Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position

Article abstract of DOI:10.1021/jm9801713

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compo

Full text of DOI:10.1021/jm9801713

3210
J . Med. Chem. 1998, 41, 3210-3219
Design a n d Syn th esis of a Ser ies of P oten t a n d Or a lly Bioa va ila ble Non cova len t
Th r om bin In h ibitor s Th a t Utilize Non ba sic Gr ou p s in th e P 1 P osition
Thomas J . Tucker,*,† Stephen F. Brady,† William C. Lumma,† S. Dale Lewis,‡ Stephen J . Gardell,‡
Adel M. Naylor-Olsen,∇ Youwei Yan,‡ J ack T. Sisko,† Kenneth J . Stauffer,† Bobby J . Lucas,‡ J oseph J . Lynch,§
J acquelynn J . Cook,§ Maria T. Stranieri,§ Marie A. Holahan,§ Elizabeth A. Lyle,§ Elizabeth P. Baskin,§
I-Wu Chen, Kimberly B. Dancheck, J ulie A. Krueger,‡ Carolyn M. Cooper,‡ and J oseph P. Vacca†
Departments of Medicinal Chemistry, Biological Chemistry, Pharmacology, Drug Metabolism, and Molecular Design and
Diversity, Merck Research Laboratories, West Point, Pennsylvania 19486
Received March 19, 1998
As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors,
we have synthesized a series of compounds that utilize nonbasic groups in the P1 position.
The work is based on our previously reported lead structure, compound 1, which was discovered
via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3
group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position
of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit
subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability
profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and
in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin
active site.
Ch a r t 1
In tr od u ction
The serine protease thrombin plays a critical role in
the blood coagulation cascade. Thrombin is responsible
for the conversion of fibrinogen to fibrin and is also the
most potent known stimulator of platelet aggregation.
In an attempt to develop novel, orally bioavailable
antithrombotic agents with improved therapeutic po-
tential over existing therapies, we have focused on the
design and synthesis of selective thrombin inhibitors.
A number of other research groups have also reported
their efforts toward the design and synthesis of novel
thrombin inhibitors.1 We have detailed our strategies
in a series of manuscripts2-5 describing a number of
potent, selective, and orally bioavailable tripeptide
thrombin inhibitors. Most recently, we reported a series
of potent thrombin inhibitors derived from a resin-based
approach that utilized 2,5-disubstituted benzylamines
as novel P1 ligands.5 The key compound 1 (Chart 1)
emerged as a novel lead structure from this work.
Compound 1 is a potent and selective thrombin inhibitor
and exhibited oral bioavailability of 45% in dogs with a
half-life of 100 min. Unfortunately, compound 1 is
extremely lipophilic (log P > 3.1), and the associated
high level of plasma protein binding compromises the
performance of the compound in the rat FeCl3-induced
thrombosis model. Despite its shortcomings, compound
1 became a crucial starting point for the design of a
series of more potent and less lipophilic analogues.
Crystallographic analysis6 of 1 bound in the active site
of thrombin indicated a novel binding interaction with
the S1 pocket of thrombin (Figure 1). Most previously
reported thrombin inhibitors rely on the interaction of
a basic amino group in the P1 position with Asp 189
located at the bottom of the S1 pocket. However, in the
case of 1 this key interaction with the S1 pocket is
obviously lacking. Instead, the chlorine atom in the
5-position of the P1 aromatic ring makes a direct
lipophilic contact with the aromatic ring of Tyr 228
located on the back wall of the S1 binding pocket. The
binding energy derived from this key lipophilic contact
appears to compensate for the lack of a direct interaction
with Asp 189 to produce a novel thrombin inhibitor of
nanomolar potency. The other key interactions of the
D-diphenylAla-Pro series of thrombin inhibitors with the
thrombin active site that we have detailed in our
previous publications2-5 are maintained by the P2 and
P3 regions of this inhibitor.
Refinement of this lead compound 1 to increase its
inhibitory potency, while simultaneously making the
compound less lipophilic, became the goals of this study.
In this manuscript, we describe our progress toward
these goals and detail a series of structurally unique
thrombin inhibitors that utilize nonbasic groups in the
P1 position.
† Department of Medicinal Chemistry.
‡ Department of Biological Chemistry.
§ Department of Pharmacology.
Syn th etic Ch em istr y
The Boc-D-cyclohexylglycine-proline starting material
(2) (Scheme 1) was prepared by standard amino acid
Department of Drug Metabolism.
∇ Department of Molecular Design and Diversity.
S0022-2623(98)00171-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/13/1998

Design, Synthesis of Noncovalent Thrombin Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3211
F igu r e 1. Stereoview of thrombin-bound inhibitor 1 (green with red oxygen and blue nitrogen atoms) with the inhibited thrombin
active site. Active site residues are color-coded as follows: lipophilic P3 residues (Leu 99, Ile 174, Trp 215) are colored magneta;
the thrombin â sheet is colored white (shown from residues Ser 214 to Gly 219); the catalytic triad is colored green; the S1 Asp
189 is colored red; the thrombin insertion loop is colored yellow; the lipophilic P1 residue Tyr 228 is colored blue. Some active site
residues have been omitted for clarity.
coupling of Boc-D-cyclohexylglycine with proline methyl
ester, followed by ester hydrolysis with LiOH/DME.
Standard amino acid coupling of 2 with the appropriate
2,5-disubstituted benzylamines provided 3a -c. Treat-
ment of 3a with gaseous HCl in ethyl acetate at 0 °C
provided a precipitate of compound 4, which was found
to be analytically pure after filtration and drying in
vacuo. The N-terminal Boc group of 3a -c was also
efficiently removed by treatment with trifluoroacetic
acid in methylene chloride (1:1). The crude amine salts
were alkylated with R-bromo-N,N-diethylacetamide7
and purified via reversed-phase preparatory LC to
provide compounds 5a -c.
The 2-hydroxy-5-chlorobenzylamine (7) was prepared
in two steps from the aldehyde 68 via reduction of the
oxime (Scheme 2). Standard amino acid coupling of 7
with 2 provided the key tripeptide intermediate 8.
Treatment of 8 with trifluoroacetic acid in methylene
chloride (1:1) followed by reversed-phase preparatory LC
purification provided 9. The phenol group of 8 was
alkylated with ethyl bromoacetate/cesium carbonate in
dioxane to give the ester 10 in good yield. Compound
10 was treated with TFA/CH2Cl2 as above to give the
N-terminally deprotected ester compound 11. The ester
group of 10 was hydrolyzed with 1 M LiOH/DME to
provide the acid intermediate 12. As before, 12 was
treated with TFA/CH2Cl2 and the crude product purified
via reversed-phase preparatory LC to give compound
13. Standard amino acid coupling of compound 12 with
the appropriate amine, followed by TFA/CH2Cl2 depro-
tection and reversed-phase preparatory LC purification,
provided the deprotected amides 14a -c.
focus of our initial efforts. Previous resin-based P3
modification work from our laboratories9 had shown
that, in a related series of thrombin inhibitors, the
amino acid D-cyclohexylglycine could serve as a reason-
able P3 group. Incorporation of this amino acid into the
P3 position of our lead structure provided the essentially
equipotent and selective compound 4 (Tables 1 and 2).
