A novel class of achiral seco-analogs of CC-1065 and the duocarmycins: Design, synthesis, DNA binding, and anticancer properties

Article abstract of DOI:10.1016/j.bmc.2004.08.051

The synthesis, DNA binding properties, and in vitro and in vivo anticancer activity of fifteen achiral seco-cyclopropylindoline (or achiral seco-CI) analogs (5a-o) of CC-1065 and the duocarmycins are described. The achiral seco-CI analogs contain a 4-hydr

Full text of DOI:10.1016/j.bmc.2004.08.051

Bioorganic & Medicinal Chemistry 12 (2004) 6221–6236
A novel class of achiral seco-analogs of CC-1065 and the
duocarmycins: design, synthesis, DNA binding, and
anticancer properties
Stanley Kupchinsky,a Sara Centioni,a Tiffany Howard,a John Trzupek,a Shane Roller,a
Virginia Carnahan,a Heather Townes,a Bethany Purnell,a Carly Price,a Heather Handl,a
Kaitlin Summerville,a Kimberly Johnson,a James Toth,a Stephen Hudson,a
Konstantinos Kiakos,b John A. Hartleyb and Moses Leea,*
aDepartment of Chemistry, Furman University, 3300 Poinsett Highway, Greenville, SC 29613, USA
bCancer Research UK Drug–DNA Interactions Research Group, Department of Oncology,
Royal Free Hospital and London Medical School, 91 Riding House St., London W1W 7BS, UK
Received 14 June 2004; revised 31 August 2004; accepted 31 August 2004
Available online 23 September 2004
Abstract—The synthesis, DNA binding properties, and in vitro and in vivo anticancer activity of fifteen achiral seco-cyclopropylin-
doline (or achiral seco-CI) analogs (5a–o) of CC-1065 and the duocarmycins are described. The achiral seco-CI analogs contain a
4-hydroxyphenethyl halide moiety that is attached to a wide range of indole, benzimidazole, pyrrole, and pyridyl-containing nonco-
valent binding components. The 4-hydroxyphenethyl halide moiety represents the simplest mimic of the seco-cyclopropylpyrroloin-
doline (seco-CPI) pharmacophore found in the natural products, and it lacks a chiral center. The sequence and minor groove
specificity of the achiral compounds was ascertained using a Taq DNA polymerase stop assay and a thermal induced DNA cleavage
experiment using either a fragment of pBR322 or pUC18 plasmid DNA. For example, seco-CI-InBf (5a) and seco-CI-TMI (5c) dem-
onstrated specificity for AT-rich sequences, particularly by reacting with the underlined adenine-N3 position of 50-AAAAA(865)-30.
This is also the sequence that CC-1065 and adozelesin prefer to alkylate. The achiral seco-CI compounds were subjected to cyto-
toxicity studies against several human (K562, LS174T, PC3, and MCF-7) and murine cancer cell lines (L1210 and P815). Following
continuous drug exposure, the achiral compounds were found to be cytotoxic, with IC50 values in the lM range. Interestingly, the
carbamate protected compound 5p was significantly less cytotoxic than agent 5c, supporting the hypothesis that loss of HCl and
formation of a spiro[2,5]cyclopropylcyclohexadienone intermediate is necessary for biological activity. The achiral seco-CI com-
pounds 5a and 5c were submitted to the National Cancer Institute for further cytotoxicity screening against a panel of 60 different
human cancer cell lines. Both compounds showed significant activity, particularly against several solid tumor cell lines. Flow cytom-
etry studies of P815 cells that were incubated with compound 5c at its IC50 concentration for 24h showed induction of apoptosis in a
large percentage of cells. Compounds 5a and 5c were selected by the NCI for an in vivo anticancer hollow-fiber test, and received
composite scores of 18 and 22, respectively. These two compounds were subsequently evaluated for in vivo anticancer activity
against the growth of a human advanced stage SC UACC-257 melanoma in skid mice. At a dose of 134mg/kg administered IP,
compound 5c gave a T/C value of 40% (for day 51), and the median number of days of doubling tumor growth was 27.7, versus
15.8 for untreated animals. For compound 5a, at 200mg/kg, the T/C was 58% and the median number of days of doubling tumor
growth was 20.0 versus 8.7 for untreated animals. At these doses no toxicity or weight loss was observed for either compound. Fur-
thermore, compound 5c was not toxic to murine bone marrow cell growth in culture, at a dose that was toxic for the previously
reported seco-CBI (cyclopropylbenzoindoline)-TMI (4).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Minor groove and AT sequence selective alkylating
agents,1a,b such as (+)-CC-1065 (1)1c–e,2 and the duocar-
mycins,2,3 exemplified by (+)-duocarmycin SA (or
DUMSA, 2) and depicted in Figure 1, belong to a group
Keywords: CC-1065; Duocarmycins; DNA alkylation; Sequence spe-
cificity; Cytotoxicity.
*
Corresponding author. Tel.: +1 864 294 3368; fax: +1 864 294
3559; e-mail: moses.lee@furman.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.051

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S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
development of novel analogs of CC-1065 and the
NH2
N
duocarmycins that effectively kill cancer cells and have
reduced toxicity to the host. In attempts to design novel
analogs of the duocarmycins and CC-1065 with reduced
toxicity to bone marrow cells, a wide range of analogs of
the cyclopropylpyrrolo[e]indolone (CPI) subunit were
synthesized and tested.1 Examples of such analogs are,
cyclopropylbenzo[e]indolone (CBI),2,14 cyclopropylpyr-
azolo[e]indolone (CPzI),15 cyclopropylfurano[e]indolone
(CFI),16 and cyclopropylindolone (CI).17 Our laborato-
ries have recently reported that seco-iso-CFI-TMI (iso-
cyclopropylfurano[e]indoline) analog 3 is endowed with
potent anticancer activity and is relatively nontoxic to
murine bone marrow cells, when compared to seco-
CBI-TMI 4.18
O
OH
N
N
OCH3
H
O
OH
H3C
OCH3
N
N
OCH3
H
O
OCH3
OCH3
N
H
N
N
CH3O2C
O
H
O
N
H
(+)-CC1065, 1
O
OCH3
OCH3
OCH3
(+)-DUMSA, 2
Cl
OCH3
OCH3
OCH3
Cl
N
N
O
O
H
N
N
H
O
OH
seco-iso-CFI-TMI, 3
OH
As part of our efforts to design novel analogs of CC-
1065 and the duocarmycins, we are undertaking a pro-
gramto investigate whether the chiral center present in
the natural products is needed for DNA sequence recog-
nition and biological activity. To date, there has been
little activity on the development of achiral analogs of
CC-1065 and the duocarmycins. One report describes
the synthesis and DNA binding studies of a bischlo-
romethyl seco-CBI-TMI analog.19 The bulky prochiral
molecule was found to alkylate DNA poorly and dem-
onstrated weak cytotoxicity due to the inefficient pro-
duction of interstrand crosslinks. Another report
described the synthesis of a spiro[2,5]cyclopropanecyclo-
hexadienone derivative, but no biochemical studies were
conducted.20
seco-CBI-TMI, 4
Cl
H
N
Cl
OCH3
O
H
O
H
N
OCH3
OCH3
N
N
N
H
O
H
O
OH
Achiral seco-CI-InBf, 5a
OH
Achiral seco-CI-TMI, 5c
Figure 1. Structures of (+)-CC-1065 (1), (+)-duocarmycin SA
(DUMSA, or 2), seco-iso-CFI-TMI (3), and seco-CBI-TMI (4).
of natural products that have potent cytotoxic proper-
ties. These compounds, which were isolated from the
fermentation broth of Streptomyces zelensis4 and Strep-
tomyces sp.,5 respectively, derive their anticancer activity
by reaction of the cyclopropylpyrroloindole or CPI moi-
ety with adenine-N3 groups in the minor groove of spe-
cific sequences of DNA. This mechanism of action was
confirmed by the isolation of adenine-CC-10656 and
adenine–duocarmycin SA products7 fromthermally
cleaved DNA-drug adducts. (+)-CC-1065 preferentially
binds to 50-PuNTTA-30 and 50-AAAAA-30 sequences
and reacts with the adenine-N3 position of the under-
lined residue.6,8 (+)-DUMSA shows similar sequence
selectivity, but, some subtle differences were observed,
such as the lack of alkylation at 50-GCAAA by CC-
1065.2,3,7 The cytotoxic potency of the CPI pharma-
cophore is directly related to its solvolytic stability.
DUMSA, one of the most cytotoxic analogs, is the
most solvolytically stable member of this class of
compounds.2,3,9
As illustrated in Figure 2, we have designed an achiral
analog 5 (seco-CI-TMI, 5c) that contains a 4-hydroxy-
phenylethyl halide functionality. Similar to the loss of
HCl in seco-CC-1065 and seco-duocarmycin compounds
in biological media,2,3 compound 5c should lose HCl to
produce the putative spiro[2,5]cyclopropanecyclohexa-
dienone DNA alkylating agent 6, which should react
with an adenine-N3 group in AT-rich sequences.
Hydroxyphenethyl halides are known to readily lose a
hydrogen halide to generate the corresponding
spiro[2,5]cyclopropanecyclohexadienones, which react
with DNA and possess cytotoxic properties.21 In this
OMe
OMe
Cl
H
N
N
OMe
H
O
5c
OH
Even though (+)-CC1065 has potent cytotoxic proper-
ties, its usefulness as an anticancer drug is hampered
by delayed lethal toxicity to the animals at therapeutic
doses.4b DUMSA is devoid of this toxicity,2a but it is
toxic to the bone marrow.5 As a result, several analogs
of CC-1065 and the duocarmycins have been developed
for clinical evaluation, including adozelesin,1b,4c,10 carz-
elesin,11 bizelesin,12 and KW2189.13 Presently, only biz-
elesin remains under clinical evaluation.12 Clinical
studies on the other three compounds were discontinued
due to adverse toxicity to the bone marrow.10–12 Conse-
quently there is a strong interest in the design and
OMe
OMe
OMe
DNA
H
N
N
H
O
O
6
H+
Figure 2. Proposed mechanism of activation and DNA alkylation of
the achiral seco-CI moiety in compound 5.

