Synthesis and antitumor activity of 5-(9-acridinylamino)anisidine derivatives

Article abstract of DOI:10.1016/j.bmc.2005.07.018

A series of 5-(9-acridinylamino)anisidines were synthesized by condensing methoxy-substituted 1,3-phenylenediamines (10 and 11) with 9-chloroacridine derivatives to form 5-(9-acridinylamino)-m-anisidines (AMAs, 14a-e) and 5-(9-acridinylamino)-o-anisidines

Full text of DOI:10.1016/j.bmc.2005.07.018

Bioorganic & Medicinal Chemistry 13 (2005) 6513–6520
Synthesis and antitumor activity of
5-(9-acridinylamino)anisidine derivatives
Valeriy A. Bacherikov,a Jang-Yang Chang,b Yi-Wen Lin,a Ching-Huang Chen,a
Wen-Yu Pan,b Huajin Dong,c Rong-Zau Lee,a Ting-Chao Chouc and Tsann-Long Sua,*
aLaboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
bDivision of Cancer Research, National Health Research Institutes, Taipei, Taiwan
cMolecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
Received 27 February 2005; revised 1 July 2005; accepted 5 July 2005
Available online 1 September 2005
Abstract—A series of 5-(9-acridinylamino)anisidines were synthesized by condensing methoxy-substituted 1,3-phenylenediamines
(10 and 11) with 9-chloroacridine derivatives to form 5-(9-acridinylamino)-m-anisidines (AMAs, 14a–e) and 5-(9-acridinylami-
no)-o-anisidines (AOAs, 15a–e). 5-(9-Acridinylamino)-p-anisidines (APAs, 17a–e) were synthesized by reacting 2-methoxy-5-nitro-
aniline (12) with 9-anilinoacridines, followed by reduction. The cytotoxic inhibition of growth of various human tumor cells in
culture, inhibitory effects against topoisomerase II, and DNA interaction of these agents were studied. The structure–activity rela-
tionship studies revealed the following degree of potency: AOAs > AMAs > APAs. They also revealed that the newly synthesized
derivatives bearing CONH2NH2NMe2 and Me substituents at C4 and C5 positions of the acridine chromophore (i.e., AMA 14e,
AOA 15e, and APA 17e) exhibited significant cytotoxicity against human tumor cell growth in vitro. AOA (15e) was the most potent
among these derivatives, which resulted in 60% suppression of tumor volume at a dose of 20 mg/kg (Q2D · 9), intravenous injection
on day 26 in nude mice bearing human breast carcinoma MX-1 xenografts.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
As reviewed by Gao et al.,5 the QSAR studies of 9-ani-
linoacridine congeners have demonstrated that the elec-
tronic effect of substituent(s) is associated with the
drugꢀs binding site, while hydrophobicity is associated
with drugꢀs entrance to the active site and steric effect
of the substituent is associated with drugꢀs binding to
an active site on a macromolecule. These points were
the three consistent critical conclusions drawn from
these QSAR studies. However, there is a need to have
more systematical investigations using these complicat-
ed QSAR studies to develop more desirable agents for
potential clinical applications.
Among antitumor DNA-intercalating agents and topoi-
somerase II (Topo II) inhibitors, 9-anilinoacridines have
been extensively investigated. Numerous attempts have
been made to design and synthesize compounds with po-
tent antitumor therapeutic efficacy and bioavailability for
clinical application by modifying the substituent(s) on the
anilino ring and/or acridine chromophore,1,2 in addition
to quantitative structure–activity relationships (QSAR)
studies.3–5 Among these agents, m-amsacrine (m-AMSA)
and its 4-methyl-5-methylcarboxamide derivative
(CI-921) were of particular interest in that m-AMSA
was subjected to clinical investigations for the treatment
of acute leukemia,6,7 and CI-921 was in phase II clinical
trials for treatment of solid tumors.8–11 The results of
these studies showed that both agents were too toxic or
did not sustain human antitumor efficacy.12,13
Previous studies examining the development of potential
anticancer agents, namely 3-(9-acridinylamino)-5-
hydroxymethylaniline (AHMA, 1, Fig. 1) and its deriv-
atives, have demonstrated that AHMA exhibited both
potent in vitro and in vivo antitumor efficacies2 and
superior antitumor efficacy when compared to both m-
AMSA and VP-16 in mice bearing mammary carci-
noma.14 Additionally, the alkylcarbamates of AHMA
(AHMA-alkylcarbamates, e.g., 2) were more effective
than their corresponding parent AHMA derivatives,
perhaps due to increased hydrophobicity of these
Keywords: Acridines; Antitumor compounds; Synthesis; Chemotherapy;
Substituent effects.
