Mitigating hERG inhibition: Design of orally bioavailable CCR5 antagonists as potent inhibitors of R5 HIV-1 replication

Article abstract of DOI:10.1021/ml2002604

A series of CCR5 antagonists representing the thiophene-3-yl-methyl ureas were designed that met the pharmacological criteria for HIV-1 inhibition and mitigated a human ether-a-go-go related gene (hERG) inhibition liability. Reducing lipophilicity was the

Full text of DOI:10.1021/ml2002604

Letter
pubs.acs.org/acsmedchemlett
Mitigating hERG Inhibition: Design of Orally Bioavailable CCR5
Antagonists as Potent Inhibitors of R5 HIV-1 Replication
Renato Skerlj,*,† Gary Bridger,‡ Yuanxi Zhou,‡ Elyse Bourque,† Ernest McEachern,‡ Sanjay Danthi,†
Jonathan Langille,‡ Curtis Harwig,‡ Duane Veale,‡ Bryon Carpenter,‡ Tuya Ba,‡ Michael Bey,‡ Ian Baird,‡
Trevor Wilson,‡ Markus Metz,† Ron MacFarland,‡ Renee Mosi,‡ Veronique Bodart,‡ Rebecca Wong,‡
Simon Fricker,† Dana Huskens,§ and Dominique Schols§
†Genzyme Corporation, 153 Second Avenue, Waltham, Massachusetts 02451, United States
‡Anormed Inc., 200-20353 64th Avenue, Langley, British Columbia, V2Y 1N5 Canada
§Rega Institute for Medical Research, Katholieke Universitett Leuven, Minderbroedersstraat 10, B-3000, Belgium
S
* Supporting Information
ABSTRACT: A series of CCR5 antagonists representing the thiophene-3-yl-methyl
ureas were designed that met the pharmacological criteria for HIV-1 inhibition and
mitigated a human ether-a-go-go related gene (hERG) inhibition liability. Reducing
lipophilicity was the main design criteria used to identify compounds that did not inhibit
the hERG channel, but subtle structural modifications were also important. Interestingly,
within this series, compounds with low hERG inhibition prolonged the action potential
duration (APD) in dog Purkinje fibers, suggesting a mixed effect on cardiac ion channels.
KEYWORDS: HIV-1, hERG, CCR5, chemokine receptor
he marketing approvals of the first in class chemokine
receptor antagonists, maraviroc1 in 2007 targeting CCR5
addition, we also utilized a gp120/CCR5 cell fusion assay10 as a
first pass screen to select compounds for antiviral screening
since the fusion data were found to be a reliable predictor of
antiviral activity.
T
and plerixafor2,3 in 2008 targeting CXCR4, mark an important
milestone for chemokine receptor drug development. Although
both of these receptors were identified as important
coreceptors required by the HIV-1 virus to infect cells,4 most
of the research effort has focused on developing inhibitors of
CCR5, since the most commonly transmitted HIV-1 strains
(M-tropic) utilize the CCR5 coreceptor.
On the basis of the recognition that compound 1a containing
an anilino nitrogen (R1 = phenyl) had potent antiviral activity,
we reasoned that the most expedient way to address the hERG
liability was to replace the substituted phenyl group on the left
had side with an amide or urea that would reduce the
lipophilicity11 as measured by cLog P. Initially, the 4-methyl
pyridine moiety on the top left-hand side was retained, but
these compounds were substantially less potent than the
corresponding thiophene analogues,5 so the thiophene moiety
was utilized for further optimization (Table 1). For example,
comparison of the tetrahydropyranyl esters 1b and 2b showed
that the thiophene 2b provided a 44-fold enhancement in
potency as compared to the 4-methyl pyridine 1b based on
CCR5 fusion inhibition. Similarly, comparison of the pyridinyl
ureas 1c and 2c showed a 130-fold increase in potency for 2c as
compared to the 4-methyl pyridine 1c.
Recently, we reported5 on the identification of pyridin-2-
ylmethylaminopiperidin-1-ylbutyl amides (1) as a novel class of
CCR5 antagonists that exhibited potent inhibition of R5 HIV-1
(BaL) replication in PBMC (Figure 1). These compounds were
Several amide analogues of compound 2 were prepared
(Table 2). Aside from maintaining antiviral potency, the
important properties that were measured to distinguish the
analogues was fusion inhibition shift in the presence of α-1-acid
glycoprotein (AGP), human plasma protein binding, and hERG
inhibition based on % inhibition and/or IC50. The simple
methyl amide 2d containing the optimum 2-chloro-4,6-
Figure 1. Structure of compounds 1 and 2.
