Aziridinyl quinone antitumor agents based on indoles and cyclopent[b]indoles: Structure-activity relationships for cytotoxicity and antitumor activity

J . Med. Chem. 2001, 44, 3545-3562

3545

Articles

Azir id in yl Qu in on e An titu m or Agen ts Ba sed on In d oles a n d

Cyclop en t[b]in d oles: Str u ctu r e-Activity Rela tion sh ip s for Cytotoxicity a n d

An titu m or Activity

Edward B. Skibo* and Chengguo Xing

Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604

Robert T. Dorr

Arizona Cancer Center, University of Arizona, Tucson, Arizona 85721

Received February 23, 2001

A large number of aziridinyl quinones represented by series 1-9 were studied with respect to

their DT-diaphorase substrate activity, DNA reductive alkylation, cytostatic/cytotoxic activity,

and in vivo activity. As a result, generalizations have been made with respect with respect to

the following: DT-diaphorase substrate design, DT-diaphorase-cytotoxicity quantitative

structure-activity relationship (QSAR), and DNA reductive alkylating agent design. A

saturating relationship exists between the substrate specificity for human recombinant DT-

diaphorase and the cytotoxicity in the human H460 non-small-cell lung cancer cell line. The

interpretation of this relationship is that reductive activation is no longer rate-limiting for

substrates with high DT-diaphorase substrate specificities. High DT-diaphorase substrate

specificity is not desirable in the indole and cylopent[b]indole systems because of the result is

the loss of cancer selectivity along with increased toxicity. We conclude that aziridinyl quinones

of this type should possess a substrate specificity (V_max/K_M) < 10 × 10^-4s^-1for DT-diaphorase

in order not to be too toxic or nonselective. While some DNA alkylation was required for

cytostatic and cytotoxic activity by series 1-9, too much alkylation results in loss of cancer

selectivity as well as increased in vivo toxicity. Indeed, the most lethal compounds are the

indole systems with a leaving group in the 3R-position (like the antitumor agent EO9). We

conclude that relatively poor DNA alkylating agents (according to our assay) show the lowest

toxicity with the highest antitumor activity.

Ch a r t 1

Many of the clinically used antitumor agents that

have withstood the test of time are DNA-directed

alkylating agents utilizing the aziridine alkylating

center. Noteworthy examples include the nitrogen mus-

tards,¹nitrosoureas,²thiotepa,³AZQ,³triethylene-

melamine,³and mitomycin C.⁴These compounds have

been thoroughly studied and have been subjected to

intense analogue development for over 40 years. Ex-

amples of aziridinyl quinone antitumor agents devel-

oped recently are shown in Chart 1.

The pyrrolo[1,2-a]benzimidazoles (PBIs) were devel-

oped in this laboratory and were found to possess

cytotoxicity with minimal antitumor activity.^5,6The

indoloquinone EO9 was considered to be a promising

antitumor agent,^7,8but phase I clinical trials revealed

short plasma half-lives as well as toxicity.^9-11The

cyclopent[b]indoles, reported in the past year,¹²possess

promising antitumor activity.

This report describes a comprehensive structure-

activity study of cyclopent[b]indole-based (series 1-5)

and indole-based (series 6-9) aziridinyl quinones rep-

resented by the nine general structures shown in Chart

2. Diverse structures were generated by varying the

* To whom correspondence should be addressed: Telephone 480-

965-3581; fax 480-965-2747; e-mail ESkibo@ASU.edu.

Published on Web 10/02/2001

3546 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

Ch a r t 2

Sch em e 1

Sch em e 2

position of the aziridinyl and quinone methyl groups (if

present) as well as the indole N-substituent. These

structural features can influence the interaction of

quinone antitumor agents with DNA,^12,13and this study

would clarify the structural requirements for such

binding. The structure of EO9^14-17inspired the synthe-

sis of indole-based diol derivatives and their acetylated

analogues.

The study of a large number of compounds required

use of a chemical/biochemical prescreen that could

predict biological activity. We successfully used the

quinone substrate specificity (V_max/K_M) for human DT-

diaphorase and the percent DNA alkylation to assess

cytotoxic and antitumor capability. From this study, we

were able to elucidate structure activity relationships

valuable in future antitumor design.

obtained by borohydride reduction of the 3-one group.

Acetylation of the 3-hydroxy group afforded a mixture

of O- and N-acetylated compounds that constituted the

1b-4b and 1c-4c series. N-Methylation of the cyclo-

pent[b]indol-3-one system, followed by quinone and

3-hydroxyl or 3-acetoxy elaboration, provided series 5.

Shown in Schemes 4-6 are the synthetic methodolo-

gies for the preparation of the indoles (series 6-9).

Preparation of the 2-(ethylcarboxyl)indole substituted

systems were carried out by the J app-Klingemann/

Fischer indole reaction as previously described.^12,20

Vilsmeier formylation and borohydride reduction af-

forded the 3-hydroxymethyl derivative of these indole

systems. The 2-ethylcarboxyl substituent was either

retained or reduced with lithium aluminum hydride

Resu lts a n d Discu ssion

Syn th esis. The preparation of series 1-9 was carried

out as outlined in Schemes 1-6. Many of the synthetic

procedures utilized for these preparations are straight-

forward and are briefly outlined below.

Shown in Schemes 1-3 are the synthetic methodolo-

gies for the preparation of the cyclopent[b]indoles (series

1-5). Preparation of the substituted cyclopent[b]indol-

3-one system was carried out by the Fischer indole

reaction as previously described.¹²Elaboration of the

quinone functional group was carried out by nitration,

catalytic reduction of the nitro group, and finally Fremy

oxidation.^18,19The 3-hydroxy group (1a -4a series) was

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3547

Sch em e 3

Sch em e 5

Sch em e 4

Sch em e 6

(LAH) to the 2-hydroxymethyl group. The hydroxy-

methyl groups were either left as is or acetylated to

afford the acetate leaving group. Finally, quinone

elaboration and aziridination were carried out as previ-

ously described.^18,19

DT-Dia p h or a se Su b st r a t e Scr een in g of Ser ies

1-9. The enzyme DT-diaphorase is an NAD(P)H-de-

pendent reducing enzyme that converts quinones and

other substrates to the corresponding two-electron

reduction products. The hydroquinone reduction product

is cytotoxic because its aziridinyl nitrogen is readily

protonated at physiological pH, resulting in alkylation

reactions (Scheme 7). The phosphate backbone²¹and the

purine N(7)-position^22,23of DNA are the usual nucleo-

3548 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

Ta ble 2. DT-Diaphorase Kinetic Parameters and DNA

Sch em e 7

Alkylation Percentages for Series 3-4

Ta ble 3. DT-Diaphorase Kinetic Parameters and DNA

Alkylation Percentages for Series 5-6

Ta ble 1. DT-Diaphorase Kinetic Parameters and DNA

Alkylation Percentages for Series 1-2

phile targets. This enzyme is elevated in some cancers

and the reduction process leads to activation of antitu-

mor agents specifically in these cancers.^24-26The reduc-

tive activation process is well-known with substrates

such as mitomycin C,^27,28EO9,^15,29and the PBIs.^6,30

Listed in Tables 1-4 are the human DT-diaphorase

kinetic parameters for series 1-9, along with the DNA

alkylation percentages. These parameters include the

apparent dissociation constant K_M, the apparent maxi-

reduction of pyrrolo[1,2-a]benzimidazoles (PBIs), the

enzyme-bound PBI structure was obtained by superim-

position into the human DT-diaphorase active site.³⁴

Shown in Figure 1 is the active site of DT-diaphorase

(1D4A) as well as the same active site containing a PBI

aziridinyl quinone. Although the structure shown in

Figure 1B was not crystallographically determined, it

permitted the rationalization of indole and cyclopent-

[b]indole substrate specificity as illustrated below.

Inspection of the substrate specificity data in Tables

1-7 reveals the following features:

(1) The presence of a methyl substituent on the

quinone ring substantially slows DT-diaphorase reduc-

tion. Comparison of series 1 and 2 or of series 6 and 7

shows up to 10-fold changes in specificity when the

methyl substituent is present. Steric interactions be-

mal velocity V_max, and the substrate specificity, V_max

/

K_M. The designation apparent refers to the presence of

productive and nonproductive constants in K_Mand

31

V_max

.

These constants cancel in the ratio V_max/K_M,

which is the best parameter to compare substrates.

Human DT-diaphorase (from the non-small-cell lung

NCI-H460 cell line)³²was used to obtain these kinetic

parameters. This enzyme is the same as that present

in other human tissues, cancerous or otherwise.

The human DT-diaphorase enzyme has been cloned

and its crystal structure determined (1D4A in the

Protein Data Bank).³³In a study of the DT-diaphorase

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3549

Ta ble 4. DT-Diaphorase Kinetic Parameters and DNA

Alkylation Percentages for Series 7-9

tween residues W105 and F106 (Figure 1) and the

methyl substituent are very likely responsible for the

lower substrate specificity of methyl-substituted quino-

nes for DT-diaphorase. Indeed, PBI analogues bearing

a 7-butyl instead of a 7-methyl were poor substrates for

the enzyme.³⁰

F igu r e 1. Model A: active sites of human DT-diaphorase from

NCI-H460 non-small-cell lung cancer obtained from the Pro-

tein Data Bank (1D4A). Important amino acid residues are

labeled. Model B: S-amino PBI substrate in the DT-diaphorase

from NCI-H460 non-small-cell lung cancer.

(2) The position of the aziridinyl substituent on the

quinone ring influences substrate specificity to a lesser

degree than the methyl substituent; compare 1a and

3a or 6a and 9a . In these cases, the parallel walls

formed by the W105 and F106 residues can accom-

modate the aziridinyl substituent regardless of its

position. These residues likewise accommodate the

aziridinyl substituent of EO9-like indole analogues.¹⁷

(3) The presence of a 3-acetoxy substituent on some

of the cyclopent[b]indole systems increases substrate

specificity markedly over the 3-hydroxy derivatives;

compare 1a and 1b or 3a and 3b. The explanation is

that the acetate carbonyl is a hydrogen-bond acceptor

for the N-H of His194, Figure 1. Similarily, the

3-acetate and 3-carbamido derivatives of the PBI system

can hydrogen-bond with this amino acid.³⁴However, if

a methyl substituent is present on the quinone ring, the

substrate changes position in the active site and the

acetate does not hydrogen-bond with His 194; compare

2a and 2b or 4a and 4b.

(4) The highest specificities for DT-diaphorase ob-

served in this study were for the indoles bearing an

ethoxycarbonyl substituent in the 2-position, 6c and 9b.

The explanation for the high substrate specificities is

resonance stabilization of the anion resulting from the

Michael transfer of hydride by the FADH₂cofactor.

Stabilization of this anionic intermediate by resonance

would also stabilize the transition state for hydride

transfer, resulting in a high V_maxvalue and accordingly

a high substrate specificity. Both the crystal structure

of DT-diaphorase³³and the reported hydride transfer

mechanism to quinones³⁵support the Michael-type

hydride transfer to the 5-position as shown in Scheme

8. In both 6c and 9b, the resulting anion (not to be

confused with the radical anion arising from hydrogen

atom transfer) can be resonance-delocalized into the

ethoxycarbonyl substituent. The 3R-acetoxy substituent

of 6d very likely shifts the substrate in the active site

so that hydride transfer occurs to the 6-position rather

than the 5-position. In this case, the anion resulting

from Michael addition is not stabilized by resonance

withdrawal by the ethoxycarbonyl substituent and its

substrate specificity is substantially lower than that of

6c.

The ideal DT-diaphorase substrate appears to be an

N-unsubstituted indoloquinone with an aziridinyl sub-

stituent at either the 5- or the 6-position, with a

carbonyl substituent at the 2-position and a hydroxy-

methyl substituent in the 3-position. A following section

will present evidence that designing an outstanding DT-

diaphorase substrate does not necessarily translate to

a good antitumor agent.

DNA Alk yla tion Scr een in g of Ser ies 1-9. The

extent of DNA reductive alkylation was determined by

catalytic reduction of the aziridinyl quinones in the

presence of 600 bp calf thymus DNA. Oxidative workup

afforded DNA colored blue as a result of the amino-

3550 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

Ta ble 5. Cytostatic (GI₅₀and TGI) and Cytotoxic (LC₅₀) Parameters and the Cancer Specificity for Select Members of Series 1-9

log GI₅₀

NSC H460

,

log TGI,

NSC H460

log LC₅₀

NSC H460

,

average log LC₅₀

60-cell line

,

cancer specificity

1a

2a

-8

-6.99

-7.67

-7.20

-7.24

-6.97

-6.53

-6.07

-5.45

-5.75

-4

-5.66 ( 1.52

-4.74 ( 2.11

-5.15 ( 2.14

-5.59 ( 1.95

-4.14 ( 1.04

-4.94 ( 2.85

-4.67 ( 1.04

-4.60 ( 1.12

-4.49 ( 1.31

-4

melanoma, CNS, renal

CNS, melanoma

melanoma, renal

melanoma, CNS

none

melanoma, renal

none

NSC lung, CNS, melanoma, renal

CNS, melanoma, renal

none

NSC lung, melanoma

melanoma

-8

-8.14

-8

3a

4a

4b

4c

-6.95

-7.08

-6.70

-5.9

-8

-6.17

-6.36

-5.88

-5.12

-8

-7

-6.95

-6.77

-7.49

-7.99

-7.96

-6.75

-6.71

-6.66

-5.94

-5.55

-5.39

-5.91

-5.30

-4.14

-4.30

-8

-6.44

-6.20

5a

6b

-5.35 ( 2.65

-5.50 ( 2.5

-5.79 ( 1.98

-8

6c

-8

6d

7a

7b

-7.43

-8

-6.22

-5.93

-7.06

-6.11

-6.22

-5.61

-6.02

-6.21

-4.93

-5.21

-4.95

-5.62 ( 1.05

-5.54 ( 1.89

-5.69 ( 2.31

-5.75 ( 2.25

-4.52 ( 1.7

-4.61 ( 2.5

-4.61 ( 1.64

-4.67 ( 2.48

-5.07 ( 1.43

-5.20 ( 1.04

-5.32 ( 1.14

-8

8b

8c

-7.45

-7.63

-7.41

-7.27

-6.86

-5.90

-5.80

melanoma

none

9a

9b

Ta ble 6. In Vivo T/C (Treated/Control) Data from the B16

Ta ble 7. In Vivo T/C (Treated/Control) Data from the B16

Melanoma Model^a

a

NA, not active; toxic, g50% lethality before any control growth

deaths.

quinone chromophore. A spectrophotometric determi-

nation of absorbance at 550 nm (ꢀ ) 800 M^-1cm^-1

)

permitted the determination of percent alkylation.^21,36

The anaerobic conditions employed for DNA reductive

alkylation assays are not meant to mimic the environ-

ment of tumor cells. These conditions do permit an

accurate assessment of the structural requirements for

DNA alkylation by the hydroquinone species. To be sure,

agents incapable of akylating DNA under these ideal

conditions will not do so in the typical cellular environ-

ment. Inspection of the DNA alkylation results in Tables

1-4 revealed the following trends:

(1) The presence of a 6-methyl substituent in the

quinone ring of the cyclopent[b]indoles substantially

reduces the percent DNA alkylation compared to ana-

logues bearing only an aziridinyl substituent (compare

1 with 2). Noteworthy exceptions are cyclopent[b]indoles

3 and 4, where a 7-methyl does not greatly influence

the percent DNA alkylation. Similarly, the PBIs could

reductively alkylate DNA with a methyl and even an

n-butyl in the 7-position.³⁰The cyclopent[b]indole DNA

binding model shown in Figure 2 rationalizes the bulk

a

NA, not active; toxic, g50% lethality before any control growth

deaths.

tolerance at the 7-position and the utility of this system

in major-groove recognition.

(2) N-Methylation of the cyclopent[b]indole series 5

results in complete loss of DNA alkylation. This obser-

vation is consistent with the binding model shown in

Figure 2, where the indole NH has a hydrogen-binding

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3551

Sch em e 8

-log LC₅₀value of 6.2, is at saturating cytotoxicity (not

shown in Figure 3). The solid curve in Figure 3 was

computer-generated from

-log LC₅₀) A(V_max/K_m)/[B + (V_max/K_m)]

(1)

where -LC₅₀is the concentration required for 50% cell

kill, V_max/K_Mis the substrate specificity for DT-diapho-

rase, and A and B are constants. At high substrate

specificities the value of constant A approaches 6.6, the

-log LC₅₀value at saturation.

The presence of a saturating relationship suggests

that reductive activation is cytotoxicity-limiting only for

substrate specificities lower than 50 × 10^-4s^-1. At

substrate specificities somewhat higher than 50 × 10^-4

s^-1, reduction is rapid and processes such as DNA

alkylation become cytotoxicity-limiting. The DT-diapho-

rase saturating relationship has also been observed at

constant antitumor agent doses, while the concentration

of the enzyme was increased in the tumor cell.⁴⁰

Increasing the enzyme concentration no doubt results

in increasingly rapid reductive activation, eventually

leading to constant cytotoxicity.

role. N-Acetylation of the cyclopent[b]indole system

(1c-4c) only diminishes DNA alkylation somewhat,

perhaps because the carbonyl has a hydrogen-bonding

role in the DNA major groove.

