Dose-dependent antithrombotic activity of an orally active tissue factor/factor VIIa inhibitor without concomitant enhancement of bleeding propensity

Article abstract of DOI:10.1016/j.bmc.2006.03.042

The discovery of a highly potent and selective tissue factor/factor VIIa inhibitor is described. Upon oral administration of its double prodrug in the guinea pig, a dose-dependent antithrombotic effect is observed in an established model of arterial throm

Full text of DOI:10.1016/j.bmc.2006.03.042

Bioorganic & Medicinal Chemistry 14 (2006) 5357–5369
Dose-dependent antithrombotic activity of an orally active tissue
factor/factor VIIa inhibitor without concomitant
enhancement of bleeding propensity
*
´
Katrin Groebke Zbinden, David W. Banner, Kurt Hilpert, Jacques Himber, Thierry Lave,
Markus A. Riederer, Martin Stahl, Thomas B. Tschopp and Ulrike Obst-Sander
F. Hoffmann – La Roche Ltd, CH-4070 Basel, Switzerland
Received 14 December 2005; revised 15 March 2006; accepted 22 March 2006
Available online 18 April 2006
Abstract—The discovery of a highly potent and selective tissue factor/factor VIIa inhibitor is described. Upon oral administration of
its double prodrug in the guinea pig, a dose-dependent antithrombotic effect is observed in an established model of arterial throm-
bosis without prolonging bleeding time. The pharmacodynamic properties of this selective inhibitor are compared to the behaviour
of a mixed factor VIIa/factor Xa inhibitor.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitor Melagatran.4 Ximelagatran has been launched
in France in 2004 under the tradename Exanta, but in
the meantime has been withdrawn from the market
due to concerns about long-term liver damage and
possible risk of heart attacks.
Thromboembolic disorders are among the leading caus-
es for morbidity and mortality in the industrialized
world. Thrombosis of either the venous or the arterial
vascular system may cause pulmonary embolism, myo-
cardial infarction or ischaemic stroke.1 The development
of new anticoagulant therapies therefore represents an
important medical need. For a long period of time the
coumarins have been the only orally active anticoagu-
lants interfering with the coagulation cascade which
have been available on the market. They act indirectly,
inhibiting the c-carboxylation in the biosynthesis of
coagulation factors prothrombin, VII, IX and X. This
mechanism leads to a slow on-set of action. Additional-
ly, the coumarins suffer from substantial food and drug
interaction and high protein binding2, rendering it very
difficult to maintain a balanced plasma exposure. Care-
ful and regular monitoring of the patient is therefore
required.3 During the last decades substantial efforts
have been devoted to the search for an orally bioavail-
able replacement of the coumarins. Out of numerous
research programmes, the only compound to have
reached the market is Ximelagatran from AstraZeneca,
which is a double prodrug of the direct thrombin
It is known that inhibition of the coagulation cascade at
the final stages (factor Xa, thrombin) can lead to bleed-
ing complications.5 An alternative target would be the
inhibition of the tissue factor/factor VIIa (TF/F.VIIa)
complex which is the main trigger of thrombotic events.6
This complex is part of the extrinsic pathway of the
coagulation cascade and causes the activation of factor
X and factor IX, ultimately resulting in the generation
of thrombin and the thrombin-mediated conversion of
fibrinogen to fibrin as well as platelet activation.
Proof-of-concept experiments in animal models from
our laboratories7 and from other research groups8 dem-
onstrate that specific inhibition of the TF/F.VIIa com-
plex results in an antithrombotic effect without
enhancing bleeding propensity. These results demon-
strated that selective inhibition of the extrinsic pathway
of the coagulation might be a very promising mecha-
nism for a novel anticoagulant and therefore stimulated
many discovery programmes in the pharmaceutical
industry aiming at the identification of low molecular
weight TF/F.VIIa inhibitors.9
Keywords: Anticoagulant; Coagulation cascade; Tissue factor/factor
VIIa inhibitor; Bleeding time prolongation; Double prodrug.
*
Recently, we have described the design, synthesis and
profiling of the first highly potent, orally bioavailable
Corresponding author. Tel.: +41 61 688 83 70; fax: +41 61 688 97
48; e-mail: katrin.groebke_zbinden@roche.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.042

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K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
TF/F.VIIa inhibitors from our laboratories.10 Despite
their promising oral activity and good half-life, a plasma
exposure sufficient to achieve a sustained anticoagulant
effect was not reached with these compounds. Here we
report the design and profiling of an orally active TF/
F.VIIa inhibitor which exhibits dose-dependent anti-
thrombotic activity in a guinea pig model of recurrent
arterial thrombosis. The efficacy and safety of this selec-
tive TF/F.VIIa inhibitor is compared with the pharma-
codynamic behaviour of a competitor compound with
mixed TF/F.VIIa and factor Xa (F.Xa) inhibitory
activity.
prodrug, by an excellent bioavailability (F = 100%). De-
spite this excellent bioavailability, very high
dose would be required to cover the plasma exposure
needed for sustained anticoagulant activity in vivo be-
cause the activity of the parent compound 1 is relatively
low.
a
Extensive efforts focused on improving the functional
activity of the phenylglycine class while maintaining
their selectivity. The targeted value for PT activity lies
around 2 lM. With respect to selectivity an aPTT/PT
ratio above 4 was regarded as optimal to warrant low
bleeding propensity and thus a large safety window.
The aPTT (2x prolongation of activated partial throm-
boplastin time) serves as a measure for the ability of
the inhibitor to interfere with the coagulation cascade
via the intrinsic pathway.11
2. Results and discussion
In a previous publication10 compound 1 (Table 1) was
described as a TF/F.VIIa inhibitor with moderate
in vitro inhibitory activity and good selectivity against
related serine proteases. It has moderate functional
activity in the PT assay which is a measure for the abil-
ity of an inhibitor to prevent clotting via the extrinsic
pathway of the coagulation cascade.11 It is characterized
by a promising pharmacokinetic profile in the rat (low
clearance and volume of distribution, long half-life)
and, when administered as amidoxime/ethylester double
It was observed that upon introduction of a fluorine
substituent in position 2 (compound 2, Table 1), the
in vitro binding affinity for F.VIIa remains unchanged,
while the functional activity (2x PT) is improved by a
factor 2. Previously it was found that a tetrahydrofura-
nyloxy substituent results in good binding affinity and
functional activity as illustrated by compound 3. Com-
bination of both the fluorine substituent and the tetra-
Table 1. Inhibition of TF/F.VIIa by phenylglycine derivatives 1–6
O
R2
R1
Cα
HO
NH
O
H2N
NH
Compound Configuration R1 R2
at Ca
Ki (lM)
F.VIIa Thrombin F.Xa Trypsin
2x PTa (lM) 2x aPTTb (lM) aPTT/PT
Thrombin
F:VIIa
1
2
RS
RS
H
F
EtO
EtO
0.19
0.20
>35
8.8
5.6
3.0
16.9
6.8
>184
44
9.1
4.4
36.8
21.7
4.0
4.9
R/S
O
O
O
O
3
4
5
6
RS
RS
R
H
F
F
F
0.14
10.7
7.1
4.5
3.7
2.9
1.2
1.9
0.41
>6.8
3.1
77
80
5.5
2.5
2.0
1.1
20.6
10.4
10.8
3.6
3.7
4.2
5.4
3.3
O
O
O
O
R/S
R
0.088
0.081
0.035
3.2
55
s
R
1.5
105
a Human citrated plasma is spiked with at least six concentrations of inhibitor. Clotting is initiated by addition of exogenous tissue factor (Innovin).
Clotting time is determined by a turbidity measurement. The concentration of inhibitor necessary to double control clotting time is determined by
fitting the data to an exponential regression.11
b Human citrated plasma is spiked with at least 6 concentrations of inhibitor. Clotting via the intrinsic pathway is initiated by addition of Actinꢁ FS
(ellagic acid in soy phosphatides). Clotting time is determined by a turbidity measurement. The concentration of inhibitor necessary to double
control clotting time (EC50) is determined by fitting the data to an exponential regression.11

