Synthesis and in vitro biological evaluation of aryl boronic acids as potential inhibitors of factor XIa

Article abstract of DOI:10.1016/j.bmcl.2006.07.043

A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal structures of the protein-inhibitor complexes led to the design and synthesis of second generation compounds showing single digit m

Full text of DOI:10.1016/j.bmcl.2006.07.043

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5022–5027
Synthesis and in vitro biological evaluation of aryl boronic acids as
potential inhibitors of factor XIa
Tsvetelina I. Lazarova,a,* Lei Jin,b Michael Rynkiewicz,b Joan C. Gorga,c Frank Bibbins,c
Harold V. Meyers,a Robert Babineb and James Stricklerc
aMedicinal Chemistry, Daiichi Asubio Medical Research Laboratories, LLC, One Kendall Square,
Building 700, Cambridge, MA 02139, USA
bStructural Chemistry, Daiichi Asubio Medical Research Laboratories, LLC, One Kendall Square,
Building 700, Cambridge, MA 02139, USA
cBiochemistry, Daiichi Asubio Medical Research Laboratories, LLC, One Kendall Square, Building 700,
Cambridge, MA 02139, USA
Received 6 June 2006; accepted 14 July 2006
Available online 28 July 2006
Abstract—A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal
structures of the protein–inhibitor complexes led to the design and synthesis of second generation compounds showing single digit
micromolar inhibition against FXIa and selectivity against thrombin, trypsin, and FXa.
Ó 2006 Elsevier Ltd. All rights reserved.
Thromboembolitic diseases are a major cause of death
and disability in the western world.1 Current antithrom-
botic therapies rely on the use of anticoagulant agents,
such as heparin, low molecular weight heparins
(LMWH), and warfarin, as well as antiplatelet agents
such as aspirin. Some of the limitations of these thera-
pies are a lack of oral bioavailability (heparin, LMWH)
and the need to constantly monitor blood parameters
(warfarin).2 Most of the current research efforts have fo-
cused on the development of inhibitors of thrombin and
factor Xa (FXa), and much progress has been reported
in these research areas, including compounds in
advanced clinical development.3–5
rently there are few reports concerning FXIa as a poten-
tial target for the development of small molecule
antithrombotic drugs.8 Since FXIa plays a role in the
amplification pathway in coagulation and not in initia-
tion of clotting, an inhibitor specific for FXIa rather
than for the other coagulation pathway proteases, such
as FXa and thrombin, may be effective at reducing the
risk of thrombosis without a significant risk of bleeding. In-
deed, in a recent publication, FXIa null mice (fXIÀ/À)
were found to be resistant to clot induction by FeCl3
in an arterial thrombosis model, but had normal bleed-
ing times.9 In this letter, we report the synthesis and
in vitro biological activity of aryl boronic acid deriva-
tives as potential inhibitors of FXIa.
In our laboratories, we have focused on a relatively
unexplored target in the blood coagulation cascade,
namely factor XIa (FXIa).6 Factor XI is a serine prote-
ase expressed as a zymogen that is converted to its active
form, FXIa, by factor XIIa and by thrombin. Upon
activation, FXIa promotes coagulation by activating
factor IX. There is evidence that a high level of FXIa
increases the risk for venous thrombosis,7 although cur-
Aryl boronic acids have found applications in diverse
chemical areas, such as organometallic chemistry,
organic synthesis, host–guest chemistry, and medicinal
chemistry. Simple phenyl boronic acids,10 as well as
some functionalized arylboronic acids11 have been
shown to have weak serine protease inhibitory activity.
More potent protease inhibition has been observed for
boronic acids incorporated within a peptidomimetic
framework.12 These boronic acid peptidomimetics can
function as covalent reversible or irreversible inhibitors
of the protease by forming a tetrahedral boronate ester
with the serine hydroxyl of the catalytic triad. A number
of peptidyl boronate inhibitors have been reported,13 the
Keywords: Aryl boronic acids; Factor XIa; Factor Xa; Thrombin;
Trypsin.
