Tetrahydroquinoline derivatives as potent and selective factor XIa inhibitors

Article abstract of DOI:10.1021/jm401670x

Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors. Compound 1 was ide

Full text of DOI:10.1021/jm401670x

Article
pubs.acs.org/jmc
Tetrahydroquinoline Derivatives as Potent and Selective Factor XIa
Inhibitors
Mimi L. Quan,* Pancras C. Wong, Cailan Wang, Francis Woerner, Joanne M. Smallheer,
Frank A. Barbera, Jeffrey M. Bozarth, Randi L. Brown, Mark R. Harpel, Joseph M. Luettgen, Paul E. Morin,
Tara Peterson, Vidhyashankar Ramamurthy, Alan R. Rendina, Karen A. Rossi, Carol A. Watson,
Anzhi Wei, Ge Zhang, Dietmar Seiffert, and Ruth R. Wexler
Discovery Chemistry and Cardiovascular Biology, Research and Development, Bristol-Myers Squibb Company, 311
Pennington-Rocky Hill Road, Pennington, New Jersey 08543, United States
S
* Supporting Information
ABSTRACT: Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the
potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we
describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors.
Compound 1 was identified as a potent and selective tool compound for proof of
concept studies. It exhibited excellent antithrombotic efficacy in rabbit thrombosis
models and did not prolong bleeding times. This demonstrates proof of concept for the
FXIa mechanism in animal models with a reversible, small molecule inhibitor.
bleeding. One hypothesis is that targeting proteases upstream
from the common pathway provides a reduction in thrombin
sufficient to impede occlusive thrombosis, yet allows enough
thrombin generation to support hemostasis.7 Since FXIa is
located upstream in the intrinsic pathway,8 we envision that by
inhibiting FXIa, we can disrupt the intrinsic coagulation
pathway without affecting either the extrinsic or the common
pathways.
Genetic evidence indicates that FXI is not required for
normal hemostasis in humans.9 Mice that are homozygous for a
targeted disruption of the FXI locus undergo normal
development and have a normal life span, and no evidence
for spontaneous or provoked bleeding has been noted.10
Furthermore, FXI deficient mice are protected from thrombosis
in a number of experimental models.10b,11 In contrast, targeted
disruption of other coagulation proteases in mice, except factor
XII (FXII), is associated with perinatal death due to bleeding or
bleeding disorders similar to the human phenotype.12 Many
recent studies have indicated that elevated FXI levels are
associated with venous thrombosis, myocardial infarction,
increased odds ratio for cerebrovascular events, and coronary
artery disease.13 Reduced incidence of ischemic stroke in
subjects with severe FXI deficiency has also been observed.14
Together, this evidence implies that FXIa may be an ideal target
for pharmaceutical intervention for the treatment and
prevention of thromboembolic diseases with minimal therapy-
associated increase in bleeding risk.7
INTRODUCTION
■
The process of unwanted or uncontrolled thrombosis is
causative for many life-threatening conditions, with arterial
thrombosis being the leading cause of morbidity and mortality.1
The most commonly used oral anticoagulant warfarin2 has a
narrow therapeutic window. Warfarin exposure is affected by
diet and cytochrome P450 enzyme polymorphisms leading to
multiple drug−drug interactions. Coagulation monitoring and
dose titration are required to maintain warfarin therapy in a
proper therapeutic range. Tremendous progress has been made
in developing new antithrombotic agents.3−5 The recently
approved novel oral anticoagulants dabigatran,3f rivaroxaban,4
apixaban,5 and edoxaban3e have addressed many of the issues
associated with warfarin. With these new anticoagulants, we
have entered a new era in the treatment and prevention of
unwanted thrombosis.
Blood coagulation is the coordinated activation of plasma
proteases, their cofactors, and platelets. The end product is the
protease thrombin, which cleaves fibrinogen to generate a fibrin
clot. The cascade has been classically divided into the intrinsic,
extrinsic, and common pathways.6 In the extrinsic pathway,
vessel injury initiates the process by converting factor VII
(FVII) to factor VIIa (FVIIa). Tissue factor-FVIIa (TF-FVIIa)
complex catalyzes the formation of factor Xa (FXa), which in
turn cleaves prothrombin to generate thrombin. In the intrinsic
pathway, factor XIIa (FXIIa), formed by contact activation,
catalyzes the formation of FXIa, which leads to the sequential
activation of factor IX (FIX) and factor X (FX). Thrombin can
also activate factor XI (FXI) to FXIa by feedback activation.6b−d
The key to a successful anticoagulant is achieving an
appropriate therapeutic balance between anticoagulation and
Received: October 28, 2013
© XXXX American Chemical Society
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Figure 1. Small molecule FXIa inhibitors.
a
Small molecule inhibitors of FXIa have been reported
(Figure 1). Racemic boronate 2 is a weak inhibitor of FXIa
(IC50 = 1400 nM) with 8- and 30-fold selectivity over thrombin
and FXa, respectively.15a Clavatadine A (3), a marine natural
product, was found to have a XIa IC50 of 1300 nM.15b A series
of potent, irreversible ketoarginine-based peptidomimetics has
also been reported.15c,d Compound 4a has a FXIa IC50 of 6 nM
and aPTT EC2x of 2.4 μM and shows good selectivity over
FVIIa, FXa, and thrombin. Pyridyl analogue 4b (FXIa IC50 = 12
nM) was evaluated in a rat mesenteric bleeding model and at 4-
fold the efficacious dose (1 mg/kg, continuous iv infusion) did
not alter bleeding time.15d Modifications to reduce the peptidic
character and molecular weight of these inhibitors led to 5 with
reduced affinity (FXIa IC50 = 116 nM).
We have previously reported compound 6 as a potent
irreversible FXIa inhibitor (Figure 1) that shows efficacy in rat
and rabbit thrombosis models with minimal bleeding time
prolongation.16 Herein we report the discovery of a series of
tetrahydroquinoline (THQ) derivatives as reversible FXIa
inhibitors and the identification of compound 1 as a tool
compound for proof of concept studies.17
Table 1. Tetrahydroquinoline SAR
Ki (nM)
compd
R1
R2
FXIa
FVIIa
FXa
7
CO2H
CO2H
CO2H
H
CONH-i-Bu
H
25
95
1100
1940
970
8
110
8.5
2200
290
NT
9
CO2H
CO2H
10
2400
NT
a
All compounds shown are racemic.
One major issue with the 1,2-phenylene THQ compounds
was atropisomerism; the rotation of the biaryl moiety is
restricted by its two ortho substituents. This complicated the
synthesis and purification of these compounds. Therefore, we
decided to explore the 1,3-phenylene analogues in order to
eliminate the atropisomers. The 1,3-phenylene compounds in
general have equal or better FXIa inhibitory potency compared
with the 1,2-phenylene compounds. However, this series of
compounds is less selective relative to FXa and FVIIa (Table
2). Diacid analogue 12 has a FXIa Ki of 3.9 nM but only 20-
and 30-fold selectivity over FVIIa and FXa, respectively.
A problem encountered with the 1,3-phenylene series was
instability due to aromatization of the THQ ring. The
aromatized compounds were 50- to 80-fold less active
compared with the corresponding THQs. One of our strategies
to solve this stability issue was to introduce substitution at the
C-4 position on the THQ ring. This approach successfully
blocked aromatization; however, the newly introduced alkyl
group reduced FXIa affinity. FXIa affinity decreased as the size
of the alkyl group increased, 15-fold for methyl (13), 200-fold
RESULTS AND DISCUSSION
■
Compound 7 (Table 1) was identified through screening of our
compound collection of serine protease inhibitors. It was
originally prepared as part of our FVIIa program18 but found to
have a FXIa Ki of 25 nM with about 4-fold selectivity over
FVIIa and 40-fold selectivity over FXa. Removal of the amide at
the R2 position reduced FXIa affinity by 4-fold but increased
selectivity versus FVIIa (8). Truncating the amide to a
carboxylic acid group resulted in 9, which showed a 3-fold
improvement in FXIa affinity. The carboxylic acid group at R1
was shown to be more crucial for FXIa activity than the
carboxylic group at R2, since removing the R1 carboxylic group
decreased FXIa activity by >280-fold (10), whereas only a 13-
fold loss was observed when the R2 carboxylic acid group was
removed (8). Single-digit nanomolar FXIa affinity was achieved
with compound 9 which has 30-fold and 100-fold selectivity
over FVIIa and FXa, respectively.
B
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a
Table 2. Comparison of 1,2- and 1,3-Phenylene
Ki (nM)
compd
R2
FXIa
FVIIa
FXa
7 (1,2)
11 (1,3)
9 (1,2)
12 (1,3)
CONH-i-Bu
CONH-i-Bu
CO2H
25
95
1100
24
23
25
8.5
3.9
290
89
970
130
CO2H
a
All compounds shown are racemic.
a
for ethyl (14), and 500-fold for n-propyl (15) as shown in
Table 4. Biaryl Acid SAR
Table 3.
a
Table 3. SAR of THQ Stabilization by C4-Substitution
Ki (nM)
compd
R2
FXIa
FVIIa
FXa
16
17
18
19
13
20
21
22
23
24
25
26
27
CONH2
NHCONHMe
NHSO2Me
CN
1.5
15
280
3000
compd
R
FXIa Ki (nM)
451
1140
>15000
2060
9300
3980
606
12
13
14
15
H
3.9
37
237
Me
Et
61
48
867
830
1960
CO2H
61
2600
275
n-Pr
NH2
64
a
CO2Me
79
442
All compounds shown are racemic.
