Indolequinone antitumor agents: Correlation between quinone structure, rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase, and in vitro cytotoxicity

DOI: 10.1021/jm980328r

Source and publish data:

Journal of Medicinal Chemistry p. 4755 - 4766 (1998)

Update date:2022-08-05

Topics:

Authors:

Beall, Howard D.

Winski, Shannon

Swann, Elizabeth

Hudnott, Anna R.

Cotterill, Ann S.

O'Sullivan, Noeleen

Green, Stephen J.

Bien, Richard

Siegel, David

Ross, David

Moody, Christopher J.

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Article abstract of DOI:10.1021/jm980328r

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H: quinone oxidoreductase (NQO1) were studied. Thus 5-methoxyindolequinones wer

Full text of DOI:10.1021/jm980328r

J . Med. Chem. 1998, 41, 4755-4766

4755

In d olequ in on e An titu m or Agen ts: Cor r ela tion betw een Qu in on e Str u ctu r e,

Ra te of Meta bolism by Recom bin a n t Hu m a n NAD(P )H:Qu in on e Oxid or ed u cta se,

a n d in Vitr o Cytotoxicity¹

Howard D. Beall,^†,∇Shannon Winski,^†Elizabeth Swann,^§Anna R. Hudnott,^§Ann S. Cotterill,^‡

Noeleen O’Sullivan,^‡Stephen J . Green,^§Richard Bien,^§David Siegel,^†David Ross,*^,†and Christopher J . Moody*^,§

School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Box C-238, 4200 East Ninth Avenue,

Denver, Colorado 80262, School of Chemistry, University of Exeter, Stocker Road, Exeter, Devon EX4 4QD, U.K., and

Department of Chemistry, Loughborough University, Loughborough, Leicestershire LE11 3TU, U.K.

Received May 27, 1998

A series of indolequinones bearing various functional groups has been synthesized, and the

effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:

quinone oxidoreductase (NQO1) were studied. Thus 5-methoxyindolequinones were prepared

by the Nenitzescu reaction, followed by functional group interconversions. The methoxy group

was subsequently displaced by amine nucleophiles to give a series of amine-substituted

quinones. Metabolism of the quinones by NQO1 revealed that, in general, compounds with

electron-withdrawing groups at the indole 3-position were among the best substrates, whereas

those with amine groups at the 5-position were poor substrates. Compounds with a leaving

group at the 3-indolyl methyl position generally inactivated the enzyme. The toxicity toward

non-small-cell lung cancer cells with either high NQO1 activity (H460) or no detectable activity

(H596) was also studied in representative quinones. Compounds which were good substrates

for NQO1 showed the highest selectivity between the two cell lines.

Ch a r t 1

In tr od u ction

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.^2,3The enzyme, a

flavoprotein, exists as a homodimer with 1 mol of FAD/

mol of NQO1,⁴and the human enzyme has been cloned

and sequenced.⁵The crystal structure of the rat liver

enzyme, which shows about 85% homology with human

NQO1, has been determined to 2.1-Å resolution.⁶

NQO1 catalyzes the 2-electron reduction of quinones

and can protect cells against the toxic effects of quino-

nes. However NQO1 is also involved in the reductive

activation of anticancer agents such as mitomycin C

(MMC) (1) (Chart 1) which operate by the so-called

bioreductive mechanism,^7-9and although other en-

zymes have also been implicated in the reductive

bioactivation process,⁹NQO1 has generated consider-

able interest because of its elevated levels in many

tumors and tumor cell lines.^8,10

It has historically been problematic to identify those

compounds which are the most efficient substrates for

NQO1. One approach has been to attempt to correlate

reduction potentials with rates of reduction. A relation-

ship between 1-electron reduction potential and rates

of reduction by cytochrome P-450 reductase^11,12and

cytochrome b₅reductase¹²has been reported. For the

1-electron reductase, xanthine oxidase, Clarke et al.

determined that there was a good correlation between

1-electron reduction potentials and rates of reduction

of nitroimidazoles.^13,14However, when a similar ap-

proach was attempted for NQO1, no correlation was

found between reduction potential and rates of reduction

by NQO1 for a series of naphthoquinones¹⁵or a series

of aziridinylbenzoquinones.¹⁶In the naphthoquinone

study,¹⁵rates of reduction by NQO1 also did not

correlate with octanol-water partition coefficients which

further limits the ability to predict substrate efficiency

for NQO1.

* Corresponding authors for biology (D.R.) and chemistry (C.J .M.).

C. J . Moody: tel, 44 1392 263429; fax, 44 1392 263434; e-mail,

c.j.moody@exeter.ac.uk.

^†University of Colorado.

^§University of Exeter.

^‡Loughborough University.

The role of NQO1 in the bioactivation of MMC (1) is

somewhat controversial, although recently a clear cor-

^∇Present address: Department of Pharmaceutical Sciences, The

University of Montana, Missoula, MT 59812.

Published on Web 10/30/1998

4756 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24

Beall et al.

Sch em e 1^a

For compounds 7-11: a , Ar ) Ph; b, Ar ) 4-Ph-C₆H₄.

relation between NQO1 activity and MMC sensitivity

in human lung and breast cancer cell lines has been

demonstrated.¹⁷On the other hand, the importance of

NQO1 in the bioactivation of other cytotoxic indole-

quinones such as EO9 (2),^18-20aziridinylbenzoquinones

such as MeDZQ (3),^16,17and the novel cyclopropamito-

senes 4²¹has been established. However there has been

no systematic attempt to correlate quinone structure

with rate of metabolism by NQO1 and toxicity toward

human tumor cell lines, and therefore we report in

detail the results of such a study in a series of indole-

quinones 5 and mitosenes 6.¹

Of the new compounds, the indolequinone ester 5d

was obtained from commercially available ethyl 5-hy-

droxy-2-methylindole-3-carboxylate.²⁴Reduction of es-

ter 5d also provided an alternative route to the 3-(hy-

droxymethyl)indolequinone 5e, and reduction under

more forcing conditions gave the 3-methyl derivative 5f.

A range of indolequinones containing amino substitu-

ents was prepared by reaction of the corresponding

methoxy quinone with the appropriate amine, a sub-

stitution reaction widely used in the mitomycin se-

ries.^25,26Thus indolequinones 5g, 5h , and 5j-5s were

obtained from the 3-(hydroxymethyl)-5-methoxy-1,2-

dimethylindole-4,7-dione (5e) by reaction with the amine

in DMF. The quinone 5i was prepared similarly from

5d . The acetoxymethyl quinone 5t was prepared by

acetylation of the hydroxymethyl compound 5e,²⁴and

the (4-nitrophenoxy)methyl derivative 5u was obtained

by carrying out a Mitsunobu reaction on the hydroxy-

methyl compound 5e with 4-nitrophenol. Compound 5x

was prepared by oxidation of methyl 5,7-dimethoxy-1,3-

dimethylindole-2-carboxylate²⁷with ceric ammonium

nitrate.

Resu lts a n d Discu ssion

Ch em istr y. The synthesis of the 1,2-dimethylindole-

quinones 5a -5w and the mitosenes 6 has already been

developed in our laboratory, and the preparation of

compounds 5a -5c, 5e, 5v, 5w , 6a , 6b, 6d , 6e, and 6h -

6j has been described previously.^22,23Several new

compounds were included in the present study with the

aim of investigating the substituent effects at the 2-,

3-, and 5-positions. Hence a range of compounds

containing a greater variety of amine substituents at

C-5 was prepared, and the C-3 position was unsubsti-

tuted or substituted with methyl, hydroxymethyl, or

(carbamoyloxy)methyl groups. Compounds containing

electron-withdrawing groups (CHO or CO₂Et) were also

included in the study. Finally on the basis that strep-

tonigrin, an excellent substrate for NQO1,¹⁷contains a

bulky aromatic substituent at C-2 of the quinoline-

quinone, we prepared both 2-phenylindolequinones and

2-(4-biphenylyl)indolequinones for direct comparison

with their 2-methyl counterparts.

The 3-(hydroxymethyl)indolequinones 5y, 5z, 5d d ,

and 5ee containing an aromatic group at C-2 were

prepared as shown in Scheme 1. Thus Nenitzescu

reaction of the 3-amino-3-arylpropenoate 7 with 1,4-

benzoquinone gave the 5-hydroxyindole 8.²⁸Methyla-

tion of the hydroxy group and the indole nitrogen was

followed by nitration to give the 4-nitroindole 10, which

was readily reduced to the corresponding amino indole

11. The 4-aminoindole 11 was either directly oxidized

with Fremy’s salt (potassium nitrosodisulfonate) to give

the indolequinones 5y and 5d d or reduced with lithium

Indolequinone Antitumor Agents

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4757

Ta ble 1. Electrochemical Reduction Potentials^a(DMF) of

Indolequinones 5 and Mitosenes 6 versus Ferrocene

aluminum hydride and then oxidized with Fremy’s salt

to give the 3-(hydroxymethyl)indolequinones 5z and

5ee. The 2-phenylindolequinones 5a a -5cc with amine

substituents at C-5 were prepared from the 5-methoxy

compound 5z by reaction with the corresponding amine.

In the series of pyrrolo[1,2-a]indolequinones 6, the new

amine derivative 6c was prepared from 6a , 6f and 6g

from 6e, and 6k -6m from 6h . Compounds 5e, 5m , 5n ,

5z, and 5cc have also been studied independently by

another research group with a view to establishing their

activity under hypoxic conditions.^29,30

E_redox

(V) vs Fc

compd

R′

CHO

CO₂Et

OMe

aziridinyl

OMe

NHMe

NMe₂

NHCH₂CH₂OH

NHCH₂CH₂OMe

cyclopropylamino

aziridinyl

methylaziridinyl

azetidinyl

pyrrolidinyl

morpholinyl

N-methylpiperazinyl

OMe

-1.39^b

-1.19

-1.32

-1.28

-1.46

-1.40

-1.47

-1.45

-1.43

-1.26

-1.27

-1.43

-1.48

-1.31

-1.34

-1.36

-1.39^b

-1.37^b

-1.29

-1.23

-1.37

-1.22

-1.28

-1.22

-1.39

-1.61

-1.19

-1.29

-1.28

-1.50

-1.37

-1.38^b

-1.39^b

-1.59

-1.46

-1.53

Electrochemical studies were performed on a number

of the indolequinones using DMF as solvent and tetra-

n-butylammonium tetrafluoroborate as the supporting

electrolyte. E_redoxvalues, determined from the (at least)

quasi-reversible voltammograms recorded for the 1-elec-

tron reduction of the indolequinones, show little varia-

tion over the range of potential sweep rates used. Thus,

the E_redoxvalues given in Table 1 are averages of the

values determined from voltammograms recorded at

potential sweep rates of 25, 50, 100, 200, 300, 400, and

500 mV s^-1. The E_redoxvalues, tabulated with reference

to ferrocene (Fc), are shown in Table 1 and show

substituent effects in line with our previous observa-

tions.²³

CH₂OH

CO₂Et

CH₂OH

CH₂OAc

CH₂OCONH₂OMe

CH₂OCONH₂aziridinyl

CO₂Et

CH₂OH

Biology. Metabolism of the indolequinones 5 (Table

2) and mitosenes 6 (Table 3) by recombinant human

NQO1 was studied using an HPLC system designed to

quantify both NADH oxidation and quinone reduction.

NADH oxidation is irreversible in this assay and is

therefore used for comparison of reduction velocities. An

NQO1 inactivation assay was used to determine enzyme

activity remaining. The indolequinones were incubated

in the presence of NQO1 and NADH, and residual

enzyme activity was determined spectrophotometrically.

In the 1,2-dimethylindolequinone series 5a -5w , mea-

surable NADH oxidation was observed for compounds

5a -5s with the exception of the 5-pyrrolidinyl com-

pound 5p . Compounds 5b and 5m , which have an

aziridinyl substituent at the 5-position, were better

substrates for human NQO1 than the corresponding

compounds 5a and 5e, which have a methoxy group at

the 5-position. Compound 5n , the methylaziridinyl

analogue of 5m , was reduced at a slightly slower rate

than the aziridine 5m , though it was still a better

substrate than the methoxy compound 5e. Compounds

containing a basic nitrogen substituent at C-5 were poor

substrates for human NQO1, and one, the pyrrolidinyl

derivative 5p , was not metabolized at all. Reduction

rates for those compounds with hydroxymethyl substit-

uents at the 3-position, for example, 5e and 5m , were

somewhat lower than those for the corresponding un-

substituted derivatives, 5a and 5b.

