5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent New Class of Inhibitors of Rho/Myocardin-Related Transcription Factor (MRTF)/Serum Response Factor (SRF)-Mediated Gene Transcription as Potential Antifibrotic Agents for Scleroderma

Article abstract of DOI:10.1021/acs.jmedchem.8b01772

Through a phenotypic high-throughput screen using a serum response element luciferase promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated gene transcription with good potency (IC₅₀ = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism. The series nevertheless has no observable cytotoxicity up to 100 μM. Ensuing pharmacokinetic optimization resulted in the development of two potent and orally bioavailable anti-fibrotic agents that were capable of dose-dependently reducing connective tissue growth factor gene expression in vitro as well as significantly reducing the development of bleomycin-induced dermal fibrosis in mice in vivo.

Full text of DOI:10.1021/acs.jmedchem.8b01772

Subscriber access provided by IDAHO STATE UNIV
Article
5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent
New Class of Inhibitors of Rho/Myocardin-Related Transcription
Factor (MRTF)/Serum Reponse Factor (SRF)-Mediated Gene
Transcription as Potential Antifibrotic Agents for Scleroderma
Dylan Kahl, Kim M Hutchings, Erika Mathes Lisabeth, Andrew Haak, Jeffrey R Leipprandt,
Thomas S. Dexheimer, Dinesh Khanna, Pei-Suen Tsou, Phillip L Campbell, David
A. Fox, Bo Wen, Duxin Sun, Marc Bailie, Richard R. Neubig, and Scott D Larsen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01772 • Publication Date (Web): 05 Apr 2019
Downloaded from http://pubs.acs.org on April 5, 2019
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of their duties.

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5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent New Class of
Inhibitors of Rho/Myocardin-Related Transcription Factor (MRTF)/Serum
Reponse Factor (SRF)-Mediated Gene Transcription as Potential
Antifibrotic Agents for Scleroderma
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Dylan J. Kahl , Kim M. Hutchings , Erika Mathes Lisabeth , Andrew J. Haak , Jeffrey R.
d
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Leipprandt , Thomas Dexheimer , Dinesh Khanna , Pei-Suen Tsou , Phillip L. Campbell , David
c
b
b
e
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A. Fox , Bo Wen , Duxin Sun , Marc Bailie , Richard R. Neubig and Scott D. Larsen *
a
b
Vahlteich Medicinal Chemistry Core, UM Pharmcokinetics Core, College of Pharmacy,
University of Michigan, Ann Arbor, Michigan 48109,
cDepartment of Internal Medicine, Division of Rheumatology and Clinical Autoimmunity Center
of Excellence, University of Michigan Medical Center, Ann Arbor, Michigan 48109,
dDepartment of Pharmacology and Toxicology, Michigan State University, East Lansing,
Michigan 48824
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eMichigan State University In Vivo Facility, East Lansing, Michigan 48824
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Abstract:
Through a phenotypic high throughput screen (HTS) using a serum response
element luciferase (SRE.L) promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-
ylthiopropionic acid lead inhibitor of Rho/MRTF/SRF-mediated gene transcription with
good potency (IC = 180 nM). We were able to rapidly improve the cellular potency by
50
5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable
potency and depth of the SAR, as well as the relatively low molecular weight of the
series, suggests, but does not prove, that binding to the unknown molecular target may
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be occurring through a covalent mechanism. The series nevertheless has no
observable cytotoxicity up to 100 M. Ensuing pharmacokinetic optimization resulted in
the development of two potent and orally bioavailable anti-fibrotic agents that were
capable of dose-dependently reducing connective tissue growth factor (CTGF) gene
expression in vitro as well as significantly reducing the development of bleomycin-
induced dermal fibrosis in mice in vivo.