H
110
110
154
207
466
540
3060
705
6700
5810
4100
1310
>15000
6400
NHCOMe
CONHMe
CONH-i-Pr
CH2NMe2
CONH-t-Bu
Extensive SAR changes were explored at the R2 position of
1960
1770
610
the C4-methyl substituted THQ (Table 4). Methylurea (17)
and methylsulfonamide (18) analogues resulted in compounds
with slightly improved FXIa inhibitory activity. Cyano (19),
amino (20), and methyl ester (21) substitutions produced
compounds with potency similar to that of the carboxylic acid
(13). Primary amide (16) restored single-digit nanomolar
affinity for FXIa. Alkylation of the amide nitrogen resulted in
>100-fold decrease in FXIa activity (24, 25, 27).
From the SAR studies described above, compound 16 was
the most potent compound identified with a FXIa Ki of 1.5 nM.
The trans isomer (28, Figure 2) of 16 was a minor product
isolated from the reaction to form the THQ scaffold and was
found to have 17-fold less inhibitory activity for FXIa when
compared to 16. The two cis enantiomers of 16 were separated
by chiral HPLC. The dextrorotary enantiomer (+)-16 was
found to be >800-fold more potent than the levorotary
enantiomer (−)-16 (Figure 2). The dextrorotary enantiomer
(+)-16 has a FXIa Ki of 0.39 nM with >1000-fold selectivity
over FXa and 200-fold over FVIIa.
1290
a
All compounds shown are racemic.
An X-ray crystal structure of (+)-16 bound in the FXIa active
site was obtained to understand the binding mode of this series
of compounds (2.3 Å, Figure 3). The benzamidine group was
bound tightly in the S1 pocket by a typical salt bridge to
Asp189 (2.9 and 3.0 Å) and also can form an H-bond to the
backbone carbonyl of Gly218 (2.9 Å). The NH of the THQ
forms an H-bond with the side chain OH of Ser195 (3.3 Å). A
stacking interaction was observed between His57 and the inner
phenyl ring. The carboxylic group forms H-bonds with His57
(2.8 Å) and the oxyanion hole which comprises Gly193 and
Ser195. The amide nitrogen formed two H-bonds with the
backbone carbonyls of Leu41 (3.0 Å) and His40 (3.1 Å). The
amide carbonyl can interact either with Arg39 directly (3.1 Å)
C
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Figure 2. Comparison of stereoisomers.
Figure 3. X-ray crystal structure of compound 16 complexed to FXIa. The initial 2Fo − Fc electron density contoured at 1 rmsd is shown with the
final model. The protein is shown with green carbon atoms, and 16 is shown with purple carbon atoms. Nitrogen atoms are shown in blue, oxygen
atoms in red, and sulfur atoms in yellow. The red spheres represent crystallographic water molecules. Hydrogen bonds are represented by black
dashes. Figure was prepared with PyMol (Schrodinger).
̈
or through a water molecule (2.7 and 2.8 Å). These interactions
obtained on introduction of the primary amide resulted in
enhanced FXIa affinity. This is a unique interaction that has not
been previously observed in FXa or FVIIa X-ray structures.
To further improve selectivity over FVIIa, we decided to
probe the interactions in the S2 pocket, since FXIa has a larger
S2 pocket than FVIIa. We first obtained an X-ray crystal
structure of compound 29 (2.2 Å, FXIa Ki = 7.6 nM and FVIIa
Ki = 41 nM) in the FVIIa active site to confirm the binding
mode in FVIIa. The amino group was found to be pointing
toward the S2 pocket (Figure 4). As expected, the rest of the
molecule interacts similarly as that observed in the FXIa
structure of (+)-16.
As shown in Table 5, a series of amides were prepared using
the amino group as a linker for substitutions. Acylation of the
amino group maintained and in some cases further improved
the FXIa potency. In general, selectivity relative to FVIIa
increased as the size of the alkyl group increased while the
selectivity over FXa decreased. Compound 35 showed the best
profile in terms of potency and selectivity in this series. The two
enantiomers of 35 were separated by chiral HPLC. The
dextrorotatory enantiomer 1 has a FXIa Ki of 0.20 nM, and the
levorotary enantiomer 36 was much less potent with a XIa Ki of
230 nM. Compound 1 has >1000-fold selectivity for FXIa
versus most of the enzymes tested except for plasma kallikrein
(23-fold) and activated protein C (365-fold). Compared with
(+)-16, the selectivity for FVIIa was improved from 220-fold to
>1000-fold (Table 6). An X-ray crystal structure of the 1−XIa
complex was obtained (2.8 Å, Figure 5). Its binding mode is
very similar to that of (+)-16 and shows the same interactions
in the P1, P2′, and oxyanion hole regions. The only difference
is that the carbonyl of the isobutylamide in 1 forms an
additional H-bond with the hydroxyl of Tyr58b (2.8 Å),
extending the isobutyl moiety deeper into the P2 pocket.
Compound 1 exhibits excellent in vitro anticoagulant activity
in the activated partial thromboplasmin time (aPTT) assay with
an EC2x of 2.2 μM. Consistent with inhibition of the intrinsic
coagulation system, no activity in the prothrombin time (PT)
assay was observed (IC2x > 20 μM). Compound 1 is
competitive with small molecule FXIa substrates and shows
reversible inhibition kinetics. It shows mixed type inhibition
using FIX as a substrate, where FIX activation was monitored
by using a novel sensitive assay.19 Similar potency and
selectivity were observed with human and rabbit enzymes
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Table 6. Human Enzyme Selectivity Profile for Compound 1
human enzyme Ki (nM) for 1
FXIa
0.20
4.6
plasma kallikrein
activated protein C
FXa
73
340
FVIIa
630
trypsin
740
plasmin
910
tPA
1700
2100
12600
14000
>20000
urokinase
thrombin
chymotrypsin
FIXa
Figure 4. X-ray crystal structure of compound 29 complexed to FVIIa.
The initial 2Fo − Fc electron density contoured at 1 rmsd is shown
with the final model. The protein is shown with green carbon atoms,
and 29 is shown with orange carbon atoms. Nitrogen atoms are shown
in blue, oxygen atoms in red, and sulfur atoms in yellow. Hydrogen
bonds are represented by black dashes. Figure was prepared with
PyMol (Schrodinger).
̈
Table 5. Probing the S2 Pocket To Improve FVIIa
Selectivity
Figure 5. X-ray crystal structure of compound 1 complexed to FXIa.
The initial 2Fo − Fc electron density contoured at 1 rmsd is shown
with the final model. The protein is shown with green carbon, and 1 is
shown with pink carbon atoms. Nitrogen atoms are shown in blue,
oxygen atoms in red, and sulfur atoms in yellow. Hydrogen bonds are
represented by black dashes. Figure was prepared with PyMol
(Schrodinger).
̈
Table 7. Comparison of Human and Rabbit in Vitro Potency
of Compound 1
enzyme
FXIa
human Ki (nM)
rabbit Ki (nM)
0.20
12600
343
0.42
FXIa
FVIIa
(nM)
FXa
aPTT
thrombin
FXa
7900
3200
compd
R
(nM)
(nM)
(μM)
( )-16
( )-30
( )-31
( )-32
( )-33
( )-34
( )-35
(−)-36
(+)-1
H
1.5
280
3000
2250
1810
1480
641
8.6
a
FVIIa
629
200
NH2
0.64
1.0
56
1.2
plasma kallikrein
aPTT EC2x
4.6
4.6
NHCOMe
NHCOEt
230
2.1
2200
2400
0.69
0.30
0.72
0.59
230
0.20
450
3.4
a
Experiment at 37 °C.
NHCO-n-Pr
NHCO-c-Pr
NHCO-i-Bu
NHCO-i-Bu
NHCO-i-Bu
520
4.1
474
880
4.5
1010
>10900
630
784
6.1
loading dose plus continuous infusion. A dose-dependent
antithrombotic effect was observed with an ID50 of 0.95 mg
kg−1 h−1 (Figure 6). Bleeding time was not increased compared
to vehicle treated animals.21 The ex vivo aPTT was prolonged
(the expected pharmacodynamic effect of a FXIa inhibitor) but
not the PT (indicative of selectivity over FVIIa, FXa, and FIIa).
5670
340
not tested
2.2
(Table 7). Compound 1 was studied in the rabbit AV shunt
thrombosis model20 by iv administration at five doses given by
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(racemate) as the major product.18a Hydrolysis or hydro-
genation of I-6 produced the final compounds (7−10).
The meta-substituted compounds described in Tables 2−5
were prepared in a similar manner using meta-substituted
boronic acids as the starting material. Compounds where R2 is
CONH2 were prepared using the procedures described in
Scheme 2. 2-Bromo-5-iodobenzoic acid II-1 was reacted with
benzyl bromide in KHCO3/DMF to give the benzyl ester II-2.
The iodo was converted to the cyano group (II-3) by reaction
with Zn(CN)2 in Pd(PPh3)4/DMF at 80 °C. Oxidation with
hydrogen peroxide in K2CO3/DMSO afforded the amide II-4.
Suzuki coupling of II-4 with boronic acid II-5 or 3-
formylphenylboronic acid II-6 in K3PO4/ Pd(PPh3)4/DMF at
100 °C produced biaryl compound II-7 or II-8, respectively.
Reduction of the carboxylic group in II-7 followed by oxidation
with the Dess−Martin reagent gave aldehyde II-9. Reaction of
aldehyde II-9 with aniline II-10 and the appropriate alkene (II-
11a) in In(OTf)3/CH3CN afforded tetrahydroquinoline II-12.
Compound II-12 was reduced to the corresponding aniline (II-
13). II-13 was coupled with an appropriate acyl chloride, then
deprotected under hydrogenation conditions to afford com-
pounds 31−35. For compound 1, the two enantiomers were
separated by chiral HPLC after acylation. The dextrorotatory
enantiomer was deprotected under hydrogenation conditions
to afford 1. Compound 16 was prepared using intermediate II-
8 via the similar procedures described.