5bb

5cc

5d d

5ee

CO₂Me

OMe

cyclopropylamino

methylaziridinyl

OMe

pyrrolidinyl

OMe

methylaziridinyl

pyrrolidinyl

4-Ph-C₆H₄CO₂Et

4-Ph-C₆H₄CH₂OH

CHO

CH₂OH

CH₂OCONH₂OMe

CH₂OCONH₂aziridinyl

CH₂OCONH₂methylaziridinyl

CH₂OCONH₂pyrrolidinyl

CH₂OCONH₂piperidinyl

CH₂OCONH₂cyclopropylamino

E_redox((0.005 V) values calculated as (E_pc+ E_pa)/2 are

averages of the values determined from voltammograms recorded

at potential sweep rates of 25, 50, 100, 200, 300, 400, and 500 mV

s^-1; E_pc, cathodic peak potential; E_pa, anodic peak potential.

Reference 23.

quinones by redox cycling. Blank experiments estab-

lished that NADH alone did not reduce the quinones

5t-5v. Because of the relative concentrations involved

(more than a 1500-fold excess of quinone to NQO1 and

more than a 6000-fold excess of NADH to NQO1), an

efficient inactivator could be reduced by the enzyme and

inactivate the enzyme completely and NADH oxidation

would be undetectable. In other words, compounds 5t-

5w may actually be substrates for NQO1, but the

analytical methods are not sensitive enough to detect

the small amount of NADH oxidation required to

facilitate inactivation of the enzyme by the quinones.

Mitomycin C also has a (carbamoyloxy)methyl group at

the 3-indolyl methyl position, and mitomycin C inacti-

vation of rat NQO1 has been shown to be mechanism-

based.³¹

1,2-Dimethylindolequinones 5t-5w bearing a leaving

group at the 3-indolyl methyl position were apparently

not substrates for human NQO1, i.e., no NADH oxida-

tion was observed. Inactivation studies showed that for

compounds 5t-5v, there was 0% enzyme activity re-

maining following incubation with NQO1 and NADH

(Table 2). Inactivation was NADH-dependent suggest-

ing that it is the reductively activated indolequinone

which inactivates the enzyme, although no hydroquino-

nes could be detected by HPLC due to their very low

molar absorptivity and the rapid regeneration of the

4758 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24

Beall et al.

Ta ble 2. Metabolism of Indolequinones 5 by Recombinant Human NQO1

metabolism

(µmol/min/mg)

NADH oxidation

% enzyme

activity remaining

compd

R′

OMe

aziridinyl

OMe

NHMe

NMe₂

NHCH₂CH₂OH

NHCH₂CH₂OMe

cyclopropylamino

aziridinyl

5.31 ( 0.93

11.6 ( 0.5

94 ( 5

49 ( 4

100

96 ( 4

100

92 ( 4

CHO

CO₂Et

CH₂OH

CO₂Et

CH₂OH

CH₂OAc

CH₂OC₆H₄NO₂

CH₂OCONH₂

8.78 ( 1.91

14.3 ( 4.9

1.25 ( 0.03

0.20 ( 0.03

0.49 ( 0.06

0.46 ( 0.04

0.32 ( 0.03

0.22 ( 0.02

0.10 ( 0.01

0.23 ( 0.02

3.35 ( 0.65

2.01 ( 0.43

0.22 ( 0.03

not detected

0.22 ( 0.06

0.69 ( 0.08

0.45 ( 0.04

not detected

0.36 ( 0.04

23.4 ( 2.3

98 ( 2

100

96 ( 4

97 ( 4

methylaziridinyl

azetidinyl

98 ( 2

pyrrolidinyl

piperidinyl

morpholinyl

N-methylpiperazinyl

OMe

aziridinyl

OMe

NHCH₂CH₂OH

cyclopropylamino

methylaziridinyl

OMe

81 ( 2

100

99 ( 2

5a a

5bb

5cc

5d d

5ee

CO₂Me

100

CO₂Et

CH₂OH

CO₂Et

11.0 ( 0.9

0.075 ( 0.002

0.09 ( 0.01

0.74 ( 0.06

0.17 ( 0.03

5.40 ( 0.83

4-Ph-C₆H₄

CH₂OH

OMe

Ta ble 3. Metabolism of Mitosenes 6 by Recombinant Human

NQO1

electron-withdrawing ester group to the 2-position, as

in indolequinone 5x, resulted in poor metabolism by the

enzyme.

As anticipated on the basis of the properties of

streptonigrin, the 2-phenylindolequinones 5y and 5z

were excellent substrates for NQO1, better than their

corresponding 2-methyl derivatives 5d and 5e. As

expected this was modified by the incorporation of

amine substituents at C-5 as in compounds 5a a and

5bb. The methylaziridine derivative 5cc was surpris-

ingly a rather poor substrate. Incorporation of the

larger biphenyl substituent at C-2 resulted in a com-

pound (5ee) that was a better substrate for the enzyme

than the corresponding 2-methyl compound 5e but

worse than the corresponding 2-phenyl derivative,

though, surprisingly, the corresponding 3-ester 5d d was

a poor substrate.

metabolism

(µmol/min/mg) % enzyme

NADH

oxidation

activity

compd

remaining

CHO

OMe

5.62 ( 0.71

2.37 ( 0.06

0.04 ( 0.01

8.77 ( 1.22

4.80 ( 0.38

2.22 ( 0.41

0.06 ( 0.01

not detected

0.06 ( 0.02

not detected

93 ( 2

77 ( 5

84 ( 5

44 ( 5

100

91 ( 2

90 ( 5

methylaziridinyl

pyrrolidinyl

OMe

CH₂OH

OMe

methylaziridinyl

pyrrolidinyl

CH₂OCONH₂OMe

CH₂OCONH₂aziridinyl

CH₂OCONH₂methylaziridinyl

CH₂OCONH₂pyrrolidinyl

CH₂OCONH₂piperidinyl

Although less compounds were investigated, a similar

pattern of quinone metabolism was observed in the

mitosene series 6. Thus measurable NADH oxidation

was observed for methoxyquinones 6a and 6e, methyl-

aziridines 6b and 6f, aziridine 6i, and pyrrolidines 6c

and 6g, although the latter three compounds were only

slightly above the limits of detection. Interestingly in

the mitosenes 6 the C-7 methoxy compounds were better

substrates than the corresponding methylaziridines.

Again the substituent at the indole 3-position (9-position

in mitosene numbering) had an effect, with the unsub-

45 ( 5

16 ( 4

CH₂OCONH₂cyclopropylamino not detected

However, compounds containing an electron-with-

drawing group such as an ester at C-3 were excellent

substrates; within the 5-methoxyindolequinones the

rate of metabolism by the enzyme decreased as the

3-substituent was altered in the order: CO₂Et > CHO

> H > CH₂OH > CH₃> CH₂OCONH₂. Switching the

Indolequinone Antitumor Agents

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4759

Ta ble 4. Cytotoxicity of Representative Indolequinones 5 and Mitosenes 6 toward Non-Small-Cell Lung Cancer (NSCLC) Cell Lines^a

cytotoxicity IC₅₀(µM)

H460 H596

compd

R′

selectivity ratio

5cc

OMe

aziridinyl

OMe

>25

0.96 ( 0.24

>25

0.018 ( 0.004

0.11 ( 0.02

>25

9.22 ( 1.51

>25

>26

CO₂Et

CH₂OH

OMe

aziridinyl

methylaziridinyl

azetidinyl

OMe

510

>220

0.7

0.3

1.6

CH₂OH

CH₂OC₆H₄NO₂

CH₂OCONH₂

CH₂OH

2.81 ( 0.52

7.45 ( 0.37

0.15 ( 0.01

8.0 ( 5.1

2.00 ( 0.20

2.16 ( 0.47

0.24 ( 0.03

OMe

aziridinyl

methylaziridinyl

OMe

methylaziridinyl

OMe

>25

0.093 ( 0.018

5.40 ( 0.58

0.36 ( 0.08

>270

1.2

7.5

CH₂OCONH₂

6.48 ( 1.58

2.71 ( 0.37

aziridinyl

H460 has high NQO1 activity, and H596 has no measurable NQO1 activity.

stituted compounds 6a and 6b being more rapidly

metabolized than their 9-hydroxymethyl counterparts

6e and 6f. The 9-[(carbamoyloxy)methyl]-substituted

mitosenes 6h -6m were either very poor substrates for

NQO1 (6i) or not substrates for NQO1 (6h , 6j-6m ). As

with the indolequinone series 5, this was likely due to

NADH-dependent inactivation of NQO1 by the reduced

quinone. Enzyme activity remaining was 0% for com-

pounds 6h -6j, 45% for compound 6l, and 16% for

compound 6m . The compound 6d containing the elec-

tron-withdrawing formyl group was an excellent sub-

strate, and as before, most of the compounds containing

the (carbamoyloxy)methyl side chain were not sub-

strates for human NQO1. Quinone reduction was

undetectable for all of the compounds (with the excep-

tion of 6f which was removed at a rate of 0.7 µmol/min/

mg) suggesting that the reduced quinones (hydroquino-

nes) reoxidize rapidly in air to regenerate the parent

quinones.

It is clear that the substituent on the quinone ring

(5-position in 5, 7-position in 6) has a marked effect on

the rate of metabolism of the indolequinones by NQO1.

In general those compounds bearing amine substituents

(other than aziridines) at C-5(7) are not substrates for

the enzyme, either as a result of steric effects (in the

case of substituents such as pyrrolidine or piperidine)

or because such substituents render the quinone more

difficult to reduce by virtue of the donation of the

nitrogen lone pair into the quinone. The fact that these

substrates are more difficult to reduce is clearly evi-

denced by electrochemical experiments (Table 1). Con-

versely the indolequinones that are easiest to reduce

electrochemically, i.e., those bearing an aziridine sub-

stituent, are among the best substrates for NQO1, with

the methylaziridine and methoxy compounds being

somewhat less efficient. The aziridine ring, because of

its inability to donate electron density into the quinone

which would involve an unfavorable flattening of the

aziridine nitrogen, has a similar electronic effect to the

methoxy group. However, although the ease of reduc-

tion is a key factor, just as with other quinones,^15,16

there is no overall correlation between reduction poten-

tial and rate of metabolism by NQO1.

The substituent at the indole 3-position (9-position

in mitosene numbering in 6) also has a considerable

effect on metabolism by NQO1. The carbamates and

other compounds bearing a leaving group at the 3-in-

dolyl methyl position are generally much poorer sub-

strates, although this is possibly due to inactivation of

the enzyme by these compounds, since elimination of

the leaving group would generate a reactive electrophile

capable of alkylating the enzyme.^24,31Compounds with

an electron-withdrawing substituent at this position are

the best substrates of those 1,2-dimethylindolequinones

and mitosenes studied, although the 3(or 9)-unsubsti-

tuted compounds and the hydroxymethyl derivatives are

also efficiently metabolized. The fact that the 2-arylin-

dolequinones 5y, 5z, and 5ee were all good substrates

was anticipated (see above) and presumably reflects the

fact that the enzyme can accommodate a large aromatic

group in its active site; further studies to probe this

aspect are underway.

However, being an efficient substrate for NQO1 does

not necessarily mean that the quinone will be selectively

cytotoxic to cells containing elevated NQO1 activity.

Representative compounds were therefore tested in vitro

against tumor cell lines. Cytotoxicity (cell survival) was

measured using the MTT colorimetric assay. Two non-

small-cell lung cancer (NSCLC) cell lines were used:

H460 with high NQO1 activity (1360 nmol/min/mg) and

H596 which has no measurable NQO1 activity due to a

polymorphism in the NQO1 gene.³²Activities of other

relevant reductases have been measured and found to

be similar as reported previously.¹⁷IC₅₀values (con-

centration at which cell survival equals 50% of control)

are reported for the indolequinones 5 and the mitosenes

6 in Table 4. A compound with an IC₅₀value which is

lower in the H460 cells than in the H596 cells suggests

4760 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24

Beall et al.

