Protecting-group strategies for the synthesis of N4-substituted, and N1,N8-disubstituted spermidines, exemplified by hirudonine

Article abstract of DOI:10.1039/a806355i

Methods are described for the preparation of derivatives of the polyamines 1,4-diaminobutane (putrescine), and N′-(3-amihopropyl)-l,4-diaminobutane (spermidine) in which a particular amino group is modified with, e.g., a guanidino function. Specific amino groups in these polyamines were protected as ;V-trifluoroacetyl, and yV-4-azidobenzyloxycarbonyl derivatives, which were unmasked chemoselectively using methanolic ammonia, and dithiothreitol-triethylamine, respectively. Guanidino functions were introduced by an improved procedure in which an amino group was treated with 3,5-dimethyl-Ar-nitro-!//-pyrazole-l-carboximidamide in methanol to give a nitroguanidine derivative, from which the nitro group was removed by catalytic transfer hydrogenation. The methodology is exemplified by the development of efficient preparative routes to agmatine, and hirudonine. The integrity of the sequence of protection/deprotection leading to hirudonine was confirmed by a crystal-structure analysis of its sulfate. The effect of selected compounds on the uptake of putrescine into rat lung cells was determined, and showed that N⁴-(4-azidobenzyloxycarbonyl)spermidine was the best inhibitor (K_i = 3.4 μM).

Full text of DOI:10.1039/a806355i

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Protecting-group strategies for the synthesis of N 4-substituted and
N 1,N 8-disubstituted spermidines, exemplified by hirudonine
Bernard T. Golding,a* Andrew Mitchinson,a William Clegg,b Mark R. J. Elsegoodb and
Roger J. Griffina
a Department of Chemistry, Bedson Building, The University of Newcastle upon Tyne,
Newcastle upon Tyne, UK, NE1 7RU
b Chemical Crystallography Unit, Department of Chemistry, Bedson Building,
The University of Newcastle upon Tyne, Newcastle upon Tyne, UK, NE1 7RU
Received (in Cambridge) 11th August 1998, Accepted 20th October 1998
Methods are described for the preparation of derivatives of the polyamines 1,4-diaminobutane (putrescine) and
N-(3-aminopropyl)-1,4-diaminobutane (spermidine) in which a particular amino group is modified with, e.g.,
a guanidino function. Specific amino groups in these polyamines were protected as N-trifluoroacetyl and
N-4-azidobenzyloxycarbonyl derivatives, which were unmasked chemoselectively using methanolic ammonia
and dithiothreitol–triethylamine, respectively. Guanidino functions were introduced by an improved procedure in
which an amino group was treated with 3,5-dimethyl-N-nitro-1H-pyrazole-1-carboximidamide in methanol to
give a nitroguanidine derivative, from which the nitro group was removed by catalytic transfer hydrogenation. The
methodology is exemplified by the development of efficient preparative routes to agmatine and hirudonine. The
integrity of the sequence of protection/deprotection leading to hirudonine was confirmed by a crystal-structure
analysis of its sulfate. The effect of selected compounds on the uptake of putrescine into rat lung cells was
determined and showed that N4-(4-azidobenzyloxycarbonyl)spermidine was the best inhibitor (Ki = 3.4 µM).
functions of the polyamine. We have previously reported a
Introduction
method for preparing spermidine derivatives by condensation
of protected C3 and C4 fragments.10,11 In this paper we describe
a method which enables N1,N8-disubstituted derivatives 9 and
16 to be prepared reliably and efficiently from spermidine,
either directly or via intermediate protection at N4 with the
4-azidobenzyloxycarbonyl group12 and/or at N1 and N8 with
the trifluoroacetyl group (see compounds 13–15). The natural
products hirudonine 17 and agmatine 7 were used as test sys-
tems for exemplifying methodology, which included the use of
the reagent 3,5-dimethyl-N-nitro-1H-pyrazole-1-carboximid-
amide 4 for introducing N-nitroguanidino functions as pre-
cursors of guanidino functions. An improved crystal-structure
determination for hydrated hirudonine sulfate is also presented.
A preliminary communication on part of this work has
appeared.13
Polyamines are essential components of mammalian and plant
cells that participate in a variety of biological functions, often
in association with nucleic acids.1 The common polyamines are
putrescine (1,4-diaminobutane, 1), spermidine [N-(3-amino-
propyl)-1,4-diaminobutane, 2] and spermine [N,NЈ-bis(3-
aminopropyl)-1,4-diaminobutane, 3] (for the numbering system
for the nitrogen atoms of spermidine see structure 2 in Scheme
3). Cellular-uptake systems for polyamines have been character-
ised2,3 and utilised for transporting toxic species into cells,4–6 for
example via their covalent attachment to N4 of spermidine.6
Numerous derivatives of polyamines have been described both
as natural products7 and as synthetic analogues.8
We were also interested in the effect that the presence of
guanidino functions in a polyamine structure would have on
their cellular uptake. It has been surmised that the transporter
system exhibits molecular recognition towards polyamines
which have two positively charged nitrogen functions separated
by ~7 Å.3,14 This can account for the known selectivity of the
transporter for diamines H2N(CH2)nNH2 where n > 3.14 As
guanidino functions bind strongly to carboxylates,15 it was
expected that polyamines bearing guanidino functions would
also be efficiently transported. Guanylated polyamines may
also be expected to compete with natural polyamines in other
biologically important processes. Indeed, certain guanylated
polyamines have been recently shown to inhibit uptake of
spermidine by deoxyhypusine synthase, an enzyme implicated
in cell proliferation.16
Primarily due to the therapeutic potential of polyamine
derivatives, many studies have been concerned with their syn-
thesis.9 The strategy employed can be either the synthesis of
the polyamine de novo or a partial synthesis starting from
putrescine, spermidine or spermine. In the latter case, a major
challenge is to discriminate efficiently between the nitrogen
We describe preliminary results on the measurement of
the uptake of hirudonine and agmatine into rat lung cells. We
have also determined the uptake of N4-(4-azidobenzyloxy-
carbonyl)spermidine 14 into cells, with a view to developing
analogous spermidine derivatives as selective, intracellular
enzyme inhibitors.
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356
349

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in 47% yield has been reported in the patent literature, using
Results and discussion
Preparation of guanidine derivatives using 3,5-dimethyl-N-nitro-
1H-pyrazole-1-carboximidamide (DMNPC) 4
similar reagents but under different reaction conditions.28
Simultaneous removal of the benzyloxycarbonyl and nitro pro-
tecting groups from compound 6 was achieved using catalytic
transfer hydrogenation,29 with formic acid as the hydrogen
donor. Addition of sulfuric acid to the reaction mixture precipi-
tated agmatine sulfate 7 in 52% yield.
Amines can be converted directly into guanidines by treatment
with O-methylisourea,17 or S-methylisothiourea.18 However,
the basicity of guanidines may present difficulties in carrying
a free guanidine group through several steps of a synthetic
sequence. An alternative approach is to use a nitroguanidine as
a masked guanidine. The nitroguanidino function is essentially
non-basic and unaffected by most reaction conditions, which
allows synthetic manipulations to be performed elsewhere in a
molecule prior to reduction of the nitroguanidine to release the
desired guanidine end-product. For the conversion of amines
into nitroguanidines, we have used 3,5-dimethyl-N-nitro-1H-
pyrazole-1-carboximidamide (DMNPC) 4.10,19
The benzyloxycarbonyl protecting group of compound 6
could be removed independently of reduction of the nitro-
guanidine group. This was originally attempted using tri-
methylsilyl iodide, either used directly,30 or generated in situ
from trimethylsilyl chloride and sodium iodide.31 Neither of
these methods was satisfactory, with the maximum yield
attained being only 39%. The reaction was best performed
using hydrobromic acid28 (see Scheme 1), which generated 1-(4-
aminobutyl)-2-nitroguanidine hydrobromide 8 in 88% yield.
Treatment of nitroguanidine with aqueous hydrazine hydrate
gave nitroaminoguanidine (1-amino-2-nitroguanidine),20 which
was condensed with pentane-2,4-dione in water, containing
acetic acid as a catalyst, to yield DMNPC 4.19 Reactions of
amines with DMNPC have been previously carried out in 1,4-
dioxane,19 which we sought to replace because of its toxicity
and low polarity. As a model for nitroguanidinations, the reac-
tion of benzylamine with DMNPC leading to N-benzyl-NЈ-
nitroguanidine was studied under a variety of conditions of
solvent, and in the presence or absence of a putative catalyst
(metal ion, acid or base). The cleanest and fastest reactions
were those in methanol (80% yield of N-benzyl-NЈ-nitro-
guanidine, which precipitated directly from the reaction mix-
ture), for which no useful catalysts were found. Indeed, the use
of either erbium() or holmium() trichloride as ‘catalyst’
diverted the reaction to afford O-methyl-N-nitroisourea.