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6223
report, we will describe the synthesis of fifteen seco-CI
compounds (5a–o) along with their DNA sequence spe-
cific alkylation and anticancer properties.
Synthesis of the achiral seco-CI analogs started from
malonate diester 7, which was synthesized according to
a literature procedure.4c Hydrolysis of the esters, fol-
lowed by acid-promoted decarboxylation afforded a
substituted acetic acid intermediate in 95% yield. The
carboxylic acid functional group was reduced to the pri-
mary alcohol (8) with borane in THF in 95% yield.
Treatment of compound 8 with carbon tetrachloride
or carbon tetrabromide and triphenylphosphine
afforded the corresponding chloride 9 or bromide 10 in
excellent yields. Catalytic hydrogenation of compounds
9 and 10 with 10% Pd–C in THF gave the aminophenols
11 and 12, which were unstable and used immediately in
the subsequent step. Coupling of the aminophenols with
the corresponding carboxylic acids in presence of 3mol
equivalents EDCI in DMF gave the target compounds
5a–l,n,o in reasonable yields. For synthesis of a potential
interstrand crosslinking agent 5m, aminophenol 11 was
condensed with 5-nitrobenzofuran-2-carboxylic acid
and EDCI. The amide intermediate 5l was collected in
14% yield, which was converted to the desired com-
pound 5m in 24% yield by reduction of the nitro group
with 5% Pd–C, followed by coupling with N,N-bis-(2-
chloroethyl)aminobenzoic acid in EDCI.
2. Results and discussion
2.1. Synthesis
Fifteen novel achiral seco-CI analogs of CC-1065 and
the duocarmycins were designed and synthesized in this
study, and the structures are given in Figure 3. Com-
pounds 5a and 5b are reminiscent of adozelesin, which
contain an indole-benzofuran noncovalent binding moi-
ety. Compounds 5c–f,l are mimics of the duocarmycins,
which contain a 5,6,7-trimethoxyindole noncovalent
binding moiety. Analogs 5g–i contain a cinnamoyl
group;22 5j,k contain a substituted pyridyl group. Agent
5m was designed to test the influence of interstrand cros-
slink formation on cytotoxicity. Compounds 5n and 5o
are hybrids of achiral seco-CI and bisbenzimide23 and
netropsin,24 respectively.
X
Cl
OCH3
OCH3
Due to the interesting biological activity of achiral seco-
CI-TMI 5c, a different strategy was needed to improve
its synthetic efficiency from11% yield using the EDCI
coupling method. Selective reduction of the nitro group
of compound 9 was achieved by catalytic hydrogenation
over Adams catalyst (PtO2) and 55 PSI of hydrogen.
The resulting amine was coupled to 5,6,7-trimehtoxyin-
dole-2-carboxylic acid in presence of PyBOP and Hu-
nigÕs base. From this reaction an amide intermediate
was obtained in 66% yield. Catalytic hydrogenation of
this intermediate over 10% Pd–C afforded the desired
compound 5c in 50% yield, after silica gel column chro-
matography. The overall yield for the synthesis of 5c,
fromcompound 9, using the PyBOP method was 33%,
which is 3 times more efficient than the EDCI method.
All intermediates and products were characterized by
FT-IR, 500MHz 1H NMR, MS, and accurate mass
measurements. Elemental analysis was done for 5c.
H
N
H
R
N
N
O
H
OCH3
O
OH
O
N
N
CH3 5p
Achiral seco-CI analogs, 5a-o
O
R =
OCH3
OCH3
H
N
O
O
N
H
OCH3
N
H
X = Cl, c
X = Br, d
X = I, e
X = Cl, a
X = Br, b
OCH3
N
H
X = Cl, f
OCH3
OCH3
N
2.2. Taq polymerase stop assay
R
X = Cl, o-OMe, g
X = Br, m-OMe, h
X = I, p-OMe, i
This PCR-based assay determines the sequence selectiv-
ity of alkylation in DNA.25 This assay works on the pre-
mise that Taq DNA polymerase activity is blocked at
the site of covalent binding. Using a singly radiolabeled
primer 50-32P-50-GCATTGGTAACTGTCAGACC-30
and a PvuII linearized pBR322 plasmid DNA, the re-
gion starting from3303 was linearly amplified. Taq
DNA polymerase stop studies were performed on com-
pounds 5a, 5b, 5l, 5m, and 5o along with CC-1065 and
adozelesin, and the results are given in Figure 4. Con-
centrations of 10lM of the achiral seco-CI compounds
were used for the studies, but a concentration of
0.005lM was used for CC-1065 and adozelesin to give
similar levels of alkylation. The results clearly show that
all compounds interact strongly at a site containing a
run of seven adenine residues [50-AAAAAAA(3114)-
30]. This finding is interesting because major changes
X = Cl, R = OCH3, j
X = Cl, R = H, k
Cl
N
NO2
H
Cl
N
O
N
H
N
X = Cl, m
H
X = Cl, l
H
N
CH3
N
H
O
N(CH2CH3)2
N
N
N
H
O
CH3
N
X = Cl, n
CH3
X = Br, o
Figure 3. Fifteen analogs of achiral seco-CI prepared in this study,
5a–o, and a carbamate derivative 5p.

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S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
using a thermal cleavage experiment, as depicted in Fig-
ure 5A, thereby providing evidence of binding of 5c to
the minor groove. This gel demonstrated an additional
alkylation site for adozelesin, A(843), which was absent
for compound 5c, suggesting an improved sequence
preference for the latter compound for the DNA
sequence studied.
2.3. Molecular modeling studies
The MM2 energy minimized complex of achiral CI-InBf
(5a) with duplex 50-dGGCGGAGTTAGG-30 in the B-
formand alkylating at the adenine N3 position of the
underlined A residue, is shown in Figure 6. This oligo-
nucleotide was used by Hurley and co-workers in their
NMR and molecular modeling studies of a DNA com-
plex with CC-1065.26 As depicted, the molecule 5a fits
snugly and isohelically in the minor groove. The angular
plane between the achiral CI and InBf units exhibits a
twist of 5ꢁ in order to accommodate the natural curva-
ture of the DNA.
2.4. Cytotoxicity studies
Although it is important to characterize the interaction
of potential anticancer agents with their DNA target,
the goal is to design compounds, which are capable of
killing tumor cells. Using a MTT based growth inhibi-
tion assay, the IC50 values for compounds 5a–p against
a range of human K562 (chronic myeloid leukemia
cells), and murine L1210 leukemia and P815 mastocy-
toma cells were determined.27 Cytotoxicity studies on
LS174T (human colorectal adenocarcinoma), PC3
(human prostate adenocarcinoma), MCF7 (human
breast adenocarcinoma) were probed using a sulforhod-
amine B assay.28 The cells were treated continuously for
three days, except for the K562 cells, which were treated
for four days. The cytotoxicity of compounds 5a–p, ex-
pressed as IC50 values are given in Table 1. For compar-
ison, the reported IC50 value of CC-1065 against L1210
cells (three days exposure) was 20pM29 The results re-
veal several interesting trends. First, compounds 5a–o
inhibited the growth of the tumor cells at lM concentra-
tions. For comparison, continuous exposure of the race-
mic seco-CI-TMI (13) and seco-CI-InBf (14) [the
structures of these compounds are given in Scheme 1]
gave IC50 values of 0.10 and 0.14lM, respectively,
against the growth of K562 cells.30 Since their achiral
counterparts 5c (1.43lM) and 5a (1.47lM) retain activ-
ity, the results suggest that the chiral center in com-
pounds 13 and 14, as well as the natural products, is
not essential for cytotoxicity.
Figure 4. Taq DNA Polymerase Stop Assay using the PvuII primer;
lane 1, control DNA; lane 2, chlorambucil; lane 3, 0.005lM CC-1065;
lane 4, 0.005lM adozelesin; lane 5, compound 5l 10lM; lane 6,
compound 5a 10lM; lane 7, compound 5b 10lM; lane 8, compound
5m 10lM; lane 9, compound 5o 10lM.
to the noncovalent binding portion of the molecules had
little effect on sequence specificity. A significantly weaker
alkylation site was seen at 50-TTG(3135)A-30, with alky-
lation at the underlined G residue. It is worthy to
note that CC-1065 produced the highest level of alkyla-
tion at the weaker site. For compound 5a, an additional
weak alkylation band was observed at 50-AAAA(3173)-
30 and 50-A7G(3115)-30.
The covalent sequence specificity of compound 5c was
ascertained using a Taq DNA polymerase stop assay
using a singly radiolabeled primer 50-32P-CTCACT-
CAAAGGCGGTAATAC-30 and a HindIII linearized
pUC18 plasmid DNA. The region of the plasmid start-
ing fromposition 749 was linearly amplified. The auto-
radiograph shown in Figure 5B demonstrates that, like
adozelesin,2 compound 5c could stop the activity of
Taq DNA polymerase at sites with contiguous adenine
residues. The sequence specific covalent reaction of com-
pound 5c with the adenine-N3 position was confirmed
Second, protection of the hydroxyl group in compound
5c led to a dramatic decrease in cytotoxicity. Carbamate
5p gave IC50 values of 75lM and >100lM against
L1210 and P815 cells. In contrast, the achiral seco-CI-
TMI agent 5c produced IC50 values for these cell lines
of 1.5 and 5.6lM, respectively. These results suggest
that the achiral hydroxyphenethyl halide compounds
eliminate HCl to form spiro[2,5]cyclopropanecyclohexa-
dienone intermediates that react with DNA and elicit
the cytotoxic activity. This observation is consistent

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6225
Figure 5. (A) Thermal cleavage gel showing purine-N3 lesions on the upper strand of a region from the HindIII linearized fragment of plasmid
pUC18. Lane 1, control; lane 2, 0.1lM adozelesin; lanes 3–5 correspond to 1, 10, 100lM of compound 5c. (B) Taq DNA polymerase stop assay on
the same fragment of DNA. Lane 1, control; lane 2, 0.1lM adozelesin; lanes 3-8 correspond to 1, 3, 10, 30, 100, and 300lM of compound 5c.
with the biological activity of seco-analogs of CC-1065
and duocarmycins.2,3 Third, even though the noncova-
lent binding portions of compounds 5a–o are very differ-
ent, their IC50 values are quite similar. This discovery,
while not surprising because compounds 5a–o have very
similar DNA covalent sequence specificity, suggests that
the DNA alkylation event is responsible for the
cytotoxic activity. Lastly, comparing the cytotoxicity
of bromo (5d, 5h) and iodo (5e) compounds to the
chloro compound, the results indicate that the nature
of the leaving group has minimal effects on biological
activity.
Compounds 5a and 5c were subjected to further cyto-
toxicity testing at the National Cancer Institute, against
a panel of 60 human cancer cell lines.31 The viability of
the cells after 48h of continuous exposure of the com-
pounds was determined using a sulforhodamine B assay.
Both compounds 5a and 5c gave comparable cytotoxic-
ity results, and Figure 7 shows the LC50 (concentration
for killing 50% of the cells) values for compound 5c
against the 60 different tumor cell lines. Bars extending
to the right indicate cells more sensitive than the average
to the particular compound, whereas bars to the left
Figure 6. MM2 energy optimized (steepest descend) structure of a
indicate less sensitive cells. Several observations were
made on the results. First, they exhibit activity in the
micromolar range. Second, agents 5a and 5c are not
complex of a covalent adduct between the chloromethyl moiety of
compound 5a and adenine-N3 of the underlined residue of duplex
dGGCGGAGTTAGG in the B-form.

