Dabigatran and dabigatran ethyl ester: Potent inhibitors of ribosyldihydronicotinamide dehydrogenase (NQO2)

Article abstract of DOI:10.1021/jm3001339

Recent studies have revealed that compounds believed to be highly selective frequently address multiple target proteins. We investigated the protein interaction profile of the widely prescribed thrombin inhibitor dabigatran (1), resulting in the identification and subsequent characterization of an additional target enzyme. Our findings are based on an unbiased functional proteomics approach called capture compound mass spectrometry (CCMS) and were confirmed by independent biological assays. 1 was shown to specifically bind ribosyldihydronicotinamide dehydrogenase (NQO2), a detoxification oxidoreductase. Molecular dockings predicted and biological experiments confirmed that dabigatran ethyl ester (2) inhibits NQO2 even more effectively than the parent 1 itself. Our data show that 1 and 2 are inhibitors of NQO2, thereby revealing a possible new aspect in the mode of action of 1. We present a workflow employing chemical proteomics, molecular modeling, and functional assays by which a compound's protein-interaction profile can be determined and used to tune the binding affinity.

Full text of DOI:10.1021/jm3001339

Article
pubs.acs.org/jmc
Dabigatran and Dabigatran Ethyl Ester: Potent Inhibitors of
Ribosyldihydronicotinamide Dehydrogenase (NQO2)
Simon Michaelis,‡ Anett Marais, Anna K. Schrey, Olivia Y. Graebner, Cornelia Schaudt, Michael Sefkow,
Friedrich Kroll, Mathias Dreger, Mirko Glinski,† Hubert Koester, Rainer Metternich,†
and Jenny J. Fischer*,‡
caprotec bioanalytics GmbH, Volmerstrasse 5, 12489 Berlin, Germany
S
* Supporting Information
ABSTRACT: Recent studies have revealed that compounds believed to be highly
selective frequently address multiple target proteins. We investigated the protein
interaction profile of the widely prescribed thrombin inhibitor dabigatran (1),
resulting in the identification and subsequent characterization of an additional
target enzyme. Our findings are based on an unbiased functional proteomics
approach called capture compound mass spectrometry (CCMS) and were
confirmed by independent biological assays. 1 was shown to specifically bind
ribosyldihydronicotinamide dehydrogenase (NQO2), a detoxification oxidoreduc-
tase. Molecular dockings predicted and biological experiments confirmed that
dabigatran ethyl ester (2) inhibits NQO2 even more effectively than the parent 1
itself. Our data show that 1 and 2 are inhibitors of NQO2, thereby revealing a
possible new aspect in the mode of action of 1. We present a workflow employing chemical proteomics, molecular modeling, and
functional assays by which a compound’s protein-interaction profile can be determined and used to tune the binding affinity.
INTRODUCTION
■
In drug discovery, the dominant paradigm is that maximally
selective ligands should be designed to act on single drug
targets.1 In the last years, it has however become evident that
many effective drugs act via modulation of multiple proteins
rather than on individual targets.1−3 For some drugs, e.g., the
atypical antipsychotics, a promiscuous pharmacological profile
is the key point for their efficacy.4 Cheminformatics have
played a crucial role in prediction of new targets for given drugs
or therapeutic areas.4−9 However, not all protein structures and
mechanisms guiding small-molecule protein interactions are
known and therefore these theoretical computational ap-
proaches currently require experimental data to formulate or
test their hypothesis. Thus, unbiased experimental methods are
needed to complete the target space. Compiling experimental
protein interaction profiles of small-molecule drugs gives novel
insights into their mode of action, thereby generating a better
understanding of the structure−activity relationship (SAR),
Figure 1. Structures of thrombin inhibitors 1−3 and tyrosine kinase
toxicology, and repurposing possibilities. Repurposing of
inhibitor 4.
established safe drugs has become a major focus of interest,
described as a particularly safe drug.16 Its prodrug dabigatran
as new drug candidates dramatically fail during clinical trials due
to toxicity or insufficient efficacy.10 Such profiling investigations
etexilate is currently in clinical trials for long-term use for the
prevention of stroke and non-CNS systemic embolism. Despite
have been carried out mainly for promiscuous drugs such as
kinase inhibitors11,12 but also for small molecules targeting
its drug safety, not all off-targets and potential novel targets of 1
histone deacetylases.13,14 Anticoagulants such as dabigatran15
have been identified. An unbiased method to compile small-
molecule−protein interaction profiles is capture compound
(1, Figure 1) are believed to have very clear-cut targets such as
inhibition of factor II (thrombin) of the blood coagulation
cascade.15
We here investigate for the first time the protein interaction
profile of an anticoagulant and chose 1, which has been
Received: January 30, 2012
Published: April 11, 2012
© 2012 American Chemical Society
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mass spectrometry (CCMS, Scheme 1), which employs small-
molecule based probes called capture compounds (CCs)
CCs of melagatran (3, Figure 1), a closely related peptide
mimetic thrombin inhibitor to compare the interaction profiles.
As chemical modifications of a parent drug may facilitate
interactions with different target proteins,18 we chose two
attachment positions to the CCs’ scaffold (Scheme 2, X) for
each drug (Supporting Information Figure S1). This resulted in
two CCs 5 and 6 derived from 1 and two CCs 7 and 8 derived
from 3 (Scheme 2A). The aim was to obtain a picture of the
compound−protein interaction profiles that should be as
complete as possible, i.e., identification of interactions occurring
at the acid and at the amidine side of the compounds.
a
Scheme 1. Schematic CCMS Workflow
The synthesis route to CC 5−8 is depicted in Scheme 2.
Briefly, commercially available dabigatran ethyl ester acetate
was equipped with an aliphatic linker to give compound 11.
Removal of the Boc protecting group followed by coupling to
the CC scaffold19 and saponification yielded CC 6 (Scheme
2B). CC 8 was available from Boc-protected amino acid 13.
Subsequent coupling of 13 with an aliphatic linker, Boc
deprotection, and coupling to the peptidic building block gave
15. Intermediate 15 was converted into 8 by subsequent Boc
d e p r o t e c t i o n , c o u p l i n g w i t h m e t h y l 2 - ( 4 -
nitrophenylsulfonyloxy)acetate, removal of the nitrobenzene-
sulfonamide protecting group, coupling to the scaffold, and
saponification (Scheme 2C). CCs 5 and 7 were prepared in one
step from 1 and 3, respectively. All CCs 5−8 were thermally
and biologically stable, and only the expected photoreaction
products were formed upon UV-irradiation.
Next, we carried out capture assays with purified
recombinant human thrombin to elucidate whether the
thrombin binding properties are retained in the CCs. As
predicted by crystal structures,20,21 both CCs 5 and 7 bearing a
terminal amidine group, specifically bound thrombin while CCs
6 and 8 with opposite attachment orientations showed no or
little interaction with thrombin (Figure 2). According to the
known binding mode of 1 into the thrombin binding pocket,
the benzamidine group forms tight interactions with Asp189
located in the bottom of the primary specificity pocket. This
pocket should not fit any modifications on the amidine group
of the inhibitor because it is very narrow. The ratio between the
signals in the lanes was 0.8:0.3:1:0 for CCs 5:6:7:8 as
determined by densitometric analysis. We believe the
unexpected detection with CC 6 is due to unspecific
background cross-linking caused by the artificial situation in
which a single purified protein is used as compared to a
complex lysate samples. As expected, no signals were observed
in the gel lanes with CC 6 and 8.
We started our investigations of the protein interaction
profile of 1 in parallel to 3 by performing CCMS experiments
(Scheme 1) in the hepatocyte derived cell line HepG2.
Enrichment and isolation of specifically interacting proteins was
carried out using CCs 5−8 in four independent replicate
experiments. The captured and trypsinized proteins were
identified using a high resolution LTQ Orbitrap hybrid mass
spectrometer. Only proteins identified with more than 99%
probability and at least two unique peptides with 95%
probability were considered to be identified with sufficient
confidence. In competition control experiments, an excess of
the respective free drugs was added that compete with the
corresponding CC for specific binding sites. Therefore, proteins
that are detected by LC-MS in the capture assay and that are
significantly diminished in competition control experiments are
defined to be specific. The specificity is quantified by the fold
change between assay and competition (Figure 3). Moreover,
a
CCs are trifunctional molecules: the selectivity function (red) binds
target proteins (green) through reversible affinity interaction, a
covalent bond between the photo-reactivity function (orange) and
the protein(s) is generated. The crosslinked proteins are isolated via
the biotin sorting function (yellow) using streptavidin magnetic beads
and identified by MS or visualized by using standard gel protocols. As
controls, additional competition experiments using the respective
drugs were carried out.
containing the compound of interest as selectivity function.
We report the synthesis of CCs and a subsequent chemical
proteomics investigation which revealed that 1 specifically binds
and functionally inhibits ribosyldihydronicotinamide dehydro-
genase (NQO2), a phase II detoxification oxidoreductase, with
similar inhibitory potential as the known NQO2 inhibitor
imatinib (4, Figure 1).17 On the basis of molecular modeling,
we then hypothesized and could indeed show experimentally
that dabigatran ethyl ester (2, Figure 1) has higher affinity than
1 to both thrombin and NQO2. We present a strategy how to
determine the protein targets of a small-molecule compound in
an unbiased way in a native biological environment and how
such data can be employed to modify interaction properties of
the drug molecule.
