Synthesis and biological evaluation of urea derivatives as highly potent and selective rho kinase inhibitors

Article abstract of DOI:10.1021/jm400062r

RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising strategy for several indications such as cardiovascular disease, glaucoma, and inflammatory disease. In 2010, our group reported urea-based ROCK inhibitors as potential antiglaucoma agents. These compounds showed potent IC₅₀ values in enzymatic and cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (～7 mmHg).(22) To develop more advanced ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis, etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process, counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in vivo pharmacokinetic properties were discovered.

Full text of DOI:10.1021/jm400062r

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Urea Derivatives as Highly
Potent and Selective Rho Kinase Inhibitors
Yan Yin,†,# Li Lin,§ Claudia Ruiz,§ Susan Khan,§ Michael D. Cameron,§,¥ Wayne Grant,‡ Jennifer Pocas,‡
Nibal Eid,‡ HaJeung Park,∞ Thomas Schroter,‡ Philip V. LoGrasso,*,‡,¥ and Yangbo Feng*,†
̈
‡
§
†Medicinal Chemistry; Discovery Biology; ∞Crystallography/Modeling Facility; Drug Metabolism and Pharmacokinetics,
¥
Translational Research Institute, and Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130
Scripps Way, #2A1, Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects
on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising
strategy for several indications such as cardiovascular disease, glaucoma, and inflammatory disease. In 2010, our group reported
urea-based ROCK inhibitors as potential antiglaucoma agents. These compounds showed potent IC50 values in enzymatic and
cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (∼7 mmHg).22 To develop more advanced
ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis,
etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process,
counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in
vivo pharmacokinetic properties were discovered.
INTRODUCTION
■
Rho-associated coiled-coil protein kinase (ROCK) is a serine/
threonine protein kinase from the AGC kinase family with
isoforms identified as ROCK-I (or ROCKβ) and ROCK-II (or
ROCKα).1 When activated by small GTP-bound RhoA, ROCK
induces phosphorylation of myosin light chain (MLC) in a dual
way by increasing MLC bis-phosphorylation directly and by
preventing its dephosphorylation through inhibition of the
MLC phosphatase thereby resulting in cellular contraction.2
The contractile effect regulates a variety of physiological
Figure 1. A previously disclosed urea-based ROCK inhibitor (1) and
processes including smooth muscle contraction,2 blood
pressure,3 endothelial permeability,4 IOP,5 and cellular
migration.6 Accumulating evidence suggests that RhoA/
ROCK signaling cascade is involved in various diseases that
involve contractile processes, such as multiple sclerosis,7
pulmonary hypertension,8 cardiovascular diseases,3,9 glaucoma,5
erectile dysfunction,10,11 and cancer.6
its key interactions in the ATP site of ROCK-II.27 In addition to hinge
binding to residues E170 and M172, the other three interactions also
contributed to the high potency of this urea-based ROCK inhibitor.
hydrophobic interactions between the benzyl moiety of
compound 1 and the hydrophobic pocket of ROCK-II located
under the P-loop appeared to be a key element responsible for
its high potency and selectivity. In addition, H-bonds between
the protonated tertiary amine and the carboxylate side chain of
Asp176 of ROCK-II (Figure 1) also contributed to high
potency and selectivity, and the tertiary amine side chain
enhanced the aqueous solubility of inhibitor 1.22
ROCK has attracted numerous and diverse interests, and
many ROCK inhibitors have been reported.12−21 We have
previously reported a urea-based ROCK inhibitor 122 (Figure
1) which showed excellent enzymatic and cellular potency, high
selectivity, good aqueous solubility, good porcine corneal
penetration, appropriate DMPK properties, and significant IOP
lowering effects in rat eyes. Molecular modeling studies showed
that the pyrazole group binds to the hinge with two H-bonds,
and the urea carbonyl group H-bonds to the side chain of
Lys121 (Figure 1). Docking studies demonstrated that
Received: January 13, 2013
© XXXX American Chemical Society
A
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a
Among the several scaffolds developed in our laborato-
ries,22−31 the urea chemotype is the most interesting. In
addition to furnishing highly selective and potent ROCK
inhibitors,22 it also provides many diversification points for
structure−activity relationship (SAR) studies and for modifying
the inhibitor’s overall pharmaceutical properties, such as
substitution to the middle phenyl ring, alkylation to both NH
moieties of the urea group, substitution to the benzyl
methylene position, and mono- or multisubstitution to the
benzyl phenyl ring (Figure 1). Moreover, the middle phenyl
ring may be replaced by a heteroaromatic group, and the tail
benzyl moiety may be replaced by other saturated or
unsaturated systems. Here we report extensive SAR studies
on all these positions in a continuing effort to develop highly
efficacious and pharmaceutically viable ROCK inhibitors from
the urea scaffold. These SAR studies are aimed at under-
standing key elements of the ROCK ATP-binding pocket and
at providing information to guide the design of ROCK
inhibitors with diversified properties.
Scheme 2. Synthesis of Inhibitors 8a−f
a
Reagents and conditions: (a) 1-(Isocyanomethyl)-3-methoxyben-
zene, DCM; (b) boronic acid pinacol ester, Ph(PPh3)4, K2CO3,
dioxane/H2O (4:1), 95 °C.
a
Scheme 3. Synthesis of Inhibitors 12a−n
CHEMISTRY
■
The preparation of unsubstituted ROCK inhibitor 5 is outlined
in Scheme 1. Urea, carbamate, or amide intermediates (4a−f
a
Scheme 1. Synthesis of Inhibitors 5a−p
a
Reagents and conditions: (a) (i) R3-NH2, MeOH, r.t; (ii) NaBH4, 0
°C; (b) 1-bromo-4-isocyanatobenzene, DCM; (c) boronic acid pinacol
ester, Ph(PPh3)4, K2CO3, dioxane/H2O (4:1), 95 °C.
the bromide intermediate 11. Finally, compound 12 was
formed via a Suzuki reaction and purified by preparative HPLC.
Application of α-position-substituted benzylamines led to
Rock inhibitor 14. As shown in Scheme 4, a urea formation
a
Reagents and conditions: (a) Primary amine or alcohol; (b) acid,
HATU, DIEA, DMF; (c) boronic acid pinacol ester, Ph(PPh3)4,
K2CO3, dioxane/H2O (4:1), 95 °C.
a
Scheme 4. Synthesis of Inhibitors 14a−d
and 4h−p) were quickly accessed through a coupling reaction
between 1-bromo-4-isocyanobenzene and a primary amine (to
form a urea) or an alcohol (to form a carbamate) in DCM, or a
condensation between 4-bromoaniline and a carboxylic acid (to
produce the carboxyl amide intermediate) in the presence of
HATU in DMF. The target compounds 5a−p were prepared
through a Suzuki coupling reaction between intermediate 4 and
boronic acid pinacol ester catalyzed by Pd(PPh3)4. The final
obtained ROCK inhibitors were all purified by preparative
reverse-phase HPLC.
To make compounds 8a−f that possess a substitution on the
urea NH group from aniline, commercial or in-house-prepared
N-substituted 4-bromoaniline 6 was reacted with 1-(isocyano-
methyl)-3-methoxybenzene in DCM to form the urea
intermediate 7. A Suzuki coupling was then applied (Scheme
2) to furnish the final inhibitor 8.
a
Reagents and conditions: (a) α-Substituted benzylamines, DCM; (b)
boronic acid pinacol ester, Ph(PPh3)4, K2CO3, dioxane/H2O (4:1), 95
°C.
between 4-bromoisocyanobenzene and a selected α-branched
benzylamine gave intermediate 13, which was subjected to a
Ph(PPh3)4-catalyzed Suzuki coupling reaction (Scheme 4) to
yield the final product 14.
Inhibitors with substitutions on both the middle phenyl ring
and on a urea NH group were synthesized as shown in Scheme
5. A SNAr reaction was first used to furnish the 4-bromo-
A short route was used to synthesize inhibitor 12 which
possessed a substitution on the urea NH group from
benzylamine (Scheme 3). Treatment of a substituted
benzaldehyde 9 with a primary amine, followed by NaBH4
reduction, gave the secondary amine 10. This amine was
reacted with 1-bromo-4-isocyanobenzene in DCM to furnish
B
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a
ROCK-I enzyme assays to screen for selectivity. After this,
potent and selective inhibitors were submitted to cell-based
assays that measured MLC bis-phosphorylation (ppMLC).32
Finally, the most advanced ROCK-II inhibitors were evaluated
in in vitro and in vivo DMPK studies.
Scheme 5. Synthesis of Inhibitors 19a and 19b
Previously we reported that the aniline−benzylamine
asymmetric urea coupled with an appropriate heteroaromatic
hinge binding moiety was a good scaffold for ROCK
inhibition.22 For example, compound 5b (Table 1), an
unsubstituted urea-based ROCK-II inhibitor, showed good
enzymatic and cellular potency and ∼32-fold selectivity against
PKA (IC50 = 18 nM, 113 nM, and 586 nM, respectively).22 To
improve upon that, a variety of structural moieties other than
benzylamines were applied to explore the structural elements
that could fit well into the hydrophobic pocket (Figure 1). The
distance between the urea carbonyl group and the terminal
phenyl ring was first studied. Compounds 5a to 5d
(corresponding to one to four backbone atoms between the
carbonyl and the phenyl ring) were thus synthesized and
evaluated. As shown in Table 1, the benzylamine-based urea 5b
is the most potent ROCK inhibitor. Replacement of the
benzylamine by aniline (5a) significantly reduced the ROCK
inhibition. Increasing the chain length from one methylene to
two (5c) slightly decreased the potency while dramatic
reduction of ROCK activity was observed when the chain
length was increased to three methylene units (5d).
Interestingly, the PKA selectivity did not change much with
the chain length (∼30-fold) except for the aniline-based urea 5a
(>60-fold). These data suggest that changing the chain length
between the urea moiety and the phenyl ring disturbed the
optimal orientation of the phenyl group assumed in 5b. It is
important to note that the ring closure analogue of 5b (5h, a
bicyclic tetrahydroisoquinoline-based urea derivative) reduced
potency of ROCK inhibition (IC50 = 280 nM). This lower
potency of 5h is in contrast to the potent ROCK inhibition
(similar to 5b) exhibited by its corresponding bicyclic carboxyl
amide analogues developed in our lab (the tetrahydronaph-
thalene 2-carboxyl amide,24 the chroman 3-carboxamide,24 the
benzadioxane 2-carboxamide,23 and the tetrahydroisoquinoline
3-carboxamide27). The most likely reason for the lower ROCK
a
Reagents and conditions: (a) 2-(Dimethylamino)ethanol, Cs2CO3,
DMF; (b) SnCl2·H2O, EtOAc; (c) (i) triphosgene, NaHCO3, CH2Cl2,
0 °C; (ii) benzylamine derivatives, CH2Cl2, 0 °C; (d) boronic acid
pinacol ester, Ph(PPh3)4, K2CO3, dioxane/H2O (4:1), 95 °C.
nitrobenzene derivative 16, followed by reduction of the nitro
group using hydrated SnCl2 to give aniline 17. Treatment of 17
with triphosgene furnished an intermediate isocyanoaryl that
was followed in one pot (without any workup) by a coupling
reaction with an N-substituted benzylamine derivative to afford
the bromide intermediate 18. Finally, the target inhibitor 19
was formed via a palladium-catalyzed Suzuki reaction and was
purified by reverse-phase preparative HPLC.
