Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK) using a small-molecule library approach

J. Med. Chem. 2005, 48, 7829-7846

7829

Discovery of Potent Chromen-4-one Inhibitors of the DNA-Dependent Protein

Kinase (DNA-PK) Using a Small-Molecule Library Approach

Ian R. Hardcastle,*^,†Xiaoling Cockcroft,^‡Nicola J. Curtin,^#Marine Desage El-Murr,^†Justin J. J. Leahy,^†

Martin Stockley,^†Bernard T. Golding,^†Laurent Rigoreau,^‡Caroline Richardson,^§Graeme C. M. Smith,^§and

Roger J. Griffin^†

Northern Institute for Cancer Research, School of Natural Sciences - Chemistry, Bedson Building,

Newcastle upon Tyne NE1 7RU, U.K., KuDOS Pharmaceuticals Ltd., 327 Cambridge Science Park, Milton Road,

Cambridge CB4 4WG, U.K., Northern Institute for Cancer Research, Medical School, Paul O’Gorman Building,

Framlington Place, University of Newcastle upon Tyne, NE2 4HH, U.K., and KuDOS Horsham Ltd., Foundry Lane,

Horsham, West Sussex RH13 5PX, U.K.

Received May 10, 2005

Structure-activity relationships for inhibition of DNA-dependent protein kinase (DNA-PK)

have been defined for substituted chromen-4-ones. For the 2-amino-substituted benzo[h]-

chromen-4-ones, a morpholine substituent at this position was essential for activity. Small

libraries of 6- and 7-alkoxy-substituted chromen-4-ones showed that a number of 7-alkoxy-

substituted chromenones displayed improved activity. Focused libraries incorporating 6-, 7-,

and 8-aryl and heteroaryl substituents were prepared. In these cases, 6- and 7-substitution

was disfavored, whereas 8-substitution was largely tolerated. Surprisingly, two compounds,

2-N-morpholino-8-dibenzofuranyl-chromen-4-one (NU7427, 32{38}) and the 2-N-morpholino-

8-dibenzothiophenyl-chromen-4-one (NU7441, 32{26}) were excellent inhibitors (IC₅₀vs DNA-

PK ) 40 and 13 nM, respectively). The ring-saturated analogue 2-N-morpholino-8-(6′,7′,8′,9′-

tetrahydrodibenzothiophene)chromen-4-one, 36, retained potent activity (IC₅₀vs DNA-PK )

23 nM). The dibenzothiophene 32{38} sensitized HeLa cells to ionizing radiation in vitro, with

dose modification factors of 2.5 at 10% survival being observed at 0.5 µM. The cytotoxicity of

the topoisomerase II inhibitor etoposide was also potentiated.

Chart 1. Structures of DNA-PK Inhibitors

Introduction

The DNA-dependent protein kinase (DNA-PK) is a

nuclear serine/threonine kinase member of the phos-

phatidylinositol (PI) 3-kinase-like (PIKK) family.¹The

DNA-PK complex comprises a large 465 kDa catalytic

subunit (DNA-PKcs), and a heterodimeric DNA-target-

ing regulatory factor Ku made up of Ku70 and Ku80

subunits. DNA-PKcs shares significant homology in the

kinase domain with the other PIKK family members:

ATR, ATM, FRAP, mTOR, hSMG-1, and to a lesser

extent the PI 3-kinase catalytic subunits p110, and

PI4K.²DNA-PK becomes catalytically active on binding

to DNA double-strand breaks (DSBs) and may phos-

phorylate a number of downstream targets including

itself and the variant histone H2AX.^3-7Cells that are

defective in either DNA-PKcs or either of the regulatory

Ku subunits are unable to effectively repair DNA DSBs

produced from either exogenous sources (e.g., ionizing

radiation) or endogenous processes (e.g., V(D)J recom-

bination). These cell lines are highly radiosensitive and

display increased sensitivity to cytotoxic drugs that

induce DNA DSBs.^8,9For this reason, we and others

have proposed that specific DNA-PK inhibitors could be

used to potentiate radiotherapy and chemotherapy in

cancer treatment.

A number of small-molecule inhibitors of DNA-PK

have been identified, including the irreversible inhibitor

wortmannin^10,11and the ATP-competitive inhibitors

SU11752,¹²IC87361,¹³and LY294002^11,14(Chart 1).

Cells treated with these compounds become radio-

sensitized and also chemosensitive to DNA DSB-causing

therapeutics, such as etoposide and bleomycin.^15,16

Previously, we employed the chromenone LY294002,

originally designed as an ATP-competitive phospha-

tidylinositol 3-kinase (PI3K) inhibitor, as a structural

lead, enabling the identification of a number of potent,

selective ATP-competitive DNA-PK inhibitors, for ex-

ample pyranone (1a, IC₅₀) 1.1 µM), thiopyranone (1b,

IC₅₀) 0.72 µM), benzo[f]chromen-4-one (2, IC₅₀) 4 µM),

* Corresponding author. Tel: +44 191 222 6645. Fax: +44 191 8591.

E-mail: I.R.Hardcastle@ncl.ac.uk.

^†Northern Institute for Cancer Research, School of Natural

Sciences.

^‡KuDOS Horsham Ltd.

^#Northern Institute for Cancer Research, Medical School.

^§KuDOS Pharmaceuticals Ltd.

Published on Web 10/29/2005

7830 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

benzo[g]chromen-4-one (3, IC₅₀) 0.4 µM), pyrimidoiso-

quinolinone (4, IC₅₀) 0.25 µM), and the benzo[h]-

chromen-4-one NU7026 (5{1}, IC₅₀) 0.23 µM).^17-19

(6) and CS₂with tert-butoxide as base. Thione (7) was

readily alkylated with chloromethylpolystyrene resin

(Merrifield, 1% divinylbenzene (DVB)) with diazabicyclo-

[5.4.0]undec-7-ene (DBU) in N,N-dimethylformamide

(DMF) at 60 °C (∼96% loading). The resin-bound alkyl-

sulfanyl compound (8) was oxidized to the sulfone (9)

with meta-chloroperbenzoic acid (mCPBA) in dichloro-

methane (DCM) and reacted with the chosen amine in

DCM to liberate the products (5{4-22}) into solution.

Excess amine was removed from the product by evapo-

ration in vacuo, or for involatile amines, an acidic

scavenger resin (Amberlite IR120+) was used. Despite

the presence of a basic group, products 5{6} and 5{9}

did not bind appreciably to the scavenger resin in the

time required for complete sequestration of the remain-

ing amine. Products were obtained in high purity and

submitted for assay without further purification.

7-Alkoxy-chromenones by Solution-Phase Syn-

thesis. The 7-hydroxychromone precursor (13b, Scheme

2) was prepared according to the method of Morris et

al.²¹Direct conversion of methyl 2,4-dihydroxybenzoate

(10) into the required â-ketoamide failed due to pre-

cipitation of the insoluble dilithium phenolate. Protec-

tion of the 4-hydroxyl group as the 2,6-dichlorobenzyl

ether (11) allowed the reaction sequence to proceed

smoothly via the â-ketoamide (12) giving protected

chromenone (13a). Removal of the protecting group to

furnish (13b) was readily achieved by hydrogenolysis.

Reaction of 13b with alkyl halides in the presence of

Triton B gave the required 7-alkoxy-2-morpholinyl-

chromenones (13{1-10}) in modest yields, while treat-

ment of 13b with benzoyl chloride afforded the corre-

sponding 7-benzoyl ester (13{11}; Table 2, method A).

6- and 7-Alkoxy-chromenones by Solid-Phase

Synthesis. The solid-phase capture and release

strategy was also employed for the synthesis of 6-

and 7-alkoxy-chromenones. The 7-p-methoxybenzyloxy-

chromene-2-thione precursor (15) was prepared from

2,4-dihydroxyacetophenone by reaction with freshly

prepared p-methoxybenzyl chloride in THF to give the

ether (14a), which upon reaction with CS₂in toluene

with potassium t-butoxide as base, followed by a careful

acidic workup, gave 15 (Scheme 3). The chromenone was

attached to the resin with DBU in DMF to give the

chromenone resin with good loading. Deprotection of the

p-methoxybenzyl group was effected with 75% TFA in

DCM at 0 °C giving the 7-hydroxy-chromene thio-resin

(16). Cleavage of the chromenone from the resin was

not observed under these conditions. Alkylation of 16

to give resin-bound 7-alkoxy-chromenone (17) was

achieved by treatment of the preswelled resin in DMF

with a variety of benzylic and alkyl halides using DBU.

Subsequent oxidation of the sulfide linker to the sulf-

oxide (18) with mCPBA in DCM, followed by cleavage

with morpholine, gave the 7-alkoxy-chromenones (13{3-

Selected compounds from these series have been dem-

onstrated to sensitize tumor cell lines in vitro to ionizing

radiation and DNA DSB-inducing chemotherapeutic

agents.^19,20

In the absence of detailed structural information for

DNA-PKcs, we have employed a pharmacophore map-

ping approach, using small focused libraries to define

more extensive structure-activity relationships (SARs)

for ATP-competitive DNA-PK inhibition by chromenone-

based inhibitors. We have previously detailed the effect

on DNA-PK-inhibitory activity of modifying or replacing

the 2-morpholinyl substituent of 4 and 5.¹⁹In this paper,

we present the results of further variations to the

2-morpholinyl function of the benzo[h]chromenone phar-

macophore. We also report the synthesis, using solution-

and solid-phase multiple-parallel approaches, and bio-

logical evaluation of chromen-4-one libraries bearing a

variety of alkoxy- and aryl-substituents at the 6-, 7-,

and 8-positions. These studies have resulted in the

discovery of 8-aryl-chromen-4-one derivatives exhibiting

potent and kinase-selective DNA-PK-inhibitory activity.

A preliminary account of selected results from this study

has been communicated.¹⁸

Chemistry

2-Aminobenzo[h]chromenones. A small library of

2-aminobenzo[h]chromenones (5{4-22}, Table 1) was

prepared using the published procedure in a modified

format.^14,19The synthesis was adapted to a solid-phase

capture and release strategy to simplify workup proce-

dures and trap the sulfur-containing byproducts on the

solid phase (Scheme 1). The chromenethione (7) was

prepared, as previously described, from naphthyl ketone

Scheme 1. Solid-Phase Synthesis of 2-Aminobenzo[h]chromen-4-ones (5{4-22})^a

^aReagents and conditions: (a) CS₂, KO^tBu, PhMe; (b) Merrifield resin, DBU, DMF, 60 °C; (c) mCPBA, DCM; (d) HNR¹R², DCM, rt.

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7831

Table 1. Chemical Structures, Synthetic Yields and Purities, and Biological Activities for the Benzo[h]chromenone Library

^aEstimated by LCMS.

Scheme 3. Solid-Phase Synthesis of

Scheme 2. Synthesis of 7-Alkoxy-chromen-4-ones

7-Alkoxy-chromen-4-ones (13{3-49})^a

(13{1-11})^a

^aReagents and conditions, Method A: (a) 2,6-dichlorobenzyl

chloride (dcbCl), K₂CO₃, NaI, acetone, reflux; (b) N-acetylamine,

LDA, THF, -78-25 °C; (c) Tf₂O, DCM, 0 °C, 16 h; (d) 10% Pd/C,

H₂, MeOH; (e) RBr, Triton B, DMF, 90 °C or (f) PhCOCl, pyridine,

DMF, 0 °C.

^aReagents and conditions: (a) PMBCl, THF; (b) (i) CS₂, KO^tBu,

PhMe, (ii) aq H₂SO₄; (c) Merrifield resin, DBU, DMF; (d) 75% TFA,

18}) in 30-90% purity as estimated by liquid chroma-

tography-mass spectrometry, LCMS (Table 2, method

B). An additional series of alkylations was performed

DCM, 0 °C; (e) method B, R²Br, DBU, DMF, 65 °C; method C,

R²OH, DIAD, PPh₃, Et₃N, THF; (f) mCPBA, DCM; (g) morpholine,

DCM.

