Phenylimidazoles as potent and selective inhibitors of coagulation factor XIa with in vivo antithrombotic activity

Article

pubs.acs.org/jmc

Phenylimidazoles as Potent and Selective Inhibitors of Coagulation

Factor XIa with in Vivo Antithrombotic Activity

Jon J. Hangeland,* Todd J. Friends, Karen A. Rossi, Joanne M. Smallheer, Cailan Wang, Zhong Sun,

James R. Corte, Tianan Fang, Pancras C. Wong, Alan R. Rendina, Frank A. Barbera, Jeﬀrey M. Bozarth,

Joseph M. Luettgen, Carol A. Watson, Ge Zhang, Anzhi Wei, Vidhyashankar Ramamurthy, Paul E. Morin,

Gregory S. Bisacchi, Srinath Subramaniam, Piramanayagam Arunachalam, Arvind Mathur,

Dietmar A. Seiﬀert, Ruth R. Wexler, and Mimi L. Quan

Research and Development, Bristol-Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543, United States

S

* Supporting Information

ABSTRACT: Novel inhibitors of FXIa containing an (S)-2-phenyl-1-(4-phenyl-1H-

imidazol-2-yl)ethanamine core have been optimized to provide compound 16b, a

potent, reversible inhibitor of FXIa (K_i= 0.3 nM) having in vivo antithrombotic

eﬃcacy in the rabbit AV-shunt thrombosis model (ID₅₀= 0.6 mg/kg + 1 mg kg⁻¹

h⁻¹). Initial analog selection was informed by molecular modeling using compounds

11a and 11h overlaid onto the X-ray crystal structure of tetrahydroquinoline 3

complexed to FXIa. Further optimization was achieved by speciﬁc modiﬁcations

derived from careful analysis of the X-ray crystal structure of the FXIa/11h complex.

Compound 16b was well tolerated and enabled extensive pharmacologic evaluation of the FXIa mechanism up to the ID₉₀for

thrombus inhibition.

inhibiting ﬁbrinolysis, and (5) converting FXI to its active

form, FXIa, which is positioned near the head of the intrinsic

coagulation pathway. FXIa subsequently catalyzes the produc-

tion of activated FIX (FIXa), which produces more FXa, thus

amplifying the amount of thrombin formed at the site of the

growing thrombus. FXIa can be formed independent of

thrombin via self-activation and by activated FXII (FXIIa),

which is activated by polyanionic surfaces (e.g., collagen platelet

polyphosphates released by activated platelets),^2eby self-

activation, or by plasma kallikrein.^2fCurrent data indicate

that nascent blood clots formed through the extrinsic pathway

are unstable and relatively short-lived. Thus, initiation of the

intrinsic pathway and formation of FXIa by thrombin or FXIIa

INTRODUCTION

■

Thromboembolic disorders such as stroke, myocardial

infarction, and deep vein thrombosis remain a leading cause

of death in developed countries.¹Normal hemostasis is a tightly

regulated process that balances the need for blood to maintain a

ﬂuid state under normal physiologic conditions while providing

a mechanism for the rapid formation of hemostatic plugs at

sites of injury to prevent life threatening loss of blood. The

coagulation cascade contains multiple plasma serine protease

zymogens organized in a cascade that serves to amplify an

initiation event such as blood vessel wall injury, leading to the

formation of a hemostatic plug under normal physiologic

conditions or the pathophysiologic formation of an interarterial

thrombus at the site of a ruptured atherosclerotic plaque.^1aThe

cascade can be divided into three interdependent pathways; the

extrinsic, intrinsic, and common pathways.^2a−dInitiation of the

extrinsic pathway occurs when injury to the blood vessel wall

exposes FVII or its activated form (FVIIa) to the integral

membrane protein tissue factor (TF) to form the TF/FVIIa

complex. This complex converts factor X and factor IX to their

active forms (FXa and FIXa, respectively) via limited

proteolysis. Factor Xa in turn catalyzes the conversion of

prothrombin to thrombin which produces ﬁbrin from

ﬁbrinogen. Thrombin has additional roles in the coagulation

cascade. These include (1) activation of factor XIII to its

activated form, FXIIIa, which catalyzes the cross-linking of

ﬁbrin to form a ﬁbrin clot, (2) ampliﬁcation of its own

generation by activating two other coagulation cofactors, FV

and FVIII, (3) activation of blood platelets, (4) activation of

thrombin-activatable ﬁbrinolysis inhibitor (TAFI), thereby

is thought to be important for maintaining clot integrity,^{3a 3c}

−

especially in tissues with high ﬁbrinolytic activity.^3b

While FXIa appears to play a key role in stable thrombus

formation, it is not required for normal hemostasis. Persons

deﬁcient in FXI (hemophilia C) may experience bleeding

associated with invasive surgical procedures but rarely

spontaneous bleeding.⁴In contrast, deﬁciencies in FIX, or its

cofactor FVIII, are associated with a severe bleeding diathesis

(hemophilia B and A, respectively).⁵Preclinical studies have

shown that FXI-deﬁcient mice were protected against ferric

chloride (FeCl₃) induced carotid artery thrombosis and venous

thrombosis while maintaining normal tail bleeding times as

compared to wild-type mice. Similar levels of protection against

FeCl₃-induced thrombus formation provided by heparin or

Received: July 15, 2014

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Figure 1. Published active site inhibitors of FXIa.

argatroban, a thrombin inhibitor, resulted in increased bleeding

times that were >10-fold longer than that observed in untreated

wild type controls.⁶Other studies indicate that elevated FXI

levels are associated with venous thrombosis, myocardial

infarction among men, increased odds ratio for cerebrovascular

events, and coronary artery disease.⁷Conversely, reduced

incidence of ischemic stroke, but not myocardial infarction, is

observed in subjects with severe FXI deﬁciency.⁸Taken

together, these data support the hypothesis that inhibition of

FXIa has the potential to eﬀectively inhibit thrombus formation

hypothesis that selective inhibition of FXIa via a small molecule

has the potential to be eﬃcacious with a low bleeding liability.

Additional small molecule FXIa inhibitors have been

reported. Two natural products isolated from the sponge

Subera clavata, clavatadine A (4a) and B (4b), were shown to

be irreversible inhibitors of FXIa (IC₅₀= 1.3 and 27 μM,

respectively).¹⁴Arylboronic ester 5 (human FXIa IC₅₀= 1.4

μM, 30- and 9-fold selective over FXa and thrombin)

represents the most potent inhibitor from an eﬀort to identify

arylboronic esters capable of bonding covalently either

reversibly or irreversibly to the active site serine.¹⁵Previous

work from our laboratories culminated in the discovery of two

series of potent, reversible FXIa inhibitors: the tetrahydroqui-

noline series (e.g., compound 3, FXIa K_i= 0.2 nM)¹²and the

indole series (e.g., compound 6, FXIa K_i= 0.3 nM).¹⁶

Compound 6 was highly selective against a broad panel of

serine proteases including thrombin (27 000-fold), FXa

(12 000-fold), FVIIa (>35 500-fold), and plasma kallikrein

(PK, 3870-fold) and showed potent in vivo antithrombotic

eﬃcacy¹⁶in the rabbit ECAT model.^18aAll the aforementioned

FXIa inhibitors bind to the active or orthosteric site of the

enzyme competing with the peptide substrate for binding.

Alternatively, binding to the heparin-binding sites (HBS) on

FXIa can inhibit the enzyme via an allosteric mechanism

through induction of conformational changes that disfavor

eﬃcient enzyme catalysis leading to full or partial inhibitors.¹⁷

In order to provide additional chemotypes to test the FXIa

mechanism in preclinical models, we developed a third, novel

chemotype built on the phenylimidazole scaﬀold. We report

without signiﬁcant bleeding liability.^{3c 3d}

−

The ﬁrst pharmacologic proof of concept studies in rats^9,10a

and rabbits^10bwere conducted with irreversible FXIa small

molecule inhibitors using ketothiazoles 1a and 1b (FXIa IC₅₀

=

6 and 12 nM, respectively)⁹and BMS-262084 (2) (FXIa IC₅₀

2.8 nM),^10aa 4-carboxy-2-azetidinone mechanism based

inhibitor (Figure 1).¹¹These studies demonstrated that

inhibition of FXIa signiﬁcantly reduced thrombus formation

in multiple models while causing minimal increase in provoked

bleeding times relative to controls. Importantly, the compounds

increased in vitro aPTT but not PT, consistent with a FXIa

inhibitory mechanism with selectivity over FVIIa, FXa, and

thrombin. More recently, tetrahydroquinoline 3,¹²a small

molecule, reversible inhibitor of FXIa (K_i= 0.2 nM), exhibited

a potent dose dependent antithrombotic eﬀect in the rabbit

AV-shunt model with no increase in provoked bleeding time

relative to untreated controls.¹³Consistent with a selective

FXIa mechanism, ex vivo aPTT was prolonged while PT

remained unchanged. These studies further support the

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Table 1. In Vitro and aPTT Data for Compounds 3, 11a−h, and 11l−s

a

K_i(nM)

b

compd

R³′

R⁴′

FXIa

FVIIa

630

FXa

340

PK

EC_2X(μM), aPTT

3

0.2

2

11a

11b

11c

11d

11e

11f

11g

11h

11l

H

120

230

800

495

1120

630

1340

30

>10900

>8180

>9000

>8180

330

>40

d

H

CN

940

NT

d

H

CO₂Me

CH₂CO₂Et

H

1924

1360

1170

1470

5540

60

NT

d

H

NT

d

CN

NT

d

CH₂CO₂Et

H

NT

c

d

H

SO₂NH₂

CONH₂

CO₂H

CONHMe

CON(Me)₂

CH₂CO₂H

CH₂CONH₂

H

NT

H

>40

d

H

590

350

2860

150

400

1100

275

545

3820

220

NT

d

11m

11n

11o

11p

11q

11r

11s

H

NT

c

d

H

5270

765

NT

d

H

NT

d

H

1310

910

NT

d

CONH₂

CH₂CO₂H

CH₂CONH₂

NT

d

H

920

NT

d

H

1902

NT

a

b

K_ivalues were obtained from puriﬁed human enzymes and are averaged from duplicate IC₅₀determinations. Activated partial thromboplastin time

c

d

(aPTT) was measured according to the method described in the Experimental Section. Values reported from a single determination (n = 1). NT =

not tested.

Figure 2. Computer model of compound 11h (salmon) overlaid onto the X-ray crystal structure of compound 3/FXIa complex (cyan). Hydrogen

bonds between the amide nitrogen located at C-4 of the terminal phenyl ring of the biphenyl contained in compound 3 are represented by a series of

gray prolate ellipsoids. Figure was prepared with PyMol (Schrodinger).

̈

herein optimization of the potency for FXIa inhibition of novel,

reversible FXIa inhibitors resulting in the identiﬁcation of trans-

N-((S)-1-(4-(3-amino-1H-indazol-6-yl)-5-chloro-1H-imidazol-

2-yl)-2-phenylethyl)-4-(aminomethyl)cyclohexanecarboxamide

(16b, Scheme 2) having potent in vivo antithrombotic activity.

RESULTS AND DISCUSSION

■

The ﬁrst example in the new series, compound 11a (S-

conﬁguration), was discovered by focused-deck screening

against FXIa. It was modestly potent against FXIa (K_i= 120

C

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Figure 3. X-ray crystal structure of compound 11h complexed to FXIa. The initial F_o− F_celectron density contoured at 3 rmsd is shown with the

ﬁnal model. The protein is shown with yellow carbon atoms and secondary structural elements, and 11h is shown with orange carbon atoms.

Nitrogen atoms are shown in blue, oxygen atoms in red, and sulfur atoms in a darker yellow. The red spheres represent crystallographic water

molecules. A single crystallographic ethylene glycol molecule is present. Hydrogen bonds are represented by a series of gray prolate ellipsoids. Figure

was prepared with PyMol (Schrodinger).

̈

nM) and plasma kallikrein (PK, K_i= 330 nM) and selective

against FVIIa (K_i> 10 900 nM) and FXa (K_i> 8180 nM) and

increased aPTT 1.4-fold at a plasma concentration of 40 μM

(Table 1). The enantiomer of 11a (R-conﬁguration) (FXIa K_i=

3500 nM) was 30-fold less potent against FXIa; hence, the SAR

was developed using the series with the S-conﬁguration.

Computer assisted modeling of compound 11a docked with

the X-ray crystal structure of compound 3 complexed to FXIa¹²

revealed that the phenylimidazole phenyl ring of 11a could

potentially overlap with the terminal phenyl ring of the

biphenyl moiety of compound 3 (model not shown).

Furthermore, the structure of the compound 3/FXIa complex

(Figure 2) showed the amide nitrogen located at C-4 of the

terminal phenyl ring of the biphenyl moiety to participate in

key hydrogen bond contacts with the backbone carbonyls of

Leu41 and His40 located in the S2′ pocket of FXIa. When this

functionality was added to the phenylimidazole phenyl ring of

the model of compound 11a giving compound 11h (R⁴′ =

CONH₂; Figure 2), the hydrogen bond interactions between

compound 3 and Leu41 and His40 were closely mimicked.

Thus, the modeling exercise provided key design insights that

focused the initial set of analogs to include functionalities

capable of hydrogen bond interactions within the S2′ pocket of

the enzyme. Selectivity versus other relevant serine proteases

was tracked to see if selectivity for FXIa inhibition was

maintained.

potent against FXIa (K_i= 30 nM), ∼2-fold more potent versus

PK (K_i= 60 nM), retained excellent selectivity against FVIIa

and FXa, and increased aPTT by 1.8-fold at a plasma

concentration of 40 μM. Extending the amide one carbon

away from the phenyl ring gave compound 11p, which was

∼10-fold less potent than 11h against FXIa. Replacing one or

both of the primary amide hydrogens of 11h with a methyl

group (compounds 11m and 11n, respectively) resulted in an

incremental loss of potency against FXIa. A subset of the

functional groups tested at C-4′ were added to C-3′

(compounds 11e, 11f, and 11q−s). All of these compounds

were less potent compared to compounds 11a and 11h.

