Hit to lead account of the discovery of bisbenzamide and related ureidobenzamide inhibitors of Rho kinase

J. Med. Chem. 2010, 53, 759–777 759

DOI: 10.1021/jm9014263

Hit to Lead Account of the Discovery of Bisbenzamide and Related Ureidobenzamide Inhibitors

of Rho Kinase

‡

†

‡

†

Tina Morwick,*^,†Frank H. Buttner, Charles L. Cywin, Georg Dahmann, Eugene Hickey, Scott Jakes, Paul Kaplita,

Mohammed A. Kashem,^†Steven Kerr,^†Stanley Kugler,^†Wang Mao,^†Daniel Marshall,^†Zofia Paw,^†Cheng-Kon Shih,^†

Frank Wu,^†and Erick Young^†

€

^†Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06801-0368, and ^‡Boehringer Ingelheim Pharma

GmbH and Co. KG, Birkendorfer Strasse 65, D-88397 Biberach an der Riss, Germany

Received September 24, 2009

A highly selective series of bisbenzamide inhibitors of Rho-associated coiled-coil forming protein kinase

(ROCK) and a related ureidobenzamide series, both identified by high throughput screening (HTS), are

described. Details of the hit validation and lead generation process, including structure-activity

relationship (SAR) studies, a selectivity assessment, target-independent profiling (TIP) results, and

an analysis of functional activity using a rat aortic ring assay are discussed.

Introduction

20 members and belongs to the Ras superfamily of monomeric

GTP/GDP-binding (GTP)ases.³Rho A, one of the most well-

characterized members, was originally identified as a homo-

logue to oncogene Ras.⁴RhoA influences multiple cellular

processes including cytoskeletal rearrangement, gene expres-

sion, and membrane trafficking, as well as cell adhesion,

migration, differentiation, proliferation, and apoptosis.⁵The

name Rho sometimes refers collectively to RhoA, RhoB, and

RhoC, which share identical effector domains, and Rho kinases

bind to all three.⁶A member of the AGC family of protein

kinases, ROCK plays a key role in regulation of smooth muscle

contraction and formation of stress fibers and focal adhesions

in nonmuscle cells.⁷Two Rho kinase isoforms have been

identified, ROCK1 and ROCK2, which share 65% overall

homology with 92% identity in their kinase domains and

absolute identity in the ATP-binding pocket.^4bThese proteins

are ubiquitously expressed, but ROCK1 is reportedly more

prominent in lung, liver, and testes, while ROCK2 expression is

enriched in brain and muscle.^4b,8ROCK1 and ROCK2 have

generally been considered to be functionally redundant. How-

ever, sporadic reports have highlighted some differences.⁹For

example, studies with primary rat embryo fibroblasts have

suggested that stress fiber and focal adhesion formation may

be dependent on ROCK1 but not ROCK2. Also, it has been

noted that ROCK1, but not ROCK2, binds and phosphory-

lates RhoE.¹⁰A unique function for ROCK2 in mediating

glucocorticoid-induced formation of tight junction sealing in

rat mammary epithelial cells has also been reported. This

apparently occurs as a consequence of differential expression

and activity of Rho kinase isoforms following treatment with

dexamethasone.¹¹

The evolution from a pharmacology-based to a target-based

approach in drug discovery has presented both opportunities

and challenges to the pharmaceutical industry.¹Utilization of

high-throughput biochemical assays for the identification of

small molecule chemical ligands which interact with distinct

protein targets greatly enhances the ability to identify potential

drug candidates, and knowledge of the specific molecular

target provides information to support mechanism of action.

Significant challenges exist, however, in the identification of

druggable, disease-relevant targets, and the selection of bio-

chemically active lead structures which can be developed into

pharmacologically active agents useful in the treatment of

disease. When high-throughput screening (HTS)^aenables the

target-based lead identification (LI) process, hit-to-lead (HtL)

groups have been utilized.²This core LI group facilitates the

validation of screening hits and selection of promising lead

candidates. In this paper, we describe our HtL efforts to identify

inhibitors of Rho kinase and our discovery of a series of

bisbenzamides and related ureidobenzamides as novel chemo-

types demonstrating potent Rho kinase inhibition.

Rho kinase, also referred to as Rho-associated coiled-coil

containing serine/threonine protein kinase (ROCK) is a down-

stream effector of RhoA G proteins following GTP-binding

and activation. In humans, the Rho family consists of at least

*To whom correspondence should be addressed. Phone: (203) 798-5751;

Fax: 203-798-5297. E-mail: tmorwick@rdg.boehringer-ingelheim.com.

^aAbbreviations: AGC, cAMP-dependent, cGMP-dependent and pro-

tein kinase C; CDC42BPB, CDC42-binding protein kinase β; DMPK,

dystrophia myotonica protein kinase; GDP, guanosine-5⁰-diphosphate;

GTP, guanosine-5⁰-triphosphate; HTS, high-throughput screening; HtL,

hit-to-lead; IMAP, immobilized metal ion affinity-based fluorescence

polarization; ITC, isothermal titration calorimetry; LHS, left-hand side;

LI, lead identification; MBS, myosin binding subunit; MLC, myosin light

chain; MLCK, myosin light chain kinase; MLCP, myosin light chain

phosphatase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase

A; PKN2, PKC-related kinase, protein kinase N2; PKCε, protein kinase C

epsilon; POC, percent of control; RBD, Rho binding domain; RHS, right-

hand side; SCX, strong cation exchanger, silica-bound p-toluenesulfonic

acid; Si-DMA, silica-bound dimethylamine; TF, tissue factor.

A key mechanism for regulation of muscle cell contractility

and nonmuscle cytoskeletal organization through the Rho/

ROCK pathway involves the phosphorylation state of the

molecular motor myosin II on Ser19 of its regulatory light

chain (MLC). Phosphorylation of this residue leads to in-

creased contractility and formation of stress fibers and focal

adhesions. Maintaining Ser19 of MLC in a phosphorylated

state is accomplished by protein kinases such as myosin light

r

2009 American Chemical Society

Published on Web 12/10/2009

pubs.acs.org/jmc

760 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Morwick et al.

Figure 1. Literature benchmark Rho kinase inhibitors with X-ray structures. ROCK1 data reported in refs 25 and 26a. ROCK2 data reported in

refs 25 and 34a.

chain kinase (MLCK) through a Ca^2þ-dependent mechanism

Table 1. Counterscreens Used for Triaging ROCK2 Actives

% ID^a

and inhibition of myosin light chain phosphatase (MLCP)

via a Ca^2þ-independent process. Activated ROCK inhibits

MLCP by phosphorylation of its regulatory myosin binding

subunit (MBS), thereby prolonging the phosphorylated state

of MLC at serine 19. Additionally, ROCK can reportedly

directly phosphorylate MLC.^4a,4b,12

kinase

PKCε

DMPK

39

50

53

34

CDC42BPB

PKN2

^aSequence ID to ROCK2 (kinase domain).

The Rho/ROCK pathway is activated by multiple stimuli

including various GPCR agonists and inflammatory cyto-

kines. ROCK activity is self-regulated by an autoinhibitory

carboxy terminus. The C-terminal region incorporates a

Rho-binding domain and the interaction between RBD and

Rho-GTP reverses autoinhibition and activates the kinase.

This autoinhibitory region also contains a pleckstrin homo-

logy domain, which may have a regulatory role by interaction

with lipid mediators, and lipids such as arachidonic acid have

been found to activate Rho kinase.^4b,13

metabolites required for post-translational protein prenyla-

tion and membrane targeting, an essential mechanism for

activation of RhoA.²³

Several ROCK inhibitors have been reported in the litera-

ture, and many are summarized in a recent review.²⁴Bench-

mark compounds used extensively to investigate the functional

outcome of Rho kinase inhibition include fasudil (HA-1077, 1)

and Y-27632 (2). Fasudil is the only marketed Rho kinase

inhibitor approved in Japan for treatment of cerebral vaso-

spasm after subarachnoid hemorrhage.^24aFasudil undergoes

oxidation in vivo to isoquinolinone 3 (hydroxyfasudil), which is

equipotent,²⁵and a dimethyl analogue (H-1152P, 4) shows

improved potency. X-ray crystal structures of 1-4 in complex

with ROCK1 dimer have been published (Figure 1).²⁶

Multiple potential therapeutic opportunities have been

suggested for Rho kinase inhibitors including cardiovascular

and cerebrovascular conditions such as hypertension, heart

failure, angina, myocardial infarction, atherosclerosis, coro-

nary and cerebral vasospasm and stroke,^{4a,4b,6a,6b,14}neuro-

logical disorders including Alzheimer’s disease, multiple

sclerosis, spinal cord injury, and neuropathic pain,¹⁵osteo-

porosis,¹⁶cancer,¹⁷bronchial asthma,¹⁸and glaucoma.¹⁹Our

interest in Rho kinase was associated with its role in cardio-

vascular function. Both Rho and ROCK are ubiquitously

expressed in vascular cells including smooth muscle, endothe-

lial, cardiac myocytes, platelets, leukocytes and monocytes/

macrophages,^4band inhibition of Rho kinase in animal

models of cardiovascular disease has demonstrated positive

results in a variety of the pathological conditions cited

above.²⁰Additionally, certain endogenous agents or xenobio-

tics associated with positive cardiovascular outcomes may

derive some of their benefit through moderation of Rho/

ROCK signaling. For example, down-regulation of Rho

kinase expression has been associated with physiological

concentrations of estrogen, and Rho/ROCK activity has also

been found to inversely correlate with eNOS expression and

NO availability.^20,21The cardiovascular benefits of HDL also

may result in part from inhibition of thrombin-induced tissue

factor (TF) upregulation via inhibition of RhoA and stimu-

lation of PI3K.²²Statins have demonstrated clinical benefits

in cardiovascular disease beyond their cholesterol-lowering

effect. These so-called pleiotropic or cholesterol-indepen-

dent effects may be mediated by reduction of isoprenoid

Results and Discussion

HTS Assay and Hit Validation Results for Inhibitor 5. The

corporate compound collection was screened in a homoge-

neous luciferase assay using ROCK2 (1-543), substrate

AKRRRLSSLRA, and a luciferin-luciferase detection re-

agent to quantify residual ATP. Dose-response data on

compounds showing reproducible activity from the screen

was generated for ROCK2 using the same assay format.

Selectivity data was acquired simultaneously on the ROCK2

actives utilizing four proteins which were selected for

counterscreening (rule-out) based on sequence homology

(DMPK, CDC42BPB), chemotype sensitivity (PKN2), and

functional significance (DMPK, PKCε, Table 1). Hits of

interest were also evaluated for ROCK1 potency, and our

objective at the time was the identification a dual inhibitor.

Generic criteria for hit validation has been recently pub-

lished.²⁷One series which passed the initial filtering exercise

is exemplified by the bisbenzamide structure 5 (Figure 2).

This hit demonstrated low micromolar potency for both

ROCK1 and ROCK2 in the luciferase assay and comparable

functional activity in our aortic ring assay, which was some-

what surprising assuming an ATP competitive mechanism of

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 761

inhibition. One attraction of 5 was the bisbenzamide motif,

which is unusual in comparison to known kinase inhibitors,

and we speculated that the scaffold might offer some ad-

vantage toward achieving selectivity. Compound 5 was not

active in the counterscreen assays (all >5 μM), but its

relatively weak potency for Rho kinase provided a small

window for selectivity assessment. Evaluation of physico-

chemical properties and in vitro pharmacokinetic and safety

parameters for 5 is shown in Table 2. The inhibitor had good

aqueous solubility but poor metabolic stability, and our

Caco-2 results suggested that permeability may be com-

promised by transporter-mediated efflux. The compound

also showed moderate inhibition of CYPs 2C9 and 3A4. This

assessment identified areas which would need to be ad-

dressed during the lead generation stage.

Lead Generation: SAR Studies. Compound 5 and addi-

tional related actives having similar potency were identified

from the screen. An analogous series of ureidoarylamides

was also found, and some examples from both series are

shown in Figure 3. Because no crystal structure for Rho

kinase was available at the time of our studies, a computa-

tional model was developed based on a crystal structure of

PKA, another member of the AGC subfamily (37% homo-

logy with ROCK2, kinase domain), in complex with the Rho

kinase inhibitor 1 (PDB code: 1Q8W).²⁸Docking studies of 5

and related bisbenzamides were not conclusive, and several

ligands including 5 did not dock using a hinge-binding

interaction as a required constraint. Studies with ligands

for which a binding hypothesis could be identified sug-

gested the ligand may form a hydrogen bond interaction

with hinge region residue Met172 (NH) via the pyridine

nitrogen. Known Rho kinase inhibitors utilize a variety

of residues for hydrogen bonding to the hinge including

pyridines, quinolines, azaindoles, indazoles, amides, and

ureas.^15aHowever, for the bisbenzamides, a search of the

screening results (actives and inactives) suggested that the

dimethoxyphenyl rather than the pyridine was important

for potency. For the urea series, screening data attributed

significance to the cyanophenyl seen in 8 and 9, and while the

pyridine fragment was also incorporated into this hit series,

the data again indicated its presence was trivial. Our initial

SAR studies targeted the bisbenzamide series probing the

relevance of these two terminal sites. For the dimethoxy-

phenyl, removal of either methoxy group, conversion to the

methylenedioxy, or replacement with a dimethyl (10-13)

was generally detrimental, indicating the intact LHS frag-

ment was important for binding (Table 3). Modifications to

the ethylpyridine substituent are shown in Table 4. Conver-

sion to the constrained isoquinoline (14) provided a modest

improvement. To assess whether the pyridine/isoquinoline

nitrogen secured the hydrogen bond donor interaction from

the hinge, the corresponding saturated derivatives with a

basic amine replacement (15, 16) were made. If the pyridine

(or isoquinoline) nitrogen participated in this interaction, the

Table 3. Dimethoxyphenyl Replacements

Figure 2. Results for resynthesized compound 5.

Table 2. Target-Independent Profiling Results for 5

MW

sol^a

Caco-2^b

Caco-2^c

HLM t_1/2(min)

CYP 1A2^d

>30

CYP 2C9^d

16

CYP 2C19^d

>30

CYP 2D6^d

>30

CYP 3A4^d,e

4/21

419

>100/63

0.5

29.1

10

c

^aSolubility (μg/mL) @ pH 4.5/7.4. ^bA to B ꢀ 10^-6cm/s. B to A ꢀ 10^-6cm/s. ^dμM. ^eSubstrates 7-benzyloxy-4-trifluoromethylcoumarine/

7-benzyloxyquinoline (BFC/BQ). (See Supporting Information for method details.)

Figure 3. Examples of bisbenzamide and ureiobenzamide actives from the screen. Compounds 7 and 9 are racemic.

762 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Table 4. RHS Optimization

Morwick et al.

Figure 4. A partial ribbon diagram shows the binding mode for

bisbenzamide 16. Key residues (backbone and/or side chain) are

shown in space-filing mode and colored by element (blue = nitro-

gen, red = oxygen, yellow = sulfur). Compound 16 uses the

˚

aromatic p-methoxy oxygen for hinge binding to Met172 (3.0 A),

while the tetrahydroisoquinoline forms an interaction with the side

˚

chain acid of Asp385 (2.6 A). Side chain fragments for a number of

hydrophobic residues in the hinge-binding region (Met169, Ile98,

Phe384, and Tyr171) are also depicted.

binding to the protein. (Supporting Information). We also

measured binding constants for 17 and 18 using isothermal

titration calorimetry (ITC) (K_d17 = 18 nM; K_d18 = 7 nM).

