Design, synthesis, and biological evaluation of novel water-soluble N-mustards as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2010.11.005

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether link

Full text of DOI:10.1016/j.bmc.2010.11.005

Bioorganic & Medicinal Chemistry 19 (2011) 471–485
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel water-soluble
N-mustards as potential anticancer agents
Naval Kapuriya a,e, Rajesh Kakadiya a, Huajin Dong b, Amit Kumar a, Pei-Chih Lee a, Xiuguo Zhang b,
Ting-Chao Chou b, Te-Chang Lee a, Ching-Huang Chen a, King Lam d, Bhavin Marvania a,e
,
Anamik Shah e, Tsann-Long Su a,c,
⇑
a Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
b Preclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
c Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
d Development Center for Biotechnology, Taipei County 221, Taiwan
e Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized.
The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position
of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that
these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xeno-
grafts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-
1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by
treating with compound 9aa0 at the maximum tolerable dose with relatively low toxicity. We also dem-
onstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline
agarose gel shift assay. A pharmacokinetic profile of the representative 9aa0 in rats was also investigated.
The current studies suggest that this agent is a promising candidate for preclinical studies.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 September 2010
Revised 1 November 2010
Accepted 2 November 2010
Available online 5 November 2010
Keywords:
Anticancer agents
Water soluble nitrogen mustards
DNA interstrand cross-linking agents
Cell cycle
1. Introduction
In one of our research projects on the discovery and develop-
ment of new anticancer agents, we have synthesized a series of
The bioavailability of drugs is one of the important factors to
determine whether a drug can be successfully developed for clini-
cal application. Consequently, the physicochemical properties,
including solubility, permeability, stability pKa, and lipophilicity/
hydrophilicity balance, are key factors that influence the bioavail-
ability of drugs. Compounds with poor solubility usually have a
higher risk of failure during the period of new drug discovery
and development because these properties may affect antitumor
evaluations in animal models as well as pharmacokinetic and phar-
macodynamic properties of the compound. Several approved drugs
possess poor water solubility, resulting in reduced bioavailability.
For example, the poor water-soluble paclitaxel (mitotic spindle
inhibitor) when converted into its water-soluble poly(glutamic
acid) conjugates shows some evidence for increased tumor
availability of the drug for targeted therapy.1–4 Similar efforts in
developing water-soluble derivatives of camptothecin (DNA
topoisomerase I inhibitors) led to the discovery of topotecan (1,
Chart 1)5 and irinotecan (CPT-11, 2),6 which have been approved
for clinical use.
DNA-directed alkylating agents, in which the phenyl N-mustard
pharmacophore (warhead) is linked to the DNA-affinic molecule
(carrier, such as 9-anilinoacridines, acridines or quinolines) via a
urea, carbamate or hydrazinecarboxamide linker.7–9 These linkers
were previously utilized for preparing antibody-directed enzyme
prodrug therapy (ADEPT),10 gene-directed enzyme prodrug ther-
apy (GDEPT),11 or melanocyte-directed enzyme prodrug therapy
(MDEPT). It demonstrated that these linkers are able to reduce
the chemical reactivity of the reactive N-mustard moiety.12–14
We also reported that the N-mustard-DNA-affinic molecule conju-
gates exhibit potent antitumor activity against various human tu-
mor xenograft models. These conjugates are able to induce more
DNA interstrand cross-linking than other alkylating agents such
as melphalan or cisplatin. However, these agents are generally
lipophilic and have poor water-solubility, compromising antitumor
and pharmacokinetic studies in animal models. To overcome the
drawback of insufficient solubility of these agents, it is necessary
to consider designing and synthesizing compounds with improved
water-solubility during the period of discovery and development.
Earlier works on the developing water-soluble N-mustard deriv-
atives by Denny and co-workers have introduced a variety of hydro-
philic side chains to the benzene ring of 4-nitroaniline-N-mustards
⇑
Corresponding author. Tel.: +886 2 2789 9045; fax: +886 2 2782 5573.
E-mail address: tlsu@ibms.sinica.edu.tw (T.-L. Su).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.005

472
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
Chart 1.
derivatives (3) via a carboxamide linker to form water-soluble bior-
eductive anticancer prodrugs.15 Other studies also clearly demon-
strated that linking a side chain containing a basic tertiary amino
function can increase the aqueous solubility of drug by forming
inorganic or organic acid salts.16–20 These studies prompted us to
design and synthesize chemical stable and water-soluble phenyl
N-mustards, yet preserving the potent antitumor activity. We
therefore prepared a series of water-soluble N-mustard-benzene
conjugates via a urea linker. The benzene moiety contains a variety
of water-soluble hydrophilic side chains including N,N-dimethyl-
amino or cyclicamino functions, which can be converted into
water-soluble derivatives by forming hydrochloride or other salts.
The hydrophilic side chain in the benzene ring is located at the para-
or meta-position to the urea linker via a carboxamide (compound
9aa0,bf0 series, Scheme 1) or an ether linkage (compound 19aa0,be0
series, Scheme 2). These conjugates were subjected to antitumor
evaluation. The results show that they exhibit potent antitumor
activity in inhibiting human tumor xenografts. The chemical
synthesis, in vitro and in vivo antitumor activity as well as DNA
cross-linking are described here in this paper.
2. Chemistry
The water-soluble phenyl N-mustard-benzene conjugates con-
taining a hydrophilic carboxamide side-chain (9a,b series) were
prepared starting from the commercially available ethyl 3- (or
4-)isocyanatobenzoate (4a,b) (Scheme 1). Thus, reactions of 4a,b
with the known N,N-bis(2-chloroethyl)benzene-1,4-diamine
hydrochloride (5)14 afforded 6a,b. The products were hydrolyzed
with concd HCl/AcOH (3:1 v/v) under reflux to yield benzoic acid
derivatives 7a,b following the literature procedure.21 Coupling of
7a,b with various amines (8a0–8f0) in dry DMF in the presence of
DDC/HOBT/Et3N afforded the desired compounds 9aa0–9bf0 in fair
to good yields after purification by column chromatography. All
final products obtained had poor water solubility, since they were
in a free base form.
Scheme 1. Reagents and conditions: (a) Et3N, THF, room temperature; (b) concd HCl/AcOH (3:1, v/v), reflux, 6 h; (c) DCC/HOBT, TEA, DMF, rt; (d) Et3N, CHCl3, rt; (e) 10% Pd/C/
H2, EAOAc; (f) triphosgene/CHCl3/Et3N, 0 °C; (g) Et3N, DMF, rt.

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
473
Scheme 2. Reagents and conditions: (a) KOH/EtOH; (b) K2CO3/toluene, alkyl chlorides, reflux; (c) 10% Pd/C, H2, 35 psi; (d) Et3N/DMF, rt.
Since compound 9aa0 was selected for further antitumor studies
based on its cytotoxicity against tumor cell lines tested (see be-
low), an alternative method was developed to optimize the synthe-
sis of this agent (Scheme 1). The reaction of 3-nitrobenzoyl
chloride (11) with N,N-dimethylethylamine hydrochloride (8a0)
yielded benzoylcarboxamides 12, which was then converted into
the corresponding aniline derivatives 13 by catalytic hydrogena-
tion (10% Pd/C, H2) in EtOH. Reaction of 13 with 4-[N,N-bis(2-chlo-
roethyl)amino]phenylisocyanate (10)22 [freshly prepared by
reacting 5 with triphosgene] in anhydrous DMF in the presence
of triethylamine (TEA) afforded the desired 9aa0 hydrochloride salt,
which was found to be soluble in water. This synthetic method can
be applied to preparing water-soluble 9aa0–9bf0 hydrochloride or
mesylate. For instance, we have synthesized 9ad0 and 9bd0 hydro-
chloride by following the same synthetic route as that for 9aa0.
However, one may obtain free compounds during purification by
column chromatography if a long column and a large amount of
silica gel is used. We have also prepared water-soluble mesylate
salt of 9aa0 by treating with methane sulfonic acid in MeOH.
The synthetic route for the preparation of phenyl N-mustard-
benzene conjugates containing a hydrophilic ether side-chain
(19a,b series) is depicted in Scheme 2. The reactions of the potas-
sium salt of 3- (or 4-)nitrophenols (15a and 15b, respectively,
freshly prepared from the corresponding 14a,b with KOH in EtOH)
hydrochloride salt. The assignment for whether compounds form
a free base or salt is further confirmed by comparison with
17aa0–17be0 and 18aa0–18be0 (see 1H NMR spectral data in Supple-
mentary data). The results demonstrated that the ether linker lo-
cated at the para-position to the urea moiety (19ba0–19be0) can
easily form hydrochloride salts under the reaction conditions, but
that has not happened in the corresponding meta-substituted
derivatives (19aa0–19ae0). Although we are able to convert the free
derivatives into its hydrochloride salts by dissolving compounds in
a mixture of CHCl3/MeOH followed with 20% HCl in ethyl acetate
(data not shown), we surprisingly found that both free base
(19aa0–19ae0) and hydrochloride salt forms (19ba0–19be0) have
poor water-solubility.
3. Biological results and discussion
3.1. In vitro cytotoxicity
Human lymphoblastic leukemia (CCRF/CEM) and its drug-resis-
tant sublines (CCRF-CEM/taxol and CCRF-CEM/VBL (330-fold resis-
tant to taxol and 680-fold resistant to vinblastine, respectively)
were used to evaluate the cytotoxicity of tested compounds and
to realize whether they had multi-drug resistance (MDR) toward
taxol or vinblastine in our antitumor screening program. The
antiproliferative activities of the newly synthesized N-mustard-
benzene conjugates are summarized in Table 1. The structure-
activity relationship study shows that the newly synthesized
compounds exhibit significant cytotoxicity with IC50 values of
sub-micromolar range in inhibiting CCRF/CEM cell growth in
culture. There is no significant difference in term of their cytotox-
icities affected by the type of side-chain (carboxamide or ether
linkage) and the location of the substituent (meta- or para-posi-
tion). In general, compounds having an ether linkage are slightly
more potent than the corresponding compounds bearing a carbox-
amide side-chain. Of this series of compounds, 9aa0 is the most cyto-
toxic. In the series of compounds having a carboxamide side-chain,
with various
x
-aminoalkyl halides (16a0–16e0) in dry toluene affor-
ded nitro substituted phenyl ether derivatives 17a,b following the
literature procedure.23 The nitro function of 17a,b was reduced to
the corresponding aniline derivatives 18a,b by catalytic hydroge-
nation (10% Pd/C/H2). The products 18a,b were then reacted with
freshly prepared phenylisocyanate 10 in the presence of triethyl-
amine to yield the final products 19aa0–19be0. Similarly, the
hydrochloride salt may be converted into its free form during
purification by liquid column chromatography.
It should be noted that compounds 9aa0–9bf0 were synthesized
as a free base form as one can identify from their 1H NMR spectro-
photometer spectrum. For example, the protons of NMe2 and
NCH2-functions in 9aa0, 9ab0, 9ba0, and 9bb0, which exist as a free
base and have poor water-solubility, are located near d 2.21 and
2.40, respectively. In contrast, the corresponding functions in
9aa0 hydrochloride or mesylate salt, which are water-soluble, have
a chemical shift at lower field, near d 2.82 and 3.26, respectively.
Similar observations were found in the series of 19aa0–19ae0, in
which the corresponding protons are located at higher fields (near
d 2.21 and 2.61, respectively), indicating that these agents exist in a
free base form. The same protons in derivatives 19ba0–19be0 have
higher chemical shift values, demonstrating that they exist as
the tertiary amino substituent (x-N,N-dimethylamino-, N-alkylpyr-
roliny- or N-alkylpiperidinyl function) does not have a big influence
to their potency. However, the N-alkylmorpholinyl derivatives (9ae0
and 9be0) are less cytotoxic against CCRF-CEM among the tested
compounds. In the series of compounds having an ether linkage,
derivatives having an ether side-chain located at the para-position
to the urea linker are more cytotoxic than or as potent as the corre-
sponding meta-substituted derivatives. Similarly, the N-alkylmorp-
holinyl derivatives (19ae0 and 19be0) are slightly less cytotoxic
against CCRF-CEM in comparison with other derivatives.

