Studies on fragment-based design of allosteric inhibitors of human factor XIa

Article abstract of DOI:10.1016/j.bmc.2020.115762

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa's active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.

Full text of DOI:10.1016/j.bmc.2020.115762

Bioorganic&MedicinalChemistry28(2020)115762
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Studies on fragment-based design of allosteric inhibitors of human factor XIa
Rio S. Boothelloa,b,c, Nehru Viji Sankaranarayanana,b, Daniel K. Afosaha,b, Rajesh Karuturia,b
,
⁎
Rami A. Al-Horania,b,d, Umesh R. Desaia,b,
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, United States
b Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, United States
c Hunter Holmes McGuire Medical Center, Richmond, VA 23249, United States
d Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA 70125, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that
promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa mo-
lecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa’s active
site is especially promising because of the possibility of controlled reduction in activity that may offer a route to
safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel al-
losteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the
heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening
linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional
wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies.
Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies
revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies
showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs
bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design
offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.
Fragment-based drug design
Coagulation factors
Anticoagulants
Heparins
Allosterism
1. Introduction
domain and the A3 domain of FXIa each possess a heparin-binding site
(HBS).13–15 Positively charged residues K252, K253 and K255 form the
Thrombin and factor Xa (FXa), two enzymes belonging to the
common pathway of coagulation, are the primary targets of inhibitors
used in the clinic today.1,2 Growing evidence points to the feasibility
and value of targeting enzymes belonging to the intrinsic and extrinsic
pathways of coagulation. One such enzyme that is gaining more en-
hanced emphasis due to its apparent safety features is factor XIa
(FXIa).3–6 Several animal model studies indicate that the inhibition of
FXIa reduces thrombosis with minimal increase in bleeding.7–10 Alter-
natively, selective inhibitors of FXIa impact the pathological con-
sequence (thrombosis) but not the physiological consequence of clot
formation (hemostasis). Hence, there is significant interest in identi-
fying novel strategies of discovering inhibitors of FXIa.
HBS of the A3 domain, whereas K259, R530, R532, K536 and K540
residues form the HBS of the catalytic domain. The former is known to
partake in the heparin-mediated antithrombin inhibition of FXIa
through the template-based mechanism,14,15 the latter is known to be
more important in the allosteric modulation of FXIa functional ac-
tivity.7,16-19
The most often used approach of targeting hFXIa is orthosteric in-
hibition. This has led to the design/discovery of several heterocyclic
agents and natural products as highly promising inhibitors.20–33 Pro-
minent among these are the tetrahydroisoquinoline- (Fig. 1), pyr-
idazine-, pyridazinone-, pyridine-, pyrimidine-and imidazole-based
agents that bind in the active site of hFXIa.21,24,25,29 Another scaffold
that is highly promising is the 10–12-atom macrocyclic scaffold (Fig. 1)
that has yielded orally active agents too.20,22,26–28 Early in the dis-
covery phase, a natural product clavatidine was identified as a
Human FXIa (hFXIa) is a 160 kDa disulfide-linked homodimer
composed of a catalytic domain and the four apple domains that are
linked through a disulfide bond to form the dimer.11,12. The catalytic
Abbreviations: AUC, analytical ultracentrifugation; BS1, binding site 1; BS2, binding site 2; DEGR-FXIa, Dansyl EGR-labeled FXIa; GAG, glycosaminoglycan; HBS,
Heparin-binding site; PMSF, Phenyl methylsulfonyl fluoride; QAO, quinazolinone; SPGG, sulfated pentagalloyl glucoside; UFH, unfractionated heparin
⁎ Corresponding author at: 800 E. Leigh Street, Suite 212, Richmond, VA 23219, United States.
E-mail address: urdesai@vcu.edu (U.R. Desai).
https://doi.org/10.1016/j.bmc.2020.115762
Received 14 April 2020; Received in revised form 24 August 2020; Accepted 6 September 2020
Availableonline13September2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Fig. 1. Examples of factor XIa (FXIa) inhibitors developed so far and sulfated dimeric quinazolinones developed in this work. Several inhibitors, e.g., BMS-962212
and pyridine-based macrocycles, have been developed to bind in the active site of FXIa. Very few allosteric inhibitors have been developed, e.g., SCI. This work
relates to the development of a library of molecules starting from monomeric sulfated QAO fragment 2S. Here, four classes of sulfated QAOs with aliphatic, aromatic
and triazole linkers were developed and studied.
reasonably potent inhibitor of FXIa.31 Interestingly, a fragment-based
strategy was implemented to discover active site inhibitors that have a
5-phenyl-1H-pyrazole-3-carboxamide scaffold.23 The class of orthos-
teric inhibitors display a wide range of potencies, of which a few, e.g.,
BMS-962212, EP-7041 and ONO-7684, are being studied in clinical
trials.
progressive variation in inhibition potency. In fact, we discovered that
the traditional wisdom of increasing linker flexibility leading to poor
potency does not hold true for this group of molecules inhibiting hFXIa.
Interestingly, the inhibition profiles could be explained by computa-
tional studies. We also show that certain related linkers, e.g., more
rigid, aromatic linkers, are not preferred, which points to the interesting
landscape of heparin-binding site of hFXIa. Overall, this work shows
that fragment-based approach offers considerable promise for the de-
sign of heparin-binding site-directed allosteric inhibitors of hFXIa.
In direct contrast orthosteric inhibitors, few allosteric inhibitors
have been designed/discovered. Whereas orthosteric inhibition affords
dose of the inhibitor as the only means of varying enzyme activity,
allosteric inhibition offers the promise of controlling FXIa activity by
modulating the efficacy of inhibition (ΔY) also. Alternatively, orthos-
teric inhibitors fully inhibit target enzyme at saturation (ΔY = > 90%);
in contrast, appropriately designed allosteric inhibitors can regulate
target enzyme at saturation (ΔY = 25–75%). Thus, allosterism is the
only means of discovering partial inhibitors, which may lead to much
higher level of safety for coagulation proteases (FXIa and others),
thereby eliminating the high risk of bleeding besetting all clinically
used anticoagulants today.34
2. Results
2.1. Fragment-based screening using molecular modeling
Our earlier studies led to identification of three allosteric inhibitors
of hFXIa including sulfated QAOs,16 sulfated pentagalloyl gluco-
sides17,18 and sulfated benzofurans.19 These molecules are structurally
different and appear to utilize different allosteric sites. Our earlier work
indicated that of these different sites, sulfated QAOs induced inhibition
through the catalytic domain alone (FXIa-CD) because the A3 domain
was not found to be important for activity.16 Hence, we assessed the
catalytic domain of hFXIa (PDB ID: 1ZOM) for fragment-based drug
design process.
Unfortunately, there are no obvious routes of discovering allosteric
partial inhibitors. We have employed various strategies to discover such
agents including the dual element strategy16,19 and the heparin-mi-
micking strategy.7,17,18 Of these, the latter group has led to the iden-
tification of SCI (Fig. 1),7 while the former have yielded sulfated qui-
nazolinone (QAO) and sulfated benzofuran scaffolds.16,19 The major
advantage of these inhibitors was their hydrophobic scaffold (Fig. 1),
which was completely different from highly hydrophilic, polysulfated,
natural ligand heparin that binds in the HBS of hFXIa. We reasoned that
a computational fragment-based approach may help improve potency
for sulfated QAO inhibitors, which offer an excellent framework for
designing/discovering allosteric regulators of FXIa. This work describes
the implementation of this fragment-based approach so as to de-
rive ~ 40-fold more potent allosteric QAOs. We developed a group of
28 dimeric sulfated QAOs with intervening linkers that displayed a
First, the entire surface of FXIa’s catalytic domain was scanned for
the plausible sites of binding for sulfated QAO 2S, a prototypic frag-
ment. Binding pockets near several positively charged residues in-
cluding K62, K107, K127, R147, K175, R185, and K202 were identified
as viable candidate sites (Fig. 2A). Using these residues as the loci and
the length of sulfated QAO as the radius (~14 Å), seven centroids were
identified as potential sites of binding (Fig. 2A). In combination, this
fragment search covered nearly 98% of the surface. Incidentally, the
surface remaining unscreened (~2%) did not encompass any positively
charged residue, although polar residues were present.
2

