Structure-based design of amidinophenylurea-derivatives for factor VIIa inhibition

Article abstract of DOI:10.1016/j.bmcl.2004.05.006

The amidinophenylurea scaffold was earlier shown to provide an excellent template for the synthesis of novel and potent inhibitors of the blood coagulation factor VIIa. In this contribution we describe the structure-based design of potent ligands guided by X-ray crystallography, molecular modeling and docking studies. The design and synthetic efforts were directed towards novel modifications to explore the protease binding region close to the S4 subsite.

Full text of DOI:10.1016/j.bmcl.2004.05.006

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3715–3720
Structure-based design of amidinophenylurea-derivatives for
factor VIIa inhibition
Otmar Klingler,* Hans Matter, Manfred Schudok, Monica Donghi, Joerg Czech,
Martin Lorenz, Hans Peter Nestler, Hauke Szillat and Herman Schreuder
Aventis Pharma Deutschland GmbH, Building G878, D-65926 Frankfurt, Germany
Received 3 February 2004; accepted 6 May 2004
Available online
Abstract—The amidinophenylurea scaffold was earlier shown to provide an excellent template for the synthesis of novel and potent
inhibitors of the blood coagulation factor VIIa. In this contribution we describe the structure-based design of potent ligands guided
by X-ray crystallography, molecular modeling and docking studies. The design and synthetic efforts were directed towards novel
modifications to explore the protease binding region close to the S4 subsite.
Ó 2004 Elsevier Ltd. All rights reserved.
Blood coagulation not only plays a crucial role in hae-
mostasis after tissue injury, but also in the pathogenesis
of many thrombotic events. The tissue factor/factor
VIIa (TF/fVIIa) complex is the physiological activator
of the coagulation system and catalyzes the proteolytic
activation of factors X and IX. The activated factor X,
in turn, activates prothrombin to thrombin that con-
verts fibrinogen to fibrin. Since the TF/fVIIa complex is
the key initiator of this process, it is a promising target
for future antithrombotic strategies.1–3
Asp102
His57
O
Thr99
S2
Ser195
Wat
CH3
O
Pro170
B
Ser190
H
N
H
N
S1
O
H
N
N
H
Trp215
O
S4
H
N+
O
R
H
O
Gly216
H
O
Asp189
O
In a previous paper, we reported the identification of a
novel series of amidinophenylureas as potent inhibitors
of factor VIIa.4 The X-ray structure of the factor VIIa/
Gly219
Figure 1. Schematic key protein–ligand interactions of compound 1 to
the fVIIa binding site, as derived from X-ray structure of 1 in complex
with human fVIIa and tissue factor (1, Ki ¼ 0:73 lM).
ꢀ
tissue factor complex with compound 1 at 2.2 A reso-
lution revealed several features, which seem important
for high affinity to factor VIIa (Fig. 1). In addition to
the amidinophenyl binding to Asp189 in S1, both urea
NH’s interact with the active triad Ser195-Oc, either
directly or via a structurally conserved water. This initial
X-ray structure displayed a phenyl ring stacked on top
of the imidazole ring from the active triad His57 directed
towards the factor VIIa S2 pocket. The following opti-
mization revealed that smaller, hydrophobic substitu-
ents fit better into this rather small pocket, while others
also reported polar interactions to key residues within
this pocket.5 The subsequent optimization strategy was
based on this X-ray structure and docking studies using
FlexX and QXP.6;7
Our initial compounds did not place substituents to-
wards the open factor VIIa S4 pocket to gain affinity
and selectivity, as it is now suggested by X-ray crystal-
lography and our binding hypothesis from docking
studies. This binding hypothesis stimulated our search
for more potent factor VIIa inhibitors in this series by
investigating further structural features to explore this
particular pocket.
Keywords: Factor VIIa; Thrombosis; Serine protease.
* Corresponding author. Tel.: +49-(0)69-305-83313; fax: +49-(0)69-
305-21618; e-mail: otmar.klingler@aventis.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.006

