Bisindolylmaleimides linked to DNA minor groove binding lexitropsins: Synthesis, inhibitory activity against topoisomerase I, and biological evaluation

Article abstract of DOI:10.1021/jm950465d

The synthesis, characterization, inhibitory activity against topoisomerase I, and biological evaluation of a series of oligopeptide-substituted bisindolylmaleimides 7-12 are described. Compounds 7-9, which contain a basic C-terminus function such as (dimethylamino)propyl and bind to DNA with C₅₀ values of 200, 160, and 135 μM, respectively, exhibited inhibition of topoisomerase I in a concentration dependent manner. Also, the relative order of observed topoisomerase I inhibition is 9 > 8 > 7 at ≤ 100 μM concentration, corresponding to the increase of the number of pyrrole units in the oligopeptide moiety. Compounds 10-12, which contain an electrostatically neutral moiety, such as methyl ester, did not bind to DNA templates nor inhibit topoisomerase I. However, the cytotoxicity activities of these compounds were 1.5 times greater than those of compounds 7-9.

Full text of DOI:10.1021/jm950465d

J . Med. Chem. 1996, 39, 1049-1055
1049
Bisin d olylm a leim id es Lin k ed to DNA Min or Gr oove Bin d in g Lexitr op sin s:
Syn th esis, In h ibitor y Activity a ga in st Top oisom er a se I, a n d Biologica l
Eva lu a tion
Guojian Xie,† Rajan Gupta,† Kevin Atchison,‡ and J . William Lown*,†
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2, and SynPhar Laboratories Inc.,
#2 Taiho Alberta Center, 4290 - 91A Street, Edmonton, Alberta, Canada T6E 5V2
Received J une 23, 1995X
The synthesis, characterization, inhibitory activity against topoisomerase I, and biological
evaluation of a series of oligopeptide-substituted bisindolylmaleimides 7-12 are described.
Compounds 7-9, which contain a basic C-terminus function such as (dimethylamino)propyl
and bind to DNA with C50 values of 200, 160, and 135 µM, respectively, exhibited inhibition of
topoisomerase I in a concentration dependent manner. Also, the relative order of observed
topoisomerase I inhibition is 9 > 8 > 7 at e100 µM concentration, corresponding to the increase
of the number of pyrrole units in the oligopeptide moiety. Compounds 10-12, which contain
an electrostatically neutral moiety, such as methyl ester, did not bind to DNA templates nor
inhibit topoisomerase I. However, the cytotoxicity activities of these compounds were 1.5 times
greater than those of compounds 7-9.
In tr od u ction
inhibit the catalytic activity of isolated topoisomerases
(both I and II), while at low concentrations distamycin
and netropsin are also able to stimulate enzymatic
activity.7 Minor groove binding effects on isolated
enzymes parallel the influence of such agents on induc-
tion of cleavable complex formation in nuclei by topoi-
somerase inhibitors.7,8 Therefore, we reasoned that the
attachment of an effector of the enzyme-DNA complex
to a minor groove vector should selectively deliver the
effector to the minor groove of unique DNA sequences
and thereby possibly influence the potency and selectiv-
ity of the antitumor agent.
To achieve this goal, bisindolylmaleimide, a derivative
of indolocarbazole, has been conjugated to novel DNA
minor groove binders, lexitropsins (i.e., information-
reading analogues of netropsin and distamycin). This
paper describes the synthesis and topoisomerase I
inhibitory function of a series of these lexitropsin
containing bisindolylmaleimides. This study reveals the
markedly high potencies of some new compounds and
also examines the effects of changing certain structural
features of the lexitropsin moieties. The first is the
consequence of increasing the inhibitory activity of the
drugs by increasing the number of pyrrole units. The
second structural feature is the type of C-terminus
group chosen. The lexitropsin C-terminus, either meth-
yl ester or dimethyl amino propyl amide, differ in two
respects. The methyl ester has a neutral function, while
dimethylamino would be protonated at physiological pH
of 7.4 to provide favorable electrostatic attraction to the
overall negative electrostatic potential of the DNA.
These and other factors possibly affecting cytotoxicity
are discussed.