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
A
B
C
R222
R147
R185
R185
K175
K202
K127
E223
BS1
R171
K170
R224
R173
Y172
E217
Q169
R173
K107
K63
BS2
I176
H174
K175
Fig. 2. Fragment-based docking and scoring approach to identify potential sites of binding on hFXIa. A) hFXIa surface was divided into putative binding domains that
could accommodate sulfated QAO 2S in the vicinity of Arg/Lys residues. Different colored domains represent different binding domains around a basic residue
(shown in spheres, green color by atom). B) Sulfated QAO 2S preferentially occupied two distinct binding sites BS1 and BS2. C) The preferential interaction arose
from multiple hydrogen bond (shown as spheres, white color by atom) and non-bonded (shown as sticks, pink color by atom) interactions with residues constituting
BS1 and BS2. These residues include basic residues as well as polar residues.
Sulfated QAO 2S was docked onto each of the seven potential sites
using a dual filter, genetic algorithm-based docking protocol developed
in our laboratory earlier for sulfated molecules.35–37 The electronic
steering engineered by the presence of the lone sulfate group of the
fragment was found to induce favorable interaction with several sites,
however, hydrophobic and steric forces generated differential binding
interactions. Two parameters, GOLDscore and RMSD (root mean square
deviation), which correspond to the strength of interaction and con-
sistency of binding, respectively, were used to parse the different geo-
metries. The results revealed that the interaction of sulfated QAO was
significantly biased towards two sites BS1 and BS2 (Fig. 2B). These two
sites are approximately 9 – 12 Å away from each other, which offers the
possibility of fragment-based design of dimeric sulfated QAO agents.
Interestingly, R173 was one of the residues making a key hydrogen
bonding interaction for both binding modes. Previous studies on human
thrombin had shown that this residue plays an important role in the
recognition of a sulfated benzofuran inhibitor and its replacement
(R173 → A) led to a defect of ~ 22-fold.37,38 This implied that the two
fragments could perhaps be linked through an appropriate linker to
develop novel inhibitors with higher potency. Thus, we embarked on
the program of synthesizing dimers of sulfated QAO 4S—21S,
24S—29S and 33S—36S that rely on either aliphatic, aromatic or
triazole linkers.
azide-alkyne cycloaddition (CuAAC) reaction (Schemes 2 and 3).16,41
Whereas the majority of synthesized library members are homo-di-
meric, 33S—36S could be considered as hetero-dimeric because one of
the monomers is a flavonoid scaffold. Likewise, diversity also arises
from the length of the different linkers. Whereas some are short (e.g.,
aromatic and alkenic linkers), others are much longer (e.g., dodecyl
linker).
For the aromatic and aliphatic linker containing dimers, the QAO
core scaffold was synthesized using a condensation reaction between
anthranilamide and suitably substituted benzaldehyde to obtain a small
group of QAO monomers (Scheme 1), which was followed by phenol
protection and substitutive dimerization with target α,ω-dibromo lin-
kers. These dimeric QAOs were then de-protected using lithium hy-
droxide and sulfated under microwave conditions with sulfur triox-
ide–amine complex in the presence of an organic base, as reported
earlier (Scheme 1).16,39 For the triazole linker containing dimers, the
copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction was
exploited. Appropriate QAO or flavonoid alkynes and azides (Schemes 2
and 3) were synthesized from corresponding monomers using acetyla-
tion of phenolic groups and nucleophilic displacement strategy fol-
lowed by deacetylation. The corresponding alkynes and azides were
coupled in the presence of aqueous CuSO4 and sodium ascorbate giving
1,2,3-triazoles 24–29 (Scheme 2) and 33–36 (Scheme 3), which were
sulfated under microwave conditions to obtain final sulfated homo-
(24S—29S, Scheme 2) or hetero- dimers (33S—36S, Scheme 3). The
final sulfated QAOs were obtained in > 95% purity as demonstrated
by UPLC analysis.
To first assess whether a sulfated QAO monomer binds and inhibits
hFXIa, we synthesized 2S in 2 steps following our earlier work em-
ploying microwave-based sulfation.39,30 Dose-dependence of hFXIa in-
hibition using the chromogenic substrate hydrolysis assay led to an IC50
of 375
21 μM (not shown). Likewise, fluorescence-based binding
affinity titrations yielded hFXIa affinity of 365
4 μM (see below).
2.3. Sulfated QAO inhibition of hFXIa and related proteases
Although this potency appears to be low, taking the small size of the
fragment into consideration, we reasoned that it bodes well for the
further design.
Sulfated QAOs were studied for inhibition of hFXIa and related
enzymes using chromogenic substrate hydrolysis assays under optimal
conditions defined for each enzyme (see Methods). The dose-depen-
dence of protease activity was fitted using the logistic equation to cal-
culate the IC50 (Table 1). The o, m and p-xylene linker containing sul-
fated QAOs, possessing 4, 5 and 6 intervening atoms, respectively,
displayed potencies in the range of 57 to 248 μM, which were sig-
nificantly better than that for the monomeric fragment. Interestingly,
the ortho- (7S, 8S) and meta- (5S, 6S) xylene containing inhibitors,
which should display bent geometries, displayed better potency than
the para- substituted molecule, which represents a linear linker
(Table 1).
2.2. Synthesis of the library of diverse dimeric sulfated QAOs
We synthesized a total of 28 sulfated QAO dimers carrying struc-
turally diverse linkers with varying lengths and geometries. The di-
versity of linkers arises from their aromatic, aliphatic and triazole
structures. The aromatic (4S—8S) and aliphatic (9S—21S) linker con-
taining sulfated QAOs were synthesized using a nucleophilic substitu-
tion reaction (Scheme 1), whereas the triazole-based sulfated QAOs
24S—29S and 33S—36S were assembled using a copper-catalyzed
3

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Scheme 1. Synthesis of Sulfated QAOs 4S-21S. aNote: 18S contains a methoxy and a sulfate group each.
Scheme 2. Synthesis of sulfated QAOs 24S – 29S.
Considering the above result on the preference of linker with bent
geometry, we studied a rigid trans-but-2-ene linker containing sulfated
QAO (12S). However, this molecule was no better than the starting
fragment. This implied that longer linker length may be important for
recognition of both sites of binding. Hence, we synthesized three sul-
fated QAOs with aliphatic linkers containing 5 intervening atoms. Of
decrease in inhibition potency to 16.6 and 14.5 µM, respectively.
To assess whether introducing hydrogen bonding partners in the
linker alter affinity/inhibition properties, we synthesized the library of
homodimeric sulfated QAOs 24S—29S containing a triazole linker. This
also allowed us to screen extended linker lengths. However, the IC50
s
did not improve over that measured for 21S. We then synthesized
heterodimeric sulfated QAOs 33S—36S, which contain one tetra-
sulfated flavonoid fragment. Yet, the IC50s of these heterodimeric
structures did not yield a better molecule. In fact, the potencies de-
creased significantly for at least two of these agents (Table 1) sug-
gesting a strong preference for the monosulfated hydrophobic frag-
ments.
—
the three, the 4-OSO3 substituted QAO (11S) yielded ~ 7-fold in-
crease in potency as compared to the monomeric fragment, whereas
that with the 3-propoxypropane linker (12S) nearly lost all of its po-
tency. This implied that both the position of sulfate group on the two
fragments and the linker length are important.
We reasoned that an extended linker would probably serve to place
the two QAO fragments better within the two hydrophobic sites. Hence,
we synthesized 4-OSO3— substituted sulfated QAOs 13S—21S carrying
progressively longer alkylenic linkers. To our surprise, increasing the
linker length from 6 to 9 intervening atoms, the IC50 decreased from
34.3 to 8.2 μM, which represent a substantial 10- to 43-fold enhance-
ment in potency over the monomeric fragment (Table 1). Further in-
crease in linker length to 10 and 11 intervening atoms yielded a slight
To assess the selectivity profile of these compounds, we evaluated
inhibition of several serine proteases including thrombin, FVIIa, FIXa,
FXa, plasma kallikrein, trypsin and chymotrypsin. Screening was per-
formed using an appropriate chromogenic substrate (see Methods).
Briefly, no inhibition of these proteases was observed at concentration
as high as 500 μM (> 63-fold selectivity, not shown), except for
20S—21S that were found to inhibit FXa and trypsin at > 200 μM
4

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Scheme 3. Synthesis of sulfated QAOs 33S – 36S.
(> 25-fold selectivity, not shown). This implied that the selectivity was
maximal when the linker length was 9 atoms. Further increase in linker
length led to higher potency against FXa and trypsin, thereby reducing
the selectivity. Overall, specific dimeric sulfated QAOs containing de-
fined aliphatic linkers were identified as selective inhibitors of FXIa.
QAO monomer (2S), sulfated QAO dimer (18S) and disulfated QAO
dimer (19S) interacting with active site dansylated-hFXIa (EGR-hFXIa).
Whereas the monomer displays a hyperbolic interaction profile, the two
dimers display a classic sigmoidal interaction profiles alluding to a co-
operative phenomenon. Alternatively, this suggests that one of the
monomers of a sulfated QAO dimer first binds to hFXIa followed by a
conformational change resulting in better binding of the second
monomer of the dimer. Because the dansyl group is located in the active
site of hFXIa, the conformational change disfavors substrate binding
resulting in inhibition.16
2.4. Mechanism of inhibition of sulfated QAOs
Computational studies predicted the site of binding of these in-
hibitors in the HBS of the catalytic domain of FXIa. This implies an
allosteric mode of inhibition. We studied the kinetics of Spectrozyme
FXIa hydrolysis by FXIa in the presence of 19S. A characteristic hy-
perbolic substrate hydrolysis profile was observed (Fig. 3), which could
be analyzed using the standard Michaelis–Menten to calculate the KM
and VMAX. As expected, the KM of Spectrozyme FXIa was found to be
The interaction profiles were analyzed by either the quadratic
binding equation (2S) or the three parameter Hill cooperativity equa-
tion to calculate the dissociation constant (KD) of binding and the Hill
co-operativity coefficient (n). Table 2 lists these parameters. Overall,
the KD of the monomer (2S) was ~ 3.2-fold higher than that for 18S,
which in turn was higher by ~ 18.3-fold than that for 19S. These results
further support the concept of bridging monomeric fragments. Ad-
ditionally, the results highlight the importance of one sulfate group
each on two monomers of a dimer.
invariant as the concentration of 19S was varied from
(0.27 0.01 mM) to 120 μM (0.26
decreased from a high of 36.9
0
0.32 mM), while the VMAX
1.5 mAU/min to a low of 9.7 0.37
mAU/min for the same concentration range (Fig. 3). This is char-
acteristic of a non-competitive mechanism of inhibition and follows the
profiles known for other heparin mimetics reported in the literature to
date.16–19,37,38
2.6. Sulfated QAO dimers bind in the HBS of the catalytic domain
To identify the site of binding, we utilized three mutants of hFXIa,
including a K252A, K253A, K255A mutant and a K529A, R530A,
R532A, K535A K539A mutant, which are respectively the HBSs of the
A3 and the catalytic domain.13-15 The third chimeric mutant combined
these replacements in both HBSs. Fig. 5 shows the inhibition profile of
sulfated dimer 19S against the three mutants. Whereas the 19S potency
against hFXIaA3 (14 μM) and wild type hFXIa (FXIaWT) (20 μM) were
2.5. Sulfated QAO dimers induce allosteric change in hFXIa
To further understand whether the non-competitive mechanism
results from remote changes induced in the active site of hFXIa, we
studied fluorescence properties of active site labeled hFXIa in the pre-
sence and absence of sulfated QAOs. Fig. 4 shows titrations of sulfated
5

