Bis-aryl urea derivatives as potent and selective LIM kinase (Limk) inhibitors

Article abstract of DOI:10.1021/jm501680m

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC₅₀ < 25 nM), excellent selectivity against ROCK and JNK kinases (>400-fold), potent inhibition of cofilin phosphorylation in A7r5, PC-3, and CEM-SS T cells (IC₅₀ < 1 μM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 μM inhibited only Limk1 and STK16 with ≥80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors.

Full text of DOI:10.1021/jm501680m

Article
pubs.acs.org/jmc
Bis-aryl Urea Derivatives as Potent and Selective LIM Kinase (Limk)
Inhibitors
Yan Yin,†,# Ke Zheng,† Nibal Eid,‡,∥ Shannon Howard,‡,∥ Ji-Hak Jeong,⊥ Fei Yi,∞ Jia Guo,∞
Chul Min Park,†,○ Mathieu Bibian,† Weilin Wu,⊥ Pamela Hernandez,‡,∥ HaJeung Park,§ Yuntao Wu,∞
Jun-Li Luo,⊥ Philip V. LoGrasso,*,‡,∥ and Yangbo Feng*,†
‡
§
∥
†Medicinal Chemistry, Discovery Biology, Crystallography/Modeling Facility, Translational Research Institute, Department of
Molecular Therapeutics, and ⊥Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, No.
2A1, Jupiter, Florida 33458, United States
#School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Hai Quan Road, Shanghai, 201418, P. R.
China
∞National Center for Biodefense and Infectious Diseases, School of System Biology, George Mason University, Manassas, Virginia
20110, United States
S
* Supporting Information
ABSTRACT: The discovery/optimization of bis-aryl ureas as Limk inhibitors to
obtain high potency and selectivity and appropriate pharmacokinetic properties
through systematic SAR studies is reported. Docking studies supported the observed
SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM),
excellent selectivity against ROCK and JNK kinases (>400-fold), potent inhibition of
cofilin phosphorylation in A7r5, PC-3, and CEM-SS T cells (IC50 < 1 μM), and good
in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61
kinases, compound 18b at 1 μM inhibited only Limk1 and STK16 with ≥80%
inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/
migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective
on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class
of bis-aryl urea derived potent and selective Limk inhibitors.
expression of Limk1 retarded PC3ASL cells’ proliferation by
arresting cells at G2/M phase,26 altered expression of Limk1
changed cell morphology and organization of actin cytoskeleton
in PC3 cells,26 increased expression of Limk1 was associated
with accumulation of chromosomal abnormalities and develop-
ment of cell cycle defects in cells that naturally express lower
concentrations of Limk1,27 reduced expression of Limk1
abolished the invasive behavior of prostate cancer cells,27 and
expression of Limk1 is higher in prostate tumors with higher
Gleason scores and incidence of metastasis.27 All these
observations suggest the possibility of up-regulated Limk1 as
a cellular oncogene, and inhibition of Limk1 activity in
cancerous prostate cells and tissues could lead to reduction of
phosphorylated cofilin and decrease of the cells’ motility and
thus the invasiveness of tumor cells and their evolution to
metastasis. Therefore, small molecule inhibitors of Limk1 could
be potential therapeutic agents for prostate cancers. Recent
studies also suggest that use of Limk inhibitors may provide a
novel way to target the invasive machinery in GBM
(glioblastoma multiforme).32−34
INTRODUCTION
■
LIM kinase (Limk) is a serine-threonine protein kinase. Two
isoforms were identified as LIM kinase 1 (Limk1) and LIM
kinase 2 (Limk2).1−4 Limk1 and Limk2 are highly homologous
and share 50% overall identity. Both isoforms consist of two
amino-terminal LIM domains, adjacent PDZ and proline/
serine-rich regions, followed by a carboxyl-terminal protein
kinase domain.5 Limk1 was found to be expressed widely in
embryonic and adult tissues, with notably high expression in
the brain, kidney, lung, stomach, and testis.6 Limk2 was found
to be expressed in almost all embryonic and adult tissues
examined with the exceptions of glial cell, the testis, and kidney
glomeruli.7 Upon activation by upstream signals, Limk
phosphorylates its substrate cofilin at the Ser-3 residue, thereby
inactivating it and leading to dynamic regulation of actin
cytoskeleton.8−12 Accumulated evidence suggests that Limk
activity is associated with a variety of diseases including
Williams syndrome,13 Alzheimer’s disease (AD),14,15 psoriatic
epidermal lesions,16 primary pulmonary hypertension
(PPH),17,18 intracranial aneurysms (IA),19 ocular hyper-
tension/glaucoma,20 HIV and other viral infections,21−24 and
cancers and cancer cell migration/invasion.25−31
Recent molecular biology studies reported that Limk1 was
Received: October 30, 2014
overexpressed in cancerous prostate cells and tissues,26 reduced
© XXXX American Chemical Society
A
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HIV-1 binding and entry into host cells are strongly impaired
by the inhibition of actin polymerization.24,35 Wu et al.
demonstrated that HIV-mediated Limk activation is through
gp120-triggered transient activation of the Rac-PAK-Limk
pathway and that knockdown of Limk through siRNA
decreased filamentous actin, increased CXCR4 trafficking, and
diminished viral DNA synthesis.23 Wen et al. showed that LIM
kinases modulate retrovirus particle release and cell−cell
transmission events.24 This research suggests that HIV hijacks
Limk to control the cortical actin dynamics for the onset of viral
infection of CD4 T cells. Therefore, Limk inhibitors are
supposed to have high potentials as therapeutics in anti-HIV
infection applications.23
To the best of our knowledge, few small molecule Limk
inhibitors have been reported in the literature.28 Bristol-Myers
Squibb Pharmaceuticals (BMS) disclosed potent Limk1
inhibitors based on an aminothiazole scaffold.36,37 Tel-Aviv
University recently published an oxazole based Limk1/2
inhibitor (T56-Limki) from computer-aided drug design,
which was found to be effective against cancer metastasis for
treatment of neurofibromatosis.34 A group of scientists from
Australia reported 4-aminobenzothieno[3,2-d]pyrimidine based
Limk1 inhibitors from a high-throughput screen (HTS)
showing activity in the micromolar range.38,39 Recently, a
Japanese group also reported a Limk inhibitor (Damnacanthal
or Dam, natural product based) from HTS campaigns, and this
compound (Dam) has a Limk1 inhibition IC50 of ∼800 nM.31
Lexicon Pharmaceuticals revealed a class of Limk inhibitors
based on a piperidine urea or guanidine scaffold for the
treatment of ocular hypertension and associated glaucoma.20
More recently, the same group of Lexicon scientists reported a
novel class of type III binding Limk2 inhibitors that are based
on a sulfonamide scaffold.40
even weaker ROCK-II affinity (IC50 = 1365 nM) but improved
Limk1 biochemical potency (IC50 = 201 nM). Interestingly, the
4-ylpyrrolopyrimidine moiety in 2 and 3 is also present in
Lexicon’s piperidine urea/guanidine based Limk inhibitors and
is believed to be involved in hinge-binding interactions.20
Encouraged by the selectivity bias of compound 3 against
Limk1 and ROCK-II, we carried out further optimization for
this bis-aryl urea scaffold (starting from 3) in the hope of
discovering highly potent, selective, and proprietary Limk
inhibitors for various applications. Herein, we report the
synthesis and structure−activity relationship (SAR) studies for
this series of bis-aryl urea based Limk inhibitors.
CHEMISTRY
■
Inhibitors 3 and 7 were accessed through a short route as
shown in Scheme 1. Coupling 3-aminobenzoic acid with
a
Scheme 1. Synthesis of Inhibitors 3 and 7
Our group reported a novel pyrazole phenyl urea scaffold 1
(Figure 1) as potent and selective Rho kinase (ROCK)
a
Reagents and conditions: (a) propan-2-amine, HATU, DIEA, DMF,
rt; (b) isocyanatobenzene derivatives, DCM; (c) boronic acid pinacol
ester, Pd(PPh3)4, dioxane/H2O, 95 °C; (d) bis(pinacolato)diboron,
PdCl2dppf, dioxane, reflux; (e) Ar-Cl, Pd(PPh3)4, dioxane/H2O, 95
°C.
propan-2-amine gave carbonyl amide 4 in the presence of
HATU as coupling reagent and DIEA as base. Mixing
intermediate 4 with 1-bromo-4-isocyanatobenzene derivatives
in dichloromethane (DCM) produced bromides 5. Finally,
targeted inhibitors 3 and 7 were synthesized through a Suzuki
coupling with an appropriate arylboronic acid pinacol ester or
alternatively via a two-step palladium catalyzed borylation/
Suzuki coupling sequence with an aryl halide. Final targeted
Limk inhibitors were all purified by the high pressure reverse-
phase liquid chromatograph (HPLC) methodology to give a
purity of ≥95% based on UV absorption (254 nm).
Pyrrolopyrimidines 10 were synthesized through the reaction
of substituted anilines 8 with isocyanatobenzene derivatives in
DCM at room temperature, followed by Pd-catalyzed
borylation/Suzuki coupling reaction with 4-chloro-5-methyl-
7H-pyrrolo[2,3-d]pyrimidine (Scheme 2).
Figure 1. Transition from ROCK inhibition to Limk inhibition for the
phenyl urea based scaffold of kinase inhibitors.
inhibitors and their significant intraocular pressure (IOP)
lowering effects on rat eyes.41,42 Compound 1 had low Limk
inhibition in counterscreen studies (IC50 > 10 μM). However,
SAR investigation revealed that replacement of the hinge-
binding moiety pyrazole in 1 with a 4-ylpyrrolopyrimidine
(compound 2) significantly decreased its ROCK-II affinity
(ROCK-II IC50 = 188 nM of 2 vs 2 nM of 1). On the other
hand, compound 2 gained a modest Limk1 inhibition (Limk1
IC50 = 642 nM vs IC50 > 10 μM for 1), revealing an interesting
hinge-binder dependent kinase selectivity profile for this phenyl
urea based scaffold. Further modification of compound 2 on its
urea terminal side led to compound 3 (Figure 1) which had an
The synthesis of N-substituted (on the urea NH attached to
the central phenyl ring) compounds 14 is described in Scheme
3. To make 12b and 12c (12a is commercially available, where
a methyl group is attached to the aniline), N-(4-bromophenyl)-
oxazolidin-2-one 11 was first prepared by reacting 4-bromoani-
B
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
4. Addition of 2-chloroethyl carbonochloridate to a mixture of
anilines and pyridine in DCM gave N-phenyloxazolidin-2-one
derivative 15.44 Then refluxing 15 and KOH in EtOH
produced N-hydroxylethylaniline 16. Heating a mixture of
iodobenzene, an N′,N′-disubstituted ethanamine, Pd(dba)2,
BINAP, and Cs2CO3 in dioxane gave secondary aniline 17.45
Finally, inhibitors 18 were synthesized from 16 or 17 by
following the synthetic procedures described in Scheme 3.
Scheme 2. Synthesis of Inhibitor 10
RESULTS AND DISCUSSION
■
Compounds prepared were first screened in biochemical assays
against Limk1 (Reaction Biology Corporation, http://www.
reactionbiology.com) and ROCK-II.46,47 Selected potent Limk
inhibitors were also counterscreened against ROCK-I,
PKA,42,46,48 and JNK3,49 as well as four selected P450 isoforms
(1A1, 2C9, 2D6, and 3A4).46,48,50 Potent and selective Limk
inhibitors were then evaluated in cell-based assays for their
inhibition of cofilin phosphorylation in A7r5 cells. Because of
the potential applications of Limk inhibitors for treatment of
cancer and HIV-infection, selected lead inhibitors were also
assessed in prostate carcinoma (PC-3) cell lines stimulated by
hepatocyte growth factor (HGF)51 and in HIV related CEM-SS
T cells23 using Western blot analysis. To assess the druggability
of these bis-aryl urea based Limk inhibitors, a few potent,
selective, and membrane permeable compounds were further
evaluated in in vitro and in vivo drug metabolism and
pharmacokinetics (DMPK) studies.41,46,50,52−55
a
Reagents and conditions: (a) isocyanatobenzene derivatives, DCM,
rt; (b) (i) bis(pinacolato)diboron, PdCl2dppf, dioxane, reflux; (ii) 4-
chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine, Pd(PPh3)4, dioxane/
H2O, 95 °C.
a
Scheme 3. Synthesis of Inhibitor 14
Since the variation of hinge-binding moieties could induce
significant differences in kinase inhibition potency and
selectivity, as indicated in Figure 1, we started SAR studies by
varying the heteroaryl ring of 3 in order to discover the best
hinge binding moiety for this bis-aryl urea scaffold of Limk
inhibitors. As shown in Table 1, compounds with a simple five-
or six-membered heteroaromatic ring as the hinge-binding
moiety, such as pyrazole, pyridine, and aminopyrimidine, are all
basically ROCK inhibitors (7a−c, IC50 < 200 nM) with low
Limk1 inhibition (IC50 > 10 μM). This observation was in
accordance with our previous reports that pyrazole, pyridine,
and aminopyrimide were suitable hinge-binding moieties for
developing ROCK inhibitors.41,46,50,53,54 Application of [5,6]-
fused aromatic rings, such as pyrrolopyridine (7d) and
purinone (7e), still yielded compounds with good ROCK-II
inhibition (IC50 = 132 and 247 nM for 7d and 7e, respectively)
but low Limk1 inhibition (IC50 > 10 μM). However, the use of
a 4-ylpurine moiety reversed the kinase selectivity between
ROCK and Limk. Compound 7f exhibited a slightly higher
potency for Limk1 inhibition (IC50 = 1.5 μM) than for ROCK-
a
Reagents and conditions: (a) 2-chloroethyl carbonochloridate,
K2CO3, CH3CN, reflux; (b) pyrrolidine or piperidine, DMSO,
microwave, 110 °C, 1 h; (c) 1-isocyanato-4-methoxybenzene, DCM;
(d) (i) bis(pinacolato)diboron, PdCl2dppf, dioxane, reflux; (ii) 4-
chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine, Pd(PPh3)4, dioxane/
H2O, 95 °C.
line with 2-chloroethyl carbonochloridate in the presence of
K2CO3 in CH3CN. Intermediate 11 and a secondary amine
pyrrolidine or piperidine were then dissolved in DMSO and
heated at 110 °C for 1 h in a microwave reactor to give N-
substituted 4-bromoaniline 12b (from pyrrolidine) and 12c
(from piperidine).43 Mixing 12 and 1-isocyanato-4-methox-
ybenzene in DCM with stirring gave urea 13. Finally, bromide
13 underwent a Pd-catalyzed borylation/Suzuki coupling
reaction sequence to produce 14.
