Design, synthesis and optimization of novel Alk5 (activin-like kinase 5) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.07.030

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the di

Full text of DOI:10.1016/j.bmcl.2016.07.030

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4334–4339
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and optimization of novel Alk5 (activin-like kinase
5) inhibitors
⇑
Haixia Wang , J. David Lawson, Nick Scorah, Ruhi Kamran, Mark S. Hixon, Joy Atienza,
⇑
Douglas R. Dougan, Mark Sabat
Takeda California, 10410 Science Center Drive, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are
potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized
to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one
drug like Alk5 inhibitors.
Received 7 June 2016
Revised 13 July 2016
Accepted 14 July 2016
Available online 16 July 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Alk5
Inhibitors
Activin
Kinase
Transforming
Growth
Factor
SBDD
Transforming growth factor b (TGF- b) belongs to the TGF- b
protein superfamily involved in cellular proliferation, cellular dif-
ferentiation and other regulatory cell functions. It plays a role in
immunity, cancer, heart disease and fibrosis.1,2 TGF-b signals
through a family of transmembrane serine/threonine kinase recep-
tors. These receptors can be divided into two classes, the type I or
activin-like kinase (Alk5) receptors and type II receptors.3,4
Signaling through these receptors starts when TGF-b ligand first
binds to a type II receptor which then recruits and phosphorylates
a type I receptor, Alk5. The Alk5 receptor then recruits and phos-
phorylates cytoplasmic protein SMADs which form heteromeric
complexes that enter the cell nucleus to affect gene transcrip-
tion.5,6 Modulating Alk5 is thought to have therapeutic potential
for metastatic cancer7 and fibrotic disease.8 Therefore we sought
to develop a series of small molecule inhibitors of this target.
Screening of our internal compound collection afforded the
early hit molecule 1 which displayed good Alk5 enzymatic potency
as well as strong cellular activity (Fig. 1). The compound however,
had poor drug like properties including elevated CYP inhibitory
activity and metabolic instability. A discovery program was initi-
ated to expand the SAR and improve the drug-likeness of 1. These
efforts are the focus of this publication.
N
Alk5 pKd = 8
NH
Alk5 pEC50 = 7.3
CYP1A2 pIC50 = 6.14
Clint(rat) (mL/min/kg) = 642
Eh (rat) = 0.9
N
H
N
F
1
Figure 1. Screening hit 1.
A synthetic sequence to access compound 1 was developed and
used to explore this series (Scheme 1). Starting with commercially
available Boc protected bromo azaindole 1a, Buchwald amination
with 4-amino pyridine afforded 1b. Further Suzuki coupling of this
intermediate with 2-fluorophenyl boronic acid gave compound 1.
Variations of this scheme were then used to access a range of dif-
ferentially substituted analogs.
Modeling of compound 1 into a crystal structure of the active
site of the Alk5 kinase domain9 (Fig. 2) suggested that the com-
pound was anchored to the kinase hinge via a hydrogen bond
between the ligand’s pyridyl nitrogen and the backbone NH of
His-283. Additionally the adjacent carbon CH forms a pseudo
hydrogen bond10 to the backbone carbonyl of Asp-281. The azain-
dole nitrogens appear to insert into a hydrogen bond network
⇑
Corresponding authors.
http://dx.doi.org/10.1016/j.bmcl.2016.07.030
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

H. Wang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4334–4339
4335
N
Br
N
N
NH
N
NH
b
a
N
Cl
N
H
O
O
N
H
Cl
N
F
1a
1b
1
Scheme 1. Reagents and conditions: (a) pyridin-4-amine, sodium t-butoxide, tris(dibenzylideneacetone)dipalladium(0), Xantophos, 1,4-dioxane, 95 °C; (b) 2-fluorophenyl-
boronic acid, bis(triphenylphosphine)palladium chloride, sodium carbonate (2 M aq), DMF, 175 °C.
fluorophenyl or 5-chloro-2,4-difluorophenyl groups at position
C6 on the azaindole core led to potent analogs 4, 5 and 6.
Compounds 2 and 6 showed good enzymatic and cellular potency
with moderate in vitro clearance (rat). Subsequent PK studies
indicated that compound 2 had good bioavailability (F = 65%) while
compound 6 had low bioavailability (F = 13%). However compound
2 and 6 suffered from elevated CYP inhibitory activity which may
at least partly attributed to the unblocked basic pyridine site.
