Optimization of novel nipecotic bis(amide) inhibitors of the Rho/MKL1/SRF transcriptional pathway as potential anti-metastasis agents

Article abstract of DOI:10.1016/j.bmcl.2013.04.080

CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthesis of conformationally restricted analogs (e.g

Full text of DOI:10.1016/j.bmcl.2013.04.080

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3826–3832
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of novel nipecotic bis(amide) inhibitors
of the Rho/MKL1/SRF transcriptional pathway as potential
anti-metastasis agents
Jessica L. Bell a, Andrew J. Haak b, Susan M. Wade b, Paul D. Kirchhoff a, Richard R. Neubig b,c,d
,
Scott D. Larsen a,d,
⇑
a Vahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, College of Pharmacy, MI, USA
b Department of Pharmacology, University of Michigan Medical School, MI, USA
c Center for Chemical Genomics, University of Michigan, Ann Arbor, MI 48109, USA
d Center for Discovery of New Medicines, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of
PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthe-
sis of conformationally restricted analogs (e.g., 2) with improved selectivity for inhibiting invasion versus
acute cytotoxicity. In this study we conducted a survey of aromatic substitution with the goal of improv-
ing physicochemical parameters (e.g., ClogP, MW) for future efficacy studies in vivo. Two new com-
pounds were identified that attenuated cytotoxicity even further, and were fourfold more potent than
2 at inhibiting PC-3 cell migration in a scratch wound assay. One of these (8a, CCG-203971, IC50 = 4.2 lM)
was well tolerated in mice for 5 days at 100 mg/kg/day i.p., and was able to achieve plasma levels exceed-
ing the migration IC50 for up to 3 h.
Received 13 March 2013
Revised 22 April 2013
Accepted 29 April 2013
Available online 7 May 2013
Keywords:
Anti-metastasis
Rho
MKL1
Ó 2013 Elsevier Ltd. All rights reserved.
SRF
Cancer
Gene transcription inhibitor
Cell migration inhibition
Metastasis is the major driver in cancer-related deaths. Metas-
tases involve dysregulation of numerous cellular processes that al-
low malignant cells to escape their point of origin and establish
themselves in distant sites. Altered programs of gene transcription
play a key role in these processes, and recent evidence points to an
important role for RhoA/C-stimulated gene transcription.1,2 The
small G proteins in the RhoA subfamily are almost never mutated
in cancer; rather, their activity is increased by overexpression or by
increases in the activity of upstream activators. Studies of human
cancer cell lines selected for high metastatic character in xenograft
models show upregulation of RhoC expression.3 Upregulation of
RhoC in human cancers is also associated with poor clinical out-
come.4 In breast and prostate cancer models, blockade of RhoC
by toxins or dominant negative approaches prevents cellular inva-
sion.5,6 Importantly, a RhoC knockout mouse shows complete sup-
pression of in vivo metastasis of polyoma T-antigen-induced
mammary tumors.1 Signals downstream of RhoC have been impli-
cated in the invasiveness of aggressive prostate cancers6,7 and in
the in vivo metastasis of breast cancer1 and melanoma.3 RhoC
activation induces transcriptional responses through the actin-reg-
ulated cytosolic-to-nuclear translocation of megakaryocytic leuke-
mia 1 protein (MKL1), which binds to the serum-response factor
(SRF), and the resulting complex activates transcription of serum
response element (SRE) regulated target genes.8,9 Recently, both
MKL1 and SRF have been shown to play important roles in metas-
tasis of melanoma and breast cancer in vivo,10 and SRF has been
shown to be clinically associated with castration-resistant prostate
cancer.11 Thus transcriptional signals, rather than actin cytoskele-
tal changes per se, are critical for the role of RhoC in metastasis.
Few drugs are known to target such transcriptional signals.
In 2007, Evelyn et al. reported the identification of small mole-
cule CCG-1423 (1, Fig. 1) as an inhibitor of SRE-driven luciferase
(SRE.L) gene transcription initiated by the RhoA/C signaling path-
ways.12 Discovered using a high-throughput screen in the Univer-
sity of Michigan Center for Chemical Genomics s(CCG), 1 possessed
considerable potency (<1 lM), but also significant cytotoxicity.