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Introduction:
Scleroderma is a chronic connective tissue disease that is characterized by
thickening and fibrosis of dermal epithelial tissue and vascular abnormalities. It has
been designated as an orphan disease, and, while the exact number affected is
unknown, estimates suggest ~100,000-300,000 individuals have some form of
1
scleroderma in the U.S. There are two classifications of the disease: localized and
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systemic. Localized scleroderma does not involve internal organs, but systemic
sclerosis (SSc) is associated with pathology in multiple organs including skin,
gastrointestinal tract, lungs, kidneys, heart, etc.3-4 The incidence of SSc in the U.S. is 9–
46 cases per million per year, and roughly 50% of these individuals have a highly
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aggressive form: diffuse systemic sclerosis (dSSc).1, 5 Patients suffering with dSSc have
a higher risk of developing fibrosis within the internal organs, greatly increasing the
patient’s mortality risk.2, 5 Current treatments are limited to immunosuppressants and
anti-inflammatories that only address the symptoms rather than the underlying causes
of the disease.2, 5 Currently in the U.S., the only FDA-approved antifibrotics are
pirfenidone and nintedanib for the treatment of idiopathic pulmonary fibrosis (IPF).3, 6
Although the etiology of fibrosis is unknown, the activation of fibroblasts and their
transition to myofibroblasts are key characteristics of the fibrotic process (Figure 1).7-8
Myofibroblasts produce excess extracellular matrix (ECM) components (e.g. collagen
and fibronectin) and stress fibers, e.g. α-smooth muscle actin (α-SMA).8-9 Many of these
components undergo cross-linking and maturation, resulting in a highly stable matrix
that accounts for epithelial tissue stiffness.10 Dysregulation of ECM homeostasis is
thought to contribute to mortality associated with SSc.1
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There are several known cell surface receptors involved in the fibrotic process,
including various integrins, GPCRs, and growth factor receptors that activate the
production of ECM components (Figure 1).9, 11-13 These surface receptors, such as
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those for transforming growth factor β (TGFβ), connective tissue growth factor (CTGF),
and lysophosphatidic acid (LPA), activate intracellular signaling through a common
mediator (Rho GTPase), which leads to activation of Rho-associated protein kinase
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(
ROCK).11, 14-18 Activation of ROCK induces the polymerization of globular (G)-actin to
filamentous (F)-actin, which releases, and thereby promotes, the nuclear localization of
myocardin-related transcription factor (MRTF).14 In the nucleus, MRTF binds to the
serum response factor (SRF) on serum response elements (SREs). Binding of
MRTF/SRF to these promoter regions of DNA activates expression of multiple genes
associated with the fibrotic process, including: COL1A1, CTGF, and ACTA2.9, 14, 16
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Figure 1: The fibroblast-to-myofibroblast transition is tightly regulated by Rho/MRTF/SRF
signaling. Both soluble and mechanical stimuli promote MRTF nuclear translocation in
fibroblasts. In the resting state, actin is not polymerized, and MRTF is bound to G-actin and
sequestered into the cytoplasm. When stress fibers form in response to these stimuli, G-actin
polymerizes into F-actin and MRTF is released. This allows MRTF to translocate into the
nucleus via binding to importins followed by MRTF/importin/nuclear pore complex (NPC)
formation. The MRTF/SRF coactivator complex increases ECM and pro-fibrotic gene
expression, contributing to the fibroblast-to-myofibroblast transition.
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While Rho/MRTF/SRF signaling plays an important role in normal wound healing,
over-stimulation or dysregulation of this pathway can lead to pathological fibrosis as well
as increased cancer cell migration and metastasis.7, 19-22 Several attempts to target
some of the aforementioned surface receptors for this signaling pathway have been
made (e.g. STX-100 for integrins, AM152 for LPA receptors, and LY2157299 for TGFβ
receptors); unfortunately, therapeutics exploiting these targets have not been
successfully developed for fibrotic diseases to date.19 As an alternative approach, a
MRTF-A knockout mouse model has demonstrated reduced fibrosis and scarring in
heart, lung, dermal, and vascular fibrosis in response to decreased collagen production
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23
and tissue stiffness. The development of an inhibitor that blocks the Rho/MRTF/SRF
transcription pathway downstream of surface receptors, which in principle could block
multiple extracellular inputs, is a promising new approach toward the treatment of SSc
and other fibrotic diseases.
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Original HTS Hit
Cl
Cl
O
O
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R
F3C
O
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H
H
H
Me
X X2
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X1
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CF3
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CCG-1423
CCG-203971: X = H; X = H; R = 2-furyl
1
2
CCG-222740: X = H; X = F; R = 2-furyl
1
2
CCG-257081: X = F; X = F; R = 4-pyridyl
Expanded HTS Hit
O
S
COOH
N
N
Me
1
PC-3 SRE.L IC50 = 180 nM
Figure 2: Original hit (CCG-1423) from SRE.L HTS and series development to CCG-257081.
New chemotype hit 1 from expanded SRE.L HTS of Rho/MRTF/SRF-pathway.
