Novel quinolinequinone antitumor agents: Structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1)

Bioorganic & Medicinal Chemistry 12 (2004) 1667–1687

Novel quinolinequinone antitumor agents: structure-metabolism

studies with NAD(P)H:quinone oxidoreductase (NQO1)

Tara Fryatt,^aHanna I. Pettersson,^aWalter T. Gardipee,^bKurtis C. Bray,^b

Stephen J. Green,^aAlexandra M. Z. Slawin,^cHoward D. Beall^b,*

and Christopher J. Moody^a,*

^aDepartment of Chemistry, University of Exeter, Stocker Road, Exeter EX4 4QD, UK

^bDepartment of Biomedical and Pharmaceutical Sciences, The University of Montana, 32 Campus Drive #1552, Missoula,

MT 59812-1552, USA

^cSchool of Chemistry, University of St. Andrews, Fife, Scotland KY16 9AJ, UK

Received 21 November 2003; accepted 13 January 2004

Abstract—A series of quinolinequinones bearing various substituents has been synthesized, and the eﬀects of substituents on the

metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (hNQO1) was studied. A range of quino-

linequinones were selected for study, and were speciﬁcally designed to probe the eﬀects of aryl substituents at C-2. A range of 28

quinolinequinones 2–29 was prepared using three general strategies: the palladium(0) catalyzed coupling of 2-chloroquinolines, the

classical Friedlander synthesis and the double-Vilsmeier reaction of acetanilides. One example of an isoquinolinequinone 30 was

also prepared, and the reduction potentials of the quinones were measured by cyclic voltammetry. For simple substituents R²at the

quinoline 2-position, the rates of quinone metabolism by hNQO1 decrease for R²=Cl>HꢀMe>Ph. For aromatic substituents,

the rate of reduction decreases dramatically for R²=Ph>1-naphthyl>2-naphthyl>4-biphenyl. Compounds containing a pyridine

substituent are the best substrates, and the rates decrease as R²=4-pyridyl>3-pyridyl>2-pyridyl>4-methyl-2-pyridyl>5-methyl-2-

pyridyl. The toxicity toward human colon carcinoma cells with either no detectable activity (H596 or BE-WT) or high NQO1

activity (H460 or BE-NQ) was also studied in representative quinones. Quinones that are good substrates for hNQO1 are more

toxic to the NQO1 containing or expressing cell lines (H460 and BE-NQ) than the NQO1 deﬁcient cell lines (H596 and BE-WT).

1. Introduction

quinones such as EO9,^8ꢁ10azidiridinylbenzoquinones

such as MeDZQ,^7,11and the novel cyclopropamito-

senes¹²is now well established, and the subject has been

reviewed recently.^13,14

NAD(P)H:quinone oxidoreductase (NQO1, EC

1.6.99.2), also known as DT-diaphorase, is an obligate

2-electron reductase that is characterized by its ability to

use either NADH or NADPH as cofactor.^1,2NQO1

catalyses the 2-electron reduction of quinones thereby

bypassing the potentially toxic semiquinone and hence

can protect cells against the toxic eﬀects of quinones.³

However NQO1 is also involved in the reductive acti-

vation of anticancer agents such as mitomycin C

(MMC) which operate by the so-called bioreductive

mechanism,^4ꢁ6and a clear correlation between NQO1

activity and MMC sensitivity in human lung and breast

cancer cell lines has been demonstrated.⁷The impor-

tance of NQO1 in the bioactivation of other cytotoxic

We recently reported the results of two studies designed

to correlate the rate of metabolism by NQO1 and toxi-

city towards human tumor cell lines with a series of

indolequinones,^15,16and NQO1 continues to generate

interest because of its elevated levels in many tumors

and tumor cell lines,^5,17and its possible role in the sta-

bilization of the tumor suppressor gene wild-type

p53.^18ꢁ20Thus other researchers have also studied the

metabolism of quinones by NQO1,^21ꢁ27and the induc-

tion and inhibition of the enzyme.^28ꢁ30Advances have

also been made in the immunohistochemical detection

of NQO1 in normal and tumor tissue,^31ꢁ33in under-

standing NQO1 gene expression,^34ꢁ36and the poly-

morphism, resulting from a C to T base change at

position 609, that occurs.^37,38

* Corresponding authors. Fax: +1-406-243-5228 (H.D.B. for biology);

fax: +1-44-1392-263434 (C.J.M. for chemistry); e-mail addresses:

beallh@selway.umt.edu; c.j.moody@ex.ac.uk

doi:10.1016/j.bmc.2004.01.021

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T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

Recently the 3-dimensional structure of human NQO1

has been solved by two research groups, and resolved to

2. Results and discussion

2.1. Chemistry

2.3 A³⁹and 1.7 A⁴⁰respectively. The structure of the

complex of enzyme with duroquinone has also been

solved,⁴⁰and the studies highlight the structural changes

that are necessary for control of access to the catalytic

site that is required by the ping-pong mechanism in

which NAD(P)H enters the catalytic site and reduces

the ﬂavin, NAD(P)⁺leaves thereby allowing the sub-

strate to enter and be subsequently reduced by FADH₂.

Although molecular modeling studies using a model of

the human enzyme based on its homology to the pre-

viously determined structure of the rat enzyme have

been useful in the design of new quinone sub-

strates,^21,41,42co-crystallization of an indolequinone

substrate with the human protein has provided detailed

structural information.⁴³Likewise, structural studies on

a related indolequinone have helped characterize a

mechanism of NQO1 inhibition.⁴⁴

A range of quinolinequinones was designed to explore

the eﬀect of substituents at C-2, and in particular vari-

ations in the aromatic ring at this position bearing in

mind the presence of a substituted 2-pyridyl group at C-

2 in SN 1. We also looked at a smaller number of var-

iations at C-6, and at the eﬀect of introducing a sub-

stituent at C-3, a position that is unsubstituted in SN.

In order to prepare such a range of quinolinequinones,

three strategies were adopted: the palladium(0) cata-

lyzed coupling of 2-chloroquinoline derivatives, the

classical Friedlander quinoline synthesis, and the ‘dou-

ble-Vilsmeier’ reaction of acetanilides. The starting

material for the ﬁrst strategy was the commercially

available 6-methoxyquinoline 31. Thus nitration at C-5,

followed by reduction of the nitro compound 32 and

oxidation of the resulting 5-aminoquinoline 33 with

Fremy’s salt [potassium nitrosodisulfonate-(KO₃S)₂NO]

gave the 6-methoxyquinoline-5,8-dione 2 (Scheme 1).

The chlorine substituent at C-2 was introduced by oxi-

dation of 6-methoxy-5-nitroquinoline 32 and treatment

of the resulting N-oxide 34 with sulfuryl chloride to give

the 2-chloroquinoline 35. Subsequent reduction of the

nitro group gave the 5-aminoquinoline 36, oxidation of

which gave the key 2-chloroquinolinequinone 3 (Scheme 1),

a compound previously prepared by Kametani et al.⁵⁶

One of the best substrates for NQO1 remains the natu-

rally occurring antitumor antibiotic streptonigrin (SN)

1. Streptonigrin, isolated from Streptomyces ﬂocculus

over 40 years ago, has activity against a broad range of

tumors. It was studied clinically in the 1960s and 1970s

as an antitumor agent, but its use was limited by reports

of delayed myelotoxicity. Nevertheless, positive results

were reported for SN both as a single agent and in

combination chemotherapy. The biological properties

of SN have been reviewed recently.⁴⁵Hydroxyl radical

(HO^ꢂ) production following reduction of the quinoline-

quinone moiety of SN leads to DNA degradation and

cytotoxicity, and an SN–metal–DNA complex is

thought to be involved.^46,47SN is an excellent substrate

for NQO1,⁴⁸and we have previously shown that it is

selectively toxic to colon⁴⁹and lung⁵⁰cell lines with

elevated NQO1, although its facile 2e-reduction by

NQO1 may be independent from the hydroxyl radical

production referred to above.

The 2-methylquinolinequinone 4 was prepared slightly

diﬀerently using the known reaction of 4-methoxy-2-

We now report the details of a study designed to exam-

ine the eﬀects of functional group substitutions on the

metabolism of a range of novel quinolinequinones 2–29

and one isoquinoline derivative 30 (Fig. 1) by recombi-

nant hNQO1.⁵¹Although others have investigated the

biological properties of quinolinequinones,^52,53includ-

ing analogues of SN,^47,54,55the current work represents

the ﬁrst detailed study of the metabolism of such qui-

nones by NQO1.

Figure 1.

Scheme 1.

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1669

nitroaniline 37 with paraldehyde to give the 2-methyl-

quinoline 38 albeit in poor yield.⁵⁷Subsequent reduc-

tion of the nitro group, and treatment of the resulting 8-

aminoquinoline 39 with Fremy’s salt gave the desired

quinone 4 (Scheme 2).

lowed by the oxidation of the 5-aminoquinoline 51 to

give the desired quinone 18 (Scheme 5).

The synthesis of a series of compounds possessing sub-

stituents other than a methoxy group at C-6 began with

the displacement of the methoxy group in quinones 2

and 5 with secondary amines to give the quinones 19–21

as shown in Scheme 6.

The 2-aryl-and-hetaryl-quinolinequinones 5–11, 13, 16

and 17 were prepared by palladium(0) catalyzed cou-

pling reactions of the 2-chloroquinolinequinone 3 with

areneboronic acids or arylstannanes under typical

Suzuki or Stille reaction conditions (Scheme 3),

although the yield in many of these reactions was poor

to modest (Table 1).

The simple 2-phenyl-6-unsubstituted quinolinequinone

22 was prepared from 8-benzyloxy-2-quinolone 52 as

shown in Scheme 7, by chlorination, Suzuki coupling of

the resulting 2-chloroquinoline 53, deprotection and

oxidation of the quinolin-8-ol 55 with Fremy’s salt.

The 2-(2-pyridyl)quinolinequinone 11 was also prepared

using the Friedlander reaction. Thus catalytic hydro-

genation of 2-benzenesulfonyloxy-3-methoxy-6-nitro-

benzaldehyde 40 gave the corresponding ortho-

aminobenzaldehyde which was immediately condensed

with 2-acetylpyridine under Friedlander conditions.⁵⁸

This gave the 2-substituted quinolin-5-ol 41 in poor

yield, the benzensulfonate ester being hydrolysed by

treatment with aqueous ethanolic sodium hydroxide,

readily oxidised to the quinone 11 (Scheme 4). Similarly,

reaction of the ortho-aminobenzaldehyde with 2-acetyl-

4-methylpyridine, 3-acetylpyridine and 4-acetylpyridine

gave the 2-pyridylquinolin-5-ols 42–44 respectively in

poor yield, oxidation of which gave the quinolinequi-

nones 12, 14 and 15 (Scheme 4).

The synthesis of quinones bearing a substituent at C-3

started with 2,5-dimethoxyacetanilide 56 which under-

went the ‘double Vilsmeier’ reaction upon treatment

with phosphorus oxychloride in DMF to give the 2-

chloroquinoline-3-carbaldehyde 57.⁵⁹Reduction of the

aldehyde, reductive removal of the chlorine, and oxida-

tive demethylation with cerium(IV) ammonium nitrate

then gave the quinone 23 (Scheme 8).

The above sequence was readily modiﬁed to incorporate

aryl or hetaryl groups at C-2. Thus Suzuki coupling of

the 2-chloroquinoline-3-carbaldehyde 57 with benzene-

boronic acid gave the 2-phenyl derivative 60. Reduction

Table 1. Palladium(0) catalyzed coupling reactions of 2-chloro-6-

methoxyquinoline-5,8-dione 3

The 4-indolyl substituted quinolinequinone 18 was also

prepared by a palladium catalyzed coupling reaction,

although it was carried out on the 2-chloro-6-methoxy-

5-nitroquinoline 35 rather than the quinone 3. Thus the

indole-4-boronate 48, prepared from 2-bromobenzalde-

hyde in four steps as shown in Scheme 5, was coupled to

the 2-chloroquinoline 35 to give the indolylquinoline 49.

Reduction of the nitro and ester groups was then fol-

Ar

Method^a

Product

Yield/%

Ph

2-Tolyl

4-Tolyl

4-Biphenyl

1-Naphthyl

2-Naphthyl

2-Pyridyl

5-Me-2-pyridyl

2-Thienyl

2-Benzofuranyl

A

B

A

5

6

7

8

9

10

11

13

16

17

24

44

42

39

18

16

88

28

25

20

^aMethod: A, Suzuki coupling with ArB(OH)₂; B, Stille coupling with

ArSnMe₃.

Scheme 2.

Scheme 3.

Scheme 4.

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T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

of the ester and oxidative demethylation of the 5,8-

dimethoxyquinoline 61 gave the corresponding quinone

24 (Scheme 9). A similar sequence of reactions using 2-

thienyl- and 2-benzofuranyl- boronic acids gave the

corresponding quinones 25 and 26 (Scheme 9).

The acetate esters of quinones 24–26 were also prepared

by acetylation of the alcohols 61, 63 and 65 followed by

Scheme 7.

Scheme 8.

Scheme 5.

Scheme 6.

Scheme 9.

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1671

oxidation of the 5,8-dimethoxy compounds 66–68 to

give the quinones 27–29 (Scheme 10). The structure of

quinolinequinone 29 was conﬁrmed by X-ray crystal-

lography (Fig. 2).

were performed on a number of the quinolinequinones

using DMF as solvent and tetra-n-butyl ammonium

tetraﬂuoroborate as the supporting electrolyte. E_1/2

values, determined from the (at least) quasi-reversible

voltammograms recorded for the one-electron reduction

of the quinolinequinones, show little variation over the

range of potential sweep rates used. Thus, the E_1/2

values given in Table 2 are averages of the values deter-

mined from voltammograms recorded at potential

Finally, a single example of an isoquinolinequinone was

prepared following a literature procedure for related

compounds.⁶⁰The known phenethylamine derivative

69⁶¹was benzoylated to give amide 70 which underwent

Bischler–Napieralski cyclisation and debenzylation

under the acidic conditions to give the isoquinolin-5-ol

71 in poor yield, oxidation of which gave the desired

isoquinoline 30 (Scheme 11).

sweep rates of 50, 100, 200, 300, 400 and 500 mV s^ꢁ1

.

The E_1/2values, tabulated with reference to E_1/2for

ferrocene (Fc^+/0) to avoid liquid junction potential, are

shown in Table 2.

With the exception of the three compounds indicated,

all the quinolinequinones and the one isoquinolinequin-

one exhibited chemically reversible electrochemistry. All

the 6-methoxyquinolinequinones have similar E_1/2

values, between ꢁ1.06 and ꢁ1.15 V, with those posses-

sing the more electron-deﬁcient rings at C-2, for exam-

ple the various pyridyl derivatives, having the most

positive E_1/2, that is, easiest to reduce. Not surprisingly,

compounds 22–25 lacking the electron-releasing meth-

oxy group at C-6 are somewhat easier to reduce than

their 6-methoxy counterparts. Although electrochemical

studies quickly identify compounds which are diﬃcult

to reduce, unfortunately, as discussed previously for

indolequinones,^15,16,62there is often little correlation

between reduction potential and rate of reduction by

NQO1. Nevertheless all the quinolinequinones investi-

gated are easier to reduce than the previously studied

indolequinones which have E_1/2relative to Fc^+/0in the

range ꢁ1.20 to ꢁ1.50 V.^15,16

2.2. Electrochemistry

The main aim of this work was to study the enzymic 2e-

reduction of quinolinequinones by NQO1. However,

the reduction of quinones can also be readily studied in

the laboratory using electrochemical techniques, and

although this gives data related to the 1e-reduction, it

does provide data on the relative ease of reduction of a

series of quinones. Therefore electrochemical studies

2.3. Biology

The metabolism of the quinones 1–30 by puriﬁed

recombinant hNQO1 was studied using a spectro-

photometic assay that employs cytochrome c as the

terminal electron acceptor.²¹In our preliminary studies

we used an alternative HPLC based assay,⁵¹but we now

use the spectrophotometric assay because it gives initial

rates of quinone metabolism. The assay uses cyto-

chrome c as the terminal electron acceptor, and the

initial reduction rates (mmol cytochrome c reduced/min/

Scheme 10.

Figure 2. X-Ray crystal structure of 3-acetoxymethyl-2-(benzofuran-

2-yl)quinoline-5,8-dione 29.

Scheme 11.

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T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

Table 2. Electrochemical reduction potentials^a(DMF) (versus ferrocene) of quinolinequinones and their metabolism by recombinant human NQO1

R²

R³

R⁶

E_1/2(V) v Fc

Reduction rate by hNQO1 (mmol/min/mg)

1

2

3

4

5

6

7

8

—

H

Cl

Me

Ph

2-Tolyl

4-Tolyl

—

H

—

OM

—

411ꢃ36

337.3ꢃ42.2

420.3ꢃ40.1

342.3ꢃ22.9

313.7ꢃ58.8

383.3ꢃ61.9

236.7ꢃ33.5

0.4ꢃ0.1

ꢁ1.11e

ꢁ1.01

ꢁ1.15

ꢁ1.11

ꢁ1.10

ꢁ1.09

ꢁ1.10

ꢁ1.09

ꢁ1.08

ꢁ1.09

ꢁ1.08

ꢁ1.05

ꢁ1.10

ꢁ1.06

ꢁ1.13

ꢁ1.29

nr

OMe

Pyrrolidinyl

Morpholinyl

H

4-Biphenyl

1-Naphthyl

2-Naphthyl

2-Pyridyl

4-Me-2-Pyridyl

5-Me-2-Pyridyl

3-Pyridyl

4-Pyridyl

2-Thienyl

2-Benzofuranyl

1-Me-2-CH₂OH-4-indolyl

H

9

87.4ꢃ8.0

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

16.8ꢃ15.5

446ꢃ39

354.0ꢃ16.4

279.5ꢃ13.5

568.7ꢃ59.7

653.0ꢃ51.2

307.7ꢃ57.1

191.2ꢃ62.9

31.1ꢃ1.3

H

46.3ꢃ2.5

Ph

H

Ph

5.8ꢃ0.6

nr

6.6ꢃ0.5

ꢁ0.98

ꢁ0.97

ꢁ0.95

nr

—

ꢁ1.04

13.4ꢃ7.7

CH₂OH

CH₂OAc

H

OMe

56.5ꢃ9.7

34.8ꢃ3.1

2-Thienyl

2-Benzofuranyl

Ph

2-thienyl

2-Benzofuranyl

Ph

17.2ꢃ1.5

16.1ꢃ4.6

61.9ꢃ10.8^b

4.2ꢃ2.2^b

2.9ꢃ0.9^b

446.3ꢃ35.9

^aE_1/2(ꢃ0.005V) values calculated as (E_pc+E_pa)/2 are averages of the values determined from voltammograms recorded at potential sweep rates of

50, 100, 200, 300, 400 and 500 mVs^ꢁ1; E_pc, cathodic peak potential; E_paanodic peak potential; nr=electrochemical reduction not reversible.

