Cheno-, urso- and deoxycholic acid spermine conjugates: Relative binding affinities for calf thymus DNA

Article abstract of DOI:10.1016/S0040-4020(00)00265-9

Cationic lipid polyamine amides (cholan-24-amides) have been prepared from chenodeoxycholic (3α,7α-dihydroxy), ursodeoxycholic (3α,7β- dihydroxy), and deoxycholic (3α,12α-dihydroxy) bile acids (5β-cholanes) by acylation of tri-Boc protected spermine. Their relative binding affinities for calf thymus DNA were determined using an ethidium bromide displacement assay. These lipopolyamine amides are synthetic vectors for non-viral gene delivery and models for lipoplex formation with respect to lipofection, a key first step in gene therapy. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00265-9

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3439±3447
Cheno-, Urso- and Deoxycholic Acid Spermine Conjugates:
Relative Binding Af®nities for Calf Thymus DNA
*
Ian S. Blagbrough, Dima Al-Hadithi and Andrew J. Geall
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK
Received 20 December 1999; revised 10 March 2000; accepted 23 March 2000
AbstractÐCationic lipid polyamine amides (cholan-24-amides) have been prepared from chenodeoxycholic (3a,7a-dihydroxy),
ursodeoxycholic (3a,7b-dihydroxy), and deoxycholic (3a,12a-dihydroxy) bile acids (5b-cholanes) by acylation of tri-Boc protected
spermine. Their relative binding af®nities for calf thymus DNA were determined using an ethidium bromide displacement assay. These
lipopolyamine amides are synthetic vectors for non-viral gene delivery and models for lipoplex formation with respect to lipofection, a key
®rst step in gene therapy. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
groups on the cholane ring system in¯uence the relative
binding af®nity of these compounds for CT DNA using an
ethidium bromide (Eth Br) displacement assay. Therefore,
using our orthogonal protection strategy for ef®cient synthe-
Polycations interact readily with the DNA phosphate
anionic backbone, causing condensation by charge
neutralisation and this effect is a key ®rst step in minimising
1
2,13
ses of unsymmetrical polyamine amides,
we have
1
±6
the size of foreign DNA for gene therapy. The triamine
spermidine and the tetraamine spermine are naturally occur-
ring linear amines found in most living cells and playing
synthesised the novel bile acid polyamine amides 11±13
of spermine 6 as they mimic the positive charge distribution
of spermidine. Polyamines are ideally suited to bind to and
7
6
6
important roles in vivo. These molecules are essentially
fully protonated at physiological pH (i.e. ammonium ions
then condense DNA. In order to reinforce these effects, it is
apparently bene®cial if a lipid moiety is covalently bound
7
14±16
at pHˆ7.4). For a review of the plethora of roles played by
to the polyamine, such a lipid can be cholesterol,
17
a
As part of our con-
8
spermidine and spermine in vivo, see Blagbrough et al. One
of their key roles is in maintaining the 3D structure of
1,4,18
bile acid, or two aliphatic chains.
tinuing SAR studies on polyamine-mediated DNA conden-
1
±4,9
5,6,8
11±13,16,19,20
DNA
interactions are readily reversible under physiological
by condensation,
though polyamine±DNA
sation,
we have determined the binding of
lipopolyamines to CT DNA. In this paper, we report the
practical synthesis of novel unsymmetrical polyamine
amides 11±13 using tri¯uoroacetyl as a protecting group
whose introduction and removal can be controlled under
1
0
conditions.
In our current structure±activity relationship (SAR) studies
of lipopolyamine amides, we are investigating the role of
the substituted lipid moiety in the DNA condensation
process. Chenodeoxycholic 1, deoxycholic 2 and urso-
deoxycholic 3 acids (Fig. 1) were chosen as the lipid
moieties because they allow controlled changes in the regio-
chemical substitution of the two hydroxyl groups on the
cholane ring system. Previously, we have shown that the
binding af®nities for calf thymus (CT) DNA of spermine
covalently attached to lithocholic acid 4 (one alcohol func-
tional group) and cholic acid 5 (three secondary alcohols) is
1
2,13,21,22
facile conditions and on a gram scale.
Results and Discussion
Synthesis
The facile introduction of tri¯uoroacetyl using ethyl
tri¯uoroacetate, reported recently,
2
1,22
and its ready removal
2
3
with aqueous ammonia (pHˆ11) or with methanolic
2
4
aqueous K CO3 solution makes it a good protecting
1
1
profoundly different. We have now investigated if changes
in the stereochemistry and position of the alcohol functional
2
group for the gram scale synthesis of unsymmetrical poly-
amine conjugates. Therefore, using our orthogonal protec-
tion strategy, we prepared unsymmetrically protected
1
2
3
N , N , N -tri-Boc-spermine 7, from spermine 6 with di-
tert-butyl dicarbonate after initial reaction with ethyl
Keywords: polyamine amides; spermine; bile acids; DNA binding af®nity;
DNA condensation; lipoplex.
* Corresponding author. Tel.: 144-1225-826795; fax: 144-1225-826114;
e-mail: prsisb@bath.ac.uk
1
2,13
tri¯uoroacetate. Selective deprotection of the tri¯uoro-
acetamide was then achieved by increasing the pH of the
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00265-9

3
440
I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
2
and ursodeoxycholic 3 acids to afford tri-Boc protected
polyamine amides 9 and 10, respectively. Deprotection and
puri®cation by RP-HPLC afforded the target amides 12 and
1
3, as their polytri¯uoroacetate salts (Fig. 2). We have
named the target compounds as their corresponding
spermine (1,12-diamino-4,9-diazadodecane) derivatives.
Fig. 3 outlines the numbering system used in the NMR
1
assignment of N -(3a,7a-dihydroxy-5b-cholan-24-carbonyl)-
,12-diamino-4,9-diazadodecane 11 (poly-TFA salt).
