Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage

Article abstract of DOI:10.1016/j.bmc.2008.04.024

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.

Full text of DOI:10.1016/j.bmc.2008.04.024

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5413–5423
Synthesis and biological activity of stable and potent
antitumor agents, aniline nitrogen mustards linked
to 9-anilinoacridines via a urea linkage
Naval Kapuriya,a Kalpana Kapuriya,a Xiuguo Zhang,b Ting-Chao Chou,b
Rajesh Kakadiya,a Yu-Tse Wu,c Tung-Hu Tsai,c Yu-Ting Chen,a Te-Chang Lee,a
Anamik Shah,d Yogesh Naliaparad and Tsann-Long Sua,*
aInstitute of Biomedical Sciences, Laboratory of Bioorganic Chemistry, Academia Sinica, Taipei 115, Taiwan
bPreclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program,
Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
cInstitute of Traditional Medicine, National Yang-Ming University, Taipei 112, Taiwan
dDepartment of Chemistry, Saurashtra University, Rajkot, Gujarat, India
Received 26 February 2008; revised 8 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-
affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard
derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-
aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to
taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed
that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat
plasma.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
An effective strategy to overcome the drawback of using
N-mustards is to design and synthesize DNA-directed
alkylating agents by linking the N-mustard residue to
DNA-affinic molecules. These conjugates were found
to have higher cytotoxicity and therapeutic efficacy than
the corresponding untargeted N-mustard derivatives.6–10
Another strategy to minimize the side-effects caused by
the drug is to prepare N-mustard prodrugs, which can
be activated selectively at tumor site after enzymatic
hydrolysis. Among N-mustard prodrugs, Springer et
al. have synthesized a series of N-mustard prodrugs by
attaching the aniline mustards to L-glutamic acid moiety
through a urea, carbamate (1, Chart 1)11 or carboxam-
ide (2, CMDA)12,13 linker for antibody-directed enzyme
prodrug therapy (ADEPT). After enzymatic cleavage by
bacterial enzyme carboxypeptidase G2 (CPG2), they can
be transformed into their corresponding active metabo-
lite phenol or aniline N-mustard drugs. The prodrugs,
3,14 4,15 and 5,16 were also synthesized by linking the
aniline N-mustard to the trigger unit tyramine, 3-
hydroxytyramine, or catecholamine, respectively, via a
Bifunctional alkylating agents particularly N-mustards
have played an important role in anticancer drug devel-
opment.1,2 However, they are highly reactive species
resulting in loss of drug’s therapeutic activity against
malignancy by reacting with other cellular components
such as proteins, thiols, or genes3 and producing many
unwanted side-effects including bone marrow toxicity.4
In addition, the N,N-bis(2-chloroethyl)amine pharma-
cophore required for bifunctional cross-linking of
DNA generally lacks intrinsic DNA-binding affinity.
Consequently, DNA alkylation by N-mustards forms
higher ratio for genotoxic mono-adducts than the bi-ad-
ducts, the latter was found to be essential for their full
cytotoxicity.5
Keywords: Cytotoxic; Aniline nitrogen mustards; 9-Anilinoacridines;
DNA alkylating agents.
*
Corresponding author. Tel.: +886
27827685; e-mail: tlsu@ibms.sinica.edu.tw
2 27899045; fax: +886 2
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.024

5414
N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
Cl
N(CH2CH2Cl)2
N
H
N
R
Z
OSO2Me
COOH
O
H
HO
N(CH2CH2Cl)2
Z
N
COOH
O
N
H
COOH
3 Z = NH, R = H
4 Z = O, R = OH
COOH
O
2 (CMDA)
1 Z = NH, O
H
N
H
N
HO
HO
O
N(CH2CH2Cl)2
5
NH2
NH2
OCH2CH2N(CH2CH2Cl)2
HN
OMe
HN
CH2OH
HN
CH2OH
N
N
N
OCH2CH2N(CH2CH2Cl)2
8 AHMA
7
6 BO-0742
Chart 1. Chemical structures of N-mustard derivatives.
urea or carbamate linker for melanocyte-directed en-
zyme prodrug therapy (MDEPT).16 Upon exposure to
tyrosinase, these conjugates can also release the active
aniline or phenol N-mustard. Since the prodrugs are sta-
ble before enzymatic hydrolysis, it suggests that the urea
or carbamate linker is capable of lowering the reactivity
of aniline or phenol N-mustard pharmacophore leading
to form rather stable N-mustard derivatives.
erty of phenyl ring. Therefore, in our continual develop-
ment efforts on new potent DNA-targeted alkylating
agents having an improved pharmacokinetics, it is of
great interest to design and synthesize phenyl N-mus-
tards linked to DNA-affinic molecule such as 9-anilino-
acridines through a urea linker instead of alkyl
N-mustard residue and carbon-chain spacer, since
phenyl N-mustards having a urea linker at para-position
are chemically less reactive than phenol or aniline
N-mustards.11,14,15 We found that the newly synthesized
compounds were much more stable than BO-0742 and
displayed potent therapeutic efficacy against several
human xenografts in animal model. Herein, we describe
the synthesis and antitumor activity of the new
conjugates.
Recently, we have reported a series of DNA-directed
alkylating agents in which the alkyl N-mustard was
linked to the anilino ring or acridine chromophore of
9-anilinoacridines, such as BO-0742 (6, Chart 1)9 and
BO-0944 (7).10 These agents were about >100-fold more
cytotoxic than AHMA (8)17,18 in inhibiting human acute
lymphoblastic leukemia (CCRF-CEM) in vitro.
Remarkably, BO-0742 exhibited a broad spectrum of
antitumor activity against various human solid tumor
xenografts in vivo. Total tumor remission was achieved
in nude mice bearing human breast MX-1 xenograft.
Our unpublished results showed that BO-0742 is chem-
ically unstable and has a short half-life (< 25 min.) in
mice. The chemical instability of BO-0742 and the re-
lated compounds can be explained by the fact that these
agents are considered as alkyl N-mustard derivatives.
The inductive effect of the alkyl function is thought to
be able to enhance the formation of the reactive aziridi-
um cation intermediate, which reacts rapidly with nucle-
ophile, such as the deoxyguanosine (dG) residue of
DNA. While in the case of phenyl N-mustards, they
are rather stable due to the electron-withdrawing prop-
2. Chemistry
The newly synthesized compounds are phenyl N-mus-
tards linked to 9-anilinoacridines via a urea spacer. In
general, the urea linker can be prepared by reacting an
amine derivative with either substituted carbamoyl chlo-
rides or isocyanate derivatives (Scheme 1). We found
that the desired compounds (24a–n and 26) can be
synthesized in better yields by treating 4-bis(2-chloroeth-
yl)aminophenyl isocyanate 10 with a variety 9-anilinoac-
ridines (8, 11–23). Following the reported procedure
with modification14,15 aniline N-mustard 9 was synthe-
sized, which was further converted into isocyanate 1021
by treating with triphosgene. The freshly prepared 10

N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
5415
H2N
N(CH2CH2Cl)2
9
a
O
C
N
N(CH2CH2Cl)2
NH2
3'
10
4'
R1
2'
1'
NH2
5'
HN
N
6'
b
b
N
5
4
25
R3
R2
8, 11-23
N(CH2CH2Cl)2
N(CH2CH2Cl)2
O
O
HN
N
HN
N
H
H
R1
HN
N
26
N
R3
R2
24a-n
R1 = CH2OH, Me, OMe
R2, R3 = H, Me, CONHCH2CH2NMe2
Scheme 1. Synthesis of N-mustard linked to 9-anilinoacridines and 9-aminoacridine via a urea linkage. Reagents and conditions: (a) triphosgene/
Et3N/CHCl3, 0 ꢁC; (b) Et3N or pyridine/DMF, room temperature.
