Design and characterization of a selenium-containing inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), a zinc-containing metalloprotease

Article abstract of DOI:10.1021/jm300735t

Available therapies for thromboembolic disorders include thrombolytics, anticoagulants, and antiplatelets, but these are associated with complications such as bleeding. To develop an alternative drug which is clinically safe, we focused on activated thrombin-activatable fibrinolysis inhibitor (TAFIa) as the target molecule. TAFIa is a zinc-containing carboxypeptidase that significantly inhibits fibrinolysis. Here we designed and synthesized selenium-containing compounds 5-13 to discover novel TAFIa inhibitors having a superior zinc-coordinating group. Compounds 5-13 significantly inhibited TAFIa activity (IC₅₀ 2.2 × 10^-12 M - 2.6 × 10^-6 M). We found that selenol is a better functional group than thiol for coordinating to zinc at the active site of TAFIa. Furthermore, compound 12, which has an amino-chloro-pyridine ring, was found to be a potent and selective TAFIa inhibitor that lacks carboxypeptidase N inhibitory activity. Therefore, compound 12 is a promising candidate for the treatment of thromboembolic disorders. This is the first report of a selenium-containing inhibitor for TAFIa.

Full text of DOI:10.1021/jm300735t

Article
pubs.acs.org/jmc
Design and Characterization of a Selenium-Containing Inhibitor of
Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa), a Zinc-
Containing Metalloprotease
Nobuko Yoshimoto,†,‡ Tomoyuki Sasaki,†,§ Katsuyoshi Sugimoto,†,§ Hidemi Ishii,†,§
and Keiko Yamamoto*,†,‡
‡
§
†High Technology Research Center, Laboratory of Drug Design and Medicinal Chemistry and Laboratory of Molecular and
Cellular Pathophysiology, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan
ABSTRACT: Available therapies for thromboembolic disor-
ders include thrombolytics, anticoagulants, and antiplatelets,
but these are associated with complications such as bleeding.
To develop an alternative drug which is clinically safe, we
focused on activated thrombin-activatable fibrinolysis inhibitor
(TAFIa) as the target molecule. TAFIa is a zinc-containing
carboxypeptidase that significantly inhibits fibrinolysis. Here
we designed and synthesized selenium-containing compounds
5−13 to discover novel TAFIa inhibitors having a superior zinc-coordinating group. Compounds 5−13 significantly inhibited
TAFIa activity (IC50 2.2 × 10−12 M − 2.6 × 10−6 M). We found that selenol is a better functional group than thiol for
coordinating to zinc at the active site of TAFIa. Furthermore, compound 12, which has an amino-chloro-pyridine ring, was found
to be a potent and selective TAFIa inhibitor that lacks carboxypeptidase N inhibitory activity. Therefore, compound 12 is a
promising candidate for the treatment of thromboembolic disorders. This is the first report of a selenium-containing inhibitor for
TAFIa.
tion with t-PA appear to enhance the efficiency of thrombolysis,
enabling lower dosing with t-PA and consequently fewer
bleeding problems.6,7 Therefore, the development of TAFIa
inhibitors as profibrinolytic agents is an attractive concept.
The research of TAFIa inhibitors is active worldwide, but
none of these inhibitors are currently used as therapeutic
agents. Several groups have reported synthetic small molecules
that act as a TAFIa inhibitor.7−13 All of these have three
characteristic functional groups (Figure 1). Because TAFIa
recognizes the C-terminal basic amino acid on the surface of
fibrin, it is reasonable that the inhibitors would have (i) a basic
group corresponding to the lysine side chain to bind to Asp256
at the bottom of the S1′ pocket, (ii) a carboxylic acid
corresponding to the lysine C-terminal carboxylic acid, and (iii)
a functional group to coordinate to the catalytic Zn (Figure 1).
Indeed, the amine group mimicking the arginine or lysine side
chain has been used as a basic group to bind to Asp256. Various
zinc-coordinating groups have been reported, including
carboxylic acid, thiol, imidazole, phosphonic acid, phosphinic
acid, hydroxamic acid, sulfonamide, and α-hydroxyketone. We
focused on a zinc-coordinating group and aimed to design
novel TAFIa inhibitors having a superior zinc-coordinating
group. Here, we report the design, synthesis, and biological
evaluation of novel TAFIa inhibitors bearing a potential zinc-
coordinating functional group.
INTRODUCTION
■
Thromboembolic disorders are a major cause of morbidity and
mortality in developed countries. Available therapies include
thrombolytics, anticoagulants, and antiplatelets, although these
therapies are associated with complications such as bleeding.
To develop a clinically safe drug, we focused on thrombin-
activatable fibrinolysis inhibitor (TAFI) as the target molecule.
TAFI is an important regulator of fibrinolysis1 and is also
termed plasma procarboxypeptidase B, R, or U.2 TAFI is an
inactive 60 kDa zymogen glycoprotein that is produced by the
liver and is present in plasma.3 In the coagulation cascade,
cleavage by thrombin at Arg92 converts TAFI from its inactive
form to activated TAFI (TAFIa). TAFIa, a labile enzyme with a
half-life of less than 10 min at 37 °C,4 is a zinc-containing basic
carboxypeptidase found in plasma that cleaves the C-terminal
arginine and lysine residues from peptide and protein.
Fibrin is initially degraded by plasmin to produce fibrin
containing newly exposed surface C-terminal lysine and
arginine residues. These basic residues function as binding
sites for tissue plasminogen activator (t-PA) and its substrate,
plasminogen, therefore positioning t-PA and plasminogen in
proximity and increasing plasmin production.5 TAFIa stabilizes
clots by eliminating these binding sites; consequently, TAFIa
significantly inhibits fibrinolysis.
Inhibition of TAFIa should modulate clotting and lysis,
without resorting to direct action on the coagulation cascade. A
TAFIa inhibitor would therefore likely diminish the risk of side
effects compared with other potential mechanisms for treating
thrombotic disease. At present, TAFIa inhibitors in combina-
Received: May 25, 2012
Published: August 14, 2012
© 2012 American Chemical Society
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Carboxypeptidase N (CPN) is a plasma zinc-containing
metalloprotease that cleaves C-terminal arginines and lysines
from peptides found in the bloodstream such as vasoactive
peptide hormones, growth factors, and cytokines.22 By cleaving
only one basic residue, CPN can change peptide activity and
receptor binding. CPN has an important function as an
inactivator of complement anaphylatoxines. From these
functions of CPN, it is important to design inhibitors that are
selective for TAFIa over CPN. Compounds 10−13 were
designed as inhibitors with good selectivity for TAFIa but not
for CPN. CPN has a spatially narrower active site cavity than
TAFIa,23 so CPN would likely have difficulty accommodating
an aromatic ring such as an amino-chloro-pyridine ring. A 2-
aminopyridine ring instead of aniline was selected because 2-
aminopyridine is more basic than aniline.
SYNTHESIS
■
The synthetic scheme for thiol compounds 1−4 is shown in
Scheme 1. 5-Amino-1-pentanol derivative 14 was derived from
commercially available 5-amino-1-pentanol by the reported
procedure.24 Mesylate 14 was converted to alkyl malonate 15
by condensation with diethyl malonate under basic conditions.