RESULTS AND DISCUSSION
■
The goal of this work was to develop potent and selective urea-
based ROCK inhibitors possessing appropriate DMPK proper-
ties that would make them suitable for various potential
applications by targeting the ROCK pathway. To do this, we
evaluated these compounds in biochemical ROCK-II and
protein kinase A (PKA) assays (PKA was selected as a routine
counterscreen to gauge the general kinase selectivity of
synthesized ROCK inhibitors22−31), and a few selected
inhibitors were further assessed in JNK3, MRCKα, p38, and
Table 1. SAR Studies for the Tail Moiety
a
b
c
IC50 values are means of two or more experiments with errors within 30% of the mean. Not determined. Published results.22
C
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potency of 5h was that the conformation assumed by the
tetrahydroisoquinoline phenyl moiety was not as favored for
hydrophobic interactions with the P-loop pocket as those in the
carboxyl amide systems.
inhibitor 5i) between the phenyl moiety and the hydrophobic
pocket under the P-loop. Alternatively, it is possible that the
disubstitutions could not be accommodated in the pocket due
to steric bulk.
The effect of substitutions on the urea NH group from the
aniline side was the next focus of SAR studies. As shown in
Table 3, any substitution to this position greatly reduced the
We next explored the feasibility of replacing the benzylamine
with a benzyl alcohol or a phenylpropyl group while still
maintaining potent ROCK inhibition. Table 1 shows the benzyl
alcohol-derived analogue carbamate 5e had a slightly improved
ROCK potency (7 nM vs 18 nM) and a much better PKA
selectivity (>300-fold vs 33-fold) as compared to urea 5b.
However, 5e had lower cell activity (IC50 > 2700 nM). Just like
in the urea series 5a−d, addition of a methylene unit to the
carbamate (5f) reduced ROCK inhibition. Although the
corresponding carboxyl amide analogue 5g presented ROCK
inhibition similar to that of 5b, its PKA selectivity was lower
(∼11-fold). On the other hand, 5g exhibited good cell potency
with an IC50 = 91 nM in the ppMLC assays (Table 1). In
compound 5i, a saturated cyclohexyl group was used to replace
the aromatic phenyl ring in 5b. As shown in Table 1, 5i was a
much weaker ROCK inhibitor than 5b (IC50: 751 nM vs 18
nM), which was likely due to a much weaker hydrophobic
interaction with the P-loop for the cyclohexyl ring in 5i than for
the phenyl group in 5b. The lower affinity for 5i also
demonstrated that hydrophobic interactions with the P-loop
were likely a key contributor to ROCK inhibition for these
urea-based inhibitors (and for inhibitors based on many other
scaffolds developed in our laboratories22−31). Overall, data in
Table 1 demonstrated that the benzylamine based urea was the
best in terms of ROCK inhibition, cell potency, and PKA
selectivity.
Table 3. Effects of Substitutions to the Urea NH of Aniline
a
IC50 values are means of two or more experiments with errors within
30% of the mean.
ROCK affinity, and the deterioration of ROCK activity was in
direct relation to the size of the substituents. For example, a
simple methyl substitution resulted in compound 8a which had
moderate ROCK potency (IC50 = 87 nM, vs 2 nM in 5j), while
large substituents represented by compounds 8c−e had ROCK
IC50 values in the micromolar range. The major reason for the
decreased ROCK potency was that substitution on this urea
NH significantly disturbed the optimal binding conformation
(see Figure 4, docking mode). Therefore, it was decided that no
substitutions to this urea NH group would be made in future
optimizations.
The effect of multisubstitutions on the benzyl phenyl ring
was studied next. It was disclosed in our previous
communication22 that a 3-methoxy group was the best
substituent in monosubstituted (on the benzyl phenyl ring)
urea-based ROCK inhibitors (5j, IC50 = 2 nM in enzyme assay,
4 nM in cell assay, and 882 nM in PKA enzyme assay, Table 2).
Table 2. Effects of Disubstitutions on the Benzyl Phenyl
Ring
The effect of alkylation on the urea NH from the
benzylamine side was previously communicated,22 and results
showed that this position was quite tolerant to simple methyl
and ethyl groups. A more detailed set of SAR studies for
substitutions on this position is summarized in Table 4. As
expected, simple alkylation using methyl, ethyl, cyclopropyl,
and isopropyl groups (12a to 12d) furnished ROCK inhibitors
exhibiting high potency in both enzyme and ppMLC cell assays
and high selectivity against PKA (>1000-fold). Substitutions
with oxygen-containing building blocks such as 2-hydroxylethyl
and 2-methoxylethyl (12e and 12f) also exhibited excellent
enzymatic potency (IC50 was 1 nM and 17 nM, respectively),
good to fair cellular activity (19 nM and 180 nM, respectively),
and low PKA inhibition (2988 nM and 10530 nM,
respectively). Interestingly, the compound containing a free
NH2 group (12g) exhibited modestly potent PKA inhibition
(72 nM) in addition to high ROCK potency in both enzyme
and cell-based assays (<1 nM and 8 nM, respectively).
Remarkably, dimethylation of the NH2 in 12g resulted in an
inhibitor (12h) with much lower PKA inhibition (456 nM)
while still retaining excellent ROCK potency (IC50 < 1 and <4
nM in enzyme and cellular assay, respectively). Encouraged by
12h, substitutions containing other tertiary amino alkyl groups
(inhibitors 12i−n) were prepared and evaluated. Like 12h,
these inhibitors had excellent ROCK potency and good to fair
a
a
compd
R1
ROCK-II (nM)
PKA (nM)
b
5j
3-OCH3
2
253
331
570
924
425
281
357
882
>20000
11170
>20000
13480
>20000
>20000
518
5k
5l
2,3-di-OCH3
2,4-di-OCH3
2,5-di-OCH3
2,6-di-OCH3
3,4-di-OCH3
3,5-di-OCH3
3-F, 4-OCH3
5m
5n
5o
5p
5q
a
IC50 values are means of two or more experiments with errors within
30% of the mean. Published results.22
b
Several compounds with dimethoxy (5k to 5p) or fluoro-
methoxy (5q) substitutions were thus prepared and evaluated.
As shown in Table 2, these compounds all possessed lower
ROCK inhibition as compared to 5i, regardless of the specific
disubstitution pattern. One possible explanation for this
observation is that these disubstitutions disturbed optimal
hydrophobic interactions (exhibited in the monosubstituted
D
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Table 4. Inhibitory and Metabolic Stability of Inhibitors 12 with Substitutions on Nitrogen of Benzylamine Moiety
a
IC50 values were means of two or more experiments with errors within 30% of the mean.
Table 5. SAR of Substitutions on the α-Position of Benzylamine
a
a
a
compd
R1
R4
ROCK-II (nM)
ppMLC (nM)
PKA (nM)
HLM t1/2 (min)
5j
OCH3
H
2
2
2
1
2
2
4
40
882
4871
580
34
112
14a
14b
14c
14d
14e
OCH3
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2OH
F
32
>120
>120
>120
>60
H
H
H
48
438
150
40
389
CH2CH2N(CH3)2
238
a
IC50 values are means of two or more experiments with errors within 30% of the mean.
cell activity and PKA selectivity. The only exception was 12j
which exhibited a PKA selectivity of only 25-fold.
metabolic stability. For example, inhibitor 12e exhibited a
human microsomal stability of t1/2 = 24 min, while the
corresponding inhibitors without the methoxy substitution
(12o and 12p) had a much higher HLM stability of t1/2 > 120
min. However, as shown in Table 4, the m-methoxy
substitution benefited both the ROCK potency and the PKA
selectivity.
The α-position of the benzylamine moiety is also an
important point for diversification to fine-tune the pharma-
ceutical properties of this urea-based scaffold. Thus, ROCK
inhibitors containing α-substitutions such as 2-hydroxyethyl,
dimethylaminoethyl, and hydroxymethyl were synthesized and
assessed (Table 5). Substitution of a 2-hydroxylethyl group to
The stability of these compounds in human liver microsomes
(HLM) was also evaluated. Briefly, the microsomal stability of
most inhibitors with simple alkyl or oxygen-containing alkyl
substitutions (12a to 12f) was good with the exception of the
isopropyl-substituted compound 12d (half-life was only 11
min). While the stability of compounds with substitutions
containing NH2, dimethylamino-, or pyrrolidino- (12g to 12j)
groups were good to excellent, inhibitors with substituents
containing six-membered piperidine or morpholine moieties
(12k−n) exhibited poor microsomal stability. The 3-methoxy
substitution on the benzyl phenyl ring was clearly a liability for
E
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the α-position of 5j resulted in inhibitor 14a which showed
equal biochemical potency and a much improved PKA
selectivity (2 nM and 4871 nM, respectively) compared to 5j.
However, the cellular activity of 14a (IC50 = 40 nM) was
deteriorated and was probably due to the free OH group
introduced with this substitution. Replacing the methoxy group
in 14a with a fluoride or hydrogen resulted in inhibitors (14b
and 14c) having similar biochemical and cellular potency but
higher PKA inhibition (580 and 438 nM vs 4871 nM in 14a).
Similar enzyme and cellular potency was also observed for
inhibitor 14e which contained a dimethylaminoethyl sub-
stitution. Interestingly, shortening the chain length of the
substitution in 14a by one methylene unit (14d) did not affect
the enzyme activity (ROCK-II and PKA) but led to slightly
lower cellular potency (Table 5). Remarkably, introduction of a
substitution to the benzyl α-position yielded ROCK inhibitors
all possessing much higher microsomal stability (14a to 14e vs
5j). Overall, substitution on the α-position of the benzylamine
moiety furnished ROCK inhibitors with excellent potency
(both enzyme and cell), good PKA selectivity, and much
improved microsomal stability (in contrast to substitutions on
the urea NH group, Table 4, and substitutions on the middle
phenyl ring22). Therefore, this type of ROCK inhibitor will be
the focus for future optimizations.
2 had a ROCK-II IC50 value which was >170-fold worse than
that of the corresponding monosubstituted counterparts (12h
in Table 4 and compound 1r in ref 27).