Attempts to replace the pyridine ring with the pyrimidine ring
afforded only a modest decrease in CYP2C8 and CYP3A4 inhibition
(data not shown). Blocking pyridine ring at C-2 position resulted in
loss of potency significantly (data not shown).
To decrease CYP inhibition, we have investigated incorporation
of electron withdrawing group (EWG) at the C-3 position of the
pyridine ring (Table 2). Starting with commercially available com-
Figure 2. Model of compound 1 in the active site of Alk5.
pound 7a (Scheme 2), the reaction with cyclopropylamine afforded
7b, followed by Buchwald amination with tert-butyl 4,6-dichloro-
1H-pyrrolo[2,3-b]pyridine-1-carboxylate to give 7c. Suzuki
defined by the conserved salt bridge residues Lys-232, Asp-351,
Glu-245, Tyr-249 through a conserved water. Finally, the 2-fluo-
rophenyl group is directed into the back hydrophobic pocket of
the Alk5 enzyme which is delineated by Tyr-249, Phe-262,
Leu-278, and gatekeeper Ser-280.
With this model, the hydrophobic pocket was initially targeted
using key moieties previously disclosed within Alk5 literature.11
This was accomplished by varying the group at position C-6 of
the azaindole core while holding the aminopyridine group at posi-
tion C-4 constant (Table 1).
coupling of this intermediate with 2,4-difluorophenyl boronic acid
yielded 7d, which upon treatment with TFA afforded target mole-
cule 7.
Installation of electron withdrawing group R1 at C-3 of the pyr-
idine ring dramatically decreased CYP inhibition as shown in ana-
logs 7–12 while the potency was maintained (Table 2). This was
attributed to the decreased basicity of the pyridine and a lower
overall lipophilicity (Compound 9: calculated basic pKa 6.7, cLogD
1.7; compound 2: calculated basic pKa 7.9, cLogD 2.7). 5-Chloro-2-
fluorophenyl group was also explored at R2 on position C-6 of
azaindole ring (compound 10).
Installation of 2,4-difluorophenyl group at position C6 on the
azaindole core gave good enzymatic potency (compound 2).
However exchange of 2,4 for 3,4-difluoro (compound 3) was
detrimental to enzymatic activity. This suggested that ortho
Based on the cellular potency, CYP profile and in-vitro clear-
ance, compound 10 was selected to be further studied for PK in
rats. Unfortunately this molecule had low bioavailability
(F = 17%). So we further explored the ring cyclization strategy in
hope of identifying a better molecule, which gave analogs 13 to
substitution aided the biaryl structure adopting
a favorable
conformation. Incorporation of 6-methylpyridin-2-yl, 5-chloro-2-
Table 1
Selected data for pyrrolopyridine analogs
2
3
N
NH
4
6
1
N
R
N
H
12
13
*
Compd
R
pIC50
pEC50
CYP1A2 pIC50
CYP2C8 pIC50
CYP3A4 pIC50
Clint(rat)
(mL/min/kg)
1
2
3
4
5
6
2-Fluorophenyl
2,4-Difluorophenyl
3,4-Difluorophenyl
6-Methylpyridin-2-yl
5-Chloro-2-fluorophenyl
5-Chloro-2,4-difluorophenyl
—
7.3
7.6
—
6.8
7.7
7.7
6.1
6.5
6.7
<4.3
—
<4.3
7.1
6.2
5.4
—
5.6
5.9
5.7
4.6
—
642
41
—
1188
118
29
7.5
4.2
7.1
8.5
7.4
6.0
7.0
<6.1
*
In vitro intrinsic clearance (microsomes).

