Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal model of glaucoma

Article abstract of DOI:10.1016/j.bmcl.2010.04.020

We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used to derive 39, which substantially reduced intr

Full text of DOI:10.1016/j.bmcl.2010.04.020

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3361–3366
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal
model of glaucoma
Robert L. Davis a, , Mehmet Kahraman a,c, Thomas J. Prins a, Yan Beaver a, , Travis G. Cook a,à, Jessica Cramp a,§
,
*
Charmagne S. Cayanan a,–, Elisabeth M. M. Gardiner a,||, Marsha A. McLaughlin b, Abbot F. Clark b,
,
Mark R. Hellberg b, Andrew K. Shiau a,àà, Stewart A. Noble a, Allen J. Borchardt a
a Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
b Alcon Research, Ltd, 6201 South Freeway, Fort Worth, TX 76134, USA
c Aragon Pharmaceuticals, Inc., 4215 Sorrento Valley Blvd., Suite 215, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 February 2010
Revised 7 April 2010
Accepted 8 April 2010
Available online 11 April 2010
We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput
enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used
to derive 39, which substantially reduced intraocular pressure in a monkey model of glaucoma-associ-
ated ocular hypertension.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
ROCK
Rho
Kinase
Adhesion
Impedance
Benzothiophene
ATP competitive
Inhibitor
Glaucoma
Rho-associated coiled-coil containing protein kinases (Rho ki-
nases, or ROCKs) are key effectors of intracellular signaling by
the monomeric GTPase RhoA.1 Activation of RhoA, generally by
extracellular stimuli, enables its binding to ROCKs, which in turn
stimulates their kinase activity.2 Active ROCKs then phosphorylate
secondary effectors of RhoA signaling, which modulate cell motil-
ity, size, division, and differentiation.3
ROCKs are pharmaceutical targets based on their roles in apop-
tosis4–8 and cytoskeleton dynamics.9–11 Many structurally diverse
small molecule ROCK inhibitors have been identified; a number
are in clinical development, and there is one launched drug (Fasu-
dil).12–19 ROCK inhibition protects cells from apoptotic stimuli,
suggesting therapeutic utility for neural injury and neurodegener-
ative diseases.20–22 Because ROCK inhibitors can trigger cytoskele-
ton remodeling and reduce cellular contractility, they can relax
vascular smooth muscle and lower blood pressure in models of
systemic and pulmonary hypertension.23–25 Analogous ROCK
inhibitor mediated cytoskeleton changes in the aqueous humor
outflow tract lead to increased aqueous humor outflow and de-
creased intraocular pressure. This observation has stimulated the
use of ROCK inhibitors as novel agents for the treatment of glau-
coma.15,26–28 Here, we describe optimization of a new series of
benzothiophene-based ROCK inhibitors and the in vivo activity of
39 in a model of glaucoma-associated ocular hypertension.
* Corresponding author. Tel.: +1 858 754 3323; fax: +1 858 754 3301.
E-mail address: rdavis@kalypsys.com (R.L. Davis).
Present address: Dart NeuroScience LLC, 7473 Lusk Blvd., San Diego, CA 92121,
United States.
à
Present address: Oregon Health and Science University, 2525 SW 1st Ave, Suite
120, Portland, OR 97201, United States.
§
Present address: Thermo Fisher Scientific, 10010 Mesa Rim Rd., San Diego, CA
92121, United States.
–
Present address: Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla,
CA 92037, United States.
||
Present address: Genomics Institute of the Novartis Research Foundation, 10675
Benzothiophene 1 (Fig. 1) was identified in an ultra high-
throughput screen (uHTS) of greater than 850,000 compounds. 1
John Jay Hopkins Dr., San Diego, CA 92121, United States.