K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
5359
hydrofuranyloxy moiety resulted in compound 4 with a
functional activity approaching the targeted value.
Compound 4 consists of 2 diastereomeric racemates
which were separated into the 4 individual stereoiso-
mers. The two isomers with configuration S at Ca of
the phenylglycine moiety were almost inactive (data
not shown). The two isomers with configuration R at
Ca (compounds 5 and 6) were both potent inhibitors
and had excellent functional activity. The plasma
potency of compound 6 even exceeds the targeted value
of 2 lM for 2x PT prolongation. Its selectivity in the
functional assays (aPTT/PT ratio) however is some-
what lower than that for compound 5. This might be
explained by the residual factor Xa inhibitory activity
(Ki (F.Xa) = 410 nM) of compound 6. Since F.Xa is
located at the intersection of the intrinsic and the
extrinsic pathways, its inhibition prolongs both PT
and aPTT.
forms the expected salt bridge with the carboxylate of
Asp 189 at the bottom of the S1 pocket. A hydrogen
bond is formed between the aniline NH and the hydrox-
yl group of the active site serine (Ser 195). The ethoxy
group in position 5 of the phenylglycine moiety is locat-
ed in the small S2 pocket of F.VIIa. It is arranged in an
extended conformation. The fluorine points towards the
solvent and is not involved in any specific interaction.
Rather its presence most likely influences the conforma-
tion of the tetrahydrofuranyloxy substituent in such a
way that it is rotated away from the fluorine substituent
and thus is accommodated optimally in the shallow S3
pocket. A detailed discussion of the influence of the
fluorine substituent on the in vitro and functional activ-
ity of the phenylglycine TF/F.VIIa inhibitors will be the
subject of a further publication. The carboxylate is
located in close proximity of the side chain of Lys 192
and might thus be engaged in a favorable electrostatic
interaction, although Lys 192 is quite disordered in
most X-ray crystal structures of complexes between
F.VIIa and inhibitors.
Since compound 5 fulfills both targeted requirements for
functional activity and selectivity, it was chosen for fur-
ther profiling. Its absolute configuration was corrobo-
rated by an X-ray structural analysis.
The PK profile upon iv administration of compound 5
in the rat and the guinea pig (Table 2) is characterized
by a long half-life (ꢀ6 h) in the rat, a low clearance
and a moderate volume of distribution (corresponding
Figure 1 shows the X-ray analysis of compound 5
bound to the active site of TF/F.VIIa. The amidine
A
B
S2 Pocket
His57
Asp102
Ser195
Lys192
Cα
Gln143
S1 Pocket
Trp215
S3 Pocket
Asp189
Figure 1. Crystal structure (2bz6.pdb) of compound 5 bound to the active site of F.VIIa. (A) Ball-and-stick representation. (B) Surface
representation. Crystals of compound 5 in complex with short form recombinant F.VIIa were produced as described.10 A crystal was frozen and
˚
measured on the Swiss Norwegian Beam Lines (SNBL) at the ESRF, Grenoble, to a resolution of 1.47 A. Experimental details were: temperature
˚
100 K, wavelength 0.80 A, detector MAR300, pixel size 0.15 mm, detector distance 260 mm, frame size 0.5ꢂ and number of frames 173. Data were
processed with XDS2000.12 The unit cell is P41212, with a = b = 94.93 A, c = 115.64 A. Ice rings were observed at 3.90, 3.67, 3.44, 2.67, 2.25 and
˚
˚
˚
1.91 A and removed using the EXCLUDE_RESOLUTION_RANGE command. To 1.5 A resolution, for 482714 observations of 69242 unique
˚
reflections the overall merging R-factor is 12.5% with 81.6% completeness and mean Intensity/standard deviation of 10.15, and in the outer shell from
˚
1.6 to 1.5A the R-factor is 80.7% with 36.5% completeness and mean Intensity/standard deviation of 1.78. The structure was refined, starting from
coordinates of other in-house structures, initially using CNX13 and then using Refmac514 and ARP/wARP15 from the CCP4 program suite16 with
cycles of model building using Moloc.17 The outer resolution limit was finally reduced to 1.6 A and the inner cut-off of the outer ice-ring reduced in
˚
˚
resolution from 1.92 to 1.94 A. The final refinement cycles with Refmac5 used TLS and restrained refinement, and hydrogens were included at riding
positions for amino acids as calculated by Refmac5, and for the chemical ligand as calculated by Moloc, but not for water molecules. For 307 amino
acids (10 with alternative conformations), the ligand, one sulfate ion, one calcium ion and 500 water molecules there were 2434 hydrogen atoms and
˚
2985 non-hydrogen atoms. For 58180 reflections (90.2% completeness to 1.60 A), overall crystallographic R-factors were 16.6% (working) and 19.3%
˚
(free), with values in the outer shell (1.64–1.60 A) of 25.3% and 27.4%, respectively. The inhibitor density is clear. The peptide Lys192-Gly193 is in the
‘inactive’ conformation previously reported.10 Coordinates have been deposited in the Protein Data Bank as 2bz6.pdb.

5360
K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
to extracellular water) in both rat and guinea pig. As
expected for a zwitterionic compound the bioavailability
of 5 in the rat is very low. As described previously for
compound 1, charged functional groups (amidine, car-
boxylic acid) can be masked resulting in the amidox-
ime/ethyl ester double prodrug 7. Such prodrug was
shown to exhibit 20% oral bioavailability in the rat.
The conversion efficiency from double prodrug to parent
upon iv administration of 7 amounts to 50%.
These results confirm previous findings from analogous
experiments with an inactive tissue factor mutant
hTFAA7 and a small molecule inhibitor from Pfizer8a,b
which demonstrated that a specific inhibitor of TF/
F.VIIa can produce an antithrombotic effects, while
only minimally disturbing hemostasis. It therefore seems
that TF/F.VIIa is an especially promising target for the
development of an anticoagulant drug. Furthermore, it
was demonstrated that it is possible to produce an anti-
thrombotic effect upon po administration of a double
prodrug of an active TF/F.VIIa inhibitor which by itself
is unsuitable for oral dosing. It remains to be shown
how a double prodrug TF/F.VIIa inhibitor would per-
form in clinical investigations.
A dose-dependent plasma concentration of 5 was
observed upon po administration of 7 in the guinea
pig. High doses between 60 and 500 mg/kg had to be
administered to see a pharmacological effect in the
guinea pig (Fig. 2) since the functional activity of 5 in
guinea pig plasma is much lower than in human plasma
(Table 3). Nevertheless a dose-dependent PT prolonga-
tion was observed as an in vivo effect, while aPTT
remained almost unchanged even at the highest dose.
3. Chemistry
The fluorinated diethoxy derivative 2 was synthesized
starting with the tert-butyldimethylsilyl protection of
4-fluorophenol (Scheme 1). Regioselective lithiation in
the ortho position of the fluorine atom followed by reac-
tion with trimethylborate and subsequent oxidation with
hydrogen peroxide led to phenol 9. The phenol was
again protected with a tert-butyldimethyl silyl group.
Regioselective lithiation in the ortho position of the
fluorine substituent followed by quenching with DMF
provided benzaldehyde 10. In a one-pot procedure both
silyl groups were removed with potassium fluoride and
then alkylated with ethyl iodide. Benzaldehyde 11 was
reacted in a Lewis-acid catalyzed condensation with
benzylisonitrile and 4-aminobenzonitrile. The intermedi-
ate iminoether 12 was treated in situ with an excess of
water and hydrolyzed to phenylglycine ester 13. Conver-
sion of the nitrile into the respective amidine by a Pinner
reaction and hydrolysis of the ester functionality led to
the zwitterionic phenylglycine derivative 2.
Compounds 5 and 7 were characterized in a guinea pig
model of recurrent arterial thrombosis. Furthermore
their propensity to enhance bleeding was assessed. In
parallel both experiments were also performed with a
mixed F.VIIa/F.Xa inhibitor, namely compound 8 from
Ono (Table 3)18, in order to confirm that selective F.VIIa
inhibition ensures a large safety window. In this study, an
established model of arterial thrombosis was used.7a
Bleeding propensity was quantified by determining the
nail cuticle bleeding time as previously described.7a
Upon continuous iv infusion of 5 and po administration
of 7, dose-dependent prolongation of PT was observed,
while aPTT remains unaffected. Furthermore, a dose-de-
pendent reduction of the thrombosis index, which is a
measure for antithrombotic efficacy7a, is observed, while
nail cuticle bleeding is only slightly prolonged at the
highest doses.
The synthesis of the unfluorinated tetrahydrofuranyloxy
derivative 3 started with the monoethylation of 5-hy-
droxymethyl-benzene-1,3-diol, followed by a benzylation
of the remaining phenol and oxidation of the hydroxy-
methyl group to give benzaldehyde 14 which was con-
densed with 4-amino-benzonitrile and benzylisonitrile to
Upon continuous iv infusion of the Ono compound 8,
both PT and aPTT were prolonged to the same extent.
An antithrombotic effect comparable to that of com-
pound 5 was observed. The nail cuticle bleeding time
however was prolonged substantially at higher doses.
Table 2. Pharmacokinetic parameters of parent compound 5 as well as of the corresponding prodrug 7
O
O
F
O
O
R'
NH
O
H2N
N
R''
Compound
R0
R00
Rat
Guinea pig
Dose
(mg/kg)
T1/2 iv
(h)
Cl
(ml/min/kg)
Vdss
F
(%)
Dose
(mg/kg)
T1/2 iv
(h)
Cl
(ml/min/kg)
Vdss
(L/kg)
(L/kg)
5
7
H
Et
H
OH
3
6.3
7.2
1.2
2
20
1
3.4
3.8
0.7

K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
APTT Thrombosis Index
5361
Cuticle Bleeding Time
PT
A
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
100
75
50
25
0
0
10 30 60 100 200
0
10 30 60 100 200
0
10 30 60 100 200
0
10 30 60 100 200
μg/kg/min
μg/kg/min
μg/kg/min
μg/kg/min
2.0
B
PT
APTT
Cuticle Bleeding Time
Thrombosis Index
3.0
2.5
2.0
1.5
1.0
0.5
0
3.0
100
75
50
25
0
1.5
1.0
0.5
0
2.5
2.0
1.5
1.0
0.5
0
0
10 20 60 125 250 500
0
10 20 60 125 250 500
0
10 20 60 125 250 500
0
10 20 60 125 250 500
mg/kg
mg/kg
mg/kg
mg/kg
C
PT
APTT
Thrombosis Index
Cuticle Bleeding Time
8
6
4
2
0
8
6
4
2
0
14
12
10
8
100
75
50
25
0
6
4
2
0
μg/kg/min
μg/kg/min
μg/kg/min
μg/kg/min
Figure 2. Dose-dependent pharmacodynamic effects (PT and aPTT prolongation), antithrombotic efficacy (expressed by percent reduction of
thrombosis index7) and nail cuticle bleeding time reduction7 in guinea pig (n = 6 per dose level) upon (A) iv administration of compound 5; (B) po
administration of compound 7; (C); iv administration of compound 8.
Table 3. Inhibitory activity of compounds 5 and 8 (mixed F.VIIa/F.Xa inhibitor from Ono) towards TF/F.VIIa and related serine proteases as well
as in human and guinea pig plasma
O
O
F
O
O
OH
N
O
H
HO
N
NH
O
NH
O
O
HO
5
8
H2N
NH
H2N
NH
Compound
Ki (lM)
Human plasma
Guinea pig plasma
F.VIIa Thrombin F.Xa Trypsin 2x PT (lM) 2x aPTT (lM) aPTT/PT 2x PT (lM) 2x aPTT (lM) aPTT/PT
5
8
0.081
0.010
4.5
4.3
1.9
0.15
3.2
0.24
2.0
1.8
10.8
1.4
5.4
0.8
17
3.5
61
1.9
3.6
0.5
furnish phenylglycine ester 16 (Scheme 2). After removal
of the benzyl group, phenol 17 was derivatized by a Mits-
unobu reaction with (RS)-3-hydroxyfurane. Conversion
of the nitrile into the respective amidine by a Pinner reac-
tion and hydrolysis of the ester functionality led to the
zwitterionic phenylglycine derivative 3.
The synthesis of the fluorinated tetrahydrofuranyloxy
compounds 4–7 started with the regioselective lithiation
of 4-ethoxyfluorobenzene in the ortho position of the
fluorine atom, quenching with trimethylborate and sub-
sequent oxidation with hydrogen peroxide to give phe-
nol 18. After protection as tert-butyldimethyl silyl

