DNA-dependent protein kinase (DNA-PK) inhibitors. synthesis and biological activity of quinolin-4-one and pyridopyrimidin-4-one surrogates for the chromen-4-one chemotype

Article abstract of DOI:10.1021/jm100608j

Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H- chromen-4-one (NU7441) (Leahy, J. J. J.; Golding, B. T.; Griffin, R. J.; Hardcastle, I. R.; Richardson, C.; Rigoreau, L.; Smith, G. C. M. Bioorg. Med. Chem. Lett. 2004, 14, 6083-6087) as a potent inhibitor (IC₅₀ = 30 nM) of DNA-dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the 1-position with water-solubilizing groups [NHCO(CH ₂)_nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors were synthesized by employing a multiple-parallel approach in which the two heterocyclic components were assembled by Suzuki-Miyaura cross-coupling. Potent DNA-PK inhibitory activity was generally observed across the compound series, with structure activity studies indicating that optimal potency resided in pyridopyrimidin-4- ones bearing a substituted dibenzothiophen-4-yl group. Several of the newly synthesized compounds (e.g., 2-morpholin-4-yl-N-[4-(2-morpholin-4-yl-4-oxo-4H- pyrido[1,2-a]pyrimidin-9-yl)dibenzothiophen-1-yl]acetamide) combined high potency against the target enzyme (DNA-PK IC₅₀ = 8 nM) with promising activity as potentiators of ionizing radiation-induced cytotoxicity in vitro.

Full text of DOI:10.1021/jm100608j

8498 J. Med. Chem. 2010, 53, 8498–8507
DOI: 10.1021/jm100608j
DNA-Dependent Protein Kinase (DNA-PK) Inhibitors. Synthesis and Biological Activity
of Quinolin-4-one and Pyridopyrimidin-4-one Surrogates for the Chromen-4-one Chemotype
Celine Cano,*,† Olivier R. Barbeau,† Christine Bailey,‡ Xiao-Ling Cockcroft,‡ Nicola J. Curtin,† Heather Duggan,‡
ꢀ
Mark Frigerio,‡ Bernard T. Golding,† Ian R. Hardcastle,† Marc G. Hummersone,‡ Charlotte Knights,‡ Keith A. Menear,‡
David R. Newell,† Caroline J. Richardson,‡ Graeme C. M. Smith,‡ Ben Spittle,‡ and Roger J. Griffin†
†Newcastle Cancer Centre, Northern Institute for Cancer Research, School of Chemistry, Bedson Building, Newcastle University,
Newcastle upon Tyne, NE1 7RU, United Kingdom, and ‡KuDOS Pharmaceuticals, Ltd., 410 Cambridge Science Park, Milton Road,
Cambridge, CB4 0PE, United Kingdom
Received May 19, 2010
Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441)
(Leahy, J. J. J.; Golding, B. T.; Griffin, R. J.; Hardcastle, I. R.; Richardson, C.; Rigoreau, L.; Smith,
G. C. M. Bioorg. Med. Chem. Lett. 2004, 14, 6083-6087) as a potent inhibitor (IC50 = 30 nM) of DNA-
dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core
template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]-
pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents
were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the
1-position with water-solubilizing groups [NHCO(CH2)nNR1R2, where n=1 or 2 and the moiety
R1R2N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors
were synthesized by employing a multiple-parallel approach in which the two heterocyclic components
were assembled by Suzuki-Miyaura cross-coupling. Potent DNA-PK inhibitory activity was generally
observed across the compound series, with structure-activity studies indicating that optimal potency
resided in pyridopyrimidin-4-ones bearing a substituted dibenzothiophen-4-yl group. Several of the
newly synthesized compounds (e.g., 2-morpholin-4-yl-N-[4-(2-morpholin-4-yl-4-oxo-4H-pyrido[1,2-a]-
pyrimidin-9-yl)dibenzothiophen-1-yl]acetamide) combined high potency against the target enzyme
(DNA-PK IC50 = 8 nM) with promising activity as potentiators of ionizing radiation-induced
cytotoxicity in vitro.
Introduction
incorporation of a dibenzothiophen-4-yl (2, NU7441)12 or
dibenzofuran-4-yl substituent at the chromenone 8-position
conferred high inhibitory activity against DNA-PK.13 In addi-
tion, 2 exhibited good selectivity for DNA-PK (IC50 = 30 nM)
over other PIKK family members and a panel of diverse
kinases. Chromenone 2 has also been demonstrated to sensitize
a human tumor cell line to both ionizing radiation and the
topoisomerase II inhibitor etoposide in vitro and in vivo.16
With a view to improving the pharmaceutical properties of 2
and related compounds, the introduction of water-solubilizing
substituents at the dibenzothiophene 1-position was investi-
gated, leading to the identification of 3 as a highly potent
(IC50 = 6 nM) DNA-PK inhibitor with promising biological
activity, and further studies with this compound are in pro-
gress.17 However, we have accrued some evidence to suggest
that 3 does not entirely resemble 2 with regard to kinase
selectivity, and potentially problematic off-target activity has
been observed for 3. This has prompted the investigation of
alternative heterocycles to the chromen-4-one of 2and 3, aswell
as the introduction of water-solubilizing groups elsewhere on
the molecule.
