Novel DNA-directed alkylating agents: Design, synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker

Article abstract of DOI:10.1016/j.bmc.2008.12.022

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these

Full text of DOI:10.1016/j.bmc.2008.12.022

Bioorganic & Medicinal Chemistry 17 (2009) 1264–1275
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel DNA-directed alkylating agents: Design, synthesis and potent
antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates
via a carbamate or carbonate linker
Naval Kapuriya a, Kalpana Kapuriya a, Huajin Dong b, Xiuguo Zhang b, Ting-Chao Chou b, Yu-Ting Chen a,
Te-Chang Lee a,d, Wen-Chuan Lee c, Tung-Hu Tsai c, Yogesh Naliapara e, Tsann-Long Su a,f,
*
a Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
b Preclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
c Institute of Traditional Medicine, National Yang-Ming University, Taipei 112, Taiwan
d National Research Institute of Chinese Medicine, Taipei 112, Taiwan
e Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
f Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was syn-
thesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the
phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro
studies revealed that these derivatives possessed significant cytotoxicity with IC50 in sub-micromolar
range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carci-
noma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a,
10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-
1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only
one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days.
This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299
cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 22 October 2008
Revised 8 December 2008
Accepted 9 December 2008
Available online 24 December 2008
Keywords:
DNA-directed alkylating agents
Cytotoxic
Phenyl nitrogen mustards
9-Anilinoacridines
1. Introduction
er cytotoxicity and potency than their corresponding untargeted
alkylating agents. Another approach to minimize these drawbacks
Using DNA alkylating N-mustards (e.g., chlorambucil and mel-
phalan) for chemotherapy have a number drawbacks including
(1) high chemical reactivity resulting in loss of drug’s therapeutic
efficacy by reacting with other cellular nucleophiles such as pro-
teins and low molecular weight thiols (inducing cellular resistance
by increased levels of glutathione);1–3 (2) lack of intrinsic DNA
binding affinity of the alkylating pharmacophore (High ratio of
genotoxic mono-adducts to cross-links (20:1), carcinogenicity or
bone marrow toxicity);4 and (3) reducing cytotoxicity via a DNA
repair mechanism.5 To overcome these drawbacks, one of the strat-
egies is to design DNA-directed alkylating agents by linking the
alkylating pharmacophore with DNA-affinic molecules (such as
acridines, anthraquinones, quinolines, phenanthridines, or 9-anili-
noacridines).6–11 It has been demonstrated that these conjugates
are able to modify the pattern of DNA alkylation and possess high-
is to prepare prodrug. Early reports showed that prodrug cyclo-
phosphamide exhibited improved pharmacokinetic and has long
half-life in animal model.12 Apart from cyclophosphamide and
the related derivatives, Springer et al. have synthesized aniline or
phenyl mustards linked to L-glutamic acid moiety through a urea
(1, Z = NH, Chart 1), carbamate (1, Z = O)13 or carboxamide (2,
CMDA)14,15 linker for antibody-directed enzyme prodrug therapy
(ADEPT). The enzymatic hydrolysis by cyclopepetidase (CPG2) of
these prodrugs leads to release the active N-mustard pharmaco-
phore at the tumor site. Prodrugs, 316, 417 and 518 were also pre-
pared for melenocyte directed enzyme prodrug therapy (MDEPT).
The reactivation of these prodrugs requires enzymatic hydrolysis
by tyrosinase. These studies demonstrated that the urea, carba-
mate, or carboxamide linker is capable of lowering the reactivity
of aniline or phenol N-mustard pharmacophore resulting in forma-
tion of rather stable N-mustard derivatives.
One of our ongoing projects on the research and development of
new potential antitumor agents, we focus on designing and devel-
oping DNA-directed alkylating agents. We have previously synthe-
* Corresponding author. Present address: Institute of Biomedical Sciences,
Academia Sinica, Taipei 115, Taiwan. Tel.: +886 2 27899045; fax: +886 2 27827685.
E-mail address: tlsu@ibms.sinica.edu.tw (T.-L. Su).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.022

N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
1265
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
1 Z = NH, O
2 (CMDA)
H
N
H
N
HO
HO
O
N(CH2CH2Cl)2
5
NHCONH
Me
N(CH2CH2Cl)2
OCH2CH2N(CH2CH2Cl)2
CH2OH
HN
N
HN
N
Me
6 (BO-0742)
7 (BO-1051)
Chart 1.
sized a series of alkyl N-mustards linked to DNA-affinic molecules
(e.g., 9-anilinoacridines or acridine). The N-mustard pharmaco-
phore was linked to the aniline ring or acridine chromophore of
9-anilinoacridines with alkoxy (O–C1–4) or alkyl (C1) linker.19–21
It revealed that these agents exhibited potent antitumor effects
in inhibiting various human acute lymphoblastic leukemia cells
(CCRF-CEM) and solid tumors in cell culture.20 Among these
agents, compound 6 (BO-0742) was found to have potent thera-
peutic efficacy against human breast MX-1 and ovarian SK-VO3
xenografts in animal model. However, our unpublished results
showed that BO-0742 has a short half-life (<25 min) in mice. To
improve the poor pharmacokinetics of BO-0742, we have recently
synthesized phenyl N-mustard-9-anilinoacridine conjugates via a
urea linker.22 It was shown that these conjugates exhibited a broad
spectrum of antitumor activity. Of these agents, BO-1051 (7) pos-
sessed potent therapeutic efficacy against human tumor xenografts
in nude mice. Total tumor remission (or complete remission, CR)
was achieved in nude mice bearing human breast MX-1 xenograft
and showed no relapse over 70 days. Human glioma U87 MG in
nude mice was also significantly suppressed. We also reported that
this agent is capable of inducing DNA interstrand cross-linking.
Notably, BO-1051 was found to be more stable than BO-0742 in
intravenous injection vehicle and in rat plasma.
While, the reactivity of phenyl N-mustards is relatively weaker
than alkyl N-mustards due to the electron-withdrawing property
of the phenyl ring. Earlier works on the development of DNA-direc-
ted alkylating agents have demonstrated that these agents have
distorted patterns of DNA alkylation by the attached DNA-affinic
molecules.23 In our studies,19–22 we utilized 9-anilinoacridines
such as 3-(9-acridinylamino)-5-hydroxymethylanilines (AH-
MAs),24 3-(9-acridinylamino)toluidines (ATs),25 3-(9-acridinylami-
no)anisidines (AAs),26 and AHMA-alkylcarbamates27 previously
synthesized in our laboratory as the DNA-affinic molecules for
linking with N-mustard residue.19–22 As a result, these N-mus-
tard-9-anilinoacridine conjugates were found to have significantly
improved cytotoxicity and potent therapeutic efficacy against var-
ious human tumor xenografts suggesting that these DNA-interca-
lating agents are very useful as DNA-affinic molecules (or
carriers) for designing DNA-directed alkylating agents. As for the
spacer used for linking N-mustard pharmacophore with DNA-affi-
nic molecules, the length and types of linker are also critical since
they may also influence both the DNA–drug interaction capability
and chemical stability. The advantage of using the urea, carbamate,
or carboxamide linker is that they are able to stabilize the reactive
phenyl N-mustards as described previously. Based on these find-
ings, we may generate new and potent DNA-directed alkylating
agents having improved bioavailability and therapeutic efficacy.
