Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa)

Article abstract of DOI:10.1016/j.bmc.2014.02.010

A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase.

Full text of DOI:10.1016/j.bmc.2014.02.010

Bioorganic & Medicinal Chemistry 22 (2014) 2261–2268
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of conformationally restricted inhibitors
of active thrombin activatable fibrinolysis inhibitor (TAFIa)
⇑
Mikael Brink, Anders Dahlén, Thomas Olsson, Magnus Polla , Tor Svensson
Cardiovascular and Metabolic Disorders Research Area, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo
Received 6 November 2013
Revised 15 January 2014
Accepted 10 February 2014
Available online 18 February 2014
[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a
common hydrogenation–alkylation sequence starting from the appropriate benzimidazole and imidazo-
pyridine system. We present a successful design strategy using a conformational restriction approach
resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with
a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the
observed potency increase.
Keywords:
Carboxypeptidase U
TAFIa
Ó 2014 Elsevier Ltd. All rights reserved.
AstraZeneca
Rigidification
Conformational restriction
Imidazole
1. Introduction
the C-terminal lysine residues are cleaved off. This diminishes the
amount of plasminogen and tPA able to bind to fibrin and thereby
Different thrombotic diseases are one of the more common
causes of disease and death in the Western world. Therapy avail-
able today includes thrombolytics, anticoagulants and antiplat-
elets. The only thrombolytics on the market are the plasminogen
activators, which are associated with more or less severe bleeding
complications. The anticoagulants and antiplatelets also have sig-
nificant drawbacks, including varying degrees of adverse hemor-
rhagic side-effects. Clearly, there is a need for antithrombotic or
thrombolytic drugs that are safer than established therapies. Pro-
carboxypeptidase U1 (proCPU, EC 3.4.17.20, thrombin activatable
fibrinolysis inhibitor, TAFI,2 procarboxypeptidase R3 and plasma
procarboxypeptidase B4,5) is a metallopeptidase, circulating in
plasma. The function of this enzyme in fibrinolysis was elucidated
in the mid 90s.6 After activation, the enzymatic activity of TAFIa is
to cleave off C-terminal basic amino acids, that is, lysine and
arginine residues, from peptides and proteins.7 During fibrinolysis,
C-terminal lysine and arginine residues are formed by the action of
plasmin on the fibrin network and thereby high-affinity binding
sites for plasminogen and tPA (tissue-type plasminogen activator)
are created. Upon binding to the clot, plasminogen is efficiently
converted to plasmin and acceleration in the rate of fibrinolysis
is achieved. Through the action of TAFIa on partly degraded fibrin,
the amount of plasmin formed. This proposed mechanism for sup-
pression of fibrinolysis provides the rationale for inhibiting TAFIa
in order to enhance clot lysis. Furthermore, since the coagulation
is unaffected, TAFIa inhibition may result in less bleeding compli-
cations than conventional therapy.8 This fact may contribute to
the interest in the pharmaceutical industry for TAFIa as a potential
drug target.9,10 The concept of TAFIa inhibition for stimulation of
fibrinolysis is supported by in vitro studies in human blood and
plasma as well as in vivo studies in animals.11 Recently, a phase
II study showing a positive effect of the novel TAFIa inhibitor
AZD9684 (compound 1 in Table 1) in patients with pulmonary
embolism was presented.12
The lead generation work that culminated in the selection of
AZD9684 has been published earlier.13 The AstraZeneca thiol series
was optimized to be absorbed via the transcellular route generating
compounds with good oral availability, see for example compound 1
in Table 1. However, a drawback with most compounds in the thiol
series was that the short elimination half-life ruled out a once or
twice daily administration in patients. In a follow-up program we
turned our attention to the imidazole-type inhibitors reported by
Pfizer (compound 2 (UK-396,082) and 3, Table 1).14 UK-396,082 is
claimed to be absorbed via the paracellular route and that oral
bioavailability is ꢀ20% in the rat and 23% in man. We reasoned that
paracellular absorption could lead to a later Tmax and longer elimi-
nation half-life resulting in a profile suitable for once or twice daily
⇑
Corresponding author. Tel.: +46 31 776 22 69; fax: +46 31 776 37 24.
E-mail address: magnus.polla@astrazeneca.com (M. Polla).
http://dx.doi.org/10.1016/j.bmc.2014.02.010
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
Table 1
Enzyme inhibition and PK data on compounds 1–5
Compound
1
2
3
4
5
TAFIa IC50 (nM)a
Ligand efficiencyb
87
450
0.43
5.0
620
0.37
8.0
1500
0.34
31
8
0.47
0.4
75
27
0.5
0.43
1.0
CLA IC50 (l
M)c
Oral bioavailability (%, rat)
Clearance (mL/min/kg, rat)
Tmax (h, rat)
8–13d
2–8
1–2
2,d 8e
6–12
0.2–2
a
b
c
The assays was performed as described earlier.16
pIC50/number of heavy atoms.
Clot lysis assay in human plasma.17
d
e
Dose 20
lmol/kg.
Dose 6.8
lmol/kg.
administration. The imidazole inhibitors 2–4 seem to be less potent,
when compared in the same assay, than their thiol counterparts
(Table 1).15 However, based on docking experiments (Fig. 1) we
envisioned that reducing conformational flexibility by forming a
six-membered ring would result in a substantial increase in potency
while keeping size sufficiently low to allow for paracellular absorp-
tion. Indeed this turned out to be the case as illustrated by com-
pound 5.
2.1. Synthesis of the 4,5,6,7-tetrahydro-1H-benzimidazoles
The 4,5,6,7-tetrahydro-1H-benzimidazole scaffold was pre-
pared starting from 4-fluoro-3-nitrobenzoic acid (Scheme 1).
Fischer esterification was followed by nucleophilic aromatic
substitution using an amine containing the desired imidazole sub-
stituent. The nitro functionality was then reduced to give a diamine
intermediate which after treatment with trimethyl orthoformate
In this paper, we describe the design and synthesis of a series of
novel, potent and selective small molecule inhibitors of TAFIa using
this rigidification approach.
gave a N-substituted benzimidazole derivative. The desired
4,5,6,7-tetrahydro-1H-benzimidazole scaffold was then formed
via a harsh hydrogenation at a pressure of 20 bar in a steel-auto-
clave using stochiometric amounts of Pd/C and Pd(OAc)2 in AcOH
at 115 °C. Milder conditions or catalytic amounts of catalysts gave
lower or no yield. Now, a critical step including LiHMDS and 3-bro-
mo-N-tritylpropan-1-amine was applied, which resulted in addi-
2. Chemistry
The imidazole propionic acids 2 and 3 and the imidazole acetic
acid 4, representing recent examples of potent TAFIa inhibitors,
were synthesized according to literature procedures (Table 1).14,18
The 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and
5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid deriv-
atives described in this study were prepared via a common hydro-
genation–alkylation sequence starting from the appropriate
benzimidazole and imidazopyridine system.
tion of the aminopropyl side-chain at the
a-position of the ester.
