Potent antitumor 9-anilinoacridines bearing an alkylating N-mustard residue on the anilino ring: Synthesis and biological activity

Article abstract of DOI:10.1016/j.bmc.2005.03.057

A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure-activity relationship studies. The alkylating N-mustard residue was linked to the C-3′ or C-4′ position of the anilino ring with an O-ethylene (O-C₂

Full text of DOI:10.1016/j.bmc.2005.03.057

Bioorganic & Medicinal Chemistry 13 (2005) 3993–4006
Potent antitumor 9-anilinoacridines bearing
an alkylating N-mustard residue on the anilino ring:
synthesis and biological activity
Valeriy A. Bacherikov,a Ting-Chao Chou,b Hua-Jin Dong,b
Xiuguo Zhang,b Ching-Huang Chen,a Yi-Wen Lin,a Tsong-Jen Tsai,a
Rong-Zau Lee,a Leroy F. Liuc and Tsann-Long Sua,*
aLaboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
bMolecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
cDepartment of Pharmacology, University of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
Received 18 February 2005; revised 31 March 2005; accepted 31 March 2005
Available online 28 April 2005
Abstract—A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure–activity relationship
studies. The alkylating N-mustard residue was linked to the C-30 or C-40 position of the anilino ring with an O-ethylene (O–C2), O-
butylene (O–C4), and methylene (C1) spacer. All of the new N-mustard derivatives exhibited significant cytotoxicity in inhibiting
human lymphoblastic leukemic cells (CCRF–CEM) in culture. Of these agents, (3-(acridin-9-ylamino)-5-{2-[bis(2-chloro-
ethyl)amino]ethoxy}phenyl)methanol (10) was subjected to antitumor studies, resulting in an approximately 100-fold more potent
effect than its parent analogue 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA) in inhibiting the growth of human lympho-
blastic leukemic cells (CCRF–CEM) in vitro. This agent did not exhibit cross-resistance against vinblastine-resistant (CCRF–CEM/
VBL) or Taxol-resistant (CCRF–CEM/Taxol) cells. Remarkably, the therapeutic effect of 10 at a dose as low as one tenth of the
Taxol therapeutic dose [i.e., 1–2 mg/kg (Q3D · 7) or 3 mg/kg (Q4D · 5); intravenous injection] on nude mice bearing human breast
carcinoma MX-1 xenografts resulted in complete tumor remission in two out of three mice. Furthermore, 10 yielded xenograft
tumor suppression of 81–96% using human T-cell acute lymphoblastic leukemia CCRF–CEM, colon carcinoma HCT-116, and
ovarian adenocarcinoma SK-OV-3 tumor models.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
hydrolysis, the high reactivity of these agents lack the
affinity to bind to DNA, resulting in the loss of their
antitumor activity. The prevalence of forming the
mono-alkylation has proven to produce genotoxicity
and resulted in less cytotoxicity.5
The alkylating nitrogen mustard derivatives exert their
cytotoxic effects by interstrand cross-linking of DNA.1
Sequences with contiguous guanines have the lowest
molecular electrostatic potentials and therefore are more
vulnerable to nucleophilic attacks.2,3 Earlier works on
the isolation of cross-linked fragments from digested
alkylator-treated DNA showed that these cross-links oc-
curred predominantly on the guanine N7 position.4
However due to the formation of mono-alkylation or
interaction with other cellular components via partial
Simple alkylators have little ability to recognize se-
quences larger than a single nucleotide.6 Additionally,
resistance to such reactive electrophiles is easily devel-
oped by an increase in the cellular levels of low-molecu-
lar-weight thiol (particularly glutathione).7 In principle,
many of these drawbacks could be ameliorated by link-
ing the N-mustard to a carrier molecule having an affin-
ity for DNA, which by reacting with other cell
components should result in less diversion of the active
drug. The use of sequence-specific carriers should direct
the pattern of alkylation sites on the DNA and ulti-
mately provide specific targets to certain genes. An
Keywords: Acridines; Antitumour compounds; Alkylating agents;
Synthesis; Chemotherapy.
*
Corresponding author. Tel.: +886
27827685; e-mail: tlsu@ibms.sinica.edu.tw
2 27899045; fax: +886 2
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.057

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V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
improvement in the proportion of lethal cross-linking
with respect to more genotoxic monoalkylation events
may limit the known carcinogenicity of alkylators.8
Finally, the development of resistance to such
compounds by elevating cellular thiol levels may be of
less concern.
chromophore and the anilino ring, respectively. At the
optimal dose, these analogues exhibited smaller percent-
age increase in lifespan (%ILS) than the parent m-
AMSA (5) in mice bearing murine leukemia p388.13
The 4-linked analogues (3) showed slightly higher in
vivo antileukemic activity than their corresponding 10-
linked analogues (4), suggesting that cytotoxic potencies
of these compounds may depend on the precise location
of the N-mustard moiety on acridine. The decrease in
effectiveness of compounds 3 and 4 is probably due to
the long linker between the alkylating N-mustard resi-
due and the anilino ring or the acridine chromophore,
leading to alternation of the binding site(s) on the
DNA and/or drug/topoisomerase II (Topo II) interac-
tions. Taking these into consideration, it may be possible
to optimize the cytotoxic activity of these DNA-directed
alkylating agents by designing a drug that exhibits
both cytotoxic and molecular processes. For example,
to enhance the interaction of 9-anilinoacridine with
Topo II and/or DNA, one can introduce an alkylating
N-mustard residue to the anilino and/or the acridine
chromophore with a short spacer between the N-mustard
residue and the 9-anilinoacridine core or alter the position
of the alkylating moiety on the anilino ring.
Thus, designing and synthesizing DNA-directed alkyla-
tors have been one of the attractive approaches in the
development of potent anticancer drugs. Earlier works
on the development of DNA-directed alkylating agents
have employed DNA-intercalating chromophore (e.g.,
heterocyclic compounds,9,10 9-aminoacridine,11,12 9-ani-
linoacridine,13 4-anilinoquinoline,14,15 anthraquinone,16
and cyclopentanthraquinone17) as a DNA affinic carrier.
For example, N-mustard targeted by intercalating carri-
ers such as acridine (1, Chart 1), and anthraquinones (2)
can achieve 10- to 100-fold improvement in potency and
improve antileukemic activities compared with their cor-
responding untargeted mustards of similar chemical
reactivity,10,12,16,18 showing altered patterns of DNA
alkylation.6 Most of the recent works, however, have
utilized DNA minor groove binders (such as distamycin
A and related analogues) as the carriers to offer higher
regio- and sequence selectivity and to minimize the limi-
tations of nitrogen mustards.19–26 In most cases, the
DNA-directed alkylating agents have higher cytotoxi-
city and potency than their corresponding untargeted
alkylating agents. Nevertheless, only a limited number
of compounds (e.g., tallimustin27) were found to have
potential clinical application.
Our recent research on the development of new potential
antitumor agents demonstrated that 3-(9-acridinyl-
amino)-5-hydroxymethylaniline (AHMAÆHCl, 6), a po-
tent Topo II inhibitor, was more cytotoxic than
m-AMSA (5) both in vitro and in vivo.28–30 Presumably,
AHMA exhibits its antitumor activity by stabilizing the
DNA–drug–Topo II ternary complex, the cleavable
complex,31 which is highly effective in killing tumor cells.
The acridine chromophore of these compounds is gener-
ally proposed to be the DNA-binding domain, while the
Fan et al.13 synthesized aniline mustard analogues of the
DNA-intercalating agent m-AMSA (i.e., compounds 3
and 4) by linking a N-mustard chain to the acridine
Figure 1. Therapeutic effect of compound 10 (3 mg/kg, Q3D · 8, iv infusion) against human ovarian adenocarcinoma xenograft SK-OV-3 (A) and
the body weight changes during treatment as indicated by arrows (B). Therapeutic effect of compound 10 (2 mg/kg, Q3D · 6, iv infusion) and Taxol
(20 mg/kg, Q3D · 8, 6 h) (C and E), respectively. The corresponding body weight changes are shown in D and F, respectively.