4336
H. Wang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4334–4339
Table 2
Selected analogs with electron withdrawing group R1 at position C-3 of pyridine ring
3
R1
N
NH
6
R2
N
N
H
Compd
R1
H
R2
pIC50
pEC50
CYP1A2 pIC50
CYP3A4 pIC50
Clint (rat)
(mL/min/kg)
2
7
2,4-Difluorophenyl
2,4-Difluorophenyl
7.5
7.9
7.62
7.78
6.5
5.9
41
78
O
<4.3
<4.3
N
H
O
O
8
9
5-Chloro-2-fluorophenyl
2,4-Difluorophenyl
7.7
7.8
7.65
7.75
<4.3
<4.3
4.8
70
80
N
H
<4.3
N
H
OH
OH
O
10
5-Chloro-2-fluorophenyl
7.9
8.04
<4.3
<4.3
37
N
H
O
O
H
N
11
12
2,4-Difluorophenyl
2,4-Difluorophenyl
7.9
6.4
7.29
—
<4.3
<4.3
<4.3
<4.3
117
34
N
H
NH2
N
H
O
O
O
N
N
H
N
N
H
O
O
N
N
H
NH
NH
d
c
b
a
N
O
N
N
H
NH
NH2
NH2
N
N
N
N
H
7a
7b
N
Cl
N
O
O
F
F
F
F
O
O
7c
7
7d
Scheme 2. Reagents and conditions: (a) AlMe3, cyclopropanamine, DCE, 0°-70 °C; (b) tert-butyl 4,6-dichloro-1H-pyrrolo[2,3-b]pyridine-1-carboxylate, dicyclohexyl(20,40,60-
triisopropyl-[1,10-biphenyl]-2-yl)phosphine, Pd2(dba)3, potassium carbonate, t-BuOH, 100 °C; (c) 2,4-difluorophenylboronic acid, bis(triphenylphosphine)palladium chloride,
sodium carbonate (2 M aq), DMF, 175 °C; (d) TFA, DCM.
Table 3
Selected data for ring cyclized pyrrolopyridine analogs
R1
R2
N
N
H
Compd
R1
R2
pIC50
pEC50
7.62
CYP1C2 pIC50
CYP3A4 pIC50
Clint
(rat)
(mL/min/kg)
2
H
N
2,4-Difluorophenyl
2,4-Difluorophenyl
7.5
7.9
6.5
5.9
41
NH
NH
13
7.75
<4.3
<4.3
160
197
272
204
O
O
O
N
N
N
N
14
15
16
5-Chloro-2-fluorophenyl
2,4-Difluorophenyl
8.4
7.7
8.2
8.02
—
5.2
4.7
5.8
5.5
5.4
6
N
N
N
3
N
5-Chloro-2-fluorophenyl
—
O
N

H. Wang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4334–4339
4337
O
OH
Br
Br
N
N
OH
NH
N
O
a
b
NH2
N
NH
NH2
a
N
H
Cl
N
N
SEM
b
Cl
Cl
N
N
Cl
Cl
13a
13b
N
SEM
Br
N
13c
F
F
18a
18b
NH
18c
H
N
NH2
H
N
H
N
N
O
N
N
O
N
O
O
N
e
d
N
c
N
N
N
N
O
d
c
N
O
N
N
H
N
N
Cl
N
N
Cl
SEM
SEM
N
F
F
Cl
F
F
N
13
13d
13e
F
F
18d
Scheme 3. Reagents and conditions: (a) (2-(chloromethoxy)ethyl)trimethylsilane,
sodium hydride, DMF, 0 °C–rt; (b) methyl 4-aminonicotinate, Tris(dibenzylide-
neacetone)dipalladium(0), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphos-
phine), sodium t-butoxide, 1,4-dioxane; 130 °C; (c) DPPA, TEA, Toluene, reflux;
(d) 2,4-difluorophenylboronic acid, sodium carbonate, Bis(triphenylphosphine)pal-
ladium(II) dichloride, 1,4-dioxane, 130 °C; (e) TBAF, ethylene diamine, THF, 130 °C.
18
Scheme 4. Reagents and conditions: (a) (5-chloro-2-fluorophenyl)boronic acid,
sodium carbonate, Bis(triphenylphosphine)palladium(II) dichloride, 1,4-dioxane,
95 °C; (b) 4-chloronicotinic acid, Tris(dibenzylideneacetone)dipalladium(0),
(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), sodium t-butoxide,
1,4-dioxane; 95 °C; (c) diphenyl phosphorazidate, triethylamine, toluene, reflux;
(d) 2-chloroacetamide, cesium carbonate, DMF, 120 °C.
