Macrocyclic MEK1/2 inhibitor with efficacy in a mouse model of cardiomyopathy caused by lamin A/C gene mutation

Article abstract of DOI:10.1016/j.bmc.2016.12.014

Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of ERK1/2, converting it into an active kinase that re

Full text of DOI:10.1016/j.bmc.2016.12.014

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Macrocyclic MEK1/2 inhibitor with efficacy in a mouse model
of cardiomyopathy caused by lamin A/C gene mutation
Wei Wu a,b, Mahendra D. Chordia c,g, Barry P. Hart d, E. Sathyajith Kumarasinghe c, Min K. Ji c,
Ajay Bhargava e, Michael W. Lawlor f, Ji-Yeon Shin a,b, Fusako Sera a, Shunichi Homma a, Antoine Muchir a,b,h
,
Uday R. Khire c,d, , Howard J. Worman
a,b,
⇑
⇑
a Department of Medicine, College of Physicians & Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, United States
b Department of Pathology and Cell Biology, College of Physicians & Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, United States
c Cheminpharma LLC, 23 Business Park Drive, Branford, CT 06405, United States
d AlloMek Therapeutics LLC, 400 Farmington Avenue, Farmington, CT 06032, United States
e Shakti BioResearch LLC, 1 Bradley Road, Suite 401, Woodbridge, CT 06525, United States
f Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, WI 53226, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Signaling mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) is involved in numerous
cellular processes. Mitogen-activated protein kinase kinases (MEK1/2) catalyze the phosphorylation of
ERK1/2, converting it into an active kinase that regulates the expression of numerous genes and cellular
processes. Inhibitors of MEK1/2 have demonstrated preclinical and clinical efficacy in certain cancers and
types of cardiomyopathy. We report the synthesis of a novel, allosteric, macrocyclic MEK1/2 inhibitor
that potently inhibits ERK1/2 activity in cultured cells and tissues of mice after systemic administration.
Mice with dilated cardiomyopathy caused by a lamin A/C gene mutation have abnormally increased car-
diac ERK1/2 activity. In these mice, this novel MEK1/2 inhibitor is well tolerated, improves left ventricular
systolic function, decreases left ventricular fibrosis, has beneficial effects on skeletal muscle structure and
pathology and prolongs survival. The novel MEK1/2 inhibitor described herein may therefore find clinical
utility in the treatment of this rare cardiomyopathy, other types of cardiomyopathy and cancers in
humans.
Received 11 October 2016
Revised 6 December 2016
Accepted 8 December 2016
Available online xxxx
Keywords:
Cardiomyopathy
Emery-Dreifuss muscular dystrophy
Extracellular signal-regulated kinase
Lamin
MEK inhibitor
Mitogen-activated protein kinase
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
cellular substrates, including other kinases and transcription fac-
tors that regulate various cellular processes and gene expression.1,2
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are
structurally similar protein-serine/threonine kinases that regulate
a variety of cellular processes including adhesion, migration, sur-
vival, differentiation, metabolism, proliferation, transcription,
cytoskeletal remodeling and cell cycle progression.1 Mitogen-acti-
vated protein kinase kinases 1 and 2 (MEK1/2) catalyze the phos-
phorylation of ERK1/2, which is required for enzyme activation.2
Activated ERK1/2 in turn catalyzes the phosphorylation of various
Several non-ATP-competitive, allosteric MEK1/2 inhibitors have
been developed and assessed in clinical studies, primarily for can-
cers in which ERK1/2 signaling is aberrantly activated.3–5
In addition to cancer, MEK1/2 inhibitors have potential to be
useful in other diseases. RASopathies, a group of genetic syn-
dromes that includes neurofibromatosis 1, Noonan syndrome, LEO-
PARD syndrome, Costello syndrome and cardiofaciocutaneous
syndrome, are caused by germline mutations in genes that encode
components or regulators of the Ras-ERK1/2 signaling pathway
that may lead to abnormally increased ERK1/2 activity.6,7 Increased
ERK1/2 signaling in heart in RASopathies can cause hypertrophic
cardiomyopathy and MEK1/2 inhibitor treatment has been shown
to be beneficial in a mouse model of Noonan syndrome.8,9 Data
from transgenic mice with cardiomyocyte-specific overexpression
of FasL also suggest that MEK1/2 inhibitors may be useful in the
preventing the progression of dilated cardiomyopathy and heart
failure.10
⇑
Corresponding authors at: Cheminpharma LLC, 23 Business Park Drive, Bran-
ford, CT 06405, United States (U.R. Khire). Department of Medicine, College of
Physicians & Surgeons, Columbia University, 630 West 168th Street, New York, NY
10032, United States (H.J. Worman).
E-mail addresses: ukhire@allomek.com (U.R. Khire), hjw14@columbia.edu
(H.J. Worman).
g
Present address: Department of Radiology, University of Virginia, Charlottesville,
VA 22908, United States.
h
Present address: Center of Research in Myology, UPMC-Inserm UMR974, CNRS
FRE3617, Institut de Myologie, G.H. Pitié Salpêtrière, 75651 Paris Cedex 13, France.
http://dx.doi.org/10.1016/j.bmc.2016.12.014
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wu W., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.014

2
W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Mutations in the lamin A/C gene (LMNA) cause a variety of dis-
nucleophilic displacement with 2-fluoro, 4-iodoaniline provided
advanced intermediate 4. Reduction of Nitro group afforded the
aniline followed by coupling with sulfonyl chloride, yielded diene
5. Olefin metathesis reaction afforded the cis-olefin as a major pro-
duct 6. Dihydroxylation catalyzed by osmium tetraoxide in the
presence of N-methyl morpholine-N-oxide, of macrocyclic olefin
6 provided cis diol 7. The lead molecule, 8, was then obtained as
a single stereoisomer from chiral chromatography.
We used an in vitro biochemical assay to test the racemic diol 7
for inhibition of MEK1 activity. The compound 7 was potent with
IC50 = 21 nM. After chiral separation, we tested the individual
enantiomeric diols for anti-proliferative activity on HT-29 cells,
the proliferation of which is slowed by inhibiting MEK1/2. The fast
moving isomer 8 on chiral HPLC was approximately 25-fold more
potent as compared to the slow moving one (IC50 = 16 nM and
400 nM, respectively). We chose the more potent faster moving
isomer for further evaluation.
eases called laminopathies.11 Most often, LMNA mutations cause
dilated cardiomyopathy with variable skeletal myopathy, includ-
ing autosomal Emery-Dreifuss muscular dystrophy, limb-girdle
muscular dystrophy type 1B and congenital muscular dystro-
phy.12–17 ERK1/2 activity is increased in hearts of mice with dilated
cardiomyopathy caused by Lmna mutation as well as in hearts of
human subjects with LMNA mutations.18–20 ERK1/2 signaling is
also increased in hearts of mice lacking emerin, which occurs in
humans with X-linked Emery-Dreifuss muscular dystrophy that
has dilated cardiomyopathy as a prominent feature.21,22 MEK1/2
inhibitors have beneficial effects in mice with an Lmna mutation
that causes cardiomyopathy.20,23,24 Given the high incidence of
heart failure in patients with LMNA mutation and poor efficacy of
standard therapies,17 we synthesized
a novel macrocyclic
MEK1/2 inhibitor with improved pharmacological profile and eval-
uated its therapeutic potential in mice with dilated cardiomyopa-
thy caused by Lmna mutation.