was then reacted with various 9-anilinoacridines (8, 11–
23), prepared by following the method previously devel-
oped in our laboratory,17,19,20 in dry DMF in the pres-
ence of triethylamine or pyridine at room temperature
to furnish the desired phenyl N-mustards linked to 9-
anilinoacridine conjugates via a urea linker (24a–n). In
a similar manner treatment of 10 with 9-aminoacridine
(25) afforded acridine-N-mustard conjugate 26 in good
yield. The yields and physical properties of the new N-
mustard conjugates are shown in Table 1.
carcinoma HCT-116 and was compared with BO-
0742 (6) and the untargeted N-mustard 9. It was
revealed that these conjugates possessed significant
cytotoxicity with IC50 values in submicro molar range
and did not exhibit cross-resistance to either vinblas-
tine or taxol. The structure–activity relationships stud-
ies of the newly synthesized derivatives showed that
the C40-OMe and C50-OMe substituted compounds
were more cytotoxic than the corresponding C40-Me
and C50-Me derivatives (24h vs 24c, 24i vs 24d, 24j
vs 24e) against CCRF-CEM cell growth in vitro. In
contrast, the C40-Me and C50-Me substituted com-
pounds were more potent than the corresponding
C40-OMe and C50-OMe derivatives (24c vs 24h, 24d
vs 24i, 24e vs 24j) in inhibiting MX-1 cell growth.
Although, the C60-Me substituted compounds were
more cytotoxic than the corresponding C60-OMe
derivatives (24f vs 24m and 24g vs 24n) against
CCRF-CEM cell growth, they (24f vs 24m) were
equally potent against MX-1 cell growth. The cytotox-
icity of the series of OMe substituted compounds
against CCRF-CEM showed that the C40-OMe deriv-
atives (24h and 24i) were about 2- to 5-fold more po-
tent than the corresponding C50-OMe (24j and 24k)
and C60-OMe (24m and 24n) conjugates. As for the
inhibitory effect of these conjugates against HCT-116
cell growth in culture, it showed that C40-Me and
C40-OMe derivatives were more cytotoxic than the
3. Biological results and discussion
3.1. In vitro cytotoxicity
We have previously demonstrated that antitumor
9-anilinoacridines including 3-(9-acridinylamino)-5-
hydroxymethylanilines (AHMAs),17 5-(9-acridinylami-
no)toluidines,19 and 5-(9-acridinylamino)anisidines20
are potent inhibitors of topoisomerase II and capable
of intercalating into DNA doubled strands. Hence,
they are suitable as a carrier for constructing the
new DNA-targeted compounds. Table 2 showed the
cytotoxicity of the newly synthesized N-mustards
(24a–n and 26) against human lymphoblastic leukemia
(CCRF-CEM), breast carcinoma (MX-1), and colon

5416
N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
Table 1. Analytical data and yields of N-mustard linked to 9-anilinoacridines (24a–n and 26)
N(CH2CH2Cl2)2
O
HN
3'
N
H
2'
1'
4'
5'
R1
HN
N
6'
4
R2
5
R3
Compound
R1
R2
R3
Mp (ꢁC)
Yield (%)
Analysis
24a
24b
24c
24d
24e
24f
24g
24h
24i
50-CH2OH
50-CH2OH
40-Me
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
174–175
251–252
271–272
267–268
>280
53.8
55.2
57.1
83.8
39.0
44.0
32.0
70.2
60.2
64.5
63.7
61.8
80.4
48.8
60
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
Me
H
40-Me
Me
H
50-Me
60-Me
Me
CONH(CH2)2NMe2
255–256
>280
60-Me
40-OMe
40-OMe
50-OMe
50-OMe
50-OMe
60-OMe
60-OMe
H
Me
263–264
255–256
260–261
253–254
188–190
268–269
270–272
184–186
24j
H
Me
24k
24l
CONH(CH2)2NMe2
24m
24n
26
H
Me
C50-Me and C50-OMe conjugates (24c vs 24e, 24h vs
24j 24i vs 24k). It is of great interest to note that
the cytotoxicity of these agents can be increased by
addition of Me group at C4 (24b, 24f, 24k, and
24n), or CONHCH2CH2NMe2 and Me at C4 and
C5 (24h and 24l), respectively (except for compounds
Table 2. Cytotoxicity of new N-mustards against human lymphoblastic leukemia (CCRF-CEM) and its drug-resistant sublines (CCRF-CEM/Taxol
and CCRF-CEM/VBL) and solid tumors (MX-1 and HCT-116) cell growth in vitroa
Compound
IC50 (nM)
CCRF-CEM
CCRF-CEM/Taxolb
CCRF-CEM/VBLb
MX-1
HCT-116
c
24a
24b
24c
24d
24e
24f
56.2 1.1
31.1 0.08
16.7 0.60
77.8 2.3
141.0 3.3
11.4 0.46
11.9 0.30
7.7 0.14
9.2 0.24
37.5 1.2
29.6 0.60
13.2 0.43
44.2 0.32
29.7 0.30
228.9 23.9
43.4 0.50
3.33 1.5
1.3 0.36
0.73 0.12
17180 142[304·]
64140 2000[1141·]
1789 331[57.5·]
33.4 1.0[2.00·]
127.9 2.2[1.64·]
593.2 2.0[4.20·]
24.5 1.1[2.15·]
303.1 0.30[25.4·]
54.9 1.8[7.13·]
24.6 0.07[2.76·]
54.8 0.10[1.46·]
43.3 0.50 [1.46·]
951.4 3.0[72.0·]
54.8 3.7[1.24·]
30.6 2.8[1.30·]
378.1 17.8[1.65·]
25.7 0.10[0.59·]
12.8 0.70[3.84·]
1.274 468[980·]
496.0 280.9[679.5·]
369.0 11
124.7 4.5
583.6 26.4
239.7 35.6
529.0 21.9
588.6 52.3
60.1 1.1
216.0 19
262.6 6.0
264.5 4.7
437.4 1.8
815.5 10.1
158.1 3.2
60.0 2.8
393.5 6.6[12.7·]
34.7 0.80[2.08·]
91.8 15.7[1.18·]
384.2 2.0[2.72·]
23.0 0.5[2.02·]
175.7 5.6[14.7·]
33.8 1.4[4.39·]
11.5 0.50[1.25·]
50.4 0.80[1.34·]
49.8 1.1[1.68·]
30.2 0.20[2.29·]
58.8 0.20[1.33·]
57.4 0.10[1.93·]
385.4 21.3[1.68·]
33.8 0.40[0.78·]
3.20 0.40[0.96·]
429.0 112.6[330·]
78.0 14.8[106.2·]
24g
24h
24i
1203 40.6
367.3 12.5
1109 61.7
868.0 47.8
638.0 2.0
590.6 4.2
1810.4 100
810.1 19.3
84.4 1.7
256.3 8.1
74.9 2.7
24j
495.6 0.53
242.0 1.6
110.2 5.3
187.8 6.1
571.2 18.5
899.8 23.6
340.8 4.9
6.5 1.2
24k
24l
24m
24n
26
9
6
3.5 0.60
35.0 0.51
2.9 0.083
Taxol
Vinblastine
1.3 0.45
1.8 0.37
a Cell growth inhibition was measured by the XTT assay22 for leukemic cells and the SRB assay23 for solid tumor cells after 72-h incubation using a
microplate spectrophotometer as described previously.24 IC50 values were determined in duplicate or triplicate from dose–effect relationship at six
or seven concentrations of each drug by using the CompuSyn software by Chou and Martin25 based on the median-effect principle and plot26,27 and
serial deletion analysis. Ranges given for taxol and vinblastine were mean SE (n = 4).
b CCRF-CEM/Taxol and CCRF-CEM/VBL are subcell lines of CCRF-CEM cells that are 330-fold resistant to taxol, and 680-fold resistant to
vinblastine, respectively, when comparing with the IC50 of the parent cell line.
c Numbers in the brackets are fold of cross-resistant determined by comparison with the corresponding IC50 of the parent cell line.

