The synthesis of novel polyamine-nitroimidazole conjugates designed to probe the structural specificities of the polyamine uptake system in A549 lung carcinoma cells

Article abstract of DOI:10.1039/a903293b

Synthetic routes were developed to synthesise an N⁴-mono-derivatised spermidine-nitroimidazole conjugate and two novel structural isomers (N¹ and N⁸-spermidine-nitroimidazole conjugates). A synthetic method was developed t

Full text of DOI:10.1039/a903293b

View Article Online / Journal Homepage / Table of Contents for this issue
The synthesis of novel polyamine–nitroimidazole conjugates
designed to probe the structural specificities of the polyamine
uptake system in A549 lung carcinoma cells
Adam Q. Siddiqui, Louise Merson-Davies and Paul M. Cullis*
Department of Chemistry, University Road, Leicester University, Leicester, UK LE1 7RH.
E-mail: pmc@leicester.ac.uk
Received (in Cambridge, UK) 26th April 1999, Accepted 16th September 1999
Synthetic routes were developed to synthesise an N4-mono-derivatised spermidine–nitroimidazole conjugate and
two novel structural isomers (N1- and N8-spermidine–nitroimidazole conjugates). A synthetic method was developed
to synthesise an N1,N7-bis-derivatised norspermidine–nitroimidazole conjugate and further applied to the synthesis
of an N1,N8-bis-derivatised spermidine–nitroimidazole conjugate. The compounds were examined for their ability
to serve as substrates for the polyamine uptake system in A549 lung carcinoma cells, by measuring their inhibition
of [14C]spermidine uptake. Marked differences were observed between the nitroimidazole–polyamine conjugates.
For maximum recognition as a substrate by the polyamine transport system, the aminobutyl unit of spermidine
should remain underivatised. The preferred site(s) for spermidine amino derivatisation was in the order:
N1 > N8 ≈ N1, N8 > N4.
imidazoles, by virtue of the high affinity of the polyammonium
Introduction
cation for DNA (the binding constants of polyammonium
Hypoxic regions in certain tumour types often show problem-
cations to DNA are in the region of 103 MϪ1).6,7
atic resistance to damage by ionising radiation due to the
Polyamine derivatives and other structurally related com-
absence of the sensitising action of oxygen, known as the
pounds are known to be able to utilise cellular polyamine
‘oxygen effect’.1 Nitroimidazole-based drugs are able to
transport systems to cross the cell membrane.5,11,12 The poly-
sensitise hypoxic cells to the damaging effects of ionising
amine carrier protein has not yet been isolated in mammalian
cells but its properties have been characterised for a variety of
radiation.2 Misonidazole (1), for example, has been shown to
cell types including NB-15 mouse neuroblastomer cells, LoVo
human colon adenocarcinoma cells and L1210 mouse leukemia
cells.9,13,14 Kinetic studies (reviewed by Seiler and Dezeure15)
have shown the polyamine transport system to obey saturation
kinetics and to be energy dependent. Furthermore, competition
studies appear to suggest that a single transport mechanism
is responsible for the uptake of putrescine, spermidine and
spermine.15 There have been a few previous studies that have
made direct comparisons between the abilities of polyamines
and structurally related compounds to be recognised as sub-
strates.5,12,16,17 Porter and co-workers using L1210 leukaemia
cells were able to demonstrate a marked preference for certain
behave as an oxygen mimetic. Under the conditions of hypoxia,
polyamine inter-amino chain lengths by measuring the inhib-
misonidazole may directly substitute for an oxygen molecule
ition constants for a variety of di- and triamines.16 Substrate
in the radical reaction pathway resulting from the oxidation
recognition was found to be greatest for triamines containing a
of DNA, leading to DNA strand breakage.3 Furthermore,
unit of two amino centres separated by four methylene groups.
nitroimidazoles can also exhibit a selective toxicity towards
In this study we report the synthesis of a range of spermidine–
hypoxic cells, an effect possibly originating from their reduction
nitroimidazole derivatives and one norspermidine–nitro-
by cellular nitroreductases.4
imidazole conjugate, via novel synthetic routes and, the common
It has been proposed by ourselves and others that polyamines
structural properties that optimise recognition by the poly-
may be used as vectors for the cellular delivery of chemothera-
amine uptake system in A549 lung carcinoma cells. In par-
peutic and/or DNA targeted drugs.5,6 Found in all eukaryotic
ticular, we highlight the major differences that exist between
cells, polyamines are essential for the processes of cellular
regioisomeric spermidine derivatives, with a view to establish-
growth and differentiation, although their precise roles remain
ing the optimum site of spermidine derivatisation for these
poorly defined. It is known that polyamines are required for the
types of drug–polyamine conjugates.
structural modification of DNA in the processes of transcrip-
tion and recombination7 and the reduction of repulsive forces
between segments of DNA to facilitate DNA packaging, for
example in phage heads.8 The conjugation of nitroimidazoles to
polyamines may target the drug to tumour cells by virtue of the
high specific activity of the polyamine uptake system observed
in a number of tumour cell lines.9,10 The polyamine moiety
would also be expected to increase the local concentration of
the drug around DNA, the proposed site of action of nitro-
Results
Synthesis of polyamine conjugates
To compare the effects of polyamine derivatisation at the three
amino-centres of spermidine, we sought to synthesise the
N4-nitroimidazole–polyamine conjugate 717 and two novel
structural analogues, compounds 5 and 6. To examine the
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252
This journal is © The Royal Society of Chemistry 1999
3243

View Article Online
the Boc group was removed by acidolysis to give the desired
compound as a hydrochloride salt 5. For the synthesis of
compound 6 both the N1 and N8 centres of 12 were phosphinyl-
ated using diphenylphosphinic chloride (DPPCl). This novel,
orthogonally protected spermidine derivative was subsequently
alkylated with epichlorohydrin using NaH as base. Addition of
2-nitroimidazole to the epoxide moiety of 18 under basic con-
ditions afforded the correct regioisomeric adduct 19. Deprotec-
tion was again accomplished in two steps, initially by removal
of the methylene bridge under Knoevenagel conditions
followed by acidolysis of the remaining DPP groups, to afford
the desired product 6. Synthesis of the N4-nitroimidazole–
spermidine conjugate was accomplished via alkylation of the
N1 N8 Boc-protected spermidine derivative 21 with 14, followed
by deprotection under acidic conditions (Scheme 2). The
N1,N7-norspermidine–nitroimidazole conjugate 8 was syn-
thesised by alkylation of the triphosphinylated spermidine
derivative 23 with two equivalents of epichlorohydrin (Scheme
3). One equivalent of 2-nitroimidazole was added to each
epoxide moiety under basic conditions, followed by deprotec-
tion under acidic conditions to afford the desired compound 8.
The N1,N8-spermidine–bis-nitroimidazole conjugate 9 was syn-
thesised by similar synthetic procedures. Both bis-epoxypropyl
polyamine adducts 24 and 27 were formed as 1:1 diastereo-
meric mixtures resulting from chirality at the 2Ј-C and 2Љ-C
centres of the hydroxypropyl linker. Evidence for this ratio
derives from the 31P NMR spectra of the subsequently formed
bis-nitroimidazole adducts 25 and 28. The diastereomers of 8
and 9 could not be separated by reverse-phase HPLC, con-
sequently both 8 and 9 were evaluated as 1:1 diastereomeric
mixtures.
The synthesis of N1-mercaptoethylspermidine 10 made use
of the orthogonally protected spermidine derivative 13 (Scheme
4). Following alkylation of 13 with episulfide, the thiol func-
tionality was protected by oxidation to the disulfide to prevent
unwanted side-reactions during deprotection. Successive depro-
tection of the N1,N4-methylene bridge and N8-Boc group was
followed by reduction of the disulfide bond using dithiothreitol
to afford the desired compound as a hydrochloride salt. The
synthesis of N4-mercaptoethylspermidine 11 was accomplished
by a similar synthetic strategy, using the N1,N8-bis-Boc pro-
tected spermidine derivative 21 as a starting point (synthetic
details given elsewhere20).
importance of inter-polyamino chain-length a novel, efficient
route was devised to synthesise compound 8, an N1,N7-bis-
derivatised norspermidine–nitroimidazole conjugate. This
synthetic method was also applied to the synthesis of its longer
chain homologue, the N1,N8-bis-derivatised spermidine–
nitroimidazole conjugate 9.17 To provide evidence that the
uptake inhibition studies on these compounds may provide a
general guide for the optimal site of spermidine derivatisation,
we also synthesised the known radioprotecting agents, N1- and
N4-mercaptoethylspermidine, compounds 10 and 11 respect-
ively, using similar synthetic procedures.
The synthesis of compounds 5 and 6 was accomplished via
routes comprising of entirely novel synthetic intermediates. The
starting point in both cases was the methylenespermidine
derivative 12 synthesised previously by Ganem.18 For com-
pound 5 the introduction of the nitroimidazole moiety at the
N1 position was achieved via selective acylation of the primary
N8 nitrogen of 12 with tert-butoxycarbonyloxyimino-2-
phenylacetonitrile (BOC-ON) at low temperature (Scheme 1).
The resulting novel, orthogonally protected spermidine deriv-
ative 13 was alkylated using 1-(2Ј,3Ј-epoxypropyl)-2-nitro-
imidazole 14 (synthesised using the procedure described by
Hoffmann19) at the N1 position to give compound 15. Depro-
tection of the compound was achieved in two steps and in high
yield. Initially the N1,N8 methylene bridge was removed under
Knoevenagel reaction conditions affording 16. Subsequently,
Inhibition of polyamine uptake
The inhibition of [14C]spermidine uptake by the nitro-
imidazole–polyamine conjugates was determined as described
in the Experimental section and the results are summarised in
Table 1. As with simple enzyme inhibition, the lower the value
of the inhibition constant (Ki) the more potent the conjugate
was as an inhibitor of spermidine uptake. In all cases
Lineweaver–Burk plots indicated simple competitive inhibition
with respect to spermidine.