RESULTS AND DISCUSSION
■
To investigate the protein interaction profile of 1 for the
identification of novel specific binders, we rely on CCMS
experiments (Scheme 1) with both CCs derived from 1 and
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a
Scheme 2. (A) Structures of CCs 5 and 6, CCs 7 and 8, and CCs 9 and 10; (B) Synthesis of CC 6; (C) Synthesis of CC 8
a
Conditions: (a) tert-butyl 4-aminobutylcarbamate, EtOH, 78 °C, 12 h, 91%; (b) TFA, DCM, 0 °C, 1 h, used as crude; (c) X−OH, HATU, DIPEA,
DMA, 23 °C, 2 d, 35% over two steps; (d) LiOH, THF/water, 4 °C, 17 h, 48%. Conditions for (C): (e) N-(4-aminobutyl)-4-
nitrobenzenesulfonamide, HATU, DIPEA, DMAP, DMA, 23 °C, 1.5 h, 85%; (f) TFA, DCM/MeOH, 40 °C, 2 h, used as crude; (g) (S)-1-((R)-2-
(Boc-amino)-2-cyclohexylacetyl)azetidine-2-carboxylic acid, HATU, DIPEA, DMA, 23 °C, 2 d, 96% over two steps; (h) TFA, DCM, 0 °C, 0.5 h,
used as crude; (i) methyl 2-(4-nitrophenylsulfonyloxy)acetate, K2CO3, MeCN, 60 °C, 1.5 h, non-pure product used as such; (k) 2-mercaptoacetic
acid, DBU, MeCN, 0 °C, 1 h, used as crude; (l) X−OH, HATU, DIPEA, DMA, 23 °C, 17 h, 43% over two steps; (n) LiOH, THF/water, 0 °C, 1 h,
78%.
experiments were carried out in quadruplicate, allowing the
determination of significance (p-value from Student's t test).
Only proteins fulfilling stringent enrichment criteria of high
specificity (fold change ≥2) and at the same time high
significance (p-value ≤0.05, corresponding to a −log10 (p-value
≥1.3) were considered as specific binders of the respective drug
(these specific binders are indicated in the boxed area, Figure 3
and also listed in Table 1).
Seven proteins fulfilled these criteria for at least one of the
investigated CCs: NQO2, HYOU1, HNMT, HNRPC, and
ADK specifically bound CCs 5 or 6 and VPS35 and LRC59
showed specific interaction with CC 8, but CC 7 showed no
significantly enriched proteins. An overview of the significantly
enriched proteins is given in Table 1. In a previous study,13 we
analyzed a quantity of tryptically digested HepG2 input cell
lysate, comparable to the protein amounts obtained in the
CCMS experiments presented here. Out of the seven proteins
enriched here, only HYOU1 and HNRPC were among the 360
proteins identified from HepG2 input lysate. This shows that
NQO2, HNMT, ADK, VPS35, and LRC59 are in too low
abundance for the detection limit of the used LC-MS method
without prior specific enrichment by CCMS. NQO2 belongs to
proteins not expressed at sufficient level to be identified by LC-
MS from HepG2 whole cell lysate, at least when using our
standard setting. However, NQO2 can be identified robustly
and specifically after enrichment by CCMS. This shows that
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and this facilitates enrichment even of weak binders and low
abundant proteins.22
Robust and specific identification of NQO2 as interactor of 1
caught our particular interest: the oxidoreductase NQO2 has
been suggested11 to be the direct target in resveratrol induced
inhibition of cancer cell growth23−25 and to play an important
role in metabolism of xenobiotics.26 Therefore, different
inhibitors for NQO2 have been developed and investigated in
recent years.27
We decided to confirm the enrichment of NQO2 by CC 5 in
a second cell line. Since the leukemia cell line K562 has been
used in several previous studies investigating NQO2, we chose
K562 for our investigations.11,17,28 In one of these studies,
Bantscheff et al.11 identified NQO2 as the first nonkinase off-
target of 4 through pull down experiments with immobilized 4.
Consequently, we chose 4 and its CCs 9 and 10 (Scheme 2A)
as positive controls for the following experiments. We repeated
the capture experiments followed by LC-MS based protein
identification with CCs 5−8 in parallel with CCs 9 and 10 in
independent duplicate experiments in K562 cell lysate
(Supporting Information Table S2). We confirmed NQO2 as
specific target of 1 and 4 by specific enrichment of NQO2
through CCs 5 and 9 followed by mass spectrometry
identification. In addition, we carried out Western Blots using
an NQO2 specific antibody to confirm the identification of
NQO2 as specifically enriched protein by an independent
detection method in both cell types (Figure 4A and Supporting
Information Figure S4A). Specific interaction of 1 with NQO2
was also confirmed by Western Blot in microsomes derived
from a pool of human liver donor samples (Supporting
Information Figure S4B).
Figure 2. Purified human recombinant thrombin is captured by CCs 5
and 7 but not by 6 and 8. (A) Silver stained SDS-PAGE shows specific
enrichment. (B) Anti-biotin blot of the same experimental setup
demonstrates covalent cross-linking of CCs 5 and 7 to thrombin.
Competition controls (Comp.) were preincubated with 1 or 3 as
indicated. P = 0.3 μg thrombin protein input control.
To show direct binding of 1, we then carried out capture
experiments with pure recombinant NQO2 (Figure 4B). Cross-
competition experiments using a CCs 5 and 6 and competing
with 4 or vice versa (Figure 4C) demonstrated that the
inhibitors 1 and 4 target the same binding pocket.
Orientation specificity of CCs 5 and 6 and cross-competition
with 4 gave first experimental insights into the binding mode of
1 to NQO2. On the basis of our data and published NQO2
crystal structures, we computed a molecular model. The active
form of NQO2 is dimeric and comprises two active sites
(Supporting Information Figure S5). The quinone binding
pocket is formed by FAD bound to one chain and residues
Figure 3. Volcano plot of quadruplicate CCMS experiments using
CCs 5−8 in HepG2 lysate. Specific proteins (boxed area) are
reproducibly identified in capture assays but not in competition
controls and therefore exhibit high fold change (x-axis) and significant
p-value (y-axis).
CCMS can identify even proteins expressed at low level
because of the enrichment procedure. In the case of CCMS, the
covalent bond formed between the target protein and the CC
allows for stringent washing steps during the isolation process,
a
Table 1. Proteins Significantly Enriched by CCMS Experiments using CCs 5−8 in HepG2 Cell Lysate
SwissProt
ID
Mw
fold
enriched in
f
b
c
d
e
protein
NQO2
full name
function
dehydrogenase
[kDa]
PI
compound change
p-value
K652 lysate
yes
P16083
ribosyldihydronicotinamide
dehydrogenase
26
5.88
5
∞
0.0016
HNMT
HYOU1
HNRPC
P50135
Q9Y4L1
P07910
histamine N-methyltransferase methyltransferase
33
111
34
5.18
5.07
4.95
6
6
6
∞
∞
∞
0.0099
0.0024
0.0025
no
no
no
hypoxia up-regulated protein 1 hypoxia up-regulated protein 1
heterogeneous nuclear
ribonucleoproteins C1/C2
function in mRNA processing
and splicing
ADK
P55263
adenosine kinase
kinase
41
35
6.25
9.61
6
8
2.8
2
0.0321
0.0240
no
no
LRC59
Q96AG4 leucine-rich repeat-containing
protein 59
membrane protein of the ER with
unknown function
VP35
Q96QK1 vacuolar protein sorting-
associated protein 35
essential component of the
retromer complex
92
5.32
8
2.8
0.0321
no
a
b
c
All protein properties were retrieved from SwissProt Database if not indicated otherwise. Calculated isoelectric point. CC with which the named
d
protein was enriched from HepG2 cell lysate. Ratio of the spectral counts with which the protein was identified in the four capture assays and in the
four competition controls (A/C). In cases where the number of spectral counts in the competition was zero, this value is ∞. Significance of
enrichment in the four capture assays compared to the four competition controls as determined by Student's t test. The capture experiments in
e
f
K652 cells were carried out in duplicate as additional controls.
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Figure 4. NQO2 is specifically captured and enriched by CC 5 and
CC 9. (A) Enrichment of endogenous NQO2 from HepG2 lysate
detected by anti-NQO2 Western Blot. (B) Silver stained SDS-PAGE
showing direct interaction of 1 with NQO2 by capturing of human
purified recombinant NQO2 protein. (C) 1, 2, and 4 occupy the same
NQO2 binding site, as demonstrated by cross-competition experi-
ments visualized by silver stained SDS-PAGE. Final concentrations of
CCs 5 μM and competitors 100 μM, P = 0.3 μg of recombinant
NQO2.