1
Changes in binding af®nity for CT DNA with respect to
variations in the position of the hydroxyl groups of cheno-
deoxycholic 1 (3a, 7a-dihydroxy-5b-cholanic acid), deoxy-
cholic 2 (3a, 12a-dihydroxy-5b-cholanic acid) and
ursodeoxycholic acids 3 (3a, 7b-dihydroxy-5b-cholanic
acid), covalently attached to spermine have been investi-
gated. Condensation of CT DNA was monitored using a
1
3,16,20
re®ned Eth Br ¯uorescence quenching assay. The
pK s of these polyamines amides 11±13 (spermidine
a
1
mimics) were assumed to be the same as (N -cholesteryl-
oxy-3-carbonyl)-1,12-diamino-4,9-diazadodecane charac-
2
0
terized potentiometrically, and therefore the positive
charge carried at physiological pH (7.4) was also assumed
to be the same (12.4). Prevention of Eth Br binding to DNA
is one method of studying the DNA binding behaviour of
2
5±32
small molecule ligands, e.g. polyamines,
though the
DNA binding modes of aliphatic polyamines and Eth Br,
a polyaromatic intercalator dye, are certainly different;
however, a qualitative comparison of DNA binding af®nity
between related chemical structures is possible.2
6±32
There-
fore, lipopolyamine amides 11±13 can be critically
3
3
compared as a function of the charge ratio required to
displace Eth Br binding to DNA.
NMR spectroscopic structural assignments
The NMR assignments of the polyamine headgroups in this
series of polyamine amides 11±13 are based upon our
1
previous results, H, C chemical shift correlation spec-
3 1
13
7
,34±38
troscopy, and literature data.
The detailed assignments
of the cholane ring structures are based on literature data
3
9
1
3
and the expected changes in the C chemical shifts due to
substituent effects are consistent with these assignments.
Methylene groups a to a secondary amine have larger
down®eld chemical shifts than those a to a primary
Figure 1. Structures of chenodeoxycholic 1, deoxycholic 2, ursodeoxy-
cholic 3, lithocholic 4 and cholic 5 acids.
4
0,41
solution above 11 with conc. aqueous ammonia, to afford
unsymmetrically protected polyamine 7 with one free
primary amine unmasked. N-Acylation of amine 7 with
chenodeoxycholic acid 1, mediated by DCC and catalytic
1-hydroxybenzotriazole (HOBt), afforded the tri-Boc
protected lipospermine 8. Deprotection by treatment with
amine.
Protonation of amines causes shielding in the
vicinity of the ammonium ions resulting in an up®eld shift
1
3
in these C signals. This shift on protonation of amines is
detectable as far as ®ve carbon atoms away, the greatest
4
0,42
effect being at the b-position.
N-Acylation of one of
the primary amines of spermine leads to an unsymmetrical
polyamine and therefore loss of symmetry of the chemical
tri¯uoroacetic acid in CH Cl (1:9) and puri®cation by
2
2
1
3
RP-HPLC afforded the target amide 11, as its polytri¯uoro-
acetate salt (Fig. 2). Microanalysis of these salts was not
within ^0.4%. However, the presence of polyamines in the
cationic lipids makes elementary analysis an inadequate
method of measuring the purity of these compounds. Poly-
amines are highly hygroscopic and can adopt a different salt
shifts in the two propylene chains. C1, C2 and C3 are
in¯uenced by an amide rather than a protonated primary
amine, and therefore are relatively more shielded, resonat-
ing further up®eld than their counterparts, C10, C11 and
C12, on the other propylene chain. Thus, unambiguous
1
3
total C NMR spectroscopic assignments of the spermidine
headgroup are based on comparison with literature
1
8
degree. Thus, the proposed structure was unambiguously
1
13
13,36
34
assigned using accurate MS, IR, H, C and HETCOR
NMR after puri®cation by RP-HPLC to homogeneity.
Protected spermine 7 was also N-acylated with deoxycholic
compounds,
calculations using additivity rules,
1
13
and also by H, C NMR chemical shift correlation
spectroscopy.

I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
3441
Figure 2. Synthesis of target polyamine amides 11±13.
DNA binding af®nities
only carry 2.4 positive charges. Compared to multicationic
polylysine they are poor condensers of CT DNA, as a large
excess of positive charge is required to displace Eth Br and
complete exclusion was never achieved within the para-
meters of the experiment. If the binding af®nities for
DNA, of these three polyamine amides 11±13, are
expressed as the charge ratio at which 50% (CR50) of the
Eth Br was quenched, then conjugate 12 (CR50ˆ1.6) has the
greatest af®nity and this can be attributed to the position and
stereochemistry of the hydroxyl groups. Amide 11
The relative DNA binding af®nities of the target compounds
11±13 were measured using a modi®ed Eth Br ¯uorescence
quenching assay, as part of our continuing SAR
1
1±13,16,19,20
studies.
cally compared against polylysine (average molecular
The decrease in ¯uorescence was criti-
weight 9600 Da) and spermine 6 (Fig. 4) for compounds
11±13 at 20 mM NaCl as a function of charge ratio
(
positive/negative charges).
(
CR ˆ2.3) has two hydroxyls at position 3 and 7, which
5
0
At physiological pH, spermine 6 carries a net positive
are both on the a-face of the cholane ring structure and
shows a weaker binding af®nity relative to amide 12,
which has the hydroxyl at position 12 on the a-face rather
than at position 7. Amide 13 (CR50ˆ2.6) has the hydroxyl at
position 3 on the a-face, but the hydroxyl at position 7 is
2
0
1
charge of 3.8. Fig. 4 shows that N -acylation of spermine
with chenodeoxycholic 1, deoxycholic 2 and ursodeoxy-
cholic 3 acids, makes these amides 11±13 slightly more
6
potent binders of CT DNA than spermine although they
1
Figure 3. Structure and numbering system for N -(3a,7a-dihydroxy-5b-cholan-24-carbonyl)-1,12-diamino-4,9-diazadodecane 11.