The X-ray crystal structure of compound 11h complexed to

FXIa, determined to 1.85 Å resolution, revealed key hydrogen

bonds and hydrophobic contacts between the inhibitor and

enzyme and an extensive hydrogen bond network between the

inhibitor and enzyme formed by crystallographic water

molecules and one ethylene glycol molecule (Figure 3). The

nitrogen of the primary amide at C-4′ of the phenylimidazole

moiety formed a hydrogen bond to the backbone carbonyl of

His40, located in the S2′ pocket, and to the backbone nitrogen

of the same residue via two intervening water molecules. The

carbonyl formed a hydrogen bond to the backbone nitrogen of

Ile151 via one intervening water molecule and to the hydroxyl

group of Tyr143 via two intervening water molecules. The

phenyl ring formed a hydrophobic contact with side chain of

Ile151 and Gly193 within the same pocket. The N-3 nitrogen

of the imidazole participated in a hydrogen bond with the

backbone carbonyl of Leu41 via an intervening water molecule,

whereas N-1 formed a hydrogen bond with a water molecule

that was part of a network of three water molecules and one

A focused set of functional groups able to form hydrogen

bonds appended onto the terminal phenyl ring at C-4′ generally

provided compounds with weaker potency than 11a (see

compounds 11b−d, 11g, and 11l−p). The single exception

from this set was compound 11h, which was 4-fold more

D

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Table 2. In vitro and aPTT Data for Compounds 11h−k and 16a−c

a

b

K_ivalues were obtained from puriﬁed human enzymes and are averaged from duplicate IC₅₀determinations. Activated partial thromboplastin time

c

(aPTT) was measured according to the method described in the Experimental Section. Rabbit aPTT value.

Table 3. Serine Protease Selectivity and aPTT and PT Data for Compound 16b

a

b

K_i(nM)

FIXa

>34900

EC_2x(μM)

aPTT

FXIa

0.3

FXa

FVIIa

4220

FXIIa

9218

thrombin

>12610

PK

5

trypsin

23

chymotrypsin

PT

6390

>20800

1

>40

a

b

K_ivalues were obtained from puriﬁed human enzymes and are averaged from duplicate IC₅₀determinations. Activated partial thromboplastin time

(aPTT) and prothrombin time (PT) were measured according to the method described in the Experimental Section.

ethylene glycol molecule linking the inhibitor to backbone

nitrogens of Gly216 and Gly218. The C-5 methine of the

imidazole was located nearby the hydrophobic side chain of

Lys192. The benzyl side chain phenyl ring participated in a

hydrophobic edge-on contact with the disulﬁde bridge formed

between Cys42 and Cys58 in the S1′ pocket. The NH of the P1

group amide linker formed a hydrogen bond to Ser214 via a

single intervening water molecule and Gly216 via two

intervening water molecules. The carbonyl oxygen of the

amide linker was positioned over the oxyanion hole formed by

Ser195, Asp194, and Gly193. The tranexamic acid moiety was

located in the S1 pocket where the primary amine formed a salt

bridge with the carboxylate of Asp189 and the backbone

carbonyl of Gly218. Thus, compound 11h formed a hydrogen

bond network with the same two S2′ pocket protein backbone

carbonyls as compound 3 (His40 and Leu41), albeit in a

diﬀerent manner owing to the phenylimidazole phenyl ring of

11h penetrating deeper into the S2′ binding pocket compared

to the terminal phenyl ring of the biphenyl moiety of

compound 3.

Closer examination of hydrogen bond opportunities available

in the S2′ pocket revealed the backbone carbonyl of His40, the

C-4′ amide of the inhibitor, and the hydroxyl group of Tyr143,

located opposite the backbone carbonyl in the S2′ pocket, to be

coplanar. We hypothesized that fusing the C-4′ amide onto the

phenyl ring to form a ﬁve- or six-membered ring containing a

heteroatom able to form a hydrogen bond to the hydroxyl

group of Tyr143 might improve potency. This hypothesis was

tested by modifying the terminal phenyl ring to give

aminoindazole 16a (Table 2) having a FXIa K_i= 3 nM,

which was 10-fold more potent than 11h against FXIa, ∼10-

fold selective versus PK (K_i= 50 nM), and showed potent

aPTT activity (EC_2x= 2 μM). Thus, two novel modiﬁcations to

the original compound in this series (11a) provided a 40-fold

overall improvement to FXIa potency.

The eﬀect of substitution at the C-5 position of the imidazole

was ﬁrst investigated by adding bromine to compound 11h

giving compound 11i (Table 2). A 3-fold improvement in

potency was realized (FXIa K_i= 9 nM), and the aPTT assay

showed it to have good in vitro anticoagulant activity (EC_2x

=

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Figure 4. X-ray crystal structure of compound 16b complexed to FXIa. The initial F_o− F_celectron density contoured at 4 rmsd is shown with the

ﬁnal model. The protein is shown with yellow carbon atoms and secondary structural elements, and 16b is shown with green carbon atoms. Nitrogen

atoms are shown in blue, oxygen atoms in red, sulfur atoms in a darker yellow, and the chlorine atom in orange. The red spheres represent

crystallographic water molecules. Hydrogen bonds are represented by a series of gray prolate ellipsoids. Figure was prepared with PyMol

(Schrodinger).

̈

10 μM). Substituting the bromine with chlorine gave

compound 11j (FXIa K_i= 4 nM, aPTT EC_2x= 6 μM),

which showed a similar improvement in potency compared to

compound 11h. The C-5 ﬂuoride (11k) was as potent against

FXIa as compound 11j but was less selective against FXa (K_i=

1370 nM) and PK (K_i= 8 nM). Combining all the

modiﬁcations that improved potency against FXIa into a single

molecule gave compound 16b having subnanomolar potency

against FXIa (K_i= 0.3 nM), 17-fold selectivity versus PK,

potent in vitro aPTT activity (EC_2x= 1 μM), and >1000-fold

selectivity versus other relevant proteases (see Table 3 for a

compilation of data for 16b). The enantiomer of 16b (R-

conﬁguration) (FXIa K_i= 21 nM) was 70-fold less potent. The

C-5 ﬂuoride analog of 16b, compound 16c, was equally potent

against FXIa (K_i= 0.3 nM) and equally selective against FVIIa,

FXa, and PK.

X-ray crystal data (2.09 Å resolution) showed the binding

modes of compounds 16b and 11h to be virtually identical

(Figure 4). The exocyclic nitrogen of the aminoindazole moiety

formed a hydrogen bond to the backbone carbonyl of His40

and, via two intervening water molecules, to the backbone

nitrogen of the same residue (cf. the amine of the primary

amide of 11h). The N-1 nitrogen of the indazole ring was

within hydrogen bonding distance to the hydroxy group of

Tyr143 as designed. The N-2 nitrogen participated in a

hydrogen bond with the backbone nitrogen of Ile151 via an

intervening water molecule (cf. the carbonyl of the primary

amide of 11h). In addition, the C-5 chlorine formed a

hydrophobic contact to the aliphatic portion the Lys192 side

chain. Combined, the modiﬁcations made to compound 11a,

guided by key computer modeling exercises, resulted in a 400-

fold improvement in potency for FXIa. In addition, compound

16b maintained excellent selectivity against a panel of serine

proteases found in the coagulation pathway and had good

selectivity against PK.

Other ﬁve-membered ring heterocycle replacements of the

imidazole were surveyed in parallel with the SAR development

of the phenylimidazole series (Table 4). 5-Phenyloxazole 20

(FXIa K_i= 1440 nM) was 12-fold less potent for FXIa

compared to compound 11a (FXIa K_i= 120 nM) with good

selectivity over FVIIa and FXa and moderate selectivity over PK

(2-fold). Replacing the C-5 methine of compound 11a with

nitrogen to give 3-phenyltriazole 22 resulted in an 8-fold loss in

potency while maintaining a similar selectivity proﬁle versus

FVIIa, FXa, and PK. Interchanging the C-5 methine with the N-

3 nitrogen of the imidazole provided 3-phenylpyrazole 27

(FXIa K_i= 6940 nM) resulting in signiﬁcant loss of potency for

FXIa (∼60-fold). The 5-oxo-1-phenyltriazole 30 was ∼25-fold

less potent than imidazole 11a but retained good selectivity

over FVIIa and FXa. The 2-phenylimidazole regioisomer of

11a, compound 33 (FXIa K_i= 1640 nM), was similar in

potency and selectivity to oxazole 20. In conclusion, the 5-

phenylimidazole regioisomer contained in 11a remained the

most potent ﬁve-membered ring heterocycle analog among the

small set surveyed.

Compound 16b exhibited reversible enzyme inhibition

kinetics at 37 °C (FXIa K_i= 0.82 nM, on rate = 3.0 μM⁻¹

s⁻¹, t_1/2oﬀ = 4.6 min), with similar potency for rabbit

coagulation factor orthologs (Table 5). Pharmacokinetic

proﬁling of compound 16b in rabbits by iv administration at

0.5 mg/kg showed the compound to be relatively rapidly

cleared and to have a relatively low volume of distribution

F

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inhibition is modest (35-fold) and PK KO mice have been

shown to be resistant to thrombus formation in mouse venous

and arterial FeCl₃-induced thrombosis models.¹⁹

Table 4. In Vitro Data for Compounds 11a, 20, 22, 27, 30,

33

CHEMISTRY

■

Phenylimidazoles 9a−g were synthesized using a three-step

sequence²⁰(Scheme 1). The cesium salt of (L)-N-Boc-

phenylalanine (7), formed using cesium carbonate in

ethanol/water (1:1), was combined with the appropriately

substituted bromoacetophenones 8a−g. The resulting β-keto

esters were condensed with ammonium acetate (reﬂuxing

xylenes, Dean−Stark trap) to give the phenylimidazoles 9a−g.

Yields for this three-step sequence ranged between 40% and

60%. Removal of the Boc group with 10% triﬂuoroacetic acid

(v/v) in methylene chloride at room temperature was followed

by amide bond formation (BOP reagent or EDC/HOBT) with

N-Boc-tranexamic acid to give intermediates 10a−g. Nitriles

10b and 10e were hydrolyzed to the primary amides 10h and

10q, respectively, using 30% hydrogen peroxide and potassium

carbonate in DMSO. Addition of magnesium oxide to the

reaction with 10h helped to accelerate an otherwise sluggish

hydrolysis. Addition of bromine to C-5 of the imidazole ring

was accomplished by reacting intermediate 10h with bromine

in methylene chloride at room temperature to give 10i.

Synthesis of the C-5 chloro analog 10j began with hydrolysis of

nitrile 9b using potassium carbonate, hydrogen peroxide, and

magnesium oxide followed by chlorination of the C-5 carbon of

the imidazole with N-chlorosuccinimide in reﬂuxing acetoni-

trile. Removal of the Boc group with TFA and addition of N-

Boc-tranexamic acid using BOP/pyridine gave 10j. The C-5

ﬂuoro analog 10k was obtained by reaction of 10h with

SelectFluor in DMF/H₂O (10:1). Esters 10c, 10d, and 10f

were hydrolyzed to the corresponding acids 10l, 10o, and 10r

using methanolic sodium hydroxide at room temperature.

Again, use of magnesium oxide accelerated the otherwise slow

hydrolysis of ester 10c. Carboxylic acid 10l was converted to

monomethylamide 10m or dimethylamide 10n using methyl-

amine or dimethylamine, respectively (EDC/HOBt). The

primary phenylacetamides 10p and 10s were synthesized

from the corresponding phenylacetic acids 10o and 10r using

EDC/HOAt and N-methylmorpholine followed by concen-

trated aqueous ammonia. The Boc group on the tranexamic

acid moiety for intermediates 10a−s was removed using 10%

triﬂuoroacetic acid (v/v) in methylene chloride at room

temperature to provide compounds 11a−s as bis-triﬂuoroacetic

acid salts.

a

K_ivalues were obtained from puriﬁed human enzymes and are

b

averaged from duplicate IC₅₀determinations. K_i> 10 900 nM and K_i

> 8180 nM for FVIIa and FXa, respectively.

(Table 6). The compound was 85% protein bound in rabbit

serum. Compound 16b was not orally bioavailable; however,

the bis-HCl salt had ample solubility (946 μg/mL in 100 mM

phosphate buﬀer, pH 7.4; amorphous solid) for iv admin-

istration. When tested using the rabbit AV-shunt model^18aat

three doses administered as a single loading dose followed by

continuous iv infusion to maintain steady state blood levels of

drug, the bis-HCl salt of compound 16b showed potent dose

dependent in vivo antithrombotic activity relative to untreated

controls with a calculated ID₅₀= 0.6 mg/kg +1 mg kg⁻¹h⁻¹

(Figure 5). Inhibition of plasma kallikrein (PK) may contribute

to the antithrombotic eﬀect of compound 16b, since the

selectivity of compound 16b for rabbit FXIa versus rabbit PK

Aminoindazoles 16a−c were accessed starting from

commercially available 4-bromo-3-ﬂuorobenzoic acid (12),

which was treated with zinc(II) cyanide and tetrakis-

(triphenylphosphine)palladium(0) in dimethylformamide at

90 °C to give 4-cyano-3-ﬂuorobenzoic acid (Scheme 2). The

carboxylic acid was converted to α-bromoketone 13 by a three-

step, one-pot transformation: (1) formation of the acid chloride

Table 5. Comparison of Human and Rabbit Selectivity Proﬁle and in Vitro aPTT for Compound 16b

a

K_i(nM)

b

species

FXIa

FXa

thrombin

FVIIa

4220

PK

EC_2x(μM), aPTT

c

human

rabbit

0.3 (0.8)

0.6 (1.2)

6390

>12610

>20000

5

1

2

c

>10000

10000

21

a

b

K_ivalues were obtained from puriﬁed human enzymes and are averaged from duplicate IC₅₀determinations. Activated partial prothrombin time

c

(aPTT) was measured according to the method described in the Experimental Section. Values were determined at 37 °C.