To continue our research on developing DNA-directed alkylat-
ing agents, we have synthesized a series of new N-mustards-9-ani-
linoacridin conjugates using the carbamate or carbonate groups as
a spacer to realize whether these conjugates have improved antitu-
mor profiles and chemical stability. The phenol N-mustard residue
was attached to the amino function of the aniline ring of AHMAs,
ATs and AAs via a carbamate spacer. We also coupled the aniline
From these studies, it clearly suggested that the selection of N-
mustard (alkyl or phenyl N-mustard), DNA-affinic molecule and
linker are important since they may affect the antitumor activity,
DNA-drug interaction and stability of these DNA-directed alkylat-
ing agents. In general, alkyl N-mustards are very reactive and
unstable due to the inductive effect of alkyl function leading to ra-
pid generation of the reactive aziridium cation intermediate, which
readily interacts with DNA forming interstrand cross-linking.

1266
N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
Table 1
or phenol N-mustard residue with the hydroxyl substituent on the
aniline ring of AHMA-alkylcarbamates (11a–c) via a carbamate or
carbonate linker. The results showed that they exhibited promising
antitumor activity against various human tumor xenografts. In this
paper we describe the synthesis, antitumor effect, DNA interstrand
cross-linking and the stability in plasma of the new N-mustard
derivatives.
Analytical data and yields of new N-mustards conjugate (10a–k, 14a–c and 15a)
Compd.
Mp (°C)
Yield (%)
Molecular formula
Analysis
10a
10b
10c
10d
10e
10f
10g
10h
10i
241–242
240–242
202–203
138–139
154–156
104–105
190–192
195–196
179–180
194–195
186–188
178–179
120–122
228–230
131–133
79
61
69
59
52
49
63
47
21
78
52
60
13
34
25
C31H28Cl2N4O3
C32H30Cl2N4O3
C32H30Cl2N4O2
C31H28Cl2N4O2
C32H30Cl2N4O2
C32H30Cl2N4O2
C31H28Cl2N4O3
C32H30Cl2N4O3
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
2. Chemistry
9-Anilinoacridines such as AHMAs (8a,b),24 ATs (8c,d,e,f),25 AAs
(8g,h,i,j,k),26 and AHMA-alkylcarbamates (11a–c)27 were synthesized
by the methods previously developed in our laboratory. These agents
were used as the DNA-directed carriers for constructing the new
C31H28Cl2N4O3
10j
C31H28Cl2N4O3
C32H30Cl2N4O3
C34H33Cl2N5O4
C36H37Cl2N5O4
C37H39Cl2N5O4
C34H32Cl2N4O5
10k
14a
14b
14c
15a
N-mustard-9-anilinoacridine
conjugates.
The
key
reagents,
4-(bis(2-chloroethyl)amino)phenyl 4-nitrophenyl carbonate (9),17,28,29
N,N-bis(2-chloroethyl)benzene-1,4-diamine hydrochloride (12),16 and
4-[N,N-bis(2-chloroethyl)-amino]phenylisocyanate (13)30 were pre-
pared by following the literature methods. The synthesis of N-mustards
linked to the amino function of aniline ring of 9-anilinoacridine conju-
gates via a carbamate linker (10a–k) is shown in Scheme 1. Reaction of
9-anilinoacridine (8a–k) with 9 in dry DMF in the presence of pyridine
at room temperature afforded 10a–k in a fair to good yield (Table 1).
The preparation of N-mustards linked to the hydroxymethyl function
of AHMA-alkylcarbamate (11a–c) via a carbamate or carbonate linkage
is shown in Scheme 2. The freshly prepared 13 (generated from 12 by
treating with triphosgene) in anhydrous DMF in presence of TEA gave
the corresponding N-mustard-AHMA-alkylcarbamate conjugates hav-
ing a carbamate spacer (14a–c) in moderate yields (Table 1). While,
the coupling of 11a–c with 9 to prepare N-mustard-AHMA-alkylcarba-
mate conjugates bearing a carbonate linker was carried out in anhydrous
DMF in presence of pyridine/DMAP at room temperature. However, only
15a was obtained in 25% after purification by liquid flash column chro-
matography. Attempts to prepare 15b,c under various reaction condi-
tions were failed; a mixture of complex decomposition products was
observed by silica gel thin-layer chromatography.
and 15a) against human lymphoblastic leukemia (CCRF-CEM) cell
growth in vitro. It demonstrated that the newly synthesized conju-
gates possess significant cytotoxicity with IC50 in submicro molar
range. The structure-activity relationship (SAR) studies of these
agents against the CCRF-CEM cell growth revealed that in ATs ser-
ies, C40–Me substituted compound (10d) was more cytotoxic than
the corresponding C20–Me (10c) or C60–Me (10f). In the AAs series,
the C40–OMe derivatives (10g and 10h) were about 3–20-fold more
potent than the C50–OMe (10i) or C60–OMe (10j and 10k) deriva-
tives. It is interesting to note that in the series of AA conjugates,
the cytotoxicity was increased by adding the methyl group at C4
position of the acridine ring (10g vs 10h, 10j vs 10k). In contrast,
the C4–Me substituted compound (10e) was less active than the
C4-unsubtituted derivative (10d). The N-mustard derivatives bear-
ing CH2OH substituent (10a and 10b) were as potent as C40–Me
(10d) and C40–OMe (10g) derivatives. Of these derivatives, 10h
(C40–OMe–C4–Me) was the most cytotoxic with the IC50 value of
0.024 lM. The SAR study of the N-mustard-AHMA-alkylcarbamate
conjugates (14a–c) showed that AHMA-ethylcarbamate (14a) was
more potent than the corresponding iso-propyl and tert-butylcar-
bamates (14b and 14c). In comparison of the cytotoxicity of the
conjugates having carbamate and carbonate linker (14a vs 15a,
respectively), 14a was more cytotoxic than 15a. Table 2 also shows
that the newly synthesized conjugates are more cytotoxic than
AHMA (8a), but less potent than BO-0742 (6) suggesting that N-
mustard masked derivatives are more cytotoxic than the corre-
3. Biological results and discussion
3.1. In vitro cytotoxicity
Table 2 shows the antiproliferative activities of the newly syn-
thesized N-mustard-9-anilinoacridine conjugates (10a–k, 14a–c
N(CH2CH2Cl)2
O
NH2
HN
O
3'
O
2'
1'
4'
R2
5'
R2
O2N
O
O
N(CH2CH2Cl)2
HN
HN
N
6'
9
DMF/Pyridine, rt
N
R1
R1
10a-k
8a-k
a: R1 = H, R2 = 5’-CH2OH
b: R1 = Me, R2 = 5’-CH2OH
c: R1 = Me, R2 = 2’-Me
d: R1 = H, R2 = 4’-Me
g: R1 = H, R2 = 4’-OMe
h: R1 = Me, R2 = 4’-OMe
i: R1 = H, R2 = 5’-OMe
j: R1 = H, R2 = 6’-OMe
k: R1 = Me, R2 = 6’-OMe
e: R1 = Me, R2 = 4’-Me
f: R1 = Me, R2 = 6’-Me
Scheme 1. Synthesis of N-mustards linked to the amino function of 9-anilinoacridines (8a–k) via a carbamate linker.