For the middle phenyl ring of this urea scaffold, introduction
of substituents containing a tertiary amino group improved
ROCK potency, increased kinase selectivity, and enhanced
aqueous solubility.22 In the current study we found that
replacement of the middle phenyl ring by a heteroaromatic
moiety was also an important diversification strategy. For
instance, a 2-yl-pyridine was used as a surrogate for the
unsubstituted phenyl ring in compound 12h to give 20 (Figure
3). This ROCK inhibitor (20) was highly potent (both enzyme
Figure 3. Replacement of the middle phenyl ring by a pyridine moiety
was tolerated, presenting highly potent ROCK inhibitors.
and cell based) and had PKA selectivity similar to that of its
counterpart analogue 12h (Table 4). On the other hand, the
introduction of the pyridine ring reduced HLM stability (from
a half-life of 34 min in 12h to a half-life of 17 min in 20).
Molecular modeling was used to guide and explain the SAR
and the optimizations for this urea-based scaffold. Several
inhibitors were docked into the catalytic domain of a homology
model of human ROCK-II enzyme developed by the methods
previously described.22−24,27 As shown in Figure 4A, the
binding motif of compound 12e revealed two H-bond
interactions between residues Glu170/Met172 and the pyrazole
group in the hinge region. A third H-bond interaction was
formed between the urea N−H and the carbonyl group of the
DFG domain residue Asp232. A fourth H-bond was formed
between the OH moiety in the side chain of 12e and the
backbone carboxyl group of Asn219. In addition to H-bonding
interactions, an important contributor to the high potency of
12e was the hydrophobic interaction between the benzyl
moiety of ligand and the enzyme hydrophobic pocket under the
P-loop (Figure 4B). Similar docking modes were also observed
for those α-position-substituted ROCK inhibitors listed in
Table 5. As demonstrated by the binding motif of 14a shown in
Figure 4C, the OH moiety from the α-substituted side chain
bound in the same direction as that in 12e and formed an H-
bond with residue Asn219 (not shown in figure).
In our previous communication,22 simultaneous substitutions
to both the middle phenyl ring with a large linear alkoxy group
and the urea NH (from the benzylamine side) with a small
methyl or ethyl group were well tolerated. This bis-substitution
also improved the cell potency, the kinase selectivity, and the
aqueous solubility. These beneficial effects were further
confirmed in inhibitors with other bis-substitution combina-
tions. For example, compound 19a had a pyrrolidine-alkoxy
group on the middle phenyl ring and an ethyl substituent on
the urea NH (Figure 2). Compound 19b possessed a
The obtained structures of ligands bound to ROCK-II in the
ATP-binding pocket explained the observed SAR quite well. To
help analyze the SAR, a surface view for the binding mode of
12e was presented in Figure 4B, which clearly showed areas on
the inhibitor that could accommodate/tolerate substitutions.
On the basis of these observations, it appeared that there was
not enough space around the urea NH of the aniline side to
accommodate a large substitution. Therefore, a small alkyl
group such as a methyl (8a, Table 3) was tolerated and
exhibited moderate ROCK inhibition, while a large substitution
like those in 8b to 8e significantly deteriorated the ROCK
affinity. Indeed, a large substitution at this position totally
changed the binding motif as shown by that of 8c in Figure 4D.
The benzyl phenyl moiety in this inhibitor did not bind toward
the P-loop, thus losing important hydrophobic interactions
which were critical for effective ROCK inhibition. These modes
Figure 2. Substitution effects on both the middle phenyl ring and the
urea NH of the benzylamine side. Simultaneous substitution by
tertiary containing groups were not tolerated as demonstrated by
compound 19c.
hydroxyethyl substitution on both the middle phenyl ring and
the urea NH (Figure 2). Although large, linear tertiary amine-
containing substitutions on the urea NH were also well
tolerated as demonstrated by compounds in Table 4,
simultaneous bis-substitution of these two positions with two
large groups (each containing a tertiary amine) was found to
lessen ROCK inhibition. For example, compound 19c in Figure
F
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Figure 4. Docking modes of representative ROCK inhibitors bound to the ATP binding pocket of a homology model of human ROCK-II. A: 12e
with key H-bond interactions; B: the surface view for the mode of 12e; C: the binding motif 14a in ROCK-II which is very similar to that of 12e; D:
the binding motif 8c in ROCK-II, which shows that the benzyl aromatic moiety is not interacted to the P-loop, thus resulting low ROCK potency.
could also explain the deteriorated ROCK activity of 5c, 5d, 5h,
5i (Table 1), and 5k to 5q (Table 2) as compared to that of 5b
and 5j. Basically, the non-benzylamine structures (in Table 1)
or the disubstituted benzylamine structures (in Table 2) had
disturbed the optimal hydrophobic interactions between the
benzyl phenyl moiety and the hydrophobic pocket under the P-
loop. Finally, the surface view shown in Figure 4B clearly
demonstrated that large substitutions could be well tolerated on
the α-position of the benzyl moiety, on the urea NH of the
benzylamine side, and on the 2′-position of the middle phenyl
ring.
To evaluate the general selectivity of these urea-based ROCK
inhibitors, some selected compounds were subjected to
additional counter screening against ROCK-I, MRCKα,
JNK3, and p38α in addition to PKA. These results are outlined
in Table 6, and they demonstrate that these compounds had
activity against MRCKα, the most closely related kinase to
ROCK in the micromolar range and had no inhibition on JNK3
and p38α at concentrations >20 μM. Screening against ROCK-
I showed that they were typically pan-ROCK inhibitors with
potent ROCK-I inhibition. Inhibition of four selected purified
cytochrome P450 isoforms (1A2, 2C9, 2D6, and 3A4) was
tested at 10 μM and also demonstrated that these urea-based
ROCK inhibitors had generally low inhibition for CYP1A2 and
the most important CYP3A4 isoform. Given the low nanomolar
ROCK potency of these compounds the relatively high %
inhibition for some compounds seen at 10 μM for CYP2C9
and CYP2D6 was not viewed as overly concerning but will be
the focus of further optimizations in this class.
Table 6. Inhibition over Chosen Kinases and P450 Enzymes
a
a
JNK
p38α
1A2/2C9/2D6/3
A4% inhib at
10 μM
a
a
MRCKα
compd IC50 (nM)
IC50
(μM)
IC50
(μM)
ROCK-I
IC50 (nM)
b
12a
12e
12h
12i
1853
4306
8695
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
4
9/96/45/27
b
6
−23/95/38/37
−29/35/85/34
b
6
c
n.d
13
58
14
33
11
10
6
0/24/88/34
−37/68/82/51
18/56/87/61
9/93/29/15
3/67/37/37
−2/71/41/23
−1/16/59/10
9/65/83/53
−21/5/51/8
c
12k
12m
12o
14a
14b
14e
19a
20
n.d
c
n.d
c
c
c
n.d
n.d
n.d
c
c
c
n.d
n.d
n.d
6400
>20
>20
c
c
c
n.d
n.d
n.d
1550
>20
>20
4
c
c
c
n.d
n.d
n.d
10
a
Data were averaged from two or more measurements with errors
b
within 30% of the average. IC50s (μM) of 1A2/2C9/3D6/3A4:
>100/0.6/>100/>100 for 12a; >100/2/40/35 for 12e; >100/30/3/80
for 12h. Not determined.
c
Several inhibitors were also evaluated in rats in vivo to
determine pharmacokinetic properties (Table 7). In summary,
ROCK inhibitors with a simple alkyl substitution or a
substitution containing a hydroxyl group normally exhibited
low clearance and low volume of distribution, high AUC and
C
max, and good oral bioavailability (12a, 12b, 12o, and 14b).
On the other hand, compounds containing an amino moiety
generally had high clearance and high volume of distribution,
low AUC and Cmax, and zero to very low oral bioavailability
G
dx.doi.org/10.1021/jm400062r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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a
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 eluents. The preparative HPLC was performed on
SunFire C18 OBD 10 μm (30 × 250 mm) with CH3CN + 50%
MeOH/H2O + 0.1% TFA as eluents 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 eluents, 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
spectra (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.
General Synthetic Procedures. A primary amine or alcohol (0.2
mmol) was added to a solution of 1-bromo-4-isocyanatobenzene (2,
0.2 mmol) in DCM (1 mL). The mixture was stirred at room
temperature until 2 was completely conversed. Then the solvent was
removed in vacuo to obtain the crude urea or carbamate intermediate
4. In an alternative route, 4-bromoaniline (3, 0.2 mmol) was added to
a mixture of 3-phenylpropanoic acid (0.2 mmol), HATU (0.2 mmol),
and DIEA (0.6 mmol) in DMF. The mixture was stirred at room
temperature until 3 was completely conversed. Then the reaction was
quenched by saturated NaHCO3 and extracted with ethyl acetate (3 ×
5 mL). The combined organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo to give the crude carboxamide
intermediate 4.
Table 7. In Vivo PK Data in Rats
T1/2
C
%
b
b
b
b
mabx
c
Cl (iv)
Vd (iv)
(iv)
AUC (iv)
(iv)
F,
compd (mL/min/kg) (L/kg)
(h)
(μM·h)
(μM)
po
12a
12e
12h
12i
9.4
1.1
1.0
1.0
1.6
1.0
1.0
2.2
1.0
1.9
0.7
2.1
2.4
1.0
5.9
4.2
0.2
0.6
0.6
0.4
3.6
7.9
9.5
0.5
1.3
2.6
3.8
48
51
10
0
11.2
176
14
0.33
0.48
1.2
71
63
86
31
4.4
3.5
4.3
1.9
0.25
0.7
12k
12m
12o
14a
14b
14e
19a
4
0.53
1.1
0
46
4
5.9
4.9
91
30
12
9.2
28
0
20
2.6
0.2
1.1
11
a
Data reported were the mean of three determinations, and the
standard error is within 40% of the mean. iv, 1 mg/kg. po, 2 mg/kg.
b
c
(12h, 12i, 12k, 12m, and 14a). The low oral bioavailability
property of the amino group containing ROCK inhibitors was
probably due to the protonation of the amino group under the
physiological environment.
CONCLUSION
■
The detailed SAR studies (presented here and in our previous
communication22) demonstrated that urea structures derived
from anilines and benzylamines are very good for ROCK
inhibition. More importantly, this urea scaffold can provide
multipoint diversification which could be applied to manipulate
the inhibitor’s pharmaceutical properties such as selectivity,
solubility, and in vitro and in vivo DMPK properties. Through
our SAR studies, good ROCK inhibitors with various DMPK
properties suitable for different applications were discovered.
Basically, these lead compounds can be grouped into two
classes. One class of compounds are those ROCK inhibitors
that possess good in vivo PK properties (systemic) such as
compounds 12a, 12e, 12o, and 14b in Table 7. A common
feature of these compounds is that they do not have a tertiary
amino moiety on their side chains. These compounds could
serve as good candidates for systemic applications such as for
the treatment of MS, cardiovascular diseases, ED, pain, etc. The
second class of compounds are those ROCK inhibitors that
have poor (systemic) PK properties such as compounds 12h,
12i, 12k, 12m,14a, 14e, and 19a in Table 7 (and 20). These
compounds all possess a tertiary amine group on their side
chains and generally have good aqueous solubility. This class of
compounds is good for topical applications such as in the
treatment of glaucoma, ED, and pulmonary hypertension, etc.