Several different protecting groups on the 3-bromopropan-1-amine
were investigated in this addition reaction, for example, Boc, Cbz,
dibenzyl, diboc and phtalimide. However, it was concluded after
extensive studies that the trityl protecting group regularly gave bet-
ter yields. Additionally, the trityl group is easily removed using TFA/
MeOH within a couple of minutes and allows for simple purification
on cationic exchange columns. Once the trityl group has been re-
moved the intermediate amino ester cyclized spontaneously, or
was treated with NH3/MeOH to induce cyclization, to form the cor-
responding spirolactam. After purification of this lactam, the de-
sired product is finally received as an HCl salt after ring-opening
Scheme 1. General synthetic route for the 4,5,6,7-tetrahydro-1H-benzimidazoles.
Reagents and conditions: (a) (1) MeOH, H2SO4 (concd), (2) isoamylamine, EtOH, (3)
Figure 1. The more active enantiomer of compound 2 docked in the active site of a
CPB homology model. Zinc atom in magenta and hydrogen bonds in dotted yellow
lines. The green arrow show attachment points for the conformational restriction.
H2 (1 bar), Pd/C, MeOH/AcOH; (b) (1) trimethyl orthoformate, PTSA, (2) H2 (20 bar),
Pd/C, Pd(OAc)2, AcOH; (c) LiHMDS, 3-bromo-N-tritylpropan-1-amine, THF (d) (1)
TFA, DCM/MeOH, (2) NH3/MeOH, (3) chiral separation (e) HCl (6 M).

M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
2263
using aqueous HCl. Separation of enantiomers was performed using
chiral chromatography on either the lactam intermediate or on the
alkylation product with the trityl protecting group still attached.
all compounds. For example, compound 2 show prolonged lysis at
low concentrations and enhanced lysis due to TAFIa inhibition at
higher concentrations. The prolonged lysis observed at low
concentrations may be due to interaction with other macromole-
cules present in plasma or the previously reported TAFIa stabilizing
effect.19 At even higher concentrations of compound 2 lysis is again
prolonged perhaps due to inhibition of plasmin or tPa. Compound
1 on the other hand does not show the same behavior at low or at
high concentrations resulting in a more predictable behavior in the
CLA. Compound 5 display prolonged lysis at lower concentrations
probably resulting in an underestimation of the potency in the
CLA in plasma. Selectivity towards carboxypeptidase N (CPN, a car-
boxypeptidase with an important role as an inactivator of ana-
phylatoxins in plasma)16 was high with CPN IC50 >1 mM for all
compounds in Table 2. As expected, permeability was low in a
Caco2 cell permeability assay with a Papp <0.1. Studies in the rat
showed highly variable pharmacokinetics as compared to com-
pound 2 (Table 1). A possible explanation for the dose-dependent
bioavailability observed for compound 5 is that an active uptake
or efflux mechanism is involved. However, bioavailability in rat
for compound 5 is in the same range as for compound 2 (23% in
man)14, indicating that bioavailability in man could be acceptable.
Attempts to increase permeability by increasing lipophilicity in the
4,5,6,7-tetrahydro-1H-benzimidazole series proved unsuccessful.
Compounds 6–9 in Table 2 with increasingly larger and more lipo-
philic alkyl substituents on the imidazole nitrogen still had very
low permeability in the Caco2 cell assay. Oral bioavailability in
rat was also low for compounds 6–9 indicating that size may be
too large for absorption via the paracellular route. TAFIa potency
remained fairly constant in the alkyl-substituted 4,5,6,7-tetrahy-
dro-1H-benzimidazole series. N-Aryl substitution on the 4,5,6,
7-tetrahydro-1H-benzimidazole scaffold resulted in somewhat
decreased TAFIa potency (compound 10 in Table 2), a finding that
may be attributed to the lower basicity of the imidazole in this
compound. Calculated pKa is 6.5 for the phenyl-substituted com-
pound 10 while the alkyl-substituted analogs 5–9 has a calculated
pKa of 7.7–8.2. In the isomeric 5,6,7,8-tetrahydroimidazo[1,2-
a]pyridine series there seems to be a reverse relationship. The al-
kyl-substituted compound 11 having a higher basicity (calculated
pKa = 8.7) have only micromolar TAFIa potency while the aryl-
substituted compounds 12–13 with calculated pKa of 7.8–7.9 are
much more potent. Although we have limited data to support this
finding, there may be a pKa optimum with respect to zinc binding
in imidazole type compounds. The most lipophilic compounds, 8, 9
and 13, show a decreased potency in the CLA due to increased
binding to plasma proteins.To verify the predicted binding mode
of the conformationally restricted compound 5 in the active site
of TAFI, soaking experiments in crystals of a H333Y/H335Q double
mutant human TAFI were carried out.21 It is known that some pro-
carboxypeptidases display enzymatic activity indicating that the
pro-peptide can move and allow small substrates in to the active
site, and data that this is true for TAFI as well motivated us to
try soaking inhibitors in crystals of our mutant TAFI.22
2.2. Synthesis of the 5,6,7,8-tetrahydroimidazo[1,2-a]pyridines
The synthesis of the 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine
scaffold started with oxidation of the appropriate 2-phenylethanol
to form the corresponding aldehyde (Scheme 2).
Whereas other oxidizing reagents, such as PCC, did not give
satisfying results, DMP (Dess–Martin periodinane) gave a clean
conversion to the aldehyde. The
by reacting the aldehyde with bromine and a few drops of dioxane in
diethyl ether. The -bromoaldehyde degraded upon standing and
a-bromoaldehyde was then formed
a
was therefore taken directly to the next step where it was reacted
with 2-aminopyridine-4-carboxylate in a microwave reactor to give
the corresponding imidazopyridine. The desired 5,6,7,8-tetrahydro-
imidazo[1,2-a]pyridine scaffold was then formed by hydrogenation.
Several different catalysts and conditions were tested and it was
found that the best mode for this conversion was to use PtO2 under
basic conditions. The aminopropyl side-chain was introduced as de-
scribed above for the 4,5,6,7-tetrahydro-1H-benzimidazole series.
An evaluation of different amino protecting groups again showed
that the trityl group was superior when it comes to yield and ease
of deprotection. Removal of the trityl group using TFA in DCM gave
the primary amine which spontaneously cyclized to form the corre-
sponding spirolactam. A Suzuki-coupling with the appropriate
phenylboronic acid followed by ring-opening of the spirolactam
with hydrochloric acid concluded the synthesis and gave the desired
products as their hydrochloride salt. Again separation of enantio-
mers using chiral chromatography was done at the spirolactam
stage.
