Development of Selective Clk1 and -4 Inhibitors for Cellular Depletion of Cancer-Relevant Proteins

Article abstract of DOI:10.1021/acs.jmedchem.6b01915

In cancer cells, kinases of the Clk family control the supply of full-length, functional mRNAs coding for a variety of proteins essential to cell growth and survival. Thus, inhibition of Clks might become a novel anticancer strategy, leading to a selective depletion of cancer-relevant proteins after turnover. On the basis of a Weinreb amide hit compound, we designed and synthesized a diverse set of methoxybenzothiophene-2-carboxamides, of which the N-benzylated derivative showed enhanced Clk1 inhibitory activity. Introduction of a m-fluorine in the benzyl moiety eventually led to the discovery of compound 21b, a potent inhibitor of Clk1 and -4 (IC₅₀ = 7 and 2.3 nM, respectively), exhibiting an unprecedented selectivity over Dyrk1A. 21b triggered the depletion of EGFR, HDAC1, and p70S6 kinase from the cancer cells, with potencies in line with the measured GI₅₀ values. In contrast, the cellular effects of congener 21a, which inhibited Clk1 only weakly, were substantially lower.

Full text of DOI:10.1021/acs.jmedchem.6b01915

Article
pubs.acs.org/jmc
Development of Selective Clk1 and -4 Inhibitors for Cellular
Depletion of Cancer-Relevant Proteins
†
†
†
,‡
Ahmed K. ElHady, Mohammad Abdel-Halim, Ashraf H. Abadi, and Matthias Engel*
†Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
‡Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbru
cken, Germany
S Supporting Information
̈
*
ABSTRACT: In cancer cells, kinases of the Clk family control the
supply of full-length, functional mRNAs coding for a variety of
proteins essential to cell growth and survival. Thus, inhibition of Clks
might become a novel anticancer strategy, leading to a selective
depletion of cancer-relevant proteins after turnover. On the basis of a
Weinreb amide hit compound, we designed and synthesized a diverse
set of methoxybenzothiophene-2-carboxamides, of which the N-
benzylated derivative showed enhanced Clk1 inhibitory activity.
Introduction of a m-fluorine in the benzyl moiety eventually led to
the discovery of compound 21b, a potent inhibitor of Clk1 and -4
(
IC50 = 7 and 2.3 nM, respectively), exhibiting an unprecedented
selectivity over Dyrk1A. 21b triggered the depletion of EGFR,
HDAC1, and p70S6 kinase from the cancer cells, with potencies in
line with the measured GI50 values. In contrast, the cellular effects of congener 21a, which inhibited Clk1 only weakly, were
substantially lower.
INTRODUCTION
that in cells expressing more than one Clk isoform,
simultaneous inhibition of several Clks might be more effective
in modulating particular splicing events.
■
The Clks (Cdc2-like kinases) are dual specificity protein
kinases that are implicated in the regulation of pre-mRNA
splicing. In the nucleus, the Clks phosphorylate serine/arginine
SR)-rich proteins, triggering the release of the latter into the
nucleoplasm, where they modulate the selection of splice sites
Direct links between Clk1 activity and cancer growth were
established, such as the multisite phosphorylation of the
alternative splicing factor SPF45, which is overexpressed in
cancer and stimulates cell migration and invasion through exon
skipping in several target mRNAs, including Fas mRNA.
Phosphorylation by Clk1 increased SPF45 protein expression,
whereas inhibition of Clk1 enhanced SPF45 degradation
(
1
during pre-mRNA processing. The Clk family consists of four
highly homologous members: Clk1/STY and Clks 2−4. While
Clk1, -2, and -4 are expressed in a variety of tissues, the highest
levels of Clk3 protein are found in mature spermatozoa,
7
2
through a proteasome-dependent pathway.
suggesting a role in the fertilization process. With respect to
Clk1 not only regulates the production of different proteins
Clk1, -2, and -4, distinct physiological roles were barely
identified so far. However, when the involvement of the Clk
isoforms was probed in a pathological context, functional
differences were found: e.g., overexpressed Clk1 increased the3
production of viral gag protein, whereas Clk2 suppressed it.
Compared with the other major family of SR protein kinases,
the splicing-related protein kinases (SRPKs), Clks have a less
constrained substrate recognition, allowing them to perform the
functionally important hyperphosphorylation of splicing factors
derived from splice variants but also maintains the supply of
8
correct mRNAs coding for full-length proteins. In this case,
Clk1 prevents exon skipping which would lead to frameshifts
and premature termination codons downstream of the
junctions. Consequently, inhibition of Clk1 results in non-
sense-mediated decay (NMD) of faulty mRNAs, finally leading
to depletion of the corresponding proteins after cellular protein
turnover. Interestingly, Araki et al. had demonstrated in a whole
transcriptome analysis that the majority of genes whose splicing
was affected by Clk1/2 inhibition play roles in metabolic
processes and cell cycle pathways, all of which promoted cell
4
,5
such as ASF/SF2. Therefore, in cells with different active
Clks, it can be expected that they have at least partially
overlapping substrate specificities. Indeed, it was previously
shown that in endothelial HUVEC cells, which express all Clk
isoforms, siRNA-mediated downregulation of both Clk1 and -4
was required to decrease the generation of mature full-length
tissue factor (TF) mRNA from pre-mRNA. On the other hand,
the alternatively spliced TF mRNA was depleted through
Special Issue: Inducing Protein Degradation as a Therapeutic
Strategy
Received: January 10, 2017
6
downregulation of either Clk1 or -4. These data demonstrated
©
XXXX American Chemical Society
A
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8
growth and survival. Recent studies provided evidence that
Clk1 and also Clk4 are mediators of cell survival especially in
9
,10
hypoxic tumor cells.
Under hypoxic conditions, hypoxia-
inducible factors (HIFs) up-regulate Clk1/4 at mRNA and
protein levels. It was shown that siRNA-mediated knock-down
of Clk1 decreases hypoxia-dependent full-length carbonic
anhydrase IX mRNA and protein levels, and modulates the
9
hypoxia-dependent cysteine-rich angiogenic inducer 61.
Although Clk4 was less well studied so far, it may have even
greater importance for cancer development, as it was found,
along with Clk2, to be one of the most frequently overex-
11
pressed kinases in a variety of human tumors. The closely
related isoenzyme Clk2 was already demonstrated to have
oncogenic potential and was found amplified and overexpressed
12
in a significant fraction of human breast tumors.
Among the previously published Clk inhibitors, (1Z)-1-(3-
ethyl-5-methoxy-2(3H)-benzothiazolylidene)-2-propanone
Figure 1. From previous hit compounds against Clk1 to the design of
a diverse library of methoxybenzothiophene amides. Previously
identified co-inhibitors of Clk1 and Dyrk1A such as 28 could be
downsized to the fragment-like compound 29 without loss of potency,
and the carboxamide derivative 30 was also tolerated by the Clk1
binding site. A hypothetical binding model was derived, in which
both N- and O-methyl groups of 30 pointed outside the ATP-binding
pocket (marked by small yellow circles); thus, several H-bond donor/
acceptor functions (e.g., from the peptide backbones of Ala171 and
Glu292), an acidic (Asp325), and an aromatic moiety (Phe172) were
all located within a 4.5 Å radius (large yellow circle), which could
potentially be addressed by amide-linked molecule extensions.
Following these considerations, a focused library of methoxybenzo-
thiophene amides was designed and synthesized (see Table 1).
(
31), known as TG003, found widespread use as a tool
compound that inhibits Clk1 and Clk4 with comparable
potencies (IC reported between 15 and 49 nM in different
50
1
3,14
studies).
However, 31 also inhibited nonsplicing-related
1
7
kinases such as CK1δ and CK1γ2 (K values of 150 and 270
d
nM, respectively; see Figure 3 in ref 15) as well as Dyrk2 and
haspin (see Table S3, Supporting Information in ref 16). The
most frequently reported off-targets of Clk inhibitors,
irrespective of the chemotype, are mainly Dyrk1A/1B but
17
also haspin, STK17, CK2, and MLCK2. Although Dyrk1A is
also involved in the regulation of alternative splicing, another
confirmed role is to counteract cardiac hypertrophy through
18
phosphorylation of NFAT, suggesting that pharmacological
inhibition might implicate severe side effects. Another
compound, ethyl 3-[(E)-2-amino-1-cyanoethenyl]-6,7-di-
chloro-1-methylindole-2-carboxylate (KH-CB19), while show-
ing good overall selectivity, equally inhibited Clk1, Clk4, and
suggested that the ether oxygen of 30 might interact with the
backbone NH of Leu244 and the amide carbonyl with the ε-
amino function of Lys191 (cf. Figure 1). In the resulting
binding orientation, the side chains of Phe172, Asp325, and
Asn293, as well as several backbone amides, were all located
within reach of the amide-linked moieties, potentially enabling
a variety of interactions (Figure 1). On the basis of this model,
we chose a diversification strategy for our library design,
envisaging the introduction of various structural extensions at
the amide function, which could establish different types of
interactions (Figure 1, Table 1). This should increase the
chances to improve the potency while maintaining or even
enhancing the selectivity for Clks.
14
Dyrk1A in the two digit nM range. Further Clk/Dyrk1A co-
19
inhibitors comprise imidazo[1,2-b]pyridazines and benzobis-
2
0
thiazoles, which were not tested against further typical off-
target kinases. Araki et al. identified in a high-throughput
screening three imidazopyridine derivatives as potent inhibitors
8
of Clk2 and, to a lesser extent, of Clk1. However, in the
extended selectivity profiling, neither Clk4 nor the above-
mentioned challenging off-targets were included; hence the true
selectivity cannot be evaluated.
In the present study, we describe the development of new
Clk1/4 inhibitors with high selectivity over Dyrk1A and other
major off-target kinases. Furthermore, selective depletion of cell
growth-related proteins is demonstrated following Clk
inhibition in tumor cell lines.
Chemistry. The synthesis of the methoxybenzothiophene-
2
-carboxamides was accomplished through a three-step syn-
thesis (Scheme 1), where ethyl thioglycolate was reacted with
-fluoro-5-methoxybenzaldehyde in the presence of potassium
2
carbonate to afford the 5-methoxybenzothiophene-2-carboxylic
acid ethyl ester (1) in good yield. After alkaline hydrolysis of
the ethyl ester, the methoxybenzothiophene-2-carboxamides
(1a−27a) were synthesized by direct coupling of the 5-
methoxybenzothiophene-2-carboxylic acid (2) with different
amines using HBTU in the presence of triethylamine. The N-
methylation of some secondary amide derivatives was done
through deprotonation using potassium bis(trimethylsilyl)-
amide (KHMDS) at 0 °C, followed by trapping of the resulting
anion by the addition of iodomethane (Scheme 2). An
additional step of ester hydrolysis was required to deprotect
the carboxyl group to prepare the amide derivatives of the
glycine, L-valine and L-alanine amino acids (Scheme 3).
RESULTS AND DISCUSSION
Compound Design. In a previous study, we described the
■
development of the fragment-like hydroxybenzothiophene
1
7
ketone 29 from the larger ketone 28 as a Dyrk1A/Clk1 co-
inhibitor (Figure 1). 29 displayed potencies in the higher nM
1
7
range. Interestingly, the Weinreb amide derivative 30 was also
well tolerated by the Clk1 binding site. On the other hand, the
modifications in 30, also including a replacement of the free
hydroxyl group by methoxy, had decreased the potency toward
Dyrk1A. Hence 30 was identified as moderately selective hit for
Clk1 (9-fold selectivity over Dyrk1A), with an IC of about
5
0
1
7
1
00 nM. In order to optimize the hit compound, molecular
modeling was performed based on the Clk1 cocrystal structure
with hymenialdisine (PDB code 1Z57); the binding model
Biological Evaluation. When the diverse set of probe
compounds was screened against human Clk1, it became clear
4
B
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Table 1. Screening of a Diversified Library of Methoxybenzothiophene-2-carboxylamides Derivatives against Clk1
a
b
Data shown are the mean of at least two independent experiments; SD ≤ 10%. IC ± SD.