(3) N-Methylation of the indole series (7c, 8c) also

results in complete loss of DNA alkylation. It is con-

cluded that the NH of the indole series also plays a role

in DNA alkylation.

There have been other DT-diaphorase cytotoxicity

structure-activity studies reported in the literature.^16,17

These studies found that cytotoxicity correlated with the

velocity of DT-diaphorase reduction but without dis-

cernible saturation. Our study was carried out differ-

ently in that substrate specificity (V_max/K_M) rather than

velocity was employed to generate the relationship. The

use of velocity terms in enzyme kinetic studies is risky

because such terms can include nonproductive equilibria

constants, whereas such constants cancel out in the

(4) The presence of an acetate leaving group at the

3R-position of the indole series (6d , 6e, 8b) results in

high DNA alkylation percentages. A recent publication³⁷

has provided evidence that elimination of the acetate

affords an enimine species capable of trapping DNA

nucleophiles (Scheme 9). If an ethoxycarbonyl is present

at the 2-position of the indole, the DNA alkylation

percent increases with either an acetate or hydroxide

leaving group at the 3R-position. The ethoxycarbonyl

group lowers the pK_aof the indole NH, facilitating

formation of the imine species, even when a weak

hydroxide leaving group is present (Scheme 9).

By using the guidelines above, it has been possible

to design excellent DNA reductive alkylating agents

with a high substrate specificity for DT-diaphorase, such

as 6c and 6d .

Cyt ot oxic a n d Cyt ost a t ic Scr een in g of Ser ies

1-9. Provided in Table 5 are the cytostatic/cytotoxic

parameters and cancer specificities for select compounds

from series 1-9. The cytostatic parameters include GI₅₀

and TGI, which are the concentrations of drug required

for 50% growth inhibition and total growth inhibition,

respectively. The cytotoxic parameter is the LC₅₀, which

is the concentration required for 50% cell kill. These in

vitro data were obtained under the In Vitro Cell Line

Screening Project at the National Cancer Institute.^38,39

By comparing the data in Table 5 with those in Tables

1-4, the following relationships were observed:

(1) The data in Table 5 were used to determine the

relationship between H460 DT-diaphorase substrate

specificity and cytotoxicity against the H460 cell line.

The -log LC₅₀vs V_max/K_Mplot shown in Figure 3

includes only DNA alkylating agents exhibiting g10%

alkylation in our assay. This plot reveals that a saturat-

ing relationship exists wherein the -log LC₅₀value of

6.6 was approached with increasingly large values for

substrate specificity (V_max/K_M> 50 × 10^-4s^-1). Com-

pound 6c, with a V_max/K_Mvalue of 430 × 10^-4s^-1and a

V

_max/K_Mexpression.³¹

(2) The TGI and GI₅₀parameters are constant even

with increasing DT-diaphorase specificity, except for

those compounds incapable of reductively alkylating

DNA. The cytostatic parameters (TGI and GT₅₀) for the

NSC H460 cell line shown in Table 5 are all in the range

of -log ) 7-8. The approximately constant cytostatic

activity could be explained by the saturation phenom-

enon where reductive activation is not the limiting

process. The limiting process would be the step following

reductive activation, DNA alkylation.

(3) There is another cytotoxic/cytostatic mechanism

beside DNA alkylation. Compounds with low DNA

alkylation capability, such as 5a and 9b, generally

exhibit low cytostatic and cytotoxic activity (-log )

5-6). The bar graph in Figure 4 compares the cytostatic/

cytotoxic activities of 5a with the DNA reductive alky-

lating agent 3a . Although both compounds possess

nearly the same substrate specificity for DT-diaphorase,

compound 5a is inactive against all histological cancer

types while 3a exhibits significant cytostatic/cytotoxic

activity. In contrast, some poor DNA alkylating agents,

such as quinones 7a and 9b, possess significant cyto-

toxic and cytostatic properties. In addition, a -log LC₅₀

vs V_max/K_Mplot of only the compounds with low DNA

alkylation capability revealed no clear correlation.

Clearly DT-diaphorase reductive activation and DNA

reductive alkylation is not required for cytotoxic and

cytostatic activity. These quinones may be activated by

one-electron reduction and afford oxygen radicals as was

observed for EO9.^41,42

3552 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

F igu r e 2. Model of an N-unsubstituted cyclopent[b]indole hydroquinone in the major groove at a GC base pair with the aziridinyl

group reacted with a phosphate. The 3-substituent is a hydrogen-bond acceptor of the cytosine amino NH; length is 2.21 Å. The

indole NH is a hydrogen-bond donor to the guanine 6-carbonyl or guanine N(7); lengths are 2.57 and 1.86 Å, respectively. The

hydroquinone hydroxyl is a hydrogen-bond donor to the guanine N(7); length is 1.86 Å.

Sch em e 9

(4) High DT-diaphorase specificity and/or high DNA

alkylation percentages do not result in high cancer

specificity. Some members of series 1-9 possess high

F igu r e 3. Plot of -log LC₅₀for series 1-9 compounds in NCI-

DT-diaphorase specificity and/or high DNA alkylation

H460 non-small-cell lung cancer cell lines versus the specificity

capability. Examples include 6d (91.2% of DNA bases

reductively activated, 72 × 10^-4M^-1s^-1DT-diaphorase

specificity), 9b (2% of DNA bases reductively activated,

554 × 10^-4M^-1s^-1DT-diaphorase specificity), and 6c

(37% of DNA bases reductively activated, 430 × 10^-4

M^-1s^-1DT-diaphorase specificity). The best DT-dia-

phorase substrate, 9b, and the best DNA alkylating

agent, 6d , possess no specificity for the histological

cancer types.

for recombinant DT-diaphorase from the same cell line. The

solid line was generated from eq 1.

Cancer specificity is measured from the ( value under

the average log LC₅₀60-cell line heading of Table 5. A

( value greater than 2 indicates a greater than 100-

fold higher/lower cytotoxicity in some histological cancer

types compared to the mean cytotoxicity.

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3553

F igu r e 4. Bar graph of -log TGI, GI₅₀, and LC₅₀for 5a and 3a versus histological cancer type. The -log parameter values are

the average of 6-8 cell lines within each histological cancer.

The DT-diaphorase levels in the National Cancer

Institute 60-cell line in vitro screen have been deter-

mined and correlated with the cytotoxicity of antitumor

agents activated by two-electron reduction.⁴³Sensitive

cancers usually include non-small-cell (NSC) lung and

colon cancers [and to a lesser extent central nervous

system (CNS), melanoma, and renal cancers] by virtue

of their elevated DT-diaphorase levels. An excellent DT-

diaphorase substrate such as 9b will be rapidly reduced

regardless of the DT-diaphorase concentration in the

cell, resulting in broad cytotoxicity. Generally quinones

3-position, hydroxide of 4a or methoxide of 32 in Table

6, possess poor in vivo activity.

The in vivo results for the indole-based systems 6, 7,

and 8 are provided in Table 7 along with the results of

simpler indole analogues 33-37.¹²These data clearly

show that the presence of hydroxymethyl substituents,

particularly in the 3-position, dramatically increases

toxicity. In contrast, the simpler methylated indole

analogues 33 and 34 exhibit substantial antitumor

activity with toxicity only seen at the highest dose. If a

hydroxymethyl is present at the 2-position of the indole,

35, antitumor activity is retained without much toxicity.

The ester derivative 36 is inactive at all doses. It is only

when the 3-hydroxymethyl group or its ester is present,

series 6, 7, and 9, that toxicity overwhelms antitumor

activity. The simplest explanation for the above findings

is that the “enimine” alkylating agent arising from

quinone reduction³⁷and then loss of water from 3-hy-

droxymethyl derivatives, as illustrated in Scheme 9, is

largely responsible for toxicity. DNA alkylation studies

now underway indicate that this alkylating species

reacts with the guanine N(7)- positions randomly. In

contrast, the iminium ion that will arise from N-

methylated indoles 6b and 8c will randomly alkylate

oxygen and nitrogen centers on DNA.⁴⁵

with DT-diaphorase specificities below 50 × 10^-4M^-1

,

such as 2a , 3a , 7a , and 7b, possess high specificities

for high DT-diaphorase cancers.

A high percentage of DNA reductive alkylation by 6d

as well as 4b also results in loss of cancer specificity ((

values of ∼1). These substrates are highly efficiently

DNA reductive alkylating agents and function even at

low DT-diaphorase concentrations, because only a low

concentration is needed for DNA alkylation, resulting

in the loss of cancer specificity. An exception seems to

be 6c, since it possesses high cancer specificity even

though it is an excellent DT-diaphorase substrate and

DNA reductive alkylating agent.

In Vivo Scr een in g Resu lts. Shown in Tables 6 and

7 are B16 melanoma syngraft assays⁴⁴of some members

of series 1-9, as well as related compounds previously

reported from this laboratory.¹²Each agent was evalu-

ated at three doses in C57/bl mice: 2, 3, or 5 mg kg^-1

day^-1, on days 1, 5, and 9 after subcutaneous tumor

implantation of 10⁵cells in the front flank on day 0.

Toxic means that there was early lethality, or g50%

lethality prior to any deaths in the control group. The

treated over control values (T/C) were measured at day

25 of the study. A T/C value <40% is considered active.

The data in Table 6 reveals that cyclopent[b]indoles

3b and 3c were toxic particularly at the 5 mg kg^-1day^-1

dose. In contrast, cyclopent[b]indole 4c was active at

all doses while 4a possessed modest activity, all without

any toxicity. The toxicity of series 3 cyclopent[b]indoles

may be due to their higher DT-diaphorase substrate

specificity and DNA alkylating capability compared to

the series 4 analogues, see Table 2. However, the

presence of a good leaving group in the 3-position

(acetate of 4c) is required for antitumor activity. There-

fore, analogues possessing a poor leaving group at the

Con clu sion s

A large number of aziridinyl quinones represented by

series 1-9 were studied with respect to their DT-

diaphorase substrate activity, DNA reductive alkylation,

cytostatic/cytotoxic activity, and in vivo activity. As a

result, a number of generalizations were made that will

be useful in future drug design efforts.

DT-Dia p h or a se Su bstr a te Design . DT-diaphorase

substrate specificity of indole- and cyclopent[b]indole-

based aziridinyl quinones is highly dependent on the

substituents present. Methylation of the quinone ring

reduces substrate specificity while the position of the

aziridinyl ring is less critical. Hydrogen-bond acceptors

at the 3-position of the cyclopent[b]indole system will

also increase substrate specificity, perhaps due to a

hydrogen-bonding interaction with the N-H of His194.

The presence of an ethoxycarbonyl group at the 2-posi-

tion of the indole system results in large increases in

DT-diaphorase specificity, perhaps due to resonance

stablization of the anion arising from hydride transfer.

3554 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

and the strongest absorbances reported. ¹H NMR spectra were

obtained from a 300 MHz spectrometer. All chemical shifts

are reported relative to that of TMS.

The synthesis of new compounds is outlined below in the

order found in Schemes 1-6.

By varying substituents in a systematic fashion, it was

possible to design substrates with a 100-fold range of

DT-diaphorase specificity.

DT-Dia p h or a se-Cytotoxicity QSAR. A compari-

son was made between the substrate specificity for

human recombinant DT-diaphorase and the cytotoxicity

of DNA alkylating and nonalkylating agents.

The DNA alkylating data were fit to a saturating

relationship wherein the cytotoxicity became constant

with DT-diaphorase substrate specificities (V_max/K_M) >

50 × 10^-4s^-1; see eq 1 and Figure 3. The interpretation

of this relationship is that reductive activation is no

longer rate-limiting at high DT-diaphorase substrate

specificities. The evidence presented in this report

indicates that high DT-diaphorase substrate specificity,

defined as >50 × 10^-4s^-1, is not a desirable feature in

the indole and cyclopent[b]indole aziridinyl quinones.

High substrate specificity results in a loss of selectivity

for histological cancer types with high DT-diaphorase

levels as well as toxicity in vivo. The application of these

conclusions to other aziridinyl quinones awaits study

in our DT-diaphorase and DNA reductive alkylation

assays.

Su bstitu ted 1,4-d ih yd r ocyclop en t[b]in d ol-3(2H)-on es

(10) were prepared as previously described for 10b.¹²

6-Meth oxy-1,4-dih ydr ocyclopent[b]indol-3(2H)-on e (10a).

Yield 20%; mp 190 °C; TLC (dichloromethane/MeOH 95:5) R_f

) 0.75; ¹H NMR (CDCl₃) δ 9.11 (1H, br s, indole proton), 7.57

(1H, d, J ) 9.0 Hz, 8-proton), 6.89 (1H, d, J ) 2.4 Hz, 5-proton),

6.83 (1H, dd, J ) 2.4 and 9.0 Hz, 7-proton), 3.89 (3H, s,

6-methoxy), 3.09 and 3.00 (4H, m, methylenes of cyclopentyl);

IR (KBr pellet) 3450, 3176, 3090, 3026, 2957, 2924, 2843, 1662,

1620, 1535, 1257, 1197, 1136, 1047, 912, and 821 cm^-1; MS

(EI) 201 (M⁺), 186 (M⁺- CH₃), 173, 158, and 130. Anal. Calcd

(C₁₂H₁₁NO₂): C, H, N.

7-Meth oxy-1,4-dih ydr ocyclopen t[b]in dol-3(2H)-on e (10c).

Yield 40%; mp 252 °C; TLC (dichloromethane/MeOH 95:5) R_f

) 0.70; ¹H NMR (CDCl₃) δ 8.42 (1H, br s, indole proton), 7.36

(1H, dd, J ) 1.5 and 9.0 Hz, 6-proton), 7.09 (1H, d, J ) 1.5

Hz, 8-proton), 7.06 (1H, d, J ) 9.0 Hz, 5-proton), 3.87 (3H, s,

7-methoxy), 3.09 and 3.00 (4H, m, methylenes of cyclopentyl);

IR (KBr pellet) 3435, 3159, 2997, 2926, 2854, 1589, 1651, 1626,

1537, 1491, 1450, 1404, 1309, 1276, 1217, 1099, and 1045 cm^-1

;

MS (EI) 201 (M⁺), 186 (M⁺- CH₃), 173, 158, and 130. Anal.

Calcd (C₁₂H₁₁NO₂): C, H, N.

The DNA nonalkylating agents did not show a cor-

relation between the cytotoxicity and DT-diaphorase

substrate specificities (V_max/K_M). These quinones are not

cytotoxic as a result of two-electron reduction and may

be activated by one-electron reduction as was observed

for EO9.^41,42

7-Me t h oxy-6-m e t h yl-1,4-d ih yd r ocyclop e n t [b]in d ol-

3(2H)-on e (10d). Yield 23%; mp 222 °C; TLC (dichloromethane/

MeOH 95:5) R_f) 0.80; ¹H NMR (CDCl₃) δ 8.30 (1H, br s, indole

proton), 7.19 and 6.99 (2H, 2s, 5,8-protons), 3.89 (3H, s,

7-methoxy), 3.09 and 3.00 (4H, m, methylenes of cyclopentyl),

and 2.35 (3H, s, 6-methyl); IR (KBr pellet) 3418, 3176, 2920,

2836, 1666, 1556, 1487, 1446, 1307, 1207, 1095, 1047, and 815

cm^-1; MS (EI) 215 (M⁺), 200 (M⁺- CH₃), 187, 172, and 115.

Anal. Calcd (C₁₃H₁₃NO₂): C, H, N.

Nitr o-1,4-d ih yd r ocyclop en t[b]in d ol-3(2H)-on es (11). To

a solution of 10 mmol of 10 in 200 mL of concentrated H₂SO₄,

cooled at -20 °C, was added a solution of 10.5 mmol of KNO₃

in 20 mL of concentrated H₂SO₄. The solution was stirred at

that temperature for 40 min and was poured over 500 g of ice,

and the solution was extracted five times with 200 mL portions

of CH₂Cl₂. The extracts were washed with NaHCO₃, dried over

Na₂SO₄, and vacuum-dried. The product was crystallized from

CH₂Cl₂and hexane.

6-Meth oxy-5-n itr o-1,4-dih ydr ocyclopen t[b]in dol-3(2H)-

on e (11a ). Yield 32%; mp 228 °C; TLC (dichloromethane/

MeOH 95:5) R_f) 0.68; ¹H NMR (CDCl₃) δ 10.01 (1H, br s,

indole proton), 7.94 and 7.00 (2H, 2d, J ) 8.7 Hz, 7,8-protons),

4.11 (3H, s, 6-methoxy), 3.12 and 3.04 (4H, m, methylenes of

cyclopentyl); IR (KBr pellet) 3460, 3375, 3269 2926, 2850, 1672,

1626, 1550, 1514, 1330, 1246, 1182, 1118, 1006, 962, and 812

cm^-1; MS (EI) 246 (M⁺), 231 (M⁺- CH₃), 218, 199, 186, 170,

158, and 142. Anal. Calcd (C₁₂H₁₀N₂O₄): C, H, N.