Eth yl 5-Meth oxy-1,2-d im eth yl-4,7-d ioxoin d ole-3-ca r -

a compound which is selectively toxic to the cell line

with elevated NQO1 activity. The compounds with the

greatest degree of selective toxicity were the 1,2-

dimethylindolequinones 5b, 5m , and 5n , each with

greater than a 25-fold difference in IC₅₀values between

the two cell lines, with 5m being more than 500-fold

selective. All three compounds were good substrates for

human NQO1, and each has either an aziridinyl sub-

stituent or a methylaziridinyl substituent at the 5(7)-

position. The only mitosenes which exhibited selective

toxicity were 6f and 6i, with 6f being greater than 250-

fold selective for the H460 cell line. Both compounds

have aziridinyl substituents.

Overall toxicity in non-small-cell lung cancer cell lines

was greatest for those indolequinones that, in principle,

can act as bifunctional alkylating agents, namely, the

hydroxymethyl and (carbamoyloxy)methyl aziridines

5m , 5n , 5w , 6f, and 6i. Compounds that are likely to

function as monoalkylating agents were less cytotoxic,

and 5a and 5d , which have no alkylating centers, were

not toxic, although they were efficient redox-cycling

compounds when activated by NQO1. In agreement

with their ability to act as efficient substrates for NQO1,

the aziridinyl quinones were usually the most selective

agents in each series, exhibiting much greater toxicity

toward the high-NQO1 H460 cells than to the NQO1-

deficient H596 cells.

boxyla te, 5d . Prepared as previously described.²⁴

3-(Hyd r oxym eth yl)-5-m eth oxy-1,2-d im eth ylin d ole-4,7-

d ion e, 5e. (a) Prepared as previously described.²²(b) Alter-

native method: To a solution of ethyl 5-methoxy-1,2-dimethyl-

4,7-dioxoindole-3-carboxylate (5d ) (3.4 g, 12.3 mmol) in

chloroform (370 mL) and EtOH (125 mL) was added a solution

of sodium dithionite (25 g) in water (160 mL). The biphasic

mixture was stirred vigorously overnight. The organic layer

was separated, washed with brine, dried (Na₂SO₄), and

concentrated to give ethyl 4,7-dihydroxy-5-methoxy-1,2-di-

methylindolecarboxylate as a solid that was used directly.

To a stirred suspension of the hydroquinone in dichlo-

romethane (500 mL) was added DIBAL (1 M solution in

dichloromethane; 13.9 g, 97.9 mmol), keeping the temperature

below -30 °C. The mixture was stirred at this temperature

for a further 2 h. The reaction mixture was quenched by

dropwise addition of an iron(III) chloride solution (1 M FeCl₃/

0.1 M HCl, 125 mL) while keeping the temperature below -30

°C. The mixture was filtered through Celite and the residue

washed with hot dichloromethane. The organic layer was

separated, washed with saturated ammonium chloride, dried

(Na₂SO₄), and concentrated. The crude product was recrystal-

lized (dichloromethane/light petroleum) to yield the title

compound as an orange/red crystalline solid (2.0 g, 71%): mp

200-202 °C (lit.²³mp 199-200 °C); H NMR (CDCl₃) δ 5.60

(1 H, s, 6-H), 4.59 (2 H, d, J ) 6.7 Hz, CH₂OH), 3.87 (1 H, t,

J ) 6.7 Hz, OH), 3.85 (3 H, s, OMe), 3.80 (3 H, s, NMe), and

2.21 (3 H, s, Me).

5-Meth oxy-1,2,3-tr im eth ylin d ole-4,7-d ion e, 5f. If the

above reaction mixture in the reduction of 5d was allowed to

warm to 0 °C, the product was 5-methoxy-1,2,3-trimethylin-

dole-4,7-dione, 5f: mp 225-226 °C (dichloromethane/light

petroleum); UV (MeOH) 473 (log ꢀ 3.31), 361 (3.42), 286 (4.24),

and 227 (4.15) nm; IR (KBr) 2981, 2944, 1664, 1654, 1636, and

1622 cm^-1; ¹H NMR (CDCl₃) δ 5.46 (1 H, s, 6-H), 3.75 (3 H, s,

OMe), 3.70 (3 H, s, NMe), 2.14 (3 H, s, Me), and 2.06 (3 H, s,

Me); ¹³C NMR (CDCl₃) δ 178.7 (CO), 178.5 (CO), 160.0, 135.5,

128.4, 122.2, 118.9, 107.4 (CH), 56.6 (OMe), 32.6 (NMe), 10.3

(Me), and 9.2 (Me); HRMS found (M⁺) 219.0895, C₁₂H₁₃NO₃

requires M 219.0895. Anal. (C₁₂H₁₃NO₃) C, H, N.

In conclusion in studying over 40 indolequinones, we

have established the relationship between quinone

structure, rate of metabolism by human NQO1, and

cytotoxicity toward cells containing elevated levels of

NQO1. Some of the compounds identified by this study

may have potential uses as, for example, irreversible

inactivators of NQO1 (e.g., 5t-5v) or highly selective

NQO1-directed antitumor agents (e.g., 5m , 5n , 6f).

Exp er im en ta l Section

3-(H yd r oxym et h yl)-5-(m et h yla m in o)-1,2-d im et h ylin -

d ole-4,7-d ion e, 5g. Purple crystalline solid in 93% yield: mp

235-237 °C; UV (MeOH) 524 (log ꢀ 3.34), 316 (4.19), and 238

Ch em istr y. Commercially available reagents were used

throughout without further purification; solvents were dried

by standard procedures. Analytical thin-layer chromatography

was carried out using aluminum-backed plates coated with

Merck Kieselgel 60 GF₂₅₄. Plates were visualized under UV

light (at 254 and/or 360 nm). Flash chromatography was

carried out using Merck Kieselgel 60 H silica or Matrex silica

60. Pressure was applied at the column head with hand

bellows. Samples were applied preadsorbed on silica. Fully

characterized compounds were chromatographically homoge-

neous. ¹H and ¹³C NMR spectra were recorded at 250, 300,

or 400 MHz (¹H frequencies) on Bruker instruments. Full

spectroscopic data are given for quinones; data for intermedi-

ates are provided in the Supporting Information.

Gen er a l Meth od for Su bstitu tion Rea ction s a t C-5

P osition w ith Am in es. The following general method was

used unless otherwise stated: To a stirred solution of 3-(hy-

droxymethyl)-5-methoxy-1,2-dimethylindole-4,7-dione (5e) (0.080

g, 34.0 mmol) in DMF (5 mL) was added the amine (40 equiv),

and the mixture was stirred for 48 h at room temperature.

Water was added (30 mL), the crude product was extracted

with dichloromethane (3 × 30 mL), and the combined extracts

were washed with water (12 × 30 mL). The organic layer was

dried (Na₂SO₄). The crude material was purified by column

chromatography (EtOAc/light petroleum, 1:1) to yield the

product.

(4.27) nm; IR (CH₂Cl₂) 3474, 3270, 1663, 1597, and 1269 cm^-1

;

¹H NMR (CDCl₃) δ 5.85 (1 H, br s, NH), 5.13 (1 H, s, 6-H),

4.60 (2 H, d, J ) 6.7 Hz, CH₂), 3.99 (1 H, t, J ) 7.1 Hz, OH),

3.90 (3 H, s, NMe), 2.87 (3 H, d, J ) 5.4 Hz, MeNH), and 2.20

(3 H, s, Me); ¹³C NMR (CDCl₃) δ 179.7 (CO), 178.7 (CO), 149.1,

132.5, 131.9, 121.7, 120.0, 96.9 (CH), 56.1 (CH₂), 32.3 (Me),

29.4 (Me), and 9.4 (Me); HRMS found (M⁺) 234.1003, C₁₂H₁₄

N₂O₄requires M 234.1004.

3-(Hyd r oxym eth yl)-5-(d im eth yla m in o)-1,2-d im eth ylin -

d ole-4,7-d ion e, 5h . Purple solid in 59% yield: mp 138-139

°C (EtOAc/light petroleum); UV (MeOH) 534 (log ꢀ 3.41), 324

(4.04), and 246 (4.18) nm; IR (CH₂Cl₂) 3440, 3062, 1654, 1620,

1560, and 1276 cm^-1; H NMR (CDCl₃) δ 5.16 (1 H, s, 6-H),

4.51 (3 H, m, CH₂OH), 3.81 (3 H, s, NMe), 3.08 (6 H, s, 2 ×

NMe), and 2.19 (3 H, s, Me); ¹³C NMR (CDCl₃) δ 181.3 (CO),

177.3 (CO), 152.8, 135.6, 129.1, 121.6, 121.1, 107.1 (CH), 53.9

(CH₂), 42.9 (NMe), 32.0 (NMe), and 9.6 (Me); HRMS found (M⁺)

248.1154, C₁₃H₁₆N₂O₃requires M 248.1161. Anal. (C₁₃H₁₆N₂O₃‚

0.5H₂O) C, H, N.

Eth yl 5-[(2-Hyd r oxyeth yl)a m in o]-1,2-d im eth yl-4,7-d i-

oxoin d ole-3-ca r boxyla te, 5i. Purple crystalline solid in 61%

yield: mp 161-162 °C (EtOAc/light petroleum); UV (MeOH)

506 (log ꢀ 3.21), and 312 (4.02) nm; IR (CH₂Cl₂) 3396, 3338,

2981, 1731, 1665, 1598, 1578, 1506, 1440, 1245, and 1214 cm^-1

;

5-Meth oxy-1,2-d im eth ylin d ole-4,7-d ion e, 5a . Prepared

¹H NMR (CDCl₃) δ 6.22 (1 H, br s, NH), 5.21 (1 H, s, 6-H),

4.37 (2 H, q, J ) 7.1 Hz, CH₂), 3.93 (3 H, s, NMe), 3.88 (2 H,

q, J ) 5.3 Hz, CH₂), 3.31 (2 H, q, J ) 5.5 Hz, CH₂), 2.43 (3 H,

s, Me), 1.94 (1 H, t, J ) 5.4 Hz, OH), and 1.39 (3 H, t, J ) 7.1

Hz, Me); ¹³C NMR (CDCl₃) δ 181.2 (CO), 179.0 (CO), 157.2,

138.8, 130.6 (2 × CH), 130.2, 129.3 (CH), 128.8 (2 × CH), 128.6,

as previously described.²³

5-(Azir id in -1-yl)-1,2-d im eth ylin d ole-4,7-d ion e, 5b. Pre-

pared as previously described.²³

3-F or m yl-5-m eth oxy-1,2-d im eth ylin d ole-4,7-d ion e, 5c.

Prepared as previously described.²³

Indolequinone Antitumor Agents

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4761

123.9, 122.8, 116.8 (CH), 56.2 (CH₂OH), 36.3 (CH), 34.7 (CH₂),

33.9 (NMe), and 17.7 (Me); HRMS found (M⁺) 306.1216,

1660, 1629, and 1582 cm^-1; H NMR (CDCl₃) δ 5.72 (1 H, s,

6-H), 4.61 (2 H, d, J ) 6.5 Hz, CH₂OH), 4.16 (1 H, t, J ) 6.5

Hz, OH), 3.86 (3 H, s, NMe), 2.21 (4 H, m, azir-CH, 2-Me),

2.10 (2 H, m, azir-CH₂), and 1.42 (3 H, d, J ) 5.4 Hz, NCHMe);

¹³C NMR (CDCl₃) δ 181.6 (CO), 179.1 (CO), 157.2, 134.4, 130.1,

123.0, 122.9, 117.0 (CH), 56.3 (CH₂), 36.5, 34.9 (CH₂), 32.6,

30.0, 18.0, and 9.8.

₁₅H₁₈N₂O₅requires M 306.1217. Anal. (C₁₅H₁₈N₂O₅‚0.4H₂O)

C, H, N.