In our previous communication, the optimization focused
more on substitutions to the central phenyl ring.22 These
compounds generally exhibited poor systemic PK properties
mainly due to destruction to the symmetry of the central
phenyl ring.33 Therefore, they are basically more suitable for
topical applications. In the full SAR studies described in this
manuscript, potent ROCK inhibitors possessing good in vivo
systemic PK properties have been discovered, and they are
mainly derived from those compounds without a large
substitution on their central phenyl ring.
1-(Isocyanomethyl)-3-methoxybenzene (0.2 mmol) was added to a
solution of N-substituted 4-bromoaniline 6 (0.2 mmol) in DCM (1
mL). The mixture was stirred at room temperature until 6 was totally
converted. Then the solvent was removed in vacuo to obtain the crude
bromide intermediate 7.
To a solution of 3-substituted aldehyde (9, 10 mmol) in methanol
(20 mL) was added a primary amine (10 mmol). After being stirred at
room temperature for 15 min, the solution was cooled to 0 °C prior to
the addition of sodium borohydride (5 mmol) portionwise. The
resulting solution was stirred at room temperature for 1 h. After the
addition of water (3 mL), methanol was removed under reduced
pressure and the resulting aqueous phase was extracted with EtOAc (3
× 15 mL). The combined extracts were dried over anhydrous Na2SO4
and concentrated in vacuo to give the secondary benzylamine. Then
secondary benzylamine (0.2 mmol) was then added to a mixture of 1-
bromo-4-isocyanatobenzene (2, 0.2 mmol) in DCM at 0 °C. After
being stirred at room temperature for 0.5−12 h, the mixture was
extracted with DCM (3 × 5 mL), dried over anhydrous Na2SO4, and
concentrated in vacuo to give the crude bromide intermediate 11
which was used in the next step without further purification.
α-Substituted benzylamine (0.2 mmol) was added to a solution of
1-bromo-4-isocyanatobenzene (2, 0.2 mmol) in DCM (1 mL). The
mixture was stirred at room temperature until 2 was completely
conversed. Then the solvent was removed in vacuo to obtain the crude
intermediate 13.
To a mixture of 4-bromo-2-fluoro-nitrobenzene (15, 1 mmol) and
Cs2CO3 (3 mmol) in DMF (5 mL) was added N, N-
dimethylaminoethanol or methanol (1.05 mmol). After the complete
conversion of the starting material detected by TLC, the mixture was
quenched by water (2 mL) and extracted with EtOAc (3 × 5 mL). The
combined organic extracts were washed with saturated brine, dried
over anhydrous Na2SO4, concentrated under reduced pressure, and
purified through silica gel to give 16. To a solution of 16 (1 mmol) in
EtOAc (5 mL) was added tin chloride dihydrate (3 mmol). The
stirring was continued at room temperature until the complete
disappearance of 16 as detected by LC/MS, and the mixture was then
EXPERIMENTAL SECTION
■
Commercially available reagents and anhydrous solvents were used
without further purification unless otherwise specified. Thin layer
chromatography (TLC) analyses were performed with precoated silica
H
dx.doi.org/10.1021/jm400062r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
quenched by water (2 mL) and extracted with EtOAc (3 × 5 mL). The
combined organic extracts were washed with saturated brine, dried
over anhydrous Na2SO4, concentrated under reduced pressure, and
purified through silica gel to give aniline 17. Triphosgene (0.07 mmol)
was added to a mixture of 17 (0.2 mmol) and saturated NaHCO3 (0.5
mL) in DCM (1 mL) at 0 °C to produce the isocyano intermediate.
Then N1-(3-methoxybenzyl)-N2,N2-dimethylethane-1,2-diamine (0.2
mmol) in DCM (1 mL) was added to the mixture of isocyano
intermediate at 0 °C. After being stirred at room temperature for 0.5 h,
the mixture was extracted with DCM (3 × 5 mL), dried over
anhydrous Na2SO4, and concentrated in vacuo to give the crude
bromide intermediate 18 which was used in the next step without
further purification.
Finally, the 1H-pyrazoleboronic acid pinacol ester (0.3 mmol) and
the crude intermediates 4, 7, 11, 13, and 18 (0.2 mmol) were
dissolved in degassed 5:1 dioxane/H2O. Pd(PPh3)4 (0.02 mmol) and a
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 being cooled
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 subjected to preparative HPLC to give the target products 5,
8, 12, 14, 19, and 20 as a white powder.
H+) 308; HRMS (ESI-Orbitrap) calcd for C18H18N3O [M + H+]:
308.1399, found 308.1405.
N-(4-(1H-Pyrazol-4-yl)phenyl)-3-phenylpropanamide (5g). 75%
1
yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 12.86 (s, br,
1H), 9.88 (s, 1H), 8.10 (, 1H), 7.85 (s, 1H), 7.57−7.50 (m, 4H),
7.31−7.24 (m, 4H), 7.20−7.16 (m, 1H), 2.91 (t, J = 7.6 Hz, 2H), 2.62
(t, J = 7.6 Hz, 2H)); 13C NMR (DMSO-d6, 100 MHz) δ 170.16,
141.18, 137.14, 135.93, 135.67, 128.28, 128.21, 127.80, 125.90, 125.31,
120.92, 119.40, 37.92, 30.82; LC/MS (M + H+) 292; HRMS (ESI-
Orbitrap) calcd for C18H18N3O [M + H+]: 292.1450, found 292.1451.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(cyclohexylmethyl)urea (5i). 72%
1
yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.35 (s, 1H),
7.94 (s, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 6.14−
6.12 (m, 1H), 2.95−2.92 (m, 2H), 1.70−1.62 (m, 5H), 1.38−1.37 (m,
1H), 1.25−1.12 (m, 3H), 0.94−0.86 (m, 2H); 13C NMR (DMSO-d6,
100 MHz) δ 155.22, 138.60, 130.04, 125.67, 125.40, 121.15, 117.83,
111.63, 45.25, 37.96, 30.31, 26.06, 25.39; LC/MS (M + H+) 299;
HRMS (ESI-Orbitrap) calcd for C19H20FN4O2 [M + H+]: 299.1872,
found 299.1877.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-methoxybenzyl)urea (5j). 86%
1
yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.52 (s, 1H),
7.94 (s, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.25
(m, 1H), 6.87 (m, 2H), 6.80 (m, 1H), 6.57 (t, J = 5.6 Hz, 1H), 4.27 (d,
J = 5.6 Hz, 2H), 3.74 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ
159.30, 155.20, 142.00, 138.43, 137.56, 130.15, 129.34, 125.91, 125.42,
121.13, 119.22, 118.06, 112.76, 111.99, 54.95, 42.67; LC/MS (M +
H+): 323; HRMS (ESI-Orbitrap) calcd for C18H19N4O2 [M + H+]:
323.1508, found 323.1496.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-phenylurea (5a). 56% yield in two
1
steps. H NMR (DMSO-d6, 400 MHz) δ 12.85 (s, br, 1H), 8.64 (s,
1H), 8.09 (s, 1H), 7.85 (s, 1H), 7.52−7.42 (m, 6H), 7.29−7.25 (m,
2H), 6.98−6.94 (m, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 152.51,
139.73, 137.63, 135.96, 128.74, 126.65, 125.50, 124.86, 121.72, 121.03,
118.55, 118.13; LC/MS (M + H+) 279. HRMS (ESI-Orbitrap) calcd
for C16H15N4O [M + H+]: 279.1246, found 279.1244.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(2,3-dimethoxybenzyl)urea (5k).
1
75% yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.53 (s,
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-benzylurea (5b). 75% yield in two
steps. H NMR (DMSO-d6, 400 MHz) δ 12.81 (br, s, 1H), 8.52 (s,
1H), 7.94 (s, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H),
7.05−7.01 (m, 1H), 6.97−6.95 (m, 1H), 6.88−6.86 (m, 1H), 6.43 (t, J
= 6.0 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 3.80 (s, 3H), 3.78 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ 155.08, 154.37, 152.24, 146.23, 138.46,
133.42, 130.13, 125.84, 125.43, 123.80, 121.15, 120.16, 117.96, 111.73,
60.04, 55.65, 37.78; LC/MS (M + H+): 353. HRMS (ESI-Orbitrap)
calcd for C18H19N4O [M + H+]: 353.1614, found 353.1620.
1
1H), 7.93 (s, 2H), 7.45 (dd, J = 6.8, 2.0 Hz, 2H), 7.38 (dd, J = 6.8, 2.0
Hz, 2H), 7.36−7.30 (m, 4H), 7.26−7.22 (m, 1H), 6.58 (t, J = 5.6 Hz,
1H), 4.30 (d, J = 5.6 Hz, 2H); 13C NMR (DMSO-d6, 100 MHz) δ
158.67, 155.19, 150.36, 140.35, 138.40, 128.27, 127.08, 126.67, 125.95,
125.41, 121.10, 118.07, 42.71; LC/MS (M + H+): 293; HRMS (ESI-
Orbitrap) calcd for C17H17N4O: 293.1402 [M + H+], found 293.1393.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-phenethylurea (5c). 78% yield in
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(2,4-dimethoxybenzyl)urea (5l).
1
77% yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.51 (s,
1
two steps. H NMR (DMSO-d6, 400 MHz) δ 8.45 (s, 1H), 7.93 (s,
1H), 7.93 (s, 2H), 7.44 (dd, J = 6.8, 2.0 Hz, 2H), 7.35 (dd, J = 6.8, 2.0
Hz, 2H), 7.12 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.48 (dd, J
= 8.4, 2.4 Hz, 1H), 6.31 (t, J = 5.6 Hz, 1H), 4.16 (d, J = 5.6 Hz, 2H),
3.81 (s, 3H), 3.74 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 159.72,
158.33, 157.81, 155.07, 138.52, 130.05, 129.02, 125.78, 125.43, 121.16,
119.82, 117.87, 104.22, 98.25, 55.39, 55.16, 37.84; LC/MS (M + H+):
353. HRMS (ESI-Orbitrap) calcd for C18H19N4O [M + H+]:
353.1614, found 353.1609.
2H), 7.44 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.0
Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.21−7.20 (m, 1H), 6.08 (t, J = 5.6
Hz, 1H), 3.35 (dt, J = 6.8, 5.6 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 155.13, 139.54, 138.52, 130.15, 129.86,
128.65, 128.33, 126.04, 125.81, 125.42, 121.17, 117.97, 40.60, 35.86;
LC/MS (M + H+): 307. HRMS (ESI-Orbitrap) calcd for C18H19N4O:
307.1559 [M + H+], found 307.1566.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-phenylpropyl)urea (5d). 78%
yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.39 (s, 1H),
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(2,5-dimethoxybenzyl)urea (5m).
