Discovery of a class of potent gap-junction modifiers as novel antiarrhythmic agents

Article abstract of DOI:10.1016/j.bmcl.2009.07.014

In an effort to discover potent, orally bioavailable compounds for the treatment of atrial fibrillation (AF) and ventricular tachycardia (VT), we developed a class of gap-junction modifiers typified by GAP-134 (1, R¹ = OH, R² = NH

Full text of DOI:10.1016/j.bmcl.2009.07.014

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4551–4554
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a class of potent gap-junction modifiers as novel
antiarrhythmic agents
a,*
b
b
c
c
Eugene L. Piatnitski Chekler , John A. Butera , Li Di , Robert E. Swillo , Gwen A. Morgan ,
c
d
e
c
Eric I. Rossman , Christine Huselton , Bjarne D. Larsen , James K. Hennan
Wyeth Research, Chemical Sciences, 500 Arcola Road, Collegeville, PA 19426, USA
Wyeth Research, Chemical Sciences, CN 8000, Princeton, NJ 08543, USA
Wyeth Research, Cardiovascular and Metabolic Disease Research, 500 Arcola Road, Collegeville, PA 19426, USA
a
b
c
d
Wyeth Research, Drug Safety and Metabolism, 500 Arcola Road, Collegeville, PA 19426, USA
e
Zealand Pharma A/S, Glostrup, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to discover potent, orally bioavailable compounds for the treatment of atrial fibrillation (AF)
Received 24 May 2009
Revised 30 June 2009
Accepted 2 July 2009
Available online 9 July 2009
and ventricular tachycardia (VT), we developed a class of gap-junction modifiers typified by GAP-134 (1,
R = OH, R = NH ), a compound currently under clinical evaluation. Selected compounds with the desired
2
1
2
in-vitro profile demonstrated positive in vivo results in the mouse CaCl
administration.
2
arrhythmia model upon oral
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Gap-junction
AF
Atrial fibrillation
VT
Ventricular tachycardia
Antiarrhythmic peptide
Antiarrhythmic agent
The conduction of electrical impulses in the heart is established
through gap-junctions. Targeting gap-junctions represents a dis-
tinct approach for the treatment of a variety of diseases involving
gap-junction modifiers had to be balanced by the requirement to
establish in vivo activity for each synthetic target. The untradi-
tional screening paradigm created limits on the scope of struc-
ture–activity relationship (SAR) studies we report in this
manuscript.
The initial effort to study SAR around the clinical candidate 1 was
focused on modifying hydrophilic areas of the molecule. We rea-
soned that by altering the polar groups such as carboxylic acid and
amino functionalities, we could modulate compound lipophilicity.
Blocking the N- and/or C-terminus of compound 1 should afford
analogs with potentially better in vivo absorption. The C-terminal
1
dis-regulation of heart rhythm such as AF and VT. Gap-junctions
uncouple during ischemia leading to impulse slowing, unidirec-
2
tional block and re-entry arrhythmias. The molecular mechanism
of action of gap-junction modulators including the clinical candi-
date GAP-134 (1, (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrroli-
3
dine-2-carboxylic acid, Fig. 1) remains unknown,
thus
precluding the use of high through-put methods to screen for addi-
tional compounds. We chose to utilize an in vivo mouse CaCl
2
arryhythmia assay to screen analogs of GAP-134.4
Here, we report efficacious gap-junction modifiers that re-
establish intercellular communications and delay time to cardiac
conduction block in mice treated with intravenous CaCl . Starting
2
O
O
R1
with the glycine-proline dipeptide motif that is derived from com-
pound 1, we focused on modulating the lipophilic/hydrophilic bal-
HN
1
2
N
O
ance by varying R and R groups (Fig. 1) to generate analogs with
modified physicochemical properties and improved oral bioavail-
ability. The effort to optimize the parameters and the activity of
R2
1
*
Corresponding author. Tel.: +1 484 865 8180; fax: +1 484 965 8199.
