Design of benzamidine-type inhibitors of factor Xa

Article abstract of DOI:10.1021/jm980227t

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, th

Full text of DOI:10.1021/jm980227t

4240
J . Med. Chem. 1998, 41, 4240-4250
Design of Ben za m id in e-Typ e In h ibitor s of F a ctor Xa
Bernhard Gabriel,† Milton T. Stubbs,†,§ Andreas Bergner,† J o¨rg Hauptmann,‡ Wolfram Bode,†
J o¨rg Stu¨rzebecher,‡ and Luis Moroder*,†
Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany, Institut fu¨r Pharmazeutische
Chemie, Philipps-Universita¨t Marburg, D-35032 Marburg, Germany, and Zentrum fu¨r Vaskula¨re Biologie und Medizin,
Friedrich-Schiller-Universita¨t J ena, D-99089 Erfurt, Germany
Received April 13, 1998
A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a
combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was
synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4′-amidino-
benzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest
affinity for fXa despite the unfavored location of the amidino substituent in the para position.
X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed
a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes
with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance
rates in rats, if compared to those of other benzamidine derivatives, suggests this compound
as an interesting new lead structure for the design of fXa inhibitors.
In tr od u ction
Extrinsic and intrinsic pathways of the blood coagula-
tion cascade intersect at factor Xa (fXa) making this
enzyme directly responsible for thrombin activation
while allowing the thrombin-catalyzed activation of the
anticoagulant APC.1 Because of this key position in the
enzymatic cascade, fXa represents an attractive target
for anticoagulant drug development. It is a trypsin-like
enzyme whose architecture2 is very similar to that of
other procoagulant proteinases such as thrombin,3,4
factor IXa,5 and factor VIIa6 as well as of the antico-
agulant APC7 despite major differences exhibited by
these enzymes in natural substrate specificities and
related biological functions.
Among the synthetic inhibitors of fXa reported so far,
the most potent ones consist of two basic moieties linked
by spacers of appropriate length like in the Daiichi
compound DX-9065a8 or the related YM-608289 as well
as in the bis-benzamidine compounds 2,7-bis(4-amidi-
nobenzylidene)cycloheptan-1-one or the 3-amidine iso-
mer.10 Initial efforts to identify the structural deter-
minants of these low-mass synthetic compounds for
specific inhibition of fXa were hampered by occlusion
of the active site cleft of des(1-45)human fXa by the
crystal packing.2 Recently, however, it was reported
that the binding mode of the synthetic inhibitor DX-
9065a to des-Gla-fXa11 is identical to that previously
determined by X-ray structural analysis of the DX-
9065a/trypsin complex,12 thus suggesting the indirect
approach based on crystallographic analyses of trypsin/
inhibitor complexes as promising as was previously
found to be in the case of thrombin inhibitors.13-17
Recently, we have applied this indirect approach with
a series of 10 different mono- and bis-benzamidine
F igu r e 1. Structures and inhibitory potencies of compounds
1a ,b10 and DX-9065a.8
compounds of different inhibitory potencies and speci-
ficities toward fXa and the plasminogen activators uPA
and sc-tPA.18 The binding mode of the inhibitors to the
active-site cleft of trypsin, as determined by X-ray
analysis, was then used for analogy modeling of the
related complexes with des-Gla-fXa. By correlating the
binding affinities and particularly the inhibitory speci-
ficities shown by this set of inhibitors10 with the
information gained in the structural analyses and
modeling experiments, the NR-tosyl-glycyl-D,L-3-amidi-
nophenylalanine alkyl esters 1a ,b appeared to be in-
teresting lead structures (Figure 1).
* Author for correspondence: Prof. Luis Moroder. Tel: 49-89-8578-
3905. Fax: 49-89-8578-2847. E-mail: moroder@biochem.mpg.de.
† Max-Planck-Institut fu¨r Biochemie.
The synthetic inhibitor DX-9065a binds in an ex-
§ Philipps-Universita¨t Marburg.
‡ Friedrich-Schiller-Universita¨t J ena.
tended conformation to the unprimed sites of fXa.11
10.1021/jm980227t CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/24/1998

Benzamidine-Type Inhibitors of Factor Xa
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4241
Sch em e 1. Syntheses of Bis-Substituted Derivatives of
Racemic NR-Benzenesulfonyl-glycyl-phenylalanine Ethyl
Estersa
F igu r e 2. Schematic representation of the putative binding
mode of compound 1a to fXa as derived from the X-ray
structure of the trypsin/NR-tosyl-glycyl-D,L-3-amidinophenyl-
alanine methyl ester complex18 and the des-Gla-fXa/DX-9065a
complex.11
Thereby the bulky naphthylamidine moiety is inserted
into the hydrophobic S1 subsite with its amidine group
salt-bridged to Asp-189 on the bottom of the pocket. The
aromatic benzyl spacer is oriented parallel to Trp-215
and the acetimidoyl-pyrrolidinyl moiety interacts with
the hydrophobic box formed by the side chains of
residues Tyr-99, Phe-174, and Trp-215, whereas the
acetimido group maps to an electronegative cavity built
up by the carbonyl oxygens of Lys-96, Thr-98, and Glu-
97 and by the carboxylate group of the latter residue.
Modeling of the X-ray structure of compound 1a ,
when bound to crystalline trypsin, onto the active-site
cleft of des-Gla-fXa revealed a very similar interaction
pattern whereby the restriction imparted by the side
chain of Tyr-99 for the P2 residues in substrates and
inhibitors of fXa is fulfilled with the glycine residue of
compound 1a (Figure 2). Conversely, the nitrogen
cation hole identified in the DX-9065a/des-Gla-fXa
complex11 is not exploited with this inhibitor. In the
present study we have, therefore, focused our attention
primarily on the effect of hydrogen-bonding partners or
positively charged groups in position 4 of the benzene-
sulfonyl group of the lead structure on the inhibitory
potency and selectivity toward a series of trypsin-like
proteinases.
a
(a) TEA, DMF; (b) H2S, TEA, pyridine; (c) CH3I, acetone; (d)
NH4OAc, MeOH, ∆; HPLC.
none imine of glycine ethyl ester with 3- and 4-cy-
anobenzyl bromide according to the method described
by O′Donnell and Bennett,20 followed by mild acid
hydrolysis of the Schiff base.
Subsequent conversion of the bis-cyano to the bis-
amidino compounds was carried out by standard pro-
cedures based on addition of hydrogen sulfide, methy-
lation of the resulting thioamido intermediates, and
aminolysis of the thioimidates to the corresponding
amidino derivatives. This conversion occurred almost
quantitatively for the cyano group of the phenylalanine
moiety, but in a significantly less efficient manner in
the case of the 4′-cyanobenzenesulfonyl moiety. Indeed,
aminolysis of the related 4′-thioimidate derivative,
besides producing the desired 4′-amidino compound, led
to its reconversion to the 4′-thioamido and 4′-cyano
compounds with a product to side-product distribution
of about 60:20:20. The side products were recovered
from the reaction mixture, thus allowing for concomitant
isolation of the 4′-cyano- and 4′-thioamidobenzenesulfo-
nyl derivatives as interesting mono-amidino compounds.
Resu lts
Ch em ist r y. NR-Tosyl-glycyl-D,L-3-amidinophenyl-
alanine ethyl ester (1b) is slightly more potent than the
related methyl ester 1a (Figure 1). Correspondingly,
the bis-benzamidine derivatives of 1b with the amidino
groups located in position 4′ of the benzenesulfonyl
group and in position 3 or 4 of the phenylalanine moiety,
respectively, were synthesized following the general
route outlined in Scheme 1. For this purpose glycine
was acylated with 4-cyanobenzenesulfonyl chloride, and
the resulting NR-(4-cyanobenzenesulfonyl)glycine was
then converted to the N-hydroxysuccinimide ester which
in turn was used to acylate D,L-4- and D,L-3-cyanophe-
nylalanine as well as the related ethyl esters. The two
cyanophenylalanine derivatives were prepared following
essentially known procedures,19 whereas D,L-4- and D,L-
3-cyanophenylalanine ethyl esters were obtained by
catalytic phase-transfer alkylation of the dibenzophe-
In h ibition of fXa . The bis-benzamidine compounds
as ethyl esters 8a ,b or free carboxylic acids 14a ,b, the
mixed cyano/amidino compounds 9a ,b, and thioamido/
amidino compounds 10a ,b were assayed for their in-
hibitory potencies against a larger series of trypsin-like
proteinases, and the resulting Ki values are reported

4242 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Gabriel et al.
Ta ble 1. Inhibition of Various Trypsin-like Enzymes by Bis-Substituted Derivatives of Racemic
NR-Benzenesulfonyl-glycyl-phenylalanine
Ki (µM)a
no.
R1
R2
R3
hfXa
bfXa
thrombin
APC
PK
plasmin uPA
sc-tPA trypsin tryptaseb
8a
8b
9a
9b
4-amidine 4′-amidine
3-amidine 4′-amidine
4-amidine 4′-cyano
3-amidine 4′-cyano
CH2CH3 0.50 (0.04) 0.53 (0.10) 41 (5)
43 (6)
29 (3)
>1000
80 (2)
8.6 (2.2) 27 (2)
7.6 (0.7) 19 (4)
>1000 70 (7)
4.2 (1.2) 29 (3)
15 (4) 110 (12) 3.6 (1.1) 12 (1)
CH2CH3 0.82 (0.10) 1.6 (0.2)
25 (2)
11 (1)
15 (1)
CH2CH3 8.7 (1.7)
CH2CH3 5.2 (1.6)
4.8 (0.5)
1.8 (0.5)
4.2 (0.2)
18 (2)
14 (1)
29 (3)
12 (1)
>1000
>1000 >1000
>1000 >1000
20 (4)
14 (3)
19 (4)
13 (2)
18 (4)
>1000
57 (9)
33 (8)
10a 4-amidine 4′-thioamide CH2CH3 7.1 (0.9)
10b 3-amidine 4′-thioamide CH2CH3 0.64 (0.09) 0.58 (0.05) 15 (1)
14a 4-amidine 4′-amidine
14b 3-amidine 4′-amidine
0.45 (0.09) >1000
160 (45) >1000 >1000
>1000
21 (4)
65 (7)
48 (8)
180 (28) 19 (3)
180 (12) 36 (5)
52 (4)
57 (2)
>1000
>1000 >1000
>1000 >1000
>1000 180 (24) 20 (1)
H
H
3.6 (0.7)
18 (2)
2.6 (0.7)
29 (3)
>1000
>1000
100 (27)
a
b
Ki values were calculated according to Dixon32 using a linear regression program; mean values ((SD, n ) 3-5). IC50 values.
in Table 1. The bis-benzamidine compound 8b contain-
ing the 3-amidinophenylalanine moiety was found to
exhibit the lowest degree of specificity, since it was
capable of inhibiting in the micromolar range all the
enzymes analyzed. A replacement of the 4′-amidino
with the thioamido substituent in the benzenesulfonyl
moiety (10b) did not affect the binding affinity, thus
strongly suggesting the absence of any significant
interaction of both these substituents with the nitrogen
cation hole. With the related cyano derivative 9b, only
a moderate reduction in inhibitory potency toward
bovine fXa is observed, if compared to the 4′-amidino
compound, although inhibition of human fXa is sensibly
affected.
By comparing the series of compounds with a 3-ami-
dinophenylalanine as constituent (8b, 9b, 10b) with
F igu r e 3. Plasma levels of the fXa inhibitors 14a (9) and 14b
(b) administered iv at 1 mg/kg doses in anesthetized rats in
comparison with NAPAP (2).
those containing the 4-amidinophenylalanine (8a , 9a ,
10a ), the most potent inhibitor of fXa was found to be
the 4,4′-bis-amidino derivative 8a with a Ki value of 0.5
µM for human fXa and 0.53 µM for bovine fXa. This
observation contrasts previous findings that a 3-amidino
group as substituent of aromatic moieties in the P1
position favors binding to fXa significantly more than
the related 4-amidino isomer, whereas in the case of
thrombin an opposite effect is generally observed.21 Most
surprising was the finding that for this 4,4′-series a
replacement of the 4′-amidino substituent with the
cyano (9a ) or thioamido (10a ) group leads to strongly
reduced binding affinities. Both these observations
suggest a different binding mode of the 3,4′- and 4,4′-
series of compounds to fXa.