One of the two ester moieties of 15 was saponified with
potassium hydroxide in ethanol to provide half ester 16 in
excellent yield. Half ester 16 was subjected to a Mannich
reaction in the presence of formaldehyde and diethylamine to
afford enoate 17. The key intermediate 17 was subjected to
Michael addition: 17 was treated with thioacetic acid in the
presence of triethylamine to afford conjugate addition product
18 in good yield. Thioacetate 18 was refluxed in conc HCl to
provide desired product 1, and 18 treated with trifluoroacetic
acid (TFA) provided 2. Furthermore, treatment of thioacetate
18 with lipase PS gave racemic thiol 19, which was then treated
with TFA to give ester 3. Target compound 4 was obtained by
hydrolysis of ester 17 followed by Michael addition using
thioacetic acid and deprotection of the N-Boc amine.
Selenium was introduced using a method recently developed
by Knapp and Darout:25 They prepared selenocarboxylic acid
from the corresponding carboxylic acid and Woollins’s
reagent26,27 in toluene and used the selenocarboxylic acids in
substitution, addition, and amidation reactions. We treated
enoate 17 with selenopropionic acid 23 prepared in situ from
propionic acid and Woollins’s reagent 22 to give the Michael
addition product, selenoester 24 in 32% yield. Selenoester 24
was refluxed in conc HCl to provide a single compound, 5,
which is a dimer and an oxidized form of selenol produced by
hydrolysis. Treatment of ester 24 with TFA provided N-Boc-
deprotected product 6 (Scheme 2).
Figure 1. Consensus structure of TAFIa inhibitor and its interactions
with TAFIa. Coordinate bonds to the zinc, and intermolecular salt
bridges and hydrogen bonds, are shown as dotted green lines.
DESIGN
■
Although thiol effectively coordinates zinc in the active site of
various zinc metalloproteases, we chose to use the selenol
rather than the thiol group. Previously, Suzuki et al. reported
that (S)-7-amino-2-[[[(R)-2-methyl-1-(3-phenylpropanoylami-
no) propyl]hydroxyphosphinoyl]methyl]heptanoic acid
(EF6265), which contains a phosphinic acid, is a potent
inhibitor of TAFIa.7 EF6265 specifically inhibits TAFIa activity,
with an IC50 value of 8.3 nM, and enhances t-PA-mediated clot
lysis concentration dependently. However, EF6265 is orally
inactive. We hypothesized that oral inactivity is due to the
presence of a phosphinic acid moiety because phosphinic acid is
hydrophilic not hydrophobic. We modified the phosphinic acid
moiety to a common functional group, thiol, and also to an
innovative group, selenol. Figure 2 shows compounds 1−13,
designed using EF6265 as a lead compound. Note that the thiol
and selenol compounds shown in Figure 2 are homologues of
cysteine and selenocysteine, respectively.
Selenium is an essential element for humans and animals. For
instance, selenocysteine is recognized as the 21st naturally
occurring amino acid in proteins, and glutathione peroxidases,
which are selenium-containing enzymes, are well-known for
playing an important role in redox processes.14−16 Various
small organoselenium compounds have been studied as
biological models.17−21 Of these, the most promising
compound is 2-phenyl-1,2-benzisoselenazol-3(2H)-one, ebse-
len, which has been demonstrated to act as a glutathione
peroxidase mimic and as a scavenger of peroxynitrite.19,20 The
sulfur analogue of ebselen shows 15-fold lower activity than
ebselen, indicating that ebselen holds a promise as an
antioxidant drug. As reviewed by Nogueira and Rocha,20b
ebselen was apparently not toxic for humans after short-term
intake, whereas dietary overexposure to selenium compounds
can increase the incidence of chronic degenerative diseases such
as type 2 diabetes, amyothrophic lateral sclerosis, and some
types of cancer. We should note that because potential
therapeutic use of organoselenium compounds has not yet
been sufficiently explored, detailed toxicological studies of
selenium are needed.
Selenoesters 7−9 were prepared by a Michael addition to
alkenoic acid 20 (Scheme 3). Treatment of 20 with
selenopropionic acid 23 afforded selenoester 25 in 44% yield.
Selenoester 25 was treated with TFA to provide N-Boc-
deprotected product 7. Seleno-4-phenylbutyric acid ester 27
was obtained using seleno-4-phenylbutyric acid 26 prepared
from the corresponding carboxylic acid and Woollins’s reagent
22. Seleno-3-phenylpropionic acid ester 29 was also obtained
using a procedure similar to that used for the preparation of 27.
Compounds 27 and 29 were treated with TFA to provide 8 and
9, respectively.
Alkenoic acid ester with an aminopyridine-ring in the side
chain, 30, and its chloro-analogue, 32, were prepared by the
method reported previously.9 Selenylation followed by
deprotection were accomplished using a procedure similar to
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Figure 2. Structures of EF6265 and TAFIa inhibitor candidates.
that described above (Scheme 4). Thus, we synthesized all
target compounds 10−13 in excellent yields.
active proteases available for digestion.11 CPN is a basic
carboxypeptidase circulating in plasma that is critical for
regulating the complement system.22 Inhibition of CPN results
in undesirable side effects. Therefore, it is critical to design
TAFIa selective inhibitors that have no CPN inhibition
activity.11
The assay results for enzyme inhibition by thiocompounds
1−4 are shown in Table 1. Compounds 1−4 inhibited TAFIa
activity in order of 1, 4, 3, and 2. These compounds inhibited
ppCPB activity with potency similar to their inhibition of
TAFIa, and compounds 1, 3, and 4 inhibited CPN activity with
40, 0.4, and 40 times less potency compared to their inhibition
of TAFIa, respectively.
ESI-mass and 13C NMR spectra confirmed that thiol
compounds 1 and 3 exist as monomers whereas selenol
compounds 5, 10, and 12 exist as dimers. The dimer form of
selenol compounds is reasonable because selenol is susceptible
to oxidation in air.
TAFIa INHIBITION ACTIVITY
■
The inhibition of human TAFIa, porcine pancreatic carbox-
ypeptidase B (ppCPB), and human CPN were assayed using a
procedure similar to that reported previously.7 ppCPB was used
instead of human pancreatic CPB, which is a digestive basic
carboxypeptidase. Pancreatic CPB is closely related to TAFIa;
indeed, TAFIa is also known as plasma CPB. Transient
inhibition of pancreatic CPB has been thought to hardly have
side effects. Inhibition of pancreatic CPB alone should not
diminish intestinal absorption due to the presence of alternate
Because diselenide is an oxidized form of the selenol
compound, we tested the TAFIa inhibition activity of selenium-
containing compounds in the presence of 10 μM dithiothreitol
(DTT). DTT is a strong reducing agent because, once oxidized,
it forms a stable six-membered ring with an internal disulfide
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Scheme 1. Synthetic Scheme of Thio-Acyl and Thiol Compounds 1−4
Therefore, compounds 12 and 13 exhibited desirable inhibition
profiles for the enzymes tested.
Scheme 2. Synthetic Scheme of Selenium-Containing
Compounds 5 and 6
DISCUSSION
■
Here we presented the design and synthesis of novel TAFIa
inhibitors containing an original element, “Se”, and provide the
TAFIa inhibition activity of these synthetic compounds.
Synthetic compounds 1−13 showed significant inhibition
activity against TAFIa. Carboxylic acid compounds 1 and 7
showed stronger activity than the corresponding carboxy ester
compounds 3 and 6, indicating that the carboxylic acid group is
superior to the corresponding ester group. This is reasonable
because two Arg residues in TAFIa recognize a carboxylic acid
in the substrate or inhibitor and form a salt bridge. This also
explains the lower activity of ethyl ester compounds 11 and 13.
