Novel oxa-spermine homologues: synthesis and cytotoxic properties.

Article abstract of DOI:10.1016/S0968-0896(01)00317-0

New oxa-spermine homologues 5-9 were synthesised and their anticancer properties were evaluated against a broad spectrum of cancer cells. All compounds, except 9 showed average GI(50) values in the range of 1.89-7.56 microM. SAR studies showed that the cytotoxic activity of these novel oxa-spermines depended on the length of the alkyl chain, the position of the oxa-amino functionality and also, on the type of sulphonamido group in the molecule. Although the mechanism of action of these compound remains to be elucidated, it would appear that direct drug-DNA interactions are not involved in the mode of action of these drugs.

Full text of DOI:10.1016/S0968-0896(01)00317-0

Bioorganic & Medicinal Chemistry 10 (2002) 691–697
Novel Oxa-Spermine Homologues: Synthesis and Cytotoxic
Properties
Vladimir A. Kuksa,a Valentin A. Pavlovb and Paul Kong Thoo Linc,*
aDepartment of Ophthalmology, School of Medicine, University of Washington, 1957 NE Pacific Avenue, Seattle,
WA 98195-6485, USA
bDepartment of Human and Animal Physiology, Faculty of Biology, University of Sofia ‘‘St. Kliment Ohridski’’,
Dr. Tzankov Blvd. 8, Sofia, 1164, Bulgaria
cThe Robert Gordon University, School of Life Sciences, St. Andrew Street, Aberdeen AB25 1HG, Scotland, UK
Received 27 June 2001; accepted 8 September 2001
Abstract—New oxa-spermine homologues 5–9 were synthesised and their anticancer properties were evaluated against a broad
spectrum of cancer cells. All compounds, except 9 showed average GI50 values in the range of 1.89–7.56 mM. SAR studies showed
that the cytotoxic activity of these novel oxa-spermines depended on the length of the alkyl chain, the position of the oxa-amino
functionality and also, on the type of sulphonamido group in the molecule. Although the mechanism of action of these compound
remains to be elucidated, it would appear that direct drug–DNA interactions are not involved in the mode of action of these drugs.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
spermine homologues and derivatives with aminooxy
functionality (i.e., oxa-polyamines) within the poly-
amine chain.9ꢀ11 We previously reported that oxa-poly-
amine derivatives affect polyamine metabolism in 3T3
Swiss cells.12,13 Furthermore, in a recent communica-
tion, we described the synthesis of a series of novel oxa-
spermidine homologues and derivatives which showed
good anticancer activity.14 We have since extended our
studies in the design and synthesis of oxa-spermine
analogues and homologues. In this paper we report the
synthesis and the in vitro cytotoxic properties of novel
oxa-spermine homologues (5–9) (Fig. 1) against a num-
ber of different panels of cancer cell lines.
Natural polyamines such as spermidine (1,8-diamino-4-
azaoctane), spermine (1,12-diamino-4,9-diazadodecane)
and putrescine (1,4-diaminobutane), play an important
role in cell growth and differentiation. The metabolism
of these polyamines is a promising target for cancer
chemotherapy and chemoprevention.1ꢀ3 During the last
20years, several types of polyamine analogues which
possess growth inhibitory activity and also are able to
affect the natural polyamine metabolism have been
synthesized and studied.3ꢀ8 We have for a number of
years been synthesizing novel type of spermidine and
Figure 1. Oxa-spermine homologues.
*Corresponding author. Tel.: 44-1224-262828; fax: +44-1224-262828; e-mail: p.kong@rgu.ac.uk
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00317-0

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V. A. Kuksa et al. / Bioorg. Med. Chem. 10 (2002) 691–697
Results and Discussion
OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3); renal
cancer (786-0, A498, ACHN, CAKI-1, RXF 393, TK-
10, UO-31); prostate cancer (PC-3, DU-145); breast
cancer (MCF-7, NCI/ADR-RES, MDA-MB-231/
ATCC, HS 578T, MDA-MB-435, MDA-N, BT-549).
Each compound was routinely tested at five 10-fold
dilutions with a highest concentration of 100 mM. The
anticancer potency of oxa-spermine homologues is
summarized in Table 1 and is expresssed as their GI50
(drug concentrations required to produce 50% growth
inhibition), TGI (drug concentrations required to pro-
duce total growth inhibition) and LC50 values (drug
concentrations that are lethal to the survival of 50%
cells). The selectivity in the anticancer activity (as GI50,
TGI and LC50 values) of all the compounds against
different cell lines in each panel is presented in Table 2.