N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
1267
NHCOOR2
NHCOOEt
OH
O
O
HN
HN
O
9
N(CH2CH2Cl)2
DMF/Pyridine/DMAP
N
N
R1
N(CH2CH2Cl)2
Triphosgene/TEA
11a-c
15a
N(CH2CH2Cl)2
DMF/TEA
CHCl3, -10 oC
NCO
NH2
12
13
NHCOOR2
H
N
O
HN
O
N(CH2CH2Cl)2
N
R1
14a-c
a: R1 = H, R2 = Et
b: R1 = H, R2 = iso-Pro
c: R1 = Me, R2 = tert-Bu
Scheme 2. Synthesis of N-mustards linked to the hydroxymethyl function of AHMA-alkylcarbamate (11a–c) via a carbamate or carbonate linker.
Table 2
Cytotoxicity of new N-mustard conjugates, 10a–k, 14a–c and 15a 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 (lM)
CCRF-CEM
CCRF-CEM/Taxolb
CCRF-CEM/VBLb
MX-1
HCT-116
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
14a
0.075 0.016
0.067 0.013
0.16 0.026
0.076 0.014
0.132 0.016
0.132 0.053
0.085 0.034
0.024 0.013
0.200 0.002
0.541 0.025
0.205 0.052
0.085 0.001
0.189 0.047
0.750 0.17
0.140 0.032[1.9ꢀ]c
0.097 0.018[1.4ꢀ]
0.174 0.004[1.09ꢀ]
0.216 0.004[2.8ꢀ]
0.169 0.011[1.3ꢀ]
0.138 0.062[1.05ꢀ]
0.16 0.042[1.9ꢀ]
0.027 0.006[1.1ꢀ]
0.309 0.003[1.5ꢀ]
0.583 0.003[1.08ꢀ]
0.210 0.001[1.0ꢀ]
0.243 0.001[2.9ꢀ]
0.648 0.057[3.4ꢀ]
3.358 0.22[4.5ꢀ]
0.509 0.004[1.6ꢀ]
0.0075[1.1ꢀ]
0.184 0.028[2.5ꢀ]
0.127 0.019[1.9ꢀ]
0.142 0.003[0.89ꢀ]
0.158 0.006[2.1ꢀ]
0.157 0.011[1.2ꢀ]
0.093 0.014[0.7ꢀ]
0.119 0.013[1.4ꢀ]
0.19 0.001[7.9ꢀ]
0.108 0.001[0.54ꢀ]
0.254 0.004[0.14ꢀ]
0.11 0.004[0.54ꢀ]
0.319 0.006[3.9ꢀ]
0.753 0.022[4ꢀ]
1.416 0.018[1.9ꢀ]
0.482 0.004[1.5ꢀ]
0.0340[4.9ꢀ]
0.099 0.004
0.084 0.003
0.181 0.008
0.214 0.004
0.286 0.025
0.163 0.014
0.181 0.005
0.138 0.012
0.340 0.007
1.555 0.034
0.338 0.02
0.597 0.007
1.13 0.045
5.933 0.657
0.089 0.001
0.0035
0.568 0.037
0.438 0.036
0.320 0.010
0.434 0.021
0.607 0.038
0.263 0.055
0.186 0.003
0.238 0.006
1.712 0.012
0.709 0.017
0.841 0.044
0.752 0.009
0.301 0.053
2.403 0.106
0.639 0.005
0.0055
14b
14c
15a
0.315 0.004
0.007
0.753
0.0031 0.0001
0.00073 0.0009
620
8a20
Taxol
Vinblastine
0.600[0.8ꢀ]
1.60[2.70ꢀ]
0.0035
0.046 0.0007
0.0022 0.0002
NDd
0.429 0.042[330ꢀ]
0.078 0.011[106.2ꢀ]
1.274 0.052[980ꢀ]
0.496 0.121[679.5ꢀ]
0.0011 0.0003
0.0011 0.0003
a
Cell growth inhibition was measured by the XTT31 assay for leukemic cells and the SRB assay32 for solid tumor cells after 72 h incubation using a microplate spectro-
photometer as described in experimental section. 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 Martin35 based on the median-effect principle and plot 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.
ND = not determined.
d

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N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
sponding parent compounds. Moreover, it also demonstrates that
the alkyl N-mustard derivatives are more potent than the corre-
sponding phenyl N-mustard counter part (6 vs 10a).
We have also evaluated the antiproliferative activities of new N-
mustard conjugates (10a–k, 14a–c and 15a) against MX-1 and
HCT-116 solid tumor cell growth (Table 2). In general, the position
Table 3
Cytotoxicity of new N-mustard conjugates, 10a, 10b, 10f, 10h, 14b and 15a against non-small cell lung cancer (H1299), human prostate cancer (PC3), human lung
adenocarcinoma (CL1-0 and CL1-5) and human breast adenocarcinoma (MCF-7) cell growth in vitroa
Compound
IC50
CL1-0
(l
M)b
H1299
PC3
1.609 0.340
5.977 1.912
4.469 1.796
1.576 0.585
15.980 4.062
23.331 4.974
11.803 5.775
CL1-5
MCF-7
10a
10b
10f
10h
14b
15a
Cisplatin
0.284 0.083
0.457 0.040
0.623 0.023
0.357 0.043
9.171 0.195
0.527 0.071
4.707 0.574
2.695 0.773
1.869 0.789
4.657 1.432
2.251 0.806
3.996 0.507
0.410 0.242
56.917 16.984
5.064 1.459
12.648 1.723
10.588 2.476
8.85 1.28
6.629 1.352
4.56 3.313
4.797 0.531
9.01 0.918
5.713 0.972
3.433 0.991
7.012 0.773
19.802 6.596
19.872 1.885
27.741 3.913
a
Cell growth inhibition was measured by the WST-1 assay34 after 72 h incubation using a microplate spectrophotometer as described in Section 5.
The values are averages of at least two separate determinations.
b
Figure 1. The therapeutic effects of 10a, 10b, 10i, and 15a in nude mice bearing MX-1 (iv injection, n = 5). (A) For average tumor size changes; p values for 10a, 10b, 10i, and
15a are <0.0001 (D14-20), <0.0008 (D14-20), <0.0001 (D16-18), and <0.0005 (D16-18), respectively. (B) For average body weight changes.