7832 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

Table 2. Chemical Structures, Synthetic Yields and Purities, and Biological Activities for 7-Alkoxy-chromenone Libraries

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7833

Table 2 (Continued)

7834 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Table 2 (Continued)

Hardcastle et al.

^aScheme 2. ^bScheme 3. ^cEstimated by LCMS. ^dAlkylation repeated 3 times. ^eValues determined from compound prepared by method

A.

Scheme 4. Solid-Phase Synthesis of 6-Alkoxy-chromen-4-ones (21a-t)^a

^aReagents and conditions: (a) PMBCl, THF; (b) (i) CS₂, KO^tBu, PhMe, (ii) aq H₂SO₄; (c) Merrifield resin, DBU, DMF, 60 °C; (d) 75%

TFA, DCM, 0 °C; (e) R²Br, DBU, DMF, 65 °C; (f) mCPBA, DCM; (g) morpholine, DCM.

under Mitsunobu conditions with a set of 36 alcohols,

which included 21 benzyl alcohols. Oxidation with

mCPBA, followed by cleavage with morpholine, gave the

products 13{3,5,12,16,17,19-49} in 28-100% purity

(Table 2, method C). Concomitant N-oxidation was

observed in the cases where the alcohol contained a

pyridine group (13{46-49}).

The resin-bound 6-hydroxy-chromenone (20) was

prepared using a similar sequence of reactions starting

from 2,5-dihydroxyacetophenone via thione (19) (Scheme

4). Alkylation of 20 was achieved by treatment of the

preswelled resin in DMF with a variety of benzylic and

alkyl halides using DBU as base. Subsequent oxidation

with mCPBA in DCM followed by cleavage with mor-

pholine gave the 6-alkoxy-chromenones (21a,b) in good

purity as determined by LCMS. Reactions with other

substituents failed to give the product in greater than

80% purity and were not submitted for assay. This

result suggests that the 6-hydroxyl position is substan-

tially less reactive in the alkylation reactions than the

7-position of the alkylthiochromenones.

6-, 7-, and 8-Aryl and Vinyl Chromenones: The

6-bromochromenone intermediate for the Suzuki-

Miyura coupling library was prepared by condensation

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7835

triflate 29b gave the desired 8-substituted chromenone

(36) in acceptable yield.²⁵

Results and Discussion

Structure-Activity Relationships for DNA-PK

Inhibition. 2-Aminobenzo[h]chromenones. Pre-

vious studies in the benzochromenone and related

pyrimidoisoquinolinone series suggested that the 2-mor-

pholino substituent was optimal for DNA-PK inhibition

and that this position was intolerant to substitution.¹⁹

To validate this observation, compounds 5{4-22} were

prepared. The amine substituents included a set chosen

using a 2D-similarity search with morpholine as the

“seed” compound. It was anticipated that compounds

able to accept the hydrogen bond postulated to form

between DNA-PK and the oxygen of the morpholino

substituent would retain some activity. In fact, as

proposed previously,¹⁹none of the compounds showed

any inhibitory activity, thus confirming morpholine as

the optimal substituent at this position. The morpholino

oxygen of LY294002 makes important interactions with

the backbone NH of Val882 of PI3Kγ by accepting a

hydrogen bond, as observed in the X-ray structure of

LY294002 bound to PI3Kγ.²⁶It is likely that analogous

important interactions arise between our inhibitors and

the homologous region of the DNA-PKcs ATP-binding

site.

6- and 7-Alkoxy-chromenones: A small series of

7-alkoxy-chromenones, 13{1-11}, prepared via the

solution-phase route, was evaluated (Table 2, Scheme

2-method A). In comparison with the parent 13b (IC₅₀

vs DNA-PK ) 0.45 µM),¹⁹the introduction of alkyl

substituents, for example, 13{1}, resulted in a loss of

activity; however, compounds with benzylic substituents

displayed comparable activity in the majority of ex-

amples. The series of compounds was expanded by the

synthesis of further analogues using the solid-phase

route (Table 2, Scheme 3-methods B and C). None of

the compounds evaluated displayed improved activity

over 13b, benzylic substitution generally resulting in a

modest loss of potency. In the case of bulky substituents,

such as 4-phenylbenzyl, 13{30}, and tert-butylbenzyl,

13{33}, a significant loss of activity was observed,

indicating a limit to the amount of steric bulk able to

be accommodated by the enzyme. Compounds bearing

the polar pyridine N-oxides, 13{46-49}, were markedly

less potent than the parent 13b.

of 5-bromo-2-hydroxyacetophenone (22) with CS₂in

toluene, with potassium t-butoxide as base, followed by

treatment with aqueous sulfuric acid giving the 6-bromo-

thione (23) (Scheme 5). Alkylation with ethyl iodide gave

the thioether (24). Finally treatment of 24 with mor-

pholine in ethylene glycol at 140 °C gave the desired

6-bromochromenone derivative (25).

Selective monotriflation at the 3- and 4-hydroxyl

groups of the diphenols (26a and 26b, respectively) was

possible using triflic anhydride under standard condi-

tions due to the reduced reactivity of the 2-hydroxyl

group, which is believed to be due to intramolecular

hydrogen bonding to the ketone (Scheme 6). The triflates

27a and 27b were reacted with the lithium enolate of

N-acetamidomorpholine to give the respective diketones

(28a and 28b). Cyclization to the corresponding mor-

pholinochromenones (29a and 29b) was effected with

triflic anhydride.

The 6-bromo- and 7- and 8-triflyl-chromenones (25,

29a, and 29b) were reacted with a range of aryl and

vinyl boronic acids (B1-53) under standard conditions

(dioxane, Pd(PPh₃)₄, K₂CO₃, 80 °C) in a parallel format

to give the 6-, 7-, and 8-aryl and vinyl chromenones (30,

31, and 32), respectively (Scheme 7, Table 3).

The tetrahydrodibenzothiophene boronate (35) was

prepared in three steps from 2-chlorocyclohexanone

(Scheme 8). Nucleophilic substitution with 2-bromo-

thiophenolate gave the thioether (33), which was cy-

clized under forcing conditions (P₄O₁₀, polyphosphoric

acid, 180 °C) to give the 6-bromotetrahydrodibenzo-

thiophene derivative (34) in good yield.^22,23Coupling of

the 6-bromo compound with bispinacolatodiboron under

standard conditions gave a moderate yield of the bor-

onate (35).²⁴A second coupling with the chromenone

Scheme 5. Synthesis of 6-Bromo-2-N-morpholinochromen-4-one (25)^a

^aReagents and conditions: (a) (i) CS₂, KO^tBu, PhMe, (ii) aq H₂SO₄; (b) EtI, K₂CO₃, acetone; (c) morpholine, ethylene glycol, 140 °C.

Scheme 6. Synthesis of 7- and 8-Triflyl-2-N-morpholinochromen-4-ones (29a,b)^a

^aReagents and conditions: (a) Tf₂O; (b) LDA, THF, 0 °C; (c) Tf₂O, DCM.

7836 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

Table 3. Chemical Structures, Synthetic Yields, and Biological Activities for 6-Bromochromenone (25), 7-Triflyl Chromenone (29a),

and 8-Triflyl Chromenone (29b) Libraries

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7837

Table 3 (Continued)

7838 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Table 3 (Continued)

Hardcastle et al.

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7839

Scheme 7. Synthesis of 6-, 7-, and 8-Substituted

2-N-Morpholinochromen-4-one Libraries (30, 31, and

32)^a

^aReagents and conditions: RB(OH)₃(B1-53), K₂CO₃,

Pd(PPh₃)₄, dioxane, 90 °C, 18 h.

Scheme 8. Synthesis of 8-(6′,7′,8′,9′-

Tetrahydrodibenzothiophen-4′-yl)-2-N-

morpholinochromen-4-one (36)^a

Figure 1. Characterization of the inhibition of DNA-PK

activity by compound 32{38}: (a) Lineweaver-Burk analysis

of the inhibition of DNA-PK by 32{38}. Inhibition kinetics were

performed with 0 (9), 0.1 (2), 0.5 (1), 1 ([), 2.5 (b) or 5 nM

(0) of 32 in a range of ATP concentrations (5-100 µM). Data

shown are the mean ( sem from at least three independent

experiments. (b) K_idetermination for compound 32. Slopes

from the Lineweaver-Burk plots were plotted against the

range of concentration of 32 used. Intersection of the plot on

the compound concentration axis gave a K_ivalue of 0.65 nM.

^aReagents and conditions: (a) 2-bromobenzenethiol, NaOH,

EtOH, reflux; (b) PO₅, polyphosphoric acid, 180 °C; (c) bis(pina-

colato)diboron, PdCl₂(dppf), KOAc, THF, reflux; (d) 29, PdCl₂(dppf),

CsCO₃, THF, reflux.

Interestingly, two compounds, the dibenzofuran 32{26}

and the dibenzothiophene 32{38} (NU7441), exhibited

a marked improvement in potency. Compound 32{38}

was resynthesized to analytical purity and reassayed,

giving an IC₅₀of 13 nM against DNA-PK. This inhibitor

was assigned the house number NU7441.

To explore further the importance of the terminal ring

of the dibenzothiophene in 32{38} and its interaction

with DNA-PKcs, the 6′,7′,8′,9′-tetrahydrodibenzo-

thiophene derivative 36 was synthesized. Interestingly,

36 was essentially equipotent with the parent 32{38}

(IC₅₀) 23 nM), suggesting a degree of steric tolerance

in this region of the DNA-PKcs active site. The potent

activity of the chromenones bearing dibenzofuran or

dibenzothiophene substituents at the 8-position (32{26},

32{38}, and 36) is remarkable in the light of the

relatively flat SARs for other aromatic and hetero-

aromatic substitutions in this series.

Characterization of DNA-PK Inhibition by (32-

{38}). Lineweaver-Burk plots for DNA-PK inhibition

by 32{38} revealed that this inhibitor acted in an ATP-

competitive fashion (Figure 1a), as seen previously for

LY294002 and NU7163.^11,19Notably, when the slopes

of the plots shown in Figure 1a were plotted against

the inhibitor concentration (Figure 1b), it was found that

32{38} exhibited a K_ivalue of 0.65 nM, confirming this

chromenone as the most potent DNA-PK inhibitor

reported to date.

Characterization of Kinase Selectivity of (32-

{38}). NU7441 was further tested for its ability to

inhibit the DNA-PK-related enzymes ATM (ataxia-

telangiectasia mutated), ATR (ATM and Rad-3 related),

The results from the 6-alkoxy-chromenone library

were disappointing as the purity of the majority of the

compounds was below the 85% standard required for

biological evaluation. Two compounds were assayed,

namely, the benzonitrile 21a (IC₅₀) 4.0 µM) and the

phthalimide 21b (IC₅₀) 4.3 µM), both of which were

an order of magnitude less potent than the correspond-

ing 7-substituted analogues, 13{13} and 13{8}, respec-

tively.

6-, 7-, and 8-Substituted Chromenones. The 6- and

7-substituted products from the Suzuki libraries (30{1-

42} and 31{1-51}, respectively) were assayed against

DNA-PK at a single concentration of 0.5 µM. None of

the compounds displayed significant inhibition at this

concentration. Consequently, IC₅₀determinations were

not performed on this library.

The 8-substituted products from the Suzuki libraries,

32{1-55}, were initially assayed at a single concentra-

tion of 0.5 µM. In this case, a number of compounds

showed improved activity over the benchmark com-

pound LY294002 (IC₅₀) 1.47 µM). Consequently, IC₅₀

determinations were carried out on the entire library.

Eleven compounds showed a significant loss of activity,

in particular, those bearing an ortho-substituted phenyl

group, for example, 32{2,7,11,14,40}, and the bis-

trifluoromethyl compound 32{28}, indicating a lack of

steric tolerance within the active site at this position.