3
442
I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
Figure 4. Ethidium bromide displacement assay of compounds 11±13 compared to spermine 6 and polylysine at low salt (20 mM NaCl). CT DNA (6 mg) in
buffer (3 ml, 20 mM NaCl, 2 mM HEPES, 10 mM EDTA, pH 7.4) was mixed with ethidium bromide (3 ml of 0.5 mg/ml) and aliquots of compound (5 ml of
0
.25 mg/ml, 1 min equilibration time) were added and the ¯uorescence (%) determined.
Figure 5. Ethidium bromide displacement assay of compounds 11±13 compared to amides 14 and 15 at low salt (20 mM NaCl). CT DNA (6 mg) in buffer
3 ml, 20 mM NaCl, 2 mM HEPES, 10 mM EDTA, pH 7.4) was mixed with ethidium bromide (3 ml of 0.5 mg/ml) and aliquots of compound (5 ml of 0.25 mg/
ml, 1 min equilibration time) were added and the ¯uorescence (%) determined.
(
now on the b-face, and this conjugate shows the weakest
binding af®nity for DNA. Comparison of the Eth Br exclu-
sion data of these amides 11±13 (Fig. 5) with the spermine
conjugates of lithocholic 14 (CR ˆ0.7) and cholic 15
(CR ˆ2.6) acids (Fig. 6) shows differences in binding
5
0
af®nity for DNA for these compounds (Fig. 5). The binding
af®nities are expressed as the charge ratio at which 50%
(CR ) of the Eth Br was quenched.
5
0
50
1
Figure 6. Structure of N -(3a-hydroxy-5b-cholan-24-carbonyl)-1,12-diamino-4,9-diazadodecane 14 and N -(3a,7a,12a-trihydroxy-5b-cholan-24-carbonyl)-
1
1
,12-diamino-4,9-diazadodecane 15.

I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
3443
3
0±32
Figure 7. Structures of steroidal polyamines 16±18 from Burrows and co-workers
There are only a few literature reports of the binding to
DNA of steroids substituted with amines.26,30±32
Compounds with the strongest interaction with DNA
appeared to be those that presented not only a large cationic
surface area, but also an extended hydrophobic region, Fig.
small increase in binding af®nity of these molecules
compared to cholanamide 13.
At elevated salt concentrations, e.g. 150 mM (Fig. 8), the
binding af®nity for DNA of polylysine is unaffected, but that
of spermine 6 shows salt-dependent binding to DNA.
Amides 11±13 (Fig. 4), which contain the cholane ring
structure with two hydroxyl moieties, mimic the salt-
dependent behaviour of spermine 6 and the displacement
of Eth Br is almost completely inhibited at elevated salt
concentrations (Fig. 8). We have previously observed that
the spermine conjugate of lithocholic acid 14 (Fig. 5)
7
af®nity for DNA.
shows steroidal polyamines 16±18 which display high
3
0,31
Tetraamine 18 had the highest af®nity,
measured by Eth Br displacement, a structure that maintains
large hydrophobic regions as well as four positive charges.
3
0±32
These reports concluded that disruption of the hydro-
phobic surface of the steroid diminished the binding af®nity
3
for DNA. The dihydroxy cholanamide derivatives 11 and
2
1
2 are facially amphiphilic molecules, that is the steroidal
7
11
1
showed a degree of salt-dependent binding.
nucleus contains both a hydrophilic (a-face) and hydro-
phobic (b-face) domain, compared to conjugate 13 which
contains a hydroxyl moiety on both the a- and b-faces. The
amphiphilic nature of conjugates 11 and 12 may explain the
These data support our hypothesis that DNA binding af®nity
and condensation are a sensitive function of both the
charge
1
6,20
11
and hydrophobicity of this type of ligand. We
Figure 8. Ethidium bromide displacement assay of compounds 11±13 compared to spermine 6 and polylysine at high salt (150 mM NaCl). CT DNA (6 mg) in
buffer (3 ml, 150 mM NaCl, 2 mM HEPES, 10 mM EDTA, pH 7.4) was mixed with ethidium bromide (3 ml of 0.5 mg/ml) and aliquots of compound (5 ml of
0
.25 mg/ml, 1 min equilibration time) were added and the ¯uorescence (%) determined.

3
444
I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
have used an adaptation of an Eth Br displacement assay
based on the work of LePecq and Paoletti, modi®ed by
Experimental
2
5
2
Cain et al. and by Gershon et al. The displacement assay
8
29
General details
Analytical TLC, column chromatography and RP-HPLC
were performed as described in our previous paper. Spec-
troscopy (IR, NMR and MS), DNA binding af®nities and
other general details are also as previously described. All
2
8
of Cain et al. has previously been used to compare the
binding af®nity of both intercalating and non-intercalating
drugs and provides comparable results rapidly without any
signi®cant variability in ¯uorescence measurement at inter-
1
3
2
8
13
mediate concentrations. In the assay of Cain et al., the
uorescence enhancement was due to direct excitation of
¯
chemicals, reagents and buffers were purchased from SAF;
solvents (HPLC grade) were purchased from Fisons.
the intercalated Eth Br (lexcitˆ546 nm, l
ˆ595 nm). In
emiss
our adaptation, we have indirectly excited the Eth Br by
energy transfer from the DNA, in a similar manner to that
General procedure A: amine acylation
2
9
used by Minsky and co-workers, lexcitˆ260 nm, this
produces a greater (10-fold) ¯uorescence enhancement.
