiophene moieties
was prepared as outlined in Scheme 3. Commercially
available 4-carboxypiperidine was protected and the
acid converted in two steps to aldehyde 15. Addition of
the lithium anion of 2-(2-dioxolanyl)thieno[2,3-b]thio-
phene11 gave alcohol 16. The aldehyde was then un-
masked and converted to the acid 18. Reduction of the
secondary alcohol to 19 with triethylsilyl hydride was
accompanied by loss of the BOC group, which was
reinstalled to give 20.
The R-isomer of 26a (26h ) was accessed through
Hoffman rearrangement of 2-(R)-N-CBZ-asparagine to
give the R-isomer of 21 which was then derivatized as
shown in Scheme 4. The (R)-phenylsulfonamide ana-
logue was prepared by Hoffman rearrangement of 2-(R)-
N-(phenylsulfonyl)asparagine to give 2-(R)-N-phenyl-
sulfonyl-3-aminopropionic acid (26i), which was used
without further derivatization.
With the appropriate derivatized thienothiophene
acids 9, 10, 12, and 20 and 2,3-diaminopropionic acid
R-sulfonamides 26 in hand, coupling proceeded cleanly
to give 27. Deprotection of the coupled products was
accomplished with different methods. In method A, the
C-terminal methyl ester and the N-terminal BOC were
removed in one step with aqueous acid to give the final
product (illustrated for 4, Scheme 5). This method was
superior to method B (basic hydrolysis of the ester,
followed by BOC removal with HCl/EtOAc) as it mini-
mized racemization at the R-stereocenter (see Experi-
mental Section).
Resu lts a n d Discu ssion
In our efforts to screen other types of bicyclic central
constraints for potency and optimal oral profile, we first
considered the physical characteristics of the isoindoli-
none lead compounds. The isoindolinone group contrib-
utes polarity to the overall structure; the isoindolinone
analogues of 3, for example, commonly exhibit log P
values of -3 or less. The N-terminal amide may be
important for hydrogen-binding interactions. The vec-
tors of the side chains are not directed in a perfectly
linear fashion.12 We decided to consider other bicyclic
systems as central constraints that would differ from
the isoindolinone in their polarity and side-chain-
directing characteristics.
Thienothiophenes offer a number of interesting fea-
tures as bicyclic central constraints. First, the vectors
of the side chains in a 2,5-substituted thienothiophene
would be similar to, but more linearly disposed than,
those of the isoindolinone. The absence of the N-
The 2,3-diaminopropanoic acid derivatives 26 were
prepared from commercially available 21 through a
sequence of esterification, BOC protection of the 3-ami-

Non-Peptide GPIIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2411
Sch em e 4a
a
(a) MeOH/SOCl2 (99%); (b) di-tert-butyl dicarbonate/NaHCO3/CH2Cl2 (95%); (c) H2, 10% Pd/C, EtOH (99%); (d) R1SO2Cl/CH2Cl2/
pyridine (∼60%); (e) HCl/EtOAc, -70 to 0 °C (100%).
Sch em e 5a
Ta ble 1. Comparison of Backbone Potencies
inhibition of platelet
no.
R
aggregation IC50 (nM)a
27
2
28
29
410
500
a
(a) THF/BOP/NMM, -40 °C to room temperature (87%); (b) 6
N HCl/dioxane (71%).
a
terminal amide provided an opportunity to test our
assumptions about the role of this functional group in
determining potency. Also, the [2,3-b] and [3,2-b] re-
gioisomers provided an additional probe of polar het-
eroatom interactions in the central region. The thieno-
thiophene substitution was therefore studied in both
substituted and unsubstituted R-carbon analogues to
provide comparison of the potency-contributing abilities
of the two types of central constraints and to test the
effect of R-sulfonamide substitution in this series.
The R-unsubstituted isoindolinone 2 inhibits ADP-
induced platelet aggregation with an IC50 of 27 nM
(Table 1).6 Both the [2,3-b] (28) and [3,2-b] (29)
thienothiophenes were less potent, with IC50 values of
410 and 500 nM, respectively. A moderate change in
polarity was observed (log P 28 was -2.3 versus <-3
for 2). These results suggested that the overall polarity
of the structures was due to the presence of the polar
C- and N-terminal substituents and that the presence
of the N-terminal amide bond and the side-chain vectors
in the isoindolinone compounds may be important
binding features for R-unsubstituted compounds that
are lacking in thienothiophene backbone compounds.
However, when the thienothiophene central con-
straints were combined with R-sulfonamide substitu-
tion, very potent compounds resulted (Tables 2 and 3).
Inhibition of platelet aggregation for both [2,3-b]- and
[3,2-b]thienothiophenes substituted with either alkyl-
sulfonamides (30-32, 40-42) or arylsulfonamides (33-
37, 43-45) occurred at low-nanomolar concentrations,
comparable to the results obtained for R-arylsulfona-
Inhibition of aggregation of human gel-filtered platelets (GFP)
was measured by a light transmittance method at 37 °C.5a Human
gel-filtered platelets were adjusted to a concentration of 2 × 108/
mL and mixed with 0.1 mg/mL human fibrinogen, 1 mM CaCl2,
and the compound of interest. Aggregation was then initiated by
addition of the agonist (10 mM adenosine diphosphate (ADP)).
Inhibition of platelet aggregation was determined by comparison
of light transmittance values for the control and subject samples.
The IC50 was determined as the concentration necessary to inhibit
the change in light transmittance by 50%. At least two determina-
tions were made for each compound, and the IC50 values were
calculated by fitting to a four-parameter equation. The average
standard error of the IC50 determinations was (20%.
by the preparation of the despiperidinylethyl analogue
of 4, 46 (structure not shown), which was ∼1000-fold
less potent inhibitor of platelet aggregation (IC50 of 46
) 1.6 µM). The integrin specificity of compounds 4 and
30-45 was determined by comparison of their inhibition
of fibrinogen binding to platelets (Tables 2 and 3) to
their inhibition of fibrinogen, vitronectin, and fibronec-
tin binding to HUVEC (human umbilical vein endothe-
lial cells),13 for which the compounds showed IC50’s
greater than 200 µM. Compound 4 was at least 35 000-
fold selective between platelet and HUVEC fibrinogen
receptors. Thus the thienothiophene central constraints,
despite their lack of potency as R-unsubstituted ana-
logues, were shown to combine in a synergistic manner
with the R-sulfonamide substitution to give very potent
and selective analogues.
This result was rewarding, since many but not all
RGD mimetics demonstrate a large enhancement in
potency upon substitution at the R-carbon.14 Without
X-ray data to examine the different binding geometries
of different series, it is difficult to explain why this is
so. However, empirically one can draw a distinction
between compounds that are more peptide-like in
structure and that have more flexibility in their back-
bone in the region next to the C-terminal amide bond
(the glycine-mimicking portion) and those that are more
mide compounds in the isoindolinone series (3, IC50
)
15 nM, for example). The 3-pyridylsulfonamide 4 showed
an IC50 of 8 nM. The R-enantiomers of both the 3-pyridyl
and phenyl analogues appeared slightly less potent (37
and 34, 2- and 4-fold, respectively) than the S-enanti-
omers. That the piperidinylethyl side chain was still a
necessary requirement for optimal potency was shown

2412 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Ta ble 2. Sulfonamide Derivatives of [2,3-b]Thienothiophenes
Egbertson et al.
not benefit the second group as much.7i The work of
Gartner and co-workers in the RGD peptides indicated
that the glycine portion is sensitive to substitution and
that bulky substitution is especially detrimental to
potency.15 The different steric demands that arise from
more or less flexibility in the center of the molecule may
have a profound effect on the accessibility of binding
pocket(s) to the substituents at the C-terminus.
inhibition
platelet
flow
fold
aggregation
SPA-A cytometry difference
no. n
R
IC50 (nM)a ED50 (nM)b KD (nM)c KD/ED50
d
We found the high potency of all the thienothiophene
R-sulfonamide analogues, despite their obvious differ-
ences in structural features, an interesting result.
Previous work in the isoindolinone series8 had suggested
that the potency of tightly binding R-substituted com-
pounds was not being accurately assessed by the in vitro
platelet aggregation assay.16 To better measure the
intrinsic differences in potency between very potent
compounds, we have isolated the unactivated form of
platelet receptor GPIIb/IIIa and have developed an
assay based on purified receptor. In this assay, ED50
values are calculated from the competitive binding
between compounds of interest and the fibrinogen
receptor antagonist [125I]L-692,884 to purified GPIIb/
IIIa activated by coating onto yttrium silicate scintil-
lation proximity assay fluomicrospheres (SPA-A assay,
see In Vitro Pharmacology, Experimental Section).
Binding measurements with fibrinogen indicate that the
SPA-A assay provides a close estimate of binding affinity
for an activated form of GPIIb/IIIa.17
1
2
3
see Figure 1
see Figure 1
see Figure 1
11
27
15
15
32
7
0.97
13
0.38
1.2
0.12
0.13
0.03
15
550
2.2
52
5.6
15
42
5.7
43
46
9.2
30 2 CH3
31 2 (CH2)3CH3
32 2 (CH2)2OCH2CH3
33 2 C6H5
34 2 C6H5 (R-isomer)
35 2 4-ClC6H5
36 2 2-thienyl
1.2
0.023
8
0.76
36
11
9
8
19
0.09
0.04
0.047
0.47
0.026
0.15
0.07
0.28
3.75
1.48
4
2 3-pyridyl
37 2 3-pyridyl
(R-isomer)
28
59
38 0 3-pyridyl
39 3 3-pyridyl
21
23
0.45
0.07
22
17
48
242
See Table 1. Measured by competition with [125I]L-692,884
for binding to purified GPIIb/IIIa activated by coating onto yttrium
silicate scintillation proximity assay fluomicrospheres. An average
standard error of mean of (20% was obtained for all compounds.
For details, see the In Vitro Pharmacology section of the Experi-
mental Section. c Measured by displacement of the fluorescent
ligand L-762,745 from human platelets by flow cytometry from at
least three different platelet donors. The standard deviation of the
measurements was for all compounds <20%. The value for 4 is
0.07 ( 0.006 nM (mean ( STD) with n ) 20. For details, see the
a
b
Using the more sensitive SPA-A assay, the R-3-
pyridylsulfonamide 3 was shown to be 34-fold more
potent than 2 (ED50 ) 0.38 versus 13 nM). The
thienothiophene analogues also proved to be more
potent than 2, and many were revealed to be more
potent than 3 (Table 2). In addition, it became apparent
that analogues at the sulfonamide side chain exert a
structural influence on potency that closely parallels the
results seen in the isoindolinone series.8 While the
methylsulfonamide 30 was approximately 3-fold less
potent than 3 (ED50 ) 1.2 vs 0.38 nM), longer alkyl
chain length was associated with improved potency, (n-
butylsulfonamide 31 ED50 ) 0.12 nM, a 2-fold improve-
ment), and incorporation of an oxygen in the chain did
not negatively affect potency (32, ED50 ) 0.13 nM).
Arylsulfonamides 33-39 were, in general, even more
potent than the alkylsulfonamides (ED50’s ) 0.03-0.09
nM), and direct comparison of 3 and 4 reveals the
contribution of the [2,3-b]thienothiophene backbone: 4
was ∼8-fold more potent than 3 (ED50 ) 0.047 vs 0.38
nM). Similarly, the phenylsulfonamide 33 was ∼12-fold
more potent than 3.10 Thus the combination of the
R-arylsulfonamides and the [2,3-b]thienothiophene back-
bone acts in a synergistic manner to give compounds
with approximately 10-fold improvements in potency
over the isoindolinone series.
d
In Vitro Pharmacology section of the Experimental Section. Fold
difference in activity as determined by ratio of flow cytometry KD
divided by SPA-A ED50
.
