Small molecule inhibitors of plasma kallikrein

Article abstract of DOI:10.1016/j.bmcl.2005.12.060

Plasma kallikrein is a serine protease that is involved in pathways of inflammation, complement fixation, coagulation, and fibrinolysis. Herein, we describe the SAR and structural binding modes of a series of inhibitors of plasma kallikrein as well as the

Full text of DOI:10.1016/j.bmcl.2005.12.060

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2034–2036
Small molecule inhibitors of plasma kallikrein
*
Wendy B. Young, Roopa Rai, William D. Shrader, Jana Burgess-Henry,
Huiyong Hu, Kyle C. Elrod, Paul A. Sprengeler, Bradley A. Katz,
Juthamas Sukbuntherng and Joyce Mordenti
Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 10 November 2005; revised 13 December 2005; accepted 15 December 2005
Available online 18 January 2006
Abstract—Plasma kallikrein is a serine protease that is involved in pathways of inflammation, complement fixation, coagulation,
and fibrinolysis. Herein, we describe the SAR and structural binding modes of a series of inhibitors of plasma kallikrein as well
as the pharmacokinetics of a lead analog 11 in rat.
Ó 2006 Elsevier Ltd. All rights reserved.
Plasma kallikrein (p-kallikrein) is a serine protease
which plays a central role in the contact activation and
Table 1. SAR of selected analogs in the 5-amidino-2-(2-hydroxy-
9
biphenyl-3-yl)-benzimidazole series
1
,2
kinin generation pathways. Once p-kallikrein is acti-
vated from the zymogen precursor prekallikrein (Flet-
CO2H
HO2C
3
5'
chor Factor), it initiates surface-mediated activation
of coagulation, fibrinolysis, and kinin generation. Once
activated, p-kallikrein levels are controlled by endoge-
R1
3'
R2
R3
N
H N
2
nous C1 inhibitor as well as a -macroglobulin and anti-
NH OH
2
4
thrombin III. An imbalance between p-kallikrein and
its naturally occurring plasma inhibitors is associated
HN
1
2
R
3
R
5
Compound R
p-kall
(nM)
Selectivity for p-kall
versus
with several disease states, such as hereditary angioede-
K
i
ma (HAE), inflammatory bowel disease (IBD), systemic
lupus, rheumatoid arthritis (RA), allergic rhinitis, and
others. Accordingly, research groups have pursued the
development of inhibitors of p-kallikrein as potential
Xa
VIIa Trypsin
1
2
3
4
5
6
7
8
9
0
1
2
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8
13
6
300
460
9
1
830
1075
2710
500
6
,7
therapeutics. To date, no small molecule inhibitors
have made it to market, although large proteins are
currently in clinical trials for the treatment of HAE
OMe H
F
1190 22
200 12
110 10
1200 30
780 25
H
12
6
Me
H
H
H
H
H
H
H
H
H
H
H
H
140
7
OH
OMe
COCH
Me
3
2900
1430
1015
1075
530
and for reduced blood loss during surgery.
5
3
6
375
600 10
150
5
While developing inhibitors for our internal protease
8
programs, we routinely screened analogs against p-kal-
likrein. In doing so, we discovered many compounds in
several different structural classes which were very po-
tent against this enzyme. In particular, analogs of the
4
10
1
1
1
CN
CONH
7
2
0.5
10,300 200 23,600
Cl
H
H
H
H
3
530 22
190 14
250 85
240 25
80 12
1300
135
75
13
14
15
OH 65
OMe 27
Me 18
5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1)
scaffold stood out as the most potent and selective.
200
25
1
6
Cl
37
Table 1 outlines the SAR of 16 selected compounds with
small substituents at the ortho, meta, and para positions
on the 3 -phenyl ring of scaffold 1. The inhibition of all
Keywords: Plasma kallikrein; p-kallikrein; Serine protease; Coagula-
tion; Fibrinolysis; Kinin generation; Small molecule inhibitor.