We have previously reported the development and execution of a SRE promoter-driven
luciferase (SRE.L) HTS for inhibitors of the Rho/MRTF/SRF pathway.19 Although SRF
responds to at least two different co-activator mechanisms, Rho/MRTF and Ras/Ternary
Complex Factor (TCF), this SRE.L luciferase assay is selective for MRTF as the TCF
sites in the promoter have been eliminated.24 This led to the discovery of CCG-1423,
which we subsequently optimized to CCG-203971, CCG-222740, and CCG-257081—
modestly potent inhibitors that are active in multiple animal models of fibrosis.19, 22, 25-32
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An expanded HTS of 80,000 compounds produced a second, more potent class of
Rho/MRTF/SRF inhibitors based on the confirmed hit (3-((5-(m-tolyl)-1,3,4-oxadiazol-2-
yl)thio)propanoic acid 1 (SRE.L IC = 180 nM, Figure 2). The ensuing SAR of this class
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proved to be remarkably deep (over 5 orders of magnitude range of activity), suggesting
that the currently unknown biological target has a very well-defined binding site.
Furthermore, we report indirect evidence (from the SAR) that suggests engagement
with the unknown target may be covalent; nevertheless, the series appears to have very
low cytotoxicity (no impact on cell viability up to 100 µM). Herein, we report the
synthesis, biological activity, pharmacokinetic profile, and initial in vivo efficacy of this
structurally distinct new series of Rho/MRTF/SRF-mediated gene transcription
inhibitors.
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Chemistry
Scheme 1. General Synthesis of 5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acidsa
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SH
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N
(CH2)n CO R'
2
O
O
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R4
R3
R4
R3
R4
R3
R4
R3
NH2
OH
N
N
N
a,b
c,d
e
H
R1
R1
R1
R1
R2
R2
R2
R2
5
6
: R' = Me
: R' = Et
7: R' = tBu
n = 1-4
2
3
4
S
(CH2)n CO2H
S
N
(CH ) CONHCH3
2 2
O
O
R4
N
N
N
f or g
h, i
R3
R1
Cl
Cl
R2
n = 1-4
9
8
a
Reagents and conditions: (a) H SO /MeOH, 85 °C 6 h. (b) H N •H O, 85 °C 14 h. (c)
2
4
4
2
2
KOH/H O/EtOH, CS , 95 °C 16 h. (d) 1N HCl workup (e) acetone, K CO , Br-(CH ) -CO R’, 25
2
2
2
3
2 n
2
°
C, 5-24 h. (f) DCM, TFA, 25 °C 3 h. (g) 1M NaOH, THF, 25 °C 16 h. (h) oxalyl chloride, DMF,
CH Cl , 25 °C, 2 h. (i) MeNH in EtOH, 25 °C, 16 h.
2
2
2
The general synthesis of new 5-aryl-1,3,4-oxadiazol-2-ylthioalkanoic acids and
derivatives is summarized in Scheme 1. Various aromatic benzoic acids (2) were
converted to their respective methyl esters under standard Fischer esterification
conditions, and then converted to the corresponding hydrazides (3) by refluxing in
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MeOH with excess hydrazine hydrate. Refluxing 3 and carbon disulfide in EtOH under
basic conditions, followed by acidic workup, generated 2-mercapto-5-aryl-1,3,4-
oxadiazoles (4). S-alkylation with either methyl, ethyl, or t-butyl -bromoalkanoates
provided the thioether esters (5-7). Hydrolysis of the esters was accomplished with
either trifluoroacetic acid (t-butyl esters) or sodium hydroxide (methyl and ethyl esters)
to give final carboxylic acids 8 (Table 1). The tert-butyl esters became the preferred
precursor when n = 2 because basic hydrolysis of the methyl ester intermediates
resulted in competing retro-Michael elimination to regenerate the 1, 3, 4-oxadiazole-2-
thiols 4. Amide 9 was prepared by conversion of the corresponding acid 8 to the acid
chloride with oxalyl chloride in DMF/CH Cl at room temperature, followed by treatment
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with methylamine.
Scheme 2. Synthesis of Thioalkanoate-modified Analogs 11 and 13a
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CO H
2
OH
O
O
O
N
N
c, d
N
N
Cl
Cl
Cl
Cl
a, b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10
11
O
NH2
N
H
CO H
Cl
Cl
2
Br
e
S
3j
O
O
N
N
N
f
N
Cl
Cl
Cl
Cl
12
13
a
Reagents and conditions: (a) K PO , CS , H O, 106 °C, 2 h. (b) propylene oxide, 25 °C, 16 h.