Figure 5. Molecular model showing 2 (gray) bound to NQO2. Protein
residues are depicted in green, the cofactor FAD in purple. (top)
Crucial binding residues: Glu193 exhibits a salt bridge to the amidine
groups of 1 and 2. An aromatic cage consisting of several Phe and Tyr
residues interacts with the central aromatic core. Met154 and Ile128
interact via Van der Waals interactions. The interaction of Ile128 with
the ethyl group of 2 (Van der Waals radii of these moieties are shown
as translucent surfaces) should stabilize binding of 2 rather than 1 in
accordance with the experimental data. (bottom) Surface of the
binding pocket colored by lipophilic potential (green, hydrophilic;
yellow, lipophilic).
from both chains and can harbor a flat, aromatic ligand lying
coplanar to the flavones redox center, stabilized by π-
interactions. This is the case for the central core of 1, but
not of 3, thus explaining selective binding of 1 over 3. The
amidine of 1 is part of a hydrogen-bond network, whereas the
acid functionality protrudes into the solvent, explaining the
orientation specificity. Our model also clarifies why the closest
relative of NQO2, which is NQO1 with 49% sequence identity,
was not specifically isolated: NQO1s Phe233 and Phe237 make
the catalytic pocket too narrow for 1. Because the acid
functionality of 1 is surrounded by hydrophobic residues, we
speculated that the more lipophilic ester 2 would further
stabilize the interaction between NQO2 and 1 (Figure 5)
(additional models as well as sequence alignment of NQO2 and
NQO1 are given in the Supporting Information).
We decided to experimentally follow up the modification
suggested by our model and investigated competition of CC 5
binding to recombinant NQO2 using 1 and 2 as competitors
(Supporting Information Figure S8). The concentrations at
which half of the NQO2 signal was lost were 10 μM for 1 and
1.5 μM for its ester 2, demonstrating almost 1 order of
magnitude stronger interaction of the ester over acid 1.
Figure 6. Michaelis Menten plots showing functional inhibition of
NQO2 catalysis by 1 (A) and by 2 (B). 4 (C) was used as positive
control. Experiments were carried out twice acquiring duplicate data
points per experiment.
We then investigated the functional inhibition of NADH-
dependent mitomycin C metabolism17 by NQO2 using 1 and 2
as inhibitors as well as 4 as positive control (Figure 6). The Ki
values were computed using Sigma Plot 10. IC50 values were
calculated based on Michaelis Menten kinetics for competitive
inhibition. 1 competitively and concentration-dependently
inhibited activated NQO2. The Ki could be approximated to
70 μM and the IC50 to 60 μM. Notably, 2 exhibits a much
higher inhibitory potential on NQO2 in the assay described,
with a Ki = 0.9 μM and an IC50 = 0.8 μM. The half maximal
inhibitory concentration of 4 was 0.34 μM in accordance with
summary of all affinity and inhibition data generated is given
in Table 2.
1 is administered in the form of an ester to increase the
bioavailability,15 therefore both forms (the ester and the drug)
are present. We determined the Ki of 2 toward NQO2 to be 0.9
μM and the IC50 to be 0.8 μM, thus we expect a contribution to
the mode of action. Because we could show functional
inhibition of recombinant NQO2 by 1 and 2 and also binding
of 1 and 2 to endogenous NQO2 within whole cell lysates and
lysates derived from human liver microsomes (Figure 4A and
the literature (Supporting Information Figure S10).17
A
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calculations and interaction surfaces see Supporting Informa-
tion).
Table 2. Affinities and Functional Inhibition between 1, 2,
and 4 and NQO2 or Thrombin
Taken together, our experimental and modeling results
clearly show the direction in which to modify the structure of 1
in order to tune its binding properties toward higher affinity.
This was indeed reflected by the inhibitory effect on enzyme
functions in vitro. While at present the physiological effects of
these interactions remain open, the strategy devised here
indicates that capture compound mass spectrometry-driven
drug profiling not only reveals the interaction profile of the
drug molecule but also can generate critical data that can guide
medicinal chemistry efforts to redesign a drug molecule toward
new properties.
CC
assay
1
2
4
half maximal competition of NQO2 binding through 11
1.5
nd
a
CC 5 (μM)
b
IC50, functional inhibition of NQO2 (μM)
>10
0.8
0.9
1.2
0.40
nd
c
Ki, functional inhibition of NQO2 (μM)
60
d
IC50, functional inhibition of thrombin (nM)
13.8
nd
a
Details are given in the Supporting Information, Figures S8 and S9.
b
c
Details are given in Supporting Information, Figure S10. Details are
d
given in Figure 6. Details are given in Supporting Information, Figure
S11.
CONCLUSION
■
In summary, we here identified NQO2 as novel target for the
thrombin inhibitor 1 by using a workflow combining medicinal
chemistry, proteomics, and molecular modeling. Our study is in
line with the notion that small-molecule drugs specifically
interact not only with one target protein but with a set of target
proteins. At least one of the previously unknown interactions
we identified for 1 is functionally relevant in vitro. Furthermore,
we were able to show that the ethyl ester of 1 has improved the
binding properties toward both NQO2 and thrombin, resulting
in higher affinity. In the context of NQO2 inhibition by
resveratrol,32 prevention of toxic effects by metabolism of
xenobiotics as well as reduction of cancer cell growth through
NQO2 inhibitors have been described.32−35 Our findings
suggest that it might be interesting to further investigate the
possible effect of the interaction between 1 and NQO2 on the
overall mode of action of 1. The results also show for the first
time how CCMS can be employed, not only to identify a drug’s
interactors and elucidate its mechanism of action but also how
these results can be used to tune the binding properties of a
given drug in the desired direction.
Supporting Information Figure S4), these compounds should
also be active within the cellular context of the cytosol where
NQO2 is located. Although it is to be expected that the ester
will be hydrolyzed, the more active precursor 2 will in part
contribute to the mode of action of dabigatran. The extent of
this contribution will depend strongly on its pharmacokinetics,
protein binding, and on the patients (e.g., with decreased liver
function) treated with the drug molecule.
To investigate whether the modification of 1 also affected its
inhibitory potential toward thrombin, we analyzed the
proteolytic properties of thrombin in the presence of 1 and
2, respectively. For 1, we determined an IC50 value of 14 nM
toward thrombin, which is in good accordance with the value of
10 nM reported previously.20,29 In the case of 2, the inhibitory
potential was even increased by 1 order of magnitude (1.2 nM,
Supporting Information Figure S11). NQO2 is a flavoprotein
that catalyzes the 2-electron reduction of various quinones,
redox dyes, and the vitamin K menadione. NQO2 predom-
inantly uses dihydronicotinamide riboside (NRH) as the
electron donor. The possible physiological relevance of
NQO2 inhibition by 1 or 2 remains to be tested. In fact, the
finding that 2 is 1 order of magnitude more active on thrombin
than 1 is quite surprising considering the X-ray studies of 1 the
thrombin system published by Hauel et al.20 Here, the
carboxylate group protrudes into bulk solvent, exhibiting no
interactions with the protein. However, a more hydrophobic
moiety like an ethyl ester could adopt a conformation that
would allow hydrophobic contacts of the whole chain to the
rim of the pocket.
One single heavy atom can increase the overall binding
energy by max 1.5 kcal/mol, which correlates to approximately
1 order of magnitude in the binding constant, although the
average binding per atom decreases with increasing heavy atom
count.30,31 We calculated the ligand binding efficiencies (LE:
atom-count dependent binding description)30 and the fit
qualities (FQ: normalized LEs)31 for both 1 and 2. For both
compounds, we calculated FQ values of around 0.6 for
thrombin and FQ values of around 0.4 for NQO2.
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise noted, all reactions were performed
in dry glassware and under argon. Commercially available reagents and
solvents were used as received without further purification. Column
chromatography was carried out by using Geduran Si 60 silica gel from
Merck. MPLC purification was performed on a Buechi system (Buechi
control unit C-620; 2 Buechi pump modules C-605; column, omnifit
(230 × 15 mm); stationary phase, LiChroprep RP-select B 25−40 μm;
Buechi UV photometer C-635; Buechi fraction collector C-660;
mobile phase A, 0.1% AcOH Millipore grade; mobile phase B, MeCN
prepsolv grade). LCT was carried out on a Waters system (HPLC
pump Waters 1525; autosampler CTC HTS PAL; column oven
Techlab K7 (40 °C); diode array detector Waters 2998 (200−600
nm); MS detector Waters LCT mass spectrometer equipped with an
electrospray ion source). LCT method A: column, Phenomenex
Mercury MS (20 mm × 2 mm); stationary phase, Phenomenex
Synergi Fusion RP, 2.5 μm; guard column Phenomenex Security
Guard, Synergi Fusion RP; flow 0.4 mL/min; mobile phase A, 0.1%
HCOOH Millipore grade; mobile phase B, MeCN LC-MS grade; 10%
B to 40% B in 9 min; 40% B to 100% B in 2 min; 100% B for 4 min.
