A novel class of in vivo active anticancer agents: Achiral seco-amino- and seco-hydroxycyclopropylbenz[e]indolone (seco-CBI) analogues of the duocarmycins and CC-1065

Article abstract of DOI:10.1021/jm050179u

One achiral seco-hydroxycyclopropylbenz[e]indolone (seco-CBI) (12) and seven achiral seco-amino-CBI (11a-g) analogues of CC-1065 and the duocarmycins were designed, synthesized and evaluated for their DNA-binding and anticancer properties. These compounds

Full text of DOI:10.1021/jm050179u

J. Med. Chem. 2005, 48, 3903-3918
3903
A Novel Class of in Vivo Active Anticancer Agents: Achiral seco-Amino- and
seco-Hydroxycyclopropylbenz[e]indolone (seco-CBI) Analogues of the
Duocarmycins and CC-1065
Atsushi Sato,†,‡ LuAnne McNulty,† Kari Cox,† Susan Kim,† Adrienne Scott,† Kristen Daniell,†
Kaitlin Summerville,† Carly Price,† Stephen Hudson,† Konstantinos Kiakos,§ John A. Hartley,§
Tetsuji Asao,‡ and Moses Lee*,†
Department of Chemistry, Furman University, 3300 Poinsett Highway, Greenville, South Carolina 29613, Cancer Research UK
Drug-DNA Interactions Research Group, Department of Oncology, University College London, 91 Riding House Street,
London, W1W 7BS, U.K., and Taiho Pharmaceutical Co., Ltd., 1-27, Misugidai Hanno-City, Saitama, 357-8527, Japan
Received February 24, 2005
One achiral seco-hydroxycyclopropylbenz[e]indolone (seco-CBI) (12) and seven achiral seco-
amino-CBI (11a-g) analogues of CC-1065 and the duocarmycins were designed, synthesized
and evaluated for their DNA-binding and anticancer properties. These compounds contain a
core 2-chloroethylnaphthalene structure and they do not have a stereocenter. From thermal
cleavage gel analyses, compounds 11a-g and 12 demonstrated similar covalent sequence
specificity to adozelesin 3 and the racemic seco-CBI-TMI 4 for binding to the 5′-AAAAA(865)-
3′ site. Continuous exposure of human (K562) and murine (B16, L1210 and P815) cancer cell
lines to the compounds demonstrated their significant cytotoxicity, with IC50 values in the
sub-micromolar range. Generally, a good leaving group on the ethyl moiety and a free amino
or hydroxyl group on the naphthyl moiety are essential for activity. According to NCI’s
cytotoxicity screen, compounds 11a and 12 were active against human cancer cell lines derived
from lung, colon, melanoma, renal system, and breast. At the respective doses of 15 and 20
mg/kg (administered via an ip route), compounds 11a and 12 inhibited the growth of murine
B16-F0 melanoma in C57BL/6 mice, with minimal toxicity, and 11a gave a significant anticancer
effect. The in vivo anticancer activity of compound 11a was confirmed in a human tumor
xenograft study (advanced stage SC-OVCAR-3 ovarian cancer growing in scid mice). Finally,
compound 11a was not toxic to murine bone marrow cell growth in culture at a dose that was
toxic for the previously reported compound 4.
Introduction
clinical studies,9-11 and presently, only bizelesin re-
mains in clinical trial.11 Consequently, there is a strong
interest in the design and development of novel ana-
logues of CC-1065 and the duocarmycins that maintain
antitumor activity but have reduced toxicity. In numer-
ous attempts to design novel duocarmycins and CC-1065
analogues with reduced toxicity to bone marrow cells,
a wide range of compounds related to the CPI subunit
have been synthesized and tested.1 Most drug design
studies were done on the basis that the cytotoxic potency
of the CPI-containing compounds was directly related
to their solvolytic stability. DUMSA, one of the most
cytotoxic analogues, is also the most solvolytically stable
member of this class of compounds.2,3,13 Examples of
some analogues that have been investigated are cyclo-
propylbenz[e]indolone (CBI),1.14 cyclopropylpyrazolo[e]-
indolone (CPzI),15 cyclopropylfurano[e]indolone (CFI),16
and cyclopropylindolone (CI).17 seco-Hydroxy-CBI-TMI
(4)14 and its amino congener (5) are examples of CBI
analogues of the duocarmycins that have been re-
ported.18 Furthermore, our laboratories have recently
reported a seco-iso-CFI-TMI (isocyclopropylfurano[e]-
indolone) analogue to have potent anticancer activity
but with relatively low toxicity to murine bone marrow
cells, when compared to compound 4.19 These results
suggest the potential of designing compounds with more
(+)-CC-1065 (1)1,2 and duocarmycin SA (DUMSA, 2)2,3
(Figure 1) are cyclopropylpyrrolo[e]indolone (CPI)-
containing alkaloids isolated from the fermentation
broth of Streptomyces zelensis4 and Streptomyces sp.5
These potent cytotoxic compounds derive their biological
activity by binding the minor groove of AT-rich se-
quences and covalently reacting between the CPI moiety
and adenine-N3. This mechanism of action was con-
firmed by the isolation of adenine-CC-10656 and adenine-
duocarmycin SA products7 from thermally cleaved
DNA-drug adducts. (+)-CC-1065 and (+)-DUMSA pref-
erentially bind 5′-PuNTTA-3′ and 5′-AAAAA-3′ se-
quences and they react with the N3-position of the
underlined adenine residue.6-8
Due to the severe toxicity of (+)-CC-1065 to mice4b
and the myelotoxicity of DUMSA,5 neither compound
was developed further. Following detailed structure-
activity relationship studies on these compounds, ado-
zelesin (3),1b,4c,9 carzelesin,10 bizelesin,11 and KW218912
have been evaluated clinically. Because of severe my-
elotoxicity, three of the compounds were dropped from
* Corresponding author. Phone: (864) 294-3368. Fax: (864) 294-
3559. E-mail: moses.lee@furman.edu.
† Furman University.
‡ Taiho Pharmaceutical Co., Ltd.
§ University College London.
10.1021/jm050179u CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/05/2005

3904 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
Figure 1. Structures of (+)-CC-1065 (1), (+)-duocarmycin SA (2), adozelesin (3), seco-hydroxy-CBI-TMI (4), and seco-amino-
CBI-TMI (5). Also depicted are structures of the achiral seco-amino and hydroxy analogues of CC-1065 and the duocarmycins
6-8, along with their chiral counterparts 9 and 10.
favorable toxicity profiles than the currently investi-
gated analogues of the duocarmycins and CC-1065.
We have undertaken a program to investigate whether
the chiral center present in the natural products is
needed for DNA sequence recognition and biological
activity. Our group has reported studies on the design,
synthesis, and biological properties of three types of
achiral analogues represented by seco-hydroxy-CI-TMI
(6),20 seco-amino-CI-TMI (7),21 and seco-duocarmycin
(8).22a These compounds, which lack a stereocenter, were
found to have similar DNA-binding preference, as well
as having comparable cytotoxic potency to their racemic
counterparts, agents 9 (IC50 0.15 µM with K562 cells)20,21
and 10 (IC50 1.38 nM with P388 cells).22b Moreover,
compound 6 was able to inhibit the growth of a human
advanced stage SC UACC-257 melanoma grown in
severe combined immunodeficiency (scid) mice.20 These
results have suggested that achiral analogues of CC-
1065 and the duocarmycins could form a novel class of
anticancer agents. Because CBI-containing compounds,
e.g. 414 and 5,18 are significantly more cytotoxic than
Figure 2. Target achiral seco-amino-CBI analogues (11a-g)
their corresponding CI-containing analogues, e.g. 9, it
was important to study the DNA-binding and biological
properties of a novel class of achiral seco-CBI analogues.
In this report, we describe the synthesis of seven seco-
amino-CBI analogues (11a-g) and a seco-hydroxy-CBI
analogue (12) (Figure 2), which contain a substituted
2-chloroethylnaphthalene core. The DNA sequence spe-
cific alkylation and anticancer properties of the target
compounds are also reported.
and an achiral seco-hydroxy-CBI-TMI analogue (12).
Results and Discussion
Synthesis. The synthesis of compounds 11a-e is
shown in Scheme 1, and it follows a similar strategy
that was used for the preparation of compounds 620 and
7.21 Reaction of 1-chloro-2,4-dinitronaphthalene (13)23
with tert-butyl ethyl malonate provided diester 14 in
54% yield. Subsequent acid-promoted removal of the

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3905
Scheme 1
tert-butyl group, followed by decarboxylation of com-
pound 14, gave ester 15 in 70% yield, which was
selectively reduced using sodium sulfide. The amino
product 16 was isolated in 11% yield after silica gel
chromatography, and it was allowed to react with benzyl
chloroformate to give the N-benzyl-protected compound
17 in 29% yield. Hydrolysis of the ester, followed by
selective reduction of the carboxylic acid group in 18
with borane, generated alcohol 19 in 49%. Reaction of
alcohol 19 with acetic anhydride provided acetate 20 in
55% yield. Reduction of the nitro group by catalytic
hydrogenation over Adam’s catalyst at 55 psi, followed
by coupling of the amine with 5,6,7-trimethoxyindole-
2-carboxylic acid in the presence of PyBOP and Hunig’s
base, gave compound 21a in 81% yield. Methanolysis
of the acetate group in compound 21a gave alcohol 22a
in 82% yield. Mesylation of compound 22a, followed by
chlorination, gave product 23a in 62% yield. Final
catalytic hydrogenation of compound 23a over 10%
palladium-on-carbon provided the target achiral com-
pound 11a in 74% yield.
moval of the acetate group, chlorination, and deprotec-
tion of the benzyl group afforded the target compounds
11d,e in an overall yield of 5 and 24%, respectively.
The pyrido compounds 11f,g were prepared using a
different strategy and it is shown in Scheme 2. Amino
ester 16 was reduced by DiBAL-H to give an alcohol
intermediate that was protected by reaction with acetic
anhydride and di-tert-butyl dicarbonate [(BOC)2O] to
afford compound 24 in an overall 10% yield. Reduction
of the nitro group on compound 24, followed by coupling
to the appropriate pyrido carboxylic acids,12e,24 gave
amides 25f,g in 56 and 61% yields, respectively. Sub-
sequent transformation of the acetate group to a chlo-
ride and removal of the BOC protecting group gave the
desired products 11f,g as hydrochloride salts in an
overall yield of 79 and 84%, respectively.
Our strategy for synthesizing compound 12 is il-
lustrated in Scheme 3. Diazotization of amino ester 16
and subsequent protection of the naphthol by reaction
with benzyl bromide produced compound 27 in 26%
overall yield. Reaction of ester 27 with DIBAL-H,
followed by treatment of the resultant product 28 with
acetic anhydride gave acetate 3 in an overall 62% yield.
Selective reduction of the nitro group followed by
coupling of the amine with 5,6,7-trimethoxyindole-2-
carboxylic acid in the presence PyBOP and Hunig’s base
gave amide 30 in 36% yield. Standard conversion of the
acetate into a chloride 31, followed by removal of the
benzyl protecting group by catalytic hydrogenation, gave
Synthesis of compound 11b was accomplished in 74%
by catalytic hydrogenation of intermediate 21a. Like-
wise, hydrogenation of intermediate 22a gave compound
11c in 71% yield. The preparation of compounds 11d,e
was achieved by reduction of compound 20, followed by
coupling of the amine with the appropriate carboxylic
acids in the presence of EDCI. The intermediates 21d,e
were obtained in quantitative yields. Subsequent re-

3906 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
Scheme 2
Scheme 3
compound 12 in an overall yield of 63%. All intermedi-
ates and products were characterized by FT-IR, 500
MHz 1H NMR, MS, and accurate mass measurements.
Compounds 11a-e and 12 were also characterized by
elemental analysis.
mation).25 The studies were performed using a singly
radiolabeled primer 5′-32P-CTCACTCAAAGGCGGTA-
ATAC-3′ and Hind III linearized pUC18 plasmid DNA.
Thermal Cleavage Analysis. The sequence specific-
ity of alkylation by compounds 11a,d-g and 12 was
assessed by a thermally induced DNA strand cleavage
experiment, which is commonly used to probe sequence
specific covalent bonding with purine-N3 in the minor
groove.2,3,14,19 The 191 base pair DNA fragment used in
these studies was obtained by PCR amplification from
the pUC18 plasmid that was linearized with Hind III.
A 5′-32P labeled primer was used as the forward primer
so that each final probe copy was singly end-labeled.
Representative results from the thermally induced DNA
strand break experiment on compounds 11a, 12 along
with 3 and 4 are depicted in Figure 3. The results
indicate that the achiral seco-CBI compounds 11a and
12 have similar DNA sequence selectivity for the 3′-A-
(865)AAAA cluster. Like compound 4, the achiral com-
pounds displayed a dose-response DNA alkylation
effect. However, the seco-hydroxy analogue 12 was
around 10-fold less reactive than 11a. It is also worthy
to note that the achiral compounds were more sequence
discriminating than compounds 3 and 4. The latter
compounds produced additional minor bands on the
gel: A(764), A(772), and A(843).
Figure 3. Thermal cleavage gel analysis on compounds 11a
and 12, compared to seco-CBI-TMI (4) and adozelesin (3). A
pUC18 plasmid was linearized with Hind III and amplified
using a 5′-32P-CTGTCGGGTTT-3′ primer. The drug-DNA
adducts were heated at 90 °C for 30 min prior to loading onto
the gel.
Taq Polymerase Stop Assay. The covalent se-
quence specificity of compounds 11a, 12, and adozelesin
(3)1b-d was confirmed by the results obtained from a
PCR-based assay (data given in the Supporting Infor-

