Creating novel activated factor XI inhibitors through fragment based lead generation and structure aided drug design

Article abstract of DOI:10.1371/journal.pone.0113705

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1'-S2' FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1'-S2' binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC₅₀ of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1'-S2' binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.

Full text of DOI:10.1371/journal.pone.0113705

RESEARCH ARTICLE
Creating Novel Activated Factor XI Inhibitors
through Fragment Based Lead Generation
and Structure Aided Drug Design
Ola Fjellström1*, Sibel Akkaya1, Hans-Georg Beisel1, Per-Olof Eriksson2, Karl Erixon1,
David Gustafsson3, Ulrik Jurva4, Daiwu Kang3, David Karis1, Wolfgang Knecht3,
Viveca Nerme3, Ingemar Nilsson1, Thomas Olsson1, Alma Redzic1, Robert Roth2,
Jenny Sandmark2, Anna Tigerström2, Linda Öster2
1 Medicinal Chemistry, Cardiovascular & Metabolic Diseases Innovative Medicines, AstraZeneca R&D,
Mölndal, Sweden, 2 Discovery Sciences, AstraZeneca R&D, Mölndal, Sweden, 3 Bioscience,
Cardiovascular & Metabolic Diseases Innovative Medicines, AstraZeneca R&D, Mölndal, Sweden, 4 Drug
Metabolism and Pharmacokinetics, Cardiovascular & Metabolic Diseases Innovative Medicines,
AstraZeneca R&D, Mölndal, Sweden
* ola.fjellstrom@astrazeneca.com
Abstract
OPEN ACCESS
Citation: Fjellström O, Akkaya S, Beisel H-G,
Eriksson P-O, Erixon K, Gustafsson D, et al. (2015)
Creating Novel Activated Factor XI Inhibitors through
Fragment Based Lead Generation and Structure
Aided Drug Design. PLoS ONE 10(1): e0113705.
doi:10.1371/journal.pone.0113705
Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrino-
lytic effects with a low bleeding risk. This motivated a structure aided fragment based lead
generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on dock-
ing experiments, was performed to generate a FXIa targeted fragment library for an NMR
screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket.
The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine
structures are novel FXIa P1 fragments. The expansion of these fragments towards the
FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa in-
hibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray
structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one
fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking
and expansion work to achieve one of the most potent and selective FXIa inhibitors reported
to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface
area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work
to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to
yield molecules with less hydrophilicity shows promise to afford potent, selective and orally
bioavailable compounds.
Academic Editor: Alessio Lodola, University of
Parma, ITALY
Received: July 31, 2014
Accepted: October 27, 2014
Published: January 28, 2015
Copyright: © 2015 Fjellström et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Protein Databank Accession Numbers are as follows:
4CRG, 4CR5, 4CR9, 4CRA, 4CRB, 4CRC, 4CRD,
4CRE, 4CRF.
Funding: The authors have no support or funding to
report.
Introduction
A well balanced haemostasis system is important to both minimize blood loss and disturbances
of blood flow. Upon injury of the vessel wall, blood is exposed to tissue factor which via a cas-
cade reaction leads to thrombin generation and a fibrin cross-linked clot to mend the injury
Competing Interests: The authors have read the
journal's policy and have the following competing
interests: All authors are, or were, employees of
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
1 / 42

Creation and Characterization of Novel FXIa Inhibitors
AstraZeneca. This does not alter adherence to PLOS
ONE policies on sharing data and materials.
and stop bleeding. Factor XI (FXI) has an important role in thrombin generation in the ampli-
fication phase of the coagulation process. However, over-production of thrombin may lead to
excessive clots resulting in thrombosis. Also, high levels of thrombin cause activation of throm-
bin activated fibrinolysis inhibitor which hinders fibrinolysis. Therefore, decreased levels of
thrombin will indirectly increase the rate of fibrinolysis. Inhibition of activated FXI (FXIa)
should decrease thrombin generation in the amplification phase, but not in the initiation
phase, and thus yield an antithrombotic and profibrinolytic effect with minimal risk of bleeding
(see reviews [1–3]). Bleeding is a serious concern with current antithrombotic drugs and FXIa
inhibitors could address this issue.
The role of FXIa in haemostasis and thrombosis in human has been extensively studied.
Human haemophilia C patients who are severely deficient in FXI display reduced incidence of
ischemic stroke [4]. Unlike haemophilia A and B patients, who are deficient in FVIII and FIX,
respectively, haemophilia C patients seldom experience spontaneous bleeding [5]. The bleeding
associated with FXI deficiency usually occurs after trauma or surgery in the tissues with high fi-
brinolytic activity [6,7]. An increased level of factor XI has been reported as a risk factor for
deep venous thrombosis [8,9], myocardial infarction [10] and ischemic stroke [11,12]. There is
also much research on the role of FXI in animals. Several studies have demonstrated that FXI-
null mice are protected against venous and arterial thrombosis without an adverse effect on
bleeding time [13–18]. Recent reports present similar effects in mice [19] and primates [20]
using antisense oligonucleotides to inhibit FXI production [19]. Antibodies against FXI/FXIa
have been shown in one study to reduce thrombus growth in the rabbit iliac artery in the pres-
ence of repeated balloon injury [21], and in another study to increase endogenous thrombolysis
in rabbit about two-fold in comparison to control antibodies [22]. Also, an anti-human anti-
body, aXIMab, prevented vascular graft occlusion in baboons [23]. In summary, there is ample
evidence in support of FXIa as an attractive antithrombotic and profibrinolytic target.
FXIa small molecule inhibitors have not reached the same level of maturity as thrombin and
activated factor X (FXa) inhibitors. The thrombin inhibitor dabigatran [24] and the FXa inhibi-
tor rivaroxaban and apixaban [25] are approved anticoagulant drugs in several markets, but ad-
verse bleeding remains an area where improvement is requested. In contrast, inhibitors of FXIa
are still in preclinical development. Daiichi Sankyo Co has reported on potent and selective
peptidomimetic alpha-ketothiazole arginine based covalent FXIa inhibitors [26,27], and one
compound was shown to display similar antithrombotic efficacy as heparin in a rat venous
thrombosis model [26]. Similarly, Bristol Myers Squibb (BMS) demonstrated in vivo antith-
rombotic efficacy in rat models with BMS-262084, a potent and selective beta-lactam arginine
that irreversibly inhibits FXIa with an IC50 of 2.8 nM [28]. Recently, BMS also showed antith-
rombotic efficacy without increased bleeding in a rabbit model with a reversible selective small
molecule FXIa inhibitor [29]. Patent applications from BMS display lists of selective FXIa in-
hibitors, or dual FXIa and plasma kallikrein inhibitors, with IC50 values in the low nM range
[30–32]. These examples encourage further work with the aim of reaching the clinical setting
for small molecule FXIa inhibitors.
In-house high throughput screening (HTS) attempts had previously failed to identify viable
leads. Therefore, structure aided fragment based lead generation (FBLG) was chosen as a rescue
strategy to create new FXIa inhibitor leads. The choice was supported by the feasibility to gen-
erate X-ray structures, to perform a fragment screen using nuclear magnetic resonance (NMR)
and to apply structure based virtual screening to select a fragment library.
A typical fragment campaign involves screening of a relatively small set (< 5000) of small
molecules of low structural complexity against a biological target. Binding to the target is iden-
tified using a robust assay, usually a biophysical technique like NMR [33–35], surface plasmon
resonance (SPR) [36–40], mass spectroscopy (MS) [41] or X-ray crystallography [42–44].
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
2 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 1. Nomenclature for FXIa substrates and corresponding binding sites. (A) FIX sequences that are substrates for FXIa. The scissile bonds cleaved
by FXIa are marked with a red dashed line. Residues N- and C-terminal of the scissile bond are referred to as P1, P2 etc. and P1’, P2’ etc., respectively. (B)
Depiction of FXIa active site in complex with FIXa substrate residues (from PDB entry 1XXD [82]). According to standard nomenclature, the substrate P1
residue binds the enzyme S1 site, the P1’ residue binds the S1’ site, and so on. The scissile bond is marked with a red dashed line.
doi:10.1371/journal.pone.0113705.g001
These techniques are suitable in fragment campaigns, as they allow for detection of the very
weak binding (dissociation binding constant (KD) in the mM range) that can be expected for
the small molecules screened. Fragment based approaches to drug discovery have during the
last years presented many successful examples [45,46], and at least one campaign has lead to a
product on the market [46,47].
Fig. 1 depicts the FXIa active site and describes the standard nomenclature according to
Schechter and Berger [48] for substrate and ligand binding to this site. P1 and P1’ denote the
peptide residues N- and C-terminal of the scissile bond, and S1 and S1’ denote the correspond-
ing enzyme binding sites.
The aim of the present study was to create novel FXIa inhibitors with potential to be devel-
oped into potent, selective and orally bioavailable compounds by targeting the S1-S1’-S2’ FXIa
binding pockets. In the FXIa FBLG campaign presented here, virtual screening, NMR, SPR and
X-ray crystallography together with enzymatic inhibition assays were successfully applied. We
report the identification of two novel FXIa S1 binding fragments and the evolution of these
into nM inhibitors using X-ray structures to guide the design. Initial fragment expansion work
shows promise to afford potent, selective and orally bioavailable compounds, which motivates
further optimization work on presented compounds.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
3 / 42

