Aligning Potency and Pharmacokinetic Properties for Pyridine-Based NCINIs

Article abstract of DOI:10.1021/acsmedchemlett.6b00194

Optimization of pyridine-based noncatalytic site integrase inhibitors (NCINIs) based on compound 2 has led to the discovery of molecules capable of inhibiting virus harboring N124 variants of HIV integrase (IN) while maintaining minimal contribution of enterohepatic recirculation to clearance in rat. Structure-activity relationships at the C6 position established chemical space where the extent of enterohepatic recirculation in the rat is minimized. Desymmetrization of the C4 substituent allowed for potency optimization against virus having the N124 variant of integrase. Combination of these lessons led to the discovery of compound 20, having balanced serum-shifted antiviral potency and minimized excretion in to the biliary tract in rat, potentially representing a clinically viable starting point for a new treatment option for individuals infected with HIV.

Full text of DOI:10.1021/acsmedchemlett.6b00194

Letter
pubs.acs.org/acsmedchemlett
Aligning Potency and Pharmacokinetic Properties for Pyridine-Based
NCINIs
Lee D. Fader,*,† Murray Bailey, Eric Beaulieu, Franco
̧
is Bilodeau, Pierre Bonneau, Yves Bousquet,
Rebekah J. Carson, Catherine Chabot, Rene Coulombe, Jianmin Duan, Craig Fenwick, Michel Garneau,
́
Ted Halmos, Araz Jakalian, Clint James, Stephen H. Kawai, Serge Landry, Steven R. LaPlante,
Stephen W. Mason, Sebastien Morin, Nathalie Rioux, Bruno Simoneau, Simon Surprenant,
Bounkham Thavonekham, Carl Thibeault, Thao Trinh, Youla Tsantrizos, Jennifer Tsoung,
Christiane Yoakim, and Dominik Wernic
́
Research and Development, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
S
* Supporting Information
ABSTRACT: Optimization of pyridine-based noncatalytic
site integrase inhibitors (NCINIs) based on compound 2
has led to the discovery of molecules capable of inhibiting virus
harboring N124 variants of HIV integrase (IN) while
maintaining minimal contribution of enterohepatic recircula-
tion to clearance in rat. Structure−activity relationships at the
C6 position established chemical space where the extent of
enterohepatic recirculation in the rat is minimized. Desymmet-
rization of the C4 substituent allowed for potency optimization
against virus having the N124 variant of integrase. Combina-
tion of these lessons led to the discovery of compound 20, having balanced serum-shifted antiviral potency and minimized
excretion in to the biliary tract in rat, potentially representing a clinically viable starting point for a new treatment option for
individuals infected with HIV.
KEYWORDS: HIV integrase, NCINI, enterohepatic recirculation
ince the advent of combination antiretroviral therapy
on minimizing the contribution of enterohepatic recirculation
S(cART) for the treatment of HIV infected individuals, to in vivo clearance. This resulted in the pyridine series of
NCINIs exemplified by compound 2, a prototype molecule
exhibiting minimal excretion into the biliary tract but suffering a
lack of potency against the clinically relevant N124 variants of
HIV integrase (ca. 17% of patient population).5,6 Herein, we
describe our effort to optimize potency against the N124
variants of integrase while maintaining minimal contribution of
enterohepatic recirculation to in vivo clearance.
Ideally, evolution of the pyridine-based NCINIs would result
in a molecule with favorable alignment between serum-shifted
potency against the N124 variants of IN, metabolic stability,
and minimal contribution of enterohepatic recirculation to in
vivo clearance. Preliminary data using compound 2 as a
prototype molecule indicated that pyridine-based NCINIs
initially suffered from a two log shift in potency against N124
variants compared to A124 or T124 variants of IN.
Furthermore, compound 2 exhibited high serum shift, which
when combined with antiviral potency and human PK
prediction led to an unacceptable dose projection.7 Our
optimization strategy began with an evaluation of the impact
there has been a sustained effort to discover new mechanistic
treatment options for patients failing therapy due to emerging
resistance or poor tolerability to first line regimens. This wave
of innovation has seen the mechanistic options available to
patients expand beyond inhibitors of HIV protease or HIV
reverse transcriptase (NRTIs and NNRTIs) to include
inhibitors of viral entry (CCR5 antagonists, fusion inhibitors)1
and HIV integrase (strand transfer inhibitors).2 We have
previously reported on our progress toward a new HIV
treatment option resulting in the discovery of a new
mechanistic class that we have termed noncatalytic site
integrase inhibitors (NCINIs).3−5 The most advanced molecule
from this mechanistic class is BI 224436 (1), a potent inhibitor
of HIV replication that was advanced into a phase Ia clinical
trial. This molecule exhibited low apparent clearance in rat, in
part due to enterohepatic recirculation of both the parent
molecule and its corresponding acyl glucuronide (Table 1).