To a solution of the poly-Boc protected spermine (1 equiv.)
in CH Cl2 (10 ml) was added the bile acid (1 equiv.),
2
5
Wilson and Bloom®eld predicted, using the polyelectrolyte
1-HOBt (0.2 equiv.) and DCC (1.5 equiv.). Then the reac-
tion mixture was stirred at 258C, under nitrogen, for 24 h
when the precipitate of DCU was removed by ®ltration. The
®ltrate was concentrated in vacuo and the residue puri®ed
by column chromatography over silica gel (CH Cl ±
4
3
theory of Manning, that when ,90% of the anionic phos-
phate charge on DNA is neutralised by the positive charges
along the polyammonium ion moiety, DNA condensation
5
,6,44±46
will occur.
Nearly complete exclusion of Eth Br
2
2
occurs before the charge ratio of the complex reaches one.
Aggregation of the DNA probably accounts for the incom-
MeOH) to afford the title compound as a white foam.
4
4
plete exclusion of Eth Br. DNA condensation is clearly an
inef®cient process with polyamine amides 11±13, as an
excess of positive charges is required to bring about a
decrease in the intensity of ¯uorescence of the Eth Br.
These polyamine amide steroids 11±13 are relatively less
lipophilic compared to our other carbamates and amide
derivatives. Therefore, the relative decrease in DNA
binding af®nity may be re¯ected by their increase in hydro-
philicity. Complete inhibition of ¯uorescence in the binding
assay, as seen with polylysine, is never achieved within the
parameters of the experiments, and this is similar to the
results obtained with unconjugated spermine 6. Basu et al.
have shown that the concentration of spermine 6 required to
release all the Eth Br is too high to be used without causing
General procedure B: Boc removal
To a stirring solution of lipopolyamine dissolved in CH Cl2
2
(180 ml), under nitrogen, at 258C was added TFA (20 ml).
After 2 h, the solution was concentrated in vacuo, the
residue lyophilized and then puri®ed by semi-preparative
RP-HPLC over Supelcosil ABZ1Plus (5 mm, 25 cm£
10 mm, MeCN±0.1% aq. TFA) to afford the title compound
as a clear glass, its polytri¯uoroacetate salt.
1
4
9
(N , N , N -Tri-tert-butoxycarbonyl)-1,12-diamino-4,9-
diazadodecane 7. 1,12-Diamino-4,9-diazadodecane
6
(spermine, 3.4.3) (1.0 g, 4.95 mmol) was reacted as
previously described and puri®ed over silica gel (CH Cl ±
MeOH±conc. aq. NH 70:10:1 to 50:10:1 v/v/v) to afford
2
2
4
4
DNA aggregation, and complete release of Eth Br from the
complex and the resultant decrease in ¯uorescence is never
seen. The small differences in the binding af®nity between
these molecules 11±13 could be due to the amphiphilic
nature of amides 11 and 12 compared to amide 13. Although
the exact mode of binding of a steroid moiety to DNA is not
known, the literature precedent is for minor-groove
3
the title compound 7 as a colourless homogeneous oil
(1.24 g, 50%), R 0.5 (CH Cl ±MeOH±conc. aq. NH
3
f
2
13
2
1
3
1
50:10:1 v/v/v). IR, H and C NMR and MS as previously
described.
1
3
1
N -(3a,7a-Dihydroxy-5b-cholan-24-carbonyl)-(N , N ,
4
9
3
2
12
N -tri-tert-butoxycarbonyl)-1,12-diamino-4,9-diazado-
binding, which is in¯uenced by hydrophobicity of the
steroid.
3
0,31
decane 8. Poly-Boc protected polyamine 7 (500 mg,
1
.0 mmol) and chenodeoxycholic acid (469 mg, 1.2 mmol)
were reacted according to the general procedure A to afford
the title compound 8 as a white foam (814 mg, 93%). Puri®ed
by column chromatography over silica gel (CH Cl ±MeOH;
Conclusion
2
2
2
3340 (OH), 1690 and 1670 (CO±N). H NMR, 400 MHz,
5:1 v/v), R 0.14 (CH Cl ±MeOH; 18:1 v/v). IR (KBr)
f 2 2
1
In this paper, we have designed novel unsymmetrical lipo-
polyamine amides, based upon the controlled acylation of
0
CDCl : 0.66 (s, 3H, 18 -CH ); 0.84±2.23 (m, 67H, 2-CH ,
3
3
2
4
7±53
0
4 -CH , 5 -CH, 6 -CH , 8 -CH, 9 -CH, 11 -CH , 12 -CH ,
0
protected spermine with carboxylic acids.
The products
6-CH , 7-CH , 11-CH , 3£O±C±(CH ) , 1 -CH , 2 -CH ,
2
2
2
3 3
2
2
0
0
0
0
0
0
0
14 -CH, 15 -CH , 16 -CH , 17 -CH, 19 -CH , 20 -CH,
have been designed to incorporate the positive charge distri-
bution of spermidine. Using a modi®ed Eth Br displacement
assay, we have established that the relative binding af®nity
of these compounds to CT DNA is subtly dependent upon
the substitution pattern within the lipid covalently attached
to the polyamine. These results give further support to our
hypothesis that DNA binding and DNA condensation are a
function of the lipid attached to the polyamine, as well as the
positively charged polyamine moiety in lipopolyamine
conjugates for use in non-viral gene therapy.