In this assay, the inhibitors demonstrated strong enthalpic

binding with relative potencies consistent with the enzyme

assay, which further precluded the likelihood of any non-

specific mechanism of inhibition.

Docking studies with optimized analogues featuring the

basic amine moiety remained somewhat inconclusive, and

again imposing the hinge-binding constraint, several struc-

tures did not dock. However, basic analogues which did

provide a binding hypothesis suggested a potential hinge-

binding interaction with the oxygen of the p-methoxy moiety

in contrast to the pyridine identified earlier. This alternative

binding mode is exemplified by the tetrahydroisoquinoline

16 (Figure 4). As can be seen in the figure, the p-methoxy

oxygen of 16 forms a hydrogen bond interaction with the NH

of hinge region residue Met172. The tetrahydroisoquinoline

nitrogen forms an interaction with the side chain acid of

Asp385. The docking hypothesis generated for 16 seemed

more reliable, but taken as a whole, the evidence from

modeling studies to support this hinge-binding interaction

was not compelling since many structures failed to dock and

we therefore used our SAR studies to further probe the

binding mode of our inhibitors.

The next series of structures were designed to evaluate the

linker regions. For these studies, 17 is the direct comparison.

We had previously looked at methylation of the linker

amides on hit structure 5 and found this to have a modest

effect on potency (∼2 fold reduction, data not shown).

Beyond simple alkylation, most other modifications to the

linker atoms resulted in a substantial to complete loss of

potency as can be seen from the data in Table 5. The presence

of a methylene component in the LHS linker adjacent to the

central aromatic seemed important as various modifications

to the 3-atom fragment which altered or eliminated this point

of flexibility were not tolerated (23-26). This indicated that

rotational freedom was important in this region possibly due

to a bent bioactive conformation. Replacing the LHS amide

^aAssay values reported as the mean ( SEM of n g 2 independent

replicates. ^bRacemic.

corresponding basic amines should lose potency as these

would be protonated and unable to accept a hydrogen bond.

On the contrary, a moderate to significant improvement was

observed, inconsistent with the docking hypothesis but

compatible with the screening data analysis. Known Rho

kinase inhibitors such as 1-4 also have a basic amine moiety.

Having demonstrated a potentially optimizable binding

interaction with a basic amine, further exploration of this

area was carried out. Regioisomeric versions of 15 and 16

provided further improvement (17, 18), and methylation of

these analogues (19, 20) was either mildly adverse (19) or had

a negligible effect on potency (20). Having an aromatic ring

instead of the acyclic ethyl group linking the amine fragment

to the right-side amide apparently offered some advantage,

albeit modest (5 vs 14, 15 vs 16, 17 vs 18). Consequently,

we evaluated some substituted benzylic amines, and found

the p-dimethylaminomethylphenyl to be optimal (21). These

studies indicated a specific binding interaction for the

basic amine. Further confirmation came from the benzyl

alcohol 22, which was expectedly less potent. ATP competi-

tion was evaluated for 15, 16, and 18 because these analogues

provided a larger window than the hit structure 5 for observ-

ing the effects of increasing concentrations of ATP (above

K_M,ATP). Using an IMAP assay format, these analogues

were found to be ATP competitive, which documented a

mechanism of inhibition and provided support for specific

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 763

Table 6. Modifications to the Dimethoxyphenyl Fragment

Table 5. Linker Modifications

entry

R

hROCK2 IC₅₀(nM)^a

21

30

31

32

33

34

35

36

37

3,4-diOMe

3-OMe

4-OMe

69 ( 8.9

>7000

1300 ( 58

>6000

3,4-diOH

3-Me-4-OMe

3-Cl-4-OMe

3-CF₃-4-OMe

3-Cl-4-OCF₃

4-CN

266 ( 61

92 ( 13

160 ( 42

670 ( 110

1700 ( 385

^aAssay values reported as the mean ( SEM of n g 2 independent

replicates.

shown), were also not tolerated. Interestingly, keeping the

p-methoxy and substituting a methyl, chloro, or trifluorometh-

yl for the m-methoxy group recaptured some of the loss in

potency (33-35), further indicating that hydrophobic inter-

actions likely contributed to binding in this region. However

the reverse tactic employing the m-methoxy fragment was not

useful (data not shown). Because the alkoxy group at the para

position consistently demonstrated its significance, we further

probed its hinge-binding potential generating 36 with an

electron-withdrawing trifluoromethyl bonded to the oxygen.

A moderate reduction in potency was seen (7-fold) providing

some support for a hinge-binding hypothesis. However, com-

paring 33 to 34 and 35, it was noted that no loss in potency

occurred as a result of an adjacent electron-withdrawing group

as might be expected if oxygen was a hydrogen bond acceptor.

We also tried to incorporate other groups at this posi-

tion, which may be more capable of mediating a hydrogen

bond interaction including a nitrile (37), amide, and urea (not

shown), but nothing proved useful. While these SAR results did

not clearly support the hinge-binding hypothesis suggested by

docking studies, they definitively established the significance of

LHS aromatic substituents for binding. Furthermore, our

studies afforded some evidence to suggest that hydrophobic

interactions may be important. In support of these findings, it

was noted that the region occupied by these fragments in

docked structure 16 (Figure 4) contained several hydrophobic

residues in close proximity including Met169 (gatekeeper),

Ile98, Phe384, and Tyr171.

In summary, our SAR studies for the bisbenzamide series

demonstrated structure-based inhibition and identified opti-

mizable regions. We could not determine with confidence

what moiety, if any, was involved in hydrogen-bonding with

the hinge. Because this is a critical feature for most kinase

inhibitors, the potential absence of hinge-binding was seen as

an opportunity to gain selectivity. Fragments which were

deemed important included a right-hand side (RHS) aro-

matic linking to a saturated, basic amine moiety, the two

central amide linkers, one of which incorporated an essential

adjacent methylene, and the LHS aromatic attachments.

We next turned our attention to the ureidobenzamide series

guided by the information acquired from the bisbenzamide

work. Our starting point here focused on structure 8, which

had similar potency to hit compound 5. Results are shown in

Table 7. Replacing the pyridine of 8 with the ethylpiperidine

^aAssay values reported as the mean ( SEM of n g 2 independent

replicates.

with a sulfonamide was also detrimental (27). What was

completely tolerated, however, was inversion of the amides

(28, 29). These results highlight the importance of the linker

components. The amides in the linker regions were potential

hinge-binding candidates, and this capacity has been asso-

ciated with amide functionality in known Rho kinase in-

hibitors.^15aHowever, the tolerance associated with reversing

these fragments diminished the likelihood than these resi-

dues engaged the critical hinge-binding interaction.

We then revisited the dimethoxyphenyl fragment, and for

these studies we enlisted the dimethylaminomethylphenyl

RHS moiety, making 21 the direct comparison as it had good

potency and offered some synthetic advantages (Table 6).

Previously we had found that removal of either methoxy

group from 5 resulted in inactive compounds (10, 11).

However, using a more potent basic amine replacement for

the pyridine, we could now differentiate between the two,

and found the p-methoxy to be somewhat more significant

(30, 31) as might be expected from the modeling studies.

However, a considerable reduction in potency was observed

on removal of either of these functional groups, affirming the

importance of both. Conversion to the dihydroxy (32) was

not tolerated nor was a methylenedioxy substitution (data

not shown) as seen previously for 12, suggesting a potential

hydrophobic interaction for one or both of the methyl groups.

Other replacements such as dimethyl or dichloro, or regio-

chemical modifications to the methoxy substituents (data not

764 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Table 7. SAR Studies for the Ureidobenzamide Series

Morwick et al.

substitution of the LHS aromatic contributed more signifi-

cantly than meta, in this case using the nitrile substituent

(38 vs 40). We looked at inversion of the RHS amide of 40, an

inconsequential modification in the bisbenzamide series. In

this case, a moderate reduction in potency was observed (41).

We then explored the RHS amine of 40 incorporating some

of the interesting groups identified from the previous SAR.

Similar regiochemical preference for the piperidine was

observed comparing 42 to the preferred 40, but incorporat-

ing the aromatic into the RHS linker had a more negligible

effect on binding (43). A comparable tolerance was also seen

for the dimethyl-substituted p-aminomethylphenyl analogue

44, which was equipotent to its unsubstituted counterpart,

45. We used the p-dimethylaminomethylphenyl RHS motif

to further evaluate the LHS aromatic substitution. This

group not only provided a synthetic advantage as before,

but also, having a more moderate level of potency, it allowed

us more room to quantitate improvements. Again, it was

found that the substitution on the LHS aromatic was some-

what uncompromising, and most replacements for the nitrile

were not tolerated. These included monosubstitution at the

3 or 4 position by methoxy, trifluoromethyl, or halogen, or a

para-substituted ester or primary urea, all of which showed

no inhibition up to a concentration of 5 μM (data not

shown). However, incorporation of chlorine adjacent to

the nitrile provided a boost in potency (46) as seen previously

for 34, consistently indicating the importance of hydrophobic

interactionsinthis region. However, unlike before, onreplace-

ment of the nitrile with an amide (47) in an attempt to identify

any hinge-binding potential for the substituent at this posi-

tion, we observed an 8-fold advantage, suggesting that this

series may have better access to the hinge, assuming a

proximal location. Interestingly, incorporation of a chlorine

substituent adjacent to the purported hinge-binding amide

erased its contribution (48) unlike what was observed with

the nitrile, suggesting these two substituents align differently

to the protein. It was also noted that shifting this amide to

the adjacent meta position (49) evoked a reduction in potency

to a greater extent than seen for the nitrile. Combining the

optimal RHS tetrahydroisoquinoline fragment with the pre-

ferred LHS amide substituent provided the most potent

analogue 50.

Our selection of the dimethylaminomethylphenyl motif

for evaluation of LHS SAR for both the bisbenzamide and

urea series led to one additional finding of interest. Some

analogues incorporating this fragment demonstrated un-

usual inhibition curves with decreasing potency at high

concentrations (above the IC₅₀), suggesting a possible solu-

bility problem, which was not substantiated by our target-

independent profiling. It was subsequently found that com-

pounds employing this motif were in fact weak inhibiters of

the luciferase enzyme used to quantify residual ATP in the

assay. Increasing utilization of luminescence technology in

HTS has led to an awareness of this aspect of assay inter-

ference, and an analysis of common HTS scaffolds which act

as inhibitors of firefly luciferase has recently been reported.²⁹

We subsequently evaluated these analogues and some com-

parators with alternative RHS fragments using an IMAP

assay and verified that the relative potencies determined

from the luciferase assay were not impacted by this level of

assay interference (Supporting Information).

^aAssay values reported as the mean ( SEM of n g 2 independent

replicates.

fragment having the preferred regiochemistry led to a

significant improvement in potency as seen previously (38).

However replacement of the nitrile of 38 with the 3,4-

dimethoxy pattern found optimal for the bisbenzamides

had the opposite effect (39). This was not too surprising, as

utilization of the nitrile in the bisbenzamide series also was

not profitable (37), indicating some differences in binding for

the two series. However, similar to previous findings, para

Lead Generation: Chemistry. Standard coupling techni-

ques were employed for synthesis of the analogues described

herein. The synthesis of common intermediates 51-54 is

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 765

Scheme 1^a

^a(i) N-(3-Dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride, benzotriazole-1-ol, diisopropylethylamine, or N-methylmorpholine, DMF;

(ii) SCX, dichloromethane; (iii) ammonia in methanol; (iv) Amberlyst A26 (OH^-form), methanol/DMF; (v) formic acid, methanol; (vi) Si-DMA,

DMF; (vii) SCX; (viii) LiOH, THF, water.

Scheme 2^a

^a(i) N-(3-Dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride, benzotriazole-1-ol, diisopropylethylamine, DMF; (ii) trifluoroacetic acid,

dichloromethane; (iii) formic acid, formaldehyde.

shown in Scheme 1. Preparation of these intermediates and

many of the reported analogues was facilitated by use of

solid-phase reagents and scavengers including deprotection

strategies incorporating catch and release technologies.³⁰

The general sequence for synthesis of hit compound 5 and

analogues 10-13 (Table 3) is exemplified by method 1

(Scheme 2) demonstrating the synthesis of 5. This directional

approach, installing the RHS amide first, provided an

efficient method for variation of the LHS substituents.

Alternatively, initial formation of the LHS dimethoxyaryl

amide fragment 52 provided a common intermediate for

synthesis of analogues in Table 4. The process is exemplified

by the synthesis of 17 (method 2; Scheme 2). Methylated

analogues 19 and 20 were prepared from the corresponding

amines using the Eschweiler-Clarke procedure.³¹

are exemplified in Scheme 3. Analogous couplings and depro-

tection strategies engaging the appropriate amine and acid

fragments were employed. Standard urea formation using an

aniline and arylisocyanate initiated the synthesis of 23.

Using common intermediate 53, analogues 30-37 (Table 6)

were synthesized by direct coupling of an aromatic acid.

Scheme 4 outlines the method exemplifying the synthesis of 30.

Methods used for synthesis of urea analogues reported in

Table 7 are outlined in Scheme 5. Synthesis of 40 was

prosecuted using intermediate 54, which was coupled to the

RHS amine fragment. Deprotection provided the desired

analogue. This general approach was also used to generate

38-39 and 42-45 employing the appropriate fragments.

Linker-modified 41 featuring a reversed RHS amide was

generated similarly by initial formation of the urea followed

by deprotection of the aniline, amide coupling, and final

deprotection. Preparation of 46 employed an inverted seq-

uence utilizing intermediate 53, which was heated in the

Variation of the linker region did not lend itself to utiliza-

tion of common synthetic intermediates, and therefore several

independent approaches to the synthesis of these analogues

766 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Morwick et al.

Scheme 3^a

^a(i) 4-Isocyanato-1,2-dimethoxy-benzene, DMF; (ii) Amberlyst A26 (OH^-form), methanol, DMF, or methanol/DMF; (iii) formic acid, methanol;

(iv) N-(3-dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride, benzotriazole-1-ol, N-methylmorpholine or N,N-diisopropylethylamine, DMF;

(v) 3-(2-amino-ethyl)-piperidine-1-carboxylic acid tert-butyl ester (vi) SCX, dichloromethane; (vii) ammonia, methanol; (viii) isophthalic acid

monomethyl ester; (ix) 3-aminobenzoic acid methyl ester; (x) SCX; (xi) Si-DMA; (xii) HCl, dioxane; (xiii) 3,4-dimethoxy-benzenesulfonyl chloride,

Si-DMA, DMF; (xiv) 3-(aminomethyl)-1-N-boc-aniline; (xv) 3-(2-carboxy-ethyl)-piperidine-1-carboxylic acid tert-butyl ester; (xvi) (3-nitrophenyl)-

acetic acid; (xvii) ammonium formate, Pd/C, methanol.