3716
O. Klingler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3715–3720
tively, for example, 34 and is in accordance to the
methods described in our previous paper.4
O
H
N
H
N
NH2
OC2H5
a
O
NC
NC
Aminobenzonitrile 2 was reacted with ethoxycarbon-
ylmethyl isocyanate to provide the corresponding urea
3, which was subjected to a Pinner reaction to give
amidinophenylurea 4 and subsequently converted to the
acid 5.8 Coupling of the key intermediate 5 with amine 7
proceeded readily to give compound 34.9 Most of the
amine building blocks were generated analogously
to amine 7 by reductive amination of ketone precursor
6.
2
3
O
H
N
H
N
OR
4: R = C2H5
5: R = H
b, c, d
O
HN
NH2
CH3
O
CH3
O
CH3
CH3
O
O
Our initial studies unveiled the influence of the S2 di-
rected substituent R1, as summarized in Table 1. Bind-
ing affinities for the inhibitors 9–36 are expressed as
inhibition constants Ki towards fVIIa and structurally
related serine proteases.10
e
H2N
O
O
O
6
7
CH3
O
The first compounds in Table 1 support our previous
results.4 Introduction of a methyl group in the benzylic
position (a-position) of compound 9 resulted in the
racemic compound 10 with a marked increase in potency
(10, Ki ¼ 0:077 lM). For selected molecules, both
enantiomers were separated and tested. In general, the
S-enantiomer is more active. This, for example, is indi-
cated by comparing compound 11 and 12. The S-
enantiomer 11 is found to be ꢀ300-fold more potent
than the R-enantiomer 12, which is in agreement with
modeling studies and subsequently confirmed by the X-
ray structure. The small a-methyl substituent of com-
pound 11 in its complex with factor VIIa/tissue factor
thoroughly fits into the S2 pocket.
O
CH3
H
H
N
N
N
H
O
O
f
O
HN
5
NH2
34
Scheme 1. Synthesis of compound 34. Reagents: (a) ethoxycarbonyl-
methyl isocyanate, DMF; (b) HCl, EtOH; (c) NH3, EtOH; (d) NaOH,
H2O; (e) MeOH, NaCNBH3, NH4OAc; (f) TOTU, DMF, N-ethyl-
morpholine.
The synthetic pathway used for the preparation of the
compounds 1–36 is outlined in Scheme 1 representa-
Table 1. In vitro activity for compounds 9–17
O
H
H
N
R1
N
N
H
O
HN
NH2
Compounds
R1
Ki (lM)
fVIIa
0.97
fXa
Thrombin
10.1
Cl
Cl
Cl
Cl
9
14.5
CH3
10
0.077
0.026
ND
6.23
ND
3.18
CH3
CH3
CH3
11
12
13
7
ND
ND
50.7
0.43
>10
CH3
Br
14
0.027
>10
15.9

O. Klingler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3715–3720
3717
Table 1 (continued)
Compounds
R1
Ki (lM)
fVIIa
0.156
fXa
Thrombin
6.76
CH3
15
16
>10
Br
CH3
O
CH3
0.035
0.256
>10
18.2
ND
H3C
17
ND
O
ND: not determined.
This encouraging result prompted us to design a novel
series of S4 directed amidinophenyl ureas containing the
essential methyl group in a-position. Since the S4 pocket
was found to be relatively flat, solvent exposed and
featureless, a diverse set of substituents at the a-methyl
benzylamide core was selected to explore this region.
Ser214-Trp215-Gly216-Gln217 b-sheet in the factor
VIIa binding site and confirms that small and hydro-
phobic substituents are well accepted in the meta-posi-
tion. The meta-methoxy substituent in 16 still can
undergo favourable hydrophobic interactions (Ki ¼
0:035 lM), while the bulkiness of the larger meta-ben-
zyloxy substituent in 17 decreases affinity to 0.256 lM.
Compared to the a-S-methyl benzylamide 13
(Ki ¼ 0:43 lM), the replacement of phenyl by naphthyl
yielded a marked increase in binding affinity of com-
pound 11 (Ki ¼ 0:026 lM), which may be attributed to
favourable hydrophobic interactions of the second aro-
A second hydrophobic or polar ring R2 was introduced
in para-position of the a-methyl benzylamide by direct
link or linkers like carboxamides and ethers. The
biological activities of these derivatives are given in
Table 2.
ꢀ
matic ring in the factor VIIa binding site area 4.5 A from
Gly216. This is consistent with the finding that bromine
at this position in 14 also increases affinity
(Ki ¼ 0:027 lM) while bromine in para-position led to
compound 15 with a Ki of 0.156 lM. The subsequent
exploration by additional meta- and para-substituted
analogues revealed steric constraints imposed by the
The para-benzyloxy derivative 18 (Ki ¼ 0:133 lM) and
para-phenyloxy derivative 19 (Ki ¼ 0:078 lM) demon-
strate the favourable effect of larger hydrophobic sub-
stituents directed towards S4 in comparison to 13. The
replacement of the ether oxygen in 19 by CH2 in 20
Table 2. In vitro activity for compounds 18–33
O
CH3
H
N
H
N
N
H
HN
O
R2
NH2
Compounds
R2
Ki (lM)
fVIIa
fXa
Thrombin
ND
18
0.133
ND
O
O
19
0.078
>10
9.1
20
21
0.190
0.083
ND
10.7
ND
9.8
O
O
CH3
O
Cl
22
0.483
ND
ND
(continued on next page)
Cl