2
2
2
2
0
0
0
0
0
0
21 -CH , 22 -CH , 23 -CH ); 3.00±3.40 (m, 12H, 1-CH ,
0
2
2
3
0
3-CH , 5-CH , 8-CH , 10-CH , 12-CH ); 3.40±3.53
0
3
2
2
2
2
2
2
2
2
0
s, 1H, CH ±NH±CO±O); 6.75±6.90 (br s, 1H, CH ±NH±
0
(m, 1H, 3 -CH); 3.83±3.86 (m, 1H, 7 -CH); 5.25±5.40 (br
2
2
1
CO). C NMR, 100 MHz, CDCl : 11.7 (18 -CH ); 18.3
3
0
3
3
0
CH ); 25.7, 25.8, 26.0 (6-CH , 7-CH , overlapping); 27.6,
0
0
0
(21 -CH ); 20.5 (11 -CH ); 22.7 (19 -CH ); 23.7 (15 -
3
3
2
2
2
2
0
28.1, 28.4 (2-CH , 11-CH , 16 -CH , 3£O±C±(CH ) ,
2
2
2
3 3

I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
3445
0
3.7 (23 -CH ); 34.5 (6 -CH ); 35.0 (10 -Cq); 35.3, 35.5
0
0
0
6.90 (br s, 1H, CH ±NH±CO). C NMR, 100 MHz,
overlapping); 30.6 (2 -CH ); 31.7 (22 -CH ); 32.8 (9 -CH);
2
7 -CH); 5.35±5.45 (br s, 1H, CH ±NH±CO±O); 6.75±
2
2
13
0
0
0
12-CH , 1 -CH , 20 -CH, overlapping); 37.3, 37.4 (1-
3
(
2
2
2
0
CH ); 39.4 (8 -CH); 39.6 (4 -CH ); 39.8 (12 -CH ); 41.4
0
0
0
0
CDCl : 12.1 (18 -CH ); 18.4 (21 -CH ); 21.1 (11 -CH );
2
2
3
3
3
2
0
5 -CH ); 42.6 (13 -Cq); 43.2, 43.7 (3-CH , 10-CH , over-
0
0
0
23.3 (19 -CH ); 25.4, 25.6, 26.0 (6-CH , 7-CH , over-
3 2 2
2
2
2
0
lapping); 46.6 (5-CH , 8-CH , overlapping); 50.4 (14 -CH);
0
0
lapping); 26.9 (15 -CH ); 27.6, 28.4, 28.6 (2-CH ,
2 2
(
2
2
2
0
0
11-CH , 16 -CH , 3£O±C±(CH ) , overlapping); 30.3
2
2
2
2
3 3
0
0
0
0
0
34.9 (1 -CH ); 35.3 (12-CH , 20 -CH, overlapping); 36.9,
0
0
5
5.8 (17 -CH); 68.4 (7 -CH); 71.9 (3 -CH); 79.7 (3£quat
(2 -CH ); 31.8 (22 -CH ); 33.7 (23 -CH ); 34.0 (10 -Cq);
2
2
2
0
0
OC, overlapping); 156.0, 156.4 (3£NH±CO±O±C±
2
2
1
CH ) ); 173.6 (CH ±CO±NH). MS, FAB found 877,
0
0
0
(
37.3 (1-CH , 4 -CH , 6 -CH , overlapping); 39.1 (9 -CH);
3
3
2
2
2
2
1
0 0
40.1 (12 -CH ); 42.4 (5 -CH ); 43.3, 43.7 (3-CH , 10-CH ,
2 2 2 2
6
% (M 11), C H N O requires Mˆ876. High-resolution
4
1
9
88
4
9
1
MS m/z, FAB found 877.6605, (M 11), C H N O
0 0
8 -CH, 13 -Cq, overlapping); 46.4, 46.6 (5-CH , 8-CH ,
2 2
4
9
89
4
9
1
0 0
overlapping); 54.9 (14 -CH); 55.7 (17 -CH); 71.2, 71.3
requires M 11ˆ877.6630.
4
N -(3a,12a-Dihydroxy-5b-cholan-24-carbonyl)-(N , N ,
N -tri-tert-butoxycarbonyl)-1,12-diamino-4,9-diazado-
decane 9. Poly-Boc protected polyamine 7 (500 mg,
0
0
(
3 -CH, 7 -CH); 79.5, 79.7 (3£quat OC, overlapping);
1
9
156.0, 156.4 (3£NH±CO±O±C±(CH ) ); 173.7 (CH ±
3
3
2
1
2
1
1
found 877, 6% (M 11),
CO±NH). MS, FAB
1
C H N O requires M ˆ876. High-resolution MS m/z,
4
9
88
4
9
1
FAB found 877.6616, (M 11), C H N O requires
1
1.0 mmol) and deoxycholic acid (469 mg, 1.2 mmol) were
4
9
89
4
9
1
reacted according to the general procedure A to afford the
title compound 9 as a white foam (640 mg, 73%). Puri®ed
by column chromatography over silica gel (CH Cl ±MeOH;
M 11ˆ877.6630.
1
N -(3a,7a-Dihydroxy-5b-cholan-24-carbonyl)-1,12-
diamino-4,9-diazadodecane 11. Boc protected polyamine
amide 8 (732 mg, 0.84 mmol) was deprotected according to
the general procedure B. The residue was lyophilized to
produce 995 mg of a white powder, 400 mg was puri®ed
by RP-HPLC (Supelcosil ABZ1Plus, 5 mm, 25 cm£
10 mm, MeCN±0.1% aq. TFA, 25:75 v/v) to afford the
title polyamine amide 11 as a clear glass (polytri¯uoro-
2
2
3
(
0:1 to 15:1 v/v), R 0.13 (CH Cl ±MeOH; 18:1 v/v). IR
f 2 2
1
KBr) 3330 (OH), 1690 and 1670 (CO±N). H NMR,
0
4
6
1
9
1
00 MHz, CDCl : 0.67 (s, 3H, 18 -CH ); 0.84±2.40 (m,
3
3
7H, 2-CH , 6-CH , 7-CH , 11-CH , 3£O±C±(CH ) ,
2
2
2
2
3 3
0
0
0
0
0
0
0
-CH, 11 -CH , 14 -CH, 15 -CH , 16 -CH , 17 -CH,
0
0
-CH , 2 -CH , 4 -CH , 5 -CH, 6 -CH , 7 -CH , 8 -CH,
2
2
2
2
2
0
0
0
0
2
2
2
0
0
0
0
0
9 -CH , 20 -CH, 21 -CH , 22 -CH , 23 -CH ); 3.00±3.40
3
3
2
2
(
m, 12H, 1-CH , 3-CH , 5-CH , 8-CH , 10-CH , 12-CH );
2
.48±3.66 (m, 1H, 3 -CH); 3.95±4.03 (m, 1H, 12 -CH);
acetate salt, 146 mg, 47%), t 5.7 min by RP-HPLC (Supel-
R
2
2
2
2
2
0
.35±5.50 (br s, 1H, CH ±NH±CO±O); 6.75±6.90 (br s,
0
3
5
1
cosil ABZ1Plus, 5 mm, 25 cm£10 mm, MeCN±0.1% aq.