The comparison between the three structurally isomeric
monoderivatised spermidine–nitroimidazole conjugates 5, 6
and 7 was of particular interest. Both the N8 and N1 terminally-
derivatised isomers 6 and 5 were substantially superior to the
N4 derivatised isomer 7 at inhibiting uptake of the normal sub-
strate. Their Ki values (Ki 6, 0.5 µM; Ki 5, 0.09 µM) were 6 and
36 times lower respectively than the inhibition constant for the
N4 derivatised compound (Ki 7, 3.1 µM). The adverse effects of
derivatising at the N4 position of spermidine could also be seen
from comparison with the Ki value for the bis-derivatised
nitroimidazole–spermidine conjugate 9 (Ki 9, 0.6 µM), although
in structure 9 both terminal amino centres were derivatised the
inhibition constant was 5.3 times lower than that of the N4
derivatised conjugate 7 (Ki 7, 3.1 µM).
The difference between the Ki values of the two terminally
mono-derivatised nitroimidazole–spermidine conjugates is also
3244
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252

View Article Online
Scheme 1 Reagents and conditions: i, formaldehyde (37% aqueous soln.); ii, BOC-ON; iii, 1-(2Ј,3Ј-epoxypropyl)-2-nitroimidazole 14, triethylamine;
iv, malonic acid, pyridine; v, trifluoroacetic acid, triethylsilane; vi, DPPCl, triethylamine; vii, NaH, epichlorohydrin; viii, 2-nitroimidazole, triethyl-
amine; ix, malonic acid, pyridine; x, HCl soln.–methanol.
of note. The N1-spermidine derivatised isomer 5 had a Ki value
approximately 6 times lower than the Ki value for the N8-
spermidine derivatised isomer 6. This suggests that to maximise
substrate recognition, neither the N4 nor N8 position of the
aminobutyl unit of spermidine should be derivatised.
position of spermidine, derivatising at the N8 position has only
a minimal effect on the derivatives ability to be recognised as a
substrate.
A comparison between the inhibition constants for the two
bis-derivatised polyamine–nitroimidazole conjugates 8 and 9
based on spermidine and norspermidine (Ki 9, 0.6 µM; Ki 8, 5.0
µM) show that spermidine-derived conjugates have a greater
affinity for the polyamine receptor involved in uptake compared
to those based on norspermidine. Unlike spermidine, norsperm-
The Ki values of the bis-derivatised N1,N8-spermidine–nitro-
imidazole conjugate 9 and the monoderivatised N8-spermidine–
nitroimidazole conjugate 6 were similar (Ki 9, 0.6 µM; Ki 6, 0.5
µM). This suggests, that in contrast to derivatisation at the N1
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252
3245

View Article Online
Scheme 2 Reagents and conditions: i, BOC-ON; ii, 1-(2Ј,3Ј-epoxy-
propyl)-2-nitroimidazole, triethylamine; iii, trifluoroacetic acid, ion
exchange resin, HCl aqueous soln.–methanol.
idine does not contain an aminobutyl unit, again emphasising
the importance of this unit for substrate recognition.
Finally, a comparison between N1-mercaptoethylspermidine
10 and N4-mercaptoethylspermidine 11 further indicated
that N1-spermidine derivatives compete favourably with regio-
isomeric N4-spermidine derivatives for recognition by the
polyamine uptake system (Ki 10, 0.6 µM; Ki 11,1.1 µM). The
difference between the inhibition constants of the N1- and the
N4-mercaptoethylspermidine derivatives 10 and 11 was smaller
than the difference observed between the N1- and the N4-
nitroimidazole spermidine derivatives 5 and 7. This may reflect
differences in other properties involved in substrate binding,
such as steric bulk, solvation/desolvation energies and possible
differences in substrate–carrier intermolecular interactions.
Scheme 3 Reagents and conditions: i, DPPCl, triethylamine; ii, NaH,
epichlorohydrin; iii, 2-nitroimidazole, triethylamine; iv, HCl aqueous
soln.
spermidine uptake. Our previous studies have suggested that
the number of positive charges on the polyamine represents
the single most important contribution to recognition by the
polyamine receptor.21 Hence, acylation at the terminal amine
centres of spermidine would be expected to reduce affinity for
the polyamine receptor.
Evidence that the inhibition constants reported in this study
may reflect actual cellular uptake comes from several sources.
Seppanen has reported the preference for N1-spermidine deriv-
atisation compared to N8-spermidine derivatisation for actual
cellular uptake in Ehrlich ascites tumour cells.22 N1-Acetyl-
spermidine was shown to accumulate to levels approximately
three times greater than those reached by N8-acetylspermidine
for both untreated and DFMO (α-difluoromethylornithine)
treated cells. Similarly, direct evidence for the preferential
uptake of N1-mercaptoethylspermidine over N4-mercaptoethyl-
spermidine was provided in a study by Newton and co-
workers.23 The intracellular concentrations of various amino-
thiols were determined in DFMO pre-treated V79 cells. The
intracellular concentrations of N1-mercaptoethylspermidine
and N4-mercaptoethylspermidine after incubation with the
pre-treated cells (50 µM drug, 30 minutes, 37 ЊC) were found to
be 610 and 250 µM respectively.
Discussion
Porter and co-workers reported that the preferred site(s) of
spermidine amino derivatisation for recognition by the
polyamine uptake system in L1210 leukaemia cells was
N4 > N8 > N1 > N1, N8.17 Their conclusions were based on
comparisons made between derivatives having alkylated
secondary amines and those where the terminal amines are
acylated. However, the loss of basicity resulting from amine
acylation would be expected to be of major significance in
substrate recognition. Unpublished studies20 have shown that
successive methylation at the nitrogen centres of spermidine
was tolerated by the polyamine transport system of Ehrlich
ascites tumour cells up until but not including the quaternary
ammonium derivative, which showed no inhibition of [3H]-
3246
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252

View Article Online
Notwithstanding the above discussion, it is worth noting
once more that despite the considerable structural variation in
the conjugates described above, the variation in Ki values are
comparatively modest. This again confirms the broad substrate
tolerance of the mammalian polyamine transporter.24
Experimental
Chemicals
Radiolabelled spermidine (112 µCi mmolϪ1) was purchased
from Amersham International (Amersham, UK). All other
chemicals unless otherwise stated were obtained from Sigma-
Aldrich Company Ltd (Poole, UK). Compounds were analysed
by HPLC using a reverse-phase column (C18, 5 µ, BDS col-
umn, Hypersil) connected to a Gilson 715 system. A Dynamax
absorbance detector (model UV-1, Rainin) was used to detect
the nitroimidazole–polyamine compounds. The solvent system
used was eluent A; 0.1 M ammonium acetate pH 6, eluent B;
acetonitrile (Far UV, HPLC grade, Fisher, Loughborough,
UK), gradient 0 min (20% B); 20 min (60% B); 23 min (80% B);
26 min (80% B); 28 min (20% B); 30 min (20% B); flow rate was
1 cm3 minϪ1 at room temperature. Fluorescence was detected
at 326 nm. NMR spectra were recorded on a Bruker AM300
NMR spectrometer (1H at 300 MHz and 13C at 75 MHz) and a
Bruker 250 ARX NMR spectrometer (1H at 250 MHz, 13C at 62
MHz and 31P at 101 MHz). 13C NMR assignments (CH3, CH2,
CH and C) have been made on the basis of interpretation of the
DEPT spectra. J Values are given in Hz throughout. Mass
spectra (low and high resolution) were recorded on a Kratos
Concept 1H double focusing forward geometry mass spec-
trometer. IR spectra were recorded on a Perkin-Elmer 16 PC
FT-IR spectrophotometer. UV spectra were recorded on a
Beckman DU 7500 spectrophotometer.
Cell lines and bacterial strains
A549 human epithelial lung carcinoma cells were used
throughout this study and were a gift from Dr C. Courage,
CMHT, Leicester University. Cells were seeded at (5–10) × 104
cells cmϪ3 and maintained in Nut. Mix. F-12 (Hams) with
Glutamax (Gibco BRL, cat 31765-027) supplemented with
foetal calf serum, penicillin (100 iu cmϪ3) and streptomycin (100
µg cmϪ3).
Scheme 4 Reagents and conditions: i, ethylene sulfide; ii, O2; iii,
malonic acid, pyridine; iv, HCl aqueous soln. (5 M), dithiothreitol.
Table 1 Inhibition of spermidine uptake in A549 human lung
carcinoma cells by polyamine conjugatesa
Compound Ki/µM
Structural characteristics
Inhibition of polyamine uptake
5
6
0.09 0.09
Spermidine N1 derivatisation
Spermidine N8 derivatisation
Spermidine N4 derivatisation
Norspermidine N1 and N7 derivatisation
Spermidine N1 and N8 derivatisation
N1-mercaptoethyl spermidine
N4-mercaptoethyl spermidine
To study the ability of a range of polyamines to inhibit the
uptake of [14C]spermidine in vitro, A549 cells were seeded into
24 well tissue culture plates (Nunc - 1 × 105 cells wellϪ1) and
incubated for 16 h to form a monolayer. Cells were incubated
0.5 0.3
3.1 0.4
6.0 1.6
0.6 0.2
0.6 0.1
1.1 0.9
7
8
9
10
11
for 30 min with 14C radiolabelled spermidine (112 µCi mmolϪ1
,
Amersham International) at various concentrations (0.125,
0.25, 0.5, 1 and 2 µM) alone or in the presence of a range
polyamines/polyamine conjugates. After incubation, the plates
were placed on ice and the cells were washed with cold 0.9%
NaCl plus 1 mM spermidine, to displace any radiolabelled
spermidine still attached to the cell surface. Cells were digested
by the addition of 1 M NaOH (400 µl) and incubation at 60 ЊC
for 30 min to 1 h. Samples were neutralised by the addition of
an equal volume of 1 M HCl. Duplicate samples (400 µl) were
added to 4 cm3 Optiphase ‘Safe’ and radioactivity determined
in a Wallac scintillation counter. The results were expressed as
pmol spermidine uptake per min per 105 cells. The mechanism
of inhibition of uptake was determined using Lineweaver–Burk
plots.
a A549 cells were incubated at 37 ЊC for 30 min with [14C]spermidine
alone or in the presence of the conjugate. The accumulation of radio-
label into the cells was calculated and Ki values determined from
Lineweaver–Burk plots. Values are the mean of at least three separate
experiments and the errors are the standard deviations.
