Minimizing the contribution of enterohepatic recirculation to clearance in rat for the NCINI class of inhibitors of HIV

Article abstract of DOI:10.1021/ml500110j

A scaffold replacement approach was used to identifying the pyridine series of noncatalytic site integrase inhibitors. These molecules bind with higher affinity to a tetrameric form compared to a dimeric form of integrase. Optimization of the C6 and C4 po

Full text of DOI:10.1021/ml500110j

Letter
pubs.acs.org/acsmedchemlett
Minimizing the Contribution of Enterohepatic Recirculation to
Clearance in Rat for the NCINI Class of Inhibitors of HIV
Lee D. Fader,*,† Rebekah Carson, Sebastien Morin, Franco
̧
is Bilodeau, Catherine Chabot, Ted Halmos,
́
Murray D. Bailey, Stephen H. Kawai,‡ Rene Coulombe, Steven Laplante, Kevork Mekhssian, Araz Jakalian,
́
Michel Garneau, Jianmin Duan, Stephen W. Mason,§ Bruno Simoneau, Craig Fenwick, Youla Tsantrizos,∥
and Christiane Yoakim
́
Research and Development, Boehringer Ingelheim (Canada), Ltd., 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
S
* Supporting Information
ABSTRACT: A scaffold replacement approach was used to
identifying the pyridine series of noncatalytic site integrase
inhibitors. These molecules bind with higher affinity to a
tetrameric form compared to a dimeric form of integrase.
Optimization of the C6 and C4 positions revealed that viruses
harboring T124 or A124 amino acid substitutions are highly
susceptible to these inhibitors, but viruses having the N124
amino acid substitution are about 100-fold less susceptible.
Compound 20 had EC50 values <10 nM against viruses having
T124 or A124 substitutions in IN and >800 nM in viruses
having N124 substitions. Compound 20 had an excellent in
vitro ADME profile and demonstrated reduced contribution of biliary excretion to in vivo clearance compared to BI 224436, the
lead compound from the quinoline series of NCINIs.
KEYWORDS: NCINI, enterohepatic recirculation, biliary excretion, integrase tetramer
the carboxylic acid and the backbone amide protons of residues
ince the discovery of HIV in 1983, it is estimated that about
1
S36 million people have died from AIDS worldwide. There E170 and H171 of IN. It was found that this interaction was
critical to antiviral potency and that there was no tolerated
isosteric replacement for the acid. The presence of a carboxylic
acid can complicate the development of a lead series into a
marketed drug for a number of reasons.16,17 In the case of BI
224436, we found that the extremely low in vivo clearance of
this molecule in rat is attributable to enterohepatic recirculation
involving excretion of parent and the corresponding acyl
glucuronide into the bile, reentry into the gastrointestinal tract,
and reabsorption of parent back into the hepatic portal system.
This phenomenon was common to all members of the NCINI
class that were evaluated in rat pharmacokinetic experiments.
The potential variability of the contribution of enterohepatic
recirculation to in vivo clearance across species introduces
uncertainty in allometric scaling and prediction of human PK
parameters, which can complicate the development of drug
candidates.18 As BI 224436 progressed into clinical develop-
ment, our medicinal chemistry back-up effort focused on biliary
excretion as the key optimization parameter to mitigate the risk
of attrition of the front runner in clinical trials.