Scheme 4^a

series. The compounds showed similar potency for ROCK

isoforms, with most analogues exhibiting a slight preference

for ROCK2 including benchmark 1. Good selectivity for

ROCK over the counterscreen panel was observed, with

three analogues showing weak and one moderate inhibition

of PKN2. However, the degree of cross reactivity for these

^a(i) N-(3-Dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochlor-

ide, benzotriazole-1-ol, diisopropylethylamine, DMF.

analogues was not significant as nearly a 100-fold level of

selectivity or greater was maintained, and all analogues had

improved selectivity as compared to literature benchmarks

at the time of our studies.

microwave with the preformed symmetrical urea to generate

the desired analogue. SCX enabled a catch and release

purification. Amides 47-50 were acquired by hydrolysis of

the corresponding nitriles. Having evaluated several condi-

tions to effect this transformation, we ultimately found

sulfuric acid in TFA to be most effective and is exemplified

for the synthesis of 47.

Lead Generation: Selectivity Assessment. We monitored

selectivity for the bisbenzamide and ureidobenzamide series

during the course of our SAR studies evaluating the potency

of selected examples against three of the counterscreening

proteins which had shown significant cross-reactivity from

the hit set, including PKCε, CDC42BPB, and PKN2. We

also tracked ROCK1 activity to confirm that our inhibitors

maintained dual potency for the two isoforms. Table 8 shows

the selectivity data for representative analogues from both

General kinase selectivity is important for compounds

intended for chronic indications, as these proteins are key

regulators of cell signaling and homeostasis. As mentioned

earlier, the scaffold represented a unique chemotype con-

sidering known kinase inhibitors, and we were optimistic

that it might provide an opportunity for the development of

selective inhibitors. Broad kinase profiling for bisbenzamide

18 (33 nM ROCK2) and urea 50 (4 nM ROCK2) provided

evidence to support our optimism. Compound 18 showed no

inhibition of an additional 72 kinases tested (IC₅₀>10 μM

or >50 POC at 3-6 μM) and weak potency for PRKG1

(IC₅₀, 5.6 μM). Urea 50, inactive against an additional 44

kinases tested (>50 POC at 3 μM), also afforded modest

inhibition of PRKG1 (IC₅₀, 3 μM), and marginal results for

PRK2 and PKCR (33 and 50 POC at 3 μM, respectively).

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 767

Scheme 5^a

^a(i) N-(3-Dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride, benzotriazole-1-ol, N,N-diisopropylethylamine or N-methylmorpholine,

DMF; (ii) HCl, dioxane; (iii) Si-DMA, DMF; (iv) p-cyanophenylisocyanate, SCX, DMF; (v) SCX; (vi) trifluoroacetic acid, dichloromethane;

(vii) 3-(2-carboxy-ethyl)-piperidine-1-carboxylic acid tert-butyl ester; DMF; (viii) SCX, dichloromethane; (ix) ammonia, methanol; (x) carbonyldi-

imidazole, DMF; (xi) SCX, methanol/acetonitrile; (xii) H₂SO₄, trifluoroacetic acid.

Table 8. Selectivity Data for Selected Inhibitors

Rho kinase activity and we therefore felt that this was a

manageable problem. However, permeability/efflux remai-

ned an issue. We found that passive permeability (PAMPA

analysis) could be improved by utilizing a 3° rather than a

2° amine or replacing the amine with an alcohol (20, 21, 22).

But as can be seen in the table, these changes alone did not

significantly alter the efflux ratio as determined by Caco-2.

An in vivo rat PK study was done on 21 because this

analogue showed high permeability in the PAMPA assay.

The results revealed very low bioavailability (1.8%) for this

compound, apparently resulting from low intestinal absorp-

tion possibly due to transport mediated efflux. A potential

solution to the unbalanced efflux ratio as measured by Caco-2

was ultimately found when analogues with modifications to

the dimethoxy phenyl fragment were evaluated. Replacing

the m-methoxy moiety with methyl or halogen led to more

permeable compounds with a more acceptable BfA/AfB

ratio (33, 34). These analogues suffered a modest to mode-

rate loss in potency in the molecular assay, but the modifica-

tions provided at least one mechanism to address this

lingering issue. The series was also evaluated for hERG

activity (18), and no inhibition was observed up to 70 μM.

These studies helped to define options to improve properties

and enabled the series to meet target-independent profiling

criteria.

Lead Generation: Functional Analysis. Inhibition of Rho

kinase moderates smooth muscle contraction leading to

blood vessel relaxation. Our functional assay measured the

extent to which our inhibitors could reverse phenylephrine-

induced contraction of rat aortic rings.³³Selected functional

results are reported in Table 10. Hit compound 5 demons-

trated functional activity equivalent to its molecular potency.

Assuming ATP competitive inhibition of Rho kinase is the only

enabling mechanism, the absence of a potency shift was un-

expected considering the reported K_M,ATPfor Rho kinase

IC ₅₀nM

entry ROCK2

IC₅₀nM

ROCK1

IC₅₀nM

PKCε

IC₅₀nM

CDC42BPB

IC₅₀nM

PKN2

15

17

18

20

21

28

34

38

40

43

44

50

1

176

53

33

46

69

73

92

89

18

14

65

4

180

97

>5000

3000

>5000

>5,000

>5000

4400

68

86

>5000

1700

205

190

220

74

31

40

1600

91

>5000

770

780

6

660

430

Again, these values afford >100-fold selectivity for the

additional kinases evaluated (Supporting Information).

Lead Generation: Target Independent Profiling. As men-

tioned earlier, one of the key challenges associated with the

HtL process is the development of ligands identified from

biochemical screens into safe and effective pharmacologi-

cally active agents. As a response to this challenge, various in

vitro profiling tools have been incorporated into the HtL

process. For hit compound 5, we had identified specific issues

as a result of this profiling process, which included poor

metabolic stability, moderate CYP inhibition (2C9, 3A4),

and an apparent efflux problem. As can be seen from the

profiling data in Table 9, replacement of the pyridine moiety

of 5 with piperidine not only improved the potency but also

improved metabolic stability and eliminated CYP inhibition

(15).³²However, adding an aromatic into the RHS linker in

some cases reestablished variable levels of 3A4 (20, 21, 33,

43) and 2C9 (33, 43) activity, but the SAR for CYP inhibition

did not correlate to other modifications which improved

768 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Table 9. Profiling Data for Rho Kinase Inhibitors

Morwick et al.

ROCK2

IC₅₀(nM)

sol

(μg/mL)^a

PAMPA ꢀ

HLM

_1/2(m)

CYP

1A2^d

CYP

2C9^d

CYP

2C19^d

CYP

2D6^d

CYP

3A4^d

entry

10^-6

Caco-2^b

Caco-2^c

t

15

17

18

20

21

22

43

33

34

176

53

67

16

0.6

BQL^g

3.6

16

0.1

0.6

0.8

87

>30^e

>30^f

>30^e

>30^f

>30^e

>30^f

>30^e

>30^f

>30^e

NT^h

>30^e

>30^f

>30^e

>30^f

>30/>30^e

>30^f

1.7/>30^e

8.5/>30^e

>30^f

5.9/>30^e

13.5^f

>30^f

0.1

139

146

55

33

46

49

BQL^g

0.3

5.4

>85

>100

48

27.1

14.6

16.1

8.8

69

10

BQL^g

0.6

66

41

870

14

10

>100

>88

>96

BQL^g

21

BQL^g

7.9

189

NT^h

22^e

266

92

30.8

45.3

12.5^f

NT^h

24

7.7

^aHT solubility @ pH 7.4. ^bA to B ꢀ 10^-6cm/s. ^cB to A ꢀ 10^-6cm/s. ^dμM. ^eFluorescence assay; 3A4 substrates BFC/BQ. ^fLCMS/MS assay. ^gBelow

quantifiable limit. ^hNot tested. (See Supporting Information for method details.)

Table 10. Cellular Potency of Selected Inhibitors and Literature Standard 1

aortic rings IC₅₀(μM)^a

entry

hROCK2 IC₅₀(nM)

hROCK1 IC₅₀(nM)

shift^b

PAMPA ꢀ 10^-6

Caco-2 (BfA/AfB)

5

1.9

2300

33

4300

68

0.82;0.44

194;94

26;17

NT^c

3.6

29.1/0.5 (58)

5.4/<0.1 (>54)

27.1/0.3 (90.3)

14.6/<0.1 (>146)

45.3/7.7 (5.9)

8.8/<0.1 (>88)

NT^c

18

20

21

34

43

1

6.4 ( 3.9

1.2 ( 0.2

6.0 ( 2.8

1.6

46

69

70

16

10

205

220

40

87;29

92

14

17.4;7.3

607;212

14.0;9.1

24

8.5 ( 2.5

6.1 ( 1.8

BQL^d

NT^c

430

660

^aEach individual result is the mean from four different segments from four different rats. Multiple individual results (n g 2) are reported as the mean (

SEM. ^bValues represent the shift between luciferase molecular data and the aortic ring functional results (ROCK2;ROCK1). ^cNot tested. ^dBelow

quantifiable limit.

isoforms (1.9 μM ROCK1, 20 μM ROCK2).³⁴For the opti-

mized bisbenzamides, we noted an overall average shift of ∼40-

fold between the ROCK-II molecular and aortic ring functional

results. However, the correlation was not always consistent, and

we expected that permeability/efflux was at least partially re-

sponsible. As can be seen in Table 10, the level of passive

permeability as measured by PAMPA in conjunction with the

efflux ratios calculated from Caco-2 studies shows a rough

correlation to the observed shift. One of our most permeable

compounds 34 gave a 17/7-fold (ROCK2/ROCK1) shift similar

to the literature benchmark 1, a significant improvement over

related analogue 21, which had a greater efflux ratio. Likewise,

an improvement in passive permeability (PAMPA) for 20 as

compared to its demethylated analogue 18 also translated into a

smaller shift. Generally, ureidobenzamides demonstrated a

greater shift as seen for 43, and while not as broadly profiled

as the bisbenzamide series, permeability/efflux constraints for

the ureidobenzamides again appeared to be partially responsible.

potency in our functional assay. We have also identified

features of the original hit compound which contributed to

poor target-independent properties and have investigated

optionswhichimprovethesedeficienciesproviding a potential

path forward. Overall, these studies supported advancement

of the series to lead optimization (LO) whereby further

development has led to highly selective, orally bioavailable

inhibitors of Rho kinase with potent pharmacological effects.

These studies will be reported in due course.

Experimental Section

General Methods. ¹H and ¹³C NMR spectra were recorded on

a Bruker UltraShield-400 MHz spectrometer in solvents as

noted. Mass spectra were obtained on a Waters ZQ single

quadrupole mass spectrometer using electrospray positive/

negative ionization. Analytical LC/MS data were recorded on

an analytical system consisting of a Waters ZQ mass spectro-

meter, Agilent 1100 DAD, and Sedex 85 ELS detectors, and an

Agilent 1100 HPLC system, equipped with an Eclipse XDB C18

2.5 mm ꢀ 100 mm, 3.5 μm particle diameter, column. A linear

gradient from 95% water/acetonitrile (0.1% formic acid) to

95% acetonitrile/water (0.1% formic acid) was used at a flow

rate of 2.5 mL/min for 7 min followed by a 2 min hold at 95%

acetonitrile/water (0.1% formic acid). Purity for all final com-

pounds (HPLC/MS) is >95%.

Purification by flash chromatography was accomplished

using RediSep prepacked disposable columns run on the Isco

Combiflash. Preparative TLC was done using Uniplate silica gel

plates purchased from Analtech. Microwave reactions were

run in a Smith Synthesizer microwave reactor. Purification by

reverse-phase HPLC utilized a mass-directed preparative scale

LC/MS FractionLynx system controlled by MassLynx version

3.5. The system consisted of a 2525 high performance liquid

chromatography (HPLC) pump, a ZQ single quadrupole mass

spectrometer, two 515 high performance liquid chromatography

(HPLC) pumps for at column dilution and a makeup pump, a

2767 Sample Manager, and a 2996 photo diode array detector

(Waters Corporation, Milford, MA). All samples were purified

Conclusion

In summary, we have identified a bisbenzamide and related

ureidobenzamide series of inhibitors of Rho kinase from an

HTS discovery approach. The involvement of Rho kinase in

various pathological processes renders significance to the

development of potent and selective inhibitors as potential

drug candidates, as well as agents for evaluating discrete

biochemical mechanisms. Our series features a unique kinase

platform demonstrating structure-based activity and afford-

ing notable selectivity within the kinase gene family (near/

>100-fold selectivity against all kinases evaluated: 76 results

for 18 and 50 results for 50). A level of selectivity may be

accrued as a result of unorthodox binding features in the

region we hypothesized to be associated with the hinge, as our

SAR studies suggest hydrophobic interactions may predomi-

nate. Optimized analogues have shown significant inhibition

of the protein, and several have demonstrated moderate

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 769

on an Atlantis prep dC18 (5 μm particle diameter)

19 mm ꢀ 50 mm column. The mobile phases consisted of (A)

water with 0.1% formic acid and (B) acetonitrile with 0.1%

formic acid. The at column dilution solvent used was methanol

with 0.1% formic acid at a constant flow rate of 1 mL/min and

an overall system flow rate of 21 mL/min. All solvents were

purchased from EM Science. All reagents were purchased from

the Aldrich Chemical Co. with the following exceptions: SCX

from Varian (Bond Elut SCX) or Silicycle (SiliaBond SCX),

Si-DMA (SiliaBond DMA) from Silicycle, N-(3-dimethyl-

aminopropyl)-N⁰-ethylcarbodiimide hydrochloride (EDC) from

Fluka, 2-pyridin-4-yl-ethylamine from Lancaster, 3-methyl-

4-methoxybenzoic acid from Matrix, 4-dimethylaminomethyl-

aniline from J&W Pharma, 4-hydroxymethylaniline from Lancaster,

3-aminomethyl-benzoic acid methyl ester hydrochloride from

Maybridge, 3-(2-amino-ethyl)-piperidine-1-carboxylic acid tert-

butyl ester from Astatech, 3-amino-benzoic acid methyl

ester from TCI, 3-chloro-4-trifluoromethoxybenzoic acid from

Indofine, 3-(aminomethyl)-1-N-boc-aniline from Peakdale, 3-t-butoxy-

carbonylamino-benzyl-ammonium chloride from Peakdale,

3-(2-carboxy-ethyl)-piperidine-1-carboxylic acid tert-butyl ester

from Astatech, 3-(tert-butoxycarbonylamino-methyl)-benzoic

acid from Fluka, 3-(3-carboxy-ethyl)-piperidine-1-carboxylic

acid tert-butyl ester from Astatech, and 7-amino-3,4-dihydro-

1H-isoquinoline-2-carboxylic acid tert-butyl ester from J&W

Pharma. The acronym TFA is used throughout to designate

trifluoroacetic acid and DCM to designate dichloromethane.

Reported methods are unoptimized.