3. Results and discussion
The first compound made using the rigidification approach de-
scribed in Figure 1, adding a 2-carbon bridge to compound 2,
resulting in a 4,5,6,7-tetrahydro-1H-benzimidazole scaffold, was
compound 5. The strategy proved successful resulting in ꢀ30 times
increase in potency in the TAFIa assay (IC50 = 8 nM) as compared to
the racemic compound 2 (see Table 1). This was also translated to a
potency increase in a clot lysis assay (CLA) in human plasma. How-
ever, the CLA does not correlate very well with TAFIa inhibition for
The soaking experiment was successful and the X-ray structure
of the complex was determined to a resolution of 2.0 Å (Fig. 2). The
key interactions with the imidazole moiety binding to the catalytic
zinc ion, the primary amine interacting with Asp349 and the car-
boxylate forming a salt bridge with Arg235 as well as the binding
geometry was in good agreement with the modelling results.23 The
X-ray structure confirmed the stereochemistry of compound 5 as
predicted from the docking experiments. We believe that the ob-
served potency difference between compounds 2 and 5 is due to
the conformational restriction built into compound 5. An overlay
of the X-ray structures of compound 5 in complex with a H333Y/
H335Q double mutant TAFI and compound 2 in complex with car-
boxypeptidase B is shown in Figure 3. The binding conformations
Scheme 2. General synthetic route for the substituted 5,6,7,8-tetrahydro[1,2-
a]pyridines. Reagents and conditions: (a) (1) DMP, DCM, (2) Br2, DEE, dioxane; (b)
(1) methyl-2-aminopyridine-4-carboxylate, EtOH, (2) PtO2, K2CO3, H2, MeOH; (c)
(1) LiHMDS, 3-bromo-N-tritylpropan-1-amine, THF, (2) TFA, DCM/MeOH; (d) (1)
2,4-dichlorophenylboronic acid, Pd(PPh3)4, K2CO3, DME, water, (2) chiral separa-
tion; (e) HCl (6 M).

2264
M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
Table 2
SAR around the imidazole templates
a
b
c
e
Compound
Template
R
TAFIa IC50 (nM)
CLA IC50
(lM)
LogD7.4
Caco2 (Papp
)
Fd (%)
pKa
5
6
7
8
9
10
11
12
13
A
A
A
A
A
A
B
B
B
Propyl
3-Methylbutyl
Cyclohexyl
76
1.0
<0
<0
<0
1.0
3.6
<0.1
0.2
0.2
0.1
<0.1
2,f 8g
1.4
1.4
8.1
8.2
8.1
7.7
8.0
6.5
8.7
7.9
7.8
<41
117
106
124
585
2270
66
0.10
0.08
2.5
16
7.1
>100
1.3
3.2
Biphenyl-3-ylmethyl
2-[1,10-Bi(cyclohexyl)-4-yl]ethylh
Phenylh
1.7
Ethylh
Phenyl
<0
2.4
0.3
0.8
4.9
0.9
20,40-Dichlorobiphenyl-3-yl
<41
a
b
c
The assay was performed using PefakitÒ TAFI from Pentapharm.20
Clot lysis assay in human plasma.17
Measured at pH 6.5.
F = oral bioavailability in the rat.
pKa for the imidazole was calculated using ACD/Labs v12.01.
d
e
f
Dose 20
lmol/kg.
g
h
Dose 6.8
lmol/kg.
Racemic mixture.
Figure 2. X-ray structure and key interactions of compound 5 in complex with a
H333Y/H335Q double mutant TAFI. Zinc atom in magenta and hydrogen bonds in
dotted yellow lines.
Figure 3. Overlay of the X-ray structures of compound 5 in complex with a H333Y/
H335Q double mutant TAFI (green bonds) and compound 2 in complex with
carboxypeptidase B (2jew, yellow bonds). Zinc atom in light blue.
of the two compounds are virtually identical and only C-2 in the
flexible and solvent exposed propyl side-chain has changed
position. All polar interactions are comparable and the only differ-
ence between compounds 2 and 5, the 2-carbon bridge, does not
have a close contact with the enzyme. The shortest distance be-
tween the 2-carbon bridge and Leu340 in TAFI is 3.7 Å indicating
that the conformational restriction is responsible for the majority
of the potency increase.
Compounds 7 and 12 were selected for further studies. DMPK
studies show that both compounds are characterized by low plas-
ma clearance in the rat (compound 7, CL = 6.2 mL/min/kg and com-
pound 12, CL = 12.0 mL/min/kg). Compound 7 also displayed a low
clearance in dog (CL = 3.5 mL/min/kg). Oral bioavailability in rat
was low for both compounds indicating that size may be too large
for absorption via the paracellular route. To investigate the general
selectivity a set of 98 enzymes and receptors was screened at MDS
Pharma showing that both compounds 7 and 12 are very selective
with only one hit, PDE4, with 79% inhibition at 10 lM.
In conclusion, we have described a conformational restriction
approach which resulted in a set of highly potent and selective
TAFIa inhibitors. The identified series consist of 4,5,6,7-tetrahy-
dro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydro-
imidazo[1,2-a]pyridine-7-carboxylic acid derivatives. The data
indicate that the novel TAFIa inhibitors described in this article
has potential both as pre-clinical tools for further understanding
of the role for TAFIa in disease as well as for the prevention and
treatment of thrombosis and related fibrin-dependent disorders
in human.

M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
2265
and AcOH (5 mL). The mixture was hydrogenated over night at rt
and 8 bar. The catalyst was filtered off through a pad of Celite
and the solvents were evaporated. The residue was dissolved in
EtOAc and washed with NaHCO3 (3 times) and brine. The organic
layer was dried over Na2SO4, filtered and evaporated leaving the
crude product (2.37 g, 100%) as a greyish solid. The product was
taken to the next step without further purification. Rf = 0.4
(petroleum ether/EtOAc, 1:1).
Step 4: p-TsOH (0.19 g, 0.1 equiv) was added to a stirred solu-
tion of the product from step 3 (2.37 g, 10.0 mmol) in trimethyl
orthoformate (15 mL) in a microwave approved vessel at room
temperature. The mixture was heated for 10 min at 155 °C in a Bio-
tage microwave reactor. The solvents were evaporated and the
residue was dissolved in DCM and NaHCO3 (satd). The phases were
separated and the organic layer was washed with brine. The organ-
ic layer was dried over Na2SO4, filtered and evaporated. Further
purification was performed by adding the compound onto an
SCX-2 column and washing with MeOH and eluating with
NH3/MeOH. The solvents were evaporated leaving the crude prod-
uct as a colourless oil (1.99 g, 80%). Rf = 0.4, (CHCl3/MeOH, 9:1).
Optional: The crude product can be further purified by column
chromatography over basic alumina using 1% MeOH in DCM as
eluent.
