Structural determinants for histamine H1 affinity, hERG affinity and QTc prolongation in a series of terfenadine analogs

Article abstract of DOI:10.1016/j.bmcl.2009.07.047

In the late 1980's reports linking the non-sedating antihistamines terfenadine and astemizole with torsades de pointes, a form of ventricular tachyarrhythmia that can degenerate into ventricular fibrillation and sudden death, appeared in the clinical literature. A substantial body of evidence demonstrates that the arrhythmogenic effect of these cardiotoxic antihistamines, as well as a number of structurally related compounds, results from prolongation of the QT interval due to suppression of specific delayed rectifier ventricular K+ currents via blockade of the hERG-IKr channel. In order to better understand the structural requirements for hERG and H₁ binding for terfenadine, a series of analogs of terfenadine has been prepared and studied in both in vitro and in vivo hERG and H₁ assays.

Full text of DOI:10.1016/j.bmcl.2009.07.047

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5043–5047
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural determinants for histamine H1 affinity, hERG affinity and QTc
prolongation in a series of terfenadine analogs
*
Robert Aslanian , John J. Piwinski, Xiaohong Zhu, Tony Priestley, Steve Sorota, Xiao-Yi Du, Xue-Song Zhang,
Robbie L. McLeod, Robert E. West, Shirley M. Williams, John A. Hey
The Schering Plough Research Institute, 2015 Galloping Hill Rd. Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In the late 1980’s reports linking the non-sedating antihistamines terfenadine and astemizole with tor-
sades de pointes, a form of ventricular tachyarrhythmia that can degenerate into ventricular fibrillation
and sudden death, appeared in the clinical literature. A substantial body of evidence demonstrates that
the arrhythmogenic effect of these cardiotoxic antihistamines, as well as a number of structurally related
compounds, results from prolongation of the QT interval due to suppression of specific delayed rectifier
ventricular K+ currents via blockade of the hERG-IKr channel. In order to better understand the structural
requirements for hERG and H1 binding for terfenadine, a series of analogs of terfenadine has been pre-
pared and studied in both in vitro and in vivo hERG and H1 assays.
Received 20 April 2009
Revised 6 July 2009
Accepted 8 July 2009
Keywords:
Antihistamine
Terfenadine
Astemizole
Torsades de pointes
hERG
Ó 2009 Elsevier Ltd. All rights reserved.
H1
In the late 1980’s reports linking the non-sedating antihista-
mines terfenadine and astemizole with torsades de pointes (‘twist-
ing of the points’), a form of ventricular tachyarrhythmia that can
degenerate into ventricular fibrillation and sudden death, appeared
in the clinical scientific literature. This led to the eventual removal
of these drugs from the market in 1997 for terfenadine and in 1999
for astemizole.1 A substantial body of evidence indicates that the
arrhythmogenic effect of these cardiotoxic antihistamines, as well
as a number of structurally related compounds, results from pro-
longation of the QTc interval due to suppression of specific delayed
rectifier ventricular K+ currents via blockade of the hERG-IKr chan-
nel.1–3 The question arose as to whether hERG activity could be
separated from the antihistaminic activity of this structural class.
In order to define, and therefore better understand, the structural
requirements for hERG binding as compared to H1 binding for ter-
fenadine, a series of compounds that are analogs of terfenadine
was prepared and studied in both in vitro and in vivo hERG and
H1 assays. The present report describes our findings.4
benzylic hydroxyl and t-butyl groups of terfenadine to hydrogen
and methyl, respectively. hERG activity was initially determined
in a high throughput patch clamp screening assay (Ionworks)5 while
a human H1 binding assay was used to determine H1 binding affin-
ity.6 Selected results were confirmed in vitro using an IonWorks
Quattro patch clamp assay and in vivo in the guinea pig.7,8 Hista-
mine H1 activity was confirmed in vivo in the guinea pig.7
Most of the analogs in this study were conveniently prepared
starting with an unsubstituted piperidine and the side chain was
introduced via either reductive amination or alkylation. For exam-
ple, commercially available amine 1 (R = Ph2COH) was coupled
with 4-(p-tolyl)butyric acid to give the amide 2 (n = 3) in 68% yield.
The amide carbonyl was reduced using LiAlH4 to yield amine 3 in
59% yield. Alternatively, reaction of 1 with an alkyl halide, such
OH
A
To determine the contribution of the different structural regions
of terfenadine to hERG binding and antihistamine activity, the par-
ent molecule was divided into three segments, the aromatic benz-
hydryl region A, the basic piperidine region B and the lipophilic
tail portion C and analogs of each were prepared (Fig. 1). Further-
more, in order to simplify the synthesis, we converted the secondary
CH3
CH3
CH3
Terfenadine
N
B
OH
C
* Corresponding author. Tel.: +1 908 740 3514; fax: +1 908 740 7152.
E-mail address: robert.aslanian@spcorp.com (R. Aslanian).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.047