Synthesis of N4-substituted spermidines 10–12 and 14
Reaction of spermidine with four equivalents of ethyl trifluoro-
acetate, in the presence of one equivalent of water in
acetonitrile under reflux, afforded N1,N8-bis(trifluoroacetyl)-
spermidine trifluoroacetate 9 as a crystalline solid in 89%
yield.13 Selective trifluoroacetylations of other amines and
polyamines by this technique have been reported.9,32–34
Although a number of methods have been reported35–45 for the
selective protection or reaction of primary amino groups in
polyamines, our procedure offers technical simplicity and the
use of readily available reagents. The reason for the selectivity
observed in these reactions is not clear. Nagao and Fujita35
observed selectivity for the primary amino groups of spermid-
ine in reactions with 3-acyl-1,3-thiazolidine-2-thiones and
explained this by postulating a conformation for spermidine
that would reduce the nucleophilicity of N4. However, the
observed selectivities may be merely a reflection of the different
basicities and hence nucleophilicities of the amino functions
(with the first protonation occurring predominantly at the N8
position, then at N1 and finally at N4).46,47 The direct synthesis
of N1,N8-disubstituted spermidines is further illustrated by the
synthesis of hirudonine 17, described below. However, the
success of this method for preparing N1,N8-disubstituted sper-
midines depends on achieving direct crystallisation and/or easy
separation of the desired product. Our method for preparing
N1,N8-disubstituted spermidines, which necessarily has more
steps, is completely reliable for securing the desired product.
Conversion of compound 9 into N1,N8-bis(trifluoroacetyl)-
N4-(4-azidobenzyloxycarbonyl) spermidine 13 was achieved in
excellent yield by treatment of compound 9 with N-ethyl-
diisopropylamine (Hünig’s base) and 4-azidobenzyl 4-nitro-
phenyl carbonate12 in THF. Reaction of compound 13 with
conc. ammonia in methanol gave N4-(4-azidobenzyloxy-
carbonyl)spermidine 14 in 82% yield (see Scheme 3).
Synthesis of agmatine 7
Agmatine is the mono-guanidinated derivative of putrescine,
formed by arginine decarboxylation, and is distributed widely
throughout Nature. It has been found in plants,21 bacteria22 and
some higher life forms, including mammals.23 Recently, this
compound and analogues have assumed considerable import-
ance because of the ability of agmatine to inhibit nitric oxide
synthase,24 its affinity for α-adrenoceptors, e.g. the I1 receptor,25
and its suppression of proliferation by reducing cellular levels
of polyamines.26
To exemplify our methodology we have prepared agmatine as
shown in Scheme 1. Nitroguanidination of N-benzyloxy-
Compound 9 is an excellent starting material for the prepar-
ation of N4-alkylated spermidines from epoxides. Although salt
9 reacts directly with epoxides in methanol, the yields are low,
and catalysing the reaction using lithium perchlorate48 in
acetonitrile was found to give much better yields. Thus, alkyl-
ation of compound 9 with styrene oxide in the presence of
catalytic lithium perchlorate and Hünig’s base in acetonitrile
gave a mixture of regioisomers 10 and 11 (ratio of 3:1, respect-
ively) in 89% total yield (see Scheme 2). Similarly, reaction of
salt 9 with propylene oxide gave compound 12 in 83% yield.
Scheme
1
Synthesis of agmatine sulfate 7 and intermediate 8.
Reagents and conditions: (i) Na2CO3 (10% w/v); 1.2 equiv. DMNPC 4,
MeOH, 25 ЊC, 3 days; (ii) HCO2H–MeOH (5% v/v), 10% Pd/C (1 mass
equiv.), 25 ЊC, 2 h; 1 equiv. H2SO4; (iii) HBr in AcOH (45% w/v), 25 ЊC,
1 h.
Synthesis of hirudonine 17
‘Orthogonally’ protected polyamines, in which each amino
function is protected with a different group capable of being
removed independently, are required for the synthesis of com-
plex polyamine derivatives. Spermidine and related compounds
are attractive templates for combinatorial synthesis,49 but their
exploitation, especially in parallel syntheses, requires efficient
carbonyl-1,4-diaminobutane 5 (obtained from the correspond-
ing hydrochloride27) with DMNPC 4 in methanol gave the
nitroguanidine 6 in 85% yield. The preparation of compound 6
350
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356

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Scheme 2 Alkylation of salt 9 with epoxides. Reagents and conditions: (i) EtNiPr2, styrene oxide, LiClO4, CH3CN, 25 ЊC, 24 h; (ii) EtNiPr2,
propylene oxide, LiClO4, CH3CN, 25 ЊC, 24 h (nb all compounds in this scheme are racemic).
Scheme 3 Synthesis of hirudonine sulfate 17. Reagents and conditions: (i) 4 equiv. CF3CO2Et, 1 equiv. water, CH3CN, reflux, 7 h; (ii) 1.1 equiv. 4-
azidobenzyl 4-nitrophenyl carbonate, 2.1 equiv. EtNiPr2, THF, 25 ЊC, dark, 24 h; (iii) conc. aq. NH3, MeOH, 25 ЊC, dark, 6 days; (iv) 2 equiv.
DMNPC 4, MeOH, 25 ЊC, dark; (v) 4 equiv. DTT, 4 equiv. Et3N, MeOH–water (9:1 v/v), 25 ЊC, dark, 4 h; (vi) HCO2H–MeOH (5% w/v), 10% Pd/C
(0.5 mass equiv.), 25 ЊC, 3 h; H2SO4.
methods for stepwise manipulation of the polyamine function-
alities. Various syntheses of orthogonally protected polyamines
have been reported, and are exemplified by the work of
Bergeron and McManis.50 Protecting groups with unusual
conditions for removal are of great potential in polyamine
synthesis. The use of the protecting group 4-azidobenzyl-
oxycarbonyl for this purpose is illustrated (see Scheme 3) in the
synthesis of the N1,N8-bis-guanidinated derivative of spermi-
dine (hirudonine 17), isolated as its sulfate. This substance has
been found in the central nervous system of the leech Hirudo
medicinalis L.51,52
Reaction of compound 14 (Scheme 3) with two equivalents
of DMNPC in methanol, at saturating concentrations of
the reactants, yielded N1,N8-bis(nitroguanidinyl)-N4-(4-azido-
benzyloxycarbonyl)spermidine 15 in 81% yield, and sufficiently
pure for the next stage. Removal of the N4-protecting group
from carbamate 15 by reduction with dithiothreitol (DTT)
in aq. methanol afforded N1,N8-bis(nitroguanidinyl)spermidine
16 in 50–60% yield. Compound 16 was reduced by catalytic
transfer hydrogenation to give hirudonine 17, isolated as its
sulfate. This could be obtained by addition of sulfuric acid to
the reaction mixture, once the Pd/C catalyst had been removed
by filtration. In this way the desired product was isolated in 89%
yield. The structure of this material was confirmed by an X-ray
analysis (see below).
With an authentic specimen of hirudonine sulfate in hand, it
was possible to explore two direct approaches to this com-
pound. In one approach, spermidine was allowed to react with
two equivalents of DMNPC in methanol. A solid precipitated
from the reaction solution, which was subjected to catalytic
transfer hydrogenation as before, to give hirudonine sulfate
with an overall yield of 66%. The synthesis of hirudonine has
been reported in a one-step procedure, by the reaction of
spermidine directly with O-methylisothiourea sulfate.53 We
confirm that this reaction does indeed give hirudonine sulfate,
which we obtained in 47% yield.
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356
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Table 1 Geometry of the triprotonated hirudonine molecule, exclud-
ing H atoms
Bond length/Å
N(1)–C(1)
C(1)–N(3)
N(5)–C(9)
C(9)–N(7)
1.323(7)
1.323(7)
1.322(8)
1.343(7)
N(2)–C(1)
C(9)–N(6)
1.335(7)
1.309(8)
Bond angle (Њ)
N(1)–C(1)–N(3)
N(3)–C(1)–N(2)
N(6)–C(9)–N(7)
119.6(5)
120.9(5)
118.5(6)
N(1)–C(1)–N(2)
N(6)–C(9)–N(5)
N(5)–C(9)–N(7)
119.5(5)
122.8(6)
118.7(6)
Fig. 1 Structure of the triprotonated hirudonine molecule with hydro-
gen bonds to twelve adjacent oxygen atoms of sulfate anions and water
molecules, and crystallographic numbering scheme.