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Table 6. Rabbit Pharmacokinetic and Protein Binding Data for Compound 16b

a

dose (mg/kg)

0.5

C_max(nM)

CL (L h⁻¹kg⁻¹

1.2 0.1

)

V_ss(L/kg)

t_1/2(h)

MRT (h)

0.6 0.1

protein binding (%)

2472 456

0.7 0.1

1.0 0.3

85

a

Determined using rabbit plasma.

using oxalyl chloride in methylene chloride, (2) conversion of

the acid chloride to the methyl ketone with trimethylsilyldiazo-

methane in acetonitrile (1 h at −15 °C, then reﬂux for 1 h), and

(3) bromination of the methyl ketone (33% hydrobromic acid

in acetic acid, −15 °C). The sequence carrying bromoketone

13 forward to imidazole 14a (R⁵=H) was the same as

described for compounds 10a−g. Reﬂuxing 14a with N-

chlorosuccinimide in acetonitrile gave chloroimidazole 14b.

SelectFluor in DMF/H₂O (10:1) gave ﬂuoroimidazole 14c

from 14a. Imidazoles 14a−c were carried forward to 15a−c,

respectively, using the reaction sequence described for

converting compounds 9a−g to 11a−g. Compounds 15a−c

were treated with hydrazine hydrate in n-butanol at 120 °C for

1 h to give the corresponding aminoindazoles 16a−c, which

were isolated as bis-triﬂuoroacetic acid salts. Alternatively, the

ﬁnal two steps in the sequence (Scheme 2, steps i and j) could

be reversed. Formation of the bis-HCl salt of 16b used in the

rabbit AV-shunt model was obtained using reverse phase HPLC

with a mobile phase containing HCl.

Figure 5. Rabbit AV-shunt data. Compound 16b was given as a single

loading dose followed by continuous iv infusion at three doses: 0.1

mg/kg + 0.16 mg kg⁻¹h⁻¹, 0.5 mg/kg + 0.8 mg kg⁻¹h⁻¹, 2.5 mg/kg +

4 mg kg⁻¹h⁻¹. Thrombi formed were removed from the shunt and

weighed. % inhibition values are relative to thrombus formation prior

to drug treatment. ID₅₀= 0.6 mg/kg + 1 mg kg⁻¹h⁻¹.

Synthesis of 5-phenyloxazole 20 (Scheme 3) began with

amide coupling between (S)-2-(1,3-dioxoisoindolin-2-yl)-3-

phenylpropanoic acid (17) and 2-amino-1-phenylethanone

a

Scheme 1. Synthesis of Phenylimidazoles 11a−s

a

(a) 7, Cs₂CO₃, EtOH/H₂O (1:1), rt, 1 h; (b) Cs salt from (a), 8a−g, DMF, rt, 1 h; (c) NH₄OAc, xylenes, reﬂux (Dean−Stark), 3 h; (d) 10−30%

(v/v) TFA, CH₂Cl₂, rt; (e) N-Boc-tranexamic acid, BOP, pyridine, DMF, rt, 16 h or EDC, HOBt, N-methylmorpholine, DMF, rt, 16 h; (f) K₂CO₃,

30% aq H₂O₂, MgO, DMSO, rt, 4 h; (g) NaOH, MgO, CH₃OH/EtOAc (5:1), rt, 5 h; (h) NaOH, MeOH, rt, 16 h; (i) Br₂, CHCl₃, rt, 16 h; (j) NCS,

ACN, reﬂux, 7 h; (k) Selectﬂuor, DMF/H₂O (10:1), rt, 2 h; (l) methylamine (10m) or dimethylamine (10n), HOBt, EDC, N-methylmorpholine,

DMF, rt, 16 h; (m) conc NH₄OH, HOAt, EDC, N-methylmorpholine, DMF, rt, 16 h.

H

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a

Scheme 2. Synthesis of Aminoindazole Analogs 16a−c

a

(a) Zn(CN)₂, Pd(PPh₃)₄, DMF, 90 °C, 3 h; (b) (i) oxalyl chloride, CH₂Cl₂, rt, 1 h, then reﬂux, 1 h, (ii) TMSCH₂N₂, ACN, −15 °C, 1 h, (iii) HBr

(33% by mass) in HOAc, −15 °C, 20 min (39% over two steps); (c) 7, Cs₂CO₃, DMF, 15 °C, 1 h (61%); (d) NH₄OAc, xylenes, reﬂux (Dean−

Stark), 2.5 h (41%); (e) NCS, ACN, reﬂux, 7 h (99%); (f) Selectﬂuor, DMF/H₂O (10:1), rt, 2 h (27%); (g) 20% TFA (v/v), CH₂Cl₂, rt, 30 min;

(h) N-Boc-tranexamic acid, BOP, TEA, THF, 75 °C, 15 min; (i) 20% TFA (v/v), CH₂Cl₂, rt, 30 min (>67% for three steps for 15b); (j) NH₂NH₂·

H₂O, n-BuOH, 120 °C, 1 h (54% for 16b).

a

Scheme 3. Synthesis of trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(5-phenyloxazol-2-yl)ethyl)cyclohexanecarboxamide (20)

a

(a) BOP, anhyd pyridine, rt, 16 h (100%); (b) POCl₃, DMF, 90 °C, 30 min (41%); (c) hydrazine, EtOH, reﬂux, 2 h (90%); (d) N-Boc-tranexamic

acid, HOAt, EDC, N-methylmorpholine, DMF, rt, 1 h; (e) 10% (v/v) TFA, CH₂Cl₂, rt, 16 h (26% over two steps).

Scheme 4. Synthesis of trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(3-phenyl-1H-1,2,4-triazol-5-

a

yl)ethyl)cyclohexanecarboxamide (22)

a

(a) Hydrazine, BOP-Cl, anhydrous pyridine, rt, 16 h (100%); (b) ethyl benzimidate·HCl, Et₃N, ACN, reﬂux, 24 h (42%); (c) 10% (v/v) TFA,

CH₂Cl₂, rt, 16 h; (d) N-Boc-tranexamic acid, BOP-Cl, anhydrous pyridine, rt, 12 h; (e) 30% (v/v) TFA, CH₂Cl₂, rt, 3 h (28% over three steps).

(18; Bop/pyridine), followed by dehydration of the inter-

mediate diketoamide with POCl₃in DMF to give (S)-2-(2-

phenyl-1-(5-phenyloxazol-2-yl)ethyl)isoindoline-1,3-dione

(19). Removal of the phthalimide group with hydrazine in

reﬂuxing EtOH was followed by acylation of the free amine

with the tranexamic side chain as previously described. The

four-step synthesis of 3-phenyltriazole 22 (Scheme 4) began

with coupling hydrazine and N-Boc-^L-phenylalanine 7 using

BOP and anhydrous pyridine. The intermediate acyl hydrazide

was condensed with ethyl benzimidate hydrochloride in

reﬂuxing acetonitrile and TEA to give triazole 21. Removal of

the Boc group with TFA and subsequent conversion of the free

amine to compound 22 were carried out as before. Synthesis of

3-phenylpyrazine 27 (Scheme 5) began with hydrolysis of ethyl

3-oxo-3-phenylpropanoate (23) to give 3-oxo-3-phenylpropa-

noic acid, followed by formation of magnesium 3-oxo-3-

phenylpropanoate using magnesium ethoxide in methanol. The

salt was combined with 1,1′-carbonyldiimidazole activated N-

CBz-^L-phenylalanine (24), which resulted in the decarbox-

ylative addition of acetophenone enolate to the mixed

anhydride of 24 to give (S)-benzyl (3,5-dioxo-1,5-diphenyl-

pentan-2-yl)carbamate (25). The diketone 25 was then

condensed with hydrazine in EtOH to give pyrazine 26.

Hydrogenation with 10% palladium on carbon followed by

installation of the transexamic side chain provided 3-phenyl-

pyrazine 27. (S)-Benzyl (1-cyano-2-phenylethyl)carbamate 28

was converted to the oxotriazine 29 by a three-step process

(Scheme 6). First, the methyl imidate of compound 28 was

I

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Scheme 5. Synthesis of trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(3-phenyl-1H-pyrazol-5-yl)ethyl)cyclohexanecarboxamide

a

(27)

a

(a) (i) 23, 1 N NaOH/EtOH (1:1), rt, 72 h; (ii) Mg(OEt)₂, MeOH, 4 h; (b) (i) 24, CDI, DMF, 2 h, rt; (ii) intermediate from (a), DMF, rt, 24 h

(13%); (c) anhydrous hydrazine, EtOH, 60 °C, 3 h (100%); (d) 10% Pd/C, MeOH, 1 atm H₂, 4 h (57%); (e) N-Boc-tranexamic acid, BOP-Cl,

anhydrous pyridine, rt, 12 h; (f) 30% (v/v) TFA, CH₂Cl₂, rt, 3 h (46% over two steps).

Scheme 6. Synthesis of trans-4-(Aminomethyl)-N-((S)-1-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazol-3-yl)-2-

a

phenylethyl)cyclohexanecarboxamide (30)

a

(a) (i) NaOCH₃, MeOH, 30 °C, 3 h, (ii) HOAc, phenylhydrazine, rt, 18 h (28%); (b) carbonyl dimidazole, THF, reﬂux, 48 h (66%); (c) 10% Pd/

C, MeOH, 1 atm H₂, 4 h (57%); (d) N-Boc-tranexamic acid, BOP-Cl, anhydrous pyridine, rt, 12 h; (e) 30% (v/v) TFA, CH₂Cl₂, rt, 3 h (46% over

two steps).

Scheme 7. Synthesis of trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(2-phenyl-1H-imidazol-4-yl)ethyl)cyclohexanecarboxamide

a

33

a

(a) (i) sodium formate, EtOH, reﬂux, 14 h, (ii) add benzamidine, NaHCO₃, reﬂux, 24 h (8%); (b) 10% Pd/C, MeOH, 1 atm H₂, 4 h (57%); (c) N-

Boc-tranexamic acid, BOP-Cl, anhydrous pyridine, rt, 12 h; (d) 30% (v/v) TFA, CH₂Cl₂, rt, 3 h (46% over two steps).

formed using sodium methoxide. Next, the imidate was

condensed with phenylhydrazine to give an iminohydrazide.

Third, the iminohydrazide was combined with carbonyl

diimidazole in reﬂuxing THF to give the oxotriazole 29.

Synthesis of 5-oxo-1-phenyltriazine 30 from compound 29 was

carried out as previously described. Synthesis of 2-phenyl-

imidazole 33 (Scheme 7) began by combining bromoketone 31

with sodium formate in reﬂuxing EtOH, followed by addition of

benzamidine and sodium bicarbonate to give the imidazole 32.

Removal of the CBz group by hydrogenation as described was

followed by acylation with N-Boc-tranexamic acid, and

subsequent removal of the Boc group with TFA completed

the synthesis of 2-phenylimidazole 33.

identiﬁcation of compound 16b, a subnanomolar, reversible

inhibitor of FXIa. Compound 16b showed potent dose

dependent antithrombotic activity in the rabbit AV-shunt

model. It was used extensively in preclinical rabbit eﬃcacy

models (AV-shunt,^18aECAT,^18bcuticle bleeding^{18c 18d}) to

−

benchmark in vivo eﬃcacy (antithrombotic eﬀect) and safety

(bleeding) parameters for this mechanism of action. Com-

pound 16b was well tolerated in these models and enabled the

studies to be conducted up to the ID₉₀for thrombus inhibition.

A full description of the pharmacology of 16b and studies

identifying orally bioavailable analogs will be published

separately.

EXPERIMENTAL SECTION

■

CONCLUSIONS

■

All reactions were run under an atmosphere of dry nitrogen or argon

unless otherwise noted. Solvents and reagents were obtained from

commercial vendors in the appropriate grade and used without further

Optimization of compound 11a for FXIa potency and

selectivity over other relevant serine proteases resulted in the

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puriﬁcation unless otherwise indicated. NMR spectra were obtained on

Joel ECP500 (500 MHz) or ECP400 (400 MHz) NMR spectrometers

with chemical shift in ppm downﬁeld from TMS as an internal

reference standard. ¹H assignment abbreviations are the following:

singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), broad

singlet (bs), doublet of doublets (dd), doublet of triplets (dt), and

multiplet (m). Flash chromatography was done using Teledyne-ISCO

automated ﬂash chromatography instruments. Reverse phase analytical

HPLC was performed on Shimadzu LC10AS systems and reverse

phase analytical HPLC/MS on Shimadzu LC10AS systems coupled

with Waters ZMD mass spectrometers using MeOH/H₂O containing

0.01% TFA or 10 mM NH₄OAc as the mobile phase. HPLC

puriﬁcation was performed using a Shimadzu preparative HPLC

system composed of a SPD-20A prominence detector, two LC-8A

pumps, a SCL-10 vp controller, and a FRC-10 fraction collector using

C18 reverse phase columns with MeOH/H₂O containing 0.05% TFA

as the mobile phase. HPLC purity was determined on a Shimadzu

LC10AT system equipped with Sunﬁre C18 and Xbridge phenyl

reverse phase columns (3.0 mm × 150 mm) operating at a ﬂow rate of

0.5 mL/min with a mobile phase consisting of H₂O/acetonitrile

containing 0.05% TFA and detector wavelengths set to 220 and 254

nm. All ﬁnal compounds were found to be >95% pure by HPLC

analysis unless otherwise noted.

Enzyme Aﬃnity Assays. Factors IXa, Xa, and XIa were purchased

from Haematologic Technologies. Factor XIIa was purchased from

American Diagnostica. Plasma kallikrein and α-thrombin were

purchased from Enzyme Research Laboratories. Trypsin and

chymotrypsin were purchased from Sigma-Aldrich. Recombinant

factor VIIa was purchased from Novo Nordisk. Recombinant soluble

tissue factor residues 1−219 was produced at Bristol-Myers Squibb.

Factor XIa, factor XIIa, and chymotrypsin assays were conducted in

50 mM HEPES, pH 7.4, 145 mM sodium chloride, 5 mM potassium

chloride, and 0.1% PEG 8000. Factor Xa, thrombin, trypsin, and

plasma kallikrein assays were conducted in 100 mM sodium

phosphate, pH 7.5, 200 mM sodium chloride, and 0.5% PEG 8000.

Factor VIIa assays were conducted in 50 mM HEPES, pH 7.4, 150

mM sodium chloride, 5 mM calcium chloride, and 0.1% PEG 8000.