2.2. Effects of 8 on heart and skeletal muscle of LmnaH222P/H222P mice
2. Results and discussion
We evaluated the effects of 8 on heart and skeletal muscle in
LmnaH222P/H222P mice, a validated model of cardiomyopathy caused
by LMNA mutation in humans.27 We used male mice because
female mice develop pathology at a significantly later age. Male
LmnaH222P/H222P mice develop initial signs of cardiomyopathy at 8
to 10 weeks with progressive left ventricular dilatation and
decreased left ventricular fractional shortening. By approximately
20 weeks of age, they also develop significant skeletal myopathy.
Median survival is approximately 26 weeks. ERK1/2 signaling is
abnormally increased of LmnaH222P/H222P mice, starting as early as
4 weeks of age.18,19 We have previously shown that treatment with
MEK1/2 inhibitors such as PD98059 and selumetinib has a benefi-
cial effect on left ventricular and skeletal muscle function and pro-
longs survivial.20,22–24,28
2.1. Chemistry
Several allosteric inhibitors of MEK1/2 are currently in clinical
development and two of them are FDA approved drugs.3–5 These
inhibitors are selective towards MEK1/2, as they bind to non-
ATP-competitive allosteric sites. However, the poor pharmacoki-
netic profile of these MEK1/2 inhibitors, such as short half-life
can be attributed to metabolically labile structural features. Our
rational design of a novel class of MEK1/2 inhibitors sought to
improve pharmacokinetic profile over existing molecules. We
designed a macrocyclic inhibitor that maintains critical protein/
ligand contacts through sulfonamide, iodine but does not possess
a
primary alcohol or hydroxamate functionality, a known
metabolic liability.25 The structure of lead molecule 8 (Scheme 1)
is distinct from other MEK1/2 inhibitors in that it is a macrocycle.
It has been reported that macrocyclic scaffolds improve drug-like
properties including target binding, selectivity and oral
bioavailability.26
Prior to administering 8 to LmnaH222P/H222P mice, we tested its
activity by examining phosphorylation (activation) of ERK1/2 in
cultured HEK-293 cells. Addition of 1–0.001 lM of 8 to culture
media essentially abolished ERK1/2 phosphorylation (Fig. 1A). We
next administered 3 mg/kg/day or 6 mg/kg/day of 8 to male
LmnaH222P/H222P mice by oral gavage starting at 14 weeks of age,
when there are symptoms of cardiomyopathy, up to 20 weeks of
age. After six weeks of systemic administration, both doses led to
The synthesis of a lead molecule 8 is depicted in Scheme 1.
Nitration of commercially available 3,4,5-trifluorophenol provided
nitrophenol 2. Allylation of the phenol 2, followed by selective
Scheme 1. Synthesis of macrocyclic MEK1/2 inhibitor 8.
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W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
We examined the effects of six weeks of treatment with 8 on
echocardiographic parameters that correlate with left ventricular
function. Treatment was started at 14 weeks of age, when signifi-
cant left ventricular dysfunction is already present, and mice were
analyzed six weeks later at 20 weeks of age. Treatment with both
3 mg/kg/day and 6 mg/kg/day of 8 resulted in significant increases
in left ventricular fractional shortening (the percentage the left
ventricular diameter decreases with each contraction) and the
higher dose also provided a significant decrease in left ventricular
end diastolic diameter (Fig. 2; Table 1). These echocardiographic
changes were consistent with improved left ventricular systolic
function.
We previously reported pathological up-regulation of the gene
encoding natriuretic peptide A (Nppa), the synthesis of which is
increased in dilated hearts, and genes encoding proteins involved
in sarcomere organization in the hearts of LmnaH222P/H222P
mice.18,23,24 We therefore assayed expression of mRNAs encoded
by Nppa and by the gene encoding cardiac myosin light chain 7
(Myl7) in mice treated with 8. Treatment with both 3 mg/kg/day
and 6 mg/kg/day significantly reduced cardiac Nppa mRNA com-
pared to placebo treatment (Fig. 3A). Myl7 mRNA was significantly
decreased after treatment with 6 mg/kg/day (Fig. 3B).
Cardiomyopathy in LmnaH222P/H222P mice is characterized by
profound myocardial fibrosis, which is prominent in male mice
by 20 weeks of age.20,23,24,27 This recapitulates the significant fibro-
sis that occurs in hearts of human subjects with cardiomyopathy
caused by LMNA mutations.29–32 In humans with LMNA mutations,
fibrosis in the interventricular septum may be the mechanism
behind the early atrioventricular block and ventricular
arrhythmias that frequently occur and can lead to sudden death.33
We therefore assessed the effects of 8 treatment on cardiac
fibrosis in LmnaH222P/H222P mice. Staining with Masson trichrome
of fixed sections of left ventricles showed that treatment of
LmnaH222P/H222P mice with both 3 mg/kg/day and 6 mg/kg/day of
8 lead to decreased fibrosis (Fig. 4A). A grading system was con-
structed based on the presence of two abnormalities that were
found to be variably present between specimens: cytoplasmic
vacuolation and cardiac fibrosis. Low-grade specimens had no
fibrosis and either no vacuolation (grade 0), mild vacuolation
(grade 1) or moderate vacuolation (grade 2). Higher-grade
specimens possessed vacuolation and mild fibrosis (grade 3) or
severe fibrosis (grade 4). This analysis showed that treatment
Fig. 1. 8 reduces ERK1/2 activity in cultured cells and mouse tissues. Representative
immunoblots using antibodies against phosphorylated (activated) ERK1/2 (pERK1/
2) and total ERK1/2 (ERK1/2). (A) Immunoblot of proteins extracted from HEK-293
cells cultured in the presence of diluent (Control) or varying concentrations of 8 for
24 h. Concentrations of 8 used are indicated above each lane. (B) Immunoblots of
proteins extracted from hearts, quadriceps and livers of LmnaH222P/H222P mice
treated with diluent (placebo) or 8 at doses of 3 mg/kg/day or 6 mg/kg/day. The bar
graphs at right show quantification of the pERK1/2 to total ERK1/2 ratio determined
from scanned immunoblots (n = 3); values shown are means standard errors.
