2-Alkyloxazoles as potent and selective PI4KIIIβ inhibitors demonstrating inhibition of HCV replication

Article abstract of DOI:10.1016/j.bmcl.2014.07.015

Synthesis and SAR of 2-alkyloxazoles as class III phosphatidylinositol-4- kinase beta (PI4KIIIβ) inhibitors is described. These compounds demonstrate that inhibition of PI4KIIIβ leads to potent inhibition of HCV replication as observed in genotype (GT) 1a and 1b replicon and GT2a JFH1 virus assays in vitro.

Full text of DOI:10.1016/j.bmcl.2014.07.015

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3714–3718
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Alkyloxazoles as potent and selective PI4KIIIb inhibitors
demonstrating inhibition of HCV replication
a
a
a
a
a
Erin P. Keaney a, , Michael Connolly , Markus Dobler , Rajeshri Karki , Ayako Honda , Samantha Sokup ,
Subramanian Karur a, Shawn Britt a, Anup Patnaik a, Prakash Raman a, Lawrence G. Hamann a,
Brigitte Wiedmann b, Matthew J. LaMarche a
⇑
a Global Discovery Chemistry, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA
b Infectious Disease Area, Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 February 2014
Revised 3 July 2014
Accepted 7 July 2014
Available online 12 July 2014
Synthesis and SAR of 2-alkyloxazoles as class III phosphatidylinositol-4-kinase beta (PI4KIIIb) inhibitors is
described. These compounds demonstrate that inhibition of PI4KIIIb leads to potent inhibition of HCV
replication as observed in genotype (GT) 1a and 1b replicon and GT2a JFH1 virus assays in vitro.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
PI4K
HCV
Hepatitis C virus (HCV) afflicts >150 million people worldwide
and leads to chronic liver disease. Over time, untreated HCV infec-
tion can lead to liver diseases such as cirrhosis and liver cancer that
contribute to more than 350,000 fatalities annually as a result of
the infection.1 Until recently, a once a week injection of pegylated
to PEG-INFa and RBV alone, which can prevent the use of this reg-
imen in special populations, including transplant patients, and
finally, the three-times-per-day dosing schedule can be inconve-
nient and negatively affect compliance.7 In addition, these and
most other direct acting antiviral (DAA) approaches have been
shown to select for resistant mutations in the targeted proteins.8,9
Targeting host proteins that are essential for viral replication may
provide an alternative strategy to overcome this resistance mech-
anism and complement DAA agents in combination treatment reg-
imens. In order to identify host proteins with the potential for
therapeutic utility in blocking HCV replication, we and others exe-
cuted high-throughput siRNA screens.10–17 As previous studies
have also demonstrated, we identified the mammalian lipid
interferon alpha (PEG-IFNa) in combination with ribavirin (RBV)
had been the standard of care for treatment of HCV infection,
and while 50% of genotype 1a (GT1a) infected patients are treated
successfully, this treatment can result in severe side effects such as
fever, fatigue, nausea, depression, and neutropenia.2–4 Due to these
side effects, many patients do not complete the full treatment
course and others do not even seek diagnosis.
A large number of small molecule therapies targeting HCV viral
proteins are currently in clinical development and the first of these
drugs have recently been approved by the FDA.5,6 Both approved
by the FDA in May 2011, telaprevir (Vertex) and boceprevir
(Merck) are HCV NS3 protease inhibitors that, in combination with
kinases phosphatidylinositol 4-kinase alpha and beta (PI4KIII
a
and b) as vital proteins involved in hepatitis C virus replica-
tion.11–15 The mode of action of these two lipid kinase isoforms
was not known at the time of the screens nor was much informa-
tion on the cellular functions available. (Since that time, PI4KIIIb
has been shown to be specifically recruited by the hepatitis C virus
to synthesize phosphatidylinositol-4-phosphate as a means to con-
struct membranes that are utilized for viral replication18). There-
fore, we next sought to identify advanced tool molecule
inhibitors of PI4KIIIb in order to assess the potential effectiveness
and safety of targeting this mechanism for treatment of HCV.
Cyclopropyl-oxazole compound 1 (Table 1, originally purchased
PEG-IFN
a
and RBV, are effective at treating HCV in patients who
and
have failed prior therapy. In combination with PEG-INF
a
RBV, the addition of a protease inhibitor increases sustained viro-
logic response (SVR) rates up to 75% and can shorten the duration
of treatment. However, significant limitations remain: triple ther-
apy is currently only approved for GT1-infected patients, side
effects (such as anemia and rash) are often exacerbated compared
from CSC Chem Divers. Moscow) was identified in
throughput screen as a potent inhibitor of PI4KIIIb. Compound 1
a high-
⇑
Corresponding author. Tel.: +1 617 871 3878.