DNA-dependent protein kinase (DNA-PKa), a member of
the phosphatidylinositol (PI) 3-kinase related kinase (PIKK)
family, is a multicomponent serine/threonine protein kinase
that plays a key role in the repair of mammalian DNA double-
strand breaks (DSBs) via the nonhomologous end joining
pathway of DNA repair.1,2 Human cell lines defective in
DNA-PK function are hypersensitive to agents that elicit
DNA DSBs.3,4 By impeding DNA DSB repair, selective
DNA-PK inhibitors have potential application as radio-
and chemopotentiators in the treatment of cancer.5-9 By use
of 2-morpholino-8-phenyl-4H-chromen-4-one (1, LY294002)10
as a template for inhibitor design, a number of potent DNA-PK
inhibitors have been developed and structure-activity relation-
ships (SARs) have evolved for these structural classes.11-15 In
these inhibitors the 2-morpholino-4H-chromen-4-one moiety
is connected at the 8-position to an aryl or heteroaryl ring,
which can be substituted or unsubstituted.12,13,15 Notably, the
*To whom correspondence should be addressed. Phone: 44 (0)
1912227060. Fax: þ44 (0) 191 222 5934. Email: celine.cano@ncl.ac.uk.
a
Abbreviations: DNA-PK, DNA-dependent protein kinase; IC50
,
Early SARs conducted with benzo[h]chromen-4-ones
(e.g., 4, IC50 = 230 nM) and the isosteric pyrimido[1,2-a]iso-
quinolin-4-one (5, IC50 = 280 nM) indicated a comparable
pattern of DNA-PK inhibitory activity for a series of derivatives,
concentration of inhibitor leading to 50% inhibition; SAR, structure-
activity relationship; PI, phosphatidylinositol; PIKK, phosphatidylino-
sitol 3-kinase related kinase; DSBs, double-strand breaks; DMR, dose-
modification ratio; IR, ionizing radiation.
r
pubs.acs.org/jmc
Published on Web 11/16/2010
2010 American Chemical Society

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8499
suggesting that these molecules make similar binding interac-
tions within the ATP-binding domain of the kinase.11
Nitroboronate ester 22 was coupled to 9-hydroxy-2-morpho-
lin-4-ylpyrido[1,2-a]pyrimidin-4-one 9-O-triflate18 and 8-bromo-
2-morpholin-4-yl-1H-quinolin-4-one18 via Suzuki-Miyaura re-
actions to give 24 and 25. The nitro intermediates were reduced to
the required amines 26 and 27 in excellent yields using zinc in
acetic acid (Scheme 2).
A similar sequence was utilized with 1-nitrodibenzofuran-
4-yl boronate 23. The Suzuki-Miyaura reactions were per-
formed in a microwave reactor, giving better yields of com-
pounds 28 and 29 in shorter reaction times than under
classical conditions. The nitro intermediates 28 and 29 were
reduced to the required amines 30 and 31 in excellent yields
employing zinc in acetic acid (Scheme 3).
Library Syntheses. The effect upon biological activity of
substitution at the 1-position of the dibenzothiophen-4-yl
and dibenzofuran-4-yl moieties was investigated through the
preparation of focused libraries employing a solution multi-
ple-parallel approach. Acylation of arylamines 26, 27, 30,
and 31 with a suitably functionalized acid chloride (chloro-
acetyl chloride or bromopropionyl chloride) enabled access
to eight compound libraries, following reaction with a range
of amines (Scheme 4 and Table 1).
Results and Discussion
The principal objective of the studies described in this paper
was to evaluate the biological activities of DNA-PKinhibitors
incorporating a quinolin-4-one or pyridopyrimidin-4-one
scaffold and bearing substituted acylamino groups at the
1-position on the pendent dibenzothiophen-4-yl or dibenzo-
furan-4-yl ring. We also wished to compare the biological
activity of these two series with the isosteric chromen-4-ones
from which 3 is derived. The DNA-PK inhibitory activity and
dose-modification ratio (DMR) values for library compounds
of suitable purity are shown in Tables 1 and 2.
We have subsequently reported the synthesis and DNA-
PK-inhibitory activity of derivatives of the pyridopyrimidin-
4-ones (6, 7) and quinolin-4-ones (8, 9).18 Following previous
studies with chromen-4-ones, indicating that the 1-position of
the dibenzothiophen-4-yl moiety was tolerant to substitution,
the introduction of water-solubilizing groups was investigated
at this position on the pyridopyrimidin-4-one and quinolin-4-
one series. In the present paper, we describe the synthesis and
biological evaluation of focused libraries of pyridopyrimidin-
4-one and quinolin-4-one DNA-PK inhibitors bearing water-
solubilizinggroupsatthe dibenzothiophene anddibenzofuran
1-position.
Structure-Activity Relationships for DNA-PK Inhibition.
The introduction of amine-substituted acylamino groups at
the dibenzothiophene 1-position on the pyridopyrimidin-4-
one (32-42, 52-58) and quinolin-4-one (43-51, 59-64)
systems was generally well tolerated, with most compounds
exhibiting DNA-PK inhibitory activity in the nanomolar
range (Table 1). This compared favorably with the activities
of the parent pyridopyrimidin-4-one (6, IC50=13 nM) and
quinolin-4-one (8, IC50 =8 nM). Where comparisons were
possible, inhibitors based on the pyridopyrimidin-4-one
template were generally more potent than the corresponding
quinolinones (compare 32 with 44, and 52 with 59), although
some exceptions are evident (e.g., 57 and 62, and 38 and 50).
The nature of the alkyl group between the acylamino sub-
stituent and the amine function did not appear to markedly
influence potency against DNA-PK, with a 1-carbon (32-51)
or 2-carbon (52-64) spacer conferring comparable activity
across the library series in most cases. However, homologation
of the spacer group for pyridopyrimidin-4-ones bearing a
morpholin-4-yl or cis-2,6-dimethylmorpholin-4-yl group
(compare 32 and 40 with 52 and 58) compromised the high
potency of the parent compounds 32 (IC50 = 8 nM) and 40
(IC50 = 7 nM) in both cases. Similar trends were also observed
for the small compound library where an isosteric dibenzofuran-
4-yl group replaced dibenzothiophen-4-yl on the pyridopyrimi-
dinone (65-81) and quinolinone (71-83) templates (Table 2).