474
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
Table 1
The cytotoxicity of new N-mustards against human lymphoblastic leukemia (CCRF/CEM) and its drug-resistant sublines (CCRF-CEM/taxol and CCRF-CEM/VBL)a
O
R
OR
N
H
H
N
H
N
HN
HN
O
O
N(CH2CH2Cl)2
N(CH2CH2Cl)2
9aa'-9bf'
19aa'-19be'
Compd
R
Cytotoxicity, IC50
CCRF-CEM/taxolb
(lM)
CCRF-CEM
CCRF-CEM/VBLb
9aa0
9ab0
(CH2)2NMe2
(CH2)3NMe2
0.13 0.002
0.29 0.012
1.22 0.02 [9.4ꢀ]c
21.20 0.15 [9.4ꢀ]
0.80 0.01 [6.2ꢀ]
44.03 0.08 [151.8ꢀ]
9ac0
9ad0
9ae0
9af0
0.34 0.001
0.27 0.02
0.58 0.002
0.28 0.003
25.20 0.45 [73.7ꢀ]
4.62 0.01 [17.0ꢀ]
8.16 0.18 [14.1ꢀ]
12.19 0.66 [43.5ꢀ]
36.56 0.24 [106.9ꢀ]
10.49 0.46 [43.7ꢀ]
16.29 0.11 [28.1ꢀ]
20.04 0.48 [71.6ꢀ]
(CH2)2N
(CH2)2N
(CH2)2N
N
O
N
9ba0
9bb0
(CH2)2NMe2
(CH2)3NMe2
0.23 0.002
0.26 0.01
4.80 0.07 [21.1ꢀ]
20.39 0.80 [77.5ꢀ]
6.74 0.29 [29.6ꢀ]
32.82 0.34 [124.8ꢀ]
9bc0
9bd0
9be0
9bf0
0.27 0.010
0.36 0.001
0.73 0.01
0.22 0.01
19.67 0.45 [71.8ꢀ]
14.97 1.47 [41.8ꢀ]
19.82 0.19 [27.1ꢀ]
20.47 0.66 [91.4ꢀ]
30.82 0.34 [112.5ꢀ]
24.28 0.29 [67.8ꢀ]
29.76 0.26 [16.8ꢀ]
32.18 0.12 [143.7ꢀ]
(CH2)2N
(CH2)2N
(CH2)2N
N
O
N
19aa0
19ab0
(CH2)2NMe2
(CH2)3NMe2
1.32 0.001
0.32 0.01
0.81 0.001 [0.6ꢀ]
2.05 0.01 [6.4ꢀ]
1.48 0.003 [1.1ꢀ]
2.37 0.35 [7.4ꢀ]
19ac0
19ad0
19ae0
0.35 0.01
0.19 0.003
0.41 0.10
1.20 0.002 [3.5ꢀ]
0.57 0.02 [3.0ꢀ]
4.20 0.01 [12.1ꢀ]
0.73 0.01 [3.8ꢀ]
1.13 0.58 [2.8ꢀ]
(CH2)2N
(CH2)2N
(CH2)2N
0.73 0.07 [1.8ꢀ]
1.50 0.01 [7.6ꢀ]
1.36 0.001 [11.8ꢀ]
O
19ba0
19bb0
(CH2)2NMe2
(CH2)3NMe2
0.10 0.002
0.12 0.001
1.19 0.04 [6.02ꢀ]
1.26 0.001 [10.9ꢀ]
19bc0
19bd0
19be0
0.14 0.001
0.12 0.003
0.35 0.01
1.57 0.001 [11.5ꢀ]
2.05 0.10 [15.0ꢀ]
0.72 0.03 [7.47ꢀ]
0.64 0.01 [1.8ꢀ]
(CH2)2N
(CH2)2N
(CH2)2N
0.50 0.002 [4.02ꢀ]
0.84 0.01 [2.4ꢀ]
O
Taxol
—
—
0.003 0.0001
0.0007 0.0001
0.43 0.04 [330ꢀ]
0.08 0.01 [106.2ꢀ]
1.27 0.05 [980ꢀ]
0.50 0.12 [679.5ꢀ]
Vinblastine
a
Cell growth inhibition was measured by the XTT assay26 for leukemic cells and the SRB assay27 for solid tumor cells after 72-h incubation using a microplate spectro-
photometer as described previously.28 Similar in vitro results were obtained by using the Cell Counting Kit-8 for the CCK-8 assays as described by technical manual of Dojindo
Molecular Technologies, Inc. (Gaithersburg, MD; website: www.dojindo.com). IC50 values were determined from dose-effect relationship at six or seven concentrations of
each drug by using the CompuSyn software by Chou and Martin30 based on the median-effect principle and plot using the serial deletion analysis.31,32 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.
We investigated whether the newly synthesized compounds are
multidrug resistant to the distinct drugs, such as taxol or vinbla-
stin. The in vitro cytotoxicity of these derivatives against CCRF-
CEM/taxol and CCRF-CEM/VBL reveal that they generally have no
or little cross-resistance to these two natural products except com-
pounds 9ab0, 9ac0, 9af0, 9bb0, 9bc0, and 9bf0, which have certain ex-
tent of cross-resistance (Table 1). It also demonstrates that
compounds having an ether side-chain (compound 19a,b series)

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
475
Table 2
Cytotoxicity of new N-mustards against human solid tumors (H1299, CL1–0, CL1–5, PC3, HCT-116, MX-1 and MCF-7) cell growth in vitro
Compd
Cytotoxicity, IC50 (lM)
a
a
a
b
b
a
H1299a
CL1–0
CL1–5
PC3
HCT-116
MX-1
MCF-7
9aa0
10.37 2.67
ND
ND
6.22 1.91
ND
11.47 2.99
ND
12.70 2.99
ND
ND
14.59 2.01
ND
ND
8.67 1.02
ND
6.23 1.67
ND
16.2 4.32
ND
ND
6.08 1.01
ND
ND
4.64 1.43
ND
8.09 1.49
ND
9.79 2.45
ND
ND
1.88 0.73
ND
ND
1.70 0.67
ND
3.19 0.99
ND
5.25 1.77
ND
ND
0.69 0.03
0.85 0.01
0.47 0.001
0.31 0.002
1.21 0.072
0.81 0.072
0.39 0.002
4.54 0.02
0.36 0.01
0.91 0.02
2.74 0.001
0.64 0.01
0.27 0.01
0.27 0.001
0.29 0.001
0.70 0.0002
0.27 0.001
0.57 0.02
2.33 0.05
2.17 0.01
0.73 0.004
1.45 0.01
2.24 0.01
1.09 0.001
1.11 0.023
2.33 0.07
4.40 0.05
3.39 0.16
3.64 0.01
0.19 0.01
0.28 0.003
0.26 0.01
0.49 0.01
0.15 0.01
0.63 0.11
ND
ND
1.18 0.95
ND
3.09 1.01
ND
5.17 1.91
ND
ND
9ab0
9ac0
9ad0
9ae0
9af0
9ba0
9bb0
9bc0
9bd0
9be0
9bf0
ND
ND
ND
ND
ND
9.63 2.01
1.52 0.79
4.38 1.06
9.79 1.56
2.25 0.88
1.84 0.91
15.84 3.10
2.73 0.91
4.07 1.59
7.09 1.42
3.01 0.94
2.87 0.68
8.51 1.56
3.25 1.01
3.01 0.53
5.11 1.34
5.92 1.56
4.75 1.12
1.91 0.99
1.13 0.89
1.67 0.73
0.94 0.23
1.78 0.29
0.93 0.14
1.54 0.85
1.82 0.95
1.25 0.99
1.32 0.32
1.84 0.77
1.24 0.39
19ad0
19ba0
19bb0
19bc0
19bd0
a
Cell growth inhibition was determined by the Alamar blue assay29 in a 72 h incubation using a microplate spectrophotometer as described previously.
b
Cell growth inhibition was measured by the SRB assay27 for solid tumor cells after 72-h incubation using a microplate spectrophotometer as described previously.28
Figure 1. Therapeutic effect of 9aa0 in nude mice bearing human breast carcinoma MX-1 xenograft (30 mg/kg, Q2D ꢀ 5, iv injection, n = 5); Treatment started on Day 8 after
tumor implantation. All treatments were carried out via iv injection. CR is referred as complete tumor remission. (A) Average tumor size changes; (B) average body weight
changes.