^bThe quinones inactivate the enzyme- see text.

mg NQO1) (Table 2) were calculated from the linear

portion (0–30 s) of the reaction graphs.

(BE-NQ) was greater than 500 nmol min^ꢁ1mg^ꢁ1total

cell protein using dichlorophenolindophenol as the

standard electron acceptor. For this report we have

compared the toxicity of representative quinones (Table

3) using both sets of cells.

Three of the compounds evaluated caused inactivation

of the enzyme: thus the 3-acetoxymethyl quinolinequi-

nones 27–29 inactivated the enzyme to the extent of 78,

63 and 68%, respectively (data not shown). Since these

compounds all contain a potential leaving group (ace-

tate) that could be eliminated upon reduction of the

quinone, it is likely that the inactivation is due to

mechanism based inhibition of NQO1, a feature of pre-

viously studied indolequinones bearing potential leaving

groups.^15,21,44

Although the metabolism of streptonigrin itself by

hNQO1 has been investigated,^48ꢁ50there has been no

similar study of quinolinequinones in general. Therefore

the current work represents the ﬁrst detailed study of

the metabolism of such quinones by hNQO1. In gen-

eral, the quinolinequinones studied were much better

substrates for recombinant hNQO1 than related indole-

quinones,^15,16this greater ease of reduction being borne

out by the electrochemical data (see above). Within the

series of 6-methoxy substituted quinones several trends

are apparent from the metabolism data (Table 2). For

simple substituents at the 2-position as in quinones 2–5,

the rates of metabolism by hNQO1 are R²=Cl>

HꢀMe>Ph. For aromatic substituents at C-2, as in

quinones 5–10, the compounds possessing the smaller

substituents are metabolized faster. Thus the rate of

reduction decreases dramatically for R²=Ph>1-

naphthyl>2-naphthyl>4-biphenyl, with the last com-

pound 8 with the large 4-biphenyl substituent being a

Cytotoxicity studies were also performed on repre-

sentative quinones with cell survival being measured

using the MTT colorimeteric assay. In our previous

work, we have used the non-small cell lung cancer

(NSCLC) cell lines H460 (with high NQO1 activity) and

H596 (with no measurable NQO1 activity). However, a

better model has been developed by Winski et al. that

utilizes the BE human colon carcinoma cell line stably

transfected with human NQO1 cDNA.³⁴Like the H596

cell line, the BE cells (BE-WT) have no measurable

NQO1 activity whereas activity in the transfected cells

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1673

Table 3. Cytotoxicity of representative quinolinequinones towards NSCLC cell lines H596 (nomeasurable NQO1 activity) and H460 (high NQO1

activity), and BE human colon carcinoma cell lines BE-WT (no NQO1 activity) and BE-NQ (high NQO1 activity)

R²

R³

R⁶

H596 IC₅₀(mM)

H460 IC₅₀(mM)

BE-WT IC₅₀(mM)

BE-NQ IC₅₀(mM)

1

2

3

5

7

8

9

10

11

13

16

17

18

19

22

27

28

29

—

H

Cl

Ph

4-Tolyl

—

H

—

OMe

Pyrrolidinyl

H

1.36ꢃ0.10

>50

30.1ꢃ2.0

49.0ꢃ1.7

>50

0.0143ꢃ0.001

14.7ꢃ0.3

16.8ꢃ0.4

15.3ꢃ0.4

14.7ꢃ0.3

>50

2.12ꢃ0.20

>50

41.0ꢃ4.0

0.11ꢃ0.02

32.8ꢃ6.1

12.5ꢃ1.3

4-Biphenyl

1-Naphthyl

2-Naphthyl

2-Pyridyl

5-Me-2-pyridyl

2-Thienyl

2-Benzofuranyl

1-Me-2-CH₂OH-4-indolyl

H

>50

28.0ꢃ0.7

31.3ꢃ0.8

>50

25.9ꢃ1.3

11.5ꢃ0.4

36.5ꢃ1.2

>50

7.2ꢃ0.6

11.0ꢃ0.3

14.3ꢃ0.3

>50

25.2ꢃ0.8

>50

8.2ꢃ0.6

>50

13.0ꢃ0.8

15.9ꢃ1.5

>50

14.5ꢃ0.1

3.72

12.4ꢃ0.5

Ph

H

CH₂OAc

H

15.1ꢃ0.5

15.1ꢃ0.7

34.6ꢃ1.3

16.2ꢃ0.8

15.3

37.0ꢃ0.2

2-Thienyl

2-Benzofuranyl

very poor substrate. The two tolyl substituted deriva-

tives 6 and 7 exhibit rates of reduction by hNQO1 that

are much closer to that of the phenyl derivative 5. In the

6-methoxyquinolinequinones 11–18 containing hetero-

aromatic groups, the 2-thienyl derivative 16 is close to

the 2-phenyl compound 5 both in terms of electro-

chemistry and rate of metabolism, a result that is per-

haps not surprising given the similarity in electronic

properties of benzene and thiophene rings. However it is

the pyridine derivatives 11–15 that are the best sub-

strates, with the 2-, 3- and 4-pyridyl compounds 11, 14,

15 being better substrates than SN itself. The reason for

investigating such compounds was, of course, the pre-

sence of a 2-pyridyl group in SN, but it is clear that the

3- and 4-pyridyl substituents lead to faster metabolism.

Within the series of ﬁve pyridyl quinolinequinones, the

rates of metabolism are R²=4-pyridyl>3-pyridyl>2-

pyridyl>4-methyl-2-pyridyl>5-methyl-2-pyridyl.

27–29 bearing acetate leaving groups inactivate the

enzyme (data not shown).

Finally the single example of an isoquinolinequinone 30

was investigated and found to be an excellent substrate.

Cytotoxicity data were obtained for selected quinoline-

quinones (Table 3). In comparison to SN, all the syn-

thetic analogues are much less cytotoxic, since

presumably they do not possess the metal-binding fea-

tures that lead, after reduction, to hydroxyl radical

production, and toxicity. As expected, quinones such as

2, 5, 11, and 16 that are good substrates for hNQO1 are

more toxic to the NQO1 containing or expressing cell

lines (H460 and BE-NQ) than the NQO1 deﬁcient

cell lines (H596 and BE-WT). Quinones such as the

biphenyl and naphthyl derivatives 8–10 which are poor

substrates show no selectivity or have no measurable

cytotoxicity (IC₅₀>50 mM).

A small number of variations at C-6 were investigated.

For compounds which share a phenyl group at the 2-

position (quinones 5, 20–22), the 6-methoxy compound

5 is by far the best substrate; the 6-unsubstituted com-

pound 22 and the compounds possessing a secondary

amine are all reduced less eﬃciently. The fact that

replacement of a methoxy group by a more electron-

releasing secondary amine deactivates the quinone towards

reduction is hardly surprising, and was also observed

throughout our studies on indolequinones.^15,16,62

In conclusion, these ﬁndings further advance our

understanding of the relationship between quinone

structure and metabolism by NQO1. Based on the

structure of streptonigrin, a naturally occurring cyto-

toxin known to be an excellent substrate for the enzyme,

we have started to probe for the ﬁrst time, the structure-

metabolism relationships in simple quinolinequinones,

thereby complementing the data on the better studied

indolequinones.

The quinones 23–29 bearing a substituent at the 3-posi-

tion are all poor substrates by comparison with simi-

larly 2-substituted derivatives. However, whether this is

due to the presence of the 3-substituent or the absence

of the 6-methoxy substituent is not clear, although the

fact that 24 is a better substrate than 22 suggests

the latter explanation. As discussed above, the quinones

3. Experimental

3.1. Chemistry

3.1.1. General information. Commercially available

solvents and reagents were used throughout without

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T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

further puriﬁcation unless otherwise mentioned. Light

petroleum refers to the fraction that boils between 40 ^ꢄC

and 60 ^ꢄC and was distilled over calcium chloride

(anhydrous). Acetonitrile was distilled from CaH₂

under N₂.

added. The mixture was stirred overnight in dichloro-

methane. The mixture was ﬁltered through Celite and

the layers were separated. The organic phase was dried

(Na₂SO₄) and concentrated in vacuo to give 5-amino-6-

methoxyquinoline 33 as a brown solid (0.50 g, 59%)

(lit.,⁶⁴mp 154–156 ^ꢄC) which was used without pur-

iﬁcation, d_H(300 MHz; CDCl₃) 3.98 (3H, s), 4.25 (2H,

br), 7.29 (1H, dd, J=8.4, 3.9 Hz), 7.43 (1H, d, J=9.0

Hz), 7.59 (1H, d, J=9.0 Hz), 8.12 (1H, d, J=8.4 Hz),

8.76 (1H, m); d_C(100 MHz; CDCl₃) 56.5 (Me), 116.4

(CH), 118.7 (C), 119.5 (CH), 129.0 (CH), 129.3 (C),

142.5 (C), 144.0 (C), 148.1 (CH); one CH not observed.

Analytical thin layer chromatography (TLC) was car-

ried out using Merck silica gel 60A plates. The plates

were visualized under ultra-violet light and/or alkaline

potassium permanganate or ninhydrin. Column chro-

matography was carried out using Merck Kieselgel 60,

230–400 mesh, with the speciﬁed eluent. Pressure was

applied at the column head with hand bellows. Samples

were applied as a saturated solution in an appropriate

solvent or were pre-adsorbed onto a small quantity of

silica.

(c) To a solution of 5-amino-6-methoxyquinoline 33

(0.45 g, 2.6 mmol) in acetone (85 mL) was added a

solution of Fremy’s salt (1.14 g, 4.3 mmol) in dihydro-

gen phosphate buﬀer (0.3M; 100 mL). The mixture was

stirred at room temperature for 1 h. A further solution

of Fremy’s salt (1.14 g, 4.3 mmol) in sodium dihydrogen

phosphate buﬀer (0.3M; 100 mL) was added and the

mixture was stirred for 1 h. The acetone was removed in

vacuo, the resulting mixture was extracted with di-

chloromethane, the organic phase was dried (Na₂SO₄)

and concentrated. The crude mixture was recystallized

from ethyl acetate/light petroleum to give the title com-

pound 2 as a light brown solid (0.27 g, 57%), mp 251–

252 ^ꢄC (from methanol) (lit.,⁶⁵mp 250–251 ^ꢄC), d_H

(300 MHz; CDCl₃) 3.95 (3H, s), 6.37 (1H, s), 7.67 (1H,

dd, J=7.8, 4.8 Hz), 8.47 (1H, dd, J=7.8, 1.8 Hz), 9.05

(1H, dd, J=4.8, 1.8 Hz); d_C(100 MHz; CDCl₃) 56.5

(Me), 110.6 (CH), 127.2 (CH), 127.9 (C), 134.6 (CH),

147.6 (C), 154.9 (CH), 160.1 (C), 179.5 (C), 182.9 (C).

Infra red spectra were recorded as KBr discs or using

NaCl plates as stated, using a Nicolet Magna infra-red

spectrometer 550. Ultra-violet spectra were recorded as

solutions in spectroscopic grade methanol or aceto-

nitrile as stated, on an ATI Unicam UV/Visible spec-

trometer (UV4). Proton magnetic resonance (¹H NM R)

were recorded using Bruker AC300 (300 MHz), Bruker

AV300 (300 MHz) and Bruker DRX400 (400 MHz)

spectrometers. Carbon magnetic resonance (¹³C NM R)

were recorded on Bruker AC300 (75 MHz) and Bruker

DRX400 (100 MHz) instruments. NMR spectra are

referenced against residual undeuterated solvent. In the

case of deuterated chloroform, this is 7.260 ppm. Sig-

nals are described as singlets (s), broad (br), doublets

(d), triplets (t), quartets (q), multiplets (m), double

doublets (dd) and double double doublets (ddd).

3.1.3. 2-Chloro-6-methoxyquinoline-5,8-dione (3). (a) To

a suspension of 6-methoxy-5-nitroquinoline 32 (5 g, 24

mmol) in acetic acid (25 mL) was added hydrogen per-

oxide (35%; 4 mL). The mixture was heated to 80 ^ꢄC for

4 h. Further hydrogen peroxide (35%; 2 mL) was added

and heating was continued for 3 h. After cooling the

mixture was basiﬁed with NaOH (1 M) and extracted

with dichloromethane. The combined organic phases

were dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

(ethyl acetate/2-propanol elution) to give 6-methoxy-5-

nitroquinoline-N-oxide 34 as a yellow solid (2.65 g,

50%), mp 174–175 ^ꢄC (from toluene/light petro-

leum)(lit.,⁶⁶mp 182–184 ^ꢄC), d_H(300 MHz; CDCl₃) 4.10

(3H, s), 7.38 (1H, dd, J=9.0, 6.0 Hz), 7.56 (2H, m), 8.44

(1H, d, J=6.0 Hz), 8.91 (1H, d, J=9.6 Hz); d_C

(100 MHz; CDCl₃) 57.2 (Me), 116.4 (CH), 118.2 (CH),

123.9 (CH), 124.2 (C), 124.3 (CH), 134.4 (C), 135.9 (C),

150.8 (C); one CH unobserved.

Mass spectra [electron impact (EI), chemical ionisation

(CI), and ﬁeld ionisation (FI)] were recorded on a

Micromass GCT spectrometer, [electrospray (ES)] on a

Micromass Platform LC Electrospray Mass Spectro-

meter or on a VAG Analytical ZAB-E instrument

(EPSRC Mass Spectrometry Service at Swansea).

3.1.2. 6-Methoxyquinoline-5,8-dione (2). (a) 6-Methoxy-

quinoline 31 (10.0 g, 62.8 mmol) was added dropwise to

fuming HNO₃(126 mL) keeping the temperature

between 0 and 10 ^ꢄC. The mixture was stirred for 30 min

before being poured onto ice. The precipitate was

collected by ﬁltration, washed with water and dried

under vacuum. The crude product was recrystallized

from methanol to give 6-methoxy-5-nitroquinoline 32 as

pale yellow crystals (10.9 g, 85%), mp 198–199 ^ꢄC (lit.,⁶³

mp 104–105 ^ꢄC); d_H(400 MHz; DMSO) 4.08 (3H, s),

7.80 (1H, dd, J=8.8, 4.4 Hz), 8.01 (1H, d, J=9.5 Hz),

8.28 (1H, d, J=8.8 Hz), 8.32 (1H, d, J=9.5 Hz), 9.01

(1H, dd, J=4.4, 1.3 Hz); d_C(100 MHz; DMSO) 58.0

(Me), 119.3 (CH), 121.0 (C), 124.8 (CH), 131.6 (CH),

132.3 (CH), 134.3 (C), 139.4 (C), 148.9 (CH), 149.9 (C).

(b) Sulfuryl chloride (37 mL) was added to 6-methoxy-

5-nitroquinoline-N-oxide 34 (5.3 g, 24 mmol) and the

mixture was heated to 60 ^ꢄC for 3 h. After cooling

the mixture was dissolved in dichloromethane and stirred

in water for 1 h to dissolve any insoluble material. The

layers were separated and the organic phase was dried

(Na₂SO₄) and concentrated in vacuo. The crude product

was puriﬁed by column chromatography (dichloro-

methane elution) to give 2-chloro-6-methoxy-5-nitro-

quinoline 35 as a yellow solid (3.99 g, 69%), mp 152–

(b) Tin powder (2.5 g) and 3MHCl were added to a

solution of 6-methoxy-5-nitroquinoline 32 (1.00 g, 4.9

mmol) in ethanol (150 mL). The mixture was heated

under reﬂux for 1 h. After cooling the solution was

decanted from the excess tin, neutralized with saturated

NaHCO₃solution and an equal volume of water was

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1675

153 ^ꢄC (from methanol) (lit.,⁵⁶mp 151–153 ^ꢄC), d_H

(300 MHz; CDCl₃) 4.07 (3H, s), 7.50 (1H, d, J=8.7 Hz),

7.59 (1H, d, J=9.3 Hz), 8.03 (1H, dd, J=8.7, 0.9 Hz),

8.17 (1H, dd, J=9.3, 0.9 Hz); d_C(100 MHz; CDCl₃)

57.2 (Me), 117.3 (CH), 120.1 (C), 125.0 (CH), 132.2

(CH), 133.0 (CH), 134.7 (C), 141.7 (C), 149.5 (C), 150.0

(C).

(C), 134.7 (CH), 135.0 (C), 155.5 (C), 159.3 (C), one C

unobserved.

(b) Tin powder (0.1 g) and HCl (3 M; 3 mL) were added

to a solution of 6-methoxy-2-methyl-8-nitroquinoline 38

(0.09 g, 0.40 mmol) in ethanol (10 mL). The reaction

mixture was heated under reﬂux for 20 min. After cool-

ing, the reaction mixture was neutralized with saturated

NaHCO₃solution and an equal volume of water was

added. The mixture was stirred in dichloromethane for

2 h. The separated organic phase was dried (Na₂SO₄)

and concentrated in vacuo. The crude product was

puriﬁed by column chromatography (dichloromethane

elution) to give 8-amino-6-methoxy-2-methylquinoline

39 as a yellow solid (0.05 g, 69%) (lit.,⁵⁷mp 102 ^ꢄC),

used without further puriﬁcation, d_H(300 MHz; CDCl₃)

2.69 (3H, s), 3.88 (3H, s), 5.00 (2H, br), 6.47 (1H, d,

J=2.5 Hz), 6.58 (1H, d, J=2.5 Hz), 7.22 (1H, d, J=8.5

Hz), 7.86 (1H, d, J=8.5 Hz).