Creation and Characterization of Novel FXIa Inhibitors
Results and Discussion
Fragment library selection
The structural characteristics of the target protein binding site and the quality of the initial li-
brary of fragments to be screened are two critical determinants for the success of an FBLG cam-
paign. The most prominent and probable fragment binding site of FXIa is the S1 pocket. A key
feature of the S1 pocket is Asp189 (trypsin numbering) deep down in the cavity and most known
ligands contain a strongly basic group that forms ionic interactions with this residue [26,27,49].
However, it has been shown that the basicity is not required [50,51], and since strongly basic P1
groups often pose high hurdles in achieving adequate oral bioavailability, we steered our frag-
ment selection towards non-basic or weakly basic compounds. Importantly, the S1 pocket in
FXIa harbors an alanine in position 190, a feature shared with thrombin and FXa whereas the
corresponding residue in activated factor IX (FIXa) and activated factor VIIa is serine. The
Ala190 provides a pocket where a neutral P1 is likely to be more easily accommodated than
when this residue is a serine. Apart from S1, cavity analyses using the Schrödinger Sitemap tool
[52] suggested that favorable ligand-protein interactions could be picked up towards the S1’-S2’
sites in FXIa. Also, comparisons of primary sequences and structural features around the S1’-S2’
sites for thrombin, FXa and FXIa suggested opportunities for achieving selective FXIa inhibitors.
Thus, the project set out to primarily target the S1-S1’-S2’ binding pockets.
The initial fragment selection focused on targeting the S1 site. A total of about 65000 frag-
ments from the AstraZeneca screening collection with MW < 250 g/mol were docked using
the Glide software from Schrödinger [53–55] to the FXIa X-ray structure (PDB id 1ZSJ), with
the N-(7-Carbamimidoyl-naphthalen-1-yl)-3-hydroxy-2-methyl-benzamide ligand and all
water molecules removed. Docked fragments with a halogen and/or nitrogen interacting with
S1 pocket residues were then manually inspected for a selection of about 1200 structures. The
same 65000 fragments were also docked into an in-house structure of FXIa catalytic domain
(CD) in complex with compound 1, see Fig. 2, with the ligand and waters removed. From these
docking experiments, an additional 600 structures were selected based on Glide score and
chemical tractability. In total, 1800 structures were submitted to experimental screening.
Screening cascade
Ligand detected one dimensional (1D) NMR spectroscopy [56–58] was used as the primary
fragment screening tool, followed by SPR and enzymatic assays to determine KD and IC50 val-
ues, respectively. Finally, structures of the FXIa catalytic domain, containing the four mutations
described by Jin et al., [59] (herein referred to as FXIa CD) in complex with ligands were solved
by X-ray crystallography.
Ligand detected 1D NMR. Compounds were screened at 100 μM in mixtures of six. Weakly
binding fragments were identified from intensity changes through measurements on three
samples viz. (1) mixtures of fragments at 100 μM in buffer; (2) with addition of 5 μM FXIa CD;
(3) with subsequent addition of 10 μM of the high affinity inhibitor compound 2 (see Fig. 2).
The signal from a compound binding with low affinity to FXIa CD decreased upon addition of
protein and regained upon addition of a high affinity reference compound. Comparison with
reference spectra of individual compounds enabled identification of the binding fragment in a
mixture. Compound 2 binds to the S1-S2-S3-S4 pockets of FXIa CD with an IC50 of 110 nM,
and is expected to displace any fragment binding weakly to the S1 site as well as to the non-
prime side of the binding pocket, i.e. S2-S3-S4. As a positive control, the S1 binding ligand
para-methyl-benzamidine (pMeBza), compound 3, was used, and in Fig. 3 the doublet at
7.66 ppm is shown.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
4 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 2. Structures with associated FXIa potencies. aKi values are shown in parenthesis. bPrecipitates at higher concentration than 111 μM. cKD value from
Biacore binding experiment.
doi:10.1371/journal.pone.0113705.g002
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
5 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 3. Ligand detected one dimensional NMR spectroscopy. Part of 1H T1rho NMR spectra of p-methyl-benzamidine (pMeBza): (A) 100 μM pMeBza in
buffer; (B) after addition of 5 μM FXIa CD; and (C) after addition of 10 μM compound 2. Shown is the doublet from the protons in the ortho position. The extra
signals in C are from the inhibitor.
doi:10.1371/journal.pone.0113705.g003
SPR. Compounds that displayed binding to FXIa CD in the NMR screen were followed up
by KD determinations using SPR. The SPR data were then used to rank and select compounds
for X-ray crystallography experiments. The SPR method was based on an inhibition in solution
assay in which compound 4, immobilized on the biosensor surface, served as a probe for the
FXIa catalytic site. The advantages of inhibition in solution assay as compared to the more con-
ventional direct binding assays where the protein is immobilized include (1) substantially in-
creased sensitivity, (2) generic and rapid assay development and (3) immediate verification of
competitive binding. Fig. 4 illustrates the design of compound 4 based on the crystal structure
of FXIa in complex with compound 1. Since any ligand that binds to the S1 pocket competes
with compound 4 for binding to FXIa CD, it is possible to derive the KD for that ligand [60].
The initial binding rate of the protein to compound 4, Kon, was used to determine the percent-
age of free protein in solution which changed by varying the concentration of the competing li-
gand. The free protein concentration was plotted against the logarithm of the ligand
concentration and a sigmoidal dose–response curve-fit model was applied to determine the
KD-value.
Enzymatic assay. All fragments that showed competitive binding in the NMR assay (frag-
ment hit compounds) and all compounds resulting from expansions of fragments were
screened in an enzymatic assay to determine their inhibitory activity against FXIa. Selected
compounds were also screened against other serine proteases to evaluate their selectivity pro-
files. All protease activities were determined by chromogenic assays monitoring the release of
para-nitroanilide at 405 nm.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
6 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 4. Crystal structure of compound 1 in complex with FXIa. The surface exposed point for linker attachment used to develop compound 4 is
highlighted. The KD of the compound 4 was determined to 390 nM. Compound 4 was linked to the sensor chip via the primary amine.
doi:10.1371/journal.pone.0113705.g004
X-ray crystallography. Selected fragments and expanded compounds that had been con-
firmed to be binding to the active site by either NMR or SPR were attempted for structure de-
termination by X-ray crystallography. To improve crystallisability, the quadruple mutant
(Ser75Ala, Lys78Ala, Thr97Ala and Cys123Ser), first reported by Jin et al., [59] was generated.
The mutated residues in this construct (FXIa CD) are located at least 16 Å from the catalytic
residue Ser195 and the alterations are therefore not anticipated to impact ligand binding in the
active site. The selected fragments were either co-crystallized with FXIa CD, or the protein
crystals were soaked in a solution containing the ligand. The crystals typically diffracted to
around 2 Å on an in-house X-ray source. Fragments were considered for chemical expansion if
their binding mode could be determined by X-ray crystallography.
Screening results
NMR screening of the 1800 structures selected by virtual screening resulted in 13 neutral or
weakly basic fragment hits (calculated pKa < 9.0, Advanced Chemistry Development, Inc.
ACD/LabspKaDB (version 6.0) software [61]). These hits were FXIa S1 binding fragments as
demonstrated by pMeBza competition in NMR experiments. About 600 near neighbours to the
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
7 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 5. Crystal structures of compounds 5 (A) and 6 (B) in complex with FXIa. Hydrogen bonds are depicted as dashed lines and the refined 2FoFc
electron density maps are contoured at 1σ. Compound 6 is depicted in the charged form to satisfy the hydrogen bond to Gly218.
doi:10.1371/journal.pone.0113705.g005
original 13 hits were selected and screened by NMR to yield a total of 50 hits. Several of the
fragment hit compounds were only weakly basic, such as aminopyridines and aminoquino-
lines, and neutral compounds, such as chloroquinolinones and the known p-chlorophenylte-
trazoles [32], were also identified. Two prioritized fragment hits, 6-chloro-3,4-dihydro-1H-
quinolin-2-one (5) and 4-methylquinolin-6-amine (6) are shown in Fig. 5. To our knowledge,
5 is a novel serine protease P1 structure, and 6 is here described for the first time to bind FXIa.
The crystal structures of compounds 5 and 6 in complex with FXIa were solved to 2.0 and 1.7
Å resolution, respectively, and the fragments could be unambiguously placed in the electron
density maps (Fig. 5). The two fragments display distinct binding modes as depicted in Fig. 5.
These binding modes were predicted from the GLIDE dockings of compounds 12 and 19 in
the virtual screen. After alignment of the proteins using the SSM function in Coot [62], a root
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
8 / 42

Creation and Characterization of Novel FXIa Inhibitors
mean square deviation (r.m.s.d.) of 0.44 Å for the compound 12 docking pose and crystal
structure was calculated using heavy atom positions only. For compound 6, the binding mode
was predicted using the docking pose of compound 19 (r.m.s.d. of 0.61 Å including all heavy
atoms except for the amino group in the 6-position).
Fragment 5 displayed a FXIa IC50 of 140 μM. The chloro atom is directed almost perpendic-
ular to the aromatic ring of Tyr228 and is located at a distance of 3.8 Å to the closest carbon
atom of the phenyl ring. This chloro atom replaces a highly conserved water molecule (see
Fig. 5B) and its position is similar to the chloro atom in chloro-phenyl containing P1s of FXIa,
thrombin and FXa inhibitor [63,64]. The halogen bonding between electron rich groups in-
cluding pi systems and the σ-hole of the halogen with a clear directionality has recently re-
ceived attention and has been reviewed in several articles [65–68]. The chloroquinolinone
compound 5 picks up an additional interaction with FXIa, i.e. the quinolinone NH group
forms a 2.7 Å hydrogen bond with the backbone carbonyl of Gly218. Notably, the chloroquino-
linone is slightly more potent than benzamidine, compound 7 (see Fig. 2), despite the fact that
the former is neutral and does not form polar interactions with Asp189. This suggests that the
chloroquinolinone P1 is a promising FXIa fragment inhibitor, with potential to be expanded
and developed into an orally active drug.
The aminoquinoline fragment 6 shown in Fig. 5 binds to the S1 pocket with an IC50 of 160
μM. The calculated pKa 7.9 (ACD/LabspKaDB [61]), is supported by an experimental pKa
value of 7.1 from the expanded analogue, compound 8. The amino group of the ligand interacts
with the carboxyl group in Asp189 through a 3.1 Å hydrogen bond, and the quinoline nitrogen
forms a 2.9 Å hydrogen bond with the carbonyl group of Gly218 (Fig. 5). Both the quinolinone
and the aminoquinoline fragments form hydrogen bonds to Gly218, even though their overall
binding geometries in the S1 pocket are quite different. The conserved water molecule stacking
against Tyr228 is bound in the S1 pocket close to the fragment with a distance of 3.6 Å between
the ligand amine nitrogen and the water oxygen.
Structure aided fragment expansion
In parallel with the fragment hit identification work, compounds 9–11 from patent applica-
tions from Bristol Myers Squibb [30,31] were synthesized and characterized. The compounds
are potent FXIa and plasma prekallikrein inhibitors and selective against e.g. thrombin and
FXa as seen in Fig. 6. The crystal structures of 9–11 in complex with FXIa CD were not avail-
able in the public domain and were therefore solved by X-ray crystallography. Given our aim
to target the prime side, it was exciting to note the S1-S1’-S2’ binding mode for all three com-
pounds, as shown for compound 9 in Fig. 7. Superposition of the crystal structures showed that
the chloro-phenyl group of compound 9 overlays with the corresponding group of the chloro-
quinolinone fragment (compound 5), and the chloro-phenyltetrazole group binds as anticipat-
ed from previously reported thrombin structures in complex with ligands carrying the same P1
[63,64]. The P1-P1’ amide group, extended from the P1 via an ethyl linker, interacts with the
active site Ser195 where the carbonyl oxygen forms hydrogen bonds with the amide nitrogen
of Ser195 and a central tightly bound water molecule (Fig. 7). The benzyl group acts as P1’ and
binds in the S1’ cavity and the amine of the amide group linking P1’ and P2’ forms a hydrogen
bond with the central water molecule that is not only located within hydrogen bond distances
to both amide groups of the inhibitor, but also to the hydroxyl of Ser195 and to the backbone
carbonyl of Leu39. The carbonyl from the P1-P1’ amide is positioned in the oxyanion hole
with hydrogen bonds to the main chain nitrogens of Gly193 and Ser195. The P2’ hydroxyqui-
nolinone moiety interacts with His38 and Tyr143 in the S2’ pocket. Two glycerol molecules,
originating from the cryo solution from the crystal freezing step, were bound in close vicinity
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
9 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 6. Structures with associated selectivity data. aKi values are shown in parenthesis. b% inhibition of the compounds at 99 μM are given if the IC50 was
above 99 μM.
doi:10.1371/journal.pone.0113705.g006
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
10 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 7. Crystal structure of compound 9 [31] in complex with FXIa. Compound 9 is displayed in thick sticks with green carbons, selected binding site
residues in thin sticks with grey carbons, water oxygens in red spheres, and hydrogen bond interactions with dashed lines. The arrows indicate: (1) the
central water molecule, (2) the hydrogen acceptor of the P1-P1’ amide and (3) the hydrogen donor of the P1’-P2’ amide.
doi:10.1371/journal.pone.0113705.g007
to the ligand, one stacking against the hydroxyquinolinone in the S2’ region and the second
was bound close to the benzyl moiety (not shown). However, since the S2’ substituent of com-
pound 11 shows a highly similar pose (not shown), the glycerol molecules do not appear to
alter the binding mode. It should be noted that after completion of the experimental work pre-
sented here, the structures of two FXIa inhibitors with closely analogous P1’-P2’ moieties that
show similar binding modes have been published [51].
The crystal structures of compounds 9–11 in complex with FXIa CD gave a clear handle on
directed expansion of fragments towards the prime side and allowed linking exercises. Frag-
ment 5 was selected as the primary target for linking/expansion work. Disregarding overall
physicochemical properties in general and ADME properties in particular, the first key ques-
tion addressed was if the novel chloroquinolinone P1 could be linked to the P1’-P2’ parts of
compound 9 to yield highly potent and selective FXIa inhibitors. Fig. 8 outlines the structural
basis and outcome of this exercise. The X-ray structures of the FXIa CD complexes with the
chloroquinolinone methylated in the 4-position (compound 12) and compound 9 were aligned
in order to guide linking. Based on X-ray structure information and docking studies, we de-
duced that the hydrogen bond acceptor in the defined space as exemplified by the P1-P1’
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
11 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 8. Crystal structures of compounds 9, 12 and 13 in complex with FXIa. Compounds 9 (green), 12 (magenta) and compound 13 (yellow) are overlaid.
The protein surface from the FXIa CD:compound 9 complex is shown as grey surface and the central water molecule that interacts with both amides is shown
as sphere.
doi:10.1371/journal.pone.0113705.g008
amide carbonyl oxygen in compound 9 (see Fig. 7) was favorable and thus judged critical to
maintain in any designed new compounds. Another key feature is a hydrogen donor moiety,
exemplified by the P1’-P2’ amide nitrogen that interacts with a central water molecule that is
highly conserved in position (see Fig. 7). Thus, linkers were designed to preserve the experi-
mentally determined positions of both the P1 fragment, the P1-P1’ amide carbonyl oxygen and
the P1’-P2’ amide nitrogen, without introducing steric strain. A range of possible linker sizes
and geometries were conceived by visual inspection of the overlay of available protein-ligand
crystal structures, and subsequently tested and prioritized by docking experiments of linked
full length compounds using GOLD [69] and GLIDE [53–55]. The most promising compounds
with unstrained geometry, maintaining the linked moieties in place, were chosen for synthesis.
No estimate for the free energy of binding was used, but the approach rather selected com-
pounds for synthesis based on good predicted geometry, leaving the actual binding affinity to
be determined experimentally. The first linker chosen to connect compound 12 and the P1’-
P2’ parts of compound 9 resulted in compound 13, which binds FXIa CD with a KD of 0.5 nM
and is one of the most potent FXIa inhibitors (IC50 = 1.0 nM) reported to date.
The potency SAR observed in this first expansion/linking work is summarized in Fig. 9. The
FXIa IC50 of the chloroquinolinone 5 P1 binder is 140 μM with a ligand efficiency [70] (LE) of
0.44. The P1-P1’ compound 14 increasing in MW from 180 to 398 resulted in only minor
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
12 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 9. Illustration of potency SAR for initial expansion/linking.
doi:10.1371/journal.pone.0113705.g009
improvement in potency with an IC50 of 33 μM and a drop in LE to 0.22. Adding on the P2’
part boosted the potency more than four orders of magnitude to an IC50 of 1 nM and a LE of
0.32 (compound 13). The isolated P1’-P2’ moiety, compound 15, showed a poor binding affini-
ty (KD = 1.4 mM), and the N,N-dimethylated analogue, compound 16, confirmed S1’-S2' bind-
ing with a nearly perfect overlap with the identical P1’-P2’ groups of compound 13 (X-ray data
not shown). The SAR outlined above can be regarded as an example of superadditivity energet-
ics achieved by fragment linking, similarly as recently described in detail by Nazaré et al. for
FXa inhibitors [71]. Rigid body entropy loss is expected to be similar for the individual frag-
ments (here, fragments 5 and 15) as for the final fragments linked molecule (here, compound
13). This rigid body entropy loss has been estimated as 15–20 kJ/mol at 298 K by Murray and
Verdonk [72]. Thus, superadditivity with respect to the starting fragments binding affinities, in
the range of three orders of magnitude, is expected when fragments are optimally linked.
Adapting the following equation from Nazaré et al. [71],
DGlink ¼ DGfinal ꢀ DGfrag1 ꢀ DGfrag2
to the present example yields:
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
13 / 42