It is possible to conclude from the results presented here that,
for maximum recognition of the substrate by the polyamine
uptake system of A549 lung carcinoma cells, where charges are
preserved, the preferred site(s) of conjugation to spermidine is
in the order: N1 > N8 ≈ N1, N8 > N4. It would therefore appear
to be preferable to derivatise spermidine at the N1 position
and not the N4 position as has been suggested by others,16 pre-
serving the integrity of the aminobutyl unit, which appears to
maximise recognition of the substrate. This study suggests that
the conjugation of drugs to polyamines continues to have
potential in the targeting of cells with active polyamine uptake
systems, with conjugation at the N1 position producing the
most promising results.
N1,N4-Methylenespermidine 1218
Spermidine (4.00 g, 27 mmol) was dissolved in distilled water
(10 cm3). Aqueous formaldehyde solution (2.14 cm3, 37%, 27
mmol) was added drop-wise to the rapidly stirring solution over
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252
3247

View Article Online
a period of 5 minutes. After stirring for an additional 20
minutes the product was removed from the reaction mixture
by washing with chloroform (8 × 10 cm3). The combined
chloroform extracts were dried over Na2SO4 and the solvent
removed in vacuo to leave an oily residue. This was further
purified by vacuum distillation (150 ЊC, 2 mbar) to leave a clear,
colourless oil (2.55 g, 60%); δH(250 MHz; CDCl3) 4.80 (3 H, s,
NH), 3.45 (2 H, s, 9-H), 2.92 (2 H, t, J 2, 1-H), 2.73 (4 H, m,
5-H, 7-H), 2.39 (2 H, t, J 3, 4-H), 1.66 (2 H, m, 6-H), 1.52 (4 H,
m, 2-H, 3-H); δC(63 MHz; CDCl3) 70.52 (9-C), 56.74, 54.52,
46.16, 43.00 (1-C, 3-C, 5-C, 8-C), 32.49 (2-C), 27.62, 25.61 (6-C,
7-C).
ethanol (10 cm3). The reaction mixture was refluxed for 2 h at
100 ЊC. Following the removal of volatiles in vacuo, the residue
was taken up in water (5 cm3) and washed with chloroform
(3 × 5 cm3). The aqueous layer was adjusted to pH 11 by the
addition of a few drops of 10% NaOH aqueous solution. The
aqueous layer was then extracted with chloroform (5 × 5 cm3)
and the combined chloroform extracts were dried over Na2SO4.
Removal of the solvent in vacuo isolated the product as a light
yellow gum (56 mg, 82%); νmax(CHCl3)/cmϪ1 3050m (aromatic
C–H stretch) 2990s (aliphatic C-H stretch), 1420s (C᎐O
᎐
stretch), 1260s, 890m, 750s; δH(250 MHz; CDCl3) 7.22 (1 H, s,
imidazole-ring CH), 7.04 (1 H, s, imidazole-ring CH), 4.85 (1
H, br s, N8-H), 4.59 (1 H, dd, J2 13.6 and J3 12.4, 3Ј-HaHb), 4.17
(1 H, dd, J2 13.6 and J3 8.0, 3Ј-HaHb), 3.92 (1 H, br m, OH),
3.02 (2 H, br m, 8-H), 2.58 (8 H, br m, 1-H, 3-H, 5-H, 1Ј-H),
1.58 (2 H, m, 2-H), 1.44 (4 H, m, 6-H, 7-H), 1.36 [9 H, s,
N1,N4-Methylene-N8-(tert-butoxycarbonyl)spermidine 13
N1,N4-Methylenespermidine 12 (3.54 g, 22.5 mmol) was dis-
solved in dichloromethane (80 cm3) and the solution suspended
in an ice bath. After stirring for 15 minutes, BOC-ON (5.55 g,
22.5 mmol) dissolved in dichloromethane (40 cm3) was added
drop-wise to the solution over 1 h. After a further 1 h of stirring
the solvent was removed in vacuo and the residue taken up in
diethyl ether (80 cm3). The solution was washed with saturated
NaOH (4 × 10 cm3) until removal of all yellow coloration. The
solution was dried over Na2SO4, solvent evaporated in vacuo
and the resulting residue was purified by flash chromatography
[over silica eluting with 0.5% (concentrated NH4OH solution)–
MeOH]. The combined fractions had solvent removed in vacuo
to leave a white crystalline solid (4.41 g, 76%); νmax(neat)/cmϪ1
C(CH ) ]; δ (63 MHz; CDCl ) 156.5 (C᎐O), 128.3, 128.4
᎐
3
3
C
3
(2 × aromatic CH), 79.5 [C(CH3)3], 68.8 (2Ј-C), 54.1, 52.5, 49.5,
48.7, 48.5, 40.7, (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 28.9, 28.1, 26.8
(2-C, 6-C, 7-C), 28.8 [C(CH3)3]; m/z (FAB) 415 (MHϩ, 59%),
368 (26, loss of HNO2), 307 (10), 268 (7), 207 (10), 154 (100),
136 (90) (Found: MHϩ, m/z 415.2669. C18H35N6O5 requires
415.2669).
N1-[2Ј-Hydroxy-3Ј-(2Љ-nitroimidazol-1Љ-yl)propyl]spermidine
hydrochloride 5
Compound 16 (0.761 g, 1.84 mmol) and triethylsilane (0.75
cm3, 4.6 mmol) were dissolved in dichloromethane. Trifluoro-
acetic acid (15 cm3) was added to the rapidly stirring solution
and, after 3 h stirring, the volatiles were removed in vacuo. The
product was then isolated by ion-exchange chromatography
using Dowex 50W anionic exchange resin, eluting with a linear
gradient of increasing concentration of aqueous HCl–MeOH
(0.5–3.0 M, 100 cm3 each). The product was isolated as a light
yellow, brittle foam (0.725 g, 93%); δH(250 MHz; CDCl3) 7.51
(1 H, s, imidazole-ring CH), 7.25 (1 H, s, imidazole-ring CH),
4.77 (1 H, m, 3Ј-HaHb), 4.42 (2 H, m, 3Ј-HaHb, 2Ј-H), 3.44 (1 H,
pseudo d, 1Ј-HaHb), 3.15 (9 H, m, 1-H, 3-H, 5-H, 8-H,
1Ј-HaHb), 2.18 (2 H, m, 2-H), 1.78 (4 H, m, 6-H, 7-H); δC(63
MHz; CDCl3) 129.2, 127.7 (2 × aromatic CH), 66.0 (2Ј-C), 53.1,
49.9, 47.4, 45.0, 44.7, 39.1 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 24.2,
23.0, 22.8 (2-C, 6-C, 7-C); m/z (FAB) 315 (MHϩ, 100%), 268
(21), 205 (15), 176 (43), 146 (13) (Found: MHϩ, m/z 315.2145.
C13H27N6O3 requires 315.2146).
3300m br (NH stretch), 2950s (C–H stretch), 1700s (C᎐O
᎐
stretch), 1550m, 1450m, 1365m, 1300m, 1160m; δH(250 MHz;
CDCl3) 5.95 (1 H, br s, N8-H), 3.22 (2 H, s, 9-H), 2.96 (2 H, br
pseudo q, 8-H), 2.63 (2 H, t, J 7, 1-H), 2.40 (2 H, br t, 3-H), 2.09
(2 H, t, J 11, 5-H), 1.67 (1 H, br s, N1-H), 1.47 (2 H, m, 2-H),
1.38 (4 H, m, 6-H, 7-H), 1.31 [9 H, s, C(CH3)3]; δC(63 MHz;
CDCl ) 156.4 (C᎐O), 79.0 [C(CH ) ], 70.1 (9-C), 55.6, 53.4,
᎐
3
3 3
45.5, 40.9 (1-C, 3-C, 5-C, 8-C), 28.8 [C(CH3)3], 28.5, 27.4, 24.8
(2-C, 6-C, 7-C); m/z (CI) 258 (MHϩ, 100%), 200 (17, loss of tert-
butyl ϩ Hϩ), 99 (44), 85 (23), 70 (23), 57 (12, tert-butylϩ
(Found: MHϩ, m/z 256.2024. C13H26N3O2 requires 256.2025).
)
ؒ
N1-[2Ј-Hydroxy-3Ј-(2Љ-nitroimidazol-1Љ-yl)propyl]-N1,N4-meth-
ylene-N8-(tert-butoxycarbonyl)spermidine 15
Compound 13 (0.910 g, 3.54 mmol) and 1-(2Ј,3Ј-epoxypropyl)-
2-nitroimidazole (595 mg, 3.54 mmol) were dissolved in meth-
anol (5 cm3) in a Young’s tube. The reaction mixture was heated
at 120 ЊC for 5 h. The volatiles were removed in vacuo and the
residue was purified by flash chromatography (silica; 6%
MeOH–CH2Cl2). The combined fractions had solvent removed
in vacuo to leave a light yellow, viscous gum (1.359 g, 90%);
νmax(CHCl3)/cmϪ1 3450m (O–H stretch), 3040m (aromatic C–H
N1,N8-Bis(diphenylphosphinoyl)-N1,N4-methylenespermidine 17
N1,N4-Methylenespermidine 12 (0.946 g, 6.02 mmol) and tri-
ethylamine (2.1 cm3, 15.1 mmol) were dissolved in dichloro-
methane (30 cm3). The reaction mixture was suspended in an ice
bath and DPPCl (2 equiv., 2.3 cm3) was added by injection.
After allowing the reaction mixture to warm to room temper-
ature over 15 minutes, water (10 cm3) was added to the reaction
mixture and the organic solvent was removed in vacuo. The
residue was partitioned between water and dichloromethane
(40 cm3 each) and the aqueous layer washed with dichloro-
methane (3 × 40 cm3). The combined organic layers were dried
over Na2SO4, after which the solvent was removed in vacuo.
