DNA interaction of bromomethyl-substituted acridines

Article abstract of DOI:10.1007/s00044-017-2030-7

A series of acridines with bifunctional substituents was synthesized with the dual properties of DNA alkylation and intercalation. 4,5-Bis(bromomethyl)acridine (1) was previously reported to crosslink and intercalate with DNA. In this study, 1,8-bis(bromomethyl)acridine (2) and 2,7-bis(bromomethyl)acridine (3), monofunctional 2-(bromomethyl)-7-methylacridine (4) and 2,7-dimethylacridine (5) were synthesized, and their crosslinking and intercalative activities were investigated to assess the reactivity of bromomethyl acridines with DNA. Interstrand crosslinking activity was similar among the three bis(bromomethyl)acridines. The acridines exhibited intercalation activity for calf thymus DNA as follows: 3 > 4 > 2 = 1 5. Intracellular DNA-crosslinking and DNA-intercalating activities were evaluated using the Ames assay. 4 was mutagenic in Salmonella typhimurium TA100 and TA98, indicating that the bromomethyl group alkylated DNA bases. All three bis(bromomethyl)acridines were mutagenic in S. typhimurium TA92 and TA94, which can detect intracellular crosslinking DNA damage, whereas 5 was not mutagenic in these strains. The results showed that the bis(bromomethyl)acridines crosslinked DNA and intercalated between DNA bases, and 3 exhibited the highest crosslinking and intercalating activity.

Full text of DOI:10.1007/s00044-017-2030-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2030-7
ORIGINAL RESEARCH
DNA interaction of bromomethyl-substituted acridines
1 ●
1 ●
2 ●
1 ●
Kazuya Harada Takahiro Imai Junko Kizu Masataka Mochizuki
Keiko Inami
1
Received: 12 June 2017 / Accepted: 11 August 2017
©
Springer Science+Business Media, LLC 2017
Abstract A series of acridines with bifunctional sub-
stituents was synthesized with the dual properties of DNA
alkylation and intercalation. 4,5-Bis(bromomethyl)acridine
intercalated between DNA bases, and 3 exhibited the
highest crosslinking and intercalating activity.
●
●
●
(
1) was previously reported to crosslink and intercalate with
DNA. In this study, 1,8-bis(bromomethyl)acridine (2) and
,7-bis(bromomethyl)acridine (3), monofunctional 2-(bro-
momethyl)-7-methylacridine (4) and 2,7-dimethylacridine
5) were synthesized, and their crosslinking and inter-
Keywords Acridine Halomethyl group Crosslink
●
Intercalation Mutagenicity
2
(
calative activities were investigated to assess the reactivity
of bromomethyl acridines with DNA. Interstrand cross-
linking activity was similar among the three bis(bromo-
methyl)acridines. The acridines exhibited intercalation
activity for calf thymus DNA as follows: 3 > 4 > 2 = 1
Introduction
Most cancer cells divide more often than normal cells, and
the cell division process can be targeted to treat cancer
patients. DNA is a primary cancer therapy target because
DNA replication is an essential phase of the cell cycle.
Many cytotoxic agents commonly used to treat cancer
patients cause a high level of DNA damage (Huang and Li
2013).
DNA crosslinkers have been widely used in cancer
treatment, including nitrogen mustard derivatives such as
melphalan and cyclophosphamide, platinum-containing
drugs such as cisplatin and carboplatin, and antibiotics
such as mitomycin C and actinomycin D (Brulikova et al.
>>> 5. Intracellular DNA-crosslinking and DNA-
intercalating activities were evaluated using the Ames
assay. 4 was mutagenic in Salmonella typhimurium TA100
and TA98, indicating that the bromomethyl group alkylated
DNA bases. All three bis(bromomethyl)acridines were
mutagenic in S. typhimurium TA92 and TA94, which can
detect intracellular crosslinking DNA damage, whereas 5
was not mutagenic in these strains. The results showed that
the bis(bromomethyl)acridines crosslinked DNA and
2012; Noll et al. 2006).