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Table 1
2.7. Do sulfated QAOs induce oligomerization of hFXIa?
Inhibition of hFXIa by sulfated QAOs.a
Although the results described above clearly demonstrate direct
allosterism through the HBS as the reason for inhibition, we reasoned
that the unusual structure of sulfated QAOs, especially the long length
and aliphatic nature of the linker, could induce oligomerization of
hFXIa. Fig. 6A shows this mechanism at a theoretical level. Briefly, one
monomer of the sulfated QAO dimer could bind to one hFXIa dimer,
whereas the other monomer of the sulfated QAO dimer could rope in
another hFXIa dimer. Alternatively, sulfated QAO dimers could induce
higher order oligomers, e.g., tetramers, hexamers, etc.
Inhibitor
LLb
IC50 (μM)
ΔY (%)
4S
6
248
60
3c
100
3c
5S
5
1
1
2
2
92
5
6S
5
76
96
6
4
3
7
2
3
7S
4
94
93
8S
4
57
87
9S
4
415
89
10
96
10S
11S
12S
13S
14S
15S
16S
17S
18S
19S
20S
21S
24S
25S
26S
27S
28S
29S
33S
34S
35S
36S
5
1
1
96
5
49
92
5
401
53
22
94
16
5
3
3
4
2
4
8
2
6
2
2
4
4
2
4
3
6
8
6
To test this, we performed native gel electrophoresis. We studied the
interaction of 19S with both hFXIa and hFXIa-PMSF, a covalently in-
hibited enzyme. Fig. 6B shows the gel image of hFXIa, FXIa-19S, hFXIa-
PMSF and hFXIa-PMSF-19S. Each protein was observed as a single band
corresponding to the enzyme’s expected dimeric molecular weight
suggesting the absence of higher order oligomers. We also utilized
analytical ultracentrifugation (AUC) to assess the oligomerization state
of hFXIa and its complex with 19S. hFXIa alone was found to ex-
clusively exist as a dimer (s = 6.6S), which is in line with litera-
ture.11,42 More importantly, the hFXIa-19S complex also exhibited di-
meric form (s = 6.6S) (Fig. 6C). Thus, both native PAGE and AUC
experimentation indicate that sulfated QAOs, especially 19S, do not
induce dysfunction in hFXIa through induction of higher order oligo-
mers.
6
1
2
1
1
1
96
6
34
87
6
35
95
7
23
96
8
20
81
9
173
8.2
16.6
14.5
51
1
93
9
0.5
101
95
10
11
11
12
13
14
15
16
11
12
13
14
0.3
0.8
96
2
2
2
2
2
2
103
100
100
112
98
39
34
22
30
35
112
93
139
133
37
10
13
92
4
6
110
106
51
2.8. Computational studies indicate preference for aliphatic linkers of
defined length
a
The IC50 and ΔY values were calculated from nonlinear regression analysis
of direct inhibition of hFXIa (see Methods).
b
LL is linker length, the minimum number of intervening atoms in the
To assess whether the unique linker length dependence could be
computationally explained based on the predicted sites of binding of
the monomeric fragment 2S, we studied 18 QAOs with aliphatic linkers
and 3 QAOs with the triazole linker. Inhibitors 4S—21S and 24S—26S
were modeled and docked onto FXIa’s CD using GOLD.35–38 For each
inhibitor, the highest scoring 20 poses were selected and analyzed in
detail. Table 4 lists the average GOLDScore of poses. Inhibitor 19S
displayed the best score supporting the solution experiments on its FXIa
inhibition potential. More importantly, as a group a significant corre-
lation was noted between the GOLDScore and IC50 with a correlation
coefficient of ~ 0.6 (Fig. 7A).
linker.
c
Errors represent
1 SE.
The preferred binding pose of each inhibitor was further analyzed to
ascertain whether linkers play a biophysical role in interaction.
Hydrogen bond interactions and the distance between N atoms of the
two QAO were measured. The best sampled pose of each inhibitor is
shown in Fig. 7B–G. Inhibitors 4S—12S displayed multiple orientations
(Fig. 7B and E) and tended to primarily occupy BS1 (see Fig. 2B). In-
hibitors 13S—19S (Fig. 7C and F) displayed similar binding orienta-
tion. More specifically, one sulfated QAO monomer in these dimers
displayed strong binding to BS1; however, the second monomer pre-
ferred to dock ~ 3–5 Å away from the BS2 (Figs. 7C and 2B). Finally,
inhibitors 20S and 21S with slightly longer linkers displayed a ~ 60°
rotated binding orientation and significantly lower consistency (Fig. 7D
and G). These results support the IC50 results that the optimal number
of intervening atoms is in the range of 8 to 10 atoms (Table 4).
To further assess the role of linkers, we also studied inhibitors
24S—26S containing triazole linkers. Each of the three inhibitors ex-
hibited lower average GOLDScores in comparison to aliphatic linkers
(Table 4). More importantly, the binding poses derived from multiple
docking and scoring runs showed large inconsistency of binding. In fact,
the RMSD of the top docked poses were greater than 12 Å, whereas the
aliphatic linkers tended to display significantly high consistency
(RMSD < 3 Å). In combination, these results suggest that hFXIa pre-
fers inhibitors with aliphatic linker of defined length.
Fig. 3. Michaelis-Menten kinetics of Spectrozyme FXIa hydrolysis by hFXIa in
the presence of sulfated QAO 19S. The initial rate of hydrolysis at various
substrate concentrations was measured spectrophotometrically in pH 7.4 buffer
at 37 °C. Solid lines represent nonlinear regressions using the standard
Michaelis equation to yield KM and VMAX
.
very similar, that against hFXIaCD and hFXIaA3-CD decreased nearly 3-
fold (Table 3). This indicated that for 19S to bind to hFXIa, the CD is
important but A3 domain is not. The ~ 3-fold loss in potency is not a
large change and suggests that other residues that provide binding
energy may be involved. These residues could be nearby basic residues
as well as hydrophobic or polar residues. Identifying the cohort of re-
sidues that contribute binding forces for selective recognition of 19S is
important but was not undertaken. Overall, the mutagenesis results
support the computational prediction that sulfated QAO fragments
would target the HBS of the hFXIa’s CD. The results also support the
allosteric mechanism of inhibition deduced through enzyme kinetics.
6

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Fig. 4. Measurement of affinity of A) sulfated QAO
dimers 16S (triangles), 17S (diamonds) and 19S
(squares) and B) sulfated QAO monomer 2S (cir-
cles) for human plasma factor XIa (FXIa). Titrations
were performed using the change in fluorescence of
dansyl group present in the active site of FXIa. The
emission of dansyl group was measured at 547 nm
(λEX = 345 nm) in 50 mM Tris–HCl buffer, pH 7.4,
containing 150 mM NaCl and 0.1% PEG8000 at
37 °C. Solid lines represent non-linear regressions
using either the Hill equation (1) (16S, 17S and
19S) or the standard hyperbolic equation (2S) to
obtain the ΔFMAX and KD of binding.
A
B
300
0
-5
250
200
150
100
50
-10
-15
-20
-25
0
0
20
40
60
80
0
200
400
600
800
[Inhibitor] µM
[Inhibitor] µM
3. Discussion
Table 2
Binding parameters of sulfated QAOs interacting with hFXIa.a
Human factor XIa (hFXIa) has attracted high attention lately be-
cause of multiple lines of evidence that indicate a strong possibility of
devising a much safer anticoagulation therapy. Several reviews have
been published touting the promise of targeting hFXIa.3–6 A 2015 study
with a FXI-directed antisense oligonucleotide on 300 patients under-
going elective knee replacement gave sufficient evidence that this
promise may hold true.8 This development gave new impetus to the
discovery of active site inhibitors of hFXIa.20–30 Many of these studies,
especially using animal models of thrombosis, continue to support the
promise that targeting hFXIa is likely to yield reduced bleeding com-
plications. In fact, two small molecules labeled EP-7041 and ONO-7684
are currently being studied in phase I clinical trials (NCT02914353 and
NCT03919890, respectively; www.clinicaltrials.gov).
Inhibitor
ΔFMAX (%)
n
KD (μM)
2S
−34
301
265
1.8b
n/ac
4.3
365
115
19.9
4.2
6.5
0.5
18S
19S
1.4
3.1
0.5
0.4
5.0
a
Titrations were performed by adding aliquots of a solution of a sulfated
QAO 2S, 18S or 19S to 250 nM FXIa–DEGR in 50 mM Tris–HCl buffer, pH 7.4,
containing 150 mM NaCl and 0.1% PEG8000 at 37 °C and monitoring the
change in fluorescence at 547 nm (λEX 345 nm). The maximal fluorescence
change (ΔFMAX), Hill coefficient ‘n’, and KD were calculated using nonlinear
regressional analysis (see Methods).
b
Error = 1 SE. cnot applicable.
Unfortunately, none of the active site inhibitors that are being de-
veloped so far offer the possibility of intrinsic regulation such as partial
antagonism. Active site inhibitors can either bind or not bind in the
active site and induce either full inhibition (100%) or no inhibition
(0%). Intermediate efficacies of inhibition, e.g., 30 – 60%, are not
available. For this reason, we have pursued allosteric inhibitors of
hFXIa to identify partial antagonists, or partial inhibitors, which may
potentially lead to much safer anticoagulants. Our search has led to
design of several allosteric hFXIa inhibitors including SPGG,17,18 SCI7
and sulfated benzofurans.19 Although some of these molecules, espe-
cially SCI, show that excellent inhibition potency and selectivity
without much bleeding risk, scaffolds that offer partial antagonism re-
main to be discovered.
Fig. 5. Dose-response profiles for 19S inhibition of recombinant wild-type FXIa
(FXIaWT) and FXIa proteins carrying alanine replacements of heparin-binding
residues in the catalytic domain (FXIaCD), A3 domain (FXIaA3) and both cata-
lytic and A3 domains (FXIaCD&A3) in 50 mM Tris-HCl buffer, pH 7.4, containing
150 mM NaCl, 0.1% PEG8000 and 0.02% Tween80 at 37 °C. Solid lines re-
present dose response fits to calculate the potency (IC50) and maximal inhibi-
tion efficacy (ΔY).
Rational design of partial antagonists is difficult. As of today, no
specific principles have been put forward to discover partial antago-
nists. Majority of partial antagonists, especially of multi-domain cell
surface receptors, have been derived from screening of natural pro-
ducts. In fact, partial antagonists of freely soluble enzymes are ex-
tremely difficult to find. We recently discovered that a sulfated ben-
zofuran monomer bound in the heparin-binding site of human alpha-
thrombin to induce ~ 50% inhibition.43,44 This molecule is the first
partial allosteric inhibitor of a soluble enzyme identified to date and
fueled the expectation that partial inhibitors of hFXIa should be pos-
sible to discover too.
Table 3
Inhibition of hFXIa variants by 19S.a
Mutant
IC50 (μM)
ΔY (%)
FXIaWT
20.0
14.0
34.0
32.0
0.8b
93
3
2
3
This work stems from this expectation and employs the strategy of
fragment-based design starting from a small molecule that is known to
bind in the target binding site. Our earlier work had shown that
monomeric sulfated QAO binds in heparin-binding site of hFXIa.16 Here
we utilized molecular modeling to assess whether the heparin-binding
site of hFXIa can accommodate more than one small sulfated fragments.
We found that 2S, the monosulfated QAO fragment, is likely to bind in
two unique sub-sites that are separated by ~ 10 Å, which is particularly
amenable for fragment-based drug design. Thus, an optimal inhibitor
FXIaA3
0.4
1.5
1.0
99
FXIaCD
96
FXIaCD&A3
105
3
a
The IC50 and ΔY were calculated following nonlinear regression analysis of
direct inhibition of hFXIa mutants (see Methods). Inhibition was monitored by
spectrophotometric measurements of residual hFXIa activity.
b
Errors represent
1 SE.
7