Creation and Characterization of Novel FXIa Inhibitors
ΔGlink = ΔGcompound 13 - ΔGfragment 5 - ΔGfragment 15 = -53.1 kJ/mol + 23.7 kJ/mol + 16.3
kJ/mol = -13.1 kJ/mol. This linker contribution of -13.1 kJ/mol corresponds to an improve-
ment by about 2.3 orders of magnitude relative to the individual fragment affinities, and agrees
well with the estimated rigid body entropy loss of 15–20 kJ/mol [72]. This example shows the
reward from superadditive affinity gain by joining two weakly binding fragments with a near
optimal linker.
Fragment expansion/linking, based on compound 9, with different P1 fragments are sum-
marized in Fig. 2. It is worth noting that the 6-methyl-3,4-dihydro-1H-quinolin-2-one P1 frag-
ment 17 was not identified as a hit in NMR but after the expansion that resulted in compound
18 yielded a FXIa IC50 of 10 nM, emphasizing that fragments with very weak binding affinities
can provide useful starting points for expansion. The expansion/linking of the aminoquinoline
fragment 6 with the P1’-P2’ part of compound 9 resulted in compound 8 with an IC50 of 120
nM. The choice of the 5-position of 6 as site for initial expansion of the aminoquinoline was
based on synthetic feasibility considerations. However, analyses of the aligned structures sug-
gested that analogous expansion/linking from the 4-position might be more favorable, i.e. ex-
panding from the methyl position in compound 19 (an equipotent analogue to compound 6).
Analogous expansion/linking work was also performed based on compound 10 with amide
bonds reversed as compared to compound 9, see data in Fig. 2. None of the designed linked
compounds, 20–24, reached as high potency as compound 10. However, compound 20 with
an IC50 of 330 nM, suggests feasibility of including reverse amides in expanded structures.
The structure aided expansion/linking work described above is briefly summarized as fol-
lows: Both the X-ray structures and docking studies presented here showed that the hydrogen
bond acceptor and donor positions highlighted in Fig. 7 are clearly defined in space and thus
key features to utilize in modeling. The novel FXIa P1s, chloroquinolinone, dihydroquinoli-
none and aminoquinoline, can upon expansion yield potent and selective P1-P1’-P2’ com-
pounds. The 4-position in chloroquinolinones and dihydroquinolinones is a favorable point
for expansion. Amides and reverse amides are both acceptable, but with compounds synthe-
sized so far, the amide nitrogen closer to the P1 part yield more potent binders.
Synthesis
The main synthetic schemes to achieve the expanded P1-P1’ and the P1-P1’-P2’ structures are
depicted in Fig. 10. One key intermediate was compound 13C (4-(bromomethyl)-6-chloroqui-
nolin-2(1H)-one), from which the final screen compounds 13, 14, 25, 26 and 28
were synthesized.
Fig. 11 outlines the synthesis of P2’ and P1’-P2’ structures that were used for complete frag-
ment expansion originating from P1 structures.
Figs. 12–16 describe the synthetic steps to arrive at remaining screening compounds.
Compound characterization
It is important to achieve potent FXIa inhibitors with high selectivity vs. other serine proteases
in order to minimize risk for adverse effects, particularly with regard to bleeding, which limits
the utility of current antithrombotic drugs, e.g. dabigatran and rivaroxaban. Selectivity data for
FXa, thrombin, trypsin, plasma kallikrein (PKK), FIXa, tissue plasminogen activator (tPA) and
plasmin were therefore generated and are summarized in Fig. 6. Assessment of fragment selec-
tivity is limited both due to the low potencies of the fragments and that the highest compound
concentration used in the assay was 99 μM. The P1’ expanded chloroquinolinone, compound
14, with a FXIa IC50 of 33 μM, displayed IC50 values above 99 μM for all other proteases in this
panel, indicating that some selectivity can be achieved through the P1-P1’ parts. The P1-P1’-
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
14 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 10. Synthesis of intermediate 13C and routes to compounds 13, 14, 25, 26 and 28. i) Methyl acetoacetat, DMAP, Pyridine, xylenes, reflux, 8h, ii)
AcOH, Br2, r.t, 3h, iii) Neat H2SO4, 45°C, 46h, iv) Hexamine, DCM, reflux, 5h, then conc. HCl, reflux, 2h, v) Diethylmalonate, THF, NaH, reflux, 1h, vi) 20%
HCl, reflux, 12h, vii) TBTU, TEA, DMF, 20B, r.t, 16h, viii) 15B, T3P, 13E, TEA, 120°C, 20 min., ix) HOBt, EDAC, 13E, BOC-L-phenylalanine, TEA, DMF, r.t,
16h, x) TFA, DCM, r.t, 16h, xi) AcOH, TBTU, 14B, pyridine, DMF, r.t, 16h, xii) 2M MeNH2, TEA, THF, 50°C, 1h, xiii) (S)-2-(tert-butoxycarbonylamino)-3-
phenylpropanoic acid, HOBt, EDAC, DIPEA, DMF, r.t, 16h, xiv) 4M HCl in dioxane, r.t, 2h, xv) 15A, HOBt, EDAC, DIPEA, DMF, xvi) (S)-2-(tert-
butoxycarbonyl(methyl)amino)-3-phenylpropanoic acid, HOBt, EDAC, DIPEA, DMF, r.t, 16h, xvii) 4M HCl in dioxane, r.t, 3h, xviii) 15A, TBTU, EDAC, DIPEA,
DMSO, r.t, 48h, xix) 4-methyl-3-phenyl-1H-pyrazole-5-carboxylic acid, HOBt, EDAC, DIPEA, DMSO, r.t, 16h.
doi:10.1371/journal.pone.0113705.g010
P2’ compound 13 displayed more than five orders of magnitude selectivity vs. all proteases in
the panel, except PKK where an 11-fold selectivity based on Ki values was observed. A similar
selectivity pattern was seen for compound 18 containing the chlorodihydroquinolinone P1
fragment. The full length aminoquinoline 8 displayed about 25-fold selectivity vs. PKK.
Regarding species selectivity, it was noted that compound 13 was about 30000-fold less po-
tent against rat FXIa than against human FXIa. We ascribed this discrepancy primarily due to
the replacement of Ala190 in human FXIa by Thr190 in rat, which makes the S1 pocket more
narrow and hydrophilic and thus disfavor binding of the neutral chloroquinolinone P1. In con-
trast, benzamidine containing FXIa inhibitors show similar FXIa potencies between human
and rat. This finding has implications on choice of species for in vivo studies and highlights the
importance of doing species analyses early in projects.
The next stage of the lead identification work focused on a more balanced approach to ad-
dress not only potency and selectivity but also physicochemical and ADME properties. As seen
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
15 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 11. Synthesis of P1’-P2’ fragments. i) DCM, r.t, 16h, then LiOH, water, THF, r.t, 16h, then PPA, 120°C, 2h, ii) TBTU, DIPEA, DMF, L-phenylalanine
methylester, r.t, 16h, iii) TBTU, pyridine, MeNH2xHCl, DMF, r.t, 16h, iv) TBTU, (S)-2-amino-N,N-dimethyl-3-phenylpropanamide hydrochloride, TEA, DMF,
r.t, 16h, v) TBTU, TEA, DCM, DMF, r.t, 16h, vi) neat TFA, r.t, 0.5h.
doi:10.1371/journal.pone.0113705.g011
in Fig. 17, compound 13 displays high polar surface area (PSA), low permeability, high efflux
and high in vivo clearance. The high rat in vivo clearance despite the low/moderate in vitro in-
trinsic clearance in rat hepatocytes may suggest active transport into bile and/or urine in vivo.
We hypothesized that, relative to compound 13, improved permeability and decreased efflux
Fig 12. Synthesis of the dihydroquinolinone derivatives 18 and 20. i) DCM, TEA, r.t, 2h, ii) Methyl 2-(triphenylphosphoranylidene)acetate, toluene, reflux,
2h, iii) NaOMe, MeOH, r.t, 1h, iv) DMSO, water, NaCl, 150°C, 16h, v) 4M NaOH, r.t, 4h, vi) TBTU, TEA, 20B, THF, r.t, 16h, vii) DPPA, TEA, DMF, then 2-
(trimethylsilyl)ethanol, 100°C, 10 min., viii) TBAF, CH3CN, 60°C, 16h, ix) 15B, TBTU, pyridine, DMF, r.t, 16h.
doi:10.1371/journal.pone.0113705.g012
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
16 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 13. Synthesis of 3 substituted dihydroquinolinone 21. i) SnBu3H, DMSO, 100°C, 16h, ii) 4-Methoxybenzyl chloride, NaH, DMF, r.t, 2h, iii) LDA, tert-
butyl 2-bromoacetate, THF, N2, -78°C, iv) Neat TFA, 80°C, 2h, v) 20B, TBTU, TEA, DMF, r.t, 16h.
doi:10.1371/journal.pone.0113705.g013
should increase bioavailability and lower clearance in vivo. Initial ADME exploration targeted
minor modifications of compound 13 to first methylate the P1-P1’ amide nitrogen, as in com-
pound 25, and subsequently adding a second methyl group on the P1’-P2’ amide nitrogen,
yielding compound 26. Minor improvements in permeability and efflux were observed with
compound 25, and compound 26 provided no further benefit. From the structure of FXIa in
complex with compound 11, the methyl group on the P1-P1’ amide in compound 25 is pre-
dicted to point out towards the solution, whereas the methyl group on the P1’-P2’ in com-
pound 26 is predicted to disturb important hydrogen bond interactions with the tightly bound
water molecule shown in Fig. 7 and would therefore be detrimental for potency. The IC50 val-
ues of <0.005 and 1.7 μM for compound 25 and 26, respectively, support this notion. After the
methylation exercise, it was decided to keep the methylated P1-P1’ amide as first step expan-
sion of the P1 fragment and search for further expansion replacing the P1’-P2’ amide with less
hydrophilic structures. In this context, 13 compounds available in our corporate compound
collection with structures containing the N-methyl-N-[(2-oxo-1H-quinolin-4-yl)methyl]form-
amide scaffold were screened. Compound 27 with a FXIa IC50 of 99 μM was identified, which
displayed high permeability (see Fig. 17), but still showed relatively high efflux. The low poten-
cy of compound 27 was improved by approximately two orders of magnitude upon addition of
the chloro atom to the aminoquinolinone P1 to afford compound 28 with an IC50 of 0.82 μM.
The ADME properties of compound 28 with high permeability (Caco2 A to B = 19 ꢁ 10–6 cm/s),
moderate rat in vivo clearance (18 mL/min/kg) and a measured oral bioavailability in rat of
27% show promise towards the goal of developing an oral drug. Potency improvements of this
lead with maintained favorable ADME properties remain to be shown. However, comparing
the potencies of the P1-P1’ compounds 28 and 14 (IC50 values of 0.82 and 33 μM, respectively),
and realizing the potency boost feasible by adding a P2’ moiety (compare compounds 14 and
13 in Fig. 9), suggest exciting opportunities for development of a potent, selective and oral
FXIa inhibitor drug.
Fig 14. Synthesis of the 3-substituted chloroquinolinone 22. i) Pd(OAc)2, PPh3, NaOAc, dry DMF, N2,
110°C, 16h, ii) 4M NaOH, r.t, 1h, iii) 20B, TBTU, TEA, THF, r.t, 12h.
doi:10.1371/journal.pone.0113705.g014
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
17 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 15. Synthesis of 3-substituted quinolinone 23. i) Piperidine, EtOH, reflux, 6h, ii) DIBAL-H, Et2O, N2, r.t, iii) Neat SOCl2, reflux, 6h, iv) DEM, NaH, THF,
N2, reflux, 2h, v) Conc. HCl, reflux, 16h, vi) 20B, TBTU, DIPEA, DMF, r.t, 16h.
doi:10.1371/journal.pone.0113705.g015
Summary and Conclusion
A virtual screen based on docking experiments was performed to select a fragment library, and
NMR was used as the primary screen with minimal risk of false positives. SPR and enzymatic
screening quantified the affinities of the NMR hits, and X-ray structures provided the basis for
fragment expansion and linking design.
The structure aided fragment based lead generation strategy adopted in this work success-
fully identified two novel FXIa P1 fragments that were expanded towards the S1’-S2’ pockets to
yield potent and selective inhibitors. The chloroquinolinone is neutral and the aminoquinoline
is only weakly basic and should provide opportunities to be developed into bioavailable full
length FXIa inhibitors without relying on a prodrug approach. It remains to be explored
whether the FXIa chloroquinolinone and aminoquinoline P1s will have more general utility as
serine protease P1s. The very potent and selective compound 13 gives promise for further opti-
mization. The future challenge will be to improve the physicochemical properties of the chloro-
quinolinone and aminoquinoline P1 based compounds presented here to optimize the
combined potency, selectivity, bioavailability and clearance profiles. Compound 28 indicates
Fig 16. Synthesis of aminoquinoline 8. i) (E)-3-Ethoxyacryloyl chloride, THF, 50°C, 16h, ii) Conc. HCl,
40°C, 1h, iii) Neat POCl3, 80°C, 3h, iv) Xantphos, Pd2(dba)3, tBuOK, PhMe, 100°C, 1h, then 2M HCl and
THF, r.t, 2h, v) AcCl, TEA, DCM, N2, 0°C, 2h, then excess MeNH2 r.t, 16h, vi) NBS, benzoyl peroxide,
(trifluoromethyl)-benzene, 80°C, 16h, then reflux 2h, vii) NaN3, DMF, 80°C, 1h, viii) Pd(OH)2 on carbon,
EtOH, H2, r.t, 16h, ix) 20% NaOH, 120°C, 1h, x) DMF, DIPEA, 15B, TBTU r.t, 16h.
doi:10.1371/journal.pone.0113705.g016
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
18 / 42