identified based on our initial redesign efforts of Schering C.6
Although these compounds met most of the criteria for further
development, they were found to have a significant hERG
liability (also reported by the GSK7 and Pfizer8 groups), which
we sought to mitigate by reducing the lipophilicity through the
design of compounds 2 (Figure 1). The antiviral activity was
used to determine a structure−activity relationship (SAR), and
it was measured utilizing the R5 HIV-1 PBMC assay.9 In
Received: November 2, 2011
Accepted: January 25, 2012
Published: January 25, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Table 1. Comparative in Vitro Pharmacology and hERG Inhibition of 4-Methyl Pyridine and Thiophene Compounds
a
Assays were performed in duplicate, and values represent the mean with standard deviations <30% of the mean. Bracketed values represent the
b
number of experiments. Viral gp120/CCR5 binding was assessed by a cell fusion assay between P4R5 cells expressing CCR5, CD4, and a LTR-β-gal
construct (LacZ gene under the control of the HIV-1 LTR promoter) and CHO-tat cells expressing the HIV-1 JRFLenv and HIV-1 Tat. The P4R5/
CHO-tat, CCR5/gp120 interaction was assessed by measuring the resultant β-galactosidase activity. IC50 is the concentration of compound required
c
to inhibit cell fusion by 50%. Fold AGP fusion shift is the shift in the CCR5 fusion assay in the presence of 1 mg/mL of α-acid glycoprotein.
d
Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers and infected by R5 HIV-1 (BaL strain) as reported in ref 9. IC50
e
is the concentration of the compound required to inhibit viral replication by 50% as measured by p-24 HIV-1-specific Ag ELISA. CC50 is the
concentration required to reduce the viability of PBMC by 50%. Plasma protein binding was calculated by diluting a 25 mM stock solution in
f
DMSO to 1 μM in human plasma, dialyzing for 5 h against PBS (pH 7.4) at 37 °C, and determining the concentration remaining in each
g
compartment by LC. hERG inhibition was measured at 10 μM in CHO cells stably expressing the recombinant human hERG channel subunit (IKr)
using an automated patch clamp platform (QPatch-Sophion Biosciences).
dimethyl pyridine amide group5 demonstrated that activity
could still be maintained in the absence of a hydrophobic
aromatic group. Extending the amide using a methoxymethyl
group 2e provided an approximate 4-fold enhancement in
antiviral activity and a hERG IC50 of 8.4 μM. The cyanomethyl
moiety (2f) further reduced lipophilicity and resulted in a 2-
fold increase in potency and a hERG IC50 of 16.0 μM as
compared to 2e. Other amide modifications included
compounds 2b and 2g, h. Of these, the cyclic carbamate 2h
had the best overall profile with hERG inhibition of 21% and an
anti-HIV-1 activity of 1.6 nM. Interestingly, use of the cyano
pyridine amide 2g provided a 1 Log reduction in cLog P as
compared to 2b but only a marginal decrease in hERG
inhibition (61 vs 67%), suggesting that discrete structural
modifications also played an important role.12 However,
because many of these compounds showed evidence of
conformational isomerism around the amide bond, the amide
analogues were not further pursued.
As a consequence, we shifted our attention to preparing a
series of urea analogues (Table 2). It was encouraging to
observe that the methyl urea 2i was found to have good
potency, although the hERG IC50 of 1.6 μM (cLog P of 2.6)
suggested that even in this series other factors were impacting
hERG. Interestingly, the dimethyl analogue 2j was inactive,
indicating the importance of NH in binding to CCR5.
Incorporation of a hydroxyl urea 2k was tolerated based on
the CCR5 fusion assay, but this did not correlate to anti-HIV-1
activity (IC50 = 344 nM). However, the corresponding methoxy
urea analogue 2l provided encouraging in vitro properties
including a hERG IC50 of 5.5 μM and an anti-HIV-1 activity of
14.8 nM. Additional analogues incorporating the meythoxy
methyl urea moiety (2m−p) were prepared in which the right-
hand side pyridyl amide was modified in an attempt to further
improve the antiviral potency and hERG inhibition. An 8-fold
increase in potency (IC50 = 1.9 nM) was achieved using the 2,6-
dicholoro-4-methyl pyridine amide 2m (cLog P = 2.6), but this
was accompanied by a 20-fold increase in hERG inhibition
(IC50 = 0.3 μM). However, this liability was overcome by using
the corresponding N-oxide 2o that displayed a hERG inhibition
of 21% while still maintaining favorable in vitro properties. In
addition, using either an iso-propyl amide 2n or a cyano group
2p in place of the 2-chloro substituent of the pyridyl amide
improved the hERG inhibition with IC50 values of >30 and 10.8
μM, respectively, and anti-HIV-1 potencies of 3−5 nM. The
hERG liability could also be mitigated by modifications to the
left-hand side urea moiety. For instance, the methoxyethoxy
urea 2q hERG IC50 was >40 μM. On the basis of a combined
modeling mutagenesis approach13 where it was postulated that
addition of an appropriately tethered carboxylic acid to the urea
would pick up an ionic interaction with Lys191, compound 2r
was prepared in which antiviral potency was maintained at 6.9
nM, but more importantly, the hERG IC50 was >50 μM.
