Synthesis and evaluation of pyridazinylpiperazines as vanilloid receptor 1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.09.010

A series of pyridazinylpiperazines were synthesized and evaluated for VR1 antagonist activity in order to improve upon the pharmaceutical and pharmacological properties of BCTC. A structurally biased chemical library of pyridazinylpiperazine analogs was prepared in an effort to improve the pharmaceutical and pharmacological profile of the lead compound N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H) -carboxamide (BCTC). The library was evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH 5.5-induced (pH) FLIPR assays in a human VR1-expressing HEK293 cell line. The most potent VR1 antagonists were found to have IC₅₀ values in the range of 9-200 nM with improved pharmaceutical and pharmacological profiles versus the lead BCTC. These compounds represent possible second-generation BCTC analogs.

Full text of DOI:10.1016/j.bmcl.2004.09.010

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5513–5519
Synthesis and evaluation of pyridazinylpiperazines as
vanilloid receptor 1 antagonists
Laykea Tafesse,* Qun Sun, Lori Schmid, Kenneth J. Valenzano, Yakov Rotshteyn,
Xin Su and Donald J. Kyle
Discovery Research, Purdue Pharma L.P., 6 Cedar, Brook Drive, Cranbury, NJ 08512, USA
Received 9 July 2004; accepted 3 September 2004
Available online 22 September 2004
Abstract—A structurally biased chemical library of pyridazinylpiperazine analogs was prepared in an effort to improve the pharma-
ceutical and pharmacological profile of the lead compound N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-
1(2H)-carboxamide (BCTC). The library was evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH5.5-induced
(pH) FLIPR assays in a human VR1-expressing HEK293 cell line. The most potent VR1 antagonists were found to have IC50 values
in the range of 9–200nM with improved pharmaceutical and pharmacological profiles versus the lead BCTC. These compounds
represent possible second-generation BCTC analogs.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The vanilloid receptor 1 (VR1, recently renamed
TRPV1) is a member of the superfamily of transient
receptor potential (TRP) cation channels, that is, ex-
pressed in peripheral sensory neurons.1,2 VR1 is a non-
selective cation channel (although it has a preference
for Ca++ ions) that has been cloned from rat dorsal root
ganglia3 and human.4 It is activated by various chemical
stimulants, such as capsaicin (CAP) and resiniferatoxin
(RTX)2,5 (Fig. 1), as well as noxious heat and low
pH.3,6 Activation of VR1 induces excitation of primary
sensory neurons and the sensation of burning pain in
animals and humans. However, prolonged nociceptor
excitation via chronic agonist administration is followed
by a long-term desensitization, ultimately showing an
analgesic effect.5,7 Several potent VR1 agonists have
been identified, but their initial excitatory effects on pri-
mary sensory neurons have led to poor patient compli-
ance thus hindering their development.8 For this
OH
OH
OH
OCH3
O
O
H
O
NH
H
N
H
S
O
O
HO
HN
O
O
OCH3
OH
Cl
Resiniferatoxin
Capsaicin
Capsazepine
Figure 1. Structures of capsaicin, resiniferatoxin, and capsazepine.
*
Corresponding author. Tel.: +1 609 409 5137; fax: +1 609 409 6930; e-mail: laykea.tafesse@pharma.com
Present address: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.010

5514
L. Tafesse et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5513–5519
scribed the design and synthesis of a series of 4-(2-pyr-
idyl)piperazine-1-carboxamide analogs as potent VR1
antagonists.15 Our group reported that N-(4-tertiarybut-
ylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-
1(2H)-carboxamide (BCTC), a member of that new
chemical series, was a highly potent VR1 antagonist that
effectively reverses the behavioral effects of inflamma-
tory and neuropathic pain in rats.15–17 From a pharma-
ceutical development perspective, BCTC has several
possible shortcomings including poor metabolic stabil-
ity, short half-life, poor aqueous solubility, and moder-
ate oral bioavailability. In addition, BCTC did not
produce dose-proportionate pharmacokinetics when
dosed orally to rats. In our continuing efforts to improve
upon the pharmaceutical and pharmacological proper-
ties of BCTC, here we report the synthesis and evalua-
tion of a series of pyridazinylpiperazine compounds as
VR1 antagonists (Scheme 1).