3-(Hyd r oxym eth yl)-5-[(2-h yd r oxyeth yl)a m in o]-1,2-d i-

m eth ylin d ole-4,7-d ion e, 5j. Purple solid in 54% yield: mp

211-213 °C (EtOAc/light petroleum); UV (MeOH) 524 (log ꢀ

3.26), 314 (4.13), and 244 (4.30) nm; IR (CH₂Cl₂) 3472, 3359,

1654, 1612, and 1274 cm^-1; ¹H NMR (CDCl₃) δ 6.13 (1 H, br s,

NH), 5.17 (1 H, s, 6-H), 4.60 (2 H, d, J ) 6.8 Hz, CH₂), 4.03 (1

H, t, J ) 6.8 Hz, OH), 3.89 (5 H, br s, NMe, CH₂), 3.31 (2 H,

q, J ) 5.5 Hz, CH₂), 2.20 (3 H, s, Me), and 1.92 (1 H, br s,

OH); ¹³C NMR (CDCl₃) δ 179.4 (CO), 178.6 (CO), 149.3, 135.2,

130.7, 120.4, 119.6, 95.2 (CH), 58.8 (CH₂), 54.0 (CH₂), 44.5

(CH₂), 31.2 (NMe), and 7.8 (Me); HRMS found (M⁺) 264.1116,

5-(Azetidin -1-yl)-3-(h ydr oxym eth yl)-1,2-dim eth ylin dole-

4,7-d ion e, 5o. Purple crystalline solid in 88% yield: mp 190-

191 °C (EtOAc/light petroleum); UV (MeOH) 548 (log ꢀ 3.64),

328 (4.25), and 244 (4.36) nm; IR (CH₂Cl₂) 3474, 2980, 1670,

1604, 1578, and 1270 cm^-1; H NMR (CDCl₃) δ 4.87 (1 H, s,

6-H), 4.58 (4 H, m, CH₂), 4.40 (1 H, t J ) 7.0 Hz, OH), 4.04 (2

H, t, J ) 7.4 Hz, CH₂), 3.89 (3 H, s, NMe), 2.48 (2 H, m, CH₂),

and 2.17 (3 H, s, Me); ¹³C NMR (CDCl₃) δ 181.5 (CO), 177.4

(CO), 148.6, 132.0, 131.9, 121.4, 120.6, 97.7 (CH), 56.1 (CH₂),

55.8 (br CH₂), 52.0 (br CH₂), 32.1 (NMe), 17.7 (CH₂), and 9.4

(Me); MS m/z (EI) 260 (M⁺, 75%), 215 (81), 199 (28), and 159

(20); HRMS found (M⁺) 260.1166, C₁₄H₁₆N₂O₃requires M

260.1161. Anal. (C₁₄H₁₆N₂O₃‚0.5H₂O) C, H, N.

₁₃H₁₆N₂O₄requires M 264.1110. Anal. (C₁₃H₁₆N₂O₄‚0.5H₂O)

C, H, N.

3-(Hyd r oxym eth yl)-5-[(2-m eth oxyeth yl)a m in o]-1,2-d i-

m eth ylin d ole-4,7-d ion e, 5k . Purple crystalline solid in 93%

yield: mp 141-142 °C (EtOAc/light petroleum); UV (MeOH)

526 (log ꢀ 3.46), 314 (4.20), and 244 (4.36) nm; IR (CH₂Cl₂)

3-(Hyd r oxym eth yl)-1,2-d im eth yl-5-(p yr r olid in -1-yl)in -

d ole-4,7-d ion e, 5p . To a stirred solution of 3-(hydroxy-

methyl)-5-methoxy-1,2-dimethylindole-4,7-dione (5e) (0.038 g,

0.16 mmol) in DMF (2 mL) was added pyrrrolidine (0.23 g,

3.24 mmol), and the mixture was stirred at room temperature

overnight. The mixture was diluted with dichloromethane (20

mL) and washed with water (5 × 20 mL). The organic layer

was dried (Na₂SO₄) and evaporated, and the crude material

was purified by column chromatography (EtOAc) to yield the

title compound (0.038 g, 84%) as a purple solid: mp 196-198

°C; UV (MeOH) 547 (log ꢀ 3.45), 328 (4.02), 250 (4.23), and

224 (4.18) nm; IR (KBr) 3422, 2980, 2732, 1639, 1612, and 1550

cm^-1; ¹H NMR (CDCl₃) δ 5.11 (1 H, s, 6-H), 4.51 (2 H, s, CH₂-

OH), 3.82 (3 H, s, NMe) 3.55 (4 H, br s, NCH₂), 2.12 (3 H, s,

2-Me), and 1.89 (4 H, m, CH₂); ¹³C NMR (CDCl₃) δ 184.5 (CO),

180.5 (CO), 151.2, 134.7, 133.5, 124.0, 123.7, 104.2 (CH), 53.6

(CH₂), 50.3 (CH₂), 34.4 (NMe), and 11.8 (Me); HRMS found

(M⁺) 274.1317, C₁₅H₁₈N₂O₃requires M 274.1317.

3690, 3380, 2996, 1661, 1627, 1607, 1506, and 1276 cm^-1; H

NMR (CDCl₃) δ 6.07 (1 H, br s, NH), 5.12 (1 H, s, 6-H), 4.58 (2

H, d, J ) 3.7 Hz, CH₂OH), 3.99 (1 H, s, OH), 3.87 (3 H, s,

OMe), 3.60 (2 H, t, J ) 5.3 Hz, MeOCH₂), 3.38 (3 H, s, NMe),

3.27 (2 H, q, J ) 5.4 Hz, NHCH₂), and 2.18 (3 H, s, Me); ¹³C

NMR (CDCl₃) δ 179.6 (CO), 178.8 (CO), 148.0, 132.6, 131.6,

121.8, 120.1, 97.2 (CH), 69.4 (CH₂), 58.9 (OMe), 56.0 (CH₂),

42.4 (CH₂), 32.2 (NMe), and 9.34 (Me); HRMS found (M⁺)

278.1265, C₁₄H₁₈N₂O₄requires M 278.1267. Anal. (C₁₄H₁₈N₂O₄‚

0.2H₂O) C, H, N.

5-(Cyclop r op yla m in o)-3-(h yd r oxym eth yl)-1,2-d im eth -

ylin d ole-4,7-d ion e, 5l. Purple crystalline solid in 51%

yield: mp 192-193 °C (EtOAc/light petroleum); UV (MeOH)

528 (log ꢀ 3.39), 316 (4.22), and 242 (4.38) nm; IR (CH₂Cl₂)

3697, 3393, 2989, 1627, 1593, 1506, and 1283 cm^-1; H NMR

(CDCl₃) δ 5.89 (1 H, br s, NH), 5.50 (1 H, s, 6-H), 4.58 (2 H, d,

J ) 6.7 Hz, CH₂OH), 3.98 (1 H, t, J ) 6.7 Hz, OH), 3.88 (3 H,

s NMe), 2.44 (1 H, m, NHCH), 2.18 (3 H, s, Me), 0.85 (2 H, m,

CH₂), and 0.65 (2 H, m, CH₂); ¹³C NMR (CDCl₃) δ 180.0 (CO),

178.9 (CO), 148.9, 132.5, 131.6, 121.7, 120.2, 99.1 (CH), 56.0

(CH₂), 32.2 (Me), 24.2 (CH), 9.4 (Me), and 7.1 (2 × CH₂); HRMS

found (M⁺) 260.1166, C₁₄H₁₆N₂O₃requires M 260.1161. Anal.

(C₁₄H₁₆N₂O₃‚0.2H₂O) C, H, N.

3-(Hyd r oxym et h yl)-1,2-d im et h yl-5-(p ip er id in -1-yl)in -

d ole-4,7-d ion e, 5q. Purple glassy solid in 44% yield: UV

(MeOH) 534 (log ꢀ 3.30), 324 (3.96), and 240 (4.20) nm; IR (CH₂-

Cl₂) 3455, 2945, 1624, 1556, 1503, and 1268 cm^-^{1 1}H NMR

;

(CDCl₃) δ 5.48 (1 H, s, 6-H), 4.57 (2 H, s, CH₂), 4.10 (1 H, m,

OH), 3.86 (3 H, s, NMe), 3.39 (4 H, m, 2 × NCH₂), 2.20 (3 H,

s, Me), and 1.68 (6 H, m, 3 × CH₂); ¹³C NMR (CDCl₃) δ 182.3

(CO), 178.4 (CO), 154.1, 133.3, 129.9, 122.8, 122.1, 109.5 (CH),

55.9 (CH₂), 51.1 (2 × CH₂), 32.0 (Me), 25.7 (2 × CH₂), 24.2

(CH₂), and 9.4 (Me); HRMS found (MH⁺) 289.1552, C₁₆H₂₀N₂O₃

+ H requires M 289.1552.

5-(Azir idin -1-yl)-3-(h ydr oxym eth yl)-1,2-dim eth ylin dole-

4,7-d ion e, 5m . To a stirred solution of 3-(hydroxymethyl)-5-

methoxy-1,2-dimethylindole-4,7-dione (5e) (0.037 g, 0.15 mmol)

in DMF (2 mL) was added aziridine (CAUTION) (0.13 g, 3.1

mmol), and the mixture was stirred at room temperature

overnight. The mixture was diluted with dichloromethane (20

mL) and washed with water (5 × 20 mL). The organic layer

was dried (Na₂SO₄) and evaporated, and the crude material

was purified by column chromatography (EtOAc) to yield the

title compound (0.029 g, 77%) as a red solid: mp 164-166 °C

(lit.²⁸mp 173-174 °C); UV (MeOH) 483 (log ꢀ 3.26), 305 (4.15),

3-(Hyd r oxym eth yl)-1,2-d im eth yl-5-(m or p h olin -1-yl)in -

d ole-4,7-d ion e, 5r . Purple crystalline solid in 65% yield: mp

199-200 °C (EtOAc/light petroleum); UV (MeOH) 522 (log ꢀ

3.40), 326 (4.05), and 240 (4.28) nm; IR (CH₂Cl₂) 3427, 1639,

1577, 1515, 1267, and 1221 cm^-1; H NMR (CDCl₃) δ 5.50 (1

H, s, 6-H), 4.58 (2 H, d, J ) 7.0 Hz, CH₂), 3.97 (1 H, t, J ) 7.0

Hz, OH), 3.87 (3 H, s, NMe), 3.84 (4 H, t, J ) 4.8 Hz, OCH₂),

3.40 (4 H, t, J ) 4.8 Hz, NCH₂), and 2.22 (3 H, s, Me); ¹³C

NMR (CDCl₃) δ 182.0 (CO), 178.3 (CO), 153.3, 133.9, 129.6,

123.0, 122.4, 111.0 (CH), 66.5 (2 × CH₂), 55.9 (CH₂), 49.9 (2 ×

CH₂), 32.1 (NMe), and 9.4 (Me); HRMS found (M⁺) 290.1262,

and 231 (4.23) nm; IR (KBr) 3422, 1636, 1588, and 1501 cm^-1

;

¹H NMR (CDCl₃) δ 5.7 (1 H, s, 6-H), 4.62 (2 H, d, J ) 7.1 Hz,

CH₂OH), 4.12 (1 H, t, J ) 7.1 Hz, OH), 3.87 (3 H, s, NMe),

2.22 (3 H, s, 2-Me), and 2.20 (4 H, s, azir-CH₂); ¹³C NMR

(CDCl₃) δ 181.4 (CO), 179.0 (CO), 157.2, 134.7, 130.0, 123.0,

122.9, 117.8 (CH), 56.3 (CH₂), 32.6 (CH₂), 28.0, and 9.9.

₁₅H₁₈N₂O₄requires M 190.1267. Anal. (C₁₅H₁₈N₂O₄‚0.3H₂O)

C, H, N.

3-(Hyd r oxym eth yl)-1,2-d im eth yl-5-(4-m eth ylp ip er a zin -

3-(Hyd r oxym eth yl)-1,2-d im eth yl-5-(2-m eth yla zir id in -

1-yl)in d ole-4,7-d ion e, 5n . To a stirred solution of 3-(hy-

droxymethyl)-5-methoxy-1,2-dimethylindole-4,7-dione (5e) (0.04

g, 0.17 mmol) in DMF (2 mL) was added 2-methylaziridine

(0.19 g, 3.4 mmol), and the mixture was stirred at room

temperature overnight. The mixture was diluted with dichlo-

romethane (20 mL) and washed with water (5 × 20 mL). The

organic layer was dried (Na₂SO₄) and evaporated, and the

crude material was purified by column chromatography (EtOAc)

to yield the title compound (0.024 g, 56%) as a red solid: mp

124-125 °C (lit.²⁸mp 120-122 °C); UV (MeOH) 484 (log ꢀ

3.26), 305 (4.15), and 232 (4.24) nm; IR (KBr) 3422, 2980, 2732,

1-yl)in d ole-4,7-d ion e, 5s. Purple crystalline solid in 75%

yield: mp 166-167 °C (EtOAc/light petroleum); UV (MeOH)

522 (log ꢀ 3.41), 326 (4.07), and 240 (4.28) nm; IR (CH₂Cl₂)

3460, 2993, 1664, 1637, 1571, 1512, and 1269 cm^-1; H NMR

(CDCl₃) δ 5.49 (1 H, s, 6-H), 4.57 (2 H, s, CH₂), 3.86 (3 H, s,

NMe), 3.42 (4 H, m, 2 × CH₂), 2.54 (4 H, m, 2 × CH₂), 2.33 (3

H, s, NMe), and 2.20 (3 H, s, Me); ¹³C NMR (CDCl₃) δ 182.1

(CO), 178.3 (CO), 153.5, 133.7, 129.7, 122.9, 122.2, 110.8 (CH),

55.9 (CH₂), 54.6 (2 × CH₂), 49.6 (2 × CH₂), 46.0 (NMe), 32.1

(NMe), and 9.4 (Me); HRMS found (M⁺) 303.1587, C₁₆H₂₁N₃O₃

requires M 303.1583. Anal. (C₁₆H₂₁N₃O₃0.1H₂O) C, H, N.