476
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
have less MDR in comparison with the corresponding derivatives
bearing a carboxamide side-chain (compound 9a,b series).
compounds 19ad0 and 19bd0 are shown to have a broad spectrum
of antitumor activity against the tested tumor cell growth in
culture.
To further explore the antiproliferative activity of the new syn-
thesized N-mustards, the selected compounds were studied for
their cytotoxicity in inhibiting other human solid tumors such as
human non-small cell lung cancer (H1299), lung adenocarcinoma
(CL1–0 and CL1–5), colon cancer (HCT-116), prostate cancer
(PC3) and human breast tumor (MX-1 and MCF-7) cell growth
in vitro (Table 2). The results showed that these agents, in general,
are more cytotoxic toward human prostate, colon and breast tu-
mors among the tumor cell lines tested. It is of great interest to
note that the tested compounds have almost equal potency against
both MX-1 and resistant MCF-7 cell lines in vitro. Of these agents,
3.2. In vivo therapeutic effect
In the present study, we evaluated the antitumor activity of
compound 9aa0 since this agent shows to be the most cytotoxic
against solid tumor cell lines tested. Figure 1 shows the thera-
peutic efficacy of this agent in nude mice bearing human adeno-
carcinoma MX-1 xenograft. The complete tumor remission (CR)
was achieved at the maximum tolerable doses of 30 mg/kg
(Q2D ꢀ 5, intravenous injection), and maintained for over
Figure 2. Therapeutic effect of 9aa0 in nude mice bearing human prostate cancer PC3 xenograft (30 mg/kg (Q2D ꢀ 4), followed by 40 mg/kg (Q2D ꢀ 3), iv injection n = 4).
(A) Average tumor size changes; (B) average body weight changes. Note that for PC3 xenograft, tumor-growth led to body weight decreases in the control experiment.

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
477
70 days. The therapeutic effect of compound 9aa0 was further
3.3. DNA cross-linking study
investigated against human prostate carcinoma PC3 xenograft
in nude mice. As shown in Figure 2, more than 99% of tumor
suppression was observed at the dose of 30 mg/kg (Q2D ꢀ 4),
followed by 40 mg/kg (Q2D ꢀ 3). We have also evaluated the
antitumor activity of compound 9aa0 in nude mice bearing hu-
man colon cancer HCT-116 xenograft (Fig. 3). More than 95% tu-
mor suppression was observed at the dose of 30 mg/kg (Q2D ꢀ 5,
iv injection). In comparison, the antitumor effects of 9aa0
(30 mg/kg, Q2D ꢀ 6, iv injection n = 4, where n = number of mice
tested per experiment) with cyclophosphamide (80 mg/kg,
Q2D ꢀ 6, iv injection) in nude mice bearing human glioma U87
MG xenograft (Fig. 4) revealed that the former compound was
more potent, but less toxic than that of the latter drug. In all
in vivo xenograft experiments, body weight is referred to total
body weight minus tumor weight assuming 1 mm3 = 1 mg.
To realize whether the newly synthesized compounds are capa-
ble of cross-linking with DNA double strands, pEGFP-N1 DNA was
reacted with compounds, 9aa0, 9ad0, 19bd0, and 19bb0 at various
concentrations as indicated (1, 5, and 10 lM) and subjected to
alkaline agarose gel shifting assay after BamH1 digestion
(Fig. 5).24 Melphalan was used as the positive control. As shown
in Fig. 5, all the tested compounds were able to induce DNA inter-
strand cross-linking, suggesting that DNA cross-linking may be the
main mechanism of action for these agents.
3.4. Cell cycle inhibition
Previous studies demonstrated that DNA-interacting agents
damage DNA, inducing G2 arrest in the cell cycle through the G2
Figure 3. Therapeutic effects of 9aa0 in nude mice bearing HCT-116 xenograft (30 mg/kg (Q2D ꢀ 5, iv injection n = 4). (A) Average tumor size changes; (B) average body
weight changes. Note that for HCT-116 xenograft, tumor-growth led to body weight decreases in the control experiment.

478
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
DNA-damage checkpoint pathway.25 We studied the inhibitory ef-
fect of 9aa0 on cell cycle distribution (Table 3). The human non-
small lung carcinoma H1299 cells were treated with 9aa0 at the
Dawley rats. A single intravenous dose was administered via
an indwelling catheter in jugular vein to a group of two male
rats at a dose level of 1.0 mg/kg. The formulations were prepared
as a solution in 5.0% w/v DMSO with 10% w/v Cremophor in di-
distilled water. Serial blood samples were collected from jugular
vein catheter up to 24 h post-dose from all animals in Group.
Concentration levels of 9aa0 were determined in plasma using
a validated LC–MS/MS assay with a lower limit of quantification
(LLOQ) of 2.5 ng/mL. The plasma concentration-time data above
the LLOQ at each dose level were used in the calculation of phar-
macokinetic parameters of 9aa0 using the validated program
WinNonlin™, version 5.2.1. Pharmacokinetic parameters are
summarized in Table 4. The result showed that the mean
terminal half-life (t1/2) and mean residence time (MRT) of 9aa0
were 0.58 h and 0.11 h, respectively. The mean apparent plasma
concentrations of 2.5, 5, and 10 lM for 24 h. The cells were har-
vested, stained with propidium iodide (PI) and analyzed with a
flow cytometer. As shown in Table 3, 9aa0 treatment induced sig-
nificant G2/M arrest in H1299 cells. Similar G2/M arrest was previ-
ously observed in SW626 cells treated with melphalan.26
Furthermore, we also found that increased sub-G1 populations
were noticed in H1299 cells treated with 9aa0 (Table 3).
3.5. Pharmacokinetic profile of 9aa0
Prior to clinical studies, our leading compound 9aa0 were
subjected to pharmacokinetic studies in healthy male Sprague
Figure 4. Therapeutic effect of 9aa0 (30 mg/kg, Q2D ꢀ 6, iv injection n = 4) and cyclophosphamide (80 mg/kg, Q2D ꢀ 6, iv injection n = 4) in nude mice bearing human glioma
U87 MG xenograft. (A) Average tumor size changes; (B) average body weight changes.

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
479
xenograft and significant suppression against prostate adenocarci-
noma PC3 xenograft were achieved with acceptable toxicity. We
also demonstrate that the newly synthesized compounds are able
to induce DNA cross-linking by alkaline agarose gel shift assay.
Furthermore, the pharmacokinetic study reveals that 9aa0 has a
good pKa profile in rats with a half-life of 0.58 h. The present stud-
ies suggest that this agent is a promising candidate for preclinical
studies.
5. Experimental section
5.1. Chemistry: general methods
All commercial chemicals and solvents were reagent grade and
were used without further purification unless otherwise specified.
Melting points were determined on a Fargo melting point appara-
tus and are uncorrected. Column chromatography was carried out
on Silica Gel G60 (70–230 mesh, ASTM; Merck and 230–400 mesh,
Silicycle Inc.). Thin-layer chromatography was performed on Silica
Gel G60 F254 (Merck) with short-wavelength UV light for visualiza-
tion. All reported yields are isolated yields after chromatography or
crystallization. Elemental analyses were done on a Heraeus CHN-O
Rapid instrument. 1H NMR spectra were recorded on a 600 MHz,
Brucker AVANCE 600 DRX and 400 MHz, Brucker Top-Spin spec-
trometers in the indicated solvent. The chemical shifts were re-
ported in ppm (d) relative to TMS. High performance liquid
chromatography analyses for checking purity of synthesized com-
pounds were recorded on a Hitachi D-2000 Elite instrument: col-
Figure 5. Representative DNA cross-linking gel shift assay for 9aa0, 9ad0, 19bd0, and
19bb0 at various concentrations as indicated. Control lane shows single stranded
DNA (SL), while cross-linking (CL) shown in all tested lanes is DNA double-stranded
cross-linking. Melphalan (1 and 10 lM) was used as a positive control.
clearance of 9aa0 was 18.0 mL/min/kg with the apparent volume
of distribution at steady state of 0.15 L/kg.
4. Conclusion
In the present studies, we have designed and synthesized a ser-
ies of water-soluble DNA alkylating N-mustard derivatives, in
which the phenyl N-mustard pharmacophore is attached to a ben-
zene ring via a urea linker. We have demonstrated that these con-
jugates can be easily converted into water-soluble derivatives by
forming hydrochloride or mesylate salts. The newly synthesized
compounds exhibited significant antitumor activity both in vitro
and in vivo against various human tumor xenografts. Detailed
structure-activity relation studies revealed that the types of the
hydrophilic side-chain (carboxamide or ether) linked to the ben-
zene ring does not greatly affect their cytotoxicities. Moreover,
we showed that these derivatives have little or no cross-resistance
to either taxol or vinblastine. Among the newly synthesized deriv-
atives, we selected compound 9aa0 for further in vivo antitumor
evaluation since this agent shows to be the most cytotoxic against
solid tumor cell lines tested. Remarkably, complete tumor
remission in nude mice bearing human breast carcinoma MX-1
umn, Mightysil RP-18 GP 250-4.6 (5
5% B, and 5% C in 25 min (mobile phase A = acetonitrile, B = THF,
and C = H2O); flow rate, 1 mL/min; injected sample 10 L, column
lm); mobile phase, 90% A,
l
temp, 27 °C; wavelength, 254 nm. The purity of all compounds
was P95% based on analytical HPLC.
5.2. Synthesis of water-soluble N-mustards having a carbox-
amide linker (9aa0,bf0 series)
5.2.1. Ethyl 3-[3-(4-(bis(2-chloroethyl)amino)phenyl)ureido]-
benzoate (6a)
To a suspension of N,N-bis(2-chloroethyl)benzene-1,4-diamine
hydrochloride (5, 6.12 g, 20 mmol) in dry chloroform (100 mL)
Table 3
Cell cycle inhibition in human non-small cell lung adenocarcinoma H1299 by treating with compound 9aa0
Concentration (
lM)
0
2.5
5
10
Sub-G1
G1
S
14.1 0.7
42.9 0.8
22.5 0.3
20.5 0.9
20.0 4.9
2.3 1.0
1.8 2.6
55.9 3.6
19.7 4.5
10.7 3.9
8.8 1.4
26.0 4.7
6.5 3.2
5.2 4.6
62.3 1.5
G2/M
60.8 2.5
Table 4
Summary of pharmacokinetic parameters of 9aa0 following intravenous injection to rats (n = 2)
Dose (mg/kg)
1
C0 (ng/mL)
t1/2 (h)
MRT (h)
0.11
AUC(0–last) (h ng/mL)
925.81
AUC(0–1) (h ng/mL)
934.93
CLss (mL/min/kg)
18.00
Vss (L/kg)
11,980
0.58
0.15