*
Corresponding author. Tel.: +886 2 2789 9045; fax: +886 2 782
9142; e-mail: tlsu@ibms.sinica.edu.tw
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.018

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V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
NHR
NH2
HN
N
CH2OH
HN
N
Me
R2
R1
R2
R1
4 R1 = R2 = H (AMT)
5 R1 = CONHCH2CH2NMe2, R2 = Me,
1 R = R1 = R2 = H (AHMA)
2 R = COOEt, R1 = R2 = H
3 R = H, R1 = CONHCH2CH2NMe2, R2 = Me,
NH2
Me
Me
HN
N
HN
N
NH2
R2
R1
R2
R1
8 R1 = R2 = H (AOT)
9 R1 = CONHCH2CH2NMe2, R2 = Me,
6 R1 = R2 = H (APT)
7 R1 = CONHCH2CH2NMe2, R2 = Me,
Figure 1.
agents.15 The structure–activity relationship studies of
AHMA derivatives have demonstrated that the CH2OH
functional group on the anilino ring played an impor-
tant role in their antitumor activity, probably via
involvement in drug/DNA binding. More recently, we
found that conversion of the CH2OH group to an ester
increases its hydrophobicity and the cytotoxicity of 2
was enhanced further. Replacing the CH2OH substitu-
ent with a Me group at the meta-, para- or ortho-posi-
tion to the NH2 group, 5-(9-acridinylamino)-m-
toluidines (AMTs, 4 and 5), 5-(9-acridinylamino)-p-tol-
uidines (APTs, 6 and 7), or 5-(9-acridinylamino)-o-tolui-
dines (AOTs, 8 and 9) were formed, respectively, which
were more hydrophobic than the parent AHMA.16 The
results showed that compounds 4, 6, and 8 were less
cytotoxic than AHMA, indicating that either removal
of the CH2OH or replacement of this functional group
with Me reduced its cytotoxicity further and weakened
its inhibitory effect against Topo II. However, 5-(9-
acridinylamino)toluidine analogs (5, 7, and 9) bearing
CONHCH2CH2NMe2 and Me functions at C4 and C5
of the acridine chromophore, respectively, were more
potent than AHMA (1) and AHMA-ethylcarbamate
(2) or as potent as AHMA derivative 3 depending on
the tumor cell line tested. These three agents exhibited
potent therapeutic effects in mice bearing human breast
carcinoma MX-1 xenografts. Moreover, APTs and
AOTs were more potent than AMTs, perhaps due to
the inductive effect of the Me function.
cytotoxicity. For example, the substituents (CON-
HCH2CH2NMe2 and Me functions) on the acridine
chromophore may have influenced the drug/DNA inter-
action and hence altered the cytotoxicity of the 9-anilino-
acridines. Earlier reports have demonstrated that adding
an electron-donor OMe group on the anilino ring of 9-
anilinoacridine increased its antitumor potency.17 Based
on these findings, one can envisage that the cytotoxicity
of 5-(9-acridinylamino)toluidines can be enhanced
further by replacing the Me function with the elevated
electron-donating OMe group to increase the electroneg-
ativity of the anilino ring. In this paper, we described the
synthesis and biological evaluations of a series of 5-(9-
acridinylamino)-m-anisidines (AMAs), 5-(9-acridinyla-
mino)-p-anisidines (APAs), and 5-(9-acridinylamino)-o-
anisidines (AOAs). The structure–activity relationship
studies concluded that the target AOAs and AMAs were
more cytotoxic than the corresponding AOTs and AMTs
in inhibiting growth of various human tumor cells in vi-
tro, with the exception of APAs.
