Synthesis and evaluation of unsymmetrical polyamine derivatives as antitumor agents

Article abstract of DOI:10.1016/j.bmc.2008.05.035

A series of unsymmetrically substituted polyamine derivatives were prepared and their cytotoxicities in mouse leukemia L1210, melanoma B16, and HeLa cells were investigated. The in vitro cytotoxicity revealed that these conjugates could recognize the poly

Full text of DOI:10.1016/j.bmc.2008.05.035

Bioorganic & Medicinal Chemistry 16 (2008) 7005–7012
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of unsymmetrical polyamine derivatives
as antitumor agents
Jianhong Wang a,b, Songqiang Xie a, Yanjie Li c, Yongjun Guo d, Yuanfang Ma c, Jin Zhao a, Otto Phanstiel IV e,
Chaojie Wang a,f,
*
a Institute of Natural Products & Medicinal Chemistry, Henan University, Henan, Kaifeng 475001, China
b Key Laboratory of Special Function Material, Henan, Kaifeng 475001, China
c Medical College, Henan University, Henan, Kaifeng 475001, China
d Lankenau Institute for Medical Research, Wynnewood, PA 19096, USA
e Chemistry Department, University of Central Florida, Orlando, FL 32816, USA
f College of Chemistry and Chemical Engineering, Henan University, Henan, Kaifeng 475001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of unsymmetrically substituted polyamine derivatives were prepared and their cytotoxicities in
mouse leukemia L1210, melanoma B16, and HeLa cells were investigated. The in vitro cytotoxicity revealed
that these conjugates could recognize the polyamine transporter, and the N-ethyl modified homospermi-
dine moiety may be another efficient carrier as homospermidine even though the introduction of terminal
alkyl groups led to reduced cytotoxicity in comparison with the un-substituted counterpart 1. The ornithine
decarboxylase and topoisomerase II inhibition experiments indicated that ODC and TOPO II were potential,
but not unique targets of these conjugates. Furthermore, the in vivo antitumor activities illustrated that the
representative conjugate 2f and the homospermidine analogue 1 evidently inhibited the tumor growth and
significantly increased the survival time of mice-bearing sarcoma 180 cells.
Received 12 March 2008
Revised 14 May 2008
Accepted 15 May 2008
Available online 20 May 2008
Keywords:
Unsymmetrical polyamine conjugate
Synthesis
Cytotoxicity
Antitumor
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
natural polyamine (putrescine, spermidine, and spermine) were
able to be ferried via PTS, but had limitations in terms of the size
During the past decades, the pathways for polyamine metabo-
lism have been gradually elucidated and this has provided new
avenues for research focusing on polyamine derivatives.1–5 It is
certainly well established that disturbance of cellular polyamine
uptake will be a potent cancer chemotherapeutic strategy in drug
development. In fact, highly proliferating cells (e.g., tumor cells)
have up-regulated polyamine transport systems (PTS) for the up-
take of exogenous polyamines.6,7 Tumor cells with up-regulated
PTS can accumulate polyamines more effectively than normal
cells,6–9 and the structural requirements of the PTS are not strin-
gent although the precise mechanism has not been completely de-
fined.3,9 Thus, polyamine–drug conjugates, which structurally
mimic the natural polyamines, will be readily ferried into cells
by the polyamine transporter. These findings provide an attractive
strategy using the polyamine backbone as a potential carrier for
drug delivery. Previously, the non-native triamine, homospermi-
dine, was reported to facilitate the uptake of relatively large N1-
aryl groups via the PTS.10–13 In addition, Delcros et al. described
that heterocyclic derivatives of various sizes once conjugated to a
of N1-aryl substituent.14 However, the bis-arylsubstituted diamine
derivatives reported by Burns were shown to behave in a mode
distinct from the polyamine analogues mentioned above.15
Previous work revealed that the N1-anthracenylhomospermi-
dine conjugate 1 (Fig. 1) exhibited a higher PTS recognition and
selectivity in Chinese hamster ovary (CHO) cells.10–12 Recent mech-
anistic studies indicated that 1 induced apoptosis in several cell
* Corresponding author. Tel.: +86 136 1981 0550.
E-mail address: wcjsxq@yahoo.com (C. Wang).
Figure 1. Chemical structures of compounds 1 and 2a–g.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.035

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J. Wang et al. / Bioorg. Med. Chem. 16 (2008) 7005–7012
lines such as B16 cells and IL-3-dependent FL5.12A pro-B cells,16,17
while its biological properties at whole animal level have been
lacking. These findings prompted us to design novel polyamine
skeletons as effective vectors.
In the course of conjugate generation, the anthracene, one DNA
intercalator, was attached to the triamine architectures with vari-
ous carbon chain lengths, and an alkyl group was introduced on the
other terminal-N atom, with the hope that simple structural mod-
ification would be identified for additional metabolism and might
exhibit better antitumor activity both in vitro and in vivo.18 The
anthracene was also chosen as the terminal substituent because
of its accepted toxic response on cellular entry, which facilitated
comparison of the potency of synthetic polyamine architec-
tures.10–12
In this report, we describe the synthesis of a series of unsym-
metrically substituted polyamine conjugates 2 (Fig. 1), the evalua-
tion of their capability for PTS utilization, and their in vitro
antitumor activity against several cancer cell lines. Furthermore,
our investigations extended to other potential targets that may ex-
plain the drug’s cytotoxicity such as ornithine decarboxylase (ODC)
and Topoisomerase inhibition. In order to comprehend the in vivo
anticancer activity of potent compounds, tumor growth inhibition
and prolongation of survival time were investigated on tumor-
bearing mice.
by flash column chromatography (chloroform/methanol 20:1),
and immediately deprotected using 48% HBr in acetic acid and
phenol in CH2Cl2 to give the hydrochloride salt 2a–g (according
to the previously reported procedures).19 The structures of target
compounds were confirmed by 1H NMR, MS and elemental analy-
sis, which were consistent with the proposed structures. The syn-
thetic route turned out to be convenient and inexpensive in
comparison with the amino alcohol strategy reported in the litera-
ture.10 Also the control compound 1 was prepared to compare the
cytotoxicity.