69% yield in two steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.81 (s, br,
1H), 8.57 (s, 1H), 8.05 (s, 1H), 7.82 (s, 1H), 7.44 (dd, J = 6.8, 2.0 Hz,
2H), 7.36 (dd, J = 6.8, 2.0 Hz, 2H), 6.92−6.90 (m, 1H), 6.82−6.78
(m, 2H), 6.41 (t, J = 6.0 Hz, 1H), 4.22 (d, J = 6.0 Hz, 2H), 3.77 (s,
3H), 3.68 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 155.13, 153.03,
150.84, 138.43, 128.89, 127.73, 125.92, 125.43, 121.15, 117.98, 114.64,
111.61, 111.35, 99.49, 55.74, 55.32, 38.16; LC/MS (M + H+): 353.
HRMS (ESI-Orbitrap) calcd for C18H19N4O [M + H+]: 353.1614,
found 353.1610.
1
7.93 (s, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.31−
7.2 (m, 2H), 7.23−7.16 (m, 3H), 6.17 (t, J = 5.6 Hz, 1H), 3.12−3.07
(m, 2H), 2.63−2.59 (m, 2H), 1.77−1.70 (m, 2H); 13C NMR (DMSO-
d6, 100 MHz) δ 156.20, 142.66, 139.54, 131.47, 130.89, 129.24,
129.23, 126.76, 126.67, 126.38, 122.19, 118.96, 39.63, 33.45, 32.54;
LC/MS (M + H+): 321. HRMS (ESI-Orbitrap) calcd for C18H19N4O:
321.1715 [M + H+], found 321.1722.
Benzyl 4-(1H-pyrazol-4-yl)phenylcarbamate (5e). 67% yield in
1
two steps. H NMR (DMSO-d6, 400 MHz) δ 12.85 (s, br, 1H), 9.72
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(2,6-dimethoxybenzyl)urea (5n).
1
(s, 1H), 8.08 (s, 1H), 7.84 (s, 1H), 7.52−7.50 (m, 2H), 7.45−7.32 (m,
7H), 5.15 (s, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 153.32,
136.92, 136.64, 135.86, 128.42, 128.05, 127.99, 127.29, 125.45, 124.82,
120.89, 118.54, 65.66; LC/MS (M + H+) 294. HRMS (ESI-Orbitrap)
calcd for C18H18N3O [M + H+]: 294.1243, found 294.1238.
Phenethyl 4-(1H-pyrazol-4-yl)phenylcarbamate (5f). 61% yield in
two steps. 1H NMR (DMSO-d6, 400 MHz) δ 9.58 (s, br, 1H), 7.97 (s,
1H), 7.65−7.60 (m, 2H), 7.58−7.57 (m, 1H), 7.56−7.54 (m, 2H),
7.51−7.42 (m, 1H), 7.35−7.30 (m, 3H), 7.26−7.22 (m, 1H), 4.31 (t, J
= 6.8 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H); 13C NMR (DMSO-d6, 100
MHz) δ 153.43, 138.08, 137.00, 132.15, 131.50, 128.83, 128.78,
128.67, 128.35, 126.31, 125.42, 118.59, 64.63, 34.72; LC/MS (M +
67% yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.44 (s,
1H), 7.93 (s, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H),
7.26 (t, J = 8.4 Hz, 1H), 6.67 (d, J = 8.4 Hz, 2H), 6.01 (t, J = 5.2 Hz,
1H), 4.29 (d, J = 5.2 Hz, 2H), 3.81 (s, 6H); 13C NMR (DMSO-d6, 100
MHz) δ 158.10. 154.83, 138.58, 130.15, 129.87, 128.96, 125.70,
125.44, 121.20, 117.74, 114.23, 104.04, 55.81, 31.84; LC/MS (M +
H+): 353. HRMS (ESI-Orbitrap) calcd for C18H19N4O [M + H+]:
353.1614, found 353.1608.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3,4-dimethoxybenzyl)urea (5o).
1
76% yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.48 (s,
1H), 7.94 (s, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H),
6.92−6.90 (m, 2H), 6.84−6.82 (m, 1H), 6.49 (t, J = 5.6 Hz, 1H), 4.21
I
dx.doi.org/10.1021/jm400062r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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(d, J = 5.6 Hz, 2H), 3.74 (s, 3H), 3.72 (s, 3H); 13C NMR (DMSO-d6,
100 MHz) δ 155.14, 150.03, 148.64, 147.72, 138.46, 132.68, 130.26,
125.91, 125.42, 121.25, 119.26, 118.05, 111.78, 111.32, 55.55, 55.42,
42.57; LC/MS (M + H+): 353. HRMS (ESI-Orbitrap) calcd for
C18H19N4O [M + H+]: 353.1614, found 353.1608.
(m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 159.14, 158.44, 158.09,
157.06, 142.59, 138.82, 132.04, 129.08, 128.88, 126.39, 120.52, 118.97,
112.46, 111.63, 54.87, 54.29, 45.80, 43.56, 42.11, 23.18; LC/MS (M +
H+): 408; HRMS (ESI-Orbitrap) calcd for C23H30N5O2 [M + H+]:
408.2400, found 408.2409.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3,5-dimethoxybenzyl)urea (5p).
79% yield in two steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.82 (s, br,
1H), 8.53 (s, 1H), 7.96−7.94 (m, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.38
(d, J = 8.8 Hz, 2H), 6.56 (t, J = 5.6 Hz, 1H), 6.47−6.46 (m, 2H), 6.37
(s, 1H), 4.22 (d, J = 5.6 Hz, 2H), 3.72 (s, 6H); 13C NMR (DMSO-d6,
100 MHz) δ 160.47, 155.19, 142.80, 138.42, 134.32, 130.29, 125.96,
125.42, 118.07, 105.02, 98.77, 98.33, 55.08, 42.79; LC/MS (M + H+):
353; HRMS (ESI-Orbitrap) calcd for C18H19N4O [M + H+]:
353.1614, found 353.1611.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-fluoro-4-methoxybenzyl)urea
(5q). 76% yield in two steps. 1H NMR (DMSO-d6, 400 MHz) δ 12.82
(s, br. 1H), 8.53 (s, 1H), 8.06 (s, 1H), 7.82 (s, 1H), 7.45 (dd, J = 6.8,
2.0 Hz, 2H), 7.37 (dd, J = 6.8, 2.0 Hz, 2H), 7.15−7.06 (m, 3H), 6.57
(t, J = 6.0 Hz, 1H), 4.22 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz) δ 155.17, 152.50, 150.07, 145.87, 138.37,
133.52, 125.98, 125.41, 123.24, 121.11, 118.11, 114.79, 114.61, 113.71,
55.99, 41.84; LC/MS (M + H+): 341; HRMS (ESI-Orbitrap) calcd for
C18H19N4O [M + H+]: 341.1414, found 341.1412.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-methylurea
1
(12a). 69% yield in three steps. H NMR (DMSO-d6, 400 MHz) δ
8.38 (s, 1H), 7.96 (s, 2H), 7.48−7.47 (m, 4H), 7.29−7.25 (m, 1H),
6.84−6.82 (m, 3H), 4.52 (s, 2H), 3.74 (s, 3H), 2.92 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz) δ 159.40, 155.63, 140.11, 138.49, 129.55,
126.58, 124.99, 121.10, 120.20, 119.36, 113.01, 112.15, 54.93, 51.18,
34.36; LC/MS (M + H+) 337; HRMS (ESI-Orbitrap) calcd for
C19H21N4O2 [M + H+]: 337.1665, found 337.1653.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-ethyl-1-(3-methoxybenzyl)urea
1
(12b). 72% yield in three steps. H NMR (DMSO-d6, 400 MHz) δ
12.82 (br, s, 1H), 8.33 (s, 1H), 8.08 (s, 1H), 7.84 (s, 1H), 7.47−7.46
(m, 4H), 7.28−7.24 (m, 1H), 6.86−6.81 (m, 3H), 4.54 (s, 2H), 3.73
(s, 3H), 3.35 (q, J = 3.2 Hz, 2H), 1.07 (t, J = 3.2 Hz, 3H); 13C NMR
(DMSO-d6, 100 MHz) δ 159.35, 155.01, 140.66, 138.49, 135.80,
129.46, 126.57, 124.96, 124.56, 121.11, 120.34, 119.33, 113.00, 112.05,
54.93, 48.66, 40.80, 13.36; LC/MS (M + H+) 351.06; HRMS (ESI-
Orbitrap) calcd for C20H23N4O2 [M + H+]: 351.1821, found 351.1809.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-cyclopropyl-1-(3-
methoxybenzyl)urea (12c). 75% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 8.30 (s, 1H), 7.98 (s, 2H), 7.54−7.48 (m,
4H), 7.27−7.23 (m, 1H), 6.84−6.81 (m, 3H), 4.50 (s, 2H), 3.73 (s,
3H), 2.62−2.58 (m, 1H), 0.89−0.86 (m, 2H), 0.76−0.73 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 159.32, 158.45, 156.04, 140.82, 138.01,
130.26, 129.46, 126.78, 125.08, 121.11, 120.06, 119.28, 112.96, 111.88,
54.90, 49.59, 28.20, 8.95; LC/MS (M + H+) 363; HRMS (ESI-
Orbitrap) calcd for C21H23N4O2 [M + H+]: 363.1821, found 363.1808.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-isopropyl-1-(3-methoxybenzyl)-
urea (12d). 70% yield in three steps. 1H NMR (DMSO-d6, 400 MHz)
δ 8.22 (s, 1H), 7.95 (s, 2H), 7.46−7.40 (m, 4H), 7.25−7.21 (m, 1H),
6.87−6.84 (m, 2H), 6.79−6.76 (m, 1H), 4.51 (s, 2H), 4.50−4.49 (m,
1H), 3.72 (s, 3H), 1.09 (s, 6H); 13C NMR (DMSO-d6, 100 MHz) δ
159.20, 158.00, 155.22, 142.28, 138.49, 130.30; 129.22, 126.51, 124.94,
121.09, 120.28, 118.64, 112.32, 111.49, 54.88, 46.42, 43.92, 20.75; LC/
MS (M + H+) 365; HRMS (ESI-Orbitrap) calcd for C21H25N4O2 [M
+ H+]: 365.1978, found 365.1965.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(2-hydroxyethyl)-1-(3-
methoxybenzyl)urea (12e). 68% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz)δ 12.82 (br, s, 1H), 8.67 (s, 1H), 7.95 (br, s,
2H), 7.47 (dd, J = 6.8, 2.0 Hz, 2H), 7.40 (dd, J = 6.8, 2.0 Hz, 2H),
7.28−7.24 (m, 1H), 6.87−6.81 (m, 3H), 5.26 (br, s, 1H), 4.57 (s, 2H),
3.74 (s, 3H), 3.57 (t, J = 5.6 Hz, 2H), 3.38 (t, J = 5.6 Hz, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 159.35, 157.63, 155.81, 157.20, 140.57,
138.44, 129.46, 126.47, 125.13, 121.08, 119.67, 119.41, 113.07, 112.06,
59.90, 54.92, 49.91, 49.00; LC/MS (M + H+) 367; HRMS (ESI-
Orbitrap) calcd for C20H23N4O3 [M + H+]: 367.1770, found 367.1758.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(2-
methoxyethyl)urea (12f). 72% yield in three steps. 1H NMR (DMSO-
d6, 400 MHz) δ 8.41 (s, 1H), 7.96 (s, 2H), 7.47 (d, J = 8.8 Hz, 2H),
7.41 (d, J = 8.8 Hz, 2H), 7.28−7.24 (m, 1H), 6.85−6.82 (m, 3H), 4.58
(s, 2H), 3.73 (s, 3H), 3.47 (s, 3H), 3.28−3.27 (m, 4H); 13C NMR
(DMSO-d6, 100 MHz) δ 159.37, 158.50, 155.45, 140.42, 138.32,
130.68, 129.49, 126.58, 125.08, 121.08, 119.95, 119.25, 112.92, 112.11,
70.80, 58.28, 54.93, 49.88, 46.06; LC/MS (ESI, M + H+) 381; HRMS
calcd for C21H25N4O3 [M + H+]: 381.1927, found 381.1913.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-methoxybenzyl)-1-methylurea
1
(8a). 82% yield in two steps. H NMR (DMSO-d6, 400 MHz) δ 8.06
(s, 2H), 7.62 (d, J = 6.8, 2.0 Hz, 2H), 7.26 (d, J = 6.8, 2.0 Hz, 2H),
7.22−7.18 (m, 1H), 6.82−6.75 (m, 3H), 6.48 (t, J = 5.6 Hz, 1H), 4.18
(d, J = 5.6 Hz, 2H), 3.73 (s, 3H), 3.17 (s, 3H); 13C NMR (DMSO-d6,
100 MHz) δ 159.15, 158.48, 156.80, 142.69, 141.66, 130.77, 130.61,
129.06, 126.98, 125.93, 120.69, 119.09, 112.55, 111.66, 54.86, 43.53,
37.05; LC/MS (M + H+) 337; HRMS (ESI-Orbitrap) calcd for
C19H21N4O2 [M + H+]: 337.1665, found 337.1653.