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S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
G0 peak33 using propidiumiodide staining. Following
a 24 h incubation of P815 cells at 10-times the IC50 con-
centration of compound 5c (56lM), the results given in
Figure 8 showed a substantial increase in the percentage
of sub-G0 stage (67%), compared to 0.7% for untreated
control cells, indicating that compound 5c was capable
of damaging DNA and inducing the cells to undergo
apoptosis. For comparison, at a concentration of
1000lM, cisplatin produced 59% of sub-G0 population
of cells, which is consistent with a literature report.33
Results fromour studies suggest that like their seco-
CC-1065 and seco-duocarmycin counterparts, achiral
seco-CI compounds are likely to exert their cytotoxic
activity through the induction of apoptosis.
Table 1. Cytotoxicity of compounds 5a–p against the growth of cancer
cells grown in culture
Compound K562 LS174T PC3 MCF7 L1210 P815
5a
5b
5c
5d
5e
5f
1.47
1.61
1.43
—
2.27
3.0
3.43
23
1.5
43
5.6
0.39
1.25
0.29
0.46 0.94
—
1.11
2.56
1.41
1.52
2.22
2.86
2.37
2.70
1.81
3.64
—
5g
5h
5i
4.04
1.14
1.62
5j
5k
5l
5m
5n
5o
5p
2.6. Hollow fiber assay for preliminary in vivo testing34
75
>100
In this study conducted at the NCI, human tumor cells
were cultivated in polyvinylidene fluoride (PVDF) hol-
low fibers, and a sample of each cell line was implanted
into each of two physiologic compartments (intraperito-
neal and subcutaneous) in mice. Each test mouse re-
ceived a total of six fibers (three intraperitoneally and
three subcutaneously) representing three distinct cancer
cell lines. A total of 12 different tumor cell lines were
used for these studies. Three mice were treated with
potential antitumor compounds (dissolved in DMSO)
at each of two test doses by the intraperitoneal route
using a QD · 4 treatment schedule. Vehicle controls
consisted of 6 mice receiving the compound diluent only.
The fiber cultures were collected on the day following
the last day of treatment. To assess anticancer effects,
viable cell mass was determined for each of the cell lines
using a formazan dye (MTT) conversion assay. From
this, the %T/C values were calculated using the average
optical density of the compound treated samples divided
by the average optical density of the vehicle controls.
The results were converted to scores of IP and SC, rep-
resenting activity against cells implanted by the two
The IC50 values are given in lM, and the cells were continuously
exposed to the compounds for 3days continuously, except 4days for
K562 cells.
indiscriminately toxic to cells, for example, they were
not active against leukemic cells. Third, these com-
pounds show activity against cells from many solid tum-
ors, and in particular 5c appears to be very active
against melanoma cells. Specific reasons for the unique
patterns of cytotoxicity for these compounds are un-
known, and experiments are underway to address this
issue.
2.5. Cell cycle studies
Duocarmycin analogs have been demonstrated to in-
duce leukemic cells to undergo apoptosis. The cells dem-
onstrated morphological abnormalities and genomic
DNA degradation associated with apoptosis.32 Flow
cytometry was used to detect apoptotic cells as a sub-
X
X
X
EtO C
2
CO Et
2
OH
NO
H
NO
2
NH
NO
2
N
2
2
R
RCO H, EDCI
2
DMF
CCl or CBr
4
H , 10% Pd/C,
2
THF
4
1. NaOH
2. HCl-THF-H O
O
Ph P
3
2
OBn
OBn
heat
OH
OBn
9, X = Cl
10, X = Br
OH
5a-j, l-o
7
3. BH -THF
3
8
11, X = Cl
12, X = Br
1. 5-nitroindole-2-
carboxylic acid
EDCI, DMF
OCH
Cl
3
1. H , PtO ,
THF, 55 PSI
2
2
OCH
3
2. H , 5% Pd/C
2
N
2. TMI-CO H
2
3. benzoic acid mustard
acid chloride, Et N,
N
H
OCH
3
PyBOP, iPr EtN,
2
Cl
3
O
CH Cl
2
2
CH Cl
2
2
3. H , 10% Pd/C,
2
13
OH
THF, NH HCO
Cl
4
2
N
H
N
H
N
Cl
OCH
Cl
Cl
O
3
O
O
OCH
3
H
N
H
N
N
N
N
H
OCH
N
H
3
H
O
O
O
5m
14
OH
OH
OH
5c
Scheme 1. Synthesis of the achiral seco-CI analogs of CC-1065 and the duocarmycins.

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6227
Figure 7. LC50 values of compound 5c against the NCIÕs panel of 60 human cancer cell lines.
routes. Compounds with a combined IP + SC score
P20, a SC score P8, or a net cell kill of one or more cell
lines were referred for xenograft testing. The composite
IP+SC score for compound 5a was 18, and cell kill was
observed. For compound 5c, the IP+SC score was 22,
and no cell kill was observed. Based on these composite
scores as well as the unique structure of the achiral
seco-CI compounds, both compounds were selected by
the NCI for further in vivo testing using human tumor
xenografts grown in nude mice.

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2.7. In vivo tumor xenograft studies35
This in vivo tumor model assay involved the subcutane-
ous implantation of the human UACC-257 melanoma
fragments (30mg) into the axillary region of pathogen-
free immunodeficient mice on day 0 of the experiment.
Drug treatments were initiated on day 10 for compound
5a and day 16 for compound 5c. In both cases the com-
pounds were dissolved in DMSO and were administered
via an IP route on a QD · 3 schedule. The volume of in-
jected was adjusted according to a ratio of 0.02mL for
every 10g of body weight. Compounds 5a and 5c were
administered at their maximum tolerated doses of 200
and 134mg/kg/dose, respectively. Tumor size and body
weights are obtained approximately two times per week.
The results for compounds 5a and 5c obtained fromthe
xenograft studies are given in Table 2. It is apparent that
both compounds were able to inhibit the growth of the
melanoma. For the achiral seco-CI-InBf compound 5a,
a T/C value was obtained at 58% at day 51. The mean
time for doubling of the tumor weight was 20.0 days,
compared to 8.7days for the control untreated animals.
For compound 5c, the T/C value was 40% at day 51,
which according the NCIs evaluation criteria is deemed
an ÔactiveÕ compound. The mean time for the tumor to
double in weight was 27.7 days, compared to 15.8 days
for the untreated control. The results provide evidence
that compound 5c possesses anticancer activity, and
more importantly, at the dose administered to the ani-
mals, no toxicity was observed. Compound 5c was also
found to be nontoxic to murine bone marrow cells in
culture at a dose that produced no colonies with the pre-
viously reported compound 4 (0.0084lM, the IC50 value
for 4 against L1210 cells).14,18
3. Conclusion
A novel class of achiral seco-CI analogs of CC-1065 and
the duocarmycins has been designed and synthesized.
Overall, the favorable cytotoxicity of the achiral seco-
CI compounds against tumor cell lines, their low toxic-
ity, and their activity against a human melanoma
xenograft strongly indicated that the chiral center pre-
sent in the CC-1065 and the duocarmycins is not needed
for antitumor activity. Furthermore, elimination of
the chiral center makes the achiral analogs a simpler
Figure 8. Propidiumiodide stained flow cytometry analysis of P815
mastocytoma cells that were incubated for 24h with compounds 5c (B)
and cisplatin (C) at 10 times their IC50 concentrations. Part A
represents untreated cells.
Table 2. In vivo anticancer properties of compounds 5a and 5c against the growth of human advanced stage SC UACC-257 melanoma grown in skid
mice
Compound
Dose
T/C (%)
Mean doubling time
Net log cell kill
5aa
200mg/kg/dose
Q4D · 3, day 10
58
(at day 51)
20.0days versus
8.7days for control
ꢀ0.60
5ca
134mg/kg/dose
Q4D · 3, day 16
40
(at day 51)
27.7days versus
15.8days for control
0.10
The compounds were administered via an intraperitoneal (IP) route.
a Both compounds did not cause significant weight loss in the animals indicating that they were relatively not toxic. Twenty skid mice were used in the
control group, and six mice were used in the test experiments. The compounds were dissolved in DMSO, and the injections were made using
volumes that corresponded to 0.02mL/10g of body weight.