Factor IXa assays were conducted in 50 mM Tris, pH 7.4, 100 mM

sodium chloride, 5 mM calcium chloride, 0.5% PEG 8000, and 2%

DMSO. The peptide substrates were the following: pyro-Glu-Pro-Arg-

pNA(p-nitroaniline) (Diapharma) for factor XIa and thrombin; N-

benzoyl-Ile-Glu-(OH, OMe)-Gly-Arg-pNA (Diapharma) for factor Xa

and trypsin; H-(D)-Ile-Pro-Arg-pNA (Diapharma) for factor VIIa; H-

(D)-CHT-Gly-Arg-pNA (American Diagnostica) for factor XIIa; H-

(D)-Leu-Ph-Gly-Arg-AMC(7-amino-4-methylcoumarin) (Penta-

pharm) or H-(D)-Pro-Phe-Arg-pNA (Diapharma) for plasma

kallikrein; MeO-Suc-Arg-Pro-Tyr-pNA (Diapharma) for chymotryp-

sin.

All assays were conducted at room temperature except where noted

in 96-well microtiter plate spectrophotometers or spectroﬂuorimeters

(Molecular Devices) with simultaneous measurement of enzyme

activities in control and inhibitor containing solutions. Compounds

were dissolved and diluted in DMSO and analyzed at a ﬁnal

concentration of 1% DMSO except where noted. Assays were initiated

by adding enzyme to buﬀered solutions containing substrate in the

presence or absence of inhibitor. Hydrolysis of the substrate resulted

in the release of pNA (p-nitroaniline), which was monitored

spectrophotometrically by measuring the increase in absorbance at

405 nm, or the release of AMC (7-amino-4-methylcoumarin), which

was monitored spectroﬂuorometrically by measuring the increase in

emission at 460 nm with excitation at 380 nm. The rate of absorbance

or ﬂuorescence change is proportional to enzyme activity. A decrease

in the rate of absorbance or ﬂuorescence change in the presence of

inhibitor is indicative of enzyme inhibition. Assays were conducted

under conditions of excess substrate and inhibitor over enzyme. The

Michaelis constant, K_m, for substrate hydrolysis by each protease was

determined by ﬁtting data from independent measurements at several

substrate concentrations to the Michaelis−Menten equation:

V

_max[S]

v =

K_m+ [S]

where v is the observed velocity of the reaction, V_maxis the maximal

velocity, [S] is the concentration of substrate, and K_mis the Michaelis

constant for the substrate.

Values of IC₅₀were determined by allowing the protease to react

with the substrate in the presence of the inhibitor. Reactions were

allowed to go for periods of 10−120 min (depending on the protease),

and the velocities (rate of absorbance or ﬂuorescence change versus

time) were measured.

The following relationship was used to calculate IC₅₀values:

v

B − A

s

= A +

n

IC₅₀

v

o

1 +

(

)

I

where v_ois the velocity of the control in the absence of inhibitor, v_sis

the velocity in the presence of inhibitor, I is the concentration of

inhibitor, A is the minimum activity remaining (usually locked at zero),

B is the maximum activity remaining (usually locked at 1.0), n is the

Hill coeﬃcient, a measure of the number and cooperativity of potential

inhibitor binding sites, and IC₅₀is the concentration of inhibitor that

produces 50% inhibition.

When negligible enzyme inhibition was observed at the highest

inhibitor concentration tested, the value assigned as a lower limit for

IC₅₀is the value that would be obtained with either 25% or 50%

inhibition at the highest inhibitor concentration. In all other cases IC₅₀

values represent the average of duplicate determinations obtained over

8−11 concentrations. Competitive inhibition was assumed for all

proteases. IC₅₀values were converted to K_ivalues by the the following

relationship:

IC₅₀

K_i=

[S]

1 +

K_m

aPTT and PT in Vitro Coagulation Assays. Standard clotting

assays were performed in a temperature-controlled automated

coagulation device (Sysmex CA-6000, Dade-Behring). Blood was

obtained from healthy volunteers by venipuncture and anticoagulated

with one-tenth volume 0.11 M buﬀered sodium citrate (Vacutainer,

Becton Dickinson). Plasma was obtained after centrifugation at 2000g

for 10 min and kept on ice prior to use. An initial stock solution of the

inhibitor at 10 mM was prepared in DMSO. Subsequent dilutions

were done in plasma. Clotting time was determined on control plasma

and plasma containing up to seven diﬀerent concentrations of

inhibitor. The activated partial thromboplastin time (aPTT) was

performed using Alexin (Trinity Biotech) according to the reagent

instructions. Plasma (50 μL) was warmed to 37 °C for 1 min before

adding aPTT reagent (50 μL). Two minutes later calcium chloride (50

μL) was added. The prothrombin time (PT) was performed using

Thromboplastin C-Plus (Dade-Behring) according to the reagent

instructions. Plasma (50 μL) was warmed to 37 °C for 3 min before

adding PT reagent (100 μL). Determinations were performed in

duplicate and expressed as a mean ratio of treated vs baseline control.

The concentrations required to prolong clotting time by 2-fold (EC_2x)

were calculated by linear interpolation (Microsoft Excel, Redmond,

WA, USA) and are expressed as total plasma concentrations, not ﬁnal

assay concentrations after addition of clotting assay reagents.

Recombinant FXI Catalytic Domain Cloning and Expression.

A gene encoding the expression of amino acids D357 to V607 of FXI

catalytic domain with the following modiﬁcations was made: point

mutations N473G, T475G to mitigate lattice hindrance, an N-terminal

addition of MDDDD which improved solubility and handling of the

protein, and a six membered poly-histidine tail on the C-terminus to

enable metal chelation aﬃnity chromatography (sequence 1). The

gene was inserted into pET14b T7 expression plasmid (EMD-

Millapore). The sequence conﬁrmed plasmid was transformed into

competent Origami B, BL21(DE3) E. coli cells. The catalytic domain

of FXI has eight cysteines and four disulﬁde bonds. Use of Origami B

enabled a small portion of the protein product to have the correctly

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oxidized pairings of these cysteines, which are ultimately puriﬁed from

mismatched cousins by selection on a benzamidine aﬃnity resin.

Fermentation of cells was undertaken at 37 °C until the logarithmic

growth phase was reached. Expression was induced with IPTG at 20

°C. Cells were harvested 12−16 h later by centrifugation, and the cell

pellets were frozen for future use.

Sequence 1. The amino acid sequence 1 of the FXI catalytic

domain used to clone and express the protein is the following:

MDDDDKMDNECTTKIKPR^∧IVGGTASVRGEWPWQVTLHT-

TSPTQRHLCGGSIIGNQWILTAAHCFYGVESPKILRVYSG-

ILNQSEIKEDTSFFGVQEIIIHDQYKMAESGYDIALLKLE-

TTVGYGDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDK-

IQNTLQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGG-

KDACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGV-

YTNVVEYVDWILEKTQAVHHHHHH

CNX (Accelrys), and inhibitor restraint dictionaries were built with

QUANTA (Accelrys), which was also used for modeling. Later the

structures were rereﬁned using BUSTER/TNT²²(GlobalPhasing,

Ltd.), MakeTNT, or GRADE (GlobalPhasing, Ltd.) for inhibitor

restraint dictionaries, and COOT²³and PROCHECK²⁴for modeling.

The PDB deposition numbers for compounds 11h and 16b complexed

to FXIa are 4TY6 and 4TY7, respectively.

Molecular Modeling Studies. Compounds 11a and 11h were

docked into the crystal structure of FXIa complexed to compound 3

using Glide XP (Schrodinger LLC, New York, NY) and OPLS2001

force ﬁeld. Twenty-ﬁve docked poses were requested as output and

were visually inspected. Binding poses were selected from these sets

based on previous knowledge of docking outcomes and SAR.

Additional reﬁnement was completed by minimizing the selected

molecule poses using MacroModel (Schrodinger LLC, New York,

NY) with the OPLS2001 force ﬁeld constraining all atoms outside 5 Å

from the ligand binding site.

In the amino acid sequence, the ∧ denotes the thrombin activation

cleavage site. Underlined amino acids are modiﬁcations and termini

additions employed.

AV-Shunt Thrombosis Model. For a full description of the model

see Wong et al.^18aArterial blood from the femoral artery of

anesthetized (ketamine, 50 mg/kg im, and xylazine, 10 mg/kg im;

supplemented as needed) New Zealand male white rabbits was

diverted through a PE60 cannula to a AV-shunt device, which is

composed of a double tygon tubing with the inner tubing containing a

2.5 cm long 2-0 silk thread, and returned to the animal via a PE60

cannula inserted into the opposite femoral vein. Signiﬁcant thrombus

formation was initiated through contact with the silk thread. After 40

min the AV-shunt was disconnected from the arterial and venous

catheters. The silk thread, which was covered with the thrombus, was

removed and weighed. The thrombus weight formed on the thread

was calculated by subtracting the average weight of a clean 2.5 cm long

2-0 silk thread. After obtaining the thrombus formation induced by the

AV-shunt in the control period, the compound or vehicle was given as

a bolus iv followed by a continuous iv infusion via the jugular vein

started 30 min prior to the insertion of a new AV-shunt. The thrombus

formation induced by the AV-shunt in the treatment period was

measured as described above. Data are reported as % inhibition of

thrombus formation in the treatment period versus in control period.

Three doses of compound 16b were tested: 0.1 mg/kg + 0.16 mg kg⁻¹

h⁻¹, 0.5 mg/kg + 0.8 mg kg⁻¹h⁻¹, 2.5 mg/kg + 4 mg kg⁻¹h⁻¹using a

dosing vehicle of saline. The saline vehicle did not signiﬁcantly alter

the AV-shunt-induced thrombus formation. A total of three rabbits

were used for each dose of 16b. The ID₅₀represents the dose that

produced 50% inhibition of thrombus formation and was estimated by

linear regression.

Recombinant FXIa Catalytic Domain Puriﬁcation. An amount

of 40 g of cells (wet packed cell weight) was thawed and suspended in

150 mL of lysis buﬀer (50 mM Na/KPO₄, pH 8.0, 0.3 M NaCl, 20

mM imidazole) per gram of packed cells and lysed mechanically in an

Emulsiﬂex homogenizer. The lsyate suspension was clariﬁed by

centrifugation at 10 °C, and the supernatant was combined with 6 mL

of Ni-NTA Superﬂow resin (Qiagen Inc.) pre-equilibrated with buﬀer

A (50 mM Na/KPO₄, pH 8.0, 0.3 M NaCl, and 20 mM imidazole) and

the mixture incubated for 2 h at 4 °C. The mixture was then packed

into a column and was washed with 50 mL of buﬀer A. Crude FXI

catalytic domain protein was eluted from the column with the buﬀer A

supplemented with 300 mM imidazole. Fractions containing the

protein were identiﬁed by molecular weight using SDS−PAGE and

were pooled and dialyzed overnight against buﬀer B (50 mM

triethanolamine, 100 mM NaCl, 1 mM EDTA, pH 7.9) at 4 °C. The

next morning, dextran sulfate sodium salt was added to a ﬁnal

concentration of 20 μg/mL. Thrombin (1000 units; Calbiochem,

catalog no. 605160) was added, and the mixture was incubated for 30

min at 37 °C to convert the FXI catalytic domain to the active form

(FXIa catalytic domain). To arrest the reaction, polybrene and MgCl₂

were added up to 100 ug/mL and 20 mM, respectively. Imidazole was

then added up to 10 mM, and the solution was loaded onto a 5 mL

column of Ni-NTA Superﬂow, washed with buﬀer A, and eluted in

buﬀer A supplemented with 300 mM imidazole. Fractions containing

FXIa catalytic domain as determined by SDS−PAGE were pooled and

ﬁltered. Final puriﬁcation of FXIa catalytic domain from inactive

contaminants was accomplished by loading the Ni-NTA eluate onto a

5 mL benzamidine Sepharose (GE, Healthcare) column pre-

equilibrated in 50 mM Tris, pH 7.4, 1 M NaCl, and the column

was washed with this buﬀer until baseline was achieved. FXIa catalytic

domain protein able to bind to benzamidine was eluted in the same

buﬀer supplemented with 40 mM benzamidine sulfate. The puriﬁed

yield from this process was 2−4 mg of protein.

Formation of Crystals Using Puriﬁed Recombinant FXIa

Catalytic Domain Complexed to Compound 11h or 16b.

Crystals of FXIa catalytic domain complexed to benzamidine were

grown at 4 °C by the hanging-drop vapor diﬀusion method. FXIa

catalytic domain from the last step of puriﬁcation (10 mg/mL) was

saturated with benzamidine. A solution of 0.2 M (NH₄)₂SO₄, 25%

polyethylene glycol methyl ether-2000, and 100 mM NaOAc, pH 4.6,

was used as the precipitant. Benzamidine was replaced by compound

11h or 16b by soaking the FXIa catalytic domain/benzamidine crystals

in a solution containing 2 mM replacement inhibitor in matched

precipitant. Crystals were cryoprotected in 20% ethylene glycol for

mounting prior to X-ray diﬀraction.

X-ray Crystal Structure Data Collection and Structure

Reﬁnement (See Supporting Information for Tabulated

Parameters). Data for FXIa crystals in complex with compound

11h or 16b were collected at the Advanced Photon Source (APS)

beamline 17-ID or in the laboratory. Raw data were processed with the

program HKL2000.²¹The atomic coordinates of human factor XIa¹²

were used as a search model. Original reﬁnement was carried out with

Preparation of Compounds 11a−s. Compounds 11a−s were

prepared by application of the methods described for the preparation

of 11a. 11i−s required additional functional group transformations as

described.

(S)-tert-Butyl (2-Phenyl-1-(4-phenyl-1H-imidazol-2-yl)ethyl)-

carbamate (9a). To a solution of N-Boc-^L-phenylalanine (7) (750

mg, 3.7 mmol) in 1:1 EtOH/H₂O (7.4 mL) was added Cs₂CO₃(650

mg, 1.89 mmol). The reaction mixture was stirred at rt for 1 h. The

solvent was removed under vacuum, and the solids were suspended in

DMF (4.7 mL). 2-Bromoacetophenone (1.0 g, 3.77 mmol) was added

in a single portion, and the reaction mixture was stirred at rt for 1.5 h.