50
that hydrophobic and/or aromatic interactions rather than
hydrogen (H−) bond formation enhanced the potency of the
methoxybenzothiophene amides, as clearly seen with 2a, 5a, 6a,
predicted binding mode in Figure 2). (iii) Replacement of the
phenyl by heterocycles generally decreased the activity (cf.,
18b, 19b, 25a−27a), suggesting that the heteroatoms did not
form H-bonds but rather caused polar repulsion from the
assumed binding pocket (Figure 2).
1
4a, and 15a (Table 1). With 59% inhibition at 100 nM, the
benzylamide 2a was the most potent compound of this first
series; hence a small focused library of benzyl, arylmethyl, and
phenylethyl amides was synthesized in the next step (Table 2).
After determination of the Clk1 inhibition at 100 nM, the
following general SAR could be deduced: (i) Among the
homologous pairs of compounds, the one-carbon spacer
between the aromatic ring and the nitrogen gave more potent
analogs than the two-carbon spacer (compare 2a and 17a, 16a,
and 17b; Tables 2 and 3). (ii) N-methylation of the amide
greatly enhanced the inhibitory activity with all matched pairs
of methylated vs unmethylated analogs (e.g., compare 2a with
Having identified the benzylamide derivative 16a as the most
promising scaffold, we aimed at fine-tuning of the Clk1
inhibitory potency through modulation of the π-electron
density. The obtained SAR indicated that this parameter
strongly influenced the activity, with the electron withdrawing
m-fluorine substitution being most advantageous. The resulting
inhibitor 21b was more potent than the unsubstituted analog
16a, showing an IC50 of 7 nM (Table 3). In contrast, the
analogs with electron-donating methyl (24a) or methoxy
substituents (23b) exhibited a substantially reduced activity
compared with 16a. Interestingly, fluorine was only favorable in
the meta-position, since the ortho- and para-regioisomers (20b
and 22b, respectively) were also less active than 16a. In our
binding model, the m-fluorophenyl ring was buried in a small,
partially hydrophobic pocket in the lid of the ATP-binding site
1
6a, 19a with 19b, 23a with 23b, etc.). This suggested that the
N-methylation increased the rotational flexibility of the amide
bond by mitigating the tautomerism and/or allowing cis-
configurations; it is assumed that this enabled more favorable
interactions of the phenyl ring with its binding site (cf.
C
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a
Scheme 1. Synthesis of Compounds 1a−27a
a
Reagents and conditions: (i) K CO in DMF at 0 °C, 1.2 equiv of ethyl thioglyoclate, 20 min, 1 equiv of 2-fluoro-5-methoxybenzaldehyde in DMF,
2
3
7
0 °C, 4 h; (ii) 4 equiv of KOH in ethanol/water, 80 °C, 3 h; (iii) 2 equiv of HBTU, 4 equiv of triethylamine, 4 equiv of the appropriate amine in
DCM, room temperature, overnight.
2
2
(
Figure 2). While such a pocket was not induced by the type of
structures. In a previous study, introduction of a single
fluorine in a benzyl ring of a thrombin inhibitor led to a
comparable 6-fold increase in potency owing to C−F···CO
compounds bound in the four available Clk1 cocrystal
structures, it was observed before in cocrystals of the highly
homologous Dyrk1A with compounds containing a branched 3-
chlorobenzyl motif at the corresponding position (cf. PDB
entries 2WO6 and 2VX3). Due to the limited size of this
pocket, the m-methyl (24a), m-methoxy (23b), but also the p-
fluorine substituent (22b) would be expected to cause steric
collisions (p-fluorine with the carbonyl of Gly173), in
agreement with the markedly lower activity of these three
compounds compared with the unsubstituted 16a (cf. Table 2).
Placing the fluorine in the meta-position (21b) apparently
avoided too close contacts and moreover increased the potency
23
interactions.
The methyl group of 21b was expected to induce an out-of-
plane rotation of the amide group, thus favoring the biologically
active conformation as predicted by the docking simulation
(Figure 2). In addition, the methyl was placed in a van der
Waals distance to Val324, suggesting an additional contribution
to the binding affinity. Further major contributions were
expected from the H-bond of the ether oxygen with Leu244,
the CH−π interaction of the benzothiophene with Phe241, and
the H-bond between the carbonyl and Lys191, altogether
explaining the high potency of 21b (Table 3). Overall, this
binding model agreed well with the observed SAR for the Clk1
inhibitors.
Kinase Selectivity Profiling. As described in the
Introduction, a quite reliable way to assess the selectivity of
Clk inhibitors (apart from a full kinome scan) is to check the
kinases that are supposed to be the most similar in the ATP
binding pocket, which were frequently identified as off-targets
in previous studies. A strong co-inhibition of Clk4 was
21
6
.5-fold compared with the unsubstituted analog 16a (Table 2).
This improvement in potency might at least partially be
attributed to an enhancement of hydrophobic interactions. In
addition, our docking model suggested that a C−F···CO
interaction with the backbone of Glu169 might also occur. In
some docking poses, the fluorine was found in a favorable
position for such an orthogonal dipolar interaction (see Figure
2
∠
), with the distance (2.9 Å) and angles (∠F···CO, 96°;
C−F···C, 152°) being in typical ranges found in cocrystal
D
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a
Scheme 2. Synthesis of N-Methylated Derivatives
inhibitors described to date, with somewhat weaker effect on
Clk2 as well.
How is the selectivity over Dyrk1A achieved? It is likely that
the methoxy group in the 5-position of the benzothiophene
forms a markedly stronger H-bond with the hinge region of
Clk1 than of Dyrk1A. In our previous benzothiophene ketone
series, a free 5-hydroxy group was necessary for potent
inhibition of Dyrk1A, whereas the corresponding methoxy
17
derivative was inactive. Similarly, the hydroxyl derivative of
the benzothiazoylidene-2-propanone 31, termed “INDY”, was
24
3
-fold more potent toward Dyrk1A than 31 itself. Molecular
docking of 21b in the Dyrk1A crystal structure from PDB entry
21
2
W06 also supported this notion, since no H-bond between
the NH of Leu241 (corresponding to Leu244 in Clk1) and the
-methoxy was formed using the same docking conditions as
5
for Clk1 (Supporting Information, Figure S1).
Inhibition of Tumor Cell Growth. In previous studies,
important anticancer effects of Clk inhibitors were shown to be
mediated through the modulation of pre-mRNA splicing, which
in several cases led to NMD of the mRNAs of genes important
8
,9
for cell growth and survival. Thus, treatment of cancer cells
by Clk1/4 inhibitors cuts off the supply of intact mRNA,
triggering the disappearance of the affected proteins depending
on their cellular half-life.
First, we determined the potency of our best Clk1/4
inhibitors to inhibit the growth of different cancer cell lines. To
this end, we selected five human cell lines of different tissue
origin which differed in the expression levels of Clk1 mRNA as
published in the Expression Atlas database (https://www.ebi.
ac.uk/gxa/home): two cell lines with low expression (T47D
a
Reagents and conditions: (i) 3 equiv of KHMDS, 0 °C, 1 h, 1.5 equiv
of CH I, room temperature, 24 h.
3
a
Scheme 3. Synthesis of Carboxylic Acids 8c−10c
(
breast carcinoma) and THP-1 (monocytic leukemia)) and
three cell lines with higher Clk1 expression (T24 (urinary
bladder carcinoma), MDA-MB-231 (breast adenocarcinoma),
and HepG2 (hepatocellular carcinoma)). Hence, a differential
sensitivity toward the Clk1/4 inhibitors was expected. As
shown in Table 4, the sensitivity of the cell lines toward the
most potent compounds 21b and 16a varied, with T24 and
HepG2 cells being most responsive (GI for 21b: 0.63 μM and
50
2
.8 μM, respectively). Intriguingly, these two cell lines also
exhibited the highest mRNA and protein expression levels of
Clk1. In contrast, both THP-1 and T47D were considerably
less sensitive toward our inhibitors, which correlated with lower
mRNA and protein expression levels of Clk1. In line with this
relationship, the MDA-MB-231 cell line showed moderate
sensitivity toward 21b and intermediate Clk1 expression (Table
a
Reagents and conditions: (i) 4 equiv of KOH in ethanol/water, 80
°C, 3 h.
4
). Altogether, our results suggested that enhanced Clk1
expression may indicate a stronger dependence of tumor cell
growth and survival on this kinase, translating into higher
sensitivity toward chemical Clk1 inhibition. In further support
of this notion, the efficacy of the chosen compound series 21b,
21a, 16a, and 19b to suppress cell growth of a specific cell line
correlated with their relative potency against Clk1 (cf. T24 and
MDA-MB-231 cells, Table 4), suggesting that the compounds
act through inhibition of Clk1/4 rather than by another
mechanism.
Lack of Cytotoxicity against Nontumor Cells. In order
to assess the potential therapeutic window of our Clk1/4
inhibitors, we performed a cytotoxicity assay using normal
peripheral blood lymphocytes (PBLs) and human embryonic
kidney cells (HEK293). Neither 21b nor the second most
potent congener 16a showed a significant cytotoxicity toward
the nonproliferating PBLs and the proliferating HEK293 cells
(Table 5). This result suggested that in nontumor cells, the
anticipated for our compounds 16a and 21b (Table 3) because
Clk1 and Clk4 possess fully identical amino acid residues in and
around the ATP binding pocket, suggesting that selective
inhibition of either of the isoenzymes might not be possible
using small molecules. Similarly, the still highly homologous
Clk2 was markedly inhibited by 21b, although with somewhat
reduced potency. Our selectivity profiling revealed that the
notorious off-targets haspin, STK17, and MLCK2 were not
inhibited by 16a and 21b. The most strongly inhibited non-Clk
kinases were Dyrk1A/1B; however, determination of the IC50
values toward Dyrk1A revealed that 16a was still 11.5-fold
selective for Clk1 (Table 3), and notably, 21b inhibited Clk1
4
8.5 times and 30 times more strongly than Dyrk1A and
Dyrk1B, respectively (IC : Clk1 = 6.9 nM; Dyrk1A and
5
0
Dyrk1B = 340 nM and 206 nM, respectively). Thus, compound
1b turned out to be one of the most selective Clk1/4
2
E
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Table 2. Inhibition of Clk1 by the Optimized Series of Methoxybenzothiophene-2-carboxylamides
a
b
Data shown are the mean of at least two independent experiments; SD ≤ 10%. IC ± SD; n.d., not determined.
50
activity of the Clk1 and -4 isoforms might not be essential to
ensure cell viability and/or proliferation.
Selective Depletion of Cancer-Relevant Proteins. Next,
we analyzed the effect of 21b on the levels of selected cancer-
associated proteins in the three most sensitive tumor cell lines
higher concentrations of 21b were needed to decrease protein
levels (cf. IC50 values in Table 4); in this case it concerned
EGFR and, surprisingly, not HDAC1 but S6K. However, the
IC50 for S6K reduction in the MDA-MB-231 cells was not
reached at the highest concentration of 21b (20 μM). Again, all
other proteins in the analyzed panel remained unchanged, with
respect to amount and size. Thus, in both T24 and MDA-MB-
231 cells, the effect of 21b on EGFR might be a major reason
for the suppression of cell growth because the IC50 values for
EGFR depletion were in good agreement with the respective
GI50 values. In the light of this finding, it was interesting to
analyze the effects of 21b in the HepG2 cells, which lack a
(cf. Table 4); again, the substantially less active congener 21a
served as a control. After treatment of T24 cells with different
concentrations of 21b, the protein levels of EGFR and HDAC1
were significantly diminished in a concentration-dependent
manner (Table 6, Figure 3A). A significant reduction of EGFR
protein was already observed at 1.25 μM (IC = 2.2 μM, Table
5
0
4
), which was in a good agreement with the observed GI .
