2-cyclopropylindoloquinones and their analogues as bioreductively activated antitumor agents: Structure-activity in vitro and efficacy in vivo

DOI: 10.1021/jm9608422

Source and publish data:

Journal of Medicinal Chemistry p. 2335 - 2346 (1997)

Update date:2022-08-03

Topics:

Authors:

Naylor, Matthew A.

Jaffar, Mohammed

Nolan, John

Stephens, Miriam A.

Butler, Susan

Patel, Kantilal B.

Everett, Steven A.

Adams, Gerald E.

Stratford, Ian J.

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Article abstract of DOI:10.1021/jm9608422

A series of 2-cycloalkyl- and 2-alkyl-3-(hydroxymethyl)-1- methylindoloquinones and corresponding carbamates have been synthesized and substituted in the 5-position with a variety of substituted and unsubstituted aziridines. Cytotoxicity against hypoxic c

Full text of DOI:10.1021/jm9608422

J . Med. Chem. 1997, 40, 2335-2346

2335

2-Cyclop r op ylin d oloqu in on es a n d Th eir An a logu es a s Bior ed u ctively Activa ted

An titu m or Agen ts: Str u ctu r e-Activity in Vitr o a n d Effica cy in Vivo

Matthew A. Naylor,*^,†Mohammed J affar,^‡J ohn Nolan,^§Miriam A. Stephens,^§Susan Butler,^§Kantilal B. Patel,^†

Steven A. Everett,^†Gerald E. Adams,^†and Ian J . Stratford^‡

Gray Laboratory Cancer Research Trust, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex, HA6 2J R,

United Kingdom, Department of Pharmacy, University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom,

Departments of Medicinal Chemistry and Experimental Cancer Therapy, Division of Experimental Oncology, Medical Research

Council, Harwell, Oxon OX11 ORD, United Kingdom

Received December 11, 1996^X

A series of 2-cycloalkyl- and 2-alkyl-3-(hydroxymethyl)-1-methylindoloquinones and corre-

sponding carbamates have been synthesized and substituted in the 5-position with a variety

of substituted and unsubstituted aziridines. Cytotoxicity against hypoxic cells in vitro was

dependent upon the presence of a 5-aziridinyl or a substituted aziridinyl substituent for

3-hydroxymethyl analogues. The activity of 5-methoxy derivatives was dependent upon the

presence of a 3-(carbamoyloxy)methyl substituent. Increasing the steric bulk at the 2-position

reduced the compounds’ effectiveness against hypoxic cells. A 2-cyclopropyl substituent was

up to 2 orders of magnitude more effective than a 2-isopropyl substituent, suggesting possible

radical ring-opening reactions contributing to toxicity. Nonfused 2-cyclopropylmitosenes were

more effective than related fused cyclopropamitosenes reported previously. The reduction

potentials of the quinone/semiquinone one-electron couples were in the range -286 to -380

mV. The semiquinone radicals reacted with oxygen with rate constants 2-8 × 10⁸dm³mol^-1

s^-1. The involvement of the two-electron reduced hydroquinone in the mediation of cytotoxicity

is implicated. The most effective compounds in vitro were the 2-cyclopropyl and 5-(2-

methylaziridinyl) derivatives, and of these, 5-(aziridin-1-yl)-2-cyclopropyl-3-(hydroxymethyl)-

1-methylindole-4,7-dione (21) and 3-(hydroxymethyl)-5-(2-methylaziridin-1-yl)-1,2-dimethylindole-

4,7-dione (54) were evaluated in vivo. Both compounds showed antitumor activity both as

single agents and in combination with radiation, with some substantial improvements over

EO9 (3) at maximum tolerated doses and as single agents against the RIF-1 tumor model and

comparable efficacy in the KHT tumor model.

In tr od u ction

Fused cyclopropamitosenes and closely related in-

doloquinones have recently been evaluated as novel

bioreductive anticancer agents targeted toward both

solid tumors with defined hypoxic fractions and tumor

tissues that may be rich in the required activating

enzymes.^1-3The reduction of quinones bearing ap-

propriate leaving groups to generate alkylating species,

originally termed bioreductive alkylation,⁴is now well

established, although the novel cyclopropamitosenes

were originally designed as analogues of the prototype

quinone bioreductive alkylating agent mitomycin c

(MMC, 1, Figure 1).⁵These compounds were expected

to exhibit much reduced electophilicty at C-1 due to the

inertness of the 1,2-cyclopropane compared to the aziri-

dine in MMC. Certain indoloquinones (e.g. 2, Figure

1) in this series were found to be highly potent cytotox-

ins compared with MMC and in some cases with

substantially higher hypoxic cytotoxicty ratios (HCR).^1-3

The cyclopropamitosenes were found to be more rapidly

reduced than MMC by DT-diaphorase (NAD(P)H:

(quinone acceptor) oxidoreductase (EC 1.6.99.2)),³an

important activator of mitosenes and an enzyme hyper-

expressed in some tumors.^6-8However, this could not

explain fully the greater potency (aerobic and hypoxic)

F igu r e 1. Structures of known lead compounds.

of the compounds compared to the current lead clinical

agent of this general type EO9 (3, Figure 1),⁹which is

reduced 2 orders of magnitude more rapidly than 2 by

DT-diaphorase.^3,10The nature of the 5-substituent (in

particular aziridines and substituted aziridines) and of

the potential leaving group on the 3-methylene sub-

stituent was therefore identified as an important feature

both in terms of oxic and hypoxic potency and ability to

act as substrates for reductase enzymes. The reduction

potential does not vary greatly among 5-aziridinyl and

5-methoxy derivatives.^2,11However, the relative rates

of reduction of this type of compound by one-electron

reductases, which will be important under hypoxic

conditions, is unknown. This may be related to the one-

electron reduction potential of the quinone/semiquinone

couple (E(Q/ Q^•-)), but there are little published data

on indoloquinones in aqueous solution. Such data would

also be an indicator of the redox-cycling propensities of

such compounds and thus be related to aerobic toxicity

^†Gray Laboratory.

^‡University of Manchester.

^§Medical Research Council.

^XAbstract published in Advance ACS Abstracts, J une 15, 1997.

2336 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15

Naylor et al.

Sch em e 1. Synthesis of 2-Cycloalkyl- and

mediated through reactive oxygen species as well as a

measure of ease of reduction by one-electron reducing

enzymes such as NADPH:cytochrome P-450 reductase

and xanthine oxidase.

2-Alkylindoloquinones^a

Intramolecular ionic ring opening of the fused cyclo-

propane was considered unlikely,³but radical ring

opening of the cyclopropane following one-electron

reduction to give a reactive H atom abstractor has been

suggested as a possible explanation for hypoxic potency,

although there was no direct evidence for this mecha-

nism.³We have therefore designed and synthesized the

analogues of 2 in which the cyclopropane rings are not

fused with the indoloquinone ring system and for which

isopropyl analogues could be synthesized that are

structurally very closely related but unable to undergo

radical ring-opening reactions. These derivatives were

also designed as closer analogues of EO9 (3), initially

retaining a hydroxymethyl substituent at the 3-position

together with an alkyl substituent at the 2-position, to

give a series of compounds which have remained un-

evaluated to date. We have sought to obtain further

structure-activity data for this series of bioreductively

activated drugs as hypoxic cell cytotoxins with the aim

of optimizing the hypoxic-cytotoxicity ratios and achiev-

ing activity in vivo, hitherto unseen with the fused

cyclopropamitosenes such as 2. The relationship of

these biological effects to the redox properties of the

drugs was also studied.

Syn th etic Ch em istr y

2-Alkyl- and 2-cycloalkyl-substituted indoles were

synthesized in 14 steps from the common precursor

3-chlorophenol as shown in Scheme 1. The crucial step

was the 1,5-electrocyclization of the imine (e.g. 8) to the

2,3-dihydroindole derivatives, which was successfully

carried out using isobutyraldehyde or cycloalkane car-

boxaldeydes, but not with acrolein in a proposed alter-

native route, using zinc acetate in methanol as has been

employed in the synthesis of 3 via a 2-acrylate deriva-

tive.⁹Nitration at the desired 4-position could be

achieved only subsequent to the N-methylation step in

order to avoid increasing yields of the 6-nitro isomer.

The subsequent six steps, including nitration, oxidations

(DDQ and Fremy’s salt), and reductions (Sn/HCl,

Na₂S₂O₄, and DIBAL-H or LiAlH₄), left the 2-cyclopropyl

moiety intact, and the desired indoloquinones were

obtained. Substitution of the 5-methoxy substituent

was successful in high-yielding reactions with aziridine

and 2-methylaziridine.

Comparable 1,2-dimethyl analogues 52-56 were syn-

thesized from commercially available 2-methyl-5-meth-

oxyindole in seven steps (Scheme 2). Substitution of the

5-position with aziridine and 2-methylaziridine was

again successful in high yield, as was the substitution

with cis-2,3-dimethylaziridine. However, 2,2-dimeth-

ylaziridine reacted with difficulty, and the resulting

5-(2,2-dimethylaziridinyl) analogue was unstable in

aqueous solution and on silica gel, ring opening via an

S_N1 mechanism to give 57 (Scheme 2). The 2-unsub-

stituted analogues 62-66 were obtained in six steps

from 5-methoxyindole-3-carboxaldehyde (Scheme 3).

Reagents: (i) H₂SO₄/NaNO₂/H₂O/C₅H₅N; (ii) K₃Fe(CN)₆/KOH/

H₂O; (iii) NaH/THF/(MeO)₂SO₂; (iv) NCCH₂CO₂Et; (v) HCl/EtOH/

H₂O; (vi) H₂/PtO₂/PhMe/EtOH; (vii) RCHO/MeOH; (viii) Zn(OAc)₂/

MeOH; (ix) Ac₂O; (x) KOH/EtOH/H₂O/0 °C; (xi) (MeO)₂SO₂/DMF/

K₂CO₃; (xii) DDQ/PhMe/reflux; (xiii) 4%KOH/MeOH; (xiv) NaH/

DMF/MeI/60 °C; (xv) fuming HNO₃/AcOH/4 °C; (xvi) Sn/HCl/

EtOH/H₂O; (xvii) Fremy’s salt/Me₂CO/NaH₂PO₄/Na₂HPO₄/pH 6.0;

(xviii) 1H-aziridine; (xix) Na₂S₂O₄/H₂O/EtOH/CHCl₃then LiAlH₄/

THF/30 °C (DIBAL-H/PhMe/-30 °C then 0 °C for 20) then FeCl₃/

Resu lts a n d Discu ssion

HCl/H₂O/0 °C; (xx) R₁-CHCH₂NH; (xxi) PhCO₂Cl/C₅H₅N/0 °C;

In previous studies the presence of a hydroxymethyl

substituent at the 3-position of indoloquinone anticancer

(xxii) NH₃/CH₂Cl₂/-78 °C; (xxiii) R₁-CHCH₂NH.

Bioreductively Activated Antitumor Agents

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2337

Sch em e 2. Synthesis of 2-Methyl Analogues^a

Sch em e 4. Potential Mechanisms of Toxicity for

2-Cyclopropyl- and 2-Alkyl-Substituted Indoloquinones

potency (see compounds 21 and 25, 38 and 42) while in

other cases potency shows the expected increase (e.g.

compounds 20 and 24, 22 and 26, 61 and 66). Excep-

tional compounds are always 5-aziridinyl derivatives,

suggesting that the dominance of aziridine-mediated

toxicity may outweigh effects due to the nature of the

potential leaving group at the 3-position in many cases.

5-Methoxy derivatives generally showed improved po-

tency and HCR when carbamate replaced hydroxy on

the 3-methylene substituent, indicating that the nature

of this potential leaving group^1-3,12-14can dominate the

bioreductive properties of non-aziridinyl analogues.

That the 3-hydroxymethyl substituent is required for

hypoxia-selectivity is indicated when comparing the

5-aziridinyl-3-methyl derivative 44 (HCR ) 1.77) with

the corresponding 3-hydroxymethyl analogue 37 (HCR

) 32.5).

Reagents: (i) NaH/DMF/MeI/60 °C; (ii) HCON(Me)Ph/POCl₃

then NaOAc/H₂O; (iii) fuming HNO₃/AcOH/4 °C; (iv) Sn/HCl/H₂O/

EtOH; (v) Fremy’s salt/Me₂CO/NaH₂PO₄/Na₂HPO₄/pH 6.0; (vi)

NaBH₄/MeOH/Ar then air; (vii) R(R₁)CCH(R₂)NH; (viii) H₂O/45

°C.

Sch em e 3^a

There is a clear trend of increasing aerobic and

hypoxic potency on reducing the steric bulk of substitu-

ent R₂(see series of compounds 21, 37, 46, and 53 for

example). Thus, the least potent 5-aziridinyl analogue

tested (both hypoxic and oxic) was the 2-cyclohexyl

derivative 46 while the most potent compound tested

(both hypoxic and oxic) was 66, which has both aziridine

and carbamate functionalities and no 2-alkyl substitu-

ent. The low HCR for this latter compound, however,

demonstrates the clear need for these structure-activity

studies to obtain both optimal potency and optimal

hypoxia-selectivity.

