Mechanism and structure-activity relationships of norspermidine-based peptidic inhibitors of trypanothione reductase

Article abstract of DOI:10.1016/j.bmc.2005.04.039

A library of polyamine-peptide conjugates based around some previously identified inhibitors of trypanothione reductase was synthesised by parallel solid-phase chemistry and screened. Kinetic analysis of library members established that subtle structural

Full text of DOI:10.1016/j.bmc.2005.04.039

Bioorganic & Medicinal Chemistry 13 (2005) 4513–4526
Mechanism and structure–activity relationships
of norspermidine-based peptidic inhibitors of
trypanothione reductase
Mark J. Dixon, Richard I. Maurer, Cristina Biggi, Julen Oyarzabal,à
Jonathan W. Essex and Mark Bradley*
School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK
Received 3 August 2004; revised 11 April 2005; accepted 15 April 2005
Available online 25 May 2005
Abstract—A library of polyamine–peptide conjugates based around some previously identified inhibitors of trypanothione reductase
was synthesised by parallel solid-phase chemistry and screened. Kinetic analysis of library members established that subtle structural
changes altered their mechanism of action, switching between competitive and non-competitive inhibition. The mode of action of the
non-competitive inhibitors was investigated in detail by a variety of techniques including enzyme kinetic analysis (looking at both
NADPH and trypanothione disulfide substrates), gel filtration chromatography and analytical ultracentrifugation, leading to the
identification of an allosteric mode of inhibition.
ꢀ 2005 Published by Elsevier Ltd.
1. Introduction
and host has opened up a highly promising avenue of
research into the development of novel drugs for the
treatment of these diseases.3,4
Infection by Trypanosoma and Leishmania parasites is
the cause of a number of tropical diseases in humans
and domestic livestock that represent a major threat to
Third World health. These diseases include African
sleeping sickness (caused by Trypanosoma brucei rhodes-
iense and Trypanosoma brucei gambiense), ChagasÕ dis-
ease (caused by Trypanosoma cruzi), mucocutaneous
and cutaneous leishmaniasis (caused by various species
of Leishmania) and Nagana (caused by Trypanosoma
congolense). Currently available treatments suffer from
resistance and severe side effects and the need for more
effective drugs is acute.1,2 The discovery of a significant
difference in the redox defence mechanism of parasite
The maintenance of an intracellular reducing environ-
ment is essential in protecting the cell from the highly
reactive oxygen species that arise as a result of aerobic
respiration and host immune response to infection. In
most organisms, the abundant thiol glutathione (GSH)
is utilised for this purpose, the level of which is main-
tained by the action of glutathione reductase (GR),
which regenerates GSH from glutathione disulfide
([GS]2). The GR/GSH system is replaced in trypano-
somes and leishmania by an analogous system based
on trypanothione (N1,N8-bis(glutathionyl)-spermidine,
T[SH]2) and trypanothione reductase (TR), which regen-
erates T[SH]2 from trypanothione disulfide (T[S]2). GR
and TR are similar in many respects, both being
NADPH-dependent homo-dimeric flavoproteins with
approximately 40% homology in their amino acid se-
quence and essentially identical mechanisms.5,6 How-
ever, the two enzymes display a remarkable degree of
specificity for their respective substrates due to critical
differences between their active sites.7 In GR, the active
site is positively charged to accommodate the carboxy-
late groups of [GS]2, whereas the active site of TR
is larger and has more hydrophobic character to
Keywords: Trypanothione reductase; Parallel synthesis; Mechanism of
action; Structure–activity relationships.
*
Corresponding author at present address: School of Chemistry,
University of Edinburgh, Kings Buildings, West Mains Road,
Edinburgh EH9 3JJ, UK. Tel.: +44 1316 504820; fax: +44 2380
596766; e-mail: mbradley@staffmail.ed.ac.uk
Current address: Division of Biological Chemistry & Molecular
Microbiology, School of Life Sciences, University of Dundee, Dundee
DD1 5EH, UK.
à
Current address: Molecular Informatics, Johnson & Johnson Phar-
maceutical R&D, 45007 Toledo, Spain.
0968-0896/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2005.04.039

