Addressing hERG activity while maintaining favorable potency, selectivity and pharmacokinetic properties of PPARδ modulators

Article abstract of DOI:10.1016/j.bmcl.2019.126928

One of the most commonly used strategies to reduce hERG (human ether-a-go-go) activity in the drug candidates is introduction of a carboxylic acid group. During the optimization of PPARδ modulators, some of the compounds containing a carboxylic acid were found to inhibit the hERG channel in a patch clamp assay. By modifying the basicity of the imidazole core, potent and selective PPARδ modulators that do not inhibit hERG channel were identified. Some of the modulators have excellent pharmacokinetic profiles in mice.

Full text of DOI:10.1016/j.bmcl.2019.126928

Journal Pre-proofs
Addressing hERG activity while maintaining favorable potency, selectivity and
pharmacokinetic properties of PPARδ Modulators
Bharat Lagu, Ramesh S. Senaiar, Arthur F. Kluge, B. Mallesh, M. Ramakrishna,
Raveendra Bhat, Michael A. Patane
PII:
S0960-894X(19)30911-4
DOI:
https://doi.org/10.1016/j.bmcl.2019.126928
BMCL 126928
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 November 2019
16 December 2019
20 December 2019
Please cite this article as: Lagu, B., Senaiar, R.S., Kluge, A.F., Mallesh, B., Ramakrishna, M., Bhat, R., Patane,
M.A., Addressing hERG activity while maintaining favorable potency, selectivity and pharmacokinetic properties
of PPARδ Modulators, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126928
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Addressing hERG activity while maintaining favorable potency,
selectivity and pharmacokinetic properties of PPAR Modulators
Bharat Lagu,*a Ramesh S. Senaiar,bArthur F. Kluge,a B. Mallesh,b M. Ramakrishna,b Raveendra Bhat, b Michael A.
Patanea
aMitobridge, Inc. (a wholly owned subsidiary of Astellas Pharma.), 1030 Massachusetts Avenue, Cambridge MA 02138.
bAurigene Discovery Technologies, Ltd., Bengaluru and Hyderabad, India.
KEYWORDS PPAR modulators, hERG, patch clamp assay, cLogP, TPSA, pKa
ABSTRACT: One of the most commonly used strategies to reduce hERG (human ether-a-go-go) activity in
the drug candidates is incorporating a carboxylic acid group. During the optimization of PPAR modulators
some of the compounds containing a carboxylic acid were found to inhibit the hERG channel in a patch
clamp assay. By modifying the basicity of the imidazole core, potent and selective PPAR modulators that do
not inhibit the hERG channel were identified. Some of the modulators have excellent pharmacokinetic
profiles
in
mice.
We have recently disclosed a series (“benzamide series”) of PPAR modulators such as 1a-b that show good
selectivity for PPAR over PPAR and PPAR (Figure 1).1,2 By replacing the cis amide conformer found in the
x-ray structure of benzamides in the ligand binding domain of PPAR receptor, a second series (“imidazole
series”) of PPAR modulators was designed (Figure 1).3 The compounds in the imidazole series such as 2a were
found to be more potent and selective modulators of PPAR receptor. Modifications to the hexanoic acid moiety
in both series significantly improves plasma exposures after oral dosing as compared to their unsubstituted
counterparts.1,3 We have shown that MA-0204 (2c), a derivative in the imidazole series, may be an effective
therapeutic for Duchene Muscular Dystrophy (DMD).3
Benzamide series
Imidazole series
F3CO
O
O
Replace "cis-amide"
with imidazole
O
N
N
N
Me
N
N
Me
O
R
Me
Me
HO2C
O
HO2C
O
2a
2c MA-0204
( )
1
1a R = HO2C
PPAR EC50 = 0.6 nM
hERG IC50 = 5.5 M
PPAR EC50 = 0.4 nM
PPAR EC50 = 13 nM
hERG 12% inh @10M
1b R = HO2C
Me
PPAR EC50 = 37 nM
hERG 8% inh @10M