N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
1269
of the N-mustard residue either linked to the amino function of
AHMAs, ATs or AAs (10a–k) or to the hydroxymetnyl of AHMA-
alkylcarbamates (14a–c or 15a) did not greatly affect their cytotox-
icity against both cell lines. Among the new conjugates tested
against MX-1 cell growth in vitro, 10a, 10b, and 15a were shown
to have significant cytotoxicity with IC50 values of 0.099, 0.084,
We used CCRF-CEM/Taxol and CCRF-CEM/VBL, which are CCRF-
CEM sub-cell lines resistant to Taxol and Vinblastine, for compari-
son to determine whether the newly synthesized compounds are
multi-drug resistant toward these two cell lines. As shown in Table
2, the newly synthesized derivatives have little or no cross-resis-
tance to either taxol or vinblastine. This suggests that all new con-
jugates are neither a good substrate of membrane multi-drug
resistance transporters (i.e., p-glycoprotein) nor mutated tubulin.
Thus, the newly synthesized compounds are effective against mul-
tiple drug resistant tumors.
and 0.089 lM, respectively. In addition, all new conjugates (10a–
k, 14a–c and 15a) possessed a comparable cytotoxicity against
HCT-116 cell line. To explore further, compounds 10a, 10b, 10f,
10h, 14b and 15a were selected to study their cell growth inhibi-
tory effect against other solid tumor such as non-small cell lung
cancer (H1299), human prostate cancer (PC3), human lung adeno-
carcinoma (CL1-0), and CL1-5 and human breast adenocarcinoma
(MCF-7) (Table 3). The SAR study revealed that these agents were
more cytotoxic in inhibiting H1299 cell growth with IC50 value in
sub micro molar range than PC3, CL1-0, CL1-5 or MCF-7 cell lines.
Importantly, all the selected derivatives were about 1-140-fold
more potent than the cisplatin against CL1-0, CL1-5 and MCF-7 cell
growth. Among these agents, compound 10a was the most potent
3.2. In vivo therapeutic effect
Compounds 10a, 10b, 10e, 10i, and 15a were selected for eval-
uating their antitumor therapeutic efficacy in animal model which
is based on their in vitro cytotoxicity, solubility and toxicity to the
host. Figure 1 shows the therapeutic effects of 10a, 10b, 10i and
15a in nude mice bearing mammary MX-1 xenografts at the max-
imal tolerated doses [50 mg/kg, Q2D ꢀ 5; 40 mg/kg, Q2D ꢀ 5,
100 mg/kg, QD ꢀ 6 and 10 mg/kg, Q2D ꢀ 3, respectively, via intra-
in inhibiting H1299 cell growth with IC50 value of 0.284 lM.
Figure 2. The therapeutic effects of 10e in nude mice bearing MX-1 (iv injection, n = 5). (A) For average tumor size changes; p value <0.0001 (D27-29); (B) for average body
weight changes.

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N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
venous injection (iv injection)]. Remarkably, in all cases, total tu-
mor remission (or complete remission, CR) was achieved with only
one cycle treatment (Fig. 1A). No tumor relapse was found in mice
treated with 10a and partial mice treated with 10b on Day 129 of
experimentation. Compound 10e also led to complete tumor
remission at the dose of 50 mg/kg, Q2D ꢀ 9, iv injection. (Fig. 2A
and B). Figure 3 shows the therapeutic effects of 10a and 10e in
nude mice bearing colon HCT-116 xenografts. Compound 10a
and 10e showed, at the doses of 50 and 50 mg/kg, respectively, sig-
nificant tumor growth inhibition (98% and 89%, respectively) in
comparison to untreated control. Although 8–15% body-weight
change was found during maximal tolerated treatment (Fig. 1B,
2B, and 3B), the body-weight of mice was readily recovered after
cession of treatment suggesting that the tested compounds have
relatively low toxicity to the hosts.
3.3. DNA interstrand cross-linking study
To realize whether the newly synthesized conjugates are able to
cross-link to DNA doubled strands, the single cell gel electrophore-
sis assay (modified comet assay)28 was employed to determine the
level of DNA interstrand cross-linking. The representative com-
pound 10a was subjected to DNA cross-linking studies in human
non-small lung cancer H1299 cells by the modified comet assay
and was compared with melphalan. The results showed that 10a
was capable of inducing DNA interstrand cross-linking in a dose-
Figure 3. The therapeutic effects of 10a and 10e in nude mice bearing HCT-116 xenografts (iv injection, n = 4). (A) For average tumor size changes; p value <0.0001; (B) for
average body weight changes.

N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
1271
to the hydroxymethyl function of AHMA-alkylcarbamates via a car-
bamate or carbonate spacer (14a–c and 15a, respectively). The SAR
studies showed that, compounds 10a–k are more cytotoxic or as po-
tent as 14a–c. The antitumor therapeutic efficacy studies against hu-
man tumor xenografts demonstrated that the newly synthesized
conjugates exhibited potent antitumor efficacy against breast carci-
noma MX-1 and colon carcinoma HCT-116 xenografts. For MX-1
xenograft, complete tumor remission was achieved after treatment
of 10a, 10b, 10e, 10i, and 15a and maintain no relapse on Day 129
by 10a and 10b. For HCT-116 xenografts, tumor growth inhibition
was 98% and 89% by 10a and 10e, respectively. Moreover, we also
found that 10a is able to induce DNA interstrand cross-linking and
the DNA cross-linking capability is much more effective than that
of melphalan. In our recent studies, we demonstrated that urea lin-
ker in phenyl N-mustard-9-anilinoacridine conjugates plays a very
important role, especially for stabilizing the reactive N-mustard res-
idue. The current studies also show that the carbamate or carbonate
linkers play an important role in enhancing drug’s therapeutic ef-
fects, DNA interstrand cross-linking and stability.
Figure 4. DNA cross-linking in H1299 cells measured by the modified comet assay.
Results were generated as described in Section 5. The experiments were performed
in duplicate and repeated three times. Data points represent the mean SE of each
test condition.
dependent manner (Fig. 4). At the dose of 10
duced 74.2% DNA cross-linking, while, melphalan induced 39.7%
DNA cross-linking at the dose of 200 M under the same experi-
lM, this agent in-
l
5. Experimental
mental conditions. Figure 5 shows the distribution of tail moment
in H1299 cells. The production of an essentially random distribu-
tion of single-strand breaks is obtained by means of 20 Gy X rays.
As cross-linking agents connect DNA with DNA and or histone pro-
teins, they decreased DNA migration in the comet assay. The comet
images clearly show the concentration-dependent reduction of
DNA migration in all H1299 cells with increasing concentrations
of the cross-linker 10a. The results suggested that DNA interstand
cross-linking may be the main mechanism of action of 10a and the
related compounds.