N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
5417
24a and 24d against HCT-116 cell growth where the
cytotoxicity of 24a > 24b and 24c > 24d). These results
confirm our previous studies17,19,20 and clearly demon-
strated that the changes in the cytotoxicity profile by
modifying substituent(s) in the anilino or acridine ring
may attribute to the appropriate increasing lipophilic-
ity and DNA-binding affinity. Of these derivatives,
compound 24h was the most cytotoxic with IC50 value
of 7.7 nM. As for the in vitro cytotoxicity of N-mus-
tard linked to acridine, it showed that compound 26
was less cytotoxic than compounds having 9-anilinoac-
ridines as a carrier. It is worthwhile to note that most
compounds linking to 9-anilinoacridines were equally
potent or in some cases more cytotoxic than the un-
masked N-mustard 9, but this finding was not ob-
served in compound 26. This suggested that the 9-
anilinoacridines were more favorable for using as a
DNA-affinic carrier than acridine.
complete tumor remission (CR) in nude mice bearing
MX-1 xenograft at the doses of 30 and 100 mg/kg, intra-
venous injection (iv), Q2D · 5 (n = 3). Remarkably,
both compounds (24b and 24d) with only one cycle 5-
dose-treatments, complete remission was achieved and
maintained for over 70 days without any relapse in 3
out of 3 mice (Fig. 1A). Compound 24a also led to com-
plete tumor suppression but not complete tumor remis-
sion at the dose of 50 mg/kg, Q2D · 8 (n = 3). The
maximal toxicity of these agents as shown in Figure
1B by body weight decrease was about 10% drop from
the initial pretreatment body weight (on day 8), after
four treatments. However, the body weight showed
recovery after cession of treatment. Similar result was
found for 24e, which also resulted in tumor complete
remission at the dose of 75 mg/kg (QD · 8, iv injection)
in nude mouse bearing MX-1 xenograft (figure not
shown). Compound 24d was further selected to evaluate
its therapeutic effect in nude mice bearing human glioma
U87 MG xenograft (Fig. 2). The results showed that 24d
was more potent than cyclophosphamide with low tox-
icity to the host (15% body-weight drop). These studies
3.2. In vivo therapeutic activity
The therapeutic effects of the representative new N-mus-
tards (24a, 24b, 24d, 24e) against human breast carci-
noma MX-1 and human Glioma U87 MG xenografts
in nude mice were evaluated. Under the experimental
conditions as indicated, 24b and 24d (Fig. 1) achieved
Figure 1. The therapeutic effects of 24a (50 mg/kg, Q2D · 8, iv
injection), 24b (30 mg/kg, Q2D · 5, iv injection), and 24d (100 mg/kg,
Q2D · 5, iv injection) in nude mice bearing MX-1 xenograft (n = 3);
Figure 2. The therapeutic effects of 24d (100 mg/kg) and cyclophos-
phamide (80 mg/kg) in nude mice bearing human glioma U87 MG
xenograft (iv inj., Q2D · 5, n = 3); control (d), 24d ( ) and
ꢀ
control (d), 24a (m), 24b ( ), and 24d (·); average tumor size changes
cyclophosphamide (Æ); average tumor size changes (A); and body
weight changes (B). The values for the treated versus the untreated
group from day 18 to day 30 are <0.0001 and <0.0012 for
cyclophosphamide and 24d, respectively.
ꢀ
(A); and average body weight changes (B). The values for the treated
versus the untreated group from day 16 to day 24 are <0.0006, <0.0003
and <0.0003 for 24a, 24b, and 24d, respectively.

5418
N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
demonstrated that the newly synthesized compounds
possess potent antitumor therapeutic efficacy with a rel-
atively mild toxicity. Interestingly, we found that the
complete tumor suppression was observed in mice on
day-16 (last dose) and remained continuously for over
70 days without relapse.
cancer H1299 cells by modified comet assay.28 The
DNA cross-linking caused by 24d was compared with
that of mephalan and cisplatin. It revealed that 24d
was capable of inducing DNA cross-linking in a dose-
dependent manner (Fig. 3). At the dose of 10 lM, this
agent induced 39.2% DNA cross-linking, while, mepha-
lan and cisplatin induced 47.3% and 47.4% DNA
cross-linking at the dose of 200 and 100 lM, respec-
tively, under the same experimental conditions. The
results suggested that DNA interstrand cross-linking
may be the main mechanism of action of 24d and the
related compounds.
3.3. Chemical stability
To realize whether the new conjugates are more stable
than BO-0742, we further investigated the comparative
chemical stability of the 24a, 24b, 24d, and BO-0742 in
intravenous injection vehicle (1 mg of compound in
DMSO/Tween 80/normal saline: 0.5:0.4:1.6 v/v/v) by
thin-layer chromatography (SiO2, solvent: CHCl3/
MeOH, 10:1 v/v) and parallel confirmed by HPLC
(Mightysil RP-18; mobile phase: acetonitril/H2O 80:20,
elution rate: 1 mL/min). It revealed that the half-life
(t 1/2, time required for 50% decomposition of com-
pound) of 24a, 24b, and 24d were 55, 59, and 36 days,
respectively. We could not detect the t1/2 value for BO-
0742 by HPLC analysis, since this agent decomposed
during eluation from column. However, TLC analysis
showed that BO-0742 had a t1/2 value of 2 h demonstrat-
ing that the newly synthesized compounds were signifi-
cantly more stable than BO-0742 although they were
less cytotoxic than the latter.
4. Conclusion
In this study, we have synthesized a series of chemically
stable DNA-directed alkylating agents, in which the
phenyl N-mustard residue is linked to DNA-intercalat-
ing 9-anilinoacridines via a urea spacer, demonstrating
that these agents exhibited potent antitumor efficacy in
vivo with a relatively low toxicity. Among these deriva-
tives, compound 24d was revealed to have potent antitu-
mor effect in nude mice bearing human breast MX-1
xenograft; complete remission was achieved and main-
tained for over 70 days without any relapse with only
one cycle of treatments. Compound 24d also effectively
suppressed human glioma U87 MG xenograft in nude
mice. Moreover, we also found that this agent is able
to cross-link with DNA and has a long half-life in rat
plasma suggesting that this agent is a promising candi-
date for preclinical studies.