480
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
was added TEA (3.5 mL) at ꢁ10 °C. A solution of ethyl 3-iso-
cyanatobenzoate (4a, 3.32 mL, 20 mmol) in dry chloroform was
added dropwise to the above mixture at the same temperature.
The reaction mixture was then allowed to stir at room temperature
for 12 h. The reaction mixture was evaporated reduced pressure.
The product was purified by column chromatography using
CH2Cl2/MeOH (100:2, v/v) as an eluent. The fraction containing
the main product were combined and evaporated to dryness. The
white solid residue was triturated with hexane, collected by filtra-
tion, and dried to give 6a, yield: 6.81 g (80%); mp 164–165 °C; 1H
NMR (DMSO-d6) d 1.32 (3H, t, J = 7.2 Hz, Me), 3.67–3.72 (8H, m,
4 ꢀ CH2), 4.31 (2H, q, J = 7.2 Hz, CH2), 6.71 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.28 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.40 (1H, t, J = 7.2 Hz,
ArH), 7.53 (1H, d, J = 8.0 Hz, ArH), 7.63–7.65 (1H, m, ArH), 8.12
(1H, s, ArH), 8.34 and 8.80 (each 1H, br s, exchangeable, 2 ꢀ NH).
Anal. Calcd for C20H23Cl2N3O3: C, H, N.
chromatographed over a silica gel column using CH2Cl2/MeOH
(100:9) as an eluent. The fractions containing the main product
were combined and evaporated in vacuo to dryness. The residue
was crystallized from ethyl acetate to give white solid. The solid
was suspended in ethyl acetate (50 mL) and treated with 2.5 M
HCl in ethyl acetate (5.0 mL) at 0 °C. The excess solvent was evap-
orated to dryness and solid separated was dried in vacuo to give
desired product 9aa0, 580 mg (58%); mp 156–157 °C; 1H NMR
(DMSO-d6) d 2.20 (6H, s, 2 ꢀ NMe), 2.42 (2H, t, J = 6.8 Hz, CH2),
3.32–3.35 (2H, m, CH2), 3.68–3.70 (8H, m, 4 ꢀ CH2), 6.71 (2H, d,
J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.34–7.39
(2H, m, 2 ꢀ ArH), 7.59 (1H, d, J = 7.6 Hz, ArH), 7.83 (1H, s, ArH),
8.29 (1H, t, J = 5.2 Hz, exchangeable, CONH), 8.40 and 8.72 (each
1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for C22H29Cl2N5O2: C,
H, N.
By following the same procedure as described for 9aa0, the fol-
lowing compounds were synthesized.
5.2.2. Ethyl 4-[3-(4-(bis(2-chloroethyl)amino)phenyl)ureido]-
benzoate (6b)
5.2.6. 1-[3-((3-(Dimethylamino)propyl)carbamoyl)phenyl]-3-[4-
(bis(2-chloroethyl)amino)-phenyl]urea (9ab0)
Compound 3b was synthesized by following the same proce-
dure as that for 3a and prepared from N,N-bis(2-chloroethyl)ben-
zene-1,4-diamine hydrochloride (5, 7.65 g, 25 mmol) and ethyl
4-isocyanatobenzoate (4b, 4.78 g, 25 mmol). Yield 8.11 g (76%);
mp 221–223 °C; 1H NMR (DMSO-d6) d 1.30 (3H, t, J = 7.2 Hz, Me),
3.70–3.71 (8H, m, 4 ꢀ CH2), 4.27 (2H, q, J = 7.2 Hz, CH2), 6.72 (2H,
d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.56 (2H,
d, J = 8.8 Hz, 2 ꢀ ArH), 7.86 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.46 and
8.96 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
Compound 9ab0 was prepared from 7a (0.792 g, 2 mmol), DCC
(0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and
N,N-dimethylpropane-1,3-diamine (8b0, 0.305 g, 3 mmol). Yield,
645 mg (65%); mp 138–140 °C; 1H NMR (DMSO-d6) d 1.61–1.66
(2H, m, CH2), 2.13 (6H, s, 2 ꢀ NMe), 2.25 (2H, t, J = 6.8 Hz, CH2),
3.27 (2H, q, J = 6.8 Hz, CH2), 3.66–3.72 (8H, m, 4 ꢀ CH2), 6.71 (2H,
d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.32–7.38
(2H, m, 2 ꢀ ArH), 7.58 (1H, d, J = 8.0 Hz, ArH), 7.84 (1H, s,
ArH), 8.40 and 8.72 (each 1H, br s, exchangeable, 2 ꢀ NH),
8.46 (1H, t, J = 5.2 Hz, exchangeable, CONH). Anal. Calcd for
C20H23Cl2N3O3: C, H, N.
5.2.3. 3-[3-(4-(Bis(2-chloroethyl)amino)phenyl)ureido]benzoic
acid (7a)
C23H31Cl2N5O2ꢂ2H2O: C, H, N.
A solution of 6a (4.253 g, 10 mmol) in mixture of concd HCl/
AcOH (100 mL, 3:2 v/v) was heated at 100 °C for 6 h. The reaction
mixture was cooled to room temperature, the separated white so-
lid was collected by filtration, the solid cake was washed with
water and dried to give 7a; yield: 3.52 g (89%); mp 191–192 °C;
1H NMR (DMSO-d6) d 3.70 (8H, s, 4 ꢀ CH2), 6.74 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.30 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.30 (1H, t, J = 8.0 Hz,
ArH), 7.51 (1H, d, J = 7.6 Hz, ArH), 7.63–7.65 (1H, m, ArH), 8.09
(1H, s, ArH), 8.09 and 8.82 (each 1H, br s, exchangeable, 2 ꢀ NH).
Anal. Calcd for C18H19Cl2N3O3ꢂ2H2O: C, H, N.
5.2.7. 1-[3-((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)phenyl]-3-[4-
(bis(2-chloroethyl)amino)-phenyl]urea hydrochloride (9ac0)
Compound 6ac0 was prepared from 7a (0.792 g, 2 mmol), DCC
(0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 1-
(2-aminoethyl)pyrolidine (8c0, 0.341 g, 3 mmol). Yield, 591 mg
(56%); mp 129–131 °C; 1H NMR (DMSO-d6) d 1.87–1.91 (5H, m,
CH), 3.0 (2H, t, J = 5.2 Hz, CH2), 3.32 (2H, d, J = 6.0 Hz, CH2), 3.62–
3.64 (3H, m, CH), 3.72 (8H, s, 4 ꢀ CH2), 6.73 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.30 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.34–7.36 (2H, m,
2 ꢀ ArH), 7.62 (1H, d, J = 9.2 Hz, ArH), 7.94 (1H, s, ArH), 8.78–8.79
(1H, m, exchangeable, CONH), 9.0 and 9.33 (each 1H, br s,
exchangeable, 2 ꢀ NH). Anal. Calcd for C24H31Cl2N5O2ꢂ2H2O: C,
H, N.
5.2.4. 4-[3-(4-(Bis(2-chloroethyl)amino)phenyl)ureido]benzoic
acid (7b)21
Compound 4b was synthesized by following the same proce-
dure as that for 7a and was prepared from 6b (2.13 g, 5 mmol) in
a mixture of conc. HCl/AcOH (70 mL, 3:2 v/v). Yield 1.78 g (90%);
mp 238–241 °C (reported >300 °C, decomp.);21 1H NMR (DMSO-
d6) d 3.69–3.73 (8H, m, 4 ꢀ CH2), 6.74 (2H, d, J = 8.8 Hz, 2 ꢀ ArH),
7.30 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.54 (2H, d, J = 8.8 Hz, 2 ꢀ ArH),
7.83 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.88 and 9.39 (each 1H, br s,
exchangeable, 2 ꢀ NH). Anal. Calcd for C18H19Cl2N3O3ꢂ2H2O: C,
H, N.
5.2.8. 1-[3-((2-(Piperidin-1-yl)ethyl)carbamoyl)phenyl]-3-(4-
(bis(2-chloroethyl)amino)-phenyl)urea hydrochloride (9ad0)
Compound 9ad0 was prepared from 7a (0.792 g, 2 mmol), DCC
(0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 1-
(2-aminoethyl)piperidine (8d0, 0.384 g). Yield, 683 mg (63%); mp
151–152 °C; 1H NMR (DMSO-d6) d 1.38–1.51 (7H, m, CH), 2.41–
2.45 (5H, m, CH), 3.34–3.38 (2H, m, CH), 3.66–3.72 (8H, m,
4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.32–7.38 (2H, m, 2 ꢀ ArH), 7.58 (1H, d, J = 7.6 Hz, ArH),
7.84 (1H, s, ArH), 8.28 (1H, t, J = 5.2 Hz, exchangeable, CONH),
8.40 and 8.70 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd
for C25H33Cl2N5O2: C, H, N.
5.2.5. 1-[3-((2-(Dimethylamino)ethyl)carbamoyl)phenyl]-3-[4-
(bis(2-chloroethyl)amino)-phenyl]urea hydrochloride (9aa0)
A mixture of 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT
(0.505 g, 3 mmol), TEA (0.4 mL) and N,N-dimethylethylenediamine
(8a0, 0.264 g, 3 mmol) in dry DMF (25 mL) was stirred at room tem-
perature for 72 h. The reaction mixture was filtered to remove the
by-product urea and the filtrate was evaporated in vacuo to dry-
ness. The residue was dissolved in CH2Cl2 (250 mL) and succes-
sively washed with water (100 mL), satd NaHCO3 aqueous
solution (100 mL) and brine (100 mL), dried over Na2SO4 and fil-
tered. The filtrate was evaporated to dryness and the residue was
5.2.9. 1-[3-((2-Morpholinoethyl)carbamoyl)phenyl]-3-[4-(bis(2-
chloroethyl)amino)phenyl]- urea hydrochloride (9ae0)
Compound 9ae0 was prepared from 7a (0.792 g, 2 mmol), DCC
(0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 1-
(2-aminoethyl)morpholine (8e0, 0.260 g, 3 mmol). Yield, 588 mg
(54%); mp 167–169 °C; 1H NMR (DMSO-d6) d 2.41–2.47 (5H, m,
CH), 3.36–3.38 (2H, m, CH), 3.57 (5H, br s, CH), 3.69 (8H, s,