Reagents: (i) NaH/DMF/MeI; (ii) concentrated HNO₃/AcOH/0

°C; (iii) Sn/HCl/H₂O/EtOH; (iv) Fremy’s salt/Me₂CO/NaH₂PO₄/

Certain aziridinyl 2-cyclopropyl analogues do not fit

into this pattern and also show much increased potency,

particularly under hypoxic conditions, compared to

corresponding isopropyl derivatives (e.g. compare 21

with 37 and 25 with 41). Reactions of the semiquinone

radical can dominate under severe hypoxia following

one-electron reduction, and these data provide some

indirect, though compelling, evidence for a contribution

from semiquinone induced radical ring opening of the

cyclopropane under such hypoxic conditions, possibly

leading to further cytotoxic reactions. The full elucida-

tion of the mechanism of such toxicity, hypothesized in

Scheme 4, clearly requires further study.

Na₂HPO₄/pH 6.0; (v) NaBH₄/MeOH/Ar; (vi) R-CHCH₂NH; (vii)

PhCO₂Cl/C₅H₅N/0 °C; (viii) 1H-aziridine.

drugs has been thought to give inactive drugs, with the

majority of compounds evaluated consequently possess-

ing carbamate or acetate derived leaving groups.^12-14

The success of the mitosenediol EO9 (3) and earlier in

vivo activity of related compounds, however,^7,9,14-16

suggests the importance of this class of compound, the

structure of which has yet to be optimized. The in vitro

biological data presented in this study (Table 1) add

credence to this suggestion, indeed substitution with the

carbamate moiety has in many cases actually reduced

2338 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15

Naylor et al.

Ta ble 1. Structures of Compounds and in Vitro Biological Data Comparing Differential Aerobic/Hypoxic Cytotoxicities

compd

type

R₁

R₂

C₅₀(air), µM

C₅₀(N₂), µM

HCR^a

MMC

EO9

0.8^b

0.4^b

2.0^b

1.0^d

50.3

1.8

13.2

1.7

0.65

65.9

1.8

103.5

45.0

23.4

27.23

1.0

4.0

32.5

9.2

178.6

8.7

23.7

1.88

1.77

6.0

37.0

3.78

12.8

188

14.2

2.5

0.92

15.4

24.7

83.8

Az^c

CH₂OCONH₂

0.003^d

0.19 ( 0.027

108.4 ( 5.9

0.965 ( 0.11

103.5 ( 5.2

540 ( 52

0.0038 ( 0.00057

60.6 ( 7.4

0.073 ( 0.011

59.8 ( 11.4

820 ( 83

0.44 ( 0.023

0.93 ( 0.067

0.0058 ( 0.0013

0.074 ( 0.015

5.6 ( 0.6

0.459 ( 0.096

150 ( 15

200 ( 20

0.79 ( 0.14

13.9 ( 1.5

0.117 ( 0.018

5.46 ( 0.43

0.87 ( 0.215

0.657 ( 0.063

0.188 ( 0.011

20.5 ( 5.3

MeO

CH₂OH

CO₂Me

CH₂OH

CH₂OCONH₂

CO₂Me

CH₂OH

CH₂OCONH₂

CH₂OH

CH₂OCONH₂

CO₂Me

CH₂OH

CH₂OCONH₂

CH₂OH

c-Pr

CH(Me)₂

c-Hex

29 ( 5.6

1.72 ( 0.18

0.603 ( 0.099

3.33 ( 0.75

130 ( 9.8

2-Me-Az

MeO

2-Me-Az

MeO

2-Me-Az

12.5 ( 2.9

150 ( 15

800 ( 80

25.7 ( 3.0

127.8 ( 13.5

20.9 ( 1.67

47.5 ( 14.6

20.6 ( 2.5

1.236 ( 0.189

0.333 ( 0.014

122.4 ( 17.5

34.5 ( 6.0

1077 ( 44

0.149 ( 0.011

94 ( 7.6

202 ( 11

1260 ( 126

220 ( 20

CH₂OH

0.93 ( 0.078

284.8 ( 37.6

0.0116 ( 0.0008

0.5 ( 0.07

2,3-Me₂-Az

Me₂C(OH)CH₂NH-

14.2 ( 1.7

500 ( 120

MeO

2-Me-Az

MeO

240 ( 23

0.153 ( 0.013

4.42 ( 1.17

3.1 ( 0.24

0.0086 ( 0.0009

0.0179 ( 0.02

0.037 ( 0.007

CH₂OH

CH₂OCONH₂

0.00019 ( 0.000032

0.00013 ( 0.000025

1.46

HCR ) hypoxic cytotoxicity ratio (C₅₀(air)/C₅₀(N₂)). Reference 32. ^cAz ) aziridin-1-yl. Reference 2.

There is evidence in the present results for clear

advantages in terms of both hypoxia-selectivity and

hypoxic potency to compounds with a 2-cyclopropyl

substituent rather than a 1,2-fused cyclopropane system

(compare compounds 5 and 21). This effect seems to

be lessened when more potent leaving groups are

present such as in carbamate 24, which is comparable

in its potencies to its fused analogue²(compare 2 with

25).

An increase in HCR upon alkyl substitution of the

aziridinyl moiety has been demonstrated in this study

with some (compare 41 and 42, 53 and 54, 63 and 64)

but not all types of compound (compare 21 and 22, 25

and 26). Significantly, the compounds which do not fall

into this pattern are again the 2-cyclopropane deriva-

tives, where toxicity is not dominated by the reactivity

of the aziridine moiety to the same degree as other

2-alkyl derivatives, possibly due to a contribution from

the cyclopropane ring (e.g. Scheme 4). Generally, a

single methyl substituent on the 5-aziridine ring is

optimal. With multiple substitution, the potency and

HCR is much reduced due to progressive deactivation

of the aziridine counteracting increasing pK_a.

However EO7⁹(the 5-methoxy analogue of EO9 (3)) and

other 5-methoxy derivatives selected from this study 20,

24, 40, and 62 were less electron-affinic than corre-

sponding 5-aziridinyl compounds and reacted typically

2-4 times faster with oxygen. Thus 5-aziridinyl semi-

quinone radicals will be longer-lived than 5-methoxy

analogues in the presence of oxygen, which, in addition

to reductive aziridine activation, may also contribute

to their greater effectiveness compared to 5-methoxy-

indoloquinones following one-electron reduction. This

will also be relevant following two-electron reduction

since it will slow down the rate of reoxidation of the

hydroquinone in sequential one-electron steps, and

influence the lifetime of semiquinones formed through

a comproportionation reaction between an indoloqui-

none and its hydroquinone.

The one-electron reduction potentials at pH 8.5 for

the aziridinylindoloquinones 53 (-294 mV) and 21

(-286 mV) were similar to that of EO9 (3), where E (Q/

^•-) ) -265 mV (lit.¹⁷-253 mV). The redox potentials

of 5-methoxyindoloquinones were generally 25-50 mV

lower than the corresponding 5-aziridinyl analogues.

This is consistent with recent half-wave potential reduc-

tion data on related indoloquinones²(e.g. compound 2

Redox chemical studies (Table 2) demonstrated that

analogues containing an aziridine group exhibited a

similar reactivity toward oxygen as EO9 (3)¹⁷(k₂(see

Experimental Redox Chemistry Section, eq 2) was

generally found to be in the order of 2 × 10⁸M^-1s^-1).

E_redox) -1.360 V and its 5-MeO analogue E_redox

)

-1.395 V). These differences in reduction potential

were reflected in the corresponding rates of reaction of

semiquinone radicals with oxygen (see Table 2). How-

Bioreductively Activated Antitumor Agents

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2339

Ta ble 2. One-Electron Reduction Potentials at pH 8.5 (E Q/Q^•-) for Representative Compounds and Rate Constants for Reaction of

Semiquinone Radicals with Oxygen

E (Q/Q^•-),

10⁸k₂(Q^•-+ O₂),

dm³mol^-1s^-1

compd

R₁

R₂

K₃

EO9(3)

EO7

Az^b

2-Me-Az

MeO

CH₂OH

CH₂OCONH₂

CHdCHCH₂OH

c-Pr

CHdCHCH₂OH

c-Pr

CH(CH₃)₂

c-Pr

39.6 ( 5.7

41.2 ( 1.4

30.1 ( 1.5

19.7 ( 1.7

15.5 ( 1.5

6.8 ( 0.4

-265 ( 5^c

-286 ( 4^c

-294 ( 4^c

-305 ( 4^c

-309 ( 5^c

-332 ( 4^d

-334 ( 4^c

-380 ( 4^d

-377 ( 9^d

1.7 ( 0.1

2.3 ( 0.1

2.8 ( 0.1

2.4 ( 0.1

4.5 ( 0.1

4.4 ( 0.1

5.1 ( 0.2

6.3 ( 0.1

8.2 ( 0.2

6.2 ( 0.2

20.3 ( 0.7

19.8 ( 1.7

Means of measurements using five different concentrations of indoloquinones. Az ) aziridin-1-yl. Potentials vs E(BV²⁺/BV^•-) )

-374 mV at pH 8.5. Potentials vs E(MV²⁺/MV^•-) ) -450 mV at pH 8.5 (will be the same at pH 7.4).

Ta ble 3. Comparison of Indoloquinones 21 and 54 with EO9

ever, 5-methoxy compounds bearing a good 3-methylene

leaving group also have exquisite hypoxia-selectivity

(e.g. 40 and 65); therefore there appears to be little

relationship between reduction potential and HCR as

determined by the MTT assay in this study, particularly

in view of the low reduction potentials determined for

the hypoxia-selective carbamates 24 and 40 compared

with EO9 (3). However, it should be noted that under

conditions of greater oxygen concentration, reduction

potential is likely to have a greater influence on reduced

(3) When Used in Combination with X-rays (20 and 50 mg

kg^-1) and Alone (MTD) in the RIF-1 and KHT Tumors

X-ray

dose,

mean relative

surviving

fraction

drug dose,

mg kg^-1

MTD,

tumor

drug

mg kg^-1

control

RIF-1

KHT

1.0

2.0 × 10^-3

5.0 × 10^-3

2.4 × 10^-5

EO9 (3)

100

<10^-5

2.0 × 10^-2

drug reactivity. The trends between k₂and E (Q/Q^•-

)

<10^-5

for the indoloquinones in this study are comparable with

those published for quinones of similar redox potential.¹⁸

Thus although the indoloquinones should be easily

reduced by enzymes such as NADPH-cytochrome P-450

and DT-diaphorase, the high values measured for k₂

would indicate that even under modest tumor hypoxia

(ca. 10 µmol dm^-3O₂) the half-life of the semiquinone

radicals {∼0.7/(k₃[O₂]} will be short (∼0.4 ms).

<10^-5

0.7

1.2 × 10^-3

1.6 × 10^-3

1.0 × 10^-4

9 × 10^-3

EO9 (3)

100

6.0 × 10^-2

8.0 × 10^-3

7.0 × 10^-4

1.5 × 10^-1

8.0 × 10^-3

5.0 × 10^-3

0.6^a

Lead compounds 21 and 54 with HCR values in excess

of 100 (2-3-fold higher than was obtained for the lead

clinical candidate EO9 (3)) were evaluated in vivo in

the RIF-1 and KHT rodent tumor models. Both com-

pounds exhibited substantial cell killing both as single

agents, with substantially greater effectiveness than

EO9 (3) in both tumor models at drug MTDs (an MTD

dose of 21 alone was as effective as a radiation dose of

15 Gy alone in the KHT tumor) and in particular after

a single dose (up to 15 Gy) of radiation (up to 3.5 log

cell kills against the RIF-1 tumor, Table 3) at drug doses

well below MTDs (MTD 21 ) 100 mg kg^-1and MTD 54

) 90 mg kg^-1). A lesser effect in the RIF-1 tumor with

EO9 (3) could only be achieved at its MTD. These

compounds are therefore now being evaluated against

human tumor xenografts. A full study of the in vivo

biological effects of these compounds will be reported

elsewhere.

1.0 × 10^{-2 a}

3.0 × 10^{-3 a}

1.0 × 10^{-4 a}

KHT

Data taken from ref 33.

generated by a two-electron reducing enzyme (such as

DT-diaphorase) and/or by sequential one-electron reduc-

tions by other enzymes and/or disproportionation reac-

tions. In this case reductive cytotoxicity will be less

oxygen dependent since hydroquinones oxidize in air

much less rapidly than do semiquinone radicals, and

therefore hypoxia-selectivity is still evident against

these experimental tumors even when the oxygen

concentration is sufficiently high to render the lifetime

of the semiquinone extremely short. The likely involve-

ment of the hydroquinone species has also been sug-

gested following the study of reduction of some mito-

senes in aqueous and nonaqueous environments by

electron spin resonance and cyclic voltammetry.¹¹How-

ever, a recent study indicates that an o-aziridinyl group

may actually be deactivated as an electrophilic center

following reduction to a hydroquinone through a com-

peting 1,5-sigmatropic rearrangement.¹⁹Further, other

studies have previously indicated that hydroquinone-

generating enzyme DT-diaphorase may protect cells

Since despite the oxygen reactivity of the semiquinone

radicals the lead indoloquinone compounds 21 and 54

showed antitumor effects in vivo against hypoxic cell

populations, and these effects compare favorably with

EO9 (3) (Table 3), little relationship between bioreduc-

tive cytotoxicity and the oxygen reactivity of one-

electron-reduced radicals is evident. This provides some

evidence that the antitumor bioreductive effect is likely

to be mediated principally through the hydroquinone,

2340 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15

Naylor et al.