2. Chemistry
5-(9-Acridinylamino)-m-anisidines (AMAs, 14a–e) were
synthesized by the condensation of 3,5-diaminoanisole
dihydrochloride (10) with the known 9-chloroacridines
(13a–e) in CHCl3/EtOH in the presence of 4-meth-
ylmorpholine in an ice/MeOH bath by following a pro-
cedure previously developed in our laboratory.14,16
Under such conditions, good yield of 14a–e was
obtained as hydrochloride salts. In a similar manner,
the reaction of 2,4-diaminoanisole dihydrochloride (11)
The studies indicated that the electronegativity of the
anilino ring induced by the Me group may affect the
drug/enzyme interaction and consequently the drugꢀs

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
6515
with the requisite 9-chloroacridines (13a–e) afforded 5-
(9-acridinylamino)-o-anisidines (AOAs, 15a–e) in good
yield. It is interesting to note that the reaction of 11 with
13a–e may afford the isomers AOAs (15a–e) and/or 5-(9-
acridinylamino)-p-anisidines (APAs, 17a–e). To identify
the structure of 15a–e, we first treated 2-methoxy-5-nit-
roaniline (12) with 9-chloroacridines (13a–e) in a mix-
ture of CHCl3/EtOH in the presence of a catalytic
amount of concd hydrochloric acid. The product, acri-
din-9-yl-(2-methoxy-5-nitrophenyl)amines (16a–e), was
then converted to APAs (17a–e) upon catalytic hydroge-
nation (5% Pd/C, H2) (see Scheme 1).
to the Me groups in APTs and AOTs, however, show
no apparent difference.
3. Biological results and discussion
3.1. In vitro cytotoxicity
Our previous study has demonstrated that 5-(9-acridi-
nylamino)toluidine derivatives bearing CONHCH2-
CH2NMe2 and Me substituents at C4 and C5 of the
acridine moiety, respectively, exhibited remarkable cyto-
toxicity both in vitro and in vivo, while, the simple 5-(9-
acridinylamino)toluidines were less cytotoxic than the
parent AHMA.16 The results demonstrated that the
electronegativity on the anilino ring, the substituent(s)
on the acridine chromophore, as well as the hydropho-
bicity of the molecule, greatly influenced the cytotoxicity
of 9-anilinoacridines. In the present studies, we evaluat-
ed the cytotoxic effects of target (9-acridinylamino)anisi-
dines (AMAs, 14a–e; AOAs, 15a–e; and APAs, 17a–e)
against numerous human tumor cell (including mouth
KB, nasopharyngeal carcinoma HONE-1, lung adeno-
carcinoma H460, colon HT-29, gastric carcinoma
TSGH, hepatoma Hepa-G2, brain tumor DBTRG,
and breast carcinoma MX-1) growth in culture (Table
1). The data clearly revealed that AOA 15e was the most
potent compound among the 5-(9-acridinylamino)anisi-
dines bearing CONCH2CH2NMe2 and Me substituents
(AMA 14e, AOA 15e, and APA 17e) at C4 and C5,
respectively. The order of cytotoxicity was as follows:
The structures of AOAs (15a–e) and APAs (17a–e) were
1
determined by the H NMR spectroscopy method. The
1H NMR (DMSO-d6) spectra of the series of AOAs
(15a–e) and APAs (17a–e) revealed that chemical shifts
for their OMe substituent can be clearly distinguished;
the signal for protons of the OMe functional group in
AOAs (15a–e) locates appears at a lower field than that
of APAs (17a–e) with d ꢀ3.84–3.91 and ꢀ3.36–3.45,
respectively. While the chemical shifts of OMe groups
in AMAs (15a–e) are about d ꢀ3.64–3.67. Apparently,
the NH2 function at C4 of 2,4-diaminoanisole (11) is
more reactive than the one at C2, resulting in the forma-
tion of AOAs (15a–e) as the prominent products. In
contrast, our previous study showed that the reaction
of 2,4-diaminotoluene with 9-chloroacridines (13a)
afforded APTs (i.e., 6). In this case, the C2-NH2 is more
reactive than the C4-NH2 due to a weaker inductive
effect of the Me function. The chemical shifts assigned
NH2
NH2
HN
N
OMe
H2N
OMe
OMe
NH2
10
R2
R1
OMe
(i)
14a-e
NH2
Cl
HN
(i)
(i)
+
H2N
N
R2
R1
N
11
R2
R1
13a-e
15a-e
NH2
MeO
HN
MeO
HN
OMe
NH2
NO2
O2N
12
(ii)
N
N
R2
R1
R2
R1
17a-e
16a-e
Scheme 1. Reagents and reaction conditions: (i) 4-methylmorpholine (or catalytic concd HCl)/EtOh/CHCl3, À10–0ꢁC, 1–5; (ii) 5% Pd/C, H2,
Catalytic concd HCl, 50 psi, 25 min. (a) R1 = R2 = H; (b) R1 = Me, R2 = H; (c) R1 = CONHCH2CH2NMe2, R2 = H; (d) R1 = CONHMe, R2 = Me;
(e) R1 = CONHCH2CH2NMe2, R2 = Me.