7-Meth oxy-8-n itr o-1,4-dih ydr ocyclopen t[b]in dol-3(2H)-

on e (11c). Yield 65%; mp 293 °C; TLC (dichloromethane/

MeOH 95:5) R_f) 0.60; ¹H NMR (CDCl₃) δ 9.49 (1H, br s, indole

proton), 7.69 and 7.23 (2H, 2d, J ) 8.7 Hz, 5,6-protons), 4.02

(3H, s, 7-methoxy), 3.30 and 3.00 (4H, m, methylenes of

cyclopentyl); IR (KBr pellet) 3437, 3192, 2928, 2854, 2660,

1672, 1628, 1568, 1518, 1329, 1271, 1244, 1182, 1091, 1049,

1001, and 815 cm^-1; MS (EI) 246 (M⁺), 229 (M⁺- OH), 216,

198, and 170. Anal. Calcd (C₁₂H₁₀N₂O₄): C, H, N.

DNA Red u ctive Alk yla tin g Agen t Design . The

presence of a 6-methyl in the cyclopent[b]indoles sub-

stantially reduces DNA reductive alkylation for steric

reasons. Likewise, an N-methyl in either the indole or

the cyclopent[b]indole systems hinders DNA reductive

alkylation resulting in nearly 0% alkylation in some

cases. Perhaps the most important prerequisite for

efficient DNA reductive alkylation by the indole system

is a leaving group in the 3R-position, either a hydroxide

or acetate. When the indole system is N-unsubstituted,

leaving group elimination affords the highly reactive

enimine functional group. Ongoing studies have re-

vealed that indole systems able to form the enimine

upon reductive activation are efficient DNA cross-

linkers. The members of series 1-9 possess a wide range

of DNA reductive alkylation capability, from near 0%

to overalkylation at ∼90%. While some DNA alkylation

was required for cytostatic and cytotoxic activity, too

much alkylation results in loss of cancer selectivity as

well as increased in vivo toxicity. Indeed, the most lethal

compounds are the indole systems with an acetate or

hydroxyl leaving group at the 3R-position. We conclude

that poor DNA alkylating agents (according to our

assay) show the lowest toxicity with the highest anti-

tumor activity. For these compounds, redox recycling

with oxygen radical generation⁴²may be responsible for

cytotoxicity.

7-Meth oxy-6-m eth yl-8-n itr o-1,4-d ih yd r ocyclop en t[b]-

in d ol-3(2H)-on e (11d ). Yield 55%; mp 240 °C; TLC (dichlo-

Exp er im en ta l Section

1

romethane/MeOH 90:10) R_f) 0.85; H NMR (CDCl₃) δ 9.45

All solutions and buffers for kinetic, DNA, and electrophore-

sis studies used doubly distilled water. All analytically pure

compounds were dried under high vacuum in a drying pistol

over refluxing toluene. Elemental analyses were run at

Atlantic Microlab, Inc., Norcross, GA. All TLCs were performed

on silica gel plates with a variety of solvents and a fluorescent

indicator for visualization. IR spectra were taken as thin films

(1H, br s, indole proton), 7.55 (H, s, 5-protons), 3.95 (3H, s,

7-methoxy), 3.30 and 3.00 (4H, m, methylenes of cyclopentyl),

and 2.49 (3H, s, 6-methyl); IR (KBr pellet) 3448, 3203, 3059,

2960, 2926, 2856, 2652, 1672, 1489, 1352, 1276, 1230, 1093,

1022, 972, 864, and 823 cm^-1; MS (EI) 260(M⁺), 243 (M⁺

-

OH), 230, 213, 185, 156, 143, and 128. Anal. Calcd

(C₁₃H₁₂N₂O₄): C, H, N.

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3555

3-Hyd r oxy-(5 or 8)-n itr o-1,2,3,4-tetr a h yd r ocyclop en t-

[b]in d oles (12). To a solution of 5 mmol of 11 in 100 mL of

MeOH was added 400 mg of NaBH₄and the solution was

stirred at room temperature for 20 min. The solution was then

mixed with 400 mL of H₂O and extracted four times with 100

mL of CH₂Cl₂. The combined extracts were dried over Na₂SO₄

and vacuum-dried. The product was crystallized from CH₂Cl₂

and hexane.

3-H yd r oxy-7-m et h oxy-6-m et h yl-1,2,3,4-t et r a h yd r ocy-

clop en t[b]in d ole-5,8-d ion e (13d ). Yield 13%; mp 215 °C;

TLC (dichloromethane/MeOH 90:10) R_f) 0.60; ¹H NMR

(CDCl₃) δ 9.10 (1H, br s, indole proton), 5.18 (1H, m, 3-hy-

droxymethylene proton), 4.02 (3H, s, 7-methoxy), 3.00, 2.78

and 2.32 (4H, m, methylenes of cyclopentyl), and 1.97 (3H, s,

6-methyl); IR (KBr pellet) 3435, 3207, 3013, 2926, 2852, 1637,

1498, 1450, 1408, 1373, 1317, 1290, 1217, 1168, 1105, 1039,

985, 949, and 808 cm^-1; MS (EMI) 247 (M⁺), 229 (M⁺- H₂O),

214, 204, 201, 190, 186, 176, 172, 158, and 144. Anal. Calcd

(C₁₃H₁₃NO₄): C, H, N.

3-Hyd r oxy-6-m eth oxy-5-n itr o-1,2,3,4-tetr a h yd r ocyclo-

p en t[b]in d ole (12a ). Yield 94%; mp 192 °C; TLC (dichlo-

romethane/MeOH 95:5) R_f) 0.45; ¹H NMR (CDCl₃) δ 9.80 (1H,

br s, indole proton), 7.68 and 6.86 (2H, 2d, J ) 8.7 Hz, 7,

8-protons), 5.36 (1H, m, 3-hydroxymethylene proton), 4.05 (3H,

s, 6-methoxy), 3.02, 2.75, and 2.38 (4H, m, methylenes of

cyclopentyl), and 1.80 (1H, d, J ) 7.2 Hz, 3-hydroxymethylene

hydroxy); IR (KBr pellet) 3435, 2928, 2852, 1626, 1566, 1508,

1334, 1290, 1184, 1112, 1057, 947, and 804 cm^-1; MS (EI) 248

(M⁺), 231 (M⁺- OH), 215, 200, 184, 172, 154, and 144. Anal.

Calcd (C₁₂H₁₂N₂O₄): C, H, N.

Azir id in yl-3-h yd r oxy-1,2,3,4-t et r a h yd r ocyclop en t [b]-

in d ole-5,8-d ion es (Ser ies a ). To a solution of 0.3 mmol of

13 in 30 mL of MeOH was added 0.5 mL of ethylenimine, and

the solution was stirred at room temperature (RT) for 8 h. The

solvent was vacuum-removed and the residue was subjected

to flash chromatography with 20% acetone in CH₂Cl₂as eluent.

The product was crystallized from CH₂Cl₂and hexane.

7-Azir id in yl-3-h yd r oxy-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (1a ). Yield 93%; mp 145 °C; TLC (dichlo-

3-Hyd r oxy-7-m eth oxy-8-n itr o-1,2,3,4-tetr a h yd r ocyclo-

p en t[b]in d ole (12c). Yield 85%; mp 230 °C; TLC (dichlo-

romethane/MeOH 95:5) R_f) 0.25; ¹H NMR (CDCl₃) δ 8.19 (1H,

br s, indole proton), 7.45 and 6.93 (2H, 2d, J ) 8.7 Hz, 5,

6-protons), 5.32 (1H, m, 3-hydroxymethylene proton), 3.97 (3H,

s, 7-methoxy), 3.00, 2.82, and 2.33 (4H, m, methylenes of

cyclopentyl); IR (KBr pellet) 3551, 3464, 3416, 3238, 3063,

2972, 2870, 1635, 1572, 1506, 1313, 1276, 1240, 1182, 1082,

954, and 787 cm^-1; MS (EI) 248 (M⁺), 231 (M⁺- OH), 213,

200, 172, 154, and 143. Anal. Calcd (C₁₂H₁₂N₂O₄): C, H, N.

1

romethane/MeOH 90:10) R_f) 0.55; H NMR (CDCl₃) δ 9.26

(1H, br s, indole proton), 5.81 (1H, 6-proton), 5.20 (1H, m,

3-hydroxymethylene proton), 2.96, 2.74, and 2.33 (4H, m,

methylenes of cyclopentyl), and 2.22 (4H, s, 7-aziridinyl); IR

(KBr pellet) 3418, 3283, 2946, 2870, 1626, 1578, 1476, 1364,

1254, 1156, 1078, 946, 846, and 808 cm^-1; MS (EI) 244 (M⁺),

226 (M⁺- H₂O), 215, 200, 192, 176, and 137. Anal. Calcd

(C₁₃H₁₂N₂O₃): C, H, N.

7-Azir id in yl-3-h yd r oxy-6-m eth yl-1,2,3,4-tetr a h yd r ocy-

clop en t[b]in d ole-5,8-d ion e (2a ). Yield 95%; mp 220 °C; TLC

(dichloromethane/MeOH 90:10) R_f) 0.55; ¹H NMR (CDCl₃) δ

9.23 (1H, br s, indole proton), 5.18 (1H, m, 3-hydroxymethylene

proton), 2.96, 2.72, and 2.36 (4H, m, methylenes of cyclopentyl),

2.32 (4H, s, 7-aziridinyl), and 2.53 (3H, s, 6-methyl); IR (KBr

pellet) 3420, 3192, 2928, 2856, 1664, 1629, 1560, 1498, 1458,

1377, 1292, 1230, 1074, 1039, 981, and 856 cm^-1; MS (EI) 258

(M⁺), 240 (M⁺- H₂O), 229, 214, 204, 190, and 151. Anal. Calcd

(C₁₄H₁₄N₂O₃): C, 65.10; H, 5.46; N, 10.85. Found: C, 65.16;

H, 5.44; N, 10.89.

3-Hyd r oxy-7-m eth oxy-6-m eth yl-8-n itr o-1,2,3,4-tetr a h y-

d r ocyclop en t[b]in d ole (12d ). Yield 92%; mp 185 °C; TLC

(dichloromethane/MeOH 90:10) R_f) 0.65; ¹H NMR (CDCl₃) δ

8.16 (1H, br s, indole proton), 7.33 (1H, s, 4-protons), 5.30 (1H,

m, 3-hydroxymethylene proton), 3.91 (3H, s, 7-methoxy), 3.00,

2.82, and 2.33 (4H, m, methylenes of cyclopentyl), and 2.43

(3H, s, 6-methyl); IR (KBr pellet) 3408, 3175, 3047, 2943, 2856,

1637, 1570, 1516, 1427, 1329, 1288, 1219, 1149, 1076, 1041,

976, and 866 cm^-1; MS (EI) 262 (M⁺), 244 (M⁺- H₂O), 227,

215, 197, 186, 168, 154, 143, 127, and 115. Anal. Calcd

(C₁₃H₁₄N₂O₄): C, H, N.

6-Azir id in yl-3-h yd r oxy-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (3a ). Yield 89%; mp 180 °C; TLC (dichlo-

romethane/MeOH 90:10) R_f) 0.50; ¹H NMR (DMSO-d₆) δ

12.12 (1H, br s, indole proton), 5.73 (1H, s, 7-proton), 5.20 (1H,

m, 3-hydroxymethylene proton), 2.70, 2.46, and 2.11(4H, m,

methylenes of cyclopentyl), and 2.12 (4H, s, 6-aziridinyl); IR

(KBr pellet) 3447, 3198, 3003, 2930, 2864, 1666, 1614, 1577,

3-H yd r oxy-1,2,3,4-t et r a h yd r ocyclop en t [b]in d ole-5,8-

d ion es (13). A solution of 0.5 mmol of the product above in

10 mL of MeOH and 40 mL of H₂O with 150 mg of 5% Pd on

carbon was reduced under 50 psi H₂for 30 min, and the

catalyst was filtered off through Celite. The filtrate was mixed

with a solution of 400 mg of KH₂PO₄and 800 mg of Fremy

salt in 150 mL of H₂O, stirred at room temperature for 3 h.

The solution was extracted five times with 50 mL portions of

CH₂Cl₂to remove the quinone product. The extracts were dried

over Na₂SO₄and vacuum-dried. The solid was purified by flash

chromatography with 10% acetone in CH₂Cl₂as the eluent.

The product was recrystallized from CH₂Cl₂and hexane.

1475, 1406, 1294, 1238, 1112, 1041, 949, 852, and 806 cm^-1

;

MS (EI) 244 (M⁺), 226 (M⁺- H₂O), 217, 199, 189, and 171.

Anal. Calcd (C₁₃H₁₂N₂O₃): C, H, N.

3-Acetoxy-(6 or 7)-a zir id in yl-1,2,3,4-tetr a h yd r ocyclo-

p en t[b]in d ole-5,8-d ion es (Ser ies b) a n d (6 or 7)-Azir id i-

n yl-3-a cetoxy-4-a cetyl-1,2,3,4-tetr a h yd r ocyclop en t[b]in -

d ole-5,8-d ion es (Ser ies c). To a solution of 0.4 mmol of 1-3a

in 10 mL of 10% acetone in CH₂Cl₂with 50 mg of DMAP

present was added 3 × 60 µL of acetic anhydride every 3 min.

The solution was then directly applied to flash chromatography

with 10% acetone in CH₂Cl₂as the eluent. The solution was

washed with saturated NaHCO₃, dried over Na₂SO₄, and

vacuum-dried. The product was precipitated from CH₂Cl₂and

hexane.

3-Hyd r oxy-6-m eth oxy-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (13a ). Yield 15%; mp 167 °C; TLC (dichlo-

romethane/MeOH 90:10) R_f) 0.70; ¹H NMR (DMSO-d₆) δ

12.34 (1H, br s, indole proton), 5.72 (1H, s, 5-proton), 5.27 (1H,

m, 3-hydroxymethylene proton), 3.74 (3H, s, 6-methoxy), 2.77,

2.46, and 2.15 (4H, m, methylenes of cyclopentyl); IR (KBr

pellet) 3493, 3136, 2989, 2939, 2858, 1664, 1628, 1591, 1543,

1402, 1323, 1238, 1120, 1055, 935, and 848 cm^-1; MS (EI) 233

(M⁺), 215 (M⁺- H₂O), 202, 190, 174, and 158. Anal. Calcd

(C₁₂H₁₁NO₄): C, H, N.

3-Acetoxy-7-a zir id in yl-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (1b). Yield 42%; mp 165 °C; TLC (dichlo-

1

romethane/MeOH 90:10) R_f) 0.65; H NMR (CDCl₃) δ 9.20

(1H, br s, indole proton), 5.83 (1H, 6-proton), 5.66 (1H, m,

3-hydroxymethylene proton), 2.99, 2.76, and 2.21 (4H, m,

methylenes of cyclopentyl), 2.21 (4H, s, 7-aziridinyl), and 2.06

(3H, s, 3-acetyl methyl); IR (KBr pellet) 3448, 3225, 3065, 2926,

2872, 1720, 1641, 1585, 1510, 1475, 1367, 1288, 1251, 1163,

1018, 985, and 812 cm^-1; MS (EI) 286 (M⁺), 258 (M⁺- CO),

243, 226, 211, 199, 170, 142, and 118. Anal. Calcd

(C₁₅H₁₄N₂O₄): C, H, N.

3-Hyd r oxy-7-m eth oxy-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (13c). Yield 18%; mp 200 °C; TLC (dichlo-

romethane/MeOH 95:5) R_f) 0.20; ¹H NMR (CDCl₃) δ 9.12 (1H,

br s, indole proton), 5.69 (1H, 6-protons), 5.20 (1H, m, 3-hy-

droxymethylene proton), 3.83 (3H, s, 7-methoxy), 3.01, 2.85,

and 2.29 (4H, m, methylenes of cyclopentyl); IR (KBr pellet)

3431, 3205, 3109, 3067, 2937, 2868, 1678, 1645, 1591, 1483,

1458, 1408, 1342, 1292, 1248, 1211, 1180, 1082, 1030, 949, 868,

and 842 cm^-1; MS (EI) 233 (M⁺), 215 (M⁺- H₂O), 200, 190,

3-Acetoxy-7-a zir id in yl-6-m eth yl-1,2,3,4-tetr a h yd r ocy-

clop en t[b]in d ole-5,8-d ion e (2b). Yield 45%; mp 165 °C; TLC

187, 176, 172, 162, 158, 144, and 130. Anal. Calcd (C₁₂H₁₁

NO₄): C, H, N.

-

1

(dichloromethante:MeOH 90:10) R_f) 0.75; H NMR (CDCl₃)

3556 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

δ 9.13 (1H, br s, indole proton), 5.66 (1H, m, 3-hydroxymeth-

ylene proton), 2.99, 2.59, and 2.30 (4H, m, methylenes of

cyclopentyl), 2.30 (4H, s, 7-aziridinyl), and 2.06 (6H, 2s,

3-acetyl methyl and 6-methyl); IR (KBr pellet) 3454, 3311,

3043, 2926, 2843, 1732, 1663, 1572, 1473, 1382, 1239, 1009,

964, and 800 cm^-1; MS (EI) 300 (M⁺), 272 (M⁺- CO), 257,

240, 225, 213, 184, 156, and 132. Anal. Calcd (C₁₆H₁₆N₂O₄):

C, H, N.