Ta ble 3. Sulfonamide Derivatives of [3,2-b]Thienothiophenes
inhibition
platelet
SPA-A
flow
fold
aggregation ED50 cytometry difference
IC50 (nM)a
(nM)b
KD (nM)c KD/ED50
d
no.
R
1
2
3
see Figure 1
see Figure 1
see Figure 1
11
27
15
14
10
10
0.97
13
0.38
15
550
15
42
0.5
0.2
40 CH3
41 (CH2)3CH3
42 (CH2)2-
OCH2CH3
43 C6H5
44 2-thienyl
45 3-pyridyl
0.21
0.27
3.0
1.1
14
4
7
13
15
0.08
0.04
0.11
0.15
0.092
0.08
1.8
2.3
0.72
a
b
See Table 1. Measured by competition with [125I]L-692,884
for binding to purified GPIIb/IIIa activated by coating onto yttrium
silicate scintillation proximity assay fluomicrospheres. An average
standard error of mean of (20% was obtained for all compounds.
For details, see the In Vitro Pharmacology section of the Experi-
mental Section. c Measured by displacement of the fluorescent
ligand L-762,745 from human platelets by flow cytometry. The
standard deviation of the measurements was for all compounds
<20%. For details, see the In Vitro Pharmacology section of the
The more sensitive assay also allowed a better dif-
ferentiation between the S- and R-enantiomers of 4. The
R-enantiomer 37 was shown to be approximately 10-
fold less potent than the S-enantiomer 4 (ED50 ) 0.42
vs 0.047 nM, respectively). Also, longer separation of
the N-terminal piperidine from the central constraint
appeared to be tolerated better than chain shortening,
as was shown by comparison of 38 and 39 (ED50 ) 0.07
vs 0.45 nM, respectively). The [3,2-b]thienothiophene
backbone appears to be somewhat less potency-enhanc-
d
Experimental Section. Fold difference in activity as determined
by ratio of flow cytometry KD divided by SPA-A ED50
.
constrained, usually by a flat, aromatic ring, in the same
region. The first group tends not to benefit as much from
R-substitution as the latter does. Conversely, substitu-
tion at the â-carbon that benefits the first group does

Non-Peptide GPIIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2413
ing than is the [2,3-b] central constraint, as shown by
compounds 42 and 45 (ED50 ) 0.27 and 0.11 nM,
respectively).
We had, therefore, identified a series of compounds
that showed high affinity for a purified, activated form
of the receptor. The affinity of thienothiophene ana-
logues for resting human platelets was then measured
by flow cytometry using competitive displacement of
fluorescein-containing isoindolinone GPIIb/IIIa antago-
nist L-762,745.9,18 Similar trends in potency were
observed on resting platelets, but the differences were
more pronounced (Tables 2 and 3). For example, 4 was
31-fold more potent than 3 on resting platelets (as
compared to 8-fold more potent against the activated
receptor), and 33 was 95-fold more potent than 3 (vs
12-fold). Interestingly, the R-enantiomer 37, which is
10-fold less potent than 4 in binding to activated
receptor, is 400-fold less potent than 4 in binding to
resting platelets. The longer chain analogue 39, which
is well-tolerated against the activated receptor (only a
1.5-fold decrease relative to 4) compared to the short
chain analogue 38 (9.5-fold decrease), is similarly as
poor as the short chained analogue 38 in resting
platelets (242- and 312-fold decreases, respectively).
Thus, these variations in structure seem to have more
effect on the potency against resting platelets than
against an activated form of the receptor.
F igu r e 2. Single-dose intravenous and oral administration
of 3 and 4 to conscious dogs and effects on inhibition of ex
vivo ADP-stimulated platelet aggregation (PRP, n ) 2-5/
group).
(Table 2), the flow cytometric measurements18 provided
the dissociation rate constant koff of 2.3 × 10-4 s-1, which
gives the t1/2 for dissociation of the compound from the
receptor of about 50 min. Using these binding param-
eters one can calculate the association rate constant kon
as 3.3 × 106 M-1 s-1. This value for kon indicates that
binding of 4 to the resting form of the receptor is a
diffusion-controlled process.
Flow cytometric measurements of the dissociation
constant on resting dog platelets provided a Kd value of
0.04 nM. This value indicates that the affinity of 4 for
receptors on resting dog platelets is comparable to the
affinity for the receptors on resting human platelets (Kd
) 0.07 nM). The high affinity of 4 for nonhuman
platelets has made this compound a useful tool for the
exploration of platelet function.
Sin gle-Dose In tr a ven ou s a n d Or a l Ad m in istr a -
tion of 4 to Con sciou s Dogs. The effects of the single-
dose intravenous bolus administration of 3 and 5 µg/kg
and the oral administration of 50, 100, and 200 µg/kg 4
on ex vivo platelet aggregation responses to ADP
(maximum extent of aggregation) in conscious dogs are
depicted in Figure 2 and compared to the iv and po
traces for 3. For 4, a potent, dose-related effect was
observed with iv and oral administration; complete
inhibition of platelet aggregation was achieved with the
extremely low dose of 5 mg/ kg iv. In addition, similar
effects were observed with the more potent platelet
agonist, collagen (data not shown). Error bars have been
omitted from Figure 2 for clarity. It should be noted,
however, that there was variability among animals with
the lowest iv (inhibition ) 61( 21 at 3 mg/kg at 4 h
postdose, approximately steady state, compound 4) and
po (peak inhibition ) 48 ( 15 at 50 mg/kg, compound
4) doses presumably due to the steep dose reponse with
these compounds. With increasing antiplatelet effects
(90-100% inhibition) at the higher doses, results were
very consistent (peak inhibition ) 99 ( 6 at 100 mg/kg
po, compound 4) among dogs. When high levels of
inhibition were achieved, the effects were maintained
for the duration of the 8-h protocol, suggesting extended
duration of activity. These results suggest minimal dose
requirement as well as low frequency of administration.
Recently, we have demonstrated that GPIIb/IIIa
antagonists can be separated into two classes15 based
on their affinity for resting and activated forms of
GPIIb/IIIa. One class binds to both resting and activated
forms of purified GPIIb/IIIa and resting and activated
platelets, with similar KD values. The other class of
compounds binds with much higher affinity to the
activated form of GPIIb/IIIa (purified or on platelets)
as compared with the resting form.19 Thienothiophene
analogues can also be separated into these two classes.
Compounds in the first class, represented by arylsul-
fonamide compounds 4, 33, 35, and 43-45, have similar
ED50 and Kd values indicating similar affinity for both
forms of the receptor (see fold difference columns, Tables
2 and 3). The affinity of the second class of compounds
(which includes the alkylsulfonamides and length-varied
analogues) for the resting form of the receptor is at least
5-fold less than the affinity for the activated form of the
receptor.20 Previous experiments suggested that the
binding site for RGD peptides on resting platelets may
resemble a narrow cavity buried 10-20 Å below the
surface of the receptor.21 Thus, the differences in the
affinities of 3-pyridyl derivatives 4 and 37-39 (Table
2) in binding to the resting and activated forms of the
receptor may indicate that the activation of the receptor
results in an increase in the size of the binding cavity
or exposure of the binding site to a position closer to
the surface of the receptor.
The high affinity of 4 for both resting and activated
forms of the receptor suggested a unique potential of
this compound in blocking GPIIb/IIIa. Compared to the
previously reported, very potent pyrazolodiazepinone
analogue L-738,167,7j,22 4 is approximately 1.5-fold more
potent against resting human platelets. Therefore, we
became interested in further characterization of the
binding parameters of this compound to human plate-
lets. In addition to the equilibrium dissociation constant

2414 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Egbertson et al.
In comparison, the iv administration of 3 at 10 mg/kg
also resulted in 100% inhibition, but this activity
decreased rapidly over 8 h.
It is likely that the very high affinity of 4 for GPIIb/
IIIa on resting platelets produces the long duration of
effect observed in dogs. However, the high affinity of 4
cannot fully explain the long duration since the t1/2 for
dissociation of the drug from the receptors is only 50
min. Therefore, the mechanism of long duration for this
highly potent fibrinogen receptor antagonist must reflect
involvement of other parameters in the clearance of the
drug from the blood. The first and most critical param-
eter for high occupancy of the receptors by the drug is
the concentration of receptors, typically 20-40 nM,
which is more than 100-fold higher than the dissociation
constant of the drug. Thus, 4 titrates the receptors, and
once all receptors are occupied (a fast event since the
on-rate for 4 is diffusion-controlled), the excess of free
drug is cleared from the blood with a t1/2 of ∼30 min.7j
The critical part of the mechanism of clearance of the
drug from blood for a long duration compound is the
competition between the clearance of free drug and
rebinding of the drug to unoccupied receptors. Since the
rebinding of the drug is much faster than the clearance
of the free drug, the resulting duration of the drug is
much longer than what would be predicted based purely
on the off-rate. Thus, the duration of action for fibrino-
gen receptor antagonists depends on the dissociation
constant and the rate of the clearance of the free drug
from the plasma. If the clearance of free drug is similar
for different low-molecular-weight FRAs, the critical
parameter to compare pharmacodynamic duration for
different FRAs would be the dissociation constant. And
indeed 3, which has a dissociation constant 31-fold
higher than that for 4 (Table 2), has a much shorter
duration of action when compared with the highly
potent 4 (Figure 2).
F igu r e 3. Once-daily oral administration of 4 for 6 days to
conscious dogs and effects on inhibition of ex vivo ADP-
stimulated platelet aggregation (PRP). Individualized loading
doses of 50-90 µg/kg 4 were followed by individualized doses
of 0, 10, 20, or 30 µg/kg doses for days 2-3 and 10 µg/kg for
days 4-6. For further details, see Experimental Section.
F igu r e 4. Ex vivo platelet aggregation and template bleeding
time relationship. Effect of 4 on inhhibition of ex vivo ADP-
stimulated platelet aggregation (PRP) and simultaneous mea-
surement of effect of 4 on buccal mucosal template bleeding
time.
On ce-Da ily Or a l Ad m in istr a tion of 4 for 6 Da ys
to Con sciou s Dogs by Ga str ic La va ge: Ma in te-
n a n ce of In h ibition of P la telet F u n ction . To deter-
mine if the extended duration of 4 would permit a once-
daily dosing regime without loss of efficacy, trough levels
of inhibition of the extent of ADP-induced ex vivo
platelet aggregation were monitored for 6 days of once
daily oral dosing (a single bolus followed by low-dose
maintenance) and for 12 days following the termination
of dosing. The results of this study demonstrated that
despite normal variability among individual animals,
submaximal levels of inhibition of platelet function could
be maintained with low (10 mg/kg) daily doses of
compound. Results for two groups of dogs are shown in
Figure 3: the maintenance group (six once-daily doses)
received 6 days of dosing and is compared to a separate
group of dogs that received a single dose only. A clear
benefit of the low, daily dose can be seen in the
maintenance group in comparison to the control group
by day 4 of the study.
of 77 ( 7% corresponded to a moderate (2.5-fold base)
elevation in bleeding time. Although several bolus
infusions could achieve complete inhibition of ADP-
induced platelet aggregation and were accompanied by
approximately 10-min template bleeding time, addi-
tional infusions beyond these were required for the
prolongation of bleeding time to the maximum time
allowed in this protocol (15 min). Figure 4 shows that
additional doses were required to inhibit aggregation
by the more potent agonist collagen. This could suggest
that additional GP IIb/IIIa receptors exposed upon
activation by the more potent agonist require increased
inhibitor concentrations to block aggregation and that
their additional exposure is associated with elevated
bleeding. For example, 80% inhibition of ADP-induced
aggregation is accompanied by bleeding times of ∼6 min,
whereas 80% of collagen-induced aggregation results in
∼10-min bleeding times. This separation between ef-
ficacy, as indicated by inhibition of aggregation, and
bleeding time prolongation suggests the potential for
safe, efficacious therapy with this compound.