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
3995
Chart 1. Structures of N-Mustard analogues and AHMA.
anilino moiety is the enzyme-binding domain in these
ternary complexes. In spite of this proposed mechanism
of action for 9-anilinoacridines, there is a lack of com-
plete understanding of the ternary complex formation.
ene (O–C2), O-butylene (O–C4), and methylene (C1)
units. These new target agents allowed us to study the
effects of the substituted position of the N-mustard on
the anilino ring and the role of spacer length on their
antitumor activity. The results revealed that all com-
pounds described in this paper exhibited significant anti-
tumor effects. Here, we described the synthesis,
antitumor efficacy and the effects on Topo II-mediated
DNA cleavage of the new target compounds.
In view of the intriguing AHMA structure, one can
envisage that AHMA may have better interactions with
both Topo II and DNA, since AHMA has superior anti-
tumor effects over m-AMSA. Therefore, we designed
and synthesized AHMA analogues bearing an alkylating
N-mustard residue, which was linked to the anilino ring
with a short spacer. To introduce a N-mustard alkyla-
ting moiety to AHMA, we synthesized a bioisostere of
AHMA 3-(9-acridinylamino)-5-hydroxymethylphenol
(9). Compound 9 was then converted into 9-anilinoacri-
dine bearing a N-mustard moiety (compound 10) by
treating it with tris(2-chloroethyl)amine. We found that
compound 10 was approximately 100-fold more cyto-
toxic than AHMA in inhibiting the cell growth of hu-
man lymphoblastic leukemia cells (CCRF–CEM) in
culture.32 This impressive result prompted us to synthe-
size a series of 9-anilinoacridines bearing a N-mustard,
in which the N-mustard was linked to the C-30 or C-40
of the anilino ring with a short spacer, such as O-ethyl-
2. Chemistry
The 9-anilinoacridines bearing an alkylating N-mustard
on the anilino ring with an O–C2 spacer were synthe-
sized by directly introducing a N-mustard chain to the
OH function of 3-(9-acridinylamino)phenol derivatives
(Scheme 1, method 1). The known 3-amino-5-hydroxy-
benzyl alcohol (7) was condensed with 9-chloroacridine
(8a0) to give 3-(9-acridinylamino)phenol derivative (9),
which was then treated with 0.2 N potassium hydroxide
in methanol to form the solid potassium salt. The salt
was then treated with tris(2-chloroethyl)amine in
DMF in the presence of K2CO3 and KF to produce a
Scheme 1. Reagents and conditions: (i) CHCl3/EtOH/concd HCl (catalytic amount); (ii) K2CO3/KF/DMF, 35 ꢁC.

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V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
moderate yield of compound 10. Alternatively, com-
pounds containing an O–C2 or O–C4 spacer between
the anilino ring and the N-mustard moiety located at
the C-30 or C-40 position of the anilino ring were synthe-
sized as shown in Scheme 2. Reacting nitro substituted
phenol derivatives (11a–e) with tris(2-chloroethyl)amine
hydrochloride in DMF in the presence of K2CO3 and
KF in refluxing acetone afforded bis(2-chloroethyl)-[2-
(3- or 4-nitrophenoxy)]ethylamines (14a–e), which were
then reduced (SnCl4/concd HCl) to the corresponding
aniline derivatives 16a–e in low yield. The unstable
crude aniline intermediates were used directly without
purification for the condensation with the requisite 9-
chloroacridines (8a0,b0) and the desired 9-anilinoacridine
derivatives (18aa0,ab0,ba0,bb0,ca0,da0, and 18ea0) bearing
an O–C2 spacer between the anilino ring and the N-mus-
tard residue were smoothly obtained in moderate yield.
Another synthetic route to construct compounds bear-
ing an O–C4 spacer between the anilino ring and N-mus-
tard was developed (Scheme 2). Thus, treatment of
nitrophenols (11a,b) with excess of 1,4-dibromobutane
under similar conditions (K2CO3/KF/Me2CO) gave 1-
(4-bromobutoxy)-3- or 4-nitrobenzenes (12a,b), which
were further reacted with diethanolamine to afford
bis(2-hydroxyethyl)-[4-(3- or 4-nitrophenoxy)butyl]amines
(13a,b). Prolongation of the reaction of 13a,b with
methanesulfonyl chloride in the presence of triethyl-
amine (2–3 days) led to the formation of bis(2-chloro-
ethyl)-[4-(3- or 4-nitrophenoxy)butyl]amines (15a,b).
Apparently, the chlorine anion generated from metha-
nesulfonyl chloride attacked the O-methanesulfonyl
function of the intermediate to furnish the desired
bis(2-chloroethyl) substituted products. The nitro
substituted derivatives 15a,b were further reduced
(SnCl4/concd HCl) to the corresponding aniline deriva-
tives 17a,b, which were then condensed with 9-chloroac-
ridines (8a0,b0) to afford the desired 9-anilinoacridines
(19aa0,ab0,ba0, and 19bb0). The N-mustard derivatives
of 9-anilinoacridine bearing a C1 spacer were prepared
starting from 3- or 4-nitrobenzyl chloride (20a and
20b, respectively) by the reaction with diethanolamine
(Scheme 3). The products (21a,b) were further converted
into the bis(2-chloroethyl)-3- or 4-aminobenzylamines
(23a,b) by treating them with methanesulfonyl chloride
followed with reduction as described previously. Simi-
larly, condensation of the crude anilines (23a,b) with
9-chloroacridines (8a0,b0) afforded the desired 9-anilino-
acridine derivatives (24aa0,ab0,ba0, and 24bb0).
3. Biological results and discussion
3.1. In vitro cytotoxicity
The cytotoxicity of the 9-anilinoacridines bearing an
alkylating N-mustard residue on the anilino ring against
human lymphoblastic leukemic cells (CCRF–CEM) is
shown in Table 1. AHMA (6) was used for comparison.
This clearly demonstrated that all of the new com-
pounds were significantly more potent than AHMA
(6). Compound 10 carrying a CH2OH function with
an IC50 value of 0.007 lM was about 100-fold more
cytotoxic than AHMA (IC50 = 0.753 lM), while, com-
pound 18aa0 lacking the CH2OH substituent was
approximately 14-fold less active that 10. The result,
similar to previous observations, confirmed that the
CH2OH function played an important role in the cyto-
toxicity of AHMA derivatives.29 In general, compounds
with a O–C2 spacer between the anilino ring and the N-
mustard residue were more cytotoxic than their corre-
sponding compounds with a O–C4 linker located at
either C-30 or C-40. With one exception, 19bb0 was more
Scheme 2. Reagents and conditions: (i) Tris(2-chloroethyl)amine hydrochloride/K2CO3/KF/acetone, reflux; (ii) Br(CH2)4Br/K2CO3/acetone, reflux;
(iii) NH(CH2CH2OH)2/DMF, 110 ꢁC; (iv) MeSO2Cl/Et3N, 0 ꢁC to rt, 2 days; (iv) CHCl3/EtOH/concd HCl (catalytic amount).