E-mail address: piatnits@gmail.com (E.L. Piatnitski Chekler).
Figure 1. GAP-134 dipeptide in clinical evaluation: R1 = OH; R2 = NH
.
2
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.014

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E. L. Piatnitski Chekler et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4551–4554
proline carboxylic acid group was converted to an amide functional-
ity using simple coupling procedures. The primary amide deriva-
O
O
O
5
O
O
NH
O
tive 2 demonstrated significant in vivo efficacy in the mouse CaCl
2
NH
HN
HN
model (Table 1). It was also discovered that amide 2 had poor stabil-
ity in rat plasma. Therefore, despite potentially rapid clearance upon
administration into mice, the primary amide analog 2 showed sim-
ilar in vivo activity versus the parent compound 1. This fact indicated
that the substitution of the carboxylic acid group in 1 with an amide
functionality does not lead to the loss of efficacy. This observation
encouraged us to further investigate amide derivatives of compound
N
N
3
5
H N
2
1
20
00
80
pH 1
1
pH 4.5
pH 6.6
pH 7.4
pH 9
1. Initially, it became our goal to identify the reason for the low sta-
bility of 2 and, thus, improve the properties of amide analogs. A dras-
tic improvement in stability was observed when unsubstituted
amide 2 was followed up with methyl amide 3 and ethyl amide 4.
Mass spectroscopy analysis of the plasma incubation samples of
amides 2 and 3 suggested the formation of a single degradation
product. Amide 3 was subjected to basic media over a 24 h time per-
iod to generate piperazine-dione 5 (Fig. 2) that was further con-
firmed to be identical to the degradation product in all plasma
6
0
0
4
SGF
20
0
SIF
0
5
10
15
20
25
Time (hours)
Figure 2. Degradation profile of 3 in buffers at 37 °C. SGF-simulated gastric fluid;
SIF-simulated intestinal fluid.
samples. Piperazine-dione 5 demonstrated no in vivo activity at
À11
the 10
mol/kg dose tested. Thus, amides 2 and 3 can have some-
what reduced potency due to their partial decomposition. The loss of
activity for the ethyl amide compound 4, however, cannot be attrib-
uted to stability issues since it showed no signs of decomposition.
Structural considerations may have played a role in the reduced
activity. Full plasma stability was achieved with bulkier amide
groups in analogs 6 and 7, where larger substituents prevented
intramolecular cyclization. Stable amide derivatives showed
in vivo activity that was comparable to that of parent compound 1.
Table 1
Effect of GAP-134 analogs on time to cardiac conduction block (DT) in mice receiving
an intravenous infusion of calcium chloride
O
O
R1
HN
N
O
R2
2
Figure 3. Mouse CaCl data for 1, 6, 7, 17 and 18 after IV administration.
Compound R1
R2
Increase in time to Plasma
conduction block, stability (%
These compounds were further investigated in dose response exper-
iments. Other amide compounds at the C-terminus (8–12) demon-
strated various degrees of activity and stability. It proved that
large substituents (12) are tolerated to some degree and are suited
for further studies. Methyl ester compound 13 showed activity in
the mouse model despite having poor stability. It was determined
that 13 undergoes hydrolysis to afford Boc derivative 14 with a sim-
ilar activity as 13.