Compound 24d was further selected to study its chemi-
cal stability in rat plasma. The degradation of this agent
was detected by HPLC. The detection limit is 20 ng/mL
for the authentic 24d in the rat plasma. It revealed that
24d is a very stable N-mustard derivative in rat plasma
with a long half-life (t1/2 = 54.18 0.96 h, n = 4). These
results demonstrate that the newly prepared N-mustards
are chemically and metabolically stable derivatives.
5. Experimental
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
3.4. DNA interstrand cross-linking study
Compound 24d was found to be cytotoxic to human
non-small lung cancer H1299 cell line with IC50 value
of 0.51 lM. This agent was selected and subjected to
DNA cross-linking studies in human non-small lung
60
50
40
30
20
10
0
Control
1
5
10
200
100
Melphalan
Cisplatin
Compound 24d
Concentration (μM)
Figure 3. DNA interstrand cross-linking study. H1299 cells were used to determine the DNA cross-linking study by a modified comet assay.
Mephalan and cisplatin were used as positive controls. Data represents the means of three individual experiments (mean SD).

N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
5419
on a Heraeus CHN-O Rapid instrument. HPLC was
performed on Waters Delta Prep4000 using Mightysil
RP-18 reverse phase column (250 · 4.6 mm). Com-
pounds were detected by UV at 260 nm. The mobile
phase was MeCN/H2O (80:20 v/v) with flow rate of
1 mL/min. 1H NMR spectra were recorded on a
600 MHz, Brucker AVANCE 600 DRX and 400 MHz,
Brucker Top-Spin spectrometers. The chemical shifts
were reported in ppm (d) relative to TMS.
ArH), 6.81 (1H, s, ArH), 7.01–7.05 (3H, m, 3· ArH),
7.24 (2H, d, J = 9.1 Hz, 2· ArH), 7.54 (4H, br s, 4·
ArH) 8.05 (2H, br s, 2· ArH), 8.25, 8.46 and 10.84 (each
1H, br s, exchangeable, 3· NH). Anal. (C31H29Cl2N5
O2Æ0.5H2O) C, H, N.
By following the same procedure the following com-
pounds were synthesized.
5.1.3. 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[3-hydro-
xymethyl-5-(4-methylacridin-9-ylamino)phenyl]urea (24b).
Compound 24b was synthesized from 10 (freshly pre-
pared from 9, 1.377 g, 4.5 mmol) and 5-hydroxymethyl
5.1. General procedure for the preparation of new N-
mustards
N,N-Bis(2-chloroethyl)benzene-1,4-diamine hydrochlo-
ride (9) was prepared by following the procedure devel-
oped by Jordan et al.14 Compound 9 was converted into
isocyanate 10, which was then condensed with appropri-
ate 9-anilinoacridines (8, 11–23) previously synthesized
from our laboratory17,19,20 and the commercially avail-
able 9-aminoacridine hydrochloride (13) in dry DMF in
the presence of triethylamine or pyridine to give 24a–n
and 26. The final products were purified either by recrys-
tallization from an appropriate solvent or by column
chromatography using (SiO2, CHCl3/MeOH, v/v 100:2).
The detailed procedure was described as follows:
3-(4-methyl-9-acridinylamino)aniline
(11,
0.988 g,
3.0 mmol)17 in dry DMF (25 mL) containing pyridine
(2.0 mL): yield 986 mg (55.2%); mp 251–252 ꢁC; 1H
NMR (DMSO-d6) d 2.79 (3H, s, Me), 3.66–3.69 (8H,
m, 4· CH2), 4.45 (2H, d, J = 5.9 Hz, CH2), 5.30 (1H,
br s, exchangeable, OH), 6.68 (2H, d, J = 8.8 Hz, 2·
ArH), 6.87 (1H, s, ArH), 7.24 (2H, d, J = 8.8 Hz, 2·
ArH), 7.34 (1H, s, ArH), 7.39–7.41 (1H, m, ArH),
7.47–7.48 (1H, m, ArH), 7.57 (1H, s, ArH), 7.86 (1H,
d, J = 7.0 Hz, ArH), 8.00 (1H, s, ArH), 8.21–8.26 (2H,
m, 2· ArH), 8.45 (1H, d, J = 8.5 Hz, ArH), 8.82, 9.62
and 11.49 (each 1H, br s, exchangeable, 3· NH). Anal.
(C32H31Cl2N5O2Æ3H2O) C, H, N.
5.1.1. 4-[N,N -Bis(2-chloroethyl)amino]phenylisocyanate
(10). To a suspension of N,N-bis(2-chloroethyl)benzene-
1,4-diamine hydrochloride (9)14 (1.683 g, 5.4 mmol) in
dry chloroform (30 mL) was added triethylamine
(2.5 mL) at 0 ꢁC. The clear solution obtained was then
added dropwise into a solution of triphosgene (0.623 g,
2.1 mmol) in dry chloroform (10 mL) at ꢁ10 ꢁC. The
reaction mixture was allowed to stand at room temper-
ature. After being stirred for 30 min, the reaction mix-
ture was evaporated to dryness under reduced
pressure. The solid residue was triturated with dry
THF (100 mL), filtered, and washed with small amount
of THF. The combined filtrate and washings was evap-
orated to dryness to give the crude isocyanate 1021
which was used directly for the next reaction without
further purification.
5.1.4. 1-(5-(Acridin-9-ylamino)-2-methylphenyl)-3-(4-(bis-
(2-chloroethyl)amino)phenyl)urea (24c). Compound 24c
was synthesized from 10 (freshly prepared from 9,
1.683 g, 5.4 mmol) and N1-(acridin-9-yl)amino-4-meth-
ylbenzene-1,3-diamine (12, 0.898 g 3.0 mmol)19 in dry
DMF (15 mL) containing triethylamine (2.5 mL): yield
771 mg (57.1%); mp 271–272 ꢁC (dec); 1H NMR
(DMSO-d6) d 2.35 (3H, s, Me), 3.66–3.69 (8H, m, 4·
CH2), 6.69 (2H, d, J = 8.8 Hz, 2· ArH), 6.86–6.88 (1H,
m, ArH), 7.25–7.27 (1H, m, ArH), 7.28 (2H, d,
J = 8.8 Hz, 2· ArH), 7.42–7.45 (2H, m, 2· ArH), 7.90–
8.00 (4H, m, 4· ArH), 8.19 (1H, s, ArH), 8.26 (2H, m,
2· ArH), 8.30, 9.37 and 11.49 (each 1H, br s, exchange-
able, 3· NH). Anal. (C31H29Cl2N5OÆ2.5H2O) C, H, N.