Rela tion sh ip betw een Effects of In tr a ven ou s 4
on ex Vivo P la t elet Aggr ega t ion R esp on ses a n d
Bleed in g Tim es in An esth etized Dogs. The effect of
increasing iv bolus doses of 4 on template bleeding times
relative to inhibition of the extent of ex vivo platelet
aggregation induced by ADP in anesthetized dogs is
depicted in Figure 4. Inhibition of platelet aggregation

Non-Peptide GPIIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2415
(s, 1H), 6.14 (s, 1H), 4.08 (m, 8H), 2.90 (t, J ) 8 Hz, 2H), 2.65
(app. t, J ) 13 Hz, 2H), 1.70 (m, 5H), 1.46 (s, 9H), 1.14 (m,
2H).
Su m m a r y
Compound 4 is a potent inhibitor of in vitro and in
vivo platelet aggregation and demonstrated an 8-fold
improvement in affinity for isolated GPIIb/IIIa receptors
over analogues possessing an isoindolinone backbone
and a one-and 1.5-fold improvement over the pyrazolo-
diazepinone-containing compound L-738,167. The very
tight binding of 4 revealed by flow cytometry studies
makes 4 a useful tool for the study of platelet function.
Low doses of 4 were sufficient to maintain ∼80%
inhibition of ex vivo platelet aggregation over several
days in dogs. Significant levels of inhibition of ADP-
induced platelet aggregation resulted in a moderate 2.5-
fold increase in bleeding time, while additional doses
were required to increase the bleeding time to the
maximum time allowed in the protocol (15 min), thus
indicating a potentially useful and safe separation of
efficacy and bleeding time.
5-[2-[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in -4-yl]eth yl]-
th ien o[3,2-b]th iop h en e-2-ca r boxya ld eh yd e (8). A solution
of 7 (0.458 g, 1.08 mmol) in acetone (20 mL) was treated with
2.2 g of trifluoroacetic acid for 5 min and then promptly diluted
with EtOAc (100 mL). This solution was extracted with
saturated Na2CO3 solution (25 mL) and H2O, dried (MgSO4),
and concentrated to give 8 (0.38 g, 93%). NMR (300 MHz,
CDCl3): δ 9.93 (s, 1H), 7.86 (s, 1H), 7.03 (s, 1H), 4.10 (m, 4H),
2.96 (t, J ) 8 Hz, 2H), 2.69 (app. t, J ) 13 Hz, 2H), 1.71 (m,
4H), 1.45 (s, 9H), 1.15 (m, 2H).
5-[2-[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in -4-yl]eth yl]-
th ien o[3,2-b]th iop h en e-2-ca r boxylic Acid (9). A solution
of 8 (0.046 g, 0.1 mmol) in tert-butyl alcohol (3 mL) at room
temperature was treated with a solution of NaClO2 (0.1 g, 1.1
mmol) and NaH2PO4‚H2O (0.1 g, 0.72 mmol) in 1 mL of H2O
and 1 mL of 2-methyl-2-butene with stirring at room temper-
ature for 16 h. The reaction mixture was concentrated and
partitioned between EtOAc (10 mL) and 3 mL of 1 N NaHSO4.
The organic phase was dried (MgSO4), and the solvent was
removed. The residue was triturated with hexane to give pure
9 (0.040 g, 83%). NMR (300 MHz, CDCl3): δ 7.99 (s, 1H), 7.00
(s, 1H), 4.10 (bd, J ) 13 Hz, 2H), 2.93 (t, J ) 8 Hz, 2H), 2.69
(app. t, J ) 13 Hz, 2H), 1.70 (m, 3H), 1.46 (s, 9H), 1.18 (m,
2H).
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. 1H NMR
spectra were recorded on a Varian XL-300 or Varian XR-400
spectrometer as noted. Chemical shifts are reported in parts
per million relative to tetramethylsilane as the internal
standard. Elemental analyses for carbon, hydrogen, and ni-
togen were determined with Leeman Labs CE440 elemental
analyzer and are within (0.4% of the theoretical value unless
noted otherwise. Low-mass spectra were recorded with a Vg
Micromass MM7035 analytical spectrometer. Normal phase
silica gel (EM Science silica gel 60 (230-400 mesh)) was used
for chromatography. HPLC separations were done using a
semipreparative Vydac protein and peptide C18 column (cat.
#218TP1022). TLCs were developed with ninhydrin or iodine
stain. All starting materials and solvents were commercially
available and used as received. Yields are unoptimized.
2-[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in yl]eth yl Iod id e
(5). 2-[N-[(tert-Butyloxy)carbonyl]amino]ethyl alcohol5 (68.8 g,
0.3 mol), diphenylchlorophosphine (86.04 g, 0.39mol), and
imidazole (61.27 g, 0.9 mol) were placed in a 3-L flask equipped
with an argon line and an overhead stirrer, dissolved in 1000
mL of toluene, and cooled to 0 °C. Iodine (99 g, 0.39 mol) was
added in portions and the reaction stirred for 1.5 h. The
reaction mixture was added to 1000 mL of saturated Na2CO3
solution; the organic layer was separated and washed with 250
mL of saturated Na2CO3 solution. The combined Na2CO3
washes were extracted with 300 mL of toluene. The organic
layers were combined and washed successively with 2 × 250
mL of 5% Na2S2O3, 250 mL of H2O, 3 × 250 mL of 10% KHSO4,
and 200 mL of brine, dried (Na2SO4), filtered, and evaporated.
The crude product was passed through a silica gel pad using
4000 mL of 20% EtOAc/hexanes as an eluent. The solution
was concentrated to give 82 g of 5 as a viscous oil (80%). Rf
(40% EtOAc/hexanes): 0.6. NMR (300 MHz, CDCl3): δ 4.1 (d,
J ) 12.8 Hz, 2H), 3.25 (t, J ) 7.2 Hz, 2H), 2.7 (t, J ) 12.8 Hz,
2H), 1.8 (m, 2H), 1.65 (m, 3H), 1.5 (s, 3H), 1.15 (m, 2H).
5-[2-[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in -4-yl]p r op -
yl]th ien o[2,3-b]th iop h en e-2-ca r boxylic Acid (10). Pre-
pared as described for 9 except that 3-[N-[(tert-butyloxy)-
carbonyl]amino]propyl iodide5 was used to alkylate thieno[2,3-
b]thiophene-2-carboxaldehyde ethylene glycol acetal.11 NMR
(300 MHz, CDCl3): δ 7.99 (s, 1H), 6.95 (s, 1H), 4.08 (bd, J )
13 Hz, 2H), 2.87(app. t, J ) 7 Hz, 2H), 2.67 (app.t, J ) 14 Hz,
2H), 1.7 (m, 4H), 1.45 (s, 9H), 1.4 (m, 3H), 1.1 (m, 2H).
P r epar ation of In ter m ediate Acid 12. 2-Car boxyth ien o-
[2,3-b]th iop h en e (11). A suspension of methylthieno[2,3-b]-
thiophene-2-carboxylic acid11 (10 g, 0.05 mol) in THF/CH3OH/
H2O (1:1:1, 300 mL) was treated with LiOH‚H2O (4.2 g, 0.1
mol) over 24 h. The THF was then removed and the residue
acidified. The resultant precipitate was collected by vacuum
filtration, the filtrate was reacidified, and a second crop of
precipitate was collected. The combined solids were dried on
a drying pistol to yield 5.1 g (55%) of 11. The filtrate was
extracted with EtOAc, dried (MgSO4), and concentrated. The
residue was suspended in EtOAc, filtered, and the solids
washed with hexanes to obtain an additional 4.2 g (45%) of
11 as a white solid (combined yield, 100%). Rf (97/3/1 CHCl3/
CH3OH/HOAc): 0.29. NMR (400 MHz, CDCl3): δ 7.90 (s, 1H),
7.36 (d, J ) 5 Hz, 1H), 7.23 (d, J ) 5 Hz, 1H).
5-[2-[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in -4-yl]eth yl]-
th ien o[2,3-b]th iop h en e-2-ca r boxylic Acid (12). Acid 11 (5
g, 0.027 mol) was dissolved in THF (190 mL) and HMPA (19
mL, 0.11 mol) and cooled to -78 °C under argon. n-Butyl-
lithium (25.6 mL, 2.12 M in hexanes, 0.054 mol) was added
dropwise and the reaction was stirred at -78 °C for 1 h. Iodide
5 (9.3 g, 0.027 mol) in 10 mL of THF was added dropwise with
vigorous stirring while the temperature of the reaction was
kept below -65 °C. The reaction was allowed to warm slowly
to room temperature overnight and then concentrated and the
residue suspended in H2O. The solution was acidified with 10%
KHSO4 and extracted with 5 × 50 mL of CH2Cl2. The organic
extracts were washed with dilute sodium thiosulfate solution
and brine, dried over MgSO4, filtered, and evaporated to give
24 g of a dark oil that was suspended/dissolved in 10:1 Et2O/
EtOAc and washed three times with 10% KHSO4 and brine
and evaporated to give 11.7 g of a tan solid. The solid was
triturated with 1% EtOAc/hexanes and the solid collected and
dried under vacuum to give 8.66 g of 12 as a pale tan solid,
94% pure by HPLC (80% yield). This material was used
directly in the next step without further purification. Rf (9:
0.5:0.5 CH2Cl2/MeOH/HOAc): 0.66. NMR (400 MHz, CD3-
OD): δ 7.8 (s, 1H), 7.0 (s, 1H), 4.0 (d, J ) 13 Hz, 2H), 2.9 (t,
P r ep a r a t ion of In t er m ed ia t e Acid 9. 5-[2-[N-[(ter t-
Bu t yloxy)ca r b on yl]p ip er id in -4-yl]et h yl]t h ien o[3,2-b]-
th iop h en e-2-ca r boxa ld eh yd e Eth ylen e Glycol Aceta l (7).
A solution of thieno[3,2-b]thiophene-2-carboxaldehyde ethylene
glycol Acetal11 (0.212 g, 1.0 mmol) in THF (10 mL) cooled to
-78 °C was treated with n-butyllithium (1.1 mmol in hexane)
with stirring for 2 h. A solution of 5 (0.339 g 0.96 mmol) in
THF (2 mL) was added, and the resulting solution was allowed
to slowly warm to room temperature over 16 h. The solvent
was removed, the residue was quenched with ether/H2O, and
the organic phase was separated, dried (MgSO4), and concen-
trated. The residue was purified using flash chromatography
on silica gel eluting with 10-20% EtOAc/hexane to give pure
7 (0.207 g, 45%). NMR (300 MHz, CDCl3): δ 7.26 (s, 1H), 6.91

2416 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Egbertson et al.