3.81 (s, 3 H, CH₃O), 4.05-4.3 (m, 4 H, CH₂CH₃), 4.5 (d, 1 H,

J ) 2.7 Hz, 2-H), and 6.9-7.2 (m, 3 H, Ar-4,6,7H).

against damage by this type of agent under hypoxic

conditions, and this has been demonstrated for EO9

(3),²⁰again suggesting the probable importance of other,

possibly radical-mediated mechanisms of cytotoxicity

under such conditions.

1-Acetyl-3-ca r boxy-2-cyclop r op yl-2,3-d ih yd r o-5-m eth -

oxyin d ole (11). The acetylindole 10 (1.0 g, 2.9 mmol) was

dissolved in EtOH (10 mL) and cooled to 0 °C in an ice/salt

bath, and a cold (0 °C) solution of KOH (aqueous, 10%, 10 mL)

was added. The solution was stirred at -5 °C for 4 h and then

for 18 h at 4 °C. The solution was poured onto ice/water (25

mL) and washed with Et₂O. The aqueous layer was then

acidified with 2.0 M HCl, extracted with CHCl₃(6 × 50 mL),

dried, and evaporated to give 0.68 g (97%) of 11 as a yellow

oil (R_f) 0.45, Me₂CO), which was used in the next step without

further purification: ¹H-NMR (CDCl₃) δ 0.5-0.7 (m, 4 H, 2 ×

cyclopropyl-CH₂), 1.25-1.4 (m, 1 H, cyclopropyl-H), 2.33 (s, 3

H, COCH₃), 3.78 (s, 3 H, CH₃O), 3.85 (br, 1 H, 3-H), 4.5 (br s,

1 H, 2-H), 6.9-7.0 (m, 2 H, Ar-4,6H), 8.05-8.15 (m, 1 H, Ar-

7H), and 10.81 (s, 1 H, CO₂H).

Meth yl 1-Acetyl-2-cyclop r op yl-2,3-d ih yd r o-5-m eth oxy-

in d ole-3-ca r boxyla te (12). To a solution of 11 (0.68 g, 2.8

mmol) in DMF (10 mL) was added K₂CO₃(0.83 g, 6 mmol)

and (MeO)₂SO₂(2 g, 15.8 mmol) and the solution stirred at

room temperature for 4 h. The solution was then poured onto

HCl (2.0 M, 20 mL), extracted with CHCl₃(4 × 25 mL), washed

with saturated NaCl, dried, and evaporated. The residue was

purified on silica, eluting with EtOAC/hexane (1:2, R_f) 0.7

(EtOAc)) to give a pale yellow oil of 12 (0.7 g, 98%), which

solidified on standing: ¹H-NMR (CDCl₃) δ 0.58-0.64 (m, 4 H,

2 × cyclopropyl-CH₂), 1.25-1.33 (m, 1 H, cyclopropyl-H), 2.33

(s, 3 H, COCH₃), 3.68 (s, 3 H, CO₂CH₃), 3.79 (s, 3 H, CH₃O),

3.85 (br, 1 H, 3-H), 4.54 (br, 1 H, 2-H), 6.78-6.95 (m, 2 H,

Ar-4,6H), and 8.2 (br s, 1 H, Ar-7H).

Meth yl 1-Acetyl-2-cyclop r op yl-5-m eth oxyin d ole-3-ca r -

boxyla te (13). A solution of 12 (1.0 g, 4 mmol) was stirred

under reflux with DDQ (0.96 g, 4.2 mmol) in toluene (12.5 mL)

for 7 h. The DDQH₂was removed by filtration and the filtrate

evaporated in vacuo. The residue was purified on silica,

eluting with hexane/EtOAc (1:1, R_f) 0.7) to give 13 (0.88 g,

87%) as a pale yellow oil: ¹H-NMR (CDCl₃) δ 0.73-0.79 (m, 2

H, cyclopropyl-CH₂), 1.22-1.3 (m, 2 H, cyclopropyl-CH₂), 2.15-

2.25 (m, 1 H, cyclopropyl-H), 2.83 (s, 3 H, COCH₃), 3.86 (s, 3

H, CO₂CH₃), 3.97 (s, 3 H, CH₃O), 6.99 (dd, 1 H, J ) 2.7 and 9

Hz, Ar-6H), 7.5 (d, 1 H, J ) 2.7 Hz, Ar-4H), and 7.96 (d, 1 H,

J ) 9 Hz, Ar-7H).

Meth yl 2-Cyclop r op yl-5-m eth oxyin d ole-3-ca r boxyla te

(14). A solution of 13 (0.88 g, 3.46 mmol) in KOH (4% in

MeOH, 50 mL) was stirred at room temperature for 1.5 h,

neutralized with 6.0 M HCl, and extracted with EtOAc (3 ×

25 mL). The organic layer was washed with H₂O (50 mL),

dried, and evaporated to give 14 (0.51 g, 70%) as a white

solid: mp 128-131 °C; ¹H-NMR (CDCl₃) δ 0.82-0.92 (m, 2 H,

cyclopropyl-CH₂), 1.05-1.2 (m, 2 H, cyclopropyl-CH₂), 2.2-3.0

(m, 1 H, cyclopropyl-H), 3.85 (s, 3 H, CO₂CH₃), 3.95 (s, 3 H,

CH₃O), 6.79 (dd, 1 H, J ) 2.7 and 9 Hz, Ar-6H), 7.22 (d, 1 H,

J ) 6.3 Hz, Ar-7H), 7.58 (d, 1H, J ) 2.7 Hz, Ar-4H), and 8.2

(br s, 1 H, NH).

Exp er im en ta l Section

NMR spectra were obtained at 90 MHz with a J EOL FX90Q

spectrometer using SiMe₄as internal standard. Elemental

analyses were determined by Butterworth Laboratories Ltd.,

Teddington, Middlesex, U.K. Solutions in organic solvents

were dried by treatment with MgSO₄or Na₂SO₄and filtration.

Dichloromethane (CH₂Cl₂) was dried over calcium chloride,

and chloroform (CHCl₃) was passed through neutral alumina

prior to use. Dimethylformamide (DMF), toluene, and tet-

rahydrofuran (THF) were anhydrous commercial grades.

Silica gel for flash column chromatography was Merck grade

(230-400 mesh). Melting points were determined on

Thomas-Hoover melting point apparatus and on a Ther-

mogallen microscope and hot stage apparatus and are uncor-

rected. Stereochemically pure cis-2,3-dimethylaziridine and

2,2-dimethylaziridine were synthesized from the appropriately

substituted 2-aminoethanols by O-sulfation and elimination

with KOH.²¹Fused cyclopropamitosenes 4 and 5 were syn-

thesized as described previously.^1,2,22EO9 (3) and its 5-meth-

oxy analogue EO7 were synthesized as described previously.⁹

5-Methoxy-2-methylindole, 5-methoxyindole-3-carboxaldehde,

methyl viologen, and benzyl viologen were purchased from

Sigma-Aldrich, U.K.

Dieth yl (5-Meth oxy-2-n itr op h en yl)m a lon a te (6). A so-

lution of 17 g (0.064 mol) of ethyl (5-methoxy-2-nitrophenyl)-

cyanoacetate, prepared in four steps from 3-chlorophenol as

previously described,⁹in EtOH (100 mL), was saturated with

HCl gas on cooling in an ice/salt bath. The solution was stirred

for 2 days, ice/water (100 mL) added, and the solution stirred

for a further 24 h at 4 °C before the crystalline solid formed

was collected by filtration, dried, and recrystallized from EtOH

to give 17 g (85%) of 6 as a white solid: mp 92-93 °C (lit.⁹mp

92-93 °C).

2-Cyclop r op yl-3,3-bis(eth oxyca r bon yl)-2,3-d ih yd r o-5-

m eth oxyin d ole (9). Compound 6 (5.0 g, 16.1 mmol) was

dissolved in a mixture of toluene (62.5 mL) and EtOH (3.75

mL) and reduced with H₂at atmospheric pressure over PtO₂

(75 mg) catalyst. After 7 h at room temperature the solution

was filtered through Celite and evaporated to dryness below

30 °C, and the resulting pale green oil (7) dissolved im-

mediately in MeOH (75 mL). To this methanolic solution was

added cyclopropanecarboxaldehyde (1.3 g, 18.6 mmol) dissolved

in MeOH (10 mL) and the solution stirred for 15 min. The

resulting solution of imine 8 was treated in situ with Zn-

(OAc)₂‚2H₂O (1.13 g, 5.15 mmol) and stirred for 18 h at room

temperature. The solution was evaporated to dryness at 30

°C, HCl (2.0 M, 50 mL) was added, and the mixture was

extracted with CH₂Cl₂(2 × 150 mL), washed with saturated

NaHCO₃(aqueous, 150 mL) and saturated NaCl (aqueous, 100

mL), dried, and evaporated. The residue was purified on silica,

eluting with hexane/EtOAc (1:1, R_f) 0.75) to give 9 (3.3 g,

68%) as a yellow oil: ¹H-NMR (CDCl₃) δ 0.48-0.52 (m, 4 H, 2

× cyclopropyl-CH₂), 1.03-1.08 (m, 1 H, cyclopropyl-H), 1.2 (t,

6 H, J ) 7.2 Hz, CH₂CH₃), 1.3 (t, 6 H, J ) 7.2 Hz, CH₂CH₃),

3.75 (s, 3 H, CH₃O-), 3.85 (d, 1 H, J ) 2.7 Hz, 2-H), 4.1-4.35

(m, 4 H, CH₂CH₃), 6.63 (s, 1 H, Ar-4H), 6.71 (d, 1 H, J ) 1.5

Hz, Ar-6H), and 7.03 (d, 1 H, J ) 1.5 Hz, Ar-7H).

Met h yl 2-Cyclop r op yl-5-m et h oxy-1-m et h ylin d ole-3-

ca r boxyla te (15). Compound 14 (9.0 g, 42.6 mmol) was added

under argon to a stirred suspension of NaH (6.0 g, 0.13 mol)

in DMF (150 mL). The solution was heated at 45 °C for 0.5 h

and cooled to 0-10 °C and MeI (33 mL, 0.23 mol) added. The

solution was then gradually heated to 60 °C, stirred at this

temperature for 1 h, cooled, poured onto cold (0 °C) NaHSO₄

(aqueous, 10%, 500 mL), and extracted with EtOAc (5 × 75

mL). The organic extracts were washed with saturated NaCl

(150 mL), dried, and evaporated. The residue was purified

on silica, eluting with EtOAc/hexane (1:2, R_f) 0.45) to give

15 (8.3 g, 86%) as a pale yellow waxy solid: ¹H-NMR (CDCl₃)

δ 0.77-0.84 (m, 2 H, cyclopropyl-CH₂), 1.21-1.29 (m, 2 H,

cyclopropyl-CH₂), 1.85-2.05 (m, 1 H, cyclopropyl-H), 3.8 (s, 3

H, CH₃N), 3.86 (s, 3 H, CO₂CH₃), 3.9 (s, 3 H, CH₃O), 6.86 (dd,

1 H, J ) 2.7 and 9 Hz, Ar-6H), 7.23 (d, 1 H, J ) 9 Hz, Ar-7H),

and 7.62 (d, 1 H, J ) 2.7 Hz, Ar-4H).