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
481
4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.33–7.40 (2H, m, 2 ꢀ ArH), 7.59 (1H, d, J = 7.2 Hz, ArH),
7.91 (1H, s, ArH), 8.30 (1H, t, J = 5.6 Hz, exchangeable, CONH),
8.34 and 8.66 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd
for C24H31Cl2N5O3: C, H, N.
3.67–3.71 (8H, m, 4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29
(2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.49 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.75
(2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.30–8.31 (1H, m, exchangeable,
CONH), 8.35 and 8.66 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal.
Calcd for C25H33Cl2N5O2: C, H, N.
5.2.10. 1-[4-(Bis(2-chloroethyl)amino)phenyl]-3-[3-(4-(piperi-
din-1-yl)piperidine-1-carbonyl] phenyl)urea hydrochloride
(9af0)
5.2.15. 1-[4-((2-Morpholinoethyl)carbamoyl)phenyl]-3-[4-(bis-
(2-chloroethyl)amino)phenyl]- urea hydrochloride (9be0)
Compound 9be0 was prepared from 7b (0.594 g, 1.5 mmol), DCC
(0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and
8e0 (0.195 g, 2.25 mmol). Yield, 538 mg (61%); mp 234–235 °C; 1H
NMR (DMSO-d6) d 2.41–2.47 (6H, m, CH), 3.35–3.39 (2H, m, CH),
3.56–3.57 (4H, m, CH), 3.70–3.71 (8H, m, 4 ꢀ CH2), 6.71 (2H, d,
J = 8.8 Hz, 2 ꢀ ArH), 7.28 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.49 (2H, d,
J = 8.8 Hz, 2 ꢀ ArH), 7.75 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.20–8.23
(1H, m, exchangeable, CONH), 8.42 and 8.79 (each 1H, br s,
exchangeable, 2 ꢀ NH). Anal. Calcd for C24H31Cl2N5O3: C, H, N.
Compound 6af0 was prepared from 7a (0.792 g, 2 mmol), DCC
(0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 4-
piperidino-piperidine (8f0, 0.504 g, 3 mmol). Yield, 700 mg (60%);
mp 118–119 °C; 1H NMR (DMSO-d6) d 1.37–1.48 (8H, m, CH),
1.68–1.79 (2H, m, CH), 2.47–2.50 (3H, m, CH), 2.73 (1H, br s, CH),
2.98 (1H, br s, CH), 3.37 (4H, br s, CH), 3.66–3.72 (8H, m,
4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 6.90–6.92 (1H, d,
J = 7.2 Hz, ArH) 7.27 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29–7.32 (1H, m,
ArH), 7.41 (1H, d, J = 8.0 Hz, ArH), 7.51 (1H, s, ArH), 8.42 and 8.71
(each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
5.2.16. 1-[4-(Bis(2-chloroethyl)amino)phenyl]-3-[4-(4-(piperi-
din-1-yl)piperidine-1-carbonyl]phenyl)urea hydrochloride
(9bf0)
C28H37Cl2N5O2: C, H, N.
5.2.11. 1-[4-((2-(Dimethylamino)ethyl)carbamoyl)phenyl]-3-[4-
(bis(2-chloroethyl)amino)-phenyl]urea hydrochloride (9ba0)
Compound 9ba0 was prepared from 7b (0.396 g, 1 mmol), DCC
(0.309 g, 1.5 mmol), HOBT (0.202 g, 1.5 mmol), TEA (0.2 mL) and
8a0, (0.132 g, 1.5 mmol). Yield, 340 mg (72%); mp 189–190 °C; 1H
NMR (DMSO-d6) d 2.17 (6H, s, 2 ꢀ NMe), 2.38 (2H, br s, CH2),
3.28 (2H, br s, CH2), 3.69 (8H, br s, 4 ꢀ CH2), 6.71 (2H, d,
J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.49 (2H, d,
J = 8.8 Hz, 2 ꢀ ArH), 7.76 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.18, 8.47
and 8.83 (each 1H, br s, exchangeable, 3 ꢀ NH). Anal. Calcd for
Compound 9bf0 was prepared from 7b (0.594 g, 1.5 mmol), DCC
(0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and
8f0 (0.378 g, 2.25 mmol). Yield, 638 mg (73%); mp 222–224 °C; 1H
NMR (DMSO-d6) d 1.38–1.50 (9H, m, CH), 1.75 (2H, br s, CH),
2.51 (2H, br s, CH), 2.84 (2H, br s, CH), 3.32–3.40 (4H, m, CH),
3.68–3.72 (8H, m, 4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH),
7.27–7.31 (4H, m, 4 ꢀ ArH), 7.48 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.46
and 8.81 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
C
28H37Cl2N5O2ꢂ0.8H2O: C, H, N.
C22H29Cl2N5O2: C, H, N.
5.3. Alternative method for preparing 9aa0, 9ad0, and 9bd0
hydrochloride
5.2.12. 1-[4-((3-(Dimethylamino)propyl)carbamoyl)phenyl]-3-
[4-(bis(2-chloroethyl)amino)-phenyl]urea (9bb0)
5.3.1. N-(2-(Dimethylamino)ethyl)-3-nitrobenzamide (12aa0)
A solution of N,N-dimethylethylene diamine (8a0, 13.2 mL) in
dry THF was added to a solution of 3-nitrobenzoyl chloride (11a,
18.5 g, 100 mmol) in dry THF at 0 °C. After being stirred at room
temperature for 3 h, the solvent was evaporated to dryness. The
oily residue was dissolved in minimum amount of water and then
evaporated to dryness. The solid separated was recrystallized from
water and washed with hexane to give 12aa0, 21 g, (89%); mp 89–
90 °C; MS (ESI) m/z: 238 [M+H]+. 1H NMR (DMSO-d6) d 2.19 (6H, s,
2 ꢀ NMe), 2.43 (2H, t, J = 6.8 Hz, CH2), 3.40 (2H, q, J = 12.6 and
6.4 Hz, CH2), 7.78 (1H, t, J = 7.9 Hz, ArH), 8.29 (1H, d, J = 7.8 Hz,
ArH), 8.37–8.39 (1H, m, ArH), 8.68 (1H, s, ArH), 8.79–8.82 (1H, m,
exchangeable, NH).
Compound 9bb0 was prepared from 7b (0.594 g, 1.5 mmol), DCC
(0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and
8b0 (0.229 g, 2.25 mmol). Yield, 534 mg (69%); mp 203–205 °C; 1H
NMR (DMSO-d6) d 1.62–1.67 (2H, m, CH2), 2.13 (6H, s, 2 ꢀ NMe),
2.25 (2H, t, J = 6.8 Hz, CH2), 3.24 (2H, q, J = 6.8 Hz, CH2), 3.69–
3.72 (8H, m, 4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H,
d, J = 8.8 Hz, 2 ꢀ ArH), 7.49 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.75 (2H,
d, J = 8.8 Hz, 2 ꢀ ArH), 8.39 (1H, t, J = 5.2 Hz, exchangeable, CONH),
8.49 and 8.86 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
C23H31Cl2N5O2ꢂ2H2O: C, H, N.
5.2.13. 1-[4-((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)phenyl]-3-[4-
(bis(2-chloroethyl)amino) phenyl]urea hydrochloride (9bc0)
Compound 9bc0 was prepared from 7b (0.594 g, 1.5 mmol), DCC
(0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and
8c0 (0.256 g, 2.25 mmol). Yield, 419 mg (53%); mp 183–185 °C; 1H
NMR (DMSO-d6) d 1.05–2.00 (4H, m, CH), 3.02–3.04 (2H, m, CH),
3.30–3.33 (2H, m, CH), 3.42–3.45 (4H, m, CH), 3.70 (8H, s,
4 ꢀ CH2), 6.72 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.52 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.85 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 8.67–8.69 (1H, m, exchangeable, CONH), 9.01 and 9.48
(each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
By following the same procedure as described for 12aa0, the fol-
lowing compounds were synthesized.
5.3.2. 3-Nitro-N-(2-(piperidin-1-yl)ethyl)benzamide (12ad0)
Compound 12ad0 was synthesized from 1-(2-aminoethyl)piper-
idine (8d0, 3.31 g, 22.0 mmol) and 3-nitrobenzoyl chloride (11ad0,
4.0 g, 21.5 mmol). Yield, 5.5 g, (92%); mp 203–204 °C; MS (ESI) m/
z: 278 [M+H]+. 1H NMR (DMSO-d6) d 1.67–1.69 (6H, m, 3 ꢀ CH2),
2.91–2.92 (2H, m, CH2), 3.22–3.32 (2H, m, CH2), 3.50–3.70 (2H,
m, CH2), 3.72–3.74 (2H, m, CH2), 7.76–7.80 (1H, m, ArH), 8.37–
8.43 (2H, m, 2 ꢀ ArH), 8.72–8.73 (1H, m, ArH), 9.38 (1H, t,
J = 5.4 Hz, exchangeable, NH).
C24H31Cl2N5O2ꢂ2H2O: C, H, N.
5.2.14. 1-[4-((2-(Piperidin-1-yl)ethyl)carbamoyl)phenyl]-3-(4-
(bis(2-chloroethyl)amino)-phenyl)urea hydrochloride (9bd0)
Compound 9bd0 was prepared from 7b (0.594 g, 1.5 mmol), DCC
(0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and
8d0 (0.288 g, 2.25 mmol). Yield, 400 mg (49%); mp 198–200 °C; 1H
NMR (DMSO-d6) d 1.37–1.39 (1H, m, CH), 1.67–1.79 (5H, m, CH),
2.98 (2H, s, CH), 3.43–3.50 (4H, m, CH), 3.60–3.63 (2H, m, CH2),
5.3.3. 4-Nitro-N-(2-(piperidin-1-yl)ethyl)benzamide (12bd0)
Compound 12bd0 was synthesized from 2-(piperidin-1-yl)eth-
anamine (8d0, 4.23 g, 33 mmol) and 3-nitrobenzoyl chloride (11b,
5.56 g, 30 mmol). Yield, 7.2 g, (86%); mp 165–167 °C (lit.27 97–
99 °C). MS (ESI) m/z: 278 [M+H]+. 1H NMR (DMSO-d6) d 1.54–
1.57 (2H, m, CH), 1.81–1.82 (4H, m, CH), 3.09–3.22 (4H, m, CH),