DNA topoisomerases are ubiquitous enzymes that
perform essential cellular functions involved in replica-
tion, recombination, and packaging and unfolding of
DNA in chromatin.1 In addition to their normal cellular
functions, both topoisomerase I and topoisomerase II
have recently emerged as important cellular targets for
chemical intervention in the development of anticancer
agents. Eukaryotic topoisomerase I is the target for the
antitumor plant alkaloid camptothecin2 and its syn-
thetic derivatives such as CPT-113 and topotecans.4
Recently certain synthetic derivatives of indolocarbazole
antibiotics have been found to exhibit anticancer activity
in vivo, and this activity correlates with their topoi-
somerase I inhibition activity.5 The studies on several
new camptothecin and indolocarbazole derivatives in-
dicate that the antitumor activity is correlated with
their abilities to induce topoisomerase I mediated DNA
cleavage in vitro.6 Thus the identification of new drugs
which induce cleavable complex formation with topoi-
somerase I is now viewed as a promising approach to
develop clinically effective antitumor agents.
Indolocarbazoles KT6006 and KT6528 (Figure 1)
represent a distinct class of in vivo active mammalian
DNA topoisomerase I inhibitory antitumor drugs.5 In
contrast to camptothecin and VP-16-based agents that
do not interact with the DNA template,7 the indolocar-
bazoles intercalate.5 This property implies that the
indolocarbazoles may interact with the template sig-
nificantly in the process of inhibiting topoisomerase I.
Thus functionalizing the indolocarbazoles so as to
promote their DNA binding and sequence selectivity
appears to be a worthwhile objective.
Syn th esis
A number of minor groove binding drugs (distamycin,
netropsin, Hoechst 33258, and DAPI) have proven to
The target conjugates 7-12 were synthesized by
condensation of the bisindolylmaleimide moiety, as its
carboxylic acid, with the amino function of distamycin
analogues (Figure 2). As a typical reaction sequence,
the synthesis of compound 7 is outlined in Scheme 1.
We recently established an efficient synthetic route to
the staurosporine aglycon,9 in which compound 13 was
* Address correspondence to: J . W. Lown, Department of Chemistry,
University of Alberta, Edmonton, Alberta, Canada T6G 2G2. Tel. 403-
492-3646; Fax 403-492-8231.
† University of Alberta.
‡ SynPhar Laboratories Inc.
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1049$12.00/0 © 1996 American Chemical Society

1050 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Xie et al.
F igu r e 1. Structures of camptothecin and staurosporine analogues.
converted to the desired carboxylic acid function. Sub-
sequently, conversion of the anhydride to the imide was
achieved by heating with ammonium acetate overnight,
yielding the key intermediate 16 in good yield. Finally,
16 was coupled with the amine-containing oligopeptide
1, prepared from N-methylpyrrole, according to the
method previously developed in this laboratory,11 in the
presence of 1,3-dicyclohexylcarbodiimide (DCC) and
4-(dimethylamino)pyridine (DMAP) to give the target
compound 7 in a reasonable yield. Similarly, coupling
of 16 with other oligopeptides 2-6 afforded the products
8-12 in moderate to good yields.
Resu lts a n d Discu ssion
The inhibitory activity of these novel conjugates
toward the topoisomerase I relaxation reaction was
evaluated by both agarose gel electrophoresis (Table 1)
and an ethidium bromide fluorescence end point assay
(Table 2).12
F igu r e 2. Structures of lexitropsin carriers (1-6) used in this
study and the corresponding bisindolylmaleimide-lexitropsin
conjugates (7-12).
The ability of these compounds to inhibit topoi-
somerase I was quantified by measuring the action on
supercoiled pBR322 DNA substrate as a function of
increasing concentration of the ligands (Figure 3a).
Supercoiled DNA (Figure 3a, lanes 1 and 2) was
converted to relaxed DNA in the presence of topoi-
somerase I (Figure 3a, lanes 3 and 4). For comparison,
camptothecin, a recognized inhibitor of topoisomerase
I, was included. As expected, the supercoiled DNA
remaining in the assay system increased with the
increasing concentration of camptothecin. The percent-
age of supercoiled DNA remaining was determined by
densitometry (Figure 3b). Also, it was observed that
conveniently prepared in two steps from the easily
available dibromomaleic acid. In the present study, we
took 13 as the starting material and treated it with
sodium hydride, followed by addition of ethyl bromoac-
etate, to obtain 14 in a moderate yield. Since the
deprotection of the N-benzyl group has not proved to
be possible,9,10 we utilized an indirect method.9 Thus,
treatment of 14 with strong base, 5 M potassium
hydroxide, readily afforded the anhydride 15 in high
yield, and simultaneously, the ester group of 14 was
Sch em e 1. Synthetic Route to Representative Bisindolylmaleimide-Lexitropsin Conjugatesa
a
Reaction condition: (a) NaH in THF then BrCH2CO2Et; (b) 5 M KOH; (c) NH4OAc, 140 °C; (d) 1, DCC, DMAP.