*P < 0.05, **P < 0.01 compared to placebo.
significant decreases in phosphorylated ERK1/2 relative to total
ERK1/2 in heart and liver, whereas only the higher dose produced
a significant decrease in quadriceps muscle (Fig. 1B). There were no
differences in body mass between the three groups at the end of
treatment
[placebo = 23.19 0.43
(n = 22);
3 mg/kg/day
8 = 24.06 0.52 (n = 19); 6 mg/kg/day 8 = 22.93 0.71 (n = 16);
values are means standard errors].
Fig. 2. Effect of 8 on echocardiographic parameters in LmnaH222P/H222P mice. (A) Representative transthoracic M-mode echocardiographic tracings from LmnaH222P/H222P mice
treated with placebo or 8 at 3 mg/kg/day or 6 mg/kg/day. Left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD) are indicated. (B) Bar
graphs show means and standard errors of means for left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD) and left ventricular
fractional shortening (FS) in LmnaH222P/H222P mice treated with placebo (n = 22), 8 3 mg/kg/day (n = 19) or 8 6 mg/kg/day (n = 16). *P < 0.05 compared to placebo.
Please cite this article in press as: Wu W., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.014

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W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Table 1
Echocardiographic data for LmnaH222P/H222P at age of 20 weeks of age treated with placebo or 8.
Treatment
n
Heart rate (beats/min)
LVEDD (mm)
LVESD (mm)
FS (%)
Placebo
8 (3 mg/kg/day)
8 (6 mg/kg/day)
22
19
16
502.73 0.46
503.63 0.54
501.56 0.61
3.96 0.08
4.00 0.06
3.77 0.11
3.32 0.10
3.24 0.09
3.02 0.16*
16.36 1.08
19.39 1.25*
20.65 1.72*
LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional shortening. Values are means standard errors.
*
P < 0.05 compared to placebo.
3 mg/kg/day and 6 mg/kg/day of 8 lead to dose-dependent statisti-
cally significant decreases in fibrosis (Fig. 4B). Hence, treatment
with 8 may prevent irreversible cardiac fibrosis in patients with
LMNA mutations and possible the atrioventricular block and ven-
tricular arrhythmias that can result from it.
Human subjects with LMNA mutations that cause cardiomyopa-
thy frequently have associated muscular dystrophy.12,14–17 Abnor-
mally increased ERK1/2 activity appears to contribute to the
development of skeletal muscle pathology in LmnaH222/H222P mice
and MEK1/2 inhibition may have beneficial effects.28 To further
assess this possibility, we examined the effects of 8 on a serum cre-
atine phosphokinase, a biomarker of striated muscle breakdown,
and skeletal muscle fatigue. Compared to placebo, treatment with
6 mg/kg/day but not 3 mg/kg/day of 8 significantly reduced serum
creatine phosphokinase activity in LmnaH222P/H222P mice (Fig. 5A).
Treatment with 6 mg/kg/day of 8 also significantly increased the
forelimb grip fatigue index, consistent with decreased fatigability
(Fig. 5B). While the beneficial effects on skeletal muscle may have
occurred secondary to improvement in heart function, the fact that
only 6 mg/kg/day provided reduced serum creatine phosphokinase
activity whereas both 3 mg/kg/day and 6 mg/kg/day had improved
heart function suggests a direct effect. Histological examination of
hematoxylin and eosin-stained sections of quadriceps muscle by a
pathologist blind to treatment showed internally nucleated fibers
in 3 of 6 samples from mice treated with placebo, in 1 of 5 samples
from mice treated with 3 mg/kg/day or 8 and 0 of 5 samples from
mice treated with 6 mg/kg/day of 8. Internally nucleated fibers are
indicative of chronic myofiber injury and regeneration, as can be
seen in a variety of muscular dystrophies and other disease states.
2.3. Toxicology analyses of 8 in LmnaH222P/H222P mice
We carried out a preliminary analysis of potential tissue toxicity
of 8 in male LmnaH222P/H222P mice treated from 14 to 20 weeks of
age. We measured serum alkaline phosphatase activity, alanine
aminotransferase activity and bilirubin concentration to assess
possible hepatic injury and liver function. We also measured serum
creatinine and blood urea nitrogen concentrations as indicators of
renal function and serum amylase activity as a marker of pancre-
atic injury. There were no statistically significant differences in
any of these parameters between male LmnaH222P/H222P mice trea-
ted with 3 mg/kg/day or 6 mg/kg/day or placebo (Table 2). These
results suggested that 6 weeks of treatment with 8 at a dose up
to 6 mg/kg/day did not produce significant liver, kidney or pancre-
atic toxicity.
Fig. 3. Effect of 8 on expression of natriuretic peptide A and myosin light chain 7
mRNAs in hearts of LmnaH222P/H222P mice. (A) Quantitative real-time
RT–PCR analysis of mRNA encoding natriuretic peptide A (Nppa) in hearts from
LmnaH222P/H222P mice treated with placebo or 8 at 3 mg/kg/day or 6 mg/kg/day. (B)
Quantitative real-time RT–PCR analysis of mRNA encoding myosin light chain 7
(Myl7) in hearts from LmnaH222P/H222P mice treated with placebo or 8 at 3 mg/kg/day
or 6 mg/kg/day. Results for placebo treatment (n = 5) are normalized to 1 and those
for treatment with 3 mg/kg/day (n = 5) or 6 mg/kg/day (n = 6) of 8 represent as fold
change compared to placebo treatment. Bar graphs show means and standard
errors. *P < 0.05 compared to placebo.
A pathologist who was blind to drug treatment and mouse
genotype examined histological section of livers, kidneys and
spleens from LmnaH222P/H222P mice after 6 weeks of receiving pla-
cebo or 8 (Table 3). Examination of the livers did not show hepato-
cyte necrosis, significant inflammation or cholestasis; only mild
non-specific changes were observed in livers from mice treated
with placebo, 3 mg/kg/day of 8 or 6 mg/kg/day of 8. The kidneys
showed no evidence of glomeruli abnormalities or interstitial
nephritis and were mostly normal except for some non-specific
inflammation in mice treated with either placebo or 8. Spleens
from some mice treated with either placebo or 8 contained hemo-
siderin-laden macrophages. These are normally present in rodent
spleens34 but may also suggest old hemorrhage somewhere in
the animals. One mouse receiving 8 had evidence of a splenic
infarct, which is not typical for a drug reaction but can be a throm-
boembolic event in cardiomyopathy.35 Overall, there were no con-
sistent or specific abnormalities in liver, kidney or spleen
histopathology in mice receiving 8 and no alterations that typically
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W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
Fig. 4. Effect of 8 on cardiac fibrosis in LmnaH222P/H222P mice. (A) Photomicrographs of representative left ventricle sections stained with Masson trichrome from
LmnaH222P/H222P mice treated with placebo or 8 at 3 mg/kg/day or 6 mg/kg/day. Scale bar: 70
m. The placebo image corresponds to grade 4 histology (severe fibrosis), the
l
3 mg/kg image corresponds to grade 2 histology (moderate vacuolation, no fibrosis) and the 6 mg/kg image corresponds to grade 1 histology (mild vacuolation, no fibrosis).