50
25
Notably, the other analyzed proteins, p70S6K, PARP-1, ERK2,
CD44, PCNA, and GRP94, were not influenced (exemplarily
shown are the CD44 signals). The control compound 21a,
which was much less potent against recombinant Clk1 (see
Table 2) and did not reach the GI50 at the highest
concentration tested (40 μM), showed a substantially weaker
effect on the protein levels of both EGFR and HDAC1 (Figure
strong expression of EGFR. Indeed, we could not detect any
EGFR protein signals in this cell line, indicating a rather low
expression. On the other hand, although there was a 44%
reduction of both HDAC1 and S6K protein levels at 20 μM
21b, the main target of the Clk1/4-triggered protein depletion
was not found within the analyzed set of proteins (Figure S2,
Supporting Information). Thus, in cell lines that overexpress
EGFR, the selective depletion of EGFR is likely a major
3A, Table 6). With the less sensitive cell line MDA-MB-231,
F
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Table 3. Selectivity Profiling of Compounds 16a and 21b
mechanism through which Clk inhibitors lead to cell growth
inhibition, whereas in cell lines displaying weak or no EGFR
expression, the mainly affected proteins remain to be identified
in future studies.
a
b
%
inhibition at 1 μM (IC50 ± SD)
16a 21b
kinase
DYRK1A
54
520 ± 28.3 nM)
58
72
It is further noteworthy that only a fraction of the analyzed
cancer-relevant proteins was actually affected in the three cell
lines, which was rather unexpected given the supposed
ubiquitous role of the Clk isoenzymes in the regulation of
alternative splicing. It could be speculated that in a given cell
line, the activities of the remaining SR protein kinases, such as
SRPKs and Dyrk1A, might differentially modulate the impact of
Clk inhibition on exon splicings of particular pre-mRNAs. In
addition to potential modulating or compensating effects of
other SR protein kinases, the selection of exon-splicing sites
specifically regulated through Clks seems to be determined
already at the level of the branch sequence composition and
length of polypyrimidine tracts in the exons. In a recent
comparative transcriptomic analysis which employed the model
Clk inhibitor 31, targeting rules for exon skipping were derived
from treated skeletal muscle cells. Exons skipped after
treatment by 31 were shorter, had fewer splicing factor binding
sites and shorter stretches of polypyrimidine tracts than the
(
(340 ± 21.2 nM)
77
DYRK1B
(206 ± 10 nM)
DYRK3
DYRK4
Clk1
n.i.
n.i.
81
n.i.
n.i.
92
(45 ± 6 nM)
(7 ± 0.12 nM)
86
Clk2
73
(19.1 ± 0.5 nM)
Clk3
Clk4
44
58
101
101
(2.3 ± 0.24 nM)
Haspin
MLCK2
STK17A
SRPK1
SRPK2
CK2
12
n.i.
23
4
12
n.i.
32
2
10
n.d.
7
26
1.4
insensitive exons.
a
The screening list was especially composed to include all kinases that
were frequently reported as off-targets for diverse chemical classes of
CONCLUSIONS AND PERSPECTIVES
■
17,27,29−32
Dyrk inhibitors.
Screenings were performed as a service at
We developed a novel series of benzylated N-methylbenzo-
thiophene-2-carboxamides as potent inhibitors of Clk1/4 and,
to a somewhat lower extent, of Clk2. The introduction of m-
fluorine in the benzyl moiety was key to the discovery of
compound 21b, which exhibited previously unreported
selectivity over Dyrk1A, a highly homologous kinase also
involved in the regulation of alternative splicing. This is
considered an important advancement since Dyrk1A inhibition
was previously shown to induce cardiac hypertrophy as a
deleterious side effect, related to its role in antagonizing
Thermo Fisher Scientific at an ATP concentration of 100 μM. n.i., no
inhibition. n.d., not determined. Data represent mean values of
b
duplicates that differed by less than 7%. IC50 values were determined
mainly for 21b for inhibition values of >50% in the primary screening.
ATP concentration was 15 μM.
1
8
calcineurin signaling. While it might be assumed that
inhibition of Clk isoforms will have detrimental effects due to
the vast number of influenced splicing events, our results
suggest that when selective Clk inhibitors are used, the number
of modulated pre-mRNA splicings might be lower than
expected. Thus, although further studies are needed that
specifically address this issue, it could be speculated that the
application of selective Clk inhibitors in vivo might have fewer
adverse side effects than one might have anticipated. In a future
potential application of Clk inhibitors for anticancer therapy,
prior analysis of Clk isoenzyme overexpression might be
performed to identify tumors that are particularly responsive
toward Clk inhibition.
Figure 2. Model for the hypothetical interaction of 21b with Clk1 as
supported by the SAR. 21b (cyan) was docked into the ATP binding-
pocket of Clk1 (PDB code 1Z57) using MOE. Some residues that
might be important for the binding are labeled. In the binding model,
EXPERIMENTAL SECTION
■
Chemistry. Solvents and reagents were obtained from commercial
suppliers and used as received. Melting points were determined on a
Stuart SMP3 melting point apparatus. All final compounds had a
percentage purity of at least 95%, and this could be verified by means
of HPLC coupled with mass spectrometry. Mass spectra (HPLC−
ESIMS) were obtained using a TSQ quantum (Thermo Electron
Corp.) instrument prepared with a triple quadrupole mass detector
2
1b is anchored between the conserved Lys191 and the hinge region
residue Leu244 via two hydrogen bonds (indicated in red). In
addition, two CH−π interactions involving Val324 and an edge-to-face
CH−π interaction with the Phe241 benzene ring were predicted. The
3
-fluorophenyl ring probably binds in a partially lipophilic pocket
enframed by the glycine-rich loop, also involving an orthogonal dipolar
interaction of the 3-fluorine with the Glu169 carbonyl. Interactions are
indicated by dashed lines, and distances between the heavy atoms are
given in Å (H-bonds, red; CH−π and orthogonal dipolar interactions,
brown). In the color code of the ATP binding pocket surface, green
denotes the most lipophilic and magenta the most hydrophilic areas.
(Thermo Finnigan) and an ESI source. All samples were injected using
an autosampler (Surveyor, Thermo Finnigan) by an injection volume
of 10 μL. The MS detection was determined using a source CID of 10
V and carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas
5
pressure of 4.0 × 10 Pa, a capillary temperature of 400 °C, a capillary
5
voltage of 35 V, and an auxiliary gas pressure of 1.0 × 10 Pa. The
stationary phase used was an RP C18 NUCLEODUR 100-3 (125 mm
G
DOI: 10.1021/acs.jmedchem.6b01915
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 4. Correlation between Clk1 Inhibitory Potency, GI50 Values, and Relative Clk1 Expression Levels in Different Cancer
Cell Lines
cell line
T24
MDA-MB-231
HepG2
THP-1
3.3
T47D
,
ab
Clk1 mRNA levels
Clk1 protein levels
compd
6.5
1.5
5.4
1.3
8
1
1
,
ac
3.2
1.2
d
d
d
d
d
e
GI50 (μM)
0.6
GI50 (μM)
GI50 (μM)
2.8
GI50 (μM)
27.2
GI50 (μM)
15.3
Clk1 inhibition IC50 ± SD (nM)
21b
21a
16a
19b
9.6
6.9 ± 0.12
>100
>40
>40
24.2
n.d.
n.d.
n.d.
2.7
3.7
>30
28.8
45 ± 6
12.5
25.7
n.d.
n.d.
25.4
81.6 ± 2.5
a
b
c
Levels relative to the expression in T47D (set = 1). Data taken from the Expression Atlas Database (https://www.ebi.ac.uk/gxa/home). Values
obtained by densitometry (SD < 8%). Data shown are the mean of at least two independent experiments, SD ≤ 12%. n.d., not determined. Values
d
e
taken from Table 2.
Table 5. Evaluation of Cytotoxicity of Compounds 16a and
mL) was added ethyl thioglycolate (31.2 mmol, 1.2 equiv). The
reaction mixture was stirred for 20 min at 0 °C under nitrogen, and
this was followed by the gradual addition of 2-fluoro-5-methoxy-
benzaldehyde (26 mmol, 1 equiv) in DMF (10 mL). The mixture was
heated to reflux at 70 °C for 4 h. Afterward the suspension was cooled
to room temperature and poured over 100 mL of 2 M HCl, the
aqueous layer was extracted by DCM (5 × 10 mL), and the combined
organic layers were thoroughly washed with water and brine, dried
2
1b against Nontumor Cells
cell type: PBLs
cell type: HEK293
a
a
compd
LC50 (μM)
GI50 (μM)
2
1b
6a
29.4
>30
>30
>30
1
a
Data shown are the mean of at least two independent experiments;
over anhydrous MgSO , evaporated under reduced pressure, and the
4
SD ≤ 12%.
resulting residue was purified by column chromatography (CC) using
a solvent system of (petroleum ether/DCM 3:2), giving the pure
Table 6. Correlation between Clk1 Inhibitory Potency,
Reduction of Cellular Proteins, and Relative Clk1
Expression Levels in the Cell Lines MDA-MB-231 and T24
carboxylic acid ethyl ester 1 as a yellow solid in a yield of 2.9 g (47%).
1
H NMR (500 MHz, DMSO-d ) δ 8.09 (s, 1H), 7.93 (d, J = 8.9 Hz,
6
1
=
H), 7.53 (d, J = 2.5 Hz, 1H), 7.17 (dd, J = 8.9, 2.6 Hz, 1H), 4.34 (q, J
7.1 Hz, 2H), 3.82 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (126
MHz, DMSO-d ) δ 161.98, 157.48, 139.64, 133.92, 133.80, 130.35,
cell line (Clk1 rel
expression
cell line (Clk1 rel
expression levels):
6
1
23.75, 118.04, 107.07, 61.36, 55.33, 14.11.
Procedure B, General Procedure for Ester Hydrolysis. The
levels): T24 (17) MDA-MB-231 (14)
a
IC50 of protein reduction (μM)
ethyl/methyl ester was suspended in a mixture of 50 mL of ethanol
and 25 mL of water; this was followed by the addition of 4 equiv of
KOH. The mixture was heated to reflux at 80 °C for 3 h. Afterward,
the solvent was removed under reduced pressure, and the resulting
aqueous layer was cooled to 0 °C, followed by gradual addition of conc
HCl until the pH was adjusted to 1. The aqueous layer was then
extracted using DCM (5 × 50 mL), and the combined organic extracts
compd EGFR HDAC EGFR S6 kinase Clk1 IC50 ± SD (nM)
2
2
1b
1a
2.2
9.5
4.2
>20
>20
6.9 ± 0.12
>100
>15
>15
>20
a
IC50 values were derived by densitometric quantification of the
signals from the immunoblot shown in Figure 3 after normalization.
The values represent an average of two independent determinations;
SD ≤ 15%.
were filtered over anhydrous MgSO and evaporated under reduced
4
pressure giving the carboxylic acid derivative. The product was used in
the next step without further purification.
Procedure C, General Procedure for the Synthesis of 5-
Methoxybenzo[b]thiophene-2-carboxylic Acid Amide Deriva-
tives. 5-Methoxybenzo[b]thiophene-2-carboxylic acid (2) (0.1 g, 0.5
mmol) was dissolved in DCM (30 mL), and then 0.3 g of HBTU and
2 mL of TEA were added. The reaction mixture was stirred for 30 min.
Following this, the appropriate amine (4 equiv) was added dropwise,
and the reaction mixture was stirred at room temperature overnight.
The solvent was evaporated under reduced pressure. The residue was
partitioned between 50 mL of ethyl acetate and 20 mL of water, and
then the aqueous layer was extracted with three 20 mL portions of
ethyl acetate. The combined organic extracts were filtered over
×
3 mm) column (Macherey & Nagel). The solvent system consisted
of water containing 0.1% TFA (A) and 0.1% TFA in acetonitrile (B).
The HPLC method used a flow rate of 400 μL/min. The percentage of
B started at 5%, was increased up to 100% during 7 min, was kept at
1
5
00% for 2 min, and was flushed back to 5% in 2 min and was kept at
% for 2 min. High resolution precise mass spectra were recorded on
ThermoFisher Scientific (TF, Dreieich, Germany) Q Exactive Focus
system equipped with heated electrospray ionization (HESI)-II source
and Xcalibur (version 4.0.27.19) software. Before analysis external
mass calibration was done according to the manufacturer’s
recommendations. The samples were dissolved and diluted in
methanol in a concentration of 3 μM and directly injected onto the
Q Exactive Focus using the integrated syringe pump. All the data
analyses were done in positive ion mode using voltage scans and the
data collected in continuous mode. A Bruker DRX 500 spectrometer
was used to obtain the H NMR and C NMR spectra, and a Varian
Mercury VX 300 spectrometer was used to obtain spectra for two of
the final amide derivatives. The chemical shifts are referenced to the
residual protonated solvent signals. The IR spectra were measured on
a PerkinElmer Spectrum 100 FT-IR spectrometer, PerkinElmer,
Rodgau, Germany.
anhydrous MgSO , the solvent was removed under reduced pressure,
4
and the product was purified by CC to give the 5-methoxybenzo[b]-
thiophene-2-carboxylic acid amide derivatives in different yields.