Bisindolylmaleimides Linked to Lexitropsins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1051
Ta ble 1. Effects of the Bisindolylmaleimides on Topoisomerase
I DNA Relaxation Activity, Estimated from Agarose Gel
Electrophoresis
Ta ble 2. Effects of the Bisindolylmaleimides on Topoisomerase
I DNA Relaxation Activity, Estimated from Ethidium Bromide
Assay
compound
concn, µM
topo I inhibna
% changea
camptothecin
50
100
25
+
Topo I (+)
Topo I (-)
BH AH
+
compound
concn, µM
BH
AH
7
0
50
0
camptothecin
25
50
75
100
25
50
100
250
25
-6
+12
+9
+7
+7
+9
+3
+6
0
-
-
-
100
250
25
+/-
-
-
+
-
8
+/-
+3
-
-
50
+
7
+3
+1
+12
+7
100
250
25
++
+5
-1
+++
-6
0
-6
-3
9
+
-10
-7
+3
+3
-4
-15
-9
-3
-7
-15
+13
+18
+18
+12
+9
+13
+7
+12
+3
+6
+9
0
-18
-2
+2
50
++
8
+5
100
250
25
+++
50
-10
-18
-14
-11
-15
-20
-14
+1
-14
-21
-32
-28
-26
-24
-28
-2
-3
++++
100
250
25
-12
-14
-6
10
11
12
0
0
0
0
0
0
0
0
0
0
0
0
50
9
100
250
25
50
-15
-13
-4
+15
+12
+14
+17
+4
+15
+15
+10
+14
+17
+17
+20
100
250
25
50
10
11
12
100
250
25
50
100
250
50
-1
-3
100
250
25
+3
+6
+1
+2
-3
+5
50
+2
+3
100
250
25
50
100
250
+1
+2
+24
+1
+33
+11
+25
+33
+52
a
Topoisomerase I inhibition was estimated from the agarose
gel electrophoresis results. The scoring system is as follows:
<10%, 10-25%, 25-50%, 50-75%, and 75-100% for +/-, +, ++,
+++, and ++++, respectively.
+9
+15
+36
+3
a
Percent change: BH, before heat denaturation of pBR322
DNA; AH, after heat denaturation of pBR322 DNA. (-) and (+)
values indicate decrease and increase in fluorescence, respectively.
Topo I (+) indicates the presence of enzyme in the reaction
mixture. Topo I (-) indicates absence of enzyme, i.e., only DNA
and compounds in the reaction mixture. Excitation and emission
wavelengths were 525 and 600 nm, respectively.
ligands having an N,N-dimethylpropyldiamine side
chain exhibited topoisomerase I inhibition which in-
creased with the number of N-methyl-2-formamido-4-
pyrrolamides in the molecule. Thus the relative order
of the observed topoisomerase I inhibition is 9 > 8 > 7
at e100 µM concentration corresponding to the increase
in the number of pyrrole units in the oligopeptide units.
It should be noted, however, that at much higher
extreme concentrations (250 µM) anomalous behavior
was observed (Figure 3b). Most likely these compounds
which were shown to bind to DNA (vide infra) inhibit
the binding of topoisomerase I to DNA. Further con-
firmation for the lack of binding of the agents 10, 11,
and 12 (vide infra) with DNA was observed in control
samples (Figure 3a, lanes 36-40), whereby in the
absence of topoisomerase I, the percentage of super-
coiled DNA remained unaltered.
Since a topoisomerase inhibitor can cause a reduction
in topoisomerase-DNA/ethidium bromide fluorescence,
the ethidium bromide fluorescence end point assay is a
useful method for quantitative determination of topoi-
somerase inhibition. These novel conjugates caused a
significant reduction in DNA/ethidium bromide fluores-
cence, below control levels (both in the presence and
absence of topoisomerase I; Table 2) in a manner which
was concentration dependent and proportional to the
number of N-methyl-2-formamido-4-pyrrolamides in the
molecule. The maximum decrease in fluorescence was
about 30% below control levels for compound 9 at a
concentration of 25 µM and remained unaltered at
higher concentrations tested. Since these compounds
were incubated together with the DNA for 30 min prior
to the addition of ethidium bromide solution, it could
be expected that their binding to the DNA minor groove
interferes with ethidium bromide intercalation, result-
ing in the concentration dependent fluorescence reduc-
tion. The fluorescence values returned to approximate
Ta ble 3. Cytotoxicities to KB (ATCC CCL 17) Cells in Culture
compound
TD50a, µg/mL
compound
11
TD50a, µg/mL
7
8
9
84.0 ( 0.4
92.3 ( 0.5
>100 ( 0.5
53.2 ( 0.3
58.0 ( 0.3
77.3 ( 0.4
0.01
12
camptothecin
10
a
Concentration required to reduce KB cell population by 50%.