DNA crosslinking agents do not react specifically with
DNA; thus, new anticancer drugs with activity only towards
DNA are desired (Hurley 2002). One strategy to improve
the selectivity and toxicity profile of DNA crosslinking
agents is to design a drug with a carrier system using a
DNA-intercalating moiety. Acridines can be considered as
the archetype of intercalators and are the most extensively
used intercalators in anticancer drug design (Demeunynck
et al. 2001, Ehsanian et al. 2011). Studies on acridine
derivatives yielded amsacrine, an aminoacridine derivative
that has been used clinically to treat acute leukemia and
Electronic supplementary material The online version of this article
(
doi:10.1007/s00044-017-2030-7) contains supplementary material,
which is available to authorized users.
*
Keiko Inami
inami@rs.noda.tus.ac.jp
1
2
Faculty of Pharmaceutical Sciences, Tokyo University of Science,
Yamazaki 2641, Noda-shi, Chiba 278-8510, Japan
Faculty of Pharmacy, Keio University, Shibakoen 1-5-30, Minato-
ku, Tokyo 105-8512, Japan

Med Chem Res
lymphoma (Louie and Issell 1985, Nitiss 2009); therefore,
acridine derivatives have been previously applied to the
search for potential anticancer agents (Demeunynck et al.
2
2
2
001; Ehsanian et al. 2011; Louie and Issell 1985; Nitiss
009; Gao et al. 2015; Schmidt and Liu 2015; Ketron et al.
012; Su et al. 2006).
Fig. 1 Structure of substituted acridines at various positions
We previously described the synthesis and crosslinking
activity of bisfunctional aromatic compounds (Higashi et al.
009; Higashi et al. 2008a). N-Nitroso-N-(arylcarbonylox-
2
ymethyl)-3-chloropropylamines is an acridine analog that
acts as an effective DNA crosslinker (Ishikawa et al. 2004;
Inami et al. 2009). In addition, 4,5-bis(bromomethyl)acri-
dine (1) has cytotoxic effects on a human T cell leukemia
cell line due to DNA interstrand-crosslinking and DNA
interstrand-intercalating activities (Higashi et al. 2008b). In
this study, 1,8- (2) and 2,7-bis(bromomethyl)acridine (3), 2-
peak. High-resolution mass spectra were collected using a
JEOL JMS-SX102A mass spectrometer (Tokyo, Japan).
Fluorescence spectra were obtained using a JASCO FP-
6500 spectrophotofluorometer (Tokyo, Japan).
Compound 1 [158.5 °C for sublimation (mp 156 °C)
(Higashi et al. 2008b; Chiron and Galy 2003)], 3 [202 °C
for sublimation] (Bartoli et al. 2009), and 5 [173.6–173.9 °C
(187 °C) (Dimroth and Criegee 1957)] were prepared
according to previously reported procedures. Melting point
for 1 has been reported previously, however, the melting
point was found to be the sublimation point. The structure
(
bromomethyl)-7-methylacridine (4) and 2,7-dimethylacri-
dine (5) were prepared to investigate the effect of sub-
stituent positions in the acridine ring on DNA crosslinking
and intercalating properties (Fig. 1).
1
1
of 1 was confirmed by H NMR and HRMS. H NMR
CDCl , 400 MHz): δ = 8.79 (1 H, s, H-9), 8.00 (2 H, d,
(
3
Experimental
J = 8.5 Hz, H-3, H-6), 7.94 (2 H, dd, J = 8.5, 6.8 Hz, H-1,
H-8), 7.68 (2 H, d, J = 6.8 Hz, H-2, H-7), 5.43 (4 H, s,
7
9
Materials and methods
CH Br); HREIMS: m/z 362.9258 C H N Br (calcd.
2
15 11
2
362.9258).