Potential species differences in the extent of the contribution of
enterohepatic recirculation to clearance introduced a large
degree of uncertainty in predicting human pharmacokinetic
parameters. Therefore, our medicinal chemistry strategy to
provide a second compound from the NCINI class of inhibitors
in order to mitigate the risk of attrition of BI 224436 focused
Received: May 11, 2016
Accepted: June 9, 2016
© XXXX American Chemical Society
A
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Table 1. Profiles of Compounds 1 and 2
Table 2. Preliminary SAR at the C6-Position
1
2
a
EC50 (nM)
T124
N124
19
7.7
810
22
b
EC50 (nM)
30
% excreted into bile @ T3h
rat in vivo CL (%QH)
rat in vivo t1/2 (h)
60
0.7
8.8
1.6
0.6
8.9
5.4
bile duct cannulated rat in vivo t1/2 (h)
a
b
NL4.3 virus (T124/T125). NL4.3 virus (N124/T125).
of a range of C6-substitutions on these parameters.8 At the
outset of this effort, most potent analogues, such as compounds
2 and 3 use the phenyl group at C6 to project a substituent (an
isoxazole in these cases) in a conformationally restricted way.
We hypothesized that varying conformational flexibility of this
linker may have an impact on the potency against virus
harboring the N124 variant of IN. Introduction of five-
membered heterocycles to give analogues such as compound 4
or an amide linker to give compound 5 gave inhibitors with
similar potency (cf. compounds 3−5, Table 2), although serum
shifts remained unacceptably high. An early investigation of the
amide N-substituent revealed that aliphatic groups were very
well tolerated and often led to improvements in potency, such
as was the case for trans-4-methylcyclohex-1-yl analogue 6.
When the symmetrical chlorophenyl C4-substituent replaced
the chromane, potency and serum shift were maintained at
similar levels (cf. compounds 6 and 7), but metabolic stability
improved. Based on these findings, the structure−activity
relationships (SARs) were expanded to include a large diversity
of amide analogues. Through this effort, numerous examples of
aliphatic amides that retain the general potency profile of
compound 7 were identified, but independent SARs around
serum shifted potency also emerged. In many cases, serum shift
was reduced to a very low level, as exemplified by compound 8.
This effort also revealed that polarity within the aliphatic
substituent was not tolerated, as demonstrated by the alcohol
and amine analogues 9 and 10, respectively. Anilide based
inhibitors were also explored, and many examples with very
similar potency profiles to the aliphatic amides (cf. compounds
7, 11, and 12) were found. Interestingly, the reversed amide 13
was well tolerated, exhibiting a profile very similar to compound
12. Cyclization of the amide found in compound 11 gave
benzimidazole 14, which showed excellent potency against virus
having the T124 variant of IN, although serum shift for this
compound was found to be somewhat high. Substitution of the
N1 atom of the benzimidazole had a marginal impact on
antiviral potency, but generally provided analogues with lower
serum shift values. Taken together, these results indicated that
variation of the C6 position could not improve potency against
the N124 IN containing virus to an appreciable extent, but
serum shift could be improved in the context of both the C6-
amide and C6-benzimidazole subseries.
a
b
c
NL4.3 virus (T124/T125). NL4.3 virus (N124/T125). Deter-
mined by measurement of EC50 values 50% human serum.
Based on the existing X-ray crystallographic information
available at the time,3 the substituent at the C6-position was
assumed to be in close proximity to residue 124.9 Due to the
B
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Letter
significant differences between the threonine and asparagine
side chains, we hypothesized that the shift in potency between
viruses with the N124 or T124 variants of IN maybe due to
inferior affinity of the inhibitors for the N124 variant.10 In an
attempt to increase binding affinity, the C6-substituents with
the most favorable properties were selected and held constant
for an attempt to optimize the C4-substituent. A similar
strategy with quinoline based inhibitors had been successful in
improving potency across multiple IN variants, and the key
tactic employed in that effort was desymmetrization of the C4-
arene. Utilizing the C4-substituent of compound 1, which was
highly optimized for potency across all major variants of
integrase, and a promising C6-amide such as that found in
compound 6 gave the stable atropisomer 16,11 which exhibited
good potency against the T124 variant but was inactive up to a
concentration of 2.5 μM against the N124 variant. However,
morpholine analogue 17, which exhibits free rotation about the
C4-axis, regained potency against the N124 variant containing
virus and exhibited good serum shift and excellent metabolic
stability. Relocking the conformation about the C4-axis by
addition of a methyl group at the ortho position of the C4-
arene gave compound 18, which showed a slight improvement
in serum-shifted antiviral potency accompanied by some loss in
metabolic stability. Methylation of the morpholine nitrogen of
compounds 17 and 18 produced compounds 19 and 20,
respectively, which regained excellent metabolic stability and
maintained good serum-shifted antiviral potency. Similar SAR
was observed in the reversed amide subseries (e.g. compounds
21 and 22). With benzimidazoles installed at C6 (compounds
23−25), these SAR trends were also observed with antiviral
potency against N124 containing virus breaching the 100 nM
level, although metabolic stability was moderately compro-
mised.