The 2- and 3-thienyl isosteres of LY294002, 32{16} and

32{17}, displayed a modest improvement in activity

over the parent compound, as did the 2-benzothiophene

compound 32{44}, the 4-methoxyphenyl derivative

32{13}, and the 4-hydroxymethyl analogue 32{32}.

7840 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

potent and specific DNA-PK inhibitor. It was also

observed that at concentrations up to 10 µM the DNA-

PK inhibitor showed no inherent toxicity in this cell-

based assay system.

Conclusions

We have successfully employed a pharmacophore

mapping approach to establish structure-activity re-

lationships for chromen-4-one-based DNA-PK inhib-

itors. Consistent with our previous results,¹⁹replace-

ment of the 2-morpholinyl substituent in the benzo[h]-

chromenone series 5{4-22} was not tolerated, providing

further evidence for a crucial interaction between this

group and the ATP-binding site. The introduction of

alkoxy substituents at the chromenone 6-position was

detrimental to DNA-PK inhibitory activity, whereas

7-alkoxy substitution was generally tolerated and in

some examples produced a modest improvement in

potency. Aryl and heteroaryl substitutions were achieved

at the 6-, 7-, and 8-positions by palladium-mediated

coupling with the appropriate chromenone (25, 29a, and

29b). Aryl substituents at the chromenone 6- or 7-posi-

tions dramatically reduced or abolished activity, whereas

analogous groups at the 8-position were generally well

tolerated. Strikingly, the 8-dibenzofuran, 8-dibenzo-

thiophene, and 8-tetrahydrodibenzothiophene deriva-

tives (32{26}, 32{38}, and 36) were found to be nano-

molar inhibitors of DNA-PK.

Compound 32{38} exhibits excellent selectivity for

DNA-PK over other PIKK family members and shows

good cellular radio- and chemopotentiation properties

at pharmacologically relevant concentrations. Prelimi-

nary studies to explore additional SARs in this series

have revealed a degree of tolerance to modification of

the dibenzothiophene ring of 32{38}. Studies are in

progress to optimize cellular and in vivo activities with

a view to identifying a clinical candidate.

Figure 2. In vitro radio- and chemosensitization of HeLa cells

by the DNA-PK inhibitor 32{38}: (a) Effects of increasing

doses of ionizing radiation (Gy) in the absence (9) or presence

of 0.2 (1) or 0.5 µM ([) 32{38}, on the clonogenic survival of

HeLa B cells. Cells were preincubated with 32{38} for 1 h

before exposure to ionizing radiation and incubated for a

further 16 h prior to fresh media being added, and colony

formation was determined after 8 days. Data are the mean (

sem of at least three independent experiments. (b) Effects of

increasing doses of etoposide in the absence (9) or presence of

0.2 (2), 0.5 (1), or 1 µM ([) 32{38} on the clonogenic survival

of HeLa B cells. Cells were preincubated with 32{38} for 1 h

before exposure to etoposide and incubated for a further 16 h

prior to fresh media being added, and colony formation was

determined after 8 days. Data are the mean ( sem of at least

three independent experiments.

mTOR (mammalian target of rapamycin), and PI 3-

kinase p110R (phosphatidylinositol 3-kinase). At con-

centrations up to 100 µM, 32{38} did not inhibit the

ATM or ATR protein kinases but did exhibit activity

against mTOR and PI 3-kinase, with IC₅₀values of 1.7

and 5.0 µM, respectively. This represents a selectivity

window of at least 100-fold against the kinases most

closely related to DNA-PK. This dramatic selectivity

was further highlighted when the compound was found

to have no significant inhibitory activity against the

Upstate panel of 60 kinases at a concentration of 10 µM.

Radiosensitization and Enhancement of Etopo-

side Cytotoxicity by 32{38} in HeLa Cells. Previous

work has revealed that inhibition of DNA-PK by a small

molecule approach leads to radio- and chemopotentia-

tion through the inhibition of DNA DSB break repair.²⁷

We therefore evaluated the potential of 32{38} to

potentiate the cytotoxicity of both ionizing radiation (IR)

and etoposide in a cell-based assay. As shown in Figure

2a, sub-micromolar concentrations of 32{38} were able

to radiosensitize HeLa cells over a wide range of IR

doses. Relative to the controls, 32{38} was found to have

dose modification factors at 10% survival of 1.3 and 2.5

at doses of 0.2 and 0.5 µM, respectively. When HeLa

cells were treated with the DNA-PK inhibitor in com-

bination with etoposide, significant potentiation of cell

kill was seen at inhibitor concentrations as low as 0.1

µM (Figure 2b). Coupled with the in vitro IC₅₀data,

these cell-based data further highlight 32{38} as a

Experimental Section

Reagents were purchased from fine chemicals vendors, and

used as received unless otherwise stated. Solvents were

purified and stored according to standard procedures. Petro-

leum ether refers to that fraction in the boiling range 40-60

°C. Melting points were obtained on a Stuart Scientific SMP3

apparatus and are uncorrected. Thin-layer chromatography

was performed using silica gel plates (Kieselgel 60F₂₅₄; 0.2 mm)

and visualization with UV light or potassium permanganate.

Chromatography was conducted under medium pressure on

silica (BDH silica gel 40-63 µm). HPLC refers to purification

on Gilson LC instruments with a 15 min gradient of 0.1%

aqueous TFA and 10-97% acetonitrile at a flow rate of 6 mL/

min using as the stationary phase a Jones Chromatography

Genesis 4µ C18 column, 10 mm × 250 mm, and peak

acquisition based on UV detection at 254 nm. Solution-phase

Suzuki reactions (general procedure L) were conducted in

“Greenhouse” reactors (Radley’s Ltd., U.K.) in batches of 24

reactions under an argon atmosphere. Proton (¹H) and carbon

(¹³C) nuclear magnetic resonance (NMR) spectra were recorded

on a Bruker Spectrospin AC 200E spectrometer at 200 and

50 MHz, respectively, or on a Bruker Avance 300 spectrometer

at 300 or 75 MHz, repectively, employing tetramethylsilane

(TMS) or the solvent as internal standard. Unless indicated

otherwise, spectra were recorded in [²H₆]-DMSO as solvent.

NH signals appeared as broad singlets (br s) exchangeable

with D₂O. Mass spectra were determined on a Micromass

Autospec M spectrometer in electron impact (EI) mode. Liquid

chromatography-mass spectrometry (LCMS) was carried out

on either a Micromass Platform instrument operating in

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7841

positive 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 5µ 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. IR spectra were recorded on a Bio-Rad FTS 3000MX

diamond ATR. Elemental analyses were performed by Butter-

worth Laboratories, Middlesex, U.K., and are within (0.4%

of theory unless otherwise specified.

General Procedure A: Alkylation of Dihydroxy Ben-

zoates and Acetophenones. A solution of the appropriate

phenol (1.0 equiv) in acetonitrile was treated with potassium

carbonate (2.2 equiv), followed by the appropriate alkylating

agent (1.0 equiv) under nitrogen. After being heated to reflux

for 16 h, the mixture was cooled and treated with 1 M

hydrochloric acid. The mixture was extracted into ethyl

acetate, washed with 1 M hydrochloric acid and brine, dried

over sodium sulfate, and evaporated in vacuo. The product was

purified by recrystallization from suitable solvents.

4-(4-Methoxybenzyloxy)-2-hydroxyacetophenone (14a).

General procedure A was followed with 2,4-dihydroxyaceto-

phenone (7.30 g, 53 mmol) and 4-methoxybenzyl chloride,

prepared by stirring 4-methoxybenzyl alcohol (6.0 mL, 48

mmol) in concentrated hydrochloric acid (40 mL) for 4 h and

extracting into DCM. Beige powder (6.36 g, 49%). Mp 82-83

°C. ¹H NMR (200 MHz, DMSO-d₆) δ 2.67 (3H, s, COCH₃); 3.87

(3H, s, OCH₃); 5.21 (2H, s, OCH₂); 6.66 (1H, d, J ) 2.4 Hz,

3-H); 6.69 (1H, dd, J ) 2.4, 8.6 Hz, 5-H); 7.06 (2H, d, J ) 8.6

Hz); 7.49 (2H, d, J ) 8.6 Hz); 7.95 (1H, d, J ) 8.6 Hz, 6-H),

12.7 (1H, bs, OH). LCMS (ESI+) m/z ) 273 [M + H]⁺. HRMS

(EI) for C₁₆H₁₆O₄calcd 272.1049, obsd 272.1054.

7-(4-Methoxybenzyloxy)-4-hydroxy-chromen-2-

thione (15). General procedure B was followed with 4-(4-

methoxybenzyloxy)-2-hydroxyacetophenone (5.44 g, 20.0 mmol).

Recrystallization (ethyl acetate) gave 15 as a yellow powder

(2.04 g, 32%). Mp 150-152 °C. ¹H NMR (200 MHz, DMSO-d₆)

δ 3.87 (3H, s, OCH₃); 5.28 (2H, s, OCH₂); 6.68 (1H, s, 3-H);

7.07 (2H, d, J ) 8.5 Hz); 7.18 (1H, dd, J ) 2.1, 8.8 Hz, 6-H);

7.34 (1H, d, J ) 2.1 Hz, 8-H); 7.53 (2H, d, J ) 8.5 Hz); 7.89

(1H, d, J ) 8.8 Hz, 5-H). LCMS (ESI+) m/z ) 315 [M + H]⁺.

HRMS (EI) for C₁₇H₁₄O₄S calcd 314.0613, obsd 314.0606.

6-(4-Methoxybenzyloxy)-4-hydroxy-chromen-2-

thione (19). General procedure B was followed with 5-(4-

methoxybenzyloxy)-2-hydroxyacetophenone (4.00 g, 14.7 mmol).

Recrystallization (ethyl acetate) gave (19) as a yellow powder

(2.06 g, 44%). Mp 157-158 °C. ¹H NMR (200 MHz, DMSO-d₆)

δ 3.99 (3H, s, OCH₃); 5.33 (2H, s, CH₂Ar); 6.90 (1H, s, 3-H);

7.18 (2H, m); 7.62 (4H, m); 7.77 (1H, m). LCMS (ESI+) m/z )

315 [M + H]⁺. HRMS (EI) for C₁₇H₁₄O₄S calcd 314.0613, obsd

314.0608.

6-Bromo-4-hydroxy-chromene-2-thione (23). General

procedure B was followed with 5-bromo-2-hydroxyaceto-

phenone (4.30 g, 20 mmol). Recrystallization (THF) gave 23

as a pale orange crystalline solid (1.85 g, 36%). Mp 122-123

°C. ¹H NMR (300 MHz, DMSO-d₆) δ 6.89 (1H, s, 3-H); 7.45

(1H, d, J ) 8.8 Hz, 8-H); 7.86 (1H, dd, J ) 2.4, 8.8 Hz, 7-H);

8.02 (1H, d, J ) 2.4 Hz, 5-H); MS ESI+ m/z ) 258 [M + H]⁺.

General Procedure C: Resin Loading Procedure.

Merrifield resin (1% cross-linked, 1.2 mmol/g; 0.70 g, 0.84

mmol) was swelled in anhydrous DMF (4 mL). The mixture

was shaken gently for 15 min and then treated with a solution

of the appropriate chromen-2-thione (2.2 mmol) in DMF (3

mL). After shaking for a further 15 min, the mixture was

treated with DBU (0.4 mL, 2.7 mmol). The reaction mixture

was heated to 70 °C and gently shaken for 24 h. The resin

was collected by filtration, washed with DMF (5 mL), methanol

(5 mL), and DCM (2 × 5 mL), and dried in vacuo.

5-(4-Methoxybenzyloxy)-2-hydroxyacetophenone (14b).