(c) Tin powder (2.4 g) and HCl (3 M; 58 mL) were

added to a suspension of 2-chloro-6-methoxy-5-nitro-

quinoline 35 (2 g, 8.4 mmol) in ethanol (200 mL) and

the mixture was heated under reﬂux for 10 min. After

cooling the mixture was neutralized with saturated

NaHCO₃solution and an equal volume of water was

added. The mixture was stirred in dichloromethane for

3 h. The layers were separated and the organic phase

was dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

(dichloromethane elution) to give 5-amino-2-chloro-6-

methoxyquinoline 36 as a dark yellow solid (1.1 g,

63%), mp 93–95 ^ꢄC (from ethyl acetate/light petroleum)

(lit.,⁵⁶mp 98–101 ^ꢄC), d_H(300 MHz; CDCl₃) 3.97 (3H,

s), 4.27 (2H, br), 7.25 (1H, d, J=9.0 Hz), 7.40 (1H, d,

J=9.0 Hz), 7.48 (1H, d, J=9.0 Hz), 8.06 (1H, d, J=9.0

Hz); d_C(100 MHz; CDCl₃) 56.5 (Me), 116.9 (CH), 117.0

(C), 118.5 (CH), 120.4 (CH), 129.8 (C), 132.1 (CH),

143.0 (C), 143.4 (C), 148.3 (C).

(c) To a solution of 8-amino-2-methyl-6-methoxy-

quinoline (0.05 g, 0.30 mmol) in acetone (11 mL) was

added a solution of Fremy’s salt (0.1 g, 0.49 mmol) in

dihydrogen phosphate buﬀer (pH 6, 0.3 M; 11 mL). The

reaction mixture was stirred at room temperature for 2

h. The acetone was removed in vacuo, the resulting

mixture was extracted with dichloromethane (3ꢅ10

mL), the organic phase was dried (Na₂SO₄) and con-

centrated. The crude product was puriﬁed by column

chromatography [dichloromethane/ethyl acetate (9:1)

elution] to give the title compound 4 as a dark yellow

solid (0.03 g, 49%), mp 211–212 ^ꢄC (lit.,⁶⁷mp 204–

206 ^ꢄC), d_H(300 MHz; CDCl₃) 2.79 (3H, s), 3.94 (3H, s),

6.34 (1H, s), 7.53 (1H, d, J=8.0 Hz), 8.35 (1H, d, J=8.0

Hz); d_C(100 MHz; CDCl₃) 25.4 (Me), 56.6 (Me), 110.3

(CH), 125.8 (C), 127.3 (CH), 134.9 (CH), 147.2 (C),

160.0 (C), 165.5 (C), 179.6 (C), 183.5 (C).

(d) To a solution of 5-amino-2-chloro-6-methoxyquino-

line 36 (1.4 g, 6.7 mmol) in acetone (250 mL) was added

a solution of Fremy’s salt (3.1 g, 11 mmol) in sodium

dihydrogen phosphate buﬀer (0.3M; 250 mL). The mix-

ture was stirred at room temperature for 1.5 h. The

acetone was removed in vacuo, the residue extracted

with dichloromethane, the organic phase dried

(Na₂SO₄) and concentrated. The crude product was

puriﬁed by column chromatography (dichloromethane

elution) to give the title compound 3 as a yellow solid

(1.0 g, 60%), mp 227–229 ^ꢄC (from ethyl acetate/light

petroleum) (lit.,⁵⁶mp 226–227 ^ꢄC), d_H(300 MHz;

CDCl₃) 3.95 (3H, s), 6.36 (1H, s), 7.68 (1H, d, J=8.4

Hz), 8.39 (1H, d, J=8.4 Hz); d_C(100 MHz; CDCl₃) 56.7

(Me), 110.4 (CH), 126.6 (C), 128.5 (CH), 137.2 (CH),

147.7 (C), 157.6 (C), 160.0 (C), 178.6 (C), 181.4 (C).

3.2. Palladium(0) catalyzed coupling reactions of 2-

chloro-6-methoxyquinoline-5,8-dione 3; preparation of

quinones 5–11, 13, 16, 17

3.2.1. General procedure for Suzuki coupling. A solution

of 2-chloro-6-methoxyquinoline-5,8-dione 3 (0.3 g, 1.3

mmol) in 1,2-dimethoxyethane (32 mL) was degassed

under reduced pressure. Pd(PPh₃)₄(0.08 g) was added

and the solution was degassed further. The mixture was

stirred under a nitrogen atmosphere for 10 min.

Na₂CO₃solution (2 M; 3 mL) was added followed by

the boronic acid (2.0 mmol). The mixture was heated

under reﬂux for 24 h. After cooling the mixture was

poured into dichloromethane and washed with water

(ꢅ3). The organic phase was dried (Na₂SO₄) and con-

centrated in vacuo. The crude product was puriﬁed by

column chromatography.

3.1.4. 6-Methoxy-2-methylquinoline-5,8-dione (4). (a)

Concentrated HCl (2 mL) was added to a mixture of

paraldehyde (0.80 mL, 6.0 mmol) and 4-methoxy-2-

nitroaniline 37 (0.5 g, 3.0 mmol). The reaction mixture

was heated until an exothermic reaction occurred and

the heating was stopped. The reaction mixture was stir-

red at room temperature for 0.5 h followed by heating

under reﬂux for 4 h. The reaction mixture was cooled to

room temperature and neutralized with NaOH solution

(1 M). The precipitate formed was collected by ﬁltration

and puriﬁed by column chromatography (dichloro-

methane elution) to yield 6-methoxy-2-methyl-8-nitro-

quinoline 38 as a pale yellow solid (0.1 g, 18%), mp

184–185 ^ꢄC (lit.,⁵⁷mp 186–187 ^ꢄC), d_H(300 MHz;

CDCl₃) 2.74 (3H, s), 3.97 (3H, s), 7.25 (1H, d, J=2.8

Hz), 7.37 (1H, d, J=8.5 Hz), 7.63 (1H, d, J=2.8 Hz),

8.01 (1H, d, J 8.5 Hz); d_C(100 MHz; CDCl₃) 25.5 (Me),

56.1 (Me), 109.4 (CH), 115.7 (CH), 124.0 (CH), 128.2

3.2.2. 6-Methoxy-2-phenylquinoline-5,8-dione (5). 24%

yield, mp 225–227 ^ꢄC, (Found: C, 70.5; H, 4.0; N, 5.0.

C₁₆H₁₁NO₃.0.4H₂O requires C, 70.5; H, 4.4; N, 5.1%);

(Found: M⁺, 265.0734. C₁₆H₁₁NO₃requires 265.0739);

l

_max(MeOH)/nm 228 (log e 4.47), 262 (4.66), 304 (4.72);

n_max(Nujol)/cm^ꢁ11683, 1657, 1611, 1591; d_H

(300 MHz; CDCl₃) 3.95 (3H, s), 6.37 (1H, s), 7.50–7.53

1676

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

(3H, m), 8.07 (1H, d, J=8.4 Hz), 8.17–8.20 (2H, m),

8.49 (1H, d, J=8.4 Hz); d_C(100.6 MHz; CDCl₃) 56.6

(Me), 110.6 (CH), 123.5 (CH), 126.3 (C), 127.8 (CH),

129.0 (CH), 130.8 (CH), 135.5 (CH), 137.3 (C), 147.7

(C), 160.1 (C), 162.3 (C), 179.5 (C), 183.1 (C); m/z (EI)

265 (M⁺, 9%), 252 (3), 236 (2), 224 (3), 207 (3), 179 (3),

83 (100).

126.6 (CH), 126.7 (C), 127.8 (CH), 128.2 (CH), 128.8

(CH), 129.9 (CH), 130.7 (C), 133.9 (C), 134.9 (CH),

136.7 (C), 160.9 (C), 164.0 (C), 183.4 (C); two ArC

unobserved; m/z (EI) 315 (M⁺, 100%), 302 (29), 272

(20), 228 (6), 204 (5), 152 (8), 83 (20), 69 (18).

3.2.7. 6-Methoxy-2-(2-naphthyl)quinoline-5,8-dione (10).

16% yield, mp 279–280 ^ꢄC, (Found: C, 72.6; H, 4.1; N,

4.2. C₂₀H₁₃NO₃.0.8H₂O requires C, 72.8; H, 4.5; N,

4.3%); (Found: M⁺, 315.0893. C₂₀H₁₃NO₃requires

315.0895); l_max(MeOH)/nm 236 (log e 4.29), 260 (4.38),

316 (4.26); n_max(CHCl₃)/cm^ꢁ11675, 1659, 1608, 1582;

d_H(400 MHz; CDCl₃) 3.95 (3H, s), 6.38 (1H, s), 7.54–

7.56 (2H, m), 7.81–7.91 (1H, m), 7.96–8.04 (2H, m), 8.22

(1H, d, J=8.0 Hz), 8.29 (1H, dd, J=8.0, 2.0 Hz), 8.52

(1H, d, J=8.8 Hz), 8.70 (1H, s); d_C(100 MHz; CDCl₃)

56.5 (Me), 110.6 (CH), 123.7 (CH), 124.5(CH), 126.3

(C), 126.6 (CH), 127.5 (CH), 127.7 (CH), 128.1 (CH),

128.8 (CH), 129.1 (CH), 133.2 (C), 134.5 (C), 134.6 (C),

135.5 (CH), 147.7 (C), 160.2 (C), 162.1 (C), 179.4 (C),

183.1 (C); m/z (EI) 315 (M⁺, 100%), 257 (44), 216 (25),

153 (37).

3.2.3. 6-Methoxy-_ꢄ2-(2-tolyl)quinoline-5,8-dione (6). 44%

yield, mp 206–207 C, (Found: M⁺, 279.0892. C₁₇H₁₃NO₃

requires 279.0895); l_max(MeCN)/nm 310 (log e 4.01),

357 (3.23); n_max(KBr)/cm^ꢁ11672, 1607, 1585; d_H

(300 MHz; CDCl₃) 2.43 (3H, s), 3.97 (3H, s), 6.39 (1H,

s), 7.30–7.39 (3H, m), 7.47–7.50 (1H, m), 7.78 (1H, d,

J=8.2 Hz), 8.52 (1H, d, J=8.2 Hz); d_C(100 MHz;

CDCl₃) 20.5 (Me), 56.7 (Me), 110.7 (CH), 126.0 (C),

126.1 (CH), 127.6 (CH), 129.5 (CH), 129.9 (CH), 131.1

(CH), 134.9 (CH), 136.3 (C), 138.7 (C), 147.2 (C), 160.1

(C), 165.6 (C), 179.6 (C), 183.1 (C); m/z (EI) 279 (M⁺,

76%), 278 (72), 264 (28), 250 (34), 236 (100) 167 (28),

166 (59), 139 (34).

3.2.4. 6-Methoxy-2-(4-tolyl)quinoline-5,8-dione (7). 42%

yield, mp 205–207 ^ꢄC, (Found: C, 72.8; H, 4.8; N, 5.0.

C₁₇H₁₃NO₃requires C, 73.1; H, 4.7; N, 5.0%); (Found:

M⁺, 279.0896. C₁₇H₁₃NO₃requires 279.0895); l_max

(MeOH)/nm 230 (log e 4.27), 264 (4.34), 310 (4.45);

3.2.8. 6-Methoxy-2-(2-pyridyl)quinoline-5,8-dione (11)

(Method 1). A solution of 2-chloro-6-methoxyquino-

line-5,8-dione 3 (0.25 g, 1.1 mmol) and 2-trimethyl-

stannylpyridine (0.32 g, 1.3 mmol) in 1,4-dioxane (30

mL) was degassed under reduced pressure. Pd(PPh₃)₄

(0.1 g) was added and the solution was degassed further.

The solution was heated under reﬂux for 17 h. After

cooling the mixture was poured into dichloromethane

and was washed with water (ꢅ3). The organic phase was

dried (Na₂SO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography (di-

chloromethane elution) to give the title compound 11 as

a yellow solid (0.26 g, 88%), mp 274–275 ^ꢄC (from

methanol/dichloromethane) (lit.,⁵⁸mp 260 ^ꢄC; lit.,⁶⁸mp

262–264 ^ꢄC), d_H(300 MHz; CDCl₃) 3.96 (3H, s), 6.38

(1H, s), 7.38–7.42 (1H, m), 7.88 (1H, m), 8.56 (1H, d,

J=8.4 Hz), 8.69–8.72 (2H, m), 8.80 (1H, d, J=8.4 Hz);

d_C(100 MHz; CDCl₃) 56.6 (Me), 110.7 (CH), 122.9

(CH), 124.3 (CH), 125.1 (CH), 127.6 (C), 135.6 (CH),

137.1 (CH), 147.3 (C), 149.4 (CH), 154.1 (C), 160.2 (C),

160.8 (C), 179.5 (C), 183.0 (C).

n

_max(CHCl₃)/cm^ꢁ11680, 1669, 1608, 1577; d_H

(400 MHz; CDCl₃) 2.42 (3H, s), 3.94 (3H, s), 6.35 (1H,

s), 7.31 (2H, d, J=8.4 Hz), 8.03 (1H, d, J=8.4 Hz), 8.09

(2H, d, J=8.4 Hz), 8.45 (1H, d, J=8.4 Hz); d_C

(100 MHz; CDCl₃) 21.4 (Me), 56.2 (Me), 110.6 (CH),

123.0 (CH), 126.1 (C), 127.7 (CH), 129.7 (CH), 134.5

(C), 135.4 (CH), 141.3 (C), 147.6 (C), 160.1 (C), 162.2

(C), 179.5 (C), 183.2 (C); m/z (EI) 279 (M⁺, 100%), 250

(21), 221 (31), 180 (27).

3.2.5. 2-(4-Biphenyl)-6-methoxyquinoline-5.8-dione (8).

39% yield, mp 313–314 ^ꢄC (from methanol), (Found:

M⁺, 341.1049. C₂₂H₁₅NO₃requires 341.1052); l_max

(MeOH)/nm 228 (log e 3.57), 272 (3.47), 320 (3.60);

n

_max(CH₂Cl₂)/cm^ꢁ11680, 1659, 1608, 1577; d_H

(400 MHz; CD₂Cl₂) 3.95 (3H, s), 6.36 (1H, s), 7.43 (1H,

m), 7.50 (2H, m), 7.72 (2H, m), 7.81 (2H, m), 8.16 (1H,

d, J=8.2 Hz), 8.30 (2H, m), 8.49 (1H, d, J=8.2 Hz); d_C

(100 MHz; CDCl₃) 56.1 (Me), 110.1 (CH), 122.9 (CH),

125.8 (C), 126.4 (CH), 126.9 (CH), 127.4 (CH), 127.7

(CH), 128.3 (CH), 135.0 (CH), 135.6 (C), 139.3 (C),

142.8 (C), 147.1 (C), 159.6 (C), 161.0 (C), 178.3 (C),

182.6 (C); m/z (EI) 341 (M⁺, 29%), 127 (24), 85 (72), 84

(88), 83 (100), 69 (35).

3.2.9.

6-Methoxy-2-(5-methyl-2-pyridyl)quinoline-5,8-

dione (13). A solution of 2-chloro-6-methoxyquinoline-

5,8-dione 3 (0.25 g, 1.1 mmol) and 5-methyl-2-trimethyl-

stannylpyridine (0.36 g, 1.3 mmol) in 1,4-dioxane (35

mL) was degassed under reduced pressure. Pd(PPh₃)₄

(0.1 g) was added and the solution was degassed further.

The solution was heated under reﬂux for 20 h. After

cooling, the mixture was poured into dichloromethane

and was washed with water (ꢅ3). The organic phase was

dried (Na₂SO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography (di-

chloromethane elution) to give the title compound 13 as

a yellow solid (86 mg, 28%), mp 277–278 ^ꢄC (from

methanol/dichloromethane), (Found: M⁺, 280.0856.

C₁₆H₁₂N₂O₃requires 280.0848); l_max(MeOH)/nm 260

(log e 3.56), 304 (3.79); n_max(CHCl₃)/cm^ꢁ11680, 1659,

1608, 1582; d_H(300 MHz; CDCl₃) 2.41 (3H, s), 3.94

(3H, s), 6.36 (1H, s), 7.65–7.69 (1H, m), 8.51–8.53 (2H,

3.2.6. 6-Methoxy-2-(1-naphthyl)quinoline-5.8-dione (9).

18% yield, mp 224–225 ^ꢄC (from methanol/chloro-

form), (Found: C, 75.1; H, 3.9; N, 4.3. C₂₀H₁₃NO₃.0.2H₂O

requires C, 75.3; H, 4.2; N, 4.4%); (Found: M⁺,

315.0900. C₂₀H₁₃NO₃requires 315.0895); l_max(MeOH)/

nm 242 (log e 4.29), 270 (4.19), 316 (3.92); n_max(Nujol)/

cm^ꢁ11675, 1669, 1618, 1577; d_H(300 MHz; MeOH-d₄)

3.99 (3H, s), 6.49 (1H, s), 7.51–7.53 (2H, m), 7.62–7.64

(1H, m), 7.72 (1H, d, J=9.0 Hz), 7.95–8.06 (4H, m),

8.61 (1H, d, J=9.0 Hz); d_C(100 MHz; MeOH-d₄) 55.9

(Me), 109.9 (CH), 124.7 (CH), 124.9 (CH), 125.9 (CH),

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1677

m), 8.59 (1H, d, J=8.1 Hz), 8.75 (1H, d, J=8.1 Hz); d_C

(100 MHz; CDCl₃) 20.9 (Me), 56.6 (Me), 110.6 (CH),

122.5 (CH), 124.0 (CH), 127.4 (C), 135.3 (C), 135.6

(CH), 137.6 (CH), 149.9 (CH), 151.6 (C), 160.2 (C),

161.0 (C), 171.0 (C), 183.0 (C); m/z (EI) 280 (M⁺,

100%), 267 (25), 251 (20), 222 (39), 194( 22), 181 (35).

was washed with brine (3ꢅ5 mL), dried (Na₂SO₄) and

concentrated in vacuo. The crude product was puriﬁed

by column chromatography (dichloromethane/ethyl

acetate (8:2) elution) to give 6-methoxy-2,2-pyr-

idylquinolin-5-ol 41 as a yellow solid (0.2 g, 33%) (lit.,⁵⁸

mp 184–185 ^ꢄC) used directly in the next step.