3
4
2
2
(
c) DMF, K CO , Br-(CH ) -CO tBu, 90 °C, 1 h. (d) DCM, TFA, 25 °C, 1 h. (e) POCl ,
2
3
2 3
2
3
bromoacetic acid, 106 °C, 16 h. (f) 2-mercaptoacetic acid, acetone, K CO , 25 °C, 2 h.
2
3
The syntheses of 4-((5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)oxy)butanoic
acid (11) and 2-(((5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)thio)acetic acid
(13) were accomplished starting with common intermediate 3j. Intermediate 10 was
produced through the cyclization/rearrangement of 3j with propylene oxide.33 O-
alkylation with t-butyl 4-bromobutyrate followed by acidic hydrolysis with trifluoroacetic
acid provided carboxylic acid 11. Alternatively, subjecting 3j to bromoacetic acid and
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3
4
POCl afforded intermediate 12. Basic S-alkylation with potassium carbonate and 2-
3
mercaptoacetic acid produced carboxylic acid 13.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthetic Route to Generate Thiobutanoate Analogs 19a-ia
CO Me
CO H
2
2
I
Cl
Cl
X
14
15a X = 3-bromo
15b X = 4-bromo
c, d
e, f
15c X = 5-bromo
CO H
CO Me
CO Me
2
2
2
a,b
g or h
HO
Me
Cl
Cl
Cl
G
3k
16
1
1
7a G = 4-vinyl
7b G = 4-propyl
17c G = 4-isopropyl
1
1
7d G = 4-1-fluoroisopropyl
7e G = 3-cyclopropyl
i
17f G = 4-cyclopropyl
17g G = 5-cyclopropyl
1
1
7h G = 4-O-cyclopropyl
7i G = 4-O-oxetanyl
1
7a' G = 4-ethyl
j, k
CO H
2
SH
S
N
O
N
N
O
l
N
m or n
Cl
G
Cl
G
18a-i
19a-i
aReagents and conditions: (a) LDA (2 eq.), THF, 0 °C, 10 min, then ethyl iodide, 0 °C, 1 h. (b)
MeOH, H SO , 85 °C, 16 h. (c) iPrMgCl, THF, -20 °C for 1.5 h, then acetone, 25 °C for 1.5 h. (d)
2
4
DAST, DCM, -78 °C to 25 °C 2 h. (e) MeOH, H SO , 85 °C, 16 h. (f) Pd(II)OAc Cl , K PO ,
2
4
2
2
3
4
P(Cy) , cyclopropyl boronic acid, toluene, H O, 100 °C, 3 h. (g) bromocyclopropane, Cs CO ,
3
2
2
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DMA, 155 °C, 24 h then MeOH, H SO , 85 °C, 16 h. (h) 3-bromooxetane, Cs CO , DMSO, 105
2
4
2
3
°
C, 8 h. (i) 2-nitrobenzene-1-sulfonyl chloride, H N •H O, MeCN, 0 °C to 25 °C, 16 h. (j)
2
4
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H N •H O, MeOH, 85 °C, 16 h. (k) KOH/H O/EtOH, CS , 95 °C, 16 h., followed by HCl workup
4
2
2
2
2
(
l) 4-bromo-butanoate ester, K CO , acetone, 25 °C, 16 h. (m) 1M NaOH, THF, 25 °C, 1 h. (n)
2
3
TFA, DCM, 25 °C, 1 h.
Next, alkyl, fluorinated alkyl, cycloalkyl, alkoxy, and cycloalkoxy derivatives were
synthesized as shown in Scheme 3. Routes to obtain vinyl and isopropyl methyl esters
1
7a and 17c were performed as previously described.35-36 Alkyl methyl ester 17b was
synthesized by alkylating 3k with two equivalents of LDA and ethyl iodide, followed by
esterification. 4-(1-fluoro)-isopropyl methyl ester 17d was synthesized from 14 by
Grignard reaction with acetone followed by fluorination of the tertiary alcohol with
diethylaminosulfur trifluoride (DAST). Suzuki coupling of commercially available 15a-c
with cyclopropyl boronic acid afforded cycloalkyl methyl esters 17e-g. 4-alkoxy aryl
derivatives were formed by O-alkylation of commercially available phenol 16 using
cesium carbonate and either DMA or DMSO (17h and 17i, respectively). Reduction of
vinyl intermediate 17a with 2-nitrobenzene-1-sulfonyl chloride and hydrazine hydrate
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resulted in ethyl intermediate 17a’. Using chemistry analogous to Scheme 1, the aryl
methyl esters 17a’ and 17b-i were converted to respective hydrazides, 5-aryl-2-
mercapto-1,3,4-oxadiazoles (18a-i), thioethers, and final carboxylic acids (19a-i) (Table
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3).