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3907
Table 1. Cytotoxicity of Compounds 11a-g and 12a
HCl to form spiro[2,5]cyclopropanebenzocyclohexadienone
intermediates that react with DNA to elicit the cytotoxic
activity. This observation is consistent with the biologi-
cal activity of seco-analogues of CC-1065, the duocar-
mycins,2,3 as well as the achiral seco-CI and DUMSA
compounds (6-8).20-22
compd
K562
0.045
L1210
B16
P815
0.068
11a
11b
11c
11d
11e
11f
11g
12
8
4
23a
31
0.18
53
41
0.15
54
60
0.043
0.26
0.75
0.062
0.027
6.8
0.087
0.39
0.55
0.18
0.055
4.2
Compounds 11a and 12 were subjected to further
cytotoxicity testing at the National Cancer Institute,
against a panel of 60 human cancer cell lines.28 The
viability of the cells after 48 h of continuous exposure
to the compounds was determined using a sulforhod-
amine B assay. Both compounds gave comparable
cytotoxicity results, and Figure 4 shows the LC50 values
(concentration for killing 50% of the cells) for compound
11a against the 60 different tumor cell lines. The LC50
results for compound 12 are given in the Supporting
Information. Bars extending to the right indicate cells
more sensitive than the average to the particular
compound, whereas bars to the left indicate less sensi-
tive cells. Several observations were made on the
results. First, the compounds exhibited activity in the
sub-micromolar range, and in certain colon, central
nervous system (CNS), and skin cancers, the LC50
values were in the nanomolar range for compound 12.
Second, compounds 11a and 12 were not indiscrimi-
nately toxic to cells. For example, they were not active
against leukemic cells. Third, these compounds showed
activity against cells from many human solid tumors,
including melanoma, lung, colon, CNS, ovary, and
breast. Specific reasons for the unique patterns of
cytotoxicity for these compounds are unknown, and
experiments are underway to address this issue. The
cytotoxicity profiles for agent 11a and 12 are similar to
that reported for achiral seco-CI-TMI (6).21
Cell Cycle Studies. Duocarmycin analogues have
been demonstrated to induce leukemic cells to undergo
apoptosis. The cells demonstrated morphological abnor-
malities and genomic DNA degradation associated with
apoptosis.21,29 Flow cytometry was used to detect apo-
ptotic cells as a sub-G0 peak,30 using propidium iodide
staining. Following a 24-hour incubation of P815 cells
at the IC50 concentration (0.068 µM) as well as 10-times
the IC50 concentration of compound 11a, the results
given in Figure 5 show a substantial increase in the
percentage of cells in the sub-G0 stage (11% at IC50, and
52% at 10 times IC50 concentration), compared to 1%
percent for untreated control cells. These results indi-
cate that compound 11a was capable of damaging DNA
and inducing the cells to undergo apoptosis. For com-
parison, cisplatin at 1000 µM produced 59% percent of
sub-G0 population of cells, which is consistent with a
literature report.30 Results from our studies suggest
that, like their seco-CC-1065, seco-duocarmycins, the
achiral analogues, 620 and 11a are likely to exert their
cytotoxic activity through the induction of apoptosis.
0.15
0.25
0.0028
0.025
5.4
0.0086
0.0084
0.078
5.4
>100
58
6
7
1.4320
1.3021
1.520
5.620
a IC50 values are given in µM. The cells were continuously
treated with compounds for 3 days, except K562 cells, which were
treated for 4 days.
The region of the plasmid starting from position 749 was
linearly amplified. All three compounds showed similar
covalent sequence selectivity at the A(865) cluster and
are consistent with results from the thermal cleavage
experiment. On the basis of the sequence specificity
results, it became evident that the chiral center present
in the CC-1065 and the duocarmycins did not play a
significant role in controlling the DNA interaction
properties. Coupled to their relative ease of synthesis,
the achiral seco-CBI compounds provide an attractive
platform for the design and development of a novel class
of anticancer agents.
Cytotoxicity Studies. An MTT-based growth inhibi-
tion assay was used to determine the cytotoxicity of the
target achiral compounds.26 The concentrations required
for inhibiting the growth of the human K562 chronic
myeloid leukemia, murine L1210 leukemia, P815 mas-
tocytoma, and B16-F0 melanoma cells by 50% (IC50
values) for compounds 11a-g and 12 were determined
following continuous exposure.19-22 IC50 values are given
in Table 1. For comparison, the reported IC50 value of
CC-1065 against L1210 cells (3 days exposure) was 30
pM.27 The results reveal several interesting trends.
First, compounds 11a,d-g and 12 inhibited the growth
of the tumor cells at sub-micromolar concentrations,
with compounds 11a and 12 being the most active.
These compounds were more active than the corre-
sponding achiral seco-CI analogues (e.g. 6 and 7). They
were, however, less active than the racemic seco-CBI-
TMI compound 4. Nonetheless, the achiral seco-CBI
compounds have significant activity and are worthy of
further biological testing.
Second, protection of the hydroxyl or amino group,
found in compounds 31 and 23a, led to a dramatic
decrease in cytotoxicity. These compounds gave IC50
values of >100 and 5.4 µM, respectively, against B16
cells. In contrast, their corresponding hydroxy and
amino counterparts, 12 and 11a, gave IC50 values for
B16 cells of 0.027 and 0.15 µM, respectively. Third, for
the seco-amino-CBI series of compounds, agents 11b and
11c gave poor cytotoxicity compared to their chloride-
containing counterparts, suggesting that a good leaving
group on the ethyl substituent in the naphthalene core
was necessary for inhibiting the growth of cancer cells.
These results indicate that the seco-achiral hydroxy- or
aminonaphthethyl halide compounds must eliminate
In Vivo Anticancer Studies on B16 Melanoma.
This study was conducted according to a similar proce-
dure reported by Li et al.31 For each group of animals
in the experiment, eight 5-7-week-old female C57BL/6
mice were inoculated in the left flank with cultured B16-
F0 murine melanoma cells at day 0. On days 1, 5, and
9, the mice were treated with the appropriate doses,
based on their maximum tolerated doses, of compound
11a (15 mg/kg) or compound 12 (15 and 20 mg/kg) or

3908 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
Figure 4. NCI cytotoxicity screen data (LC50 values) for compounds 11a against a panel of 60 different human cancer cell lines.
cyclophosphamide (30 mg/kg)29b or vehicle only. The
compounds were formulated in PET/glucose and admin-
istered using an interperitoneal route. The volume of
injection was adjusted according to a ratio of 0.02 mL
for every 10 g of body weight. The size (volume) of the
tumor was monitored regularly, and the results are
depicted in Figure 6, parts A (for 11a) and B (for 12).
Measurements of the tumor volumes were ceased when
four animals (50%) in the test group had died.
The in-vivo anticancer effects for compound 11a were
apparent from the results given in Figure 6A. For the
control group, four of the mice had died by day 21;
however, the fourth mice in the 11a treated group died
on day 34, indicating an extension in the life span in
the treated group of mice. Moreover, on day 21, the size
of the tumor in the treated animals was barely palpable,
compared to the large tumors (average 4500 mm3)
already visible for the control animals. Furthermore, it
is worthy to mention that compound 11a was signifi-
cantly more active than cyclophosphamide, a clinically
useful agent, under the conditions employed. As part
of this study, the weights of the animals were also
monitored regularly. It was found that at the doses
administered, compound 11a and cyclophosphamide did
not cause any significant weight loss following drug
injection.
The in vivo results for compound 12 (Figure 6B), at a
dose of 15 mg/kg, gave weak anticancer activity, com-
pared to the untreated control mice. However, at a
higher dose of 20 mg/kg, a significant reduction in the
tumor growth was observed. However, there was no
significant extension in the life span of the treated
animals. At the 15 mg/kg dose, there was no weight loss
observed for the animals following drug injections.
However, at 20 mg/kg, some weight loss was observed
immediately after the injection (data not shown), but
the animals regained their weight within 2-3 days.

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3909
Figure 6. (A) In vivo anticancer studies of compound 11a
against B16-F0 murine melanoma grown in the flank of
C57BL/6 female mice. The mice were treated intraperitoneally.
Diamonds represent the control untreated mice, squares
represent cyclophosphamide-treated mice at 30 mg/kg. Tri-
angles represent mice treated with compound 11a at a dose
of 15 mg/kg. Measurements of tumor size were stopped when
half of the animals in each cohort of eight mice died. (B) In
vivo anticancer studies of compound 12 against B16-F0 murine
melanoma grown in the flank of C57BL/6 female mice. The
mice were treated intraperitoneally. Diamonds represent the
control untreated mice, and squares represent mice treated
with 12 at a dose of 15 mg/kg. Triangles represent mice treated
with compound 12 at a dose of 20 mg/kg. Measurements of
tumor size were stopped when half of the animals in each
cohort of eight mice died.
Figure 5. Flow cytometry analysis of P815 murine masto-
cytoma cells, stained with propidium iodide: (A) control
untreated cells, (B) the cells were treated with compound 11a
for 24 h at its IC50 concentration, and (C) the cells were treated
with compound 11a for 24 h at 10 times its IC50 value.
median times to doubling of the tumor for the treated
and control groups, respectively. According to the NCI’s
evaluation criteria, compounds that produce %T/C
values of >0 and <40 are deemed to have tumor
inhibition properties.32 Accordingly, compound 11a is
considered to be an “active” compound. It is equally
important to note that, at this dose, the mice did not
experience any toxicity or weight loss.
The in vivo results provide evidence that compound
11a possesses anticancer activity, and more impor-
tantly, even at a dose of 15 mg/kg administered to the
animals, no toxicity was observed. This compound is
significantly less toxic than its chiral counterparts,
including adozelesin, which is toxic at a dose of 1 mg/
kg.9,31 The reduced in vivo activity of the seco-hydroxy
compound 12 could be due to a higher reactivity of the
naphthol moiety, which might rapidly undergo hydroly-
sis of the chloride and effectively reduce the concentra-
tion of the compound in the plasma. Compound 11a was
not toxic to murine bone marrow cells grown in culture,
at a dose (0.0084 µM) that was toxic for the previously
reported racemic compound 4 (25 versus 0 colonies,
respectively).19
In Vivo Tumor Xenograft Studies of 11a.32 This
in vivo tumor model assay conducted by the National
Cancer Institute involved subcutaneous implantation of
the advanced stage human SC-OVCAR-3 ovarian cancer
fragments (30 mg) into the axillary region of pathogen-
free scid mice on experimental day 0. Compound 11a
was dissolved in Tween 80 and saline and was admin-
istered via an ip route on a q4d × 3 schedule. Drug
treatments were initiated on day 6 for at a dose of 11.20
mg/kg. The volume injected was adjusted according to
a ratio of 0.01 mL for every 10 g of body weight. Tumor
size and body weights were obtained approximately two
times per week. From the results, it was apparent that
compound 11a was able to inhibit the growth of the
human ovarian cancer xenograft. The %T/C value,
expressed as the ratio of the change in median tumor
weight for treated and control animals, was recorded
as 24% on day 33. The net log cell kill was 0.30,
indicating that compound 11a was capable of killing the
tumor cells. The mean time for doubling of the tumor
weight was 25.3 days, compared to 13.0 days for the
control untreated animals. Furthermore, the growth
delay %T - C/C was 95%, where T and C are the