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6229
platformfromwhich more active and potentially useful
medicinal compounds could be developed in the
future.36 Such studies are in progress, and the results
will be reported when they become available.
mmol). The solution was allowed to stir overnight under
a positive pressure of nitrogen and then concentrated
under reduced pressure. The brown oily residue was
purified using silica gel column chromatography and a
10% ethyl acetate, 5% CHCl3, and 85% petroleumether
solvent system. 2-(4-Benzyloxy-2-nitrophenyl)ethyl
chloride (9) was isolated as a thick yellowish oil, which
solidified upon standing in the refrigerator (1.06g,
3.64mmol, 97% yield). Mp 38–40ꢁC; TLC (20% ethyl
4. Experimental
4.1. 2-(4-Benzyloxy-2-nitrophenyl)ethanol (8)
1
acetate/hexane) Rf = 0.53; H NMR (500MHz, CDCl3)
To a round-bottomflask containing diethyl 2-(4-benzyl-
oxy-2-nitrophenyl)malonate (7)4c (2.00g, 5.17mmol)
was added EtOH (45mL) and 10% NaOH (60mL) pro-
ducing a dark brown solution, which was refluxed for
3h. The solvent was removed under reduced pressure
to forma yellow suspension and THF (30mL) was
added to produce a clear yellow solution, which was
placed in an ice bath and stirred. 6M HCl (30mL)
was slowly added to the solution to reach pH1. The light
orange solution was refluxed for another hour, at which
time two layers formed. The top THF layer was col-
lected and the aqueous was extracted twice with EtOAc
(50mL each). The combined organic extracts were dried
with anhydrous sodiumsulfate, and concentrated to
produce 4-benzyloxy-2-nitrophenylacetic acid as an or-
ange solid (1.41g, 4.91mmol, 95%). Mp 145–152ꢁC;
7.51 (d, 2.5, 1H), 7.33 (m, 5H), 7.23 (d, 8.5, 1H), 7.10
(dd, 2.5, 8.5, 1H), 5.02 (s, 2H), 3.70 (t, 7.0, 2H), 3.20
(t, 7.0, 2H); 13C NMR (125MHz, CDCl3) 158.1, 149.5,
135.7, 143.0, 128.7, 128.4, 127.5, 125.1, 120.4, 110.7,
70.6, 44.0, 35.8; IR (neat) 3092, 3038, 2962, 2919,
2865, 1618, 1570, 1532, 1499, 1451, 1343, 696; EI-MS
m/z (rel. intensity) 291 (M+, 10); isotope pattern of M+
and M++2 agrees with the presence of one chlorine
atom. Accurate mass (EI-MS) for C15H1435ClNO3:
calcd. 291.0662, obsd. 291.0667.
4.3. 2-(4-Benzyloxy-2-nitrophenyl)ethyl bromide (10)
The procedure was similar to that used for the synthesis
of compound 9, except 2-(4-benzyloxy-2-nitrophenyl)
ethanol (8) (1.00g, 3.66mmol), CBr4 and dry CH3CN
were used. Column chromatography (SiO2, 10%
EtOAc/5% CHCl3/85% petroleumether gradient elution)
afforded 2-(4-benzyloxy-2-nitrophenyl)ethyl bromide as
a brownish solid (1.33g, 3.56mmol, 92%). Mp 30–
1
TLC (CHCl3) Rf = 0.15; H NMR (CDCl3, 300MHz)
7.76 (d, 2.7, 1H), 7.41 (m, 5H), 7.26 (d, 8.7, 1H), 7.21
(dd, 2.7, 8.7, 1H), 5.13 (s, 2H), 4.00 (s, 2H); IR (CHCl3
cast) 3400–2600 (br), 3100, 1698, 1601, 1526, 1523, 1453,
1376, 1349; EI-MS m/z (rel. intensity) 287 (M+, 10).
Accurate mass (EI-MS) for C15H13NO5: calcd.
287.0794, obsd. 287.0799.
1
34ꢁC; TLC (20% EtOAc/hexane) Rf = 0.48; H NMR
(CDCl3, 500MHz) 7.52 (d, 2.5, 1H), 7.34 (m, 5H), 7.24
(d, 8.5, 1H), 7.11 (dd, 2.5, 8.5, 1H), 5.04 (s, 2H), 3.57
(t, 7.0, 2H), 3.30 (t, 7.0, 2H); 13C NMR (CDCl3,
125MHz) 158.0, 149.4, 135.6, 133.8, 128.7, 128.4,
127.5, 125.8, 120.5, 110.8, 70.6, 35.9, 32.0; IR (neat)
3070, 3027, 2919, 2855, 1618, 1570, 1526, 1499, 1451,
1408, 1381, 1343, 695; EI-MS m/z (rel. intensity) 335
(M+, 9); isotope pattern of M+ and M++2 agrees with
the presence of one bromine atom. Accurate mass (EI-
MS) for C15H14NO3Br: calcd. 335.0157, obsd. 335.0160.
4-Benzyloxy-2-nitrophenylacetic acid (6.38g, 22.2mmol)
was dissolved in freshly distilled and dry THF (60mL)
was kept under an atmosphere of N2 and stirred in an
ice bath. Borane (55.6mL, 55.5mmol, 1M solution in
THF) was slowly added to the solution, producing much
effervescence. The red solution was left stirring in the ice
bath for 10min and then allowed to stir at room temper-
ature for 4h. Water (150mL) was slowly added to the
flask. The THF layer was collected, and the aqueous
layer was extracted twice with EtOAc (75mL each).
The combined organic extracts were dried with anhy-
drous sodiumsulfate and concentrated to give a brown
oil. Column chromatography (silica gel, 10–60% EtOAc/
5% CHCl3/hexane gradient elution) afforded 2-(4-benz-
yloxy-2-nitrophenyl)ethanol (8) (5.72g, 21.0mmol,
95%) as a brownish solid. Mp 48–52ꢁC; TLC (10%
4.4. 2-(2-Amino-4-hydroxyphenyl)ethyl chloride (11)
Pd/C 10% (45mg) was added to 2-(4-benzyloxy-2-nitro-
phenyl)ethyl chloride(132mg, 0.453mmol). The mixture
was suspended in chilled THF (30mL), degassed and
purged three times with hydrogen, and then reduced
under atmospheric pressure at room temperature. After
3h the solution the solution was filtered by suction over
Celite. The filtrate was concentrated under reduced
pressure to give 2-(2-amino-4-hydroxyphenyl)ethyl
chloride as a white solid (60.7mg, 0.354mmol, 78%).
Mp 96–100ꢁC; TLC (10% MeOH/CHCl3) Rf = 0.34;
1H NMR (CDCl3 in 1 drop DMSO-d6, 500MHz) 8.20
(br s, 1H), 6.85 (d, 8.0, 1H), 6.27 (dd, 2.5, 8.0, 1H),
6.26 (d, 2.5, 1H), 4.00 (br s, 2H), 3.66 (t, 8.5, 2H),
2.90 (t, 8.5, 2H); IR (neat) 3139, 3020, 2954, 1622,
1594, 1513, 1464, 1560, 1306, 722; EI-MS m/z (rel. inten-
sity) 171 (M+, 28); isotope pattern of M+ and M++2
agrees with the presence of one chlorine atom. Accurate
mass (EI-MS) for C8H10ClNO: calcd. 171.0451, obsd.
171.0444.
1
MeOH/CHCl3) Rf = 0.61; H NMR (CDCl3, 500MHz)
7.54 (d, 2.5, 1H), 7.42 (m, 5H), 7.31 (d, 8.5, 1H), 7.17
(dd, 2.5, 8.5, 1H), 5.10 (s, 2H), 3.91 (br q, 6.5, 2H),
3.65 (br t, 6.5, OH), 3.10 (t, 6.5, 2H); IR (neat) 3400,
3050, 2965, 2921, 2858, 1622, 1523, 1453, 1344. EI-MS
m/z (rel. intensity) 273 (M+, 14). Accurate mass (EI-
MS) for C15H15NO4: calcd. 273.1001, obsd. 273.0992.
4.2. 2-(4-Benzyloxy-2-nitrophenyl)ethyl chloride (9)
Triphenylphosphine (1.98g, 7.55mmol), CCl4 (2.18mL,
22.62mmol), and dry CH2Cl2 (13.7mL) were added to
2-(4-benzyloxy-2-nitrophenyl)ethanol (8) (1.03g, 3.77