Compounds 5–9 were synthesized according to the
methods described in our previous report10 and the
strategy adopted is depicted in Scheme 1. 2-Bis(ami-
nooxy)ethane and 1,3-bis(aminooxy)propane dihydro-
chlorides were prepared from the reaction between the
corresponding dibromoalkanes and N-hydroxy-
phthalimide followed by acid hydrolysis.15 Their sub-
sequent sulphonation with either mesitylenesulphonyl
chloride (Mts-Cl) or 2,2,5,7,8-pentamethyl-3,4-dihydro-
2H-chromen-6-sulphonylchloride (Pmc-Cl) in pyridine
gave the bis(sulphonylaminooxy) alkanes 1a–c in 60–
75% yields. N-Alkylation of 1a–c with the appropriate
o-bromoalkylphthalimide (2a,2b) gave the fully pro-
tected oxa-spermine homologues 4a–e with yields vary-
ing between 55 and 60%. The removal of the phthaloyl
groups from 4a–d was performed in hydrazine/ethanol.
The resulting diamines were converted directly to their
dihydrochloride salts to give compounds 5–8. For the
synthesis of oxa-polyamine with terminal aminooxy
groups such as in 9, bromopropyloxyphthalimide 3 was
first prepared from the reaction between 1,3-dibromopro-
pane and N-hydroxyphthalimide according to procedure
previously reported.16 Compound 3 underwent alkylation
with 1b to give a product which upon hydrazinolysis and
treatment with acid afforded 9.
The new oxa-spermine homologues showed good anti-
cancer activity against all the cell lines used. The anti-
cancer potency of these compounds generally depended
on the length of the alkyl chain and on the type of the
sulphonamido functionality (i.e., Mts or Pmc groups).
When the length of the alkyl chain between the two
oxygen atoms was increased from two (5) to three car-
bon atoms (6), the growth inhibitory activity, expressed
as mean GI50 values, showed a general increase (Table 1).
A significant decrease in the TGI and LC50 values of
compound 6 was also observed. When the sulphona-
mido group, Pmc is introduced instead of the Mts group
into the chain as shown in compound 8, this contributed
to a further increase in the growth inhibitory activity
(Table 1). Furthermore the extension in the length of
the terminal alkyl chain (7) also exhibited a modest
increase in anticancer activities (Table 1). However, the
introduction of two oxygen atoms next to the terminal
amino groups (9) resulted in a marked decrease in the
anticancer potency and was considered to be inactive by
the NCI criteria (GI50>100 mM) (data not shown). It is
interesting to mention that an increase in drug concentra-
tion from 10to 100 mM was accompanied by a decrease in
the cytotoxicity of compound 8 against most of the cell
lines tested. Similar paradoxical effect has recently been
reported for other type of compounds tested by the NCI17
and appears not to be related to any technical errors.
The growth inhibition properties of the new oxa-sper-
mine homologues 5, 6, 7, 8 and 9 against a wide range of
cancer cells, representing human leukaemia, lung, colon,
CNS, melanoma, ovarian, renal, prostate and breast
tumors were investigated at the National Cancer Insti-
tute (NCI) in the USA. The following cell lines were
used in the in vitro screen: leukaemia [CCRF-CEM,
HL-60(TB), K-562, MOLT-4, RPMI-8226, SR]; non-
small cell lung cancer (A549/ATCC, EKVX, HOP-62,
HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-
H460, NCI-H522); colon cancer (COLO 205, HCC-
2998, HCT-116, HCT-15, HT-29, KM-12, SW-620);
CNS cancer (SF-268, SF-295, SF-539, SNB-19, SNB-75,
U251); melanoma (LOXIMVI, MALME-3M, M14,
SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257,
UACC-62); ovarian cancer (IGROV1, OVCAR-3,
ꢁ
.
Scheme 1. Reagents and conditions: (i) R-Cl, pyridine, rt, 24 h; (ii) K2CO3, DMF, 85 C, 8 h; (iii) N2H4 H2O, EtOH, reflux overnight; (iv) satd HCl/
Et2O, Et2O/MeOH.

