Imidazo[1,2-a]pyridines that directly interact with hepatitis C NS4B: Initial preclinical characterization

Article abstract of DOI:10.1021/ml300090x

A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC₅₀ < 10 nM), direct binding to purified NS4B protein (IC

Full text of DOI:10.1021/ml300090x

Letter
pubs.acs.org/acsmedchemlett
Imidazo[1,2-a]pyridines That Directly Interact with Hepatitis C NS4B:
Initial Preclinical Characterization
J. Brad Shotwell,*,† Subramanian Baskaran,† Pek Chong,† Katrina L. Creech,‡ Renae M. Crosby,†
Hamilton Dickson,† Jing Fang,† Dulce Garrido,† Amanda Mathis,† Jack Maung,§ Derek J. Parks,‡
Jeffrey J. Pouliot,† Daniel J. Price,‡ Roopa Rai,∥ John W. Seal, III,‡ Uli Schmitz,⊥ Vincent W. F. Tai,†
Michael Thomson,† Mi Xie,† Zhiping Z. Xiong,† and Andrew J. Peat†
†GlaxoSmithKline, Antiviral Discovery Performance Unit, 5 Moore Drive, Research Triangle Park, North Carolina 27709-3398,
United States
‡GlaxoSmithKline, Platform Technology and Science, 5 Moore Drive, Research Triangle Park, North Carolina 27709-3398,
United States
S
* Supporting Information
ABSTRACT: A series of imidazo[1,2-a]pyridines which directly
bind to HCV Non-Structural Protein 4B (NS4B) is described. This
series demonstrates potent in vitro inhibition of HCV replication
(EC50 < 10 nM), direct binding to purified NS4B protein (IC50
<
20 nM), and an HCV resistance pattern associated with NS4B (H94N/
R, V105L/M, F98L) that are unique among reported HCV clinical
assets, suggestive of the potential for additive or synergistic com-
bination with other small molecule inhibitors of HCV replication.
KEYWORDS: NS4B, hepatitis C virus, imidazo[1,2-a]pyridines, replicon
epatitis C virus (HCV) is a serious worldwide concern,
H
infecting an estimated 170 million people (3% of the
global population), including 4 million people in the United
States.1,2 HCV leads to chronic liver disease, cirrhosis, and
hepatocellular carcinoma, and it remains the most common
indication for liver transplantation in developed countries.
Between 8,000 and 10,000 deaths per year in the United States
are attributed to complications of chronic HCV infection.1,2 The
long-time standard of care, a combination of injected interferon
and oral ribavirin, is poorly tolerated, contraindicated in a
significant portion of the HCV-infected patient population, and
affords a sustained virologic response in only half of those
treated.1,2 The addition of one of the two recently approved
protease inhibitors to the current standard of care significantly
improves the overall cure rate; however, tolerability and drug−
drug interactions remain a key concern.3,4
Identifying an oral drug cocktail that would both obviate the
need for injected interferon and lead to sustained virologic
responses over 90% remains the ultimate goal of most clinical
development plans. The majority of clinical candidates currently
undergoing investigation lead to HCV resistance arising in viral
proteins, including well-characterized clinical mutations in the
HCV protease, NS5A, and the HCV RNA-dependent RNA
polymerase (NS5B).5,6 This article discloses the initial lead
optimization of a series of potent HCV replication inhibitors
structurally related to anguizole (1e),7,8 which is reported to
directly bind to isolated HCV NS4B protein (Figure 1).8,9 These
small molecules appear to act via a novel mechanism of action,
which likely does not suffer from clinical cross-resistance to
Figure 1. Tritiated NS4B probes and anguizole.
known NS5B, NS5A, or protease inhibitors currently approved
or undergoing clinical trials and represents a potential option for
combination therapy.