Standard Coupling Procedure. A carboxylic acid, N-(3-

dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride (1.1-

1.4 equiv), and benzotriazole-1-ol (1.1-1.4 equiv) were com-

bined in a reaction vial and DMF was added (0.1-0.6 mmol acid/

mL). The mixture was agitated briefly, and then N,N-diisopropyl-

ethylamine (DIEA) or N-methylmorpholine (NMM) (2-3 equiv)

was added. The mixture was agitated for 15-30 min, then

the amine coupling fragment was added (0.6-1.4 equiv), and the

resulting mixture was agitated for 16-70 h. The reaction mixture

was diluted with water and extracted with ethyl acetate. The com-

bined extracts were washed with 1N HCl, NaHCO₃(satd aq) and

brine (as appropriate to the specific reaction), dried with Na₂SO₄,

and concentrated. The isolated amide was either purified or used

directly. Alternatively, the reaction mixture was purified directly by

preparative HPLC.

and methanol (2 ꢀ 4 mL). The product acid was eluted with a

solution of 20% formic acid in methanol. The eluent was

concentrated, and the resulting solid was triturated in diethy-

lether. The title compound was isolated by filtration (386 mg,

1

61%). H NMR (400 MHz, DMSO-d₆) δ 13.30 (br, 1H) 9.18

(t, J = 5.8 Hz, 1H), 8.47 (s, 1H), 8.13-8.08 (m, 2H), 7.61 (t, J =

7.7 Hz, 1H), 6.96-6.84 (m, 3H), 4.42 (d, J = 5.8 Hz, 2H), 3.74

(s, 3H), 3.72 (s, 3H). ESMS: m/z 316 (M þ H). HPLC: 100%.

3-Aminomethyl-N-(4-dimethylaminomethyl-phenyl)-benzamide (53).

3-(tert-Butoxycarbonylamino-methyl)-benzoic acid (377 mg,

1.5 mmol) and 4-dimethylaminomethylaniline (150 mg, 1 mmol)

were coupled using the standard procedure with NMM to

provide [3-(4-dimethylaminomethyl-phenylcarbamoyl)-benzyl]-

carbamic acid tert-butyl ester (265 mg, 69% yield). ESMS: m/z

227 (M þ H), which was used without purification. This inter-

mediate was dissolved in DCM (5 mL). and the resulting solution

was treated with SCX (2.27 g, 0.88 mmol/g, 2.0 mmol). The

mixture was agitated for 16 h. The resin was isolated by filtration

and washed with DCM, acetonitrile, and then methanol. The

product was eluted with NH₃(7 N in methanol). The eluent was

concentrated to provide the title compound (172 mg, 89.5%

1

yield). H NMR (400 MHz, methanol-d₄) δ 7.75 (s, 1H), 7.66

(d, J = 7.8 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 7.8 Hz,

1H)), 7.32 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.5 Hz, 2H), 3.74

(s, 2H), 3.32 (s, 2H), 2.12 (s, 6H). ESMS: m/z 284 (M þ H).

HPLC: 96%.

3-[3-(4-Cyano-phenyl)-ureidomethyl]-benzoic Acid (54). 3-Methoxy-

carbonyl-benzyl-ammonium chloride (605 mg, 3.0 mmol) was

dissolved in DMF (6 mL) in a reaction vial. Si-DMA (3.3 g,

5 mmol) was added, and the mixture was agitated for 1 h.

Subsequently, p-cyanophenylisocyanate (403.5 mg, 2.8 mmol)

was added and the mixture was agitated 16 h. SCX (568 mg,

0.5 mmol) was added, and the mixture was agitated for 30 min.

The sorbents were filtered and the filtrate was concentrated to

provide 3-[3-(4-cyano-phenyl)-ureidomethyl]-benzoic acid methyl

ester (843 mg, 91% yield), which was used directly. The urea

was dissolved in THF (7 mL). A solution of lithium hydroxide

(410 mg, 10 mmol) in water (3 mL) was added. The resulting

mixture was heated at 50 °C for 2 h and then cooled to room

temperature. The organic phase was separated, and the remain-

ing aqueous solution was neutralized. The title compound was

isolated by filtration (710 mg, 93% yield). ¹H NMR (400 MHz,

DMSO-d₆) δ 12.98 (s, 1H), 9.21 (s, 1H), 7.90 (s, 1H), 7.83 (d, J =

7.7 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H),

7.55 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 6.97 (t, J =

6.0 Hz, 1H), 4.37 (d, J = 6.0 Hz, 2H). ESMS: m/z 296 (M þ H).

HPLC: 92%.

3,4-Dimethoxy-N-[3-(2-pyridin-4-yl-ethylcarbamoyl)-benzyl]-

benzamide (5). 3,4-Dimethoxybenzoic acid (100 mg, 0.55 mmol)

and 51 (100 mg, 0.39 mmol) were coupled using the standard

procedure with DIEA. The isolate was purified by flash chro-

matography using a gradient of ethyl acetate in hexane to

provide the title compound (92 mg, 56% yield). ¹H NMR

(400 MHz, CDCl₃) δ 8.42 (d, J = 6.2 Hz, 2H), 7.69 (s, 1H), 7.62

(d, J= 7.7 Hz, 1H), 7.44-7.48 (m, 2H), 7.38-7.28 (m, 4H), 7.18 (t,

J = 5.9 Hz, 1H), 7.11 (t, J = 5.9 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H),

4.58 (d, J = 5.9 Hz, 2H), 3.92 (s, 3H), 3.89 (s, 3H), 3.72 (q, J =

6.3 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H). ESMS: m/z 420 (M þ H).

Using the method described for the synthesis of 5, compounds

10-13 were prepared from the corresponding substituted ben-

zoic acids.

Procedures for Synthesis of Common Intermediates 51-54. 3-

Aminomethyl-N-(2-pyridin-4-yl-ethyl)-benzamide (51). 3-(tert-

Butoxycarbonylamino-methyl)-benzoic acid (150.7 mg, 0.6 mmol)

and 2-pyridin-4-yl-ethylamine (0.06 mL, 0.5 mmol) were coupled

using the standard procedure with DIEA. The isolate, [3-(2-pyridin-

4-yl-ethylcarbamoyl)-benzyl]-carbamic acid tert-butyl ester, which

was used without purification, was dissolved in DCM (5 mL) and

treated with SCX (1.36 g, 1.2 mmol). The resulting mixture was

agitated for 24 h and then filtered. The silica was washed with DCM

and methanol. The product was eluted with NH₃(7 N in methanol),

and the eluent was concentrated to provide the title compound

1

(125 mg, 94%). H NMR (400 MHz, DMSO-d₆) δ 8.53 (t, J =

7.0 Hz, 1H), 8.49 (dt, J= 4.8, 0.8 Hz, 1H), 7.79 (s, 1H), 7.69 (dt, J=

7.6, 1.8 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H),

7.37(t,J= 7.6 Hz, 1H), 7.26 (d, J= 7.8 Hz, 1H), 7.20 (ddd, J = 7.5,

3.8, 1.0 Hz, 1H), 3.78 (s, 2H), 3.59 (dt, J = 7.0, 7.0 Hz, 2H), 2.98

(t, J = 7.0 Hz, 2H). ESMS: m/z 256 (M þ H). HPLC: 100%.

3-[(3,4-Dimethoxy-benzoylamino)-methyl]-benzoic Acid (52).

3,4-Dimethoxybenzoic acid (400.8 mg, 2.2 mmol) and 3-meth-

oxycarbonyl-benzyl-ammonium chloride (403.3 mg, 2 mmol)

were coupled using the standard procedure with DIEA to give

3-[(3,4-dimethoxy-benzoylamino)-methyl]-benzoic acid methyl

ester, which was used without purification. The ester was

dissolved in a mixture of methanol (6 mL) and DMF (1 mL)

and treated with Amberlyst A26 (OH^-form) (3730 mg, 5 mmol),

and the resulting mixturewas agitated for 36 h. The resin was then

filtered and washed with methanol (2 ꢀ 4 mL), DMF (1 ꢀ 4 mL),

4-Methoxy-N-[3-(2-pyridin-4-yl-ethylcarbamoyl)-benzyl]-benz-

amide (10). Purified by preparative HPLC using an acetonitrile/

water/formic acid gradient: 62.9% yield. H NMR (400 MHz,

1

CDCl₃) δ 8.44 (d, J = 6.2 Hz, 2H), 7.80-7.75 (m, 2H), 7.69

(s, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H),

7.40-7.26 (m, 3H), 7.05-6.94 (m, 2H), 6.93-6.88 (m, 2H), 4.58

(d, J = 5.9 Hz, 2H), 3.86 (s, 3H), 3.73 (q, J = 6.7 Hz, 2H), 3.04

(t, J = 6.8 Hz, 2H). ESMS: m/z 390 (M þ H).

770 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Morwick et al.

3-Methoxy-N-[3-(2-pyridin-4-yl-ethylcarbamoyl)-benzyl]-benz-

amide (11). Purified by preparative HPLC using an acetonitrile/

water/formic acid gradient: 58% yield. ¹H NMR (400 MHz,

CDCl₃) δ 8.42 (d, J = 6.1 Hz, 2H), 7.69 (s, 1H), 7.62 (d, J =

7.7 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.41-7.31 (m, 6H), 7.21

(t, J = 5.9 Hz, 1H), 7.12 (t, J = 5.9 Hz, 1H), 7.08-7.12 (m, 1H),

4.58 (d, J = 5.9 Hz, 2H), 3.82 (s, 3H), 3.72 (q, J = 6.7 Hz, 2H),

3.02 (t, J = 6.8 Hz, 2H). ESMS: m/z 390 (M þ H).

5.0 Hz, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 3.51-3.21 (m, 4H),

2.86-2.68 (m, 1H), 2.55-2.37 (m, 1H), 1.96-1.63 (m, 4H),

1.63-1.37 (m, 2H), 1.22-1.01 (m, 1H). ESMS: m/z 426 (M þ H).

3,4-Dimethoxy-N-[3-(1,2,3,4-tetrahydro-isoquinolin-7-ylcarba-

moyl)-benzyl]-benzamide (18). Purification by preparative

HPLC using an acetonitrile/water/formic acid gradient pro-

vided the title compound: 25% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.22 (s, 1H), 9.01 (t, J = 6.0 Hz, 1H), 7.89 (s, 1H),

7.83 (d, J = 13.3 Hz, 1H), 7.61-7.41 (m, 6H), 7.11 (d, J = 8.4 Hz,

1H), 7.04 (d, J = 8.5 Hz, 1H), 4.55 (d, J = 5.9 Hz, 2H), 4.20

(s, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.13 (t, J = 5.9 Hz, 2H), 2.79

(t, J = 5.8 Hz, 2H). ESMS: m/z 446 (M þ H).

1,3-Benzodioxole-5-carboxylic acid 3-(2-pyridin-4-yl-ethyl-

carbamoyl)-benzylamide (12). Purified by preparative HPLC

using an acetonitrile/water/formic acid gradient: 21% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.55 (d, J = 5.3 Hz, 2H), 7.72

(s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.43

(t, J = 7.6 Hz, 1H), 7.36-7.23 (m, 4H), 6.85 (d, J = 8.0 Hz, 1H),

6.50 (t, J = 5.9 Hz, 1H), 6.39 (t, J = 5.9 Hz, 1H), 6.06 (s, 2H),

4.66 (d, J = 5.9 Hz, 2H), 3.76 (q, J = 6.7 Hz, 2H), 3.01 (t, J =

6.8 Hz, 2H). ESMS: m/z 404 (M þ H).

3,4-Dimethoxy-N-{3-[2-(1-methyl-piperidin-3-yl)-ethylcarbamoyl]-

benzyl}-benzamide (19). Compound 17 (80 mg, 0.188 mmol) was

dissolved in formic acid (0.5 mL) and formaldehyde (0.5 mL,

37% aqueous solution) was added. The resulting mixture was

heated at 70 °C 16 h. The reaction mixture was concentrated,

and the isolate was purified by preparative HPLC using an

acetonitrile/water/formic acid gradient providing the title com-

3,4-Dimethyl-N-[3-(2-pyridin-4-yl-ethylcarbamoyl)-benzyl]-benz-

amide (13). Purified by preparative HPLC using an acetonitrile/

water/formic acid gradient: 59.3% yield. H NMR (400 MHz,

1

pound (41 mg, 50% yield). H NMR (400 MHz, DMSO-d₆) δ

8.97 (t, J = 5.9 Hz, 1H), 8.46 (t, J = 5.5 Hz, 1H), 7.79 (s, 1H),

7.70 (d, J = 7.4 Hz, 1H), 7.54 (dd, J = 8.4, 2.0 Hz, 1H), 7.49 (d,

J = 1.9 Hz, 1H), 7.47-7.38 (m, 2H), 7.04 (d, J = 8.5 Hz, 1H),

4.51 (d, J = 5.8 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.28 (dd, J =

12.9, 6.9 Hz, 4H), 2.92-2.76 (m, 2H), 2.28 (s, 3H), 2.13-1.99 (m,

1H), 1.91-1.69 (m, 2H), 1.68-1.55 (m, 1H), 1.56-1.34 (m, 2H),

0.88 (dd, J = 22.8, 11.1 Hz, 1H). ESMS: m/z 440 (M þ H).

3,4-Dimethoxy-N-[3-(2-methyl-1,2,3,4-tetrahydro-isoquinolin-

7-ylcarbamoyl)-benzyl]-benzamide (20). Prepared from 18 ac-

cording to the procedure described for the synthesis of 19: 22%

yield. ¹H NMR (400 MHz, CDCl₃) δ 9.15 (s, 1H), 7.87 (s, 1H),

7.77 (d, J = 7.7 Hz, 1H), 7.65 (s, 1H), 7.52-7.33 (m, 6H), 7.03

(d, J = 8.3 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 4.59 (d, J = 5.5 Hz,

2H), 3.98 (s, br, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.32 (s, br, 2H),

3.05 (s, br, 2H), 2.78 (s, 3H). ESMS: m/z 460 (M þ H).

CDCl₃) δ 8.43 (d, J = 6.0 Hz, 2H), 7.69 (s, 1H), 7.63 (d, J =

7.7 Hz, 1H), 7.58 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.44 (d, J =

7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.32-7.26 (m, 2H), 7.17 (d,

J = 7.9 Hz, 1H), 7.09 (t, J = 5.8 Hz, 1H), 7.03 (t, J = 5.8Hz, 1H),

4.58 (d, J = 6.0 Hz, 2H), 3.71 (q, J = 6.8 Hz, 2H), 3.01 (t, J =

6.9 Hz, 2H), 2.30 (s, 3H) 2.28 (s, 3H). ESMS: m/z 388 (M þ H).

N-[3-(Isoquinolin-6-ylcarbamoyl)-benzyl]-3,4-dimethoxy-benz-

amide (14). 5-Aminoisoquinoline, (50 mg, 0.35 mmol) and 52

(50 mg, 0.16 mmol) were coupled using the standard procedure

with DIEA. The isolate was purified by preparative HPLC using

an acetonitrile/water/formic acid gradient to provide the title

compound (23 mg, 33% yield). ¹H NMR (400 MHz, CDCl₃) δ

9.21 (s, 1H), 8.56 (s, 1H), 8.51-8.47 (m, 2H), 7.99 (d, J = 8.9 Hz,

1H), 7.95 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.74 (dd, J = 8.8,

2.1 Hz, 1H), 7.69 (d, J = 5.9 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H),

7.57-7.45 (m, 2H), 7.36 (dd, J = 8.4, 2.1 Hz, 1H), 6.88 (d, J =

8.4 Hz, 1H), 6.72 (t, J = 5.9 Hz, 1H), 4.74 (d, J = 6.0 Hz, 2H),

3.95 (s, 3H), 3.94 (s, 3H). ESMS: m/z 442 (M þ H).