4762 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24

Beall et al.

3-(Acetoxym eth yl)-5-m eth oxy-1,2-d im eth ylin d ole-4,7-

d ion e, 5t. To a stirred solution of 3-(hydroxymethyl)-5-

methoxy-1,2-dimethylindole-4,7-dione (0.084 g, 34.0 mmol) in

pyridine (24 mL) was added acetic anhydride (3.5 mL), and

the mixture was stirred for 24 h at room temperature. Water

was added (30 mL), the crude product was washed with 1 M

HCl and extracted with dichloromethane (2 × 40 mL), and the

combined extracts were washed with water (2 × 20 mL). The

organic layer was dried (Na₂SO₄). The crude material was

purified by column chromatography (EtOAc/light petroleum,

1:1) to yield the title compound (0.040 mg, 40%) as an orange

crystalline solid: mp 177-178 °C (EtOAc/light petroleum)

(lit.²⁴mp 185-186 °C); UV (MeOH) 450 (log ꢀ 3.32), 340 (3.60),

and 286 (4.37) nm; IR (CH₂Cl₂) 1736, 1657, 1637, 1564, 1269,

The product was purified by flash column chromatography

(EtOAc/light petroleum, 4:6) to give the title compound 8a

(0.82 g, 58%) as a colorless crystalline solid: mp 179-181 °C

(lit.²⁸mp 179 °C).

Eth yl 5-Meth oxy-1-m eth yl-2-p h en ylin d ole-3-ca r boxy-

la te, 9a . Potassium hydroxide (0.18 g, 3.2 mmol) was added

to a stirring solution of ethyl 5-hydroxy-2-phenylindole-3-

carboxylate (8a ) (0.30 g, 1.1 mmol) in DMSO (10 mL); the

reaction was stirred for 0.5 h. Methyl iodide (0.68 g, 4.8 mmol)

was added, and the reaction was stirred for 2 h. Water was

added (20 mL), and the mixture was extracted with EtOAc (3

× 15 mL), washed with 1 M hydrochloric acid (4 × 15 mL)

and water (2 × 15 mL), dried (MgSO₄), and evaporated under

reduced pressure. The crude product was purified by flash

column chromatography (light petroleum/EtOAc, 4:1) to give

the title compound 9a (0.20 g, 60%) as a colorless crystalline

solid: mp 107-108 °C. Anal. (C₁₉H₁₉NO₃) C, H, N.

E t h yl 5-Met h oxy-1-m et h yl-6-n it r o-2-p h en ylin d ole-3-

ca r boxyla te, 10a . Ethyl 5-methoxy-1-methyl-2-phenylindole-

3-carboxylate (9a ) (0.56 g, 1.7 mmol), stirred in glacial acetic

acid (7 mL), was cooled to -10 °C. A mixture of glacial acetic

acid (4 mL) and nitric acid (1.5 mL) was added at -10 °C, and

the reaction was stirred at room temperature for 2 h. The

yellow suspension was poured onto an ice/water mixture. The

reaction mixture was stirred in dichloromethane (30 mL) for

1 h. The organic layer was separated, and the aqueous layer

was extracted with dichloromethane (3 × 30 mL); the combined

extracts were washed with saturated sodium bicarbonate (3

× 40 mL) and water (2 × 40 mL), dried (Na₂SO₄), and

evaporated under reduced pressure. Flash column chroma-

tography (light petroleum/EtOAc, 7:3) gave the 6-nitro and

4-nitro compounds in the ratio 1:7. Ethyl 5-methoxy-1-methyl-

6-nitro-2-phenylindole-3-carboxylate (66 mg, 11%) was a yellow

crystalline solid: mp 167-168 °C. Anal. (C₁₉H₁₈N₂O₅‚0.2H₂O)

C, H, N. Ethyl 5-methoxy-1-methyl-4-nitro-2-phenylindole-3-

carboxylate (10a ) (0.45 g, 75%) was a yellow crystalline solid:

mp 154-155 °C. Anal. (C₁₉H₁₈N₂O₅‚0.2H₂O) C, H, N.

Eth yl 4-Am in o-5-m eth oxy-1-m eth yl-2-p h en ylin d ole-3-

ca r boxyla te, 11a . To a stirred solution of ethyl 5-methoxy-

1-methyl-4-nitro-2-phenylindole-3-carboxylate (10a ) (1.47 g, 4

mmol) in EtOH (100 mL) was added tin powder (2.94 g, 24

mmol) in hydrochloric acid (3 M; 35 mL), and the mixture was

heated under reflux for 2 h. After cooling, the solution was

decanted and neutralized with saturated sodium bicarbonate

(500 mL), water (100 mL) and dichloromethane (300 mL) were

added, and the mixture was stirred vigorously overnight. The

aqueous layer was removed and the organic layer washed with

water (2 × 200 mL), dried (Na₂SO₄), and evaporated under

reduced pressure. The crude product was purified by flash

column chromatography (light petroleum/EtOAc, 7:3) to give

the title compound 11a (0.79 g, 59%) as a colorless crystalline

solid: mp 125-126 °C. Anal. (C₁₉H₂₀N₂O₃‚0.4H₂O) C, H, N.

and 1229 cm^-1; H NMR (CDCl₃) δ 5.62 (1 H, s, 6-H), 5.25 (2

H, s, CH₂), 3.90 (3 H, s, OMe), 3.80 (3 H, s, NMe), 2.28 (3 H,

s, Me), and 2.04 (3 H, s, Me); ¹³C NMR (CDCl₃) δ 178.9 (CO),

177.6 (CO), 171.0, 159.7, 137.8, 129.2, 121.8, 155.8, 56.6 (CH₂),

56.4 (OMe), 32.4 (NMe), 22.7 (Me), and 14.1 (Me).

5-Meth oxy-1,2-d im eth yl-3-[(4-n itr op h en oxy)m eth yl]in -

d ole-4,7-d ion e, 5u . 3-(Hydroxymethyl)-5-methoxy-1,2-di-

methylindole-4,7-dione (0.46 g, 1.96 mmol), triphenylphosphine

(1.0 g, 3.8 mmol), diethyl azodicarboxylate (0.68 g, 3.9 mmol),

and 4-nitrophenol (0.46 g, 3.3 mmol) were stirred overnight

in THF at 50 °C. Excess solvent was removed and the majority

of the product triturated out with ether and filtered off. The

residue was dissolved in dichloromethane, washed with water,

dried (Na₂SO₄), and concentrated. The crude residue was

purified by column chromatography (light petroleum/EtOAc,

1:1) to yield the title compound as an orange crystalline solid

(0.4 g, 57%): mp 227-288 °C; UV (MeOH) 454 (ꢀ log 2.82),

282 (3.91), and 227 (3.82) nm; IR (KBr) 3242, 3043, 2991, 1753,

1695, 1662, 1637, 1594, and 1482 cm^-^{1 1}H NMR (CDCl₃) δ

;

8.18, 7.05 (4 H, AA′XX′, ArH), 5.64 (1 H, s, 6-H), 5.40 (2 H, s,

CH₂OAr), 3.91 (3 H, s, OMe), 3.82 (3 H, s, NMe), and 2.31 (3

H, s, Me); ¹³C NMR (CDCl₃) δ 178.7 (CO), 178.28 (CO), 163.6,

159.6, 141.7, 138.1, 129.0, 125.9 (CH), 121.3, 115.8, 115.0 (CH),

106.8 (CH), 61.1 (CH₂OAr), 56.5 (OMe), 32.4 (NMe), and 9.9

(Me); HRMS found (M⁺) 356.1013, C₁₈H₁₆N₂O₆requires M

356.1008.

3-[(Ca r ba m oyloxy)m eth yl]-5-m eth oxy-1,2-d im eth ylin -

d ole-4,7-d ion e, 5v. Prepared as previously described.²³

5-(Azir id in -1-yl)-3-[(ca r b a m oyloxy)m e t h yl]-1,2-d i-

m eth ylin d ole-4,7-d ion e, 5w . Prepared as previously de-

scribed.²³

Meth yl 5-Meth oxy-1,3-d im eth yl-4,7-d ioxoin d ole-2-ca r -

boxyla te, 5x. To a stirring solution of methyl 5,7-dimethoxy-

1,3-dimethylindole-2-carboxylate²⁷(0.56 g, 0.21 mmol) in

acetonitrile (20 mL) at 0 °C was added a solution of cerium-

(IV) ammonium nitrate (1.2 g, 2.1 mmol) in water (8 mL)

dropwise. After the addition, the mixture was allowed to stir

at room temperature for 1 h. Saturated ammonium chloride

was added and the mixture extracted with dichloromethane.

The organic layer was separated, dried (Na₂SO₄), and concen-

trated. The residue was purified by column chromatography

(dichloromethane/EtOAc, 19:1) and recrystallized (dichlo-

romethane/light petroleum) to yield the title compound (0.012

g, 21%) as a yellow crystalline solid: mp 218-220 °C; UV

(MeOH) 418 (ꢀ log 3.09), 348 (3.24), and 266 (4.32) nm; IR

(KBr) 3046, 2967, 1723, 1683, 1650, 1604, and 1505 cm^-1; ¹H

NMR (CDCl₃) δ 5.75 (1 H, s, 6-H), 4.26 (3 H, s, NMe), 3.92 (3

H, s, OMe), 3.83 (3 H, s, OMe), and 2.58 (3 H, s, Me); ¹³C NMR

(CDCl₃) δ 179.4, 177.5, 161.8, 160.7, 131.7, 130.2, 126.8, 121.1,

107.8 (CH), 56.6 (OMe), 51.7 (OMe), 35.1 (NMe), and 11.7 (Me);

HRMS found (M⁺) 263.0794, C₁₃H₁₃NO₅requires M 263.0793.

Anal. (C₁₃H₁₃NO₅‚0.3H₂O) C, H, N.

Eth yl 5-Meth oxy-1-m eth yl-4,7-d ioxo-2-p h en ylin d ole-3-

ca r boxyla te, 5y. Ethyl 4-amino-5-methoxy-1-methyl-2-phe-

nylindole-3-carboxylate (11a ) (0.21 g, 0.64 mmol) was stirred

in acetone (30 mL). Potasium nitrosodisulfonate (0.36 g, 0.14

mmol) in dihydrogen phosphate buffer (0.3 M; 30 mL) was

added, and reaction mixture was stirred at room temperature

for 3 h. The excess acetone was removed in vacuo, water was

added (40 mL), and the mixture was extracted with dichlo-

romethane (2 × 20 mL). The combined extracts were washed

with water (2 × 20 mL) and brine (2 × 20 mL), dried (Na₂-

SO₄), and evaporated under reduced pressure. The crude

product was purified by flash column chromatography (light

petroleum/EtOAc, 1:1) to give the title compound (0.21 g, 96%)

as an orange crystalline solid: mp 138-140 °C (EtOAc/light

petroleum); UV (MeOH) 430 (log ꢀ 3.12) and 326 (3.53) nm;

IR (CH₂Cl₂) 2930, 1726, 1629, 1603, 1450, 1260, 1194, and 1030

Eth yl 5-Hydr oxy-2-ph en ylin dole-3-car boxylate, 8a. Eth-

yl 3-amino-3-phenylpropenoate (7a ) (1.03 g, 5 mmol) in acetic

acid was added to a solution of benzoquinone (0.54 g, 5 mmol)

in acetic acid (3 mL) and heated under reflux for 2.5 h. The

excess acetic acid was removed in vacuo, the residue was

extracted with dichloromethane (3 × 30 mL), and the combined

extracts were washed with water (2 × 20 mL) and brine (2 ×

20 mL). The organic layer was dried (Na₂SO₄) and evaporated.

cm^-^{1 1}H NMR (CDCl₃) δ 7.47 (3 H, m, ArH), 7.36 (2 H, m,

;

ArH), 5.73 (1 H, s, 6-H), 4.19 (2 H, q, J ) 7.1 Hz, CH₂), 3.85

(3 H, s, OMe), 3.78 (3 H, s, NMe), 1.16 (3 H, t, J ) 7.1 Hz,

Me); ¹³C NMR (CDCl₃) δ 179.2, 175.5, 163.7, 160.3, 142.5, 130.3

(2 × CH), 129.6, 129.6 (CH), 128.6, 128.5 (2 × CH), 121.6,

114.8, 106.5, (CH), 61.0 (CH₂), 56.6 (OMe), 34.1 (Me), and 13.8

Indolequinone Antitumor Agents

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4763

(Me); HRMS found (M⁺) 339.1111, C₁₉H₁₇NO₅requires M

339.1107. Anal. (C₁₉H₁₇NO₅‚0.9H₂O) C, H, N.

found (M⁺) 322.1322, C₁₉H₁₈N₂O₃requires M 322.1317. Anal.