*
Corresponding author. Tel.: +1 617 470 6276; fax: +1 781 435
1112; e-mail: tlazarova@medchempartners.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.043

T. I. Lazarova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5022–5027
5023
most promising of which is VelcadeÒ, a selective boro-
nate proteosome inhibitor approved as an antineoplastic
agent.14
In our laboratory, we began the investigation of aryl
boronic acids as potential inhibitors of FXIa with the
screening of commercially available substituted phenyl
boronic acids. We selected compounds with hydrogen
bond donor substituents on the phenyl ring that could
make an electrostatic interaction with aspartate 189 of
the S1 specificity pocket. All compounds were tested in
an in vitro enzyme inhibition assay15 against FXIa,
and boronic acid 1 emerged as the only compound with
an IC50 value in the range of that of the control com-
Scheme 1. Reagents and conditions: (a) 1 equiv 6, 1 equiv i-Pr2EtN,
DMF, rt, 18 h, 90%; (b) TFA, rt, 5 h, 87%; (c) 1 equiv pinacol, 1 equiv
i-Pr2EtN, dioxane, 60 °C, 18 h, 91%.
pound benzamidine
2
(IC50 1 = 77.3 lM, IC50
2 = 120 lM, respectively).
Scheme 2. Reagents and conditions: (a) 1 equiv dipinacolato diboron,
3 equiv KOAc, 0.1 equiv Pd(dppf)Cl2, DMSO, 80 °C, 18 h, 67%; (b)
4 N HCl, rt, 30 min, 96%; (c) 1 equiv 6, 1 equiv i-Pr2EtN, DMF, rt,
18 h, 88%; (d) TFA, rt, 5 h, 94%.
We selected compound 1 for further studies. In order to
maximize the interaction of 1 with aspartate 189, the
methylene amine group was converted to a guanidinium
group. At the same time, the length of the linker be-
tween the guanidinium group and the phenyl ring was
varied in order to find the optimum spatial arrangement
for interaction with both serine 195 and aspartate 189.
For that purpose, we prepared boronic acids 3, 4, and
5. For ease of manipulation, we chose to isolate the
compounds as boron pinacol esters. This functional
group modification was not expected to interfere with
the ability of the boron to form the covalent complex
with the active site serine 195, since X-ray crystallo-
graphic studies had previously shown that the ester moi-
ety is hydrolyzed prior to binding (due to the rapid
equilibration between the boronic ester and the free
boronic acid in solution).16
Scheme 3. Reagents and conditions: (a) 1 equiv 6, 1 equiv i-Pr2EtN,
DMF, rt, 18 h, 86%; (b) TFA, rt, 5 h, 90%.
as a TFA salt in 78% overall yield. Treatment of 3 with
1 equiv of pinacol and 1 equiv of diisopropylethyl amine
in dioxane for 18 h at 60 °C afforded the pinacol ester 7
in 91% yield.
The preparation of the pinacol ester of boronic acid 4 is
outlined in Scheme 2. Miyaura boronylation17 of the
commercially available phenyl bromide 8 with 1 equiv
of dipinacolato diboron, 3 equiv of potassium acetate,
and 10 mol% palladium diphenylphosphoferrocene
dichloride (Pd(dppf)Cl2) in dimethylsulfoxide (DMSO)
for 18 h at 80 °C afforded the boron pinacol ester in
67% yield after a flash chromatography purification. A
subsequent treatment of the pinacol ester with 4 N
hydrochloric (HCl) acid for 30 min at ambient tempera-
ture gave the amine salt 9 in 96% yield. The introduction
and deprotection of the guanidinium group was accom-
plished in a two-step procedure, similar to that for com-
The syntheses of the pinacol esters of acids 3, 4, and 5
are shown in Schemes 1–3, respectively. In Scheme 1,
boronic acid 1 was treated with 1 equiv of the bis
BOC-protected guanidinylation reagent 6 and 1 equiv
of diisopropylethyl amine in dimethylformamide
(DMF) for 18 h at ambient temperature, followed by
deprotection with trifluoroacetic acid (TFA) for 5 h at
ambient temperature to give the desired compound 3

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T. I. Lazarova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5022–5027
pound 3 (Scheme 1) and the pinacol ester 10 was isolated
as the TFA salt in 83% overall yield for the last two-
steps.
nidine group in compound 12 was in a different posi-
tion than the guanidine groups in compounds 3 and
10 and formed only one hydrogen bond with aspartate
189 instead of two, yet the location of the nitrogen in-
volved in the hydrogen bonding was very close to the
location of the nitrogen atoms in compounds 3 and
10. The phenyl ring of compound 12 adopted a third
orientation in the S1 pocket. This structural informa-
tion suggested that the meta position of the phenyl ring
(relative to the boronic ester) or the benzylic carbon in
compounds 3 and 10 provided good opportunities to
access a small pocket (located just above the disulfide
bond between cysteine 191 and cysteine 219) near the
S1 subsite that is specific for FXIa.