Comparison of sulfur- and selenium-compounds (1 versus 5,
2 versus 6, 4 versus 7−9) demonstrated the more potent
activity of the selenium-containing compounds, indicating that
“Se” is superior to “S” at coordinating to zinc in enzyme. In
addition, selenoesters 7−9 showed activity comparable to
selenol 5, indicating that selenoesters 7−9 function after
hydrolysis to selenol 5. Indeed, selenoesters 7−9 in stock
solution gradually decomposed into hydrolysis product 5. Quite
recently, we obtained the preliminary result that Se of selenol
compound coordinates to zinc at the active site of CPB by the
X-ray crystal structural analysis (data not shown). After the
more detailed study, we will publish the experimental results.
Selenium is an essential trace element. In the form of
selenocysteine, the 21st amino acid, selenium is incorporated
cotranslationally into selenoproteins. As a homologue of sulfur
in the periodic table of the elements, the chemistry of selenium
has some resemblance to that of sulfur.28 Biology uses the
differences between sulfur and selenium, particularly lower pKa
value of the selenol/selenolate couple compared to that of the
thiol/thiolate couple.28 In the present study, the strong
nucleophilicity of selenol might contribute to strong inhibition
activity. As described in the Design section, detailed
toxicological studies of our selenium compounds are needed
in the next step.
bond. In animal and human cells, glutathione acts as reducing
agent in a similar manner to that of DTT. Prior to the
inhibition test, we verified that 10 μM DTT does not affect
TAFIa activity (data not shown). All selenium-containing
compounds (5−13) showed potent TAFIa inhibition activity,
with IC50 values of 10−12−10−6 M (Table 2). In particular,
diselenide compounds 5, 10, and 12 showed strong activity,
with IC50 values in the range 10−9−10−12 M. In the absence of
DTT, compounds 5, 10, and 12 showed 105 times less potent
activity than in the presence of DTT. Selenoesters 6−9, 11, and
13 showed strong/moderate TAFIa inhibition activity. They
also showed weaker activity in the absence of DTT.
Compounds with a lysine mimic side chain (5−9) inhibited
ppCPB activity with potency similar to their inhibition of
TAFIa, while compounds with an aminopyridine ring in the
side chain, 10−13, showed 3−90 times less potent inhibition of
ppCPB. As described above, it is not disadvantageous if TAFIa
inhibitors also inhibit pancreatic CPB.
All compounds with a lysine mimic side chain (5−9), and
two compounds with an aminopyridine ring in the side chain
(10 and 11), significantly inhibited CPN. Interestingly,
compounds with an amino-chloro-pyridine ring in the side
chain, 12 and 13, showed no inhibition activity against CPN.
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Scheme 3. Synthetic Scheme of Selenium-Containing Compounds 7−9
Compounds with an aminopyridine side chain, 10 and 12,
showed 105 times stronger activity than their esters, 11 and 13,
indicating that “SeH” and “COOH” are desirable functional
groups. In terms of selectivity for TAFIa, compound 12 exhibits
desirable specificity. Similar selectivity has been reported for the
corresponding sulfur compound,9 but the inhibitory activity of
the sulfur compound is more than 105 times weaker than that
of selenium compound 12. Therefore, compound 12 is a
promising candidate as a TAFIa inhibitor that lacks CPN
inhibition activity.
The crystal structures of human and bovine TAFI
demonstrated that the TAFI pro-domain is partially rotated
away from the active site, exposing the catalytic residues to the
solvent.29,30 In addition, the salt-bridge in pro-CPB, located
between the pro-domain and the substrate binding site in the
catalytic cavity, was missing in TAFI, thereby exposing the
active site.29,30 Enghild’s group showed that TAFI itself exhibits
continuous and stable carboxypeptidase activity against
substrates, and they suggested that the activity down-regulates
fibrinolysis in vivo.31 Furthermore, they recently determined
the crystal structure of bovine TAFI complexed with tick
carboxypeptidase inhibitor (TCI) and suggested that the
inhibitors, such as TCI, interact with TAFI in a substrate-like
manner.32 Therefore, inhibitors of TAFI but not of TAFIa
might be effective for fibrinolysis. Small molecules like our
synthetic inhibitors may be effective if they can easily enter to
the active site of zymogen TAFI. In particular, compound 12 is
promising candidate for the treatment of thrombosis.
compound 12, which has an amino-chloro-pyridine ring at the
side chain, is a promising and practical candidate drug for
fibrinolysis because it is a selective TAFIa inhibitor that lacks
CPN inhibitory activity. This is the first report of a selenium-
containing inhibitor for TAFIa which is a zinc-containing
metalloprotease. The present study demonstrated the potential
for developing novel inhibitors for other zinc-containing
metalloproteases.
EXPERIMENTAL SECTION
All reagents were purchased from commercial sources. Silica gel
column chromatography was carried out using 100−210 μm silica gel
(KANTO CHEMICAL silica gel 60 N). Unless otherwise stated,
■
1
NMR spectra were recorded at 300 MHz for H NMR and 75 MHz
for 13C NMR in CDCl3 solution with TMS as an internal standard,
and the chemical shifts are given in δ values. Signals arising from the
trifluoroacetate counteranions were not listed. Low-resolution mass
spectra (LRMS) and high-resolution mass spectra (HRMS) were
recorded on a JEOL AccuTOF LC-plus JMS-T100LP using electro-
spray positive ionization. All air and moisture sensitive reactions were
carried out under argon or nitrogen atmosphere. Purity was
determined by HPLC (CAPCELL PAK UG120, 4.6 mm × 150
mm, 0.2% TFA−methanol/milliQ (1:1−3:7), flow rate 1.0 mL/min),
and was >95% for all compounds whose biological activity was tested.
Diethyl {5-[(tert-Butoxycarbonyl)amino]pentyl}-
propanedioate (15). To a suspension of NaOEt (30.19 g, 0.44
mol) in dry EtOH (300 mL) was added diethyl malonate (67.5 mL,
0.44 mol) at 0 °C, and the mixture was stirred at room temperature for
10 min. A solution of mesylate 14 (24.93 g, 88.70 mmol) in dry EtOH
(120 mL) was added to the mixture, and the mixture was stirred for 17
h at 40 °C. The mixture was evaporated, and the residue was poured
into aq NH4Cl and the mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and evaporated. The
residue was chromatographed on silica gel (400 g, hexane/AcOEt = 7/
3) to give 15 (28.05 g, 91.7%) as a colorless oil. 1H NMR δ 1.27 (6 H,
t, J = 7.1 Hz, COOCH2CH3 × 2), 1.2−1.6 (4 H, m, CH2 × 2), 1.44 (9
CONCLUSIONS
■
We have designed and synthesized a novel type of TAFIa
inhibitor, compounds 5−13, that contain selenium to
coordinate to zinc in the active site of enzymes. In particular,
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Scheme 4. Synthetic Scheme of Selenium-Containing Compounds 10−13
a
Table 1. Enzyme Inhibition Data on Compounds 1−4
Table 2. Enzyme Inhibition Data on Compounds 5−13
IC50 (M)
IC50 (M)
compd
TAFIa
CPB
CPN
compd
TAFIa
CPB
CPN
8.6 × 10−9
EF6265
1; DD2
2; DD3
3; DD4
4; DD5
5.5 × 10−9
3.4 × 10−8
3.1 × 10−4
6.2 × 10−5
3.8 × 10−6
4.9 × 10−9
3.6 × 10−8
1.9 × 10−5
2.6 × 10−4
1.4 × 10−6
6.7 × 10−6
1.4 × 10−6
>3.8 × 10−3
2.5 × 10−5
1.6 × 10−4
5; DD9
2.0 × 10−9
1.1 × 10−9
(>3.8 × 10−3
3.0 × 10−8
)
(1.1 × 10−4
)
)
)
)
)
)
(>3.8 × 10−3
)
6; DD10
7; DD12
8; DD19
9; DD20
3.3 × 10−8
1.6 × 10−7
(1.4 × 10−6
)
)
)
)
)
)
)
(9.1 × 10−4
(1.4 × 10−5
)
)
)
)
)
)
1.1 × 10−9
1.1 × 10−9
(1.9 × 10−6
1.8 × 10−8
(2.0 × 10−6
(7.6 × 10−7
4.1 × 10−9
(1.6 × 10−6
3.2 × 10−9
1.6 × 10−8
H, s, Boc), 1.65 (2 H, m, CH2), 1.89 (2 H, m, CH2), 3.10 (2 H, q, J =
6.5 Hz, CH2NHBoc), 3.30 (1 H, t, J = 7.5 Hz, CH), 4.20 (4 H, q, J =
7.1 Hz, COOCH2CH3 × 2), 4.51 (1 H, bs, NH). 13C NMR δ 14.1 (2
carbons), 26.4, 27.0, 28.4 (3 carbons), 28.6, 29.8, 40.4, 51.9, 61.3 (2
carbons), 79.0, 155.9, 169.5 (2 carbons). LRMS: m/z 368.2 (MNa+).