E-mail address: erin.keaney@novartis.com (E.P. Keaney).
http://dx.doi.org/10.1016/j.bmcl.2014.07.015
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

E. P. Keaney et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3714–3718
3715
Table 1
PI3K and PI4K biochemical activity, and HCV cellular activity of HTS hit and subsequent analogs
IC50 (nM)
PI4KIII
HCV ECb (nM)
50
Compound
PI3Ka
a
PI4KIIIb
Replicon 1b
Replicon 1a
GT2a JFH1 virus
1
2
3
4
5
2400
>9100
ND
6290
8200
>10,000
ND
ND
>10,000
>10,000
135
49
>2500
18
1024
99
6730
46
1578
166
2095
59
1818
ND
ND
375
324
19
34
66
a
IC50 values were measured against PI3K
a, b, c, and d. The most potent value is shown and the actual target is only mentioned if the compound exhibits high inhibition.
b
CC50 values measured in the HCV GT1b replicon assay were all >31,000 nM.19
(and subsequent analogs) is highly selective for inhibition of
PI4KIIIb and does not significantly inhibit PI4KIII or other kinases
a
tested in biochemical assays (>50 protein kinases, >5 lipid kinases).
Our chemistry efforts were focused on enhancing the potency
against the PI4KIIIb enzyme and HCV, while maintaining this selec-
tive kinase profile. Toward that end, we investigated a number of
diversity points around the oxazole core, which ultimately led to
the identification of PI4KIIIb selective inhibitors effective at inhib-
iting GT1 replicons and GT2a virus with EC50 values in the nano-
molar range and a >1000 separation versus cytotoxicity.
In order to develop an understanding of the pharmacophore for
PI4KIIIb inhibition, early exploration of SAR around compound 1
centered on investigation of the potential overlap of the oxazole
scaffold with other chemotypes that had been identified in the
high-throughput screen. With respect to that initial goal, modifica-
tion of the sulfonamide via incorporation of aminocyclohexanol (2)
led to a slight enhancement in potency, but importantly was also
Figure 1. Compound 1 docked into the PI4KIIIb homology model.
accompanied by
a significant boost in aqueous solubility
(0.22 mM compared to <0.002 mM for 1).20 Addition of a methyl
group in the 3-position on the phenyl ring (3) was not tolerated,
while an additional methyl group on the oxazole ring (4) led to a
favorable improvement in both enzymatic potency and inhibition
in the HCV replicon. Simple replacement of the 2-methyl group
on the phenyl ring with a methoxy group (5) maintained potent
inhibition of PI4KIIIb and provided a favorable scaffold for addi-
tional investigation of both the alkyl substituent on the oxazole
as well as the aryl-sulfonamide moiety. This phenyl region SAR
mirrored that observed in related aminothiazole and aminoimidaz-
ole series of PI4KIIIb inhibitors that were explored in parallel.21,22
A homology model of PI4KIIIb (Fig. 1) was generated using the
predicted to make a monodentate hydrogen bond with Val613 in
the hinge, while the sulfonamide group may make interactions
with Asp689 and catalytic Lys564. A possible edge-to-face interac-
tion between the phenyl group of 1 and the side-chain of Tyr598
was noted, as well as hydrophobic interactions with Ile688 and
gatekeeper Ile610. The chloro-phenyl group in this model is ori-
ented towards the ribose binding pocket.
The initial synthetic route to compounds in this series
employed copper catalysis to effect the oxazole formation from
1,2-dibromoolefin 8 and cyclopropane carboxamide yielding 5-
aryloxazole 9 (Scheme 1). While this method ultimately provided
the desired compounds, low conversion rates hindered rapid deriv-
atization. Methylation of alkyne 6 proceeded smoothly in quantita-
tive yield and was subsequently followed by bromination leading
to a mixture of E and Z isomers (8). A one-pot successive CAN/
CAO bond forming reaction reported by both Schuh and Martin
crystal structure of PI3Kc solved in house (PDB entry 3SD5) as a
template in the program Prime (version 2.0).23,24 Docking analysis
was used in combination with the SAR data generated to predict
the possible binding mode of the oxazole compounds in the ATP
binding site of PI4KIIIb.25 The oxazole nitrogen of compound 1 is

3716
E. P. Keaney et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3714–3718
Scheme 1. Reagents and conditions: (a) nBuLi, MeI, THF, ꢀ78 °C to rt (91%); (b) Br2,
CH2Cl2, rt, 45 min (Quant. carried on crude); (c) cyclopropane carboxamide, N,N0-
dimethylenediamine, CuI, K2CO3, toluene, 110 °C, 5 d (8%; undesired regioisomer
30%); (d) ClSO3H, rt, 2.5 h (87% carried on crude); (e) NH2-R, pyridine, THF, rt, 1.5 h
(42–71%).