Step 5: Pd/C (0.5 g) followed by Pd(OAc)2 (0.5 g) was added to a
solution of the product from step 4 (1.99 g, 8.1 mmol) in glacial
acetic acid (30 mL) in a steel-autoclave at room temperature. The
reaction mixture was hydrogenated at 20 bar and 115 °C for
1–2 days. The reaction mixture was filtered through a pad of Celite,
washed with acetic acid and the combined filtrate was evaporated.
The residue was dissolved in DCM and washed with NaHCO3 (satd).
Finally, the organic layer was dried over Na2SO4, filtered and con-
centrated. The residue was further purified on an SCX-2 column by
washing with MeOH and eluating with NH3/MeOH. The NH3/MeOH
was evaporated leaving the title compound as a colourless oil
(1.4 g, 69%). Rf = 0.2, (CHCl3/MeOH, 9:1). 1H NMR (400 MHz, CDCl3)
d 0.94 (d, 6H), 1.55–1.64 (m, 3H), 1.78–2 (m, 2H), 2.17–2.29 (m,
1H), 2.47–2.97 (m, 5H), 3.71 (s, 3H), 3.74–3.81 (m, 2H), 7.33 (s,
1H). MS (ESI+) [C14H22N2O2+H+]: 251.1
4. Experimental
4.1. General
All solvent and reagents used were of reagent grade purchased
from commercial resources.
Purity and characterization of compounds were established by li-
quid chromatography–mass spectroscopy (LC–MS) and NMR
analytical techniques (see method details below). Unless stated
otherwise, the purities were above 95% for all reported compounds.
High resolution MS and purity was detected on a Waters LCTp
ToF MS using electrospray ionization (ESI-MS). The LC inlet con-
sisted of a Waters Acquity UPLC system, and the separation was
performed on a Waters C18 XBridge at 45–50 °C. The separation
was obtained with a 2–95% MeCN gradient over 3 min at pH 10
(40 mM NH3 and 5 mM H2CO3). A measure of related impurities
was assessed at 210 nM.
1H NMR were recorded on a Bruker Avance DPX400 (400 MHz),
AV500 (500 MHz) or AV600 (600 MHz) and were determined in
CHCl3-d, DMSO-d6 and MeOH-d4 with trimethylsilane (TMS)
(0.00 ppm) or solvent peaks as the internal reference. Chemical
shifts are reported in ppm relative to solvent signal at 7.26 ppm
(CHCl3), 2.50 ppm (DMSO) and 3.30 ppm (MeOH) and coupling
constant (J) values are reported in Hertz (Hz). Splitting patterns
are indicated as follows: s, singlet; d, doublet; t, triplet; m, multi-
plet; br, broad peak.
Chiral separation of the lactam intermediates were performed
on Waters preparative HPLC system fitted with either Chiralpak
AD, Chiralpak AS or Chiracel OD. The enantiomeric excess was
determined using the same setup and detected by PDA at
240 nm. Generally, the first eluting compound is the desired enan-
tiomer. The desired enantiomers regularly show a negative optical
rotation in the range ꢁ20 to ꢁ50° (as lactams).
The final compounds were purified by RP-HPLC on a Waters
Fraction Lynx system equipped with a ZQ MS detector. The col-
umns used were Waters Xbridge C18 OBD 5 lm (pH 10, gradient
5–95% MeCN + 0.2% NH3) or Waters SunFire C18 OBD 5
3, gradient 5–95% MeCN + 0.1 M formic acid).
lm (pH
4.2.2. Synthesis of 3-bromo-N-tritylpropan-1-amine
4.2. General procedure for substituted 4,5,6,7-tetrahydro-1H-
A solution of tritylchloride (5.90 g, 21.2 mmol) in DCM (50 mL)
was added dropwise to a solution of 3-bromopropan-1-amine
(5.12 g, 23.4 mmol) and triethylamine (5.92 g, 58.5 mmol) in
DCM (25 mL). The reaction mixture was stirred at ambient temper-
ature for 70 h before water was added and the two phases were
separated. The aqueous phase was extracted with DCM and the
combined organic extract was washed with water, dried over
MgSO4 and evaporated. The residue was crystallised from heptane
to yield the title compound (4.92 g, 55%) as a colorless crystalline
material. 1H NMR (500 MHz, CDCl3) d 1.53 (s, 1H), 2.05 (p,
J = 6.5 Hz, 2H), 2.29 (t, J = 6.5 Hz, 2H), 3.59 (t, J = 6.5 Hz, 2H), 7.21
(t, J = 6.6 Hz, 3H), 7.30 (t, J = 6.6 Hz, 6H), 7.50 (d, J = 6.6 Hz, 6H).
benzimidazoles
4.2.1. Synthesis of methyl-1-(3-methylbutyl)-4,5,6,7-
tetrahydro-1H-benzimidazole-5-carboxylate
Step 1: 4-fluoro-3-nitrobenzoic acid (15 g, 81.0 mmol) and
H2SO4 (0.43 mL, 8.10 mmol) were dissolved in MeOH (200 mL)
and the mixture was refluxed over night. The solvent was evapo-
rated leaving a white solid. This solid was dissolved in DCM and
K2CO3 (1 M in water). The phases were separated and the aqueous
layer was extracted three times with DCM. The combined organic
layer was dried through a phase-separator and finally evaporated
leaving the product as a white solid (16 g, 99%). The product was
taken to the next step without further purification. Rf = 0.8 (CHCl3/
MeOH, 9:1).
Step 2: To a stirred solution of the product from step 1 (2 g, 10.0
mmol, 1 equiv) in dry methanol (40 mL) was added isoamylamine
(1.75 mL, 15.0 mmol, 1.5 equiv) at room temperature. The mixture
was refluxed over night. The reaction mixture was concentrated
and the residue was dissolved in EtOAc and washed with NaHCO3
(satd) and brine. Finally the organic layer was dried over anhydrous
Na2SO4, filtered and concentrated to leave the crude product (2.7 g,
100%) as an orange solid. The product was taken to the next step
without further purification. Rf = 0.5 (petroleum ether/EtOAc, 6:4).
Step 3: Pd/C (0.1 equiv) was added to a stirred solution of the
product from step 2 (2.7 g, 10.0 mmol) in dry methanol (10 mL)
4.2.3. Synthesis of (5R)-1-(3-methylbutyl)-1,4,6,7-tetrahydro-
20H-spiro[benzimidazole-5,30-piperidin]-20-one
Step 1: To a stirred solution of methyl-1-(3-methylbutyl)-4,5,6,7-
tetrahydro-1H-benzimidazole-5-carboxylate (200 mg, 0.80 mmol)
and 3-bromo-N-tritylpropan-1-amine (365 mg, 0.96 mmol) in THF
(5 mL) was LiHMDS (1.20 mL, 1.20 mmol, 1 M in THF) added in
one portion at room temperature. The reaction mixture was stirred
at room temperature for 30 min. The reaction was quenched with
NH4Cl (20%) and extracted into DCM (repeated twice). The pooled
organic layer was dried through a phase-separator and evaporated
leaving an orange oil. The residue was dissolved in DCM and added
to a silica plug (5 g). The column was washed once with DCM (i.e.