Bacto agar and bacto nutrient broth were obtained from
Becton Dickinson and Company (Franklin Lakes, NJ,
USA). Sodium ammonium hydrogen phosphate tetrahydrate
was purchased from Merck (Darmstadt, Germany). λ Hin-
dIII digest was obtained from Roche Diagnostics (Basel,
Switzerland). pBR322 DNA was purchased from New
England BioLabs Japan Inc. (Tokyo, Japan). Agarose-I and
ethylenediamine-N,N,N’′,N′′-tetraacetic acid disodium salt
1,8-Bis(bromomethyl)acridine (2)
3,5-Dinitrotoluene (912 mg, 5 mmol) and tin (3.58 g, 30
mmol) were dissolved in hydrochloric acid (15 mL) and
stirred at 65 °C for 1 h. After cooling the mixture to room
temperature, water (30 mL) was added to the mixture,
which was then washed with CH Cl (20 mL). The aqueous
2
2
dehydrate (EDTA・2Na) were purchased from Dojindo
phase was alkalized with 5 M NaOH aqueous solution
®
(
Kumamoto, Japan). Briplatin was obtained from Bristol-
(120 mL) and then was extracted with CH Cl (40 mL × 5).
2
2
Myers Company (New York City, NY, USA). Bromophe-
nol blue and p-toluidine were purchased from Tokyo Che-
mical Industry Co., Ltd. (Tokyo, Japan). Cyclohexane and
The combined organic phase was dried over anhydrous
sodium sulfate, filtered, and concentrated under reduced
pressure to produce the colorless oil 3,5-diaminotoluene.
3,5-Diaminotoluene (309 mg, 2.5 mmol), glycerol
(922 mg), hydrochloric acid (0.25 mL, 2.9 mmol), and for-
mic acid (0.05 mL, 1.25 mmol) were stirred at 155 °C for
30 min and then stirred at 170 °C for an additional 30 min.
After cooling the mixture to room temperature, water (20
mL) and 1 M NaOH aqueous solution (30 mL) were added.
A precipitate was generated, filtered under a vacuum and
washed with water to produce a brown solid, crude 1,8-
dimentylproflavine (215 mg). Crude 1,8-dimethylproflavine
(205 mg, 0.85 mmol) was dissolved in a mixture of ethanol
(9 mL) and sulfuric acid (45 mL). After the mixture was
heated to 80 °C, sodium nitrite (298 mg, 4.25 mmol) was
slowly added, and the mixture was stirred for 3 h. After
cooling the mixture to room temperature, water (50 mL) and
2
,2′-azobis(isobutyronitrile) (AIBN) were obtained from
Kanto Chemical Co., Inc. (Tokyo, Japan). The remaining
reagents were obtained from Wako Pure Chemical Indus-
tries, Ltd. (Osaka, Japan).
The reaction progress was monitored using thin-layer
chromatography (TLC) on silica gel 60 F254 (0.25 mm,
Merck, Darmstadt, Germany). Column chromatography
was performed using silica gel 60 (0.040–0.063 mm,
Merck) or aluminum oxide 90 active basic (0.063–0.200
mm, Merck). Melting points were determined using a
Yanaco micro-melting point apparatus without correction
(
Tokyo, Japan). NMR spectra were recorded with a JEOL
JNM-LA400 spectrometer (Tokyo, Japan). Chemical shifts
were expressed in ppm downfield shifted from the TMS

Med Chem Res
5
M NaOH aqueous solution (10 mL) were added. The
solid appeared and was filtered out. The filtrate was con-
centrated under reduced pressure to produce a yellow solid,
which was purified on silica gel (dichloromethane) to gen-
erate the desired product as a yellow solid (87 mg). The
solid was recrystallized from cyclohexane to yield the single
compound 4 (22 mg).
mixture was extracted with CH Cl2 (20 mL × 5). The
2
combined organic phase was dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure to
generate a brown solid. The crude product was fractionated
on aluminum oxide (in 5% water) [chloroform:n-hexane
(
1:2)] and then recrystallized from ethanol and water to
yield the single compound 1,8-dimethylacridine (10 mg).