Figure 1.
Medicinal chemists routinely screen lead compounds for hERG channel binding in order to assess their
potential to cause QT prolongation, which could lead to Torsades de pointes arrythmias.4 Several drugs have been
withdrawn from market or given “black box” labels due to risks related to hERG channel-related QT
prolongation.5 Such measures are necessary if the concentration of drug required for its therapeutic activity nears
the concentration where the hERG channel is inhibited.6
In order to address this potential safety concern, medicinal chemists optimize lead compounds for
minimal hERG inhibition (typically IC50 >10 M), while maintaining or improving activity for the molecular
target (typically IC50 or EC50<100 nM). Although not an absolute requirement, a vast majority of the hERG active
molecules possess basic amines in their structures. In order to understand the interaction of molecules with the
channel, structural information regarding the hERG K-channel is used.7-8 It is known that hERG channel binding
pocket interactions occur via the hydrophobic central cavity and two amino acid residues Y652 and F656.9 The
interactions of the hERG inhibitors with the phenylalanine moiety are more hydrophobic in nature whereas the
interactions with the tyrosine moiety are hypothesized to be -cationic in nature.9,10 Therefore, modulating
lipophilicity of compounds (TPSA, LogD or LogP) or the basicity (pKa) of nitrogens in the molecules is a
commonly used tactic to reduce hERG activity4, 11-13 A carboxyl group in the structure of a molecule can help
lower the lipophilicity and minimize hydrophobic interactions. There are several examples in the literature where
a carboxylic acid moiety was introduced into a compound structure to reduce the hERG activity.14-15 In certain
cases, adding a carboxylic acid moiety can result in a zwitterionic compound.16 Zwitterionic molecules typically
have low permeability, which reduces the probability of hERG binding.11
When lead compounds in the benzamide series,1a and 1b, were screened for hERG activity in an
automated patch clamp assay, little or no activity (<15% inhibition at 10 M) was observed.17 Such results were
consistent with our expectations as 1a and 1b lack basic nitrogen atoms and possess a carboxylic acid. Therefore,
when imidazole 2a, was found to inhibit hERG channel activity (IC50 = 5 M), the result was unexpected
because: (1) the compound contains a carboxylic acid and (2) even if the imidazole nitrogen is basic, the
compound still would exist in a zwitterionic form.
In order to further understand whether the change in the shape of the molecule resulting from replacing
an amide group in 1a and 1b with an imidazole(2a) led to the hERG acivity, two thiazoles (3 and 4) and one
pyrazole (5) were synthesized and screened for hERG activity (Figure 2). All three compounds lack hERG
inhibitory activity (<15% inhibition at 10 M).
O
F
F3C
O
Me
N
N
F
N
N
Me
S
Me
S
Me
Me
HO2C
O
HO2C
O
HO2C
O
Me
Me
5
4
3
PPAR EC50 = 29.7 nM
hERG 14% inh @10M
PPAR EC50 = 11.2 nM
hERG 4% inh @10M
PPAR EC50 = 2.0 nM
hERG 4% inh @10M
Figure 2.
Based on these results, it is unlikely that the hERG activity observed for imidazole 2a results from the
three dimensional shape of the molecule. We assessed whether lipophilicity (as characterized by cLogP or TPSA
(topological polar surface area) impacts the hERG inhibition.11-12 Calculated logP for 2a (cLogP = 5.1)18 is lower
than those for 3, 4 and 5 (cLogP = 6.1-7.5). TPSA for all four compounds are between 71A⁰ and 78A⁰. Therefore,
lipophilicity parameters do not seem to correlate with the differences in the hERG activity of these compounds.
We then examined if the basicity of the nitrogen atoms in the heterocyclic rings could explain the difference in
the hERG inhibitory properties of these compounds. In order to test this hypothesis, imidazoles 2b-2l with either
electron withdrawing or electron donating groups on the imidazole ring or on the phenyl ring that is directly
attached to the imidazole ring (Table 1) were synthesized and tested. The imidazole compounds bearing a methyl
group on the imidazole ring, were synthesized using Scheme 1.1,3 For the synthesis of compounds 2e and 2i-j, the
2