0
9
*
Corresponding author. E-mail: wendy.young@yahoo.com
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.060

W. B. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2034–2036
2035
sixteen compounds for p-kallikrein is quite good, with
inhibition constants (K ) ranging from 0.5 nM (analog
The pharmacokinetic properties of 11 were evaluated
after intravenous (iv) bolus administration to male Spra-
i
1
3
1
1) to 65 nM (analog 13). This structural class also
gue–Dawley rats (1 mg/kg).
The pharmacokinetic
potently inhibits a related enzyme factor VIIa (fVIIa).
Consequently, there is only a 1- to 200-fold separation
between the activity for p-kallikrein and fVIIa within
these selected analogs. The preference for p-kallikrein
versus factor Xa (fXa) and trypsin is fairly impressive
with roughly a 100- to 1000-fold potency separation
for most analogs. Although not noted in Table 1, the
selectivity versus another related enzyme, thrombin,
was >1000-fold for all analogs, with most being
parameters of this evaluation are provided in Table 2.
Analog 11 has impressive preclinical pharmacokinetics,
displaying low plasma clearance, a small volume of
distribution, and a long mean residence time (almost
10 h)-ideal characteristics for a drug targeting blood
components. These data suggest that once-a-day dosing
in human is possible.
Analog 11 was synthesized as outlined in Scheme 1.
Starting from the commercially available 4-iodoanisole
(17), the succinate moiety was inserted via a Heck
>100,000-fold. Within this set of compounds, analog
11 is the most potent for p-kallikrein and has the best
selectivity profile versus fXa, fVIIa, and trypsin.
We recently reported the first published crystal struc-
tures of human plasma kallikrein (complexed with ben-
Table 2. Pharmacokinetics of 11 in male rats (n = 3) following iv bolus
13
administration of 1 mg/kg
Pharmacokinetic parameter
max (lM) at 2 min
CL (mL/min/kg)
˚
zamidine at 1.85 A and 1.40 A resolution two crystal
10
˚
11
a
Mean (±SD)
1
2
forms). P-kallikrein adopts a fold typical of a trypsin-
like serine protease, with the catalytic triad, Asp102,
His57, and Ser195 at the active site, and Asp189 at the
S1 site (Fig. 1). Based on crystal structures of other tryp-
sin-like proteases bound by inhibitors similar to those
C
17.8 (1.3)
0.54 (0.03)
71 (6)
V
V
c
(mL/kg)
ss (mL/kg)
319 (5)
591 (41)
MRT (min)
AUC (lM*min)
a-t1/2 (min) [%AUC]
b-t1/2 (min) [%AUC]
c-t1/2 (min) [%AUC]
8
reported here, we constructed a model of 11 in p-kalli-
3390 (183)
2 (0.1) [1]
30 (5) [8]
449 (36) [91]
krein (Fig. 1). In this model, the amidine makes hydro-
gen bonds with Asp189 at the S1 site, the phenolate with
His57 and Ser195 at the active site, and the succinate
with Lys192. From the model shown in Figure 1 or
other models of 11 in fXa, fVIIa, and trypsin (not
shown), it is not immediately apparent what elements
are responsible for the differences in binding affinity be-
tween these enzymes for this scaffold. The excellent
selectivity observed against fIIa, however, can be ratio-
a
C
max, maximum plasma concentration at first sampling time (2 min);
CL, plasma clearance; V , volume of central compartment; Vss
volume of distribution at steady state; MRT, mean residence time;
AUC, area under the plasma concentration versus time curve; t1/2
c
,
,
half life for the three exponential phases (a, b, c); and %AUC, por-
tion of AUC associated with each half life.
nalized by the negative electronic interaction of the
0
5 -acid on the inhibitors with Glu192 of thrombin. In
addition, the model suggests that the increased potency
CO
2
Me
2
CO Me
I
MeO
C
MeO C
2
2
and selectivity observed for compound 11 is due to a
0
a-d
e-f
specific interaction between the 3 benzamide and
Asp60 in p-kallikrein.