Procedure D, General Procedure for the Synthesis of 5-
Methoxybenzo[b]thiophene-2-carboxylic Acid Methylamide
Derivatives. The respective amide derivative was dissolved in dry
THF and stirred at 0 °C. This was followed by the gradual addition of
1
13
3
equiv of potassium bis(trimethylsilyl)amide (0.5 M solution in
THF). The reaction mixture was left to stir for 1 h, and after that CH3I
(1.5 equiv) was added. The reaction mixture was left to stir for 24 h at
room temperature, and afterward, the solvent was removed under
reduced pressure, then a small amount of water was added, and
extraction was carried out using DCM (5 × 30 mL). The organic
General Synthetic Methods and Experimental Details for
Some Key Compounds. Procedure A, Synthesis of 5-
Methoxybenzo[b]thiophene-2-carboxylic Acid Ethyl Ester (1).
To an ice cooled suspension of 7.2 g (2 equiv) of K CO in DMF (20
layers were combined, dried over anhydrous MgSO , the solvent was
4
removed under reduced pressure, and the product was purified by CC
2
3
H
DOI: 10.1021/acs.jmedchem.6b01915
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. 21b induces cellular depletion of cancer-relevant proteins. T24 (A) and MDA-MB-231 cells (B) were treated by 21b and the weak Clk1
inhibitor 21a as a comparison for 3 days at the indicated concentrations. The total cellular proteins extracted from the cells were analyzed by
immunoblotting using a set of antibodies directed against eight cancer-related proteins (EGFR, p70S6 kinase α, PARP-1, ERK2, HDAC1, CD44,
PCNA, GRP94). Shown are the signals belonging to proteins that were significantly reduced depending on the 21b concentrations; for comparison,
one of the unchanged signal sets is also exemplarily shown (corresponding to CD44). In parts A and B, one representative result out of two separate
experiments is shown.
or by salting out to give the methylated 5-methoxybenzo[b]thiophene-
-carboxylic acid amide derivatives in different yields.
-Methoxybenzo[b]thiophene-2-carboxylic Acid (2). The
Hz, 1H), 7.57 (s, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 8.8, 2.5
Hz, 1H), 3.82 (s, 3H), 3.63−3.59 (m, 4H), 1.68−1.63 (m, 2H), 1.59−
2
1.54 (m, 4H); 13C NMR (126 MHz, DMSO-d ) δ 162.27, 157.37,
5
6
compound was synthesized according to procedure B, to give a
139.74, 138.24, 131.44, 124.52, 123.22, 116.15, 106.51, 55.29, 54.87,
1
+
white solid in a yield of 2.3 g (90%). H NMR (500 MHz, DMSO-d )
23.98; MS (ESI) m/z = 275.97 (M + H) . HRMS (ESI): calcd for
6
+
−1
δ 8.01 (d, J = 0.6 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 2.5
(C H NO S) 276.10582, found 276.10524 (M + H) . IR (cm ): ν
̃
15 18 2
Hz, 1H), 7.15 (dd, J = 8.9, 2.6 Hz, 1H), 3.82 (s, 3H); 13C NMR (126
1608 (CO).
MHz, DMSO-d ) δ 163.51, 157.38, 139.84, 135.64, 133.82, 129.90,
(5-Methoxybenzo[b]thiophen-2-yl)morpholin-4-ylmetha-
none (4a). The compound was synthesized according to procedure C,
using morpholine to give a yellow solid: yield 0.05 g (28%). The
6
1
23.73, 117.74, 106.93, 55.33.
(
5-Methoxybenzo[b]thiophen-2-yl)(4-methylpiperazin-1-yl)-
methanone(1a). The compound was synthesized according to
product was purified by CC (ethyl acetate/hexane 1:1); mp 109−110
1
procedure C, using 1-methylpiperazine to give a yellow solid: yield
°C; H NMR (500 MHz, DMSO-d ) δ 7.88 (d, J = 8.9 Hz, 1H), 7.65
6
0
1
8
2
4
1
4
.05 g (31.2%). The product was purified by CC (DCM/MeOH
(d, J = 0.6 Hz, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.09 (dd, J = 8.9, 2.6 Hz,
1
13
00:2); mp 84−85 °C; H NMR (500 MHz, DMSO-d ) δ 7.90 (d, J =
1H), 3.82 (s, 3H), 3.70−3.67 (m, 4H), 3.66−3.63 (m, 4H); C NMR
6
.9 Hz, 1H), 7.68 (s, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.11 (dd, J = 8.9,
.5 Hz, 1H), 3.83 (s, 3H), 3.12−3.10 (m, 4H), 3.09 (t, J = 3.4 Hz,
(126 MHz, CDCl ) δ 164.51, 158.39, 140.16, 137.69, 133.23, 125.75,
3
123.62, 117.14, 106.68, 67.42, 56.08, 26.46, 26.27; MS (ESI) m/z =
H), 2.63 (s, 3H); 13C NMR (126 MHz, DMSO-d ) δ 162.71, 157.45,
+
6
278 (M + H) . HRMS (ESI): calcd for (C H NO S) 278.08509,
14 16
3
+
−1
39.69, 136.99, 131.83, 125.81, 123.33, 116.59, 106.61, 55.32, 52.87,
found 278.08447 (M + H) . IR (cm ): ν
̃
1600 (CO).
+
5.76, 43.15; MS (ESI) m/z = 291.01 (M + H) . HRMS (ESI): calcd
5-Methoxybenzo[b]thiophene-2-carboxylic Acid Allylamide
(5a). The compound was synthesized according to procedure C, using
allylamine to give an orange solid: yield 0.05 g (42%). The product
was purified by CC (DCM); mp 130−131 °C; H NMR (300 MHz,
DMSO-d ) δ 8.94 (t, J = 5.6 Hz, 1H), 8.02 (s, 1H), 7.89 (d, J = 8.8 Hz,
+
for (C H N O S) 291.11672, found 291.11591 (M + H) . IR
15
19
2
2
−
1
(
cm ): ν
̃
1620 (CO).
1
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid Benzyla-
mide (2a). The compound was synthesized according to procedure
C (with the exception of the reaction time being limited to 2 h), using
benzylamine to give a yellow solid: yield 0.015 g (10.7%). The product
6
1H), 7.43 (d, J = 2.5 Hz, 1H), 7.10 (dd, J = 8.8, 2.5 Hz, 1H), 5.92 (ddt,
J = 17.1, 10.4, 5.3 Hz, 1H), 5.21 (dd, J = 17.2, 1.7 Hz, 1H), 5.13 (dd, J
1
3
was purified by CC (DCM/petroleum ether 3:1); mp 208−209 °C;
= 10.2, 1.6 Hz, 1H), 3.92 (t, J = 5.5 Hz, 2H), 3.84 (s, 3H); C NMR
1
H NMR (500 MHz, DMSO-d ) δ 9.31 (t, J = 6.0 Hz, 1H), 8.04 (s,
(75 MHz, DMSO-d ) δ 161.80, 157.87, 141.44, 140.78, 135.61,
6
6
1
4
6
1
1
H), 7.89 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H), 7.36−7.33 (m,
133.13, 124.96, 124.04, 117.13, 115.94, 107.21, 55.89, 42.01; MS (ESI)
+
H), 7.28−7.25 (m, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 4.49 (d, J =
m/z = 247.99 (M + H) . HRMS (ESI): calcd for (C H NO S)
1
3
14
2
.0 Hz, 2H), 3.83 (s, 3H); 13C NMR (126 MHz, DMSO-d ) δ 161.51,
+
−1
̃
248.07452, found 248.07393 (M + H) . IR (cm ): ν 3303 (NH),
6
57.37, 140.87, 140.28, 139.28, 132.67, 128.34, 127.30, 126.88, 124.61,
1617 (CO).
23.57, 116.67, 106.73, 55.39, 42.64; MS (ESI) m/z = 298.03 (M +
5-Methoxybenzo[b]thiophene-2-carboxylic Acid Cyclopro-
pylamide (6a). The compound was synthesized according to
procedure C, using cyclopropylamine to give an orange solid: yield
+
H) . HRMS (ESI): calcd for (C H NO S) 298.09017, found
2
1
7
16
2
+
−1
98.08941 (M + H) . IR (cm ): ν
̃
3279 (NH), 1609 (CO).
(
5-Methoxybenzo[b]thiophen-2-yl)piperidin-1-
0.04 g (33.7%). The product was purified by CC (DCM); mp 164−
1
ylmethanone(3a). The compound was synthesized according to
165 °C; H NMR (300 MHz, DMSO-d ) δ 8.73 (d, J = 3.8 Hz, 1H),
6
procedure C, using piperidine to give a white solid: yield 0.04 g
7.94 (s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 2.5 Hz, 1H), 7.10
(
2
30.8%). The product was purified by CC (DCM/petroleum ether
(dd, J = 8.9, 2.5 Hz, 1H), 3.83 (s, 3H), 2.85 (ddd, J = 11.1, 7.5, 3.9 Hz,
1
13
:1); mp 90.9 °C; H NMR (500 MHz, DMSO-d ) δ 7.87 (d, J = 8.9
1H), 0.73 (dt, J = 7.0, 4.9 Hz, 2H), 0.60 (dt, J = 5.7, 4.0 Hz, 2H);
C
6
I
DOI: 10.1021/acs.jmedchem.6b01915
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Journal of Medicinal Chemistry
Article
NMR (75 MHz, DMSO-d ) δ 163.17, 157.85, 141.55, 140.77, 133.10,
135.07, 126.84, 124.31, 118.48, 107.45, 62.45, 55.98, 50.28, 17.26,
6
+
1
2
24.88, 124.04, 117.01, 107.21, 55.89, 23.48, 6.25; MS (ESI) m/z =
14.48; MS (ESI) m/z = 307.95 (M + H) . HRMS (ESI): calcd for
+
+
−1
48.02 (M + H) . HRMS (ESI): calcd for (C H NO S) 248.07452,
(C H NO S) 308.09565, found 308.09479 (M + H) . IR (cm ): ν
3400 (NH), 1731 (CO), 1646 (CO).
̃
1
3
14
2
15 18 4
+
−1
found 248.07391 (M + H) . IR (cm ): ν
̃
3254 (NH), 1621 (CO).
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid (2-
(S)-2-[(5-Methoxybenzo[b]thiophene-2-carbonyl)amino]-
propionic Acid (10c). The compound was synthesized according to
procedure B, using compound 10a to give a white solid: yield 0.043 g
Hydroxyethyl)amide (7a). The compound was synthesized
according to procedure C, using ethanolamine to give a white solid:
yield 0.042 g (34.8%). The product was purified by CC (DCM/
1
(95.6%); mp 187.8 °C. H NMR (500 MHz, MeOD) δ 7.95 (d, J = 0.5
1
MeOH 100:3); mp 119−120 °C; H NMR (500 MHz, MeOD) δ 7.87
Hz, 1H), 7.76 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.09 (dd, J
= 8.9, 2.5 Hz, 1H), 4.60 (q, J = 7.3 Hz, 1H), 3.87 (s, 3H), 1.54 (d, J =
7.4 Hz, 3H); 13C NMR (126 MHz, MeOD) δ 176.32, 164.97, 159.78,
142.17, 140.88, 135.39, 127.10, 124.63, 118.77, 107.78, 56.31, 50.31,
(
s, 1H), 7.76 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.09 (dd, J
=
8.9, 2.5 Hz, 1H), 3.87 (s, 3H), 3.72 (t, J = 5.8 Hz, 2H), 3.51 (t, J =
5
1
.9 Hz, 2H); 13C NMR (126 MHz, MeOD) δ 165.10, 159.45, 141.84,
+
41.01, 134.92, 126.33, 124.29, 118.33, 107.40, 61.57, 55.97, 43.58;
17.84; MS (ESI) m/z = 279.88 (M + H) . HRMS (ESI): calcd for
+
+
−1
MS (ESI) m/z = 251.93 (M + H) . HRMS (ESI): calcd for
(C H NO S) 280.06435, found 280.06352 (M + H) . IR (cm ): ν
̃
13 14 4
+
−1
(
C H NO S) 252.06944, found 252.06878 (M + H) . IR (cm ): ν
̃
3324 (NH), 2960−2930 (OH), 1708 (CO), 1622 (CO).