Torsion angle (Њ)
Crystal structure of hydrated hirudonine sulfate
N(1)–C(1)–N(3)–C(2) Ϫ174.0(5) N(2)–C(1)–N(3)–C(2)
C(1)–N(3)–C(2)–C(3) Ϫ180.0(5) N(3)–C(2)–C(3)–C(4) Ϫ179.6(5)
C(2)–C(3)–C(4)–C(5) Ϫ178.8(5) C(3)–C(4)–C(5)–N(4) 179.9(5)
175.5(5) C(5)–N(4)–C(6)–C(7) Ϫ178.9(5)
6.0(8)
It is conceivable that intramolecular group migration can occur
between nitrogen functions in spermidine and it was therefore
considered essential to confirm the regioselectivity of the
sequences described. We also wished to determine the conform-
ation of hirudonine 17, which was of interest in the context of
protein and DNA binding studies. The structure has been pre-
viously reported,54 but the data were of poor quality and the
structural results of low precision. We have obtained more
satisfactory results from data collected at low temperature,
facilitating a reasonable modelling of the disorder in the sulfate
anions and water molecules.
C(4)–C(5)–N(4)–C(6)
N(4)–C(6)–C(7)–C(8)
179.6(5) C(6)–C(7)–C(8)–N(5)
C(7)–C(8)–N(5)–C(9) Ϫ176.0(5) C(8)–N(5)–C(9)–N(6)
C(8)–N(5)–C(9)–N(7) Ϫ178.0(5)
179.2(5)
3.4(8)
conditions, making it a poor substrate for the receptor. The best
inhibitor was compound 14, a spermidine derivative, in which
the terminal amino groups had been left intact. This was
accepted at the receptor despite the bulky group attached at the
N4-position.
The structure of the fully ordered cation is shown in Fig. 1,
together with twelve neighbouring oxygen atoms to which it is
hydrogen bonded. All the hydrogen atoms of the cation were
clearly revealed in a difference synthesis and were refined sub-
ject to the constraints of a riding model with C–H and N–H
distances appropriate to X-ray diffraction. The hirudonine
molecule is unambiguously triprotonated in such a way that the
The substrate-binding sites of polyamine transporters are
thought to contain carboxylate groups,13 which interact with
protonated polyamines. In some lactate dehydrogenase
enzyme–substrate complexes, replacement of an amino residue
in the binding site with a guanidine residue results in stronger
interactions to the carboxy group of the substrate.62 The failure
of hirudonine 17 to inhibit putrescine uptake was therefore
surprising given the result for agmatine 7, and that methylgly-
oxal bis(guanylhydrazone) (MGBG) and its analogues are well
known guanidinated compounds taken up by these trans-
porters.14 It would be interesting to test hirudonine 17 and
agmatine 7 in other models for polyamine uptake, such as the
murine B16 melanoma cell line. Further studies upon com-
pound 14 are also desirable, to test for its cytotoxic properties
and for its behaviour upon irradiation with UV light.
ϩ
central nitrogen is NH2 and all four terminal nitrogen atoms
are NH2. The essentially planar guanidine groups have com-
pletely delocalised bonding with all their C–N bonds of similar
length (Table 1). All twelve N–H bonds are involved in hydro-
gen bonding to oxygen atoms of sulfate anions and water
molecules, with H ؒ ؒ ؒ O distances in the range 1.81–2.10 Å,
N ؒ ؒ ؒ O 2.71–2.96 Å, and N–H ؒ ؒ ؒ O angles of 162–174Њ except
for one value of 139Њ.
The cation adopts a fully extended (all-anti) conformation in
this crystal structure, with all torsion angles (excluding H-
atoms) within 6.2Њ of 180Њ (or 0Њ in two N–C–N–C cases). A
similar extended conformation has been found in several
derivatives of spermine and spermidine,55–58 while a single
gauche segment occurs in others.59–61
Experimental
General procedures
All commercial chemical reagents used were of analytical grade
or the highest available purity. When necessary, reagents and
solvents were purified by standard procedures.12,63 Light petrol-
eum was the fraction boiling between 40 and 60 ЊC and ether
refers to diethyl ether. Solvents were removed either on a rotary
evaporator (ambient temperature; 10–15 mmHg) or by short-
path distillation (ambient temperature; 0.001–0.01 mmHg).
Water was removed with a freeze-dryer (LSL Secfroid, 0.05–0.2
mmHg).
The sulfate anions are each two-fold disordered. Disorder
also affects most of the water molecules in the crystal structure.
Our disorder model is consistent with a tetrahydrate formu-
lation,54 and with normal hydrogen-bonding geometries.
Inhibition of putrescine uptake into lung cells by spermidine
derivatives
The inhibitory effect upon the uptake of 14C-putrescine into rat
lung slices was determined for agmatine 7, hirudonine 17, 1-(4-
aminobutyl)-2-nitroguanidine 8 and N4-(4-azidobenzyloxy-
carbonyl)spermidine 14 by using the method of Smith and
Wyatt.14 All of the compounds were inhibitors of putrescine
uptake except for hirudonine 17, which showed no significant
effect (KI-values/µM: 5.3 0.2 for 7, 65 12 for 8 and 3.4 1.3
for 14). Identical Vmax-values were obtained when calculated in
the absence or presence of the compounds tested, indicating
that competitive inhibition was occurring. Pre-incubation
studies with compounds 7 and 14 confirmed the competitive
nature of their inhibition of putrescine uptake.
TLC was performed on silica gel 60F254 (Merck Art. No.
1.05554) with visualisation of spots by UV light (254 nm), or by
exposure to ninhydrin, potassium permanganate or molybdic
acid solution, followed by heating to ~80 ЊC. For ‘flash’ chrom-
atography, Fisons Matrix silica 60, mesh 0.035–0.070 mm was
used. Mps were measured on a Kofler hot stage or Gallenkamp
apparatus and are uncorrected. CHN analyses were performed
with a Carlo-Erba Instrumentazione model 1106 analyser. IR
spectra were recorded on a Nicolet 20PC Fourier transform
spectrometer. NMR spectra were recorded on Bruker instru-
1
ments, at 200, 300 or 500 MHz for H, and with broad-band
Compound 8 was a much less effective inhibitor of putrescine
uptake than was agmatine 7, its close analogue. This was pre-
sumably because compound 8 contains a non-basic nitroguani-
dine group, which would not be protonated under physiological
decoupling at 50, 75 or 125 MHz for 13C. Chemical shifts and
coupling constants are recorded in units of ppm and Hz,
respectively. Chemical shifts labelled with an asterisk are
exchangeable with D2O. Electron impact (EI) and fast-atom
352
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356

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bombardment (FAB) mass spectra were recorded on a Kratos
MS80RF spectrometer. FAB spectra were recorded using a
matrix of 3-nitrobenzyl alcohol unless otherwise stated. The
relative intensities of the peaks are given in parentheses.
Agmatine sulfate 7
To 1-[4-(benzyloxycarbonylamino)butyl]-2-nitroguanidine
6
(0.25 g, 0.81 mmol) in a formic acid–methanol mixture (5% w/v,
20 ml) was added 10% Pd/C (0.25 g). The mixture was stirred
at room temperature under nitrogen for 2 h. The catalyst was
filtered off and sulfuric acid (0.5 M; 1.61 ml, 0.81 mmol) was
added to the solution. Upon cooling at 5 ЊC, crystals of agma-
tine sulfate 7 were produced (96 mg, 52%), homogeneous by
TLC (silica gel; 4:1:1:2 v/v butan-1-ol–acetic acid–pyridine–
water; ninhydrin detection, Rf 0.21), mp 227–228 ЊC (lit.,64
Uptake of compounds into rat lung slices
Measurements of the uptake of compounds into rat lung slices
were performed at the MRC Toxicology Unit, Leicester using
the methods of Smith, Wyatt and co-workers.3,14
ϩ
229 ЊC); νmax/cmϪ1 (KBr disc) 3335 (NH2 of guanidine), 3172
Nitroaminoguanidine (1-amino-2-nitroguanidine)20
ϩ
(NH of guanidine), 3023 (NH3 of amine), 2965 and 2881
To an agitated slurry of 2-nitroguanidine (2.83 g, 27 mmol) in
water (40 ml) was added aq. hydrazine hydrate (1.32 ml, 1.36 g,
27.2 mmol) in water (20 ml) dropwise. The temperature in the
flask was maintained at 60–65 ЊC until all the hydrazine had
been added. The temperature was then held at 55–60 ЊC for a
further 15 min with constant stirring. The slurry cleared to
a solution, which turned from colourless to yellow, and finally
to orange. The mixture was cooled rapidly to below 45 ЊC,
neutralised with hydrochloric acid (10 M) to prevent further
reaction, and cooled at 5 ЊC overnight. A precipitate was pro-
duced which was collected by filtration, washed with ice-cold
water and air-dried. The crude solid was recrystallised twice
from water to give nitroaminoguanidine as a solid (1.04 g,
32%) homogeneous by TLC (silica gel; 20% v/v methanol in
dichloromethane; UV detection) Rf 0.34, mp 187–188 ЊC
(decomp.) lit.,20 190 ЊC); δH(200 MHz; d6-DMSO) 4.80 (2H, s,
NH2),7.67(1H,brs,NHNH2),8.39(1H,brs,oneofNH2)and9.45
(1H, br s, one of NH2); δC(50 MHz; d6-DMSO) 161.8
(CH ) and 1636 (C᎐N); δ (200 MHz; D O) 1.69 (4H, m,
᎐
2 H 2
CH2CH2CH2CH2), 3.01 (2H, br m, CH2NH3ϩ), 3.28 [2H, br m,
CH NHC(᎐NH )NH ϩ], 7.73* (3H, br s, CH2NH3ϩ), 8.02* [2H,
᎐
᎐
2
2
2
br s, NHC(᎐NH )NH ϩ], 8.67* [1H, br s, NHC(᎐NH )NH ϩ];
᎐
2
2
2
2
δC(50 MHz; D2O) 25.5 and 26.5 (CH2CH2CH2CH2), 40.5
ϩ
and 42.0 [CH2NH3 and CH NHC(᎐NH )NH ϩ] and 158.3
᎐
2
2
2
[NHC(᎐NH )NH ϩ]; m/z (FAB) 131 (M ϩ 1, 100%).