3910 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
solution of ester 15 (10.0 g, 32.87 mmol) in EtOH (200 mL)
and the mixture was stirred for 0.5 h. The mixture was poured
into water (200 mL), concentrated, and extracted with AcOEt
(300 mL). The organic layer was washed with H2O (100 mL)
and saturated aqueous NaCl (50 mL). The aqueous layer was
extracted with AcOEt (100 mL). The combined organic layer
was washed with saturated aqueous NaCl (50 mL), dried (Na2-
SO4), and concentrated under reduced pressure. Flash chro-
matography (20% AcOEt-petroleum ether) afforded amine 16
In conclusion, the favorable DNA interaction proper-
ties, cytotoxicity against tumor cell lines, low systemic
toxicity, significant activity against murine melanoma
grown in mice as a solid tumor (especially for 11a), and
demonstrated activity against a human ovarian cancer
xenograft grown in scid mice strongly indicated that the
achiral seco-amino-CBI-TMI is worthy of further devel-
opment as an anticancer agent. Major efforts are cur-
rently underway in our laboratories to further establish
the antitumor effects of compound 11a using a wide
range of human tumor xenograft models and adminis-
tration strategies, as well as to gain insight into its
selective activity against the 60 different human cancer
cell lines in the NCI cytotoxicity screen. The results from
the achiral seco-CBI compounds effectively demon-
strated that analogues of CC-1065 and the duocarmcy-
ins, which lack a stereocenter, would still exhibit DNA
sequence specificity and potent antitumor activity.
1
(1.0 g, 11%) as a dark purple solid: mp 93-100 °C (dec); H
NMR (CDCl3, 500 MHz) δ 8.08 (d, 8.0, 1H), 7.86 (d, 7.5, 1H),
7.66 (t, 7.0, 1H), 7.62 (t, 7.0, 1H), 7.20 (s, 1H), 4.40 (brs, 2H),
4.26 (s, 2H), 4.20 (q, 7.0, 2H), 1.26 (t, 7.0, 3H); IR (neat) 3459,
3379, 3255, 2980, 1728, 1634, 1590, 1515, 1466, 1439, 1368,
1337, 1249, 1200, 1156, 1027, 841, 756 cm-1; FABMS (NBA)
m/z 274 (M+).
Ethyl [4-(N-Benzylamino)-2-nitronaphthalen-1-yl]ac-
etate (17). Benzyl chloroformate (1.56 mL, 10.94 mmol) was
slowly added to a solution of amine 16 (1.0 g, 3.65 mmol) and
Et3N (0.56 mL, 4.01 mmol) in dry CH2Cl2 (20 mL) that was
kept at 0 °C and under N2 atmosphere. The mixture was
heated to reflux for 3 days and then concentrated. The residue
was dissolved in AcOEt (50 mL) and washed with H2O (10
mL) and saturated aqueous NaCl (25 mL). The organic layer
was dried (Na2SO4) and concentrated under reduced pressure.
Flash chromatography (20% AcOEt-petroleum ether to CHCl3)
afforded compound 17 (0.39 g, 29%) as an orange solid: mp
Experimental Section
Melting points were determined on a Mel-Temp apparatus
and are uncorrected. The 1H NMR spectra were recorded on a
Varian INOVA 500 MHz spectrometer. The appropriate deu-
terated solvents are indicated in the procedure, with TMS as
the internal standard, and line positions are recorded in ppm
from the reference signal. Infared spectra were recorded on a
Perkin-Elmer Paragon 500 FT-spectrophotometer and only the
principle sharply defined bands are reported in cm-1. Mass
spectra and accurate mass measurements were recorded at
the University of South Carolina. Elemental analyses were
performed at Midwest Microlabs.
1
160-164 °C; H NMR (CDCl3, 500 MHz) δ 8.09 (d, 8.5, 1H),
7.88 (d, 8.0, 1H), 7.66 (t, 8.0, 1H), 7.60 (t, 7.0, 1H), 7.46 (d,
7.5, 2H), 7.41 (t, 7.5, 2H), 7.35 (t, 7.5, 1H), 7.07 (s, 1H), 4.92
(brs, 1H), 4.52 (d, 4.0, 2H), 4.24 (s, 2H), 4.19 (q, 7.0, 2H), 1.26
(t, 7.5, 3H); IR (neat) 3397, 2927, 1723, 1594, 1537, 1515, 1439,
1333, 1200, 1124, 1027, 823, 752, 703 cm-1; FABMS (NBA)
m/z 364 (M+).
[4-(N-Benzylamino)-2-nitronaphthalen-1-yl]acetic Acid
(18). A solution of NaOH (0.17 g, 4.31 mmol) in H2O (20 mL)
was added to a suspension of ester 17 (0.8 g, 2.20 mmol) in
EtOH (20 mL), and the mixture was heated to reflux for 2 h.
The EtOH was evaporated and washed with Et2O (10 mL).
The aqueous layer was acidified (pH 1) with 6 M HCl. The
resulting precipitate was filtered and left out to dry overnight
to provide acid 18 (0.66 g, 89%) as an orange solid: mp 170-
172 °C (dec); 1H NMR (DMSO-d6, 500 MHz) δ 8.40 (d, 8.5, 1H),
8.13 (d, 9.0, 1H), 7.67 (m, 2H), 7.55 (m, 1H), 7.41 (d, 7.5, 2H),
7.32 (t, 7.0, 2H), 7.22 (t, 8.0, 1H), 6.71 (s, 1H), 4.55 (d, 5.5,
2H), 4.04 (s, 2H); IR (Nujol) 3405, 2652, 1696, 1590, 1515, 1461,
Commercial grade solvents and reagents were used without
further purification with the following exceptions: triethyl-
amine, acetonitrile, acetic anhydride, formic acid, CDCl3,
pyridine, methanol, and deuterated DMSO-d6 were dried over
molecular sieves 3Å. THF was dried by distillation over sodium
and benzophenone. DMSO was distilled over NaOH under
vacuum and stored over molecular sieves 3Å. DMF was
distilled over BaO under vacuum and stored over molecular
sieves 3Å.
tert-Butyl Ethyl (2,4-Dinitronaphthalen-1-yl)malonate
(14). K2CO3 (4.04 g, 29.22 mmol) was added to a solution of
1-chloro-2,4-dinitronaphthalene (6.04 g, 23.91 mmol) and tert-
butyl ethyl malonate (5.0 g, 26.50 mmol) in THF (50 mL), and
the mixture was heated to reflux overnight. The mixture was
cooled and filtered, and then the filtrate was concentrated
under reduced pressure. Flash chromatography (20-50%
AcOEt-petroleum ether gradient elution) afforded compound
14 (7.6 g, 54%) as a pale red solid: mp 86-88 °C; 1H NMR
(CDCl3, 500 MHz) δ 8.65 (s, 1H), 8.55 (dd, 0.5, 8.5, 1H), 8.36
(dd, 0.5, 9.0, 1H), 7.89 (dt, 1.0, 8.0, 1H), 7.81 (dt, 1.5, 9.0, 1H),
5.64 (s, 1H), 4.26 (q, 7.0, 2H), 1.44 (s, 9H), 1.24 (t, 7.0, 3H); IR
(neat) 2980, 1745, 1537, 1368, 1346, 1306, 1253, 1147, 1027,
850, 832, 801, 774, 761 cm-1; FABMS (NBA) m/z 405 (M +
H+, 5).
Ethyl (2,4-Dinitronaphthalen-1-yl)acetate (15). Con-
centrated HCl (1.5 mL) was added to a solution of malonate
14 (0.60 g, 1.48 mmol) in AcOEt (6 mL), and the mixture was
stirred overnight. The mixture was diluted with AcOEt (30
mL) and washed with H2O (10 mL), saturated aqueous
NaHCO3 (10 mL), and saturated aqueous NaCl (10 mL). The
organic layer was dried (Na2SO4) and concentrated under
reduced pressure. Recrystallization (ether) afforded ester 15
(0.31 g, 69%) as a pale orange solid: mp 132-134 °C; 1H NMR
(CDCl3, 500 MHz) δ 8.71 (s, 1H), 8.61 (d, 8.5, 1H), 8.30 (d, 8.5,
1H), 7.92 (dt, 1.0, 8.5, 1H), 7.86 (dt, 1.5, 8.5, 1H), 4.51 (s, 2H),
4.22 (q, 7.5, 2H), 1.27 (t, 7.5, 3H); 304 (M-); IR (neat) 3087,
2989, 1732, 1528, 1422, 1355, 1204, 1169, 1027, 889, 832, 779
cm-1; FABMS (NBA) m/z 305 (M + H+).
1439, 1377, 1333, 1249, 1120, 929, 832, 792, 752, 699 cm-1
;
FABMS (NBA) m/z 336 (M+); ESMS m/z 337 (M + H+).
2-[4-(N-Benzylamino)-2-nitronaphthalen-1-yl]etha-
nol (19). BH3-THF complex (1.0 M, 0.13 mL, 0.13 mmol) was
added to a solution of acid 18 (40.0 mg, 0.12 mmol) in dry THF
(5 mL) under N2 atmosphere and the mixture was stirred
overnight. The reaction mixture was treated with saturated
aqueous NH4Cl (1 mL) and extracted with AcOEt (20 mL). The
organic layer was washed with saturated aqueous NaCl (5
mL), dried (Na2SO4), and concentrated under reduced pres-
sure. Flash chromatography (25% AcOEt-petroleum ether)
afforded alcohol 19 (21.2 mg, 55%) as a dark red solid: mp
1
128-130 °C; H NMR (CDCl3, 500 MHz) δ 8.24 (d, 9.0, 1H),
7.88 (d, 8.0, 1H), 7.66 (t, 7.5, 1H), 7.60 (t, 7.5, 1H), 7.45 (d,
7.5, 2H), 7.41 (t, 7.5, 2H), 7.35 (t, 7.5, 1H), 6.92 (s, 1H), 4.87
(br s, 1H), 4.50 (d, 5.0, 2H), 4.05 (t, 8.5, 2H), 3.40 (t, 7.0, 2H),
1.84 (br s, 1H); IR (neat) 3406, 2936, 2892, 1594, 1519, 1435,
1342, 1124, 1036, 912, 823, 752, 699 cm-1; FABMS (NBA) m/z
322 (M+); ESMS m/z 323 (M + H+).
[4-(N-Benzylamino)-2-nitronaphthalen-1-yl]ethyl Ac-
etate (20). Acetic anhydride (0.8 mL, 8.73 mmol) was added
to a solution of 19 (1.6 g, 4.96 mmol) in dry pyridine (16 mL)
at room temperature, and the solution was stirred overnight.
The mixture was concentrated and dissolved in AcOEt (50 mL).
The organic layer was washed with 1 M HCl (10 mL), H2O
(10 mL), saturated aqueous NaHCO3 (10 mL), and saturated
aqueous NaCl (10 mL), and the organic layer was dried (Na2-
SO4) and concentrated under reduced pressure. Flash chro-
Ethyl (4-Amino-2-nitronaphthalen-1-yl)acetate (16).
Na2S (60%, 4.27 g, 32.87 mmol) was added to a refluxing