3718
O. Klingler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3715–3720
Table 2 (continued)
Compounds
R2
Ki (lM)
fVIIa
0.023
fXa
Thrombin
2.3
O
23
>10
Cl
O
O
N
24
25
0.020
0.142
>10
>10
2.3
3.2
CH3
O
H
N
26
27
28
29
0.034
0.020
0.012
0.026
4.6
14.8
12.1
33.2
10.9
O
O
>10
>10
>10
N
H
H
N
O
O
H
N
CH3
O
O
O
30
31
0.043
0.196
>10
>10
11.9
N
H
H
N
CH3
>50
O
H
N
32
0.047
0.013
>10
>10
14.5
68.5
O
O
H
N
NH
33
ND: not determined.
reduces binding affinity to 0.19 lM. Affinity could not
be improved by introduction of further hydrophobic
residues in 19 leading to the phenyloxy derivatives 21
and 22. However, as demonstrated by 23 and 24,
further improvement in potency is observed when
modifications are carried out with benzyloxy analogue
18. The meta-chloro and methoxy derivatives 23 and 24
turned out to be approximately 6-fold more potent than
its analogue 18. This is in agreement with our models,
suggesting that additional hydrophobic substituents
preferably at the meta- or para-position of the distal
benzyloxy ring of 18 interact with another hydrophobic
subpocket on top of Ser170H-Pro170I. Additional
substituents at the meta-position of the benzylamide ring
in 18 also improved binding affinity, as exemplified for
34 (Table 3) with a Ki value of 0.033 lM in comparison
to 18 (0.133 lM).
in meta-position of the central ring increase activity as
illustrated by compounds 35 and 36.
Changing the benzylether linker to a carboxamide
slightly increase activity as demonstrated by compounds
26–33. In addition, the high potency of 33 suggests
favourable contributions by additional polar interaction
at the edge of the factor VIIa binding pocket.
The complex of 29 with human factor VIIa/tissue factor
ꢀ
was solved by X-ray structure analysis at 2.7 A (Fig. 2),
thus validating our docking hypothesis about filling the
neighbourhood of the serine protease S4 subsite by
hydrophobic substituents.12 No major changes in the
binding mode for the amidinophenylurea moiety of 29
were observed, compared to our previously reported
X-ray structure.4 However, the S2 pocket is now filled with
a smaller, more favourable methyl substituent, which
slightly affects the position of the neighbouring ligand
amide bond and additionally tightens the central phenyl
ring system. This central phenyl group is well ordered in
Interestingly, the more polar morpholine 25 (Table 2)
displayed similar activity (Ki ¼ 0:142 lM) than the
unsubstituted ether 18. Again, hydrophobic substituents

O. Klingler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3715–3720
Table 3. In vitro activity for compounds 34–36
3719
O
H
N
H
N
R3
N
H
O
HN
NH2
Compounds
R3
Ki (lM)
fVIIa
0.033
fXa
Thrombin
0.5
O
OMe
O
34
>10
H3C
H3C
O
O
N
N
35
36
0.015
0.030
>10
>10
0.65
1.9
Br
H3C
CF3
ꢀ
Figure 2. X-ray structure of compound 29 in factor VIIa/tissue factor complex at 2.7 A resolution. Structural water molecules are indicated by cyan
coloured spheres. The protein cavity on the right is represented by a MOLCAD surface coloured by subpocket depth.11
our electron density maps, in contrast to the crystal
structure of the complex with 1, where this phenyl group
showed significant disorder. The added meta-methyl-
phenyl residue does not enter the S4 pocket, but points
straight ahead towards the factor VIIa loop encom-
passing amino acids 170B–170I. The amide carbonyl
group has favourable interactions with a bound water
molecule, which, in turn, is fixed by hydrogen bonds to
the side chain hydroxyl and main chain nitrogen of Thr99
separating S2 and S4. This additional water mediated
hydrogen bond might explain the increasing effect on
affinity by replacing ether by amide linkage. The corre-
sponding amide nitrogen is also involved in a hydrogen
bond with a structurally conserved water molecule in the
solvent exposed binding site region. The second methyl
group again accommodates the small hydrophobic re-
gion on top of Ser170H-Pro170I, while the distal phenyl
group is favourably stacked on top of Pro170I.
inhibitors with variations directed towards the serine
protease S4 pocket and its vicinity. The best compounds
(24, 27, 28, 33, 35) show Ki values between 12 and 20 nM
without significant inhibition of the related serine pro-
teases factor Xa and thrombin. The use of structure-
based design and modeling in close combination
with parallel synthesis allowed us to explore rapidly
structural requirements for this particular factor VIIa
binding site subregion, demonstrating that the amidin-
ophenylurea scaffold indeed constitutes a promising
series for factor VIIa inhibition.
Acknowledgements
The authors would like to thank Peter Below, Silke
Herok, Iris Rein, Alexander Liesum and Andrea
Struppmann for their technical assistance.
In summary, we have extended the structure–activity
relationship of an interesting series of factor VIIa