Present address: University of North Texas Health Science Center, 3500 Camp
Bowie Blvd., Fort Worth, TX 76107, United States.
exhibited an enzymatic IC50 of 1.5
isoforms, showed 5 to 10-fold selectivity over protein kinases A
and C , and appeared to have an ATP competitive mechanism of
lM against ROCK1 and ROCK2
àà
Present address: Ludwig Institute for Cancer Research, UCSD, 9500 Gilman Dr.,
a
#0660, La Jolla, CA 92093, United States.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.020

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R. L. Davis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3361–3366
the expanding interest in label-free technologies for cellular
assays. Since ROCK inhibition profoundly changes cell shape and
decreases cell adhesion32,36, we reasoned that the ACEA/Roche
RT-CESÒ impedance system would enable the quantitative mea-
surement of cellular ROCK inhibition.37
Briefly, cells are seeded on 96-well microtiter plates containing
microelectrode arrays in each well. As adherent cells either prolif-
erate or increase relative adhesion, and consequently resist current
flow, the impedance at constant voltage increases. Conversely,
impedance will decrease as adhesion decreases.38
Figure 1. Structure of compound 1 and reference compounds.
The response of GTM-339 cells to the known ROCK inhibitor
action.29 In a subsequent optimization campaign, substitutions at
the benzothiophene 2 and 5-positions were studied to improve po-
tency and solubility.30 Substitutions at the 3-position will be re-
ported elsewhere. SAR was driven by enzymatic assays of ROCK1
and ROCK2, and a functional cellular assay with a novel label-free
readout.31
Among the confirmed uHTS hits, cellular activity assessed by
visualization of stress fibers in HeLa cells11,32 generally showed lit-
tle correlation with enzymatic IC50s (data not shown), likely a re-
sult of variable cellular permeability and the non-quantitative
nature of the assay. To support cellular SAR, a simple, reliable,
easily quantified assay with throughput to support multiple com-
pounds in dose–response was required. Traditional, label-depen-
dent approaches to study ROCK inhibition in cells include
manual/automated fluorescence microscopy of cytoskeleton rear-
rangements33, or Western blot to detect changes in substrate phos-
phorylation.9 None of these existing approaches met our criteria.
Transepithelial electrical impedance has long been used to as-
sess the integrity of cell monolayers in culture.34,35 More recently,
higher throughput impedance platforms have emerged to address
HMN-115240 (2, Fig. 1 ROCK1 IC50 0.03 0.002
lM) at three con-
centrations is shown in Figure 2A. There is a rapid, dose dependent
decrease in impedance. About one hour after HMN-1152 addition,
the impedance differential stabilizes, and the trace follows a paral-
lel path to the DMSO control. These kinetics are consistent with the
effects of ROCK inhibition studied by cell imaging.33
To correlate the impedance changes with cell morphology
changes, GTM-3 cells were treated with HMN-1152, followed by
indirect immunofluorescence of vinculin, a cytoskeleton protein
associated with cellular focal adhesions (Fig. 2B).41,42 HMN-1152
treatment decreased the vinculin staining density and intensity,
as well as induced a cell shape change, compared to DMSO control-
treated cells. Notably, the myosin light-chain kinase inhibitor ML-
7, which is structurally similar to HMN-1152 but a very weak ROCK
inhibitor (3, Fig. 1, ROCK1 IC50 38
4 lM), showed no similar effect
in the impedance or immunofluorescence assays (data not shown).
We further validated the impedance assay with a panel of small
molecule drugs or drug-like compounds of wide structural diver-
sity and function.43 Based on the high specificity of the impedance
Figure 2. Validation of an impedance assay to study ROCK inhibition in living cells. (A) Impedance (reported as ‘Normalized Cell Index’, Y axis) of GTM-3 cells measured over
time (X axis) after compound addition at about 18 h (arrow). HMN-1152 in DMSO was added, in duplicate, to a final concentration of 1 M (1), 3 M (3), or 10 M (10), versus
DMSO (D) only as negative control. Error bars represent 1 standard deviation. (B) Indirect immunofluorescence of vinculin in GTM-3 cells treated for 3 h with 0.1% DMSO (a)
or 0.1% DMSO and 10 M HMN-1152 (b) in culture media. Bar = 20 m.