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3911
matography (20% AcOEt-petroleum ether) of the residue
afforded acetate 20 (1.0 g, 55%) as a red solid: mp 75-90 °C;
1H NMR (CDCl3, 500 MHz) δ 8.33 (d, 8.5, 1H), 7.88 (d, 8.0,
1H), 7.69 (t, 7.5, 1H), 7.61 (t, 7.5, 1H), 7.45 (d, 7.5, 2H), 7.41
(t, 8.0, 2H), 7.35 (t, 8.0, 1H), 6.93 (s, 1H), 4.89 (t, 5, 1H), 4.50
(d, 5.5, 2H), 4.44 (t, 7.0, 2H), 3.46 (t, 7.5, 2H), 2.06 (s, 3H); IR
(neat) 1735, 1592, 1521, 1435, 1343, 1239, 1129, 1035, 752
cm-1; FABMS (NBA) m/z 364 (M+); EIMS m/z 364 (M+); HR-
EIMS m/z 364.1424 (M+, C21H20N2O4 requires 364.1423).
an orange foam (700 mg, 76%): 1H NMR (CDCl3, 500 MHz) δ
9.26 (s, 1H), 8.34 (s, 1H), 7.88 (d, 8.0, 1H), 7.86 (d, 8.0, 1H),
7.56 (t, 7.5, 1H), 7.47 (m, 3H), 7.39 (t, 8.0, 2H), 7.33 (t, 7.5,
1H), 7.16 (s, 1H), 7.13 (brs, 1H), 6.87 (s, 1H), 4.59 (t, 6.5, 2H),
4.49 (s, 2H), 4.08 (s, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 3.52 (t, 6.5,
2H), 2.84 (s, 3H).
To a solution of the above mesylate (700 mg, 1.16 mmol) in
dry DMF (7 mL) was added LiCl (920 mg, 21.61 mmol) at room
temperature under N2 atmosphere. The reaction mixture was
stirred for 3 days and then diluted with AcOEt (100 mL). The
organic solution was washed with H2O (20 mL) and saturated
aqueous NaCl (5 mL) and then dried (Na2SO4) and concen-
trated under reduced pressure. Flash chromatography (30%
AcOEt-petroleum ether) of the residue afforded chloride 23a
(0.52 g, 82%, overall yield of 62% from 22a) as a yellow foam:
mp 58-62 °C; 1H NMR (CDCl3, 500 MHz) δ 9.31 (s, 1H), 8.51
(s, 1H), 7.85 (d, 8.0, 1H), 7.83 (d, 7.5, 1H), 7.30-7.54 (m, 8H),
7.15 (s, 1H), 6.85 (s, 1H), 4.68 (brs, 1H), 4.07 (s, 3H), 3.96 (t,
6.0, 2H), 3.95 (s, 5H), 3.91 (s, 3H), 3.52 (t, 6.5, 2H); IR (neat)
3406, 3308, 2936, 1772, 1648, 1590, 1532, 1493, 1466, 1413,
1306, 1240, 1226, 1107, 1049, 996, 907, 827, 756, 734, 699
cm-1; FABMS (NBA) m/z 544 (M+); ESMS m/z 544 (M+).
4-(2-Chloroethyl)-3-(5,6,7-trimethoxyindolyl-2-carbox-
amido)-1-naphthylamine (11a). A solution of compound 23a
(400 mg, 0.74 mmol) in THF (10 mL) was added to a
suspension of 10% Pd/C (50 mg) in chilled THF (10 mL), and
the suspension was hydrogenated under H2 atmosphere at
room temperature for 3 days. The suspension was filtered over
Celite and the filtrate was concentrated under reduced pres-
sure. The product was crystallized from diisopropyl ether to
give amine 11a (250 mg, 74%) as a pale yellow solid: mp 182
°C (dec); 1H NMR (CDCl3, 500 MHz) δ 9.40 (s, 1H), 8.54 (s,
1H), 7.85 (d, 8.5, 1H), 7.83 (d, 8.0, 1H), 7.53 (t, 7.5, 1H), 7.46
(t, 8.5, 1H), 7.32 (s, 1H), 6.97 (s, 1H), 6.86 (s, 1H), 4.25 (br s,
2H) 4.08 (s, 3H), 3.98 (t, 6.5, 2H), 3.95 (s, 3H), 3.92 (s, 3H),
3.54 (t, 6.5, 2H); IR (neat) 3326, 2927, 2856, 1626, 1590, 1536,
1501, 1466, 1408, 1302, 1240, 1107, 1049, 1000, 907, 832, 756
cm-1; HR-FABMS (NBA) m/z 454.1549 (M + H+, C24H24ClN3O4
requires 454.1534). Anal. (C24H24N3O4Cl‚1/2H2O) C, H, N.
2-[4-Benzylamino-2-(5,6,7-trimethoxyindole-2-carbox-
amido)naphthalen-1-yl]ethyl Acetate (21a). A solution of
20 (0.90 g, 2.47 mmol) in THF (30 mL) was added to a
suspension of PtO2 (0.2 g) in chilled THF (10 mL), and the
suspension was hydrogenated (with shaking) at 55 psi at room
temperature for 2 h. The suspension was filtered over Celite
and the filtrate was concentrated under reduced pressure to
provide as a pale brown solid (0.80 g, 97%). The solid (0.8 g,
2.39 mmol) was dissolved in CH2Cl2 (40 mL), and 5,6,7-
trimethoxy-2-carboxylic acid (0.64 g, 2.54 mmol), benzotriazol-
1-yloxy-tripyroridinophosphonium hexafluorophosphate (Py-
BOP) (1.32 g, 2.54 mmol), and N,N-diisopropylethylamine (1.5
mL, 8.46 mmol) were added. The solution was stirred under
N2 atmosphere at room temperature for 2 days. The reaction
mixture was concentrated, and the residue was dissolved in
AcOEt (100 mL) and washed with 10% HCl (20 mL), saturated
aqueous NaHCO3 (20 mL), and saturated aqueous NaCl (20
mL). The organic layer was dried (Na2SO4) and concentrated
under reduced pressure. Flash chromatography (33% AcOEt-
petroleum ether) of the crude material afforded compound 21a
as a brown foam (1.1 g, 81%): mp 176-178 °C; 1H NMR
(CDCl3, 500 MHz) δ 9.38 (s, 1H), 8.62 (s, 1H), 7.96 (d, 8.5, 1H),
0.7.85 (d, 8.5, 1H), 7.55 (t, 8.0, 1H), 7.49 (d, 8.0, 2H), 7.44 (m,
2H), 7.40 (t, 8.0, 2H), 7.33 (t, 7.5, 1H), 7.18 (brs, 1H), 6.88 (s,
1H), 4.66 (brs, 1H), 4.52 (d, 5.0, 2H), 4.40 (t, 7.0, 2H), 4.09 (s,
3H), 3.95 (s, 3H), 3.93 (s, 3H), 3.41 (t, 7.0, 2H), 2.15 (s, 3H);
13C NMR (DMSO-d6, 300 MHz) δ 170.41, 159.88 149.06,
143.21, 139.84, 139.53, 139.06, 134.60, 132.92, 131.61, 128.27,
126.77, 125.70, 124.13, 123.79, 122.99, 122.50, 122.14, 115.97,
105.17, 104.07, 97.92, 63.40, 61.03, 55.93, 46.37, 25.93, 20.69;
IR (neat) νmax 3307, 1729, 1648, 1592, 1534, 1466, 1410, 1304,
1241, 1104, 1046, 1000, 912, 832, 756, 733 cm-1; FABMS
(NBA) m/z 568 (M + H+); ESMS m/z 568 (M + H+); HR-
FABMS (NBA) m/z 568.2204 (M + H+, C33H34N3O6 requires
568.2210).
2-[4-Benzylamino-2-(5,6,7-trimethoxyindole-2-carbox-
amido)naphthalen-1-yl]ethanol (22a). To a solution of ester
21a (4.1 g, 7.22 mmol) in 1:1 methanol:THF (200 mL) was
added potassium carbonate (0.93 g, 6.70 mmol) at room
temperature and the mixture was stirred overnight. The
reaction mixture was neutralized (pH 6) with 1 M HCl and
concentrated. The residue was diluted with AcOEt (100 mL)
and washed with saturated aqueous NaCl (20 mL), and the
organic layer was dried (Na2SO4) and concentrated under
reduced pressure to yield compomund 22a as a brown foam
(3.1 g, 82%): mp 96-114 °C; 1H NMR (CDCl3, 500 MHz) δ
9.83 (s, 1H), 9.26 (s, 1H), 7.86 (d, 8.5, 1H), 7.84 (d, 9.0, 1H),
7.49 (m, 3H), 7.44 (s, 1H), 7.38 (m, 3H), 7.32 (t, 7.0, 1H), 7.02
(brs, 1H), 6.82 (s, 1H), 4.60 (brs, 1H), 4.51 (s, 2H), 4.16 (brs,
2H), 4.07 (s, 3H), 3.94 (s, 3H), 3.89 (s, 3H), 3.32 (t, 5.0, 2H),
2.34 (brs, 1H); IR (neat) νmax 3294, 2934, 1722, 1644, 1594,
1530, 1466, 1411, 1304, 1266, 1224, 1104, 1046, 996, 908, 831,
756, 700 cm-1; FABMS (NBA) m/z 525 (M+); ESMS m/z 526
(M + H+).
2-[4-Amino-2-(5,6,7-trimethoxyindole-2-carboxamido)-
naphthalen-1-yl]ethyl Acetate (11b). A solution of 2-[4-(N-
benzylamino)-2-(5,6,7-trimethoxyindole-2-carboxamido)naph-
thalen-1-yl]ethyl acetate 21a (0.10 g, 0.18 mmol) in THF (10
mL) was added to a suspension of 10% Pd/C (0.1 g) in chilled
THF (10 mL), and the suspension was hydrogenated under
H2 atmosphere at room temperature for 3 days. The suspension
was filtered over Celite and the filtrate was concentrated under
reduced pressure. The residue was crystallized with diisopro-
pyl ether to yield amine 11b (0.064 g, 74%) as a white solid:
1
mp 182-186 °C (dec); H NMR (CDCl3, 500 MHz) δ 9.40 (s,
1H), 8.65 (s, 1H), 7.94 (d, 8.5, 1H), 7.86 (d, 8.5, 1H), 7.57 (s,
1H), 7.55 (t, 8.0, 1H), 7.46 (t, 8.0, 1H), 7.19 (brs, 1H), 6.88 (s,
1H), 4.39 (t, 7.0, 2H), 4.23 (br s, 2H), 4.10 (s, 3H), 3.96 (s, 3H),
3.93 (s, 3H), 3.41 (t, 7.0, 2H), 2.10 (s, 1.5, 3H); IR (neat) νmax
3341, 1727, 1639, 1591, 1536, 1505, 1466, 1410, 1304, 1243,
1106, 1046, 758 cm-1; FABMS (NBA) m/z 478 (M + H+). Anal.
(C26H27N3O6) C. H. N.
2-[4-Amino-2-(5,6,7-trimethoxyindole-2-carboxamido)-
naphthalen-1-yl]ethanol (11c). A solution of 2-[4-(N-benz-
ylamino)-2-(5,6,7-trimethoxyindole-2-carboxamido)naphtha-
len-1-yl]ethanol 22a (0.14 g, 0.27 mmol) in THF (10 mL) was
added to a suspension of 10% Pd/C (0.1 g) in chilled THF (10
mL) and the suspension was hydrogenated under H2 atmo-
sphere at room temperature for 3 days. The suspension was
filtered over Celite and the filtrate was concentrated in reduced
pressure. The residue was crystallized with diisopropyl ether
to yield product 11c (0.083 g, 76%) as a pale yellow solid: mp
2-[4-(N-Benzylamino)-2-(5,6,7-trimethoxyindole-2-car-
boxamido)naphthalen-1-yl]ethyl Chloride (23a). To an
ice-chilled solution of 22a (800 mg, 1.52 mmol) and dry
triethylamine (0.4 mL, 2.98 mmol) in CH2Cl2 (8 mL) was added
methanesulfonyl chloride (0.28 mL, 2.98 mmol), and the
solution was stirred for 1 h. The reaction mixture was diluted
with CH2Cl2 (50 mL) and washed with H2O (10 mL) and
saturated aqueous NaCl (20 mL). The organic layer was dried
(Na2SO4) and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (50%
AcOEt-petroleum ether) to yield a mesylate intermediate as
1
172-180 °C (dec); H NMR (CDCl3, 500 MHz) δ 9.85 (s, 1H),
9.28 (s, 1H), 7.86 (d, 8.5, 1H), 7.86 (d, 8.0, 1H), 7.54 (s, 1H),
7.50 (t, 8.0, 1H), 7.43 (t, 8.5, 1H), 7.03 (d, 2.0, 1H), 6.84 (s,
1H), 4.18 (t, 5.5, 2H), 4.09 (s, 3H), 3.95 (s, 3H), 3.90 (s, 3H),
3.33 (t, 7.0, 2H); IR (neat) νmax 3291, 2936, 1638, 1595, 1541,
1509, 1464, 1432, 1410, 1305, 1264, 1106, 1047, 996, 909, 833,