As expected from the X-ray structure of DX-9065a
complexed to des-Gla-fXa where the carboxylate group
is directed toward the bulk solvent, the compounds
14a ,b with the free C-terminal carboxyl group exhibit
a high degree of selectivity for inhibition of fXa (Table
1). Again the 4,4′-bis-benzamidine compound 14a is
more active than the 3,4′-bis-benzamidine isomer 14b,
supporting the hypothesis of a putative different binding
mode.
view of these crucial requirements we have examined
the inhibitory activity of our set of compounds toward
different serine proteinases.
Compared to fXa, the inhibitory activity of the most
potent inhibitor of this series, i.e., of compound 8a ,
toward thrombin is lower by nearly 2 orders of magni-
tude (Table 1). A similarly poor inhibition was observed
in the case of the anticoagulantly effective APC as well
as of PK, trypsin, and tryptase. For most of the
compounds listed in Table 1 the Ki values were found
to be 1 or 2 orders of magnitude higher than those
related to inhibition of fXa. The same holds for inhibi-
tion of fibrinolytic enzymes such as plasmin and the
plasminogen activators uPA and sc-tPA. Only com-
pound 10a showed a thrombin inhibition with a sub-
micromolar Ki value possibly indicating a NAPAP-like
binding mode.14
P h a r m a cokin etic Stu dies. Esters of 3-amidinophe-
nylalanine are known to be hydrolyzed in blood or
plasma of experimental animals.25 We have therefore
limited the pharmacokinetic studies to 14a ,b with their
free C-terminal carboxyl function. The plasma levels
of these two inhibitors were monitored after iv admin-
istration at a dose of 1 mg/kg of body weight to
anesthetized rats in comparison to NAPAP. Figure 3
shows the plasma levels of the inhibitors over a period
of 2 h. For NAPAP, as reported previously,26,27 the
In h ibition of Oth er Tr yp sin -lik e P r otea ses. For
an in vivo use of fXa inhibitors as antithrombotic drugs,
the inhibitors are required to act in a highly selective
manner. Particularly important is a minimal inhibition
of enzymes of the fibrinolytic system and of APC which
are counterbalancing prothrombotic stimuli.22-24 In

Benzamidine-Type Inhibitors of Factor Xa
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4243
F igu r e 4. X-ray structure of the trypsin/8a complex.
plasma level declines rapidly reaching the detection
limit at about 60 min after administration; thereby
almost no distinction is observable between the initial
distribution and subsequent elimination phase. Con-
versely, for the inhibitors 14a ,b significantly higher
values were obtained in the initial distribution phase
which is followed by a slow decline of the plasma level.
For comparison, 14a shows after 30 min a 32-fold and
14b a 21-fold higher plasma level than NAPAP; after
120 min the concentrations of 14a ,b in plasma were still
about 1 µg/mL, respectively, whereas the concentration
of NAPAP dropped below the detection limit already
after 60 min.
X-r a y Str u ctu r es of th e 8a ,b/Tr yp sin a n d 10b/
Tr yp sin Com p lexes. To answer the question of
whether the different substitution patterns affect the
binding mode of the inhibitors to fXa, bovine â-trypsin
crystals were soaked with the 4,4′- and 3,4′-bis-benza-
midine derivatives 8a ,b as well as the mixed 3-amidino/
4′-thioamido derivative 10b. In the case of the trypsin
complexes with 8b and 10b, the electron densities of
the inhibitors were too weak to allow for refinement of
the structures, a fact that has to be correlated with the
relatively weak inhibition of trypsin by the two com-
pounds.
For the 8b/trypsin complex the electron density was
very weak except for the region around the S1 pocket
which allowed to identify unambiguously the 3-amidi-
nophenylalanine side chain inserted into the S1 subsite;
for the rest of the molecule possible interaction sites
could not be assigned. Similarly, in the 10b/trypsin
complex the 3-amidinophenylalanine is inserted into the
S1 pocket in full agreement with the previous findings
obtained with compound 1a .18 Again assignment of the
rest of the molecule was prevented by the weak differ-
ence electron density map.
The difference electron density map of the 8a /trypsin
complex is well-defined, thus allowing unambiguous
identification of the whole inhibitor molecule as well as
the assignment of the chirality R of the 4-amidinophe-
nylalanine moiety when bound to the enzyme (Figure
4). Most surprising was the finding that the 4,4′-bis-
benzamidine compound 8a binds to the active site cleft
of trypsin in a clearly different mode than the 3,4′-bis-
benzamidine derivative 8b as hypothetically postulated
on the basis of the inhibition constants. At the entrance
of the S1 pocket the unusual bulky electron density
matches well with the sulfonyl group and thus with
insertion of the 4′-amidinobenzene moiety into the S1
pocket. The typical salt bridge between the amidino
function and the carboxylate oxygens of Asp-189 is
formed, and an additional hydrogen bond is established
with the carbonyl oxygen of Gly-219, as shown in Figure
4. One oxygen of the sulfonyl group is hydrogen-bonded
to the hydroxyl group of the catalytic Ser-195 and to
one water molecule that bridges to Ser-195Oγ and His-
57Nꢀ2. As expected from the inverted binding mode of
the inhibitor 8a , the hydrogen-bonding pattern of the
glycyl residue to the enzyme differs from the pattern
observed so far for similar inhibitor/trypsin complexes.18
In fact, the glycine NH binds to the Ser-214 carbonyl
oxygen, whereas its carbonyl oxygen is hydrogen-bonded
to the backbone NH of Gly-216. The well-defined ethyl
ester group points toward bulk solvent with a water
molecule bound to its carbonyl group. Two more water
molecules are hydrogen-bonded to the second oxygen of
the sulfonyl group, to the oxycarbonyl oxygen of the
ester moiety and to the amide proton of the (R)-4-
amidinophenylalanine residue. The 4-amidino sub-
stituent of the C-terminal phenylalanine is anchored via
a hydrogen-bonding network, mediated by two water
molecules, to the backbone oxygens of Asn-97, Thr-98,
and Glu-175.
Discu ssion
Modeling of the newly synthesized bis-benzamidine
compounds 8a ,b to fXa on the basis of the coordinates
of the trypsin/compound 1a complex suggested a better
fitting of the 3- than the 4-amidinophenylalanine moiety
into the S1 pocket for salt bridging to Asp-189. In both
cases an additional interaction of the second 4′-amidino
moiety with the electronegative cavity generated by the
carbonyl oxygens of Lys-96, Thr-98, and Glu-97 and the
side-chain carboxylate function of Glu-97 was highly
probable. Surprising was, therefore, the finding that
the 4,4′-bis-benzamidine compound 8a showed an inhi-
bition of fXa higher than that of compound 8b despite
the highly unfavored location of the amidino group in
the para position as recently further confirmed with a
series of differently substituted bis-benzamidine com-
pounds.28 Somehow expected was therefore the retro-
binding mode of compound 8a to trypsin in crystals, i.e.,

4244 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Gabriel et al.
experiments. Our present findings fully confirm this
hypothesis.
A retro-binding mode of inhibitors has previously been
reported for thrombin when complexed, e.g., with
hirudin,29-31 ornithodorin,32 the natural product Na-
zumamide A,33 or hirudin-derived synthetic inhibi-
tors.34-36 The X-ray analysis of the trypsin/8a complex
yielded an additional example of such retro-binding of
synthetic inhibitors, and both the inhibitory data and
modeling experiments are strongly supportive for this
type of binding of 8a even to fXa. Thus a new template
molecule was disclosed which could serve for the devel-
opment of low-mass fXa inhibitors with possibly selec-
tive interactions at the specificity sites of the enzyme.
For a synthetic inhibitor as an anticoagulant drug,
the oral administration would certainly be the preferred
one. However, most of the known synthetic inhibitors
of thrombin and fXa show poor absorption after oral/
intraduodenal administration and a marked hepatic
first-pass effect causing low oral bioavailability. Ad-
ditionally, the half-life of the compounds is relatively
short, thus requiring iv infusion. In this context, we
have examined the structure-pharmacokinetic rela-
tionships of various benzamidine-derived inhibitors in
order to improve their oral bioavailability. In compari-
son with NAPAP, the two bis-benzamidine compounds
14a ,b that contain a free C-terminal carboxyl group
were found to exhibit, upon iv administration, signifi-
cantly reduced plasma clearance rates in rats that differ
also from those of several other benzamidine-derived
inhibitors.26,27,37 The prolonged half-life measured for
the two compounds represents the first example of a
decisive improvement of pharmacokinetic properties in
benzamidine-based inhibitors. This property taken
together with the new binding mode may represent an
interesting indication for further synthetic studies using
compounds 8a and 14a as new lead structures.
F igu r e 5. Schematic representation of the retro-binding mode
of 8a to human fXa and the potential interaction sites as
resulting from modeling the 8a /fXa complex on the basis of
the X-ray structure of des-Gla-fXa11 and the 8a /trypsin com-
plex.
with the 4′-amidinobenzenesulfonyl group inserted into
the S1 pocket.
Using the X-ray structure of human des-Gla-fXa11 and
the coordinates of 8a when bound to trypsin, a model
of the fXa/8a complex was generated. As schematically
outlined in Figure 5, the overall hydrogen-bonding
pattern of this inhibitor is essentially retained even in
fXa. The sulfonyl oxygen is hydrogen-bonded to the
side-chain hydroxyl function of Ser-195, and this inter-
action leads to a different orientation of the benzamidine
moiety in the S1 pocket that allows for a salt-bridge
formation between the 4′-amidino group and Asp-189
with an additional hydrogen bond to the carbonyl group
of Gly-219. The glycine carbonyl is hydrogen-bonded
to the NH of Gly-216 thus orienting the aromatic ring
of the C-terminal 4-benzamidine moiety into the aryl
binding site (S4) of fXa, which is flanked by the side
chains of Phe-174 and Tyr-99 not present in trypsin.
The 4-amidino moiety is located behind the aryl binding
site, points toward the carbonyl oxygens of Glu-97, Thr-
98, and Lys-96, and is located within interaction dis-
tance to the side-chain carboxylate of Glu-97. This
positioning of the amidino group within the nitrogen
cation hole detected with the DX-9065a inhibitor cannot
rationally explain the higher affinity of compound 8a
than 8b for fXa, since modeling experiments with the
latter compound, even when bound in the conservative
mode, indicated a similar interaction in the electrone-
gative cavity. This electrostatic interaction apparently
does not suffice for increasing binding affinities, and in
trypsin crystals, where the electronegative cavity is
absent, immobilization of this portion of the molecule
was not achieved as well-evidenced by the poor differ-
ence electron density map.
Exp er im en ta l Section
Abbr evia tion s: fXa, factor X activated; APC, activated
protein C; DX-9065a, (+)-2-[4-[((3S)-1-acetimidoyl-3-pyrrolidi-
nyl)oxy]phenyl]-3-(7-amidino-2-naphthyl)propionic acid; YM-
60828, [N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-
amidino-2-naphthyl)methyl]sulfamoyl]acetic acid dihydro-
chloride; PK, plasma kallikrein; uPA, urokinase-type plasmi-
nogen activator; sc-tPA, single-chain tissue-type plasminogen
activator; NAPAP, NR-(2-naphthylsulfonyl-glycyl)-D,L-p-ami-
dinophenylalanyl-piperidine; HOSu, N-hydroxysuccinimide;
DCCI, N,N′-dicyclohexylcarbodiimide; DMF, N,N′-dimethyl-
formamide; TEA, triethylamine; pNA, 4-nitroanilide; TFA,
trifluoroacetic acid; Cbz, benzyloxycarbonyl.