Although 4 showed reasonable potency as a thrombin
inhibitor, the compound exhibited only moderate oral
bioavailability and poor performance in the rat FeCl3
model of arterial thrombosis (Tables 3 and 4). We were
convinced that additional inhibitory potency was re-
quired to obtain suitable compounds from this series.
Molecular modeling studies10 with 4 indicated that it
might be possible to alkylate groups onto the N-
terminus of the molecule that had the potential to make
hydrogen-bonding interactions with the N-H of Gly 219
on the thrombin â sheet and also fill our previously
reported lipophilic N-terminus binding site. We initially
prepared benzylsulfonamide N-terminal versions of 4
based on our earlier reported work;2 however, these
compounds were much too insoluble and too lipophilic
to pursue further. Instead, we chose to prepare com-
pounds which were alkylated on the N-terminal nitro-
gen atom with the N,N-diethylacetamido group. Mod-
eling indicated that the carbonyl functionality of this
group could potentially interact with the N-H of Gly
219, while the N,N-diethyl group could serve to fill the
lipophilic N-terminus binding site in a manner similar
to our previously reported N-terminal benzylsulfona-
mides.2 This group would have the clear advantage of
adding far less lipophilicity to the compounds than an
N-terminal benzylsulfonamide moiety. Synthesis of the
prototype amide 5a confirmed our assumptions, and this
compound showed an approximately 12-fold increase in
inhibitory potency (Table 1).
Resu lts a n d Discu ssion
In an effort to reduce the lipophilicity of the lead
structure 1, the P3 diphenylalanine moiety became the

3212 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Tucker et al.
Sch em e 1a
a
Reagents: (a) EDC, HOBT, triethylamine/DMF, 18 h; (b) 1 M LiOH/DME; (c) EDC, HOBT, triethylamine, appropriate 2,5-disubstituted
benzylamine/DMF, 18 h; (d) HCl(g), EtOAc, -10 °C; (e) 1:1 TFA/CH2Cl2; (f) R-bromo-N,N-diethylacetamide, DIEA/DMF, 50 °C, 18 h;
purification by reversed-phase preparatory LC.
Removal of either the 2-chloro or the 5-chloro sub-
stituent from 5a resulted in an 18-fold loss of inhibitory
potency (5b,c), confirming the importance of the 2,5-
disubstitution pattern. In addition to the critical in-
teraction of the 5-chloro substituent with Tyr 228 in the
S1 pocket of thrombin, it appears that the substituent
in the 2-position of the aromatic ring also plays a crucial
role in the unique binding mode exhibited by these
inhibitors. The 2-position substituent may act as a
conformational lock, helping to properly position the
aromatic ring to allow maximum contact between the
5-chloro atom and the aromatic ring of Tyr 228. Un-
fortunately, 5a exhibited only moderate performance in
bioavailability (Table 4) and efficacy models (Table 3),
forcing us to rethink our approach. This compound
remained quite lipophilic (log P ) 2.67), and on the basis
of these data as well as our previously reported results,
we concluded that further reductions in the lipophilicity
of the molecules would be necessary to obtain good
performance in our antithrombotic model. We therefore
decided to explore replacement of the 2-chloro substitu-
ent on the P1 phenyl ring with more polar groups.
In considering potential substitutions at the 2-position
of the P1 aromatic ring, an examination of the crystal-
lographic results obtained with 1 indicated that the
substituent on the 2-position of the aromatic ring fits
in such a manner as to point directly at the N-terminal
region around the amino acid residue Gly 219. We
hypothesized that it might be possible to append po-
tential hydrogen-bonding groups to this position of the
P1 aromatic ring that could occupy the same region of
space as the N-terminal diethylacetamide group of 5a .
Molecular modeling studies10 (based on the crystal
structure of 1) on various derivatives indicated that a
phenol moiety at the 2-position of the P1 ring alkylated
with the appropriate hydrogen-bonding group might
have the potential to achieve the desired interaction
with Gly 219. The replacement of the 2-chloro substitu-
ent with a phenol or an appropriately alkylated phenol
moiety would also address the key goal of making the
compounds less lipophilic. On the basis of these hy-

Design, Synthesis of Noncovalent Thrombin Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3213
Sch em e 2a
a
Reagents: (a) NH2OH‚HCl, NaOAc/EtOH-water, room temperature, 4 h; (b) 5% Rh on C/EtOH-H2SO4, Parr apparatus; (c) EDC,
HOBT, triethylamine, 2/DMF, 18 h; (d) HCl(g)/EtOAc, -10 °C; (e) ethyl bromoacetate, Cs2CO3/1,4-dioxane, 36 h; (f) TFA/CH2Cl2, 30 min
followed by preparatory LC purification; (g) 1 M LiOH/DME; (h) EDC, HOBT, NMM, appropriate amine/DMF, 18 h.
potheses, the 2-phenol derivative 9 was synthesized and
was 2-fold more potent than its 2-chloro analogue 4
(Table 1). Compound 9 (log P ) 2.05) was also one-half
log less lipophilic than 4 (log P ) 2.63). Most impor-
tantly, 9 provided a platform for continued synthetic
efforts in this series. Guided by the previously described
modeling studies on 9, we hypothesized that an aceta-
mide derivative similar to that used on the N-terminus
of compound 5a , but instead alkylated onto the phenolic
oxygen of 9, might achieve the desired hydrogen-
bonding interaction with Gly 219. Preparation of the
ester derivative 11 seemed to confirm the potential of
this approach, as this compound exhibited an 8-fold
increase in potency versus the phenol 9 and a 16-fold
enhancement versus the 2-chloro derivative compound
4. Unfortunately, the ester function of 11 was subject
to rapid hydrolysis in vivo to the corresponding acid 13,
which exhibited a large loss in inhibitory potency. The
reduced potency of 13 is consistent with its reduced
capacity to make a hydrogen-bonding interaction with
the thrombin â sheet. In an effort to produce stable
compounds capable of making this key hydrogen-bond-
ing interaction, we chose to focus on the preparation of
a series of amide derivatives. We wanted to keep the
alkyl groups on the amide of minimal size in an effort
to keep the lipophilicity of the compounds in a reason-
able range, so we chose to avoid bulky, disubstituted
amides of the type used on the N-terminus of 5a .