3912 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
757, 731 cm-1; FABMS (NBA) m/z 435 (M + H+). Anal.
7.55 (dt, 1.0, 7.5, 1H), 7.46 (d, 7.5, 2H), 7.39 (t, 7.5, 2H), 7.33
(t, 7.0, 1H), 7.31 (s, 1H), 7.17 (s, 1H), 7.11 (d, 2.5, 1H), 6.98 (d,
9.0, 1H), 6.98 (d, 8.5, 1H), 4.68 (br s, 1H), 4.48 (s, 2H), 4.00 (t,
6.5, 2H), 3.87 (s, 3H), 3.57 (t, 6.5, 2H); IR (neat) νmax 3406,
3282, 1645, 1591, 1529, 1475, 1453, 1422, 1399, 1249, 1213,
1164, 1029, 758, 733, 701 cm-1; FABMS (NBA) m/ z 484 (M+).
(C24H25N3O5) C. H. N.
2-[4-(N-Benzylamino)-2-(5-methoxyindole-2-carboxa-
mido)naphthalen-1-yl]ethyl Acetate (21e). To a solution
2-[4-(N-benzylamino)-2-aminonaphthalen-1-yl]ethyl acetate (0.30
g, 0.90 mmol), prepared from the reduction of acetate 20 by
hydrogenation over PtO2 at 55 psi, in pyridine (3 mL) were
added 5-methoxy-2-carboxylic acid (0.23 g, 1.19 mmol), 1-hy-
droxy-benzotriazole (0.16 g, 1.19 mmol), and 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.23 g,
1.19 mmol) under N2 atmosphere at room temperature. The
mixture was stirred for 2 days at room temperature and then
heated at 60 °C for 2 days. The reaction mixture was diluted
with AcOEt (50 mL); washed with 10% HCl (10 mL), saturated
aqueous NaHCO3 (10 mL), and saturated aqueous NaCl (20
mL); dried (Na2SO4); and concentrated under reduced pressure
to yield compound 21e as a brown foam (0.52 g, quant.): mp
98-110 °C; 1H NMR (CDCl3, 500 MHz) δ 9.41 (s, 1H), 8.67 (s,
1H), 7.96 (d, 8.5, 1H), 7.85 (d, 8.0, 1H), 7.55 (t, 7.0, 1H), 7.47
(d, 8.0, 2H), 7.43 (t, 9.0, 1H), 7.39 (t, 7.5, 2H), 7.34 (m, 2H),
7.20 (br s, 1H), 7.13 (d, 1.5, 1H), 6.99 (dd, 9.0, 1.0, 1H), 6.99
(dd, 9.0, 1.5, 1H), 4.69 (br s, 1H), 4.50 (s,2H), 4.41 (t, 7.0, 2H),
3.88 (s, 3H), 3.42 (t, 7.0, 2H), 2.15 (s, 3H); IR (neat) νmax 3272,
1728, 1647, 1592, 1530, 1476, 1453, 1236, 1213, 1162, 1030,
909, 841, 807, 759, 733, 699 cm-1; FABMS (NBA) m/z 507 (M+).
4-(2-Chloroethyl)-3-(5-methoxyindole-2-carboxamido)-
1-naphthylamine (11e). A solution of chloride 23e (68 mg,
0.15 mmol) in THF (10 mL) was added to a suspension of 10%
Pd/C (100 mg) in chilled THF (10 mL), and the suspension
was hydrogenated under H2 atmosphere at room temperature
overnight. The suspension was filtered over Celite and the
filtrate was concentrated under reduced pressure. The residue
was crystallized with diethyl ether to yield compound 11e (40
mg, 68%) as a white solid: mp 228 °C (dec); 1H NMR (DMSO-
d6, 500 MHz) δ 9.95 (s, 1H), 8.11 (d, 8.5, 1H), 7.96 (d, 8.0, 1H),
7.52 (t, 7.0, 1H), 7.41 (t, 7.0, 1H), 7.35 (d, 9.0, 1H), 7.28 (s,
1H), 7.13 (s, 1H), 6.89 (d, 9.0, 1H), 6.67 (s, 1H) 5.83 (s, 2H),
3.77 (s, 3H), 3.73 (t, 8.0, 2H), 3.37 (t, 8.0, 2H); IR (Nujol) νmax
3296, 1652, 1592, 1533, 1462, 1260, 1094, 1020, 800 cm-1
;
FABMS (NBA) m/z 394 (M + H+). Anal. (C22H20N3O2Cl) C. H.
N.
2-[4-(N-Benzylamino)-2-(5-(5-benzofuran-2-carboxami-
do)indole-2-carboxamido)naphthalen-1-yl]ethyl Acetate
(21d). The procedure was similar to that used for the prepara-
tion of 21e. Compound 21d was isolated as a pale brown solid
(0.64 g, quant.): mp 150-156 °C; 1H NMR (CDCl3, 500 MHz)
δ 9.52 (s, 1H), 8.74 (s, 1H), 8.44 (s, 1H), 8.31 (s, 1H), 7.97 (d,
8.5, 1H), 7.85 (d, 8.5, 1H), 7.72 (d, 8.0, 1H), 7.62 (s, 1H), 7.59
(d, 8.5, 1H), 7.55 (t, 7.5, 1H), 7.31-7.49 (m, 11H), 7.22 (s, 1H),
4.52 (s, 1H), 4.40 (t, 7.0, 2H), 3.49 (s, 2H), 3.42 (t, 7.0, 2H),
2.16 (s, 3H); IR (neat) νmax 3402, 1728, 1654, 1592, 1534, 1475,
1448, 1426, 1302, 1245, 1142, 1076, 1031, 881, 810, 739, 699
cm-1; FABMS (NBA) m/z 636 (M +); ESMS m/z 637 (M + H+).
2-[4-(N-Benzylamino)-2-(5-(5-benzofuran-2-carboxami-
do)indole-2-carboxamido)naphthalen-1-yl]ethyl Chlo-
ride (23d). The procedure was similar to that used for the
preparation of 23e. 2-[4-(N-Benzylamino)-2-(5-(5-benzofuran-
2-carboxamido)indole-2-carboxamido)naphthalen-1-yl]etha-
nol (0.32 g, 54%) was obtained as a pale yellow solid: mp 180-
185 °C; 1H NMR (DMSO-d6, 500 MHz) δ 8.26 (d, 8.5, 1H), 8.13
(s, 1H), 7.97 (d, 8.5, 1H), 7.82 (d, 7.5, 1H), 7.74 (t, 8.0, 1H),
7.71 (s, 1H), 7.49 (t, 7.5, 2H), 7.34-7.41 (m, 9H), 7.29 (t, 7.5,
2H), 7.19 (t, 7.0, 2H), 6.88 (br s, 1H), 4.47 (s, 2H), 3.96 (br s,
2H), 3.69 (br s, 2H); IR (Nujol) νmax 3263, 1642, 1595, 1542,
1456, 1404, 1377, 1330, 1267, 1231, 1169, 1138, 1029, 872, 827,
805, 740, 704, 668 cm-1; FABMS (NBA) m/z 595 (M +H+);
ESMS m/z 595 (M + H+).
2-[4-(N-Benzylamino)-2-(5-methoxyindole-2-carboxa-
mido)naphthalen-1-yl]ethyl Chloride (23e). To a solution
of acetate 21e (0.50 g, 0.99 mmol) in 1:1 methanol:tetra-
hydrofuran (10 mL) was added potassium carbonate (0.13 g,
0.91 mmol) at room temperature, and the mixture was stirred
overnight. The reaction mixture was diluted with AcOEt (100
mL) and washed with H2O (20 mL) and saturated aqueous
NaCl (20 mL), and then the organic layer was dried (Na2SO4)
and concentrated under reduced pressure to yield 2-[4-(N-
benzylamino)-2-(5-methoxyindole-2-carboxamido)naphthalen-
1-yl]ethanol as a brown foam (0.42 g, 91%): mp 198 °C (dec);
1H NMR (DMSO-d6, 500 MHz) δ 10.18 (s, 1H), 8.26 (d, 8.5,
1H), 7.97 (d, 8.5, 1H), 7.51 (t, 7.0, 1H), 7.43 (d, 8.5, 1H), 7.39
(d, 7.5, 2H), 7.33 (s, 1H), 7.31 (s, 1H), 7.28 (t, 7.5, 2H), 7.18 (t,
7.5, 1H), 7.11 (br s, 1H), 7.08 (d, 2.5, 1H), 6.91 (t, 6.0, 1H),
6.86 (dd, 9.0, 2.5, 1H), 6.81 (s, 1H), 5.35 (t, 4.5, 1H), 4.47 (d,
5.5, 2H), 3.76 (s, 3H), 3.69 (m, 2H), 3.11 (t, 6.0, 2H); IR (neat)
νmax 3272, 2945, 2874, 1646, 1594, 1528, 1479, 1453, 1422,
1404, 1261, 1213, 1162, 1030, 841, 805, 758, 734, 699 cm-1
;
FABMS (NBA) m/z 465 (M+).
To an ice-chilled solution of the above alcohol (0.40 g, 0.86
mmol) and triethylamine (0.44 mL, 3.14 mmol) in THF (20
mL) was added methanesulfonyl chloride (0.24 mL, 3.14
mmol), and the mixture was stirred for 1 h. The reaction
mixture was diluted with CH2Cl2 (50 mL) and washed with
H2O (10 mL) and saturated aqueous NaCl (20 mL), and then
the organic layer was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (30% AcOEt-petroleum ether) to yield 2-[4-
(N-benzylamino)-2-(5-methoxyindole-2-carboxamido)naphtha-
len-1-yl]ethyl methanesulfonate (0.28 g, 60%) as a yellow
foam: mp 160 °C (dec); 1H NMR (CDCl3, 500 MHz) δ 9.09 (brs,
1H), 8.36 (brs, 1H), 7.89 (d, 9.0, 1H), 7.87 (d, 9.0, 1H), 7.57 (t,
8.5, 1H), 7.48 (d, 7.5, 2H), 7.40 (t, 7.5, 2H), 7.34 (m, 2H), 7.17
(s, 1H), 7.13 (s,1H), 7.00 (d, 8.5, 1H), 7.00 (d, 9.0, 1H), 4.70 (br
s, 2H), 4.61 (t, 6.0, 2H), 4.50 (s, 2H), 3.88 (s, 3H), 3.54 (t, 6.0,
2H), 2.84 (s, 3H); IR (neat) νmax 3400, 1643, 1591, 1530, 1479,
1453, 1351, 1231, 1213, 1173, 1142, 1076, 1031, 974, 947, 841,
805, 761, 734, 699 cm-1; FABMS (NBA) m/z 543 (M+).
2-[4-(N-Benzylamino)-2-(5-(5-benzofuran-2-carboxamido)in-
dole-2-carboxamido)naphthalen-1-yl]ethyl methanesulfonate
(0.32 g, 95%) was produced as a brown oil: 1H NMR (CDCl3,
500 MHz) δ 9.33 (s, 1H), 8.76 (s, 1H), 8.42 (s, 1H), 8.19 (s,
1H), 7.90 (d, 9.0, 1H), 7.87 (d, 8.5, 1H), 7.71 (d, 8.5, 1H), 7.44-
7.60 (m, 9H), 7.39 (t, 7.5, 2H), 7.22-7.35 (m, 4H), 7.12 (s, 1H),
4.60 (t, 6.5, 2H), 4.48 (s, 2H), 3.54 (t, 6.0, 2H), 2.88 (s, 3H); IR
(neat) νmax 3202, 1639, 1590, 1536, 1404, 1351, 1258, 1231,
1171, 1076, 1045, 969, 947, 748 cm-1; ESMS m/z 673 (M +
H+).
Flash chromatography (30% AcOEt-petroleum ether) af-
forded chloride 23d (84 mg, 29%) as a yellow solid: mp 180
1
°C (dec); H NMR (DMSO-d6, 500 MHz) δ 10.43 (s, 1H), 9.99
(s, 1H), 8.31 (d, 8.5, 1H), 8.15 (s, 1H), 8.00 (d, 8.5, 1H), 7.82
(d, 8.0, 1H), 7.74 (s, 1H), 7.72 (d, 8.5, 1H), 7.58 (t, 8.5, 2H),
7.49 (t, 7.0, 2H), 7.43 (d, 8.5, 1H), 7.39 (s, 1H), 7.37 (d, 7.0,
2H), 7.28 (t, 8.0, 2H), 7.20 (d, 7.0, 1H), 7.06 (t, 5.5, 1H), 6.37
(s, 1H), 4.48 (d, 5.5, 2H), 3.74 (t, 7.5, 2H), 3.33 (t, 7.5, 2H); IR
(Nujol) νmax 3365, 3206, 3022, 1609, 1639, 1516, 1406, 1403,
1260, 748 cm-1; ESMS m/z 613 (M+).
To a solution of the sulfonate (0.27 g, 0.50 mmol) in dry DMF
(3 mL) was added LiCl (0.39 g, 9.19 mmol) at room tempera-
ture under N2 atmosphere, and the mixture was stirred for 3
days. The reaction mixture was diluted with AcOEt (100 mL)
and washed with H2O (20 mL) and saturated aqueous NaCl
(5 mL), and then the organic layer was dried (Na2SO4) and
concentrated under reduced pressure. Flash chromatography
(30% AcOEt-petroleum ether) afforded compound 23e (0.16
4-(2-Chloroethyl)-3-[5-(5-benzofuran-2-carboxamido)-
indole-2-carboxamido]-1-naphthylamine (11d). The pro-
cedure was similar to that used in the synthesis of compound
11e. The residue was crystallized with diethyl ether to yield
compound 11d (16 mg, 29%) as a pale yellow solid: mp 216
°C (dec); 1H NMR (DMSO-d6, 500 MHz) δ 10.44 (s, 1H), 10.01
1
g, 66%) as a yellow foam: mp 182 °C (dec); H NMR (CDCl3,
500 MHz) δ 9.28 (br s, 1H), 8.55 (br s, 1H), 7.87 (d, 8.5, 2H),

