Synthesis and biological evaluation of 7-substituted-1-(3-bromophenylamino) isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase and epidermal growth factor receptor

Article abstract of DOI:10.1016/j.bmc.2010.11.007

Here we present the synthesis and biological activity of a series of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase (MLCK) and the epidermal growth factor receptor kinase (EGFR). The inhibitory

Full text of DOI:10.1016/j.bmc.2010.11.007

Bioorganic & Medicinal Chemistry 19 (2011) 429–439
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of
7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles
as inhibitors of myosin light chain kinase and epidermal
growth factor receptor
Haridas B. Rode a, Martin L. Sos b, Christian Grütter a, Stefanie Heynck b, Jeffrey R. Simard a, Daniel Rauh a,
⇑
a Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany
b Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Köln,
Gleueler Strasse 50, 50931 Köln, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we present the synthesis and biological activity of a series of 7-substituted-1-(3-bromophenylami-
no)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase (MLCK) and the epidermal
growth factor receptor kinase (EGFR). The inhibitory effect of these molecules was found to be dependent
on the nature of the substituents at the 7-position of the isoquinoline scaffold.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 September 2010
Revised 2 November 2010
Accepted 4 November 2010
Available online 10 November 2010
Keywords:
Kinase inhibitors
Inhibitor profiling
Medicinal chemistry
1. Introduction
Thus, the phosphorylation of myosin is a major regulatory pathway
in smooth muscle contraction and is complemented by Ca2+-inde-
Protein kinases are crucial enzymes for the regulation of nearly
every cellular signaling event, including proliferation and apopto-
sis. Kinase function has been found to be disregulated in many dis-
eases such as cancer, inflammation, diabetes and central nervous
system disorders.1 The detailed understanding of kinase malfunc-
tion in the onset and progression of cancer led to the development
of potent kinase inhibitors as a means of novel targeted therapies
currently in clinical use.2 Furthermore, kinase inhibitors serve as
powerful tool compounds in chemical biology research to probe
and dissect enzymatic kinase function in complex cellular sys-
tems.3 An increasing number of biological studies investigating
the role of the Ca2+/calmodulin dependent kinase smMLCK
(smooth muscle myosin light chain kinase, also referred to as
MLCK) in metastatic cancer cell invasion, regulation of epithelial
cell survival, hypertension and inflammatory bowel diseases re-
flect its emergence as a potentially novel therapeutic target.4–8
pendent Rho kinases (ROCK I and ROCK II), which phosphorylate
and inactivate myosin light chain phosphatase to prevent uncon-
trolled muscle fiber relaxation.10,11 Interestingly, MLCK-dependent
increases in myosin ATPase activity have also been observed in the
contraction and migration of non-muscle cells, highlighting an
important role for MLCK in muscle as well as non-muscle
cells.12–14
Despite these many biological roles, only a handful of small or-
ganic molecules have been reported so far to be active as MLCK
inhibitors and might qualify as probe molecules to study the diverse
functional roles of this key kinase in more detail (Fig. 1).15–19 The
naphthalene sulfonamides W-7, ML-7, and ML-9, which are close
analogs of the marketed vasodilator and ROCK II kinase inhibitor
Fasudil (HA-1077), as well as the microbial metabolites K-252a18
and Wortmannin19 have been shown to be ATP-competitive
inhibitors of MLCK.15 Additionally, studies on the auto-inhibition
mechanism of MLCK led to the identification of potent, peptide-
based inhibitors.17 Here we report the identification and further
development of 7-substituted-1-(3-bromophenylamino)isoquino-
line-4-carbonitriles as a novel class of selective MLCK inhibitors.
Interestingly, one of these compounds was also found to be an inhib-
itor of a clinically relevant target in cancer, namely the epidermal
growth factor receptor (EGFR), both in biochemical and cellular
assays.
MLCK is a serine/threonine kinase and transfers the c-phosphate
from ATP to a serine residue of the 20-kD regulatory light chain
of myosin (MLC20).4,5 Phosphorylated myosin interacts with actin
filaments, resulting in the contraction of smooth muscle fibers.9
⇑
Corresponding author. Tel.: +49 (0)231 9742 6480; fax: +49 (0)231 9742 6479.
E-mail address: daniel.rauh@cgc.mpg.de (D. Rauh).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.007

430
H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
Figure 1. Structures of known ATP-competitive MLCK inhibitors. W-7, ML-7 and ML-9 are close analogs of the Rho kinase (ROCK II) inhibitor Fasudil (HA-1077). Wortmanin
and K-252a are metabolites obtained from microorganisms and are potent but unselective inhibitors of MLCK.15–19
ATP binding pocket and adjacent to the hinge region. The size
and polarity of this amino acid is well-known for influencing inhib-
itor affinity and selectivity profiles amongst kinases.2
2. Results and discussion
2.1. Screening for inhibitors of MLCK
2.3. Synthesis and SAR of a focused library of 7-substituted-3-
bromophenylamino-isoquinoline-4-carbonitriles
In order to identify novel inhibitors of MLCK, we screened a fo-
cused compound library in a 384-well format by detecting sub-
strate phosphorylation in the presence of Ca2+/calmodulin and
using homogeneous time-resolved fluorescence (HTRF) as a read-
out. We identified 7-substituted-1-(3-bromophenylamino)iso-
quinoline-4-carbonitriles 8 and 9 as weak primary hits. Although
these two molecules differ in the extent of saturation of the side
chain attached at the 7-position of the isoquinoline core, they were
Based on this initial SAR and to further optimize compound
potency, we synthesized a focused library of 3-bromophenylami-
no-isoquinoline-4-carbonitriles with various substituents at the
7-position of the isoquinoline core (Scheme 1). The synthesis com-
menced with the nitration of commercially available isoquinoline-
1-one 1 to produce the 5- and 7-nitro positional isomers 2 and 3,
which were separated by repeated column chromatography. The
7-nitro isomer 3 was brominated to give 4. Palladium-catalyzed
cyanation of 4 with Zn(CN)2 as the cyanide source and S-Phos as
the ligand produced 5 in moderate yield. Alternatively, the cyana-
tion reaction could be performed with the assistance of micro-
waves, but resulted in significantly lower yields. Chlorination of 5
with phosphorous oxychloride followed by treatment with m-bro-
moaniline resulted in analog 6. Selective reduction of the nitro
group in the presence of bromine was then successfully fulfilled
by treating 6 with SnCl2. The resulting amine 7 was coupled with
different acids or treated with the corresponding acid chlorides to
generate a focused library of 7-substituted-1-(3-bromophenylami-
no)isoquinoline-4-carbonitriles. All compounds were tested for
inhibition of MLCK (Table 1). The introduction of a N-methylpiper-
azine in the 7-position of the isoquinoline core (11) significantly
enhanced potency (threefold) against MLCK when compared to
primary hits 8 and 9 while introduction of aromatic amine function-
alities (17, 18) led to loss of potency. Similarly, modification of the
7-position with alkyl chains (10, 13) or oxygen containing ring
systems such as furan (15, 16), morpholino (12) or oxopyrrolidine
equipotent with IC50 values of 25 and 24 lM, respectively (Table 1).
To the best of our knowledge, 7-amino substituted isoquinoline-4-
carbonitriles were not previously reported as MLCK inhibitors in
literature so we set out to further develop these hits into more po-
tent inhibitors.