LCT method B: column, Phenomenex Kinetex (50 mm × 3 mm);
stationary phase, Phenomenex Kinetex C18, 2.6 μm, 100 Å; guard
column Phenomenex Security Guard, Synergi Fusion RP; flow 0.8
mL/min; mobile phase A, 0.1% HCOOH Millipore grade; mobile
phase B, MeCN LC-MS grade; 10% B to 40% B in 2 min; 40% B to
100% B in 1.5 min; 100% B for 1.5 min. Accurate mass spectrometric
analysis was performed on an LTQ Orbitrap XL mass spectrometer
(Thermo Electron, Bremen, Germany). For the analysis, a 5 μM
solution of the corresponding sample in water/MeCN (50/50, 0.1%
Bigger ligands in general30,31 and 1 in particular20 are known
to interact mainly by hydrophobic interactions. Thus the ratio
of the interacting surfaces of 1 and its ethyl ester should be in
the range of the ratio of the pK values. We calculated a ratio of
0.88−0.91, which is in good correlation with the pK ratio from
the NQO2 binding assay (0.86). Supporting Information
Figure S12 shows that the ethyl ester group of 2 significantly
extends the interaction surface especially with hydrophobic
amino acids such as Ile 128 and Met 154 (for details of the LE
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Journal of Medicinal Chemistry
Article
formic acid) was directly injected via the syringe pump with a flow rate
of 1 μL/min. The singly charged polydimethylcyclosiloxane back-
ground ion (Si(CH3)2O)6H+ (m/z 445.120025) generated during the
electrospray process from ambient air was used as lock mass for real
time internal recalibration. Further mass spectrometric settings were as
follows: spray voltage was set to 1.6 kV, temperature of the heated
transfer capillary was set to 200 °C. MS spectra (from m/z 400−2000)
were acquired in the orbitrap with a resolution of r = 60000 at m/z
400 (after accumulation to a target value of 500000 charges in the
linear ion trap). Proton nuclear magnetic resonance (1H NMR)
spectra and carbon nuclear magnetic resonance (13C NMR) spectra
were recorded on an Avance 400 (400 MHz) spectrometer from
Bruker. Chemical shifts are reported in ppm relative to CHCl3 (δ 7.26)
133.4, 131.9, 130.5, 129.4, 127.7, 124.8, 124.4, 123.1, 120.6, 117.1,
113.5, 110.7, 71.5, 71.4, 71.2, 71.2, 69.9, 63.4, 61.6, 57.0, 52.7, 48.0,
43.6, 41.2, 41.1, 39.6, 38.7, 38.2, 37.9, 37.2, 36.8, 30.6, 30.4, 30.3, 29.8,
29.5, 27.9, 26.9, 26.0. LCT method B: tR = 3.12 min; calcd for
C62H79F3N15O11S [MH]+, 1298.57508; obsd, 1299.2 (20%); 650.1
(100%). HRMS: obsd, 1298.57603 (+0.7 ppm).
Capture Compound 7. At 0 °C, a solution of 3 (5.0 mg, 12 μmol)
and scaffold X-NH-C4H8-NH2 (9.3 mg, 11 μmol) in dry DMA (0.3
mL) was treated with DIPEA (9.7 μL, 55 μmol) and HATU (4.64 mg,
12 μmol). The reaction solution was stirred for 10 min at 0 °C, was
allowed to warm to 23 °C, and was stirred additionally 17 h with
exclusion of light. As LCT indicated incomplete conversion, HATU
(1.9 mg, 5 μmol) and 3 (1.0 mg, 2 μmol) were added and stirring was
continued for 17 h with exclusion of light. The solvent was removed
under reduced pressure, and the crude product was directly purified by
MPLC (eluent A, 0.1% AcOH; eluent B, MeCN; 0% B to 45% B in 20
min) to yield a pure fraction of 7 (9.3 mg, 7 μmol, 67%) as a colorless
1
for H NMR and the central resonance of CDCl3 (δ 77.0) for 13C
NMR. The purity of compounds (≥95%) was confirmed by elution as
a single peak by LCT and NMR spectra. The synthesis of the capture
compound scaffolds19 4, 9, and 10 is published elsewhere.12
1
Capture Compound 5. A solution of 1 (34.0 mg, 72 μmol) and
scaffold X-NH-C4H8-NH2 (73.0 mg, 87 μmol) in dry DMA (10 mL)
was treated with DIPEA (63 μL, 361 μmol), HOBt (16.6 mg, 108
μmol), DMAP (44.0 mg, 361.0 μmol), and DCC (22.3 mg, 108
μmol). The reaction mixture was stirred for 17 h at 50 °C with
exclusion of light. The solvent was removed under reduced pressure,
and the crude product was directly purified by MPLC (eluent A, 0.1%
AcOH; eluent B, MeCN; 20% B to 50% B in 20 min) to yield a pure
fraction of 5 (52.0 mg, 40 μmol, 56%). 1H NMR (400 MHz, MeOD):
δ (ppm) = 8.36 (ddd, J = 4.9, 1.9, and 0.8 Hz, 1H), 7.93/7.32 (AA′BB′,
J = 8.5 Hz, 4H), 7.61/6.85 (AA′BB′, J = 8.9 Hz, 4H), 7.58 (dd, J = 1.5
and 0.6 Hz, 1H), 7.53 (ddd, J = 8.1, 7.5, and 1.9 Hz, 1H), 7.37 (dd, J =
8.5 and 0.6 Hz, 1H), 7.21 (dd, J = 8.5 and 1.5 Hz, 1H), 7.12 (ddd, J =
7.5, 4.9, and 0.8 Hz, 1H), 6.97 (dt, J = 8.1 and 0.8 Hz, 1H), 4.9 (m
below H2O signal), 4.69 (br, s, 2H), 4.47 (ddd, J = 7.8, 4.9, and 0.8 Hz,
1H), 4.34 (t, J = 7.0 Hz, 2H), 4.29 (dd, J = 7.9 and 4.5 Hz, 1H), 3.82
(s, 3H), 3.61−3.52 (m, 8H), 3.50 (t, J = 6.1 Hz, 2H), 3.49 (t, J = 6.1
Hz, 2H), 3.27 (t, J = 6.8 Hz, 2H), 3.24 (t, J = 6.8 Hz, 2H), 3.18 (ddd, J
= 8.9, 5.7 and 4.5, 1H), 3.17−3.13 (m, 2H), 3.10−3.05 (m, 2H), 2.91
(dd, J = 12.7 and 4.9 Hz, 1H), 2.77 (dd, J = 14.9 and 5.8 Hz, 1H), 2.70
(dd, J = 14.9 and 7.8 Hz, 1H), 2.69 (br d, J = 12.4 Hz, 1H), 2.63 (t, J =
6.8 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.77−1.52 (m, 8H), 1.50−1.37
(m, 6H). 13C NMR (100.6 MHz, MeOD): δ (ppm) = 175.9, 173.4,
173.2, 172.9, 172.3, 168.4, 167.2, 166.1, 157.4, 155.1, 154.7, 149.9,
141.8, 139.5, 138.7, 136.6, 133.4, 131.4, 130.7, 129.3, 127.7, 124.9,
124.3, 123.4 (q, J = 274.1 Hz), 123.1, 120.8, 115.7, 113.4, 110.8, 71.5,
71.5, 71.2, 71.2, 69.9, 69.9, 63.4, 61.6, 57.0, 52.7, 47.0, 41.1, 41.1, 40.1,
40.0, 38.5, 38.1, 37.8, 36.8, 35.8, 30.6, 30.4, 30.3, 29.8, 29.5, 29.5 (q, J =
41.2 Hz), 27.7, 27.6, 26.9. LCT method B: tR = 3.15 min; calcd for
C62H80F3N16O10S [MH]+, 1297.59106; obsd, 1298.2 (75%); 649.6
(100%). HRMS: obsd, 1297.58853 (−1.9 ppm).