The preparation of N-substituted (on the urea NH attached
to the terminal phenyl ring) compound 18 is shown in Scheme
a
Scheme 4. Synthesis of Inhibitor 18
a
Reagents and conditions: (a) 2-chloroethyl carbonochloridate, Py, DCM, rt; (b) KOH, EtOH, reflux; (c) Pd(dba)2, BINAP, Cs2CO3, dioxane; (d)
(i) 1-bromo-4-isocyanatobenzene, DCM, rt; (ii) bis(pinacolato)diboron, PdCl2dppf, dioxane, reflux; (iii) 4-chloro-5-methyl-7H-pyrrolo[2,3-
d]pyrimidine, Pd(PPh3)4, dioxane/H2O, 95 °C.
C
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. SAR Studies of the Hinge-Binding Moiety
Table 2. Effects of Substitutions on the Central Phenyl Ring
a
IC50 values are the mean of ≥2 experiments with errors within 40% of
the mean.
ring is the best choice for preparing a highly potent and
selective Limk inhibitor.
SAR was next investigated on the terminal phenyl ring of
compound 7g, where a 5-methylpyrrolopyrimidine is used as
the hinge-binding moiety and the central phenyl group is
nonsubstituted for the convenience of organic synthesis. As
shown in Table 3, removal of the 3-carbonyl amide from the
Table 3. SAR Studies on the Terminal Aromatic Ring
a
IC50 values are the mean of ≥2 experiments with errors within 40% of
the mean.
II inhibition (IC50 = 5.6 μM). Interestingly, changing hinge-
binding group from the purine in 7f to a pyrrolopyrimidine ring
in 3 significantly enhanced the Limk1 inhibition potency and
the selectivity against ROCK. Moreover, substitution of a
methyl group on the 5-position of the pyrrolopyrimidine ring
(7g) further improved the inhibition potency over Limk1 (IC50
= 62 nM vs 201 nM for 3) and the selectivity against ROCK-II
(IC50 = 1608 nM). Interestingly, application of 6-
methylpyrrolopyrimidine and 5,6-dimethylpyrrolopyrimidine
rings (7h and 7i) gave slightly lower Limk1 inhibition (IC50
= 80 nM) but better selectivity over ROCK-II. Therefore,
further optimizations for other parts of 3 will use 5-
methylpyrrolopyrimidine as the hinge-binding moiety. How-
ever, the 5,6-dimethylpyrrolopyrimidine moiety will also be
used in preparing drug candidate Limk inhibitors, since it could
lead to higher selectivity and better DMPK properties (Tables
5, 6, and 7).
For the convenience of compound synthesis, SAR studies for
the central phenyl ring were mainly based on substitutions at its
ortho-position (to the urea moiety). As shown in Table 2, three
substitutions were evaluated. Compared to the nonsubstituted
inhibitor 7g, the trifluoromethyl substitution yielded a
compound (7j) that had a similar Limk1 inhibition potency
(IC50 = 60 nM vs 62 nM for 7g) but lower selectivity (ROCK-
II IC50 = 976 nM vs 1608 nM for 7g). However, substitution by
a small F group (with a size close to that of a proton,
a
IC50 (nM)
compd
R
Limk1
ROCK-II
10a
10b
10c
10d
10e
10f
H
142
315
283
75
2358
5421
3-F
2-OCH3
3-OCH3
4-OCH3
6652
2572
35
>10000
2290
203
4507
10g
>10000
a
IC50 values are the mean of ≥2 experiments with errors within 40% of
the mean.
terminal phenyl ring of 7g yielded a compound (10a) with a
lower Limk1 inhibitory activity (IC50 = 142 nM for 10a vs 62
nM for 7g). Interestingly, replacing the 3-carboxyl amide with a
F group (10b) significantly reduced the Limk1 inhibition (IC50
= 315 nM), which is probably due to special F-bonding
interactions56 between this F group and its surrounding protein
residues under the P-loop (see Figure 3 of docking studies).
This special F-bonding interaction might disturb the optimal
binding conformation of these urea based Limk inhibitors. The
same effects were also observed in several other Limk inhibitors
(see Table 4). It is important to point out that this special effect
of F-bonding interactions was not observed for F-substitutions
on the central phenyl ring (7k, Table 2), indicating that this
effect is dependent on the position of F-substitutions. Actually,
we have observed similar negative effects (reducing kinase
inhibition potency) of F-bonding interactions in developing our
ROCK-II inhibitors53 and JNK3 inhibitors,55 where the F-
compound 7k) led to both enhanced Limk1 inhibition (IC50
=
18 nM) and improved selectivity against ROCK-II (based on
IC50 values, the selectivity over ROCK-II is 26-fold and 43-fold
for 7g and 7k, respectively, Table 2). On the other hand,
substitution by a large dimethylaminoethoxy side chain (7l)
significantly decreased both the Limk1 inhibition (IC50 = 710
nM vs 62 nM for 7g) and the selectivity over ROCK-II (Table
2). Therefore, an ortho-F-substitution on the central phenyl
D
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
substituted aromatic moieties are all bound to an area under the
P-loop inside the ATP-binding pocket of proteins kinases.
Unlike F-substitutions, replacing the 3-carboxyl amide with a
methoxy group resulted in a Limk inhibitor (10d) that had a
similar Limk1 inhibitory potency (IC50 = 75 nM vs 62 nM for
7g) and a slightly better selectivity over ROCK-II (Table 3).
However, the 2-methoxy substitution (10c) significantly
reduced the Limk1 inhibition activity (IC50 = 283 nM). On
the other hand, the 4-methoxy substitution (10e) enhanced
both Limk1 inhibition (IC50 = 35 nM) and selectivity against
ROCK-II (IC50 > 10 μM, selectivity > 285-fold). Similar SAR
patterns were also obtained for F, Cl, and methyl substitutions
on this terminal phenyl group (see Table 4), indicating that 4-
substitution is the best fit for this scaffold in Limk inhibitions.
Heteroaryl rings other than the benzene ring were also
evaluated as the terminal aromatic moieties. For example,
application of a 2-ylthiazole (10f) resulted in lower Limk1
inhibition (compared to 10a), and the use of a 2-ylpyridine
moiety (10g) almost inactivated the compound against both
Limk1 and ROCK-II.
Table 4. SAR of the Urea NH Group Attached to the
Terminal Phenyl Moiety
Investigation of the substitution effects on the two urea NH
groups was the next focus in our SAR studies. For the urea NH
attached to the central phenyl ring, neither small nor large
substitutions including pyrrolidinoethyl and piperidinoethyl
could be tolerated. As shown in Figure 2, a simple methyl
a
IC50 values are the mean of ≥2 experiments with errors within 40% of
b
the mean. Not determined.
terminal phenyl ring (2-, 3-, 4-positions), were prepared and
evaluated (compounds 18c−n, Table 4). Generally, the 4-
substitution exhibited the highest and the 2-substitution gave
the lowest Limk1 inhibitory activity. Selectivity against ROCK-
II followed the same pattern with the 4-substitution being the
highest and 2-substitution the lowest, no matter what was the
substitution group. Among the 4-substituted Limk inhibitors,
the 4-Cl analog had the best Limk1 inhibitory potency (18h,
IC50 = 25 nM) and its 4-F counterpart (18e, IC50 = 86 nM) had
the lowest Limk1 affinity probably due to the special F-bonding
interactions,56 while the Limk1 inhibitory activity and the
selectivity against ROCK-II for the methyl and methoxy
analogs (18k and 18n) were in between.
To confirm that alkylation to this urea NH group could be
well tolerated, two more substitutions were explored. As shown
in Table 4, aminoethyl and N′,N′-dimethylaminoethyl sub-
stitutions were applied to both 4-Cl- and 4-methoxyphenylur-
eas, and the resulting 4 compounds 18o−r all exhibited high
Limk1 inhibitions. Compounds 18p−r were assessed in
counterscreen studies, and 18q and 18r were found to have
high selectivity against ROCK-II (IC50 > 10 μM, selectivity is
>210-fold and >500-fold for 18q and 18r, respectively) while
that for 18p was only ∼21-fold. The lower Limk1 inhibition
potency observed for 18a, as compared to 18q and 18r, might
be due to its bulky pyrrolidine ring which might have disturbed
the optimal binding conformation.
Figure 2. Substitutions on the urea NH attached to the central phenyl
group were not tolerated.
substitution (14a) would significantly reduce the Limk1
inhibitory potency (IC50 = 1090 nM vs 35 nM for 10e).
Larger substitutions to this NH group gave even lower Limk1
inhibitions, as evidenced by the Limk1 IC50 values of
compounds 14b and 14c (Figure 2). These results demon-
strated that alkylation to this urea NH disturbed the optimal
binding conformations, or the NH is involved in H-bonding
interactions to the protein, thus resulting in a low Limk affinity
(also see docking modes in Figure 3).
In contrast to observations in Figure 2, SAR studies
demonstrated that substitutions on the urea NH group attached
at the terminal phenyl ring were well tolerated, and excellent
Limk inhibitors could be obtained through this modification. As
shown in Table 4, a pyrrolidinoethyl substitution yielded a
compound (18a) with slightly lower Limk1 inhibition (IC50
=
Computer modeling studies of lead compounds demon-
strated that these bis-aryl urea based Limk inhibitors are all type
I ATP-competitive kinase inhibitors. The docking mode of
compound 18b in the crystal structure of Limk1 protein (PDB
code 3S95) is shown in Figure 3. Key interactions in this motif
include two H-bonds between the pyrrolopyrimidine N/NH
(N1 and N7) and hinge residue I416; one plausible H-bond
between N3 of pyrrolopyrimidine ring and the side chain OH
368 nM vs 142 nM for 10a). However, replacing the
pyrrolidine ring with a hydroxyl group (18b) led to both a
high Limk1 inhibitory activity (IC50 = 43 nM vs 142 nM for
10a) and a good selectivity over ROCK-II (IC50 = 6565 nM vs
2358 nM for 10a). Inspired by 18b, a small library of 4 × 3 =
12 analogs (of 18b), based on four functional groups (F, Cl,
methyl, and methoxy) and three substitution patterns on the
E
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
group of residue T413 (not labeled in Figure 3, since this H-
bonding requires rotation movement of the T413 side chain);
one H-bond between the urea carbonyl moiety and the side
chain amino group of K368; one H-bond between the OH
group and residue D478; cation−π interactions between the
terminal phenyl ring and the side chain amino group of K368;
hydrophobic interactions between the terminal phenyl ring and
its surrounding residues under the P-loop. It is important to
point out that hydrophobic interaction between the aromatic
rings of pyrrolopyrimidine/central phenyl moieties and their
surrounding side chains of protein residues also contributed to
the high affinity of these Limk inhibitors.
the side chain OH of T413 explained why compound 3 (Table
1) was a good Limk inhibitor while compound 7d had low
Limk1 inhibition. The significant decrease of Limk1 inhibition
in 7f as compared to 3 (Table 1) is probably due to the extra
H-bonding interactions between N5 (of 7f) and surrounding
protein residues, which might disturb the optimal binding
conformation of the ligand, thus reducing its affinity toward
Limk1.
To summarize, our SAR analysis and docking studies for this
bis-aryl urea based scaffold of Limk inhibitors showed that both
5- and 6-methyl-4-ylpyrimidines and the 5,6-dimethyl-4-
ylpyrrolopyrimidine could serve well as hinge-binding moieties
for Limk inhibition. Among them, the 5,6-dimethylpyrrolopyr-
imidine was the best, considering that it could render much
better selectivity (against ROCK) and higher microsomal
stability (see Table 6). An ortho-F-substitution on the central
phenyl ring (to the urea moiety) could improve the Limk
inhibitory potency while still keeping high microsomal stability
(Table 6). On the other hand, an F-substitution on the terminal
phenyl ring reduced inhibitory potency against Limk1 probably
because of the special F-bonding interactions (under the P-
loop). SAR analysis also indicated that a 4-Cl or a 4-methyl
substitution on the terminal phenyl group gave overall best
Limk inhibitors. Remarkably, a substitution to the urea NH
attached on the terminal phenyl side could improve both
biochemical and cell potency, enhance selectivity, and more
importantly, increase the inhibitor’s DMPK properties and
bioavailability (see Table 7).
The binding motif of compound 18b supported our observed
SAR. For example, both mono- and bis-methyl substituted (to
To take advantage of the important SAR information above,
Limk inhibitors that combine the best structural elements from
SAR analysis were thus prepared and evaluated. Table 5 lists
Table 5. Biochemical and Cell Potency for Optimized Limk
Inhibitors
Figure 3. Docking of 18b to the crystal structure of Limk1 (PDB code
3S95): (A) schematic view showing key interactions; (B) surface view
showing the binding pockets.
the 5- and/or 6-position) or even larger group substituted
(unpublished results) pyrrolopyrimidine rings were well
tolerated because of the open space around this area, and
these substitutions could enhance the inhibitor’s Limk
inhibition because of the extra interactions introduced by
substitution(s). Substitution to the urea NH attached to the
central phenyl ring led to inactive compounds because this
substitution could disturb the orientation of the urea carbonyl
group, thus weakening its H-bonding to K368. On the other
hand, substitutions to the urea NH adjacent to the terminal
phenyl ring were well tolerated and could lead to enhanced
Limk inhibition, since there is enough space around this area
and the substitution is directed toward the solvent. 4-
Substitutions on the terminal phenyl group gave the most
active Limk inhibitors (compared to the 2- and 3-substitutions)
because there is a deep hydrophobic pocket around there. The
H-bonding interaction between the pyrrolopyrimidine N3 and
a
IC50 values are the mean of ≥2 experiments with errors within 50% of
b
the mean. Not determined.
the structures and biochemical potency data for four
representative compounds. In compounds 18s−w, a 4-yl-5,6-
dimethylpyrrolopyrimidine was used as the hinge binding
moiety for optimal microsomal stability and better selectivity.
An ortho-F-substitution on the central phenyl ring and a 4-Cl
substitution on the terminal phenyl group were employed in
order to achieve higher Limk1 potency. Representative
substitutions on the terminal urea NH were applied to further
investigate the DMPK properties (see discussion for Tables 6
and 7). Indeed, these compounds all had excellent Limk1
potency (IC50 ≤ 21 nM) and good selectivity against ROCK-II
(IC50 > 20 μM for 18w and 18x).
F
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 6. Selectivity, Microsomal Stability, and Cell Potency Data for Selected Compounds
microsomal stability
a
biochemical inhibition IC50 (nM)
t1/2 (min)
a
compd
ROCK-I
JNK3
P450 % inh at 10 μM 1A2, 2C9, 2D6, 3A4
human
rat
44
cofilin phosphorylation in A7r5 cells, IC50 (nM)
7g
1283
7738
33, 49, 16, 34
77, 42, 34, 42
29, 35, 13, 32
−16, 14, 5, 13
−4, 13, 3, 25
−4, 39, 1, 37
−19, 31, 2, 42
−12, 23, 4, 27
−5, 24, −4, 5
3, 17, 3, 44
33
>1000
b
b
7i
>20000
2390
nd
47
>120
55
nd
b
7k
nd
27
4000
470
b
18b
18e
18f
18g
18h
18k
18m
18n
18s
18t
18w
18x
5536
nd
87
30
b
3920
>10000
44
52
nd
b
4390
nd
90
38
420
372
118
190
b
15050
5915
nd
22
20
>10000
>10,000
>120
56
73
7628
39
b
b
4317
nd
38
21
nd
>20000
>10000
>10000
>10000
−8, 8, −4, 2
56
40
730
210
b
nd
40, 13, 8, 16
24, 24, 52, −11
15, −23, 20, −9
23, 50, 15, 52
99
44
b
b
nd
>120
19
>120
23
nd
b
b
nd
nd
320
250
b
nd
>10000
15
13
a
b
IC50 values are the mean of ≥2 experiments with errors within 40% of the mean. Not determined.