V. A. Kuksa et al. / Bioorg. Med. Chem. 10 (2002) 691–697
693
Table 1. Cytotoxicity of oxa-spermine homologues 5–8 against different panels of human cancer cell lines
Panel of cell linesa,b
Compound/Anticancer activity (mM)c,d,e
5
6
7
8
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
L (6)
NSCLC (9)
CC (7)
CNSC (6)
M (8)
OC (6)
RC (7)
PC (2)
BC (7)
Mean Valueg
2.73
5.19
7.73
5.8
5.32
4.52
16.5
13.5
6.8
84.3
40.7
49.5
43.2
55.5
21.7
65.7
ND
>100
>100
2.46
2.36
1.94
2.93
2.205.21
2.39
9.13
1.77
4.27
4.29
5.39
9.87
6.98
12.4
4.28
18.9
76.9
14.9
34.5
15.3
1.73
22.7
49.2
2.97
1.69
1.89
1.79
>100
2.99
23.1
>100
1.85
1.80
1.72
1.88
3.52
3.39
3.32
3.18
ND
3.21
19.5
NDf
ND
ND
ND
6.08
38.4
6.07
1.93
6.79
25.7
5.44
13.8
86.7
>100
85.6
22.9
>100
3.22
3.57
6.2
3.78
1.67
2.00
1.61
3.51
2.09
3.48
19.0
2.77
7.22
1.7
ND
37.2
ND
68.6
2.21
1.81
2.12
1.89
>100
>100
3.40
9.79
6.09
22.8
3.42
5.97
56.4
7.56
3.27
aL, leukemia; NSCLC, non-small cell lung cancer; CC, colon cancer; CNSC, CNS cancer; M, melanoma; OC, ovarian cancer; RC, renal cancer; PC,
prostate cancer; BC, breast cancer.
bThe number of cell lines routinely tested is shown in parentheses.
cData obtained from NCI’s in vitro tumour cells screen.
dData are mean values of the corresponding panel.
eThe value of each cell line (not shown) is an average of at least two determinations.
fNot determined.
gMean GI50 values over all cell lines tested.
Table 2. Cytotoxicity of oxa-spermine homologues against different human cancer cell lines
Cell linesa
Compound/Anticancer activity (mM)b,c
5
6
7
8
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
L
K-562
3.32
3.78
>100
>100
>100
>100
2.01
1.35
5.05
3.09
>100
ND
4.74
3.00
>100
>100
>100
>100
1.93
1.68
NDd
ND
ND
ND
MOLT-4
NSCLC
NCI-H226
NCI-H522
CC
9.74
2.39
62.5
4.91
>100
>100
1.46
1.16
3.16
2.42
6.82
5.08
1.81
1.76
4.10
3.27
ND
6.07
1.75
1.89
3.46
3.42
ND
ND
HCT-15
KM-12
CNSC
22.5
2.31
59.3
5.28
>100
>100
21.0
1.88
41.4
3.56
81.6
6.72
1.82
2.12
3.60
4.35
7.12
8.91
1.77
1.74
3.31
2.40
ND
ND
SF-268
SF-295
M
LOX IMVI
UACC-257
OC
2.08
22.7
5.93
83.6
>100
>100
1.21
10.9
2.49
26.0
5.12
1.83
1.83
3.42
3.41
6.37
6.33
1.61
1.98
3.18
ND
ND
ND
62.4
ND
2.97
2.92
>100
>100
>100
>100
2.03
1.73
4.04
3.23
1.74
1.75
3.19
3.27
5.83
6.10
1.62
2.02
3.03
4.21
ND
ND
6.04
IGROV-1
OVCAR-8
RC
1.75
18.9
5.67
79.2
>100
>100
2.05
2.70
4.00
4.22
7.80
8.98
1.73
1.78
3.22
3.34
5.99
6.22
1.71
1.81
ND
3.45
ND
ND
TK-103.74
UO-31
PC
35.4
27.7
>100
>100
3.32
16.9
14.5
30.6
40.4
55.3
1.65
2.16
3.07
4.83
5.73
12.3
1.71
1.95
3.29
3.97
ND
ND
76
PC-3
DU-145
BC
1.46
25.6
3.83
>100
>100
>100
1.88
1.68
3.41
3.39
6.19
5.99
1.76
1.46
3.15
2.38
5.64
5.23
1.7
1.91
ND
3.42
ND
ND
NCI/ADR-RES
HS 578T
24.6
2.67
51.3
30.3
>100
>100
21.7
1.08
44.0
2.53
89.3
5.92
14.7
2.13
27.9
4.32
52.8
8.72
3.59
1.88
15.8
3.70
ND
ND
aL, leukemia; NSCLC, non-small cell lung cancer; CC, colon cancer; CNSC, CNS cancer; M, melanoma; OC, ovarian cancer; RC, renal cancer; PC,
prostate cancer; BC, breast cancer.
bData obtained from NCI’s in vitro tumour cells screen.
cData are an average of at least two determinations.
dNot determined.