2.2. Profiling for kinase inhibitor selectivity
In order to gain more insights into their selectivity profiles and
SAR, we profiled the identified hit molecules 8 and 9 against a pa-
nel of 95 kinases at a concentration of 10 lM (National Centre for
Protein Kinase Profiling, University of Dundee, UK) (Fig. 2). Despite
the different substituents at the 7-position of the isoquinoline ring,
the compounds show similar selectivity profiles with preferred
inhibition of MLCK. While 8 also strongly inhibits the Ca2+/calmod-
ulin dependent kinases PHK and CAMK1, 9 shows preferential inhi-
bition of the receptor tyrosine kinase HER4. Interestingly, both
molecules fail to inhibit the Ca2+/calmodulin dependent protein ki-
nase kinase b (CAMKKb). With the exception of HER4, all kinases
inhibited by 8 or 9 posses a large hydrophobic amino acid at the
gatekeeper position. The gatekeeper is located at the back of the

H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
431
Table 1
of inhibition of MLCK by our newly developed isoquinoline in more
detail, we performed a Lineweaver–Burk analysis. A double-reci-
procal plot of 1/V versus 1/[ATP] showed that 11 inhibits MLCK in
an ATP-competitive manner, further suggesting that this com-
pound class binds to the ATP pocket of the kinase domain (Fig. 3).
Focused library of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitrilesa
HN
Br
R
N
2.4. Biochemical characterization of 11
CN
Being the most potent inhibitor on MLCK in vitro, we then pro-
filed 11 against the same panel of 95 kinases (Fig. 2). Compound 11
inhibited MLCK and CAMK1 potently and revealed a similar profile
when compared to 8 and 9. The two Src kinase family members Lck
and YES1, each which possess a Thr gatekeeper, and the serine/
threonine specific kinase PHK, which has a large Phe at the gate-
keeper position, are also inhibited. The activity of Aurora B (Leu
gatekeeper) is moderately inhibited by 11 while the activity of
Ca2+/calmodulin dependent kinase CAMKKb is not perturbed. Un-
like in the case of 9, HER4, PKBb, FGF-R1 and MKK1 are not strongly
inhibited by 11.
8-18
Compound
R
IC50 in lM
MLCK
EGFR
H
N
N
N
8
9
25.1 2.1
23.6 0.1
NI
4.1 0.6
O
O
H
N
0.15 0.1
20.8 1.1
H
N
10
2.5. Modeling 11 to the ATP binding pocket of MLCK
O
N
H
To get deeper insights into the possible binding mode of 7-
substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles
in target kinases and to better understand the preference for ki-
nases with larger or hydrophobic gatekeeper residues, we modeled
the most potent inhibitor 11 to the ATP pocket of MLCK (Fig. 4b).
Compound 11 is a close structural analog of a class of well known
N
N
11
12
8.7 1.0
1.3 0.4
6.9 0.8
O
O
H
N
N
NI
4-anilino-quinazoline
and
4-anilino-quinoline-3-carbonitrile
O
based EGFR and HER2 inhibitors such as erlotinib, gefitinib and
EKB-569 and differs by the nitrile in the 4-position of the inhibitor
core (Fig. 4a).20 The crystal structures of various 4-anilino-quinaz-
oline and 4-anilino-quinoline-3-carbonitrile based inhibitors in
complex with protein kinases are known from literature and high-
light that the N1 nitrogen of the heteroaromatic ring system forms
a crucial hydrogen bonding interaction to the backbone of the ki-
nase hinge region.21 However, such inhibitors were designed to
mainly target protein kinases which hold a Thr at the gatekeeper
position since the polar side chain of the Thr gatekeeper serves
as a hydrogen bond donor and is essential for inhibitor binding
and selectivity. In case of 11, we argue that the nitrile in the 4-po-
sition of the inhibitor core would serve as the hydrogen bond
acceptor to allow for interactions with the hinge region of the ki-
nase and would position the inhibitor core and the bromo-anilino
moiety more distant from the gatekeeper position. This binding
mode could explain why the isoquinoline-4-carbonitrile core is
well tolerated by larger gatekeeper residues (Leu in MLCK, Phe in
PHK and Met in CAMK1). To test this hypothesis, we first generated
a homology model of MLCK (family member 1) using the crystal
structure of MLCK (family member 4; PDB code: 2X4F) as a tem-
plate and manually docked compound 11 to the ATP binding site
(Fig. 4b). The proposed binding mode of 11 shows the predicted
hydrogen bond between the 4-substituted nitrile and the backbone
of Val341 of the hinge region. It also reveals a potential charged
interaction of the protonated amine of 11 with the side chain of
Glu348. We recently observed a similar interaction for (2E)-N-{4-
[(3-bromophenyl)amino]-6-quinazolinyl}-4-(dimethylamino)-2-
butenamide in cSrc-S345C.22 This additional charged interaction
could account for the higher potency of 8, 9 and 11 over 10 and
13 against MLCK. The similar profiles of compounds 8, 9 and 11
against the selected kinase panel indicates that the selectivity of
these compounds is gained from conserved features such as the
isoquinoline scaffold together with the presence of the terminal
tertiary amino group at the 7-position of the isoquinoline core.
Thus, further modifications of 8, 9 and 11 aided by structure-based
H
N
O
13
14
NI
NI
12.8 0.2
2.2 0.5
O
H
N
O
O
N
H
O
H
N
15
16
NI
NI
55.6 4.0
45.2 2.6
O
O
O
H
N
O
17
18
NI
NI
28.0 2.2
HN
N
H
O
NI
N
H
HN
a
The structures of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-car-
bonitriles. Compound 9 is an irreversible inhibitor for EGFR. IC50 values were
measured with an activity-based phosphorylation assay (HTRF) and are presented
as an average of at least two independent experiments performed in duplicate. NI
indicates ‘no inhibition’ up to a concentration of 40 lM.
(14) was not tolerated by MLCK and no inhibition could be ob-
served up to a concentration of 40 M. In light of the initial hits 8
l
and 9 and the newly synthesized derivative 11, the observed SAR
underscores the importance of tertiary amines in the 7-position of
the isoquinoline core for MLCK activity and suggests that the pro-
tonated tertiary amine may serve as a critical hydrogen bond donor
when bound to the target kinase. In order to characterize the mode

432
H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
Figure 2. Kinase selectivity profiles. Kinase selectivity profiles were determined for 8, 9 and 11 against a panel of 95 different protein kinases at an inhibitor concentration of
10 M (National Centre for Protein Kinase Profiling, University of Dundee, UK). The screening was carried out using a radioactive assay. The scores represent the %-activity
l
remaining relative to the 100% DMSO controls. The inhibition is color coded from red (strong inhibition, 0–25% score) to dark yellow (medium inhibition, 25–60% score) to
white (weak/no inhibition, >60% score). The size and hydrophobicity of the gatekeeper residue turned out to be an important selectivity filter for this class of compounds.
design may result in even more potent and selective inhibitors ac-
tive against MLCK.
formation of the Michael-acceptor with this unique cysteine resi-
due. Based on this hypothesis, we decided to analyze the effect
of 9 on the clinically relevant receptor tyrosine kinase EGFR.
Activating mutations of EGFR are found in 10–20% of patients suf-
fering from non-small cell lung cancer (NSCLC), thus highlighting
EGFR as an important target in cancer therapy.26,27 To investigate
the effect of the synthesized 7-substituted 3-bromophenylamino-
isoquinoline-4-carbonitriles on the inhibition of EGFR, we carried
out phosphorylation assays in the presence and absence of inhibi-
tors (Table 1). Compound 9 was singled out from a set of 11 tested
inhibitors and showed nanomolar inhibition of EGFR while all other
inhibitorswithsaturated side chains in the 7-positionof the isoquin-
oline core produced moderate or no inhibition. This finding
highlights that the formation of a covalent bond by the Michael-
acceptor of 9 is key for potent inhibition of EGFR by the isoquino-
line-4-carbonitriles described above.