HRMS calcd for C17H31NNaO6 368.2049, found 368.2082.
7-[(tert-Butoxycarbonyl)amino]-2-(ethoxycarbonyl)-
heptanoic Acid (16). To a solution of alkyl malonate 15 (27.82 g,
80.63 mmol) in EtOH (100 mL) was added a solution of KOH (6.01
g, 107.08 mmol) in EtOH (140 mL), and the mixture was stirred at
room temperature for 13 h. The mixture was concentrated under
reduced pressure and the residue dissolved in water. The solution was
washed with AcOEt, acidified by addition of 1N HCl, and extracted
with AcOEt. The organic layer was washed with water, dried over
MgSO4, and evaporated. The residue was dried in vacuo to afford half
ester 16 (24.82 g, 97.1%) as a colorless oil. 1H NMR (CD3OD) δ 1.26
(3 H, t, J = 7.2 Hz, COOCH2CH3), 1.34 (4 H, m, CH2 × 2), 1.43 (9
H, s, Boc), 1.46 (2 H, m, CH2), 1.85 (2 H, m, CH2), 3.01 (2 H, t, J =
6.8 Hz, CH2NHBoc), 3.34 (1 H, m, CH), 4.18 (2 H, q, J = 7.2 Hz,
COOCH2CH3). 13C NMR (CD3OD) δ 14.5, 27.7, 28.3, 28.9 (3
carbons), 30.0, 30.9, 41.4, 53.2, 62.5, 80.0, 158.7, 171.6, 173.0. LRMS:
(1.5 × 10−6
(6.0 × 10−6
5.3 × 10−9
3.2 × 10−9
(1.4 × 10−6
1.4 × 10−8
(1.7 × 10−6
(2.0 × 10−6
10; DD22 2.2 × 10−12
(2.0 × 10−7
11; DD23 8.9 × 10−7
1.9 × 10−10
2.9 × 10−10
(2.8 × 10−7
(4.2 × 10−6
2.5 × 10−6
1.7 × 10−6
(1.5 × 10−5
(1.8 × 10−4
(>3.8 × 10−3
1.5 × 10−9
)
)
12; DD28 2.5 × 10−11
(3.3 × 10−6
13; DD29 2.6 × 10−6
>3.8 × 10−3
(3.7 × 10−4
1.3 × 10−4
)
(>3.8 × 10−3
)
)
>3.8 × 10−3
(>3.8 × 10−3
)
(>3.8 × 10−3
(>3.8 × 10−3
a
Parentheses indicate IC50 values in the absence of DTT.
m/z 340.2 (MNa+). HRMS calcd for C15H27NNaO6 340.1736, found
340.1746.
Ethyl 7-[(tert-Butoxycarbonyl)amino]-2-methylidenehepta-
noate (17). A solution of half ester 16 (24.03 g, 75.80 mmol),
diethylamine (30 mL), and 36% formaldehyde (30 mL) was stirred at
room temperature for 15 h. The mixture was poured into H2O and
extracted with AcOEt. The organic layer was dried over MgSO4 and
evaporated. The residue was chromatographed on silica gel (120 g,
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hexane/AcOEt = 9/1) to give 17 (16.00 g, 74.1%) as a colorless oil.
1H NMR δ 1.30 (3 H, t, J = 7.1 Hz, COOCH2CH3), 1.31 (4 H, m,
LRMS: m/z 220.2 (MH+). HRMS calcd for C10H22NO2S 220.1371,
found 220.1414.
7-[(tert-Butoxycarbonyl)amino]-2-methylideneheptanoic
Acid (20). A solution of enoate 17 (542.2 mg, 1.90 mmol) in 5%
KOH/EtOH-H2O (19:1, 5 mL) was stirred at 50 °C for 3.5 h. The
mixture was evaporated, and the residue was dissolved in water. The
mixture solution was washed with AcOEt, acidified by addition of satd
citric acid, and extracted with AcOEt. The organic layer was dried over
MgSO4 and evaporated. The residue was chromatographed on silica
gel (50 g, hexane/AcOEt = 1/1) to give 20 (482.2 mg, 98.8%) as a
CH2 × 2), 1.44 (9 H, s, Boc), 1.49 (2 H, m, CH2), 2.30 (2 H, t, J = 7.1
Hz, CH2), 3.11 (2 H, m, CH2NHBoc), 4.20 (2 H, q, J = 7.1 Hz,
COOCH2CH3), 4.52 (1 H, bs, NH), 5.51 and 6.13 (each 1 H, s, C
CH2). 13C NMR δ 14.2, 26.3, 28.1, 28.4 (3 carbons), 29.9, 31.7. 40.5,
60.6, 79.0, 124.4, 140.8, 155.9, 167.3. LRMS: m/z 308.2 (MNa+).
HRMS calcd for C15H28NO4 286.2018, found 286.2029.
Ethyl 2-[(Acetylsulfanyl)methyl]-7-[(tert-butoxycarbonyl)-
amino]heptanoate (18). Triethylamine (320 μL, 2.30 mmol) was
added to a solution of enoate 17 (542.7 mg, 1.90 mmol) in thioacetic
acid (3 mL) at 0 °C, and the mixture was stirred at room temperature
for 45 h. The reaction mixture was poured into satd NaHCO3 and
ice−water, and the mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and evaporated. The
residue was chromatographed on silica gel (40 g, benzene/AcOEt =
19/1) to give 18 (525.8 mg, 76.5%) as a colorless oil. 1H NMR δ 1.26
(3 H, t, J = 7.1 Hz, COOCH2CH3), 1.2−1.8 (8 H, m, CH2 × 4), 1.44
(9 H, s, Boc), 2.33 (3 H, s, SAc), 2.56 (1 H, m, CH), 3.00 (1H, dd, J =
13.5, 8.6 Hz, CH2SAc), 3.11 (2 H, m, CH2NHBoc), 3.12 (1 H, dd, J =
13.5, 5.5 Hz, CH2SAc), 4.15 (2 H, q, J = 7.1 Hz, COOCH2CH3), 4.52
(1 H, bs, NH). 13C NMR δ 14.2, 26.5, 26.6, 28.4 (3 carbons), 29.8,
30.3, 30.5, 31.8, 40.4, 45.5, 60.6, 79.0, 155.9, 174.2, 195.4. LRMS: m/z
384.2 (MNa+). HRMS calcd for C17H31NNaO5S 384.1821, found
384.1859.