The mixture was ﬁltered to remove solids. The solids were washed

with DMF. The combined washings and ﬁltrate were concentrated in

vacuo to yield (S)-2-oxo-2-phenylethyl 2-((tert-butoxycarbonyl)-

amino)-3-phenylpropanoate (I-1) (650 mg). I-1 was placed in a

ﬂask ﬁtted with a Dean−Stark trap and dissolved in xylenes (10 mL).

NH₄OAc (2.64 g, 30 mmol) was added to the ﬂask, and the mixture

was heated to reﬂux for 3 h. The reaction mixture was cooled to rt and

the solvent removed in vacuo. The residue was redissolved in EtOAc,

washed with saturated aq NaHCO₃and brine, dried over Na₂SO₄,

ﬁltered, and evaporated in vacuo to provide 9a (780 mg, 57%). m/z

364 [M + H]⁺. Alternatively, I-1 and 10 equiv of NH₄OAc were

dissolved in ethanol and heated to 160 °C in a sealed tube for 30 min

using microwave irradiation. The reaction was cooled to rt, the ethanol

was evaporated in vacuo, and the residue was redissolved in EtOAc.

The organic layer was washed with brine, dried over MgSO₄, and

L

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evaporated to dryness. The product thus formed was suﬃciently pure

to carry onto the next step.

2H), 7.22−7.26 (m, 1H), 7.27−7.32 (m, 2H), 7.68 (t, J = 8.0 Hz, 1H),

7.81 (d, J = 7.7 Hz, 1H), 7.91 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 8.07

(s, 1H). m/z 428 [M + H]⁺.

tert-Butyl ((trans-4-(((S)-2-Phenyl-1-(4-phenyl-1H-imidazol-

2-yl)ethyl)carbamoyl)cyclohexyl)methyl)carbamate (10a). To

a solution of 9a (100 mg, 0.28 mmol) in CH₂Cl₂(2.8 mL) was added

neat TFA (0.2 mL). The reaction mixture was stirred at rt for 16 h.

The solvent and TFA were removed in vacuo. The residue was twice

redissolved in CH₃OH and the solvent removed in vacuo to provide

(S)-2-phenyl-1-(4-phenyl-1H-imidazol-2-yl)ethanamine, bis-triﬂuoro-

acetic acid salt (I-2) (124 mg, 92%), which was carried onto the

next step without puriﬁcation. m/z 262 (M − H)⁻. To a solution of I-

2 (75 mg, 0.15 mmol) and N-Boc-tranexamic acid (75 mg, 0.29 mmol)

in pyridine (1.7 mL) was added BOP reagent (152 mg, 0.34 mmol).

The reaction mixture was stirred at rt for 12 h. The mixture was

diluted with H₂O and extracted with EtOAc. The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

evaporated in vacuo to give 10a (100 mg, 71%). 10a was carried onto

the next step without puriﬁcation. m/z 503 [M + H]⁺. Alternatively, I-

2 was coupled with 1.0 equiv of N-Boc-tranexamic acid, 1.2 equiv of

HOAt, 5.0 equiv of N-methylmorpholine, and 1.2 equiv of EDC.

trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(4-phenyl-1H-imi-

dazol-2-yl)ethyl)cyclohexanecarboxamide, Bis-triﬂuoroacetic

Acid Salt (11a). 10a (100 mg, 0.20 mmol) was dissolved in 30%

TFA in CH₂Cl₂(2.0 mL). The reaction mixture was stirred at rt for 3

h. The mixture was twice dissolved in toluene and dried in vacuo. 11a

was isolated by preparative HPLC (46 mg, 49%). ¹H NMR (500 MHz,

CD₃OD) δ 1.02−1.11 (m, 2H), 1.32−1.45 (m, 2H), 1.54−1.61 (m,

1H), 1.80 (br d, J = 12 Hz, 1H), 1.86 (br d, J = 12 Hz, 3H), 2.28 (dt, J

= 12, 3.3 Hz, 1H), 2.77 (d, J = 7.1 Hz, 2H), 3.31 (dd, 1H, overlap with

CH₃OH), 3.38 (dd, J = 13, 8.0 Hz), 5.31 (t, J = 8.2 Hz, 1H), 7.18 (d, J

= 7.1 Hz, 2H), 7.22−7.30 (m, 3H), 7.43−7.50 (m, 3H), 7.62 (d, J =

6.6 Hz, 2H), 7.75 (s, 1H). m/z 403 [M + H]⁺.

trans-4-(Aminomethyl)-N-((S)-1-(4-(4-cyanophenyl)-1H-imi-

dazol-2-yl)-2-phenylethyl)cyclohexanecarboxamide, Bis-tri-

ﬂuoroacetic Acid Salt (11b). ¹H NMR (500 MHz, CD₃OD) δ

1.02−1.12 (m, 2H), 1.30−1.46 (m, 2H), 1.53−1.61 (m, 1H), 1.80 (d, J

= 12 Hz, 1H), 1.86 (d, J = 11 Hz, 3H), 2.23−2.32 (m, 1H), 2.64 (d, J

= 9.4 Hz, 1H), 2.78 (d, J = 6.6 Hz, 2H), 3.32 (dd, J = 8.2 Hz, 1 H,

overlap with CH₃OH), 3.38 (dd, J = 13, 8.2 Hz, 1H), 5.33 (t, J = 8.2

Hz, 1H), 7.19 (d, J = 6.6 Hz, 2H), 7.22−7.26 (m, 1H), 7.26−7.32 (m,

2H), 7.85 (s, 4H), 7.94 (s, 1H). HRMS calcd for C₂₆H₂₉N₅O [M +

H]⁺428.2450, found 428.2443.

M e t h y l 4 - ( 2 - ( ( S ) - 1 - ( t r a n s - 4 - ( A m i n o m e t h y l ) -

cyclohexanecarboxamido)-2-phenylethyl)-1H-imidazol-4-yl)-

benzoate, Bis-triﬂuoroacetic Acid Salt (11c). ¹H NMR (500

MHz, CD₃OD) δ 1.00−1.14 (m, 2H), 1.30−1.47 (m, 2H), 1.51−1.64

(m, 1H), 1.80 (d, J = 12 Hz, 1H), 1.86 (d, J = 12 Hz, 3H), 2.23−2.34

(m, 1H), 2.78 (d, J = 6.6 Hz, 2H), 3.32 (dd, J = 8.2 Hz, 1 H, overlap

with CH₃OH), 3.39 (dd, J = 13, 8.2 Hz, 1H), 3.93 (s, 3H), 5.33 (t, J =

8.2 Hz, 1H), 7.19 (d, J = 7.2 Hz, 2H), 7.22−7.26 (m, 1H), 7.27−7.32

(m, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.90 (s, 1H), 8.12 (d, J = 8.8 Hz,

2H). HRMS calcd for C₂₇H₃₂N₄O₃[M + H]⁺461.2553, found

461.2570.

Ethyl 2-(3-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)phenyl)-

1

acetate, Bis-triﬂuoroacetic Acid Salt (11f). H NMR (500 MHz,

CD₃OD) δ 1.02−1.12 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H), 1.33−1.46

(m, 2H), 1.57 (m, 1H), 1.80 (d, J = 12 Hz, 1H), 1.86 (d, J = 12 Hz,

3H), 2.23−2.32 (m, 1H), 2.77 (d, 2H), 3.30 (dd, 1H, overlap with

CH₃OH), 3.38 (dd, J = 13, 8.2 Hz,1H), 3.72 (s, 2H), 4.15 (q, J = 7.2

Hz, 2H), 5.31 (t, J = 8.2 Hz, 1H), 7.16−7.21 (m, 2H), 7.25 (d, J = 5.5

Hz, 1H), 7.26−7.32 (m, 2H), 7.37−7.41 (m, 1H), 7.46 (t, J = 7.7 Hz,

1H), 7.52−7.58 (m, 2H), 7.76 (s, 1H). m/z 489 [M + H]⁺.

trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(4-(4-sulfamoyl-

phenyl)-1H-imidazol-2-yl)ethyl)cyclohexanecarboxamide, Bis-

1

triﬂuoroacetic Acid Salt (11g). H NMR (500 MHz, CD₃OD) δ

1.00−1.12 (m, 2H), 1.30−1.47 (m, 2H), 1.51−1.63 (m, 1H), 1.79 (d, J

= 12 Hz, 1H), 1.86 (d, J = 10 Hz, 3H), 2.21−2.33 (m, 1H), 2.77 (d, J

= 7.2 Hz, 2H), 3.31 (dd, J = 8.25 Hz, 1H, overlap with CH₃OH), 3.38

(dd, J = 13, 8.2 Hz, 1H), 5.32 (t, J = 8.2 Hz, 1H), 7.19 (d, J = 7.2 Hz,

2H), 7.21−7.26 (m, 1H), 7.26−7.32 (m, 2H), 7.82 (d, J = 8.2 Hz,

2H), 7.89 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H). m/z 482 [M + H]⁺.

tert-Butyl ((trans-4-(((S)-1-(4-(4-Carbamoylphenyl)-1H-imida-

zol-2-yl)-2-phenylethyl)carbamoyl)cyclohexyl)methyl)-

carbamate (10h). 10b (117 mg, 0. 22 mmol) was dissolved in

DMSO (2 mL) and treated with K₂CO₃(90.0 mg, 0.65 mmol) under

Ar. 30% aq H₂O₂(0.8 mL, 2.4 mmol) and MgO (44 mg, 1.11 mmol)

were added sequentially. The reaction mixture was stirred at rt for 4 h.

The reaction mixture was diluted with EtOAc, washed with 1 N aq

HCl and brine, dried over MgSO₄, ﬁltered, and evaporated to dryness

in vacuo to provide 10h (109 mg, 90%) as a yellow solid. 10h was

carried onto the next step without puriﬁcation. m/z 546 [M + H]⁺.

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)benzamide,

Bis-triﬂuoroacetic Acid Salt (11h). 10h was treated with TFA

according to the procedure described for the conversion of compound

1

10a to 11a to provide 11h (17.3 mg, 20%). H NMR (500 MHz,

CD₃OD) δ 1.00−1.13 (m, 2H), 1.30−1.47 (m, 2H), 1.52−1.63 (m,

1H), 1.80 (d, J = 12 Hz, 1H), 1.86 (d, J = 11 Hz, 3H), 2.28 (t, J = 12

Hz, 1H), 2.77 (d, J = 6.6 Hz, 2H), 3.31 (dd, J = 8.8 Hz, 1H, overlap

with CH₃OH), 3.38 (dd, J = 13, 8.2 Hz, 1H), 5.33 (t, J = 8.2 Hz, 1H),

7.19 (d, J = 7.2 Hz, 2H), 7.21−7.26 (m, 1H), 7.26−7.32 (m, 2H), 7.76

(d, J = 8.8 Hz, 2H), 7.88 (s, 1H), 7.99 (d, J = 8.2 Hz, 2H). HRMS

calcd for C₂₆H₃₀N₅O₂[M + H]⁺446.2556, found 446.2561.

tert-Butyl ((trans-4-(((S)-1-(5-Bromo-4-(4-carbamoylphenyl)-

1H-imidazol-2-yl)-2-phenylethyl)carbamoyl)cyclohexyl)-

methyl)carbamate (10i). To a solution of 10h (70 mg, 0.13 mmol)

in CHCl₃(10 mL) was added bromine (21 mg, 0.13 mmol). The

reaction mixture was stirred at rt for 16 h. The reaction mixture was

evaporated to dryness in vacuo to yield 10i (93 mg, 100%), which was

carried onto the next step without puriﬁcation. m/z 622/624 (1:1) [M

− H]⁻.

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-5-bromo-1H-imidazol-4-yl)-

benzamide, Bis-triﬂuoroacetic Acid Salt (11i). 10i was converted

to 11i (35.8 mg, 36%) using the method described for the conversion

of compound 10a to 11a. ¹H NMR (500 MHz, CD₃OD) δ 0.99−1.11

(m, 2H), 1.29−1.48 (m, 2H), 1.55−1.63 (m, 1H), 1.75 (d, 1H), 1.85

(d, J = 9.9 Hz, 3H), 2.16−2.27 (m, 1H), 2.77 (d, J = 6.6 Hz, 2H), 3.17

(dd, J = 13, 8.2 Hz, 1H), 3.25 (dd, J = 13, 7.7 Hz, 1H), 3.98 (s, 1H),

5.22 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 7.2 Hz, 2H), 7.20 (d, J = 7.2 Hz,

1H), 7.23−7.28 (m, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.94 (d, J = 8.2 Hz,

2H). HRMS calcd for C₂₆H₃₀BrN₅O₂524.1661 [M + H]⁺, found

524.1653.

Ethyl 2-(4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexan-

ecarboxamido)-2-phenylethyl)-1H-imidazol-4-yl)phenyl)-

1

acetate, Bis-triﬂuoroacetic Acid Salt (11d). H NMR (500 MHz,

CD₃OD) δ 1.01−1.13 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H), 1.33−1.44

(m, 2H), 1.51−1.64 (m, 1H), 1.80 (d, J = 13 Hz, 1H), 1.86 (d, J = 11

Hz, 4H), 2.23−2.32 (m, 1H), 2.77 (d, J = 7.1 Hz, 2H), 3.32−3.38 (dd,

1H, overlap with CH₃OH), 3.38 (dd, J = 13, 8.2, 1H), 3.70 (s, 2H),

4.15 (q, J = 7.2 Hz, 2H), 5.31 (t, J = 8.5 Hz, 1H), 7.17 (d, J = 7.2 Hz,

2H), 7.21−7.26 (m, 1H), 7.27−7.32 (m, 2H), 7.41 (d, J = 8.2 Hz,

2H), 7.59 (d, J = 8.2 Hz, 2H), 7.74 (s, 1H). HRMS for calcd for

C₂₉H₃₆N₄O₃[M + H]⁺489.2866, found 489.2861.