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V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
Table 1. The cytotoxicity of 5-(9-acridinylamino)anisidine derivatives on the inhibition of growth various human tumor cells in vitro
Compound
Inhibition of cell growth (IC50, lM)
KB
HONE-1
H460
HT-29
TSGH
HEPA-G2
DBTRG
MCF-7
1 (AHMA)
3
NDa
NDa
NDa
3.20
2.83
0.70
2.71
0.09
NDa
NDa
0.32
0.32
0.31
0.39
0.03
NDa
NDa
6.45
6.36
0.88
2.55
0.13
NDa
0.30
0.04
3.40
3.32
2.90
0.73
3.11
0.06
0.09
0.90
0.33
0.34
0.15
0.50
0.03
0.09
0.80
6.50
4.82
2.12
3.07
0.10
0.07
NDa
NDa
NDa
2.57
1.45
0.44
2.06
0.03
NDa
NDa
0.33
0.29
0.08
0.19
0.01
NDa
NDa
3.31
2.40
0.36
0.43
0.02
NDa
0.90
0.12
3.50
3.00
2.78
0.47
2.73
0.05
0.09
2.50
0.34
0.38
0.12
0.60
0.02
0.09
2.50
7.79
8.62
0.88
4.53
0.27
0.05
0.50
0.40
7.20
2.96
2.67
0.38
2.83
0.03
0.12
2.50
0.38
0.38
0.14
0.62
0.03
0.08
4.10
8.28
7.73
0.91
3.32
0.10
0.04
1.40
0.20
3.20
3.56
3.24
0.41
3.91
0.04
0.06
4.0
3.50
0.16
10.0
4.70
5.00
1.69
14.77
0.18
2.8
NDa
NDa
NDa
4.89
4.15
1.16
6.67
0.18
NDa
NDa
2.08
2.60
1.19
3.24
0.01
NDa
NDa
7.92
26.19
3.37
17.50
2.00
NDa
AMT4
14a
14b
14c
14d
14e
AMT5
AOT8
15a
4.30
2.93
2.45
1.32
2.33
0.08
0.30
4.00
5.77
21.67
1.59
14.88
2.23
0.86
0.35
0.36
0.15
0.97
0.02
0.10
2.50
5.93
10.75
2.06
8.00
0.29
0.10
15b
15c
15d
15e
AOT9
APT6
17a
17b
17c
17d
17e
APT7
a Not determined.
AOA 15e > AMA 14e > APA 17e. Among compounds
with or without other substituent(s) on the acridine
chromophore (i.e., AMA 14a–d, AOAs 15a–d, and
APAs 17a–d), the C4-CONH2NH2NMe2-substituted
derivatives (i.e., AMA 14c, AOA 15c, and APA 17c)
were approximately 2- to 10-fold more potent than their
corresponding parent AMA 14a, AOA 15a, and APA
17a, which depends on the tumor cell lines tested. The
C4-Me-substituted (i.e., AMA 14b, AOA 14b, and
APA 17b) and C4-CONHMe, C5-Me-disubstituted
compounds (i.e., AMA 14d, AOA 15d, and APA 17d)
did not have any improved cytotoxic effect in compari-
son with their corresponding parent compounds 14a,
15a, and 17a, respectively. This demonstrated that the
CONH2NH2NMe2 substituent played an important role
for their antitumor activity, perhaps due to better bind-
ing affinity with DNA double strands.8,16 In general, the
order of potency in the series of 5-(9-acridinylami-
no)anisidines was as follows: AOAs > AMAs > APAs.
To elicit the effect of the OMe versus Me substituent at-
tached on the anilino ring, we compared the cytotoxicity
of 5-(9-acridinylamino)anisidines with those of previ-
ously synthesized AHMAs (1 and 3) and 5-(9-acridinyla-
mino)toluidines (AMTs, 4, 5; AOTs, 6, 7; and APTs, 8,
9). The structure–activity relationship studies of these
agents concluded that: (1) Both AMA 14e and AOA
15e were more cytotoxic than AHMA derivative 3, while
APA 17e was less potent than 3 and (2) The degree of
cytotoxicity was AMAs > AMTs; AOAs > AOTs; APTs
> APAs. Our previous study had demonstrated that
APTs were as potent as AOTs, but were significantly
more cytotoxic than AMTs, with the exception of those
compounds having Me and CONH2NH2NMe2 substitu-
ents on the acridine chromophore (i.e., 5, 7, 9, 14e, 15e,
and 17e). These findings have suggested that the Me
group (possessing an inductive effect) and the electron-
donating OMe group affect the electronegativity of the
anilino ring and consequently alter the drug/enzyme-
binding and antitumor activity. The effect of the substi-
tuent on the cytotoxicity of 9-anilinoacridines can be
clearly observed in APT and APA families. The reason
for the low cytotoxicity of APAs (especially, APA
17a–d) remains unclear. Nevertheless, these studies have
demonstrated that the drug/DNA binding of 9-anilino-
acridines appears to be one of the most important fac-
tors for increasing the 9-anilinoacridineꢀs cytotoxicity,
as exhibited by 14e, 15e, and 17eꢀs potent antitumor
activity in vitro.