Instead, we focused on a series of more polar monosub-
stituted amide derivatives. Preparation of the amide
derivatives 14a -c strongly supported our hydrogen-
bonding hypothesis, as each of these derivatives re-
tained in large measure the potency of the ester 11
(Table 1). Crystallographic analysis11 of 14b bound in
the thrombin active site (Figure 2) confirmed the
presence of the hydrogen-bonding interaction with the
N-H of Gly 219 on the thrombin â sheet. The aceta-
mide side chain on the phenolic oxygen bends toward

3214 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Ta ble 1. Nonbasic P1-Containing Thrombin Inhibitors
Tucker et al.
a
Ki values for all serine proteases are the average of at least two determinations, where the variation from the mean value is (10%
b
or less. Experimental protocols are provided in ref 2 and references cited therein. Yield refers to the final isolated and purified compound.
Ta ble 2. Selectivity of Thrombin Inhibitors versus Various
worth only approximately 5-fold in potency enhance-
ment. While initially this appears to be a somewhat
Serine Proteasesa
Ki (µM)
lower enhancement in potency than might be expected
for a hydrogen-bonding interaction, it is consistent with
the 10-fold potency enhancement observed for the
incorporation of the benzylsulfonamide group onto the
N-terminus in our previously reported work.2 In this
previously reported work,2 crystallographic results in-
dicated that one of the N-terminal sulfonamide oxygens
was participating in a hydrogen-bonding interaction
with the same N-H of Gly 219. These observations may
be due to the fact that both of the aforementioned
hydrogen-bonding interactions occur in a highly solvated
region close to the water interface. As expected and
consistent with our previously reported data,2 the P3
cyclohexyl ring lies in the lipophilic S3 pocket, with the
proline ring filling the S2 pocket. The usual three
hydrogen-bonding interactions with amino acid residues
Gly 216 and Ser 214 on the thrombin â sheet are
maintained.
Ki
compd thrombin
plasma
chymo-
no.
(nM)
trypsin TPA plasmin kallikrein trypsin
1
4
5a
14a
14b
14c
3
5
0.31
0.50
0.74
0.61
7
29
27
9
23
18
ND
19
29
11
ND
36
ND
691
244
152
ND
113
ND
586
399
816
ND
79
>50
49
174
131
71
67
a
Ki values for all serine proteases are the average of at least
two determinations, where the variation from the mean value is
(10% or less. ND ) not determined.
the N-terminus of the inhibitor, with the carbonyl group
making a hydrogen-bonding interaction with the N-H
of Gly 219 and the ethyl group occupying the lipophilic
N-terminus binding site region analogously to the
benzyl group of our previously described sulfonamide
inhibitors.2 The hydrogen-bonding interaction of the
amide carbonyl with the N-H of Gly 219 appears to be

Design, Synthesis of Noncovalent Thrombin Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3215
Ta ble 3. Antithrombotic Efficacy of Thrombin Inhibitors: Rat Carotid Artery FeCl3-Induced Thrombosis Modela,b
2× APTT (µM)c
% free
human
compd no.
log P
r
no. of occlusions
rat
human
rat
final plasma concn (nM)
4
5a
14a
14b
14c
argatroban
2.63
2.67
1.96
2.07
2.04
ND
6
6/6
3/6
8/10
1/6
1/6
0/6
ND
ND
0.31
0.38
0.42
ND
0.95
0.50
0.25
0.41
0.35
0.28
9 ( 2
ND
7 ( 1
14 ( 2
11 ( 1
ND
4 ( 1
ND
26 ( 4
15 ( 2
12 ( 2
ND
329
245
726
794
709
ND
6
10
6
6
6
a
b
ND indicates that the parameter was not determined for that compound. Compounds were infused at 10 µg/kg/min iv for 120 min
prior to the FeCl3 insult. Vehicle for compound administration was saline. Vehicle control animals showed complete occlusion. c The 2×
APTT value is defined as the concentration of inhibitor in plasma required to double the activated partial thromboplastin time.
Ta ble 4. Oral Bioavailability of Thrombin Inhibitorsa
ited the most consistent performance across species,
showing oral bioavailabilities of 20%, 40%, and 63% in
rats, dogs, and cynomolgous monkeys, respectively.
Examination of the pharmacokinetic parameters for
14b,c (Table 5) in all three species showed that the
N-ethyl amide 14b clearly exhibited a better overall
performance than the N-cyclopropyl amide 14c. Un-
fortunately, both compounds exhibited half-lives that
were too short to be considered candidates for develop-
ment as clinically useful oral thrombin inhibitors.
Initial studies with these inhibitors indicate that the
key issue contributing to the less than optimal in vivo
half-life appears to be biotransformation.12 Oxidative
metabolism of the P3 cyclohexane ring appears to be
one of the key biotransformations observed with these
compounds.12 Further investigation of the metabolic
liabilities associated with this series of inhibitors is
currently underway.
In summary, we have developed a series of potent and
selective thrombin inhibitors based on the use of non-
basic groups in the P1 position. We have demonstrated
that noncovalent thrombin inhibitors of subnanomolar
potency can be designed that do not make hydrogen-
bonding interactions with Asp 189 at the bottom of the
S1 pocket of thrombin. The key interactions in the S1
pocket are instead lipophilic in nature, involving pri-
marily a critical lipophilic interaction with the aromatic
ring of Tyr 228 in the back wall of the S1 pocket. We
have also demonstrated that additional binding energy
can be achieved in this series by direct hydrogen-
bonding interaction with the N-H of Gly 219, using
groups appended to either the N-terminus of the mol-
ecules or the 2-position of the P1 aromatic ring. The
properties of this series of molecules can be modified to
produce thrombin inhibitors such as compound 14b that
exhibit excellent overall profiles of potency, selectivity,
bioavailability, and antithrombotic efficacy. Lengthen-
ing the half-life of these compounds without compromis-
ing the overall profile has become the key challenge for
this series of compounds, and efforts toward meeting
these goals will be the subject of future publications
from our laboratories.
F (%) (dose iv, mg/kg; dose po, mg/kg)
compd
no.
cynomolgous
monkeye
log Pb
ratc
dogd
1
4
5a
14b
14c
>3.10 ND
45 ( 5 (1; 5)
ND
ND
ND
2.63 23 ( 4 (2; 10) ND
2.67 10 ( 2 (2; 10) ND
2.07 10 ( 4 (5; 20) 40 ( 10 (2; 5) 63 ( 9 (1; 5)
2.04 20 ( 5 (5; 12) ND
34 ( 8 (1; 5)
a
Details of the experimental protocols are described in the
Experimental Section. All values are the mean of at least three
determinations. ND means not determined for that compound.
b Log P values were determined experimentally using the standard
octanol/water procedure.2,3 c Compounds were dosed orally to rats
d
as a suspension in 1% methocel. Compounds were dosed orally
to dogs as a 1% methocel suspension. e Compounds were dosed
orally to monkeys as a 1% methocel suspension.