Scheme 4: Synthetic Route to Generate Thiobutanoic Acid Sulfone Analog 21 a
COOH
SH
N
O
S
COOH
3
SO Me
2
O
S
N
3
O
O
O
a
N
O
d
N
e
N
N
b or c
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
21
4j
20
8q
a
Reagents and conditions: (a) MeI, Et N, THF, 25 °C, 2 hr (b) (NH ) Mo O , H O , EtOH, 25 °C,
3
4 6
7
24
2
2
2
hr (c) mCPBA, DCM, 25 °C, 2 hr (d) 4-mercaptobutanoic acid, K CO , acetone, 25 °C, 1 hr,
2
3
(e) mCPBA, DCM, 25 °C, 2 hr.
Sulfone 21 was prepared as shown in Scheme 4, which also includes an
alternative synthetic route for 8q. 1,3,4-oxadiazole thioether 4j was converted to
electrophilic sulfone 20 in two steps. First, thiol 4j was methylated with methyl iodide
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and triethylamine. This thioether intermediate was oxidized to 20 using either mCPBA or
37
ammonium molybdate tetrahydrate and hydrogen peroxide. Nucleophilic displacement
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of sulfone 20 using 4-mercaptobutanoic acid efficiently produced thiobutanoic acid 8q,
which was subsequently oxidized to thiobutanoic acid sulfone analog 21 using mCPBA.
Results and Discussion
CO H
O
N
2
S
N
NH
O
CO H
1
: Ar = 3-Me-Ph: SRE.L IC50 = 180 nM
2
S
22: Ar = 3-pyr: SRE.L IC50 = 87,000 nM
23: Ar = 4-pyr: SRE.L IC50 > 100,000 nM
24: Ar = 2-Me-3-furanyl: SRE.L IC50 > 100,000 nM
Ar
N
25: SRE.L IC50 > 100,000 nM
O
CO H
2
S
N
N
CO H
O
S
2
N
S
N
O
N
N
N
MeO
26: SRE.L IC50 > 100,000 nM
27: SRE.L IC50 > 100,000 nM
28: SRE.L IC50 > 100,000 nM
Figure 3. Preliminary SAR investigating the importance of the heterocycle and carboxylic acid.
SRE.L activity (mean of n = 3, SEM < 10%).
We began our SAR investigation by probing the importance of the phenyl-
substituted oxadiazole ring system and the carboxylic acid with commercially available
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compounds (Figure 3). Pyridyl (22 and 23) and furanyl (24) substituted oxadiazole
analogs led to complete loss in activity. Attempts to replace the oxadiazole core with
other heterocycles (25-27) were similarly unsuccessful. Replacement of the carboxylic
acid with an acetal isostere (28) also abolished activity. Based on these strongly
negative results, we focused our initial synthetic SAR effort on 5-aryl-1,3,4-oxadiazaole-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-thioalkanoic acids.
Table 1. SRE.L Activity of 5-Aryl-1,3,4-Oxadiazole-2-thiopropionic acids
CO H
2
S
N
O
R4
N
R3
R1
R2
SRE.L
IC50
Cmpd
R1
R2
R3
R4
nM)a,
(
b
8
a
b
H
H
H
H
H
H
H
H
H
Cl
H
Cl
H
H
H
H
H
3,500
340
8
8
c
Me
OMe
H
34
8d
160
8e
1,500
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H
H
Me
OMe
H
H
H
H
180
3,600
25
8f
H
H
8g
Cl
H
8h
Me
OMe
Cl
H
H
H
6,300
3,100
4 (2c)
1.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8i
H
H
H
8j
H
Cl
H
8k
Cl
H
Me
OMe
Me
Me
Cl
H
8l
Cl
H
H
2.6
8m
Cl
Me
H
H
0.02c
0.23c
0.32c
8n
Cl
Me
Me
8o
Cl
H
aInhibition of Gα12-stimulated SRE.L activity (mean of n = 3, SEM < 10%) in PC3 cells unless
b
otherwise noted. Cell viability and proliferation assay with WST-1 < 5% inhibition up to 100 M
c
inhibitor (mean of n = 3, SEM < 10%). SRE.L assay performed in HEK293T cells (mean of n =
3
, SEM < 10%).
