Potent antitumor N-mustard derivatives of 9-anilinoacridine, synthesis and antitumor evaluation

Article abstract of DOI:10.1016/j.bmcl.2004.06.080

A series of 9-anilinoacridine N-mustard derivatives, in which the alkylating N-mustard residue was linked to the C-3′ or C-4′ position of the anilino ring with an O-ethylene spacer, was synthesized and evaluated for cytotoxicity against human lymphoblasti

Full text of DOI:10.1016/j.bmcl.2004.06.080

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4719–4722
Potent antitumor N-mustard derivatives of
9-anilinoacridine, synthesis and antitumor evaluation
Valeriy A. Bacherikov,a Ting-Chao Chou,b Hua-Jin Dong,b Ching-Huang Chen,a
Yi-Wen Lin,a Tsong-Jen Tsaia and Tsann-Long Sua,
*
aLaboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
bMolecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
Received 20 May 2004; revised 23 June 2004; accepted 25 June 2004
Available online 19 July 2004
Abstract—A series of 9-anilinoacridine N-mustard derivatives, in which the alkylating N-mustard residue was linked to the C-30 or
C-40 position of the anilino ring with an O-ethylene spacer, was synthesized and evaluated for cytotoxicity against human lympho-
blastic leukemic cells (CCRF-CEM) in culture. The results showed that all of the new compounds exhibited potent cytotoxicity with
IC50 values ranging from 0.002 to 0.7lM, which were as potent or significantly more potent than 3-(9-acridinylamino)-5-hydroxy-
methylaniline (AHMA). Compound 9 did not exhibit cross-resistance against both vinblastine-resistant (CCRF-CEM/VBL) and
taxol-resistant (CCRF-CEM/taxol) cells. Additionally, compound 9 demonstrated potent antitumor effect in nude mice bearing
human breast carcinoma MX-1 xenografts, resulting in complete tumor remission in two out of three mice at the maximal dose
of 1–2mg/kg (Q3D·7) or 3mg/kg (Q4D·5) via intravenous injection.
ꢀ 2004 Elsevier Ltd. All rights reserved.
DNA bifunctional alkylating agents containing nitrogen
mustard moiety are an important class of anticancer
drugs.1,2 Although the mustard derivatives are capable
of cross-linking to DNA doubled strands, the high react-
ivity of these agents lack the affinity to bind to DNA,
resulting in the loss of their antitumor activity. This is
perhaps due to either the formation of mono-alkylation
via partial hydrolysis or interaction with other cellular
components. The formation of the mono-alkylation
has proven to produce genotoxicity resulting in less
cytotoxicity.3 The drawback of N-mustard drugs has
been improved by linking the alkylating moiety to a
DNA affinic carrier such as DNA-intercalating chromo-
phore (e.g., 9-aminoacridine,4,5 9-anilinoacridine,6
anthraquinone,7 cyclopentanthraquinone8) or DNA
minor groove binder (such as distamycin A and related
analogues)9 to allow the drug to target the DNA, to of-
fer higher regio- and sequence selectivity, and to mini-
mize the limitations of nitrogen mustards.10,11
N-Mustards targeted by intercalating carriers such as
5
7
acridine (1) and anthraquinones (2) can achieve 10-
to 100-fold improvements in potency and improved an-
tileukemic activities compared with their corresponding
untargeted mustards of similar reactivity. Fan et al.6
synthesized aniline mustard analogues of the DNA-
intercalating agent m-AMSA (i.e., compounds 3 and 4)
by linking a mustard residue to the anilino ring or acr-
idine chromophore, respectively. These agents, however,
were less potent than the parent m-AMSA in mice bear-
ing murine leukemia p388. The low potency of 3 and 4 is
perhaps due to altering the drugÕs binding site(s) on the
DNA and drug/topoisomerase II (Topo II) interactions.
To enhance the interaction between 9-anilinoacridine
and Topo II and/or DNA, one can introduce the N-
mustard residue to 9-anilinoacridine with a short spacer.
Based on this hypothesis, we designed and synthesized
N-mustard derivatives of 9-anilinoacridine, in which
the N-mustard residue was linked to the anilino ring
with a short spacer (O–C2) and located at the C-30 or
C-40 position (Fig. 1).
Keywords: Acridines; Antitumor compounds; Alkylating agents; Syn-
thesis; Chemotherapy.
3-(9-Acridinylamino)-5-hydroxymethylaniline (AHMA,
5), was previously demonstrated as a potent topoiso-
merase II inhibitor and exhibited significant antitumor
*
Corresponding author. Tel.: +886-2-2789-9045; fax: +886-2-2782-
7685; e-mail: tlsu@ibms.sinica.edu.tw
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.080

