Antitumor AHMA linked to DNA minor groove binding agents: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm0200714

DNA minor groove binder hybrid molecules, netropsin derivatives such as N-[2-(dimethylamino)-ethyl]-1-methyl-4-aminopyrrolo-2-carboxamide (MePy) or its derivatives containing two units of N-methylpyrrolecarboxamide (diMePy) and bisbenzimidazole (Ho33258),

Full text of DOI:10.1021/jm0200714

J . Med. Chem. 2002, 45, 4485-4493
4485
An titu m or AHMA Lin k ed to DNA Min or Gr oove Bin d in g Agen ts: Syn th esis a n d
Biologica l Eva lu a tion
Kamesh Rastogi,†,| J ang-Yang Chang,‡,| Wen-Yu Pan,‡ Ching-Huang Chen,† Ting-Chao Chou,§
Li-Tzong Chen,‡ and Tsann-Long Su*,†
Laboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan,
Division of Cancer Research, National Health Research Institutes, Taipei, Taiwan, and Molecular Pharmacology and
Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received February 12, 2002
DNA minor groove binder hybrid molecules, netropsin derivatives such as N-[2-(dimethylamino)-
ethyl]-1-methyl-4-aminopyrrolo-2-carboxamide (MePy) or its derivatives containing two units
of N-methylpyrrolecarboxamide (diMePy) and bisbenzimidazole (Ho33258), were linked to the
NH2 function of AHMA or to the CH2OH group of AHMA-ethylcarbamate to form AHMA-N-
netropsins (13-16) and AHMA-ethylcarbamate-O-netropsins (19-22), and AHMA-bisbenz-
imidazole (AHMA-Ho33258, 25), respectively. These conjugates’ in vitro antitumor activity,
inhibition of a variety of human tumor cell growth, revealed that AHMA-ethylcarbamate-O-
netropsin derivatives were more cytotoxic than AHMA-N-netropsin compounds. In the same
studies, all compounds bearing MePy were more potent than those compounds linked with
diMePy. Moreover, AHMA-netropsin derivatives bearing a succinyl chain as the linking spacer
were more potent than those compounds having a glutaryl bridge. Among these hybrid
molecules, AHMA-ethylcarbamate-O-succinyl-MePy (19) was 2- to 6-fold more cytotoxic than
the parent compound AHMA (5) in various cell lines, whereas compound 25 had very poor
solubility and was inactive. Studies on the inhibitory effect against topoisomerase II (Topo II)
and DNA interaction of these conjugates showed no correlation between the potency of DNA
binding and inhibitory activity against Topo II.
In tr od u ction
logical activities. A variety of natural products and
synthetic ligands have been developed and used as
molecular probes for studying the drug-DNA binding.
These agents selectively inhibit gene transcription and
can be applied for designing ligand-drug conjugate as
DNA specific targeting antitumor agents. The oligopep-
tide, N-methylpyrrolecarboxamide derivatives [dista-
mycin A (1), and netropsin (2)],9-11 and synthetic
bisbenzimidazole dye (Ho33258 and Ho33342, 3 and 4,
respectively, Chart 1)7,8 are known to bind to the minor
groove of DNA with A-T specificity and cause widening
of the minor groove.12,13 Ho33342 induces protein-DNA
cross-links and DNA strand break in cultured mam-
malian cells.14 G2-phase and chromosome endoredupli-
cation are also prominent effects of Ho33342 treat-
ment.15,16 In addition, these minor groove binders are
able to impede the catalytic activity of Topo I17,18 and
Topo II.19,20
The macromolecular DNA embodies the genetic speci-
fication of an organism, directs cellular proliferation and
differentiation, and is a potential cellular target for drug
action. Natural antitumor antibiotics such as actino-
mycin D,1 doxorubicin,2 daunomycin,3 and quinoxaline
derivatives (echinomycin, a bifunctional intercalator)4
are examples of drugs that bind to short DNA sequences
via two processes, intercalation and groove binding.
Both processes contribute to the affinity and sequence-
specific recognition to the DNA. The antitumor anti-
biotic dynemicin A5 and neocarzinostatin6 bind to DNA
by intercalating their chromophore (anthraquinone for
dynemicin and naphthoate for neocarzinostatin). A free
radical mechanism enables their active enediyne core
to cleave the DNA at a specific sequence. The mode of
interaction, sequence-specificity of binding, uptake, and
pharmacokinetics of the drug affect these agents’ anti-
tumor selectivity.
In view of these minor groove binding characteristics,
these hybrid molecules possess several properties for
rational drug design including (i) enhanced DNA-
binding strength and selectivity, (ii) interference with
topoisomerases, and (iii) facilitation of the cellular
transport of the drugs, thus promoting their potential
use in cancer chemotherapy.21 Consequently, the minor
groove binding ligands are applied widely for the design
and synthesis of DNA specific targeting antitumor
agents. DNA-alkylating agent (i.e., N-mustard deriva-
tives and anthramycin) linked with Ho33258 or dista-
mycin A22-28 and photoactivable isoalloxazine (flavin)
or bioreductive mitomycin C linked with distamycin or
netropsin were synthesized to improve the selectivity
Footprinting techniques have aided in better under-
standing of the gene’s molecular biology and established
various binding properties for DNA interacting anti-
tumor agents. As a result, many research efforts have
focused on the agent’s ability to target specific sequences
on the DNA and improve its selectivity and pharmaco-
*To whom correspondence should be addressed. Tel: +886-2-2789
9045. Fax: +886-2-2782 7685. E-mail: tlsu@ibms.sinica.edu.tw.
† Academia Sinica.
‡ National Health Research Institutes.
§ Memorial Sloan-Kettering Cancer Center.
| K. Rastogi and J .-Y. Chang contributed equally to the work
reported in this study.
10.1021/jm0200714 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/21/2002

4486 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Rastogi et al.
Ch a r t 1
Our computer modeling study of AHMA-DNA bind-
ing reveals that the NH2 function, which lies outside
the drug-DNA complex, may be favorable for inter-
action with Topo II at the active binding site. DNA
cleavage assay has been used to show that AHMA
inhibits Topo II-mediated relaxation of supercoiled DNA
and promotes DNA breaks at a subset of the cleavage
sites of m-AMSA.41 Both drugs cleave DNA at sites that
are different from those of the other Topo II inhibitors.
Although AHMA and m-AMSA have similar mecha-
nisms of action, there is a greater selectivity of DNA
cleavage sites by AHMA. The substituent(s) on the
anilino ring alter the strength of the DNA interaction
and the sequence-specific binding to poly(dA-dT)2 and
poly(dG-dC)2 of the drug. The intrinsic equilibrium
binding data show a larger binding constant for the
more cytotoxic derivatives for binding to poly(dA-dT)2
compared to that for binding to poly(dG-dC)2. However,
the circular dichroism (CD) assays demonstrate that the
NH2-substituted derivatives have a better intercalated
binding geometry to the poly(dG-dC)2, suggesting that
specific intercalations with GC-rich areas in natural
DNA for AHMA and derivatives with a free NH2 group
may stabilize the drug-DNA complex. Although the
DNA-drug sequence-specific binding of AHMA deriva-
tives may be affected by the substituent(s) on the anilino
ring, it is still unclear which substituent (NH2 or CH2-
OH) on the anilino ring of AHMA is the critical element
for Topo II inhibition and cytotoxicity. Therefore, linking
a DNA minor groove binding ligand (i.e., netropsin
derivatives or Ho33258) to the NH2 or CH2OH function
of AHMA may reveal the role of enzyme-drug interac-
tion and DNA-drug sequence-specific binding. In this
paper we describe the syntheses, antitumor effects, Topo
II inhibition, and DNA interaction of AHMA derivatives
linked to DNA minor groove binder. The results show
that the AHMA derivatives linked with one or two
unit(s) of N-methylpyrrolecarboxamide ligand (MePy or
diMePy, respectively) to the CH2OH function of AHMA-
ethylcarbamate resulted in an increase in cytotoxicity,
whereas these two ligands linked to the NH2 function
of AHMA decreased the cytotoxicity of the parent
compound.
of the anticancer agents.29,30 Several DNA-intercalators
linked to DNA minor groove binding agents, such as
ellipticine-distamycin,31,32 anthracycline-distamycin,33
and acridine-netropsin34-37 hybrid molecules, were also
designed to improve their DNA sequence-specific bind-
ing and antitumor activities.
