Pyrrolidine amides of pyrazolodihydropyrimidines as potent and selective KV1.5 blockers

Article abstract of DOI:10.1016/j.bmcl.2009.12.085

Design and synthesis of pyrazolodihydropyrimidines as K_V1.5 blockers led to the discovery of 7d as a potent and selective antagonist. This compound showed atrial selective prolongation of effective refractory period in rabbits and was selected for clinical development.

Full text of DOI:10.1016/j.bmcl.2009.12.085

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1436–1439
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrolidine amides of pyrazolodihydropyrimidines as potent
and selective KV1.5 blockers
*
John Lloyd , Heather J. Finlay, Wayne Vacarro, Tram Hyunh, Alexander Kover, Rao Bhandaru, Lin Yan,
Karnail Atwal, Mary Lee Conder, Tonya Jenkins-West, Hong Shi, Christine Huang, Danshi Li, Huabin Sun,
Paul Levesque
Bristol-Myers Squibb Pharmaceutical Research and Development, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Design and synthesis of pyrazolodihydropyrimidines as KV1.5 blockers led to the discovery of 7d as a
potent and selective antagonist. This compound showed atrial selective prolongation of effective refrac-
tory period in rabbits and was selected for clinical development.
Received 29 October 2009
Revised 21 December 2009
Accepted 22 December 2009
Available online 4 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Kv1.5
IKur
Atrial fibrillation
Potassium channel blocker
Atrial fibrillation (AF) is the most common cardiac arrhythmia
and affects over 2.2 million patients in the United States alone.1
In this condition the atria beat rapidly and inefficiently which al-
lows for blood stasis that can lead to clot formation. A higher inci-
dence of clot formation puts AF patients at increased risk of stroke
and 15% of strokes are in patients with AF.2 Treatment options for
patients with AF include surgical procedures, radiofrequency abla-
tion, implanted devises and drug treatments. One approach to drug
treatment is agents that prolong refractory period in atrial tissue.
Such drugs currently available include sodium channel blockers,
potassium channel blockers or some combination of these activi-
ties either in a single drug or in multiple drugs.3 All the current
drugs target ion channels that are expressed in both atrium and
ventricle. Prolongation of refractory period in the ventricle has
been shown to increase the incidence of the potentially life threat-
ening arrhythmia torsades de points.4 The ultra rapid potassium
current (IKur) is a current conducted by the KV1.5 potassium chan-
nel that in humans is only expressed in the atrium.5 Block of IKur
would therefore be a way to treat AF without the risk of ventricular
arrhythmia.
our efforts to further optimize this series of pyrrolidine amides
with regard to KV1.5 potency, pharmacokinetic properties and
pharmacodynamic effects.
The synthesis of pyrazolodihydropyrimidines has been
described previously. The pyrazolodihydropyrimidine intermedi-
ate (4) was available from the Biginelli reaction of 3-aminopyra-
zole, 3,4-dichlorobenzaldehyde and t-butylacetoacetate. The ester
was hydrolyzed to provide the acid (5). Amide coupling under
standard conditions with substituted pyrrolidines provided
compounds 6–23 (Scheme 1). The final compounds were initially
obtained as mixtures of diastereomers with the stereochemistry
uncontrolled at the 7-position of the dihydropyrimidine. In some
cases the diastereomers where separated into isomers a and b.
The pyrrolidines used in the synthesis of compounds 6, 8, 9, and
11 were commercially available. Several aryl and heteroaryl substi-
tuted pyrrolidines, including the 2-(4-fluorophenyl)pyrrolidine,
the 2-pyridylpyrrolidine and the 4-pyridyl pyrrolidine, were
synthesized using known methods7 (Scheme 2).