Ma ter ia ls a n d Meth od s. All solvents and reagents used
in the syntheses were of the highest quality commercially
available and when required were further purified and dried
by standard methods. Melting points were determined on a
Bu¨chi apparatus and are uncorrected. TLC silica gel 60 plates
were from Merck AG (Darmstadt, Germany), and compounds
were visualized with the chlorine/tolidine or permanganate
reagent. Analytical HPLC was performed on either Nucleosil
300/C8 or C18 columns (Macherey-Nagel, Du¨ren, Germany)
using linear gradients of CH3CN/2% H3PO4 from 5:95 (A) to
80:20 (B). Preparative reversed-phase chromatography was
carried out on Lichroprep RP 18 (25-40 µm; Merck AG,
Darmstadt, Germany) and preparative HPLC on Nucleosil RP
8 (Macherey-Nagel, Du¨ren, Germany). FAB-MS spectra were
recorded on a Finnigan MAT 900 spectrometer and EI-MS on
a Finnigan MAT 312 spectrometer. IR spectra were recorded
on a Perkin-Elmer FT-IR spectrometer (model 1760 X), 1H
Recently, with a series of bis-benzamidine compounds
of shorter spacers than the glycyl spacer used in the
present study, Maduskuie et al.28 succeeded to obtain
nanomolar inhibition constants. Thereby the 4′,3-bis-
benzamidine pattern proved to be essential for interac-
tions of the 3-benzamidine group with the S1 pocket.
Correspondingly, a retro-binding mode was postulated
for the inverted 3′,4-substitution pattern on the basis
of the experimental binding affinity data and modeling

Benzamidine-Type Inhibitors of Factor Xa
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4245
NMR spectra in DMSO-d6 on a Bruker AM 400 or AMX 500
spectrometer, and 13C NMR spectra on the AM 400 spectro-
meter. Chemical shifts are reported in ppm (δ) using tetram-
ethylsilane as reference.
(yield 99% of yellow oil) and subsequent hydrolysis with 1 M
HCl: yield 78% of yellowish oil (lit.23 56%); FAB-MS m/z: 219.1
1
[M + H+]; Mr ) 218.11 calcd for C12H14N2O2; H NMR δ 1.12
3
(t, 3H, OCH2CH3, J ) 7.18 Hz), 2.83 (dd, 1H, Ar-CH2-CRH,
3
2
2J ) 13.50 Hz, J ) 7.67 Hz), 2.92 (dd, 1H, Ar-CH2-CRH, J
Nr-(4-Cya n oben zen esu lfon yl)glycin e (2). A suspension
of 4-cyanobenzenesulfonyl chloride (10.0 g, 49.7 mmol) in 120
mL ether was added dropwise to a solution of glycine (3.39 g,
45.2 mmol) in 45 mL 1 M NaOH. The reaction mixture was
stirred vigorously at room temperature for 20 h at pH 9. The
organic layer was washed with water, and the combined
aqueous extracts were acidified with HCl to pH 1. The
precipitate was filtered off, washed with water, and dried at
60 °C over KOH pellets: yield 10.4 g (96%) of white powder;
mp 181-182 °C; EI-MS m/z ) 240.0 [M+]; Mr ) 240.02 calcd
for C9H8N2O4S; IR (KBr) ν 3249 (s), 3196 (s, NH); 3097 (w),
3071 (w), 3048 (w), 3007 (w, dCH); 2936 (w, CH2); 2516 (w,
OH); 2248 (s, CtN); 1752 (s), 1730 (s, CdO); 1493 (w, CdC);
1441 (s), 1427 (m, δCH2); 1341 (s), 1165 (s, ArSO2N<); 1H NMR
3
3
3
) 13.48 Hz, J ) 6.07 Hz), 3.58 (m, 1H, CRH, J ) 7.53 Hz, J
) 6.13 Hz), 4.03 (m, 2H, OCH2CH3, 3J ) 7.10 Hz), 7.48 (t, 1H,
3
3
4
Ar-H5, J ) 7.97 Hz), 7.56 (dvt, 1H, Ar-H4, J ) 7.82 Hz, J )
1.19 Hz), 7.66 (m, 1H, Ar-H6), 7.67 (t, 1H, Ar-H2, 4J ) 1.22
Hz); 13C NMR δ 13.91 (OCH2CH3), 40.46 (Ar-CH2-CRH), 55.20
(CRH), 59.96 (OCH2CH3), 110.91 (Ar-C3), 118.80 (CtN), 129.11
(Ar-C4), 129.95 (Ar-C5), 132.82 (Ar-C2), 134.27 (Ar-C6), 139.83
(Ar-C1), 174.58 (CRH-COOCH2CH3).
Nr-(4′-Cya n ob en zen esu lfon yl)glycyl-D,L-4-cya n op h e-
n yla la n in e Eth yl Ester (5a ). Compound 4a (1.52 g, 6.96
mmol) was reacted in DMF with the active ester 3 (1.85 g,
5.48 mmol) and 0.76 mL (5.48 mmol) TEA for 3 days. The
bulk of the solvent was evaporated and the residue distributed
between AcOEt and 5% KHSO4. The organic layer was
washed with water, dried over Na2SO4, and evaporated to
dryness. The residue was recrystallized from CHCl3/n-hex-
ane: yield 2.38 g (99%) of orange foam; FAB-MS m/z: 441.3
[M + H+]; Mr ) 440.12 calcd for C21H20N4O5S; IR (KBr) ν 3355
(m, NH); 3095 (w, dCH); 2984 (w), 2936 (w, CH2/3); 2232 (m,
CtN); 1738 (s, CdO); 1671 (s, N-CdO); 1610 (w, δNH); 1536
(m, CdC); 1447 (w, δCH2); 1377 (w, δCH3); 1342 (m, ArSO2N<);
1217 (m, C-O); 1165 (s, ArSO2N<); 1H NMR δ 1.11 (t, 3H,
OCH2CH3, 3J ) 7.0 Hz), 2.95 (dd, 1H, Ar-CH2-CRH, 2J ) 14.0
δ 3.67 (s, 2H, NH-CH2-COOH), 7.95 (m, 2H, Ar-H3/5
,
3J )
,
3J ) 8.53 Hz, AA′), 8.35
8.49 Hz, BB′), 8.06 (d, 2H, Ar-H2/6
(br s, 1H, CH2-NH-SO2); 13C NMR δ 43.66 (NH-CH2-
COOH), 114.66 (Ar-C4), 117.69 (CtN), 127.19 (Ar-C2/6), 133.11
(Ar-C3/5), 144.97 (Ar-C1), 169.98 (CH2-COOH).
Nr-(4-Cya n oben zen esu lfon yl)glycin e N-Hyd r oxysu c-
cin im id a te (3). To an ice-cold solution of 2 (1.2 g, 5.0 mmol)
and HOSu (0.58 g, 5.05 mmol) in 35 mL dioxane was added
DCCI (1.03 g, 5.0 mmol), and the reaction mixture was stirred
overnight at 4 °C. The urea was filtered off, the solution
evaporated, and the residue crystallized from 2-propanol: yield
1.53 g (91%) of white powder; mp 184-185 °C; FAB-MS m/z
338.2 [M + H+]; Mr ) 337.04 calcd for C13H11N3O6S; IR (KBr)
ν 3385 (m, NH); 3070 (w), 3044 (w, dCH); 2980 (w), 2939 (w,
CH2); 2234 (m, CtN); 1827 (s), 1784 (m), 1748 (s), 1712 (sh,
CdO); 1630 (s, δNH2); 1491 (w, CdC); 1434 (m), 1416 (s, δCH2);
1341 (s Ar-SO2N<); 1215 (s, C-O); 1175 (s, ArSO2N<); 1079
(s, C-O); 1H NMR δ 2.60 (s, 4H, OC-CH2-CH2-CO), 4.85 (s,
2H, dN-CH2-COO), 8.16 (d, 2H, Ar-H3/5, 3J ) 7.96 Hz, BB′),
3
2
Hz, J ) 9.0 Hz), 3.08 (dd, 1H, Ar-CH2-CRH, J ) 14.0 Hz,
3J ) 6.0 Hz), 3.53 (m, 2H, CO-CH2-NH, J ) 17.0 Hz, J )
2
3
8.0 Hz), 4.05 (q, 2H, OCH2CH3, 3J ) 7.0 Hz), 4.43 (dvt, 1H,
3
3
3
CRH, J ) 8.0 Hz, J ) 6.0 Hz), 7.38 (d, 2H, Ar-H3/5, J ) 8.2
3
Hz, BB′), 7.73 (d, 2H, Ar-H2/6, J ) 8.2 Hz, AA′), 7.91 (d, 2H,
3
3
Ar-H3′/5′, J ) 8.5 Hz, BB′), 8.03 (d, 2H, Ar-H2′/6′, J ) 8.5 Hz,
AA′), 8.27 (br s, 1H, CH2-NH-SO2), 8.38 (d, 1H, CRH-NH-
CO, 3J ) 8.0 Hz); 13C NMR δ 13.82 (OCH2CH3), 36.60 (Ar-
CH2-CRH), 44.53 (CO-CH2-NH), 52.81 (CRH), 60.68 (OCH2-
CH3), 109.44 (Ar-C4), 114.69 (Ar-C4′), 117.66 (SO2-Ar-CtN),
118.74 (CtN), 127.23 (Ar-C2′/6′), 130.19 (Ar-C2/6), 131.99 (Ar-
3
8.21 (d, 2H, Ar-H2/6, J ) 7.96 Hz, AA′), 10.50 (s, 1H, S-OH);
13C NMR δ 25.13 (OC-CH2-CH2-CO), 41.41 (NH-CH2-
COOH), 114.94 (Ar-C4), 117.63 (CtN), 127.09 (Ar-C2/6), 133.15
(Ar-C3/5), 144.71 (Ar-C1), 165.71 (CH2-CO-Nd), 172.59 (dN-
CO-CH2).
C
3/5), 133.06 (Ar-C3′/5′), 142.90 (Ar-C1), 144.65 (Ar-C1′), 167.30
(NH-CO-CH2), 170.59 (CRH-COOCH2CH3).
Nr-(4′-Cya n ob en zen esu lfon yl)glycyl-D,L-3-cya n op h e-
n yla la n in e Eth yl Ester (5b). The title compound was
prepared by reacting 4b with 3 as described for 5a : yield 96%
of yellowish foam; FAB-MS m/z: 441.2 [M + H+]; Mr ) 440.12
calcd for C21H20N4O5S; IR (KBr) ν 3370 (m, NH); 3098 (w, d
CH); 2984 (w), 2936 (w, CH2/3); 2233 (m, CtN); 1738 (s, Cd
O); 1667 (s, N-CdO); 1535 (m), 1485 (w, CdC); 1446 (w,
δCH2); 1377 (w, δCH3); 1342 (m, ArSO2N<); 1213 (m, C-O);
1164 (s, ArSO2N<); 1H NMR δ 1.11 (t, 3H, OCH2CH3, 3J )
D,L-4-Cya n op h en yla la n in e Eth yl Ester (4a ). To a solu-
tion of 4-cyanobenzyl bromide (8.8 g, 44.9 mmol), N-diphenyl-
methylene-glycine ethyl ester38 (10.0 g, 37.4 mmol), and
tetrabutylammonium bromide (1.21 g, 3.74 mmol) in CH3CN
was added powdered K2CO3 (12.9 g, 93.5 mmol). After 48 h
under vigorous stirring the K2CO3 was filtered off and the
solution evaporated. The residue was dissolved in ether, and
the organic layer was washed with brine, dried over Mg2SO4,
and then evaporated to dryness: yield 14.2 g (99%) of yellowish
powder; mp 69-72 °C; the IR (KBr) and 1H and 13C NMR
spectra were consistent with the assigned structure.