In order to examine the selectivity profile of these bis-aryl
significant reduction of microsomal stability, as compared to its
4-substituted counterpart (18g vs 18h and 18m vs 18n), to the
nonsubstituted analog (18b), and even to its 2-substituted
analog (18f).
urea based Limk1 inhibitors, selected lead compounds were
subjected to counterscreening against ROCK-I, JNK3, and four
representative cytochrome P450 isoforms. As summarized in
Table 6, these Limk inhibitors all exhibited low inhibitory
activity over tested kinases and P450 enzymes except that 7i
showed modest inhibition against enzyme 1A2 (77%) at 10
μM. In addition to counterscreens against ROCK and JNK3,
lead inhibitor 18b was also profiled against a panel of 61 kinases
(Reaction Biology Corporation, http://www.reactionbiology.
com/webapps/site/). Results showed that 18b at 1.0 μM
inhibited only Limk1 and STK16 with ≥80% inhibition (∼3%
hit ratio) and hit also Aurora-a, Flt3, LRRK2, and RET with
>50% inhibition (∼10% hit ratio). Detailed profiling data for
18b is provided in Supporting Information. The profiling data
demonstrated that selective Limk inhibitors can be obtained
from this bis-aryl urea based scaffold.
These Limk inhibitors also had good to excellent stability in
human and rat liver microsomes (Table 6) with good to
excellent half-lives. It is important to point out that, compared
to the monomethyl substituted pyrrolopyrimidine based analog
7g, the 5,6-dimethylpyrrolopyrimidine based Limk inhibitors 7i,
18s, and 18t exhibited a higher stability in both human and rat
microsomes and a higher selectivity against ROCK (see also
Tables 2 and 5). However, when the hydroxyl or the amino
group on 18s and 18t was methylated, as shown in 18w and
18x, there was a significant drop in the microsomal stability
(Table 6). Apparently, the lower stability of 18w and 18x was
mainly due to demethylation on their side chain dimethylamino
or methoxy groups. Other important SAR information from the
selectivity profiling and stability data in Table 6 include the
following: (1) all hydroxyethyl substituted (to the urea NH)
compounds (18 series) had excellent stability in human liver
microsomes with the exception of 18g (t1/2 = 22 min only); (2)
F-substitution on the central phenyl ring did not reduce the
microsomal stability while still keeping the excellent selectivity
(7k vs 7g); (3) F-substitution on the terminal phenyl ring not
only reduced the Limk1 inhibitory potency (compared to its Cl,
methyl, and methoxy substituted counterparts) but also
deteriorated the microsomal stability (18e vs 18b, 18h, 18k,
and 18n); (4) 3-substitution on the terminal phenyl ring led to
In an effort to investigate the cell-based activity of these
Limk1 inhibitors, we monitored the phosphorylation state of
cofilin in several cell lines. Data in A7r5 cells (Table 6) showed
that inhibitors without any substitutions on their urea NH
group (7g, 7i, 7k) had a cell activity of IC50 values only in the
micromolar range. On the other hand, Limk inhibitors with
their urea NH group (the one attached to the terminal aryl
ring) substituted by a hydroxyethyl or an aminoethyl or a
methoxyethyl or a dimethylaminomethyl group (18b to 18x)
had IC50 values all in the submicromolar range, with the best
one close to 100 nM (18h). In addition, SAR patterns shown in
the cell-based potency were similar to those observed in
biochemical potency and selectivity assays. For example, 4-Cl
(18h) and 4-methyl (18k) substitutions produced compounds
with better cell activity than 4-methoxy (18n) substitutions,
and the 4-substitution exhibited the highest cell activity among
2-, 3-, and 4-substitutions (18f, 18g, and 18h) on the terminal
phenyl ring.
Since Limk inhibitors could find wide applications, such as in
glaucoma,20 cancer,27,28,57,58 infection,21−24,59 and Alzheimer’s
disease (AD),14,15 etc., cofilin phosphorylation assays were also
carried out for a few selected lead compounds in prostate
carcinoma (PC3) cell lines stimulated by hepatocyte growth
factor (HGF) and in HIV-related CEM-SS T cell lines.60 As
shown in Figure 4, inhibitor 18b exhibited significant inhibition
even at a concentration of only 50 nM in Western blot analysis
of cofilin phosphorylation in PC-3 cells (Figure 4A). Similar
cell-based potency was also observed for 18f and 18h in PC-3
cells (see Supporting Information). The phosphorylation status
of p-cofilin in CEM-SS T cells for inhibitors 18p, 18r, and 18x
is shown in Figure 4B. Again >50% inhibition was seen for all
these compounds at 1 μM, an inhibitory potency similar to that
obtained in A7r5 cells. The results from these three tested cell
lines demonstrated that the optimized Limk inhibitors had
good cell permeation. Compounds 18p and 18r had almost the
same biochemical Limk1 potency (IC50 values were both ∼20
nM, Table 4). Apparently, the better cell potency observed for
G
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. (A) Inhibition of cofilin phosphorylation in PC-3 cell lines stimulated by HGF treated with 18b. Similar cell potency was also observed for
compound 18f in PC-3 cells. (B) Western blot analysis of p-cofilin in CEM-SS T cell lines for 18p, 18r, and 18x.
a
Table 7. Data for Plasma Pharmacokinetics Studies on Rats
b
b
b
b
b
c
compd
Cl (iv) (mL min−1 kg−1
)
Vd (iv) (L/kg)
T1/2 (iv) (h)
AUC (iv) (μM·h)
Cmax (iv) (μM)
F (%) (po)
7g
9.0
7.1
0.3
0.7
0.5
0.4
0.7
0.5
0.3
0.3
6.0
4.0
17.1
0.5
1.5
1.2
1.0
1.5
1.6
2.2
1.5
2.6
1.8
2.2
4.6
5.1
6.1
3.2
3.3
3.4
4.3
5.5
8.5
5.1
0
0
7k
10a
18b
18f
18h
18k
18n
18o
18p
18r
18s
18w
18x
3.4
14.3
8.4
7.0
16
36
21
20
24
24
0
5.2
7.7
7.7
5.1
4.7
3.0
13.2
15.6
19.4
1.1
10.5
9.4
2.7
2.1
10.6
2.2
36.0
55.9
42.2
4.5
0.7
1.1
0
0.6
0.2
0
14.5
3.0
12.3
2.4
29
d
14.2
6.9
nd
d
5.1
3.5
nd
a
b
c
Data reported are the mean of three determinations, and the standard error was within 40% of the mean. iv dosing: 1 mg/kg. po dosing: 2 mg/kg.
Not determined.
d
18r than for 18p (Figure 4B) is due to the free NH2 group
present in 18p, a structural element normally associated with
deteriorated cell penetration.
compared to those for 18r (and also for 18o and 18p) further
demonstrated that an F-substitution on the central phenyl ring
and the application of a 5,6-dimethylpyrrolopyrimidine as the
hinge-binding moiety can improve the inhibitor’s PK proper-
ties.
In vivo pharmacokinetics (PK) studies were conducted for
selected compounds during the whole optimization at various
stages in order to identify structural elements that are favorable
for in vivo applications and/or to evaluate the feasibility of
optimized Limk inhibitors for animal studies. PK properties of
iv dosing (1 mg/kg) and the oral bioavailability (% F) for
selected lead Limk inhibitors are listed in Table 7. Generally, a
2-hydroxyethyl side chain reduced the clearance (Cl) compared
to the nonsubstituted (NH) urea derivatives (10a, 18b, 18h,
18k, 18n vs 7g and 7k). In contrast, a side chain containing a
terminal amino group increased the clearance significantly
(18o, 18p, 18r vs 18h, 18k, and 18n). Remarkably, the high
clearance of compounds with an amino side chain could be
reduced dramatically by introducing an F-substitution on the
central phenyl ring or by using a 5,6-dimethylpyrrolopyrimidine
(instead of the 5-methylpyrrolopyrimidine) as the hinge-
binding moiety or a combination of both (18w vs 18r). All
Limk1 inhibitors listed in Table 7 had reasonable volume of
distribution (Vd) values except a few which possessed an amino
side chain and in which a 5-methylpyrrolopyrimidine was used
as the hinge binding moiety (18o, 18p, and 18r). The much
lower Cl and Vd values and higher AUC value for 18w as
The PK data in Table 7 showed that substitution to the urea
NH group could generally increase the half-lives of these urea
based Limk inhibitors (the 10 and 18 series vs the 7 series).
The AUC and Cmax properties for these compounds were also
excellent except for the three inhibitors (18o, 18p, and 18r)
which contained an amino side chain and no F-substitutions on
their central phenyl ring and in which a 5-methylpyrrolo-
pyrimidine was used as the hinge-binding moiety. It is
important to point out that, even with an amino side chain,
inhibitor 18w still exhibited good AUC and Cmax values,
probably because of the presence of both an F-substitution on
the central phenyl ring and a 5,6-dimethylpyrrolopyrimidine
moiety in its structure. Data in Table 7 also indicated that while
the nonsubstituted urea compounds (7g and 7k) had no oral
bioavailability (% F) at all, all inhibitors containing a
hydroxyethyl side chain could exhibit reasonable oral
bioavailability. However, those inhibitors containing an amino
side chain (18o, 18p, and 18r) had no oral bioavailability
either, probably because of the high clearance (Cl) exhibited by
these compounds.
H
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Since Limk1 expression is highly expressed in cancerous
prostate cells and predominantly found in metastatic prostate
tumor tissues and is required for cancer cell migration and
invasion,61,62 Limk1 is considered as a biomarker for prostate
cancer progression.63 Limk1 is involved in Rac-induced actin
cytoskeleton reorganization through inactivating phosphoryla-
tion of cofilin and also mediated with focal adhesion
complexes.8,64 Reorganization of cytoskeleton is an essential
feature of motility, detachment, and invasion of cancer cells.
Moreover, Limk1 expression is correlated with the aggressive-
ness of cancer cells, and Limk1 expression in metastatic PC-3
cells is higher than less-aggressive LNCaP and M21 cells.26 To
confirm the role of Limk inhibitors on the invasion and
migration of prostate cancers, we examined the effect of
optimized Limk inhibitors in PC-3 cells using an in vitro
invasion assay or in vitro migration assay. Thus, Transwell
chambers were coated with GFR Matrigel, and PC-3 cells were
seeded in the inset of the chamber as described in the
Experimental Section. After incubating for 48 h, the invasive
PC-3 cells were counted and analyzed by hematoxylin staining
under microscope. As shown in Figure 5 for two representative
indicating migrated cells, was analyzed by ImageJ software
(version 1.48). As shown in Figure 6, the migrated PC-3 cells
were decreased significantly even at a concentration as low as
0.1 μM, and the migration was inhibited 74% by 1 μM inhibitor
18b (16.5% (NS) by 0.1 μM, 74.0% (p < 0.01) by 1 μM, and
77.5% (p < 0.01) by 10 μM compared to the control). Similar
inhibition potency was also obtained for inhibitor 18f (13.0%
(NS) by 0.1 μM, 76.1% (p < 0.01) by 1 μM, and 81.0% (p <
0.01) by 10 μM, compared to the control). These results
indicated that 18b or 18f had inhibitory effects on invasion and
migration of metastatic PC-3 cells. Considering that both
inhibitors had low inhibition against ROCK-I and ROCK-II
(Tables 4 and 6), the inhibition of which could also lead to
suppression of cell migration/invasion,65,66 results in Figures 5
and 6 also demonstrated that 18b and 18f must have played a
role in Limk inhibitions in vitro.
To demonstrate the potential application of these Limk
inhibitors for the treatment of glaucoma, the intraocular
pressure (IOP) lowering effect of compound 18w was
monitored after applying it topically on rat eyes (brown
Norway rats, n = 6/group, housed under constant low-light
conditions),67 following a protocol described previously by our
groups.41,53 Thus, compound 18w was applied to the right eyes
of an elevated IOP rat model (initial IOP was ∼28 mmHg)
using a dose of 50 μg (20 μL drop of a 0.25% solution). As
shown in Figure 7, significant decreases in IOP were detected at
Figure 5. Effect of Limk inhibitors on invasion of PC-3 cell.
Comparison of cell invasion (left panel) and phase contrast images
(right panel) by treatment of 18b (1 μM) or 18f (1 μM) for 48 h in
PC-3 cells. The results are shown as mean SD of one representative
experiment (from three independent experiments) performed in
triplicate. Statically significant differences are indicated: (∗) p < 0.05.
Figure 7. IOP lowering effect of 18w on rat eyes. Topical dosing at 50
μg. Data were averaged from six determinations (based on six rats).
inhibitors 18b or 18f, the invasion of PC-3 cells was
significantly inhibited by the treatment of 1 μM Limk inhibitors
(76% for 18b and 83% for 18f, compared to the control).
To verify the role of Limk inhibitors on migration of PC-3
cells, a wound was created by scratching in a cell monolayer as
described in the Experimental Section. After incubating for 24 h
with treatment of inhibitor 18b or 18f, the closed wound area,
4 h, slightly weakened at 7 h, and IOP returning to baseline at
24 h as compared to the vehicle. It must be pointed out that the
IOP drop could not be due to ROCK inhibition, since 18w had
a high selectivity against ROCK (Table 5).
Figure 6. Effect of Limk1 inhibitors on migration of PC-3 cells. Comparison of the average (%) of wound closure (left panel) and phase contrast
images (right panel) by treatment with indicated concentration of 18b (A) or 18f (B) for 24 h in PC-3 cells. The results are shown as the mean
SD of one representative experiment (from three independent experiments) performed in triplicate. Statically significant differences are indicated:
(NS) no significance; (∗) p < 0.01. Scale bar: 20 μm.
I
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mixture was stirred at room temperature for 2 h. Then the solvent was
removed in vacuo to give the crude bromide 5 for next step without
further purification.
The mixture of substituted anilines 8 (0.2 mmol) and
isocyanatobenzene derivatives (0.2 mmol) in DCM (1 mL) was
stirred at room temperature for 2 h. Then the solvent was removed in
vacuo to give the crude bromides 9 for next step without further
purification.
