Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the Rho/MRTF/SRF transcriptional pathway as potential antifibrotic therapeutics for systemic scleroderma

Article abstract of DOI:10.1016/j.bmcl.2017.02.070

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50?mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.

Full text of DOI:10.1016/j.bmcl.2017.02.070

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the
Rho/MRTF/SRF transcriptional pathway as potential antifibrotic
therapeutics for systemic scleroderma
Kim M. Hutchings a,e, Erika M. Lisabeth d,e, Walajapet Rajeswaran a, Michael W. Wilson a,
Roderick J. Sorenson a, Phillip L. Campbell c, Jeffrey H. Ruth c, Asif Amin c, Pei-Suen Tsou c,
Jeffrey R. Leipprandt d, Samuel R. Olson d, Bo Wen b, Ting Zhao b, Duxin Sun b, Dinesh Khanna c,
David A. Fox c, Richard R. Neubig d, Scott D. Larsen a,
⇑
a Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
b UM Pharmacokinetics Core, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
c Department of Internal Medicine, Division of Rheumatology and Clinical Autoimmunity Center of Excellence, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
d Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1,
CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when
given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, how-
ever, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal
chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identifi-
cation of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently
showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced
dermal fibrosis in mice when administered orally at 50 mg/kg, an effect that was comparable to what we
had observed earlier with 1 at a 4-fold higher IP dose.
Received 10 February 2017
Revised 24 February 2017
Accepted 26 February 2017
Available online xxxx
Keywords:
Scleroderma
Fibrosis
Antifibrotic
Myofibroblast
Rho
Ó 2017 Elsevier Ltd. All rights reserved.
MRTF
It has been estimated that fibrotic diseases are responsible for
up to 40% of all deaths worldwide.1 Although the progression
and pathology of fibrosis are well described (relentless and exces-
sive accumulation of collagen-rich extracellular matrix (ECM)), the
precise etiology of the disease remains obscure. Systemic sclerosis
(scleroderma, SSc) is a fibrotic disease of the connective tissues
that involves both the vascular and immune systems. The hallmark
of SSc, and in fact of all fibrotic disease, is the transition of normal
fibroblasts into myofibroblasts, which are characterized by the
Evidence is growing that fibroblast activation to myofibroblasts
results from gene transcription stimulated by a common Rho-
mediated signaling pathway that originates from divergent extra-
cellular pro-fibrotic stimuli.5,6 Specifically, Rho mediates the con-
version of G-actin to F-actin, which releases G-actin-bound
myocardin-related transcription factor (MRTF), resulting in accu-
mulation of MRTF in the nucleus, where it binds to serum response
factor (SRF) on the serum response element (SRE) promoter. This
Rho/actin/MRTF/SRF pathway may in fact serve as a regulator of
the fibrotic process used in wound healing,7 but dysregulation
and/or overstimulation can lead to fibrosis. Recently, MRTF-A
expression in the nucleus of dermal fibroblasts from SSc patients
was shown to be increased relative to normal.8
We have discovered and developed a novel class of inhibitors of
the Rho/MRTF/SRF pathway.9–11 A lead compound from this effort,
CCG-203971 (1, Table 1), has exhibited promising anti-fibrotic
activity both in vitro and in vivo in several animal models of
disease, including intestinal fibrosis,12 pulmonary fibrosis13 and
dermal fibrosis.14 Despite these encouraging preliminary results,
expression of alpha smooth muscle actin (a-SMA) and the produc-
tion of ECM.2 SSc-associated fibrosis is currently treated with
immunosuppressants, which are of limited efficacy, have untoward
side effects, and do not address the underlying pathology.3 Recent
research suggests that targeting fibroblast activation is likely to be
a more effective strategy.4
⇑
Corresponding author.
E-mail address: sdlarsen@med.umich.edu (S.D. Larsen).
These authors contributed equally to this work.
e
http://dx.doi.org/10.1016/j.bmcl.2017.02.070
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hutchings K.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.070

2
K.M. Hutchings et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 1
Piperidine analogs.