Cl
Cl
Cl
Cl
N
Cl
N
Cl
N
In our previous publications we disclosed KV1.5 blockers based
on a pyrazolodihydropyrimidine template.6 We noted the pyrroli-
dine amides (1 and 2) and how substitution of the appropriate ste-
reochemistry enhanced potency (Fig. 1). This paper will describe
O
O
O
O
O
H
N
H
N
H
N
N
N
N
N
H
N
H
N
H
1 KV1.5 IC50 0.66 uM
2 KV1.5 IC50 0.20 uM
3 KV1.5 IC50 >1.0 uM (32% inh)
* Corresponding author. Tel.: +1 609 818 5327; fax: +1 609 818 3331.
E-mail address: john.lloyd@bms.com (J. Lloyd).
Figure 1. Pyrazolodihydropyrimidine KV1.5 antagonists.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.085

J. Lloyd et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1436–1439
1437
Cl
Cl
Cl
reaction of Boc-L-proline with hydroxyacetamidine followed by
Cl
N
Cl
N
Cl
N
dehydration and deprotection (Scheme 3).
The tetrazoles were synthesized by dehydration of the Cbz-L-
a
b
O
O
O
H
N
H
N
H
N
proline amide to the nitrile followed by reaction with sodium
azide. The methyl tetrazoles were then formed by alkylation with
methyl iodide then deprotection (Scheme 4).
O
OH
R
N
H
N
H
N
H
4
5
6-21
The thiazoles were synthesized by formation of the thioamide
Compd
R
Compd
R
from the Cbz-
butane or chloroacetaldehyde, then deprotection (Scheme 5).
The triazole was synthesized by reaction of Cbz- -proline amide
L-proline amide followed by reaction with 3-bromo-
N
O
N
L
13a
13b
N
1
with DMF-dimethylacetal followed by reaction with hydrazine.
Deprotection was performed with HBr in acetic acid. The triazolone
N
was synthesized by reaction of N-benzyl-L-proline ethyl ester with
N
N
N
O
N
N
hydrazine to form the acylhydrazide. Reaction of the hydrazide
with methylisocyanate provided the acylsemicarbazide that could
be cyclized under basic conditions. Hydrogenation provided the
substituted pyrrolidine (Scheme 6.).
The aminothiadiazole was synthesized by coupling of Fmoc-L-
proline with thiosemicarbazide followed by dehydration. The Fmoc
group was removed using piperidine (Scheme 7).
NH
14a
14b
2
3
N
N
N
N
N
N
O
15
16
N
N
N
N
S
O
O
6
7
N
O
O
N
H
CO2H
N
N
N
O
O
N
N
HN
c
a,b
F
N
Scheme 3. Reagents and conditions: (a) CH3C(NOH)NH2, EDC, HOBt, CH2Cl2; (b)
Cs2CO3, THF, 89% (over 2 steps); (c) TFA, CH2Cl2, 70%.
NH
17a
17b
H
N
N
N
O
N
N
N
H
O
NH2
N
O
HN
N
O
NH
N
H
a, b
O
N
c
N
N
O
N
8
9
18
19
HN
F
N
d
NH2
N
O
N
N
N
N
N
O
N
N
S
N
N
O
O
N
N
O
N
O
N
N
N
O
N
20a
20b
e
e
10
11
12
H
N
N
N
N
N
N
N
N
N
N
N
N
N
O
N
HN
HN
21a
21b
H
Scheme 4. Reagents and conditions: (a) POCl3, pyridine, À10 °C, 75%; (b) NaN3,
AcOH, NH4OAc, 95 °C, 88%; (c) H2/Pd/C, AcOH, H2O, 20%; (d) MeI, K2CO3, DCM, 57%
(1:1 endo: exo); (e) 5%Pd/C Degussa, EtOH, H2, 100%.
N
H
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0–25 °C, 2 h, 88%; (b)
pyrrolidine, EDC, HOBt, CH2Cl2, 14–68%.
S
b, c
N
O
S
HN
O
HBr
NH2
O
NH2
Ar
O
Ar
O
N
a
O
N
a, b, c
d
HN
N
N
S
N
d, c
Scheme 2. Reagents and conditions: (a) NaH, ArCO2Me THF, 65 °C, 1 h; (b) HCl,
HN
HBr
reflux, 12 h; (c) NaOH, 64% over 3 steps; (d) NaBH4, EtOH, 40%.