5362
K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
Si
O
Si
O
O
OBn
Si
O
HO
O
OH
F
a,b
a,c
HO
OH
O
OBn
F
d
a,b,c
F
NH
O
d
H
N
O
9
10
HO
O
H
14
N
S
15
O
O
O
F
e
O
O
OH
O
OBn
O
O
O
e
H
11
O
O
HO
NH
NH
g,h,i
f
NH
O
O
O
O
O
O
O
F
O
O
F
g,h
f
F
H2N
NH
N
N
O
HO
O
NH
NH
NH
3
17
16
N
O
O
Scheme 2. Preparation of unfluorinated tetrahydrofuranyl derivative
3. Reagents and conditions: (a) ethylbromide, K2CO3, DMF, 60 ꢂC;
(b) benzylbromide, K2CO3, DMF, 65 ꢂC; (c) MnO2, 1,2-dichloroeth-
ane, 50 ꢂC; (d) i—14, 4-aminobenzonitrile, MeOH; ii—toluene-4-
sulfonylisocyanide, BF3-OEt2, 0 ꢂC; (e) 20 equiv H2O; (f) H2, Pd/C,
THF, EtOH; (g) 3-hydroxy-tetrahydrofurane, PPh3, DEAD, THF; (h)
HCl(gas), CHCl3/MeOH 3:1, ꢁ10 ꢂC; evaporate, then 2 N NH3 in
MeOH; i—LiOH, THF.
HN
NH2
N
N
12
2
13
Scheme 1. Preparation of fluorinated diethoxy derivative 2. Reagents
and conditions: (a) t-BDMSCl, imidazole, DMF, 0 ꢂC; (b) i—sec-butyl
lithium (1.3 M in hexanes), THF, ꢁ78 ꢂC, 30 min; ii—B(OMe)3,
ꢁ78 ꢂC, 30 min; iii—HOAc, H2O2, 0 ꢂC ! rt, overnight; (c) sec-butyl
lithium (1.3 M in hexanes), DMF, THF, ꢁ78 ꢂC; (d) ethyl iodide, KF,
DMF, rt; (e) i—4-aminobenzonitrile, EtOH; ii—benzylisonitrile, BF3–
OEt2, 0 ꢂC; (f) 20 equiv H2O; (g) HCl(gas), CHCl3/MeOH 3:1, ꢁ10 ꢂC;
evaporate, then 2N NH3 in MeOH; (h) LiOH, THF.
4. Experimental
4.1. General
All reagents and solvents were purchased from Fluka
and used directly without further purification. Column
chromatography was performed on Merck Kieselgel 60
ether, compound 19 was lithiated regioselectively in the
ortho position of the fluorine atom and quenched with
DMF. Lewis-acid catalyzed condensation of benzalde-
hyde 20 with 4-aminobenzonitrile and 2-morpholinoe-
thylisocyanide gave iminoether 21 which was
hydrolyzed in situ with an excess of water to give phe-
nylglycine ester 22. The silyl-protecting group was lost
under these reaction conditions.
1
(230–400 mesh) silica gel. H NMR spectra were ob-
tained using a Bruker Avance 300 MHz instrument.
Chemical shifts are reported in parts per million (d) rel-
ative to TMS using residual chloroform or dimethyl
sulfoxide as an internal reference. Coupling constants
(J) are reported in hertz (Hz). The peak shapes are
denoted as follows: s, singlet; d, doublet; t, triplet; q,
quartett; m, multiplet; br, broad. ESI mass spectra were
recorded on an Sciex API 150 instrument from Applied
Biosystems using Analyst 1.3 software. EI mass spectra
were recorded on a Finnigan MAT SSQ 7000 instru-
ment using Xcalibur software.
Mitsunobu reaction of phenol 22 with (RS)-3-hydrox-
ytetrahydrofurane, conversion to the respective amidine
by a Pinner reaction and ester hydrolysis provided the
zwitterionic product 4.
The analogous reaction sequence using (S)-3-hydroxy-
tetrahydrofurane in the Mitsunobu step led to derivative
24 which was separated into the active inhibitor 5 and its
corresponding inactive epimer by chiral HPLC.
4.2. Synthesis of (RS)-(4-carbamimidoyl-phenylamino)-
(3,5-diethoxy-2-fluoro-phenyl)-acetic acid (2) (Scheme 1)
4.2.1. 5-(tert-Butyl-dimethyl-silanyloxy)-2-fluoro-phenol
(9). A solution of 4-fluorophenol (50 g, 0.45 mol) in
DMF (225 ml) was cooled under an argon atmosphere
to 0 ꢂC and treated with tert-butyldimethylsilyl chloride
(73.86 g, 0.49 mol). Then imidazole (33.36 g, 0.49 mol)
was added slowly. A colourless precipitate formed.
The reaction mixture was stirred for 1 h. Then H2O
(360 ml) was added. The solution was extracted with
n-hexane. The organic layers were washed with water,
10% Na2CO3 solution and again water, then dried over
In an analogous manner using (R)-3-hydrox-
ytetrahydrofuran in the Mitsunobu step the active inhib-
itor 6 and its corresponding inactive epimer were
obtained.
Reaction of the nitrile 23 with hydroxylamine hydro-
chloride and subsequent separation of the epimeric mix-
ture by chiral HPLC led to the double prodrug 7.

K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
5363
O
O
O
F
Si
O
O
F
O
O
F
O
OH
F
O
d
c
b
a
Si
Si
F
NH
O
H
N
N
20
19
18
O
21
N
O
OH
F
O
O
O
O
F
O
O
O
O
O
F
F
O
HO
HO
HO
NH
NH
NH
NH
e,f,g
h,f,g
i
O
O
O
O
O
F
22
OH
F
F
H2N
NH
H2N
NH
H2N
NH
N
22
4
25
6
j
O
O
F
O
O
F
O
O
O
O
O
O
O
O
O
F
F
O
HO
HO
k,l
f,g
NH
NH
i
NH
NH
23
O
O
O
O
H2N
NH
H2N
NH
H2N
N
N
OH
23
24
5
7
Scheme 3. Preparation of fluorinated tetrahydrofuranyl derivatives 4–7. Reagents and conditions: (a) i—pentamethyldiethylentriamine,
n-butyllithium (1.6 M in hexanes), THF, ꢁ78 ꢂC, 2 h; ii—B(OMe)3, ꢁ78 ꢂC ! rt, 2 h; iii—AcOH, 0 ꢂC, 30 min; H2O2 (aq, 35%), 0 ꢂC ! rt,
overnight; (b) t-BDMSCl, imidazole, DMF, 0 ꢂC; (c) i—pentamethylethylenetriamine, n-butyllithium (1.6 M in hexanes), THF, ꢁ78 ꢂC ! ꢁ50 ꢂC,
5 h; ii—DMF, ꢁ78 ꢂC ! rt, overnight; (d) i—20, 4-aminobenzonitrile, EtOH, 1 h, rt; ii—2-morpholinoethylisocyanide, then BF3–OEt2, 0 ꢂC ! rt,
3 h; iii—20 equiv H2O, 50 ꢂC, overnight; (e) (RS)-3-hydroxy-tetrahydrofurane, PPh3, DEAD, THF; (f) HCl(gas), CHCl3/MeOH 3:1, ꢁ10 ꢂC;
evaporate, then 2 N NH3 in MeOH; (g) LiOH, THF; (h) (R)-3-hydroxy-tetrahydrofurane, PPh3, DEAD, THF; (i) separation of epimers on
ChiralPak AD with heptane/isopropanol/trifluoroacetic acid 75:25:0.2 as eluent; (j) (S)-3-hydroxy-tetrahydrofurane, PPh3, DEAD, THF; (k) H2N-
OH hydrochloride, TEA, EtOH, rt; (l) separation of epimers on ChiralPak AD with heptane/isopropanol/trifluoroacetic acid 70:30:0.2 as eluent.
MgSO4 and filter. The filtrate was concentrated and
dried to give tert-butyl-(4-fluoro-phenoxy)-dimethyl-si-
lane (92.22 g, 91%) as a colourless liquid. 1H NMR
(300 MHz, CDCl3) d = 0.01 (6 H, s, CH3), 0.80 (9H, s,
t-butyl), 6.59 (2H, dd, J = 4.6 Hz, 9.1 Hz, 2-CH, 20-
CH), 6.73 (2H, t, J = 9.1 Hz, 3-CH, 30-CH).
then dried over MgSO4. The crude product was purified
by chromatography on silica gel using cyclohexane/
EtOAc 18:1 as eluent. The title compound 9 (38.86 g,
73%) was obtained as a yellow liquid. 1H NMR
(300 MHz, DMSO-d6) d = 0.00 (6H, s, CH3), 0.77 (9H,
s, t-butyl), 6.01 (1H, dt, J = 3.2 Hz, 8.7 Hz, 4-CH),
6.28 (dd, J = 2.9 Hz, 7.6 Hz, 6-CH), 6.81 (1H, dd,
J = 8.7 Hz, 11.2 Hz, 3-CH), 9.61 (1H, s br, OH); MS
(ESI) m/z 240.5 ([MꢁH]ꢁ, 100%).
A solution of tert-butyl-(4-fluoro-phenoxy)-dimethyl-si-
lane (50.0 g, 0.22 mol) in THF (220 ml) was cooled un-
der an argon atmosphere to ꢁ65 ꢂC and treated within
30 min with sec-butyllithium in hexane (1.3 M,
188.5 ml, 0.245 mol). The obtained off-white suspension
was stirred for 30 min at ꢁ65 ꢂC. Then a solution of
trimethylborate (25 ml, 0.224 ml) in THF (45 ml) was
added within 30 min. The reaction mixture was stirred
for 30 min at ꢁ65 ꢂC, then warmed to 0 ꢂC and treated
with acetic acid (19 ml, 0.332 mol). Hydrogen peroxide
solution (35% in water, 21 ml, 0.244 mol) was added
within 30 min. A white precipitate formed. After the
addition of H2O (500 ml), the obtained solution was
extracted with Et2O. The combined organic layers were
washed with H2O, 10% NaOH solution and again H2O,
4.2.2. 3,5-Bis-(tert-butyl-dimethyl-silanyloxy)-2-fluoro-
benzaldehyde (10). Using the same procedure as de-
scribed for the preparation of tert-butyl-(4-fluoro-phen-
oxy)-dimethyl-silane (Section 4.2.1, first reaction step),
compound 9 (4.5 g, 18.6 mmol) was reacted with
tert-butyldimethylsilyl chloride (3.1 g, 20.4 mmol) and
imidazole (1.4 g, 20.4 mmol) in DMF (10 ml) to give
2,4-bis-(tert-butyl-dimethyl-silanyloxy)-1-fluoro-benzene
(6.54 g, 99%) as a light yellow liquid. 1H NMR
(300 MHz, DMSO-d6) d = 0.00 (6H, s, CH3), 0.01 (6H,
s, CH3), 0.77 (9H, s, t-butyl), 0.79 (9H, s, t-butyl), 6.24
(1H, dd, J = 2.7 Hz, 7.4 Hz, 3-CH), 6.27 (dt,