In general, members of this series exhibited excellent
selectivity versus five other human serine proteases
(Table 2). We were especially concerned about selectiv-
ity versus human chymotrypsin, as substrates of this
enzyme do not contain charged groups in the P1
position. All compounds tested showed excellent selec-
tivity versus this enzyme.
Key compounds in this series were evaluated for their
in vivo antithrombotic efficacy in the rat carotid artery
FeCl3-induced thrombosis model (Table 3). The 2×
APTT values determined in plasma for these compounds
were in general quite low, suggesting that reasonable
performance in an antithrombotic model could be ex-
pected from at least some members of this series. The
more lipophilic compounds 4 and 5a were poor perform-
ers in this model, while the less lipophilic amides 14b,c
performed much better. However, the unsubstituted
amide 14a showed results that were contrary to this
trend, and these results clearly make any attempt at
specific quantitative correlation of log P values and
antithrombotic activity difficult. While 14a is the least
lipophilic compound tested, exhibits rat plasma protein
binding essentially identical to the other compounds
tested, and possesses a 2× APTT value suggesting that
it should have antithrombotic activity, it performs
poorly in the rat model. Further investigation of this
behavior is ongoing.
Key compounds in this series were also evaluated for
oral bioavailability in several species. The prototype
compound 1 exhibited good oral bioavailability in dogs
(F ) 45%), and the second-generation compound 4 also
showed reasonable oral bioavailability in rats (Table 4).
The N-terminal acetamide compound 5a showed oral
bioavailability of only 10% in rats. However, the
phenoxyacetic amides 14b,c both showed reasonable
performance in several species. Compound 14b exhib-
Exp er im en ta l Section
Melting points were determined in open capillary tubes in
a Thomas-Hoover apparatus and are uncorrected. 1H NMR
spectra were recorded on a Varian Unity 400 spectrometer.
Chemical shifts are reported in δ (ppm) relative to tetra-
methylsilane. All reagents used were of commercial synthetic
grade. Aldrich Sure-Seal dimethylformamide was used as
solvent in all amino acid coupling reactions. All reversed-
phase preparatory HPLC purifications were performed on a
Waters Prep 4000 instrument, using a Waters C18 Prep-Pac

3216 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Tucker et al.
F igu r e 2. Stereoview of thrombin-bound inhibitor 14b (green with red oxygen and blue nitrogen atoms) with the inhibited
thrombin active site. Active site residues are color-coded as follows: lipophilic P3 residues (Leu 99, Ile 174, Trp 215) are colored
magneta; the thrombin â sheet is colored white (shown from residues Ser 214 to Gly 219); the catalytic triad is colored green; the
S1 Asp 189 is colored red; the thrombin insertion loop is colored yellow; the lipophilic P1 residue Tyr 228 is colored blue. Some
active site residues have been omitted for clarity.
Ta ble 5. Pharmacokinetic Parametersa for Compounds 14b,c in Rat,b Dog,c and Monkeyd
compd no.
log P
dose (mg/kg)
route
AUC (µM h)
t1/2 (min)
Cl (mL/min/kg)
F (%)
Rat
14b
14c
2.07
2.04
5
12
5
iv
po
iv
3.88 ( 0.40
1.96 ( 0.20
8.98 ( 0.94
3.20 ( 0.21
51 ( 6
35 ( 11
17 ( 6
10 ( 2
20 ( 5
68 ( 14
20
po
Dog
14b
2.07
2
5
iv
po
4.76 ( 0.50
4.85 ( 0.51
61 ( 6
73 ( 12
14 ( 4
40 ( 10
Cynomolgous Monkey
2.23 ( 0.10
14b
14c
2.07
2.04
1
5
0.5
5
iv
po
iv
102 ( 10
35 ( 5
29 ( 4
4.00 ( 0.40
63 ( 9
34 ( 8
0.42 ( 0.05
1.46 ( 0.12
40 ( 10
po
a
Details of the experimental protocols are described in the Experimental Section. Results are the mean of at least three determinations.
Dosed po as 1% methocel suspension, iv in saline. c Dosed po as 1% methocel suspension, iv in saline. Dosed po as 1% methocel
b
d
suspension, iv in 20% PEG 200.
and various gradients. Mass spectroscopy was performed on
a VG HF mass spectrometer with resolving powers of 1000
for low resolution and 5000 for high resolution and FAB
ionization using a glycerol matrix. Reagent abbreviations:
HOBT, 1-hydroxybenzotriazole; EDC, 1-ethyl-3-(3-(dimethyl-
amino)propyl)carbodiimide HCl; DME, 1,2-dimethoxyethane;
DIEA, diisopropylethylamine; TFA, trifluoroacetic acid.
Boc-D-cycloh exylglycin e-p r olin e (2). A solution of 8.00
g (31 mmol) of Boc-D-cyclohexylglycine, 5.80 g (35 mmol) of
proline methyl ester hydrochloride, 5.80 g (38 mmol) of HOBT,
and 10.45 mL (75 mmol) of triethylamine in 100 mL of
anhydrous DMF was treated with 7.90 g (40 mmol) of EDC,
and the resulting solution stirred at room temperature under
argon for 18 h. The reaction mixture was diluted with a 2-fold
excess of 10% aqueous citric acid and the resulting mixture
extracted with 2 × 150 mL of ethyl acetate. The combined
extracts were washed with brine, dried (MgSO4), filtered, and
concentrated to give a clear oil. The crude oil was chromato-
graphed over silica gel with 1:1 ethyl acetate/hexane to give
the product ester as a clear oil. The oil was immediately
dissolved in 100 mL of DME, and the solution was treated with
100 mL of 1 M LiOH. The mixture was stirred at room
temperature for 18 h and was acidified with dilute aqueous
KHSO4 to pH 4. The mixture was extracted with 2 × 100 mL
of ethyl acetate, and the combined extracts were washed with
brine, dried (MgSO4), filtered, and concentrated to give 6.45 g
(72% for two steps) of pure title compound 2 as a foam. 400
MHz 1H NMR (CDCl3): 0.93-1.31 (complex, 6H), 1.44 (s, 9H),
1.56-1.89 (complex, 5H), 2.02 (complex, 3H), 2.52 (m, 1H), 3.56
(q, 1H), 3.94 (t, 1H), 4.25 (t, 1H), 4.59 (t, 1H), 5.09 (d, J ) 9
Hz, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
3a -c a n d Com p ou n d 4. Boc-D-cycloh exylglycin e-p r o-
lin e-N-(2,5-d ich lor oben zyl)a m id e (3a ) a n d D-Cycloh exyl-
glycin e-p r olin e-N-(2,5-d ich lor oben zyl)a m id e (4). A solu-
tion of 100 mg (0.28 mmol) of compound 2, 50 mg (0.28 mmol)
of 2,5-dichlorobenzylamine, 42 mg (0.31 mmol) of HOBT, and
43 mL (0.31 mmol) of triethylamine in 2 mL of anhydrous DMF
was treated with 60 mg (0.31 mmol) of EDC, and the resulting
solution stirred at room temperature under argon for 18 h.