Cl
O
O
O
R1
Y
N
N
H
X
Cmpd
R1
X
Y
SRE.L IC50
(
l
M)a
MLM T1/2 (min)b
Sol (mg/mL)c
ACTA2 (%ctrl)d
ClogPe
TPSAf
1
8a
H
H
H
Me
F
H
H
H
H
H
H
H
CH2
CF2
CH2
CH2
CH2
CH2
CH2
CHNHAc
CHNHCO2Me
C@CH2
CHNMe2
C@O
CH2
CH2
O
0.64
0.60
3.1
0.55
0.80
21
2.7
5.0
5.0
0.098
5.8
1.6
4.9
13.4
2.3
37
9.3
4.25
4.27
3.49
4.80
4.39
2.78
3.40
2.88
3.50
4.34
3.88
3.43
62.6
62.6
71.8
62.6
62.6
82.9
92.1
91.7
100.9
62.6
65.8
79.6
11a
11b
11c
12d
13d
22a
22b
23
CH2
CH2
NAc
NCO2Me
CH2
CH2
CH2
CH2
CH2
0.84
2.8
1.4
3.3
9.5
24
25
3.9
a
b
c
d
e
f
Inhibition of G
Half-life in mouse liver microsomes.
Thermodynamic solubility in water, determined by Analiza, Inc, using quantitative nitrogen detection.
Inhibition of ACTA2 expression (percent control) in TGF-b-stimulated human dermal fibroblasts (10 mM, 24 h, mean of n = 3).
a12-stimulated SRE. Luciferase activity (mean of n ꢀ 3) in HEK293T cells.
Calculated log partition coefficient.
Topological polar surface area (Å2).
Cl
1 has modest potency and poor pharmacokinetic (PK) properties
that would make it a poor candidate for further development.
We report here our efforts to improve the solubility and metabolic
stability of 1 to facilitate additional in vivo studies, and the suc-
cessful identification of an analog exhibiting oral antifibrotic activ-
ity in a murine model of bleomycin-induced dermal fibrosis.
The half-life of 1 in mouse liver microsomes (MLM) is only
1.6 min (Table 1), indicating high susceptibility to oxidative meta-
bolism. An analysis by SMARTCyp15,16 predicted that the primary
Cl
Cl
O
O
O
O
a
Br
HN
N
H
N
N
N
H
Het
3
2
c
b
O
O
Het
N
H
sites for metabolism are the furan (a known structural alert17
)
4
and the 5-carbon of the piperidine-3-carboxylic acid. Metabolite
id experiments on several analogs indicated oxidation of the piper-
idine ring and the left-hand aromatic ring as well as hydrolysis of
the secondary carboxamide as major pathways (Supplemental).
Our medicinal chemistry strategy thus had three major objectives:
1) stabilize the 3-furanylphenyl moiety to oxidation through sub-
stitution or replacement of one or both of the rings with heterocy-
cles; 2) block oxidation of the piperidine carbons sterically or
through fluorination; and 3) explore alternatives to the labile sec-
ondary amide. In most cases these changes would reduce ClogP,
which we expected would also improve the modest aqueous solu-
bility of 1.
Schemes 1 and 2 summarize the preparation of analogs that
replace the 3-furanylphenyl of 1 with various heteroaromatics,
and the inclusion of geminal difluoro on the piperidine ring. Piper-
idine carboxamide 211 was coupled with commercially available 3-
bromo-heteroaryl carboxylic acids to provide amides 3 which
could be reacted with 2-furanylboronic acid under Suzuki condi-
tions to give final amides 4 (Scheme 1). If the requisite furan-2-
yl-heteroaryl carboxylic acids were commercially available, they
could be coupled directly with 2. Hydroxyacid 5 could be con-
verted to anilide 6 in two steps (Scheme 2). The initial HATU cou-
Scheme 1. Reagents and Conditions: (a) EDC, DIPEA, DMAP, Br-Het-CO2H, THF,
63%; (b) 2-furanylboronic acid, Pd(PPh3)4,Cs2CO3, Dioxane:H2O (4:1), 80 °C, 62%; (c)
EDC, DIPEA, DMAP, furan-2-yl-Het-CO2H, THF, 15–82%.
Cl
Cl
O
O
O
O
a,b
BocN
N
H
BocN
OH
BocN
N
H
c
O
7
OH
5
OH
6
Cl
O
O
Cl
Het
N
N
H
f
d - f
HN
N
H
Y
X
R
R
2
8:
R = F
9: R = H
Scheme 2. Reagents and Conditions: (a) 4-chloroaniline, HATU, Et3N, DMF; (b) 4-
chloroaniline, Me3Al, CH2Cl2, 0 °C to RT, 74% over 2 steps; (c) (ClCO)2, DMSO, TEA,
CH2Cl2, 69%; (d) DAST, CH2Cl2 À78 °C to 0 °C, 23%; (e) TFA, CH2Cl2, 84%; (f) Ar-CO2H,
EDC, DIPEA, DMAP, THF, 46–98%.
pling with 4-chloroaniline gave
a mixture of lactone and
hydroxyamides 6 that had to be further reacted under Weinreb
conditions to drive to completion (Supplemental). Swern oxidation
of the alcohol gave ketone 7 that was converted to the geminal
difluoride with DAST. N-deprotection and amide coupling with
diverse aromatic acids under standard conditions provided the
difluoro analogs 8. Coupling of unsubstituted piperidine amide 2
with various aromatic acids gave the simple nipecotic amide
derivatives 9.