1-(4-(1H-Pyrazol-4-yl)phenyl)-1-(2-hydroxyethyl)-3-(3-
methoxybenzyl)urea (8b). 28% yield in four steps. 1H NMR (DMSO-
d6, 400 MHz) δ 8.08 (s, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4
Hz, 2H), 7.21−7.17 (m, 1H), 6.80−6.75 (m, 3H), 6.24 (t, J = 6.0 Hz,
1H), 4.16 (d, J = 6.0 Hz, 2H), 3.73 (s, 3H), 3.62 (t, J = 6.0 Hz, 2H),
3.46 (t, J = 6.0 Hz, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 159.42,
159.13, 157.78, 156.83, 143.88, 142.71, 140.20, 131.40, 129.06, 128.61,
126.08, 118.98, 112.35, 111.70, 58.99, 54.85, 51.56, 43.45; LC/MS (M
+ 1) 367; HRMS (ESI-Orbitrap) calcd for C20H23N4O3 [M + H+]:
367.1770, found 367.1758.
1-(4-(1H-Pyrazol-4-yl)phenyl)-1-(2-(dimethylamino)ethyl)-3-(3-
methoxybenzyl)urea (8c). 26% yield in four steps. 1H NMR (DMSO-
d6, 400 MHz) δ 9.33 (br, s, 1H), 8.11 (s, 2H), 7.71 (d, J = 8.4 Hz, 2H),
7.34 (d, J = 8.4 Hz, 2H), 7.36−7.17 (m, 1H), 6.79−6.76 (m, 3H), 6.43
(t, J = 6.0 Hz, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.92 (dd, J = 11.6, 6.4 Hz,
2H), 3.72 (s, 3H), 3.16 (dd, J = 11.6, 6.4 Hz, 2H), 2.84 (s, 3H), 2.83
(s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 159.14, 158.44, 157.68,
142.39, 138.49, 132.40, 129.08, 128.85, 126.50, 119.01, 117.79, 112.51,
111.68; LC/MS (M + H+) 394; HRMS (ESI-Orbitrap) calcd for
C22H28N5O2 [M + H+]: 394.2243, found 394.2240.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-methoxybenzyl)-1-(2-(pyrroli-
1
din-1-yl)ethyl)urea (8d). 39% yield in four steps. H NMR (DMSO-
d6, 400 MHz) δ 9.46 (br, s, 1H), 8.11 (s, 2H), 7.72 (d, J = 8.4 Hz, 2H),
7.35 (d, J = 8.4 Hz, 2H), 7.22−7.18 (m, 1H), 6.80−6.76 (m, 3H), 6.44
(t, J = 6.0 Hz, 1H), 4.17 (d, J = 6.0 Hz, 2H), 3.92 (dd, J = 12.4, 6.4 Hz,
2H), 3.72 (s, 3H), 3.68−3.61 (m, 2H), 3.24 (dd, J = 12.4, 6.4 Hz, 2H),
3.08−3.01 (m, 2H), 2.02−1.97 (m, 2H), 1.87−1.84 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 159.13, 158.30, 157.97, 157.01, 142.42,
138.57, 132.32, 129.08, 128.82, 126.49, 120.46, 119.04, 112.53, 111.67,
54.87, 53.63, 52.47, 45.51, 43.60, 22.52; LC/MS (M + H+) 420;
HRMS (ESI-Orbitrap) calcd for C24H30N5O2 [M + H+]: 420.2400,
found 420.2396.
1-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-(dimethylamino)propyl)-3-(3-
methoxybenzyl)urea (8e). 64% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.40 (s, br, 1H), 8.10 (s, 2H), 7.69 (d, J =
8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.22−7.19 (m, 1H), 6.80−6.75
(m, 3H), 6.35 (t, J = 6.0 Hz, 1H), 4.15 (d, J = 6.0 Hz, 2H), 3.73 (s,
3H), 3.67−3.63 (m, 2H), 3.08−3.02 (m, 2H), 2.74 (s, 6H), 1.80−1.73
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(2-aminoethyl)-1-(3-
methoxybenzyl)urea (12g). The synthesis started from N-Boc-
protected ethyl diamine. After Suzuki reaction, the organic solvent
of the reaction mixture was removed under reduced pressure and
remaining residue was treated with TFA (0.4 mL) in DCM (0.6 mL).
One half hour later the mixture was submitted for the preparative
HPLC to give 12g as a white solid in 57% overall yield (four steps). 1H
NMR (DMSO-d6, 400 MHz) δ 8.54 (s, 1H), 7.97 (s, 1H), 7.74 (br, s,
3H), 7.51−7.46 (m, 4H), 7.33−7.29 (m, 1H), 6.88−6.83 (m, 3H),
4.65 (s, 2H), 3.75 (s, 3H), 3.48 (t, J = 6.0 Hz, 2H), 2.98 (t, J = 6.0 Hz,
2H); 13C NMR (DMSO-d6, 100 MHz) δ 159.46, 158.47, 158.13,
J
dx.doi.org/10.1021/jm400062r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
155.59, 139.64, 138.02, 129.73, 126.99, 124.97, 121.01, 120.64, 119.08,
112.94, 112.28, 54.98, 49.73, 43.90, 37.46; LC/MS (M + H+) 366;
HRMS (ESI-Orbitrap) calcd for C20H24N5O2 [M + H+]: 366.1930,
found 366.1918.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(2-(dimethylamino)ethyl)-1-(3-
methoxybenzyl)urea (12h). 75% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.17 (br, s, 1H), 8.61 (s, 1H), 7.98 (s, 2H),
7.50−7.47 (m, 4H), 7.34−7.30 (m, 1H), 6.89−6.83 (m, 3H), 4.67 (s,
2H), 3.75 (s, 3H), 3.59 (t, J = 6.4 Hz, 2H), 3.26 (t, J = 6.4 Hz, 2H),
2.84 (s, 3H), 2.83 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 159.50,
158.21, 157.90, 155.64, 139.34, 137.86, 129.77, 127.14, 124.98, 120.96,
120.70, 119.03, 112.86, 112.35, 54.99, 54.55, 49.26, 42.71, 41.24; LC/
MS (M + 1) 394; HRMS (ESI-Orbitrap) calcd for C22H28N5O2 [M +
H+]: 394.2243, found 394.2229.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(2-(pyrroli-
din-1-yl)ethyl)urea (12i). 78% yield in three steps. 1H NMR (DMSO-
d6, 400 MHz) δ 9.41 (br, s, 1H), 8.61 (s, 1H), 7.96 (s, 1H), 7.50−7.45
(m, 4H), 7.34−7.30 (m, 1H), 6.89−6.83 (m, 3H), 4.68 (s, 2H), 3.75
(s, 3H), 3.61−3.57 (m, 4H), 3.39−3.31 (m, 2H), 3.09−3.01 (m, 2H),
2.02−2.00 (m, 2H), 1.90−1.84 (m, 2H); 13C NMR (DMSO-d6, 100
MHz) δ 159.46, 158.60, 158.25, 155.36, 139.56, 138.04, 129.70,
126.91, 124.97, 121.06, 120.62, 119.14, 112.94, 112.34, 54.97, 53.31,
51.58, 49.49, 42.36, 22.64; LC/MS (M + 1) 420; HRMS (ESI-
Orbitrap) calcd for C24H30N5O2 [M + H+]: 420.2400, found 420.2394.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(3-(pyrroli-
din-1-yl)propyl)urea (12j). 75% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.53 (s. br, 1H), 8.47 (s, 1H), 7.96 (s, 2H),
7.50−7.44 (m, 4H), 7.32−7.28 (m, 1H), 6.87−6.83 (m, 3H), 4.61 (s,
2H), 3.74 (s, 3H), 3.54−3.50 (m, 2H), 3.37−3.34 (m, 2H), 3.14−3.09
(m, 2H), 3.01−2.93 (m, 2H), 2.01−1.99 (m, 2H), 1.90−1.84 (m, 4H);
13C NMR (DMSO-d6, 100 MHz) δ 159.44, 158.53, 158.18, 155.36,
7.50−7.45 (m, 4H), 7.33−7.29 (m, 1H), 6.88−6.84 (m, 3H), 4.62 (s,
2H), 3.75 (s, 3H), 3.42−3.40 (m, 2H), 3.36−3.33 (m, 2H), 3.05−3.00
(m, 2H), 2.90−2.81 (m, 2H), 1.95−1.87 (m, 2H), 1.83−1.79 (m, 2H),
1.71−1.57 (m, 3H), 1.42−1.34 (m, 1H); 13C NMR (DMSO-d6, 100
MHz) δ 159.44, 158.38, 158.05, 155.39, 139.99, 138.14, 129.66,
126.89, 124.97, 121.04, 120.59, 119.15, 112.94, 112.17, 54.97, 53.51,
52.05, 49.21, 43.40, 22.54, 22.35, 21.21; LC/MS (M + H+): 448;
HRMS (ESI-Orbitrap) calcd for C26H34N5O2 [M + H+]: 448.2713,
found 448.2718.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-benzyl-1-(2-hydroxyethyl)urea
1
(12o). 62% in three steps. H NMR (400 MHz, DMSO-d6) δ 8.69 (s,
1H), 7.96 (s, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H),
7.37 − 7.31 (m, 2H), 7.27 (dd, J = 14.6, 7.1 Hz, 3H), 4.60 (s, 2H),
3.56 (t, J = 5.4 Hz, 3H), 3.37 (t, J = 5.3 Hz, 2H), 2.07 (s, 1H). LC/MS
calcd for C19H20N4O2 (M + H+): 337, found 337.