3.2.10. 6-Methoxy-2-(2-thienyl)quinoline-5,8-dione (16).

25% yield, mp 208–209 ^ꢄC, (Found: C, 61.7; H, 3.2; N,

5.0. C₁₄H₉NO₃S requires C, 62.0; H, 3.3; N, 5.2%);

(Found: M⁺, 271.0301. C₁₄H₉NO₃S requires 271.0303;

(b) A solution of the above pyridylquinoline 41 (0.07 g,

0.3 mmol) in methanol (40 mL) was added to a stirred

solution of Fremy’s salt (1.0 g, 3.5 mmol) in dihydrogen

phosphate buﬀer (pH 6, 0.3 M; 40 mL). The solution

was stirred at room temperature overnight, diluted with

water and the crystalline solid which precipitated was

collected. The crude product was puriﬁed by column

chromatography (dichloromethane/ethyl acetate (8:2)

elution) to give the title compound 11 as a yellow solid

(0.08 g, 74%), data given above.

l_max(MeOH)/nm 258 (log e 4.18), 324 (4.38), 400 (3.43);

n_max(CHCl₃)/cm^ꢁ11675, 1659, 1613, 1577; d_H

(400 MHz; CDCl₃) 3.94 (3H, s), 6.33 (1H, s), 7.18 (1H,

dd, J=5.2, 3.6 Hz) 7.57 (1H, dd, J=5.2, 1.2 Hz), 7.84

(1H, dd, J=3.6, 1.2 Hz), 7.91 (1H, d, J=8.4 Hz), 8.41

(1H, d, J=8.4 Hz); d_C(100 MHz; CDCl₃) 56.5 (Me),

110.4 (CH), 121.8 (CH), 125.9 (C), 128.0 (CH), 128.6

(CH), 131.0 (CH), 135.4 (CH), 143.0 (C), 147.8 (C),

157.3 (C), 160.1 (C), 179.1 (C), 182.7 (C); m/z (EI) 271

(M⁺, 100%), 258 (54), 213 (32), 172 (50), 83 (58), 69

(55).

3.3.2.

6-Methoxy-2-(4-methyl-2-pyridyl)quinoline-5,8-

dione (12). (a) A solution of 2-benzenesulfonyloxy-3-

methoxy-6-nitrobenzaldehyde 40 (1.0 g, 3.0 mmol) in

THF (40 mL) was hydrogenated over 10% Pd/C (0.5 g).

After 2 h the mixture was ﬁltered and the ﬁltrate was

used immediately in the Friedlander condensation. To a

stirred solution of 2-acetyl-4-methylpyridine (0.6 g, 4.5

mmol) and Triton B (10 drops) was added a solution of

the aminoaldehyde in THF under an atmosphere of

nitrogen. The solution was stirred at room temperature

for 19 h, during which additional Triton B (10 drops)

was added. The reaction mixture was neutralized with

HCl (1 M) and extracted with chloroform (3ꢅ20 mL).

The chloroform extract was washed with brine (3ꢅ10

mL), dried (Na₂SO₄) and concentrated in vacuo. A

stirred mixture of the residue and 15% NaOH solution

(20 mL) in ethanol (20 mL) was heated under reﬂux

overnight. The solution was cooled, diluted with water,

and washed with chloroform (3ꢅ10 mL). The aqueous

layer was separated, neutralized with dilute HCl, and

extracted with chloroform (3ꢅ20 mL). The chloroform

extract was washed with brine (3ꢅ10 mL), dried

(Na₂SO₄) and concentrated in vacuo. The crude product

was puriﬁed by column chromatography (dichloro-

methane/ethyl acetate (8:2) elution) to give 6-methoxy-

2-(4-methyl-2-pyridyl)quinolin-5-ol 42 as a yellow solid

(61.8 mg, 8%), used directly in the next step.

3.2.11. 2-(2-Benzofuranyl)-6-methoxy-quinoline-5,8-dione

(17). 20% yield, mp 299–301 ^ꢄC, (Found: C, 67.3; H,

3.6; N, 4.2. C₁₈H₁₁NO₄.0.8H₂O requires C, 67.6; H, 3.9;

N, 4.4%); (Found: M⁺, 305.0689. C₁₈H₁₁NO₄requires

305.0688); l_max(MeOH)/nm 246 (log e 4.23), 336 (4.36);

n_max(CHCl₃)/cm^ꢁ11680, 1654, 1608, 1577; d_H

(300 MHz; CDCl₃) 3.95 (3H, s), 6.37 (1H, s), 7.29 (1H,

m), 7.37–7.43 (1H, m), 7.57 (1H, dd, J=8.4, 0.9 Hz),

7.68–7.71 (1H, m), 7.87 (1H, d, J=0.9 Hz), 8.20 (1H, d,

J=8.4 Hz), 8.52 (1H, d, J=8.4 Hz); d_C(100 MHz;

CDCl₃) 56.1 (Me), 108.7 (CH), 110.0 (CH), 111.1 (CH),

121.8 (CH), 121.9 (CH), 123.1 (CH), 125.9 (C), 126.0

(CH), 127.8 (C), 135.2 (CH), 147.4 (C), 152.9 (C), 155.2

(C), 159.7 (C), 182.1 (C); two C unobserved; m/z (EI)

305 (M⁺, 100%), 247 (22), 206 (26), 84 (37).

3.3. Preparation of quinones 11, 12, 14, 15 by the Fried-

¨

lander reaction

3.3.1. 6-Methoxy-2-(2-pyridyl)quinoline-5,8-dione (11)

(Method 2). (a) A solution of 2-benzenesulfonyloxy-3-

methoxy-6-nitrobenzaldehyde 40⁶⁹(1.0 g, 3.0 mmol) in

THF (25 mL) was hydrogenated over 10% Pd/C (0.5 g).

After 5 h the mixture was ﬁltered and the ﬁltrate was

immediately used in the Friedlander condensation. To a

stirred solution of 2-acetylpyridine (0.5 mL, 4.5 mmol)

and Triton B (10 drops) was added a solution of the

aminoaldehyde in THF under an atmosphere of nitro-

gen. The solution was stirred at room temperature for

20 h, during which additional Triton B (10 drops) was

added. The reaction mixture was neutralized with HCl

(1 M) and extracted with chloroform (3ꢅ10 mL). The

chloroform extract was washed with brine (3ꢅ5 mL),

dried (Na₂SO₄) and concentrated in vacuo. A stirred

mixture of the residue and 15% NaOH solution (20 mL)

in ethanol (20 mL) was heated under reﬂux overnight.

The solution was cooled, diluted with water, and

washed with chloroform (2ꢅ10 mL). The aqueous layer

was separated, neutralized with dilute HCl, and extrac-

ted with chloroform (3ꢅ10 mL). The chloroform extract

(b) A solution of the pyridylquinoline 42 (0.06 g, 0.23

mmol) in methanol (32 mL) was added to a stirred

solution of Fremy’s salt (0.8 g, 2.9 mmol) in dihydrogen

phosphate buﬀer (pH 6, 0.3 M; 32 mL). The solution

was stirred at room temperature overnight and diluted

with water, and the crystalline solid which precipitated

was collected. The solid was recrystallized from di-

chloromethane to yield the title compound 12 as a yellow

solid (0.04 g, 59%), mp 250 ^ꢄC, (Found: M⁺, 280.0850.

C₁₆H₁₂N₂O₃requires 280.0848); l_max(MeCN)/nm 288

(log e 4.04), 304 (4.01), 356 (3.15), 428 (2.14);

n

_max(KBr)/cm^ꢁ13062, 2938, 2850, 1680, 1675, 1607,

1585; d_H(400 MHz; CDCl₃) 2.47 (3H, s) 3.95 (3H, s),

6.38 (1H, s), 7.22 (1H, s), 8.53 (3H, m), 8.79 (1H, d,

J=8.0 Hz); d_C(100 MHz; CDCl₃) 21.2 (Me) 56.7 (Me),

110.7 (CH), 123.7 (CH), 124.6 (CH), 126.2 (CH), 127.6

(C), 135.7 (CH), 147.3 (C), 148.7 (C), 149.2 (CH), 153.7

1678

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

(C), 160.3 (C), 161.0 (C), 179.5 (C), 183.2 (C); m/z (EI)

280 (M⁺, 100%), 265 (13), 252 (17), 196 (11), 181 (28),

170 (11), 128 (14), 118 (13), 115 (23), 104 (63), 91 (54).

266 (M⁺, 67%), 209 (11), 177 (19), 149 (100), 71 (15), 57

(22).

3.3.4. 6-Methoxy-2-(4-pyridyl)quinoline-5,8-dione (15).

(a) A solution of 2-benzenesulfonyloxy-3-methoxy-6-

nitrobenzaldehyde 40 (1.0 g, 3.0 mmol) in THF (40 mL)

was hydrogenated over 10% Pd/C (0.5 g). After 2 h the

mixture was ﬁltered and the ﬁltrate was used immedi-

ately in the Friedlander condensation. To a stirred

solution of 4-acetylpyridine (0.5 g, 4.5 mmol) and Triton

B (10 drops) was added a solution of the aminoaldehyde

in THF under an atmosphere of nitrogen. The solution

was stirred at room temperature for 19 h, during which

additional Triton B (10 drops) was added. The reaction

mixture was neutralized with HCl (1 M) and extracted

with chloroform (3ꢅ20 mL). The chloroform extract

was washed with brine (3ꢅ10 mL), dried (Na₂SO₄) and

concentrated in vacuo. A stirred mixture of the residue

and 15% NaOH solution (20 mL) in ethanol (20 mL)

was heated under reﬂux overnight. The solution was

cooled, diluted with water, and washed with chloroform

(3ꢅ10 mL). The aqueous layer was separated, neu-

tralized with dilute HCl, and extracted with chloroform

(3ꢅ20 mL). The chloroform extract was washed with

brine (3ꢅ10 mL), dried (Na₂SO₄) and concentrated in

vacuo. The crude product was puriﬁed by column

chromatography [dichloromethane/ethyl acetate (8:2)

elution] to give 6-methoxy-2-(4-pyridyl)quinolin-5-ol 44

as a yellow solid (0.05 g, 7%), used directly in the next

step.

3.3.3. 6-Methoxy-2-(3-pyridyl)quinoline-5,8-dione (14).

(a) A solution of 2-benzenesulfonyloxy-3-methoxy-6-

nitrobenzaldehyde 40 (1.0 g, 3.0 mmol) in THF (35 mL)

was hydrogenated over 10% Pd/C (0.5 g). After 5 h the

mixture was ﬁltered and the ﬁltrate was used immedi-

ately in the Friedlander condensation. To a stirred

solution of 3-acetylpyridine (0.5 g, 4.5 mmol) and Triton

B (10 drops) was added a solution of the aminoaldehyde

in THF under an atmosphere of nitrogen. The solution

was stirred at room temperature for 20 h, during which

additional Triton B (10 drops) was added. The reaction

mixture was neutralized with HCl (1 M) and extracted

with chloroform (3ꢅ15 mL). The chloroform extract

was washed with brine (3ꢅ10 mL), dried (Na₂SO₄) and

concentrated in vacuo. A stirred mixture of the residue

and 15% NaOH solution (20 mL) in ethanol (20 mL)

was heated under reﬂux overnight. The solution was

cooled, diluted with water, and washed with chloroform

(3ꢅ10 mL). The aqueous layer was separated, neu-

tralized with dilute HCl, and extracted with chloroform

(3ꢅ15 mL). The chloroform extract was washed with

brine, dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

(dichloromethane/ethyl acetate (8:2) elution) to give 6-

methoxy-2-(3-pyridyl)quinolin-5-ol 43 as a yellow solid

(0.2 g, 27%), (Found: M⁺, 252.0901. C₁₅H₁₂N₂O₂

requires 252.0899); n_max(KBr)/cm^ꢁ13287, 3069, 2996,

2938, 2836, 1621, 1578; d_H(400 MHz; CDCl₃) 4.03 (3H,

s), 6.46 (1H, br), 7.46 (1H, ddd, J=0.7, 4.8, 7.9 Hz),

7.51 (1H, d, J 9.1 Hz), 7.76 (1H, d, J=9.1 Hz), 7.83

(1H, d, J=8.9 Hz), 8.50 (1H, ddd, J=1.6, 2.2, 7.9 Hz),

8.61 (1H, d, J=8.9 Hz), 8.69 (1H, dd, J=1.6, 4.8 Hz),

9.35 (1H, dd, J=0.7, 2.2 Hz); d_C(100 MHz; CDCl₃)

57.0 (Me), 116.6 (CH), 117.6 (CH), 118.2 (C), 121.2

(CH), 123.7 (CH), 131.3 (CH), 135.0 (CH), 135.4 (C),

139.2 (C), 141.9 (C), 143.8 (C), 148.5 (CH), 149.7 (CH),

152.9 (C); m/z (EI) 252 (M⁺, 97%), 237 (100), 149 (15),

97 (17), 85 (32), 71 (37), 57 (44), 55 (26).

(b) A solution of the pyridylquinoline 44 (0.05 g, 0.20

mmol) in methanol (27 mL) was added to a stirred

solution of Fremy’s salt (0.7 g, 2.5 mmol) in dihydrogen

phosphate buﬀer (pH 6, 0.3 M; 27 mL). The solution

was stirred at room temperature overnight and diluted

with water, and the crystalline solid which precipitated

was collected. The solid was recrystallized from

dichloromethane to yield the title compound 15 as a

yellow solid (0.03 g, 64%), mp >260 ^ꢄC, (Found:

M⁺, 266.0695. C₁₅H₁₀N₂O₃requires 266.0691); l_max

(MeCN)/nm 288 (log e 3.82), 304 (3.77), 356 (2.85), 428

(1.90); n_max(KBr)/cm^ꢁ13076, 2982, 2945, 2844, 1687,

1651, 1607, 1578; d_H(400 MHz; CDCl₃) 3.97 (3H, s),

6.42 (1H, s), 8.05 (2H, dd, J=1.6, 4.5 Hz), 8.14 (1H, d,

J=8.2 Hz), 8.58 (1H, d, J=8.2 Hz), 8.80 (2H, dd,

J=1.6, 4.5 Hz); d_C(100 MHz; CDCl₃) 56.8 (Me), 111.0

(CH), 121.6 (CH), 124.1 (CH), 127.5 (C), 136.2 (CH),

144.4 (C), 147.8 (C), 150.7 (CH), 159.7 (C), 160.2 (C),

179.2 (C), 182.6 (C); m/z (EI) 266 (M⁺, 100%), 209

(19), 180 (14), 140 (14).

(b) A solution of the pyridylquinoline 43 (0.1 g, 0.50

mmol) in methanol (70 mL) was added to a stirred

solution of Fremy’s salt (1.9 g, 6.7 mmol) in dihydrogen

phosphate buﬀer (pH 6, 0.3 M; 70 mL). The solution

was stirred at room temperature overnight, diluted with

water and the crystalline solid which precipitated was

collected. The crude product was puriﬁed by column

chromatography (dichloromethane/ethyl acetate (8:2)

elution) to give the title compound 14 as a yellow solid

(0.06 g, 45%), mp 182–184 ^ꢄC, (Found: M⁺, 266.0689.

C₁₅H₁₀N₂O₃requires 266.0691); l_max(MeCN)/nm 288

(log e 3.98), 304 (3.95), 356 (3.15), 428 (2.39);

3.3.5. 2-(2-Hydroxymethyl-1-methylindol-4-yl)-6-meth-

oxyquinoline-5,8-dione (18). (a) Sodium metal (0.97 g,

42.3 mmol) was added to methanol (40 mL), and the

mixture was cooled to ꢁ30 ^ꢄC. A solution of 2-bromo-

benzaldehyde (2.00 g, 11 mmol), methyl azidoacetate

(4.87 g, 42.3 mmol) in methanol (10 mL) was added and

the mixture was stirred for 3 h at ꢁ30 ^ꢄC and then at

4 ^ꢄC overnight. Water was added and the solvent was

removed in vacuo. The residue was extracted with ethyl

acetate, the combined organic phases were washed with

water and brine, dried (MgSO₄) and concentrated in

n

_max(KBr)/cm^ꢁ13040, 2938, 2836, 1680, 1658, 1607,

1570; d_H(300 MHz; CDCl₃) 3.96 (3H, s), 6.39 (1H, s),

7.47 (1H, dd, J=4.8, 7.7 Hz), 8.11 (1H, d, J=8.2 Hz),

8.54 (2H, m), 8.74 (1H, d, J=3.5 Hz), 9.29 (1H, d,

J=1.5 Hz); d_C(100 MHz; CDCl₃) 56.8 (Me), 111.8

(CH), 123.7 (CH), 123.9 (CH), 126.8 (C), 133.2 (C),

135.6 (CH), 136.0 (CH), 147.9 (C), 148.6 (CH), 151.5

(CH), 159.8 (C), 160.2 (C), 179.3 (C), 182.8 (C); m/z (EI)

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1679

vacuo. The product was puriﬁed by column chromato-

graphy (light petroleum/ethyl acetate (gradient) elution)

to give methyl 2-azido-3-(2-bromophenyl)propenoate 45

as a yellow solid (1.58 g, 51%), mp 85–86 ^ꢄC, (Found:

M⁺, 280.9798. C₁₀H₈⁷⁹BrN₃O₂requires 280.9800);

methyl 4-bromo-1-methylindole-2-carboxylate 47 (0.38

g, 1.4 mmol) in dioxane (20 mL) was added and the

mixture was heated to 80 ^ꢄC for 18 h. After cooling

the mixture was diluted with ether and washed with brine.