has been a continuous search for antiretroviral (ARV) agents to
combat HIV, which has led to the discovery of a number of
mechanistic classes of replication inhibitors. These include
nucleoside2 and non-nucleoside3 inhibitors of reverse tran-
scriptase (RT), inhibtors of HIV protease,4 strand transfer
inhibitors of integrase (IN),5,6 chemokine receptor 5 (CCR5)
antagonists, and HIV entry inhibitors,7 all of which have
marketed drugs available to patients. Additionally, a number of
investigational mechanisms of action have been identified,8
including noncatalytic site integrase inhibitors (NCINIs), first
disclosed in 2007.9 Recent work has shown that this class of
inhibitor binds to an allosteric pocket on the catalytic core
domain (CCD) of IN, shifting the oligomerization equilibrium
of IN toward the tetrameric state.10−12 This aberrant
multimerization negatively impacts viral maturation, which
leads to the NCINI antiviral effect.13,14
Recently we disclosed our discovery of the NCINIs,
culminating in the identification of BI 224436, the first
compound from this mechanistic class to advance into clinical
trials.15 Compound 1 exemplifies the structural features of the
prototypical quinolone series. The methyl substitution at C2
orients the C3 acetic acid group, favoring the bioactive form,
and C4 bears an aromatic core. We demonstrated the
importance of the C3 and C4 substituents in binding to IN
CCD, which included hydrogen bonding interactions between
One approach widely used by medicinal chemists to improve
the properties of a lead series is scaffold hopping.19 During the
Received: March 15, 2014
Accepted: April 16, 2014
Published: April 16, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
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course of lead optimization efforts, it was recognized that
replacement of the quinoline core of the NCINI scaffold
impacted properties other than potency. Herein we describe
our scaffold replacement effort that led to the discovery of the
pyridine series of NCINIs, whose salient feature is a reduced
contribution of enterohepatic recirculation to in vivo
clearance.20
During our investigation of B-ring substitution of compound
1, we synthesized compound 2 and noted that the additional
ring fused to the C7 and C8 positions was well tolerated in
antiviral assays, in spite of a 7-fold loss in binding affinity in our
displacement assay (Table 1).21 The antiviral assays used to
and 4 introduced different length linkers between the A- and C-
rings, and while compound 3, having the shorter linker, had
similar antiviral potency to compound 2, compound 4 was
about 10-fold less potent in terms of antiviral potency and had
6-fold less affinity for IN. We speculated that one difference
between these molecules might be the degree of coplanarity
between the A- and C-rings. Pyridine analogue 5 was made to
test this hypothesis since we believed the A- and C-rings would
be close to orthogonal. To our surprise, compound 5 was about
8-fold more potent than compound 3 in the antiviral assays
using viruses having the T124 IN substitution, in spite of having
similar binding affinity for IN. Equally surprising was the lack of
antiviral potency against viruses having the N124 substitution.
We later found that these were general trends with all pyridine-
based NCINIs. This prompted us to explore the biophysical
interaction of these inhibitor series with integrase and
modifications of the displacement assay. Isothermal titration
calorimetry and NMR revealed that the pyridine series of
inhibitors did not show significant binding to the dimeric CCD
form of integrase, while the quinoline series bound in a manner
that reflected the antiviral potency of the inhibitor. Both series
bound tightly to full length integrase at micromolar protein
concentrations. Chemical cross-linking experiments followed by
SDS-PAGE revealed that monomer and dimer forms of
integrase could be captured at the 100 nM His6-IN
concentrations used in the displacement assay. In contrast,
cross-linking studies with a thioredoxin (Trx)-tagged IN
construct also gave bands associated with trimer and tetramer
forms of integrase. This indicates that the Trx-tagged IN
construct exists to a greater extent in the higher order tetramer
form of integrase compared to the His6-IN construct under
assay conditions. When this construct was used in a
modification of the displacement assay, compound 5 bound
to IN with 25-fold more affinity than to His-IN. The level of
affinity was also similar in magnitude to antiviral potency
against viruses having the T124 substitution on IN, highlighting
the importance of the tetrameric form to the antiviral potency
of NCINIs.
Binding of the quinoline and pyridine NCINIs to Trx-tagged
IN was also compared using 19F NMR spectroscopy. To do
this, we titrated the same full length construct used in the new
displacement assay with fluoro analogues 6 or 7 (Figure 1).
Under these conditions, resonances for bound inhibitors are
extremely broad and are not observable, consistent with slow
binding on the NMR time scale to a very large biomolecular
complex. Thus, after the protein is saturated with ligand, the
resonance for free inhibitor will appear in the 19F NMR
spectrum as excess inhibitor is added. When this titration is
performed with compound 6, saturation was observed at a 1:1
ratio of IN to inhibitor, consistent with binding of the quinoline
NCINI to each available pocket on the IN CCD, in the context
of full length IN. In contrast, saturation was only observed at a
1:2 ratio of pyridine analogue 7 to full length IN. One possible
explanation for these results is that the pyridine NCINI’s bind
selectively to tetrameric IN, while quinoline NCINIs bind to
both dimeric and tetrameric IN forms.