l
l
l
l
l

R. L. Davis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3361–3366
3363
Table 1
Exploration of 2-position SAR
Compound
R1
ROCK1
IC50 (lM)
a
1
1.5 (0.2)
Scheme 1. Synthesis of 1. Reagents and conditions: (a) n-BuLi, B(Oi-Pr)3, ꢀ78 °C;
then aq. HCl, rt, 60%; (b) Pd(PPh3)2Cl2, K2CO3, THF, 70 °C, 75%; (c) NH4OH, EtOH,
sealed tube, 80 °C, 82%.
7
8
9
1.2 (0.1)b
>100c
13 (2)
effect observed with ROCK inhibitors, and the complete reversibil-
ity upon compound washout (data not shown), this assay met our
criteria, with sufficient throughput to build cellular SAR. Recently,
Schroter et al. developed a 96-well phospho-myosin light-chain
indirect immunofluorescence assay for ROCK inhibitor SAR devel-
opment.44 The reported EC50s of known ROCK inhibitors indicates
that their assay is more sensitive, which is an advantage in rank
ordering lower potency compounds. By contrast, the complemen-
tary impedance assay has the advantage of speed (e.g., most assays
completed in one to two hours) and simplicity, requiring no special
reagents, fixation, or post-fixation procedures.
Compound 1 was prepared as shown in Scheme 1. Commer-
cially available 4 was converted to boronic acid 5 by lithium halo-
gen exchange of bromine, followed by trapping of the resulting
anion with tri-isopropyl borate according to the procedure of Li
et al.45 Suzuki coupling of 5 and 2,4-dichloropyrimidine yielded 6
in 75% yield, which was then treated with aqueous NH4OH in eth-
anol to give 1 in 82% yield.
10
11
8.3 (2.0)d
28 (9)
12
13
7.1 (1.4)
>10e
a
Values are means of two or more independent experiments; values for ROCK2
enzymatic assays were comparable. The standard error of the mean is shown in
parentheses.
b
By analogy with the pyridine moiety of ROCK inhibitor Y-27632,
compounds 7–13 were prepared to optimize the 2-aminopyrimi-
dine putative donor–acceptor interactions with residues Glu154
and Met156 in the ATP binding site of ROCK1.46,47 The 2-position
of the benzothiophene was elaborated by Suzuki coupling of the
corresponding heteroaryl halides to intermediate 5.
17% maximum efficacy.
No activity at maximum tested concentration (96
44% maximum efficacy.
c
lM).
d
e
Not tested above 10 lM (no activity).
Surprisingly, relatively small changes to the 2-aminopyrimidine
were not tolerated (Table 1). For instance, N-methyl (7) or N,N-di-
methyl (8) 2-aminopyrimidines had negligible activity, while 4-
aminopyrimidine (9) and 2-aminopyridine (10) were much weaker
than 1. Even a 4-pyridine (11), common to several ROCK inhibitors,
was very weak. A 4-azaindole (12) showed comparable, albeit less
potent, activity, while a 3-azaindole (13) was very weak. None of
the compounds showed significant cellular activity up to 10 lM.
Because the 2-aminopyrimidine of 1 did not tolerate changes, this
area of the molecule was not explored further.
To improve potency, we examined potential hydrophobic inter-
actions around the benzothiophene 5-position. Compounds 19–29
were synthesized from 16 as shown in Scheme 2. Intermediate 16
was prepared in five steps, starting with alkylation of 14 with bro-
moacetone, followed by sulfuric acid mediated cyclization to give
15. Acylation of 15 under Friedel–Crafts conditions gave the 2-
acetyl derivative, which was reacted with DMF/DMA to give 16.
Cyclo-condensation of 16 with guanidine hydrochloride under ba-
sic conditions48 formed the 2-aminopyrimidine ring of 17 in 54%
yield. Intermediate 17 was coupled under palladium catalysis with
organo-zinc reagents49, phenol, or aniline50 to give 19–27, 28, and
29, respectively. Analogous chemistry with 6 was inefficient, due
to the lower reactivity at the chlorine.