Capture Compound 6. At 0 °C, a solution of compound 12 (1.0
mg, 0.75 μmol) in a solvent mixture of THF and water (15 μL, 2/1 v/
v) was treated with a solution of lithium hydroxide (0.054 mg, 2.3
μmol) in water. The reaction mixture was stirred for 17 h at 4 °C. At 0
°C, the pH was adjusted to 4 by addition of diluted acetic acid
solution. The crude product was directly purified by HPLC (eluent A,
0.1% HCOOH; eluent B, MeCN; 10% B for 7 min; 10% B to 100% B
in 1 min; 100% B for 16 min) to give 6 (0.47 mg, 0.36 μmol, 48%). 1H
NMR (400 MHz, MeOD): δ (ppm) = 8.34 (ddd, J = 4.9, 1.9, and 0.7
Hz, 1H), 7.93/7.31 (AA′BB′, J = 8.5 Hz, 4H), 7.59 (br s, 1H), 7.55 (td,
J = 7.6 and 1.9 Hz, 1H), 7.49/6.83 (AA′BB′,J = 8.8, 4H), 7.35 (br d, J =
8.5 Hz, 1H), 7.29 (dd, J = 8.5 and 1.5 Hz, 1H), 7.12 (ddd, J = 7.5, 4.9,
and 0.9 Hz, 1H), 7.06 (dt, J = 8.1 and 0.8 Hz, 1H), 4.9 (m below H2O
signal), 4.68 (br s, 2H), 4.47 (ddd, J = 7.9, 5.0, and 0.8 Hz, 1H), 4.33−
4.27 (m, 3H), 3.82 (s, 3H), 3.60−3.50 (m, 8H), 3.49 (t, J = 6.1 Hz,
2H), 3.47 (t, J = 6.1 Hz, 2H), 3.39−3.33 (m, 2H), 3.27−3.21 (m, 6H),
3.18 (ddd, J = 8.9, 5.8, and 4.5 Hz, 1H), 2.90 (dd, J = 12.8 and 5.0 Hz,
1H), 2.83 (dd, J = 14.9 and 6.0 Hz, 1H), 2.69 (dd, J = 14.9 and 7.5 Hz,
1H), 2.69 (br d, J = 12.6 Hz, 1H), 2.63−2.57 (m, 2H), 2.17 (t, J = 7.4
Hz, 2H), 1.76−1.52 (m, 12H), 1.46−1.37 (m, 2H). 13C NMR (100.6
MHz, MeOD): δ (ppm) = 178.7, 175.9, 173.1, 172.8, 172.7, 168.5,
166.1, 164.9, 157.5, 155.0, 154.1, 149.9, 141.8, 139.5, 138.5, 136.6,
solid. H NMR (400 MHz, MeOD): δ (ppm) = 7.95/7.36 (AA′BB′, J
= 8.3 Hz, 4H), 7.76/7.57 (AA′BB′, J = 8.5 Hz, 4H), 4.9 (m below H2O
signal, C(x)-H), 4.8 (m below H2O signal, C(x)-H), 4.63/4.47 (AB, J
= 15.9 Hz, 2H), 4.49 (ddd, J = 7.7, 5.0, and 0.9 Hz, 1H), 4.30 (dd, J =
7.9 and 4.4 Hz, 1H), 4.32−4.28 (m, 2H), 3.62−3.53 (m, 8H), 3.52−
3.49 (m, 4H), 3.35−3.12 (m, 9H), 3.19/3.08 (AB, J = 16.1 Hz, 2H),
2.94 (dd, J = 6.5 and 1.3 Hz, 1H), 2.92 (dd, J = 12.5 and 4.9 Hz, 1H),
2.80 (dd, J = 14.9 and 6.0 Hz, 1H), 2.70 (br d, J = 12.5 Hz, 1H), 2.69
(dd, J = 14.9 and 7.6 Hz, 1H), 2.59 (dtd, J = 11.4, 9.4, and 6.3 Hz,
1H), 2.31 (ddt, J = 11.5, 9.4, and 5.7 Hz, 1H), 2.18 (t, J = 7.4 Hz, 2H),
2.00 (br d, J = 12.5 Hz, 1H), 1.90 (s, 3H), 1.83−1.38 (m, 19H), 1.33−
1.14 (m, 4H), 1.05 (m, 1H). 13C NMR (100.6 MHz, MeOD): δ
(ppm) = 176.1, 176.0, 174.1, 173.0, 172.9, 172.3, 168.4, 166.1, 146.9,
136.6, 133.4, 129.4, 129.2, 129.1, 127.7, 71.5, 71.5, 71.2, 71.2, 70.0,
69.9, 63.5, 63.4, 62.6, 61.6, 57.0, 52.7, 51.5, 50.4, 43.6, 42.0, 41.1, 40.0,
39.7, 38.5, 38.2, 37.9, 36.9, 30.9, 30.4, 30.3, 29.8, 29.5, 27.8, 27.7, 27.5,
27.3, 27.2, 26.9. LCT method B: tR = 3.05 min; calcd for
C59H86F3N14O11S [MH]+, 1255.62678; obsd, 1256.2 (15%); 628.6
(100%). HRMS: obsd, 1255.62629 (−0.4 ppm).
Capture Compound 8. At 0 °C, a solution of compound 17 (8.6
mg, 7 μmol) in a solvent mixture of THF and water (1.5 mL, 1/1 v/v)
was treated with lithium hydroxide (0.49 mg, 20 μmol). The reaction
mixture was stirred for 1 h at 0 °C. THF (1.5 mL) was added, and the
pH was adjusted to 7 by addition of 2 M HClaq at 0 °C. The solvents
were removed under reduced pressure, and the crude product was
directly purified by MPLC (eluent A, 0.1% AcOH; eluent B, MeCN;
10% B to 40% B in 5 min; 40% B for 7 min) to give 8 (6.6 mg, 5 μmol,
78%). 1H NMR (400 MHz, MeOD): δ (ppm) = 7.95/7.33 (AA′BB′, J
= 8.5 Hz, 4H), 7.77/7.42 (AA′BB′, J = 8.2 Hz, 4H), 4.9 (m below H2O
signal), 4.8 (m below H2O signal), 4.53/4.46 (AB, J = 15.5 Hz, 2H),
4.48 (ddd, J = 7.9, 5.0, and 0.7 Hz, 1H), 4.37 (td, J = 9.0 and 6.4 Hz,
1H), 4.29 (dd, J = 7.9 and 4.5 Hz, 1H), 4.23 (td, J = 9.0 and 5.6 Hz,
1H), 3.62−3.53 (m, 8H), 3.51 (t, J = 6.2 Hz, 2H), 3.50 (t, J = 6.0 Hz,
2H), 3.40−3.20 (m, 11H), 3.18 (m, 1H), 2.91 (dd, J = 12.8 and 5.0
Hz, 1H), 2.79 (dd, J = 14.8 and 5.9 Hz, 1H), 2.72 (dd, J = 14.8 and 7.9
Hz, 1H), 2.70 (br d, J = 12.7 Hz, 1H), 2.62 (dtd, J = 11.4, 9.2, and 6.4
Hz, 1H), 2.31 (ddt, J = 11.4, 9.2, and 5.6 Hz, 1H), 2.18 (t, J = 7.4 Hz,
2H), 1.90 (br d, J = 12.4 Hz, 1H), 1.86−1.51 (m, 17H), 1.46−1.36 (m,
2H), 1.33−1.11 (m, 4H), 1.06 (m, 1H). 13C NMR (100.6 MHz,
MeOD): δ (ppm) = 176.0, 175.1, 173.0, 172.3, 172.2, 170.0, 168.5,
166.1, 161.6, 143.6, 136.6, 134.6, 133.4, 129.4, 128.6, 128.5, 127.7,
71.5, 71.5, 71.2, 71.2, 70.0, 69.9, 63.4, 63.0, 62.4, 61.6, 57.0, 52.8, 50.7,
43.8, 41.1, 40.9, 40.5, 40.1, 38.6, 38.2, 37.9, 36.9, 30.4, 30.3, 29.9, 29.8,
29.8, 29.5, 27.7, 27.0, 26.9. LCT method B: tR = 3.42 min; calcd for
[MH]+, 1257.59481; obsd, 1258.2 (100%); 629.6 (30%). HRMS:
obsd, 1257.59488 (−0.055 ppm).
Ethyl 3-(2-((4-(N-(4-(tert-Butoxycarbonylamino)butyl)-
carbamimidoyl)phenylamino)methyl)-1-methyl-N-(pyridin-2-
yl)-1H-benzo[d]imidazole-5-carboxamido)propanoate (11). In
a screw top vial, a solution of dabigatran ethyl ester acetate (10.0 mg,
18 μmol) (from Suzhou Unite PharmTech Co.) in dry EtOH (25 μL)
was treated with tert-butyl 4-aminobutylcarbamate (33.6 mg, 180
μmol). The vial was tightly screwed, and the reaction mixture was
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Journal of Medicinal Chemistry
Article
stirred at 78 °C for 12 h. As LCT analyses showed almost complete
conversion, the solvent was removed by argon stream and the crude
product was directly purified by MPLC (eluent A, 0.1% AcOH; eluent
B, MeCN; 30% B to 40% B in 6 min; 40% B for 4 min) to give desired
124.6, 77.9, 43.1, 42.3, 38.5, 28.2, 26.6, 26.2. LCT method B: tR = 3.85
min; calcd for C23H31N4O7S [MH]+, 507.19080; obsd, 507.4 (20%);
451.3 (100%). HRMS: obsd, 507.19043 (−0.73 ppm).
t e r t - B u t y l ( S ) - 1 - C y c l o h e x y l - 2 - ( ( R ) - 2 - ( 4 - ( 4 - ( 4 -
nitrophenylsulfonamido)butylcarbamoyl)benzylcarbamoyl)-
azetidin-1-yl)-2-oxoethylcarbamate (15). At 0 °C, a suspension of
compound 14 (858 mg, 1.69 mmol) in a solvent mixture of DCM and
MeOH (18 mL, 1/1 v/v) was treated with TFA (4 mL). The reaction
mixture was stirred at 0 °C for 1 h, was allowed to warm to 23 °C, and
was stirred additionally 2 h at 40 °C. As LCT indicated full conversion,
all volatile materials were removed under reduced pressure. The crude
product was coevaporated twice with DCM, dried under high vacuum,
and dissolved in dry DMA (0.2 mL). At 0 °C, this solution was added
1
product 11 (10.9 mg, 16 μmol, 91%) as a colorless solid. H NMR
(400 MHz, MeOD): δ (ppm) = 8.39 (dd, J = 5.0 and 1.8 Hz, 1H),
7.57 (br d, J = 1.2 Hz, 1H), 7,52 (m, 1H), 7.52/6.84 (AA′BB′, J = 8.8
Hz, 4H), 7.37 (d, J = 8.5 Hz, 1H), 7.28 (dd, J = 8.4 and 1.3 Hz, 1H),
7.14 (dd, J = 7.3 and 5.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 4.68 (br s,
2H), 4.36 (t, J = 7.0 Hz,2H), 4.05 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H),
3.40 (t, J = 7.2 Hz, 2H), 3.09 (t, J = 6.9 Hz, 2H), 2.75 (t, J = 7.0 Hz,
2H), 1.75−1.67 (m, 2H), 1.61−1.54 (m, 2H), 1.43 (s, 9H), 1.20 (t, J =
7.2 Hz, 3H). 13C NMR (100.6 MHz, MeOD): δ (ppm) = 173.2, 173.1,
165.1, 158.8, 157.3, 155.2, 154.1, 150.0, 141.8, 139.4, 138.7, 131.3,
130.4, 124.9, 124.4, 123.1, 120.7, 117.3, 113.4, 110.7, 80.0, 61.8, 46.2,
43.6, 41.2, 40.5, 34.1, 30.6, 28.8, 28.5, 26.1, 14.4. LCT method B: tR =
3.16 min, calcd for C36H47N8O5 [MH] +, 671.36639; obsd, 671.6
(80%). HRMS: obsd, 671.36462 (−2.6 ppm).