(B) Cardiac fibrosis scores for LmnaH222P/H222P mice treated with placebo (n = 5) or 3 mg/kg/day (n = 5) or 6 mg/kg/day (n = 6) of 8. Bar graph shows means and standard errors.
*P < 0.05, **P < 0.01 compared to placebo.
occur with drug toxicity. The fact that minor abnormalities were
present in both placebo-treated and drug-treated LmnaH222P/H222P
mice suggested that they were a result of underlying disease and
not drug toxicity.
tional models of cardiomyopathy caused by LMNA mutations.36–38
Mouse models also do not exactly recapitulate the corresponding
cardiomyopathy caused by LMNA mutations, as the human disease
is generally inherited in an autosomal dominant fashion whereas
heterozygous mice are mostly asymptomatic or develop relatively
less severe disease only at advanced ages.27,36–41 In addition, the
current experiments were all performed in male LmnaH222P/H222P
mice and studies in female mice, which develop pathology at older
age,27 should be considered. Finally, future molecular docking
studies and crystallization of the novel MEK1/2 inhibitor we
describe will improve our understanding of its structure and mech-
anism of action.
Regardless of these limitations, the preclinical efficacy and lack
of observed significant adverse events in male LmnaH222P/H222P
mice indicates that clinical development of novel MEK1/2 inhibitor
described in this paper is warranted. In addition to cardiomyopa-
thy caused by LMNA mutations, this inhibitor may have utility in
the treatment of cardiomyopathy in X-linked Emery-Dreifuss mus-
cular dystrophy22 and in the treatment of RASopathies, including
the hypertrophic cardiomyopathy in Noonan syndrome.8,9 A potent
MEK1/2 may also find utility in the treatment of various cancers.3–5
2.4. Effects of 8 on survival of LmnaH222P/H222P mice
To assess the effects of 8 on survival, LmnaH222P/H222P mice
started treatment with either placebo, 8 3 mg/kg/day or 8 6 mg/
kg/day and were followed until death or euthanasia was recom-
mended by a veterinarian blind to treatment group (Fig. 6). Mice
treated with placebo had a median survival of 202 days whereas
median survival was 227 days for mice treated with 8 3 mg/kg/day
and 225 days for mice treated with 8 6 mg/kg/day. The median sur-
vivals of mice treated with both doses of 8 were statistically signif-
icantly (P < 0.05) longer than that for mice treated with placebo.
3. Conclusions
We have synthesized a novel, allosteric, macrocyclic MEK1/2
inhibitor that potently inhibits ERK1/2 activity in cultured cells
and in tissues of mice after systemic administration. Other
MEK1/2 inhibitors are currently approved for or in late clinical
development for various cancers.3–5 Our preclinical results suggest
that this novel MEK1/2 inhibitor will be effective for treating car-
diomyopathy caused by LMNA mutations.
4. Experimental
4.1. General chemistry information
There are several limitations to the present study that could be
addressed in future research. We used only one mouse model,
LmnaH222P/H222P mice, and the results could be generalized to addi-
NMR spectra were recorded on Agilent DD2 400 (400 MHz)
instrument. Column chromatography was carried out using Combi-
Flash over RediSep column cartridges employing Merck silica gel
Please cite this article in press as: Wu W., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.014

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W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
(Kieselgel 60, 63–200
l
m). Pre-coated silica gel plates F-254 were
4.1.4. 1,2-Benzenediamine, 3,4-difluoro-N2-(2-fluoro-4-iodophenyl)-
6-(2-propen-1-yloxy)-
used for thin-layer analytical chromatography. Mass determina-
tions were performed using electrospray ionization on Sciex API
365 LC/MS/MS system. Purity (>95%) was determined by using
reverse phase using Gilson analytical HPLC, using Phenomenex
Compound 4 (12 g, 26.66 mmol) was dissolved in ethanol
(330 mL) by heating at 90 °C. The solution was refluxed for
30 min and then sodium dithionite (37.13 g, 213 mmol) in water
(110 mL) was added and heating was continued for additional
1 h. The mixture was cooled to room temperature, concentrated
to remove ethanol, extracted with mix of 20% heptanes in ethyl
acetate, dried (Na2SO4), filtered and concentrated. The resulting
solid was used in the next step without any further purification.
Yield: quantitative. Thin-layer chromatography (10% ethyl acet-
ate:hexanes) Rf = 0.18 MS: (M+H)+ 421.4.
Lune 5
l
C18(2) 250 ꢀ 4.6 mm column. Samples were run at
1 mL/min using gradient mixtures of 5–100% water with 0.1% tri-
fluoroacetic acid (A) and acetonitrile with 0.1% trifluoroacetic acid
(B) for 10 min.
4.1.1. Phenol, 3,4,5-trifluoro-2-nitro- (2)
Nitric acid (16 mL, 247 mmol) was added dropwise to an ice-
cold stirred solution of 3,4,5-trifluorophenol (20 g, 135 mmol) in
glacial acetic acid (40 mL) in a 2 L flask. The reaction mixture
was slowly brought to room temperature and stirred for 1 h.
Thin-layer chromatography was used to monitor the progress of
reaction. After completion, the reaction mixture was poured into
ice water and the aqueous layer was extracted with 10% ethyl acet-
ate in heptanes. The organic layer was washed with saturated
NaHCO3 solution, water (X3), brine, dried over anhydrous Na2SO4
and concentrated to yield benzene, 1,2,3-trifluoro-4-nitro-5-(2-
propen-1-yloxy)- 1, as yellow oil. Yield: 25.31 g (131 mmol, 97%).
Thin-layer chromatography (30% ethyl acetate:hexanes) Rf = 0.22
1H NMR (CDCl3): d 6.84 (m, 1H), 10.28 (br s, 1H).
4.1.5. (1-Allyl-N-(6-(allyloxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)
amino)phenyl)cyclopropane-1-sulfonamide) (5)
1,2-Benzenediamine, 3,4-difluoro-N2-(2-fluoro-4-iodophenyl)-
6-(2-propen-1-yloxy)- from the previous step (30.28 g, 72 mmol)
was taken in a seal tube and 4-dimethylaminopyridine (500 mg),
pyridine (9.1 mL), methylene chloride (15 mL) and 1-allylcyclo-
propane-1-sulfonyl chloride (13 g, 72 mmol) were added sequen-
tially. The reaction mixture was then heated at 50 °C in a sealed
tube for 3 days. After cooling to room temperature, the solvent
was evaporated and the residue was dissolved in ethyl acetate
and filtered through a plug of silica gel to remove baseline
impurities. The organic layer was evaporated and diethyl ether
and hexanes added to the residue. The gray solid was filtered
and dried to provide a clean product (1-allyl-N-(6-(allyloxy)-3,4-
difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)cyclopropane-1-
sulfonamide). Yield: 37 g (92%). Thin-layer chromatography (10%
ethyl acetate:hexanes) Rf = 0.35 1H NMR (CDCl3): d 0.78 (m, 2H),
1,24 (m, 2H), 2.71 (d, J = 7.2 Hz, 2H), 4.59 (d, J = 5.6 Hz 2H), 5.06
(d, J = 18 Hz 1H), 5.11 (d, J = 10 Hz 1H), 5.41 (d, J = 10 Hz 1H),
5.47 (d, J = 14 Hz, 1H), 5.66–5.68 (m, 1H), 6.05–6.08 (m, 2H),
6.51–6.56 (m, 2H), 7.09 (d, J = 8.8 Hz, 1H), 7.21–7. 28 (m, 1H),
7.33 (s, 1H).