Scheme 2. Synthesis of 19–29. Reagents and conditions: (a) 1-Bromoacetone,
pyridine, Et2O, rt, 80%; (b) H2SO4 (aq), 110 °C, 10 h, 42%; (c) AlCl3, acetyl chloride,
CS2, 3 h, 51%; (d) DMF-DMA, 80 °C, 12 h, 59%; (e) guanidine hydrochloride, NaOEt,
EtOH, reflux 36 h, 54%; (f) Ar2ZnX, Pd(PPh3)2Cl2, CuI, THF, 70 °C, 50–90%; (g)
Pd(OAc)2, Ar-OH, K3PO4, 2-(di-t-butylphosphino)biphenyl, toluene, reflux, 24 h, 20–
40%; (h) ArNH2, Pd2(dba)3, 1,3-bis(2,6-di-i-propylphenyl)-imidazolium chloride, t-
BuONa, toluene, 100 °C, 40–75%.
Exploration at the 5-position used the comparative reference
1851, with an enzymatic IC50 of 2.9
lM (Table 2). Attaching a phe-
nyl directly to the 5-position (19) resulted in a loss of potency, pos-
sibly due to steric interactions. Moving the phenyl group away

3364
R. L. Davis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3361–3366
from the core with a methylene linker carbon (20) improved po-
tency fivefold over 18 and yielded the first compound of this series
to show a low but detectable cellular effect at 10 lM. While ortho
Since solubility is a key determinant for topical ocular bioavail-
ability in our in vivo model, further optimization was directed at
increasing solubility while maintaining potency and efficacy in the
cellular assay. Initial tests revealed low solubility for compounds
such as 22 (0.04 mM), 25 (0.01 mM) and 29 (0.03 mM).56,57 To
improve solubility, amine substituents with a predicted pKa above
8 were considered. Ideally, the amine would be synthetically acces-
sible, be neutral toward or improve potency, and greatly improve
solubility. Initially, we targeted the methylene linker between the
benzothiophene core and the ‘tail’ phenyl group, since this position
would place the amine in an environment where it might be less
likely to interrupt existing interactions with the binding site.
The first compound in this series, 30, not only improved solubil-
ity (to 0.22 mM), but also improved enzymatic potency 12-fold rel-
ative to its des-amino counterpart (22), and improved cellular
potency by at least threefold (Table 3). The effect of the installed
amine on the potency of 22 prompted a re-examination of the 5-
position SAR. Compounds 30–35 were prepared by converting
intermediate 16 to the nitrile (Scheme 3), followed by reaction
with the appropriate Grignard reagent (in excess) to give an imine,
which was reduced in situ with sodium borohydride.
With the aminomethylene linker, there was now much less dif-
ference between the IC50s of the meta hydroxyl (31)58 and meth-
oxy (30) groups. While the rank difference in cellular EC50s was
preserved (compare to 25 and 22, respectively), there was a major
overall increase in potency. Remarkably, the poor activity of the
para methoxy group improved 80-fold with the aminomethylene
linker (compare 32 and 23), and gained appreciable cellular activ-
ity where there was none detected before. The potency of com-
pounds 33 and 34 provided further evidence of ‘‘flat” SAR for
phenyl substituents when combined with the aminomethylene
linker. In fact, the unmodified phenyl of 35 was just as potent, in
dramatic contrast to 20.
(21) or para (23) methoxy groups on the phenyl of 20 reduced
enzymatic potency, a meta methoxy group (22) was 2 to 3-fold
more potent than 20, although there was no comparable improve-
ment in the cellular assay. Hydroxyl (25)52 or carboxyl (26)53 meta
substituents increased enzymatic potency 10 or 4-fold, respec-
tively, beyond that of 22. Notably, 25 showed significantly greater
cellular activity. By contrast, trifluoromethyl (24) or carboxyme-
thylester (27)54 meta substituents were very weak. Replacement
of the methylene linker carbon in 25 with oxygen (28) led to a loss
of enzymatic and cellular potency, while substitution with nitro-
gen (29) resulted in activity comparable to 25. Thus, 25 repre-
sented the most optimal analog of 1 yet identified.