With a view to delineating SARs in more detail, relation-
ships were analyzed as illustrated in Figure 1. With the exclu-
sion of compounds 36 and 48, where compound 36 had
Chemistry
The structures of all compounds synthesized and evaluated
for biological activity are recorded in Tables 1 and 2. Inter-
mediates required for the synthesis of pyridopyrimidin-4-one
and quinolin-4-one DNA-PK inhibitors were prepared fol-
lowing standard procedures. The preparation of 4-hydroxy-
dibenzothiophene 12 and 4-hydroxydibenzofuran 13 was
achieved by reaction of 4-lithiodibenzothiophene or 4-lithio-
dibenzofuran, respectively, with dioxygen in the presence of
methylmagnesium bromide.19 Nitration of these phenols gave
appreciable ortho-nitration. This was avoided by methylation
of the hydroxyl groups of 12 and 13 to afford 14 and 15,
respectively, with subsequent nitration occurring exclusively
at the 1-position to afford 16 and 17. Deprotection of the
methyl ethers to the corresponding phenols followed by
reaction with triflic anhydride/triethylamine gave the required
triflates 20 and 21. The 4-O-triflates were readily converted
into the corresponding boronates 22 and 23 by treatment with
bis(pinacolato)diboron (Scheme 1).

8500 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Cano et al.
Table 1. Chemical Structures and Biological Activity of Dibenzothiophen-4-yl Substituted Quinolinones and Pyridopyrimidin-4-ones
a IC50 values were determined over a range of different concentrations, usually from 0.01 to 10 μM. Values are the mean of at least three separate
determinations.
unexpectedly poor activity (IC50 = 175 nM), there was a
linear correlation between the DNA-PK inhibitory activities
of the dibenzothiophen-4-yl pyridopyrimidin-4-ones and
quinolin-4-ones (r = 0.69, r2 = 0.48, p = 0.02, Figure 1A).
Furthermore, overall, the dibenzothiophen-4-ylpyridopyrimi-
din-4-one DNA-PK inhibitors were more potent than the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8501
Table 2. Chemical Structures and Biological Activity of Dibenzothiofuran-4-yl Substituted Quinolinones and Pyridopyrimidin-4-ones
a IC50 values were determined over a range of different concentrations, usually from 0.01 to 10 μM. Values are the mean of at least three separate
determinations.
Scheme 1a
a Reagents and conditions: (a) (i) n-BuLi, THF, reflux; (ii) MeMgBr, O2, 25 °C, 39% for 12 and 36% for 13; (b) MeI, K2CO3, acetone, reflux, 96% for
14 and 99% for 15; (c) HNO3, AcOH, 25 °C, 99% for 16 and 48% for 17; (d) pyridine hydrochloride, 150 °C, 41% for 18 and 99% for 19; (e) triflic
anhydride, NEt3, DCM, 0 °C, 98% for 20 and 94% for 21; (f) bis(pinacolato)diboron, KOAc, PdCl2(dppf), dppf, dioxane, 95 °C, 66% for 22 and 87%
for 23.
corresponding quinolin-4-one inhibitors (paired t test, p =
0.024). In an analogous manner to the dibenzothiophen-4-yl
quinolinone and pyridopyrimidin-4-one inhibitors and with the
exclusion of compounds 79 and 82 where compound 82 had

8502 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Cano et al.
Scheme 2a
a Reagents and conditions: (a) pyridopyrimidinone triflate18 or bromoquinolinone,18 PdCl2(dppf), Cs2CO3, THF, 85 °C, 92% for 24 and 20% for 25;
(b) Zn, AcOH, 25 °C, 99% for 26 and 85% for 27.
Scheme 3a
a Reagents and conditions: (a) pyridopyrimidin-4-one triflate, Pd(PPh3)4, K2CO3, dioxane, 180 °C, microwave, 30 min, 69% for 28 and 60% for 29;
(b) Zn, AcOH, 25 °C, 96% for 30 and 91% for 31.
unexpectedly poor activity (IC50 = 314 nM), there was also a
linear correlation between the DNA-PK inhibitory activities of
the dibenzofuran-4-ylquinolinones and the pyridopyrimidin-4-
ones (r = 0.85, r2 =0.73, p=0.01, Figure 1B). As with the
dibenzothiophen-4-yl DNA-PK inhibitors, the dibenzofuran-
4-ylpyridopyrimidin-4-one DNA-PK inhibitors were more
potent than the corresponding quinolinone inhibitors (paired
t test, p = 0.004), confirming that in both series pyridopy-
rimidin-4-one is the preferred core pharmacophore for DNA-
PK inhibitor potency. Lastly, the relationship between the
DNA-PK inhibitory potency of the dibenzothiophen-4-yl and
dibenzofuran-4-yl substituents on the quinolin-4-one and pyr-
idopyrimidin-4-one scaffolds was studied, data for 17 pairs of
compounds being available, i.e., 10 pyridopyrimidin-4-ones
and 7 quinolinones (Tables 1 and 2). For the pyridopyrimi-
din-4-ones there was a significant linear correlation between the
DNA-PK inhibitory potency of the dibenzothiophen-4-yl and
dibenzofuran-4-yl inhibitors (r = 0.70, r2 = 0.49, p = 0.02,
Figure 1C), with the dibenzothiophen-4-yl inhibitors being in
general more potent that the dibenzofuran-4-yl derivatives. By
contrast, there was no relationship between the inhibitory
potencies of the dibenzothiophen-4-yl and dibenzofuran-4-yl
derivatives in the quinolin-4-one series, reflecting the lack of
significant SAR for the dibenzofuran-4-yl compounds, although
this analysis was necessarily restricted to only seven pairs of
compounds (Figure 1C).