N
Cl
N
N
R
N
N
N
N
O
NH
O
NH
Ar
BCTC
Scheme 1. Proposed pyridazinylpiperazine analogs based on BCTC.
reason, the use of VR1 antagonists as potent analgesics
has attracted much attention. Capsazepine (Fig. 1) was
the first well-characterized synthetic antagonist.9–11
A biased chemical library of pyridazinylpiperazines
was prepared in a straightforward manner as shown
in Scheme 2. Pyridazine systems were explored as
Recently, several different structural classes of VR1
antagonists have been reported.12–15 Previously, we de-
Cl
6
N
R
5
N
N
3
N
Cl
R
H
N
4
b
a
N
N
N
R
+
N
N
H
N
Cl
N
H
O
NH
Ar
1: R = 6-Cl
2: R = 6-Me
3: R = 6-OMe
4: R = 4-Me, 6-Cl
5 = 5-Me, 6-Cl
6 = 6-Ph
a: Ar =
b: Ar =
7: 4-Chlorophthalazine
F
F
F
c: Ar =
d: Ar =
c
F
O
F
F
N
S
e: Ar =
F
6
5
N
N
N
R
R
N
4
3
N
b
N
N
H
N
O
NH
Ar
8: R = H
9: R = 4 -Me
10: R = 5-Me
11: Phthalazine
Scheme 2. Reagents and conditions: (a) DMSO, 100ꢁC, Et3N, 6h; (b) Ar–NCO, THF, rt, 2h; (c) Pd/C, H2, MeOH, rt, 3h.

L. Tafesse et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5513–5519
5515
Table 1. High throughput values for pyridazinylpiperazines in the crude form; IC50 values (nM) CAP/pH
N
5
N
O
3
6
N
N
HN Ar
R
4
Pyridazine
Ar
a
b
c
d
e
S
N
O
F
F
F
F
F
F
F
N
N
1
ND
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
Cl
N
N
2
3
>10,000/>10,000
ND
>10,000/>10,000
ND
>10,000/>10,000
ND
>10,000/>10,000
ND
>10,000/>10,000
>10,000/>10,000
N
N
OMe
Cl
N
N
N
4
ND
ND
2796/>25,000
ND
2178/3262
N
Cl
5
6
>10,000/>10,000
ND
>10,000/>10,000
>10,000/>10,000
ND
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
N
N
N
N
>10,000/>10,000
Ph
Cl
7
ND
ND
ND
ND
ND
N
N
N
8
340/>25,000
28/50
5745/13,286
ND
>10,000/>10,000
1465/5861
>10,000/>10,000
1386/2727
>10,000/>10,000
6169/>25,000
N
9
N
N
N
N
10
893/2642
5021/20,336
4867/13,602
>10,000/>10,000
>10,000/>10,000
11
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
>10,000/>10,000
Bold-face represents discovered hits.
replacements for the 3-chloropyridine portion of BCTC.
In order to compare the pharmacology of the proposed
library with BCTC, various 4-substituted arylisocya-
nates a–d were selected as building blocks based on
the structure of BCTC. The building block e was
included in order to increase the diversity of the pro-
posed library. The intermediates 3-piperazin-1yl-pyrida-
zines 1–7 were synthesized by reacting various
substituted 3-chloro-pyridazines with piperazine in
DMSO at 100ꢁC using triethylamine as a base. The

5516
L. Tafesse et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5513–5519
Table 2. Results of the purified pyridazine analogs
in vitro potency, consistent with BCTC series. The use
of unsubstituted pyridazine 8 resulted in analogs with
moderate potency in the CAP assay and little or no
activity when evaluated in the pH assay. When the sub-
stitution at the 6-position is removed as in analogs made
using the head groups 9 and 10, the in vitro potencies
are significantly improved in both the pH and CAP as-
says. Comparison of analogs prepared using head
groups 9 and 10 gives a clear example where substitution
at the 4-position is preferred over the 5-position. The
methyl group at the 4-position gave an analog, that is,
31-fold more potent in the CAP assay and 52-fold more
potent in the pH assay compared to the 5-methyl ana-
log. The phthalazine analogs prepared using 11 did
not show VR1 antagonist activity in either assays.
N
5
N
O
3
6
N
N
HN
R'
R
4
No.