c
D
T (% of response remaining)
a,b
in control)
1
2
3
4
6
7
8
9
0
1
OH
NH
NH–CH
NH–C
NH–i-Pr
NH–cyclic-Pr
NH–cyclic-pentyl
NH–t-Bu
NH
NH
NH
NH
NH
NH
NH
NH
2
2
2
2
2
2
2
2
2
2
206.6 ± 21.0
193.2 ± 10.9
93
27
87
d
2
145.0 ± 13.1d
3
2
H
5
137.2 ± 8.8
162.0 ± 20.8
196.0 ± 16.0
110.6 ± 13.7
135.7 ± 11.0
100
100
96
90
99
N-Terminal analogs (Table 1) generally showed weaker activity
in the mouse CaCl model. Mono- or di-methylation of the amino
2
d
1
1
(R)–NH–CH(CH
NH-tetrahydro-
H-pyran-4-yl
3
)(i-Pr) NH
120.1 ± 7.3
89
81
NH
99.4 ± 8.3d
group (15, 16) led to the reduced efficacy. Acyl derivatives 17
and 18 were more potent than alkyl analogs 15 and 16. Hence,
compounds 17 and 18 were further investigated in full dose re-
sponse studies. It was assumed that the acyl groups should signif-
icantly change the lipophilicity of the molecules to warrant further
PK studies despite their relatively weaker potency. Compounds 6,
2
1
2
(R)–NH-tetrahy
drofuran-3-yl
NH
2
143.5 ± 16.1
100
166.3 ± 12.7d
1
1
1
1
1
1
1
3
4
5
6
7
8
9
OCH
OH
OH
OH
OH
OH
OH
3
NH–Boc
NH–Boc
0
100
100
100
100
100
34
156.2 ± 15.7
120.7 ± 22.7
118.9 ± 12.3
165.7 ± 14.0
NH–CH
3
3 2
NH–(CH )
NH–COCH
NH–CHO
7, 17 and 18 were compared to the clinical candidate 1 with re-
3
spect to in vivo efficacy after IV and oral administrations (Fig. 3
and Figure 4, respectively). Dose dependency was observed for
170.4 ± 16.1
132.1 ± 17.4d
NH–COCF
3
all studied analogs of 1 with
reaching over 200% versus control (Fig. 3). The bell shaped dose re-
sponse curve is specific to the mouse CaCl model, and has been
DT values after IV administration
Compound stability in plasma.
a
Values are means of at least three experiments, standard deviation is given.
Measurements were performed at 10
Rat plasma stability -% remaining after 3 h incubation.
Data may reflect lower concentration due to compound instability.
b
c
À11
2
mol/kg compound concentration.
previously observed with other antiarrhythmic compounds by Zea-
d
6
land Pharma’s research group. Compounds 6 and 7 showed effi-

E. L. Piatnitski Chekler et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4551–4554
4553
Table 2
CD-1 mice pharmacokinetic parameters for compounds 1, 6, 7, 17 and 18a
1
6
7
17
18c
IV (1 mg/kg) CD-1 mice
Clearance (mL/min/kg)
65b
2.7
1.6
58
2
0.9
20
0.6
2.1
24
0.3
0.3
12
0.2
0.4
V
ss (L/kg)
T1/2 (h)
PO (5 mg/kg) CD-1 mice
Mouse% F
6
6
7
2
2
T
C
T
1/2 (h)
max (ng/mL)
max (h)
1.9
26
0.5
73
1.6
44
0.25
90
1.2
87
0.25
305
1.2
38
0.25
66
1.4
89
0.25
144
AUC (ng h/mL)
a
Values are obtained from the mean drug concentration. (N = 3 animals/time
point).
b
c
Actual dose was 0.77 mg/kg for compound 1.
IV Dose for compound 18 was 1.6 mg/kg.
2
Figure 4. Mouse CaCl data for 1, 6, 7, 17 and 18 after oral administration.