(C₁₉H₁₈N₂O₃) C, H, N.

3-(Hydr oxym eth yl)-5-m eth oxy-1-m eth yl-2-ph en ylin dole-

4,7-d ion e, 5z. THF (5 mL) was added to LiAlH₄(0.11 g, 3

mmol) under a nitrogen atmosphere. A solution of ethyl

4-amino-5-methoxy-1-methyl-2-phenylindole-3-carboxylate (11a)

(0.20 g, 0.06 mmol) in THF (10 mL) was added to the stirring

suspension. The reaction was stirred at room temperature for

2 h. The reaction was cooled to 0 °C; water (0.5 mL) followed

by 15 mol % NaOH (1 mL) were added dropwise. Silica and

EtOAc (40 mL) were then added, the reaction mixture was

filtered through Celite, and the aqueous layer was removed.

The organic layer was washed with water (2 × 15 mL) and

brine (2 × 15 mL), dried (MgSO₄), and evaporated under

reduced pressure, giving 4-amino-3-(hydroxymethyl)-5-meth-

oxy-1-methyl-2-phenylindole (0.13 g, 76%) as a brown solid:

mp 135-136 °C (EtOAc/light petroleum); IR (CH₂Cl₂) 3391,

3191, 2991, 1506, 1491, 1440, and 1276 cm^-1; ¹H NMR (CDCl₃)

δ 7.48 (3 H, m, ArH), 7.36 (2 H, m, ArH), 6.98 (1 H, d, J ) 8.7

Hz, ArH), 6.70 (1 H, d, J ) 8.7 Hz, ArH) 4.78 (2 H, s, CH₂),

3.91 (3 H, s, OMe), and 3.51 (3 H, s, NMe); ¹³C NMR (CDCl₃)

δ 140.6, 138.7, 134.7, 131.1, 130.8 (2 × CH), 128.4 (2 × CH),

128.3 (CH), 117.4, 111.9, 110.9 (CH), 98.6 (CH), 58.2 (OMe),

57.5 (CH₂), and 30.9 (Me); HRMS found (M⁺) 282.1373,

3-(Hyd r oxym eth yl)-5-(2-m eth yla zir id in -1-yl)-1-m eth yl-

2-p h en ylin d ole-4,7-d ion e, 5cc. Red crystalline solid in 79%

yield: mp 163-164 °C (EtOAc/light petroleum) (lit.²⁹mp 147-

149 °C); UV (MeOH) 478 (log ꢀ 3.32) and 312 (4.14) nm; IR

(CH₂Cl₂) 3370, 3047, 2976, 1619, 1578, 1486, 1450, and 1260

cm^-^{1 1}H NMR (CDCl₃) δ 7.48 (3 H, m, ArH), 7.29 (2 H, m,

;

ArH), 5.82 (1 H, s, 6-H), 4.52 (2 H, d, J ) 7.3 Hz, CH₂OH),

4.19 (1 H, t, J ) 7.2 Hz, OH), 3.78 (3 H, s, NMe), 2.30 (1 H, m,

CH), 2.09 (2 H, m, CH₂), and 1.44 (3 H, d, J ) 5.5 Hz, Me); ¹³

NMR (CDCl₃) δ 181.2 (CO), 179.0 (CO), 157.2, 138.8, 130.6 (2

× CH), 130.2, 129.3 (CH), 128.8 (2 × CH), 128.6, 123.9, 122.8,

116.8 (CH), 56.2 (CH₂OH), 36.3 (CH), 34.7 (CH₂), 33.9 (NMe),

and 17.7 (Me).

Eth yl 3-Am in o-3-(4-bip h en ylyl)p r op en oa te, 7b. Pre-

pared using a modified procedure for enamine formation from

1,3-dicarbonyl compounds.³³Ethyl (4-phenylbenzoyl)acetate

(3.59 g, 13.4 mmol), ammonium acetate (10.75 g, 0.138 mol),

benzene (110 mL), and acetic acid (22 mL) were heated under

reflux using Dean-Stark conditions for 24 h. The cooled

reaction mixture was washed with saturated sodium hydrogen

carbonate (3 × 75 mL); the benzene layer was dried (Na₂SO₄)

and concentrated to give the title compound (3.48 g, 97%) as

an off-white crystalline solid: mp 90-92 °C (EtOAc/hexane).

Anal. (C₁₇H₁₇NO₂) C, H, N.

₁₇H₁₈N₂O₂requires M 282.1368. Anal. (C₁₇H₁₈N₂O₂‚0.2H₂O)

C, H, N.

Potassium nitrosodisulfonate (1.95 g, 7 mmol) in sodium

Eth yl 2-(4-Bip h en ylyl)-5-h yd r oxyin d ole-3-ca r boxyla te,

8b. 1,4-Benzoquinone (1.71 g, 15.8 mmol) and ethyl 3-amino-

3-(4-biphenylyl)propenoate (7b) (3.5 g, 9.8 mmol) were heated

under reflux for 1 h in acetic acid (50 mL). After cooling, the

crude product was precipitated out with light petroleum and

filtered off. The crude product was purified by column

chromatography (EtOAc) and recrystallized (EtOAc) to yield

dihydrogen phosphate buffer (0.3 M; 40 mL) was added to a

stirring solution of the above compound (0.79 g, 2.8 mmol) in

acetone (40 mL). The reaction mixture was stirred at room

temperature for 1 h. The excess acetone was removed in

vacuo, water (30 mL) was added, and the mixture was

extracted with dichloromethane (3 × 20 mL). The combined

organic extracts were washed with water (2 × 20 mL) and

brine (2 × 20 mL), dried (Na₂SO₄), and evaporated under

reduced pressure. The crude product was recrystallized

(EtOAc/light petroleum) giving the title compound 5z (0.35 g,

65% yield over two steps) as a red crystalline solid: mp 206-

208 °C (lit.²⁹mp 150-152 °C); UV (MeOH) 450 (log ꢀ 3.25)

and 344 (3.48) nm; IR (CH₂Cl₂) 3519, 3421, 3053, 2986, 1639,

a colorless solid (4.21 g, 90%): mp 260-262 °C. Anal. (C₂₃H₁₉

NO₃‚0.1H₂O) C, H, N.

E t h yl 2-(4-Bip h en ylyl)-5-m et h oxy-1-m et h ylin d ole-3-

ca r boxyla te, 9b. To a stirring solution of indole 8b (2.61 g,

7.3 mmol) in DMSO (20 mL) was added potassium hydroxide

(1.64 g, 29.3 mmol). After 30 min, iodomethane (4.15 g, 29.2

mmol) was added dropwise. The mixture was stirred at room

temperature for 3 h. The crude mixture was diluted with

EtOAc and washed thoroughly with 1 M hydrochloric acid. The

organic layer was separated, dried (MgSO₄), and concentrated.

The crude material was purified by column chromatography

(petroleum ether/EtOAc, 4:1) and recrystallized (EtOAc/hex-

ane) to yield the title compound (2.39 g, 85%) as a colorless

crystalline solid: mp 171-173 °C. Anal. (C₂₅H₂₃NO₃) C, H,

1593, 1491, 1265, and 1209 cm^-1; H NMR (CDCl₃) δ 7.48 (3

H, m, ArH), 7.30 (2 H, m, ArH), 5.71 (1 H, s, 6-H), 4.50 (2 H,

s, CH₂) 3.98 (1 H, br s, OH), 3.85 (3 H, s, OMe), and 3.78 (3 H,

s, NMe); ¹³C NMR (CDCl₃) δ 179.2 (CO), 178.9 (CO), 160.0,

139.2, 130.6 (2 × CH), 129.9, 129.4 (CH), 128.8 (2 × CH), 128.3,

124.1, 122.2, 107.4 (CH), 56.6 (OMe), 56.1 (CH₂), and 34.0 (Me).

3-(Hyd r oxym eth yl)-5-[(2-h yd r oxyeth yl)a m in o]-1-m eth -

yl-2-p h en ylin d ole-4,7-d ion e, 5a a . Purple crystalline solid

in 58% yield: mp 192-194 °C (dichloromethane); UV (MeOH)

524 (log ꢀ 3.40) and 314 (4.21) nm; IR (CH₂Cl₂) 3365, 3309,

E t h yl 2-(4-Bip h en ylyl)-5-m et h oxy-1-m et h yl-4-n it r o-

in d ole-3-ca r boxyla te, 10b. To a solution of indole 9b (1.41

g, 3.7 mmol) in acetic acid (15 mL), cooled to -10 °C, was added

a mixture of concentrated nitric acid (2 mL) and acetic acid

(7.5 mL). The mixture was stirred at room temperature for 2

h. A yellow suspension was obtained which was poured onto

an ice/water mixture; the crystals obtained were filtered off

and dried. The crude product was purified by column chro-

matography (light petroleum/EtOAc, 1:1) and recrystallized

(light petroleum/EtOAc) to yield the title compound (1.22 g,

77%) as a pale yellow crystalline solid: mp 277-279 °C.

E t h yl 4-Am in o-2-(4-b ip h en ylyl)-5-m et h oxy-1-m et h yl-

in d ole-3-ca r boxyla te, 11b. To a suspension of indole 10b

(0.76 g, 1.76 mmol) in EtOH (44 mL) were added tin powder

(0.94 g, 7.90 mmol) and hydrochloric acid (3 M; 13 mL). The

mixture was heated under reflux for 30 min. Upon cooling

the solution was decanted from the excess tin and neutralized

with saturated aqueous sodium hydrogen carbonate. The

suspension obtained was added to an equal volume of water.

The precipitate and aqueous layer were stirred overnight with

dichloromethane and filtered through Celite and the layers

separated. The organic layer was dried (Na₂SO₄) and concen-

trated. The crude product was purified by column chroma-

tography (dichloromethane/EtOAc, 7:3) and recrystallized

(light petroleum/EtOAc) to yield the title compound as a pale

2934, 1669, 1583, 1506, 1435, 1214, and 1163 cm^-1; H NMR

(DMSO-d) δ 7.50 (5 H, m, ArH), 7.02 (1 H, t, J ) 5.8 Hz, NH),

5.21 (1 H, s, 6-H), 4.86 (1 H, t, J ) 5.7 Hz, OH, D₂O exch),

4.55 (1 H, t, J ) 5.2 Hz, OH, D₂O exch), 4.37 (2 H, d, J ) 5.2

Hz, CH₂OH), 3.75 (3 H, s, NMe), 3.58 (2 H, m, CH₂), and 3.19

(2 H, m, CH₂); ¹³C NMR (DMSO-d) δ 178.9 (CO), 178.3 (CO),

149.4, 138.8, 131.4, 131.0 (2 × CH), 129.3 (CH), 129.0 (2 ×

CH), 122.5, 119.7, 111.6, 96.5 (CH), 58.8 (CH₂), 53.9 (CH₂),

45.3 (CH₂), and 34.2 (NMe); HRMS found (M⁺) 326.1272,

₁₈H₁₈N₂O₄requires M 326.1267. Anal. (C₁₈H₁₈N₂O₄‚2.4H₂O)

C, H, N.