We evaluated further the effect of the newly synthesized
compounds on cells overexpressing P-gp170/MDR118
and MRP (multidrug resistance-associated protein).19
As shown in Table 2, the cytotoxicity of 14a, 14e, 15a,
15e, 17a, and 17e toward KB-Vin10 and KB-7D cells,
which displayed overexpression of MDR1, MRP, and
down-regulation of topoisomerase II, respectively, was
similar to that of parental KB cells, indicating that 5-
(9-acridinylamino)anisidines were not cross-resistant to
etoposide-resistant (KB-7D) and vincristine-resistant
(KB-Vin10) cells.
3.2. In vivo therapeutic activity
Among the agents tested, compound 15e exhibited the
most potent cytotoxic effect toward numerous human
cancer cell lines and was subjected to further in vivo
antitumor evaluation. The therapeutic efficacy of com-
pound 15e [maximal dose: 20 mg/kg (Q2D · 9); intrave-
nous injection] on nude mice (n = 4) bearing human
breast carcinoma MX-1 xenograft resulted in 60%
reduction of tumor with 20% of body weight decrease
compared with the control (n = 5) on day 26. Cain

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
6517
Table 2. The cytotoxicity of 5-(9-acridinylamino)anisidine derivatives against human tumor KB and its sublines resistant to etoposide (KB-7D) and
vincristine (KB-Vin10) cell growth in vitro
Compound
Inhibition of cell growth (IC50, lM)
KB
KB-7D
KB-Vin10
PLDB (%) at 5 lM
AHMA
14a
14e
0.50
2.45
0.08
0.31
0.02
6.0
1.50 (3.0·)a
3.06 (1.25·)
0.21 (2.65·)
0.25 (0.80·)
0.03 (1.50·)
7.1 (0.18·)
0.29 (2.42·)
78.8 (197·)
5.6 (4667·)
0.70 (1.4·)a
2.75 (1.12·)
0.25 (3.13·)
0.23 (0.74·)
0.04 (2.00·)
7.0 (1.16·)
0.32 (2.67·)
31.5 (78.8·)
75.8 (65417·)
12.0
0.0
1.2
6.5
15a
15e
0.6
4.6
17a
17e
0.12
0.40
0.0012
4.3
VP-16
Vincristine
NDb
NDb
a Numbers in the brackets are folds of resistance of the resistant cells when compared with the IC50ꢀs of the KB parent cells.
b Not determined.
et al. indicated that increasing the electronic effect of
substituents on the anilino ring of 9-anilinoacridines in-
creased vulnerability for thiol attack,20,21 thereby
decreasing its cytotoxicity. This effect may be due to
instability of the anisidine moiety in AOAs and APAs.
These agents could be easily bio-oxidized to form rela-
tively inactive compounds (i.e., ortho- or para-iminoqui-
none derivative, respectively).