2
3
7.0 Hz), 2.92 (dd, 1H, Ar-CH2-CRH, J ) 14.0 Hz, J ) 9.0
2
3
Hz), 3.05 (dd, 1H, Ar-CH2-CRH, J ) 14.0 Hz, J ) 6.0 Hz),
2
3
3.53 (m, 2H, CO-CH2-NH, J ) 16.6 Hz, J ) 6.0 Hz), 4.04
3
3
(q, 2H, OCH2CH3, J ) 7.0 Hz), 4.44 (dvt, 1H, CRH, J ) 8.0
The solution of the intermediate N-diphenylmethylene-D,L-
4-cyanophenylalanine ethyl ester (5.58 g, 14.6 mmol) in 50 mL
ether was stirred with 21.9 mL (21.9 mmol) 1 M HCl for 22 h.
The reaction mixture was washed with 1 M HCl and water,
and the combined aqueous extracts were neutralized with
Na2CO3 (pH 9.5) and reextracted with AcOEt. The organic
layer was dried over Na2SO4 and evaporated to dryness: yield
6.55 g (80%) of yellowish oil; FAB-MS m/z: 219.0 [M + H+];
Mr ) 218.11 calcd for C12H14N2O2; 1H NMR δ 1.11 (t, 3H,
OCH2CH3, 3J ) 7.15 Hz), 2.85 (dd, 1H, Ar-CH2-CRH, 2J )
13.38 Hz, 3J ) 7.61 Hz), 2.95 (dd, 1H, Ar-CH2-CRH, 2J )
3
3
Hz, J ) 6.0 Hz), 7.48 (t, 1H, Ar-H5, J ) 7.6 Hz), 7.53 (dvt,
3
4
1H, Ar-H4, J ) 7.6 Hz, J ) 1.6 Hz), 7.65 (s, 1H, Ar-H2), 7.69
(dvt, 1H, Ar-H6, 3J ) 7.6 Hz, 4J ) 1.6 Hz), 7.91 (d, 2H, Ar-
H3′/5′, 3J ) 8.5 Hz, BB′), 8.03 (d, 2H, Ar-H2′/6′, 3J ) 8.5 Hz, AA′),
3
8.26 (t, 1H, CH2-NH-SO2, J ) 6.0 Hz), 8.37 (d, 1H, CRH-
NH-CO, 3J ) 8.0 Hz); 13C NMR δ 13.82 (OCH2CH3), 36.01
(Ar-CH2-CRH), 44.52 (CO-CH2-NH), 52.91 (CRH), 60.64
(OCH2CH3), 111.14 (Ar-C4), 114.69 (Ar-C4′), 117.65 (SO2-Ar-
CtN), 118.67 (CtN), 127.22 (Ar-C2′/6′), 129.31 (Ar-C5), 130.39
(Ar-C4), 132.71 (Ar-C2), 133.05 (Ar-C3′/5′), 134.12 (Ar-C6), 138.55
(Ar-C1), 144.65 (Ar-C1′), 167.32 (NH-CO-CH2), 170.61 (CRH-
COOCH2CH3).
3
3
3
13.35 Hz, J ) 6.22 Hz), 3.59 (dd, 1H, CRH, J ) 7.77 Hz, J
) 6.24 Hz), 4.03 (q, 2H, OCH2CH3, 3J ) 7.08 Hz), 7.41 (d, 2H,
3
3
Nr-(4′-Th ioa m id ob e n ze n e su lfon yl)glycyl-D,L-4-t h io-
a m id op h en yla la n in e Eth yl Ester (6a ). Hydrogen sulfide
was bubbled through the solution of 5a (2.14 g, 4.86 mmol)
and 4.03 mL (28.1 mmol) TEA in 30 mL of dry pyridine for 15
min. The solution was stirred for 5 days and then evaporated.
The residue was washed in AcOEt with 1 M HCl and water,
dried over Na2SO4, and evaporated to dryness: yield 2.3 g
(93%) of yellowish foam; HPLC (isocratic elution with A for 5
Ar-H3/5, J ) 8.15 Hz, BB′), 7.73 (d, 2H, Ar-H2/6, J ) 8.19 Hz,
AA′); 13C NMR δ 13.92 (OCH2CH3), 40.48 (Ar-CH2-CRH),
55.21 (CRH), 59.97 (OCH2CH3), 109.02 (Ar-C4), 118.87 (CtN),
130.32 (Ar-C2/6), 131.79 (Ar-C3/5), 144.24 (Ar-C1), 174.54 (CRH-
COOCH2CH3).
D,L-3-Cya n op h en yla la n in e Eth yl Ester (4b). The title
compound was prepared as described for 4a via the intermedi-
ate N-diphenylmethylene-D,L-3-cyanophenylalanine ethyl ester

4246 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Gabriel et al.
min; linear gradient from A to B in 25 min): tR ) 20.07 min;
FAB-MS m/z: 509.2 [M + H+]; Mr ) 508.09 calcd for
C21H24N4O5S3; IR (KBr) ν 3315 (s), 3194 (s, NH2); 3089 (sh,
dCH); 2975 (w), 2915 (w, CH2/3); 1728 (s, CdO); 1659 (s, N-Cd
O); 1630 (s, δNH2); 1565 (w), 1531 (m, CdC); 1423 (m, δCH2);
1377 (w, δCH3); 1330 (m, ArSO2N<); 1275 (m, C-O); 1164 (s,
(Ar-C2/6), 134.61 (Ar-C4′), 145.18 (Ar-C1), 167.46 (NH-CO-
CH2), 170.59 (CRH-COOCH2CH3), 188.61 (C-SCH3(dNH2+)).
Nr-(4′-Meth ylth ioim id o-ben zen esu lfon yl)glycyl-D,L-3-
m eth ylth ioim ido-ph en ylalan in e Eth yl Ester Bis(h ydr oio-
d id e) (7b). The compound was obtained from 6b as described
for 7a : yield 88% of yellow foam; HPLC (conditions as for
1
3
6a ): tR ) 14.57 min; FAB-MS m/z: 537.2 [M + H+]; Mr
)
ArSO2N<); H NMR δ 1.12 (t, 3H, OCH2CH3, J ) 7.03 Hz),
2.93 (dd, 1H, Ar-CH2-CRH, 2J ) 13.76 Hz, 3J ) 8.40 Hz),
3.02 (dd, 1H, Ar-CH2-CRH, 2J ) 13.80 Hz, 3J ) 5.92 Hz),
3.48 (m, 2H, CO-CH2-NH, 2J ) 16.57 Hz, 3J ) 6.21 Hz), 4.04
(q, 2H, OCH2CH3, 3J ) 7.04 Hz), 4.44 (dvt, 1H, CRH, 3J ) 8.05
536.12 calcd for C23H28N4O5S3; IR (KBr) ν 3406 (s), 3192 (sh,
NH2); 3061 (w, dCH); 2996 (w), 2935 (sh, CH2/3); 1730 (m, Cd
O); 1666 (s, N-CdO); 1629 (sh, δNH2); 1565 (w), 1535 (m, Cd
C); 1401 (m, δCH2); 1377 (w, δCH3); 1337 (m, ArSO2N<); 1275
(w), 1210 (w, C-O); 1166 (s, ArSO2N<); 1H NMR δ 1.11 (t,
3H, OCH2CH3, 3J ) 7.12 Hz), 2.83 (s, 3H, -SCH3), 2.85 (s,
3H, -SCH3), 3.01 (dd, 1H, Ar-CH2-CRH, 2J ) 13.82 Hz, 3J )
8.61 Hz), 3.12 (dd, 1H, Ar-CH2-CRH, 2J ) 13.77 Hz, 3J )
6.04 Hz), 3.54 (d, 2H, CO-CH2-NH, 3J ) 6.09 Hz), 4.05 (q,
2H, OCH2CH3, 3J ) 7.05 Hz), 4.48 (dvt, 1H, CRH, 3J ) 8.23
Hz, 3J ) 6.10 Hz), 7.22 (d, 2H, Ar-H3/5
,
3J ) 8.32 Hz, BB′),
3
7.79 (d, 2H, Ar-H3′/5′, J ) 8.43 Hz, BB′), 7.84 (d, 2H, Ar-H2/6
,
3J ) 8.34 Hz, AA′), 7.97 (d, 2H, Ar-H2′/6′, J ) 8.37 Hz, AA′),
3
3
8.20 (t, 1H, CH2-NH-SO2, J ) 6.23 Hz), 8.32 (d, 1H, CRH-
NH-CO, 3J ) 7.72 Hz), 9.40 (s, 1H, CdS(-NH2)), 9.65 (s, 1H,
CdS(-NH2)), 9.76 (s, 1H, CdS(-NH2)), 10.06 (s, 1H, CdS(-
NH2)); 13C NMR δ 13.85 (OCH2CH3), 36.37 (Ar-CH2-CRH),
44.71 (CO-CH2-NH), 53.14 (CRH), 60.63 (OCH2CH3), 126.13
(Ar-C2′/6′), 127.24 (Ar-C2/6), 127.68 (Ar-C3′/5′), 128.54 (Ar-C3/5),
137.54 (Ar-C4), 140.30 (Ar-C4′), 142.12 (Ar-C1), 142.79 (Ar-C1′),
167.43 (NH-CO-CH2), 170.81 (CRH-COOCH2CH3), 198.84
(CdS(-NH2)), 199.55 (CdS(-NH2)).
3
3
Hz, J ) 6.05 Hz), 7.58 (t, 1H, Ar-H5, J ) 7.66 Hz), 7.64 (dvt,
1H, Ar-H4, 3J ) 7.82 Hz), 7.72 (br s, 1H, Ar-H2), 7.73 (dvt, 1H,
3
4
3
Ar-H6, J ) 7.68 Hz, J ) 1.06 Hz), 7.96 (d, 2H, Ar-H3′/5′, J )
3
8.94 Hz, BB′), 8.00 (d, 2H, Ar-H2′/6′, J ) 8.65 Hz, AA′), 8.28
3
(t, 1H, CH2-NH-SO2, J ) 6.10 Hz), 8.42 (d, 1H, CRH-NH-
CO, 3J ) 7.83 Hz); 13C NMR δ 13.88 (OCH2CH3), 15.68
(-SCH3), 36.20 (Ar-CH2-CRH), 44.67 (CO-CH2-NH), 53.14
(CRH), 60.73 (OCH2CH3), 126.34 (Ar-C4), 127.19 (Ar-C2′/6′),
128.07 (Ar-C2), 128.61 (Ar-C5), 128.70 (Ar-C3′/5′), 129.41 (Ar-
C3), 131.02 (Ar-C4′), 135.93 (Ar-C6), 138.58 (Ar-C1), 145.45 (Ar-
C1′), 167.47 (NH-CO-CH2), 170.66 (CRH-COOCH2CH3), 188.93
(C-SCH3(dNH2+)).
Nr-(4′-Th ioa m id ob e n ze n e su lfon yl)glycyl-D,L-3-t h io-
a m id op h en yla la n in e Eth yl Ester (6b). The title compound
was prepared from 5b as described for 6a : yield 91% of yellow
foam; HPLC (conditions as for 6a ): tR ) 20.20 min; FAB-MS
m/z: 509.2 [M + H+]; Mr ) 508.09 calcd for C21H24N4O5S3; IR
(KBr) ν 3315 (s), 3194 (s, NH2); 3090 (sh, dCH); 2978 (w,
CH2/3); 1729 (s, CdO); 1667 (s, N-CdO); 1630 (s, δNH2); 1534
(m, CdC); 1412 (m, δCH2); 1377 (w, δCH3); 1332 (m, ArSO2N<);
1277 (m, C-O); 1164 (s, ArSO2N<); 1H NMR δ 1.11 (t, 3H,
OCH2CH3, 3J ) 7.06 Hz), 2.93 (dd, 1H, Ar-CH2-CRH, 2J )
13.74 Hz, 3J ) 8.16 Hz), 3.02 (dd, 1H, Ar-CH2-CRH, 2J )
13.80 Hz, 3J ) 6.12 Hz), 3.50 (m, 2H, CO-CH2-NH, 2J ) 16.80
N r-(4′-Am id in ob e n ze n e su lfon yl)glycyl-D,L-4-a m id i-
n op h en yla la n in e Eth yl Ester Bis(tr iflu or oa ceta te) (8a ).