TFA, 25:75 v/v). IR (KBr) 3400 (OH) and 1670 (CO±N).
2
1
H, CH ±NH±CO). C NMR, 100 MHz, CDCl : 12.7
3
1
2
0
H NMR, 400 MHz, [ H] DMSO: 0.61 (s, 3H, 18 -CH );
6 3
2
3
0
0
0
0
0 0 0 0
0.82±1.55 (m, 20H, 1 b-CH, 2 a-CH, 2 b-CH, 5 b-CH,
(18 -CH ); 17.4 (21 -CH ); 23.1 (19 -CH ); 23.6 (15 -
CH ); 25.8, 26.1 (6-CH , 7-CH , 7 -CH , overlapping);
2
1
3
3
3
0
7.1, 27.4 (6 -CH , 16 -CH ); 27.6, 28.4, 28.6 (2-CH ,
0 0 0 0 0 0
8 b-CH, 11 a-CH, 11 b-CH, 12 a-CH, 14 a-CH, 15 a-
2
2
2
2
0
0
0 0 0 0 0 0
CH, 16 b-CH, 17 a-CH, 19 -CH , 20 -CH, 21 -CH , 22 b-
3 3
2
2
2
0
0
0
1-CH , 11 -CH , 3£O±C±(CH ) , overlapping); 30.4 (2 -
0
6.0 (8 -CH); 36.4 (4 -CH ); 37.3, 37.4 (1-CH ); 42.0 (5 -
CH); 1.55±2.25 (m, 19H, 6-CH , 7-CH , 11-CH , 2-CH ,
2
2
2
3 3
2
2
0
2
0
0
0
0
0
0
0
15 b-CH, 16 a-CH, 22 a-CH, 23 a-CH, 23 b-CH); 2.80±
0
CH ); 31.6 (22 -CH ); 33.6 (9 -CH, 23 -CH ); 34.1 (10 -
2
1 a-CH, 4 a-CH, 4 b-CH, 6 a-CH, 6 b-CH, 12 b-CH,
2
2
0
0
0
0
0
0
3.05 (m, 10H, 3-CH , 5-CH , 8-CH , 10-CH , 12-CH );
Cq); 35.2, 35.5 (12-CH , 1 -CH , 20 -CH, overlapping);
2
2
0
0
0
CH ); 43.2, 43.7 (3-CH , 10-CH , overlapping); 46.4, 46.6
3
2
2
2
2
2
2
2
0
3.05±3.15 (m, 2H, 1-CH ); 3.15±3.24 (m, 1H, 3 b-CH);
2
2
2
2
0
0
0
(
(
5-CH , 8-CH , 13 -Cq, overlapping); 47.1 (17 -CH); 48.2
2
3.58±3.66 (m, 1H, 7 b-CH); 4.40 (br s, 2£OH, [1H O]);
2
2
0
0
0
1
14
1
14 -CH); 71.7 (3 -CH); 73.0 (12 -CH); 79.7 (3£quat OC,
7.27 (1:1:1, t, Jˆ51 Hz, N± H); 8.07, 8.76, 8.93 (3£br s,
ammonium signals). C NMR, 100 MHz, [ H] DMSO:
6
1
3
2
overlapping); 156.0, 156.4 (3£NH±CO±O±C±(CH ) );
3
3
1
73.7 (CH ±CO±NH). MS, FAB found 877, 6%
0 0 0
11.6 (18 -CH ); 18.3 (21 -CH ); 20.3 (11 -CH ); 22.6,
3 3 2
1
2
1
1
0 0
22.7 (6-CH , 7-CH , 19 -CH , overlapping); 23.2 (15 -
2 2 3
(
MS m/z, FAB found 877.6620, (M 11), C H N O
requires M 11ˆ877.6630.
1
N -(3a,7b-Dihydroxy-5b-cholan-24-carbonyl)-( N , N ,
N -tri-tert-butoxycarbonyl)-1,12-diamino-4,9-diazado-
decane 10. Poly-Boc protected polyamine 7 (500 mg,
M 11), C H N O requires M ˆ876. High-resolution
4
9
88
1
4
9
1
0
CH ); 23.8 (2-CH ); 26.1 (11-CH ); 27.9 (16 -CH ); 30.6
2 2 2 2
4
9
89
4
9
1
0
0
0
(2 -CH ); 31.6 (22 -CH ); 32.3 (23 -CH ); 34.8, 34.9
2
2
0
2
0
0
(1 -CH ); 35.6 (1-CH ); 36.2 (12-CH ); 38.9, 39.7, 39.9
0
(
6 -CH , 10 -Cq); 35.1 (9 -CH, 20 -CH, overlapping); 35.3
2
4
9
0
0
2
2
2
1
2
0
0
0
0
43.9 (3-CH ); 44.7 (10-CH ); 46.0, 46.1 (5-CH , 8-CH );
(4 -CH , 8 -CH, 12 -CH ); 41.4 (5 -CH ); 42.0 (13 -Cq);
2
2
2
2
2
2
2
0 0 0 0
1
.0 mmol) and ursodeoxycholic acid (391 mg, 1.0 mmol)
50.1 (14 -CH); 55.6 (17 -CH); 66.2 (7 -CH); 70.4 (3 -CH);
1
1
were reacted according to the general procedure A to afford
the title compound 10 as a white foam (667 mg, 76%).