N-[3-(4-Dimethylaminomethyl-phenylcarbamoyl)-benzyl]-3,

4-dimethoxy-benzamide (21). 4-Dimethylaminomethylaniline

(25.5 mg, 0.17 mmol) and 52 (40 mg, 0.13 mmol) were coupled

using the standard procedure with NMM. Purification by

preparative HPLC using an acetonitrile/water/formic acid gra-

dient provided the title compound (37 mg, 65% yield). ¹H NMR

(400 MHz, DMSO-d₆) δ 10.24 (s, 1H), 9.00 (t, J = 5.9 Hz, 1H),

7.89 (s, 1H), 7.84 (d, J = 7.3 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H),

7.61-7.43 (m, 4H), 7.29 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz,

1H), 4.56 (d, J = 5.9 Hz, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.42

(s, 2H), 2.18 (s, 6H). ESMS: m/z 448 (M þ H).

General Procedure for Synthesis of 15-18. Intermediate 52

and the appropriate amine were coupled using the standard

procedure with DIEA. Deprotection was accomplished by

dissolution of the amide in DCM (0.1 mmol/mL), followed by

treatment with TFA (20% of solvent). The resulting mixture was

agitated for 2 h, then concentrated. If needed, purification was

done by preparative HPLC using an acetonitrile/water/formic

acid gradient or flash chromatography using a methanol DCM

gradient.

N-[3-(4-Hydroxymethyl-phenylcarbamoyl)-benzyl]-3,4-dimethoxy-

benzamide (22). 4-Hydroxyomethylaniline (19.7 mg, 0.16 mmol)

and 52 (45.0 mg, 0.14 mmol) were coupled using the standard

procedure with NMM purification by preparative HPLC using

an acetonitrile/water/formic acid gradient provided the title

3,4-Dimethoxy-N-[3-(2-piperidin-4-yl-ethylcarbamoyl)-benzyl]-

benzamide (15). Purification by flash chromatography using a

methanol/DCM gradient provided the title compound, TFA

salt: 41% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.60 (s, br, 1H),

8.27 (s, br, 1H), 7.76 (s, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.50 (d,

J = 7.9 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.40-7.32 (m, 2H),

7.12 (t, J = 5.5 Hz, 1H), 6.96 (t, J = 5.5 Hz, 1H), 6.88 (d, J =

8.4 Hz, 1H), 4.67 (d, J = 5.9 Hz, 2H), 3.92 (s, 3H), 3.91 (s, 3H),

3.51-3.38 (m, 4H), 2.88-2.81 (m, 2H), 2.02-1.98 (m, 2H),

1.73-1.46 (m, 5H). ESMS: m/z 426 (M þ H).

1

compound (41 mg, 68% yield). H NMR (400 MHz, DMSO-

d₆) δ 10.27 (s, 1H), 9.00 (t, J = 5.9 Hz, 1H), 7.89 (s, 1H), 7.83

(d, J = 7.3 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.56-7.47 (m, 4H),

7.27 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz, 1H), 5.14 (t, J =

5.7 Hz, 1H), 4.56 (d, J = 5.9 Hz, 2H), 4.46 (d, J = 5.9 Hz, 2H),

3.85 (s, 3H), 3.84 (s, 3H). ESMS: m/z 419 (M - H).

3-[3-(3,4-Dimethoxy-phenyl)-ureido]-N-(2-piperidin-3-yl-ethyl)-

benzamide (23). 3-Amino-benzoic acid methyl ester (75.6 mg,

0.5 mmol) was dissolved in DMF (2 mL) in a reaction vial, and

4-isocyanato-1,2-dimethoxy-benzene (98.5 mg, 0.55 mmol) was

added. The reaction mixture was agitated 7 h, diluted with DMF

(1 mL), and treated with Amberlyst A26 (OH^-form) (1.12 g,

1.5 mmol). The resulting mixture was agitated at ambient temp-

erature for 16 h. The sorbent was filtered and washed with

several portions of methanol. The product was eluted with 20%

formic acid in methanol, and the sorbent was then washed with

several portions of DMF. The eluent was concentrated to a solid

which was triturated in methanol. Filtration provided 3-[3-(3,4-

dimethoxy-phenyl)-ureido]-benzoic acid (106 mg, 67% yield).

3,4-Dimethoxy-N-[3-(1,2,3,4-tetrahydro-isoquinolin-6-ylcarba-

moyl)-benzyl]-benzamide (16). Purification by preparative

HPLC using an acetonitrile/water/formic acid gradient pro-

vided the title compound: 35% yield. ¹H NMR (400 MHz,

methanol-d₄) δ 7.81 (s, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.49-7.32

(m, 6H), 6.93-6.90 (m, 2H), 4.54 (s, 2H), 3.86 (s, 2H), 3.78 (s,

3H), 3.77 (s, 3H), 3.00 (t, J = 6.0 Hz, 2H), 2.75 (t, J = 6.0 Hz,

2H). ESMS: m/z 446 (M þ H).

3,4-Dimethoxy-N-[3-(2-piperidin-3-yl-ethylcarbamoyl)-benzyl]-

benzamide (17). TFA salt: 74% yield. ¹H NMR (400 MHz,

CDCl₃) δ 8.81 (s, br, 1H), 8.58 (s, br, 1H), 7.86 (t, J = 5.5 Hz,

1H), 7.68 (s, 1H), 7.63-7.55 (m, 2H), 7.48-7.36 (m, 3H),

7.34-7.28 (m, 1H), 6.84 (d, J = 8.3 Hz, 1H), 4.54 (d, J =

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 771

¹H NMR (400 MHz, DMSO-d₆) δ 12.93 (s, 1H), 8.80 (s, 1H),

8.53 (s, 1H), 8.14 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.54 (d, J =

7.8 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.23 (s, 1H), 6.88 (s, 2H),

3.75 (s, 3H), 3.71 (s, 3H). ESMS: m/z 317 (M þ H). HPLC: 95%.

This intermediate (47.4 mg, 0.15 mmol) and 3-(2-amino-ethyl)-

piperidine-1-carboxylic acid tert-butyl ester (34.3 mg, 0.16 mmol)

were coupled using the standard procedure with NMM. The

isolate was dissolved directly in DCM (1 mL) and treated with

SCX (398 mg, 0.35 mmol), and the resulting mixture was

agitated for 16 h. The mixture was filtered and the sorbent

was washed with several portions of DCM and methanol. The

product was eluted with NH₃(7N in methanol). The eluent was

concentrated providing the title compound (53 mg, 82.8%

yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (s, 1H), 8.62

(s, 1H), 8.39 (t, J = 5.5 Hz, 1H), 7.87 (s, 1H), 7.59 (d, J = 7.9 Hz,

1H), 7.40 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.23 (s,

1H), 6.87 (s, 2H), 3.74 (s, 3H), 3.71 (s, 3H), 3.31-3.20 (m, 2H),

2.96 (d, J = 11.2 Hz, 1H), 2.87 (d, J = 12.0 Hz, 1H), 2.45-2.38

(m, 1H), 2.24-2.08 (m, 1H), 1.80 (d, J = 14.8 Hz, 1H), 1.59-1.52

(m, 1H), 1.47-1.31 (m, 4H), 1.08-0.94 (m, 1H). ESMS: m/z 427

(M þ H).

directly. This intermediate was dissolved in methanol (5 mL) and

treated with Amberlyst A26 (OH^-form) (1.5 g, 2 mmol). The

resulting mixture was agitated at ambient temperature for 72 h.

The sorbent was filtered and washed with several portions of

methanol and DMF. The product was eluted with 20% formic

acid in methanol and the sorbent was then washed with methanol

and DMF. The eluent was concentrated to 3-[2-(3,4-dimethoxy-

phenyl)-acetylamino]-benzoic acid (137 mg, 43% yield). ¹H

NMR (400 MHz, DMSO-d₆) δ 13.00 (br, 1H), 10.30 (s, 1H),

8.23 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H),

7.42 (t, J = 7.8 Hz, 1H), 6.96 (s, 1H), 6.91-6.84 (m, 2H), 3.75

(s, 3H), 3.72 (s, 3H), 3.57 (s, 2H). ESMS: m/z 316 (M þ H).

HPLC: 100%. This intermediate (37.8 mg, 0.12 mmol) and 3-(2-

amino-ethyl)-piperidine-1-carboxylic acid tert-butyl ester (32 mg,

0.14 mmol) were coupled using the standard procedure with

DIEA. The reaction mixture was purified directly by prepara-

tive HPLC using an acetonitrile/water/formic acid gradient to

provide 3-(2-{3-[2-(3,4-dimethoxy-phenyl)-acetylamino]-benzoyl-

amino}-ethyl)-piperidine-1-carboxylic acid tert-butyl ester

(53 mg, 84% yield). ESMS: m/z 526 (M þ H). HPLC: 100%.

Thisintermediate(53mg, 0.1 mmol) was taken upin0.5 M HCl in

dioxane (1 mL) and methanol (0.5 mL). The mixture was agitated

for 1 h then concentrated. The isolate was purified by preparative

HPLC using an acetonitrile/water/formic acid gradient to pro-

vide the title compound (19 mg, 45% yield). ¹H NMR (400 MHz,

DMSO-d₆) δ 10.39 (s, 1H), 8.47 (t, J = 5.5 Hz, 1H), 8.04 (s, 1H),

7.78 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.37 (t, J =

7.9 Hz, 1H), 6.97 (s, 1H), 6.95-6.78 (m, 2H), 3.75 (s, 3H), 3.72

(s, 3H), 3.56 (s, 2H), 3.27 (dd, J = 12.8, 6.7 Hz, 2H), 3.16 (d, J =

11.9 Hz, 1H), 3.09 (d, J = 12.4 Hz, 1H), 2.64 (t, J = 12.2 Hz, 1H),

2.47-2.34 (m, 1H), 1.83 (d, J = 10.9 Hz, 1H), 1.77-1.64 (m, 1H),

1.55-1.35 (m, 4H), 1.18-1.01 (m, 1H). ESMS: m/z 426 (M þ H).

3,4-Dimethoxy-N-[3-(2-piperidin-3-yl-ethylcarbamoyl)-phenyl]-

benzamide (26). 3,4-Dimethoxybenzoic acid (364.3 mg, 2.0 mmol)

and 3-aminobenzoic acid methyl ester (302.3 mg, 2.0 mmol)

were coupled using the standard procedure with NMM to pro-

vide 3-(3,4-dimethoxy-benzoylamino)-benzoic acid methyl ester:

ESMS: m/z 316 (M þ H). HPLC: 95%, which was used directly.

This intermediate was dissolved in DMF (3 mL) and treated with

Amberlyst A26 (OH^-form) (3.0 g, 4.0 mmol). The resulting

mixture was agitated at ambient temperature for 16 h. The sorbent

was filtered and washed with several portions of methanol. The

product was eluted with 20% formic acid in methanol, and the

sorbent was then washed with methanol. The eluent was concen-

trated to 3-(3,4-dimethoxy-benzoylamino)-benzoic acid (130 mg,

22% yield). ESMS: m/z 302 (M þ H). HPLC: 85%. This inter-

mediate (44 mg, 0.15 mmol) and 3-(2-amino-ethyl)-piperidine-

1-carboxylic acid tert-butyl ester (36.5 mg, 0.16 mmol) were

coupled using the standard procedure with NMM. The reaction

mixture was purified directly by preparative HPLC using an

acetonitrile/water/formic acid gradient to provide 3-{2-[3-(3,4-

dimethoxy-benzoylamino)-benzoylamino]-ethyl}-piperidine-1-carbo-

xylic acid tert-butyl ester (48 mg, 64.2% yield). ESMS: m/z 512

(M þ H). HPLC: 100%. This intermediate (48 mg, 0.094 mmol)

was dissolved in DCM (1 mL) and treated with SCX (200 mg,

0.175 mmol), and the resulting mixture was agitated for 16 h.

The mixture was filtered, and the sorbent was washed with

several portions of DCM and methanol. The product was eluted

with NH₃(7N in methanol). The eluent was concentrated then

taken up in 1N HCl aq (1 mL) and methanol (1 mL). The

mixture was agitated briefly and then concentrated to provide

N-(3,4-Dimethoxy-benzyl)-N-(2-piperidin-3-yl-ethyl)-isophthal-

amide (24). Isophthalic acid monomethyl ester (180.2 mg,

1.0 mmol) and 3,4-dimethoxybenzylamine (0.167 mL, 1.1

mmol) were coupled using the standard procedure with NMM

to provide N-(3,4-dimethoxy-benzyl)-isophthalamic acid meth-

yl ester (350 mg, 98% yield). ESMS: m/z 330 (M þ H). HPLC:

95%, which was used directly. This intermediate was dissolved

in methanol/DMF (4/1, 5 mL) and treated with Amberlyst A26

(OH^-form) (1.9 g, 2.5 mmol). The resulting mixture was

agitated at ambient temperature for 36 h. The sorbent was

filtered and washed with several protions of methanol and

DMF. The product was eluted with 20% formic acid in metha-

nol, and the sorbent was then washed with methanol and DMF.

The eluent was concentrated to N-(3,4-dimethoxy-benzyl)-iso-

phthalamic acid (240 mg, 76% yield). ¹H NMR (400 MHz,

DMSO-d₆) δ 12.96 (br, 1H), 9.00 (t, J = 5.9 Hz, 1H), 7.96

(s, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.58-7.44 (m, 4H), 7.04 (d, J =

8.4 Hz, 1H), 4.53 (d, J = 5.9 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H).

ESMS: m/z 316 (M þ H). HPLC: 100%. This intermediate

(44.1 mg, 0.14 mmol) and 3-(2-amino-ethyl)-piperidine-1-car-

boxylic acid tert-butyl ester (34.3 mg, 0.16 mmol) were coupled

using the standard procedure with NMM. The isolate was

dissolved directly in DCM (1 mL) and treated with SCX

(398 mg, 0.35 mmol), and the resulting mixture was agitated

for 16 h. The mixture was filtered and the sorbent was washed

with several portions of DCM and methanol. The product was

eluted with NH₃(7N in methanol). The eluent was concentrated

to the title compound (48 mg, 59.6% yield). ¹H NMR (400 MHz,

DMSO-d₆) δ 9.08 (t, J = 5.8 Hz, 1H), 8.56 (t, J = 5.5 Hz, 1H),

8.33 (s, 1H), 8.05-7.90 (m, 2H), 7.56 (t, J = 7.7 Hz, 1H), 6.97

(d, J = 1.5 Hz, 1H), 6.94-6.83 (m, 2H), 4.42 (d, J = 5.8 Hz,

2H), 3.74 (s, 3H), 3.72 (s, 3H), 3.33-3.23 (m, 2H), 2.95 (d, J =

11.9 Hz, 1H), 2.86 (d, J = 11.8 Hz, 1H), 2.49-2.36 (m, 1H),

2.24-2.12 (m, 1H), 1.80 (d, J = 13.9 Hz, 1H), 1.59-1.50

(m, 1H), 1.51-1.30 (m, 4H), 1.09-0.93 (m, 1H). ESMS: m/z

426 (M þ H).