Recently, we synthesized a series of DNA topo-
isomerase II (Topo II)-mediated anticancer 9-anilino-
acridine derivatives, namely, 3-(9-acridinylamino)-5-
hydroxymethylaniline (AHMA, 5) derivatives and their
AHMA-alkylcarbamates (6).38,39 AHMA (5) is one of the
representative compounds in the new 9-anilinoacridines
generation. This agent possesses an intriguing chemical
structure, where the substituents on the anilino ring
are in the positions meta to each other. Unlike m-
amsacrine (m-AMSA), AHMA can avoid bio-oxidation
to form quinonediimine, thus having a longer half-life
in human plasma than m-AMSA. AHMA is a potent
Topo II inhibitor and has antitumor efficacy superior
to that of m-AMSA and VP-16 in mice bearing E0771
mammary adenocarcinoma or B-16 melanoma.38 Simi-
larly, AHMA-alkylcarbamates are more potent than
their parent compounds.39 Both AHMA-ethylcarbamate
and AHMA-tert-butylcarbamate have antitumor efficacy
better than that of either m-AMSA or adriamycin in
nude mice bearing human breast tumor MX-1 and its
resistant tumor MCF-7/Ad xenografts with less toxicity
toward the host.40 Our structure-activity relationship
(SAR) study indicates that modification of the NH2 or
CH2OH function(s) with long side chains does not
greatly alter the antitumor activity of the parent
compound (AHMA).
Ch em istr y
The minor groove binder netropsin derivatives, N-[2-
(dimethylamino)ethyl]-1-methyl-4-aminopyrrole-2-car-
boxamide (7, MePy) or compound containing two units
of N-methylpyrrolecarboxamide (8, diMePy) were linked
to AHMA (5). The AHMA-netropsin molecules were
constructed using a spacer, either a 4-carbon unit
(succinyl) or a 5-carbon unit (glutaryl group), to link the
intercalator AHMA and the minor groove binder. The
known MePy (7) and diMePy (8) were linked to the NH2
function of AHMA (5) or the CH2OH group of AHMA-
ethylcarbamate (6) by following the method described
previously.31 Thus, treating AHMA (5) with succinic
anhydride (9) or glutaric anhydride (10) in the presence
of pyridine gave AHMA-N-carbonylpropionic acid mono-
carboxamide (11) and AHMA-N-carbonylbutyric acid
monocarboxamide (12) (Chart 1), respectively, in good
yield. However, occasionally, thin-layer chromatography
(TLC) showed a trace of N,O-diacyl byproduct. The
O-acyl function of the byproduct can be converted into
acid 11 or 12 by base hydrolysis. The carboxylic function

Synthesis of Antitumor AHMA Linked to DNA Binder
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4487
Sch em e 1a
a
Conditions: a, DMPA/pyridine, 85-90 °C, 12 h; b, PyBOP/pyridine, Hunig’s base at -5 °C, and then rt 12 h.
Sch em e 2a
a
Conditions: a, DMPA/pyridine, 85-90 °C, 12 h; b, PyBOP/pyridine, Hunig’s base at -5 °C, and then rt 12 h.
of 11 or 12 was condensed with MePy (7) or diMePy (8)
in a mixture of benzotriazol-1-yloxytripyrrolidinophos-
phonium hexafluorophosphate (PyBOP) and Hunig’s
base in dried DMF to afford the desired corresponding
AHMA-N-succinyl-MePy (13), AHMA-N-glutaryl-Me-
Py (14), AHMA-N-succinyl-diMePy (15), and AHMA-
N-glutaryl-diMePy (16), in good yield (50-70%).
pressure and washing the residue well with ether or
CHCl3 to remove byproducts followed by triturating
with EtOH. However, following the same procedure did
not yield pure 21. In contrast with compounds contain-
ing a succinyl linking spacer (19 and 21), compounds
20 and 22, which bear a glutaryl spacer, were isolated
in good yield by liquid chromatography.
The hybrid molecule (Ho33258, 3) was also linked to
the NH2 function of AHMA (5). The synthesis of
AHMA-N-(Ho33258-O-butyl)carboxamide (25) is shown
in Scheme 3. Treatment of Ho33258 (3) with ethyl
bromobutyrate (K2CO3/DMF, 60 °C) gave O-alkylated
compound 23, which was saponified in ethanolic NaOH
solution to yield acid 24. Condensation of 24 with
AHMA (5) in dried DMF in the presence of 2,2′-
dithiopyridine/triphenylphosphine42 gave 25 in 70%
yield.
Utilizing the same AHMA-netropsin derivatives
synthesis, AHMA-ethylcarbamate-netropsin deriva-
tives (19-22) were obtained in good yield (85%) by
treating AHMA-ethylcarbamate (6) with 9 or 10 in dry
pyridine in the presence of 4-(dimethylamino)pyridine
(DMAP) to give the AHMA-ethylcarbamate-succinylic
acid (17) or AHMA-ethylcarbamate-glutaric acid mo-
noester (18), respectively. Condensation of 17 or 18 with
MePy (7) or diMePy (8) was carried out by the same
procedure as described for the synthesis of compounds
13-16. The free products, AHMA-ethylcarbamate-O-
succinyl-MePy (19) and AHMA-ethylcarbamate-O-
succinyl-diMePy (21) were extremely unstable. Puri-
fication of these two compounds either by liquid
chromatography or preparative thin-layer chromatog-
raphy (SiO2, in CHCl3/MeOH gradient system) led to
the hydrolysis of the ester bond and liberated AHMA-
ethylcarbamate (5). Although pure 19 and 21 were
obtained after chromatography, they decomposed during
concentration or standing at room temperature. There-
Biologica l Resu lts a n d Discu ssion
In Vitr o Cytotoxicity. The cytotoxicity of AHMA
derivatives linked with DNA minor groove binder (13-
16, 19, 20, 22, and 25) against various human tumor
cells (colon HT-29, nasopharyngeal carcinoma HONE-1
and BM-1, Hepatoma Hepa-G2, Glioblastoma multifomi
DBTRG, breast carcinoma MX-1, and T-cell acute lym-
phocytic leukemia CCRF-CEM) grown in culture were
determined and the IC50 values are provided in Table
1. AHMA-N-netropsin derivatives (13-16) displayed
less cytotoxicity than either AHMA (5) or AHMA-
ethylcarbamate (6) in inhibiting of cell growth of the
1
fore, we could not obtain a uniform H NMR spectrum
and elemental analysis. However, we were able to obtain
pure 19 after removing the solvent under reduced

4488 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Rastogi et al.
Sch em e 3a
a
Conditions: a, K2CO3/DMF, 60 °C, 12 h; b, 1 N NaOH/EtOH, 60 °C, 1 h; c, 2,2′-dithiopyridine/Ph3P/DMF, -5 °C, and then rt 24 h.
Ta ble 1. In Vitro Cytotoxicity of AHMA Derivatives Linked with Minor Groove Binder Against a Variety of Human Tumor Cell
Growth
percentage of
inhibition of cell growth (IC50 µM)
protein-linked
compd
HT-29
HONE-1
BM-1
0.86
0.38
7.0
73.3
8.6
77.8
0.08
0.08
0.49
>100
TSGH
0.45
Hepa-G2
DBTRG
MX-1
CCRF-CEM
DNA breaksa
5
6
0.88
0.48
1.7
0.31
0.07
0.85
39
1.4
0.46
4.2
3.5
5.0
8.1
NDb
ND
ND
ND
ND
ND
0.039
0.085
0.156
0.36
0.057
ND
ND
ND
10.5
15.0
3.3
0.0
3.3
0.0
16.5
4.6
0.34
4.6
13
14
15
16
19
20
22
25
>100
46.8
4.8
100
7.0
73.6
20
4.4
2.4
>100
0.23
0.75
0.64
>100
63.9
0.06
0.80
0.13
>100
36.4
0.08
1.2
1.2
>100
68
>100
0.80
0.40
7.5
ND
0.09
0.08
0.63
>100
6.85
17.6
2.04
>100
4.2
>100
>100
NDb
a
The K-SDS coprecipitation assay: the percentage of protein-linked DNA breaks (PLDBs) generated by AHMA-netropsin derivatives
b
at concentration of 10 µM. Not determined.
tested tumor cell lines in culture. This clearly demon-
strates that AHMA-N-succinyl-MePy (13) and AHMA-
N-succinyl-diMePy (15) are more cytotoxic than the
corresponding AHMA-N-glutaryl-MePy (14) and AH-
MA-glutaryl-diMePy (16). The hybrid molecules bear-
ing a succinyl function (4-carbon chain) as linking spacer
were more cytotoxic than those compounds bearing a
glutaryl spacer (5-carbon chain) (13 vs 14 and 15 vs 16).