Therefore, we carried out molecular modifications of 1 with the
aim of improving its potency and/or selectivity while mitigating
cytotoxicity. Through replacement of the potentially labile N–O
bond and conformational restriction of the flexible tether region
between the two aromatic rings, we arrived at our lead compound,
⇑
Corresponding author. Tel.: +1 734 615 0454.
E-mail address: sdlarsen@umich.edu (S.D. Larsen).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.080

J. L. Bell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3826–3832
3827
Cl
Cl
Cl
O
O
O
O
O
F3C
O
F3C
N
H
N
H
N
N
H
CF3
CF3
1
(CCG-1423)
O
2
O
O
R1
F3C
N
N
H
R2
N
H
N
H
Ring B
CF3
Ring A
19
3
Figure 1.
nipecotic bis(amide) 2 (Fig. 1), which offered an improved biolog-
ical profile over compound 1.13 Although there was a 10-fold de-
crease in potency, 2 retained inhibition of Ga12QL-stimulated
SRE.L expression with similar efficacy to 1, but with greatly re-
duced cytotoxicity. Unfortunately, compound 2 exhibits poor phys-
coupled to 4-chloroaniline with EDC/DMAP to afford intermediate
amide 6. Following deprotection, the resulting common piperidine
intermediate 7 was coupled with diverse m-substituted benzoic
acids, again using EDC/DMAP.15 The resulting compounds were
purified using acidic Amberlyst-15 resin to remove excess 7, fol-
lowed by a water wash to remove residual DMF, affording library
analogs 8a–t. This synthesis afforded compounds with overall
yields ranging between 39% and 100% and purities greater than
90%. For the aniline library (Scheme 2), ethyl nipecotate 9 was acyl-
ated with 3,5-bis(trifluoromethyl)benzoyl chloride followed by
saponification to afford free acid 11. The acid was then coupled
with diverse m-anilines using EDC/DMAP. Compounds were puri-
fied by aqueous workup and, if necessary, chromatographed to af-
ford aniline library analogs 12a–w. This synthesis afforded
compounds with yields ranging between 32 and 100% and with
purities between 81 to greater than 95%.
icochemical properties, including low solubility (<10
apparently low permeability (Table 4).
lg/mL) and
We elected to further explore diversity at each of the terminal
aromatic rings of 2 with the goals of improving its potency and/
or physicochemical parameters (e.g., reduced lipophilicity and/or
molecular weight). Our initial survey of aromatic substitution13
indicated that some degree of lipophilicity on the aromatic rings
is necessary for activity, likely due to the need for cell permeability.
Therefore, we designed libraries 3 to explore diversity on the aro-
matic rings while modestly reducing lipophilicity with the goal of
increasing solubility (Fig. 1).
We first explored ring A of 2 (Fig. 1). We utilized MScreen14 to
select commercially available m-benzoic acids with calculated logP
(ClogP) values less than that of 3,5-bis(trifluoromethyl)benzoic
acid, and which would produce final analogs with total molecular
weights below 550. We initially chose only meta-substitutions, as
they have greater rotational freedom to interact with various areas
in the binding site than do ortho- or para-substitutions. Similarly,
we explored ring B (Fig. 1), again using MScreen to select commer-
cially available meta-substituted anilines with ClogPs less than
that of 4-chloroaniline and that would result in molecular weights
of 550 or less. In choosing only starting acids and anilines with
lower lipophilicities than those in 2, we hoped to decrease the
overall lipophilicities of our new analogs to enhance solubility.
The synthetic routes to new analogs of 2 are presented in
Schemes 1 and 2. For the acid library (Scheme 1), the starting
nipecotic acid 4 was BOC protected, and the resulting acid 5 was
The effects on Rho-mediated gene transcription and cytotoxic-
ity of all newly synthesized analogs were determined in PC-3 cells
transiently transfected with a luciferase reporter gene driven by
the SRE promoter (SRE.L) at an initial concentration of 100
For compounds showing greater than 50% inhibition at 100
l
l
M.13
M, a
full dose-response curve (DRC) was generated. Table 1 summarizes
the effects of benzamide analogs 8. Also included in the table are
efficacies at the maximum concentration (100 lM) as we expect
that both potency and overall efficacy will be important indicators
of therapeutic potential. In general, the new analogs demonstrated
little to no cytotoxicity, with the exception of 8d. A majority of
analogs, however, had lower potency than lead 2, with the
exception of analogs 8a and 8b that had equivalent or slightly bet-
ter potency and similar maximal efficacies.