The selectivity of compound 25 was determined at 10
against a panel of 39 kinases.55 Only two kinases (Aurora B and
MSK1) showed inhibition consistent with low single-digit M po-
lM
l
tency, supporting the notion that this series is not especially indis-
criminate. However, the modest cellular activity of 25 compared to
the benchmark Y-3998317 (cellular EC50 0.79 0.06
support in vivo testing.
lM) did not
Table 2
Exploration of 5-position SAR
Compound R2
ROCK1 IC50
2.9 (0.1)
(l
M) GTM-3 EC50
>10
(lM)
a
a
18
19
20
21
22
23
4.2 (0.4)b
0.58 (0.13)
9.7 (1.2)
>10
>10c
>10
>10d
>10
Table 3
Exploration of tail group SAR with aminomethylene linker
0.23 (0.04)
7.2 (1.1)
R3
ROCK1 IC50
(l
M)
GTM-3 EC50
3.6 (1.1)b
(lM)
a
a
Compound
30
0.02 (0.001)
0.01 (0.001)
24
25
26
27
28
8.6
>10
31
32
0.9 (0.04)
2.6 (0.6)c
0.02 (0.002)
0.06 (0.01)
10 (0.6)
2.9 (0.5)e
>10
0.09 (0.01)
>10
33
34
0.03 (0.002)
0.05 (0.01)
2.7 (0.7)
0.36 (0.003)
>10
1.9 (0.1)d
29
0.02 (0.002)
1.0 (0.2)f
35
0.02 (0.003)
1.4 (0.4)
a
Values are means of two or more independent experiments (except for enzy-
matic assay of 24, n = 1, R2 = 0.99); values for ROCK2 enzymatic assays were com-
a
Values are means of two or more independent experiments; values for ROCK2
enzymatic assays were comparable. The standard error of the mean is shown in
parentheses.
parable. The standard error of the mean is shown in parentheses.
b
35% maximum efficacy at 96
11% maximum efficacy at 10
15% maximum efficacy at 10
50% maximum efficacy at 10
35% maximum efficacy at 10
lM.
lM.
lM.
lM.
lM.
c
d
e
f
b
46% maximum efficacy.
56% maximum efficacy.
67% maximum efficacy.
c
d

R. L. Davis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3361–3366
3365
Table 5
In vivo activity of compound 39
Time (h)
D
IOP (%, L)
D
IOP (%, NL)
0
1
3
6
0
0
ꢀ19.6 4.4
ꢀ24.1 6.0
ꢀ26.0 5.5
ꢀ20.0 6.1
ꢀ13.9 6.2
ꢀ12.0 7.2
Mean and standard error of percentage change in intraocular pressure [IOP; lasered
eye (L), non-lasered eye (NL)] for treated animals [2 ꢁ 25 l of a 0.5% (w/v) sus-
pension, 250 g/eye total] were measured up to 6 h after topical instillation. For
reference, Y-39983 [1 ꢁ 30 l of a 0.1% (w/v) suspension, 30 g/eye total] yielded a
l
Scheme 3. Synthesis of 30–41. Reagents and conditions: (a) Zn(CN)2, Zn, Pd[P(t-
Bu)3]2, DMAC, 90%; (b) ArMgX, THF, 70 °C, 16 h; then NaBH4, MeOH 0 °C to rt, 29–
90%.
l
l
l
28.1% and a 20.1% maximum IOP reduction in the lasered and non-lasered eye,
respectively. Mean baseline measurements for the lasered and non-lasered eyes
were 35.8 mm Hg and 26.1 mm Hg, respectively.