16 (Table 3). The synthesis of these analogs is described in
Scheme 3. The commercially available 4-bromo-6-chloro-1H-
pyrrolo[2,3-b]pyridine (13a) was protected with SEM group first
and then subjected to Buchwald amination with methyl 4-aminon-
icotinate to provide 13c. A Curtius rearrangement then afforded
the imidazolidinone ring (13d). Suzuki coupling with 2,4-difluo-
rophenyl boronic acid, followed by SEM deprotection with TBAF
gave the target 13.
Compounds 13–16 showed high enzymatic and cellular
potency. However, they suffered from high clearance.14 Based on
the activities of compounds 15 and 16, a methyl group at 3 position
on imidazopyridinone ring was tolerated. This provides a vector for
ADME property optimization. Molecular modeling study also pre-
dicted that there was sufficient space to explore this area.
In an attempt to reduce MW, ring count, HBD and TPSA the
azaindole core of compound 16 was replaced with a methyl pyri-
dine moiety (compound 17). Further introduction of a carboxamide
group significantly improved metabolic stability (compound 18)
(Table 4).
Chemistry access to compound 18 is described in Scheme 4.
Suzuki coupling of commercially available 18a with (5-chloro-2-
fluorophenyl)boronic acid gave the intermediate 18b. Buchwald
amination of 18b with 4-chloronicotinic acid gave 18c.
Intermediate 18c was converted to 18d through Curtius rearrange-
ment and cyclization. Alkylation of 18d with 2-chloroacetamide
afforded target 18.
Compound 18 was selected as our lead molecule because of its
overall favorable profiles. It showed a clean CYP profile, low clear-
ance, good enzymatic and cellular potency, excellent bioavailabil-
ity in rat PK (F = 62%) and low hERG liability (21% inhibition @
10 lM). Additionally compound 18 possessed good kinase and
ALK5 family isozyme selectivity.15 Molecular modeling suggests
Table 4
Selected analogs of imidazopyridinone
R
N 3
N
O
N
Cl
A
F
Compd
A
R
pIC50
8.2
pEC50
CYP1C2 pIC50
CYP3A4 pIC50
Clint (rat)
(mL/min/kg)
16
Methyl
—
5.8
6
204
N
H
N
17
18
19
Methyl
7.5
7.6
7.9
6.34
6.63
6.48
5.7
4.3
5.1
5.8
105
29
N
N
N
–CH2CONH2
Ethyl
<4.3
>5.8
110

4338
H. Wang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4334–4339
Acknowledgments
We are grateful to Matthew J. Cukierski, Mary E. Carsillo, Ron
Eydelloth and Vic Kadambi for their work on toxicology studies.
References and notes
1. Yingling, J. M.; Blanchard, K. L.; Sawyer, J. S. Nat. Rev. Drug Disc. 2004, 3, 1011.
2. Massague, J.; Blain, S. W.; Lo, R. S. Cell 2000, 103, 295.
3. Attisano, L.; Wrana, J. L. Science 2002, 296, 1646.
4. Miyazano, K. Int. J. Hematol. 1997, 65, 97.
5. Huse, M.; Chen, Y. G.; Massague, J.; Kuriyan, J. Cell 1999, 96, 425.
6. Derynck, R.; Zhang, Y.; Feng, X. H. Cell 1998, 95, 737.
7. Muraoka-Cook, R. S.; Shin, I.; Yi, J. Y.; Easterly, E.; Barcellos-Hoff, M. H.;
Yingling, J. M.; Zent, R.; Arteaga, C. L. Oncogene 2006, 25, 3408.
8. Moon, J. A.; Kim, H. T.; Cho, I. S.; Sheen, Y. Y.; Kim, D. K. Kidney Int. 2006, 7,
1234.
9. Sawyer, J. S.; Beight, D. W.; Britt, K. S.; Anderson, B. D.; Campbell, R. M.;
Goodson, T., Jr.; Herron, D. K.; Li, H. Y.; McMillen, W. T.; Mort, N.; Parsons, S.;
Smith, E. C.; Wagner, J. R.; Yan, L.; Zhang, F.; Yingling, J. M. Bioorg. Med. Chem.
Lett. 2004, 14, 3581. PDB ID: 1RW8 (Research Collaboratory for Structural
Bioinformatics).