The residue was purified by flash chromatography (over silica
eluting with 5% MeOH–CH2Cl2). The combined fractions had
solvent removed in vacuo to leave a white, brittle foam (2.551 g,
stretch) 2950s (aliphatic C–H stretch), 1700s (C᎐O stretch),
᎐
1480m, 1360m, 1365m, 1250m, 1160m, 900m, 7735m br; δH(250
MHz; CDCl3) 7.24 (1 H, s, imidazole-ring CH), 7.06 (1 H, s,
imidazole-ring CH), 4.91 (1 H, br s, N8-H), 4.62 (1 H, dd, J2 13.6
and J3 1.6, 3Ј-HaHb), 4.56 (1 H, br s, D2O exchange, OH), 4.08
(2 H, m, 3Ј-HaHb, 2Ј-H), 3.22 (2 H, pseudo q, 8-H), 3.04 (2 H, d,
J 4.9, 9-H), 2.65 (4 H, m, 1-H, 1Ј-H), 1.59 (2 H, m, 2-H), 1.42
(4 H, m, 6-H, 7-H), 1.36 [9 H, s, C(CH3)3]; δC(63 MHz; CDCl3)
156.0 (C᎐O), 128.4, 128.1 (2 × aromatic CH), 79.0 [C(CH ) ],
᎐
3
3
75.7 (9-C), 67.5 (2Ј-C), 59.1, 54.8, 53.9, 52.9, 40.7, 40.2 (1-C,
3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 28.8 [C(CH3)3], 28.2, 24.3, 24.1 (2-C,
6-C, 7-C); m/z (EI) 425 (M Ϫ Hϩ, 13.9%), 409 (6.4), 369 (11.0,
loss of tert-butyl), 254 (6.2), 149 (6.1), 114 (21), 70 (29), 57
(50.1), 51 (100) (Found: M Ϫ Hϩ, m/z 425.2511. C19H33N6O5
requires 425.2512).
76%); νmax(CHCl3)/cmϪ1 3370w, 2920m, 1440m, 1190s (P᎐O
᎐
stretch), 1120s (P᎐O stretch), 1065m, 960w; δ (250 MHz;
᎐
H
CDCl3) 7.88 (8 H, m, meta-ring CH), 7.40 (12 H, m, ortho-ring
CH, para-ring CH), 3.70 [2 H, d (s, 31P decoupled), JP-H 8.18,
9-H], 3.64 [1 H, q (t, 31P decoupled), J 6.73, NH], 3.08 [2 H, q
(t, 31P decoupled), J 6.87, 1-H], 2.89 [2 H, m (q, 31P decoupled),
8-H], 2.65 (2 H, t, J 4.99, 3-H), 2.36 (2 H, t, J 6.93, 5-H), 1.55
(4 H, m, 7-H, 2-H), 1.26 (2 H, m, 6-H); δC(63 MHz; CDCl3)
133.76, 130.372 (Ar-C), 132.43, 132.38, 132.24, 132.14, 132.00,
N1-[2Ј-Hydroxy-3Ј-(2Љ-nitroimidazol-1Љ-yl)propyl]-N8-(tert-
butoxycarbonyl)spermidine 16
Compound 15 (70 mg, 0.17 mmol), malonic acid (62.5 mg, 0.60
mmol) and pyridine (0.042 cm3, 0.52 mmol) were dissolved in
3248
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252

View Article Online
131.82, 131.70, 131.66, 128.62, 128.54, 128.42, 128.34 (Ar-CH),
66.32 (9-C), 52.73, 52.72, 44.82, 40.47 (1-C, 3-C, 5-C, 8-C),
29.91 (d, JP-C 6.9), 24.36, 23.20 (d, JP-C 4.6) (2-C, 6-C, 7-C);
δP(101 MHz; CDCl3) 27.91 (s, N1-P), 23.52 (s, N8-P); m/z (FAB)
558.22 (Mϩ, 35%), 356.17 (21; loss of Ph P᎐O ϩ Hϩ), 270.09
28.47 (s, N1-P); m/z (FAB) 727 (Mϩ, 41%), 646 (18), 258 (8), 201
(100, Ph P᎐Oϩ ).
ؒ
᎐
2
N1,N8-Bis(diphenylphosphinoyl)-N8-[2Ј-hydroxy-3Ј-(2Љ-nitro-
imidazol-1Љ-yl)propyl]spermidine 20
᎐
2
(14), 201 (100, Ph P᎐Oϩ ), 69.95 (11).
ؒ
᎐
2
Compound 19 (100 mg, 0.14 mmol), malonic acid (52.2 mg,
0.50 mmol) and pyridine (0.035 cm3, 0.43 mmol) were dissolved
in ethanol (10 cm3). The reaction mixture was refluxed for 2 h at
100 ЊC. Following the removal of volatiles in vacuo, the residue
was taken up in water (5 cm3) which was adjusted to pH 11 by
the addition of a few drops of aqueous NaOH (10% solution).
The aqueous layer was then extracted with chloroform (5 × 5
cm3) and the combined chloroform extracts were dried over
Na2SO4. Removal of the solvent in vacuo isolated the product
as a light yellow, viscous oil (97 mg, 98%); νmax(CHCl3)/cmϪ1
3220m br (N–H), 3050m, 2960m (aromatic or aliphatic C–H
N1,N8-Bis(diphenylphosphinoyl)-N1,N4-methylene-N8-(2Ј,3Ј-
epoxypropyl)spermidine 18
Compound 17 (0.268 g, 0.48 mmol) and NaH (29 mg, 0.72
mmol) were added to THF (12 cm3). After stirring the reaction
mixture for 10 minutes, epichlorohydrin (0.2 cm3, 2.4 mmol)
was added and the mixture was heated at 90 ЊC for 3 h. Allow-
ing the reaction mixture to cool to room temperature, further
additions of NaH (0.72 mmol) and epichlorohydrin (5 equiv.)
were made. The reaction mixture was then heated at 90 ЊC for a
further 3 h. The mixture was allowed to cool to room temper-
ature whereupon ethyl acetate and water (5 cm3 each) were
added. After 5 minutes stirring the solvent was removed in
vacuo. The residue was partitioned between water and chloro-
form (30 cm3 each) and the aqueous layer was washed with
chloroform (3 × 20 cm3). The combined organic layers were
dried over Na2SO4, then solvent was removed in vacuo. The
residue was purified by flash chromatography (over silica elut-
ing with 5% MeOH–CH2Cl2). The combined fractions had
solvent removed in vacuo to leave a colourless, viscous gum
stretch), 2300w, 1530m, 1480s, 1425s, 1360s, 1210s (P᎐O
᎐
stretch), 1125s (P᎐O stretch), 1120s, 950s, 710s br (out of plane
᎐
aromatic C–H stretch); δH(250 MHz; CDCl3) 7.74 (8 H, m,
meta-ring CH), 7.37 (12 H, m, ortho-ring CH, para-ring CH),
7.19 (1 H, d, J 0.7, imidazole-ring C-H), 6.97 (1 H, d, J 0.7,
imidazole-ring C-H), 6.18 (1 H, br s, D2O exchange, OH), 4.59
(1 H, dd, Jgem 13.4 and Jvic 1.8, 3Ј-HaHb), 4.28 [1 H, pseudo q
(t, 31P decoupled), N1-H], 4.08 (1 H, br m, 2Ј-H), 3.91 (1 H, dd,
Jgem 13.4 and Jvic 8.6, 3Ј-HaHb), 2.95 (6 H, m, 1-H, 8-H, 1Ј-H),
2.58 (2 H, t, J 6.2, 3-H), 2.33 (2 H, t, J 6.7, 5-H), 1.62 (2 H, m,
2-H), 1.43 (2 H, m, 7-H), 1.12 (2 H, m, 6-H); δC(63 MHz;
CDCl3) 144.96, 132.1, 129.3 (Ar-C), 132.8, 132.7, 132.5, 132.4,
132.3, 132.1, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7, 128.5,
128.3 (Ar-CH), 68.03 (2Ј-C), 54.15, 50.67, 49.37, 48.72, 47.57,
40.50 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 31.42 (d, JP-C 7.0), 27.22,
26.53 (d, JP-C 4.2) (2-C, 6-C, 7-C); δP(101 MHz; CDCl3) 35.36
(215 mg, 73%); νmax(CHCl3)/cmϪ1 3375w, 2925m, 1190s (P᎐O
᎐
stretch), 1120s (P᎐O stretch), 1035m, 960w; δ (250 MHz;
᎐
H
CDCl3) 7.80 (8 H, m, meta-ring CH), 7.31 (12 H, m, ortho-ring,
para-ring CH), 3.55 [2 H, d (s, 31P decoupled), JP-H 8.25, 9-H],
3.26 [1 H, m (dd, 31P decoupled, Jgem 9.73 and Jvic 3.53),
3Ј-HaHb], 3.03 (4 H, m, 1-H, 7-H), 2.85 (2 H, m, 2Ј-H, 3Ј-HaHb),
2.59 (1 H, pseudo t, J 4.45, 1Ј-HaHb), 2.52 (2 H, br t, 5-H or
3-H), 2.23 (1 H, dd, Jgem 4.45 and Jvic 2.57, 1Ј-HaHb), 2.14 (2 H,
br t, 3-H or 5-H), 1.49 (4 H, m, 6-H, 7-H), 0.97 (2 H, m, 2-H);
δC(63 MHz; CDCl3) 131.1, 131.0 (Ar-C), 136.3, 133.2, 133.0,
132.24, 132.8, 132.7, 132.6, 132.3, 132.2, 130.7, 129.1, 129.0
(Ar-CH), 67.1 (9-C), 51.8 (2Ј-C), 53.9, 53.3, 49.0, 47.4, 45.6,
45.2 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 24.7, 23.9, 21.6 (2-C, 6-C,
7-C); δP(101 MHz; CDCl3) 31.31 (s, N8-P), 28.03 (s, N1-P); m/z
(FAB) 614.25 (Mϩ, 63%), 412.20 (22, loss of Ph P᎐O ϩ Hϩ),
(s, N8-P), 23.84 (s, N1-P); m/z (FAB) 715 (Mϩ, 64%), 634 (6),
ϩ
307 (82), 201 (100, Ph P᎐Oϩ ) (Found: M , m/z 715.2925.
ؒ
᎐
2
C37H45O5N6P2 requires 715.2926).