In silico docking of 30 into the ATP binding site of ROCK2 (PDB:
2F2U) suggested an explanation for the altered SAR relative to
compounds without the aminomethylene linker. With the 2-ami-
nopyrimidine positioned in the ATP binding site to make hydrogen
bonds with Glu170 and Met172, the methylene linker amine could
be positioned within hydrogen bonding distance of the carboxyl
side chain of Asp232 (analogous to Asp216 of ROCK1). This strong
polar interaction could reduce the importance of the phenyl sub-
stituents and account for the shift in SAR (model not shown).
We introduced 5-position tail groups smaller than phenyl with
the goal of retaining potency in the context of the aminomethylene
linker, and possibly improving corneal permeability.59 While sub-
stitution of the phenyl group with hydrogen (36) greatly reduced
enzymatic and cellular potency compared to 35, increasing the size
of an attached alkyl group from methyl (37) to ethyl (38) to n-pro-
pyl (39) lead to corresponding improvements in enzymatic po-
tency of about fivefold, 15-fold, and 100-fold relative to 36 (Table
4). In addition, the rank order of cellular activity showed a similar
effect. Branched alkyl groups were well tolerated, both alpha (40)
and beta (41) to the aminomethylene linker.
pressure was achieved by 1 h after dosing. The IOP reduction was
sustained in the hypertensive eye through at least six hours. The
efficacy of 39 (250
lg/eye) was comparable to the efficacy of Y-
39983 (30 g per eye), providing in vivo proof-of-activity.
l
In conclusion, we have discovered a new benzothiophene con-
taining series of ROCK inhibitors based on an uHTS hit. The devel-
opment of a novel medium throughput label-free live-cell assay for
ROCK inhibition allowed us to optimize cellular SAR in parallel
with enzymatic assays, and guided compound selection for
in vivo pharmacology. In principle, this application of electrical
impedance should translate to other targets with dose-propor-
tional effects on cell adhesion, such as G12/13 coupled G-protein
receptors.
Installation of an aminomethylene linker between the benzo-
thiophene core and the tail group had a profound impact on both
the enzymatic and cellular SAR, leading to compounds suitable
for topical application to the eye. A compound with in vitro activity
comparable to the clinical candidate Y-39983 significantly lowered
intraocular pressure in vivo, validating this series for further
medicinal chemistry optimization.
Based on their low lM (37, 38, 40, 41) or sub-lM (39) cellular
EC50s, low molecular weight, and improved solubility (>0.45 mM),
these compounds have attractive properties comparable to other
advanced ROCK inhibitors, such as the clinical candidate Y-
39983.15,17 To demonstrate in vivo activity, we tested compound
39 by topical application to the eyes of conscious cynomolgus
monkeys with laser-induced trabeculoplasty and ocular hyperten-
sion (Table 5).60 Statistically significant reduction of intraocular
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R3
ROCK1 IC50
2.2 (0.5)
(
lM)
GTM-3 EC50
>10
(lM)
a
a
Compound
36
37
38
39
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4.2 (0.6)
1.7 (0.3)
0.6 (0.1)
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a
Values are means of two or more independent experiments; values for ROCK2
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42. GTM-3 cells were plated on glass coverslips one day before use, treated with
compound or DMSO negative control for 3 h, fixed with 2% paraformaldehyde
in PBS for 10 min at rt, and immuno-stained with an anti-vinculin mAb (Sigma)
and goat anti-mouse (Fab0)2 Alexa-488 secondary antibody (Invitrogen) under
conventional conditions. Images were acquired on a Zeiss Axiovert 200 M
28. Rao, P. V.; Deng, P. F.; Kumar, J.; Epstein, D. L. Invest. Ophthalmol. Vis. Sci. 2001,
42, 1029.