Received: April 11, 2012
Accepted: May 24, 2012
Published: May 24, 2012
© 2012 American Chemical Society
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a
Imidazo[1,2-a]pyridines 1a−b are potent inhibitors of HCV in
the in vitro replicon10 system, with measured EC50s for wild type
(WT) genotype 1a and 1b replicons of <55 nM. Both 1a and 1b
were recently disclosed in conjunction with other novel chemical
matter optimized from a replicon-based HTS screen, with 1b
having been identified following extensive optimization of
1a.11−15 Resistance passaging for compounds within this series
(including 21 and anguizole7) revealed that key mutants arise in
the HCV NS4B protein, specifically H94N/R, F98L, and
V105L/M (genotype 1b). Imidazo[1,2-a]pyridines 1a−b show
significant shifts in EC50s for replicons bearing these mutations
(>20×). Titration of 1c with purified HCV NS4B protein affords
a Kapp of 76 nM (data not shown) while an identical experiment
with 1d affords a Kapp of 30 nM (Figure 2). Displacement of 1d
Scheme 1. Synthesis of Core and Elaboration
a
Conditions: (i) α-bromopyruvate, DMF, 50 °C (83%);11 (ii) NCS,
DMF, 50 °C, 1 h, (91%);11 (iii) 3-furanylboronic acid, K3PO4, MeCN,
Pd(dppf)Cl2, (82%);12 (iv) amine, HATU, DIPEA, DMF (20−
85%).11−15
followed by selective chlorination with NCS and tandem
Suzuki cross-coupling/hydrolysis, affords furan 4. Subsequent
HATU mediated amide formation affords novel compounds
5−20. We found that a 3-substituted furan at C(5) and chlorine
for bromine replacement at C(3) were well-tolerated relative to 1
( 6, Table 1), and we chose to focus first on the optimization of
the amide moiety in the context of C(3) chloro and C(5) 3-furyl
substituents.
Figure 2. Equilibirum binding of 1d for isolated His-Tagged NS4B. Kd =
A range of 3-aryl (5) and 3-heteroaryl (6, 7) substituted
pyrrolidines were well tolerated, giving submicromolar inhibitors
of the HCV WT1a and WT1b replicons with retained ability to
displace radioligand 1d from purified NS4B (Table 1). Ring
expansion to the corresponding piperidine (i.e., 6 → 8) gave
similarly equipotent HCV replication inhibitor 8.
30 nM (see Supporting Information).
with 1b affords a measured IC50 = 30 nM (Figure 3) as expected,
and direct displacement of 1d by anguizole (1e) affords a
Eliminating the chiral center proved most efficient for rapid
assessment of novel amides. As such, we chose initially to further
pursue the piperidine subseries. Basic pyrrolidine (9) exibited no
HCV inhibition or NS4B binding, while incorporation of the
corresponding cyclic amide (10) rescued both to a significant
extent. Replacing the cyclic amide with the corresponding oxa-
zolidinone or oxazolidine dione afforded 11 and 12, respectively.
Oxazolidinone 11 was 5−10 times more potent than the
corresponding amide (10). Direct substitution of the oxazolidi-
none ring was not well tolerated (see 13 and 14). In fact, the size,
substitution, and electronics of the oxazolidinone ring appeared
critical, as dozens of ring-opened and ring-substituted oxa-
zolidinone analogues showed a much reduced ability to inhibit
HCV replication in vitro and/or to bind directly to purified
NS4B.11−15
With potent oxazolidinone 11 and oxazolidine dione 12 in
hand, we examined standard DMPK properties, including P450
inhibition, reactive metabolite potential, and in vivo rat PK. We
found 11 had no significant P450 liabilities or reactive metabolite
potential in a glutathione trapping assay, but exhibited rapid
in vivo clearance in rat (Table 2). Importantly, high circulating
levels of 12 were observed following IV or PO dosing of 11.
Although dione 12 represented a marginal improvement in
in vivo clearance, potency, and overall exposure relative to 11, it
was positive in our assessments of reactive metabolite. With these
data in hand, we set out to identify a suitable oxazolidinone
replacement, which could not be oxidized in vivo to reactive
oxazolidine diones such as 12.
Figure 3. IC50 determination for 1b via 1d displacement from purified
NS4B.
measured IC50 = 22 nM. The affinities of additional analogues for
purified NS4B protein (see Supporting Information) are re-
ported using the direct displacement of 1d (see NS4B binding in
subsequent tables16). These data are consistent with a direct
interaction between small molecules within this class and the
HCV NS4B protein.17
The development of initial lead molecule 1a was complicated
by apparent thiophene-related, rapid in vitro/in vivo metabolism
and irreversible trapping of glutathione in in vitro reactive
metabolite assays. We set out to systematically optimize the
DMPK and potency of 1a.