1-Acetyl-2-cyclop r op yl-3,3-bis(eth oxyca r bon yl)-2,3-d i-

h yd r o-5-m eth oxyin d ole (10). Compound 9 (3.0 g, 10 mmol)

was dissolved in Ac₂O (5 mL) and stirred for 3 h at room

temperature. The anhydride was then evaporated in vacuo

and the residue purified on silica, eluting with hexane/EtOAc

(2:1, R_f) 0.4) to give a pale yellow oil which was redissolved

in Et₂O and evaporated. Trituration of the resulting oil with

Et₂O gave a white solid which was collected by filtration and

washed with cold Et₂O to give 10 (3.0 g, 87%) as a white

Meth yl 1-Meth yl-2-cyclop r op yl-5-m eth oxy-4-n itr oin -

d ole-3-ca r boxyla te (16). To a solution of 15 (8.0 g, 34.66

mmol) in AcOH (150 mL) cooled to 0 °C was added dropwise

a cold (0 °C) mixture of fuming HNO₃(27 mL) in AcOH (100

solid: mp 104-105 °C; H-NMR (CDCl₃) δ 0.5-0.57 (m, 4 H,

2 × cyclopropyl-CH₂)), 1.0-1.15 (m, 1 H, cyclopropyl-H), 1.2

(t, 3 H, J ) 7.2 Hz, CH₂CH₃), 1.3 (t, 3 H, J ) 7.2 Hz, CH₂CH₃),

Bioreductively Activated Antitumor Agents

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2341

mL). The solution was stirred for 3 h while warming to room

temperature, poured onto 300 g of crushed ice, and after 15

min the resulting yellow solid was collected by suction filtra-

tion. The dried residue was purified on silica, eluting with

EtOAc/hexane (1:1, R_f) 0.45) to give 7.5 g (71%) of 16 as a

yellow solid, recrystallized from EtOAc: mp 129-131 °C; ¹H-

NMR (CDCl₃) δ 0.74-0.81 (m, 2 H, cyclopropyl-CH₂), 1.22-

1.3 (m, 2 H, cyclopropyl-CH₂), 1.85-2.05 (m, 1 H, cyclopropyl-

H), 3.8 (s, 3 H, CH₃N-), 3.81 (s, 3 H, CO₂CH₃), 3.89 (s, 3 H,

CH₃O), 6.94 (d, 1 H, J ) 9 Hz, Ar-7H), and 7.32 (d, 1 H, J )

9 Hz, Ar-6H).

Meth yl 4-Am in o-2-cyclop r op yl-5-m eth oxy-1-m eth ylin -

d ole-3-ca r boxyla te (17). To a suspension of 2.5 g (9.25

mmol) of 16 in EtOH (180 mL) were added tin powder (5.25 g,

44.2 mmol) and HCl (3.0 M, 70 mL), and the solution was

stirred at room temperature for 1 h. The solution was then

decanted from the excess tin and neutralized with saturated

NaHCO₃(aqueous), the resulting red suspension was added

to an equal volume of H₂O and extracted with CHCl₃(5 × 50

mL) and then EtOAc (5 × 50 mL), and the combined extracts

were evaporated. The residue was purified on silica, eluting

with EtOAc/hexane (1:1, R_f) 0.5) to give 17 (2.2 g, 87%) as a

white solid which was used immediately in the next step: mp

49-50 °C; ¹H-NMR (CDCl₃) δ 0.65-0.72 (m, 2 H, cyclopropyl-

CH₂), 1.22-1.3 (m, 2 H, cyclopropyl-CH₂), 1.8-1.9 (m, 1 H,

cyclopropyl-H), 3.73 (s, 3 H, CH₃N), 3.85 (s, 3 H, CO₂CH₃), 3.9

(s, 3 H, CH₃O-), 5.6 (br s, 1 H, NH₂), 6.5(d, 1 H, J ) 9 Hz,

Ar-7H), and 6.9 (d, 1 H, J ) 9 Hz, Ar-6H).

2-C y c lo p r o p y l-3-(m e t h o x y c a r b o n y l)-5-m e t h o x y -1-

m eth ylin d ole-4,7-d ion e (18). To a solution of 17 (2.0 g, 7.2

mmol) in Me₂CO (250 mL) was added a solution of potassium

nitrosodisulfonate ((KSO₃)₂NO, Fremy’s salt, 9.7 g, 36.2 mmol))

in NaH₂PO₄/Na₂HPO₄buffer (250 mL, 0.3 M, pH 6.0) and the

solution stirred at room temperature for 1 h. The Me₂CO was

removed in vacuo and the resulting orange precipitate collected

by suction filtration, washed with H₂O, and dried in a vacuum

oven at 45 °C to afford 18 as an orange solid which was

recrystallized from EtOAc: mp 169-170 °C; ¹H-NMR (CDCl₃)

δ 0.69-0.77 (m, 2 H, cyclopropyl-CH₂), 1.1-1.2 (m, 2 H,

cyclopropyl-CH₂), 1.48-1.58 (m, 1 H, cyclopropyl-H), 3.80 (s,

3 H, CH₃N), 3.90 (s, 3 H, CO₂CH₃), 4.01 (s, 3 H, CH₃O), and

5.64 (s, 1 H, 6-H). Anal. (C₁₅H₁₅NO₅) C, H, N.

CH₂), 1.2-1.33 (m, 2 H, cyclopropyl-CH₂), 1.61-1.71 (m, 1 H,

cyclopropyl-H), 3.81 (s, 3 H, CH₃N), 3.98 (s, 3 H, CH₃O), 4.0

(br s, 1 H, CH₂OH), 4.69 (br d, 2 H, CH₂OH), and 5.64 (s, 1 H,

6-H). Anal. (C₁₄H₁₅NO₄) C, H, N.

5-(Azir id in -1-yl)-2-cyclop r op yl-3-(h yd r oxym et h yl)-1-

m eth ylin d ole-4,7-d ion e (21). Compound 20 (0.1 g, 0.38

mmol) was dissolved, stirred in freshly distilled 1H-aziridine

(3 mL, ca.70 mmol, CAUTION!) for 0.75 h, and evaporated in

vacuo, and the residue was redissolved in EtOAc and evapo-

rated until a red precipitate appeared and the solid collected.

The red solid was recrystallized from EtOAc to give 21 (80

mg, 77%): mp 177-179 °C; ¹

H-NMR (CDCl₃) δ 0.7-0.8 (m, 2

H, cyclopropyl-CH₂), 1.22-1.3 (m, 2 H, cyclopropyl-CH₂), 1.6-

1.7 (m, 1 H, cyclopropyl-H), 2.18 (s, 4 H, 2 × azir-CH₂), 3.97

(s, 3 H, CH₃N), 4.73 (s, 2 H, CH₂OH), and 5.77 (s, 1 H, 6-H).

Anal. (C₁₅H₁₆N₂O₃) C, H, N.

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

1-yl)-1-m eth ylin d ole-4,7-d ion e (22). Compound 20 (0.1 g,

0.38 mmol) was dissolved and stirred in freshly distilled

2-methylaziridine (3 mL, ca.50 mmol) for 2.5 h. The solution

was evaporated in vacuo and the residue redissolved in EtOAc,

evaporated until a red precipitate appeared and the solid

collected. The red solid was recrystallized from EtOAc to give

22 (85 mg, 78%): mp 130-131 °C;

¹H-NMR (CDCl₃) δ 0.68-

0.74 (m, 2 H, cyclopropyl-CH₂), 1.08-1.2 (m, 2 H, cyclopropyl-

CH₂), 1.42 (d, 3 H, J ) 4.5 Hz, azir-CH₃), 1.5-1.6 (m, 1 H,

cyclopropyl-H), 2.01-2.15 (m, 3 H, azir-CHCH₂), 3.97 (s, 3 H,

CH₃N), 4.73 (s, 2 H, CH₂OH), and 5.74 (s, 1 H, 6-H). Anal.

(C₁₆H₁₈N₂O₃) C, H, N.

2-Cyclop r op yl-5-m et h oxy-1-m et h yl-3-[[(p h en oxyca r -

bon yl)oxy]m eth yl]in d ole-4,7-d ion e (23). To a solution of

20 (0.1 g, 0.38 mmol) in anhydrous pyridine (6 mL) at 0 °C

was added dropwise phenyl chloroformate (0.1 g, 0.64 mmol)

and the solution then allowed to reach room temperature and

stirred for 2 h. The solution was then extracted with CH₂Cl₂

(25 mL), washed with H₂O (25 mL) and saturated NaCl (25

mL), dried, and evaporated. The residue was purified on silica,

eluting with EtOAc (R_f) 0.75) to give 23 as an orange solid

which was used without further purification: mp 136-139 °C;

¹H-NMR (CDCl₃) δ 1.02-1.18 (m, 2 H, cyclopropyl-CH₂), 1.21-

1.28 (m, 2 H, cyclopropyl-CH₂), 1.78-1.88 (m, 1 H, cyclopropyl-

H), 3.79 (s, 3 H, CH₃N), 4.01 (s, 3 H, CH₃O), 5.27 (s, 2 H,

CH₂OCOPh), 5.51 (s, 1 H, 6-H), and 7.15-7.3 (m, 5 H, Ar).

3-[(Ca r ba m oyloxy)m et h yl]-2-cyclop r op yl-5-m et h oxy-

1-m et h ylin d ole-4,7-d ion e (24). The phenyl carbonate 23

5-(Azir id in -1-yl)-2-cyclop r op yl-3-(m eth oxyca r bon yl)-1-

m eth y1in d ole-4,7-d ion e (19). Compound 18 (0.29 g, 1

mmol) was dissolved and stirred in freshly redistilled 1H-

aziridine (3 mL, ca.70 mmol, CAUTION!) for 1 h and evapo-

rated in vacuo and the residue redissolved in EtOAc. The

solvent was then partially evaporated until a red precipitate

appeared. The red solid was then collected by suction filtration

and recrystallized from EtOAc to afford 19 (0.25 g, 83%) as a

(0.3 g, 0.78 mmol) was dissolved in anhydrous CH₂

Cl₂(38 mL)

and the solution cooled to -78 °C. The solution was then

saturated with NH₃and stirred at -78 °C until reaction was

complete (ca. 2 h). The solution was then allowed to reach

room temperature and evaporated in vacuo. The residue was

redissolved in CH₂Cl₂(100 mL), washed with H₂O (2 × 100

mL) and saturated NaCl (50 mL), dried, and evaporated, and

the residue was recrystallized from EtOAc to afford 220 mg

(92%) of 24 as an orange solid: mp 240-242 °C dec; ¹

red solid: mp 138-140 °C; H-NMR (CDCl₃) δ 0.65-0.72 (m,

2 H, cyclopropyl-CH₂), 1.2-1.35 (m, 2 H, cyclopropyl-CH₂),

1.7-1.8 (m, 1 H, cyclopropyl-H), 2.18 (s, 4 H, 2 × azir-CH₂),

3.92 (s, 3 H, CH₃N), 3.99 (s, 3 H, CO₂CH₃), and 5.77 (s, 1 H,

6-H). Anal. (C₁₆H₁₆N₂O₄) C, H, N.

H-NMR

(CDCl₃

) δ 1.02-1.13 (m, 2 H, cyclopropyl-CH ), 1.22-1.26 (m,

2 H, cyclopropyl-CH₂), 1.51-1.55 (m, 1 H, cyclopropyl-H), 3.78

(s, 3 H, CH₃N), 3.99 (s, 3 H, CH₃O), 4.79 (br s, 2 H, NH₂), 5.3

(s, 2 H, CH₂OCONH₂), and 5.62 (s, 1 H, 6-H). Anal.

(C₁₅H₁₆N₂O₅) C, H, N.

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

ylin d ole-4,7-d ion e (20). To a solution of 18 (0.3 g, 1.03 mmol)

in CHCl₃(30 mL) and EtOH (11 mL) was added a solution of

Na₂S₂O₄(2.1 g, 12 mmol) in H₂O (13 mL). The solution was

stirred at room temperature for 0.5 h and the organic layer

separated, washed with saturated NaCl (50 mL), dried, and

evaporated. The crude hydroquinone was then dissolved in

anhydrous CH₂Cl₂(30 mL) under argon and cooled to -30 °C,

and DIBAL-H (5 mL of a 1.5 M solution in toluene) was added

dropwise such that the solution temperature remained below

-30 °C. The solution was then allowed to reach 0 °C and

stirred for 2.5 h at this temperature, and a solution of FeCl₃

(9 mL, 1.0 M (0.1 M HCl)) was added. The solution was stirred

for 10 min at 0 °C, and then CHCl₃(150 mL) and H₂O (150

mL) were added. The aqueous layer was extracted with CHCl₃

(5 × 50 mL) and then EtOAc (5 × 50 mL), and the combined

organic phases were washed with saturated NaCl (250 mL),

dried, and evaporated. The residue was purified on silica,

eluting with EtOAc (R_f) 0.5) to give 20 as an orange solid

after recrystallization from EtOAc (125 mg, 47%): mp 200-

202 °C; ¹H-NMR (CDCl₃) δ 0.71-0.82 (m, 2 H, cyclopropyl-

5-(Azir id in -1-yl)-3-[(ca r ba m oyloxy)m eth yl]-2-cyclop r o-

p yl-1-m eth ylin d ole-4,7-d ion e (25). The carbamate 24 (0.3

g, 1.0 mmol) was stirred at room temperature in 1H-aziridine

(2 mL, CAUTION!) for 15 min, evaporated, and redissolved

in EtOAc (5 mL). The solution was then evaporated to 50%

volume and the resulting red precipitate filtered and washed

well with cold EtOAc to afford 25 (210 mg, 63%) as a red

solid: mp 235-238 °C dec; H-NMR ((CD₃)₂SO) δ 0.65-0.73

(m, 2 H, cyclopropyl-CH₂), 1.05-1.15 (m, 2 H, cyclopropyl-CH₂),

1.73-1.85 (m, 1 H, cyclopropyl-H), 2.18 (s, 4 H, 2 × azir-CH₂),

3.93 (s, 3 H, CH₃N), 5.06 (s, 2 H, CH₂OCONH₂), 5.78 (s, 1 H,

6-H), and 6.42 (br s, 2 H, NH₂). Anal. (C₁₆H₁₇N₃O₄) C, H, N.

3-[(Ca r ba m oyloxy)m eth yl]-2-cyclop r op yl-1-m eth yl-5-

(2-m eth yla zir id in -1-yl)in d ole-4,7-d ion e (26). The carbam-

ate 24 (0.05 g, 0.164 mmol) was stirred at room temperature

in 2-methylaziridine (1.5 mL) for 4 h, evaporated, and redis-

solved in EtOAc (5 mL). The solution was then evaporated to

2342 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15

Naylor et al.