R
R0
IC50 (nM)
pH
IC50 (nM)
CAP
12
13
14
15
16
17
18
19
20
21
H
t-Bu
t-Bu
t-Bu
>10,000
>10,000
679.4 141.4
3825.8 2727.9
47.2 9.9
6-Cl
4-Me
220.7 50.5
300.7 50.5
>10,000
4-Me, 6-Cl t-Bu
72.6 58.5
>10,000
>10,000
5-Me
5-Me
4-Me
4-Me
4-Me
4-Me
t-Bu
CF3
i-Pr
Ph
>10,000
2783.7 1072.2
5727.7 1321.8
2263.0 409.6
4332.0 499.4
176.6 66.7
279.0 120.5
2154.0 351.3
4222.7 501.0
CF3
OCF3
Guided by the initial screening results of the library
compounds, some compounds were selected and puri-
fied to greater than 98% purity via flash chromatogra-
phy on silica gel as demonstrated by two differing
RP-HPLC methods.19 The correct molecular weights
were confirmed by mass spectroscopy19 and structures
IC50 values are the mean of at least three determinations.
piperazin-1yl-pyridazines 1, 4, 5, 7 were further reduced
with Pd/C in methanol using H2 to give additional head
groups 8–11 thereby increasing diversity of the proposed
library. The intermediates 1–11 were then reacted with
isocyanates a–e for 3h in THF at room temperature to
give the desired products.
1
were further confirmed by H NMR spectroscopy.20
Upon purification and reassay of the library hits, no sig-
nificant change to the IC50 values was observed as com-
pared to the crude molecules. These results are shown in
Table 2. The use of an unsubstituted pyridazine head
group, as in 12, gave a product that has moderate po-
tency in the CAP assay and no activity in the pH assay.
Introduction of 6-chloro, as in 13, resulted in a 5-fold
decrease in potency in the CAP assay compared to 12.
The introduction of a 4-methyl group on the pyridazine
ring, 14, resulted in a significant increase in potency in
both assays, >50-fold in the pH assay and >14-fold in
the CAP assay compared to 12. The introduction of
a 6-chloro substitution, as in 15, did not change the po-
tency in the pH assay but reduced the potency by 2-fold
in the CAP assay compared to 14. The use of 5-methyl
substitutions on pyridazine head groups, as in 16 and
17, resulted in analogs with no VR1 antagonist activity.
The use of i-propyl or phenyl substitution in the tail
group instead of the t-butyl in 14, as in 18 and 19, re-
duced the potency by 12–26-fold in the pH assay and
by 4–6-fold in the CAP assay. Replacing the t-Bu group
in 14 with CF3 or OCF3, resulted in analogs 20 and 21
that have much reduced in vitro potency, 10–20-fold in
the pH assay and 45–90-fold in the CAP assay.
The compounds were evaluated for human VR1 anta-
gonist activity in a HEK293 cell line against CAP and
pH5.5 activation. The assays were conducted essentially
as described previously.16 The tests were carried out
measuring 100nM CAP- or pH5.5-mediated calcium
influx with a Fluorometric Imaging Plate Reader
(FLIPR).18
Our library purity cutoff for the initial pharmacological
assay was set to be greater than 70% (determined by LC/
MS19). A total of 39 compounds, out of 55 attempted
syntheses, passed the library purity criteria and were as-
sayed at 10lM concentration. The results are as shown
in Table 1. The wells showing >50% inhibition (10 hits
total) of the maximal CAP or pH response were consid-
ered positive and were followed up by additional experi-
ments to determine their IC50 values. The pyridazine
analogs made using head groups 1, 2, 3, 5, and 6 with
a substitution at the 6-position showed no antagonist
activity in either the pH or CAP assays. However, mod-
erate in vitro potency was observed in the CAP assay
when a substituent is added at the 4-position, as in ana-
logs made using head group 4. This result showed that
having substitution at the 4-position is crucial for the
From the SAR study (unpublished) that was developed
for BCTC series, we had observed that introduction of a
methyl group in the R stereoisomer configuration on the
6
N
R
5
N
N
3
N
R
H
4
N
b
a
N
N
N
R
+
N
N
H
N
Cl
N
H
O
NH
Ar
Scheme 3. Reagents and conditions: (a) DMSO, 100ꢁC, Et3N, 6h; (b) Ar–NCO, THF, rt, 2h.

L. Tafesse et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5513–5519
Table 3. Results of the R-methyl pyridazine analogs
5517
N
N
O
3
6
N
N
HN Ar
R
5
4
No.