Creation and Characterization of Novel FXIa Inhibitors
Fig 17. Structures with associated FXIa potencies and selected physicochemical and ADME parameters. alogD measured by liquid chromatography.
bCaco2 A to B permeability measured at pH 6.5. cEfflux ratio determined in Caco2 cells by dividing Papp B to A with Papp A to B permeabilities measured at pH
7.4 at side A. dRat intrinsic clearance determined from measurements in rat hepatocytes. eRat in vivo clearance. fCaco2 A to B permeability measured at pH
7.4 at side A.
doi:10.1371/journal.pone.0113705.g017
progress towards this goal. A success in this endeavor could be well rewarded in the form of an
effective and safe antithrombotic drug.
Materials and Methods
Protein expression and purification
Protein expression and purification of the catalytic domain (residues 370–607) was carried out
as described by Jin et al. [59]. To improve crystallisability this protein contains the four muta-
tions: S434A, K437A, T475A and C482S (S75A, K78A, T115A, C123S, trypsin numbering).
This protein was used for crystallisation, NMR and SPR measurements.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
19 / 42

Creation and Characterization of Novel FXIa Inhibitors
NMR screening
NMR samples were prepared in aqueous buffer containing 50 mM deuterated TRIS, pH 7.4,
3 mM NaN3, and 10% D2O. FXIa CD stock solution was buffer exchanged and concentrated to
approximately 100 μM (2.7 mg/mL) (UV absorption ε280 = 60390 cm-1) using 10 kDa cut-off
Millipore filter centrifugation tubes. Control experiments with pMeBza and compound 2 were
done on all protein preparations (Fig. 3). Stock solutions of test compounds were prepared in
randomised mixtures of six by pooling 10 μL aliquots of 10 mM DMSO solutions from the AZ
compound library in microtiter plates followed by vacuum evaporation of the solvent. The
solid compound mixtures were then redissolved in 20 μL DMSO-d6 yielding 5 mM compound
stock solutions. Samples for retesting of individual compounds were prepared from original
compound library solution in regular DMSO, necessitating the simultaneous suppression of
solvent and DMSO peaks (see below). Samples were prepared “just in time” using a Genesis liq-
uid handling robot (TECAN, CH) and transferred to the NMR spectrometer via a SampleRail
system (Bruker Biospin, DE). Mixtures were screened at 100 μM compound concentration in
aqueous buffer with 5 μM FXIa CD added in the second step. At the third step, 10 μM inhibitor
(compound 2) was added.
NMR data were collected at 20oC on a Bruker Avance III 600 MHz NMR spectrometer
equipped with a cryo probe head. 1D 1H T1ρ NMR spectra [56] were recorded with 256 scans,
3.2 s interscan delay, 180 ms spin lock time and 64 kHz spin lock field. Solvent suppression was
achieved by applying an 8 ms excitation sculpting sequence [58] before acquisition. For sam-
ples prepared from non-deuterated DMSO stock solutions, simultaneous suppression of the
solvent and DMSO peaks was achieved by replacing the selective 180 degree flip back pulses
with bilinear pulses created with the Pulse tool module in TopSpin (Bruker, CH). Spectra were
analyzed in TopSpin and compounds were considered hits if they displayed significant signal
amplitude changes upon addition of protein and inhibitor.
FXIa surface plasmon resonance assay
The tethered free amine ligand 4 was covalently linked to the dextran layer on the CM5 sensor
chip (GE Healthcare/Biacore) using the Amine Coupling Kit (Healthcare/Biacore) and per-
formed according to the manufacturer’s recommendations. Carboxyl groups on the dextran
layer were activated by injecting a 1:1 mixture of 0.5 M N-ethyl-N’ (3-dimethylaminopropyl)-
carbodiimide and 0.5 M N-hydroxysuccinimide at 10 μL/min for 7 min. The activated carboxyl
groups on the control channels were blocked by injecting a 1 M ethanolamine solution for 7
min at 10 μL/min. The tethered ligand was coupled to the enzyme binding channels by inject-
ing a 100 μM solution of the compound (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfo-
nic acid, pH 7.4, 0.15 M NaCl, 0.005% (v/v) polysorbate 20, 2% DMSO) over the chip surface
at 10 μL/min for 1 min. The remaining activated groups were blocked with 1 M ethanolamine.
Compounds were tested in a concentration response curve. The compounds were serially dilut-
ed 1:3 in DMSO to form a 10-point concentration response curve with 5 mM for the highest
concentration. The FXIa CD was added to the plate at a final concentration of 50 nM and the
mixture was incubated for 15 min at RT. The plate was then sealed and placed in the Biacore
3000 instrument. Samples were injected at 20 μL/min for 2 min. The signal generated on the
control channel was subtracted from the signal of the channel with the tethered ligand. The
value used to calculate percent inhibition of binding was the slope of the response measured 8 s
after the start of the injection. The surface of the chip was regenerated by injecting 0.5% SDS
for a period of 36 s.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
20 / 42