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
ii
3997
i
CH2N(CH2CH2Cl)2
22a,b (R = NO2)
23a,b (R = NH2)
R
CH2N(CH2CH2OH)2
R
O2N
O2N
20a,b
21a,b
a: R = 3-CH2Cl
b: R = 4-CH2Cl
8a',b'
iii
CH2N(CH2CH2Cl)2
HN
N
R2
R1
24aa', 24ab', 24ba', 24bb'
Scheme 3. Reagents and conditions: (i) NH(CH2CH2OH)2, 120 ꢁC; (ii) MeSO2Cl/Et3N, 0 ꢁC to rt, 2 days; (iii) CHCl3/EtOH/concd HCl (catalytic
amount).
Table 1. The in vitro cytotoxicity of N-mustard derivatives of 9-anilinoacridine against human lymphoblastic leukemic cells (CCRF–CEM)
Compound
R1
R2
R3
R4
R5
R6
IC50 (lM)
AHMAÆHCl 6
10
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
O(CH2)2N(CH2CH2Cl)2
H
CH2OH
CH2OH
H
H
H
0.753
0.0070
0.095
H
H
H
18aa0
19aa0
18ba0
19ba0
18ab0
19ab0
18bb0
19bb0
18ca0
18da0
18ea0
24aa0
24ba0
24ab0
24bb0
O(CH2)2N(CH2CH2Cl)2
O(CH2)4N(CH2CH2Cl)2
H
H
H
H
O(CH2)2N(CH2CH2Cl)2
H
H
H
0.4555
0.0200
0.0610
0.0030
0.0235
0.7730
0.0230
0.0263
0.0388
0.0026
0.0074
0.0300
0.0017
0.0081
H
H
H
H
H
O(CH2)2N(CH2CH2Cl)2
O(CH2)4N(CH2CH2Cl)2
H
H
H
CONH(CH2)2NMe2
H
H
Me
Me
Me
Me
H
O(CH2)4N(CH2CH2Cl)2
H
H
H
CONH(CH2)2NMe2
CONH(CH2)2NMe2
O(CH2)2N(CH2CH2Cl)2
O(CH2)4N(CH2CH2Cl)2
O(CH2)2N(CH2CH2Cl)2
O(CH2)2N(CH2CH2Cl)2
OMe
H
H
H
CONH(CH2)2NMe2
H
H
Me
H
H
O(CH2)2N(CH2CH2Cl)2
H
H
H
H
H
CH2N(CH2CH2Cl)2
H
H
H
H
H
CH2N(CH2CH2Cl)2
H
CH2N(CH2CH2Cl)2
H
H
H
CH2N(CH2CH2Cl)2
H
H
CONH(CH2)2NMe2
CONH(CH2)2NMe2
Me
Me
H
potent than the corresponding 18bb0. We also found that
the substituent(s) on the anilino or acridine ring affected
the cytotoxicity of the target compounds: (1) addition of
an OMe group to C-40 of 18ba0 resulted in increased po-
tency (18ba0 vs 18ea0); (2) introduction of a Me group to
the anilino ring did not affect the cytotoxicity of the par-
ent 18aa0 (18ca0 or 18da0 vs 18aa0); (3) addition of a
dimethyaminoethylcarboxamido and a Me function to
the C4 and C5 of the acridine chromophore further in-
creased the cytotoxicity of the corresponding parent
compound with an exception of compound 18bb0
(18aa0 vs 18ab0; 18ba0 vs 18bb0; 19aa0 vs 19ab0). In the
series of compounds without substituents on the acri-
dine chromophore (i.e., 18aa0, 19aa0, 18ba0, and 19ba0),
compounds with alkylating chain at C-40 were about
8-fold more cytotoxic than the corresponding C-30-
substituted agents. However, the position of the N-mustard
residue did not affect the potency of those compounds
having substituents on the acridine chromophore. The
low cytotoxicity of compound 18bb0 was probably due
to its instability in the presence of moisture or water.
In contrast to the above observations, in the series of

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V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
compounds with a C1 spacer between the anilino ring
and the N-mustard residue, compound 24aa0 (alkylating
chain substituted at C-30) was about 4-fold more potent
than the corresponding 24ba0 (C-40 substitution). Simi-
larly, the substituents (dimethyaminoethylcarboxamido
and methyl functions) on the acridine chromophore en-
hanced the cytotoxicity (3- to 4-fold) of compounds in
this series (24aa0 vs 24ab0 and 24ba0 vs 24bb0). The most
potent compounds in the current studies were 18ab0 and
24ab0 with an IC50 value of 0.0028 and 0.0017 lM,
respectively.
reduced potency by 12-, 205-, and 12-fold, respectively,
suggesting covalent binding to the target (probably bind
to DNA, Topo II or/and other cell components) in tu-
mor cells by 10.
The targeting specificity of compound 10 and AHMA
(6) against topoisomerase I and topoisomerase II was
evaluated using three pairs of drug-resistant cell lines,
which are known to express reduced levels of topoisome-
rases. HL-60/MX2 is a topoisomerase II-deficient sub-
line of HL-60 while P388/CPT45 is a topoisomerase
I-deficient subline of P388. U937/CR is a camptothe-
cin-resistant subline of U937 with altered topoisomerase
I activity. As shown in Table 3, compound 10 exhibited
the same in vitro cytotoxicity against the topoisomerase-
deficient cells compared to their wild type cells, suggest-
ing that neither topoisomerase I nor topoisomerase II is
the primary cytotoxic target of compound 10. As posi-
tive controls, both AHMAÆHCl (6) and VP-16 were
shown to be less cytotoxic against topoisomerase II-defi-
cient HL-60/MX2 cells as compared to their wild type
HL-60 cells, consistent with the fact that both are
known topoisomerase II-targeting anticancer drugs.
Table 2 shows the growth inhibition of human lympho-
blastic leukemic cells (CCRF–CEM) and its drug-resis-
tant sublines (resistant to vinblastine and Taxol,
CCRF–CEM/VBL and CCRF–CEM/Taxol, respec-
tively) by 10, 18ba0,ca0,da0,ea0, 24aa0,ba0,ab0, and 24bb0.
The results demonstrated that these compounds did
not develop cross-resistance to either vinblastine or Tax-
ol. It suggested that the newly synthesized N-mustard
derivatives were neither a good substrate of p-glycopro-
tein nor mutated tubulin. It is notable to show that there
was no change in the potency when 10 was washed away
after incubation for 3 h. Whereas, in the same experi-
ment, the cytotoxicity of AHMA, Taxol, and vinblastine
Topotecan, a topoisomerase I-specific drug, was
also used as a negative control. As shown in Table 3,
Table 2. The cytotoxicity of N-mustard derivatives against human lymphoblastic leukemic cells (CCRF–CEM) and its drug-resistant sublines
(CCRF–CEM/VBL and CCRF–CEM/Taxol) and human tumor cell growth in vitroa
Compound
IC50 (lM)
Lymphoblastic leukemic
CCRF–CEM/VBL
Solid tumors
HCT-116
CCRF–CEM
CCRF–CEM/Taxol
A549
MX-1
AHMAÆHCl (6)
10
0.753
0.0070
0.020
1.60 [2.1·]
0.600 [0.8·]
0.0340 [4.9·]
0.061 [3.7·]
0.175.6 [6.7·]
0.147 [3.8·]
0.0222 [8.5·]
0.0151 [2.7·]
0.0610 [3.7·]
0.0012 [1.0·]
0.018 [2.4·]
0.143 [95.3·]
0.029 [24.2·]
0.0470 [0.55]b
0.0056 [0.0050]b
ND
NDc
0.0035
0.0035
ND
0.0075 [1.1·]
0.0665 [4.1·]
0.137 [5.2·]
0.114 [2.9·]
0.00165 [6.3·]
0.0306 [5.5·]
0.0665 [4.1·]
0.0013 [1.1·]
0.022 [2.9·]
1.62 [1080·]
0.540 [450·]
0.0055
ND
0.071
18ba0
18ca0
0.0263
0.0388
0.0026
0.0056
0.0184
0.0012
0.0076
0.0015
0.0012
0.0769
0.0900
0.0344
0.0350
0.0046
0.0053
0.0134
0.0017
0.0071
ND
18da0
0.0694
0.0068
0.0062
0.0369
0.0032
0.0165
0.0013
0.0014
18ea0
0.0045
0.0071
24aa0
24ba0
0.0209
0.0017
24ab0
24bb0
Taxol
Vinblastine
0.0129
0.0019 [0.390]b
0.0081 [0.095]b
ND
a XTT assays were used for leukemia cells and SRB assays were used for solid tumor cells. Incubation was 72 h, as described previously.42 Numbers in
the brackets are folds of resistance of the resistant cells when compared with the IC50ꢀs of the CCRF–CEM parent cells.
b Incubation for 3 h, washed, and then incubated for a total of 72 h. Washing did not affect BO-742 (10) efficacy, whereas efficacy for AHMA, Taxol,
and vinblastine were reduced 11.7-, 205-, and 11.7-folds, respectively, due to washing.
c ND: not determined.
Table 3. In vitro cytotoxicity of compound 10 and AHMAÆHCl (6) against various topoisomerase-deficient drug-resistant cell linesa
Compound
IC50 (lM)b
HL-60
HL-60/MX2
P388
P388/CPT45
U937
U937/CR
10
0.02
0.2
0.02 [1·]c
1.0 [5·]
2.0 [33.3·]
0.03 [1.0·]
0.02
0.02
0.02
0.03
0.02 [1·]
0.03
0.3
0.03 [1·]
AHMAÆHCl (6)
VP-16
Topotecan
0.003 [0.15·]
0.002 [0.1·]
13 [433·]
0.05 [0.17·]
0.04 [0.8·]
0.03 [1.0·]
0.06
0.03
0.05
0.03
a HL-60/MX2 cells, a subline of HL-60 selected for resistance to the Topo II drug mitoxantrone, express a lower level of Topo IIa and an
undetectable level of Topo IIb. P388/CPT45 cells and U937/CR cells were camptothecin-resistant variants of P388 and U937, respectively.
b Cell growth inhibition IC50ꢀs were determined by a 4 day MTT assay.
c Numbers in the brackets are folds of resistance of the resistant cells when compared with the IC50ꢀs of the corresponding parent cells.