3-[2-(3,4-Dimethoxy-phenyl)-acetylamino]-N-(2-piperidin-3-yl-

ethyl)-benzamide (25). (3,4-Dimethoxy-phenyl)-acetic acid (215.8 mg,

1.1 mmol) was dissolved in DMF (7 mL). N-(3-dimethylamino-

propyl)-N⁰-ethylcarbodiimide hydrochloride (249 mg, 1.3 mmol)

and benzotriazole-1-ol (175.7 mg, 1.3 mmol) were added,

followed by DIEA (0.23 mL, 1.32 mmol). The resulting mix-

ture was agitated for 1 h, and 3-aminobenzoic acid methyl ester

(151.2 mg, 1.0 mmol) was added. The reaction mixture was

agitated for 16 h, then treated with SCX (1.7 g, 1.5 mmol) and

Si-DMA (1.33 g, 2.0 mmol) and the resulting mixture was

agitated for 2 h and then filtered. The filtrate was concentrated

to 3-[2-(3,4-dimethoxy-phenyl)-acetylamino]-benzoic acid methyl

ester. ESMS: m/z 330 (M þ H). HPLC: 100%, which was used

1

the title compound, HCl salt (29 mg, 72.5% yield). H NMR

(400 MHz, DMSO-d₆) δ 10.21 (s, 1H), 9.03 (br, 1H), 8.72 (br,

1H), 8.53 (t, J = 5.9 Hz, 1H), 8.22 (s, 1H), 7.96 (d, J = 7.9 Hz,

1H), 7.68 (d, J = 8.4 Hz, 1H), 7.60-7.52 (m, 2H), 7.42 (t, J = 7.9

Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 3.85 (s, 3H), 3.85 (s, 3H),

3.40-3.12 (m, 4H), 2.80-2.68 (m, 1H), 2.60-2.48 (m, 1H),

1.92-1.72 (m, 3H), 1.70-1.40 (m, 3H), 1.22-1.08 (m, 1H).

ESMS: m/z 412 (M þ H).

772 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Morwick et al.

3-[(3,4-Dimethoxy-benzenesulfonylamino)-methyl]-N-(2-piperi-

din-3-yl-ethyl)-benzamide (27). 3-Aminomethyl-benzoic acid

methyl ester, hydrochloride (201.6 mg, 1.0 mmol) was dissolved

in DMF (3 mL) and Si-DMA (2.0 g, 3.0 mmol) was added, and

the resulting mixture was agitated for 30 min, after which time,

3,4-dimethoxybenzenesulfonyl chloride (236.7 mg, 1.0 mmol)

was added and the reaction mixture was agitated for 16 h. The

sorbent was filtered and washed with DMF, and the filtrate was

concentrated. The isolate was dissolved in DCM (10 mL) and

the solution was washed with 1N HCl (5 mL) and brine

(5 mL), dried with Na₂SO₄, and concentrated to 3-[(3,4-di-

methoxy-benzenesulfonylamino)-methyl]-benzoic acid methyl

ester. ESMS: m/z 366 (M þ H). HPLC: 100%, which was used

directly. This intermediate was dissolved in methanol/

DMF (1:1, 3 mL) and treated with Amberlyst A26 (OH^-form)

(1.5 g, 2.0 mmol). The resulting mixture was agitated at ambient

temperature for 16 h. The sorbent was filtered and washed with

several portions of methanol. The product was eluted with 20%

formic acid in methanol. The eluent was concentrated to 3-[(3,4-

dimethoxy-benzenesulfonylamino)-methyl]-benzoic acid (192 mg,

54.6% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 12.95 (br, 1H),

8.09 (t, J = 6.4 Hz, 1H), 7.82 (s, 1H), 7.78 (d, J = 7.7 Hz,

1H), 7.47 (d, J = 7.7 Hz, 1H), 7.41-7.34 (m, 2H), 7.25

(d, J = 2.1 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 4.03 (d, J = 6.4

Hz, 2H), 3.82 (s, 3H), 3.78 (s, 3H). ESMS: m/z 352 (M þ H).

HPLC: 100%. This intermediate (49.2 mg, 0.14 mmol) and 3-(2-

amino-ethyl)-piperidine-1-carboxylic acid tert-butyl ester (34.3

mg, 0.15 mmol) were coupled using the standard procedure with

NMM. The reaction mixture purified directly by preparative

HPLC using an acetonitrile/water/formic acid gradient to pro-

vide 3-(2-{3-[(3,4-dimethoxy-benzenesulfonylamino)-methyl]-

benzoylamino}-ethyl)-piperidine-1-carboxylic acid tert-butyl

ester (53 mg, 67.4% yield). ESMS: m/z 562 (M þ H). HPLC:

100%. This intermediate (53 mg, 0.094 mmol) was dissolved in

DCM (1 mL) and treated with SCX (200 mg, 0.175 mmol), and

the resulting mixture was agitated for 16 h. The mixture was

filtered and the sorbent was washed with several portions of

DCM and methanol. The product was eluted with NH₃(7N in

methanol). The eluent was concentrated then taken up in 1 M

HCl aq (1 mL) and methanol (1 mL). The mixture was agitated

briefly and then concentrated to provide the title compound,

HCl salt (30 mg, 68.9% yield). ¹H NMR (400 MHz, DMSO-d₆)

δ 9.05 (br, 1H), 8.72 (br, 1H), 8.52 (t, J = 5.5 Hz, 1H), 8.09 (t,

J = 5.8 Hz, 1H), 7.77 (s, 1H), 7.71 (d, J = 7.0 Hz, 1H), 7.44-

7.28 (m, 4H), 7.10 (d, J = 8.5 Hz, 1H), 3.97 (d, J = 5.6 Hz, 2H),

3.83 (s, 3H), 3.78 (s, 3H), 3.42-3.12 (m, 4H), 2.80-2.62 (m, 1H),

2.58-2.43 (m, 1H), 1.92-1.70 (m, 3H), 1.70-1.38 (m, 3H),

1.21-1.08 (m, 1H). ESMS: m/z 462 (M þ H).

agitated 16 h. The reaction mixture was filtered, and the solids were

washed with DCM (2 ꢀ 2 mL) and methanol (2 ꢀ 2 mL). The

product was eluted with NH₃(7N in methanol), and the eluent was

concentrated to provide the title compound (24 mg, 35% yield). ¹H

NMR (400 MHz, DMSO-d₆) δ 9.87 (s, 1H), 8.92 (t, J = 5.9 Hz,

1H), 7.57-7.48 (m, 4H), 7.23 (t, J = 7.8 Hz, 1H), 7.03 (d, J =

8.5 Hz, 1H), 6.98 (d, J= 7.6 Hz, 1H), 4.43 (d, J= 5.9 Hz, 2H), 3.81

(s, 3H), 3.80 (s, 3H), 2.92 (d, J = 11.3 Hz, 1H), 2.85 (d, J = 11.8

Hz, 1H), 2.39 (td, J = 11.8, 2.7 Hz, 1H), 2.28 (t, J = 7.6 Hz, 2H),

2.18-2.08 (m, 1H), 1.75 (d, J = 13.8 Hz, 1H), 1.60-1.21 (m, 5H),

1.04-0.93 (m, 1H). ESMS: m/z 426 (M þ H).

N-{3-[(3,4-Dimethoxy-phenylcarbamoyl)-methyl]-phenyl}-3-pipe-

ridin-3-yl-propionamide (29). 3-Nitrophenylacetic acid (136 mg,

0.75 mmol) and 3,4-dimethoxyaniline (115 mg, 0.75 mmol) were

coupled using the standard procedure with NMM. The isolate

was purified by preparative HPLC using an acetonitrile/water/

formic acid gradient to provide N-(3,4-dimethoxy-phenyl)-2-(3-

nitro-phenyl)-acetamide (36 mg, 15% yield). This intermediate

(36 mg, 0.114 mmol) was dissolved in methanol (1 mL) and

ammonium formate (69 mg, 1.1 mmol) then Pd/C (5 mg) were

added. The reaction vial was sealed and agitated at 45 °C 16 h.

The reaction mixture was filtered, and the filtrate was diluted

with ethyl acetate (10 mL). This mixture was washed with

water (2 ꢀ 5 mL) and brine (1 ꢀ 5 mL), dried with Na₂SO₄,

and concentrated to provide the intermediate 2-(3-amino-

phenyl)-N-(3,4-dimethoxy-phenyl)-acetamide (10 mg, 31%

yield). ESMS: m/z 287 (M þ H). HPLC: 95%. 3-(2-Carboxy-

ethyl)-piperidine-1-carboxylic acid tert-butyl ester (12.9 mg,

0.05 mmol) and the intermediate prepared above (10 mg,

0.035 mmol) were coupled using the standard procedure with

NMM to provide 3-(2-{3-[(3,4-dimethoxy-phenylcarbamoyl)-

methyl]-phenylcarbamoyl}-ethyl)-piperidine-1-carboxylic acid

tert-butyl ester (19 mg, 72% yield). This intermediate

was dissolved directly in DCM (1 mL) and treated with SCX

(136 mg, 0.12 mmol). The resulting mixture was agitated 16 h,

filtered, and the sorbent was washed with DCM (2 ꢀ 2 mL) and

methanol (2 ꢀ 2 mL). The product was eluted with NH₃(7N in

methanol), and the eluent was concentrated. The isolate was

purified by preparative HPLC using an acetonitrile/water/for-

mic acid gradient providing the title compound (6.5 mg, 38%

1

yield). H NMR (400 MHz, methanol-d₄) δ 7.57 (s, 1H), 7.46

(d, J = 8.1 Hz, 1H), 7.35-7.22 (m, 2H), 7.09 (d, J = 7.6 Hz, 1H),

7.02 (dd, J = 8.6, 2.4 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 3.79

(s, 3H), 3.78 (s, 3H), 3.63 (s, 2H), 3.39-3.26 (m, 2H), 2.87 (td,

J = 12.8, 3.1 Hz, 1H), 2.65 (t, J = 12.0 Hz, 1H), 2.49-2.37 (m,

2H), 1.95 (t, J = 16.3 Hz, 2H), 1.85-1.53 (m, 4H), 1.32-1.15

(m, 1H). ESMS: m/z 426 (M þ H).

3,4-Dimethoxy-N-[3-(3-piperidin-3-yl-propionylamino)-benzyl]-

benzamide (28). 3,4-Dimethoxybenzoic acid (100 mg, 0.55

mmol) and 3-(aminomethyl)-1-N-boc-aniline (111 mg, 0.5 mmol)

were coupled using the standard procedure with NMM to

provide {3-[(3,4-dimethoxy-benzoylamino)-methyl]-phenyl}-car-

bamic acid tert-butyl ester (190 mg, 89% yield). This intermediate

was dissolved in DCM (2 mL). SCX (1.14 g, 1.0 mmol) was added

to the solution, and the resulting mixture was agitated for 48 h. The

reaction mixture was filtered and the sorbent was washed with

DCM (2 ꢀ 2 mL) and methanol (2 ꢀ 2 mL). The product was

eluted with NH₃(7N in methanol). The eluent was concentrated to

provide N-(3-amino-benzyl)-3,4-dimethoxy-benzamide (112 mg,

78% yield). ESMS: m/z 287 (M þ H). HPLC: 100%. 3-(2-Carboxy-

ethyl)-piperidine-1-carboxylic acid tert-butyl ester (41.2 mg, 0.16

mmol) and the intermediate prepared above, N-(3-amino-benzyl)-

3,4-dimethoxy-benzamide (45.8 mg, 0.16 mmol), were coupled using

the standard procedure with NMM to provide 3-(2-{3-[(3,4-di-

methoxy-benzoylamino)-methyl]-phenylcarbamoyl}-ethyl)-piper-

idine-1-carboxylic acid tert-butyl ester (84 mg, 100% yield).

ESMS: m/z 526 (M þ H). HPLC: 100%. This intermediate

(84 mg 0.16 mmol) was dissolved directly in DCM (1 mL) and

treated with SCX (400 mg, 0.35 mmol). The resulting mixture was

N-[3-(4-Dimethylaminomethyl-phenylcarbamoyl)-benzyl]-3-

methoxy-benzamide (30). 3-Methoxybenzoic acid (40 mg, 0.26

mmol) and 53 (40 mg, 0.14 mmol) were coupled using the

standard procedure with DIEA. The isolate was purified by

preparative HPLC using an acetonitrile/water/formic acid gra-

dient providing the title compound (16 mg, 27% yield). ¹H

NMR (400 MHz, CDCl₃) δ 8.75 (s, 1H), 7.91 (s, 1H), 7.83

(d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 7.8 Hz,

1H), 7.47 (d, J = 7.7 Hz, 1H), 7.46-7.42 (m, 1H), 7.41-7.30

(m, 4H), 7.06 (ddd, J = 7.6, 2.6, 1.6 Hz, 1H), 7.01 (t, J = 5.8 Hz,

1H), 4.70 (d, J = 5.9 Hz, 2H), 3.92 (s, 2H), 3.85 (s, 3H), 2.55

(s, 6H). ESMS: m/z 418 (M þ H).

Compounds 31-37 were prepared from 53 and the corres-

ponding substituted benzoic acid according to the procedure

described for the synthesis of 30.

N-[3-(4-Dimethylaminomethyl-phenylcarbamoyl)-benzyl]-4-

methoxy-benzamide (31). Yield 14%. ¹H NMR (400 MHz,

CDCl₃) δ 8.82 (br, 1H), 7.90 (s, 1H), 7.82 (d, J = 8.8 Hz, 3H),

7.75 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 7.2 Hz, 1H), 7.47 (d, J =

7.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.7 Hz, 3H),

4.69 (d, J = 5.8 Hz, 2H), 3.96 (s, 2H), 3.86 (s, 3H), 2.57 (s, 6H).

ESMS: m/z 418 (M þ H).

Article

N-[3-(4-Dimethylaminomethyl-phenylcarbamoyl)-benzyl]-3,4-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 773

reaction mixture was purified directly by preparative HPLC

using an acetonitrile/water/formic acid gradient to provide

3-(2-{3-[3-(3-cyano-phenyl)-ureidomethyl]-benzoylamino}-ethyl)-

piperidine-1-carboxylic acid tert-butyl ester (36 mg, 71% yield).

ESMS: m/z 506 (M þ H). HPLC: 100%. This intermediate

(36 mg, 0.07 mmol) was treated with 0.5 M HCl in dioxane

(1 mL, 0.5 mmol). Methanol (0.5 mL) was added to solublize,

and the resulting mixture was agitated for 1 h then concentrated.