dropwise to
a solution of (S)-1-((R)-2-(Boc-amino)-2-
cyclohexylacetyl)azetidine-2-carboxylic acid (576 mg, 1.69 mmol),
DIPEA (2.95 mL, 16.9 mmol), and HATU (708 mg, 1.86 mmol) in
dry DMA (0.2 mL). The reaction mixture was stirred for 5 min at 0
°C, was allowed to warm to 23 °C, and was stirred for additional 2 d
with exclusion of light. The solvent was removed under reduced
pressure, and the crude product was directly purified by column
chromatography (eluent A, DCM; eluent B, MeOH; 1% B to 4% B) to
yield 15 (1.18 g, 1.62 mmol, 96%) as a colorless foam. 1H NMR (400
MHz, CDCl3): δ (ppm) = 8.29/8.03 (AA′BB′, J = 8.7 Hz, 4H), 8.25
(br t, J = 6 Hz, 1H), 7.64/7.25 (AA′BB′, J = 8 Hz,4H), 6.9 (br s, 1H),
6.27 (br t, J = 5.7 Hz, 1H), 5.16 (br d, J = 5 Hz, 1H), 4.86 (dd, J = 9.0
and 5.7 Hz, 1H), 4.57 (dd, J = 15.6 and 6.3 Hz, 1H), 4.42 (td, J = 8.4
and 5.8 Hz, 1H), 4.32 (dd, J = 15.7 and 5.1 Hz, 1H), 4.18 (td, J = 8.5
and 5.7 Hz, 1H), 3.77 (t, J = 7.6 Hz, 1H), 3.36−3.26 (m, 2H), 2.99−
2.91 (m, 2H), 2.64 (m, 1H), 2.44 (m, 1H), 1.83 (br d, J = 12.2 Hz,
1H), 1.80−1.44 (m, 9H), 1.30 (s, 9H), 1.26−0.96 (m, 5H). 13C NMR
(100.6 MHz, CDCl3): δ (ppm) = 172.9, 171.3, 168.4, 156.3, 150.0,
146.3, 142.4, 132.8, 128.4, 127.5, 127.2, 124.4, 80.4, 61.8, 55.3, 48.9,
43.1, 42.9, 39.9, 39.5, 29.7, 29.1, 28.4, 26.7, 26.0, 25.9, 20.3. LCT
method A: tR = 10.88 min; calcd for C35H49N6O9S [MH]+, 729.32762;
obsd, 729.0 (5%); 629.0 (100%). HRMS: obsd, 729.32700 (−0.85
ppm).
M e t h y l 2 - ( ( S ) - 1 - C y c l o h e x y l - 2 - ( ( R ) - 2 - ( 4 - ( 4 - ( 4 -
nitrophenylsulfonamido)butylcarbamoyl)benzylcarbamoyl)-
azetidin-1-yl)-2-oxoethylamino)acetate (16). At 0 °C, a solution
of compound 15 (250 mg, 343 μmol) in dry DCM (20 mL) was
treated with TFA (5 mL) and stirred for 30 min at 0 °C. As TLC
indicated full conversion, all volatile materials were removed under
reduced pressure. The crude product was coevaporated twice with
DCM and dried under high vacuum. Under an atmosphere of argon,
the crude product was dissolved in dry MeCN (17 mL) and treated
with methyl 2-(4-nitrophenylsulfonyloxy)acetate (113 mg, 412 μmol)
and potassium carbonate (119 mg, 858 μmol). The reaction mixture
was stirred at 60 °C. After 30 min, additional methyl 2-(4-
nitrophenylsulfonyloxy)acetate (113 mg, 412 μmol) was added and
stirring was continued for 1 h. The solvent was removed under
reduced pressure, and the crude product was directly purified by
column chromatography (eluent A, DCM; eluent B, MeOH; 1% B to
10%) to yield compound 16 as a nonpure product (168 mg) which
was used as such for the next step. LCT method A: tR = 6.37 min;
calcd for C33H45N6O9S [MH]+, 701.29632; obsd, 701.2 (100%).
Compound 17. Under an atmosphere of argon, a solution of
compound 16 (108 mg, 154 μmol) in dry MeCN (5 mL) was treated
with 2-mercaptoacetic acid (32.1 μL, 462 μmol) and DBU (116 μL,
771 μmol) and was stirred at 0 °C for 1 h. All volatile materials were
removed under reduced pressure, and the crude product was enriched
via MPLC (eluent A, 0.1% AcOH; eluent B, MeCN; 10% B to 40% B
in 30 min). All product containing fractions were combined, and the
solvents were removed under reduced pressure. The crude product
was dissolved in dry DMA (0.3 mL) and treated with scaffold X−OH
(27.2 mg, 35 μmol), DIPEA (55 μL, 320 μmol), and HATU (13.4 mg,
35 μmol). The reaction mixture was stirred for 10 min at 0 °C, was
allowed to warm to 23 °C, and was stirred additionally 17 h with
exclusion of light. The solvent was removed under reduced pressure
and the crude product was directly purified by MPLC (eluent A, 0.1%
AcOH; eluent B, MeCN; 10% B to 60% B in 30 min) to yield
Compound 12. At 0 °C, a solution of compound 11 (10.0 mg, 15
μmol) in dry DCM (1.0 mL) was treated with TFA (0.2 mL) and
stirred at 0 °C for 1 h. All volatile materials were removed under
reduced pressure. The crude product was coevaporated twice with
DCM, dried under high vacuum, and dissolved in dry DMA (0.3 mL).
At 0 °C, the solution was successively treated with scaffold X−OH
(13.8 mg, 18.0 μmol), DIPEA (26 μL, 15 μmol), and HATU (6.2 mg,
16 μmol). The reaction mixture was stirred for 10 min at 0 °C, was
allowed to warm to 23 °C, and was stirred for additional 2 d with
exclusion of light. The solvent was removed under reduced pressure,
and the crude product was directly purified by MPLC (eluent A, 0.1%
AcOH; eluent B, MeCN; 30% B to 45% B in 9 min; 45% B for 3 min)
1
to yield 12 (6.9 mg, 5 μmol, 35%). H NMR (400 MHz, MeOD): δ
(ppm) = 8.39 (ddd, J = 4.9, 1.9, and 0.7 Hz, 1H), 7.92/7.31 (AA′BB′, J
= 8.4 Hz, 4H), 7.58 (dd, J = 1.5 and 0.6 Hz, 1H), 7.51 (ddd, J = 8.1,
7.6, and 1.9 Hz, 1H), 7.49/6.83 (AA′BB′, J = 8.9 Hz, 4H), 7.37 (dd, J =
8.5 and 0.6 Hz, 1H), 7.28, J = 8.5 and 1.5 Hz, 1H), 7.13 (ddd, J = 7.5,
4.9, and 0.8 Hz,1H), 6.93 (dt, J = 8.0 and 0.8 Hz,1H), 4.92 (dd, J = 5.8
and 1.5 Hz, 1H), 4.68 (br s, 2H), 4.47 (ddd, J = 7.8, 4.9, and 0.8 Hz,
1H), 4.36 (t, J = 7.0 Hz, 2H), 4.28 (dd, J = 7.8 and 4.4 Hz, 1H), 4.05
(q, J = 7.1 Hz, 2H), 3.83 (s, 3H), 3.60−3.50 (m, 8H), 3.49 (t, J = 6.1
Hz, 2H), 3.47 (t, J = 6.1 Hz, 2H), 3.39−3.35 (m, 2H), 3.27−3.21 (m,
6H), 3.18 (ddd, J = 9.0, 5.8, and 4.5 Hz,1H), 2.91 (dd, J = 12.8 and 4.9
Hz,1H), 2.83 (dd, J = 14.9 and 6.1 Hz,1H), 2,75 (t, J = 7.0 Hz,2H),
2.68 (dd, J = 14.8 and 7.4 Hz, 1H), 2.68 (br d, J = 12.8 Hz, 1H), 2.17
(t, J = 7.4 Hz, 2H), 1.78−1.49 (m, 12H), 1.46−1.37 (m, 3H), 1.20 (t, J
= 7.2 Hz,3H). 13C NMR (100.6 MHz, MeOD): δ (ppm) = 175.9,
173.2, 173.1, 172.8, 172.7, 168.4, 166.1, 164.9, 157.3, 155.2, 154.1,
150.0, 141.8, 139.5, 138.7, 136.6, 133.4, 131.3, 130.5, 129.3, 127.7,
124.9, 124.4, 123.1, 120.7, 117.1, 113.4, 110.8, 71.5, 71.4, 71.2, 71.2,
69.9, 63.4, 61.8, 61.6, 57.0, 52.6, 46.2, 43.6, 41.2, 41.0, 39.5, 38.6, 38.2,
37.9, 36.9, 34.1, 30.7, 30.4, 30.3, 29.8, 29.5, 27.9, 26.9, 26.0, 14.4. LCT
method B: tR = 3.34 min; calcd for C64H83F3N15O11S [MH]+,
1326.60638; obsd, 1327.2 (50%), 664.1 (100%). HRMS: obsd,
1326.60599 (−0.3 ppm).