The organic phase was dried (MgSO₄) and concentrated

in vacuo. The residue was puriﬁed by column chroma-

tography (light petroleum/ethyl acetate (95:5) elution)

to give the boronate ester of methyl 4-bromo-1-methyl-

indole-2-carboxylate 48 as a colorless solid (0.24 g,

54%), mp 161–162 ^ꢄC (from light petroleum), (Found:

C, 65.0; H,7.3; N, 4.3. C₁₇H₂₂BNO₄requires C, 64.8; H,

7.0; N, 4.4%); (Found: M⁺, 315.1643. C₁₇H₂₂BNO₄

requires 315.1642); n_max(Nujol)/cm^ꢁ11710, 1562, 1506,

1367, 1234; d_H(300 MHz; CDCl₃) 1.39 (12H, s), 3.92

(3H, s), 4.07 (3H, s), 7.35 (1H, dd, J=8.4, 6.9 Hz), 7.47

(1H, d, J=8.4 Hz), 7.66 (1H, dd, J=6.9, 0.9 Hz), 7.72

(1H, d, J=0.9 Hz); d_C(100 MHz; CDCl₃) 24.9 (Me),

31.5 (Me), 51.5 (Me), 83.6 (C), 112.1 (CH), 113.0 (CH),

124.3 (CH), 127.9 (C), 128.8 (CH), 130.1 (C), 139.1 (C),

162.9 (C); one C unobserved; m/z (EI) 315 (M⁺, 100%),

269 (34), 242 (25), 215 (37), 198 (17).

n

_max(CHCl₃)/cm^ꢁ12130, 1716; d_H(300 MHz; CDCl₃)

3.86 (3H, s), 7.10 (1H, td, J=7.8, 1.8 Hz), 7.17 (1H, s),

7.24–7.29 (1H, m), 7.53 (1H, dd, J=7.8, 1.8 Hz), 8.03

(1H, dd, J=7.8, 1.8 Hz); d_C(100 MHz; CDCl₃) 53.1

(Me), 123.5 (CH), 125.2 (C), 127.1 (CH), 127.2 (C),

130.3 (CH), 131.2 (CH), 132.91 (C), 132.93 (CH), 163.7

(C); m/z (EI) 281 (M⁺, 2%), 255 (6), 223 (7), 209 (22),

194 (100), 115 (67), 89 (48), 59 (39).

(b) A solution of methyl 2-azido-(2-bromophenyl)pro-

penoate 45 (1.5 g, 5.3 mmol) in dry xylene (45 mL) was

added to boiling dry xylene (120 mL) and heating con-

tinued for 3 h. After cooling, the xylene was removed in

vacuo. The crude product was puriﬁed by column

chromatography (light petroleum/ethyl acetate (95:5)

elution) to give methyl 4-bromoindole-2-carboxylate 46

as a colorless solid (1.13 g, 84%), mp 164–165 ^ꢄC (from

ethyl acetate/light petroleum), (Found: C, 47.3; H, 3.0;

N, 5.4. C₁₀H₈BrNO₂requires C, 47.3; H, 3.2; N, 5.5%);

(Found: M⁺, 252.9738. C₁₀H₈⁷⁹BrNO₂requires

252.9739); n_max(Nujol)/cm^ꢁ13324, 1700, 1567, 1521; d_H

(300 MHz; CDCl₃) 3.97 (3H, s), 7.16 (1H, dd, J=7.5,

8.1 Hz), 7.27 (1H, dd, J=2.4, 0.9 Hz), 7.32 (1H, dd,

J=7.5, 0.9 Hz), 7.37 (1H, dt, J=8.1, 0.9 Hz), 9.33 (1H,

br); d_C(100 MHz; CDCl₃) 52.2 (Me), 108.9 (CH), 111.1

(CH), 116.4 (C), 123.7 (CH), 126.1 (CH), 127.4 (C),

128.4 (C), 137.0 (C), 162.3 (C); m/z (EI) 253 (M⁺,

74%), 221 (100), 195 (8), 169 (15), 114 (63), 88 (9).

(e) A ﬂask was charged with 2-chloro-6-methoxy-5-

nitroquinoline 35 (0.57 g, 2.4 mmol), dichloro[1,1⁰-bis-

(diphenylphosphino)ferrocene]palladium(II) (114 mg,

0.14 mmol) and potassium phosphate (3.1 g, 15 mmol)

and was ﬂushed with nitrogen. A solution of the indole

boronate 48 (0.7 g, 2.2 mmol) in dioxane (70 mL) was

added and the mixture was heated to 80 ^ꢄC for 24 h.

After cooling the mixture was diluted with ether and

was washed with brine. The organic phase was dried

(MgSO₄) and concentrated in vacuo. The residue was

puriﬁed by column chromatography (light petroleum/

ethyl acetate(gradient) elution) to give methyl 4-(6-

methoxy-5-nitroquinolin-2-yl)-1-methylindole-2-carboxyl-

ate 49 as a pale yellow solid (0.37 g, 44%), mp 189–

191 ^ꢄC (from ethyl acetate/light petroleum), (Found:

C, 64.7; H, 4.4; N, 10.4. C₂₁H₁₇N₃O₅requires C,

64.4; H, 4.4; N, 10.7%); (Found: M⁺, 391.1164.

C₂₁H₁₇N₃O₅requires 391.1168); n_max(Nujol)/cm^ꢁ1

1721, 1618, 1593, 1516, 1352; d_H(300 MHz; CDCl₃)

3.92 (3H, s), 4.10 (3H, s), 4.13 (3H, s), 7.48–7.50 (2H,

m), 7.58 (1H, d, J=9.6 Hz), 7.67 (1H, dd, J=6.0, 2.4

Hz), 7.90 (1H, s), 8.03 (1H, d, J=9.0 Hz), 8.15 (1H, dd,

J=9.0, 0.6 Hz), 8.37 (1H, dd, J=9.6, 0.6 Hz); d_C

(100 MHz; CDCl₃) 31.8 (Me), 51.6 (Me), 57.2 (Me),

110.6 (CH), 111.7 (CH), 116.3 (CH), 120.0 (C), 121.4

(CH), 123.6 (CH), 124.1 (C), 125.0 (CH), 128.8 (C),

129.6 (CH), 133.1 (C), 134.3 (CH), 134.9 (C), 140.5 (C),

142.5 (C), 149.1 (C), 157.5 (C), 162.5 (C); m/z (EI) 391

(M⁺, 100%), 346 (23), 318 (20), 285 (51), 257 (30), 256

(55), 255 (35).

(c) A solution of methyl-4-bromoindole-2-carboxylate

46 (1.65 g, 6.5 mmol) in DMF (30 mL) was added to a

solution of sodium hydride (0.24 g, 10 mmol) in DMF

(80 mL) at 0 ^ꢄC and the mixture was stirred at room

temperature for 45 min. Methyl iodide (1.37 g, 10

mmol) was added at 0 ^ꢄC and stirring was continued for

2 h at room temperature. Water was added and the

mixture was extracted with ether. The combined organic

phases were washed with water, dried (Na₂SO₄) and

concentrated in vacuo. The residue was puriﬁed by col-

umn chromatography (light petroleum/ethyl acetate

(1:1) elution) to give methyl 4-bromo-1-methylindole-2-

carboxylate 47 as a colorless solid (1.56 g, 90%), mp 86–

88 ^ꢄC (from light petroleum), (Found: C, 49.5; H, 3.6;

N, 5.1. C₁₁H₁₀BrNO₂requires C, 49.3; H, 3.8; N,

5.2%); (Found: M⁺, 266.9894. C₁₁H₁⁷₀⁹BrNO₂requires

266.9895); n_max(Nujol)/cm^ꢁ11716, 1552, 1506, 753; d_H

(300 MHz; CDCl₃) 3.93 (3H, s), 4.07 (3H, s), 7.19 (1H, t,

J=8.1 Hz), 7.31–7.33 (3H, m); d_C(100 MHz; CDCl₃)

32.0 (Me), 51.7 (Me), 109.4 (CH), 110.3 (CH), 116.5 (C),

123.4 (CH), 125.6 (CH), 126.7 (C), 128.1 (C), 139.7 (C),

162.2 (C); m/z (EI) 267 (M⁺, 100%), 254 (15), 236 (35),

209 (18), 188 (23), 169 (23).

(f) Tin powder (0.83 g) and HCl (3 M; 17 mL) were

added to a solution of methyl 4-(6-methoxy-5-nitroquin-

olin-2-yl)-1-methylindole-2-carboxylate 49 (0.46 g, 1.2

mmol) in ethanol (35 mL) and the mixture was heated

under reﬂux for 30 min. After cooling the mixture was

decanted from the excess tin and neutralized with satu-

rated NaHCO₃solution. An equal volume of water was

added and the mixture was stirred in dichloromethane

for 2 h. The layers were separated and the organic

phase was dried (Na₂SO₄) and concentrated in vacuo.

(d)

Dichloro[1,1⁰-bis(diphenylphosphino)ferrocene]-

palladium (II) (32 mg, 0.04 mmol), diphenylphosphino-

ferrocene (24 mg, 0.04 mmol), potassium acetate (0.4 g,

4 mmol) and bis(pinacolato)diboron (0.38g, 1.5 mmol)

in a ﬂask was ﬂushed with nitrogen. A solution of

1680

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

The residue was puriﬁed by column chromatography

(dichloromethane/ethyl acetate (98:2) elution) to give

methyl 4-(5-amino-6-methoxyquinolin-2-yl)-1-methyl-

indole-2-carboxylate 50 as an orange sticky solid (0.11

g, 26%), n_max(Nujol)/cm^ꢁ13457, 3365, 1751, 1613,

1557; d_H(300 MHz; CDCl₃) 3.93 (3H, s), 4.02 (3H, s),

4.14 (3H, s), 7.47–7.49 (3H, m), 7.67 (1H, dd, J=6.0,

2.4 Hz), 7.74 (1H, dd, J=9.0, 0.6 Hz), 7.84 (1H, d,

J=8.7 Hz), 7.92 (1H, s), 8.24 (1H, dd, J=9.0, 0.6 Hz);

NH₂not observed; d_C(100 MHz; CDCl₃) 31.7 (Me),

51.5 (Me), 56.7 (Me), 110.9 (CH), 111.0 (CH), 116.5

(CH), 117.4 (C), 119.7 (CH), 120.1 (CH), 121.0 (CH),

124.3 (C), 125.1 (CH), 128.4 (C), 129.3 (C), 129.4 (CH),

134.5 (C), 140.5 (C), 142.7 (C), 144.0 (C), 155.9 (C),

162.7 (C); m/z (EI) 361 (M⁺, 48%), 346 (60), 217 (35),

216 (34), 59 (88), 52 (47), 45 (100).

(MH⁺, 3%), 335 (2), 146 (47), 132 (33), 110 (47), 108

(55), 96 (52), 72 (49), 69 (40), 58 (100).

3.3.6. 6-(Pyrrolidin-1-yl)quinoline-5,8-dione (19). Pyrrol-

idine (0.4 mL, 5.3 mmol) was added to a solution of

6-methoxyquinoline-5,8-dione 2 (50 mg, 0.3 mmol) in

DMF (5 mL) and the mixture was stirred at room

temperature for 3 days. The mixture was diluted with

dichloromethane and washed with water (ꢅ5). The

organic phase was dried (Na₂SO₄) and concentrated in

vacuo. The product was puriﬁed by column chromato-

graphy (ethyl acetate elution) to give the title compound

19 as a red solid (42 mg, 71%), mp 196–197 ^ꢄC (from

ethyl acetate/light petroleum), (Found: M⁺, 228.0901.

C₁₃H₁₂N₂O₂requires 228.0898); l_max(MeOH)/nm 232

(log e 4.34), 268 (4.11), 310 (3.90), 472 (3.73); n_max

(Nujol)/cm^ꢁ11680, 1618, 1577, 1552; d_H(300 MHz;

CDCl₃) 1.99–2.04 (4H, m), 3.38 (2H, br), 3.98 (2H, br),

5.94 (1H, s), 7.54 (1H, dd, J=7.8, 4.5 Hz), 8.32 (1H, dd,

J=7.8, 1.8 Hz), 8.97 (1H, dd, J=4.5, 1.8 Hz); d_C

(100 MHz; CDCl₃) 25.2 (br, CH₂), 51.1 (br, CH₂), 106.0

(CH), 125.8 (CH), 128.2 (C), 134.2 (CH), 148.3 (C),

148.8 (C) 154.5 (CH), 180.5 (C), 182.7 (C); m/z (EI) 228

(M⁺, 26%), 209 (30), 114 (17), 83 (33), 70 (100), 57 (77).

(g) A solution of the methyl 4-(5-amino-6-methoxy-

quinolin-2-yl)-1-methylindole-2-carboxylate 50 (0.11 g,

0.4 mmol) in THF (15 mL) was added to a solution of

lithium aluminium hydride (15 mg, 1.4 mmol) in THF

(10 mL) at 0 ^ꢄC. The mixture was warmed and stirred at

room temperature for 30 min. The mixture was cooled

to 0 ^ꢄC and water (1 mL) was added followed by NaOH

solution (1 M; 1 mL) and ﬂash silica. The mixture was

ﬁltered through Celite, and the ﬁltrate was dried

(Na₂SO₄) and concentrated in vacuo to give 4-(5-amino-

6-methoxyquinolin-2-yl)-1-methylindole-2-methanol 51

as a yellow oily solid (88 mg, 75%), which was used

without puriﬁcation or characterisation, d_H(300 MHz;

CDCl₃) 3.82 (3H, s), 4.01 (3H, s), 4.82 (2H, s), 7.01 (1H,

s), 7.36 (2H, m), 7.44 (1H, d, J=9.0 Hz), 7.58 (1H, dd,

J=6.0, 2.4 Hz), 7.71 (1H, d, J=9.0 Hz), 7.79 (1H, d,

J=8.7 Hz), 8.17 (1H, d, J=8.7 Hz).

3.3.7.

2-Phenyl-6-(pyrrolidin-1-yl)-quinoline-5,8-dione

(20). Pyrrolidine (0.5 mL, 4.20 mmol) was added to a

solution of 6-methoxy-2-phenylquinoline-5,8-dione 5

(21 mg, 0.08 mmol) in DMF (2 mL) and the mixture

was stirred at room temperature for 3 days. The mixture

was diluted with dichloromethane and washed with

water (3ꢅ5 mL). The organic phase was dried (Na₂SO₄)

and concentrated in vacuo. The crude product was

puriﬁed by column chromatography (ethyl acetate elu-

tion) to give the title compound 20 as a red solid (24 mg,

100%), mp 202–203 ^ꢄC (from ethyl acetate), (Found:

MH⁺, 305.1290. C₁₉H₁₆N₂O₂+H requires 305.1290);

(h) A solution of Fremy’s salt (36 mg, 0.12 mmol) in

sodium dihydrogen phosphate buﬀer (0.3 M; 5 mL) was

added to a solution of 4-(5-amino-6-methoxyquinolin-2-

yl)-1-methylindole-2-methanol 51 (26 mg, 0.08 mmol) in

acetone (10 mL) and the mixture was stirred at room

temperature for 1.5 h. A further solution of Fremy’s salt

(36 mg, 0.12 mmol) in the buﬀer (0.3 M; 5 mL) was

added and the mixture was stirred for a further 1.5 h.

The mixture was extracted with dichloromethane, and

the combined organic phases were dried (Na₂SO₄)

and concentrated in vacuo. The residue was puriﬁed by

column chromatography (dichloromethane/ethyl ace-

tate (3:1) elution) to give the title compound 18 as a

pink/red solid (19 mg, 69%), mp 261 ^ꢄC (from chloro-

form/methanol), (Found: C, 66.4; H, 4.4; N, 7.6.

C₂₀H₁₆N₂O₄.0.7H₂O requires C, 66.5; H, 4.9; N, 7.8%);

(Found: M⁺, 348.1120. C₂₀H₁₆N₂O₄requires 348.1110);

l_max(MeOH)/nm 300 (log e 4.11), 316 (4.09), 336 (4.05),

480 (3.61); n_max(KBr)/cm^ꢁ13047, 2955, 2868, 1680,

1567, 1552; d_H(400 MHz; CDCl₃, ꢁ20 ^ꢄC) 2.00–2.05

(4H, m), 3.40 (2H, t, J=6.0 Hz), 4.01 (2H, t, J=6.0

Hz), 6.01 (1H, s), 7.49–7.52 (3H, m), 8.00 (1H, d, J=8.4

Hz), 8.18 (2H, d, J=1.6 Hz), 8.38 (1H, d, J=8.4 Hz);

d_C(100 MHz; CDCl₃, ꢁ20 ^ꢄC) 23.9 (CH₂), 26.8 (CH₂),

51.0 (CH₂), 51.9 (CH₂), 105.6 (CH), 122.7 (CH), 126.6

(C), 127.9 (CH), 129.0 (CH), 130.6 (CH), 135.6 (CH),

137.6 (C), 148.5 (C), 148.9 (C), 161.9 (C), 181.0 (C),

182.5 (C); m/z (EI) 304 (M⁺, 9%), 178 (9), 154 (20), 102

(34), 77 (35), 53 (60), 49 (47), 41 (100).

3.3.8. 6-(Morpholin-4-yl)-2-phenylquinoline-5,8-dione (21).