1-Benzyl-3-(2-fluoro-4-(1H-pyrazol-4-yl)phenyl)-1-(2-
1
hydroxyethyl)urea (12p). 56% in three steps. H NMR (400 MHz,
DMSO-d6) δ 12.91 (s, 1H), 8.86 (s, 1H), 8.17 (s, 1H), 7.92 (s, 1H),
7.72 (t, J = 8.5 Hz, 1H), 7.47 (dd, J = 12.5, 1.9 Hz, 1H), 7.39 − 7.19
(m, 5H), 5.62 (s, 1H), 4.56 (s, 2H), 3.58 (dd, J = 9.0, 4.3 Hz, 2H),
3.41 − 3.26 (m, 6H), 2.08 (d, J = 4.2 Hz, 1H). LC/MS calcd for
C19H19FN4O2 (M + H+): 355, found 355.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-hydroxy-1-(3-
1
methoxyphenyl)propyl)urea (14a). 79% yield in two steps. H NMR
(DMSO-d6, 400 MHz) δ 8.45 (s, 1H), 7.98 (s, 2H), 7.44 (d, J = 8.8
Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.27−7.23 (m, 1H), 6.90−6.87 (m,
2H), 6.81−6.79 (m, 1H), 6.69−6.66 (m, 1H), 4.84−4.79 (m, 2H),
3,75 (s, 3H), 3.46−3.38 (m, 2H), 1.86−1.81 (m, 2H); 13C NMR
(DMSO-d6, 100 MHz) δ 159.23, 154.58, 146.07, 138.42, 129.27,
125.83, 125.52, 125.41, 121.14, 118.56, 118.42, 117.90, 112.13, 111.63,
57.75, 54.92, 50.44, 32.46; LC/MS (M + H+): 367; HRMS (ESI-
Orbitrap) calcd for C23H30N5O2 [M + H+]: 367.1770, found 367.1762.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(1-(3-fluorophenyl)-3-
hydroxypropyl)urea (14b). 76% yield in two steps. 1H NMR (DMSO-
d6, 400 MHz) δ 8.48 (s, 1H), 7.93 (s, 2H), 7.43 (d, J = 8.8 Hz, 2H),
7.40−7.36 (m, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.16−7.11 (m, 2H),
7.07−7.02 (m, 1H), 6.72 (d, J = 7.6 Hz, 1H), 4.89−4.83 (m, 1H),
3.46−3.37 (m, 2H), 1.86−1.81 (m, 2H); LC/MS (M + H+) 355;
HRMS (ESI-Orbitrap) calcd for C19H20FN4O2 [M + H+]: 355.1570,
found 355.1587.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-hydroxy-1-phenylpropyl)urea
(14c). 81% yield in two steps. 1H NMR (DMSO-d6, 400 MHz) δ 8.44
(s, 1H), 7.93 (s, 2H), 7.44−7.42 (m, 2H), 7.35−7.30 (m, 7H), 7.24−
7.20 (m, 1H), 6.67 (d, J = 8.0 Hz, 1H), 4.86−4.81 (m, 1H), 3.46−3.35
(m, 2H), 1.86−1.81 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ
161.25, 158.42, 158.06, 154.55, 144.36, 138.41, 130.10, 128.20, 126.51,
126.18, 125.79, 125.40, 121.12, 117.86, 57.73, 50.44, 31.58; LC/MS
(M + H+) 337; HRMS (ESI-Orbitrap) calcd for C19H21N4O2 [M +
H+]: 337.1665, found 337.1660.
140.06, 138.16, 129.64, 126.86, 124.98, 121.04, 120.60, 119.14, 112.17,
111.88, 54.95, 53.18, 51.72, 49.31, 43.38, 24.36, 22.56; LC/MS (M +
1) 434; HRMS (ESI-Orbitrap) calcd for C25H32N5O2 [M + H+]:
434.2556, found 434.2559.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(2-
morpholinoethyl)urea (12k). 73% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.59 (br, s, 1H), 8.64 (s, 1H), 7.97 (s, 2H),
7.51−7.45 (m, 4H), 7.34−7.30 (m, 1H), 6.89−6.83 (m, 3H), 4.68 (s,
2H), 4.02−3.97 (m, 2H), 3.75 (s, 3H), 3.66−3.60 (m, 4H), 3.57−3.52
(m, 2H), 3.33−3.30 (m, 2H), 3.13−3.11 (m, 2H); 13C NMR (DMSO-
d6, 100 MHz) δ 159.51, 158.35, 158.02, 155.63, 139.29, 137.83,
129.78, 127.14, 124.99, 120.97, 120.71, 119.02, 112.85, 112.35, 63.33,
55.00, 53.77, 51.50, 49.38, 40.58; LC/MS (M + H+) 436; HRMS (ESI-
Orbitrap) calcd for C24H30N5O3 [M + H+]: 436.2349, found 436.2333.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(3-
morpholinopropyl)urea (12l). 64% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.62 (s, br, 1H), 8.49 (s, 1H), 7.96 (s, 2H),
7.50−7.44 (m, 4H), 7.32−7.28 (m, 1H), 6.88−6.83 (m, 3H), 4.62 (s,
2H), 3.99−3.96 (m, 2H), 3.74 (s, 3H), 3.66−3.60 (m, 2H), 3.43−3.40
(m, 2H), 3.37−3.33 (m, 2H), 3.10−3.04 (m, 4H), 1.95−1.90 (m, 2H);
13C NMR (DMSO-d6, 100 MHz) δ 159.45, 158.32, 157.99, 155.38,
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(2-hydroxy-1-phenylethyl)urea
1
(14d). 75% yield in two steps. H NMR (DMSO-d6, 400 MHz) δ
12.81 (s, br, 1H), 8.62 (s, 1H), 7.83−7.78 (m, 2H), 7.45−7.43 (m,
2H), 7.35−7.32 (m, 6H), 7.25−7.22 (m, 1H), 6.67 (d, J = 7.6 Hz,
1H), 4.97 (s, br, 1H), 4.77−4.72 (m, 1H), 3.70−3.59 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 154.71, 142.12, 138.40, 130.10, 128.03,
126.68, 126.61, 125.87, 125.43, 121.09, 117.79, 113.79, 64.94, 55.00;
LC/MS (M + H+) 323; HRMS (ESI-Orbitrap) calcd for C18H19N4O2
[M + H+]: 323.1508, found 323.1500.
139.93, 138.11, 129.68, 126.91, 124.97, 121.01, 120.59, 119.10, 112.91,
112.17, 66.31, 63.36, 54.98, 53.74, 51.11, 49.27, 22.12; LC/MS (M +
H+) 450; HRMS (ESI-Orbitrap) calcd for C25H32N5O3 [M + H+]:
450.2505, found 450.2509.
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(2-(piperi-
din-1-yl)ethyl)urea (12m). 70% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.00 (s, br, 1H), 8.63 (s, 1H), 7.97 (s, 2H),
7.51−7.45 (m, 4H), 7.34−7.30 (m, 1H), 6.89−6.83 (m, 3H), 4.67 (s,
2H), 3.75 (s, 3H), 3.62−3.59 (m, 2H), 3.53−3.50 (m, 2H), 3.25−3.21
(m, 2H), 2.95−2.87 (m, 2H), 1.83−1.80 (m, 2H), 1.70−1.56 (m, 3H),
1.41−1.35 (m, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 159.51,
158.22, 157.89, 155.63, 139.36, 137.84, 129.79, 127.14, 125.00, 120.96,
120.68, 119.04, 112.87, 112.35, 55.01, 53.60, 52.51, 49.50, 41.00,
22.54, 21.14; LC/MS (M + H+): 434; HRMS (ESI-Orbitrap) calcd for
C25H32N5O2 [M + H+]: 434.2556, found 434.2565.
1-(4-(1H-Pyrazol-4-yl)phenyl)-3-(3-(dimethylamino)-1-
1
phenylpropyl)urea (14e). 69% yield in two steps. H NMR (DMSO-
d6, 400 MHz) δ 9.45 (s, br, 1H), 8.59 (s, 1H), 7.94 (s, 2H), 7.46−7.42
(m, 2H), 7.40−7.35 (m, 6H), 7.32−7.28 (m, 1H), 4.85−4.79 (m, 1H),
3.17−3.01 (m, 2H), 2.79 (s, 6H), 2.14−2.07 (m, 2H); 13C NMR
(DMSO-d6, 100 MHz) δ 158.71, 158.36, 154.71, 142.78, 138.29,
128.54, 127.22, 126.29, 125.97, 125.41, 121.08, 117.94, 54.67, 50.84,
42.49, 31.13; LC/MS (M + H+) 364; HRMS (ESI-Orbitrap) calcd for
C21H26N5O [M + H+]: 364.2137, found 364.2129.