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V. A. Kuksa et al. / Bioorg. Med. Chem. 10 (2002) 691–697
Table 3. Drug-induced alterations in calf thymus DNA melting
behavior (ÁTm) at different Drug: DNA molar ratio
The data from the in vitro screening of oxa-spermines
(5–8) were processed by the NCI COMPARE analysis
which allowed a prediction of the biochemical mechan-
ism of novel drugs.20 The analysis showed no correla-
tion with the mode of action of any of the compounds
in the NCI Standard Agent Database (Pearson correla-
tion coefficients <0.6), suggesting the involvement of
yet unknown biochemical targets in the cytotoxicity of
our compounds.
Molar ratio (drug/DNAa)
Induced ÁTm (ꢁC )b
Compound 6
Spermine
9.2
28.3
17.0
1:200
1:100
1:5
2.4
a[DNA]=100 mM.
bÁTm=Tm (DNA+drug)ꢀTm (DNA).
The negative NCI COMPARE analysis of our com-
pounds, showing no correlation with drugs targeting
DNA was in accordance with the results from the ther-
mal denaturation experiments with calf thymus DNA,
since only small effects of compound 6 were observed at
high drug concentration.
Oxa-spermine homologues 5–8 were also shown to pos-
sess some degree of selectivity in their action against
different cell lines in each panel (Table 2). Although an
increase in the anticancer activity of oxa-spermine
homologues was observed with the elongation of either
the terminal or internal alkyl chain (Table 1), this was
accompanied by a decrease in their selectivity (Table 2).
For example, in the panel of renal tumors the GI50
values for compound 5 were 3.74 and 27.7 mM, for
compound 6, its GI50 were 3.32 and 16.9 mM and for
compound 7 its GI50 were 1.65 and 2.16 mM against
TK-10and UO-31 cell lines, respectively. It is interest-
ing to note that Pmc containing compound such as 8
exhibited low anticancer selectivity. It is worth men-
tioning that, with regard to their further development as
anticancer agents, oxa-spermine homologue 6 appears
to be the most interesting one since it demonstrated high
anticancer activity and at the same time, retained a
relatively high selectivity. When the cytotoxic properties
of the recently described oxa-spermidine homologues
and derivatives14 and the oxa-spermine homologues
presented here were compared, the latter group of
compounds showed a higher cytotoxicity profile.
Conclusion
We have designed and synthesized novel oxa-spermine
homologues (5–9). These compounds were tested by the
NCI in an in vitro preclinical screening program against
60human tumor cell lines. Oxa-spermine homologues
(5–8) showed good anticancer activity against all the cell
lines used with GI50 values in the low micro-molar
range (1.89–7.56 mM). The cytotoxic activity of these
compounds generally depended on the length of the
alkyl chain and on the type of the sulphonamido func-
tionality (i.e., Mts or Pmc groups). On the basis of
thermal denaturation studies and the negative NCI
COMPARE analysis, it appears that direct interactions
with DNA are not involved in the mechanism of action
of these compounds. The most promising compound 6
was selected for further in vivo testing by the NCI Bio-
logical Evaluation Committee. The precise mechanism
of action of the oxa-spermine derivatives is currently
under investigation in our laboratory. These compounds
appear to be good inducers of apoptosis in MCF-7
human breast cancer cells treated with compound 6
(unpublished). The optimization of these compounds
from the SAR point of view and the elucidation of the
mechanism of their action might lead to the develop-
ment of a novel type of anticancer drugs.
It is well known that spermidine and spermine interact
with DNA and this is at least partially responsible for
their multifunctional roles in biology.1 Furthermore in
vitro studies had shown that spermidine and spermine
cause strong stabilization of calf thymus DNA duplex.
We recently synthesized a series of bis-naphthalimido-
propyl spermidine and spermine derivatives possessing
anticancer properties18 and these compounds were
shown to strongly stabilize the thermal helix!coil
transition of calf thymus DNA duplex, when compared
with spermidine and spermine.19 The cytotoxic activity
of the bis-naphthalimidopropyl polyamines was due, at
least in part, to their effects on DNA.
Experimental
All reagents were purchased from Aldrich, Fluka and Lan-
caster and were used without purification. 2,2,5,7,8-Penta-
methyl-3,4-dihydro-2H-chroman-6-sulphonylchloride was
synthesized by previously reported method21 and can
also be purchased from Novabiochem. Melting points
(mp) were determined on Gallenkamp melting point
apparatus in open capillaries and are uncorrected. TLC
Considering the possibility DNA–drug interactions to
be part of the mechanism of action of the oxa-spermine
homologues, we investigated the influence of oxa-sper-
mine homologue 6 on the DNA double helix stability
using thermal denaturation experiments. In addition,
natural polyamine spermine was used as a control. The
results from these experiments are shown in Table 3. In
contrast with spermine, which exhibits strong stabiliza-
tion of DNA duplex against thermal denaturation with
all the concentrations used, only a weak stabilizing
effect of compound 6 was observed at 1:5 (drug/DNA)
molar ratio.
was performed on Kieselgel plates (Merck) 60F
in
254
chloroform/methanol (97:3 or 99:1). IR spectra were
recorded on a Nicolet 5ZDX FT-IR spectrophotometer
or a Perkin–Elmer 781 IR spectrophotometer. FAB-
mass spectra were obtained on a VG Analytical
AutoSpec (25Kv) spectrometer, EC/CI spectra were
performed on a Micromass Quatro II (low resolution)
or a VG Analytical ZAB-E instrument (accurate mass).