1
2
14
3
3
537 (OH), 3311 (NH), 1615 (CO).
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (2-
Aminoethyl)amide (11a). The compound was synthesized accord-
ing to procedure C, using ethylenediamine to give an orange solid:
yield 0.06 g (38.4%). The product was purified by CC (DCM/
[
(5-Methoxybenzo[b]thiophene-2-carbonyl)amino]acetic
Acid Methyl Ester (8a). The compound was synthesized according
to procedure C, using glycine methyl ester to give a white solid: yield
1
0
1
.035 g (26.2%). The product was purified by CC (DCM); mp 121−
MeOH/TEA 100:2:1); mp 100.2 °C; H NMR (500 MHz, MeOD) δ
1
22 °C; H NMR (500 MHz, MeOD) δ 7.90 (s, 1H), 7.78 (d, J = 8.9
7.87 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.08
(dd, J = 8.9, 2.5 Hz, 1H), 3.85 (s, 3H), 3.74−3.72 (m, 2H), 3.47 (t, J =
Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 4.13
s, 2H), 3.87 (s, 3H), 3.76 (s, 3H); 13C NMR (126 MHz, DMSO-d6)
δ 170.18, 162.00, 157.42, 140.20, 139.97, 132.73,125.15, 123.60,
13
(
6.4 Hz, 2H), 3.02 (s, 1H), 2.98 (s, 2H); C NMR (126 MHz, MeOD)
δ 165.19, 159.43, 141.84, 140.96, 134.91, 126.39, 124.31, 118.36,
+
+
1
16.92, 106.80, 55.41, 51.83, 41.14 ; MS (ESI) m/z = 279.01 (M) .
107.44, 56.01, 43.41, 41.99; MS (ESI) m/z = 250.94 (M + H) . HRMS
HRMS (ESI): calcd for (C H NO S) 280.06435, found 280.06357
(ESI): calcd for (C H N O S) 251.08542, found 251.08457 (M +
13
14
4
12 15
2
2
+
−1
+
−1
(
M + H) . IR (cm ): ν
̃
3390 (NH), 1728 (CO), 1645 (CO).
H) . IR (cm ): ν
̃
3260−3200 (NH ), 3065 (NH), 1620 (CO).
2
[
(5-Methoxybenzo[b]thiophene-2-carbonyl)amino]acetic
5-Methoxybenzo[b]thiophene-2-carboxylic Acid Phenyla-
mide (12a). The compound was synthesized according to procedure
C, using aniline to give a yellow solid: yield 0.03 g (25%). The product
Acid (8c). The compound was synthesized according to procedure B,
using compound 8a to give a white solid: yield 0.015 g (95%); mp
1
1
1
21.2 °C; H NMR (500 MHz, MeOD) δ 7.90 (d, J = 0.6 Hz, 1H),
was purified by CC (DCM/petroleum ether 3:1); mp 169.8 °C; H
7
.77 (d, J = 8.9 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.10 (dd, J = 8.9, 2.5
NMR (500 MHz, MeOD) δ 8.08 (d, J = 0.5 Hz, 1H), 7.80 (d, J = 8.9
Hz, 1H), 7.71−7.70 (m, 1H), 7.70−7.68 (m, 1H), 7.42 (d, J = 2.5 Hz,
1H), 7.40−7.36 (m, 2H), 7.19−7.15 (m, 1H), 7.12 (dd, J = 8.9, 2.5
13
Hz, 1H), 4.09 (s, 2H), 3.87 (s, 3H); C NMR (126 MHz, MeOD) δ
1
1
for (C H NO S) 266.04870, found 266.04776 (M + H) . IR (cm ):
ν
̃
73.11, 165.31, 159.60, 141.91, 140.47, 135.18, 126.95, 124.46, 118.65,
+
13
07.60, 56.13, 42.38; MS (ESI) m/z = 265 (M) . HRMS (ESI): calcd
Hz, 1H), 3.89 (s, 3H); C NMR (126 MHz, MeOD) δ 163.17,
+
−1
159.50, 141.91, 141.68, 139.50, 135.27, 129.88, 126.77, 125.76, 124.34,
12
12
4
+
3324 (NH), 3000−2980 (OH), 1736 (CO), 1618 (CO).
122.26, 118.56, 107.53, 56.00; MS (ESI) m/z = 283.95 (M + H) .
(
S)-Methyl 2-(5-Methoxybenzo[b]thiophene-2-carboxami-
HRMS (ESI): calcd for (C H NO S) 284.07452, found 284.07371
16
14
2
+
do)-3-methylbutanoate (9a). The compound was synthesized
according to procedure C, using L-valine methyl ester to give a yellow
solid: yield 0.14 g (91%). The product was purified by CC (DCM);
(M + H) .
(R)-5-Methoxybenzo[b]thiophene-2-carboxylic Acid (1,2,2-
Trimethylpropyl)amide (13a). The compound was synthesized
according to procedure C, using (R)-3,3-dimethyl-2-butylamine to give
1
mp 124.7 °C; H NMR (500 MHz, MeOD) δ 8.00 (s, 1H), 7.76 (d, J
=
4
6
8.9 Hz, 1H), 7.38 (d, J = 2.5 Hz, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H),
.49 (d, J = 7.0 Hz, 1H), 3.87 (s, 3H), 3.76 (s, 3H), 2.28 (dq, J = 13.7,
.8 Hz, 1H), 1.06 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H); 13
a white solid: yield 0.1 g (71.4%). The product was purified by CC
1
(DCM); mp 155.3 °C; H NMR (500 MHz, DMSO-d ) δ 8.19 (d, J =
6
C
9.4 Hz, 1H), 8.09 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.5 Hz,
1H), 7.09 (dd, J = 8.8, 2.5 Hz, 1H), 3.94 (dq, J = 13.9, 6.9 Hz, 1H),
3.82 (s, 3H), 1.11 (d, J = 6.9 Hz, 3H), 0.91 (s, 9H); 13C NMR (126
NMR (126 MHz, MeOD) δ 173.63, 165.05, 159.49, 141.82, 140.30,
1
1
35.12, 126.93, 124.31, 118.48, 107.57, 60.19, 56.00, 52.55, 31.79,
9.62, 19.15; MS (ESI) m/z = 322.03 (M + H) . HRMS (ESI): calcd
+
MHz, CDCl ) δ 161.06, 157.32, 141.48, 140.31, 132.63, 124.35,
3
+
for (C H NO S) 322.11130, found 322.11071 (M + H) .
123.54, 116.27, 106.83, 55.38, 52.63, 34.76, 26.36, 15.47; MS (ESI) m/
16
20
4
+
(
S)-2-[(5-Methoxybenzo[b]thiophene-2-carbonyl)amino]-3-
z = 291.99 (M + H) . HRMS (ESI): calcd for (C H NO S)
1
6
22
2
+
−1
methylbutyric Acid (9c). The compound was synthesized according
to procedure B, using compound 9a to give a yellow solid: yield 0.08 g
̃
292.13712, found 292.13625 (M + H) . IR (cm ): ν 3310 (NH),
1616 (CO).
1
(
60.2%); mp 137.6 °C; H NMR (500 MHz, MeOD) δ 8.00 (s, 1H),
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (2,2,2-
Trifluoroethyl)amide (14a). The compound was synthesized
according to procedure C, using 2,2,2-trifluoroethylamine to give a
yellow solid: yield 0.02 g (14.8%). The product was purified by CC
7
2
6
.97 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.37 (s, 1H), 7.08 (dd, J = 8.9,
.5 Hz, 1H), 4.50 (d, J = 6.4 Hz, 1H), 3.86 (s, 3H), 2.30 (dq, J = 13.5,
.8 Hz, 1H), 1.07 (d, J = 3.9 Hz, 3H), 1.06 (d, J = 3.9 Hz, 3H); 13
C
1
NMR (126 MHz, DMSO-d ) δ 172.88, 161.91, 157.36, 140.32, 140.29,
(DCM/petroleum ether 2:1); mp 130.8 °C; H NMR (500 MHz,
6
1
1
32.77, 125.40, 123.55, 116.63, 106.85, 58.40, 55.38, 29.53, 19.27,
MeOD) δ 7.92 (s, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.38 (d, J = 2.5 Hz,
1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 4.09 (q, J = 9.3 Hz, 2H), 3.86 (s,
+
8.73; MS (ESI) m/z = 308.01 (M + H) . HRMS (ESI): calcd for
+
−1
13
(
C H NO S) 308.09565, found 308.09483 (M + H) . IR (cm ): ν
̃
3H); C NMR (126 MHz, MeOD) δ 163.57, 157.99, 140.18, 138.27,
1
5
18
4
1
3
305 (NH), 2960−2930 (OH), 1718 (CO), 1631(CO).
133.68, 125.64, 124.40 (d, J
= 278.4 Hz), 122.82, 117.24, 106.00,
C−F
+
(
S)-2-[(5-Methoxybenzo[b]thiophene-2-carbonyl)amino]-
54.47, 41.89; MS (ESI) m/z = 289.05 (M) . HRMS (ESI): calcd for
(C H F NO S) 290.04626, found 290.04572 (M + H) . IR (cm ):
+
−1
propionic Acid Ethyl Ester (10a). The compound was synthesized
according to procedure C, using L-alanine ethyl ester to give a
yellowish white solid: yield 0.09 g (57%). The product was purified by
12 11 3 2
ν
̃
3283 (NH), 1619 (CO).
5-Methoxybenzo[b]thiophene-2-carboxylic Acid Thiazol-2-
1
CC (DCM); mp 101.5 °C; H NMR (500 MHz, MeOD) δ 7.95 (s,
ylamide (15a). The compound was synthesized according to
1
8
3
H), 7.76 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.09 (dd, J =
procedure C, using 2-aminothiazole to give a yellow solid: yield
.9, 2.5 Hz, 1H), 4.60 (q, J = 7.3 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H),
0.023 g (16.5%). The product was purified by CC (DCM/MeOH
.86 (s, 3H), 1.53 (d, J = 7.3 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H); 13
C
100:1); mp 177.4 °C; H NMR (500 MHz, acetone-d ) δ 8.39 (s, 1H),
1
6
NMR (126 MHz, MeOD) δ 174.24, 164.72, 159.44, 141.81, 140.39,
7.92 (d, J = 8.9 Hz, 1H), 7.50 (d, J = 3.6 Hz, 1H), 7.48 (d, J = 2.5 Hz,
J
DOI: 10.1021/acs.jmedchem.6b01915
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
3
1
5
H), 7.20 (d, J = 3.6 Hz, 1H), 7.17 (dd, J = 8.9, 2.5 Hz, 1H), 3.89 (s,
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (Pyridin-3-
ylmethyl)amide (19a). The compound was synthesized according to
procedure C, using 3-picolylamine to give a white solid: yield 0.051 g
H); 13C NMR (126 MHz, acetone-d ) δ 161.27, 159.30, 159.09,
6
41.49, 139.44, 138.18, 135.13, 127.69, 124.36, 118.79, 114.51, 107.61,
+
(
44.5%). The product was purified by CC (DCM/MeOH 100:2); mp
5.89; MS (ESI) m/z = 290 (M) . HRMS (ESI): calcd for
1
+
1
8
1
67.3 °C; H NMR (500 MHz, MeOD) δ 8.47 (d, J = 1.7 Hz, 1H),
.35 (dd, J = 4.9, 1.5 Hz, 1H), 7.79−7.76 (m, 2H), 7.65 (d, J = 8.9 Hz,
H), 7.33 (ddd, J = 7.9, 4.9, 0.8 Hz, 1H), 7.26 (d, J = 2.5 Hz, 1H), 6.98
(
(
C H N O S ) 291.02619, found 291.02539 (M + H) . IR
cm ): ν
1
3
11
2
2 2
−
1
̃
3159 (NH), 1670 (CO).