A solution of 7a (0.8 g, 1.01 mmol) and ammonium acetate
(195 mg, 2.53 mmol) in dry MeOH (25 mL) was refluxed for 4
h and then evaporated. The oily residue was chromatographed
on a Lichroprep RP-18 column (2 × 200 cm) by elution with a
linear gradient from 1% TFA/CH3CN (95:5) to 1% TFA/CH3-
CN (55:45) in 10 h at a flow rate of 380 mL/h. The elution
pattern exhibited three peaks which were separately pooled,
evaporated, and dried over KOH pellets.
3
3
Hz, J ) 6.64 Hz), 4.04 (q, 2H, OCH2CH3, J ) 7.01 Hz), 4.44
(dvt, 1H, CRH, 3J ) 7.80 Hz, 3J ) 6.30 Hz), 7.30-7.32 (m, 2H,
Ar-H4 and Ar-H5), 7.72-7.74 (m, 2H, Ar-H2 and Ar-H6), 7.79
3
3
(d, 2H, Ar-H3′/5′, J ) 8.35 Hz, BB′), 7.97 (d, 2H, Ar-H2′/6′, J )
8.41 Hz, AA′), 8.04 (t, 1H, CH2-NH-SO2, J ) 6.14 Hz), 8.32
3
Peak 1 corresponds to the title compound: yield 0.26 g (37%)
of white powder; HPLC (linear gradient from 100% A to 60%
B in 18 min): tR ) 7.62 min; FAB-MS m/z: 475.2 [M + H+];
Mr ) 474.17 calcd for C21H26N6O5S; IR (KBr) ν 3333 (m, NH2);
3104 (m, dCH); 2945 (sh), 2867 (sh, CH2/3); 1738 (sh, CdO);
1670 (s, N-CdO); 1618 (w, δNH2); 1537 (m), 1492 (m, CdC);
1442 (m, δCH2); 1377 (w, δCH3); 1340 (m, ArSO2N<); 1206 (s,
(d, 1H, CRH-NH-CO, 3J ) 7.70 Hz), 9.40 (s, 1H, CdS(-NH2)),
9.65 (s, 1H, CdS(-NH2)), 9.80 (s, 1H, CdS(-NH2)), 10.06 (s,
1H, CdS(-NH2)); 13C NMR δ 13.83 (OCH2CH3), 36.62 (Ar-
CH2-CRH), 44.71 (CO-CH2-NH), 53.35 (CRH), 60.62 (OCH2-
CH3), 125.41 (Ar-C4), 126.14 (Ar-C3′/5′), 127.69 (Ar-C2′/6′ and Ar-
C2), 128.20 (Ar-C5), 131.83 (Ar-C6), 136.50 (Ar-C1), 139.32 (Ar-
C3), 142.12 (Ar-C4′), 142.79 (Ar-C1′), 167.40 (NH-CO-CH2),
170.87 (CRH-COOCH2CH3), 198.84 (CdS(-NH2)), 199.95 (Cd
S(-NH2)).
1
C-O); 1136 (s, ArSO2N<); H NMR δ 1.13 (t, 3H, OCH2CH3,
2
3
3J ) 7.20 Hz), 3.01 (dd, 1H, Ar-CH2-CRH, J ) 13.85 Hz, J
2
3
) 8.71 Hz), 3.13 (dd, 1H, Ar-CH2-CRH, J ) 13.84 Hz, J )
5.87 Hz), 3.52 (dd, 2H, CO-CH2-NH, 2J ) 16.58 Hz), 4.06 (q,
2H, OCH2CH3, 3J ) 7.09 Hz), 4.50 (dvt, 1H, CRH, 3J ) 8.36
Hz, 3J ) 5.99 Hz, 3J ) 5.67 Hz), 7.45 (d, 2H, Ar-H3/5, 3J ) 8.40
Nr-(4′-Meth ylth ioim id o-ben zen esu lfon yl)glycyl-D,L-4-
m eth ylth ioim ido-ph en ylalan in e Eth yl Ester Bis(h ydr oio-
d id e) (7a ). To 6a (0.79 mg, 1.55 mmol) in 25 mL dry acetone
was added methyl iodide (0.58 mL, 9.31 mmol) under nitrogen
in the dark. After 2 days the reaction mixture was evaporated
to dryness: yield 0.87 g (97%) of yellowish foam; HPLC
(conditions as for 6a ): tR ) 14.69 min; FAB-MS m/z: 537.2
[M + H+]; Mr ) 536.12 calcd for C23H28N4O5S3; IR (KBr) ν 3400
(sh), 3192 (sh, NH2); 3048 (w, dCH); 2973 (w, CH2/3); 1730 (m,
CdO); 1659 (s, N-CdO); 1608 (m, δNH2); 1566 (w), 1531 (m,
CdC); 1400 (m, δCH2); 1377 (w, δCH3); 1335 (m, ArSO2N<);
1277 (w), 1207 (sh, C-O); 1165 (s, ArSO2N<); 1H NMR δ 1.11
3
Hz, BB′), 7.75 (d, 2H, Ar-H2/6, J ) 8.38 Hz, AA′), 7.96 (d, 2H,
Ar-H3′/5′
,
3J ) 9.19 Hz, BB′), 7.98 (d, 2H, Ar-H2′/6′
,
3J ) 9.15
3
Hz, AA′), 8.46 (d, 1H, CRH-NH-CO, J ) 7.82 Hz), 9.29 (br
s, 7H, C-NH2(dNH2+) and CH2-NH-SO2); 13C NMR δ 13.86
(OCH2CH3), 36.40 (Ar-CH2-CRH), 44.69 (CO-CH2-NH), 53.07
(CRH), 60.72 (OCH2CH3), 126.25 (Ar-C4), 126.76 (Ar-C2′/6′),
127.92 (Ar-C3/5), 129.00 (Ar-C3′/5′), 129.66 (Ar-C2/6), 131.79 (Ar-
C4′), 143.47 (Ar-C1), 144.88 (Ar-C1′), 164.77 (C-NH2(dNH2+)),
165.21 (C-NH2(dNH2+)), 167.50 (NH-CO-CH2), 170.71 (CRH-
COOCH2CH3). Anal. (C21H26N6O5S(CF3CO2H)2) H, N; C: calcd,
42.73; found, 42.08.
3
(t, 3H, OCH2CH3, J ) 7.15 Hz), 2.84 (s, 3H, -SCH3), 2.85 (s,
3H, -SCH3), 3.02 (dd, 1H, Ar-CH2-CRH, 2J ) 13.80 Hz, 3J )
8.81 Hz), 3.13 (dd, 1H, Ar-CH2-CRH, 2J ) 13.81 Hz, 3J )
5.89 Hz), 3.54 (d, 2H, CO-CH2-NH, 3J ) 5.89 Hz), 4.05 (q,
2H, OCH2CH3, 3J ) 7.05 Hz), 4.48 (dvt, 1H, CRH, 3J ) 8.23
Hz, 3J ) 6.24 Hz, 3J ) 5.86 Hz), 7.49 (d, 2H, Ar-H3/5, 3J ) 8.25
Hz, BB′), 7.80 (d, 2H, Ar-H3′/5′, 3J ) 8.22 Hz, BB′), 7.97 (d, 2H,
Peak 2 corresponds to NR-(4′-cyanobenzenesulfonyl)glycyl-
D,L-4-amidinophenylalanine ethyl ester mono(trifluoroacetate)
(9a ): yield 58 mg (10%) of white powder; HPLC (conditions
as for 8a ): tR ) 12.32 min; FAB-MS m/z: 458.2 [M + H+]; Mr
) 457.14 calcd for C21H23N5O5S; IR (KBr) ν 3333 (m, NH2);
3099 (m, dCH); 2973 (sh), 2940 (sh, CH2/3); 2235 (m, CtN);
1736 (sh, CdO); 1671 (s, N-CdO); 1617 (w, δNH2); 1539 (m),
1492 (m, CdC); 1442 (m, δCH2); 1378 (w, δCH3); 1340 (m,
ArSO2N<); 1208 (s, C-O); 1162 (s, ArSO2N<); 1H NMR δ 1.13
Ar-H2/6
,
3J ) 8.93 Hz, AA′), 8.01 (d, 2H, Ar-H2′/6′
,
3J ) 8.89
3
Hz, AA′), 8.28 (t, 1H, CH2-NH-SO2, J ) 6.10 Hz), 8.44 (d,
1H, CRH-NH-CO, 3J ) 7.80 Hz); 13C NMR δ 13.88 (OCH2CH3),
15.68 (-SCH3), 36.48 (Ar-CH2-CRH), 44.66 (CO-CH2-NH),
52.92 (CRH), 60.75 (OCH2CH3), 126.42 (Ar-C1′), 127.22 (Ar-
3
(t, 3H, OCH2CH3, J ) 7.06 Hz), 2.96 (dd, 1H, Ar-CH2-CRH,
3
2
C
2′/6′), 127.96 (Ar-C3/5), 128.79 (Ar-C3′/5′), 129.23 (Ar-C4), 130.22
2J ) 13.81 Hz, J ) 8.90 Hz), 3.10 (dd, 1H, Ar-CH2-CRH, J

Benzamidine-Type Inhibitors of Factor Xa
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4247
3
2
) 13.85 Hz, J ) 5.79 Hz), 3.53 (dd, 2H, CO-CH2-NH, J )
16.73 Hz, 3J ) 8.82 Hz), 4.06 (q, 2H, OCH2CH3, 3J ) 7.09 Hz),
as for 8a ): tR ) 12.32 min; FAB-MS m/z: 458.4 [M + H+]; Mr
) 457.14 calcd for C21H23N5O5S; IR (KBr) ν 3488 (sh), 3335
(w, NH2); 3087 (w, dCH); 2983 (sh), 2936 (sh), 2867 (sh, CH2/3);
2236 (w, CtN); 1741 (sh, CdO); 1673 (s, N-CdO); 1529 (m),
1481 (w, CdC); 1432 (w, δCH2); 1383 (w, δCH3); 1338 (m,
ArSO2N<); 1205 (s, C-O); 1164 (s, ArSO2N<); 1H NMR δ 1.11
4.44 (dvt, 1H, CRH, 3J ) 8.57 Hz, J ) 5.80 Hz, J ) 5.32 Hz),
3
3
3
7.44 (d, 2H, Ar-H3/5, J ) 8.32 Hz, BB′), 7.74 (d, 2H, Ar-H2/6
,
3J ) 8.35 Hz, AA′), 7.91 (d, 2H, Ar-H3′/5′, J ) 8.44 Hz, BB′),
3
8.03 (d, 2H, Ar-H2′/6′
,
3J ) 8.63 Hz, AA′), 8.42 (d, 1H, CRH-
3
NH-CO, J ) 7.85 Hz), 9.05 (br s, 4H, C-NH2(dNH2+) and
CH2-NH-SO2); 13C NMR δ 13.87 (OCH2CH3), 36.40 (Ar-
CH2-CRH), 44.58 (CO-CH2-NH), 53.01 (CRH), 60.69 (OCH2-
CH3), 116.36 (Ar-C4′), 117.57 (CtN), 126.21 (Ar-C4), 127.23 (Ar-
C2′/6′), 127.93 (Ar-C3/5), 128.95 (Ar-C1′), 129.64 (Ar-C2/6), 133.09
(Ar-C3′/5′), 143.47 (Ar-C1), 165.10 (C-NH2(dNH2+)), 167.87
3
(t, 3H, OCH2CH3, J ) 7.09 Hz), 2.95 (dd, 1H, Ar-CH2-CRH,
3
2
2J ) 13.79 Hz, J ) 8.68 Hz), 3.07 (dd, 1H, Ar-CH2-CRH, J
3
3
) 13.81 Hz, J ) 5.76 Hz), 3.54 (m, 2H, CO-CH2-NH, J )
3
6.02 Hz), 4.06 (q, 2H, OCH2CH3, J ) 7.10 Hz), 4.47 (m, 1H,
3
3
3
CRH, J ) 8.36 Hz, J ) 8.00 Hz, J ) 5.84 Hz), 7.52 (t, 1H,
3
Ar-H5, J ) 7.42 Hz), 7.55 (m, 1H, Ar-H4), 7.68 (m, 2H, Ar-H2
and Ar-H6), 7.90 (m, 2H, Ar-H3′/5′, 3J ) 8.41 Hz, BB′), 8.02 (m,
(NH-CO-CH2),
(C21H23N5O5S(CF3CO2H)1) H; C: calcd, 48.32; found, 46.62.