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Table 4
Fragment based structural design of sulfated QAO inhibitors for Factor XIa.
Inhibitor LBDa (Å) GOLDScoreb Hbondsc Interacting residuesc
4S
6.4
105.3
106.6
100.0
93.7
6
R171, R185, R224
R171, H174, R185, R224
R173, R224
5S
6.7
6
6S
6.4
9
7S
5.8
9
K170, R171, R224
R171, R173, R185, R224
R171, Q221, R224
R171, R173, H174, R224
K170, R173, R185, R224
R171, R185, R224
K170, R171, R173, R185, R224
R171, R173, R185, R224
R171, R173, R185, R224
R171, R173, R185, R224
R171, R173, R224
R173, R224
8S
5.8
105.0
102.6
104.6
109.1
103.7
109.2
108.3
107.3
109.6
108.6
94.3
9
9S
4.6
8
10S
11S
12S
13S
14S
15S
16S
17S
18S
19S
20S
21S
24S
25S
26S
6.4
7
7.1
10
7
5.9
8.9
10
10
10
11
9
8.4
8.6
9.9
10.9
11.6
11.4
12.9
12.5
NAd
NA
4
114.7
107.1
108.5
102.3
99.0
10
6
K170, R171, R173, R185, R224
R171, R173, R224
R171, R173, Q221, R224
NA
8
NA
NA
NA
NA
NA
96.1
NA
a
LBD is the average distance between the two nitrogen atoms of each QAO
fragment for the top 20 poses.
b
c
Average GOLDScore of top 20 poses from 1000 GA runs of each molecule.
The number of consistent hydrogen bonds and interacting residues for top
20 docked poses.
d
Not applicable because of inconsistency of binding.
exactly opposite behavior is observed here. The reason appears to be
based on the mode of binding suggested by computational studies
(Fig. 2). These indicate that the orientation of the two monomeric
sulfated QAOs in the two sub-sites is not face-to-face, which necessi-
tates a round-about, flexible linker, thereby requiring 9 intervening
atoms. In fact, molecular modeling studies show a preference for this
linker length (Fig. 7), while not completely ruling out longer linkers.
Yet, the potencies of homodimeric sulfated QAOs (i.e., 20S and 21S,
Table 1) start to decrease as linker length increases to 10 and 11 atoms.
This suggests enhanced flexibility of the linker is likely to be detri-
mental. The FXIa inhibition potency of the best molecule discovered in
this study, i.e., 19S, is reasonable (IC50 ~ 8 μM). Although not attrac-
tive for in-depth animal model studies, it would be worth the effort to
study anti-thrombotic as well as bleeding consequences of this scaffold
so as to direct future design efforts.
Fig. 6. A) Two possible models of sulfated QAO dimer-dependent inhibition of
FXIa including allosteric modulation of the FXIa dimer or induction of oligo-
merization of FXIa dimer. Gradient native PAGE (B) and analytical ultra-
centrifugation (C) analysis of hFXIa and hFXIa–19S complex. A) Electrophoresis
of hFXIa (lane 1), hFXIa–19S complex (lane 2), fFXIa–PMSF (lane 3), FXIa–19S
complex–PMSF (lane 4), hFXIa treated with DTT, and hFXIa–19S complex
treated with DTT was performed using 4 – 20% gradient gel. B) AUC profiles of
hFXIa alone (black) or hFXIa–19S (red) were monitored at A280 and trans-
formed into relative concentrations of each species (CS) at varying sedimenta-
tion co-efficient (S). Note: PMSF = phenylmethylsulfonyl fluoride.
These fundamental results lead to the concept that better agents
could be discovered by replacing part of the homodimer with a frag-
ment that binds in a more favorable, direct, face-to-face orientation. A
key conclusion here is that alternative fragment(s) of QAOs should be
designed/discovered. One such fragment is the sulfated flavonoid
scaffold, which could be thought of as a polysulfated version of the
monosulfated QAO scaffold. In fact, we had evaluated this scaffold
earlier for hFXIa. However, the extent of studies was restricted to linker
lengths of 4 – 8 atoms.16
should be possible to discover by linking the two monosulfated QAO
fragments.
To assess whether the higher sulfated flavonoid fragment coupled
with longer linkers may engineer higher potency, we compared
homodimers 24S—29S with heterodimers 33S—36S. Unfortunately,
none of the agents were found to be better than the aliphatic homo-
dimers. This was a little surprising. Yet, the results convey the value of
aliphatic linkers, while also perhaps suggesting that the triazole-based
linker introduces unfavorable interactions and/or limited flexibility,
thereby reducing potency. Our molecular modeling studies also support
these conclusions (Table 4). A key conclusion from these results is that
sulfated heterodimers with aliphatic linkers of 8 – 10 intervening atoms
may yield agents with IC50s lower than 10 µM.
To a large extent, this work supports the expectation originating
from fragment-based design strategy. Synthesis, enzyme activity assays,
enzyme kinetics and biophysical studies of more than two dozen sul-
fated QAOs show that unique dimeric sulfated QAOs display more than
40-fold increase in potency over the monomeric fragment, bind pre-
ferentially with basic residues of the heparin-binding site present in
catalytic domain, and induce co-operative disruption of the active site.
An extremely interesting result is the increase in inhibition potency
as the length of the linker increases to 9 intervening atoms (i.e., 4
through 11). A priori, common principles of medicinal chemistry state
that longer alkyl chains should enhance flexibility of the molecule,
which should theoretically reduce affinity for protein targets. Yet, an
Finally, the efficacy of each inhibitor studied in this work was >
90% (Table 1). Alternatively, no partial allosteric inhibitor was
8

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
Fig. 7. Computational results on sulfated QAOs support biochemical inhibition results. A) The GOLDScores following genetic algorithm docking and scoring studies
show significant correlation with log(IC50) (r2 of ~ 0.6) suggesting a good possibility of inhibition arising from dual mode binding of the sulfated QAO dimers. Note:
Sulfated QAOs with IC50 > 200 were not included in the analysis. The best binding poses of sulfated QAOs 4S – 12S, 13S – 19S, and 20S – 21S are shown in panels
B, C and D, respectively. Each molecule is shown in a different color representation. Inhibitor 19S is orange colored and it is also shown as reference in panel C.
Panels E, F and G represent zoomed in versions of panels B, C, and D, respectively. As the linker length increased to 10 atoms and above, there was a noticeable ~ 60°
difference in orientation of binding and interactions became weaker.
discovered. Yet, the results convincingly support the rationale that
fragment-based strategy could rapidly lead to significant enhancement
in inhibition potency. As noted earlier by us in the discovery of the sole
partial allosteric inhibitor of human alpha-thrombin,43,44 we predict
that screening a much larger library of such agents is necessary to
identify the first partial inhibitor of hFXIa.
St. Louis, MO), as described in our earlier publications on studies of
non-saccharide glycosaminoglycan mimetics.35–37 Inhibitors 2S,
4S—21S and 24S—26S were docked in the catalytic domain of hFXIa
using GOLD.35 The first step of fragment-based design utilized a region
of 20 Å around each of Lys63, Lys107, Lys127, Arg147, Lys125, Arg185
and Lys202 (chymotrypsinogen numbering system) residues as the site
of binding (Fig. 2). For each site, 1000 genetic algorithm runs were
employed. Each resulting pose was scored and best two poses with
highest GOLDScores were retained. Then, triplicate docking runs were
employed to assess consistency of binding, which equates to selectivity
of recognition. The six docked poses per site were analyzed for RMSD
(root mean square deviation) using in-house Sybyl script and the site
with lowest RMSD value was selected as the preferred site of binding.
To assess the aliphatic dimers with variable linker lengths, a similar
4. Experimental methods
4.1. Molecular modeling studies
The coordinates for the activated catalytic domain of hfXIa were
obtained from the protein data bank (PDB ID: 1ZOM).12 Molecular
modeling was performed using Tripos Sybyl-X v2.1 (Tripos Associates,
9