General procedure A was followed with 2,5-dihydroxyaceto-

phenone (3.65 g, 24.0 mmol) and 4-methoxybenzyl chloride

(prepared by stirring 4-methoxybenzyl alcohol (3.0 mL, 24

mmol) in concentrated hydrochloric acid (20 mL) for 4 h and

extracting into DCM). Off-white powder (4.03 g, 62%). Mp 86-

(Benzo[h]-4H-chromen-2-yl)thiomethylpolystyrene

resin (8). General procedure C was followed with 4-hydroxy-

benzo[h]chromen-2-thione (0.50 g, 2.2 mmol) in DMF (3 mL)

yielding a pale yellow resin.

1

87 °C. H NMR (200 MHz, DMSO-d₆) δ 2.73 (3H, s, COCH₃);

3.86 (3H, s, OCH₃); 5.12 (2H, s, OCH₂); 7.00 (1H, d, J ) 9.1

Hz, 3-H); 7.05 (2H, d, J ) 8.7 Hz); 7.33 (1H, dd, J ) 3.0, 9.1

Hz, 5-H); 7.49 (2H, d, J ) 8.7 Hz); 7.53 (1H, d, J ) 3.0 Hz,

6-H), 11.6 (1H, bs, OH). LCMS (ESI+) m/z ) 273 [M + H]⁺.

HRMS (EI) for C₁₆H₁₆O₄calcd 272.1049, obsd 272.1051.

S-(7-Hydroxy-4-oxo-4H-chromen-2-yl)thiomethylpoly-

styrene resin (16). General procedure C was followed with

7-(4-methoxybenzyloxy)chromen-2-thione (15) (1.20 g, 3.82

mmol) giving a pale yellow resin. The resin was swelled in

DCM (2.5 mL) and gently agitated for 15 min. The mixture

was cooled (ice-acetone), TFA (7.5 mL) was added, and the

mixture was agitated for 4 h, then filtered, washed (DCM,

methanol and DCM) and dried yielding a pale yellow resin,

1.37 g (∼1.38 mmol, ∼96%, based on mass).

Methyl 4-(2,6-dichlorobenzyloxy)-2-hydroxybenzoate

(11). General procedure A was followed with methyl 2,4-

dihydroxybenzoate (10)(1.68 g, 10.0 mmol), 2,6-dichlorobenzyl

chloride (1.95, 10.0 mmol), and sodium iodide (1.50 g, 10.0

mmol). Off-white powder (2.07 g, 63%). Mp 127-129 °C. IR

(Diamond ATR) υ_max(cm^-1): 2951, 1671, 1616, 1580, 1435,

S-(6-Hydroxy-4-oxo-4H-chromen-2-yl)thiomethylpoly-

styrene resin (20). General procedure C was followed with

6-(4-methoxybenzyloxy)-4-hydroxy-chromen-2-thione (0.70 g,

2.2 mmol). The resin was swelled in DCM (1.5 mL) and gently

agitated for 15 min. The mixture was cooled (ice-acetone), TFA

(4.5 mL) was added, and then the mixture was agitated for 4

h, filtered, and washed (DCM, methanol, DCM) yielding a pale

yellow resin, 0.74 g (∼0.74 mmol, ∼89%, based on mass).

GeneralProcedureD: SynthesisofaBenzo[h]chromen-

4-ones Library. Benzo[h]-4-oxo-4H-chromen-2-yl-thiomethyl-

polystyrene resin (maximum 0.036 mmol) was swelled in

anhydrous DCM (1 mL) and gently shaken for 15 min. The

reaction mixture was treated with a solution of the appropriate

amine (0.36 mmol) in DCM (0.2 mL). The mixture was

vigorously shaken at room temperature for 16 h, followed by

addition of Amberlite IR120+ resin and shaking for a further

1 h. The reaction mixture was filtered, and the resin was

washed successively with DCM (2 × 5 mL), MeOH (2 × 5 mL),

and DCM (5 mL). The filtrate was evaporated in vacuo to

provide the title compounds. The products were submitted for

analysis by LC-MS without further purification.

1

1344, 1248, 1215, 1184, 1134, 1092, 773. H NMR (200 MHz,

DMSO-d₆) δ 3.97 (3H, s, CH₃); 5.38 (2H, s, OCH₂Ar); 6.73 (1H,

dd, J ) 2.4, 8.8 Hz, 5-H); 6.82 (1H, d, J ) 2.4 Hz, 3-H); 7.62

(3H, m, C₆H₃Cl₂); 7.84 (1H, d, J ) 8.8 Hz, 6-H); 10.88 (1H, br,

OH). MS (EI+) m/z ) 330, 328, 326 [M]⁺. HRMS (EI) for

C₁₅H₁₂Cl₂O₄calcd 326.0113, obsd 326.0122.

General Procedure B: Synthesis of 4-Hydroxythio-

coumarins. A solution of potassium tert-butoxide (3.2 equiv)

in THF was slowly treated with a mixture of the appropriate

2-hydroxyacetophenone (1.0 equiv) and carbon disulfide (1.0

equiv) in THF at 10 °C under nitrogen. After the reaction

mixture was stirred for 16 h at room temperature, the thick

mixture was treated with water and washed twice with ether.

The aqueous solution was adjusted to pH 4 by treatment with

10% sulfuric acid and stirred for 16 h, passing nitrogen

through the flask, with an aqueous bleach trap fitted to trap

the hydrogen sulfide generated (CAUTION: hydrogen sulfide

is a highly toxic colorless gas). The precipitate was allowed to

settle, and the supernatant liquid was decanted. The yellow

solid residue was stirred vigorously in petrol until a fine

precipitate had formed, which was collected by filtration and

washed with copious amounts of cold petrol. The crude product

was purified by recrystallization from the appropriate solvent.

1-(4-(2,6-Dichlorobenzyloxy)-2-hydroxyphenyl)-3-(mor-

pholin-4-yl)-propan-1,3-dione (12). To a solution of diiso-

propylamine (3.2 mol equiv) in THF at -78 °C was added

n-BuLi (2.5 M in hexanes, 3.0 mol equiv). The resulting

7842 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

mixture was allowed to warm to 0 °C over 15 min under N₂

and cooled to -10 °C, acetyl morpholine (0.92 mL, 8.0 mmol)

was added, and stirring was continued for 90 min. Methyl

4-(2,6-dichlorobenzyloxy)-2-hydroxybenzoate (11; 1.64 g, 5.00

mmol) was added and stirring was continued for 16 h at room

temperature. Water (5 mL) was added, and the solution was

adjusted to pH 1.0 with hydrochloric acid (2 M) and extracted

(DCM, 3 × 80 mL). The combined organic layers were dried

(Na₂SO₄) and evaporated in vacuo to give the crude product.

Trituration with ether gave 12 as an off-white powder (1.68

g, 78%). Mp 117-121 °C. IR (Diamond ATR) υ_max(cm^-1): 3071,

2968, 2853, 1627, 1599, 1439, 1360, 1220, 1196, 1114, 994. ¹H

NMR (200 MHz, DMSO-d₆) δ 3.57 (4H, m); 3.67 (4H, m); 4.31

(2H, s, COCH₂); 5.42 (2H, s, OCH₂Ar); 6.76 (1H, dd, J ) 2.3,

8.8 Hz, 5-H); 6.79 (1H, d, J ) 2.3 Hz, 3-H); 7.64 (3H, m,

C₆H₃Cl₂); 7.94 (1H, d, J ) 8.8 Hz, 6-H); 12.2 (1H, br, OH). MS

(ESI+) m/z ) 426, 424 [M + H]⁺. HRMS (EI) for C₂₀H₁₉-

Cl₂NO₅calcd 423.0640, obsd 423.0649.

CH₃); 5.51 (1H, s, 3-H); 7.04 (1H, dd, J ) 2.0, 8.7 Hz, 6-H);

7.18 (1H, d, J ) 2.0 Hz, 8-H); 7.89 (1H, d, J ) 8.7 Hz, 5-H).

LCMS (ESI+) m/z ) 290 [M + H]⁺. HRMS (EI) for C₁₆H₁₉NO₄

calcd 289.1314, obsd 289.1311.

7-Cyclohexylmethoxy-2-(morpholin-4-yl)-chromen-4-

one (13{2}). General procedure E with 13b (124 mg, 0.5 mmol)

and cyclohexylmethyl bromide (0.28 mL, 2.0 mmol) gave 13{2}

as white crystals (55 mg, 32%). Mp 187-188 °C. ¹H NMR (200

MHz, DMSO-d₆) δ 1.16 (5H, m, cyclohexyl); 1.87 (6H, m,

cyclohexyl); 3.60 (4H, m, morpholine); 3.80 (4H, m, morpho-

line); 3.97 (2H, s, CH₂); 5.50 (1H, s, 3-H); 7.12 (1H, dd, J )

2.1, 8.7 Hz, 6-H); 7.18 (1H, d, J ) 2.1 Hz, 8-H); 7.88 (1H, d, J

) 8.7 Hz, 5-H). LCMS (ESI+) m/z ) 344 [M + H]⁺. HRMS

(EI) for C₂₀H₂₅NO₄calcd 343.1784, obsd 343.1793.

7-Benzyloxy-2-(morpholin-4-yl)-chromen-4-one (13{3}).

General procedure E with 13b (124 mg, 0.5 mmol) and benzyl

bromide (0.50 mL, 2.0 mmol) gave 13{3} as white crystals (98

1

mg, 48%). Mp 170-172 °C. H NMR (200 MHz, DMSO-d₆) δ

7-(2,6-Dichlorobenzyloxy)-2-(morpholin-4-yl)-chromen-

4-one (13a). A solution of 1-(morpholin-4-yl)-3-(4-(2,6-dichloro-

benzyloxy)-2-hydroxyphenyl)-propan-1,3-dione (8.60 g, 20.3

mmol) in DCM was treated with a solution of trifluoromethane-

sulfonic anhydride (3.6 equiv) in DCM under nitrogen at 0 °C.

After stirring at room temperature for 20 h, the reaction

mixture was evaporated in vacuo. The residue was redissolved

in methanol and stirred for 4 h before treatment with an equal

volume of water. The mixture was stirred for 1 h, evaporated

in vacuo to provide an aqueous mixture, and adjusted to pH 8

by treatment with saturated sodium hydrogen carbonate

solution. The mixture was extracted three times into DCM,

dried (Na₂SO₄), and evaporated in vacuo. The crude product

was vigorously stirred in ether for 16 h, and the precipitate

was collected by filtration. Recrystallization gave 13a as tan

3.59 (4H, m); 3.82 (4H, m); 5.31 (2H, s, CH₂); 5.52 (1H, s, 3-H);

7.13 (1H, dd, J ) 2.3, 8.7 Hz, 6-H); 7.28 (1H, d, J ) 2.3 Hz,

8-H); 7.45-7.60 (5H, m, C₆H₅); 7.91 (1H, d, J ) 8.7 Hz, 5-H).

LCMS (ESI+) m/z ) 338 [M + H]⁺, 179. C, H, N.

7-(2-Chlorobenzyloxy)-2-morpholin-4-yl-chromen-4-

one (13{4}). General procedure E with 13b (124 mg, 0.5 mmol)

and 2-chlorobenzyl bromide (0.26 mL, 2.0 mmol) gave 13{4}

as white crystals (52 mg, 28%). Mp 167-168 °C. ¹H NMR (200

MHz, DMSO-d₆) δ 3.61 (4H, m); 3.81 (4H, m); 5.36 (2H, s, CH₂);

5.54 (1H, s, 3-H); 7.15 (1H, dd, J ) 2.3, 8.7 Hz, 6-H); 7.35 (1H,

d, J ) 2.3 Hz, 8-H); 7.50-7.76 (4H, m); 7.93 (1H, d, J ) 8.7

Hz, 5-H). LCMS (ESI+) m/z ) 374, 372 [M + H]⁺. HRMS (EI)

for C₂₀H₁₈ClNO₄calcd 371.0924, obsd 371.0929.