tert-Butyl 4-(4-(4-Nitrophenylsulfonamido)butylcarbamoyl)-
benzylcarbamate (14). A solution of 4-(tert-butoxycarbonylamino-
methyl)-benzoic acid (1.0 g, 3.98 mmol) in dry DMA (30 mL) was
treated with DIPEA (3.47 mL, 19.9 mmol) and HATU (1.59 g, 4.18
mmol). After stirring for 2 min, a solution of N-(4-aminobutyl)-4-
nitrobenzenesulfonamide (1.54 g, 3.98 mmol) in dry DMA (10 mL)
and DMAP (486 mg, 3.98 mmol) were added and the reaction mixture
was stirred for 1.5 h at 23 °C. DCM (250 mL) was added, and the
organic layer was washed with brine (2 times 100 mL). The organic
layer was dried over MgSO4, the solvents were removed under reduced
pressure, and the crude product was recrystallized from pure DCM to
1
yield compound 14 (1.72 g, 3.38 mmol, 85%) as a colorless solid. H
NMR (400 MHz, DMSO-d): δ (ppm) = 8.40/8.03 (br AA′BB′, J = 8.4
Hz, 4H), 8.35 (br s, 1H), 7.98 (br s, 1H), 7.74/7.28 (br AA′BB′, J = 7.4
Hz, 4H), 7.44 (br s, 1H), 4.16 (br d, 2H), 3.18 (br q, 2H), 2.82 (br q,
2H), 1.52 −1.28 (m, 13H). 13C NMR (100.6 MHz, DMSO-d): δ
(ppm) = 165.9, 155.8, 149.5, 146.2, 143.3, 133.0, 128.0, 127.1, 126.6,
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compound 17 (17.4 mg, 15 μmol, 43%) as a colorless solid. 1H NMR
(400 MHz, MeOD): δ (ppm) = 7.94/7.34 (AA′BB′, J = 8.5 Hz, 4H),
7.77/7.41 (AA′BB′, J = 8.2 Hz, 4H), 4.9 (m below H2O signal), 4.81
(dd, J = 9.4 and 5.6 Hz, 1H), 4.53/4.47 (AB, J = 15.7 Hz, 2H), 4.48
(dd, J = 7.7 and 5.0, 1H), 4.29 (dd, J = 7.8 and 4.5 Hz, 1H), 4.31−4.18
(m, 2H), 3.64 (s, 3H), 3.61−3.52 (m, 8H), 3.50 (t, J = 6.2 Hz, 2H),
3.50 (t, J = 6.0 Hz, 2H), 3.37−3.16 (m, 11H), 3.08 (d, J = 7.4 Hz, 1H),
2.91 (dd, J = 12.7 and 4.9 Hz, 1H), 2.80 (dd, J = 14.9 and 5.9 Hz, 1H),
2.72 (dd, J = 14.9 and 7.6 Hz, 1H), 2.70 (br d, J = 12.7 Hz, 1H), 2.60
(dtd, J = 11.4, 9.2, and 6.5 Hz, 1H), 2.28 (ddt, J = 11.4, 9.2, and 5.7
Hz, 1H), 2.18 (t, J = 7.4 Hz, 2H), 1.96 (br d, J = 12.7 Hz, 1H), 1.81−
1.51 (m, 17H), 1.46−1.38 (m, 2H), 1.32−1.12 (m, 4H), 1.04 (m, 1H).
13C NMR (100.6 MHz, MeOD): δ (ppm) = 175.9, 175.8, 174.2, 172.9,
172.7, 172.3, 169.8, 168.4, 166.1, 143.5, 136.6, 134.6, 133.4, 129.3,
128.6, 128.5, 127.7, 123.4 (q, J = 274.1 Hz), 71.5, 71.5, 71.2, 71.2, 69.9,
69.9, 63.4, 62.9, 62.5, 61.6, 57.0, 52.7, 52.3, 50.3, 43.7, 42.1, 41.1, 40.5,
40.1, 38.5, 38.1, 37.8, 36.8, 30.6, 30.4, 30.3, 29.8, 29.5 (q, J = 40.6 Hz),
29.5, 27.8, 27.4, 27.3, 27.2, 26.9. LCT method B: tR = 3.27 min; calcd
for C60H86F3N12O13S [MH]+, 1271.61046; obsd, 1272.0 (75%); 636.5
(100%). HRMS: obsd, 1271.60929 (−0.92 ppm).
Stability Tests for the Capture Compounds. The stability of all
CCs was evaluated for the following conditions: thermal stress
(incubation of CC native or denatured for 30 min at 4 and 80 °C
respectively) and biological stress (incubation of CC in HepG2 cell
lysates for 30 min at 4 and 37 °C, respectively). Moreover, it was
determined that after UV irradiation for 10 and 30 min in the
caproBox, only one defined reaction product is formed. The caproBox
is a device that enables controlled UV irradiation with simultaneous
cooling. The characteristics of the caproBox are as follows: capture
temperature 0.5−4 °C, λ = 275−375 nm with λmax = 312 nm,
irradiance Ie 10−12 mW/cm2, irradiation energy for each sample ca. 1
J. No degradation of the CC was observed when it was exposed under
normal overhead light for 1 h. The protocol for the experimental
determination of stability has been published.36
Silver Stained SDS-PAGE and Western Blots. were carried out
according to the standard laboratory procedures as published
previously.13 For silver staining, the ProteoSilver Kit (Sigma Aldrich,
Germany) was used according to the manufacturer’s instructions.
Antibodies were diluted as follows: anti-NQO2 1:500 (abcam,
Cambridge, UK) and secondary antirabbit antibody conjugated to
horseradish peroxidase 1:1000 (abcam, Cambridge, UK). In the case of
blots for the detection of biotinylated proteins, streptavidin−
horseradish peroxidase (Sigma Aldrich, Germany) was used instead
of a first antibody at a dilution of 1:1000 and blots were developed
directly after washing. Intensity of gel bands was determined using the
ImageJ software (Wayne Rasband, National Institues of Health, USA,
http://rsb.info.nih.gov/ij/freeware).
Protein Digestion. After protein capturing, streptavidin magnetic
beads were washed twice with 200 μL of LC-MS grade water (Fluka,
St. Louis, MO, USA). For tryptic digestion, the proteins bound to the
streptavidin magnetic beads were incubated with 9 μL of 50 mM
ammonium bicarbonate and 1 μL of trypsin (0.5 μg/μL) (Roche,
Germany) for 16 h at 37 °C on a temperature-controlled shaker. The
supernatant was removed and evaporated to dryness in a miVac DNA
vacuum centrifuge (Genevac, UK) and stored at −20 °C until mass
spectrometric analysis.
Mass Spectrometric Analysis. Tryptic digests were analyzed by
an online nanoflow liquid chromatography system (nLC Proxeon,
Biosystems, AIS, Denmark) connected to an LTQ Orbitrap XL mass
spectrometer (Thermo Electron, Bremen, Germany) utilizing a
nanoelectrospray ion source (Proxeon Biosystems A/S, Denmark).
First, 5μL of the peptide solution were loaded directly onto a
precolumn (nanoflow Biosphere C18, 5 μm, 120 Å, 20 mm × 0.1 mm;
NanoSeparations, Netherlands) coupled to an analytical column
(nanoflow Biosphere C18, 5 μm, 120 Å, 100 mm × 0.075 mm;
NanoSeparations, Netherlands) using 5% MeCN/0.1% HCOOH.
Then during the LC run, peptides were eluted during an 80 min linear
gradient from 5% MeCN/0.1% HCOOH to 40% MeCN/0.1%
HCOOH followed by an additional 2 min to 100% MeCN/0.1%
HCOOH and remaining at 100% MeCN/0.1% HCOOH for another
8 min with a controlled flow rate of 400 nL/min. The LC-MS analysis
was performed in the data-dependent mode to automatically switch
between orbitrap-MS (profile mode) and LTQ-MS/MS (centroid
mode) acquisition. The instrument was set to sequentially isolate the
most intense ions (up to five, depending on signal intensity) for
fragmentation in the linear ion trap using collision-induced
dissociation (CID) at a target value of 10000 charges. The resulting
fragment ions were recorded in the LTQ. Phosphorylation at serine,
threonine, and tyrosine, oxidation of methionines, deamidation at
asparagines and glutamine, acetylation at lysine and serine, formylation
at lysine, and methylation at arginine, lysine, serine, threonine, and
asparagine were allowed as variable modifications. No fixed
modifications were used in the database search. Mass spectrometric
settings: spray voltage to 1.6 kV, temperature of the heated transfer
capillary 200 °C, and normalized collision energy is 35% for MS2. The
minimal signal required for MS2 is 500 counts. An activation q = 0.25
and an activation time of 30 ms for MS2 acquisitions was applied.