Puri®ed by column chromatography over silica gel
173.2 (CO±NH). MS, FAB found 577, 100% (M 11),
C H N O requires Mˆ576. High-resolution MS m/z,
3
4
64
4
3
1
FAB found 577.5060, (M 11), C H N O requires
1
3
4
65
4
3
1
(
MeOH; 18:1 v/v). IR (KBr) 3320 (OH), 1690 and 1670
CH Cl ±MeOH; 30:1 to 15:1 v/v), R 0.25 (CH Cl ±
M 11ˆ577.5057.
2
2
f
2
2
1
CO±N). H NMR, 400 MHz, CDCl : 0.66 (s, 3H, 18 -
0
1
(
CH ); 0.84±2.40 (m, 67H, 2-CH , 6-CH , 7-CH , 11-CH ,
N -(3a,12a-Dihydroxy-5b-cholan-24-carbonyl)-1,12-
diamino-4,9-diazadodecane 12. Boc protected polyamine
amide 9 (595 mg, 0.68 mmol) was deprotected according to
the general procedure B. The residue was lyophilized to
produce 760 mg of a white powder, 460 mg was puri®ed
by RP-HPLC (Supelcosil ABZ1Plus, 5 mm, 25 cm£
10 mm, MeCN±0.1% aq. TFA, 25:75 v/v) to afford the
3
3
2
2
2
2
0
0
0
0
0
3
8
1
2
8
£O±C±(CH ) , 1 -CH , 2 -CH , 4 -CH , 5 -CH, 6 -CH ,
0 0 0 0 0 0
-CH, 9 -CH, 11 -CH , 12 -CH , 14 -CH, 15 -CH ,
2 2 2
3
3
2
2
2
2
0
0
0
0
0
0
6 -CH , 17 -CH, 19 -CH , 20 -CH, 21 -CH , 22 -CH ,
2
3
3
2
0
-CH , 10-CH , 12-CH ); 3.54±3.66 (m, 2H, 3 -CH,
3 -CH ); 3.00±3.35 (m, 12H, 1-CH , 3-CH , 5-CH ,
2
2
2
2
0
2
2
2

3
446
I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
1
resolution MS m/z, FAB found 577.5066, (M 11),
1
title polyamine amide 12 as a clear glass (polytri¯uoro-
acetate salt, 158 mg, 43%), t 5.6 min by RP-HPLC (Supel-
1
C H N O requires M 11ˆ577.5057.
R
34 65
4
3
cosil ABZ1Plus, 5 mm, 25 cm£10 mm, MeCN±0.1% aq.
TFA, 25:75 v/v). IR (KBr) 3400 (OH) and 1670 (CO±N).
1
2
0
H NMR, 400 MHz, [ H] DMSO: 0.59 (s, 3H, 18 -CH );
Acknowledgements
6
3
0 0 0 0
.79±1.07 (m, 8H, 1 b-CH, 15 a-CH, 19 -CH , 21 -CH );
3 3
0
1
0
.07±2.20 (m, 32H, 2-CH , 6-CH , 7-CH , 11-CH , 1 a-
2
We thank the EPSRC and Celltech Chiroscience (CASE
award to A. J. G.) for ®nancial support. We also thank Dr
Michael A. W. Eaton (Celltech Chiroscience) and Dr Ian S.
Haworth (University of Southern California) for useful
discussions and for their interest in these studies. I. S. B.
and I. S. Haworth are joint holders of a NATO grant (CRG
970290).
2
2
0
2
0
0
0
0
0
b-CH, 7 a-CH, 7 b-CH, 8 b-CH, 9 a-CH, 11 a-CH,
CH, 2 a-CH, 2 b-CH, 4 a-CH, 4 b-CH, 5 b-CH, 6 a-CH,
6
1
0
0
0
0
0
0
1 b-CH, 14 a-CH, 15 b-CH, 16 a-CH, 16 b-CH, 17 a-
0
0
0
0
0
0
0
0
0
0
0
CH, 20 -CH, 22 a-CH, 22 b-CH, 23 a-CH, 23 b-CH);
.80±3.04 (m, 10H, 3-CH , 5-CH , 8-CH , 10-CH , 12-
2
CH ); 3.04±3.15 (m, 2H, 1-CH ); 3.30±3.42 (m, 1H, 3 b-
2
2
2
2
0
2
2
0
CH); 3.60±4.60 (m, 12 b-CH, 2£OH, [1H O]); 7.27 (1:1:1,
2
1
14
1
t, Jˆ51 Hz, N ± H); 8.07, 8.76, 8.94 (3£br s, ammonium
1
3
2
0
signals). C NMR, 100 MHz, [ H] DMSO: 12.3 (18 -CH );
References
6
3
0 0
7.0 (21 -CH ); 22.5, 22.6 (6-CH , 7-CH ); 23.0 (19 -CH );
3 2 2 3
3.4 (15 -CH ); 23.7 (2-CH ); 26.1 (11-CH , 7 -CH , over-
2
1
2
0
0
2
2
2
1. Behr, J.-P.; Demeneix, B.; Loef¯er, J.-P.; Perez-Mutul, J. Proc.
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0
0
0
lapping); 26.9, 27.1 (6 -CH , 16 -CH ); 28.5 (11 -CH );
2
2
2
0
0
0
3
(
0.1 (2 -CH ); 31.6 (22 -CH ); 32.3 (23 -CH ); 32.8
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2
2
2
0 0 0 0
9 -CH); 33.7 (10 -Cq); 35.0 (1 -CH , 20 -CH, overlap-
2
0
0
ping); 35.5 (8 -CH); 35.6 (1-CH ); 36.1, 36.2 (12-CH , 4 -
2
CH ); 41.5 (5 -CH); 43.8 (3-CH , 13 -Cq, overlapping);
2
4
4
2
0
4.5 (10-CH ); 45.9, 46.0, 46.1 (5-CH , 8-CH , 17 -CH);