1
colorless oil. H NMR (CD3OD) δ 1.2−1.6 (6 H, m, CH2 × 3), 1.43
(9 H, s, Boc), 2.29 (2 H, m, CH2), 3.02 (2 H, t, J = 6.8 Hz,
CH2NHBoc), 5.57 and 6.12 (each 1 H, d, J = 1.3 Hz, CCH2). 13C
NMR (CD3OD) δ 27.6, 28.9 (3 carbons), 30.0, 30.9, 33.0, 41.4, 79.9,
125.5, 142.8, 158.7, 170.7. LRMS: m/z 280.2 (MNa+). HRMS calcd
for C13H23NNaO4 280.1525, found 280.1552.
2-[(Acetylsulfanyl)methyl]-7-[(tert-butoxycarbonyl)amino]-
heptanoic Acid (21). A solution of 20 (453.5 mg, 1.76 mmol),
thioacetic acid (500 μL), and triethylamine (295 μL, 2.12 mmol) in
dry CH2Cl2 (3 mL) was stirred at room temperature for 22 h. The
reaction mixture was poured into ice−water and extracted with AcOEt.
The organic layer was washed with brine, dried over MgSO4, and
evaporated. The residue was chromatographed on silica gel (70 g,
hexane/AcOEt = 3/2) to give compound 21 (490.8 mg, 83.4%) as a
1
colorless oil. H NMR (CD3OD) δ 1.2−1.6 (6 H, m, CH2 × 3), 1.43
(9 H, s, Boc), 1.61 (2 H, mCH2), 2.31 (3 H, s, SAc), 2.52 (1 H, m,
CH), 2.99 (3 H, m, CH2NHBoc and CH2SAc), 3.11 (1 H, dd, J = 13.5,
5.5 Hz, CH2SAc). 13C NMR (CD3OD) δ 27.8, 27.9, 28.9 (3 carbons),
30.6, 30.9, 31.4, 33.1, 41.4, 47.1, 80.0, 158.7, 178.1, 197.1. LRMS: m/z
356.2 (MNa+). HRMS calcd for C15H27NNaO5S 356.1508, found
356.1550.
2-[(Acetylsulfanyl)methyl]-7-aminoheptanoic Acid (4). In a
similar manner to that for the synthesis of 2 from 18, target compound
4 was obtained as the TFA salt (33.6 mg) from 21 (31.2 mg, 0.094
mmol) in quantitative yield.. 1H NMR (CD3OD) δ 1.42 (4 H, m, CH2
× 2), 1.66 (4 H, m, CH2 × 2), 2.31 (3 H, s, SAc), 2.54 (1 H, m, CH),
2.92 (2 H, t, J = 7.5 Hz, CH2NH2), 3.00 (1 H, dd, J = 13.6, 8.5 Hz,
CH2SAc), 3.12 (1 H, dd, J = 13.6, 5.5 Hz, CH2SAc). 13C NMR
(CD3OD) δ 27.3, 27.6, 28.4, 30.6, 31.3, 32.7, 40.7, 46.9, 180.0, 197.2.
LRMS: m/z 234.1 (MH+). HRMS calcd for C10H20NO3S 234.1164,
found 234.1206.
Ethyl 7-[(tert-Butoxycarbonyl)amino]-2-[(propanoylselanyl)-
methyl]heptanoate (24). A heterogeneous mixture of Woollins’s
reagent 22 (225.8 mg, 0.42 mmol) and propionic acid (190 μL, 2.55
mmol) in dry toluene (1 mL) was refluxed for 2 h. The resulting
yellow solution containing the selenopropionic acid 23 was cooled to
room temperature, and then to this solution methylene compound 17
(240.6 mg, 0.84 mmol) in dry toluene (1 mL) was added. The mixture
was stirred at 70 °C for 41 h. The reaction mixture was allowed to cool
to room temperature and chromatographed on silica gel (30 g,
hexane/AcOEt = 3/1) to give selenoester 24 (114.5 mg, 32.1%) as a
colorless oil. 1H NMR δ 1.17 (3 H, t, J = 7.5 Hz, SeCOCH2CH3), 1.26
(3 H, t, J = 7.1 Hz, COOCH2CH3), 1.2−1.8 (8 H, m, CH2 × 4), 1.44
(9 H, s, Boc), 2.63 (3 H, m, CH and SeCOCH2CH3), 3.08 (4 H, m,
CH2NHBoc and CH2Se), 4.14 (2 H, q, J = 7.1 Hz, COOCH2CH3),
4.53 (1 H, bs, NH). 13C NMR (CDCl3) δ 9.7, 14.5, 26.3, 26.8, 26.9,
28.6 (3 carbons), 30.1, 33.0, 40.7, 41.7, 46.5, 60.8, 79.3, 156.2, 174.9,
202.6. LRMS: m/z 446.1 (MNa+). HRMS calcd for C18H33NNaO580Se
446.1422, found 446.1419.
2,2′-(Diselane-1,2-diyldimethanediyl)bis(7-aminoheptanoic
Acid) (5). In a similar manner to that for the synthesis of 1 from 18,
target compound 5 was obtained as the HCl salt from 24 (36.9 mg,
0.087 mmol). Purification was performed with ion exchange
chromatography (adande:l PCX5006) using methanol/28% ammonia
(1:1) as an elution solvent. To the eluent containing target compound
was added 4 M HCl (495 μL), and the mixture was dried in vacuo to
give 5 as the HCl salt (21.1 mg, 44.1%). 1H NMR (D2O) δ 1.34 (8 H,
m, CH2 × 4), 1.61 (8 H, m, CH2 × 4), 2.80 (2 H, m, CH × 2), 2.93 (4
H, t, J = 7.5 Hz, CH2NH2 × 2), 3.08 (4 H, dd, J = 7.2, 2.5 Hz, CH2Se
× 2). 13C NMR (D2O) δ 25.3 (2 carbons), 25.7 (2 carbons), 26.4 (2
7-Amino-2-(sulfanylmethyl)heptanoic Acid (1). A solution of
thioacetate 18 (341.6 mg, 0.95 mmol) in conc HCl (1 mL) was stirred
at 100 °C for 1 h. The mixture was allowed to cool to room
temperature and evaporated. The residue was purified by MPLC
(ODS-SM, 26 mm × 300 mm, 50 μm, 50% MeOH/Milli-Q + 0.2%
TFA, 12 mL/min) to give target compound 1 as TFA salt (187.1 mg,
1
64.8%). H NMR (D2O) δ 1.39 (4 H, m, CH2 × 2), 1.62 (4 H, m,
CH2 × 2), 2.65 (1 H, m, CH), 2.73 (2 H, m, CH2SH) 2.98 (2 H, t, J =
7.6 Hz, CH2NH2). 13C NMR δ 25.3, 25.7, 26.4, 29.2, 31.2, 39.3, 45.9,
179.2. LRMS: m/z 192.1 (MH+). HRMS calcd for C8H18NO2S
192.1058, found 192.1090.
Ethyl 2-[(Acetylsulfanyl)methyl]-7-aminoheptanoate (2). A
solution of 18 (29.5 mg, 0.082 mmol) in TFA/H2O (19:1, 1 mL) was
stirred at 0 °C for 1 h, and then the mixture was concentrated in vacuo
to afford compound 2 as the TFA salt (33.1 mg, quantitative yield). 1H
NMR (CD3OD) δ 1.25 (3 H, t, J = 7.1 Hz, COOCH2CH3), 1.40 (4 H,
m, CH2 × 2), 1.62 (4 H, m, CH2 × 2), 2.31 (3 H, s, SAc), 2.56 (1 H,
m, CH), 2.91 (2 H, t, J = 7.5 Hz, CH2NH2), 3.01 (1 H, dd, J = 13.6,
5.4 Hz, CH2SAc), 4.14 (2 H, q, J = 7.1 Hz, COOCH2CH3). 13C NMR
δ 14.7, 27.2, 27.6, 28.4, 30.5, 31.3, 32.6, 40.7, 47.0, 62.0, 176.0, 197.0.