The route to analogs incorporating heteroatoms into the
piperidine ring or bearing substitution
a to the carboxamide is
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K.M. Hutchings et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Cl
presented in Scheme 3. Commercial acids 10a-d were converted to
O
bis(amides) 11a-d through sequential EDC-mediated coupling
with 4-chloroaniline, N-deprotection and further amide coupling
with 3-furan-2-ylbenzoic acid. Hydrogenolysis of 11d followed
by reaction with acetic anhydride or methyl chloroformate
afforded the N-acyl analogs 12d and 13d. Replacements of the sec-
ondary carboxamide of 1 with reverse amide (16) and ether (19)
were accomplished as described in Schemes 4 and 5, respectively.
Finally, analogs bearing substitution at the 5-carbon of the
piperidine-3-carboxamide were prepared as summarized in
Schemes 6 and 7. 5-hydroxy ester 20 was subjected to mesylation,
displacement with azide, saponification, EDC-mediated amide cou-
pling and N-deprotection to give 5-azido piperidine 21. Amidation
of the secondary amine, Staudinger reduction and subsequent N-
acylation provided amide 22a or carbamate 22b as mixtures of
diastereomers. Ketone 7 could be methylenated under Wittig con-
CO2Me
a - e
BocN
HN
N
H
20 OH
21 N3
Cl
O
O
N
O
f, g, h
N
H
22a:
22b:
R = Me
R = OMe
NHCOR
Scheme 6. Reagents and Conditions: (a) CH3SO2Cl, DIPEA, CH2Cl2; (b) NaN3, DMF,
80 °C, 16 h, 59% (2 steps); (c) 1:1 2 N NaOH/MeOH, 95%; (d) 4-chloroaniline, EDC,
DIPEA, DMAP, THF 75%; (e) TFA, CH2Cl2, 99%; (f) 3-(furan-2-yl)benzoic acid, EDC,
DIPEA, DMAP, THF, 83%; (g) PPh3, THF/H2O (2:1), 70 °C, 6 h, 72%; (h) CH3COCl, Et3N,
CH2Cl2, quant. or CH3CO2Cl, Et3N, CH2Cl2, 84%.
ditions, followed by N-deprotection and amidation, to give exo-
methylene derivative 23. The carbonyl of 7 could also be reduc-
tively aminated, providing dimethylamino derivative 24 after N-
deprotection/amidation. Finally, the carbonyl of 7 could be left in
place during N-deprotection/acylation to give keto analog 25.
All new analogs were tested for their ability to block the Rho/
MRTF/SRF pathway in HEK293T cells transfected with a luciferase
reporter driven by the MRTF-selective SRE.L promoter, a modifica-
tion of an assay that has previously been described by us (see Sup-
plemental). Selected active analogs were then assayed for stability
to incubation with MLM and for thermodynamic aqueous solubil-
ity. Data are summarized in Tables 1–4 along with key calculated
physical properties: ClogP and topological polar surface area
(TPSA).
Cl
O
O
O
R1
Y
R1
Y
CO2H
a - c
N
N
H
BocN
1 = H, Y = O
R
R
1 = H, Y = O
1
10a:
11a:
11b:
R
R
1
10b:
= Me, Y = CH2
= Me, Y = CH2
10c: R1 = F, Y = CH2
10d: R1 = H, Y = NCbz
11c: R1 = F, Y = CH2
11d: R1 = H, Y = NCbz
Cl
O
O
O
R1
d,e
N
N
H
Y
1
12d:
13d:
R
= H, Y = NAc
= H, Y = NCO2Me
1
R
Substitution (F or Me) alpha to the secondary carboxamide was
first explored to attenuate hydrolysis (11b and 11c). Although SRE.