Figure 3. Model of compound 18 in the active site of Alk5.
10. Pierce, A. C.; Sandretto, K. L.; Bemis, G. W. Proteins 2002, 49, 567.
11. (a) Li, H. Y.; McMillen, W. T.; Heap, C. R.; McCann, D. J.; Yan, L.; Campbell, R. M.;
Mundla, S. R.; King, C. H.; Dierks, E. A.; Anderson, B. D.; Britt, K. S.; Huss, K. L.;
Voss, M. D.; Wang, Y.; Clawson, D. K.; Yingling, J. M.; Sawyer, J. S. J. Med. Chem.
2008, 51, 2302; (b) Ref. 4.; (c) Kim, D.; Kim, J.; Park, H. Bioorg. Med. Chem. 2013,
2004, 12; (d) Singh, J.; Chuaqui, C. E.; Boriack-Sjodin, P. A.; Lee, W.; Pontz, T.;
Corbley, M. J.; Cheung, H. K.; Arduini, R. M.; Mead, J. N.; Newman, M. N.;
Papadatos, J. L. Bioorg. Med. Chem. Lett. 2003, 13, 4355.
Table 5
Rat toxicity study of compound 18
Group Test article and dosagey Hearts evaluated Incidence Severity**
1
2
3
4
0.5%MC
6
6
6
6
0
0
6
6
—
vehicle control
Compound 18
50 mg/kg/day
Compound 18
150 mg/kg/day
Compound 18
500 mg/kg/day
12. Enzymatic activity assay summary: Inhibition of ALK5 activity was tested by the
—
use of
a
LanthaScreenTM activity assay. TGFBR1 ALK5, fluorescein-labeled
Peptide substrate, and ATP were allowed to react for 90 minutes at room
temperature with a concentration gradient of test compounds. Then EDTA (to
stop the reaction) and terbium-labeled phospho-specific antibody (to detect
phosphorylated peptide product) were added. The antibody association with
the phosphorylated fluorescein labeled substrate resulted in an increased TR-
FRET value. ALK5 activity was quantified by measuring an increase in TR-FRET
on a BMG LABTECH PHERAstar plus instrument.
Minimal
Moderate
**
Severity grade represents an average grade of all lesions from all affected valves
13. Cell assay summary: Alk5 inhibitor activity was tested in CellSensorÒ SBE-bla
HEK 293T (Thermo Fisher Scientific) cells contain a beta-lactamase reporter
gene under control of the Smad binding element (SBE) which was stably
integrated into HEK 293T cells. Cells were plated and treated with compounds
and stimulated with TGFb1. Cells were then incubated with LiveBLAzer FRET B/
G CCF4-AM (Thermo Fisher Scientific) and read in Spectramax.
14. MS metabolic soft spot analysis of compound 13 in rat liver microsome
samples was conducted. The poor metabolic stability of compound 13 (high
clearance in rat, 160 mL/min/kg) was attributed to formation of the oxidation
product as below.
per animal. There was no apparent valve predilection.
y
All rats treated with compound 18 daily for 5 consecutive days groups 3 and 4
developed discernable heart valve lesions.
that compound 18 maintains the hinge and back hydrophobic
pocket interactions of compound 1 (Fig. 3). The addition of an
acetamide group does generate a hydrogen bond to the backbone
NH of Ser-287. It is not surprising that potency is not increased
(relative to compound 17) as it is solvent exposed.
At this point we were apprised of a potential cardiac toxicity
associated with modulating Alk5.16 The team wanted to determine
if compound 18 had such liability. In mice, dosing of compound 18
at the 50 and 200 mg/kg modulated pSMAD for up to 8 hours trig-
gering our decision to advance the compound into rat toxicity
studies. The oral administration of our lead molecule (18) in a fully
empowered exploratory toxicology study revealed an induced car-
diovalvulopathy in all study rats at both the medium and high dose
animal groups (Table 5). The observed cardiovascular toxicity was
characterized by valvular interstitial cell proliferation, neutrophil
presence, hemorrhage and fibrin deposition in the heart valves of
the treated animals.17
This toxicity was deemed to have severe functional implications
resulting in potential permanent structural and functional changes
to heart valves. The toxicity was also fast onset and irreversible.