Morpholine (1 mL, 11 mmol) was added to a solution of

6-methoxy-2-phenylquinoline-5,8-dione 5 (23 mg, 0.10

mmol) in DMF (2 mL) and the mixture was stirred at

room temperature for 3 days. The mixture was diluted

with dichloromethane and washed with water (3ꢅ5

mL). The organic phase was dried (Na₂SO₄) and con-

centrated in vacuo. The crude product was puriﬁed by

column chromatography (ethyl acetate elution) to give

the title compound 21 as a red solid (33 mg, 100%), mp

203–204 ^ꢄC (from ethyl acetate), (Found: MH⁺,

321.1233. C₁₉H₁₆N₂O₃+H requires 321.1239); n_max(KBr)/

cm^ꢁ13057, 2945, 2919, 2852, 1675, 1572; d_H(400 MHz;

l_max(MeOH)/nm 242 (log e 4.28), 278 (4.17), 352 (3.96),

454 (3.45); n_max(Nujol)/cm^ꢁ13472, 1675, 1644, 1603,

1572; d_H(300 MHz; CDCl₃) 3.86 (3H, s), 4.01 (3H, s),

4.89 (2H, d, J=3.9 Hz), 6.36 (1H, s), 7.33–7.38 (2H, m),

7.48 (1H, d, J=8.1 Hz), 7.72 (1H, d, J=8.1 Hz), 8.17

(1H, d, J=8.4 Hz), 8.48 (1H, d, J=8.4 Hz); OH not

observed; d_C(100 MHz; CDCl₃) 29.9 (Me), 56.6 (Me),

56.7 (CH₂), 101.8 (CH), 110.2 (CH), 111.9 (CH), 120.6

(CH), 121.5 (CH), 125.52 (C), 125.58 (CH), 125.8 (C),

129.5 (C), 135.0 (CH), 138.9 (C), 141.3 (C), 147.3 (C),

160.3 (C), 164.2 (C) 179.5 (C), 183.6 (C); m/z (CI) 349

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1681

CDCl₃) 3.56 (4H, t, J=4.8 Hz), 3.87 (4H, t, J=4.8 Hz),

6.20 (1H, s), 7.49 (3H, m), 7.99 (1H, d, J=8.3 Hz), 8.17

(2H, d, J=7.9 Hz), 8.36 (1H, d, J=8.3 Hz); d_C

(100 MHz; CDCl₃) 49.2 (CH₂), 66.4 (CH₂), 112.4 (CH),

122.9 (CH), 127.78 (C), 127.82 (CH), 128.9 (CH), 130.6

(CH), 135.6 (CH), 137.6 (C), 147.9 (C), 152.7 (C), 162.0

(C), 182.2 (C), 182.3 (C); m/z (EI) 320 (M⁺, 75%), 264

(84), 263 (100), 223 (55), 179 (81).

methane (10 mL) and the mixture was stirred at room

temperature for 24 h. The reaction was quenched with

saturated NaHCO₃solution. The resulting mixture was

extracted with dichloromethane, the combined organic

phases were dried (Na₂SO₄) and concentrated in vacuo.

The residue was puriﬁed by column chromatography

[light petroleum/ethyl acetate (7:3) elution] to give 2-

phenylquinolin-8-ol 55 as a colorless oil (0.18 g, 84%)

(lit.,⁷¹mp 55 ^ꢄC), n_max(CHCl₃)/cm^ꢁ13411 (OH), 1598,

1562; d_H(300 MHz; CDCl₃) 7.19 (1H, dd, J=7.2, 1.2

Hz), 7.33 (1H, dd, J=8.1, 1.2 Hz), 7.42 (1H, d, J=7.2

Hz), 7.46–7.56 (3H, m), 7.91 (1H, d, J=8.7 Hz), 8.14–

8.17 (2H, m), 8.21 (1H, d, J=8.7 Hz); d_C(100 MHz;

CDCl₃) 110.1 (CH), 117.6 (CH), 119.5 (CH), 127.3

(CH), 127.5 (CH), 128.7 (CH), 128.8 (CH), 129.5 (CH),

136.9 (CH), 138.0 (C), 138.8 (C), 152.3 (C), 154.9 (C).

3.3.9. 2-Phenylquinoline-5,8-dione (22). (a) A solution of

8-benzyloxy-2(1H)-quinolone 52⁷⁰(1.00 g, 4.0 mmol) in

phosphorus oxychloride (110 mL) was heated under

reﬂux for 4 h. The phosphorus oxychloride was removed

in vacuo. The residue was poured into ice water and

neutralized with saturated K₂CO₃solution. This mix-

ture was extracted with dichloromethane, the combined

organic phases were dried (Na₂SO₄) and concentrated in

vacuo. The crude product was recrystallized from ethyl

acetate/light petroleum to give 8-benzyloxy-2-chloro-

quinoline 53 as a colorless solid (0.91 g, 85%), mp 96–

97 ^ꢄC, (Found: C, 70.9; H, 4.3; N, 5.1. C₁₆H₁₂ClNO

(d) To a solution of 2-phenylquinolin-8-ol 55 (0.17 g,

0.75 mmol) in acetone (30 mL) was added a solution of

Fremy’s salt (0.35 g, 1.3 mmol) in sodium dihydrogen

phosphate buﬀer (0.3 M; 30 mL) and the mixture was

stirred at room temperature for 1 h. A further solution

of Fremy’s salt (0.35 g, 1.3 mmol) in the buﬀer (0.3 M;

30 mL) was added and stirring was continued for 1 h.

The acetone was removed in vacuo. The residue was

extracted with dichloromethane and the combined

organic phases were dried (Na₂SO₄) and concentrated in

vacuo. The residue was puriﬁed by column chromato-

graphy (dichloromethane elution) to give the title com-

pound 22 as a yellow/brown solid (0.10 g, 58%), mp

139–141 ^ꢄC (lit.,⁷²mp 121–123 ^ꢄC), l_max(MeOH)/nm

230 (log e 4.26), 266 (4.39), 302 (4.06), 354 (3.52); n_max

(CHCl₃)/cm^ꢁ11685, 1669, 1582; d_H(300 MHz; CDCl₃)

7.06 (1H, d, J=10.2 Hz), 7.16 (1H, d, J=10.2 Hz),

7.51–7.53 (3H, m), 8.11 (1H, d, J=8.1 Hz), 8.16–8.19

(2H, m), 8.45 (1H, d, J=8.1 Hz); d_C(100 MHz; CDCl₃)

124.1 (CH), 127.5, 127.8 (CH), 129.0 (CH), 130.8 (CH),

135.3 (CH), 137.3, 137.9 (CH), 139.1 (CH), 147.4, 162.1,

183.2 (C), 184.5 (C).

requires C, 71.2; H, 4.5; N, 5.2%); (Found: M⁺, 269.0609.

35

12

C₁₆H ClNO requires 269.0607);

n

_max(Nujol)/cm^ꢁ1

1591, 1564, 847; d_H(300 MHz; CDCl₃) 5.43 (2H, s), 7.07

(1H, dd, J=6.0, 3.0 Hz), 7.28–7.42 (6H, m), 7.50 (2H, d,

J=7.2 Hz), 8.06 (1H, d, J=8.7 Hz); d_C(100 MHz;

CDCl₃) 70.9 (CH₂), 111.7 (CH), 119.6 (CH), 122.9

(CH), 126.9 (CH), 127.0 (CH), 127.8 (CH), 128.1 (C),

128.5 (CH), 136.8 (C), 138.7 (CH), 139.9 (C), 149.8 (C),

153.6 (C); m/z (EI) 269 (M⁺, 19%), 163 (7), 91 (100).

(b) A solution of 8-benzyloxy-2-chloroquinoline 53 (1.1

g, 4.0 mmol) in 1,2-dimethoxyethane (71 mL) was

degassed under reduced pressure and ﬂushed with

nitrogen. Pd(PPh₃)₄(0.24 g) was added and the mixture

was degassed further. The mixture was stirred for 10

min then Na₂CO₃solution (2 M; 8.1 mmol) was added

followed by phenylboronic acid (0.71 g, 5.9 mmol). The

mixture was heated under reﬂux for 18 h. After cooling

the mixture was poured into dichloromethane and

washed with water, the organic phases were dried

(Na₂SO₄) and concentrated in vacuo. The crude product

was puriﬁed by column chromatography (light petro-

leum/ethyl acetate (gradient) elution) to give 8-benzyl-

oxy-2-phenylquinoline 54 as a colorless solid (0.96 g,

78%), mp 90–91 ^ꢄC (from ethyl acetate/light petro-

leum), (Found: C, 84.2; H, 5.3; N, 4.4. C₂₂H₁₇NO.

0.1H₂O requires C, 84.4; H, 5.5; N, 4.5%); (Found:

M⁺, 311.1310. C₂₂H₁₇NO requires 311.1310); n_max(Nu-

jol)/cm^ꢁ11597, 1558, 1255, 1104; d_H(300 MHz, CDCl₃)

5.45 (2H, s), 7.12 (1H, dd, J=7.2, 1.8 Hz), 7.31–7.46

(6H, m), 7.48–7.53 (2H, m), 7.60 (2H, d, J=7.2 Hz),

7.92 (1H, d, J=8.7 Hz), 8.18 (1H, d, J=8.7 Hz), 8.22–

8.25 (2H, m); d_C(100 MHz; CDCl₃) 71.3 (CH₂), 111.6

(CH), 119.1 (CH), 120.0 (CH), 126.3 (CH), 127.1 (CH),

127.5 (CH), 127.6 (CH), 128.5 (CH), 128.7 (CH), 129.2

(CH), 136.7 (CH), 137.4 (C), 139.7 (C), 140.7 (C), 154.8

(C), 155.9 (C); one C unobserved; m/z (EI) 311 (M⁺,

34%), 234 (25), 205 (39), 191 (39), 91 (100).

3.3.10. 3-Hydroxymethylquinoline-5,8-dione (23). (a)

Phosphorus oxychloride (7.0 mL, 72 mmol) was added

to DMF (2 mL, 26 mmol) at 0 ^ꢄC, N-(2⁰,5⁰-dimethoxy-

phenyl)acetamide 56 (2.0 g, 10.3 mmol) was added and

the mixture was stirred for 5 min at 0 ^ꢄC and was then

heated under reﬂux for 2 h. After cooling the mixture

was poured into ice water. The mixture was extracted

with dichloromethane, the combined organic phases

were dried (Na₂SO₄) and concentrated in vacuo. The

residue was puriﬁed by column chromatography (light

petroleum/ethyl acetate (7:3) elution) to give 2-chloro-

5,8-dimethoxyquinoline-3-carbaldehyde 57 as an orange

solid (1.1 g, 41%), mp 157–159 ^ꢄC (lit.,⁷³mp 159 ^ꢄC), d_H

(300 MHz; CDCl₃) 3.99 (3H, s), 4.05 (3H, s), 6.84 (1H,

d, J=8.7 Hz), 7.14 (1H, d, J=8.7 Hz), 9.15 (1H, s),

10.56 (1H, s).

(b) A solution of 5,8-dimethoxy-2-chloroquinoline-3-

carbaldehyde 57 (1.0 g, 3.9 mmol) in THF (120 mL) was

added to a solution of lithium aluminium hydride (0.6 g,

16 mmol) in THF (80 mL) at ꢁ60 ^ꢄC. The mixture was

stirred at ꢁ60 ^ꢄC for 1 h. Water (8 mL) was added fol-

lowed by NaOH (1M; 8 mL) and silica (1 g), and the

(c) Boron trichloride dimethyl sulﬁde complex (2.0M; 10

mL, 20 mmol) was added to a solution of 8-benzyloxy-

2-phenylquinoline 54 (0.3g, 1.0 mmol) in dichloro-

1682

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

mixture was then ﬁltered through Celite. The ﬁltrate

was dried (Na₂SO₄) and concentrated in vacuo. The

residue was recrystallized from dichloromethane/light

petroleum to give 2-chloro-5,8-dimethoxyquinoline-3-

methanol 58 as a colorless solid (1.0 g, 100%), mp 151–

154 ^ꢄC, (Found: C, 54.1; H, 4.6; N, 5.2. C₁₂H₁₂ClNO₃.

0.7H₂O requires C, 54.1; H, 5.0; N, 5.3%); (Found:

M⁺, 253.0505. C₁₂H₁³₂⁵ClNO₃requires 253.0506); n_max

(Nujol)/cm^ꢁ13416, 3318, 1613, 1598, 725; d_H(300 MHz;

CDCl₃) 2.38 (1H, t, J=5.7 Hz), 3.94 (3H, s), 4.01 (3H,

s), 4.91 (2H, d, J=5.7 Hz), 6.76 (1H, d, J=8.4 Hz), 6.94

(1H, d, J=8.4 Hz), 8.62 (1H, s); d_C(100 MHz; CDCl₃)

55.8 (Me), 56.1 (Me), 62.2 (CH₂), 104.5 (CH), 108.1

(CH), 120.7, 131.5 (CH), 132.1, 139.0, 148.5, 148.6,

148.9; m/z (EI) 253 (M⁺, 40%), 238 (100), 224 (29), 160

(8), 116 (8), 75 (10), 63 (10).

3.3.11. 3-Hydroxymethyl-2-phenylquinoline-5,8-dione (24).

(a) A solution of 2-chloro-5,8-dimethoxyquinoline-3-

carbaldehyde 57 (1.0 g, 3.9 mmol) in 1,2-dimethoxy-

ethane (70 mL) was degassed under reduced pressure.

Pd(PPh₃)₄(0.44 g) was added followed by further

degassing. The mixture was stirred under a nitrogen

atmosphere for 10 min. Na₂CO₃solution (2 M; 10 mL)

was added followed by phenylboronic acid (0.7 g, 5.7

mmol). The mixture was heated under reﬂux for 18 h.

After cooling the mixture was poured into dichloro-

methane and washed with brine. The organic phase was

dried (Na₂SO₄) and concentrated in vacuo. The residue

was puriﬁed by column chromatography (dichloro-

methane elution) to give 5,8-dimethoxy-2-phenylquino-

line-3-carbaldehyde 60 as a yellow solid (0.8 g, 71%), mp

219–221 ^ꢄC (from ethyl acetate), (Found: M⁺, 293.1060.

C₁₈H₁₅NO₃requires 293.1052); n_max(Nujol)/cm^ꢁ11690,

1608, 1572; d_H(300 MHz; CDCl₃) 4.00 (3H, s), 4.03

(3H, s), 6.80 (1H, d, J=8.4 Hz), 7.09 (1H, d, J=8.4

Hz), 7.49–7.55 (3H, m), 7.68–7.72 (2H, m), 9.23 (1H, s),

10.17 (1H, s); d_C(100 MHz; CDCl₃) 55.9 (Me), 56.4

(Me), 104.3 (CH), 110.8 (CH), 120.0 (C), 127.1 (C),

128.5 (CH), 129.2 (CH), 130.6 (CH), 133.4 (CH), 137.9

(C), 141.5 (C), 149.3 (C), 150.0 (C), 159.6 (C), 191.5

(CH); m/z (EI) 293 (M⁺, 56%), 292 (32), 278 (91), 264

(20), 85 (65), 83 (100).

(c) Tin (0.45 g) and HCl (3M; 7 mL) was added to a

solution of 2-chloro-5,8-dimethoxyquinoline-3-metha-

nol 58 (0.17 g, 0.7 mmol) in ethanol (20 mL) and the

mixture was heated under reﬂux for 2 h. After cooling

the mixture was decanted from the excess tin. The mix-

ture was neutralized with saturated NaHCO₃solution,

an equal volume of water was added and the mixture

was stirred in dichloromethane for 1 h. The mixture was

ﬁltered through Celite and the layers were separated, the

organic phase was dried (Na₂SO₄) and concentrated in

vacuo. The crude product was puriﬁed by column

chromatography (dichloromethane/ethyl acetate (gra-

dient) elution) to give 5,8-dimethoxyquinoline-3-metha-

nol 59 as a colorless oily solid (80 mg, 55%), (Found:

M⁺, 219.0905. C₁₂H₁₃NO₃requires 219.0895); n_max

(CHCl₃)/cm^ꢁ13385 (OH), 1629, 1506; d_H(300 MHz;

CDCl₃) 3.94 (3H, s), 4.02 (3H, s), 4.90 (2H, s), 6.74 (1H,

d, J=8.4 Hz), 6.90 (1H, d, J=8.4 Hz), 8.49–8.50 (1H,

m), 8.89 (1H, d, J=2.1 Hz); OH not observed; d_C

(100 MHz, CDCl₃) 55.7 (Me), 56.0 (Me), 63.1 (CH₂),

104.0 (CH), 106.7 (CH), 121.2, 128.6 (CH), 133.3, 139.8,

148.6, 149.2 (CH), 149.4; m/z (EI) 219 (M⁺, 39%), 211

(21), 204 (100), 190 (21), 91 (84), 57 (47).

(b) Sodium borohydride (0.01 g, 0.34 mmol) was added

to a solution of 5,8-dimethoxy-2-phenylquinoline-3-car-

baldehyde 60 (0.2 g, 0.7 mmol) in 1,2-dimethoxyethane

(30 mL) and the mixture was heated under reﬂux for 2

h. After cooling water was added followed by HCl

(2 M). The mixture was extracted with ether and the

combined organic phases were dried (MgSO₄) and con-

centrated in vacuo. The crude product was puriﬁed by

column chromatography (dichloromethane/ethyl acet-

ate (gradient) elution) to give 5,8-dimethoxy-2-phenyl-

quinoline-3-methanol 61 as a colorless solid (0.11 g,

55%), mp 170–172 ^ꢄC (from dichloromethane/light

petroleum), (Found: C, 72.5; H, 5.7; N, 4.6.

C₁₈H₁₇NO₃.0.2H₂O requires C, 72.3; H, 5.9; N, 4.7%);

(Found: M⁺, 295.1203. C₁₈H₁₇NO₃requires 295.1208);

n_max(Nujol)/cm^ꢁ13457, 1618, 1593; d_H(300 MHz;

CDCl₃) 3.99 (3H, s), 4.01 (3H, s), 4.82 (2H, br), 6.77

(1H, d, J=8.4 Hz), 6.94 (1H, d, J=8.4 Hz), 7.42–7.49

(3H, m), 7.64–7.67 (2H, m), 8.73 (1H, s); OH not

observed; d_C(100 MHz; CDCl₃) 55.8 (Me), 56.1 (Me),

62.8 (CH₂), 103.8 (CH), 107.2 (CH), 120.7 (C), 128.2

(CH), 128.4 (CH), 129.1 (CH), 130.5 (CH), 131.8 (C),

139.5 (C), 140.0 (C), 148.6 (C), 149.5 (C), 158.1 (C); m/z

(EI) 295 (M⁺, 60%), 280 (100), 266 (29), 139 (16), 83

(61).