5.1.5. 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-
5-(4-methylacridin-9-ylamino)phenyl]urea (24d). Com-
pound 24d was synthesized from 10 (freshly prepared
from 9, 1.836 g, 6 mmol) and 4-methyl-N1-(4-methylac-
ridin-9-yl)benzene-1,3-diamine (13, 1.065 g, 3.4 mmol)19
in dry DMF (50 mL) containing pyridine (2.0 mL): yield
5.1.2. 1-[3-(Acridin-9-ylamino)-5-hydroxymethyl-phenyl]-
3-{4-[bis(2-chloroethyl)amino]phenyl}urea (24a). A solu-
tion of isocyanate 10 (freshly prepared from 9, 0.672
g, 2.2 mmol) in dry DMF (10 mL) was added dropwise
to a solution of 3-(9-acridinylamino)-5-hydroxymethy-
laniline (8, 0.752 g, 2.0 mmol)17 in dry DMF (40 mL)
containing triethylamine (2.0 mL) at 0 ꢁC. After being
stirred for 18 h at room temperature, the reaction mix-
ture was evaporated to dryness in vacuo, the residue
was dissolved in a mixture of CHCl3/MeOH containing
silica gel (5.0 g) and then evaporated to dryness. The res-
idue was put on the top of a silica gel column (2 · 20 cm)
and purified by using CHCl3/MeOH (100:5 v/v) as an
eluent. The fractions containing the main product were
combined and evaporated in vacuo to dryness and the
residue was recrystallized from CHCl3/MeOH to give
24a, 618 mg (53.8%); mp 174–175 ꢁC; 1H NMR
(DMSO-d6) d 3.65–3.71 (8H, m, 4· CH2), 4.41 (2H, d,
J = 6.0 Hz, CH2), 5.16 (1H, t, J = 6.0 Hz, exchangeable,
OH), 6.37 ( 1H, s, ArH), 6.68 (2H, d, J = 9.1 Hz, 2·
1
1.631 g (83.8%); mp 267–268 ꢁC; H NMR (DMSO-d6)
d 2.34 (3H, s, Me), 2.78 (3H, s, Me), 3.65–3.70 (8H,
m, 4· CH2), 6.68 (2H, d, J = 9.2 Hz, 2· ArH), 6.86–
6.89 (1H, m, ArH), 7.24–7.27 (1H, m, ArH), 7.28 (2H,
d, J = 9.2 Hz, 2· ArH), 7.37–7.41 (1H, m, ArH), 7.43–
7.48 (1H, m, ArH), 7.86–7.87 (1H, m, ArH), 7.97–8.01
(1H, m, ArH), 8.20–8.23 (3H, m, 3· ArH), 8.33 (1H,
br s, exchangeable, NH), 8.42–8.44 (1H, m, ArH), 9.35
and 11.51 (each 1H, br s, exchangeable, 2· NH). Anal.
(C32H31Cl2N5OÆ2H2O) C, H, N.
5.1.6. 1-[3-(Acridin-9-yl)amino-5-methylphenyl]-3-{4-
[bis(2-chloroethyl)amino]phenyl}urea (24e). Compound
24e was synthesized from 10 (freshly prepared from
9, 0.918 g, 3.0 mmol) and N1-(acridin-9-yl)amino-

5420
N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
5-methylbenzene-1,3-diamine (14, 0.517 g, 1.7 mmol)19 in
dry DMF (25 mL) containing pyridine (2 mL): yield
5.1.10. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(2-meth-
oxy-5-(4-methylacridin-9-ylamino)phenyl)urea (24i).
Compound 24i was synthesized from 10 (freshly pre-
pared from 9, 0.841 g, 2.7 mmol) and 4-methoxy-N1-
(4-methylacridin-9-yl)benzene-1,3-diamine (18, 0.493 g
1.5 mmol)20 in dry DMF (10 mL) containing triethyl-
amine (2.0 mL): yield 428 mg (60.2%); mp 255–256 ꢁC
1
375 mg (39%); mp > 280 ꢁC; H NMR (DMSO-d6) d
2.26 (3H, s, Me), 3.68 (8H, s, 4· CH ), 6.68 (2H, d,
2
J = 9.0 Hz, 2· ArH), 6.79 (1H, s, ArH), 7.25–7.27
(3H, m, 3· ArH), 7.46–7.49 (2H, m, 2· ArH), 7.54
(1H, s, ArH), 8.00–8.07 (4H, m, 4· ArH), 8.28–8.30
(2H, m, 2· ArH), 8.95, 9.38 and 11.51 (each 1H, br
s, exchangeable, 3· NH). Anal. (C31H29Cl2N5OÆ2.9-
H2O) C, H, N.
1
(dec); H NMR (DMSO-d6) d 2.50 (3H, s, Me), 3.65–
3.68 (8H, m, 4· CH2), 3.94 (3H, s, OMe), 6.68 (2H, d,
J = 9.0 Hz, 2· ArH), 6.93–6.95 (1H, m, ArH), 7.10
(1H, m, ArH), 7.26 (2H, d, J = 9.0 Hz, 2· ArH), 7.35–
7.37 (1H, m, ArH), 7.41–7.43 (1H, m, ArH), 7.82–7.83
(1H, s, ArH), 7.94–7.96 (1H, m, ArH), 8.21–8.22 (2H,
m, 2· ArH), 8.31–8.32 (1H, m, ArH), 8.50 (1H, br s,
ArH), 8.51, 9.44 and 11.55 (each 1H, br s, exchangeable,
3· NH). Anal. (C32H31Cl2N5O2Æ2.4H2O) C, H, N.
5.1.7. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-methyl-
3-(4-methylacridin-9-ylamino)phenyl)urea (24f). Com-
pound 24f was synthesized from 10 (freshly prepared
from 9, 1.683 g, 5.4 mmol) and 6-methyl-N1-(4-methyl-
acridin-9-yl)benzene-1,3-diamine (15, 0.910 g 3.0 mmol)19
in dry DMF (15 mL) containing triethylamine (2.5 mL):
yield 576 mg (44%); mp 255–256 ꢁC (dec); 1H NMR
(DMSO-d6) d 2.34 (3H, s, Me), 2.78 (3H, s, Me), 3.65–
3.71 (8H, m, 4·CH2), 6.69 (2H, d, J = 9.0 Hz, 2· ArH),
6.81–6.83 (1H, m, ArH), 7.21–7.24 (1H, m, ArH), 7.28
(2H, d, J = 9.0 Hz, 2· ArH), 7.34–7.38 (1H, m, ArH),
7.41–7.45 (1H, m, ArH), 7.82–7.83 (1H, m, ArH), 7.93–
7.97 (1H, m, ArH), 8.15–8.22 (3H, m, 3· ArH), 8.31
(1H, br s, exchangeable, NH), 8.39–8.42 (1H, m, ArH),
9.40 and 12.45 (each 1H, br s, exchangeable, 2· NH).
Anal. (C32H31Cl2N5OÆ2.7H2O) C, H, N.
5.1.11. 1-(3-(Acridin-9-ylamino)-5-methoxyphenyl)-3-(4-
(bis(2-chloroethyl)amino)phenyl)urea (24j). Compound
24j was synthesized from 10 (freshly prepared from 9,
0.841 g, 2.7 mmol) and N1-(acridin-9-yl)-5-methoxyben-
zene-1,3-diamine (19 , 0.473 g 1.5 mmol)20 in dry DMF
(10 mL) containing triethylamine (2.0 mL): yield 452 mg
1
(64.5%); mp 260–261 ꢁC (dec); H NMR (DMSO-d6) d
3.67 (3H, s, OMe), 3.68–3.70 (8H, m, 4· CH2), 6.56
(1H, s, ArH), 6.69 (2H, d, J = 8.9 Hz, 2· ArH), 7.15
(1H, s, ArH), 7.19 (1H, s, ArH), 7.25 (2H, d,
J = 8.9 Hz, 2· ArH), 7.47–7.50 (2H, m, 2· ArH),
7.98–8.00 (2H, m, 2· ArH), 8.04–8.06 (2H, m, 2·
ArH), 8.29–8.31 (2H, m, 2· ArH), 8.88, 9.39 and
11.45 (each 1H, br s, exchangeable, 3· NH). Anal.