was then utilized to form the oxazole ring (9).26,27 They report lim-
ited work on the formation of 2,4,5-trisubstituted oxazoles, though
they did highlight one example albeit with reduced yield compared
to that for the synthesis of disubstituted oxazoles. We also
observed low conversion to the desired tri-substituted oxazole 9
and isolated a mixture of both oxazole regioisomers. From the oxa-
zole intermediate, synthesis of the desired compounds was
straightforward, where regioselective chlorosulfonylation followed
by reaction with various amines (R) provided sulfonamides 10.
An alternate synthetic route provided a streamlined synthesis
and allowed for rapid elaboration of both the oxazole alkyl substi-
tuent R0 and the sulfonamide R (Scheme 2). Chlorosulfonylation
and subsequent reaction with a variety of amines led to sulfona-
mides 13. Bromination was accomplished by reaction with carb-
Scheme 2. Reagents and conditions: (a) ClSO3H, 0 °C to rt, 2 h (quant.); (b) NH2-R,
Et3N, CH2Cl2, 0 °C to rt, 5 h (16%-Quant.); (c) carboxyethyltriphenylphosphonium
tribromide, THF, 0 °C to rt, 18 h (27–95%); (d) R0COOH, K2CO3, acetone, rt, 5 min
(43%-Quant. carried on crude); (e) AcOH, NH4OAc, 120 °C, 15 min (8–70%).
set of compounds (up to 5-fold in compound 26) and a correlation
between enzyme and cellular activity was observed. Kinase selec-
tivity was also maintained, exemplified by the lack of compounds
20 and 22 to inhibit PI4K
trations of 9 M. Importantly, the compounds showed no evidence
of cytotoxicity in replicon containing cells at concentrations up to
30 M, suggesting that a therapeutic window may be achievable
a and PI3 kinases a, b, and d up to concen-
l
oxyethyltriphenylphosphonium
tribromide
to
give
the
corresponding -bromoketones 14, which then underwent dis-
a
l
placement with various carboxylic acids to provide esters 15. As
the penultimate step, the displacement reaction proved to be a
useful point with which to introduce diversity, given the high
yields, lack of purification required, and wide availability of car-
boxylic acids. A subsequent simple cyclization with ammonium
acetate in acetic acid led to oxazoles 16.28
with this mode of inhibition of a host cell factor. Additionally,
the inhibitory effects of these compounds on HCV replication
extends to the GT1a replicon and the GT2a live virus settings as
well (Compound 19 HCV replicon 1a EC50 = 86 nM and GT2a JFH1
EC50 = 147 nM; Compound 26 HCV replicon 1a EC50 = 53 nM and
GT2a JFH1 EC50 = 383 nM).
Compounds were evaluated for inhibition of the PI4KIIIb
enzyme in a biochemical assay as well as for inhibitory activity
against HCV replication using a subgenomic GT1b replicon assay
(Table 2).29–31 With a structure close to that of the HTS hit com-
pound 1, we sought to increase potency of 5 against both the
PI4KIIIb enzyme as well as HCV in the GT1b replicon assay. Initial
modification of the 2-oxazole position (R0) highlighted a tolerabil-
ity for variation, providing further support to our docking model.
We envisioned this moiety to be somewhat solvent exposed how-
ever, and were consequently hopeful that introduction of a hetero-
atom would provide the basis for an additional interaction below
the hinge region. Interestingly, in this putative solvent exposed
region, some hydrophobic contact flanking the hinge binding
region is required for enzymatic potency (compounds 5 and 18)
as compared to unsubstituted oxazole 17. Despite this, neither
increasing bulk (compounds 19, 20, 21) nor introducing heteroat-
oms (22, 23) within this moiety provided any incremental
enhancement in enzymatic potency, therefore supporting the
hypothesis that this region of the molecule is solvent exposed.