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3913
(s, 1H), 8.18 (s, 1H), 8.11 (d, 8.5, 1H), 7.97 (d, 8.5, 1H), 7.83
(d, 7.5, 1H), 7.75 (s, 1H), 7.72 (d, 8.5, 1H), 7.58 (dd, 2.0, 8.5,
1H), 7.53 (t, 8.0, 1H), 7.50 (t, 7.5, 1H), 7.35-7.46 (m, 5H), 6.68
(s, 1H), 5.83 (brs, 1H), 3.75 (t, 7.5, 2H), 3.38 (t, 7.5, 2H); IR
(Nujol) νmax 3434, 1707, 1593, 1534, 1462, 1378, 1260, 1122,
1074, 1022, 953, 914, 861, 801, 743 cm-1; ESMS m/z 523 (M +
H+). Anal. (C30H23N4O3Cl) C. H. N.
1601, 1537, 1497, 1384, 1287, 1236, 1161, 1026, 830, 756, 732
cm-1; HR-FABMS (NBA) m/z 506.2300 (M + H+, C28H32N3O6
requires 506.2291).
2-[4-(N-tert-Butoxycarbonylamino)-2-(3-aza-2,4-
dimethoxycinnamoylamido)naphthalen-1-yl]ethyl Ac-
etate (25g). The procedure was similar to that used for the
synthesis of compound 25f. Compound 25g was collected as a
1
white solid (0.22 g, 61%): mp 228-230 °C; H NMR (CDCl3,
2-[4-(N-tert-Butoxycarbonylamino)-2-nitronaphthalen-
1-yl]ethyl Acetate (24). Acetic anhydride (4.0 mL, 42.39
mmol) was added to a solution of 2-[4-amino-2-nitronaphtha-
len-1-yl]ethanol 16 (3.3 g, 14.21 mmol) in dry pyridine (33 mL)
at room temperature, and the mixture was stirred for 2 h. The
mixture was diluted with AcOEt (100 mL). The organic layer
was washed with H2O (30 mL), saturated aqueous NaHCO3
(30 mL), and saturated aqueous NaCl (30 mL), and then the
organic layer was dried (Na2SO4) and concentrated under
reduced pressure. Flash chromatography (33% AcOEt-
petroleum ether) afforded 2-[4-amino-2-nitronaphthalen-1-yl]-
ethyl acetate (1.0 g, 26%) as an orange solid: mp 108-110
500 MHz) δ 8.56 (br s, 1H), 8.45 (br s, 1H), 7.98 (d, 8.0, 1H),
7.91 (d, 8.0, 1H), 7.87 (d, 15.5, 1H), 7.75 (d, 8.0, 1H), 7.54 (t,
7.0, 1H), 7.48 (t, 7.5, 1H), 6.94 (d, 15.5, 1H), 6.77 (br s, 1H),
6.37 (d, 8.0, 1H), 4.30 (t, 8.0, 2H), 4.07 (s, 3H), 3.97 (s, 3H),
3.41 (t, 7.5, 2H), 2.17 (s, 3H), 1.55(s, 9H); IR (neat) νmax 3252,
2979, 1736, 1688, 1656, 1620, 1595, 1537, 1503, 1484, 1455,
1422, 1387, 1366, 1321, 1280, 1241, 1162, 1098, 1040, 1014,
903, 812, 756, 732 cm-1; FABMS (NBA) m/z 536.2392 (M +
H+, C29H34N3O7 requires 536.2397).
2-[4-(N-tert-Butoxycarbonylamino)-2-(3-aza-4-meth-
oxycinnamoylamido)naphthalen-1-yl]ethyl Chloride (26f).
To a solution of acetate 25f (0.19 g, 0.38 mmol) in methanol
(4 mL) and tetrahydrofuran (10 mL) was added potassium
carbonate (0.06 g, 0.45 mmol) at room temperature and the
mixture was stirred overnight. The reaction mixture was
diluted with AcOEt (100 mL) and washed with H2O (20 mL)
and saturated aqueous NaCl (20 mL), and then the organic
layer was dried (Na2SO4) and concentrated under reduced
pressure to yield 2-[4-(N-tert-butoxycarbonylamino)-2-(3-aza-
4-methoxycinnamoylamido)naphthalen-1-yl]ethanol as a pale
brown foam (0.17 g, 97%): mp 116-120 °C; 1H NMR (CDCl3,
500 MHz) δ 9.21 (br s, 1H), 8.26 (s, 1H), 8.19 (s, 1H), 7.83 (br
s, 2H), 7.68 (d, 8.0, 1H), 7.54 (d, 15, 1H), 7.40 (m, 2H), 6.81
(br s, 1H), 6.72 (d, 8.5, 1H), 6.36 (d, 15.5, 1H), 4.02 (br s, 2H),
3.98 (s, 3H), 3.25 (br s, 2H), 2.88 (br s, 1H), 1.55 (s, 9H); IR
(neat) νmax 3271, 2979, 1694, 1662, 1601, 1538, 1497, 1384,
1311, 1288, 1231, 1160, 1026, 909, 830, 732 cm-1; FABMS
(NBA) m/z 464 (M + H+).
1
°C; H NMR (CDCl3, 500 MHz) δ 8.33 (d, 8.5, 1H), 7.84 (dd,
8.0, 0.5, 1H), 7.70 (dt, 7.5, 0.5, 1H), 7.64 (dt, 8.5, 1.5, 1H), 7.11
(s, 1H), 4.44 (t, 8.0, 2H), 4.38 (br s, 2H), 3.49 (t, 8.0, 2H), 2.06
(d, 0.5, 3H); IR (neat) 3381, 1728, 1636, 1589, 1516, 1469, 1437,
1342, 1241, 1041, 754 cm-1; FABMS (NBA) m/z 274 (M+).
Di-tert-butyl dicarbonate (3.18 g, 14.57 mmol) was added
to a solution of 2-[4-amino-2-nitronaphthalen-1-yl]ethyl acetate
(1.0 g, 3.65 mmol) in dry pyridine (10 mL) at room temperature
and the mixture was stirred overnight. The mixture was
concentrated and diluted with AcOEt (100 mL). The organic
layer was washed with H2O (30 mL) and saturated aqueous
NaCl (30 mL), and then the organic layer was dried (Na2SO4)
and concentrated under reduced pressure. Flash chromatog-
raphy (25% AcOEt-petroleum ether) afforded compound 24
(1.0 g, 73%) as a yellow solid: mp 122-124 °C; 1H NMR
(CDCl3, 500 MHz) δ 8.40 (d, 8.0, 2H), 7.94 (d, 8.0, 1H), 7.73 (t,
7.0, 1H), 7.70 (t, 7.5, 1H), 7.00 (br s, 1H), 4.45 (t, 7.5, 2H),
3.55 (t, 7.5, 2H), 2.05 (s, 3H), 1.58 (s, 9H); IR (neat) 1735, 1598,
1527, 1367, 1342, 1234, 1156, 1040, 893, 760 cm-1; HR-FABMS
(NBA) m/z 374.1487 (M+, C19H22N2O6 requires 374.1478).
2-[4-(N-tert-Butoxycarbonylamino)-2-(3-aza-4-meth-
oxycinnamoylamido)naphthalen-1-yl]ethyl acetate (25f).
A solution of acetate 24 (1.0 g, 2.67 mmol) in THF (40 mL)
was added to a suspension of 10% Pd/C (0.1 g) in chilled THF
(10 mL) and the suspension was hydrogenated (with shaking)
at 55 psi at room temperature for 1 h. The suspension was
filtered over Celite and the filtrate was concentrated under
reduced pressure to afford an orange foam. The compound was
washed with diisopropyl ether to afford 2-[2-amino-4-(N-tert-
butoxycarbonylamino)naphthalen-1-yl]ethyl acetate a pale yel-
low solid (0.48 g, 52%): mp 138-140 °C; 1H NMR (CDCl3, 500
MHz) δ 7.86 (d, 9.0, 1H), 7.72 (d, 8.5, 1H), 7.56 (br s, 1H), 7.46
(t, 7.0, 1H), 7.27 (m, 1H), 6.85 (br s, 1H), 4.26 (t, 8.0, 2H), 4.22
(br s, 2H), 3.24 (t, 7.5, 2H), 2.10 (s, 3H), 1.56 (s, 9H); IR (neat)
νmax 3379, 2977, 1727, 1626, 1534, 1504, 1456, 1391, 1367,
1238, 1158, 1043, 894, 758, 730 cm-1; FABMS (NBA) m/z 344
(M+).
To an ice-chilled solution of 2-[4-(N-tert-butoxycarbonyl-
amino)-2-(3-aza-4-methoxycinnamoylamido)naphtha-
len-1-yl]ethanol (0.16 g, 0.35 mmol) and triethylamine (0.2 mL,
1.43 mmol) in 1:1 CH2Cl2:THF (6 mL) was added methane-
sulfonyl chloride (0.1 mL, 1.29 mmol), and the mixture was
stirred for 1 h. The reaction mixture was diluted with AcOEt
(50 mL) and washed with H2O (10 mL) and saturated aqueous
NaCl (10 mL), and then the organic layer was dried (Na2SO4)
and concentrated under reduced pressure. The residue was
washed with diethyl ether to yield 2-[4-(N-tert-butoxycarbo-
nylamino)-2-(3-aza-4-methoxycinnamoylamido)naphthal en-1-
yl]ethyl methanesulfonate (0.17 g, 90%) as a white solid: mp
1
224 °C (dec); H NMR (CDCl3, 500 MHz) δ 8.34 (s, 1H), 8.29
(br s, 1H), 8.10 (br s, 1H), 7.92 (d, 8.0, 1H), 7.88 (d, 8.0, 1H),
7.84 (d, 8.5, 1H), 7.79 (d, 16.0, 1h), 7.56 (t, 7.0, 1H), 7.52 (t,
6.5, 1H), 6.85 (s, 1H), 6.79 (d, 8.5, 1H), 6.65 (d, 15.5, 1H), 4.55
(t, 6.5, 2H), 3.99 (s, 3H), 3.54 (t, 6.5, 2H), 2.94 (s, 3H), 1.56 (s,
9H); IR (neat) νmax 3248, 2980, 1688, 1660, 1626, 1601, 1532,
1497, 1384, 1356, 1288, 1249, 1229, 1173, 1023, 974, 953, 828,
756, 733 cm-1; FABMS (NBA) m/z 542 (M + H+).
To a solution of 2-[4-(N-tert-butoxycarbonylamino)-2-(3-aza-
4-methoxycinnamoylamido)naphthalen-1-yl]ethyl methane-
sulfonate (0.15 g, 0.28 mmol) in dry DMF (3 mL) was added
LiCl (0.69 g, 16.28 mmol) at room temperature under N2
atmosphere, and the mixture was stirred for 3 days. To the
reaction mixture was added H2O (20 mL), and the precipitate
was filtered. The solid was washed with MeOH to yield
chloride 26f (0.12 g, 91%) as a white solid: mp 220 °C (dec);
1H NMR (CDCl3, 500 MHz) δ 8.31 (s, 1H), 8.05 (brs, 1H), 7.89
(brs, 1H), 7.82 (d, 8.5, 1H), 7.72 (m, 2H), 7.48 (m, 2H), 6.94 (s,
1H), 6.78 (d, 7.0, 1H), 6.58 (d, 16.0, 1H), 3.99 (s, 3H), 3.74(br
s, 2H), 3.49 (br s, 2H), 3.40 (br s, 1H), 1.57 (s, 9H); IR (neat)
νmax 3224, 2997, 1715, 1687, 1652, 1617, 1602, 1544, 1501,
1402, 1384, 1310, 1289, 1242, 1165, 1024, 979,831, 754, 734
cm-1; HR-FABMS (NBA) m/z 482.1841 (M + H+, C26H29ClN3O4
requires 482.1847).
To a solution of 2-[2-amino-4-(N-tert-butoxycarbonylamino)-
naphthalen-1-yl]ethyl acetate (0.23 g, 0.67 mmol) in pyridine
(4 mL) were added (2E)-3-[6-methoxy(3-pyridyl)]prop-2-enoic
acid (0.18 g, 1.00 mmol), 1-hydroxy-benzotriazole (0.14 g, 1.00
mmol), and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI) (0.19 g, 1.00 mmol) under N2 atmosphere
at room temperature. The mixture was stirred for 2 days at
ambient temperature and then heated at 60 °C for 3 days. The
reaction mixture was diluted AcOEt (50 mL) and washed with
H2O (10 mL) and saturated aqueous NaCl (20 mL), and then
the organic layer was dried (Na2SO4) and concentrated under
reduced pressure. The residue was washed with diethyl ether
to yield compound 25f as a white solid (0.19 g, 56%): mp 208-
1
212 °C; H NMR (CDCl3, 500 MHz) δ 8.62 (br s, 2H), 8.38 (s,
1H), 7.95 (d, 8.0, 1H), 7.92 (d, 8.5, 1H), 7.87 (dd, 8.5, 2.5, 1H),
7.79 (d, 15.5, 1H), 7.55 (t, 7.5, 1H), 7.49 (t, 7.5, 1H), 6.81 (m,
3H), 4.27 (t, 7.5, 2H), 3.99 (s, 3H), 3.40 (t, 8.0, 2H), 2.19 (s,
3H), 1.55 (s, 9H); IR (neat) νmax 3256, 2980, 1736, 1688, 1661,
2-[4-(N-tert-Butoxycarbonylamino)-2-(3-aza-2,4-di-
methoxycinnamoylamido)naphthalen-1-yl]ethyl Chlo-