Creation and Characterization of Novel FXIa Inhibitors
Crystallisation and data collection
In order to obtain ligand complexes with compounds 5, 6, 8, 10, 11, 12 and 13, co-crystallisa-
tion was used. FXIa (ꢂ 1 mg/ml in 20 mM Tris pH 7.4, 200 mM NaCl) was mixed with respec-
tive compound to a concentration of 1–5 mM and incubated at room temperature for around 1
h. The complexes were then concentrated to about 20 mg/ml. Crystallisations was set up at 20°
C using the hanging-drop vapour diffusion method with the precipitation solution 1.8–2.2 M
(NH4)2SO4 and 0.1 M Tris pH 8.5. Cubic shaped crystals appeared after a few days. For cryo
protection a quick dip in 3.1 M (NH4)2SO4 and 0.1 M Tris pH 8.5 was done for compound 5,
6, 8, 12 and 13 and in 20% glycerol, 2 M (NH4)2SO4 and 0.1 M Tris pH 8.5 for compound 10
and 11 before flash-freezing in liquid nitrogen. For compound 5, 6, 8, 12 and 13 data was col-
lected on a Rigaku FRE+ rotating anode with a CCD-A200-CU detector. Data for compound
10 was collected at the ID23–1 beam line and data for 11 was collected at the ID14–4 beam line
of the European Synchrotron Radiation Facility in Grenoble, France. Summary of data collec-
tion parameters and statistics are listed in Table 1.
For obtaining ligand complexes with compounds 1 and 9, powder soaks to preformed
FXIa-benzamidine crystals were performed. FXIa-benzamidine crystals were obtained by mix-
ing diluted FXIa (ꢂ 1 mg/mL in 20 mM Tris pH 7.4, 200 mM NaCl) with benzamidine to a
concentration of 10 mM and incubating at room temperature for 1 h. The complex was con-
centrated to ꢂ 20 mg/mL and crystallisation set up with the precipitate solution of 1.8–2.2 M
(NH4)2SO4 and 0.1 M Tris pH 8.5. Single crystals were transferred to a solution consisting
of ꢂ 2 M (NH4)2SO4 and 0.1 M Tris pH 8.5 to which excess of compound powder was added.
The soaking lasted for 24 h for compound 9 and 10 days for compound 1. The crystals were
briefly soaked in a cryoprotection solution containing 20% glycerol, 2 M (NH4)2SO4 and 0.1 M
Tris pH 8.5 before being flash-frozen in liquid nitrogen. The data was collected at the ID14–4
beam line of the European Synchrotron Radiation Facility in Grenoble, France. A summary of
data collection parameters and statistics are listed in Table 1.
Data processing and structure determination
The crystals belong to the spacegroup I23 with one copy of the FXIa CD in the asymmetric
unit. The data for the FXIa:compound 1 complex was processed using autoPROC [73] and
XDS [74] and scaled with Aimless [75]. All other data was processed with the programs
Mosflm [76] and Scala [75,77]. Structure determination by molecular replacement was con-
ducted with the program Molrep [75,78], Phaser [79] or rigid body refinement with Refmac
[75,80], using an in house high-resolution crystal structure as search model. Rebuilding of the
model and addition of water molecules were done in the program Coot [62]. The structure of
the FXIa:compound 1 complex was refined with Refmac and autoBUSTER (BUSTER version
2.11.5. Cambridge, United Kingdom: Global Phasing Ltd) and the final protein model com-
prised residues 16–130 and 133–244 (chymotrypsin numbering). Compound 1 was well de-
fined in the electron density, except for the benzyl group which was modelled in two different
orientations, the predominant orientation is depicted in Fig. 4. All other datasets were refined
using Refmac [80] and all protein residues, 16–245, were included in the final models. The li-
gands were placed in the FoFc difference maps during the last steps of refinement. Details from
data collection, refinement and the final models are summarised in Table 1. The atomic coordi-
nates and structure factors have been deposited in the protein data bank with accession codes
4CR5, 4CR9, 4CRA, 4CRB, 4CRC, 4CRD, 4CRE, 4CRF and 4CRG.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
21 / 42

Creation and Characterization of Novel FXIa Inhibitors
Table 1. X-ray crystallography.
PDB ID
FXIa:compound 1
4CRG
FXIa:compound 5
4CR5
FXIa:compound 6
4CR9
Space group
Cell dimensions (Å)
Resolution (Å)
Rmerge
I23
I23
I23
a = b = c = 120.9
49.3–1.3 (1.33–1.25)1
0.071 (0.745)
14.8 (1.8)
a = b = c = 121.0
24.7–2.0 (2.05–2.0)1
0.153 (0.623)
11.1 (3.3)
a = 121.3
21.5–1.7 (1.74–1.7)1
0.079 (0.83)
15.2 (2.4)
<I / σI>
Completeness (%)
Redundancy
92.8 (75.0)
99.9 (99.9)
8.7 (8.1)
100.0 (100.0)
7.7 (7.2)
5.2 (2.5)
Refinement
No. atoms
Measured / unique refl.
388302/75396
0.176/ 0.195
174104 / 20045
0.203 / 0.247
236553 / 32745
0.179 / 0.208
Rwork / Rfree
Protein
1986
1894
1914
Water/ Ligand
333/58
197/12
298/13
Average B-factors
Protein (Å2)
Water (Å2)
Ligand (Å2)
18.3
36.0
18.4
14.8
21
18.3
29.8
16.6
17.2
R.m.s deviations
Bond lengths (Å)
0.010
0.008
0.006
Bond angles (°)
1.1
1.2
1.1
FXIa:compound 8
4CRA
FXIa:compound 9
4CRB
FXIa:compound 10
4CRC
PDB ID
Space group
Cell dimensions (Å)
Resolution (Å)
Rmerge
I23
I23
I23
a = b = c = 121.1
23.8–1.8 (1.85–1.80)1
0.087 (0.943)
12.3 (1.6)
a = b = c = 120.8
50–1.85 (1.9–1.85)1
0.08 (0.63)
13.9 (2.2)
99.5 (100.0)
5.1 (5.1)
a = b = c = 117.8
41.6–1.6 (1.64–1.60)1
0.08 (0.66)
12.9 (2.6)
<I / σI>
Completeness (%)
Redundancy
99.1 (98.1)
4.6 (4.3)
99.6 (100.0)
7.0 (7.0)
Refinement
No. atoms
Measured / unique refl.
125895 / 27223
0.177 / 0.204
127473 / 25023
0.19 / 0.213
248924 / 35732
0.185 / 0.213
Rwork / Rfree
Protein
1918
1881
1918
Water/Ligand
215/38
122/40
195/39
Average B-factors
Protein (Å2)
Water (Å2)
Ligand (Å2)
23.0
32.5
21.9
28.6
34.8
25.5
21.8
32.4
17.1
R.m.s deviations
Bond lengths (Å)
0.007
0.007
0.006
Bond angles (°)
1.1
1.2
1.2
FXIa:compound 11
4CRD
FXIa:compound 12
4CRE
FXIa:compound 13
4CRF
PDB ID
Space group
Cell dimensions (Å)
I23
I23
I23
a = b = c = 120.5
a = b = c = 121.1
a = b = c = 121.3
(Continued)
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
22 / 42

Creation and Characterization of Novel FXIa Inhibitors
Table 1. (Continued)
FXIa:compound 1
4CRG
FXIa:compound 5
4CR5
FXIa:compound 6
4CR9
PDB ID
Resolution (Å)
Rmerge
50–2.1 (2.15–2.10)1
0.082 (0.508)
11.4 (2.4)
23.8–1.7 (1.77–1.73)1
0.068 (0.559)
21.4 (2.9)
32.4–2.3 (2.36–2.30)1
0.227 (1.12)
9.5 (2.1)
<I / σI>
Completeness (%)
Redundancy
99.5 (100.0)
5.8 (4.3)
99.9 (98.7)
100.0 (100.0)
9.9 (9.5)
9.3 (6.3)
Refinement
No. atoms
Measured / unique refl.
98743 / 17077
0.203 / 0.234
288638 / 31069
0.172 / 0.197
131774 / 13350
0.181 / 0.244
Rwork / Rfree
Protein
1881
1928
1892
Water/Ligand
76/35
287/13
95/39
Average B-factors
Protein (Å2)
Water (Å2)
Ligand (Å2)
41.7
39.9
32.9
17.2
29.0
21.0
28.6
29.2
22.6
R.m.s deviations
Bond lengths (Å)
0.010
1.4
0.005
1.1
0.011
1.4
Bond angles (°)
Data collection and refinement statistics.
1Values in parentheses refer to highest-resolution shell.
doi:10.1371/journal.pone.0113705.t001
Enzymatic assays
Once the compounds were synthesized and characterized, enzymatic assays were performed to
evaluate the inhibitory activity of the resulting compounds against FXIa and other serine prote-
ases. All protease activities were determined by chromogenic assays monitoring the release of
para-nitroanilide at 405 nm. IC50 values for the compounds were determined using equation:
n
Q ¼ V=ð1 þ ð½Iꢃ=IC50Þ Þ
Q denotes the assay readout, V denotes the uninhibited velocity of the enzyme, [I] denotes the
inhibitor concentration, n denotes the slope and IC50 is the inhibitor concentration at V/2.
The IC50 values were converted into Ki values for competitive inhibitors using the equation:
Ki ¼ IC50=ð1 þ ½Sꢃ=KmÞ
and the respective Km values for the substrate in the assay. [S] denotes the
substrate concentration.
FXIa. Human FXIa was from HTI, Essex Junction, USA. Inhibition by compounds was
measured using 420 μM pyroGlu-Pro-Arg-pNAꢁHCl (Bachem, Bubendorf, Switzerland,
Km = 420 μM) as substrate in 50 mM Tris pH 7.4, 100 mM NaCl, 5 mM CaCl2, 0.1 mg/mL
BSA, 1% DMSO (v/v).
Trypsin. Recombinant human trypsin was from Polymun, Vienna, Austria. Inhibition by
compounds was measured using 300 μM Bz-Val-Gly-Arg-pNA (Bachem, Bubendorf, Switzer-
land, Km = 230 μM) as substrate in 50 mM Tris pH 7.4, 40 mM NaCl, 20 mM CaCl2, 0.1%
Tween 20 (v/v), 1% DMSO (v/v).
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
23 / 42