The reaction mixture was diluted with three times its volume
of 10% citric acid solution, and the suspension stirred vigor-
ously at room temperature for 2 h. The suspension was
filtered to give 125 mg (87%) of compound 3a as a tacky

Design, Synthesis of Noncovalent Thrombin Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3217
amorphous white solid after drying. FAB MS: MH+ ) 512.
mL (0.48 mmol) of DIEA was obtained 42 mg (29%) of desired
product 5c as an amorphous fluffy white lyophilisate. HR FAB
MS: theor MH+ 491.27889, obsd 491.27720. 400 MHz 1H
NMR (CDCl3): 1.06 (m, 6H), 1.10-1.36 (complex, 5H), 1.73
(t, 2H), 2.79 (t, 2H), 1.99 (m, 4H), 3.17 (m, 2H), 3.26 (m, 2H),
3.36 (m, 2H), 3.56 (m, 1H), 3.83 (d, 1H), 3.88 (m, 1H), 4.08 (d,
1H), 4.29 (d, 1H), 4.49 (m, 1H and m, 2H), 6.85 (br t, 1H NH),
7.16-7.38 (complex, 4H arom). Anal. for C26H38N4O3Cl2‚
1.25TFA‚0.35H2O: C (calcd 53.49, obsd 53.46); H (calcd 6.45,
obsd 6.43); N (calcd 8.76, obsd 8.79).
2-Hyd r oxy-5-ch lor oben zyla m in e Su lfa te (7). A mixture
of 4.70 g (30 mmol) of 5-chloro-2-hydroxybenzaldehyde (6), 2.10
g (30 mmol) of hydroxylamine hydrochloride, and 2.50 g (30
mmol) of sodium acetate in 50 mL of absolute ethanol/25 mL
of water was stirred at room temperature for 4 h. The reaction
mixture was diluted with 75 mL of water and the suspension
filtered to give 4.50 g of the crude oxime which was used as
recovered in the next step.
1
400 MHz H NMR (CDCl3): 0.90-1.21 (m, 5H), 1.25 (s, 9H),
1.58-1.83 (complex, 6H), 1.89 (m, 1H), 2.00 (m, 2H), 2.42 (m,
1H), 3.58 (q, 1H), 3.96 (t, 1H), 4.09 (t, 1H), 4.43 (dd, J ) 6, 16
Hz, 1H), 4.58 (dd, J ) 6, 16 Hz, 1H), 4.69 (d, J ) 6 Hz, 1H),
5.02 (d, J ) 6 Hz, 1H), 7.16 (d, J ) 3 Hz, 1H), 7.24 (d, J ) 3
Hz, 1H), 7.37 (s, 1H), 7.58 (br s, 1H, NH).
A 125-mg (0.24 mmol) sample of compound 3a was dissolved
in 2 mL of ethyl acetate, and the solution was cooled to -10
°C. The reaction mixture was bubbled with HCl gas for several
minutes and was stirred in the cold for an additional 15 min.
The reaction mixture was bubbled with argon to remove HCl,
and the white precipitate was collected by filtration, washed
with a little cold ethyl acetate, and dried in vacuo over P2O5;
105 mg (97%) of the desired product 4 was recovered after
1
drying. HPLC purity ) >98% at 214 nm. 400 MHz H NMR
(CD3OD): 1.11-1.39 (complex, 5H), 1.64-1.96 (complex, 6H),
2.02 (m, 2H), 2.09 (m, 1H), 2.31 (m, 1H), 3.63 (m, 1H), 3.78
(m, 1H), 4.02 (d, 1H), 4.42 (q, 2H), 4.50 (m, 1H), 7.29 (d, 1H
arom), 7.39 (d, 1H arom), 7.52 (s, 1H arom). Anal. for
The crude oxime from above was dissolved in 45 mL of
absolute ethanol by carefully adding 4.5 mL of concentrated
H2SO4 without cooling. The resulting solution was treated
with 450 mg of 5% Rh on C catalyst and was hydrogenated on
a Parr apparatus for 36 h at 60 psi. The resulting solution
was filtered through Celite and the filtrate diluted with 1
volume of water. The pH was adjusted to 7-8, at which point
the product began to crystallize. Filtration and drying pro-
vided 4.00 g (67%) of the crude desired product as a tan solid.
The material was 90-95% pure by HPLC analysis and was
C
20H27N3O2Cl2‚HCl: C (calcd 47.99, obsd 47.94); H (calcd 5.93,
obsd 6.20); N (calcd 8.40, obsd 8.49).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
5a -c. N-(N′,N′-Dieth yla ceta m id o)-D-cycloh exylglycin e-
p r olin e-N-(2,5-d ich lor oben zyl)a m id e (5a ). A 125-mg (0.24
mmol) sample of compound 3a was dissolved in 1 mL of CH2-
Cl2/1 mL TFA, and the solution stirred at room temperature
for 18 h. The reaction mixture was concentrated in vacuo and
the oily residue placed on a high-vacuum pump for 3 h to
remove any traces of solvent. A 50-mg (0.11 mmol) sample of
the deprotected material was dissolved in 1 mL of anhydrous
DMF and was treated with 38 mL (0.22 mmol) of DIEA,
followed by 22 mg (0.11 mmol) of R-bromo-N,N-diethylaceta-
mide. The resulting solution was stirred at room temperature
for 18 h, at which time HPLC indicated that the reaction was
only 50% complete. An additional 0.50 mol equiv of the
bromide was added, and the reaction mixture was heated at
60 °C for 5 h. The reaction mixture was concentrated in vacuo
and the tan oily residue purified via reversed-phase prepara-
tory LC. Suspected product fractions were combined, concen-
1
used crude in the next reaction. 400 MHz H NMR (DMSO-
d6): 3.78 (s, 2H), 6.69 (dd, 1H), 7.05 (br d, 1H), 7.14 (br s, 1H).
Boc-D-cycloh exylglycin e-p r olin e-N-(2-h yd r oxy-5-ch lo-
r oben zyl)a m id e (8) a n d D-Cycloh exylglycin e-p r olin e-N-
(2-h yd r oxy-5-ch lor oben zyl)a m id e (9). A solution of 408 mg
(1.15 mmol) of Boc-D-cyclohexylglycine-proline (2), 151 mg
(0.96 mmol) of 2-hydroxy-5-chlorobenzylamine sulfate (7), 191
mg (1.25 mmol) of HOBT, and 268 mL (1.92 mmol) of
triethylamine in 4 mL of anhydrous DMF was treated with
258 mg (1.34 mmol) of EDC, and the resulting solution stirred
at room temperature for 18 h. The reaction mixture was
diluted with several volumes of water and the mixture
extracted with ethyl acetate. The ethyl acetate extract was
washed with dilute aqueous KHSO4, water, dilute aqueous
NaHCO3, and brine and was dried (MgSO4). Filtration and
concentration provided a crude oily product which was purified
via flash chromatography over silica gel with methylene
chloride. Concentration of the product fractions gave 437 mg
(92%) of desired product 8 as a foam.