LRMS: m/z 262.2 (MH+). HRMS calcd for C12H24NO3S 262.1477,
found 262.1521.
Ethyl 7-[(tert-Butoxycarbonyl)amino]-2-(sulfanylmethyl)-
heptanoate (19). A solution of 18 (35.3 mg, 0.098 mmol) and
Amano Lipase PS (immobilized on diatomite) (21.3 mg) in 20 mM
NaOAc (pH 5.9, 1.5 mL) was stirred at 55 °C for 17 h. The reaction
mixture was poured into water, and the mixture was extracted with
AcOEt. The organic layer was washed with brine, dried over MgSO4,
and evaporated. The residue was chromatographed on silica gel (10 g,
hexane/AcOEt = 3/1) to give 19 (23.8 mg, 76.2%) as a colorless oil.
1H NMR δ 1.1−1.8 (8 H, m, CH2 × 4), 1.26 (3 H, dt, J = 7.0, 1.1 Hz,
COOCH2CH3), 1.42 (9 H, s, Boc), 2.60 (2 H, m, CH2SH), 2.74 (1 H,
m, CH), 3.08 (2 H, m, CH2NHBoc), 4.16 (2 H, dq, J = 7.0, 1.1 Hz,
COOCH2CH3), 4.54 (1 H, bs, NH). 13C NMR δ 14.3, 26.0, 26.5, 26.7,
28.4 (3 carbons), 29.8, 31.3, 40.4, 49.2, 60.5, 79.0, 155.9, 174.2. LRMS:
m/z 342.2 (MNa+). HRMS calcd for C15H29NNaO4S 342.1715, found
342.1747.
Ethyl 7-Amino-2-(sulfanylmethyl)heptanoate (3). In a similar
manner to that for the synthesis of 2 from 18, target compound 3 was
obtained as the TFA salt (23.6 mg) from 19 (22.1 mg, 0.069 mmol) in
1
quantitative yield. H NMR (CD3OD) δ 1.26 (3 H, t, J = 7.2 Hz,
COOCH2CH3), 1.38 (4 H, m, CH2 × 2), 1.64 (4 H, m, CH2 × 2),
2.56 (1 H, m, CH), 2.68 (2 H, m, CH2SH), 2.91 (2 H, t, J = 7.6 Hz,
CH2NH2), 4.17 (2 H, q, J = 7.2 Hz, COOCH2CH3). 13C NMR
(CD3OD) δ 14.8, 26.7, 27.4, 27.8, 28.5, 32.4, 40.7, 50.7, 61.9, 176.2.
7702
dx.doi.org/10.1021/jm300735t | J. Med. Chem. 2012, 55, 7696−7705

Journal of Medicinal Chemistry
Article
carbons), 30.2, 30.3, 31.65, 31.74, 39.4 (2 carbons), 46.7 (2 carbons),
179.4 (2 carbons). LRMS: m/z 477.1 (MH+). HRMS calcd for
C16H33N2O480Se2 477.0771, found 477.0764.
(CD3OD) δ 27.1, 27.8, 27.9, 29.0 (3 carbons), 30.9, 32.4, 33.8, 41.4,
47.5, 50.4, 80.0, 127.5, 129.5 (2 carbons), 129.6 (2 carbons), 141.3,
158.6, 178.3, 202.1. LRMS: m/z 494.2 (MNa+). HRMS calcd for
C22H33NNaO580Se 494.1422, found 494.1433.
Ethyl 7-Amino-2-[(propanoylselanyl)methyl]heptanoate (6).
In a similar manner to that for the synthesis of 2 from 18, target
compound 6 was obtained as the TFA salt (19.9 mg) from 24 (19.2
7-Amino-2-{[(3-phenylpropanoyl)selanyl]methyl}heptanoic
Acid (9). In a similar manner to that for the synthesis of 2 from 18,
target compound 9 was obtained as the TFA salt (29.4 mg) from 29
1
mg, 0.045 mmol) in quantitative yield. H NMR (CD3OD) δ 1.13 (3
1
(27.1 mg, 0.058 mmol) in quantitative yield. H NMR (CD3OD) δ
H, t, J = 7.5 Hz, SeCOCH2CH3), 1.24 (3 H, t, J = 7.1 Hz,
COOCH2CH3), 1.40 (4 H, m, CH2 × 2), 1.65 (4 H, m, CH2 × 2),
2.63 (1 H, m, CH), 2.65 (2 H, q, J = 7.5 Hz, SeCOCH2CH3), 2.91 (2
H, t, J = 7.5 Hz, CH2NH2), 3.07 (2 H, m, CH2Se), 4.13 (2 H, q, J = 7.1
Hz, COOCH2CH3). 13C NMR (CDCl3) δ 9.9, 14.7, 26.8, 27.3, 27.8,
28.5, 33.6, 40.8, 42.4, 47.7, 62.0, 176.3, 203.6. LRMS: m/z 324.1
(MH+). HRMS calcd for C13H26NO380Se 324.1078, found 324.1084.
7-[(tert-Butoxycarbonyl)amino]-2-[(propanoylselanyl)-
methyl]heptanoic Acid (25). In a similar manner to that for the
synthesis of 24 from 17, target compound 25 (235.3 mg) was obtained
from 20 (346.3 mg, 1.35 mmol) in 44.3% yield. 1H NMR δ 1.18 (3 H,
t, J = 7.5 Hz, SeCOCH2CH3), 1.2−1.8 (8 H, m, CH2 × 4), 1.44 (9 H,
s, Boc), 2.64 (3 H, m, CH, SeCOCH2CH3), 3.09 (4 H, m,
CH2NHBoc, CH2Se), 4.60 (1 H, bs, NH). 13C NMR δ 9.7, 25.9,
26.7, 26.8, 28.6 (3 cabons), 30.0, 32.8, 40.6, 41.7, 46.3, 79.4, 156.3,
179.6, 202.6. LRMS m/z 418.1 (MNa+). HRMS calcd for
C16H29NNaO580Se 418.1109, found 418.1088.
1.39 (4 H, m, CH2 × 2), 1.60 (4 H, m, CH2 × 2), 2.59 (1 H, m, CH),
2.92 (2 H, m, CH2NH2), 2.95 (4 H, m, CH2Ph, CH2CO), 3.06 (2 H,
m, CH2Se), 7.17 (3 H, m, Ph-H), 7.26 (2 H, m, Ph-H). 13C NMR
(CD3OD) δ 26.8, 27.1, 27.6, 28.3, 32.3, 33.4, 40.6, 47.3, 50.3, 127.4,
129.4 (2 carbons), 129.5 (2 carbons), 141.2, 178.1, 202.1. LRMS: m/z
372.1 (MH+). HRMS calcd for C17H26NO380Se 372.1078, found
372.1110.