5044
R. Aslanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5043–5047
R
N
R
R
N
a
b
CH3
CH3
N
H
O
n
n
c
1
2
3
O
R = H, CO2Et,
or
n
N
HO
R
CH3
4
Scheme 1. Reagents and conditions: (a) (p-tolyl)alkyl acid, HBTA, DEC, CH2Cl2; (b) LAH, THF; (c) p-tolylalkyl bromide, Et3N, DMF,
temperature (for n = 4).
D (for n = 1) or K2CO3, i-Pr2NEt, room
as 4-methylbenzyl bromide, in the presence of a base such as tri-
ethylamine gave the tertiary amine 4 directly in 86% yield (see
Scheme 1).
O
b
O
a
OH
OH
H3C
H3C
The synthesis of the simplified analog of terfenadine carboxyl-
ate, compound 9, first required the preparation of aldehyde 7. Wit-
tig olefination of aldehyde 5 gave olefin 6. The double bond in 6
was reduced using 5% Pd/C and H2 and the aldehyde unmasked
with pyridinium p-toluene sulfonate. Reductive amination of alde-
hyde 7 with amine 1 followed by saponification gave the desired
product 9 (Scheme 2).
10
11
OH
OH
c
CH3
CH3
N
N
Many of the compounds that are known to interact with the
hERG channel possess a lipophilic tail consisting of an aromatic
moiety tethered to a basic amine via a carbon chain of various
lengths (for example, ebastine, droperidol and haloperidol in addi-
tion to terfenadine). To determine if the binding interaction was
the result of a specific interaction with the aromatic group or sim-
ply a lipophilic effect, the cyclohexyl analog 13 was prepared
(Scheme 3). 4-(p-Tolyl)butyric acid (10) was reduced to the cyclo-
hexane 11 using 5% Rh/C. Compound 11 was coupled to 1 and the
amide 12 reduced to give the target 13.
In order to determine the contribution of the tertiary amine of
the central piperidine ring to hERG binding and H1 activity, the
cyclohexyl analog 17 was prepared (Scheme 4). Formation of the
Wittig reagent 15 from alcohol 14 proceeded in 62% overall yield.
Reaction of the ylide of 15 with ethyl 4-oxocyclohexanecarboxyl-
ate followed by reduction with H2 over Pd/C gave cyclohexyl com-
pound 16 in 50% yield over two steps. Reaction with excess phenyl
O
12
13
Scheme 3. Reagents: (a) 5% Rh/C, H2, MeOH, 59%; (b) 1, HBTA, DEC, CH2Cl2, 74%; (c)
LAH, THF, 56%.
Grignard gave the tertiary alcohol 17 in 70% yield. The relative ste-
reochemistry about the cyclohexane ring was determined to be
trans by NMR spectroscopy.
Table 1 lists the results from the patch clamp assay as a % inhi-
bition at 10 lM and human H1 binding affinity for the terfenadine
analogs prepared in this study. Entry 3, which is the simplified ana-
log of terfenadine, has an identical in vitro profile to the parent
compound (entry 1) for both hERG activity and H1 binding affinity.
Interestingly, the simplified acid analog (entry 4) of terfenadine
carboxylate is slightly more potent at the hERG channel than
would be anticipated. Based on these data, it seems that excising
the secondary hydroxyl and t-butyl groups of terfenadine may
not significantly impact the hERG and H1 profiles of the analogs
compared to the parent, but the effect is less pronounced if a car-
boxylic acid is present.
CO2CH3
CO2CH3
CO2CH3
b, c
a
d
Considering the benzhydryl region of terfenadine next (A in
Fig. 1), the data in Table 1 imply that both hERG affinity as well
as H1 binding affinity are dependent on the nature of the substitu-
ents in this area. Complete removal of the benzhydryl alcohol moi-
ety as in entry 10 causes a decrease in both hERG affinity as well as
H1 affinity, although H1 affinity seems to be effected to a greater
extent. Reintroduction of the alcohol moiety as in entry 11 does
not significantly increase affinity for the H1 receptor, although it
does result in a slight improvement in hERG affinity, perhaps due
to the polar nature of the hydroxyl group. However, reintroduction
of a single phenyl ring in addition to the hydroxyl group as in entry
12 causes an increase in both hERG and H1 affinity. This is not sur-
prising. Many hERG active compounds such as droperidol and hal-
operidol contain a piperidine ring substituted with a single phenyl
ring at the 4-position. It is also consistent with site directed
mutagenesis studies9 and pharmacophore models of the hERG
channel.10 These studies show that an aromatic group capable of
O
CHO
CHO
O
7
5
6
OH
OH
e
CO2H
CO2CH3
N
N
8
9
Scheme 2. Reagents and conditions: (a) [2-(1,3-dioxolan-2-yl)ethyl]triphenylphos-
phonium bromide, KN(SiMe3)2, THF, 54%; (b) 5% Pd/C, H2, MeOH, 91%; (c) PPTS,
acetone, 50 °C, 62%; (d) 1, NaBH(OAc)3, CH2Cl2, 60%; (e) KOH, MeOH, 80%.

R. Aslanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5043–5047
5045
H3C
H3C
a, b
c, d
OH
PPh3Br
15
14
OH
EtO2C
e
CH3
CH3
16
17
Scheme 4. Reagent: (a) PBr3, room temperature, 76%; (b) PPh3, toluene, reflux, 81%; (c) n-BuLi, ethyl 4-oxocyclohexanecarboxylate, THF, À78 °C to room temperature, 54%;
(d) H2, Pd/C, MeOH, 92%; (e) PhMgBr, THF, 0 °C to room temperature, 70%.
a
p
-stacking interaction with Phe656 in the hERG channel contrib-
points of the aromatic rings of H1 antagonists.11,12 Therefore, in
this region of terfenadine it would appear that hERG affinity and
H1 binding affinity parallel each other due to similar binding inter-
actions in the channel and receptor.
The contribution to hERG activity and H1 binding affinity of the
basic, tertiary amine in region B was then evaluated by converting
utes significantly to the binding interaction of terfenadine and
related compounds. Interestingly, site directed mutagenesis and
pharmacophore modeling studies of the H1 receptor have also
demonstrated the importance of three aromatic amino acid resi-
dues, Trp167, Phe433 and Phe436, as the putative interaction
Table 1
Ionworks patch clamp and H1 binding data for terfenadine analogsa
Entry Structure
Patch
H1
Binding Ki
Entry Structure
Patch
H1
Binding Ki
clampb (%)
clampb
HO
HO
OH
OH
1
96
99
85
40 nM
39 nM
61 nM
2
3
27 nM
N
N
N
CO2H
CH3
CH3
CH3
CH3
CH3
HO
HO
3
4
56
14
203 nM
659 nM
N
N
CH3
CO2H
HO
HO
5
6
NH
CH3
HO
HO
7
100
20 nM
8
11
71
97
NA
N
N
O
CO2CH3
CH3
HO
N
9
35
NA
10
3.2 lM
CH3
OH
CH3
HO
N
N
11
54
NA
12
121 nM
CH3
CH3
(continued on next page)

5046
R. Aslanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5043–5047
Table 1 (continued)
Entry Structure
Patch
H1
Binding Ki
Entry Structure
Patch
H1
Binding Ki
clampb (%)
clampb
HO
HO
13
100
16 nM
14
100
64 nM
CH3
N
N
N
CH3
HO
15
99
155nM
CH3
NA = not active.
a
Data are the results of at least two independent determinations.
% Inhibition @ 10 lM.
b
Table 2
In vitro patch clamp data and in vivo data in the guinea pig
Entry
Structure
Whole cell patch
QTc MED
(mg/kg)
Antihistamine
ED50 (mg/kg)
clamp (IC50
)
HO
OH
OH
1
2
3
4
5
6
312 nM
0.03
0.8
N
N
CH3
CH3
CH3
HO
>100
lM
NA@ 100 mg/kg
0.08
CO2H
CH3
CH3
HO
HO
HO
HO
396 nM
0.1
0.9
N
N
N
CH3
CO2H
CH3
11
lM
NA@ 50 mg/kg
0.1
>100
>100
l
M
M
N.T.
N.T.
N.T.
N.T.
O
l
CH3
NA = not active.
N.T. = not tested.
MED = minimum effective dose.

R. Aslanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5043–5047
5047
the amine either to a non-basic amide group or completely remov-
ing the basic center by substitution with carbon (entries 8 and 9).
Both analogs showed decreased hERG and H1 binding affinity.
These results were anticipated. It is well known for antihistamines
that a basic amine significantly contributes to H1 binding affinity
due to interaction with Asp116 in transmembrane domain III of
the histamine H1 receptor.13 Furthermore, significant evidence ex-
ists that a cationic binding site in the hERG channel, most probably
Tyr652,9 contributes to binding affinity of hERG blockers via a cat-
terfenadine carboxylate analogs (entries 3 and 4) paralleled the
activity of their counterparts. Interestingly, the carboxylic acid
analogs (Table 2, entries 2 and 4) both appear to be significantly
more potent antihistamines in vivo in the guinea pig than their
corresponding reduced analogs. The data in Table 2 also confirm
the importance of a basic nitrogen to hERG affinity (entries 5 and
6).
Based on a simplified structural model, a structure–activity pro-
file for hERG activity and H1 binding in a series of terfenadine ana-
logs was elucidated. Structural features that appear to contribute
to both hERG activity and H1 binding include a basic, tertiary
amine, a lipophilic tail incorporating either an aromatic moiety
or a cyclohexyl ring, and at least one aromatic ring approximately
6 Å from the basic nitrogen center. Overall, with the exception of
the carboxylic acid analogs, it appears that in this series of terfen-
adine analogs, hERG activity generally parallels the antihistamine
activity.
ion–p interaction, although a recent modeling study suggests that
Tyr652 may be too far away from the basic center in molecules that
block hERG to interact.9,10,14 These authors propose instead a polar
interaction with Ser624. These data do in fact show a decreased
affinity for the hERG channel for both analogs. Our data seem to
support the former hypothesis since the amide analog, which
may be able to maintain a polar interaction, but not a cationic
one, is weaker at the hERG channel than the parent.
Turning last to the lipophilic tail of terfenadine (region C), our
data indicates that a phenyl ring is not required for hERG affinity
since the cyclohexyl analog is also a potent hERG blocker (entry
5). This suggests that a hydrophobic interaction may be all that
is required for binding in this region of the pore, consistent with
some models.14 Moreover, an aromatic group in this region does
not appear to be necessary to H1 binding affinity, either. The cyclo-
hexyl analog is nearly equipotent as an H1 ligand compared to its
aromatic counterpart. Complete removal of the lipophilic side
chain does cause a loss of binding affinity for both the hERG chan-
nel and the H1 receptor (entry 6). This is qualitatively similar to re-
sults obtained with sertindole wherein removal of the sertindole
side chain decreased but did not abolish hERG affinity.10b The effect
of the spatial relationship of the terminal aromatic residue to the
central piperidine ring on hERG and H1 binding affinity was also
evaluated. hERG affinity remains consistent with that of the parent
regardless of the chain length as in examples 13, 14, and 15. How-
ever, the best H1 binding affinity appears to reside in the shorter
chain analog.
Interestingly, in contrast to the carboxylic acid, the methyl ester
(entry 7) shows surprisingly high hERG affinity, a result that is con-
sistent with sertindole analogs.10b This implies that a species that
is negatively charged at physiological pH may be necessary to abol-
ish hERG activity. Alternatively, the ester may not be able to adopt
the same conformation as the carboxylic acid does to decrease its
hERG activity.10b
Having identified the structural features of these terfenadine
analogs that contribute to hERG and H1 binding affinity, we next
looked to confirm these observations. Towards this, the whole cell
patch clamp assay (Ionworks) was used to determine hERG IC50
values in vitro and the guinea pig hERG model was used to confirm
hERG activity in vivo. Antihistaminic activity was confirmed in vivo
in the guinea pig histamine-induced bronchospasm assay. Table 2
presents the results for selected compounds.
Acknowledgements
The authors would like to thank the Analytical Chemistry group
of the Schering-Plough Research Institute for determination of the
configuration of compound 17 as well as for mass spectral
determinations.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.047.
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These data in Table 2 demonstrate that the in vitro data from
Table 1 is predictive of the cell-based and in vivo data, and also
help to demonstrate the validity of simplifying the analogs. For
example, terfenadine is a potent hERG blocker using the patch
clamp assay (IC50 = 312 nM) which was further confirmed in vivo
in the guinea pig (MED = 0.03 mg/kg). Additionally, as expected,
terfenadine was a potent antihistamine (ED50 = 0.8 mg/kg). Con-
versely, terfenadine carboxylate was inactive both in vitro and
in vivo against hERG but displayed excellent antihistaminic activ-
ity (ED50 = 0.08 mg/kg). Moreover, the simplified terfenadine and
14. Farid, R.; Day, T.; Friesner, R. A.; Pearlstein, R. A. Biorg. Med. Chem. 2006, 14,
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