Depending upon the tumor cell lines tested, it was also
revealed that compounds with MePy were either as
potent as or more potent than compounds bearing
diMePy moiety (i.e., 13 vs 15 and 14 vs 16). For
example, compound 13 was approximately 2.5-fold more
potent than 15 in inhibition of HT-29 and HONE-1
tumor cell growth in culture. In the series of AHMA-
ethylcarbamate-O-netropsin derivatives (19, 20, and
22), we found that these compounds were significantly
more potent than the corresponding AHMA-N-netrop-
sin derivatives and, in some cases, were as potent as or
more potent than AHMA (5) or AHMA-ethylcarbamate
(6). Similarly, it revealed that compounds with a suc-
cinyl linker were more potent than compounds with a
glutaryl linker (19 vs 22), and compounds linked with
MePy were more cytotoxic than those linked with
diMePy (21 vs 22). Among these compounds, AHMA-
ethylcarbamate-O-succinyl-MePy (22) was about 1-10
times more potent than AHMA (5) or AHMA-ethylcar-
bamate (6) depending on the tumor cell lines tested. The
in vitro cytotoxicity study showed that AHMA-N-
Ho33258 (25) was inactive at the concentration up to
100 µM on the inhibition of human tumor cells tested
in culture, perhaps because of its poor solubility and
inability to penetrate into the cell.
Previously, Rene et al.37 synthesized acridine-netrop-
sin and demonstrated that the 1′-substituent on the
anilino ring of amsacrine was a critical element for Topo
II inhibition and cytotoxicity. Additionally, 9-anilino-
acridine linked with MePy was more cytotoxic than the
corresponding compound linked with diMePy. Similar
findings were shown in our present study. Furthermore,
our recent work on the synthesis of AHMA-EDTA
conjugates revealed that AHMA linked with EDTA, a
metal chalator, to the NH2 function was significantly
less cytotoxic than compounds bearing an EDTA at CH2-
OH function.43 These results demonstrated that AHMA
derivatives bearing a bulky substituent on the NH2
function decreased their cytotoxicty, indicating that the
NH2 may have played an important role in drug-Topo
II interaction.
In ter a ction of AHMA-Netr op sin Der iva tives
w ith Top o II. We had previously demonstrated that
AHMA was a potent inhibitor of Topo II.38 To investi-
gate the inhibitory activity of AHMA-netropsin deriva-
tives against Topo II, an in vitro Topo II relaxation

Synthesis of Antitumor AHMA Linked to DNA Binder
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4489
F igu r e 2. Effect of DNA unwinding by AHMA derivatives
linked with minor groove binder measured as described
previously.50 Different concentrations (0.1, 0.5, 2.5, and 10 µM)
of the following compounds were tested: lane 1, supercoiled
DNA; lane 2, DNA; lane 3, linear DNA plus T4 ligase; lanes
4-7, compound 5; lanes 8-11, compound 6; lanes 12-15,
compound 13; lanes 16-19, compound 14; lanes 20-23,
compound 15; lanes 24-27, compound 18; lanes 28-31,
compound 19; lanes 32-34, compound 20; lanes 35-39,
compound 22.
F igu r e 1. Inhibition of the DNA topoisomerase II catalytic
activity by AHMA derivatives linked with minor groove binder.
The experiment was performed by the method described
previously.48 Different concentrations (5, 10, and 25 µM) of the
following compounds were tested: lane 1, DNA alone; lane 2,
DNA plus 2 units of enzyme; lanes 3-5, compound 5; lanes
6-8, compound 6; lanes 9-11, compound 13; lanes 12-14,
compound 14; lanes 15-17, compound 15; lanes 18-20,
compound 16; lanes 21-23, compound 19; lanes 24-26,
compound 20; lanes 27-29, compound 22.
14, 22 > 18 > 5, 6, 19, and 20, suggesting that no
correlation exists between the potency of DNA binding
and inhibitory activity against Topo II. Compounds 5,
6, 19, 20, and 22 were more cytotoxic toward various
human cancer cells than other derivatives (Ta ble 1).
The present studies revealed that increasing DNA
intercalating activity did not correlate with enhancing
inhibitory activity against Topo II and cytotoxicity of
these compounds.
assay using pBR322 supercoiled DNA was performed.
As shown in Figure 1, the AHMA netropsin derivatives
were tightly bound to the double helical DNA at
concentrations between 5 and 25 µM, making it difficult
to compare the relative potency of these compounds on
Topo II inhibition. To overcome this problem, an in vitro
k-SDS coprecipitation assay was carried out to measure
the amount of protein-linked DNA breaks (PLDBs)
generated by these compounds. As shown in Table 1,
after a 30-min exposure to increasing concentrations of
these compounds, steady-state levels of PLDBs were
increased in a dose-dependent manner, followed by a
plateau of PLDBs levels (data not shown). The order of
ability to generate the maximal amount of PLDBs of
these compounds was 19, 6 > 5 > 22, 20 > 13, 15 > 14,
16 (see Table 1). The study demonstrated that AHMA-
N-netropsin derivatives (13-16) were poor inhibitors of
Topo II and therefore had less cytotoxicity.
In ter a ction of AHMA-Netr op sin Der iva tives
w ith DNA. To investigate whether enhancement of
DNA binding activity of AHMA-netropin derivatives
could increase the inhibitory activity against Topo II and
subsequently lead to an improvement in their cytotox-
icity, a DNA circular-ligation assay with linearized
pBR322 DNA and T4 ligase was performed. The assay
allowed for the detection of tertiary structure alteration
caused by DNA binding of both intercalating and
nonintercalating drugs. As shown in Figure 2, com-
pound 5 (lanes 4-7) produced a concentration-depend-
ent band shift, indicating a change in the DNA linking
number. Compound 5 produced positive supercoiled
DNA at a concentration of greater than 2.5 µM. Fur-
thermore, in the presence of compounds 13, 14, 15, and
22 at 10 µM, substrate linear DNA remained un-
changed, indicating that strong intercalating activity of
these compounds caused inhibition of T4 DNA ligase.
The order of DNA intercalating activity was 13, 15 >
Con clu sion
We have synthesized a series of AHMA-minor groove
binder hybrid molecules, AHMA-N-netropsins (13-16),
AHMA-ethylcarbamate-O-netropsins (19, 21, and 22),
and AHMA-bisbenzimidazole dye (25) (AHMA-Ho33258)
for evaluation of their in vitro antitumor activity,
inhibitory effect against Topo II, and DNA interaction.
Our study reveals that AHMA-N-netropsin derivatives
are less active than AHMA-ethylcarbamate-O-netrop-
sin derivatives. In addition, all compounds bearing
MePy were found to be more cytotoxic than those
compounds linked to diMePy in the same study. More-
over, AHMA-netropsin derivatives using succinyl chain
as the linking spacer are more potent than those
compounds having a glutaryl bridge. Among these
hybrid molecules, compound 19 is 2- to 6-fold more
cytotoxic than the parent compound, AHMA (5), in
various human tumor cell lines. Compound 25 has very
poor solubility and is inactive. The Topo II relaxation
assay reveals AHMA-N-netropsin derivatives (16-19)
as poor inhibitors of Topo II in comparison with the
AHMA-ethylcarbamate-O-netropsine derivatives. There-
fore, the former has weak in vitro cytotoxicity in all
tumor cell lines tested. In contrast, we find that there
is no correlation between the cytotoxicity of drugs tested
and their interaction with double helical DNA.