4.1.2. Benzene, 1,2,3-trifluoro-4-nitro-5-(2-propen-1-yloxy)- (3)
Potassium carbonate (51 g, 370 mmol) and allyl bromide
(25.6 mL, 296 mmol) were added to a solution of phenol, 3,4,5-tri-
fluoro-2-nitro- 1 (47.6 g, 247 mmol) in acetone (350 mL) at room
temperature. The reaction mixture was heated at 70 °C for 3 h,
and cooled to room temperature and continued to stir overnight.
Thin-layer chromatographic analysis indicated completion of reac-
tion. Solvent was evaporated and the residue was dissolved on
water and extracted with ethyl acetate. Organic layer was washed
with brine and dried (Na2SO4). Organic solvents were evaporated
and the residue was purified on silica gel column using 10–25%
ethyl acetate/ heptanes as eluent. Yield: 49.2 g (211 mmol, 85%).
Thin-layer chromatograph (10% ethyl acetate:hexanes) Rf = 0.35
1H NMR (CDCl3): d 4.65 (dt, 2H), 5.39 (d, 1H), 5.45 (d, 1H), 5.98
(m, 1H), 6.72 (m, 1H).
4.1.6. (Z)-10,11-Difluoro-12-((2-fluoro-4-iodophenyl)amino)-4,7-
dihydro-1H-spiro[benzo[b][1,5,4]oxathiazecine-3,10-cyclopropane]
2,2-dioxide (6)
The catalyst (3-phenyl-1H-inden-1-ylidene[bis(i-butylphoban)]
ruthenium(II) dichloride (671 mg, 5 mol%) was added to a solution
of (1-allyl-N-(6-(allyloxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)
amino)phenyl)cyclopropane-1-sulfonamide) 5 (10 g) in dichloro-
methane (3 L) and the mixture was degassed for 10 min by bub-
bling nitrogen gas. The mixture was then stirred at 65 °C for
15 h. The next day, the solvent was evaporated and the crude pro-
duct purified by silica gel column (5%/50% ethyl acetate/heptanes).
The desired cis isomer, (Z)-10,11-difluoro-12-((2-fluoro-4-iodo-
phenyl)amino)-4,7-dihydro-1H-spiro[benzo[b][1,5,4]oxathiazecine-
3,10-cyclopropane] 2,2-dioxide was isolated (7 g, 74%) and a minor
4.1.3. Benzenamine, 2,3-difluoro-N-(2-fluoro-4-iodophenyl)-6-nitro-
5-(2-propen-1-yloxy)- (4)
A solution of 2-fluoro-4-iodoaniline (31.04 G, 131 mmol) in
anhydrous tetrahydrofuran (600 mL) was cooled to ꢁ78 °C and
stirred for 1 h. Lithium bis(trimethylsilyl)amide (1 M in tetrahy-
drofuran solution) was added to the reaction mixture through
addition funnel dropwise (over 30 min). To this mixture was then
added solution of Benzene, 1,2,3-trifluoro-4-nitro-5-(2-propen-1-
yloxy)- 3 (30.7 g, 132 mmol) in tetrahydrofuran (50 mL) was added
dropwise, and the resulting purple mixture was stirred at ꢁ78 °C
for 4 h and then warmed to room temperature overnight. The mix-
ture was quenched with water. Tetrahydrofuran was removed
under vacuum and the resulting slurry was diluted with ethyl acet-
ate and water, organic separated and washed with brine. After con-
trans
product
(E)-10,11-difluoro-12-((2-fluoro-4-iodophenyl)
amino)-4,7-dihydro-1H-spiro[benzo[b][1,5,4]oxathiazecine-3,10-
cyclopropane] 2,2-dioxide (1.92 g, 20%) was also isolated. Rf for the
desired product was 0.45 (thin-layer chromatography in 40% ethyl
acetate/heptanes) and for the trans product was 0.2 under same
conditions. MS: (M+H)+ 537, 1H NMR (CDCl3): d 0.74 (br s, 2H),
1,14 (br s, 2H), 3.14 (m, 2H), 4.92 (s, 2H), 5.46 (dd, J = 12.0 Hz, 1
H), 5.72 (dd, J = 8.0, 12.0 Hz), 6.27 (s, 1H), 6.51 (m, 2H), 7.18 (s,
1H), 7.29 (d, J = 8.0 Hz, 1H). 7.41 (d, J = 12 Hz, 1H).
centration,
a solid residue was obtained. This residue was
triturated with heptanes and a small amount of ether. The solid
was filtered and washed with heptanes and dried. Yield: 53 g
(89%). Thin-layer chromatography (10% ethyl acetate:hexanes)
Rf = 0.23 1H NMR (CDCl3): d 4.62 (dt, 2H), 5.33–5.36 (d, 1H), 5.48
(d, 1H), 5.98–6.02 (m, 1H), 6.22 (dd, 1H), 6.36 (dd, 1H), 7.04–7.08
(m, 1H), 7.45–7.52 (m, 2H), 7.79 (s, 1 H).
4.1.7. 10,11-Difluoro-12-((2-fluoro-4-iodophenyl)amino)-5,6-dihy-
droxy-4,5,6,7-tetrahydro-1H-spiro[benzo[b][1,5,4]oxathiazecine-3,10-
cyclopropane] 2,2-dioxide (7)
N-Methyl morpholine N-oxide (16.6 g, 3.3 equivalents) at room
temperature followed by an aqueous solution of osmium tetraox-
ide (6 mL of 5 mol% solution) were added to a 20-g solution of 2
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W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
Table 3
Histological findings in livers, kidney and spleens from LmnaH222P/H222P mice at
20 weeks of age treated for 6 weeks with placebo or 8.
Treatment
Sample
#
Histology
Livers
Placebo
Placebo
Placebo
Placebo
7603
7604
7605
7606
Normal
Normal
Normal
Greater vacuolation than in other specimens;
uncertain significance; could be preparation
artifact
Placebo
Placebo
8 3 mg/kg/day
8 3 mg/kg/day
8 3 mg/kg/day
8 3 mg/kg/day
7607
7608
7609
7610
7611
7612
Normal
Normal
Normal
Normal
Normal
Slightly greater vacuolation in some zones;
similar to 7606 but milder; could be preparation
artifact
8 3 mg/kg/day
8 6 mg/kg/day
7613
7614
Normal
Occasional hemosiderin laden macrophages.