Melting points were determined on a Fargo melting point appa-
ratus and are uncorrected. Column chromatography was carried
out on Silica Gel G60 (70–230 mesh and 230–400 mesh). Thin-
layer chromatography was performed on Silica Gel G60 F254 with
short-wavelength UV light for visualization. Elemental analysis
was done on a Heraeus CHN–O Rapid instrument. 1H NMR spectra
were recorded on 400 MHz spectrometers. The chemical shifts
were reported in ppm (d) relative to TMS.
5.1. General procedure for the preparation of N-mustard linked
to the amino function of 9-anilinoacridines via a carbamic acid
ester linkage (10a–k)
3.4. Stability in rat plasma
Compound 10a was further selected to study its chemical sta-
bility in rat plasma. The degradation of this agent was detected
by HPLC. The detection limit is 20 ng/ml of the authentic 10a in
the rat plasma. It revealed that 10a is a stable N-mustard derivative
in rat plasma with long half-life (t1/2 = 6.41 0.86 h, n = 3). These
results demonstrate that the newly prepared N-mustards are
chemically and metabolically stable derivatives.
9-Anilinoacridines such as AHMAs (8a,b),24 ATs (8c,d,e,f),25 AAs
(8g,h,i,j,k),26 and AHMA-alkylcarbamates (11a–c)27 were synthe-
sized by the methods previously developed in out laboratory.
These agents were used as the DNA-directed carriers for construct-
ing the N-mustard-9-anilinoacridine conjugates. The key interme-
diate, 4-[bis-(2-chloroethyl)amino] phenyl ester 4-nitro-phenyl
ester (9),17,28,29 N,N-Bis(2-hloroethyl)benzene-1,4-diamine hydro-
chloride (12),16 and 4-[N,N-bis(2-chloroethyl)amino]phenyl-isocy-
anate (13)30 were prepared by following the literature methods.
The reaction of 9 with appropriate 9-anilinoacridines (8a–k)24–26
was carried out in dry DMF in the presence of pyridine to give
10a–k. The final products were purified either by recrystallization
from an appropriate solvent or by column chromatography using
4. Conclusion
In the current studies, we have designed and synthesized a series
of DNA-directed alkylating agents by linking the phenyl N-mustard
pharmacophore with the amino function of the aniline ring of vari-
ous 9-anilinoacridine derivatives via a carbamate linker (10a–k) or
Figure 5. Typical comet images from H1299 cell treated ex vivo with 10a and melphalan. The presence of cross-links retards the electrophoretic mobility of alkaline
denatured cellular DNA. Cross-links are, therefore, quantitated as the decrease in comet tail moment compared with irradiated control. (a) Control; (b) 20 Gy irradiation; (c)
melphalan (200 lM); (d) 10a 0.05 lM; (e) 10a 1 lM; (f) 10a 5 lM; (g) 10a 10 lM.

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N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
(SiO2, CHCl3/MeOH, v/v 100:1). The detailed procedure was de-
scribed as follows.
(0.940 g, 3 mmol):25 Yield 0.905 g (52%); mp 154–156 °C; 1H
NMR (DMSO-d6) d 2.36 (3H, s, Me), 2.81 (1H, s, Me), 3.71 (8H, s,
4 ꢀ CH2), 6.72 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 6.96–7.00 (2H, m,
2 ꢀ ArH), 7.12–7.13 (1H, m, 2 ꢀ ArH), 7.33–7.44 (3H, m, 3 ꢀ ArH),
7.62 (1H, br s, ArH), 7.83–7.84 (1H, m, ArH), 7.96–7.97 (1H, m,
ArH), 8.21–8.27 (2H, m, 2 ꢀ ArH), 8.57–8.58 (1H, m, ArH), 9.52
and 11.65 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal.
(C32H30Cl2N4O2) C, H, N.
5.1.1. [3-(Acridin-9-ylamino)-5-hydroxymethyl-phenyl]
carbamic acid 4-[bis(2-chloroethyl)-amino]-phenyl ester (10a)
Compound 9 (1.123 g, 2.7 mmol) was added into a solution of [3-
(acridin-9-ylamino)-5-amino-phenyl] methanol (8a) (1.130 g,
2.73 mmol)24 in dry DMF (20 mL) containing pyridine (5 mL) at room
temperature and stirred for 48 h. The reaction mixture was evapo-
ratedunderreducedpressuretodrynessandthesolidresiduewasdis-
solved in a mixture of CHCl3/MeOH containing silica gel (5 g) and then
evaporated invacuo to dryness. The residuewasput onthe top of a sil-
ica gel column (20ꢀ4 cm) and purified by using CHCl3/MeOH (100/1
v/v) as an eluent. The fraction containing the main product were
combined and evaporated in vacuo to dryness and the residue was
recrystallized from CHCl3/MeOH to give 10a, 1.169 g (79%); mp
241–242 °C; 1H NMR (DMSO-d6) d 3.72 (8H, s, 4 ꢀ CH2), 4.46 (2H, s,
CH2), 5.36 (1H, br s, exchangeable, OH), 6.74 (2H, d, J = 9.0 Hz,
2 ꢀ ArH), 7.02 (1 H, br s, ArH), 7.03 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 7.46–
7.51 (4H, m, 4 ꢀ ArH), 7.99–8.02 (2H, m, 2 ꢀ ArH), 8.11–8.12 (2H, m,
2 ꢀ ArH), 8.28–8.30 (2H, m, 2 ꢀ ArH), 10.38 and 11.53 (each 1H, br
s, exchangeable, 2 ꢀ NH). Anal. (C31H28Cl2N4O3) C, H, N.
5.1.6. [4-Methyl-3-(4-methyl-acridin-9-ylamino)-phenyl]
carbamic acid 4-[bis(2-chloroethyl)-amino] phenyl ester (10f)
Compound 10f was prepared from 9 (1.197 g, 3 mmol) and 4-
methyl-N1-(4-methyl-acridin-9-yl)-benzene-1,3-diamine
(8f)
(0.627 g, 2 mmol):25 Yield 0.563 g (49%); mp 104–105 °C; 1H
NMR (DMSO-d6) d 2.21 (3H, s, Me), 2.80 (3H, s, Me), 3.70 (8H, s,
4 ꢀ CH2), 6.46–6.47 (1H, m, ArH), 6.70–6.72 (2H, m, 2 ꢀ ArH),
6.96 (5H, br s, 5 ꢀ ArH), 7.32–7.42 (2H, m, 2 ꢀ ArH), 7.60–7.72
(2H, m, 2 ꢀ ArH), 8.01–8.11 (2H, m, 2 ꢀ ArH), 9.04 and 9.15 (each
1H, br s, exchangeable, 2 ꢀ NH). Anal. (C32H30Cl2N4O2) C, H, N.