CONCLUSION
■
Through the application of a 4-ylpyrrolopyrimidine as the
hinge-binding moiety to replace the pyrazole group in ROCK
inhibitor 1, we identified compounds with high Limk1
inhibition potency. Systematic SAR studies around this bis-
aryl urea scaffold (3) have led to a series of potent and selective
Limk inhibitors. Docking studies demonstrated that these bis-
aryl urea Limk inhibitors exhibited a typical type I kinase
binding motif. The optimized Limk inhibitors had high
biochemical potency and high selectivity over ROCK-I,
ROCK-II, and JNK3. Inhibitor 18b (also coded as SR-7826)
was found to hit only Limk1 and STK16 with ≥80% inhibition
at 1 μM against a panel of 61 kinases. The lead Limk inhibitors
also had good cell-based potency in cofilin phosphorylation
assays and in cell-based migration/invasion assays. In addition,
they had fair to excellent in vitro and in vivo DMPK properties,
such as a clean inhibition profile against select CYP-450
isoforms, a high stability in human and rat liver microsomes,
and favorable PK properties in iv dosing (high AUC/Cmax, low
Cl, and long half-lives) and fair to good oral bioavailability
(18b, 18k, 18n, and 18s) in rats. For example, compounds
18s−x (18x also coded as SR-11157) all had excellent potency
against Limk1 (IC50 ≤ 21 nM), good cell-based activity against
cofilin phosphorylation in A7r5 cells (IC50 ≤ 320 nM), and
high selectivity over ROCK and JNK. The optimized inhibitors,
such as 18b and 18f, showed excellent activities in migration/
invasion cell-based assays. In addition, significant IOP drop on
rat eyes (>20%) was achieved for inhibitor 18w (also coded as
SR-11124) after topical administration (at a dose of 50 μg).
Applications of optimized Limk inhibitors on other indications
are under investigation and will be reported in due course.
2-Chloroethyl carbonochloridate (10 mmol) was added to a mixture
of 4-bromoaniline (10 mmol) and K2CO3 (30 mmol) in CH3CN (100
mL), and the reaction was stirred for 24 h. Then solvent was removed
in vacuo and the remaining residue redissolved in water and ethyl
acetate. The organic layers were combined, dried over anhydrous
Na2SO4, concentrated in vacuo, and purified through silica gel to give
crude N-(4-bromophenyl)oxazolidin-2-one 11. Then 11 (0.2 mmol)
and secondary amine (0.6 mmol) including pyrrolidine and piperidine
were dissolved in DMSO (1 mL) and heated at 110 °C in microwave.
After the complete conversion of 11, the mixture was diluted with
water and extracted with ethyl acetate. The organic layers were
combined, dried over anhydrous Na2SO4, and concentrated under
reduced pressure to give the intermediates 12a−c. The mixture of
12a−c (0.2 mmol) and 1-isocyanato-4-methoxybenzene (0.2 mmol) in
DCM (1 mL) was stirred at room temperature for 2 h. The solvent
was then removed in vacuo to give the crude bromide 13a−c for next
step without further purification.
Finally, the boronic acid pinacol ester (0.3 mmol) and the crude
bromide 5 (0.2 mmol) were dissolved in degassed 5:1 dioxane/H2O.
Pd(PPh3)4 (0.02 mmol) and 2 M solution of K2CO3 (0.6 mmol) were
added sequentially under argon, and the mixture was heated at 95 °C
for 2 h. After cooling to room temperature, the mixture was diluted
with water and extracted with ethyl acetate (3 × 5 mL). The organic
layers were combined, dried over anhydrous Na2SO4 and concentrated
in vacuo. The residue was then purified by preparative HPLC to give
the targeted products 7a and 7b as white solid.
In an alternative route, bis(pinacolato)diboron (0.24 mmol), crude
5, 9, and 13 (0.2 mmol), and PdCl2(dppf) (0.02 mmol) were dissolved
in degassed dioxane (5 mL). After refluxing for 2 h, the mixture was
diluted with water and extracted with ethyl acetate (3 × 5 mL). The
organic layers were combined, dried over anhydrous Na2SO4, and
concentrated in vacuo to give crude boronic acid pinacol ester.
Followed the synthesis procedure of 7a, 7c−k, 10a−f, 14a−c were
synthesized form crude boronic acid pinacol ester (0.2 mmol) and Ar-
Cl (0.2 mmol).
EXPERIMENTAL SECTION
■
Commercially available reagents and anhydrous solvents were used
without further purification unless otherwise specified. Thin layer
chromatography (TLC) analyses were performed with precolated silica
gel 60 F254. The mass spectra were recorded by LC/MS with
Finnigan LCQ Advantage MAX spectrometer of Thermo Electron.
Flash chromatography was performed on prepacked columns of silica
gel (230−400 mesh, 40−63 μm) by CombiFlash with EtOAc/hexane
or MeOH/DCM as eluent. The preparative HPLC was performed on
SunFire C18 OBD 10 μm (30 mm × 250 mm) with CH3CN + 50%
MeOH/H2O + 0.1% TFA as eluent to purify the targeted compounds.
Analytic HPLC was performed on Agilent Technologies 1200 series
with CH3CN (solvent B)/H2O + 0.9% CH3CN + 0.1% TFA (solvent
A) as eluent, and the targeted products were detected by UV in the
detection range of 215−310 nm. All compounds were determined to
be >95% pure by this method. NMR spectra were recorded with a
Bruker 400 MHz spectrometer at ambient temperature with the
residual solvent peaks as internal standards. The line positions of
multiplets were given in ppm (δ), and the coupling constants (J) were
given in hertz. The high-resolution mass spectrmetry (HRMS,
electrospray ionization) experiments were performed with Thermo
Finnigan orbitrap mass analyzer. Data were acquired in the positive ion
mode at resolving power of 100 000 at m/z 400. Calibration was
performed with an external calibration mixture immediately prior to
analysis.
2-Chloroethyl carbonochloridate (1 mmol) was added to a mixture
of substituted anilines (1 mmol) and pyridine (3 mmol) in DCM (10
mL), and the mixture was stirred for 24 h. Then solvent was removed
in vacuo and the remaining residue redissolved in water and ethyl
acetate. The organic layers were combined, dried over anhydrous
Na2SO4, and concentrated in vacuo to give crude 15. KOH (10 mmol)
was added to the mixture of crude 15 (1 mmol) in EtOH (10 mL).
Then the mixture was refluxed until the complete conversion of 15.
The solvent was removed in vacuo, and the remaining residue was
redissolved in water and ethyl acetate. The organic layers were
combined, dried over anhydrous Na2SO4, concentrated in vacuo, and
purified by silica gel to give intermediates 16. The mixture of
iodobenzene (0.2 mmol), 2-(pyrrolidin-1-yl)ethanamine (0.6 mmol),
Pd(dba)2 (0.01 mmol), BINAP (0.01 mmol), and Cs2CO3 (0.6 mmol)
in dioxane (1 mL) was refluxing for 24 h. After cooling to room
temperature, water and ethyl acetate were added. Then the organic
layers were combined, dried over anhydrous Na2SO4, concentrated in
vacuo, and purified by silica gel to give intermediates 17. Then 18a and
18b−n were synthesized from 17 and 16, respectively, following the
synthetic procedure of 10a−f from 8.
General Synthetic Procedures. The mixture of 3-aminobenzoic
acid (10 mmol), propan-2-amine (10 mmol), HATU (10 mmol), and
DIEA (30 mmol) in DMF (10 mL) was stirred at room temperature
until the complete conversion of the starting material. Then saturated
NaHCO3 was added to quench the reaction and extracted with ethyl
acetate (3 × 15 mL). The organic layers were combined, dried over
anhydrous Na2SO4, and concentrated in vacuo to give crude aniline
carboxamide 4. Aniline 4 (0.2 mmol) was then added to the solution
of isocyanatobenzene derivatives (0.2 mmol) in DCM (1 mL). The
3-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)ureido)-N-
1
isopropylbenzamide (3). 45% yield in 4 steps. H NMR (DMSO-
d6, 400 MHz) δ 12.28 (s, br, 1H), 9.06 (s, 1H), 8.96 (s, 1H), 8.81 (s,
1H), 8.18−8.16 (m, 3H), 7.87 (s, 1H), 7.71−7.64 (m, 4H), 7.46−7.44
(m, 1H), 7.38−7.36 (m, 1H), 6.95 (s, 1H), 4.14−4.07 (m, 1H), 1.18
(d, J = 5.2 Hz, 6H); 13C NMR (DMSO-d6, 100 MHz) δ 165.50,
152.70, 152.38, 152.19, 147.81, 143.00, 139.47, 135.77, 130.01, 129.56,
J
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
128.51, 127.01, 120.81, 120.71, 118.10, 117.70, 113.78, 101.66, 40.95,
22.27. LC/MS (M + H+): 415.11. HRMS (ESI-Orbitrap) calcd for
C23H23N6O2: 415.1882 [M + H+]. Found: 415.1872.
8.73 (m, 1H), 8.19−8.17 (m, 1H), 7.87 (s, 1H), 7.68−7.61 (m, 5H),
7.46−7.44 (m, 1H), 7.38−7.34 (m, 1H), 4.11 (q, J = 6.8 Hz, 1H), 1.97
(s, 3H), 1.17 (d, J = 6.8 Hz, 6H). LC/MS (M + H+): 443.16.
3-(3-(2-(2-(Dimethylamino)ethoxy)-4-(5-methyl-7H-pyrrolo-
[2,3-d]pyrimidin-4-yl)phenyl)ureido)-N-isopropylbenzamide
3-(3-(4-(1H-Pyrazol-4-yl)phenyl)ureido)-N-isopropylbenza-
1
mide (7a). 68% yield in 3 steps. H NMR (DMSO-d6, 400 MHz) δ
1
(7l). 46% yield in 4 steps. H NMR (DMSO-d6, 400 MHz) δ 12.3 (s,
12.86 (s, br, 1H), 8.82 (s, 1H), 8.69 (s, 1H), 8.18−8.16 (m, 1H),
7.96−7.93 (m, 1H), 7.84−7.79 (m, 1H), 7.78−7.74 (m, 1H), 7.72−
7.68 (m, 1H), 7.65−7.60 (m, 1H), 7.53−7.51 (m, 2H), 7.45−7.41 (m,
3H), 7.36−7.32 (m, 1H), 4.08 (q, J = 6.4 Hz, 1H), 1.17 (d, J = 6.4 Hz,
6H). HRMS (ESI-Orbitrap) calcd for C20H22N5O2: 364.1773 [M +
H+]. Found: 364.1792.
N-Isopropyl-3-(3-(4-(pyridin-4-yl)phenyl)ureido)benzamide
(7b). 65% yield in 3 steps. 1H NMR (DMSO-d6, 400 MHz) δ 9.12 (s,
1H), 9.04 (s, 1H), 8.78 (d, J = 5.6 Hz, 2H), 8.19−8.17 (m, 1H), 8.12
(d, J = 5.6 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.89−7.88 (m, 1H), 7.70
(d, J = 8.8 Hz, 2H), 7.64−7.61 (m, 1H), 7.46−7.44 (m, 1H), 7.38−
7.34 (m, 1H), 4.08 (q, J = 6.4 Hz, 1H), 1.17 (d, J = 6.4 Hz, 6H); 13C
NMR (DMSO-d6, 100 MHz) δ 165.46, 152.57, 152.35, 144.23, 142.99,
139.46, 135.77, 128.52, 128.49, 127.52, 121.84, 120.82, 120.68, 118.42,
117.74, 40.94, 22.28. LC/MS (M + H+): 375.14.
br, 1H), 9.73 (s, 1H), 9.60 (s, 1H), 8.84 (s, 1H), 8.45 (s, 1H), 8.34−
8.32 (m, 1H), 8.20−8.18 (m, 1H), 7.90 (s, 1H), 7.71−7.70 (m, 1H),
7.51 (s, 1H), 7.48−7.47 (m, 1H), 7.40−7.33 (m, 2H), 4.51 (t, J = 4.6
Hz, 2H), 4.10 (q, J = 6.4 Hz, 1H), 3.63 (t, J = 4.6 Hz, 2H), 2.94 (s,
6H), 2.12 (s, 3H), 1.17 (d, J = 6.4 Hz, 6H); 13C NMR (DMSO-d6, 100
MHz) δ 165.44, 158.91, 158.58, 154.45, 152.33, 152.23, 146.68,
145.76, 139.52, 135.78, 131.26, 128.52, 127.95, 123.80, 120.68, 118.34,
117.72, 114.64, 113.18, 111.10, 62.96, 55.44, 42.65, 40.94, 22.26,
12.78. LC/MS (M + H+): 516.13.
N-Isopropyl-3-(3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
yl)-2-(trifluoromethyl)phenyl)ureido)benzamide (7j). 47% yield
1
in 4 steps. H NMR (DMSO-d6, 400 MHz) δ 12.19 (s, br, 1H), 9.71
(s, 1H), 8.83 (s, 1H), 8.33−8.26 (m, 2H), 8.21−8.19 (m, 1H), 8.01−
8.00 (m, 2H), 7.85 (s, 1H), 7.70−7.68 (m, 1H), 7.48−7.46 (m, 2H),
7.41−7.37 (m, 1H), 4.10 (q, J = 6.8 Hz, 1H), 2.09 (s, 3H), 1.16 (d, J =
6.8 Hz, 6H); 13C NMR (DMSO-d6, 100 MHz) δ 165.37, 155.31,
152.57, 152.17, 149.46, 139.24, 137.50, 135.83, 134.08, 131.87, 128.70,
127.18, 126.48, 125.15, 124.24, 122.44, 120.96, 120.58, 117.53, 114.95,
108.93, 40.93, 22.27, 12.81. HRMS (ESI-Orbitrap) calcd for
C25H24F3N6O2: 497.1913 [M + H+]. Found: 497.1902.
3-(3-(4-(2-Aminopyrimidin-4-yl)phenyl)ureido)-N-isopropyl-
1
benzamide (7c). 52% yield in 4 steps. H NMR (DMSO-d6, 400
MHz) δ 9.43 (s, br, 2H), 8.92−8.80 (m, 1H), 8.32−8.17 (m, 1H),
8.24−8.17 (m, 1H), 8.12−8.10 (m, 2H), 7.90 (s, 1H), 7.66−7.61 (m,
3H), 7.45−7.34 (m, 3H), 7.28 (s, 1H), 4.09 (q, J = 6.8 Hz, 1H), 1.16
(d, J = 6.8 Hz, 6H); 13C NMR (DMSO-d6, 100 MHz) δ 166.79,
165.51, 159.06, 152.36, 152.00, 143.87, 139.51, 135.78, 128.67, 128.46,
128.19, 120.74, 120.66, 117.72, 117.64, 105.05, 40.93, 22.27. HRMS
(ESI-Orbitrap) calcd for C21H23N5O2: 391.1882 [M + H+]. Found:
391.1889.