Some portal triads have mild lymphocytic
inflammation; significance uncertain
Normal
8 6 mg/kg/day
8 6 mg/kg/day
8 6 mg/kg/day
7615
7616
7617
Normal
Some intracellular vacuolation in some zones;
similar to 7612; could be preparation artifact
Normal
8 6 mg/kg/day
7618
Kidneys
Placebo
Placebo
Placebo
Placebo
7619
7620
7621
7622
Normal
Normal
Normal
Some lymphocytic inflammation around the
renal pelvis (around the ureter); otherwise
normal
Placebo
7623
Some lymphocytic inflammation around the
renal pelvis (around the ureter); otherwise
normal
Placebo
7624
7625
7626
7627
7628
7629
7630
7631
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Fig. 5. Effect of 8 on skeletal muscle in LmnaH222P/H222P mice treated with placebo, 8
3 mg/kg/day or 6 mg/kg/day. (A) Serum creatine phosphokinase (CPK) activities in
LmnaH222P/H222P mice treated with placebo (n = 10), 8 3 mg/kg/day (n = 10) or 8
6 mg/kg/day (n = 10). Values are means standard errors. (B) Forelimb grip fatigue
index of LmnaH222P/H222P mice treated with placebo (n = 7) or 8 6 mg/kg/day (n = 6).
Values are means standard errors. *P < 0.05, **P < 0.01 compared to mice treated
with placebo.
8 3 mg/kg/day
8 3 mg/kg/day
8 3 mg/kg/day
8 3 mg/kg/day
8 3 mg/kg/day
8 6 mg/kg/day
8 6 mg/kg/day
Some lymphocytic inflammation around the
renal pelvis (around the ureter) and occasional
medium-sized arteries; more inflammation
than 7623 and 7622
Mostly normal but some mild lymphocytic
inflammation around small arteries with
occasional hemosiderin-laden macrophages
Normal
Table 2
8 6 mg/kg/day
7632
Selected serum chemistry values in LmnaH222P/H222P mice at 20 weeks of age treated
for 6 weeks with placebo or 8.
8 6 mg/kg/day
8 6 mg/kg/day
7633
7634
Treatment
Placebo
8 (3 mg/kg/day)
8 (6 mg/kg/day)
Normal
Alkaline Phosphatase
(U/L)
ALT (U/L)
Total bilirubin (mg/L)
Amylase (U/L)
BUN (mg/dL)
Creatinine (mg/dL)
BUN/Creatinine
91.5 6.99
104.5 7.43
113.0 6.55
Treatment
Sample
#
Histology results
56 10.55
69.6 10.81
0.32 0.02
1372 282.9
18.04 0.48
0.46 0.01
19.66 0.61
69.6 14.31
0.37 0.03
1086 40.18
19.12 1.83
0.46 0.01
20.77 1.92
0.36 0.02
1102 53.63
17.98 0.94
0.45 0.01
20.14 1.25
Spleens
Placebo
Placebo
Placebo
Placebo
Placebo
Placebo
7651
7652
7653
7654
7655
7656
Normal
Normal
Normal
Normal
Normal
ALT, alanine aminotransferase; BUN, blood urea nitrogen. Values are
means standard errors for n = 10 mice per group.
Moderately increased number of hemosiderin-
laden macrophages suggestive of old
hemorrhage somewhere
8 3 mg/kg/day
8 3 mg/kg/day
7657
7658
Mildly increased number of hemosiderin-laden
macrophages suggestive of old hemorrhage
somewhere
Normal (hemosiderin-laden macrophages
present but at approximately normal levels)
Normal
((Z)-10,11-difluoro-12-((2-fluoro-4-iodophenyl)amino)-4,7-dihydro-
1H-spiro[benzo[b][1,5,4]oxathiazecine-3,10-cyclopropane] 2,2-diox-
ide) in tetrahydrofuran (500 mL) and water (5 mL). The mixture
was stirred at room temperature for 16 h and then quenched by
adding 10 mL of 1 M aqueous sodium sulfite solution and stirred
for 1 h. The mixture was concentrated under vacuum and the resi-
due was diluted with ethyl acetate and washed with 5 M sodium
8 3 mg/kg/day
8 3 mg/kg/day
8 3 mg/kg/day
7659
7660
7661
Normal
Normal
(continued on next page)
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8
W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
noethyl ether)-N,N,N0,N0-tetraacetic acid, 0.4 mM ethylenedi-
Table 3 (continued)
aminetetraacetic acid, 5 mM MgCl2 and 0.05 mM dithiothreitol.
Reaction mixture was incubated at room temperature for 90 min.
Treatment
Sample
#
Histology results
At the end of the reaction, 20
was added and incubated at room temperature for 40 min. Forty
L of Kinase Detection Reagent (Promega) was then added and
incubated at room temperature for 1 h. Chemiluminescence was
measured in a luminometer and IC50 calculated using SoftMax
software.
lL of ADP-Glo reagent (Promega)
8 6 mg/kg/day
7662
Area of necrosis consistent with splenic infarct;
moderately increased number of hemosiderin-
laden macrophages suggestive of hemorrhage
old somewhere
Normal
Normal
l
8 6 mg/kg/day
8 6 mg/kg/day
8 6 mg/kg/day
8 6 mg/kg/day
7663
7664
7665
7666
Normal
Moderately increased number of hemosiderin-
laden macrophages suggestive of old
hemorrhage somewhere
4.3. Cell culture experiments
4.3.1. Anti-proliferative activity
HT-29 (American Type Culture Collection) were cultured at
37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supple-
mented with l-glutamine and 10% fetal bovine serum (Invitrogen).
Proliferation was assessed by plating 2000 cells/well in 100 lL of
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum or Roswell Park Memorial Institute medium supple-
mented with 10% fetal bovine serum in a 96-well plate and incu-
bated overnight at 37 °C with 5% CO2. After overnight incubation,
the culture media were replaced with 100 lL of the same media
containing various concentrations of the compounds. Compounds
were added at 3-fold dilutions with concentrations ranging from
3.3
l
M to 4.5 nM. After 72 h of incubation at 37 °C with 5% CO2, cell
M/well
viability was measured in a luminometer after adding 100
l
CellTiterGlo reagent (Promega). IC50s were calculated using Soft-
Max software.
Fig. 6. Effect of 8 on survival of LmnaH222P/H222P mice. Kaplan-Meier plots for
LmnaH222P/H222P mice treated with placebo (n = 23), 8 3 mg/kg/day (n = 17) or 8
6 mg/kg/day (n = 15) are shown.