5.1.7. [5-(Acridin-9-ylamino)-2-methoxy-phenyl] carbamic
acid 4-[bis(2-chloroethyl)-amino] phenyl ester (10g)
Compound 10g was prepared from 9 (0.800 g, 2 mmol) and N1-
By following the same procedure, the following compounds
were synthesized.
acridin-9-yl-4-methoxy-benzene-1,3-diamine
(8 g)
(0.630 g,
2 mmol):26 Yield 0.718 g (63%); mp 190–192 °C; 1H NMR (DMSO-
d6) d 3.70 (8H, s, 4 ꢀ CH2), 3.92 (3H, s, OMe), 6.73 (2H, d,
J = 8.3 Hz, 2 ꢀ ArH), 7.00 (2H, d, J = 8.7 Hz, 2 ꢀ ArH), 7.19 (2H, s,
2 ꢀ ArH), 7.39–7.44 (2H, m, 2 ꢀ ArH), 7.84 (1H, s, ArH), 7.94–7.98
(2H, m, 2 ꢀ ArH), 8.10–8.12 (2H, m, 2 ꢀ ArH), 8.26–8.29 (2H, m,
2 ꢀ ArH), 9.25 and 11.62 (each 1H, br s, exchangeable, 2 ꢀ NH).
Anal. (C31H28Cl2N4O3) C, H, N.
5.1.2. [3-Hydroxymethyl-5-(4-methyl-acridin-9-ylamino)-
phenyl] carbamic acid 4-[bis(2-chloroethyl)-amino] phenyl
ester (10b)
Compound 10b was prepared from 9 (1.196 g, 3 mmol) and [3-
amino-5-(4-methyl-acridin-9-ylamino)-phenyl]methanol
(8b)
(0.987 g, 3 mmol):24 Yield 1.053 g (61%); mp 240–242 °C; 1H
NMR (DMSO-d6) d 2.81 (3H, s, Me), 3.72 (8H, s, 4 ꢀ CH2), 4.57
(2H, s, CH2), 5.35 (1H, br s, exchangeable, OH), 6.74 (2H, d,
J = 9.0 Hz, 2 ꢀ ArH), 6.98 (1H, br s, ArH), 7.03 (2H, d, J = 9.0 Hz,
2 ꢀ ArH), 7.38–7.40 (2H, m, 2 ꢀ ArH), 7.46–7.49 (2H, m, 2 ꢀ ArH),
7.86–7.87 (1H, m, ArH), 7.99–8.00 (1H, m, ArH), 8.21–8.27 (2H,
m, 2 ꢀ ArH), 8.54–8.56 (1H, m, ArH), 10.37 and 11.77 (each 1H,
br s, exchangeable, 2 ꢀ NH). Anal. (C32H30Cl2N4O3), C, H, N.
5.1.8. [2-Methoxy-5-(4-methyl-acridin-9-ylamino)phenyl]
carbamic acid 4-[bis(2-chloroethyl)amino] phenyl ester (10h)
Compound 10h was prepared from 9 (1.197 g, 3 mmol) and 4-
methoxy-N1-(4-methyl-acridin-9-yl)-benzene-1,3-diamine (8 h)
(0.66 g, 2 mmol):26 Yield 0.559 g (47%); mp 195–196 °C; 1H NMR
(DMSO-d6) d 2.78 (3H, s, Me), 3.71 (8H, s, 4 ꢀ CH2), 3.79 (3H, s,
OMe), 6.48 (1H, s, ArH), 6.70–6.72 (2H, m, 2 ꢀ ArH), 6.96–6.98 (3H,
m, 3 ꢀ ArH), 7.28–7.39 (3H, m, 3 ꢀ ArH), 7.58–7.69 (2H, m, 2 ꢀ ArH),
8.04–8.13 (2H, m, 2 ꢀ ArH), 8.86 (1H, s, ArH), 8.99 and 9.67 (each 1H,
br s, exchangeable, 2 ꢀ NH). Anal. (C32H30Cl2N4O3) C, H, N.
5.1.3. [2-Methyl-3-(4-methyl-acridin-9-ylamino)-phenyl]
carbamic acid 4-[bis(2-chloroethyl)-amino] phenyl ester (10c)
Compound 10c was prepared from 9 (2.0 g, 5 mmol) and 2-
methyl-N-(4-methyl-acridin-9-yl)-benzene-1,3-diamine
(8c)
(0.689 g, 2.2 mmol):25 Yield 0.786 g (69%); mp 202–203 °C; 1H
NMR (DMSO-d6) d 2.02 (3H, s, Me), 2.49 (3H, s, OMe), 3.73 (8H, s,
4 ꢀ CH2), 6.40–6.42 (1H, m, ArH), 6.74–6.77 (4H, m, 4 ꢀ ArH),
7.03 (2H, d, J = 8.6 Hz, 2 ꢀ ArH), 7.06–7.10 (3H, m, 3 ꢀ ArH),
7.33–7.45 (2H, m, 2 ꢀ ArH), 7.69–7.70 (1H, m, ArH), 8.06 (1H, br
s, ArH), 9.33 and 9.71 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal.
(C32H30Cl2N4O2) C, H, N.
5.1.9. [3-(Acridin-9-ylamino)-5-methoxyphenyl] carbamic acid
4-[bis(2-chloroethyl)-amino] phenyl ester (10i)
Compound 10i was prepared from 9 (1.19 g, 3 mmol) and N1-
acridin-9-yl-5-methoxy-benzene-1,3-diamine
(8i)
(0.945 g,
3 mmol):26 Yield 0.317 g (21%); mp 179–180 °C; 1H NMR (DMSO-
d6) d 3.66 (3H, s, OMe), 3.70–3.72 (8H, m, 4 ꢀ CH2), 6.13 (1H, br
s, ArH), 6.57 (1H, br s, ArH), 6.74 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 6.90
(1H, br s, ArH), 7.02 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 7.10 (2H, br s,
2 ꢀ ArH), 7.51–7.59 (4H, m, 4 ꢀ ArH), 7.95 (2H, br s, 2 ꢀ ArH),
10.05 and 11.23 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal.
(C31H28Cl2N4O3) C, H, N.
5.1.4. [5-(Acridin-9-ylamino)-2-methyl-phenyl] carbamic acid
4-[bis(2-chloroethyl)-amino] phenyl ester (10d)
Compound 10d was prepared from 9 (1.20 g, 3 mmol) and
N1-acridin-9-yl-4-methyl-benzene-1,3-diamine (8d) (0.600 g,
2 mmol):25 Yield 0.663 g (59%); mp 138–139 °C; 1H NMR (DMSO-
d6) d 2.26 (3H, s, Me), 3.71 (8H, s, 4 ꢀ CH2), 6.47–6.50 (1H, m,
ArH), 6.76–6.78 (3H, m, 3 ꢀ ArH), 6.94 (2H, br s, 2 ꢀ ArH), 7.00
(2H, d, J = 8.7 Hz, 2 ꢀ ArH), 7.11–7.13 (2H, m, 2 ꢀ ArH), 7.35–7.50
(4H, m, 4 ꢀ ArH), 8.21 (1H, m, ArH), 9.24 and 10.84 (each 1H, br
s, exchangeable, 2 ꢀ NH). Anal. (C31H28Cl2N4O2) C, H, N.