V. A. Kuksa et al. / Bioorg. Med. Chem. 10 (2002) 691–697
695
1H and 13C NMR spectra were recorded on a JEOL
JNM-EX90FT NMR spectrometer.
chloroform (50mL). The resulting solution was washed
with 2M HCl, saturated bicarbonate solution, water,
dried over Na2SO4 and filtered through silica gel. The
solvent was removed under vacuo followed by the
addition of Et2O. The mixture was allowed to stir for 20
min at room temperature, cooled to give white crystals.
The latter was filtered off and washed with Et2O.
1,2-Bis(aminooxy)ethane and 1,3-bis(aminooxy)propane
dihydrochlorides. There were synthesized according to
our method previously reported.15
1,12 - Diphthalimido - 4,9- di(2 - meitylenesulphonyl) - 4,9-
diaza-5,6-dioxadodecane 4a. This was obtained in 60%
as a white solid; mp 183–184 ꢁC (dec) (EtOH). 1H NMR
(CDCl3): d 7.70–7.93 (m, 8H), 7.01 (s, 4H), 3.83 (t, 4H),
3.55 (s, 4H), 3.35 (t, 4H), 2.66 (s, 12H), 2.37 (s, 6H),
1.79–2.46 (m, 4H). 13C NMR (CDCl3): d 168.0(C ¼O),
143.7, 141.9, 133.9, 132.0, 129.6, 132.1 (arom C), 73.5,
47.5, 35.7, 25.6, 23.1, 21.0. IR cmꢀ1 (Nujol): 3100, 1710
(C¼O), 1600, 1325, 1165 (SO2). HRMS (FAB): calcd
for C42H46N4O10S2 830.27, [MH]+ 831.2734, found:
831.2747 [MH]+.
Bis[(sulphonyl)aminooxy]alkanes 1a and 1b. General
procedure.
Bis(aminooxy)alkane
dihydrochloride
(0.0302 M) was dissolved in pyridine (120 cm3). Aryl
sulphonylchloride (0.0757 mol) was added to the solu-
tion and the mixture was left stirring overnight at room
temperature. All the solvent was removed under vacuo
to give a precipitate. This was dissolved in chloroform
(200 cm3) and transferred in a separating funnel (500
cm3). The organic phase was washed with (i) dilute
hydrochloric acid (1 M) (ii) water (iii) saturated solution
of sodium bicarbonate (iv) water. The chloroform layer
was dried with sodium sulphate and filtered. Removal
of the solvent gave a solid which was recrystallised from
toluene.
1,13-Diphthalimido-4,10-di(2-meitylenesulphonyl)-4,10-
diaza-5,9-dioxatridecane 4b. This was obtained in 82%
as a white solid; 1H NMR (CDCl3): d 7.57–7.81 (m, 8H,
arom CH, Phth), 6.92 (s, 4H, arom H of Mts group),
3.79 (t, 4H, 2ꢂCH2O), 3.14–3.71 (m, 8H, 4ꢂCH2N),
2.60(s, 12H, 4 ꢂCH3), 2.26 (s, 6H, 2ꢂCH3), 2.02 (m,
4H, 2ꢂCH2), 1.47 (m, 2H, CH2). 13C NMR (CDCl3): d
168.0(C ¼O), 143.9, 141.9, 133.9, 132.0, 129.6, 123.2
(arom C), 73.0(CH 2–O), 47.5, 35.8, 27.2, 26.0, 23.1,
1,2-Bis[N,N0-(2-mesitylsulphonyl)aminooxy]ethane
1a.
This was obtained in 91% yield. Mp 116–117 ꢁC (dec,
toluene) 1H NMR (CDCl3): d 7.32 (s, 2H, NH), 7.04 (s,
4H), 7.32 (s, 2H, NH), 4.00 (s, 4H), 2.65 (s, 12H), 2.30
(s, 6H). ). 13C NMR (CDCl3): d 143.6, 140.8, 132.1, 130.8
(arom C), 73.8 (CH2O), 23.0 , 21.0 (CH). IR cmꢀ1 (Nujol):
21.0(CH ). IR cmꢀ1 (Nujol): 3100, 1710 (C¼O), 1600,
3
3
3250 , 30 60 , 2820 , 160 0 , 1350 , 1160 . LRMS (FAB): calcd1325, 1165 (SO2). HRMS (FAB): calcd for
for C20H28N2O6S2 456.14, found: 457 [MH]+.