᎐
2
2
1-(4-Aminobutyl)-2-nitroguanidine hydrobromide 8
1-[4-(Benzyloxycarbonylamino)butyl]-2-nitroguanidine 6 (0.50
g, 1.6 mmol) in hydrobromic acid–acetic acid (45% w/v,
4.4 ml) was stirred at rt for 1 h. Ether was added dropwise
to the solution to give a precipitate, and the mixture was
chilled overnight at 5 ЊC. The solid was filtered off, and re-
crystallised from methanol–ether, to give (4-aminobutyl)-
nitroguanidine hydrobromide 8 as crystals (0.37 g, 88%),
homogeneous by TLC (silica gel; 4:1:1:2 v/v butan-1-ol–acetic
acid–pyridine–water, ninhydrin detection, Rf 0.40), mp 172–
174 ЊC (lit.,28 173–175 ЊC); δH(200 MHz; d6-DMSO) 1.63
(4H, m, CH2CH2CH2CH2), 2.91 (2H, m, H3NϩCH2CH2), 3.28
[H NC(᎐NNO )NHNH ].
᎐
2
2
2
3,5-Dimethyl-N-nitro-1H-pyrazole-1-carboximidamide 4
[2H, m, CH NHC(᎐NNO )NH ], 7.73* (2H, br s, H NϩCH ),
᎐
2
2
2
3
2
To a solution of nitroaminoguanidine (0.70 g, 5.9 mmol) in
boiling water (130 ml) were added glacial acetic acid (1 ml) and
pentane-2,4-dione (0.77 ml, 0.75 g, 7.5 mmol) with vigorous
stirring. The solution was allowed to cool to rt, and was stirred
for 7 h. A precipitate was formed. This was filtered off, and
recrystallised from ethanol to give 3,5-dimethyl-N-nitro-1H-
pyrazole-1-carboximidamide DMNPC 4 as crystals (0.86 g,
80%), homogeneous by TLC (silica gel; 20% v/v methanol
in dichloromethane; UV detection) Rf 0.79, mp 127–128 ЊC
(lit.19 125–126 ЊC); δH(200 MHz; CDCl3) 2.18 (3H, s, CH3), 2.55
(3H, s, CH3), 5.98 (1H, s, CH), 8.14 (1H, br s, one of NH2)
and 9.04 (1H, br s, one of NH2); δC(50 MHz; CDCl3) 13.7 and
15.9 (2 × CH3), 112.6, 144.8 and 153.8 (ring carbons) and 155.7
8.02* [2H, br s, NHC(᎐NNO )NH ] and 8.67* [1H, br s,
᎐
2
2
NHC(᎐NNO )NH ]; m/z (FAB) 176 (M ϩ 1, 100%), 102
᎐
2
2
(M Ϫ H2NNO2, 18), 89 [M Ϫ C(NNO2)NH2, 25] (Found: C,
23.7; H, 5.4; N, 26.9. C5H13N5O2ؒHBr requires C, 23.4; H, 5.5;
N, 27.3%).
N1,N8-Bis(trifluoroacetyl)spermidine trifluoroacetate 9
To a stirred solution of spermidine (1.01 g, 7.0 mmol) in
acetonitrile (25 ml) were added water (0.126 ml, 7.0 mmol) and
ethyl trifluoroacetate (3.4 ml, 4.1 g, 29 mmol). The mixture was
heated at reflux for 7 h, and then cooled to 5 ЊC. The pure
product which precipitated was collected by filtration under
reduced pressure. The filtrate was concentrated to give a crude
product, which was recrystallised from acetonitrile to give
pure N1,N8-bis(trifluoroacetyl)spermidine trifluoroacetate 9 as
crystals, which were combined with the initially precipitated
material (combined yield 2.81 g, 89%). The substance was
homogeneous by TLC (silica gel, 4:1 v/v dichloromethane–
methanol; potassium permanganate detection, Rf 0.65), mp
145–146 ЊC: νmax/cmϪ1 (KBr disc) 3319 (NH), 2960 and 2877
[H NC(᎐NNO )NHNH ].
᎐
2
2
2
1-[4-(Benzyloxycarbonylamino)butyl]-2-nitroguanidine 6
N-Benzyloxycarbonyl-1,4-diaminobutane
hydrochloride27
(0.70 g, 2.7 mmol) was dissolved in aq. sodium carbonate (10%
w/v; 10 ml) and the resultant solution was extracted with
dichloromethane (3 × 5 ml). The organic layers were combined
and concentrated to give the free amine. This was taken up in
methanol (10 ml) and DMNPC 4 (0.57 g, 3.1 mmol) was added
with stirring. The mixture was stirred at rt for 3 days. The
solvent was removed and the crude product was purified by
‘flash’ chromatography (silica, elution with 5% v/v methanol in
dichloromethane) to give 1-[4-(benzyloxycarbonylamino)butyl
2-nitroguanidine 6 as a solid (0.71 g, 85%) homogeneous by
TLC (silica gel; 20% v/v methanol in dichloromethane; UV
detection, Rf 0.72), mp 127–129 ЊC (lit.,28 123–124.5 ЊC); δH(200
MHz; d6-acetone) 1.66 (4H, m, CH2CH2CH2CH2), 3.24 (2H, br
(CH ), 1706 and 1671 (C᎐O), 1561 (CO Ϫ) and 1193 (CF3);
᎐
2
2
δH(200 MHz; d6-DMSO) 1.65 (4H, m, CH2CH2CH2CH2),
1.92 (2H, m, NH2ϩCH2CH2CH2NH), 3.01 (4H, br t, CH2-
NH2ϩCH2), 3.32 (4H, m, 2 × CF3CONHCH2), 8.81 (2H, m,
2 × CF3CONH) and 9.68 (2H, br s, CH2NH2ϩCH2); δC(50
MHz; d6-DMSO) 26.9, 29.1 and 29.3 (CH2CH2CH2-
NH2ϩCH2CH2), 40.6 and 42.2 (2 × CF3CONHCH2), 48.5 and
50.4 (CH2NH2ϩCH2), 120 (2 × overlapping q, JCF 288,
2 × CF3CONH), 120.7 (q, JCF 296, CF3COOϪ), 160.4 (two
overlapping q, JCF 44, 2 × CF3CONH) and 162.8 (q, JCF 32,
CF3COOϪ); m/z (EI) 338 (Mϩ, 100%), 197 (M Ϫ CH3CH2-
NHCOCF3, 11) and 183 (M Ϫ CF3CONHCH2CH2CH3, 21)
(Found: C, 34.7; H, 3.8; N, 9.1. C13H18F9N3O4 requires C, 34.6;
H, 4.0; N, 9.3%).
s, NH ), 3.41 [2H, m, CH NHC(᎐NNO )NH ], 4.74 (2H, br s,
᎐
2
2
2
2
NHCO2), 6.52 [0.4H, br s, CH2NHC(NNO2)NH2 tautomer A],
7.37 (5H, m, ArH) and 7.66 [0.6H, br s, CH2NC(NHNO2)NH2,
tautomer B]; m/z (FAB) 310 (M ϩ 1, 100%) (Found: C, 50.9; H,
6.1; N, 22.6. C13H19N5O4 requires C, 50.5; H, 6.15; N, 22.65%).