2.6. Biochemical characterization of compounds and
the inhibition of EGFR
An interesting result from the inhibitor profiling outlined above
was the observation that 9, but not its closely related analogs 8 and
11, is an inhibitor of the receptor tyrosine kinase HER4. The only
structural difference between 8 and 9 is the presence of a trans
double bond (Michael-acceptor) in the N,N-dimethylamino moiety
attached to the 7-position of the inhibitor core. Thus, the difference
in HER4 inhibition is likely related to the presence of this Michael
acceptor. Members of the HER kinase family such as EGFR (HER1),
HER2 and HER4 are known for a conserved cysteine residue that
can be targeted by irreversible inhibitors equipped with Michael-
acceptors.23 Cys803 in HER4 is isostructural to Cys797 in EGFR
(or Cys773 in an alternative EGFR sequence numbering system)
and Cys805 in HER2 and is located at the N-terminal end of a short
helix at the lip of the ATP pocket, in which the helix dipole serves
to activate the Cys sulfhydryl.24,25 We hypothesized that the
potency of 9 against HER4 might be linked to covalent bond
2.7. Modeling of 9 into the ATP binding pocket of EGFR
In order to understand its possible binding mode, we modeled 9
into the ATP binding pocket of EGFR (Fig. 4c). As described above,
isoquinoline-4-carbonitriles can be placed in the ATP binding

H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
433
O
O
O
O2N
a)
NH
NH
NH
+
NO2
1
2
3
O
O
O
O2N
O2N
O2N
NH
NH
b)
NH
c)
d), e)
Br
CN
3
4
5
HN
Br
HN
Br
HN
Br
O2N
H2N
R
g)
f)
N
N
N
CN
CN
CN
6
7
8-18
Scheme 1. Synthesis of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles. Reagents and conditions: (a) H2SO4, KNO3; (b) Br2, DCM; (c) Zn(CN)2, cat.
Pd2(dba)3, cat. S-Phos, DMF/H2O; (d) POCl3; (e) 3-bromoaniline, pyridine, ethanol; (f) SnCl2, EtOH; (g) acid chloride, Et3N, THF for 10, 15, 16; acid analog, EDCIÁHCl, Et3N, DMF
for 8; acid analogs, (COCl)2, cat. DMF, THF, Et3N, for 9, 11, 12, 14, 17 and 18.
nificantly increases inhibitor binding.22,28 In a publication by
Wissner et al., two 6,7-dimethoxyisoquinoline-4-carbonitrile ana-
logs were tested and were shown to be very weak inhibitors of
EGFR. Based on modeling studies, the authors proposed that the
very weak inhibition by 6,7-dimethoxyisoquinoline-4-carbonitr-
iles could be attributed to their inability to form a direct hydrogen
bond with backbone atoms in the hinge region of the kinase do-
main.29 However, this is at odds with our own modeling studies
and in case of 8, 9 and 11, we believe that additional potency
against EGFR can be gained by the incorporation of a terminal ter-
tiary amine functionality at the 7-position of the isoquinoline core
in order to form an extra charged interaction with Asp800 in EGFR.
This trend was also observed for MLCK with the exception of 14,
which showed clear preferential activity against EGFR. Interest-
ingly, MLCK has a charged Glu348 side chain at the analogous po-
sition of Asp800 of EGFR which is capable of forming the hydrogen
bonding interactions described above. Considering this similarity,
the discrepancy in potency for 14 against EGFR and MLCK could
be the result of two factors. First, the activity-based assays used
to study MLCK activity require an additional activator molecule,
calmodulin, to attain full MLCK activity, while no such activator
is required to measure full EGFR activity. Additionally, it is likely
that the flexibility of the various substituents at the 7-position of
the isoquinoline core also plays an important role in kinase speci-
ficity. In the case of 14, the substituent is much more rigid when
compared to that in 8, 9 and 11. However, the carbonyl of the lac-
tam of 14 may also act as a hydrogen bond acceptor. The lack of
inhibition observed against MLCK therefore suggests that EGFR
can better accommodate the more rigid substituent of 14.
Figure 3. Kinetic analysis of inhibition of MLCK by 11 (Lineweaver-Burk plot). The
double reciprocal plot of 1/V against 1/[ATP] shows that 11 inhibits MLCK in an ATP-
competitive manner. The reaction velocities at 375 nM substrate, three different
ATP concentrations (80, 110 and 140
10 M) were measured and plotted.
lM) with varying concentrations of 11 (0, 1, 5,
l
pocket of the kinase domain with the nitrile making a key hydro-
gen bond to the backbone of the hinge region of the kinase domain.
When modeled into EGFR, the Michael acceptor of 9 is in close
proximity to the conserved cysteine (Cys797), which would allow
for covalent bond formation. The observation that 9 is a better
EGFR inhibitor than 8 is in line with previously published results
which have shown that irreversible inhibitors are generally more
potent inhibitors of EGFR than their reversible counterpart.22,28
The structural model of 9 in complex with EGFR also highlights po-
tential hydrogen bonding interactions of protonated amine func-
tionalities in the 7-position of the inhibitor with the side chain of
Asp800 in EGFR (Glu348 in MLCK). We have recently described a
similar interaction for 4-anilino-quinazolines when bound to EGFR
or the tyrosine kinase cSrc and observed that this interaction sig-
2.8. Cellular activity of 9
In order to determine cellular activities of the most potent
inhibitor 9, we performed a cell-based screen using 81 genetically
defined NSCLC cell lines.30 The viability of these cells upon
inhibitor treatment was determined by measuring the cellular

434
H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
Figure 4. Proposed binding mode of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles to the ATP-binding site of MLCK and EGFR. (a) 4-Anilinoquinaz-
olines and quinoline-3-carbonitrile are known tyrosine kinase inhibitors. Complex crystal structures and detailed SAR studies show that the N1 of the 10-membered ring is
crucial for activity and forms a key hydrogen bond to the hinge region of the kinase domain. (b) A structural model of MLCK was generated based on the crystal structure of
MLCK family member 4 (PDB code: 2X4F). The proposed binding mode of 11 shows the nitrile of the isoquinoline core within hydrogen bonding distance to the backbone of
the hinge region (pink) of the kinase domain. The terminal tertiary amine of the piperazine ring is likely to be protonated under physiological conditions and can be
positioned within hydrogen bonding distance to the side chain of Glu348 (black dotted lines). (c) The irreversible inhibitor 9 is modeled to the ATP-binding site of EGFR based
on the crystal structure of a 4-anilinoquinazoline bound to EGFR (PDB code: 2J5F). The nitrile of the isoquinoline ring forms a hydrogen bond to the backbone of the hinge
region of EGFR and helps to position the electrophile in the 7-position within close proximity to the side chain of Cys797 to allow covalent bond formation. The protonated
tertiary amine of the inhibitor side chain forms a charged interaction with the side chain of Asp800, analogous to the type of the extra charged interaction seen in the model
for MLCK.
ATP content. The sensitivity profile of 9 shows preferred inhibition
of EGFR-mutated and EGFR-dependent cell lines such as PC9 and
H3255 (Fig. 5a and b).30 The viability of cells independent of EGFR
signaling such as the KRAS mutant cell line A549 was not inhibited
fect the reversible binding of 9 to EGFR-T790M, thereby explaining
the weak activity of 9 in H1975 cells which harbor this mutation.