2.2. Cytotoxic activity
The in vitro cytotoxicities of the synthesized compounds were
evaluated against mouse leukemia L1210, melanoma B16, and
HeLa cells by MTT assays. Since many alkylpolyamine analogues
induce spermidine/sperm-N1-acetyltransferase (SSAT) and act as
a substrate for SSAT and polyamine oxidase (PAO), toxic oxidation
products produced in the metabolic pathway (e.g., aldehydes,
H2O2) would add to the cytotoxic effect of the analogue.20,21 Thus,
experiments were run in the presence of aminoguanidine to avoid
the disadvantages. In general, the unsymmetrically substituted
polyamine derivatives 2a–g exhibited significant to moderate cyto-
toxicity with IC50 values varying from 1.71–16.41 lM, but none of
the seven compounds showed higher potency than the reference
compound 1 (Table 1). However, inspection of the results revealed
that compound 2f displayed more potent inhibitory activity
in HeLa and L1210 cells than the other conjugates tested.
Interestingly, the homospermidine moiety containing an N-ethyl
group is an important fragment existing in bis-ethyl polyamine
derivatives.2,3,20
2. Results and discussion
2.1. Chemistry
The synthesis of the unsymmetrically substituted polyamine
derivatives involved the preparation of polyamine motifs and con-
jugation of these intermediates with 9-anthraldehyde. As shown in
Scheme 1, the ordinary alkyl amines reacted with mesitylenesulfo-
nyl chloride in a biphasic solution of CH2Cl2/NaOH gave the pro-
tected compounds, and then anion formation with NaH in DMF
and coupling with N-bromoalkylphthalimide in DMF generated
the phthalimide intermediate. Unmasking of the primary amino
functionality with hydrazine hydrate in refluxing ethanol afforded
the primary amines, 3a–d. Using the same procedures described
above provided the polyamine motif 4a–g.
The targeted polyamine derivatives were prepared using the
straightforward one-pot route shown in Scheme 1. The reductive
amination of 4a–g to 5a–g was achieved in two steps by condensa-
tion of the primary amine with one equivalent of aldehyde and re-
duced by treatment with excess NaBH4 in CH2Cl2/MeOH. Since
purification of 5 was important, the intermediate was separated
To investigate whether these conjugates could enter cells via
PTS, the growth inhibitory effect was determined in B16 cells co-
incubated with excess-free spermidine (SPD) or
a-difluoromethyl-
ornithine (DFMO). Cells grown for 48 h with 500
lM SPD or 5 mM
DFMO were exposed to various concentrations of the conjugates.
Also, aminoguanidine (1 mM) was added to prevent oxidation of
drugs by the enzyme present in the culture medium. As shown
in Table 1, IC50 values showed moderate increases (r1, ratio of
IC50 values) for assays run in the presence of SPD. Interestingly,
compounds 2b, 2f, and 2g had higher levels of SPD protection than
the control compound 1. SPD, as reported, provides competition
with polyamine conjugates for PTS utilization, and inhibits the up-
take of structural analogues via the PTS in tumor cells.22,23 The re-
sults illustrate that SPD protected the B16 cells from the conjugates
tested and thus it can be rationalized that these conjugates com-
Scheme 1. Preparation of unsymmetrically substituted polyamine conjugates. Reagents and conditions: (a) MtsCl (1.1 equiv), NaOH, CH2Cl2/H2O; (b) N-bromoalkylph-
thalimide, NaH, DMF; (c) H2NNH2ꢀH2O, C2H5OH, reflux, 12 h; (d) MtsCl (1.1 equiv), NaOH, CH2Cl2/H2O; (e) N-bromoalkylphthalimide, NaH, DMF; (f) H2NNH2ꢀH2O, C2H5OH,
reflux, 6 h; (g) 9-anthraldehyde, CH2Cl2/CH3OH; (h) NaBH4, CH2Cl2/CH3OH; (i) 48% HBr/HOAc, phenol, NaOH, CH2Cl2, rt; HCl, C2H5OH, 0 °C.

J. Wang et al. / Bioorg. Med. Chem. 16 (2008) 7005–7012
7007
Table 1
Cytotoxicity assay results for polyamine derivatives in HeLa, L1210, and B16 cells
Compound
HeLa IC50
,
l
M
L1210 IC50
,
lM
B16 IC50
,
l
M
B16+SPD IC50
,
l
M
ra1 IC50 ratio
B16+DFMO IC50
,
l
M
ra2 IC50 ratio
1
1.19 0.07
10.36 0.20
13.95 0.36
11.71 0.02
10.15 0.11
11.09 0.07
2.26 0.04
7.16 0.16
1.04 0.06
7.89 0.13
16.41 0.36
6.60 0.09
7.99 0.12
7.38 0.17
1.71 0.09
7.79 0.17
1.40 0.04
7.02 0.15
7.74 0.17
5.43 0.14
5.93 0.06
4.05 0.05
5.08 0.10
9.66 0.50
2.46 0.03
10.55 0.15
16.49 0.14
8.85 0.25
8.85 0.19
6.17 0.06
18.68 0.55
25.73 0.35
1.76
1.50
2.13
1.63
1.49
1.52
3.68
2.66
0.97 0.02
2.73 0.03
2.70 0.01
1.02 0.01
1.99 0.03
1.31 0.01
2.69 0.07
4.50 0.04
1.44
2.57
2.87
5.32
2.98
3.10
1.89
2.15
2a
2b
2c
2d
2e
2f
2g
IC50 values from three-independent experiments were given as means SD.