The high activity of the AHMA-ethylcarbamate-O-
netropsin derivatives shows that the hydroxymethyl
function of AHMA faces the DNA helix, whereas the
NH2 function is located outside the helix. Increasing the
size of the substituent on the NH2 function may
interfere with the drug-Topo II interaction and de-

4490 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Rastogi et al.
the residue was chromatographed over a column of silica gel
(2 × 30 cm). Compound 13 was eluted from CHCl3/MeOH/NH4-
OH (v/v 90:9:1): 226 mg (57%); mp 186-187 °C. 1H NMR
(DMSO-d6) δ 2.54 (4H, m, 2 × COCH2), 2.77 (6H, s, NMe2),
3.14 (2H, m, NCH2), 3.46 (2H, m, NHCH2), 3.76 (3H, s, NMe),
4.40 (2H, s, ArCH2), 5.17 (1H, brs, OH), 6.54 and 6.72 (each
1H, m, 2 × ArH), 7.08-7.21 (3H, m, 3 × ArH), 7.27 (1H, m,
ArH), 7.39-7.64 (4H, m, 4 × ArH), 7.97 (2H, m, 2 × ArH),
8.14 (2H, m, CONH and ArH), 9.84 and 9.91 (each 1H, s, 2 ×
CONH). Anal. (C34H37N7O4) C, H, N.
crease the cytotoxicity. Because drug-DNA binding has
no correlation with the drug’s cytotoxicity, linking MePy
moiety to the CH2OH group of AHMA-ethylcarbamate
may stabilize the formation of DNA-drug-Topo II
ternary complexes and, hence, have potent cytotoxicity.
Exp er im en ta l Section
Melting points were determined on a Fargo melting points
apparatus and are uncorrected. Column chromatography was
carried out over silica gel G60 (70-230 mesh, ASTM; Merck).
Thin-layer chromatography was performed on silica gel G60
F 254 (Merck) with short wavelength UV light for visualiza-
tion. Elemental analyses were done on a Heraeus CHN-O
rapid instrument. 1H NMR spectra were recorded on a Brucker
400 spectrophotometer with Me4Si as internal standard.
AHMA-N-Su ccin ic Acid Mon oca r boxa m id e (11). A
mixture of 5 (1.1 g, 3.48 mmol), succinic anhydride (350 mg,
3.49 mmol), and DMAP (122 mg, 0.1 mmol) in pyridine (15
mL) was heated at 85-90 °C for 12 h. The solvent was
evaporated in vacuo to dryness, and the residue was chro-
matographed over silica gel column (3 × 55 cm). Gradient
elution with CHCl3/MeOH/NH4OH (15:84:1) afforded the
compound 11: 940 mg (65.2%); mp 235-236 °C. 1H NMR
(DMSO-d6) δ 2.49-2.53 (4H, m, 2 × COCH2), 4.43 (2H, s,
ArCH2), 5.19 (1H, brs, OH), 6.44 (1H, s, ArH), 6.91 (2H, m, 2
× ArH), 7.24 (2H, m, 2 × ArH), 7.32 (2H, m, 2 × ArH), 7.54
(3H, m, 3 × ArH), 8.24 (1H, m, ArH), 9.89 (1H, s, CONH),
10.89 (1H, s, NH). Anal. (C24H21N3O4‚2H2O) C, H, N.
Through the use of the same procedure as that used for the
synthesis of 11 the following compounds were synthesized.
AHMA-N-Glu ta r ic Acid Mon oca r boxa m id e (12). Com-
pound 12 was prepared from 5 (950 mg, 3.01 mmol), glutaric
anhydride (345 mg, 3.02 mmol), and 4-(dimethylamino)-
pyridine (DMAP, 122 mg, 1.0 mmol) in pyridine (15 mL): yield
920 mg (71%); mp 179-180 °C. 1H NMR (DMSO-d6) δ 1.76
(2H, m, CH2), 2.27 (4H, m, 2 × COCH2), 4.42 (2H, s, ArCH2),
5.0 (1H, brs, OH), 6.40 (1H, s, ArH), 6.91 (2H, m, 2 × ArH),
7.20 (1H, s, ArH), 7.30 (2H, m, 2 × ArH), 7.46 (3H, m, 3 ×
ArH), 8.16 (2H, m, ArH), 9.77 (1H, s, NHCO), 10.90 (1H, brs,
NH). Anal. (C25H23N3O4‚2.5 H2O) C, H, N.
AHMA-Eth ylca r ba m a te-O-Su ccin ic Acid Mon oester
(17). Compound 17 was prepared from 5 (950 mg, 2.45 mmol),
succinic anhydride (260 mg, 2.59 mmol), and DMAP (100 mg,
0.81 mmol) in pyridine (15 mL): yield 1.0 g (84%); mp 228-
229 °C. 1H NMR (DMSO-d6) δ 1.21 (3H, t, J ) 6.4 Hz, Me),
2.23 and 2.43 (each 2H, m, 2 × COCH2), 4.07 (2H, q, J ) 6.4
Hz, CH2), 4.98 (2H, s, ArCH2), 6.38 (1H, m, ArH), 6.83 (2H,
m, 2 × ArH), 7.12 (2H, m, 2 × ArH), 7.33 (2H, m, 2 × ArH),
7.46-7.50 (3H, m, 3 × ArH), 8.14 (1H, m, ArH), 9.57 (1H, brs,
NHCO), 10.9 (1H, brs, NH). Anal. (C27H25N3O6) C, H, N.
AHMA-E t h ylca r ba m a t e-O-Glu t a r ic Acid Mon oest er
(18). Compound 18 was prepared from 5 (930 mg, 2.40 mmol),
glutaric anhydride (290 mg, 2.54 mmol), and DMAP (100 mg,
0.81 mmol) in pyridine (15 mL): yield 1.0 g (83%); mp 181-
182 °C. 1H NMR (DMSO-d6) δ 1.21 (3H, t, J ) 7.2 Hz, Me),
1.72 (2H, m, CH2), 2.23 (2H, m, CH2CO), 2.35 (2H, m, COCH2),
4.06 (2H, q, J ) 7.2 Hz, CH2), 4.98 (2H, m, ArCH2), 6.39 (1H,
s, ArH), 6.84 (1H, s, ArH), 6.90 (2H, m, 2 × ArH), 7.37 (2H,
m, 2 × ArH), 7.38-7.61 (3H, m, 3 × ArH), 8.11 (2H, m, 2 ×
ArH), 9.59 (1H, s, NHCO), 11.13 (1H, brs, NH). Anal.
(C28H27N3O6) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]-1-m eth yl-4-[[[3-(9-a cr id in -
yla m in o)-5-h yd r oxym eth yla n ilin o]ca r bon ylp r op a n oyl]-
a m in o]p yr r ole-2-ca r boxa m id e (AHMA-N-su ccin yl-Me-
P y) (13). A mixture of 11 (273 mg, 0.65 mmol) and PyBOP
(357 mg, 0.68 mmol) in dry DMF (10 mL) was stirred at -5
°C for 2 h. A solution of freshly prepared 7 (151 mg, 0.72 mmol)
in DMF (5 mL) was added slowly into the above reaction
mixture, followed by addition of Hunig’s base (0.11 mL, 0.63
mmol) at -20 °C under argon atmosphere. The temperature
was then allowed to rise to room temperature and the reaction
mixture was continuously stirred for an additional 12 h. The
solvent was evaporated under reduced pressure to dryness and
Following the same procedure as that for the synthesis of
16, the following compounds were synthesized.
N-[2-(Dim eth yla m in o)eth yl]-1-m eth yl-4-[[[3-(9-a cr id in -
yla m in o)-5-h yd r oxym et h yla n ilin o]ca r b on ylb u t a n oyl]-
a m in o]p yr r ole-2-ca r boxa m id e (AHMA-N-glu ta r yl-Me-
P y) (14). Compound 14 was prepared from 12 (230 mg, 0.53
mmol), PyBOP (313 mg, 0.60 mmol), and freshly prepared 7
(124 mg, 0.59 mmol): 182 mg (55%); mp 218-219 °C. 1H NMR
(DMSO-d6) δ 1.85 (2H, m, CH2), 2.16 (6H, s, NMe2) 2.26 (4H,
m, 2 × COCH2), 2.33 (2H, m, NCH2), 3.24 (2H, m, NHCH2),
3.77 (3H, s, NMe), 4.43 (2H, s, ArCH2), 5.15 (1H, brs, OH),
6.46 (1H, m, ArH), 6.85, 6.92, and 7.09 (6H, each 2H, 6 × ArH),
7.23-7.30 (3H, m, 3 × ArH), 7.46 (2H, m, 2 × ArH), 7.86 (2H,
m, CONH and ArH), 8.13 (1H, m, ArH), 9.76 and 9.77 (each
1H, s, 2 × CONH), and 10.87 (1H, brs, NH). Anal. (C35H39N7O4‚
1.3H2O) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]-1-m eth yl-4-[1-m eth yl-4-[1-
m eth yl-4-[[[3-(9-a cr id in yla m in o)-5-h yd r oxym eth yla n ili-
n o]ca r bon ylp r op a n oyl]a m in o]p yr r ole-2-ca r boxa m id o]-
pyr r ole-2-car boxam ide (AHMA-N-su ccin yl-diMeP y) (15).