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
3999
topotecan exhibited the same in vitro cytotoxicity
against topoisomerase II-deficient HL-60/MX2 cells as
compared to their wild type HL-60 cells. As expected,
topotecan was less cytotoxic against topoisomerase
I-deficient P388/CPT45 cells and U937/CR cells as com-
pared to their respective wild type cells. Taken together,
these results suggest that the main mechanism of cyto-
toxicity of compound 10 is not through its inhibitory
effect on topoisomerases.
age tumor size. Based on these studies, compound 10 ap-
peared to be more effective in treating human breast
tumor cells than human T-cell lymphoblastic leukemic
cells.
However, the therapeutic efficacies of compounds
24aa0,ab0,ba0, and 24bb0 were shown to be less potent than
that of 10 in the same experimental model (Table 5).
About 80% of tumor suppression was observed when
mice (n = 2) were treated with 24aa0 and 24ab0 at the
dose of 5 mg/kg (Q4D · 4), and 5 mg/kg (Q3D · 3) by
intravenous injection, respectively. The low potency of
24aa0,ab0,ba0, and 24bb0 was probably due to rather
the production of inactive bis(2-hydroxyethyl)amine
products in vivo system via easy hydrolysis. We have
also compared the therapeutic effects of the N-mustard
derivatives with that of AHMA derivatives in nude mice
bearing MX-1 xenograft (Table 6). This revealed that
68%, 84%, and 81% of average tumor volume was re-
duced on day 18 when mice were treated with AHMA
hydrochloride (6ÆHCl), AHMA–ethylcarbamate (25),
and AHMA–tert-butylcarbamate (26)28 at the dose of
15, 12, and 12 mg/kg (Q2D · 5, intravenous injection),
respectively. This clearly demonstrated that 10 was
significantly more therapeutically effective than AHMA
analogues.
3.2. In vivo therapeutic activity
Compound 10 and selected derivatives were subjected to
comparative in vivo antitumor studies with derivatives
of AHMA. Nude mice (n = 4) bearing human ovarian
carcinoma (SK-OV-3) xenograft were treated with 10
via intravenous infusion for 6 h, Q3D · 8, at the doses
of 3.0 mg/kg (on day 9, 12, 15, 18, 21, 24, 30, and 33)
upon subcutaneous tumor cell implantation. The study
revealed that there was a 96% reduction in the tumor
volume with only about 5% of body weight changes (Ta-
ble 4 and Fig. 1A and B). Table 4 also revealed that the
therapeutic efficacy of compound 10 [maximal dose:
2 mg/kg (Q3D · 7); intravenous injection] on nude mice
(n = 3) bearing human breast carcinoma MX-1 xeno-
graft resulted in complete remission in two out of three
mice (Fig. 1C and D). It is worth noting that at the opti-
mal dose, compound 10 attained complete tumor remis-
sions at one tenth of the optimal therapeutic dose of
paclitaxol (Taxolꢂ), which is one of the most important
agents currently used in clinics, in the MX-1 xenograft
model (Fig. 1E and F). Similarly, compound 10 attained
81% suppression of tumor in nude mice bearing human
colon carcinoma HCT-116 at 5 mg/kg (Q3D · 5, iv infu-
sion, 6 h). In another experiment, the therapeutic effects
and toxicity of compound 10 in nude mice bearing hu-
man T-cell acute lymphoblastic leukemic CCRF–CEM
xenografts were studied. Table 4 reveals that at 2 mg/
kg (Q3D · 6, iv) dose, there was 81% reduction in aver-
3.3. Topo II-mediated DNA relaxation and DNA
cleavage
Comparison of Topo II-mediated relaxation of pRYG–
DNA induced by VP-16, compound 10 and AHMA–
tert-butylcarbamate revealed that these agents inhibited
DNA relaxation (Fig. 2). However, compound 10 bind
tightly to DNA indicating that 10 might covalently bind
to DNA as indicated by retardation in gel mobility and
by resistant to washing when A549 tumor cells were
treated with this agent. As shown in Figure 3, treatment
of 32P-end labeled linear DNA with compound 10
Table 4. Therapeutic effects of compound 10 in nude mice bearing human xenografts
Tumor useda
Dose (mg/kg)
Route and schedule
Maximal body
weightb (%)
Maximal tumor
suppression (%)
Complete tumor
disappearance
Toxicity death
CCRF–CEM
HCT-116
SK-OV-3
MX-1
2
5
3
2
iv injection, Q3D · 6
iv infusion, 6 h, Q3D · 5
iv infusion, 6 h, Q3D · 8
iv injection, Q3D · 7
28
24
8
80.6
81.5
0/3
0/4
0/4
2/3
0/3
0/4
0/4
0/3
96.0
>99.1
19
a Human tumor tissue 50 mg/nude mouse was implanted subcutaneously on day 0. The dates of starting the treatments were: CCRF–CEM (T-cell
acute lymphoblastic leukemia), day 10; HCT-116 (colon carcinoma), day 12; SK-OV-3 (ovarian adenocarcinoma), day 9; and MX-1 (breast
carcinoma), day 11.
b Body weight = total body weight ꢀ tumor weight.
Table 5. Therapeutic effects and toxicity of N-mustard derivatives of 9-anilinoacridine in nude mice bearing human mammary carcinoma (MX-1)
xenograftsa
Compound
Dose (mg/kg)
Schedule
Maximal body weightb (%)
Maximal tumor suppression (%)
Tumor free
Death
24aa0
24ba0
24ab0
24bb0
5
Q4D · 3
Q4D · 4
Q3D · 3
Q4D · 4
29
11
29
10
82
0/2
0/2
0/2
0/4
0/2
0/2
0/2
0/4
5
64.8
80
1.5
1.5
27
a MX-1 tissue 50 mg was implanted subcutaneously on day 0. Treatment (iv injection) began on day 11 when tumor size were 80–120 mm3.
b Body weight = total body weight ꢀ tumor weight.