Cell-Based Studies. The dose modification ratio (DMR) is
defined as the ratio of the number of cells that survive a single
2 Gy dose of ionizing radiation (IR) to that of the number of
cells that survive the same dose of (IR) in combination with a
given concentration of DNA-PK inhibitor. This value pro-
vides an indirect measure of the ability of a particular
compound to potentiate the DNA damage elicited by IR
and also indicates whether or not the compound in question
is cell permeable. IR alone at the dose used (2 Gy) caused
40-60% survival. The DNA-PK inhibitors alone, at the
concentrations used (0.5 and 0.1 μM), were not cytotoxic,
and hence, the dose modification observed represents radio-
potentiation and not additive toxicity.
In cell-based studies (HeLa cells), the DNA-PK inhibitors
potentiated IR-induced cell killing at 0.5 μM, with DMRs of
over 10 being observed for selected compounds (i.e., >10-fold

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8503
Scheme 4a
a Reagents and conditions: (a) chloroacetyl chloride or bromopro-
pionyl chloride, NEt3, DMA, 25 °C; (b) HNR1R2, DMA, 25 °C.
enhanced cell killing at a dose of 2 Gy radiation). However, the
dibenzothiophen-4-yl-substituted pyridopyrimidin-4-one inhi-
bitors demonstrated superior activity in the cell-based assay
and hence are the preferred chemotype for further investiga-
tion. To investigate the impact of DNA-PK inhibitory potency
on the potentiation of IR-mediated cytotoxicity, the relation-
ship between DNA-PK inhibition (IC50) and the DMR was
studied. When the dibenzothiophen-4-yl-substituted pyrido-
pyrimidin-4-one inhibitors were used, there was a trend toward
greater potentiation (i.e., higher DMR values) with the more
potent DNA-PK inhibitors. Thus, compounds 34, 38, 40, and
53 produced a greater than 10-fold potentiation of IR-induced
cytotoxicity (Table 1, Figure 2A). However, although there was
a trend toward higher DMR values for the more potent
dibenzothiophen-4-yl-substituted pyridopyrimidin-4-one
DNA-PK inhibitors, this trend was not significant at the
5% level (r = 0.37, p = 0.14), indicating that there are
additional determinants of the cellular activity, compound
solubility and cell permeability most probably being key
factors. A similar analysis was performed for the diben-
zothiophen-4-yl-substituted quinolin-4-one inhibitors
(Figure 2B). Notably, for this series there was no relation-
ship between DNA-PK inhibition and potentiation of
IR-induced cytotoxicity, the majority of compounds generat-
ing DMR values of only 2-4 (Table 1). Two dibenzothio-
phen-4-yl-substituted quinolinone DNA-PK inhibitors,
namely, 46 and 48, produced notable IR potentiation with
DMR values of 8.2 and 5.8, respectively. However, as a class,
none of these DNA-PK inhibitors generated DMR values of
>10, and the lack of even a tentative relationship between
potency of DNA-PK inhibition and potentiation of IR-induced
cytotoxicity further limits interest in this class of inhibitors. The
promising results observed for DNA-PK inhibitors in the IR
potentiation studies at 0.5 μM (Table 1) prompted the evalua-
tion of selected compounds at 0.1 μM. Encouragingly, 32, 34,
and 52 also displayed pronounced potentiation (DMR > 3) at
the lower concentration, identifying these pyridopyrimidin-4-
ones as potential candidates for further investigations.
Figure 1. (A) Correlation between DNA-PK inhibition for dibenzo-
thiophen-4-ylpyridopyrimidin-4-ones and quinolinones. Each point
represents data for a pair of compounds with otherwise identical
structures. Data are from Table 1, and the line is that given by
unweighted linear regression analysis. (B) Correlation between the
DNA-PK inhibition for dibenzofuran-4-ylpyridopyrimidin-4-ones
and quinolinones. Each point represents data for a pair of compounds
with otherwise identical structures. Data are from Table 2, and the line
is that given by unweighted linear regression analysis. (C) Relationship
between the DNA-PK inhibition for dibenzothiophen-4-yl- and dibenzo-
furan-4-ylquinolinone and pyridopyrimidin-4-one DNA-PK inhibi-
tors. Each point represents data for a pair of compounds with otherwise
identical structures. Data are from Tables 1 and 2, and the line is that
given by unweighted linear regression analysis of the relationship
between the DNA-PK inhibitory activity of dibenzothiophen-4-yl-
and dibenzofuran-4-ylpyridopyrimidin-4-ones.
Again, with regard to cellular activity, quinolinone and
the pyridopyrimidin-4-one DNA-PK inhibitors bearing a

8504 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Cano et al.
Conclusions
In this paper, we have investigated the possibility of replacing
the chromen-4-one scaffold of previously identified DNA-PK
inhibitors, with isosteric pyridopyrimidin-4-one and quinolin-
4-one heterocycles, and have elucidated the effects of introdu-
cing prospective water-solubilizing groups on the pendent
dibenzothiophen-4-yl and dibenzufuran-4-yl substituents. Sub-
stitution at the dibenzothiophene and dibenzofuran 1-positions
by a range of acylamino groups was generally tolerated without
detriment to DNA-PK inhibitory activity. Delineation of SARs
for DNA-PK inhibition indicated a preference for the pyrido-
pyrimidin-4-one core heterocycle over the corresponding qui-
nolin-4-one, with 1-substituted dibenzothiophen-4-yl proving
superior to the dibenzofuran-4-yl isostere. This trend also
extended to cell-based radiopotentiation studies in vitro, where
pyridopyrimidin-4-ones bearing 1-substituted dibenzothio-
phen-4-yl groups exhibited excellent potentiation of IR-induced
cytotoxicity in the HeLa tumor cell line [e.g., 32, DNA-PK
IC50=8 nM, DMR (0.1 μM)=3.5 and 34, DNA-PK IC50
=
22 nM, DMR (0.1 μM) = 4]. Encouragingly, these values are
comparable with those obtained for the analogous chromen-
4-one DNA-PK inhibitor 3 [IC50=6 nM, DMR (0.1 μM)=
3.2],17 and further studies are in progress.