Table 3
Dog pharmacokinetic parameters for compounds 1, 6, 7, 17 and 18a
cacy that is either similar to the activity of compound 1 or exceeds
it at some doses. Oral administration of compounds 6, 7, 17 and 18
with doses 1–30 mg/kg significantly prolonged the time to conduc-
1
6
7b
17
18c
IV (1 mg/kg) dogs
Clearance
5 ± 0.5
5 ± 0.4
4 ± 0.5
8 ± 2.2
5 ± 0.9
2
tion block in mice after infusion of CaCl (Fig. 4), thus, establishing
(
mL/min/kg)
oral activity of the compound class. The oral dose response curve
for 17 showed differences with the other compounds’ curves
where much higher efficacy was observed at 10 mg/kg, while the
efficacy at the highest dose was lower than for compounds 1, 6,
V
ss (L/kg)
8 ± 2.2
6.3 ± 0.4
1.1 ± 0.1
8.6 ± 0.5
0.6 ± 0.1
6.9 ± 0.4
0.4 ± 0.1
1.2 ± 0.2
0.4 ± 0.1
1.5 ± 0.2
T1/2 (h)
PO (5 mg/kg) dogs
Dog% F
14
16
18
8
13
7
and 18.
T
C
T
1/2 (h)
max (ng/mL)
max (h)
4.0 ± 0.5
799 ± 392
1.1 ± 0.6
4791 ± 0.5
8.3 ± 1.8
650 ± 201
1.2 ± 0.8
2849 ± 385
6.3
658
2.0
4.5 ± 2.8
258 ± 117
0.8 ± 0.3
972 ± 197
6.2 ± 3.6
737 ± 136
1.4 ± 0.8
3820 ± 461
Additional structure–activity relationship points were obtained
in the course of the initial medium through-put screening of the
Zealand Pharma small pepetide library via IV administration at
2
ties precluded us from reproducing multiple compounds in dose
response measurements. Nevertheless, activity was unambigu-
AUC (ng h/mL)
4471
a
À11
Values are means of at least three animals.
 10
mol/kg (n = 7–11). The lack of robust screening capabili-
b
c
Where there is no SD, the number of animals is two.
For compound 18, IV dose administered was 2.3 mg/kg and oral was 7.8 mg/kg.
ously established for multiple analogs of compound 1 that demon-
À11
strated
D
T value of 149.8% of saline control at 2 Â 10
mol/kg
lipophilicity may be necessary. The oral bioavailability of com-
pounds 6 and 7 was found to be similar to 1, suggesting that mod-
ification of only one terminus of the de-peptide motif is not
sufficient to improve bioavailability of the scaffold.
In summary, we report the development of the first orally bio-
available scaffold of small molecule gap-junction modifiers. Se-
lected compounds prolonged the time to AV conduction block in
the mouse model upon oral administration. Compounds 6, 7, 17
and 18 showed no hERG or CYP450 inhibition. Further modifica-
tions of this unique dipeptide scaffold to improve PK parameters
may be of interest for future studies.
dose. It was found that the presence of electron-donating and elec-
tron-withdrawing para-substituents is tolerated. For example, the
D
1
T values for p-nitro-group and p-methyl-group analogs are
66.0% and 168.3% of control, respectively. The enantiomer of com-
pound 1, (2R,4S)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-
carboxylic acid, showed similar in vivo activity in the mouse CaCl
model (134.9%). One of the diastereomers of compound 1, (2S,4S)1-
2-amino-acetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid,
has a T = 168.3% of control.
2
(
D
Having established the oral activity of compounds 6, 7, 17 and
8, our efforts focused on the evaluation of their PK properties
1
compared to clinical candidate 1. The aforementioned amides have
either the C- or N-terminus modified with a more lipophilic func-
tionality than present in molecule 1. All analogs were dosed orally
and IV in CD-1 mice (Table 2) since the mouse model was a pri-
References and notes
1.
(a) Aonuma, S.; Koama, Y.; Akai, K.; Komiyama, Y.; Nakajima, S.; Wakabayashi,
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mary screening tool for the program. However, clinical candidate
2. (a) Guerrero, P. A.; Schuessler, R. B.; Davis, L. M.; Beyer, E. C.; Johnson, C. M.;
Yamada, K. A.; Saffitz, J. E. J. Clin. Invest. 1997, 998, 1991; (b) Lerner, L. B.;
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7
1
was previously evaluated in a dog atrial fibrillation model.