Having established that antiviral potency against N124 IN
containing virus could be optimized, a range of structurally
diverse analogues were evaluated in bile-duct cannulated rat
pharmacokinetic experiments. For these studies, compounds
with aligned potency and stability in human and rat liver
microsome assays were selected. We evaluated the plasma
exposure and the percent of parent excreted into the biliary
tract as the acyl glucuronide at 3 h post i.v. dose.12 For
promising compounds, full pharmacokinetic profiles were
determined. For these compounds, the results comparing the
extent to which the acyl glucuronide of parent was excreted into
the biliary tract to the ratio of terminal half-life in rat to bile
duct-cannulated rat are presented in Figure 1. In general, a
trend toward a ratio of 1 for half-life in rat to half-life in bile
duct cannulated rat as the extent of acyl glucuronide excreted
into the biliary tract decreases is observed. For compounds with
a ratio near unity, a clearance in vitro−in vivo correlation was
observed, confirming the generality of this observation for the
pyridine series.
Figure 1. Ratio of i.v. t1/2 (rat) and i.v. t1/2 (bile duct cannulated rat) as
a function of %parent excreted as the acyl glucuronide into the biliary
tract.
the expected range for a carboxylic acid. However, while
compound 20 showed very low total excretion into the biliary
tract, compound 23 was highly excreted indicating the low
clearance of this compound likely has a significant contribution
from enterohepatic recirculation. This was a general observa-
tion for the C6-benzimidazole derivatives, and so, inhibitors
related to compound 23 were deprioritized.
The profiling of compound 20 was expanded to include its
effectiveness against infection of peripheral blood mononuclear
cells with viruses harboring all of the major variants of IN. As
illustrated in Table 5, compound 20 showed excellent potency
against viruses with T124 and A124 variants of IN and EC50
values of 27 and 42 nM against the N124/T125 and N124/
A125 strains, respectively.13 No detectable inhibition of the
cytochrome P450 enzymes was observed, indicating a low risk
for P450-mediated drug−drug interactions. The half-lives for
compound 20 were similar for intact and bile-duct cannulated
rats, consistent with results presented in Table 4, in view of the
data summarized by Figure 1. Metabolic stability in incubations
with dog and monkey hepatocytes indicated low to moderate
clearance across species. As with rat, these observations
translated to low to moderate clearance in vivo. Taken together,
the data presented for compound 20 indicates that favorable
alignment of serum shifted EC50s and a pharmacokinetic profile
that includes minimized excretion into the biliary tract can be
achieved for pyridine-based NCINIs.
In conclusion, the continued optimization of pyridine-based
NCINIs has led to the discovery of molecules capable of
inhibiting virus harboring N124 variants of IN while
maintaining minimal contribution of enterohepatic recircula-
tion to clearance in rat, which may have implications on the
development of the NCINI class for therapeutic use in treating
HIV infected individuals. Following the original disclosure of
C6-phenyl and C6-benzimidazole modified NCINIs in 2010,8
the Kvaratskhelia group selected resistance mutants of HIV
bearing the T124N amino acid substitution under selection
pressure from a close analogue of compound 14.9 Given that
variants of HIV having the N124 substitution are naturally
occurring in the patient population, an appropriate level of
potency against these viruses is critical to the use of pyridine-
based NCINIs in any new treatment regimen. Compounds 20
and 23 demonstrate that it is possible to significantly inhibit
these viruses with pyridine-based NCINIs, providing evidence
Based on their preliminary antiviral profiles, compounds 20
and 23 were characterized in an expanded battery of in vitro and
in vivo experiments. Both compounds exhibit excellent antiviral
potency against virus with T124 variants of IN and reasonable
serum shift values (Table 3). Further profiling revealed these
two compounds showed favorable permeability in the Caco-2
assay and excellent aqueous solubility (Table 4). Metabolic
stability was assessed in human and rat hepatocyte incubations,
and generally low levels of hepatic clearance were observed.