J ) 7 Hz, 2H), 2.7 (bt, 2H), 1.7 (bd, 2H), 1.6 (m, 2H) 1.5 (m,
followed by a solution of NaClO2 (0.76 g, 8.4 mmol) and
NaHPO4 (0.76 g, 5.5 mmol) in 7.6 mL of H2O. The mixture
was stirred over 18 h, then the organic phase was separated,
and the solvent was removed. The residue was partitioned
between EtOAc and KHSO4 and the organic phase washed
with water, dried (MgSO4), filtered, and concentrated to yield
crude 18 (0.23 g, 76%). NMR (300 MHz, DMSO): δ 7.72 (s,
1H), 7.14 (s, 1H), 4.59 (d, J ) 6 Hz, 1H), 3.95 (m, 2H), 2.61
(m, 1H), 1.71 (m, 3H), 1.11 (s, 9H), 1.0 (m, 3H).
5-[(P iper idin -4-yl)m eth yl]th ien o[2,3-b]th ioph en e-2-car -
boxylic Acid (19). A mixture of 18 (0.141 g, 0.456 mmol) and
triethylsilane (1.0 g, 8.6 mmol) was treated with 3 mL of TFA
and capped with a balloon to trap the CO2 released while
stirring over 18 h. The TFA was then removed in vacuo and
the residue triturated with Et2O to give crude 19 containing
residual triethylsilane and Et2O. This material was taken on
without further purification. NMR (300 MHz, CD3OD): δ 7.84
(s, 1H), 7.10 (s, 1H), 3.48 (d, J ) 7 Hz, 2H), 2.99 (m, 4H), 1.96
(m, 3H).
1H), 1.44 (s, 9H), 1.1 (m, 2H).
P r ep a r a tion of In ter m ed ia te Acid 20. N-[(ter t-Bu tyl-
oxy)ca r bon yl]p ip er id in e-4-ca r boxylic Acid (13). A solu-
tion of Piperidine-4-carboxylic acid (12.9 g, 100 mmol) and
NaOH (4.0 g, 100 mmol) in 100 mL of H2O was diluted with
100 mL of dioxane and cooled to 0 °C. Di-tert-butyl dicarbonate
(24.0 g, 110 mmol) was added, followed by an additional 30
mL of dioxane and 130 mL of H2O. The mixture was stirred
at 0 °C for 30 min and then at ambient temperature for 2 h.
Triethylamine (10 g, 100 mmol) and di-tert-butyldicarbonate
(10 g, 46 mmol) were added, and stirring was continued over
18 h. The dioxane was removed in vacuo and the residue
partitioned betweened EtOAc and 10% KHSO4. The organic
phase was dried (MgSO4), filtered, and concentrated. The
residue was triturated with hexanes and collected by filtration
to afford 21 g (92%) of 13 as a white solid. NMR (300 MHz,
CDCl3): δ 4.02 (bd, J ) 13 Hz, 2H), 2.86 (app. t, J ) 12.5 Hz,
2H), 2.49 (m, 1H), 1.93 (m, 2H), 1.66 (m, 2H), 1.46 (s, 9H).
[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in -4-yl]m eth yl Al-
coh ol (14). A solution of 13 (21.2 g, 92.3 mmol) in 250 mL of
THF was cooled to 0 °C and treated with borane-THF (1.0
M, 277 mL, 277 mmol) over 1 h. After an additional 1 h at 0
°C, 1 N NaOH (305 mL, 305 mmol) was added, keeping the
temperature between 5 and 10 °C. The cooling bath was then
removed and the reaction allowed to reach ambident temper-
ature, at which time TLC analysis showed all 13 was con-
sumed. The THF was removed in vacuo and the residue
extracted with Et2O, washed with brine, dried (MgSO4),
filtered, and concentrated to give 14 (21.5 g, >100%) as an
oil. NMR (300 MHz, CDCl3): δ 4.12 (app. d, J ) 13 Hz, 2H),
3.50 (d, J ) 6 Hz, 2H), 2.70 (app. t, J ) 13 Hz, 2H), 1.65 (m,
3H), 1.46 (s, 9H), 1.14 (m, 2H).
N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in e-4-ca r boxya ld e-
h yd e (15). A solution of 14 (2.15 g, 10 mmol) in 17 mL of CH2-
Cl2 was added in one portion to a stirred suspension of PCC
(3.23 g, 15 mmol), freshly fused NaOAc (1.23 g, 15 mmol), and
2 g of powdered 3 Å molecular sieves. Stirring was continued
for 2 h; then the reaction was triturated with Et2O and
decanted onto a column of SiO2. The eluant was collected and
concentrated. Chromatography (SiO2, 30% EtOAc/hexanes)
gave 15 (1.46 g, 69%). NMR (300 MHz, CDCl3): δ 9.67 (s, 1H),
3.9 (app. d, J ) 13 Hz, 2 H), 2.93 (m, 2H), 2.43 (m, 1H), 1.89
(m, 2H), 1.56 (m, 2H), 1.46 (s, 9H).
5-[[N -[(t er t -B u t y lo x y )c a r b o n y l]p ip e r id in -4-y l]h y -
d r o x y m e t h y l]t h ie n o [2,3-b ]t h io p h e n e -2-c a r b o x a ld e -
h yd e Eth ylen e Glycol Aceta l (16). A solution of 2-(dioxola-
nyl)thieno[2,3-b]thiophene11 (0.212 g, 1 mmol) in 10 mL of THF
was cooled to -78 °C. n-Butyllithium (2.01 M in hexanes, 0.50
mL, 1 mmol) was added dropwise, and the resulting mixture
stirred at -78 °C for 2 h. A solution of 15 (0.213 g, 1 mmol) in
1 mL of THF was added, and the mixture stirred at -78 °C
for 3.5 h. The cooling bath was then allowed to expire and the
mixture to gradually warm to ambident temperature over 18
h. The reaction was poured into EtOAc, washed with water,
dried (MgSO4), filtered, and concentrated. Chromatography
(SiO2, 46% EtOAc/hexanes) afforded 16 (0.32 g, 77%). NMR
(300 MHz, CDCl3): δ 7.22 (s, 1H), 7.01 (s, 1H), 6.13 (s, 1H),
4.62 (m, 1H), 4.10 (m, 6H), 2.66 (m, 2H), 2.25 (m, 1H), 2.03
(m, 1H), 1.79 (m, 1H), 1.46 (s, 9H), 1.22 (m, 2H).
5-[[N -[(t er t -B u t y lo x y )c a r b o n y l]p ip e r id in -4-y l]h y -
d r o x y m e t h y l]t h ie n o [2,3-b ]t h io p h e n e -2-c a r b o x a ld e -
h yd e (17). A solution of 16 (0.32 g, 0.77 mmol) in 50 mL of
EtOAc and 20 mL of THF was treated with 1 N KHSO4 (25
mL) over 48 h. The layers were then separated, and the organic
phase was washed with H2O and saturated NaHCO3, dried
(MgSO4), filtered, and concentrated to afford 17 (0.29 g, 98%).
NMR (300 MHz, CDCl3): δ 9.91(s, 1H), 7.82 (s, 1H), 7.15 (s,
1H), 4.69 (d, J ) 7.5 Hz, 1H), 4.18 (m, 2H), 3.68 (m, 2H), 2.00
(m, 1H), 1.79 (m, 2H), 1.45 (s, 9H), 1.26 (m, 2H).
5-[[N-[(ter t-Bu tyloxy)ca r bon yl]p ip er id in -4-yl]m eth yl]-
th ien o[2,3-b]th iop h en e-2-ca r boxylic Acid (20). To a sus-
pension of crude 19 (0.357 mmol) and triethylamine (1 mL, 7
mmol) in 20 mL of THF was added 5 mL of H2O. The resultant
solution was treated with di-tert-butyl dicarbonate (0.15 g, 0.68
mmol) for 18 h. The THF was removed in vacuo, and the
residue was partitioned between 1 N KHSO4 and EtOAc. The
organic phase was dried (MgSO4), filtered, and concentrated
to yield 0.226 g of crude 20 containing residual triethylsilane
and Et2O. This material was used to prepare 38 without
further purification.
Gen er a l Meth od for th e P r ep a r a tion of 2-(S)-Su lfon a -
m id e 3-Am in op r op ion ic Acid Meth yl Ester (26). Meth yl
2-(S )-[N -[(Be n zyloxy)ca r b on yl]a m in o]-3-a m in op r op i-
on a te Hyd r och lor id e (22). To a cooled suspension of 2-(S)-
[N-[(Benzyloxy)carbonyl]amino]-3-aminopropionic acid (21)
(Fluka) (10 g, 0.042 mol) in 150 mL of CH3OH was added 5.47
g (0.046 mol) of thionyl chloride over 20 min. The resulting
solution was allowed to stir at room temperature overnight.
After ∼18 h, the solvent was removed in vacuo, and the
residual solid was stirred with 150 mL of Et2O for 0.5 h. The
resulting white solid was collected and air-dried to give 11.98
g (99%) of ester 22. Mp: 168.5-171 °C. Rf (CHCl3 saturated
with ammonia): 0.37. NMR (400 MHz, CD3OD): δ 7.33 (m,
5H), 5.13 (s, 2H), 4.50 (dd, J ) 5, 9 Hz, 1H), 3.71 (s, 3H), 3.43
(dd, J ) 5, 13 Hz, 1H), 3.21 (dd, J ) 9, 13 Hz, 1H).
Met h yl 2-(S)-[N-[(Ben zyloxy)ca r b on yl]a m in o]-3-[N-
[(ter t-bu tyloxy)ca r bon yl]a m in o]p r op ion a te (23). To a
two-phase mixture of CH2Cl2 (500 mL) and saturated NaHCO3
solution (300 mL) was added 28.87 g (0.10 mol) of 22. After a
few minutes, 21.83 g (0.10 mol) of di-tert-butyl dicarbonate was
added in one portion, and the resulting mixture was stirred
at room temperature for 4 h. The CH2Cl2 layer was then
separated from the aqueous layer, and the aqueous layer was
extracted with 300 mL of CH2Cl2. The combined organic
extracts were washed with brine, dried and the solvent
removed in vacuo to provide the product as a viscous oil.
Trituration of this oil with 300 mL of hexane gave 33.54 g
(95%) of 23 as a white solid. Mp: 85-87 °C. Rf (5% MeOH/
CH2Cl2): 0.68. NMR (400 MHz, CDCl3): δ 7.35 (s, 5H), 5.78
(bs, 1H), 5.11 (s, 2H), 4.83 (bs, 1H), 4.41 (bs, 1H), 3.76 (s, 3H),
3.55 (bs, 2H), 1.41 (s, 9H).
Meth yl 2-(S)-Am in o-3-[N-[(ter t-bu tyloxy)ca r bon yl]a m i-
n o]p r op ion a te (24). To a solution of 23 (6.60 g, 0.0187 mol)
in 150 mL of EtOH was added 0.5 g of 10% Pd/C. The resulting
mixture was hydrogenated under balloon pressure at room
temperature for 4 h. The catalyst was filtered off and the
solvent removed in vacuo to provide 4.05 g (99%) of 24 as a
viscous oil. Rf (10% MeOH/CHCl3/NH3): 0.6. NMR (400 MHz,
CDCl3): δ 5.0 (bs, 1H), 3.72 (s, 3H), 3.56 (t, J ) 5.7 Hz, 1H),
3.46 (m, 1H), 3.23 (m, 1H), 1.55 (bs, 2H), 1.42 (s, 9H).