Selected structure–activity relationships (SAR) suggest that
lipophilicity and/or topological polar surface area (tPSA) may be
Cl
O
O
O
b
a
BocN
OH
HN
OH
HN
BocN
N
H
5
4
6
Cl
Cl
O
O
O
R1
c
d
N
H
N
N
H
7
8
Scheme 1. Reagents and conditions: (a) BOC2O, NaOH, H2O, dioxane; (b) 4-ClPhNH2, EDC, DMAP, DCM; (c) TFA, DCM, À10 °C; (d) (i) m-R1PhCO2H, EDC, DMAP, DCM/DMF; (ii)
Amberlyst-15, DCM.

3828
J. L. Bell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3826–3832
O
O
O
O
O
F3C
F3C
b
a
N
OH
N
OEt
HN
OEt
11
10
CF3
CF3
9
O
O
F3C
c
N
N
H
R2
CF3
12
Scheme 2. Reagents and conditions: (a) 3,5-Bis(CF3)PhCOCl, DIPEA, DCM; (b) LiOH, EtOH; (c) m-R2PhNH2, EDC, DMAP, DCM.
Table 1
Effects of ring A substitution on transcription and cytotoxicity in transfected PC-3 cellsa
Cl
O
O
R1
N
N
H
8
b
c
Compd no.
R1
IC50 SRE.L (
l
M)b
% Inh SRE.L (100
l
M)
% Inh WST-1 (100 lM)
2
3,5-Bis(CF3)
Furan-2-yl
PhCO
Thiazol-2-yl
t-BuCH2CONH
CH2@CH
2-Me-thiazol-4-yl
Me
MeS
Oxazol-5-yl
Et2NSO2
MeO2C
5-Me-1,2,4-oxadiazol-3-yl
MeO
MeOCH2
9.8
6.4
9.9
27
26
33
33
35
35
43
51
67
78
ND
ND
ND
ND
ND
ND
ND
78
87
75
92
69
84
81
63
76
72
69
64
59
38
32
32
20
17
15
8
14
0
0
32
0
0
1
0
0
0
0
0
0
0
0
3
0
2
0
8a
8b
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
MeSO2NH
–CN
MeSO2
–NO2
MeCONH–
4
a
b
c
For assay descriptions, see Ref. 12.
Inhibition of Rho-pathway selective serum response element-luciferase reporter. Values are mean of n P 3 independent experiments. ND: not determined.
Inhibition of mitochondrial reduction of WST-1. Values are mean of n P 3 independent experiments.
correlated with activity. Oxazole 8j adds a nitrogen to the furan
ring of 8a, resulting in diminished activity. This substitution in-
creases the tPSA (from 59 to 71 Å2) and decreases the ClogP from
4.88 to 3.46. A similar trend is observed when comparing sulfide 8i
with sulfone 8r, styrene 8f with methoxy 8n, and oxazole 8j with
oxadiazole 8m. In fact, overall we found that potency correlated
weakly with decreasing tPSA (R2 = 0.32, data not shown), and mod-
erately with increasing ClogP (R2 = 0.78, data not shown). In sum-
mary, the best analog from the carboxylic acid library was furan 8a,
which had both improved potency and efficacy in the SRE.L assay,
as well as lower acute cytotoxicity. Also of interest was analog 8b,
which incorporates a photoactivatable benzophenone group with-
out loss of activity. This suggests that photoaffinity probes based
on 8b may be of possible use in identifying the unknown molecular
target.16
12a and 12b (not included as part of the original library design,
but later as analogs of 8b), showed a nearly 10-fold increase in po-
tency with slight decreases in efficacy, perhaps due to poor solubil-
ity at higher concentrations. Pyridine 12c showed a similar
increase in potency while maintaining better efficacy. Analogs
12d–12i all showed similar or slight increases in potency.