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-5-chloro-1H-imidazol-4-yl)-

benzamide, Bis-triﬂuoroacetic Acid Salt (11j). 9b was converted

to 11j by sequentially applying the procedures used to convert 10b to

10h, 14a to 14b, and 9a to 11a. H NMR (400 MHz, CD₃OD) δ

0.94−1.13 (m, 2H), 1.30−1.49 (m, 2H), 1.51−1.64 (m, 1H), 1.75 (d, J

= 13 Hz, 1H), 1.86 (d, J = 11 Hz, 3H), 2.16−2.27 (m, 1H), 2.77 (d, J

trans-4-(Aminomethyl)-N-((S)-1-(4-(3-cyanophenyl)-1H-imi-

dazol-2-yl)-2-phenylethyl)cyclohexanecarboxamide, Bis-tri-

ﬂuoroacetic Acid Salt (11e). ¹H NMR (500 MHz, CD₃OD) δ

0.99−1.13 (m, 2H), 1.26−1.47 (m, 2H), 1.52−1.63 (m, 1H), 1.79 (d, J

= 12 Hz, 1H), 1.85 (d, J = 12 Hz, 3H), 2.21−2.34 (m, 1H), 2.77 (d, J

= 7.1 Hz, 2H), 3.32 (dd, J = 8.0 Hz, 1 H, overlap with CH₃OH), 3.38

(m, J = 13, 8.2 Hz, 1H), 5.34 (t, J = 8.2 Hz, 1H), 7.19 (d, J = 7.1 Hz,

1

M

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= 7.0 Hz, 2H), 3.14 (dd, J = 13, 7.2 Hz, 1H), 3.24 (dd, J = 13, 7.5 Hz,

1H), 5.21 (t, J = 7.9 Hz, 1H), 7.14−7.22 (m, 3H), 7.22−7.29 (m, 2H),

7.74 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H). m/z 480 [M + H]⁺.

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-5-ﬂuoro-1H-imidazol-4-yl)-

benzamide, Bis-triﬂuoroacetic Acid Salt (11k). 10h was

converted to 11k by sequential application of the methods descibed

for the conversion of 14a to 14c and 10a to 11a. ¹H NMR (400 MHz,

CD₃OD) δ 7.90 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.27−

7.20 (m, 2H), 7.20−7.14 (m, 3H), 5.17 (t, J = 7.7 Hz, 1H), 3.20 (dd, J

= 13.2, 7.7 Hz, 1H), 3.12 (dd, J = 13.2, 7.7 Hz, 1H), 2.77 (d, J = 7.1

Hz, 2H), 2.26−2.16 (m, 1H), 1.89−1.80 (m, 3H), 1.78−1.70 (m, 1H),

1.64−1.51 (m, 1H), 1.49−1.29 (m, 2H), 1.12−0.98 (m, 2H). ¹⁹F

NMR (376 MHz, CD₃OD) δ −77.47 (s), −133.62 (s). m/z 464 [M +

H]⁺.

4-(2-((S)-1-(trans-4-((tert-Butoxycarbonylamino)methyl)-

cyclohexanecarboxamido)-2-phenylethyl)-1H-imidazol-4-yl)-

benzoic Acid (10l). To a solution of 10c (162 mg, 0.28 mmol) in

CH₃OH (5 mL) and EtOAc (1 mL) were added 1 N aq NaOH (2

mL) and MgO (112 mg, 2.8 mmol). The reaction mixture was stirred

for 5 h at rt. The solvent was evaporated to dryness in vacuo. The

residue was diluted with water and treated with 1 N aq HCl. The

aqueous solution was diluted with EtOAc, washed with brine, dried

over MgSO₄, ﬁltered, and evaporated in vacuo to yield 10l (75 mg,

60%), which was carried onto the next step without puriﬁcation. m/z

548 [M + H]⁺.

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)benzoic Acid,

Bis-triﬂuoroacetic Acid Salt (11l). 10l was treated according to

the method described for compound the conversion of compound 10a

to 11a to provide 11l (34 mg, 18%). ¹H NMR (500 MHz, CD₃OD) δ

1.02−1.11 (m, 2H), 1.32−1.46 (m, 2H), 1.53−1.62 (m, 1H), 1.80 (d, J

= 12 Hz, 1H), 1.86 (d, J = 12 Hz, 3H), 2.28 (tt, J = 12, 3.3 Hz, 1H),

2.77 (d, J = 7.1 Hz, 2H), 3.33 (d, J = 8.2 Hz, 1 H, overlap with

CH₃OH), 3.39 (dd, J = 13, 8.2 Hz, 1H), 5.33 (t, J = 8.2 Hz, 1H), 7.19

(d, J = 7.1 Hz, 2H), 7.24 (t, J = 7.1 Hz, 1H), 7.29 (t, J = 7.1 Hz, 2H),

7.76 (d, J = 8.2 Hz, 2H), 7.89 (s, 1H), 8.12 (d, J = 8.8 Hz, 2H). HRMS

calcd for C₂₆H₂₉N₄O₃447.2396 [M + H]⁺, found 447.2397.

tert-Butyl ((trans-4-(((S)-1-(4-(4-(Methylcarbamoyl)phenyl)-

1H-imidazol-2-yl)-2-phenylethyl)carbamoyl)cyclohexyl)-

methyl)carbamate (10m). 10l (45 mg, 0.08 mmol) and methyl-

amine (0.2 mL, 0.09 mmol) were converted to 10m (44.0. mg, 93%)

using the alternative procedure described for conversion of I-2 to 10a.

10m was carried to the next step without puriﬁcation. m/z 560 [M +

H]⁺.

2-(4-(2-((S)-1-(trans-4-((tert-Butoxycarbonylamino)methyl)-

cyclohexanecarboxamido)-2-phenylethyl)-1H-imidazol-4-yl)-

phenyl)acetic Acid (10o). 10d (200 mg, 0.34 mmol) was dissolved

in CH₃OH (3 mL) and treated with 1 N aq NaOH (0.34 mL, 0.34

mmol). The reaction mixture was stirred at rt for 16 h. The solvent

was removed in vacuo. The residue puriﬁed by preperative HPLC to

provide 10o (90 mg, 47%). m/z 561 [M + H]⁺.

2-(4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)phenyl)acetic

Acid, Bis-triﬂuoroacetic Acid Salt (11o). 10o was treated

according to the method described for conversion of 10a to 11a to

1

provide 11o (15 mg, 81%). H NMR (500 MHz, CD₃OD) δ 0.99−

1.13 (m, 2H), 1.52−1.63 (m, 1H), 1.80 (d, J = 12 Hz, 1H), 1.86 (d, J =

10 Hz, 3H), 2.22−2.33 (m, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.31 (dd, 1

H, overlap with CH₃OH), 3.38 (dd, J = 13, 8.2 Hz, 1H), 3.67 (s, 2H),

5.31 (t, J = 8.2 Hz, 1H), 7.18 (d, J = 6.6 Hz, 2H), 7.21−7.26 (m, 1H),

7.26−7.32 (m, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H),

7.74 (s, 1H). HRMS calcd for C₂₇H₃₂N₄O₃461.2553 [M + H]⁺, found

461.2572.

tert-Butyl (trans-4-(((S)-1-(4-(4-(2-Amino-2-oxoethyl)-

phenyl)-1H-imidazol-2-yl)-2-phenylethyl)carbamoyl)-

cyclohexyl)carbamate (10p). To a solution of 10o (33 mg, 0.060

mmol) and HOAt (9.7 mg, 0.071 mmol) in DMF (1 mL) were added

sequentially N-methylmorpholine (18.2 mg, 0.18 mmol) and EDC

(13.6 mg, 0.071 mmol). The reaction mixture was stirred for 30 min at

rt. Concentrated aq NH₃(5 drops from a Pasteur pipet) was added,

and the reaction mixture was stirred at rt for 16 h. The mixture was

diluted with EtOAc and washed with a 1:1 mixture of H₂O and brine,

1 N aq HCl, and brine, dried over MgSO₄, ﬁltered, and dried in vacuo.

The product was isolated by preparative HPLC to provide 10p (9 mg,

22%). m/z 560 [M + H]⁺.

trans-N-((S)-1-(4-(4-(2-Amino-2-oxoethyl)phenyl)-1H-imida-

zol-2-yl)-2-phenylethyl)-4-(aminomethyl)cyclohexane-

carboxamide, Bis-triﬂuoroacetic Acid Salt (11p). 10p (9 mg,

0.016 mmol) was converted to 11p (11 mg, 99%) according the

1

method described for the conversion of compound 10a to 11a. H

NMR (500 MHz, CD₃OD) δ 1.02−1.11 (m, 2H), 1.32−1.44 (m, 2H),

1.53−1.61 (m, 1H), 1.79 (d, J = 13 Hz, 1H), 1.85 (d, J = 11 Hz, 3H),

2.28 (tt, J = 3.0, 12 Hz, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.31 (dd, 1 H,

overlap with CH₃OH), 3.38 (dd, J = 13, 8.0 Hz, 1H), 3.57 (s, 2H),

5.31 (t, J = 8.2 Hz, 1H), 7.17 (d, J = 7.2 Hz, 2H), 7.22−7.25 (m, 1H),

7.27−7.30 (m, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H),

7.74 (s, 1H). HRMS calcd for C₂₇H₃₃N₅O₂460.2713 [M + H]⁺, found

460.2714.

3-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)benzamide,

Bis-triﬂuoroacetic Acid Salt (11q). 11q was prepared from 10e

according to the method used for conversion of 10b to 11h. ¹H NMR

(500 MHz, CD₃OD) δ 1.00−1.13 (m, 2H), 1.30−1.47 (m, 2H), 1.52−

1.64 (m, 1H), 1.80 (d, J = 12 Hz, 1H), 1.86 (d, J = 12 Hz, 3H), 2.28 (t,

J = 12 Hz, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.32 (dd, J = 8.2 Hz, overlap

with CH₃OH), 3.39 (dd, J = 13, 8.2 Hz, 1H), 5.34 (t, J = 8.2 Hz, 1H),

7.19 (d, J = 6.6 Hz, 2H), 7.22−7.26 (m, 1H), 7.27−7.32 (m, 2H), 7.60

(t, J = 7.7 Hz, 1H), 7.83 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 8.18 (s,

1H). HRMS calcd for C₂₆H₃₁N₅O₂446.2556 [M + H]⁺, found

446.2535.

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)-N-methylben-

zamide, Bis-triﬂuoroacetic Acid Salt (11m). 10m was converted

to 11m (1.0 mg, 3%) according to the method for the conversion of

1

10a to 11a. H NMR (500 MHz, CD₃OD) δ 1.02−1.11 (m, 1H),

1.32−1.46 (m, 1H), 1.53−1.62 (m, 1H), 1.79 (d, J = 13 Hz, 1H), 1.86

(d, J = 11 Hz, 1H), 2.30−2.65 (m, 1H), 2.77 (d, J = 7.2 Hz, 1H), 2.92

(s, 3H), 3.38 (dd, J = 13, 8.2 Hz, 2H, overlap with CH₃OH), 5.29 (t, J

= 8.2 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.28

(t, J = 7.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.86 (s, 1H), 7.92 (d, J =

8.8 Hz, 1H). HRMS calcd for C₂₇H₃₂N₅O₂460.2713 [M + H]⁺, found

460.2735.

2-(3-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)phenyl)acetic

Acid, Bis-triﬂuoroacetic Acid Salt (11r). 11r was synthesized from

4-(2-((S)-1-(trans-4-(Aminomethyl)cyclohexane-

carboxamido)-2-phenylethyl)-1H-imidazol-4-yl)-N,N-dimethyl-

benzamide, Bis-triﬂuoroacetic Acid Salt (11n). 11n was prepared

from 10l according to the method for 11m from 10l by substituting

1

10f using the methods for the conversion of 10d to 11o. H NMR

(500 MHz, CD₃OD) δ 1.01−1.13 (m, 2H), 1.29−1.47 (m, 2H), 1.52−

1.63 (m, 1H), 1.80 (d, J = 12 Hz, 1H), 1.86 (d, J = 10 Hz, 4H), 2.23−

2.32 (m, J = 12, 12, 3.1, 3.0 Hz, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.31

(dd, 1H, overlap with CH₃OH), 3.38 (dd, J = 13, 8.2 Hz, 1H), 5.31 (t,

J = 8.2 Hz, 1H), 7.18 (d, J = 7.2 Hz, 2H), 7.22−7.27 (m, 1H), 7.27−

7.32 (m, 2H), 7.39 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.53

(d, J = 7.7 Hz, 1H), 7.56 (s, 1H), 7.76 (s, 1H). m/z 461 [M + H]⁺.

trans-N-((S)-1-(4-(3-(2-Amino-2-oxoethyl)phenyl)-1H-imida-

zol-2-yl)-2-phenylethyl)-4-(aminomethyl)cyclohexane-

carboxamide, Bis-triﬂuoroacetic Acid Salt (11s). 11s was

synthesized from 10f using the methods for the conversion of 10d

1

dimethylamine for methylamine. H NMR (500 MHz, CD₃OD) δ

1.03−1.11 (m, 2H), 1.32−1.46 (m, 2H), 1.54−1.61 (m, 1H), 1.81 (d, J

= 15 Hz, 1H), 1.86 (d, J = 10 Hz, 3H), 2.28 (tt, J = 12, 3.3 Hz, 1H),

2.77 (d, J = 7.1 Hz, 2H), 3.01 (s, 3H), 3.12 (s, 3H), 3.31 (dd, 1 H,

overlap with CH₃OH), 3.39 (dd, J = 13, 8.2 Hz, 1H), 5.31 (t, J = 8.2

Hz, 1H), 7.18 (d, J = 7.2 Hz, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.29 (t, J =

7.4 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.85 (s,

1H) ppm. HRMS calcd for C₂₈H₃₄N₅O₂474.2870 [M + H]⁺, found

474.2875.