3914 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
ride (26g). The procedure was similar to that used in
the synthesis of compound 26f. 2-[4-(N-tert-butoxycarbon-
ylamino)-2-(3-aza-2,4-dimethoxycinnamoylamido)naphtha-
len-1-yl]ethanol was isolated as a pale brown foam (0.20 g,
99%): mp 164-168 °C; 1H NMR (CDCl3, 500 MHz) δ 8.92 (br
s, 1H), 8.27 (br s, 1H), 7.90 (d, 8.0, 1H), 7.80 (d, 15, 1H), 7.68
(d, 8.0, 1H), 7.47 (m, 2H), 6.73 (br s, 1H), 6.59 (d, 15.5, 1H),
6.33 (d, 8.0, 1H), 4.08 (br s, 2H), 4.03 (s, 3H), 3.96 (s, 3H),
3.33 (t, 5.5, 2H), 1.55 (s, 9H); IR (neat) νmax 3272, 1692, 1656,
1594, 1484, 1457, 1422, 1388, 1321, 1280, 1248, 1160, 1040,
1015, 761, 730 cm-1; FABMS (NBA) m/z 494 (M + H+).
2-[4-(N-tert-butoxycarbonylamino)-2-(3-aza-2,4-dimethoxy-
cinnamoylamido)naphthalen-1-yl]ethyl methanesulfonate (0.18
g, 93%) was isolated as a yellow solid: mp 200 °C (dec); 1H
NMR (CDCl3, 500 MHz) δ 8.27 (s br, 1H), 7.91 (d, 8.5, 2H),
7.84 (d, 15.5, 1H), 7.72 (d, 8.0, 1H), 7.56 (t, 9.0, 1H), 7.51 (t,
7.5, 1H), 7.28 (m, 1H), 6.84 (s br, 1H), 6.79 (d, 15.5, 1H), 6.36
(d, 8.0, 1H), 4.55 (t, 6.5, 2H), 4.06 (s, 3H), 3.97 (s, 3H), 3.55 (t,
6.5, 2H), 2.92 (s, 3H), 1.55 (s, 9H); IR (neat) νmax 3388, 3246,
2980, 1688, 1656, 1594, 1532, 1485, 1457, 1422, 1388, 1355,
1321, 1280, 1249, 1226, 1173, 1098, 1040, 1014, 952, 814, 761,
725 cm-1; FABMS (NBA) m/z 572 (M + H+).
Chloride 26g (0.12 g, 84%) was collected as a white solid:
mp 202 °C (dec); 1H NMR (CDCl3, 500 MHz) δ 8.14 (br s, 1H),
7.93 (t, 7.5, 2H), 7.85 (d, 15.5, 1H), 7.72 (d, 8.5, 1H), 7.69 (br
s, 1H), 7.57 (t, 7.5, 1H), 7.53 (t, 8.0, 1H), 6.83 (br s, 1H), 6.68
(d, 15, 1H), 6.36 (d, 7.5, 1H), 4.06 (br s, 3H), 3.97 (s, 3H), 3.88
(t, 7.0, 2H), 3.57 (t, 7.0, 2H), 1.55 (s, 9H); IR (neat) νmax 3219,
1688, 1656, 1621, 1594, 1541, 1501, 1484, 1453, 1404, 1386,
1320, 1284, 1250, 1162, 1098, 1075, 1016, 810, 752 cm-1; HR-
FABMS (NBA) m/z 512.1965 (M + H+, C27H31ClN3O5 requires
512.1952).
4-(2-Chloroethyl)-3-(3-aza-4-methoxycinnamoylamido)-
1-naphthylamine Dihydrochloride (11f). To a suspension
of chloride 26f (0.12 g, 0.25 mmol) in dry CH2Cl2 (10 mL) was
added 3 N HCl in AcOEt (3 mL, 9 mmol) at room temperature,
and the resulting solution was stirred overnight. The precipi-
tate was filtered to yield product 11f (0.11 g, 100%) as a pale
yellow solid: mp 230 °C (dec); 1H NMR (DMSO-d6, 500 MHz)
δ 10.20 (s, 1H), 8.45 (d, 2.5, 2H), 8.19 (d, 8.0, 1H), 8.08 (d, 8.0,
1H), 8.03 (dd, 8.5, 2.5, 1H), 7.81 (s, 1H), 7.68 (m, 1H), 7.62 (d,
15.5, 1H), 7.05 (d, 15.5, 1H), 6.93 (d, 8.5, 1H), 5.27 (br s, 2H),
3.90(s, 3H), 3.81 (t, 8.5, 2H), 3.59 (t, 8.5, 2H); IR (Nujol) νmax
3370, 1671, 1636, 1606, 1556, 1511, 1461, 1377, 1329, 1233,
1186, 1008, 974, 839, 764, 721 cm-1; HR-FABMS (NBA) m/z
382.1334 (M + H+, C21H21N3O2Cl requires 382.1322).
hydroxy-2-nitronaphthalen-1-yl]acetate (0.42 g, 42%) as a
yellow solid: mp 96-100 °C; 1H NMR (CDCl3, 500 MHz) δ
7.99 (d, 8.5, 1H), 7.86 (d, 8.0, 1H), 7.66 (t, 7.0, 1H), 7.55 (t,
7.5, 1H), 6.90 (s, 1H), 4.34 (q, 7.0, 2H), 4.29 (s, 2H), 1.38 (t,
7.5, 3H); IR (neat) νmax 3351, 2983, 1708, 1595, 1517, 1430,
1374, 1338, 1251, 1215, 1156, 1081, 1023, 847, 772, 757
cm-1
.
Anhydrous K2CO3 (0.50 g, 3.68 mmol) was added to a
solution of ethyl 2-[4-hydroxy-2-nitronaphthalen-1-yl]acetate
(0.42 g, 1.53 mmol) and benzyl bromide (0.44 mL, 3.68 mmol)
in acetone (4 mL) at room temperature, and the mixture was
stirred overnight. The reaction mixture was filtered, and the
filtrate was concentrated. The residue was diluted with AcOEt
(50 mL), and to the mixture was added N-methypiperazine (3
mL) to remove any excess benzyl bromide. The mixture was
washed with water (10 mL), 1 M HCl (10 mL), saturated
aqueous NaHCO3 (10 mL), and saturated aqueous NaCl (10
mL) and then dried (Na2SO4) and evaporated to yield com-
pound 27 (0.35 g, 63%) as a yellow solid: mp 128-130 °C; 1H
NMR (CDCl3, 500 MHz) δ 8.44 (dd, 0.5, 8.0, 1H), 8.10 (d, 8.5,
1H), 7.71 (t, 7.0, 1H), 7.66 (t, 7.0, 1H), 7.54 (d, 7.0, 2H), 7.46
(t, 7.5, 2H), 7.43 (s, 1H), 7.40 (t, 7.0, 1H), 5.32 (s, 2H), 4.33 (s,
2H), 4.21 (q, 7.0, 2H), 1.27 (t, 7.0, 3H); IR (neat) νmax 2980,
1725, 1592, 1524, 1512, 1469, 1451, 1426, 1371, 1331, 1252,
1202, 1159, 1111, 1022, 842, 770, 756, 724, 690 cm-1; EIMS
m/z 365 (M+).
2-[4-Benzyloxy-2-nitronaphthalen-1-yl]ethanol (28).
DIBAL-H (1.0 M, 3.84 mL, 3.84 mmol) was slowly added to a
solution of acetate 27 (0.35 g, 0.96 mmol) in THF (4 mL) under
N2 atmosphere at 0 °C, and the mixture was stirred for 15
min. The mixture was slowly poured into chilled 1 N HCl (10
mL), and the mixture was extracted with AcOEt (50 mL). The
organic layer was washed with 1 M HCl (10 mL), saturated
aqueous NaHCO3 (10 mL), and saturated aqueous NaCl (10
mL), and then the organic layer was dried (Na2SO4) and
evaporated to yield alcohol 28 (0.33 g, quant.) as a dark brown
1
solid: mp 82-90 °C ; H NMR (CDCl3, 500 MHz) δ 8.43 (d,
8.5, 1H), 8.25 (d, 9.0, 1H), 7.71 (t, 7.0, 1H), 7.65 (t, 7.0,1H),
7.54 (d, 8.0, 2H), 7.45 (t, 7.5, 2H), 7.40 (t, 7.0, 1H), 7.19 (s,
1H), 5.29 (s, 2H), 4.08 (t, 6.5, 2H), 3.49 (t, 6.5, 2H); IR (neat)
νmax 3368, 2929, 1593, 1526, 1513, 1463, 1423, 1363, 1337,
1271, 1240, 1161, 1103, 1039, 829, 755, 697 cm-1; EIMS m/z
323 (M+).
2-[4-Benzyloxy-2-nitronaphthalen-1-yl]ethyl Acetate
(29). Acetic anhydride (0.35 mL, 3.71 mmol) was added to a
solution of alcohol 28 (0.30 g, 0.93 mmol) in dry pyridine (3
mL) at room temperature and the mixture was stirred
overnight. The mixture was concentrated and dissolved in
AcOEt (50 mL). The organic layer was washed with 1 M HCl
(10 mL), saturated aqueous NaHCO3 (10 mL), and saturated
aqueous NaCl (10 mL), and then then the organic layer was
dried (Na2SO4) and concentrated. The residue was purified by
silica gel column (1:6 AcOEt-petroleum ether) to yield agent
29 (0.21 g, 62%) as a yellow solid: mp 82-87 °C; 1H NMR
(CDCl3, 500 MHz) δ 8.43 (d, 8.5, 1H), 8.33 (d, 8.5, 1H), 7.33
(dt, 1.0, 7.5, 1H), 7.66 (dt, 1.0, 7.0, 1H), 7.54 (d, 7.5, 2H), 7.45
(t, 7.5, 2H), 7.40 (t, 7.0, 1H), 7.28 (s, 1H), 5.30 (s, 2H), 4.47 (t,
7.0, 2H), 3.55 (t, 7.0, 2H), 2.06 (s, 3H); IR (neat) νmax 1738,
1593, 1527, 1514, 1466, 1424, 1365, 1337, 1235, 1104, 1043,
770, 752, 698 cm-1; EIMS m/z 388 (M + Na+).
2-[4-Benzyloxy-2-(5,6,7-trimethoxyindole-2-carboxami-
do)naphthalen-1-yl]ethyl Acetate (30). A solution of acetate
29 (0.20 g, 0.55 mmol) in THF (30 mL) was added to a
suspension of PtO2 (100 mg) in THF (10 mL), and the
suspension was hydrogenated (with shaking) at 55 psi at room
temperature for 1 h. The suspension was filtered over Celite
and the filtrate was concentrated in reduced pressure to yield
2-[4-benzyloxy-2-aminonaphthalen-1-yl]ethyl acetate as a brown
solid (0.18 g, 98%): mp 76-82 °C; 1H NMR (CDCl3, 500 MHz)
δ 8.24 (d, 8.0, 1H), 7.80 (d, 8.5, 1H), 7.22-7.53 (m, 7H), 6.41
(s, 1H), 5.20 (s, 2H), 4.25 (t, 8.0, 2H), 3.21 (t, 8.0, 2H),2.11 (s,
3H); IR (neat) νmax 3378, 2927, 1731, 1624, 1600, 1516, 1454,
1410, 1366, 1239, 1158, 1133, 1028, 827, 759, 698 cm-1; EIMS
m/z 335 (M+).
4-(2-Chloroethyl)-3-(3-aza-2,4-dimethoxycinnamoyl-
amido)-1-naphthylamine Dihydrochloride (11g). The pro-
cedure was similar to that used in the synthesis of 11f. The
precipitate was filtered to yield compound 11g (0.11 g, 97%)
1
as a yellow solid: mp 150-160 °C (dec); H NMR (DMSO-d6,
500 MHz) δ 9.93 (s, 1H), 8.13 (d, 8.5, 1H), 8.05 (d, 7.5, 1H),
7.95 (d, 8.5, 1H), 7.66 (d, 16.0, 1H), 7.66 (m, 1H), 7.60 (t, 7.0,
1H), 7.53 (brs, 1H), 6.97 (d, 16.0, 1H), 6.50 (d, 7.5, 1H), 4.01(s,
3H), 3.92 (m, 5H), 3.77 (t, 7.5, 2H), 3.52 (t, 8.0, 2H); IR (Nujol)
νmax 1594, 1543, 1461, 1385, 1322, 1250, 1097, 1018, 751 cm-1
;
HR-FABMS (NBA) m/z 412.1413 (M + H+, C22H23N3O3Cl
requires 412.1428).
Ethyl 2-[4-Benzyloxy-2-nitronaphthalen-1-yl]acetate
(27). To a 35% H2SO4 (50 mL) was added ethyl ester 16 (1.0
g, 3.65 mmol) at room temperature, and the mixture was
stirred overnight. To the suspension was added a solution of
NaNO2 (0.33 g, 4.74 mmol) in chilled water (1 mL) below 3
°C, and the mixture was stirred for 5 min. A few crystals of
urea were added to the mixture to decompose any excess
NaNO2. To the cold mixture was added a solution of Cu(NO3)2‚
3H2O (14.09 g, 58.33 mmol) in water (total volume was 140
mL) below 10 °C. With vigorous stirring, to the mixture was
added Cu2O (0.48 g, 3.35 mmol) and the mixture was stirred
for 3 h. The mixture was extracted with AcOEt (200 mL). The
organic layer was washed with water (50 mL) and saturated
aqueous NaCl (50 mL) and then dried (Na2SO4) and evapo-
rated. The residue was purified by silica gel column chroma-
tography (1:6 AcOEt-petroleum ether) to yield ethyl 2-[4-

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3915
To a solution of 2-[4-benzyloxy-2-aminonaphthalen-1-yl]-
ethyl acetate (0.16 g, 0.48 mmol) in pyridine (3 mL) were added
5,6,7-trimethoxy-2-carboxylic acid (0.18 g, 0.72 mmol), 1-hy-
droxybenzotriazole (0.10 g, 0.72 mmol), and EDCI (0.27 g, 1.43
mmol) under N2 atmosphere at room temperature. The mix-
ture was stirred for 3 days at room temperature and then
heated to 60 °C for 2 days. The reaction mixture was diluted
with AcOEt (50 mL); washed with 10% HCl (10 mL), saturated
aqueous NaHCO3 (10 mL), and saturated aqueous NaCl (10
mL); dried (Na2SO4); and concentrated under reduced pres-
sure. The residue was purified by silica gel column (1:4
AcOEt-petroleum ether) to yield as an orange foam compound
30 (0.10 g, 37%): mp 55-65 °C; 1H NMR (CDCl3, 500 MHz) δ
9.40 (s, 1H), 8.74 (s, 1H), 8.39 (d, 8.0, 1H), 7.93 (d, 8.5, 1H),
7.78 (s, 1H), 7.71 (dd, 3.5, 5.5, 1H), 7.57 (d, 7.5, 2H), 7.54 (dd,
3.5, 5.8, 1H), 7.44 (t, 7.5, 2H), 7.37 (t, 7.5, 1H), 7.24 (s, 1H),
6.89 (s, 1H), 5.31 (s, 2H), 4.42 (t, 7.0, 2H), 4.10 (s, 3H), 3.96 (s,
3H), 3.94 (s, 3H), 3.44 (t, 7.0, 2H), 2.17 (s, 3H); IR (neat) νmax
3293, 2963, 2933, 1731, 1634, 1593, 1538, 1503, 1465, 1409,
1370, 1306, 1239, 1111, 1075, 1048, 999, 909, 832, 761, 736,
699 cm-1; HR-FABMS (NBA) m/z 568.2204 (M + H+, C33H32-
N2O7 requires 568.2210).
4-(2-Chloroethyl)-3-(5,6,7-trimethoxyindole-2-carboxa-
mido)-1-naphthol (12). To a solution of acetate 30 (0.10 g,
0.18 mmol) in MeOH (2 mL) was added potassium carbonate
(29.1 mg, 0.21 mmol) at room temperature and the mixture
was stirred overnight. The reaction mixture was diluted with
AcOEt (50 mL) and washed with H2O (10 mL) and saturated
aqueous NaCl (20 mL), and then the organic layer was dried
(Na2SO4) and concentrated under reduced pressure to yield
2-[4-benzyloxy-2-(5,6,7-trimethoxyindole-2-carboxamido)naph-
thalen-1-yl]ethanol (82 mg, 87%) as a yellow oil: 1H NMR
(CDCl3, 500 MHz) δ 9.96 (s, 1H), 9.25 (s, 1H), 8.39 (d, 8.0, 1H),
7.85 (d, 8.5, 1H), 7.75 (s, 1H), 7.71 (dd, 3.5, 5.8, 1H), 7.57 (d,
7.5, 2H), 7.53 (m, 1H), 7.43 (t, 7.5, 2H), 7.36 (t, 7.0, 1H), 7.06
(s, 1H), 6.84 (s, 1H), 5.30 (s, 2H), 4.21 (t, 6.0, 2H), 4.08 (s, 3H),
3.95 (s, 3H), 3.91 (s, 3H), 3.37 (t, 5.0, 2H); IR (neat) νmax 3272,
2932, 1726, 1644, 1594, 1540, 1505, 1464, 1409, 1370, 1263,
1236, 1127, 1103, 1049, 997, 831, 760, 701 cm-1; EIMS m/z
527 (M + H+).
To a solution of 2-[4-benzyloxy-2-(5,6,7-trimethoxyindole-2-
carboxamido)naphthalen-1-yl]ethanol (80 mg, 0.15 mmol) and
triethylamine (0.09 mL, 0.61 mmol) in CH2Cl2 (2 mL) kept in
an ice bath was added methanesulfonyl chloride (0.05 mL, 0.61
mmol), and then the mixture was stirred for 1 h. The reaction
mixture was diluted with CH2Cl2 (50 mL) and washed with
H2O (10 mL) and saturated aqueous NaCl (20 mL), and then
the organic layer was dried (Na2SO4) and concentrated under
reduced pressure to yield 2-[4-benzyloxy-2-(5,6,7-trimethoxy-
indole-2-carboxamido)naphthalen-1-yl]ethyl methanesulfonate
(0.11 g, quant.) as a brown oil: 1H NMR (CDCl3, 500 MHz) δ
9.24 (s, 1H), 8.47 (s, 1H), 8.41 (d, 8.0, 1H), 7.85 (d, 8.5, 1H),
7.71 (dd, 3.5, 5.5, 1H), 7.60 (t, 7.0, 1H), 7.48-7.56 (m, 3H),
7.44 (t, 8.0, 2H), 7.37 (t, 7.5, 1H), 7.18 (s, 1H), 6.88 (s, 1H),
5.28 (s, 2H), 4.63 (t, 6.5, 2H), 4.09 (s, 3H), 3.95 (s, 3H), 3.93 (s,
3H), 3.56 (t, 6.5, 2H), 2.86 (s, 3H); IR (neat) νmax 3330, 3099,
2933, 2874, 1725, 1649, 1592, 1537, 1502, 1466, 1411, 1369,
1306, 1238, 1174, 1127, 1076, 1048, 998, 972, 951, 833, 796,
751, 702 cm-1; EIMS m/z 509 (M+ - CH3SO3H).
4.05 (t, 6.0, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.60 (t, 6.0, 2H); IR
(neat) νmax 3271, 2932, 1722, 1634, 1592, 1537, 1503, 1464,
1409, 1370, 1306, 1258, 1237, 1111, 1076, 1050, 998, 910, 830,
761, 736, 700 cm-1; EIMS m/z 545 (M+).
A solution of chloride 31 (70 mg, 0.13 mmol) in THF (10
mL) was added to a suspension of 10% Pd/C (50 mg) in chilled
THF (10 mL), and the suspension was hydrogenated under
H2 atmosphere at room temperature overnight. The suspension
was filtered over Celite and the filtrate was concentrated in
reduced pressure. The residue was crystallized with diethyl
ether to yield compound 12 (54 mg, 91%) as a white solid: mp
1
212 °C (dec); H NMR (CDCl3, 500 MHz) δ 9.55 (s, 1H), 8.76
(s, 1H), 8.33 (d, 8.0, 1H), 7.82 (d, 8.5, 1H), 7.78 (brs, 1H), 7.71
(s, 1H), 7.57 (t, 7.0, 1H), 7.48 (t, 7.5, 1H), 7.03 (s, 1H), 6.88 (s,
1H), 4.13 (s, 3H), 4.06 (t, 6.0, 1H), 3.97 (s, 3H), 3.93 (s, 3H),
3.61 (t, 6.0, 2H); IR (neat) νmax 3416, 3193, 3149, 2936, 1621,
1592, 1524, 1435, 1411, 1383, 1328, 1267, 1244, 1197, 1126,
1096, 1054, 852, 821, 758, 743 cm-1; FABMS (NBA) m/z 455
(M + H+), FABHR (NBA) m/z 454.1290 (M + H+, C24H23ClN2O5
requires 454.1296). Anal. (C24H23N2O5Cl) C, H, N.
Cytotoxicity Studies on Human Chronic Myeloid
Leukemia K562 Cells. The compounds were dissolved in
DMSO to obtain 1.75 × 10-2 M stock solutions, which were
diluted with DMSO to prepare standard solutions with con-
centrations from 1.75 × 10-3 to 1.75 × 10-10 M. These solutions
were then further diluted with DMEM (24 µL of standard
solution in 176 µL of media). Addition of these new drug
solutions (5 µL) to wells containing 100 µL media/cell suspen-
sion resulted in final drug concentrations ranging from 1.0 ×
10-12 M to 1.0 × 10-4 M.
The K562 human chronic myeloid leukemia cells were
maintained in RPM1 1640 medium supplemented with 10%
fetal calf serum and 2 mM glutamine at 37 °C in a humidified
atmosphere containing 5% CO2 and were incubated with a
specified dose of drug continuously for 4 days at 37 °C in the
dark. The incubation was terminated by centrifugation (5 min,
300g), and the cells were washed once with drug-free medium.
Following the appropriate drug treatment, the cells were
transferred to 96-well microtiter plates, 104 cells per well, eight
wells per sample. Plates were then kept in the dark at 37 °C
in a humidified atmosphere containing 5% CO2. The assay was
based on the ability of viable cells to reduce a yellow soluble
tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT, Sigma Chemical Co.) to an insoluble
purple formazan precipitate. Following incubation of the plates
for 4 days (to allow control cells to increase in number by 10-
fold), 20 µL of a 5 mg/mL solution of MTT in phosphate-
buffered saline was added to each well and the plates further
incubated for 5 h. The plates were then centrifuged for 5 min
at 300g, and the bulk of the medium was pipetted from the
cell pellet, leaving 10-20 µL per well. Then 200 µL of DMSO
was added to each well, and the samples were agitated to
ensure complete mixing. The optical density was then read at
a wavelength of 550 nm on a Titertek Multiscan ELISA plate
reader, and the dose-response curve was constructed. For each
curve, an IC50 value was read as the dose required to reduce
the final optical density to 50% of the control value.
Cytotoxicity Studies on Murine L1210 Leukemia, B16
Melanoma, and P815 Mastocytoma Cells. The P815, B16-
F0, and L1210 cell lines were obtained from American Type
Tissue Culture Collection (ATCC). The cell lines were grown
in Delbecco’s Modified Eagle Medium (DMEM, Atlanta Bio-
logicals) supplemented with 10% fetal bovine serum, Hepes
buffer (2 mM, Mediatech Cellgro, 25-060-Cl), L-glutamine (2
mM, Mediatech Cellgro), and penicillin/streptomycin (50 000
units penicillin, 50 000 µg streptomycin, Atlanta Biologicals).
Cells were maintained at 37 °C in a 5% humidified CO2
atmosphere. Cultured cells were counted using a hemocytom-
eter and resuspended in fresh DMEM at a concentration of 8
× 105 cells/mL. This cell suspension (100 µL) was added to
96-well flat-bottom cell 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 (5
µL/well), resulting in final concentrations ranging from 1 ×
To a solution of 2-[4-benzyloxy-2-(5,6,7-trimethoxyindole-2-
carboxamido)naphthalen-1-yl]ethyl methanesulfonate (0.11 g,
0.17 mmol) in dry DMF (2 mL) was added LiCl (0.29 g, 6.94
mmol) at room temperature under N2 atmosphere and the
mixture was stirred for 3 days. The reaction mixture was
diluted AcOEt (100 mL) and washed with H2O (20 mL) and
saturated aqueous NaCl (10 mL) and then dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatogaphy (1:4 AcOEt-petroleum
ether) to yield 2-[4-benzyloxy-2-(5,6,7-trimethoxyindole-2-car-
boxamido)naphthalen-1-yl]ethyl chloride 31 (75 mg, 81%) as
1
a white solid: mp 180-184 °C; H NMR (CDCl3, 500 MHz) δ
9.23 (s, 1H), 8.63 (s, 1H), 8.42 (d, 7.5, 1H), 7.84 (d, 8.5, 1H),
7.53-7.60 (m, 4H), 7.49 (t, 8.0, 1H), 7.44 (t, 7.0, 2H), 7.38 (t,
7.5, 1H), 6.99 (s, 1H), 6.88 (s, 1H), 5.29 (s, 2H), 4.10 (s, 3H),