0
2
0
7.4 (14 -CH); 69.8 (3 -CH); 70.9 (12 -CH); 173.2 (CO±
2
2
2
5. Wilson, R. W.; Bloom®eld, V. A. Biochemistry 1979, 18, 2192.
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0
0
0
1
1
NH). MS, FAB found 577, 100% (M 11), C H N O
3
3
4
64
1
4
1
requires M ˆ576. High-resolution MS m/z, FAB found
1
1
5
5
77.5063, (M 11), C H N O requires M 11ˆ
8. Blagbrough, I. S.; Carrington, S.; Geall, A. J. Pharmaceutical
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3
4
65
4
3
77.5057.
9
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1
N -(3a,7b-Dihydroxy-5b-cholan-24-carbonyl)-1,12-
diamino-4,9-diazadodecane 13. Boc protected polyamine
amide 10 (618 mg, 0.71 mmol) was deprotected according
to the general procedure B. The residue was lyophilized to
produce 840 mg of a white powder, 330 mg was puri®ed by
RP-HPLC (Supelcosil ABZ1Plus, 5 mm, 25 cm£10 mm,
MeCN±0.1% aq. TFA, 22:78 v/v) to afford the title poly-
amine amide 13 as a clear glass (poly-TFA salt, 124 mg,
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4
5
9%), tR 5.2 min by RP-HPLC (Supelcosil ABZ1Plus,
mm, 25 cm£10 mm, MeCN±0.1% aq. TFA, 25:75 v/v).
1
IR (KBr) 3400 (OH) and 1670 (CO±N). H NMR,
2
0
00 MHz, [ H] DMSO: 0.62 (s, 3H, 18 -CH ); 0.82±1.52
6 3
4
(
6
1
0 0 0 0 0
m, 22H, 1 b-CH, 2 a-CH, 2 b-CH, 4 a-CH, 5 b-CH,
0
4 a-CH, 15 a-CH, 16 b-CH, 17 a-CH, 19 -CH , 21 -
0 0 0 0 0
a-CH, 6 b-CH, 8 b-CH, 11 a-CH, 11 b-CH, 12 a-CH,
0
0
0
0
0
0
3
0
-CH , 11-CH , 1 a-CH, 4 b-CH, 12 b-CH, 15 b-CH,
CH , 22 b-CH); 1.52±2.20 (m, 17H, 2-CH , 6-CH ,
3
16. Geall, A. J.; Eaton, M. A. W.; Baker, T.; Catterall, C.; Blag-
brough, I. S. FEBS Lett. 1999, 459, 337.
2
2
0
6 a-CH, 20 -CH, 22 a-CH, 23 a-CH, 23 b-CH); 2.80±
0
0
0
7
1
3
3
2
2
0
0
0
0
.05 (m, 10H, 3-CH , 5-CH , 8-CH , 10-CH , 12-CH );
0
17. Walker, S.; So®a, M. J.; Kakarla, R.; Kogan, N. A.; Wierichs,
L.; Longley, C. B.; Bruker, K.; Axelrod, H. R.; Midha, S.; Babu, S.
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Rangara, R.; Pitard, B.; Crouzet, J.; Wils, P.; Schwartz, B.;
Scherman, D. J. Med. Chem. 1998, 41, 224.
2
2
2
2
2
0
.05±3.13 (m, 2H, 1-CH ); 3.22±3.37 (m, 2H, 3 b-CH,
2
0
7
a-CH); 5.00 (br s, 2£OH [1H O]); 7.25 (1:1:1, t,
2
1
14
1
Jˆ51 Hz, N± H); 8.04, 8.73, 8.91 (3£br s, ammonium
1
3
2
0
signals). C NMR, 100 MHz, [ H] DMSO: 12.1 (18 -CH );
6
3
0 0
8.5 (21 -CH ); 20.9 (11 -CH ); 22.6, 22.7 (6-CH , 7-CH );
3 2 2 2
1
2
0
0
3.3 (19 -CH ); 23.8 (2-CH ); 26.1 (11-CH ); 26.8 (15 -
3
19. Geall, A. J.; Blagbrough, I. S. Tetrahedron Lett. 1998, 39, 443.
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Blagbrough, I. S. Chem. Commun. 1998, 1403.
2
2
0
0
0
23 -CH ); 33.8 (10 -Cq); 34.9 (1 -CH ); 35.1 (20 -CH);
CH ); 28.2 (16 -CH ); 30.3 (2 -CH ); 31.7 (22 -CH ); 32.4
2
2
2
0
2
0
0
0
5.6 (1-CH ); 36.2 (12-CH ); 37.3 (6 -CH ); 37.7 (4 -
(
3
2
2
0
CH ); 38.8 (9 -CH); 39.9 (12 -CH ); 42.2 (5 -CH); 43.0,
0
2
2
2
21. O'Sullivan, M. C.; Dalrymple, D. M. Tetrahedron Lett. 1995,
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0
3.1 (8 -CH, 13 -Cq); 43.9 (3-CH ); 44.7 (10-CH ); 46.1,
0
0
2
2
0
6.2 (5-CH , 8-CH ); 54.7 (14 -CH); 55.9 (17 -CH); 69.5
0
4
4
(
22. Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron
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2
2
0
0
2
2
0
0
1
7 -CH); 69.7 (3 -CH); 173.2 (CO±NH). MS, FAB found
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1
1
5
77, 60% (M 11), C H N O requires M ˆ576. High-
3
4
64
4
3

I. S. Blagbrough et al. / Tetrahedron 56 (2000) 3439±3447
3447
2
2
2
2
2
2
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4
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1
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4
4
4
1
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4
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