2-(bromomethyl)-7-methylacridine
(4) Yellow
solid
(
recrystallized from cyclohexane); yield 30.1%; TLC
1
1
,8-dimethylacridine Yellow needle crystal; yield, 4%;
(dichloromethane) R 0.4; sublimation point, 156 °C; H
f
TLC (hexane/ethyl acetate 3:1) R 0.5; mp 166.8–170.0 °C;
NMR (CDCl , 400 MHz): δ = 8.62 (1 H, s, H-9), 8.22 (1 H,
d, J = 8.8 Hz, H-4), 8.15 (1 H, d, J = 8.8 Hz, H-5), 7.96 (1
H, d, J = 2.0 Hz, H-1), 7.78 (1 H, dd, J = 2.2, 9.0 Hz, H-3),
f
3
1
H NMR (CDCl , 400 MHz): δ = 9.01 (1 H, s, H-9), 8.10
3
(
2 H, d, J = 8.8 Hz, H-4, H-5), 7.69 (2 H, dd, J = 8.8, 8.8
Hz, H-3, H-6), 7.38 (2 H, d, J = 8.8 Hz, H-2, H-7), 2.85 (6
7.75 (1 H, d, J = 0.48 Hz, H-8), 7.65 (1 H, dd, J = 1.7, 8.8
1
3
13
H, s, CH3); C NMR (CDCl , 100 MHz,): δ = 148.9 (C, C-
Hz, H-6), 4.71 (2 H, s, CH Br), 2.59 (3 H, s, CH );
C
3
2
3
4
a, C-5a), 134.9(C), 130.0 (CH, C-9), 128.8 (CH, C-3, C-
NMR (CDCl , 100 MHz): δ = 148.4 (CH, C-9), 148.0 (C,
3
6
), 127.6 (CH, C-4,C-5), 126.0 (CH, C-2, C-7), 125.7 (C,
C-4a), 136.0 (C, C-5a), 135.1 (C, C-2), 134.8 (C, C-7),
133.7 (CH, C-3), 130.9 (CH, C-6), 130.3 (CH, C-4), 129.1
(CH, C-5), 127.7 (CH, C-1), 127.0 (CH, C-8), 126.3 (C, C-
9a), 126.2 (C, C-8a), 33.6 (CH, CH Br), 21.8 (CH, CH );
−
1
C-8a, C-9a), 19.0 (CH ); IR (cm ): 2729, 1378; HREIMS:
m/z 207.1049 C H N (calcd. 207.1048).
A suspension of 1,8-dimethylacridine (60 mg, 0.29
mmol), NBS (52 mg, 0.29 mmol), and AIBN (2.5 mg,
3
1
5 13
2
3
−1
IR (cm ): 2577, 1432, 1390, 816; HREIMS: m/z 285.0154
7
9
0
.015 nmol) in carbon tetrachloride (80 mL) was refluxed
C H
BrN (calcd. 285.0153).
12
1
5
for 30 min. Then, more NBS (53 mg, 0.29 mmol) was
added, and the mixture was refluxed for 1 h. After cooling
the mixture in an ice bath, a solid appeared that was
collected by filtration. The filtrate was dried over sodium
sulfate, filtered, and concentrated under reduced pressure to
produce a yellow solid. The crude product was purified on
silica gel [dichloromethane:ethyl acetate (40:1)] to generate
the desired product as a yellow solid. The solid was
recrystallized from cyclohexane to yield the single com-
pound 2 (13 mg).
Agarose gel interstrand-crosslink assay
The assay procedure was performed according to Hartley
et al (Hartley et al. 1981). Test compounds were diluted to
the desired concentrations per 2 μL DMSO. Linearized
pBR322 DNA (40 ng/2 μL) was treated with a test com-
pound at pH 7.4, 37 °C for 2 h. After the reaction, the DNA
was precipitated and lyophilized to dryness. The DNA was
dissolved in separation buffer (30% DMSO, 1 mM EDTA),
was heated for 5 min at 95 °C and was immediately placed
in an ice bath. The samples were electrophoresed in a 1.2%
agarose gel at 50 V for 2 h, and the DNA bands were
visualized by staining with ethidium bromide. All of the
data are representative of at least two separate experiments.