A 200-mg (0.41 mmol) sample of 8 was dissolved in 2 mL of
CH2Cl2/2 mL of TFA, and the resulting solution stirred under
argon for 30 min. The reaction mixture was concentrated in
vacuo and the crude oil product purified via reversed-phase
preparatory LC. Suspected product fractions were concen-
trated to remove volatiles, and the residue was lyophilized.
Lyophilization gave 100 mg (63%) of desired product 9 as an
amorphous fluffy white powder. FAB MS: MH+ ) 394. HPLC
purity ) >99% at 214 nm. 400 MHz 1H NMR (CD3OD): 1.09-
1.38 (complex, 5H), 1.64-1.95 (complex, 6H), 2.00 (m, 2H), 2.12
(m, 1H), 2.26 (m, 1H), 3.64 (m, 1H), 3.78 (m, 1H), 3.99 (d, 1H),
4.36 (s, 2H), 4.98 (m, 1H), 4.81 (m, 1H), 6.76 (d, 1H arom),
7.09 (d, 1H arom), 7.12 (s, 1H arom). Anal. for C20H28N3O3-
Cl‚1.45TFA‚0.45H2O: C (calcd 48.40, found 48.41); H (calcd
5.40, found 5.35); N (calcd 7.40, found 7.50).
trated to remove volatiles, and placed on
a lyophilizer.
Lyophilization gave 30 mg (43%) of the desired product 5a as
a tacky white amorphous powder. HR FAB MS: theor MH+
525.23992, obsd 525.23820. HPLC purity ) >99% at 215, 254
nm. 400 MHz 1H NMR (CDCl3): 1.04-1.33 (complex, 5H),
1.12 (m, 6H), 1.56-1.77 (complex, 4H), 1.83-2.11 (complex,
4H), 2.21 (m, 2H), 3.20 (m, 2H), 3.38 (q, 2H), 3.52 (m, 1H),
3.88 (m, 1H), 4.02 (q, 2H), 4.09 (d, J ) 7 Hz, 1H), 4.34 (dd, J
) 5, 14 Hz, 1H), 4.58 (m, 2H), 4.61 (dd, J ) 5, 14 Hz, 1H),
7.11 (d, 1H arom), 7.22 (m, 2H arom), 7.39 (s, 1H, NH). Anal.
for
C26H39N4O3Cl‚1.20TFA‚0.15H2O: C (calcd 54.09, obsd
54.12); H (calcd 6.47, obsd 6.47); N (calcd 8.89, obsd 9.09).
N-(N′,N′-Dieth yla ceta m id o)-D-cycloh exylglycin e-p r o-
lin e-N-(5-ch lor oben zyl)a m id e (5b). In a manner identical
to that described above for compound 5a , from 200 mg (0.48
mmol) of compound 3b after Boc removal and treatment with
93 mg (0.50 mmol) of R-bromo-N,N-diethylacetamide and
167.00 mL (0.96 mmol) of diisopropylethylamine was obtained
119 mg (41%) of desired product 5b as an amorphous fluffy
white lyophilisate. FAB MS: MH+ ) 491. HPLC purity )
>99% at 215, 254 nm. 400 MHz 1H NMR (CDCl3): 1.04-1.33
(complex, 5H), 1.05 (t, 3H), 1.22 (t, 3H), 1.65 (d, 2H), 1.81 (t,
2H), 1.96 (d, 2H), 2.01 (m, 1H), 2.11 (m, 1H), 2.19 (m, 2H),
3.19 (m, 2H), 3.35 (m, 2H), 3.51 (m, 1H), 3.88 (m, 1H), 4.14 (q,
2H), 4.08 (d, 2H), 4.31 (dd, J ) 6, 15 Hz, 1H), 4.48 (dd, J ) 6,
15 Hz, 1H), 4.52 (m, 1H), 7.12-7.30 (complex, 4H arom), 7.34
(br t, 1H NH). Anal. for C26H38N4O3Cl2‚1.65TFA‚0.65H2O: C
(calcd 48.51, obsd 48.51); H (calcd 5.69, obsd 5.70); N (calcd
7.72, obsd 7.96).
Boc-D-cycloh exylglycin e-p r olin e-N-[2-(O-ca r beth oxy-
m et h yl)-5-ch lor ob en zyl]a m id e (10) a n d D-Cycloh exyl-
glycine-proline-N-[2-(O-carbethoxymethyl)-5-chlorobenzyl]-
a m id e (11). A solution of 237 mg (0.48 mmol) of Boc-D-
cyclohexylglycine-proline-N-(2-hydroxy-5-chlorobenzyl)amide
(8) in 7 mL of anhydrous 1,4-dioxane was treated with 172
mg (0.53 mmol) of Cs2CO3 and 53 mg (0.53 mmol) of ethyl
bromoacetate, and the mixture stirred at room temperature
in an argon atmosphere overnight. An additional 0.50 mol
equiv of base and bromide were added, and stirring was
continued for an additional 18 h. The reaction mixture was
partitioned between ethyl acetate and water and the organic
N-(N′,N′-Dieth yla ceta m id o)-D-cycloh exylglycin e-p r o-
lin e-N-(2-ch lor oben zyl)a m id e (5c). In a manner identical
to that described above for compound 5a , from 115 mg (0.24
mmol) of compound 3c after Boc removal and treatment with
47 mg (0.24 mmol) of R-bromo-N,N-diethylacetamide and 84

3218 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Tucker et al.
layer washed twice with brine and dried over anhydrous
MgSO4. Concentration provided the crude product, which was
purified by flash chromatography over silica gel with ethyl
acetate. Concentration of suspected product fractions gave 194
mg (78%) of Boc-protected product 10 as a foam.
C24H35N4O4Cl‚1.30TFA‚0.15H2O: C (calcd 50.71, obsd 50.72);
H (calcd 5.86, obsd 5.84); N (calcd 8.89, obsd 9.21).
D-Cycloh exylglycin e-pr olin e-N-[2-(O-acetam ido)-5-ch lo-
r oben zyl]a m id e (14a ). In a manner identical to that de-
scribed above for compound 14b, from 110 mg (0.20 mmol) of
compound 10, 12 mg (0.23 mmol) of ammonium chloride, 37
mg (0.24 mmol) of HOBT, 28 mL (0.26 mmol) of N-methyl-
morpholine, and 50 mg (0.26 mmol) of EDC was obtained 15
mg (17%) of the desired product 14a as a fluffy white
amorphous lyophilisate. HR FAB MS: MH+ theor 451.21120,
obsd 451.21195. 400 MHz 1H NMR (CD3OD): 1.09-1.39
(complex, 5H), 1.67-1.95 (complex, 6H), 1.98 (m, 2H), 2.11 (m,
1H), 2.25 (m, 1H), 3.64 (m, 1H), 3.78 (m, 1H), 4.00 (d, 1H),
4.42 (m, 1H), 4.43 (d, 2H), 4.60 (s, 2H), 6.95 (d, 1H arom), 7.28
(d, 1H arom), 7.36 (s, 1H arom). Anal. for C22H33N4O4Cl‚
1.50TFA‚0.35H2O: C (calcd 48.39, obsd 48.42); H (calcd 5.42,
obsd 5.46); N (calcd 9.10, obsd, 8.93).