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S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
4.5. 2-(2-Amino-4-hydroxyphenyl)ethyl bromide (12)
7.5, 2H), 3.12 (t, 7.5, 2H); IR (neat) 3346, 3120, 2943,
1660, 1616, 1589, 1512, 1423, 1414, 1305, 739, 619;
FAB-MS (NBA) m/z (rel. intensity) 438 (M+H+ꢀHBr,
3). Accurate mass for C26H20N3O4: calcd. 438.1454,
obsd. 438.1440.
The procedure was similar to that used for the synthesis
of compound 11, except 2-(4-benzyloxy-2-nitrophen-
yl)ethyl bromide (540mg, 1.6mmol) was used. 2-(2-
Amino-4-hydroxyphenyl)ethyl bromide was isolated as
an off-white solid (540mg, 2.50mmol, 100%). Mp 160–
4.8. 4-(2-Chloroethyl)-3-(5,6,7-trimethoxyindole-2-car-
boxamino)phenol (5c)
1
166ꢁC; TLC (10% MeOH/CHCl3) Rf = 0.27; H NMR
(CDCl3 in 1 drop DMSO, 500MHz) 7.21 (br s, 1H),
7.14 (d, 8.0, 1H), 7.10 (d, 2.0, 1H), 6.87 (dd, 2.0, 8.0),
3.83 (t, 7.5, 2H), 3.17 (t, 7.5, 2H); IR (neat) 3335,
3008, 2921, 1622, 1561, 1502, 1464, 1442, 698; EI-MS
m/z (rel. intensity) 135 (M+ꢀHBr, 85). Accurate mass
(EI-MS) for C8H9NO: calcd. 135.0684, obsd. 135.0678.
A mixture of 2-(2-amino-4-hydroxyphenyl)ethyl chlo-
ride (11) (449mg, 1.72mmol), 5,6,7-trimethoxyindole-
2-carboxylic acid (429.0mg, 1.71mmol), and EDCI
(987.7mg, 5.16mmol) was dissolved in dry DMF
(15mL). The solution was stirred under a N2 atmos-
phere at roomtemperature for 3days. The DMF was re-
moved using a Kugelrohr apparatus (60ꢁC, 1mmHg),
and the oily residue was partitioned between CHCl3
(200mL) and water (75mL). The organic layer was dried
(Na2SO4), filtered, and concentrated. Purification of the
residue by column chromatography (1–5% MeOH/
CHCl3 gradient elution) afforded compound 5c as an
off-white solid (77.8mg, 11% yield). Mp = 48–50ꢁC;
TLC (10% MeOH/CHCl3) Rf = 0.43; 1H NMR
(500MHz, CDCl3) 9.71 (s br, 1H), 8.32 (s, 1H), 7.99
(d, 2.5, 1H), 7.18 (s br, 1H), 7.09 (d, 8.5, 1H), 6.95 (d,
2.0, 1H), 6.86 (s, 1H), 6.72 (dd, 2.5, 8.5, 1H), 4.11 (s,
3H), 3.96 (s, 3H), 3.91 (s, 3H), 3.83 (t, 6.0, 2H), 3.09
(t, 6.0, 2H); IR (KBr) 3362, 3062, 2958, 2916, 2854,
1649, 1540, 1503, 1457, 1410, 1374, 1306, 798, 756;
FAB-MS (NBA) m/z (rel. intensity) 404 (M+, 2), 405
(M+H+, 3). Accurate mass for C20H22N2O535Cl2: calcd.
405.1217, obsd. 405.1216. Anal. Calcd for
C20H21N2O5Cl: C, 59.33; H, 5.23; N, 6.92. Found: C,
59.56; H, 5.33; N, 7.13.
4.6. 4-(2-Chloroethyl)-3-[5-(5-benzofuran-2-carboxam-
ido)indole-2-carboxamido]phenol (5a)
2-(2-amino-4-hydroxyphenyl)ethyl chloride (11), pro-
duced fromcatalytic hydrogenation of compound
10
(0.4068g, 1.40mmol) with 10% Pd/C (0.200g), was
mixed with 5-(Benzofuran-2-carboxamido)indole-2-
carboxylic acid (0.36g, 1.12mmol) and EDCI (0.81g,
4.20mmol). The mixture was dissolved in dry DMF
(15mL) and stirred under a N2 atmosphere at room tem-
perature for 4days. The DMF was removed using a Ku-
gelrohr apparatus (40ꢁC, 0.1mmHg). The oily residue
was dissolved in chloroform(200mL) and washed with
water (50mL) and 5% sodium bicarbonate (50mL).
The organic layer was collected, dried with sodiumsul-
fate, and concentrated under reduced pressure. The
product was purified on a silica gel column. The column
was eluted with ethyl acetate (0–50%)/chloroformto
give the product as an off-white solid (299mg,
0.632mmol, 28%). Mp = 233–235ꢁC; TLC (10%
MeOH/CHCl3) Rf = 0.37; 1H NMR (DMSO-d6,
500MHz) 11.74 (d, 1.5, 1H, 1H), 10.44 (s, 1H), 9.87
(s, 1H), 9.48 (s, 1H), 8.17 (d, 1.0, 1H), 7.83 (d, 8.0,
1H), 7.76 (s, 1H), 7.73 (d, 8.5, 1H), 7.58 (dd, 2.0, 8.5,
1H), 7.51 (dt, 1.0, 8.0, 1H), 7.44 (d, 8.0, 1H), 7.38 (t,
8.0, 1H), 7.33 (d, 1.0, 1H), 7.17 (d, 9.0, 1H), 6.79 (d,
2.5, 1H), 5.67 (dd, 2.5, 9.0 1H), 3.75 (t, 7.5, 2H),2.98
(t, 7.5, 2H); IR (neat) 3401, 3259, 3041, 2965, 2921,
1643, 1594, 1534, 1446, 1301, 739; UV–vis (ethanol)
4.8.1. An alternative synthesis of 5c. A mixture of 2-(4-
benzyloxy-2-nitrophenyl)ethyl
chloride
(1.00g,
3.44mmol), platinum oxide (PtO2) (200mg) and chilled
THF (50mL) was degassed and purged with H2 three
times, and allowed to shake at 55 PSI for an hour.
The solution was filtered over Celite, concentrated,
and the residue was coevaporated twice with dry CH2Cl2
(5mL each). The resulting 2-(2-amino-4-benzyloxyphen-
yl)ethyl chloride was dissolved in dry CH2Cl2 (50mL),
and added to a stirring suspension of 5,6,7-trim-
ethoxyindole-2-carboxylic acid (961mg, 3.83mmol)
218
M
(e = 1.1 · 106 Mꢀ1 cmꢀ1),
290
(e = 1.7 · 104
ꢀ1 cmꢀ1); FAB-MS (NBA) m/z (rel. intensity) 474
(M+H+, 2). Accurate mass for C26H21N3O435Cl: calcd.
474.1221, obsd. 474.1219.
and
benzotriazol-1-yloxy-tripyrrolidinophosphonium
hexafluorophosphate (PyBOP) (2.00g, 3.84mmol) in
dry CH2Cl2 (440mL). The reaction mixture was flushed
with N2 and allowed to stir at roomtemperature for
10min. Dry N,N-diisopropylethylamine (1.5mL,
8.6mmol) was added, and the solution was stirred over-
night at roomtemperature. The solution was diluted
with 100mL of CH2Cl2 and washed once with water
(100mL), once with 10% HCl (1M) (100mL), once with
saturated aqueous NaHCO3 (100mL) and once with
saturated NaCl (100mL). The organic layer was dried
with anhydrous sodiumsulfate, and concentrated. The
product was purified using a 16:1 CH2Cl2/EtOAc silica
gel column. The product (benzyl 4-(2-chloroethyl)-3-
(5,6,7-trimethoxyindole-2-carboxamino)phenyl ether)
was isolated as a light yellow foamy solid (1.13g,
2.29mmol, 66%). TLC (16:1 CH2Cl2/EtOAc) Rf = 0.38;
4.7. 4-(2-Bromoethyl)-3-[5-(5-benzofuran-2-carboxam-
ido)indole-2-carboxamido]phenol (5b)
The procedure was similar to that used for the synthesis
of compound 5a, except 2-(4-benzyloxy-2-nitrophen-
yl)ethyl bromide (300mg, 0.893mmol) was used. Prod-
uct 5b was isolated as an off-white solid (8.1mg,
0.016mmol, 2%). Mp = 285–290ꢁC; TLC (10% MeOH/
CHCl3) Rf = 0.50; 1H NMR (DMSO-d6, 500MHz)
11.70 (s, 1H), 10.45 (s, 1H), 9.32 (s, 1H), 8.18 (d, 1.5,
1H), 7.83 (d, 8.0, 1H), 7.76 (s, 1H), 7.73 (dd, 1.0, 8.0,
1H), 7.60 (dd, 2.0, 8.5, 1H), 7.51 (dt, 1.0, 8.0, 1H),
7.47 (d, 8.5, 1H), 7.38 (dt, 1.0, 8.0, 1H), 7.15 (d, 2.0
1H), 7.07 (d, 8.0, 1H), 6.47 (dd, 2.0, 8.0, 1H), 4.52 (t,