Figure 6. Antithrombotic activity of compound 1 in the rabbit A-V
shunt model. Compound 1 was given as a single bolus followed by
continuous iv infusion at five doses. Thrombi formed were removed
from the shunt and weighed. The % inhibition values are relative to
control animals that did not receive 1.
A fuller description of the pharmacology will be published
separately.21
CHEMISTRY
■
The syntheses of ortho-substituted biaryl compounds are
shown in Scheme 1. Boronic acid I-1 was coupled either with
bromide I-2a−c or triflate I-2d22 to give biarylaldehyde I-3.
Reaction of a mixture of aldehyde I-3, styrene I-4, and aniline I-
5 with indium triflate in acetonitrile afforded cis-THQ I-6
Meta-substituted compounds 17, 18, 20, and 23 were
prepared as described in Scheme 3. Bromide III-1 was coupled
with 3-formylphenylboronic acid II-6 via Suzuki coupling to
a
Scheme 1. Synthesis of the Ortho-Substituted Biaryl THQ Compounds
a
Reagents: (a) Pd(PPh3)4, PhCH3/EtOH, reflux, 65−90%; (b) In(OTf)3, CH3CN, 70 °C; (c) LiOH, THF/H2O, reflux; (d) Pd/C, H2, 20−40% for
the last three steps combined.
F
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a
Scheme 2. Synthesis of the Meta-Substituted Biaryl THQ Compounds
a
Reagents: (a) KHCO3, PhCH2Br, DMF, rt, 100%; (b)Zn (CN)2, Pd(PPh3)4, DMF, 90 °C, 74%; (c) K2CO3, 30% H2O2, DMSO, rt, 75%; (d)
Pd(PPh3)4, K3PO4, DMF, 100 °C, 93%; (e) (1) isobutyl chloroformate, Et3N, THF, (2) NaBH4/H2O, 36%; (f) Dess−Martin, EtOAc, 72%; (g)
In(OTf)3, Et3N, CH2Cl2, 70 °C, 61%; (h) Fe, EtOH/HOAc/H2O, 80 °C, 91%; (i) RCOCl, CH3CN, 64−90%; (j) Raney Ni/H2; (k) 10% Pd/C/H2,
13−90% for steps j and k combined.
give biaryl III-2. III-2 was then reacted with alkene II-11a and
aniline II-10 using the conditions described above to afford
THQ III-4. The nitro group of III-4 was reduced to aniline III-
5, which was reacted with acetyl chloride, isobutyl carbamate,
or methanesulfonyl chloride to give the desired products after
deprotection. Compound 26 was prepared as described in
Scheme 4. Benzoic acid IV-1 was protected with benzyl
bromide. Reductive amination with dimethylamine and sodium
cyanoborohydride in MeOH gave the dimethylbenzylamine
(IV-2). The benzyl ester was exchanged to the methyl ester
during this step. The phenol was then reacted with pyridine and
triflic anhydride to give IV-3. Suzuki coupling of IV-3 with 3-
formylphenylboronic acid II-2 afforded biaryl IV-4, which was
converted to compound 26 following the procedures described
above.
CONCLUSION
■
A series of potent, selective, and reversible FXIa inhibitors
incorporating a tetrahydroquinoline scaffold was explored, and
a suitable tool compound (1) was identified for in vivo
evaluation. Compound 1 has an outstanding in vitro profile
with an FXIa Ki of 0.20 nM and >1000-fold selectivity over
related serine proteases except plasma kallikrein and activated
protein C. It exhibited excellent antithrombotic efficacy in
rabbit thrombosis models and did not prolong bleeding times.21
To our knowledge, this is the first report demonstrating proof
of concept for the FXIa mechanism in animal models with a
reversible, small molecule inhibitor.
EXPERIMENTAL SECTION
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
■
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a
Scheme 3. Synthesis of the Meta-Substituted Biaryl THQ Compounds
a
Reagents: (a) Pd(PPh3)4, K3PO4, PhCH3/EtOH, reflux, 85%; (b) In(OTf)3, Et3N, CH2Cl2, 70 °C, 61%; (c) NaSH, THF/dioxane, 92%; (d)
RNCO or RCOCl or MeSO2Cl, Et3N/CH2Cl2, 40−90%; (e) LiOH, THF/H2O, reflux, 90−100%; (f) Raney Ni/H2, 8−26%.
a
Scheme 4. Synthesis of the Meta-Substituted Biaryl THQ Compounds
a
Reagents: (a) NaHCO3/BnBr/DMF; (b) NHMe2/NaCNBH3/MeOH; (c) pyridine/triflate anhydride/CH2Cl2; 22% for steps a, b, and c
combined; (d) Pd(PPh3)4, toluene/EtOH, reflux, 86%; (e) In(OTf)3, Et3N, CH2Cl2, 70 °C; (f) LiOH, THF/H2O, reflux, 77% for steps e and f
combined; (g)10% Pd/C/H2, 38%.
purification unless otherwise indicated. NMR spectra (1H, 13C, 19F)
were obtained on VXR or Unity 300 MHz instruments (Varian
Instruments, Palo Alto, CA) with chemical shifts in ppm downfield
from TMS as an internal reference standard. 1H assignment
abbreviations are the following: singlet (s), doublet (d), triplet (t),
quartet (q),quintet (quin), broad singlet (bs), doublet of doublets
(dd), doublet of triplets (dt), and multiplet (m). Elemental analyses
were performed by Quantitative Technologies, Inc., Whitehouse, NJ
08888, and were within 0.4% of the theoretical values. Mass spectra
were measured with a HP 5988A mass spectrometer with particle
beam interface using NH3 for chemical ionization or a Finnigan MAT
8230 mass spectrometer with NH3-DCI or VG TRIO 2000 for ESI.
High-resolution mass spectra were measured on a VG 70-VSE
instrument with NH3 ionization. Flash chromatography was performed
using EM Science silica gel 60 (230−400 mesh). Preparative thin layer
chromatography was done on EM Science 60 plates F254 (2 mm, 20
cm × 20 cm). HPLC purification was performed on a Jasco 900 series
instrument or a Rainin Dynamax SD200 using a C18 reverse phase
column with acetonitrile/H2O (containing 0.05% TFA) as a mobile
phase. All compounds were found to be >95% pure by HPLC analysis
unless otherwise noted. Melting points were determined on a Thomas-
Hoover melting point apparatus and were uncorrected.
X-ray Crystal Structure Data Collection and Structure
Refinement (See Supporting Information Table 1). Data for
factor VIIa and factor XIa crystals in complex with inhibitors were
collected at the Advanced Photon Source (APS) beamline 17-BM or
in the laboratory. Raw data were processed with the program
HKL2000.23 The atomic coordinates of human factor VIIa (PDB code
1DAN) or factor XIa were used as a search model. Original refinement
was carried out with CNX (Accelrys), and inhibitor restraint
H
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dictionaries were built with QUANTA (Accelrys), which was also used
for modeling. Later the structures were rerefined using BUSTER/
TNT24 (GlobalPhasing, Ltd.), MakeTNT or GRADE (GlobalPhasing,
Ltd.) for inhibitor restraint dictionaries, and COOT25 for modeling.
Coordinates for the enzyme/inhibitor structures, data collection and
refinement statistics, and the X-ray diffraction data have been
deposited with the Protein Data Bank (PDB). The PDB deposition
number for compounds 1, (+)-16, and 29 are 4NA7, 4NA8, and
4NA9, respectively.
Enzyme Affinity Assays. The affinity of compounds for various
enzymes was determined using purified enzymes and synthetic
substrates from commercial sources. Assays were conducted at room
temperature (∼25 °C) or at 37 °C in temperature controllable, 96-well
microtiter plate spectrophotometers (Molecular Devices, Spectramax)
with simultaneous measurement of enzyme activities in control and
inhibitor-containing solutions. Assays were initiated by adding enzyme
to buffered solutions containing synthetic 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. The rate of
absorbance change is proportional to enzyme activity. A decrease in
the rate of absorbance in the presence of inhibitor is indicative of
enzyme inhibition. Assays were conducted under conditions of excess
substrate and inhibitor over enzyme. The Michaelis−Menten constant,
Km, for substrate hydrolysis was determined by nonlinear fitting of the
reaction velocity data from multiple substrate concentrations to the
Michaelis−Menten equation,
Purified thrombin was obtained after prothrombin activation with
rabbit prothrombinase complex (rabbit FXa/human FVa/phospholi-
pids) followed by affinity chromatography. The resulting proteins were
>95% pure as judged by sodium dodecyl sulfate−polyacrylamide gel
electrophoresis. Aliquots of each enzyme were stored at −70 °C and
were rapidly thawed in a 37 °C water bath just prior to use. Rabbit
FVIIa activity was determined via the use of a coupled enzyme
assay.18c Activated rabbit plasma kallikrein was generated following a
procedure described for human plasma kallikrein.27 The factor IX
activation methods are described in ref 19.
In Vitro Coagulation Assays (aPTT). Standard clotting assays
were performed in a temperature-controlled automated coagulation
device (Sysmex CA-6000, Dade-Behring). Blood was obtained from
1
healthy volunteers by venipuncture and anticoagulated with
/
10
volume of 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 on plasma containing different concentrations of inhibitor.
Determinations at each plasma concentration were done in duplicate.
The clotting time at each concentration was compared with the
control clotting time for each pooled plasma. The activated partial
thromboplastin time (aPTT) was performed using Alexin (Sigma)
according to the reagent instructions. Plasma (50 μL) was warmed to
37 °C for 1 min before adding aPTT reagent (50 μL). At 3 min later
calcium chloride (50 μL) was added.