(d) Cerium(IV) ammonium nitrate (0.47 g, 0.8 mmol)

was added to a solution of 5,8-dimethoxyquinoline-3-

methanol 59 (80 mg, 0.4 mmol) in acetonitrile (20 mL)

and water (7 mL) and the mixture was stirred at room

temperature for 3 h. The mixture was diluted with water

and was extracted with dichloromethane. The combined

organic phases were dried (Na₂SO₄) and concentrated in

vacuo. The residue was puriﬁed by column chromato-

graphy (dichloromethane elution) to give the title com-

pound 23 as a pale pink solid (66 mg, 87%), mp 144–

146 ^ꢄC (from ethyl acetate/light petroleum), (Found: C,

.

62.7; H, 3.6; N, 7.2. C₁₀H₇NO₃.0.1 H₂O requires C,

62.9; H, 3.8; N, 7.3%); (Found: M⁺, 189.0424. C₁₀H₇NO₃

requires 189.0426); l_max(MeOH)/nm 242 (log e 4.36),

320 (3.12); n_max(Nujol)/cm^ꢁ13354, 1680, 1660, 1588;

d_H(300 MHz; CDCl₃) 2.39 (1H, br), 4.96 (2H, br),

7.06 (1H, d, J=10.5 Hz), 7.15 (1H, d, J=10.5 Hz),

8.43–8.44 (1H, m), 9.03 (1H, d, J=2.1 Hz); d_C

(100 MHz; CDCl₃) 60.9 (CH₂), 128.0, 132.0 (CH), 138.1

(CH), 139.1 (CH), 143.1, 146.1, 153.0 (CH), 183.3 (C),

185.8 (C); m/z (EI) 189 (M⁺, 100%), 160 (60), 133 (51),

104 (16), 91 (63).

(c) Cerium(IV) ammonium nitrate (0.4 g, 0.8 mmol)

was added to a solution of 5,8-dimethoxy-3-hydroxy-

methyl-2-phenylquinoline 61 (0.1 g, 0.34 mmol) in aceto-

nitrile (10 mL) and water (6 mL). The mixture was stirred

at room temperature for 1.5 h. The mixture was diluted

with water and extracted with dichloromethane. The

combined organic phases were dried (Na₂SO₄) and

concentrated in vacuo. The crude product was puriﬁed

by column chromatography (dichloromethane/ethyl

acetate (gradient) elution) to give the title compound 24

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1683

as a pale yellow solid (51 mg, 57%), mp 185–187 ^ꢄC

(from ethyl acetate/light petroleum), (Found: M⁺,

265.0741. C₁₆H₁₁NO₃requires 265.0739); l_max

(MeOH)/nm 252 (log e 4.38), 344 (3.30); n_max(Nujol)/

cm^ꢁ13308, 1675, 1669, 1582; d_H(400 MHz; DMSO)

4.62 (2H, d, J=4.4 Hz), 7.14 (1H, d, J=10.4 Hz), 7.18

(1H, d, J=10.4 Hz), 7.52–7.53 (3H, m), 7.60–7.62 (2H,

m), 8.53 (1H, s); OH not observed; d_C(100 MHz;

DMSO) 60.4 (CH₂), 128.0 (C), 128.7 (CH), 129.4 (CH),

129.7 (CH), 133.6 (CH), 138.3 (CH), 138.5 (C), 139.8

(CH), 140.9 (C), 145.8 (C), 161.2 (C), 183.5 (C), 185.4

(C); m/z (EI) 265 (M⁺, 86%), 264 (68), 133 (42), 91

(100).

at room temperature for 1.5 h. The mixture was diluted

with water and extracted with dichloromethane (3ꢅ5

mL). The combined organic phases were dried (Na₂SO₄)

and concentrated in vacuo. The crude product was

puriﬁed by column chromatography (dichloromethane/

ethyl acetate (gradient) elution) to give the title com-

pound 25 as a yellow solid (0.05 g, 94%), mp 148–

149 ^ꢄC, (Found; M⁺, 271.0312. C₁₄H₉NO₃S requires

271.0303); l_max(MeOH)/nm 292 (log e 4.02), 324 (3.99),

388 (3.48); n_max(KBr)/cm^ꢁ13426, 1669, 1665, 1608,

1577; d_H(400 MHz; DMSO-d₆) 4.87 (2H, d, J=5.4 Hz),

5.87 (1H, t, J=5.4 Hz), 7.11 (1H, d, J=10.4 Hz), 7.16

(1H, d, J=10.4 Hz), 7.25 (1H, dd, J=5.1, 3.8 Hz), 7.73

(1H, dd, J=3.8, 1.0 Hz), 7.86 (1H, dd, J=5.1, 1.0 Hz),

8.53 (1H, s); d_C(100 MHz; DMSO-d₆) 61.0 (CH₂), 127.1

(C), 129.3 (CH), 130.6 (CH), 131.9 (CH), 133.9 (CH),

138.4 (CH), 138.9 (C), 139.7 (CH), 143.0 (C), 145.8 (C),

153.6 (C), 183.2 (C), 185.1 (C); m/z (EI) 271 (M⁺,

100%), 242 (32), 238 (31), 210 (31).

3.3.12. 3-Hydroxymethyl-2-(thien-2-yl)quinoline-5,8-dione

(25). (a) A solution of 2-chloro-5,8-dimethoxyquino-

line-3-carbaldehyde 57 (0.1 g, 0.40 mmol) in 1,2-di-

methoxyethane (10 mL) was degassed under reduced

pressure and ﬂushed with nitrogen. Pd(PPh₃)₄(50 mg,

0.05 mmol) was added and the system was degassed

again. The mixture was stirred for 10 min. Sodium car-

bonate solution (2 M; 1 mL) was added followed by

thiophene-2-boronic acid (0.06 g, 0.50 mmol) and the

mixture was heated under reﬂux for 19 h. After cooling,

the mixture was poured into dichloromethane (10 mL)

and washed with water (3ꢅ5 mL). The organic phase

was dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

(dichloromethane elution) to give 5,8-dimethoxy-2-

(thien-2-yl)quinoline-3-carbaldehyde 62 as a yellow

solid (0.08 g, 69%), used without further puriﬁcation,

3.3.13. 2-(Benzofuran-2-yl)-3-hydroxymethylquinoline-

5,8-dione (26). (a) A solution of 2-chloro-5,8-dimethoxy-

quinoline-3-carbaldehyde 57 (0.1 g, 0.40 mmol) in 1,2-

dimethoxyethane (15 mL) was degassed under reduced

pressure and ﬂushed with nitrogen. Pd(PPh₃)₄(47 mg,

0.05 mmol) was added and the system was degassed

again. The mixture was stirred for 10 min. Sodium car-

bonate solution (2 M; 1 mL) was added followed by

benzofuran-2-boronic acid (0.08 g, 0.50 mmol) and the

mixture was heated under reﬂux for 19 h. After cooling,

the mixture was poured into dichloromethane (10 mL)

and washed with water (3ꢅ10 mL). The organic phase

was dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

(dichloromethane elution) to give 2-(benzofuran-2-yl)-

5,8-dimethoxyquinoline-3-carbaldehyde 64 as a yellow

solid (113 mg, 85%), used without further puriﬁcation,

n

_max(KBr)/cm^ꢁ11670, 1608, 1567; d_H(300 MHz;

CDCl₃) 3.98 (3H, s), 4.04 (3H, s), 6.75 (1H, d, J=8.5

Hz), 7.06 (1H, d, J=8.5 Hz), 7.19 (1H, dd, J=5.2, 3.6

Hz), 7.36 (1H, dd, J=3.6, 1.1 Hz), 7.59 (1H, dd, J=5.2,

1.1 Hz), 9.15 (1H, s), 10.45 (1H, s), d_C(100 MHz;

CDCl₃) 55.9 (Me), 56.5 (Me), 104.4 (CH), 111.2 (CH),

119.7 (C), 126.8 (C), 128.0 (CH), 129.7 (CH), 130.9

(CH), 134.0 (CH), 141.4 (C), 141.5 (C), 149.0 (C), 149.9

(C), 152.4 (C), 191.2 (CH).

n

_max(KBr)/cm^ꢁ11680, 1613, 1577; d_H(300 MHz;

CDCl₃) 4.00 (3H, s), 4.08 (3H, s), 6.80 (1H, d, J=8.5

Hz), 7.10 (1H, d, J=8.5 Hz), 7.32 (1H, ddd, J=7.7, 7.4,

0.8 Hz), 7.41 (1H, ddd, J=8.2, 7.4, 1.4 Hz), 7.62 (1H,

dd, J=8.2, 0.8 Hz), 7.68 (1H, d, J=0.8 Hz), 7.73 (1H,

dd, J=7.7, 0.8 Hz), 9.23 (1H, s), 10.88 (1H, s); d_C

(100 MHz; CDCl₃) 55.8 (Me), 56.3 (Me), 104.8 (CH),

110.2 (CH), 110.8 (CH), 111.8 (CH), 120.0 (C), 122.1

(CH), 123.5 (CH), 125.9 (CH), 127.1 (C), 128.2 (C),

133.8 (CH), 141.3 (C), 147.7 (C), 149.0 (C), 149.8 (C),

154.0 (C), 155.9 (C), 191.5 (CH).

(b) Sodium borohydride (4 mg, 0.10 mmol) was added

to a solution of 5,8-dimethoxy-2-(thien-2-yl)quinoline-3-

carbaldehyde 62 (77 mg, 0.26 mmol) in 1,2-dimethoxy-

ethane (15 mL) and the mixture was heated to reﬂux for

2 h. After cooling, water was added followed by HCl

(2 M). The mixture was extracted with ether (3ꢅ5 mL)

and the combined organic phases were dried (Na₂SO₄)

and concentrated in vacuo. The crude product was

puriﬁed by column chromatography (dichloromethane/

ethyl acetate (gradient) elution) to give 5,8-dimethoxy-

2-(thien-2-yl)quinoline-3-methanol 63 as a colorless

solid (62 mg, 79%), used without further puriﬁcation,

d_H(300 MHz; CDCl₃) 2.66 (1H, br), 3.90 (3H, s), 4.02

(3H, s), 4.95 (2H, s), 6.64 (1H, d, J=8.5 Hz), 6.87 (1H,

d, J=8.5 Hz), 7.11 (1H, dd, J=5.2, 3.8 Hz), 7.47 (1H,

dd, J=5.2, 1.1 Hz), 7.60 (1H, dd, J=3.8, 1.1 Hz), 8.54

(1H, s).

(b) Sodium borohydride (0.007 g, 0.19 mmol) was added

to

a solution of 2-benzofuran-2-yl-5,8-dimethoxy-

quinoline-3-carbaldehyde 64 (0.1 g, 0.34 mmol) in 1,2-

dimethoxyethane (20 mL) and the mixture was heated

to reﬂux for 2 h. After cooling water was added fol-

lowed by HCl (2 M). The mixture was extracted with

ether (3ꢅ10 mL) and the combined organic phases were

dried (Na₂SO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography [di-

chloromethane/ethyl acetate (gradient) elution] to give

2-(benzofuran-2-yl)-5,8-dimethoxyquinoline-3-methanol

65 as a colorless solid (0.1 g, 92%), used without further

puriﬁcation, n_max(KBr)/cm^ꢁ13400, 1598, 1557; d_H

(300 MHz; CDCl₃) 3.00 (1H, br), 3.88 (3H, s), 4.04 (3H,

(c) Cerium(IV) ammonium nitrate (0.2 g, 0.49 mmol)

was added to a solution of 5,8-dimethoxy-2-(thien-2-

yl)quinoline-3-methanol 63 (0.06 g, 0.21 mmol) in aceto-

nitrile (6 mL) and water (4 mL). The mixture was stirred

1684

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

s), 5.11 (2H, s), 6.65 (1H, d, J=8.5 Hz), 6.87 (1H, d,

J=8.5 Hz), 7.26 (1H, ddd, J=7.4, 7.1, 1.1 Hz), 7.34

(1H, ddd, J=8.2, 7.1, 1.1 Hz), 7.56 (1H, dd, J=8.2, 0.8

Hz), 7.61 (1H, d, J=0.8 Hz), 7.65 (1H, dd, J=7.4, 0.8

Hz), 8.61 (1H, s); d_C(100 MHz; CDCl₃) 55.6 (Me), 56.1

(Me), 63.1 (CH₂), 104.0 (CH), 107.2 (CH), 108.9 (CH),

111.7 (CH), 120.5 (C), 122.8 (CH), 123.2 (CH), 125.4

(CH), 128.4 (C), 131.2 (CH), 131.5 (C), 139.2 (C), 146.5

(C), 148.4 (C), 149.0 (C), 154.7 (C), 155.2 (C). No fur-

ther characterisation was carried out, and the com-

pound was used directly in the next step.

(b) Cerium(IV) ammonium nitrate (90 mg, 0.18 mmol)

was added to a solution of 3-acetoxymethyl-5,8-di-

methoxy-2-phenylquinoline 66 (27 mg, 0.08 mmol) in

acetonitrile (2.3 mL) and water (1.4 mL). The reaction

mixture was stirred for 1.5 h, diluted with water and

extracted with dichloromethane (3ꢅ3 mL). The com-

bined organic phases were dried (Na₂SO₄) and con-

centrated in vacuo. The crude product was puriﬁed by

column chromatography [dichloromethane/ethyl acetate

(9:1) elution] to give the title compound 27 as a yellow

solid (24 mg, 98%), mp 141–143 ^ꢄC, (Found: MH⁺,

308.0922. C₁₈H₁₃NO₄+H requires 308.0923); l_max

(MeCN)/nm 265 (log e 4.20), 348 (3.44); n_max(KBr)/

cm^ꢁ11730, 1665, 1585, 1432; d_H(300 MHz; CDCl₃) 2.17

(3H, s), 5.31 (2H, s), 7.10 (1H, d, J=10.4 Hz), 7.19 (1H,

d, J=10.4 Hz), 7.49–7.54 (3H, m), 7.57–7.61 (2H, m),

8.55 (1H, s); d_C(75 MHz; CDCl₃) 20.8 (Me), 62.7

(CH₂), 127.5 (C), 128.7 (CH), 129.0 (CH), 130.0 (CH),

134.8 (C), 134.9 (CH), 137.7 (C), 138.0 (CH), 139.3

(CH), 146.2 (C), 163.4 (C), 170.3 (C), 182.9 (C), 184.4

(C); m/z (ESI) 330 ([M+Na]⁺, 100%), 308 (MH⁺,

58%), 271 (12), 239 (48), 151 (27).

(c) Cerium(IV) ammonium nitrate (0.4 g, 0.73 mmol)

was added to a solution of 2-(benzofuran-2-yl)-5,8-

dimethoxyquinoline-3-methanol 65 (0.1 g, 0.31 mmol)

in acetonitrile (10 mL) and water (6 mL). The mixture

was stirred at room temperature for 1.5 h and then

diluted with water and extracted with dichloromethane

(3ꢅ10 mL). The combined organic phases were dried

(Na₂SO₄) and concentrated in vacuo. The crude product

was puriﬁed by column chromatography (dichloro-

methane/ethyl acetate (9:1) elution) to give the title

compound 26 as a yellow solid (0.04 g, 39%), mp 197–

198 ^ꢄC (Found; M⁺, 305.0689. C₁₈H₁₁NO₄requires

305.0688); l_max(MeOH)/nm 300 (log e 4.09), 338 (4.04),

390 (3.61); n_max(KBr)/cm^ꢁ13472, 1680, 1654, 1577; d_H

(400 MHz; DMSO-d₆) 5.07 (2H, d, J=4.9 Hz), 5.86

(1H, t, J=5.5 Hz), 7.15 (1H, d, J=10.4 Hz), 7.20 (1H,

d, J=10.4 Hz), 7.21 (1H, ddd, J=7.4, 7.1, 1.1 Hz), 7.34

(1H, ddd, J=8.2, 7.1, 1.1 Hz), 7.74–7.76 (2H, m), 7.81

(1H, dd, J=7.4, 0.8 Hz), 8.61 (1H, s); d_C(100 MHz;

DMSO-d₆) 60.4 (CH₂), 111.2 (CH), 112.2 (CH), 122.9

(CH), 124.2 (CH), 126.9 (CH), 127.9 (C), 128.2 (C),

133.4 (CH), 138.5 (CH), 139.8 (CH), 140.7 (C), 146.1

(C), 149.1 (C), 153.8 (C), 155.4 (C), 183.2 (C), 185.0 (C);

m/z (FI) 305 (M⁺, 100%).

3.3.15. 3-Acetoxymethyl-2-(thien-2-yl)quinoline-5,8-dione

(28). (a) DMAP (a few crystals) was added to a solu-

tion of the alcohol (51 mg, 0.17 mmol) in pyridine (1

mL, 12 mmol) and acetic anhydride (1 mL, 10 mmol).

The reaction mixture was stirred at room temperature

for 3 days. Water was added and the mixture was

extracted with ethyl acetate (3ꢅ3 mL). The combined

organic phases were washed with CuSO₄(10% solu-

tion), dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

[dichloromethane/ethyl acetate (9:1) elution] to yield 3-

acetoxymethyl-5,8-dimethoxy-2-(thien-2-yl)quinoline 67

as a yellow solid (54 mg, 92%), mp 116–117 ^ꢄC, (Found:

MH⁺, 344.0962. C₁₈H₁₇NO₄S+H requires 344.0956);

3.3.14.

3-Acetoxymethyl-2-phenylquinoline-5,8-dione

n

_max(KBr)/cm^ꢁ11723, 1600, 1571; d_H(300 MHz;

(27). (a) DMAP (a few crystals) was added to a

solution of 5,8-dimethoxy-2-phenylquinoline-3-metha-

nol 61 (50 mg, 0.17 mmol) in pyridine (1 mL, 12 mmol)

and acetic anhydride (1 mL, 10 mmol). The reaction

mixture was stirred at room temperature for 3 days.