N

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1

to 11p. H NMR (500 MHz, CD₃OD) δ 0.99−1.13 (m, 2H), 1.29−

(S)-4-(2-(1-Amino-2-phenylethyl)-5-chloro-1H-imidazol-4-

yl)-2-ﬂuorobenzonitrile, Bis-triﬂuoroacetic Acid Salt (I-5). 14b

(0.20 g, 0.45 mmol) was stirred with CH₂Cl₂(6 mL) and TFA (1.5

mL) under N₂for 0.5 h. The solvent and TFA were removed in vacuo

to give I-5 (0.26 g, 99%). I-5 was used without puriﬁcation. ¹H NMR

(400 MHz, CD₃OD) δ 3.33 (m, 2H), 4.56 (dd, J = 8.6, 6.4 Hz, 1H),

7.12 (d, J = 6.6 Hz, 2H), 7.25−7.30 (m, 3H), 7.67 (m, 2H), 7.81 (m,

1H). m/z 341 [M + H]⁺.

1.48 (m, 2H), 1.53−1.62 (m, 1H), 1.80 (d, 1H), 1.86 (d, J = 10 Hz,

3H), 2.21−2.33 (m, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.33 (dd, 1H,

overlap with CH₃OH), 3.38 (dd, J = 13, 8.2 Hz, 1H), 3.59 (s, 2H),

5.32 (t, J = 8.2 Hz, 1H), 7.18 (d, J = 7.15 Hz, 2H), 7.22−7.26 (m, 1H),

7.26−7.32 (m, 2H), 7.37−7.41 (m, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.52

(d, 1H), 7.57 (s, 1H), 7.75 (s, 1H). m/z 460 [M + H]⁺.

4-(2-Bromoacetyl)-2-ﬂuorobenzonitrile (13). 4-Bromo-3-ﬂuo-

robenzoic acid (12) (7.5 g, 34 mmol), Zn(CN)₂(4.0 g, 34 mmol), and

Pd(PPh₃)₄(3.95 g, 34 mmol) were combined in 60 mL of DMF that

had been sparged with N₂. The reaction mixture was heated at 90 °C

under N₂for 3 h. The mixture was cooled to rt and ﬁltered. The ﬁltrate

was diluted with water and extracted with EtOAc. The combined

organic layers were washed with H₂O, brine, dried over MgSO₄, and

concentrated in vacuo to yield 4-cyano-3-ﬂuorobenzoic acid (I-3) (4.0

g, 20 mmol). To a solution of I-3 in CH₂Cl₂(50 mL) was added oxalyl

chloride (2.3 mL, 26 mmol) dropwise over 15 min. The reaction

mixture was stirred at rt for 1 h, then heated at reﬂux for 1 h under N₂.

The solvent was removed and the residue dissolved in CH₃CN (50

mL). This solution was cooled to −15 °C. TMSCH₂N₂(2.0 M in

hexane; 11 mL, 22 mmol) was added dropwise over 20 min. The

reaction mixture was stirred at −15 °C for 1 h under N₂. A solution of

HBr in HOAc (33% by mass; 4.25 mL) was added dropwise over 20

min, and the reaction mixture was stirred at −15 °C for an additional

20 min. The solvent was removed, and the residue dissolved in EtOAc,

washed with water and brine, dried over MgSO₄, and concentrated to

provide 13 (3.2 g, 39% yield over two steps), which was used in the

tert-Butyl (trans-4-(((S)-1-(5-Chloro-4-(4-cyano-3-ﬂuoro-

phenyl)-1H-imidazol-2-yl)-2-phenylethyl)carbamoyl)-

cyclohexyl)carbamate (I-6). N-Boc-tranexamic acid (0.14 g, 0.54

mmol), BOP reagent (0.24 g, 0.54 mmol), and TEA (0.38 mL, 2.7

mmol) were combined in THF (10 mL). The reaction mixture was

stirred at rt for 15 min under N_2,followed by the addition of I-5 (0.26

g, 0.45 mmol). The resulting mixture was heated at 75 °C for 15 min

under N₂. The mixture was cooled to rt and the solvent removed in

vacuo. The residue was dissolved in EtOAc. The solution was washed

with H₂O and brine, dried over MgSO₄, concentrated in vacuo, and

puriﬁed by ﬂash chromatography (40 g silica, 10−100% EtOAc in

1

hexane) to give intermediate I-6 (0.21 g, 67%). H NMR (400 MHz,

CD₃OD) δ 0.94 (m, 2H), 1.25−1.37 (m, 4H), 1.42 (s, 9H), 1.76−1.79

(m, 3H), 2.15 (m, 1H), 2.85 (m, 2H), 3.20−3.30 (m, 2H), 5.17 (m,

1H), 7.16−7.23 (m, 5H), 7.67−7.80 (m, 3H). m/z 580 [M + H]⁺.

trans-4-(Aminomethyl)-N-((S)-1-(5-chloro-4-(4-cyano-3-ﬂuo-

rophenyl)-1H-imidazol-2-yl)-2-phenylethyl)cyclohexane-

carboxamide, Bis-triﬂuoroacetic Acid Salt (15b). I-6 (0.21 g, 0.36

mmol) was stirred with CH₂Cl₂(8 mL) and TFA (2 mL) under N₂for

0.5 h. The solvent and TFA were removed in vacuo to give 15b (0.25

g, >100%). 15b was carried onto the next step without puriﬁcation. m/

z 480 [M + H]⁺.

1

next step without puriﬁcation. H NMR (400 MHz, CD₃OD) δ 2.42

(s, 2H), 7.76−7.85 (m, 3H). m/z 240, 242 (1:1) [M + H]⁺.

(S)-tert-Butyl 1-(4-(4-Cyano-3-ﬂuorophenyl)-1H-imidazol-2-

yl)-2-phenylethylcarbamate (14a). To a solution of 13 (3.2 g,

13 mmol) and 7 (3.5 g, 13 mmol) in DMF (20 mL) was added

Cs₂CO₃(2.6 g, 8 mmol). The reaction mixture was stirred at 15 °C for

1 h under N₂. The mixture was diluted with 100 mL of EtOAc, washed

with H₂O, brine, dried over MgSO₄, concentrated in vacuo, and

puriﬁed by ﬂash chromatography (240 g silica, 10−55% EtOAc in

hexane) to yield (S)-2-(4-cyano-3-ﬂuorophenyl)-2-oxoethyl 2-(tert-

butoxycarbonylamino)-3-phenylpropanoate (I-4) (3.5 g, 63%). m/z

425 [H + H⁺]⁺. I-4 was combined with NH₄OAc (12 g, 155 mmol)

and suspended in xylenes (100 mL). The mixture was heated under N₂

at 150 °C for 2.5 h in a ﬂask equipped with a Dean−Stark trap. The

xylenes were removed in vacuo. The residue was dissolved in EtOAc,

washed with H₂O and brine, dried over MgSO₄, concentrated in

vacuo, and puriﬁed by ﬂash chromatography (240 g silica, 15−70%

trans-N-((S)-1-(4-(3-Amino-1H-indazol-6-yl)-5-chloro-1H-imi-

dazol-2-yl)-2-phenylethyl)-4-(aminomethyl)cyclohexane-

carboxamide, Bis-triﬂuoroacetic Acid Salt (16b-2TFA). 15b

(0.21 g, 0.36 mmol) and hydrazine monohydrate (0.69 mL) were

combined in n-BuOH (8 mL). The reaction mixture was heated at 120

°C under N₂for 1 h. The solvent was removed in vacuo and the

product was puriﬁed by preperative HPLC to give 16b-2TFA (0.14 g,

1

54%). H NMR (500 MHz, CD₃OD) δ 0.98−1.12 (m, 2H), 1.30−

1.49 (m, 2H), 1.53−1.63 (m, 1H), 1.75 (d, J = 12 Hz, 1H), 1.85 (d, J =

12 Hz, 3H), 2.18−2.27 (m, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.16 (dd, J =

13, 7.7 Hz, 1H), 3.25 (dd, J = 13, 7.7 Hz, 1H), 5.21 (t, J = 8.0 Hz, 1H),

7.15−7.21 (m, 3H), 7.23−7.28 (m, 2H), 7.53 (dd, J = 8.5, 1.4 Hz,

1H), 7.71 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H). HRMS calcd for

C₂₆H₃₀ClN₇O 491.2200 [M + H]⁺, found 492.2283.

Alternative Synthesis of Compound 16b Leading to the Bis-

hydrochloric Acid Salt. tert-Butyl (trans-4-((S)-1-(4-(3-Amino-

1H-indazol-6-yl)-5-chloro-1H-imidazol-2-yl)-2-phenylethyl-

carbamoyl)cyclohexyl)methylcarbamate (I-7). To a slurrry of I-6

(4.4 g, 7.8 mmol) in n-BuOH (45 mL) was added hydrazine (14.5 mL,

462 mmol). The reaction mixture was heated to 120 °C under Ar for 1

h. The solvent and excess hydrazine were removed in vacuo. The

solids were washed with Et₂O, collected by ﬁltration, and rinsed with

additional Et₂O and then dried in vacuo to provide I-7 (4.57 g, 99%).

I-7 was carried onto the next step without puriﬁcation. m/z 592 [M +

H]⁺.

1

EtOAc in hexane) to give 14a (2.2 g, 41%). H NMR (400 MHz,

CDCl₃) δ 1.39 (s, 9H), 3.30 (m, 2H), 4.86 (d, J = 6.6 Hz, 1H), 5.32

(d, J = 7.5 Hz, 1H), 7.14−7.24 (m, 6H), 7.53−7.61 (m, 3H). m/z 407

[M + H]⁺.

(S)-tert-Butyl (1-(5-Chloro-4-(4-cyano-3-ﬂuorophenyl)-1H-

imidazol-2-yl)-2-phenylethyl)carbamate (14b). To a solution of

14a (2.2 g, 5.4 mmol) in CH₃CN (100 mL) was added NCS (0.80 g,

6.7 mmol). The reaction mixture was heated at reﬂux for 7 h under N₂.

The solvent was removed in vacuo. The residue was dissolved in

EtOAc, washed with H₂O, saturated aq NaHCO₃, and brine, dried

over MgSO₄, and concentrated in vacuo to give 14b (2.4 g, 99%). 14b

was used without puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 1.27 (s,

9H), 3.23 (m, 2H), 4.89 (m, 1H), 5.46 (d, J = 7.0 Hz, 1H), 7.07 (d, J =

6.2 Hz, 2H), 7.25−7.26 (m, 5H), 7.54 (m, 1H). m/z 441 [M + H]⁺.

(S)-tert-Butyl 1-(4-(4-Cyano-3-ﬂuorophenyl)-5-ﬂuoro-1H-imi-

dazol-2-yl)-2-phenylethylcarbamate (14c). To a solution of 14a

(406 mg, 1 mmol) in DMF (10 mL) and H₂O (1 mL) was added

Selecﬂuor (390 mg, 1.1 mmol). The reaction mixture was stirred at rt

for 2 h, then diluted with H₂O and extracted with EtOAc. The

combined organic extracts were washed with brine, dried over Na₂SO₄,

ﬁltered, and evaporated in vacuo. Puriﬁcation by silica gel

chromatography (40 g silica gel; EtOAc in hexanes) provided 14c

trans-N-((S)-1-(4-(3-Amino-1H-indazol-6-yl)-5-chloro-1H-imi-

dazol-2-yl)-2-phenylethyl)-4-(aminomethyl)cyclohexane-

carboxamide, Bis-hydrochloric Acid Salt (16b-2HCl). Intermedi-

ate I-7 (4.55 g, 7.7 mmol) was suspended in 4 N HCl in dioxane (45

mL). The reaction mixture was stirred at rt for 1.5 h. The mixture was

dried in vacuo and the product puriﬁed by prep HPLC using buﬀer

1

containing 0.05% HCl to aﬀord 16b-2HCl (1.88 g, 43%). H NMR

(400 MHz, CD₃OD) δ 1.01−1.15 (m, 2H), 1.29−1.50 (m, 2H), 1.53−

1.65 (m, 1H), 1.76−1.92 (m, 4H), 2.32 (tt, J = 12, 3.3 Hz, 1H), 2.78

(d, J = 7.0 Hz, 2H), 3.35 (dd, J = 8.1, 2.9 Hz, 2H), 5.26 (t, J = 8.1 Hz,

1H), 7.21−7.27 (m, 3H), 7.27−7.33 (m, 2H), 7.55 (dd, J = 8.8, 1.3

Hz, 1H), 7.80 (s, 1H), 8.10 (d, J = 8.8 Hz, 1H). m/z 492 [M + H]⁺.

trans-N-((S)-1-(4-(3-Amino-1H-indazol-6-yl)-1H-imidazol-2-

yl)-2-phenylethyl)-4-(aminomethyl)cyclohexanecarboxamide,

Bis-triﬂuoroacetic Acid Salt (16a). Intermediate 14a was converted

to compound 16a by using the methods used to convert 14b to 16b.

1

(113 mg, 27%). H NMR (400 MHz, CDCl₃) δ 1.37 (s, 9H), 3.10−

3.30 (m, 2H), 4.81−4.89 (m, 1H), 5.42 (d, J = 7.1 Hz, 1H), 6.90−7.00

(m, 2H), 7.08 (m, 2H), 7.21−7.27 (m, 3H), 7.45−7.52 (m, 1H), 11.01

(s, 1H). m/z 425 [M + H]⁺.

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¹H NMR (500 MHz, CD₃OD) δ 1.00−1.13 (m, 2H), 1.30−1.47 (m,

NMR (400 MHz, CD₃OD) δ 1.02−1.06 (m, 2H), 1.29−1.42 (m, 2H),

1.56 (m, 1H), 1.75 (m, 1H), 1.83−1.86 (m, 3H), 2.21 (m, 1H), 2.77

(d, J = 7.0 Hz, 2H), 3.18−3.21 (m, 1H), 3.31 (m, 1H), 5.42 (t, J = 8.1

Hz, 1H), 7.22−7.26 (m, 5H), 7.51−7.52 (m, 3H), 7.96−7.98 (m, 2H).

m/z 404 [M + H]⁺.