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M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
one column volume). The product was then eluted with MeOH/
EtOAc (1:10) and evaporated leaving the product as a pale yellow oil.
Step 2: The trityl protecting group was removed by dissolving
the residue in DCM and adding TFA (2 mL), followed by addition
of MeOH after 15 min. After additionally 5 min the solvents were
removed by evaporation. The residue was dissolved in DCM and
added onto an SCX-2 cation exchange column. The column was
washed with DCM followed by MeOH and the product was then
eluated with NH3/MeOH (lactam formation often occurs spontane-
ously but can be induced by treatment with NH3/MeOH or Et3N
and heating). The NH3/MeOH layer was evaporated leaving the
racemate of the title compound (144 mg, 58% yield over 3 steps).
Step 3: The enantiomers were separated on preparative HPLC fitted
Step 3: Two vessels of bromo(3-bromophenyl)acetaldehyde
(2.55 g, 9.17 mmol) and methyl 2-aminopyridine-4-carboxylate
(1.47 g, 9.66 mmol) in EtOH (15 mL) were heated to 120 °C in a sin-
gle node microwave oven for 0.5 h. The two reaction mixtures were
pooled and DCM and an aqueous solution of NaHCO3 (satd) were
added. The two phases were separated and the aqueous phase
was extracted with DCM. The combined organic extract was
washed with water, dried over MgSO4 and evaporated. The residue
was crystallized from EtOH to yield methyl 3-(3-bromophenyl)imi-
dazo[1,2-a]pyridine-7-carboxylate (5.10 g, 84%) as colorless nee-
dles. 1H NMR (500 MHz, CDCl3) d 3.98 (s, 3H), 7.40–7.47 (m, 2H),
7.50–7.54 (m, 1H), 7.57–7.62 (m, 1H), 7.72–7.75 (m, 1H), 7.87 (s,
1H), 8.31–8.34 (m, 1H), 8.41–8.44 (m, 1H).
with Chiralpak AD (250 ꢂ 20 mm, 5
l
m); Mobile phase EtOH/Et3N,
Step 4: Methyl 3-(3-bromophenyl)imidazo[1,2-a]pyridine-7-
carboxylate (4.50 g, 13.6 mmol) was dissolved in MeOH (100 mL)
and K2CO3 (5.63 g, 40.7 mmol) and PtO2 (0.50 g, 2.2 mmol) were
added. The reaction mixture was stirred for 20 h at ambient tem-
perature under hydrogen atmosphere (8 bar). The solvent was dec-
anted and H2SO4 (2 mL, concd) was added and the mixture was
refluxed for 3 h to convert the acid into its methyl ester (acid
formed to some extent during the reduction). The reaction mixture
was allowed to reach ambient temperature before it was poured
into an ice-cold aqueous solution of NaHCO3 (satd). DCM was
added and the two phases were separated. The aqueous phase
was extracted with DCM and the combined organic extract was
washed with water, dried over MgSO4 and evaporated. The residue
was purified by column chromatography (silica gel, gradient of
MeOH in DCM as eluent) to yield the title compound (3.15 g,
69%) as a colorless solid. 1H NMR (300 MHz, CDCl3) d 2.05–2.20
(m, 1H), 2.31–2.42 (m, 1H), 2.88–3.03 (m, 1H), 3.08-3.20 (m, 1H),
3.25–3.35 (m, 1H), 3.77 (s, 3H), 3.86–3.97 (m, 1H), 4.02–4.11 (m,
1H), 7.06–7.10 (m, 1H), 7.25–7.31 (m, 2H), 7.44–7.50 (m, 1H),
7.51–7.54 (m, 1H).
100:0.1; Flow 10 mL/min; UV 240 nm; Temperature 20 °C. Optical
rotation: ꢁ28° (of the desired (R)-enantiomer with 99.9% ee). 1H
NMR (400 MHz, CDCl3) d 0.82–0.92 (m, 6H), 1.43–1.59 (m, 4H),
1.61–1.84 (m, 3H), 2.19–2.32 (m, 1H), 2.32–2.45 (m, 1H), 2.47–
2.6 (m, 2H), 3.02 (d, J = 15.8 Hz, 1H), 3.18–3.29 (m, 1H), 3.36 (s,
2H), 3.67–3.78 (m, 2H), 6.62 (s, 1H), 7.28 (s, 1H). MS (ESI+) [C16H25-
N3O+H+]: 276.1
4.2.4. Synthesis of (5R)-5-(3-aminopropyl)-1-(3-methylbutyl)-
4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid
(compound 6)
A
solution of (5R)-1-(3-methylbutyl)-1,4,6,7-tetrahydro-20H-
spiro[benzimidazole-5,30-piperidin]-20-one (72 mg, 0.26 mmol) in
aqueous HCl (0.5–1 mL, 6 M) was heated at 100 °C for 3 h. The sol-
vents were evaporated leaving the title compound as the HCl salt
(86 mg, 100% yield). 1H NMR (600 MHz, CD3OD) d 0.94–1.03 (m,
6H), 1.55–2 (m, 9H), 2.3–2.41 (m, 1H), 2.62 (d, J = 16.1 Hz, 1H),
2.64–2.72 (m, 1H), 2.77–2.86 (m, 1H), 2.91–3 (m, 2H), 3.21 (d,
1H), 3.27–3.31 (m, 1H), 4.05–4.22 (m, 2H), 8.82 (s, 1H). HRMS
(ESI+) m/z calcd for [C16H27N3O2+H+]: 294.2182, found 294.2171.
4.3.2. Synthesis of 3-(3-bromophenyl)-5,6-dihydro-20H,8H-
spiro[imidazo[1,2-a]pyridine-7,30-piperidin]-20-one
4.3. General procedure for substituted 5,6,7,8-tetrahydro[1,2-
a]pyridines
Step 1: LiHMDS (1.64 mL, 1.64 mmol, 1 M in hexanes) was added
to a solution of methyl 3-(3-bromophenyl)-5,6,7,8-tetrahydroimi-
dazo[1,2-a]pyridine-7-carboxylate (500 mg, 1.49 mmol) in THF
(8 mL) immediately followed by the addition of a solution of 3-bro-
mo-N-tritylpropan-1-amine (851 mg, 2.24 mmol) in THF (2 mL).
The reaction mixture was stirred at ambient temperature for 0.5 h
before an aqueous solution of NH4Cl (5%) and DCM were added.