N: calcd, 12.26; found, 11.67.
170.90
(CRH-COOCH2CH3).
Anal.
3
2H, Ar-H2′/6′, J ) 8.48 Hz, AA′), 8.29 (t, 1H, CH2-NH-SO2,
3
3J ) 6.10 Hz), 8.39 (d, 1H, CRH-NH-CO, J ) 7.82 Hz), 9.12
(s, 2H, C-NH2(dNH2+)), 9.25 (s, 2H, C-NH2(dNH2+)); 13C
NMR δ 13.85 (OCH2CH3), 36.34 (Ar-CH2-CRH), 44.55 (CO-
CH2-NH), 53.16 (CRH), 60.66 (OCH2CH3), 114.69 (Ar-C4′),
117.66 (CtN), 126.31 (Ar-C4), 127.22 (Ar-C2′/6′), 128.02 (Ar-
C2), 128.68 (Ar-C5), 128.83 (Ar-C3), 133.08 (Ar-C3′/5′), 134.52
(Ar-C6), 137.97 (Ar-C1), 144.62 (Ar-C1′), 165.41 (C-NH2(d
NH2+)), 167.39 (NH-CO-CH2), 170.74 (CRH-COOCH2CH3).
Anal. (C21H23N5O5S(CF3CO2H)1) H; C: calcd, 48.32; found,
45.87. N: calcd, 12.26; found, 11.56.
Peak 3 corresponds to Nr-(4′-thioamidobenzenesulfonyl)-
glycyl-D,L-4-amidinophenylalanine ethyl ester mono(trifluoro-
acetate) (10a ): yield 40 mg (6%) of white powder; HPLC
(conditions as for 8a ): tR ) 12.00 min; FAB-MS m/z: 492.2
[M + H+]; Mr ) 491.13 calcd for C21H25N5O6S2; IR (KBr) ν 3329
(m, NH2); 3119 (sh, dCH); 2936 (sh), 2855 (sh, CH2/3); 1734
(w, CdO); 1671 (s, N-CdO); 1539 (m), 1492 (m, CdC); 1418
(m, δCH2); 1377 (w, δCH3); 1333 (m, ArSO2N<); 1207 (s), 1187
(s, C-O); 1136 (s, ArSO2N<); 1H NMR δ 1.13 (t, 3H, OCH2CH3,
2
3
3J ) 7.14 Hz), 2.99 (dd, 1H, Ar-CH2-CRH, J ) 13.80 Hz, J
Peak 3 corresponds to Nr-(4′-thioamidobenzenesulfonyl)-
glycyl-D,L-3-amidinophenylalanine ethyl ester mono(trifluoro-
acetate) (10b): 61 mg (10%) of white powder; HPLC (conditions
as for 8a ): tR ) 12.02 min; FAB-MS m/z: 492.4 [M + H+]; Mr
) 491.13 calcd for C21H25N5O6S2; IR (KBr) ν 3329 (w), 3202
(w, NH2); 3098 (sh, dCH); 2983 (sh), 2936 (sh, CH2/3); 1736
(sh, CdO); 1675 (s, N-CdO); 1531 (m), 1480 (w, CdC); 1425
(m, δCH2); 1378 (w, δCH3); 1334 (m, ArSO2N<); 1204 (s, C-O);
1166 (s, ArSO2N<); 1H NMR δ 1.12 (t, 3H, OCH2CH3, 3J )
7.03 Hz), 2.98 (dd, 1H, Ar-CH2-CRH, 2J ) 13.81 Hz, 3J )
8.64 Hz), 3.09 (dd, 1H, Ar-CH2-CRH, 2J ) 13.83 Hz, 3J )
2
3
) 8.79 Hz), 3.12 (dd, 1H, Ar-CH2-CRH, J ) 13.83 Hz, J )
5.77 Hz), 3.47 (t, 2H, CO-CH2-NH, 3J ) 5.79 Hz), 4.06 (q,
2H, OCH2CH3, 3J ) 7.15 Hz), 4.49 (m, 1H, CRH, 3J ) 8.02 Hz,
3J ) 5.66 Hz), 7.45 (d, 2H, Ar-H3/5, 3J ) 8.33 Hz, BB′), 7.74 (d,
2H, Ar-H2/6, 3J ) 8.28 Hz, AA′), 7.79 (d, 2H, Ar-H3′/5′, 3J ) 8.51
Hz, BB′), 7.97 (d, 2H, Ar-H2′/6′, 3J ) 8.44 Hz, AA′), 8.08 (t, 1H,
3
3
CH2-NH-SO2, J ) 6.10 Hz), 8.40 (d, 1H, CRH-NH-CO, J
) 7.85 Hz), 9.03 (s, 1H, C-NH2(dNH2+)), 9.24 (s, 1H, C-NH2-
(dNH2+)), 9.67 (s, 1H, CdS(-NH2)), 10.07 (s, 1H, CdS(-NH2));
13C NMR δ 13.88 (OCH2CH3), 36.42 (Ar-CH2-CRH), 44.71
(CO-CH2-NH), 53.02 (CRH), 60.73 (OCH2CH3), 126.12 (Ar-
C4 and Ar-C2′/6′), 127.70 (Ar-C3′/5′), 127.93 (Ar-C3/5), 129.66 (Ar-
C2/6), 142.06 (Ar-C1), 142.82 (Ar-C4′), 143.50 (Ar-C1′), 165.21
(C-NH2(dNH2+)), 167.49 (NH-CO-CH2), 170.70 (CRH-
COOCH2CH3), 198.81 (CdS(-NH2)). Anal. (C21H25N5O5S2(CF3-
CO2H)1) H; C: calcd, 45.61; found, 44.73. N: calcd, 11.57;
found: 11.08.
3
5.72 Hz), 3.47 (m, 2H, CO-CH2-NH, J ) 6.16 Hz), 4.06 (q,
2H, OCH2CH3, 3J ) 7.08 Hz), 4.52 (m, 1H, CRH, 3J ) 8.21 Hz,
3
3
3J ) 8.08 Hz, J ) 5.91 Hz), 7.52 (t, 1H, Ar-H5, J ) 7.45 Hz),
3
4
7.55 (dvt, 1H, Ar-H4, J ) 7.53 Hz, J ) 1.25 Hz), 7.67 (dvt,
1H, Ar-H6, 3J ) 7.64 Hz, 4J ) 1.38 Hz), 7.69 (br s, 1H, Ar-H2),
3
7.78 (d, 2H, Ar-H3′/5′, J ) 8.46 Hz, BB′), 7.97 (d, 1H, Ar-H2′/6′
,
3J ) 8.45 Hz, AA′), 8.07 (t, 1H, CH2-NH-SO2, 3J ) 6.19 Hz),
8.36 (d, 1H, CRH-NH-CO, 3J ) 7.84 Hz), 9.07 (s, 2H, C-NH2-
(dNH2+)), 9.24 (s, 2H, C-NH2(dNH2+)), 9.66 (s, 1H, CdS(-
NH2)), 10.07 (s, 1H, CdS(-NH2)); 13C NMR δ 13.86 (OCH2CH3),
36.36 (Ar-CH2-CRH), 44.69 (CO-CH2-NH), 53.16 (CRH),
60.69 (OCH2CH3), 126.11 (Ar-C4), 126.31 (Ar-C2′/6′), 127.69 (Ar-
C3′/5′), 128.03 (Ar-C2), 128.71 (Ar-C5), 128.82 (Ar-C3), 134.55
(Ar-C6), 137.98 (Ar-C1), 142.08 (Ar-C4′), 142.81 (Ar-C1′), 165.42
(C-NH2(dNH2+)), 167.53 (NH-CO-CH2), 170.77 (CRH-
COOCH2CH3), 198.81 (CdS(-NH2)). Anal. (C21H25N5O5S2(CF3-
CO2H)1) H; C: calcd, 45.61; found: 44.02. N: calcd, 11.57;
found, 10.93.
N r-(4′-Am id in ob e n ze n e su lfon yl)glycyl-D,L-3-a m id i-
n op h en yla la n in e Eth yl Ester Bis(tr iflu or oa ceta te) (8b).
After aminolysis of 7b (0.80 g, 1.01 mmol) reversed-phase
chromatography on Lichroprep RP-18 as described for 8a led
again to three peaks.
Peak 1 corresponds to the title compound: 225 mg (32%) of
white powder; HPLC (conditions as for 8a ): tR ) 7.82 min;
FAB-MS m/z: 475.5 [M + H+]; Mr ) 474.17 calcd for
C
21H26N6O5S; IR (KBr) ν 3320 (m), 3100 (m, NH2); 3050 (sh,
dCH); 2902 (sh), 2876 (sh, CH2/3); 1730 (sh, CdO); 1670 (s,
N-CdO); 1612 (w, (NH); 1527 (m), 1483 (w, CdC); 1444 (w,
δCH2); 1378 (w, δCH3); 1340 (m, ArSO2N<); 1206 (s, C-O);
1143 (s, ArSO2N<); 1H NMR δ 1.12 (t, 3H, OCH2CH3, 3J )
7.09 Hz), 3.00 (dd, 1H, Ar-CH2-CRH, 2J ) 13.85 Hz, 3J )
8.66 Hz), 3.10 (dd, 1H, Ar-CH2-CRH, 2J ) 13.79 Hz, 3J )
5.79 Hz), 3.52 (d, 2H, CO-CH2-NH, 3J ) 5.87 Hz), 4.06 (q,
2H, OCH2CH3, 3J ) 7.07 Hz), 4.52 (m, 1H, CRH, 3J ) 8.18 Hz,
Nr-(4′-Cya n ob en zen esu lfon yl)glycyl-D,L-4-cya n op h e-
n yla la n in e (11a ). D,L-4-Cyanophenylalanine19 (1.2 g, 6.31
mmol) was reacted with the active ester 3 (2.13 g, 6.31 mmol)
in DMF with TEA (0.875 mL, 6.31 mmol) as auxiliary base.
After 4 days the reaction mixture was evaporated, and the
residue was distributed between AcOEt and 5% KHSO4. The
organic layer was dried over Na2SO4 and evaporated, and the
residue was recrystallized from 2-propanol: yield 1.95 g (75%)
of white powder; mp 202-203 °C; FAB-MS m/z: 413.1 [M +
H+]; Mr ) 412.08 calcd for C19H16N4O5S; IR (KBr) ν 3386 (m),
3310 (m, NH); 3095 (w), 3046 (w, dCH); 2936 (w, CH2); 2518
(w, OH); 2236 (m, CtN); 1729 (s, CdO); 1619 (s, (NH); 1539
3
3
3J ) 7.61 Hz, J ) 5.86 Hz), 7.52 (t, 1H, Ar-H5, J ) 7.49 Hz),
3
7.57 (dvt, 1H, Ar-H4, J ) 7.67 Hz), 7.68-7.70 (m, 2H, Ar-H2
and Ar-H6), 7.97 (s, 4H, Ar-H2′/3′/5′/6′), 8.28 (t, 1H, CH2-NH-
SO2, 3J ) 6.05 Hz), 8.43 (d, 1H, CRH-NH-CO, 3J ) 7.76 Hz),
9.25 (s, 3H, C-NH2(dNH2+)), 9.47 (s, 3H, C-NH2(dNH2+));
13C NMR δ 13.85 (OCH2CH3), 36.33 (Ar-CH2-CRH), 44.66
(CO-CH2-NH), 53.23 (CRH), 60.68 (OCH2CH3), 126.30 (Ar-
C4), 126.75 (Ar-C2′/6′), 128.06 (Ar-C2), 128.70 (Ar-C5), 128.84
(Ar-C3), 129.00 (Ar-C3′/5′), 131.77 (Ar-C4′), 134.52 (Ar-C6), 138.00
(Ar-C1), 144.92 (Ar-C1′), 164.84 (C-NH2(dNH2+)), 165.48 (C-
NH2(dNH2+)), 167.53 (NH-CO-CH2), 170.80 (CRH-COOCH2-
CH3). Anal. (C21H26N6O5S1(CF3CO2H)2) C, H, N.