Ar
R
IC50 (nM) pH
29.8 9.3
IC50 (nM) CAP
BCTC
34.0 11.0
8.5 3.6
22
23
24
25
26
4-Me
5-Me
15.2 3.5
>25,000
8590.7 477.2
10.0 1.0
4-Me, 6-Cl
5-Me, 6-Cl
4-Me
67.5 26.9
>25,000
8118.8 1449.0
57.7 23.4
F
F
250.7 14.3
F
F
27
28
29
5-Me
>25,000
>10,000
F
F
N
S
N
S
4-Me, 6-Cl
5-Me, 6-Cl
226.3 61.7
13,469.7 4385.2
103.3 30
1623.0 477.0
F
F
IC50 values are the mean of at least three determinations.
piperazine ring generally increases the potency of ana-
logs by at least 10-fold. Therefore, pyridazine analogs
that showed promising in vitro potency from the screen-
ing library were selected and resynthesized with an R-
methyl group on the piperazine ring. The syntheses of
the R-methyl analogs were accomplished in a similar
manner to the library synthesis by using R-2-methylpip-
erazine (AstaTech) instead of simple piperazine as
shown in Scheme 3.15 The results are as shown in Table
3. The introduction of the R-methyl substitution on the
piperazine ring dramatically improved the in vitro po-
tency of the resulting analog 22 compared to 14, 14-fold
in the pH assay and 5-fold in the CAP assay. Compound
22 is more potent than BCTC, 2-fold in the pH assay
and 4-fold in the CAP assay. In agreement with the pre-
vious observation, all of the analogs with 5-methyl sub-
stitutions on the pyridazine ring 23, 25, 27, and 29 were
inactive whereas the analogs with 4-methyl substitutions
22, 24, 26, and 28 were significantly more potent. The
introduction of the R-methyl substitution on the piper-
azine ring did not improve the in vitro potency of 23
and 27 when compared to the unsubstituted analogs
16 and 17. The R-methyl substitution did improve the
potency of 24 by 4-fold in the pH assay and 7-fold in
the CAP assay when compared to the unsubstituted
piperazine analog 15. Introduction of the 6-chloro on
the pyridazine ring, as in compound 24, did not affect
the potency in the CAP assay when compared to 22.
However, the pH assay indicated a 4-fold decrease in
potency. The in vitro potency of 26 also showed
improvement over the unsubstituted piperazine analog
20, 9-fold in the pH assay and 37-fold in the CAP assay.
Comparison of benzothiazole analog 28 with a similar
analog in the screening library revealed once again that
the introduction of the R-methyl group on the pipera-
zine ring improves the potency significantly, 21-fold
in the CAP assay and 14-fold in the pH assay. Mov-
ing the methyl substitution on the pyridazine ring from
the 4- to the 5-position, as in 28 and 29, was again
shown to significantly decrease the potency in the benzo-
thiazole analogs. Even though the benzothiazole analog
28 is not the most potent pyridazine analog, it is struc-
turally different from the usual aniline tail groups used
and it was presumed that it would result in a different
pharmaceutical profile.
Compounds that inhibit the human ether-a-go-go re-
lated gene (hERG) channel have a potential liability of
prolonging the cardiac QT interval causing ventricular
arrhythmias and fibrillation.21 BCTC and compounds
22, 24, and 28 were screened for their abilities to block
hERG channel expressed in HEK-293 cells at 1lM con-
centration using a standard whole cell voltage-clamp
protocol.22 The results are as shown in Table 4. It was
discovered that BCTC has a significantly high inhibition
(87%) whereas the pyridazine analogs 22 and 24 show

5518
L. Tafesse et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5513–5519
only 7% and 6% inhibitions, respectively. Compound 28
has 36% inhibition at 1lM concentration. The use of the
pyridazine head groups has significantly decreased the
ability of the piperazine-1-carboxamide analogs to inhi-
bit the hERG channel.
10000
1000
100
10
The analogs with IC50 values <500nM were selected and
their rat liver microsomal (RLM) stability was evalu-
ated. This was done by incubating the analogs with rat
liver microsomes in the presence of NADPH and oxy-
gen. After 30min, the amount of the parent compound
was measured by LC/MS. The results are as shown in
Table 4. The use of pyridazine groups instead of the
pyridine in BCTC seems to have improved RLM stabil-
ity of the resulting analogs. The RLM stabilities of 22
and 24 were 35% and 26%, respectively. These are signifi-
cant improvements over BCTC that only had 0.3% of
parent compound remaining after a 30min incubation.
Changing the t-butyl group of 22 to a CF3, as in 26,
has improved the RLM significantly to 74%. The use
of benzothiazole, as in 28, instead of the usual aniline
also had a favorable effect in the RLM assay (63%).