1
,8-bis(bromomethyl)acridine (2) Yellow needle crystal;
yield, 13%; TLC (dichloromethane/hexane 1:1) R 0.9;
f
1
sublimation point, 168 °C; H NMR (CDCl , 400 MHz,): δ
3
=
8.78 (1 H, s, H-9), 8.01 (2 H, d, J = 8.8 Hz, H-4, H-5),
7
8
1
1
2
1
3
.95 (2 H, dd, J = 8.8, 8.6 Hz, H-3, H-6), 7.54 (2 H, d, J =
1
3
.6 Hz, H-2, H-7), 5.43 (4 H, s, CH Br); C NMR (CDCl3,
2
DNA intercalation assay
00 MHz): δ = 145.8 (C, C-4a, C-5a), 136.5 (C, C-1 C-8),
31.1 (CH, C-4, C-5), 129.0 (CH, C-3, C-6), 126.8 (CH, C-
−
1
DNA intercalation activity was assayed with a competitive
ethidium displacement assay (Baguley et al. 1981). A
DMSO aliquot of a test compound was added to a mixture
of calf thymus DNA (0.5 mg/mL, 4 μL) and ethidium bro-
mide (1 mg/mL, 1 μL) in 0.01 M SHE buffer (pH 7.0, 2 mL).
The reaction mixture was incubated at 25 °C for 30 min. The
fluorescence intensity (EX: 520 nm; EM: 600 nm) was
recorded. The remaining fluorescence was expressed as a
percentage of the initial fluorescence. The final fluorescence
was obtained by subtracting the value in the absence of
ethidium bromide from the value in the presence of ethi-
dium bromide. All of the data are expressed as the
, C-7), 125.8 (C, C-8a, C-9a), 30.1 (CH Br); IR (cm ):
2
7
9
430, 756; HREIMS: m/z 362.9265 C H N Br (calcd.
1
5
11
2
62.9258).
2
-(Bromomethyl)-7-methylacridine (4)
A suspension of 5 (59 mg, 0.28 mmol), NBS (26 mg,
.14 mmol), and AIBN (2.8 mg, 0.015 nmol) in carbon
tetrachloride (60 mL) was refluxed for 1 h. More NBS
26 mg, 0.14 mmol) was added, and the mixture was
refluxed for 1 h. After cooling the mixture on an ice bath, a
0
(

Med Chem Res
Scheme 1 Synthesis of 2.
Reagents and conditions: (a)
Ac
SO
NaNO
Sn, HCl, 99%; (e) 1) HCOOH,
glycerol, HCl, 2) NaNO
SO , EtOH, 4%; (f) NBS,
AIBN, CCl4, 24%
2
O, pyridine, 91%; (b) HNO
, 84%; (c) 1) H SO , 2)
, H SO , EtOH, 72%; (d)
3
,
H
2
4
2
4
2
2
4
2
,
H
2
4
mean ± standard error (SE) of at least three separate
experiments.
were formed between two bromomethyl groups and two
nucleophilic positions in the DNA bases.
Bacterial mutation assay
Results and discussion
Chemistry
The bacterial mutation assay was performed according to
the pre-incubation method reported by Ames (Maron and
Ames 1983; Mortelmans and Zeiger 2000). Professor B. N.
Ames (University of California, Berkeley, USA) provided
the Salmonella typhimurium TA98 strain, and Dr. T. Nohmi
To enhance the DNA affinity of bis(bromomethyl) com-
pounds, an acridine ring was selected as a DNA carrier.
Bromomethyl aromatic compounds have two benzylic
positions that are highly reactive to DNA nucleophiles.
Using this knowledge, the bis(bromomethyl) groups were
introduced into DNA-intercalative acridines.
(
National Institute of Health Sciences, Tokyo, Japan) pro-
vided the S. typhimurium TA100, TA94 and TA92 strains.
The test compounds were diluted to the desired concentra-
tion in 50 μL DMSO. The DMSO solution for each con-
centration was placed in a test tube to which 0.5 mL 0.1 M
sodium phosphate buffer (pH 7.4) was added, followed by a
Crosslinkable bis(bromomethyl)acridines substituted at
1,8-positions (2) and 2,7-positions (3) were synthesized
for comparison with 4,5- bis(bromomethyl)acridine (1).