2d, J ) 8.4 Hz, 5, 6-protons), 5.36 (1H, m, 3-hydroxymethylene

proton), 3.82 (3H, s, 4-methyl), 3.09, 2.88, and 2.33 (4H, m,

methylenes of cyclopentyl), 2.39 (3H, s, 7-methyl), and 1.65

(1H, d, J ) 9.0 Hz, 3-hydroxymethylene hydroxy); IR (KBr

pellet) 3435, 3302, 3202, 2962, 2930, 1637, 1560, 1516, 1460,

1321, 1269, 117, 1045, 952, 908, and 808 cm^-1; MS (EI) 246

(M⁺), 229 (M⁺- OH), 212, 199, 182, 167, and 156. Anal. Calcd

(C₁₃H₁₄N₂O₃): C, H, N.

3-Acetoxy-6-a zir id in yl-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (3b). Yield 32%; mp 155 °C; TLC (dichlo-

romethane/acetone 80:20) R_f) 0.45; H NMR (CDCl₃) δ 9.31

4,7-Dim eth yl-3-h yd r oxy-1,2,3,4-tetr a h yd r ocyclop en t[b]-

in d ole-5,8-d ion e (16). The catalytic reduction and Fremy

oxidation of 15 to afford 16 was carried out as described for

the preparation of 13: Yield 14%; mp 160 °C; TLC (dichlo-

romethane/MeOH 96:4) R_f) 0.35; ¹H NMR (CDCl₃) δ 6.35 (1H,

q, J ) 1.5 Hz, 6-proton), 5.22 (1H, m, 3-hydroxymethylene

proton), 3.95 (3H, s, 4-methyl), 3.02, 2.73, and 2.34 (4H, m,

methylenes of cyclopentyl), 2.04 (3H, d, J ) 1.5 Hz, 7-methyl),

and 1.72 (1H, d, J ) 8.4 Hz, 3-hydroxymethylene hydroxy);

IR (KBr pellet) 3469, 2943, 2872, 1639, 1604, 1475, 1384, 1221,

1

(1H, br s, indole proton), 5.92 (1H, 7-proton), 5.64 (1H, m,

3-hydroxymethylene proton), 3.00, 2.56, and 2.28 (4H, m,

methylenes of cyclopentyl), 2.24 (4H, s, 6-aziridinyl), and 2.05

(3H, s, 3-acetyl methyl); IR (KBr pellet) 3437, 3219, 3103, 1995,

1945, 2864, 1724, 1643, 1583, 1481, 1369, 1296, 1236, 1112,

1018, 991, 956, 891, and 850 cm^-1; MS (EI) 286 (M⁺), 258 (M⁺

- CO), 244, 226, 215, 199, and 170. Anal. Calcd (C₁₅H₁₄N₂O₄):

C, H, N.

1159, 1039, 947, and 877 cm^-1; MS (EI) 231 (M⁺), 214 (M⁺

-

7-Azir id in yl-3-a cet oxy-4-a cet yl-1,2,3,4-t et r a h yd r ocy-

OH), 203, 188, 174, and 160. Anal. Calcd (C₁₃H₁₃NO₃): C, H,

N.

clop en t[b]in d ole-5,8-d ion e (1c). Yield 45%; mp 140 °C; TLC

1

(dichloromethane/acetone 80:20) R_f) 0.65; H NMR (CDCl₃)

6-Azir id in yl-4,7-d im et h yl-3-h yd r oxy-1,2,3,4-t et r a h y-

d r ocyclop en t[b]in d ole-5,8-d ion e (5a ) was prepared by the

procedure described for series a: Yield 32%; mp 187 °C; TLC

(dichloromethane/MeOH 96:4) R_f) 0.60; ¹H NMR (DMSO-d₆)

δ 5.41 (1H, d, J ) 6.6 Hz, 3-hydroxyl), 5.04 (1H, m, 3-hy-

droxymethylene proton), 3.82 (3H, s, 4-methyl), 2.65, 2.50, and

2.10 (4H, m, methylenes of cyclopentyl), and 2.17 (4H, s,

6-aziridine); IR (KBr pellet) 3431, 2995, 2949, 2922, 2858,

1653, 1620, 1479, 1377, 1340, 1153, 1051, and 983 cm^-1; MS

(EI) 272 (M⁺), 255 (M⁺- OH), 243, 227, and 217. Anal. Calcd

(C₁₅H₁₆N₂O₃): C, H, N.

δ 6.20 (1H, m, 3-hydroxymethylene proton), 5.90 (1H, s,

6-proton), 2.99, 2.41, and 2.20 (4H, m, methylenes of cyclo-

pentyl), 2.74 (3H, s, 4-acetyl methyl), 2.22 (4H, s, 7-aziridinyl),

and 2.06 (3H, s, 3-acetyl methyl); IR (KBr pellet) 3431, 3005,

2928, 2870, 1734, 1672, 1639, 1593, 1491, 1371, 1246, 1186,

1157, 1095, 1020, and 856 cm^-1; MS (EI) 328 (M⁺), 300 (M⁺

-

CO), 286, 244, 226, 199, 171, and 132. Anal. Calcd (C₁₇

₁₆N₂O₅): C, H, N.

7-Azir idin yl-3-acetoxy-4-acetyl-6-m eth yl-1,2,3,4-tetr ah y-

H

d r ocyclop en t[b]in d ole-5,8-d ion e (2c). Yield 48%; mp 156

1

°C; TLC (dichloromethane/acetone 80:20) R_f) 0.70; H NMR

3-Acet yl-6-a zir id in yl-4,7-d im et h yl-1,2,3,4-t et r a h yd r o-

cyclop en t[b]in d ole-5,8-d ion e (5b). Yield 50%; mp 157 °C;

TLC (dichloromethane/acetone 96:4) R_f) 0.62; ¹H NMR

(CDCl₃) δ 6.10 (1H, m, 3-hydroxymethylene proton), 3.89 (3H,

s, 4-methyl), 2.96, 2.79, and 2.40 (4H, m, methylenes of

cyclopentyl), 2.30 (4H, s, 6-aziridinyl), 2.09 and 2.08 (6H, 2s,

7-methyl and 3-acetyl methyl); IR (KBr pellet) 3447, 3020,

2922, 2854, 1734, 1653, 1626, 1583, 1375, 1336, 1232, 1151,

1030, and 856 cm^-1; MS (EI) 314 (M⁺), 299 (M⁺- CH₃), 272,

255, 239, 227, and 215. Anal. Calcd (C₁₇H₁₈N₂O₄): C, H, N.

(CDCl₃) δ 6.26 (1H, m, 3-hydroxymethylene proton), 2.97, 2.43,

and 2.22 (4H, m, methylenes of cyclopentyl), 2.72 (3H, s,

4-acetyl), 2.25 (4H, s, 7-aziridinyl), 2.08 and 2.05 (6H, 2s,

6-methyl and 3-acetyl methyl); IR (KBr pellet) 3433, 3011,

2945, 2854, 1734, 1666, 1637, 1593, 1496, 1381, 1350, 1240,

1217, 1157, 1022, 983, and 933 cm^-1; MS (EI) 342 (M⁺), 300

(M⁺- acetyl), 283, 258, 240, and 225. Anal. Calcd (C₁₈

₁₈N₂O₅): C, H, N.

6-Azir id in yl-3-a cet oxy-4-a cet yl-1,2,3,4-t et r a h yd r ocy-

-

H

clop en t[b]in d ole-5,8-d ion e (3c). Yield 56%; mp 167 °C; TLC

2-(Eth oxyca r bon yl)in d oles (17). The synthesis of 17a and

17c was reported in the literature,⁴⁶and 17b was synthesized

following the procedure described for 16a .

1

(dichloromethane/acetone 80:20) R_f) 0.65; H NMR (CDCl₃)

δ 5.97 (1H, m, 3-hydroxymethylene proton), 5.91 (1H, s,

7-proton), 2.97, 2.43, and 2.21 (4H, m, methylenes of cyclo-

pentyl), 2.77 (3H, s, 4-acetyl methyl), 2.25 (4H, s, 6-aziridinyl),

and 2.04 (3H, s, 3-acetyl methyl); IR (KBr pellet) 3448, 3007,

2941, 2862, 1736, 1668, 1637, 1585, 1473, 1361, 1251, 1230,

2-(Eth oxyca r bon yl)-5-m eth oxy-6-m eth ylin d ole (17b).

Yield 63%; mp 165 °C; TLC (CHCl₃) R_f) 0.55; ¹H NMR

(CDCl₃) δ 8.65 (1H, br s, indole proton), 7.26, 7.11, and 6.99

(3H, 3s, 3, 4, 7-proton), 4.38 (2H, q, J ) 7.2 Hz, methylene of

2-ethoxyl), 3.87 (3H, s, 5-methoxyl), 2.33 (3H, s, 6-methyl), and

1.39 (3H, t, J ) 7.2 Hz, methyl of 2-ethoxyl); IR (KBr pellet)

3445, 3325, 2982, 2941, 2839, 1682, 1520, 1246, 1213, 1168,

1136, 1026, 864, and 833 cm^-1; MS (EI) 233 (M⁺), 218, 204,

187, 172, and 144. Anal. Calcd (C₁₃H₁₅NO₃): C, H, N.

1112, 1033, 981, and 893 cm^-1; MS (EI) 328 (M⁺), 300 (M⁺

-

CO), 286, 244, 226, 199, 171, and 132. Anal. Calcd

(C₁₇H₁₆N₂O₅): C, 62.19; H, 4.91; N, 8.53. Found: C, 62.16; H,

4.88; N, 8.50.

4,7-Dim e t h yl-8-n it r o-1,4-d ih yd r ocyclop e n t [b]in d ol-

3(2H)-on e (14). To 0.2 mmol of 11b and 400 mg of KOH in

20 mL of acetone was added 1.0 mL of MeI, followed by stirring

at RT for 1 h. The reaction mixture was then neutralized with

concentrated HCl and extracted four times with 30 mL

portions of CH₂Cl₂. The combined extracts were dried (Na₂-

SO₄), concentrated to a residue, and chromographed with CH₂-

Cl₂as eluent. The product was crystallized from methylene

chloride by use of hexane. Yield 92%; mp 207 °C; TLC

(dichloromethane) R_f) 0.45; ¹H NMR (CDCl₃) δ 7.49 and 7.28

(2H, 2d, J ) 8.7 Hz, 5, 6-protons), 3.96 (3H, s, 4-methyl), 3.14

and 2.97 (4H, m, methylenes of cyclopentyl), and 2.62 (3H, s,

5-methyl); IR (KBr pellet) 3437, 3063, 2939, 2849, 1683, 1521,

14581373, 1338, 1265, 1201, 1047, and 833 cm^-1; MS (EI) 244

(M⁺), 227 (M⁺- OH), 197, 184, 169, and 154. Anal. Calcd

(C₁₃H₁₂N₂O₃): C, H, N.

2-(Eth oxyca r bon yl)-3-for m ylin d oles (18). A solution of

6 mL of distilled DMF with 2 mL of distilled POCl₃in 40 mL

of dried CH₂Cl₂was stirred at 0 °C for 30 min. To this solution

was slowly added 50 mmol of 18 in 30 mL of dried CH₂Cl₂,

and the reaction mixture was stirred at 0 °C for 30 min. To

the completed reaction was added NaHCO₃saturated solution

until neutral. The product was removed from the neutralized

reaction by extracting five times with 100 mL portions of

methylene chloride. The product was crystallized from dichlo-

romethane and hexane.

2-(Eth oxyca r bon yl)-3-for m yl-5-m eth oxyin d ole (18a ).

Yield 87%; mp 231 °C; TLC (CHCl₃) R_f) 0.30; ¹H NMR

(CDCl₃) δ 10.74 (1H, s, 3-formyl), 9.20 (1H, br s, indole proton),

7.90 (1H, d, J ) 2.7 Hz, 4-proton), 7.35 (1H, d, J ) 8.7 Hz,

7-proton), 7.07 (1H, dd, J ) 2.7 and 8.7 Hz, 6-proton), 4.52

(2H, q, J ) 7.2 Hz, methylene of 2-ethoxy), 3.91 (3H, s,

5-methoxy), and 1.45 (3H, t, J ) 7.2 Hz, methyl of 2-ethoxy);

IR (KBr pellet) 3448, 3157, 2985, 2833, 1709, 1637, 1581, 1531,

1467, 1433, 1398, 1371, 1244, 1201, 1122, 1028, 983, and 804

4,7-Dim et h yl-3-h yd r oxy-8-n it r o-1,2,3,4-t et r a h yd r ocy-

clop en t[b]in d ole (15). The borohydride reduction of 14 to

afford 15 was carried out as described above for the prepara-

tion of 12: Yield 90%; mp 171 °C; TLC (dichloromethane/

MeOH 95:5) R_f) 0.80; ¹H NMR (CDCl₃) δ 7.37 and 7.05 (2H,

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3557

cm^-1; MS (EI) 247 (M⁺), 218 (M⁺- HCO), 200, 186, 173, 158,

144, 130, and 119. Anal. Calcd (C₁₃H₁₃NO₄): C, H, N.

2-(E t h oxyca r b on yl)-3-for m yl-5-m et h oxy-6-m et h ylin -

d ole (18b). Yield 90%; mp 223 °C; TLC (CHCl₃) R_f) 0.45; ¹H

NMR (CDCl₃) δ 10.72 (1H, s, 3-formyl), 9.20 (1H, br s, indole

proton), 7.81 and 7.21 (2H, 2s, 4,7-protons), 4.51 (2H, q, J )

7.2 Hz, methylene of 2-ethoxy), 3.93 (3H, s, 5-methoxy), 2.34

(3H, s, 6-methyl), and 1.45 (3H, t, J ) 7.2 Hz, methyl of

2-ethoxy); IR (KBr pellet) 3435, 3105, 3053, 2993, 2939, 2835,

1718, 1639, 1572, 1531, 1469, 1433, 1390, 1371, 1263, 1205,

1147, 1093, 1060, 962, 866, and 812 cm^-1; MS (EI) 261 (M⁺)

232 (M⁺- HCO), 214, 200, 187, 172, 158, 144, and 133. Anal.

Calcd (C₁₄H₁₅NO₄): C, H, N.

2d, J ) 9 Hz, 6,7-protons), 5.30 and 4.35 (2H, 2t, J ) 5.1 Hz,

hydroxyls of 2,3-hydroxymethyls), 4.63 and 4.38 (4H, 2d, J )

5.1 Hz, methylenes of 2,3-hydroxymethyls), and 3.92 (3H, s,

6-methoxyl); IR (KBr pellet) 3348, 2960, 2752, 1633, 1579,

1514, 1464, 1435, 1357, 1321, 1261, 1199, 1103, 1039, 979, and

829 cm^-1; MS (EI) 252 (M⁺) 234 (M⁺- H₂O), 219, 205, 191,

177, 163, 159, and 121. Anal. Calcd (C₁₁H₁₂N₂O₅): C, H, N.

2,3-Di(h yd r oxym eth yl)-5-m eth oxy-6-m eth yl-4-n itr oin -

d ole (20b). Yield 44%; mp 158 °C; TLC (ethyl acetate/

1

methanol 95:5) R_f) 0.50; H NMR (CDCl₃) δ 8.52 (1H, br s,

indole proton), 7.30 (H, 1s, 7-protons), 4.90 and 4.65 (4H, 2d,

J ) 5.7 Hz, methylenes of 2,3-hydroxymethyls), 4.02 (3H, s,

5-methoxyl), 2.48 (3H, s, 6-methyl), 2.23 and 2.04 (2H, 2t, J )

5.7 Hz, hydroxyls of 2,3-hydroxymethyls); IR (KBr pellet) 3447,

3385, 2924, 2852, 1637, 1560, 1475, 1429, 1356, 1327, 1278,

1228, 1176, 1101, 1001, 916, 866, and 815 cm^-1; MS (EI) 266

(M⁺) 248 (M⁺- H₂O), 231, 219, 215, 201, 187, 162, 158, 143,

and 130. Anal. Calcd (C₁₂H₁₄N₂O₅): C, H, N.

3-F or m yl-2-(m eth oxyca r bon yl)-6-m eth oxyin d ole (18c).

Yield 90%; mp 185 °C; TLC (CHCl₃/acetone 95:5) R_f) 0.65;

¹H NMR (CDCl₃) δ 10.69 (1H, s, 3-formyl), 9.10 (1H, br s, indole

proton), 8.32 (1H, d, J ) 8.7 Hz, 4-proton), 7.01 (1H, dd, J )

2.7 and 8.7 Hz, 5-proton), 6.85 (1H, d, J ) 2.7 Hz, 7-proton),

4.04 and 3.87 (6H, 2s, 6-methoxy and ester methoxy); IR (KBr

pellet) 3311, 3173, 3130, 2953, 2926, 2856, 1712, 1641, 1579,

1444, 1250, 1213, 1161, 1097, 1026, and 827 cm^-1; MS (EI)

2,3-Di(h yd r oxym eth yl)-6-m eth oxy-7-n itr oin d ole (20c).

Yield 35%; mp 172 °C; TLC (dichloromethane/methanol 95:5)

R_f) 0.18; ¹H NMR (DMSO-d₆) δ 11.05 (1H, br s, indole proton),

7.91 and 7.01 (2H, 2d, J ) 9 Hz, 4,5-protons), 5.07 and 4.76

(2H, 2t, J ) 6.0 Hz, hydroxyls of 2,3-hydroxymethyls), 4.63

and 4.62 (4H, 2d, J ) 6.0 Hz, methylenes of 2,3-hydroxy-

methyls), and 3.94 (3H, s, 6-methoxyl); IR (KBr pellet) 3443,

3221, 3013, 2924, 2881, 2852, 1629, 1570, 1510, 1421, 1345,

1300, 1253, 1199, 1091, 1012, 958, 846, and 894 cm^-1; MS (EI)

252 (M⁺), 235 (M⁺- OH), 221, 205, and 158. Anal. Calcd

(C₁₁H₁₂N₂O₅): C, H, N.