(C31H29Cl2N5O2Æ2.2H2O): C, H, N.
5.1.8. 9-[5-(3-{4-[Bis(2-chloroethyl)amino]phenyl}ureido)-
2-methylphenylamino]-5-methylacridine-4-carboxylic acid
(2-dimethylaminoethyl)amide (24g). Compound 24g was
synthesized from 10 (freshly prepared from 9, 0.918 g,
3.0 mmol) and 9-(5-amino-2-methylphenylamino)-5-
methylacridine-4-carboxylic acid (2-dimethylamino-
ethyl)amide (16, 0.732 g, 1.7 mmol)19 in dry DMF
(25 mL) containing pyridine (2 mL): yield 385 mg
5.1.12. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-meth-
oxy-5-(4-methylacridin-9-ylamino)phenyl)urea (24k).
Compound 24k was synthesized from 10 (freshly pre-
pared from 9, 0.841 g, 2.7 mmol) and 5-methoxy-N1-
(4-methylacridin-9-yl)benzene-1,3-diamine (20, 0.493 g
1
(32%); mp > 280 ꢁC; H NMR (DMSO-d6) d 2.19 (3H,
s, Me), 2.67 (6H, br s, 2· NMe), 2.85 (3H, s, Me),
3.14 (2H, br s, CH2), 3.63–3.70 (8H, m, 4· CH2), 3.85
(2H, s, CH2), 6.40–6.41 (1H, m, ArH), 6.65 (2H, d,
J = 9.0 Hz, 2· ArH), 6.97–6.99 (1H, s, ArH), 7.24 (2H,
d, J = 9.0 Hz, 2· ArH), 7.45 (1H, s, ArH), 7.51 (1H,
m, ArH), 7.74 (2H, br s, 2· ArH), 7.93 (1H, br s,
exchangeable, NH), 8.08–8.10 (1H, m, ArH), 8.39–8.41
(1H, m, ArH), 8.67–8.69 (1H, m, ArH), 9.04, 9.45 and
12.18 (each 1H, br s, exchangeable, 3· NH). Anal.
(C37H41Cl2N7O2Æ5.7H2O) C, H, N.
20
1.5 mmol), in dry DMF (10 mL) containing triethyl-
amine (2.0 mL): yield 459 mg (63.7%); mp 253–254 ꢁC
1
(dec); H NMR (DMSO-d6) d 2.80 (3H, s, Me), 3.66
(3H, s, OMe), 3.67–3.70 (8H, m, 4· CH2), 6.54 (1H, s,
ArH), 6.69 (2H, d, J = 9.0 Hz, 2· ArH), 7.14 (1H, s,
ArH), 7.17 (1H, s, ArH), 7.25 (2H, d, J = 9.0 Hz, 2·
ArH), 7.40–7.42 (1H, m, ArH), 7.47–7.50 (1H, m,
ArH), 7.85–7.86 (1H, m, ArH), 7.98–8.00 (1H, m,
ArH), 8.23–8.27 (2H, m, 2· ArH), 8.48–8.50 (1H, m,
ArH), 8.97, 9.51 and 11.47 (each 1H, br s, exchangeable,
3· NH). Anal. (C32H31Cl2N5O2Æ2.5H2O) C, H, N.
5.1.9. 1-(5-(Acridin-9-ylamino)-2-methoxyphenyl)-3-(4-
(bis(2-chloroethyl)amino)phenyl)urea (24h). Compound
24h was synthesized from 10 (freshly prepared from 9,
1.683 g, 5.4 mmol) N1-(acridin-9-yl)-4-methoxybenzene-
1,3-diamine (17, 0.946 g, 3.0 mmol),20 in dry DMF
(15 mL) containing triethylamine (2.5 mL): yield 923 mg
5.1.13. 1-(3-(4-((2-(Dimethylamino)ethyl)carbamoyl)acri-
din-9-ylamino)-5-methoxyphenyl)-3-(4-(bis(2-chloroeth-
yl)amino)phenyl)urea (24l). Compound 24l was
synthesized from 10 (freshly prepared from 9, 0.550 g,
1.8 mmol) and 9-(3-amino-5-methoxyphenylamino)-N1-
(2-(dimethylamino)ethyl)acridine-4-carboxamide (21,
0.429 g 1.0 mmol)20 in dry DMF (10 mL) containing tri-
ethylamine (2.0 mL): yield 359 mg (61.8%); mp 188–
1
(70.2%); mp 263–264 ꢁC (dec); H NMR (DMSO-d6) d
3.67–3.68 (8H, m, 4· CH2), 3.96 (3H, s, OMe), 6.68 (2H,
d, J = 8.5 Hz, 2· ArH), 6.95–6.96 (1H, m, ArH), 7.12–
7.13 (1H, m, ArH), 7.26 (2H, d, J = 8.5 Hz, 2· ArH),
7.42–7.44 (2H, m, 2· ArH), 7.95–7.97 (2H, m, 2· ArH),
8.00–8.02 (2H, m, 2· ArH), 8.24–8.27 (2H, m, 2· ArH),
8.33 (1H, br s, ArH), 8.44, 9.29 and 11.47 (each 1H, br
s, exchangeable, 3· NH). Anal. (C31H29Cl2N5O2Æ2.6H2O)
C, H, N.
1
190 ꢁC (dec); H NMR (DMSO-d6) d 2.88 (6H, br s,
2· NMe), 3.44 (2H, s, CH2), 3.60–3.68 (11H, m, 4·
CH2, OMe), 3.73 (1H, br s, CH), 3.97 (1H, s, CH),
6.04 (1H, s, exchangeable, NH), 6.67–6.68 (2H, m, 2·

N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
5421
ArH), 6.78–6.87 (1H, m, ArH), 7.21 (2H, br s, 2· ArH),
7.53–7.57 (2H, m, 2· ArH), 7.87 (1H, s, ArH), 8.24 (1H,
s, ArH), 8.34–8.43 (1H, m, ArH), 8.68 (1H, s, ArH), 8.86
(1H, br s, ArH), 9.11–9.33 (1H, m, ArH), 9.57 (1H, s,
ArH), 10.48 and 12.01 (each 1H, br s, exchangeable,
2· NH). Anal. (C36H39Cl2N7O3Æ6.9H2O) C, H, N.
the addition of phenazine methosulfate-XTT solution at
37 ꢁC for 6 h, absorbance at 450 and 630 nm was de-
tected on a microplate reader (EL 340; Bio-Tek Instru-
ments Inc., Winooski, VT). IC50 values were
determined from dose–effect relationship at six or seven
concentrations of each drug by using the CompuSyn
software by Chou and Martin25 based on the median-ef-
fect principle and plot.26,27 Ranges given for taxol and
vinblastine were mean SE (n = 4).