Experimental Section
Solvents were purified and stored according to standard
procedures. Petrol refers to that fraction of hexanes boiling in
the range 40-60 °C. Melting points were obtained on a Stuart
Scientific SMP3 apparatus and are uncorrected. TLC was per-
formed with Merck 60 F254 silica gel plates. “Flash” column
chromatography was conducted under medium pressure on silica
(Merck silica gel 40-63 μm). HPLC purification were performed
on Gilson LC instruments, with a 15 min gradient of 0.1% aque-
ous TFA and 10-97% acetonitrile, at a flow rate of 6 mL/min,
using as the stationary phase a Jones Chromatography Genesis
4 μm C18 column, 10 mm ꢀ 250 mm, and peak acquisition based
on UV detection at 254 nm. Solution-phase palladium-mediated
coupling reactions were conducted in greenhouse reactors
1
(Radley’s Ltd., U.K.) under an argon atmosphere. H NMR
and 13C NMR spectra were recordedona Bruker Spectrospin AC
300E spectrometer (300 MHz for 1H, 75 MHz for 13C) or a
Bruker AMX (500 MHz for 1H, 126 MHz for 13C) using CDCl3
as solvent, unless indicated otherwise. LCMS was carried out
either on a Micromass Platform instrument operating in posi-
tive and negative ion electrospray mode, employing a 50 mm ꢀ
4.6 mm C18 column (Supelco Discovery or Waters Symmetry)
and a 15 min gradient elution of 0.05% formic acid and methanol
(10-90%), or on a Finnegan LCQ instrument in positive ion
mode with a Phenomenex 5 μm Luna C18 column, 4.6 mm ꢀ
50 mm, and an 8 min gradient of 0.1% aqueous formic acid and
acetonitrile (5-98%) with a flow rate of 2 mL/min. Library
compounds were found to be >95% pure by HPLC and NMR
analyses. IR spectra were recorded on a Bio-Rad FTS 3000MX
diamond ATR. Accurate masses were measured using a Finnigan
MAT 95 XP or a Finnigan MAT 900 XLT by the EPSRC
National Mass Spectrometry Service Centre, Swansea, SA2
8PP, U.K.
Figure 2. Relationship between potentiation of IR-induced cytotoxi-
city (DMR) and DNA-PK inhibitory potency (IC50). (A) Relationship
between DNA-PK inhibition and potentiation of IR-induced cytotoxi-
city by dibenzothiophen-4-ylpyridopyrimidin-4-one DNA-PK inhibi-
tors. Each point represents data for an individual compound and are
from Table 1. (B) Relationship between DNA-PK inhibition and
potentiation of IR-induced cytotoxicity by dibenzothiophen-4-ylquino-
linone DNA-PK inhibitors. Each point represents data for an individual
compound and are from Table 1. (C) Relationship between DNA-PK
inhibition and potentiation of IR-induced cytotoxicity by dibenzofuran-
4-ylpyridopyrimidin-4-one DNA-PK inhibitors. Each point represents
data for an individual compound and are from Table 2.
9-(1-Nitrodibenzothiophen-4-yl)-2-morpholin-4-ylpyrido[1,2-a]-
pyrimidin-4-one (24). In a Schlenk tube, 1-nitro-4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)dibenzothiophene 22 (0.98 g,
2.77 mmol) and cesium carbonate (2.71 g, 8.30 mmol) were mixed
in THF (8 mL) and degassed. Concurrently, 9-hydroxy-2-mor-
pholin-4-ylpyrido[1,2-a]pyrimidin-4-one 9-O-triflate16 (1.15 g,
3.05 mmol) and PdCl2dppf (0.11 g, 0.14 mmol) were suspended
in THF (8 mL) and degassed. The solutions were mixed together
in the Schlenk tube, stirred, and heated at 80 °C for 18 h. When
the mixture was cooled, DCM (20 mL) was added. The solution
dibenzofuran-4-yl substituent generally produced lower le-
vels of potentiation of IR (Table 2) than the analogous
dibenzothiophen-4-yl series (Table 1). Indeed the only occa-
sion when DMR values of over 2 were consistently observed
for the dibenzofuran-4-yl substituted pyridopyrimidin-4-
ones arose at the higher inhibitor concentration of 0.5 μM
(Table 2). Importantly, for this series of compounds there
was no relationship between potency of DNA-PK inhibition
and the DMR value (Figure 2C).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8505
was washed with water (20 mL), dried over magnesium sulfate,
and concentrated under reduced pressure. The residue was
purified by flash chromatography using AcOEt/DCM (1:1) as
eluent. After evaporation, the product (1.17 g, 92%) was ob-
tained as a yellow solid: Rf = 0.37 (AcOEt/DCM 1:1); mp 259-
260 °C; IR (cm-1) 3095, 2980, 2858, 1709, 1628, 1580, 1539, 1516,
739, 713; 1H NMR (300 MHz, MeOD) δ 3.32-3.35 (4H, m, 2 ꢀ
CH2N-morpholine), 3.66-3.68 (4H, m, 2 ꢀ CH2O-morpho-
line), 7.01 (1H, d, J = 8.0 Hz, H-Ar), 7.29 (1H, d, J = 7.8 Hz,
H-Ar), 7.48-7.58 (4H, m, H-Ar), 7.78 (1H, d, J = 7.5 Hz, H-Ar),
7.84 (1H, d, J = 6.9 Hz, H-Ar), 8.26-8.28 (2H, m, H-Ar); 13C
NMR (75 MHz, MeOD) δ 47.7 (2 ꢀ CH2N-morpholine), 67.3
(2 ꢀ CH2-O-morpholine), 115.2, 124.5, 125.3, 126.1, 127.9,
130.1, 136.3, 137.2; HRMS calcd for C25H21N3O2S [M þ H]þ
428.1427, found 428.1431.