1
0
4
8
12
Time (h)
16
20
24
(A)
100000
10000
1000
100
3 mg/kg
10 mg/kg
30 mg/kg
10
1
0
4
8
12
Time (h)
16
20
24
In order to facilitate the compound selection process,
initial pharmacokinetic (PK) screening studies were con-
ducted. The PK profiles were determined following
intravenous (iv) administration (3mg/kg) of compounds
in 25% aqueous HPBCD. The mean plasma concentra-
tions (N = 3 rats) were used to calculate the PK para-
meters utilizing noncompartmental analysis (NCA)
algorithm of WinNonlin software. Analysis of the in
vivo PK parameters revealed that compounds 22, 24,
and 26 have very short half-lives and showed no
improvement over BCTC, as shown in Table 5. Surpris-
ingly, compound 28 showed a significantly longer half-
life of 6h compared to the usual aniline analogs.
(B)
Figure 2. In vivo pharmacokinetic profile of compound 28. Com-
pound 28 was dosed to rats intravenously (A) or orally (B) and plasma
samples withdrawn at time points indicated.
doses under fasted conditions, the corresponding bio-
availability was calculated as the ratio of the oral
dose-adjusted AUC to the dose-adjusted AUC for iv
administration (Fig. 2). The bioavailability of the
10mg/kg oral dose was calculated to be 62% (with a
maximum plasma concentration of 3957 755ng/mL
at 4h) whereas the bioavailability of 3mg/kg oral dose
was calculated to be 70% (with a maximum plasma con-
centration of 2689 143ng/mL at 3h). This is a signifi-
cant improvement over BCTC that showed marginal
oral bioavailability only at high oral dose (40mg/kg).
Unlike BCTC, compound 28 showed good aqueous sol-
ubility and high RLM stability that most likely led to its
marked increases in bioavailability and dose propor-
tional PK.
Additional studies were done in order to investigate the
oral bioavailability and dose proportionality of com-
pound 28. Following a single iv and escalating oral
Table 4. Rat liver metabolic stability and hERG channel inhibitions
for BCTC and potent pyridazine VR1 antagonists
No.
Metabolic stability
(% remaining)
HERG
(% inhibition)
In summary, we have synthesized and evaluated a struc-
turally biased chemical series of pyridazinylpiperazines
analogs as VR1 antagonists. The most potent VR1
antagonists were found to have IC50 values in the range
of 9–200nM. Moreover, we have identified a potent ana-
log 28 that has demonstrated an improved pharmaceuti-
cal and pharmacological profile versus the lead
compound BCTC. This compound represents a sec-
ond-generation BCTC analog.
BCTC
22
24
0.3
35
87
8
4
2
5
26
74
7
26
28
ND
36
63
3
Table 5. In vivo pharmacokinetic profile of BCTC, 22, 24, 26, and 28
No.
Half-life
(h)
Clearance
(L/h/kg)
Volume of distribution
(L/kg)
BCTC
22
24
0.9
0.5
0.2
0.9
6.0
4.6
7.0
5.8
5.1
Acknowledgements
6.5
1.44
4.26
0.93
The authors would like to thank Dr. Victor Ilyin for re-
view of the manuscript and Dr. Scott Nolan for techni-
cal assistance.
26
28
3.35
0.11

L. Tafesse et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5513–5519
5519
J.; Limberis, J.; Malik, S.; Whittemore, E. R.; Hodges, D.
J. Pharmacol. Exp. Ther. 2003, 306, 377.
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18. PCR amplification of human VR1 from spinal cord
cDNA, subcloning into the pIND/V5-His-TOPO vector
and generation of an inducible, stable cell line were
performed as previously described for rat VR1.16 Primers
flanking the coding region of human VR1 were designed
as follows: forward primer, GAAGATCTTCGCT-
GGTTGCACACTGGGCCACA, and reverse primer,
GAAGATCTTCGGGGACAGTGACGGTTGGATGT.
In addition, the capsaicin- and acid (pH5.5)-based assays
were also as previously described for the rat VR1.16
19. Liquid chromatography/mass spectrometry (LC/MS) was
performed on Agilent Series 1100 MSD instrument with
an electrospray sample inlet system. ZORBAXEclipse
XDB column (4.6 · 50mm) was used with a gradient of
15–90% B over 4.3min (A = 0.1% TFA in H2O, B = 0.1%
TFA/CH3CN). Two unique HPLC methods were used to
access purity of the purified compounds. The first method
uses Discovery HS C-18 (3l, 4.6 · 150mm) column with a
gradient of 15–95% B over 12.1min (A = 0.1% TFA in
H2O, B = 0.1% TFA/CH3CN). The second HPLC method
uses LUNA C-18 (3l, 4.6 · 150mm) column with a
gradient of 10–95% B over 43min (A = 0.05% TFA in
H2O, B = 0.05% TFA/CH3CN).