The thiophene compounds (Tables 1 and 2) were prepared
as outlined in Scheme 1 other than compound 2a.5 The
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ACS Medicinal Chemistry Letters
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a
Table 2. In Vitro Pharmacology and hERG Inhibition of Compounds 2
a
See Table 1.
substituted cyano pyridine 4a, used for the preparation of
compounds 2g and 2p, was prepared from commercially
available 2-chloro-4-methyl nicotinic acid by nucleophilic
addition of the cyano group to the corresponding N-oxide
3a. The advanced intermediate 8, prepared by reductive
amination of 3-aminomethyl thiophene 714 with ketone 6,
was used to prepare the analogues 2a−r. Nitrile reduction of
enantiomerically pure 55,15 under Raney-Ni-catalyzed hydro-
genation conditions followed by Boc protection of the amine
afforded 6 in good yield. Installation of the amide moiety was
readily accomplished using standard peptide coupling proce-
dures, whereas the urea moiety was installed using
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ACS Medicinal Chemistry Letters
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a
Scheme 1.
a
Reagents: (a) CO(NH2)2·H2O2, TFAA, 1,2-dichloroethane, 65 °C, 18 h, 39%. (b) (Me)2NCOCl, TMSCN, 1,2-dichloroethane/DMF (3:1), 50 °C,
18 h, 43%. (c) H2 (45 psi), Raney Ni, Boc2O, NaOH, MeOH, 1 h, 60%. (d) NaBH(OAc)3, CH2Cl2, 98%. (e) HOBt, EDCI, DIPEA, RCO2H, DMF,
74−89%. (f) RNCO, CH2Cl2, RT. (g) RNH2, CDI, Et3N, CH2Cl2 or R2NCOCl, Et3N, 1,2-dichloroethane, 60 °C. (h) TFA, CH2Cl2, RT, 100%. (i)
NaOH (10 N, aq.)/EtOH (1:3), 100 °C, 16 h, 75%. (j) HOBt, EDCI, DIPEA, iPrNH2, DMF, 40%.
commercially available isocyanates or amines. Boc deprotection
under mildly acidic conditions then afforded the compounds
2b−m and 2o−r in good overall yields. The iso-Pr amide 2n
was prepared by base hydrolysis of the precursor nitrile
followed by standard coupling procedures (Scheme 1).
The pharmacokinetic parameters of some compounds that
had an acceptable hERG profile were evaluated in rat and/or
dog (Table 3). Many of the ureas that were evaluated in dog
>10 μM. Compounds 2e, 2l, and 2q progressed to a 7 day
safety study in rat. The NOAEL for all three compounds was
determined to be at ≥400 mg/kg (the highest test dose).
In addition to 7 day safety dog studies (no telemetry studies
were conducted), these compounds and others were evaluated
in the canine Purkinje fiber assay16 to further understand the
risk for drug-induced arrhythmias (Table 4). Surprisingly, all of
the thiophene compounds tested at 10 μM using the three
standard stimulation frequencies resulted in significant
prolongation of the APD in a reverse rate-dependent fashion,
which is consistent with blocking the hERG potassium
channel.17 However, this observation was not supported by
the in vitro hERG inhibitory data. For example, compounds 2f,
2h, and 2q inhibited hERG at IC50 values of 16 to >40 μM, but
the % change in APD60 at a basic cycle length (BCL) of 2 s was
significant (18−43%). In addition, 2l and 2n also showed a
significant % change in APD60, 55 and 37%, respectively, with a
hERG IC50 of 5.5 and >30 μM. There was no occurrence of
early after depolarizations (EADs), and as the prolongation of
APD60 and APD90 was similar at every stimulation cycle, there
was no evidence of triangulation of the action potential. Given
this observed discrepancy between hERG channel inhibition
and APD, we postulated that these compounds were likely
having a mixed effect on cardiac ion channels. The lack of effect
on the resting membrane potential (RMP) or the rate of
depolarization (Vmax) suggests that sodium channels are not
inhibited at the concentrations tested. In addition to hERG
(IKr), there is significant contribution to phase III repolariza-
tion provided by the slowly activating delayed rectifier
potassium channel (IKs).18 Inhibition of this channel provides
a possible mechanism for the APD observed in this study;
however, none of these compounds were specifically tested for
inhibition of this channel.