H
O
30%
75%
Br
OMe
OH
18
OH
17
19
CN
2
0
CO
Me
21
2
MeO
2
C
CN
(
2
HO) B
h
g
H
O
OH
88%
95%
2
CO H
2
HO C
N
R
10: R=CN
11: R = CONH
2
i
HN
NH OH
2
H N
Scheme 1. Reagents and conditions: (a) dimethylfumarate, PdCl
NEt , MeCN; (b) Pd(OH) /H , MeOH; (c) HBr, reflux; (d) SOCl
MeOH; (e) paraformaldehyde, MgCl
cinimide, DMF; (g) Pd(PPh , Na
benzamidine-HCl, O , EtOH; (i) 2 M NaOH.
2
,
,
3
2
2
2
2
, TEA, MeCN; (f) N-bromosuc-
CO , toluene; (h) 3,4-diamino-
3
)
4
2
3
Figure 1. Model of 11 in plasma kallikrein.
2

2036
W. B. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2034–2036
reaction with dimethylfumarate and the resulting olefin
was reduced by catalytic hydrogenation. Subsequent
treatment with HBr followed by reesterification of the
acid moieties led to diester 18. Next, compound 18
was treated with magnesium chloride and paraformalde-
hyde followed by reaction with n-bromosuccinimide to
give aryl bromide 19. A Suzuki reaction of 19 and 20
afforded the biaryl analog 21. The benzimidazole was
then installed by treatment of 21 with 3,4-diaminobenz-
amidine to give 10. Finally, the nitrile of 10 was hydro-
lyzed to the amide with sodium hydroxide and the
resulting crude material was purified by reverse-phase
HPLC to give 11 as a racemic mixture of enantiomers.
6. Okada, Y.; Tsuda, Y.; Tada, M.; Wanaka, K.; Okamoto,
U.; Hijikata-Okunomiya, A.; Okamoto, S. Chem. Pharm.
Bull. 2000, 48, 1964.
7
. As of this writing, Dyax and Genzyme are codeveloping
DX-88, a proteinacious kallikrein inhibitor, which is in
Phase II for the treatment of HAE and for reduced
bleeding during Coronary Artery Bipass Graft (CABG)
surgery.
8
. Young, W. B.; Mordenti, J.; Torkelson, S.; Shrader, W.
D.; Kolesnikov, A.; Rai, R.; Liu, L.; Hu, H.; Leahy, E.
M.; Green, M. J.; Sprengeler, P. A.; Katz, B.; Yu, C.; Janc,
J.; Elrod, K. Marzec, U.; Hanson, S. Biorg. Med. Chem.
Lett. in press; Shrader, W. D.; Kolesnikov, A.; Burgess-
Henry, J.; Rai, R.; Hendrix, J.; Hu, H.; Torkelson, S.;
Ton, T.; Young, W. B.; Katz, B. A.; Yu, C.; Cabuslay, R.;
Sanford, E.; Janc, J.; Sprengeler, P. Biorg. Med. Chem.
Lett. in press; Young, W. B.; Kolesnikov, A.; Rai, R.;
Sprengeler, P. A.; Leahy, E. M.; Shrader, W. D.; Sanga-
lang, J.; Burgess-Henry, J.; Spencer, J.; Elrod, K.; Cregar,
L. Bioorg. Med. Chem. Lett. 2001, 11(17), 2253; Katz, B.
A.; Elrod, K.; Luong, C.; Rice, M.; Mackman, R. L.;
Sprengeler, P. A.; Spencer, J.; Hatay, J.; Janc, J.; Link, J.;
Litvak, J.; Rai, R.; Rice, K.; Sideris, S.; Verner, E.;
Young, W. B. J. Mol. Biol. 2001, 307(5), 1451; Katz, B. A.;
Elrod, K.; Verner, E.; Mackman, R. L.; Luong, C.;
Shrader, W. D.; Sendzik, M.; Spencer, J. R.; Sprengeler, P.