8. (a) Urban, L.; Campbell, E. A.; Panesar, M.; Patel, S.;
Chaudhry, N.; Kane, S.; Buchheit, K.; Sandells, B.; James,
I. F. Pain 2000, 89, 65; (b) Brand, L.; Berman, E.; Schwen,
R.; Loomans, M.; Janusz, J.; Bohne, R.; maddin, C.;
Gardner, J.; Lahann, T.; Farmer, R. Drugs Exp. Clin. Res.
1987, 13, 259.
20. NMR spectra were obtained on a Bruker Avance 400
(1H at 400MHz). The NMR data for compounds 22, 24,
1
26, 28 is as follows; 22: H NMR (CDCl3): d 8.77 (d, 1H,
J = 4.60Hz), 7.34–7.32 (m, 4H), 7.20 (d, 1H, J = 4.60Hz),
6.46 (br s, 1H), 4.50–4.39 (m, 1H), 3.98 (br d, 1H,
J = 12.50Hz), 3.69–3.61 (m, 1H), 3.53–3.44 (m, 2H), 3.30
(dd, 1H, J = 12.50Hz, J = 3.73Hz), 3.16 (dt, 1H,
J = 12.50Hz, J = 3.50Hz), 2.38 (br s, 3H), 1.45 (d, 3H,
J = 6.58Hz), 1.32 (s, 9H). Compound 24: 1H NMR
(CDCl3): d 7.379–7.29 (m, 4H), 7.25–7.23 (m, 1H), 6.42
(br s, 1H), 4.48–4.39 (m, 1H), 3.97 (br d, 1H, J =
13.15Hz), 3.66–3.58 (m, 1H), 3.66–3.58 (m, 1H), 3.53–
3.41 (m, 2H), 3.26 (dd, 1H, J = 3.72Hz, J = 12.28Hz), 3.13
(dt, 1H, J = 3.72Hz, 12.72Hz), 2.37 (br d, 3H, J =
1.09Hz), 1.43 (d, 3H, J = 6.57Hz), 1.31 (s, 9H). Com-
pound 26: 1H NMR (CDCl3): d 8.77 (d, 1H, J =
4.60Hz), 7.60–7.53 (m, 4H), 7.22 (d, 1H, J = 4.60Hz),
6.89 (br s, 1H), 4.55–4.45 (m, 1H), 4.03 (br d, 1H,
J = 12.93Hz), 3.70–3.64 (m, 1H), 3.57–3.45 (m, 2H), 3.31
(dd, 1H, J = 3.73Hz, 12.71Hz), 3.18 (dt, 1H, J = 3.44Hz,
J = 12.28Hz), 1.47 (d, 1H, J = 6.79Hz). Compound 28: 1H
NMR (CDCl3): d 8.84 (br s, 1H), 7.61–7.53 (m, 1H), 7.49–
7.42 (m, 1H), 7.25–7.21 (m, 1H), 7.16–7.07 (m, 1H), 4.54–
4.43 (m, 1H), 4.08 (br d, 1H, J = 12.93Hz), 3.64–3.56 (m,
1H), 3.54–3.37 (m, 2H), 3.23 (dd, 1H, J = 3.73Hz,
J = 12.49Hz), 3.11 (dt, 1H, J = 3.51Hz, J = 12.71Hz),
2.34 (br d, 3H, J = 0.44Hz), 1.44 (d, 3H, J = 6.79Hz).
21. Roden, D. M.; Lazzara, R.; Rosen, M.; Schwartz, P. J.;
Towbin, J.; Vincent, G. M. Circulation 1996, 94, 1996.
22. The assays were conducted on hERG channel expressed in
HEK-293 cells (obtained from Wisconsin Alumni Research
Foundation) at 1lM concentration using a standard whole
cell voltage-clamp protocol, that is, pre-pulsing from
ꢀ80mV to +20mV for 2s and then pulsing to ꢀ40mV for
1s every 15s. The peak amplitude of the tail current elicited
by the second, test, pulse was used to assess the degree of
inhibition. Percent inhibition was measured at least in three
cells for each compound, with astemizole, 100nM as a
positive control; data is presented as mean SEM.
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