D-Cycloh exylglycin e-p r olin e-N-[2-(O-cyclop r op yla ce-
ta m id o)-5-ch lor oben zyl]a m id e (14c). In a manner identical
to that described above for compound 14b, from 140 mg (0.25
mmol) of compound 10, 19 mL (0.28 mmol) of cyclopropy-
lamine, 46 mg (0.30 mmol) of HOBT, 60 mL (0.60 mmol) of
N-methylmorpholine, and 62 mg (0.33 mmol) of EDC was
obtained 94 mg (62%) of the desired product 14c as a fluffy
white amorphous lyophilisate. FAB MS: MH+ ) 491. 400
MHz 1H NMR (CD3OD): 0.59 (m, 2H), 0.78 (m, 2H), 1.10-
1.38 (complex, 5H), 1.64-1.91 (complex, 6H), 1.99 (m, 2H), 2.08
(m, 1H), 2.24 (m, 1H), 2.77 (m, 1H), 3.64 (m, 1H), 3.76 (m,
1H), 4.01 (d, 1H), 4.42 (m, 1H), 4.43 (m, 2H), 4.57 (s, 2H), 6.88
(d, 1H arom), 7.25 (d, 1H arom), 7.34 (s, 1H arom). Anal. for
C25H35N4O4Cl‚1.00TFA‚0.10H2O: C (calcd 51.98, obsd 51.92);
H (calcd 5.76, obsd 5.77); N (calcd 8.72, obsd, 8.72).
P h a r m a cok in etic Stu d ies in Ra ts, Dogs, a n d Mon k eys.
Pharmacokinetic studies in rats, dogs, and cynomolgus mon-
keys were conducted in the fully conscious state in animals
which had been previously surgically prepared with indwelling
arterial cannulae for repeated blood sampling. Also, pharma-
cokinetic studies in all species were conducted in the fasted
state: i.e., animals were deprived of food for 24 h prior to test
agent administration. Oral test agent administration in all
species was by gavage; intravenous test agent administration
was by indwelling jugular vein cannula in rat and by periph-
eral venipuncture in dogs and cynomolgus monkeys. Blood
samples, collected on citrate (final concentration 0.38%), were
obtained prior to treatment and at multiple time points for
up to 24 h after treatment. Samples were analyzed by HPLC.
A 139-mg (0.24 mmol) sample of 10 was dissolved in 2 mL
of CH2Cl2/2 mL of TFA, and the solution was stirred under
argon for 30 min. The reaction mixture was concentrated in
vacuo and the oily residue purified via reversed-phase prepa-
ratory LC. Suspected product fractions were combined and
concentrated to remove volatiles and placed on a lyophilizer
for 18 h. Lyophilization provided 80 mg (70%) of the desired
product 11 as an amorphous fluffy white powder. FAB MS:
1
MH+ ) 480. HPLC purity ) >98% at 214 nm. 400 MHz H
NMR (CD3OD): 1.11-1.38 (complex, 5H), 1.29 (t, 3H), 1.64-
1.96 (complex, 6H), 2.00 (m, 2H), 2.09 (m, 1H), 2.19 (m, 1H),
3.64 (m, 1H), 3.79 (m, 1H), 3.99 (d, 1H), 4.24 (q, 2H), 4.44 (d,
2H), 4.45 (m, 1H), 4.79 (m, 1H), 4.82 (m, 1H), 6.88 (d, 1H
arom), 7.21 (d, 1H arom), 7.27 (s, 1H arom). Anal. for
C
25H32N3O5Cl‚0.45TFA‚1.00H2O: C (calcd 48.78, found 48.80);
H (calcd 5.41, found 5.42); N (calcd 6.35, found 6.39).
Boc-D -cycloh e xylglycin e -p r olin e -N -[2-(O-ca r b oxy-
m et h yl)-5-ch lor ob en zyl]a m id e (12) a n d D-Cycloh exyl-
glycin e-p r olin e-N-[2-(O-ca r boxym eth yl)-5-ch lor oben zyl]-
a m id e (13). A 110-mg (0.24 mmol) portion of Boc-D-cyclo-
hexylglycine-proline-N-[2-(O-carbethoxymethyl)-5-chloroben-
zyl]amide (10) was dissolved in 3 mL of 1 M LiOH/3 mL of
DME, and the solution stirred at room temperature for 18 h.
The reaction mixture was concentrated to remove DME, and
the pH was adjusted to approximately 6 with 10% aqueous
citric acid. The material was extracted with ethyl acetate and
the extract washed with brine, dried (MgSO4), and concen-
trated in vacuo to 100 mg (96%) of the crude Boc acid product
12 as a foam.
A 50-mg (0.11 mmol) sample of compound 12 was dissolved
in 1 mL of CH2Cl2/1 mL of TFA, and the resulting solution
stirred at room temperature for 30 min. The reaction mixture
was concentrated in vacuo and the oily residue purified via
reversed-phase preparatory LC. Suspected product fractions
were combined and concentrated to remove volatiles and
placed on a lyophilizer for 18 h. Lyophilization gave 41 mg
(82%) of the desired product 13 as a fluffy white amorphous
1
powder. FAB MS: MH+ ) 452. 400 MHz H NMR (CD3OD):
1.09-1.38 (complex, 5H), 1.63-1.94 (complex, 6H), 1.99 (m,
2H), 2.09 (m, 1H), 2.26 (m, 1H), 3.63 (m, 1H), 3.78 (m, 1H),
3.99 (d, 1H), 4.45 (m, 2H), 4.48 (m, 1H), 4.82 (m, 1H), 6.92 (d,
1H arom), 7.23 (d, 1H arom), 7.32 (s, 1H arom). Anal. for
C
22H30N3O5Cl‚TFA‚0.45H2O: C (calcd 45.79, obsd 45.81); H
(calcd 4.89, obsd 4.89); N (calcd 6.21, obsd 6.41).
Ack n ow led gm en t. The authors thank Dr. Graham
Smith, Ms. Patrice Ciecko, Mr. Ken Anderson, and Mr.