Scheme 3 shows the preparation of the pinacol ester of
boronic acid 5. Commercially available 4-amino phenyl
boronic pinacol ester 11 was converted to the target
pinacol ester 12 in 77% overall yield by the same two-
step procedure used in the preparation of compound 3
(Scheme 1).
Pinacol esters 7, 10, and 12, as well as boronic acid 3
were tested in in vitro enzyme inhibition assays against
FXIa, FXa, thrombin, and trypsin.15 The results are
shown in Table 1. Compounds 10 and 12 showed the
greatest potency against FXIa with IC50 values of 7.3
and 5.9 lM, respectively, while compounds 3 and 7 were
less potent (IC50 3 = 22 lM and IC50 7 = 24.7 lM), but
showed a promising selectivity trend, especially against
FXa and trypsin (approximately 10-fold). Both com-
pounds 10 and 12 had good selectivity against FXa,
but no selectivity against thrombin and trypsin. As
expected, there was no difference in the IC50 values for
boronic acid 3 and its pinacol ester 7, since the pinacol
moiety was hydrolyzed prior to binding.18 We decided
to obtain the crystal structures of compounds 3, 10,
We chose to modify the benzylic position in compound
10. Since such a modification would create a chiral cen-
ter, we decided to prepare the racemic compound and let
the biologically active enantiomer co-crystallize selec-
tively with the protein.
The synthesis of boronic ester 13, a representative mem-
ber of the new series of compounds, is shown in Scheme
4. Commercially available amine 14 was treated with
1 equiv of tert-butylcarbonyl (Boc) anhydride, 3 equiv
of triethyl amine in dichloromethane (DCM) for 18 h
at ambient temperature to provide the protected Boc
derivative in 96% yield. The ketone was then reduced
to the alcohol with excess sodium borohydride in meth-
anol for one hour at ambient temperature in 83% yield.
Reaction of the alcohol intermediate with 1 equiv of
nicotinoyl chloride and 3 equiv of diisopropylethyl
amine in DCM for 18 h at ambient temperature gave
the nicotinoyl ester 15 in 76% yield. The boronic pinacol
ester was introduced by treating 15 with 1 equiv of dip-
inacolato diboron, 3 equiv of potassium acetate, and
0.1 equiv of Pd(dppf)Cl2 in DMSO at 80 °C for 18 h
and isolated in 73% yield after a flash chromatography
purification. The Boc group was removed by treatment
with 4 N HCl for 30 min (97% yield). The resulting free
amine was converted to a guanidinium group by reac-
tion with 1 equiv of pyrazole reagent 6 and 1 equiv of
diisopropylethyl amine in DMF for 18 h at ambient
temperature. Final deprotection with excess TFA for
5 h at ambient temperature gave the target compound
13 as a racemate in 78% overall yield for the last two-
steps.
and 12 with the FXIa catalytic domain rhaFXI370–607
-
S434A, T475A, C482S, K437A (FXIac)19 in order to
rationalize the observed selectivity and look for oppor-
tunities to introduce additional points of contact with
the enzyme.
In the crystal structure20 (Fig. 1A–C), all three com-
pounds showed a glycerol boronate ester, instead of
the original pinacol ester for compounds 10 and 12
and the free boronic acid for compound 3. Since glyc-
erol was only present in the cryo-protecting solutions,
the transesterification reaction could have occurred
when the crystal was prepared for X-ray analysis.
The formation of the glycerol boronate ester had no ef-
fect on the biological properties of compounds 3, 10,
and 12, since there was no change in their IC50 values
in the presence of glycerol. All three compounds bound
in the active site with the guanidine group interacting
with aspartate 189 in the S1 pocket of the FXIac.