The two phases were separated and the aqueous phase was
extracted with DCM. The organic extracts were combined and
washed with water, dried over MgSO4 and evaporated. The residue
was purified by column chromatography (silica gel, EtOAc/hep-
tane/Et3N, 15:5:1 as eluent) to yield methyl 3-(3-bromophenyl)-7-
[3-(tritylamino)propyl]-5,6,7,8-tetrahydroimidazo-[1,2-a]pyridine-
7-carboxylate (907 mg, 96%) as a colorless solid. 1H NMR (300 MHz,
CDCl3) d 1.36–1.59 (m, 3H), 1.61–1.80 (m, 2H), 1.88–2.02 (m, 1H),
2.08–2.17 (m, 2H), 2.31–2.41 (m, 1H), 2.82 (d, J = 17 Hz, 1H), 3.49
(d, J = 17 Hz, 1H), 3.70 (s, 3H), 3.90–3.96 (m, 2H), 7.07 (s, 1H),
7.14–7.22 (m, 3H), 7.24–7.31 (m, 8H), 7.42–7.48 (m, 7H), 7.51
(s, 1H).
Step 2: Trifluoroacetic acid (6 mL) was added to a solution of
methyl 3-(3-bromophenyl)-7-[3-(tritylamino)propyl]-5,6,7,8-tetra-
hydro-imidazo[1,2-a]pyridine-7-carboxylate (907 mg, 1.43 mmol)
in DCM (10 mL). The strongly yellow reaction mixture was stirred
at ambient temperature for 5 min before MeOH (5 mL) was added
and the resulting colorless mixture was stirred for 15 min before it
was concentrated in vacuo. MeOH (10 mL) was added and the solu-
tion was evaporated again (this procedure was repeated one more
time). The residue was dissolved in DCM and was eluted through an
SCX-2 column, first with DCM to remove by-products, then with
NH3/MeOH to eluate the product. The MeOH solution was evaporated
4.3.1. Synthesis of methyl 3-(3-bromophenyl)-5,6,7,8-
tetrahydroimidazo[1,2-a]pyridine-7-carboxylate
Step 1: 2-(3-Bromophenyl)ethanol (4.00 g, 19.9 mmol) was dis-
solved in DCM (30 mL) and Dess–Martin periodinane (9.28 g,
21.9 mmol) was added in portions. The reaction mixture was stirred
at ambient temperature for 2 h before an aqueous solution of
NaHCO3 (3.3 equiv) and Na2S2O3 (6 equiv) in water (30 mL) was
added and the resulting solution was stirred for five minutes before
the two phases were separated. The aqueous phase was extracted
with DCM. The organic extracts were combined and washed with
water, dried over MgSO4 and evaporated to yield (3-bromophe-
nyl)acetaldehyde (3.81 g, 96%) as
a
colorless oil. 1H NMR
(500 MHz, CDCl3) d 3.68 (d, J = 2.1 Hz, 2H), 7.13–7.17 (m, 1H),
7.22–7.25 (m, 1H), 7.37–7.40 (m, 1H), 7.43–7.47 (m, 1H), 9.75 (t,
J = 2.1 Hz, 1H).
Step 2: (3-Bromophenyl)acetaldehyde (3.80 g, 19.1 mmol) was
dissolved in diethyl ether (50 mL) and three drops of dioxane and
three drops of bromine were added. The reaction mixture was stir-
red at ambient temperature until the bromine color had disap-
peared (a few minutes) and then bromine (3.05 g, 19.1 mmol)
was added dropwise during 0.5 h. An aqueous solution of NaHCO3
(satd) was added and the two phases were separated. The aqueous
phase was extracted with diethyl ether and the combined organic
extract was washed with water, dried over MgSO4 and evaporated
to yield bromo(3-bromophenyl) acetaldehyde (5.06 g, 95%) as an
oil. 1H NMR (500 MHz, CDCl3) d 5.20 (d, J = 3.1 Hz, 1H), 7.27–7.31
(m, 1H), 7.34–7.37 (m, 1H), 7.51–7.54 (m, 1H), 7.57–7.60 (m,
1H), 9.54 (d, J = 3.1 Hz, 1H).

M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
2267
to yield the title compound (501 mg, 97%) as a colorless oil. 1H NMR
(300 MHz, CD3OD) d 1.76–2.11 (m, 5H), 2.38–2.54 (m, 1H), 2.82–
2.90 (m, 1H), 3.23–3.37 (m, 3H), 3.98–4.16 (m, 2H), 7.20 (s, 1H),
7.34–7.43 (m, 1H), 7.45–7.50 (m, 1H), 7.54–7.59 (m, 1H), 7.66-7.69
(m, 1H).
(m, 2H), 2.79–2.88 (m, 1H), 2.89–2.99 (m, 2H), 3.18–3.26 (m,
1H), 3.70 (s, 2H), 4.06–4.2 (m, 1H), 8.85 (s, 1H).
HRMS (ESI+) m/z calcd for [C17H27N3O2+H+]: 306.2181, found
306.2172.
4.4.3. 5-(3-Aminopropyl)-1-(biphenyl-3-ylmethyl)-4,5,6,7-
tetrahydro-1H-benzimidazole-5-carboxylic acid (compound 8)
1H NMR (400 MHz, CD3OD) d 1.55–1.96 (m, 5H), 2.31 (d,
J = 13.0 Hz, 1H), 2.44–2.78 (m, 3H), 2.86–2.98 (m, 2H), 3.16–3.36
(m, 3H), 5.45 (q, J = 15.3, 15.3, 15.3 Hz, 2H), 7.25 (d, J = 7.6 Hz,
1H), 7.31–7.39 (m, 1H), 7.4–7.54 (m, 3H), 7.55–7.68 (m, 4H), 8.91
(s, 1H).
4.3.3. Synthesis of (7R)-3-(20,40-dichlorobiphenyl-3-yl)-5,6-
dihydro-20H,8H-spiro[imidazo[1,2-a]pyridine-7,30-piperidin]-20-
one
Step 1: Tetrakis(triphenylphosphine)palladium(0) (50 mg,
0.043 mmol) was added to a solution of 3-(3-bromophenyl)-5,6-
dihydro-20H,8H-spiro[imidazo[1,2-a]pyridine-7,30-piperidin]-20-
one (225 mg, 0.62 mmol) in DME (5 mL). The mixture was stirred
for five minutes before 2,4-dichlorophenylboronic acid (131 mg,
0.69 mmol) and an aqueous solution of K2CO3 (2 mL, 1 M) were
added. The reaction mixture was refluxed for 2 h before water
and DCM were added and the two phases were separated. The
aqueous phase was extracted with DCM and the organic extracts
were combined and washed with water, dried over MgSO4 and
evaporated. The residue was purified by column chromatography
(silica gel, DCM/MeOH, 10:1 as eluent) to yield a product fraction
that was purified further on an SCX-2 column (product eluted with
10% NH3/MeOH in DCM). This yielded the racemate of the title
compound (159 mg, 60%) as a colorless solid.
HRMS (ESI+) m/z calcd for [C24H27N3O2+H+]: 390.2181, found
390.2194.