Ethyl 3-{6-[(tert-Butoxycarbonyl)amino]pyridin-3-yl}-2-
[(propanoylselanyl)methyl]propanoate (31). Compound 30
(150.7 mg, 0.49 mmol) was subjected to selenylation reaction in a
similar manner to that for the synthesis of 24 from 17. The reaction
mixture was allowed to cool to room temperature and chromato-
graphed on silica gel [25 g, CH2Cl2/28% ammonia solution−MeOH
(1:9) = 39/1] to give compound 31 (192.6 mg, 88.3%) as a white
solid. 1H NMR δ 1.16 (3 H, t, J = 7.1 Hz, COOCH2CH3), 1.17 (3 H,
t, J = 7.5 Hz, COCH2CH3), 1.53 (9 H, s, Boc), 2.63 (2 H, q, J = 7.5
Hz, COCH2CH3), 2.80 (1 H, m, CH), 2.91 (2 H, m, Py-CH2), 3.07 (2
H, d, J = 6.2 Hz, CH2Se), 4.07 (2 H, q, J = 7.1 Hz, COOCH2CH3),
7.48 (1 H, dd, J = 8.6, 2.3 Hz, Py-4), 7.88 (1 H, d, J = 8.6 Hz, Py-5),
8.02 (1 H, bs, NH), 8.08 (1 H, d, J = 2.3 Hz, Py-2). 13C NMR δ 9.7,
14.4, 26.0, 28.6 (3 carbons), 35.5, 41.7, 48.0, 61.1, 81.1, 112.2, 128.4,
138.9, 148.2, 151.1, 152.7, 173.7, 202.0. LRMS: m/z 445.1 (MH+).
HRMS calcd for C19H29N2O580Se1 445.1242, found 445.1251.
2,2′-(Diselane-1,2-diyldimethanediyl)bis[3-(6-aminopyridin-
3-yl)propanoic Acid] (10). A solution of compound 31 (24.3 mg,
0.055 mmol) in conc. HCl (1 mL) was stirred at 100 °C for 1 h. The
reaction mixture was evaporated. The residue was purified by ion
exchange chromatography (OASIS MCX) using methanol/28%
ammonia (13:1) as an elution solvent. To the eluent containing
target compound was added 4 M HCl (900 μL), and the mixture was
7-Amino-2-[(propanoylselanyl)methyl]heptanoic Acid (7). In
a similar manner to that for the synthesis of 2 from 18, target
compound 7 was obtained as the TFA salt (25.3 mg) from 25 (24.3
1
mg, 0.062 mmol) in quantitative yield. H NMR (CD3OD) δ 1.14 (3
H, t, J = 7.5 Hz, SeCOCH2CH3), 1.42 (4 H, m, CH2 × 2), 1.66 (4 H,
m, CH2 × 2), 2.58 (1 H, m, CH), 2.65 (2 H, q, J = 7.5 Hz,
SeCOCH2CH3), 2.92 (2 H, t, J = 7.5 Hz, CH2NH2), 3.06 (2 H, m,
CH2Se). 13C NMR (CD3OD) δ 9.8, 26.6, 27.1, 27.6, 28.3, 33.4, 40.6,
42.2, 47.4, 178.2, 203.7. LRMS: m/z 296.0 (MH+). HRMS calcd for
C11H22NO380Se 296.0765, found 296.0754.
7-[(tert-Butoxycarbonyl)amino]-2-{[(4-phenylbutanoyl)-
selanyl]methyl}heptanoic Acid (27). In a similar manner to that
for the preparation of selenopropionic acid 23, selenocarboxylic acid
26 was prepared. To the solution containing 26, compound 20 (153.6
mg, 0.60 mmol) in dry toluene (1 mL) was added and the mixture was
stirred at 70 °C for 18 h. The reaction mixture was allowed to cool to
room temperature and chromatographed on silica gel (15 g, hexane/
AcOEt = 7/3) to give compound 27 (135.7 mg, 46.9%) as a colorless
1
dried in vacuo to give 10 as the HCl salt (10.8 mg, 66.7%). H NMR
(CD3OD) δ 2.90 (4 H, d, J = 6.7 Hz, Py-CH2 × 2), 3.13 (6 H, m, CH
× 2, CH2Se × 2), 7.01 (2 H, d, J = 9.1 Hz, Py-4 × 2), 7.71 (2 H, s, Py-
5 × 2), 7.87 (2 H, d, J = 9.1 Hz, Py-2 × 2). 13C NMR (CD3OD) δ
31.5, 31.6, 34.5, 34.6, 48.8, 48.9, 114.9 (2 carbons), 125.0 (2 carbons),
135.1 (2 carbons), 147.0 (2 carbons), 154.7 (2 carbons), 176.2 (2
carbons). LRMS: m/z 519.0 (MH+). HRMS calcd for
C18H23N4O480Se2 519.0050, found 519.0060.
1
oil. H NMR (CD3OD) δ 1.2−1.8 (8 H, m, CH2 × 4), 1.42 (9 H, s,
Boc), 1.95 (2 H, m, CH2CH2Ph), 2.56 (1 H, m, CH), 2.63 (4 H, m,
CH2Ph, CH2CO), 3.03 (4 H, m, CH2NH, CH2Se), 7.16 (3 H, m, Ph-
H), 7.26 (2 H, m, Ph-H). 13C NMR (CD3OD) δ 27.1, 27.8, 28.0, 28.5,
29.0 (3 carbons), 30.9, 33.9, 35.8, 41.4, 47.6, 48.2, 80.0, 127.2, 129.6 (2
carbons), 130.0 (2 carbons), 142.7, 158.7, 178.4, 202.8. LRMS: m/z
508.2 (MNa+). HRMS calcd for C23H35NNaO580Se 508.1578, found
508.1578.
Ethyl 3-(6-Aminopyridin-3-yl)-2-[(propanoylselanyl)methyl]-
propanoate (11). A solution of compound 31 (21.6 mg, 0.049
mmol) in TFA/H2O (19:1, 1 mL) was stirred at 0 °C for 1 h. The
reaction mixture was evaporated, to which H2O was added. The
aqueous solution was washed with hexane and concentrated in vacuo
7-Amino-2-{[(4-phenylbutanoyl)selanyl]methyl}heptanoic
Acid (8). In a similar manner to that for the synthesis of 2 from 18,
target compound 8 was obtained as the TFA salt (19.3 mg) from 27
1
to afford 11 as the TFA salt (12.5 mg, 56.1%). H NMR (CD3OD) δ
1.14 (3 H, t, J = 7.4 Hz, COCH2CH3), 1.19 (3 H, t, J = 7.1 Hz,
COOCH2CH3), 2.66 (2 H, q, J = 7.4 Hz, COCH2CH3), 2.79 (1 H, m,
CH), 2.82 and 2.90 (each 1 H, d, J = 6.1 Hz, Py-CH2), 3.10 (2 H, m,
CH2Se), 4.09 (2 H, q, J = 7.1 Hz, COOCH2CH3), 6.98 (1 H, d, J = 9.1
Hz, Py-5), 7.67 (1 H, d, J = 2.1 Hz, Py-2), 7.84 (1 H, dd, J = 9.1, 2.1
Hz, Py-4). 13C NMR (CD3OD) δ 9.7, 14.5, 26.1, 34.7, 42.2, 48.2, 62.1,
114.8, 124.8, 135.3, 147.0, 154.9, 174.6, 203.1. LRMS: m/z 345.1
(MH+). HRMS calcd for C14H21N2O378Se 343.0725, found 343.0754.