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
docking and scoring strategy was followed except for few differences.
The binding site was defined as an area of 28 Å around the BS1 and BS2,
two loci obtained from sulfated QAO monomer binding studies. Al-
though the number of genetic algorithm runs for each molecule re-
mained the same, we retained all 1000 poses for analysis of preferred
orientation of the linker and two monomer fragments.
reaction between anthranilamide and suitably substituted benzalde-
hyde in presence of sodium hydrogen sulfite and catalytic amounts of p-
toluenesulfonic acid, as previously described.16 The free hydroxyl
group(s) were acetylated in dichloromethane in the presence of N,N-
diisopropylethylamine (DIPEA) (2.0 equiv per hydroxyl group) and
acetic anhydride (1.0 equiv per hydroxyl group), as described earlier.16
4.2. Chemicals, reagents and analytical chemistry
4.6. General procedure for dimerization of QAO monomers
Anhydrous CH2Cl2, THF, CH3CN, DMF, DMA, acetone and other
solvents were reagent grade and purchased from Sigma-Aldrich
(Milwaukee, WI) or Fisher (Pittsburgh, PA). Analytical TLC was per-
formed using UNIPLATETM silica gel GHLF 250 μm pre-coated plates
(ANALTECH, Newark, DE). Column chromatography was performed
using silica gel (200–400 mesh, 60 Å) from Sigma-Aldrich. Chemical
reactions sensitive to air or moisture were carried out under nitrogen
atmosphere in oven-dried glassware. Reagent solutions, unless other-
wise noted, were handled under a nitrogen atmosphere using syringe
techniques. Flash chromatography was performed using Teledyne ISCO
(Lincoln, NE) Combiflash RF system and disposable normal silica car-
tridges of 30–50 µ particle size, 230–400 mesh size and 60 Å pore size.
The flow rate of the mobile phase was in the range of 18 to 35 mL/min
and mobile phase gradients of ethyl acetate/hexanes and CH2Cl2/
CH3OH were used to elute compounds.
K2CO3 (2.5 eqv.) was added to solution of 1 – 3 in DMF under
stirring (Scheme 1), which was followed by addition and agitation of an
appropriate aryl/alkyl dibromide (0.5 eqv.) for 12 h. Following com-
pletion of reaction, confirmed by TLC, the mixture was acidified with
water and extracted with ethyl acetate. The products were purified
using flash chromatography on silica gel (10 – 50% ethyl acetate in
hexanes) in 70 – 80% yield.
4.7. General procedure for deprotection of acetylated QAO dimers
Acetylated dimers were first solubilized in THF and exposed to
LiOH·H2O (4 eqv.) under vigorous stirring at room temperature for 4 –
6 h. The reaction mixture was extracted using ethyl acetate and product
purified using flash chromatography on silica gel (20 – 70% ethyl
acetate in hexanes) to give the deacetylated QAO dimers 7–21 in 70 –
80% yields.
4.3. Proteins and chromogenic substrates
4.8. General procedure for synthesis of sulfated QAOs
Human plasma proteases including thrombin, FXa, FIXa, and FXIa
were obtained from Haematologic Technologies (Essex Junction, VT).
Active site-labeled hFXIa, i.e., FXIa-DEGR, was also obtained from
Haematologic Technologies. Bovine α-chymotrypsin and bovine trypsin
were obtained from Sigma-Aldrich (St. Louis, MO). Stock solutions of
FXIa, thrombin, trypsin, FXIa-DEGR and chymotrypsin were prepared
in 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.1%
PEG8000 and 0.02% Tween80. Stock solutions of FXa were prepared in
20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM
CaCl2, 0.1% PEG8000 and 0.02% Tween80. Chromogenic substrates,
Sulfation of phenolic precursors was achieved using microwave
assisted chemical sulfation, as described earlier.16,39,40 Briefly, to a
stirred solution of the appropriate polyphenol in an. CH3CN (1–5 mL) at
room temperature, Et3N (10 eqv. per –OH group) and Me3N:SO3 com-
plex (6 eqv. per –OH group) was added. The reaction vessel was sealed
and micro-waved at 50 W (CEM Discover, Cary, NC) for 30 min at 90 °C.
The reaction mixture was cooled and transferred to a round bottom
flask, volume reduced as much as possible, and then directly loaded on
to
a flash chromatography column for purification using di-
Spectrozyme
TH
(H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide),
chloromethane and methanol solvent system (5–20%) to obtain the
quaternary amine conjugated sulfated QAOs. Flash chromatography
when performed in this manner helps remove inorganics and aids
purification. For sodium exchange, the samples were loaded onto a SP
Sephadex C-25 column and lyophilized to obtain a white powder.
Spectral characteristics of all the sulfated compounds 2S, 4S—21S,
24S—29S and 33S—36S are as follows.
Spectrozyme FXa (methoxycarbonyl-D-cyclohexylglycyl-Gly-Arg-p-ni-
troanilide), and Spectrozyme CTY were from American Diagnostica
(Greenwich, CT). FXIa chromogenic substrate S-2366 (H-D-Val-Leu-Arg-
p-nitroanilide.2HCl) and trypsin substrate S-2222 (benzyl-Ile-Glu(–OH
and –OCH3)-Gly-Arg-p-nitroanilide.HCl) were from Diapharma (West
Chester, OH). Pooled normal human plasma was from Valley
Biomedical (Winchester, VA). Activated partial thromboplastin time
reagent containing ellagic acid, thromboplastin-D, and 25 mM CaCl2
were from Fisher Diagnostics (Middletown, VA). Precast gels (4 – 20%
gradient) were from Bio-Rad Laboratories (Hercules, CA). Recombinant
human FXIa proteins, i.e., FXIaWT, FXIaCD, FXIaA3 and FXIaCD&A3, were
a gift from Professor David Gailani from the Vanderbilt University.
(2S). 1H NMR (DMSO‑d6, 400 MHz) δ: 12.44 (s, 1H, NH), 8.15–8.13
(m, 3H, ArH), 7.85 (s, 1H, ArH), 7.74 (s, 1H, ArH), 7.52–7.48 (m, 1H,
ArH), 7.33 (d, J = 8.8 Hz, 2H, ArH). 13C NMR (DMSO‑d6, 100 MHz):
156.4, 152.2, 134.4, 128.7, 127.3, 126.9, 126.1, 125.8, 120.7, 119.6.
MS (ESI) calculated for C36H24N4Na2O10S2 [(M−Na)]-, m/z 317.02,
found [(M−Na)]-, m/z 317.48.
(4S). 1H NMR (DMSO‑d6, 400 MHz): 8.37 (s, 2H, ArH), 8.3 (d,
J = 7.7 Hz, 2H, ArH), 8.22 (d, J = 7.9 Hz, 2H, ArH), 8.01–7.94 (m, 4H,
ArH), 7.7 (s, 4H, ArH), 7.67–7.63 (m, 2H, ArH), 7.51–7.4 (m, 2H, ArH),
7.37–7.36 (m, 2H, ArH), 5.83 (s, 4H, PhCH2). 13C NMR (DMSO‑d6,
100 MHz): 165.9, 158.9, 157.6, 151.3, 138.7, 136.4, 134.3, 129.5,
128.3, 127.6, 127.2, 123.3, 119.1, 117.9, 114.6, 67.7. MS (ESI) calcu-
lated for C36H24N4Na2O10S2 [M]-, m/z 782.07, found [(M−2Na)]2-, m/
z 367.89.
4.4. Chemical characterization of compounds
1H and 13C NMR were recorded using Bruker 400 MHz spectrometer
in either CDCl3, CD3OD, acetone‑d6, DMSO-D6 or D2O. Signals, in parts
per million (ppm), were relative either to the internal standard or the
residual peak of the solvent. NMR data are being reported as chemical
shift (ppm), multiplicity of signal (s = singlet, d = doublet, t = triplet,
q = quartet, dd = doublet of doublet, m = multiplet), coupling con-
stants (Hz), and integration. ESI-MS were recorded using Waters Ac-
quity TQD MS spectrometer in either positive or negative ion mode.
Samples were dissolved in methanol and infused at a rate of 20 μL/min.
(5S). 1H NMR (DMSO‑d6, 400 MHz): 8.38 (s, 2H, ArH), 8.29 (d,
J = 7.8 Hz, 2H, ArH), 8.18 (d, J = 7.9 Hz, 2H, ArH), 8.02–7.95 (m, 5H,
ArH), 7.68–7.60 (m, 4H, ArH), 7.53–7.44 (m, 3H, ArH), 7.37 (d,
J = 7.56 Hz, 2H, ArH), 5.87 (s, 4H, PhCH2). 13C NMR (DMSO‑d6,
100 MHz): 166.0, 158.7, 153.9, 151.2, 138.3, 136.7, 134.3, 128.9,
128.8, 127.7, 127.2, 123.3, 123.0, 120.4, 114.6, 67.9. MS (ESI) calcu-
lated for C36H24N4Na2O10S2 [M]-, m/z 782.07, found [(M−2Na)]2-, m/
z 367.93.
4.5. General procedure for synthesis of acetyl protected QAOs
The QAO core structure was synthesized using a condensation
10