N8-[2Ј-Hydroxy-3Ј-(2Љ-nitroimidazol-1Љ-yl)propyl]spermidine
hydrochloride 6
Compound 20 (89.1 mg, 0.125 mmol) was dissolved in 0.5 M
HCl (16 cm3; 50:50 MeOH–H2O). The solution was sub-
sequently stirred at 60 ЊC for 2 h. The product was then isolated
by ion-exchange chromatography using Dowex 50W anionic
exchange resin, eluting with a linear gradient of increasing con-
centration of aqueous HCl–MeOH (0.5–3.0 M, 100 cm3 each).
The product was isolated as a light yellow, brittle foam (39.0
mg, 99%); δH(250 MHz; CDCl3) 7.56 (1 H, s, imidazole-ring
CH), 7.30 (1 H, s, imidazole-ring CH), 4.82 (1 H, pseudo q, 3Ј-
HaHb), 4.45 (2 H, m, 3Ј-HaHb, 2Ј-H), 3.46 (1 H, pseudo d, 1Ј-
HaHb), 3.20 (9 H, m, 1-H, 3-H, 5-H, 8-H, 1Ј-HaHb), 2.15 (2 H,
m, 2-H), 1.86 (4 H, m, 6-H, 7-H); δC(63 MHz; CDCl3) 131.61,
130.29 (Ar-CH), 66.37 (2Ј-C), 55.43, 52.06, 49.61, 49.61, 47.15,
39.19 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 26.36, 25.37, 25.16 (2-C,
6-C, 7-C); m/z (FAB) 315 (MHϩ, 100%), 299 (5), 268 (6), 234
(5), 158 (6) (Found: MHϩ, m/z 315.2148. C13H27N6O3 requires
315.2146).
᎐
2
201.05 (100, Ph P᎐Oϩ ), 84.01 (17).
ؒ
᎐
2
N1,N8-Bis(diphenylphosphinoyl)-N1,N4-methylene-N8-[2Ј-
hydroxy-3Ј-(2Љ-nitroimidazol-1Љ-yl)propyl]spermidine 19
Compound 18 (0.539 g, 0.88 mmol) and 2-nitroimidazole (45
mg, 1.23 mmol) were dissolved in methanol (10 cm3) in a sealed
tube. Following the addition of triethylamine (0.12 cm3, 2.64
mmol) the reaction mixture was heated at 120 ЊC for 5 h. The
volatiles were then removed in vacuo and the residue was
purified by flash chromatography (over silica eluting with 5%
MeOH–CH2Cl2). The combined fractions had solvent removed
in vacuo to leave a light yellow, viscous gum (0.454 g, 71%);
νmax(CHCl3)/cmϪ1 3050s, 2950m, 2300m, 1430m, 1370s, 1310s,
1200s (P᎐O stretch), 1125m (P᎐O stretch), 1070s, 903w, 804s,
᎐
᎐
670s br (out of plane aromatic CH stretch); δH(250 MHz;
CDCl3) 7.65 (8 H, m, meta-ring CH), 7.29 (12 H, m, ortho-ring
CH, para-ring CH), 7.19 (1 H, d, J 0.7, imidazole ring C-H),
6.92 (1 H, d, J 0.7, imidazole ring C-H), 6.19 (1 H, br s, D2O
exchange, OH), 4.71 (1 H, dd, Jgem 13.6 and Jvic 1.95, 3Ј-HaHb),
4.27 (1 H, m, 2Ј-H), 3.98 (1 H, dd, Jgem 13.6 and Jvic 8.8,
3Ј-HaHb), 3.68 (1 H, pseudo t, J 10.1, 1Ј-HaHb), 3.52 (1 H,
pseudo t, J 10.1, 1Ј-HaHb), 3.06 (6 H, m, 1-H, 8-H, 9-H), 2.55
(2 H, m, 3-H), 2.20 (2 H, m, 5-H), 1.55 (4 H, m, 6-H, 7-H), 1.07
(2 H, m, 2-H); δC(63 MHz; CDCl3) 145.0, 130.7, 130.6 (Ar-C),
132.8, 132.7, 132.6, 132.4, 132.3, 132.1, 129.3, 129.2, 129.1,
129.0, 128.9, 128.6, 128.3 (Ar-CH), 67.7 (2Ј-C), 67.1 (9-C), 54.1,
53.3, 52.7, 50.3, 46.8, 45.7 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 25.5,
24.7, 23.8 (2-C, 6-C, 7-C); δP(101 MHz; CDCl3) 34.59 (s, N8-P),
N1,N8-Bis(tert-butoxycarbonyl)spermidine 21
Spermidine (3.50 g, 24.13 mmol) was dissolved in THF (120
cm3), to which was added BOC-ON (11.95 g, 48.5 mmol) at
0 ЊC. After 1 h stirring the THF was removed in vacuo and the
residue taken up in diethyl ether (80 cm3). The solution was
washed with saturated NaOH (4 × 10 cm3) until removal of
yellow coloration. The solution was dried over Na2SO4, evapor-
ated and the resulting white crystalline solid was recrystallised
from diisopropyl ether (5.92 g, 71%) (Found: C, 59.0; H, 10.2;
N, 12.2. C17H35N3O4 requires C, 59.1; H, 10.2; N, 12.2%); mp
82–84 ЊC; δH(300 MHz; CDCl3) 5.39 (1 H, br t, NHCO), 5.37
(1 H, br t, NHCO), 3.19 (2 H, m, 1-H), 3.11 (2 H, m, 8-H), 2.65
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252
3249

View Article Online
(2 H, t, J 6.6, 3-H), 2.60 (2 H, t, J 6.5, 5-H), 1.65 (2 H, m, 2-H),
brittle foam (4.31 g, 84%); νmax(CHCl3)/cmϪ1 3400w (N–H
1.52 (4 H, m, 6-H, 7-H), 1.44 (18 H, br s, CMeaMeb, N1-H,
stretch), 3200w, 2930w, 1730w, 1410m, 1190s (P᎐O stretch),
᎐
N8-H); δ (75 MHz; CDCl ) 156.10, 156.03 (C᎐O × 2), 78.78
1125s (P᎐O stretch); δ (250 MHz; CDCl ) 7.72 (12 H, m, meta-
᎐
᎐
C
3
H
3
[C(CH3)3 × 2], 49.42, 47.64, 40.40, 39.12 (1-C, 3-C, 5-C, 8-C),
29.87, 27.87, 27.35 (2-C, 6-C, 7-C), 28.43 [C(CH3)3 × 2]; m/z
(CI) 346 (MHϩ, 100%), 289 (31, loss of tert-butyl), 99 (28), 86
ring CH), 7.38 (18 H, m, ortho-ring, para-ring CH), 4.06 (2 H,
q, 3JH-H 7.4 and 2JH-P 7.4, N1-H, N7-H), 3.07 (4 H, m, 1-H, 7-H),
2.89 (4 H, m, 3-H, 5-H), 1.74 (4 H, m, 2-H, 6-H); δH{31P}(250
MHz; CDCl3) 7.72 (12 H, m, meta-ring CH), 7.38 (18 H, m,
(21), 57 (27, tert-butylϩ ).
ؒ
3
ortho-ring CH, para-ring CH), 4.06 (2 H, t, JH-H 7.4, N1-H,
N7-H), 3.07 (4 H, t, J 6.9, 1-H, 7-H), 2.89 (4 H, q, J 6.4, 3-H,
5-H), 1.74 (4 H, m, 2-H, 6-H); δC(63 MHz; CDCl3) 133.74,
132.51, 130.46 (Ar-C), 132.28. 132.13, 132.09, 131.94, 131.86,
131.69, 131.65, 131.61, 128.68, 128.53, 128.50, 128.34 (Ar-CH)
42.91 (d, 3JC-P 3.4) (1-C, 7-C), 38.06 (3-C, 5-C), 30.28 (2-C, 6-C);
δP(101 MHz; CDCl3) 32.27 (s, N4-P), 23.81 (s, N1-P, N7-P);
N1,N8-Bis(tert-butoxycarbonyl)-N4-[2Љ-hydroxy-3Љ-(2Ј-nitro-
imidazol-1Ј-yl)propyl]spermidine 22
Compound 21 (1.9 g, 5.5 mmol) and 14 (0.87 g, 5.2 mmol) were
dissolved in methanol (40 cm3). Triethylamine (0.73 cm3, 5.5
mmol) was added and the mixture was subsequently refluxed at
100 ЊC for 1 hour. The solvent was removed in vacuo and the
residue purified by flash chromatography (silica; 10% MeOH–
CH2Cl2). The yellow–brown oil crystallised on standing to form
a solid of the same colour (1.42 g, 50%); mp 58–61 ЊC; δH(300
MHz; CDCl3) 7.31 (1 H, d, J 1.0, ring-CH), 7.10 (1 H, d, J 1.0,
ring-CH), 4.98 (1 H, br s, N1-H), 4.83 (1 H, br s, N8-H), 4.75
(1 H, dd, J 2.0 and J 13.7, 3Љ-CHaHb), 4.16 (1 H, dd, J 8.3 and
J 13.7, 3Љ-CHaHb), 3.13 (4 H, br m, 1-H, 8-H), 2.55 (4 H, m,
3-H, 5-H), 2.44 (2 H, m, 1Љ-CHaHb), 1.62 (2 H, m, 2-H), 1.46
(4 H, br s, 6-H, 7-H), 1.43 [9 H, s, C(CH3)3], 1.40 [9 H, s,
m/z (FAB) 732 (MHϩ, 56%), 475 (9), 201 (100, Ph P᎐Oϩ ), 154
ؒ
᎐
2
(55), 77 (25, Phϩ ).