50% volume and the resulting red precipitate filtered, washed

well with cold EtOAc, and then recrystallized from EtOAc to

afford 26 (30 mg, 56%) as a red solid: mp 209-211 °C dec;

¹H-NMR ((CD₃)₂SO) δ 0.63-0.72 (m, 2 H, cyclopropyl-CH₂),

1.01-1.09 (m, 2 H, cyclopropyl-CH₂), 1.29 (d, 3 H, J ) 5.4 Hz,

azir-CH₃), 1.75-1.85 (m, 1 H, cyclopropyl-H), 1.98-2.05 (m, 3

H, azir-CHCH₂), 3.93 (s, 3 H, CH₃N), 5.06 (s, 2 H, CH₂-

OCONH₂), 5.76 (s, 1 H, 6-H), and 6.41 (br s, 2 H, NH₂). Anal.

(C₁₇H₁₉N₃O₄) C, H; N: calcd,12.76; found, 12.24.

3.82 (s, 3 H, CO₂CH₃), 3.91 (s, 3 H, CH₃O), 3.95-4.15 (m, 1 H,

CH(CH₃)₂), 6.97 (d, 1 H, J ) 9 Hz, Ar-6H), and 7.35 (d, 1 H, J

) 9 Hz, Ar-7H).

2-Isop r op yl-3-(m et h oxyca r b on yl)-5-m et h oxy-1-m et h -

ylin d ole-4,7-d ion e (35). Compound 33 was reduced as

described for 17 to give 34 (85%) and the crude material

oxidized with Fremy’s salt as described for 18. After workup

the resulting orange precipitate was collected by suction

filtration, washed with H₂O, and dried in a vacuum oven at

45 °C to afford 35 as an orange solid (75%), recrystallized from

EtOAc: mp 192-194 °C; ¹H-NMR (CDCl₃) δ 1.34 (d, 6 H, J )

7.2 Hz, CH(CH₃)₂), 3.12-3.28 (m, 1 H, CH(CH₃)₂), 3.81 (s, 3

H, CH₃N), 3.90 (s, 3 H, CO₂CH₃), 3.97 (s, 3 H, CH₃O), and

5.64 (s, 1 H, 6-H). Anal. (C₁₅H₁₇NO₅) H, N, C; calcd., 61.86;

found, 62.31.

3-(Hyd r oxym eth yl)-2-isop r op yl-5-m eth oxy-1-m eth ylin -

d ole-4,7-d ion e (36). To a solution of 35 (0.5 g, 1.7 mmol) in

CHCl₃(50 mL) and EtOH (18 mL) was added a solution of

Na₂S₂O₄(3.5 g, 20 mmol) in H₂O (22 mL). The solution was

stirred at room temperature for 1 h, and the organic layer was

separated, washed with saturated NaCl (50 mL), dried, and

evaporated. The crude hydroquinone was dried over 18 h in

vacuo and then dissolved in anhydrous THF (5 mL) under

argon and added to a solution of LiAlH₄(12 mL of a 1.0 M

solution in THF) dropwise at room temperature and under

argon. The solution was then stirred for 1 h at 30 °C and

cooled to 0 °C, and H₂O (15 mL) was added dropwise, followed

by a solution of FeCl₃(12 mL, 1.0 M (0.1 M HCl)) added at 0

°C. The solution was stirred for 10 min at 0 °C, and then

EtOAc (150 mL) and H₂O (150 mL) were added. The aqueous

layer was extracted with EtOAc (5 × 50 mL) and the organic

phase washed with saturated NaCl (250 mL), dried, and

evaporated. The residue was purified on silica, eluting with

EtOAc/hexane (1:1, R_f) 0.22) to give, after recrystallization

from EtOAc, 36 as an orange solid (140 mg, 31%): mp 160-

161 °C; ¹H-NMR (CDCl₃) δ 1.37 (d, 6 H, J ) 7.2 Hz, CH(CH₃)₂),

3.1-3.28 (m, 1 H, CH(CH₃)₂), 3.82 (s, 3 H, CH₃N), 3.97 (s, 3

H, CH₃O), 4.71 (br, 2 H, CH₂OH), and 5.64 (s, 1 H, 6-H). Anal.

(C₁₄H₁₇NO₄) C, H, N.

1-Acetyl-3,3-bis(eth oxyca r bon yl)-2,3-d ih yd r o-2-isop r o-

p yl-5-m eth oxyin d ole (27). This compound was prepared

(56%) by the method described for compound 9 but using

isobutyraldehyde in place of cyclopropanecarboxaldehyde. The

residue after workup was dissolved in Ac₂O (5 mL) and stirred

for 3 h at room temperature. The anhydride was then

evaporated in vacuo and the residue purified on silica, eluting

with hexane/EtOAc (1:1, R_f) 0.5) to give 27 (87%) as a white

solid: mp 77.5-78.5 °C; H-NMR (CDCl₃) δ 0.6 (d, 3 H, J )

7.2 Hz, CHCH₃), 0.9 (d, 3 H, J ) 7.2 Hz, CHCH₃), 1.2 (t, 6 H,

J ) 7.2 Hz, CH₂CH₃), 1.3 (t, 6 H, J ) 7.2 Hz, CH₂CH₃), 2.12-

2.28 (m, 1 H, CH(CH₃)₂), 2.36 (s, 3 H, COCH₃), 3.81 (s, 3 H,

CH₃O), 4.1-4.33 (m, 4 H, 2 × CH₂CH₃), 4.8 (br, 1 H, 2-H),

and 6.9-7.8 (m, 3 H, Ar-4,6,7H).

1-Acetyl-3-ca r boxy-2,3-d ih yd r o-2-isop r op yl-5-m eth ox-

yin d ole (28). The acetylindole 27 was hydrolyzed as de-

scribed for the preparation of 11 to give 28 (90%) as an off-

white foam (R_f) 0.45, Me₂CO), which was used in the next

step without further purification: ¹H-NMR (CDCl₃) δ 0.59 (d,

3 H, J ) 7.2 Hz, CHCH₃), 0.9 (d, 3 H, J ) 7.2 Hz, CHCH₃),

2.15-2.22 (m, 1 H, CH(CH₃)₂), 2.2 (s, 3 H, COCH₃), 3.72 (s, 3

H, CH₃O), 3.98 (br, 1 H, 3-H), 4.62 (br, 1 H, 2-H), 6.8-6.98

(m, 2 H, Ar-4,7H), and 7.78-7.85 (m, 1 H, Ar-6H).

Meth yl 1-Acetyl-2,3-d ih yd r o-2-isop r op yl-5-m eth oxyin -

d ole-3-ca r boxyla te (29). In a procedure identical to that

carried out on 11, compound 29 was prepared as a pale brown

oil (93%): ¹H-NMR (CDCl₃) δ 0.71 (d, 3 H, J ) 7.2 Hz, CHCH₃),

1.0 (d, 3 H, J ) 7.2 Hz, CHCH₃), 2.1-2.2 (m, 1 H, CH(CH₃)₂),

2.32 (s, 3 H, COCH₃), 3.69 (s, 3 H, CO₂CH₃), 3.79 (s, 3 H,

CH₃O), 3.8 (br, 1 H, 3-H), 4.6 (br, 1 H, 2-H), 6.8-6.98 (m, 2 H,

Ar-4,7H), and 7.78-7.85 (m, 1 H, Ar-6H).

5-(Azir id in -1-yl)-3-(h yd r oxym e t h yl)-2-isop r op yl-1-

m eth ylin d ole-4,7-d ion e (37). A solution of 36 (50 mg, 0.19

mmol) in 1H-aziridine (0.5 mL, ca.11.7 mmol, CAUTION!) was

stirred for 0.5 h at room temperature and then evaporated to

dryness, and the residue was purified on silica, eluting with

EtOAc (R_f) 0.55) to a give, after recrystallization from EtAOc,

37 (42 mg, 81%) as a red solid: mp 144-145 °C; ¹H-NMR

(CDCl₃) δ 1.37 (d, 6 H, J ) 7.2 Hz, CH(CH₃)₂), 2.18 (s, 4 H, 2

× azir-CH₂), 3.1-3.28 (m, 1 H, CH(CH₃)₂), 3.95 (s, 3 H, CH₃N),

4.68 (br s, 1 H, CH₂OH), 4.76 (br, 2 H, CH₂OH), and 5.76 (s,

1 H, 6-H). Anal. (C₁₅H₁₈N₂O₃) C, H, N.

Met h yl 1-Acet yl-2-isop r op yl-5-m et h oxyin d ole-3-ca r -

boxyla te (30). A solution of 29 (1.0 g, 4.0 mmol) was stirred

under reflux with DDQ (0.96 g, 4.2 mmol) in toluene (12.5 mL)

for 2 days. The DDQH₂was removed by filtration and the

filtrate evaporated in vacuo. The residue was purified on

silica, eluting with hexane/Me₂CO (3:1, R_f) 0.65) to give 30

(0.44 g, 44%) as a pale red oil: ¹H-NMR (CDCl₃) δ 1.45 (d, 6

H, J ) 7.2 Hz, CH(CH₃)₂), 2.78 (s, 3 H, COCH₃), 3.87 (s, 3 H,

CO₂CH₃), 3.96 (s, 3 H, CH₃O), 3.88-3.98 (m, 1 H, CH(CH₃)₂),

6.89 (dd, 1 H, J ) 2.7 and 9 Hz, Ar-6H), 7.44 (d, 1 H, J ) 9

Hz, Ar-7H), and 7.55 (d, 1 H, J ) 2.7 Hz, Ar-4H).

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

yl)-1-m eth ylin d ole-4,7-d ion e (38). Compound 36 (0.1 g, 0.38

mmol) was dissolved and stirred in freshly distilled 2-meth-

ylaziridine (3 mL, ca.50 mmol) for 2.5 h. The solution was

evaporated in vacuo and the residue redissolved in EtOAc,

evaporated, and purified on silica (eluting with EtOAc) to

afford a red glass (38, 85 mg, 78%): ¹H-NMR (CDCl₃) δ 1.37

(d, 6 H, J ) 7.2 Hz, CH(CH₃)₂), 1.42 (d, 3 H, J ) 4.5 Hz, azir-

CH₃), 2.01-2.15 (m, 3 H, azir-CHCH₂), 3.1-3.28 (m, 1 H,

CH(CH₃)₂), 3.95 (s, 3 H, CH₃N), 4.68 (br s, 1 H, CH₂OH), 4.76

(br, 2 H, CH₂OH), and 5.76 (s, 1 H, 6-H). Anal. (C₁₆H₂₀N₂-

O₃‚0.5H₂O) C, H, N.

Meth yl 2-Isopr opyl-5-m eth oxyin dole-3-car boxylate (31).

This compound was prepared from 30 as described for 14 (70%)

as a pale red oil: ¹H-NMR (CDCl₃) δ 1.43 (d, 6 H, J ) 7.2 Hz,

CH(CH₃)₂), 3.87 (s, 3 H, CO₂CH₃), 3.92 (s, 3 H, CH₃O), 4.08-

4.25 (m, 1 H, CH(CH₃)₂), 6.9 (dd, 2 H, J ) 2.7 and 9 Hz, Ar-

6H), 7.1-7.6 (m, 2 H, Ar-4,7H), and 8.2 (br s, 1 H, NH).

Meth yl 2-Isop r op yl-5-m eth oxy-1-m eth ylin d ole-3-ca r -

boxyla te (32). In a procedure identical to that carried out

on 14, compound 32 was prepared (80%) and purified on silica,

eluting with EtOAc/hexane (1:2, R_f) 0.74) as a pale yellow

solid: mp 105-106 °C; ¹H-NMR (CDCl₃) δ 1.45 (d, 6H, J )

7.2 Hz, CH(CH₃)₂), 3.79 (s, 3 H, CH₃N), 3.88 (s, 3 H, CO₂CH₃),

3.92 (s, 3 H, CH₃O), 4.22-4.45 (m, 1 H, CH(CH₃)₂), 6.9 (dd, 2

H, J ) 2.7 and 9 Hz, Ar-6H), and 7.1-7.64 (m, 1 H, Ar-4,7H).