MS-Data Annotation and Analysis. MS data were analyzed
using SEQUEST implemented in Bioworks Browser 3.3.1 SP1
(Thermo Fisher Scientific) and X!Tandem (www.thegpm.org, version
2007.01.01.1). Automated database search against the human
UniProtKB/Swiss-Prot database (release 57.6) was performed with 5
ppm precursor tolerance, 1 Da fragment ions tolerance, and full trypsin
specificity allowing for up to two missed cleavages. The estimated false
discovery rate of peptide identifications was determined using the
reversed protein database approach and was <0.5%. Only proteins
identified with more than 99% probability and at least two unique
peptides with 95% probability were considered to be identified with
sufficient confidence. Furthermore, these protein hits were manually
validated by inspection of the MS2 spectra. For statistical analysis,
Student's t test was carried out between the number of unweighted
spectral counts in the four capture assays and the four competition
controls. For the fold change, the average number of unweighted
spectral counts in the assays was divided by the average number of
Cell Lysates. The HepG2 and K562 cell lines were purchased from
the German collection of microorganisms and cell lines (DSMZ,
Braunschweig, Germany) and cultivated according to the provided
instructions. The protocol for the preparation of cell lysates has been
described elsewhere.13
CCMS Experiments. Protein concentrations were determined
according to Bradford.37 Protein input material was either 400 μg of
HepG2 or K562 cell lysate (400 μg) or 1 μg of human recombinant
NQO2 (Sigma Aldrich, Germany) or 2 μg of human recombinant
thrombin (Sigma Aldrich, Germany). The protein input was
supplemented with 20 μL of 5× capture buffer (caprotec bioanalytics
GmbH, Berlin, Germany). In the case of cell lysates, a preclearing step
was then carried out: beads from 50 μL streptavidin coated DynaBead-
Suspension (Invitrogen) slurry were added, incubated for 30 min at 4
°C, and removed to reduce naturally biotinylated proteins from the
lysates. For the competition control, additional 10 μL of a 1 mM
solution of either 1, 2, 3, or 4 was added and the reaction volumes of
both the capture reaction and competition control were adjusted to 90
μL with Milli-Q water and incubated for 30 min at 4 °C on a rotation
wheel. A solution of either one of the CCs 5 or 6, CCs 7 or 8, or CCs
9 and 10 (10 μL of a 50 μM solution) were added to the capture, and
respective competition experiments and equilibration was carried out
for 1 h at 4 °C. Then the reaction mixture was irradiated for 10 min
using the caproBox. Subsequently, buffer conditions were adjusted by
adding 25 μL of 5× wash buffer (caprotec bioanalytics GmbH, Berlin,
Germany) to each reaction. The samples were then incubated with 50
μL Dynabeads MyOne Streptavidin C1, (Invitrogen, Karlsruhe,
Germany) for 1 h at 4 °C on a rotation wheel. The beads containing
the CC−protein conjugates were then collected using the caproMag, a
device for the convenient handling of magnetic beads (caprotec
bioanalytics GmbH, Berlin, Germany), and washed first six times with
200 μL 1× WB, then three times with 80% MeCN and finally once
with MS grade water. MeCN was used to remove polymers from the
beads. The beads were stored at 4 °C in deionized water until further
analysis by either SDS-PAGE or protein digestion followed by LC-MS
analysis.
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unweighted spectral counts in the competition control. We considered
a protein to be significantly enriched in the capture assays compared to
the competition controls if the fold change was greater than 2 and at
the same time the p-value was smaller than 0.05 (corresponding to a
negative log10 in the volcano plot of greater than 1.3, y-axis).
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
NQO2 Inhibition Assay. Using the NADH-dependent mitomycin
C metabolism by NQO2, we examined 1 as a novel NQO2 inhibitor.
NQO2 (0.5 μM) was incubated with the substrate mitomycin C (50
μM) and four different drug concentrations as indicated in 100 mM
potassium phosphate buffer (pH 5.8) at room temperature for 5 min
prior to the addition of NADH (in increasing concentrations) as a
cosubstrate and photometric monitoring at 340 nm for 30 min at rt.
Indicated amounts display final assay concentrations. We determined
Ki values for different concentrations of 1 and 4 utilizing a
Lineweaver−Burk application in Sigma Plot10. Data generated were
used to calculate the IC50 of inhibition of NQO2 activity by 1.
Thrombin Assay. The assay used to test for percent inhibition was
a fluorescence assay utilizing Boc-Val-Pro-Arg-AMC, as first described
by Morita et al.38 and contracted to European ScreeningProt
(Hamburg, Germany). The Boc-Val-Pro-Arg-AMC (15 μM) substrate
in 5 μL of assay buffer assay buffer was supplemented with varying
concentrations of either 1 or 2 (16 2-fold dilutions of compounds in
DMSO, 2.5 mM to 75 nM). The assay buffer consisted of consisted of
50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20. Then α
thrombin was added to a final concentration of 5.5 ng/mL and the
reaction incubated for 2 h at room temperature. Subsequently,
fluorescence was measured on an Envision reader (excitation 355 nm,
emission 460 nm). Human α-thrombin was purchased from
Haematilogic Technologies Inc. (HCT-0020). Substrate Boc-Val-
Pro-Arg-AMC was from Bachem (I-1120.0050).
Molecular Docking. Coordinates were taken from the crystal
structure of human QR2 in complex with resveratrol (PDB code
1SG0) and preprocessed with the SYBYL 8.1 Package. Hydrogens
were added and minimized using the TRIPOS force field and
Gasteiger−Marsili charges (rmsd = 0.1, structure A), followed by a
staggered minimization protocol restraining (a) backbone, ligand, and
Zn pocket (Zn, His-N and Cys-SH), (b) protein Cα atoms and Zn
pocket, and (c) Zn pocket only (structure B). Docking studies were
performed using Surflex-Dock39 (SYBYL 8.1 or SYBYLX1.1.1,
TRIPOS Inc., St. Louis, MO, USA). The resveratrol ligand was
extracted and used for protomol generation. Structures of 1, 2, and
truncated CCs 5 and 6 (Me and Et amides instead of the PEG-biotin
moiety) were built, minimized (Tripos force field, Gasteiger−Marsili
charges, rmsd = 0.05) and docked to the prepared protein (structures
A and B). Docking was performed using the SurflexDock protocol
with default parameters besides starting conformations (10) and
number of resulting poses (50) or the SurflexGeom protocol with
default parameters. All docking poses were collected and sorted in
groups by orientation using an in-house script and evaluated by
experimental criteria (binding of 1, but not of 3; Coplanar to FAD;
open carboxyl terminus; amidine involved in ligand binding) and the
docking scores.
ACKNOWLEDGMENTS
■
We thank Philip Gribbon of European ScreeningPort GmbH,
Hamburg, Germany, for experimental support in the thrombin
assay.
ABBREVIATIONS USED
■
A, assay; Ac, acetyl; ACN, acetonitrile; Boc, tert-butyl
carbamate; Asp, asparagine; C, competition; CC, capture
compound; CCMS, capture compound mass spectrometry;
CNS, central nervous system; DBU, 1,8-diazabicyclo[5.4.0]-
undec-7-ene; DCC, N,N′-dicyclohexylcarbodiimide; DCM,
dichloromethane; DIPEA, N,N-diisopropylethylamine; DMA,
N,N-dimethylacetamide; DMAP, 4-dimethylaminopyridine;
FAD, flavin adenine dinucleotide; FQ, fit quality of the ligand
efficiency; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluoro-phosphate; HepG2, a human cell
line derived from hepatocellular carcinoma; HOBt, 1-
hydroxybenzotriazole; IC50, concentration at which half the
enzyme is inhibited; Ile, isoleucine; K562, a human cell line
derived from myelogenous leukemia; Ki, the measure of bond-
tightness between an enzyme and a corresponding enzyme
inhibitor; LE, ligand efficiency; LTQ, linear trap quadrupole;
Met, methionine; mRNA, messenger ribonucleic acid; NADH,
nicotinamide adenine dinucleotide; NMR, nuclear magnetic
resonance; NQO2, ribosyldihydronicotinamide dehydrogenase;
p-value, probability value; PAGE, polyacrylamide gel electro-
phoresis; PEG, polyethylene glycol; Phe, phenylalanine; PI,
isoelectric point; pK, negative logarithm of the dissociation
constant; SAR, structure−activity relationship; SDS, sodium
dodecylsulfate; TFA, trifluoroacetic acid; THF, tetrahydrofur-
an; UV, ultraviolet
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S
* Supporting Information
Additional computational results and figures and tables
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 30 6392 3976. Fax: +49 30 6392 3985. E-mail:
jenny.fischer@caprotec.com.
Present Address
†M.G.: Inovalab AG, Kagenstrasse 17, CH-4153 Reinach,
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