29. Enzymatic activity was measured by ATP consumption: Purified ROCK1 or ROCK2
(Invitrogen, cat. PR7028A or PV4048, respectively), peptide substrate (S6:
KEAKEKRQEQIAKRRRLSSLRASTSKSGGS-QKOH, Biopeptide, Inc.) and ATP (final
equipped with a Hamamatsu ORCA ER cooled CCD camera and OpenLab
software (Improvision). Images were imported into Adobe Photoshop (v.10) for
labeling and minor alterations in brightness only.
concentrations 0.82
reaction mix (20 mM Tris–HCl [pH 7.5], 10 mM MgCl2, 0.4 mM CaCl2, 0.15 mM
EGTA, 0.1 mg/ml bovine serum albumin) just prior to dispensing 25 l/well to
384-well plates. Immediately thereafter, 0.24 l of test compounds, previously
arrayed in 11 point, 1/2 log dilution dose–response, were added by passive pin
transfer (final top concentration 96 M, 2–4 replicate wells per concentration,
1% DMSO [vol/vol] in all assay wells). After 2 h at 30 °C, 15 l of Easylite
l
g/ml, 100
l
g/ml, and 3
l
M, respectively) were added to
43. The cell impedance assay was validated for ROCK inhibition by testing the
following kinase inhibitors at 10
(VEGFR, PDGFR, Raf), PD-184352 (Mek), gefitinib (EGFR), imatinib (PDGFR,
Abl, Kit), VX-745 (p38
), Ro-31-7549 (PKCs), indirubin-30-oxime (GSK3b, some
lM (targets in parentheses): sorafenib
l
l
a
CDKs), STO-609 (CAMKK), SU-6656 (Src, Fyn), LY-294,002 (PI3-kinase), and H-
89 (PKA, ROCK). Only H-89 had a similar but weaker effect than HMN-1152,
consistent with its IC50 of 0.36 0.08 lM against ROCK. The other compounds
l
l
(Perkin–Elmer) was added, and the resulting luminescence was read on a
Molecular Devices Acquest plate reader. Raw luminescence data were
normalized to negative (DMSO) and positive (HMN-1152, 2) controls. Data
analysis was performed using Spotfire (Spotfire, Inc.) and Kalypsys proprietary
software. Some compounds were also tested in kinase assays measuring ADP
production (ADPQuest, DiscoverX), under similar conditions and according to
the manufacturer’s instructions. Increasing the concentration of ATP in these
assays led to an increase in IC50 values, consistent with an ATP-competitive
mechanism of action.
had dramatically different effects quantitatively, qualitatively, or both. For
example, imatinib caused a rapid, small magnitude increase in impedance,
followed by a slow decrease to baseline over 3 h. Other compounds with
different effects than ROCK inhibitors included 6-anilinoquinoline-5,8-quinone
(guanylate cyclase inhibitor), 20,50-dideoxyadenosine (adenylate cyclase
inhibitor), and nocodazole (microtubule polymerization inhibitor, tested at
2
l
M). Treatment with blebbistatin (myosin II inhibitor, 10 or 1
dose-dependent effect similar to ROCK inhibitors, but with one-third the
magnitude, while taxol (microtubule stabilizer, 0.1 or 0.01 M) showed a
lM) showed a
l
30. All synthesized compounds displayed spectral data (MS, NMR) consistent with
their assigned structures. Compounds were tested as racemic mixtures when
present as such. Synthetic methods can be found in Kahraman, M. et al., U.S.
patent application 20080021026.
similar low magnitude, non dose-dependent effect. Only cytochalasin D (actin
polymerization inhibitor) or an RGD containing peptide with sequence
GRGDTP (integrin inhibitor; a control peptide with sequence GRADSP was
inactive) showed dose dependent effects at 10 and 1 lM strikingly similar to
31. Cellular activity was measured with the xCelligence system (ACEA Biosciences/
Roche Applied Sciences). Briefly, 96-well E-plates were coated with fibronectin
that of ROCK inhibitors. The effects of the last four, and especially the last two,
compounds are consistent with their targets and expected effects on cell
adhesion. Sorafenib, PD-184352, gefitinib, imatinib, and VX-745 were
synthesized by Kalypsys; all other compounds in impedance validation
experiments were purchased from commercial vendors.