5-[[N -[(t er t -B u t y lo x y )c a r b o n y l]p ip e r id in -4-y l]h y -
d r oxym eth yl]th ien o[2,3-b]th iop h en e-2-ca r boxylic Acid
(18). To a solution of 17 (0.289 g, 0.759 mmol) in 19 mL tert-
butyl alcohol (Aldrich) was added 2-methyl-2-butene (19 mL)
Meth yl 2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[N-[(ter t-
b u t yloxy)ca r b on yl]a m in o]p r op ion a t e (25). 3-Pyridine-
sulfonic acid (30 g, 0.188 mol) was added to PCl5 (46.8 g, 0.225
mol), suspended in 150 mL of toluene, and heated to reflux

Non-Peptide GPIIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2417
overnight. The suspension was cooled and concentrated to yield
a yellow oil, which was diluted with benzene, filtered through
a pad of Celite, and concentrated to give 30.7 g (92%) of
3-pyridinesulfonyl chloride as a yellow oil, which was used in
the next step without purification. NMR (300 MHz, CDCl3):
δ 9.27 (s, 1H), 8.98 (d, J ) 4 Hz, 1H), 8.35 (d, J ) 8 Hz, 1H),
7.62 (dd, J ) 4, 8 Hz, 1H).
Amine 24 (16.8 g, 0.077 mole) dissolved in 330 mL of CH2-
Cl2 was treated with 3-pyridinesulfonyl chloride (20.6 g, 0.116
mol) and pyridine (12.5 mL, 0.154 mol), and the reaction was
stirred for 21 h. The reaction was concentrated, adsorbed to
[N-[(benzyloxy)carbonyl]amino]-3-amino propionic acid. This
was taken to the (R)-3-pyridylsulfonamide derivative 26h as
described for 26a . NMR (300 MHz, CD3OD): δ 9.25(s, 1H),
9.02 (d, J ) 5 Hz, 1H), 8.75 (d, J ) 9 Hz, 1H), 8.06 (dd, J ) 5,
9 Hz, 1H), 4.51 (dd, J ) 5, 9 Hz, 1H), 3.55 (s, 3H), 3.45 (dd, J
) 5, 13 Hz, 1H), 3.16 (dd, J ) 9, 13 Hz, 1H).
2-(R)-[N-(P h en ylsu lfon yl)am in o]-3-am in opr opion ic Acid
(26i). A solution of d-asparagine‚HCl (4.31 g, 25.6 mmol) and
NaOH (2.10 g, 50.0 mmol) in 20 mL of H2O and 20 mL of
dioxane was cooled to 0 °C. Benzenesulfonyl chloride (3.65 mL,
28.5 mmol) was added, followed by an additional 1.14 g (28.5
mmol) of NaOH in 20 mL of H2O. After 30 min at 0 °C, the
dioxane was removed in vacuo. The residue was basified with
20% NaOH to pH 10 and washed with EtOAc. The aqueous
layer was then acidified with concentrated HCl and cooled to
0 °C. The resultant precipitate was collected by vacuum
filtration and dried in a drying pistol for 18 h to yield 2.20 g
(32%) of 2-(R)-[N-(phenylsulfonyl)amino]asparagine as a white
solid. NMR (300 MHz, D2O + NaOD): δ 7.63 (m, 5H), 3.98
(m, 1H), 2.64 (m, 1H), 2.49 (dd, J ) 9, 15 Hz, 1H).
To a solution of NaOH (2.40 g, 60 mmol) in 10 mL of H2O
at 0 °C was added 0.54 mL (10 mmol) of bromine. After 5 min
at 0 °C, a solution of 2-(R)-[N-(phenylsulfonyl)amino]aspar-
agine (2.20 g, 8.08 mmol) and NaOH (0.68 g, 17 mmol) in 5
mL H2O was added, and the solution stirred for 15 min at 0
°C. The reaction was then heated to 90 °C for 30 min, cooled
to 0 °C, and neutralized with concentrated HCl. The resultant
precipitate was collected by vacuum filtration to yield 1.48 g
(65%) of the hydrochloride salt of 26i as a white solid. NMR
(300 MHz, D2O + NaOD): δ 7.85 (m, 2H), 7.61 (m, 3H), 3.55
(dd, J ) 4.7, 7.5 Hz, 1H), 2.82 (dd, J ) 4.7, 13 Hz, 1H), 2.65
(dd, J ) 7.5, 13 Hz, 1H).
Meth od for P r ep a r a tion of Cou p led In ter m ed ia tes.
Meth yl 2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[[2-ca r bo-
n yl-5-[2-[N-[(ter t-bu tyloxy)ca r bon yl]p ip er id in -4-yl]eth yl]-
th ien o[2,3-b]th iop h en eyl]a m in o]p r op ion a te (27). Com-
pound 12 (8.5 g, 0.0215 mol), amine 26a (R ) 3-pyridyl, 7.36
g, 0.025 mol), and BOP reagent (14.73 g, 0.033 mol) were
suspended in 150 mL of CH3CN and cooled to -40 °C.
N-Methylmorpholine (7.05 mL, 0.075 mol) was added, the cold
bath was removed, and the reaction mixture was stirred for
16 h. The resulting homogeneous solution was concentrated
to one-half its original volume, diluted with 300 mL EtOAc,
and washed successively with 10% KHSO4, H2O, saturated
NaHCO3, and brine, dried (Na2SO4), filtered, and concentrated.
The crude product was chromatographed on SiO2 using a
gradient (15-45% acetone/hexanes) to give 12 g (87%) of 27.
Rf (45% acetone/hexanes): 0.28. NMR (300 MHz, CDCl3): δ
9.0 (s,1H), 8.6 (d, J ) 4 Hz, 1H), 8.1 (d, J ) 8 Hz, 1H), 7.5 (s,
1H), 7.4 (m, 1H), 7.3 (dd, J ) 4, 8 Hz, 1H), 7.2 (m, 1H), 6.8 (s,
1H), 4.3 (m, 1H), 4.0-4.1 (bs, 2H), 3.9 (m, 2H), 3.6 (s, 3H), 2.9
(m, 2H), 2.7 (bt, 2H), 1.6-1.5 (m, 5H), 1.45 (s, 9H), 1.1 (m,
2H).
silica and chromatographed with
a gradient of 30-70%
acetone/hexanes to give crude 25 which was swished with hot
EtOAc, cooled, and filtered to give 25 (17.5 g, 63%) as a pale-
yellow solid. Rf (30% acetone/hexanes): 0.29. NMR (300 MHz,
CDCl3): δ 9.0(s,1H), 8.8 (d, J ) 4 Hz, 1H), 8.6 (d, J ) 4 Hz,
1H), 8.1 (d, J ) 8 Hz, 1H), 7.45 (dd, J ) 4, 8 Hz, 1H), 7.3 (m,
1H), 4.1(m, 1H), 4.1 (s, 3H), 3.4-3.5 (m, 2H), 1.4 (s, 9H).
Met h yl 2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-a m in o-
p r op ion a te (26a ). A suspension of 25 (17.5 g, 0.049 mol) in
200 mL of EtOAc was cooled to -78 °C. HCl gas was bubbled
through the solution for 10 min, and the solution was then
placed in an ice bath. After stirring for 40 min at 0 °C, no
starting material could be detected by TLC. The solution was
concentrated, first at room temperature and then at 40 °C to
yield 16 g (100%) of 26a as an off-white solid. Rf (9:1:1 EtOH/
H2O/NH4OH): 0.34. NMR (300 MHz, CD3OD) d 9.3(s, 1H), 9.0
(dd, J ) 1, 5 Hz, 1H), 8.9 (d, J ) 9 Hz, 1H), 8.2 (dd, J ) 5, 9
Hz, 1H), 4.6 (dd, J ) 5, 9 Hz, 1H), 3.6 (s, 3H), 3.5 (dd, J ) 5,
13 Hz, 1H), 3.3 (dd, J ) 9, 13 Hz, 1H).
Meth yl 2-(S)-[N-(Meth ylsu lfon yl)a m in o]-3-a m in op r o-
p ioa te Hyd r och lor id e (26b). The procedure for 26a was
used except that methanesulfonyl chloride was used in the
sulfonylation step. NMR (400 MHz, CD3OD): δ 4.46 (dd, J )
4.7, 9 Hz, 1H), 3.84 (s, 3H), 3.43 (dd, J ) 4.7, 13 Hz, 1H), 3.10
(dd, J ) 9, 13 Hz, 1H), 3.06 (s, 3H).
Meth yl 2-(S)-[N-(n -Bu tylsu lfon yl)a m in o]-3-a m in op r o-
p ion a te Hyd r och lor id e (26c). The procedure for 26a was
used except that n-butanesulfonyl chloride was used in the
sulfonylation step. Rf (9:1 EtOH/H2O): 0.6. NMR (300 MHz,
CDCl3): δ 8.1 (bs, 2H), 7.2 (m, 1H), 4.65 (m, 1H), 3.82 (s, 3H),
3.65 (m, 1H), 3.54 (m, 1H), 3.20 (bs, 2H), 1.8 (m, 2H), 1.45 (m,
2H), 0.95 (t, J ) 7 Hz, 3H).
Meth yl 2-(S)-[N-(Eth oxyleth ylsu lfon yl)a m in o]-3-a m i-
n op r op ion a te Hyd r och lor id e (26d ). The procedure for 26a
was used except that ethoxyethanesulfonyl chloride, prepared
from oxidation of the commercially available sulfide with
chlorine,23 was used in the sulfonylation step. NMR (300 MHz,
CD3OD): δ 3.89 (m, 5H), 3.65-3.76 (m, 6H), 1.19 (t, J ) 7 Hz,
3H).
Meth yl 2-(S)-[N-(P h en ylsu lfon yl)a m in o]-3-a m in op r o-
p ion a te Hyd r och lor id e (26e). The procedure for 26a was
used except that benzenesulfonyl chloride was used in the
sulfonylation step. Rf (10% MeOH/CHCl3 saturated with
NH3): 0.4. NMR (300 MHz, CD3OD): δ 7.88 (m, 2H), 7.60 (m,
3H), 4.24 (m, 1H), 3.40 (s, 3H), 3.36 (m, 1H), 3.30 (m, 2H),
3.09 (m, 1H).
Met h yl 2-(S)-[N-(2-Th ien ylsu lfon yl)a m in o]-3-a m in o-
p r op ion a te Hyd r och lor id e (26f). The procedure for 26a was
used except that 2-thiophenesulfonyl chloride was used in the
sulfonylation step. NMR (300 MHz, CD3OD): δ 7.86 (m, 1H),
7.66 (m, 1H), 7.17 (m, 1H), 4.33 (dd, 1H), 3.54 (s, 3H), 3.37
(dd, 1H), 3.12 (dd, 1H).
Met h yl 2-(S)-[N-(4-Ch lor op h en ylsu lfon yl)a m in o]-3-
a m in op r op ion a te Hyd r och lor id e (26g). The procedure for
26a was used except that 4-chlorobenzenesulfonyl chloride was
used in the sulfonylation step. NMR (300 MHz, CD3OD): δ
7.85 (d, J ) 8.5 Hz, 2H), 7.60 (d, J ) 8.5 Hz, 2H), 4.27 (dd,
1H), 3.47 (s, 3H), 3.35 (dd, 1H), 3.08 (dd, 1H).
P r ep a r a tion of 2-(R)-Su lfon a m id e 3-a m in op r op ion ic
Acid s 26h ,i. Meth yl 2-(R)-N-[(3-P yr id ylsu lfon yl)a m in o]-
3-a m in op r op ion a te (26h ). 2-(R)-[N-[(Benzyloxy)carbonyl]-
amino]asparagine was treated with bis(trifluoroacetoxy)phenyl
iodide and pyridine using the method of Waki24 to afford 2-(R)-
Meth od A for Dep r otection of Cou p led In ter m ed ia tes.