Creation and Characterization of Novel FXIa Inhibitors
Thrombin. Human recombinant thrombin was prepared in house. Inhibition by com-
pounds was measured using 300 μM pyroGlu-Pro-Arg-pNAꢁHCl (Km = 280 μM) as substrate
in 50 mM Tris/HCl pH 7.4, 100 mM NaCl, 0.2% BSA (w/v), 1% DMSO (v/v).
Plasmin. Human plasmin was from Enzyme Research Laboratories, Swansea, UK. Inhibi-
tion by compounds was measured using 300 μM pyroGlu-Pro-Arg-pNAꢁHCl (Km = 275 μM)
as substrate in 50 mM Tris pH 7.4, 100 mM NaCl, 0.1% Tween 80 (v/v), 1% DMSO (v/v).
FIXa. Human FIXa was from Enzyme Research Laboratories, Swansea, UK. Inhibition by
compounds was measured using 500 μM CH3SO2-(D)-CHG-Gly-Arg-pNAꢁAcOH (HTI,
Essex Junction, USA, Km = 1300 μM) as substrate in 33 mM Tris/HCl pH 7.4, 66 mM NaCl,
3.3 mM CaCl2, 33% Ethylene glycol (v/v), 0.3% BSA (w/v), 1% DMSO (v/v).
FXa. Human FXa was from HTI, Essex Junction, USA. Inhibition by compounds was mea-
sured with 100 μM Z-D-Arg-Gly-Arg-pNA (Bachem, Bubendorf, Switzerland, Km = 50 μM) as
substrate in 50 mM Tris pH 7.4, 100 mM NaCl, 5 mM CaCl2, 0.2% BSA (w/v), 1% DMSO (v/v),
1% DMSO (v/v).
tPa. Human tPA was from Biopool, Umeå, Sweden. Inhibition by compounds was measured
with 250 μM CH3SO2-D-HHT-Gly-Arg-pNA (Pentapharm, Basel, Switzerland, Km = 140 μM)
in 50 mM Tris/HCl pH 7.4, 100 mM NaCl, 0.032% Tween 80 (v/v), 1% DMSO (v/v).
PKK. Human PKK was from Calbiochem, Darmstadt, Germany. Inhibition by compounds
was measured with 400 μM D-Pro-Phe-Arg-pNAꢁHCl (Hyphen Biomed, Neuville-Sur-Oise,
France, Km = 105 μM) in 50 mM Tris/HCl pH 7.4, 100 mM NaCl, 5 mM CaCl2, 0.2% BSA (v/w),
1% DMSO (v/v).
Chromatographic determination of lipophilicity
The chromatographic data was obtained utilizing a Waters Aquity UPLC chromatographic sys-
tem running a gradient from 5–95% acetonitrile at pH7.4. The stationary phase was a Waters
BEH C18 2.1x50 mm. Standardized retention data were obtained by normalization against a
set of compounds with precisely determined (octanol/water) logD-values. Lipophilicity values
for samples were derived from a corresponding calibration curve.
Caco2 permeability and efflux
A monolayer of Caco2 cells, cultured on semi-permeable polycarbonate surfaces, was used to
study the permeability in the apical to basolateral direction. The process was atomized by a ro-
botic Tecan EVO platform (Männerdorf, Switzerland) and 24-transwell plates from Costar
(Cambridge, MA, USA). HBSS buffer pH 7.4 was dispensed to the basal side of the monolayer.
The assay was initiated by adding the test substrate (10 μmol/l in HBSS buffer pH 6.5) to the
apical side of the monolayer. Samples for quantitative analysis by LC/MS/MS were withdrawn
before the addition of the test substrate and at 45 and 120 min post addition of the test sub-
strate. During incubation the transwell plates were placed in a shaking incubator at 37°C be-
tween sampling. The peak areas were exported to Excel where Papp-values and recoveries were
calculated. Twenty-two reference compounds with known human oral bioavailability were
used to establish a correlation curve with Papp versus the fraction of the oral dose absorbed (fa).
Efflux was studied in the same Caco2 cells with the modification that HBSS buffer pH 7.4
was used on both sides of the monolayer. The permeability was studied in the apical to basolat-
eral direction (Papp AB) as well as in the basolateral to apical direction and (Papp BA). Efflux ra-
tios were derived from the following equation: Papp BA/Papp AB.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
24 / 42

Creation and Characterization of Novel FXIa Inhibitors
Rat hepatocyte metabolic stability assay
Rat hepatocyte metabolic stability was determined in accordance with the method described by
Jacobson et al. [81]. Cryo preserved hepatocytes at a concentration of 106 viable cells/mL were
used. After thawing, hepatocytes were incubated for 10 min to warm to 37°C and test com-
pounds, dissolved in acetonitrile, were added to give a final concentration of 1 μM. At 0.5, 5,
15, 30, 45, 60, 80, 100 and 120 min, the incubation system was mixed and 20 μL aliquots were
transferred at each time point to wells in a separate plate filled with 80 μL acetonitrile to stop
the reaction. The quenching plate was then vortexed followed by centrifugation, and superna-
tants were analyzed by LC/MS/MS. Peak areas were determined from extracted ion chromato-
grams, and the in vitro intrinsic clearance (in vitro CLint, in μL/min/106 cells) of parent
compound was calculated from the slope in the regression analysis of the natural logarithm of
parent concentration vs. time curve.
In vivo rat PK
Two days prior to dosing, female Sprague Dawley rats were prepared by cannulation of the left
carotid artery for blood sampling and by cannulation of the right jugular vein for intravenous
administration. The catheters were filled with heparin (100 IU/mL), exteriorized at the nape of
the neck and sealed. The surgery was performed under isoflurane (Forene1, Abbott) anesthe-
sia. After surgery the rats were housed individually and had free access to food and water.
About 16 h prior to dosing the animals were deprived of food, and fasted until 4 h after dosing.
The rats had free access to drinking water during the experiment. On the experiment day, the
test item formulation was administered orally by gavage or intravenously in the jugular vein.
At pre-defined time points, blood samples of about 0.150 mL were withdrawn from the carotid
artery up to 24h after dosing. A total of 10 samples were withdrawn. The blood samples were
collected in heparinized plastic tubes and centrifuged, within 30 min, for five min at 10 000 g
and +4°C. The plasma was transferred to a 96 well plate and stored at -20°C until analysis by
LC/MS/MS. The studies were approved by the Göteborg Animal Research Ethical Board.
Chemistry
Chemicals and solvents from commercially available sources was purchased and used without
further purification. 1H NMR spectra were recorded on a Bruker biospin GmbH 400, 500 or
600 MHz spectrometer. Chemical shifts are reported in parts-per-million (δ) relative to
DMSO-d6 at 2.50 ppm, CDCl3 at 7.26 ppm or CD3OD at 3.31 ppm as an internal standard.
High-resolution mass spectrometry (HRMS) analysis was performed using a Waters XEVO-
qTOF instrument (Waters, Milford, USA) with MassLynx 4.1 software. If necessary, the purity
was determined by ultra high performance liquid chromatography (U(H)PLC). Purity of all
final compounds was 95% or higher at 210 nm. The instrument was a Waters U(H)PLC
Acquity system (Binary pump, Sample Manager, Column Compartment and UV-PDA detec-
tor). The column was a Waters Acquity BEH C18, 1.7 μm particle size (100 mm × 2.1 mm).
Heating of reactions with microwaves was performed on a Biotage Initiator 2.45 GHz,
400W microwave for the times indicated. Compounds 1, 2, 4 and 27 were obtained from the
AstraZeneca corporate compound collection, compounds 3, 5, 6, 7, 12, 17, and 19 were com-
mercially available and compounds 9 [31], 10 [31] and 11 [30] were synthesized according to
literature. Experimental and spectroscopic details for all other non-commercially available
compounds are given below or are available in S1 File.
In general, optimizations of the reaction procedures were not performed and yields are
given for isolated materials.
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
25 / 42

Creation and Characterization of Novel FXIa Inhibitors
N-(3-(2-(2-aminoethoxy)ethoxy)phenyl)-6-carbamimidoyl-4-(pyrimidin-2-ylamino)-2-
naphthamide (4). Compound 4 was synthesized in an 8 step sequence from methyl 6-cyano-2-
naphthoate as described in the S1 File. 1H NMR (500 MHz, CD3OD) δ 3.16 (s, 2H), 3.74–3.87
(m, 2H), 3.92 (s, 2H), 4.22 (d, 2H), 6.78 (d, 1H), 6.90 (t, 1H), 7.22 (d, 1H), 7.29 (t, 1H), 7.61 (s,
1H), 7.91 (d, 1H), 8.27 (d, 1H), 8.38 (s, 1H), 8.43–8.55 (m, 3H), 8.76 (s, 1H).
(E)-3-Ethoxy-N-m-tolylacrylamide (8A). To a solution of (E)-3-ethoxyacryloyl chloride
(8.20 g, 60.9 mmol) in THF (275 mL) was added 3-Methylaniline (13.1 g, 122 mmol), forming
a pale orange precipitate. The reaction mixture was heated at 50°C for 16 h. The reaction was
allowed to cool to room temperature and then filtered. The filtrate contained crude compound
8A (12.5 g, 100%) sufficiently pure to be used in the next reaction step without further purifica-
tions. 1H NMR (400 MHz, CDCl3) δ 1.24–1.32 (3H, m), 2.28 (3H, d, J = 6.9 Hz), 3.82 (2H, q,
J = 7.1, 7.1, 7.1 Hz), 5.38 (1H, d, J = 12.1 Hz), 6.87 (1H, d, J = 7.5 Hz), 7.15 (1H, t, J = 7.8, 7.8
Hz), 7.32 (1H, d, J = 7.8 Hz), 7.39 (1H, s), 7.59 (1H, d, J = 12.1 Hz), 7.67 (1H, d, J = 16.3 Hz).
5-Methylquinolin-2(1H)-one (8B). (E)-3-Ethoxy-N-m-tolylacrylamide (8A) was dissolved
in warm (40°C) concentrated HCl (40 mL) and stirred for 1 h, allowing the reaction to slowly
reach room temperature. The reaction mixture was then poured into a beaker with crushed ice,
forming an orange precipitate. The precipitate was isolated by filtration, washed with cold
water and then dried under reduced pressure. The crude solid contained a mixture of regioi-
somers (40:60, 3.60 g, 41%) with the desired product in minority. The regioisomer mixture was
separated by preparative HPLC to give the desired regioisomer 8B (0.601 g, 17%). 1H NMR
(400 MHz, CDCl3) δ 2.44 (3H, s), 6.63 (1H, d, J = 9.4 Hz), 7.03 (1H, d, J = 7.4 Hz), 7.14 (1H, s),
7.43 (1H, d, J = 8.0 Hz), 7.76 (1H, d, J = 9.5 Hz), 11.35 (1H, s).
2-Chloro-5-methylquinoline (8C). 5-Methylquinolin-2(1H)-one (8B) (0.50 g, 3.15 mmol)
was heated in POCl3 (7.43 g, 48.4 mmol) at 80°C for 3 h. The reaction was allowed to reach
room temperature and poured into a beaker containing crushed ice and stirred until all ice had
melted. Resulting suspension was extracted with chloroform (3 x 15 mL). The combined organ-
ic fractions was dried (MgSO4), filtered, and then concentrated under reduced pressure to af-
ford compound 8C as a crude black solid (0.480 g, 87%).
5-Methylquinolin-2-amine (8D). A mixture of 2-chloro-5-methylquinoline 8C (0.484 g,
2.72 mmol), Xantphos (0.126 g, 0.220 mmol), tris(dibenzylideneacetone)dipalladium (0.100 g,
0.110 mmol) and potassium tert-butoxide (0.917 g, 8.17 mmol) in Toulene (15 mL) was heated
at 100°C for 1 h using a microwave reactor. The reaction was quenched with water and the
phases separated. The aqueous phase was extracted with DCM (3 x 20 mL), the combined or-
ganic fractions was passed through a phase separator to remove water and palladium residues.
The filtrate was concentrated under reduced pressure, the residue re-dissolved in a mixture of
THF (10 mL) and 2M HCl (10 mL), and stirred at room temperature for 2 h. The reaction was
concentrated under reduced pressure to give compound 8D as a crude solid (0.427 g, 99%).
The crude product was used in the next reaction step without further purifications.
N-(5-Methylquinolin-2-yl)acetamide (8E). A mixture of 5-methylquinolin-2-amine (8D)
(0.427 g, 2.72 mmol) and triethylamine (TEA) (3.79 mL, 27.2 mmol) in DCM (10 mL) was stir-
red at 0°C under an inert atmosphere of nitrogen. Acetyl chloride (1.94 mL, 27.2 mmol) was
added drop wise and resulting mixture stirred for 2 h. A large excess methylamine (2M in
THF) was then added and resulting mixture stirred for 16 h to cleave di-acetylated products.
The reaction was concentrated under reduced pressure and resulting residue filtered through a
plug of silica using EtOAc/ heptane (1:3) as the eluent. The crude compound 8E (0.270 g, 50%)
was sufficiently pure to be used in the next reaction step without further purifications.
N-(5-(Bromomethyl)quinolin-2-yl)acetamide (8F). A solution of N-(5-methylquinolin-2-
yl)acetamide 8E (0.270 g, 1.35 mmol), NBS 0.528 g, 2.97 mmol), benzoyl peroxide (0.098 g,
0.400 mmol) was dissolved in (trifluoromethyl)-benzene (6 mL) and the resulting mixture
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
26 / 42