2,3-Di(h yd r oxym eth yl)in d ole-4,7-d ion es (21). A solution

of 0.2 mmol of 20 in 15 mL of methanol and 60 mL of water in

the presence of 80 mg of 5% Pd on carbon was hydrogenated

at 50 psi H₂for 25 min. The catalyst was filtered off through

Celite, and the filtrate was combined with a solution consisting

of 50 mg of KH₂PO₄and 100 mg of Fremy salt in 50 mL of

H₂O. The solution was stirred at room temperature for 6 h

and then extracted six times with 50 mL portions of ethyl

acetate. The combined extracts were dried over Na₂SO₄and

vacuum-dried to a residue that was purified by flash chroma-

tography with ethyl acetate as the eluent. The product was

recrystallized from ethyl acetate and hexane.

233 (M⁺) 218, 201, 186, 173, 158, and 144. Anal. Calcd (C₁₂H₁₁

NO₄): C, H, N.

-

2-(Eth oxyca r bon yl)-3-for m yl-(4 or 7)-n itr oin d oles (19).

A solution of 2 mmol of 18 was stirred in 25 mL of 70% HNO₃

for 25 min, followed by addition of 100 g of ice. The resulting

mixture was extracted three times with 100 mL portions of

methylene chloride. The combined extracts were washed with

saturated NaHCO₃and then dried over Na₂SO₄. The product

was crystallized from CH₂Cl₂and hexane.

2-(E t h oxyca r b on yl)-3-for m yl-5-m e t h oxy-4-n it r oin -

d ole (19a ). Yield 73%; mp 220 °C; TLC (CHCl₃) R_f) 0.15; ¹H

NMR (CDCl₃) δ 10.6 (1H, s, 3-formyl), 9.42 (1H, br s, indole

proton), 7.58 and 7.24 (2H, 2d, J ) 8.7 Hz, 6,7-protons), 4.54

(2H, q, J ) 7.2 Hz, methylene of 2-ethoxy), 3.97 (3H, s,

5-methoxy), and 1.47 (3H, t, J ) 7.2 Hz, methyl of 2-ethoxy).

IR (KBr pellet) 3437, 3269, 3080, 2999, 2928, 1689, 1637, 1547,

1523, 1477, 1444, 1390, 1269, 1219, 1184, 1126, 1085, 1012,

869, and 833 cm^-1; MS (EI) 292 (M⁺) 274 (M⁺- H₂O), 263,

246, 229, 216, 198, 188, 170, 160, 144, and 130. Anal. Calcd

(C₁₃H₁₂N₂O₆): C, H, N.

2,3-Di(h ydr oxym eth yl)-5-m eth oxyin dole-4,7-dion e (21a).

Yield 15%; mp 208 °C; TLC (ethyl acetate) R_f) 0.20; ¹H NMR

(DMSO-d₆) δ 12.8 (1H, br s, indole proton), 5.76 (1H, s,

6-proton), 4.99 and 4.63 (2H, 2t, J ) 5.4 Hz, hydroxyls of 2,3-

hydroxymethyls), 4.58 and 4.46 (4H, 2d, J ) 5.4 Hz, methyl-

enes of 2,3-hydroxymethyls), and 3.95 (3H, s, 5-methoxy); IR

(KBr pellet) 3425, 2926, 2856, 1647, 1599, 1533, 1465, 1398,

1340, 1249, 1167, 1122, 1037, and 880 cm^-1; MS (EI) 237 (M⁺),

219 (M⁺- H₂O), 204, 190, 176, 162, 148, 134, and 106. Anal.

Calcd (C₁₁H₁₁NO₅): C, H, N.

2-(Eth oxyca r bon yl)-3-for m yl-5-m eth oxy-6-m eth yl-4-n i-

tr oin d ole (19b). Yield 50%; mp 198 °C; TLC (CHCl₃) R_f)

1

0.40; H NMR (CDCl₃) δ 10.61 (1H, s, 3-formyl), 9.40 (1H, br

s, indole proton), 7.41 (1H, s, 7-proton), 4.53 (2H, q, J ) 7.2

Hz, methylene of 2-ethoxy), 3.91 (3H, s, 5-methoxy), 2.48 (3H,

s, 6-methyl), and 1.47 (3H, t, J ) 7.2 Hz, methyl of 2-ethoxy);

IR (KBr pellet) 3367, 3105, 2985, 2941, 1726, 1658, 1539, 1464,

1429, 1394, 1253, 1197, 1178, 1016, 977, 864, and 808 cm^-1

;

MS (EI) 306 (M⁺) 288 (M⁺- H₂O), 277, 261, 243, 230, 202,

196, 174, 156, 144, and 130. Anal. Calcd (C₁₄H₁₄N₂O₆): C, H,

N.

2,3-Di(h yd r oxym eth yl)-5-m eth oxy-6-m eth ylin d ole-4,7-

d ion e (21b). Yield 26%; mp 178 °C; TLC (ethyl acetate/

3-F or m yl-2-(m e t h ylca r b oxyl)-6-m e t h oxy-7-n it r oin -

d ole (19c). Yield 92%; mp 197 °C; TLC (acetone/dichlo-

1

methanol 95:5) R_f) 0.48; H NMR (CDCl₃) δ 9.43 (1H, br s,

1

indole proton), 4.75 and 4.67 (4H, 2d, J ) 6.0 Hz, methylenes

of 2,3-hydroxymethyls), 4.02 (3H, s, 5-methoxy), 3.82 and 2.20

(2H, 2t, J ) 6.0 Hz, hydroxyls of 2,3-hydroxymethyls), and 1.98

(3H, s, 6-methyl); IR (KBr pellet) 3437, 3227, 3122, 2961, 2852,

1693, 1635, 1604, 1492, 1444, 1375, 1303, 1240, 1188, 1103,

1051, 1024, and 929 cm^-1. MS (EI) 251 (M⁺), 233 (M⁺- H₂O),

romethane 20:80) R_f) 0.80; H NMR (CDCl₃) δ 10.74 (1H, s,

3-formyl), 10.61 (1H, br s, indole proton), 8.71 and 7.15 (2H,

2d, J ) 8.7 Hz, 4,5-protons), 4.12 and 4.08 (6H, 2s, 6-methoxyl

and 2-ester methyl); IR (KBr pellet) 3396, 2960, 2883, 1716,

1662, 1545, 1442, 1411, 1342, 1253, 1192, 1089, 962, and 810

cm^-1; MS (EI) 278 (M⁺) 263, 260, 245, 218, 202, 187, and 169.

Anal. Calcd (C₁₂H₁₀N₂O₆): C, H, N.

218, 104, 190, 176, 162, 146, and 134. Anal. Calcd (C₁₂H₁₃

NO₅): C, H, N.

-

2,3-Di(h yd r oxym eth yl)n itr oin d oles (20). To a solution

of 100 mg of lithium aluminum hydride (LAH) in 15 mL of

dry tetrahydrofuran (THF) cooled to -15 °C was added a

solution of 0.5 mmol of 19 in 2 mL of dry THF. The solution

was stirred at the same temperature for another 5 min and

then 5 mL of ethyl acetate was added, followed by the addition

of 2 mL of water. The solid was filtered off and the solution

was vacuum-dried, followed by flash chromatography with

ethyl acetate as the eluent. The product was recrystallized

from ethyl acetate and hexane.

2,3-Di(h ydr oxym eth yl)-6-m eth oxyin dole-4,7-dion e (21c).

Yield 10%; mp 217 °C; TLC (dichloromethane/methanol

1

90:10) R_f) 0.30; H NMR (DMSO-d₆) δ 12.6 (1H, br s, indole

proton), 5.75 (1H, s, 5-proton), 5.07 and 4.73 (2H, 2t, J ) 6.0

Hz, hydroxyls of 2,3-hydroxymethyls), 4.60 and 4.47 (4H, 2d,

J ) 6.0 Hz, methylenes of 2,3-hydroxymethyls), and 3.75 (3H,

s, 6-methoxy). IR (KBr pellet) 3433, 3173, 3117, 2951, 2876,

1670, 1633, 1597, 1248, 1112, 997, and 852 cm^-1; MS (EI) 237

(M⁺), 219 (M⁺- H₂O), 204, 190, 176, 162, 148, and 134. Anal.

Calcd (C₁₁H₁₁NO₅): C, H, N.

2,3-Di(h yd r oxym eth yl)-5-m eth oxy-4-n itr oin d ole (20a ).

Yield 40%; mp 127 °C; TLC (ethyl acetate) R_f) 0.30; ¹H NMR

(DMSO-d₆) δ 11.4 (1H, br s, indole proton), 7.51 and 7.02 (2H,

Azir id in yl-2,3-d i(h yd r oxym eth yl)in d ole-4,7-d ion es (6,

7, 9a ). To 0.2 mmol of 21 in 20 mL of methanol was added 0.6

3558 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

mL of ethylenimine, and the resulting mixture was stirred at

room temperature for 10 h. The solvent was removed and the

residue was purified by flash chromatography with 20%

acetone in ethyl acetate as the eluent. The product was

recrystallized from ethyl acetate and hexane.

1s, 7-proton), 5.13 and 4.44 (2H, 2t, J ) 5.4 Hz, hydroxyls of

2,3-hydroxymethyls), 4.61 and 4.42 (4H, 2d, J ) 5.4 Hz,

methylenes of 2,3-hydroxymethyls), 3.77 and 3.75 (6H, 2s,

1-methyl and 5-methoxy), and 2.40 (3H, s, 6-methyl). IR (KBr

pellet) 3447, 2945, 2891, 1637, 1525, 1477, 1377, 1329, 1174,

1124, 993, 854, and 765 cm^-1; MS (EI) 280 (M⁺), 262, 245, 229,

215, 186, and 173. Anal. Calcd (C₁₃H₁₆N₂O₅): C, H, N.

5-Azir idin yl-2,3-di(h ydr oxym eth yl)in dole-4,7-dion e (6a).

Yield 74%; mp 167 °C; TLC (ethyl acetate/methanol 95:5) R_f

1

2,3-Di(h yd r oxym eth yl)-1,6-d im eth yl-5-m eth oxyin d ole-

4,7-d ion e (24). Yield 16%; mp 160 °C; TLC (ethyl acetate/

methanol 95:5) R_f) 0.56; ¹H NMR (DMSO-d₆) δ 5.13 and 4.67

(2H, 2t, J ) 5.4 Hz, hydroxyls of 2,3-hydroxymethyls), 4.59

and 4.54 (4H, 2d, J ) 5.4 Hz, methylenes of 2,3-hydroxy-

methyls), 3.90 and 3.87 (6H, 2s, 1-methyl and 5-methoxy), and

1.85 (3H, s, 6-methyl); IR (KBr pellet) 3335, 3194, 2943, 2852,

1682, 1643, 1506, 1464, 1313, 1122, 1008, 989, 900, and 740

cm^-1; MS (EI) 265 (M⁺), 247 (M⁺- H₂O), 232, 218, 204, 190,

176, 148, 132, and 120. Anal. Calcd (C₁₃H₁₅NO₅): C, H, N.

5-Azir idin yl-2,3-di(h ydr oxym eth yl)-1,6-dim eth ylin dole-

4,7-d ion e (7c). Yield 50%; mp 165 °C; TLC (ethyl acetate/

methanol 95:5) R_f) 0.48; ¹H NMR (DMSO-d₆) δ 5.08 and 4.59

(2H, 2t, J ) 5.4 Hz, hydroxyls of 2,3-hydroxymethyls), 4.58

and 4.52 (4H, 2d, J ) 5.4 Hz, methylenes of 2,3-hydroxy-

methyls), 3.89 (3H, s, 1-methyl), 2.23 (4H, s, 5-aziridinyl), and

1.93 (3H, s, 6-methyl); IR (KBr pellet) 3435, 3335, 3057, 2958,

) 0.35; H NMR (DMSO-d₆) δ 12.3 (1H, br s, indole proton),

5.78 (1H, s, 6-proton), 4.99 and 4.65 (2H, 2t, J ) 5.7 Hz,

hydroxyls of 2,3-hydroxymethyls), 4.60 and 4.44 (4H, 2d, J )

5.7 Hz, methylenes of 2,3-hydroxymethyls), and 2.14 (4H, s,

5-aziridinyl); IR (KBr pellet) 3445, 3209, 3047, 2926, 2856,

1631, 1579, 1498, 1465, 1361, 1271, 1159, 1095, 1003, 846, and

804 cm^-1; MS (EI) 248 (M⁺), 230 (M⁺- H₂O), 215, 201, 189,

and 174. Anal. Calcd (C₁₂H₁₂N₂O₄): C, H, N.

5-Azir id in yl-2,3-d i(h yd r oxym et h yl)-6-m et h ylin d ole-

4,7-d ion e (7a ). Yield 75%; mp 165 °C; TLC (ethyl acetate/

1

methanol 95:5) R_f) 0.38; H NMR (DMSO-d₆) δ 12.3 (1H, br

s, indole proton), 4.94 and 4.62 (2H, 2t, J ) 5.1 Hz, hydroxyls

of 2,3-hydroxymethyls), 4.58 and 4.40 (4H, 2d, J ) 5.1 Hz,

methylenes of 2,3-hydroxymethyls), 2.23 (4H, s, 5-aziridinyl),

and 1.92 (3H, s, 6-methyl); IR (KBr pellet) 3404, 3209, 2949,

2879, 1626, 1587, 1562, 1500, 1458, 1377, 1350, 1248, 1188,

1149, 1103, 1020, 995, and 819 cm^-1; MS (EI) 262 (M⁺), 247

(M⁺- CH₃), 244 (M⁺- H₂O), 229, 215, and 203. Anal. Calcd

(C₁₃H₁₄N₂O₄): C, H, N.

2852, 1666, 1631, 1508, 1336, 1151, 1043, 989, and 875 cm^-1

;

MS (EI) 276 (M⁺), 258, 243, 229, 214, 202, and 188. Anal. Calcd

(C₁₄H₁₆N₂O₄): C, H, N.

6-Azir idin yl-2,3-di(h ydr oxym eth yl)in dole-4,7-dion e (9a).

2-(Eth oxycar bon yl)-3-(h ydr oxym eth yl)n itr oin doles (25).

To a solution of 2 mmol of 19 in 100 mL of MeOH was added

1 g of NaBH₄, and the solution was stirred at room tempera-

ture for 20 min, followed by the addition of 200 mL of water.

The solution was then extracted five times with 100 mL

portions of chloroform. The extract was dried over Na₂SO₄and

vacuum-dried to a residue. The product was crystallized from

CH₂Cl₂and hexane.

Yield 52%; mp 175 °C; TLC (ethyl acetate/methanol 95:5) R_f

) 0.38; H NMR (DMSO-d₆) δ 12.4 (1H, br s, indole proton),

1

5.76 (1H, s, 5-proton), 5.04 and 4.72 (2H, 2t, J ) 5.7 Hz,

hydroxyls of 2,3-hydroxymethyls), 4.57 and 4.46 (4H, 2d, J )

5.7 Hz, methylenes of 2,3-hydroxymethyls), and 2.14 (4H, s,

6-aziridinyl); IR (KBr pellet) 3394, 3232, 3038, 2928, 2874,

1660, 1610, 1500, 1392, 1275, 1132, 1020, and 879 cm^-1; MS

(EI) 248 (M⁺), 230, 202, 189, 174, 160, and 146. Anal. Calcd

(C₁₂H₁₂N₂O₄): C, H, N.

2-(E t h oxyca r b on yl)-3-(h yd r oxym et h yl)-5-m et h oxy-4-

n itr oin d ole (25a ). Yield 90%; mp 165 °C; TLC (chloroform/

5-Azir idin yl-2,3-di(acetoxym eth yl)in dole-4,7-dion e (6e).

To a solution of 0.2 mmol of 6a in 5 mL of CH₂Cl₂and 1 mL

of acetone containing 50 mg of DMAP was added 100 µL of

acetic anhydride. The solution was stirred at room tempera-

ture for 5 min and directly flash-chromatographed with ethyl

acetate as the eluent. The eluted solution containing the

product was washed with NaHCO₃, dried over Na₂SO₄, and

vacuum-dried. The product was recrystallized from ethyl

acetate and hexane. Yield 82%; mp 167 °C; TLC (dichlo-

romethane/methanol 95:5) R_f) 0.6; ¹H NMR (CDCl₃) δ 8.8

(1H, br s, indole proton), 5.72 (1H, s, 6-proton), 4.63 and 4.46

(4H, 2s, methylenes of 2,3-hydroxymethyls), 2.24 (4H, s,

5-aziridinyl), 2.10 and 2.08 (6H, 2s, 2,3-acetyl methyls); IR

(KBr pellet) 3447, 3420, 3194, 3047, 2966, 1739, 1674, 1628,

1570, 1506, 1475, 1377, 1246, 1165, 1145, 1112, 1033, 977, 949,

850, and 804 cm^-1; MS (EI) 332 (M⁺), 290 (M⁺- acetyl), 279,

261, 243, 226, 217, and 188. Anal. Calcd (C₁₆H₁₆N₂O₆): C, H,

N.