2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid (4). Compound 27 (17.3 g, 0.027 mol) was
dissolved in 900 mL of 6 N HCl and stirred for 20 h at which
time HPLC analysis indicated all 27 was consumed. The
solvent was removed to yield 16.4 g of yellow solid hydrochlo-
ride salt. Addition of 500 mL of H2O to the crude product
resulted in dissolution and then crystallization. The crystalline
product was filtered off to give 11 g of a wet solid which was
dried in a drying pistol overnight at room temperature to yield
6.21 g of 4 as the free base. The filtrate was concentrated to
dryness to yield 7.5 g, which was dissolved in 70 mL of H2O.
One equivalent of 1 N NaOH (12 mL) was added slowly
dropwise while the solution was stirred vigorously, and a
crystalline precipitate formed. After the mixture cooled for 1
h, the solid was collected and dried on the drying pistol
overnight at room temperature to yield 3.87 g of 4 as the free
base. The combined yield of the two crops was 10.08 g (71%).
Mp 245-247 °C (>99% ee (CHIRAL AGP 100 × 4.0 mm,
Advanced Separation Technology)). Rf (9:1:1 EtOH/H2O/NH4-

2418 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Egbertson et al.
OH): 0.31. NMR (300 MHz, D2O + NaOD): δ 8.7 (s,1H), 8.0
(d, J ) 8 Hz, 1H), 7.7 (d, J ) 5 Hz, 1H), 7.2 (s, 1H), 6.8 (dd, J
) 5, 8 Hz, 1H), 6.8 (s, 1H), 4.8 (dd, J ) 2, 3 Hz,1H), 4.7 (bd,
2H), 3.1 (m, 2H), 2.8 (bd, 2H), 2.6 (bt, 2H), 2.1 (bt, 2H), 1.6
(m, 4H), 1.1 (m, 1H), 1.9 (m, 2H). Exact mass (FAB, m + 1):
calcd, 523.1144; found, 523.1163. Anal. (C22H26N4O5S3‚1.95
H2O) C, H, N.
2-(S)-[N-(Met h ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (30). Acid 12 and amine 26b
were coupled as described for 27. The coupled intermediate
was deprotected using method A as described for 4 except that
the crystallization step was omitted (50% for two steps). Rf
(9:1:1 EtOH/H2O/NH4OH): 0.47. NMR (400 MHz, D2O): δ 7.56
(s, 1H), 6.85 (s, 1H), 4.23 (dd, J ) 5, 8 Hz, 1H), 3.71 (dd, J )
5, 14 Hz, 1H), 3.45 (dd, J ) 8, 14 Hz, 2H), 3.24 (bd, J ) 13 Hz,
2H), 2.93 (s, 3H), 2.74 (m, 4H), 1.78 (bd, J ) 14 Hz, 2H), 1.50
(m, 3H), 1.25 (m, 2H). Exact mass (FAB, m + 1): calcd,
460.1035; found, 460.1028. Anal. (C18H25N3O6S3‚1.8 HCl‚
0.25H2O).
2-(S)-[N-(2-Eth oxyeth ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-
5-[2-(p ip er id in -4-yl)eth yl]th ien o[2,3-b]th iop h en eyl]a m i-
n o]p r op ion ic Acid Hyd r och lor id e (32). Acid 12 and amine
26d were coupled as described for 27. The coupled intermedi-
ate was deprotected using Method A as described for 30 (38%
for two steps). NMR (300 MHz, D2O): δ 7.62 (s, 1H), 6.80 (s,
1H), 4.27 (dd, J ) 4.6, 9 Hz, 1H), 3.75 (m, 3H), 3.50 (m, 1H),
3.34 (m, 6H), 2.73 (bt, J ) 12 Hz, 2H), 2.60 (bs, 2H), 1.75 (bd,
2H), 1.20-1.53 (m, 5H), 0.99 (t, J ) 6 Hz, 3H). Exact mass
(FAB, m + 1): calcd, 518.1453; found, 518.1444. Anal.
(C21H31N3O6S3‚1.25HCl‚0.10Et2O) C, H, N.
2-(R)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid (37). Acid 12 and amine 26h (R-isomer) were
coupled as described for 27. The coupled intermediate was
deprotected using method A as described for 30 (45% for two
steps) (>99% ee (CHIRAL AGP 100 × 4.0 mm, Advanced
Separation Technology)). NMR (300 MHz, D2O+NaOD): δ 8.74
(s, 1H), 7.98 (d, J ) 8 Hz, 1H), 7.81 (d, J ) 5 Hz, 1H), 7.30 (s,
1H), 6.92 (m, 1H), 6.81 (s, 1H), 3.70 (m, 2H), 3.10 (m, 1H),
2.81 (bd, J ) 12 Hz, 2H), 2.50 (t, J ) 7 Hz, 2H), 2.26 (t, J )
11.5 Hz, 2H), 1.44 (m, 4H), 1.10 (m, 1H), 0.92 (m, 2H). Exact
mass (FAB, m + 1): calcd, 523.1144; found, 523.1135. Anal.
(C22H26N4O5S3‚2.00 HCl‚0.30H2O) C, N; H: calcd, 9.32; ob-
served, 8.80.
2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-
[(p ip er id in -4-yl)m eth yl]th ien o[2,3-b]th iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (38). Acid 20 and amine 26a
were coupled as described for 27. The coupled intermediate
was deprotected using method A as described for 30 (29% for
two steps). NMR (300 MHz, CD3OD): δ 8.86 (m, 2H), 8.05 (m,
1H), 7.72 (s, 1H), 7.10 (s, 1H), 4.43 (dd, J ) 5, 9 Hz, 1H), 3.81
(dd, J ) 5, 14 Hz, 1H), 3.53 (dd, J ) 9, 14 Hz, 1H), 3.39 (bd,
J ) 13 Hz, 2H), 2.92 (m, 4H), 1.98 (m, 3H), 1.48 (m, 2H). Anal.
(C21H24N4O5S3‚2HCl‚H2O) C, H, N.
2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-5-[3-
(p ip er id in -4-yl)p r op yl]th ien o[2,3-b]th iop h en yl]a m in o]-
p r op ion ic Acid (39). The acid 10 and amine 26a were coupled
as described for 27. The coupled intermediate was deprotected
using method A as described for 30 (36% for two steps). NMR
(300 MHz, CD3OD): δ 9.24 (s, 1H), 8.84 (m, 1H), 8.02 (m, 1H),
7.69 (s, 1H), 7.05 (s, 1H), 4.42 (dd, J ) 5, 8.5 Hz, 1H), 3.81
(dd, J ) 5, 14 Hz, 1H), 3.52 (dd, J ) 8.5, 14 Hz, 1H), 3.36 (bd,
J ) 13 Hz, 2H), 2.93 (m, 4H), 1.95 (bd, J ) 13 Hz, 2H), 1.70
(m, 2H), 1.39 (m, 4H). Anal. (C23H28N4O5S3‚2.55HCl‚0.05H2O)
C,H,N.
3H), 2.84-2.74 (m, 4H), 1.81 (m, 2H), 1.57 (m, 3H), 1.26 (m,
2H). Exact mass (FAB, m + 1): calcd, 460.1035; found,
460.1017. Anal. (C18H25N3O5S3‚1.65HCl) C, H, N.
2-(S)-[N-(2-Eth oxyeth ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-
5-[2-(p ip er id in -4-yl)et h ylt h ien o[2,3-b]t h iop h en eyl]a m i-
n o]p r op ion ic Acid Hyd r och lor id e (42). Acid 9 and amine
26d were coupled as described for 27. The coupled intermedi-
ate was deprotected using method A as described for 30 (42%
for two steps). NMR (300 MHz, D2O): δ 7.78 (s, 1H), 6.90 (s,
1H), 4.32 (m, 1H), 3.73 (m, 3H), 3.40 (m, 7H), 2.78 (m, 4H),
1.85 (bd, 2H), 1.6-1.24 (m, 5H), 1.02 (t, 3H). Exact mass (FAB,
m + 1): calcd, 518.1453; found, 518.1452. Anal. (C21H31N3O6S3‚
1.50HCl) C, H, N.
2-(S)-[N-(3-P yr id ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[3,2-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (45). Acid 9 and amine 26a
were coupled as described for 27. The coupled intermediate
was deprotected using method A as described for 30 (34% for
two steps). NMR (300 MHz, D2O + NaOD): δ 8.68 (s, 1H),
7.97 (s, 1H), 7.83 (d, 1H), 7.39 (s, 1H), 7.01 (m, 2H), 3.73 (m,
1H), 3.61 (dd, 1H), 3.19 (dd, 1H), 2.85 (m, 4H), 2.36 (t, 3H),
1.58 (m, 4H), 1.30 (m, 1H), 1.03 (m, 2H). Exact mass (FAB, m
+ 1): calcd, 523.1144; found, 523.1135. Anal. (C22H26N4O5S3‚
1.90HCl‚1.05H2O) C,H,N.
Meth od B for Dep r otection of Cou p led In ter m ed ia tes.
2-(S)-[N-(n -Bu tylsu lfon yl)a m in o]-3-[[2-ca r bon yl-5-[2-(p i-
p er id in -4-yl)eth yl]th ien o[2,3-b]th iop h en eyl]a m in o]p r o-
p ion ic Acid (31). Acid 12 and amine 26c were coupled as
described for 27. The coupled intermediate was deprotected
with LiOH in 1:1:1 THF/H2O/MeOH as described for 11; then
the BOC group was removed with HCl-saturated EtOAc at 0
°C as described for 26a . The HCl salt was converted to the
free base by recrystallization from water as described for 4
(71% for three steps) NMR (300 MHz, CD3OD): δ 7.76 (s, 1H),
7.05 (s, 1H), 4.30 (m, 1H), 3.82 (m, 1H), 3.56 (m, 1H), 3.00 (m,
3H), 2.00 (bd, J ) 12 Hz, 2H), 1.72 (m, 4H), 1.48 (m, 4H), 0.85
(t, J ) 7 Hz, 3H). Anal. (C21H31N3O5S3‚0.5H2O) C, H, N.
2-(S)-[N-(P h en ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid (33). Acid 12 and amine 26e were coupled
as described for 27. The coupled intermediate was deprotected
as described for 31 and recrystallized from water (60% for
three steps). NMR (300 MHz, DMSO-d6): δ 8.61 (m, 2H), 7.79
(m, 3H), 7.49 (m, 3H), 7.14 (s, 1H), 3.20 (m, 3H), 2.80 (m, 4H),
1.82 (d, 2H), 1.58 (m, 3H), 1.30 (m, 2H). Anal. (C23H27N3O5S3)
C, H, N.
2-(R)-[N-(P h en ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid (34). Acid 12 in DMF was activated with CDI
in DMF and then treated with amine 26i and N,N-diisopro-
pylethylamine to afford the coupled BOC acid intermediate.
