2,6-Disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-dependent protein kinase (DNA-PK)

Article abstract of DOI:10.1016/S0960-894X(03)00652-8

6-Aryl-2-morpholin-4-yl-4H-pyran-4-ones and 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones were synthesised and evaluated as potential inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). Several compounds in each series exhibited super

Full text of DOI:10.1016/S0960-894X(03)00652-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3083–3086
2,6-Disubstituted Pyran-4-one and Thiopyran-4-one Inhibitors of
DNA-Dependent Protein Kinase (DNA-PK)
Jonathan J. Hollick,a Bernard T. Golding,a Ian R. Hardcastle,a Niall Martin,b
Caroline Richardson,b Laurent J. M. Rigoreau,a,b Graeme C. M. Smithb
and Roger J. Griffina,*
aNorthern Institute for Cancer Research, School of Natural Sciences-Chemistry, Bedson Building, University of Newcastle,
Newcastle Upon Tyne NE17RU, UK
bKuDOS Pharmaceuticals Limited, 327 Cambridge Science Park, Milton Road, Cambridge CB4 0WG, UK
Received 9 April 2003; accepted 16 May 2003
Abstract—6-Aryl-2-morpholin-4-yl-4H-pyran-4-ones and 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones were synthesised and eval-
uated as potential inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). Several compounds in each
series exhibited superior activity to the chromenone LY294002, and were of comparable potency to the benzochromenone NU7026
(IC50=0.23 mM). Importantly, members of both structural classes were found to be selective inhibitors of DNA-PK over related
phosphatidylinositol 3-kinase-related kinase (PIKK) family members. A multiple-parallel synthesis approach, employing Suzuki
cross-coupling methodology, was utilised to prepare libraries of thiopyran-4-ones with a range of aromatic groups at the 30- and 40-
positions on the thiopyran-4-one 6-aryl ring. Screening of the libraries resulted in the identification of 6-aryl-2-morpholin-4-yl-4H-
thiopyran-4-ones bearing naphthyl or benzo[b]thienyl substituents at the 40-position, as potent DNA-PK inhibitors with IC50 values
in the 0.2–0.4 mM range.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
been shown to potentiate the in vitro cytotoxicity of
ionising radiation5,6 and a number of anticancer
drugs.7À9 DNA-PK inhibitors may thus have clinical
utility as radio- and chemo-potentiators in the treat-
ment of cancer.
The nuclear serine/threonine protein kinase DNA-
dependent protein kinase (DNA-PK), a member of the
phosphatidylinositol 3-kinase-related kinase (PIKK)
family, comprises a large catalytic subunit (DNA-
PKcs), and a heterodimeric subunit (Ku).1À3 DNA-PK
is activated and assembles at the site of DNA double
strand breaks (DSBs), and is a crucial component of the
cellular DNA DSB repair machinery.4 Phosphorylation
of a number of target proteins has been observed in
vitro, including those involved in the cell cycle and
apoptosis.5 DNA-PK plays an essential role in the
physiological process of V(D)J recombination,2 and is
also involved in the repair of DNA DSBs induced by
ionising radiation and certain cancer chemotherapeutic
agents. Importantly, cells without competent DNA-PK
are hypersensitive to ionising radiation and radio-
mimetic agents, and inhibition of DNA-PK activity has
Although reportedly a selective ATP-competitive PI 3-
kinase (PI 3-K) inhibitor (IC50=1.5–2.0 mM),10,11 the
chromenone LY294002 (1) has also been shown to
exhibit comparable inhibitory activity against DNA-PK
(IC50=1.4 mM, Ki=6.0 mM).12 As part of a research
programme to develop potent and selective DNA-PK
inhibitors suitable for eventual clinical evaluation, we
have utilised 1 as a structural lead. In the absence of
suitable crystal structure information for DNA-PK, a
*Corresponding author. Tel.: +44-191-222-8591; fax: +44-191-222-
8591; e-mail: r.j.griffin@ncl.ac.uk
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00652-8