We started by systematically investigating prototype electron donating (methoxy),
withdrawing (chloro) and alkyl (methyl) groups at each of the three aryl positions (8a-i,
Table 1). Remarkably, this revealed an over 250-fold span of potency over just these
first 9 analogs; SRE.L IC s ranged from 6300 nM (8h) to 25 nM (8g). Within this small
50
initial group we observed several clear activity cliffs, including 2-Cl 8g (25 nM) vs 3-Cl
8
e (1500 nM) and 4-Me 8c (34 nM) vs 3-Me 1 (180 nM) vs 2-Me 8h (6300 nM). We
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were then interested to see if the individual optimum substitutions would be synergistic.
From the data in Table 1, it is clear that, among the four substituents investigated at R1,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl is optimum. We thus combined this substitution with all three non-H substituents at
3
R (8j-l) and observed a significant increase in potency in each case; all three of these
analogs had single digit nM SRE.L activities. Despite these significant improvements in
SRE.L activity, all analogs tested did not affect cell proliferation/viability up to 100 µM as
measured by WST-1, highlighting the series lack of acute cytotoxicity even at high
concentrations. For comparison, the clinically approved antifibrotics pirfenidone and
nintedanib were tested in the SRE.L assay. Pirfenidone was inactive up to a
concentration of 100 M, above which it was cytotoxic (as determined by CellTiter-
TM
Fluor ). Nintedanib had an IC of 1.3 M, but showed some cytotoxicity at 10 uM and
50
an IC of under 30 M.
50
It should be noted that the phenotypic screening SRE.L assay was originally
designed as a HTS for cancer therapeutics, and it was developed using PC-3 cells.19
However, to improve the throughput of the assay, the transfected cell line was switched
to HEK-293T cells. To ensure equivalency in SRE.L potency from one cell line to the
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
other, various analogs were tested under both assay conditions (e.g. 8j, 8q, and 19f).
We did not observe any notable difference in activity between the two transfected cell
lines, so the assay was permanently switched to HEK-293T cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We were able to improve activity even further with trisubstituted analogs (8m-o)
1
3
2
4
that combined the best R , R -disubstitution analogs (8j & 8k) with the best R /R
substituent (Me). This resulted in our first sub-nanomolar analogs with SRE.L potency
as high as IC50 = 20 pM (8m). These extremely tight and potent SAR trends suggest
that these inhibitors likely interact with a very well-defined binding site on the unknown
molecular target.
Table 2. Carboxylic Acid Sidechain SAR.
G
S Z
O
N
N
Cl
Cl
SRE.L
IC50
MLM T1/2
(min)d
Compd
Z
G
(
nM)a, b
4 (2c)
2.6
8j
(CH2)2
(CH2)2
CO H
>60
2
6
9
CO Et
-
-
2
(CH2)2 CONHCH3 >100,000
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p
q
CH2
CO H
>100,000
1.8 (0.84c)
-
>60
-
2
8
(CH2)3
(CH2)4
CO H
2
8r
CO H
43
2
a
b
Inhibition of Gα12-stimulated SRE.L (mean of n = 3, SEM < 10%) in PC3 cells. Cell viability
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and proliferation assay with WST-1 < 5% inhibition up to 100 M inhibitor (mean of n = 3, SEM
c
< 10%) up to 100 M inhibitor. SRE.L assay performed in HEK293T cells (mean of n = 3, SEM
d
<
10%). Half-life in mouse liver microsomes.
We then focused on elucidating how the propionic acid sidechain modulated
activity (Table 2). Replacing the carboxylic acid functionality with neutral methyl amide
(
9) was unsuccessful; however, ethyl ester 6 maintained activity, likely due to facile
hydrolysis by non-specific esterases in the cells. This is consistent with the lack of
activity of neutral acetal 28 (Figure 3), suggesting that the acid functionality is crucial for
target engagement. To determine the optimal carbon chain length between the
oxadiazole and the carboxylic acid, analogs bearing linkers from one to four methylenes
were synthesized (8p-r). We found that the optimal carbon chain length was three (8q),
and that activity was completely lost when there was only one carbon separating the
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
oxadiazole and carboxylic acid (8p). This steep SAR is further evidence of the
importance of the carboxylic acid group and its position relative to the oxadiazole ring. A
three-carbon chain was used for all subsequent analogs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CO H
2
Nu Target
N
S
N
Target-NuH
O
O
8j
NuH = SH, OH, NH2
MW = 319
N
N
ClogP = 2.25
SRE.L IC50 = 2 nM
Cl
Cl
Cl
Cl
CO H
2
CO H
2
O
N
S
O
O
N
N
N
Cl
Cl
Cl
Cl
1
1
13
CLogP: 1.83
SRE.L IC50 = 11,500 nM
CLogP: 2.53
SRE.L IC50 > 100,000 nM
CO H
2
CO H
O
2
O S
O
N
N
O
N
N
Cl
Cl
Cl
Cl
29
21
ClogP: 1.50
CLogP: 1.81
SRE.L IC50 > 100,000 nM
SRE.L IC50 > 100,000 nM
Figure 4. Hypothetical reaction of 8j with a target nucleophile, and close structural analogs
designed to be less (11, 13 and 29) or more (21) susceptible to attack by cellular nucleophiles.