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V. A. Bacherikov et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4719–4722
O
MeO
CH2N(CH2CH2Cl)2
(CH2)nX
N(CH2CH2Cl)2
HN
N
OMe
O
OMe
NHSO2 Me
MeO
2
1
HN
N
CONH(CH2)3
N(CH2CH2Cl)2
3
NH2
MeO
NHCO(CH2)3
N(CH2CH2Cl)2
HN
N
CH2OH
HN
N
4
5 AHMA
Figure 1. Structures of N-mustard analogues and AHMA.
activity both in vitro and in vivo.12–14 To introduce the
N-mustard moiety to AHMA, we synthesized a bioiso-
steric isomer of AHMA namely, 3-(9-acridinylamino)-
5-hydroxymethylphenol (8) (Scheme 1). The known
3-amino-5-hydroxybenzyl alcohol (6) was condensed
with 9-chloroacridine (7a0) to give 8, which was then
converted to potassium salt by treating with 0.2N
KOH in MeOH. The solid salts were then treated with
tris(2-chloroethyl)amine hydrochloride in DMF in the
presence of K2CO3/KF to produce moderate yield
(50%) of compound 9. Alternatively, reacting m-nitro-
phenol (10a) or p-nitrophenol (10b) with tris(2-chloro-
ethyl)amine hydrochloride afforded 11a,b, which were
then reduced (SnCl2/concd HCl) to the corresponding
aniline derivatives 12a,b. The unstable crude anilines
12a,b were used directly without purification for the
condensation with the requisite 9-chloroacridines
(7a0,b0) and the desired 9-anilinoacridine derivatives
(13aa0, 13ab0, 13ba0, and 13bb0) bearing an O–C2 spacer
between the anilino ring and the N-mustard residue were
smoothly obtained in moderate yield (Scheme 2).
The cytotoxicity of the new N-mustard derivatives of
9-anilinoacridine against human lymphoblastic leukemic
cells (CCRF-CEM), A549 lung carcinoma, and
HCT-116 colon tumor was shown in Table 1 and was
compared with AHMA (5). This clearly demonstrated
that all of the new compounds were significantly more
potent than AHMA (5). Compound 9 carrying a
CH2OH function with IC50 value of 0.007lM was
approximately 107-fold more cytotoxic than AHMA
(IC50 =0.753lM). While compound 13aa0, lacking the
CH2OH substituent, was approximately 14-fold less
active that 9, thus indicating the CH2OH function
played an important role in the cytotoxicity of
AHMA derivatives. It also revealed that introduction
of a dimethyaminoethylcarboxamido and a methyl
function to the C-4 and C-5 of the acridine chromo-
phore (i.e., compound 13ab0) further increased the cyto-
toxicity of the parent compound 13aa0. Compounds
with the alkylating chain at C-40 (13ba0) were about
5-fold more cytotoxic than the corresponding C-30-sub-
stituted one (13aa0). The low cytotoxicity of compound
OR
OH
Cl
N
HN
N
CH2OH
i
+
H2N
CH2OH
R2
R1
6
8 R = H
7a' (R1 = R2 = H)
ii
a: R = CH2OH
b: R = H
9 R = CH2CH2N(CH2CH2Cl)2
Scheme 1. Reagents and conditions: (i) CHCl3/EtOH/concd HCl (catalytic amount); (ii) K2CO3/KF/DMF, 35ꢁC.