4.3.2. Effects on phosphorylated ERK1/2
HEK-293 cells (American Type Culture Collection) were cul-
tured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum. 8 was dissolved in 10%
v/v Chremophor EL (BASF)/normal saline at a concentration of
sulfite solution and brine. The organic layer was concentrated
and the residue was purified on a small (plug) silica gel using
50%/90% ethyl acetate/hexanes to provide a quantitative yield of
10,11-difluoro-12-((2-fluoro-4-iodophenyl)amino)-5,6-dihydroxy-
4,5,6,7-tetrahydro-1H-spiro[benzo[b][1,5,4]oxathiazecine-3,10-cyclo-
propane] 2,2-dioxide. Thin-layer chromatography (neat ethyl
acetate) Rf = 0.4; MS: (M+H)+ 570.9, 1H NMR (CDCl3): d 0.68–0.85
(br m, 3H), 1.17 (br m, 1H), 1,4 (s, 1H), 2.25 (d, 1H), 3.25–3.65
(m, 3H), 3.75 (m, 1H), 3.85 (d, 1H), 4.20 (br s, 1H), 4.42 (br t, 1H),
6.25 (m, 1H), 6.50–6.55 (m, 1H), 7.28 (s, 1H), 7.30–7.40 (d,
J = 8.0 Hz, 1H), 7.60 (m, 1H).
100 lM. Cells were seeded in 6-well plate at a density of
5.0 ꢀ 105. After 24 h of incubation, the cells were rinsed twice with
culture medium. Cells were then incubated for 24 h in culture
medium with 100
trol or culture medium with serial dilutions of 8 at finial concentra-
tions of 1–0.001 M. Cells were then washed three times with
ll 10% v/v Chremophor EL/normal saline as con-
l
phosphate-buffered saline and collected for protein extraction
and immunoblotting as described below.
4.4. Mouse experiments
4.4.1. Mice
4.1.8. 10,11-Difluoro-12-((2-fluoro-4-iodophenyl)amino)-5,6-
dihydroxy-4,5,6,7-tetrahydro-1H-spiro[benzo[b][1,5,4]oxathiazecine-
3,10-cyclopropane] 2,2-dioxide (8)
The active enantiomer was obtained by chiral separation of the
racemic diol (26.64 g) using Thar 350 preparative supercritical
fluid chromatography (column: ChiralPak AD-10u, 300 ꢀ 50 mm
I.D., mobile phase: A for CO2 and B for Methanol(0.05%DEA), gradi-
ent B(45%), flow rate: 200 mL/min. The faster moving enantiomer,
8 (RT 7.00) was isolated (12.27 g), the slower moving isomer was
also isolated (RT 10.92, 12.60 g).
The Institutional Animal Care and Use Committee at Columbia
University Medical Center approved the use of animals and the
study protocol. LmnaH222P/H222P mice were bred and genotyped as
described previously.23,25 Mice were fed a chow diet and housed
in
a barrier facility with 12 h/12 h light/dark cycles. Male
LmnaH222P/H222P mice were used in all experiments because they
develop signs and symptoms of cardiomyopathy at an earlier age
and have a shorter lifespan than female LmnaH222P/H222P mice.25
4.4.2. Treatment of mice with 8
8 was dissolved in 10% v/v Chremophor EL/normal saline at con-
centrations of 0.75 mg/ml or 1.5 mg/ml and 0.1 ml was adminis-
tered 6 days a week by oral gavage for doses of 3 mg/kg/day or
6 mg/kg/day. Placebo was the same volume of 10% v/v Chremophor
EL/normal saline given by oral gavage. For biochemical analysis of
tissue, echocardiographic analysis, analysis of fibrosis, analysis of
skeletal muscle function and toxicology analyses, treatment was
started at 14 weeks of age and continued until 20 weeks of age.
For survival analyses, treatment was started at 10 weeks of age
and continued either until death or a veterinarian blind to treat-
4.2. In vitro biochemical assay of MEK1 activity
We used an in vitro biochemical assay to test 7 for inhibition of
MEK1 activity. The reaction mixture contained 80 ng of a recombi-
nant fusion protein of glutathione-S-transferase and full-length
human MEK1 (Cell Signaling), 4
lg of ERK1/ERK2 peptide (Enzo
Life Sciences), 100 M or 1 mM ATP, 1
l
lM to 1.37 nM of 7 diluted
in dimethyl sulfoxide and buffer containing 5 mM MOPS (pH 7.2),
2.5 mM b-glycerophosphate, 1 mM methylene glycol-bis(b-ami-
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W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
9
ment group determined that euthanasia was necessary. Euthanasia
was performed in a CO2 chamber followed by cervical dislocation,
according to the protocol of the Institute of Comparative Medicine
at Columbia University Medical Center. Euthanasia was confirmed
by checking for lack of response to limb and tail pinch. Immedi-
ately after euthanasia, mouse organs were quickly excised and
divided with one portion snap frozen in liquid nitrogen and the
other portion placed in 4% formaldehyde solution.
on the presence of two abnormalities that were found to be vari-
ably present between specimens: cytoplasmic vacuolation and car-
diac fibrosis. Specimens were scored as no fibrosis and no
vacuolation (grade 0), no fibrosis and mild vacuolation (grade 1),
no fibrosis and moderate vacuolation (grade 2), vacuolation and
mild fibrosis (grade 3) or vacuolation and severe fibrosis (grade
4). The same blinded pathologist provided descriptive assessments
of sections of other tissues stained with hematoxylin and eosin.
4.4.3. Protein extraction and immunoblotting
4.4.7. Serum biochemical analysis
Methods for protein extraction and immunoblotting have been
described previously.18,23 Briefly, proteins were extracted from cul-
tured cells and mouse tissues by homogenization in extraction buf-
fer as described previously. Extracted proteins were separated by
SDS-polyacrylamide slab gel electrophoresis, transferred to nitro-
cellulose membranes and blotted with primary antibodies against
total ERK1/2 (No. Sc-94, Santa-Cruz) and phosphorylated ERK1/2
(No. 9101, Cell Signaling). Secondary antibodies were horseradish
peroxidase-conjugated (GE Healthcare). SuperSignal West Pico
Chemiluminescent Substrate (Thermo Fisher Scientific) was used
to visualize recognized proteins. For quantification of phosphory-
lated ERK1/2 compared to total ERK1/2, immunoblots were
scanned and analyzed using ImageJ64 software.42
Serum was separated from blood drawn from mice and stored
at -80 °C until analyzed. Routine clinical chemistry analysis was
performed on an AutoAnalyzer at the Comparative Pathology Lab-
oratory at Columbia University Medical Center.
4.4.8. Skeletal muscle fatigability analysis
Forelimb grip strength was assessed using a published proto-
col.44 An investigator blind to treatment performed the experi-
ments. Each mouse was placed in front of a Chatillon DFIS-2
digital force gauge pull meter with angled mesh assembly and
allowed to grasp the pull bar with forelimbs and then pulled back-
ward by the tail until the grip was broken. Force applied to the bar
the moment the grasp was released was automatically recorded.