3.3. Interaction of AHMA, APT, and AMT derivatives
with topoisomerase II
Inour previous study, AHMA and itsanalogs were shown
to be potent Topo II inhibitors.14,15 To study whether
these compounds also inhibited Topo II catalytic activity,
an ATP-dependent Topo II-mediated DNA relaxation
assay was performed. The results for the representative
compounds are shown in Figure 2, where AHMA, AMTs,
and APTs bound tightly to the DNA at concentrations of
5–25 lM. Since Topo II inhibitors are capable of inducing
double-stranded breaks, an in vitro K-SDS co-precipita-
tion assay for measuring the amount of protein-linked
DNA breaks (PLDBs) induced by AHMA, 14a, 14e,
15a, 15e, 17a, and 17e, was performed. After a 30 min
exposure to an increased concentration of these com-
pounds, except for compound 15b, steady-state levels of
PLDBs were increased in a dose-dependent manner, fol-
lowed by a plateau at a concentration of 5 lM (data not
shown). It reveals that there is a slight difference in PLDB
level in the AMA compounds (0.0 and 1.2% for 14a and
14e, respectively) and no difference among APA com-
pounds (4.6 and 4.3% for 17a and 17e, respectively). How-
ever, the amount of PLDBs generated by 15a was
significantly higher than 15e. Thus, a direct correlation
between the PLDB values of compounds tested and their
cytotoxicity was not seen. A similar result was found in
the series of (9-acridinylamino)toluidine derivatives.16
Figure 2. Inhibition of the DNA topoisomerase II catalytic activity by
5-(9-acridinylamino)anisidine derivatives. The experiment was per-
formed by the method described previously.26
intercalating and non-intercalating compounds. The re-
sult showed that AHMA caused a dose-dependent band
shift and produced a positive supercoiled DNA at con-
centrations greater than 2.5 lM (Fig. 3), indicating a
change in the drug/DNA-linking number. Additionally,
the DNA remained unchanged in the presence of 14e,
15e, or 17e at 10 lM, suggesting that there was strong
DNA intercalation and T4 ligase inhibition in the pres-
ence of these agents. The DNA-binding affinities for 14e,
15e, or 17e are greater than those for 14a, 15a, and 17a,
respectively, and were more cytotoxic than the other
tested compounds. The present results together with pre-
vious findings have demonstrated that increase in drug/
DNA-binding activity enhanced the cytotoxicity of 9-
anilinoacridines.
3.4. Interaction of AHMA, APT, and AMT derivatives
with DNA
To study the correlation between cytotoxicity and the
DNA-binding affinity of AHMA, APTs, and AMTs, a
DNA circle-ligation assay with linearized DNA and
T4 ligase was performed. The assay detected tertiary
structure changes resulting from DNA binding to both
4. Conclusion
We have synthesized a series of 5-(9-acridinylami-
no)anisidine derivatives for antitumor studies against a

6518
V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
Figure 3. Effect of DNA unwinding by 5-(9-acridinylamino)anisidine derivatives measure, as described previously.28,29
variety of human tumor cell growth in vitro. AOT 15e
appeared to be the most potent agent among the com-
pounds tested in this series. The SAR studies showed
that compounds bearing CONCH2CH2NMe2 and Me
substituents (AMA 14e, AOA 15e, and APA 17e) at
C4 and C5 of the acridine chromophore, respectively,
displayed potent cytotoxic effect in inhibiting growth
of various human tumor cells in culture. Additionally,
the present studies have demonstrated that the most
important factor that affects the cytotoxicity of 5-(9-
acridinylamino)anisidine (or 9-anilinoacridines) is the
drug/DNA-binding affinity. However, electronegativity
generated by the substituent (Me or OMe) on the anilino
ring and hydrophobicity of the drug may have some
effect on the drugꢀs biological activity. The current
systematic SAR studies will be beneficial for designing
future 9-anilinoacridines with increased potent antitu-
mor therapeutic efficacy.
appropriate MeO-substituted 1,3-phenylenediamines
(10 and 11) or 2-methoxy-5-nitroaniline (12) in a mix-
ture of CHCl3 and EtOH in the presence of 4-meth-
ylmorpholine (for 14a–e and 15a–e) or concd HCl (for
16a–e) by following the method described previous-
ly.14,16 The desired products were purified either by
recrystallization or chromatography on a silica gel col-
umn. Compounds 17a–e were synthesized by the reac-
tion of 16a–e under catalytic hydrogenation (5% Pd/C,
H2) in EtOH solution in the presence of concd HCl.
The synthesis of representative compounds is given be-
low. The analytic data and yield of other new derivatives
are shown in Table 3.
5.2.1.
N-Acridin-9-yl-5-methoxybenzene-1,3-diamine
(AMA 14a). A suspension of 3,5-diaminoanisole dihy-
drochloride (2.11 g, 10 mmol) and 4-methylmorpholine
(4.8 mL, 43.6 mmol) in ethanol (30 mL) was stirred in
an ice-methanol bath for 10 min. A solution of 9-chloro-
acridine (13a, 3.44 g, 16 mmol) in CHCl3 (50 mL) was
then added dropwise to the above mixture and stirred
at À5 ꢁC for 2 h and then at room temperature over-
night. The resulting solid product was collected by filtra-
tion and then recrystallized from EtOH to give 14a,
5. Experimental
5.1. Materials and methods
1
Melting points were determined on a Fargo melting
point apparatus and are uncorrected. Column chroma-
tography was carried out on silica gel G60 (70–230
mesh, ASTM, Merck and 230–400 mesh, Silicycle
Inc.). Thin-layer chromatography was performed on sil-
ica gel G60 F254 (Merck) with short-wavelength UV
light for visualization. Elemental analyses were done
on a Heraeus CHN-O Rapid instrument. 1H NMR spec-
tra were recorded in DMSO-d6 solution on a 600 MHz
Brucker AVANCE 600 DRX spectrometer, and chemi-
cal shifts are reported in ppm, relatively to TMS. Split-
ting patterns are designated as follows: s, singlet; br s,
broad singlet; d, doublet; t, triplet; q, quartet; m,
multiplet.