Therefore, all advanced compounds were tested in dog PK both oral
and IV routes to compare them to 1 (Table 3).
3.
(a) Butera, J. A.; Larsen, B. D.; Hennan, J. K.; Kerns, E.; Di, L.; Alimardanov, A.;
Swillo, R. E.; Morgan, Gwen A.; Liu, K.; Wang, Q.; Rossman, E. I.; Unwalla, R.;
McDonald, L.; Huselton, C.; Petersen, J. S. J. Med. Chem. 2009, 52, 908; b Larsen, B.
D.; Petersen, J. S.; Haugan, K. J.; Butera, J. A.; Hennan, J. K.; Kerns, E. H.; Piatnitski,
E. L. U.S Pat. Appl. 0149460-A1, 2007.
As some PK parameters of compounds 6, 7, 17 and 18 were
found to be similar to GAP-134 (1), the stability of these analogs re-
quired further investigation. All compounds showed T1/2 >30 min
upon incubation with human and rat microsomes. The stability
in aqueous solutions of various pH values, simulated intestinal
fluid and simulated gastric fluid was also established for these four
compounds. However, compound 18 underwent in vivo metabo-
lism resulting in formation of small amount of GAP-134 (1). As a
result, further development of 18 was stopped. Absorption limited
PK profile suggests that additional efforts to modulate compounds
4.
Lynch, J. J.; Rahwan, R. G.; Witiak, D. T. J. Cardiovasc. Pharmacol. 1981, 3, 49–60.
5. (2S,4R)-4-benzamido-1-(2-(tert-butoxycarbonylamino)acetyl)pyrrolidine-2-
carboxylic acid (0.05 g, 0.1 mmol), 1-hydroxybenzotriazole monohydrate
(
Aldrich, 0.021 g, 0.15 mmol, 1.2 equiv) and 1-(3,3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (Aldrich, 0.029 g, 0.15 mmol, 1.2 equiv) were
dissolved in acetonitrile (15 mL) under a nitrogen atmosphere with ice cooling.
The temperature was gradually increased to room temperature over 2 h time
period, and the mixture was then stirred at room temperature overnight. The
reaction solution was again cooled to 0 °C, a 25–30% aqueous solution of the
corresponding amine (prepared from a pure reagent obtained from Aldrich)

4
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E. L. Piatnitski Chekler et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4551–4554
(
0.1 mL) was added, and stirring was continued with cooling for 30 min and
nitrogen atmosphere. The precipitate was filtered, washed with
dichloromethane (2 mL) and diethyl ether (2 mL) and dried under high
vacuum to afford a hydrochloride salt of the corresponding compounds in 75–
84% yield and at least 98% purity.
then at room temperature for 2 h. Acetonitrile (5 mL) was added to the reaction
mixture, and the volatiles were removed in vacuo. The semi-solid residue was
purified by silica-gel (EMD, 0.040–0.063 mm) chromatography (developing
solvent: 3–5% gradient methanol–dichloromethane) to afford the corresponding
amides in 80–87% yield. The products from the previous step were dissolved in
dry dichloromethane (10 mL) under a nitrogen atmosphere, and a 1 M ethereal
solution of hydrogen chloride (Aldrich) (1 mL) was added while keeping the
temperature below 30 °C. The reaction mixture was stirred overnight under a
6. Kjolbye, A. L.; Knudsen, C. B.; Jepsen, T.; Larsen, B. D.; Petersen, J. S. J. Pharmacol.
Exp. Ther. 2003, 306, 1191–1199.
7. Rossman, E. I.; Liu, K.; Morgan, G. A.; Swillo, R. E.; Buter, J. A.; Gruver, M.;
Kantrowitz, J.; Feldman, H. S.; Haugan, K.; Petersen, J. S.; Hennan, J. K. Circulation
2007, 116, 392.