The BOC group was removed with HCl-saturated EtOAc at 0
°C as described for 26a . The crude product was chromato-
graphed on silica eluting with 9:1:1 EtOH/H2O/NH4OH to give
the free base (25% for two steps). NMR (300 MHz, D2O +
NaOD): δ 7.57 (d, J ) 7.5 Hz, 2H), 7.27 (s, 1H), 7.00 (m, 2H),
6.88 (s, 1H), 6.81 (m, 1H), 3.60 (m, 2H), 3.05 (m, 1H), 2.80 (m,
2H), 2.78 (m, 2H), 2.28 (m, 2H), 1.48 (m, 4H), 1.20 (m, 1H),
0.92 (m, 2H). Exact mass (FAB, m + 1): calcd, 522.1191; found,
522.1200. Anal. (C23H27N3O5S3‚1.35 H2O‚0.55SiO2) C,H,N.
2-(S)-[N-(4-Ch lor op h en ylsu lfon yl)a m in o]-3-[[2-ca r bo-
n yl-5-[2-(p ip er id in -4-yl]eth yl]th ien o[2,3-b]th iop h en eyl]-
a m in o]p r op ion ic Acid Hyd r och lor id e (35). Acid 12 and
amine 26g were coupled as described for 27. The coupled
intermediate was deprotected as described for 31 (80% for
three steps). NMR (300 MHz, CD3OD): δ 7.77 (d, J ) 9 Hz, 2
Hz), 7.59 (s, 1H), 7.35 (d, J ) 9 Hz, 2H), 7.05 (s, 1H), 4.20 (dd,
J ) 5, 9 Hz, 1H), 3.71 (dd, J ) 5, 14 Hz, 1H), 3.38 (m, 3H),
2.98 (m, 4H), 2.00 (bd, J ) 14 Hz, 2H), 1.73 (m, 3H), 1.45 (m,
2H). Anal. (C23H26ClN3O5S3‚HCl‚0.10EtOAc‚0.95H2O) C, H, N.
2-(S)-[N-(2-Th ien ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (36). Acid 12 and amine 26f
were coupled as described for 27. The coupled intermediate
2-(S)-[N-(Met h ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[3,2-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (40). Acid 9 and amine 26b
were coupled as described for 27. The coupled intermediate
was deprotected using method A as described for 30 (50% for
two steps). NMR (400 MHz, D2O): δ 7.70 (s, 1H), 6.98 (s, 1H),
4.18 (dd, J ) 5, 8.5 Hz, 1H), 3.72 (dd, J ) 5, 14 Hz, 1H), 3.46
(dd, J ) 8.5, 14 Hz, 1H), 3.26 (bd, J ) 12.5 Hz, 2H), 2.93 (s,

Non-Peptide GPIIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2419
was deprotected as described for 31 and then chromatographed
on silica eluting with 9:1:1 EtOH/H2O/NH4OH to give the free
base (72% for three steps). NMR (300 MHz, CD3OD): δ 7.68
(s, 1H), 7.61 (m, 2H), 7.05 (s, 1H), 7.00 (m, 1H), 4.20 (dd, J )
5, 8 Hz, 1H), 3.75 (dd, J ) 5, 13.5 Hz, 1H), 3.52 (dd, J ) 8,
13.5 Hz, 1H), 3.37 (bd, J ) 12 Hz, 2H), 2.96 (m, 4H), 1.99 (bd,
J ) 11 Hz, 2H), 1.72 (m, 3H), 1.43 (m, 2H). Exact mass (FAB,
m + 1): calcd, 528.0750; found, 528.0771. Anal. (C21H25N3O5S3‚
HCl‚0.60EtOAc‚0.85SiO2) C, H, N.
2-(S)-[N-(n -Bu t ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[3,2-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (41). Acid 9 and amine 26c
were coupled as described for 27. The coupled intermediate
was deprotected as described for 31 (56% for three steps). NMR
(300 MHz, CD3OD): δ 7.86 (s, 1H), 7.13 (s, 1H), 4.32 (dd, J )
5, 9 Hz, 1H), 3.82 (dd, J ) 5, 18.5 Hz, 1H), 3.58 (dd, J ) 9 Hz,
1H), 3.32 (m, 2H), 3.00 (m, 6H), 2.00 (m, 3H), 1.72 (m, 5H),
1.30 (m, 6H), 0.86 (t, J ) 7 Hz, 3H). Anal. (C21H31N3O5S3‚HCl)
C, H, N.
2-(S)-[N-(P h en ylsu lfon yl)a m in o]-3-[[2-ca r b on yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[3,2-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (43). Acid 9 and amine 26e
were coupled as described for 27. The coupled intermediate
was deprotected as described for 31 (65% for three steps). NMR
(300 MHz, CD3OD): δ 7.81 (m, 2H), 7.69 (s, 1H), 7.40 (m, 3H),
7.12 (s, 1H), 4.15 (m, 1H), 3.71 (dd, J ) 5, 13.5 Hz, 1H), 3.36
(m, 3H), 2.98 (m, 7H), 2.00 (m, 3H), 1.72 (m, 3H), 1.42 (m,
3H). Anal. (C23H27N3O5S3‚HCl) C, H, N.
2-(S)-[N-(2-Th ien ylsu lfon yl)a m in o]-3-[[2-ca r bon yl-5-[2-
(p ip er id in -4-yl)et h yl]t h ien o[3,2-b]t h iop h en eyl]a m in o]-
p r op ion ic Acid Hyd r och lor id e (44). Acid 9 and amine 26f
were coupled as described for 27. The coupled intermediate
was deprotected as described for 31 and then chromatographed
on silica eluting with 9:1:1 EtOH/H2O/NH4OH to give the free
base (60% for three steps). NMR (300 MHz, CD3OD): δ 7.76
(s, 1H), 7.61 (m, 2H), 7.12 (s, 1H), 6.98 (m, 1H), 4.24 (dd, J )
5, 8 Hz, 1H), 3.75 (dd, J ) 5, 13.5 Hz, 1H), 3.52 (dd, J ) 8,
13.5 Hz, 1H), 3.38 (bd, J ) 12.5 Hz, 2H), 3.00 (m, 4H), 2.01
(m, 2H), 1.75 (m, 3H), 1.44 (m, 2H). Exact mass (FAB, m + 1):
calcd, 528.0750; found, 528.0764. Anal. (C21H25N3O5S3‚HCl‚
1.15H2O‚0.75SiO2) C, H, N.
Meth od C for Dep r otection of Cou p led In ter m ed ia tes.
3-[[2-Ca r b on yl-5-[2-(p ip er id in -4-yl)et h yl]t h ien o[2,3-b]-
th iop h en eyl]a m in o]p r op ion ic Acid Hyd r och lor id e (28).
tert-Butyl-3-aminopropionic acid was coupled with 12 as
described for 27. The protecting groups were removed in one
step with HCl-saturated EtOAc at 0 °C as described for 26a
(62% for two steps). NMR (300 MHz, CD3OD): δ 7.73 (s, 1H),
7.05 (s, 1H), 3.62 (t, 2H), 3.38 (bd, 2H), 2.95 (m, 4H), 2.62 (t,
2H), 2.01 (bd, 2H), 1.74 (m, 3H), 1.95 (m, 2H). Anal.
(C17H22N2O3S2‚HCl‚0.6H2O) C, H, N.
3-[[2-Ca r bon yl-5-[2-(p ip er id in -4-yl)eth yl]th ien o[3,2-b]-
th iop h en eyl]a m in o]p r op ion ic Acid Hyd r och lor id e (29).
Prepared as described for 28 except that 9 was used in the
coupling step (58% for two steps). NMR (400 MHz, CD3OD):
δ 7.81 (s, 1H), 7.12 (s, 1H), 3.61 (t, J ) 7 Hz, 2H), 3.37 (bd, J
) 12.5 Hz, 2H), 3.0 (m, 4H), 2.62 (t, J ) 7 Hz, 2H), 2.00 (bd,
J ) 14 Hz, 2H), 1.75 (m, 3H), 1.41 (m, 2H). Anal. (C17H22N2O3S2‚
HCl‚0.35H2O) C, H, N.
2-(S)-[N-(3-pyr idylsu lfon yl)am in o]-3-[(2-car bon ylth ien o-
[2,3-b]th iop h en eyl]a m in o]p r op ion ic Acid (46). Coupled as
described for 27, deprotected as described for 31, then chro-
matographed in 45:1:1 EtOH/H2O/NH4OH, and further puri-
fied by preparative HPLC eluting with 95:5 0.1% TFA in H2O/
CH3CN (25% for two steps). Rf (45:1:1 EtOH/H2O/NH4OH):
0.45. NMR (300 MHz, D2O + NaOD): δ 8.78 (s, 1H), 8.09 (d,
J ) 7.5 Hz, 1H), 7.95 (d, J ) 5 Hz, 1H), 7.56 (d, J ) 5, Hz,
1H), 7.38 (s, 1H), 7.31 (d, J ) 5 Hz, 1H), 7.17 (m, 1H), 3.98
(m, 1H), 3.65 (m, 1H), 3.32 (m, 1H). Exact mass (FAB, m + 1):
calcd, 412.0096; found, 412.0098. Anal. (C15H13N3O5S3‚0.55H2O)
C, H, N.
by passing platelet lysates sequentially over concanavalin A
affinity, Sepharose 4B-hexyl-RGDS, affinty and Sephacryl
S-300HR size exclusion columns by a modification of the
method of Kouns et al.25 Purified GPIIb/IIIa was coated onto
yttrium silicate scintillation proximity assay fluomicrospheres
(Amersham RPN 143) and is abbreviated IIb/IIIa/SPA. The
binding of the RGD-containing heptapeptide [125I]L-692,884
(New England 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]L-692,-
884 to IIb/IIIa/SPA at pH 7.5 (20 mM HEPES, 0.15 M NaCl,
room temperature) with ∼0.3 nM IIb/IIIa/SPA, ∼0.3 nM
[
125I]L-692,884, and a wide range of concentrations of the
competing nonradiolabeled compounds. After equilibration, the
bound CPM were measured and the ED50 value was deter-
mined by nonlinear least-squares fit to 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 nonspe-
cific binding signal. The average standard error of mean for
ED50 determinations was ( 20%.
Equ ilibr iu m Dissocia tion Con sta n t (KD) for Com p eti-
t ive Bin d in g of An a logu es w it h F lu or escen t Liga n d
L-762,745. Fluorescein-containing L-762,7459,18 in flow cyto-
metric buffer at a final concentration of 110 nM was added to
wells of a 96-well microtiter plate that contained test com-
pounds at concentrations ranging from 0.05 to 1000 nM. GFP
were then added to each well to give a final concentration of
2 × 106 cells/mL, and the microtiter plate was incubated for 2
h at room temperature. The samples were analyzed in a
FACScan, as described previously.17 The flow cytometric
measurements of the affinity of the compounds for the recep-
tors on resting platelets were measured from at least three
different platelet donors. The standard deviation of the
measurements was for all compounds <20%. The value for 4
is 0.07 ( 0.006 nM (mean ( STD) with n ) 20.
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 institu-
tional animal care and use committee (MRL-20P).
Sin gle-Dose In tr a ven ou s a n d Or a l Ad m in istr a tion of
4 to Con sciou s Dogs. The potency of antiplatelet activity of
4 was assessed following single-dose intravenous administra-
tion. Two groups of conscious, purpose-bred mongrel dogs of
either sex (10.5-13 kg) were administered 4 as an intravenous
bolus (vehicle ) saline) of 3 (n ) 3) or 5 (n ) 3) µg/kg. In
addition, oral antiplatelet activity of 4 was assessed following
single-dose oral administration in aqueous solution by gastric
lavage to conscious dogs. Three groups of dogs of either sex
(9.2-12.7 kg) were administered 4 orally (5-mL volume,
vehicle ) sterile water): 50 (n ) 5), 100 (n ) 4); 200 (n ) 2)
µ/kg. For all treatment groups described above, blood samples
were obtained before compound administration (baseline) and
at multiple timepoints up to 8 h after compound administra-
tion from either saphenous or cephalic veins (0.38% sodium
citrate, final concentration) for the measurement of ex vivo
platelet aggregation responses to ADP and collagen and for
whole blood platelet counts, as described below.