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356
353

View Online
N4-(4-Azidobenzyloxycarbonyl)-N1,N8-bis(trifluoroacetyl)-
spermidine 13
tography (silica gel; 15% v/v methanol in dichloromethane;
UV detection) to give N4-(4-azidobenzyloxycarbonyl)-N1,N8-
bis(nitroguanidino)spermidine 15 as a pale yellow solid (0.690 g,
81%) homogeneous by TLC (silica gel; 10% v/v methanol in
To a mixture of N1,N8-bis(trifluoroacetyl)spermidine trifluoro-
acetate 9 (0.44 g, 0.97 mmol) in dry, stirred THF (6 ml) was
added N-ethyldiisopropylamine (0.35 ml, 2.0 mmol), followed
by a solution of 4-azidobenzyl 4-nitrophenyl carbonate (0.35 g,
1.11 mmol) in THF (3 ml) over a period of 5 min. The mixture
was stirred at rt under nitrogen in the dark for 24 h. The sol-
vent was removed to give a brown oil, which was taken up in
dichloromethane (5 ml). The resulting solution was washed
successively with aq. sodium hydroxide (0.1 M; 3 × 15 ml) fol-
lowed by saturated sodium aq. chloride (3 × 30 ml). The
organic layer was concentrated to give the crude product, which
was purified by ‘flash’ chromatography (silica gel; 2% v/v
methanol in dichloromethane; UV detection) to give N1,N8-
bis(trifluoroacetyl)-N4-(4-azidobenzyloxycarbonyl)spermidine
13 as a yellow oil (0.45 g, 92%), homogeneous by TLC (silica
gel; 2% methanol in dichloromethane v/v; UV detection; Rf
0.31); νmax/cmϪ1 (cap film) 3310 (NH), 3104 (ArH), 2946 and
dichloromethane; UV detection; Rf 0.26), mp 144–146 ЊC: νmax
cmϪ1 (KBr disc) 3407 (NH2), 2951 and 2881 (CH2), 2119 (N3),
1685 (CO), 1642 (C᎐N) and 1595 (NO ); δ (200 MHz; d -
/
᎐
2
H
6
DMSO) 1.55 (4H, br m, CH2CH2CH2CH2), 1.79 (2H, m,
NHCH2CH2CH2N), 3.25 (4H, m, CH2NCH2), 3.48 [4H, m,
2 × CH2NHC(NNO2)NH2], 5.13 (2H, s, CH2OCO), 7.21 (2H,
d, J 8.3, 2 × ArH), 7.48 (2H, d, J 8.3, 2 × ArH), 8.03* (4H, br s,
2 × NH2) and 8.67* (2H, br s, 2 × NH); δC(50 MHz; d6-DMSO)
25.2, 25.9 and 28.2 (CH2CH2CH2CH2NCH2CH2), 44.1, 45.0,
46.5 and 47.1 [2 × CH2NHC(NNO2)NH2 and CH2NCH2],
119.4 (ArC), 129.7 (ArC), 134.2 (ArC), 139.2 (ArC), 155.6 (CO)
and 159.5 [2 × NHC(NNO2)NH2]; m/z (FAB) 495 (M ϩ 1,
100%) and 320 (M Ϫ CO2CH2C6H4N3, 17) (Found: C, 41.4; H,
5.2; N, 33.3. C17H26N12O6 requires C, 41.3; H, 5.3; N, 34.0%).
N1,N8-Bis(nitroguanidino)spermidine 16
2888 (CH ), 2120 (N ), 1705 (C᎐O) and 1169 (CF ); δ (200
᎐
2
3
3
H
MHz; CDCl3) 1.48 (4H, s, CH2CH2CH2CH2), 1.63 (2H, m,
NCH2CH2CH2N), 3.23 (8H, m, 2 × CF3CONHCH2, CH2-
NCH2), 5.03 (2H, s, OCH2Ar), 6.36 [1H, br s, CONH(CH2)4],
6.95 (2H, d, J 8.5, 2 × ArH) and 7.27 [1H, d, J 8.5, 2 × ArH),
7.92 (1H, br s, CONH(CH2)3]; δC(50 MHz; CDCl3) 25.4, 26.3
and 27.1 (CH2CH2CH2CH2NCH2CH2), 36.4 and 39.6 (2 ×
CF3CONHCH2), 44.2 and 46.5 (CH2NCH2), 67.3 (CO2CH2),
115.9 (two overlapping q, JCF 289, 2 × CF3CONH), 119.2
(ArC), 130.0 (ArC), 133.0 (ArC), 140.5 (ArC), 157.1 (CO2CH2),
157.5 (two overlapping q, JCF 34, 2 × CF3CONH); m/z (FAB)
513 (M ϩ 1, 2%), 469 (M Ϫ N3, 3), 364 (M Ϫ OCH2C6H4N3,
15) and 338 (M Ϫ CO2CH2C6H4N3, 13) (Found: C, 45.6; H, 4.1;
N, 16.5. C19H22F6N6O4 requires C, 44.5; H, 4.3; N, 16.4%).
To a suspension of N4-(4-azidobenzyloxycarbonyl)-N1,N8-
bis(nitroguanidino)spermidine 15 (0.318 g, 0.643 mmol) in a
methanol–water mixture (9:1 v/v; 5 ml) were added DTT (0.398
g, 2.58 mmol) and triethylamine (0.260 g, 2.57 mmol). The mix-
ture was stirred at rt in the dark for 4 h, to give a yellow solu-
tion. The solvent was removed and the residue was purified by
‘flash’ chromatography (silica gel; 10% v/v methanol in conc.
ammonia; UV detection) to give an oil. This crystallised
upon trituration with water, giving N1,N4-bis(nitroguanidio)-
spermidine 16 as a solid (0.103 g, 50%) homogeneous by TLC
(silica gel; 4:1:1:2 v/v butan-1-ol–acetic acid–pyridine–water;
UV detection; Rf 0.44), mp 75–77 ЊC; νmax/cmϪ1 (KBr disc) 3406
(NH ), 2932 and 2860 (CH ), 1636 (C᎐N) and 1598 (NO );
᎐
2
2
2
δH(200 MHz; d6-DMSO) 1.55 (4H, m, CH2CH2CH2CH2), 1.72
(2H, m, NCH2CH2CH2N), 2.60 (4H, m, CH2NHCH2), 3.27
[4H, m, 2 × CH2NHC(NNO2)NH2], 8.00* (4H, br s, 2 × NH2),
8.63* [2H, br s, 2 × NHC(NH2)(NNO2)]; δC(50 MHz; d6-
DMSO) 26.4, 26.8 and 28.9 (CH2CH2CH2CH2NHCH2CH2),
39.4 (NHCH2CH2CH2NHCH2), 40.9 (NHCH2CH2CH2CH2-
NHCH2), 46.3 and 49.0 [2 × CH2NHC(NNO2)NH2] and 159.6
and 160.3 [2 × NHC(NH2)(NNO2)]; m/z (FAB) 320 (M ϩ 1,
100%), 303 (M Ϫ NH2, 16), 275 (M Ϫ NO2, 27) and 258 (M Ϫ
H2NNO2, 43) (Found: C, 34.2; H, 6.7; N, 39.9. C9H21N9O4
requires C, 33.9; H, 6.6; N, 39.5%).