The PI4KIIIb inhibitory potency was modestly enhanced within this
Further exploration of the scaffold revealed a requirement for
the sulfonyl moiety for potent PI4KIIIb inhibition (Table 3). Based
on our docking model, we envisioned both the O and NH of the sul-
fonamide to be engaging in significant interactions with the pro-
tein with the catalytic lysine and DFG loop respectively (Fig. 1).
To probe this hypothesis, the des-sulfonamide (27), sulfones (28)
and (29), and amide (30) analogs were synthesized. From this set
of compounds, PI4KIIIb IC50 values suggested a key role for the sul-
fonyl oxygens, where removal of the sulfonamide nitrogen did not
have a detrimental effect on activity, therefore suggesting that the
proposed interaction with Asp689 is either non-existent or not
critical for compound binding. Complete removal of the sulfon-
amide or replacement with an amide (30) led to significant loss
in PI4KIIIb inhibition. Substitution in this position is not limited
to hydroxylated alkyls, as aryl analog 31 also potently inhibits
PI4KIIIb. In general a correlation is observed between PI4IIIKb
and replicon activity and the window over cytotoxicity in replicon
containing cells is maintained.
In an effort to expand the breadth of this class of compounds to
investigate other heterocyclic cores, both the corresponding

E. P. Keaney et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3714–3718
3717
Table 2
Table 3
PI4KIIIb biochemical and HCV replicon activity of 2-substituted oxazoles
PI4KIIIb biochemical and HCV replicon activity of sulfonyl moiety SAR
a
Compound
R
IC50 (nM) PI4KIIIb
EC50 (nM)
HCV replicon 1b
a
Compound
R0
IC50 (nM) PI4KIIIb
EC50 (nM)
27
28
>2500
76
>30,000
HCV replicon 1b
120
67
5
19
34
17
18
1610
414
>10,000
3211
29
30
79
19
20
28
9
41
30
9727
3557
<30
21
22
19
9
108
32
31
11
32
33
27
64
43
67
23
24
36
37
402
193
34
33
37
114
193
25
26
191
4
410
41
35b
a
CC50 values measured in the HCV GT1b replicon assay were all >31,000 nM.19
a
CC50 values measured in the HCV GT1b replicon assay were all >31,000 nM.
Contains a hydrogen in place of the methyl group in the 4-position of the
b
pyrazole and imidazole analogs were synthesized (Table 4). As
compared to oxazole 5, the pyrazole analog 36 showed equipotent
activity against PI4KIIIb and provides a potential path towards
broadening this class of PI4KIIIb inhibitors. In contrast, imidazole
analog 37 was much less active than the oxazole or pyrazole
counterparts.
This 2-alkyloxazole series represents a new class of compounds
that are highly potent and selective inhibitors of PI4KIIIb. These
compounds can be accessed through a straightforward synthetic
sequence that provides a facile route to diversification. Data
obtained with molecules in this series further illustrate that inhibi-
tion of PI4KIIIb leads to robust inhibition of HCV replication across
multiple genotypes. Ultimately this approach highlights the
oxazole.
strategy of targeting cellular proteins as a potential route toward
novel HCV therapies. Towards that end, initial resistance frequency
experiments with compound 2 in separate combinations with an
HCV NS3 protease inhibitor and with an HCV NS5B polymerase
inhibitor illustrated that the combination did modestly suppress
the emergence of resistance. This initial data lends support to the
stated hypothesis that targeting host proteins that are essential
for viral replication may be a viable strategy to combat resistance
mechanisms that emerge in the presence of DAA agents. An

3718
E. P. Keaney et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3714–3718
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a
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R
IC50 (nM) PI4KIIIb
EC50 (nM)
HCV replicon 1b
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19. Cytotoxic effects of compounds were monitored by determining viability of
cells after 48 h compound treatment using a tetrazolium compound (MTS)-
based assay (CellTiter 96 AQueous One Solution cell proliferation assay,
Promega).