Compound 15 was prepared from 11 (264 mg, 0.63 mmol),
PyBOP (357 mg, 0.68 mmol), and freshly prepared 8 (246 mg,
0.74 mmol): yield 250 mg (54%); mp 181-182 °C. 1H NMR
(DMSO-d6) δ 2.50 (4H, m, 2 × COCH2), 2.59 (6H, s, NMe2),
2.89 (2H, m, NCH2), 3.53 (2H, m, NHCH2), 3.81 (6H, s, 2 ×
NMe), 4.40 (2H, s, ArCH2), 5.16 (1H, brs, OH), 6.37 (1H, m,
ArH), 6.86-6.91 (4H, m, 4 × ArH), 7.13-7.22 (3H, m, 3 ×
ArH), 7.35 (1H, m, ArH), 7.40-7.58 (4H, m, 4 × ArH), 7.85
(1H, m, ArH), 8.08 (1H, s, CONH), 9.88 (3H, brs, 3 × CONH).
Anal. (C40H41N9O5) C, H, N.
N-[2-(Dim et h yla m in o)et h yl]-1-m et h yl-4-[1-m et h yl-4-
[[[3-(9-a cr id in yla m in o)-5-h yd r oxym eth yla n ilin o]ca r bo-
n ylb u t a n oyl]a m in o]p yr r ole-2-ca r b oxa m id o]-p yr r ole-2-
car boxam ide (AHMA-N-glu tar yl-diMeP y) (16). Compound
16 was prepared from 12 (304 mg, 0.70 mmol), PyBOP (400
mg, 0.76 mmol), and freshly prepared 8 (233 mg, 0.70 mmol):
1
yield 270 mg (51.8%); mp 178-180 °C. H NMR (DMSO-d6) δ
1.83 (2H, m, CH2), 2.17 (6H, s, NMe2), 2.24 (4H, m, 2 ×
COCH2), 2.41 (2H, m, J ) 2.4 Hz, NCH2), 3.28 (2H, m, J )
2.4 Hz, NHCH2), 3.79 and 3.81 (each 3H, s, 2 × NMe), 4.42
(2H, s, ArCH2), 5.14 (1H, brs, OH), 6.39 (1H, s, ArH), 6.82-
6.92 (4H, m, 4 × ArH), 7.17-7.30 (5H, m, 5 × ArH), 7.66 (3H,
m, 3 × ArH), 7.86 (2H, m, CONH and ArH), 8.15 (2H, m, 2 ×
ArH), 9.81, 9.83, and 9.84 (each 1H, s, 3 × CONH), 10.90 (1H,
s, NH). Anal. (C41H45N9O5‚2.5H2O) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]-1-m eth yl-4-[[[3-(9-a cr id in -
yla m in o)-5-(eth oxyca r bon yla m in o)ben zyloxy]ca r bon yl-
pr opan oyl]am in o]pyr r ole-2-car boxam ide (AHMA-Eth yl-
ca r ba m a te-O-su ccin yl-MeP y) (19). Compound 19 was pre-
pared from 17 (307 mg, 0.72 mmol), PyBOP (391 mg, 0.75
mmol), and freshly prepared 7 (151 mg, 0.72 mmol). The
reaction mixture was evaporated in vacuo to dryness and the
residue was washed well with ether (20 mL × 4), followed with
CHCl3 (20 mL × 3). The solid residue was then triturated with
EtOH and the resulted solid product 19 was collected by
1
filtration and dried: 260 mg (53%); mp 159-161 °C. H NMR
(DMSO-d6) δ 1.20 (3H, t, J ) 7.3 Hz, Me), 2.20 (6H, s, NMe2),
2.41 and 2.53 (each 2H, m, 2 × COCH2), 2.59 and 3.36 (each
2H, m, NCH2 and NHCH2), 3.76 (3H, s, NMe), 4.08 (2H, q,
J ) 7.3 Hz, CH2), 4.98 (2H, s, ArCH2), 6.41, 6.65, and 6.83
(each 1H, s, 3 × ArH), 7.10 (2H, m, 2 × ArH), 7.21-7.61 (5H,
m, 5 × ArH), 7.88 (2H, m, CONH and ArH), 8.05 (2H, m, 2 ×
ArH), 9.57 and 9.83 (each 1H, s, 2 × CONH), 10.93 (1H, s,
NH). MS: 679 (M+). Anal. (C37H41N7O6‚H2O) C, H, N.

Synthesis of Antitumor AHMA Linked to DNA Binder
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4491
N-[2-(Dim eth yla m in o)eth yl]-1-m eth yl-4-[[[3-(9-a cr id in -
yla m in o)-5-(eth oxyca r bon yla m in o)ben zyloxy]ca r bon yl-
bu ta n oyl]a m in o]p yr r ole-2-ca r boxa m id e (AHMA-Eth yl-
ca r b a m a t e-O-glu t a r yl-MeP y) (20). Compound 20 was
prepared from 18 (300 mg, 0.59 mmol), PyBOP (358 mg, 0.69
mmol), and fresh prepared 7 (143 mg, 0.68 mmol). The product
20 was purified by column chromatography (SiO2, CHCl3/
MeOH, 10:1, v/v): 310 mg (78%); mp 147-148 °C. 1H NMR
(DMSO-d6) δ 1.20 (3H, t, J ) 7.2 Hz, Me), 1.80 (2H, m, CH2),
2.18 (6H, s, NMe2), 2.26 (2H, m, CH2CONH), 2.37 (4H, m,
COCH2 and NCH2), 3.25 (2H, m, NHCH2), 3.76 (3H, s, NMe),
4.07 (2H, q, J ) 7.2 Hz, CH2), 5.0 (2H, s, ArCH2), 6.40 (1H, m,
ArH), 6.66 and 6.84 (each 2H, m, 4 × ArH), 7.03 (3H, m, 3 ×
ArH), 7.10 and 7.32 (each 2H, m, 4 × ArH), 7.46 (3H, m, 3 ×
ArH), 7.90 (2H, m, CONH and ArH), 9.61 and 9.78 (each 1H,
s, 2 × CONH), 10.95 (1H, s, NH). Anal. (C38H43N7O6‚2H2O) C,
H, N.
N-[2-(Dim et h yla m in o)et h yl]-1-m et h yl-4-[1-m et h yl-4-
[[[3-(9-a cr id in yla m in o)b en zyloxy]ca r b on ylp r op a n oyl]-
a m in o]p yr r ole-2-ca r b oxa m id o]p yr r ole-2-ca r b oxa m id e
(AHMA-Eth ylca r ba m a te-O-su ccin yl-d iMeP y) (21). Com-
pound 21 was prepared from 17 (300 mg, 0.61 mmol), PyBOP
(358 mg, 0.69 mmol), and freshly prepared 8 (229 mg, 0.69
mmol). The reaction mixture was evaporated to dryness, and
the residue was washed well with CHCl3 to remove the major
byproduct. The residue was triturated with EtOH to give 24,
yield 240 mg (49%), which was ca. 85% pure detected by its
1H NMR spectrum. 1H NMR (DMSO-d6) δ 1.20 (3H, t, J ) 7.3
Hz, Me), 2.55 and 2.60 (each 2H, m, 2 × COCH2), 2.73 (6H, s,
NMe2), 3.06 and 3.51 (each 2H, NCH2 and NHCH2), 3.80 and
3.81 (each 3H, s, 2 × NMe), 4.09 (2H, q, J ) 7.3 Hz, CH2),
4.99 (2H, s, ArCH2), 6.42, 6.83, and 6.89 (each 1H, s, 3 × ArH),
6.94 (2H, m, 2 × ArH), 7.12 and 7.18 (each 2H, m, 4 × ArH),
7.38-7.52 (5H, m, 5 × ArH),8.11 (2H, m, CONH and ArH),
9.57, 9.88, and 9.90 (each 1H, s, 3 × CONH).
precipitate was collected by filtration. The solid cake was
washed sequentially with water, acetone, and ether, and dried
to afford 24: 1.10 g (90%); mp 261-262 °C. H NMR (DMSO-
d6) δ 1.99 (2H, m, CH2), 2.42 (2H, t, COCH2), 2.45 (3H, s, NMe),
2.84 (4H, m, 2 × NCH2), 3.44 (4H, s, 2 × NCH2), 4.10 (2H, t,
OCH2), 6.99 (1H, d, J ) 8.6 Hz, ArH), 7.11 (3H, m, 3 × ArH),
7.50 (1H, d, J ) 8.8 Hz, ArH), 7.66 (1H, m, ArH), 8.06 (1H, d,
J ) 7.8 Hz, ArH), 8.24 (2H, d, J ) 8.8 Hz, 2 × ArH), 8.40 (1H,
s, ArH). Anal. (C29H33N5O3) C, H, N.