The boron atom was tetracoordinate and covalently at-
tached to serine 195. There were structural differences
between the ligands in order to accommodate their dif-
ferent lengths. The two ends (guanidine and glycerol
boronate) of compounds 3 and 10 were superimposed
on each other. To accommodate the extra methylene
group in compound 10, the phenyl ring tilted away
from the position it occupied in compound 3. The gua-
The in vitro biological data for compound 13 and its
precursor 10 are shown in Table 2. The IC50 value for
the racemic 13 against FXIa was 1.4 lM. Compound
13 also showed an improved selectivity against trypsin
(approximately 200-fold) as compared to the precursor
compound 10 (3-fold). There was no significant change
in the selectivity against FXa (approximately 30-fold
for compound 13 vs 25-fold for compound 10). There
was also a modest improvement in the selectivity against
thrombin (8-fold for compound 13 as compared to 4-
fold for compound 10). In order to find a possible expla-
nation for the observed results, as well as find out, which
of the two enantiomers is responsible for the biological
activity, we solved the crystal structure of compound
13 with FXIac.
Table 1. In vitro biological data
Compound IC50 (lM) IC50 (lM) IC50 (lM) IC50 (lM)
FXIa
FXa
Thrombin Trypsin
3
7
22
>200
>200
146
129
>200
>200
24.7
7.3
5.9
10
12
175.4
129
30.8
5.5
20.3
2.3

T. I. Lazarova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5022–5027
5025
Figure 1. Panels A–D are electron density maps calculated using Fourier coefficients Fobs–Fcalc using phases from the final refined models omitting
the atoms for the ligand and contoured at 2.5 sigma for FXIa complexes with compounds 3, 10, 12, and 13, respectively. Panel E is a wall-eyed
stereodiagram of the superposition of the bound conformations of compounds 3 (magenta), 10 (cyan), and 12 (green) in the active site of FXIa. The
solvent accessible protein surface is from the FXIa-compound 3 complex.
The crystal structure (Fig. 1D) showed that the S enan-
tiomer of compound 13 was bound in the active site.
Assuming only this enantiomer is active against FXIa,
this effectively results in a submicromolar IC50 for com-
pound 13. As previously seen, the compound crystal-
lized as the glycerol boronate ester and formed a
covalent bond between its boron atom and the oxygen
of serine 195, as well as a salt bridge between its guani-
dine group and the side-chain carboxylate of aspartate
189. There are, however, differences between the FXIa-
bound structures of compound 13 and its precursor,
compound 10 (Fig. 1B). The ester carbonyl in com-
˚
pound 13 is within hydrogen bonding distance (3.0 A)
of and oriented toward the backbone nitrogen of lysine
192. The pyridyl group makes van der Waals contacts
with the side-chain of leucine 146, and this binding
mode results in a shift of the position of residues 143–
˚
147 of up to 1.7 A in the FXIa-compound 13 structure
compared to the FXIa-compound 10 structure
(Fig. 1B). While the hydrogen bond made by the ester

5026
T. I. Lazarova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5022–5027
Scheme 4. Reagents and conditions: (a) 1.0 equiv (Boc)2O, 2.5 equiv Et3N, DCM, rt, 18 h, 96%; (b) 5 equiv NaBH4, methanol, rt, 1 h, 83%; (c)
1.0 equiv nicotinoyl chloride, 3 equiv i-Pr2EtN, DCM, rt, 18 h, 76%; (d) 1 equiv dipinacolato diboron, 3 equiv KOAc, 0.1 equiv Pd(dppf)Cl2, DMSO,
80 °C, 18 h, 73%; (e) 4 N HCl, rt, 30 min, 97%; (f) 1 equiv 6, 1 equiv i-Pr2EtN, DMF, rt, 18 h, 85%; (g) TFA, rt, 5 h, 92%.
5. (a) Gould, W. R.; Leadley, R. J. Curr. Phar. Des. 2003, 9,
2337; (b) Samana, M. Throm. Res. 2002, 106, 267; (c) Rai,
R.; Sprengeler, P. A.; Elrod, K. C.; Young, W. B. Curr.
Med. Chem. 2001, 8, 101.