V. A. Bacherikov et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4719–4722
4721
(i)
R
O(CH2)2N(CH2CH2Cl)2
O2N
R
10a,b
a: 3-OH; b: 4-OH
11a,b R = NO2
(ii)
12a,b R = NH2
(iii)
O(CH2)2N(CH2CH2Cl)
Cl
HN
N
N
R2
R1
R2
R1
7a' (R1 = R2 = H)
13aa',13ab',13ba',13bb'
7b' (R1 = CONHCH2CH2NMe2;
R2 = Me)
Scheme 2. Reagents and conditions: (i) tris(2-chloroethyl)amine hydrochloride/K2CO3/KF/acetone, reflux; (ii) SnCl2/concd HCl, 90ꢁC; (iii) CHCl3/
EtOH/concd HCl (catalytic amount); a series: N-mustard residue substituted at C-30; b series: N-mustard residue substituted at C-40; a0 series:
R1 =R2 =H; b0 series: R1 =CONHCH2CH2NMe2, R2 =Me.
Table 1. The cytotoxicity of N-mustard analogues of 9-anilinoacridine against human tumor cell growth in culturea
Compound
IC50 (lM)
CCRF-CEM/taxol
CCRF-CEM
CCRF-CEM/VBL
A549
HCT-116
AHMA 5
9
0.753
0.0070
0.095
1.60 [2.1·]b
0.0075 [1.1·]
ND
0.600 [0.8·]
0.0340 [4.9·]
ND
0.0470
0.0056
ND
NDc
0.0055
ND
13aa0
13ba0
0.0200
0.0030
0.7730
0.0015
0.0012
0.066 [3.3·]
ND
ND
0.061 [3.1·]
ND
ND
ND
ND
ND
ND
13ab0
13bb0
ND
0.0029
0.0099
ND
0.0026
0.0087
Taxol
Vinblastine
1.62 [1080·]
0.540 [450·]
0.143 [95.3·]
0.029 [24.2·]
a XTT assays were used for leukemia cells and SRB assays were used for solid tumor cells. Incubation was 72h, as described previously.15
b Numbers in the bracket are folds of resistance of the resistant cells when compared with the IC50Õs of the CCRF-CEM parent cells.
c ND: not determined.
13bb0 was an exception. Additional compounds are
currently being studied in our laboratory to gain a
better understanding of their structure–activity
relationship.
revealed that the therapeutic efficacy of compound
9 [maximal dose: 1–2mg/kg (Q3D·7) or 3mg/kg
(Q4D·5); intravenous injection] on nude mice (n=3)
bearing human breast carcinoma MX-1 xenografts
resulted in complete tumor remission in two out of three
mice.
At present we merely took compound 9 for further anti-
tumor investigation since this agent is structurally closer
to AHMA. Table 1 showed the growth inhibition of hu-
man lymphoblastic leukemic cells (CCRF-CEM) and its
drug-resistant sublines (resistant to vinblastine and
taxol, CCRF-CEM/VBL, and CCRF-CEM/taxol,
respectively) by compound 9. The results demonstrated
that 9 did not develop cross-resistance to vinblastine
or taxol. It suggests that compound 9 is neither a good
substrate of p-glycoprotein nor mutated tubulin. Table 2
The current studies resulted in finding potent antitumor
N-mustard derivatives of 9-anilinoacridine, in which the
alkylating N-mustard residue is linked to the anilino
ring with a short spacer (O–C2). The significant cytotox-
ic effects of all new N-mustard derivatives indicated that
these agents may possess dual effects: DNA cross-link-
ing and Topo II inhibition. The present study also dem-
onstrated that compound
9 exhibited significant
Table 2. Therapeutic effects and toxicity of compound 9 in nude mice bearing human mammary carcinoma (MX-1) xenograftsa
Dose (mg/kg) Schedule Average body weightb change (g)
Average tumor size (T/C) Tumor free Toxicity
D11 D14 D17 D20 D23
(death)
D26
D17 D20 D23 D26
1.0 1.0 1.0 1.0
0.37 0.17 0.14 ND
Control
9
29.2 +1.6 +1.5 +1.5 +1.9 ––c
0/3c
2/3d
0/3
0/3
1–2
Q3D·7
28.3 +1.2 ꢀ1.4 ꢀ3.0 ꢀ3.0 ꢀ3.9
a MX-1 tissue 50mg was implanted S.C. on Day 0. Treatment (iv injection) began on D11 when tumor size were 80 ꢁ 120mm3.
b Body weight=total body weightꢀtumor weight.
c Animals were sacrificed when there was excessive tumor burden (e.g., tumor size >3500mm3).
d Animals were sacrificed on day 32.

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Acknowledgements
This work was supported by the National Science Coun-
cil, Taiwan (Grant No. NSC90-2320-B-001-032). 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.
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Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.bmcl.2004.06.080.
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