5.1.10. [3-(Acridin-9-ylamino)-4-methoxyphenyl] carbamic
acid 4-[bis(2-chloroethyl)-amino] phenyl ester (10j)
Compound 10j was prepared from 9 (2.0 g, 5 mmol) and N1-
acridin-9-yl-4-methoxy-benzene-1,3-diamine
(8j)
(0.701 g,
2.2 mmol):26 Yield 0.912 g (78%); mp 194–195 °C; 1H NMR
(DMSO-d6) d 3.41 (3H, s, OMe), 3.73 (8H, s, 4 ꢀ CH2), 6.76 (2H, d,
J = 9.0 Hz, 2 ꢀ ArH), 7.06 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 7.18–7.20
(1H, m, ArH), 7.43–7.47 (2H, m, 2 ꢀ ArH), 7.58–7.60 (1H, m, ArH),
7.76 (1H, s, ArH), 7.98–8.02 (2H, m, 2 ꢀ ArH), 8.11–8.13 (2H, m,
2 ꢀ ArH), 8.26–8.28 (2H, m, 2 ꢀ ArH), 10.31 and 11.37 (each 1H,
br s, exchangeable, 2 ꢀ NH). Anal. (C31H28Cl2N4O3) C, H, N.
5.1.5. [2-Methyl-5-(4-methyl-acridin-9-ylamino)-phenyl]
carbamic acid 4-[bis(2-chloroethyl)-amino] phenyl ester (10e)
Compound 10e was prepared from 9 (1.197 g, 3 mmol) and 4-
methyl-N1-(4-methyl-acridin-9-yl)-benzene-1,3-diamine
(8e)

N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
1273
5.1.11. [4-Methoxy-3-(4-methyl-acridin-9-ylamino) phenyl]
carbamic acid 4-[bis(2-chloroethyl)-amino] phenyl ester (10k)
Compound 10k was prepared from 9 (1.50 g, 3.7 mmol) and 4-
Yield 0.158 g (13%), mp 120–122 °C; 1H NMR (DMSO-d6) d 0.89
(6H, d, J = 6.4 Hz, 2 ꢀ Me), 1.86–1.90 (1H, m, CH), 3.67 (8H, s,
4 ꢀ CH2), 3.82 (2H, d, J = 6.1 Hz, CH2), 5.01 (2H, s, CH2), 6.49 (1H,
m, ArH), 6.67–6.69 (2H, m, 2 ꢀ ArH), 6.84 (2H, m, 2 ꢀ ArH), 7.18–
7.26 (5H, m, 5 ꢀ ArH), 7.52 (3H, m, 3 ꢀ ArH), 8.30 (2H, m, 2 ꢀ ArH),
9.37, 9.57 and 10.94 (each 1H, br s, exchangeable, 3 ꢀ NH). Anal.
(C36H37Cl2N5O4) C, H, N.
methoxy-N1-(4-methyl-acridin-9-yl)-benzene-1,3-diamine
(8k)
(0.548 g, 1.6 mmol):26 Yield 0.489 g (52%); mp 186–189 °C; 1H
NMR (DMSO-d6) d 2.58 (3H, s, Me), 3.58 (3H, s, OMe), 3.71 (8H, s,
4 ꢀ CH2), 6.70–6.72 (4H, m, 4 ꢀ ArH), 6.95–6.97 (4H, m, 4 ꢀ ArH),
7.12 (2H, s, 2 ꢀ ArH), 7.44–7.56 (2H, m, 2 ꢀ ArH), 8.00–8.02 (2H,
m, 2 ꢀ ArH), 8.22 and 9.80 (each 1H, br s, exchangeable, 2 ꢀ NH).
Anal. (C32H30Cl2N4O3) C, H, N.
5.2.4. [3-{4-[Bis-(2-chloroethyl)amino]phenylcarbamoyl-
oxymethyl}-5-(4-methylacridin-9-ylamino)phenyl] carbamic
acid tert-butyl ester (14c)
5.2. General procedure for the preparation of N-mustard linked
to the hydroxyl function of AHMA-alkylcarbamates via a
carbamic acid ester linkage (14a–c)
Compound 14c was synthesized from 13 (freshly prepared from
12, 1.530 g, 5 mmol) and [3-hydroxymethyl-5-(4-methylacridin-9-
ylamino)phenyl] carbamic acid tert-butyl ester (0.858 g,
2 mmol):27 Yield 0.462 g (34%), mp 228–230 °C; 1H NMR (DMSO-
d6) d 0.90 (6H, d, J = 6.7 Hz, 2 ꢀ Me), 1.82–1.89 (1H, m, CH), 2.81
(3H, s, Me), 3.68–3.70 (8H, m, 4 ꢀ CH2), 3.85 (2H, d, J = 6.6 Hz,
CH2), 5.05 (2H, s, CH2), 6.69 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 6.99 (1H,
s, ArH), 7.25 (2H, d, J = 6.1 Hz, 2 ꢀ ArH), 7.35–7.39 (1H, m, ArH),
7.44–7.54 (3H, m, 3 ꢀ ArH), 7.82 (1H, d, J = 7.0 Hz, ArH), 7.95–
7.99 (1H, m, ArH), 8.19–8.25 (2H, m, 2 ꢀ ArH), 8.51–8.53 (1H, m,
ArH), 9.38, 9.95 and 11.54 (each 1H, br s, exchangeable, 3 ꢀ NH).
Anal. (C37H39Cl2N5O4) C, H, N.
N,N-Bis(2-chloroethyl)benzene-1,4-diamine hydrochloride (12)
was prepared by following the procedure developed by Jordan
et al. 16 Isocyanate 1330 (freshly prepared from 12 by treating with
triphosgene in CHCl3 at ꢁ10 °C) was then reacted with appropriate
9-anilinoacridines carbamates (11a– c)27 in dry DMF in the pres-
ence of triethylamine to give 14a–c. The final products were puri-
fied either by recrystallization from an appropriate solvent or by
column chromatography using (SiO2, CHCl3/MeOH, v/v 100:2).
The detailed procedure for the synthesis of 14a–c is described
as follows:
5.2.5. Carbonic acid 3-(acridin-9-ylamino)-5-
ethoxycarbonylamino-benzyl ester 4-[bis-(2-chloro-ethyl)-
amino] phenyl ester (15a)
5.2.1. 4-[N,N-Bis(2-chloroethyl)amino]phenylisocyanate (13)
To a suspension of N,N-bis(2-chloroethyl)benzene-1,4-diamine
hydrochloride (12) (1.683 g, 5.4 mmol)16 in dry chloroform
(30 mL) was added triethylamine (2.5 mL) at 0 °C. The clear solu-
tion obtained was then added dropwise into a solution of triphos-
gene (0.623 g, 2.1 mmol) in dry chloroform (10 mL) at ꢁ10 °C. The
reaction mixture was allowed to stand at room temperature. After
being stirred for 30 min, the reaction mixture was evaporated to
dryness under reduce 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 evaporated to dry-
ness to give the crude isocyanate 1330 which was used directly for
next reaction without further purification.