1

acetone 80:20) R_f) 0.40; H NMR (CDCl₃) δ 10.45 (1H, br s,

indole proton), 7.54 and 7.28 (2H, 2d, J ) 8.7 Hz, 6,7-protons),

5.12 (2H, d, J ) 6.6 Hz, 3-methylene), 5.00 (1H, t, J ) 6.6 Hz,

3-hydroxyl), 4.54 (2H, q, J ) 7.2 Hz, methylene of ethoxy),

4.01 (3H, s, 5-methoxy), and 1.48 (3H, t, J ) 7.2 Hz, methyl of

ethoxy); IR (KBr pellet) 3562, 3452, 3313, 2993, 2949, 2849,

1682, 1533, 1475, 1384, 1255, 1180, 1120, 1012, and 827 cm^-1

;

MS (EI) 294 (M⁺), 276, 248, 231, 212, and 186. Anal. Calcd

(C₁₃H₁₄N₂O₆): C, H, N.

2-(E t h oxyca r b on yl)-3-(h yd r oxym et h yl)-5-m et h oxy-6-

m eth yl-4-n itr oin d ole (25b). Yield 90%; mp 147 °C; TLC

(chloroform/acetone 80:20) R_f) 0.50; ¹H NMR (CDCl₃) δ 8.91

(1H, br s, indole proton), 7.35 (1H, s, 7-proton), 4.97 (2H, d, J

) 6.3 Hz, 3-methylene), 4.45 (2H, q, J ) 7.2 Hz, methylene of

ethoxy), 3.90 (3H, s, 5-methoxy), 2.49 (1H, t, J ) 6.3 Hz,

3-hydroxyl), 2.48 (3H, s, 6-methyl), and 1.44 (3H, t, J ) 7.2

Hz, methyl of ethoxy); IR (KBr pellet) 3431, 3238, 3001, 2957,

2889, 1718, 1701, 1527, 1465, 1261, 1188, 1155, 1006, and 779

cm^-1; MS (EI) 308 (M⁺), 291 (M⁺- OH), 279, 261, 245, 226,

215, 200, 187, and 174. Anal. Calcd (C₁₄H₁₆N₂O₆): C, H, N.

3-(Hyd r oxym eth yl)-6-m eth oxy-2-(m eth oxyca r bon yl)-7-

n itr oin d ole (25c). Yield 93%; mp 150 °C; TLC (chloroform/

methanol 90:10) R_f) 0.35; ¹H NMR (CDCl₃) δ 10.25 (1H, br s,

indole proton), 8.03 and 7.01 (2H, 2d, J ) 9.3 Hz, 4,5-protons),

5.07 (2H, d, J ) 6.9 Hz, 3-methylene), 4.90 (1H, t, J ) 6.9 Hz,

3-hydroxyl), 4.10 and 4.02 (6H, 2s, 6-methoxy and ester

methyl); IR (KBr pellet) 3433, 2920, 2850, 1736, 1629, 1560,

1521, 1464, 1253, 1180, 1091, 970, and 804 cm^-1; MS (EI) 280

(M⁺), 263 (M⁺- OH), 247, 231, 219, 200, 172, and 157. Anal.

Calcd (C₁₂H₁₂N₂O₆): C, H, N.

2-(Eth oxyca r bon yl)-3-for m yl-1,6-d im eth yl-5-m eth oxy-

4-n itr oin d ole (22). To 0.2 mmol of 19b in 20 mL of acetone

with 500 mg of KOH was added 1.0 mL of MeI; the mixture

was stirred at room temperature for 1 h, neutralized with HCl,

and extracted four times with 30 mL portions of CH₂Cl₂. The

combined extracts was dried with Na₂SO₄and dried, followed

by chromatography with CH₂Cl₂as the eluent. The product

was crystallized from CH₂Cl₂and hexane. Yield 55%; mp 148

1

°C; TLC (CHCl₃) R_f) 0.55; H NMR (CDCl₃) δ 10.42 (1H, s,

3-formyl), 7.40 (H, s, 7-proton), 4.52 (2H, q, J ) 7.2 Hz,

methylene of ethoxy), 4.06 and 3.91 (6H, 2s, 1-methyl and

5-methoxyl), 2.51 (3H, s, 6-methyl), and 1.46 (3H, t, J ) 7.2

Hz, methyl of ethoxy); IR (KBr pellet) 3435, 2999, 2930, 1712,

1666, 1545, 1475, 1404, 1238, 1161, 1031, 979, 908, and 779

cm^-1; MS (EI) 320 (M⁺), 307, 287, 275, 244, and 216. Anal.

Calcd (C₁₅H₁₆N₂O₆): C, H, N.

2-(E t h oxyca r b on yl)-3-(h yd r oxym et h yl)in d ole-4,7-d i-

on es (26). A solution of 2 mmol of 25 in 80 mL of methanol

with 300 mg of 5% Pd on carbon was reduced under 50 psi H₂

for 25 min, and the catalyst was filtered off through Celite.

The solvent was vacuum-dried and the residue was dissolved

in 5 mL of acetone, which was then mixed with a solution of

800 mg of KH₂PO₄and 1.6 g of Fremy salt in 200 mL of H₂O.

2,3-Di(h yd r oxym et h yl)-1,6-d im et h yl-5-m et h oxy-4-n i-

tr oin d ole (23). Synthesis of this compound followed the same

procedure as the synthesis of 21. Yield 55%; mp 197 °C; TLC

(EtOAc/MeOH 95:5) R_f) 0.65; ¹H NMR (DMSO-d₆) δ 7.58 (1H,

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3559

The solution was stirred at room temperature for 5h and

extracted five times with 200 mL of methylene chloride. The

extract was dried and purified by flash chromatography with

10% acetone in methylene chloride as the eluent. The product

was precipitated from CH₂Cl₂and hexane.

2-(E t h oxyca r bon yl)-3-(h yd r oxym et h yl)-5-m et h oxyin -

d ole-4,7-d ion e (26a ). Yield 64%; mp 213 °C; TLC (chloroform/

methanol 90:10) R_f) 0.30; ¹H NMR (CDCl₃) δ 9.77 (1H, br s,

indole proton), 5.85 (1H, s, 6-proton), 5.07 (2H, d, J ) 7.2 Hz,

3-methylene), 4.41 (2H, q, J ) 7.2 Hz, methylene of ethoxy),

4.22 (1H, t, J ) 7.2 Hz, 3-hydroxyl), 3.89 (3H, s, 5-methoxy),

and 1.41 (3H, t, J ) 7.2 Hz, methyl of ethoxy); IR (KBr pellet)

3433, 3122, 3072, 2989, 2924, 2785, 1712, 1649, 1597, 1560,

1487, 1467, 1340, 1280, 1161, 1031, and 844 cm^-1; MS (EI)

279 (M⁺), 261, 250, 233, 218, 205, 190, and 177. Anal. Calcd

(C₁₃H₁₃NO₆): C, H, N.

MS (EI) 276(M⁺), 244 (M⁺- CO), 229, 216, 188, 160, and 132.

Anal. Calcd (C₁₃H₁₂N₂O₅): C, H, N.

3-(Acetoxym eth yl)-5-a zir id in yl-2-(eth oxyca r bon yl)in -

d ole-4,7-d ion e (6d ). Synthesis of this compound followed the

procedure described for the acetylation of 6e. Yield 73%; mp

185 °C; TLC (chloroform/acetone 90:10) R_f) 0.45; ¹H NMR

(CDCl₃) δ 9.76 (1H, br s, indole proton), 5.96 (1H, s, 6-proton),

5.54 (2H, s, 3-methylene), 4.42 (2H, q, J ) 7.2 Hz, methylene

of ethoxy), 2.29 (4H, s, 5-aziridinyl), 2.07 (3H, s, 3-acetoxy-

methyl), and 1.39 (3H, t, J ) 7.2 Hz, methyl of ethoxy); IR

(KBr pellet) 3445, 3234, 2995, 2920, 2852, 1726, 1701, 1676,

1583, 1556, 1500, 1363, 1280, 1244, 1153, 1024, and 819 cm^-1

;

MS (EI) 332 (M⁺), 318, 290, 279, 260, 244, and 217. Anal. Calcd

(C₁₆H₁₆N₂O₆): C, H, N.

2-(E t h oxyca r b on yl)-5-m e t h oxy-3-m e t h yl-4-n it r oin -

d ole (27a ). To 1.17 g (5 mmol) of 2-(ethylcarboxyl)-5-methoxy-

3-methylindole⁴⁶in 180 mL of CH₂Cl₂, cooled by a dry ice bath

at -20 °C, was added 0.5 mL of 70% HNO₃, and the solution

was stirred for 10 min. The solution was then neutralized with

NaHCO₃aqueous solution and extracted four times with 50

mL portions of methylene chloride. The extracts were dried

over Na₂SO₄and vacuum-dried to a residue that was purified

by flash chromotography on silica gel with CH₂Cl₂as the

eluent. Yield 880 mg (69%); mp 175-177 °C; TLC (CHCl₃) R_f

) 0.5; ¹H NMR(CDCl₃) δ 8.81(1H, br s, indole proton), 7.45

and 7.12 (2H, d, J ) 9 Hz, 6,7-protons), 4.44 (2H, q, J ) 7.5

Hz, methylene of ethoxy), 3.94 (3H, s, 5-methoxy), 2.46 (3H,

s, 3-methyl), and 1.43 (3H, t, J ) 7.5, methyl of ethoxy); IR

(KBr pellet) 3423, 3340, 2933, 2847, 1680, 1628, 1541, 1475,

1384, 1251, 1182, 1120, 1049, 1016, and 796 cm^-1; MS (EI)

278 (M⁺), 261 (M⁺- OH), 249, 232, 215, 203, 187, 174, and

156. Anal. Calcd (C₁₃H₁₄N₂O₅): C, H, N.

2-(E t h ylca r b oxyl)-3-(h yd r oxym e t h yl)-5-m e t h oxy-6-

m eth ylin d ole-4,7-d ion e (26b). Yield 41%; mp 183 °C;

1

TLC (CHCl₃/MeOH 90:10) R_f) 0.45; H NMR (CDCl₃) δ 9.75

(1H, br s, indole proton), 5.05 (2H, d, J ) 7.2 Hz, 3-methylene),

4.41 (2H, q, J ) 7.2 Hz, methylene of ethoxy), 4.34 (1H, t, J )

7.2 Hz, 3-hydroxyl), 4.08 (3H, s, 5-methoxy), 2.01 (3H, s,

6-methyl), and 1.41 (3H, t, J ) 7.2 Hz, methyl of ethoxy); IR

(KBr pellet) 3435, 3211, 3063, 2991, 2920, 2852, 1710, 1647,

1606, 1496, 1294, 1232, 1112, 1074, 1033, and 866 cm^-1; MS

(EI) 293 (M⁺), 275, 264, 247, 232, 219, 204, and 186. Anal.

Calcd (C₁₄H₁₅NO₆): C, H, N.

3-(H yd r oxym et h yl)-6-m et h oxy-2-(m et h oxyca r b on yl)-

in d ole-4,7-d ion e (26c). Yield 42%; mp 150 °C; TLC (chloro-

form/methanol 90:10) R_f) 0.35; ¹H NMR (CDCl₃) δ 9.70 (1H,

br s, indole proton), 5.91 (1H, s, 5-proton), 5.04 (2H, d, J ) 7.8

Hz, 3-methylene), 4.59 (1H, t, J ) 7.8 Hz, 3-hydroxyl), 3.96

and 3.88 (6H, 2s, 6-methoxy and ester methyl); IR (KBr pellet)

3431, 3238, 3001, 2957, 2889, 1718, 1701, 1527, 1465, 1398,

1261, 1188, 1155, 1006, and 779 cm^-1; MS (EI) 265 (M⁺), 250

(M⁺- Me), 233, 218, 205, 190, 162, 148, and 120. Anal. Calcd

(C₁₂H₁₁NO₆): C, H, N.

Azir idin yl-2-(eth oxycar bon yl)-3-(h ydr oxym eth yl)in dole-

4,7-d ion es. To a solution of 1 mmol of product above in 50

mL of methanol was added 1 mL of aziridine, and the reaction

was stirred at room temperature for 20 h. The solvent was

evaporated and the residue was purified by flash chromatog-

raphy on silica gel with 10% acetone in CH₂Cl₂as the eluent.

The product was recrystallized from CH₂Cl₂and hexane.

5-Azir idin yl-2-(eth ylcar boxyl)-3-(h ydr oxym eth yl)in dole-

4,7-d ion e (6c). Yield 86%; mp 170 °C; TLC (chloroform/

methanol 90:10) R_f) 0.32; ¹H NMR (CDCl₃) δ 9.78 (1H, br s,

indole proton), 5.96 (1H, s, 6-proton), 5.07 (2H, d, J ) 7.2 Hz,

3-methylene), 4.41 (2H, q, J ) 7.2 Hz, methylene of ethoxy),

4.34 (1H, t, J ) 7.2 Hz, 3-hydroxyl), 2.30 (4H, s, 5-aziridinyl),

and 1.41 (3H, t, J ) 7.2 Hz, methyl of ethoxy); IR (KBr pellet)

3450, 3259, 3024, 2899, 2862, 1703, 1656, 1581, 1550, 1491,

1363, 1278, 1240, 1149, 1028, and 842 cm^-1; MS (EI) 290 (M⁺),

279, 260, 244, 233, 226, 215, 199, 188, and 171. Anal. Calcd

(C₁₄H₁₄N₂O₅): C, H, N.

2-(Eth oxyca r bon yl)-1,3-d im eth yl-4-n itr oin d oles (28).

To 2 mmol of 27 in 20 mL of acetone containing 400 mg of

KOH was added 1.0 mL of MeI, and the mixture was stirred

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

neutralized with HCl and extracted four times with 30 mL

portions of CH₂Cl₂. The combined extracts were dried over Na₂-

SO₄and evaporated to a residue that was purified by chro-

matography on silica gel with CH₂Cl₂as the eluent. The

purified product was recrystallized from CH₂Cl₂and hexane.

2-(Eth oxyca r bon yl)-5-m eth oxy-1,3-d im eth yl-4-n itr oin -

d ole (28a ). Yield ∼100%; mp 120-122 °C; TLC (CHCl₃) R_f)

0.45; ¹H NMR (CDCl₃) δ 7.42 and 7.13 (2H, d, J ) 9.3 Hz,

6,7-protons), 4.42 (2H, q, J ) 7.2 Hz, 2-methylene of ethyl),

3.98 and 3.94 (6H, 2s, 1-methyl and 5-methoxy), 2.40 (3H, s,

3-methyl), and 1.43 (3H, t, J ) 7.2, 2-methyl of ethyl); IR (KBr

pellet) 3441, 3003, 2947, 2912, 1709, 1637, 1527, 1465, 1375,

1236, 1165, 1112, 1045, 912, and 794 cm^-1; MS (EI) 292 (M⁺),

275 (M⁺- OH), 263, 244, 216, 188, 163, and 149. Anal. Calcd

(C₁₄H₁₆N₂O₅): C, H, N.

2-(Eth oxycar bon yl)-1,3,5-tr im eth yl-4-n itr oin dole (28b).

Yield 90%; mp 65-68 °C; TLC (CHCl₃) R_f) 0.50; ¹H NMR

(CDCl₃) δ 7.38 and 7.18 (2H, d, J ) 9 Hz, 6,7-protons), 4.46

(2H, q, J ) 7.5 Hz, 2-methylene of ethoxy), 3.99 (3H, s,

1-methyl), 2.42 and 2.39 (6H, 2s, 3,5-methyls), and 1.43 (3H,

t, J ) 7.5, 2-methyl of ethoxy); IR (KBr pellet) 3338, 2978,

2928, 2854, 1699, 1521, 1373, 1290, 1251, 1155, 1085, 1014,

and 802 cm^-1; MS (EI) 276 (M⁺), 259 (M⁺- OH), 247, 229,

5-Azir id in yl-2-(et h ylca r b oxyl)-3-(h yd r oxym et h yl)-6-

m eth ylin d ole-4,7-d ion e (7b). Yield 76%; mp 197 °C; TLC

(chloroform/methanol 90:10) R_f) 0.40; ¹H NMR (CDCl₃) δ 9.61

(1H, br s, indole proton), 5.04 (2H, d, J ) 7.2 Hz, 3-methylene),

4.45 (2H, q, J ) 7.2 Hz, methylene of ethoxy), 4.38 (1H, t, J )

7.2 Hz, 3-hydroxyl), 2.39 (4H, s, 5-aziridinyl), 2.10 (3H, s,

6-methyl), and 1.41 (3H, t, J ) 7.2 Hz, methyl of ethoxy); IR

(KBr pellet) 3448, 3252, 3088, 2997, 2922, 2854, 1709, 1651,

1587, 1550, 1492, 1377, 1209, 1261, 1176, 1147, 1035, 997, 962,

and 871 cm^-1; MS (EI) 304 (M⁺), 286 (M⁺- H₂O), 275, 258,

240, 229, 212, and 202. Anal. Calcd (C₁₅H₁₆N₂O₅): C, H, N.