5364
K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
J = 3.0 Hz, 8.5 Hz, 5-CH), 6.91 (1H, dd, J = 8.7 Hz,
10.8 Hz, 6-CH); MS (ESI) m/z 355.2 ([MꢁH]ꢁ).
ꢁ15 ꢂC. A stream of dry HCl gas was passed through
the solution for 15 min. The flask was stoppered well
and stored at 4 ꢂC overnight. Then, the reaction mixture
was concentrated. The residue was taken up in EtOH
(4 ml) and treated with 2 M NH3 in EtOH (5.2 ml,
10.4 mmol). The mixture was heated for 2 h at 60 ꢂC
and then concentrated. The crude product was purified
by chromatography on silica gel using a gradient of
CH2Cl2/MeOH 19:1 ! 2:1 as eluent to give (4-carbam-
imidoyl-phenylamino)-(3,5-diethoxy-2-fluoro-phenyl)-
acetic acid ethyl ester hydrochloride (535 mg, 94%) as an
off-white solid. 1H NMR (300 MHz, DMSO-d6) d = 1.14
(3H, t, J = 7.0 Hz, CH3), 1.28 (3H, t, J = 6.8 Hz, CH3),
1.33 (3H, t, J = 6.8 Hz, CH3), 3.95 (2H, q, J = 7.0 Hz,
CH2), 4.09 (4H, m, 2· CH2), 5.56 (1H, d, J = 8.2 Hz,
CHa), 6.53 (1H, s br, ar-CH), 6.66 (1H, d, J = 6.5 Hz,
ar-CH), 6.82 (2H, d, J = 8.4 Hz, ar-CH), 7.39 (1H, d,
J = 8.2 Hz, NH), 7.64 (2H, d, J = 8.4 Hz, ar-CH), 8.77
(4H, br, H2N+@C–NH2); MS (ESI) m/z 404.5
([M+H]+, 100%).
A solution of 2,4-bis-(tert-butyl-dimethyl-silanyloxy)-1-
fluoro-benzene (6.3 g, 17.7 mmol) in THF (18 ml) was
cooled to ꢁ70 ꢂC and treated within 1 h with sec-butylli-
thium solution in hexane (1.3 M, 15 ml, 19.4 mmol).
Then DMF (1.5 ml) in THF (3 ml) was added within
30 min. Stirring was continued for 1 h at ꢁ70 ꢂC and
90 min at rt, followed by the addition of H2O (30 ml).
The reaction mixture was extracted with Et2O. The com-
bined organic layers were washed with H2O, dried over
MgSO4, filter and concentrated to give 10 (6.3 g, 92%) as
a yellow liquid. 1H NMR (300 MHz, DMSO-d6)
d = 0.18 (6H, s, CH3), 0.20 (6H, s, CH3), 0.93 (9H, s,
t-butyl), 0.96 (9H, s, t-butyl), 6.78 (2H, m), 10.12 (1H,
s, CHO); MS (ESI) m/z 385.2 2 ([M+H]+).
4.2.3. 3,5-Diethoxy-2-fluoro-benzaldehyde (11). A solu-
tion of 10 (3.0 g, 7.8 mmol) in DMF (25 ml) was treated
with potassium fluoride (1.81 g, 31.2 mmol) and ethyl
iodide (1.51 ml, 18.72 mmol). The reaction mixture
was stirred for 2 h at rt, and then quenched with H2O
(25 ml). The obtained suspension was extracted with
Et2O. The combined organic layers were washed with
H2O, dried over MgSO4 and concentrated. The crude
product was purified by chromatography on silica gel
using a gradient of cyclohexane/EtOAc 18:1 ! 1:1 as
To a suspension of (4-carbamimidoyl-phenyl-amino)-
(3,5-diethoxy-2-fluoro-phenyl)-acetic acid ethyl ester
hydrochloride (320 mg, 0.73 mmol) in THF (4 ml) was
added 1 N LiOH solution (3.6 ml). The reaction mixture
was stirred for 3 h at rt and then neutralized with 1 N
HCl solution. The precipitate was filtered off and dried
to give 2 (234 mg, 86%) as white solid. 1H NMR
(300 MHz, DMSO-d6 + 1 drop DCl 20 wt% in D2O)
1H NMR d = 1.28 (3H, t, J = 7.0, CH3), 1.34 (3H, t,
J = 7.0, CH3), 3.95 (2H, q, J = 7.0 Hz, CH2), 4.08 (2H,
q, J = 7.0, CH2), 5.46 (1H, s, CHa), 6.53 (1H, t br, ar-
CH), 6.64 (1H, dd, J = 2.5 Hz, 6.7 Hz, ar-CH), 6.86
(2H, d, J = 8.7 Hz, ar-CH), 7.64 (2H, d, J = 8.7 Hz, ar-
CH), 8.72 (<2H, s, H2N+@C–NH2), 8.93 (<2H, s,
H2N+@C–NH2); MS (ESI) m/z 376.5 ([M+H]+, 100%).
1
eluent to give 11 (904 mg, 55%) as a white solid. H
NMR (300 MHz, DMSO-d6) d = 1.33 (3H, t,
J = 7.0 Hz, CH3), 1.35 (3H, t, J = 7.0 Hz, CH3), 4.05
(2H, q, J = 7.0 Hz, CH2), 4.14 (2H, q, J = 7.0 Hz, CH2),
6.77 (1H, dd, J = 3.0 Hz, 4.2 Hz, ar-CH), 7.03 (1H, dd,
J = 3.0 Hz, 7.4 Hz, ar-CH), 10.19 (1H, s, CHO).
4.2.4.
(4-Cyano-phenylamino)-(3,5-diethoxy-2-fluoro-
phenyl)-acetic acid ethyl ester (13). To a solution of 11
(880 mg, 4.2 mmol) in 15 ml EtOH were added under
an argon atmosphere 4-aminobenzonitrile (493 mg,
4.3. Synthesis of (4-carbamimidoyl-phenylamino)-[3-eth-
oxy-5-(tetrahydro-furan-3-yloxy)-phenyl]-acetic acid (3)
(Scheme 2)
˚
4.2 mmol) and 4 A molecular sieves. The reaction mix-
ture was cooled to 0 ꢂC and treated with benzyl isonitrile
(0.51 ml, 4.2 mmol), followed by the slow addition of
1.57 ml of boron trifluoride diethyl etherate (1.57 ml,
12.5 mmol) while maintaining a temperature between
0 ꢂC and 5 ꢂC. The reaction was stirred for 15 min at
0 ꢂC and for 90 min at rt, then filtered and concentrated.
The obtained iminoether 12 was dissolved in EtOH
(15 ml) and treated with H2O (1.5 ml). After stirring at
rt overnight, a precipitate had formed. It was filtered
off, washed with cold EtOH/H2O 1:1 and dried to give
13 (1.15 g, 72%) as an off-white solid. 1H NMR
(300 MHz, DMSO-d6) d = 1.15 (3H, t, J = 7.1 Hz,
CH3), 1.30 (3H, t, J = 7.0 Hz, CH3), 1.35 (3H, t,
J = 7.0 Hz, CH3), 3.98 (2H, q, J = 7.0 Hz, CH2), 4.09
(4H, m, 2· CH2), 5.49 (1H, d, J = 8.2 Hz, CHa), 6.51
(1H, dd, J = 2.9 Hz, 4.6 Hz, ar-CH), 6.66 (1H, dd,
J = 2.7 Hz, 6.9 Hz, ar-CH), 6.76 (2H, d, J = 8.7 Hz, ar-
CH), 7.33 (1H, d, J = 8.2 Hz, NH), 7.48 (2H, d,
J = 8.7 Hz, ar-CH); MS (EI) 386 ([M]+).
4.3.1. 3-Benzyloxy-5-ethoxy-benzaldehyde (14). To a
solution of 3,5-dihydroxy benzylalcohol (125 g,
0.89 mol) in DMF (1250 ml) were added under an argon
atmosphere K2CO3 (246.6 g, 1.78 mol) and ethylbro-
mide (73.2 ml, 0.98 mol). The suspension was stirred
for 4 h at 65 ꢂC, then filter. The filtrate was concentrat-
ed. The residue was taken up in EtOAc and washed with
water and brine. The organic layer was dried over
MgSO4, filtered and concentrated. The crude product
was purified by chromatography on silica gel using hex-
ane/EtOAc 3:1 as eluent to give 3-ethoxy-5-hydroxy-
methyl-phenol (45.8 g, 31%) as a yellow solid. 1H
NMR (300 MHz, DMSO-d6) d = 1.28 (3H, t,
J = 7.0 Hz, CH3), 3.93 (2H, q, J = 7.0 Hz, CH2), 4.35
(2H, d br, J = 4.2 Hz, CH2-OH), 5.06 (1H, t br,
J = 5.3, HO-CH2), 6.15 (1H, t, J = 2.2 Hz, ar-CH),
6.30 (2H, m, ar-CH), 9.27 (1H, s br, ar-OH). MS (EI)
m/z 168 ([M]+).
4.2.5. (4-Carbamimidoyl-phenylamino)-(3,5-diethoxy-2-
fluoro-phenyl)-acetic acid (2). A solution of 13 (500 mg,
1.3 mmol) in CHCl3/EtOH 3:1 (5 ml) was cooled to
To a solution of 3-ethoxy-5-hydroxymethyl-phenol
(45.8 g, 0.27 mol) in DMF (470 ml) were added under
an argon atmosphere K2CO3 (75.3 g, 0.54 mol) and ben-