2H), 1.51−1.64 (m, 1H), 1.80 (d, J = 13 Hz, 1H), 1.86 (d, J = 12 Hz,

3H), 2.29 (tt, J = 12, 3.3 Hz, 1H), 2.77 (d, J = 7.2 Hz, 2H), 3.33 (dd, J

= 8.8 Hz, 1 H, overlap with CH₃OH), 3.38 (dd, J = 13, 8.2 Hz, 1H),

5.35 (t, J = 8.2 Hz, 1H), 7.20 (d, J = 7.2 Hz, 2H), 7.22−7.27 (m, 1H),

7.27−7.32 (m, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.72 (s, 1H), 7.91 (s,

1H), 7.98 (d, J = 8.8 Hz, 1H). HRMS calcd for C₂₆H₃₁N₇O 458.2650

[M + H]⁺, found 458.2700.

(S)-Benzyl (3,5-Dioxo-1,5-diphenylpentan-2-yl)carbamate

(25). To a solution of ethyl benzolyacetate (23) (2.88 g, 15 mmol)

in EtOH (30 mL) was added 1 N aq NaOH (30 mL). The reaction

mixture was stirred at rt for 72 h. The mixture was cooled to 0 °C and

acidiﬁed with 1 N aq HCl. The EtOH was removed under reduced

pressure. The aqueous layer was extracted with CH₂Cl₂. The

combined organic extracts were washed with brine, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo to give 3-oxo-3-

phenylpropanoic acid (I-11) (600 mg, 24%). m/z 165 [M + H]⁺.

To a solution of I-11 (600 mg, 3.65 mmol) in THF (20 mL) and

CH₃OH (20 mL) was added magnesium ethoxide (417 mg, 3.65

mmol). The mixture was stirred at rt for 4 h. The solvents were

removed in vacuo. The residue was dissolved in DMF (2 mL) and

added to a mixture of N-CBz-^L-phenylalanine (24) (800 mg, 2.68

mmol) and 1,1′-carbonyldiimidazole (520 mg, 3.21 mmol) in DMF (4

mL), which had been stirred at rt for 2 h. The combined reaction

mixture was stirred at rt for 16 h, diluted with EtOAc, washed with

dilute aq HCl, water, and brine, dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The product was isolated by prep HPLC to

give 25 (140 mg, 13%). m/z 402 [M + H]⁺.

trans-N-((S)-1-(4-(3-Amino-1H-indazol-6-yl)-5-ﬂuoro-1H-imi-

dazol-2-yl)-2-phenylethyl)-4-(aminomethyl)cyclohexane-

carboxamide, Bis-triﬂuoroacetic Acid Salt (16c). Intermediate

14c was converted to compound 16c using the methods used to

convert 14b to 16b. ¹H NMR (400 MHz, CD₃OD) δ 7.94 (d, J = 8.2

Hz, 1H), 7.51 (s, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.28−7.21 (m, 2H),

7.21−7.14 (m, 3H), 5.18 (t, J = 8.0 Hz, 1H), 3.23 (dd, J = 13.7, 7.7 Hz,

1H), 3.14 (dd, J = 13.2, 8.2 Hz, 1H), 2.77 (d, J = 7.1 Hz, 2H), 2.28−

2.17 (m, 1H), 1.91−1.79 (m, 3H), 1.79−1.70 (m, 1H), 1.65−1.51 (m,

1H), 1.50−1.29 (m, 2H), 1.13−0.98 (m, 2H). ¹⁹F NMR (471 MHz,

CD₃OD) δ −77.15 (s), −131.90 (s). m/z 476 [M + H]⁺.

(S)-2-(2-Phenyl-1-(5-phenyloxazol-2-yl)ethyl)isoindoline-

1,3-dione (19). To a solution of (S)-2-(1,3-dioxoisoindolin-2-yl)-3-

phenylpropanoic acid (17) (290 mg, 1 mmol) and 2-amino-1-

phenylethanone (18) (171 mg, 1 mmol) in anhydrous pyridine (8

mL) was added BOP reagent (996 mg, 2.2 mmol). The reaction was

stirred at rt for 16 h. The reaction was diluted with water and extracted

with EtOAc. The combined organic extracts were washed with brine,

dried over anhydrous Na₂SO₄, ﬁltered, and evaporated to dryness in

vacuo to yield 412 mg (100%) of crude (S)-2-(1,3-dioxoisoindolin-2-

yl)-N-(2-oxo-2-phenylethyl)-3-phenylpropanamide (I-8). m/z 413 [M

+ H]⁺. To a solution of I-8 (412 mg, 1 mmol) in anhydrous DMF (1.7

mL) was added phosphorus oxychloride (460 mg, 3 mmol). The

solution was heated to 90 °C for 20 min, cooled to rt, and poured onto

ice. The resulting slurry was stirred for 30 min, then added to saturated

aq NaHCO₃and extracted with EtOAc. The combined organic

extracts were washed with H₂O and brine, dried over Na₂SO₄, ﬁltered,

and dried in vacuo. The product was puriﬁed by ﬂash chromatography

to yield 19 (160 mg, 41%). m/z 395 [M + H]⁺.

trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(5-phenyloxazol-

2-yl)ethyl)cyclohexanecarboxamide, Bis-triﬂuoroacetic Acid

Salt, Triﬂuoroacetic Acid Salt (20). To a solution of compound

19 (160 mg, 0.4 mmol) in ethanol (10 mL) was added hydrazine (39

mg, 1.2 mmol). The reaction mixture was heated to reﬂux for 2 h.

After cooling to rt, the solvent was removed in vacuo. The residue was

dissolved in CH₂Cl₂, and insoluble solids were removed by ﬁltration.

The clear ﬁltrate was concentrated to yield 95 mg (90%) of (S)-2-

phenyl-1-(5-phenyloxazol-2-yl)ethanamine (I-9). m/z 265 [M + H]⁺.

I-9 (95 mg, 0.5 mmol) was converted to compound 20 (48 mg, 26%)

using the procedures described for the conversion of compound I-2 to

11a. ¹H NMR (500 MHz, CD₃OD) δ 1.04−1.10 (m, 2H), 1.29−1.42

(m, 2H), 1.56 (m, 1H), 1.75 (m, 1H), 1.83−1.86 (m, 3H), 2.22 (m,

1H), 2.77 (d, J = 7.2 Hz, 2H), 3.20 (m, 1H), 3.33 (m, 1H), 5.41 (m,

1H), 7.22−7.27 (m, 5H), 7.34 (t, J = 7.2 Hz, 1H), 7.42 (m, 3H), 7.63-

7.65 (d, J = 8.2 Hz, 2H). m/z 404 [M + H]⁺.

(S)-tert-Butyl (2-Phenyl-1-(3-phenyl-1H-1,2,4-triazol-5-yl)-

ethyl)carbamate (21). Compound 7 (265 mg, 1 mmol) and

hydrazine (64 mg, 2 mmol) were combined using the conditions

describe to convert I-2 to 10a and substituting hydrazine for N-Boc-

tranexamic acid to give (S)-tert-butyl (1-hydrazinyl-1-oxo-3-phenyl-

propan-2-yl)carbamate (I-10) (279 mg, 100%). m/z 280 [M + H]⁺.

To a solution of I-10 (279 mg, 1.0 mmol) and Et₃N (161 mg, 1.6

mmol) in ACN (10 mL) was added ethyl benzimidate hydrochloride

(278 mg, 1.5 mmol). The reaction was heated to reﬂux for 24 h,

cooled to rt, and diluted with EtOAc. The mixture was washed with

H₂O and brine, dried over Na₂SO_4,ﬁltered, and evaporated to dryness

in vacuo. The product was puriﬁed by preparative HPLC to yield 21

(154 mg, 42%). m/z 365 [M + H]⁺.

(S)-Benzyl (2-Phenyl-1-(3-phenyl-1H-pyrazol-5-yl)ethyl)-

carbamate (26). To a solution of compound 25 (32 mg, 0.08

mmol) in EtOH (3 mL) was added hydrazine hydrate (3 mg, 0.09

mmol). The reaction mixture was heated at 60 °C for 3 h. EtOH was

removed in vacuo to give compound 26 (32 mg, 100%). m/z 398 [M

+ H]⁺.

trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(3-phenyl-1H-pyr-

azol-5-yl)ethyl)cyclohexanecarboxamide, Bis-triﬂuoroacetic

Acid Salt (27). To a solution of compound 26 (32 mg, 0.06

mmol) in CH₃OH (3 mL) was added 10% Pd/C (5 mg). The reaction

mixture was stirred under 1 atm of H₂at rt for 4 h. The catalyst was

removed by ﬁltration. The product was isolated by preparative HPLC

to give (S)-2-phenyl-1-(3-phenyl-1H-pyrazol-5-yl)ethanamine (I-12)

(12 mg, 76%). m/z 262 [M − H]⁻. I-12 (12 mg, 0.046 mmol) was

converted to compound 27 (11 mg, 46%) using the procedures

described for the conversion of intermediate I-2 to 11a. ¹H NMR (500

MHz, CD₃OD) δ 1.02−1.06 (m, 2H), 1.30−1.33 (m, 1H),1.43−1.46

(m, 1H), 1.56 (m, 1H), 1.64 (m, 1H), 1.83 (m, 3H), 2.16−2.18 (m,

1H), 2.75 (d, J = 7.0 Hz, 2H), 3.08−3.10 (m,1H), 3.27 (m, 1H), 5.35

(t, J = 8.1 Hz, 1H), 6.61 (s, 1H), 7.22 (m, 1H), 7.23 (m, 4H), 7.35−

7.36 (t, J = 7.2 Hz, 1H), 7.41−7.44 (t, J = 7.7 Hz, 2H), 7.69−7.71 (d, J

= 7.2 Hz, 2H). m/z 403 [M + H]⁺.

(S)-Benzyl (1-(5-Oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazol-

3-yl)-2-phenylethyl)carbamate (29). A solution of (S)-benzyl (1-

cyano-2-phenylethyl)carbamate (28) (140 mg, 0.5 mmol) and sodium

methoxide (13 mg, 0.24 mmol) in CH₃OH (4 mL) was stirred at 30

°C for 3 h. Acetic acid (14 mg, 0.24 mmol) was added followed by

phenylhydrazine (108 mg, 1 mmol). The reaction mixture was stirred

at rt for 18 h. The mixture was cooled to 0 °C, and solids were

removed by ﬁltration. The product was isolated by ﬂash chromato-

graph to give (S)-benzyl (1-imino-3-phenyl-1-(2-phenylhydrazinyl)-

propan-2-yl)carbamate (I-13) (55 mg, 28%). m/z 389 [M + H]⁺. A

solution of I-13 (55 mg, 0.14 mmol) and 1,1′-carbonyldiimidazole (68

mg, 0.42 mmol) in THF (6 mL) was heated to reﬂux for 48 h. The

reaction was cooled to rt, diluted with CH₂Cl₂, washed with water and

brine, dried over Na₂SO_4,ﬁltered, and concentrated in vacuo to give

29 (38 mg, 66%). 29 was carried onto the next step without

puriﬁcation. m/z 413 [M − H]⁻.

trans-4-(Aminomethyl)-N-((S)-1-(5-oxo-1-phenyl-4,5-dihy-

dro-1H-1,2,4-triazol-3-yl)-2-phenylethyl)cyclohexane-

carboxamide, Triﬂuoroacetic Acid Salt (30). Compound 29 (38

mg, 0.09 mmol) was converted to 30 (8 mg, 17%) by using the

trans-4-(Aminomethyl)-N-((S)-2-phenyl-1-(3-phenyl-1H-

1,2,4-triazol-5-yl)ethyl)-cyclohexanecarboxamide, Bis-tri-

ﬂuoroacetic Acid Salt (22). Compound 21 (154 mg, 0.42 mmol)

was converted to compound 22 (75 mg, 28%) using the methods

1

procedures described for the conversion of compound 26 to 27. H

NMR (500 MHz, CD₃OD) δ 1.02−1.06 (m, 2H), 1.30−1.33 (m, 1H),

1.43−1.46 (m, 1H), 1.56 (m, 1H), 1.67 (m, 1H), 1.81- 1.86 (m, 3H),

1

described for the conversion of compound 9a to compound 11a. H

P

dx.doi.org/10.1021/jm5010607 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2.16−2.18 (m, 1H), 2.75 (d, J = 7.2 Hz, 2H), 3.08−3.10 (m, 1H), 3.31

(m, 1H), 5.15−5.17 (m, 1H), 7.19−7.21 (m, 2H), 7.27−7.28 (m, 4H),

7.40−7.43 (t, J = 8.0 Hz, 2H), 7.85−7.87 (d, J = 7.7 Hz, 2H). m/z

420.1 [M + H]⁺.

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̈

1

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AUTHOR INFORMATION

■

Corresponding Author

*Phone: +1 609 818 4958. Fax: +1 609 818 3560. E-mail: jon.

hangeland@bms.com.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Use of the IMCA-CAT beamline 17-ID at the Advanced

Photon Source was supported by the companies of the

Industrial Macromolecular Crystallography Association through

a contract with the Illinois Institute of Technology. Use of the

Advanced Photon Source was supported by the U.S. Depart-

ment of Energy, Oﬃce of Science, Oﬃce of Basic Energy

Sciences, under Contract DE-AC02-06CH11357. We thank Dr.

Steven Sheriﬀ for ﬁnal reﬁnement of the X-ray crystal structures

for the FXIa/11h and FXIa/16b complexes and for depositing

the coordinates and X-ray data in the Protein Data Bank, Dr.

William R. Ewing and Dr. R. Michael Lawrence for assistance

editing the manuscript, and Jergen Bauman, Dr. Eileen Bird, Dr.

Baomin Xin, and Gang Luo for obtaining the rabbit

pharmacokinetic data.

ABBREVIATIONS USED

■

aPTT, activated partial thromboplastin time; AV, arterovenous;

ECAT, electrically mediated carotid arterial thrombosis; FVIIa,

activated coagulation factor VII; FVIIIa, activated coagulation

factor VIII; FIXa, activated coagulation factor IX; FXa, activated

coagulation factor X; FXIa, activated coagulation factor XI;

FXIIa, activated coagulation factor XII; IPTG, isopropyl β-^D-1-

thiogalactopyranoside; PK, plasma kallikrein; PT, prothrombin

time; TAFI, thrombin-activatable ﬁbrinolysis inhibitor; TF,

tissue factor; TT, thrombin time; VT, venous thombosis

Q

dx.doi.org/10.1021/jm5010607 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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R

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