ra1, r2a represent the ratio of IC50 values, (B16+SPD/B16) and (B16/B16+DFMO), respectively.
petitively penetrate into cells via the PTS. DFMO, which depletes
intracellular polyamine level by inhibition of ornithine decarboxyl-
activity (about 67% inhibited compared to no treatment), while 1
reduced 31% of ODC activity, and DFMO, a direct ODC inhibitor,
ase (ODC), could increase uptake of exogenous polyamines. There-
fore, cells treated with DFMO should be more susceptible to
polyamine analogues and should provide lower IC50 values. As a re-
sult, exposure of B16 cells to the conjugates in the presence of
5 mM DFMO enhanced their growth inhibitory activity (e.g., r2 in
Table 1). In DFMO-treated cells, IC50 values for 2a–g were signifi-
cantly lower when compared to their respective counterparts.10
Interestingly, the synergistic effects of DFMO on these terminal al-
kyl-substituted derivatives were even stronger than that on the
control 1.10–12 However, their cytotoxicity was attenuated by the
introduction of terminal alkyl groups. This might be explained by
the different intracellular targets effected by these drug types.
With 2f in hand, we evaluated how this simple structural modifi-
cation of 1 influenced its biological behavior. In this regard, we
evaluated their ODC and topoisomerase II inhibition activity. In
short, N-ethylation had some interesting effects.
at 5 lM inhibited 90%. The results demonstrate the intricate influ-
ence of structural alterations on the pharmacological outcome.
2.4. Topoisomerase inhibitory activity
Topoisomerases are essential enzymes which regulate the
topology and supercoiling of DNA in the course of transcription
and replication. Topoisomerase I and II (TOPO I, TOPO II) can,
respectively, catalyze single- and double-strand breakage during
changes in the topological state of duplex DNA.22,26,27 Conse-
quently, these enzymes are important molecular targets of anti-
neoplastic drugs.
To test whether our drugs influenced TOPO II activity, a simple
electrophoretic assay28 was used for assessing TOPO II mediated
DNA relaxation activity. During electrophoresis, catenated kDNA
has limited migratory ability and remains near the top well due
to its high molecular weight, while its decatenated forms migrate
further down the gel. As shown in Figure 3, when compounds 1
2.3. Ornithine decarboxylase inhibitory activity
and 2f were carried out at concentrations of 5, 10, and 20 lM,
For example, it is known that bis-ethyl polyamine analogues
can inhibit the activity of ODC.24 To assess the effect of selected
conjugates on ODC activity, ODC 4293, (transformed human cells
in which the expression of the ODC gene is greatly induced by
estrogen), provided a negative control, while cells treated with
DFMO (which inhibits ODC activity) provided the positive con-
respectively, they all displayed strong TOPO II inhibitory activity.
However, the terminal ethyl substituted anthracene–polyamine
conjugate 2f exhibited greater inhibitory activity than 1. This dif-
ference is clearly observed at the concentration of 5 lM, wherein
compound 1 (lane 8) markedly prevented kDNA from being decat-
enated by TOPO II, while compound 2f completely inhibited the
decatenation reaction with most of the kDNA remaining in the
top well (lane 4). The TOPO I mediated DNA relaxation activity
was also evaluated with the same compounds, but they did not
show inhibitory activity at any of the applied concentrations (data
trol.25 The conjugates were tested at 5
lM and reduced ODC activ-
ity to different extents ranging from 33% to 56% compared with the
negative control (Fig. 2). Compound 2g exerted a higher inhibitory
Figure 3. Effects of 1 and 2f on the decatenation of kDNA by TOPO II, lane 1, TOPO
II; lane 5, 9, kDNA with 1.5 U of TOPO II (decatenated form); lanes 2–4, kDNA with
Figure 2. The effects of selected conjugates on ODC activity in ODC 4293 cells. Data
from three separate experiments were given as means SD.
1.5 U of TOPO II in the presence of 2f at 20, 10, and 5
kDNA with 1.5 U of TOPO II in the presence of 1 at 20, 10, and 5
l
M, respectively; lanes 6–8,
M, respectively.
l

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J. Wang et al. / Bioorg. Med. Chem. 16 (2008) 7005–7012
not shown). The results are consistent with the TOPO II inhibitory
capability of polyamine derivatives reported previously,22 and
indicate that the presence of the terminal alkyl group (e.g., ethyl)
can enhance TOPO II inhibitory activity.
2.5. In vivo antitumor activity against subcutaneously
transplanted sarcoma 180 cells
In vivo research has an advantage to discover drugs. Compound
1, as reported previously,10–12 had potent cytotoxicity against sev-
eral tumor cell lines and exhibited a higher PTS recognition. How-
ever, its in vivo antitumor activity has not been reported till now.
Therefore, compound 1 and 2f, the representative conjugate above
tested, were selected for a preliminary in vivo investigation. The
acute maximum tolerated dose (MTD) was firstly determined fol-
lowing administration of increasing drug doses to healthy female
Kunming mice. Therefore, 2f and 1 were found to have a MTD of
20 mg/kg and 10 mg/kg, respectively.