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6231
1
1H NMR (500MHz CDCl3) 9.19 (s, 1H), 8.16 (s, 1H),
7.62 (d, 3.0, 1H), 7.45 (d, 7.5, 2H), 7.39 (t, 7.5, 2H),
7.33 (t, 7.5, 1H), 7.15 (d, 8.5, 1H), 6.90 (s, 1H), 6.85
(dd, 3.0, 8.5, 1H), 6.84 (s, 1H), 5.09 (s, 2H), 4.08 (s,
3H), 3.94 (s, 3H), 3.91 (s, 3H), 3.83 (t, 6.5, 2H), 3.10
(t, 6.5, 2H); IR (neat) 3296, 3073, 2970, 2924, 2850,
1751, 1709, 1644, 1579, 1537, 1499, 1467, 1411, 1304,
797, 732; EI-MS m/z (rel. intensity) 494 (M+, 20), 458
(M+ꢀHCl, 100).
500MHz H NMR (CDCl3) 9.48 (s, 1H), 7.97 (s, 1H),
7.67 (d, 2.0, 1H), 7.09 (d, 8.0, 1H), 6.96 (d, 2.0,
1H), 6.86 (s, 1H), 6.72 (dd, 2.0, 8.0, 1H), 6.28 (s br,
1H), 4.11 (s, 3H), 3.95 (s, 3H), 3.91 (s, 3H), 3.38
(t, 6.5, 2H), 3.22 (t, 6.5, 2H); IR (Neat) 3274, 3079,
2928, 2848, 1696, 1643, 1589, 1541, 1497, 1465, 1412,
1364; FAB-MS (NBA) m/z (rel. intensity) 497 (M+H+,
2), 496 (M+, 1). Accurate mass for C20H21N2O5I: calcd.
496.0495, obsd. 496.0501.
To a flask containing benzyl 4-(2-chloroethyl)-3-(5,6,7-
trimethoxyindole-2-carboxamino)phenyl ether (2.17g,
4.38mmol) was added an aqueous solution of 25%
NH4HCO2 (17.60mL, 0.0697mmol) and THF
(100mL). The solution was allowed to stir in an ice bath
for 10min and Pd/C 10% (450mg) was added. The mix-
ture was stirred under an atmosphere of H2 at room
temperature overnight. The reaction mixture was filtered
over Celite and the filtrate was concentrated. The result-
ing yellowish oil was purified using a silica gel column,
beginning with 0.5% MeOH/CHCl3. The MeOH per-
centage was increased by 0.5% increments every
100mL. The product was collected and concentrated
on a concentrated to produce compound 5c as a white
foamy solid (880mg, 2.17mmol, 50%).
4.11. 4-(2-Chloroethyl)-3-(5-methoxyindole-2-carbox-
amino)phenol (5f)
The procedure was similar to that used for the synthesis
of compound 5a, except 5-methoxyindole-2 carboxylic
acid (180mg, 0.941mmol) was used. Compound 5f was
obtained as an off-white residue (29mg, 0.0842mmol,
10%). Mp = 64–70ꢁC; TLC (1% MeOH/CHCl3)
Rf = 0.42; 1H NMR (500MHz, CDCl3) 9.08 (s, 1H),
8.26 (s, 1H), 7.56 (d, 2.5, 1H), 7.36, (d, 9.0, 1H), 7.12
(d, 8.5, 1H), 7.10 (d, 2.0, 1H), 7.01 (dd, 3.0, 8.0, 1H),
6.95 (s, 1H), 6.73 (dd, 3.0, 8.0, 1H), 5.38 (s, 1H), 3.87
(s, 3H), 3.84 (t, 6.5, 2H), 3.11 (t, 6.5, 2H); IR (neat)
3290, 3050, 2957, 2916, 1653, 1618, 1534, 1508, 1472,
1451, 715; EI-MS m/z (rel. intensity) 344 (M+, 20), 308
(M+ꢀHCl, 100); Accurate mass for C18H17N2O335Cl:
calcd. 334.0928, obsd. 344.0914.
4.9. 4-(2-Bromoethyl)-3-(5,6,7-trimethoxyindole-2-car-
boxamino)phenol (5d)
4.12. 4-(2-Chloroethyl)-3-(2-methoxycinnamoylam-
ido)phenol (5g)
A solution of compound 5c (58mg, 0.14mmol) and lith-
ium bromide (39mg, 0.45mmol) in dry DMF (2mL) and
was stirred under a N2 atmosphere at 60ꢁC for 4days.
At that time, ethyl acetate (50mL) was added to the
reaction mixture. The solution was washed with water
(2 · 50mL) and dried (Na2SO4). The residue was puri-
fied by preparative TLC (0.5% MeOH/CHCl3) to give
the desired product 5d as a yellowish film(10mg,
The procedure was similar to that used for the synthesis
of compound 5a, except 2-methoxycinnamic acid
(168mg, 0.942mmol) was used. Agent 5k was isolated
as an off-white foamy solid (70.4mg, 0.213mmol,
25%). Mp = 70ꢁC; TLC (10% MeOH/CHCl3) Rf =
1
0.61; H NMR (500MHz, CDCl3) 8.01 (d, 16.0, 1H),
1
16%). TLC (2% MeOH/CHCl3) Rf = 0.35. 500MHz H
7.53 (d, 6.5, 1H), 7.49 (s br, 1H), 7.36 (t, 7.0, 1H),
7.27 (s, 1H), 7.08 (d, 8.0, 1H), 6.98 (t, 7.5, 1H), 6.93
(d, 7.5, 1H), 6.69 (dd, 2.0, 6.5, 1H), 6.68 (d, 16.0, 1H),
5.35 (s br, 1H), 3.91 (s, 3H), 3.76 (t, 6.5, 2H), 3.05 (t,
6.5, 2H); IR (neat) 3253, 3071, 3011, 2925, 2839, 1648,
1614, 1541, 1485, 1459, 753; EI-MS m/z (rel. intensity)
331 (M+, 2), 295 (M+ꢀHCl, 35); Accurate mass for
C18H18NO335Cl: calcd. 331.0975, obsd. 331.0970.
NMR (CDCl3) 9.42 (s, 1H), 8.23 (s, 1H), 7.66 (s, 1H),
7.64 (s br, 1H), 7.03 (d, 8.5, 1H), 6.87 (d, 2.0, 1H),
6.77 (s, 1H), 6.65 (dd, 2.0, 8.5, 1H), 4.04 (s, 3H), 3.88
(s, 3H), 3.85 (s, 3H), 3.77 (t, 6.5, 2H), 3.04 (t, 6.5, 2H);
IR (Neat) 3317, 3054, 2957, 2931, 2852, 1712, 1651,
1616, 1537, 1506, 1462, 1410, 1370, 1339, 1305, 735,
699; EI-MS m/z (rel. intensity) 449 (M+H+, <1), 368
(MꢀHBr, 65). Accurate mass for C20H20N2O5: calcd.
368.1372, obsd. 368.1373.
4.13. 4-(2-Chloroethyl)-3-(3-methoxycinnamoylam-
ido)phenol (5h)
4.10. 4-(2-Iodoethyl)-3-(5,6,7-trimethoxyindole-2-carbox-
amino)phenol (5e)
The procedure was similar to that used for the synthesis
of compound 5a, except 3-methoxycinnamic acid
(168mg, 0.942mmol, 25%) was used. Product 5h was
obtained as a light yellow solid (45.9mg; total yield
100.6mg, 0.303mmol, 35%). Mp = 220ꢁC; TLC (10%
A solution of compound 5c (50mg, 0.12mmol) and so-
dium iodide (25mg, 0.17mmol) in dry acetone (4.0mL,
over Molecular sieves 3A) was heated to reflux under
a Drierite drying tube overnight. The solvent was re-
moved under reduced pressure, and the residue was dis-
solved in CH2Cl2 (50mL), washed with water (10mL),
and then dried over anhydrous sodiumsulfate. Removal
of the solvent gave a brown oil, which was purified by
preparative silica gel TLC (5% MeOH/CHCl3). The de-
sired product 5e was obtained as an off-white film
(3.5mg, 6%). TLC (5% MeOH/CHCl3) Rf = 0.39.
1
MeOH/CHCl3) Rf = 0.64; H NMR (500MHz, CDCl3)
7.73 (d, 16.0, 1H), 7.48 (s br, 1H), 7.32 (t, 8.0, 1H),
7.31 (s, 1H), 7.22 (d, 8.0, 1H), 7.10 (d, 8.5, 1H), 7.08
(d, 2.0, 1H), 6.94 (dd, 2.0, 8.0, 1H), 6.70 (d, 7.0, 1H),
6.53 (d, 16.0, 1H), 4.90 (s, 1H), 3.85 (s, 3H), 3.77 (t,
6.5, 2H), 3.05 (t, 6.5, 2H); IR (nujol) 3356, 3252, 2727,
1709, 1640, 1597, 1541, 718; EI-MS m/z (rel. intensity)
331 (M+, 5), 295 (M+ꢀHCl, 32).