N-Isopropyl-4-[3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl]-
ureido]benzamide (7d). 40% yield in 4 steps. 1H NMR (DMSO-d6,
400 MHz) δ 12.26 (s, br, 1H), 9.08 (s, 1H), 8.97 (s, 1H), 8.80 (s, 1H),
8.25−8.14 (m, 3H), 8.12 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.76 (d, J =
8.8 Hz, 2H), 7.46−6.36 (m, 2H), 6.96−6.95 (m, 2H), 4.08 (m, 1H),
1.17 (d, J = 6.4 Hz, 6H). LC/MS (M + H+): 414.15.
4-[3-[4-(7-Ethyl-8-oxo-8,9-dihydro-7H-purin-6-yl)phenyl]-
ureido]-N-isopropylbenzamide (7e). 45% yield in 4 steps. 1H
NMR (DMSO-d6, 400 MHz) δ 12.08 (s, br, 1H), 9.42 (s, 1H), 8.84−
8.82 (m, 1H), 8.76 (s, 1H), 8.35−8.14 (m, 3H), 8.12 (d, J = 8.8 Hz,
2H), 7.96 (d, J = 8.8 Hz, 2H), 7.69−7.59 (m, 1H), 4.08 (m, 1H), 3.35
(q, J = 3.2 Hz, 2H), 1.17 (d, J = 6.4 Hz, 6H), 1.07 (t, J = 3.2 Hz, 3H).
LC/MS (M + H+): 460.17.
3-(3-(2-Fluoro-4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
phenyl)ureido)-N-isopropylbenzamide (7k). 42% yield in 4 steps.
1H NMR (DMSO-d6, 400 MHz) δ 12.20 (s, br, 1H), 9.46 (s, 1H),
8.89 (s, 1H), 8.81 (s, 1H), 8.52−8.49 (m, 1H), 8.25−8.19 (m, 1H),
7.88−7.84 (m, 1H), 7.68−7.64 (m, 2H), 7.52−7.48 (m, 3H), 7.42−
7.39 (m, 1H), 4.10 (q, J = 6.8 Hz, 1H), 2.10 (s, 3H), 1.17 (d, J = 6.8
Hz, 6H); 13C NMR (DMSO-d6, 100 MHz) δ 165.44, 154.88, 152.40,
152.10, 149.96, 148.37, 139.27, 135.83, 129.21, 128.61, 126.88, 126.43,
120.84, 120.56, 119.50, 117.45, 116.19, 115.99, 114.82, 109.82, 40.95,
22.26, 12.75. HRMS (ESI-Orbitrap) calcd for C24H24FN6O2: 447.1945
[M + H+]. Found: 447.1934.
1-(4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-3-
1
phenylurea (10a). 62% yield in 3 steps. H NMR (DMSO-d6, 400
MHz) δ 12.56 (s, br, 1H), 9.18 (s, 1H), 8.94−8.90 (m, 2H), 7.78−
7.67 (m, 4H), 7.62−7.58 (m, 1H), 7.50−7.48 (m, 2H), 7.32−7.28 (m,
2H), 7.01−6.98 (m, 1H), 2.10 (s, 3H); HRMS calcd for C20H18N5O:
344.1511 [M + H+]. Found: 344.1526.
N-Isopropyl-4-[3-[4-(9H-purin-6-yl)phenyl]ureido]-
1-(3-Fluorophenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
1
1
benzamide (7f). 29% yield in 4 steps. H NMR (DMSO-d6, 400
pyrimidin-4-yl)phenyl)urea (10b). 51% yield in 3 steps. H NMR
MHz) δ 11.98 (s, br, 1H), 9.48 (s, 1H), 8.81−8.79 (m, 1H), 8.76 (s,
1H), 8.35−8.14 (m, 3H), 8.12 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz,
2H), 7.69−7.59 (m, 2H), 6.95 (s, 1H), 4.08 (m, 1H), 1.17 (d, J = 6.4
Hz, 6H). HRMS (ESI-Orbitrap) calcd for C22H22N7O2: 416.1835 [M
+ H+]. Found: 416.1849.
(DMSO-d6, 400 MHz) δ 12.55 (s, br, 1H), 9.17−9.12 (m, 2H), 8.88
(s, 1H), 7.69−7.68 (m, 4H), 7.60−7.50 (m, 2H), 7.36−7.31 (m, 1H),
7.18−7.16 (m, 1H), 6.84−6.79 (m, 1H), 2.10 (s, 3H). LC/MS (M +
H+): 362.11.
1-(2-Methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
1
pyrimidin-4-yl)phenyl)urea (10c). 60% yield in 3 steps. H NMR
N-Isopropyl-3-(3-(4-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
1
yl)phenyl)ureido)benzamide (7g). 40% yield in 4 steps. H NMR
(DMSO-d6, 400 MHz) δ 12.49 (s, 1H), 9.66 (s, 1H), 8.88 (s, 1H),
8.35 (s, 1H), 8.16−8.14 (m, 1H), 7.69−7.68 (m, 4H), 7.56 (s, 1H),
7.06−7.03 (m, 1H), 7.00−6.98 (m, 1H), 6.96−6.89 (m, 1H), 3.90 (s,
3H), 2.10 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 154.87, 152.24,
152.18, 147.80, 146.90, 142.24, 130.81, 128.35, 127.60, 126.70, 122.12,
120.52, 118.45, 117.12, 114.61, 110.93, 110.77, 55.75, 12.66. LC/MS
(M + H+): 374.09.
(DMSO-d6, 400 MHz) δ 12.63 (s, 1H), 9.21 (s, 1H), 9.10 (s, 1H),
8.92 (s, 1H), 8.19−8.18 (m, 1H), 7.89 (s, 1H), 7.74−7.68 (m, 4H),
7.65−7.61 (m, 2H), 7.46−7.44 (m, 1H), 7.39−7.35 (m, 1H), 4.10 (q, J
= 6.4 Hz, 1H), 2.10 (s, 3H), 1.17 (d, J = 6.4 Hz, 6H); 13C NMR
(DMSO-d6, 100 MHz) δ165.52, 159.07, 158.74, 153.86, 152.50,
152.09, 145.81, 142.76, 139.59, 135.78, 130.90, 128.46, 125.16, 120.78,
117.71, 117.44, 114.54, 111.64, 40.94, 22.27, 12.51. HRMS (ESI-
Orbitrap) calcd for C24H25N6O2: 429.2039 [M + H+]. Found:
429.2029.
1-(3-Methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
1
pyrimidin-4-yl)phenyl)urea (10d). 57% yield in 3 steps. H NMR
(DMSO-d6, 400 MHz) δ 12.61 (s, br, 1H), 9.14 (s, 1H), 8.93−8.91
(m, 2H), 7.72−7.67 (m, 4H), 7.60 (s, 1H), 7.23−7.18 (m, 2H), 6.98−
6.96 (m, 1H), 6.59−6.57 (m, 1H), 3.74 (s, 3H), 2.10 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ 159.64, 153.71, 152.42, 152.07, 145.64,
142.89, 140.85, 130.91, 129.49, 128.52, 124.83, 117.37, 114.51, 111.75,
110.63, 107.32, 104.13, 54.88, 12.50. LC/MS (M + H+): 374.09.
1-(4-Methoxyphenyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
N-Isopropyl-3-(3-(4-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
1
yl)phenyl)ureido)benzamide (7h). 35% yield in 4 steps. H NMR
(DMSO-d6, 400 MHz) δ 12.22 (s, br, 1H), 9.07 (s, 1H), 8.96 (s, 1H),
8.74 (s, 1H), 8.20−8.18 (m, 1H), 8.14−8.12 (m, 2H), 7.88 (s, 1H),
7.70−7.68 (m, 2H), 7.66−7.61 (m, 1H), 7.46−7.44 (m, 1H), 7.38−
7.34 (m, 1H), 6.69 (s, 1H), 4.10 (q, J = 6.8 Hz, 1H), 1.17 (d, J = 6.8
Hz, 6H). LC/MS (M + H+): 429.17.
1
pyrimidin-4-yl)phenyl)urea (10e). 45% yield in 3 steps. H NMR
(DMSO-d6, 400 MHz) δ 11.89 (s, br, 1H), 8.86 (s, 1H), 8.71 (s, 1H),
8.60 (s, 1H), 7.61−7.60 (m, 4H), 7.38 (d, J = 8.8 Hz, 2H), 7.35 (s,
1H), 6.88 (d, J = 8.8 Hz, 2H), 3.73 (s, 3H), 2.10 (s, 3H). LC/MS (M
3-(3-(4-(5,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
phenyl)ureido)-N-isopropylbenzamide (7i). 32% yield in 4 steps.
1H NMR (DMSO-d6, 400 MHz) δ 9.04 (s, 1H), 8.99 (s, 1H), 8.74−
K
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+ H+): 374.14. HRMS (ESI-Orbitrap) calcd for C21H20N5O2:
374.1617 [M + H+]. Found: 374.1608.
144.40, 142.65, 130.67, 130.58, 130.36, 128.04, 123.44, 118.83, 114.75,
114.52, 112.97, 111.26, 58.81, 52.25, 12.58. HRMS (ESI-Orbitrap)
calcd for C22H21FN5O2: 406.1679 [M + H+]. Found: 406.1686.
1-(4-Fluorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18e). 41% yield in 5
1-[4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl]-3-
1
thiazol-2-ylurea (10f). 40% yield in 3 steps. H NMR (DMSO-d6,
400 MHz) δ 11.95 (s, br, 1H), 8.76 (s, 1H), 8.71 (s, 1H), 8.60 (s, 1H),
7.71−7.68 (m, 4H), 7.53−7.48 (m, 1H), 6.99−6.97 (m, 1H), 6.58−
6.56 (m, 1H), 2.10 (s, 3H). LC/MS (M + H+): 351.14.
1-[4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl]-3-
pyridin-2-ylurea (10g). 48% yield in 3 steps. LC/MS (M + H+):
345.12.
1
steps. H NMR (DMSO-d6, 400 MHz) δ 12.51 (s, br, 1H), 8.86 (s,
1H), 8.37 (s, 1H), 7.66−7.62 (m, 4H), 7.60 (s, 1H), 7.44−7.41 (m,
2H), 7.29−7.25 (m, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.55 (t, J = 6.0 Hz,
2H), 2.06 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 161.76, 159.34,
154.39, 152.13, 146.35, 142.72, 138.65, 130.30, 130.14, 128.01, 118.83,
116.20, 115.98, 114.55, 111.24, 58.70, 52.33, 12.57. LC/MS (M + H+):
406.06. HRMS (ESI-Orbitrap) calcd for C22H21FN5O2: 406.1679 [M
+ H+]. Found: 406.1670.
3-(4-Methoxyphenyl)-1-methyl-1-(4-(5-methyl-7H-pyrrolo-
1
[2,3-d]pyrimidin-4-yl)phenyl)urea (14a). 55% yield in 3 steps. H
NMR (DMSO-d6, 400 MHz) δ 12.40 (s, br, 1H), 8.87 (s, 1H), 8.40 (s,
1H), 7.73 (d, J = 8.8 Hz, 2H), 7.55−7.54 (m, 1H), 7.52 (d, J = 8.8 Hz,
2H), 7.36 (d, J = 6.8 Hz, 2H), 6.85 (d, J = 6.8 Hz, 2H), 3.38 (s, 3H),
3.17 (s, 3H), 2.11 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 159.21,
154.89, 152.28, 147.29, 146.42, 134.02, 132.78, 130.47, 127.57, 127.29,
124.54, 122.05, 114.80, 113.53, 110.66, 55.12, 37.10, 12.67. LC/MS
(M + H+): 388.18. HRMS (ESI-Orbitrap) calcd for C22H22N5O2:
388.1773 [M + H+]. Found: 388.1764.
1-(2-Chlorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18f). 40% yield in 5
1
steps. H NMR (DMSO-d6, 400 MHz) δ 12.70 (s, br, 1H), 8.92 (s,
1H), 8.72 (s, 1H), 7.69−7.63 (m, 5H), 7.53−7.52 (m, 1H), 7.47−7.43
(m, 1H), 7.36−7.33 (m, 1H), 3.78 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 6.0
Hz, 2H), 2.07 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 158.42,
158.07, 154.22, 152.08, 145.63, 144.25, 142.95, 133.22, 130.69, 130.46,
128.58, 127.53, 126.23, 126.19, 118.86, 114.51, 111.70, 58.82, 52.31,
12.49. HRMS (ESI-Orbitrap) calcd for C22H21ClN5O2: 422.1384 [M +
H+]. Found: 422.1369.
1-(3-Chlorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18g). 45% yield in 5
steps. 1H NMR (DMSO-d6, 400 MHz) δ 8.85 (s, 1H), 7.64−7.58 (m,
5H), 7.57−7.56 (m, 1H), 7.52 (s, 1H), 7.47−7.45 (m, 1H), 7.44−7.39
(m, 2H), 3.58 (t, J = 6.0 Hz, 2H), 3.42 (t, J = 6.0 Hz, 2H), 2.06 (s,
3H); 13C NMR (DMSO-d6, 100 MHz) δ 158.28, 157.95, 154.22,
152.12, 146.21, 142.75, 139.25, 132.59, 131.76, 130.35, 130.24, 129.21,
128.36, 128.15, 118.99, 114.53, 111.33, 58.79, 51.67, 12.58. LC/MS
(M + H+): 422.06.
3-(4-Methoxyphenyl)-1-(4-(5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)phenyl)-1-(2-(pyrrolidin-1-yl)ethyl)urea (14b).
1
18% yield in 5 steps. H NMR (DMSO-d6, 400 MHz) δ 9.59 (s, br,
1H), 8.79 (s, 1H), 8.12 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.57 (d, J =
8.4 Hz, 2H), 7.43 (s, 1H), 7.32 (d, J = 6.8 Hz, 2H), 6.82 (d, J = 6.8 Hz,
2H), 4.08−4.04 (m, 2H), 3.70 (s, 3H), 3.65−3.64 (m, 2H), 3.37−3.32
(m, 2H), 3.12−3.06 (m, 2H), 2.14 (s, 3H), 2.04−2.02 (m, 2H), 1.90−
1.86 (m, 2H). HRMS (ESI-Orbitrap) calcd for C27H31N6O2: 471.2508
[M + H+]. Found: 471.2516.
3-(4-Methoxyphenyl)-1-(4-(5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)phenyl)-1-(2-(piperidin-1-yl)ethyl)urea (14c).
1
19% yield in 5 steps. H NMR (DMSO-d6, 400 MHz) δ 12.02 (s,
br, 1H), 8.77 (s, 1H), 8.15 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.56 (d, J
= 8.4 Hz, 2H), 7.41 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8
Hz, 2H), 4.09−4.06 (m, 2H), 3.70 (s, 3H), 3.56−3.54 (m, 2H), 3.28−
3.24 (m, 2H), 2.97−2.91 (m, 2H), 2.14 (s, 3H), 1.85−1.81 (m, 2H),
1.68−1.62 (m, 4H). LC/MS (M + H+): 485.15.