(S)-1-Ethyl-1-(3-methoxybenzyl)-3-(2-(1-methylpyrrolidin-3-
yloxy)-4-(1H-pyrazol-4-yl)-phenyl)urea (19a). 65% yield in four
3-(4-(1H-Pyrazol-4-yl)phenyl)-1-(3-methoxybenzyl)-1-(3-(piperi-
din-1-yl)propyl)urea (12n). 72% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.08 (s, br, 1H), 8.49 (s, 1H), 7.97 (s, 2H),
1
steps. H NMR (DMSO-d6, 400 MHz) δ 10.04 (s, br, 1H), 8.05 (s,
2H), 7.68−7.54 (m, 2H), 7.32−7.28 (m, 1H), 7.19−7.15 (m, 2H),
K
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6.90−6.86 (m, 3H), 5.32−5.22 (m, 1H), 4.58−4.55 (m, 2H), 4.05−
3.98 (m, 1H), 3.76 (s, 3H), 3.43−3.37 (m, 2H), 3.18−3.11 (m, 2H),
2.89−2.85 (m, 3H), 2.59−2.54 (m, 1H), 2.26−2.14 (m, 1H), 2.08−
2.01 (m, 1H, 1.15−1.10 (m, 3H); 13C NMR (DMSO-d6, 100 MHz) δ
159.43, 158.50, 158.17, 154.98, 148.09, 147.01, 140.37, 129.64, 126.86,
122.62, 120.97, 119.02, 117.75, 112.90, 112.04, 109.48, 75.19, 59.70,
54.98, 53.91, 53.16, 49.04, 41.55, 31.08, 13.25; LC/MS (M + H+) 450;
HRMS (ESI-Orbitrap) calcd for C25H32N5O3 [M + H+]:450.2505,
found 450.2513.
3-(2-(2-Hydroxyethoxy)-4-(1H-pyrazol-4-yl)phenyl)-1-(2-hydrox-
yethyl)-1-(3-methoxybenzyl)-urea (19b). 1H NMR (400 MHz,
DMSO-d6) δ 8.46 (d, J = 24.5 Hz, 1H), 8.02 (s, 2H), 7.85 (t, J =
6.0 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.21 (d, J = 1.8 Hz, 1H), 7.12
(dd, J = 8.3, 1.8 Hz, 1H), 6.90 (d, J = 7.3 Hz, 2H), 6.83 (dd, J = 7.3,
1.8 Hz, 1H), 4.53 (s, 2H), 4.07 (t, J = 5.1 Hz, 2H), 3.77 − 3.70 (m,
5H), 3.60 (t, J = 4.9 Hz, 2H), 3.37 (t, J = 4.7 Hz, 2H). LC/MS calcd
for C22H26N4O5 (M + H+): 427, found 427.
blocked with Licor Blocking Buffer (LI-COR Biosciences, diluted 1:1
in PBS) for 1.5 h, and incubated with anti-PP-MLC (Cell Signaling) at
1:200 dilution overnight at 4 °C with rocking. Plates were then washed
twice with PBS/0.05% Tween 20 and once with Licor Blocking
Buffer/PBS/0.05% Tween 20. Secondary antibody incubation was
performed with Goat-anti-Rabbit IR 800 (LI-COR Biosciences) used
at 1:500 dilution in Licor Blocking Buffer/PBS/0.05% Tween 20 for 1
h at RT with rocking, followed by washing twice with PBS/0.05%
Tween 20 and once with Licor Blocking Buffer/PBS/0.05%. Nuclear
staining was performed with TO-PRO-3 iodide (642/661) (Invi-
trogen) diluted at 1:4000 in LI-COR Buffer/PBS for 30 min at RT
with rocking, followed by washing twice with PBS. The ppMLC and
nuclear signal was determined by reading the plate on an Odyssey LI-
COR Infrared Scanner (LI-COR Biosciences at 800 and 700 nm
channels, respectively, and collecting the RAW data. The final
percentage of ppMLC phosphorylation was derived by normalizing
the signal in each well to its corresponding nuclear fluorescence.
Molecular Modeling. The homology model of human Rock2 was
constructed using Prime v3.1 (Schrodinger, LLC, New York, NY) with
the option of a single template to build a single chain. The crystal
structure of Bovine Rock2 (PDB ID 2F2U), which shows sequence
identity over 92%, was used as a template for the model, and the final
model also included the ligand of the template, Fasudil. The model
was further prepared through protein preparation wizard in Maestro
v9.3 (Schrodinger, LLC). Docking studies were performed using Glide
SP v5.8 (Schrodinger, LLC) with one hydrogen bond constraint to
either the carbonyl oxygen of Glu 170 or amino group of Met 172 in
the hinge region. The docking grid was generated around the model
ligand Fasudil with a box size of 20 × 20 × 20 Å3. Each inhibitor
compound was prepared for Glide docking with LigPrep v2.5
(Schrodinger, LLC) to include different conformational states. The
docking pose with the highest docking score for each inhibitor
compound was then merged to the human Rock2 model for energy
minimization using the OPLS2005 force field (Schrodinger, LLC).
3-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)phenyl)-1-(2-
(dimethylamino)ethyl)-1-(3-methoxybenzyl)urea (19c). 57% yield in
1
four steps. H NMR (DMSO-d6, 400 MHz) δ 9.77−9.45 (m, 1H),
8.05 (s, 2H), 7.80 (s, 1H), 7.59−7.57 (m, 1H), 7.40−7.36 (m, 1H),
7.29−7.28 (m, 1H), 7.22−7.19 (m, 1H), 6.94−6.87 (m, 3H), 4.69 (s,
2H), 4.41−4.38 (m, 2H), 3.78 (s, 3H), 3.65−3.62 (m, 2H), 3.36−3.35
(m, 2H), 3.29−3.28 (m, 2H), 2.85 (s, 6H), 2.77 (s, 6H); 13C NMR
(DMSO-d6, 100 MHz) δ 160.52, 159.43, 159.10, 156.68, 150.14,
139.96, 130.95, 130.39, 126.72, 124.92, 121.87, 119.75, 118.64, 113.80,
113.28, 110.17, 63.28, 56.40, 56.03, 55.42, 50.49, 43.65, 43.49, 42.69;
LC/MS (M + H+): 481; HRMS (ESI-Orbitrap) calcd for C26H37N6O3
[M + H+]: 481.2927, found 481.2935.
3-(5-(1H-Pyrazol-4-yl)pyridin-2-yl)-1-(2-(dimethylamino)ethyl)-1-
(3-methoxybenzyl)urea (20). 70% yield in three steps. 1H NMR
(DMSO-d6, 400 MHz) δ 9.17 (br, s, 1H), 8.61 (s, 1H), 7.98 (s, 2H),
7.50−7.47 (m, 4H), 6.89−6.83 (m, 3H), 4.67 (s, 2H), 3.75 (s, 3H),
3.59 (t, J = 6.4 Hz, 2H), 3.26 (t, J = 6.4 Hz, 2H), 2.84 (s, 3H), 2.83 (s,
3H); LC/MS (M + 1) 395; HRMS (ESI-Orbitrap) calcd for
C21H27N6O2 [M + H+]: 395.2195, found 395.2176.
ASSOCIATED CONTENT
* Supporting Information
■
ROCK-I/II Assays.34 Assays were performed using the STK2 kinase
system from Cisbio. A 5 μL mixture of a 1 μM STK2 substrate and
ATP (ROCK-I: 4 μM, ROCK-II: 20 μM) in STK-buffer was added to
the wells of the 384-well plate using a BioRAPTR FRD Workstation
(Aurora Discovery, Carlsbad, CA). Twenty nanoliters of test
compound was then dispensed using a 384-head offline Pintool
system (GNF Systems, San Diego, CA). The reaction was started by
adding either 5 μL of 2.5 nM ROCK-I (Upstate #14-601) or 5 μL of
0.5 nM ROCK-II in STK-buffer. After 4h at RT the reaction was
stopped by adding 10 μL of 1X antibody and 62.5 nM Sa-XL in
Detection Buffer. After 1 h incubation at RT, the plates were read on
the Viewlux in HTRF mode.
S
Details for the synthetic procedure of 6b−f; LC purity data,
protocols for JNKs, MRCKα, and p38α enzyme assays;
procedures for DMPK studies. This material is available free
of charge via the Internet at http://pubs.acs.org/
AUTHOR INFORMATION
Corresponding Author
■
*(Y.F.) E-mail: yfeng@scripps.edu; phone: 561-228-2201; fax:
561-228-3089. (P.L.) E-mail: lograsso@scripps.edu; phone:
561-228-2230; fax: 561-228-3089.
PKA Assay.35 A 5 μL mixture of 60 μM kemptide and 20 μM ATP
in kinase buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 0.1% BSA, 2
mM DTT) was added to the wells using a BioRAPTR FRDTM
Workstation (Aurora Discovery, Carlsbad, CA). Twenty nanoliters of
test compound was then dispensed using a 384-head offline Pintool
system (GNF Systems, San Diego, CA). The reaction was started by
adding 5 μL of 0.5 nM PKA (Upstate #14-440) in kinase buffer (5 μL
of kinase buffer was used for high wells). After 70 min incubation at
RT, the reaction was stopped by adding 10 μL Kinase-Glo reagent and
the plate was read after 10 min incubation at RT on the Viewlux in
luminescence mode.
ppMLC Assay. A7r5 cells obtained from ATCC (Manassas, VA)
were maintained in 20% FBS supplemented DMEM at 5% CO2, 37
°C, in a humid environment and plated in clear-bottomed Packard
View plates ((Perkin-Elmer, Waltham, MA) at 5000 cells/well and
allowed to attach overnight. Serum starvation was performed by media
change to DMEM/0.05% FBS for 4 h. The cells were then treated with
13333 nM to 18 nM final compound concentration along with DMSO
control for 1 h, followed by treatment with 10 μM L-α-
lysophosphatidic acid (LPA, Sigma-Aldrich) for 10 min. Cells were
fixed in 4% paraformaldehyde for 25 min, washed with 0.1 M glycine,
permeabilized with 0.2% Triton-X for 20 min, washed with PBS,
Present Address
#School of Chemical and Environmental Engineering, Shanghai
Institute of Technology, 100 Hai Quan Rd., Shanghai, P.R.
China 201418.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Dr. Derek Duckett and Ms. Weimin Chen
■
for p38α and JNK assays, Dr. Michael Chalmers for HRMS,
1
and Dr. Ke Zheng for the H NMR characterization of 12o,
12p, and 19b. This research was also partially supported by the
Shanghai Municipal Natural Science Foundation
(12ZR1431100, to Y.Y).
ABBREVIATIONS USED
AUC, pharmacokinetic area under curve; Cl, pharmacokinetic
clearance; Cmax, pharmacokinetic maximum concentration; F%,
■
L
dx.doi.org/10.1021/jm400062r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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and selective azaindole-based Rho kinase (ROCK) inhibitors.
ChemMedChem 2008, 3, 1893−1904.
oral bioavailability; HLMS, human liver microsomal stability;
ppMLC, bis-phospho myosin light chain; Vd, volume of
distribution; IOP, intraocular pressure; ROCK, Rho-associated
coiled-coil protein kinase; SAR, structure−activity relationship;
DMPK, drug metabolism and pharmacokinetics; DCM,
dichloromethane; HATU, 2-(1H-7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate methanami-
nium; DMF, N,N-dimethylformamide; HPLC, high pressure
liquid chromatography; DCM, dichloromethane
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Journal of Medicinal Chemistry
Article
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dx.doi.org/10.1021/jm400062r | J. Med. Chem. XXXX, XXX, XXX−XXX