Table 1. Scaffold Modification of NCINIs
Kd‑app
(nM)
Kd‑app
(nM)
EC50
EC50
EC50
(nM)
(nM)
(nM)
a
b
c
His-IN
Trx-His6-IN
TT
TA
NT
1
2
3
4
5
24
51
62
30
77
180
160
1100
1000
63
72
110
34
560
68
40
78
800
a
b
Determined with NL4.3 virus (T124/T125). Determined with
c
recombinant NL4.3 virus (T124/A125). Determined with recombi-
nant NL4.3 virus (N124/T125).
profile NCINIs utilized a panel of wild-type and recombinant
viruses that differ in the amino acid substitutions at positions
124 and 125 on IN since these residues are naturally
polymorphic in the HIV patient population.15 In our profiling
panel, we included six variants comprising an estimated 93% of
the potential patient population, according to an analysis of
1532 sequences from the Los Alamos database. Table 1 shows
data from three of these IN variants intended to exemplify
general trends. The displacement assay we used to profile
compounds measures apparent dissociation constants of
inhibitors from IN.22 Throughout the course of our effort to
optimize the quinoline series of NCINIs, the displacement
assay correlated with the antiviral assays (i.e., R2 = 0.80 for the
TT variant). On the basis of these results, we began modifying
the B ring of compound 2 by replacing the linker between the
A- and C-rings made up of C5 and C6 methines. Compounds 3
Having identified the pyridine scaffold as a possible
replacement for the quinoline core of compounds such as 1,
the process of optimizing potency and in vitro ADME
properties was initiated. Initial focus was placed on establishing
SAR around the C6 position with the expectation that much of
the SAR from the rest of the molecule would parallel what was
known from the quinoline series. Simple substitutions at the
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ACS Medicinal Chemistry Letters
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substitution at the meta-position led to improvements in
antiviral potency against the TT variant, with no appreciable
change against the NT variant, as exemplified by comparison of
compound 9 to compound 5. In general, gains made by
substitution at the meta- and para-positions could be combined,
although the gains were not completely additive (compare
increase from compound 10 to those of 8 and 9). Heterocyclic
rings were also explored at the meta- and para-positions. In the
case of the meta-ring biaryl, gains in antiviral potency against
the TT variant could be realized with introduction of 5-
membered heterocyclic rings (i.e., compounds 12 and 13), with
no appreciable change in potency against the NT variant. When
the para-biaryl motif was explored, excellent gains in potency
against the TT variant were accompanied for the first time by
gains in potency against the NT variant. It was noted that
introduction of the ortho-fluoro modification to the para-biaryl
motif had a modest, negative impact on antiviral potency
against the NT variant (cf. compounds 14 and 16). Taken
together, these results indicated that optimization of antiviral
potency is possible through structural changes at the R6
position. In addition to the difficulty encountered in improving
antiviral potency against the NT variant of IN, it was noted that
all of the pyridine compounds made to this point suffered from
modest to poor metabolic stability when incubated with human
liver microsomes. In our earlier work, we discovered that the
presence of a saturated ring on the C4 substituent was generally
met with poor metabolic stability. It was also discovered that
Figure 1. 19F NMR titration of Trx-His6-IN with inhibitors 6 and 7.
para-position generally led to improvements in antiviral
potency (Table 2). For example, the addition of a methyl
group to give compound 8 resulted in a 4-fold improvement in
antiviral potency against the virus harboring the TT variant of
IN, but no change in potency against the NT variant. Similarly,
Table 2. SAR at the R6 Position
a
b
c
Determined with NL4.3 virus (T124/T125). Determined with recombinant NL4.3 virus (N124/T125). Denotes n = 1.
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Table 3. SAR at the R4 Position
a
b
c
Determined with NL4.3 virus (T124/T125). Determined with recombinant NL4.3 virus (N124/T125). Denotes n = 1.
the introduction of polarity into the C4 substituent was one
successful strategy toward balancing antiviral potency with
good metabolic stability. To evaluate this approach in the
pyridine series, a range of C4 modified analogues of compound
10 were made (Table 3). Stable atropisomers 17 and 18 were
prepared and showed similar potency compared to compound
10, improved HLM stability, but decreased RLM stability. Our
goal was to identify compounds with balanced potency and
metabolic stability not only in human but also in rat
microsomal preparations in order to more accurately evaluate
the relative contribution of enterohepatic recirculation to in
vivo clearance. Although this strategy to improving metabolic
stability showed signs of being successful, it was also noted that
simplifying the C4 substituent showed similar promise. An
unanticipated result was that the simple 4-ClPh group at C4
gave compounds with similar virological profiles to analogues
with more complex C4 substituents (cf. compounds 10 and
19). We explored combinations of this simplified C4 group
with the SAR summarized in Table 2 and discovered
compound 20, a compound with excellent antiviral potency
against the TT variant and improved metabolic stability in
HLM and RLM assays.
contribution of enterohepatic recirculation to PK parameters
such as t1/2. In addition to having good metabolic stability in
the RLM assay, the compound showed good permeability in
the caco-2 assay, moderate to low inhibition of the CYP450
isozymes, acceptable logD7.4, and excellent solubility at pH 6.8.