ؒ
N1,N7-Bis(2Ј,3Ј-epoxypropyl)-N1,N4,N7-tris(diphenylphos-
phinoyl)norspermidine 24
Compound 23 (1.573 g, 2.16 mmol) was dissolved in THF (40
cm3). Additions of NaH (6 equiv., 12.96 mmol, 0.518 g) and
epichlorohydrin (21.60 mmol, 1.68 cm3) were made to the
stirred solution and the resulting mixture was heated at 90 ЊC
for 3 hours. The mixture was allowed to cool to room tem-
perature and a further addition of NaH (12.96 mmol) was
made. Following 15 minutes of stirring a further addition of
epichlorohydrin (21.6 mmol) was made and the reaction
mixture was re-heated to 90 ЊC where it was maintained for a
further 3 h. The mixture was allowed to cool to room temper-
ature whereupon ethyl acetate and water (5 cm3 each) were
added. After 5 minutes stirring the solvent was removed in
vacuo. The residue was partitioned between water and chloro-
form (30 cm3 each) and the aqueous layer was washed with
chloroform (3 × 20 cm3). The combined organic layers were
dried over Na2SO4, then solvent was removed in vacuo. The
residue was purified by flash chromatography (silica; 5%
MeOH–CH2Cl2). The combined fractions had solvent removed
in vacuo to leave a colourless, viscous gum (1.261 g, 69%);
C(CH ) ]; δ (75 MHz; CDCl ) 156.1, 144.7 (C᎐O × 2), 127.8,
᎐
3
3
C
3
127.7 (2 × aromatic-CH), 79.2, 79.15 [C(CH3)3 × 2], 66.9
(2Ј-C), 57.7, 53.7, 53.6, 51.6, 40.2, 38.4 (1-C, 3-C, 5-C, 8-C,
1Ј-C, 3Ј-C), 28.4, 28.35 [C(CH3)3 × 2], 27.7, 27.4, 23.9 (2-C, 6-C,
7-C); m/z (FAB) 515 (MHϩ, 100%), 468 (23, loss of HNO2),
458 (9, loss of tert-butyl), 70 (21), 57 (8, tert-butylϩ ) (Found
ؒ
Mϩ ϩ 1, m/z (CI) 399.2971. C20H39N4O4 requires 399.2971).
N4-[2Љ-Hydroxy-3Љ-(2Ј-nitroimidazol-1Ј-yl)propyl]spermidine
hydrochloride 7
Compound 22 (0.13 g, 0.25 mmol) was dissolved in trifluoro-
acetic acid (2 cm3) and the resulting solution stirred for 1 h. The
acid was removed in vacuo and the residue dissolved in 0.5 M
HCl (2 cm3). Ion-exchange purification was accomplished using
Dowex 50W anionic exchange resin (17 cm3), eluting with a
linear gradient of increasing concentration of aqueous HCl
(0.5–3.0 M, 340 cm3 each). Elution of the product was moni-
tored by UV spectroscopy (315 nm). Combined fractions had
solvent removed in vacuo to leave a light yellow foam (86 mg,
82%); δH(300 MHz; CDCl3) 7.62 (1 H, br s, ring CH), 7.36 (1 H,
br s, ring CH), 4.88 (1 H, dd, J2 14.8 and J3 2.4, 3Ј-HaHb), 4.61
(1 H, br m, 2Ј-H), 4.52 (1 H, dd, J2 14.8 and J3 8.7, 3Ј-HaHb),
3.65 (1 H, dd, J2 13.0 and J3 2.2, 1Ј-HaHb), 3.44 (5 H, br m, 1-H,
8-H, 1Ј-HaHb), 3.16 (4 H, m, 3-H, 5-H), 2.28 (2 H, m, 2-H), 1.91
(4 H, br m, 6-H, 7-H); δC(75 MHz; CDCl3) 147.1 (Ar-C), 79.2,
131.5, 130.4 (Ar-CH), 66.8 (2Ј-C), 58.5, 56.6, 55.8, 54.0, 42.0,
41.0 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 3Ј-C), 27.4, 25.7, 24.8 (2-C, 6-C,
7-C); m/z (FAB) 315 (MHϩ, 100%), 307 (73), 289 (20), 259 (9),
234 (5), 158 (6) (Found: MHϩ, m/z 315.21450. C13H27N6O3
requires 315.21446).
νmax(CHCl3)/cmϪ1 3400m, 2960m, 1440m, 1260m, 1190s (P᎐O
᎐
stretch), 1120s (P᎐O stretch), 900m, 700m br (out of plane
᎐
Ar–CH stretch); δH(250 MHz; CDCl3) 7.79 (12 H, m, meta-ring
CH), 7.42 (18 H, m, ortho-ring CH, para-ring CH), 3.27 (2 H,
m, 2Ј-H, 2Љ-H), 3.01 (4 H, m, 1Ј-H, 1Љ-H), 2.79 (10 H, m, 1-H,
3-H, 5-H, 7-H, 3Ј-HaHb, 3Љ-HaHb), 2.27 (2 H, m, 3Ј-HaHb,
3Љ-HaHb), 1.76 (4 H, m, 2-H, 6-H); δC(63 MHz; CDCl3) 132.9,
132.6, 132.2, 131.9 (Ar-C), 132.5, 132.4, 132.3, 130.9, 130.6,
130.5, 128.7, 128.6, 128.5, 128.4 (Ar-CH), 53.5 (2Ј-C, 2Љ-C),
51.3, 48.6, 45.1, 44.8, 42.8 (1-C, 2-C, 3-C, 5-C, 6-C, 7-C, 1Ј-C,
1Љ-C, 3Ј-C, 3Љ-C); δP(101 MHz; CDCl3) 31.32 (s, N1-P, N7-P),
30.45 (s, N4-P); m/z (FAB) 844 (MHϩ, 100%), 642 (8, loss of
ϩ
Ph P᎐O), 302 (6), 201 (84, Ph P᎐Oϩ ), 77 (21, Ph ).
ؒ
ؒ
᎐
᎐
2
2
N1,N7-Bis[2Љ-hydroxy-3Љ-(2Ј-nitroimidazol-1Ј-yl)propyl]-
N1,N4,N8-tris(diphenylphosphinoyl)norspermidine 25
Compound 24 (539 mg, 0.64 mmol) and 2-nitroimidazole (3
equiv., 217 mg) were dissolved in methanol (10 cm3) in a sealed
tube. Following the addition of triethylamine (0.54 cm3, 3.84
mmol) the reaction mixture was heated at 120 ЊC for 5 h. The
volatiles were then removed in vacuo and the residue was puri-
fied by flash chromatography (silica; 5% MeOH–CH2Cl2). The
combined fractions had solvent removed in vacuo to leave a
light yellow, viscous gum (556 mg, 81%); δH(250 MHz; CDCl3)
7.69 (12 H, m, meta-ring CH), 7.43 (18 H, m, ortho-ring CH,
para-ring CH), 7.23 (2 H, s, imidazole-ring CH), 7.02 (2 H, s,
imidazole-ring CH), 5.94 (2 H, br s, OH), 4.66 (2 H, pseudo d,
3Ј-HaHb, 3Љ-HaHb), 4.12 (2 H, br m, 2Ј-H, 2Љ-H), 3.95 (2 H, m,
3Ј-HaHb, 3Љ-HaHb), 3.09 (4 H, m, 1Ј-H, 1Љ-H), 2.88 (4 H, m, 1-H
and 7-H or 3-H and 5-H), 2.70 (4 H, m, 3-H and 5-H or 1-H
and 7-H), 1.75 (4 H, m, 2-H, 6-H); δC(63 MHz; CDCl3) 144.9,
N1,N4,N7-Tris(diphenylphosphinoyl)norspermidine 23
Norspermidine (0.923 g, 7.0 mmol) was dissolved in THF (50
cm3). Triethylamine (3.4 cm3, 21 mmol) was added to the solu-
tion which was subsequently stirred for 5 min. DPPCl (4.0 cm3,
21 mmol) was added by injection to the rapidly stirring solution
over a 5 minute period. After a further 1 h stir, water (10 cm3)
was added to the reaction mixture and the organic solvent was
removed in vacuo. The residue was partitioned between water
and dichloromethane (50 cm3 each) and the aqueous layer
washed with dichloromethane (3 × 50 cm3). The combined
organic layers were dried over Na2SO4, after which the solvent
was removed in vacuo. The residue was purified by flash
chromatography (silica; 5% MeOH–CH2Cl2). The combined
fractions had their solvent removed in vacuo to leave a white,
3250
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252

View Article Online
131.8, 131.5, 130.3, 129.7 (Ar-C), 132.7, 132.6, 132.5, 129.4,
129.3, 129.2, 129.1, 129.0, 128.5, 128.3 (Ar-CH), 68.4 (2Ј-C,
2Љ-C), 54.2 (3Ј-C, 3Љ-C), 50.5, 45.4, 43.6, 27.7 (1-C, 2-C, 3-C,
5-C, 6-C, 7-C, 1Ј-C, 1Љ-C); δP(101 MHz; CDCl3) 34.96 (s, N1-P),
for a further 3 h. The mixture was allowed to cool to room
temperature, whereupon ethyl acetate and water (1 cm3 each)
were added. After 5 minutes stirring the solvent was removed
in vacuo. The residue was partitioned between water and
chloroform (5 cm3 each) and the aqueous layer was washed
with chloroform (3 × 5 cm3). The combined organic layers
were dried over Na2SO4, then solvent was removed in vacuo.
The residue was purified by flash chromatography (silica; 5%
MeOH–CH2Cl2). The combined fractions had solvent removed
in vacuo to leave a colourless, viscous gum (65 mg, 58%);
34.82 (s, N8-P), 31.64 (d, N4-P); m/z (FAB) 1070 (MHϩ, 29%),
ϩ
915 (27), 834 (6), 289 (18), 201 (100, Ph P᎐Oϩ ), 77 (92, Ph ).