4000
V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
Table 6. Therapeutic effects of AHMA derivatives in nude mice bearing MX-1 xenografta
Compound
R
Dose (mg/kg)b
Average weight change (gm)
Average tumor volume (T/C)
N
Died of toxicity
iv, Q2D · 5
Day 8 Day 14 Day 16 Day 18 Day 14 Day 16 Day 18
Control
6ÆHCl
29.6
27.6
15.3
28.3
+0.6
ꢀ0.2
+0.4
+0.0
+1.5
+0.4
+1.0
+0.2
+2.7
+0.5
+2.3
+0.0
1.00
1.08
1.12
0.56
1.00
0.89
0.95
0.42
1.00
0.82
0.85
0.33
5
5
5
4
0
0
0
0
H
5
10
15
25
26
COOEt
COOCMe3
4
8
27.1
28.2
25.6
+0.0
ꢀ0.5
ꢀ0.2
+0.6
+0.4
ꢀ0.5
ꢀ0.1
ꢀ1.1
ꢀ0.7
+1.0
+1.0
ꢀ1.2
+0.9
ꢀ0.7
ꢀ1.8
+2.0
+2.3
ꢀ1.1
1.12
0.91
0.34
0.98
0.72
0.26
0.88
0.71
0.16
5
5
3
0
0
0
12
4
8
27.9
25.3
27.6
1.06
1.03
0.24
0.90
1.00
0.22
1.00
0.99
0.19
5
4
4
0
0
0
12
a MX-1 tissue 50 lg was implanted subcutaneously in mice on day 0. Every other day iv treatments were given on day 8, 10, 12, 14, and 16. The
average tumor volume of control group on day 14, 16, and 18 were 179 15, 299 39, and 492 49 mm3 (mean SEM), respectively.
b Vehicle used was 20 lL DMSO + 180 lL Saline.
Figure 3. Topoisomerase II-mediated DNA cleavage by AHMAÆHCl
(6), compound 10 and VP-16.
Figure 2. Comparison of Topo II-mediated relaxation of pRYG–
DNA induced by VP-16, compound 10 and AHMA–tert-butylcarba-
mate (26). These agents inhibited DNA relaxation. However,
compound 10 bind tightly to DNA.
4. Conclusions
The alkylating N-mustard derivatives exhibit potent
antitumor activity and have been applied for cancer
chemotherapy. However, these agents have a num-
ber of drawbacks due to their high reactivity, resulting
in interacting with other cell components and, hence,
having less diversion of active drug. Additionally, the
majority of N-mustard derivatives are kinetically unsta-
ble, leading to the formation of mono-alkylation. Such
mono-adducts are considered to be genotoxic rather
than cytotoxic. Recently, numerous DNA directed
N-mustard derivatives were synthesized for potential
therapeutic application. Although, most of these agents
resulted in reduced gel mobility consistent with the for-
mation of a cross-linked DNA dimer, suggesting that
compound 10 is a DNA cross-linking agent. The result
shown in Figure 3 also demonstrated that compound
10, unlike AHMAÆHCl (6), did not induce Topo II-medi-
ated DNA cleavage. These results together with the
cytotoxicity results suggest that compound 10, unlike
AHMA, may exert its cytotoxicity primarily through
its DNA cross-linking activity.

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
4001
have improvements in potency compared with the corre-
5.1. 3-(Acridin-9-ylamino)-5-hydroxymethylphenol (9)
sponding untargeted mustards, only limited compounds
were found to have clinical application potential. Most
DNA directed N-mustard derivatives possess se-
quence-specific binding to DNA. However, the DNA se-
quence-specific binding of these agents, generally, have
no direct correlation with their cytotoxicity. This could
be due to the loss of their Topo II inhibiting properties.
It clearly demonstrates that DNA binding and Topo II
inhibition (either caused by Topo II poison or topoiso-
merase catalytic inhibition) may be viewed as two dis-
tinct molecular processes that contribute separately to
cytotoxic activity. Solo DNA-alkylation with less or
lacking of Topo II inhibitory effect may discriminate
the antitumor efficacy of the drug.13
A solution of 9-chloroacridine (8a0, 8.56 g, 40 mmol) in
CHCl3 (30 mL) was added dropwise to a solution of 3-
amino-5-hydroxymethylphenol33 (7.03 g, 40 mmol) and
4-methylmorpholine (4.2 mL, 4.05 g, 40 mmol) in EtOH
(150 mL) at ꢀ5 ꢁC for 2.5 h. The reaction mixture was
stirred for an additional 1 h in an ice bath and then con-
centrated in vacuo to dryness and the residue was crystal-
lized from ethanol to give 4.73 g of 9. Additional product
(4.97 g) was obtained from the mother liquid after chro-
matography (SiO2, 6 · 30 cm, eluent CHCl3–MeOH,
10:1 v/v); total 9.7 g (75.6%); mp 201–202 ꢁC; 1H
NMR (DMSO-d6): d 4.43 (2H, s, CH2), 5.23 (1H, br s,
exchangeable, OH), 6.69 (1H, s, ArH), 6.80 (1H, s,
ArH), 6.83 (1H, s, ArH), 7.45 (2H, m, 2 · ArH), 7.99
(2H, m, 2 · ArH), 8.10 (2H, m, 2 · ArH), 8.29 (2H, m,
2 · ArH), 9.88 and 11.43 (each 1H, br s, exchangeable,
NH or OH). Anal. (C20H15N2O2ÆHClÆ0.6H2O) C, H, N.
The present 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, O–C4, and C1 unit).
We demonstrated that compound 10 exhibits significant
cytotoxicity in inhibiting human lymphoblastic leuke-
mia cells (CCRF–CEM) and its drug-resistant sublines
(resistant to vinblastine and Taxol, CCRF–CEM/VBL
and CCRF–CEM/Taxol, respectively). The efficacious
in vivo therapeutic effect of this agent suggested that
other compounds reported herein might also have po-
tent therapeutic activity. The studies on the inhibitory
effect against Topo II and the cytotoxicity against Topo
II and Topo I mutant cell lines of 10 clearly demon-
strated that AHMA is a Topo II drug but not Topo I
drug whereas 10 is neither. The results also indicated
that DNA is the main target for 10. The interaction of
10 or other derivatives with DNA doubled strands is
currently underway in our laboratories to realize the for-
mation of DNA cross-linking or mono-adduct and their
sequence-specific binding.
5.2. (3-(Acridin-9-ylamino)-5-{2-[bis(2-chloroethyl)-
amino]ethoxy}phenyl)methanol (10)
A solution of 9 (2.68 g, 8.46 mmol) in 0.2 N KOH/MeOH
(42.3 mL, 8.46 mmol) was stirred at room temperature
for 10 min and the precipitated orange potassium salt
was collected by filtration and dried. The salt was added
to a mixture of tris(2-chloroethyl)amine hydrochloride
(3.03 g, 12.6 mmol), dry powdered K2CO3 (5.80 g,
42 mmol) and dry KF (0.487 mg, 8.46 mmol) in anhy-
drous DMF (50 mL). The mixture was then gradually
heated at 50 ꢁC for 19 h, cooled and then filtered through
a pad of Celite, washed with DMF (10 mL). The com-
bined filtrate and washings were evaporated in vacuo to
dryness. The residue was dissolved in EtOAc (150 mL),
washed with water (50 mL · 3), dried over Na2SO4, and
evaporated in vacuo to dryness. The residue was chro-
matographed on a silica gel column (4 · 17 cm) using
CHCl3–MeOH (10:1 v/v). The fractions containing the
product were combined and evaporated in vacuo to dry-
ness and the solid residue was recrystallized from ace-
tone–hexane (5:1) to give 10, 2.04 g (50 %); mp 153–
154 ꢁC; 1H NMR (DMSO-d6): d 2.93 (6H, br s,
3 · NCH2), 3.60 (4H, br s, 2 · CH2Cl), 3.96 (2H, br s,
OCH2), 4.43 (2H, s, CH2OH), 5.05 (1H, s, OH), 6.13
(1H, m, ArH), 6.28 (1H, m, ArH), 6.82 (1H, s, ArH),
7.00–7.12 (1H, m, ArH), 7.29 (2H, m, ArH), 7.46 (3H,
m, 3 · ArH), 7.72 (1H, m, ArH), 8.13 (1H, m, ArH),
8.16 (2H, m, ArH), 10.84 (1H, br s, exchangeable, NH).
Anal. (C26H27Cl2N3O2) C, H, N.
Studies with 10 in nude mice bearing human ovarian
carcinoma (SK-OV-3) xenograft possessed potent anti-
tumor therapeutic effect with little toxicity against
human tumor xenografts when the agent was
administered by iv infusion. Further in vivo therapeutic
efficacies of 10 and other derivatives against human
tumor xenografts by iv infusion are currently being
investigated.
5. Experimental
Melting points were determined on a Fargo melting
point apparatus and are uncorrected. Column chroma-
tography was carried out over silica gel G60 (70–
230 mesh, ASTM; Merck). Thin layer chromatography
was performed on silica gel G60 F254 (Merck) plates
with short wave length UV light for visualization. Ele-
mental analyses were done on a Heraeus CHN–O Rapid
instrument. 1H NMR spectra were recorded on a
Brucker AMX-400 spectrophotometer with Me4Si as
an internal standard. The following are the synthesis
of the representative compounds. The analytical
data and the yield of other new derivatives are shown
in Table 7.
5.3. Bis(2-chloroethyl)-[2-(3-nitrophenoxy)ethyl]amine (14a)
A mixture of m-nitrophenol (4.0 g, 28.8 mmol), tris(2-
chloroethyl)amine hydrochloride (8.34 g, 34.5 mmol),
KF (1.66 g, 28.8 mmol) and K2CO3 (19.84 g, 143.8
mmol) in dry acetone (200 mL) was refluxed for 2 days.
After cooling, the reaction mixture was filtered and
the solid was washed with acetone. The combined
filtrate and washings were evaporated in vacuo to dry-
ness and the residue was dissolved in CHCl3 (200 mL),
washed with water (3 · 100 mL), dried over Na2SO4,
and evaporated in vacuo to dryness. The residue was