The isolate was purified by preparative HPLC using an aceto-

nitrile/water/formic acid gradient providing the title compound

(20 mg, 70% yield). ¹H NMR (400 MHz, DMSO) δ 9.96 (s, 1H),

8.53 (t, J = 5.6 Hz, 1H), 8.07-7.94 (m, 1H), 7.90-7.76 (m, 2H),

7.70 (d, J = 7.5 Hz, 1H), 7.65 (dq, J = 8.4, 1 Hz, 1H), 7.48-7.37

(m, 3H), 7.33-7.30 (m, 1H), 4.34 (d, J = 5.8 Hz, 2H), 3.49-3.27

(m, 2H), 3.19 (d, J = 13.0 Hz, 1H), 3.11 (d, J = 11.9 Hz, 1H),

2.75-2.61 (m, 1H), 2.55-2.45 (m, 1H), 1.84 (d, J = 14.6 Hz,

1H), 1.78-1.60 (m, 2H), 1.59-1.40 (m, 3H), 1.17-1.04 (m, 1H).

ESMS: m/z 406 (M þ H).

dihydroxy-benzamide (32). Yield 10%. ¹H NMR (400 MHz,

methanol-d₄) δ 7.93 (s, 1H), 7.90-7.82 (m, 3H), 7.61 (d, J = 7.7

Hz, 1H), 7.55-7.47 (m, 3H), 7.35 (d, J = 2.2 Hz, 1H), 7.29 (dd,

J = 8.3, 2.2 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 4.64 (s, 2H), 4.28

(s, 2H), 2.86 (s, 6H). ESMS: m/z 420 (M þ H).

N-[3-(4-Dimethylaminomethyl-phenylcarbamoyl)-benzyl]-4-

1

methoxy-3-methyl-benzamide (33). Yield 29%. H NMR (400

MHz, CDCl₃) δ 8.80 (s, 1H), 7.63 (s, 1H), 7.56 (d, J = 7.7 Hz,

1H), 7.51 (d, J = 8.5 Hz, 2H), 7.45 (dd, J = 8.5, 2.2 Hz, 1H), 7.39

(s, 1H), 7.27 (d, J = 7.7 Hz, 1H), 7.16 (t, J = 7.7 Hz, 1H), 7.09

(d, J = 8.5 Hz, 2H), 6.79 (t, J = 5.9 Hz, 1H), 6.57 (d, J = 8.6 Hz,

1H), 4.39 (d, J = 5.9 Hz, 2H), 3.78 (s, 2H), 3.62 (s, 3H), 2.39

(s, 6H), 1.97 (s, 3H). ESMS: m/z 432 (M þ H).

3-Chloro-N-[3-(4-dimethylaminomethyl-phenylcarbamoyl)-

1

benzyl]-4-methoxy-benzamide (34). Yield 35%. H NMR (400

MHz, CDCl₃) δ 9.18 (s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.85

(s, 1H), 7.81-7.74 (m, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.49 (d, J =

7.7 Hz, 1H), 7.45 (t, J = 5.8 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H),

7.31 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.7 Hz, 1H), 4.60 (d, J =

5.8 Hz, 2H), 4.03 (s, 2H), 3.93 (s, 3H), 2.65 (s, 6H). ESMS: m/z

452 (M þ H).

3-[3-(3,4-Dimethoxy-phenyl)-ureidomethyl]-N-(2-piperidin-3-yl-

ethyl)-benzamide (39). 3-Methoxycarbonyl-benzyl-ammonium

chloride (101 mg, 0.5 mmol) was dissolved in DMF (2 mL) in a

reaction vial. Si-DMA (500 mg, 0.75 mmol) was added, and

the mixture was agitated for 1 h. Subsequently, 3,4-dimethoxy-

phenylisocyanate (0.067 mL, 0.45 mmol) was added, and the

mixture was agitated 16 h. SCX (100 mg, 0.09 mmol) was added,

and the mixture was agitated for 15 min. The sorbents were

filtered and washed with methanol and DMF, and the filtrate

was concentrated to provide 3-[3-(3,4-dimethoxy-phenyl)-

ureidomethyl]-benzoic acid methyl ester (155 mg, 90% yield).

ESMS: m/z 345 (M þ H). HPLC: 100%. The urea (110 mg, 0.32

mmol) was dissolved in DMF (1 mL), and Amberlyst A26 (OH^-

form) (746 mg, 1.0 mmol) was added. The resulting mixture was

agitated for 16 h. The sorbent was filtered and washed with

several portions of DMF then methanol. The product was

eluted with a solution of 20% formic acid in methanol, and

the eluent was concentrated to provide 3-[3-(3,4-

dimethoxyphenyl)-ureidomethyl]-benzoic acid (72 mg, 68%

yield). ¹H NMR (400 MHz, DMSO) δ 13.00 (br, 1H),

8.48 (s, 1H), 7.88 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.52 (d,

J = 7.7 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.16 (s, 1H), 6.81 (s,

2H), 6.65 (t, J = 5.9 Hz, 1H), 4.33 (d, J = 5.8 Hz, 2H), 3.69 (s,

3H), 3.66 (s, 3H). ESMS: m/z 331 (M þ H). HPLC: 100%. This

intermediate (48 mg, 0.145 mmol) and 3-(2-amino-ethyl)-piper-

idine-1-carboxylic acid tert-butyl ester (34.3 mg, 0.15 mmol)

were coupled using the standard procedure with NMM to

provide 3-(2-{3-[3-(3,4-dimethoxy-phenyl)-ureidomethyl]-benzoyl-

amino}-ethyl)-piperidine-1-carboxylic acid tert-butyl ester.

ESMS: m/z 541 (M þ H). HPLC: 100%, which was used

directly. The intermediate was dissolved in DCM (1 mL) and

treated with SCX (454 mg, 0.4 mmol), and the resulting mixture

was agitated for 16 h. The mixture was filtered, and the sorbent

was washed with several portions of DCM and methanol. The

product was eluted with NH₃(7N in methanol). The eluent was

concentrated to provide the title compound (56 mg, 88% yield).

¹H NMR (400 MHz, DMSO) δ 8.53-8.39 (m, 2H), 7.77 (s, 1H),

7.70 (d, J = 5.3 Hz, 1H), 7.48-7.37 (m, 2H), 7.19 (s, 1H), 6.82

(s, 2H), 6.63 (t, J = 5.9 Hz, 1H), 4.33 (d, J = 5.7 Hz, 2H), 3.70

(s, 3H), 3.68 (s, 3H), 3.41-3.25 (m, 4H), 3.00 (d, J = 13.7 Hz,

2H), 1.68 (d, J = 13.7 Hz, 2H), 1.48-1.40 (m, 3H), 1.18-1.04

(m, 2H). ESMS: m/z 441 (M þ H).

N-[3-(4-Dimethylaminomethyl-phenylcarbamoyl)-benzyl]-

4-methoxy-3-trifluoromethyl-benzamide (35). Yield 26%. ¹H

NMR (400 MHz, CDCl₃) δ 9.22 (s, 1H), 8.13 (d, J = 1.9 Hz,

1H), 8.10 (dd, J = 8.7, 2.1 Hz, 1H), 7.93 (s, 1H), 7.80 (d, J =

7.8 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.62 (t, J = 5.8 Hz, 1H),

7.53 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.32 (d, J =

8.4 Hz, 2H), 7.01 (d, J = 8.7 Hz, 1H), 4.65 (d, J = 5.8 Hz, 2H),

3.97 (s, 2H), 3.94 (s, 3H), 2.58 (s, 6H). ESMS: m/z 486 (M þ H).

3-Chloro-N-[3-(4-dimethylaminomethyl-phenylcarbamoyl)-

benzyl]-4-trifluoromethoxy-benzamide (36). Yield 30%. ¹H NMR

(400 MHz, DMSO) δ 10.32 (s, 1H), 9.36 (t, J = 5.8 Hz, 1H), 8.21

(d, J = 2.0 Hz, 1H), 8.14 (s, 1H), 8.00 (dd, J = 8.6 Hz, 2.0 Hz,

1H), 7.89 (s, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.78-7.70 (m, 3H),

7.57-7.48 (m, 2H), 7.32 (d, J = 8.4 Hz, 1H), 4.58 (d, J = 5.8 Hz,

2H), 2.32 (s, 6H), buried CH₂. ESMS: m/z 506 (M þ H).

4-Cyano-N-[3-(4-dimethylaminomethyl-phenylcarbamoyl)-

benzyl]-benzamide (37). Yield 18%. ¹H NMR (400 MHz,

CDCl₃) δ 8.83 (s, 1H), 7.99 (d, J = 8.6 Hz, 2H), 7.92 (s, 1H),

7.82 (d, J = 7.8 Hz, 1H), 7.79-7.69 (m, 4H), 7.57 (d, J = 7.8 Hz,

1H), 7.47 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 5.9 Hz, 1H), 7.38

(d, J = 8.6 Hz, 2H), 4.71 (d, J = 5.8 Hz, 2H), 3.98 (s, 2H), 2.60

(s, 6H). ESMS: m/z 413 (M þ H).

3-[3-(3-Cyano-phenyl)-ureidomethyl]-N-(2-piperidin-3-yl-ethyl)-

benzamide (38). 3-Methoxycarbonyl-benzyl-ammonium chlor-

ide (111 mg, 0.55 mmol) was dissolved in DMF (2 mL) in a

reaction vial. Si-DMA (570 mg, 0.85 mmol) was added, and the

mixture was agitated for 1 h. Subsequently, m-cyanophenyliso-

cyanate (72 mg, 0.5 mmol) was added and the mixture was

agitated 16 h. SCX (114 mg, 0.1 mmol) was added and the

mixture was agitated for 30 min. The solids were filtered and the

solvent was concentrated to provide 3-[3-(3-cyano-phenyl)-

ureidomethyl]-benzoic acid methyl ester: ESMS: m/z 310 (M

þ H). HPLC: 100%, which was used directly. The urea was

dissolved in methanol (2 mL) and DMF (2 mL), and Amberlyst

A26 (OH^-form) (746 mg, 1.0 mmol) was added. The resulting

mixture was agitated for 16 h, and then the sorbent was filtered

and washed with several portions of methanol. The product was

eluted with formic acid (20% in methanol), and the eluent was

concentrated to provide 3-[3-(3-cyano-phenyl)-ureidomethyl]-

benzoic acid (85 mg, 58% yield). ¹H NMR (400 MHz, DMSO) δ

12.90 (br, 1H), 9.13 (s, 1H), 7.96 (t, J = 1.7 Hz, 1H), 7.90 (s, 1H),

7.83 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.4 Hz, 1H), 7.55 (d, J =

7.7 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H),

7.35 (d, J = 7.6 Hz, 1H), 7.04 (t, J = 5.9 Hz, 1H), 4.37 (d, J =

5.8 Hz, 2H). ESMS: m/z 296 (M þ H). HPLC: 95%. This

intermediate (29.5 mg, 0.1 mmol) and 3-(2-amino-ethyl)-pipe-

ridine-1-carboxylic acid tert-butyl ester (27.4 mg, 0.12 mmol)

were coupled using the standard procedure with DIEA. The

3-[3-(4-Cyano-phenyl)-ureidomethyl]-N-(2-piperidin-3-yl-ethyl)-

benzamide (40). Common intermediate 54 (38.4 mg, 0.13 mmol)

and 3-(2-amino-ethyl)-piperidine-1-carboxylic acid tert-butyl

ester (34.3 mg, 0.15 mmol) were coupled using the standard

procedure with DIEA. The reaction mixture was purified directly

by preparative HPLC using an acetonitrile/water/formic acid

gradient to provide 3-(2-{3-[3-(4-cyano-phenyl)-ureidomethyl]-

benzoylamino}-ethyl)-piperidine-1-carboxylic acid tert-butyl

ester (26 mg, 40% yield). ESMS: m/z 506 (M þ H). HPLC:

100%. The intermediate (26 mg, 0.051 mmol) was treated with

774 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2

Morwick et al.

0.5 M HCl in dioxane. Methanol (0.5 mL) was added to solublize,

and the resulting mixture was agitated for 1 h then concentrated

to provide the title compound, HCl salt (20.5 mg, 99% yield).

¹H NMR (400 MHz, DMSO) δ 9.63 (s, 1H), 8.82 (d, J = 10.4 Hz,

1H), 8.58-8.45 (m, 2H), 7.81 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H),

7.65 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.49-7.39

(m, 2H), 7.18 (t, J = 5.8 Hz, 1H), 4.37 (d, J = 5.7 Hz, 2H),

3.40-3.09 (m, 5H), 2.74 (dd, J = 24.0, 11.2 Hz, 1H), 1.86 (d, J =

14.0 Hz, 1H), 1.80-1.70 (m, 2H), 1.65-1.37 (m, 3H), 1.14 (dd,

J = 25.8, 11.0 Hz, 1H). ESMS: m/z 406 (M þ H).

5.9 Hz, 2H), 4.17 (s, 2H), 3.31-3.21 (m, 2H), 2.88 (t, J = 5.5 Hz,

2H). ESMS: m/z 426 (M þ H).

3-[3-(4-Cyano-phenyl)-ureidomethyl]-N-(4-dimethylaminomethyl-

phenyl)-benzamide (44). Common intermediate 54 (23 mg, 0.078

mmol) and4-dimethylaminomethylaniline (14.3mg, 0.095 mmol)

were coupled using the standard procedure with DIEA. The

isolate was purified by preparative HPLC using an acetonitrile/

water/formic acid gradient to provide the title compound (28 mg,

76% yield). ¹H NMR (400 MHz, DMSO) δ 10.10 (s, 1H), 9.17

(s, 1H), 7.70 (s, 1H), 7.66 (d, J = 7.0 Hz, 1H), 7.55 (d, J = 8.5 Hz,

2H), 7.50 (d, J = 8.9 Hz, 2H), 7.43 (d, J = 8.9 Hz, 2H), 7.37-

7.29 (m, 2H), 7.10 (d, J = 8.5 Hz, 2H), 6.93 (t, J = 5.9 Hz, 1H),

4.23(d, J = 5.9 Hz, 2H), 3.26 (s, 2H), 2.02 (s, 6H). ESMS: m/z 428

(M þ H).

N-{3-[3-(4-Cyano-phenyl)-ureidomethyl]-phenyl}-3-piperidin-

3-yl-propionamide (41). 3-t-Butoxycarbonylamino-benzyl-am-

monium chloride (103.5 mg, 0.4 mmol) was dissolved in DMF

(1 mL) in a reaction vial, and Si-DMA (350 mg, 0.525 mmol) was

added. The resulting mixture was agitated for 1 h, after which

time p-cyanophenylisocyanate(52mg, 0.36 mmol) was addedand

the reaction mixture was agitated 16 h. SCX (113 mg, 0.1 mmol)

was added, and the mixture was agitated an additional 30 min.