Meth yl 2-Isop r op yl-5-m eth oxy-1-m eth yl-4-n itr oin d ole-

3-ca r boxyla te (33). To a solution of 32 (8.0 g, 34.66 mmol)

in AcOH (150 mL) cooled to 0 °C was added dropwise a cold (0

°C) mixture of fuming HNO₃(27 mL) in AcOH (100 mL). The

solution was stirred for 3 h while warming to room tempera-

ture and then poured onto 300 g of crushed ice, and the

resulting yellow solid was collected by suction filtration. The

dried residue was purified on silica, eluting with EtOAc/hexane

(1:2, R_f) 0.26) to give 7.5 g (71%) of 33 as a yellow solid,

recrystallized from EtOAc: mp 149-150.5 °C; ¹H-NMR (CDCl₃)

δ 1.44 (d, 6 H, J ) 7.2 Hz, CH(CH₃)₂), 3.78 (s, 3 H, CH₃N),

2-Isop r op yl-5-m eth oxy-1-m eth yl-3-[[(p h en oxyca r bon -

yl)oxy]m eth yl]in d ole-4,7-d ion e (39). To a solution of 36

(0.55 g, 2.1 mmol) in anhydrous pyridine (25 mL) at 0 °C was

added dropwise phenyl chloroformate (0.5 g, 3.2 mmol), the

solution was then allowed to reach room temperature and

stirred for 1 h, and then a further 0.25 g (1.6 mmoL) of phenyl

chloroformate was added. After a further 1 h, H₂O (200 mL)

was added, and the solution was extracted with EtOAc (4 ×

50 mL), washed with H₂O (100 mL) and saturated NaCl (100

mL), dried, and evaporated. The residue was purified on silica,

eluting with EtOAc (R_f) 0.8) to give 39 (0.6 g, 75%) as an

orange solid: mp 126-127 °C; H-NMR (CDCl₃) δ 1.37 (d, 6

H, J ) 7.2 Hz, CH(CH₃)₂), 3.1-3.28 (m, 1 H, CH(CH₃)₂), 3.79

Bioreductively Activated Antitumor Agents

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2343

(s, 3 H, CH₃N), 4.01 (s, 3 H, CH₃O), 5.27 (s, 2 H, CH₂OCOPh),

5.51 (s, 1 H, 6-H), and 7.15-7.3 (m, 5 H, Ar).

2-Cycloh exyl-3-(h yd r oxym et h yl)-5-m et h oxy-1-m et h -

ylin d ole-4,7-d ion e (45). The corresponding 3-methoxycar-

bonyl precursor compound was prepared as described for 18

and 35 but using cyclohexanecarboxaldehyde in the imine

cyclization step, and the subsequent oxidation step with DDQ

required a much prolonged reaction time of 12 h. The crude

4-amino compound was again oxidized with Fremy’s salt as

described for 18. After workup the resulting orange precipitate

was collected by suction filtration, washed with H₂O, and dried

in a vacuum oven at 45 °C to give 2-cyclohexyl-3-(methoxy-

carbonyl)-5-methoxy-1-methylindole-4,7-dione as an orange

3-[(Ca r b a m oyloxy)m et h yl]-2-isop r op yl-5-m et h oxy-1-

m eth ylin d ole-4,7-d ion e (40). The phenyl carbonate 39 (0.1

g, 0.26 mmol) was dissolved in anhydrous CH₂Cl₂(15 mL) and

the solution cooled to -78 °C. The solution was then saturated

with NH₃and stirred at -78 °C until reaction was complete

(ca. 2.5 h). The solution was then allowed to reach room

temperature and evaporated in vacuo. The residue was

redissolved in CH₂Cl₂(50 mL), washed with H₂O (2 × 50 mL)

and saturated NaCl (25 mL), dried, and evaporated, and the

residue was recrystallized from EtOAc to afford 55 mg (69%)

of 40 as an orange solid: mp 244-246 °C dec; ¹H-NMR (CDCl₃)

δ 1.37 (d, 6 H, J ) 7.2 Hz, CH(CH₃)₂), 3.1-3.28 (m, 1 H,

CH(CH₃)₂), 3.78 (s, 3 H, CH₃N), 3.99 (s, 3 H, CH₃O), 4.79 (br

s, 2 H, NH₂), 5.3 (s, 2 H, CH₂OCONH₂), and 5.62 (s, 1 H, 6-H).

Anal. (C₁₅H₁₈N₂O₅) C, H, N.

solid (75%) recrystallized from EtOAc: mp 190-192 °C; H-

NMR (CDCl₃) δ 1.27 (m, 6 H, 6 × cyclohexyl-H), 1.83 (m, 5 H,

5 × cyclohexyl-H), 3.79 (s, 3 H, CH₃N), 3.91 (s, 3 H, CO₂CH₃),

3.96 (s, 3 H, CH₃O), and 5.63 (s, 1 H, 6-H). This compound

was then reduced with LiAlH₄as described for 36 to give 45

as an orange solid (59%): mp 214-215 °C; H-NMR (CDCl₃)

δ 1.25 (m, 6 H, 6 × cyclohexyl-H), 1.78 (m, 5 H, 5 × cyclohexyl-

H), 3.81 (s, 3 H, CH₃N), 3.97 (s, 3 H, CH₃O), 4.76 (s, 2 H, CH₂-

OH), and 5.63 (s, 1 H, 6-H). Anal. (C₁₇H₂₁NO₄) C, H, N.

5-(Azir id in -1-yl)-2-cycloh e xyl-3-(h yd r oxym e t h yl)-1-

m eth ylin d ole-4,7-d ion e (46). A solution of 45 (30 mg,0.1

mmol) in freshly distilled 1H-aziridine (0.5 mL, ca.11.7 mmol,

CAUTION!) was stirred for 0.5 h at room temperature and

then evaporated to dryness and the residue purified on silica,

eluting with EtOAc/hexane (1:1, R_f) 0.3) to give, after

recrystallization from EtOAc, 46 (22 mg, 70%) as a red solid:

mp 128-130 °C; ¹H-NMR (CDCl₃) δ 1.26 (m, 6 H, 6 ×

cyclohexyl-H), 1.78 (m, 5 H, 5 × cyclohexyl-H), 2.18 (s, 4 H, 2

× aziridine-CH₂), 3.95 (s, 3 H, CH₃N), 4.76 (s, 2 H, CH₂OH),

and 5.77 (s, 1 H, 6-H). Anal. (C₁₈H₂₂N₂O₃‚1.5H₂O) C, H, N.

5-Meth oxy-1,2-d im eth ylin d ole (47). 5-Methoxy-2-meth-

ylindole (10 g, 0.062 mol) was added gradually and under dry

argon to a stirred suspension of NaH (2.73 g of a 60%

dispersion, 0.068 mol) in DMF (150 mL). The suspension was

heated at 45 °C for 10 min and cooled to room temperature,

and MeI (33 mL, 0.23 mol) was added over 5 min. The solution

was then heated at 60 °C for 1 h, cooled, poured onto cold (0

°C) NaHSO₄(aqueous, 10%, 150 mL), extracted with EtOAc

(3 × 100 mL), dried, and evaporated. The residue was purified

on silica, eluting with 3%EtOAc/hexane (R_f) 0.5) to give 4.5

5-(Azir idin -1-yl)-3-[(car bam oyloxy)m eth yl]-2-isopr opyl-

1-m eth ylin d ole-4,7-d ion e (41). The carbamate 40 (0.1 g,

0.33 mmol) was stirred at room temperature in 1H-aziridine

(2 mL, CAUTION!) for 25 min, evaporated, and redissolved

in EtOAc (5 mL). The solution was then evaporated to 50%

volume, and the resulting red precipitate was filtered and

washed well with cold EtOAc to afford 41 (55 mg, 53%) as a

red solid: mp 230-233 °C dec; ¹H-NMR ((CD₃)₂SO) δ 1.37 (d,

6 H, J ) 7.2 Hz, CH(CH₃)₂), 2.18 (s, 4 H, 2 × azir-CH₂), 3.1-

3.28 (m, 1 H, CH(CH₃)₂), 3.93 (s, 3 H, CH₃N), 5.06 (s, 2 H,

CH₂OCONH₂), 5.78 (s, 1 H, 6-H), and 6.42 (br s, 2 H, NH₂).

Anal. (C₁₆H₁₉N₃O₄) C, H, N.

3-[(Ca r ba m oyloxy)m eth yl]-2-isop r op yl-1-m eth yl-5-(2-

m eth yla zir id in -1-yl)in d ole-4,7-d ion e (42). The carbamate

40 (0.1 g, 0.32 mmol) was stirred at room temperature in

2-methylaziridine (1.5 mL) for 2.5 h, evaporated, and redis-

solved in EtOAc (5 mL). The solution was then evaporated to

50% volume, and the resulting red precipitate was filtered,

washed well with cold EtOAc, and then recrystallized from

EtOAc to afford 42 (55 mg, 52%) as a red solid: mp 204-205

°C dec; H-NMR ((CD₃)₂SO) δ 1.37 (d, 6 H, J ) 7.2 Hz, CH-

(CH₃)₂), 1.42 (d, 3 H, J ) 5.4 Hz, azir-CH₃), 1.98-2.05 (m, 3

H, azir-CHCH₂), 3.1-3.28 (m, 1 H, CH(CH₃)₂), 3.93 (s, 3 H,

CH₃N), 5.06 (s, 2 H, CH₂OCONH₂), 5.76 (s, 1 H, 6-H), and 6.41

(br s, 2 H, NH₂). Anal. (C₁₇H₂₁N₃O₄) C, H, N.

g (41%) of 47 as a pale brown solid: mp 73-74 °C; H-NMR

(CDCl₃) δ 2.37 (s, 3 H, 2-CH₃), 3.59 (s, 3 H, CH₃N), 3.82 (s, 3

H, CH₃O), 6.16 (s, 1 H, 3-H), and 6.9-7.28 (m, 3 H, Ar-4,6,7H).

5-Meth oxy-1,2-d im eth ylin d ole-3-ca r boxa ld eh yd e (48).

N-Methylformanilide (0.95 g, 7.04 mmol) and POCl₃(1.08 g,

7.05 mmol) were stirred at room temperature until the yellow

solid chloroimmonium Vilsmeier compound formed. This

yellow solid was then added to a solution of 47 (0.7 g, 4 mmol)

in 1,2-dichloroethane (15 mL), the solution was heated under

reflux for 1.5 h and cooled, NaOAc (1.0 M, 50 mL) was added,

and the solution was stirred for 2.5 h. The solution was

extracted with EtOAc (4 × 100mL), dried, and evaporated. The

residue was purified on silica, eluting with EtOAc/hexane (1:

1, R_f) 0.5(EtOAc)) to give 0.35 g (43%) of 48 as an off-white

solid: mp 108-110 °C; ¹H-NMR (CDCl₃) δ 2.62 (s, 3 H, 2-CH₃),

3.63 (s, 3 H, CH₃N), 3.89 (s, 3 H, CH₃O), 6.94-7.2 (m, 2 H,

Ar-6,7H), 7.8 (d, 1 H, J ) 2 Hz, Ar-4H), and 10.1 (s, 1 H, CHO).

5-Met h oxy-1,2-d im et h yl-4-n it r oin d ole-3-ca r b oxa ld e-

h yd e (49). Compound 48 (6.0 g, 0.03 mol) was dissolved in

AcOH (480 mL) and cooled to 5 °C and fuming HNO₃(18 mL)

in AcOH (72 mL) added dropwise with stirring over 5 min.

The solution temperature was allowed to rise to 20 °C over 3

h, the mixture was poured onto crushed ice (500 g), and the

yellow precipitate was collected by suction filtration and dried

in vacuo at 50 °C. The yellow solid was purified on silica,

eluting with EtOAc/hexane (2:1, R_f) 0.42 (EtOAc)) to give

4.37 g (59%) of 49 as a pale yellow solid: mp 236-238 °C dec;

¹H-NMR ((CD₃)₂SO) δ 2.71 (s, 3 H, 2-CH₃), 3.76 (s, 3 H, CH₃N),

3.89 (s, 3 H, CH₃O), 7.25 (d, 1 H, J ) 9 Hz, Ar-7H), 7.75 (d, 1

H, J ) 9 Hz, Ar-6H), and 9.9 (s, 1 H, CHO).

1,3-Dim et h yl-2-isop r op yl-5-m et h oxyin d ole-4,7-d ion e

(43). To a solution of 35 (0.5 g,1.7 mmol) in CHCl₃(50 mL)

and EtOH (18 mL) was added a solution of Na₂S₂O₄(3.5 g, 20

mmol) in H₂O (22 mL). The solution was stirred at room

temperature for 1 h, and the organic layer was separated,

washed with saturated NaCl (50 mL), dried, and evaporated.

The crude hydroquinone was dried over 18 h in vacuo,

dissolved in anhydrous THF (5 mL) under argon, and added

to a solution of DIBAL-H (10 mL of a 1.5 M solution in toluene)

dropwise at -30 °C and under argon. The solution was then

stirred for 18 h at 4 °C and cooled to 0 °C, and H₂O (15 mL)

was added dropwise, followed by a solution of FeCl₃(12 mL,

1.0 M (0.1 M HCl)) added at 0 °C. The solution was stirred

for 10 min at 0 °C, and then EtOAc (150 mL) and H₂O (150

mL) were added. The aqueous layer was extracted with EtOAc

(5 × 50 mL), and the organic phase was washed with saturated

NaCl (250 mL), dried, and evaporated. The residue was

purified on silica, eluting with EtOAc/hexane (1: 2, R_f) 0.44)

to give, after recrystallization from EtOAc, 43 as an orange

solid (48 mg, 11%): mp 168-169 °C; H-NMR (CDCl₃) δ 1.35

(d, 6 H, J ) 7.2 Hz, CH(CH₃)₂), 2.38 (s, 3 H, 3-CH₃), 3.1-3.28

(m, 1 H, CH(CH₃)₂), 3.78 (s, 3 H, CH₃N), 3.95 (s, 3 H, CH₃O),

and 5.57 (s, 1 H, 6-H). Anal. (C₁₄H₁₇NO₃) C, H, N.