To evaluate the effects of 2f and 1 on tumor growth, Kunming
mice inoculated with sarcoma 180 cells were treated with these
analogues, and the tumor weights of mice were measured after 7
days of the administration. As shown in Table 2, preliminary re-
sults demonstrated promising tumor growth inhibition. For exam-
ple, at the end of treatment, 2f reduced tumor weight by 56.9%
(0.69 0.08 g), 44.4% (0.89 0.10 g), and 38.0% (0.99 0.10 g) at
the dose of 10, 5, and 2.5 mg/kg, respectively, while 5-fluorouracil
(5-Fu), the positive control, decreased tumor weight by 63.8%
(0.58 0.10 g) at the dose of 20 mg/kg, compared with the negative
control (1.60 0.13 g). Interestingly, compound 1, which exhibited
stronger toxicity than 2f both in vitro and in vivo, inhibited tumor
growth by equal degree at half-dose of 2f. In this regard, N-ethyla-
tion also resulted in the reduced cytotoxicity in vivo.
Figure 4. Typical morphological illustration (HE staining; 200ꢁ) of the S180 sarc-
oma. Sarcoma was treated without drug (control, A) or with 20 mg/kg 5-Fu (B),
5 mg/kg 2f (C), 5 mg/kg 1 (D), respectively.
The morphological change was also observed in the S180 sar-
coma (Fig. 4). In control group, tumor cells grew prosperously with
typical characteristics, such as nucleus anachromasis, chromato-
spherite hypertrophia, and muscle infiltration (Fig. 4A). After treat-
ment of 2f and 1, many focal necrosis (NA, see the black arrows,
Fig. 4C and D) were found in the sarcoma.
2.6. Survival time in sarcoma 180-bearing mice
The effects of 2f and 1 on survival time in sarcoma 180-bearing
mice were evaluated by measuring the increase of the lifespan.
Thus the mouse sarcoma 180 cells were transplanted into Kunming
mice by intraperitoneal injection (ip), and the administration of
test compounds was started 24 h after inoculation (day 1) and
the results are shown in Figure 5. The median survival time
(MST) in the control mice was 13.0 1.2 days, while it was in-
creased in a dose-dependent manner by treatment with 2f and 1.
Table 2
The effect of 2f and 1 on tumor growth in mice-bearing sarcoma 180 cellsa
Group
Dose (mg/kg)
Tumor weight (g)
Inhibitory rate (%)
MTDb (mg)
Control
5-Fu
1
—
1.60 0.13
0.58 0.10
1.03 0.11
0.82 0.09
0.76 0.06
0.99 0.10
0.89 0.10
0.69 0.08
—
—
—
10
20
1.25
2.5
5
2.5
5
63.8
35.6
48.8
52.5
38.0
44.4
56.9
Figure 5. The effect of 2f and 1 on the increase of the lifespan in mice-bearing
sarcoma 180 cells.
After the compound 2f was ip administrated at the doses of 2.5,
5, 10 mg/kg, the lifespan increased by 1.33 (17.25 1.4 days),
1.80 (23.38 1.9 days), 2.29 (29.75 1.8 days)-fold compared with
that of the control group, respectively, while compound 1 in-
creased the lifespan by 1.26 (16.38 1.1 days), 1.94 (25.25 1.4
days), 2.35 (30.5 1.9 days)-fold at the doses of 1.25, 2.5, 5 mg/
kg, and 2.64 (34.38 2.1 days)-fold of 5-Fu at the dose of 20 mg/
kg, respectively.
2f
20
10
a
Sarcoma 180 cells were implanted subcutaneously into Kunming mice and the
tumor weight was measured on the 7th day (ip) after the treatment of chemicals.
Data from three separate experiments were given as means SD.
b
Acute maximum tolerated dose (MTD) was defined as the dose just below the
lowest dose level that killed at least one female Kunming mouse in a treatment
group after a maximum of 15 days.

J. Wang et al. / Bioorg. Med. Chem. 16 (2008) 7005–7012
7009
4.1.1. N-(3-Aminopropyl)-N-ethyl-mesitylenesulfonamide (3a)
3. Conclusion
Colorless oil (90% yield), 1H NMR (400 MHz, CDCl3, d): 1.06 (t,
J = 7.1 Hz, 3H), 1.33 (m, 2H), 1.59–1.64 (m, 2H), 2.29 (s, 3H), 2.60
(s, 6H), 3.23–3.26 (m, 4H), 6.93 (s, 2H); ESI-MS m/z: 285.1
[M+1]+.
In summary, a series of unsymmetrically substituted polyamine
derivatives were prepared and evaluated for their antitumor activ-
ities both in vitro and in vivo. The present survey revealed that the
synthesized conjugates were able to recognize the PTS, and the N-
ethyl modified homospermidine moiety had the potential to be an
efficient carrier as homospermidine even though the introduction
of N-alkyl groups reduced the cytotoxicity in comparison with
the un-substituted counterparts. The ODC and TOPO II inhibition
experiments demonstrated that there is no direct relationship be-
tween ODC and TOPO II inhibition and cytotoxicity of these drugs
because compound 1 (the control) was the most cytotoxic in our
cell assays and yet the less potent inhibitor in these assays (vs
2f). This indicated that these enzymes are potential targets, but
not unique targets for the antitumor activity of this series. The in
vivo evaluation on mice inoculated with sarcoma 180 cells demon-
strated that 2f, as well as 1, significantly inhibited the tumor
growth and increased the survival time. However, it was important
to note that the introduction of N-alkyl groups also resulted in re-
duced toxicity in vivo, which might be of clinical benefit. For exam-
ple, the maximum tolerated dose in mice for 2f was found to be
twice over 1. Additional studies are required to determine the
mechanism by which these polyamine conjugates exert their cyto-
toxicity, which is an ongoing progress in our laboratory.