4002
V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
Table 7. Analytical data and yield of the new compounds
Compound
Chemical formulaa
20H15N2O2Æ1HClÆ0.6H2O
C26H27Cl2N3O2
Mp (ꢁC)
Yield (%)
Analysis
9
10
C
201–202
153–154
Syrup
75.6
50
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
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
12a
12b
C10H12NO3Æ0.1H2O
C14H22BrN2O5Æ1/2H2O
60
Syrup
Syrup
Syrup
67.7
79.3
62
13a
13b
C
C
14H22N2O5Æ1.8H2O
14H22BrN2O5Æ1/2H2O
14a
14b
C12H16Cl2N2O3Æ2HClÆ2H2O
12H16Cl2N2O3ÆHCl
C13H18Cl2N2O3ÆHCl
13H18Cl2N2OÆHCl
C13H18Cl2N2O4ÆHCl
14H20Cl2N2O3ÆHCl
119–120
188–189
158–159
152–153
181–182
120–121
166–167
123–124
129–130
225–226
185–186
235–236
197–198
134–145
126–127
131–132
109–110
166–167
71–72
25
21
C
14c
14d
24
29
C
14e
15a
16
60
C
15b
C14H20Cl2N2O3ÆHCl
85
36
18aa0
18ab0
18ba0
18bb0
18ca0
18da0
18ea0
19aa0
19ab0
19ba0
19bb0
21a
C25H25Cl2N3OÆ1.HClÆ5H2O
C31H37N5O2Cl2Æ4HClÆ3H2O
C25H25Cl2N3OÆ2HClÆ0.5H2O
C31H37Cl2N5O2Æ5/2HClÆ4H2O
C26H27Cl2N3OÆ3/2HClÆ5/2H2O
C26H27Cl2N3OÆ3/2HClÆH2O
C26H27Cl2N3O2Æ5/4HClÆH2O
C27H29Cl2N3OÆ2ÆHClÆ5H2O
C33H41Cl2N5O2Æ3HClÆ7H2O
C27H29Cl2N3OÆ3HClÆ0.5H2O
C33H41Cl2N5O2Æ1.5HClÆ4H2O
C11H16N2O4
41
29
67
50
55
58
30
26
22
71
58
21b
22a
C11H16N2O4
11H14Cl2N2O2ÆHCl
74–75
153–154
45–46
80.5
89
C
22b
C11H14Cl2N2O2ÆHCl
73
85
24aa0
24ab0
24ba0
24bb0
C24H23Cl2N3Æ2HClÆ0.5H2O
C30H35Cl2N5OÆ3HClÆ2H2O
C24H23Cl2N3Æ2HClÆ0.5H2O
C30H35Cl2N5OÆ3HClÆ2H2O
207–208
198–199
220–223
204–205
92
35
76.3
a Compounds are hygroscopic and contain crystal water.
chromatographed on a silica gel column (5 · 7 cm) using
CH2Cl2 as the eluent. The fractions containing the de-
sired product were combined and evaporated under
reduced pressure to give 14a as syrup, 2.2 g (25%); mp
119–120 ꢁC (HCl salt, EtOH); 1H NMR (CDCl3): d,
3.05 (4H, t, J = 6.9 Hz, 2 · NCH2), 3.10 (2H, t,
J = 5.5 Hz, NCH2), 3.57 (4H, t, J = 6.9 Hz, 2 · CH2Cl),
4.12 (2H, t, J = 5.5 Hz, OCH2), 7.21 (1H, dq, J = 2.5,
8.3 Hz, ArH), 7.41 (1H, t, J = 8.8 Hz, ArH), 7.69 (1H,
t, J = 2.5 Hz, ArH), 7.79 (1H, dq, J = 2.5, J = 8.3 Hz,
ArH). Anal. (C12H16Cl2N2O3Æ2HClÆ2H2O) C, H, N.
reduced pressure to dryness. The residue was chromato-
graphed on a silica gel column (5 · 20 cm) using CHCl3
as an eluent. The fractions containing the desired
product were combined, evaporated in vacuo to give
12a, yield 5.95 g (60%) as syrup; 1H NMR (CDCl3):
d 2.02 (2H, m, CH2), 2.08 (2H, m, CH2), 3.50 (2H, t,
J = 6.2 Hz, CH2Br), 4.78 (2H, t, J = 6.2 Hz, OCH2),
7.22 (1H, dq, J = 2.7, 8.3 Hz, ArH), 7.43 (1H, t,
J = 8.1 Hz, ArH), 7.69 (1H, dq, J = 2.7, 8.3 Hz, ArH),
7.89 (1H, m, ArH). Anal. (C10H12NO3Æ0.1H2O) C, H,
N.
By following the same procedure as that for 14a, com-
pounds 14b–e (Table 7) were synthesized.
By following the same procedure as that for compound
12a, compound 12b (Table 7) was prepared.
5.4. 1-(4-Bromobutoxy)-3-nitrobenzene (12a)
5.5. 2-{(2-Hydroxyethyl)-[4-(3-nitrophenoxy)butyl]-
amino}ethanol (13a)
A mixture of m-nitrophenol (11a, 4.99 g, 35.9 mmol),
1,4-dibromobutane (9.30 g, 43.1 mmol), and K2CO3
(7.43 g, 53.9 mmol) in acetone (200 mL) was refluxed
for 4 h. After cooling, the reaction mixture was filtered
through a pad of Celite and the filter cake was washed
with acetone. The combined filtrate and washings were
evaporated in vacuo to dryness. The residue was diluted
with water (100 mL) and extracted with CHCl3
(6 · 100 mL). The organic extracts were combined,
washed successively with 1% NaOH (50 mL) and water
(30 mL), dried over Na2SO4, and evaporated under
A mixture of 12a (5.0 g, 21.7 mmol) and diethanolamine
(6.85 g, 65.1 mmol) in DMF was heated at 115 ꢁC with
vigorous stirring for 30 min. After cooling, the mixture
was concentrated under reduced pressure to remove
the solvent. The residue was washed successively with
hexane (5 · 50 mL) and ether (2 · 30 mL) and was then
dissolved in CHCl3 (200 mL). The CHCl3 solution was
washed with water (6 · 80 mL) to remove excess dietha-
nolamine, dried over Na2SO4, and evaporated in vacuo
to dryness. Compound 13a, 5.23 g (79.3%), was