Scheme 5. Reagents and conditions: (a) Lawesson’s reagent, THF, 65 °C, 100%; (b)
3-bromobutan-2-one EtOH, rt to 80 °C, 100%, (c) 30% HBr, AcOH, ether trituration,
70–80%; (d) 2-chloroacetaldehyde, CHCl3, 75 °C, 45%.
The remaining heterocyclic pyrrolidines were synthesized from
-proline. The 1,2,4-oxadiazole pyrrolidine was synthesized by
L

1438
J. Lloyd et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1436–1439
N
Table 1
N
O
HN
Inhibition of KV1.5 of substituted pyrrolidine amides
O
NH2
O
N
HN
N
a
a, b
Compds
KV1.5 inhibition IC50 (lM)
O
N
c
O
N
HN
HBr
1
2
3
6
7
8
9
0.66
0.20
>1.0 (32% inh)
0.15
0.07
HN
H
O
N
HN
NH
N
O
O
O
N
OEt
0.16
0.15
f, g
d, e
N
N
HN
a
Values are means of 2–4 experiments.
Scheme 6. Reagents and conditions: (a) 2,2-dimethoxy-N,N-dimethylacetamide,
90 °C, 100%; (b) NH2NH2, AcOH, 95 °C, 74%; (c) 30% HBr, AcOH, ether trituration,
94%; (d) NH2NH2, MeOH, 65 °C, 16 h, 95%; (e) MeNCO, THF, 24 h, 86%; (f) NaOH
(1.0 M, aq), 100 °C, 1 h, 95%; (g) HCO2NH4, 10% Pd-C, MeOH, 80 °C, 1 h, 88%.
in 2-heterocyclo-substituted pyrrolidine amides. In an attempt to
add an ionizable group to increase solubility, we investigated a
series of pyridyl substituted pyrrolidines. The 4-pyridyl (10) and
2-pyridyl (12) compounds were three–sixfold less active than the
phenyl substituted compound. The 3-pyridyl compound (11) was
even less active. The oxadiazole (13b) synthesized from L-proline
showed similar activity as the ester (9). The corresponding oxadi-
NH2
NH2
N
N
N
N
O
S
S
OH
Fmoc
FMOC
b
a
N
N
HN
azole from D-proline was >6-fold less active confirming the prefer-
ence for the S-isomer seen with compounds 2 and 3 (Table 2).
The tetrazole compounds (14a and 14b) had very little activity
probably due to the charged functionality. The methyl tetrazole
(15) recovered some activity but was still about threefold less ac-
tive than the ester (9). The thiazole (16) had similar potency to
the methyl tetrazole. The triazoles (17a and 17b) and triazolone
(18) had very little potency as did the aminothiadiazole (19). Some
of the most potent heterocycles were the isoxazoles (Table 3). The
more active diastereomers (20b and 21b) had activity similar but
not superior to the 4-fluorophenyl compound (7) as a mixture of
diastereomers.
Of the compounds synthesized in this study, the 2-(4-fluoro-
phenyl)pyrrolidine amide (7) was one of the most active against
KV1.5 and was one of the first compounds indentified that met
our ion channel selectivity criteria for progression to further
studies. It was resynthesized and the four isomers separated by a
combination of silica gel chromatography (eluting with 75% ethyl
Scheme 7. Reagents and conditions: (a) NH2NHCSNH2, POCl3, 100 °C, 5 min; (b)
20% piperidine, CH2Cl2, 20% (over 2 steps).
The isoxazole was synthesized from Cbz-L-proline methyl ester
by reaction with the anion of acetone oxime followed by cycliza-
tion and deprotection of the amine in hydrochloric acid and TFA.
The isomeric isoxazole was synthesized by a known route8 from
the L-prolinal (Scheme 8).