E t h y l 3 - ( 6 - A m i n o - 5 - c h l o r o p y r i d i n - 3 - y l ) - 2 -
[(propanoylselanyl)methyl]propanoate (13). Compound 32
(88.4 mg, 0.26 mmol) was subjected to selenylation reaction in a
similar manner to that for the synthesis of 24 from 17. The mixture
was allowed to cool to room temperature and chromatographed on
silica gel [30 g, CH2Cl2/28% ammonia solution−MeOH (1:9) = 99/
1] to give compound 13 (84.9 mg, 86.5%) as a colorless oil. 1H NMR
δ 1.18 (3 H, t, J = 7.5 Hz, COCH2CH3), 1.19 (3 H, t, J = 7.1 Hz,
COOCH2CH3), 2.65 (2 H, q, J = 7.5 Hz, COCH2CH3), 2.78 (1 H, m,
CH), 2.86 (2 H, m, Py-CH2), 3.07 (2 H, d, J = 6.1 Hz, CH2Se), 4.09 (2
1
(17.2 mg, 0.036 mmol) in quantitative yield. H NMR (CD3OD) δ
1.42 (4 H, m, CH2 × 2), 1.66 (4 H, m, CH2 × 2), 1.94 (2 H, m,
CH2CH2Ph), 2.58 (1 H, m, CH), 2.64 (4 H, m, CH2Ph, CH2CO),
2.91 (2 H, m, CH2NH2), 3.07 (2 H, m, CH2Se), 7.16 (3 H, m, Ph-H),
7.26 (2 H, m, Ph-H). 13C NMR (CD3OD) δ 26.8, 27.2, 27.6, 28.3,
28.4, 33.4, 35.7, 40.6, 47.4, 48.0, 127.1, 129.45 (2 carbons), 129.49 (2
carbons), 142.5, 178.1, 202.8. LRMS: m/z 386.1 (MH +). HRMS calcd
for C18H28NO380Se 386.1234, found 386.1259.
7-[(tert-Butoxycarbonyl)amino]-2-{[(3-phenylpropanoyl)-
selanyl]methyl}heptanoic Acid (29). In a similar manner to that
for the preparation of selenopropionic acid 23, selenocarboxylic acid
28 was prepared from hydrocinnamic acid and Woollins’s reagent.
Using selenocarboxylic acid 28, target compound 29 (108.4 mg) was
1
obtained from 20 (158.1 mg, 0.61 mmol) in 37.5% yield. H NMR
(CD3OD) δ 1.2−1.8 (8 H, m, CH2 × 4), 1.43 (9 H, s, Boc), 2.56 (1 H,
m, CH), 2.94 (4 H, m, CH2Ph, CH2CO), 3.03 (4 H, m, CH2NHBoc,
CH2Se), 7.17 (3 H, m, Ph-H), 7.25 (2 H, m, Ph-H). 13C NMR
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H, q, J = 7.1 Hz, COOCH2CH3), 4.78 (2 H, bs, NH), 7.35 (1 H, d, J =
2.0 Hz, Py-4), 7.79 (1 H, d, J = 2.0 Hz, Py-2). 13C NMR δ 9.4, 14.2,
25.7, 34.6, 41.5, 47.8, 60.9, 114.8, 124.9, 137.5, 146.3, 153.5, 173.4,
201.8. LRMS: m/z 379.0 (MH+). HRMS calcd for C14H20ClN2O380Se
379.0328, found 379.0331.
ABBREVIATIONS USED
■
TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa,
activated thrombin-activatable fibrinolysis inhibitor; t-PA, tissue
plasminogen activator; CPN, carboxypeptidase N; CPB,
carboxypeptidase B; TFA, trifluoroacetic acid; DTT, dithio-
threitol
2,2′-(Diselane-1,2-diyldimethanediyl)bis[3-(6-amino-5-
chloropyridin-3-yl)propanoic Acid] (12). In a similar manner to
that for the synthesis of 5 from 24 followed by the purification, target
compound 12 was obtained as the HCl salt (30.0 mg, quantitative
yield) from 13 (32.6 mg, 0.086 mmol). 1H NMR (CD3OD) δ 2.94 (4
H, d, J = 7.0 Hz, CH2CH × 2), 3.09 (2 H, m, CH × 2), 3.19 (4 H, m,
CH2Se × 2), 7.81 (2 H, s, Py-4 × 2), 8.14 (2 H, d, J = 1.7 Hz, Py-2 ×
2). 13C NMR (CD3OD) δ 31.6, 31.7, 34.37, 34.41, 48.71, 48.74, 119.8
(2 carbons), 125.8 (2 carbons), 134.6 (2 carbons), 145.9 (2 carbons),
152.0 (2 carbons), 176.06, 176.08. LRMS: m/z 586.9 (MH+). HRMS
calcd for C18H20 Cl2N4O478Se80Se 583.9200, found 583.9239.
TAFIa Inhibition. TAFI was purified from normal human plasma
using plasminogen-depleted plasma as described previously.33
Thrombomodulin (TM) was purchased from American Diagnostica,
(Greenwich, CT, USA). Human plasma thrombin, hippuryl-arginine
(Hip-Arg), and hippuryl-lysine (Hip-Lys) were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Porcine pancreas carboxypeptidase B
(ppCPB) was purchased from Worthington Biochemical (Freehold,
NJ, USA). Human plasma carboxypeptidase N (CPN) was purchased
from Elastin Products (St. Louis, MO, USA).
To activate TAFI, TAFI from human plasma (4.3 μg/mL TAFI, 20
μL) was mixed with 20 μL of 300 ng/mL TM in buffer A (50 mM
Tris-HCl buffer, pH 7.4, supplemented with 0.1% BSA and 0.15 M
sodium chloride) and incubated at 25 °C for 3 min. The mixture was
additionally mixed with 20 μL of 3 U/mL thrombin in buffer A and
TAFI in the mixture was activated by incubation at 25 °C for 30 min.
A part of prepared TAFIa solution (25 μL) was transferred into
another tube and incubated with 25 μL of 3.2 mM Hip-Arg substrate
in buffer B (0.1 M Tris-HCl buffer containing or not containing a final
concentration of 10 μM DTT, pH 7.6) at 25 °C for 30 min (the final
volume was 80 μL). The reaction was stopped by adding 100 μL of 0.2
M PIPES−NaOH buffer, pH 7.6, containing 12.5% Tween 20. Color
was developed by adding 100 μL of 1% cyanuric acid in 2-
methoxyethanol, followed by measurement of absorbance at 405 nm.34
For determination of inhibitor activities of various compounds
against TAFIa and ppCPB activity, 25 μL of 20 U/L of TAFIa or
ppCPB was transferred into other tubes and then incubated with 30
μL of various concentrations of compounds at 25 °C for 10 min.
TAFIa and ppCPB activities were measured by same method as
described above. In measurement of inhibitor activity of various
compounds against CPN, 50 U/L CPN was transferred into other
tubes and incubated with 30 μL of various concentrations of inhibitors
at 37 °C for 10 min. The mixture was incubated with 25 μL of 3.2 mM
Hip-Lys substrate at 37 °C for 30 min in buffer B. Stopping of the
reaction, development of color, and measurement of absorbance were
same as described above. IC50 (50% inhibitory concentration) values
were calculated from the sigmoidal inhibition curves by using
Microsoft Excel software.
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AUTHOR INFORMATION
■
Corresponding Author
(10) Wang, Y. X.; Zhao, L.; Nagashima, M.; Vincelette, J.; Sukovich,
D.; Li, W.; Subramanyam, B.; Yuan, S.; Emayan, K.; Islam, I.; Hrvatin,
P.; Bryant, J.; Light, D. R.; Vergona, R.; Morser, J.; Buckman, B. O. A
novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor
(TAFIa), part I: pharmacological characterization. Thromb. Haemost.
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*Phone: +81 42 721 1580. Fax: +81 42 721 1580. E-mail:
yamamoto@ac.shoyaku.ac.jp.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Part of this work was supported by a Grant-in-Aid for High
Technology Research Center Project (no. 19-8) from the
Ministry of Education, Culture, Sports, Science and Technol-
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