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
4003
obtained as syrup; 1H NMR (CHCl3): d, 1.67 (2H,
m, CH2), 1.83 (2H, m, CH2), 2.62 (2H, t, J = 7.2 Hz,
NCH2), 2.67 (4H, t, J = 5.4 Hz, 2 · NCH2), 3.62 (4H,
m, 2 · CH2OH), 4.04 (2H, t, J = 5.3 Hz, OCH2), 7.21
(1H, dq, J = 2.7, 8.3 Hz, ArH), 7.41 (1H, t, J = 8.2 Hz,
ArH), 7.69 (1H, s, ArH), 7.78 (1H, dq, J = 2.7, 8.3 Hz,
ArH). Anal. (C14H22N2O5Æ1.8H2O) C, H, N.
m, ArH), 6.56 (1H, m, ArH), 7.17 (1H, m, ArH), 7.22
(2H, br s, ArH), 7.60 (2H, m, ArH), 7.94 (2H, m,
ArH), 8.04 (2H, m, ArH), 11.21 (1H, br s, NH). Anal.
(C25H25Cl2N3OÆHClÆ5H2O) C, H, N.
By following the same procedure as that for the synthe-
sis of compound 18aa0, compounds 18ab0,ba0,bb0,
ca0,da0,ea0, 19aa0,ab0,ba0,bb0, 24aa0,ab0,ba0, and 24bb0
(Table 7) were prepared.
By following the same procedure as that for compound
13a, compounds 13b, 21a, and 21b (Table 7) were
synthesized.
6. Biological experiments
6.1. Cytotoxicity assays
5.6. Bis(2-chloroethyl)-[4-(3-nitrophenoxy)butyl]amine (15a)
Methanesulfonyl chloride (5.75 g, 50.3 mmol) was
added dropwise to a solution of 13a (5.0 g, 16.8 mmol)
and triethylamine (6.78 g, 67.0 mmol) in dry CHCl3
(25 mL) in an ice bath. The reaction mixture was stirred
for 3 days at room temperature and then diluted with
CHCl3 (150 mL). The solution was washed successively
with cold aqueous solution of 10% NaHCO3 (50 mL)
and ice water (100 mL), dried over Na2SO4, and evapo-
rated in vacuo to dryness. The residue was recrystallized
from EtOH to give pale yellow crystals, 3.4 g (60%); mp
The effects of the compounds on cell growth were deter-
mined in all human tumor cells (i.e., lung adenocarci-
noma A549, colon carcinoma HCT-116, breast
carcinoma MX-1, and T-cell acute lymphocytic leuke-
mia CCRF–CEM), in a 72 h incubation, by XTT–tetra-
zolium assay, as described by Scudiero et al.34 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 concen-
trations of each compound were used. The IC50 and
dose–effect relationships of the compounds for antitu-
mor activity were calculated by a median–effect plot,35,36
using a computer program on an IBM-PC worksta-
tion.37 The cytotoxicity of compound 10 and AH-
MAÆHCl against a number of drug-resistant cell lines
with an altered expression of Topo I or Topo II was
measured by the MTT assay as described.38
1
120–121 ꢁC; H NMR (CHCl3): d, 1.97 (2H, m, CH2),
2.15 (2H, m, CH2), 3.37 (2H, t, J = 8.2
Hz, NCH2), 3.57 (4H, s, 2NCH2), 4.10 (6H, m, OCH2
and 2 · CH2Cl), 7.22 (1H, dq, J = 8.3 Hz, ArH), 7.44
(1H, t, J = 2.7, 8.2 Hz, ArH), 7.77 (1H, s, ArH), 7.84
(1H, dq, J = 2.7, 8.3 Hz, ArH). Anal. (C14H20Cl2-
N2O3Æ1.8H2O) C, H, N.
By following the same procedure as that for compound
15a, compounds 15b, 22a, and 22b (Table 7) were
synthesized.
6.2. In vivo studies
5.7. Acridin-9-yl-(3-{2-[bis(2-chloroethyl)amino]ethoxy}-
phenyl)amine (18aa0)
Athymic nude mice bearing the nu/nu gene were used
for human breast tumor MX-1, human T-cell acute lym-
phoblastic leukemia CCRF-CEM, human colon carci-
noma HCT-116, and human ovarian adenocarcinoma
SK-OV-3 xenografts. Outbred Swiss-background mice
were obtained from Charies River Breeding Laborato-
ries. Male mice 8 weeks old or older weighing 22 g or
more were used for most experiments. Drug was admin-
istrated via the tail vein by iv injection. Tumor volumes
were assessed by measuring length · width · height (or
width) by using a caliper. Vehicle used was 20 lL
DMSO in 180 lL saline. All animal studies were con-
ducted in accordance with the guidelines of the National
Institutes of Health Guide for the Care and Use of Ani-
mals and the protocol approved by the Memorial Sloan-
Kettering Cancer Centerꢀs Institutional Animal Care
and Use Committee.
A mixture of bis(2-chloroethyl)-[2-(3-nitrophenoxy)-
ethyl]amine (14a) (307 mg, 1.0 mmol) and SnCl2Æ
2H2O (675 mg, 3.0 mmol) in concd HCl (4 mL) was stir-
red at 60 ꢁC for 30 min. The clear solution was poured
into ice (25 g) and neutralized slowly with 25% NH4OH.
The mixture was extracted with CHCl3 (4 · 50 mL),
dried over Na2SO4, and evaporated in vacuo to dryness
to give crude 3-bis(2-chloroethyl)aminoethoxyaniline
(16a), which was dissolved in CHCl3 (20 mL) and added
to a solution of 9-chloroacridine (8a, 106 mg, 0.5 mmol)
in CHCl3 (20 mL) containing two drops of concd HCl in
an ice bath. After being stirred at room temperature for
6 h, the mixture was evaporated in vacuo to dryness and
the residue was chromatographed on silica gel column
(1 · 20 cm) using CHCl3–methanol (10:1 v/v) as the elu-
ant. The main fractions containing the desired product
were combined and evaporated in vacuo to dryness.
The residue was treated with excess dry HCl in EtOAc
(2.5 N) and then evaporated under reduced pressure to
dryness. The solid residue was recrystallized from ace-
tone/ethyl acetate to give 18aa0, 164 mg (36%) as HCl
salt; mp 123–124 ꢁC; 1H NMR (DMSO-d6): d 2.97
(6H, t, J = 7.1 Hz, 3 · NCH2), 3.49 (4H, t, J = 7.1 Hz,
2 · CH2Cl), 3.94 (2H, t, J = 5.4 Hz, OCH2), 6.51 (2H,
6.3. Topoisomerase II–DNA relaxation assay
Topo II–DNA relaxation activities were determined by
following the procedures described by Liu and Miller39
and Hirose et al.40 The reaction mixture (50 lL)
containing 10 mM Tris, pH 7.9, 50 mM KCl, 100 mM
NaCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA,
1 mM ATP, 30 lg/mL BSA, 15 lg/mL SV40 DNA, or

