Concise SAR Exploration Based on the "head-to-Tail" Approach: Discovery of PI4KIIIα Inhibitors Bearing Diverse Scaffolds

Article abstract of DOI:10.1021/acsmedchemlett.6b00232

In typical kinase inhibitor programs, a hinge binder showing best potency with preferential specificity is initially selected, followed by fine-tuning of the accompanying substituents on its core module. A shortcoming of this approach is that the exclusiv

Full text of DOI:10.1021/acsmedchemlett.6b00232

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Letter
Concise SAR Exploration Based on the “Head-to-Tail” Approach:
Discovery of PI4KIII# inhibitors Bearing Diverse Scaffolds
Satoru Noji, Noriyoshi Seki, Takaki Maeba, Takayuki Sakai, Eiichi Watanabe,
Katsuya Maeda, Kyoko Fukushima, Toru Noguchi, Kazuya Ogawa, Yukiyo
Toyonaga, Tamotsu Negoro, Hisashi Kawasaki, and Makoto Shiozaki
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00232 • Publication Date (Web): 03 Aug 2016
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Concise SAR Exploration Based on the “Head-to-Tail” Approach:
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Discovery of PI4KIIIα inhibitors Bearing Diverse Scaffolds
Satoru Noji,† Noriyoshi Seki,† Takaki Maeba,† Takayuki Sakai,† Eiichi Watanabe,† Katsuya
Maeda,† Kyoko Fukushima,† Toru Noguchi,§ Kazuya Ogawa,§ Yukiyo Toyonaga,§ Tamotsu Nego-
ro¶ Hisashi Kawasaki† and Makoto Shiozaki*,†
†Chemical Research Laboratories, §Biological Pharmacological Research Laboratories, ¶Drug Metabolism & Pharma-
cokinetics Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho,
Takatsuki Osaka, 569-1125, Japan
KEYWORDS . HCV, PI4KIIIα,”Head-to-Tail” Approach, Diverse Scaffolds
ABSTRACT: In typical kinase inhibitor programs, a hinge binder showing best potency with preferential specificity is ini-
tially selected, followed by fine-tuning of the accompanying substituents on its core module. A shortcoming of this ap-
proach is that the exclusive focus on a single chemotype can endanger all the analogues in the series if a critical short-
coming is revealed. Thus, an early evaluation of structure-activity relationships (SAR) can mitigate unforeseen out-
comes within a series of multiple compounds, although there have been very few examples to follow such a policy.
PI4KIIIα is one of four mammalian phosphatidylinositol-4 kinases and has recently drawn significant attention as an
emerging target for hepatitis C virus (HCV) treatment. In this communication, a novel “head-to-tail” approach to dis-
cover a diverse set of PI4KIIIα inhibitors is reported. We believe this method will generate distinct core scaffolds is a ra-
tional strategy to circumvent potential risks in general kinase programs.
Hepatitis C virus (HCV) is a leading cause of chronic
liver disease and 130–150 million people are considered to
be infected worldwide.1,2 Patients with persistent infection
of HCV are at high risk to develop serious liver damage
typified by hepatocellular carcinoma, which can eventual-
ly necessitate liver transplantation.3,4 Although the thera-
peutic options are being improved after the approval of
newly-developed direct acting antivirals,5 significant con-
cerns remain about drug resistance as well as inadequate
coverage of all existing HCV genotypes. Due to these un-
met needs, development of a new broad-spectrum antivi-
ral strategy managing such potential problems are of
great importance. To achieve such goals, the disruption of
the viral lifecycle is a viable option so that several host
proteins which is implicated in HCV reproduction have
been intensely pursued.6
production.8,9 Therefore, a strategy targeting this enzyme
is a rational approach for treatment of this virus based on
the emerging mechanism of action.
While the concept of targeting this lipid kinase seems
valid, one must take into account that interference with
such an essential cellular function might cause some seri-
ous damage to the host cells themselves. Fortunately,
there are other PI4 kinases such as PI4KIIIβ which are
responsible for maintenance of PI4P homeostasis,10 and
thus specific inhibition of PI4KIIIα holds great promise to
suppress HCV replication while keeping basal cell traf-
ficking functions via PI4P intact through bypassing such
redundant machinery.
PI4KIIIα is known as one of four mammalian phospha-
tidylinositol-4 kinases, and responsible for formation of
phosphatidylinositol 4-phosphate (PI4P) at cytoplasmic
membranes, cis-Golgi compartments, and nucleus.7 One
of the non-structural HCV protein, NS5A, was reported to
hijack the PI4P production machinery, which facilitates
the viral replication by forming unique membranous web-
like structures. Subsequently, silencing the PI4KIIIα gene
was confirmed to abrogate HCV’s replication as well as its
Figure 1. Structure of PI4KIIIα inhibitor reported by GSK.