N4-(4-Azidobenzyloxycarbonyl)spermidine 14
To a solution of N4-(4-azidobenzyloxycarbonyl)-N1,N8-bis-
(trifluoroacetyl)spermidine 13 (0.114 g, 0.22 mmol) in methanol
(8 ml) was added conc. ammonia (2 ml). The mixture was
stirred at rt in the dark for 24 h and more conc. ammonia was
added (2 ml). The reaction mixture was stirred as before for 5
days. The methanol was removed in vacuo, and the resultant
aqueous solution made up to 5 ml with distilled water, washed
with dichloromethane (3 × 10 ml) and freeze-dried. The crude
product was purified by ‘flash’ chromatography (silica gel;
10% v/v methanol in conc. ammonia; UV detection) to give N4-
(4-azidobenzyloxycarbonyl)spermidine 14 as an oil (58 mg,
82%) homogeneous by TLC (silica gel; 10% v/v methanol in
conc. ammonia; UV detection; Rf 0.23): νmax/cmϪ1 (cap film)
Hirudonine sulfate 17 by catalytic transfer hydrogenation
To a slurry of N1,N8-bis(nitroguanidino)spermidine 16 (0.100
g, 0.314 mmol) in a 5% w/v formic acid–methanol mixture (7.5
ml) was added 10% Pd/C (50 mg). The mixture was stirred
under nitrogen for 3 h. The catalyst was filtered off and the
solvent was removed from the filtrate. The resultant residue was
taken up in water (1.5 ml) and sulfuric acid (1 M) was added
dropwise to the solution until the pH was ~2. The solution was
cooled to 0 ЊC and crystals of hirudonine sulfate 17 were
deposited. These were filtered off, and ethanol was added to the
supernatent until the cloud point was reached. Upon cooling
to 5 ЊC, a second batch of crystals was produced. These were
filtered off, combined with the first batch, and the combined
product was recrystallised from water, to yield hirudonine
sulfate 17 as crystals (0.135 g, 89%) homogeneous by TLC
(silica gel; 4:1:1:2 v/v butan-1-ol–acetic acid–pyridine–water;
ninhydrin detection; Rf 0.20), mp 167–168 ЊC (lit.,53 169 ЊC):
νmax/cmϪ1 (KBr disc) 3405 (NH3ϩ), 3173 (NH), 2958 and 2877
3395 (NH), 2942 and 2870 (CH ), 2114 (N ) and 1684 (C᎐O);
᎐
2
3
δH(200 MHz; d6-DMSO) 1.59 (4H, br s, CH2CH2CH2CH2),
1.86 (2H, m, NCH2CH2CH2N), 2.86 (4H, m, 2 × CH2NH2),
3.32 (4H, m, CH2NCH2), 5.14 (2H, s, OCH2), 7.22 (2H, d, J 8.4,
Ar meta protons), 7.50 (2H, d, J 8.5, Ar ortho protons) and 7.96
(4H, br s, 2 × NH2); δC(50 MHz; D2O) 26.5, 28.5 and 30.0
(CH2CH2CH2CH2NCH2CH2), 40.1 and 40.9 (two CH2NH2),
45.9 and 47.9 (CH2NCH2), 68.4 (OCH2), 120.5 (Ar meta car-
bons), 131.0 (Ar ortho carbons), 134.6 (Ar ipso carbon), 141.1
(Ar para carbon), 158.7 (CO); m/z (FAB) 321 (M ϩ 1, 30%)
(Found: C, 56.7; H, 7.5; N, 25.9. C15H24N6O6 requires C, 56.25;
H, 7.5; N, 26.25%).
N4-(4-Azidobenzyloxycarbonyl)-N1,N8-bis(nitroguanidino)-
spermidine 15
(CH ) and 1640 (C᎐N); δ (200 MHz; D O) 1.72 (4H, m,
᎐
2
H
2
To
a
stirred solution of N4-(4-azidobenzyloxycarbonyl)-
CH2CH2+CH2CH2), 2.00 (2H, m, NCH2CH2CH2N), 3.10 (4H,
+
spermidine 14 (0.551 g, 1.72 mmol) in methanol (10 ml) was
added DMNPC 4 (0.632 g, 3.45 mmol). The mixture was stirred
in the dark at rt for 3 days. A precipitate of crude product was
formed. This was filtered off, and purified by ‘flash’ chroma-
m, CH2NH2CH2) and 3.27 [4H, m, 2 × CH2NHC(NH)NH3];
δC(50 MHz; D2O) 24.3 (NCH2CH2CH2N), 26.5 (CH2CH2-
+
CH2CH2), 39.7 and 41.9 (CH2NH2CH2), 46.3 and 48.7
+
+
[2 × CH2NHC(NH)NH3] and 158.1 [2 × NHC(NH)NH3); m/z
354
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356

View Online
(FAB) 230 (M ϩ 1, 66%) and 100 [M Ϫ NH(CH2)4NHC-
(NH)NH2, 42] (Found: C, 24.0; H, 7.6; N, 21.5. C9H26-
N7O6S1.5ؒ4H2O requires C, 24.1; H, 7.6; N, 21.9%).
N4-(2-Hydroxy-1-phenylethyl)-N1,N8-bis(trifluoroacetyl)-
spermidine 11: νmax/cmϪ1 (cap film) 3150–3500 (OH), 3092 and
3033 (Ar CH), 2947 and 2871 (sp3 CH), 1707 (CO), 1160, 1186
and 1210 (CF); δH(200 MHz; CDCl3) 1.54–1.64 (4H, m,
CH2CH2CH2CH2), 1.66–1.90 (2H, m, NCH2CH2CH2N), 2.23–
2.36 (2H, m, CH2NCH2), 2.36 (1H, br s, OH), 2.53–2.75 (2H,
m, CH2NCH2), 3.35–3.52 (4H, m, 2 × CH2NH), 3.85 -3.99 (2H,
m, CH2OH), 5.88 (1H, dd, J1 9.28, J2 3.9, NCHPhCH2OH),
6.89 (1H, br s, NH), 7.12–7.17 (2H, m, Ar ortho protons), 7.28–
7.38 (3H, Ar meta and para protons) and 7.82 (1H, br s, NH);
δC(50 MHz; CDCl3) 24.6, 26.2 and 26.5 (CH2CH2NCH2CH-
2CH2CH2), 38.7 and 39.7 (CH2NCH2), 47.4 and 49.1 (2 ×
CH2NH), 61.2 (CH2OH), 64.9 (NCHPhCH2OH), 114.0 (two
overlapping q, JCF 290, 2 × CF3), 128.1 (ArC), 128.5 (ArC),
128.8 (ArC), 136.0 (ArC) and 157.5 (two overlapping q, JCF 37,
2 × CO); m/z (FAB) 458 (M ϩ 1, 5%), 426 (M Ϫ CH2OH, 100),
338 (M Ϫ CHPhCH2OH, 5), 126 (CF3CONHCH2ϩ, 10) and 91
(PhCH2ϩ, 10) (Found: C, 49.3; H, 5.1; N, 8.7. C19H25F6N3O3
requires C, 49.9; H, 5.5; N, 9.2%).
Hirudonine sulfate 17 by direct guanidination of spermidine with
S-methylisothiourea
To a solution of spermidine (0.315 g, 2.17 mmol) in aq. ammo-
nia (20% w/v; 5 ml) was added S-methylisothiourea sulfate
(0.946 g, 3.40 mmol) in portions at rt. The reaction solution was
stirred at rt for 24 h and the resultant precipitate was filtered off,
washed successively with ice-cold water and then ethanol, and
air-dried. The solid was recrystallised from water–ethanol to
give hirudonine sulfate 17 as crystals (0.488 g, 47%) homo-
geneous by TLC (silica gel; 4:1:1:2 v/v butan-1-ol–acetic acid–
pyridine–water; ninhydrin detection; Rf 0.20), mp 169–170 ЊC
(lit.,53 169 ЊC): νmax/cmϪ1 (KBr disc) 3405 (NH3ϩ), 3173 (NH),
2958 and 2877 (CH ) and 1640 (C᎐N); δ (50 MHz; D O) 1.72
᎐
2
H
2
(4H, m, CH2CH2CH2CH2), 2.00 (2H, m, NCH2CH2CH2N),
+
3.10 (4H, m, CH2NH2CH2) and 3.27 [4H, m, 2 × CH2NHC-
+
(NH)NH3]; δC(50 MHz; D2O) 24.3 (NCH2CH2CH2N), 26.5
N4-(2-Hydroxypropyl)-N1,N8-bis(trifluoroacetyl)spermidine 12
+
(CH2CH2CH2CH2), 39.7 and 41.9 (CH2NH2CH2), 46.3 and
+
+
48.7 [2 × CH2NHC(NH)NH3] and 158.1 [2 × NHC(NH)NH3];
m/z (FAB) 230 (M ϩ 1, 70%) and 100 [M Ϫ NH(CH2)4NHC-
(NH)NH2, 39] [Found (again for the tetrahydrate): C, 24.0; H,
7.45; N, 21.6%].
To a stirred suspension of N1,N8-bis(trifluoroacetyl)spermidine
trifluoroacetate 9 (0.802 g, 1.77 mmol) and lithium perchlorate
(0.191 g, 1.77 mmol — see warning above) in dry acetonitrile
(1.3 ml) was added N-ethyldiisopropylamine (0.310 ml, 0.232
g, 1.77 mmol) at rt. A solution formed immediately, to which
was added propylene oxide (0.124 ml, 0.103 g, 1.77 mmol). The
reaction mixture was stirred at rt for 24 h. Water (5 ml) was
added and the solution was extracted with ether (3 × 10 ml).