The comparison of BI 224436 and compound 20 is shown in
Table 4 and shows that the in vitro ADME profiles are very
similar. The main difference between these two compounds is
their virological profiles, where viruses harboring all six variants
of IN are susceptible to BI 224436, while viruses harboring the
NT and NA variants of IN, which are estimated to be present in
about 12% of the patient population, are far less susceptible to
compound 20 than the TT, TA, AT, and AA variants of IN.
Additionally, BI 224436 has been optimized for low serum shift,
but serum shift remains as an optimization parameter for
compound 20. Compound 20 was evaluated in a rat PK study
and showed an in vivo half-life of 8.9 h, very similar to that
observed for BI 224436. However, in a bile duct cannulated rat
experiment 60% of BI 224436 was excreted into the bile, while
only 22% of compound 20 was excreted into the bile, as
measured at the 3 h time point. This translated into an in vivo
half-life in the bile duct cannulated rat of 1.6 h for BI 22436,
compared to a 5.4 h half-life for compound 20. These results
provided the proof of concept for our optimization strategy by
The in vitro ADME profile of compound 20 was supportive
of a further in vivo PK experiment in bile duct cannulated rat
allowing the assessment of the biliary excretion and
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ACS Medicinal Chemistry Letters
Letter
Table 4. Comparison of BI 224436 and 20
ACKNOWLEDGMENTS
■
We gratefully acknowledge the contribution of the following
colleagues: Celine Plouffe for Kd‑app determinations, Elizabeth
BI 224 436
20
a
́
EC50 range, nM
11−27
2.1
6.5−810
9.9
b
Wardrop and Sonia Tremblay for EC50 determinations, Hugo
Poirier for Caco-2 permeability data, Josie DeMarte for
microsomal stability data, Laibin Luo, Danhui Sun, and Eduard
Bugan for logD and solubility determinations. We thank
Michael Cordingley, Richard Bethell, and Paul Edwards for
guidance and support.
serum shift (50% HS), fold-change
HLM/RLM (t1/2), min
Caco-2 (Papp), ×106, cm/s
210/>300
14
230/160
17
CYP450 inh. (IC50, 3A4/2D6), μM
23/>30
0.44
>0.85
60
17/>30
3.1
logD7.4
c
solubility (pH = 6.8), mg/mL
0.25
22
% excreted into bile @ T3h
rat in vivo CL (%QH)
ABBREVIATIONS
0.7
0.6
■
rat in vivo t1/2 (h)
8.8
8.9
CCD, catalytic core domain; CyP450, cytochrome P450;
NCINI, noncatalytic site integrase inhibitor; HLM, human
liver microsomes; RLM, rat liver microsomes; PK, pharmaco-
kinetic
bile duct cannulated rat in vivo t1/2 (h)
1.6
5.4
a
Determined with HxB2 virus (A124/T125 IN variant), NL4.3 virus
(T124/T125), or recombinant NL4.3 virus (A124/T125, A124/A125,
N124/T125, or N124/A125 IN variants) as previously described.
b
Determined by measurement of EC50 values 50% human serum.
c
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AUTHOR INFORMATION
Corresponding Author
*(L.D.F.) E-mail: lee.fader@boehringer-ingelheim.com. Phone:
+1 (203) 791-6766.
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†(L.D.F.) Boehringer Ingelheim Pharmaceuticals, Inc., 900
Ridgebury Road, Ridgefield, Connecticut 06877, United States.
‡(S.H.K.) Department of Chemistry and Biochemistry,
Concordia University, 7141 Sherbrooke Street West, Montreal,
QC H4B 1R6, Canada.
§(S.W.M.) Virology, Bristol-Myers Squibb, 5 Research Parkway,
Wallingford, Connecticut 06492, United States.
∥(Y.T.) Department of Chemistry, McGill University, 801
Sherbrooke Street West, Montreal, QC H3A 0B8, Canada.
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The authors declare no competing financial interest.
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ACS Medicinal Chemistry Letters
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