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid Benzyl-
13
methylamide (16a). The compound was synthesized according to
procedure C, using N-benzylmethylamine to give a white solid: yield
(dd, J = 8.9, 2.5 Hz, 1H), 4.51 (s, 2H), 3.75 (s, 3H); C NMR (126
MHz, MeOD) δ 164.92, 159.49, 149.48, 148.87, 141.79, 140.52,
0
.103 g (68.9%). The product was purified by CC (DCM); mp 121.9
137.80, 136.75, 135.00, 126.63, 125.33, 124.33, 118.51, 107.45, 55.98,
+
1
4
(
2.07; MS (ESI) m/z = 298.98 (M + H) . HRMS (ESI): calcd for
°
7
C; H NMR (500 MHz, CDCl ) δ 7.69 (d, J = 8.8 Hz, 1H), 7.45−
3
+
−1
C H N O S) 299.08542, found 299.08473 (M + H) . IR (cm ): ν
̃
.37 (m, 3H), 7.36−7.28 (m, 3H), 7.19 (s, 1H), 7.04 (dd, J = 8.7, 1.7
16 15 2 2
13
3
276 (NH), 1610 (CO).
Hz, 1H), 4.81 (s, 2H), 3.84 (s, 3H), 3.13 (s, 3H); C NMR (126
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid Methyl-
MHz, CDCl ) δ 165.22, 157.70, 139.72, 138.34, 136.54, 132.86,
3
pyridin-3-ylmethylamide (19b). The compound was synthesized
according to procedure D, using compound 19a to give a yellow solid:
yield 0.02 g (47.77%). The compound was purified by salting out,
through addition of 3 mL of DCM, then addition of 30 mL of
petroleum ether to salt out compound 19a leaving the product in
1
3
28.87, 127.65, 126.74, 124.87, 123.00, 116.57, 106.08, 55.49, 36.96,
4.36; MS (ESI) m/z = 313.01 (M + H) . HRMS (ESI): calcd for
+
+
−1
(
C H NO S) 312.10582, found 312.10479 (M + H) . IR (cm ): ν
̃
18 18 2
1
613 (CO).
5-Methoxybenzo[b]thiophene-2-carboxylic Acid Phenethy-
filtrate, which was then filtered over anhydrous MgSO . The solvent
lamide (17a). The compound was synthesized according to
procedure C, using 2-phenylethylamine to give a white solid: yield
4
was then removed under reduced pressure to give the final product;
1
mp 132−133 °C; H NMR (500 MHz, acetone-d ) δ 8.62 (s, 1H),
0
.11 g (81.7%). The product was purified by CC (DCM/petroleum
6
1
8.54 (dd, J = 4.7, 1.6 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 7.3
Hz, 1H), 7.69 (s, 1H), 7.41 (dd, J = 7.4, 4.9 Hz, 2H), 7.09 (dd, J = 8.9,
ether 4:1); mp 153.8 °C; H NMR (500 MHz, CDCl ) δ 7.69 (d, J =
3
8
7
1
2
1
1
.9 Hz, 1H), 7.61 (s, 1H), 7.36−7.33 (m, 2H), 7.28−7.26 (m, 1H),
.26−7.24 (m, 2H), 7.22 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 8.9, 2.5 Hz,
H), 3.86 (s, 3H), 3.73 (dd, J = 12.9, 6.9 Hz, 2H), 2.95 (t, J = 6.9 Hz,
2
.5 Hz, 1H), 4.86 (s, 2H), 3.85 (s, 3H), 3.25 (s, 3H); 13C NMR (126
MHz, MeOD) δ 159.56, 149.45, 149.42, 141.40, 137.85, 134.84,
H); 13C NMR (126 MHz, DMSO-d ) δ 161.41, 157.36, 141.14,
134.81, 125.60, 125.56, 125.39, 124.06, 124.01, 118.20, 107.43, 55.97,
6
+
4
9.85, 30.77; MS (ESI) m/z = 313.03 (M + H) . HRMS (ESI): calcd
40.27, 139.35, 132.58, 128.66, 128.35, 126.13, 124.27, 123.55, 116.58,
+
+
for (C17
H
17
N
2
O
2
S) 313.10107, found 313.10021 (M + H) . IR
1609 (CO).
5-Methoxybenzo[b]thiophene-2-carboxylic Acid 2-
Fluorobenzylamide (20a). The compound was synthesized
according to procedure C, using 2-fluorobenzylamine to give a white
06.69, 55.39, 40.91, 35.03; MS (ESI) m/z = 312 (M + H) . HRMS
−1
(cm ): ν
̃
(
ESI): calcd for (C H NO S) 312.10582, found 312.10484 (M +
18 18 2
+
−1
H) . IR (cm ): ν
̃
3337 (NH), 1629 (CO).
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid Methyl-
phenethylamide (17b). The compound was synthesized according
to procedure D, using compound 17a, to give a yellow solid: yield
0
solid: yield 0.134 g (74%). The product was purified by CC (DCM);
1
mp 152.8 °C; H NMR (500 MHz, DMSO-d ) δ 9.30 (t, J = 5.8 Hz,
6
.012 g (57%). The product was purified by CC (DCM/petroleum
1
7
7
1
H), 8.06 (s, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H),
.41 (td, J = 7.7, 1.6 Hz, 1H), 7.33 (ddd, J = 9.6, 6.4, 1.8 Hz, 1H),
.22−7.20 (m, 1H), 7.19−7.17 (m, 1H), 7.10 (dd, J = 8.9, 2.5 Hz,
1
ether 4:1); mp 180−181 °C; H NMR (500 MHz, DMSO-d ) δ 7.87
6
(
dd, J = 8.8, 5.5 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H), 7.32−7.27 (m, 3H),
7
1
6
1
1
.24 (ddd, J = 15.9, 9.2, 4.4 Hz, 3H), 7.08 (ddd, J = 8.4, 5.8, 2.5 Hz,
13
H), 4.53 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H); C NMR (126 MHz,
H), 3.82 (s, 3H), 3.70 (t, J = 7.2 Hz, 2H), 3.17 (s, 3H), 2.91 (t, J =
1
DMSO-d ) δ 161.62, 160.04 (d, J
1
= 244.5 Hz), 157.37, 140.58,
.9 Hz, 2H); 13C NMR (126 MHz, DMSO-d ) δ 161.45, 157.37,
6
C−F
3
3
6
40.25, 132.69, 129.63 (d, J
= 4.4 Hz), 129.01 (d, J
= 14.7 Hz), 124.81 (s), 124.36 (d, J
= 8.1 Hz),
= 3.4 Hz),
C−F
C−F
41.15, 140.30, 139.38, 132.60, 128.78, 128.45, 126.34, 124.30, 123.22,
16.61, 106.59, 55.42, 55.34, 40.94, 35.06; MS (ESI) m/z = 326 (M +
2
4
1
25.76 (d, J
C−F
C−F
2
1
23.56, 116.71, 115.12 (d, J
= 21.1 Hz), 106.74, 55.38, 36.51 (d,
+
C−F
H) . HRMS (ESI): calcd for (C H NO S) 326.12147, found
3
3
+
1
9
20
2
JC−F = 4.6 Hz); MS (ESI) m/z = 315.98 (M + H) . HRMS (ESI):
+
26.12052 (M + H) .
+
calcd for (C H FNO S) 316.08075, found 316.08001 (M + H) . IR
17
15
2
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (Pyridin-2-
−1
(cm ): ν
̃
3288 (NH), 1612 (CO).
ylmethyl)amide (18a). The compound was synthesized according to
procedure C, using 2-picolylamine to give a white solid: yield 0.064
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid (2-
Fluorobenzyl)methylamide (20b). The compound was synthesized
according to procedure D using compound 20a, to give a yellowish
(
55.85%). The product was purified by CC (DCM/MeOH 100:1);
1
mp 137−138 °C; H NMR (500 MHz, MeOD) δ 8.50 (ddd, J = 5.0,
1
1
white solid: yield 0.065 g (62.5%). The product was purified by CC
.8, 0.9 Hz, 1H), 7.94 (d, J = 0.5 Hz, 1H), 7.82 (td, J = 7.8, 1.8 Hz,
H), 7.77 (d, J = 8.9 Hz, 1H), 7.46 (dt, J = 7.9, 1.0 Hz, 1H), 7.38 (d, J
2.4 Hz, 1H), 7.34−7.31 (m, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 4.70
1
(
DCM); mp 110−111 °C; H NMR (500 MHz, DMSO-d ) δ 7.88 (d,
6
J = 8.9 Hz, 1H), 7.78 (s, 1H), 7.43 (s, 1H), 7.38 (ddd, J = 12.7, 8.9, 4.3
=
(
Hz, 2H), 7.24 (t, J = 9.0 Hz, 2H), 7.11−7.07 (m, 1H), 4.80 (s, 2H),
s, 2H), 3.86 (s, 3H); 13C NMR (126 MHz, MeOD) δ 165.01, 159.49,
13
3
.81 (s, 3H), 3.22 (s, 3H); C NMR (126 MHz, DMSO-d ) δ 160.37
6
1
1
59.24, 149.82, 141.83, 140.63, 138.94, 135.03, 126.70, 124.34, 123.87,
22.95, 118.50, 107.45, 55.98, 45.94; MS (ESI) m/z = 299.01 (M +
1
(d, J
= 245.9 Hz), 157.37, 140.59, 139.90, 138.40, 131.85, 129.52
= 4.6 Hz), 129.47, 124.72, 123.73 (d, J
123.57, 123.23, 116.58, 115.46 (d, J
5.30, 45.92; MS (ESI) m/z = 329.99 (M + H) . HRMS (ESI): calcd
C−F
3
2
(d, J
= 14.4 Hz),
C−F
C−F
+
H) . HRMS (ESI): calcd for (C H N O S) 299.08542, found
2
1
6
15
2
2
= 20.9 Hz), 106.63, 55.39,
C−F
+
−1
+
2
99.08468 (M + H) . IR (cm ): ν
̃
3281 (NH), 1610 (CO).
5
+
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid Methylpyr-
for (C H FNO S) 330.09640, found 330.09567 (M + H) . IR
18
17
2
idin-2-ylmethylamide (18b). The compound was synthesized
according to procedure D, using compound 18a, to give a yellow
solid: yield 0.025 g (47%). The product was purified by CC (DCM/
−1
(cm ): ν
̃
1601 (CO).
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid 3-
Fluorobenzylamide (21a). The compound was synthesized
according to procedure C, using 3-fluorobenzylamine to give a white
1
MeOH 100:2); mp 144.9 °C; H NMR (500 MHz, acetone-d ) δ 8.56
6
(
s, 1H), 7.79 (d, J = 9.2 Hz, 2H), 7.71−7.63 (m, 1H), 7.38−7.35 (m,
solid: yield 0.13 g (71.5%). The product was purified by CC (DCM);
1
2
3
1
1
H), 7.28 (s, 1H), 7.04 (d, J = 9.4 Hz, 1H), 4.86 (s, 2H), 3.81 (s, 3H),
mp 164.9 °C; H NMR (500 MHz, DMSO-d ) δ 9.35 (t, J = 6.0 Hz,
6
.27 (s, 3H); 13C NMR (126 MHz, acetone-d ) δ 164.64, 158.87,
50.45, 141.15, 140.18, 137.71, 133.42, 126.11, 123.83, 123.33, 122.56,
17.40, 114.67, 107.20, 56.05, 55.80, 49.80; MS (ESI) m/z = 313 (M +
6
1H), 8.05 (d, J = 0.6 Hz, 1H), 7.91−7.88 (m, 1H), 7.44 (d, J = 2.4 Hz,
1H), 7.39 (ddd, J = 10.7, 7.0, 5.1 Hz, 1H), 7.20−7.17 (m, 1H), 7.17−
7.13 (m, 1H), 7.12−7.07 (m, 2H), 4.50 (d, J = 6.0 Hz, 2H), 3.83 (s,
+
13
1
H) . HRMS (ESI): calcd for (C H N O S) 313.10107, found
3H); C NMR (126 MHz, DMSO-d ) δ 162.20 (d, J
= 243.4
1
7
17
2
2
6
C−F
+
−1
3
3
13.10015 (M + H) . IR (cm ): ν
̃
1604 (CO).