4.1.2. N-(4-Aminobutyl)-N-ethyl-mesitylenesulfonamide (3b)
Colorless oil (100% yield), 1H NMR (400 MHz, CDCl3, d): 1.06 (t,
J = 7.1 Hz, 3H), 1.32–1.36 (m, 4H), 1.51–1.55 (m, 2H), 2.29 (s, 3H),
2.60 (s, 6H), 3.16–3.25 (m, 4H), 6.93 (s, 2H); ESI-MS m/z: 299.1
[M+1]+.
4.1.3. N-(3-Aminopropyl)-N-ethyl-mesitylenesulfonamide (3c)
Colorless oil (85% yield), 1H NMR (400 MHz, CDCl3, d): 0.77 (t,
J = 7.3 Hz, 3H), 1.23–1.26 (m, 2H), 1.45–1.51 (m, 2H), 1.63–1.67
(m, 2H), 2.29 (s, 3H), 2.60 (s, 6H), 3.11 (t, J = 7.6 Hz, 2H), 3.26 (t,
J = 7.3 Hz, 2H), 6.93 (s, 2H); ESI-MS m/z: 299.1 [M+1]+.
4.1.4. N-(4-Aminobutyl)-N-propyl-mesitylenesulfonamide (3d)
Colorless oil (100% yield), 1H NMR (400 MHz, CDCl3, d): 0.78 (t,
J = 7.4 Hz, 3H), 1.31–1.36 (m, 4H), 1.47–1.56 (m, 4H), 2.29 (s, 3H),
2.60 (s, 6H), 3.11 (t, J = 7.6 Hz, 2H), 3.17 (t, J = 7.6 Hz, 2H), 6.95 (s,
2H); ESI-MS m/z: 313.1 [M+1]+.
4.2. General procedure for the preparation of N,N0-bis(mes-
itylenesulfonyl)-N-(w-aminoalkyl)-N0-alkyl-alkyldiamines (4a–g)
4. Experimental
Mesitylenesulfonyl chloride (3.6 g, 16.6 mmol), 3 (15.1 mmol),
N-bromoalkylphthalimide (15.6 mmol) and hydrazine hydrate
(55%, 3.46 g, 59.4 mmol) were reacted according to the procedure
for preparation of 3 to afforded 4a–g as thick oil.
The reagents used were of commercial origin and were em-
ployed without further purification. Solvents were purified and
dried by standard procedures. Purifications by column chromatog-
raphy were carried out over silica gel (230–400 mesh). 1H NMR
spectra were recorded on a Bruker AV-400 at 400 MHz. Mass spec-
tra were determined by ESI recorded on a Esquire 3000 LC–MS
mass instrument. Elemental analyses were performed by the Vario
Elemental CHSN-O microanalyzer.
4.2.1. N1,N4-Bis(mesitylenesulfonyl)-N1-(3-aminopropyl)-N4-
ethyl-1,4-butyl-diamine (4a)
1H NMR (400 MHz, CDCl3, d): 0.99 (t, J = 7.1 Hz, 3H), 1.39–1.40
(m, 4H), 1.64 (m, 2H), 1.80 (br s, 4H), 2.29 (s, 6H), 2.57 (s, 12H),
3.11–3.16 (m, 6H), 3.21 (t, J = 7.1 Hz, 2H), 6.93 (s, 4H); ESI-MS m/
z: 538.2 [M+1]+.
4.1. General procedure for the preparation of N-(w-
aminoalkyl)-N-alkyl-mes-itylenesulfonamides (3a–d)
4.2.2. N1,N3-Bis(mesitylenesulfonyl)-N1-(4-aminobutyl)-N3-
ethyl-1,3-propyl-diamine (4b)
A solution of mesitylenesulfonyl chloride (10 g, 45.7 mmol) in
100 mL CH2Cl2 was added to
1H NMR (400 MHz, CDCl3, d): 0.98 (t, J = 7.1 Hz, 3H), 1.26–1.31
(m, 4H), 1.34 (br s, 2H), 1.45 (m, 2H), 1.69–1.70 (m, 2H), 2.29 (s,
6H), 2.55 (s, 12H), 3.03–3.12 (m, 8H), 6.92 (s, 4H); ESI-MS m/z:
538.2 [M+1]+.
2 (41.7 mmol) in 2 N NaOH
(100 mL) with ice-bath cooling. The mixture was stirred for 18 h
at room temperature, the layers were separated, and the organic
layers were washed with 0.5 N HCl (100 mL) and 1:1 H2O/brine
(100 mL) and were concentrated in vacuo. Recrystallization from
aqueous EtOH gave the protected alkylamines.
Sodium hydride (60%, 0.99 g, 24.8 mmol) was added to the pro-
tected alkylamine (16.5 mmol) in DMF (30 mL) under ice-bath con-
ditions and was stirred for 30 min, then stirring at room
temperature for an additional 30 min. N-Bromoalkylphthalimide
(24.8 mmol) was added followed by heating at 45 °C for 4 h. The
reaction was quenched with EtOH (5 mL) and H2O (10 mL), and
solvents were removed in vacuo. The residue was dissolved in
CHCl3 and washed with H2O, dried with anhydrous sodium sulfate,
and then the solvent was removed in reduced pressure. Flash chro-
matography (20% EtOAc/petroleum ether) gave phthalimide
intermediates.