(US Biological, cat. C2605) by adding 35
ll undiluted reagent to each well for
30 min at 37 °C. After aspiration, 50 l of cell media (DMEM, Pen/Strep, 1% fetal
l
bovine serum) was added to each well, and background impedance was
measured. Then 100
l
l of GTM-3 cells in media (0.25 ꢁ 106/ml) was dispensed
44. Schroter, T.; Griffin, E.; Weiser, A.; Feng, Y.; LoGrasso, P. Biochem. Biophys. Res.
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P. J. J. Org. Chem. 2002, 67, 5394.
to each well, and the cells were allowed to settle and spread for 30 minutes at
rt. The detector was mounted in a culture incubator, and impedance was
measured every hour until the following day, when test compounds were
added to each well (final top concentration 10
l
M [0.1% DMSO] or 15
l
M
46. Jacobs, M.; Hayakawa, K.; Swenson, L.; Bellon, S.; Fleming, M.; Taslimi, P.;
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[0.15% DMSO], duplicate wells per concentration). Impedance was then
measured every minute for 2–3 h, followed by every 15 min for several
hours more, or up to 2 days to monitor long-term effects. The average minimal
impedance per compound per concentration was used to generate a dose–
response curve. Data normalization and analysis were similar to that for the
enzymatic assay. The positive control was Y-39983 (ROCK1 IC50
0.03 0.002
concentration of 0.2% or greater induced impedance effects without test
compound, we were limited to a top concentration of about 10 M for test
lM, GTM-3 EC50 0.79 0.06 lM). Since DMSO at a final
51. Prepared as described in Scheme 1; des-chloro 4 was used as an intermediate.
52. Prepared from 22 by treatment with BBr3 in DCM.
l
compounds, based on a standard 10 mM stock in 100% DMSO. This limitation
and the overall sensitivity limit of the assay often yielded less than 100%
efficacy at the highest tested concentration, where we report an EC50 but less
than 100% efficacy. In separate experiments, reference compounds dissolved in
53. Prepared from 17: n-BuLi, THF, ꢀ78 °C; then solid CO2, 3 h, 20%.
54. Prepared from 26: (trimethylsilyl)diazomethane (2 M in Et2O), THF/MeOH
(1:1), rt, 43%.
55. Compound 25 was tested at 10
following kinases (Invitrogen,% inhibition in parentheses if P50%): ALK4,
AMPK, Aurora B (82), B-Raf, CaMK2 , CaMK4, CDK2/cyclinA, CDK5/p35, CK1 1,
CK1 1, CK2 1, DAPK3, EGFR, EphB4, Erk1, FGFR1, GSK3b, INSR, Jak2, Jnk1, Lck,
Mek1, MKK6, MLK1, MAPKAPK2, MSK1 (94), p38 , p70S6K (55), Pak6, PDGFR
PKA (67), PKC , PKC , PKN1 (53), RSK1, SGK1 (61), Src, TrkA, ZAP70.
lM for inhibition in functional assays of the
water at 100 mM allowed us to test concentrations up to 100 lM and confirm
100% efficacy compared to the most potent control, Y-39983. We tested
reversibility by aspirating compound-containing media and replacing with
compound-free media after multiple washes. Reversibility was tested up to
24 h after compound addition, and was complete about 3 h after media
replacement. In parallel 24 h cytotoxicity assays, there was no significant effect
a
a
e
a
a
a,
a
e
56. Solubility was assessed by conventional kinetic assays, measuring absorbance
after precipitate filtration from compounds initially in 100% DMSO, with
calculation from known standards.
57. Chen, T. M.; Shen, H.; Zhu, C. Comb. Chem. High Throughput Screen. 2002, 5, 575.
58. Prepared from 30 by treatment with BBr3 in DCM.
of compounds up to at least 30 lM.
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