Toward this end, we started a SAR investigation to ob-
tain selective PI4KIIIα inhibitors. Among the inhibitors
reported to date, we focused our attention on a com-
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pound 1 reported by a GSK research group11 since this
compound showed good PI4KIIIα inhibitory activity
(pIC50 = 8.3) with preferable selectivity over PI4KIIIβ as
high as 200-fold (Figure 1).
Comparison of the affinity pocket of PI4KIIIα (cyan) and
PI4KIIIβ (white) with 5.
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4
5
6
7
8
To gain a structure-based inspiration, a homology
model of PI4KIIIα was generated by using MOE (Chemi-
cal Computing Group, Montreal, Canada) using the
known crystal structure15 of PI3Kγ as a template (Figure
2a). Compound 2 was placed manually into the ATP-
binding pocket of PI4KIIIα with the 1-nitrogen atom of
the quinazolinone acting as a hinge binder and the other
parts of the molecule forming hydrophobic contacts in
the pocket. The resultant complex model suggested that
the substituted pyridine moiety of 2 projected toward a
site corresponding to the affinity pocket so that explora-
tion of this part of 2 was prioritized. In the preceding pa-
per, the GSK group reported that reversal of the amino-
sulfonyl linkage resulted in an improvement of potency
against PI4KIIIα,11 and so we synthesized two analogues
having the reversed orientation (3 and 4, Table 1). The
results provided quite a contrast. Compound 3 showed
significant improvement in PI4KIIIα inhibitory potency
while its sulfone analogue 4 showed complete loss of the
potency. It is quite plausible that the NH part of the sul-
fonamide moiety acquired a crucial electrostatic interac-
tion with some residues in the pocket, suggesting that the
exploration of core motifs could be feasible once we
strengthened the compound’s PI4KIIIα selectivity without
losing its robust potency. Actually, our homology and
sequence comparison model pointed out a pocket adja-
cent to the methoxy group of 3 in PI4KIIIα. Compared to
other closely-related enzymes, this binding pocket was
more spacious, suggesting that this difference in binding
site might be a source of improved isomer selectivity.
Driven by this hypothesis, we synthesized two com-
pounds possessing a slightly larger substituent as the R1
group. Gratifyingly, compound 5 completely satisfied our
expectation and > 1000-fold selectivity over PI4KIIIβ
along with further improvement of PI4KIIIα inhibitory
potency was achieved.
The compound possessed
a 2-aminoquinazolinone
skeleton as a hinge binder, on which a disubstituted pyri-
dine and an atropisomeric benzene ring were attached.
Although the selectivity profile of this compound was
reasonably assured, we felt confident in implementing
another core module having reduced affinity, since the
hinge domain is known to be a highly conserved region
across many kinases. So, we decided to initiate investiga-
tion from its monodentate12 analogue 2 (Table 1), on
which a simple benzene ring was implemented to avoid
any stereoisomeric complications. To further mitigate an
unexpected risk,13 we undertook an exploration of distinct
classes of hinge-binding modules because we expected
such an approach to subsequently result in generation of
a diverse set of the inhibitors. In typical kinase programs,
a hinge binder showing best potency with preferential
specificity is initially recruited, followed by fine-tuning of
the substituents on this core motif. A shortcoming of
such a classical approach is the excessive commitment to
a sole chemotype, which, once a critical problem is re-
vealed during the screening, could endanger further in-
vestigation of all the corresponding congeners. It is quite
evident that a wide variety of compounds will be generat-
ed if the core scaffold can be modified, particularly if
there are numerous potential substituents available.
Aside from the hinge region, the affinity pocket14 is an
ideal place to obtain good binding. Therefore, as the ini-
tial step of our investigation, we searched for a motif
which displayed a favorable affinity for this pocket .
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(a)
Tail
Head
Core
Table 1. SAR of the Head Motif
Ile1841
Affinity Pocket
Lys1792
Enzyme (IC50, nM)a
Replicon
Fold
(EC50, nM)a
R1
R2
Compd
PI4K
( / )
α β
Ⅲ
(b)
1b
α
β
2
3
190
>30000 >160
99
ꢀ
β→Glu584
α→Asp1800
14
780
56
4
>1000 >30000 >30
>30000
20
Lys1792
5
4.1
4900
1200
β→Leu581
α→Cys1797
6b
100
>30000 >3000
400
Figure 2. (a) A PI4KIIIα homology model with compound 2
based on using the PI3Kγ crystal structure as a template. (b)
a Values of IC50 and EC50 are mean values determined
from at least 3 replicates. bRacemic.