Values given are an average of three of more determinations.
control levels after heat denaturation of the DNA
treated with the topoisomerase I inhibitors. This de-
crease in fluorescence is due to irreversible heat dena-
turation of open circular DNA (OC DNA), because the
alkaline pH of ethidium bromide assay medium (pH 12)
prevents self-annealing of OC DNA so that it is only
the covalently closed circular DNA (CC DNA) which
readily renatures upon cooling. This net loss of duplex
DNA is observed as a decrease in fluorescence. We,
however, observed a significant increase in fluorescence.
It is reasonable to conclude that the heat-denatured
DNA released the minor groove binders, and upon
cooling and renaturation of the CCC DNA, more ethid-
ium bromide intercalated the DNA, resulting in an
increased fluorescence. Among the compounds with the
neutral carbomethoxy side chain (10-12), 10 and 11
had little effect on DNA fluorescence other than a slight
increase after heating, consistent with no binding.
Compound 12 caused an increase in fluorescence prior
to heating. This increased fluorescence was substan-
tially reduced after heat denaturation. Agarose gel

1052 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Xie et al.
F igu r e 3. (a) Topoisomerase I relaxation assays as described in methodology; 0.5 unit of topoisomerase I was used in each
assay. Lane 1 and 2: supercoiled pBR322 DNA, no enzyme, no drug. Lanes 3 and 4: no drug. Lanes 5-9: 25, 50, 75, 100, and
125 µM camptothecin, respectively. Lanes 10-13: 250, 100, 50, and 25 µM of 7, respectively. Lanes 14-17: 25, 50, 100, and
250 µM of 8, respectively. Lanes 18-21: 25, 50, 100, and 250 µM of 9, respectively. Lanes 22-33: 25, 50, 100, and 250 mM of
10, 11, and 12, respectively. Lanes 35-40: no enzyme, 250 µM of 7, 8, 9, 10, 11, and 12, respectively. (b) Densitometric analysis
of percent supercoiled DNA remaining as a function of increased concentration of the ligands.
(
)
F igu r e 4. Fluorescence values of ethidium and lambda DNA (0.7 at A260) complex in the presence of compounds 7-9 at
concentrations indicated. The fluorescence was measured at 605 nm with excitation at 540 nm.
electrophoresis results confirmed that there was no
DNA nicking by these compounds.
The percent decrease in fluorescence seen with added
drug concentrations permitted the calculation of C50
values for compounds 7-9 as 200, 160, and 135 µM,
respectively. The increase in observed DNA binding
from 7 to 9 is in accord with the increasing number of
N-methylpyrrole units in the lexitropsin carrier from 1
to 3. While ethidium quenching is often a significant
factor in drug-DNA binding studies, minor groove
binding drugs show low levels of quenching in relation
The next step was to determine the comparative
binding ability of compounds 7-12. This was ac-
complished by determining the micromolar drug con-
centration necessary to reduce the fluorescence of
initially DNA-bound ethidium by 50% (i.e., C50 value17)
(Figure 4). The lower the C50 value, the greater the
binding ability of the drug under the assay conditions.

Bisindolylmaleimides Linked to Lexitropsins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1053
THF (2 mL) with exclusion of moisture (argon). After 30 min,
ethyl bromoacetate (137.4 mg, 0.78 mmol) was added at 0 °C,
the mixture was stirred at room temperature for 2 h under
argon, and then concentrated to dryness. The residue material
was taken up in ethyl acetate. After washing with brine,
drying (Na2SO4), and removal of the solvent, purification of
the crude product by column chromatography, eluting with
CHCl3-EtOAc-Et3N (97:2:1), gave 199 mg (51% yield) of the
to ethidium displacement that quenching can be ig-
nored.17 Compounds 10, 11, and 12 showed no evidence
of DNA binding.
The inability of the ligands having methyl esters (10-
12) to interfere with the action of topoisomerase I
resulted in complete relaxation of the supercoiled DNA
by the enzyme (Figure 3a, lanes 22-33). Overall, these
results indicate that the interaction between the binding
moiety of ligands (7-9) and DNA interferes with the
action of topoisomerase I. The significance of structure-
activity relationship could be deduced by a recent
study13 indicating that tyrphostin derivatives not only
block protein kinase but also bind to topoisomerase I.