1
(s, CdC); 1407 (m, δCH2); 1343 (m), 1166 (s, ArSO2N<); H
2
3
NMR δ 2.92 (dd, 1H, Ar-CH2-CRH, J ) 13.78 Hz, J ) 8.96
Hz), 3.11 (dd, 1H, Ar-CH2-CRH, 2J ) 13.82 Hz, 3J ) 4.98
Hz), 3.51 (dd, 2H, CO-CH2-NH, 2J ) 16.70 Hz, 3J ) 5.45
3
3
Hz), 4.40 (dvt, 1H, CRH, J ) 8.51 Hz, J ) 5.01 Hz), 7.38 (d,
2H, Ar-H3/5, 3J ) 8.15 Hz, BB′), 7.73 (d, 2H, Ar-H2/6, 3J ) 8.16
Hz, AA′), 7.90 (d, 2H, Ar-H3′/5′, 3J ) 8.39 Hz, BB′), 8.02 (d, 2H,
Peak 2 corresponds to NR-(4′-cyanobenzenesulfonyl)glycyl-
D,L-3-amidinophenylalanine ethyl ester mono(trifluoroacetate)
(9b): yield 66 mg (11%) of white powder; HPLC (conditions
3
3
Ar-H2′/6′, J ) 8.40 Hz, AA′), 8.24 (d, 1H, CRH-NH-CO, J )
3
8.11 Hz), 8.26 (t, 1H, CH2-NH-SO2, J ) 5.80 Hz), 12.91(s,

4248 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Gabriel et al.
1H, CRH-COOH); 13C NMR δ 36.66 (Ar-CH2-CRH), 44.60
(CO-CH2-NH), 52.71 (CRH), 109.32 (Ar-C4), 114.68 (Ar-C4′),
117.66 (SO2-Ar-CtN), 118.77 (CtN), 127.21 (Ar-C2′/6′), 130.17
(Ar-C2/6), 131.93 (Ar-C3/5), 133.05 (Ar-C3′/5′), 143.32 (Ar-C1),
144.64 (Ar-C1′), 167.12 (NH-CO-CH2), 171.99 (CRH-COOH).
Nr-(4′-Cya n ob en zen esu lfon yl)glycyl-D,L-3-cya n op h e-
n yla la n in e (11b). The title compound was prepared from D,L-
3-cyanophenylalanine19 as described for 11a : yield 69% of
white powder; mp 181-183 °C; FAB-MS m/z: 413.1 [M + H+];
Mr ) 412.08 calcd for C19H16N4O5S; IR (KBr) ν 3373 (s), 3198
(s, NH); 3094 (w, dCH); 2971 (sh), 2927 (sh, CH2); 2234 (m,
CtN); 1718 (s), 1679 (s, CdO); 1551 (m), 1487 (w, CdC); 1450
(w), 1417 (m, δCH2); 1363 (m), 1169 (s, ArSO2N<); 1H NMR δ
2.90 (dd, 1H, Ar-CH2-CRH, 2J ) 13.96 Hz, 3J ) 8.82 Hz),
3.09 (dd, 1H, Ar-CH2-CRH, 2J ) 13.91 Hz, 3J ) 5.06 Hz),
3.52 (dd, 2H, CO-CH2-NH, 2J ) 16.74 Hz, 3J ) 6.11 Hz),
131.80 (Ar-C6), 136.93 (Ar-C1), 139.40 (Ar-C3), 142.09 (Ar-C4′),
142.87 (Ar-C1′), 167.26 (NH-CO-CH2), 172.25 (CRH-COOH),
198.91 (CdS(-NH2)), 200.09 (CdS(-NH2)).
Nr-(4′-Meth ylth ioim id o-ben zen esu lfon yl)glycyl-D,L-4-
m eth ylth ioim id o-p h en yla la n in e Bis(h yd r oiod id e) (13a ).
The compound was prepared from 12a as described for 7a :
yield 92% of yellow foam; HPLC (conditions as for 12a ): tR
)
21.07 min; FAB-MS m/z: 509.2 [M + H+]; Mr ) 508.09 calcd
for C21H24N4O5S3; 1H NMR δ 2.81 (s, 3H, -SCH3), 2.82 (s, 3H,
-SCH3), 2.98 (dd, 1H, Ar-CH2-CRH, 2J ) 13.81 Hz, 3J ) 9.10
Hz), 3.16 (dd, 1H, Ar-CH2-CRH, 2J ) 13.90 Hz, 3J ) 5.10
Hz), 3.51 (dd, 2H, CO-CH2-NH, 3J ) 6.11 Hz), 4.46 (dvt, 1H,
3
3
3
CRH, J ) 8.57 Hz, J ) 8.27 Hz, J ) 5.35 Hz), 7.47 (d, 2H,
3
3
Ar-H3/5, J ) 8.38 Hz, BB′), 7.79 (d, 2H, Ar-H2/6, J ) 8.33 Hz,
AA′), 7.92 (d, 2H, Ar-H3′/5′
Ar-H2′/6′, J ) 8.64 Hz, AA′), 8.20 (t, 1H, CH2-NH-SO2, J )
,
3J ) 8.22 Hz, BB′), 7.97 (d, 2H,
3
3
3
3
3
6.16 Hz), 8.29 (d, 1H, CRH-NH-CO, J ) 8.04 Hz).
4.41 (dvt, 1H, CRH, J ) 8.46 Hz, J ) 4.84 Hz), 7.48 (t, 1H,
3
3
4
Nr-(4′-Meth ylth ioim id o-ben zen esu lfon yl)glycyl-D,L-3-
m eth ylth ioim id o-p h en yla la n in e Bis(h yd r oiod id e) (13b).
Prepared as described for 13a : yield 94% of yellow foam;
HPLC (conditions as for 12a ): tR ) 21.22 min; FAB-MS m/z:
509.2 [M + H+]; Mr ) 508.09 calcd for C21H24N4O5S3; 1H NMR
δ 2.75 (s, 3H, -SCH3), 2.83 (s, 3H, -SCH3), 2.97 (dd, 1H, Ar-
CH2-CRH, 2J ) 13.78 Hz, 3J ) 8.98 Hz), 3.15 (dd, 1H, Ar-
Ar-H5, J ) 7.58 Hz), 7.53 (dvt, 1H, Ar-H4, J ) 7.85 Hz, J )
3
1.35 Hz), 7.63 (s, 1H, Ar-H2), 7.68 (dvt, 1H, Ar-H6, J ) 7.62
Hz, 4J ) 1.52 Hz), 7.90 (d, 2H, Ar-H3′/5′ 3J ) 8.39 Hz, BB′),
,
8.02 (d, 2H, Ar-H2′/6′,
3J ) 8.35 Hz, AA′), 8.23 (d, 1H, CRH-
NH-CO, 3J ) 8.21 Hz), 8.26 (t, 1H, CH2-NH-SO2, 3J ) 6.11
Hz), 12.86 (s, 1H, CRH-COOH); 13C NMR δ 36.07 (Ar-CH2-
CRH), 44.59 (CO-CH2-NH), 52.78 (CRH), 111.09 (Ar-C4),
114.69 (Ar-C4′), 117.67 (SO2-Ar-CtN), 118.73 (CtN), 127.22
(Ar-C2′/6′), 129.28 (Ar-C5), 130.30 (Ar-C4), 132.65 (Ar-C2), 133.05
(Ar-C3′/5′), 134.13 (Ar-C6), 138.91 (Ar-C1), 144.65 (Ar-C1′), 167.16
(NH-CO-CH2), 172.02 (CRH-COOH).
2
3
CH2-CRH, J ) 13.80 Hz, J ) 4.91 Hz), 3.52 (dd, 2H, CO-
3
3
3
CH2-NH, J ) 6.08 Hz), 4.45 (dvt, 1H, CRH, J ) 8.50 Hz, J
) 8.42 Hz, 3J ) 5.07 Hz), 7.56 (t, 1H, Ar-H5, 3J ) 7.66 Hz),
3
7.63 (dvt, 1H, Ar-H4, J ) 7.68 Hz), 7.71 (s, 1H, Ar-H2), 7.72
3
3
Nr-(4′-Th ioa m id ob en zen esu lfon yl)glycyl-D,L-4-t h ioa -
m id op h en yla la n in e (12a ). Compound 11a was reacted with
hydrogen sulfide and worked up as described for 6a : yield 2.1
g (98%) of yellow foam; HPLC (isocratic elution with A for 5
min; linear gradient from A to B in 40 min): tR ) 23.52 min;
FAB-MS m/z: 481.3 [M + H+]; Mr ) 480.06 calcd for
C19H20N4O5S3; IR (KBr) ν 3315 (s), 3188 (s, NH2); 3064 (sh,
dCH); 2938 (sh, CH2/3); 1726 (w, CdO); 1659 (w, N-CdO);
1630 (s, δNH2); 1547 (w), 1531 (m, CdC); 1423 (m, δCH2); 1328
(dvt, 1H, Ar-H6, J ) 7.46 Hz), 7.93 (d, 2H, Ar-H3′/5′, J ) 8.49
Hz, BB′), 7.98 (d, 2H, Ar-H2′/6′, 3J ) 8.68 Hz, AA′), 8.23 (t, 1H,
3
3
CH2-NH-SO2, J ) 6.12 Hz), 8.27 (d, 1H, CRH-NH-CO, J
) 8.09 Hz).
N r-(4′-Am id in ob e n ze n e su lfon yl)glycyl-D,L-4-a m id i-
n op h en yla la n in e Bis(tr iflu or oa ceta te) (14a ). Aminolysis
of 13a and subsequent reversed-phase chromatography on
Lichroprep RP-18 were carried out as reported for 8a . Frac-
tions of the main peak were collected, evaporated, and dried
over KOH pellets: yield 35 mg (40%) of white powder; HPLC
(conditions as for 8a ): tR ) 5.40 min; FAB-MS m/z: 447.1 [M
+ H+]; Mr ) 446.14 calcd for C19H22N6O5S; 1H NMR δ 2.98
(dd, 1H, Ar-CH2-CRH, 2J ) 13.95 Hz, 3J ) 8.92 Hz), 3.16
(dd, 1H, Ar-CH2-CRH, 2J ) 13.88 Hz, 3J ) 5.12 Hz), 3.51
1
(m), 1163 (s, ArSO2N<); H NMR δ 2.91 (dd, 1H, Ar-CH2-
CRH, 2J ) 13.80 Hz, 3J ) 8.50 Hz), 3.06 (dd, 1H, Ar-CH2-
2
3
CRH, J ) 13.83 Hz, J ) 5.20 Hz), 3.57 (dd, 2H, CO-CH2-
NH, 2J ) 16.56 Hz, 3J ) 6.23 Hz), 4.43 (dvt, 1H, CRH, 3J )
8.10 Hz, 3J ) 5.43 Hz), 7.22 (d, 2H, Ar-H3/5, 3J ) 8.30 Hz, BB′),
3
3
3
7.79 (d, 2H, Ar-H3′/5′, J ) 8.46 Hz, BB′), 7.82 (d, 2H, Ar-H2/6
,
(dd, 2H, CO-CH2-NH, J ) 5.77 Hz), 4.45 (dvt, 1H, CRH, J
3J ) 8.30 Hz, AA′), 7.96 (d, 2H, Ar-H2′/6′, J ) 8.45 Hz, AA′),
8.06 (t, 1H, CH2-NH-SO2, J ) 6.16 Hz), 8.17 (d, 1H, CRH-
3
3
3
) 8.34 Hz, J ) 8.20 Hz, J ) 5.32 Hz), 7.45 (d, 2H, Ar-H3/5
,
3
3J ) 8.25 Hz, BB′), 7.74 (d, 2H, Ar-H2/6
,
3J ) 8.42 Hz, AA′),
NH-CO, 3J ) 7.94 Hz), 9.40 (s, 1H, CdS(-NH2)), 9.65 (s, 1H,
CdS(-NH2)), 9.75 (s, 1H, CdS(-NH2)), 10.06 (s, 1H, CdS(-
NH2)), 12.82 (s, 1H, CRH-COOH); 13C NMR δ 36.39 (Ar-CH2-
CRH), 44.79 (CO-CH2-NH), 52.98 (CRH), 126.14 (Ar-C2′/6′),
127.21 (Ar-C2/6), 127.69 (Ar-C3′/5′), 128.55 (Ar-C3/5), 137.45 (Ar-
C4), 140.70 (Ar-C4′), 142.09 (Ar-C1), 142.89 (Ar-C1′), 167.29
(NH-CO-CH2), 172.21 (CRH-COOH), 198.90 (CdS(-NH2)),
199.59 (CdS(-NH2)).