Interestingly, there was no correlation overall between potency
and either tPSA or ClogP in this library (R2 < 0.1). This perhaps sug-
gests that local polarity/dipole changes on ring A are actually
responsible for the SAR, rather than overall physical properties.
However, some interesting comparisons can still be made between
similar compounds. Inactive analog 12s differs from highly active
analog 12b only in the presence of a ketone between the aromatic
rings, suggesting that conformation could be important for activity.
Replacement of the methyl ester of 12j with t-butyl ester in 12w
resulted in complete loss of activity, as did replacing the methoxy
of 12k with allyloxy in 12r, both suggesting some steric limita-
tions. However, these latter results are not consistent with the
favorable activity of bulky analogs 12a–c, possibly indicative of
The effects of aromatic substitution on ring B are summarized in
Table 2. As with the ring A library, most compounds demonstrated
little to no cytotoxicity, with the exception of amine 12p, which
had significant cytotoxicity of 51% at 100 lM. The best analogs,

J. L. Bell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3826–3832
3829
Table 2
Effects of ring B substitution on transcription and cytotoxicity in transfected PC-3 cellsa
O
O
F3C
N
N
H
R2
CF3
12
M)
Compd no.
R2
IC50 SRE.L (
l
% Inh SRE.L (100
lM)
% Inh WST-1 (100 lM)
2
4-Cl
PhO
PhCH2
2-Pyr-CH2CH2O
Pyrrolidin-1-yl-SO2
2-Me-thiazol-4-yl
Et
CH2@CH
Oxazol-5-yl
MeS
MeO2C
MeO
–CN
Morpholin-1-yl-CH2
–NO2
MeCO
Pyrrolidin-1-yl-CH2
2-CO2Me-furan-4-yl
CH2@CHCH2O
PhCO
4-Methyl-1,2,4-triazol-3-yl
2-Oxopyrrolidin-1-yl
3-Cl-4-CF3-pyridin-2-yl-O
tBuO2C
9.8
1.5
1.6
2.3
5.6
6.1
8.1
11
12
12
26
30
32
32
66
76
ND
ND
ND
ND
ND
ND
ND
ND
78
59
59
82
70
73
66
53
87
60
63
51
77
73
65
73
94
39
38
38
36
35
25
24
14
1
0
5
0
1
0
1
9
0
0
0
0
0
9
0
51
0
0
0
0
0
0
0
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
a
Assays and abbreviations defined in Table 1.
multiple binding modes. Overall, however, it must be emphasized
that any interpretation of SAR needs to be undertaken with the ca-
veat that activity in the cell-based SRE.L assay will be dependent
on multiple variables in addition to intrinsic activity, including cell
permeability, distribution inside the cell, and perhaps even
metabolism.
only realized with the most lipophilic analogs 12a and 12b, which
may account for their increased potency in the cell-based SRE-luc as-
say. One interesting observation from this dataset is that the supe-
rior activity of conformationally restrained analog 2 versus acyclic
analog 19 (Fig. 1, IC50 = 38 l
M)13 is likely not due to increased pas-
sive permeability and therefore probably reflects a true increase in
Based on the results from the acid- and aniline-substituted li-
braries, additional analogs were examined, as illustrated in Table 3.
Replacement of the furan of 8a with a thiophene (8u) slightly in-
creased potency, but introduced some detectable cytotoxicity.
Movement of the furan of 8a from the meta- to the para-position
(13) caused a loss in potency with introduction of slight cytotoxic-
ity. 12x moved the furan from ring A to ring B, which led to a loss of
both potency and efficacy. Compounds 12y and 12z shortened the
pyridyl tether of 12c by one and two carbons, respectively. Both
had inferior potency. Analogs 15 and 16 were prepared to deter-
mine if a pyridine B-ring could improve the solubility of 2. Unfor-
tunately all showed unacceptable losses in potency.
Next, we examined ‘hybrid’ analogs 17 and 18 of our most
promising compounds, combining the A-ring furan moiety of 8a
with the B-ring moieties of 12a and 12b. Surprisingly, neither hy-
brid retained any activity. To determine if this reflects a steric
intolerance for aromatic substitution on both rings, we prepared
hybrid 14 containing the B-ring thioether of 12i. In this case activ-
ity was not lost, but there also was no improvement.