6-Azir id in yl-3-(h yd r oxym eth yl)-2-(m eth ylca r boxyl)in -

d ole-4,7-d ion e (9b). Yield 52%; mp 258 °C; TLC (chloroform/

methanol 90:10) R_f) 0.45; ¹H NMR (CDCl₃) δ 9.78 (1H, br s,

indole proton), 6.02 (1H, s, 5-proton), 5.02 (2H, d, J ) 6.9 Hz,

3-methylene), 4.59 (1H, t, J ) 6.9 Hz, 3-hydroxyl), 3.93 (3H,

s, ester methyl), and 2.28 (4H, s, 6-aziridinyl); IR (KBr pellet)

3420, 3257, 3074, 2997, 2958, 2922, 2852, 1705, 1674, 1626,

213, 202, 174, 157, 142, 131, and 115. Anal. Calcd (C₁₄

H₁₆N₂O₄): C, H, N.

-

2-(E t h o x y c a r b o n y l)-3-(h y d r o x y m e t h y l)-4-n it r o in -

d oles (29). To 1.50 mmol of 28 in 15 mL of CCl₄at reflux were

added 258 mg of N-bromosuccinimide (NBS) and 26 mg of 2,2′-

azo-bis-isobutyrylnitrile (AIBN). The reaction was refluxed for

1.5 h with additional AIBN added every 30 min (13 mg × 3).

Hexane was added to the cooled reaction mixture in order to

precipitate the crude product as a light yellow solid. A solution

of the crude product above in 25 mL of THF, 20 mL of H₂O,

and 4 mL of HCl was stirred at room temperature for 1.5 h.

This mixture was neutralized with saturated NaHCO₃solution

and then extracted three times with 50 mL portions of CH₂-

1587, 1545, 1267, 1157, 1078, 1030, 952, 868, and 804 cm^-1

;

3560 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

Cl₂. The extracts were dried over Na₂SO₄and concentrated to

a residue. The product was recrystallized from CH₂Cl₂and

hexane.

1373, 1342, 1300, 1182, 1069, 1055, 989, and 802 cm^-1; MS

(EI) 250 (M⁺), 233 (M⁺- OH), 221, 191, 177, 163,149, and

105. Anal. Calcd (C₁₂H₁₄N₂O₄): C, H, N.

2,3-Di(h yd r oxym eth yl)in d ole-4,7-d ion es (31). A solution

of 0.5 mmol of product above with 295 mg of 5% Pd-C in 40

mL of methanol was reduced under 50 psi H₂for 30 min. The

catalyst was Celite-filtered off, and the filtrate was vacuum-

dried. The solid residue was dissolved in 4 mL of acetone and

mixed with a solution of 250 mg of KH₂PO₄and 500 mg of

Fremy salt in 25 mL of water. The solution was stirred at room

temperature for 4 h and extracted five times with 50 mL

portions of ethyl acetate. The extracts were dried over Na₂-

SO₄and vacuum-dried and then purified by flash column

chromatography on silica gel with ethyl acetate as the eluent.

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

d ion e (31a ). Yield 25%; mp 191-193 °C; TLC (chloroform/

methanol 9:1) R_f) 0.45; ¹H NMR (CDCl₃) δ 6.23 (1H, s,

6-proton), 4.70 and 4.65 (4H, 2d, J ) 6 Hz, 2,3-methylenes of

hydroxymethyls), 4.11 and 1.98 (2H, 2t, J ) 6 Hz, hydroxys of

2,3-hydroxymethyls), 4.04 and 3.84 (6H, 2s, 1-methyl and

5-methoxy); IR (KBr pellet) 3465, 3332, 2945, 2931, 1649, 1537,

1419, 1374, 1233, 950, and 811 cm^-1; MS (EI) 251 (M⁺), 233

(M⁺- H₂O), 218, 204, 190, 176, 162, and 120. Anal. Calcd

(C₁₂H₁₃NO₅): C, H, N.

2-(E t h oxyca r b on yl)-3-(h yd r oxym et h yl)-5-m et h oxy-1-

m eth yl-4-n itr oin d ole (29a ). Yield 34%; mp 120-122 °C; TLC

(CHCl₃/MeOH 9:1) R_f) 0.55; ¹H NMR (CDCl₃) δ 7.48 and 7.17

(2H, d, J ) 8.7 Hz, 6,7-protons), 4.87 (2H, d, J ) 6.6 Hz,

3-hydroxymethyl), 4.47 (2H, q, J ) 7.2 Hz, 2-methylene of

ethyl), 4.01 and 3.96 (6H, 2s, 1-methyl and 5-methoxyl), 2.31

(1H, t, J ) 6.6 Hz, 3-hydroxy), and 1.46 (3H, t, J ) 7.2,

2-methyl of ethyl); IR (KBr pellet) 3497, 3412, 3232, 2947,

1685, 1621, 1609, 1534, 1287, 1174, 1000, and 823 cm^-1; MS

(EI) 308 (M⁺), 291 (M⁺- OH), 261, 244, 235, 216, 201, 177,

163, 149, and 105. Anal. Calcd (C₁₄H₁₆N₂O₆): C, H, N.

2-(Eth oxyca r bon yl)-3-(h yd r oxym eth yl)-5-m eth yl-4-n i-

tr oin d ole (29b). Yield 80%; mp 134-136 °C; TLC (chloroform/

methanol 9:1) R_f) 0.45; ¹H NMR (CDCl₃) δ 9.08 (1H, br s,

indole proton), 7.46 and 7.23 (2H, d, J ) 9 Hz, 6,7-protons),

5.01 (2H, d, J ) 6 Hz, 3-hydroxymethyl), 4.46 (2H, q, J ) 7.5

Hz, 2-methylene of ethoxy), 2.48 (3H, s, 5-methyl), 2.35 (1H,

t, J ) 6 Hz, 3-hydroxy), and 1.47 (3H, t, J ) 7.5, methyl of

ethoxy); IR (KBr pellet) 3447, 3194, 2980, 2928, 1718, 1678,

1527, 1465, 1365, 1265, 1180, 1116, 989, and 808 cm^-1; MS

(EI) 278 (M⁺), 261 (M⁺- OH), 245, 231, 215, 186, 171, and

149. Anal. Calcd (C₁₃H₁₄N₂O₅): C, H, N.

2,3-Di(h yd r oxym eth yl)-5-m eth ylin d ole-4,7-d ion e (31b).

Yield 52%; mp 181-183 °C; TLC (chloroform/methanol 9:1)

R_f) 0.24; ¹H NMR (DMSO-d₆) δ 12.4 (1H, br s, 1-indole

proton), 6.44 (1H, q, J ) 1.5 Hz, 6-proton), 5.03 and 4.66 (2H,

2t, J ) 6 Hz, hydroxys of 2,3-hydroxymethyls), 4.59 and 4.46

(4H, 2d, J ) 6 Hz, methylenes of 2,3-hydroxymethyls), and

1.94 (3H, s, 5-methyl); IR (KBr pellet) 3423, 3101, 2953, 2930,

2-(E t h oxyca r bon yl)-3-(h yd r oxym et h yl)-1,5-d im et h yl-

4-n itr oin d ole (29c). Yield 31%; mp 113-115 °C; TLC (chlo-

1

roform/methanol 9:1) R_f) 0.50; H NMR (CDCl₃) δ 7.43 and

7.23 (2H, d, J ) 9 Hz, 6,7-protons), 4.88 (2H, d, J ) 6 Hz,

3-hydroxymethyl), 4.48 (2H, q, J ) 7.5 Hz, methylene of

ethoxy), 4.01 and 2.48 (6H, 2s, 1,5-methyl), 2.35 (1H, t, J ) 6

Hz, 3-hydroxy), and 1.46 (3H, t, J ) 7.5 Hz, methyl of ethoxy);

IR (KBr pellet) 3489, 3425, 3244, 2928, 1685, 1637, 1618, 1525,

1707, 1523, 1465, 1384, 1253, 1159, 1109, 991, and 806 cm^-1

;

MS (EI) 221 (M⁺), 203 (M⁺- H₂O), 190, 174, 147, 118, and

107. Anal. Calcd (C₁₁H₁₁NO₄): C, H, N.

1263, 1168, 1014, and 810 cm^-1; MS (EI) 292 (M⁺), 275 (M⁺

-

OH), 245, 228, 219, 200, 185, and 172. Anal. Calcd (C₁₄

₁₆N₂O₅): C, H, N.

2,3-Di(h yd r oxym eth yl)-4-n itr oin d oles (30). To a solution

2,3-Di(h yd r oxym et h yl)-1,5-d im et h ylin d ole-4,7-d ion e

(31c). Yield 32%; mp 136-138 °C; TLC (chloroform/methanol

9:1) R_f) 0.50; ¹H NMR (CDCl₃) δ 6.40 (1H, q, J ) 1.5 Hz,

6-proton), 4.72 and 4.67 (4H, 2d, J ) 6 Hz, methylenes of 2,3-

hydroxymethyls), 4.04 and 1.94 (2H, 2t, J ) 6 Hz, hydroxys of

2,3-hydroxymethyls), 4.04 (3H, s, 1-methyl), and 2.34 (3H, s,

5-methyl); IR (KBr pellet) 3439, 3377, 2955, 2928, 1643, 1608,

1508, 1465, 1377, 1242, 1020, 993, and 802 cm^-1; MS (EI) 235

(M⁺), 217(M⁺- H₂O), 202, 188, 174, 161, 146, and 133. Anal.

Calcd (C₁₂H₁₃NO₄): C, H, N.

H

of 0.3 mmol of 29 in 4 mL of dry THF cooled to -10 °C was

added 60 mg of LAH, and the solution was stirred for 5 min.

The unreacted LAH was quenched by gradually adding 5 mL

of ethyl acetate followed by 2 mL of H₂O. The ethyl acetate

layer was separated and the aqueous layer was washed five

times with 20 mL portions of ethyl acetate. The combined

extracts were dried over Na₂SO₄and concentrated to a residue.

Purification was carried out by flash chromatography on silica

gel with ethyl acetate as the eluent.

2,3-Di(h yd r oxym eth yl)-5-m eth oxy-1-m eth yl-4-n itr oin -

d ole (30a ). Yield 66%; mp 163-165 °C; TLC (chloroform/

methanol 9:1) R_f) 0.35; ¹H NMR (CDCl₃) δ 7.42 and 7.02 (2H,

2d, J ) 8.7 Hz, 6,7-protons), 4.85 and 4.69 (4H, 2d, J ) 6 Hz,

methylenes of 2,3-hydroxymethyls), 3.95 and 3.84 (6H, 2s,

1-methyl and 5-methoxy), 2.33 and 2.19 (2H, 2t, J ) 6 Hz,

hydroxys of 2,3-hydroxymethyls); IR (KBr pellet) 3425, 2924,

2847, 1628,1521, 1475, 1421, 1365, 1300, 1263, 1105, 1062,

1001, 974, and 891 cm^-1; MS (EI) 266 (M⁺), 249 (M⁺- OH),

237, 208, 194, 189,165, and 121. Anal. Calcd (C₁₂H₁₄N₂O₅): C,

H, N.

2,3-Di(h yd r oxym eth yl)-5-m eth yl-4-n itr oin d ole (30b).

Yield 60%; mp 132-134 °C; TLC (chloroform/methanol 9:1)

R_f) 0.3; ¹H NMR (DMSO-d₆) δ 11.5 (1H, br s, indole proton),

7.45 and 7.03 (2H, d, J ) 9 Hz, 6,7-protons), 5.31 and 4.38

(2H, 2t, J ) 6 Hz, hydroxys of 2,3-hydroxymethyls), 4.64 and

4.40 (4H, 2d, J ) 6 Hz, methylenes of 2,3-hydroxymethyls),

and 2.34 (3H, s, 5-methyl); IR (KBr pellet) 3394, 2995, 2926,

1637, 1510, 1475, 1363, 1321, 1151, 1014, and 974 cm^-1; MS

(EI) 236 (M⁺), 219 (M⁺- OH), 207, 177, 163, 149, and 105.

Anal. Calcd (C₁₁H₁₂N₂O₄): C, H, N.

5-Azir id in yl-2,3-d i(h yd r oxym et h yl)-1-m et h ylin d ole-

4,7-d ion e (6b). Yield 87%; mp 145-147 °C; TLC (chloroform/

methanol 9:1) R_f) 0.40; ¹H NMR (CDCl₃) δ 5.82 (1H, s,

6-hydrogen), 4.38 and 2.22 (2H, 2t, J ) 5.4 Hz, hydroxys of

2,3-hydroxymethyls), 4.71 and 4.66 (4H, 2d, J ) 5.4 Hz,

methylenes of 2,3-hydroxymethyls), 4.01 (3H, s, 1-methyl), and

2.22 (4H, s, 5-aziridinyl); IR (KBr pellet) 3539, 3362, 2954,

2919, 1683, 1623, 1514, 1472, 1376, 1250, 1031, and 810 cm^-1

;

MS (EI) 262 (M⁺), 244 (M⁺- H₂O), 229, 215, 201, 169, 159,

and 146. Anal. Calcd (C₁₃H₁₄N₂O₄): C, H, N.

6-Azir id in yl-2,3-d i(h yd r oxym et h yl)-5-m et h ylin d ole-

4,7-d ion e (8a ). Yield 33%; mp 193-194 °C; TLC (chloroform/

methanol 9:1) R_f) 0.20; ¹H NMR (DMSO-d₆) δ 12.3 (1H, br s,

1-indole proton), 5.03 and 4.70 (2H, 2t, J ) 5.4 Hz, hydroxys

of 2,3-hydroxymethyls), 4.57 and 4.44 (4H, 2d, J ) 5.4 Hz,

methylenes of 2,3-hydroxymethyls), 2.26 (4H, s, 6-aziridinyl),

and 1.91 (3H, s, 5-methyl); IR (KBr pellet) 3465, 3097, 2943,

2928, 1617, 1543, 1465, 1375, 1234, 1108, 994, and 812 cm^-1

;

MS (EI) 262 (M⁺), 244 (M⁺- H₂O), 229, 215, 201, 188, 169,

159, 146, 134, and 108. Anal. Calcd (C₁₃H₁₄N₂O₄): C, H, N.

6-Azir id in yl-2,3-d i(a cetoxym eth yl)-5-m eth ylin d ole-4,7-

d ion e (8b). Yield 88%; mp 135-137 °C; TLC (chloroform/

acetone 9:1) R_f) 0.60; ¹H NMR (CDCl₃) δ 9.4 (1H, br s, 1-indole

proton), 5.31 and 5.19 (4H, 2s, methylenes), 2.28 (4H, s,

6-aziridinyl), 2.10, 2.08, and 2.05 (9H, 3s, methyls); IR (KBr

pellet) 3390, 3088, 2956, 2917, 1635, 1498, 1364, 1211, 1134,

940, and 825 cm^-1; MS (EI) 346 (M⁺), 303 (M⁺- acetate), 287,

244, 226, and 215. Anal. Calcd (C₁₇H₁₈N₂O₆): C, H, N.

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

4,7-d ion e (8c). Yield 69%; mp 140-141 °C; TLC (chloroform/

2,3-Di(h ydr oxym eth yl)-1,5-dim eth yl-4-n itr oin dole (30c).

Yield 59%; mp 119-121 °C; TLC (chloroform/methanol 9:1)

1

R_f) 0.34; H NMR (DMSO-d₆) δ 7.63 and 7.16 (2H, d, J ) 9

Hz, 6,7-protons), 5.18 and 4.40 (2H, 2t, J ) 6 Hz, hydroxys of

2,3-hydroxymethyls), 4.63 and 4.45 (4H, 2d, J ) 6 Hz,

methylenes of 2,3-hydroxymethyls), 3.78 and 2.34 (6H, 2s, 1,5-

methyls); IR (KBr pellet) 3425, 2972, 2925, 1647, 1518, 1465,

Aziridinyl Quinone Antitumor Agents

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3561

methanol 9:1) R_f) 0.46; ¹H NMR (CDCl₃) δ 4.66 and 4.63 (4H,

2d, J ) 5.4 Hz, methylenes), 4.49 and 2.15 (2H, 2t, J ) 5.4

Hz, hydroxyls), 3.99 (3H, s, 1-methyl), 2.32 (4H, s, 6-aziridinyl),

and 1.91 (3H, s, 5-methyl); IR (KBr pellet) 3512, 3384, 2959,

2926, 1678, 1622, 1514, 1467, 1381, 1235, 1017, 992, and 810

cm^-1; MS (EI) 276 (M⁺), 258 (M⁺- H₂O), 243, 230, 215, 202,

Refer en ces

(1) Struck, R. F. Nitrogen Mustard and Related Structures. In

Cancer Chemotherapeutic Agents; Foyle, W. O., Ed.; American

Chemical Society: Washington, DC, 1995; pp 112-120.

(2) Elliott, R. D. Nitrosoureas. In Cancer Chemotherapeutic Agents;

Foye, W. O., Ed.; American Chemical Society: Washington, DC,

1995; pp 134-143.

(3) Reynolds, R. C. Aziridines. In Cancer Chemotherapeutic Agents;

Foye, W. O., Ed.; American Chemical Society: Washington, DC,

1995; pp 187-197.

188, 174, 160, 145, 132, 122, and 108. Anal. Calcd (C₁₄

₁₆N₂O₄): C, H, N.

Kin etics Stu d ies w ith Recom bin a n t DT-Dia p h or a se.