Benzyl 2′-Formyl-4-(isobutylcarbamoyl)biphenyl-2-carbox-
ylate (I-3d). Benzyl 5-(isobutylcarbamoyl)-2-(trifluoromethyl-
sulfonyloxy)benzoate22 (2.1 g, 4.6 mmol), 2-formylphenylboronic
acid (1.0 g, 6.8 mmol), K3PO4 (1.5 g, 6.8 mmol), and Pd[PPh3]4 (0.53
g, 10%) were added together in 30 mL of DMF. The mixture was
degassed and heated at 100 °C under N2 for 2 h. The reaction mixture
was cooled and poured into water. It was extracted with EtOAc. The
combined organic solution was washed with brine and dried over
MgSO4. It was concentrated and purified by chromatography (silica
gel, 10% EtOAc in CH2Cl2) to give 1.75 g of the biarylaldehyde I-3d
Vmax
Km + S
S
v =
where v is the observed velocity of the reaction, Vmax is the maximal
velocity, S is the concentration of substrate, and Km is the Michaelis−
Menten constant for the substrate.
Values of Ki were determined by allowing the enzyme to react with
the substrate in the presence of the inhibitor. Reactions were allowed
to go for periods of 10−120 min, and the velocities were measured
when a linear steady state rate was observed. The following
relationships were used to calculate Ki values using nonlinear fitting
routines
1
(91%). MS: 416.4 (M + 1)+. H NMR (CDCl3) δ 9.72 (s, 1H), 8.38
(s, 1H), 8.00 (dd, 1H), 7.84 (dd, 1H), 7.53−7.24 (m, 6H), 7.14 (d,
1H), 7.06 (m, 2H), 6.29 (m, 1H), 5.01 (s, 2H), 3.32 (m, 2H), 1.94 (m,
1H), 0.98 (d, 6 H).
v
B − A
s
= A +
n
IC50
v
Dimethyl 2′-Formylbiphenyl-2,4-dicarboxylate (I-3b). Di-
methyl 4-bromoisophthalate (0.53 g, 2.0 mmol), 2-formylphenyl-
boronic acid (0.66 g, 4.4 mmol), K3PO4 (0.59 g, 2.8 mmol), and
Pd[PPh3]4 (0.23 g, 10 mol %) were reacted using the same procedure
described for I-3d to give 0.35 g of the biarylaldehyde I-3b (65%). 1H
NMR (CDCl3) δ 10.01 (s, 1H), 8.59 (d, 1H), 8.24 (d, 1H), 7.93 (d,
1H), 7.84 (s, 1H), 7.60 (m, 3H), 7.40 (d, 1H), 3.99 (s, 3H), 3.71 (s,
3H).
o
1 +
(
)
I
and
Ki =
IC50
S
1 +
Km
for a competitive inhibitor, where vo is the velocity of the control in the
absence of inhibitor, vs is 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, IC50 is the
concentration of inhibitor that produces 50% inhibition, Ki is the
dissociation constant of the enzyme−inhibitor complex, S is the
concentration of substrate, and Km is the Michaelis−Menten constant
for the substrate.
Ethyl 2′-Formylbiphenyl-2-carboxylate (I-3c). This compound
was prepared by the same method described for I-3b using ethyl 2-
bromobenzoate and 2-formylphenylboronic acid. MS: 255.1 (M + 1)+.
Ethyl 2′-Formylbiphenyl-4-carboxylate (I-3a). This compound
was prepared by the same method described for I-3b using ethyl 4-
bromobenzoate and 2-formylphenylboronic acid. MS: 255.1 (M + 1)+.
2′-(6-Carbamimidoyl-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)-4-isobutylcarbamoylbiphenyl-2-carboxylic Acid (7). 4-
Aminobenzamidine mono-HCl salt (0.13 g, 0.77 mmol) was
suspended in acetonitrile (30 mL). Benzyl 2′-formyl-4-(isopentyl-
carbamoyl)biphenyl-2-carboxylate (I-3d) (0.32 g, 0.77 mmol) and
styrene (0.88 mL, 7.7 mmol) were added, followed by indium triflate
(0.45 g, 0.77 mmol). The mixture was heated at 70 °C under N2 for 12
h. The mixture was concentrated and dissolved in 30 mL of MeOH
and 5 mL of acetic acid. Catalytic amount of 10% Pd/C was added,
and the mixture was stirred under 1 atm of H2 for 3 h. The reaction
mixture was filtered through Celite, concentrated, and purified by
reverse phase HPLC (0.5% TFA in CH3CN/H2O) to give 100 mg of
the desired product TFA salt as a mixture of atropisomers (20%). MS:
Ki values were determined in this manner at a fixed substrate
concentration. The IC50 values were obtained over 4−11 inhibitor
concentrations assayed in duplicate with a standard error of <20%. For
several of the enzymes negligible inhibition was observed at the highest
inhibitor concentration tested. In these cases the value assigned as a
lower limit for Ki is the value that would be obtained with 50%
inhibition at the highest inhibitor concentration assuming competitive
inhibition.
By use of established protein purification procedures,26 FX and
prothrombin were isolated from citrated plasma obtained from healthy
New Zealand white rabbits. Purified FXa was obtained after activation
with Russell’s viper venom followed by affinity chromatography.
1
547.5 (M + 1)+. Structure was confirmed by variable temperature H
NMR (DMSO-d6, 60−140 °C).
I
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2′-(6-Carbamimidoyl-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)biphenyl-2,4-dicarboxylic Acid (9). This compound was
prepared using the same procedures described for compound 7. MS:
492.3 (M + 1)+.
2′-(6-Carbamimidoyl-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)biphenyl-2-carboxylic Acid (8). This compound was prepared
using the same procedures described for compound 7. MS: 448.5 (M
+ 1)+.
DMF. The reaction mixture was cooled at 0 °C. Potassium carbonate
(0.20 g, 1.5 mmol) was added, followed by dropwise addition of H2O2
(1.2 mL of 30%). The cooling bath was removed, and the mixture was
stirred at room temperature for 12 h. The reaction was quenched with
aqueous NaHSO3 and water. The precipitate formed was filtered and
dried to give 1.1 g of the desired amide (75%). MS: 334.2, 336.3 (M +
1)+.
Benzyl 4-Carbamoyl-3′-formylbiphenyl-2-carboxylate (II-8).
Benzyl 2-bromo-5-carbamoylbenzoate (II-4) (7.2 g, 22 mmol), 3-
formylphenylboronic acid (4.8 g, 32 mmol), K3PO4 (7.5 g, 36 mmol),
and Pd[PPh3]4 (2.4 g, 10%) were reacted using the same procedure
described for I-3d to give 7.2 g of the biarylaldehyde (93%). 1H NMR
(CDCl3) δ 9.93 (s, 1H), 8.35 (d, 1H), 8.05 (dd, 1H), 7.83 (dd, 1H),
7.75 (s, 1H), 7.55−7.43 (m, 4H), 7.28 (m, 3H), 7.08 (m, 2H), 3.10 (s,
2H).
2′-(6-Carbamimidoyl-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)biphenyl-4-carboxylic Acid (10). This compound was
prepared using the same procedures described for compound 7. MS:
1
448.4 (M + 1)+. H NMR (DMSO-d6) δ 8.62 (s, 2H), 8.18 (s, 2H),
8.04−8.02 (d, 2H), 7.74−7.72 (d, 1H), 7.54−7.22 (m, 11H), 6.84 (s,
1H), 6.73−6.70 (d, 1H), 4.61−4.57 (d, 1H), 4.35 (m, 1H), 4.05−3.99
(m, 1H), 2.16−2.01 (m, 2H).
3′-(6-Carbamimidoyl-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)-4-isobutylcarbamoylbiphenyl-2-carboxylic Acid (11). This
compound was prepared using benzyl 3′-formyl-4-(isopentyl-
carbamoyl)biphenyl-2-carboxylate (prepared by the same procedures
described for I-3d) following the same procedure described for
compound 7. MS: 547.5 (M + 1)+. Analytical HPLC purity, 91.4%. 1H
NMR (DMSO-d6) δ 8.70 (m, 3H), 8.38 (s, 2H), 8.22 (s, 1H), 8.04
(d,1H), 7.53−7.26 (m, 6H), 4.78 (m, 1H), 4.35 (m, 1H), 3.12 (t, 2H),
2.22 (m, 1H), 2.08 (m, 1H), 1.86 (m, 1H), 0.90 (d, 6H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-carbamoylbiphenyl-2-carboxylic Acid (16). 4-
Aminobenzamidine mono-HCl salt (0.11 g, 0.58 mmol) was
suspended in acetonitrile (30 mL). II-8 (0.21 g, 0.58 mmol) and α-
methylstyrene (0.34 mL, 2.9 mmol) were added, followed by indium
triflate (0.17 g, 0.29 mmol). Following the procedure described for
compound 7, the desired product was isolated as the TFA salt (80 mg,
22%). MS: 505.3 (M + 1)+. Analytical HPLC purity, 94.1%. 1H NMR
(DMSO-d6) δ 8.61 (s, 2H), 8.18−8.15 (m, 3H), 8.00 (d, 1H), 7.79−
7.50 (m, 5H), 7.25 (m, 5H), 7.01 (s, 1H), 6.78 (d, 1H), 4.66 (d, 1H),
1.80−2.10 (m, 2H), 1.74 (s, 3H).
Benzyl 4-Carbamoyl-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-
oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)-5′-
biphenyl-2-carboxylate (II-15). 2-(4-Aminophenyl)-5-methyloxa-
diazole (0.49 g, 2.8 mmol) was suspended in acetonitrile (20 mL).