central imidazole rings bearing a trifluoromethyl group were constructed via 2-(4-substituted-phenyl)-4-(2,2,2-
trifluoroacetyl)oxazol-5(4H)-one as shown in Scheme 2.19 Compounds with a trifluoromethyl (2f), a chloro (2h)
or a cyano (2g) substituent on the imidazole ring were synthesized via a common intermediate 23 as shown in
Scheme 3.19
Scheme 1. Synthesis of Compounds 2a-d
R1
R1
O
N
N
COOH
a
b
c
N
N
H
N
R1
R1
Me
8
MeO
Me
9
MeO
6
7
R1
R1
N
N
e
d
N
O
R3
N
O
R3
Me
O
Me
O
EtO
HO
10
2a-d
R1 = OCF3, CN,
R3 = H or Me
2c
was synthesized by the same route starting from 2-
O
6
methylbenzofuran-5-carboxylic acid instead of
Reagents and conditions: a) Prop-2-yn-1-amine, EDCI.HCl, HOBt, Et3N, DMF, RT, 12h; b) 2-Methoxybenzyl
amine, Zn(OTf)2, toluene, 110⁰C, 12h; c) BBr3, DCM, 0⁰C-RT, 4h; d) Ethyl (3R)-6-bromo-3-methylhexanoate or
ethyl-6-bromohexanoate, K2CO3, DMF, RT, 12h; e) LiOH.H2O, THF, EtOH, H2O, RT, 12h.
Scheme 2. Synthesis of Compounds 2e, 2i-j
R1
R1
R1
R1
O
a
b
c
d
O
O
N
O
O
O
N
O
N
COOH
11
H
H
F3C
OMe
OH
12
13
14
R1
R1
R1
R1
N
N
N
e
f
g
N
N
N
O
R3
CF3
CF3
O
NH
OH
OH
CF3
15
CF3
O
MeO
HO
EtO
16
17
18
R1
R1
N
N
h
i
N
N
Only when R1 = I
O
R3
O
R3
CF3
CF3
O
O
EtO
HO
19
2e, 2i-j
R3 = H or Me
R1 = OCF3, CN, Cl, I, 2-furyl
3

Reagents and conditions: a) Methyl glycinate hydrochloride, EDCI.HCl, HOBt, Et3N, DMF, 12h, RT; b)
LiOH.H2O, THF, EtOH, H2O, RT, 12h; c) 2,2,2-Trifluoroacetic anhydride, acetone, 0⁰C-RT, 12h; d) 1,4-
Dioxane, H2O, 100⁰C, 3h; e) 2-Methoxyl benzyl amine, AcOH, toluene, 120⁰C, 12h; f) BBr3, DCM, -78⁰C-RT;
g) Ethyl 6-bromohexanoate, K2CO3, DMF, RT, 12h; h) In the case where R1 = I, furan-2-boronic acid, Pd(PPh3)4,
Na2CO3, DME, EtOH, H2O, 90⁰C, 12h; i) LiOH.H2O, THF, EtOH, H2O, RT, 12h.
Scheme 3. Synthesis of 2f, 2g and 2h
F3C
N
N
CHO
N
a
b
c
N
N
N
H
H
F3C
F3C
F3C
MeO
20
21
22
23
F3C
F3C
F3C
N
I
N
N
d
e
N
N
N
O
Me
CF3
CF3
MeO
MeO
O
HO
2f
24
25
F3C
F3C
10% over
3 steps
N
N
f
24
N
N
O
Me
CN
MeO
CN
O
HO
2g
26
F3C
F3C
37% over
3 steps
N
N
g
23
N
N
O
Me
Cl
Cl
MeO
O
HO
2h
27
Reagents and conditions: a) Ethane-1,2-diamine, I2, K2CO3, t-BuOH, 85⁰C, 5h, 85% yield; b)
(Diacetoxyiodo)benzene, K2CO3, DMSO, RT, 12h, 55% yield; c) 2-Methoxybenzyl bromide, NaH (60%
dispersion), DMF 0⁰C-RT, 4h, 83% yield; d) NIS, DMF, 80⁰C, 12h, 36% yield; e) TMSCF3, Ag2CO3, 1,10-
phenanthroline, KF, CuI, DMF, 100⁰C, 12h, 59% yield; f) CuCN, Pd(PPh3)4, DMF, microwave, 150⁰C, 2h, 45%
yield; g) NCS, CH3CN, 70⁰C, 12h, 40% yield.
All the compounds in Table 1 show excellent PPAR activity (EC50 = 0.4 - 30 nM). The comparison of
hERG activity and cLogP or TPSA for a set of compounds (Table 1) revealed no correlation between the hERG
activity and physicochemical properties. However, a clear relationship was observed between nitrogen basicity in
the heteroaromatic ring and the hERG activity with an infliction point around pKa = 6.0 (Figure 3).20 Decreasing
electron-donation to the phenyl ring that is attached to the imidazole (2c) lowered hERG activity. Adding a
stronger electron withdrawing (cyano) group at the same position (2d), reduced hERG inhibition below 50% at 10
M. When electron withdrawing groups were placed directly on the imidazole ring (2e - 2l), hERG activity was
4