The organic layers were combined, and the solvent was
removed in vacuo. The crude product was purified by ‘flash’
chromatography (silica gel; 10% v/v methanol in dichlorometh-
ane; molybdic acid detection) to give N4-(2-hydroxypropyl)-
N1,N8-bis(trifluoroacetyl)spermidine 12 as a pale yellow oil
(0.582 g, 83%) homogeneous by TLC (silica gel; 10% v/v
methanol in dichloromethane; molybdic acid detection; Rf
0.22); νmax/cmϪ1 (cap film) 3302 and 3099 (NH), 3100–3500
(OH), 2946 and 2826 (sp3 CH), 1708 (CO), 1210, 1187 and 1159
(CF); δH(200 MHz; CDCl3) 1.10 (3H, d, J 6.1, CH3), 1.42–1.60
(4H, m, CH2CH2CH2CH2), 1.64–1.77 (2H, m, NCH2CH2-
CH2N), 2.27 [2H, d, J 6.1, NCH2CH(OH)CH3], 2.37–2.70 (4H,
m, CH2CH2NCH2CH2), 3.02 (1H, br s, OH), 3.28–3.47 (4H, m,
2 × CH2NHCO), 3.78 [1H, sextet, J 6.3, CH2CH(OH)CH3] and
7.27 and 8.01 (2H, 2 br s, 2 × NHCOCF3); δC(50 MHz; CDCl3)
20.5 (CH3), 24.0, 25.9 and 26.5 (CH2CH2CH2CH2NCH2CH2),
38.8 and 39.6 (2 × CH2NHCO), 52.4 and 53.7 (CH2CH2CH2-
CH2NCH2CH2), 62.3 [NCH2CH(OH)CH3], 64.2 [CH2CH-
(OH)CH3], 116.2 (two overlapping q, JCF 287, 2 × CF3) and
157.8 (two overlapping q, JCF 60.4, 2 × CO); m/z (EI) 394
(M Ϫ 1, 2%), 364 (M Ϫ CH3OH, 1), 350 (M Ϫ CH3CHϩOH,
100), 211 [CF3CONH(CH2)4NHCH2CH2ϩ, 18], 197 [CF3CON-
H(CH2)4NHCH2ϩ, 12], 154 [CF3CONH(CH2)2CH2ϩ, 35] and
126 (CF3COCH2ϩ, 26) (Found: C, 42.1; H, 5.7; N, 11.0.
C14H23F6N3O3 requires C, 42.5; H, 5.8; N, 10.6%).
N4-(2-Hydroxy-2-phenylethyl)-N1,N8-bis(trifluoroacetyl)-
spermidine 10
To a stirred mixture of lithium perchlorate (0.107 g, 1.01 mmol)
in dry acetonitrile (0.75 ml) were added styrene oxide (0.114 ml,
0.120 g, 1.00 mmol), N1,N8-bis(trifluoroacetyl)spermidine tri-
fluoroacetate 9 (0.452 g, 1.00 mmol), and N-ethyldiisopropyl-
amine (0.175 ml, 0.130 g, 1.01 mmol) at rt. (Warning: lithium
perchlorate is potentially explosive, and should be handled with
extreme caution at all times. Safety screens and additional facial
protection must be used.) The mixture was stirred under nitro-
gen at rt for 24 h. The solution was diluted with water (4 ml)
and then extracted with ether (3 × 10 ml). The organic layers
were combined, and the solvent was removed to give a crude
oil. This was purified by ‘flash’ chromatography (silica gel; 5%
v/v methanol in dichloromethane; molybdic acid detection.
Note that a large excess of silica is required for good separ-
ation)togiveN4-(2-hydroxy-2-phenylethyl)-N1,N8-bis(trifluoro-
acetyl)spermidine 10 as an oil (0.309 g, 67%) homogeneous
by TLC (silica gel; 10% v/v methanol in dichloromethane;
molybdic acid detection) Rf 0.40. Isomeric N4-(2-hydroxy-1-
phenylethyl)-N1,N8-bis(trifluoroacetyl)spermidine 11 was also
produced as an oil (0.103 g, 22%), homogeneous by TLC (silica
gel; 10% v/v methanol in dichloromethane; molybdic acid
detection) Rf 0.45. The overall yield of both isomers was 0.412
g, 89%.
N4-(2-Hydroxy-2-phenylethyl)-N1,N8-bis(trifluoroacetyl)-
spermidine 10: νmax/cmϪ1 (cap film) 3200–3400 (OH), 3100 and
3066 (Ar CH), 2949 and 2870 (sp3 CH), 1708 (CO), 1162, 1185
and 1209 (CF); δH(200 MHz; CDCl3) 1.41–1.60 (4H, m,
CH2CH2CH2CH2), 1.61–1.74 (2H, m, NCH2CH2CH2N), 2.32–
2.69 [6H, m, N(CH2)3], 2.91 (1H, br s, OH), 3.24–3.45 (4H, m,
2 × CH2NH), 2.84 [1H, dt, J1 8.7, J2 4.5, CH2CH(OH)Ph], 6.90
(1H, br s, NH), 7.21–7.32 (5H, m, ArH) and 7.82 (1H, br s,
NH); δC(50 MHz; CDCl3) 24.0, 25.9 and 26.5 (CH2CH2NCH2-
CH2CH2CH2), 38.9 and 39.6 (CH2CH2NCH2CH2), 52.4 and
53.6 (2 × CH2NH), 62.8 [NCH2CH(OH)Ph], 70.8 [CH2CH-
(OH)Ph], 115.9 (two overlapping q, JCF 293, 2 × CF3), 125.9
(Ar ortho carbons), 127.9 (Ar para carbon), 128.5 (Ar meta
carbons), 142.2 (Ar ipso carbon) and 157.5 and 157.6 (two over-
lapping q, JCF 38, 2 × CO); m/z (FAB) 458 (M ϩ 1, 37%), 440
(M Ϫ OH, 25) and 350 (M Ϫ PhCH2OH, 100) (Found: C, 48.1;
H, 5.5; N, 8.7. C19H25F6N3O3ؒH2O requires C, 48.0; H, 5.7;
N, 8.8%).
Crystal-structure determination
Crystal data: (C9H26N7)3ϩؒ3/2 (SO4)2Ϫؒ4H2O, Mr = 448.52, tri-
¯
clinic, P1, a = 7.092(5), b = 11.736(9), c = 14.767(12) Å, α =
103.65(3), β = 103.42(3), γ = 106.15(6)Њ, V = 1087.3(14) Å3, Z =
2, Dcalcd = 1.370 g cmϪ3, µ = 0.26 mmϪ1 (Mo-Kα, λ = 0.710 73Å),
F(000) = 482, T = 160 K. Crystal size 0.44 × 0.21 × 0.18 mm,
Stoe-Siemens four-circle diffractometer, cell parameters from
2θ-values of 26 reflections measured at ω; 3045 intensities
measured by ω/θ scans, θmax = 22.5Њ, maximum indices 7, 12, 15;
2821 unique reflections, Rint = 0.159 (based on relatively weak
high-angle data), corrections for ~1% intensity decay.
The structure was determined by direct methods and
2
refined65 on all F2-values with weighting wϪ1 = σ2(Fo ) ϩ
(0.1388P)2 ϩ 1.6381P, where P = (Fo2 ϩ 2Fc2)/3. Cation
J. Chem. Soc., Perkin Trans. 1, 1999, 349–356
H
355

View Online
23 W. Raasch, S. Regunathan, G. Li and D. J. Reis, Life Sci., 1995, 56,
atoms were located in a difference synthesis and refined with a
riding model; other atoms were assigned anisotropic displace-
ment parameters. Both sulfate anions were found to be dis-
ordered, one as two orientations with a common S-atom
[occupancies 61:39(2)%], the other over an inversion centre
with two O-atoms in common. One ordered and seven dis-
ordered (variously 50% and 25%) positions were resolved and
refined for water oxygen atoms, but H-atoms for these were not
located; the total water content is 4H2O per cation.
2319.
24 E. Galea, S. Regunathan, V. Eliopoulos, D. L. Feinstein and
D. J. Reis, Biochem. J., 1996, 316, 247.
25 R. R. Ruffolo, W. Bondinell and J. P. Hieble, J. Med. Chem., 1995,
38, 3681.
26 J. Satriano, S. Matsufuji, Y. Murakami, M. J. Lortie, D. Schwartz,
C. J. Kelly, S. Hayashi and R. C. Blantz, J. Biol. Chem., 1998, 273,
15313.
27 W. B. Lawson, M. D. Leafer, A. Tewes and G. J. S. Rao, Hoppe-
Seyler’s Z. Physiol. Chem., 1968, 349, 251.
28 S. Yaginuma, A. Asahi, M. Takada, M. Hayashi and K. Fukukawa,
Eur. Pat., 196 189, 1986 (Chem. Abstr., 1987, 106, 67676a).
29 B. ElAmin, G. M. Anantharamaiah, G. P. Royer and G. E. Means,
J. Org. Chem., 1979, 44, 3442.
30 R. S. Lott, V. S. Chauhan and C. H. Stammer, J. Chem. Soc., Chem.
Commun., 1979, 495.