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According to our homology model (Figure 2b),16 the
a diverse set of heterocycles whose substituents could
project into a different location (Table 2). Rather surpris-
ingly, compound 7 being almost identical to the reference
compound 5 showed reduced PI4KIIIα potency, while
compound 8 whose R3 substituent was directed to a dif-
ferent vector showed comparable potency. Further dis-
similar analogues having a 5,6-fused heteroaromatic
group as the core unit generally showed reduced PI4KIIIα
potency (9–13). However, significant transformations of
the R3 portion restored the inhibitory potency, allowing
even the least potent analogue 13 to reach a single-digit
nanomolar value of IC50 against PI4KIIIα (17). It was also
notable that the SAR trend of the R3 portion was different
depending on the core modules (9 to 15 vs 13 to 17), sug-
gesting that each core structure had a distinct preference
for the accompanying tail portion R3. Following a typical
optimization protocol, fine-tuning in the head and the
tail region was complementarily conducted with one of
the most potent compounds, 5.
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4
5
6
7
8
methoxymethyl group of compound 5 may be forced too
closely to the Glu584 and Leu581 residues of PI4KIIIβ.
Contrastingly, their counterparts of PI4KIIIα are Asp1800
and Cys1797, respectively, more compact amino acids that
afford more room near the identical substituent of the
inhibitor 5. Based on this analysis, we considered that the
slightly larger two residues in PI4KIIIβ would cause steric
congestion, while a favorable hydrophobic contact be-
tween the Cys1797 of PI4KIIIα and the terminal methoxy
portion of 5 might have occurred. Moreover, the size of
the methoxymethyl group was almost optimal and com-
pound 6 enlarged at a position adjacent to the pyridine
ring showed significant loss of potency against either of
the PI4KIII enzymes.
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Table 2. SAR of the Core Motif
Table 3. SAR of the Tail Motif
Enzyme (IC50, nM)a
Replicon
Fold
R3
(EC50, nM)a
Compd
5
Core
PI4KⅢ
( / )
α β
α
β
1b
4.1
4900
8200
1400
200
1200
1030
640
22
20
68
Enzyme (IC50, nM)a
Replicon
Fold
(EC50, nM)a
1b
R3
R4
Compd
PI4KⅢ
( / )
α β
α
β
7
8.0
2.2
9.1
13
ꢀPr
18
19
20
n
1.9
230
660
130
160
220
120
260
130
100
710
10
81
13
88
6.8
(CH2)2OH
2.5
1.0
8
41
Me
(CH2)2OMe
9
610
880
1000
780
700
50
21 (CH2)2SO2Me
1.6
ꢀPr
22 (CH2)2OMe
c
0.31
10
11
12
13
14
15
16
17
410
32
a Values of IC50 and EC50 are mean values determined from
at least 3 replicates.
50
3700
74
Starting from an analogue bearing a simple tail-like
substructure (18), we introduced a functional group at the
terminus (19–21) to lower the lipophilicity to increase the
drug-like properties.17 In accordance with the previous
results represented by 15 and 17, compound 20 having an
ether moiety showed better potency; by a slight modifica-
tion of the head group (22), the PI4KIIIα selectivity was
further strengthened. Toward this end, we have success-
fully discovered a novel PI4KIIIα inhibitor having superb
PI4KIIIα potency. This compound is composed of three
new modules, the head, core, and tail groups, which are
distinct from those of PI4KIIIα inhibitors reported to date
(It is also noteworthy that GSK reported that replacement
of the benzene tail part with an alkyl group such as tetra-
hydropyran resulted in reduced PI4KIIIα potency).11
Needless to say, brief optimization of the rest of the mol-
ecules such as 15 and 17 will lead to generation of more
diverse PI4KIIIα inhibitors, although the details are be-
yond the scope of this report.
93
>10000 >110
260
3.1
2.0
16
18000
98
96
32
20
58
53
39
29
920
78
5.7
300
31
aIC50 and EC50 are mean values determined from at least 3
replicates.