Along these lines, we recently showed that both steric hindrance
of the initial reversible binding combined with increased competi-
tion with ATP act in concert and transmit drug-resistance at least
for 4-anilino-quinazolines in EGFR-T790M.25
at concentrations up to 40 lM, thus underlining EGFR as the rele-
vant cellular target of 9 (Fig. 5b). A particular challenge in drug
development for targeted cancer therapy is the emergence of resis-
tance mutations in EGFR. The T790 M mutation at the gatekeeper
position of EGFR is found in nearly 50% of NSCLC patients that re-
lapse from initial treatment with tyrosine kinase inhibitors such as
erlotinib or gefitinib.31 Although irreversible inhibitors of the 4-
anilino-quinazoline and -quinoline scaffold were shown to be ex-
tremely potent against this EGFR drug resistant mutation in bio-
chemical assays,20,32 they are only of limited potency in cellular
studies and highlight the need for the identification of novel inhib-
itors.28 To address this, we evaluated the potency of 9 in the EGFR
dependent and drug resistant (EGFR-T790M) NSCLC cell line
H1975 and found cellular activity in the high micromolar range
(Fig. 5c). This is in accord with our previous findings where we ana-
lyzed the determinants of the activity and selectivity of clinically
relevant reversible and irreversible 4-anilino-quinazoline and
-quinoline based EGFR inhibitors by chemogenomic profiling
against genetically defined NSCLC cell lines.28 Although our model-
ing studies suggest that the nitrile pushes the inhibitor scaffold
away from the gatekeeper to avoid a steric clash with larger amino
acid side chains, it is likely that M790 in EGFR-T790M still inter-
feres sterically with the binding of 11. These effects would also af-
3. Conclusion
7-Substituted-1-(3-bromophenylamino)isoquinoline-4-carbo-
nitriles represent an interesting class of novel kinase inhibitors
with a preference for MLCK and EGFR. We have synthesized 7-
substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitrile
derivatives and evaluated their activities against MLCK using
in vitro kinase assays and showed that inhibition of the phosphor-
ylation reaction depends on the nature of the substituent at the 7-
position of the isoquinoline scaffold. The presence of a terminal
tertiary amine moiety at this position was found to be required
for biological activity. The preference of the compounds for inhib-
iting kinases which possess a bulky hydrophobic amino acid resi-
due at the gatekeeper position was explained by modeling 11
into the ATP binding pocket of MLCK. Guided by biochemical pro-
filing, we identified the irreversible isoquinoline-4-carbonitrile 9
as a potent inhibitor of HER-kinases with low micromolar cellular
activity. Modeling studies suggest that the electrophile of 9
alkylates a Cys residue at the lip of these ATP pocket known to
be sensitive for covalent inactivation by irreversible 4-anilino-

H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
435
Figure 5. Growth inhibition of NSCLC cell lines by 9. (a) The cellular activity profile of 9 is depicted in a chart presentation where GI50 values are plotted against genetically
annotated NSCLC cell lines.28,30 Cells were treated with various concentrations of compound and cell viability was determined by measuring the cellular ATP content. The five
most sensitive cell lines are highlighted and EGFR/HER2 dependent or mutated cell lines are marked by an asterisk. (b) EGFR mutant NSCLC cell line PC9 and the KRAS mutant
cell line A549 (negative control since independent of EGFR) were treated with increasing concentrations of 9 and the viability of the cells was again determined by measuring
the cellular ATP content. The error bars represent the standard deviation for three replicates. (c) The drug resistant NSCLC cell line H1975 expressing the EGFR-T790M
gatekeeper mutant was treated with increasing concentrations of 9 and showed only limited inhibition. Inhibition of the activation status of mutant EGFR was determined by
immunoblotting and measuring levels of phosphorylated EGFR.
quinazolines and -quinolines. In light of these biochemical and bio-
logical observations, we feel that the discussed isoquinoline-4-car-
bonitriles may serve as valuable starting points for further
compound design to generate probe molecules which allow for
the investigation of kinase biology in chemical biology research.
KNO3 (3.4 g, 33.62 mmol) was added in portions and the reaction
mixture was stirred at 0 °C for 30 min. The mixture was then al-
lowed to warm to room temperature and was stirred overnight.
The next day, the reaction mixture was poured on ice and the re-
sulted suspension was extracted three times with chloroform.
The organic phases were combined, dried with sodium sulfate
and evaporated under vacuum. The obtained viscous liquid was
repeatedly purified by column chromatography (EtOAc/petroleum
ether, 0.25:0.75 to 1:0) to furnish two positional isomers as yellow
crystalline material, 1.06 g (20%) of 5-nitroisoquinoline-1(2H)-one,
2, and 710 mg (14%) of the required 7-nitro isomer 3. The analyti-
cal data for 3: 1H NMR (400 MHz, DMSO-d6): 11.74 (br s, 1H), 8.89
(d, J = 2.6 Hz, 1H), 8.43 (dd, J = 8.8, 2.6 Hz, 1H), 7.90 (d, J = 8.8 Hz,
1H), 7.45 (d, J = 7.1 Hz, 1H), 6.72 (d, J = 7.1 Hz, 1H); 13C NMR
(101 MHz, DMSO-d6): 161.13, 145.07, 142.81, 133.47, 128.09,
126.14, 125.66, 122.62, 104.06; HRMS (ESI-MS): Calcd for
C9H7N3O3 [M+H+]: 191.04512. Found: 191.04501. The analysis for
2: 1H NMR (500 MHz, DMSO-d6): 11.73 (br s, 1H), 8.56 (d,
J = 8.0 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H),
7.42–7.47 (m, 1H), 6.95 (d, J = 7.6 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6): 160.35, 144.51, 133.18, 133.11, 130.93, 129.34,
127.75, 125.71, 98.47; HRMS (ESI-MS): Calcd for C9H7N3O3
[M+H+]: 191.04512. Found:191.04509.
4. Experimental
4.1. Synthesis of compounds
1H and 13C NMR spectra were recorded on a Varian Mercury
400, Bruker DRX 500 or Varian Inova 600 spectrometer. The spectra
refer to the residual solvent signals: dimethylsulfoxide-d6
(2.50 ppm) for 1H and (39.52 ppm) for 13C. Chemical shifts (d) are
given in parts per million (ppm) while the coupling constants (J)
are reported in hertz (Hz). The following abbreviations are used;
s = singlet, d = doublet, dd = doublet of doublet, ddd = doublet of
doublet of doublet, t = triplet, td = triplet of doublet, dt = doublet
of triplet, q = quartet, qd = quartet of doublet, qn = quintet, br
s = broad singlet, m = multiplet. LC–MS spectra were obtained on
a LTQ Orbitrap (high resolution mass spectrometer from Thermo
Electron) coupled to an ‘Accela’ HPLC System (consisting of Accela
pump, Accela autosampler and Accela PDA detector) supplied with
a ‘Hypersil GOLD’ column (50 mm  1 mm, 1.9
lm particle size)
from Thermo Electron. Analytical TLC was carried out on Merck
4.1.2. 4-Bromo-7-nitroisoquinolin-1(2H)-one (4)
60 F245 aluminium-backed silica gel plates. Compounds were puri-
fied by column chromatography using Baker silica gel (40–70 lm
particle size). Preparative HPLC was carried out on the final com-
pounds using a Varian Prostar with UV-detector (Model 340) and
Compound 3 (1.49 g, 7.84 mmol) was dissolved in DCM (25 mL)
and bromine (0.44 mL, 8.56 mmol) was added. The reaction mix-
ture was stirred at room temperature. After 5 h, the reaction was
stopped and the solvent was evaporated under reduced pressure.