A small subset of compounds was selected for evaluation of the
impact of structure on kinetic solubility17 and passive permeability
as measured by the PAMPA Explorer Kit (pION) (Table 4). For each
parameter, compounds were binned into one of three groups: high,
medium or low (described in Table 4 legend). Unfortunately none of
the new analogs had solubilities exceeding that of leads 1 or 2; all
were binned Low solubility. Marginal improvement in permeability
was observed with furan analog 8a relative to 2, but it did not rise
above the low bin. Significant improvements in permeability were
affinity for the target.
We previously demonstrated that 2 can inhibit invasion into
Matrigel by cultured PC-3 cells.13 To compare our best new analogs
with 2 for their ability to inhibit PC-3 cell migration, we employed
a scratch wound assay. PC-3 cells (5.0 Â 105) were plated in DMEM
containing 10% FBS and grown to confluence in a 12-well plate.
After 24 h, a scratch was made using a 200
was replaced with DMEM containing 0.5% FBS and either test com-
pounds (10 M) or 0.1% DMSO control. Images of the wounds were
lL pipette tip. Medium
l
taken at 0 h using a bright-field inverted microscope (Leica DM
IRB) at 2.5Â magnification. After 24 h the cells were fixed (10% for-
malin) and stained (0.5% crystal violet) to obtain high contrast
images. Area quantification of the wounds was determined compu-
tationally using ImageJÒ software (NIH). The extent of migration
was determined by subtracting the area of the wound after 24 h
from the initial area of the wound. The percent inhibition was plot-
ted by normalizing the compound treated cells to the DMSO con-
trol. Results are summarized in Fig. 2. In this assay, lead
compound 2 only inhibited PC-3 cell migration by 22%. Acyclic
analog 19 was included as a negative control with poor SRE.L activ-
ity, and showed minimal (<10%) inhibition of migration. Surpris-
ingly, none of the most potent new B-ring analogs (12a–d)
performed significantly better than 2 in the scratch assay, all inhib-
iting less than 30% at 10 lM. In fact, the only B-ring analog that
afforded any improvement was thiazole 12e (42% inhibition). On
the other hand, A-ring analogs 8a and 8u, despite their more mod-
est SRE.L potency, were much more effective in this migration as-
say, both inhibiting about 70% at 10 lM. Dose response curves

3830
J. L. Bell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3826–3832
Table 3
Effects of additional substitutions on transcription and cytotoxicity in transfected PC-3 cellsa
a
a
Compd no.
Structure
IC50 SRE.L
(
lM)
% Inh SRE.L (100
l
M)
% Inh WST-1a (100
lM)
Cl
Cl
S
O
O
O
O
8u
4.7
81
80
4
N
N
N
H
N
H
13
9.2
21
6
0
O
O
O
O
O
O
O
F3C
O
N
N
12x
12y
63
79
H
CF3
N
F3C
F3C
N
N
N
H
O
O
7.4
15
CF3
CF3
N
H
N
12z
14
10
10
60
63
0
0
O
O
O
N
N
H
S
Cl
Cl
O
O
O
O
F3C
F3C
N
N
N
H
N
15
16
ND
31
37
88
0
CF3
CF3
N
N
H
25
O
O
O
O
17
18
ND
ND
21
21
0
0
N
N
N
H
O
O
O
N
H
a
Assays and abbreviations defined in Table 1.
were acquired for the most effective compounds and are depicted
in Figure 3. Both 8a (IC50 = 4.2 M) and 8u (IC50 = 4.5 M) had
potencies significantly better than 2 (IC50 = 17 M) or the Rho-ki-
nase (ROCK) inhibitor Y-27632 (IC50 = 28 M), which was included
and SK-Mel-147.12 Therefore, we also tested 8a in the RhoC–SRE.L
assay using transiently transfected SK-Mel-147 cells. We observed
similar results to the PC-3 cell line, with 8a displaying an IC50 of
l
l
l
l
5.3 lM and no WST-1 cytotoxicity, exhibiting its potential as an
for comparison because of its role in Rho-mediated signaling and
inhibitor of metastatic melanoma, all of which will be disclosed
reported effects on cancer cell migration.18–21
in a future publication.22
Upon the initial discovery of 1, we also observed that it had an
effect on the RhoC-overexpressing melanoma cell lines A375M2
Mechanistic analysis of compound 1 has demonstrated that it
acts downstream of RhoA and targets MKL/SRF-dependent

J. L. Bell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3826–3832
3831
Table 4
Physicochemical property data for selected new analogs
c
Compd no.