Table 2. In vitro biological data
Compound IC50 (lM) IC50 (lM) IC50 (lM) IC50 (lM)
FXIa
FXa
Thrombin Trypsin
10
13
7.3
1.4
175.4
43.6
30.8
12.3
20.3
>200
6. Jin, L.; Pandey, P.; Babine, R. E.; Gorga, J. C.; Seidl, K.
J.; Gelfand, E.; Weaver, D. T.; Abdel-Meguid, S. S.;
Strickler, J. E. J. Biol. Chem. 2005, 6, 4704.
7. Salomon, O.; Zivelin, A.; Livnat, T.; Dardik, R.;
Loewenthal, R.; Avishal, O.; Steinberg, D. M.; Rosove,
M. H.; O’Connell, N.; Lee, C. A.; Seligsohn, U. Blood
2003, 101, 4783.
carbonyl is probably not contributing much to specific-
ity since the other proteases tested can make this inter-
action as well, the steric clash between leucine 146 and
the pyridyl group of 13 is not likely to be accommodated
well by all serine proteases. This observation can explain
the gain in selectivity observed with compound 13 com-
pared to compound 10. The pyridyl ring is also posi-
tioned so that additional substituents on the meta- or
ortho-positions (relative to the boronic acid) could make
additional interactions in both the S2 subsite and a flex-
ible pocket near the mouth of the S1 pocket that is
formed by lysine 192, cysteine 191, cysteine 219, and res-
idues 143–148. Compounds that optimize interactions in
these subsites, in addition to the covalent interaction
with ser195 and the electrostatic interaction with asp
189, may very well advance both the potency and selec-
tivity of this compound series.
8. Ries, U. J.; Wienen, W. Drugs Future 2003, 28, 355.
9. Wang, X.; Cheng, Q.; Xu, L.; Feuerstein, G. Z.; Hsu, M.-
Y.; Smith, P. L.; Seiffert, D. A.; Schumacher, W. A.;
Ogletree, M. L. J. Thrombosis Haemostasis 2005, 3, 695.
10. (a) Weston, G. S.; Blazquez, J.; Baquero, F.; Shoichet, B.
K. J. Med. Chem. 1998, 41, 4577; (b) Wasserthal, P. S.;
Martichonok, V.; Keller, T. H.; Chin, B.; Martin, R.;
Jones, J. B. Bioorg. Med. Chem. Lett. 1994, 2, 35; (c)
Patent WO 92/19707 1992 to Procter and Gamble
Company, US.
11. (a) Deadman, J. J.; Spencer, J.; Greenidge, P. A.;
Goodwin, C. A.; Kakkar, V. V.; Scully, M. F. WO
2002057273 A1; (b) Spencer, J.; Burd, A. P.; Goodwin, C.
A.; Merette, S. A. M.; Scully, M. F.; Adatia, T.;
Deadman, J. J. Tetrahedron 2002, 58, 1551; (c) De
Nanteuil, G.; Gloanec, P.; Lila, C.; Portevin, B.; Verbeu-
ren, T.; Rupin, A.; Simonet, S. EP 792883 A1.
12. (a) Bukhtiyarova, M.; Rizzo, C. J.; Kettner, C. A.;
Korant, B. D.; Scarnati, H. T.; King, R. W. Antiviral
Chem. Chemother. 2002, 12, 367; (b) Jagannathan, S.;
Forsyth, T. P.; Kettner, C. A. J. Org. Chem. 2001, 66,
6375; (c) Stoll, V. S.; Eger, B. T.; Hynes, R. C.;
Martichonok, V.; Jones, J. B.; Pai, E. F. Biochemistry
1998, 37, 451; (d) Davis, J. H.; Agard, D. A.; Handel, T.
M.; Basus, V. J. J. Biomol. NMR 1997, 10, 21; (e) Amparo,
E. C.; Miller, W. H.; Pacofsky, G. J.; Wityak, J.; Weber, P.
C.; Duncia, J. J. V.; Santella, J. B., III. WO 9620689 A2;
(f) Pacofsky, G. J.; Pruitt, J. R.; Weber, P. C. WO 9509859
A1; (g) Tian, Z.-Q.; Brown, B. B.; Mack, D. P.; Hutton, C.