The reaction mixture was filtered and the sorbent was washed

with DMF. The filtrate was concentrated to provide {3-[3-(4-

cyano-phenyl)-ureidomethyl]-phenyl}-carbamic acid tert-butyl

ester (140 mg, 95% yield). ESMS: m/z 367 (M þ H). HPLC:

100%. The isolate was dissolved in DCM (5 mL) and treated with

TFA (1 mL), and the resulting mixture was stirred at room

temperature for 1.5 h. The solution was concentrated to provide

1-(3-amino-benzyl)-3-(4-cyano-phenyl)-urea (96 mg, 94% yield):

ESMS: m/z 267 (M þ H). HPLC: 94%. 3-(2-Carboxy-ethyl)-

piperidine-1-carboxylic acid tert-butyl ester (38.6 mg, 0.15

mmol), and the urea prepared above (40 mg, 0.15 mmol) were

coupled using the standard procedure with NMM to provide

3-(2-{3-[3-(4-cyano-phenyl)-ureidomethyl]-phenylcarbamoyl}-

ethyl)-piperidine-1-carboxylic acid tert-butyl ester (63 mg, 83%

yield). ESMS: m/z 506 (M þ H). HPLC: 74%. This intermediate

was dissolved in DCM (1 mL) and treated with SCX (341 mg, 0.3

mmol). The resulting mixture was agitated for 16 h. The mixture

was filtered and the sorbent was washed with several portions of

DCM and methanol. The product was eluted with NH₃(7N in

methanol). The eluent was concentrated and the isolate was

purified by preparative HPLC using an acetonitrile/water/formic

acid gradient providing the title compound (9 mg, 18% yield). ¹H

N-(4-Aminomethyl-phenyl)-3-[3-(4-cyano-phenyl)-ureidomethyl]-

benzamide (45). Prepared according to the procedure used for the

synthesis of 42: 85% yield. ¹H NMR (400 MHz, DMSO) δ 10.38

(s, 1H), 9.86 (s, 1H), 7.89 (s, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.79 (d,

J = 8.5 Hz, 2H), 7.79-7.61 (m, 5H), 7.55-7.47 (m, 2H), 7.41 (d,

J = 8.5 Hz, 2H), 4.40 (d, J = 5.8 Hz, 2H), 3.94 (s, 2H). ESMS: m/

z 400 (M þ H).

3-[3-(3-Chloro-4-cyano-phenyl)-ureidomethyl]-N-(3-dimethyl-

aminomethyl-benzyl)-benzamide (46). 2-Chloro-4-aminobenzo-

nitrile (305 mg, 2 mmol) and carbonyldiimidazole (324 mg,

2 mmol) were combined in a reaction vial, and DMF (5 mL)

was added. The resulting mixture was heated at 60 °C for 16 h,

and the product, 1,3-bis-(3-chloro-4-cyano-phenyl)-urea, was

isolated by filtration and washed with several portions of 1N

HCl (220 mg, 73% yield). ESMS: m/z 331 (M þ H). HPLC:

100%. This intermediate (200 mg, 0.6 mmol) was dispersed in

DMF (2 mL) in a microwave vial, and a solution of 53 (200 mg,

0.7 mmol), in DMF (1 mL), was added. The vial was sealed and

heated in the microwave at 150 °C for 70 min. The reaction

mixture was purified directly by preparative HPLC using an

acetonitrile/water/formic acid gradient, then further purified by

catch and release with Varian SCX cartridges to provide the title

compound (60 mg, 18.4% yield). ¹H NMR (400 MHz, DMSO)

δ 10.26 (s, 1H), 9.48 (s, 1H), 7.96-7.92 (m, 1H), 7.89-7.82 (m,

2H), 7.79 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H),

7.55-7.46 (m, 2H), 7.41 (dd, J = 8.7, 2.0 Hz, 1H), 7.26 (d, J =

8.4 Hz, 2H), 7.16 (t, J = 5.9 Hz, 1H), 4.41 (d, J = 5.9 Hz, 2H),

3.39 (s, 2H), 2.51 (s, 6H). ESMS: m/z 462 (M þ H).

NMR (400 MHz, DMSO)

δ

9.97 (s, 1H), 9.95

(s, 1H), 7.76-7.59 (m, 5H), 7.53 (d, J = 8.1 Hz, 1H), 7.47 (s,

1H), 7.24 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 4.26 (d, J =

5.8 Hz, 2H), 3.16 (t, J = 12.5 Hz, 2H), 2.77-2.63 (m, 1H),

2.50-2.42 (m, 1H), 2.33 (t, J = 7.4 Hz, 2H), 1.82-1.71 (m, 2H),

1.65-1.45 (m 4H), 1.11 (dd, J = 22.9, 10.4 Hz, 1H). ESMS: m/z

406 (M þ H).

3-[3-(4-Amido-phenyl)-ureidomethyl]-N-(4-dimethylaminomethyl-

phenyl)-benzamide (47). Compound 44 (20 mg, 0.058 mmol) was

dissolved in 0.5 mL of 20% H₂SO₄in TFA. The resulting

mixture was agitated for 36 h. The reaction mixture was con-

centrated, and ice was added followed by 0.7 mL methanol

and the mixture was purified directly by preparative HPLC

using an acetonitrile/water/formic acid gradient providing

3-[3-(4-Cyano-phenyl)-ureidomethyl]-N-(2-piperidin-4-yl-ethyl)-

benzamide (42). Common intermediate 54 (30 mg, 0.10 mmol) and

4-(2-carboxy-ethyl)-piperidine-1-carboxylic acid tert-butyl ester

(27.4 mg, 0.12 mmol) were coupled using the standard procedure

with NMM to provide 4-(2-{3-[3-(4-cyano-phenyl)-ureidomethyl]-

benzoylamino}-ethyl)-piperidine-1-carboxylic acid tert-butyl ester

(50 mg, 97% yield). ESMS: m/z 506 (M þ H); HPLC: 100%. This

intermediate was dissolved in DCM (1 mL), and TFA (0.3 mL)

was added. The resulting mixture was agitated for 2 h and then

concentrated. The isolate was purified by preparative HPLC using

an acetonitrile/water/formic acid gradient providing the title com-

pound (37 mg, 91% yield). ¹H NMR (400 MHz, DMSO) δ 9.63

(s, 1H), 8.48 (t, J = 5.5 Hz, 1H), 7.78 (s, 1H), 7.75-7.57 (m, 5H),

7.49-7.34 (m, 3H), 4.35 (d, J = 5.8 Hz, 2H), 3.34-3.20 (m, 4H),

2.81 (dd, J = 14.0, 11.3 Hz, 2H), 1.85 (d, J = 12.6 Hz, 2H),

1.68-1.41 (m, 3H), 1.27 (dd, J = 23.4, 10.7 Hz, 2H). ESMS: m/z

406 (M þ H).

1

the title compound (13 mg, 50% yield). H NMR (400 MHz,

DMSO) δ 10.29 (s, 1H), 8.95 (s, 1H), 8.16 (s, 1H), 7.88 (s, 1H),

7.84 (d, J = 7.0 Hz, 1H), 7.79-7.71 (m, 4H), 7.55-7.44 (m, 3H),

7.29 (d, J = 8.4 Hz, 2H), 7.15 (s, 1H), 6.89 (t, J = 5.8 Hz, 1H),

4.40 (d, J = 5.8 Hz, 2H), 3.50 (s, 2H), 2.24 (s, 6H). ESMS: m/z

446 (M þ H).

3-[3-(3-Chloro-4-amido-phenyl)-ureidomethyl]-N-(4-dimethyl-

aminomethyl-phenyl)-benzamide (48). Prepared from 46 accord-

ing to the procedure used for the synthesis of 47: 60% yield.

¹H NMR (400 MHz, DMSO) δ 10.30 (s, 1H), 8.99 (s, 1H), 7.88

(s, 1H), 7.84 (d, J = 7.0 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H),

7.72-7.66 (m, 2H), 7.57-7.47 (m, 2H), 7.41 (s, 1H), 7.37-7.31

(m, 3H), 7.26 (dd, J = 8.4, 1.9 Hz, 1H), 6.91 (t, J = 5.3 Hz, 1H),

4.40 (d, J = 5.8 Hz, 2H), 3.63 (s, 2H), 2.33 (s, 6H). ESMS: m/z

480 (M þ H).

3-[3-(4-Cyano-phenyl)-ureidomethyl]-N-(1,2,3,4-tetrahydro-iso-

quinolin-7-yl)-benzamide (43). Prepared according to the proce-

dure used for the synthesis of 42: 70% yield. ¹H NMR

(400 MHz, DMSO) δ 10.29 (s, 1H), 9.47 (s, 1H), 7.88 (s, 1H),

7.83 (d, J = 8.6 Hz, 1H), 7.69-7.59 (m, 5H), 7.58-7.46 (m, 3H),

7.24 (t, J = 5.8 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 4.40 (d, J =

3-[3-(3-Amido-phenyl)-ureidomethyl]-N-(4-dimethylaminomethyl-

phenyl)-benzamide (49). Intermediate 53 (36.8 mg, 0.13 mmol)

was dissolved in DMF (1 mL) and 3-cyanophenylisocyanate

(21.6 mg, 0.15 mmol) was added. The reaction mixture was

agitated for 72 h then purified directly by preparative HPLC

using an acetonitrile/water/formic acid gradient to provide

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 775

3-[3-(3-cyano-phenyl)-ureidomethyl]-N-(4-dimethylaminomethyl-

phenyl)-benzamide (28 mg, 50.4% yield). This intermediate was

then converted to the title compound according to the method

described for the synthesis of 47: 45% yield. ¹H NMR

(400 MHz, DMSO) δ 10.29 (s, 1H), 8.80 (s, 1H), 7.91-

7.81 (m, 4H), 7.75 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 7.0 Hz,

1H), 7.55-7.47 (m, 2H), 7.39 (d, J = 7.7 Hz, 1H), 7.29 (d, J =

7.9 Hz, 4H), 6.81 (t, J = 6.0 Hz, 1H), 4.40 (d, J = 5.8 Hz, 2H),

3.52 (s, 2H), 2.25 (s, 6H). ESMS: m/z 446 (M þ H).

rod on one side and the other side connected to a force

transducer and placed into 37 °C temperature-controlled tissue

baths containing PBS that was constantly oxygenated with

95% O₂and 5% CO₂. Isometric force was continually mea-

sured and the data collected by a digital acquisition system.

The rings were placed under a preload of 2.5g of force, which

was continuously adjusted during a 1 h equilibration period to

serve as baseline force. This amount of force was chosen from a

series of preliminary force-force experiments, which showed

near maximum contraction with 125 mM KCl. Rings were

contracted with 50 mM KCL to obtain the maximum contrac-

tion level for normalization. Following a washout period of

30 min, the rings were preconstricted with 10^-6M phenylephr-

ine and relaxed with a bolus dose of 10^-7M acetylcholine to

check the integrity of the endothelium. Following a second

washout period of 30 min, rings were preconstricted a second

time with 10^-6M phenylephrine and the contraction allowed

to stabilize. A cumulative dose response of a Rho-kinase

inhibitor was tested in a DMSO vehicle at a 1:1000 dilution

using half log intervals. After each dose of inhibitor, the

response was allowed to stabilize before the addition of the

next dose. Following the cumulative dose response with in-

hibitor, the tissues were washed 3ꢀ in PBS and allowed to

equilibrate at resting tension. A second KCL contraction was

performed as stated above to check the viability of the tissue.

Following this, a second phenylephrine contraction and acet-

ylcholine bolus dose were given as above to check for the

integrity of the endothelium following inhibitor testing. The

effect of the Rho-kinase inhibitors were expressed as a percen-

tage relaxation from the phenylephrine-induced contraction at

each dose. The IC₅₀for each inhibitor was determined from the

concentration that produced 50% relaxation from the pheny-

lephrine-induced contraction. The data for each inhibitor

represents the mean from four different segments from four

different rats.

3-[3-(4-Amido-phenyl)-ureidomethyl]-N-(1,2,3,4-tetrahydro-

isoquinolin-7-yl)-benzamide (50). Prepared from 43 according

to the procedure used for the synthesis of 47: 72% yield.

¹H NMR (400 MHz, DMSO) δ 10.26 (s, 1H), 9.08 (s, 1H),

7.90 (s, 1H), 7.83 (d, J = 7.1 Hz, 1H), 7.80-7.73 (m, 3H),

7.59 (s, 1H), 7.56-7.43 (m, 5H), 7.19-7.08 (m, 2H), 7.03

(t, J = 5.8 Hz, 1H), 4.39 (d, J = 5.8 Hz, 2H), 4.04 (s, 2H),

3.14 (t, J = 5.8 Hz, 2H), 2.79 (t, J = 5.7 Hz, 2H). ESMS: m/z 444

(M þ H).

ROCK1/ROCK 2 Dose-Response Assay. The activity of

ROCK1 (1-477) and ROCK2 (1-543) kinases was measured

utilizing Cambrex PKLight ATP detection reagent (Cambrex

catalogue no. LT27-200), a homogeneous assay technology

using luciferin-luciferase to quantify residual ATP. The assay

was performed in 96-well half area, white, nonbinding surface

microtiter plates (Corning, catalogue no. 3642) in assay buffer

consisting of 25 mM HEPES (pH 7.5), 10 mM MgCl₂, 50 mM

KCl, 0.2% BSA, 0.01% CHAPS, 100 μM Na₃VO₄, and 200 μM

TCEP. Test compounds received as 5 mg/mL DMSO stocks

were serially diluted 1:3 in DMSO for the 10-point concentra-

tion response. The DMSO dilutions were further diluted in the

assay buffer, and 10 μL of this dilution was added to the assay

plate, for a final starting assay concentration of 3 μg/mL in 1%

DMSO. ROCK1 (12.6 μM stock) was diluted to 5 nM in assay

buffer, and 30 μL was added to the assay, for a final concentra-

tion of 3 nM in a total volume of 30 μL. ROCK2 (29.0 μM stock)

was diluted to 8.33 nM in assay buffer, and 30 μL was added to

the assay for a final concentration of 5 nM in a total volume of

30 μL. After 15 min of preincubation of the test compounds with

the kinase, a 10 μL volume of a mixture of 3.75 μM ATP, and

2.5 μM peptide substrate (AKRRRLSSLRA,^34a,34bANA-

SPEC, catalogue no. 27192) diluted in assay buffer was added

to each well for a final concentration of 750 nM ATP and

500 nM peptide. The kinase reaction mixture was incubated for

90 min at 28 °C. Following incubation, 30 μL of ATP detection

reagent diluted in reconstitution buffer was added to the assay

plates, and the resulting mixtures were incubated at room

temperature for 15 min. The relative light unit (RLU) signal

was measured on the Analyst plate reader (Molecular Devices)

in luminescence mode. The RLU signals were converted to

percent of control (POC) values using the formula POC =

100(BCTRL - Signal)/(BCTRL - PCTRL), where signal is the

test well signal, BCTRL is the average of background (negative

control) well signals on the plate, and PCTRL is the average of

positive control well signals on the plate. For the concentration-

responsive compounds, POC values as a function of test com-

pound concentration were fitted to a four parameter logistic

equation of the form: y = A þ (B - A)/[1 þ (x/C)^D], where A, B,

C, and D are fitted parameters (parameter B is fixed at zero

POC) and x and y are the independent and dependent variables,

respectively. The IC₅₀(50% inhibitory concentration) was

determined as the inflection point parameter, C.

Acknowledgment. We thank Rachel Kroe-Barrett for the

ITC experiments.

Supporting Information Available: ATP competition studies,

data comparison for luciferase inhibitors using IMAP and

luciferase assay formats, IMAP dose-response assay method,

additional kinase selectivity data, and target independent profil-

ing methods. This material is available free of charge via the

Internet at http://pubs.acs.org.

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Article Doi

Hit to lead account of the discovery of bisbenzamide and related ureidobenzamide inhibitors of Rho kinase

DOI: 10.1021/jm9014263

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