K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
5365
zylbromide (35.6 ml, 0.30 mol). The yellow suspension
was stirred at 65 ꢂC for 4 h. The reaction mixture was
concentrated, then taken up in EtOAc and washed with
water and brine. The organic layer was dried over
MgSO4, filtered and concentrated. The crude product
was purified by chromatography using hexane/EtOAc
4:1 as eluent to give (3-benzyloxy-5-ethoxy-phenyl)-
methanol (58.7 g, 83%) as a yellow liquid. 1H NMR
(300 MHz, CDCl3) d = 1.39 (3H, t, J = 7.0 Hz, CH3),
1.67 (1H, t, J = 6.0, OH), 4.01 (2H, q, J = 7.0 Hz,
CH2), 4.62 (2H, d, J = 6.0 Hz, -CH2-OH), 5.04 (2H, s,
Bn-CH2), 6.46 (1H, t, J = 2.3 Hz, ar-CH), 6.52 (1H, m,
ar-CH), 6.60 (1H, m, ar-CH), 7.31–7.44 (5H, m br,
Bn-CH); MS (EI) m/z 258 ([M]+).
J = 5.4 Hz, NH), 6.35 (1H, t, J = 2.3 Hz, ar-CH), 6.49
(1H, m, ar-CH), 6.51 (2H, d, J = 8.8 Hz, ar-CH), 6.58
(1H, m, ar-CH), 7.37 (2H, d, J = 8.8 Hz, ar-CH); MS
(ESI) m/z 327.3 ([M+H]+, 100%).
4.3.4. (4-Carbamimidoyl-phenylamino)-[3-ethoxy-5-(tet-
ra-hydrofuran-3-yloxy)-phenyl]-acetic acid (3).
A
solution of 17 (1.50 g, 4.6 mmol), 3-hydroxy-tetra-
hydrofurane (0.51 ml, 6.3 mmol) and triphenylphos-
phine (1.87 g, 7.1 mmol) in THF (30 ml) was treated
with diethyl azodicarboxylate (1.11 ml, 7.1 mmol). The
reaction mixture was stirred for 5 h and then concentrat-
ed. The crude product was purified by column chroma-
tography using cyclohexane/EtOAc 4:1 as eluent to give
(4-cyano-phenylamino)-[3-ethoxy-5-(tetrahydro-furan-
3-yloxy)-phenyl]-acetic acid methyl ester (2.0 g, 110%,
contains EtOOC-NH-NH-COOEt as impurity). 1H
NMR (300 MHz, DMSO-d6) d = 1.30 (3H, t,
J = 6.9 Hz, CH3), 1.92 (1H, m, tetrahydropyrane-
CH2), 2.22 (1H, m, THP-CH2), 3.53–3.94 (4H, m,
THP-CH2O), 3.99 (2H, q, J = 6.9, CH2), 5.04 (1H, t
br, J = 4.8 Hz, THP-CH), 5.33 (1H, d, J = 8.0 Hz,
CHa), 6.40 (1H, t, J = 2.1 Hz, ar-CH), 6.61 (1H, t br,
A solution of (3-benzyloxy-5-ethoxy-phenyl)-methanol
(58.7 g, 0.23 mol) in 1,2-dichloroethane (1180 ml) was
treated under an argon atmosphere with manganese(IV)
oxide (71.1 g, 0.81 mol). The black suspension was stir-
red overnight at 50 ꢂC and then filtered over Celite.
The filtrate was concentrated to give 47.3 g (81%) of
14 as a yellow liquid. 1H NMR (300 MHz, CDCl3)
d = 1.43 (3H, t, J = 7.0 Hz, CH3), 4.06 (2H, q,
J = 7.0 Hz, CH2), 5.10 (2H, s, Bn-CH2), 6.78 (1H, t,
J = 2.3 Hz, ar-CH), 7.01 (1H, m, ar-CH), 7.08 (1H, m,
ar-CH), 7.34–7.41 (5H, m br, Bn-CH), 9.89 (1H, s,
CHO); MS (EI) m/z 256 ([M]+).
ar-CH), 6.64 (1H,
t br, ar-CH), 6.78 (2H, d,
J = 8.7 Hz, ar-CH), 7.32 (1H, d, J = 8.2 Hz, NH), 7.46
(2H, d, J = 8.7 Hz, ar-CH).
A solution of (4-cyano-phenylamino)-[3-ethoxy-5-(tet-
rahydro-furan-3-yloxy)-phenyl]-acetic acid methyl ester
(2.0 g, 5.0 mmol) in CHCl3/EtOH 3:1 (22 ml) was
cooled to ꢁ15 ꢂC. A stream of dry HCl gas was
passed through the solution for 15 min. The flask
was stoppered well and stored at 4 ꢂC overnight.
Then, the reaction mixture was concentrated. The res-
idue was taken up in EtOH (24 ml) and treated with
2 M NH3 in EtOH (7.0 ml, 14.0 mmol). The mixture
was heated for 5 h at 65 ꢂC and then concentrated.
The crude product was purified by chromatography
on silica gel using CH2Cl2/MeOH 19:1 as eluent to
give (4-carbam-imidoyl-phenylamino)-[3-ethoxy-5-(tet-
rahydro-furan-3-yloxy)-phenyl]-acetic acid methyl ester
4.3.2. (3-Benzyloxy-5-ethoxy-phenyl)-(4-cyano-phenyl-
amino)-acetic acid methyl ester (16). A solution of 14
(47.3 g, 0.18 mol) in MeOH (900 ml) was treated under
an argon atmosphere with 4-aminobenzonitrile (21.8 g,
0.18 mol). The yellow solution was stirred for 2 h at rt,
then treated with toluene-4-sulfonylisocyanide (36.0 g,
0.18 mol) and cooled to 0 ꢂC. Then, boron trifluoride
diethyl etherate (69.5 ml, 0.55 mol) was added within
20 min, maintaining the temperature below 5 ꢂC. The
reaction mixture was stirred at 0 ꢂC for 10 min and at
rt for 1 h, then concentrated. The residue was taken
up in 660 ml MeOH and 66 ml H2O, heated until the
material was completely dissolved and then cooled to
rt. The suspension was stirred for 1 h and then filter.
The remaining solid was recrystallized from MeOH/hex-
1
hydrochloride (800 mg, 35%) as an off-white solid. H
NMR (250 MHz, CDCl3) d = 1.35 (3H, t, J = 6.8 Hz,
CH3), 2.05–2.19 (2H, m, THP-CH2), 3.73 (3H, s,
CH3), 3.73–3.98 (6H, m, CH2-O), 4.87 (1H, m,
CHa), 5.00 (1H, m, THP-CH), 5.78 (1H, m, NH),
6.31 (1H, s, ar-CH), 6.51–6.58 (4H, m, ar-CH), 7.67
1
ane to give 16 (49.1 g, 64%) as a light yellow solid. H
NMR (300 MHz, DMSO-d6) d = 1.30 (3H, t,
J = 6.9 Hz, CH3), 3.65 (3H, s, CH3-O), 3.99 (2H, q,
J = 6.9 Hz, CH2), 5.06 (2H, s, Bn-CH2), 5.32 (1H, d,
J = 7.8, CHa), 6.52 (1H, t, J = 2.1, ar-CH), 6.64 (1H, t
br, ar-CH), 6.73 (1H, t br, ar-CH), 6.78 (2H, d,
J = 8.7, ar-CH), 7.31–7.47 (7H, m, ar-CH, Bn-CH).
(2H, d, J = 8.4 Hz, ar-CH), 8.10 (<2H,
s
br,
H2N+@C–NH2), 8.72 (<2H, s br, H2N+@C–NH2);
MS (ESI) m/z 414.4 ([M+H]+, 100%).
4.3.3. (4-Cyano-phenylamino)-(3-ethoxy-5-hydroxy-phen-
yl)-acetic acid methyl ester (17). To a solution of 16
(24.9 g, 59.8 mmol) in EtOH/THF 1:1 (700 ml) was add-
ed Pd/C 10% (2.5 g). The reaction mixture was hydroge-
nated at 1.1 bar and rt for 5 h. The catalyst was filtered
off and washed with EtOH. The filtrate was concentrat-
ed. The crude product was purified by chromatography
n silica gel using hexane/EtOAc 3:1 as eluent to give 17
To a suspension of (4-carbamimidoyl-phenylamino)-[3-
ethoxy-5-(tetrahydrofuran-3-yloxy)-phenyl]-acetic acid
methyl ester hydrochloride (800 mg, 1.8 mmol) in THF
(8 ml) was added 1N LiOH solution (5.3 ml). The reac-
tion mixture was stirred for 1 h at rt, then neutralized
with 1 N HCl solution and then concentrated. The crude
product was purified by chromatography on RP-18 sili-
ca gel using a gradient of 0–40% MeCN in H2O to give 3
(330 mg, 47%) as an off-white solid. 1H NMR
(300 MHz, DMSO-d6 + 1 drop DCl 20 wt% in D2O)
d = 1.30 (3H, t, J = 6.9 Hz, CH3), 1.93 (1H, m, tetrahyd-
ropyrane-CH2), 2.21 (1H, m, THP-CH2), 3.70–3.90 (4H,
1
(13.0 g, 67%) as a yellow liquid. H NMR (300 MHz,
CDCl3) d = 1.39 (3H, t, J = 7.0, CH3(OEt)), 3.76 (3H,
s, CH3-O), 3.97 (2H, q, J = 7.0 Hz, CH2), 4.95 (1H, d,
J = 5.4 Hz, CHa), 4.97 (1H, s, OH), 5.51 (1H, d,