1
2′-[4-[3-(9-Acr id in yla m in o)-5-h yd r oxym eth yla n ilin o]-
ca r bon ylp r op oxyp h en yl]-5-(4-m eth yl-1-p ip er a zin yl)-2,5′-
bi-1H-ben zim id a zole (AHMA-Ho33258) (25). To a mixture
of 24 (350 mg, 0.68 mmol), 2,2′-thiopyridine (151 mg, 0.68
mmol), and Ph3P (178 mg, 0.68 mmol), DMF (5 mL) was added
in argon atmosphere, and the mixture was stirred for 0.5 h. A
solution of AHMA (214 mg, 0.68 mmol) in 5 mL of DMF was
added at -5 °C, and the reaction mixture was stirred for 24
h. The solvent was removed under reduced pressure. The
residue was diluted with water and ethyl acetate, and the
organic phase was washed with sodium bicarbonate solution,
dried over Na2SO4, and crystallized from ethanol to afford 25
1
(400 mg, 72%); mp >280 °C. H NMR (DMSO-d6) δ 2.13 (2H,
t, CH2), 2.55 (3H, s, NMe), 2.58 (2H, t, COCH2), 2.90 (4H, m,
2 × NCH2), 3.96 (4H, m, 2 × NCH2), 4.20 (2H, t, OCH2), 4.52
(2H, s, ArCH2), 7.07 (1H, s, ArH), 7.23-7.26 (3H, m, 3 × ArH),
7.39 (1H, d, ArH), 7.50-7.54 (2H, m, 2 × ArH), 7.63 (1H, s,
ArH), 7.77 (2H, m, 2 × ArH), 7.97 (1H, d, ArH), 8.04-8.08
(2H, m, 2 × ArH), 8.13 (2H, m, 2 × ArH), 8.29-8.34 (5H, m,
5 × ArH), 8.72 (1H, s, ArH), 10.37 (1H, s, CONH), 10.98 (1H,
s, NH). Anal. (C49H48N9O3) C, H, N.
Biologica l Assa ys
Cytotoxicity Assa ys. The effects of the compounds
on cell growth were determined in all human tumor cells
(i.e., colon HT-29, nasopharyngeal carcinoma HONE-1
and BM-1, hepatoma Hepa-G2, breast carcinoma MX-
1, gastric carcinoma TSGH, brain tumor DBTRG, and
T-cell acute lymphocytic leukemia CCRF-CEM), in a
72-h incubation, by XTT-tetrazolium assay, as described
by Scudiero et al.44 After the addition of phenazine
methosulfate-XTT solution at 37 °C for 6 h, absorbance
at 450 and 630 nm was detected on a microplate reader
(EL 340; Bio-Tek Instruments Inc., Winooski, VT). Six
to seven concentrations of each compound were used.
The IC50 and dose-effect relationships of the compounds
for antitumor activity were calculated by a median-effect
plot,45,46 using a computer program on an IBM-PC
workstation.47
N-[2-(Dim et h yla m in o)et h yl]-1-m et h yl-4-[1-m et h yl-4-
[[[3-(9-a cr id in yla m in o)-5-(eth oxyca r bon yla m in o)ben zyl-
oxy]ca r b on ylb u t a n oyl]a m in o]p yr r ole-2-ca r b oxa m id o]-
p yr r ole-2-ca r boxa m id e (AHMA-Eth ylca r ba m a te-O-glu -
ta r yl-d iMeP y) (22). Compound 22 was prepared from 18 (213
mg, 0.42 mmol), PyBOP (325 mg, 0.62 mmol), and freshly
prepared 8 (150 mg, 0.47 mmol): yield 174 mg (50.8%); mp
166-168 °C. 1H NMR (DMSO-d6) δ 1.20 (3H, t, J ) 7.0 Hz,
Me), 1.81 (2H, m, CH2), 2.20 (6H, s, NMe2), 2.29 (4H, m, 2 ×
COCH2), 2.40 (2H, m, NCH2), 3.16 (2H, m, NHCH2), 3.79 and
3.81 (each 3H, s, 2 × NMe), 4.07 (2H, q, J ) 7.0 Hz, OCH2),
4.99 (2H, s, ArCH2), 6.40 (1H, m, ArH), 6.84 and 7.15 (each
2H, m, 4 × ArH), 7.32-7.48 (5H, m, 5 × ArH), 7.94 (2H, m,
CONH and ArH), 8.10 (2H, m, 2 × ArH), 9.58, 9.81, and 9.84
(each 1H, s, 3 × CONH), 10.94 (1H, brs, NH). Anal.
(C44H49N9O7‚1.5H2O) C, H, N.
Eth yl Ho33258-O-Bu tyr a te (23). To a stirred suspension
of Ho33258 (3, 1.8 g, 3.91 mmol) and K2CO3 (1.48 g, 10.75
mmol) in DMF (20 mL) was added ethyl-4-bromobutyrate (0.8
mL, 5.55 mmol), and the reaction mixture was heated at 60
°C for 12 h. After cooling, the mixture was filtered and the
filtrate was evaporated in vacuo to dryness. The residue was
dissolved in MeOH. Silica gel (3 g) was added to the methanolic
solution and the solution was evaporated under reduced
pressure. The dry silica gel residue was moved to the top of a
silica gel column (4 × 30 cm) and chromatographed using
CHCl3 as the eluant. The product 23 was eluted from CHCl3/
MeOH (10:1, v/v) and recrystallized from EtOH: 1.4 g (68%),
mp 230-231 °C. 1H NMR (DMSO-d6) δ1.19 (3H, t, J ) 5.6
Hz, Me), 2.03 (2H, m, CH2), 2.45 (2H, m, COCH2), 2.90 (3H, s,
NMe), 3.08 and 3.16 (4H, m, 2 × NCH2), 3.90 (4H, m, 2 ×
NCH2), 4.08-4.12 (4H, m, 2 × OCH2), 7.21 (3H, m, 3 × ArH),
7.34 (1H, m, ArH), 7.73 (1H, d, J ) 9.0 Hz, ArH), 7.94 (1H, d,
J ) 8.3 Hz, ArH), 8.14 (1H, m, ArH), 8.24 (2H, m, 2 × ArH),
8.57 (1H, s, ArH). Anal. (C31H37N5O3) C, H, N.
In h ibition of Top oisom er a se II Ca ta lytic Activ-
ity by Dr u gs. Topoisomerase II catalytic activity was
assayed by the ATP-dependent relaxation of pBR322
supercoiled DNA.48 Various concentrations of the drugs
were incubated with 2 units of DNA topoisomerase II
(Topogen) and 0.25 µg of pBR322 DNA. The inhibition
of the relaxation activity is determined by comparison
with an untreated control. AHMA was used as a positive
control.
Mea su r em en t of P r ot ein -Lin k ed DNA Br ea k s.
Cells in log phase growth were labeled with [14C]-
thymidine for 24 h. After the cells were labeled, they
were trypsinized, resuspended in fresh medium at the
density of 5 × 105 cells/mL, and shaken gently in a 37
°C water bath for 1 h in suspension. Various concentra-
tions of drugs were added and incubation was continued
for an additional 0.5 h. The cells were collected and
analyzed for protein-linked DNA breaks by the potas-
sium-sodium dodecyl sulfate (K-SDS) precipitation
method, as described previously.49
Ho33258-O-Bu tyr ic a cid (24). A suspension of 23 (1.30 g,
2.46 mmol) in a mixture of 1 N NaOH (5 mL) and ethanol (5
mL) was heated at 60 °C for 1 h. The reaction mixture was
cooled and acidified with 10% HCl to pH 4, and the resulting

4492 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Rastogi et al.