A.; Kolesnikov, A.; Tai, V. W.; Hui, H. C.; Breitenbucher,
J. G.; Allen, D.; Janc, J. J. Mol. Biol. 2003, 329, 93.
. Inhibition assays for factor Xa and thrombin were
performed as described (Cregar, L.; Elrod, K. C.; Putnam,
D.; Moore, W. R. Arch. Biochem. Biophys. 1999, 366, 125)
with the pH adjusted to 7.4. The plasma kallikrein,
trypsin, and fVIIa assays were performed and analyzed as
in the above reference with the following additional
details. Factor VIIa (Enzyme Research) was incubated at
In conclusion, a series of potent 5-amidino-2-(2-hy-
droxy-biphenyl-3-yl)-benzimidazole inhibitors of plas-
ma kallikrein were identified. Within this series,
analog 11 demonstrated the best potency and selectiv-
ity profile for plasma kallikrein versus related serine
proteases. The pharmacokinetic parameters after iv
dosing to rat indicate that this compound is highly
stable in vivo and could be further developed for
the treatment of inflammatory or coagulation
disorders.
9
Acknowledgments
The authors thank Robert Booth, Michael Green, Peter
Young, and Gregory Jackson for helpful scientific dis-
cussions, and Nancy Fadis and Barbara Nielsen for
aid in preparing the manuscript.
7
nM and CH SO -D-CHA-But-Arg-pNA (Centerchem)
3 2
was used as the substrate. The buffer for the factor VIIa
assay was supplemented with 11 nM relipidated tissue
factor and 5 mM CaCl2. Trypsin (Athens Research Insti-
tute) was incubated at 10 nM with variable concentrations
of inhibitor in 50 mM Tris (pH 7.4), 150 mM NaCl,
1.5 mM EDTA, 0.05% Tween 20, and 10% DMSO. The
reaction was initiated with substrate, Tosyl-Gly-Pro-Lys-
pNA (Centerchem), supplied at the Km (25 lM). Plasma
Kallikrein (Athens Research Institute) was incubated at
540 pM with variable concentrations of inhibitor in
50 mM Tris (pH 7.4), 150 mM NaCl, 500 lM EDTA,
0.05% Tween 20, and 10% DMSO. The reaction was
initiated with substrate, Pro-Phe-Arg-AMC (Bachem),
supplied at the Km (350 lM).
References and notes
1
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2
3
1965, 26, 521.
4
5
. Bhoola, K. D.; Figuera, C. D.; Worthy, K. Pharmacol.
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Med. Res. 2004, 35, 369; Stadnicki, A.; Pastucha, E.;
Nowaczyk, G.; Mazurek, U.; Plewka, D.; Machnik, G.;
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Liver Physiol. 2005, 289, G361; Davis, A. E. Clin.
Immunol. 2005, 3; Colman, R. W. Immunopharmacol
10. The PDB access code for the X-ray coordinates is 2ANW.
11. The PDB access code for the X-ray coordinates is 2ANY.
12. Tang, J.; Yu, C.; Williams, S.R.; Springman, E.; Jeffreys,
D.; Sprengeler, P.; Estevez, A.; Sampang, J.; Shrader, W.;
Spencer, J.; Young, W.; McGrath, M.; Katz, B. J. Mol.
Biol. in press.
13. Plasma concentrations of 11 were assayed by LC/MS/MS
(LOQ = 0.00457 lM). Pharmacokinetic data were ana-
lyzed by WinNonlin-Pro (Pharsight Corp.), using a three-
compartment model.
1
999, 43, 103; Colman, R. W.; Sartor, R. B.; Adam, A.
A.; DeLa Cadena, R. A.; Stadnick, A. Clin. Rev. Allergy
Immunol. 1998, 16, 365.