The mixture was column chromatographed (EtOAc/petroleum
ether, 1:1) to produce 1.3 g (62%) of 4 as a yellow solid. 1H NMR
(400 MHz, DMSO-d6): 12.06 (br s, 1H), 8.85 (d, J = 2.4 Hz, 1H),
8.53 (dd, J = 9.0, 2.4 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.81 (s, 1H);
13C NMR (101 MHz, DMSO-d6): 160.16, 145.65, 140.24, 134.46,
127.31, 127.17, 126.24, 122.87, 96.25; HRMS (ESI-MS): Calcd for
C9H679BrN2O3 [M+H+]: 268.95563, for C9H681BrN2O3 [M+H+]:
270.95358. Found: 268.95599, 270.95357.
a VP 25-21 Nucleodur (C18 Gravity, 5 l) column (Serial No.
2105150). Anhydrous solvents were purchased from Acros Organ-
ics and Fluka. Other chemical materials were purchased from Alfa
Aesar, Fluka, and Sigma–Aldrich and were used as received.
4.1.1. 7-Nitroisoquinoline-1(2H)-one (3)
1-Isoquinolinol (4.0 g, 6.89 mmol) was added portionwise in
ice-cooled sulfuric acid (25 mL). Once the solid was dissolved,

436
H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
4.1.3. 7-Nitro-1-oxo-1,2-dihydroisoquinoline-4-carbonitrile (5)
4-Bromo-7-nitroisoquinolin-1(2H)-one (1.2 g, 4.4 mmol),
red at 0 °C for 30 min. 7-Amino-1-(3-bromophenylamino)isoquino-
line-4-carbonitrile 7 (20 mg, 0.059 mmol) was added into the
reaction mixture and the resulting mixture was stirred at room
temperature for 48 h, subsequently added to aq NaHCO3, extracted
with EtOAc (3 Â 100 mL), concentrated under vacuum and column
chromatographed (MeOH/DCM with triethylamine, 0:1 to
0.15:0.85) to obtain a yellowish powder which was further purified
by preparative HPLC affording 20 mg (74%) of 8 as an off-white
powder. 1H NMR (400 MHz, DMSO-d6): 10.45 (s, 1H), 9.95 (s, 1H),
8.85 (s, 1H), 8.47 (s, 1H), 8.07 (s, 1H), 7.99 (dd, J = 8.8, 1.5 Hz, 1H),
7.90 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 8.0 Hz,
1H), 7.28 (d, J = 8.0 Hz, 1H), 2.44 (t, J = 7.4 Hz, 2H), 2.34 (t,
J = 7.1 Hz, 2H), 2.20 (s, 6H), 1.79 (qn, J = 7.3 Hz, 2H); 13C NMR
(101 MHz, DMSO-d6): 171.52, 154.71, 147.07, 141.50, 138.78,
130.98, 130.32, 126.46, 125.82, 124.37, 124.23, 121.15, 120.86,
118.03, 117.55, 112.87, 95.47, 58.19, 44.80 (C Â 2), 33.96, 22.56;
HRMS (ESI-MS): Calcd for C22H2379BrN5O [M+H+]: 452.10805, for
4
Pd2(dba)3 (0.34 g, 0.37 mmol), S-Phos (0.34 g, 0.83 mmol) and
Zn(CN)2 (0.63 g, 5.36 mmol) were suspended in DMF/H2O (32 mL,
99:1 v/v). The reaction mixture was degassed for 20 min, heated
at 140 °C for 20 min. The temperature of the reaction mixture
was lowered to 120 °C and stirring was continued for an additional
3 h. The progress of the reaction was monitored by TLC. Once com-
pleted, the mixture was diluted with water and extracted with
EtOAc (3 Â 400 mL). The combined EtOAc layers were dried over
sodium sulfate, filtered and concentrated under vacuum. The
resulting crude was purified by column chromatography (EtOAc/
petroleum ether, 0.3:0.7 to 0.6:0.4) to obtain 476 mg (49%) of 5
as yellow crystalline material. 1H NMR (400 MHz, DMSO-d6):
12.65 (br s, 1H), 8.89 (d, J = 2.5 Hz, 1H), 8.62 (dd, J = 8.8, 2.5 Hz,
1H), 8.46 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H); 13C NMR (101 MHz,
DMSO-d6): 160.31, 146.11, 143.57, 138.94, 127.77, 125.48,
125.09, 122.84, 115.50, 87.85; HRMS (ESI-MS): Calcd for
C
22H2381BrN5O [M+H+]: 454.10600. Found: 452.10771, 454.10543.
C
10H6N3O3 [M+H+]: 216.04037. Found: 216.04049.
4.1.7. (E)-N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-
yl)-4-(dimethylamino)but-2-enamide (9)
(E)-4-(Dimethylamino)but-2-enoic acid hydrochloride (39 mg,
0.235 mmol) was added to 1.5 mL DCM and cooled to 0 °C under
4.1.4. 1-(3-Bromophenylamino)-7-nitroisoquinoline-4-
carbonitrile (6)
The 7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carbonitrile
5
(470 mg, 2.18 mmol) was added to POCl3 (25 mL) and refluxed
for 5 h. The excess of POCl3 was removed under reduced pressure
and the resulting crude intermediate, 1-chloro-7-nitro-1,2-dihy-
droisoquinoline-4-carbonitrile, was thoroughly dried under high
vacuum. The next day, the crude intermediate along with 3-bro-
argon. Oxalyl chloride (21 lL, 0.240 mmol) was added dropwise
followed by a catalytic amount of DMF. The reaction mixture was
stirred at 30 °C for 2 h (solution A). 7-Amino-1-(3-bromophenyla-
mino)isoquinoline-4-carbonitrile 7 (20 mg, 0.059 mmol) was dis-
solved in 1-methyl-2-pyrrolidinone (0.2 mL) and added to pre-
cooled solution A. The reaction mixture was stirred for 1.5 h at
room temperature and then poured into saturated aq NaHCO3, ex-
tracted with EtOAc (3 Â 100 mL), washed with brine and concen-
trated under high vacuum. The crude product was purified by
preparative HPLC to obtain 8 mg (31%) of 9 as a yellow solid. 1H
NMR (400 MHz, DMSO-d6): 10.59 (s, 1H), 10.00 (s, 1H), 8.95 (s,
1H), 8.49 (s, 1H), 8.06 (s, 1H), 7.99 (dd, J = 8.9, 1.5 Hz, 1H), 7.93
(d, J = 8.8 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H),
7.28 (d, J = 8.1 Hz, 1H), 6.84 (dt, J = 15.4, 6.0 Hz, 1H), 6.38 (d,
J = 15.4 Hz, 1H), 3.18 (d, J = 5.6 Hz, 2H), 2.25 (s, 6H); 13C NMR
(101 MHz, DMSO-d6): 163.45, 154.73, 147.27, 141.46, 138.55,
131.23, 130.36, 126.47, 125.99, 125.88, 124.51, 124.22, 121.17,
120.84, 117.99, 117.53, 113.30, 95.49, 59.48, 44.91 (C Â 2); HRMS
moaniline (260 lL, 2.40 mmol) and pyridine (0.21 mL, 2.57 mmol)
was refluxed in ethanol for 2.5 h. The solvent was removed under
vacuum and the resulting crude was column chromatographed
using EtOAc/petroleum ether (0:1 to 1:1) to obtain 460 mg (58%)
of 6 as a yellow solid. 1H NMR (400 MHz, DMSO-d6): 10.55 (br s,
1H), 9.72 (d, J = 2.1 Hz, 1H), 8.74 (s, 1H), 8.64 (dd, J = 9.1, 2.2 Hz,
1H), 8.08–8.13(m, 2H), 7.85 (dt, J = 7.0, 2.2 Hz, 1H), 7.43–7.34 (m,
2H); 13C NMR (151 MHz, DMSO-d6): 156.11, 152.39, 146.12,
140.36, 138.64, 130.49, 127.00, 126.18, 125.76, 125.04, 121.55,
121.49, 121.18, 116.85, 116.40, 94.90; HRMS (ESI-MS): Calcd for
C
16H1079BrN4O2 [M+H+]: 368.99816, for C16H1081BrN4O2 [M+H+]:
370.99612. Found: 368.99862, 370.99617.
4.1.5. 7-Amino-1-(3-bromophenylamino)isoquinoline-4-
carbonitrile (7)
(ESI-MS): Calcd for C
22H2179BrN5O [M+H+]: 450.09240, for
C22H2181BrN5O [M+H+]: 452.08999. Found: 450.09210, 452.08999.