L potency was maintained, there was no significant improvement
in stability to MLM, which contain hydrolyzing enzymes. Incorpo-
ration of heteroatoms into the piperidine ring (11a, 12d, 13d)
diminished potency in every case. We next explored substitution
on the piperidine 5-carbon, a site specifically predicted by SMART-
Cyp to be susceptible to oxidation. Geminal difluorination (8a) did
not improve potency, but lengthened the half-life in MLM by a fac-
tor of three. Acyl or alkyl nitrogen substitution, tested as mixtures
of diastereomers (22a-b, 24) induced marked losses in potency. A
carbonyl at the 5-carbon (25) was similarly detrimental, but inter-
estingly, the corresponding exo methylene (23) improved SRE.L
potency nearly 10-fold (IC50 = 98 nM). Unfortunately, stability to
MLM was not improved.
Table 2 summarizes our attempts to replace the benzamide
phenyl ring of 1 with heterocycles, which was expected to improve
metabolic stability by lowering overall ClogP. All 5-membered
heterocycles (4d-g), along with pyrimidine 4c, were significantly
less active than 1. Pyridine analogs 4a and 4b, however, main-
tained more activity, but did not possess measurably improved
metabolic stability.
Scheme 3. Reagents and Conditions: (a) 4-chloroaniline, EDC, DIPEA, DMAP, THF,
55–84%; (b) HCl in dioxane or TFA in CH2Cl2, 54–100%; (c) 3-(furan-2-yl)benzoic
acid, EDC, DIPEA, DMAP, THF, 49–94%; (d) 10% Pd/C, H2, MeOH, 40%; (d) Ac2O,
DMAP, THF or ClCO2Me, Et3N, CH2Cl2, 55%.
Cl
H
NH2
a
N
BocN
BocN
O
O
14
b,c
15
16
Cl
O
O
N
H
N
Scheme 4. Reagents and Conditions: (a) 4-chlorobenzoic acid, EDC, DIPEA, DMAP,
THF, 83%; (b) TFA, CH2Cl2; (c) 3-(furan-2-yl)benzoic acid, EDC, DIPEA, DMAP, THF,
87% (2 steps).
Cl
Additional analogs designed to avoid secondary amide hydroly-
sis are presented in Table 3. N-methylation (26) reduced activity by
10-fold. The reverse amide 16 similarly lost too much potency.
Interestingly, ether 19 retained all of the activity of amide 1, but
was also similarly unstable to MLM, suggesting that the primary
pathway for in vitro clearance is oxidative rather than hydrolytic.
We then focused our attention on replacing the oxidatively
labile phenyl furan (Table 4). Analogs 9c, 9i, 9j and 9o have pyra-
zole or imidazole installed as furan bioisosteres with lower
lipophilicity and electron density. 3-pyrazole analog 9o main-
tained strong activity in the SRE.L assay, but was only marginally
more stable in the MLM, suggesting that oxidation of the
a,b
BocN
c,d
OH
BocN
O
O
17
18
19
Cl
O
O
N
Scheme 5. Reagents and Conditions: (a) MsCl, Et3N, CH2Cl2, quant.; (b) 4-
chlorophenol, Cs2CO3, DMF, 75 °C, 65%; (c) TFA, CH2Cl2; (d) 3-(furan-2-yl)benzoic
acid, EDC, DIPEA, DMAP, THF, 77% (2 steps).
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K.M. Hutchings et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Cl Cl
O
O
O
O
b - d
N
N
H
BocN
N
H
7
O
23
c, d
a, c, d
Cl
Cl
O
O
O
O
O
O
N
N
N
N
H
H
24
N
O
25
Scheme 7. Reagents and Conditions: (a) (CH3)2NH, NaBH(OAc)3, HOAc, 4 h, 98%; (b) CH3P(Ph)3Br, KOtBu 30 min, 69% (c)TFA, CH2Cl2; (d) 3-(furan-2-yl)benzoic acid, EDC,
DIPEA, DMAP, THF 10–83% (2 steps).
Table 2
Heterocyclic replacements for benzamide.a
Cl
O
O
O
Ar
N
N
H
Cmpd
Ar
SRE.L IC50
0.64
(lM)
MLM T1/2 (min)
1.6
ClogP
4.25
TPSA
62.6
1
4a
2.9
<3
<3
3.03
75.4
N
4b
4c
1.9
3.41
3.09
75.4
88.3
N
N
15.3
N
4d
4e
4f
5.3
6.8
6.2
2.86
2.92
3.56
91.2
88.6
103.7
N
HN
O
N
N
S
S
4g
5.8
3.33
103.7
N
a
Assay descriptions provided in Table 1.
piperidine ring remained an issue. The most metabolically stable
pyrazole 9j unfortunately had diminished SRE.L activity.
last model of pro-fibrotic gene expression. Primary human dermal
fibroblasts were stimulated with TGF-b, a pro-fibrotic cytokine that
The remaining analogs in Table 4 (8b-n) represent our attempts
to combine two or more of the most successful modifications from
the earlier SAR (furan replacement, geminal difluorination of the
piperidine, replacement of the benzamide phenyl with pyridine)
into single analogs. Greatest retention of SRE.L potency was real-
ized with pyridine or N-Me imidazole replacements for the furan
in combination with difluorination of the piperidine (8b, 8d-f, 8k).