C43H48N4O10S2 844.28, [MH]+ 845.2890, found:
845.2929 [MH]+.
1,3-Bis[N,N0-(2-mesitylsulphonyl)aminooxy]propane 1b.
This was obtained in 79% yield. Mp 157–158 ꢁC
1,19-Diphthalimido-7,13-di(2-meitylenesulphonyl)-7,13-
diaza-8,12-dioxatnanodecane 4c. This was obtained in
1
(EtOH) H NMR (CDCl3): d 7.49 (s, 2H, NH), 7.24 (s,
1
4H), 4.01 (s, 4H), 2.84 (s, 12H), 2.55 (s, 6H), 1.99 (m,
2H). ). 13C NMR (CDCl3): d 143.6, 140.7, 132.1, 130.8
(aromatic C), 73.5, 26.8, 23.0, 21.0. IR cmꢀ1
(Nujol):=3200, 1330,1170. LRMS (FAB): calcd for
C21H30N2O6S2 470.15, found: 471 [MH]+.
83% as a white solid; H NMR (CDCl3): d 7.59–7.84
(m, 8H, arom CH, Phth), 6.87 (s, 4H, arom H of Mts
group), 3.56 (t, 4H, 2ꢂCH2O), 2.90–3.28 (m, 8H,
4ꢂCH2N), 2.51 (s, 12H, 4ꢂCH3), 2.33 (s, 6H, 2ꢂCH3),
1.00–1.72 (m, 20H, 10ꢂCH2). 13C NMR (CDCl3): d
168.2 (C¼O), 142.1, 140.0, 133.8, 133.4, 132.1, 131.8,
123.0(arom C), 45.3, 45.1, 37.8, 28.3, 27.0, 26.2, 26.1,
24.7, 22.7, 20.8 (CH2, CH3). IR cmꢀ1 (Nujol): 3100,
1710(C ¼O), 1600, 1325, 1165 (SO2). LRMS (FAB):
calcd for C49H60N4O10S2 928.38, found: 929 [MH]+.
1,3-Bis[N,N0-(2,2,5,7,8-pentamethyl-3,4-dihydro-2H-chro-
man-6-sulphonylchloride)amino-oxy]propane 1c. This
was obtained in 79% yield. 1H NMR (CDCl3): d 7.35 (s,
2H, NH), 3.92 (t, 4H), 2.61 (s, 12H), 2.22 (s, 6H), 1.63–
2.00 (m, 6H), 1.42 (s, 12H), 13C NMR (CDCl3): d 155.5,
138.1, 125.7, 124.8, 118.6 (arom C), 74.2, 73.5, 32.5,
26.8, 21.3, 18.5, 17.5, 12.1. IR cmꢀ1 (Nujol): 3200,
1550,1330,1155. LRMS (FAB): calcd for C31H46N2O8S2
638.82, found: 639 [MH]+.
1,13-Diphthalimido-4,10-di(2,2,5,7,8-pentamethyl-3,4-di-
hydro-2H-chromen-6-sulphonyl)-4,10-diaza-5,9-dioxatri-
decane 4d. This was obtained in 95% as a white solid.
1H NMR (CDCl3): d 7.75–7.92 (m, 8H), 3.82 (t, 4H),
3.61 (t, 4H), 3.36 (t, 4H), 2.73 (t, 4H), 2.61 (s, 12H), 2.19
(s, 6H), 1.84–2.08 (m, 10H), 1.4 (s, 12H). 13C NMR
(CDCl3): d 167.9 (C¼O), 155.7, 139.2, 139.1, 133.8,
132.0, 124.8, 124.7, 123.1, 118.7. (arom C), 74.3, 72.9,
47.5, 35.8, 32.5, 27.3, 26.6, 25.9, 21.3, 18.5, 17.5, 17.5,
12.1.(CH2 and CH3 groups). IR cmꢀ1 (Nujol): 3000,
1710(C ¼O), 1600, 1325, 1155 (SO2). LRMS (FAB):
calcd for C53H64N4O12S2 1013.22, found: 1013 [M]+.