1-(3-Bromophenylamino)-7-nitroisoquinoline-4-carbonitrile
6
(300 mg, 0.82 mmol) and tin(II) chloride (900 mg, 4.74 mmol)
were added in ethanol (60 mL) and heated at 70 °C for 3 h. Upon
completion, the reaction mixture was concentrated to 10 mL and
added to saturated aq NaHCO3 (20 mL). The aqueous layer was ex-
tracted with EtOAc (3 Â 100 mL) and dried over sodium sulfate, fil-
tered and concentrated under vacuum to obtain a crude product
which was purified by column chromatography (EtOAc/petroleum
ether, 0.2:0.8 to 0.4:0.6) to furnish 240 mg (87%) of 7 as a yellow
solid. 1H NMR (400 MHz, DMSO-d6): 9.55 (s, 1H), 8.23 (s, 1H),
8.12 (t, J = 1.8 Hz, 1H), 7.81 (dd, J = 8.2, 0.9 Hz, 1H), 7.67 (d,
J = 8.8 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.27–7.36 (m, 2H), 7.20–
7.25 (m, 1H), 5.88 (br s, 2H); 13C NMR (101 MHz, DMSO-d6):
153.10, 149.04, 143.20, 141.93, 130.28, 126.01, 125.18, 124.71,
123.48, 123.04, 121.18, 120.14, 119.53, 117.96, 102.70, 95.92;
HRMS (ESI-MS): Calcd for C16H1279BrN4 [M+H+]: 339.02399, for
C16H1281BrN4 [M+H+]: 341.02194. Found: 339.02437, 341.02175.
4.1.8. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-
yl)propionamide (10)
Propionyl chloride (5.7
ophenylamino)isoquinoline-4-carbonitrile 7 (20 mg, 0.059 mmol)
and triethylamine (18 L, 0.130 mmol) were added to 1 mL of
lL, 0.065 mmol), 7-amino-1-(3-brom-
l
THF at room temperature. After 2 h of stirring, the reaction mixture
was poured into aq. NaHCO3, extracted with EtOAc, washed with
brine and dried over sodium sulfate. The crude compound was dis-
solved in a minimum amount of acetone and a few drops of petro-
leum ether were added. The mixture was concentrated under
reduced pressure to produce a suspension. The suspended solid
was allowed to settle down and the liquid was decanted. The solid
was further washed with petroleum ether and dried to obtain
10 mg (43%) of 10 as an off-white solid. 1H NMR (400 MHz,
DMSO-d6): 10.33 (s, 1H), 9.94 (s, 1H), 8.84 (s, 1H), 8.47 (s, 1H),
8.06 (t, J = 1.8 Hz, 1H), 7.98–7.87 (m, 2H), 7.80–7.75 (m, 1H), 7.33
(t, J = 7.8 Hz, 1H), 7.30–7.25 (m, 1H), 2.43 (q, J = 7.5 Hz, 2H), 1.15
(t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, DMSO-d6): 172.33, 154.66,
147.00, 141.45, 138.72, 130.95, 130.27, 126.39, 125.78, 124.34,
124.20, 121.10, 120.81, 117.98, 117.46, 112.86, 95.46, 29.39, 9.49;
HRMS (ESI-MS): Calcd for C19H1679BrN4O [M+H+]: 395.05020, for
4.1.6. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-yl)-4-
(dimethylamino)butanamide (8)
4-(Dimethylamino)butanoic acid hydrochloride (20 mg,
0.12 mmol) and EDCIÁHCl (22 mg, 0.12 mmol) were added to 1 mL
of dry DMF followed by triethylamine (34
l
L, 0.24 mmol) and stir-
C
19H1681BrN4O [M+H+]: 397.04815. Found: 395.05001, 397.04779.

H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
437
4.1.9. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-yl)-3-
(4-methylpiperazin-1yl)propanamide (11)
3-(4-Methylpiperazin-1-yl)propanoic acid (38 mg, 0.220 mmol)
was added in 2 mL THF and cooled to 0 °C under argon. Oxalyl
4.1.12. (S)-N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-
yl)-5-oxopyrrolidine-2-carboxamide (14)
From -pyroglutamic acid (30 mg, 0.232 mmol), oxalyl chloride
(20.70 L, 0.236 mmol), catalytic DMF in was dissolved in 1.5 mL
L
l
chloride (20
l
L, 0.228 mmol) was added dropwise followed by a
DCM and stirred for 2 h at 30 °C. 7-Amino-1-(3-bromophenylami-
no)isoquinoline-4-carbonitrile 7 (20 mg, 0.059 mmol in 0.2 mL of 1-
methyl-2-pyrrolidinone) was added and stirred for 2 h at room tem-
perature as described for 9. Preparative HPLC on the crude product
produced 7 mg (27%) of 14 as an off-white solid. 1H NMR (400 MHz,
DMSO-d6): 10.56 (s, 1H), 10.03 (s, 1H), 8.86 (s, 1H), 8.49 (s, 1H),
8.11–7.98 (m, 3H), 7.94 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H),
7.34 (t, J = 7.9 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 4.30 (dd, J = 8.3,
3.8 Hz, 1H), 2.46–2.35 (m, 1H), 2.31–2.14 (m, 2H), 2.13–2.03 (m,
1H); 13C NMR (101 MHz, DMSO-d6): 177.51, 171.85, 154.77, 147.38,
141.42, 138.26, 131.36, 130.36, 126.50, 125.95, 124.60, 124.35,
121.16, 120.97, 117.95, 117.52, 113.32, 95.45, 56.38, 29.24, 25.34;
HRMS (ESI-MS): Calcd for C21H1779BrN5O2 [M+H+]: 450.05601, for
drop of DMF. The reaction mixture was stirred at 40 °C for 3 h
(solution A). 7-Amino-1-(3-bromophenylamino)isoquinoline-4-
carbonitrile 7 (25 mg, 0.073 mmol) was dissolved in 1-methyl-2-
pyrrolidinone (0.5 mL) and added to pre-cooled solution A. The
reaction mixture was stirred overnight at room temperature and
then poured into saturated aq NaHCO3 solution, extracted with
EtOAc (3 Â 100 mL), washed with brine and concentrated under
high vacuum. The crude product was first purified by column chro-
matography (MeOH/DCM, 0:1 to 0.1:0.9) to obtain a hygroscopic
compound which was further purified by preparative HPLC to yield
14 mg (39%) of 11 as an off-white powder. 1H NMR (400 MHz,
DMSO-d6): 10.56 (s, 1H), 9.99 (s, 1H), 8.83 (s, 1H), 8.48 (s, 1H),
8.06 (t, J = 1.7 Hz, 1H), 7.99 (dd, J = 8.9, 1.4 Hz, 1H), 7.92 (d,
J = 8.8 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H),
7.30–7.26 (m, 1H), 2.68 (t, J = 6.8 Hz, 2H), 2.57 (t, J = 6.8 Hz, 2H),
2.50–2.34 (m, 8H), 2.21 (s, 3H); 13C NMR (101 MHz, DMSO-d6):
170.60, 154.72, 147.13, 141.50, 138.70, 131.05, 130.35, 126.29,
125.85, 124.50, 124.23, 121.17, 120.85, 118.04, 117.55, 112.76,
95.48, 54.48 (C Â 2), 53.57, 52.10 (C Â 2), 45.36, 34.03; HRMS
C
21H1781BrN5O2 [M+H+]: 452.05397. Found: 450.05572, 452.05360.