1
1493, 1427, 1356, 1329, 1298, 1236, 1175, 1119, 1070, 995; H
NMR (300 MHz, CDCl3) δ 3.33-3.35 (4H, m, 2 ꢀ CH2N-
morpholine), 3.61-3.63 (4H, m, 2 ꢀ CH2O-morpholine), 5.68
(1H, s, H-3), 7.08 (1H, dd, J = 7.4 and 7.3 Hz, H-7), 7.48-7.65
(3H, m, H-Ar), 7.83-7.90 (3H, m, H-Ar),8.16(1H, d, J = 7.8 Hz,
H-Ar), 9.10 (1H, d, J=8.1 Hz, H-6); 13C NMR (75 MHz, CDCl3)
δ 44.8 (2 ꢀ CH2N-morpholine), 66.8 (2 ꢀ CH2O-morpholine),
81.6 (C-3), 112.3 (C-7), 120.5, 123.0, 125.4, 125.7, 127.1, 128.0,
128.9, 129.3, 131.7, 132.9, 136.5, 138.1, 140.4, 143.1, 146.4, 148.6,
159.0, 160.6; HRMS calcd for C24H18N4O4S [M þ H]þ
459.1122, found 459.1121.
2-Morpholin-4-yl-8-(1-nitrodibenzothiophen-4-yl)-1H-quinolin-
4-one (25). Inthe manner described for 24, 2-morpholin-4-yl-8-(1-
nitrodibenzothiophen-4-yl)-1H-quinolin-4-one (25) was prepared
from 1-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)di-
benzothiophene 22 (983 mg, 2.77 mmol), cesium carbonate
(2.71 g, 8.30 mmol), 8-bromo-2-morpholin-4-yl-1H-quinolin-4-
one16 (941 mg, 3.05 mmol), and PdCl2dppf (113 mg, 0.14 mmol)
and was obtained as a yellow solid (256 mg, 20%): Rf = 0.24
(AcOEt/DCM 1:1); mp 77-78 °C; IR (cm-1) 2848, 2364, 2338,
1670, 1615, 1584, 1516, 1414, 1348, 1302, 1230, 1187, 1112, 998,
932; 1H NMR (300 MHz, CDCl3) δ3.19-3.21 (4H, m, 2 ꢀ CH2N-
morpholine), 3.67-3.69 (4H, m, 2 ꢀ CH2O-morpholine), 6.21
(1H, s, H-3), 7.43-7.57 (3H, m, H-Ar), 7.60-7.72 (2H, m, H-Ar),
7.83 (1H, d, J = 7.8 Hz, H-Ar), 7.94 (1H, d, J = 8.0 Hz, H-Ar),
8.30 (1H, d, J=7.7Hz, H-Ar);13C NMR (75 MHz, CDCl3) δ46.8
(2 ꢀ CH2N-morpholine), 66.4 (2 ꢀ CH2O-morpholine), 92.8,
121.4, 123.2, 123.4, 125.5, 125.8, 126.5, 127.6, 127.7, 129.0, 131.8,
132.9, 140.7, 143.8, 146.6, 171.2; HRMS calcd for C25H19N3-
O4S [M þ H]þ 458.1169, found 458.1168.
9-(1-Nitrodibenzofuran-4-yl)-2-morpholin-4-ylpyrido[1,2-a]pyri-
midin-4-one (28). Ina Schlenktube, 1-nitro-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)dibenzofuran 23 (500 mg, 1.47 mmol)
and potassium carbonate (480 mg, 3.7 mmol) were mixed in
dioxane (10 mL) and degassed. Concurrently, 9-hydroxy-2-mor-
pholin-4-ylpyrido[1,2-a]pyrimidin-4-one 9-O-triflate18 (466 mg,
1.23 mmol) and Pd(PPh3)4 (71 mg, 0.06 mmol) were suspended
in dioxane (10 mL) and degassed. The solutions were mixed
together in a microwave vessel and heated with the microwave
reactor at 180 °C for 30 min. When the mixture was cooled, DCM
(20 mL) was added. The solution was washed with water (20 mL),
dried over magnesium sulfate, and concentrated under reduced
pressure. The residue was triturated with hot methanol, and the
product (375 mg, 69%) was filtered off as a brownsolid:Rf = 0.51
(AcOEt); mp 262-265 °C; IR (cm-1) 1706, 1673, 1631, 1599,
1541, 1511, 1430, 1338, 1306, 1230, 1194, 1116, 1070, 1028, 999,
975, 860; 1H NMR (300 MHz, CDCl3) δ 3.26-3.29 (4H, m, 2 ꢀ
CH2N-morpholine), 3.47-3.49 (4H, m, 2 ꢀ CH2O-morpholine),
5.57 (1H, s, H-3), 7.03 (1H, dd, J = 7.8 and 7.6 Hz, H-7), 7.44 (1H,
dd, J = 7.6 and 7.5 Hz, H-Ar), 7.58-7.61 (2H, m, H-Ar), 7.74
(1H, d, J = 7.6 Hz, H-Ar), 7.86 (1H, d, J = 7.5 Hz, H-Ar), 8.24
(1H, d, J = 8.0 Hz, H-Ar), 8.66 (1H, d, J = 8.0 Hz, H-Ar), 9.10
(1H, d, J=7.8Hz, H-6);13CNMR(125MHz, CDCl3) δ44.4 (2 ꢀ
CH2N-morpholine), 66.3 (2 ꢀ CH2O-morpholine), 81.1 (C-3),
111.6, 112.1, 119.3, 120.7, 123.9, 126.3, 127.6, 128.3, 128.8, 128.9,
130.0, 138.4, 142.9, 143.9, 148.5, 154.7, 157.0, 158.7, 160.3; HRMS
calcd for C24H19N4O5 [M þ H]þ 443.1350, found 443.1352.