5-(Cyclop r op yla m in o)-3-(h yd r oxym eth yl)-1-m eth yl-2-

p h en ylin d ole-4,7-d ion e, 5bb. Purple crystalline solid in

86% yield: mp 188-189 °C (EtOAc/light petroleum); UV

(MeOH) 520 (log ꢀ 3.30) and 318 (4.07) nm; IR (CH₂Cl₂) 3503,

3360, 2950, 1655, 1614, 1588, 1491, and 1178 cm^-1; H NMR

(CDCl₃) δ 7.48 (3 H, m, ArH), 7.29 (2 H, m, ArH), 5.94 (1 H,

br s, NH), 5.62 (1 H, s, 6-H), 4.50 (2 H, d, J ) 7.1, CH₂OH),

4.00 (1 H, t, J ) 7.1 Hz, OH), 3.81 (3 H, s, NMe), 2.49 (1 H, m,

CHNH), 0.88 (2 H, m, CH₂), and 0.65 (2 H, m, CH₂); ¹³C NMR

(CDCl₃) δ 180.1 (CO), 179.0 (CO), 149.2, 137.4, 132.1, 130.7 (2

× CH), 129.2 (CH), 128.8 (2 × CH), 123.3, 120.4, 99.6 (CH),

56.3 (CH₂OH), 34.0 (NMe), 24.3 (CH), and 7.1 (2 × CH); HRMS

4764 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24

Beall et al.

yellow crystalline solid (0.71 g, 97%): mp 204-205 °C. Anal.

romethane/ether) to give the title compound as a purple solid

(0.036 g, 74%): mp 260-262 °C; UV (MeOH) 546 (log ꢀ 3.50),

330 (4.12), 248 (4.36), and 224 (4.28) nm; IR (KBr) 2973, 2875,

(C₂₅H₂₄N₂O₃) C, H, N.

Eth yl 2-(4-Bip h en ylyl)-5-m eth oxy-1-m eth yl-4,7-d ioxo-

in d ole-3-ca r boxyla te, 5d d . To a solution of indole 11b

(0.161 g, 0.4 mmol) in acetone (15 mL) was added a solution

of potassium nitrosodisulfonate (0.54 g, 2.0 mmol) in sodium

dihydrogen phosphate buffer (0.3 M; 15 mL). The mixture was

stirred at room temperature for 1 h. The excess acetone was

removed in vacuo. The resulting residue was extracted with

dichloromethane and washed with water. The organic layer

was dried (Na₂SO₄) and concentrated. The crude product was

purified by column chromatography (EtOAc/dichloromethane,

1:1) and recrystallized (dichloromethane/light petroleum) to

yield the title compound as an orange solid (0.14 g, 87%): mp

204-205 °C; UV (MeOH) 434 (log ꢀ 3.19) and 278 (4.68) nm;

1677, 1624, and 1558 cm^-1; H NMR (CDCl₃) δ 6.17 (1 H, s,

9-H), 5.16 (1 H, s, 6-H), 4.22 (2 H, t, J ) 7.2 Hz, 3-CH₂), 3.61

(4 H, br s, NCH₂), 2.80 (2 H, t, J ) 7.4 Hz, 1-CH₂), 2.54 (2 H,

quintet, J ) 7.4 Hz, 2-CH₂), and 1.94 (4 H, m, CH₂); ¹³C NMR

(CDCl₃) δ 180.2 (CO), 177.1 (CO), 149.9, 142.5, 128.4, 126.9,

100.5, 98.9, 51.2 (CH₂), 46.5 (CH₂), 27.8 (CH₂), 25.3 (CH₂), and

13.4; HRMS found (M⁺) 256.1212, C₁₅H₁₆N₂O₂requires M

256.1212. Anal. (C₁₅H₁₆N₂O₂‚0.2H₂O) C, H, N.

9-F or m yl-7-m eth oxy-1,2-d ih yd r o-3H-p yr r olo[1,2-a ]in -

d ole-5,8-d ion e, 6d . Prepared as previously described.²²

9-(Hydr oxym eth yl)-7-m eth oxy-1,2-dih ydr o-3H-pyr r olo-

[1,2-a ]in d ole-5,8-d ion e, 6e. Prepared as previously de-

scribed.²²

IR (KBr) 3063, 2971, 2940, 1731, 1685, 1639, and 1598 cm^-1

;

¹H NMR (CDCl₃) δ 7.70 (2 H, m, ArH), 7.65 (2 H, m, ArH),

7.45 (4 H, m, ArH), 7.38 (1 H, m, ArH), 5.73 (1 H, s, 6-H), 4.22

(2 H, q, J ) 7.1 Hz, CH₂Me), 3.84 (3 H, s, OMe), 3.82 (3 H, s,

NMe), and 1.20 (3 H, t, J ) 7.1 Hz, CH₂Me); ¹³C NMR (CDCl₃)

δ 179.2, 175.5, 163.8, 160.3, 142.4, 142.2, 140.1, 130.7 (CH),

129.6, 128.9 (CH), 127.9 (CH), 127.3, 127.2 (CH), 127.1 (CH),

121.6, 114.9, 106.5 (CH), 61.1 (CH₂), 56.6 (OMe), 43.2 (NMe),

and 13.8 (Me); HRMS found (M⁺) 415.1420, C₂₅H₂₁NO₅re-

quires M 415.1419. Anal. (C₂₅H₂₁NO₅‚0.1H₂O) C, H, N.

2-(4-Bip h en ylyl)-3-(h yd r oxym eth yl)-5-m eth oxy-1-m e-

th ylin d ole-4,7-d ion e, 5ee. To a suspension of lithium alu-

minum hydride (0.41 g, 10.8 mmol) in THF (50 mL) at 0 °C

was added a solution of aminoindole 11b (1.12 g, 2.7 mmol) in

THF (25 mL). The reaction was allowed to warm to room

temperature and stirred for 30 min. The mixture was cooled

to 0 °C and quenched by the addition of water (0.5 mL), sodium

hydroxide (1 M; 0.5 mL), and silica gel (5 g). The granular

precipitate was filtered off through a pad of Celite. The filtrate

was dried (MgSO₄) and concentrated in vacuo to give the

alcohol which was used directly in the next step without

purification or characterization.

9-(Hyd r oxym eth yl)-7-(2-m eth yla zir id in -1-yl)-1,2-d ih y-

d r o-3H-p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6f. To a stirred

solution of 9-(hydroxymethyl)-7-methoxy-1,2-dihydro-3H-pyr-

rolo[1,2-a]indole-5,8-dione (6e) (0.108 g, 0.44 mmol) in DMF

(5 mL) was added 2-methylaziridine (0.81 g, 14.0 mmol), and

the mixture was stirred at room temperature overnight. The

reaction was shown to have gone to completion by TLC. The

excess aziridine was blown off by bubbling nitrogen directly

into the mixture. Water was added and the mixture extracted

with dichloromethane. The dichloromethane extracts were

washed with water and dried (Na₂SO₄). The solvent was

removed in vacuo and the residue purified by column chro-

matography (EtOAc/dichloromethane, 1:1) and recrystallized

(dichloromethane/light petroleum) to give the title compound

as a red solid (0.081 g, 68%): mp 133-135 °C; UV (MeOH)

486 (log ꢀ 3.28), 362 (3.56), 312 (4.19), and 232 (4.32) nm; IR

(KBr) 3572, 3375, 3276, 2980, 2927, 1624, and 1591 cm^-1; ¹H

NMR (CDCl₃) δ 5.72 (1 H, s, 6-H), 4.60 (2 H, d, J ) 6.9 Hz,

10-CH₂), 4.20 (2 H, t, J ) 7.2 Hz, 3-CH₂), 4.03 (1 H, t, J ) 6.9

Hz, OH), 2.85 (2 H, t, J ) 7.2 Hz, 1-CH₂), 2.55 (2 H, m, 2-CH₂),

2.27 (1 H, m, azir-CH), 2.10 (2 H, m, azir-CH₂), and 1.43 (3 H,

d, J ) 5.5 Hz, NCHMe); ¹³C NMR (CDCl₃) δ 181.0 (CO), 177.9

(CO), 157.8, 141.3, 127.4, 125.8, 117.8, 115.5 (CH), 56.6 (CH₂),

46.9 (CH₂), 36.3 (CH), 34.6 (CH₂), 27.4 (CH₂), 22.5 (CH₂), and

17.6 (Me); HRMS found (M⁺) 272.1161, C₁₅H₁₆N₂O₃requires

M 272.1161. Anal. (C₁₅H₁₆N₂O₃‚H₂O) C, H, N.

To a solution of the above compound in acetone (120 mL)

was added a solution of potassium nitrosodisulfonate (4.2 g,

15.7 mmol) in sodium dihydrogen phosphate buffer (0.3 M; 120

mL). The mixture was stirred at room temperature for 1 h.

The excess acetone was removed in vacuo. The resulting

residue was extracted with dichloromethane and washed with

water. The organic layer was dried (Na₂SO₄) and concen-

trated. The crude product was purified by column chroma-

tography (EtOAc/dichloromethane, 3:7) and recrystallized

(dichloromethane/light petroleum) to yield the title compound

as an orange/red solid (0.87 g, 87%): mp 200-201 °C; UV

(MeOH) 446 (log ꢀ 3.35), 350 (3.56), and 280 (4.69) nm; IR

(KBr) 3416, 3073, 2950, 1644, and 1598 cm^-1; ¹H NMR (CDCl₃)

δ 7.72 (2 H, m, ArH), 7.64 (2 H, m, ArH), 7.44 (5 H, m, ArH),

5.74 (1 H, s, 6-H), 4.57 (2 H, d, J ) 7.3 Hz, CH₂OH), 4.05 (1

H, t, J ) 7.3 Hz, OH), 3.87 (3 H, s, OMe), and 3.85 (3 H, s,

NMe); ¹³C NMR (CDCl₃) δ 179.2 (CO), 178.9 (CO), 160.0, 142.3,

140.0, 139.0, 131.1, 131.0 (CH), 130.0, 128.9 (CH), 127.9 (CH),

127.3 (CH), 127.1 (CH), 124.2, 122.2, 107.4 (CH), 56.6 (OMe),

9-(Hyd r oxym eth yl)-7-(p yr r olid in -1-yl)-1,2-d ih yd r o-3H-

p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6g. To a stirred solution of

9-(hydroxymethyl)-7-methoxy-1,2-dihydro-3H-pyrrolo[1,2-a]in-

dole-5,8-dione (6e) (0.07 g, 0.28 mmol) in DMF (4 mL) was

added pyrrolidine (0.17 g, 24 mmol), and the mixture was

stirred at room temperature overnight. Water was added and

the mixture extracted with dichloromethane. The dichlo-

romethane extracts were washed with water and dried (Na₂-

SO₄). The solvent was removed in vacuo and the residue

purified by column chromatography (EtOAc) and recrystallized

(dichloromethane/ether) to give the title compound as a purple

solid (0.07 g, 86%): mp 228-230 °C; UV (MeOH) 546 (log ꢀ

3.56), 330 (4.16), 248 (4.38), and 224 (4.29) nm; IR (KBr) 3289,

2966, 2861, 1657, 1618, and 1558 cm^-^{1 1}H NMR (CDCl₃) δ

;

56.1 (CH₂), and 34.1 (Me); HRMS found (M⁺) 373.1314, C₂₃H₁₉

5.17 (1 H, s, 6-H), 4.56 (2 H, d, J ) 6.8 Hz, 10-CH₂), 4.20 (2 H,

t, J ) 7.2 Hz, 3-CH₂), 4.08 (1 H, t, J ) 6.8 Hz, OH), 3.68 (4 H,

br s, NCH₂), 2.79 (2 H, t, J ) 7.2 Hz, 1-CH₂), 2.52 (2 H, quintet,

J ) 7.2 Hz, 2-CH₂), and 1.95 (4 H, m, CH₂); ¹³C NMR (CDCl₃)

δ 181.9 (CO), 176.8 (CO), 149.7, 139.5, 129.0, 124.4, 116.7,

100.7 (CH), 56.7 (CH₂), 51.4 (CH₂), 46.6 (CH₂), 27.5 (CH₂), 25.2

NO₄requires M 373.1314. Anal. (C₂₃H₁₉NO₄‚0.4H₂O) C, H,

9-(Hydr oxym eth yl)-7-m eth oxy-1,2-dih ydr o-3H-pyr r olo-

[1,2-a ]in d ole-5,8-d ion e, 6a . Prepared as previously de-

scribed.²²

7-(2-Meth yla zir id in -1-yl)-1,2-d ih yd r o-3H-p yr r olo[1,2-a ]-

in d ole-5,8-d ion e, 6b. Prepared as previously described.²³

7-(P yr r olidin -1-yl)-1,2-dih ydr o-3H-pyr r olo[1,2-a ]in dole-

5,8-d ion e, 6c. To a stirred solution of 7-methoxy-1,2-dihydro-

3H-pyrrolo[1,2-a]indole-5,8-dione (6a ) (0.044 g, 0.20 mmol) in

DMF (4 mL) was added pyrrolidine (0.170 g, 24 mmol), and

the mixture was stirred at room temperature overnight.