Creation and Characterization of Novel FXIa Inhibitors
heated at 80°C for 16 h. The reaction proceeded slowly and was heated at reflux for 2 h to
achieve complete conversion of the starting material. The reaction was diluted with water and
then extracted with DCM (3 x 15 mL). The combined organic fractions was dried (MgSO4), fil-
tered and then concentrated to give compound 8F as a crude pale brown oil (0.377 g, 100%).
The crude material was used without further purifications in the next reaction step.
N-(5-(Azidomethyl)quinolin-2-yl)acetamide (8G). A solution of N-(5-(bromomethyl)qui-
nolin-2-yl)acetamide 8F (0.377 g, 1.35 mmol) and sodium azide (0.176 g, 2.70 mmol) in DMF
(2 mL) was heated at 80°C for 1 h using a microwave reactor. The crude reaction mixture was
diluted with 95% ethanol (6 mL) and used directly in the next reaction step.
N-(5-(Aminomethyl)quinolin-2-yl)acetamide (8H). The crude (DMF/Ethanol solution, 2
mL:6 mL) solution of N-(5-(azidomethyl)quinolin-2-yl)acetamide 8G was transferred to a 50
mL round bottom flask and palladium hydroxide on carbon (0.050 g, 0.36 mmol) was added.
The reaction mixture was stirred under an atmosphere of hydrogen for 16 h. The resulting mix-
ture was filtered through a phase separator to remove palladium residues and then concentrat-
ed under reduced pressure to give crude compound 8H (0.291 g, 100%) as a brown oil. The
crude oil was used directly in the next reaction step without further purifications.
5-(Aminomethyl)quinolin-2-amine (8I). A mixture of the crude N-(5-(aminomethyl)qui-
nolin-2-yl)acetamide 8H (0.291 g, 1.35 mmol) was heated in 8 mL 20% NaOH at 120°C for 1
h. LCMS analysis showed full conversion of the acetamide to corresponding amino-quinoline
and a pyridone by-product in an estimated 1:1 ratio. The mixture was concentrated under re-
duced pressure, re-dissolved in methanol and filtered. The crude product was purified by pre-
parative HPLC, relevant fractions were pooled and freeze dried to give compound 8I as a white
solid (0.028 g, 12%). Compound 8I was used directly without further purifications in the next
reaction step.
(S)-N-(1-{(2-Aminoquinolin-5-yl)methylamino}-1-oxo-3-phenylpropan-2-yl)-4-hy-
droxy-2-oxo-1,2-dihydroquinoline-6-carboxamide (8). To a solution of 5-(aminomethyl)qui-
nolin-2-amine 8I (0.056 g, 0.330 mmol), 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-
tetramethylisouronium tetrafluoroborate (0.118 g, 0.370 mmol) in DMF (2 mL) was added N,
N-Diisopropylethylamine (DIPEA) (0.114 mL, 0.650 mmol) and (S)-2-(4-hydroxy-2-oxo-1,2-
dihydroquinoline-6-carboxamido)-3-phenylpropanoic acid 15B. Resulting mixture was stirred
at room temperature for 16 h. Excess DMF was removed under reduced pressure and the re-
sulting residue was taken up in methanol and purified by preparative HPLC. Fractions contain-
ing the desired product was pooled and excess solvent removed by freeze drying to give
compound 8 as a white solid (0.016 g, 10%). 1H NMR (400 MHz, CD3OD) δ 3.13 (2H, ddd,
J = 7.3, 13.5, 39.8 Hz), 4.67 (2H, dd, J = 14.7, 41.8 Hz), 4.82 (1H, t, J = 7.4, 7.4 Hz), 6.77 (1H, d,
J = 9.1 Hz), 7.03 (1H, d, J = 6.5 Hz), 7.18 (5H, s), 7.30 (1H, d, J = 8.6 Hz), 7.39–7.52 (8H, m),
7.89 (1H, dd, J = 2.1, 8.6 Hz), 8.00 (1H, d, J = 9.2 Hz), 8.39 (1H, d, J = 2.0 Hz). HRMS: calcd. for
[M+H]+ C29H25N5O4 508.1985, found 508.1988.
N-(4-Chlorophenyl)-3-oxobutanamide (13A). A mixture of methyl 3-oxobutanoate
(3.38 mL, 31.4 mmol), 4-chloroaniline (1.00 g, 7.84 mmol), DMAP (0.958 g, 7.84 mmol) and
pyridine (4 mL) was refluxed in xylenes (20 mL) for 8 h. The reaction mixture was partitioned
between 2M HCl (20 mL) and EtOAc (20 mL), the aqueous phase was extracted with EtOAc
(2x10 mL) and the combined organic fractions was dried (Na2SO4) and then concentrated to
produce compound 13A as a pale brown solid (1.330 g, 80%). 1H NMR (600 MHz, DMSO-d6)
δ 2.20 (3H, s), 3.55 (2H, s), 7.36 (2H, d, J = 8.8 Hz), 7.60 (2H, d, J = 8.8 Hz), 10.22 (1H, s).
4-Bromo-N-(4-chlorophenyl)-3-oxobutanamide (13B). To a stirred solution of N-(4-
chlorophenyl)-3-oxobutanamide (13A) (18.2 g, 86.1 mmol) in AcOH (80 mL) at room temper-
ature was dropwise added a solution of bromine (5.32 mL, 103 mmol) in AcOH (20 mL) over
3 h. Resulting mixture was stirred for 3 h, producing a brown precipitate. The reaction mixture
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
27 / 42

Creation and Characterization of Novel FXIa Inhibitors
was poured into a 1L beaker filled to 2/3 with crushed ice. A crude brown precipitate contain-
ing a mixture of various brominated species was collected by filtration. The product was puri-
fied by silica column chromatography (EtOAc/ DCM gradient from 0–5% EtOAc, with 0.5%
TEA). Relevant fractions was pooled and concentrated under reduced pressure to give com-
pound 13B as a pale brown solid (14.0 g, 56%). 1H NMR (400 MHz, DMSO-d6) δ 2.19 (2H, s),
3.53 (2H, s), 7.31–7.47 (2H, m), 7.54–7.61 (2H, m), 10.18 (1H, s).
4-(Bromomethyl)-6-chloroquinolin-2(1H)-one (13C). 4-Bromo-N-(4-chlorophenyl)-3-
oxobutanamide (13B) (12.0 g, 41.3 mmol) was heated at 45°C in sulfuric acid (60 mL) for 24
h. Due to slow conversion of the starting material another portion of sulfuric acid (60 mL) was
added and the reaction was continued until complete conversion was observed (LCMS), a total
of 46 h. The reaction was allowed to reach ambient temperature and then poured into a 2L bea-
ker filled to 2/3 with crushed ice, producing a pink precipitate. The precipitate was collected by
filtration, washed with ice-cold water and then dried on a freeze-drier giving compound 13C as
a pink solid (10.2 g, 90%). 1H NMR (500 MHz, DMSO-d6) δ 4.93 (2H, s), 6.80 (1H, s), 7.35
(1H, d, J = 8.8 Hz), 7.59 (1H, dd, J = 2.2, 8.8 Hz), 7.89 (1H, d, J = 2.2 Hz), 11.95 (1H, s).
Diethyl 2-{(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)methyl}malonate (13D). To a stir-
red solution of diethyl malonate (3.62 mL, 23.85 mmol) in THF (15 mL) at room temperature
was added NaH (0.286 g, 11.93 mmol) in portions. Resulting mixture was stirred at room tem-
perature for 10 min before a slurry of 4-(bromomethyl)-6-chloroquinolin-2(1H)-one (13C)
(0.65 g, 2.39 mmol) in THF (15 mL) was added dropwise. The resulting brown suspension was
refluxed for 1 h and then concentrated under reduced pressure to give a brown oil. Water was
then added until a precipitate formed. The precipitate was filtered and washed with cold water
and then dried on a freeze-drier, giving compound 13D as a white solid (0.688 g, 82%). 1H
NMR (500 MHz, DMSO-d6) δ 1.12 (6H, t, J = 7.1, 7.1 Hz), 3.30 (2H, d, J = 8.0 Hz), 3.93 (1H, t,
J = 8.0, 8.0 Hz), 4.10 (4H, q, J = 7.0, 7.0, 7.1 Hz), 6.38 (1H, s), 7.32 (1H, d, J = 8.8 Hz), 7.56 (1H,
dd, J = 2.2, 8.8 Hz), 7.87 (1H, d, J = 2.2 Hz), 11.83 (1H, s).
3-(6-Chloro-2-oxo-1,2-dihydroquinolin-4-yl)propanoic acid (24A). A suspension of di-
ethyl 2-{(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)methyl}malonate (13D) (0.688 g, 1.96
mmol) in 20% HCl (aq) (30 mL) was refluxed for 12 h. The reaction was then cooled under
stirring on an ice bath to form a precipitate. The precipitate was filtered, washed with cold
water and dried on a freeze-drier to give compound 24A as a white solid (0.421 g, 86%). 1H
NMR (500 MHz, DMSO-d6) δ 2.62 (2H, t, J = 7.4, 7.4 Hz), 3.03 (2H, t, J = 7.3, 7.3 Hz), 6.40
(1H, s), 7.32 (1H, d, J = 8.8 Hz), 7.55 (1H, dd, J = 2.2, 8.8 Hz), 7.80 (1H, d, J = 2.2 Hz), 11.77
(1H, s), 12.30 (1H, s).
4-(Aminomethyl)-6-chloroquinolin-2(1H)-one (13E). A mixture of 4-(bromomethyl)-6-
chloroquinolin-2(1H)-one (13C) (0.300 g, 1.10 mmol) and hexamethylene tetraamine (0.193 g,
1.38 mmol) in dichloroethane (20 mL) was refluxed for 5 h. A white precipitate formed and
was collected by filtration. The intermediate solid was then refluxed in concentrated hydro-
chloric acid (10 mL) for 2 h. The reaction was stirred on an ice-bath and quenched with water,
forming a new precipitate. The precipitate was filtered, washed with cold water and then dried
on a freeze-drier to give compound 13E as a white solid (0,213 g, 93%). 1H NMR (500 MHz,
DMSO-d6) δ 4.10 (2H, d, J = 0.9 Hz), 6.66 (1H, s), 6.97 (9H, s), 7.36 (1H, d, J = 8.8 Hz), 7.57
(1H, dd, J = 2.3, 8.8 Hz), 7.81 (1H, d, J = 2.3 Hz).
(S)-N-(1-{(6-Chloro-2-oxo-1,2-dihydroquinolin-4-yl)methylamino}-1-oxo-3-phenylpro-
pan-2-yl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamide (13). A mixture of (S)-2-
(4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamido)-3-phenylpropanoic acid (15B)
(0.233 g, 0.660 mmol), T3P (0.788 mL, 1.32 mmol), 4-(aminomethyl)-6-chloroquinolin-2
(1H)-one (13E) (0.138 g, 0.66 mmol) and TEA (0.277 mL, 1.99 mmol) was heated at 120°C for
20 min using a microwave reactor. Excess DMF was removed under reduced pressure to
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
28 / 42