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J. J. Hollick et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3083–3086
substituted aroylacetone (8) was readily prepared by
acylation of the appropriate acetophenone (7) with ethyl
acetate–sodium ethoxide. Treatment of the dianion of 8
with carbon disulfide afforded, upon carefully con-
trolled workup, the 2-mercapto-6-arylthiopyran-4-one
(9), and alkylation of the thiol group of 9 with iodo-
ethane gave 10 quantitatively. Final displacement of the
2-thioethyl substituent was effected by heating 10 with
morpholine in ethane-1,2-diol to yield the target thio-
pyran-4-ones 4a–f.15
Scheme 1. General synthesis of pyran-4-ones. Reagents and condi-
tions: (i) (a) Ph3P=CHCO2Me, PhMe, reflux; (b) 250 ꢁC; (ii) n-BuLi,
(i-Pr)2NH, N-acetylmorpholine, À78 ꢁC!0 ꢁC; (iii) MeSO3H, 25 ꢁC.
Although unambiguous SARs are not apparent from
the initial compounds synthesised, the nature and posi-
tion of the 6-aryl substituent clearly influences DNA-
PK inhibitory activity in both series (Table 1). In addi-
tion, preliminary in vitro studies with the pyran-4-ones
(3) and thiopyran-4-ones (4) suggest that the latter series
might have superior cellular activity, despite the slightly
higher potency of the pyran-4-ones against DNA-PK.
In order to investigate this further, a multiple-parallel
synthesis (MPS) approach was utilised to generate two
libraries with a range of aromatic groups at the 30- and
40-positions on the thiopyran-4-one 6-aryl ring. The
required libraries were prepared by a Suzuki cross-cou-
pling reaction between the 30- and 40-bromophenyl sub-
stituted thiopyran-4-ones (4e and 4f), and a range of
commercially available boronic acids and esters
(Scheme 2).16 A total of 97 thiopyran-4-one derivatives
(42 from 4e and 55 from 4f) were deemed of suitable
purity (ꢀ85%) for evaluation in a pre-screen DNA-PK
assay, from which six compounds emerged as suffi-
ciently potent for IC50 values to be determined (Table 2).
pharmacophore mapping approach has been exploited
for the delineation of structure–activity relationships
(SARs) around the core structure of 1. These studies
have resulted in the identification of a number of struc-
turally diverse inhibitors, which are more potent and
selective than 1, as exemplified by NU7026 (2)
(IC50=0.23 mM).13 Further refinement of the pharma-
cophore model indicated that, whereas the 2-morpholin-
4-yl substituent appears to be a prerequisite for inhibi-
tory activity, ‘minimalisation’ of the chromenone core
structure might be tolerated. In this paper we describe
the synthesis and preliminary biological evaluation of
DNA-PK inhibitors derived from the monocyclic
pyran-4-one (3) and thiopyran-4-one (4) templates.
Chemical Synthesis
The required 2,6-disubstituted pyran-4-ones (3) were
prepared by a literature procedure as summarised in
Scheme 1.14 Briefly, treatment of the appropriate ben-
zoyl chloride with methyl triphenylphosphoranylidine,
gave the phenylpropiolate ester (5), which furnished the
phenylacetylenic b-ketoamide (6) on reaction with the
O-lithio derivative of acetylmorpholine. Ring closure of
6 to the required pyran-4-one (3) was achieved with
methanesulfonic acid. To the best of our knowledge, the
corresponding 6-aryl-2-morpholin-4-ylthiopyran-4-ones
(4) have not been reported previously, and the synthesis
of this heterocycle is summarised in Scheme 2. The
Results and Discussion
Our previous studies have demonstrated that selective
inhibition of DNA-PK, over other PIKK family mem-
bers, is achievable through modification of the chrome-
none pharmacophore of 1. For example, NU7026 (2) is
approximately 6-fold more potent than 1 as a DNA-PK
inhibitor, and at least 70-fold more selective for DNA-
PK over PI 3-K (p110a), whereas 1 is essentially equi-
potent against both kinases.13,17 However, the ben-
zo[h]chromenone template of 2 is not readily amenable
Scheme 2. General synthesis of thiopyranones. Reagents and conditions: (i) NaOEt, EtOAc, THF, 25 ꢁC; (ii) LDA, THF, CS2, À78!25 ꢁC; (iii)
H2O, 0 ꢁC!25 ꢁC, HCl (aq); (iv) K2CO3, EtI, (Me)2CO, reflux; (v) morpholine, (CH2OH)2, 120!150 ꢁC; (vi) Pd(PPh3)4, K2CO3, ArB(OH)2, diox-
ane, 90 ꢁC.