Water was added and the mixture extracted with dichlo-

romethane. The dichloromethane extracts were washed with

water and dried (Na₂SO₄). The solvent was removed in vacuo

and the residue purified by column chromatography (EtOAc/

light petroleum, 7:3, to EtOAc) and recrystallized (dichlo-

(CH₂), and 22.3 (CH₂); HRMS found (MH⁺) 287.1396, C₁₃H₁₄

N₂O₃+ H requires M 287.1395. Anal. (C₁₆H₁₈N₂O₃‚0.1H₂O)

C, H, N.

9-[(Ca r ba m oyloxy)m eth yl]-7-m eth oxy-1,2-d ih yd r o-3H-

p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6h . Prepared as previously

described.²²

7-(Azir idin -1-yl)-9-[(car bam oyloxy)m eth yl]-1,2-dih ydr o-

3H-p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6i. Prepared as previ-

ously described.²³

9-[(Ca r b a m oyloxy)m et h yl]-7-(2-m et h yla zir id in -1-yl)-

1,2-d ih yd r o-3H-p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6j. Pre-

pared as previously described.²³

Indolequinone Antitumor Agents

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4765

9-[(Ca r ba m oyloxy)m eth yl]-7-(p yr r olid in -1-yl)-1,2-d ih y-

d r o-3H-p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6k . A solution of

the carbamate 6h (0.061 g, 0.21 mmol), pyrrolidine (3.4 g, 47.9

mmol), and DMF (5 mL) was stirred at room temperature for

16 h. The mixture was evaporated and the residue recrystal-

lized (MeOH/light petroleum) to give the title compound (0.057

g, 82%) as a purple solid: mp >300 °C; UV (MeOH) 538 (log

ꢀ 3.52), 326 (4.09), 248 (4.32), and 222 (4.23) nm; IR (KBr)

trolyte in DMF. Oxygen was removed from the solutions by

purging with nitrogen. All measurements were performed at

21 ( 2 °C.

Biology. NADH and MTT were obtained from Sigma

Chemical Co., St. Louis, MO. All other reagents were at least

of analytical grade.

1. Cell Cu ltu r e. H460 and H596 cells were grown in

minimum essential medium (MEM) with Earle’s salts, nones-

sential amino acids, ^L-glutamine, and penicillin/streptomycin

and supplemented with 10% fetal bovine serum. Cell culture

medium and supplements were obtained from Life Technolo-

gies, Inc., Grand Island, NY. The cells were incubated at 37

°C under a humidified atmosphere containing 5% CO₂.

2. Hu m a n Recom bin a n t NQO1. Recombinant human

NQO1 from Escherichia coli was expressed, purified, and

characterized as previously described.³⁴

3. HP LC An a lysis. Reduction of the indolequinones was

followed by HPLC using an Alltech C18 (5 µm, 250 mm × 4.6

mm) column with a Shimadzu HPLC system (SCL-6A control-

ler, SPD-6AV UV-vis detector, two LC-6A pumps, and a

C-R3A integrator). The solvent program used a linear gradient

of 5-80% B over 10 min, 80% B for 5 min, then 80-5% B over

5 min (solution A, 10 mM potassium phosphate buffer, pH 6.0;

solution B, methanol). Reactions were run in 25 mM Tris-

HCl (pH 7.4) containing 200 µM NADH, 50 µM indolequinone,

and recombinant human NQO1. NADH oxidation and quinone

removal were quantified at 340 nm following 30-min incuba-

tions at 22 °C.

4. Cytotoxicity Assa y. Cytotoxicity was determined using

the MTT colorimetric assay.³⁵Cells were plated in 96-well

plates at a density of (1-2) × 10⁴cells/mL and allowed to

attach overnight (16 h). Indolequinone solutions were applied

in serumless media for 2 h. Indolequinone solutions were

removed and replaced with complete media (with serum), and

the 96-well plates were incubated for 5-7 days. MTT (50 µg)

was added, and the cells were incubated for another 4 h.

Media/MTT solutions were removed carefully by aspiration,

the MTT formazan crystals were dissolved in 100 µL of DMSO,

and absorbance was determined on a plate reader at 550 nm.

IC₅₀values (concentration at which cell survival equals 50%

of control) were determined from semilog plots of percent of

control versus concentration.

5. In a ctiva tion of NQO1 by In d olequ in on es. The

ability of the indolequinones to inactivate NQO1 was deter-

mined using a previously reported procedure³¹with some

modifications. Inactivation was determined spectrophoto-

metrically following 30-min incubations at 22 °C in the

presence (+) or absence (-) of NADH. Incubations (0.5 mL)

contained (() 100 µM NADH, 10 µM indolequinone, and 1 µg

of recombinant hNQO1 in 25 mM Tris-HCl (pH 7.4) + 2 mg/

mL Tween-20. After 30 min, reactions were stopped with 4

volumes (2 mL) of cold 25 mM Tris-HCl (pH 7.4) + 250 mM

sucrose. Enzyme activity remaining was determined using a

standard NQO1 activity assay.²The NQO1 assay (1 mL)

contained a 50-µL sample from the incubation reaction, 40 µM

2,6-dichlorophenol-indophenol (DCPIP), and 200 µM NADH

in 25 mM Tris-HCl (pH 7.4) + 0.7 mg/mL bovine serum

albumin (BSA). DCPIP reduction was followed for 30 s at 600

nm. NQO1 activity from (+) NADH incubations was compared

to (-) NADH controls for calculations of enzyme activity

remaining (%).

3440, 3355, 3210, 2934, 2855, 1690, 1664, 1618, and 1552 cm^-1

;

¹H NMR (DMSO-d) δ 6.44 (2 H, br s, NH₂), 5.04 (1 H, s, 6-H),

5.01 (2 H, s, 10-CH₂), 4.09 (2 H, m, 3-CH₂), 3.51 (4 H, br s,

NCH₂), 2.78 (2 H, m, 1-CH₂), 2.44 (2 H, m, 2-CH₂), and 1.86 (4

H, m, CH₂); ¹³C NMR (DMSO-d) δ 180.5, 175.9, 157.2, 149.7,

142.3, 127.9, 123.1, 111.6, 100.0 (CH), 57.8 (CH₂), 51.4 (CH₂),

46.9 (CH₂), 27.4 (CH₂), and 23.0 (CH₂); HRMS found (MH⁺)

330.1454, C₁₇H₁₉N₃O₄+ H requires M 330.1454. Anal.

(C₁₇H₁₉N₃O₄‚0.4H₂O) C, H, N.

9-[(Ca r ba m oyloxy)m eth yl]-7-(p ip er id in -1-yl)-1,2-d ih y-

d r o-3H-p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6l. A solution of the

carbamate 6h (0.061 g, 0.21 mmol), piperidine (3.4 g, 40.5

mmol), and DMF (5 mL) was stirred at room temperature for

16 h. The mixture was evaporated and the residue chromato-

graphed (EtOAc) and recrystallized (dichloromethane/light

petroleum) to give the title compound (0.049 g, 73%) as a dark

purple solid: mp 231-232 °C; UV (MeOH) 534 (log ꢀ 3.52),

328 (4.11), and 238 (4.35) nm; IR (KBr) 3421, 3309, 3210, 2940,

2855, 1749, 1710, 1670, 1618, and 1552 cm^-1; ¹H NMR (DMSO-

d) δ 5.47 (1 H, s, 6-H), 5.21 (2 H, s, 10-CH₂), 4.69 (2 H, br s,

NH₂), 4.20 (2 H, m, 3-CH₂), 3.40 (4 H, m, NCH₂), 2.89 (2 H, m,

1-CH₂), 2.53 (2 H, m, 2-CH₂), and 1.68 (6 H, m, CH₂); ¹³C NMR

(DMSO-d) δ 180.7, 177.8, 156.8, 155.2, 143.3, 127.3, 125.2,

111.3, 107.8 (CH), 58.5 (CH₂), 51.1 (CH₂), 46.8 (CH₂), 27.3

(CH₂), 25.8 (CH₂), 24.3 (CH₂), and 23.0 (CH₂); HRMS found

(MH⁺) 344.1610, C₁₈H₂₁N₃O₄+ H requires M 344.1610. Anal.

(C₁₈H₂₁N₃O₄‚0.5H₂O) C, H, N.

9-[(Ca r b a m oyloxy)m et h yl]-7-(N-cyclop r op yla m in o)-

1,2-d ih yd r o-3H-p yr r olo[1,2-a ]in d ole-5,8-d ion e, 6m . A so-

lution of the carbamate 6h (0.057 g, 0.19 mmol), cyclopropyl-

amine (3.3 g, 57.8 mmol), and DMF (5 mL) was stirred at room

temperature for 16 h. The mixture was evaporated and the

residue chromatographed (EtOAc/2-propanol, 9:1) and recrys-

tallized (MeOH/light petroleum) to give the title compound

(0.050 g, 81%) as a purple solid: mp 257-258 °C; UV (MeOH)

524 (log ꢀ 3.29), 316 (3.87), and 242 (4.32) nm; IR (KBr) 3427,

3348, 3309, 3204, 2980, 1703, 1664, 1611, and 1591 cm^-1; H

NMR (DMSO-d) δ 7.30 (1 H, d, J ) 2.9 Hz, NH), 6.44 (2 H, br

s, NH₂), 5.30 (1 H, s, 6-H), 5.00 (2 H, s, 10-CH₂), 4.10 (2 H, m,

3-CH₂), 2.78 (2 H, m, 1-CH₂), 2.42 (3 H, m, 2-CH₂, cycloprop-

CH), 0.74 (2 H, m, cycloprop-CH₂), and 0.59 (2 H, m, cycloprop-

CH₂); ¹³C NMR (DMSO-d) δ 178.6, 177.1, 157.1, 151.1, 142.6,

128.4, 122.5, 111.6, 96.8 (CH), 57.5 (CH₂), 47.1 (CH₂), 27.4

(CH₂), 24.9 (CH), 22.9 (CH₂), and 6.8 (CH₂); HRMS found (M⁺)

315.1219, C₁₆H₁₇N₃O₄requires M 315.1219. Anal. (C₁₆H₁₇N₃O₄‚

0.4H₂O) C, H, N.

Electr och em ica l Mea su r em en ts. Tetrabutylammonium

tetrafluoroborate (Bu₄NBF₄) (Aldrich, 99%) was dried under

vacuum at 70 °C. Ferrocene (Aldrich, 97%) was resublimed.

Dimethylformamide (DMF) (Aldrich, 99.8%) was dried over 4-Å

molecular sieves.

For each of the indolequinones studied, cyclic voltammo-

grams were recorded over a range of potential sweep rates from

25 to 500 mV s^-1, using an E.G. & G. Princeton Applied

Research model 273 potentiostat, controlled by an IBM-

compatible PC using E.G. & G. Princeton Applied Research

model 270/250 research electrochemistry software. A three-

electrode cell was employed, with a silver quasi-reference

electrode and a platinum disk working electrode. The working

electrode was pretreated by polishing with 3-µm diamond

paste, followed by anodization and then cathodization for 2-3

min each in 0.1 M H₂SO₄, before rinsing with deionized water

and drying. Solutions for voltammetry were all 1 mM in the

indolequinone, approximately 1 mM in ferrocene (added as an

internal reference), and 0.1 M in Bu₄NBF₄supporting elec-

Ack n ow led gm en t. We acknowledge support from

the U.K. Cancer Research Campaign, the University of

Exeter, and R01 CA 51210.

Su p p or tin g In for m a tion Ava ila ble: Spectroscopic data

for intermediates (4 pages). Ordering information is given on

any current masthead page.

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J M980328R

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