On ce-Da ily Or a l Ad m in istr a tion of 4 for 6 Da ys to
Con sciou s Dogs by Ga str ic La va ge: Ma in ten a n ce of
In h ibition of P la telet F u n ction . A study in dogs was
conducted to determine if submaximal but therapeutic levels
of inhibition of platelet aggregation could be maintained with
once-daily oral doses of 4. A group of conscious, purpose-bred
mongrel dogs of either sex (n ) 4, 7.6-11.0 kg) was adminis-
tered individualized loading doses of 50-90 µg/kg 4 on the first
day of treatment, followed by individualized daily doses of 0,
10, 20, or 30 µg/kg for 2 days and identical daily doses of 10
µg/kg for 3 additional days, 6 total days of dosing. Compound
4 was administered in solution by gastric lavage (5-mL volume
with sterile water as vehicle and 10-mL flush with sterile
water). Blood samples were obtained prior to the first oral dose
In Vit r o P h a r m a cology: Scin t illa t ion P r oxim it y-
Ba sed Assa y for Affin ity of a n Activa ted F or m of GP IIb/
IIIa . GPIIb/IIIa was purified from outdated human platelets

2420 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Egbertson et al.
(baseline) at 120, 180, 240, and 330 min after the first oral
dose and at 30 min before and 60 min after compound
administration on succeeding days (i.e., 23.5 h after dosing
on the previous day) for the measurement of ex vivo platelet
aggregation responses to ADP and collagen and for whole blood
platelet counts, as described below. Blood samples were also
obtained over the next 12 days following termination of dosing
for these measurements. A separate, control group of four dogs
was studied for daily effects on ex vivo platelet aggregation
following one, single oral dose for comparison to the multiple-
dose group.
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(2) (a) Cook, N. S.; Kottirsch, G.; Zerwes, H.-G. Platelet Glycoprotein
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(3) Mckean, M.-L.; Adelman, S. J . Future Therapies for the Preven-
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(4) Hartman, G. D.; Egbertson, M. S.; Halczenko, W.; Laswell, W.
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(5) (a)Egbertson, M. S.; Chang, C. T. C.; Duggan, M. E.; Gould, R.
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J r.; Lynch, R. J .; Manno, P. M.; Naylor, A. M.; Prugh, J . D.;
Ramjit, D. R.; Sitko, G. R.; Smith, R. S.; Turchi, L. M.; Zhang,
G. Non-Peptide Fibrinogen Receptor Antagonists. 2. Optimiza-
tion of a Tyrosine Template as a Mimic for Arg-Gly-Asp. J . Med.
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(6) Egbertson, M. S.; Naylor, A. M.; Hartman, G. D.; Cook, J . J .;
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strained Inhibitor. Bioorg. Med. Chem. Lett. 1994, 4, 1835-1840.
(7) We are not alone in employing this strategy; several groups have
demonstrated the success of this approach: (a) Alig, L.; Eden-
hoger, A.; Hadva´ry, P.; Hu¨rzeler, M.; Knopp, D.; Mu¨ller, M.;
Steiner, B.; Trzeciak, A.; Weller, T. Low Molecular Weight, Non-
Peptide Fibrinogen Receptor Antagonists. J . Med. Chem. 1992,
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Eggleston, D. S.; Hwang, S. M.; Kopple, K. D.; Koster, P. F.;
Nichols, A.; Pieshoff, C. E.; Samanen, J . M.; Vasko, J . A.; Wong,
A.; Huffman, W. F. Design and Synthesis of a C7 Mimetic for
the Predicted γ-turn Conformation Found in Several Con-
strained RGD Antagonists. J . Med. Chem. 1992, 35, 3970-3972.
(c) 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. S.; 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 Structure and Conformation of a Highly Con-
strained Cyclic RGD Peptide. J . Am. Chem. Soc. 1993, 115,
8861-8862. (d) McDowell, R. S.; Blackburn, B. K.; Gadek, T.
R.; McGee, L. R.; Rawson, T.; Reynolds, M. E.; Robarg, K. D.;
Sommers, T. C.; Thorsett, E. D.; Tischler, M.; Webb, R. R.;
Venuti, M. C. From Peptide to Non-Peptide. The De Novo Design
of Potent, Non-Peptidal Inhibitors of Platelet Aggregation Based
on a Benzodiazepinedione Scaffold. J . Am. Chem. Soc. 1994, 116,
5077. (e) Ku, T. W.; Miller, W. H.; Bondinell, W. E.; Erhard, K.
F.; Keenan, R. M.; Nichols, A.; Peishoff, C. E.; Samenen, 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. (f) 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. (g) Duggan, M. E.; Naylor-
Olsen, A. M.; Perkins, J . J .; Anderson, P. S.; Chang, C. T.-C.;
Cook, J . J .; Gould, R. J .; Ihle, N. C.; Hartman, G. D.; Lynch, J .
J .; Lynch, R. J .; Manno, P. D.; Schaffer, L. W.; Smith, R. L. Non-
Peptide Fibrinogen Receptor Antagonists. 7. Design and Syn-
thesis of a Potent, Orally Active Fibrinogen Receptor Antagonist.
J . Med. Chem. 1995, 38, 3332-3341. (h) Weller, T.; Alig, L.;
Beresini, M.; Blackburn, B.; Bunting, S.; Hadva´ry, P.; Mu¨ller,
H. M.; Knopp, D.; Levet-Traift, B.; Lipari, M. T.; Modi, N. B.;
Mu¨ller, M.; Refino, C. J .; Schmitt, M.; Scho¨nholzer, P.; Weiss,
S.; Steiner, B. Orally Active Fibrinogen Receptor Antagonists.
2. Amidoximes as Prodrugs of amidines. J . Med. Chem. 1996,
39, 3139-3147. (i) Xue, C.-B.; Raafalski, M.; Roderick, J .;
Eyermann, C. J .; Mousa, S.; Olsen, R. E.; DeGrado, W. F. Design,
Synthesis and In Vitro Activities of a Series of Benzimidazole/
Benzoxazole Glycoprotein IIb/IIIa Inhibitors. Bioorg. Med. Chem.
Lett. 1996, 6, 339-344. (j) Askew, B. C.; Bednar, R. A.; Bednar,
B.; Claremon, D. A.; Cook, J . J .; McIntyre, C. J .; Hunt, C. A.;
Gould, R. J .; Lynch, R. J .; Lynch, J . J .; Gaul, S. L.; Stranieri,
M. T.; Sitko, G. R.; Holahan, M. A.; Glass, J . D.; Hamill, T.;
Rela tion sh ip betw een Acu te Effects of In tr a ven ou s 4
on ex Vivo P la telet Aggr ega tion Resp on ses a n d Bleed -
in g Tim es in An esth etized Dogs. The relationship between
the inhibition of platelet aggregation and the prolongation of
bleeding time was investigated during the intravenous ad-
ministration of increasing doses of 4 to the anesthetized dog.
Two groups of pentobarbital anesthetized dogs were evaluated
for ex vivo inhibition of platelet aggregation and template
bleeding time: 4 (3-6 increasing doses, iv bolus, n ) 6) and
vehicle (5 mL of saline, 4 bolus infusions, n ) 4). Purpose-
bred mongrels of either sex (9.2-14.8 kg) were anesthetized
with 35 mg/kg iv sodium pentobarbital, intubated, ventilated
with room air, and instrumented with arterial catheters for
blood withdrawal and venous catheters for the infusion of
experimental agents and supplemental anesthesia. Prior to the
administration of the first bolus of 4 or vehicle, a blood sample
(5 mL) was taken to determine the baseline platelet aggrega-
tion response to ADP and collagen as described below, and
buccal mucosal bleeding time was measured using a SIM-
PLATE bleeding time device (Organon Teknika Corp., Durham,
NC) as described below. One minute following each bolus, a
blood sample was obtained and a bleeding time measurement
was initiated. A second blood sample was taken at the
termination of the bleeding time measurement. For each
individual dose, the percent inhibition of aggregation was
averaged from the “pre” and “post” bleeding time samples, and
this value was used to represent the effect on platelet ag-
gregation for that particular dose and measured bleeding time.
This was repeated for 3-6 iv bolus doses of 4 (0.5 µg/kg
followed by 0.5 or 1.0 µg/kg) or for 4 iv bolus infusions of
vehicle. In the 4 treatment group the final dose was not
predetermined but was the dose at which template bleeding
time exceeded the maximum limit which was set at 15 min.
Ex Vivo P la telet Aggr ega tion Resp on ses to ADP a n d
Colla gen in P RP a n d Wh ole Blood P la telet Cou n ts. Blood
was withdrawn into sodium citrate (0.38%, final concentra-
tion). Platelet-rich plasma (PRP) was prepared by centrifuga-
tion of whole blood at 150g for 5 min, and the platelet
concentration 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 agonist. 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. Aggregation was initiated
by the addition of 10 µM ADP + 1 µM epinephrine or 10 µg/
mL collagen + 1 µM epinephrine. The extent of 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 aggregatory response. The
effect of 4 treatment on the extent and rate of aggregation is
expressed as the percent inhibition using the baseline, pre-
treatment aggregation response (day 1 in mulitple day main-
tenance studies) as 100%. Whole blood platelet counts were
determined using an automated hematology analyzer (Serono-
Baker Diagnostics, Allentown, PA).
Bu cca l Mu cosa l Tem p la te Bleed in g Tim es. Buccal
mucosal template bleeding times were measured with
a
SIMPLATE bleeding time device (Organon Teknika Corp.,
Durham, NC). Uniform incisions were made on the mucous
membrane of the inner upper lip of the dog, and the duration
of bleeding was timed to a maximum of 15 min.

Non-Peptide GPIIb/ IIIa Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2421
Gorham, L. M.; Prueksaritanont, T.; Baldwin, J . J .; Hartman,
G. D. Non-Peptide Glycoprotein IIb/IIIa Inhibitors. 17. Design
and Synthesis of Orally Active, Long-Acting Non-Peptide Fi-
brinogen Receptor Antagonists. J . Med. Chem. 1997, 40, 1779-
1788.
(16) The potency of R-substituted compounds with KD values in the
range of receptor concentration may approach the lower limit
of accuracy of the assay for inhibition of aggregation. The lowest
observed IC50 in a receptor binding assay can never be less than
50% of the concentration of receptors used in the assay, in this
case, 4-16 nM (calculated from 2 × 108 platelets/mL, 20 000 to
1000 000 receptors/platelet).
(17) Bednar, R. A.; Gaul, S. L.; Hamill, T. G.; Egbertson, M. S.;
Shafer, J . A.; Hartman, G. D.; Gould, R. J .; Bednar, B. Inden-
tification of Low-Molecular Weight GPIIb/IIIa Antagonists that
Bind Preferentially to Activated Platelets. J . Pharmacol. Exp.
Ther. 1998, 285, 1317-1326.
(18) Bednar, B.; Cunningham, M. E.; McQueney, P. A.; Egbertson,
M. S.; Askew, B. C.; Bednar, R. A.; Hartman, G. D.; Gould, R. J .
Flow Cytometric Measurement of Kinetic and Equilibrium
Binding Parameters of RGD Ligands in Binding to GPIIb/IIIa
on Platelets. Cytometry 1997, 28, 58-65.
(19) For other reports on the affinity of GPIIb/IIIa ligands for resting
or activated platelets, see: (a) Kouns, W. C.; Hadvary, P.;
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