(18) Chen, A. Y.; Yu, C.; Gatto, B.; Liu, L. F. DNA Minor Groove-
Binding Ligands: A Different Class of Mammalian DNA Topo-
isomerase I Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
8131-8135.
(19) Woynarowski, J . M.; McHugh, M.; Sigmund, R. D.; Beerman,
T. A. Modulation of Topoisomerase II Catalytic Activity by DNA
Minor Groove Binding Agents Distamycin, Hoechst 33258, and
4′,6-Diamidine-2-phenylindole. Mol. Pharmacol. 1989, 35, 177-
182.
(20) Fesen, M.; Pommier, Y. Mammalian Toposiomerase II Activity
Is Modulated by the DNA Minor Groove Binder Distamycin in
Simian Virus 40 DNA. J . Biol. Chem. 1989, 264, 11354-11359.
(21) Henichart, J . P.; Waring, M. J .; Riou, J . F.; Denny, W. A.; Bailly,
C. Copper-Dependent Oxidative and Topoisomerase II-Mediated
DNA Cleavage by a Netropsin/4′-(9-Acridinylamino)methane-
sulfon-m-anisidide Combilexin. Mol. Pharmacol. 1997, 51, 448-
461.
(22) Gupta, R.; Huang, L.; Wang, H.; Lown, J . W. Design, Synthesis,
DNA Sequence Preferential Alkylation and Biological Evaluation
of N-Mustard Derivatives of Hoechst 33258 Analogues. Anti-
Cancer Drug Des. 1995, 10, 25-41.
(23) Arcamone, F. M.; Animati, F.; Barbieri, B.; Configliacchi, E.;
D’Alessio, R.; Geroni, C.; Giuliani, F. C.; Lazzari, E.; Menozzi,
M.; Mongelli, N. Synthesis, DNA-Binding Properties, and Anti-
tumor Activity of Novel Distamycin Derivatives. J . Med. Chem.
1989, 32, 774-778.
(24) Broggini, M.; Erba, E.; Ponti, M.; Ballinari, D.; Geroni, C.;
Spreafico, F.; D’Incalci, M. Selective DNA Interaction of the
Novel Distamycin Derivative FCE 24517. Cancer Res. 1991, 51,
199-204.
(25) Baraldi, P. G.; Balboni, G.; Romagnoli, R.; Spalluto, G.; Cozzi,
P.; Geroni, C.; Mongelli, N.; Rutigliano, C.; Bianchi, N.; Gambari,
R. PNU 157977: a New Potent Antitumor Agent Exhibiting Low
In Vivo Toxicity in Mice Injected with L1210 Leukaemia Cells.
Anti-Cancer Drug Des. 1999, 14, 71-76.
DNA Un w in d in g Mea su r em en t. The DNA unwind-
ing effect of drugs was assayed using a method described
previously.50 Briefly, plasmid DNA was linearized with
HindIII restriction endonuclease and recovered by
phenol extraction and ethanol precipitation. Reaction
mixture containing 66 mM Tris-HCl (pH 7.6), 6 mM
MgCl2, 10 mM dithiotreitol, 0.7 mM ATP, and 0.6 µg of
DNA, and drugs were equilibrated at 15 °C for 10 min
and then incubated with an excess amount of T4 DNA
ligase at 15 °C for 60 min. The reaction was stopped by
addition of 20 mM EDTA. DNA was analyzed by agarose
gel electrophoresis after removal of drugs from the
reaction mixture: extraction with phenol and ether, and
precipitation with ethanol. The DNA circle-ligation
assay, using nicked DNA as substrate, was performed
by a method based on that of Montecucco et al.51
Ack n ow led gm en t. This work was supported by the
National Science Council, Taiwan (Grant NSC90-2320-
B-001-032).
Refer en ces
(1) Takusagawa, F.; Dabrow, M.; Neidle, S.; Berman, H. M. The
Structure of a Pseudo Intercalated Complex Between Actino-
mycin and the DNA Binding Sequence d(GpC). Nature 1982,
296, 466-469.
(2) Frederick, C. A.; Williams, L. D.; Ughetto, G.; Van der Marel,
G.; Van Boom, J . H.; Rich, A.; Wang, A. H. J . Structural
Comparison of Anticancer Drug-DNA Complexes: Adriamycin
and Daunomycin. Biochemistry 1990, 29, 2538-2549.
(3) Quigley, G. J .; Wang, A. H.; Ughetto, G.; Van der Marel, G. A.;
Van Boom, J . H.; Rich, A. Molecular Structure of an Anticancer
Drug-DNA Complex: Daunomycin Plus d(CpGpTpApCpG).
Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 7204-7208.
(4) Ughetto, G.; Wang, A. H.; Quigley, G. J .; Van der Marel, G. A.;
Van Boom, J . H.; Rich, A. A Comparison of the Structure of
Echinomycin and Triostin A Complexed to a DNA Fragment.
Nucleic Acids Res. 1985, 13, 2305-2323.
(5) Sugiura, Y.; Shiraki, T.; Konishi, M.; Oki, T. DNA Intercalation
and Cleavage of an Antitumor Antibiotic Dynemicin that
Contains Anthracycline and Enediyne Cores. Proc. Natl. Acad.
Sci. U.S.A. 1990, 87, 3831-3835.
(26) Turner, P. R.; Ferguson, L. R.; Denny, W. A. Polybenzamide
Mustards: Structure-Activity Relationships for DNA Sequence-
Specific Alkylation. Anti-Cancer Drug Des. 1999, 14, 61-70.
(27) Cozzi, P.; Beria, I.; Caldarelli, M.; Capolongo, L.; Geroni, C.;
Mazzini, S.; Ragg, E. Phenyl Sulfur Mustard Derivatives of
Distamycin A. Bioorg. Med. Chem. Lett. 2000, 10, 1653-1656.
(28) Walker, W. L.; Kopka, M. L.; Filipowsky, M. E.; Dickerson, R.
E.; Goodsell, D. S.. Design of B-DNA Cross-Linking and Sequence-
Reading Molecules. Biopolymers 1995, 35, 543-553.
(29) Herfeld, P.; Helissey, P.; Giorgi-Renault, S.; Goulaouic, H.; Pager,
J .; Auclair, C. Poly(pyrrolecarboxamides) Linked to Photoacti-
vable Chromophore Isoalloxazine. Synthesis, Selective Binding,
and DNA Cleaving Properties. Bioconjugate Chem. 1994, 5, 67-
76.
(6) Lee, S. H.; Goldberg, I. H. Sequence-Specific, Strand-Selective,
and Directional Binding of Neocarzinostatin Chromophore to
Oligodeoxyribonucleotides. Biochemistry 1989, 28, 1019-1026.
(7) Pjura, P. E.; Grzeskowiak, K.; Dickerson, R. E. Binding of
Hoechst 33258 to the Minor Groove of B-DNA. J . Mol. Biol. 1987,
197, 257-271.
(8) Durand, R. E.; Oliver, P. L. Cytotoxicity, Mutagenicity, and DNA
Damage by Hoechst 33342. J . Histochem. Cytochem. 1982, 30,
111-116.
(30) Paz, M. M.; Das, T. A.; Tomasz, M. Mitomycin C Linked to DNA
Minor Groove Binding Agents: Synthesis, Reductive Activation,
DNA Binding and Cross-Linking Properties and In Vitro Anti-
tumor Activity. Bioorg. Med. Chem. 1999, 7, 2713-2726.
(31) Bailly, C.; Leclere, V.; Pommery, N.; Colson, P.; Houssier, C.;
Rivalle, C.; Bisagni, E.; He´nichart, J .-P. Binding to DNA,
Cellular Uptake and Biological Activity of a Distamycin-Ellip-
ticine Hybrid Molecule. Anti-Cancer Drug Des. 1993, 8, 145-
164.
(9) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E.
The Molecular Origin of DNA-Drug Specificity in Netropsin and
Diastamycin. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376-1380.