4004
V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
pBR322 DNA, and a known amount of the Topo II,
were incubated at 30 ꢁC for 15 min. The reaction was
stopped by the addition of SDS to 1%, EDTA to
20 mM, and the proteinase K to 400 lg/mL; the mixture
was further incubated at 37 ꢁC for 30 min, mixed with
dye solution containing 5% sucrose and 0.01% xylene
cyanol, and analyzed on a 1% agarose gel using TBE
(89 mM Tris–borate, 2 mM EDTA) containing 0.1%
SDS.
1 mg/mL proteinase K followed by incubation for an
additional 60 min at 37 ꢁC. EDTA reversal was per-
formed by adding EDTA to 20 mM at the end of the
first incubation. Following a second incubation at
37 ꢁC for 10 min, reactions were then terminated by
SDS and proteinase K as described above. DNA sam-
ples were electrophoresed in 1% agarose gel containing
0.5· TPE buffer. Gels were dried onto Whatman
3MM chromatographic paper and autoradiographed
at ꢀ80 ꢁC using Kodak XAR-5 film.
6.4. Topoisomerase II-mediated DNA cleavage assay
Topo II-mediated DNA cleavages were determined by
following the procedures previously described.41 The
reaction mixture (20 lL each) containing 40 mM Tris–
HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.5 mM
EDTA, 1 mM ATP, 30 lg/mL bovine serum albumin,
20 ng of 30-end 32P-labeled YEpG DNA, 10 ng of puri-
fied hTopo IIa and a test compound was incubated at
37 ꢁC for 30 min. The reactions were terminated by
addition of 5 lL of a solution containing 5% SDS and
Acknowledgements
This work was supported by the National Science Coun-
cil, Taiwan (Grant No. NSC93-2323-B-001-001), and
NIH RO1 Grants CA39662 and CA102463 (to
L.F.L.). The NMR spectra of synthesized compounds
were obtained at High-Field Biomacromolecular NMR
Core Facility supported by the National Research Pro-
gram for Genomic Medicine (Taiwan).
Appendix A. C, H, N analysis
Compound
Chemical formula
Anal. Calcd
H (%)
Found
H (%)
C (%)
N (%)
C (%)
N (%)
9
10
12a
12b
13a
13b
14a
14b
14c
14d
14e
15a
C20H15N2O2ÆHClÆ0.6H2O
C26H27Cl2N3O2
C10H12NO3Æ0.1H2O
C14H22BrN2O5Æ1/2H2O
C14H22N2O5Æ1.8H2O
C14H22BrN2O5Æ1/2H2O
C12H16Cl2N2O3Æ2HClÆ2H2O
C12H16Cl2N2O3ÆHCl
C13H18Cl2N2O3ÆHCl
C13H18Cl2N2OÆHCl
65.84
64.24
43.53
54.71
50.60
54.71
34.45
41.94
43.65
43.65
41.78
45.55
45.07
51.68
47.60
55.98
49.86
53.45
57.70
56.98
50.24
46.84
53.97
44.87
54.99
54.99
42.13
42.13
56.93
51.62
56.93
51.62
5.07
5.62
4.46
7.21
7.28
7.21
5.30
4.99
5.35
5.35
5.12
6.03
5.67
6.25
6.06
5.26
6.41
5.78
5.68
5.56
6.40
6.91
5.53
6.21
6.71
6.71
4.82
4.82
5.18
6.07
5.18
6.07
7.68
8.66
5.07
9.11
8.43
9.11
6.69
8.15
7.83
7.83
7.50
7.58
7.53
7.23
8.95
7.84
9.38
7.19
5.68
7.67
6.51
8.28
6.99
7.92
11.66
11.66
8.93
8.93
8.30
10.03
8.30
10.03
65.86
64.20
44.10
54.58
50.65
54.58
34.45
41.78
43.44
43.45
41.69
45.55
45.25
51.70
47.60
56.06
50.21
53.54
58.01
56.76
50.61
46.91
53.34
44.95
55.03
55.14
41.29
42.36
55.63
51.50
56.86
51.39
65.86
5.88
4.52
7.50
7.02
7.50
5.31
5.04
5.38
5.40
5.07
5.76
5.75
6.30
6.45
5.33
6.67
5.88
58.01
5.52
6.53
6.75
5.43
6.24
6.72
6.74
4.82
5.05
5.20
6.20
5.31
6.26
7.51
8.28
5.09
9.00
8.18
9.00
6.53
8.02
7.69
7.73
7.50
7.50
7.50
7.26
8.91
7.84
9.65
7.07
7.73
7.70
6.27
8.22
7.03
7.84
11.63
11.64
8.80
8.78
8.29
10.19
8.16
9.97
C13H18Cl2N2O4ÆHCl
C14H20Cl2N2O3Æ1.8H2O
C14H20Cl2N2O3ÆHCl
15b
18aa0
18ab0
18ba0
18bb0
18ca0
18da0
18ea0
19aa0
19ab0
19ba0
19bb0
21a
C25H25Cl2N3OÆHClÆ5H2O
C31H37N5O2Cl2Æ4HClÆ3H2O
C25H25Cl2N3OÆ2HClÆ0.5H2O
C31H37Cl2N5O2Æ5/2HClÆ4H2O
C26H27Cl2N3OÆ3/2HClÆ5/2H2O
C26H27Cl2N3OÆ3/2HClÆH2O
C26H27Cl2N3O2Æ5/4HClÆH2O
C27H29Cl2N3OÆ2ÆHClÆ5H2O
C33H41Cl2N5O2Æ3HClÆ7H2O
C27H29Cl2N3OÆ3HClÆ0.5H2O
C33H41Cl2N5O2Æ1.5HClÆ4H2O
C11H16N2O4
C11H16N2O4
C11H14Cl2N2O2ÆHCl
C11H14Cl2N2O2ÆHCl
C24H23Cl2N3Æ2HClÆ0.5H2O
C30H35Cl2N5OÆ3HClÆ2H2O
C24H23Cl2N3Æ2HClÆ0.5H2O
C30H35Cl2N5OÆ3HClÆ2H2O
21b
22a
22b
24aa0
24ab0
24ba0
24bb0

V. A. Bacherikov et al. / Bioorg. Med. Chem. 13 (2005) 3993–4006
4005
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