Hz), 161.28, 157.38, 142.29 (d, J
= 7.0 Hz), 140.59, 140.25,
C−F
K
DOI: 10.1021/acs.jmedchem.6b01915
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4
1
2
32.69, 130.31 (d, 3JC−F = 8.3 Hz), 124.78, 123.57, 123.25 (d, J
=
removed under reduced pressure to give the final product; mp 111.7
C−F
2
2
1
.6 Hz), 116.73, 113.93 (d, J
= 21.5 Hz), 113.64 (d, J
C−F
= 20.9
°C; H NMR (500 MHz, DMSO-d ) δ 7.88 (d, J = 8.8 Hz, 1H), 7.42
C−F
C−F
6
4
Hz), 106.74, 55.38, 42.17 (d, J
(
3
= 1.2 Hz); MS (ESI) m/z = 315.98
M + H) . HRMS (ESI): calcd for (C H FNO S) 316.08075, found
(s, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.11−7.06 (m, 1H), 6.95−6.76 (m,
+
13
4H), 4.72 (s, 2H), 3.80 (s, 3H), 3.75 (s, 3H), 3.19 (s, 3H); C NMR
17
15
2
+
−1
16.08003 (M + H) . IR (cm ): ν
̃
3286 (NH), 1614 (CO).
(126 MHz, DMSO-d ) δ 161.52, 159.54, 157.37, 140.87, 139.91,
6
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid (3-
138.63, 131.79, 129.84, 124.63, 123.56, 123.22, 119.44, 116.54, 112.64,
+
Fluorobenzyl)methylamide (21b). The compound was synthesized
according to procedure D, using compound 21a to give a white solid:
106.61, 55.39, 55.30, 54.99, 42.59 ; MS (ESI) m/z = 342.02 (M + H) .
HRMS (ESI): calcd for (C H NO S) 342.11639, found
1
9
̃
20
3
+
−1
yield 0.05 g (48%). The product was purified by CC (DCM); mp
09.6 °C; H NMR (500 MHz, DMSO-d ) δ 7.89 (dd, J = 8.8, 4.3 Hz,
342.11554(M + H) . IR (cm ): ν
1617 (CO).
1
1
1
3
5-Methoxybenzo[b]thiophene-2-carboxylic Acid Methyl-(3-
methylbenzyl)amide (24a). The compound was synthesized
according to procedure C, using N-methyl-1-(m-tolyl)methanamine
6
H), 7.47−7.41 (m, 2H), 7.20−7.06 (m, 5H), 4.76 (s, 2H), 3.80 (s,
H), 3.22 (s, 3H); 13C NMR (126 MHz, DMSO-d ) δ 162.39 (d,
6
1
3
JC−F = 243.1 Hz), 161.64, 157.37, 140.13 (d, J
= 6.5 Hz), 139.93,
= 8.0 Hz), 124.79, 123.58,
to give a yellow solid: yield 0.134 g (73.6%). The product was purified
C−F
3
1
1
1
1
38.44, 132.71 131.85, 130.69 (d, J
by CC (DCM); mp 125.7 °C; H NMR (500 MHz, DMSO-d
6
) δ 7.88
C−F
2
2
16.60, 114.15 (d, J
= 20.7 Hz), 113.65 (d, J
= 21.2 Hz),
(d, J = 8.8 Hz, 1H), 7.79 (s, 1H), 7.42 (s, 1H), 7.28 (t, J = 7.5 Hz,
1H), 7.10 (dd, J = 16.0, 7.9 Hz, 4H), 4.71 (s, 2H), 3.80 (s, 3H), 3.17
C−F
C−F
+
06.63, 55.30, 42.19, 37.29; MS (ESI) m/z = 330.01 (M + H) . HRMS
ESI): calcd for (C H FNO S) 330.09640, found 330.09556 (M +
(s, 3H), 2.31 (s, 3H); 13C NMR (126 MHz, DMSO-d
) δ 163.37,
(
6
1
8
17
2
+
−1
H) . IR (cm ): ν
̃
1612 (CO).
157.36, 139.90, 138.53, 137.86, 136.90, 131.78, 128.61, 128.00, 126.09,
124.60, 123.87, 123.21, 116.51, 106.60, 55.29, 51.07, 37.19, 21.02; MS
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid 4-
Fluorobenzylamide (22a). The compound was synthesized
according to procedure C, using 4-fluorobenzylamine to give a white
solid: yield 0.11 g (73%). The product was purified by CC (DCM);
mp 185.8 °C; H NMR (500 MHz, DMSO-d ) δ 9.31 (d, J = 4.9 Hz,
H), 8.02 (d, J = 3.8 Hz, 1H), 7.88 (dd, J = 8.8, 3.9 Hz, 1H), 7.43 (d, J
2.7 Hz, 1H), 7.41−7.35 (m, 2H), 7.20−7.14 (m, 2H), 7.12−7.08 (m,
H), 4.47 (d, J = 3.9 Hz, 2H), 3.83 (s, 3H); C NMR (126 MHz,
= 241.5 Hz), 157.36, 140.75,
= 2.7 Hz), 132.67, 129.31 ( JC−F, J = 8.1 Hz),
24.66, 123.56, 116.68, 115.05 (d, JC−F = 21.3 Hz), 106.72, 55.37,
1.96; MS (ESI) m/z = 316 (M + H) . HRMS (ESI): calcd for
+
(ESI) m/z = 325.95 (M + H) . HRMS (ESI): calcd for (C H NO S)
1
9
20
2
+
−1
326.12147, found 342.12078(M + H) . IR (cm ): ν
̃
1614 (CO).
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (Furan-2-
ylmethyl)amide (25a). The compound was synthesized according
to procedure C, using furfurylamine to give a yellow solid: yield 0.1 g
(58%). The product was purified by CC (DCM); mp 118.9 °C; H
NMR (500 MHz, DMSO-d ) δ 9.24 (t, J = 5.6 Hz, 1H), 8.03 (s, 1H),
1
6
1
=
1
1
13
6
1
DMSO-d ) δ 161.51, 161.20 (d, J
7.88 (d, J = 8.9 Hz, 1H), 7.60 (dd, J = 1.8, 0.9 Hz, 1H), 7.42 (d, J = 2.5
Hz, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 6.41 (dd, J = 3.2, 1.8 Hz, 1H),
6
C−F
4
3
1
1
4
40.25, 135.46 (d, J
C−F
2
6.31 (dd, J = 3.2, 0.7 Hz, 1H), 4.47 (d, J = 5.7 Hz, 2H), 3.82 (s, 3H);
+
13
C NMR (126 MHz, DMSO-d ) δ 161.38, 157.36, 151.94, 142.16,
6
+
−1
(
C H FNO S) 316.08075, found 316.08010 (M + H) . IR (cm ): ν
̃
140.58, 140.24, 132.68, 124.79, 123.55, 116.73, 110.50, 107.16, 106.71,
1
7
15
2
+
3
279 (NH), 1615 (CO).
55.37, 35.97; MS (ESI) m/z = 288 (M + H) . HRMS (ESI): calcd for
+
−1
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid (4-
(C H NO S) 288.06944, found 288.06872(M + H) . IR (cm ): ν
3306 (NH), 1616 (CO).
̃
15 14 3
Fluorobenzyl)methylamide (22b). The compound was synthesized
according to procedure D, using compound 22a to give a yellow solid:
yield 0.05 g (53.19%). The compound was purified by salting out,
through addition of 3 mL DCM, followed by the addition of 30 mL of
petroleum ether that will salt out compound 22a leaving the product
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (2-Imida-
zol-1-ylethyl)amide (26a). The compound was synthesized
according to procedure C, using 2-(1H-imidazol-1-yl)ethanamine to
give a white solid: yield 0.025 g (34.7%). The product was purified by
1
in the filtrate, which was then filtered over anhydrous MgSO . The
CC (DCM/MeOH 100:3); mp 143 °C; H NMR (500 MHz, MeOD)
4
solvent was then removed under reduced pressure to give the final
δ 7.88 (s, 1H), 7.78 (d, J = 0.5 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.34
(d, J = 2.4 Hz, 1H), 7.24 (s, 1H), 7.08 (dd, J = 8.9, 2.5 Hz, 2H), 4.33−
1
product; mp 125−126 °C; H NMR (500 MHz, acetone-d ) δ 7.85−
6
13
7
8
3
2
8
1
.81 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.45−7.37 (m, 3H), 7.16 (t, J =
4.29 (m, 2H), 3.85 (s, 3H), 3.75−3.72 (m, 2H); C NMR (126 MHz,
MeOD) δ 165.15, 159.49, 141.74, 140.38, 138.39, 134.94, 127.83,
.7 Hz, 2H), 7.08 (dd, J = 8.9, 2.5 Hz, 1H), 4.80 (s, 2H), 3.84 (s, 3H),
.18 (s, 3H); 13C NMR (126 MHz, DMSO-d ) δ 161.47 (d, J
1
=
=
126.60, 124.31, 121.49, 118.53, 107.44, 55.99, 47.56, 41.77; MS (ESI)
6
C−F
3
+
42.9 Hz), 157.37, 140.77, 140.27, 139.92, 131.82, 129.32 (d, J
m/z = 302 (M + H) . HRMS (ESI): calcd for (C H N O S)
C−F
15 16
3
2
2
+
−1
.1 Hz), 124.68, 123.56, 123.22, 116.56, 115.47 (d, JC−F = 21.1 Hz),
06.62, 55.38, 55.30, 41.97; MS (ESI) m/z = 330 (M + H) . HRMS
ESI): calcd for (C H FNO S) 330.09640, found 330.09566 (M +
̃
302.09632, found 302.09546 (M + H) . IR (cm ): ν 3300 (NH),
+
1612 (CO).
(
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (2-Imida-
zol-1-yl-ethyl)methylamide (26b). The compound was synthesized
according to procedure D, using compound 26a to give a white solid:
1
8
17
2
+
−1
H) . IR (cm ): ν
̃
1601 (CO).
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid 3-
Methoxybenzylamide (23a). The compound was synthesized
according to procedure C, using 3-methoxybenzylamine, to give a
white solid: yield 0.12 g (63.6%). The product was purified by CC
yield 0.08 g (36%). The product was purified by CC (DCM/MeOH
1
100:2.5); mp 117−118 °C; H NMR (500 MHz, acetone-d ) δ 7.82
6
(d, J = 8.8 Hz, 1H), 7.63 (s, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.40 (s,
1H), 7.16 (dd, J = 8.9, 2.5 Hz, 1H), 7.08 (dd, J = 8.8, 2.5 Hz, 1H), 6.97
(s, 1H), 4.37 (t, J = 6.0 Hz, 2H), 3.91 (t, J = 5.9 Hz, 2H), 3.86 (s, 3H),
1
(
DCM); mp 147.1 °C; H NMR (500 MHz, DMSO-d ) δ 9.29 (t, J =
6
6
7
1
2
1
1
.0 Hz, 1H), 8.04 (d, J = 0.6 Hz, 1H), 7.89 (dd, J = 8.9, 0.6 Hz, 1H),
.43 (d, J = 2.4 Hz, 1H), 7.28−7.24 (m, 1H), 7.10 (dd, J = 8.9, 2.5 Hz,
H), 6.92−6.90 (m, 2H), 6.84−6.82 (m, 1H), 4.46 (d, J = 6.0 Hz,
3.10 (s, 3H); 13C NMR (126 MHz, DMSO-d ) δ 157.34, 139.88,
6
139.84, 137.42, 131.73, 128.18, 128.16, 123.20, 119.76, 119.72, 116.45,
H), 3.83 (s, 3H), 3.74 (s, 3H); 13C NMR (126 MHz, DMSO-d ) δ
106.58, 55.31, 54.89, 43.66, 29.00; MS (ESI) m/z = 315.97 (M + H) .