Since we had a suitable head group in hand, we em-
barked on an exploration of the core motif. We examined
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pyridine or 3-Br-5-R2-6-R1-pyridine, PdCl2(dppf)-CH2Cl2,
The PI4KIIIα inhibitors shown in this paper were syn-
thesized following a method highlighted in Scheme 1. The
route was simple and a diverse set of compounds were
prepared through Suzuki-type cross coupling in a conver-
gent manner.
1
2
3
4
5
6
7
8
Cs2CO3, 1,4-dioxane, H2O, 100 °C; (g) 29, corresponding ar-
ylbromide,18 (XPhos)Pd-G1, K3PO4, 1,4-dioxane, H2O, 100 °C,
MW (h) 28, corresponding arylBpin,19 PdCl2(amphos), 1.5 M
K2CO3, 1,4-dioxane, 100°C; (i) (1) 29, corresponding arylio-
dide,20 PdCl2(dppf)-CH2Cl2, 1.5 M K2CO3, 1,4-dioxane, 100 °C;
(2) PhB(OH)2 or 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane, PdCl2(dppf)-CH2Cl2, 1.5 M
K2CO3, 1,4-dioxane, 100 °C; (j) 10% Pd(OH)2/C, H2 (1 atm),
THF, MeOH, r.t.
Scheme 1. Synthesis of Diverse Sets of PI4KIIIα inhib-
itors
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Given its superb PI4KIIIα potency along with good on-
target selectivity, additional assays were conducted with
representative compound 22. Firstly, two additional coun-
ter assays were carried out to further confirm its non-
promiscuous character, and over 800-fold selectivity was
also observed against two typical lipid kinases, PI3Kα and
PI3Kβ. More importantly, 22 exhibited a low nanomolar
EC50 in both 1b and 1a replicon assays, thus demonstrating
broad-spectrum inhibition of HCV genotypes, which was
originally expected and now seems highly plausible. Good
physicochemical properties as well as high metabolic sta-
bility were also notable, leading to preferable plasma half-
life and excellent oral bioavailability for 22.
Table 4. Profiles of 22
(a)
Enzyme (IC50, nM)a
Replicon
LEb
(PI4KIII
(EC50, nM)a
PI4KⅢ
PI3K
)
α
1a
1b
α
β
α
β
O
N
Ph
N
Ph
O
0.31
220
240
1400
0.34
10
6.8
Ar
Ar
N
N
a Values of IC50 and EC50 are mean values determined from
g
g
7
8
b
Ph
Ph
at least 3 replicates. LE = –1.37 log IC50(PI4KIIIα)/number of
Ar
Ar
N
g
g
N
heavy atoms.
28 or 29
N
N
N
10
11
(b)
h
i
iv parameters (0.3 mg/kg)
po parameters (1.0 mg/kg)
MS in liver MS
(60 min, remain.%)
R3
R3
N
g
Ar
Ar
ClogP
t1/2
Cl
tot
Vdss
AUC
MRT
(hr)
F
β
Me
N
Ph
human
rat
(hr)
(L/hr/kg)
(L/kg)
(ꢀM·hr)
(%)
N
N
Me
Ar
9
14
15
13
16
17
N
2.4
104
91
3.3
0.040
0.20
42
5.5
91
N
N
j
j
12
(c)
F
S
F
4.5
4
H
N
i.v.
Ar =
O
O
p.o.
N
3.5
3
OMe
aReagents and conditions:
2.5
2
(a) (1) MsCl, Et3N, CH2Cl2,
0
°C – r.t.; (2) 2,4-
difluorothiophenol, i-Pr2NEt, THF, 0 °C – r.t.; (3) mCPBA,
CH2Cl2 , 0 °C – r.t.; (b) (1) NaH, CH3I, THF, 0 °C; (2) 2,4-
difluorobenzene-1-sulfonyl chloride, DMAP, pyridine, 60 °C
then 4 M NaOH, 1,4-dioxane, H2O, 90 °C; (c) (1) 2,6-
dimethoxybenzylamine, DMF, 120 °C, MW; (2) NaBH4,
MeOH, 0 °C – r.t.; (3) NaH, CH3I, THF, 0 °C – r.t.; (4) 2,4-
difluorobenzene-1-sulfonyl chloride, DMAP, pyridine, 60 °C;
(d) (1) trichloroisocyanuric acid, PhCONH2, CHCl3, 75 °C; (2)
c-PrOH, NaH, THF, r.t. then H2O, r.t.; (3) DPPA, Et3N, t-
BuOH, toluene, 100 °C; (4) TFA, CHCl3, r.t.; (5) 2,4-
difluorobenzene-1-sulfonyl chloride, pyridine, 60 °C then 4 M
NaOH, 1,4-dioxane, H2O, 60 °C; (6) (Bpin)2, PdCl2(dppf)-
CH2Cl2, KOAc, 1,4-dioxane, 100 °C; (e) (Bpin)2, PdCl2(dppf)-
CH2Cl2, KOAc, 1,4-dioxane, 100 °C; (f) 3-Bpin-5-R2-6-R1-
1.5
1
0.5
0
0
4
8
12 16 20 24 28
Time (hr)
Figure 3. (a) In vitro pharmacological profiles of 22 were
shown in tabular form. (b) Both in vitro ADME parameters
and rat PK parameters for 22 were shown in tabular form. (c)
Pharmacokinetic curve of 22 in rat. See the supporting in-
formation for detailed protocol.