diminished substantially, for example, when comparing 2a (hERG IC50 = 5.5 M) to 2k and 2l (<10% inhibition
of hERG @ 10M)or 2c to 2e.
Table 1. pKa, cLogP, TPSA and PPAR activity for compounds 2a-2l and 3-5.
O
R1
R5
2a R3 = H, R4 = Me, R5
=
N
N
N
R3
O
N
R4
Me
Me
HO2C
O
2b R3 = H, R4 = Me, R5
=
R2
HO2C
O
O
2c-j, 2l
2a, 2b and 2k
2k R3 = Me, R4 = CF3, R5
=
Calculated parametersc
TPSA
hERG (EC50)b,
%inh@10M
PPAR
R1
R2
EC50 nMa
Cpd
pKa
2.8
1.8
2.8
6.3
6.3
6.3
6.3
4.7
4.7
3.4
4.7
4.7
4.7
4.7
ND
cLogP
6.7
6.1
7.5
5.1
5.1
5.8
4.2
5.9
5.6
4.5
5.7
4.3
5.4
5.2
ND
3
---
---
---
---
---
---
---
29.7±3.4
2.0±0.1
11.2±3.9
0.6±0.3
3.9±1.3
0.4±0.1
4.6
14
4
72.6
64.4
72.6
77.5
77.5
73.6
88.1
73.6
64.4
88.1
64.4
88.1
64.4
77.5
ND
4
5
---
4
2a
2b
2c
2d
2e
2f
2g
2h
2i
---
5.5 M
51
---
OCF3
CN
Me
Me
CF3
CF3
CN
Cl
10 M
15
OCF3
CF3
2.6 ± 1.1
2.9 ±0.2
2.7
2.0
5.7
2.5
3
CF3
CF3
1.5±0.8
8.2 ± 2.6
8.0±0.4
0.7±0.2
1.2±0.9
CN
CF3
CF3
CF3
---
4
2j
2k
2l
Cl
5.0
2.3
7.0
2-Furyl
---
aTransactivation assay24 EC50 values are an average of at least two experiments (SEM shown unless single determination).
% Activation of compound at each concentration was calculated considering activity of GW501516 at 10 uM as
100%. The Emax was between 81-103% except for compound 3 (Emax = 61%). Please see reference 3 or
b
c
WO2016057660 for the details of the assay system; See reference 17; For the calculation of cLogP, TPSA and pKa,
commercially available ACD software was used.18, 21; ND = Not Determined;
5