ؒ
ؒ
᎐
2
N1,N7-Bis[2Љ-hydroxy-3Љ-(2Ј-nitroimidazol-1Ј-yl)propyl]-
norspermidine hydrochloride 8
νmax(CHCl3)/cmϪ1 3400m, 2960m, 1440m, 1260m, 1190s (P᎐O
Compound 25 (32 mg, 0.029 mmol) was dissolved in 0.5 M
HCl–MeOH (50:50; 5 cm3). The solution was subsequently
stirred at 60 ЊC for 2 h. The product was then isolated by ion-
exchange chromatography using Dowex 50W anionic exchange
resin, eluting with a linear gradient of increasing concentration
of aqueous HCl–MeOH (0.5–3.0 M, 100 cm3 each). The prod-
uct was isolated as a light yellow, brittle foam (15 mg, 87%);
δH(250 MHz; CDCl3) 7.43 (2 H, s, imidazole-ring CH), 7.15
(2 H, s, imidazole-ring CH), 4.78 (2 H, m, 3Ј-HaHb, 3Љ-HaHb),
4.31 (4 H, m, 3Ј-HaHb, 3Љ-HaHb, 2Ј-H, 2Љ-H), 3.35 (2 H, m,
1Ј-HaHb, 1Љ-HaHb), 3.16 (10 H, m, 1Ј-HaHb, 1Љ-HaHb, 1-H, 3-H,
5-H, 7-H), 2.09 (4 H, m, 2-H, 6-H); δC(63 MHz; CDCl3) 144.50
(Ar-C), 129.10, 127.90 (Ar-CH), 66.00 (2Ј-CH, 2Љ-CH), 53.10
(3Ј-C, 3Љ-C), 49.90, 44.90, 44.89, 22.80 (1-C, 2-C, 3-C, 5-C, 6-C,
7-C, 1Ј-C, 1Љ-C); m/z (FAB) 470 (MHϩ, 31%), 329 (60), 301 (14),
264 (14), 233 (16), 205 (12), 176 (100) (Found: MHϩ, m/z
470.2475. C18H32N9O6 requires 470.2476).
᎐
stretch), 1120s (P᎐O stretch), 900m, 700m br (out of plane
᎐
Ar-CH stretch); δH(250 MHz; CDCl3) 7.73 (12 H, m, meta-ring
CH), 7.42 (18 H, m, ortho-ring CH, para-ring CH), 3.29 (2 H,
m, 2Ј-H, 2Љ-H), 3.04 (4 H, m, 1Ј-H, 1Љ-H), 2.76 (10 H, m, 1-H,
3-H, 5-H, 8-H, 3Ј-HaHb, 3Љ-HaHb), 2.31 (2 H, m, 3Ј-HaHb,
3Љ-HaHb), 1.72 (2 H, m, 2-H), 1.24 (4 H, m, 6-H, 7-H); δC(75
MHz; CDCl3) 133.40, 133.07, 132.10, 131.40 (Ar-C), 133.00,
133.89, 132.80, 132.76, 132.68, 132.64, 132.59, 131.51, 129.44,
128.39, 128.31, 128.24 (Ar-CH), 51.8, 51.7 (2Ј-C, 2Љ-C), 48.94,
47.33, 45.52, 45.43, 45.24, 43.46 (1-C, 3-C, 5-C, 8-C, 1Ј-C, 1Љ-C,
3Ј-C, 3Љ-C), 27.61, 26.21, 26.23 (2-C, 6-C, 7-C); δP(101 MHz;
CDCl3) 31.58 (s, N1-P), 31.46 (s, N8-P), 30.78 (s, N4-P); m/z
(FAB) 858 (MHϩ, 100%), 802 (5), 656 (10, loss of Ph P᎐O), 338
᎐
2
ϩ
ϩ
(30), 201 (96, Ph P᎐Oϩ ), 77 (32, Ph ) (Found: MH , m/z
ؒ
ؒ
᎐
2
858.3387. C49H55N3O5P3 requires 858.3354).
N1,N8-Bis[2Љ-hydroxy-3Љ-(2Ј-nitroimidazol-1Ј-yl)propyl]-
N1,N4,N8-tris(diphenylphosphinoyl)spermidine 28
N1,N4,N8-Tris(diphenylphosphinoyl)spermidine 26
Spermidine (0.588 g, 4.0 mmol) was dissolved in THF (50 cm3).
Triethylamine (1.5 cm3, 12 mmol) was added to the solution
which was subsequently stirred for 5 minutes. DPPCl (2.4 cm3,
9 mmol) was added by injection to the rapidly stirring solution
over a 5 minute period. After a further 1 hour stir, water
(10 cm3) was added to the reaction mixture and the organic
solvent was removed in vacuo . The residue was partitioned
between water and dichloromethane (50 cm3 each) and the
aqueous layer washed with dichloromethane (3 × 50 cm3). The
combined organic layers were dried over Na2SO4, after which
the solvent was removed in vacuo. The residue was purified by
flash chromatography (silica; 5% MeOH–CH2Cl2). Combined
fractions had their solvent removed in vacuo to leave a white,
brittle foam (2.27 g, 76%); νmax(CHCl3)/cmϪ1 3405w (N–H
Compound 27 (40 mg, 0.047 mmol) and 2-nitroimidazole (21
mg, 0.188 mmol) were dissolved in methanol (1 cm3) in a sealed
tube. Following the addition of triethylamine (6 equiv., 0.39
cm3) the reaction mixture was heated at 120 ЊC for 5 h. The
volatiles were then removed in vacuo and the residue was puri-
fied by flash chromatography (silica; 5% MeOH–CH2Cl2). The
combined fractions had solvent removed in vacuo to leave a
light yellow, viscous gum (37 mg, 72%); δH(250 MHz; CDCl3)
7.68 (12 H, m, meta-ring CH), 7.39 (18 H, m, ortho-ring CH,
para-ring CH), 7.21 (2 H, s, imidazole-ring CH), 7.01 (2 H, s,
imidazole-ring CH), 4.52 (2 H, pseudo d, 3Ј-HaHb, 3Љ-HaHb),
4.05 (2 H, br m, 2Ј-H, 2Љ-H), 3.99 (2 H, m, 3Ј-HaHb, 3Љ-HaHb),
3.02 (4 H, m, 1Ј-H, 1Љ-H), 2.69 (8 H, m, 1-H, 3-H, 5-H, 8-H),
1.68 (2 H, m, 2-H), 1.23 (4 H, m, 6-H, 7-H); δC(63 MHz;
CDCl3) 144.92, 133.47, 133.03, 132.24, 131.05 (Ar-CH), 133.00,
133.89, 132.70, 132.54, 132.06, 132.00, 131.39, 129.23, 128.37,
128.34, 128.23, 126.98 (Ar-CH), 68.3 (2Ј-C, 2Љ-C), 54.20 (3Ј-C,
3Љ-C), 50.65, 47.53, 45.61, 45.22, 43.44, 27.81, 25.91, 25.83 (1-C,
2-C, 3-C, 5-C, 6-C, 7-C, 1Ј-C, 1Љ-C); δP(101 MHz; CDCl3) 35.75
(d, N1-P), 35.47 (d, N8-P), 31.14 (s, N4-P); m/z (FAB) 1084
stretch), 3205w, 2930w, 1730w, 1410m, 1190s (P᎐O stretch),
᎐
1125s (P᎐O stretch); δ (300 MHz; CDCl ) 7.75 (12 H, m, meta-
᎐
H
3
ring CH), 7.38 (18 H, m, ortho-ring CH, para-ring CH), 4.58
(1 H, br pseudo q, 3JH-H 7.4 and 2JP-H 7.4, N1-H), 3.48 (1 H, br
3
2
pseudo q, JH-H 6.9, JP-H 6.9, N8-H), 3.12 (2 H, m, 1-H), 2.86
(6 H, m, 3-H, 7-H, 8-H), 1.74 (2 H, m, 2-H), 1.52 (2 H, m, 7-H),
1.34 (2 H, m, 6-H); δC(75 MHz; CDCl3) 132.22, 132.07, 131.98,
131.48 (Ar-C), 131.92, 131.89, 131.80, 131.76, 131.68, 131.64,
128.59, 128.51, 128.44, 128.39, 128.31, 128.24 (Ar-CH), 45.61,
43.12, 40.31, 37.90 (1-C, 3-C, 5-C, 8-C), 30.30, 29.34, 25.92
(2-C, 6-C, 7-C); δP(101 MHz; CDCl3) 31.80 (s, N4-P), 23.72
(s, N1-P), 23.61 (s, N8-P); m/z (FAB) 744 (M Ϫ Hϩ, 100%), 684
(14.2), 544 (8.9, loss of Ph2PO), 473 (17.9), 416 (2.9), 305 (16.8),
(MHϩ, 22%), 1039 (2), 1003 (9), 915 (26), 834 (4), 289 (15), 201
ϩ
(100, Ph P᎐Oϩ ), 77 (92, Ph ).
ؒ
ؒ
᎐
2
N1,N8-Bis[2Љ-hydroxy-3Љ-(2Ј-nitroimidazol-1Ј-yl)propyl]sperm-
idine hydrochloride 9
Compound 28 (46 mg, 0.042 mmol) was dissolved in 0.5 M
HCl–MeOH (50:50; 5 cm3). The solution was subsequently
stirred at 60 ЊC for 2 h. The product was then isolated by
ion-exchange chromatography using Dowex 50W anionic
exchange resin, eluting with a linear gradient of increasing con-
centration of aqueous HCl–MeOH (0.5–3.0 M, 100 cm3 each).
The product was isolated as a light yellow, brittle foam (22 mg,
87%); δH(300 MHz; CDCl3) 7.46 (2 H, s, imidazole-ring CH),
7.22 (2 H, s, imidazole-ring CH), 4.75 (2 H, m, 3Ј-HaHb,
3Љ-HaHb), 4.37 (4 H, m, 3Ј-HaHb, 3Љ-HaHb, 2Ј-H, 2Љ-H), 3.38
(2 H, m, 1Ј-HaHb, 1Љ-HaHb), 3.16 (10 H, m, 1Ј-HaHb, 1Љ-HaHb,
1-H, 3-H, 5-H, 8-H), 2.14 (2 H, m, 2-H), 1.79 (4 H, m, 6-H,
7-H); δC(63 MHz; CDCl3) 131.4, 130.5 (Ar-CH), 68.4 (2Ј-C,
2Љ-C), 55.4 (3Ј-C, 3Љ-C), 52.2, 52.1 (1Ј-C, 1Љ-C), 49.7, 49.6, 47.3,
47.1, 25.4 (2-C), 25.2, 25.1 (6-C, 7-C); m/z (FAB) 484 (MHϩ,
242 (14.9), 216 (74.0, Ph2PONH ϩ).