4.1.13. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-
yl)furan-2-carboxamide (15)
7-Amino-1-(3-bromophenylamino)isoquinoline-4-carbonitrile
7 (20 mg, 0.059 mmol), furan-2-carboxylic acid chloride (6.2
lL,
0.064 mmol) and triethylamine (18 L, 0.128 mmol) in 2 mL THF
l
(ESI-MS): Calcd for
C
24H2679BrN6O [M+H+]: 493.13460, for
were stirred at 0 °C for 30 min. The reaction mixture was concen-
trated and the crude product was purified by preparative HPLC
to obtain 8 mg (32%) of 15 as an off-white solid. 1H NMR
(400 MHz, DMSO-d6): 10.72 (br s, 1H), 10.03 (br s, 1H), 8.98 (s,
1H), 8.51 (s, 1H), 8.19 (dd, J = 8.8, 1.5 Hz, 1H), 8.13–7.98 (m, 2H),
7.94 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 3.3 Hz,
1H), 7.32 (t, J = 8.0 Hz, 1H), 7.29-7.24 (m, 1H), 6.73 (m, 1H); 13C
NMR (101 MHz, DMSO-d6): 156.43, 154.71, 147.50, 147.13,
146.27, 141.40, 137.95, 131.44, 130.34, 127.47, 125.89, 124.31,
124.19, 121.18, 120.80, 117.81, 117.53, 115.38, 114.85, 112.34,
C
24H2681BrN6O [M+H+]: 495.13255. Found: 493.13409, 495.13197.
4.1.10. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-yl)-
3-morpholinopropanamide (12)
From 3-morpholinopropanoic acid hydrochloride (46 mg,
0.235 mmol), oxalyl chloride (21
was dissolved in 1 mL DCM and heated for 2 h at 30 °C. 7-Amino-
1-(3-bromophenylamino)isoquinoline-4-carbonitrile (20 mg,
lL, 0.240 mmol), catalytic DMF
7
0.059 mmol in 0.2 mL of 1-methyl-2-pyrrolidinone) was added
and stirred at room temperature for 2 h as described in the
procedure for 9. The crude product was purified by preparative
HPLC, producing 11 mg (39%) of 12 as a white solid. 1H NMR
(400 MHz, DMSO-d6): 10.52 (br s, 1H), 9.99 (br s, 1H), 8.83 (s,
1H), 8.48 (s, 1H), 8.06 (t, J = 1.7 Hz, 1H), 7.99 (dd, J = 8.9, 1.5 Hz,
1H), 7.92 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.34 (t,
J = 8.0 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 3.58 (t, J = 4.4 Hz, 4H), 2.68
(t, J = 6.7, Hz, 2H), 2.59 (t, J = 6.7 Hz, 2H), 2.47–2.38 (m, 4H); 13C
NMR (151 MHz, DMSO-d6): 170.51, 154.73, 147.06, 141.46,
138.64, 131.03, 130.29, 126.31, 125.81, 124.42, 124.22, 121.11,
120.83, 118.00, 117.46, 112.79, 95.45, 66.14 (C Â 2), 54.05, 53.05
(C Â 2), 33.77; HRMS (ESI-MS): Calcd for C23H2379BrN5O2 [M+H+]:
95.43; HRMS (ESI-MS): Calcd for
C
21H1479BrN4O2 [M+H+]:
433.02946, for
C
21H1481BrN4O2 [M+H+]: 435.02742. Found:
433.02933, 435.02715.
4.1.14. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-
yl)furan-3-carboxamide (16)
7-Amino-1-(3-bromophenylamino)isoquinoline-4-carbonitrile
7 (20 mg, 0.059 mmol), furan-3-carboxylic acid chloride (8.4 mg,
0.064 mmol) and triethylamine (18 lL, 0.128 mmol) were added
to THF at room temperature and the reaction mixture was stirred
for 30 min. The solvent was evaporated under high vacuum and
the crude was purified by preparative HPLC and crystallization, fur-
nishing 7 mg (28%) of 16 as a brown solid. 1H NMR (400 MHz,
DMSO-d6): 10.52 (br s, 1H), 10.02 (br s, 1H), 8.97 (s, 1H), 8.54–
8.49 (m, 2H), 8.21 (dd, J = 8.8, 1.7 Hz, 1H), 8.13 (t, J = 1.9 Hz, 1H),
7.96 (d, J = 8.9 Hz, 1H), 7.88–7.82 (m, 2H), 7.34 (t, J = 8.0 Hz, 1H),
7.30–7.26 (m, 1H), 7.09 (s, 1H); 13C NMR (101 MHz, DMSO-d6):
160.63, 154.67, 147.35, 146.26, 144.48, 141.41, 138.36, 131.27,
130.34, 127.20, 125.83, 124.33, 124.11, 122.61, 121.15, 120.67,
117.85, 117.47, 114.40, 109.13, 95.41; HRMS (ESI-MS): Calcd for
480.10296, for
480.10257, 482.10048.
C
23H2381BrN5O2 [M+H+]: 482.10092. Found:
4.1.11. Ethyl 1-(3-Bromopheylamino)-4-cyanoisoquinolin-7-
ylcarbamate (13)
7-Amino-1-(3-bromophenylamino)isoquinoline-4-carbonitrile
7 (10 mg, 0.030 mmol), ethyl chloroformate (3.37
lL, 0.035 mmol)
and triethylamine (9 L, 0.065 mmol) were added to pre-cooled
l
THF. The reaction mixture was stirred at room temperature for
6 days. Upon completion, the reaction mixture was concentrated
and purified by preparative HPLC to obtain 6 mg (50%) of 13 as a
white solid. 1H NMR (400 MHz, DMSO-d6): 10.13 (s, 1H), 9.95 (br
s, 1H), 8.68 (s, 1H), 8.46 (s, 1H), 8.05 (s, 1H), 7.90–7.86 (m, 2H),
7.77 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.30–7.25 (m, 1H),
4.21 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H); 13C NMR
(101 MHz, DMSO-d6): 154.57, 153.73, 146.92, 141.50, 138.95,
138.83, 130.63, 130.34, 125.82, 124.48, 124.18, 121.16, 120.87,
118.13, 117.53, 111.73, 95.42, 60.63, 14.56; HRMS (ESI-MS): Calcd
C
21H1479BrN4O2 [M+H+]: 433.02946, for C21H1481BrN4O2 [M+H+]:
435.02742. Found: 433.02921, 435.02702.
4.1.15. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-yl)-
3-(1H-indol-3-yl)propanamide (17)
Indole-3-propionic acid (44 mg, 0.232 mmol), oxalyl chloride
(20.7 lL, 0.236 mmol), catalytic DMF in 1 mL DCM was stirred for
2 h at 30 °C before and 7-amino-1-(3-bromophenylamino)isoquin-
oline-4-carbonitrile 7 (20 mg, 0.059 mmol in 0.2 mL of 1-methyl-
2-pyrrolidinone) as added and stirred for additional 2 h at room
temperature as described in the procedure for 9. Column chroma-
tography of the crude product (EtOAc/petroleum ether, 0.1:0.9 to
for
C
19H1679BrN4O2 [M+H+]: 411.04511, for C19H1681BrN4O2
[M+H+]: 413.04307. Found: 411.04492, 413.04236.