Water was added and the mixture was extracted with

ethyl acetate (3ꢅ3 mL). The combined organic phases

were washed with CuSO₄(10% solution), dried

(Na₂SO₄) and concentrated in vacuo. The crude product

was puriﬁed by column chromatography [dichloro-

methane/ethyl acetate (9:1) elution] to yield 3-acetoxy-

methyl-5,8-dimethoxy-2-phenylquinoline 66 as a yellow

solid (50 mg, 87%), mp 152–153 ^ꢄC, (Found: MH⁺,

CDCl₃) 2.18 (3H, s), 3.97 (3H, s), 4.04 (3H, s), 5.46 (2H,

s), 6.73 (1H, d, J=8.5 Hz), 6.94 (1H, d, J=8.5 Hz),

7.14–7.17 (1H, m), 7.49–7.56 (2H, m), 8.67 (1H, d,

J=0.5 Hz); d_C(75 MHz; CDCl₃) 21.1 (Me), 55.8 (Me),

56.5 (Me), 64.4 (CH₂), 103.9 (CH), 108.4 (CH), 120.0

(C), 125.6 (C), 127.80 (CH), 127.83 (CH), 128.8 (CH),

133.5 (CH), 139.8 (C), 143.7 (C), 148.6 (C), 149.2 (C),

151.4 (C), 170.7 (C); m/z (EI) 344 (MH⁺, 52%), 328

(32), 286 (100), 270 (90), 256 (35), 84 (63), 60 (100).

(b) Cerium(IV) ammonium nitrate (90 mg, 0.18 mmol)

was added to a solution of 3-acetoxymethyl-5,8-di-

methoxy-2-(thien-2-yl)quinoline 67 (27 mg, 0.08 mmol)

in acetonitrile (2.3 mL) and water (1.4 mL). The reac-

tion mixture was stirred for 1.5 h, diluted with water

and extracted with dichloromethane (3ꢅ3 mL). The

combined organic phases were dried (Na₂SO₄) and

concentrated in vacuo. The crude product was puriﬁed

by column chromatography [dichloromethane/ethyl

acetate (9:1) elution] to give the title compound 28 as a

yellow solid (24 mg, 95%), mp 147–148 ^ꢄC, (Found:

M⁺, 313.0409. C₁₆H₁₁NO₄S requires 313.0409); l_max

(MeCN)/nm 320 (log e 3.98), 376 (3.59); n_max(KBr)/

cm^ꢁ11745, 1724, 1665, 1578, 1418; d_H(400 MHz;

CDCl₃) 2.21 (3H, s), 5.46 (2H, s), 7.06 (1H, d, J=10.4

338.1396.

n

C₂₀H₁₉NO₄+H

requires

338.1392);

_max(KBr)/cm^ꢁ11738, 1607, 1469; d_H(300 MHz;

CDCl₃) 2.11 (3H, s), 4.00 (3H, s), 4.03 (3H, s), 5.24 (2H,

s), 6.79 (1H, d, J=8.5 Hz), 6.98 (1H, d, J=8.5 Hz),

7.45–7.47 (3H, m), 7.62–7.65 (2H, m), 8.67 (1H, d,

J=0.5 Hz); d_C(75 MHz; CDCl₃) 21.0 (Me), 55.8 (Me),

56.2 (Me), 64.1 (CH₂), 103.9 (CH), 107.7 (CH), 120.4

(C), 127.1 (C), 128.3 (CH), 128.6 (CH), 129.2 (CH),

132.3 (CH), 139.3 (C), 148.6 (C), 149.5 (C), 158.8 (C),

170.6 (C); one C unobserved; m/z (EI) 338 (MH⁺,

32%), 322 (20), 280 (28), 264 (22), 174 (20), 105 (50), 91

(100).

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

1685

Hz), 7.14 (1H, d, J=10.4 Hz), 7.20 (1H, dd, J=3.8, 5.1

Hz), 7.59 (1H, dd, J=0.8, 3.8 Hz), 7.63 (1H, dd, J=0.8,

5.1 Hz), 8.52 (1H, s); d_C(100 MHz; CDCl₃) 20.9 (Me),

63.0 (CH₂), 126.6 (C), 128.5 (CH), 129.7 (CH), 131.5

(CH), 132.2 (C), 135.4 (CH), 138.1 (CH), 139.1 (CH),

141.9 (C), 146.1 (C), 155.6 (C), 170.4 (C), 182.5 (C),

184.1 (C); m/z (EI) 313 (M⁺, 53%), 271 (47), 255 (77),

254 (100).

dichloromethane (30 mL) was treated dropwise with

benzoyl chloride (1.0 mL, 8.4 mmol). After 5 h, triethyl-

amine (0.8 mL, 5.6 mmol) was added and stirring was

continued for another 1.5 h, additional triethylamine

(0.4 mL, 2.8 mmol) was then added. After 1 h, water (20

mL) was added and the mixture was extracted with

ether (3ꢅ20 mL), washed with HCl (1 M) and saturated

NaHCO₃solution, dried (Na₂SO₄) and concentrated in

vacuo. The crude product was puriﬁed by column

chromatography (dichloromethane elution) to give N-

[2-(3-methoxy-2-benzyloxyphenyl)-2-methoxyethyl]benz-

amide 70 as a yellow oil (1.0 g, 43%), (Found: M⁺,

391.1801. C₂₄H₂₅NO₄requires 391.1783); n_max(ﬁlm)/

cm^ꢁ13345, 3061, 3032, 2930, 2829, 1709, 1658, 1520; d_H

(300 MHz; CDCl₃) 3.15 (3H, s), 3.49 (1H, ddd, J=4.1,

7.5, 11.7 Hz), 3.79–3.90 (1H, m), 3.92 (3H, s), 4.70 (1H,

dd, J=4.5, 7.5 Hz), 5.11 (2H, d, J=2.4 Hz), 6.63 (1H,

br), 6.92 (1H, dd, J=1.5, 7.9 Hz), 6.98 (1H, dd, J=1.3,

7.9 Hz), 7.11 (1H, dd, J=7.9, 7.9 Hz), 7.26–7.75 (9H,

m), 8.09–8.13 (1H, m); d_C(100 MHz; CDCl₃) 45.7 (CH),

56.2 (CH₂), 57.0 (Me), 57.2 (Me), 75.4 (OCH₂), 112.4

(CH), 119.0 (CH), 124.9 (CH), 127.3 (CH), 127.4 (CH),

128.5 (CH), 128.7 (C), 128.8 (CH), 128.9 (CH), 128.94

(C), 128.97 (C), 129.0 (CH), 130.5 (CH), 131.63 (C),

133.9 (C), 137.9 (CH), 146.1 (CH), 153.1 (CH), 167.7

(C); m/z (EI) 391 (M⁺, 17%), 362 (53), 361 (36), 360

(100), 257 (52), 239 (15).

3.3.16. 3-Acetoxymethyl-2-(benzofuran-2-yl)quinoline-

5,8-dione (29). (a) DMAP (a few crystals) was added to

a solution of the alcohol (57 mg, 0.17 mmol) in pyridine

(1 mL, 12 mmol) and acetic anhydride (1 mL, 10 mmol).

The reaction mixture was stirred at room temperature

for 3 days. Water was added and the mixture was

extracted with ethyl acetate (3ꢅ3 mL). The combined

organic phases were washed with CuSO₄(10% solu-

tion), dried (Na₂SO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography

[dichloromethane/ethyl acetate (9:1) elution] to yield 3-

acetoxymethyl-2-(benzofuran-2-yl)-5,8-dimethoxyquino-

line 68 as a yellow solid (47 mg, 74%), mp 157–158 ^ꢄC,

(Found: MH⁺, 378.1342. C₂₂H₁₉NO₅+H requires

378.1341);

n

_max(KBr)/cm^ꢁ11723, 1593, 1461; d_H

(300 MHz; CDCl₃) 2.19 (3H, s), 4.01 (3H, s), 4.09 (3H,

s), 5.71 (2H, s), 6.81 (1H, d, J=8.5 Hz), 6.99 (1H, d,

J=8.5 Hz), 7.27–7.30 (2H, m), 7.32–7.42 (1H, m), 7.63

(1H, d, J=6.5 Hz), 7.71 (1H, d, J=6.5 Hz), 8.71 (1H,

s); d_C(75 MHz; CDCl₃) 21.1 (Me), 55.8 (Me), 56.2 (Me),

64.3 (CH₂), 104.5 (CH), 107.8 (CH), 108.8 (CH), 111.9

(CH), 120.4 (C), 121.8 (CH), 123.2 (CH), 125.4 (CH),

126.4 (C), 128.4 (C), 132.9 (CH), 139.7 (C), 147.0 (C),

148.5 (C), 149.3 (C), 154.5 (C), 155.5 (C), 170.8 (C); m/z

(EI) 377 (M⁺, 72%), 362 (100), 304 (42), 91 (58), 84

(72).

(b) POCl₃(4 mL) was added slowly to a solution of N-

[2-(3-methoxy-2-benzyloxyphenyl)-2-methoxyethyl]benz-

amide 70 (0.9 g, 2.3 mmol) in toluene (15 mL) and the

mixture was heated to 90 ^ꢄC. After 6 h, ice was added

and the mixture was stirred for 0.5 h. The mixture was

extracted with ether (3ꢅ10 mL). The aqueous layer

was basiﬁed with NaOH solution (2 M) to pH 10 and

extracted with chloroform (3ꢅ10 mL), dried (Na₂SO₄)

and concentrated in vacuo to give a mixture of 5-benz-

yloxy-6-methoxy-1-phenylisoquinoline and 6-methoxy-

1-phenylisoquinolin-5-ol 71. The mixture was used

without further puriﬁcation. The mixture (0.2 g) was

dissolved in MeOH–HCl (6 M; 5 mL) and heated to

reﬂux overnight. After cooling, the mixture was neu-

tralized with NaOH solution (2 M), extracted with

chloroform (3ꢅ10 mL), dried (Na₂SO₄) and con-

centrated in vacuo. The crude product was puriﬁed by

column chromatography [dichloromethane/ethyl acetate

(8:2) elution] to give 6-methoxy-1-phenylisoquinolin-5-ol

71 as a yellow solid (0.06 g, 11%), d_H(300 MHz;

CDCl₃) 3.95 (3H, s), 7.18 (1H, d, J=9.2 Hz), 7.39–7.46

(3H, m), 7.57–7.62 (3H, m), 7.86 (1H, d, J=5.8 Hz),

8.45 (1H, d, J=5.8 Hz).

(b) Cerium (IV) ammonium nitrate (90 mg, 0.18 mmol)

was added to a solution of 3-acetoxymethyl-2-(benzo-

furan-2-yl)-5,8-dimethoxyquinoline 68 (30 mg, 0.08

mmol) in acetonitrile (2.3 mL) and water (1.4 mL). The

reaction mixture was stirred for 1.5 h, diluted with

water and extracted with dichloromethane (3ꢅ3 mL).

The combined organic phases were dried (Na₂SO₄) and

concentrated in vacuo. The crude product was puriﬁed

by column chromatography [dichloromethane/ethyl

acetate (9:1) elution] to give the title compound 29 as a

yellow solid (26 mg, 93%), mp 224 ^ꢄC, (Found: M⁺,

347.0802. C₂₀H₁₃NO₅requires 347.0794); l_max

(MeCN)/nm 348 (log e 4.27), 390 (3.99); n_max(KBr)/

cm^ꢁ11738, 1665, 1600, 1563; d_H(400 MHz; CDCl₃) 2.23

(3H, s), 5.77 (2H, s), 7.08 (1H, d, J=10.4 Hz), 7.17 (1H,

d, J=10.4 Hz), 7.32–7.34 (1H, m), 7.40–7.44 (1H, m),

7.59 (1H, d, J=8.0 Hz), 7.72 (1H, d, J=8.0 Hz), 7.89

(1H, s), 8.52 (1H, s); d_C(100 MHz; CDCl₃) 21.0 (Me),

62.9 (CH₂), 111.9 (CH), 112.1 (CH), 122.5 (CH), 123.9

(CH), 126.7 (CH), 127.1 (C), 127.7 (C), 133.4 (C), 134.8

(CH), 138.3 (CH), 139.1 (CH), 146.3 (C), 150.8 (C),

153.7 (C), 156.0 (C), 170.5 (C), 182.6 (C), 183.9 (C); m/z

(EI) 347 (M⁺, 19%), 289 (100), 260 (44), 207 (27).

(c) A solution of 6-methoxy-1-phenylisoquinolin-5-ol 71

(0.06 g, 0.25 mmol) in methanol (30 mL) was added to a

stirred solution of Fremy’s salt (0.9 g, 3.2 mmol) in

dihydrogen phosphate buﬀer (pH 6, 0.3 M; 30 mL). The

solution was stirred at room temperature overnight and

diluted with water and ﬁltered. The ﬁltrate was extrac-

ted with chloroform (3ꢅ15 mL), dried (Na₂SO₄) and

concentrated in vacuo. The crude product was puriﬁed

by column chromatography [dichloromethane/ethyl

acetate (9:1) elution] to give the title compound 30 as a

yellow solid (0.02 g, 35%), mp 220–221 ^ꢄC, (Found:

3.3.17. 6-Methoxy-1-phenylisoquinoline-5,8-dione (30).

(a) A stirred mixture of N-[2-(3-methoxy-2-benzyloxy-

phenyl)-2-methoxyethyl]amine 69⁶¹(1.6 g, 5.6 mmol) in

1686

T. Fryatt et al. / Bioorg. Med. Chem. 12 (2004) 1667–1687

MꢁH⁺, 264.0658. C₁₆H₁₁NO₃-H requires 264.0661);

quantiﬁed using a modiﬁcation of an assay that uses

cytochrome c as the terminal electron acceptor.²¹Reac-

tion mixtures contained 1 mMNADH (Sigma), 25 mM

quinolinequinone, 70 mMcytochrome c (Sigma) and

0.1–3.0 mg/mL hNQO1 in 25 mMTris–HCl (pH 7.4)

with 0.07% BSA and 0.1% Tween-20. Reactions were

run at least in triplicate at 22 ^ꢄC in a Beckman DU 7500

spectrophotometer at 550 nm (molar absorptivity 21.1

mM^ꢁ1cm^ꢁ1for cytochrome c). Initial reduction rates

(mmol cytochrome c reduced/min/mg NQO1) were cal-

culated from the linear portion (0–30 s) of the reaction

curves.

l_max(MeCN)/nm 280 (log

e 3.95), 335 (3.60);

n

_max(KBr)/cm^ꢁ13432, 3062, 1694, 1650, 1614, 1546; d_H

(300 MHz; CDCl₃) 3.91 (3H, s), 6.12 (1H, d, J=4.9 Hz),

7.46 (5H, s), 7.97 (1H, d, J=4.9 Hz), 9.03 (1H, d, J=4.9

Hz); d_C(100 MHz; CDCl₃) 56.5 (Me), 111.8 (CH), 118.0

(CH), 122.9 (C), 128.0 (CH), 128.7 (CH), 128.8 (CH),

138.4 (C), 140.1 (C), 153.3 (CH), 159.0 (C), 160.5 (C),

179.7 (C), 184.1 (C); m/z (EI) 264 (MꢁH⁺, 56%), 236

(64), 222 (47), 166 (24), 139 (34), 126 (38), 77 (45), 69

(100).

3.4. Electrochemical measurements

3.5.3. Cytotoxicity assay. Cytotoxicity was determined

using a MTT colorimetric assay. Cells were plated in 96-

well plates at a density of 1–2ꢅ10⁴cells/mL and allowed

to attach overnight (16 h). Quinolinequinone solutions

were applied in medium for 2 h. Quinolinequinone

solutions were removed and replaced with medium

alone, and the 96-well plates were incubated for 5–7

days. MTT (Sigma) was added to each well (50 mg), and

the cells were incubated for another 4 h. Medium/MTT

solutions were removed carefully by aspiration, the

MTT formazan crystals were dissolved in 100 mL

DMSO and absorbance was determined on a plate

reader at 550 nm. Each compound was run in triplicate,

and IC₅₀values (concentration at which cell survival

equals 50% of control) were determined from plots of

percent of control versus concentration.⁷⁴

Tetrabutylammonium tetraﬂuoroborate (Bu₄NBF₄)

(Aldrich, 99%) was dried under vacuum at 70 ^ꢄC. Fer-

rocene (Aldrich, 97%) was resublimed. DMF (Aldrich,

99.8%) was used as solvent.

For each of the quinolinequinones studied, cyclic vol-

tammograms were recorded over a range of potential

sweep rates from 50 to 500 mV s^ꢁ1, using a BAS 100B

Electrochemical Analyzer. A three-electrode cell was

employed, with a platinum counter electrode and a Ag/

AgCl in 3 MNaCl reference electrode (BAS). This

reference was separated from the main compartment of

the cell by a salt bridge containing DMF solvent and

Bu₄NBF₄supporting electrolyte. The reference, plus

bridge, was calibrated by voltammetry relative to E_1/2

for ferrocene (Fc^+/0) in DMF/Bu₄NBF₄, to allow the

measured E_1/2values for the quinolinequinones to be

quoted relative to Fc^+/0. The working electrode was a 3

mm diameter platinum disc (BAS), pretreated by pol-

ishing with 1 mm diamond paste, followed by voltam-

Acknowledgements

We acknowledge support from Cancer Research UK,

The University of Exeter, The Millhayes Trust, USPHS

NIH Grant R15 CA78232, the EPSRC Mass Spectro-

metry Centre at Swansea for mass spectra, and the

EPSRC Chemical Database Service at Daresbury.

metric cycling in 0.1 MH SO₄until the characteristic

2

voltammogram for polycrystalline platinum was

achieved, before ﬁnally rinsing in distilled water and

drying. Solutions for voltammetry were all 1 mMin

quinolinequinone and 0.1 Min Bu ₄NBF₄in DMF.

Oxygen was removed from the solutions by purging

with nitrogen. All measurements were performed at

room temperature.

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Article Doi

Novel quinolinequinone antitumor agents: Structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1)

DOI: 10.1016/j.bmc.2004.01.021

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