Furthermore, mono-alkylating 2-(bromomethyl)-7-methy-
lacridine (4) and non-alkylating 2,7-dimethylacridine (5)
were prepared to investigate their interaction mode with
double-stranded DNA. 1,8-Dimethylacridine, 2 and 4 were
newly synthesized.
p-Toluidine was nitrated after acetylation, deacetylated
by alkaline aqueous solution and diazotized under acidic
conditions to produce 3,5-dinitrotoluene (Scheme 1). The
0
.1 mL culture of the test strain. The mixture was incubated
for 20 min at 37 °C with shaking (70 strokes/min), and then
top agar (2 mL) was added. The mixture was poured onto a
minimal-glucose agar plate. After incubation at 37 °C for
4
4 h, the colonies were counted. The results were con-
sidered positive if the test produced a reproducible, dose-
related increase in the number of revertant colonies or the
number of colonies was double the background number of
colonies. All of the plates were prepared in duplicate, and
the experiments were repeated at least twice. The results are
expressed as the mean ± SE.
3,5-dinitrotoluene was reduced to 3,5-diaminotoluene with
tin under acidic conditions, was condensed with formic acid
and was subsequently deaminated by diazotization to form a
novel 1,8-dimethylacridine, which reacted with N-bromo-
succinimide (NBS, 2 eq.) in the presence of AIBN to pro-
duce the desired compound 2.
Molecular modeling
Molecular modeling was performed with Discovery Studio
p-Toluidine was nitrated followed by reduction with
tin under acidic conditions (Scheme 2). The 2,4-diamino-
toluene was condensed with formic acid and was
subsequently deaminated by diazotization to form 2,7-
dimethylacridine. The 2,7-dimethylacridine was reacted
4
.1 Client (BIOVIA, San Diego, CA, USA) to obtain
structural features. To construct the DNA-compound 3
complex, compound 3 was manually inserted into the 5-
GG-3′ and 5-CG-3′ sequence duplex, and preferential bonds

Med Chem Res
Scheme 2 Synthesis of 3 and 4.
Reagents and conditions: (a)
HNO
HCl, 84%; (c) 1) HCOOH,
glycerol, HCl, 2) NaNO
SO , EtOH, 59%; (d) NBS (2
3 2 4
, H SO , 95%; (b) Sn,
2
,
H
2
4
eq.), AIBN, CCl4, 21% for 3,
NBS (1 eq.), AIBN, CCl4, 30%
for 4
with N-bromosuccinimide (NBS, 2 eq.) in the presence of
AIBN to produce compound 3, or with NBS (1 eq.) to
produce compound 4.
DNA interstrand crosslinking activity
DNA crosslinking damage occurs when crosslinking agents
covalently connect two nucleotide residues from the same
DNA strand (intrastrand crosslink) or from opposite strands
(
interstrand crosslink). Intrastrand crosslinks are readily
removed by nucleotide excision repair (Brulikova et al.
012; Noll et al. 2006). However, an interstrand crosslink
2
constitutes an absolute block to DNA strand separation, thus
interrupting DNA replication and transcription.
Fig. 2 Crosslinking activity of 1 (a), 2 (b), and 3 (c) towards plasmid
DNA
The DNA crosslinking efficiency of acridines was
investigated using an assay involving linear double-
stranded DNA derived from the plasmid pBR322 (linear-
ized with HindIII) (Hartley et al. 1981). Following complete
denaturation to the single-stranded form, the presence of an
interstrand crosslink results in renaturation to double-
stranded DNA during electrophoresis in a neutral agarose
gel (Fig. 2).
showed no activity in this test system (data not shown). The
results indicated that bis(bromomethyl)acridines reacted
with plasmid DNA to form interstrand crosslinks.
DNA-intercalating activity
Lane 1: non-denatured DNA (double-stranded DNA);
Lane 2: denatured DNA (single-stranded DNA); Lane 3: bis
Ethidium bromide (EtBr) displacement was used to inves-
tigate the intercalating activity between acridines and DNA
(Baguley et al. 1981). The assay was based on the theory
that a highly fluorescent complex would form between the
intercalating dye EtBr and calf thymus DNA. Acridine
binding to calf thymus DNA was evaluated by the fluor-
escence emission intensity of the EtBr-DNA system fol-
lowing addition of the compound using amsacrine as a
reference intercalator (Fig. 3).
(
bromomethyl)acridine, 1 μM; Lane 4: bis(bromomethyl)
acridine, 10 μM; Lane 5: bis(bromomethyl)acridine, 25 μM;
Lane 6: bis(bromomethyl)acridine, 50 μM; Lane 7, bis
(
bromomethyl)acridine, 100 μM; Lane 8: cisplatin, 0.1 μM.