5.1.14 . 1-(3-(Acridin-9-ylamino)-4-methoxyphenyl)-3-(4-
(bis(2-chloroethyl)amino)phenyl)urea (24m). Compound
24m was synthesized from 10 (freshly prepared from 9,
0.841 g, 2.7 mmol) and N1-(acridin-9-yl)-6-methoxyben-
zene-1,3-diamine (22, 0.473 g 1.5 mmol)20 in dry DMF
(10 mL) containing triethylamine (2.0 mL): yield
563 mg (80.4%); mp 268–269 ꢁC (dec); 1H NMR
(DMSO-d6) d 3.41 (3H, s, OMe), 3.67–3.70 (8H, m, 4·
CH2), 6.70 (2H, d, J = 9.0 Hz, 2· ArH), 7.14–7.15 (1H,
m, ArH), 7.28 (2H, d, J = 9.0 Hz, 2· ArH), 7.43–7.47
(3H, m, 3· ArH), 7.81 (1H, br s, ArH), 7.98–8.00 (2H,
m, 2· ArH), 8.04–8.06 (2H, m, 2· ArH), 8.26–8.28
(2H, m, 2· ArH), 8.88, 9.30 and 11.32 (each 1H, br s,
exchangeable, 3· NH). Anal. (C31H29Cl2N5O2Æ3.
4H2O) C, H, N.
5.2.2. In vivo studies. Athymic nude mice bearing the nu/
nu gene were used for human breast tumor MX-1 and
human glioma U87 MG xenograft. Outbred Swiss-back-
ground mice were obtained from the National Cancer
Institute (Frederick, MD). Male mice 8 weeks old or
older weighing 22 g or more were used for most experi-
ments. Drug was administrated via the tail vein by iv
injection. Tumor volumes were assessed by measuring
length · width · height (or width) by using caliper.
Vehicle used was 50 lL DMSO and 40 lL Tween 80 in
160 lL saline. The maximal tolerate dose of the tested
compound was determined and applied for the in vivo
therapeutic efficacy assay. 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 Center’s Institutional Animal Care
and Use Committee.
5.1.15. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-meth-
oxy-3-(4-methylacridin-9-ylamino)phenyl)urea
(24n).
Compound 24n was synthesized from 10 (freshly pre-
pared from 9, 0.841 g, 2.7 mmol) and 6-methoxy-N1-
(4-methylacridin-9-yl)benzene-1,3-diamine (23, 0.493 g
1.5 mmol)20 in dry DMF (10 mL) containing triethyl-
amine (2.0 mL): yield 347 mg (48.8%); mp 270–272 ꢁC
5.2.3. Determination of half-life of 24d in rat plasma. The
chromatographic system consisted of a photodiode-ar-
ray system (Shimadzu SPD-M20A, Tokyo, Japan),
chromatographic pump (Shimadzu LC-20AT), an
auto-sampler (Shimadzu SIL-20AC) equipped with a
20 lL sample loop. Compound 24d was separated from
rat plasma using a revise phase column (Agilent RP-C8,
30 · 150 mm, particle size 5 lm, Palo Alto, CA, USA)
maintained at an ambient temperature (25 1 ꢁC) to
perform the ideal chromatographic system. The detector
wavelength was set at 266 nm. The mobile phase com-
prised methanol:10 mM NaH2PO4 (60:40, v/v), which
was adjusted to pH 3.0 with 85% of H3PO4. Analysis
was run at a flow rate of 0.8 mL/min and the samples
were quantified using peak area. An aliquot of plasma
sample (100 lL, with 24d 5 lg/mL) was vortex-mixed
with acetonitrile (1:2, v/v) for protein precipitation and
centrifuged at 10,000g for 10 min. The supernatant
was passed through a 0.45 lm filter for injecting into
the HPLC.
1
(dec); H NMR (DMSO-d6) d 2.79 (3H, s, Me), 3.39
(3H, s, OMe), 3.67–3.70 (8H, m, 4· CH2), 6.69 (2H, d,
J = 8.8 Hz, 2· ArH), 7.12–7.13 (1H, m, ArH), 7.28
(2H, d, J = 8.8 Hz, 2· ArH), 7.37–7.39 (1H, m, ArH),
7.42–7.45 (2H, m, 2· ArH), 7.78 (1H, br s, ArH),
7.85–7.86 (1H, m, ArH), 7.97–7.99 (1H, m, ArH),
8.18–8.23 (2H, m, 2· ArH), 8.46–8.50 (1H, m, ArH),
8.93, 9.35 and 11.32 (each 1H, br s, exchangeable, 3·
NH). Anal. (C32H31Cl2N5O2Æ3.2H2O) C, H, N.
5.1.16 .
1-Acridin-9-yl-3-{4-[bis-(2-chloroethyl)amino]-
phenyl}-urea (26). Compound 26 was synthesized from
10 (freshly prepared from 9, 0.306 g, 1 mmol) and 9-
aminoacridinehydrochloride (25, 0.248 g, 1 mmol) in
dry DMF (10 mL) containing triethylamine (0.5 mL):
yield 273 mg (60%); mp 184–186 ꢁC; 1H NMR
(DMSO-d6) d 3.69–3.71 (8H, m, 4· CH2), 6.72 (2H, d,
J = 8.8 Hz, 2· ArH), 7.10–7.14 (2H, m, 2· ArH),
7.39–7.48 (3H, m, 3· ArH), 7.58–7.62 (2H, m, 2·
ArH), 7.84–7.86 (1H, m, ArH), 8.14–8.16 (2H, m, 2·
ArH), 9.37 and 11.33 (each 1H, br s, exchangeable, 2·
NH). Anal. (C24H22Cl2N4O) C, H, N.
5.3. Determination of DNA interstrand cross-linking
The level of DNA interstrand cross-linking was deter-
mined using a modified comet assay.28,29 All steps were
carried out under subdued lighting. Briefly, H1299 cells
(2 · 105 cells) were plated in a 60 mm dish and incu-
bated at 37 ꢁC with 5% CO2 for 32 h. The growing cells
were treated with alkylating agent (24d, mephalan or
cisplatin). After being treated for 1 h, the cells were ex-
posed to 20 Gy irradiation to induce DNA strand
breaks. An aliquot of 5 · 105 cells were suspended in
50 lL of phosphate-buffered saline, mixed with a
250 lL of 1.2% low melting point agarose, and
5.2. Biological experiments
5.2.1. Cytotoxicity assays. The effects of the compounds
on cell growth were determined in T-cell acute lympho-
cytic leukemia (CCRF-CEM) and their resistant subcell
lines (CCRF-CEM/Taxol and CCRF-CEM/VBL) by
the XTT assay22 and human solid tumor cells (i.e.,
breast carcinoma MX-1 and colon carcinoma HCT-
116) the SRB assay23 in a 72 h incubation using a micro-
plate spectrophotometer as described previously.24 After

5422
N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
subjected to comet assay. The tail moment of 100 cells
were analyzed for each treatment by aid of the
COMET assay software (Perceptive instruments,
Haverhill, UK). The degree of DNA interstrand
cross-linking presented at a drug-treated sample was
determined by comparing the tail moment of the
irradiated control, which was calculated by the following
formula. Percentage of DNA with interstrand cross-link-
ing = [1 ꢁ (TMdi ꢁ TMcu/TMci ꢁ TMcu)]·100%, where
TMdi = tail moment of drug-treated irradiated sample,
TMcu = tail moment of untreated unirradiated control,
and TMci = tail moment of untreated irradiated
control.
Acknowledgments
This work was supported by the National Science Council
(Grant No. NSC-95-2320-B-001-025-MY3) and Acade-
mia Sinica (Grant No. AS-96-TP-B06). The NMR spectra
of the synthesized compounds were obtained at High-
Field Biomacromolecular NMR Core Facility supported
by the National Research Program for Genomic Medi-
cine (Taiwan). We would like to thank Dr. Shu-Chuan
Jao in the Institute of Biological Chemistry at Academia
Sinica for providing the NMR service. In addition, we
are grateful to the National Center for High-performance
computing for computer time and facilities.