Versatile imidazo[1,2-a]pyridine carboxylic acid 4 could be
prepared in four steps from commercially available 2-amino
pyridine 2 (Scheme 1). Alkylation/cyclization with α-bromopyruvate,
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a
a
Table 1. Inhibition of HCV RNA in the Replicon System
Table 2. Rat DMPK Profile for 11 and 12
11
12
46
clearance
Vdss
72
2.5
3.1
PO T1/2
AUC0−24
%F
1.9
3
700
60%
1100
60%
a
Dosed as a solution in DMSO/4% HPβCD in acetic acid (5:95); IV
dosed 1 mg/kg; PO dosed 5 mg/kg; clearance (mL/min/kg); Vdss
(L/kg); T1/2 (h), AUC (ng·h/mL). All n = 3 animals.
Recognizing that the carbonyl moiety was an important phar-
macophore (i.e., 9 → 10, Table 1), we envisioned a number of
modifications which might position this critical group in similar
space to 10. Methyl piperazinone 15 exhibited a 1.2 μM IC50 in
our radioligand displacement assay, but aryl 16 showed more
promising potency, with the addition of the pendant ring
affording 50-fold improvements in NS4B affinity and replicon
potency. In contrast to the oxazolidinone series, incorporation of
a properly placed carbonyl gave a less dramatic (i.e., 17 → 16)
change in replicon potency and NS4B binding affinity. However,
a
Table 3. Inhibition of HCV RNA in the Replicon System
a
For 5−10, 12−14 WT 1a and 1b replicon data is the average of
n = 3; for NS4B binding, data is the average of n = 6. For 11,
WT 1a, 1b, and NS4B binding represent n = 506, 523, and
24, respectively. An analysis of variance (ANOVA) model was fit
and used to link values to the compounds (see Supporting
Information).
a
For WT 1a and 1b replicon data is the average of n = 3; for NS4B binding,
data is the average of n = 6. XC50 determinations performed as for Table 1.
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further refinement to cyclohexyl afforded potent HCV replicon
inhibitor 18.
replaced by a cyclopropyl. The same trends in SAR observed with
the furan functionality were observed in the context of the
cyclopropyl as well. For example, the cis isomer corresponding to
21 shows significant reductions in both NS4B binding and
replicon inhibition (see Table SI-3, Supporting Information).
Compound 21 was equipotent to furan 20 and showed no
shift in potency in well-characterized replicon mutants arising
in NS5B, NS3, or NS5A (Table 4), consistent with a novel MOA
involving direct interaction with NS4B. In contrast, stable
replicons bearing NS4B mutations H94N or V105M showed
significant resistance to 21 (>100×; see Table 4), while
transiently transfected NS4B mutations F98L, H94R, and
V105L showed resistance as well. Further, 21 possessed no
measurable P450 (3A4, 2C9, 2D6, 1A2, 2C19) or reactive
metabolite liabilities. In vivo profiling of 21 in rat revealed
improved clearance and oral exposure relative to 11, 18, and 20
when dosed in rat or dog at 5 mg/kg (Table 5).
Bare cyclohexyl 18 suffered from rapid in vitro and in vivo
clearance in rat (data not shown, Clrat ≫ hepatic blood flow).
Metabolite ID studies with rat/human hepatocytes indicated a
principal route of metabolism of 18 was via direct oxidation of the
pendant cyclohexyl. Preoxidation of the cyclohexyl was tolerated
with respect to NS4B binding and replicon potency, with a trans
relative configuration of a pendant hydroxyl better tolerated than
the corresponding cis arrangement (20 vs 19, Table 3).
The imidazopyridine analogues, in general, display a reason-
able correlation (R2 = 0.48) between direct NS4B binding
and cell-based replicon inhibition (see Supporting Information
Table SI-4). While permeability for analogues in this series was
uniformly high, many of the outliers with reduced potency in the
replicon assays (e.g., 5 and 6) relative to the NS4B binding assay
were found to have poor measured solubility (<15 μM).