Benzyl 4-carbamoyl-3′-formyl-5′-biphenyl-2-carboxylate (II-8) (1.0 g,
2.8 mmol) and α-methylstyrene (1.8 mL, 14 mmol) were added,
followed by indium triflate (0.78 g, 1.4 mmol). The mixture was
degassed and heated at 70 °C under N2 for 6 h. The mixture was
concentrated and purified by chromatography (silica gel, 30% EtOAc
in hexane and then 3% MeOH in CH2Cl2). MS: 635.5 (M + 1)+. The
product mixture was separated by chiral HPLC (OD chiracel 250,
100%ACN) to give three products. II-15a (226 mg, first peak, Rf =
5.00 min, [α]2D0 −85.5 (c 0.26, MeOH)). II-15b (229 mg, third peak,
Rf = 5.48 min, [α]2D0 +85.7 (c 0.27, MeOH)). II-15c (70 mg, second
peak, Rf = 5.13 min, racemate trans isomer).
3′-(6-Carbamimidoyl-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)biphenyl-2,4-dicarboxylic Acid (12). This compound was
prepared using dimethyl 3′-formylbiphenyl-2,4-dicarboxylate (pre-
pared by the same procedures described for I-3b) following the
same procedure described for compound 7. MS: 492.3 (M + 1)+.
1
Analytical HPLC purity, 99.9%. H NMR (CD3OD) δ 8.43 (s, 1H),
8.20 (d, 1H), 7.53−7.26 (m, 11H), 7.01 (s, 1H), 6.76 (d, 1H), 4.78 (d,
1H), 4.35 (m, 1H), 2.37−2.10 (m, 2H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2,4-dicarboxylic Acid (13). This com-
pound was prepared following the same procedure as described for 7.
MS: 506.3 (M + 1)+. Analytical HPLC purity, 99.7%. 1H NMR
(CD3OD) δ 8.39 (d, 1H), 8.15 (d, 1H), 7.48−7.22 (m, 9H), 7.18 (m,
1H), 7.06 (s, 1H), 6.76 (d, 1H), 4.75 (m, 1H), 2.20 (t, 1H), 1.98 (m,
1H), 1.81 (s, 3H).
3′-(6-Carbamimidoyl-4-ethyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2,4-dicarboxylic Acid (14). This com-
pound was prepared following the same procedure as described for 7.
MS: 520.3 (M + 1)+. Analytical HPLC purity, 95%. 1H NMR
(CD3OD) δ 8.41 (s, 1H), 8.18 (d, 1H), 7.50−7.25 (m, 10H), 7.18 (m,
1H), 7.06 (s, 1H), 6.80 (d, 1H), 4.75 (m, 1H), 2.30−2.17 (m, 4H),
1.03 (t, 3H).
(+)-3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahy-
droquinolin-2-yl)-4-carbamoylbiphenyl-2-carboxylic Acid
[(+)-16]. II-15b (229 mg, 0.31 mmol) was dissolved in 15 mL of
MeOH/Et3N (8:1). Catalytic amount of 10% Pd/C was added, and
the mixture was stirred under 1 atm H2 for 12 h. The reaction mixture
was filtered through Celite, concentrated, and purified by reverse phase
HPLC (0.5% TFA in CH3CN/H2O) to give 110 mg of the desired
product as a TFA salt (57%). MS: 505.4 (M + 1)+. Analytical HPLC
3′-(6-Carbamimidoyl-4-propylyl-4-phenyl-1,2,3,4-tetrahy-
droquinolin-2-yl)biphenyl-2,4-dicarboxylic Acid (15). This
compound was prepared following the same procedure as described
1
for 7. MS: 534.3 (M + 1)+. Analytical HPLC purity, 95%. H NMR
purity, 99.6%. [α]2D0 +79.5 (c 0.21, MeOH). H NMR (DMSO-d6) δ
1
(CD3OD) δ 8.50 (s, 1H), 8.17 (d, 1H), 7.48−7.22 (m, 10H), 7.18 (m,
1H), 7.08 (s, 1H), 6.81 (d, 1H), 4.77 (m, 1H), 2.21−2.08 (m, 4H),
1.14 (m, 2H), 1.10 (t, 3H).
8.62 (s, 2H), 8.15 (m, 3H), 8.00 (d, 1H), 7.50−7.30 (m, 5H), 7.25 (m,
3H), 7.15 (m, 1H), 7.01 (s, 1H), 6.68 (d, 1H), 4.66 (d, 1H), 2.05 (t,
1H), 1.90 (m, 1H), 1.72 (s, 3H).
Benzyl 2-Bromo-5-cyanobenzoate (II-3). 2-Bromo-5-iodoben-
zoic acid (6.5 g, 20 mmol) was dissolved in DMF (70 mL). Potassium
bicarbonate (2.2 g, 22 mmol) was added, followed by benzyl bromide
(2.8 mL, 22 mmol). The mixture was stirred at room temperature
under N2 for 12 h. The reaction mixture was poured into water and
extracted with EtOAc. The combined organic solution was washed
with brine and dried over MgSO4. It was concentrated and dried to
give 9.0 g (100%) of the benzyl ester. The benzyl ester (2.3 g, 7.7
mmol), Zn(CN)2 (1.3 g, 12 mmol), and Pd[PPh3]4 10 mol % were
added together with 25 mL of DMF. The mixture was degassed and
heated at 90 °C for 4 h. The cooled reaction mixture was diluted with
100 mL of NH4OH/H2O and extracted with EtOAc. The combined
organic solution was washed with brine and dried over MgSO4. It was
concentrated and purified by chromatography (silica gel, 5% EtOAc in
hexane) to give 1.8 g of the cyano derivative (74%). MS: 316.0, 317.9
(M + 1)+.
(−)-3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahy-
droquinolin-2-yl)-4-carbamoylbiphenyl-2-carboxylic Acid
[(−)-16]. This compound was prepared from II-15a following the
same procedure as described for (+)-16. MS: 505.4 (M + 1)+.
Analytical HPLC purity, 99.6%. [α]2D0 −80.5 (c 0.21, MeOH). 1H
NMR (DMSO-d6) δ 8.63 (s, 2H), 8.20 (m, 3H), 8.03 (d, 1H), 7.52−
7.32 (m, 5H), 7.30 (m, 3H), 7.20 (m, 1H), 7.05 (s, 1H), 6.82 (d, 1H),
4.72 (d, 1H), 2.08 (t, 1H), 1.92 (m, 1H), 1.77 (s, 3H).
(trans)-3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tet-
rahydroquinolin-2-yl)-4-carbamoylbiphenyl-2-carboxylic Acid
(28). This compound was prepared from II-15c following the same
procedure as described for (+)-16. MS: 505.4 (M + 1)+. Analytical
1
HPLC purity, 99%. H NMR (DMSO-d6) δ 8.28−8.08 (m, 2H), 8.00
(d, 1H), 7.73 (s, 1H), 7.58 (m, 1H), 7.50−7.30 (m, 3H), 7.31−7.10
(m, 4H), 7.02 (m, 2H), 6.75 (d, 1H), 6.48 (d, 1H), 3.87 (d, 1H), 2.25
(m, 1H), 1.91 (t, 1H), 1.70 (s, 3H).
Benzyl 2-Bromo-5-carbamoylbenzoate (II-4). Benzyl 2-bromo-
5-cyanobenzoate (II-3) (1.4 g, 4.4 mmol) was dissolved in 15 mL of
Methyl 3′-Formyl-4-nitrobiphenyl-2-carboxylate (III-2).
Methyl 2-bromo-5-nitrobenzoate (III-1) (0.98 g, 3.8 mmol), 3-
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Journal of Medicinal Chemistry
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formylphenylboronic acid (II-6) (0.57 g, 3.8 mmol), K3PO4 (3.2 g, 15
mmol), and Pd[PPh3]4 (0.22 g, 5%) were reacted using the same
procedure described for I-3d to give 0.92 g of the biarylaldehyde
(85%). MS: 327.2 (M + CH3CN + H)+ .
Methyl 3′-((2S,4R)-4-Methyl-6-(5-methyl-1,2,4-oxadiazol-3-
yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)-4-nitrobiphenyl-
2-carboxylate (III-4). 2-(4-Aminobphenyl)-5-methyloxadiazole (II-
10) (0.31 g, 1.8 mmol) was suspended in acetonitrile (20 mL). Methyl
3′-formyl-4-nitrobiphenyl-2-carboxylate (III-2) (0.50 g, 1.8 mmol)
and α-methylstyrene (III-3) (1.1 mL, 8.8 mmol) were added, followed
by indium triflate (0.49 g, 0.88 mmol). The mixture was heated at 70
°C under N2 for 4 h. The mixture was concentrated and purified by
silica gel chromatography (20% EtOAc in hexane) to give 0.60 mg of
the desired product (61%). MS: 561.2 (M + 1)+.
7.41−7.10 (m, 12H), 7.05 (m, 1H), 6.77 (d, 1H), 4.73 (m, 1H), 2.97
(s, 3H), 2.20 (t, 1H), 1.98 (m, 1H), 1.79 (s, 3H).
4-Acetylamino-3′-(6-carbamimidoyl-4-methyl-4-phenyl-
1,2,3,4-tetrahydroquinolin-2-yl)biphenyl-2-carboxylic Acid
(23). Methyl 4-amino-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-oxadia-
zol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)biphenyl-2-carboxy-
late (III-5) (100 mg, 0.19 mmol) was dissolved in 5 mL of CH2Cl2.