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K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
m, THP-CH2O), 3.98 (2H, q, J = 6.9, CH2), 4.98 (1H, t
br, THP-CH), 5.26 (1H, s, CHa), 6.39 (1H, t br, ar-CH),
6.64 (1H, t br, ar-CH), 6.67 (1H, t br, ar-CH), 6.82 (2H,
d, J = 8.8 Hz, ar-CH), 7.62 (2H, d, J = 8.8 Hz, ar-CH),
8.68 (<2H, s, H2N+@C–NH2), 8.91 (<2H, s,
H2N+@C–NH2); MS (ESI) m/z 400.5 ([M+H]+, 100%).
ties) as a light yellow liquid. 1H NMR (300 MHz,
CDCl3) d = 0.22 (6H, s, CH3-Si), 1.01 (9H, s, t-butyl-
CH3), 1.40 (3H, t, J = 7.0 Hz, CH3), 3.95 (2H, q,
J = 7.0 Hz, CH2), 6.83 (2H, m, ar-CH), 10.28 (1H, s,
CHO).
4.4.4. (4-Cyano-phenylamino)-(5-ethoxy-2-fluoro-3-hy-
droxy-phenyl)-acetic acid ethyl ester (22). To a solution
of 20 (16.9 g, 56.6 mmol) was added 4-aminobenzonitri-
le (6.7 g, 56.6 mmol). The reaction mixture was stirred
for 1 h at rt, then treated with 2-morpholinoethyl isocy-
anide (7.8 ml, 56.6 mmol) and cooled to 0 ꢂC. Boron tri-
fluoride diethyl etherate (28.4 ml, 226.3 mmol) was
added slowly, maintaining the temperature between
0 ꢂC and 5 ꢂC. The reaction mixture was stirred for
15 min at 0 ꢂC and for 3 h at rt. Then, 20 ml H2O was
added. The mixture was heated to 50 ꢂC overnight and
then concentrated. The residue was taken up in water
and extracted with EtOAc. The organic layer was dried
over MgSO4, filter and concentrated. The crude product
was purified by chromatography on silica gel using
cyclohexane/EtOAc 2:1 as eluent to give 22 (12.9 g,
4.4. Synthesis of (4-cyano-phenylamino)-(5-ethoxy-2-
fluoro-3-hydroxy-phenyl)-acetic acid ethyl ester (22)
(Scheme 3)
4.4.1. 5-Ethoxy-2-fluoro-phenol (18). A solution of 4-eth-
oxy-1-fluoro-benzene (56.3 g, 0.4 mol) in THF (200 ml)
was treated under an argon atmosphere with pentameth-
yl-diethylentriamine (84 ml, 0.4 mol) and cooled to
ꢁ78 ꢂC. n-Butyllithium solution (1.6 M in hexane,
251 ml, 0.4 mol) was added slowly. The reaction mixture
was stirred for 3 h at ꢁ78 ꢂC. Then, trimethylborate
(89.6 ml, 0.8 mol) was added slowly. The mixture was
stirred for 15 min at ꢁ78 ꢂC, then for 2 h at rt, then
cooled to 0 ꢂC and treated with acetic acid (63.2 ml,
1.1 mol). Hydrogen peroxide solution (30% in water,
68.3 ml, 0.6 mol) was added slowly at 0 ꢂC. The reaction
was warmed to rt and stirred overnight. After cooling to
0 ꢂC, saturated Na2SO3 solution (200 ml), then 300 ml
H2O and 500 ml hexane were added. The organic layer
was separated, washed with water and brine, dried over
MgSO4, filtered and concentrated to give 18 (60.3 g,
96%) as a light brown liquid. 1H NMR (300 MHz,
DMSO-d6) d = 1.28 (3H, t, J = 7.0 Hz, CH3), 3.91 (2H,
q, J = 7.0 Hz, CH2), 6.29 (1H, dt, J = 3.2 Hz, 8.9 Hz,
4-CH), 6.47 (1H, dd, J = 3.0 Hz, 7.6 Hz, 6-CH), 7.00
(1H, dd, J = 8.9 Hz, 11.2 Hz, 3-CH), 9.79 (1H, s, OH).
1
64%) as a viscous oil. H NMR (300 MHz, DMSO-d6)
d = 1.14 (3H, J = 7.0 Hz, CH3), 1.27 (3H, t, J = 6.9 Hz,
CH3), 3.88 (2H, q, J = 6.9 Hz, CH2), 4.07–4.21 (2H,
m, CH2), 5.45 (1H, d, J = 8.0 Hz, CHa), 6.38 (1H, t
br, J = 3.6 Hz, ar-CH), 6.45 (1H, dd, J = 2.8 Hz,
7.0 Hz, ar-CH), 6.76 (2H, d, J = 8.5 Hz, ar-CH), 7.31
(1H, d, J = 8.2 Hz, NH), 7.48 (2H, d, J = 8.2 Hz, ar-
CH), 10.02 (1H, s, OH); MS (ESI) m/z 357.1 ([MꢁH]ꢁ)
4.5. Synthesis of (4-carbamimidoyl-phenylamino)-[3-eth-
oxy-2-fluoro-5-(tetrahydro-furan-3- yloxy)-phenyl]-acetic
acid (4) (Scheme 3)
4.4.2. tert-Butyl-(5-ethoxy-2-fluoro-phenoxy)-dimethyl-
silane (19). Using the same procedure as described for
the preparation of tert-butyl-(4-fluoro-phenoxy)-
dimethyl-silane (Section 4.2.1, first reaction step),
compound 18 (75.5 g, 0.48 mol) was reacted with tert-
butyldimethylsilyl chloride (80.2 g, 0.53 mol) and imid-
azole (36.2 g, 0.53 mol) in DMF (250 ml) to give 19
(127.0 g, 97%) as a light brown liquid. 1H NMR
(300 MHz, CDCl3) d = 0.00 (3H, s, CH3-Si), 0.01 (3H,
s, CH3-Si), 0.80 (s, 9H, tert-butyl-CH3), 1.19 (3H, t,
J = 7.0 Hz, CH3), 3.75 (2H, q, J = 7.0 Hz, CH2), 6.21
(1H, dt, J = 3.2 Hz, 9.0 Hz, 4-CH), 6.27 (1H, dd,
J = 2.9 Hz, 7.1 Hz, 6-CH), 6.73 (1H, dd, J = 8.9 Hz,
10.3 Hz, 3-CH).
A solution of 22 (358 mg, 1.0 mmol), 3-hydroxy-tetra-
hydrofurane (106 mg, 1.2 mmol) and triphenylphos-
phine (315 mg, 1.2 mmol) in THF (10 ml) was cooled
under an argon atmosphere to 0 ꢂC and treated with
diethyl azodicarboxylate (0.187 ml, 1.2 mmol). The reac-
tion mixture was stirred for 3 h and then concentrated.
The crude product was purified by column chromatog-
raphy using cyclohexane/EtOAc 1:1 as eluent to give
(4-cyano-phenylamino)-[3-ethoxy-2-fluoro-5-(tetrahy-
dro-furan-3-yloxy)-phenyl]-acetic acid ethyl ester
(405 mg, 95%, contains according to 1H NMR
1
EtOOC-NH-NH-COOEt (H2-DEAD) as impurity). H
NMR (300 MHz, DMSO-d6) d = 1.14 (3H, t, J = 6.9,
CH3), 1.17 (t, J = 6.9, H2-DEAD-CH3), 1.29 (3H, t,
J = 6.9 Hz, CH3), 1.98 (1H, m, THP-CH2), 2.22 (1H,
m, THP-CH2), 3.71–3.91 (4H, m, THP-CH2O), 3.95
(2H, q, J = 6.9 Hz, CH2), 4.03 (q, J = 6.9 Hz, H2-
DEAD-CH2), 4.15 (2H, m, CH2), 5.09 (1H, t br, THP-
CH), 5.49 (1H, d, J = 8.2 Hz, CHa), 6.55 (1H, dd,
J = 2.9 Hz, 4.6 Hz, ar-CH), 6.65 (1H, dd, J = 2.8 Hz,
6.9 Hz, ar-CH), 6.77 (2H, d, J = 8.7 Hz, ar-CH), 7.33
(1H, d, J = 8.3 Hz, ar-CH), 7.48 (2H, d, J = 8.7 Hz, ar-
CH), 8.98 (1H, H2-DEAD-NH); MS (EI) m/z 428.1
([M]+).
4.4.3. 3-(tert-Butyl-dimethyl-silanyloxy)-5-ethoxy-2-fluo-
ro-benzaldehyde (20). To a solution of 19 (43.3 g,
0.16 mol) in THF (160 ml) was added under an argon
atmosphere pentamethyl-diethylentriamine (66.9 ml,
0.32 mol). After cooling the mixture to ꢁ78 ꢂC, n-buty-
llithium solution (1.6 M in THF, 200 ml, 0.32 mol) was
added within 1 h. The obtained viscous suspension was
stirred for 5 h at a temperature between ꢁ50 and
ꢁ60 ꢂC. After cooling again to ꢁ78 ꢂC, DMF (24.7 ml,
0.32 mol) was added within 20 min. The clear yellow
solution was stirred overnight, warming to rt. The reac-
tion was quenched with ice and extracted with EtOAc.
The organic layer was dried over MgSO4, filtered and
concentrated to give 20 (51.4 g, 107%, contains impuri-
A solution of (4-cyano-phenylamino)-[3-ethoxy-2-fluo-
ro-5-(tetrahydro-furan-3-yloxy)-phenyl]-acetic acid eth-
yl ester (400 mg, 0.93 mmol) in CHCl3/EtOH 3:1 was