-

H

Kinetics studies were carried out in 0.05 M Tris buffer, pH

7.4, under anaerobic conditions, employing Thunberg cuvettes.

A 4.0 mM quinone stock solution was prepared in dimethyl

sulfoxide (DMSO). To the top port of the cuvette was added

the quinone stock solution, and to the bottom port were added

the recombinant DT-diaphorase and NADH stock solutions in

Tris buffer. Both portions were purged with argon for 20 min

and equilibrated at 30 °C for 10 min. The ports were then

mixed and the reaction was followed at 336 nm for 10 min to

obtain initial rates. The final concentrations of the mixture

were 0.3 mM NADH, 1.3 × 10^-6to 6.7 × 10^-5M quinone

substrate, and 1.4 × 10^-9M recombinant DT-diaphorase. To

calculate V_max, the value of ∆ꢀ must be obtained. ∆ꢀ was

calculated from the initial and final absorbances for complete

quinone reduction; all values are between 8000 and 9000 M^-1

cm^-1. All data were fitted to a Lineweaver-Burk plot from

which V_maxand K_Mwere obtained.

Mod elin g in to th e DT-Dia p h or a se Active Site. Crystal-

lographic coordinates for human DT-diaphorase (1D4A) were

obtained from the Protein Data Bank. The coordinates were

used as downloaded from the Protein Data Bank and are

unrefined. For modeling purposes, INSIGHT II from Molecular

Simulations, Inc. (San Diego, CA), was used as previously

described.^34,47

Alk yla tion of DNA by Red u ced In d oloqu in on es. To a

mixture of 1-2 mg of sonicated (600 bp) calf thymus DNA in

2.0 mL of 0.05 M Tris buffer, pH 7.4, and 2 mg of Pd on carbon

was added a 5:1 base pair equivalent amount of the indolo-

quinone dissolved in 0.5 mL of dimethyl sulfoxide. The

resulting solution was degassed under argon for 30 min, after

which the mixture was purged with H₂for 10 min. The solution

was then purged with argon for 10 min and placed in a 30 °C

bath for 24 h. The reaction was opened to the air and the

catalyst was removed with a Millex-PF 0.8 µM syringe filter.

The filtrate was adjusted to 0.3 M acetate with a 3 M stock

solution of acetate, pH 5.1, and then diluted with 2 volumes

of ethanol. The mixture was chilled at -20 °C for 12 h and

the DNA pellet was collected by centrifugation at 12000g for

20 min. The pellet was redissolved in water and then precipi-

tated and centrifuged again. The resulting blue or red pellet

was suspended in ethanol, centrifuged, and dried. The dried

pellet was weighed and dissolved in 1 mL of double-distilled

water, resulting in a clear colored solution with λ_max∼ 550

nm, ꢀ ∼ 750 M^-1cm^-1. This is the chromophore of the

aminoquinone resulting from nucleophile-mediated opening of

the aziridine ring. Model 2′-chloroethyl aminoquinones for

extinction coefficient determination were prepared by treat-

ment of the indoloquinone with HCl.⁴⁸

In Vivo Eva lu a tion . The B-16 melanoma in C57/bl mice

syngraft model was employed to determine in vivo activity.⁴⁴

Each agent was evaluated at three doses: 2, 3, or 5 mg kg^-1

day^-1, on days 1, 5, and 9 after subcutaneous tumor implanta-

tion of 10⁵cells in the front flank on day 0. Toxic means that

there was early lethality, or g50% lethality prior to any deaths

in the control group. The treated over control values (T/C) were

measured at day 25 of the study. A T/C value <40% is

considered active. NA means that the compound was not

active. The control was obtained with drug-free animals.

(4) Tomasz, M.; Palom, Y. The mitomycin bioreductive antitumor

agents: Cross-linking and alkylation of DNA as the molecular

basis of their activity. Pharmacol. Ther. 1997, 76, 73-87.

(5) Skibo, E. B. The Discovery of the Pyrrolo[1,2-a]benzimidazole

Antitumor Agents: The Design of Selective Antitumor Agents.

Curr. Med. Chem. 1996, 2, 900-931.

(6) Skibo, E. B. Pyrrolobenzimidazoles in cancer treatment. Expert

Opin. Ther. Patents 1998, 8, 673-701.

(7) Hendriks, H. R.; Pizao, P. E.; Berger, D. P.; Kooistra, K. L.;

Bibby, M. C.; Boven, E.; Dreef-van der Meulen, H. C.; Henrar,

H. H.; Fiebig, H. H.; Double, J . A.; Hornstra, H. W.; Pinedo, H.

M.; Workman, P.; Schwartsmann, G. EO9: A Novel Bioreductive

Alkylating Indoloquinone With Prefential Solid Tumour Activity

and Lack of Bone Marrow Toxicity in Preclinical Models. Eur.

J . Cancer 1993, 29A, 8997-906.

(8) Maliepaard, M.; Wolfs, A.; Groot, S. E.; de Mol, N. J .; J anssen,

L. H. M. Indoloquinone EO9: DNA Interstrand Cross-linking

Upon Reduction by DT-Diaphorase or Xanthine Oxidase. Br. J .

Cancer 1995, 71, 836-839.

(9) Schellens, J . H. M.; Planting, A. S. T.; Vanacker, B. A. C.; Loos,

W. J .; Deboerdennert, M.; Vanderburg, M. E. L.; Koier, I.;

Krediet, R. T.; Stoter, G.; Verweij, J . Phase I and Pharmacologi-

cal Study of the Novel Indoloquinone Bioreductive Alkylating

Cytotoxic Drug E09. J . Natl. Cancer Inst. 1994, 86, 906-912.

(10) Aamdal, S.; Lund, B.; Koier, I.; Houten, M.; Wanders, J .; Verweij,

J . Phase I trial with weekly EO9, a novel bioreductive alkylating

indoloquinone, by the EORTC Early Clinical Study Group

(ECSG). Cancer Chemother. Pharmacol. 2000, 45, 85-88.

(11) Loadman, P. M.; Phillips, R. M.; Lim, L. E.; Bibby, M. C.

Pharmacological properties of a new aziridinylbenzoquinone,

RH1 (2,5- diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzo-

quinone), in mice. Biochem. Pharmacol. 2000, 59, 831-837.

(12) Xing, C. G.; Wu, P.; Skibo, E. B.; Dorr, R. T. Design of cancer-

specific antitumor agents based on aziridinylcyclopent[b]indolo-

quinones. J . Med. Chem. 2000, 43, 457-466.

(13) Zhou, R.; Skibo, E. B. Chemistry of the Pyrrolo[1,2-a]benzimi-

dazole Antitumor Agents: Influence of the 7-Substituent on the

Ability to Alkylate DNA and Inhibit Topoisomerase II. J . Med.

Chem. 1996, 39, 4321-4331.

(14) Beall, H. D.; Hudnott, A. R.; Winski, S.; Siegel, D.; Swann, E.;

Ross, D.; Moody, C. J . Indolequinone antitumor agents: Rela-

tionship between quinone structure and rate of metabolism by

recombinant human NQO1. Bioorg. Med. Chem. Lett. 1998, 8,

545-548.

(15) Naylor, M. A.; Swann, E.; Everett, S. A.; J affar, M.; Nolan, J .;

Robertson, N.; Lockyer, S. D.; Patel, K. B.; Dennis, M. F.;

Stratford, M. R. L.; Wardman, P.; Adams, G. E.; Moody, C. J .;

Stratford, I. J . Indolequinone antitumor agents: Reductive

activation and elimination from (5-methoxy-1-methyl-4,7-dioxo-

indol-3-yl)methyl derivatives and hypoxia-selective cytotoxicity

in vitro. J . Med. Chem. 1998, 41, 2720-2731.

(16) Beall, H. D.; Winski, S.; Swann, E.; Hudnott, A. R.; Cotterill, A.

S.; O’Sullivan, N.; Green, S. J .; Bien, R.; Siegel, D.; Ross, D.;

Moody, C. J . Indolequinone antitumor agents: Correlation

between quinone structure, rate of metabolism by recombinant

human NAD(P)H:quinone oxidoreductase, and in vitro cytotox-

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

(17) Phillips, R. M.; Naylor, M. A.; J affar, M.; Doughty, S. W.; Everett,

S. A.; Breen, A. G.; Choudry, G. A.; Stratford, I. J . Bioreductive

activation of a series of indolequinones by human DT-diapho-

rase: Structure-activity relationships. J . Med. Chem. 1999, 42,

4071-4080.

(18) Zimmer, H.; Lankin, D. C.; Horgan, S. W. Oxidations with

Potassium Nitrosodisulfonate (Fremy’s Radical). The Teuber

Reaction. Chem. Rev. 1971, 71, 229-246.

(19) Skibo, E. B.; Islam, I.; Schulz, W. G.; Zhou, R.; Bess, L.; Boruah,

R. The Organic Chemistry of the Pyrrolo[1,2-a]benzimidazole

Antitumor Agents. An Example of Rational Drug Design. Synlett

1996, 297-309.

Ack n ow led gm en t. We thank the National Insti-

tutes of Health, the National Science Foundation, and

the Arizona Disease Control Research Commission for

their generous support. We also thank Professor David

Ross for the generous supply of NSC 460 recombinant

DT-diaphorase.

(20) Liu, R.; Zhang, P.; T. Gan, T.; Cook, J . M. Regiospecific

Bromination of 3-Methylindoles with NBS and Its Application

to the Concise Synthesis of Optically Active Unusual Tryp-

tophans Present in Marine Cyclic Peptides. J . Org. Chem. 1997,

62, 7447-7456.

(21) Schulz, W. G.; Nieman, R. A.; Skibo, E. B. Evidence for DNA

Phosphate Backbone Alkylation and Cleavage by Pyrrolo[1,2--

3562 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22

Skibo et al.

a]benzimidazoles, Small Molecules Capable of Causing Sequence

Specific Phosphodiester Bond Hydrolysis. Proc. Natl. Acad. Sci.

U.S.A. 1995, 92, 11854-11858.

(36) Craigo, W. A.; LeSueur, B. W.; Skibo, E. B. Design of Highly

Active Analogues of the Pyrrolo[1,2-a]benzimidazole Antitumor

Agents. J . Med. Chem. 1999, 42, 3324-3333.

(22) Hargreaves, R. H. J .; Mayalarp, S. P.; Butler, J .; McAdam, S.

R.; O’Hare, C. C.; Hartley, J . A. Cross-linking and sequence

specific alkylation of DNA by aziridinyl quinones. 2. Structure

requirements for sequence selectivity. J . Med. Chem. 1997, 40,

357-361.

(37) Skibo, E. B.; Xing, C.; Groy, T. Recognition and Cleavage at the

DNA Major Groove. Bioorg. Med. Chem. 2001, 9, 2445-2459.

(38) Paull, D. K.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,

D. A.; Rubinstein, L.; Plowman, J .; Boyd, M. R. Display and

Analysis of Differential Activity of Drugs Against Human Tumor

Cell Lines: Development of Mean Graph and COMPARE

Algorithm. J . Natl. Cancer Inst. 1989, 81, 1088-1092.

(39) Boyd, M. R. Status of the NCI Preclinical Antitumor Drug

Discovery Screen. Princ. Pract. Oncol. (PPO Updates) 1989, 3

(10).

(40) Winski, S. L.; Swann, E.; Hargreaves, R. H. J .; Butler, J .; Moody

C. J .; Ross, D. Relationship Between NQO1 Levels in a Series

of Stably Transfected Cell Lines and Susceptibility to Antitumor

Quinones. Biochem. Pharmacol. 2001, 61, 1509-1516.

(41) Butler, J .; Spanswick, V. J .; Cummings, J . The Autoxidation of

the Reduced Forms of EO9. Free Radical Res. 1996, 25, 141-

148.

(42) Butler, J . Redox cycling antitumor drugs. In DNA and Free

Radicals: Techniques, Mechanisms & Applications; Aruoma, O.

I., Halliwell, B., Eds.; Oica International: Micoud, 1998; pp 131-

159.

(43) Fitzsimmons, S. A.; Workman, P.; Grever, M.; Paull, K.; Ca-

malier, R.; Lewis, A. D. Reductase enzyme expression across the

National Cancer Institute Tumor cell line panel: correlation with

sensitivity to mitomycin C and EO9. J . Natl. Cancer Inst. 1996,

88, 259-269.

(23) Alley, S. C.; Brameld, K. A.; Hopkins, P. B. DNA Interstrand

Cross-Linking by 2,5-Bis(1-aziridinyl)-1,4-benzoquinone: Nucle-

otide Sequence Preferences and Covalent Structures of the dG-

to-dG Cross-Links at 5′-d(GN_nC) in Synthetic Oligonucleotide

Duplexes. J . Am. Chem. Soc. 1994, 116, 2734-2741.

(24) Rauth, A. M.; Goldberg, Z.; Misra, V. DT-diaphorase: Possible

roles in cancer chemotherapy and carcinogenesis. Oncol. Res.

1997, 9, 339-349.

(25) Rauth, A. M.; Melo, T.; Misra, V. Bioreductive therapies: An

overview of drugs and their mechanisms of action. Int. J . Radiat.

Oncol. Biol. Phys. 1998, 42, 755-762.

(26) Stratford, I. J .; Workman, P. Bioreductive drugs into the next

millennium. Anti-Cancer Drug Des. 1998, 13, 519-528.

(27) Siegel, D.; Beall, H.; Senekowitsch, C.; Kasai, M.; Arai, H.;

Gibson, N. W.; Ross, D. Bioreductive Activation of Mitomycin C

by DT-Diaphorase. Biochemistry 1992, 31, 7879-7889.

(28) Cummings, J .; Spanswick, V. J .; Tomasz, M.; Smyth, J . F.

Enzymology of mitomycin C metabolic activation in tumour

tissue: Implications for enzyme-directed bioreductive drug

development. Biochem. Pharmacol. 1998, 56, 405-414.

(29) J affar, M.; Naylor, M. A.; Robertson, N.; Stratford, I. J . Targeting

hypoxia with a new generation of indolequinones. Anti-Cancer

Drug Des. 1998, 13, 593-609.

(30) Skibo, E. S.; Gordon, S.; Bess, L.; Boruah, R.; Heileman, J .

Studies of Pyrrolo[1,2-a]benzimidazole Quinone DT-Diaphorase

Substrate Activity, Topoisomerase II Inhibition Activity, and

DNA Reductive Alkylation. J . Med. Chem. 1997, 40, 1327-1339.

(31) Fersht, A. R. The Hydrogen Bond in Molecular Recognition-

.Trends Biochem. Sci. 1987, 12, 301-304.

(32) Beall, H. D.; Mulcahy, R. T.; Siegel, D.; Traver, R. D.; Gibson,

N. W.; Ross, D. Metabolism of Bioreductive Antitumor Com-

pounds by Purified Rat and Human DT-Diaphorase. Cancer Res.

1994, 54, 3196-3201.

(33) Faig, M.; Bianchet, M. A.; Talalay, P.; Chen, S.; Winski, S.; Ross,

D.; Amzel, L. M. Structures of recombinant human and mouse

NAD(P)H:quinone oxidoreductases: species comparison and

structural changes with substrate binding and release. Proc.

Natl. Acad. Sci. U.S.A. 2000, 97, 3177-82.

(34) Huang, X.; Suleman, A.; Skibo, E. B. Rational Design of Pyrrolo-

[1,2-a]benzimidazole Based Antitumor Agents Targeting the

DNA Major Groove. Bioorg. Chem. 2000, 28, 324-337.

(35) Skibo, E. B.; Lee, C. H. Facile Oxidation of Methoxide to

Formaldehyde by a Heterocyclic Quinone. J . Am. Chem. Soc.

1985, 107, 4591-4593.

(44) Griswold, D. P. Consideration of the Subcutaneously Implanted

B16 Melanoma as a Screening Model for Potential Anticancer

Agents. Cancer Chemother. Rep. 1972, 3, 315-324.

(45) Ouyang, A.; Skibo, E. B. The Iminium Ion Chemistry of Mitosene

DNA Alkylating Agents. Enriched ¹³C NMR Studies. Biochem-

istry 2000, 39, 5817-5830.

(46) Allen, M. S.; Hamaker, L. K.; Laloggia, A. J .; Cook, J . M. Entry

Into 6-Methoxy-D-(+)-Tryptophans: Stereospecific Synthesis of

1-Benzenesulfonyl-6-Methoxy-D-(+)-Tryptophan Ethyl Ester.

Synth. Commun. 1992, 22, 2077-2102.

(47) Suleman, A.; Skibo, E. B. Insights into the Mechanism and

Substrate Specificty of Human DT-Diaphorase through Molec-

ular Modeling. J . Med. Chem. 2001, submitted.

(48) Skibo, E. B.; Xing, C. Chemistry and DNA Alkylation Reactions

of Aziridinyl Quinones: Development of an Efficient Alkylating

Agent of the Phosphate Backbone. Biochemistry 1998, 37,

15199-15213.

J M010085U

Article Doi

Aziridinyl quinone antitumor agents based on indoles and cyclopent[b]indoles: Structure-activity relationships for cytotoxicity and antitumor activity

DOI: 10.1021/jm010085u

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm010085u

Full text of DOI:10.1021/jm010085u

Products guided by the article

R&D Labs maybe for 379260-70-5

Relevant to this article

Hot Product