Triethylamine (100 mg, 1.0 mmol) was added, followed by acetyl
chloride (39 mg, 0.50 mmol). The mixture was stirred at room
temperature under N2 for 2 h. It was diluted with CH2Cl2, washed
with water and brine, dried over MgSO4, and concentrated (160 mg,
100%). MS: 573.4 (M + 1)+. The ester was hydrolyzed and then
converted to the benzamidine following the same procedures
described for 20 (31 mg, 26%). MS: 519.4 (M + 1)+. Analytical
1
Methyl 4-Amino-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-oxa-
diazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)-
biphenyl-2-carboxylate (III-5). A solution of methyl 3′-((2S,4R)-4-
methyl-6-(5-methyl-1,2,4-oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-nitrobiphenyl-2-carboxylate (III-4) (0.56 g, 1.0
mmol) in 15 mL of THF/dioxane mixture (1:1) was added to a
mixture of sodium hydrosulfide (0.52 g, 3.0 mmol) in 5 mL of H2O at
room temperature. The mixture was stirred at room temperature
under N2 for 2 h. The mixture was concentrated and dissolved in
CH2Cl2. It was washed with water and brine, dried over MgSO4, and
concentrated to a yellow solid (0.49 g, 92%). MS: 531.3 (M + 1)+.
4-Amino-3′-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-
tetrahydroquinolin-2-yl)-biphenyl-2-carboxylic Acid (20).
Methyl 4-amino-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-oxadiazol-3-
yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)biphenyl-2-carboxylate
(III-5) (50 mg, 0.094 mmol) was dissolved in MeOH (5 mL) and
H2O (1 mL). LiOH (21 mg, 0.50 mmol) was added. The mixture was
refluxed under N2 for 2 h. It was diluted with water and extracted with
EtOAc. The combined organic mixture was washed with water and
brine, dried over MgSO4, and concentrated (47 mg, 97%). MS: 517.3
(M + 1)+. The resulting acid (46 mg, 0.090 mmol) was dissolved in
EtOH and HOAc (21 mL/3 mL). Catalytic amount of Raney Ni was
added, and the mixture was stirred under 50 psi of H2 for 12 h. The
mixture was filtered through Celite, concentrated, and purified by
reverse phase HPLC (0.5% TFA in CH3CN/H2O) to give 13 mg of
the desired product as a TFA salt (24%). MS: 477.3 (M + 1)+.
purity, 98%. H NMR (CD3OD) δ 7.98 (m, 1H), 7.22 (m, 1H), 7.41
(m, 11H), 7.02 (m, 1H), 6.77 (d, 1H), 4.72 (m, 1H), 2.20 (m, 1H),
2.11 (s, 3H), 1.97 (m, 1H), 1.81 (s, 3H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-cyanobiphenyl-2-carboxylic Acid (19). Benzyl
2-bromo-5-cyanobenzoate (II-3) (0.32 g, 1.0 mmol), 3-formylphe-
nylboronic acid (0.23 g, 1.5 mmol), K3PO4 (0.32 g, 1.5 mmol), and
Pd[PPh3]4 (0.12 g, 10%) were reacted using the same procedure
described for I-3d to give 0.21 g of benzyl 4-cyano-3′-formylbiphenyl-
2-carboxylate. This intermediate (55 mg, 0.16 mmol) was added
together with 4-aminobenzamidine mono-HCl salt (30 mg, 0.16
mmol), α-methylstyrene (0.12 mL, 0.81 mmol), indium triflate (50
mg, 0.08 mmol) with 5 mL of acetonitrile. Following the procedure
described for compound 7, the desired product was isolated as the
TFA salt (16 mg, 17%). MS: 487.5(M + 1)+. Analytical purity, 92%.
1H NMR (DMSO-d6) δ 8.60 (s, 2H), 8.20(s, 2H), 8.11 (s, 1H), 7.98
(d, 1H), 7.58 (d, 1H), 7.40 (m, 3H), 7.26 (m, 3H), 7.15 (m, 1H), 7.00
(m, 1H), 6.77 (d, 1H), 4.68 (d, 1H), 2.05 (t, 1H), 1.88 (m, 1H), 1.72
(s, 3H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2-carboxylic Acid (22). This compound
was prepared following the same methods described for compound 8.
1
MS: 463.3 (M + 1)+. Analytical purity, 93%. H NMR (CD3OD) δ
7.78 (d, 1H), 7.50 (m, 1H), 7.40−7.10 (m, 12), 7.02 (d, 1H), 6.77 (d,
1H), 4.72 (d, 1H), 2.20 (t, 1H), 1.95 (m, 1H), 1.80 (s, 3H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2,4-dicarboxylic Acid 4-Methyl Ester
(21). This compound was prepared following the same procedures
described for compound 13. MS: 520.4 (M + 1)+. Analytical purity,
1
Analytical HPLC purity, 90%. H NMR (CD3OD) δ 7.80 (m, 2H),
7.41−7.10 (m, 9H), 7.05 (m, 2H), 6.77 (d, 2H), 4.70 (m, 1H), 2.20 (t,
1H), 1.98 (m, 1H).
1
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-(3-methylureido)biphenyl-2-carboxylic Acid
(17). Methyl 4-amino-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-oxadia-
zol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)biphenyl-2-carboxy-
late (III-5) (100 mg, 0.19 mmol) was dissolved in 5 mL of CH2Cl2.
Triethylamine (100 mg, 1.0 mmol) was added, followed by methyl
isocyanate (29 mg, 0.50 mmol). The mixture was stirred at room
temperature under N2 for 12 h. It was diluted with CH2Cl2, washed
with water and brine, dried over MgSO4, and concentrated (130 mg,
100%). MS: 588.4 (M + 1)+. The ester was hydrolyzed and then
converted to the benzamidine following the same procedures
described for compound 20 (32 mg, 26%). MS: 534.4 (M + 1)+.
100%. H NMR (CD3OD) δ 8.42 (s, 1H), 8.18 (d, 1H), 7.55−7.25
(m, 10), 7.20 (m, 1H), 7.09 (d, 1H), 6.80 (d, 1H), 4.79 (m, 1H), 3.95
(s, 3H), 2.22 (t, 1H), 2.00 (m, 1H), 1.82 (s, 3H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-methylcarbamoylbiphenyl-2-carboxylic Acid
(24). This compound was prepared following the same procedures
described compound 11. MS: 519.3 (M + 1)+. Analytical purity,
1
99.9%. H NMR (DMSO-d6) δ 8.59 (m, 2H), 8.18 (m, 2H), 7.95 (d,
1H), 7.40 (m, 4H), 7.20 (m, 4H), 7.01 (s, 1H), 6.78 (d, 1H), 4.65 (d,
1H), 2.77 (d, 3H), 2.04 (t, 1H), 1.86 (m, 1H), 1.72 (s, 3H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-isopropylcarbamoylbiphenyl-2-carboxylic
Acid (25). This compound was prepared following the same methods
described for compound 11. MS: 547.4 (M + 1)+. Analytical purity,
1
Analytical HPLC purity, 98%. H NMR (CD3OD) δ 7.79 (m, 1H),
7.66 (m, 1H), 7.40−7.10 (m, 11H), 7.02 (m, 1H), 6.75 (d, 1H), 4.72
(m, 1H), 2.75 (s, 3H), 2.20 (m, 1H), 1.98 (m, 1H), 1.80 (s, 3H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-methanesulfonylaminobiphenyl-2-carboxylic
Acid (18). Methyl 4-amino-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-
oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)biphenyl-2-
carboxylate (III-5) (100 mg, 0.19 mmol) was dissolved in 5 mL of
CH2Cl2. Triethylamine (100 mg, 1.0 mmol) was added, followed by
methanesulfonyl chloride (46 mg, 0.40 mmol). The mixture was
stirred at room temperature under N2 for 4 h. It was diluted with
CH2Cl2, washed with water and brine, dried over MgSO4, and
concentrated (48 mg, 42%). MS: 608.2 (M + 1)+. The ester was
hydrolyzed and then converted to the benzamidine following the same
procedures described for 20 (10 mg, 7.9%). MS: 555.4 (M + 1)+.
1
99.5%. H NMR (DMSO-d6) δ 8.60 (s, 2H), 8.38 (d, 1H), 8.18 (m,
2H), 7.98 (d, 1H), 7.40 (m, 4H), 7.23 (m, 3H), 7.00 (m, 1H), 6.77 (d,
1H), 4.68 (m, 1H), 4.08 (m, 1H), 2.05 (t, 1H), 1.88 (m, 1H), 1.73 (s,
3H), 1.12 (d, 6H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-tert-butylcarbamoylbiphenyl-2-carboxylic
Acid (27). This compound was prepared following the same methods
described for compound 11. MS: 561.3 (M + 1)+. Analytical purity,
1
91%. H NMR (DMSO-d6) δ 8.67 (s, 2H), 8.25 (s, 1H), 8.15 (s, H),
8.01 (m, 1H), 7.42 (m, 4H), 7.28 (m, 4H), 7.08 (s, 1H), 6.83 (d, 1H),
4.73 (m, 1H), 2.10 (m, 1H), 1.85 (m, 1H), 1.79 (s, 3H), 1.40 (s, 9H).
Methyl 5-((Dimethylamino)methyl)-2-(trifluoromethyl-
sulfonyloxy)benzoate (IV-3). 2-Hydroxy-5-formylbenzoic acid
(IV-1) (2.0 g, 12 mmol) was dissolved in 30 mL of DMF. Potassium
1
Analytical HPLC purity, >98%. H NMR (CD3OD) δ 7.62 (m, 1H),
K
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Journal of Medicinal Chemistry
Article
bicarbonate (1.3 g, 13 mmol) was added, followed by benzyl bromide
(1.6 mL, 13 mmol). The mixture was stirred at room temperature for
12 h. The reaction mixture was poured into water and extracted with
EtOAc. The combined extract was washed with brine, dried over
Na2SO4, concentrated to a light brown oil. This oil was dissolved in 10
mL of MeOH, and dimethylamine (25 mL, 2.0 N in MeOH) was
added. The mixture was stirred at room temperature for 30 min, and
the sodium cyanoborohydride (1.3 g, 20 mmol) was added. The
mixture was stirred at room temperature for 12 h. It was diluted with
water and extracted with EtOAc. The combined extract was washed
with brine, dried over Na2SO4, concentrated to light brown oil. This
material was dissolved in CH2Cl2 (100 mL) and cooled at 0 °C.