K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
5367
cooled under an argon atmosphere to ꢁ10 ꢂC. A stream
of dry HCl gas was passed through the solution for
15 min. The flask was stoppered well and stored at
4 ꢂC overnight. Then, the reaction mixture was concen-
trated. The residue was taken up in EtOH (2 ml) and
treated with 2 M NH3 in EtOH (2.4 ml, 4.7 mmol).
The mixture was heated for 2 h at 60 ꢂC and then con-
centrated. The product was isolated by chromatography
on silica gel using CHCl3/MeOH 4:1 as eluent to give (4-
carbamimidoyl-phenylamino)-[3-ethoxy-2-fluoro-5-(tet-
rahydro-furan-3-yloxy)-phenyl]-acetic acid ethyl ester
hydrochloride (135 mg, 30%). 1H NMR (300 MHz,
DMSO-d6) d = 1.14 (3H, t, J = 6.9 Hz, CH3), 1.29 (3H,
t, J = 6.9 Hz, CH3), 1.98 (1H, m, THP-CH2), 2.23 (1H,
m, THP-CH2), 3.71–3.89 (4H, m, THP-CH2O), 3.95
(2H, q, J = 6.9 Hz, CH2), 4.16 (2H, m, CH2), 5.09
(1H, t br, THP-CH), 5.56 (1H, d, J = 8.2 Hz, CHa),
6.57 (1H, t br, ar-CH), 6.66 (1H, dd, J = 2.8 Hz,
6.9 Hz, ar-CH), 6.83 (2H, d, J = 8.7 Hz, ar-CH), 7.40
(1H, d, J = 8.3 Hz, NH), 7.64 (2H, d, J = 8.7 Hz, ar-
CH), 8.64 (<2H, s, H2N+@C–NH2), 8.89 (<2H, s,
H2N+@C–NH2); MS (ESI) m/z 446.3 ([M+H]+, 100%).
(1H, t br, THP-CH), 5.49 (1H, d, J = 8.2 Hz, CHa),
6.55 (1H, dd, J = 2.9 Hz, 4.6 Hz, ar-CH), 6.65 (1H, dd,
J = 2.8 Hz, 6.9 Hz, ar-CH), 6.77 (2H, d, J = 8.7 Hz, ar-
CH), 7.33 (1H, d, J = 8.3 Hz, NH), 7.48 (2H, d,
J = 8.7 Hz, ar-CH), 8.98 (1H, H2-DEAD -NH); MS
(EI) m/z 428.1 ([M]+).
4.6.2. (4-Carbamimidoyl-phenylamino)-{3-ethoxy-2-fluo-
ro-5-[(R)-(tetrahydro-furan-3-yl)oxy]-phenyl}-acetic acid
(24). Using the same procedure as described for the
preparation of 4 (Section 4.5, second and third reaction
step), 23 (6 g, 14 mmol) was converted to 24 (3.13 g, 54%
over two steps). 1H NMR (300 MHz, DMSO-d6 + 1
drop DCl 20 wt% in D2O) d = 1.29 (3H, t, J = 6.9 Hz,
CH3), 1.98 (1H, m, THP-CH2), 2.23 (1H, m, THP-
CH2), 3.72–3.90 (4H, m, THP-CH2O), 3.95 (2H, q,
J = 6.9 Hz, CH2), 5.08 (1H, t br, THP-CH), 5.46 (1H,
s, CHa), 6.57 (1H, m br, ar-CH), 6.63 (1H, dd,
J = 2.8 Hz, 6.7 Hz, ar-CH), 6.80 (2H, d, J = 8.7 Hz, ar-
CH), 7.63 (2H, d, J = 8.7 Hz, ar-CH), 8.67 (<2H, s,
H2N+@C–NH2), 8.91 (<2H, s, H2N+@C–NH2); MS
(ESI) m/z 418.3 ([M+H]+, 100%).
A solution of (4-carbamimidoyl-phenylamino)-[3-eth-
oxy-2-fluoro-5-(tetrahydro-furan-3-yloxy)-phenyl]-ace-
tic acid ethyl ester hydrochloride (95 mg, 0.20 mmol) in
THF (2.5 ml) was cooled to 0 ꢂC and treated with 1 N
LiOH solution (0.98 mg, 0.98 mmol). The reaction mix-
ture was stirred for 2 h and then neutralized with 1 N
HCl. The precipitate was filtered and dried to give 4
(62 mg, 76%) as a white solid. 1H NMR (300 MHz,
DMSO-d6 + 1 drop DCl 20 wt% in D2O) d = 1.29 (3H,
t, J = 6.9 Hz, CH3), 1.98 (1H, m, THP-CH2), 2.23 (1H,
m, THP-CH2), 3.72–3.90 (4H, m, THP-CH2O), 3.95
(2H, q, J = 6.9 Hz, CH2), 5.08 (1H, t br, THP-CH),
5.46 (1H, s, CHa), 6.57 (1H, m br, ar-CH), 6.63 (1H,
dd, J = 2.8 Hz, 6.7 Hz, ar-CH), 6.80 (2H, d,
J = 8.7 Hz, ar-CH), 7.63 (2H, d, J = 8.7 Hz, ar-CH),
8.67 (<2H, s, H2N+@C–NH2), 8.91 (<2H, s,
H2N+@C–NH2); MS (ESI) m/z 418.3 ([M+H]+, 100%).
4.6.3. (R)-(4-Carbamimidoyl-phenylamino)-{3-ethoxy-2-
fluoro-5-[(R)-(tetra-hydro-furan-3-yl)-oxy]-phenyl}-acetic
acid (5). The epimeric mixture 24 (3.0 g, 7.2 mmol) was
separated by chiral HPLC, using ChiralPak AD as sta-
tionary phase and heptane/isopropanol/TFA 75:25:0.2
as eluent. The two fractions containing the respective
epimers were worked up separately. After distilling off
the eluent, the residue was taken up several times in
H2O and evaporated completely to remove residual
TFA. Then, the remaining solid was taken up in water
and neutralized with 1N LiOH solution. The precipitate
was collected, washed with water and diethyl ether and
dried to give 5 (first eluting fraction, 922 mg, 31%) as
a white solid, along with its epimer (S)-(4-carbamimi-
doyl-phenylamino)-{3-etho-xy-2-fluoro-5-[(R)-(tetrahy-
dro-furan-3-yl)-oxy]-phenyl}-acetic acid (second eluting
fraction, 1.20 g, 40%) as a white solid.
1
Compound 5: H NMR (300 MHz, DMSO-d6 + 1 drop
4.6. Synthesis of (R)-(4-carbamimidoyl-phenylamino)-{3-
ethoxy-2-fluoro-5-[(R)-(tetrahydro-furan-3-yl)oxy]-phen-
yl}-acetic acid (5) (Scheme 3)
DCl 20 wt% in D2O) d = 1.29 (3H, t, J = 6.9 Hz, CH3),
1.98 (1H, m, THP-CH2), 2.23 (1H, m, THP-CH2),
3.72–3.90 (4H, m, THP-CH2O), 3.95 (2H, q,
J = 6.9 Hz, CH2), 5.08 (1H, t br, THP-CH), 5.46 (1H,
s, CHa), 6.57 (1H, m br, ar-CH), 6.63 (1H, dd,
J = 2.8 Hz, 6.7 Hz, ar-CH), 6.80 (2H, d, J = 8.7 Hz, ar-
CH), 7.63 (2H, d, J = 8.7 Hz, ar-CH), 8.67 (<2H, s,
H2N+@C–NH2), 8.91 (<2H, s, H2N+@C–NH2); MS
(ESI) m/z 418.3 ([M+H]+, 100%).
4.6.1. (4-Cyano-phenylamino)-{3-ethoxy-2-fluoro-5-[(R)-
(tetrahydro-furan-3- yl)oxy]-phenyl}-acetic acid ethyl
ester (23). Using the same procedure as described for
the preparation of (4-cyano-phenylamino)-[3-ethoxy-2-
fluoro-5-(tetrahydrofuran-3-yloxy)-phenyl]-acetic acid
ethyl ester (Section 4.5, first reaction step), compound
22 (7.38 g, 20.6 mmol) was reacted with (S)-3-hydroxy-
tetrahydrofurane (1.68 ml, 24.7 mmol), triphenylphos-
phine (6.48 g, 24.7 mmol) and diethyl azodicarboxylate
(3.84 ml, 24.7 mmol) in THF (220 ml) to give 23
(9.37 g, >100%, contains according to 1H NMR
4.7. Synthesis of (R)-(4-carbamimidoyl-phenylamino)-{3-
ethoxy-2-fluoro-5-[(S)-(tetra- hydrofuran-3-yl)oxy]-phen-
yl}-acetic acid trifluoro acetate (6) (Scheme 3)
1
EtOOC-NH-NH-COOEt (H2-DEAD) as impurity). H
4.7.1. (4-Carbamimidoyl-phenylamino)-{3-ethoxy-2-fluo-
ro-5-[(S)-(tetrahydro- furan-3-yl)oxy]-phenyl}-acetic acid
(25). Using the same procedure as described for the
preparation of 24 (Section 4.5), compound 22 was first
reacted with (R)-3-hydroxytetrahydrofurane and then
converted to 25. White solid. 1H NMR (300 MHz,
DMSO-d6 + 1 drop DCl 20 wt% in D2O) d = 1.29 (3H,
NMR (300 MHz, DMSO-d6) d = 1.14 (3H, t,
J = 6.9 Hz, CH3), 1.17 (t, J = 6.9 Hz, H2-DEAD
-CH3), 1.29 (3H, t, J = 6.9 Hz, CH3), 1.98 (1H, m,
THP-CH2), 2.22 (1H, m, THP-CH2), 3.71–3.91 (4H,
m, THP-CH2O), 3.95 (2H, q, J = 6.9, CH2), 4.03 (q,
J = 6.9 Hz, H2-DEAD-CH2), 4.15 (2H, m, CH2), 5.09