Table 3. Pharmacokinetics of 2e, 2l, and 2n−p in Rat and
a
Dog
Cmax
AUC0‑inf
CL
V (L/ T1/2
F
(%)
compd species (μM) (h μM) (mL/min/kg)
kg)
(h)
2e
2l
rat
2.54
3.36
4.9
5.59
13.40
1.64
5.89
3.71
0.37
3.24
85.0
87.1
89.6
52.6
29.5
40.3
39.2
18.6
53.0
9.8
2.5
7.0
1.2
5.2
4.1
0.7
1.8
29
73
72
151
52
7
2e
2l
3.3
23.9
7.7
2n
2o
2p
dog
2.62
0.26
1.94
2.6
6.3
60
a
Clearance (CL), volume of distribution (V), and half-life (T1/2
)
calculated following a 10 μmol/kg iv dose in rat and 5 μmol/kg iv dose
in dog. Oral bioavailability (F) calculated following solution doses of
100 μmol/kg in rat and 12.5 μmol/kg in dog.
PK had good oral bioavailability and exposure other than the N-
oxide 2o. Compounds 2e and 2l had good oral bioavailability in
rat and dog and were selected for further evaluation as were
compounds 2n and 2q that had good dog PK.
The selectivity of 2e, 2l, 2n, 2q, and related analogues was
evaluated in Ca2+ flux assays against a series of other closely
related G-protein-coupled receptors (GPCRs), which included
CCR1, CCR2b, CCR4, CXCR1, CXCR2, and CXCR4, and
were found to be noninhibitory at concentrations of 5 μM.
When tested against a panel of five isoforms of CYP 450, these
compounds were found to be noninhibitory at concentrations
In conclusion, we have described our efforts to mitigate a
hERG liability present in a series of compounds by decreasing
lipophilicity. Although we were able to identify compounds
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ACS Medicinal Chemistry Letters
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a
Table 4. Dog Purkinje Fiber Data and hERG Data for Compounds 2f, 2h, 2l, 2n, and 2q
Δ%
ΔmV
b
compd
BCL (s)
APD60
APD90
RMP
APA
Vmax (Δ%)
hERG IC50 (μM)
2f
2
42.9 4.2*
31.5 1.9*
15.7 0.9*
18.4 8.3
14.5 8.4
7.7 7.1
47.6 7.5*
31.9 4.6*
17.7 2.0*
14.2 2.1*
10.6 2.1*
4.5 1.4
−1.5 1.3
−1.9 1.1
−0.9 1.6
−1.8 1.8
−2.2 2.9
−1.5 3.2
−0.6 0.7
−0.5 0.9
−1.3 0.5
−1.4 0.5
−1.9 0.6
−2.3 0.8
−2.7 3.1
−2.8 2.8
−3.6 3.2
4.3 3.1
3.4 2.3
2.9 1.5
13.7 8.2
15.2 7.9
11.3 6.4
−3.6 1.8
−3.8 2.3
−2.5 1.6
1.2 2.6
2.2 2.1
2.6 1.8
2.4 2.7
1.3 2.3
1.2 2.5
29.1 9.5
21.1 7.0
18.4 5.2*
57.1 42.5
81.7 56.9
48.4 27.4
−8.3 15.2
−8.0 15.6
−7.4 17.2
4.5 13.1
7.8 11.1
9.0 11.5
4.0 6.4
16
1
0.5
2
2h
2l
na
1
0.5
2
55.1 4.5*
42.6 5.6*
24.9 3.0*
37.1 11.2*
34.5 10.5*
24.8 5.7*
24.2 4.7*
18.1 2.7*
12.2 3.1
57.0 6.6*
42.2 5.5*
27.3 3.3*
34.2 5.9*
31.6 5.5*
24.2 3.4*
22.6 3.7*
16.5 2.7*
10.5 2.9*
5.5
33
1
0.5
2
2n
2q
1
0.5
2
>40
1
−4.0 5.4
−4.1 3.9
0.5
a
Action potential parameters are reported as means SDs percent change (Δ%) increase (+) or decrease (−) in test compound-exposed Purkinje
fibers over those measured in time-matched vehicle control. APD60 and APD90 = action potential duration at 60 and 90% repolarization, respectively;
RMP = resting membrane potential; APA = action potential amplitude; Vmax, maximum velocity of phase 0 depolarization; and BCL = basic cycle
b
length. Compounds tested at 10 μM.
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ASSOCIATED CONTENT
■
S
* Supporting Information
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Experimental procedures and characterization data for the
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This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: renato.skerlj@genzyme.com.
Notes
The authors declare no competing financial interest.
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