A solution of phthalimide intermediate (11.9 g, 27.0 mmol),
excess hydrazine hydrate in EtOH were heated at reflux for
12 h, and solvents were removed in vacuo. The residue was dis-
solved in CHCl3 and washed with H2O. The organic layer was
dried with anhydrous sodium sulfate and concentrated in vacuo.
Flash chromatography (10% CH3OH/CHCl3) afforded 3a–d as thick
oil.
4.2.3. N1,N3-Bis(mesitylenesulfonyl)-N1-(3-aminopropyl)-N3-
ethyl-1,3-propyl-diamine (4c)
1H NMR (400 MHz, CDCl3, d): 0.72 (t, J = 7.3 Hz, 3H), 1.47–1.49
(m, 2H), 1.53–1.59 (m, 6H, 4CH2+NH2), 1.69 (m, 2H), 2.29 (s, 6H),
2.55 (s, 12H), 2.96–3.06 (m, 6H), 3.19 (t, J = 7.0 Hz, 2H), 6.93 (s,
4H); ESI-MS m/z: 538.2 [M+1]+.
4.2.4. N1,N4-Bis(mesitylenesulfonyl)-N1-(3-aminopropyl)-N4-
propyl-1,4-butyl-diamine (4d)
1H NMR (400 MHz, CDCl3, d): 0.75 (t, J = 7.4 Hz, 3H), 1.34–1.45
(m, 10H, 8CH2+NH2), 1.58–1.62 (m, 2H), 2.30 (s, 6H), 2.62 (s,
12H), 3.01 (t, J = 7.6 Hz, 2H), 3.12 (m, 4H), 3.19 (t, J = 7.2 Hz, 2H),
6.94 (s, 4H); ESI-MS m/z: 552.2 [M+1]+.
4.2.5. N1,N3-Bis(mesitylenesulfonyl)-N1-(4-aminobutyl)-N3-
propyl-1,3-propyl-diamine (4e)
1H NMR (400 MHz, CDCl3, d): 0.72 (t, J = 7.4 Hz, 3H), 1.28–1.39
(m, 10H, 8CH2+NH2), 1.63–1.75 (m, 2H), 2.28 (s, 6H), 2.55 (s,
12H), 2.95–3.08 (m, 8H), 6.92 (s, 4H); ESI-MS m/z: 552.3 [M+1]+.

7010
J. Wang et al. / Bioorg. Med. Chem. 16 (2008) 7005–7012
4.2.6. N1,N4-Bis(mesitylenesulfonyl)-N1-(4-aminobutyl)-N4-
ethyl-1,4-butyldiamine (4f)
7.86 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H), 8.65 (s,
1H); ESI-MS m/z: 364.3 Anal. Calcd for
[M+1]+;
1H NMR (400 MHz, CDCl3, d): 0.96 (t, J = 7.2 Hz, 3H), 1.26–1.31
(m, 2H), 1.36–1.37 (m, 4H), 1.41–1.47 (m, 2H), 1.60 (m, 2H), 2.27
(s, 6H), 2.54 (s, 12H), 3.06–3.14 (m, 8H), 6.90 (s, 4H); ESI-MS m/
z: 552.2 [M+1]+.
C
24H36Cl3N3ꢀ0.2H2O: C, 60.49; H, 7.70; N, 8.82. Found: C,
60.27; H, 7.61; N, 8.59.
4.4.3. N1-(Anthracen-9-ylmethyl)-N3-3-(propylamino)propyl-
propane-1,3-diamine trihydrochloride salt (2c)
4.2.7. N1,N4-Bis(mesitylenesulfonyl)-N1-(4-aminobutyl)-N4-
propyl-1,4-butyl-diamine (4g)
Laurel-green powder (60.2% yield), 1H NMR (400 MHz, D2O, d):
0.88 (t, J = 7.4 Hz, 3H), 1.58–1.63 (m, 2H), 1.95–2.03 (m, 4H), 2.91–
3.05 (m, 8H), 3.15 (t, J = 8.0 Hz, 2H), 4.78 (s, 2H), 7.44 (t, J = 7.4 Hz,
2H), 7.54 (t, J = 7.5 Hz, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 7.9,
Hz 2H), 8.21 (s, 1H); ESI-MS m/z: 364.3 [M+1]+; Anal. Calcd for
1H NMR (400 MHz, CDCl3, d): 0.71 (t, J = 7.4 Hz, 3H), 1.26–1.35
(m, 2H), 1.36–1.38 (m, 6H), 1.40–1.45 (m, 2H), 1.59 (m, 2H), 2.27
(s, 6H), 2.56 (s, 12H), 2.98 (t, J = 7.6 Hz, 2H), 3.06–3.09 (m, 6H),
6.90 (s, 2H), 6.91 (s, 2H); ESI-MS m/z: 566.3 [M+1]+.
C
24H36Cl3N3ꢀ0.3H2O: C, 60.26; H, 7.71; N, 8.78. Found: C, 60.33;
H, 7.66; N, 8.62.
4.3. General procedure for the preparation of
N-bis(mesitylenesulfonyl)-N-(w-anthracen-9-ylmethyl-
aminoalkyl)-N-alkyl-alkyldiamines (5a–g)
4.4.4. N1-3-(Anthracen-9-ylmethylamino)propyl-N4-propyl-
butane-1,4-diamine trihydrochloride salt (2d)
Laurel-green powder (39.2% yield), 1H NMR (400 MHz, D2O, d):
0.92 (t, J = 7.4 Hz, 3H), 1.61–1.67 (m, 6H), 2.03–2.05 (m, 2H), 2.95–
3.01 (m, 4H), 3.03–3.11 (m, 6H), 4.73 (s, 2H), 7.46 (t, J = 7.4 Hz, 2H),
7.56 (t, J = 7.6 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.8 Hz,
2H), 8.23 (s, 1H); ESI-MS m/z: 378.2 [M+1]+; Anal. Calcd for
C25H38Cl3N3ꢀ1.5H2O: C, 58.42; H, 8.04; N, 8.18. Found: C, 58.34; H,
7.71; N, 7.82.