Pyridine (4.0 g, 50 mmol) was added, followed by triflic anhydride
(2.5 mL, 15 mmol). The reaction mixture was stirred at 0 °C for 30
min and then poured into water. The mixture was extracted with
EtOAc. The combined extract was washed with brine, dried over
Na2SO4, concentrated, and purified by flash chromatography (silica,
1:1 EtOAc/hexane) to give 0.88 g of brown oil (22%). MS: 342.2 (M
+ 1)+.
Methyl 4-((Dimethylamino)methyl)-3′-formylbiphenyl-2-
carboxylate (IV-4). Methyl 5-((dimethylamino)methyl)-2-
(trifluoromethylsulfonyloxy)benzoate (IV-3) (0.44 g, 1.3 mmol), 3-
formylphenylboronic acid (0.29 g, 1.9 mmol), K3PO4 (0.41 g, 1.9
mmol), and Pd[PPh3]4 (0.15 g, 10%) were reacted using the same
procedure described for I-3d to give 0.33 g of the biarylaldehyde
(86%). MS: 298.3 (M + 1)+.
Methyl 4-((Dimethylamino)methyl)-3′-((2S,4R)-4-methyl-6-
(5-methyl-1,2,4-oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2-carboxylate (IV-5). 2-(4-Aminophenyl)-
5-methyloxadiazole (II-10) (59 mg, 0.34 mmol) was suspended in
acetonitrile (10 mL). Methyl 4-((dimethylamino)methyl)-3′-formylbi-
phenyl-2-carboxylate (IV-4) (100 mg, 0.34 mmol) and α-methylstyr-
ene (III-3) (0.22 mL, 1.7 mmol) were added, followed by indium
triflate (95 mg, 0.17 mmol). The mixture was heated at 70 °C under
N2 for 4 h. The mixture was concentrated and taken into the next step
without purification. MS: 537.4 (M + 1)+.
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-dimethylaminomethylbiphenyl-2-carboxylic
Acid (26). Methyl 4-((dimethylamino)methyl)-3′-((2S,4R)-4-methyl-
6-(5-methyl-1,2,4-oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-
2-yl)biphenyl-2-carboxylate (IV-5) was dissolved in MeOH (5 mL),
THF (5 mL), and H2O (2 mL). LiOH (42 mg) was added. The
mixture was refluxed under N2 for 8 h. It was concentrated and
purified by reverse phase HPLC (0.5% TFA in CH3CN/H2O) to give
180 mg of TFA salt of the desired acid (77%). MS: 559.4 (M + 1)+.
Part of the product (86 mg, 0.15 mmol) was dissolved in EtOH and
HOAc (21 mL/3 mL). Catalytic amount of Raney Ni was added, and
the mixture was stirred under 50 psi of H2 for 12 h. The mixture was
filtered through Celite, concentrated, and purified by reverse phase
HPLC (0.5% TFA in CH3CN/H2O) to give 36 mg of TFA salt of the
desired product (38%). MS: 519.4 (M + 1)+. Analytical purity, 97.8%.
1H NMR (CD3OD) δ 7.94 (s, 1H), 7.68 (m, 1H), 7.50−7.25 (m, 10),
(0.28 g) was added, followed by a few drops of water. The reaction
mixture was stirred at room temperature for 1.5 h. The reaction was
quenched with 1 N HCl and water. It was extracted with EtOAc. The
combined EtOAc solution was washed with water and brine, dried
over MgSO4, concentrated, and purified by chromatography (silica gel,
1−5% MeOH in CH2Cl2) to give 0.28 g of the corresponding alcohol
(36%). MS: 407.2 (M + 1)+. This alcohol (0.25 g, 0.62 mmol) and
MnO2 (0.43 g, 4.9 mmol) were then refluxed with benzene (15 mL)
under N2 for 20 h. It was filtered through Celite, concentrated, and
purified by chromatography (silica gel, 3−4% MeOH in CH2Cl2) to
give 0.18 g of the corresponding aldehyde (72%). 1H NMR (CDCl3)δ
9.96 (s, 1H), 8.48 (d, 2H), 8.30 (d, 1H), 8.13 (m, 1H), 8.00 (m, 1H),
7.45 (dd, 1H), 7.26 (m, 3H), 7.08 (m, 2H), 5.11 (s, 2H).
5′-Amino-3′-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-
tetrahydroquinolin-2-yl)-4-carbamoylbiphenyl-2-carboxylic
Acid (30). 4-Aminobenzamidine mono-HCl salt (16 mg, 0.09 mmol),
benzyl 4-carbamoyl-3′-formyl-5′-nitrobiphenyl-2-carboxylate (II-9)
(33 mg, 0.09 mmol), α-methylstyrene (0.05 mL, 0.47 mmol), and
indium triflate (25 mg, 0.05 mmol) were reacted following the
procedure described for compound 7. The desired product was
isolated as a TFA salt (6.2 mg, 11%). MS: 520.3 (M + 1)+. Analytical
purity, 100%. 1H NMR (DMSO-d6) δ 8.59 (s, 2H), 8.16 (s, 2H), 8.11
(s, 1H), 8.09 (s, 1H), 7.97 (d, 1H), 7.45−7.30 (m, 3H), 7.22 (m, 3H),
7.17 (m, 1H), 7.01 (s, 1H), 6.77 (d, 1H), 6.71 (s, 1H), 6.61 (s, 1H),
6.53 (s, 1H), 4.50 (m, 1H), 1.95 (m, 1H), 1.82 (m, 1H), 1.72 (s, 3H).
5′-Amino-3′-(6-carbamimidoyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2-carboxylic Acid (29). This compound
was prepared following the same methods described for compound 30.
1
MS: 463.2(M + 1)+. Analytical purity, 98.6%. H NMR (DMSO) δ
8.64 (s, 2H), 8.21 (s, 2H), 7.67 (d, 1H), 7.55 (m, 1H), 7.42−7.20 (m,
8H), 6.92 (s, 1H), 6.85−6.70 (m, 4H), 6.66 (s, 1H), 6.53 (s, 1H), 4.60
(m, 1H), 4.30 (m, 1H), 2.16 (m, 1H), 1.89 (m, 1H).
Benzyl 4-Carbamoyl-3′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-
oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)-5′-
nitrobiphenyl-2-carboxylate (II-12). 2-(4-Aminobphenyl)-5-meth-
yloxadiazole (72 mg, 0.41 mmol) (II-10) was suspended in acetonitrile
(10 mL). Benzyl 4-carbamoyl-3′-formyl-5′-nitrobiphenyl-2-carboxylate
(II-9) (150 mg, 0.37 mmol) and α-methylstyrene (0.25 mL, 1.86
mmol) were added, followed by indium triflate (104 mg, 0.20 mmol).
The mixture was degassed and heated at 70 °C under N2 for 6 h. The
mixture was concentrated and purified by chromatography (silica gel,
30% EtOAc in hexane and then 3% MeOH in CH2Cl2) to give 0.17 g
of the desired product (61%). MS: 680.3 (M + 1)+.
Benzyl 3′-Amino-4-carbamoyl-5′-((2S,4R)-4-methyl-6-(5-
methyl-1,2,4-oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroqui-
nolin-2-yl)biphenyl-2-carboxylate (II-13). Benzyl 4-carbamoyl-3′-
((2S,4R)-4-methyl-6-(5-methyl-1,2,4-oxadiazol-3-yl)-4-phenyl-1,2,3,4-
tetrahydroquinolin-2-yl)-5′-nitrobiphenyl-2-carboxylate (II-12) (63
mg, 0.09 mmol) was added with Fe (28 mg) and HOAc (0.2 mL)/
EtOH (4 mL)/H2O (0.8 mL). The mixture was heated at 80 °C for 30
min under N2. It was diluted with EtOH and filtered through Celite.
The filtrate was concentrated and redissolved in EtOAc. The EtOAc
solution was washed with water and brine, dried over MgSO4,
concentrated to give 55 mg of crude amine (91%). This material was
used in the next step without further purification. MS: 650.4 (M + 1)+.
5′-Acetylamino-3′-(6-carbamimidoyl-4-methyl-4-phenyl-
1,2,3,4-tetrahydroquinolin-2-yl)-4-carbamoylbiphenyl-2-car-
boxylic Acid (31). Benzyl 3′-amino-4-carbamoyl-5′-((2S,4R)-4-
methyl-6-(5-methyl-1,2,4-oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)biphenyl-2-carboxylate (II-13) (70 mg, 0.11 mmol) was
dissolved in 10 mL of CH2Cl2. It was cooled at 0 °C, and triethylamine
(0.1 mL) was added, followed by acetyl chloride (21 μL). The mixture
was stirred at 0 °C under N2 for 1.5 h. Water and CH2Cl2 were added.
The two layers were separated. The CH2Cl2 layer was washed with
water and brine, dried over MgSO4, and concentrated to give 80 mg of
crude product (100%, MS: 692.0 (M + 1)+). This material was
dissolved in 10 mL of EtOH and HOAc (7:1). Catalytic amount of
Raney Ni was added, and the mixture was stirred under 50 psi of H2
for 2 h. The mixture was filtered through Celite and concentrated. The
resulting solid was dissolved in 10 mL of EtOH and HOAc (7:1).
Catalytic amount of 10% Pd/C was added, and the mixture was stirred
7.18 (m, 1H), 7.05 (m, 1H), 6.78 (d, 1H), 4.76 (m, 1H), 4.37 (s, 2H),
2.86 (s, 6H), 2.20 (m, 1H), 1.98 (m, 1H), 1.81 (s, 3H).