Indicated below each structure is SRE.L activity (HEK293T cells, mean of n = 3, SEM < 10%).
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Based on our unsuccessful attempts to replace the oxadiazole core with similar
heterocycles (Figure 3), the steep SAR of the series with regard to both aryl substitution
and alkanoic acid chain length, and (3) the reported ability of 5-methylsulfonyl-1,3,4-
oxadiazole probes to ligate cellular nucleophiles37-39, we hypothesized that the
oxadiazole thioether core might be covalently engaging with a binding site nucleophile
as illustrated in Figure 4. We therefore carefully selected analogs (11, 13, 29) that are
structurally very similar to 8j but predicted to be much less capable of reacting with
nucleophiles (Figure 4). In support of our hypothesis, we saw an almost complete loss
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in activity upon replacement of the S with O or CH (11, 29) or migration of the S away
2
from the heterocyclic core (13). Conversely, activity was lost when the system was
made more susceptible to nucleophilic attack by oxidizing the sulfide of 8q to the
corresponding sulfone (21). Although the inactivity of the sulfone could be explained by
simple poor cell permeability (due to lower ClogP) or rapid reaction with abundant
cellular nucleophiles, this particular result certainly suggests that oxidation of our sulfide
inhibitors in cellulo to irreversibly-binding sulfones is not occurring. Furthermore, it has
been reported that a non-covalent 2-benzylthio-1,3,4-oxadiazole inhibitor of glycogen
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
synthase kinase-3β can markedly lose potency with simple changes to the thiomethyl
linker analogous to what we made.40 Overall, therefore, our SAR does not prove or
disprove a covalent mode of binding of our compounds. Confirmation will require careful
proteome labeling studies, which are underway and will be reported in due course.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Table 3. SRE.L Activity of 5-Aryl-1,3,4-Oxadiazole-2-thiobutanoic Acids.
CO H
2
S
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
R4
N
N
R3
R1
R2
MLM
SRE.L IC50
(nM)a, b
Cmpd
R1
R2
R3
R4
T1/2
(min)b
8
q
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
Cl
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
0.84
0.1c
>60
8
s
28
8
t
H
MeO
Et
2
0.0007
<0.0001
0.0007
46
-
1
9a
19b
9c
H
-
H
Pr
-
1
H
i-Pr
CF3
H
-
8u
H
-
8v
CF3
Cl
Cl
Cl
Cl
Cl
H
10,000
0.06
-
8w
H
CF3
CF(CH3)2
Br
>60
19d
H
0.5
-
-
-
-
8x
H
0.07
8
y
H
Ph
25
19e
cPr
H
0.019
0
.001
19f
Cl
H
c-Pr
H
47
(
0.0012c)
19g
9h
9i
Cl
Cl
Cl
H
H
H
H
cPr
H
20
-
>60
-
1
O-cyclopropyl
O-oxetanyl
1.3
1
H
370
a
b
Inhibition of Gα12-stimulated SRE.L activity (mean of n = 3, SEM < 10%) in HEK cells. Cell
viability and proliferation assay with WST-1 < 5% inhibition up to 100 M inhibitor (mean of n =
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
c
3
, SEM < 10%). SRE.L data generated with transfected PC-3 cells (mean of n = 3, SEM <
d
1
0%). Half-life in mouse liver microsomes.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, we investigated the SAR on the aryl ring of the butanoic acid analog 8q
(
Table 3). As shown previously in Table 1, incorporation of a small alkyl or alkoxy group
at R2/4 or R led to marked improvements in potency with the propionic acid series. We
therefore explored expanding the size of the alkyl group and, anticipating the potential
for metabolic oxidation of the aromatic methyl group, included small cycloalkyl groups,
fluoroalkyl groups, and small cycloalkoxy groups that we expected to be less prone to
metabolic oxidation. Consistent with what we previously observed, replacement of the
3
4
-Cl group of butanoic acid 8q with Me led to a significant improvement in potency (8s).