The compounds 1–3 and 6–21 were tested for block of potas-
sium current in mouse fibroblast L929 cells expressing human
KV1.5.9 As previously published, our initial survey of pyrrolidine
amides revealed that the unsubstituted pyrrolidine (1) had moder-
ate activity that could be enhanced with substitution on the pyrrol-
idine ring.9 The 2-methoxymethyl compound (2) was threefold
more active although the stereochemistry of the substitution was
important with the S-isomer (2) favored over the R-isomer (3).
The 2-phenyl substituted compound (6) also had significantly
enhanced activity over the unsubstituted pyrrolidine amide (1)
and was similar to the methoxymethyl substituted compound
(2). Herein, we report the investigation of substituted pyrrolidine
amides. Several 2-arylpyrrolidines with substituents on the phenyl
ring were synthesized with the possibility of enhancing potency
and blocking potential sites of metabolism. Among the most potent
of these was the 4-fluorophenyl compound (7). Substitution at the
2-position of the pyrrolidine was favored over substitution at the
Table 2
Inhibition of KV1.5 of pyridyl- and oxadiazoyl-substituted pyrrolidine amides
a
Compds
KV1.5 inhibition IC50
(l
M)
10
0.31
11
12
>1.0 (35% inh)
0.66
13a
13b
>1.0 (22% inh)
0.16
3-position as in compound 8. The L-proline methyl ester amide
(9) also had significant potency (Table 1).
a
Values are means of 2–4 experiments.
With the observation that both aryl and ester substituents in
the 2-position showed acceptable activity, we became interested
Table 3
Inhibition of KV1.5 of heterocyclo-substituted pyrrolidine amides
N
N
O
a
Compds
KV1.5 inhibition IC50 (lM)
O
O
O
N
O
N
O
O
OMe
O
O
14a
14b
15
>1.0 (4% inh)
>1.0 (8% inh)
0.48
O
a, b
c
g
O
N
N
HN
16
0.30
O
17a
17b
18
>1.0 (19% inh)
>1.0 (3% inh)
>1.0 (11% inh)
>1.0 (4% inh)
>1.0 (21% inh)
0.11
OMe
d, e, f
O
O
N
N
HN
19
20a
20b
21a
21b
Scheme 8. Reagents and conditions: (a) acetone oxime, n-BuLi, THF, 5 h; (b) H2SO4,
0 °C, 1.5 h, 51% (over 2 steps); (c) triflic acid, CH2Cl2, 15 min, 42%; (d) NH2OHÁHCl,
pyridine, 100%; (e) NCS, DCE, 0–20 °C; (f) 2-bromopropene, Et3N, DCE, 46% (over 2
steps); (g) 50% TFA, CH2Cl2, 77%.
>1.0 (38% inh)
0.09
a
Values are means of 2–4 experiments.

J. Lloyd et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1436–1439
1439
hERG,10 sodium11 and L-type calcium12 ion channels. This com-
pound was crystallized and the single crystal X-ray analysis
showed the R stereochemistry in the dihydropyrimidine ring and
S stereochemistry in the pyrrolidine ring (Fig. 2).
Table 4
Inhibition of KV1.5 of 2-(4-fluorophenyl)pyrrolidine amides
a
Compds
KV1.5 inhibition IC50
(
lM)
7a
7b
7c
7d
2.1
1.8
0.15
0.05
The pharmacokinetics of 7d was investigated in rats and dogs
(Table 5).13 Compound 7d has intermediate systemic clearance in
rats. Steady-state volume of distribution was greater than total body
water, indicating significant extravascular distribution. Terminal
half-life was 0.57 h in rats. Oral bioavailability (F) was 51%. Half-life
was longer in dogs (1.5 h) with slightly lower bioavailability.
The pharmacodynamic activity of 7d was tested in a rabbit
model which measured the effective refractory period (ERP) in
both atrium and ventricle.14 Like humans, rabbits express the IKur
current in atrium but not ventricle. The compound was dosed at
0.3, 1.0, 3.0, and 10 mg/kg and prolonged atrial ERP by >20% at a
dose of 3 mg/kg. Reflecting the selectivity for KV1.5 over ventricu-
lar ion channels, 7d showed no effect on ventricular ERP up to the
highest dose of 10 mg/kg with plasma concentration of nearly
a
Values are means of 2–4 experiments.