Precipitation of compound at higher concentrations likely
confounds EC50 interpretation and reproducibility. Conversely,
analogues lacking an extra aromatic ring (9−15, 18−21) af-
forded maximum measured solubilities of >500 μM, were gen-
erally well-behaved in the replicon assay, and as such constituted
the focus of our efforts.
Overall hydroxycyclohexyl moieties 19 and 20 gave an im-
proved in vivo DMPK profile (data not shown) relative to 18.
Despite reductions in in vivo clearance, 20 suffered from positive
reactive metabolite, apparently now as a result of oxidation of the
pendant furan (not observed directly for any previous mole-
cules). A brief survey of less reactive functionality at the furan
position revealed that small, alkyl groups in addition to heteroaryl
functionality were well tolerated. This survey led to the
subsequent identification of 21, wherein the pendant furan was
Table 5. Rat and Dog DMPK Profile for 21
a
a
rat (21)
dog (21)
clearance
Vdss
20
1.1
4.1
3
0.9
PO T1/2
AUC0−24
%F
10
3800
98%
52000
65%
a
Dosed as a solution in DMSO/Solutol/20% HPβCD (10:10:80); IV
dosed 1 mg/kg; PO dosed 5 mg/kg; clearance (mL/min/kg); Vdss
(L/kg); T1/2 (h), AUC (ng·h/mL).
Imidazo[1,2-a]pyridine (21) inhibits HCV replication in vitro
with EC50 < 10 nM for genotype 1a and 1b replicons (Table 4),
demonstrates good oral exposure in rodent and dog (Table 5),
and maps its resistance to HCV NS4B (Table 4), unique among
existing HCV inhibitors undergoing clinical trials. These
properties make this class of imidazo[1,2-a]pyridines a
promising and unique class of HCV replication inhibitors. We
will report the full in vivo characterization of 21 and other related
development candidates in due course.
a
Table 4. Replicon Profile for 21
ASSOCIATED CONTENT
■
S
* Supporting Information
Replicon and NS4B binding assay protocols, synthetic
procedures, compound purities, protein purification protocols,
in vivo/in vitro DMPK protocols, details of radioligand
syntheses, and statistical methods. This material is available
free of charge via the Internet at http://pubs.acs.org.
mutant replicon
gene
EC50 (nM)
NS4B binding
WT 1a
60
0.81
5.4
200
800
53
WT 1b
AUTHOR INFORMATION
■
1B H94N
1B V105M
1B H94R*
1B V105L*
1B F98L*
1B A156T*
1B M414T*
1B L31V*
NS4B
NS4B
NS4B
NS4B
NS4B
NS3
Corresponding Author
*E-mail: jbs26900@GSK.com.
38
Present Addresses
§Kanion USA, Inc., 3916 Trust Way, Hayward, CA 94545.
∥Alios BioPharma, 260 East Grand Ave., 2nd Floor, South San
Francisco, CA 94080.
27
1.0
1.0
1.0
NS5B
NS5A
⊥Gilead Sciences, Department of Structural Chemistry, 333
Lakeside Drive, Foster City, CA 94404.
a
WT 1a and WT 1b data represents n = 137 and 138, respectively; for
NS4B binding, data represents n = 6. XC50 determinations performed
as for Table 1. For 1B replicons H94N (n = 65), for 1B V105M (n =
59), for 1B A156T/M414T/L31 V (n = 2), H94R (n = 30), F98L (n =
20), and V105L (n = 182). * denotes transiently transfected replicons;
all other replicons were stable.
Notes
The authors declare the following competing financial
interest(s): All authors are current or former employees of
GlaxoSmithKline.
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did not show a correlation between assay activity and compound
potency in replicon or binding assays. A full biological characterization
of compound 21 will be published separately.
ACKNOWLEDGMENTS
■
We thank Steven Novick for the statistical analyses described in
this communication and the Supporting Information. The
authors acknowledge Randy Bledsoe and Tom Consler for the
purification of the NS4B protein and the antibody preparation.
The authors acknowledge Todd Baughman for conducting the
reactive metabolite assays. The authors acknowledge Luz Helena
Carballo for replicon screening.
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