To a stirred solution of amine 4 (10 mmol) in 25% MeOH/CH2Cl2
(10 mL) was added a solution of 9-anthraldehyde (10 mmol) in 25%
MeOH/CH2Cl2 (10 mL) under N2. The mixture was stirred at room
temperature overnight until the imine formation was complete
(monitored by TLC). The solvent was evaporated under vacuum
to give the crude imine as a bright-green solid.
NaBH4 (30 mmol) was added in small portions to a solution of
imine 1:1 CH3OH/CH2Cl2 (20 mL) at 0 °C. The mixture was stirred
at room temperature overnight and then concentrated under vac-
uum. The residue was dissolved in CH2Cl2 and washed with aque-
ous NaOH (0.5 N, 50 mL). The organic layer was separated, dried
over anhydrous Na2SO4, filtered, and concentrated under vacuum
to afford 5. Since 5 was unstable, the intermediate was separated
by flash column chromatography (5% CH3OH/CHCl3) and immedi-
ately deprotected.
4.4.5. N1-(Anthracen-9-ylmethyl)-N4-3-(propylamino)propyl-
butane-1,4-diamine trihydrochloride salt (2e)
Laurel-green powder (40.5% yield), 1H NMR (400 MHz, D2O, d):
0.90 (t, J = 7.3 Hz, 3H), 1.59–1.62 (m, 2H), 1.95–2.02 (m, 6H), 2.93–
3.15 (m, 8H), 3.13–3.16 (m, 2H), 4.75 (s, 2H), 7.49 (t, J = 7.4 Hz, 2H),
7.60 (t, J = 7.4 Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 8.7 Hz,
2H), 8.20 (s, 1H); ESI-MS m/z: 378.3 [M+1]+; Anal. Calcd for
C25H38Cl3N3ꢀ1.4H2O: C, 58.63; H, 8.03; N, 8.20. Found: C, 58.92;
H, 7.90; N, 7.89.
4.4. General procedure for the preparation of the target
compounds (2a–g)
4.4.6. N1-(Anthracen-9-ylmethyl)-N4-4-(ethylamino)butyl-
butane-1,4-diamine trihydrochloride salt (2f)
Hydrogen bromide in HOAc (48%, 20 mL) was added to 5
(3 mmol) and phenol (10.5 g, 0.112 mol) in CH2Cl2 (30 mL) at
0 °C. The mixture was stirred overnight at room temperature and
was quenched with H2O (50 mL). The aqueous layer was separated
and washed with CH2Cl2 (50 mL), then the aqueous layer was con-
centrated in vacuo. The residue was treated with 1 N NaOH
(10 mL) and 19 N NaOH (5 mL) under ice-bath cooling and was ex-
tracted with CHCl3 (50 mL). After concentration of the CHCl3 ex-
tracts, the oil was dissolved in EtOH (5 mL) and acidified with
concentrated HCl (5 mL) in ice-bath cooling. The precipitate was
collected and washed with anhydrous EtOH to give 2 as laurel-
green powder.
Laurel-green powder (60.2% yield), 1H NMR (400 MHz, D2O, d):
1.24 (t, J = 7.3 Hz, 3H), 1.59–1.67 (m, 8H), 3.00–3.06 (m, 8H), 3.16
(t, J = 7.6 Hz, 2H), 5.15 (s, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.66 (t,
J = 7.6 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.18 (d, J = 8.8 Hz, 2H),
8.54 (s, 1H); ESI-MS m/z: 378.2 [M+1]+; Anal. Calcd for
C25H38Cl3N3ꢀ1.2H2O: C, 59.04; H, 8.01; N, 8.26. Found: C, 59.33;
H, 7.89; N, 8.31.
4.4.7. N1-(Anthracen-9-ylmethyl)-N4-4-(propylamino)butyl-
butane-1,4-diamine trihydrochloride salt (2g)
Laurel-green powder (41.5% yield), 1H NMR (400 MHz, D2O, d):
1.07 (t, J = 6.1 Hz, 3H), 1.77–1.85 (m, 10H), 3.09–3.16 (m, 8H), 3.35
(t, J = 6.3 Hz, 2H), 5.17 (s, 2H), 7.69 (t, J = 7.6 Hz, 2H), 7.79 (t,
J = 7.4 Hz, 2H), 8.18 (d, J = 8.2 Hz, 2H), 8.26 (d, J = 8.8 Hz, 2H),
8.65 (s, 1H); ESI-MS m/z: 392.4 [M+1]+; Anal. Calcd for
4.4.1. N1-3-(Anthracen-9-ylmethylamino)-propyl-N4-ethyl-
butane-1,4-diamine trihydrochloride salt (2a)
Laurel-green powder (36.0% yield), 1H NMR (400 MHz, D2O, d):
1.17 (t, J = 7.2 Hz, 3H), 1.64 (m, 4H), 1.99–2.03 (m, 2H), 2.86–3.01
(m, 8H), 3.22 (t, J = 7.9 Hz, 2H), 5.07 (s, 2H), 7.50 (t, J = 7.4 Hz,
2H), 7.61 (t, J = 7.5 Hz, 2H), 8.01 (d, J = 8.1 Hz, 2H), 8.10 (d,
J = 8.3 Hz, 2H), 8.50 (s, 1H); ESI-MS m/z: 364.3 [M+1]+; Anal. Calcd
for C24H36Cl3N3ꢀ0.8H2O: C, 59.15; H, 7.78; N, 8.62. Found: C, 59.40;
H, 7.67; N, 8.61.