5-(Azir id in -1-yl)-1,3-d im eth yl-2-isop r op ylin d ole-4,7-d i-

on e (44). A solution of 43 (30 mg, 0.12 mmol) in 1H-aziridine

(0.5 mL, ca.11.7 mmol, CAUTION!) was stirred for 0.5 h at

room temperature and then evaporated to dryness and the

residue purified on silica, eluting with EtOAc/hexane (1:2, R_f

) 0.32) to a give, after recrystallization from EtAOc, 44 (5.5

mg, 18%) as a red solid: mp 110-112 °C; H-NMR (CDCl₃) δ

4-Am in o-5-m eth oxy-1,2-d im eth ylin d ole-3-ca r boxa ld e-

h yd e (50). Nitro compound 49 (0.18 g, 0.74 mmol) was

dissolved in EtOH (15 mL), powdered tin (0.45 g, 3.8 mmol)

was added, followed by HCl (3.0 M, 5.6 mL), and the solution

was heated under gentle reflux for 1 h. Water (50 mL) was

1.35 (d, 6 H, J ) 7.2 Hz, CH(CH₃)₂), 2.15 (s, 4 H, 2 × azir-

CH₂), 2.38 (s, 3 H, 3-CH₃), 3.1-3.28 (m, 1 H, CH(CH₃)₂), 3.94

(s, 3 H, CH₃N), and 5.72 (s, 1 H, 6-H). Anal. (C₁₅H₁₈N₂O₂) H,

N; C: calcd, 65.22; found, 65.74.

2344 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15

Naylor et al.

added and the solution neutralized with NaHCO₃(aq), ex-

tracted with CHCl₃(3 × 100 mL), dried, and evaporated. The

residue was purified on silica, eluting with EtOAc/hexane (1:

1, R_f) 0.57 (EtOAc)) to give 0.1 g (62%) of 50 as a pale yellow

solid: mp 152-153 °C dec; ¹H-NMR ((CD₃)₂SO) δ 2.61 (s, 3 H,

2-CH₃), 3.59 (s, 3 H, CH₃N), 3.75 (s, 3 H, CH₃O), 6.0 (br s, 2

H, NH₂), 6.56 (d, 1 H, J ) 9 Hz, Ar-7H), 6.85 (d, 1 H, J ) 9

Hz, Ar-6H), and 9.71 (s, 1 H, CHO).

3-F or m yl-5-m eth oxy-1,2-d im eth ylin d ole-4,7-d ion e (51).

To a solution of 50 (0.074 g, 0.34 mmol) in Me₂CO (14 mL)

was added a solution of Fremy’s salt (0.45 g, 1.68 mmol) in

NaH₂PO₄/Na₂HPO₄buffer (14 mL, 0.3 M, pH 6.0) and the

solution stirred at room temperature for 1 h. The solution was

evaporated at 30 °C to remove most of the Me₂CO and the

resulting orange precipitate collected by suction filtration and

washed well with H₂O and cold MeOH to give 51 (60 mg,

75%): mp 239-242 °C; ¹H-NMR ((CD₃)₂SO) δ 2.5 (s, 3 H,

2-CH₃), 3.82 (s, 3 H, CH₃N), 3.88 (s, 3 H, CH₃O), 5.89 (s, 1 H,

6-H), and 10.37 (s, 1 H, CHO).

5-Meth oxy-3-(h yd r oxym eth yl)-1,2-d im eth ylin d ole-4,7-

d ion e (52). To a suspension of 51 (0.25 g, 0.94 mmol) in

MeOH (100 mL, degassed by boiling under argon in vacuo)

was added NaBH₄(0.36 g, 9.7 mmol) while a dry argon

atmosphere was maintained. The solution was stirred at room

temperature for 1 h, aerated prior to the addition of H₂O (40

mL), extracted with CH₂Cl₂(3 × 100 mL), dried, and evapo-

rated. The residue was purified on silica, eluting with EtOAc

(R_f) 0.4) to afford 52 (0.1 g, 33%) as an orange solid,

recrystallized from EtOAc: mp 215-216 °C (lit.²mp 199-200

°C); ¹H-NMR (CDCl₃) δ 2.21 (s, 3 H, 2-CH₃), 3.81 (s, 3 H,

CH₃N), 3.86 (s, 3 H, CH₃O), 4.65 (br d, 2 H, J ) 7.2 Hz, CH₂-

OH), and 5.61 (s, 1 H, 6-H). Anal. (C₁₂H₁₃NO₄) C, H, N.

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

4,7-d ion e (53). A solution of 52 (235 mg, 1.0 mmol) in freshly

redistilled 1H-aziridine (1.5 mL, ca. 35 mmol, CAUTION!) was

stirred for 0.5 h at room temperature and evaporated in vacuo.

The residue was redissolved in EtOAc and condensed by 75%,

and the precipitate was collected and washed with cold EtOAc

to give 225 mg (91%) of 53 as a dark red solid: mp 173-174

°C dec; ¹H-NMR (CDCl₃) δ 2.19 (s, 4 H, 2 × azir-CH₂), 2.22 (s,

3 H, 2-CH₃), 3.86 (s, 3 H, CH₃N), 3.95 (br, 1 H, CH₂OH), 4.65

(br d, 2 H, CH₂OH), and 5.76 (s, 1 H, 6-H). Anal. (C₁₃H₁₄N₂O₃)

C, H, N.

3-(H yd r oxym e t h yl)-5-(2-m e t h yla zir id in -1-yl)-1,2-d i-

m eth ylin d ole-4,7-d ion e (54). A solution of 52 (235 mg, 1.0

mmol) in 2-methylaziridine (2 mL) was stirred at room

temperature for 4 h and worked up as described for 53 to give

54 (195 mg, 75%) as a dark red solid: mp 120-122 °C; ¹H-

NMR (CDCl₃) δ 1.42 (d, 3 H, J ) 5.4 Hz, azir-CH₃), 2.08-2.21

(m, 3 H, azir-CHCH₂), 2.22 (s, 3 H, 2-CH₃), 3.89 (s, 3 H, CH₃N),

4.61 (br, 1 H, CH₂OH), 4.63 (br, 2H, CH₂OH), and 5.75 (s, 1

H, 6-H). Anal. (C₁₄H₁₆N₂O₃) H, N; C: calcd, 64.62; found,

65.09.

3-(H yd r oxym et h yl)-5-(cis-2,3-d im et h yla zir id in -1-yl)-

1,2-d im eth ylin d ole-4,7-d ion e (55). A solution of 52 (100 mg,

0.43 mmol) in cis-2,3-dimethylaziridine (2 mL) was stirred for

1 h at room temperature and then evaporated in vacuo at 30

°C. The residue was purified on silica, eluting with EtOAc

(R_f) 0.6) to give 55 (65 mg, 55%) after recrystallization from

EtOAc: mp 132-135 °C; ¹H-NMR (CDCl₃) δ 1.38 (d, 6 H, J )

5.4 Hz, 2 × azir-CH₃), 2.15-2.21 (m, 2 H, 2 × azir-CH), 2.22

(s, 3 H, 2-CH₃), 3.85 (s, 3 H, CH₃N), 4.24 (br, 1 H, CH₂OH),

4.63 (br, 2 H, CH₂OH), and 5.73 (s, 1 H, 6-H). Anal.

(C₁₅H₁₈N₂O₃) C, H, N.

3-(H yd r oxym et h yl)-5-(2,2-d im et h yla zir id in -1-yl)-1,2-

d im eth ylin d ole-4,7-d ion e (56). A solution of 52 (100 mg,

0.43 mmol) in 2,2-dimethylaziridine (2 mL) was stirred at 95

°C for 5 h. After this time (reaction does not go to completion)

the solution was cooled and evaporated in vacuo, and the

residue redissolved in EtOAc and evaporated to precipitate

56 (25 mg, 21%) as a dark red solid, which was unstable on

silica and in solution: mp 138-141 °C; ¹H-NMR (CDCl₃) δ 1.32

(s, 6 H, 2 × azir-CH₃), 2.07 (s, 2 H, azir-CH₂), 2.22 (s, 3 H,

2-CH₃), 3.87 (s, 3 H, CH₃N), 4.25 (br, 1 H, CH₂OH), 4.86 (br,

2 H, CH₂OH), and 5.68 (s, 1 H, 6-H). Anal. (C₁₅H₁₈N₂O₃‚²/

₃H₂O) C, H, N.

3-(H yd r oxym et h yl)-5-(2,2-d im et h yl-2-h yd r oxyet h yl)-

a m in o)-1,2-d im eth ylin d ole-4,7-d ion e (57). Compound 56

(25 mg, 0.09 mmol) was dissolved in 5 mL of H₂O with

warming to 45 °C and then evaporated in vacuo at 50 °C. The

residue was purified on silica, eluting with EtOAc (R_f) 0.35)

to give 57 (25 mg, 95%) after recrystallization from EtOAc:

mp 188-190 °C; ¹H-NMR ((CD₃)₂SO) δ 1.14 (s, 6 H, 2 × CH₃),

2.21 (s, 3 H, 2-CH₃), 3.02 (d, 2 H, J ) 7.2 Hz, NHCH₂C(CH₃)₂),

3.84 (s, 3H, CH₃N), 4.58 (br, 2 H, CH₂OH), 4.65 (br, 1 H,

CH₂OH), 5.14 (s, 1 H, 6-H), and 6.63 (br t, 1 H, NH). Anal.

(C₁₅H₂₀N₂O₄‚¹/₃H₂O) C, H, N.

5-Meth oxy-1-m eth ylin d ole-3-ca r boxa ld eh yd e (58). 5-

Methoxyindole-3-carboxaldehyde (2.0 g, 11.4 mmol) was added

gradually to a suspension of NaH (0.55 g, 13.7 mmol) in DMF

(50 mL) with stirring. The suspension was stirred for 0.5 h,

MeI (1.94 g, 13.7 mmol) was added, and the mixture was

stirred for 1 h at room temperature. The reaction mixture was

then poured onto NaHCO₃(10%, 300 mL), extracted with

EtOAc (4 × 75 mL), washed with NaHCO₃(10%, 3 × 50 mL)

and saturated NaCl (3 × 100 mL), dried, and evaporated in

vacuo to give 58 (1.70 g, 79%) as a white solid: mp 132-133

°C; ¹H-NMR (CDCl₃) δ 3.81 (s, 3 H, CH₃N), 3.89 (s, 3 H, CH₃O),

7.08 (dd, 1 H, J ) 2 and 9 Hz, Ar-6H), 7.31 (d, 1 H, J ) 9 Hz,

Ar-7H), 7.59 (s, 1 H, 2-H), 7.8 (d, 1 H, J ) 2Hz, Ar-4H), and

9.93 (s, 1H, CHO).

5-Met h oxy-1-m et h yl-4-n it r oin d ole-3-ca r b oxa ld eh yd e

(59). To a solution of 58 (1.50 g 7.94 mmol) dissolved in AcOH

(150 mL) was added a mixture of concentrated HNO₃(4.5 mL)

in AcOH (25 mL) dropwise at 0 °C over 3 h. After addition,

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

to crushed ice (75 g), filtered, washed with H₂O (5 × 100mL),

and dried to give 59 (1.56 g, 84%) as a pale yellow solid: mp

197-198 °C; ¹H-NMR ((CD₃)₂SO) δ 3.94 (s, 6 H, CH₃O and

CH₃N), 7.32 (d, 1 H, J ) 9 Hz, Ar-7H), 7.72 (d, 1 H, J ) 9 Hz,

Ar-6H), 8.27 (s, 1 H, 2-H), and 9.75 (s, 1 H, CHO).

4-Am in o-5-m e t h oxy-1-m e t h ylin d ole -3-ca r b oxa ld e -

h yd e (60). To a suspension of 59 (1.0 g, 4.27 mmol) in EtOH

(150 mL) was added tin (4.43 g, 37 mmol) followed by HCl

(3.0 M, 60 mL). The mixture was stirred at room temperature

for 2 h and decanted. The solution was added gradually to

saturated NaHCO₃(aqueous, 300 mL) and extracted with

EtOAc (3 × 100 mL). The organic layer was separated, washed

with saturated NaHCO₃(aqueous, 2 × 175 mL) and saturated

NaCl (3 × 75 mL), dried, and evaporated in vacuo to give 60

(0.79 g, 91%) as a dark yellow solid which was used in the

next step without further purification: R_f) 0.64 (EtOAc); ¹H-

NMR (CDCl₃) δ 3.75 (s, 3 H, CH₃N), 3.88 (s, 3 H, CH₃O), 5.79

(br s, 2 H, NH₂), 6.56 (d, 1 H, J ) 9 Hz, Ar-7H), 7.53 (d, 1 H,

J ) 9 Hz, Ar-6H), 7.64 (s, 1 H, 2-H), and 9.60 (s, 1 H, CHO).

3-F or m yl-5-m eth oxy-1-m eth ylin d ole-4,7-d ion e (61). To

60 (0.75 g, 3.68mmol) dissolved in Me₂CO (75 mL) was added

Fremy’s salt (4.0 g, 14.9 mmol) in H₂O (20 mL) followed by a

solution of Na₂HPO₄/NaH₂PO₄buffer (0.3 M, pH 6, 20 mL).