3
7.95 (d, 2H, Ar-H3′/5′, J ) 8.84 Hz, BB′), 7.98 (d, 2H, Ar-H2′/6′
,
3J ) 8.79 Hz, AA′), 8.26 (t, 1H, CH2-NH-SO2, 3J ) 5.98 Hz),
8.30 (d, 1H, CRH-NH-CO, 3J ) 8.13 Hz), 9.08 (s, 2H, C-NH2-
(dNH2+)), 9.24 (s, 2H, C-NH2(dNH2+)), 9.36 (s, 2H, C-NH2-
(dNH2+)), 9.45 (s, 2H, C-NH2(dNH2+)), 12.91 (br s, 1H, CRH-
COOH); 13C NMR δ 36.56 (Ar-CH2-CRH), 44.84 (CO-CH2-
NH), 53.06 (CRH), 126.19 (Ar-C4), 126.88 (Ar-C2′/6′), 127.96 (Ar-
C3/5), 129.11 (Ar-C3′/5′), 129.74 (Ar-C2/6), 131.88 (Ar-C4′), 144.01
(Ar-C1), 144.91 (Ar-C1′), 164.91 (C-NH2(dNH2+)), 165.33 (C-
NH2(dNH2+)), 167.42 (NH-CO-CH2), 172.23 (CRH-COOH).
Anal. (C19H22N6O5S(CF3CO2H)2) H, N; C: calcd, 40.94; found,
40.04.
Nr-(4′-Th ioa m id ob en zen esu lfon yl)glycyl-D,L-3-t h ioa -
m id op h en yla la n in e (12b). The title compound was pre-
pared from 11b as described for 6a : yield 98% of yellow foam;
HPLC (conditions as for 12a ): tR ) 23.82 min; FAB-MS m/z:
481.3 [M + H+]; Mr ) 480.06 calcd for C19H20N4O5S3; IR (KBr)
ν 3314 (s), 3192 (s, NH2); 3051 (sh, dCH); 2938 (sh, CH2/3);
1725 (m, CdO); 1665 (sh, N-CdO); 1630 (s, δNH2); 1535 (m),
1486 (w, CdC); 1414 (m, δCH2); 1329 (s), 1163 (s, ArSO2N<);
N r-(4′-Am id in ob e n ze n e su lfon yl)glycyl-D,L-3-a m id i-
n op h en yla la n in e Bis(tr iflu or oa ceta te) (14b). The title
compound was prepared and purified as described for 14a :
yield 25% of white powder; HPLC (conditions as for 8a ): tR
)
2
3
1H NMR δ 2.90 (dd, 1H, Ar-CH2-CRH, J ) 13.88 Hz, J )
8.51 Hz), 3.06 (dd, 1H, Ar-CH2-CRH, 2J ) 13.83 Hz, 3J )
5.21 Hz), 3.48 (dd, 2H, CO-CH2-NH, 2J ) 16.45 Hz, 3J )
6.17 Hz), 4.43 (dvt, 1H, CRH, 3J ) 8.10 Hz, 3J ) 5.21 Hz), 7.29
6.80 min; FAB-MS m/z: 447.2 [M + H+]; Mr ) 446.14 calcd
for C19H22N6O5S; 1H NMR δ 2.98 (dd, 1H, Ar-CH2-CRH, 2J )
13.68 Hz, 3J ) 8.33 Hz), 3.13 (dd, 1H, Ar-CH2-CRH, 2J )
13.73 Hz, 3J ) 5.13 Hz), 3.51 (m, 2H, CO-CH2-NH), 4.44 (dvt,
3
3
3
3
(s, 1H, Ar-H5, J ) 7.65 Hz), 7.30-7.32 (m, 1H, Ar-H4), 7.70-
1H, CRH, J ) 7.57 Hz, J ) 5.13 Hz), 7.51 (t, 1H, Ar-H5, J )
7.48 Hz), 7.53 (dvt, 1H, Ar-H4, 3J ) 7.37 Hz), 7.66 (dvt, 1H,
7.73 (m, 1H, Ar-H6), 7.74 (s, 1H, Ar-H2), 7.79 (d, 2H, Ar-H3′/5′
,
3
3
3J ) 8.37 Hz, BB′), 7.96 (d, 2H, Ar-H2′/6′, J ) 8.42 Hz, AA′),
Ar-H6, J ) 7.78 Hz), 7.68 (br s, 1H, Ar-H2), 7.96 (s, 4H, Ar-
3
8.02 (t, 1H, CH2-NH-SO2, J ) 6.17 Hz), 8.18 (d, 1H, CRH-
H2′/3′/5′/6′), 8.17 (br s, 1H, CH2-NH-SO2), 8.27 (br s, 1H, CRH-
NH-CO), 9.21 (s, 4H, C-NH2(dNH2+)), 9.43 (s, 4H, C-NH2(d
NH2+)), 12.86 (br s, 1H, CRH-COOH); 13C NMR δ 36.25 (Ar-
CH2-CRH), 44.70 (CO-CH2-NH), 51.93 (OCH3), 53.16 (CRH),
126.32 (Ar-C4), 127.24 (Ar-C2′/6′), 127.74 (Ar-C2), 128.69 (Ar-
C5), 128.84 (Ar-C3), 129.10 (Ar-C3′/5′), 131.96 (Ar-C4′), 134.46
NH-CO, 3J ) 7.96 Hz), 9.39 (s, 1H, CdS(-NH2)), 9.64 (s, 1H,
CdS(-NH2)), 9.79 (s, 1H, CdS(-NH2)), 10.06 (s, 1H, CdS(-
NH2)), 12.80 (s, 1H, CRH-COOH); 13C NMR δ 36.58 (Ar-CH2-
CRH), 44.78 (CO-CH2-NH), 53.21 (CRH), 125.44 (Ar-C4),
126.14 (Ar-C3′/5′), 127.67 (Ar-C2′/6′ and Ar-C2), 128.09 (Ar-C5),

Benzamidine-Type Inhibitors of Factor Xa
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4249
(Ar-C6), 138.12 (Ar-C1), 142.77 (Ar-C1′), 164.69 (C-NH2(d
NH2+)), 165.61 (C-NH2(dNH2+)), 167.85 (NH-CO-CH2),
171.40 (CRH-COOH). Anal. (C19H22N6O5S(CF3CO2H)2) H, N;
C: calcd, 40.94; found: 40.09.
pleteness 94.0%) for trypsin/8b, and 18 573 (81.3% complete-
ness) for trypsin/10b were collected. The corresponding R
merges were 3.6% (11.5% for the 2.00-1.90 Å resolution shell),
4.2% (17.2%), and 5.5% (24.3%), respectively.
Conventional crystallographic rigid body, positional, and
temperature factor refinements were carried out with XPLOR45
using the â-trypsin structure (PDB, Brookhaven; accession
code 1MTS) as starting model. Model building was carried out
with TURBO-FRODO.46 The structure was refined to a R
factor of 18.7% (free R factor 23.6%) with rms deviations from
target values of 0.009 Å for bonds and 1.952° for angles. The
rms deviation of B factors of bonded atoms was 2.23 Å2.
Modeling experiments on fXa were performed using the
coordinates of des-Gla-fXa11 (PDB entry 1FAX) and Insight II
(version 95.0; MSI Technologies, San Diego, CA).
Deter m in a tion of In h ibition Con sta n ts. The measure-
ments were carried out on a microplate reader (MR 5000,
Dynatech, Denkendorf, Germany) at 25 °C. The test medium
consisted of 200 µL Tris buffer (0.05 M; 0.154 M NaCl, 5%
ethanol, pH 8.0), 25 µL aqueous substrate solution, and 50 µL
enzyme solution. Two concentrations of the substrate and five
concentrations of the inhibitor were used; 3 min after the
addition of the enzyme 25 µL acetic acid (50%) was added to
quench the reaction and the optical density was measured at
405 nm. The Ki values were calculated according to Dixon39
using a linear regression program. In case of tryptase deter-
mination of inhibition constants was not possible because of
nonlinear Dixon kinetics as described previously.40 Therefore,
inhibitory potency was evaluated by determination of the
inhibitor concentration required to reduce the enzyme activity
by 50% (IC50). The Ki and IC50 values reported are means from
at least three determinations.
En zym es a n d Su bstr a tes for Ki Deter m in a tion . The
following enzymes and the respective substrates were used at
the final concentrations indicated: bovine thrombin prepared
according to Walsmann41 (2262 U/mg, final concentration 0.45
U/mL), substrate MeSO2-D-hexahydrotyrosyl-Gly-Arg-pNA (fi-
nal concentrations 0.18 and 0.09 mM); bovine fXa (5 U/vial,
0.11 U/mL; Diagnostic Reagents Ltd., Thame, U.K.), human
fXa (4 mg/vial, 0.18 µg/mL; Kordia Lab. Supplies, Leiden, The
Netherlands), substrate MeSO2-D-Nle-Gly-Arg-pNA (0.36 and
0.18 mM); human APC (1 mg/vial, 1.13 µg/mL; Kordia Lab.
Supplies, Leiden, The Netherlands), substrate H-D-Lys(Cbz)-
Pro-Arg-pNA (0.36 and 0.18 mM); human PK (1 mg/vial, 4 µg/
mL; Kordia Lab. Supplies, Leiden, The Netherlands), substrate
Bz-Pro-Phe-Arg-pNA (0.36 and 0.18 mM); human plasmin
(0.67 CTA-U/mg, 0.06 CTA-U/mL; Behringwerke AG, Marburg,
Germany), substrate Tos-Gly-Pro-Lys-pNA (0.18 and 0.09
mM); human uPA (500 000 U/vial, final concentration 150
U/mL; Ribosepharm GmbH, Haan, Germany), substrate Bz-
âAla-Gly-Arg-pNA (0.18 and 0.09 mM); sc-tPA purified from
CHO cells42 (4.1 mg/mL, 0.0031 µg/mL), substrate MeSO2-D-
hexahydrotyrosyl-Gly-Arg-pNA (0.54, 0.27, and 0.145 mM);
bovine pancreatic trypsin (42 U/mg, 0.0038 U/mL; Serva,
Heidelberg, Germany), substrate MeSO2-D-hexahydrotyrosyl-
Gly-Arg-pNA (0.18 and 0.06 mM), human lung tryptase
prepared according to ref 43 (8 µM, 2.4 nM), substrate Tos-
Gly-Pro-Arg-pNA (0.18 mM). The substrates were supplied
by Pentapharm Ltd., Basel, Switzerland.
Ack n ow led gm en t. The authors thank Inge Buhrow
for the mass spectra; Christa Bo¨ttner for the enzyme
kinetics; Stella Fiori, Dr. Sabine Rudolph-Bo¨hner, Dr.
Michaela Schenk, and Dr. Isolde Sonnenbichler for the
NMR measurements; Regina Scharf and Petra Schwille
for technical assistance; and Gu¨nter Wu¨nsche for el-
emental analyses. This study was supported by Grants
MO 377/8-1, BO 1279/2-1, and STU 161/1-1 of the
Deutsche Forschungsgemeinschaft.
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