+
6
61.50, 159.30, 157.36, 140.86, 140.84, 140.27, 132.67, 129.42, 124.61,
23.56, 119.42, 116.67, 113.02, 112.17, 106.72, 55.37, 54.96, 42.58;
MS (ESI) m/z = 328.02 (M + H) . HRMS (ESI): calcd for
C H NO S) 328.10074, found 328.09998 (M + H) . IR (cm ): ν
HRMS (ESI): calcd for (C H N O S) 316.11197, found 316.11123
1
6
18
3
2
+
−1
(M + H) . IR (cm ): ν
̃
1626 (CO).
+
5-Methoxybenzo[b]thiophene-2-carboxylic Acid (1H-Imida-
zol-2-ylmethyl)amide (27a). The compound was synthesized
according to procedure C, using 2-aminomethyl-1H-imidazole
dihydrochloride, giving a white solid: yield 0.1 g (60%). The
compound precipitated after reaction completion. The reaction
mixture was filtered, and the residue was washed using petroleum
+
−1
(
̃
18 18 3
3
290 (NH), 1610 (CO).
5
-Methoxybenzo[b]thiophene-2-carboxylic Acid (3-
Methoxybenzyl)methylamide (23b). The compound was synthe-
sized according to procedure D, using compound 23a to give a yellow
solid: yield 0.05 g (42%). The compound was purified by salting out,
through addition of a 3 mL of DCM, then addition of 30 mL of
petroleum ether to salt out compound 23a leaving the product in the
1
ether (3 × 10 mL) to give the desired product; mp 239.7 °C; H NMR
(500 MHz, DMSO-d ) δ 9.27 (t, J = 5.7 Hz, 1H), 8.04 (d, J = 0.6 Hz,
6
1H), 7.89 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.10 (dd, J =
filtrate. which was then filtered over MgSO . Then the solvent was
8.9, 2.5 Hz, 1H), 6.93 (s, 2H), 4.50 (d, J = 5.7 Hz, 2H), 3.83 (s, 3H);
4
L
DOI: 10.1021/acs.jmedchem.6b01915
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
3
C NMR (126 MHz, DMSO-d ) δ 161.60, 157.36, 144.55, 140.71,
system (LI-COR). All cell treatments and immunoblotting analyses
were performed two times in duplicates. The normalization of the
signals according to the amount of loaded protein was performed in
two ways: the gel used for the Western transfer was stained afterward
using Coomassie blue, and the remaining protein bands quantified
using GelQuant.NET software provided by biochemlabsolutions.com.
In addition, the relative protein load was measured using signals from
primary antibodies that were insensitive to the compound treatments.
Molecular Docking. All procedures were performed using the
Molecular Operating Environment (MOE) software package (version
2010, Chemical Computing Group). For the docking simulations,
6
1
3
(
40.66, 140.28, 132.69, 124.86, 123.54, 121.73, 116.72, 106.70, 55.38,
6.99; MS (ESI) m/z = 387.96 (M + H) . HRMS (ESI): calcd for
C H N O S) 288.08067, found 288.07992(M + H) . IR (cm ): ν
288 (NH), 1629 (CO).
+
+
−1
̃
14 14 3 2
3
Biological Assays. Protein Kinases and Inhibition Assays.
2
7
Human Dyrk1A was expressed and purified as described earlier.
Active human Clk1 was purchased from Life Technologies (lot no.
095729A, catalog no. PV3315). Woodtide substrate peptide for
1
Dyrk1A (KKISGRLSPIMTEQ) and RS repeat substrate peptide for
Clk1 (GRSRSRSRSRSRSRSR) were custom synthesized at the
Department of Medical Biochemistry and Molecular Biology, Saarland
University, Homburg, Germany. Kinase inhibition assays for Dyrk1A,
Dyrk1B, Clk1, and CK2 were performed as described previously, in
4
PDB entries 1Z57 (Clk1 cocrystallized with hymenialdisine) and
2WO6 (Dyrk1A cocrystallized with N-(5-1H-indazol-3-yl)benzamide)
2
1
were used. Molecular docking simulations with 21b as a ligand were
performed using the MMFF94x and OPLS-AA force fields and the
“triangle matcher” method (number of return poses set to 2000) and
“Force field” as refinement. The refinement setting was further
configured as follows: free side chain movements enabled, cutoff 10 Å.
By use of these settings, docking runs were performed in two different
ways: (i) the binding pocket was defined by selecting residues Leu244,
Lys191, and Gly173 (Clk1) or Leu241, Lys188, and Gly171
(Dyrk1A); (ii) after an initial run using condition (i), a
pharmacophore was defined based on a suitable pose after energy
minimization using the MOE LigX routine (settings: receptor strength
27
the presence of 15 μM ATP. The calculated IC50 values are
representative of at least two independent determinations. The larger
panel of kinases shown in Table 3 was screened by the SelectScreen
Kinase Profiling Service, Thermo Fisher Scientific, Paisley, U.K.
MTT Cell Growth Assay. The T24 bladder carcinoma cell line, the
hepatocellular carcinoma cell line HepG2, and the breast cancer cell
lines MDA-MB-231 and T47D were cultured in Dulbecco’s modified
Eagle medium (DMEM, Sigma-Aldrich). The acute monocytic
leukemia cell line THP-1 and peripheral blood lymphocytes (PBLs,
pooled samples from three healthy donors) were maintained in RPMI-
=
1, ligand strength = 5000), by selecting both the methoxy oxygen
1
1
640 medium (Sigma-Aldrich). Both media were supplemented with
% pen/strep solution (Invitrogen) and 5% fetal calf serum (FCS,
close to the NH of the leucine and the carbonyl oxygen near the
conserved lysine as acceptor points (this binding orientation prevailed
among the poses with the compound docked deep in the pocket). In
the pharmacophore definition window, the radius of the pharmaco-
phore points was raised to 1.6 Å. The subsequent docking was
performed using the pharmacophore-supported placement. The
number of retained poses was set to 500 each time. Only the poses
with the top 10 scoring values were further evaluated for plausibility.
The final selected poses were optimized again using the LigX routine.
Sigma-Aldrich). For the MTT assay, all cells except PBLs were seeded
in a 96-well plate (5000 cells per well) already containing DMSO as a
control (0.2% final concentration) or the test compounds dissolved in
DMSO. The cells were grown for 5 days at 37 °C in a humidified
incubator containing 5% CO , without further change of medium,
2
28
before the detection was carried out as described. PBLs were
suspended in 96 well plates at 30 000 cells per well and maintained in
the presence of compounds or DMSO for 2 days prior to
measurement.
Treatment of Cell Lines and Immunoblotting Analysis. T24,
MDA-MB-231, and HepG2 cells were seeded in Petri dishes (5.5 cm
diameter) and grown to 70% confluency in DMEM medium as
specified under “MTT Cell Growth Assay”. The compounds were
diluted from DMSO stocks in DMEM to the final concentrations
indicated in the figure legend, and then the medium in the plates was
exchanged by compound-containing medium. After 3 days in the
incubator, the cells were washed once with phosphate buffered saline
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01915.
■
*
S
Figures S1 and S2 (PDF)
Molecular formula strings (CSV)
(
PBS), harvested by the addition of 0.6 mL of 0.8× concentrated SDS-
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 681 302 70312. Fax: +49 681 302 70308. E-mail:
ma.engel@mx.uni-saarland.de.
■
ORCID
Mohammad Abdel-Halim: 0000-0003-1326-4219
Matthias Engel: 0000-0001-5065-8634
aminopropanesulfonic acid (CAPS), pH 9.6, 15% methanol; cathode
buffer, 60 mM Tris, 40 mM CAPS, 0.1% SDS). The membranes were
blocked in blocking buffer (3% BSA in PBS) and incubated with
suitable combinations of the following primary monoclonal antibodies
in blocking buffer (all from Santa Cruz Biotechnology, dilutions all
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
1
:660, antibody clone number in parentheses): EGFR (A-10), p70S6
kinase α (H-9), PARP-1 (F-2), ERK2 (D-2), HDAC1 (10E2), HCAM
=CD44) (DF1485), PCNA (PC10), GRP 94 (9G10) at 4 °C
The authors declare no competing financial interest.
(
overnight. When the Clk1 protein expression was analyzed, total cell
protein from untreated cells was harvested and subject to Western
Blotting as above, and anti-Clk1/4 antibody (Santa Cruz Biotechnol-
ogy catalog no. sc-515307, dilution 1:660) was applied. After
incubation with the primary antibody, the membrane was washed
four times using PBS with 0.1% Tween 20, incubated with the IRDye
ACKNOWLEDGMENTS
■
We thank Prof. Dr. R. W. Hartmann and Prof. Dr. Christian
Ducho for generously providing lab facilities to the M.E. group.
A.K.E. and M.A.-H. acknowledge The Science & Technology
Development Fund in Egypt (STDF) for funding through the
GE-SEED, Project 17391. The support by the Deutsche
Forschungsgemeinschaft (DFG) (Grant EN381/2-3) to M.E. is
greatly acknowledged. The authors are particularly thankful to
6
80-conjugated anti-mouse IgG secondary antibody (diluted 1:10 000
in blocking buffer) for 1 h, and washed again four times as above.
Finally the membrane was scanned using an Odyssey infrared imaging
M
DOI: 10.1021/acs.jmedchem.6b01915
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Dr. Jessica Hoppstadter for her constant support and supply
Article
̈
(12) Yoshida, T.; Kim, J. H.; Carver, K.; Su, Y.; Weremowicz, S.;
Mulvey, L.; Yamamoto, S.; Brennan, C.; Mei, S.; Long, H.; Yao, J.;
Polyak, K. CLK2 is an oncogenic kinase and splicing regulator in
breast cancer. Cancer Res. 2015, 75, 1516−1526.
with reagents and cells.
ABBREVIATIONS USED
CC, column chromatography; CID, collision induced dissoci-
ation; Clk, cdc2-like kinase; CK, casein kinase; Dyrk, dual
■
(
13) Muraki, M.; Ohkawara, B.; Hosoya, T.; Onogi, H.; Koizumi, J.;
Koizumi, T.; Sumi, K.; Yomoda, J.; Murray, M. V.; Kimura, H.;
Furuichi, K.; Shibuya, H.; Krainer, A. R.; Suzuki, M.; Hagiwara, M.
Manipulation of alternative splicing by a newly developed inhibitor of
Clks. J. Biol. Chem. 2004, 279, 24246−24254.
specificity protein kinase; GI , the concentration for 50% of
5
0
maximal inhibition of cell proliferation; GRP94, glucose
regulated protein 94; HBTU, N,N,N′,N′-tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate; HCAM,
(14) Fedorov, O.; Huber, K.; Eisenreich, A.; Filippakopoulos, P.;
King, O.; Bullock, A. N.; Szklarczyk, D.; Jensen, L. J.; Fabbro, D.;
Trappe, J.; Rauch, U.; Bracher, F.; Knapp, S. Specific CLK inhibitors
from a novel chemotype for regulation of alternative splicing. Chem.
Biol. 2011, 18, 67−76.
(
=CD44) homing cell adhesion molecule; HIF, hypoxia
inducible factor; HUVEC, human umbilical vein endothelial
cell; KHMDS, potassium bis(trimethylsilyl)amide; MLCK2,
myosin light chain kinase 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-
(
15) Mott, B. T.; Tanega, C.; Shen, M.; Maloney, D. J.; Shinn, P.;
Leister, W.; Marugan, J. J.; Inglese, J.; Austin, C. P.; Misteli, T.; Auld,
D. S.; Thomas, C. J. Evaluation of substituted 6-arylquinazolin-4-
amines as potent and selective inhibitors of cdc2-like kinases (Clk).
Bioorg. Med. Chem. Lett. 2009, 19, 6700−6705.
2
,5-diphenyltetrazolium bromide; NFAT, nuclear factor of
activated T-cell; NMD, nonsense-mediated decay; p70S6
kinase, ribosomal protein S6 kinase; PARP-1, poly ADP ribose
polymerase 1; PCNA, proliferating cell nuclear antigen; SPF45,
splicing factor 45; STK17, serine threonine kinase 17
(
16) Gao, Y.; Davies, S. P.; Augustin, M.; Woodward, A.; Patel, U. A.;
Kovelman, R.; Harvey, K. J. A broad activity screen in support of a
chemogenomic map for kinase signalling research and drug discovery.
Biochem. J. 2013, 451, 313−328.
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