1-(4-Chlorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18h). 48% yield in 5
1
steps. H NMR (DMSO-d6, 400 MHz) δ 12.49 (s, br, 1H), 8.87 (s,
1H), 8.56 (s, 1H), 7.66−7.60 (m, 4H), 7.55 (s, 1H), 7.50−7.47 (m,
2H), 7.43−7.40 (m, 2H), 3.75 (t, J = 6.0 Hz, 2H), 3.56 (t, J = 6.0 Hz,
2H), 2.06 (s, 3H). HRMS (ESI-Orbitrap) calcd for C22H21ClN5O2:
422.1384 [M + H+]. Found: 422.1375.
1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)phenyl)-1-o-tolylurea (18i). 45% yield in 5 steps.
1H NMR (DMSO-d6, 400 MHz) δ 12.56 (s, br, 1H), 8.88 (s, 1H),
8.18 (s, 1H), 7.65−7.58 (m, 5H), 7.36−7.29 (m, 4H), 3.74 (t, J = 6.0
Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.22 (s, 3H), 2.06 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ 158.40, 158.06, 154.37, 153.84, 152.08,
145.75, 143.02, 136.21, 131.13, 130.43, 129.30, 128.47, 127.64, 127.09,
118.82, 114.50, 111.62, 58.82, 51.71, 17.31, 12.51. LC/MS (M + H+):
402.09.
1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)phenyl)-1-m-tolylurea (18j). 43% yield in 5 steps.
1H NMR (DMSO-d6, 400 MHz) δ 12.70 (s, br, 1H), 8.92 (s, 1H),
3-(4-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-1-
phenyl-1-(2-(pyrrolidin-1-yl)ethyl)urea (18a). 46% yield in 4
1
steps. H NMR (DMSO-d6, 400 MHz) δ 12.04 (s, br, 1H), 9.57 (s,
1H), 8.74 (s, 1H), 8.20 (s, 1H), 7.63−7.55 (m, 4H), 7.53−7.51 (m,
2H), 7.48−7.46 (m, 1H), 7.42−7.39 (m, 2H), 4.04−4.00 (m, 2H),
3.68−3.65 (m, 2H), 3.31−3.29 (m, 2H), 3.08−3.07 (m, 2H), 2.04−
2.02 (m, 5H), 1.90−1.87 (m, 2H). LC/MS (M + H+): 441.00.
1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)phenyl)-1-phenylurea (18b). From commecially
available 2-phenylaminoethanol, 18b was synthesized in 62% yield
1
through 3 steps. H NMR (DMSO-d6, 400 MHz) δ 8.83−8.81 (m,
1H), 8.40 (s, 1H), 7.64−7.58 (m, 4H), 7.47−7.43 (m, 3H), 7.39−7.37
(m, 2H), 7.32−7.29 (m, 1H), 3.76 (t, J = 6.4 Hz, 2H), 3.56 (t, J = 6.4
Hz, 2H), 2.06 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 154.43,
152.14, 146.62, 142.62, 130.30, 129.37, 129.09, 127.78, 127.66, 127.59,
126.52, 119.18, 118.67, 114.56, 111.08, 58.79, 52.24, 12.61. HRMS
(ESI-Orbitrap) calcd for C22H22N5O2: 388.1773 [M + H+]. Found:
388.1764.
8.40 (s, 1H), 7.68−7.61 (m, 5H), 7.35−7.31 (m, 1H), 7.21 (s, 1H),
7.16−7.12 (m, 2H), 3.74 (t, J = 6.4 Hz, 2H), 3.53 (t, J = 6.4 Hz, 2H),
2.35 (s, 3H), 2.07 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 158.11,
154.38, 153.97, 152.09, 145.87, 142.96, 142.34, 138.77, 130.42, 129.16,
128.37, 128.20, 127.32, 124.73, 118.69, 114.50, 111.55, 58.75, 52.20,
20.95, 12.53. LC/MS (M + H+): 402.06.
1-(2-Fluorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18c). 49% yield in 5
1
steps. H NMR (DMSO-d6, 400 MHz) δ 12.59 (s, br, 1H), 8.89 (s,
1-(2-Hydroxyethyl)-3-(4-(5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)phenyl)-1-p-tolylurea (18k). 39% yield in 5 steps.
1H NMR (DMSO-d6, 400 MHz) δ 12.60 (s, br, 1H), 8.89 (s, 1H),
8.28 (s, 1H), 7.66−7.62 (m, 4H), 7.60−7.58 (m, 1H), 7.26−7.25 (m,
4H), 3.72 (t, J = 6.0 Hz, 2H), 3.54 (t, J = 6.0 Hz, 2H), 2.34 (s, 3H),
2.07 (s, 3H). LC/MS (M + H+): 402.09. HRMS (ESI-Orbitrap) calcd
for C23H24N5O2: 402.1930 [M + H+]. Found: 402.1920.
1H), 8.75 (s, 1H), 7.66−7.54 (m, 4H), 7.52−7.50 (m, 1H), 7.42−7.40
(m, 1H), 7.39−7.37 (m, 1H), 7.34−7.26 (m, 2H), 3.72 (t, J = 6.4 Hz,
2H), 3.57 (t, J = 6.4 Hz, 2H), 2.07 (s, 3H); 13C NMR (DMSO-d6, 100
MHz) δ 159.17, 158.37, 156.71, 154.39, 153.62, 152.07, 145.55,
143.00, 130.74, 130.49, 129.81, 128.64, 125.11, 119.20, 116.55, 114.51,
111.76, 58.92, 51.98, 12.47. LC/MS (M + H+): 406.07.
1-(3-Fluorophenyl)-1-(2-hydroxyethyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18d). 45% yield in 5
1-(2-Hydroxyethyl)-1-(2-methoxyphenyl)-3-(4-(5-methyl-
1
7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18l). 47% yield in
steps. H NMR (DMSO-d6, 400 MHz) δ 12.54 (s, br, 1H), 8.88 (s,
1
5 steps. H NMR (DMSO-d6, 400 MHz) δ 8.83 (s, 1H), 7.59−7.58
1H), 8.66 (s, 1H), 7.68−7.62 (m, 4H), 7.57 (s, 1H), 7.48−7.43 (m,
1H), 7.32−7.29 (m, 1H), 7.23−7.21 (m, 1H), 7.15−7.10 (m, 1H),
3.79 (t, J = 6.0 Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ 163.53, 161.11, 154.21, 152.14, 146.34,
(m, 5H), 7.49−7.48 (m, 1H), 7.38−7.32 (m, 2H), 7.16−7.14 (m, 1H),
7.04−7.00 (m, 1H), 3.80 (s, 3H), 3.62−3.51 (m, 4H), 2.06 (s, 3H);
13C NMR (DMSO-d6, 100 MHz) δ 158.30, 155.25, 154.72, 154.23,
L
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
152.10, 146.10, 143.02, 130.49, 130.32, 130.01, 128.97, 128.16, 120.86,
118.67, 114.51, 112.74, 111.39, 58.74, 55.67, 51.22, 12.54. LC/MS (M
+ H+): 418.07.
(ESI-Orbitrap) calcd for C25H26ClFN6O: 481.1919 [M + H+]. Found:
481.1909.
1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)-2-fluorophenyl)-1-(2-methoxyethyl)urea (18x).
1-(2-Hydroxyethyl)-1-(3-methoxyphenyl)-3-(4-(5-methyl-
1
50% yield in 4 steps. H NMR (400 MHz, DMSO) δ 12.63 (s, 1H),
7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18m). 46% yield
1
8.83 (s, 1H), 8.54 (s, 1H), 7.86 (t, J = 8.2 Hz, 1H), 7.68−7.29 (m,
6H), 3.94−3.84 (m, 4H), 3.66 (s, 3H), 2.39 (s, 3H), 1.93 (s, 3H). LC/
MS (ESI-Orbitrap). Found: 468.14.
in 5 steps. H NMR (DMSO-d6, 400 MHz) δ 12.50 (s, br, 1H), 8.86
(s, 1H), 8.39 (s, 1H), 7.66−7.60 (m, 4H), 7.55 (s, 1H), 7.36−7.32 (m,
1H), 6.98−6.97 (m, 1H), 6.93−6.88 (m, 2H), 3.78 (s, 3H), 3.75 (t, J =
6.0 Hz, 2H), 3.56 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz) δ 159.95, 158.32, 158.10, 154.26, 152.11,
146.22, 143.55, 142.78, 130.36, 130.05, 128.11, 119.77, 118.71, 114.53,
113.57, 112.22, 111.33, 58.75, 55.18, 52.19, 12.57. LC/MS (M + H+):
418.05.
Docking of Limk Inhibitors into a Crystal Structure of Limk1.
Inhibitor 18b was prepared for glide docking using LigPrep
(Schrodinger, LLC, NY). The chain A of PDB code 3S95 was
prepared using protein preparation wizard in Maestro, version 9.8
(Schrodinger, LLC, NY) by removing water molecules and bound
ligand and adding hydrogen atoms. The docking grid was generated
around the original ligand with a box size of 18 × 18 × 18 Å3. Docking
was conducted without any constraint. The top scored docking pose
was merged to the protein for energy minimization using Prime
(Schrodinger, LLC, NY).
Limk1 Biochemical Assays and Kinase Profiling. Biochemical
assays for all Limk inhibitors and kinase profiling were carried out in
Reaction Biology Corporation and followed the protocols described
on its Web site. Compounds were tested in 10-dose IC50 mode with 3-
fold series dilution starting at 10 μM for IC50 measurements.
Compounds were tested at 1 μM with duplicate experiments in
profiling assays. Control compound staurosporine was tested in 10-
dose IC50 mode with 3-fold serial dilution starting at 10 μM. Reactions
were carried out at 10 μM ATP, 1 μM substrate cofilin, and 50 nM
Limk1 (final concentrations).
In-Cell Western Assay in A7r5 Cells. A7r5 cells (15 000 cells/
well) were plated in a clear-bottomed Packard View black 96-well plate
in 100 μL of 10% FBS DMEM:F12 medium and were allowed to
attach overnight. The next day, the cells were serum starved in 1% FBS
DMEM medium for 2 h and then treated with the compounds for 1 h.
Cells were then fixed in 4% paraformaldehyde in PBS for 20 min at
room temperature (rt) with no shaking. They were then washed once
with 0.1 M glycine to neutralize paraformaldehyde for 5 min. Cells
were permeabilized with 0.2% Triton X-100 in PBS for 20 min at rt on
an orbital shaker after which they were washed once with PBS for 5
min. They were then incubated with Licor blocking buffer in PBS (1:1
dilution in PBS) for 1−1.5 h rocking at rt. Cells were incubated with
primary antibody p-cofilin Ab (Cell Signaling, no. 3311) 1:100 dilution
in Licor blocking buffer overnight at 4 °C. Next day, they were washed
twice with PBS−0.1% Tween 20 (PBST) washing solution for 5 min
each at room temperature on the orbital shaker, followed by one wash
with Licor blocking buffer containing 0.05% Tween 20 for 5 min on
the shaker at rt. The cells were then incubated with secondary
antibody goat anti-rabbit IR800 (1:500 dilution) for 1 h at rt in the
dark (covered the plate with foil) in Licor blocking buffer-containing
Tween-20. Following this, cells were washed twice with PBST for 5
min each at room temperature and then once with Licor blocking
buffer-containing 0.05% Tween-20. The wells were then incubated
with ToPro 3 stain (nucleic acid staining), diluted 1:4000 in Licor
blocking buffer or Licor blocking buffer with 0.05% Tween-20 for 30
min at room temperature in the dark. Finally the plates were washed
twice with PBS and analyzed using the Odyssey LICOR infrared
scanner.
1-(2-Hydroxyethyl)-1-(4-methoxyphenyl)-3-(4-(5-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18n). 48% yield in
1
5 steps. H NMR (DMSO-d6, 400 MHz) δ 12.56 (s, br, 1H), 8.88 (s,
1H), 8.13 (s, 1H), 7.66−7.59 (m, 5H), 7.30 (d, J = 6.8 Hz, 2H), 7.01
(d, J = 6.8 Hz, 2H), 3.80 (s, 3H), 3.69 (t, J = 6.4 Hz, 2H), 3.53 (t, J =
6.4 Hz, 2H), 2.06 (s, 3H). HRMS (ESI-Orbitrap) calcd for
C23H24N5O3: 418.1879 [M + H+]. Found: 418.1686.
1-(2-Aminoethyl)-1-(4-methoxyphenyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18o). 52% yield in 4
steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.64 (s, 1H), 8.86 (s, 1H),
8.35 (s, 1H), 7.82 (s, 2H), 7.59 (dt, J = 13.0, 6.5 Hz, 5H), 7.53−7.43
(m, 4H), 3.85 (t, J = 6.2 Hz, 2H), 2.93−2.82 (m, 2H), 2.00 (dd, J =
8.4, 2.5 Hz, 3H). LC/MS (M + H+): 415.11.
1-(2-Aminoethyl)-1-(4-chlorophenyl)-3-(4-(5-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18p). 45% yield in 4
steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.60 (s, 1H), 8.89 (s, 1H),
8.06 (s, 1H), 7.87 (s, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.8
Hz, 2H), 7.58 (s, 1H), 7.45−7.39 (m, 2H), 7.10−7.04 (m, 2H), 3.87
(t, J = 6.2 Hz, 2H), 3.81 (d, J = 9.0 Hz, 3H), 2.99−2.88 (m, 2H), 2.06
(d, J = 0.9 Hz, 3H). HRMS (ESI-Orbitrap) calcd for C22H22ClN5O:
421.1544 [M + H+]. Found: 421.1563.
1-(2-(Dimethylamino)ethyl)-1-(4-methoxyphenyl)-3-(4-(5-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18q).
1
50% yield in 4 steps. H NMR (DMSO-d6, 400 MHz) δ 12.55 (s,
1H), 9.42 (s, 1H), 8.88 (s, 1H), 8.10 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H),
7.62 (d, J = 8.8 Hz, 2H), 7.57 (s, 1H), 7.45−7.38 (m, 2H), 7.11−7.03
(m, 2H), 3.98 (t, J = 6.2 Hz, 2H), 3.82 (s, 3H), 3.20 (d, J = 5.2 Hz,
2H), 2.88 (t, J = 8.5 Hz, 6H), 2.05 (d, J = 0.9 Hz, 3H). LC/MS (ESI-
Orbitrap). Found: 445.21.
1-(4-Chlorophenyl)-1-(2-(dimethylamino)ethyl)-3-(4-(5-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)urea (18r). 65%
yield in 4 steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.70 (s, 1H), 9.52
(s, 1H), 8.93 (s, 1H), 8.48 (s, 0H), 7.67 (dd, J = 23.4, 8.8 Hz, 4H),
7.62−7.49 (m, 5H), 4.04 (t, J = 6.2 Hz, 2H), 3.21 (d, J = 4.7 Hz, 2H),
2.89 (d, J = 3.5 Hz, 6H), 2.06 (d, J = 0.7 Hz, 3H). LC/MS (ESI-
Orbitrap). Found: 449.21.
1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)-2-fluorophenyl)-1-(2-hydroxyethyl)urea (18s).