7-(4-Chlorobenzyloxy)-2-morpholin-4-yl-chromen-4-

one (13{5}). General procedure E with 13b (124 mg, 0.5 mmol)

and 4-chlorobenzyl bromide (0.41 g, 2.0 mmol) gave 13{5} as

white crystals (840 mg, 46%). Mp 185 °C (dec).¹H NMR (200

MHz, DMSO-d₆) δ 3.60 (4H, m); 3.82 (4H, m); 5.31 (2H, s, CH₂);

5.52 (1H, s, 3-H); 7.13 (1H, dd, J ) 2.2, 8.7 Hz, 6-H); 7.28 (1H,

d, J ) 2.2 Hz, 8-H); 7.59-7.71 (4H, m, C₆H₄Cl); 7.92 (1H, d, J

) 8.7 Hz, 5-H). LCMS (ESI+) m/z ) 374, 372 [M + H]⁺. HRMS

(EI) for C₂₀H₁₈ClNO₄calcd 371.0924, obsd 371.0931.

1

crystals (6.68 g, 81%). Mp 194-195 °C. H NMR (200 MHz,

DMSO-d₆) δ 3.62 (4H, m); 3.82 (4H, m); 5.44 (2H, s, CH2Ar);

5.55 (1H, s, 3-H); 7.13 (1H, dd, J ) 2.4, 8.8 Hz, 6-H); 7.43 (1H,

d, J ) 2.4 Hz, 8-H); 7.57-7.73 (3H, m, C6H3Cl2); 7.94 (1H, d,

J ) 8.8 Hz, 5-H). LCMS (ESI+) m/z ) 410, 408, 406 [M +

H]⁺. HRMS (EI) for C₂₀H₁₇Cl₂NO₄calcd 405.0535, obsd

405.0542.

7-Hydroxy-2-(morpholin-4-yl)-chromen-4-one (13b). A

suspension of 13a (6.60 g, 16.2 mmol) and 10% Pd/C (0.15 g)

in methanol (150 mL) was stirred under an atmosphere of

hydrogen for 40 h. The catalyst was removed by filtration

through Celite, washing with methanol. The filtrate was

concentrated in vacuo to provide an off-white solid. This was

treated with fresh catalyst, resuspended in methanol, and

stirred under hydrogen for a further 72 h. The catalyst was

removed by filtration through Celite, washing with methanol.

The filtrate was evaporated in vacuo, and the crude product

was recrystallized from methanol to provide 13b as a white

solid (2.26 g, 57%). Mp 250 °C (dec). ¹H NMR (200 MHz,

DMSO-d₆) δ 3.78 (4H, m); 3.86 (4H, m); 6.15 (1H, s, 3-H); 7.05-

7.13 (2H, m, 6,8-H); 7.93 (1H, d, 5-H); 11.3 (1H, br, OH). MS

(EI) m/z ) 247 (M⁺), 190, 105. HRMS (EI) for C₁₃H₁₃NO₄calcd

247.0845, obsd 247.0837.

7-(4-Bromobenzyloxy)-2-morpholin-4-yl-chromen-4-

one (13{6}). General procedure E with 13b (124 mg, 0.5 mmol)

and 4-bromobenzyl bromide (0.50 g, 2.0 mmol) gave 13{6} as

1

white crystals (75 mg, 36%). Mp 221-222 °C. H NMR (200

MHz, DMSO-d₆) δ 3.60 (4H, m); 3.82 (4H, m); 5.30 (2H, s, CH₂);

5.52 (1H, s, 3-H); 7.13 (1H, dd, J ) 2.0, 8.7 Hz, 6-H); 7.27 (1H,

d, J ) 2.0 Hz, 8-H); 7.53 (2H, d, J ) 8.3 Hz, 2′,6′-H); 7.72 (2H,

d, J ) 8.3 Hz, 3′,5′-H); 7.92 (1H, d, J ) 8.7 Hz, 5-H). LCMS

(ESI+) m/z ) 418, 416 [M + H]⁺. HRMS (EI) for C₂₀H₁₈BrNO₄

calcd 415.0419, obsd 415.0424.

7-(4-Methylsulfanylbenzyloxy)-2-(morpholin-4-yl)-

chromen-4-one (13{7}). General procedure E with 13b (124

mg, 0.5 mmol) and 4-thiomethylbenzyl chloride (0.48 g, 2.0

mmol) gave 13{7} as an off-white powder (96 mg, 50%). Mp

157-159 °C. IR (Diamond ATR) υ_max(cm^-1): 1589, 1550, 1410,

1

1219, 771, 490. H NMR (200 MHz, DMSO-d₆) δ 2.76 (3H, s,

General Procedure E: Solution-Phase Alkylation. A

solution of 7-hydroxy-2-(morpholin-4-yl)-chromen-4-one 13b

(124 mg, 0.5 mmol) in DMF (5 mL) was treated with a

methanolic solution of benzyltrimethylammonium hydroxide

(0.54 mL, 1.2 mmol), followed by a solution of the appropriate

alkylating agent (2.0 mmol) in DMF (1.0 mL). The reaction

mixture was heated in an aluminum block at 90 °C for 16 h.

The solution was diluted with ethyl acetate (25 mL) and

washed with water (10 mL). The ethyl acetate extract was

evaporated in vacuo to dryness, and the residue was stirred

vigorously in ether for 16 h. The solid product was collected

by filtration and purified by recrystallization from methanol.

SCH₃); 3.76 (4H, m); 3.99 (4H, m); 5.43 (2H, s, CH₂Ar); 5.70

(1H, s, 3-H); 7.29 (1H, m, 6-H); 7.44 (1H, m, 8-H); 7.55 (2H,

m, 2′,6′-H); 7.69 (2H, m, 3′,5′-H); 8.08 (1H, m, 5-H). LCMS

(ESI+) m/z ) 384 [M + H]⁺. HRMS (EI) for C₂₁H₂₁NO₄S calcd

383.1191, obsd 383.1186. C, H, N.

N-[2-(2-(Morpholin-4-yl)-4-oxo-4H-chromen-7-yloxy)-

ethyl]phthalimide (13{8}). General procedure E with 13b

(124 mg, 0.5 mmol) and N-(2-bromoethyl)-phthalimide (0.406

g, 2.0 mmol) gave 13{8} as white crystals (6 mg, 3%). Mp 230

°C (dec). LCMS (ESI+) m/z ) 421 [M + H]⁺. HRMS (EI) for

C₂₃H₂₀N₂O₆calcd 420.1321, obsd 420.1328.

N-[3-(2-(Morpholin-4-yl)-4-oxo-4H-chromen-7-yloxy)-

propyl]phthalimide (13{9}). General procedure E with 13b

(124 mg, 0.5 mmol) and N-(3-bromopropyl)phthalimide (0.538

g, 2.0 mmol) gave 13{9} as white crystals (35 mg, 16%). Mp

210-211 °C. ¹H NMR (200 MHz, DMSO-d₆) δ 2.60 (2H, m,

NCH₂CH₂CH₂O); 3.58 (4H, m, morpholine); 3.81 (4H, m,

morpholine); 3.89 (2H, m, NCH₂CH₂CH₂O); 4.22 (2H, m, NCH₂-

7-Propoxy-2-(morpholin-4-yl)-chromen-4-one (13{1}).

General procedure E with 13b (124 mg, 0.5 mmol) and

1-bromopropane (0.18 mL, 2.0 mmol) gave 13{1} as white

1

crystals (39 mg, 27%). Mp 115 °C (dec). H NMR (200 MHz,

DMSO-d₆) δ 1.08 (3H, t, CH₂CH₂CH₃); 1.86 (2H, m, CH₂-

CH₂CH₃); 3.60 (4H, m); 3.81 (4H, m); 4.12 (2H, t, CH₂CH₂-

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7843

CH₂CH₂O); 5.50 (1H, s, 3-H); 6.86 (1H, dd, J ) 2.0, 8.6 Hz,

6-H); 7.03 (1H, d, J ) 2.0 Hz, 8-H); 7.83 (1H, d, J ) 8.6 Hz,

5-H); 7.95 (4H, m, phth-H₄). LCMS (ESI+) m/z ) 435 [M +

H]⁺. HRMS (EI) for C₂₄H₂₂N₂O₆calcd 434.1478, obsd 434.1475.

7-(Naphthalen-2-ylmethoxy)-2-morpholin-4-yl-chromen-

4-one (13{10}). General procedure E with 13b (124 mg, 0.5

mmol) and 2-(bromomethyl)-naphthalene (442 mg, 2.0 mmol)

gave 13{10} as white crystals (58 mg, 30%). Mp 263-264 °C.

¹H NMR (200 MHz, DMSO-d₆) δ 3.60 (4H, m); 3.81 (4H, m);

5.49 (2H, s, CH₂); 5.53 (1H, s, 3-H); 7.19 (1H, dd, J ) 2.2, 8.7

Hz, 6-H); 7.34 (1H, d, J ) 2.2 Hz, 8-H); 7.62-7.73 (3H, m);

7.92 (1H, d, J ) 8.7 Hz, 5-H); 8.02-8.11 (4H, m). LCMS (ESI+)

m/z ) 388 [M + H]⁺. HRMS (EI) for C₂₄H₂₁NO₄calcd 387.1471,

obsd 387.1476.

7-Benzoyloxy-2-(morpholin-4-yl)-chromen-4-one(13{11}).

A solution of 7-hydroxy-2-(morpholin-4-yl)-chromen-4-one (13b)

(0.25 g, 1.0 mmol) in DMF (10 mL) was treated with benzoyl

chloride (0.13 mL, 1.1 mL), followed by pyridine (0.10 mL, 1.2

mmol) at 0 °C. The solution was warmed to room temperature,

stirred 16 h, then diluted with ethyl acetate (100 mL), and

washed with hydrochloric acid (0.5 M; 50 mL), water (50 mL),

and brine (50 mL). The organic phase was dried (Na₂SO₄) and

evaporated in vacuo. Recrystallization (EtOAc) gave 13{11}

as white crystals (0.19 g, 55%). Mp 204-206 °C. ¹H NMR (200

MHz, DMSO-d₆) δ 3.64 (4H, m); 3.83 (4H, m); 5.65 (1H, s, 3-H);

7.45 (1H, m); 7.74 (3H, m); 8.87 (1H, m); 8.09 (1H, m); 8.26

(2H, m). MS (ES) m/z ) 352 [M + H]⁺; 179. C, H, N.

General Procedure F: Solid-Phase Alkylation. The

appropriate resin-bound hydroxy-chromenone (0.030 g, <0.036

mmol) was swelled in anhydrous DMF (5 mL) in a polytetra-

fluoroethylene (PTFE) fritted vessel and gently shaken for 15

min. The mixture was treated with DBU (0.2 mL, 1.3 mmol).

After shaking for a further 15 min, the mixture was treated

with the appropriate alkylating agent (0.7 mmol). The reaction

was heated to 65 °C and shaken for 20 h. The resin was

collected by filtration and washed in order with DMF × 1,

methanol × 1, and DCM × 2. The resin was resuspended in

DCM (2 mL), and the desired compound was cleaved from the

resin according to general procedure G.

General Procedure G: Cleavage Procedure. The resin-

bound chromenone (maximum 0.036 mmol) was suspended in

DCM (2 mL), and after shaking for 10 min, the mixture was

treated with mCPBA (0.2 g, 1.1 mmol). The mixture was

shaken at room temperature for 3 h and then filtered. The

resin was washed in order with DCM × 2, methanol × 2, and

DCM × 2 and re-suspended in DCM (2 mL). After shaking for

15 min, the mixture was treated with a solution of morpholine

(0.005 mL, 0.05 mmol) in DCM (2 mL). The mixture was

shaken at room temperature for 16 h and filtered and the resin

was washed with methanol (2 × 2 mL). The filtrate was

evaporated in vacuo to provide the title compound. The product

was submitted for analysis by LCMS without further purifica-

tion.