Both compounds showed similarly low clearance in rat
pharmacokinetic experiments with volumes of distribution in
C
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Table 3. C4- and C6-Position Combinations
Table 5. Profile of Compound 20
EC50 (nM)
p24 ELISA
IN Variant
T124/T125
T124/A125
A124/T125
A124/A125
N124/T125
N124/A125
3A4
0.5
0.4
0.6
0.9
27
42
CYP450 IC50 (μM)
>30
>30
>30
>30
>30
1.3
31
2D6
1A2
2C19
2C9
rat t1/2/BDC rat t1/2
dog hepatocytes (%QH)
monkey hepatocytes (%QH)
dog i.v. Cl (%QH)
31
10
monkey i.v. Cl (%QH)
30
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00194.
■
S
Summary of synthetic approaches, characterization data
for key compounds, correlation of apparent Kd values
and EC50 values, comparison between the energy barriers
for free rotation of the quinoline and pyridine C4-biaryl
bonds, and summary of screening results in bile duct
cannulated and intact rat PK experiments (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: lee.fader@boehringer-ingelheim.com. Phone: +1
(203) 791-6766.
■
Present Address
†Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury
Road, Ridgefield, Connecticut 06877, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Sonia Tremblay and Elizabeth Wardrop for
■
EC50 determinations; Cel
́
ine Plouffe for Kd‑app determinations;
Hugo Poirier for Caco-2 permeability data; Josie DeMarte for
microsomal stability data; Federico Columbo for hepatocyte
stability determinations; Laibin Luo, Danhui Sun, and Eduard
Bugan for logD and solubility determinations; Edith Bellavance
and Marieve Dupuis for pharmacokinetic experiments; and
Michael Cordingley, Richard Bethell, and Paul Edwards for
their leadership and support.
a
b
c
NL4.3 virus (T124/T125). NL4.3 virus (N124/T125). Deter-
mined by measurement of EC50 values 50% human serum.
that, with further optimization, this class of replication inhibitor
may represent a clinically viable treatment option for
individuals infected with HIV.
Table 4. Selected in Vitro ADME and Rat Pharmacokinetic Data for Compounds 20 and 23
a
solubility pH = 6.8
rat Vss
(L/kg)
total excretion into bile
(%)
Caco-2 Papp × 10−6 cm/s
mg/mL
HHEP (%QH) RHEP (%QH) rat i.v. Cl (%QH)
20
23
39.8
10.6
0.93
>1
17
24
7
23
24
1.4
1.6
20
70
19
a
Sum of acylglucuronide and parent excreted into bile.
D
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(13) For in depth antiviral potency evaluation, EC50 values were
determined by ELISA formatted viral replication assay that quantifies
the viral protein p24. For example, see Sturino, C. F.; Bousquet, Y.;
James, C. A.; DeRoy, P.; Duplessis, M.; Edwards, P. J.; Halmos, T.;
Minville, J.; Morency, L.; Morin, S.; Thavonekham, B.; Tremblay, M.;
Duan, J.; Ribadeneira, M.; Garneau, M.; Pelletier, A.; Tremblay, S.;
Lamorte, L.; Bethell, R.; Cordingley, M. G.; Rajotte, D.; Simoneau, B.
Identification of potent and orally bioavailable nucleotide competing
reverse transcriptase inhibitors: in vitro and in vivo optimization of a
series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors. Bioorg.
Med. Chem. Lett. 2013, 23, 3967−75.
ABBREVIATIONS
■
HLM, human liver microsomes; HHEP, human hepatocyte;
RHEP, rat hepotaocyte; CYP450, cytochrome P450
REFERENCES
■
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(6) All EC50 values were determined by luciferase reporter gene assay
as previously described and are the average of at least two experiments.
See refs 4 and 5.
(7) For treatment of patients infected with virus harboring the T124
variant of IN with compound 2, the efficacious exposure at Cmin can be
estimated to be 30 × serum-shifted EC50 = 2.2 μM. Human PK can be
predicted in the following way: Cl = human hepatocyte stability; F(%)
= 100 − Cl(%QH); Vd = cross-species average (rat, dog, cyno);
assume ka = 1. Based on these assumptions, the dose required to cover
Cmin = 2.2 μM for 24 h = 31 g.
(8) All compounds presented in this manuscript were prepared as
previously described. See: Yoakim, C.; Bailey, M. D.; Bilodeau, F.;
Carson, R. J.; Fader, L.; Kawai, S.; Laplante, S.; Simoneau, B.;
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(10) Throughout the course of this work, affinity was approximated
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derived apparent Kd values correlated with EC50 values. See
Supporting Information.
(11) For a comparison between the energy barriers for free rotation
of the quinoline and pyridine C4-biaryl bonds, see Supporting
Information.
(12) See Supporting Information for a summary of screening results.
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DOI: 10.1021/acsmedchemlett.6b00194
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