2′-(Benzyloxycarbonyl)-4′-carbamoyl-5-nitrobiphenyl-3-car-
boxylic Acid (II-7). Benzyl 2-bromo-5-carbamoylbenzoate (II-4) (4.7
g, 14 mmol), 3-carboxyl-5-nitrophenylboronic acid (3.0 g, 14 mmol),
K3PO4 (4.5 g, 21 mmol), and Pd[PPh3]4 (0.82 g, 5%) were added
together with 50 mL of DMF. The mixture was degassed and heated at
100 °C under N2 for 8 h. The reaction mixture was cooled and poured
into water. It was extracted with EtOAc. The combined organic
solution was washed with brine and dried over MgSO4. It was
concentrated and recrystallized from EtOAc and CH2Cl2 to give 1.6 g
of the biaryl product (27%). MS: 421.2 (M + 1)+.
Benzyl 4-Carbamoyl-3′-formyl-5′-nitrobiphenyl-2-carboxy-
late (II-9). 2′-(Benzyloxycarbonyl)-4′-carbamoyl-5-nitrobiphenyl-3-
carboxylic acid (II-7) (0.80 g, 1.9 mmol) was dissolved in 20 mL of
THF at room temperature. Triethylamine (0.32 mL, 2.1 mmol) was
added, followed by isobutyl chloroformate (0.30 mL, 2.1 mmol). The
mixture was stirred at room temperature under N2 for 20 min. NaBH4
L
dx.doi.org/10.1021/jm401670x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
under 40 psi of H2 for 2 h. The mixture was filtered through Celite and
concentrated. The crude product was purified by reverse phase HPLC
(0.5% TFA in CH3CN/H2O) to give 10 mg of the desired product as
from the levorotary enantiomer using the same procedures as
described for 1. MS: 604.4 (M + 1)+. [α]D24 −87.0 (c 0.10, MeOH).
Analytical purity, 100%. 1H NMR (DMSO-d6) δ 9.99 (s, 1H), 8.67 (s,
2H), 8.27 (s, 2H), 8.23 (s, 1H), 8.18 (s, 1H), 8.05 (d, 1H), 7.75 (s,
1H), 7.59 (s, 1H), 7.47 (s, 1H), 7.44 (m, 3H), 7.31 (m, 4H), 7.22 (m,
1H), 7.10 (m, 2H), 6.84 (d, 1H), 4.66 (m, 1H), 2.17 (d, 2H), 2.06 (m,
2H), 1.92 (m, 1H), 1.79 (s, 3H), 0.93 (d, 6H).
1
a TFA salt (13%). MS: 562.4 (M + 1)+. Analytical purity, 93.9%. H
NMR (CD3OD) δ 8.34 (s, 1H), 8.04 (d, 1H), 7.79 (s, 1H), 7.48 (m,
3H), 7.38−7.25 (m, 4H), 7.20 (m, 2H), 7.11 (s, 1H), 6.80 (d, 1H),
4.75 (m, 1H), 2.27 (m, 1H), 2.13 (s, 3H), 2.02 (m, 1H), 1.83 (s, 3H).
3′-((2S,4R)-6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tet-
rahydroquinolin-2-yl)-4-carbamoyl-5′-propionamidobiphenyl-
2-carboxylic Acid (32). This compound was prepared followed the
same procedures described for compound 31. MS: (M + 1)+.
ASSOCIATED CONTENT
* Supporting Information
■
S
1
Crystallographic data and refinement statistics for X-ray crystal
structures of the FXIa complexes with compounds 1 and 16
and FVIIa complex with compound 29. This material is
available free of charge via the Internet at http://pubs.acs.org.
Analytical purity, 95%. H NMR (DMSO-d6) δ 10.0 (s, 2H), 8.69 (s,
2H), 8.23 (m, 3H), 8.05 (m, 1H), 7.76 (s, 1H), 7.56 (m, 2H), 7.47 (m,
2H), 7.32 (m, 2H), 7.22 (m, 1H), 7.10 (d, 2H), 6.83 (m, 1H), 4.65 (d,
1H), 2.31 (m, 2H), 2.03 (t, 1H), 1.94 (m, 1H), 1.79 (s, 3H), 1.07 (t,
3H).
3′-Butyramido-5′-((2S,4R)-6-carbamimidoyl-4-methyl-4-
phenyl-1,2,3,4-tetrahydroquinolin-2-yl)-4-carbamoylbiphenyl-
2-carboxylic Acid (33). This compound was prepared followed the
same procedures described for compound 31. MS: (M + 1)+.
Analytical purity, 94.9%. 1H NMR (DMSO-d6) δ 10.0 (s, 1H), 8.69 (s,
2H), 8.23 (m, 3H), 8.05 (d, 1H), 7.76 (s, 1H), 7.57 (m, 2H), 7.47 (m,
2H), 7.32 (m, 2H), 7.22 (m, 1H), 7.10 (d, 2H), 6.83 (m, 1H), 4.70 (d,
1H), 2.27 (t, 2H), 2.05 (t, 1H), 1.96 (m, 1H), 1.79 (s, 3H), 1.59 (m,
2H), 0.91 (t, 3H).
AUTHOR INFORMATION
Corresponding Author
*Phone 609-818-5301. Fax: (609) 818-3331. E-mail: mimi.
quan@bms.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
3′-((2S,4R)-6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tet-
rahydroquinolin-2-yl)-4-carbamoyl-5′-(cyclopropane-
carboxamido)biphenyl-2-carboxylic Acid (34). This compound
was prepared followed the same procedures described for compound
Use of the IMCA-CAT beamline 17-BM 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 deposition of the X-ray structures and data
and Dr. Yuval Blat and Donna Pedicord for factor IX activation
assays.
1
31. MS: (M + 1)+. Analytical purity, 90%. H NMR (DMSO-d6) δ
10.36 (s, 1H), 8.69 (s, 2H), 8.23 (m, 4H), 8.05 (m, 1H), 7.77 (s, 1H),
7.55 (m, 2H), 7.47 (m, 2H), 7.32 (m, 2H), 7.22 (m, 1H), 7.10 (d,
2H), 6.83 (m, 1H), 4.72 (d, 1H), 2.40 (m, 1H), 2.05 (t, 1H), 1.96 (m,
1H), 1.79 (s, 3H), 0.78 (m, 4H).
3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-
quinolin-2-yl)-4-carbamoyl-5′-(3-methylbutyrylamino)-
biphenyl-2-carboxylic Acid (35). This compound was prepared
followed the same procedures described for 31. MS: 604.4 (M + 1)+.
Analytical purity, 93.4%. 1H NMR (DMSO-d6) δ 9.88 (s, 1H), 8.31 (s,
2H), 8.00 (m, 1H), 7.80 (s, 1H), 7.35−7.48 (m, 4H), 7.20−7.32 (m,
4H), 7.15 (m, 2H), 7.08 (m, 1H), 6.78 (m, 1H), 4.68 (m, 1H), 2.20
(m, 2H), 2.10 (m, 1H), 2.00 (m, 1H), 1.96 (m, 1H), 1.79 (s, 3H), 0.96
(m, 6H).
REFERENCES
■
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Geneva, Switzerland, June 2011.
(+)-3′-(6-Carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahy-
droquinolin-2-yl)-4-carbamoyl-5′-(3-methylbutyrylamino)-
biphenyl-2-carboxylic Acid (1) and (−)-3′-(6-Carbamimidoyl-4-
methyl-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)-4-carbamo-
yl-5′-(3-methylbutyrylamino)biphenyl-2-carboxylic Acid (36).
Benzyl 3′-amino-4-carbamoyl-5′-((2S,4R)-4-methyl-6-(5-methyl-1,2,4-
oxadiazol-3-yl)-4-phenyl-1,2,3,4-tetrahydroquinolin-2-yl)biphenyl-2-
carboxylate (II-13) (2.39 g, 3.68 mmol) was dissolved in 350 mL of
CH2Cl2. It was cooled at 0 °C, and triethylamine (5.0 mL) was added,
followed by isovaleryl chloride (3.3 mL). The mixture was stirred at 0
°C under N2 for 1.5 h. Water and CH2Cl2 were added. The two layers
were separated. The CH2Cl2 layer was washed with water and brine,
dried over MgSO4, concentrated, and purified by flash chromatog-
raphy (silica, 50−100 EtOAc/hexane) to give 1.73 g of the desired
product (64%, MS: 734.4 (M + 1)+). This material was separated by
chiral HPLC (Chiralcel OD 4.6 × 250, 50% IPA/50% heptane) to give
0.90 g of the dextrorotatory enantiomer and 0.80 g of the levorotary
enantiomer. The dextrorotatory enantiomer was dissolved in 130 mL
of MeOH/Et3N (8:1). Catalytic amount of 10% Pd−C was added, and
the mixture was stirred under 1 atm H2 for 12 h. The mixture was
filtered through Celite and concentrated. The crude product was
purified by reverse phase HPLC (0.5% TFA in CH3CN/H2O) to give
0.80 g TFA salt of 1 (91%). MS: 604.4 (M + 1)+. [α]D20 +63.9 (c 0.25,
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1
MeOH). Analytical purity, 96.4%. H NMR (DMSO-d6) δ 9.92 (s,
1H), 8.60 (s, 2H), 8.15 (m, 3H), 7.98 (d, 1H), 7.69 (s, 1H), 7.51 (s,
1H), 7.40 (m, 2H), 7.38 (m, 3H), 7.25 (m, 3H), 7.13 (m, 1H), 7 (d,
2H), 6.78 (d, 1H), 4.58 (m, 1H), 2.11 (m, 2H), 2.00 (m, 2H), 1.85
(m, 1H), 1.72 (s, 3H), 0.86 (m, 6H). Compound 36 was prepared
M
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