3720
O. Klingler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3715–3720
mogenic assay as described in: Ostrem, J. A.; Al-Obeidi,
References and notes
F.; Safar, P.; Safarova, A.; Stringer, S. K.; Patek, M.;
Cross, M. T.; Spoonamore, J. C.; LoCascio, J. C.;
Kasireddy, P.; Thorpe, D. S.; Sepetov, N.; Lebl, M.;
Wildgoose, P.; Strop, P. Biochemistry 1998, 37, 1053–
1059.
1. Raghavan, S. A. V.; Dikshit, M. Drugs Future 2002, 27,
669–683.
2. Ruef, J.; Katus, H. A. Expert. Opin. Invest. Drugs 2003,
12, 669–683.
11. Heiden, W.; Goetze, T.; Brickmann, J. J. Comput. Chem.
1992, 14, 246–250.
12. The factor VIIa-tissue factor complex was crystallized in
hanging drops at pH 6.5 using conditions described by:
Kirchhofer, D.; Guha, A.; Nemerson, Y.; Konigsberg, W.
3. Golino, P. Thromb. Res. 2002, 106, V257–V265.
4. Klingler, O.; Matter, H.; Schudok, M.; Bajaj, S. P.; Czech,
J.; Lorenz, M.; Nestler, H. P.; Schreuder, H.; Wildgoose,
P. Bioorg. Med. Chem. Lett. 2003, 13, 1463–1467.
5. South, M. S.; Case, B. L.; Wood, R. S.; Jones, D. E.;
Hayes, M. J.; Girard, T. J.; Lachance, R. M.; Nicholson,
N. S.; Clare, M.; Stevens, A. M.; Stegeman, R. A.;
Stallings, W. C.; Kurumbail, R. G.; Parlow, J. Bioorg.
Med. Chem. Lett. 2003, 13, 2319–2325.
6. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol.
Biol. 1996, 261, 470–489.
7. McMartin, C.; Bohacek, R. S. J. Comput. Aided Mol. Des.
1997, 11, 333–344.
ꢁ
H.; Vilbois, F.; Chene, C.; Banner, D.; D’Arcy, A. Prot.
Struct. Funct. Gen. 1995, 22, 419–425. Data were collected
in-house on a Mar345 image plate detector mounted on
a Nonius FR571 rotating anode generator, equipped
with an Osmic multilayer mirror system. Data were
processed with XDS: Kabsch, W. J. Appl. Cryst. 1993,
ꢀ
26, 795–800. The crystal diffracted to 2.7 A with an
overall Rsym of 10.7%. The space group was P212121
ꢀ
ꢀ
ꢀ
with a ¼ 69:98 A, b ¼ 81:84 A and c ¼ 126:43 A. Initial
phases were calculated using the structure of the complex
with 14 as starting model, based on the original struc-
ture by Banner et al.13 The inhibitor was fitted
using Quanta and refined using CNX (Accelrys). The
final R-factor was 17.3% (R-free ¼ 24.3%). The com-
plete inhibitor is well defined in the electron density
maps.
8. (a) Pinner, A.; Klein, F. Chem. Ber. 1878, 11, 1825; (b)
Gautier, J. A.; Miocque, M.; Farnoux, C. C. In The
Chemistry of Amidines and Imidates; Patai, S., Ed.; Wiley:
New York, 1975; pp 283–348.
9. TOTU: O-[Ethoxycarbonyl)cyanomethylenamino]-N,N,
N0,N0-tetramethyl-uronium tetrafluoro-borate; Konig,
€
W.; Breipohl, G.; Pokorny, P.; Birkner, M. In Proceedings
of the 21st European Peptide Symposium 1990; Giralt, E.,
Andren, D., Eds.; ESCOM, Leiden, 1990; pp 143.
13. Banner, D.; D’Arcy, A.; Chene, C.; Winkler, F. K.; Guha,
A.; Konigsberg, W. H.; Nemerson, Y.; Kirchhofer, D.
Nature 1996, 380, 41–46.
10. Inhibitory activity expressed as inhibition constant Ki
towards fVIIa/tissue factor was determined using a chro-