Each mouse pulled the grid three times in a row and then returned
in the cage for a resting period of at least one minute. Each mouse
performed a total of five series of pulls, each followed by a short
resting period. In this way, each mouse had pulled a total of 15
times (3 pulls ꢀ 5 times = 15 pulls). The grip strength was normal-
ized to body mass. The fatigue index was determined by calculat-
ing the decrement between the average of the first two and the last
two series of pulls 1 + 2 + 3 = A, 4 + 5 + 6 = B, 10 + 11 + 12 = C and
13 + 14 + 15 = D. The formula (C + D)/(A + B) gives a value of 1 for
mice that are not fatigued. The mice were tested for two consecu-
tive days.
4.4.4. Echocardiography
At 20 weeks of age, mice were anaesthetized with 1.5% isoflu-
rane by inhalation and placed on a heating pad (37 °C). Echocardio-
graphy was performed using a Visualsonics Vevo 770 ultrasound
with a 30 MHz transducer applied to the chest wall. Cardiac ven-
tricular dimensions were measured in two-dimensional mode
and M-mode three times for the number of animals indicated. Left
ventricular fractional shortening (FS) was calculated from the left
ventricular end diastolic diameter (LVEDD) and left ventricular
end systolic diameter (LVESD) using the following formula: FS =
[(LVEDD ꢁ LVESD)/LVEDD] ꢀ 100.
4.4.9. Survival studies
4.4.5. Quantitative real-time RT-PCR analysis
LmnaH222P/H222P mice were treated with placebo, 3 mg/kg/day or
6 mg/kg/day of 8 as described above starting at 10 weeks of age
and until time of death or signs of significant distress requiring
euthanasia. Specific signs of significant distress included (i) diffi-
culty with normal ambulatory movement, (ii) failure to eat or
drink, (iii) weight loss of more than 20%, (iv) depression, (v) rough
or unkempt hair coat, and (vi) significant respiratory distress. A
staff veterinarian at the Institute of Comparative Medicine at
Columbia University Medical Center, blind to treatment group,
determined if euthanasia was required.
RNA was extracted from heart tissue using the RNeasy isolation
kit (Qiagen). Complementary DNA was synthesized from total RNA
using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scien-
tific) according to the manufacturer’s instructions. For each repli-
cate in each experiment, RNA from tissue samples of different
animals was used. Primers were designed corresponding to mouse
RNA sequences using Primer3 (http://frodo.wi.mit.edu/cgi-bin/pri-
mer3/primer3_www.cgi): for Nppa forward 50-gcttccaggccatattg-
gag-30 and reverse 50-ccctgcttcctcagtctgct-30; for Myl7 forward
50-tcaaggaagccttcagctgc-30 and reverse 50-cggaacacttaccctcccg-30.
Real-time RT-PCRs contained HotStart-IT SYBR green qPCR Master
4.4.10. Statistics
Mix (Affymetrix), 200 nM of each primer, and 0.2
lL of template in
Values for scanned immunoblots, echocardiographic parame-
ters, real-time RT–PCR, blood chemistry results and cardiac fibrosis
scores were compared using one-way ANOVA. To validate these
results, a nonparametric (Mann-Whitney) test was performed
and concordance checked. Mouse survival was analyzed using
the Kaplan-Meier estimator.38 Differences in median survival were
compared using a log-rank (Mantel-Cox) test with P < 0.05 consid-
ered to be statistically significant.45 Statistical analyses were per-
formed using GraphPad Prism software.
a 25 L reaction volume. Amplification was carried out using the
l
ABI 7300 Real-Time PCR System (Applied Biosystems) with an ini-
tial denaturation at 95 °C for 2 min followed by 50 cycles at 95 °C
for 30 s and 62 °C for 30 s. Relative levels of mRNA expression were
calculated using the DDCT method.43 Individual expression values
were normalized by comparison with Gapdh mRNA (forward
50-tgcaccaccaactgcttag-30 and reverse 50-ggatgcagggatgatgttc-30)
4.4.6. Histological analysis
Tissue samples from LmnaH222P/H222P mice were fixed in 4%
formaldehyde for 48 h, embedded in paraffin, sectioned at 5
lM
Declaration of interest
and stained with hematoxylin and eosin or Masson trichrome. Rep-
resentative stained sections were scanned using a Leica SCN400
scanner. Images were processed using Adobe Creative Cloud Photo-
shop CC (Adobe Systems). A pathologist blind to mouse genotype
and treatment group analyzed pathology in heart sections stained
with Masson trichrome. A grading system was constructed based
W.W. has no related interest to disclose. M.D.C. was an
employee of Cheminpharma and is an inventor on patents related
to this work. B.P. Hart has equity in AlloMek Therapeutics and is an
inventor on patents related to the work. E.S. Kumarasinghe in an
employee of Cheminpharma. M.K. Ji is an employee of Chem-
Please cite this article in press as: Wu W., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.014

10
W. Wu et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
11. Worman HJ, Fong LG, Muchir A, Young SG. J Clin Invest. 2009;119:1825.
12. Bonne G, Di Barletta MR, Varnous S, Bécane HM. Nat Genet. 1999;21:285.
13. Fatkin D, MacRae C, Sasaki T, et al. N Engl J Med. 1999;341:1715.
14. Brodsky GL, Muntoni F, Miocic S, Sinagra G, Sewry C, Mestroni L. Circulation.
2000;101:473.
15. Muchir A, Bonne G, van der Kooi AJ, et al. Hum Mol Genet. 2000;9:1453.
16. Quijano-Roy S, Mbieleu B, Bönnemann CG, et al. Ann Neurol. 2008;64:177.
17. Lu JT, Muchir A, Nagy PL, Worman HJ. Dis Model Mech. 2011;4:562.
18. Muchir A, Pavlidis P, Decostre V, et al. J Clin Invest. 2007;117:1282.
inpharma. A. Bhargava is an executive at Shakti Biorsearch and has
equity in the company. M.W. Lawlor receives research support
from Audentes Therapeutics, Solid GT and Demeter Therapeutics
and is a member of the Scientific Advisory Board of Audentes Ther-
apeutics. J.-Y. Shin has no related interest to disclose. F. Sera has no
related interest to disclose. S. Homma has not related interest to
disclose. A. Muchir is a member of the Scientific Advisory Board
of AlloMek Therapeutics, has equity in AlloMek Therapeutics and
is an inventor on a patent related to the work. U.R. Khire is an exec-
utive at AlloMek Therapeutics, an executive at Cheminpharma, has
equity in both of these companies and is an inventor on patents
related to the work. H.J. Worman previously received research sup-
port from AlloMek Therapeutics, is a member of the Scientific Advi-
sory Board of AlloMek Therapeutics, has equity in AlloMek
Therapeutics and is an inventor on a patent related to the work.
AlloMek Therapeutics owns patents related to this work and is
developing the drug.
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Please cite this article in press as: Wu W., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.014