It has also been shown previously that genistein, which
blocks tyrosine kinase, also blocks topoisomerase II.14
However, these studies could not elaborate whether the
antiproliferative effect of these derivatives is due to their
effect on either kinase or topoisomerase inhibition or
both. Our recent communication15 and the present
study indicate that while compounds of this type,
containing a template-binding dimethylamine moiety as
a peptide terminus, inhibit either kinase or topoi-
somerase I, compounds 10-12 containing nonbinding
methyl ester inhibit only protein kinase activity. The
anticancer cytotoxic potency of the compounds 10-12
exhibited approximately 1.5 times greater activity than
the former group (7-9). This is perhaps expected
because compounds with a neutral side chain (10-12)
can penetrate the cellular membrane more easily.
While more potent kinase inhibitors are evidently more
effective against the particular KB cancer cell line, the
ability of compounds 7-9 to act in parallel on cellular
targets inhibiting both kinase and topoisomerase I
activity may serve for the development of more selective
chemotherapeutic agents. Such studies are in progress
and will be reported in due course.
1
product 14 as a red solid: mp 94-95 °C; H NMR (CDCl3) δ
1.25 (3 H, d, J ) 7.5 Hz), 4.25 (2 H, q, J ) 7.5 Hz), 4.87 (2 H,
s), 4.89 (2 H, s), 6.68-6.75 (13 H, m), 7.72 (1 H, d, J ) 3.0
Hz), 7.75 (1 H, s), 8.50 (1 H, br); HRMS (EI) calcd for
C31H23O4N3 501.168 85, found 501.168 65.
3-[1-(Car boxym eth yl)-3-in dolyl]-4-(3-in dololy)-2,5-fu r an -
d ion e (15). To a solution of ester 14 (98 mg, 0.195 mmol) in
5 mL of EtOH at 0 °C was added 2 mL of 5 M KOH. The
resulting solution was stirred at room temperature for 2 h,
cooled, and acidified with 2 N HCl. The red precipitate was
collected by filtration and purified by flash chromatography,
eluting with EtOAc-CHCl-MeOH (1:1:0.8) to give 65 mg (86%
yield) of 15 as a red solid: mp 143-144 °C; 1H NMR δ 5.22 (2
H, s), 6.70 (2 H, t, J ) 7.5 Hz), 6.88-7.10 (4 H, m), 7.45 (1 H,
s), 7.46 (1 H, s), 7.97 (2 H, d, J ) 1.5 Hz), 11.02 (1 H, br);
HRMS (EI) calcd for C22H14O5N2 386.090 27, found 386.090 71.
3-[1-(Ca r boxym eth yl)-3-in d olyl]-4-(3-in d olyl)-1H-p yr -
r ole-2,5-d ion e (16). The maleic anhydride 15 (56 mg, 0.145
mmol) was heated with ammonium acetate (2 g) at 145 °C
(bath temperature) for 12 h. The mixture was allowed to cool,
water (10 mL) was added, and the mixture was extracted with
ethyl acetate. The extracts were combined, washed with
water, dried (Na2SO4), and evaporated in vacuo to give a dark
red residue which was purified by flash chromatography,
eluting with EtOAc-CHCl3-MeOH (1:1:1) to yield 46 mg (81%
yield) of 16 as a red solid: mp 155-157 °C; 1H NMR δ 5.18 (2
H, s), 6.60-6.68 (2 H, m), 6.85-7.08 (4 H, m), 7.35-7.40 (2 H,
m), 7.85 (2 H, s), 9.70 (1 H, br), 10.80 (1 H, br); HRMS (EI)
calcd for C22H15O4N3 385.106 26, found 385.106 27.
Gen er a l P r oced u r e for Cou p lin g Rea ction s betw een
Acid 16 a n d Oligop ep tid es 1-6: P r ep a r a tion of 3-[1-[[[[1-
Me t h y l-2-[[[3-(N ,N -d im e t h y la m in o )p r o p y l]a m in o ]-
ca r bon yl]-4-p yr r olyl]a m in o]ca r bon yl]m eth yl]-3-in d olyl]-
4-(3-in d olyl)-1H-p yr r ole-2,5-d ion e (7). A solution of the
nitro ester, the precursor of amino ester 1 (19 mg, 0.074 mmol),
was hydrogenated over 15 mg of 10% Pd-C in 2 mL of MeOH
and 1 mL of THF at atmospheric pressure. When TLC
indicated completion of the reduction, the catalyst was filtered
off and the solvent was evaporated in vacuo. The resulting
amine was dissolved in 2 mL of THF and cooled to 5 °C. To
this solution was added the acid 16 (26 mg, 0.06 mmol)
followed by EDCI (14.2 mg, 0.074 mmol). The mixture was
stirred at room temperature for 4 h. Five milliliters of water
was added to the solution, and the mixture was extracted with
EtOAc (2 × 2 mL). The organic layer was removed, dried on
Na2SO4, and concentrated to give a residue which was purified
by flash chromatography, eluting with EtOAc-hexane-MeOH
(3:6:1), to yield 23 mg (65% yield) of 7 as a light red solid: mp
112 °C dec; IR (CH2Cl2-MeOH) 2940, 1705, 1638, 1578, 1530,
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Fisher-
J ohns apparatus and are uncorrected. The IR spectra were
recorded on a Nicolet 7199 FT spectrophotometer, and only
1
the principal bands are reported. The H NMR spectra were
recorded on Bruker WH-300 and WH-400 spectrometers in
CDCl3 with tetramethylsilane as an internal standard or in
DMSO-d6. FAB (fast atom bombardment) mass spectra were
determined on an Associated Electrical Industries (AEI) MS-9
and MS-50 focusing high-resolution mass spectrometers. Kie-
selgel 60 (230-400 mesh) of E. Merck was used for TLC, with
the solvent system indicated in the procedure. TLC plates
were visualized by using UV light or 2.5% phosphmolybdic acid
in methanol with heating.