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S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
4.14. 4-(2-Chloroethyl)-3-(4-methoxycinnamoylam-
ido)phenol (5i)
1616, 1545, 1462, 1379, 1326, 739; FAB-MS (NBA)
m/z (rel. intensity) 360 (M+H+, 4). Accurate mass for
C17H15N3O435Cl: calcd. 360.0751, obsd. 360.0762.
The procedure was similar to that used for the synthesis
of compound 5a, except 4-methoxycinnamic acid
(168mg, 0.942mmol) was used. The product 5i was ob-
tained as a light tan/yellow solid (23.3mg; total yield
74.2mg, 0.22mmol, 26%). Mp = 210–213ꢁC; TLC
(10% MeOH/CHCl3) Rf = 0.60; 1H NMR (500MHz,
CDCl3) 8.60 (s br, 1H), 8.08 (s br, 1H), 7.58 (d, 16.0,
1H), 7.48 (m, 2H), 6.97 (d, 8.0, 1H), 6.84 (m, 2H),
6.62 (d br, 7.0, 1H), 6.49 (d br, 16.0, 1H), 3.76 (s, 3H),
3.62 (t, 7.5, 2H), 2.95 (t, 7.5, 2H); IR (nujol) 3295,
3183, 3063, 1644, 1588, 1545, 1511, 696; EI-MS m/z
(rel. intensity) 331 (M+, 3), 295 (M+, 29).
A suspension of compound 5l (250mg, 0.69mmol) and
10% Pd–C (187mg) in chilled THF (20mL) was hydro-
genated at atmospheric pressure and room temperature
overnight. The suspension was filtered over a pad of Cel-
ite, and the filtrate was concentrated to give a yellow
solid. To this solid was added p-N,N-bis-(2-chloro-
ethyl)aminobenzoic acid (219mg, 0.84mmol), EDCI
(436mg, 2.28mmol) and freshly distilled and dry DMF
(10mL). The reaction mixture was stirred under a N2
atmosphere and at room temperature for two days.
The reaction mixture was concentrated using a Kugel-
rohr apparatus (0.1mmHg, 60ꢁC), and the residue was
partitioned in chloroformand water. The water layer
was further extracted twice with ethyl acetate. The com-
bined organic layers were dried over anhydrous sodium
sulfate and concentrated. The residue was purified by
silica gel column chromatography (0–10% methanol in
chloroform). Compound 5m was obtained as a white
solid (96mg, 24%). Mp 80–90ꢁC; TLC (10% MeOH/
CHCl3) Rf = 0.52; 1H NMR (DMSO-d6, 500MHz)
11.65 (d, 1.0, 1H), 9.84 (s, 1H), 9.49 (S, 1H), 8.12 (d,
2.0, 1H), 7.90 (d, 9.0, 2H), 7.51 (dd, 2.0, 8.5, 1H), 7.39
(d, 9.0, 1H), 7.30 (d, 1.0, 1H), 7.17 (d, 8.5, 1H), 6.85
(d, 9.0, 2H), 6.78 (d, 2.5, 1H), 6.68 (dd, 2.5, 8.5, 1H),
3.78 (m, 8H), 3.75 (t, 7.5, 2H), 2.97 (t, 7.5, 2H); IR (neat)
3390, 3019, 2965, 2921, 2856, 1643, 1600, 1539, 1513,
1464, 1447, 1256, 1229, 1180, 1153, 1093, 1044, 1017,
870, 799; UV–vis (ethanol) 220 (e = 1.1 · 106 Mꢀ1 cmꢀ1),
310 (e = 3.8 · 104 Mꢀ1 cmꢀ1); FAB-MS (NBA) m/z (rel.
intensity) 573 (M+H+, 1). Accurate mass for
C28H28N4O335Cl3: calcd. 573.1227, obsd. 573.1232.
4.15. 4-(2-Chloroethyl)-3-(2,6-dimethoxy-5-pyridyl)-E-
ethen-1-ylcarboxamidophenol (5j)
The procedure was similar to that used for the synthesis
of compound 5a, except 3-(2,6-dimethoxy-5-pyr-
idyl)acrylic acid (245mg, 1.09mmol) was used. Com-
pound 5j was isolated as a light yellow solid (234mg,
74%). Mp = 153–155ꢁC; TLC (10% MeOH/CHCl3)
1
Rf = 0.55; H NMR (500MHz, CDCl3) 7.80 (d, 15.5,
1H), 7.69 (d, 8.0, 1H), 7.53 (s br, 1H), 7.44 (s br, 1H),
7.08 (d, 8.5, 1H), 6.68 (dd, 1.0, 8.0, 1H), 6.61 (d, 15.5,
1H), 6.36 (d, 8.0, 1H), 5.91 (s br, 1H), 4.05 (s, 3H),
3.96 (s, 3H), 3.75 (t, 7.5, 2H), 3.04 (t, 7.5, 2H); IR (nujol)
3273, 3180, 1652, 1594, 1483, 665; FAB-MS m/z (rel.
intensity) 363 (M+H+, 7). Accurate mass for
C18H20N2O4Cl: calcd. 363.1112: obsd. 363.1118.
4.16. 4-(2-Chloroethyl)-3-(6-methoxy-5-pyridyl)-E-ethen-
1-ylcarboxamidophenol (5k)
The procedure was similar to that used for the synthesis
of compound 5a, except 3-(6-methoxy-5-pyridyl)acrylic
acid (0.215g, 1.10mmol) was used. The product 5k
was obtained as an off-white solid. Mp 180ꢁC. TLC
4.18. 4-(2-Chloroethyl)-3-[2-(4-N,N-(diethyl)aminophen-
yl)benzimidazole-6-carboxamido]phenol (5n)
The procedure was similar to that used for the synthesis
of compound 5a, except 2-(4-(N,N-diethyl)aminophen-
yl)benzimidazole-6-carboxylic acid (0.350g, 1.13mmol)
was used. Compound 5n was isolated as a white solid
(70mg, 13% yield). Mp = 223ꢁC; TLC (10% MeOH/
1
(10% MeOH/CHCl3) Rf = 0.50. H NMR (DMSO-d6)
8.85 (s, 1H), 8.32 (s, 1H), 8.00 (s, 1H), 7.91 (s br, 1H),
7.88 (dd, 1.5, 8.0), 7.72 (d, 15.1, 1H), 7.00 (d, 8.0, 1H),
6.80 (d, 15.1, 1H), 6.52 (dd, 1.5, 8.0, 1H), 4.28 (br t,
2H), 3.97 (s, 3H), 3.15 (br t, 2H); IR (nujol) 3095,
1646, 1602, 1593, 1495, 747; FAB-MS (NBA) m/z (rel.
intensity) 297 (M+HꢀHCl, 6). Electrospray MS m/z
(rel. intensity) 333 (M+H+, 5), 297 (M+H+ꢀHCl, 100).
1
CHCl3) Rf = 0.43; H NMR (500MHz, CDCl3 and 2
drops of DMSO-d6) d 8.78 (s br, 1H), 8.65 (s br, 1H),
8.22 (s, 1H), 8.14 (d, 9.0, 2H), 7.83 (d, 8.5, 1H), 6.76
(d, 9.0, 2H), 6.73 (dd, 2.0, 8.0, 1H), 3.77 (t, 6.5, 2H),
3.45 (q, 7.5, 4H), 3.07 (t, 7.5, 2H), 1.23 (t, 7.5, 6H); IR
(nujol) 3408, 1607, 1493, 1379, 1306, 798, 663; FAB-
MS (NBA) m/z (rel. intensity) 463 (M+H+, 5). Accurate
mass for C26H28N4O235Cl2: calcd. 463.1901, obsd.
463.1901.
4.17. 4-(2-Chloroethyl)-3-[[5-(4-bis-(2-chloroethyl)-
amino)benzamido]indole-2-carboxamido]phenol (5m)
The intermediate 5l was prepared using a procedure sim-
ilar to that used for the synthesis of 5a, except 5-nitroin-
dole-2-carboxylic acid (372mg, 1.81mmol) was used.
4-(Chloroethyl)-3-(5-nitroindole-2-carboxamido)phenol
5l was isolated as a yellow solid (70mg, 14%). Mp 290–
4.19. N-(2-(2-Bromoethyl)-4-hydroxyphenyl)-1-methyl-4-
(1-methyl-4-butanamido pyrrole-2-carboxamido)pyrrole-
2-carboxamide (5o)
1
300ꢁC; TLC (10% MeOH/CHCl3) Rf = 0.43; H NMR
(CDCl3, 500MHz) 10.98 (s br, 1H), 8.87 (s br, 1H),
8.67 (d, 2.0, 1H), 8.59 (s, 1H), 8.17 (dd, 2.0, 9.0,
1H), 7.55 (d, 9.0, 1H), 7.20 (d, 2.5, 1H), 7.11 (d, 8.5,
1H), 6.77 (dd, 2.5, 8.5, 1H), 3.78 (t, 7.5, 2H), 3.08
(t, 7.5, 2H); IR (neat) 3391, 3261, 2929, 2858, 1658,
The procedure was similar to that used for the synthesis
of compound 5b, except N-methyl-4-(N-methyl-4-buta-
namidopyrrole-2-carboxamide)-pyrrole-2-carboxylic-
acid (830mg, 2.50mmol) was used. The product 5o
was obtained as an off-white foamy solid (230mg,

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6233
0.462mmol, 40%). Mp 138–150ꢁC; TLC (10% MeOH/
CHCl3) Rf = 0.13; H NMR (CDCl3 in 1 drop DMSO-
were washed once with drug-free medium. Following
the appropriate drug treatment, the cells were trans-
ferred to 96-well microtitre plates, 104 cells per well, 8
wells per sample. Plates were then kept in the dark at
37ꢁC in a humidified atmosphere containing 5% CO2.
The assay is based on the ability of viable cells to reduce
a yellow soluble tetrazoliumsalt, 3-4,5-dimethylthiazol-
2,5-diphenyltetrazolium bromide (MTT, Sigma Chemi-
cal Co.) to an insoluble purple formazan precipitate.
Following incubation of the plates for 4days (to allow
control cells to increase in number by 10 fold) 20lL of
a 5mg/mL solution of MTT in phosphate buffered saline
was added to each well and the plates further incubated
for 5h. The plates were then centrifuged for 5min at
300g and the bulk of the medium pipetted from the cell
pellet leaving 10–20lL per well. DMSO (200lL) was
added to each well and the samples agitated to ensure
complete mixing. The optical density was then read at
a wavelength of 550nmon a Titertek Multiscan ELISA
plate reader, and the dose–response curve was con-
structed. For each curve, an IC50 value was read as
the dose required to reduce the final optical density to
50% of the control value.
1
d6, 500MHz) 9.85 (s, 1H), 9.74 (s, 1H), 9.22 (s, 1H),
7.39 (br s, 1H), 7.33 (d, 2.0, 1H), 7.14 (d, 2.0, 1H),
7.02 (d, 8.0, 1H), 6.87 (d, 2.0, 1H), 6.69 (d, 2.0,
1H), 6.41 (dd, 2.5, 8.0, 1H), 4.22 (t, 8.5, 2H), 3.83
(s, 3H), 3.73 (s, 3H), 2.99 (t, 8.0, 2H), 2.21 (t, 7.0, 2H),
1.59 (sextet, 7.5, 2H), 0.895 (t, 7.5, 3H); IR (Nujol)
3368, 3270, 3125, 1632, 1600, 728; FAB-MS (NBA) m/z
(rel. intensity) 450 (M+H+ꢀHBr, 10), 449 (450ꢀH+,
10). Accurate mass (FAB-MS) for C24H28N5O4: calcd.
450.2141, obsd. 450.2147, for C24H27N5O4: calcd.
449.2063, obsd. 449.2082.
4.20. O-4-(2-Chloroethyl)-3-(5,6,7-trimethoxyindole-2-
carboxamino)phenyl-N-methylpiperazinyl carbamate (5p)
A solution of compound 5c (70mg, 0.171mmol) in dis-
tilled dry THF (1.5mL) and dry CH2Cl2 (30mL) was
chilled in an ice bath. Dry triethylamine (0.048mL,
0.342mmol) and p-nitrophenyl chloroformate (86mg,
0.428mmol, dissolved in 5mL of CH2Cl2) were added.
The solution was allowed to stir at 0ꢁC under a N2
atmosphere for three hours. 4-Methylpiperazine
(0.057mL, 0.51mmol) was added to the solution and
the reaction mixture was allowed to stir under N2 for
5h. At that time, the solvent was removed under re-
duced pressure and the resulting yellow residue was dis-
solved in CHCl3 and purified on a silica gel column
using CHCl3/MeOH (0–2.5%) as the solvent. Carbamate
5p was isolated as a white solid (78.3mg, 0.147mmol,
86%). Mp = 84ꢁC; TLC (2.5% MeOH/CHCl3)
Rf = 0.25. 500MHz 1H NMR (CDCl3) 9.09 (s, 1H),
8.12 (s, 1H), 7.65 (d, 2.5, 1H), 7.16 (d, 8.0, 1H), 6.94
(dd, 2.5, 8.0, 1H), 6.83 (d, 2.0, 1H), 6.78 (s, 1H), 4.02
(s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.77 (t, 6.5, 2H),
3.67 (m, 2H), 3.57 (m, 2H), 3.08 (t, 6.5, 2H), 2.45 (m,
4H), 2.32 (s, 3H); IR (Neat) 3299, 3140, 3007, 2936,
2794, 1719, 1661, 1590, 1532, 1497, 1461, 1426, 1368,
752; FAB-MS m/z (rel. intensity) 531 (M+H+, 10).
Accurate mass for C26H31N4O635Cl: 530.1932, obsd.
530.1907.
4.22. Cytotoxicity studies on human colon, prostate, and
breast cancer cells
The human colon adenocarcinoma cell line, LS 174T,
was obtained fromthe European Collection of Animal
Cell Cultures, CAMR, Porton Down, UK. LS174T cells
were routinely cultured in EagleÕs MEM supplemented
with 1% nonessential amino acids, 10% heat inactivated
fetal calf serum, 2mM L-glutamine, 50IU/mL penicillin,
50lg/mL streptomycin at 37ꢁC in a 5% CO2, 95% air-
humidified incubator. Cells were seeded in 96-well
microtiter plates at densities of 1000 cells/well. The opti-
mal seeding density was determined to ensure exponen-
tial growth in control wells throughout the experimental
period and to obtain a linear relationship between
absorbance at 492nmand cell number when analyzed
by the sulforhodamine B (SRB) assay. Twenty-four
hours after seeding the cells were incubated continu-
ously for 3days with the drugs (concentration range
0.01–500lM). The cells were washed once and then
incubated in drug-free medium for 6days. At the end
of the incubation cell growth was determined by the
SRB assay, which quantifies viable cells by measuring
their total cellular protein content. The IC50 (concentra-
tion of drug giving 50% survival in vitro), was calculated
fromthe dose–response curve obtained by plotting the
percentage of inhibition versus the log molar drug con-
centration. Similar studies were conducted against the
growth of PC3 (human prostate cancer cells) and
MCF-7 (human breast cancer cells).
4.21. Cytotoxicity studies on human chronic myeloid
leukemia K562 cells
The compounds were dissolved in DMSO to obtain
1.75 · 10ꢀ2 M stock solutions, which were diluted with
DMSO to prepare standard solutions fromconcentra-
tions of 1.75 · 10ꢀ3 M to 1.75 · 10ꢀ10 M. These solu-
tions were then further diluted with DMEM (24lL
standard solution in 176lL media). Addition of these
new drug solutions (5lL) to wells containing
100lL media/cell suspension resulted in final drug
concentrations ranging from1.0 · 10ꢀ12 to 1.0 · 10ꢀ4 M.
4.23. Cytotoxicity studies on murine L1210 leukemia and
P815 mastocytoma cells
The K562 human chronic myeloid leukemia cells were
maintained in RPM1 1640 medium supplemented with
10% fetal calf serum and 2mM glutamine at 37ꢁC in a
humidified atmosphere containing 5% CO2 and were
incubated with a specified dose of drug continuously
for 4days at 37ꢁC in the dark. The incubation was ter-
minated by centrifugation (5min, 300g) and the cells
The P815 and L1210 cell lines were obtained from
American Type Tissue Culture Collection (ATCC).
The cell lines were grown in DelbeccoÕs Modified Eagle
Medium(DMEM, Atlanta Biologicals) supplemented
with 10% fetal bovine serum, Hepes Buffer (2mM,