(10) Dattagupta, N.; Hogan, M.; Crothers, D. M. Interaction of
Netropsin and Distamycin with Deoxyribonucleic Acid: Electric
Dichroism Study. Biochemistry 1980, 19, 5998-6005.
(11) Coll, M.; Frederick, C. A.; Wang, A.-H.; Rich, A. A Bifurcated
Hydrogen-Bonded Conformation in the d(AT) Base Pairs of the
DNA Dodecamer d(CGCAAATTTGCG) and its Complex with
Distamycin. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8385-8389.
(12) Gilbert, D. E.; Feigon, J . Structure Analysis of Drug-DNA
Interaction. Curr. Opin. Struct. Biol. 1991, 1, 439-445.
(13) Neidle, S.; Pearl, L. H.; Skelly, J . V. DNA Structure and
Perturbation by Drug Binding. Biochem. J . 1987, 243, 1-13.
(14) Smith, P. J .; Bell, S. M.; Dee, A.; Sykes, H. Involvement of DNA
Topoisomerase II in the Selective Resistance of a Mammalian
Cell Mutant to DNA Minor Groove Ligands, Ligand-Induced
DNA-Protein Cross-linking and Responses to Topoisomerase
Poisons. Carcinogenesis 1990, 11, 659-665.
(32) Bailly, C.; O’Hugin, C.; Houssin, R.; Colson, P.; Houssier, C.;
Rivalle, C.; Bisagni, E.; He´nichart, J .-P.; Waring, M. J . DNA-
Binding Properties of Distamycin-Ellipticine Hybrid Molecule.
Mol. Pharmcol. 1995, 41, 845-855.
(33) Arcamone, F. Design and Synthesis of Anthracycline and Dis-
tamycin Derivaitves as New Sequence-Specific, DNA-Binding
Pharmacological Agents. Gene 1994, 149, 57-61.
(34) Eliadis, A.; Phillips, D. R.; Reiss, J . A.; Skorobogaty, A. The
Synthesis and DNA Footprinting of Acridine-Linked Netropsin
and Distamycin Bifunctional Mixed Ligands. J . Chem. Soc.,
Chem. Commun. 1988, 1049-1052.
(35) Bailly, C.; Collyn-D’Hooghe, M.; Lantoine, D.; Fournier, C.;
Hecquet, B.; Fosse, P.; Saucier, J .-M.; Colson, P.; Houssier, C.;
He´nichart, J .-P. Biological Activity and Molecular Interaction
of a Netropsin-Acridine Hydrid Ligand with Chromatin and
Topoisomerase II. Biochem. Pharmacol. 1992, 43, 457-466.
(36) Bourdouxhe-Housiaux, C.; Colson, P.; Houssier, C.; Waring, M.
J .; Bailly, C. Interaction of a DNA-Threading Netropsin-Amsa-
crine Combilexin with DNA and Chromatin. Biochemistry 1996,
35, 4251-4264.
(37) Rene, B.; Fosse, P.; Khelifa, T.; J acquemin-Sablon, A.; Bailly,
C. The 1′-Substituent on the Anilino Ring of the Antitumor Drug
Amsacrine is a Critical Element for Topoisomerase II Inhibition
and Cytotoxicity. Mol. Pharmacol. 1996, 49, 343-350.
(38) Su, T.-L.; Chou, T.-C.; Kim, J . Y.; Huang, J .-T.; Ciszewska, G.;
Ren, W.-Y.; Otter, G. M.; Sirotnak, F. M. Watanabe, K. A.
9-Substituted Acridine Derivatives with Long Half-Life and
Potent Antitumor Activity: Synthesis and Structure Activity
Relationships. J . Med. Chem. 1995, 38, 3226-3235.
(15) Hirschberg, J .; Lavi, U.; Goitein, R.; Marcus, M. The Pleiotropic
effects of 33258 Hoechst on the Cell Cycle in Chinese Hamster
Cells in vitro. Exp. Cell Res. 1980, 130, 63-72.
(16) Kusyk, C. J .; Hsu, T. C. Induction of High Frequencies of
Endoreduplication in Mammalian Cell Cultures with 33258
Hoechst and Rubidazone. Cytogenet. Cell Genet. 1979, 23, 39-
43.
(17) McHugh, M. M.; Woynarowski, J . M.; Sigmund, R. D.; Beerman,
T. A. Effects of Minor Groove Binding Drugs on Mammalian
Topoisomerase I Activity. Biochem. Pharmacol. 1989, 38, 2323-
2328.

Synthesis of Antitumor AHMA Linked to DNA Binder
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4493
(39) Su, T.-L.; Chen, C.-H.; Huang, L.-F.; Chen, C.-H.; Basu, M. K.;
Zhang, X.-G.; and Chou, T.-C. Synthesis and Structure-Activity
Relationships of Potential Anticancer Agents: Alkylcarbamates
of 3-(9-Acridinylamino)-5-hydroxymethylaniline J . Med. Chem.
1999, 42, 4741-4748.
(40) Su, T.-L.; Chou, T.-C. Development of 3-(9-Acridinylamino)-5-
hydroxymethylaniline as Potential Topoisomerase II-Mediated
Anticancer Agents. Cancer Detect. Prev. 2000/ Suppl. 2000, 24,
211.
(41) Scarborough, A.; Su, T.-L.; Leteutre, F. F.; Pommier, Y.; Chou,
T.-C. DNA Interaction and Topoisomerase II Inhibition by the
Antitumor Agent 3-(9-Acridinylamino)-5′-hydroxymethylaniline
and Derivatives. Bioorg. Med. Chem. 1996, 24, 229-241.
(42) Corey, E. J .; Nicolaou, K. C. Effect and Mild Lactonization
Method for the Synthesis of Macrolides. J . Am. Chem. Soc. 1974,
96, 5614-5616.
(43) Rastogi, K.; Chou, T.-C.; Ting, C.-Y.; Chen, K.-T.; Hwang, J .;
Su, T.-L. Synthesis and Biological Activity of AHMA-EDTA
Conjugates. Submitted for publication.
(44) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
Tierney, S.; Nofziger, T. H.; Currens, M. J .; Seniff, D.; Boyd, M.
R. Evaluation of Soluble Tetrazolium/Formazan Assay for Cell
Growth and Drug Sensitivity in Culture Using Human and
Other Tumor Cell Lines. Cancer Res. 1988, 48, 4827-4833.
(45) Chou, T.-C.; Talalay, P. Quantitative Analysis of Dose-Effect
Relationships: The Combined Effects of Multiple Drugs or
Enzyme Inhibitors. Adv. Enzyme Regul. 1984, 22, 27-55.
(46) Chou, T.-C. The Median-Effect Principle and the Combination
Index for Quantitation of Synergism and Antagonism. In
Synergism and Antagonism in Chemotherapy; Chou, T.-C.,
Rideout D., Eds.; Academic Press: New York, 1991; pp 61-102.
(47) Chou, J .; Chou, T.-C. Dose-Effect Analysis with Microcomput-
ers: Quantitation of ED50, LD50, Synergism, Antagonism, Low-
Dose Risk, Receptor-Ligands Binding and Enzyme Kinetics;
Biosoft: Cambridge, U.K., 1987.
(48) Yamashita, Y.; Kawada, S.; Fujii, N.; Nakano, H. Induction of
Mammalian DNA Topoisomerase I and II Mediated DNA Cleav-
age by Saintopin,
a New Antitumor Agent from Fungus.
Biochemistry 1991, 30, 5838-5845.
(49) Rowe, T. C.; Chen, G. L.; Hsiang, Y. H.; Liu, L. F. DNA Damage
by Antitumor Acridines Mediated by Mammalian DNA Topo-
isomerase II. Cancer Res. 1986, 46, 2021-2026.
(50) Camilloni, G.; Seta, F. D.; Negri, R.; Ficca, A. G.; Di Mauro, E.
Structure of RNA Polymerase II Promoters: Conformational
Alterations and Template Properties of Circularized Saccharo-
myces cerevisiae GAL1-GAL10 Divergent Promoters. EMBO J .
1986, 5, 763-771.
(51) Montecucco, A.; Pedrali-Noy, G.; Spadari, S.; Zanolin, E.; Ciar-
rocchi, G. DNA Unwinding and Inhibition of T4 DNA Ligase by
Anthracyclines. Nucleic Acids Res. 1988, 16, 3907-3918.
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