Several of these analogs also had improved half-lives in MLM,
especially 8b and 8d. Most polar replacements for the furan (e.g.
imidazole 8c, pyrimidines 8g and 8h, and 4-pyrazole 8j) resulted
in unacceptably diminished potency in the SRE.L assay. The sole
exception was 1-pyrazole 8i. Attempts to replace the benzamide
phenyl of 8a with pyridine (8m, 8n) were similarly unsuccessful.
Selected analogs that were both active in the SRE.L assay and
had improved stability to MLM were advanced to a primary fibrob-
increases ACTA2 gene expression (a-SMA protein), a marker of the
fibroblast to myofibroblast transition,2 and treated with 10 mM of
selected compounds to determine the reduction in ACTA2 mRNA
as measured by qPCR. Results are included in Tables 1 and 4. Initial
geminal difluoro analog 8a, despite equivalent potency in the pri-
mary SRE.L assay, displayed much greater inhibition of ACTA2
expression at 10 mM than 1 (9% control vs 37% control). Olefin 23
also exhibited stronger efficacy in the fibroblast assay than 1, con-
sistent with its much greater SRE.L potency. Eight new analogs in
Table 4 were tested, and their efficacy varied widely. There is no
clear correlation with SRE.L potency, although no compound with
an SRE.L IC50 > 1 mM inhibited ACTA2 expression by more than
50%. A mild correlation of ACTA2 expression inhibition with ClogP,
however, is evident (Supplemental Fig. S2), suggesting that a
minimum lipophilicity is needed for good permeability into the
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K.M. Hutchings et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Table 3
Primary carboxamide replacements.a
Cl
O
O
X
N
Y
Cmpd
X
Y
SRE.L IC50
(
lM)
MLM T1/2 (min)
1.6
ClogP
TPSA
1
C@O
NH
CH2
C@O
NH
C@O
O
0.64
4.9
0.81
8.6
4.25
3.92
4.73
4.11
62.6
62.6
42.7
53.8
16
19
26b
1.8
NMe
a
Assay descriptions provided in Table 1.
Prepared as in Scheme 1 in Ref. 9, using N-Me-4-Cl-aniline.
b
Table 4
Heteroaryl carboxamide analogs.a
Cl
O
O
HET
N
N
H
Y
X
R
R
Cmpd
HET
X
Y
R
SRE.L IC50
(
lM)
MLM T1/2 (min)
Sol (mg/mL)
ACTA2 (%ctrl)
ClogP
TPSA
1
2-Furanyl
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CF
N
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
F
F
F
F
F
F
F
F
F
F
F
F
0.64
0.37
5.5
1.6
16
13.4
14.9
37
110
4.25
3.24
3.39
4.38
4.00
4.00
4.05
3.29
3.65
3.52
4.14
2.78
3.44
3.06
2.46
3.62
3.49
3.88
62.6
67.2
78.1
62.3
62.3
62.3
75.2
75.2
67.2
78.1
62.3
75.2
75.4
75.4
67.2
67.2
78.1
78.1
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
9c
9i
1-Me-5-imidazoyl
2-Imidazolyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
2-Pyrimidinyl
5-Pyrimidinyl
1-Pyrazolyl
4-Pyrazolyl
4-Pyridyl
4-Pyridyl
2-Furanyl
2-Furanyl
1-Imidazolyl
1-Pyrazolyl
4-Pyrazolyl
3-Pyrazolyl
1.0
9.5
< 3
7.8
3.9
37
0.90
0.55
3.0
3.2
0.68
4.5
0.53
1.2
7.1
3.5
5.2
23.1
75.4
31
65
11.9
4.8
13
6.9
7.7
2.5
3.0
23
140
9.8
62
14
190
CH
CH
CH
CH
CH
F
H
H
H
H
2.9
3.0
0.92
1.7
5.6
3.4
9j
9o
73
73
a
Assay descriptions provided in Table 1.
fibroblasts. In contrast, ClogP overall correlated only very weakly
with SRE.L activity and solubility, and not at all with MLM T1/2
(Supplemental Figs. S3–S5).
bleomycin treatment alone. Significantly, these results are compa-
rable to what was achieved by 1 in our previous study but at one-
fourth the total daily dose and by oral administration.