A.; Bartlett, P. A. J. Org. Chem. 1997, 62, 514; (h) Coutts,
S. J.; Kelly, T. A.; Snow, R. J.; Kennedy, C. A.; Barton, R.
W.; Adams, J.; Krolikowski, D. A.; Freeman, D. M.;
Cambell, S. J. J. Med. Chem. 1996, 39, 2087.
13. (a) Coburn, C. A. Expert Opin. Ther. Patents 2001, 11,
721; (b) Vacca, J. P. Curr. Opin. Chem. Biol. 2000, 9, 355;
(c) Kettner, C.; Mersinger, I.; Knobb, R. J. Biol. Chem.
1990, 265, 18289; (d) Tarrapelli, C.; Metternich, R.;
Ehrhardt, C. J. Biol. Chem. 1993, 268, 4734; (e) Weber,
P. C.; Lee, S. I.; Levandowski, F. A.; Schadt, M. C.;
Chang, C. W.; Kettner, C. A. Biochemistry 1995, 34,
3750.
In conclusion, we have synthesized single digit micromo-
lar aryl boronic acid inhibitors of FXIa with selectivity
against trypsin, thrombin, and FXa. X-ray studies of
the inhibitors with the catalytic domain of FXIa identi-
fied several subsites where additional interactions with
the enzyme may lead to the design of more potent and
selective compounds. Synthesis and biological evalua-
tion of second generation aryl boronic acid inhibitors
has been initiated.
References and notes
1. Ruef, J.; Katus, H. A. Expert Opin. Ther. Patents 2003, 12,
781.
2. Hirsh, J.; Fuster, V. Circulation 1994, 89, 1449.
3. (a) Coburn, C. A. Expert Opin. Ther. Patents 2001, 11, 1;
(b) Sanderson, P. E. J. Annu. Rep. Med. Chem. 2001, 36,
79; (c) Vacca, J. P. Annu. Rep. Med. Chem. 1998, 33, 81;
(d) Ilies, M. A.; Scozzafava, A.; Supuran, C. T. Expert
Opin. Ther. Patents 2002, 12, 1181.
4. (a) Rewinkel, J. B. M.; Adang, A. E. P. Curr. Pharm. Des.
1999, 5, 1043; (b) Steinmetzer, T.; Hauptmann, J.;
Sturzebecher, J. Expert Opin. Ther. Patents 2001, 11, 845.
14. Velcade is a trademark of Millenium Pharmaceuticals,
Inc. (a) Adams, J.; Kauffman, M. Cancer Invest. 2004,

T. I. Lazarova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5022–5027
5027
22, 304; (b) Adams, J. Expert Opin. Ther. Patents 2003,
13, 45.
16. Von Matt, A.; Ehrhardt, C.; Burkhard, P.; Metternich, R.;
Walkinshaw, M.; Tarrapelli, C. Bioorg. Med. Chem. 2000,
8, 2291.
17. Ishiyama, T.; Miyaura, N. Chem. Rec. 2004, 3, 271.
18. Pinacol ester 10 was completely converted to the corre-
sponding boronic acid 4 after 30 min incubation in the
assay buffer, as shown by LS/MS.
19. Jin, L.; Pandey, P.; Babine, R. E.; Gorga, J. C.; Seidl, K.
J.; Gelfand, E.; Weaver, D. T.; Abdel-Meguid, S. S.;
Strickler, J. E. J. Biol. Chem. 2005, 6, 4704.
20. Crystallographic data for structures are on file with
CCDC. Deposition numbers: compound 3 1ZMJ, com-
pound 10 1ZML, compound 12 1ZMN, compound 13
1ZMJ.
15. Peptidolytic activities for FXIa, FXa, thrombin, and
trypsin were measured using a fluorogenic reporter group,
7-methyl-4-aminocoumarin (AMC), attached via an amide
linkage to the tripeptide pyroglutamyl-prolyl-arginyl (Glp-
Pro-Arg). The assay was carried out in 20 mM Tris–HCl,
150 mM NaCl, and 0.02% Tween 20 at pH 7.4. Enzyme and
substrate concentrations were 0.25 nM and 50 lM, respec-
tively, for factor XIa. The assay was incubated at 30 °C for
15–30 min and then released AMC measured (emission
460 nm, excitation 355 nm) on a Wallac 1420 Multilabel
Counter. The data were fit to a one-site dose response using
non-linear least squares to determine the IC50 value.