Compound 9 (0.80 g, 2 mmol) was added into a solution of [3-
(acridin-9-ylamino)-5-hydroxymethylphenyl] carbamic acid ethyl
ester (11a) (0.77 g, 2 mmol)27 in dry DMF (20 mL) containing pyr-
idine (5 mL) and DMAP (10 mg) at room temperature and stirred
for 48 h. The Reaction mixture was evaporated under reduced
pressure to dryness and the solid residue was dissolved in a mix-
ture of CHCl3/MeOH containing silica gel (5 g) and then evaporated
in vacuo to dryness. The residue was put on the top of a silica gel
column (20 ꢀ 4 cm) and purified by using CHCl3/MeOH (100/1 v/
v) as an eluent. The fraction containing the main product were
combined and evaporated in vacuo to dryness and the residue
was recrystallized from CHCl3/MeOH to give 15a; 0.31 g (25%);
mp 131–133 °C; 1H NMR (DMSO-d6) d 1.22 (3H, t, J = 7.0 Hz, Me),
3.71 (8H, s, 4 ꢀ CH2), 4.09 (2H, q, J = 7.0 Hz, CH2,), 5.13 (2H, s,
CH2), 6.53 (1H, br s, ArH), 6.73 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 6.96
(1H, br s, ArH), 7.01 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 7.04 (2H, br s,
2 ꢀ ArH), 7.25 (1H, s, ArH), 7.59 (4H, br s, 4 ꢀ ArH), 7.95 (2H, br
s, 2 ꢀ ArH), 9.66 and 11.17 (each 1H, br s, exchangeable, 2 ꢀ NH).
Anal. (C34H32Cl2N4O5) C, H, N.
5.2.2. (3-(Acridin-9-ylamino)-5-{4-[bis(2-
chloroethyl)amino]phenylcarbamoyl oxymethyl}-phenyl)
carbamic acid ethyl ester (14a)
A solution of isocyanate 13 (freshly prepared from 12, 0.44 g,
1.4 mmol) in dry DMF (5 mL) was added drop wise to a solution
of [3-(acridin-9-ylamino)-5-hydroxymethyl-phenyl] carbamic acid
ethyl ester (0.30 g, 0.8 mmol)27 in dry DMF (15 mL) containing tri-
ethylamine (1 mL) at room temperature. The reaction mixture was
stirred for 26 h at room temperature. The solid separated out was
filtered and recrystallized from CHCl3/MeOH to give 14a; 0.30 g
6. Biological experiments
6.1. Cytotoxicity assays
(60%), mp 178–179 °C; 1H NMR (DMSO-d6)
d 1.23 (3H, t,
J = 7.0 Hz, Me), 3.69 (8H, s, 4 ꢀ CH2), 4.11 (2H, q, J = 7.0 Hz, CH2),
5.06 (2H, s, CH2), 6.69 (2H, d, J = 8.4 Hz, 2 ꢀ ArH), 7.04 (1H, s,
ArH), 7.25 (2H, br s, 2 ꢀ ArH), 7.43–7.48 (2H, m, 2 ꢀ ArH), 7.54–
7.57 (2H, m, 2 ꢀ ArH), 7.96–8.00 (2H, m, 2 ꢀ ArH), 8.14–8.16 (2H,
m, 2 ꢀ ArH), 8.26–8.29 (2H, m, 2 ꢀ ArH), 9.40, 9.98 and 11.54 (each
1H, br s, exchangeable, 3 ꢀ NH). Anal. (C34H33Cl2N5O4) C, H, N.
By following the similar procedures, the following compounds
were synthesized.
The cytotoxic effects of the newly synthesized compounds
were determined in T-cell acute lymphocytic leukemia (CCRF-
CEM) and their resistant subcell lines (CCRF-CEM/Taxol and
CCRF-CEM/VBL) by the XTT assay31 and human solid tumor cells
(i.e., breast carcinoma MX-1 and colon carcinoma HCT-116) the
SRB assay32 in a 72 h incubation using a microplate spectropho-
tometer as described previously.33 After the addition of phenazine
methosulfate-XTT solution, incubated at 37 °C for 6 h and absor-
bance at 450 and 630 nm was detected on a microplate reader
(EL 340). The cytotoxicity of the newly synthesized compounds
against non-small cell lung cancer (H1299), human prostate can-
cer cell line (PC3), human breast adenocarcinoma cell line (MCF-
7), human lung adenocarcinoma (CL1-0 and CL1-5) cell lines were
5.2.3. (3-(Acridin-9-ylamino)-5-{4-[bis(2-
chloroethyl)amino]phenylcarbamoyloxymethyl}- phenyl)-
carbamic acid iso-butyl ester (14b)
Compound 14b was synthesized from 13 (freshly prepared from
12, 1.530 g, 5 mmol) and [3-(acridin-9-ylamino)-5-hydroxy-
methyl-phenyl] carbamic acid iso-butyl ester (0.830 g, 2 mmol):27

1274
N. Kapuriya et al. / Bioorg. Med. Chem. 17 (2009) 1264–1275
determined by the WST-1 assay34 in a 72 h incubation using a
microplate spectrophotometer as described previously. After the
addition of WST-1 solution, it was incubated at 37 °C for 4 h.
Absorbance at 460 and 630 nm was detected on a microplate
reader. IC50 values were determined from dose-effect relationship
at six or seven concentrations of each drug by using the COMPUSYN
software by Chou and Martin35 based on the median-effect prin-
ciple and plot.36,37 Ranges given for Taxol, Vinblastine and Cis-
platin were mean SE (n = 4).
Acknowledgments
This work was supported by the National Science Council
(Grant Number NSC-97-2320-B-001-004-MY2) and Academia Sini-
ca (Grant number AS-96-TP-B06). The NMR spectra of the synthe-
sized compounds were obtained at High-Field Biomacromolecular
NMR Core Facility supported by the National Research Program for
Genomic Medicine (Taiwan). T.-C. Chou is supported by the Sloan-
Kettering Institute General Fund. We would like to thank Dr. Shu-
Chuan Jao in the Institute of Biological Chemistry at Academia Sini-
ca for providing the NMR service. We are grateful to the National
Center for High-performance computing for computer time and
facilities.
6.2. In vivo studies
Athymic nude mice bearing the nu/nu gene were used for hu-
man breast tumor MX-1 and human colon HCT-116 xenografts.
Outbred Swiss-background 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 experiments. Drug
was administrated via the tail vein by iv injection. Tumor volumes
were assessed by measuring length ꢀ width ꢀ height (or width)
Supplementary data
Supplementary data (Elemental analysis data of compound
10a–k, 14a–c and 15a) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2008.12.022.
using caliper. Vehicle used was 50
lL DMSO and 40 lL Tween 80
in 160 L saline. The maximal tolerate dose of the tested com-
l
References and notes
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