Three new compounds possessing both greater efficacy than 1
in the fibroblasts and longer half-lives in MLM were progressed
to short (7 h) PK studies in mice to determine if they could achieve
plasma exposure superior to that of 1 (Table 5). Analyses were ini-
tially performed following single intraperitoneal (IP) injection at a
dose of 10 mg/kg. All displayed marked improvements in AUC over
1 (from 8-fold to over 20-fold). 8f and 8k had particularly high
exposures, so were re-analyzed after single oral gavage at 20 mg/
kg. The nearly equivalent dose-normalized exposures achieved by
8f following either IP or PO administration, as well as its superior
solubility, encouraged us to progress this compound to our initial
in vivo efficacy model of dermal fibrosis.
We previously reported that 1 can attenuate the development
of bleomycin-induced dermal fibrosis in mice with 14 days of IP
dosing (100 mg/kg bid).14 We repeated this fibrosis prevention
study with 8f, dosed PO at 50 mg/kg qd. Results are presented in
Fig. 1 and indicate efficacy at reducing the increases in both dermal
thickness (À55%) and hydroxyproline content (À62%) induced by
In conclusion, starting with a lead inhibitor 1 of the Rho/MRTF/
SRF signaling pathway possessing poor solubility and metabolic
stability, we undertook a systematic campaign to improve the
physical and PK properties to facilitate testing in long-term animal
models of fibrosis. Guided by both computational prediction and
actual metabolite id studies, we modified areas of the molecule
expected to be highly susceptible to metabolism in vivo, including
the piperidine core and the heteroarylbenzamide moiety. Our
major strategies centered on reducing the electron density of the
aromatic rings, incorporating fluorine and reducing overall ClogP.
We were successful in identifying several analogs with equipotent
or better SRE.L activity and markedly improved plasma exposure
levels upon IP or PO dosing in mice. In this work, analog 8f
(CCG-232601) was shown to attenuate the development of
bleomycin-induced dermal fibrosis in mice upon oral dosing at
one-fourth the daily IP dose of 1 that had demonstrated compara-
ble efficacy in an earlier experiment. Another new analog from this
effort, 8a (CCG-222740), has recently been shown by Yu-Wai-Man
Please cite this article in press as: Hutchings K.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.070

6
K.M. Hutchings et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 5
Single dose plasma exposure in mice.
Route
Cmpd
IP
PO
AUCa
AUCa
Concb (0.5 h)
Concb (7 h)
1 (CCG-203971)
8a (CCG-222740)
8f (CCG-232601)
8k (CCG-257081)
1,200
9,200
15,251
26,700
33,719
36,385
14.4
13.2
2.4
1.3
a
Estimated area under the plasma concentration curve (0–7 h, h.ng/mL) following a single dose by intraperitoneal injection (10 mg/kg) or oral gavage (20 mg/kg).
Plasma concentration at time indicated (mM).
b
Fig. 1. 8f inhibits bleomycin-induced skin fibrosis in mice. Three groups of mice (n = 8) were treated for 14 days with one of the following daily protocols: Control:
intracutaneous injections of PBS; Bleo: intracutaneous injections of bleomycin with concurrent oral gavage of vehicle; or Bleo + 8f: intracutaneous injections of bleomycin
with concurrent oral gavage of 50 mg/kg 8f. Statistical analyses were done with GraphPad Prism 7 using an unpaired Student’s t-test. ****p < 0.0001; **p < 0.01.
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Acknowledgements
The research reported in this publication was supported by the
National Institute of Arthritis and Musculoskeletal and Skin Dis-
eases of the National Institutes of Health under award numbers
R01AR066049 (SDL) and T32AR007080 (P-ST), and also by the
National Cancer Institute of the National Institutes of Health under
award number P30CA046592 (embedding and cutting of skin sam-
ples from the bleomycin study was performed by the UMCCC His-
tology/Immunohistochemistry CORE). The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health. Support
was also provided by the Scleroderma Foundation, and the Michi-
gan State University Fibrosis Research Fund.
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.02.
070.
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Please cite this article in press as: Hutchings K.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.02.070