Appendix A
C, H, N analysis
Compound
Molecular formula
MW
C, H, N analysis
Anal. Calcd
Found
C%
H%
N%
C%
H%
N%
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
24k
24l
24m
24n
26
C31H29Cl2N5O2Æ0.5H2O
C32H31Cl2N5O2Æ3H2O
C31H29Cl2N5OÆ2.5H2O
C32H31Cl2N5OÆ2H2O
C31H29Cl2N5OÆ2.9H2O
C32H31Cl2N5OÆ2.7H2O
C37H41Cl2N7O2Æ5.7H2O
C31H29Cl2N5O2Æ2.6H2O
C32H31Cl2N5O2Æ2.4H2O
C31H29Cl2N5O2Æ2.2H2O
C32H31Cl2N5O2Æ2.5H2O
C36H39Cl2N7O3Æ6.9H2O
C31H29Cl2N5O2Æ3.4H2O
C32H31Cl2N5O2Æ3.2H2O
C24H22Cl2N4O
583.53
642.59
603.54
608.57
609.78
621.18
789.38
601.36
631.75
614.15
633.56
812.96
635.77
646.17
453.38
63.81
59.81
61.69
63.15
61.06
61.87
56.21
61.92
60.83
60.62
60.66
53.19
58.56
59.48
63.58
5.18
5.80
5.20
3.42
5.23
5.43
6.69
5.23
5.24
5.05
5.22
5.57
5.22
5.32
4.89
12.00
10.89
11.60
11.00
11.48
11.27
11.42
11.64
11.08
11.40
11.05
12.06
11.01
10.84
12.36
64.07
59.74
61.83
63.38
61.08
61.84
56.25
61.96
60.89
60.64
60.64
53.18
58.50
59.49
63.35
5.26
5.79
5.17
3.52
5.11
5.44
6.72
5.21
5.25
5.05
5.23
5.56
5.24
5.35
5.05
11.67
10.67
11.53
10.97
11.32
11.43
11.44
11.59
10.93
11.24
10.95
12.03
11.09
10.82
12.09
10. Su, T.-L.; Lin, Y.-W.; Chou, T.-C.; Zhang, X.; Bacheri-
kov, V. A.; Chen, C.-H.; Liu, L. F.; Tsai, T.-J. J. Med.
Chem. 2006, 49, 3710.
11. Springer, C. J.; Dowell, R.; Burke, P. J.; Hadley, E.;
Davies, D.; Blakey, D. C.; Melton, R. G.; Niculescu-
Duvaz, I. J. Med. Chem. 1995, 38, 5051.
12. Springer, C. J.; Antoniw, P.; Bagshawe, K. D.; Searle, F.;
Bisset, G. M. F.; Jarman, M. J. Med. Chem. 1990, 33, 677.
13. Springer, C. J. Drugs Future 1993, 18, 212.
14. Jordan, A. M.; Khan, T. H.; Malkin, H.; Helen, M. I.;
Osborn, H. M. I. Bioorg. Med. Chem. 2002, 10, 2625.
15. Jordan, A. M.; Khan, T. H.; Osborn, H. M. I.; Photiou,
A.; Riley, P. A. Bioorg. Med. Chem. 1999, 7, 1775.
16. Knaggs, S.; Malkin, H.; Osborn, H. M. I.; Williams, N. A.
O.; Yaqoob, P. Org. Biomol. Chem. 2005, 3, 4002.
17. Su, T.-L.; Chou, T.-C.; Kim, J. Y.; Huang, J.-T.;
Ciszewska, G.; Ren, W.-Y.; Otter, G. M.; Sirotnak, F.
M.; Watanabe, K. A. J. Med. Chem. 1995, 38, 3226.
18. Su, T.-L. Curr. Med. Chem. 2002, 9, 1677.
References and notes
1. Gajski, S. R.; William, R. M. Chem. Rev. 1998, 98,
2723.
2. Denny, W. A. Curr. Med. Chem. 2001, 8, 533.
3. Suzukake, K.; Vistica, B. P.; Vistica, D. T. Biochem.
Pharmacol. 1983, 32, 165.
4. Rodney, M.; Carney, J. P.; Kelley, M. R.; Glassner, B. J.;
Williams, D. A.; Samson, L. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 206.
5. Kaldor, J. M.; Day, N. E.; Hemminki, K. Eur. J. Cancer
Clin. Oncol. 1988, 24, 703.
6. Gourdie, T. A.; Valu, K. K.; Gravatt, G. L.; Boritzki, T.
J.; Baguley, B. C.; Wakelin, L. P. G.; Wilson, W. R.;
Woodgate, P. D.; Denny, W. A. J. Med. Chem. 1990, 33,
1177.
7. Fan, J.-Y.; Valu, K. K.; Woodgate, P. D.; Baguley, B. C.;
Denny, W. A. Anti-Cancer Drug Des. 1997, 12, 181.
8. Wyatt, M. D.; Lee, M.; Hartley, J. A. Anti-Cancer Drug
Des. 1997, 12, 49.
19. Chang, J.-Y.; Lin, C.-F.; Pan, W.-Y.; Bacherikov, V.;
Chou, T.-C.; Chen, C.-H.; Dong, H.; Cheng, S.-Y.; Tsai,
T.-J.; Lin, Y.-W.; Chen, K.-T.; Chen, L.-T.; Su, T.-L.
Bioorg. Med. Chem. 2003, 11, 4959.
9. Bacherikov, V. A.; Chou, T.-C.; Dong, H.-J.; Zhang, X.;
Chen, C.-H.; Lin, Y.-W.; Tsai, T.-J.; Lee, R.-Z.; Liu, L.
F.; Su, T.-L. Bioorg. Med. Chem. 2005, 13, 3993.

N. Kapuriya et al. / Bioorg. Med. Chem. 16 (2008) 5413–5423
5423
20. Bacherikov, V. A.; Lin, Y. W.; Chang, J.-Y.; Chen, C. H.;
Pan, W.-Y.; Dong, H.; Lee, R.-Z.; Chou, T.-C.; Su, T.-L.
Bioorg. Med. Chem. 2005, 13, 6513.
21. Dowell, R. I.; Springer, C. J.; Davies, D. H.; Hadley, E.
M.; Burke, P. J.; Boyle, F. T.; Melton, R. G.; Connors, T.
A. J. Med. Chem. 1996, 39, 1100.
22. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks,
A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.;
Boyd, M. R. Cancer Res. 1988, 48, 4827.
24. Chou, T.-C.; O’Connor, O. A.; Tong, W. P.; Guan, Y.-B.;
Zhang, X.-G.; Stachel, S. J.; Lee, S.; Danishefsky, S. J.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8113.
25. Chou, T.-C., Martin, N. ComboSyn, Inc.: Paramus, NJ,
2005.
26. Chou, T.-C.; Talalay, P. Adv. Enzyme Regul. 1984, 22, 27.
27. Chou, T.-C. Pharmacol. Rev. 2006, 58, 621.
28. Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L.
Exp. Cell Res. 1988, 175, 184.
23. Skehan, P.; Storeng, R. H.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenny, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
29. Friedmann, B. J.; Caplin, M.; Savic, B.; Shah, T.; Lord, C.
J.; Ashworth, A.; Hartley, J. A.; Hochhauser, D. Mol.
Cancer Ther. 2006, 5, 209.