1
52% yield in 4 steps. H NMR (DMSO-d6, 400 MHz) δ 12.67 (s,
1H), 8.84 (s, 1H), 8.54 (s, 1H), 7.93 (t, J = 8.3 Hz, 1H), 7.58 (dd, J =
11.4, 1.9 Hz, 2H), 7.54−7.40 (m, 4H), 3.80−3.76 (m, 4H), 2.40 (d, J
= 10.5 Hz, 3H), 1.93 (s, 3H). HRMS (ESI-Orbitrap) calcd for
C23H21ClFN5O2: 454.1446 [M + H+]. Found: 454.1434.
Cofilin Phosphorylation Cell Assay in PC-3 Cell Lines. PC3
cells were cultured at a density of 0.5 × 106 cells/mL in 60 mm culture
dishes in 10% FBS RPMI 1640 media. Then the cells were treated with
DMSO and the indicated concentration of Limk inhibitors. After
incubating for 24 h, the cells were rinsed with ice-cold PBS twice and
collected by spinning down at 4 °C in 10 000 rpm for 5 min. Cellular
lysates were prepared by suspending cells in SDS sample buffer, 120
mmol/L Tris, 4% SDS, 20% glycerol, 0.1 mg/mL bromophenol blue,
and 100 mmol/L DTT (pH 6.8). After brief sonication, the lysates
were heated at 95 °C for 5 min. The cell lysates were separated by 12%
SDS−PAGE and transferred to Immobilon-P membranes (Millipore
Corp.). Immunostaining was done using antibodies specific for
phospho-cofilin (Cell Signaling, no. 3313) and β-actin (GeneScript,
no. A00702) antibodies and the corresponding second antibodies for
1-(2-Aminoethyl)-1-(4-chlorophenyl)-3-[4-(5,6-dimethyl-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)-2-fluorophenyl]urea (18t). 52%
1
yield in 4 steps. H NMR (DMSO-d6, 400 MHz) δ 12.30 (s, 1H),
8.76 (s, 1H), 7.85 (d, J = 9.0 Hz, 3H), 7.80 (d, J = 8.6 Hz, 2H), 7.63−
7.56 (m, 4H), 7.51 (dd, J = 11.4, 1.8 Hz, 1H), 7.45 (dd, J = 8.3, 1.7 Hz,
1H), 3.91 (t, J = 6.3 Hz, 2H), 3.10 (qd, J = 7.3, 4.8 Hz, 3H), 2.95 (dd,
J = 11.9, 6.0 Hz, 2H), 2.37 (s, 3H), 1.92 (d, J = 4.5 Hz, 3H), 1.18 (t, J
= 7.3 Hz, 4H). LC/MS (M + H+): 453.14.
1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)-2-fluorophenyl)-1-(2-(dimethylamino)ethyl)-
urea (18w). 54% yield in 4 steps. 1H NMR (DMSO-d6, 400 MHz) δ
12.38 (s, 1H), 9.33 (s, 1H), 8.76 (s, 1H), 7.86 (s, 1H), 7.78 (t, J = 8.2
Hz, 1H), 7.66−7.57 (m, 2H), 7.57−7.42 (m, 3H), 4.02 (t, J = 6.3 Hz,
2H), 3.21 (s, 2H), 2.86 (s, 6H), 2.36 (s, 3H), 1.91 (s, 3H). HRMS
M
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
whole immunoglobulins from mouse or rabbit (Amersham Bio-
sciences). Immunoreactive proteins were detected by chemolumines-
cence using the Pierce ECL Western blotting substrate (Thermo
Scientific). We quantified the actual levels of proteins by using the
Multigauge, version 3.0, software (Fujifilm). The gels were stained
with Coomassie Brilliant Blue R-250 (0.25%) for 1 h and then
destained (all solutions from Bio-Rad) to check the loading amount of
protein samples on the gels.
Cofilin Phosphorylation Cell Assay in CEM-SS T Cell Lines.
CEM-SS T cells (1.0 × 106) were treated with a Limk inhibitor at 10
and 1 μM, separately, at 37 °C for 4 h. Cells were lysed in NuPAGE
LDS sample buffer (Invitrogen) followed by sonication. Samples were
heated at 90 °C for 10 min, separated by SDS−PAGE, and then
transferred onto nitrocellulose membranes (Invitrogen). The mem-
branes were washed in TBST for 3 min and then blocked for 30 min at
room temperature with starting block blocking buffer (Pierce). The
blots were incubated with a rabbit anti-phospho cofilin (ser3) antibody
(1:500 dilution) (Cell Signaling) diluted in 2.5% milk-TBST and
rocked overnight at 4 °C. The blots were washed three times for 15
min, then incubated with goat anti-rabbit 800cw labeled antibodies
(Li-cor Biosciences) (1:5000 diluted in blocking buffer) for 1 h at 4
°C. The blots were washed three times for 15 min and scanned with
Odyssey infrared imager (LI-COR Biosciences). The same blots were
also stripped and reprobed with antibodies against GAPDH (Abcam)
as a loading control.
In Vitro Invasion Assay in PC-3 Cells. Transwell chambers
coated with GFR Matrigel (BD Biosciences) were used for
measurement of cell invasion. The Matrigel was solidified at 37 °C
in a humidified incubator the day before the assay. PC3 cells (1 × 103
cells per well) were grown in serum-free RPMI 1640 media in the
upper side of the inset. The lower well was filled with RPMI 1640
supplemented with 10% FBS with a Limk inhibitor (1 μM). PC3 cells
were incubated at 37 °C in a humidified atmosphere containing 5%
CO2 for 48 h. Then Transwell membrane was rinsed three times with
PBS, and the cells were fixed in 2.5% EM grade glutaraldehyde for 15
min at room temperature (rt) with no shaking. After removal of
glutaraldehyde by aspirating, the cells were permeabilized with 0.5%
Triton X-100 in PBS for 3 min at rt, and they were rinsed three times
with PBS. Then the cells were stained by Gill’s hematoxylin no.1 for 15
min at rt, and they were washed by distilled water three times. To
remove any residual stain, the cells were washed by acid alcohol for 3
min and then rinsed by distilled water twice. The cells were exposed
with 0.04% NH4OH until a blue color is observed on the membrane
and then rinsed by distilled water twice. The membranes were dried
overnight, and the stained cells were visualized under a Leica
DMI3000B microscope.
In Vitro Migration Assay in PC-3 Cells. PC3 cells were cultured
to confluence at >90% in 6-well culture dishes in 10% FBS RPMI 1640
media the day before the assay. Lines were drawn with a marker on the
bottom of the dishes. Using a sterile 1 mL pipet tip, the dishes were
scratched, giving three separate wounds through the cells moving
perpendicular to the line drawn in the step above. The cells were
rinsed with PBS twice very gently and the RPMI 1640 media were
added. Pictures were taken of the dishes using phase contrast under
Leica DMI3000B microscope. Then the cells were treated with the
indicated concentration of a Limk Inhibitor and incubated for 24 h.
Pictures were taken of the dishes to detect closed wound area as
describe above, and the closed wound area was analyzed by ImageJ
software (version 1.48).
ASSOCIATED CONTENT
* Supporting Information
Cofilin phosphorylation in PC-3 cells for compounds 18f and
18h, DMPK assay procedures, and detailed kinase profiling data
for 18b against a panel of 61 kinases. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
*P.V.L.: e-mail, lograsso@scripps.edu; phone, 561-228-2230.
*Y.F.: e-mail, yfeng@scripps.edu; phone, 561-228-2201.
■
Present Address
○C.M.P: Korea Research Institute of Chemical Technology
(KRICT), 141 Gajeongro, Yuseong, 305-600, Korea.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by NIH Grants R21-EY021799
(Y.F. and P.V.L.) and 1RO1CA140956 (J.-L.L), by the Science
and Technology Commission of Shanghai Municipality (P. R.
China) Grant 12ZR1431100 (Y.Y), and by the Korea Research
Institute of Chemical Technology Scholarship SK-1303
(C.M.P).
REFERENCES
■
(1) Bernard, O.; Ganiatsas, S.; Kannourakis, G.; Dringen, R. Kiz-1, a
protein with LIM zinc finger and kinase domains, is expressed mainly
in neurons. Cell Growth Differ. 1994, 5, 1159−1171.
(2) Stanyon, C. A.; Bernard, O. LIM-kinase1. Int. J. Biochem. Cell Biol.
1999, 31, 389−394.
(3) Mizuno, K.; Okana, I.; Ohashi, K.; Nunoue, K.; Kuma, K.; Miyata,
T.; Nakamura, T. Identification of a human cDNA encoding a novel
protein kinase with two repeats of the LIM/double Zinc finger motif.
Oncogene 1994, 9, 1605−1612.
(4) Osada, H.; Hasada, K.; Inazawa, J.; Uchida, K.; Ueda, R.;
Takahashi, T.; Takahashi, T. Subcellular localization and protein
interaction of the human LIMK2 gene expressing alternative
transcripts with tissue-specific regulation. Biochem. Biophys. Res.
Commun. 1996, 229, 582−589.
(5) Bernard, O. Lim kinases, regulators of actin dynamics. Int. J.
Biochem. Cell Biol. 2007, 39, 1071−1076.
(6) Acevedo, K.; Moussi, N.; Li, R.; Soo, P.; Bernard, O. LIM kinase
2 is widely expressed in all tissues. J. Histochem. Cytochem. 2006, 54,
487−501.
(7) Scott, R. W.; Olson, M. F. LIM kinases: function, regulation and
association with human disease. J. Mol. Med. (Berlin) 2007, 85, 555−
568.
(8) Arber, S.; Barbayannis, F. A.; Hanser, H.; Schneider, C.; Stanyon,
C. A.; Bernard, O.; Caroni, P. Regulation of actin dynamics through
phosphorylation of cofilin by LIM-kinase. Nature 1998, 393, 805−809.
(9) Edwards, D. C.; Sanders, L. C.; Bokoch, G. M.; Gill, B. N.
Activation of LIM-kinased by Pak1 couples Rac/Cdc42 GTPase
signaling to actin cytoskeletal dynamics. Nat. Cell Biol. 1999, 1, 253−
259.
(10) Maekawa, M.; Ishizaki, T.; Boku, S.; Watanabe, N.; Fujita, A.;
Iwamatsu, A.; Obinata, T.; Ohashi, K.; Mizuno, K.; Narumiya, S.
Signaling from Rho to the actin cytoskeleton through protein kinases
ROCK and LIM-kinase. Science 1999, 285, 895−898.
(11) Vlachos, P.; Joseph, B. The Cdk inhibitor p57(Kip2) controls
LIM-kinase 1 activity and regulates actin cytoskeleton dynamics.
Oncogene 2009, 28, 4175−4188.
(12) Thirone, A. C.; Speight, P.; Zulys, M.; Rotstein, O. D.; Szaszi,
K.; Pedersen, S. F.; Kapus, A. Hyperosmotic stress induces Rho/Rho
kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells:
In Vitro and in Vivo DMPK Assays. All in vitro (microsomal
stability, CYP-450 inhibition, etc.) and in vivo pharmacokinetics
studies were carried out in the DMPK Core Facility of Scripps Florida.
Detailed procedures for these assays have been described in previous
publications (refs 41, 42, 46, 48−50, and 52−55) and are also
provided in Supporting Information.
N
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
key role in the osmotically triggered F-actin response. Am. J. Physiol.:
Cell Physiol. 2009, 296, C463−475.
(30) Manetti, F. Recent findings confirm LIM domain kinases as
emerging target candidates for cancer therapy. Curr. Cancer Drug
Targets 2012, 12, 543−560.
(31) Ohashi, K.; Sampei, K.; Nakagawa, M.; Uchiumi, N.; Amanuma,
T.; Aiba, S.; Oikawa, M.; Mizuno, K. Damnacanthal, an effective
inhibitor of LIM-kinase, inhibits cell migration and invasion. Mol. Biol.
Cell 2014, 25, 828−840.
(32) Park, J. B.; Agnihotri, S.; Golbourn, B.; Bertrand, K. C.; Luck, A.;
Sabha, N.; Smith, C. A.; Byron, S.; Zadeh, G.; Croul, S.; Berens, M.;
Rutka, J. T. Transcriptional profiling of GBM invasion genes identifies
effective inhibitors of the LIM kinase-Cofilin pathway. Oncotarget
2014, 5, 9382−9395.
(33) Rak, R.; Kloog, Y. Targeting LIM kinase in cancer and
neurofibromatosis. Cell Cycle 2014, 13, 1360−1361.
(34) Mashiach-Farkash, E.; Rak, R.; Elad-Sfadia, G.; Haklai, R.;
Carmeli, S.; Kloog, Y.; Wolfson, H. J. Computer-based identification of
a novel LIMK1/2 inhibitor that synergizes with salirasib to destabilize
the actin cytoskeleton. Oncotarget 2012, 3, 629−639.
(35) Iyengar, S.; Hildreth, J. E.; Schwartz, D. H. Actin-dependent
receptor colocalization required for human immunodeficiency virus
entry into host cells. J. Virol. 1998, 72, 5251−5255.
(36) Ross-Macdonald, P.; de Silva, H.; Guo, Q.; Xiao, H.; Hung, C.
Y.; Penhallow, B.; Markwalder, J.; He, L.; Attar, R. M.; Lin, T. A.; Seitz,
S.; Tilford, C.; Wardwell-Swanson, J.; Jackson, D. Identification of a
nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol.
Cancer Ther. 2008, 7, 3490−3498.
(37) He, L.; Seitz, S. P.; Trainor, G. L.; Tortolani, D.; Vaccaro, W.;
Poss, M.; Tarby, C. M.; Tokarski, J. S.; Penhallow, B.; Hung, C. Y.;
Attar, R.; Lin, T. A. Modulation of cofilin phosphorylation by
inhibition of the Lim family kinases. Bioorg. Med. Chem. Lett. 2012, 22,
5995−5998.
(13) Hoogenraad, C. C.; Akhmanova, A.; Galjart, N.; De Zeeuw, C. I.
LIMK1 and CLIP-115: linking cytoskeletal defects to Williams
syndrome. BioEssays 2004, 26, 141−150.
(14) Piccioli, Z. D.; Littleton, J. T. Retrograde BMP signaling
modulates rapid activity-dependent synaptic growth via presynaptic
LIM kinase regulation of cofilin. J. Neurosci. 2014, 34, 4371−4381.
(15) Heredia, L.; Helguera, P.; de Olmos, S.; kedikian, G.; Sola Vigo,
F.; LaFerla, F.; et al. Phosphorylation of actin-depolymerizing factor/
cofilin by Lim-kinase mediates amylolid beta-induced degeneration: a
potential mechanism of neuronal dystrophy in Alzheimer’s disease. J.
Neurosci. 2006, 26, 6533−6542.
(16) Honma, M.; Benitah, S. A.; Watt, F. M. Role of LIM kinases in
normal and psoriatic human epidermis. Mol. Biol. Cell 2006, 17, 1888−
1896.