Double-stranded DNA from crosslink formation
increased in a concentration-dependent manner up to
5 μM; at higher concentrations, double-stranded DNA was
2
no longer observed. The disappearance of UV-visible bands
indicated DNA fragmentation caused by double-stranded
DNA breakage. Alkylation of the endocyclic nitrogens in
purine bases—N7G, N7A, N3G, N3A, N1A, and N3C—
make the bond between the sugar and base more labile,
potentially leading to DNA strand breakage (Gates 2009).
The capability of interstrand crosslink formation was similar
among the three bis(bromomethyl)acridines. 4 and 5
The activity was observed in the following order: 3 >
amsacrine > 4 > 2 = 1. The fluorescence intensity of EtBr-
DNA did not decrease upon addition of 5 at the same
concentration range as bis(bromomethyl)acridines. 5 inter-
acts reversibly with DNA, but the intercalative capacity of 5
was not sufficient to displace EtBr in the DNA. The acri-
dines 1–4 reduced EtBr-DNA complex fluorescence, indi-
cating that acridines with bromomethyl group(s) compete

Med Chem Res
Ames 1981a, 1981b). The mutagenic behaviors and muta-
genic capacity of acridines and MMC were evaluated
(Fig. 4).
The non-alkylating analog 5 was not mutagenic in any
Salmonella strains at a concentration below 200 nmol/plate,
at which a precipitate was observed (see the supporting
information). The monofunctional analog 4 was mutagenic
in S. typhimurium TA100 and TA98 without cytotoxicity,
indicating the formation of monoalkylated adducts (Fig. 4).
MMC was not mutagenic in S. typhimurium TA100 and
TA98 but showed mutagenicity in S. typhimurium TA92
and TA94. Thus, mutagenicity in S. typhimurium TA92 or
TA94 is necessary to detect intracellular crosslinking
activity (Haroun and Ames 1981a, 1981b).
Bis(bromomethyl)acridines 1–3 were cytotoxic after
showing mutagenicity in S. typhimurium TA100 and TA98
and were also mutagenic in S. typhimurium TA92 and TA94
(Fig. 4). The mutagenic behavior of bis(bromomethyl)
acridines was similar to that of MMC. The data indicated
that mutagenicity of bis(bromomethyl)acridines resulted
from DNA intercalation and covalent crosslinking. These
data agreed with the intercalative and plasmid DNA
interstrand-crosslink assays. The mutagenicity of 3 in S.
typhimurium TA94 and TA92 was higher than those of 1
and 2, and the data correlated well with the DNA-
intercalative activity measured using EtBr. 1 and 2
showed similar intercalation and interstrand crosslinking
properties.
S. typhimurium TA92 encodes a GGG mutation in
hisG46, whereas S. typhimurium TA94 encodes the
hisD3052 frameshift mutation, which has a repetitive
CGCGCGCG sequence (Ames et al. 1973). The mutagenic
properties of acridines on Salmonella strains depend on the
nature and positions of the aromatic ring substituents (Ames
et al. 1973; Brown et al. 1980). Thus, the substituent
positions on the acridine ring profoundly affect the muta-
genic characteristics of Salmonella.
In our previous study, 2,6-bis(bromomethyl)naphthalene
showed the highest interstrand crosslinking activity among
ten positional isomers (Higashi et al. 2009). The relative
mutagenicity of 3 in S. typhimurium TA92 (approximately
740,000 revertants/nmol) was compared with that of 2,6-bis
(bromomethyl)naphthalene (~600 revertants/μmol) (Higashi
et al. 2009). The activity of 3 was 120-fold higher than that
of naphthalene. DNA alkylation by the bis(bromomethyl)
group is driven by noncovalent association of the inter-
calative acridine ring.