2-Morpholin-4-yl-8-(1-nitrodibenzofuran-4-yl)-1H-quinolin-
4-one (29). In the manner described for 28, 2-morpholin-4-yl-
8-(1-nitrodibenzofuran-4-yl)-1H-quinolin-4-one (29) was pre-
pared from 1-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl) dibenzofuran 23 (500 mg, 1.47 mmol), potassium
carbonate (480 mg, 3.69 mmol), 8-bromo-2-morpholin-4-yl-
1H-quinolin-4-one18 (380 mg, 1.23 mmol), and Pd(PPh3)4 (71
mg, 0.06 mmol) and was obtained as a yellow solid (303 mg,
60%): Rf = 0.67 (AcOEt); mp 247-249 °C; IR (cm-1) 3421,
2852, 2360, 2333, 1614, 1573, 1500, 1435, 1429, 1348, 1309,
1233, 1199, 1152, 1124, 1039, 991, 917, 866, 823; 1H NMR (300
MHz, CDCl3) δ 3.04-3.06 (4H, m, 2 ꢀ CH2N-morpholine),
3.54-3.60 (4H, m, 2 ꢀ CH2O-morpholine), 5.92 (1H, s, H-3),
7.19-7.45 (2H, m, H-Ar), 7.51-7.58 (2H, m, H-Ar), 7.65-
7.70 (2H, m, H-Ar), 8.31 (2H, m, H-Ar), 8.67 (1H, d, J = 7.5
Hz, H-Ar); HRMS calcd for C25H20N3O5 [M þ H]þ 442.1397,
found 442.1398.
9-(1-Aminodibenzothiophen-4-yl)-2-morpholin-4-ylpyrido[1,2-
a]pyrimidin-4-one (26). Zinc powder (883 mg, 13.51 mmol) was
added to 9-(1-nitrodibenzothiophen-4-yl)-2-morpholin-4-yl-
pyrido [1,2-a]pyrimidin-4-one 24 (619 mg, 1.35 mmol) in AcOH
(10 mL) and stirred at room temperature overnight. The reac-
tion mixture was filtered through Celite and washed successively
with methanol (4 ꢀ 50 mL) and DCM (4 ꢀ 50 mL). The
combined organic layers were evaporated under reduced pres-
sure, and the residue was diluted with water (100 mL). Aqueous
ammonia (25 mL) was added to the solution, and the resultant
precipitate was collected by filtration. The product was dried
under reduced pressure and was obtained as a yellow solid (575
mg, 99%): Rf = 0.36 (AcOEt/DCM 1:1); mp 208-209 °C; IR
(cm-1) 3435, 3358, 2916, 2856, 1693, 1618, 1582, 1539, 1489,
1431, 1366, 1333, 1296, 1231, 1176, 1113, 1072, 1022, 999, 928,
1
899; H NMR (300 MHz, CDCl3) δ 3.41-3.43 (4H, m, 2 ꢀ
CH2N-morpholine), 3.59-3.61 (4H, m, 2 ꢀ CH2O-morpho-
line), 5.68 (1H, s, H-3), 6.90 (1H, d, J = 7.8 Hz, H-Ar), 7.02 (1H,
dd, J = 7.9 and 7.8 Hz, H-7), 7.43-7.53 (3H, m, H-Ar), 7.82
(1H, d, J = 8.2 Hz, H-Ar), 7.89 (1H, d, J = 7.8 Hz, H-8), 8.28
9-(1-Aminodibenzofuran-4-yl)-2-morpholin-4-ylpyrido[1,2-a]-
pyrimidin-4-one (30). In the manner described for 26, 9-(1-amino-
dibenzofuran-4-yl)-2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-one
(30) was prepared from 9-(1-nitrodibenzofuran-4-yl)-2-morpholin-
4-ylpyrido[1,2-a]pyrimidin-4-one 28 (300 mg, 0.68 mmol) and zinc
powder (445 mg, 6.8 mmol) and was obtained as a white solid (269
mg, 96%): Rf = 0.32 (AcOEt); mp 294-295 °C; IR (cm-1) 3340,
3224, 2937, 2872, 2258, 1697, 1637, 1623, 1543, 1493, 1440, 1373,
1309, 1258, 1234, 1191, 1150, 1109, 1073, 999, 909, 856, 776;
1H NMR (300 MHz, MeOD) δ 3.36-3.38 (4H, m, 2 ꢀ CH2N-
morpholine), 3.52-3.55 (4H, m, 2 ꢀ CH2O-morpholine), 6.72
(1H, d, J = 7.8 Hz, H-Ar), 6.88 (1H, dd, J = 7.6 and 7.5 Hz,
H-Ar), 7.31-7.47 (5H, m, H-Ar), 7.90-7.92 (2H, m, H-Ar), 8.90
(1H, d, J = 7.5 Hz, H-Ar); 13C NMR (75 MHz, MeOD) δ 46.2
(2 ꢀ CH2N-morpholine), 68.0 (2 ꢀ CH2O-morpholine), 110.4,
112.8, 114.6, 122.5, 124.5, 125.6, 127.5, 128.0, 131.8, 133.6,
138.9, 145.1, 150.8, 156.7, 156.9, 161.7, 162.1; LCMS m/z
(1H, d, J = 8.2 Hz, H-Ar), 9.01 (1H, d, J = 8.1 Hz, H-6); 13
C
NMR (75 MHz, CDCl3) δ 44.8 (2 ꢀ CH2N-morpholine), 66.9
(2 ꢀ CH2O-morpholine), 81.4 (C-3), 112.7, 112.9, 122.9, 123.0,
123.6, 124.9, 125.9, 127.6, 129.6, 134.9, 135.9, 137.4, 138.9,
141.5, 144.3, 149.1, 159.7, 160.5, 163.19;HRMScalcdfor C24H20-
N4O2S [M þ H]þ 429.1380, found 429.1381.