Matt Zrada for elemental analyses and log P determina-
tions. The authors also thank Dr. Harri Ramjit, Dr. Art
Coddington, and Dr. Charles Ross for mass spectroscopy
data. Also, the authors are grateful to Dr. J oel Huff and
Dr. J ules Shafer for their support and encouragement.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
14a -c. D-Cycloh exylglycin e-p r olin e-N-[2-(O-eth yla ceta -
m id o)-5-ch lor oben zyl]a m id e (14b). A solution of 91 mg
(0.17 mmol) of Boc-D-cyclohexylglycine-proline-N-[2-(O-car-
boxymethyl)-5-chlorobenzyl]amide (12), 37 mg (0.20 mmol) of
HOBT, 35 mg (0.20 mmol) of ethylamine hydrochloride, and
56 mL (0.51 mmol) of N-methylmorpholine in 5 mL of
anhydrous DMF was treated with 58 mg (0.23 mmol) of EDC,
and the resulting solution stirred at room temperature in an
argon atmosphere for 18 h. The reaction mixture was con-
centrated in vacuo, and the residue was partitioned between
ethyl acetate and water. The organic layer was washed with
brine, dried (MgSO4), filtered, and concentrated in vacuo to
give a solid residue. The residue was dissolved in 2 mL of
CH2Cl2/2 mL of TFA, and the solution stirred at room tem-
perature for 30 min. The reaction mixture was concentrated
in vacuo and the oily residue purified via reversed-phase
preparatory LC. Suspected product fractions were combined
and concentrated to remove volatiles and placed on a lyo-
philizer for 18 h. Lyophilization provided 64 mg (63%) of the
desired product 14b as an amorphous fluffy white powder.
FAB MS: MH+ ) 479. 400 MHz 1H NMR (CD3OD): 1.11-
1.39 (complex, 5H), 1.18 (t, 3H), 1.65-1.91 (complex, 6H), 1.98
(m, 2H), 2.08 (m, 1H), 2.25 (m, 1H), 3.64 (m, 1H), 3.77 (m,
1H), 4.02 (d, 1H), 4.43 (m, 1H), 4.45 (q, 2H), 4.56 (q, 2H), 6.91
(d, 1H arom), 7.25 (d, 1H arom), 7.37 (s, 1H arom). Anal. for
Su p p or t in g In for m a t ion Ava ila b le: Crystallographic
analysis of 1 and 14b (2 pages). Ordering information is found
on any current masthead page.
Refer en ces
(1) Several comprehensive reviews of thrombin inhibitor design have
recently been published: (a) Wiley, M. R.; Fisher, M. J . Small
Molecule Direct Thrombin Inhibitors. Exp. Opin. Ther. Patents
1997, 7 (11), 1265-1282. (b) Ripka, W. C. New Thrombin
Inhibitors in Cardiovascular Disease. Curr. Opin. Chem. Biol.
1997, 242-253.
(2) Tucker, T. J .; Lumma, W. C.; Mulichak, A. M.; Chen, Z.; Naylor-
Olsen, A. M.; Lewis, S. D.; Lucas, R.; Freidinger, R. M.; Kuo, L.
C. Design of Highly Potent Noncovalent Thrombin Inhibitors
that Utilize a Novel Lipophilic Binding Pocket in the Thrombin
Active Site. J . Med. Chem. 1997, 40, 830-832.
(3) Tucker, T. J .; Lumma, W. C.; Lewis, S. D.; Gardell, S. J .; Lucas,
R. J .; Baskin, E. P.; Woltmann, R.; Lynch, J . J .; Lyle, T. A.;
Appleby, S. D.; Chen, I-W.; Dancheck, K. B.; Vacca, J . P. Potent

Design, Synthesis of Noncovalent Thrombin Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3219
Non-Covalent Thrombin Inhibitors that Utilize the Unique
Amino Acid D-Dicyclohexylalanine in the P3 Position. Implica-
tions on Oral Bioavailability and Antithrombotic Efficacy. J .
Med. Chem. 1997, 40, 1565-1569.
(8) The aldehyde starting material 6 is available commercially from
Aldrich Chemical Co.
(9) Brady, S. F.; Stauffer, K. J .; Lumma, W. C.; Smith, G. M.; Ramjit,
H. G.; Lewis, S. D.; Lucas, B. J .; Gardell, S. J .; Lyle, E. A.;
Appleby, S. D.; Cook, J . J .; Holahan, M. A.; Stranieri, M. T.;
Lynch, J . J .; Lin, J . H.; Chen, I. W.; Vastag, K.; Naylor-Olsen,
A. M.; Vacca, J . P. Discovery and Development of L-372,460, a
Novel Potent Orally Active Small Molecule Inhibitor of Throm-
bin: Co-application of Structure-Based Design and Rapid Mul-
tiple Analogue Synthesis on Solid Support. J . Med. Chem. 1998,
41, 401-406.
(4) Tucker, T. J .; Lumma, W. C.; Lewis, S. D.; Gardell, S. J .; Lucas,
R. J .; Baskin, E. P.; Woltmann, R.; Lynch, J . J .; Lyle, T. A.;
Appleby, S. D.; Chen, I-W.; Dancheck, K. B.; Naylor-Olsen, A.
M.; Krueger, J . A.; Cooper, C. M.; Vacca, J . P. The Synthesis of
a Series of Potent and Orally Bioavailable Thrombin Inhibitors
That Utilize 3,3-Disubstituted Propionic Acid Derivatives in the
P3 Position. J . Med. Chem. 1997, 40, 3687-3693.
(5) Lumma, W. C.; Wtherup, K. M.; Tucker, T. J .; Brady, S. F.; Sisko,
J . T.; Naylor-Olsen, A. M.; Lewis, S. D.; Freidinger, R. M. Design
of Novel, Potent, Non-Covalent Inhibitors of Thrombin with
Nonbasic P1 Substructures. Rapid Structure Activity Studies
by Solid-Phase Synthesis. J . Med. Chem. 1998, 41, 1011-1013.
(6) For the crystal structure of compound 1, a single crystal was
used, diffracting to 2.3-Å resolution. The crystals were of space
group C2. Full experimental details along with data collection
and structure refinement parameters are provided as Supporting
Information. The data will be deposited in the Protein Data
Bank.
(10) Molecular modeling was performed as follows: Models were
constructed using AMF/C View (AMF is Merck proprietary
software). The ligands were energy-minimized within the context
of the active site using OPTiMOL with the MMFF force field.
(11) For the crystal structure of compound 14b, a single crystal was
used, diffracting to 1.9-Å resolution. The crystals were of space
group C2. Full experimental details along with data collection
and structure refinement parameters are provided as Supporting
Information. The data will be deposited in the Protein Data
Bank.
(12) Unpublished data, Department of Drug Metabolism, Merck
Research Labs, West Point, PA.
(7) Booysen, J . F.; Holzapfel, C. W. A New Facile Synthesis of a
Thromboxane B2 Precursor. Synth. Commun. 1995, 25 (10),
1461-1472.
J M9801713