These findings in conjunction with literature reports16 led the pro-
ject team to terminate the program and disclose our findings to the
scientific community.
OH
N
N
O
N
NH
N
F
F
15. Compound 18 at 1
lM concn was profiled against the following kinase panel:
ABL1, AKT1 (PKB alpha), AURKA (Aurora A), BRAF, CDK1/cyclin B, CDK2/cyclin
A, CDK5/p25, CHEK1 (CHK1), CHEK2 (CHK2), CLK1, CLK2, CSK, EGFR (ErbB1),
ERBB2 (HER2), GSK3B (GSK3 beta), IGF1R, IKBKB (IKK beta), INSR, IRAK1, JAK2,
KDR (VEGFR2), LCK, LIMK2, LYN A, MAP2K1 (MEK1), MAP2K2 (MEK2), MAP2K6
(MKK6), MAP3K10 (MLK2), MAP3K2 (MEKK2), MAP3K3 (MEKK3), MAP3K5
(ASK1), MAP3K9 (MLK1), MAPK1 (ERK2), MAPK10 (JNK3), MAPK12 (p38
gamma), MAPK14 (p38 alpha), MAPK3 (ERK1), MAPK8 (JNK1), MAPK9 (JNK2),
MAPKAPK2, MAPKAPK5 (PRAK), MET (cMet), PAK1, PAK4, PAK6, PDK1 Direct,
PRKACA (PKA), PRKCA (PKC alpha), PRKCB1 (PKC beta I), PRKCD (PKC delta),
PRKCG (PKC gamma), RPS6KB1 (p70S6K), SGK (SGK1), SRC, STK3 (MST2), STK4
(MST1). Compound 18 was inactive against all (showing 616% inhibition (est.
In summary, a novel series of Alk5 inhibitors has been identified
and developed. Starting from screening hit 1, by using SBDD and
medicinal chemistry knowledge, metabolic stability and bioavail-
ability were successfully optimized, culminating in the identifica-
tion of lead compound 18. However, inhibition of Alk5 with
compound 18 produced significant cardiac toxicity and led to the
program’s termination.
IC50 P5
l
M) except MAP3K2 (MEKK2) showing 31% inhibition at 1
lM (est.
IC50 = 2.2
l
M).
Compound 18 ALK5 family isozyme profiling selectivity: ALK4 is 43-fold, ALK2
and ALK3 (BMPR1a) are each >200-fold, based on 2pt screening done at 10 and
1 lM, and compared to the in-house determined of ALK5 IC50 value.

H. Wang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4334–4339
4339
16. Anderton, M. J.; Mellor, H. R.; Bell, A.; Sadler, C.; Pass, M.; Powell, S.; Steele, S. J.;
Roberts, R. R. A.; Heier, A. Toxicol. Pathol. 2011, 39, 916.
Clinical observations: Rats were observed once prior to the start of dosing on
Study Day 1. On Day 1–5, each rat was observed continuously for 30 min after
dosing and again at approximately 4 h post-dosing for changes in general
appearance and behavior. For each observation period, the observer was not be
blinded as to the animals’ treatment group. The rats were terminated on Day 6
(24 h after last dose).
Heart Perfusion & Collection: Rats were anesthetized, perfused through the left
ventricle with ꢀ100 mL ice cold PBS and subsequently 10% formalin fixed using
200–300mLs of ice cold formalin. Hearts and surrounding vasculature were
dissected and placed into labeled formalin jars for delivery to embedding
facility.
17. Experimental design: Six female Wistar rats from Harlan (10 weeks of
age)/group were used for the main study groups; the TK groups consisted of
N = 2 rats/dosage. Rats were administered a po (QD) dose of the compound for
5 consecutive days. The rats were observed daily for their survival and signs of
overt toxicity. The dose volume was 10 mL/kg and was based on body weight.
The body weight range of test animals at the initiation of dosing will be
between 150 and 190 g. Rats were held in the study room for approximately
7 days prior to the initiation of dosing.
Rats were assigned to treatment groups in a non-selective manner designed to
balance body weights between groups. Each rat was identified by a unique
number located on their tail.
Microscopic examination: Mitral, Tricuspid, and Pulmonic valves were
examined microscopically by 2 pathologists independently.