The mixture was stirred for 0.75 h, excess Me₂CO was

removed, and the product was filtered, washed with H₂O (50

mL), dried, and recrystallized from EtOAc to give 61 (0.61 g,

76%) as a yellow solid: mp 188-190 °C; H-NMR (CDCl₃) δ

3.87 (s, 3 H, CH₃N), 4.02 (s, 3 H, CH₃O), 5.78 (s, 1 H, 6-H),

7.44 (s, 1 H, 2-H), and 10.54 (s, 1 H, CHO).

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

on e (62). To a solution of 61 (0.5 g, 2.28 mmol) in anhydrous

argon degassed MeOH (300 mL) was added NaBH₄(0.65 g,

17 mmol). The solution was degassed with argon, stirred for

2 h under argon, and then evaporated in vacuo to give a solid

which was diluted with CH₂Cl₂(300 mL), washed with H₂O

(2 × 100 mL) and saturated NaCl (100 mL), and condensed to

give a 62 as an orange solid (0.2 g, 40%) after recrystallization

from EtOAc: mp 185-186^oC; ¹H-NMR (CDCl₃) δ 3.85 (s, 3 H,

CH₃N), 3.94 (s, 3 H, CH₃O), 4.25-4.29 (m, 2 H, CH₂OH), 5.73

(s, 1 H, 6-H), and 6.88 (s, 1 H, 2-H). Anal. (C₁₁H₁₁NO₄) C, H,

5-(Azir id in -1-yl)-3-(h yd r oxym et h yl)-1-m et h ylin d ole-

4,7-d ion e (63). A solution of 62 (200 mg, 0.9 mmol) in 1H-

aziridine (1.5 mL, ca. 35 mmol, CAUTION!) was stirred at

room temperature for 1.5 h. Excess aziridine was removed in

vacuo, and the product was recrystallized from EtOAc to give

Bioreductively Activated Antitumor Agents

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2345

63 (130 mg, 62%) as an orange solid: mp 169-171 °C; ¹H-

NMR (CDCl₃) δ 2.22 (s, 4 H, 2 × aziridine-CH₂), 3.91 (s, 3 H,

CH₃N), 4.64 (s, 2 H, CH₂OH), 5.81 (s, 1 H, 6-H), and 6.69 (s,

1 H, 2-H). Anal. (C₁₂H₁₂N₂O₃) C, H, N.

At pH 8.5 the semiquinone radical absorption (720 nm)

decayed with second-order kinetics and a half-life which

decreased with increasing radiation dose (i.e. with initial

concentration of radicals), indicating that the semiquinone

radical decays predominantly by a radical-radical reaction to

generate the hydroquinone as in reaction 1.

3-(H yd r oxym et h yl)-5-(2-m et h yla zir id in -1-yl)-1-m et h -

ylin d ole-4,7-d ion e (64). A solution of 62 (200 mg, 0.9 mmol)

in 2-methylaziridine (1 mL) was stirred at room temperature

for 2.5 h. Excess 2-methylaziridine was removed in vacuo, and

the product was purified on silica, eluting with EtOAc (R_f)

0.6) to give 64 (120 mg, 54%) as a red solid recrystallized from

2Q^•-+ 2H⁺f Q + QH₂

(1)

The reciprocal of the first half-life of the semiquinone radical

of 54 varied linearly with the initial radical concentration, and

from the slope of the fitted straight line the rate constant 2k₂

) (2.3 ( 0.1) × 10⁷M^-1s^-1was obtained, and this is

comparable with that recorded for EO9 (3).¹⁷

EtOAc: mp 89-90 °C; H-NMR (CDCl₃) δ 1.47 (d, 3 H, J )

4.5 Hz, azir-CH₃), 2.04-2.2 (m, 3 H, azir-CHCH₂), 3.91 (s, 3

H, N-CH₃), 4.67 (m, 2 H, CH₂OH), 5.79 (s, 1 H, 6-H), and 6.70

(s, 1 H, 2-H). Anal. (C₁₃H₁₄N₂O₃) C, H, N.

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

4,7-d ion e (65). To a solution of 62 (0.1 g, 0.45 mmol) in

anhydrous pyridine (5 mL) at 0 °C was added dropwise phenyl

chloroformate (0.1 g, 0.64 mmol) and the solution then allowed

to reach room temperature and stirred for 2 h. The solution

was then extracted with CH₂Cl₂(30 mL), washed with H₂O

(35 mL) and saturated NaCl (35 mL), dried, and evaporated.

The residue was purified on silica, eluting with EtOAc (R_f)

0.8) to give an orange solid of 5-methoxy-1-methyl-3-[[(phe-

noxycarbonyl)oxy]methyl]indole: mp 85-86 °C; ¹H-NMR

((CD₃)₂SO/CDCl₃) δ 3.79 (s, 3 H, CH₃N), 3.91 (s, 3 H, CH₃O),

5.43 (s, 2 H, CH₂OCOPh), 5.67 (s, 1 H, 6-H), 6.89 (s, 1 H, 2-H),

and 7.15-7.3 (m, 5 H, Ar). This material (0.1 g, 0.3 mmol)

was dissolved in anhydrous CH₂Cl₂(18 mL) and the solution

cooled to -78 °C. The solution was then saturated with NH₃

and stirred at -78 °C until the reaction was complete (0.75

h). The solution was then allowed to reach room temperature

and evaporated in vacuo. The residue was redissolved in CH₂-

Cl₂(100 mL), washed with H₂O (2 × 100 mL) and saturated

NaCl (50 mL), dried and evaporated, and the residue recrys-

tallized from EtOAc to afford 75 mg (98%) of 65 as an orange

solid: mp 231-234 °C (dec.); ¹H-NMR ((CD₃)₂SO/CDCl₃) δ 3.78

(s, 3 H, CH₃N), 3.88 (s, 3 H, CH₃O), 5.04 (s, 2 H, CH₂OCONH₂),

5.77 (s, 1 H, 6-H), 6.42 (br s, 2 H, NH₂), and 7.09 (s, 1 H, 2-H).

Anal. (C₁₂H₁₂N₂O₅) H, N; C: calcd, 54.55; found, 54.98.

3-[(Car bam oyloxy)m eth yl]-5-(azir idin -1-yl)-1-m eth ylin -

d ole-4,7-d ion e (66). Compound 65 (50 mg, 0.2 mmol) was

stirred for 0.5 h in 1H-aziridine (ca. 35 mmol, CAUTION!),

evaporated in vacuo, and redissolved in EtOAc. The solution

was evaporated again and the residue recrystallized from

EtOAc to afford 35 mg (70%) of 66 as a red solid: mp 195-

198 °C dec; ¹H-NMR ((CD₃)₂SO) δ 2.18 (s, 4 H, 2 × azir-CH₂),

3.90 (s, 3 H, CH₃N), 5.23 (s, 2 H, CH₂OCONH₂), 5.49 (br s, 2

H, NH₂) 5.76 (s, 1 H, 6-H), and 6.86 (s, 1 H, 2-H). Anal.

(C₁₃H₁₃N₃O₄) C, H, N.

The reactivities of semiquinone radicals with oxygen were

determined by saturating solutions with N₂O/O₂gas mixtures

(British Oxygen Company, U.K.) which contained 0.2-2.1%

O₂. In the presence of oxygen the semiquinone radicals

decayed (eq 2) faster with increasing oxygen concentrations.

Absolute rate constants k₂determined from the slope of the

linear plots of the first-order rate constants versus oxygen

concentration are shown in Table 2.

•-

^•-+ O₂f Q + O₂

(2)

The redox potentials of the one-electron couple E (Q/Q^•-) were

determined by establishing redox equilibria with viologens of

known redox potential in reaction 3 according to methods

descibed previously.²⁷

^•-+ V²⁺h Q + V^•+

(3)

For V²⁺/indoloquinone systems, redox equilibration usually

occurred within ∼20 µs (during which there was negligible

decay of semiquinone radicals via reaction 1), and it was

therefore possible to calculate the equilibrium constant K₃from

absorbances at equilibrium.

Estimates of K₃and E (Q/Q^•-) for the range of indoloqui-

nones studied are shown in Table 2. All E (Q/Q^•-) values

quoted have been corrected for the effect of ionic strength (I

) 0.004 ) ca. 7 mV), utilizing the Debye-Hu¨ckel expression.²⁴

Biologica l Eva lu a tion in Vitr o. Selective toxicity to

hypoxic V79-379A cells was determined for all compounds

using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-

tetrazolium bromide] assay as has been described previous-

ly.^28-30These results are presented in Table 1, where C₅₀(air)

values, the concentration required to kill 50% of the aerobic

cells under the conditions of the assay, are divided by C₅₀(N₂)

values, concentrations required to kill hypoxic cells, to give

hypoxic cytotoxicity ratios (HCR), which enable quantitative

comparisons of bioreductive activities (hypoxic cytotoxicities)

of drugs.

Red ox Ch em istr y. The properties of representative semi-

quinone radicals were studied following reduction of the

quinones by the (CH₃)₂C^•OH radical generated by pulse

radiolysis.²³Optical detection was used to determine the one-

electron reduction potentials (E (Q/ Q^•-)) of the indoloquinones

and the reactivities of the semiquinone radicals with oxygen.

Experiments were performed with a 6 MeV linear accelerator

as described previously.²⁴Solutions at 23 °C, pH 8.5, contained

0.2 M 2-propanol and 10 mM phosphate buffer (NaH₂PO₄/Na₂-

HPO₄) with Q (0 or 60 µM) and either benzyl viologen, BV²⁺

(0-4 mM), or methyl viologen, MV²⁺(0-4 mM). Absorbances

at 600 nm were measured ∼20 µs after a dose of ∼2 Gy.

Solutions were dearated by saturation with N₂O. The alcohol

Biologica l Eva lu a tion in Vivo. The RIF-1 and KHT

murine sarcoma tumor models were used for the initial in vivo

tumor toxicity evaluations of lead compounds. The maximum

tolerated doses and tumor response experiments were carried

out essentially as described previously.³⁰Tumor-bearing mice

were given doses of irradiation that were chosen to kill most

of the oxic cells in the tumors. Thus, the measured response

would reflect the survival of residual hypoxic clonogenic cells.³¹

Indoloquinones were given immediately after X-rays such that

any therapeutic response greater than that achieved by

radiation alone is a reflection of residual hypoxic cell killing.

These conditions were achieved by giving 10Gy to KHT tumors

and 25Gy to RIF-1 tumors. Each experiment also included

mice exposed to radiation without drug and drug alone.

Results obtained with the selected lead compounds are dis-

played in Table 3.

•

converts the radiolytically produced OH and H^•radicals in

<0.2 µs to the reductant (CH₃)₂C^•OH which reduces the

indoloquinones or viologens (V²⁺) to the corresponding semi-

quinone Q^•-radicals or viologen radical cations V^•+

. All

experiments were performed at pH 8.5 because the aziridinyl

indoloquinones, including EO9 (3),²⁵slowly hydrolyze at physi-

ological pH. The absorption spectra of the radicals exhibited

absorption maxima in the 340-400 nm region, similar to that

of EO9 (3).¹⁷The radical spectra did not change between pH

7 and pH 10, and it was concluded that at pH 7.4 and above

the semiquinone is deprotonated. Due to the instability of the

aziridine at low pH it was not possible to determine the pK of

the semiquinone radical. Previous studies indicate that most

semiquinone radicals have a pK of around 4-6.²⁶

Ack n ow led gm en t. This work was funded by the

financial support of the U.S. National Cancer Institute

under Grant No. CA55165-02 and the U.K. Medical

Research Council. The authors thank Dr. M. R. L.

Stratford (Gray Laboratory Cancer Research Trust) for

2346 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15

Naylor et al.

(17) Butler, J .; Spanswick, V. J .; Cummings, J . The autoxidation of

the reduced forms of EO9. Free Rad. Res. 1996, 25, 141-148.

(18) Wardman, P.; Dennis, M. F.; Everett, S. A.; Patel, K. B.;

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(19) Zhou, R.; Skibo, E. B. Chemistry of the pyrrolo[1,2-a]benzimid-

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(20) Plumb, J . A.; Gerritsen, M.; Workman, P. DT-diaphorase protects

cells from the hypoxic cytotoxicity of indoloquinone EO9. Br. J .

Cancer 1994, 70, 1136-1143.

(21) Dickey, F. H.; Fickett, W.; Lucas, H. J . Stereoisomeric 2,3-

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(23) Patel, K. B.; Wilson, R. L. Semiquinone free radicals and oxygen.

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(24) Candeias, L. P.; Folkes, L. K.; Porssa, M.; Parrick, J .; Wardman,

P. Rates of reaction of indoleacetic acids with horseradish

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the determination of the stabilities of drugs prior to

radiolytic reduction experiments and Professor P. Ward-

man (Gray Laboratory Cancer Research Trust) for

valued comments and discussion. Gray Laboratory

support from the Cancer Research Campaign (CRC) is

gratefully acknowledged.

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