General Procedure H: Mitsunobu Alkylation. Com-

pound 13b (20 mg, < 0.024 mmol) was swelled in THF (1 mL)

in a PTFE fritted vessel and gently shaken for 15 min. With

gentle agitation for 10 min between the addition of each

reagent, the vessel was sequentially treated with TEA (0.05

mL), a solution of triphenylphosphine (0.063 g) in THF (0.5

mL), and a solution of the appropriate alcohol (0.25 mmol) in

THF (0.5 mL). After a further 10 min, the vessel was treated

with a solution of diisopropyl azodicarboxylate (DIAD) (0.047

mL) in THF (0.5 mL) chilled in a dry ice/acetone bath prior to

addition. The reaction vessels were gently agitated for 20 h

and drained, the resin was washed with DCM × 2, DMF × 1,

methanol × 1, and DCM × 2, and the desired compound was

cleaved from the resin according to General procedure G.

dried (Na₂SO₄), and evaporated in vacuo to yield a brown solid.

Recrystallization (EtOAc, petrol) gave 24 as off-white crystals

1

(0.820 g, 64%). Mp 155-157 °C. H NMR (300 MHz, DMSO-

d₆) δ 1.38 (3H, t, J ) 7.4 Hz, CH₃); 3.02 (2H, q, J ) 7.4 Hz,

CH₂); 6.19 (1H, s, 3-H); 7.45 (1H, d, J ) 7 Hz, 8-H); 7.65 (1H,

dd, J ) 2.4, 7 Hz, 7-H); 8.23 (1H, d, J ) 2.4 Hz, 5-H). MS

(ESI+) m/z ) 288 [M + H]⁺.

6-Bromo-2-morpholin-4-yl-chromen-4-one (25). A mix-

ture of 24 (0.45 g, 1.58 mmol) and m-chloroperbenzoic acid (2.0

mol equiv) in DCM (10 mL) was stirred for 3 h at room

temperature, then cooled to -15 °C and filtered, and the

filtrate was collected and concentrated in vacuo. The residues

were dissolved in warm acetonitrile (25 mL), and morpholine

(5 mol equiv) was added. The mixture was stirred at room

temperature for 16 h, then concentrated in vacuo. The residues

were dissolved in DCM and washed with 2 N HCl, water, and

saturated sodium chloride and then dried (Na₂SO₄) and

concentrated in vacuo. Recrystallization (MeOH) gave 25 as

an off white solid (0.31 g, 63%). Mp 147-149 °C. λ(nm/MeOH)

) 217.5 (λ_max), 234.0, 301.5; ¹H NMR (200 MHz, CDCl₃) δ 3.44

(4H, m, CH₂N); 3.77 (4H, m, CH₂O); 5.42 (1H, s, 3-H); 7.11

(1H, d, 8-H); 7.57 (1H, dd, 7-H); 8.20 (1H, d, 5-H); MS (ESI+)

m/z ) 310.24 [M + H]⁺; IR (Diamond ATR) υ_max(cm^-1) 2968,

2909, 2868, 1639, 1604, 1556, 1408, 1246, 985, 788.

General Procedure I: 6-Position Suzuki Library. To

a mixture of the appropriate boronic acid or ester (0.071 mmol),

25 (20 mg, 0.064 mmol), and powdered potassium carbonate

(18 mg, 0.13 mmol) in degassed 1,4-dioxane (0.5 mL) was

added a solution of tetrakis(triphenylphosphine)palladium(0)

(3 mg) in degassed dioxane (0.3 mL), and the resulting mixture

was heated to 90 °C for 18 h under nitrogen. The mixture was

cooled and passed through a silica plug (isolute Si 500 mg

cartridge) and eluted with 30% methanol/DCM (8 mL). The

eluant was analyzed by LCMS and purified by preparative

HPLC to >95% purity, as estimated by LCMS.

6-(4-Methoxyphenyl)-2-morpholin-4-yl-chromen-4-

one (30{13}). To a solution of 23 (60 mg, 0.194 mmol) in DME

(1 mL) was added aq Na₂CO₃(1 M, 0.46 mL), 4-methoxy-

phenylboronic acid (32 mg, 0.21 mmol), and tetrakis(tri-

phenylphosphine)palladium(0) (7 mg, 0.006 mmol). The reac-

tion mixture was heated to reflux for 16 h, cooled, diluted with

DCM (10 mL), and washed with water (10 mL), and the

organic fraction was evaporated in vacuo. Chromatography

(10% MeOH/DCM) gave 30{13} as a white solid (32 mg, 49%).

Mp 220-223 °C. IR (KBr) υ_max(cm^-1) 3072, 2860, 1604, 1554,

1519, 1439, 1352, 1245, 1112, 812. MS (ESI+) m/z ) 338.28

[M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ 3.46 (4H, m); 3.78

(7H, m); 5.45 (1H, s); 7.92 (2H, d); 7.26 (1H, d); 7.52 (2H, d);

7.68 (1H, dd); 8.26 (1H, d).

N-[2-(2-Morpholin-4-yl-4-oxo-4H-chromen-6-yl)phenyl]-

acetamide (30{40}). General procedure I with (2-acetyl-

aminophenyl)boronic acid pinacol ester (0.019 g, 0.071 mmol)

gave 30{40}, 4.8 mg (21%). MS (ESI+) m/z ) 365 [M + H]⁺.

¹H NMR (300 MHz, CDCl₃) δ 1.93 (3H, s, CH₃); 2.25 (1H, br s,

NH); 3.51 (4H, t, J ) 4.8 Hz, NCH₂); 3.78 (4H, t, J ) 4.8 Hz,

OCH₂); 5.59 (1H, s, 3-H); 7.25-7.51 (4H, m, ArH); 7.53-7.58

(1H, m, ArH); 7.93 (1H, d, J ) 8.1 Hz, ArH); 8.01 (1H, d, J )

2.8 Hz, ArH). MS (EI), m/z (%): 364 (M⁺) (25). HRMS (EI)

(C₂₁H₂₀N₂O₄) calcd 364.142, obsd 364.141.

2-Hydroxy-4-trifluoromethanesulfonyloxybenzoic acid

methyl ester (27a). Trifluromethanesulfonic anhydride (1.31

mL, 7.81 mmol) was added dropwise to a mixture of 26a (5.0

g, 29.8 mmol), DCM (30 mL), pyridine (1.2 mL, 14.9 mmol),

and dimethylaminopyridine (DMAP) (0.07 g, 0.58 mmol) at 0

°C. The mixture was allowed to warm to room temperature,

stirred 16 h, then washed with HCl (1 M; 50 mL), dried

(Na₂SO₄) and concentrated in vacuo. Recrystallization (EtOAc)

gave 27a as a white solid (4.10 g, 46% yield). Mp 104-105 °C.

¹H NMR (300 MHz, CDCl₃) δ 4.00 (3H, s, CH₃); 6.84 (1H, dd,

J ) 2.5, 6.3 Hz,6-H); 6.94 (1H, d, J ) 2.5 Hz, 8-H); 7.95 (1H,

d, J ) 6.3 Hz, 5-H); MS (ESI+) m/z ) 300 [M + H]⁺.

6-Bromo-2-ethylsulfanyl-chromen-4-one (24). A mixture

of 23 (1.01 g, 4.43 mmol), potassium carbonate (0.70 g, 5.0

mmol), and iodoethane (1.3 mL, 16 mmol) in acetone (50 mL)

was heated to reflux for 2 h. The mixture was concentrated in

vacuo, and the residue was partitioned between water (100

mL) and DCM (100 mL). The aqueous layer was extracted into

DCM (3 × 50 mL), and the organic extracts were combined,

2-Hydroxy-3-trifluoromethanesulfonyloxybenzoic Acid

Methyl Ester (27b). To a mixture of 26b (4.00 g, 24 mmol),

pyridine (0.96 mL, 12 mmol), and DMAP (0.07 g, 0.6 mmol) in

7844 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

DCM (25 mL) was added trifluoromethanesulfonic anhydride

(1.05 mL, 6.25 mmol) dropwise at 0 °C. The mixture was

allowed to warm to room temperature and stirred for 60 h,

then washed with 1 M HCl (40 mL), dried (Na₂SO₄), and

concentrated in vacuo. Recrystallization (EtOAc) gave 27b as

white crystals (2.62 g, 37%). Mp 91.5-92 °C. ¹H NMR (300

MHz, CDCl₃) δ 7.85 (1H, d, Ar4), 7.45 (1H, d, Ar6), 6.95 (1H,

t, Ar5), 4.00 (3H, d, CH₃). MS (ESI+) m/z ) 300.0 [M + H]⁺.

General Procedure J: â-Keto Amide Formation. To a

solution of diisopropylamine (3.28 mol equiv) in THF (15 mL)

cooled to -78 °C under nitrogen was added 2.5 M n-butyl-

lithium (2.5 M in hexanes, 3.28 mol equiv), and the mixture

was stirred for 30 min at 0 °C; a solution of the appropriate of

N-acetylamine (1.7 mol equiv) in THF (20 mL) was added and

stirring continued for 1 h at 0 °C, and then a solution of the

appropriate hydroxybenzoic acid methyl ester in THF (20 mL)

was added and stirring continued for 5 h. The mixture was

adjusted to pH 7 by addition of 1 M hydrochloric acid and

extracted into DCM. The combined DCM fractions were dried

(Na₂SO₄) and concentrated in vacuo. The residues were

triturated with ether to provide the title compound.

Trifluoromethanesulfonic Acid 3-Hydroxy-4-(3-mor-

pholin-4-yl-3-oxopropionyl)phenyl Ester (28a). General

procedure J with 27a (1.65 g, 7 mmol) and N-acetylmorpholine

(1.09 mL, 9.35 mmol) afforded a brown solid (0.535 g, 20%).

¹H NMR (300 MHz, CDCl₃) δ 3.48 (2H, s, CH₂); 3.64 (8H, m,

morpholine); 6.70 (1H, dd, J ) 2.4, 8.9 Hz, Ar-6H); 7.85 (1H,

d, J ) 2.4 Hz Ar-2H); 7.35 (1H, d, J ) 8.9 Hz Ar-5H). MS

(ESI+) m/z ) 398.

Trifluoromethanesulfonic Acid 2-Hydroxy-3-(3-mor-

pholin-4-yl-3-oxopropionyl)phenyl Ester (28b). General

procedure J with 27b (2.10 g, 7 mmol) gave 28b as brown solid

(1.10 g, 36%). ¹H NMR (300 MHz, CDCl₃) δ 3.50 (8H, m,

morpholine); 4.05 (2H, s, CH₂); 6.90 (1H, dd, Ar-H); 7.85 (1H,

d, Ar-H); 7.35 (1H, d, Ar-H). MS (ESI+) m/z ) 398.25.

General Procedure K: Synthesis of 2-Amino Chromen-

4-ones. To a solution of the appropriate 1-(1-hydroxyaryl)-3-

amino propan-1,3-dione in DCM (5 mL) was added triflic

anhydride (3.0 mol equiv) under N₂at 0 °C, and the mixture

was stirred at room temperature for 72 h, then concentrated

in vacuo. The residues were dissolved in methanol (20 mL)

and stirred for 5 h. An equal volume of water (20 mL) was

added, and the mixture was stirred for 16 h, then concentrated

in vacuo. The aqueous concentrate was adjusted to pH 8 with

NaHCO₃(sat.) and extracted with DCM × 3. The DCM

extracts were combined, dried (Na₂SO₄), and concentrated in

vacuo. Triturated in ether followed by column chromatography

gave the desired product.

and purified by preparative HPLC to >95% purity, as esti-

mated by LCMS.