5368
K. G. Zbinden et al. / Bioorg. Med. Chem. 14 (2006) 5357–5369
t, J = 6.9 Hz, CH3), 1.98 (1H, m, THP-CH2), 2.23 (1H,
m, THP-CH2), 3.72–3.90 (4H, m, THP-CH2O), 3.95
(2H, q, J = 6.9 Hz, CH2), 5.08 (1H, t br, THP-CH),
5.46 (1H, s, CHa), 6.57 (1H, m br, ar-CH), 6.63 (1H,
dd, J = 2.8 Hz, 6.7 Hz, ar-CH), 6.80 (2H, d,
J = 8.7 Hz, ar-CH), 7.63 (2H, d, J = 8.7 Hz, ar-CH),
8.67 (<2H, s, H2N+@C–NH2), 8.91 (<2H, s,
H2N+@C–NH2); MS (ESI) m/z 418.3 ([M+H]+, 100%).
phenyl-amino]-acetic acid ethyl ester (8.1 g, 17.6 mmol)
was separated by chiral HPLC, using ChiralPak AD
as stationary phase and heptane/isopropanol/TFA
70:30:0.2 as eluent. The two fractions containing the
respective epimers were worked up separately. After dis-
tilling off the eluent, the residue was taken up in EtOAc
and washed with 10% KHCO3 solution and brine. The
organic layer was dried over MgSO4, concentrated and
dried to give 7 (first eluting fraction, 3.0 g, 37%) as an
off-white amorphous solid, along with its epimer
(S)-[5-ethoxy-2-fluoro-3-[(R)-tetrahydro-furan-3-yloxy]-
phenyl]-[4-(N-hydroxycarbamimidoyl)-phenyl-amino]-
acetic acid ethyl ester (second eluting fraction, 3.21 g,
4.7.2. (R)-(4-Carbamimidoyl-phenylamino)-{3-ethoxy-2-
fluoro-5-[(S)-(tetra-hydro-furan-3-yl)-oxy]-phenyl}-acetic
acid trifluoro acetate (6). The epimeric mixture 25
(128 mg, 0.31 mmol) was separated by chiral HPLC,
using ChiralPak AD as stationary phase and heptane/
isopropanol/TFA 75:25:0.2 as eluent. The two fractions
containing the respective epimers were worked up sepa-
rately. After distilling off the eluent, the residue was tak-
en up several times in H2O and evaporated completely
to remove residual TFA. Then, the remaining solid
was taken up in water and lyophilized to give 6 (first
eluting fraction, 66 mg, 41%) as a white amorphous
solid, along with its epimer (S)-(4-carbamimidoyl-phe-
nylamino)-{3-ethoxy-2-fluoro-5-[(S)-(tetrahydro-furan-
3-yl)-oxy]-phenyl}-acetic acid trifluoro acetate (second
eluting fraction, 80 mg, 49%) as a white solid.
1
40%). H NMR (300 MHz, DMSO-d6) d = 1.13 (3H, t,
J = 6.9 Hz, CH3), 1.22 (3H, t, J = 6.9 Hz, CH3), 1.98
(1H, m, THP-CH2), 2.23 (1H, m, THP-CH2), 3.71–
3.89 (4H, m, THP-CH2O), 3.95 (2H, q, J = 6.9 Hz,
CH2), 5.08 (1H,
t br, THP-CH), 5.41 (1H, d,
J = 8.7 Hz, CHa), 5.55 (2H, s br, H2N), 6.55 (1H, d,
J = 8.7 Hz, NH), 6.58–6.65 (4H, m, ar-CH), 7.38 (2H,
d, J = 8.7 Hz, ar-CH), 9.26 (1H, s, OH); MS (ESI) m/z
418.3 ([M+H]+, 100%).
Acknowledgments
1
Compound 6: H NMR (300 MHz, DMSO-d6 + 1 drop
The authors thank Bernd Brodbeck, Louis Burcklen,
Sandra Grall-Ulsemer, Heidi Hoffmann-Maiocchi,
Manfred Mulchi, Caroline Prassler, Noemi Raschetti,
¨
Bettina Roser, Ernst Schaffter, Patricia von Arx and Jit-
ka Weber for their skillfull technical assistance as well as
DCl 20 wt% in D2O) d = 1.29 (3H, t, J = 6.9 Hz, CH3),
1.98 (1H, m, THP-CH2), 2.23 (1H, m, THP-CH2),
3.72–3.90 (4H, m, THP-CH2O), 3.95 (2H, q,
J = 6.9 Hz, CH2), 5.08 (1H, t br, THP-CH), 5.46 (1H,
s, CHa), 6.57 (1H, dd, J = 2.9 Hz, 4.8 Hz, ar-CH), 6.63
(1H, dd, J = 2.9 Hz, 6.8 Hz, ar-CH), 6.81 (2H, d,
J = 8.9, ar-CH), 7.64 (2H, d, J = 8.9 Hz, ar-CH), 8.73
(<2H, s, H2N+@C–NH2), 8.93 (<2H, s, H2N+@C–
NH2); MS (ESI) m/z 418.3 ([M+H]+, 100%).
´
Allan D’Arcy, Andre Alker, Fiona Gruninger, Daniel
¨
Kirchhofer, Dirk Kostrewa and the staff of the Swiss
Norwegian Beam Lines at the ESRF for their respective
contributions to protein production, crystal growth and
data collection.
4.8. Synthesis of (R)-[5-ethoxy-2-fluoro-3-[(R)-tetrahy-
dro-furan-3-yloxy]-phenyl]-[4-(N- hydroxycarbamimi-
doyl)-phenyl-amino]-acetic acid ethyl ester (7) (Scheme 3)
References and notes
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To a solution of 24 (12.46 g, 29.1 mmol) in EtOH
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582 mmol). The reaction mixture was stirred overnight
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car-bamimidoyl)-phenyl-amino]-acetic acid ethyl ester
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CH3), 1.22 (3H, t, J = 6.9 Hz, CH3), 1.98 (1H, m,
THP-CH2), 2.23 (1H, m, THP-CH2), 3.71–3.89 (4H,
m, THP-CH2O), 3.95 (2H, q, J = 6.9 Hz, CH2), 5.08
(1H, t br, THP-CH), 5.41 (1H, d, J = 8.7 Hz, CHa),
5.55 (2H, s br, H2N), 6.55 (1H, d, J = 8.7 Hz, NH),
6.58–6.65 (4H, m, ar-CH), 7.38 (2H, d, J = 8.7 Hz, ar-
CH), 9.26 (1H, s, OH); MS (ESI) m/z 418.3 ([M+H]+,
100%).
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