All reagents obtained commercially were used without
further purification unless otherwise stated. Ethanol and
methanol were distilled from magnesium turnings, tetrahy-
drofuran (THF) was distilled from sodium/benzophenone under
an atmosphere of dry argon, and dimethylformamide (DMF)
was distilled from barium oxide and stored over molecular
sieves (3A). Hydrogenation reactions were usually conducted
under atmospheric pressure at room temperature over a
palladium catalyst (10% on charcoal) unless otherwise speci-
fied. In atmospheric hydrogenation, a hydrogen balloon was
used to furnish a constant source of hydrogen. In high-
pressure hydrogenation experiments, a Parr shaker or high-
pressure autoclave was used. As a necessary criterion of
homogeneity and purity, it was determined for each new
compound by TLC analysis prior to the determination of
accurate molecular mass by high-resolution spectrometry.
3-[1-[(Eth oxyca r bon yl)m eth yl]-3-in d olyl]-4-(3-in d olyl)-
1-(p h en ylm eth yl)p yr r ole-2,5-d ion e (14). A solution of ma-
leimide 13 (326 mg, 0.78 mmol) in 5 mL of THF was added to
a stirred suspension of sodium hydride (57 mg, 1.92 mmol) in
1
1465, 1387, 1339, 745 cm-1; H NMR δ 1.60 (2 H, m), 2.18 (6
H, s), 2.23 (2 H, t, J ) 7.5 Hz), 3.15 (2 H, q, J ) 7.5 Hz), 5.08
(2 H, s), 6.55-6.70 (4 H, m), 6.89-7.04 (3 H, m), 7.10 (1 H, d,
J ) 2.0 Hz), 7.35 (1 H, s), 7.37 (1 H, s), 7.68 (1 H, d, J ) 2.0
Hz), 7.90 (1 H, s), 8.08 (1 H, t, J ) 6.0 Hz), 10.30 (1 H, s),
10.90 (1 H, s), 11.15 (1 H, s); HRMS (FAB) calcd for
C33H33O4N7H (MH+) 592.2672, found 592.2652.
Sp ectr oscop ic a n d An a lytic Da ta on Bisin d olylm a le-
im id es. 3-[1-[[[[1-m et h yl-2-[[[1-m et h yl-2-[[[1-m et h yl-2-
[[[3-(N,N-d im eth yla m in o)p r op yl]a m in o]ca r bon yl]-4-p yr -
r o ly l]a m in o ]c a r b o n y l]-4-p y r r o ly l]a m in o ]c a r b o n y l]-
m eth yl]-3-in d olyl]-4-(3-in d olyl)-1H-p yr r ole-2,5-d ion e (8).
Prepared from 2 (32.5 mg, 0.086 mmol) and 16 (28 mg, 0.072
mmol) as a red solid in 58% yield as described above for 7: mp
138 °C dec; IR (CH2Cl2-MeOH) 3253, 3054, 2979, 2938, 1707,
1645, 1577, 1465, 1401, 1339, 1264, 1107, 744 cm-1; 1H NMR
δ 1.65 (2 H, p, J ) 7.5 Hz), 2.23 (6 H, s), 2.39 (2 H, t, J ) 7.5
Hz), 3.20 (2 H, q, J ) 7.5 Hz), 3.78 (3 H, s), 3.84 (3 H, s), 5.08
(2 H, s), 6.58-6.70 (3 H, m), 6.80 (1 H, s), 6.85-7.04 (4 H, m),
7.18-7.20 (2 H, m), 7.70 (1 H, d, J ) 2.0 Hz), 7.90 (1 H, s),

1054 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Xie et al.