6-Bromohexylphthalimide 2b and 3-bromopropyloxyph-
thalimide 3. There were synthesised according to the
method reported previously.15
ꢀ,! - Di[phthalimidoalkyl] - N,N0 - di(arylsulphonyl) -
ꢀ,!bis-bis-(aminooxy)alkanes 4a–c. General N-alkyla-
tion reaction. A mixture of arylsulphoamide (3.13
mmol), o-Bromoalkyl(oxy)phthalimide (6.4 mmol),
anhydrous K2CO3 were dissolved in anhydrous DMF (8
mL) was allowed to stir at rt for 24 h. The solvent was
removed under vacuo and the residue dissolved in
1,12-Diphthalimidooxy-4,9-di(2,2,5,7,8-pentamethy3,4-
dihydro-2H-chromen-6-sulphonyl)-4,9-diaza-5,6-dioxado-
decane 4e. This was obtained in 90% as a white solid;

696
V. A. Kuksa et al. / Bioorg. Med. Chem. 10 (2002) 691–697
1H NMR (CDCl3): d 7.85 (m, 8H, arom CH, Phth), 4.38
(t, 4H, 2ꢂCH2O), 3.51–3.58 (m, 8H, 4ꢂCH2N), 2.74 (t,
8H), 2.62 (s, 12H), 2.39 (m, 4H), 2.20(s, 6H), 1.84–2.00(m,
6H), 1.40(s, 12H). 13C NMR (CDCl3): d 163.0(C ¼O),
155.7, 139.2, 139.1, 134.4, 128.9, 124.9, 124.9, 124.8, 123.4,
118.7 (arom C), 75.9, 74.3, 72.6, 46.7, 32.6, 26.0, 21.4, 18.6,
17.5, 12.2 (CH2 and CH3). IR cmꢀ1 (Nujol): 3100, 1730
(C¼O), 1600, 1340, 1155 (SO2). LRMS (FAB): calcd for
C53H64N4O14S2 1045.22, found: 1045 [M]+.
dihydrochloride 8. This was obtained as a white solid in
1
60% yield; H NMR (DMSO-d6): d 8.42 (broad s, 6H,
2ꢂNH+3 ), 2.68–3.25 (m, 16H), 2.49 (s, 12H), 2.03 (s,
6H), 1.73–1.95 (m, 10H), 1.33 (s,12H). 13C NMR
(DNSO-d6): d 155.4, 139.0, 138.7, 124.8, 124.4, 119.3
(arom C), 74.6, 72.8, 42.6, 32.1, 32.0, 26.6, 25.5, 21.1,
18.6, 17.5, 12.2 (CH2 and CH3). IR cmꢀ1 (Nujol): 3400
(N–H), 2700, 2555, 1325,1155 (SO2). LRMS (FAB):
calcd for C37H62N4O8S2Cl2 825.92, found: 753
[Mꢀ2HCl]+.
Hydrazinolysis (removal of the phthaloyl group). Com-
pounds 4a–e (1.94 mmol) were dissolved in ethanol (30
.
mL) and N2H4 H2O (4.0mmol) was added dropwise.
4,9-Bis(2,2,5,7,8-pentamethy-3,4-dihydro-2H-chromen-6-
sulphonyl)-5,9-dioxa-4,10-diazotetradecane 9. This was
obtained in 72% yield as a white solid; 1H NMR
(CDCl3): d 6.22 (broad s, 4H), 4.14 (t, 4H), 3.24 (m,
12H), 2.49 (s, 12H), 2.12 (s, 6H), 1.87 (m, 10H), 1.34 (s,
12H). 13C NMR (CDCl3): d 155.1, 138.6, 124.4,
124.0,118.9 (arom C), 74.3, 72.4, 45.9, 34.0, 31.7, 26.2,
25.0, 20.7, 18.2, 17.2, 11.9 (CH2 and CH3). IR cmꢀ11
(Nujol): 3400, 2800, 1325, 1155 (SO2). LRMS (FAB):
calcd for C37H62N4O10S2Cl2 825.92, found: 753
[Mꢀ2HCl]+.
The mixture was left refluxing overnight whereby a
white precipitate was formed. The white solid was fil-
tered off and washed with ethanol. The filtrate was
reduced to dryness and the residue resuspended in
chloroform. The solution with a white turbidity was fil-
tered again and the filtrate was reduced to dryness to
afford the free diamine compound as a yellow oil in
quantitative yield. To form the corresponding dihy-
drochloride salt the diamine was dissolved in ether/
methanol (5:1, 10mL) followed by the addition of a
saturated solution of HCl/ether (3 mL) with cooling.
After filtration and drying, the dihydrochloride salt was
obtained as a white solid.