6234
S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
Mediatech Cellgro, 25-060-Cl), L-Glutamine (2mM,
Mediatech Cellgro), and penicillin/streptomycin
(50,000 units penicillin, 50,000lg streptomycin, Atlanta
Biologicals). Cells were maintained at 37ꢁC in a 5%
humidified CO2 atmosphere. Cultured cells were
counted using a hemocytometer and resuspended in
fresh DMEM at a concentration of 8 · 105 cells/mL.
This cell suspension (100lL) was added to 96 well flat-
bottomcell culture plates. At this concentration,
80,000 cells were seeded in each well. Drug solutions
(dissolved in DMSO and diluted in DMEM) were added
to each well (5lL/well) resulting in final concentrations
ranging from1 · 10ꢀ4 to 1 · 10ꢀ12 M. Quadruplicate
wells were prepared for each drug concentration. The
plates were incubated for 72h at 37ꢁC in a 5% CO2
atmosphere. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazoliumbromide) was dissolved in PBS
(5mg/mL). After the indicated cell incubation period,
10lL of this stock MTT solution was added to each well
and the plates were further incubated for 4h at 37ꢁC in a
5% CO2 atmosphere. After this final incubation, 100lL
acid isopropanol solution (16lL 12.1N HCl in 5mL iso-
propanol) was added to each well. The contents of the
well were mixed thoroughly by pipetting the cell suspen-
sion up and down and the plates were allowed to sit at
room temperature for 15min in order to allow for full
development of the purple color. The plates were read
on a Dynatech Plate Reader, utilizing Dynex Revelation
3.2 software, with a test wavelength of 570nmand a ref-
erence wavelength of 630nm. The dose inhibiting the
growth by 50% (IC50) was extrapolated fromcurves gen-
erated based on the averages of the absorbance data (4
points/concentration).
GCATTGGTAACTGTCAGACC-30 binds in the se-
quence 3303–3284 and was used to examine the top
strand. Linear amplification of DNA was carried out
in a total volume of 100lL containing 0.5lg of template
DNA, 50pmol of labeled primer, 250lM of each dNTP,
1U ÔRed HotÕ Taq polymerase, 20mM (NH4)2SO4.
75mM Tris–HCl, pH9.0, 0.01% Tween, 2.5mM MgCl2,
and 0.01% gelatin. After an initial denaturation at 94ꢁC
for 4min, the cycling conditions were as follows: 94ꢁC
for 1min, 60ꢁC for 1min, and 72ꢁC for 1min, for a total
of 30 cycles. After being amplified, the samples were eth-
anol precipitated and washed with 70% ethanol. Sam-
ples were dissolved in formamide loading dye, heated
for 2min at 90ꢁC, cooled on ice, and electrophoresed
at 2500–3000V for 3h on a 80cm · 20cm · 0.4mm 6%
acrylamide denaturing sequencing gel (Sequagel, Na-
tional Diagnostics). The gels were dried, and X-ray film
was exposed to the gels (Hyperfilm, Amersham, UK).
Densitometry was carried out on a Bio-Rad GS-670
imaging densitometer.
4.26. Taq polymerase stop assay and thermal cleavage
analysis for compound 5a using pUC18 plasmid DNA
For the Taq DNA polymerase stop studies, the proce-
dure was similar to that described above, except plasmid
pUC18 was linearized with HindIII, which cuts at only
one site in the plasmid (position 399) and provides
a stop for the Taq DNA polymerase downstream
from the primer. The synthetic primer 50-
CTCACTCAAAGGCGGTAATAC-30 binds to the
complementary (bottom) strand at position 749–769
and was used to examine the alkylation patterns on
the bottomstrand. The oligodeoxynucleotide primer
was 50 end labeled prior to amplification using T4
polynucleotide kinase and [c-32P]-ATP (5000Ci/mmol,
Amersham, UK).
4.24. NCI in vitro cytotoxicity screen
Compounds 5a and 5c were also tested against a panel
of 60 human cancer cell lines at the National Cancer
Institute, Bethesda, MD.31 The cytotoxicity studies were
conducted using a 48h exposure protocol, and a sulfor-
hodamine Bassay. Dose–response curves were used to
generate the GI50 (concentration of drug needed to inhi-
bit the growth by 50%), TGI (total growth inhibition),
and LC50 (concentration needed to kill 50% of cells).
For the thermal cleavage analysis, the region of the plas-
mid containing the prominent sites of damage on the
bottomstrand was PCR amplified, using the pUC1 pri-
mer and the synthetic primer 50-TGGTATCTTTA-
TAGTCCTGTCG-30,50 end labeled and binding on
the complementary (upper) strand at positions 956–
935. The 166 base pairs singly end-labeled fragment gen-
erated, was purified by agarose gel electrophoresis and
isolated using a Bio101 kit according to the manufac-
turerÕs instruction.
4.25. Taq polymerase stop assay on compounds 5a, b, l, m,
o using pBR322 plasmid DNA
All drug–DNA reactions were performed in 25mM tri-
ethanolamine, 1mM EDTA, pH7.2, at 37ꢁC for 5h.
Following incubation, DNA was precipitated by addi-
tion of 1/10 volume of 3M sodium acetate and 3vol of
95% ethanol and washed with 70% ethanol. The result-
ing pellet was dried by lyophilization.
The dry DNA pellets fromthe drug–DNA incubations
were resuspended in sodiumcitrate buffer (pH7.2) and
heated to 90ꢁC for 30min to thermally cleave at sites
of adenine or guanine N3 lesions. Samples were chilled,
precipitated, and dried.
Prior to drug/DNA incubation, plasmid pBR322
DNA was linearized with PvuII restriction enzyme to
provide a stop for the Taq downstreamfromthe
primer. The oligodeoxynucleotide primers were 50-end
labeled prior to amplification using T4 polynucleotide
kinase and [c32P]-ATP (5000Ci/mmol, Amersham,
UK). The labeled primers were purified by elution
through Bio-Rad spin columns. The primer 50-
4.27. Flow cytometry studies
The P815 cell line was used for this experimental proce-
dure. The cells were incubated with drugs in a final con-
centration equaling 10 times their IC50 values. The
10 · IC50 concentrations for cisplatin and compound
5c were 1000 and 56lM, respectively. Cisplatin was used
as a positive control. The drug stock solutions were

S. Kupchinsky et al. / Bioorg. Med. Chem. 12 (2004) 6221–6236
6235
made in DMSO so that the addition of 5lL of the solu-
tion into 5mL of media would result in the desired con-
centrations listed above. Cells were counted and
resuspended at a concentration of 1.5 · 105 cells/mL,
and 5mL of this cell suspension were pipetted into a
small culture flask. The drug solutions were added
(5lL), and the cells were incubated at 37ꢁC and 5%
CO2 for 24h. The cells were then harvested via centrifu-
gation (1000 rpm, 10min, 4ꢁC) and washed twice with
sample buffer (0.5g glucose in 500mL Ca2+/Mg2+ free
PBS; Electron Microscopy Sciences, Fort Washington,
PA). To fix the cells, the cells were vortexed vigorously
for ꢁ10s, after which the vortexing was continued at a
slower rate during the dropwise addition of 1mL of
ice-cold 70% ethanol. The tubes were capped and stored
at 4ꢁC until the day of the flow cytometry analysis.
Ms. Lillia Holmes at the Greenville Oncology Research
Center, Greenville, SC, for performing the flow cytome-
try experiments. The authors also thank Dr. Robert
Kelly of Pharmacia & Upjohn for a generous gift of
adozelesin and CC-1065.
References and notes
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Acknowledgements
Support (to M.L.) fromthe Dreyfus Foundation,
Research Corporation, National Science Foundation-
REU, Enzacta Ltd. (UK), and the National Cancer
Institute are gratefully acknowledged. J.A.H. thanks
Cancer Research UK for support. The authors thank

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