᝽
N1,N8-Bis(2Ј,3Ј-epoxypropyl)-N1,N4,N8-tris(diphenylphos-
phinoyl)spermidine 27
N1,N4,N8-Tris(diphenylphosphinoyl)spermidine 26 (100 mg,
0.13 mmol) was dissolved in THF (10 cm3). Additions of NaH
(31 mg, 0.78 mmol) and epichlorohydrin (1.1 cm3, 1.3 mmol)
were made to the stirred solution and the resulting mixture was
heated at 90 ЊC for 3 h. The mixture was allowed to cool to
room temperature and a further addition of NaH (20 mg, 0.39
mmol) was made. Following 15 minutes of stirring a further
addition of epichlorohydrin (10 equiv., 1.1 cm3) was made and
the reaction mixture re-heated to 90 ЊC where it was maintained
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252
3251

View Article Online
32%), 329 (60), 264 (23), 219 (26), 176 (100) (Found: MHϩ, m/z
484.2632. C19H34N9O6 requires 484.2632).
the residue was taken up in triethylammonium acetate buffer
(5 cm3, pH 7.4), to which was added dithiothreitol (0.32 mmol,
50 mg). The reaction mixture was then stirred for 30 minutes
before removal of water in vacuo. The product was purified by
ion-exchange chromatography using Dowex 50W anionic
exchange resin, eluting with a linear gradient of increasing con-
centration of aqueous HCl–MeOH (0.5–3.0 M, 200 cm3 each).
The product was isolated as a white, hygroscopic solid (133 mg,
78%); δH(250 MHz; CDCl3) 3.03 (2 H, t, J 7.5, 1Ј-H), 2.91 (8 H,
m, 1-H, 3-H, 5-H, 8-H), 2.62 (2 H, t, J 7.5, 2Ј-H), 1.90 (2 H, m,
2-H), 1.56 (4 H, m, 6-H, 7-H); δC(63 MHz; CDCl3) 50.20, 47.35,
44.73, 44.62, 39.10 (1-C, 3-C, 5-C, 8-C, 1Ј-C), 24.15 (2-C),
22.99, 22.84 (6-C, 7-C), 20.20 (2Ј-C); m/z (FAB) 206 (MHϩ,
100%), 180 (15), 165 (26) (Found: MHϩ, m/z 206.1691.
C9H24N3S requires 206.1691).
N1,N1Ј-(Dithiodiethylene)bis[N1,N4-methylene-N8-(tert-butoxy-
carbonyl)spermidine] 30
N1,N4-Methylene-N8-(tert-butoxycarbonyl)spermidine
13
(205.4 mg, 0.8 mmol) was dissolved in acetonitrile (3 cm3) and
transferred to a sealed tube. Following the addition of ethylene
sulfide (0.8 mmol, 48.5 cm3), the tube was sealed and the
reaction mixture heated at 80 ЊC for 3 days. After the removal
of acetonitrile in vacuo the residue was purified by flash
chromatography (silica; MeOH–1% conc. NH4OH solution).
The combined fractions had solvent removed in vacuo to leave
the desired product, a colourless viscous oil (37%). A second
product, the reduced form of the desired product, was collected
in a similar fashion as a colourless oil. This was allowed to
oxidise by stirring in dichoromethane (20 cm3) for 120 h to yield
the desired product which was subsequently combined with the
amount already collected (329 mg, 65%); νmax(CH2Cl2)/cmϪ1
Acknowledgements
We thank the Ministry of Defence for their generous financial
support.
2940s (aliphatic C–H stretch), 1710s (C᎐O stretch), 1500m,
᎐
1335m, 1250s, 1165m, 700m; δH(250 MHz; CDCl3) 4.86 (1 H, br
s, NH), 3.12 (2 H, s, 9-H), 3.07 (2 H, br m, 8-H), 2.75 (2 H, m,
1Ј-H or 2Ј-H), 2.68 (2 H, m, 2Ј-H or 1Ј-H), 2.48 (4 H, m, 3-H,
1-H), 2.30 (2 H, m, 5-H), 2.62 (2 H, m, 2-H), 1.42 (4 H, m, 6-H,
7-H), 1.37 [9 H, m, C(CH3)3]; δC(63 MHz; CDCl3) 156.43
References
1 J. D. Chapman, A. J. Franko and C. J. Koch, in Biological Bases and
Clinical Implications of Tumor Radioresistance, ed. G. H. Fletcher,
C. Nervi and H. R. Withers, Masson Publishers, New York, 1983.
2 G. E. Adams, C. Clarke, K. B. Dawson, P. W. Sheldon and I. J.
Stratford, in Biological Bases and Clinical Implications of Tumor
Radioresistance, ed. G. H. Fletcher, C. Nervi and H. R. Withers,
Masson Publishers, New York, 1983.
3 L. S. Kappen and I. H. Goldberg, Biochemistry, 1991, 30, 2034.
4 J. E. Biaglow, M. E. Varnes, J. Koch and R. Sridhar, in Free Radicals
and Cancer, ed. R. E. Floyd, Marcel Dekker, New York, 1981.
5 C. W. Porter, J. Bergeron and N. J. Stolowich, Cancer Res., 1982, 44,
4072.
(C᎐O), 79.32 [C(CH ) ], 76.15 (9-C, 9Љ-C), 54.90, 54.77, 52.68,
᎐
3
3
52.47, 40.74, 37.04, 28.84 [C(CH3)3], 28.26, 24.62, 23.44 (2-C,
6-C, 7-C); m/z (FAB) 633 (MHϩ, 48%), 631 (57, M Ϫ Hϩ), 316
(100, monomer), 282 (14), 260 (44), 215 (57, loss of Boc), 159
(50) (Found: MHϩ, m/z 633.4196. C30H61N6O4S2 requires
633.4196).
N1,N1Ј-(Dithiodiethylene)bis[N8-(tert-butoxycarbonyl)sperm-
idine] 31
6 P. M. Cullis, M. C. R. Symons, G. M. Cohen and P. Wardman,
Med. Sci. Res., 1990, 18, 87.
7 T. J. Thomas and T. Thomas, Biochem. J., 1994, 298, 485.
8 L. C. Gosule and J. A. Schellman, Nature, 1976, 259, 333.
9 D. L. Kramer, J. T. Miller, R. J. Bergeron, R. Khomutov,
A. Khomutov and C. W. Porter, J. Cell. Physiol., 1993, 155, 399.
10 C. W. Tabor and H. Tabor, Annu. Rev. Biochem., 1984, 53, 749.
11 P. Seppanen, L. Alhonen-Hongisto and J. Janne, Eur. J. Biochem.,
1980, 110, 7.
12 I. Wyatt, R. B. Moore and L. L. Smith, Int. J. Radiat. Biol., 1989, 55,
463.
13 C. A. Rinehart and K. Y. Chen, J. Biol. Chem., 1984, 259, 4750.
14 S. A. McCormack and L. R. Johnson, Am. J. Physiol., 1989, 256,
G 868.
Compound 30 (45 mg, 0.07 mmol) was dissolved in ethanol (1
cm3), followed by pyridine (0.22 mmol, 36 µl) and malonic acid
(0.26 mmol, 54 mg). The reaction mixture was refluxed for two
hours at 115 ЊC. Following the removal of volatiles in vacuo, the
residue was taken up in water (5 cm3) and washed with chloro-
form (3 × 5 cm3). The aqueous layer was adjusted to pH 11
by the addition of a few drops of 10% aqueous NaOH. The
aqueous layer was then extracted with chloroform (5 × 5 cm3)
and the combined chloroform extracts were dried over Na2SO4.
Removal of the solvent in vacuo isolated the product as a faintly
yellow, viscous oil (35 mg, 81%); νmax(CH2Cl2)/cmϪ1 2950s
15 N. Seiler and F. Dezeure, Int. J. Biochem., 1990, 22, 211.
16 C. W. Porter, J. Miller and J. Bergeron, Cancer Res., 1984, 44, 126.
17 J. L. Holley, A. Mather, R. T. Wheelhouse, P. M. Cullis, J. A.
Hartley, J. P. Bingham and G. M. Cohen, Biochem. Pharmacol.,
1992, 4, 763.
(aliphatic C–H stretch), 1710s (C᎐O stretch), 1450m, 1335m,
᎐
1250s, 710m; δH(250 MHz; CDCl3) 4.90 (1 H, br s, OCNH),
3.07 (2 H, s, 8-H), 2.89 (2 H, m, 1Ј-H or 2Ј-H), 2.79 (2 H, m,
2Ј-H or 1Ј-H), 2.62 (6 H, m, 1-H, 3-H, 5-H), 1.88 (2 H, br s,
D2O exchange, NH), 1.62 (2 H, m, 2-H), 1.43 (4 H, m, 6-H,
18 B. Ganem, Acc. Chem. Res.,1982, 15, 290.
19 K. Hoffmann, in The Chemistry of Heterocyclic Compounds, ed.
A. Weissberger, Interscience Publishers Ltd, London, 1953.
20 R. T. Wheelhouse, PhD Thesis, Leicester University, 1990.
21 P. M. Cullis, R. E. Green, L. Merson-Davies and N. G. Travis,
Chem. Biol., 1999, 6, 717.
7-H), 1.39 [9 H, m, C(CH ) ]; δ (63 MHz; CDCl ), 156.4 (C᎐O),
᎐
3
3
C
3
79.3 [C(CH3)3], 50.0, 48.8, 48.4, 40.9, 40.3, 39.1 (1-C, 3-C, 5-C,
8-C, 1Ј-C, 2Ј-C, 1Љ-C, 2Љ-C), 30.5, 28.3, 27.7 (2-C, 6-C, 7-C), 28.8
[C(CH3)3]; m/z (FAB) 610 (MH2ϩ, 100%), 304 (27, monomer),
272 (30, loss of C14H31N3O2S2), 136 (74) (Found: MH2ϩ, m/z
610.4274. C28H62N6O4S2 requires 610.4274).
22 P. Seppanen, Acta Chem. Scand., Ser. B, 1981, 35, 731.
23 G. L. Newton, J. A. Aguilera, J. F. Ward and R. C. Fahey, Radiat.
Res., 1996, 145, 776.
24 P. M. Cullis, R. E. Green, L. Merson-Davies and N. G. Travis,
Biochem. Soc. Trans., 1998, 26, 595.
N1-(2-Mercaptoethyl)spermidine hydrochloride 10
A solution of 31 (327 mg, 0.54 mmol) in 5 M HCl (5 cm3) was
stirred for 30 min. Following the removal of water in vacuo
Paper 9/03293B
3252
J. Chem. Soc., Perkin Trans. 1, 1999, 3243–3252