7-(2-Benzyloxyphenyl)-2-morpholin-4-yl-chromen-4-

one (31{2}). General procedure L was followed with 29a and

(2-benzyloxyphenyl)boronic acid (0.0132 g, 0.058 mmol) yield-

1

ing 31{2}, 4.7 mg (21%). H NMR (300 MHz, CDCl₃) δ 3.45

(4H, t, J ) 4.8 Hz, NCH₂); 3.76 (4H, t, J ) 4.8 Hz, OCH₂);

5.05 (2H, s, Ar-CH₂); 5.57 (1H, s, 3-H); 6.98-7.04 (2H, m,

ArH); 7.28-7.32 (4H, m, ArH); 7.49-7.55 (2H, m, ArH); 8.10

(1H, d, J ) 8.1 Hz, ArH). MS (ESI+) m/z ) 414 [M + H]⁺.

HRMS (C₂₆H₂₃NO₄) calcd 413.163, obsd 413.161.

7-(3-Benzyloxyphenyl)-2-morpholin-4-yl-chromen-4-

one (31{3}). General procedure L was followed with 29a and

(3-benzyloxyphenyl)boronic acid (0.0132 g, 0.058 mmol) yield-

1

ing 31{3}, 5.0 mg (23%). H NMR (300 MHz, CDCl₃) δ 3.48

(4H, t, J ) 4.8 Hz, NCH₂); 3.78 (4H, t, J ) 4.8 Hz, OCH₂);

5.07 (2H, s, Ar-CH₂); 5.49 (1H, s, 3-H); 6.94 (1H, d, J ) 9.5

Hz, ArH); 7.25-7.49 (7H, m, ArH); 8.13 (1H, d, J ) 8.1 Hz).

MS (ESI+) m/z ) 414 [M + H]⁺. HRMS (EI) (C₂₆H₂₃NO₄) calcd

413.162, obsd 413.161.

3-[3-(2-Morpholin-4-yl-4-oxo-4H-chromen-7-yl)phenyl]-

acrylic Acid Methyl Ester (31{6}). General procedure L

with 29a and [3-(E-3-methoxy-3-oxo-1-propen-1-yl)phenyl]-

boronic acid (0.0119 g, 0.058 mmol) gave 31{6}, 4.9 mg (24%).

¹H NMR (300 MHz, CDCl₃) δ 3.51 (4H, t, J ) 4.8 Hz, NCH₂);

3.76 (3H, s, OCH₃); 3.79 (4H, t, J ) 4.8 Hz, OCH₂); 5.57 (1H,

s, 3-H); 6.47 (1H, d, J ) 16 Hz, CH)CH); 7.30-7.62 (6H, m,

ArH); 7.71 (1H, d, J ) 16 Hz, CHdCH); 8.17 (1H, d, J ) 8.2

Hz, ArH). MS (ESI+) m/z ) 392 [M + H]⁺. MS (EI), m/z (%):

391 (M⁺) (100). HRMS (EI) (C₂₃H₂₁NO₅) calcd 391.063, obsd

391.067.

7-Biphenyl-2-yl-2-morpholin-4-yl-chromen-4-one(31{14}).

General procedure L with 29a and 2-biphenylboronic acid

(0.0115 g, 0.058 mmol) gave 31{14}, 5.0 mg (25%). MS (ESI+)

1

m/z ) 384 [M + H]⁺. H NMR (300 MHz, CDCl₃) δ 3.46 (4H,

t, J ) 4.9 Hz, NCH₂); 3.75 (4H, t, J ) 4.9 Hz, OCH₂); 5.69

(1H, s, 3-H); 6.98-7.21 (6H, m, ArH); 7.34-7.49 (4H, m, ArH);

7.87 (1H, d, J ) 8.1 Hz, ArH). MS (EI), m/z (%): 383 (M⁺)

(100). HRMS (EI) (C₂₅H₂₁NO₃) calcd 383.152, obsd 383.152.

2-Morpholin-4-yl-7-thiophen-2-yl-chromen-4-one(31{17}).

General procedure L with 29a and 2-thiophene boronic acid

(0.0074 g, 0.058 mmol) gave 31{17}, 1.1 mg (7%). MS (ESI+)

1

m/z ) 314 [M + H]⁺. H NMR (300 MHz, CDCl₃) δ 3.61 (4H,

t, J ) 4.8 Hz, NCH₂); 3.81 (4H, t, J ) 4.8 Hz, OCH₂); 6.20

(1H, s, 3-H); 7.08 (1H, dd, J ) 1.4 and 3.7 Hz, CH-CH-CH-

S); 7.29-7.55 (3H, m, ArH,); 8.10 (1H, d, J ) 8.3 Hz). MS (EI),

m/z (%): 313 (M⁺) (100). HRMS (EI) (C₁₇H₁₅NO₃S) calcd

313.077, obsd 313.077.

Trifluoromethanesulfonic Acid 2-Morpholin-4-yl-4-

oxo-4H-chromen-7-yl Ester (29a). General procedure K with

28a (0.32 g, 0.81 mmol) gave 29a as an off-white solid (0.127

g, 41%). Mp 143-145 °C. ¹H NMR (300 MHz, CDCl₃) δ3.45

(4H, m, CH₂N); 3.77 (4H, m, CH₂O); 5.36 (1H, s,3-H); 7.32 (2H,

m, Ar-H); 8.01 (1H, m, Ar-H). MS ESI+ m/z ) 380.21 [M +

H]⁺. IR (Diamond ATR) υ_max(cm^-1) 3084, 1639, 1599, 1571,

2-Morpholin-4-yl-7-(4-trifluoromethoxyphenyl)-

chromen-4-one (31{18}). General procedure L with 29a and

4-(trifluoromethoxy)phenyl boronic acid (0.0119 g, 0.058 mmol)

gave 31{18}, 6.3 mg (30%). MS (ESI+) m/z ) 392 [M + H]⁺.

¹H NMR (300 MHz, CDCl₃) δ 3.56 (4H, t, J ) 4.8 Hz, NCH₂);

3.80 (4H, t, J ) 4.8 Hz, OCH₂); 5.95 (1H, s, 3-H); 7.28 (2H, d,

J ) 9.8 Hz, ArH); 7.51-7.58(4H, m); 8.17 (1H, d, J ) 7.6 Hz,

ArH). MS (EI), m/z (%): 391 (M⁺) (100). HRMS (EI) (C₂₀H₁₆-

NO₄F₃) calcd 391.103, obsd 391.103.

1409, 1282, 1132, 781 cm^-1

.

Trifluoromethanesulfonic Acid 2-Morpholin-4-yl-4-

oxo-4H-chromen-8-yl Ester (29b). General procedure K with

28b (0.91 g, 2.3 mmol) gave 29b as a white solid (0.25 g, 29%).

7-Furan-2-yl-2-morpholin-4-yl-chromen-4-one (31{20}).

General procedure L with 29a and 2-furan boronic acid (0.0065

g, 0.058 mmol) gave 31{20}, 6.0 mg (38%). MS (ESI+) m/z )

1

Mp 178-179 °C. H NMR (300 MHz, CDCl₃) δ 3.50 (4H, m,

CH₂N); 3.78 (4H, m, CH₂O); 5.46 (1H, s, 3-H); 7.40 (2H, m,

Ar-H6, 7); 8.09 (1H, m, Ar-H5). MS ESI+ m/z ) 380 [M + H]⁺.

λ(nm/MeOH) ) 208.5(λ_max), 314.0. IR (Diamond ATR) υ_max

(cm^-1) 2870, 1606, 1558, 1207, 1138, 785. C, H, N.

1

298 [M + H]⁺. H NMR (300 MHz, CDCl₃) δ 3.59 (4H, t, J )

4.8 Hz, NCH₂); 3.80 (4H, t, J ) 4.8 Hz, OCH₂); 6.11 (1H, s,

3-H); 6.48 (1H, dd, J ) 1.6 and 1.8 Hz); 6.79 (1H, d, J ) 2.9

Hz); 7.48 (1H, d, J ) 1.3 Hz); 7.58-7.62 (2H, m, ArH); 8.08

(1H, d, J ) 8.3 Hz, ArH). MS (EI), m/z (%): 297 (M⁺) (100).

HRMS (EI) (C₁₇H₁₅NO₄) calcd 297.100, obsd 297.099.

General Procedure L: 7- and 8-Position Suzuki Li-

brary. To a mixture of the appropriate boronic acid (0.058

mmol), 29a or 29b (20 mg, 0.053 mmol), and powdered

potassium carbonate (14.6 mg, 0.106 mmol) in degassed 1,4-

dioxane (0.5 mL) was added a solution of tetrakis(triphenyl-

phosphine)palladium(0) (3.1 mg) in degassed dioxane (0.3 mL),

and the reaction mixture was heated to 90 °C for 18 h under

nitrogen. The mixture was cooled and passed through a silica

plug (isolute Si 500 mg cartridge) and eluted with 30%

methanol/DCM (8 mL). The solution was analyzed by LCMS

7-(3-Acetylphenyl)-2-morpholin-4-yl-chromen-4-one

(31{24}). General procedure L with 29a and 3-acetylphenyl-

boronic acid (0.0095 g, 0.058 mmol) gave 31{24}, 5.1 mg (28%).

1

MS (ESI+) m/z ) 350 [M + H]⁺. H NMR (300 MHz, CDCl₃)

δ 2.62 (3H, s, CH₃); 3.52 (4H, t, J ) 4.8 Hz, NCH₂); 3.80 (4H,

t, J ) 4.8 Hz, OCH₂); 5.61 (1H, s, 3-H); 7.49-7.60 (3H, m, ArH);

7.76-7.79 (1H, m, ArH); 7.90-7.94 (1H, m, ArH); 8.15-8.19

Chromen-4-one DNA-PK Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7845

(2H, m, ArH). MS (EI), m/z (%): 349 (M⁺) (100). HRMS (EI)

(C₂₁H₁₉NO₄) calcd 349.131, obsd 349.131.

mL) and water (5 mL) was added dropwise 2-bromoben-

zenethiol (0.46 mL, 3.8 mmol), followed by 2-chlorocyclo-

hexanone (560 mg, 42 mmol) in ethanol (3 mL). The mixture

was stirred 30 min at room temperature then heated to reflux

16 h. When the mixture cooled, water (10 mL) was added, the

organic layer was removed, and the aqueous phase was

extracted with chloroform (30 mL). The combined organic

layers were dried over magnesium sulfate, filtered, and

concentrated in vacuo. Chromatography (neutral alumina; 40%

toluene, hexane) gave 33 as a translucent oil (841 mg, 78%

2-Morpholin-4-yl-7-(3-trifluoromethoxyphenyl)-

chromen-4-one (31{25}). General procedure L with 29a and

3-(trifluoromethoxy)phenylboronic acid (0.0119 g, 0.058 mmol)

gave 31{25}, 6.8 mg (33%). MS (ESI+) m/z ) 392 [M + H]⁺.

¹H NMR (300 MHz, CDCl3) δ 3.53 (4H, t, J ) 4.8 Hz, NCH2);

3.79 (4H, t, J ) 4.8 Hz, OCH2); 5.70 (1H, s, 3-H); 7.20-7.25

(1H, m, ArH); 7.41-7.54 (5H, m, ArH); 8.17 (1H, d, J ) 8.2

Hz, ArH). MS (EI), m/z (%): 391 (M⁺) (79). HRMS (EI) (C₂₀H₁₆-

NO₄F) calcd 391.103, obsd 391.104.

1

yield). H NMR (300 MHz, CDCl₃) δ 1.53-1.98 (m, 4H, CH₂);

2.02-2.26 (m, 3H, CH₂); 2.82-2.98 (m, 1H, CH₂); 3.84 (t, J )

5.9 Hz, 1H, CHS); 6.98 (t, J ) 7.2 Hz, 1H, Ph); 7.16 (t, J ) 7.2

Hz, 1H, Ph); 7.33 (d, J ) 7.8 Hz, 1H, Ph); 7.47 (d, J ) 7.8 Hz,

1H, Ph). ¹³C NMR (75 MHz, CDCl₃) δ 22.7; 27.5; 33.8; 39.1;

54.9; 125.9; 128.0; 128.3; 132.1; 133.3; 135.4; 207.5.