482
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
3.24–3.27 (2H, m, CH), 3.72–3.76 (2H, m, CH), 8.21 (2H, d,
J = 8.7 Hz, 2 ꢀ ArH), 8.32 (2H, d, J = 8.7 Hz, 2 ꢀ ArH), 9.40 (1H, t,
J = 5.2 Hz, exchangeable, NH).
was washed with water, dried (Na2SO4), and evaporated under re-
duced pressure. The residue (800 mg, 1.55 mmol) was dissolved in
EtOH (200 mL). Methanesulfonic acid (149 mg, 155 mmol) was
also added to the solution. The mixture was stirred at room tem-
perature for 3 h and the solvent was removed under reduced pres-
sure. The solid residue was recrystallized from EtOH to yield 9aa0
mesylate, 645 mg 68%, mp 155–156 °C; 1H NMR (DMSO) d 2.36
(3H, s, CH3SO3), 2.85 (3H, s, NMe), 2.86 (3H, s, NMe), 3.27 (2H,
m, CH2), 3.60 (2H, m, CH2), 3.69 (8H, m, 4 ꢀ CH2) 6.71 (2H, d,
J = 9.0 Hz, 2 ꢀ ArH), 7.30 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 7.37 (1H, d,
J = 7.7 Hz, ArH), 7.43 (1H, d, J = 7.7 Hz, ArH), 7.56 (1H, d,
J = 7.7 Hz, ArH), 8.0 (1H, s, ArH), 8.49 (1H, s, NH), 8.62 (1H, br,
exchangeable, NH), 8.81 (1H, s, NH), 9.27 (1H, br, NH). Anal. Calcd
for C23H33N5O5Cl2Sꢂ0.25H2O: C, H, N, S.
5.3.4. 3-Amino-N-(2-(dimethylamino)ethyl)benzamide (13aa0)
10% Palladium on charcoal (1.5 g) was suspended in a solution
of 12aa0 (10.6 g, 4.5 mmol) in ethyl acetate. The mixture was
hydrogenated at 35 psi for 3 h. The reaction mixture was filtered
through a pad of Celite and the filtrate was evaporated in vacuo
to dryness to give 13aa0, 8.62 g (93%); mp 97–100 °C. 1H NMR
(DMSO-d6) d 2.16 (6H, s, 2 ꢀ NMe), 2.36 (2H, t, J = 6.8 Hz, CH2),
3.40 (2H, q, J = 13 and 6.5 Hz, CH2), 5.20 (1H, br s, exchangeable,
NH), 6.66 (1H, d, J = 7.8 Hz, ArH), 6.91 (1H, d, J = 7.7 Hz, ArH),
7.00 (1H, s, ArH), 7.05 (1H, t, J = 7.4 Hz, ArH), 8.05–8.08 (1H, m,
exchangeable, NH). The product pure enough and was used di-
rectly for the next reaction without further purification.
By following the same procedure as described for 9aa0 hydro-
chloride, the compounds 9ad0 hydrochloride and 9bd0 hydrochlo-
ride were synthesized.
By following the same procedure as described for 13aa0, the
compounds 13ad0 and 13bd0 were synthesized.
5.3.9. Compound 9ad0 hydrochloride
5.3.5. 3-Amino-N-(2-(piperidin-1-yl)ethyl)benzamide (13ad0)
Compound 13ad0 was synthesized from 12ad0 (3.0 g, 10 mmol)
and Pd/C (0.5 g). Yield was 2.0 g (75%). 1H NMR (DMSO-d6) d
1.67–1.82 (6H, m, 3 ꢀ CH2), 2.88–2.90 (2H, m, CH2), 3.15–3.30
(2H, m, CH2), 3.50–3.64 (2H, m, CH2), 3.66–3.69 (2H, m, CH2),
4.20 (2H, br s, exchangeable, NH2), 7.20 (1H, d, J = 7.8 Hz, ArH),
7.37 (1H, t, J = 7.8 Hz, ArH), 7.54 (1H, s, ArH), 7.62 (1H, d,
J = 7.8 Hz, ArH), 8.96 (1H, t, J = 5.4 Hz, exchangeable, NH). The
product pure enough and was used directly for the next reaction
without further purification.
Compound 9ad0 was prepared from 13ad0 (1.5 g, 6 mmol) and
N-mustard isocyanate 10 [freshly prepared from 5 (4.0 g, 13 mmol)
and triphosgene (2.58 g, 9.5 mmol)]. Yield, 1.4 g (46%); mp 98–99;
1H NMR (DMSO-d6) d 1.76–1.78 (6H, m, 3 ꢀ CH2), 2.90–2.91 (2H,
m, CH2), 3.18–3.20 (2H, m, CH2), 3.44–3.50 (2H, m, CH2), 3.67–
3.68 (2H, m, CH2), 3.69–3.71 (8H, m, 4 ꢀ CH2), 6.71 (2H, d,
J = 8.1 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.1 Hz, 2 ꢀ ArH), 7.34 (1H, t, J
=7.8 Hz, ArH), 7.46 (1H, d, J = 7.5 Hz, ArH), 7.60 (1H, d, J = 7.8 Hz,
ArH), 7.93 (1H, s, ArH), 8.76 (1H, br s, exchangeable, NH), 8.84,
9.15 (each 1H, s, exchangeable, 2 ꢀ NH). HRMS (FAB+) m/z calcd
for C25H33Cl2N5O2 [M+H]+: 506.4678; found: 506.2072.
5.3.6. 4-Amino-N-(2-(piperidin-1-yl)ethyl)benzamide (13bd0)
Compound 13ad0 was synthesized from 12bd0 (10.6 g,
4.5 mmol) and Pd/C (1.5 g). Yield was 8.62 g (93%); mp 109–
110 °C (lit.25 118–120 °C). 1H NMR (DMSO-d6) d 1.52 (2H, m, CH),
1.76 (4H, m, CH), 3.07 (6H, m, CH), 3.58–3.59 (2H, m, CH), 5.67
(2H, s, exchangeable, NH2), 6.54 (2H, d, J = 8.4 Hz, 2 ꢀ ArH), 7.64
(2H, d, J = 8.4 Hz, 2 ꢀ ArH), 8.43 (1H, s, exchangeable, NH). The
product pure enough and was used directly for the next reaction
without further purification.
5.3.10. Compound 9bd0 hydrochloride
Compound 9bd0 was prepared from 13bd0 (0.50 g, 2 mmol) and
N-mustard isocyanate 10 [freshly prepared from
5 (1.07 g,
3.5 mmol) and triphosgene (0.4 g, 1.36 mmol)]. Yield, 0.66 g
(64%); mp 127–128; 1H NMR (DMSO-d6) d 1.36–1.41 (1H, m, CH),
1.68–1.79 (5H, m, CH), 2.87–2.96 (2H, m, CH), 3.20–3.23 (2H, m,
CH), 3.51–3.54 (2H, m, CH), 3.63–3.66 (2H, m, CH), 3.67–3.72
(8H, m, 4 ꢀ CH2), 6.73 (2H, d, J = 8.9 Hz, 2 ꢀ ArH), 7.30 (2H, d,
J = 8.9 Hz, 2 ꢀ ArH), 7.53 (2H, d, J = 8.6 Hz, 2 ꢀ ArH), 7.85 (2H, d,
J = 8.6 Hz, 2 ꢀ ArH), 8.75 (1H, t, J = 5.3 Hz, exchangeable, CONH),
9.01 and 9.48 (each 1H, s, exchangeable, 2 ꢀ NH). HRMS (FAB+)
m/z calcd for C25H33Cl2N5O2 [M+H]+: 506.4678; found: 506.2076.
5.3.7. Compound 9aa0 hydrochloride
A solution of known 4-[N,N-bis(2-chloroethyl)-amino]phenyl-
isocyanate (10) [freshly prepared from N,N-bis(2-chloroethyl)-ben-
zene-1,4-diamine hydrochloride (5, 2.68 g, 8.75 mmol) by treating
with triphosgene (1 g, 3.4 mmol) in the presence of TEA (1.25 mL)
at ꢁ10 °C] in dry DMF was added dropwise to a solution of 13aa0
(1.22 g, 5 mmol) in dry DMF containing Et3N (1.5 mL) at room tem-
perature. After being stirred at room temperature for 8 h, the sol-
vent was evaporated to dryness in vacuo. The residue was
purified by column chromatography using CHCl3/MeOH (v/v
100:5) as the eluent. The fraction containing main products were
combined and evaporated to dryness to give water-soluble 9aa0
hydrochloride, 1.5 g (78%); mp 172–173 °C; 1H NMR (DMSO-d6) d
5.4. Synthesis of water-soluble N-mustards having an ether
linker (19aa0–19be0 series)
Detailed procedures for the synthesis of intermediates 15a,b,
17aa0–17be0, and 18aa0–18be0 along with their spectroscopic data
are provided in the Supplementary data.
5.4.1. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-
(dimethylamino)ethoxy)phenyl)urea (19aa0)
1H NMR (DMSO-d6)
d
2.82 (6H, s, 2 ꢀ NMe), 3.26 (2H, t,
Triethylamine (5 mL) was added dropwise to a solution of phen-
ylisocyanate 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and
triphosgene (1.24 g, 4.2 mmol) in dry CHCl3 (100 mL)] at ꢁ10 °C
and stirred for 30 min. A solution of 18aa0 (1.384 g, 6 mmol) in
CHCl3 (25 mL) containing TEA (3 mL) at ꢁ10 °C was added slowly
to the above solution. The reaction mixture was then allowed to
stir at room temperature for 16 h and the reaction mixture was
washed successively with water (350 mL) and brine (350 mL).
The organic layer was dried (Na2SO4) and filtered. The filtrate
was evaporated in vacuo to dryness and the residue was chromato-
graphed on a silica gel column using CH2Cl2/MeOH (100:6) as an
eluent. The fractions containing the main product were combined
and evaporated to dryness. The white solid residue was triturated
J = 5.8 Hz, CH2), 3.62–3.65 (2H, m, CH2), 3.66–3.71 (8H, m,
4 ꢀ CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.29 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 7.32–7.37 (1H, m, ArH), 7.47 (1H, d, J = 7.6 Hz, ArH),
7.61 (1H, d, J = 8.4 Hz, ArH), 7.95 (1H, s, ArH), 8.74 (1H, t,
J = 5.2 Hz, exchangeable, CONH), 8.95 and 9.26 (each 1H, br s,
exchangeable, 2 ꢀ NH). Anal. Calcd for C22H29Cl2N5O2ꢂHClꢂH2O: C,
H, N.
5.3.8. Compound 9aa0 mesylate
A solution of 9aa0 hydrochloride (900 mg) in H2O (500 mL) was
neutralized with saturated NaHCO3 aqueous solution to pH 7–8
and then extracted with CHCl3 (300 mL ꢀ 3). The organic layer