J. J. Hollick et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3083–3086
Table 1. Inhibition of DNA-PK by selected pyranone-4-ones and thiopyran-4-onesa
3085
Compd
X
R
IC50 (mM)b
Compd
X
R
IC50 (mM)b
1
2
—
—
O
O
O
O
O
O
—
—
H
3-F
1.4
3g
3h
4a
4b
4c
4d
4e
4f
O
O
S
S
S
S
S
S
4-MeO
4-tBu
H
0.22
0.48
0.72
0.53
0.28
0.92
0.80
0.68
0.23
1.10
0.53
0.35
0.18
0.38
0.54
3a
3b
3c
3d
3e
3f
4-Cl
4-F
4-MeO
4-tBu
3-Br
4-Cl
2-MeO
3-MeO
4-Br
aRef 17.
bValues are the means of at least three separate determinations.
Table 2. DNA-PK-inhibitory activity of thiopyran-4-ones identified from library screensa
Compd
Ar
IC50 (mM)a
Compd
Ar
IC50 (mM)a
11a
0.92
12a
0.33
11b
11c
0.35
0.84
12b
12c
0.19
0.26
aLibrary compounds were assayed for % inhibition at 1.0, 0.5 and 0.1 mM. IC50 values were determined for compounds exhibiting >50% inhibition
at 1.0 mM, according to ref 17.
to extensive chemical modification, and in particular the
introduction of substituents onto the fused aromatic
ring system. Accordingly, it was envisaged that a 6-
arylpyran-4-one or 6-arylthiopyran-4-one template,
bearing the essential 2-morpholin-4-yl group, would
offer more opportunities for introducing structural
diversity in the aromatic region, while retaining the core
pharmacophore elements common to 1 and 2.
that a lipophilic substituent at the 40-position of the
6-aryl ring is favoured for both series, with two pyran-4-
ones (3d and 3g) and one thiopyran-4-one (4c) exhibit-
ing activity comparable with NU7026 (2). Perhaps more
importantly, the pyran-4-one/thiopyran-4-one template
was found to retain the selectivity for DNA-PK
observed for the benzo[h]chromenone scaffold. For
example, 3g was found to be at least 200-fold more
potent an inhibitor of DNA-PK (IC50=0.22 mM) com-
pared with the other PIKK family members PI 3-K
(p110a), ATM and ATR (IC50 >50 mM). A similar
degree of selectivity has also been observed in the thio-
pyran-4-one series.
All of the pyran-4-ones (3a–3h) and thiopyran-4-ones
(4a–4f) synthesised initially are more potent than
LY294002 (1), with the pyran-4-ones generally tending
to be at least equipotent (compare 3g with 4c) or slightly
more potent (compare 3d with 4b, and 3h with 4d) than
their thiopyran-4-one counterparts. An exception arises
with the parent 6-phenyl derivatives, where the thio-
pyran-4-one (4a) is marginally more active than the
corresponding pyran-4-one (3a). Although definitive
SAR correlations are clearly not possible, it is evident
Initial evaluation of the two thiopyran-4-one compound
libraries (11 and 12) for DNA-PK inhibitory activity
revealed a wide range of activities, with library members
bearing an aryl substituent at the 40-position (12) gen-
erally proving more potent than those with a 30-aryl

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J. J. Hollick et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3083–3086
group (11). Three compounds from each library (11a–
11c) and (12a–12c) were identified from this pre-screen
as being sufficiently potent to warrant IC50 determina-
tions (Table 2). Although there are no large differences
in activity within this compound set, those thiopyran-4-
ones with a 40-aryl substituent (12a–12c) tend to be the
more potent, consistent with the overall trend observed
for the initial two libraries. Perhaps more interesting are
the clear structural similarities between the 40-aryl sub-
stituents on 12a–12c, which comprise 1-naphthyl and 1-
(4-methyl)naphthyl (12b and 12c), and the isosteric 3-
thianaphthene (3-benzo[b]thienyl) group (12a). These
three thiopyran-4-ones exhibit DNA-PK inhibitory
activity comparable with that of NU7026 (2), and are
among the most potent DNA-PK inhibitors reported to
date. In summary, we have established that pyran-4-
ones and thiopyran-4-ones are versatile platforms for
the development of potent and selective DNA-PK inhi-
bitors, and have utilised a simple library approach for
the identification of a potentially interesting class of
inhibitors derived from the thiopyran-4-one pharmaco-
phore. Further studies are underway to optimise the
biological and pharmaceutical properties of this new
series of DNA-PK inhibitors.
7. Boulton, S.; Kyle, S.; Durkacz, B. W. Eur. J. Cancer 2000,
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B. W.; Golding, B. T.; Hardcastle, I. R.; Leahy, J. J. J.; Mar-
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15. All new compounds exhibited spectral (1H NMR, IR, UV)
and analytical (elemental analysis and/or LC-MS) data fully
consistent with the assigned structures.
16. A general procedure for the MPS of thiopyran-4-ones is as
follows: A solution of 4e or 4f (20 mg, 0.057 mmol) in dioxane
(1 mL) was sonicated under N2 for 5 min, before addition to a
reaction tube containing the appropriate arylboronic acid
(0.0625 mmol) and K2CO3 (19 mg, 0.14 mmol). A solution of
tetrakis(triphenylphosphine)palladium(0) (3 mg) in dioxane
(0.3 mL) was added, and the reaction mixture was stirred
under N2 at 90 ꢁC for 18 h. After cooling, the product was
isolated by chromatography on silica (isolute Si 500 mg car-
tridge), eluting with CH2Cl2:MeOH (3:1, 3 mL). Further puri-
fication by preparative HPLC furnished the required
thiopyranones in 5–60% yield.
Acknowledgements
The authors thank Cancer Research UK and the
BBSRC (Studentship to J.J.H.) for financial support.
17. The DNA-PK used for in vitro assays was purified from
HeLa cell nuclear extract. The known ability of DNA-PK to
phosphorylate the serine-15 residue of a p53 peptide in vitro
was exploited in a classic ELISA style assay using an antibody
that only recognises the p53 serine-15 site when phosphory-
lated (Cell Signalling Technology). The primary antibody to
p53 phosphoserine-15 was detected using an HRP conjugated
goat anti-rabbit antibody (Pierce) with ECL reagent (NEN)
being used for the readout. The ability of compounds to inhi-
bit this phosphorylation event was monitored over a concen-
tration range with IC50 values generated from these results.
Full details of the assay will be published elsewhere.
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