20. Automated high-throughput equilibrium solubility measurements are
determined by HPLC and Miniaturized Shake-Flask Method. Compounds are
first solubilized at 10 mM in pure DMSO. The DMSO stock solution is then
transferred into a microtiter plate. The DMSO solvent is dried with GeneVac
solvent evaporator. After the addition of buffer solution, the plate is sealed and
shaken for 24 hours at room temperature. The plate is then sent to a centrifuge
1035
9951
CC50 values measured in the HCV GT1b replicon assay were all >31,000 nM.19
a
for phase separation and the supernatant is quantified by HPLC, using
calibration curve constructed with the same DMSO stock solution.
a
21. Connolly, M.; Patnaik, A.; Dobler, M.; LaMarche, M. J.; Wiedmann, B.;
Whitehead, L.; Fujimoto, R.; Gaither, L. A.; Knapp, M.; Drumm, J.; Ding, J.;
Keaney, E. P.; Hamann, L. G.; Dennehy, M.; Mathy, J. E.; Xiong, X.; Puyang, X.;
Jiang, X.; Mehta, A.; Damzal, M. D.; Amaral, A.; Spear, J.; Zhao, H.; Bailey, S.;
Glowienke, S.; Brichet, M.; Fischer, J.; Karki, R. J. Manuscript in preparation.
22. LaMarche, M. J.; Cappacci-Daniels, C.; Colvin, R. A.; Connolly, M.; Dennehy, M.;
Ding, J.; Dobler, M.; Drumm, J.; Hu, L.; Karki, R. J.; Karur, S.; Patnaik, A.; Keaney,
E. P.; Puyang, X.; Raman, P.; Sanchez, C.; Tommasi, R.; Whitehead, L.; Xiang, X.;
Xiong, X.; Yoon, T.; Zabawa, T. P.; Wiedmann, B. Manuscript in preparation.
23. Burger, M. T.; Pecchi, S.; Wagman, A.; Ni, Z. J.; Knapp, M.; Hendrickson, T.;
Atallah, G.; Pfister, K.; Zhang, Y.; Bartulis, S.; Frazier, K.; Ng, S.; Smith, A.;
Verhagen, J.; Haznedar, J.; Huh, K.; Iwanowicz, E.; Xin, X.; Menezes, D.; Merritt,
H.; Lee, I.; Wiesman, M.; Kaufman, S.; Crawford, K.; Chin, M.; Bussiere, D.;
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obvious consideration when targeting host-cell factors is the
importance of obtaining an appropriate therapeutic window to
avoid potential on-target cytotoxic events. Cytotoxicity of the com-
pounds was assessed in parallel to the replicon activity in Huh7
replicon containing cells and a EC50/CC50 ratio of >1000 was
achieved in all compounds of this chemotype. In addition, we have
previously demonstrated clinical proof of concept that targeting
the host-cell factor cyclophilin for treatment of HCV is therapeuti-
cally viable and therefore provides further evidence that such an
approach can be successful.32 While we have shown a lack of cyto-
toxicity in the HCV replicon assay, further investigation into the
safety of specifically targeting PI4KIIIb highlighted an antiprolifer-
ative effect on bone marrow and lymphocytes in vitro and inhibi-
tion of B-cell proliferation in vivo.33 Irrespective of these safety
challenges identified with inhibition of PI4KIIIb, there still exists
substantial opportunity to complement DAA therapeutics with
agents targeting host-cell factors in the treatment of HCV.
24. Prime, Version 2.0, Schrödinger, LLC, New York, NY, 2008.
25. Glide, Version 5.0, Schrödinger, LLC, New York, NY, 2008.
26. Schuh, K.; Glorius, F. Synthesis 2007, 2297.
27. Martin, R.; Cuenca, A.; Buchwald, S. L. Org. Lett. 2007, 9, 5521.
28. Cremlyn, R.; Swinburne, F.; Shode, O.; Lynch, J. J. J. Heterocycl. Chem. 1987, 24,
117.
29. The assay for PI4KIIIb IC50 determination was previously described in Ref.10
.
30. The activity of compounds was tested in Huh7 cells stably transfected with
subgenomic replicons of genotype 1b (Con1, ReBLikon GmbH) containing a
luciferase reporter gene and the neomycin phosphotransferase for selection
with geneticinÒ (G418).
References and notes
31. Some compounds were also tested in the genotype 2a infectious virus (JFH1)
assay and in the full-length genotype 1b (Apath, LLC) assay using quantitative
RT-PCR.
32. Ma, S.; Boerner, J. E.; TiongYip, C.; Weidmann, B.; Ryder, N. S.; Cooreman, M. P.;
Lin, K. Antimicrob. Agents Chemother. 2006, 50, 2976.
33. Lamarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.; Colvin, R.; Dennehy,
M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L. A.; Gao, J.; Jiang, X.; Lin, K.;
McKeever, U.; Puyang, X.; Raman, P.; Thohan, S.; Tommasi, R.; Wagner, K.;
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