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
483
with hexane and then collected by filtration to give 19aa0, 0.710 g
(46%); mp 143–145 °C; 1H NMR (DMSO-d6) d 2.21 (6H, s, 2 ꢀ NMe),
2.61 (2H, s, CH2), 3.69 (8H, br s, 4 ꢀ CH2), 4.00 (2H, s, CH2), 6.51–
6.53 (1H, m, ArH), 6.70–6.72 (2H, m, 2 ꢀ ArH), 6.88–6.90 (1H, m,
ArH), 7.13–7.18 (2H, m, 2 ꢀ ArH), 7.26–7.28 (2H, m, 2 ꢀ ArH),
8.32 and 8.52 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd
for C21H28Cl2N4O2: C, H, N.
3.69 (8H, s, 4 ꢀ CH2), 4.30 (2H, t, J = 4.9 Hz, CH2), 6.72 (2H, d,
J = 8.3 Hz, 2 ꢀ ArH), 6.92 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.28 (2H, d,
J = 8.5 Hz, 2 ꢀ ArH), 7.38 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.84 and
9.01 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
C21H28Cl2N4O2ꢂHClꢂH2O: C, H, N.
5.4.7. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(3-(dimethyl-
amino)propoxy)phenyl)urea hydrochloride (19bb0)
By following the same procedure as described for 19aa0, the fol-
lowing compounds were prepared.
Compound 19bb0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18bb0 (1.384 g, 6 mmol). Yield, 1.70 g (58%); mp 178–180 °C; 1H
NMR (DMSO-d6) d 2.07–2.14 (2H, m, CH2), 2.78 (6H, s, 2 ꢀ NMe),
3.17–3.22 (2H, m, CH2), 3.65–3.72 (8H, m, 4 ꢀ CH2), 3.99 (2H, t,
J = 6.0 Hz, CH2), 6.69 (2H, d, J = 8.9 Hz, 2 ꢀ ArH), 6.86 (2H, d,
J = 9.0 Hz, 2 ꢀ ArH), 7.26 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 7.35 (2H, d,
J = 9.0 Hz, 2 ꢀ ArH), 8.62 and 8.64 (each 1H, br s, exchangeable,
2 ꢀ NH). Anal. Calcd for C22H30Cl2N4O2ꢂHClꢂH2O: C, H, N.
5.4.2. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(3-(dimethyl-
amino)propoxy)phenyl)urea (19ab0)
Compound 19ab0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18ab0 (1.384 g, 6 mmol). Yield, 1.247 g (43%); mp 127–128 °C; 1H
NMR (DMSO-d6) d 1.84–1.89 (2H, m, CH2), 2.21 (6H, s, 2 ꢀ NMe),
2.44 (2H, t, J = 7.2 Hz, CH2), 3.69–3.70 (8H, m, 4 ꢀ CH2), 3.95 (2H,
t, J = 7.2 Hz, CH2), 6.48–6.51 (1H, m, ArH), 6.70 (2H, d, J = 8.8 Hz,
2 ꢀ ArH), 6.86 (1H, d, J = 8.0 Hz, ArH), 7.13 (1H, t, J = 8.0 Hz, ArH),
7.20 (1H, s, ArH), 7.27 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.40 and 8.62
(each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
5.4.8. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-(pyrrolidin-
1-yl)ethoxy)phenyl)urea (19bc0)
Compound 19bc0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18bc0 (1.456 g, 6 mmol). Yield, 1.56 g (52%); mp 205–207 °C; 1H
NMR (DMSO-d6) d 1.87–1.90 (2H, m, CH2), 2.00 (2H, br s, CH2),
3.07–3.10 (2H, m, CH2), 3.53–3.58 (4H, m, 2 ꢀ CH2), 3.65–3.72
(8H, m, 4 ꢀ CH2), 4.28 (2H, t, J = 4.9 Hz, CH2), 6.69 (2H, d,
J = 8.9 Hz, 2 ꢀ ArH), 6.93 (2H, d, J = 8.9 Hz, 2 ꢀ ArH), 7.27 (2H, d,
J = 8.8 Hz, 2 ꢀ ArH), 7.38 (2H, d, J = 8.8 Hz, 2 ꢀ ArH), 8.70 and
8.87 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
C22H30Cl2N4O2ꢂ0.6H2O: C, H, N.
5.4.3. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-(pyrrolidin-
1-yl)ethoxy)phenyl)urea (19ac0)
Compound 19ac0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18ac0 (1.456 g, 6 mmol). Yield, 1.36 g (45%); mp 124–125 °C; 1H
NMR (DMSO-d6) d 1.68 (4H, s, 2 ꢀ CH2), 2.50 (4H, s, 2 ꢀ CH2),
2.77 (2H, s, CH2), 3.69 (8H, s, 4 ꢀ CH2), 4.01 (2H, s, CH2), 6.51
(1H, d, J = 7.2 Hz, ArH), 6.70 (2H, d, J = 8.0 Hz, 2 ꢀ ArH), 6.88 (1H,
d, J = 7.6 Hz, ArH), 7.13 (1H, t, J = 8.0 Hz, ArH), 7.18 (1H, s, ArH),
7.27 (2H, d, J = 8.0 Hz, 2 ꢀ ArH), 8.37 and 8.57 (each 1H, br s,
exchangeable, 2 ꢀ NH). Anal. Calcd for C23H30Cl2N4O2: C, H, N.
C23H30Cl2N4O2ꢂ1.1HCl: C, H, N.
5.4.9. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-(piperidin-
1-yl)ethoxy)phenyl)urea (19bd0)
Compound 19bd0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18bd0 (1.54 g, 6 mmol). Yield, 1.73 g (56%); mp 211–212 °C; 1H
NMR (DMSO-d6) d 1.37–1.39 (1H, m, CH), 1.67–1.70 (1H, m, CH),
1.79 (4H, br s, 2 ꢀ CH2), 2.98 (2H, br s, CH2), 3.43–3.50 (2H, m,
2 ꢀ CH2), 3.67–3.72 (8H, m, 4 ꢀ CH2), 4.34 (2H, t, J = 4.9 Hz, CH2),
6.69 (2H, d, J = 8.9 Hz, 2 ꢀ ArH), 6.92 (2H, d, J = 8.9 Hz, 2 ꢀ ArH),
7.27 (2H, d, J = 8.9 Hz, 2 ꢀ ArH), 7.38 (2H, d, J = 8.9 Hz, 2 ꢀ ArH),
8.72 and 8.89 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd
for C24H32Cl2N4O2ꢂHCl: C, H, N.
5.4.4. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-(piperidin-
1-yl)ethoxy)phenyl)urea (19ad0)
Compound 19ad0 was prepared 10 [freshly prepared from 5
(1.683 g, 5.4 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18ad0 (0.75 g, 3 mmol). Yield, 0.850 g (55%); mp 86–88 °C; 1H
NMR (DMSO-d6) d 1.37 (2H, s, CH2), 1.49 (4H, s, 2 ꢀ CH2), 2.42
(4H, s, 2 ꢀ CH2), 2.63 (2H, s, CH2), 3.69 (8H, s, 4 ꢀ CH2), 4.00 (2H,
s, CH2), 6.50–6.52 (1H, m, ArH), 6.69–6.71 (2H, m, 2 ꢀ ArH),
6.87–6.88 (1H, m, ArH), 7.12–7.18 (2H, m, 2 ꢀ ArH), 7.26–7.28
(2H, m, 2 ꢀ ArH), 8.35 and 8.54 (each 1H, br s, exchangeable,
2 ꢀ NH). Anal. Calcd for C24H32Cl2N4O2ꢂ5H2O: C, H, N.
5.4.10. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-morpholi-
noethoxy)phenyl)urea (19be0)
Compound 19be0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18be0 (1.55 g, 6 mmol). Yield, 1.33 g (43%); mp 210–212 °C; 1H
5.4.5. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-morpholi-
noethoxy)phenyl)urea (19ae0)
Compound 19ae0 was prepared 10 [freshly prepared from 5
(3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and
18ae0 (1.55 g, 6 mmol). Yield, 0.948 g (31%); mp 168–169 °C; 1H
NMR (DMSO-d6) d 3.20 (2H, br s, CH2), 3.47–3.52 (4H, m,
2 ꢀ CH2), 3.65–3.71 (8H, m, 4 ꢀ CH2), 3.78–3.84 (2H, m, CH2),
3.95–3.98 (2H, m, CH2), 4.36 (2H, t, J = 4.8 Hz, CH2), 6.69 (2H, d,
J = 9.0 Hz, 2 ꢀ ArH), 6.93 (2H, d, J = 8.9 Hz, 2 ꢀ ArH), 7.27 (2H, d,
J = 8.9 Hz, 2 ꢀ ArH), 7.38 (2H, d, J = 9.0 Hz, 2 ꢀ ArH), 8.67 and
8.84 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal. Calcd for
NMR (DMSO-d6)
d
2.45–2.48 (4H, m, 2 ꢀ CH2), 2.68 (2H, t,
J = 6.0 Hz, CH2), 3.58 (4H, t, J = 4.8 Hz, 2 ꢀ CH2), 3.66–3.72 (8H, m,
4 ꢀ CH2), 4.04 (2H, t, J = 6.0 Hz, CH2), 6.51–6.54 (1H, m, ArH),
6.70 (2H, d, J = 9.2 Hz, 2 ꢀ ArH), 6.87 (1H, d, J = 8.0 Hz, ArH), 7.13
(1H, t, J = 8.0 Hz, ArH), 7.19 (1H, s, ArH), 7.27 (2H, d, J = 9.2 Hz,
2 ꢀ ArH), 8.31 and 8.50 (each 1H, br s, exchangeable, 2 ꢀ NH). Anal.
Calcd for C23H30Cl2N4O3ꢂHCl: C, H, N.
C23H30Cl2N4O3ꢂHCl: C, H, N.
5.5. Cytotoxicity assays
The in vitro cytotoxicity of the newly synthesized N-mustard
derivatives 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 assay28 and human solid tumor cells
(i.e., breast carcinoma MX-1 and colon carcinoma HCT-116) by
the SRB assay29 in a 72 h incubation using a microplate spectro-
photometer as previously described.30 After the addition of phena-
5.4.6. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-(dimethyl-
amino)ethoxy)phenyl)urea hydrochloride (19ba0)
Compound 19ba0 was prepared 10 [freshly prepared from 5
(1.683 g, 5.4 mmol) and triphosgene (0.623 g, 4.2 mmol)] and
18ba0 (0.65 g, 3 mmol). Yield, 0.864 g (60%); mp 203–205 °C; 1H
NMR (DMSO-d6) d 2.83 (6H, s, 2 ꢀ NMe), 3.46–3.47 (2H, m, CH2),