2.24 g (71%); mp 235–236 ꢁC; H NMR d 3.84 (3H, s,
OMe); 5.58 (2H, br s, NH2), 6.17 (1H; s, ArH), 6.23
(2H, br s, 2· ArH); 7.45 (2H, m, 2· ArH), 7.97 (2H,
m, 2· ArH); 8.11 (2H, m, 2· ArH), 8.35 (2H, m, 2·
ArH). Anal. (C20H17N3OÆHClÆ0.2H2O) C, H, N.
By following the same procedure as that for the synthe-
sis of 14a, compounds 14b–e and 15a–e were prepared.
5.2.2.
N-Acridin-9-yl-4-methoxybenzene-1,3-diamine
(APA 17a). A mixture of 16a (1.15 g, 3.0 mmol) and 5%
Pd/C in methanol (250 mL) containing concd HCl
(0.5 mL) was hydrogenated at 50 psi for 25 min. The mix-
ture was filtered through a pad of Celite and the solid cake
was washed with methanol. The filtrate and washing were
combined and evaporated under reduced pressure to dry-
ness. The residue was chromatographed on a silica gel
column (2 · 24 cm) using CHCl3/MeOH (10:1 v/v) as
the eluent. The product 17a was eluted from CHCl3/
MeOH (5:1 v/v). Fractions containing 17a were com-
bined and concentrated under reduced pressure. The res-
5.2. General procedure for the preparation of
5-(9-acridinylamino)anisidines
All new 5-(9-acridinylamino)anisidines (14a–e, 15a–e,
and 16a–e) were prepared by the condensation of the
requisite substituted 9-chloroacridines (13a–e) and the

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
Table 3. Analytic data and yield of 5-(9-acridinylamino)anisidines
6519
Compound
Chemical formula*
mp (ꢁC)
Yield (%)
Analysis
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
16a
16b
16c
16d
16e
17a
17b
17c
17d
17e
C
20H17N3OÆHClÆ0.2H2O
235–236
224–225
257–258
224–225
207–208
283–284
280–283
221–222
220–221
173–174
239–240
207–208
185–186
254–255
208–209
243–244
230–231
207–208
278–279
229–230
71
69
55
77
45
91.4
86
58.6
53
75
90
74
72
74
73
70
84
51
83
72
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C21H19N3OÆ2HClÆH2O
C25H27N5O2Æ4HClÆ0.5H2O
C23H22N4O2Æ0.25HClÆ0.25H2O
C
C
26H29N5O3Æ4HClÆ2.5H2O
20H17N3OÆ2.5HClÆ0.8H2O
C21H19N3OÆ2HClÆ0.5H2O
25H27N5O2Æ5HClÆ3.2H2O
C23H22N4O2ÆHClÆ3.7H2O
26H29N5O2Æ3HClÆ3.6H2O
C20H15N3OÆHClÆ0.1H2O
20H15N3O3Æ0.8HClÆ1.8H2O
C
C
C
C25H25N5O4ÆHClÆ2H2O
C23H22N4O2Æ1.53HClÆ0.25H2O
C
26H27N5O4Æ2HClÆ2H2O
C20H17N3OÆHClÆ0.6H2O
C21H19N3OÆ5HClÆ0.5H2O
C25H27N5O2Æ2.5HClÆ4.1H2O
C
23H22N4O2Æ2.5HCl.0.8H2O
C26H29N5O2Æ4HClÆ3.5H2O
* All compounds are hygroscopic and contain HCl and crystal water.
idue was treated with 4.2 N HCl/EtOAc (3 mL) and then
evaporated in vacuo to dryness. The residue was co-evap-
orated several times with EtOH and the solid residue was
recrystallized from EtOH/acetone to give 17a, 667 mg
0.25 lg pBR322 DNA. The inhibition of relaxation
activity was determined by comparison with an untreat-
ed control. AHMA was used as a positive control.