C
26H40Cl3N3ꢀ1.4H2O: C, 58.63; H, 8.03; N, 8.20. Found: C, 58.94;
H, 7.94; N, 8.19.
4.5. Cell culture
The cells of L1210, HeLa and B16, obtained from American
Type Culture Collection (ATCC, Rockville, MD, USA), were cul-
tured in RPMI-1640 supplemented with 10% heat-inactivated
4.4.2. N1-(Anthracen-9-ylmethyl)-N4-3-(ethylamino)-propyl-
butane-1,4-diamine trihydrochloride salt (2b)
FCS and antibiotics (100 U/mL penicillin and 100 lg/mL strepto-
mycin sulfate) at 37 °C in an atmosphere of 95% air and 5% CO2
under humidified conditions. 1 mmol/L aminoguanidine was
added as an inhibitor of amine oxidase derived from FCS and
had no effect on the various parameters of the cells measured
in this study.
Laurel-green powder (39.5% yield), 1H NMR (400 MHz,
D2O, d): 1.29 (t, J = 7.3 Hz, 3H), 1.71–1.72 (m, 4H), 2.07–
2.10 (m, 2H), 3.05 (t, J = 6.8 Hz, 2H), 3.09–3.14 (m, 8H),
4.71 (s, 2H), 7.50 (t, J = 7.4 Hz, 2H), 7.60 (t, J = 7.4 Hz, 2H),

J. Wang et al. / Bioorg. Med. Chem. 16 (2008) 7005–7012
7011
4.6. IC50 determinations and methods for transport-related
studies
ity under a 12-h light–dark cycle. The effect of drug on tumor
growth was measured by evaluating tumor weight. For solid tumor
development, the Kunming mice (6–8 weeks olds, weighing 18–
20 g) were injected subcutaneously with 2 ꢁ 106 of sarcoma 180
cells. The next day after inoculation, mice randomized into eight
groups were administrated by intraperitoneal injection with 2f
(10, 5 and 2.5 mg/kg), 1 (5, 2.5 and 1.25 mg/kg), physiologic saline
or 5-Fu (20 mg/kg) for consecutive 7 days. On day 8, the mice were
killed by cervical dissociation, and solid tumors were removed and
weighed. The inhibitory rate was calculated as follows: inhibitory
rate (%) = [(A ꢂ B)/A] ꢁ 100, where A was the mean tumor weight
of the negative control group, and B was that of the drug treated
or positive group.
Chemosensitivity was assessed using the thiazolyl blue tetrazo-
lium bromide (MTT) assay. Briefly, 5000 exponentially growing
cells (HeLa cells or B16 cells) were seeded onto 96-well, flat-bot-
tomed plates and allowed to attach overnight; 4000 exponentially
growing L1210 cells were seeded onto 96-well, flat-bottomed
plates. The cells were treated with the increasing concentrations
of synthetic polyamine conjugates in the absence and the presence
of DFMO (5 mM) or SPD (500 lM) for 48 h, and 100 lL MTT (1 mg/
mL) was added to each well. After incubation at 37 °C for 4 h, the
MTT solution was removed and the crystals of the viable cells were
dissolved with DMSO (HeLa cells or B16 cells) or 10% SDS (L1210
cells). The absorbance of each well was read at 570 nm. The inhibi-
tion rate was calculated from plotted results using untreated cells
as 100%.
4.11. Enhancement of survival time in mice-bearing sarcoma
180 cells
For calculating the survival time, Kunming mice were inocu-
lated ip with 1 ꢁ 106 sarcoma 180 cells/mouse on day 0 and the
treatment with three doses of 2f (10, 5 and 2.5 mg/kg, ip) or 1 (5,
2.5 and 1.25 mg/kg, ip) was started 24 h after inoculation for 8 con-
secutive days. The control group was treated with physiologic sal-
ine. The median survival time (MST) for each group was observed
and the antitumor activity of drug was evaluated by measuring the
increase of the lifespan.
4.7. ODC assays
ODC activity was measured by the released 14CO2 from labeled
ornithine. ODC 4293 cells were cultured in a six-well plate at a
density of 1 ꢁ 105 per well in DMEM (Cellgro) supplied with 10%
(Invitrogen), Charcaol/Dextran-treated FBS (Hyclone), 200 ng/mL
G418 (Calbiochem), 200 ng/mL Zeocin, and 5
lM Ponasterone A
(Invitrogen) for 24 h. The conjugates tested were then added into
the cells at 5
l
M for another 24 h. Sample collection and the assay
Acknowledgments
procedure were carried out as described previously.25
This work was supported by National Science Foundation of
China (20472016) and Henan Natural Science Foundations
(512001300, 072102330028). We thank Dr. Susan Gilmour (Lanke-
nau Institute for Medical Research, USA) for the generous gift of
ODC 4293 cell line.
4.8. Topoisomerases assays28
The decatenation assays were performed in buffer A contain-
ing 50 mM Tris–HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2,
30
0.5 mM ATP. Reaction mixtures contained 0.25
kinetoplast DNA, 1.5 U of human topoiosomerase II, and the
appropriate polyamine conjugate (1, 2f) at 5 M, 10 M, 20 M,
l
g/mL bovine serum albumin, 0.5 mM dithiothreitol, and
lg of catenated
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