Solubility (
l
g/mL)a
ClogPb
logPeff (cm/s)
2
8a
8u
12a
12b
19
7.99–4.50 (Low)
4.26–3.09 (Low)
2.41–1.36 (Low)
4.30–3.22 (Low)
3.85–1.89 (Low)
3.45–2.21 (Low)
5.55
4.88
5.37
6.68
6.65
5.40
À10 0.00 (Low)
À8.3 2.3 (Low)
À10 0.00 (Low)
À5.1 0.40 (Mod-low)
À4.7 0.05 (Mod)
À10 0.00 (Low)
a
b
c
Kinetic solubility; method and ranges found in Ref. 17.
Calculated log P (ChemBioDraw Ultra 12.0).
Log of effective permeability (cm/s) as determined by PAMPA Explorer. Ranges
are defined in Ref. 17. Measurements are mean of n = 3.
Figure 3. Effects of new compounds on PC-3 cell migration. Compounds were
tested at various concentrations in a scratch wound assay. IC50 values are in lM.
exceeding the PC-3 cell migration IC50 for up to 3 h, indicating its
potential use for further in vivo studies. More detailed studies will
be reported in due course.
In summary, an SAR study of 2 focusing on aromatic ring diver-
sity was undertaken with the goal of improving selectivity and/or
potency, while attenuating cytotoxicity and improving drug-like
properties. Although we were not successful at improving solubil-
ity, we did identify one analog (8a, CCG-203971) that has reduced
acute cytotoxicity and improved potency versus 2 with regard to
inhibition of PC-3 cell migration (IC50 = 4.2 lM vs 16.6 lM), as
well as reduced lipophilicity and molecular weight. Furthermore,
preliminary tolerability studies in normal mice indicate that 8a
is well tolerated up to doses of 100 mg/kg IP over 5 days, and pos-
sesses pharmacokinetic properties suitable for future xenograft
studies.
Acknowledgments
This work was supported in part by a Pharmacological Sciences
Training Program grant GM007767 from NIGMS (A.J.H.). The con-
tents of this paper are solely the responsibility of the authors and
do not necessarily represent the official views of NIGMS.
Figure 2. Effects of new compounds on PC-3 cell migration using a scratch wound
assay. (A) Example images of the wounds after 24 h taken with a bright-field
inverted microscope (Leica DM IRB, 2.5Â magnification). (B) Percent inhibition of
migration into wound area. Area quantification of the wounds was determined
computationally using ImageJÒ software (NIH).
References and notes
1. Hakem, A.; Sanchez-Sweatman, O.; You-Ten, A.; Duncan, G.; Wakeham, A.;
Khokha, R.; Mak, T. W. Genes Dev. 2005, 19, 1974.