The observed differences in the hERG activity of imidazole 2a versus other heteroaromatic ring
containing compounds (3-5) tracks the differences in the basicity of nitrogens (pKa = 6.3 versus 1.8-2.8).
Compound 2b where the basicity of nitrogen is similar to the nitrogen in 2a, also has hERG activity (50%
inhibition at 10 M). With the increased basicity of nitrogen in the heteroaromatic ring, it’s more likely that
molecule exists in zwitterionic form, which could increase hERG activity. This may contradict some reports
where zwitterionic character was introduced in the molecules as a strategy to decrease hERG activity including
the well-known example of transforming terfenadine into zwitterionic fexofenadine with reduce hERG activity.21
However, there are a few publications where zwitterionic compounds that inhibit the hERG channel were
reported.22
pKa versus hERG inh@10 uM
80
70
60
50
40
30
20
10
0
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
pKa of N in heteroaromatic ring
Figure 3: Correlation between pKa and hERG activity for compounds in Table 1. The graph was generated in
MS Excel. The dotted line represented trendline generated by the software based on the data.
While reviewing the hERG activity of compounds, it’s important to consider many points, for example,
differences in assays. The data shown here was generated using an automated patch clamp assay. For an accurate
measurement of hERG activity, especially for compounds with low aqueous solubility, a manual patch clamp
assay should be performed.23 It is important to point out that most of the the imidazole compounds described in
Table 1 exhibit low aqueous solubility (thermodynamic solubility <25 mM). Compounds such as 2c could be a
viable clinical candidate because of the large window between the PPAR potency (EC50 = 0.4 nM) and the
observed hERG activity (IC50 = 10 M, in the automated patch clamp assay).3 Typically cardiac safety is then
measured in telemetrized animals before a compound enters clinical trials in human. Therefore, it was important
to assess oral bioavailability of these new compounds in addition to their profile for activating PPAR isoforms.
Potency, selectivity and the pharmacokinetic (PK) profiles for 2f, 2h and 2i are shown in Table 2.
All the compounds are potent PPAR modulators (EC50 <10 nM) and selective over PPAR and PPAR
receptors (EC50 >100,000 nM)) in transactivation assays.24 Compounds 2f, 2h and 2i show good oral
bioavailability (F = 70 - 100%) in mice, have low to moderate clearance (5-16 mL/min/kg) and reasonable
elimination half-lives (3.5-4.1 h). The observed PK profile in mice is better than that observed for 2c and some
previously reported imidazole compounds.3
Table 2. PPAR isoform selectivity and mouse PK data for compounds 2f, 2h and 2i.
Compound
EC50 PPAR nMa
EC50 PPAR nMa
EC50 PPAR nMa
AUC(0-inf) (ng*h/mL)b
2f
2h
2i
1.5±0.8
8.2 ± 2.6
2.9 ±0.2
>100,000
>100,000
6962
>100,000
>100,000
3881
>100,000
>100,000
14735
6