1
(70%); mp 243–245 ꢁC; H NMR d 3.36 (3H, s, OMe),
5.3.3. Measurement of protein-linked DNA breaks. Cells
in log-phase growth were labeled with [14C]thymidine
for 24 h. After labeling, the cells were trypsinized, resus-
pended in fresh medium at a density of 5 · 105 cells/mL,
and shaken gently in a 37 ꢁC water bath for 1 h in sus-
pension. Various concentrations 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 potassium-sodium dodecyl sulfate (K-
SDS) precipitation method, as described previously.27
The percentage of PLDBs generated by the compounds
was calculated by radioactivity (cpm) of precipitated
DNA divided by the total radioactivity (cpm).
6.73 (1H, m, ArH), 6.78 (1H, m, ArH), 6.96 (1H, m,
ArH), 7.39 (2H, m, ArH), 7.94 (2H, m, ArH), 8.05 (2H,
m, 2· ArH), 8.27 (2H, m, 2· ArH), 10.36 (2H, br s,
exchangeable, NH2), 11.49 (1H, br s, exchangeable,
NH). Anal. (C20H17N3OÆHClÆ0.6H2O) C, H, N.
By following the same procedure as that for the synthe-
sis of 17a, compounds 17b–e were prepared.
5.3. Biological assays
5.3.1. Cytotoxicity assays. 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, oral
carcinoma KB, breast carcinoma MCF-7 and MX-1,
and T-cell acute lymphocytic leukemia CCRF-CEM),
for a 72 h incubation, by the XTT-tetrazolium assay,
as described by Scudiero et al.22 After the addition of
phenazine methosulfate-XTT solution at 37 ꢁC for 6 h,
absorbance at 450 and 630 nm was detected on a micro-
plate reader (EL 340; Bio-Tek Instruments Inc., Winoo-
ski, 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,23,24 using a computer program on
an IBM PC workstation.25
5.3.4. DNA unwinding measurement. The DNA unwind-
ing effect of drugs was assayed according to the method
described by Camilloni et al.28 Briefly, 20 lg of pBR322
DNA was linearized with HindIII restriction endonucle-
ase and recovered by phenol/chloroform extraction and
ethanol precipitation. The reaction mixtures (totally
200 lL) containing 66 mM Tris–HCl (pH 7.6), 6 mM
MgCl2, 10 mM dithiothreitol, 0.7 mM ATP, 0.6 lg
DNA, and drugs were equilibrated at 15 ꢁC for 10 min
and then incubated with excess amount of T4 DNA li-
gase at 15 ꢁC for 60 min. The reaction was stopped by
the addition of 20 mM EDTA. DNA was analyzed by
agarose-gel electrophoresis after treatment to remove
the drugs from the reaction mixture: extraction with phe-
nol and ether, and precipitation with ethanol. DNA was
resuspended in 25 lL TE buffer with 1% SDS and ana-
lyzed in 1% agarose gel in TAE overnight.29
5.3.2. Inhibition of topoisomerase II catalytic activity by
drugs. Topo-II catalytic activity was assayed by the
ATP-dependent relaxation of pBR322 supercoiled
DNA.26 Various concentrations of the drugs were incu-
bated with 2 U DNA topoisomerase II (Topogen) and
5.3.5. In vivo assay. Athymic nude mice bearing the
nu/nu gene were used for human breast tumor MX-1
xenograft. Outbred Swiss-background mice were ob-

6520
V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 6513–6520
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tained from Chares River Breeding Laboratories. Male
mice 7 weeks old or older weighing 22 g or more were
used for experiments. Drug was administered via tail
vein by iv injection. Tumor volumes were assessed by
measuring the length · width · height (or width) by
using caliper. Vehicle used was 20 lL DMSO, diluted
with 180 lL saline. All animal studies were conducted
in accordance with the Guidelines of the National Insti-
tutes of Health Guide for the Care and Use of Animals
and the protocol approved by the Institutional Animal
Care and Use Committee.
Acknowledgments
This work was supported by the National Science Coun-
cil of Taiwan (Grant No. NSC92-2320-B-001-004). The
NMR spectra of synthesized compounds were obtained
at High-Field Biomacromolecular NMR Core Facility
supported by the National Research Program for Geno-
mic Medicine (Taiwan).
17. Cain, B. F.; Atwell, G. J.; Denny, W. A. J. Med. Chem.
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Supplementary data
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The 1H NMR spectroscopic data of compounds listed in
Table 3 can be found, in the online version, at
doi:10.1016/j.bmc.2005.07.018.
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