Larger alkyl butanoic acids (19a-c, 19f) achieved even greater potency, approaching the
limit of what our SRE.L assay can accurately determine (< 0.1 pM). While the CF3
analogs 8u,v,w all maintained activity comparable to their corresponding methyl analogs
8
c,h,s, the larger fluoro containing derivative, 1-fluoroisopropyl 19d, was less potent
3
than its alkyl comparator 19c, suggesting a possible size limit at R . Interestingly,
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replacement of the 4-Cl of 8q with 4-Br (8x) led to a 10-fold improvement in activity,
consistent with the greater potency observed when increasing the size of the 4-methyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
group to small alkyls. Additional evidence for the size limitation at R was provided by
the 4-Ph analog 8y. Incorporation of oxygen (e.g. 8t, 19h and 19i) also did not afford the
same levels of activity as the corresponding aliphatic analogs, perhaps due to
decreased permeability. The narrow SAR we had been observing was once again
3
2
4
evident when the cyclopropyl at R of 19f was migrated to the R (19e) or R (19g)
positions, leading to significant reductions in SRE.L activity. Finally, with regard to in
vitro metabolic stability (as determined by half-life in the presence of mouse liver
3
microsomes (MLM T½)), replacement of the Cl at R of 8q with more potent Me (8s) was
detrimental as expected, but further replacement of the Me with c-Pr (19f) returned a
measure of metabolic stability while improving potency 100-fold.
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2
2
2
2
2
2
2
3
3
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3
3
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3
3
3
4
4
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5
5
6
Table 4. Physical and Pharmacokinetic Parameters for Selected Rho/MRTF/SRF
Inhibitors.
SRE.
L IC50
Route,
Dose
AUC
Cl
Solubility MLM T½
T ½
(hr)
Cmax
F
Compound
(hr*ng/ (mL/min/
(min)c
(ng/mL)
%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(uM)
(nM)
(mg/kg)
IV, 15
mL)
47350
63170
10716
11140
3255
361
kg)
2.0
5.3
88550
34050
35650
23150
8140
-
682a
8j
4
>60
PO, 30 2.0
IV, 10 1.4
PO, 20 1.2
-
67
-
15.5
8q
1.8
841a
>60
-
52
-
IV, 3
1.7
1.6
15.4
19f
PO, 3
-
-
-
397
11
17
29
0.001
>1000b
47
(
CCG-232964)
PO, 10 1.8
PO, 30 2.2
1826
9496
1433
10053
aThermodynamic solubility analysis was performed by Analiza Inc. using quantitative nitrogen
b
o
c
detection. (www.analiza.com). Equilibrium solubility in PBS at 25 C. Half-life in mouse liver
microsomes. PK parameters were estimated using non-compartmental analysis with
Phoenix/WINONLIN.
IV and PO PK analyses were performed on representative analogs 8j, 8q, and
19f (Table 4). Propionic acid 8j demonstrated good oral bioavailability (67%) and a
moderate half-life (2 h). Rather surprisingly, the corresponding butanoic acid 8q,
despite apparently similar metabolic stability in vitro, had inferior in vivo PK, with a
clearance rate almost 3-fold higher than 8j and lower oral bioavailability. Butanoic acid
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9f also had a higher in vivo clearance rate, suggesting that the longer alkanoic acid
moiety is a primary site of metabolism. It is interesting to note, however, that plasma
exposure of 19f was not linear with oral dose, increasing from 11% bioavailability at 3
mg/kg to 29% at 30 mg/kg, suggesting some saturation of first pass clearance.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6: 8j and 19f inhibit LPA induced CTGF gene expression. Primary dermal fibroblasts
from healthy donors were pre-treated with varying concentrations of 8j (CCG-58150) and 19f
(CCG-232964) for (A) 48 h or (B) 72 h and stimulated with 1 µM LPA for 1 h. CTGF mRNA
levels were measured by qPCR. Results are expressed as the mean ± SEM. *p<0.05,
**p<0.01 vs DMSO control using One-way ANOVA; n>3.
To assess direct effects on pro-fibrotic gene expression, primary human dermal
fibroblasts were pre-treated with 8j (CCG-58150) or 19f (CCG-232964) for 48 or 72 h
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