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
(6S). 1H NMR (DMSO‑d6, 400 MHz): 8.5 (d, J = 8.65 Hz, 4H, ArH),
8.14 (d, J = 8.1 Hz, 2H, ArH), 7.97–7.90 (m, 5H, ArH), 7.66–7.61 (m,
5H, ArH), 7.36 (d, J = 8.64, 4H, ArH), 5.87 (s, 4H, PhCH2). 13C NMR
(DMSO‑d6, 100 MHz): 165.9, 158.8, 156.1, 136.9, 134.2, 131.8, 129.0,
128.8, 127.8, 127.5, 127.3, 126.9, 123.2, 119.8, 114.3, 67.8. MS (ESI)
calculated for C36H24N4Na2O10S2 [M]-, m/z 782.07, found [(M−2Na)]2
-, m/z 367.93.
157.3, 156.9, 155.7, 146.8, 132.3, 129.3, 128.5, 125.4, 119.8, 114.9,
101.4, 66.6, 55.5, 28.1, 25.3. MS (ESI) calculated for
C36H32N4Na2O10S2 [M]-, m/z 822.13, found [(M−2Na)]2 -, m/z 387.96.
(16S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.7 Hz, 4H, ArH),
8.06 (d, J = 8.16 Hz, 2H, ArH), 7.85–7.81 (m, 4H, ArH), 7.53–7.50 (m,
2H, ArH), 7.3 (d, J = 8.7 Hz, 4H, ArH), 4.65 (t, J = 6.4 Hz, 4H, NCH2),
3.0 (s, 4H, CH2), 1.16–0.97 (m, 6H, CH2). 13C NMR (DMSO‑d6,
100 MHz): 166.2, 158.9, 156.1, 151.3, 134.0, 131.9, 128.9, 127.4,
126.7, 123.2, 119.8, 114.4, 66.7, 52.78, 28.6, 28.2, 25.6. MS (ESI)
calculated for C35H30N4Na2O10S2 [M]-, m/z 776.12, found [(M−2Na)]2
-, m/z 364.98.
(7S). 1H NMR (DMSO‑d6, 400 MHz): 8.34 (s, 2H, ArH), 8.17 (d,
J = 4.6 Hz, 2H, ArH), 8.0–7.8 (m, 8H), 8.0–7.9 (m, 8H, ArH), 7.8–7.4
(m, 8H, ArH), 6.08 (s, 4H, PhCH2). 13C NMR (DMSO‑d6, 100 MHz):
165.8, 158.6, 153.8, 151.1, 138.2, 135.2, 134.1, 130.4, 128.7, 127.5,
127.0, 123.2, 123.0, 120.4, 114.4, 66.6. MS (ESI) calculated for
C36H24N4Na2O10S2 [M]-, m/z 782.07, found [(M−2Na)]2 -, m/z 367.96.
(8S). 1H NMR (DMSO‑d6, 400 MHz): 8.3 (d, J = 8.8 Hz, 4H, ArH),
7.93 (d, J = 8 Hz, 2H, ArH), 7.8–7.7 (m, 6H, ArH), 7.43–7.41 (m, 2H,
ArH), 7.32–7.27 (m, 2H, ArH), 7.21 (d, J = 8.8 Hz, 4H, ArH), 5.98 (s,
4H, PhCH2). 13C NMR (DMSO‑d6, 100 MHz): 165.7, 158.7, 156.0,
151.3, 135.2, 134.1, 131.7, 130.1, 128.9, 128.6, 127.4, 126.6, 122.9,
119.7, 114.1, 66.6. MS (ESI) calculated for C36H24N4Na2O10S2 [M]-, m/
z 782.07, found [(M−2Na)]2-, m/z 367.89.
(17S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.8 Hz, 4H, ArH),
8.13 (d, J = 7.9 Hz, 2H, ArH), 7.94–7.88 (m, 4H, ArH), 7.61–7.54 (m,
2H, ArH), 7.34 (d, J = 8.8 Hz, 4H, ArH), 4.71 (t, J = 6.4 Hz, 4H,
NCH2), 2.09–1.89 (m, 4H, CH2), 1.56–1.47 (m, 4H, CH2), 1.08 (t,
J = 7.2 Hz, 4H, CH2). 13C NMR (DMSO‑d6, 100 MHz): 166.12, 158.9,
156.1, 151.2, 134.0, 131.9, 128.9, 127.4, 126.6, 123.1, 119.8, 114.3,
66.7, 28.6, 28.1, 25.5. MS (ESI) calculated for C36H32N4Na2O10S2
[(M−Na)]-, m/z 790.14, found [(M−2Na)]2 -, m/z 371.96.
(18S). 1H NMR (DMSO‑d6, 400 MHz): 8.47–8.42 (m, 4H, ArH), 8.11
(d, J = 8.08 Hz, 2H, ArH), 7.94–7.89 (m, 2H, ArH), 7.60–7.57 (m, 2H,
ArH), 7.34 (d, J = 8.8 Hz, 2H, ArH), 7.08 (d, J = 8.8 Hz, 2H, ArH),
4.691 (t, J = 6.4 Hz, 4H, NCH2), 3.84 (s, 3H, OCH3), 1.9–1.87 (m, 4H,
CH2) 1.53–1.39 (m, 10H, CH2). 13C NMR (DMSO‑d6, 100 MHz): 166.1,
161.5, 158.8, 156.0, 151.2, 134.0, 131.9, 129.9, 129.6, 128.9, 127.4,
127.3, 126.6, 126.5, 123.1, 119.8, 114.3, 114.3, 113.8, 66.6, 66.6,
55.2, 28.7, 28.6, 28.1, 25.4. MS (ESI) calculated for C38H37N4NaO7S
[M]-, m/z 716.23, found [(M−Na)]-, m/z 693.92.
(9S). 1H NMR (DMSO‑d6, 400 MHz): 8.35–8.20 (m, 6H, ArH), 7.9 (s,
4H, ArH), 7.64 (s, 2H, ArH), 7.43 (m, 4H, ArH), 6.5 (s, 2H, C = CH), 5.4
(s, 4H, PhCH2). 13C NMR (DMSO‑d6, 100 MHz): 165.8, 153.9, 151.2,
138.3, 134.3, 128.8, 127.6, 127.2, 123.2, 123.0, 120.4, 114.5, 66.3,
52.8. MS (ESI) calculated for C32H22N4Na2O10S2 [M]-, m/z 732.06,
found [(M−2Na)]2 -, m/z 342.91.
(10S). 1H NMR (DMSO‑d6, 400 MHz): 8.24 (s, 2H, ArH), 8.17 (d,
J = 7.7 Hz, 2H, ArH), 8.06 (d, J = 7.64 Hz, 2H, ArH), 7.91–7.84 (m,
4H, ArH), 7.53–7.52 (m, 2H, ArH), 7.38–7.28 (m, 4H, ArH), 4.72 (t,
J = 6.3 Hz, 4H, NCH2), 2.04–1.97 (m, 4H, CH2), 1.8–1.73 (m, 2H,
CH2). 13C NMR (DMSO‑d6, 100 MHz): 166.3, 158.8, 153.9, 151.1,
138.4, 134.1, 128.8, 127.6, 127.0, 123.2, 120.4, 114.6, 66.7, 27.9,
22.3. MS (ESI) calculated for C33H26N4Na2O10S2 [M]-, m/z 748.09,
found [(M−2Na)]2 -, m/z 350.94.
(19S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.8 Hz, 4H, ArH),
8.13 (d, J = 8.2 Hz, 2H, ArH), 7.95–7.89 (m, 4H, ArH), 7.62–7.58 (m,
2H, ArH), 7.34 (d, J = 8.8 Hz, 4H, ArH), 4.71 (t, J = 6.4 Hz, 4H,
NCH2), 2.1–1.87 (m, 4H, CH2), 1.54 (s, 4H, CH2), 1.4 (s, 6H, CH2). 13
C
NMR (DMSO‑d6, 100 MHz): 166.2, 158.8, 156.1, 151.1, 134.0, 131.8,
129.9, 128.9, 127.3, 126.6, 123.1, 119.8, 115.3, 114.3, 66.7, 28.8,
28.7, 28.1, 25.5. MS (ESI) calculated for C37H34N4Na2O10S2 [M]-, m/z
804.15, found [(M−2Na)]2 -, m/z 379.03.
(11S). 1H NMR (DMSO‑d6, 400 MHz): 8.38 (d, J = 8.8 Hz, 4H,
ArH), 8.04 (d, J = 8.0 Hz, 2H, ArH), 7.88 (m, 4H, ArH), 7.51–7.47 (m,
2H, ArH), 7.27 (d, J = 8.8 Hz, 4H, ArH), 4.71 (s, 4H, NCH2), 2.01–1.97
(m, 4H, CH2), 1.77–1.74 (m, 2H, CH2). 13C NMR (DMSO‑d6, 100 MHz):
166.2, 158.9, 156.1, 151.3, 138.2, 134.0, 131.9, 128.9, 127.4, 126.6,
123.1, 119.8, 66.6, 27.9, 22.3. MS (ESI) calculated for
(20S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.8 Hz, 4H, ArH),
8.14 (d, J = 8.3 Hz, 2H, ArH), 7.96–7.90 (m, 4H, ArH), 7.63–7.60 (m,
2H, ArH), 7.4 (d, J = 8.8 Hz, 4H, ArH), 4.71 (t, 6.5 Hz, 4H, NCH2),
1.93–1.86 (m, 4H, CH2), 1.54–1.49 (m, 4H, CH2), 1.4–1.34 (m, 8H,
CH2). 13C NMR (DMSO‑d6, 100 MHz): 166.2, 159.1, 154.3, 134.1,
130.0, 128.9, 126.7, 126.5, 123.2, 119.8, 115.3, 114.1, 66.8, 28.7,
C
33H26N4Na2O10S2 [M]-, m/z 748.09, found [(M−2Na)]2 -, m/z 351.00.
(12S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.7 Hz, 4H, ArH),
8.02 (d, J = 8.1 Hz, 2H, ArH), 7.92–7.85 (m, 4H, ArH), 7.47 (t,
J = 7.9 Hz, 2H, ArH), 7.33 (d, J = 8.7 Hz, 4H, ArH), 4.89 (s, 4H,
OCH2), 4.12 (s, 4H, NCH2). 13C NMR (DMSO‑d6, 100 MHz): 165.8,
153.9, 151.2, 138.3, 134.3, 128.8, 128.5, 127.6, 127.2, 123.2, 120.4,
114.5, 66.3, 52.8. MS (ESI) calculated for C32H24N4Na2O11S2 [M]-, m/z
750.07, found [(M−2Na)]2 -, m/z 351.86.
28.6, 28.1, 25.4. MS (ESI) calculated for
C38H36N4Na2O10S2
[(M−Na)]-, m/z 818.17, found [(M−2Na)]2 -, m/z 386.04.
(21S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.8 Hz, 4H, ArH),
8.13 (d, J = 8.0 Hz, 2H, ArH), 7.94–7.88 (m, 4H, ArH), 7.6 (t, J = 1.6,
2H, ArH), 7.34 (d, J = 8.8 Hz, 4H, ArH), 4.7 (t, J = 6.4 Hz, 4H, NCH2),
1.93–1.87 (m, 4H, CH2), 1.53–1.48 (m, 4H, CH2), 1.38–1.31 (m, 10H,
CH2). 13C NMR (DMSO‑d6, 100 MHz): 166.1, 158.8, 156.0, 151.2,
134.0, 131.9, 128.9, 127.4, 126.6, 123.1, 119.7, 114.3, 66.7, 28.8,
28.7, 28.1, 25.5. MS (ESI) calculated for C39H38N4Na2O10S2
[(M−Na)]-, m/z 832.18, found [(M−2Na)]2 -, m/z 392.98.
(13S). 1H NMR (DMSO‑d6, 400 MHz): 8.3 (s, 2H, ArH), 8.25 (d,
J = 7.4 Hz, 2H, ArH), 8.14 (d, J = 8 Hz, 2H, ArH), 7.98–7.93 (m, 4H,
ArH), 7.58–7.37 (m, 6H, ArH), 4.76 (s, 4H, NCH2), 2.0 (s, 4H, CH2), 1.7
(s, 4H, CH2). 13C NMR (DMSO‑d6, 100 MHz): 166.3, 158.8, 153.9,
151.1, 138.4, 134.1, 128.8, 127.6, 127.0, 123.2, 120.4, 114.6, 66.8,
28.1, 25.4. MS (ESI) calculated for C34H28N4Na2O10S2 [M]-, m/z
762.10, found [(M−2Na)]2 -, m/z 357.91.
(24S). 1H NMR (D2O, 400 MHz): 7.93 (d, J = 8.6 Hz, 2H),
7.67–7.73 (m, 3H), 7.25–7.12 (m, 9H), 6.95 (d, J = 7.9 Hz, 1H),
6.75–6.69 (m, 2H), 5.19 (s, 2H), 4.15 (bs, 2H), 3.58 (bs, 2H), 1.47 (s,
2H), 0.98 (s, 2H), 0.71–0.67 (m, 6H). 13C NMR (100 MHz, D2O): 165.7,
165.2, 158.7, 158.5, 153.6, 153.6, 150.1, 149.7, 142.8, 133.9, 133.7,
129.7, 129.6, 126.4, 125.8, 124.4, 121.1, 120.9, 113.6, 113.5, 66.7,
59.6, 50.3, 38.6, 29.3, 28.0, 27.6, 25.6, 25.1. MS (ESI) calculated for
(14S). 1H NMR (DMSO‑d6, 400 MHz): 8.4 (d, J = 8.8, 4H, ArH), 8.1
(d, J = 7.9 Hz, 2H, ArH), 7.93–7.89 (m, 4H, ArH), 7.56–7.52 (m, 2H,
ArH), 7.34 (d, J = 8.8 Hz, 4H, ArH), 4.74 (t, J = 6.3 Hz, 4H, NCH2),
2.0 (s, 4H, CH2), 1.7 (s, 4H, CH2). 13C NMR (DMSO‑d6, 100 MHz):
166.3, 158.8, 153.9, 151.1, 138.4, 134.1, 128.8, 127.6, 127.0, 123.2,
120.4, 114.6, 66.8, 28.1, 25.4. MS (ESI) calculated for
C
38H33N7Na2O10S2 [M]-, m/z 857.15, found [(M−2Na)]2 -, m/z 405.85.
(25S). 1H NMR (D2O, 400 MHz): 7.94 (d, J = 8.5 Hz, 2H),
C
34H28N4Na2O10S2 [M]-, m/z 762.10, found [(M−2Na)]2 -, m/z 357.94.
(15S). 1H NMR (DMSO‑d6, 400 MHz): 8.40 (d, J = 8.6 Hz, 4H,
7.78–7.73 (m, 3H), 7.25–7.01 (m, 9H), 7.02 (d, J = 7.9 Hz, 1H),
6.78–6.74 (m, 2H), 5.22 (s, 2H), 4.12 (bs, 2H), 3.62 (bs, 2H), 1.44 (bs,
2H), 1.02 (s, 2H), 0.71–0.60 (m, 8H). 13C NMR (100 MHz, D2O): 165.8,
165.4, 158.7, 153.8, 153.7, 153.4, 150.1, 149.5, 142.9, 134.1, 133.7,
129.8, 129.7, 126.1, 125.5, 122.6, 121.2, 120.9, 113.7, 113.3, 66.8,
ArH), 7.8 (d, J = 9.1 Hz, 2H, ArH), 7.51–7.48 (m, 2H, ArH), 7.33–7.31
(m, 6H, ArH), 4.73 (t, J = 6.4 Hz, 4H, NCH2), 3.8 (s, 6H, CH2), 2.0 (s,
4H, NCH2), 1.7 (s, 4H, NCH2). 13C NMR (DMSO‑d6, 100 MHz): 165.3,
11