4.4.4. 5-(3-Aminopropyl)-1-{2-[1,10-bi(cyclohexyl)-4-yl]ethyl}-
4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid
(compound 9)
1H NMR (600 MHz, CD3OD) d 0.82–2.13 (m, 25H), 2.24–2.42 (m,
2H), 2.59 (d, J = 15.8 Hz, 1H), 2.63–2.85 (m, 3H), 2.87–3.01 (m, 3H),
3.52–3.62 (m, 1H), 4.06–4.16 (m, 2H), 4.17–4.2 (m, 1H), 7.4–7.52
(m, 2H), 8.74–8.81 (m, 1H).
HRMS (ESI+) m/z calcd for [C25H41N3O2+H+]: 416.3277, found
416.3290.
Step 2: The enantiomers of racemic 3-(20,40-dichlorobiphenyl-3-
yl)-5,6-dihydro-20H,8H-spiro[imidazo[1,2-a]pyridine-7,30-piperi-
din]-20-one (155 mg) were separated by preparative HPLC: Column
4.4.5. 5-(3-Aminopropyl)-1-phenyl-4,5,6,7-tetrahydro-1H-
benzimidazole-5-carboxylic acid (compound 10)
1H NMR (600 MHz, DMSO-d6) d 1.15–1.78 (m, 5H), 1.89–2.17
(m, 1H), 2.40 (s, 1H), 2.62 (s, 1H), 2.68–2.79 (m, 1H), 2.89–3 (m,
2H), 3.11 (s, 1H), 3.4–3.52 (m, 1H), 3.87 (s, 1H), 7.29–7.61 (m,
5H), 8.09 (s, 1H).
Chiralpak AS (250 ꢂ 20 mm, 5
lm); Mobile phase MeOH/Et3N,
100:0.1; flow 15 mL/min; UV 270 nm; Temperature 20 °C. Optical
rotation: ꢁ48° (of the desired (R)-enantiomer in 99.9% ee). Isolated
titled compound (61 mg, 39%). 1H NMR (300 MHz, CDCl3) d 1.70–
1.98 (m, 5H), 2.50–2.60 (m, 1H), 2.96–3.04 (m, 1H), 3.30–3.44
(m, 3H), 3.89–4.00 (m, 1H), 4.12–4.21 (m, 1H), 6.67 (br s, 1H),
7.11 (s, 1H), 7.26–7.35 (m, 2H), 7.35–7.38 (m, 1H), 7.40–7.53 (m,
4H).
HRMS (ESI+) m/z calcd for [C17H21N3O2+H+]: 300.1634, found
300.1651.
4.4.6. 7-(3-Aminopropyl)-3-ethyl-5,6,7,8-tetrahydro
imidazo[1,2-a]pyridine-7-carboxylic acid (compound 11)
1H NMR (600 MHz, DMSO-d6) d 1.08–1.24 (m, 2H), 1.43–1.54
(m, 1H), 1.56–1.68 (m, 1H), 1.72–1.81 (m, 1H), 2.05–2.12 (m,
1H), 2.2–2.29 (m, 1H), 2.7–2.78 (m, 4H), 2.83–2.91 (m, 3H), 3.72–
3.83 (m, 2H), 4.12–4.19 (m, 1H), 4.3–4.6 (m, 1H), 7.32 (s, 1H),
8.06 (s, 2H).
4.3.4. Synthesis of (7R)-7-(2-aminopropyl)-3-(20,40-dichloro-
biphenyl-3-yl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-
carboxylic acid hydrochloride (Compound 13)
(7R)-3-(20,40-Dichlorobiphenyl-3-yl)-5,6-dihydro-20H,8H-spiro-
[imidazo[1,2-a]pyridine-7,30-piperidin]-20-one (61 mg, 0.14 mmol)
was dissolved in HCl (2 mL, 6 M) and the reaction mixture was
heated to 70 °C for 20 h before it was evaporated to yield the title
compound (69 mg, 100%) as a solid. 1H NMR (300 MHz, CD3OD) d
1.64–1.92 (m, 3H), 1.95–2.05 (m, 1H), 2.18–2.32 (m, 1H), 2.42–
2.53 (m, 1H), 2.95–3.02 (m, 2H), 3.07 (d, J = 17.4 Hz, 1H), 3.63 (d,
J = 17.4 Hz, 1H), 3.98–4.13 (m, 1H), 4.26–4.38 (m, 1H), 7.41–7.70
(m, 8H). HRMS (ESI+) m/z calcd for [C23H23Cl2N3O2+H+]:
444.1245, found 444.1247.
4.4.7. 7-(3-Aminopropyl)-3-phenyl-5,6,7,8-tetrahydro
imidazo[1,2-a]pyridine-7-carboxylic acid (compound 12)
1H NMR (600 MHz, CD3OD) d 1.64–1.77 (m, 1H), 1.78–1.92 (m,
2H), 1.95–2.06 (m, 1H), 2.19–2.31 (m, 1H), 2.39–2.49 (m, 1H),
2.93–3.03 (m, 2H), 3.04–3.15 (m, 1H), 3.29 (s, 2H), 3.52–3.76 (m,
1H), 3.91–4.07 (m, 1H), 4.19–4.33 (m, 1H), 7.51–7.61 (m, 5H).
HRMS (ESI+) m/z calcd for [C17H21N3O2+H+]: 300.1712, found
300.1731.
4.4. Final compounds
4.5. Biological methods
4.4.1. 5-(3-Aminopropyl)-1-propyl-4,5,6,7-tetrahydro-1H-
benzimidazole-5-carboxylic acid (compound 5)
1H NMR (400 MHz, CDCl3) d 0.89 (t, J = 7.4, 7.4 Hz, 3H), 1.45–
1.62 (m, 1H), 1.63–1.92 (m, 6H), 2.07 (s, 1H), 2.23–2.5 (m, 2H),
2.5–2.68 (m, 2H), 3.08 (d, J = 15.7 Hz, 1H), 3.22–3.37 (m, 2H),
3.65–3.77 (m, 2H), 5.88 (s, 1H), 7.33 (d, J = 15.5 Hz, 1H).
HRMS (ESIꢁ) m/z calcd for [C14H23N3O2ꢁH+]: 264.1712, found
264.1748.
4.5.1. LogD assay
A: 10 mM ammonium acetate in MeCN/H2O, 95:5.
B: 10 mM ammonium acetate in MeCN/H2O, 5:95.
Mobile phase B is adjusted to pH 7.4 with NH3 and an equal
amount NH3 is added to mobile phase A.
Gradient: 99.9% B for 0.17 min., linear gradient from 99.9% B to
99.9% A between 0.17 and 1.5 min., 99.9% A for 0.26 min. Return to
initial conditions and make sure the column is equilibrated before
next injection. Flow rate: 1.0 mL/min for a 2.1 mm id column. For
other dimensions, the flow rate was scaled appropriately.