2
2
2
2
2
¹
²
Final Rw = {Σ[w(Fo Ϫ Fc ) ]/Σ[w(Fo ) ]} = 0.239 for all data,
conventional R = 0.074 for F-values of 1830 reflections having
2
Fo2 > 2σ(Fo ), goodness of fit S = 1.062 for all F2-values and 335
refined parameters. Final difference map extremes ϩ0.93 and
Ϫ0.37 e ÅϪ3. Full crystallographic details, excluding structure
factor tables, have been deposited at the Cambridge Crystallo-
graphic Data Centre (CCDC). For details of the deposition
scheme, see ‘Instructions for Authors’, J. Chem. Soc., Perkin
Trans. 1, available via the RSC Web page (http://www.rsc.org/
authors). Any request to the CCDC for this material should
quote the full literature citation and the reference number 207/
273.
31 T. Morita, Y. Okamoto and H. Sakurai, J. Chem. Soc., Chem.
Commun., 1978, 874.
32 M. C. O’Sullivan and D. M. Dalrymple, Tetrahedron Lett., 1995, 36,
3451.
33 D. Xu, K. Prasad, O. Repic and T. J. Blacklock, Tetrahedron Lett.,
1995, 36, 7357.
34 I. S. Blagbrough and A. J. Geall, Tetrahedron Lett., 1998, 39, 439.
35 Y. Nagao and E. Fujita, Heterocycles, 1982, 17, 537.
36 R. W. Doskotch, A. B. Ray, W. Kubelka, E. H. Fairchild, C. D.
Hufford and J. L. Beal, Tetrahedron, 1974, 30, 3229.
37 A. V. Joshua and J. R. Scott, Tetrahedron Lett., 1984, 25, 5725.
38 S.-I. Murahashi, T. Naota and N. Nakajima, Chem. Lett., 1987, 879.
39 B. Garrigues and L. Vidaud, Bull. Soc. Chim. Belg., 1988, 97, 775.
40 Y. Kikugawa, K. Mitsui, T. Sakamoto, M. Kawase and H. Tamiya,
Tetrahedron Lett., 1990, 31, 243.
Acknowledgements
We thank Lilly Pharmaceuticals and the MRC Toxicology
Unit, Leicester for support of A. M., Dr R. D. Verschoyle
(MRC Toxicology Unit, University of Leicester) for assistance
with polyamine uptake assays, Professor L. L. Smith (MRC
Toxicology Unit) for helpful discussions and the EPSRC for
partial funding of the X-ray equipment.
41 G. M. Cohen, P. M. Cullis, J. A. Hartley, A. Mather, M. C. R.
Symons and R. T. Wheelhouse, J. Chem. Soc., Chem. Commun.,
1992, 298.
42 C. Morin and M. Vidal, Tetrahedron, 1992, 48, 9277.
43 V. Fauchet, L. Bourel, A. Tartar and C. Sergheraert, Bioorg. Med.
Chem. Lett., 1994, 4, 2559.
44 S. Lemaireaudoire, M. Savignac and J. P. Genet, Synlett, 1996, 75.
45 I. Levchine, P. Rajan, M. Borloo, W. Bollaert and A. Haemers,
Synthesis, 1994, 37.
46 Y. Takeda, K. Samejima, K. Nagano, M. Watanabe, H. Sugeta and
Y. Kyogoku, Eur. J. Biochem, 1983, 130, 383.
47 M. M. Kimberley and J. H. Goldstein, Anal. Chem., 1981, 53, 789.
48 M. Chini, P. Crotti and F. Macchia, Tetrahedron Lett., 1990, 31,
4661.
49 I. R. Marsh and M. Bradley, Tetrahedron, 1997, 53, 17317.
50 R. J. Bergeron and J. S. McManis, J. Org. Chem., 1988, 53, 3108.
51 Y. Robin, N. van Thoai and L.-A. Pradel, Biochim. Biophys. Acta,
1957, 24, 381.
52 A. Mori, M. Hiramatsu, A. Numoto and Y. Robin, C. R. Seances
Soc. Biol. Ses Fil., 1981, 175, 755.
53 Y. Robin and N. van Thoai, C. R. Hebd. Séances Acad. Sci., 1961,
252, 1224.
54 M. Nethaji and V. Pattabhi, Proc. Indian Acad. Sci., Chem. Sci.,
1987, 99, 403.
55 I. Labadi, R. Sillanpaa and H. Lonnberg, J. Chem. Soc., Dalton
Trans., 1992, 765.
56 Y. Huse and Y. Iitaka, Acta Crystallogr., Sect. B, 1969, 25, 498.
57 E. Giglio, A. M. Liquori, R. Puliti and A. Ripamonti, Acta
Crystallogr., 1966, 20, 683.
58 Y. Iitaka and Y. Huse, Acta Crystallogr., 1965, 18, 110.
59 M. Jaskolski, Acta Crystallogr., Sect. C, 1987, 43, 1375.
60 A. Gharbi and A. Jouini, Acta Crystallogr., Sect. C, 1996, 52, 1342.
61 E. Giglio, A. M. Liquori and A. Ripamonti, Acta Crystallogr., 1966,
20, 652.
62 K. W. Hart, A. R. Clarke, D. B. Wigley, A. D. B. Waldman,
W. N. Chia, D. A. Barstow, T. Atkinson, J. B. Jones and J. J.
Holbrook, Biochim. Biophys. Acta, 1987, 914, 294.
63 D. D. Perrin, W. L. F. Armarego and D. R. Perrin, The Purification
of Laboratory Reagents, Pergamon Press, Oxford, 2nd edn., 1980.
64 O. Keijiro, J. Chem. Soc. Jpn., 1946, 67, 132 (Chem. Abstr., 1952, 46,
90e).
References
1 A. E. Pegg, Cancer Res., 1988, 48, 759.
2 D. M. L. Morgan, Biochem. Soc. Trans., 1990, 18, 1080.
3 M. C. O’Sullivan, B. T. Golding, L. L. Smith and I. H. Wyatt,
Biochem. Pharmacol., 1991, 41, 1839.
4 R. J. Bergeron, Y. Feng, W. R. Weimar, J. S. McManis, H. Dimova,
C. Porter, B. Raisler and O. Phanstiel, J. Med. Chem., 1997, 40, 1475
and refs. cited therein.
5 W. D. W. Heston, K. A. Watanabe and K. W. Pankiewicz, Biochem.
Pharmacol., 1987, 36, 1849.
6 R. D. Verschoyle, P. Carthew, J. L. Holley, P. Cullis and G. M.
Cohen, Cancer Lett., 1994, 85, 217.
7 S. Sagner, Z. W. Shen and M. H. Zenk, Tetrahedron Lett., 1997, 38,
2443.
8 M. C. O’Sullivan, O. Zhou, Z. Li, T. B. Durham, D. Rattendi,
S. Lane and C. J. Bacchi, Bioorg. Med. Chem., 1997, 5, 2145.
9 A. J. Geall, R. J. Taylor, M. E. Earll, M. A. W. Eaton and I. S.
Blagbrough, Chem. Commun., 1998, 1403.
10 J. Boukovalas, B. T. Golding, R. W. McCabe and P. K. Slaich,
Angew. Chem., Int. Ed. Engl. Suppl., 1983, 22, 860.
11 B. T. Golding, M. C. O’Sullivan and L. L. Smith, Tetrahedron Lett.,
1988, 29, 6651.
12 R. J. Griffin, E. Evers, R. Davison, D. Layton and W. J. Irwin,
J. Chem. Soc., Perkin Trans. 1, 1996, 1205.
13 A. Mitchinson, B. T. Golding, R. J. Griffin and M. C. O’Sullivan,
J. Chem. Soc., Chem. Commun., 1994, 2613.
14 L. L. Smith and I. Wyatt, Biochem. Pharmacol., 1981, 30, 1053.
15 D. B. Wigley, A. Lyall, K. W. Hart and J. J. Holbrook, Biochem.
Biophys. Res. Commun., 1987, 149, 927.
16 Y. B. Lee, M. H. Park and J. E. Folk, J. Med. Chem., 1995, 38, 3053.
17 W. L. Hughes, H. A. Saroff and A. L. Carney, J. Am. Chem. Soc.,
1949, 71, 2476.
18 S. C. Yorke, J. W. Blunt, M. H. G. Munro, J. C. Cook and
K. L. Rinehart, Aust. J. Chem., 1986, 39, 447.
19 A. F. S. A. Habeeb, Biochim. Biophys. Acta, 1964, 93, 533.
20 R. A. Henry, R. C. Makosky and G. B. L. Smith, J. Am. Chem. Soc.,
1951, 73, 474.
21 S. Matsuzaki, K. Hamana and K. Isobe, Phytochemistry, 1990, 29,
1313.
65 G. M. Sheldrick, SHELXTL Version 5, Bruker AXS, Madison,
Wisconsin, USA, 1994.
22 K. Hamana, T. Akiba, F. Uchino and S. Matsuzaki, Can. J.
Microbiol., 1989, 35, 450.
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