Anticancer studies
The in vitro anticancer screening tests were carried out
at the National Cancer Institute. Details of the assay
procedures have been reported previously.22 A 48 h
drug exposure protocol was followed and a sulforhoda-
mine B assay was used to estimate cell viability and cell
growth.
1,12-Diamino-4,9-bis(2-meitylenesulphonyl)-4,9-diaza-
5,8-dioxadodecane dihydrochloride 5. This was obtained
1
as a white solid in 56%; H NMR (DMSO-d6): d 8.32
(broad s, 6H, 2ꢂNH+3 ), 7.17 (s, 4H), 4.26 (s, 4H), 2.78–
3.35 (m, 8H), 2.57 (s, 12H), 2.36 (s, 6H), 1.52–2.16 (m,
4H). 13C NMR (DMSO-d6): d 143.9, 141.1, 131.9, 129.1
(arom C), 73.0, 46.8, 37.3, 24.2, 22.5, 20.5, (CH2 and
CH3). IR cmꢀ1 (Nujol):=3400 (N–H), 1950 (NH+3 ),
1600, 1340,1165 (SO2). HRMS (FAB): calcd for
C26H44N4O6S2Cl2 643.25, ([Mꢀ2HCl]+ 571.2624),
found: 571.2615 [Mꢀ2HCl]+.
Thermal denaturation studies
Optical thermal denaturation experiments were
performed in stoppered quartz cuvettes using
a
Perkin-Elmer Lambda 2 UV–vis spectrophotometer
fitted with a Peltier circulating heating/cooling tem-
perature programmer and water supply, according to
the published procedure.23 Calf thymus DNA (Sigma)
working solutions (100 mM) were prepared by dilu-
tion of a DNA stock solution in SHE buffer. The
DNA–drug solutions were prepared by the addition
of the drug (5% DMSO/water) to give the desired
final concentration. The binding affinity (i.e., Tm) was
measured by determining the change in midpoint of
the thermal denaturation curves of DNA in 0.01 M
SHE buffer (pH 7.0). Drug-induced alterations in
DNA melting behaviour are given by ÁTm. Tm for
the drug-free Calf thymus DNA was determined to be
67 ꢁC.
1,13-Diamino-4,10-di(2-meitylenesulphonyl)-4,10-diaza-
5,9-dioxatridecane dihydrochloride 6. was obtained in
1
60% yield as a white solid. H NMR (DMSO-d6): d
8.40(broad s, 6H, 2 ꢂNH3+), 7.20(s, 4H), 3.14–3.52
(m, 8H, 4ꢂCH2N), 2.78–3.10(m, 4H), 2.64 (s, 12H), 2.38
(s, 6H), 2.00(m, 4H), 1.40(m, 2H). 13C NMR (DMSO-
d6): d 144.2, 141.3, 132.2, 129.5, 72.9, 47.2, 26.9, 24.6,
22.8, 20.8. HRMS (FAB): calcd for C27H44N4O6S2Cl2
658.27 ([Mꢀ2HCl]+ 585.2781), found: 585.2785
[Mꢀ2HCl]+.
1,19-Diphthalimido-7,13-di(2-meitylenesulphonyl)-7,13-
diaza-8,12-dioxananodecane dihydrochloride 7. This was
obtained in 60% yield as a white solid; 1H NMR
(DMSO-d6): d 8.28 (broad s, 6H, 2ꢂNH+3 ), 7.17 (s, 4H),
2.97–3.40(m, 8H, 4 ꢂCH2N), 2.68–2.95 (t, 4H), 2.62 (s,
12H), 2.35 (s, 6H), 1.10–1.86 (m, 18H). 13C NMR
(DMSO-d6): d 143.7, 140.8, 131.8, 129.4, 72.3, 48.9,
26.7, 26.5, 25.9, 25.7, 25.3, 22.4, 20.4. HRMS (FAB):
calcd for C33H58N4O6S2Cl2 741.37 ([Mꢀ2HCl]+
669.3720), found: 669.3738 [Mꢀ2HCl]+.
Acknowledgements
V.A.P. is a recipient of a postdoctoral NATO/Royal
Society fellowship. The authors wish to thank The
Robert Gordon University for the financial support, the
centre of Mass Spectrometry at the University of Wales,
Swansea, for mass spectroscopic analyses, the National
Cancer Institute (USA) for screening our compounds
and Mr. Sergay S. Karpeev and Miss Diane Hion for
technical assistance.
4,10-Bis(2,2,5,7,8-pentamethy-3,4-dihydro-2H-chromen-
6-sulphonyl)-4,10-diaza-5,9-dioxatridecane-1,14,diamine

V. A. Kuksa et al. / Bioorg. Med. Chem. 10 (2002) 691–697
697
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