8.08 (1 H, t, J ) 7.5 Hz), 9.88 (1 H, s), 10.38 (1 H, s), 10.92 (1
H, s), 11.15 (1 H, s); HRMS (FAB) calcd for C39H39O5N9H
(MH+) 714.3152, found 714.3148.
respectively. Fluorescence was recorded both before (BH) and
after (AH) heat denaturation of DNA.
Dr u g-DNA Bin d in g. C50 values of drugs were determined
as described.17 The assay was performed at pH 5.0 (9.3 mM
NaCl, 2 mM NaOAc buffer, pH 5.0, 0.1 mM EDTA, and 1.26
µM ethidium bromide) so that the added drugs exist in cationic
form.
3-[1-[[[[1-Meth yl-2-[[[1-m eth yl-2-[[[1-m eth yl-2-[[[3-(N,N-
d im eth yla m in o)p r op yl]a m in o]ca r bon yl]-4-p yr r olyl]a m i-
n o]car bon yl]-4-pyr r olyl]am in o]car bon yl]-4-pyr r olyl]am i-
n o]ca r bon yl]m eth yl]-3-in d olyl]-4-(3-in d olyl)-1H-p yr r ole-
2,5-d ion e (9). Prepared from 3 (37 mg, 0.075 mmol) and 16
(25 mg, 0.065 mmol) as a red solid in 52% yield as described
above for 7: mp 154 °C dec; IR (CH2Cl2-MeOH) 3245, 1706,
Cell Cu lt u r e Cyt ot oxicit y Assa y. In vitro cytotoxicity
assay of compounds was performed using KB cancer cell line
(ATCC CCL 17).18 KB cells were cultivated in Eagle’s mini-
mum essential medium supplemented with 10% calf serum
and incubated in a humidified 5% CO2 atmosphere at 37 °C.
Cells were counted on a Neubauer hemocytometer and seeded
at 100 µL of 3 × 103 cells per mL per well and allowed to
culture for 24 h. Test compounds were added in triplicate at
different concentrations. Control wells were identical except
that the test compound was absent. After 3 days, the cells
were fixed in 25% glutaraldehyde, washed with water, dried,
and then stained with 100 µL of 0.05% crystal violet. The wells
were eluted with 0.05 M NaH2PO4-ethanol (1:1 v/v) and read
at OD450 on a multiscan spectrophotometer. TD50 values were
determined as the concentrations required to reduce KB cell
count by 50%.
1
1643, 1578, 1465, 1403, 1124, 1063 cm-1; H NMR δ 1.64 (2
H, p, J ) 7.5 Hz), 2.18 (6 H, s), 2.28 (2 H, t, J ) 7.5 Hz), 3.20
(2 H, q, J ) 7.5 Hz), 3.80-3.91 (9 H, m), 5.12 (2 H, s), 6.60-
6.72 (3 H, m), 6.81 (1 H, s), 6.92-7.05 (5 H, m), 7.15-7.40 (5
H, m), 7.72 (1 H, d, J ) 2.0 Hz), 7.92 (1 H, s), 8.10 (1 H, t, J
) 7.5 Hz), 9.90 (1 H, s), 9.94 (1 H, s), 10.48 (1 H, s), 10.95 (1
H, s), 11.18 (1 H, s); HRMS (FAB) calcd for C45H45O6N11
H
(MH+) 836.3632, found 836.3629.
3-[1-[[[[1-Meth yl-2-(m eth oxyca r bon yl)-4-p yr r olyl]a m i-
n o]ca r bon yl]m eth yl]-3-in d olyl]-4-(3-in d olyl)-1H-p yr r ole-
2,5-d ion e (10). Prepared from 4 (22 mg, 0.12 mmol) and 16
(40.5 mg, 0.105 mmol) as a red solid in 69% yield as described
above for 7: mp 91-92 °C; IR (CH2Cl2-MeOH) 3304, 1705,
1577, 1532, 1449, 1406, 1340, 1258, 1141, 1062, 794 cm-1; 1H
NMR δ 3.25 (3 H, s), 3.80 (3 H, s), 5.08 (2 H, s), 6.60-6.58 (3
H, m), 6.75 (1 H, d, J ) 2.0 Hz), 6.90-7.05 (3 H, m), 7.71 (1 H,
d, J ) 2.0 Hz), 7.89 (1 H, s), 10.38 (1 H, s), 10.90 (1 H, br),
11.65 (1 H, s); HRMS (EI) calcd for C29H23O5N5 521.169 92,
found 521.169 37.