4.4.2. 5-(3-Aminopropyl)-1-cyclohexyl-4,5,6,7-tetrahydro-1H-
benzimidazole-5-carboxylic acid (compound 7)
Column: Waters ACQUITY UPLCÒ BEH C18 1.7
or column with similar selectivity.
lm, 50 ꢂ 2.1 mm
1H NMR (600 MHz, CD3OD) d 1.24–1.39 (m, 1H), 1.43–2.03 (m,
11H), 2.11 (dd, J = 37.4, 11.9 Hz, 2H), 2.32–2.4 (m, 1H), 2.55–2.77
Sample preparation: 100 lL 0.5mM in DMSO.

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M. Brink et al. / Bioorg. Med. Chem. 22 (2014) 2261–2268
4.5.2. Caco-2 cell monolayer permeability experiments
References and notes
The Caco-2 cell monolayers are washed once with HBSS prior to
start. TEER is measured both before and after performing all the
transport experiments. Experiments are made in apical A to baso-
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3. Campbell, W.; Okada, H. Biochem. Biophys. Res. Commun. 1989, 162, 933.
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1991, 266, 21833.
5. Plasma carboxypeptidase B should not be confused with the related enzyme
pancreatic carboxypeptidase B.
6. Bouma, B. N.; Marx, P. F.; Mosnier, L. O.; Meijers, J. C. M. Thromb. Res. 2001, 101,
329.
lateral B direction. Transport buffer, 800
dispensed to the basal side of the monolayer. The assay is then ini-
tiated when 200 L of each compound is added to the apical side at
10 M (all test compounds are diluted in HBSS, pH 6.5, with 1%
DMSO as co-solvent). Samples are withdrawn before and at 45
and 120 min post addition of test compound. 2 L and 200 L are
lL, (HBSS, pH 7.4) is first
l
l
l
l
7. Decleck, P. J. Hamostaseologie 2011, 3, 165.
withdrawn from the apical donor compartment and the basolateral
receiver compartment, respectively. During incubation the trans-
well plates are placed in a shaking incubator at 480 rpm and
37 °C between the sampling up to 120 min. All samples are ana-
lysed subsequently (never stored or frozen).
8. Nagashima, M.; Yin, Z.-F.; Zhao, L.; White, K.; Zhu, Y.; Lasky, N.; Halks-Miller,
M., ; Broze, G. J., Jr.; Fay, W. P.; Morser, J. J. Clin. Invest. 2002, 109, 101.
9. WO 00/66550, WO 00/66557, WO 03/013526, WO 03/027128, WO 01/19836,
WO 02/14285, WO 03/061652 and WO 03/061653.
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2009, 7, 1962.
12. Eriksson, H.; Jensen, E.; Sandset, P. M.; Wall, U.; Andersson, M.; Nerme, V.;
Eriksson-Lepkowska, M.; Wåhlander, K. J. Thromb. Haemostasis 2007, 2, P.
13. Polla, M. O.; Tottie, L.; Norden, C.; Linschoten, M.; Musil, D.; Trumpp-
Kallmeyer, S.; Aukrust, I. R.; Ringom, R.; Holm, K. H.; Neset, S. M.; Sandberg,
M.; Thurmond, J.; Yu, P.; Hategan, G.; Anderson, H. Bioorg. Med. Chem. 2004, 12,
1151.
14. Bunnage, M. E.; Blagg, J.; Steele, J.; Owen, D. R.; Allerton, C.; McElroy, A. B.;
Miller, D.; Ringer, T.; Butcher, K.; Beaumont, K.; Evans, K.; Gray, A. J.; Holland, S.
J.; Feeder, N.; Moore, R. S.; Brown, D. G. J. Med. Chem. 2007, 50, 6095.
15. An HPLC based method was used to compare the thiol containing compound 1
with the imidazole derivatives instead of using PefakitÒ TAFI from Pentapharm.
PefakitÒ TAFI contains a substrate that is cleaved by TAFIa to form a thiol
derivative. The thiol derivative subsequently reacts with Ellman’s reagent to
produce a coloured substance that is detected at 405 nM. The HPLC based
method was used to ensure that the thiol containing compound 1 would not
interfere with the measurement. The two methods show a good correlation
(R2 = 0.92) for the 25 compounds tested in both assays.
16. (a) Hendriks, D.; Scharpé, S.; van Sande, M. Clin. Chem. 1985, 31, 1936; and (b)
Wang, W.; Hendriks, D.; Scharpé, S. J. Biol. Chem. 1994, 269, 15937.
17. Leurs, J.; Wissing, B.-M.; Nerme, V.; Schatteman, K.; Björquist, P.; Hendriks, D.
Thromb. Haemost. 2003, 89, 264.
18. Nantermet, P. G.; Barrow, J. C.; Lindsley, S. R.; Young, M.-B.; Mao, S.-S.; Carroll,
S.; Bailey, C.; Bosserman, M.; Colussi, D.; McMasters, D. R.; Vacca, J. P.; Selnick,
H. G. J. Med. Chem. 2003, 46, 5294.
4.6. Crystallization and X-ray structure determination
H333Y/H335Q TAFI was concentrated to 10 mg/mL in HEPES
(20 mM) pH 7.4 and NaCl (150 mM). Crystallization was done
using the hanging drop method where 2 mL of protein solution
was mixed with 1 mL of well solution containing 10% (w/v)
PEG20k, dioxane (2%) and bicine (0.1 M) pH 9.0 and allowed to
equilibrate over well solution. Crystals were then soaked in well
solution supplemented with 1 mM compound dissolved in DMSO
(the final DMSO concentration was 1%). Data were collected at
beam line ID23-1, European Synchrotron Radiation Facility, Greno-
ble, France. The structure was solved by molecular replacement
using the structure of porcine CPB as a starting model. The R-factor
of the refined model was 19.2% and the free R-factor was 23.0%.
Acknowledgments
Excellent technical assistance by Anne Legnehed (clot-lysis
assay), Britt-Marie Wissing and Pia Berntsson (CPU and CPN hplc
assays), Johan Brengdahl (Pefakit assay) and Tove Sjögren (crystal-
lography) is gratefully acknowledged.
19. Schneider, M.; Nesheim, M. J. Thromb. Haemostasis 2003, 1, 147.
20. DSM Nutritional Products AG, Branch Pentapharm, PO Box, CH-4002 Basel,
Switzerland.
21. Knecht, W.; Willemse, J.; Stenhamre, H.; Andersson, M.; Berntsson, P.;
Furebring, C.; Harrysson, A.; Malmborg Hager, A.-C.; Wissing, B.-M.;
Hendriks, D.; Cronet, P. FEBS J. 2006, 273, 778.
22. Willemse, J. L.; Polla, M.; Hendriks, D. F. Anal. Biochem. 2006, 356, 157.
23. TAFI 1–401 numbering. During activation the activation peptide (residue 1–92)
is cleaved off generating the active enzyme, TAFIa, containing residue 93–401.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.010.
These data include MOL files and InChiKeys of the most important
compounds described in this article.