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developed to discover selective PI4KIIIα inhibitors. After
a concise investigation, we have successfully identified
several classes of compounds showing a single-digit na-
nomolar value of IC50 against PI4KIIIα. They are com-
posed of different core structures, which could further
multiply the diversity after the successive tuning of the
accompanying substituents. We believe this method is
rational yet truly simple and this overall concept will also
be applicable to general kinase programs.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting information is available free of charge on the
ACS Publications website at DOI:
Synthetic procedures, experimental data, assay procedures
and PK profiles of 22 (PDF)
AUTHOR INFORMATION
Corresponding Author
* Phone: +81-72-681-9700. E-mail:makoto.shiozaki@jt.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Hiromasa Hashimoto for support.
ABBREVIATIONS
NS5A, Hepatitis C virus (HCV) nonstructural protein 5A;
PI3Kγ, Phosphoinositide 3-kinase gamma; PK, pharmacoki-
netics; LE, ligand efficiency.
REFERENCES
1) Gower, E.; Estes, C.; Blach, S. Razavi-Shearer, K.; Razavi,
H. Global epidemiology and genotype distribution of the
hepatitis C virus infection. J. Hepatol. 2014, 61, S45–S57.
2) Hepatitis
http://www.who.int/mediacentre/factsheets/fs164/en/
(accessed by May 17, 2016).
C.
16) PI4KIIIβ homology model shown in Figure 2b is a ho-
mology model (with no ligand molecule). We observed
that when creating the PI4KIIIβ complex model with the
compound 5 in the same manner as the complex model
of PI4KIIIα, compound 5 was shifted to the opposite di-
rection from the affinity pocket, indicating that the
methoxymethyl moiety was too large for PI4KIIIβ.
17) Peters, J.-U.; Schnider, P.; Mattei, P.; Kansy, M. Pharma-
cological Promiscuity: Dependence on Compound Prop-
erties and Target Specificity in a Set of Recent Roche
Compounds. ChemMedChem 2009, 4, 680–686.
18) The corresponding arylbromide means as follows; SI-17
for 7, SI-23 for 8, SI-24 for 10, SI-25 for 11, and SI-22 for 12.
Please also see the supporting information.
19) The corresponding arylBipn means as follows; SI-27 for 9
and SI-30 for 14.
3) Perz, J. F.; Armstromg, G. L.; Farrington, L. A.; Hutin, Y. J.
F.; Bell, B. P. The contributions of hepatitis B virus and
hepatitis C virus infections to cirrhosis and primary liver
cancer worldwide. J. Hepatol. 2006, 45, 529–538.
4) Brown Jr., R. S. Hepatitis C and liver transplantation. Na-
ture, 2005, 436, 973-978.
5) Liang, T. J.; Ghany, M. G. Therapy of Hepatitis C—Back
to the Future. N. Engl. J. Med. 2014, 370, 2043–2047.
6) Hahn, T. V.; Ciesek, S.; Manns, M. P. Arrest All Accesso-
ries-Inhibition of Hepatitis C Virus by Compounds that
Target Host Factors. Discov. Med. 2011, 12, 237–244.
7) Balla, A.; Balla T. Phosphatidylinositol 4-kinases: old en-
zymes with emerging functions. Trends Cell Biol. 2006,
16, 351–361.
8) Berger, K. L.; Cooper, J. D.; Heaton, N. S.; Yoon, R.; Oak-
land, T. E.; Jordan, T. X.; Mateu, G.; Grakoui, A.; Randall,
G. Roles for endocytic trafficking and phosphatidylinosi-
tol 4-kinase III alpha in hepatitis C virus replication.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 7577–7582.
20) SI-32 is the corresponding aryliodide for synthesis of 13 as
well as 16.
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Core Motifs
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Diverse Set of Kinase Inhibitors
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