438
H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
0.6:0.4) resulted in 8 mg (27%) of 17 as a viscous liquid. 1H NMR
(400 MHz, DMSO-d6) 10.81 (s, 1H), 10.44 (s, 1H), 9.99 (s, 1H),
8.85 (s, 1H), 8.48 (s, 1H), 8.06 (t, J = 1.8 Hz, 1H), 7.98 (dd, J = 8.9,
1.6 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.59
(d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.7 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H),
7.18 (d, J = 1.6 Hz, 1H), 7.07 (t, J = 7.1 Hz, 1H), 6.99 (t, J = 7.3 Hz,
1H), 3.09 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H); 13C NMR
(101 MHz, DMSO-d6): 171.43, 154.73, 147.12, 141.49, 138.76,
136.27, 131.04, 130.35, 127.01, 126.42, 125.86, 124.45, 124.26,
122.29, 121.16, 121.01, 120.88, 118.35, 118.25, 118.04, 117.56,
113.52, 112.83, 111.40, 95.49, 37.14, 20.74; HRMS (ESI-MS): Calcd
for C27H2179BrN5O [M+H+]: 510.09203, for C27H2181BrN5O [M+H+]:
512.09035. Found: 510.09203, 512.08975.
4.3. Modeling
4.3.1. Homology modeling of the kinase domain of human
smMLCK
In order to find a suitable structural template for smMLCK, a
BLAST search against the PDB-databank was performed. From this
search, the structure of the human myosin light chain kinase do-
main (human myosin light chain kinase family member 4, PDB
code: 2X4F) was identified as the closest homolog and was chosen
as template for preparation of the homology model. The amino
acid sequences of smMLCK and the template were aligned using
ClustalW33 and further processed by Modeller 8v2 (http://salilab.
org/modeller). Ten models were built using the model
procedure of the program. The resulting models were evaluated
for stereochemical quality with PROCHECK.34 The best solution
was taken as the final model for the human smMLCK kinase
domain.
4.1.16. N-(1-(3-Bromophenylamino)-4-cyanoisoquinolin-7-yl)-
1H-indol-2-carboxamide (18)
Indole-2-acetic acid (0.038 g, 0.235 mmol), oxalyl chloride
(20.7 lL, 0.236 mmol), catalytic DMF in 1 mL DCM were stirred
for 2 h at 30 °C. 7-Amino-1-(3-bromophenylamino)isoquinoline-
4-carbonitrile 7 (20 mg, 0.060 mmol in 0.2 mL of 1-methyl-2-pyr-
rolidinone) was added and stirred for 2 h at room temperature as
described in the procedure for 9. Column chromatography
(EtOAc/petroleum ether, 0.1:0.9 to 0.4:0.6) of the crude product
afforded 4 mg of pure (14%) of 18 as a yellow solid. 1H NMR
(400 MHz, DMSO-d6): 11.85 (s, 1H), 10.73 (s, 1H), 10.06 (s, 1H),
9.00 (s, 1H), 8.53 (s, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.12 (s, 1H), 8.00
(d, J = 8.8 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H),
7.56–7.47 (m, 2H), 7.35 (t, J = 8.0 Hz, 1H),7.29 (d, J = 8.0 Hz, 1H),
7.25 (t, J = 8.0 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H); 13C NMR (151 MHz,
DMSO-d6): 159.93, 154.69, 147.35, 141.46, 138.30, 137.02, 131.36,
130.94, 130.36, 127.49, 127.02, 125.81, 124.36, 124.04, 121.88,
121.18, 120.61, 120.02, 117.92, 117.46, 114.60, 112.47, 104.41,
95.52, 40.06, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10; HRMS
4.3.2. Modeling of 11 and 9 into the ATP binding site of MLCK
and EGFR
Docking of 11 to the smMLCK homology model was performed
manually with COOT35 using the structure of Erlotinib in complex
with EGFR (PDB code: 1M17)36 as reference. Docking of the irre-
versible inhibitor 9 to EGFR was done in the same way. In this case,
the structure of the cSrc kinase domain with the covalent inhibitor
RL3 (PDB code: 2QLQ)22 was used as a reference. Images were gen-
erated with PyMol v0.98.37
4.4. Cellular studies
4.4.1. Cell lines and reagents
81 NSCLC cells were obtained from DSMZ (www.dsmz.de),
ATCC (www.atcc.org), and other cell culture collections and were
maintained as described previously.30 Mutational analysis and
SNP-arrays were used for routine authentication of the cell lines.30
Inhibitors were dissolved in DMSO and stored at À80 °C.
(ESI-MS): Calcd for
C
25H1779BrN5O [M+H+]: 482.06110, for
C
25H1781BrN5O [M+H+]: 484.05905. Found: 482.06067, 484.05867.
4.2. Biochemical characterization of inhibitor activity
4.4.2. Viability assays
MLCK and calmodulin were purchased from Millipore GmbH,
Germany. EGFR was purchased from Invitrogen GmbH, Karlsruhe,
Germany. IC50 determinations for EGFR kinase were measured
with the HTRFÒ KinEASE™-TK assay kit and for MLCK with HTRFÒ
KinEASE™-STK from Cisbio according to the manufacturer’s
instructions. For EGFR, biotinylated poly-Glu-Tyr peptide was used
as a substrate. In the case of MLCK, the phosphorylation reaction
was performed with the peptide substrate STK1. After completion
of the kinase reaction, an anti-phosphotyrosine (for EGFR) and
anti-phospo-STKS1 (for MLCK) antibody labeled with Europium
Cryptate were added to the reaction mixture together with Strep-
tavidin labeled with the fluorophore XL665. The amount of phos-
phorylation of substrate was then determined by measuring the
FRET signal between Europium Cryptate and XL665. ATP concen-
Cells were plated into sterile 384-well plates using a Multidrop
instrument (www.thermo.com). After 24 h of incubation, com-
pounds were added in 10 threefold serial dilutions using a CyBio
Vario pintool (www.cybio.com). Cellular viability was determined
after 96 h by measuring the ATP content using the CellTiter-Glo as-
say (www.promega.com). Half-maximal inhibitory concentrations
were then determined using the R package ‘GI50’.38
4.4.3. Western blotting
Western blotting was carried out as described previously.39 The
following antibodies were used: EGFR, p-EGFR (Biosource; USA),
anti-rabbit-antibody, anti-mouse (Millipore; Germany).
Acknowledgments
trations were set at their respective Km values (9.5
lM for EGFR,
18 M for MLCK) while 375 nM STK1 substrate was used for MLCK
l
We thank Christian Hedberg and Hans-Dieter Arndt for helpful
and 50 nM of poly-Glu-Tyr substrate was used for EGFR. Kinase
and inhibitor were pre-incubated for 30 min before the reaction
was started by addition of ATP and substrate peptide. A Tecan Sa-
fire2 plate reader was used to measure the fluorescence of the sam-
ples at 620 nm (Eu-labeled antibody) and 665 nm (XL665 labeled
discussions. Merck Sharp & Dohme, Bayer-Schering Pharma,
Merck-Serono and BayerCrop Science are thanked for financial
support. The work was supported by the German Federal Ministry
for Education and Research through the German National Genome
Research Network-Plus (NGFN-Plus) (BMBF Grant 01GS08102).
Streptavidin) 60 ls after excitation at 317 nm. The ratio of both
intensities was calculated in the presence of eight different inhib-
itor concentrations (also without inhibitor) and was plotted
against inhibitor concentrations and fit to a Hill 4-parameter equa-
tion to determine IC50 values. At least two independent determina-
tions of each IC50 were made, each time in duplicate.
Supplementary data
Supplementary data (GI50 values measured for 9 against 81
NSCLC cell lines) associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2010.11.007.

H. B. Rode et al. / Bioorg. Med. Chem. 19 (2011) 429–439
439
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