3916 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Sato et al.
10-4 to 1 × 10-12 M. Quadruplicate wells were prepared for
each drug concentration. The plates were incubated for 72 h
at 37 °C in a 5% CO2 atmosphere. MTT was dissolved in PBS
(5 mg/mL). After the indicated cell incubation period, 10 µL
of this stock MTT solution was added to each well, and the
plates were further incubated for 4 h at 37 °C in a 5% CO2
atmosphere. After this final incubation, 100 µL of acid 2-pro-
panol solution (16 µL of 12.1 N HCl in 5 mL of 2-propanol)
was added to each well. The contents of the well were mixed
thoroughly by pipetting the cell suspension up and down and
the plates were allowed to sit at room temperature for 15 min
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 wavelenth of 570 nm and
a reference wavelength of 630 nm. The dose inhibiting the
growth by 50% (IC50) was extrapolated from curves generated
on the basis of the averages of the absorbance data (four points/
concentration).
NCI in Vitro Cytotoxicity Screen. Compounds 11a and
12 were also tested against a panel of 60 human cancer cell
lines at the National Cancer Institute, Bethesda, MD.28 The
cytotoxicity studies were conducted using a 48 h exposure
protocol and a sulforhodamine B assay. Dose-respose curves
were used to generate the GI50 (concentration of drug needed
to inhibit the growth by 50%), TGI (total growth inhibition),
and LC50 (concentration needed to kill 50% of cells).
solutions were added (5 µL), and the cells were incubated at
37 °C and 5% CO2 for 24 h. The cells were then harvested via
centrifugation (1000 rpm, 10 min, 4 °C) and washed twice with
sample buffer (0.5 g glucose in 500 mL Ca2+/Mg2+ free PBS;
Electron Microscopy Sciences, Fort Washington, PA). To fix
the cells, the cells were vortexed vigorously for ∼10 s, after
which the vortexing was continued at a slower rate during the
dropwise addition of 1 mL of ice-cold 70% ethanol. The tubes
were capped and stored at 4 °C until the day of the flow
cytometry analysis.
Approximately 1 h before the desired time of analysis, the
cells were stained with propidium iodide (PI). The PI staining
solution was prepared by the addition of 0.6 mL of the PI stock
solution (made from 0.8 mg of PI with 0.8 mL of water) to 12
mL of sample buffer. RNase A (EC 3.2.27.5; Sigma) was added
(1200 Kunitz units, ∼17 mg). This solution was then thor-
oughly mixed before it (1 mL) was added to the cells, which
were pelleted by centrifugation at 3000 rpm for 5 min at 4 °C.
The cells were kept on ice until they were analyzed by flow
cytometry. The analysis was completed on a Becton Dickinson
FACScan.
Hematopoietic Progenitor Assay on Compound 11a.
For direct incubation of the drug solutions with bone marrow
cells, the concentration of the drug solution was chosen to be
0.0084 µM (the IC50 value for the seco-CBI-TMI compound 4
against L1210 cells). Bone marrow cells were harvested from
the femurs of healthy DBA mice by flushing the femur with
IMEM media (∼1 mL). These cells were counted and diluted
to a concentration of 2.0 × 105 cells/mL in additional IMEM
media. The drug solutions were prepared by making a 2.52 ×
10-2 M stock solution of the drug in DMSO. To make duplicate
cultures, 0.3 mL of cells in IMEM was added to 3.0 mL of the
Methocul M3434 media (StemCell Technologies) in a 15 mL
conical tube. The tube was then vortexed to ensure complete
mixing of the cells and media. Then, 11 µL of the drug stock
solution was added to the tube and thoroughly mixed via
vortexing. This cell suspension (1 mL) was then transferred
(via 16G needle) into the wells of a 6-well plate. The plates
were incubated for 12 days at 37 °C and 5% CO2. The colonies
were counted using a dissecting microscope with a blue filter
film attached to add contrast to the cells.
In Vivo Anticancer Studies of Compounds 11a and 12
on the Growth of B16-F0 Murine Melanoma in C57BL/6
Mice. Female C57BL/6J mice were obtained from the Jackson
Laboratory and were 5-7 weeks old upon arrival. Groups of
eight mice were inoculated with B16 cells that came directly
from a culture flask without first being passaged through a
mouse. The cultured cells were washed three times with PBS
and resuspended in PBS at a concentration of 4.0 × 106 cells/
mL. The cell suspensions (50 µL) were delivered subcutane-
ously (sc) to the left flank at day 0. At this concentration, each
mouse received 200 000 tumor cells. The length and width of
the tumors were measured almost every other day using
calipers (Bel-Art Products, Vernier Type H 13415) and the
tumor volume was calculated as:
Taq Polymerase Stop Assay and Thermal Cleavage
Analysis. All drug-DNA reactions were performed in 25 mM
triethanolamine, 1 mM EDTA, pH 7.2, at 37 °C for 5 h.
Following incubation, DNA was precipitated by addition of 1/10
volume of 3 M sodium acetate and 3 volumes of 95% ethanol
and washed with 70% ethanol. The resulting pellet was dried
by lyophilization.
Prior to drug-DNA incubation, plasmid pUC18 DNA was
linearized with Hind III, 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
5′-CTCACTCAAAGGCGGTAATAC-3′ binds to the comple-
mentary (bottom) strand at position 749-769 and was used
to examine the alkylation patterns on the bottom strand. The
oligodeoxynucleotide primer was 5′-end labeled prior to am-
plification using T4 polynucleotide kinase and [γ-32P]ATP
(5000 Ci/mmol, Amersham). The labeled primers were purified
by elution through Bio-Rad spin columns. Linear amplification
of DNA was carried out in a total volume of 100 µL containing
0.5 µg of template DNA, 50 pmol of labeled primer, 250 µM of
each dNTP, 1 unit “Red Hot” Taq polymerase, 20 mM (NH4)2-
SO4, 75 mM Tris-HCl, pH 9.0, 0.01% Tween, 2.5 mM MgCl2,
and 0.01% gelatin. After an initial denaturation at 94 °C for 4
min, the cycling conditions were as follows: 94 °C for 1 min,
60 °C for 1 min, and 72 °C for 1 min, for a total of 30 cycles.
After being amplified, the samples were ethanol precipitated
and washed with 70% ethanol. Samples were dissolved in
formamide loading dye, heated for 2 min at 90 °C, cooled on
ice, and electrophoresed at 2500-3000 V for 3 h on a 80 cm ×
20 cm × 0.4 mm 6% acrylamide denaturing sequencing gel
(Sequagel, National Diagnostics). The gels were dried, and
X-ray film was exposed to the gels (Hyperfilm, Amersham).
Densitometry was carried out on a Bio-Rad GS-670 imaging
densitometer.
For the thermal cleavage analysis, the dry DNA pellets from
the drug-DNA incubations were resuspended in sodium
citrate buffer (pH 7.2) and heated to 90 C for 30 min to
thermally cleave at sites of adenine or guanine N3 lesions.
Samples were chilled, precipitated, and dried.
Flow Cytometry Studies on Compound 11a. P815 cells
were incubated with compound 11a at doses of its cytotoxic
IC50 (0.068 µΜ) and 10 times its IC50 (0.68 µM). Cisplatin (1000
µM) was used as a positive control. The drug stock solutions
were made in DMSO so that the addition of 5 µL of the solution
into 5 mL of media would result in the desired concentrations
listed above. Cells were counted and resuspended at a con-
centration of 1.5 × 105 cells/mL, and 5 mL of this cell
suspension was pipetted into a small culture flask. The drug
tumor volume (mm3) ) length (mm) × [width (mm)]2
The drug solutions were formulated by mixing the appropriate
amounts for each drug with 1.6 mL of PET (poly(ethylene
glycol) 400, absolute ethanol, and Tween 80 in 6:3:1 portions)
and 3.2 mL of 5% glucose in water. In each case clear, colorless
solutions were obtained. These solutions were stored at 20 °C
and the compound’s stability was monitored by RP-HPLC
analysis. Each mouse received 100 µL of the drug solution via
an interperitoneal (ip) administration route on days 1, 5, and
9. Each injection delivers 15 mg/kg of compound 11a and 15
mg/kg and 20 mg/kg for 12. Animal weights were taken every
other day. The control group of mice was treat on a similar
schedule with 100 µL of the vehicle, and another group of mice
was treated similarly with cyclophosphamide at a dose of 30
mg/kg in the same vehicle.
Acknowledgment. Support (to M.L.) from the Drey-
fus Foundation, Research Corporation, Taiho Pharma-

Anticancer Analogues of the Duocarmycins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3917
AT-Rich Adenine N3 Alkylation Selectivity of the Enantiomeric
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ceutical Co. of Japan, National Science Foundation-
REU, and the National Cancer Institute are gratefully
acknowledged. J.A.H. thanks Cancer Research UK for
support. The authors thank Ms. Lillia Holmes at the
Greenville Oncology Research Center, Greenville, SC,
for performing the flow cytometry experiments. The
authors also thank Dr. Robert Kelly of Pharmacia &
Upjohn for a generous gift of adozelesin and CC-1065.
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Supporting Information Available: Results from the
Taq polymerase stop assay on compounds 11a and 12, data
from an NCI cytotoxicity screen for compound 12, and an
alternative synthesis of compound 23a from alcohol 19. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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