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
59.8, 50.3, 38.6, 29.4, 28.5, 28.2, 27.9, 25.8, 25.3. MS (ESI) calculated
for C39H35N7Na2O10S2 [M]-, m/z 871.17, found [(M−2Na)]2 -, m/z
412.47.
115.3, 114.3, 66.0, 52.7, 49.3, 29.7, 28.7, 28.2, 28.1, 25.8, 25.46, 25.1,
24.3, 24.8.MS (ESI) calculated for C41H34N5Na2O24S5 [(M−Na)]-, m/z
1231.98, found [(M−Na)]-, m/z 604.38.
(26S). 1H NMR (D2O, 400 MHz): 7.96 (d, J = 8.4 Hz, 2H),
7.82–7.75 (m, 3H), 7.25–7.19 (m, 9H), 7.07 (d, J = 7.7 Hz, 1H),
6.80–6.79 (m, 2H), 5.25 (s, 2H), 4.19 (bs, 2H), 3.70 (bs, 2H), 1.44 (bs,
2H), 1.1 (s, 2H), 0.70–0.56 (m, 10H). 13C NMR (100 MHz, D2O): 165.8,
165.4, 158.6, 154.0, 153.9, 153.8, 151.0, 149.8, 143.0, 134.8, 133.8,
129.6, 129.3, 126.8, 125.7, 123.7, 121.8, 120.9, 113.6, 113.3, 66.9,
60.0, 50.9, 38.6, 30.0, 28.5, 28.3, 27.8, 26.3, 25.5, 25.1. MS (ESI)
calculated for C40H37N7Na2O10S2 [M]-, m/z 885.18, found [(M−2Na)]2
-, m/z 419.96.
(36S). 1H NMR (DMSO‑d6, 400 MHz): 8.44 (d, J = 8.6 Hz, 2H),
8.30–8.27 (m, 1H), 8.17–8.13 (m, H), 7.93–7.91 (m, 2H), 7.62–7.6 (m,
2H), 7.32 (d, J = 8.8 Hz, 2H), 7.13–7.07 (m, 1H), 6.85 (d, J = 2.2 Hz,
1H), 5.23 (s, 2H), 4.72 (t, J = 6.3 Hz, 2H), 4.38 (t, J = 7.4 Hz, 2H),
1.94–1.84 (m, 4H) 1.54–1.28 (m, 12H). 13C NMR (100 MHz, D2O):
169.2, 160.1, 158.2, 156.0, 151.0, 148.5, 142.2, 134.1, 131.7, 129.8,
128.6, 127.3, 126.7, 124.0, 123.2, 119.8, 115.2, 114.3, 114.1, 66.7,
52.1, 49.3, 29.7, 28.2, 28.3, 28.2, 25.1, 25.4, 24.1. MS (ESI) calculated
for C42H36N5Na2O24S5 [(M−Na)]-, m/z 1246.00, found [(M−2Na)]2 -
m/z 612.43.
,
(27S). 1H NMR (D2O, 400 MHz): 7.97 (d, J = 7.3 Hz, 2H),
7.86–7.78 (m, 3H), 7.29–7.19 (m, 9H), 7.12 (d, J = 7.6 Hz, 1H),
6.83–6.80 (m, 2H), 5.28 (s, 2H), 4.12 (bs, 2H), 3.73 (bs, 2H), 1.45 (bs,
2H), 1.12 (s, 2H), 0.74–0.53 (m, 12H). 13C NMR (100 MHz, D2O):
165.8, 165.4, 158.6, 154.0, 153.9, 153.0, 150.1, 149.0, 142.8, 134.9,
133.9, 129.8, 129.3, 126.0, 125.0, 123.7, 121.1, 120.9, 113.8, 113.3,
66.9, 60.1, 50.2, 42.5, 29.6, 28.9, 28.5, 25.9, 25.6, 25.1, 24.7. MS (ESI)
calculated for C41H39N7Na2O10S2 [M]-, m/z 899.20, found [(M−2Na)]2
-, m/z 426.94.
4.9. Direct inhibition of hFXIa by sulfated QAOs
A microplate reader-based chromogenic substrate hydrolysis assay
was used to measure direct inhibition of hFXIa, as described ear-
lier.16–19 Briefly, each well of a 96-well microplate carrying 85 µL of pH
7.4 buffer (50 mM Tris.HCl, 150 mM NaCl, 0.1% PEG8000 and 0.02%
Tween80), 5 µL of a potential inhibitor (or reference solution), and 5 µL
of hFXIa (0.765 nM final concentration in the well) was incubated at
37 °C for 10 min, after which S2366 (5 µL, 345 μM final concentration
in the well) was added and the residual hFXIa activity was measured
from the initial rate of increase A405. The relative residual hFXIa ac-
tivity was calculated from the initial rates in the presence and absence
of the inhibitor. The dose–dependence of residual activity was analyzed
using the standard logistic equation to obtain the potency (IC50) and
efficacy (ΔY) of inhibition.
(28S). 1H NMR (D2O, 400 MHz): 7.98 (d, J = 7.9 Hz, 2H),
7.85–7.77 (m, 3H), 7.31–7.23 (m, 9H), 7.15 (d, J = 7.8 Hz, 1H),
6.78–6.72 (m, 2H), 5.39 (s, 2H), 4.18 (bs, 2H), 3.81 (bs, 2H), 1.48 (bs,
2H), 1.14 (s, 2H), 0.89–0.68 (m, 14H). 13C NMR (100 MHz, D2O):
165.8, 165.3, 158.7, 155.0, 154.1, 153.6, 150.1, 149.0, 143.7, 135.1,
134.0, 129.8, 129.1, 126.6, 125.3, 123.7, 121.0, 120.1, 113.8, 113.1,
66.7, 60.0, 50.2, 42.9, 29.8, 28.6, 28.3, 25.2, 25.1, 25.1, 24.8, 24.6. MS
(ESI) calculated for
C
42H41N7Na2O10S2 [M]-, m/z 913.22, found
[(M−2Na)]2 -, m/z 433.53.
(29S). 1H NMR (D2O, 400 MHz): 7.96 (d, J = 8.3 Hz, 2H),
7.89–7.81 (m, 3H), 7.43–7.19 (m, 9H), 7.03 (d, J = 7.8 Hz, 1H),
6.83–6.75 (m, 2H), 5.25 (s, 2H), 4.13 (bs, 2H), 3.84 (bs, 2H), 1.48 (bs,
2H), 1.25 (s, 2H), 0.88–0.65 (m, 16H). 13C NMR (100 MHz, D2O):
165.8, 165.3, 158.7, 155.0, 154.1, 153.7, 150.1, 149.0, 143.7, 135.1,
134.0, 129.7, 129.1, 126.6, 125.4, 123.7, 121.0, 120.1, 113.9, 113.1,
66.7, 60.0, 50.2, 42.9, 29.8, 28.6, 28.3, 25.2, 25.1, 25.1, 24.9, 24.6. MS
4.10. Direct inhibition of related serine proteases
The inhibition of thrombin, FIXa, FXa, kallikrein, trypsin and chy-
motrypsin by sulfated QAOs was measured using appropriate chromo-
genic substrates, as reported in the literature.16–19 These assays were
performed at 37 °C and pH 7.4, except for thrombin, which was per-
formed at 25 °C and pH 7.4. The concentrations of enzymes and sub-
strates in the wells were respectively, 6 nM and 50 μM (thrombin);
1.09 nM and 125 μM (hFXa); 20 nM and 200 μM (plasma kallikrein);
2.5 ng/ml and 80 μM (trypsin); 500 ng/ml and 240 μM (chymotrypsin);
and 89 nM and 425 μM (hFIXa). The ratio of the proteolytic activity in
the presence of sulfated QAO to that in its absence was used to calculate
percent inhibition (%).
(ESI) calculated for
C
43H43N7Na2O10S2 [M]-, m/z 927.23, found
[(M−2Na)]2 -, m/z 440.55.
(33S). 1H NMR (DMSO‑d6, 400 MHz): 8.42 (d, J = 8.7 Hz, 2H),
8.31–8.28 (m, 1H), 8.15–8.11 (m, 3H), 7.93–7.9 (m, 2H), 7.65–7.62 (m,
2H), 7.34 (d, J = 8.7 Hz, 2H), 7.13–7.07 (m, 1H), 6.85–6.81 (m, 1H),
5.24 (s, 2H), 4.71 (t, J = 6.4 Hz, 2H), 4.37 (t, J = 7.9 Hz, 2H),
1.93–1.85 (m, 4H) 1.55–1.24 (m, 6H). 13C NMR (100 MHz, D2O):
169.0, 160.3, 157.9, 155.8, 154.1, 148.6, 143.7, 135.1, 132.0, 129.8,
128.8, 127.3, 124.3, 123.1, 119.0, 115.1, 114.1, 114.0, 113.9, 66.7,
52.8, 49.4, 29.2, 28.6, 28.3, 25.2, 25.1. MS (ESI) calculated for
4.11. Michaelis–Menten kinetics of substrate hydrolysis
The initial rate of S2366 hydrolysis by hFXIa was obtained from the
linear increase in A405 corresponding to less than 10% consumption of
the substrate. The initial rate was measured at S2366 concentrations
between 0.03 and 2 mM in the presence of fixed concentration of 19S in
50 mM Tris.HCl buffer, pH 7.4, containing 150 mM NaCl, 0.1%
PEG8000 and 0.02% Tween80 at 37 °C. The data were analyzed using
the standard hyperbolic Michaelis–Menten equation (VI = VMAX × [S]/
C
39H30N5Na5O24S5 [(M−Na)]-, m/z 1203.94, found [(M−2Na)]2 -, m/z
590.38.
(34S). 1H NMR (DMSO‑d6, 400 MHz): 8.44 (d, J = 8.8 Hz, 2H),
8.32–8.27 (m, 1H), 8.15–8.12 (m, 3H), 7.94–7.92 (m, 2H), 7.64–7.61
(m, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.13–7.07 (m, 1H), 6.86–6.82 (m,
1H), 5.23 (s, 2H), 4.71 (t, J = 6.4 Hz, 2H), 4.38 (t, J = 7.9 Hz, 2H),
1.93–1.85 (m, 4H) 1.54–1.23 (m, 8H). 13C NMR (100 MHz, D2O):
169.0, 160.2, 158.8, 156.0, 151.0, 148.5, 142.2, 134.1, 131.8, 129.9,
128.9, 127.3, 126.7, 124.3, 123.2, 119.8, 115.3, 114.3, 114.1, 66.8,
52.8, 49.4, 29.7, 28.5, 28.2, 28.1, 25.8, 25.4. MS (ESI) calculated for
(KM + [S])) to calculate KM and VMAX
.
4.12. Affinity (KD) of sulfated QAOs binding to hFXIa
C
40H32N5Na5O24S5 [(M−Na)]-, m/z 1217.96, found [(M−2Na)]2 -, m/z
Fluorescence experiments were performed using a QM4 spectro-
fluorometer (Photon Technology International, Birmingham, NJ) in
50 mM Tris.HCl buffer, pH 7.4, containing 150 mM NaCl and 0.1%
PEG8000 at 37 °C. The KD of hFXIa–inhibitor complex was measured
using the change in the fluorescence of the active site dansyl group as a
function of inhibitor concentration. Titrations were performed by
597.39.
(35S). 1H NMR (DMSO‑d6, 400 MHz): 8.45 (d, J = 8.7 Hz, 2H),
8.30–8.27 (m, 1H), 8.16–8.13 (m, 3H), 7.94–7.92 (m, 2H), 7.63–7.61
(m, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.13–7.07 (m, 1H), 6.84 (d,
J = 2.1 Hz, 1H), 5.23 (s, 2H), 4.71 (t, J = 6.4 Hz, 2H), 4.37 (t,
J = 7.3 Hz, 2H), 1.93–1.84 (m, 4H) 1.54–1.28 (m, 10H). 13C NMR
(100 MHz, D2O): 169.2, 158.8, 156.1, 155.0, 154.1, 153.3, 150.1,
148.5, 143.7, 134.1, 134.3, 129.9, 128.9, 127.3, 124.2, 123.2, 119.8,
adding aliquots of sulfated QAOs to
a fixed concentration of
hFXIa–DEGR (250 nM) and monitoring the change in emission at
547 nm (λEX = 345 nm). The slit widths on the excitation and emission
12