Cl
F
Cl
N
7d
KV1.5 IC50 0.05 uM
hERG IC50 10.5 uM
O
I
Na IC50 >10 uM (38% inh)
Ca IC50 >10 uM (7% inh)
N
N
I
N
H
Figure 2. Stereochemistry and activity of 7d.
7
lM (Fig. 3). Plasma free fraction in rabbits for 7d was 3.0%.
In conclusion, pyrrolidine amides of pyrazolodihydropyrimi-
Table 5
dines were discovered as potent and selective KV1.5 blockers. A
substituent at the 2-position significantly enhanced activity and
the S-configuration is favored over the R-configuration. Aryl and
some uncharged heteroaryl substituents were well tolerated. Com-
pound 7d was chosen for more complete in vitro and in vivo eval-
uation and found to be potent in a pharmacodynamic model
measuring effective refractory period. This compound was chosen
for further pre-clinical toxicology studies and development as a
clinical candidate.
Pharmacokinetic parameters of compound 7d in rats and dogs
Rata
Boga
Dose (
lmol/kg)
10 (inf)b
10 (inf)b
20 (po)c
20 (po)c
F (%)
t1/2 (h)
Clearance (mL/min/kg)
Vdss (L/kg)
51
0.57 0.17
35
1.6 0.6
6
37
7
1.5 0.6
42 4.3
2.1 0.08
9
a
b
c
Values are means from three animals.
inf = intra-arterial infusion for 10 min.
po = oral gavage.
References and notes
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Drugs 2002, 62, 2377.
40
(n=5)
Atrial ERP
4. Roden, D. M. Br. J. Pharmacol. 2008, 154, 1502.
Ventricular ERP
6.95 2.8
30
20
10
0
5. Amos, G. J.; Wettwer, E.; Li, Q.; Himmel, H. M.; Ravens, U. Circulation 1994, 90, I-
581a; Li, G. R.; Feng, J.; Yue, L.; Carrier, M.; Nattel, S. Circ. Res. 1996, 78, 689.
6. Vacarro, W.; Huynh, T.; Lloyd, J.; Atwal, K. S.; Finlay, H. J.; Levesque, P. C.;
Conder, M. L.; Jenkins-West, T.; Shi, H.; Sun, L. Bioorg. Med. Chem. Lett. 2008, I18,
6381.
1.37 0.52
0.37 0.14
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3
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BMS-394136 Dose (mg/kg I.V.)
12. Fearon, I. M.; Ball, S. G.; Peers, C. Br. J. Pharmacol. 2000, 129, 547.
13. Compound 7d was administered to rats and dogs, as a solution in polyethylene
glycol 200: ethanol/water (1:1:1). Plasma was prepared from each blood
sample by centrifugation and analyzed by LC/MS. Plasma concentrations
versus time data were analyzed by non-compartmental methods. The total
plasma clearance, terminal half-life (t1/2), and the steady state volume of
Figure 3. Pharmacodymanic effect of 7d on prolongation of atrial and ventricular
effective refractory period in rabbits (n = 5). Plasma concentrations (lM) are noted
at each point on the AERP versus dose plot.
distribution (Vdss
) were calculated after intra-arterial administration. The
acetate and hexane) to separate the diastereomers and chiral HPLC
(Chiracel AD, 13% isopropanol, heptane with 0.1% TFA) to separate
the enantiomers (Table 4). The most active isomer (7d) showed
excellent activity in blocking KV1.5 and very good selectivity over
absolute oral bioavailability (F, expressed as %) was estimated by taking the
ratio of dose-normalized AUC value after an oral dose to that after an intra-
arterial dose.
14. Sun, H.; Lloyd, J.; Shi, H.; Li, D.; Levesque, P. Heart Rhythm. 2008, 5, S213.