transcriptional activation. However, the exact mechanism of action
has not yet been deciphered. Our findings suggest that 1 could af-
fect the functions of MKL1 in various ways, including: preventing
its release from actin, blocking translocation from the cytoplasm
to the nucleus, repressing transcription via increasing sumoylation,
disrupting the interaction between MKL1 and its transcoactivator
SRF, or inhibiting its coactivator function.12 Preliminary studies
have in fact indicated that 8a blocks MKL1 nuclear localization.22
In preliminary in vivo tolerability studies, compound 1 demon-
strated a level of toxicity that would preclude extended dosing in
xenograft models (deaths observed with repeated dosing at
7.5 mg/kg intraperitoneally (IP)). Based on its favorable activity
in the migration assay and markedly lower acute cytotoxicity, we
selected 8a for the next round of in vivo testing. A 5-day tolerabil-
ity study was conducted at 10, 20, 50, and 100 mg/kg/day dosed IP
using 3 mice per dose. Following sacrifice on the 8th day, the mice
were weighed and blood samples were collected. Dissection was
performed, and the internal organs were weighed and examined
for abnormalities. Weight was maintained over the course of the
study, with the only abnormality observed being a slight increase
in liver weight. All mice survived the 5-day study without any
noted abnormal behavior. In preliminary pharmacokinetic studies,
a single IP dose of 100 mg/kg 8a resulted in plasma levels of drug
2. Mees, C.; Nemunaitis, J.; Senzer, N. Cancer Gene Ther. 2009, 16, 103.
3. Clark, E. A.; Golub, T. R.; Lander, E. S.; Hynes, R. O. Nature 2000, 406, 532.
4. Sahai, E.; Marshall, C. J. Nat. Rev. Cancer 2002, 2, 133.
5. van Golen, K. L.; Wu, Z. F.; Qiao, X. T.; Bao, L. W.; Merajver, S. D. Cancer Res.
2000, 60, 5832.
6. Yao, H.; Dashner, E. J.; van Golen, C. M.; van Golen, K. L. Oncogene 2006, 25,
2285.
7. Evelyn, C. R.; Wade, S. M.; Wang, Q.; Wu, M.; Iniguez-Lluhi, J. A.; Merajver, S. D.;
Neubig, R. R. Mol. Cancer Ther. 2007, 6, 2249.
8. Treisman, R. Trends Biochem. Sci. 1992, 17, 423.
9. Cen, B.; Selvaraj, A.; Burgess, R. C.; Hitzler, J. K.; Ma, Z.; Morris, S. W.; Prywes, R.
Mol. Cell. Biol. 2003, 23, 6597.
10. Medjkane, S.; Perez-Sanchez, C.; Gaggioli, C.; Sahai, E.; Treisman, R. Nat. Cell
Biol. 2009, 11, 257.
11. Prencipe, M.; Madden, S. F.; O’Neill, A.; O’Hurley, G.; Culhane, A.; O’Connor, D.;
Klocker, H.; Kay, E. W.; Gallagher, W. M.; Watson, W. R. Prostate 2013, 73, 743.
12. Evelyn, C. R.; Wade, S. M.; Wang, Q.; Wu, M.; Iniguez-Lluhi, J. A.; Merajver, S.
D.; Neubig, R. R. Mol. Cancer Ther. 2007, 6, 2249.
13. Evelyn, C. R.; Bell, J. L.; Ryu, J. G.; Wade, S. M.; Kocab, A.; Harzdorf, N. L.; Hollis
Showalter, H. D.; Neubig, R. R.; Larsen, S. D. Bioorg. Med. Chem. Lett. 2010, 20,
665.
14. Jacob, R. T.; Larsen, M. J.; Larsen, S. D.; Kirchhoff, P. D.; Sherman, D. H.; Neubig,
R. R. J. Biomol. Screen. 2012, 17, 1080.
15. Lawrence, R. M.; Biller, S. A.; Fryszman, O. M.; Poss, M. A. Synthesis 1997, 1997,
553.
16. Leslie, B. J.; Hergenrother, P. J. Chem. Soc. Rev. 2008, 37, 1347.
17. Kerns, E. H.; Di, L. Drug-like Properties: Concepts, Structure Design and Methods:
From ADME to Toxicity Optimization; Academic Press: Amsterdam; Boston,
2008. p 292.

3832
J. L. Bell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3826–3832
18. Uehata, M.; Ishizaki, T.; Satoh, H.; Ono, T.; Kawahara, T.; Morishita, T.;
Tamakawa, H.; Yamagami, K.; Inui, J.; Maekawa, M.; Narumiya, S. Nature 1997,
389, 990.
20. Vial, E.; Sahai, E.; Marshall, C. J. Cancer Cell 2003, 4, 67.
21. Jeong, K. J.; Park, S. Y.; Cho, K. H.; Sohn, J. S.; Lee, J.; Kim, Y. K.; Kang, J.; Park, C.
G.; Han, J. W.; Lee, H. Y. Oncogene 2012, 31, 4279.
19. Somlyo, A. V.; Bradshaw, D.; Ramos, S.; Murphy, C.; Myers, C. E.; Somlyo, A. P.
Biochem. Biophys. Res. Commun. 2000, 269, 652.