R.S. Boothello, et al.
Bioorganic&MedicinalChemistry28(2020)115762
side were 1 mm each. The fluorescence change was fitted using the Hill
equation (1) for ligand binding to obtain the dissociation constant (KD).
In this equation, ΔF represents the change in fluorescence following
addition of sulfated QAO (i.e., [I]) from the initial fluorescence (F0),
while ΔFMAX represents the maximal change in fluorescence. Hill
coefficient ‘n’ is a measure of the cooperativity of binding.
prevention of venous thrombosis. N Eng J Med. 2015;372:232–240.
9. Crosby JR, Marzec U, Revenko AS, et al. Antithrombotic effect of antisense factor XI
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2013;33:1670–1678.
10. Schumacher WA, Seiler SE, Steinbacher TE, et al. Antithrombotic and hemostatic
effects of a small molecule factor XIa inhibitor in rats. Eur J Pharmacol.
2007;570:167–174.
11. Gailani D, Smith SB. Structural and functional features of factor XI. J Thromb
Haemost. 2009;7:75–78.
F
F0
[I]n
=
FMAX
×
12. Papagrigoriou E, McEwan PA, Walsh PN, Emsley J. Crystal structure of the factor XI
zymogen reveals a pathway for transactivation. Nat Struct Mol Biol.
2006;13:557–558.
(KD)n + [I]n
(1)
13. Geng Y, Verhamme IM, Smith SA, et al. Factor XI anion-binding sites are required for
productive interactions with polyphosphate. J Thromb Haemost. 2013;11:2020–2028.
14. Ho DH, Badellino K, Baglia FA, Walsh PN. A binding site for heparin in the apple 3
domain of factor XI. J Biol Chem. 1998;273:16382–16390.
4.13. Analytical ultracentrifugation studies
hFXIa was diluted to 0.2 mg/mL in 50 mM Tris.HCl buffer, pH 7.4,
containing 150 mM NaCl and 0.1% PEG8000 at 20 °C. Samples (420 μL)
of hFXIa alone and hFXIa-19S complex (100 μM), were run at
25,000 rpm overnight at 20 °C in a Beckman Coulter XL-I analytical
ultracentrifuge. Absorbance (λ = 280 nm) and interference scans were
recorded until the boundary moved to the bottom of the cell. The
continuous distribution C(s) distribution analysis was performed using
SEDFIT (https://sedfitsedphat.nibib.nih.gov).
15. Zhao M, Abdel-Razek T, Sun MF, Gailani D. Characterization of a heparin binding site
on the heavy chain of factor XI. J Biol Chem. 1998;273:31153–31159.
16. Karuturi R, Al-Horani RA, Mehta SC, Gailani D, Desai UR. Discovery of allosteric
modulators of factor XIa by targeting hydrophobic domains adjacent to its heparin-
binding site. J Med Chem. 2013;56:2415–2428.
17. Al-Horani RA, Ponnusamy P, Mehta AY, Gailani D, Desai UR. Sulfated penta-
galloylglucoside is a potent, allosteric, and selective inhibitor of factor XIa. J Med
Chem. 2013;56:867–878.
18. Al-Horani RA, Desai UR. Designing allosteric inhibitors of factor XIa. Lessons from
the interactions of sulfated pentagalloylglucopyranosides. J Med Chem.
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19. Argade MD, Mehta AY, Sarkar A, Desai UR. Allosteric inhibition of human factor XIa:
discovery of monosulfated benzofurans as a class of promising inhibitors. J Med
Chem. 2014;57:3559–3569.
4.14. Native polyacrylamide gel electrophoresis.
20. Fang T, Corte JR, Gilligan PJ, Jeon Y, Osuna H, Rossi KA, Myers Jr JE, Sheriff S, Lou
Z, Zheng JJ, Harper TW, Bozarth JM, Wu Y, et al. Orally bioavailable amine-linked
macrocyclic inhibitors of factor XIa. Bioorg Med Chem Lett. 2020;30:126949. https://
doi.org/10.1016/j.bmcl.2020.126949.
hFXIa and hFXIa in the presence of 19S (100 μM) were analyzed by
native PAGE (4–20%) using Mini-PROTEAN® TGX™ from Bio-Rad
(Hercules, CA). Laemmli sample buffer (10 μL, 2X concentration, Bio-
Rad) was mixed with 10 μL of hFXIa or hFXIa-19S samples and directly
loaded onto the gel without boiling. The gel was run at a constant 65 V
and stained using 30% methanol, 10% acetic acid, 0.1% brilliant blue R
solution for 20 min at room temperature. Following de-staining with
30% methanol and 10% acetic acid, an image of the gel was captured.
21. Corte JR, Pinto DJP, Fang TA, et al. Potent, orally bioavailable, and efficacious
macrocyclic inhibitors of factor XIa. Discovery of pyridine-based macrocycles pos-
sessing phenylazole carboxamide P1 groups. J Med Chem. 2020;63:784–803.
22. Clark CG, Rossi KA, Corte JR, Fang T, Smallheer JM, De Lucca I, Nirschl DS, Orwat
MJ, Pinto DJP, Hu Z, Wang Y, Yang Wu, Jeon Y, Ewing WR, Myers Jr JE, Sheriff S,
Lou Z, Bozarth JM, Wu Y, Rendina A, Harper T, Zheng J, Xin B, Xiang Q, et al.
Structure based design of macrocyclic factor XIa inhibitors: Discovery of cyclic P1
linker moieties with improved oral bioavailability. Bioorg Med Chem Lett.
2019;29:126604. https://doi.org/10.1016/j.bmcl.2019.08.008.
23. Wei QC, Zheng ZC, Zhang SJ, et al. Fragment-based lead generation of 5-phenyl-1H-
pyrazole-3-carboxamide derivatives as leads for potent factor XIa inhibitors.
Molecules. 2018;23. https://doi.org/10.3390/molecules23082002.
24. Hu Z, Wang C, Han W, et al. Pyridazine and pyridazinone derivatives as potent and
selective factor XIa inhibitors. Bioorg Med Chem Lett. 2018;28:987–992.
25. Pinto DJP, Orwat MJ, Smith LM, et al. Discovery of a parenteral small molecule
coagulation factor Xla inhibitor clinical candidate (BMS-962212). J Med Chem.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
26. Wang C, Corte JR, Rossi KA, Bozarth JM, Wu Y, Sheriff S, Myers Jr JE, et al.
Macrocyclic factor XIa inhibitors. Bioorg Med Chem Lett. 2017;27(17):4056–4060.
27. Corte JR, Yang W, Fang TA, et al. Macrocyclic inhibitors of Factor XIa: Discovery of
alkyl-substituted macrocyclic amide linkers with improved potency. Bioorg Med Chem
Lett. 2017;27:3833–3839.
We thank Professor David Gailani of Vanderbilt University for the
gift of recombinant FXIa proteins (wild-type and heparin-binding site
mutants). We also thank the computational resources made available to
us through a grant from the National Center for Research Resources
(S10 RR027411) to VCU. This work was supported in part by grants
from the NIH including HL107152, HL090586 and HL128639 (URD).
28. Corte JR, Fang TA, Osuna H, et al. Structure-based design of macrocyclic factor XIa
inhibitors: Discovery of the macrocyclic amide linker. J Med Chem.
2017;60:1060–1075.
29. Corte JR, Fang T, Pinto DJP, et al. Orally bioavailable pyridine and pyrimidine-based
Factor XIa inhibitors: Discovery of the methyl N-phenyl carbamate P2 prime group.
Bioorg Med Chem. 2016;24:2257–2272.
Appendix A. Supplementary data
30. Smith II LM, Orwat MJ, Hu Z, et al. Novel phenylalanine derived diamides as Factor
XIa inhibitors. Bioorg Med Chem Lett. 2016;26:472–478.
31. Buchanan MS, Carroll AR, Wessling D, et al. Clavatadine A, a natural product with
selective recognition and irreversible inhibition of factor XIa. J Med Chem.
2008;51:3583–3587.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115762.
32. Al-Horani RA. Factor XI(a) inhibitors for thrombosis: an updated patent review
(2016-present). Expert Opin Ther Pat. 2020;30:39–55.
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