Fig. 3 Competitive ethidium-DNA fluorescence displacement assay of
1
(●), 2 (▲), 3 (■), 4 (○), 5 (Δ), and amsacrine (◊). The SE in 1 was too
small to show (see the supplementary information)
with EtBr for binding sites and displace EtBr from the
DNA-drug complex. DNA binding activity was enhanced
by covalent modification on the acridine ring. The acridines
with bromomethyl group(s) at the 2-position(s) and/or 7-
position(s) exhibit higher activity than 1 and 2. The results
suggested that introducing a bromomethyl group at specific
substituent position(s) enhances intercalative activity.
Intracellular crosslinking and intercalating activities
using the Ames assay
The Ames assay was used to evaluate the mode of action of
acridines towards intracellular DNA (Ishikawa et al. 2004;
Inami et al. 2009; Maron and Ames 1983; Mortelmans and
Zeiger 2000). S. typhimurium TA100 can detect base pair
substitution mutations caused by DNA alkylation or
intrastrand DNA crosslinking, whereas S. typhimurium
TA98 can detect frameshift mutations caused by covalent
intercalative binding. To enhance the ability of S. typhi-
murium TA100 and TA98 to detect DNA crosslinking
agents, the uvrB gene was retained to enable bacterial DNA
repair in S. typhimurium TA92 and TA94, respectively
(
Haroun and Ames 1981a, 1981b). Mitomycin C (MMC)
To confirm the interaction of 3 with DNA at the atomic
level, molecular modeling simulations were performed
using GG and CG sequences, which are target DNA
sequences in S. typhimurium TA92 and TA94, respectively
(Fig. 5). Because 3 demonstrated DNA intercalation in the
EtBr displacement assay and mutagenicity towards S.
exerts mutagenicity by reductive activation in S. typhimur-
ium TA92 and TA94 and was used as a positive control
(
Maron and Ames 1983; Basu et al. 1984). Simple inter-
calating mutagens that do not react with DNA covalently do
not show mutagenicity in Salmonella strains (Haroun and

Med Chem Res
Fig. 4 Mutagenicity of 1–5 and
MMC in Salmonella
typhimurium TA100 a, TA92 b,
TA98 c and TA94 d: 1 (●), 2
(
▲), 3 (■), 4 (○), 5 (Δ), and
MMC (◊)
typhimurium TA92 and TA94, 3 was inserted into both the
GG and CG sequences by π–π stacking interactions. Due to
the high nucleophilic reactivity of the N7-position of gua-
nine in DNA (Gates 2009), one N7-alkylation in guanine
was formed predominantly in double-stranded DNA fol-
lowed by another nucleophilic position in the double-
stranded DNA. From the simulation data for the GG
sequence, 3 might alkylate at N7-guanine, and the reaction
sequence would occur to crosslink at N3-cytosine in another
strain. However, in the GC sequence, 3 might alkylate two
N7-guanines in the double-stranded sequence. The data
indicated that 3 might intercalate and form crosslinks with
both of the DNA sequences. The nitrogen atom in the
acridine ring might face both the major groove and minor
groove (Sakore et al. 1977; Löber and Achtert 1969; Shieh
et al. 1980; Sasikala and Mukherjee 2013). Thus, the pro-
posed models are reasonable structures to explain our
experimental results.
Overall, the introduction of a crosslinkable functional
group into the acridine ring enhanced DNA affinity through
DNA alkylation and intercalation. Additionally, non-
covalent DNA binding facilitated efficient DNA alkylation
by bromomethyl compounds.

Med Chem Res
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Conclusion
This study targeting crosslinking agents to DNA using
DNA intercalation has shown that this strategy can greatly
enhance bis(bromomethyl) moiety crosslinking reactivity
by introduction of an intercalative ring. Based on this pre-
liminary study, other hybrid molecules that combine minor
groove ligands, which have DNA sequence-recognizing
properties, would be developed. An enhanced DNA binding
affinity and selectivity at specific DNA sites are the
potential advantages of these hybrid molecules, and the
improvement of the selectivity and toxicity profiles will
extend the clinical benefit of crosslinking drugs.
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Acknowledgements This work was supported in part by a Grant-in-
Aid from the Ministry of Education, Culture, Sports, Science and
Technology of Japan and by a Grant-in-Aid for the Science Research
Promotion Fund from the Japan Private School Promotion Foundation.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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Ishikawa S, Tajima M, Mochizuki M (2004) Synthesis and properties
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