(17) Bongalon, S.; Dai, Y. P.; Singer, C. A.; Yamboliev, I. A. PDGF
and IL-1beta upregulate cofilin and LIMK2 in canine cultured
pulmonary artery smooth muscle cells. J. Vasc. Res. 2004, 41, 412−421.
(18) Dai, Y. P.; Bongalon, S.; Tian, H.; Parks, S. D.; Mutafova-
Yambolieva, V. N.; Yamboliev, I. A. Upregulation of profilin, cofilin-2
and LIMK2 in cultured pulmonary artery smooth muscle cells and in
pulmonary arteries of monocrotaline-treated rats. Vascul. Pharmacol.
2006, 44, 275−282.
(19) Akagawa, H.; Tajima, A.; Sakamoto, Y.; Krischek, B.; Yoneyama,
T.; Kasuya, H.; Onda, H.; Hori, T.; Kubota, M.; Machida, T.; Saeki,
N.; Hata, A.; Hashiguchi, K.; Kimura, E.; Kim, C. J.; Yang, T. K.; Lee, J.
Y.; Kimm, K.; Inoue, I. A haplotype spanning two genes, ELN and
LIMK1, decreases their transcripts and confers susceptibility to
intracranial aneurysms. Hum. Mol. Genet. 2006, 15, 1722−1734.
(20) Harrison, B. A.; Whitlock, N. A.; Voronkov, M. V.; Almstead, Z.
Y.; Gu, K. J.; Mabon, R.; Gardyan, M.; Hamman, B. D.; Allen, J.;
Gopinathan, S.; McKnight, B.; Crist, M.; Zhang, Y.; Liu, Y.; Courtney,
L. F.; Key, B.; Zhou, J.; Patel, N.; Yates, P. W.; Liu, Q.; Wilson, A. G.;
Kimball, S. D.; Crosson, C. E.; Rice, D. S.; Rawlins, D. B. Novel class
of LIM-kinase 2 inhibitors for the treatment of ocular hypertension
and associated glaucoma. J. Med. Chem. 2009, 52, 6515−6518.
(21) Xu, X.; Guo, J.; Vorster, P.; Wu, Y. Involvement of LIM kinase 1
in actin polarization in human CD4 T cells. Commun. Integr. Biol.
2012, 5, 381−383.
(22) Manetti, F. HIV-1 proteins join the family of LIM kinase
partners. New roads open up for HIV-1 treatment. Drug Discovery
Today 2012, 17, 81−88.
(23) Vorster, P. J.; Guo, J.; Yoder, A.; Wang, W.; Zheng, Y.; Xu, X.;
Yu, D.; Spear, M.; Wu, Y. LIM kinase 1 modulates cortical actin and
CXCR4 cycling and is activated by HIV-1 to initiate viral infection. J.
Biol. Chem. 2011, 286, 12554−12564.
(24) Wen, X.; Ding, L.; Wang, J. J.; Qi, M.; Hammonds, J.; Chu, H.;
Chen, X.; Hunter, E.; Spearman, P. ROCK1 and LIM kinase modulate
retrovirus particle release and cell-cell transmission events. J. Virol.
2014, 88, 6906−6921.
(25) Bagheri-Yarmand, R.; Mazumdar, A.; Sahin, A. A.; Kumar, R.
LIM kinase 1 increases tumor metastasis of human breast cancer cells
via regulation of the urokinase-type plasminogen activator system. Int.
J. Cancer 2006, 118, 2703−2710.
(38) Sleebs, B. E.; Nikolakopoulos, G.; Street, I. P.; Falk, H.; Baell, J.
B. Identification of 5,6-substituted 4-aminothieno[2,3-d]pyrimidines as
LIMK1 inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 5992−5994.
(39) Sleebs, B. E.; Ganame, D.; Levit, A.; Street, I. P.; Gregg, A.;
Falkabc, H.; Baell, J. B. Development of substituted 7-phenyl-4-
aminobenzothieno[3,2-d]pyrimidines as potent LIMK1 inhibitors.
Med. Chem. Commun. 2011, 2, 982−986.
(40) Goodwin, N. C.; Cianchetta, G.; Burgoon, H. A.; Healy, J.;
Mabon, R.; Strobel, E. D.; Allen, J.; Wang, S.; Hamman, B. D.;
Rawlins, D. B. Discovery of a type III inhibitor of LIM kinase 2 that
binds in a DFG-out conformation. ACS Med. Chem. Lett. 2015, 6, 53−
57.
(41) Yin, Y.; Cameron, M. D.; Lin, L.; Khan, S.; Schroter, T.; Grant,
̈
W.; Pocas, J.; Chen, Y. T.; Schurer, S.; Pachori, A.; LoGrasso, P.; Feng,
̈
Y. Discovery of highly potent and selective urea-based ROCK
inhibitors and their effects on intraocular pressure in rats. ACS Med.
Chem. Lett. 2010, 1, 175−179.
(42) Yin, Y.; Lin, L.; Ruiz, C.; Khan, S.; Cameron, M. D.; Grant, W.;
Pocas, J.; Eid, N.; Park, H.; Schroter, T.; Lograsso, P. V.; Feng, Y.
Synthesis and biological evaluation of urea derivatives as highly potent
and selective rho kinase inhibitors. J. Med. Chem. 2013, 56, 3568−
3581.
(43) Morita, Y.; Ishigaki, T.; Kawamura, K.; Iseki, K. Short and
Practical Synthesis of N′,N′-disubstituted N-aryl-1,2-ethylenediamines
by a decarboxylative ring-opening reaction under nucleophilic
conditions. Synthesis 2007, 16, 2517−2523.
(26) Davila, M.; Frost, A. R.; Grizzle, W. E.; Chakrabarti, R. LIM
kinase 1 is essential for the invasive growth of prostate epithelial cells:
implications in prostate cancer. J. Biol. Chem. 2003, 278, 36868−
36875.
(27) Davila, M.; Jhala, D.; Ghosh, D.; Grizzle, W. E.; Chakrabarti, R.
Expression of LIM kinase 1 is associated with reversible G1/S phase
arrest, chromosomal instability and prostate cancer. Mol. Cancer 2007,
6, 40.
(28) Manetti, F. LIM kinases are attractive targets with many
macromolecular partners and only a few small molecule regulators.
Med. Res. Rev. 2012, 32, 968−998.
(29) Suyama, E.; Wadhwa, R.; Kawasaki, H.; Yaguchi, T.; Kaul, S. C.;
Nakajima, M.; Taira, K. LIM kinase-2 targeting as a possible anti-
metastasis therapy. J. Gene Med. 2004, 6, 357−363.
(44) Goodacre, C. J.; Bromidge, S. M.; Clapham, D.; King, F. D.;
Lovell, P. J.; Allen, M.; Campbell, L. P.; Holland, V.; Riley, G. J.; Starr,
K. R.; Trail, B. K.; Wood, M. D. A series of bisaryl imidazolidin-2-ones
has shown to be selective and orally active 5-HT2C receptor
antagonists. Bioorg. Med. Chem. Lett. 2005, 15, 4989−4993.
(45) Beletskaya, I. P.; Bessmertnykh, A. G.; Guilard, R. Halo-
substituted aminobenzenes prepared by Pd-catalyzed amination.
Synlett 1999, 9, 1459−1461.
(46) Feng, Y.; Yin, Y.; Weiser, A.; Griffin, E.; Cameron, M. D.; Lin,
L.; Ruiz, C.; Schurer, S. C.; Inoue, T.; Rao, P. V.; Schroter, T.;
̈
̈
LoGrasso, P. Discovery of substituted 4-(pyrazol-4-yl)-phenylbenzo-
O
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dioxane-2-carboxamides as potent and highly selective Rho kinase
(64) Yang, N.; Higuchi, O.; Ohashi, K.; Nagata, K.; Wada, A.;
Kangawa, K.; Nishida, E.; Mizuno, K. Cofilin phosphorylation by LIM-
kinase 1 and its role in Rac-mediated actin reorganization. Nature
1998, 393, 809−812.
(65) Ying, H.; Biroc, S. L.; Li, W. W.; Alicke, B.; Xuan, J. A.; Pagila,
R.; Ohashi, Y.; Okada, T.; Kamata, Y.; Dinter, H. The Rho kinase
inhibitor fasudil inhibits tumor progression in human and rat tumor
models. Mol. Cancer Ther. 2006, 5, 2158−2164.
(66) Hopkins, A. M.; Pineda, A. A.; Winfree, L. M.; Brown, G. T.;
Laukoetter, M. G.; Nusrat, A. Organized migration of epithelial cells
requires control of adhesion and protrusion through Rho kinase
effectors. Am. J. Physiol.: Gastrointest. Liver Physiol. 2007, 292, G806−
817.
(67) Nine week old brown Norway rats (n = 6/group) were housed
under constant low light conditions for 65 days (illuminance ranging
from 60 to 80 lx). At 17 weeks, 20 μL of 18w (0.25%) was applied to
the right eye. The left eye was untreated and served as the control.
IOP measurements were made on both eyes prior to and at 1, 4, 7 and
24 h after administration. A Tonolab pen was used for IOP
measurements. Initial baseline IOP of the treated eye was 28
mmHg. The maximal IOP decrease of the treated eye was ∼6
mmHg from 1 to 4 h.
(ROCK-II) inhibitors. J. Med. Chem. 2008, 51, 6642−6645.
(47) Schroter, T.; Griffin, E.; Weiser, A.; Feng, Y.; LoGrasso, P.
̈
Detection of myosin light chain phosphorylationa cell-based assay
for screening Rho-kinase inhibitors. Biochem. Biophys. Res. Commun.
2008, 374, 356−360.
(48) Feng, Y.; Cameron, M. D.; Frackowiak, B.; Griffin, E.; Lin, L.;
Ruiz, C.; Schroter, T.; LoGrasso, P. Structure-activity relationships,
and drug metabolism and pharmacokinetic properties for indazole
piperazine and indazole piperidine inhibitors of ROCK-II. Bioorg. Med.
Chem. Lett. 2007, 17, 2355−2360.
(49) Feng, Y.; Chambers, J. W.; Iqbal, S.; Koenig, M.; Park, H.;
Cherry, L.; Hernandez, P.; Figuera-Losada, M.; LoGrasso, P. V. A
small molecule bidentate-binding dual inhibitor probe of the LRRK2
and JNK kinases. ACS Chem. Biol. 2013, 8, 1747−1754.
(50) Chen, Y. T.; Bannister, T. D.; Weiser, A.; Griffin, E.; Lin, L.;
Ruiz, C.; Cameron, M. D.; Schurer, S.; Duckett, D.; Schroter, T.;
̈
̈
LoGrasso, P.; Feng, Y. Chroman-3-amides as potent Rho kinase
inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 6406−6409.
(51) Ahmed, T.; Shea, K.; Masters, J. R.; Jones, G. E.; Wells, C. M. A
PAK4-LIMK1 pathway drives prostate cancer cell migration down-
stream of HGF. Cell. Signalling 2008, 20, 1320−1328.
(52) Yin, Y.; Lin, L.; Ruiz, C.; Cameron, M. D.; Pocas, J.; Wayne, G.;
Schroter, T.; Chen, W.; Duckett, D.; Schurer, S. C.; LoGrasso, P.;
̈
̈
Feng, Y. Benzothiazole as Rho-associated kinase (ROCK-II) inhibitors.
Bioorg. Med. Chem. Lett. 2009, 19, 6686−6690.
(53) Fang, X.; Yin, Y.; Wang, B.; Yao, L.; Chen, Y. T.; Schroter, T.;
̈
Weiser, A.; Pocas, J.; Wayne, G.; Cameron, M. D.; Lin, L.; Ruiz, C.;
Khan, S.; Schurer, S. C.; Pachori, A.; LoGrasso, P.; Feng, Y.
̈
Tetrahydroisoquinoline derivatives as potent and selective Rho kinase
Inhibitors. J. Med. Chem. 2010, 53, 5727−5737.
(54) Chowdhury, S.; Chen, Y. T.; Fang, X.; Grant, W.; Pocas, J.;
Cameron, M. D.; Ruiz, C.; Lin, L.; Park, H.; Schroter, T.; Bannister, T.
̈
D.; LoGrasso, P. V.; Feng, Y. Amino acid derived quinazolines as
Rock/PKA inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 1592−1599.
(55) Zheng, K.; Iqbal, S.; Hernandez, P.; Park, H.; LoGrasso, P.;
Feng, Y. Design and synthesis of highly potent and isoform selective
JNK3 inhibitors: SAR studies on aminopyrazole derivatives. J. Med.
Chem. 2014, 57, 10013−10030.
(56) Zhou, P.; Zou, J. W.; Tian, F. F.; Shang, Z. C. Fluorine
bondingHow does it work in protein−ligand interactions? J. Chem.
Inf. Model. 2009, 49, 2344−2355.
(57) Prudent, R.; Vassal-Stermann, E.; Nguyen, C. H.; Pillet, C.;
Martinez, A.; Prunier, C.; Barette, C.; Soleilhac, E.; Filhol, O.; Beghin,
A.; Valdameri, G.; Honore, S.; Aci-Seche, S.; Grierson, D.; Antonipillai,
J.; Li, R.; Di Pietro, A.; Dumontet, C.; Braguer, D.; Florent, J. C.;
Knapp, S.; Bernard, O.; Lafanechere, L. Pharmacological inhibition of
LIM kinase stabilizes microtubules and inhibits neoplastic growth.
Cancer Res. 2012, 72, 4429−4439.
(58) Yoshioka, K.; Foletta, V.; Bernard, O.; Itoh, K. A role for LIM
kinase in cancer invasion. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
7247−7252.
(59) Wu, Y.; Beddall, M. H.; Marsh, J. W. Rev-dependent lentiviral
expression vector. Retrovirology 2007, 4, 12.
(60) Wu, Y.; Beddall, M. H.; Marsh, J. W. Rev-dependent indicator T
cell line. Curr. HIV Res. 2007, 5, 394−402.
(61) Mishima, T.; Naotsuka, M.; Horita, Y.; Sato, M.; Ohashi, K.;
Mizuno, K. LIM-kinase is critical for the mesenchymal-to-amoeboid
cell morphological transition in 3D matrices. Biochem. Biophys. Res.
Commun. 2010, 392, 577−581.
(62) Horita, Y.; Ohashi, K.; Mukai, M.; Inoue, M.; Mizuno, K.
Suppression of the invasive capacity of rat ascites hepatoma cells by
knockdown of Slingshot or LIM kinase. J. Biol. Chem. 2008, 283,
6013−6021.
(63) Alers, J. C.; Rochat, J.; Krijtenburg, P. J.; Hop, W. C.; Kranse, R.;
Rosenberg, C.; Tanke, H. J.; Schroder, F. H.; van Dekken, H.
Identification of genetic markers for prostatic cancer progression. Lab.
Invest. 2000, 80, 931−942.
P
DOI: 10.1021/jm501680m
J. Med. Chem. XXXX, XXX, XXX−XXX