7-(4-Hydroxymethylphenyl)-2-morpholin-4-yl-chromen-

4-one (31{32}). General procedure L with 29a and 4-(hydroxy-

methyl)phenyl boronic acid (0.0088 g, 0.058 mmol) gave

31{32}, 3.7 mg (21%). MS (ESI+) m/z ) 338 [M + H]⁺. ¹H

NMR (300 MHz, CDCl₃) δ 3.49 (4H, t, J ) 4.6 Hz, NCH₂); 3.78

(4H, t, J ) 4.6 Hz, OCH₂); 4.71 (2H, s, CH₂OH); 5.54 (1H, s,

3-H); 7.41-7.45 (3H, m, ArH); 7.51-7.59 (3H, m, ArH); 8.12

(1H, d, J ) 8.2 Hz, ArH). MS (EI), m/z (%): 337 (M⁺) (100).

HRMS (EI) (C₂₀H₁₉NO₄) calcd 337.131, obsd 337.133.

2-Morpholin-4-yl-7-(2-trifluoromethylphenyl)chromen-

4-one (31{39}). General procedure L with 29a and 2-(trifluoro-

methyl)phenylboronic acid (0.0110 g, 0.058 mmol) gave 31{39},

6-Bromo-1,2,3,4-tetrahydrodibenzothiophene (34).²³A

mixture of polyphosphoric acid (1.12 g, 6.5 mmol) and phos-

phorus pentoxide (250 mg, 1.8 mmol) was slowly heated to 180

°C with continuous stirring. Compound 33 (203 mg, 0.71 mmol)

was added, and the reaction mixture was stirred for 30 min

at 180 °C. When the mixture cooled, water (30 mL) was added,

and the mixture was extracted with ether (90 mL). The organic

extract was dried (MgSO₄), filtered, and concentrated in vacuo.

Chromatography (silica gel; petroleum ether) gave 34 as an

oil (153 mg, 81%). ¹H NMR (300 MHz, CDCl₃) δ 1.79-1.85 (m,

4H, CH₂); 2.58-2.61 (m, 2H, CH₂); 2.73-2.76 (m, 2H, CH₂);

7.08 (t, J ) 7.8 Hz, 1H, Ph); 7.29 (d, J ) 7.7 Hz, 1H, Ph); 7.38

(d, J ) 7.9 Hz, 1H, Ph). ¹³C NMR (75 MHz, CDCl₃) δ 22.5;

23.9; 24.3; 26.0; 116.2; 119.7; 125.6; 126.8; 130.9; 138.7; 141.3.

4,4,5,5-Tetramethyl-2-(6,7,8,9-tetrahydrodibenzothio-

1

5.3 mg (27%). MS (ESI+) m/z ) 376 [M + H]⁺. H NMR (300

MHz, CDCl₃) δ 3.46 (4H, t, J ) 4.7 Hz, NCH₂); 3.77 (4H, t, J

) 4.7 Hz, OCH₂); 5.51 (1H, s, 3-H); 7.23-7.29 (3H, m, ArH);

7.44-7.56 (2H, m, ArH); 7.72 (1H, d, J ) 7.1 Hz, ArH); 8.11

(1H, d, J ) 8.2 Hz, ArH). MS (EI), m/z (%): 375 (M⁺) (100).

HRMS (C₂₀H₁₆NO₃F₃) calcd 375.108, obsd 375.109.

8-(4-Methoxyphenyl)-2-morpholin-4-yl-chromen-4-

one (32{13}). General procedure L with 29b and 4-methoxy-

phenyl boronic acid (8.8 mg, 0.058 mmol) gave 32{13}, 2.5 mg

phen-4-yl)-1,3,2]dioxaborolane (35).

A mixture of 34

(500 mg, 1.87 mmol), PdCl₂(1,1′-Bis(diphenylphosphino)ferro-

cene(dppf)) (150 mg, 0.19 mmol), potassium acetate (1.10 g,

11.23 mmol), and bis(pinacolato)diboron (713 mg, 2.81 mmol)

in THF (25 mL) was heated to reflux 16 h. Ethyl acetate (20

mL) was added, and the organic layer was washed with water

(20 mL) and brine (20 mL), then dried (MgSO₄), and concen-

trated in vacuo. Chromatography on (silica; 5% ethyl acetate,

petroleum ether) gave 35a (282 mg, 48%). ¹H NMR (300 MHz,

CDCl₃) δ 1.27 (s, 12H, C(CH₃)₂); 1.78-1.80 (m, 4H, CH₂); 2.59-

2.61 (m, 2H, CH₂); 2.74-2.76 (m, 4H, CH₂); 7.22 (t, J ) 7.5

Hz, 1H, Ph); 7.52 (d, J ) 7.9 Hz, 1H, Ph); 7.65 (d, J ) 7.0 Hz,

1H, Ph).

1

(14%). MS (ESI+) m/z ) 338 [M + H]⁺. H NMR (300 MHz,

CDCl₃) δ 3.30 (4H, t, J ) 4.8 Hz, NCH₂); 3.68 (4H, t, J ) 4.8

Hz, OCH₂); 3.81 (3H, s, CH₃); 5.48 (1H, s, 3-H); 6.90-6.93 (2H,

m, ArH); 7.29-7.37 (3H, m, ArH); 7.41-7.49 (1H, m, ArH);

8.07 (1H, d, J ) 7.8 Hz, ArH). MS (EI), m/z (%): 337 (M⁺)

(100). HRMS (EI) (C₂₀H₁₉NO₄) calcd 337.131, obsd 337.130.

2-Morpholin-4-yl-8-(4-trifluoromethoxyphenyl)-

chromen-4-one (32{18}). General procedure L with 29b and

4-(trifluoromethoxy)phenyl boronic acid (11.9 mg, 0.058 mmol)

gave 32{18}, 3.9 mg (19%). MS (ESI+) m/z ) 392 [M + H]⁺.

¹H NMR (300 MHz, CDCl₃) δ 3.34 (4H, t, J ) 4.8 Hz, NCH₂);

3.75 (4H, t, J ) 4.8 Hz, OCH₂); 5.56 (1H, s, 3-H); 7.33-7.36

(2H, m, ArH); 7.41-7.46 (1H, m, ArH); 7.54-7.57 (3H, m,

ArH); 8.21 (1H, d, J ) 7.8 Hz, ArH). MS (EI), m/z (%): 383

(M⁺) (65). HRMS (EI) (C₂₅H₂₁NO₃) calcd 383.152, obsd 383.153.

2-Morpholin-4-yl-8-(3-trifluoromethoxyphenyl)-

chromen-4-one (32{25}). General procedure L with 29b and

3-(trifluoromethoxy)phenyl boronic acid (11.9 mg, 0.058 mmol)

gave 32{25}, 7.2 mg (35%). MS (ESI+) m/z ) 392 [M + H]⁺.

¹H NMR (300 MHz, CDCl₃) δ 3.35 (4H, t, J ) 4.8 Hz, NCH₂);

3.75 (4H, t, J ) 4.8 Hz, OCH₂); 5.57 (1H, s, 3-H); 7.44-7.47

(4H, m, ArH); 7.51-7.59 (2H, m, ArH); 8.23 (1H, d, J ) 7.8

Hz, ArH). MS (EI), m/z (%): 391 (M⁺) (100). HRMS (EI)

(C₂₀H₁₆NO₄F₃) calcd 391.103, obsd 391.105.

2-Morpholin-4-yl-8-(6′,7′,8′,9′-tetrahydrodibenzothio-

phen-4′-yl)chromen-4-one (36). A mixture of 35 (48 mg, 0.75

mmol), 29b (63 mg, 0.83 mmol), PdCl₂(dppf) (4 mg, 0.023

mmol), and Cs₂CO₃(147 mg, 2.25 mmol) in degassed THF (5

mL) was heated to reflux for 16 h. Water (10 mL) was added,

and the mixture was extracted with DCM (3 × 20 mL). The

combined organic layers were dried (MgSO₄) and concentrated

in vacuo. HPLC (Genesis C18; methanol, water) gave 36 (23

mg, 25%). ¹H NMR (300 MHz, CDCl₃) δ 1.80-1.90 (m, 4H,

CH₂); 2.69-2.75 (m, 4H, CH₂); 3.05-3.08 (m, 4H, CH₂N); 3.52-

3.55 (m, 4H, CH₂O); 5.44 (s, 1H, CH); 7.22 (dd, J ) 1.0 and

7.3, 1H, Ph); 7.34-7.40 (m, 2H, Ph); 7.56 (d, J ) 7.9 Hz, 1H,

Ph); 7.69 (dd, J ) 1.7 and 7.8 Hz, 1H, Ph). ¹³C NMR (75 MHz,

CDCl₃) δ 22.7; 24.0; 24.3; 26.0; 44.9; 66.3; 87.2; 120.7; 124.4;

125.1; 125.4; 126.0; 129.3; 130.2; 133.7; 138.0; 140.6; 143.7;

147.9; 162.5; 177.6; 200.5. IR (Diamond ATR) υ_max(cm^-1) 2918,

2853, 1619, 1563, 1386, 1238, 1104, 1032, 840, 776, 728. LCMS

(ESI+) m/z ) 419 [M + H]⁺. C, H, N.

8-Dibenzothiophen-1-yl-2-morpholin-4-yl-chromen-4-

one (32{38}). To a solution of 29b (150 mg, 0.396 mmol) in

dioxane (5 mL) was added K₂CO₃(109 mg, 0792 mmol),

dibenzothiophene-4-boronic acid (108 mg, 0.475 mmol), and

tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.020 mmol).

The reaction mixture was heated to reflux for 16 h, cooled,

diluted with DCM (15 mL), and washed with water (10 mL),

and the organic fraction was evaporated in vacuo. Chroma-

tography (10% MeOH/DCM) gave 32{38} as an off-white solid

(59 mg, 35%). Mp 219.5-221 °C. UV (nm/EtOH) λ_max) 233.0.

IR (Diamond ATR) υ_max(cm^-1): 3055, 2948, 2855, 1622, 1562,

1408, 1242, 1114, 991, 867, 787, 740. ¹H NMR (300 MHz,

CDCl₃) δ 3.02 (4H, m); 3.43 (4H, m); 5.45 (1H, s); 7.41 (4H,

m); 7.51 (1H, t); 7.72 (2H, m); 8.17 (2H, m); 8.21 (1H, m); MS

(ESI+) m/z ) 414 [M + H]⁺; Anal. Calcd for C₂₅H₁₉NO₃S; C,

72.62, H, N; Found: C, 71.51.

Enzyme Inhibition and Clonogenic Survival Assays.

In vitro assays for DNA-PK, ATM, ATR, mTOR, and PI3-K

(p110R) were conducted as previously described.¹⁹Cell-based

clonogenic assays were performed as previously described.¹⁹

Acknowledgment. The authors thank Adrian Moore

for purification of the Suzuki-coupling library com-

pounds and Cancer Research U.K. and EPSRC for

funding.

Supporting Information Available: Full experimental

details for 2-alkylaminobenzo[h]chromenone library, full ex-

perimental details for the 6- and 7- alkoxy-chromenone librar-

2-(2-Bromophenylsulfanyl)cyclohexanone (33).²²To a

solution sodium hydroxide (156 mg, 3.8 mmol) in ethanol (5

7846 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Hardcastle et al.

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JM050444B

Article Doi

Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK) using a small-molecule library approach

DOI: 10.1021/jm050444b

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Article abstract of DOI:10.1021/jm050444b

Full text of DOI:10.1021/jm050444b

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