8-(1-Aminodibenzothiophen-4-yl)-2-morpholin-4-yl-1H-quino-
lin-4-one (27). In the manner described for 26, 8-(1-aminodi-
benzothiophen-4-yl)-2-morpholin-4-yl-1H-quinolin-4-one (27)
was prepared from 2-morpholin-4-yl-8-(1-nitrodibenzothio-
phen-4-yl)-1H-quinolin-4-one 25 (365 mg, 0.80 mmol) and zinc
powder (522 mg, 7.98 mmol) and was obtained as a brown oil
(291 mg, 85%): Rf = 0.35 (AcOEt/DCM 1:1); IR (cm-1) 3365,
2870, 1666, 1624, 1589, 1493, 1406, 1363, 1247, 1180, 1116, 797,

8506 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Cano et al.
413.19 ([M þ H]þ). Anal. Calcd for 0.86 mol of C24H20N4O3 þ
0.14 mol of MeOH: C, 69.48, H, 4.99, N, 13.41. Found:
C, 69.22, H, 4.79, N, 13.38. HRMS calcd for C24H21N4O3
[M þ H]þ 413.1608, found 413.1609.
UK for generous support. The EPSRC Mass Spectrometry
Service at the University of Wales (Swansea, U.K.) is also
gratefully acknowledged.
8-(1-Aminodibenzofuran-4-yl)-2-morpholin-4-yl-1H-quinolin-
4-one (31). In the manner described for 26, 8-(1-aminodibenzo-
furan-4-yl)-2-morpholin-4-yl-1H-quinolin-4-one (31) was pre-
pared from 2-morpholin-4-yl-8-(1-nitrodibenzothiophen-4-yl)-
1H-quinolin-4-one 29 (290 mg, 0.66 mmol) and zinc powder
(430 mg, 6.58 mmol) and was obtained as a brown oil (246 mg,
91%): Rf = 0.44 (AcOEt); IR (cm-1) 1708, 1572, 1374, 1278,
Supporting Information Available: Synthesis and analytical
details for compounds 12-23 and solution-phase library synth-
esis and analytical details for inhibitors 32-83. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
1
1190, 1116, 1049, 1010, 931, 880, 827, 748, 722, 688, 688; H
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morpholine), 3.58-3.60 (4H, m, 2 ꢀ CH2O-morpholine), 5.98
(1H, s, H-3), 6.85 (1H, d, J = 8.1 Hz, H-Ar), 7.25-7.51 (3H, m,
H-Ar), 7.52-7.68 (3H, m, H-Ar), 8.05 (1H, t, J = 8.1 Hz,
H-Ar), 8.67 (1H, d, J =7.8 Hz, H-Ar); 13C NMR (75 MHz,
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morpholine), 111.9, 112.6, 122.9, 125.1, 125.6, 127.6, 127.9,
131.6, 132.7, 135.3, 146.2, 156.8; HRMS calcd for C25H22N3O3
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heparin-agarose chromatography. DNA-PK (250 ng) activity
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containing 25 mM Hepes, pH 7.4, 12.5 mM MgCl2, 50 mM KCl,
1 mM DTT, 10% glycerol, 0.1% NP-40, and 1 μg of the
substrate GST-p53N66 (the amino-terminal 66 amino acid
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added varying concentrations of inhibitor (in DMSO at a final
concentration of 1%). After 10 min of incubation, ATP was
added to give a final concentration of 50 μM along with a 30mer
double-stranded DNA oligonucleotide (final concentration
of 0.5 ng/mL) to initiate the reaction. After 1 h with shaking,
150 μL of phosphate-buffered saline (PBS) was added to the
reaction and 5 μL was then transferred to a 96-well opaque
white plate containing 45 μL of PBS per well, where the GST-
p53N66 substrate was allowed to bind to the wells for 1 h at
room temperature. To detect the phosphorylation event on the
serine-15 residue of p53 elicited by DNA-PK, a rabbit phos-
phoserine-15 antibody (Cell Signaling Technology) was used in
a basic ELISA procedure. An anti-rabbit HRP conjugated
secondary antibody (Pierce) was then employed in the ELISA
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Renaissance) to detect the signal as measured by chemilumi-
nescent counting via a TopCount NXT (Packard). IC50 was
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in which the DNA-PK activity in the varying concentrations
of compounds was plotted against the concentration of
compound.
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an automated colony counter (Oxford Optronics Ltd., Oxford,
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% survival- compound= þ 2 Gy
DMR ¼
% survival
þ compound= þ 2 Gy
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Acknowledgment. We thank Adrian Moore for help with
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