Ack n ow led gm en t. The authors acknowledge the
National Cancer Institute of Canada and the Depart-
ment of Chemistry, University of Alberta, for support
of this research.
3-[1-[[[[1-Meth yl-2-[[[1-m eth yl-2-(m eth oxyca r bon yl)-4-
p yr r olyl]a m in o]ca r bon yl]-4-p yr r olyl]a m in o]ca r bon yl]m -
eth yl]-3-in d olyl]-4-(3-in d olyl)-1H-p yr r ole-2,5-d ion e (11).
Prepared from 5 (37 mg, 0.12 mmol) and 16 (38.5 mg, 0.10
mmol) as a red solid in 53% yield as described above for 7: mp
142 °C dec; IR (CH2Cl2-MeOH) 3204, 2948, 1758, 1632, 1548,
Refer en ces
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(11) (a) Xie, G.; Morgan, A. R.; Lown, J . W. Synthesis and DNA
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1
1423, 1104, 978 cm-1; H NMR δ 3.72 (3 H, s), 3.83 (3 H, s),
3.84 (3 H, s), 5.10 (2 H, s), 6.58-6.70 (3 H, m), 6.85 (1 H, d, J
) 2.0 Hz), 6.88-7.00 (5 H, m), 7.15 (1 H, d, J ) 2.0 Hz), 7.35-
7.40 (2 H, m), 7.45 (1 H, d, J ) 2.0 Hz), 7.69 (1 H, d, J ) 2.0
Hz), 7.90 (1 H, s), 9.92 (1 H, s), 10.38 (1 H, s), 10.90 (1 H, br),
11.65 (1 H, s); HRMS (FAB) calcd for C35H29O6N7H 644.2257,
found 644.2231.
3-[1-[[[[1-Met h yl-2-[[[1-m et h yl-2-[[[1-m et h yl-2-(m et h -
oxycar bon yl)-4-pyr r olyl]am in o]car bon yl]-4-pyr r olyl]am i-
n o]ca r b on yl]-4-p yr r olyl]a m in o]ca r b on yl]m et h yl]-3-in -
d olyl]-4-(3-in d olyl)-1H-p yr r ole-2,5-d ion e (12). Prepared
from 6 (32 mg, 0.074 mmol) and 16 (25.7 mg, 0.06 mmol) as a
red solid in 48% yield as described above for 7: mp 198 °C dec;
IR (CH2Cl2-MeOH) 3279, 1703, 1652, 1615, 1580, 1532, 1465,
1
1341, 1256, 1191, 1059 cm-1; H NMR δ 3.71 (3 H, s), 3.83 (9
H, s), 5.10 (2 H, s), 6.56-6.70 (3 H, m), 6.90-7.08 (6 H, m),
7.18 (1 H, d, J ) 2.0 Hz), 7.24 (1 H, d, J ) 2.0 Hz), 7.32-7.40
(2 H, m), 7.46 (1 H, d, J ) 2.0 Hz), 7.60 (1 H, d, J ) 2.0 Hz),
7.91 (1 H, s), 9.95 (2 H, s), 10.48 (1 H, s), 10.92 (1 H, s), 11.15
(1 H, s); HRMS (FAB) calcd for C41H35O7N9H (MH+) 766.2737,
found 766.2700.
Top oisom er a se I Rela xa tion Assa y a n d F lu or escen ce
Assa y by Eth id iu m Br om id e. The assays were done as
described previously.12,16 In brief, 0.25 µg of pBR322 DNA and
0.5 unit of topoisomerase I were incubated for 30 min at 37
°C in the presence of the ligands in a final volume of 10 µL.
Following the incubation, an equal volume of agarose gel
loading buffer5 (2 × TBA, 0.1% bromophenol blue, 0.2% SDS
and 20% glycerol) was added and incubated for another 30 min
at 37 °C prior to loading. The gel was run overnight and
stained in 0.5 µg/mL ethidium bromide solution, and the DNA
was visualized using 300 nm wavelength transiluminator and
photographed with Polaroid film. The negative was scanned
on an LKB ultroscan XL laser densitometer.
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incubated with ligands in the presence and absence of topoi-
somerase I (0.5 unit) for 30 min at 37 °C. Following the
incubation, 1.2 mL of ethidium bromide buffer (0.5 µg/mL
ethidium bromide, 20 mM K3PO4, and 0.5 mM EDTA, pH 12)
was added for fluorometry. The fluorescence was recorded on
a Perkin-Elmer Model 650-40 fluorescence spectrophotometer
with excitation and emission wavelengths of 525 and 600 nm,

Bisindolylmaleimides Linked to Lexitropsins
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J M950465D