484
N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
zine methosulfate–XTT solution, incubated at 37 °C for 6 h and
absorbance at 450 and 630 nm was detected on a microplate read-
er (EL 340). The cytotoxicity of the newly synthesized compounds
against non-small cell lung cancer H1299, human prostate cancer
PC3, human lung adenocarcinoma (CL 1–0 and CL 1–5) and human
breast adenocarcinoma MCF-7 were determined by the Alamar
blue assay31 in a 72 h incubation using a microplate spectropho-
tometer as previously described. After the addition of Alamar blue
solution, it was incubated at 37 °C for 6 h. Absorbance at 570 and
600 nm was detected on a microplate reader. IC50 values were
determined from dose-effect relationship at six or seven concen-
trations of each drug using the CompuSyn software by Chou and
Martin32 based on the median-effect principle and plot.33,34 Ranges
given for taxol, vinblastine and cisplatin were mean SE (n = 4).
5.9. In vivo pharmacokinetic study/drug administration and
sampling
All procedures of the present study were in accordance with the
IACUC guidelines. Prior approval of the Institutional Animal Ethics
Committee (IAEC) was obtained before initiation of the study. A
single intravenous dose of 9aa0 was administered via an indwelling
catheter in jugular vein to two male Sprague Dawley rats at a dose
level of 1.0 mg/kg. The formulation was prepared as a solution in
5.0% w/v DMSO with 10% w/v Cremophor in double distilled water.
Blood samples were collected at 0.083, 0.166, 0.333, 0.666, 1, 2, 4,
6, and 8 h after fasted from twelve hours prior to dosing. Serial
blood samples (ꢃ200
via the carotid artery cannulations. Samples were placed into tubes
containing 10 L of 350 IU/mL heparin solution. Plasma was har-
lL/each) were collected from all animals
l
5.6. In vivo studies
vested from the blood by centrifugation at 4000 rpm for 10 min
at 4 2 °C. All samples were stored at approximately ꢁ80 °C until
bioanalysis.
Athymic nude mice bearing the nu/nu gene were obtained from
NCI, Frederick, MD and used for all human tumor xenografts. Male
nude mice 6 weeks or older weighing 20–24 g or more were used.
Compound 9aa0 was administered via the tail vein for iv injection
as previously described.30 Tumor volume was assessed by measur-
ing length ꢀ width ꢀ height (or width) by using a caliper. Vehicle
5.10. Bioanalytical method
Concentration levels of 9aa0 in rat plasma samples were deter-
mined by partially validated LC–MS/MS method. The calibration
curve was linear from 2.5 to 300 ng/mL for 9aa0. The coefficient
of determination (r2) was greater than 0.999. The plasma samples
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. For tumor-
bearing nude mice during the course of the experiment, the
body-weight referred to total body weight minus the weight of
the tumor assuming 1 mm3 = 1 mg. All animal studies were con-
ducted in accordance with the guidelines for the National Institute
of Health Guide for the Care and Use of Animals and the protocol
approved by the Institutional Animal Care and Use Committee.
were quenched by adding 90
lL of acetonitrile (0.1% formic acid)
containing the internal standard (BO-1441, 100 ng/mL) to 30
lL
of sample. The plasma samples were vortex-mixed briefly at high
speed, kept on ice for 10 min, and then centrifuged at 12,000 rpm
for 5 min. Approximately 100
was transferred to an amber clean autosampler vial with insert
for analysis. A 5 L of the aliquot solution was subsequently in-
lL of the supernatant of each tube
l
jected into the LC–MS/MS system.
5.7. Alkaline agarose gel shift assay
Acknowledgements
Formation of DNA cross-linking was analyzed by alkaline aga-
rose gel electrophoresis. In brief, purified pEGFP-N1 plasmid DNA
We are grateful to the National Science council (Grant No. NSC-
99-2320-B-001-013) and Academia Sinica (Grant No. AS-96-TP-
B06) for financial support. T.-C.C. was supported by the Sloan-Ket-
tering Institute General Fund. The NMR spectra of synthesized
compounds were obtained at High-Field Biomolecular NMR Core
Facility supported by the National Research Program for Genomic
Medicine (Taiwan). We would like to thank Dr. Shu-Chuan Jao in
the Institute of Biological Chemistry (Academia Sinica) for provid-
ing the NMR service and the National Center for High-performance
computing for computer time and facilities.
(1500 ng) was mixed with various concentrations (1–20
lM) of
9aa0, 9ad0, 19bd0 and 19bb0 in 40
lL binding buffer (3 mM sodium
chloride/1 mM sodium phosphate, pH 7.4, and 1 mM EDTA). The
reaction mixture was incubated at 37 °C for 2 h. At the end of the
reaction, the plasmid DNA was linearized by digestion with BamHI
and followed by precipitation with ethanol. The DNA pellets were
dissolved and denatured in alkaline buffer (0.5 N NaOH–10 mM
EDTA). An aliquot of 20
lL of DNA solution(1 lg) was mixed with
4
lL of 6% alkaline loading dye and then electrophoretically re-
solved on a 0.8% alkaline agarose gel with NaOH–EDTA buffer at
4 °C. The electrophoresis was carried out at 18 V for 22 h. After
staining the gels with an ethidium bromide solution, the DNA
was then visualized under UV light.
Supplementary data
Supplementary data (detailed experimental procedures for the
synthesis of intermediate compounds 15a,b, 17ae0–17be0, and
18aa0–18be0 along with their spectroscopic data, and elemental
analysis data of all unknown compounds) associated with this arti-
cle can be found, in the online version, at doi:10.1016/j.bmc.
2010.11.005.
5.8. Cell cycle inhibition
The effects of 9aa0 on cell cycle distribution were analyzed with
a flow cytometer as previously described.35 Briefly, human non-
small cell lung carcinoma H1299 cells were treated with 9aa0at
2.5, 5, and 10 mM for 24 h. The attached cells were then trypsini-
zed, washed with phosphate buffer saline (PBS), and fixed with
ice-cold 70% ethanol for 30 min. The cells were stained with
References and notes
1. Gilbert, C. W.; McGowan, E. B.; Seery, G. B.; Black, K. S.; Pegram, M. D. J. Exp.
Ther. Oncol. 2003, 3, 27.
4 lg/ml propidium iodide (PI) in PBS containing 1% Triton X-100
and 0.1 mg/ml RNase A. The stained cells were then analyzed using
the FACS SCAN flow cytometer (Becton Dickinson, San Joes, CA,
USA). The percentage of the cells in each cell cycle phase was
determined using the ModFit LT 2.0 software based on the DNA
histograms.
2. Guillemard, V.; Saragovi, H. U. Cancer Res. 2001, 61, 694.
3. Ojima, I.; Geng, X.; Wu, X.; Qu, C.; Borella, C. P.; Xie, H.; Wilhelm, S. D.; Leece, B.
A.; Bartle, L. M.; Goldmacher, V. S.; Chari, R. V. J. Med. Chem. 2002, 45, 5620.
4. Safavy, A.; Bonner, J. A.; Waksal, H. W.; Buchsbaum, D. J.; Gillespie, G. Y.;
Khazaeli, M. B.; Arani, R.; Chen, D. T.; Carpenter, M.; Raisch, K. P. Bioconjugate
Chem. 2003, 14, 302.

N. Kapuriya et al. / Bioorg. Med. Chem. 19 (2011) 471–485
485
5. Kingsbury, W. D.; Boehm, J. C.; Jakas, D. R.; Holden, K. G.; Hecht, S. M.;
Gallagher, G.; Caranfa, M. J.; McCabe, F. L.; Faucette, L. F.; Johnson, R. K.;
Hertzberg, R. P. J. Med. Chem. 1991, 34, 98.
6. Sawada, S.; Okajima, S.; Aiyama, R.; Nokata, K.; Furuta, T.; Yokokura, T.; Sugino,
E.; Yamaguchi, K.; Miyasaka, T. Chem. Pharm. Bull. 1991, 39, 1446.
7. Kapuriya, N.; Kapuriya, K.; Zhang, X.; Chou, T.-C.; Kakadiya, R.; Wu, Y.-T.; Tsai,
T.-H.; Chen, Y.-T.; Lee, T.-C.; Shah, A.; Naliapara, Y.; Su, T.-L. Bioorg. Med. Chem.
2008, 16, 5413.
19. Tercel, M.; Wilson, W. R.; Denny, W. A. J. Med. Chem. 1993, 36, 2578.
20. Davidson, S. K.; Summers, J. B.; Albert, D. H.; Holms, J. H.; Heyman, H. R.;
Magcoc, T. J.; Conway, R. G.; Rhein, D. A.; Carter, G. W. J. Med. Chem. 1994, 37,
4423.
21. Benn, M. H.; Creighton, A. M.; Owen, L. N.; White, G. R. J. Chem. Soc. 1961, 2365.
22. 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.
23. Tietze, L. F.; Major’, F. Eur. J. Org. Chem. 2006, 2314.
8. Kapuriya, N.; Kapuriya, K.; Dong, H.; Zhang, X.; Chou, T.-C.; Chen, Y.-T.; Lee, T.-
C.; Lee, W.-C.; Tsai, T.-H.; Naliapara, Y.; Su, T.-L. Bioorg. Med. Chem. 2009, 17,
1264.
24. Hartley, J. A.; Berardini, M. D.; Souhami, R. L. Anal. Biochem. 1991, 193, 131.
25. Matthew, J.; O’Connell, M. J.; Walworth, N. C.; Carr, A. M. Trends Cell Biol. 2000,
10, 296.
9. Kakadiya, R.; Dong, H.; Kumar, A.; Dodiya, N.; Kapuriya, N.; Zhang, X.; Chou, T.-
C.; Lee, T.-C.; Shah, A.; Su, T.-L. Bioorg. Med. Chem. 2010, 18, 2285.
10. Denny, W. A.; Wilson, W. R. J. Pharm. Pharmacol. 1998, 50, 387.
11. Roth, J. A.; Cristiano, R. G. J. Natl. Cancer Inst. 1997, 89, 21.
12. Jordan, A. M.; Khan, T. H.; Malkin, H.; Helen, M. I.; Osborn, H. M. I. Bioorg. Med.
Chem. 2002, 10, 2625.
13. Knaggs, S.; Malkin, H.; Osborn, H. M. I.; Williams, N. A. O.; Yaqoob, P. Org.
Biomol. Chem. 2005, 3, 4002.
14. Jordan, A. M.; Khan, T. H.; Osborn, H. M. I.; Photiou, A.; Riley, P. A. Bioorg. Med.
Chem. 1999, 7, 1775.
15. Palmer, B. D.; Wilson, W. R.; Cliffe, S.; Denny, W. A. J. Med. Chem. 1992, 35,
3214.
16. Bodor, N.; Kaminski, J. J. J. Med. Chem. 1980, 23, 566.
17. Vinogradova, N. D.; Kuznetsov, S. G.; Chigareva, S. M. Khim.-Farm. Zh. 1980, 14,
41.
26. Erba, E.; Mascellani, E.; Pifferi, A.; D’Incalci, M. Int. J. Cancer 1995, 62, 170.
27. Thyrum, P.; Day, A. R. J. Med. Chem. 1965, 8, 107.
28. 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.
29. 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.
30. Chou, T.-C.; O’Connor, O. A.; Tong, W. P.; Guan, Y.-B.; Zhang, X.-G.; Stachel, S. J.;
Lee, S.; Danishefsky, S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8113.
31. Al-Nasiry, S.; Hanssens, M.; Luyten, C.; Pijnenborn, R. Hum. Reprod. 2007, 22,
1304.
32. Chou, T.-C.; Martin, N. CompuSyn Software; ComboSyn: Paramus, NJ, 2005.
33. Chou, T.-C.; Talalay, P. Adv. Enzyme Regul. 1984, 22, 27.
34. Chou, T.-C. Pharmacol. Rev. 2006, 58, 621.
35. Kakadiya, R.; Dong, H.; Lee, P.-C.; Kapuriya, N.; Zhang, X.; Chou, T.-C.; Lee, T.-C.;
Kapuriya, K.; Shah, A.; Su, T.-L. Bioorg. Med. Chem. 2009, 17, 5614.
18. Bogardus, J. B.; Higuchi, T. J. Pharm. Sci. 1982, 71, 153.