Creation and Characterization of Novel FXIa Inhibitors
produce a crude brown oily residue, containing the desired product and an inseparable phos-
phorous ester of the product and the coupling reagent. The crude mixture was treated with 6M
HCl at room temperature for 1h to cleave phosphorous ester byproducts. The reaction was
concentrated and then purified by preparative HPLC to give compound 13 as a pale pink solid
(0.109 g, 30%). 1H NMR (400 MHz, DMSO-d6) δ 2.97–3.08 (1H, m), 3.13 (1H, dd, J = 4.5, 13.7
Hz), 4.41–4.56 (2H, m), 4.67–4.76 (1H, m), 6.46 (1H, s), 6.72 (1H, s), 7.11 (1H, t, J = 7.2, 7.2
Hz), 7.19 (2H, t, J = 7.4, 7.4 Hz), 7.24–7.31 (3H, m), 7.34 (1H, d, J = 8.8 Hz), 7.47 (1H, s), 7.53
(1H, dd, J = 2.1, 8.8 Hz), 7.78 (1H, d, J = 2.1 Hz), 7.88 (1H, dd, J = 1.9, 8.6 Hz), 8.29 (1H, d,
J = 1.8 Hz), 8.71 (1H, t, J = 5.7, 5.7 Hz), 8.77 (1H, d, J = 8.1 Hz). 2 protons were not observed.
HRMS: calcd. for [M+H]+ C29H23ClN4O5 543.1435, found 543.1447.
(S)-Tert-butyl 1-{(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)methylamino}-1-oxo-3-phe-
nylpropan-2-ylcarbamate (14A). 1H-Benzo[d][1,2,3]triazol-1-ol hydrate (0.468 g, 3.06 mmol)
and N1-{(ethylimino)methylene}-N3,N3-dimethylpropane-1,3-diamine hydrochloride (0.693
g, 3.61 mmol) were added to a suspension of 4-(aminomethyl)-6-chloroquinolin-2(1H)-one
(13E) (2.00 g, 2.78 mmol), (S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoic acid (0.738 g,
2.78 mmol), and TEA (1.16 mL, 8.34 mmol) in DMF (15 mL). The mixture was stirred at room
temperature for 16 h, diluted with ethanol (10 mL) and then filtered through a pad of Celite1.
Excess solvent was removed under reduced pressure and the crude residue was purified by
recrystallisation from EtOAc. Compound 14A was isolated as a white solid (0.965 g, 76%). 1H
NMR (400 MHz, DMSO-d6) δ 1.30 (8H, s), 2.79 (1H, dd, J = 10.4, 13.5 Hz), 2.96 (1H, dd, J = 4.6,
13.7 Hz), 4.21 (1H, td, J = 4.6, 10.2, 10.3 Hz), 4.49 (2H, d, J = 5.5 Hz), 6.44 (1H, s), 7.08 (1H, d,
J = 8.6 Hz), 7.18 (1H, dd, J = 2.6, 5.8 Hz), 7.24 (4H, dd, J = 5.4, 7.5 Hz), 7.33 (1H, d, J = 8.8 Hz),
7.55 (1H, dd, J = 2.2, 8.8 Hz), 7.84 (1H, t, J = 9.7, 9.7 Hz), 8.53 (1H, t, J = 5.8, 5.8 Hz), 11.71 (1H, s).
(S)-2-Amino-N-{(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)methyl}-3-phenylpropana-
mide (14B). A solution of (S)-tert-butyl (1-[{(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)meth-
yl}amino]-1-oxo-3-phenylpropan-2-yl)carbamate (14A) (0.086 g, 0.19 mmol) in DCM (8 mL)
and TFA (3 mL) was stirred at room temperature for 16 h. Excess solvents was removed under
reduced pressure to give compound 14B as a white solid (0.075 g, 100%). 1H NMR (600 MHz,
DMSO-d6) δ 3.05 (2H, ddd, J = 7.1, 13.7, 32.2 Hz), 4.11 (1H, d, J = 4.5 Hz), 4.44–4.56 (2H, m),
6.43 (1H, s), 7.22 (5H, ddd, J = 6.7, 11.0, 14.2 Hz), 7.34 (1H, dd, J = 5.7, 17.9 Hz), 7.57 (1H, dd,
J = 2.2, 8.8 Hz), 7.81 (1H, t, J = 9.2, 9.2 Hz), 8.32 (2H, s), 9.01 (1H, t, J = 5.8, 5.8 Hz), 11.84 (1H, s).
(S)-2-Acetamido-N-{(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)methyl}-3-phenylpropa-
namide (14). Acetic acid (0.017 mL, 0.30 mmol) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-
1,1,3,3-tetramethylisouronium tetrafluoroborate (0.095 g, 0.30 mmol) was dissolved in DMF (4
mL) and stirred at room temperature for 10 min. Pyridine (0.032 mL, 0.39 mmol) was added
and the reaction mixture was stirred for another 10 min. (S)-2-amino-N-{(6-chloro-2-oxo-1,2-
dihydroquinolin-4-yl)methyl}-3-phenylpropanamide (14B) (0.035 g, 0.10 mmol) was added
and the resulting mixture was stirred at room temperature for 16 h. Excess DMF was removed
under reduced pressure and resulting crude material was purified by preparative HPLC to give
compound 14 as a white solid (0.023 g, 59%). 1H NMR (400 MHz, DMSO-d6) δ 1.78 (3H, s),
2.78 (1H, dd, J = 10.0, 13.5 Hz), 2.99 (1H, dd, J = 4.8, 13.6 Hz), 4.52 (3H, ddd, J = 5.2, 14.1, 23.7
Hz), 6.38 (1H, s), 7.16 (1H, dd, J = 4.3, 8.6 Hz), 7.23 (4H, d, J = 4.3 Hz), 7.33 (1H, d, J = 8.8 Hz),
7.55 (1H, dd, J = 1.9, 8.8 Hz), 7.82 (1H, d, J = 1.9 Hz), 8.26 (1H, d, J = 8.3 Hz), 8.62 (1H, t, J = 5.5,
5.5 Hz), 11.69 (1H, s). HRMS: calcd. for [M+H]+ C21H20ClN3O3 398.1271, found 398.1270.
4-Hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A). To a solution of methyl
4-aminobenzoate (2.34 g, 15.5 mmol) in DCM (40 mL) was dropwise added ethyl 3-chloro-3-
oxopropanoate (2.58 mL, 20.1 mmol) and resulting mixture was stirred at room temperature
for 16 h. The reaction mixture was washed with 1M NaOH (2 x 40 mL) and brine (40 mL). The
organic layer was dried (Na2SO4), filtered and then concentrated under reduced pressure. The
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
29 / 42

Creation and Characterization of Novel FXIa Inhibitors
residue was hydrolyzed directly by stirring in a mixture of THF (10 mL), water (10 mL) and
lithium hydroxide (1.85 g, 77.4 mmol) at room temperature for 16 h. Excess THF was removed
under reduced pressure before the pH was adjusted to 3 with 2M HCl. The product precipitat-
ed and was collected by filtration. The solid material was then heated in polyphosphoric acid
(5.24 mL, 15.5 mmol) at 120°C for 2 h. The reaction mixture was cooled on an ice bath and
water (100 mL) was added under stirring. A white solid was formed and collected by filtration
and washed with ice-cold water giving compound 15A as a white solid (2.30 g, 72%). 1H NMR
(400 MHz, DMSO-d6) δ 5.77 (1H, s), 7.32 (1H, t, J = 13.8, 13.8 Hz), 7.96–8.07 (1H, m), 8.38
(1H, d, J = 1.8 Hz), 11.50 (1H, s), 11.61 (1H, s).
(S)-2-(4-Hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxamido)-3-phenylpropanoic acid
(15B). A mixture of 4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (15A) (0.57 g,
2.78 mmol), 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluorobo-
rate (1.07 g, 3.33 mmol), and N-ethyl-N-isopropylpropan-2-amine (1.94 mL, 11.1 mmol) in
DMF (15 mL) was stirred at room temperature for 10 min, giving a yellow suspension. (S)-
methyl 2-amino-3-phenylpropanoate hydrochloride (0.719 g, 3.33 mmol) was then added and
resulting mixture stirred at room temperature for 16 h. The yellow mixture was partitioned be-
tween water and EtOAc, the aqueous layer was washed with DCM, dried (Na2SO4), filtered
and concentrated under reduced pressure to give an orange oil. Ice-cold water was added to the
crude oil forming an orange solid. The solid was washed with cold water and dried under re-
duced pressure to give the crude ester intermediate. The crude solid was hydrolyzed directly by
stirring in a mixture of water, THF and lithium hydroxide for 2 h. Excess THF was removed
under reduced pressure and 1M HCl was then added to the residue, forming a white precipitate.
Compound 15B was collected by filtration (0.430 g, 44%). 1H NMR (500 MHz, DMSO-d6) δ 3.09
(1H, dd, J = 10.5, 13.8 Hz), 3.19 (1H, dd, J = 4.6, 13.8 Hz), 4.63 (1H, ddd, J = 4.6, 8.2, 10.6 Hz),
5.76 (1H, s), 7.17 (1H, t, J = 7.2, 7.2 Hz), 7.22–7.34 (5H, m), 7.90 (1H, dt, J = 7.3, 7.3, 14.6 Hz),
8.31 (1H, d, J = 1.9 Hz), 8.78 (1H, d, J = 8.1 Hz), 11.39 (1H, s), 11.54 (1H, s), 12.74 (1H, s).
(S)-4-Hydroxy-N-(1-(methylamino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydroquinoline-
6-carboxamide (15). A mixture of (S)-2-(4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxa-
mido)-3-phenylpropanoic acid (15B) (0.070 g, 0.20 mmol), O-(7-azabenzotriazol-1-yl)-N,N,
N',N'-tetramethyluronium hexafluorophosphate (0.189 g, 0.50 mmol) was stirred in DMF
(5 mL) at room temperature for 10 min before pyridine (0.321 mL, 3.97 mmol) and methylamine
hydrochloride (0.268 g, 3.97 mmol) was added, resulting mixture was stirred at room tempera-
ture for 16 h. DMSO (5 mL) was added and excess DMF was then removed under reduced pres-
sure. The remaining DMSO solution was purified by preparative HPLC to give compound 15 as
a white solid (0.0683 g, 94%). 1H NMR (600 MHz, DMSO-d6) δ 2.60 (3H, t, J = 5.8, 5.8 Hz),
2.88–2.97 (2H, m), 3.04 (2H, dd, J = 3.2, 13.3 Hz), 4.49–4.55 (1H, m), 7.12–7.21 (2H, m), 7.25–
7.31 (4H, m), 7.39 (1H, s), 7.41–7.47 (2H, m), 8.00 (1H, d, J = 4.7 Hz), 8.43 (1H, d, J = 8.4 Hz),
10.40 (1H, s). HRMS: calcd. for [M+H]+ C20H19N3O4 366.1454, found 366.1462.
(S)-N-(1-(Dimethylamino)-1-oxo-3-phenylpropan-2-yl)-4-hydroxy-2-oxo-1,2-
dihydroquinoline-6-carboxamide (16). A suspension of 4-hydroxy-2-oxo-1,2-dihydroquino-
line-6-carboxylic acid (15A) (0.050 g, 0.24 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate (0.190 g, 0.50 mmol) was stirred in DMF (5 mL) at
room temperature for 10 min before (S)-2-amino-N,N-dimethyl-3-phenylpropanamide hydro-
chloride (0.067 g, 0.29 mmol) and TEA (0.085 mL, 0.61 mmol) was added, resulting mixture
was stirred at room temperature for 16 h. DMSO (5 mL) was added and excess DMF was then
removed under reduced pressure. The remaining DMSO solution was purified by preparative
HPLC to give compound 16 as a white solid (0.051 g, 55%). 1H NMR (500 MHz, DMSO-d6)
δ 2.79 (3H, s), 2.94 (3H, s), 3.01 (2H, t, J = 7.5, 7.5 Hz), 5.07 (1H, d, J = 6.6 Hz), 5.73 (1H, s),
7.17 (1H, d, J = 7.3 Hz), 7.2–7.32 (5H, m), 7.93 (1H, d, J = 8.6 Hz), 8.34 (1H, s), 8.82 (1H, d,
PLOS ONE | DOI:10.1371/journal.pone.0113705 January 28, 2015
30 / 42