CL (mL/min/kg)b
t1/2 (h)b
5.0
5.8
70
16
3.5
100
2.5
4.1
73
%Fb
aTransactivation assay24 EC50 values are an average of at least two experiments (SEM shown unless single
determination); bExposure data for compounds dosed i.v. at 1 mg/kg and orally at 3 mg/kg in CD-1 mice25
In summary, we have demonstrated that the hERG activity of the imidazole PPAR modulators can be
attenuated by tuning the basicity of the nitrogen in the imidazole ring. We effectively decreased the hERG
activity while maintaining favorable PPAR potency, selectivity and oral bioavailability. This study serves as
another reminder for the medicinal chemists not to be overconfident that hERG activity can be ameliorated by
adding a carboxylic acid moiety to a structure.
AUTHOR INFORMATION
Corresponding Author
* Tel. 1(617)401-9122. E-mail: Bharat.lagu@astellas.com
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
ACKNOWLEDGMENT
Authors thank Drs. Takashi Ogiyama and Taisuke Takahashi for the calculation of pKa and logP. Authors also
thank Dr. Mahaboobi Jaleel for the PPAR data, Dr. Nirbhay K. Tiwari for the pharmacokinetic data, Mr. M. S.
Sudheer for the hERG data. We thank Mr. G. Styanarayana and Mr. Jeswin Jose for technical support.
ABBREVIATIONS
DMSO, Dimethyl Sulfoxide; BBr3, boron tribromide; DCM, dichloromethane; RT, room temperature; h, hour;
Pd(PPh3)4, Tetrakis(triphenylphosphine)palladium(0); Na2CO3, sodium carbonate; DME, 1,2-dimethoxyethane;
EtOH, ethyl alcohol; LiOH.H2O, lithium hydroxide monohydrate; THF, tetrahydrofuran; EDCI.HCl,
ethylcarbodiimide hydrochloride; HOBt, 1-hydroxy benzotriazole; Et3N, triethylamine; DMF, N,N-dimethyl
formamide; Zn(OTf)2, zinc trifluoromethanesulfonate.
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J. K.; Thornberry, N. A.; Weber, A. E. Discovery of potent and selective phenylalanine based dipeptidyl
peptidase IV inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 2533-2536.
16. Choi, Y. J.; Seo, J.; Shin, K. J. “Successful reduction of off-target hERG toxicity by structural
modification of a T-type calcium channel blocker” Bioorg. Med. Chem. Lett. 2014, 24, 880–883.
17. Semi-automatic patch clamp method was used using Port-A- Patch (from Nanion technologies,
Germany). HEK293 cells stably expressing hERG potassium channels. 0.1% DMSO in extracellular
HEPES buffer was used as the vehicle. Assay conditions were room temperature and the compound
addition was done manually. Aspirin at 10M concentration was used as a negative control. Cisapride or
verapamil at 1M concentration were used as positive controls.
18. TPSA
and
cLogP
were
calculated
using
ACD/ToxSuite
2.95
ACD/Labs.
(http://www.acdlabs.com/products/admet/tox).
19. The description of the synthesis and the spectroscopic data has been described in WO2017180818.
20. pKa were calculated using ACD/ToxSuite 2.95 ACD/Labs. Comparable pKa numbers were obtained
using Marvin suite (https://chemaxon.com/products/marvin).
21. Rampe, D.; Wibble, B.; Brown, A. M.; Dage, R. C. Effects of terfenadine and its metabolites on a
delayed rectifier K+ channel cloned from human heart. Mol. Pharmacol. 1993, 44, 1240-1245.
22. Nikolov, N. G.; Dybdahl, M.; Jónsdóttir, S. Ó.; Wedebye, E. B. “hERG blocking potential of acids and
zwitterions characterized by three thresholds for acidity, size and reactivity” Bioorg. Med. Chem. 2014,
22, 6004-6013.
23. Yajuan, X.; Xin, L.; Zhiyuan, L. “A Comparison of the Performance and Application Differences
between Manual and Automated Patch-Clamp Techniques” Current Chemical Genomics, 2012, 6, 87-92.
24. In the transactivation assay CV-1 cells are transfected with a PPAR ligand binding domain fused to a
GAL4 promoter to generate a hormone-inducible activator. A test ligand is added and activity is
measured in a luciferase assay. See WO2016057660 for further details.
25. All the animal experiments were carried out as per the guidelines of the Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA), Government of India and approved by
the Institutional Animal Ethics Committee (IAEC), Aurigene Discovery Technologies Ltd,, Bengaluru,
India.
8

O
R1
Tune basicity
of imidazole
N
N
N
Me
N
R2
HO2C
O
Me
HO2C
O
2
hERG IC50 >10
M

R
1 and/or R2 = electron withdrawing groups
PPAR EC50 = 0.6 nM
hERG IC50 = 5.5
M

Good oral bioavailability
O
R1
Tune basicity
of imidazole
N
N
N
Me
N
R2
HO2C
O
Me
HO2C
O
2
hERG IC50 >10
M

R
1 and/or R2 = electron withdrawing groups
PPAR EC50 = 0.6 nM
hERG IC50 = 5.5
M

Good oral bioavailability
9