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M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
accommodate the positively charged spermidine portion
of T[S]2.6,8,9 The key role of TR in protecting the para-
site from oxidative stress and the specificities of TR and
GR make the enzyme a potential target for anti-trypano-
somal drugs.
group of various sizes or left as a primary amino group.
The library was completed with the dipeptides H-Trp-
Arg-OH (32) and H-Trp-Arg(Pmc)-OH (33), included
to evaluate the importance of the polyamine backbone.
Compounds 4–6 and 10–12 were prepared from immobi-
lised, bis-protected norspermidine 9, attached to the resin
via an acid labile, Wang-type linker (Scheme 1). Com-
pound 9 was prepared by protection of the primary amino
groups of norspermidine 7 by reaction with 2 equiv of 2-
acetyl dimedone, prior to reaction with 40-nitrophenyl-
(4-hydroxymethyl-phenoxy-allylacetate)carbonate28 to
give 8. The allyl ester of 8 was removed upon treatment
with Pd(PPh3)4 and thiosalicylic acid, and the resulting
carboxylic acid coupled to aminomethyl polystyrene resin
via standard HOBt/DIC activation to give 9. Synthesis of
4–6and 10–12 was completed from 9 using standard solid-
phase peptide chemistry and compounds were purified by
semi-preparative HPLC. In order to avoid the total
deprotection of the Pmc group during cleavage from the
Numerous inhibitors of TR have been reported, based
on tricyclic antidepressants,10–14 polyamine conju-
gates15–20 and substrate analogues21,22 amongst others.
Within our group, combinatorial and solid-phase chem-
istry has been utilised in the search for novel inhibitors
of TR,23–26 and one such investigation identified the
spermidine-based compounds 1–3 (Fig. 1).27 Com-
pounds 1–3 represent promising lead structures with
the most active, 2, having a Ki of 100 nM. Surprisingly,
1–3 displayed non-competitive behaviour despite their
structural similarity to T[S]2 and other reported competi-
tive inhibitors. Here, we describe our investigations into
the mechanism of action of this class of inhibitors and
the development of structure–activity relationships
based on the screening of structurally simplified
analogues.
resin,
a
cocktail of TFA/thioanisole/DCM/water
(10:2:7:1) for 4 · 15 min was used. HPLC analysis of the
crude cleavage mixture (of 4, 5 and 6) is shown Figure 2.
2. Results
Compounds 17–31 were prepared from immobilised,
orthogonally protected norspermidine 1329in a multiple
parallel fashion (Scheme 2). Following Dde deprotec-
tion of 13, blocking of the first arm was achieved by
reaction with either di-tert-butyl dicarbonate, acetic
anhydride or hexanoic anhydride to give 14–16. The tri-
fluoroacetyl group was removed and derivatisation on
the second arm was accomplished using standard
solid-phase chemistry.
In order to avoid the use of a mixture of regioisomers (as
with 2), norspermidine analogues of the lead compounds
were used as direct replacements (4–6, Fig. 1). It was
envisaged that this structural modification, whilst sim-
plifying synthesis and purification, would have little
effect on the inhibitory properties of the compounds,
although it was necessary to confirm this experimentally.
The lead compounds contain several structural compo-
nents of potential importance to binding, including the
arginine and tryptophan residues, the 2,2,5,7,8-penta-
methylchroman-6-sulfonamide (Pmc) group and the
polyamine. A library of simplified structural analogues
of 4–6 was investigated with the aim of identifying key
binding elements and developing SAR. The library con-
sisted of norspermidine based compounds derivatised
with structural elements on both arms (10–12, Scheme
1 and Table 1) and one arm only (17–31, Scheme 2
and Table 1). The one-armed compounds were deriva-
tised on the second arm with either a simple amide
Initial screening of the entire library at a concentration
of 100 lM revealed a range of activities. Compounds
17–22, 32 and 33 showed little or no activity and were
not investigated further. Compounds 4–6, 10–12 and
23–31 exhibited significant activity and were subjected
to full kinetic analysis (Table 1 and Fig. 3). The most
potent inhibitors were two of the lead compounds, 5
and 6, which had measured Ki values of 220 nM and
160 nM, respectively. The other library members exhib-
ited varying potency with Ki values in the range
3–131 lM. As with the spermidine compounds 1–3,
NH
NH
O
O
O
O
H
H
N
S
N
H2N
N
H
N
H
N
H
NH2
Pmc =
n
O
O
O
NH
NH
HN
NHR1
HN
NHR2
1 n =2, R1 = R2 = H
4 n =1, R1 = R2 = H
5 n = 1, R1 = H, R2 = Pmc
6 n = 1, R1 = R2 = Pmc
2 n = 2, R1 = H, R2 = Pmc/
n = 2, R1 = Pmc, R2 = H
3 n = 2, R1 = R2 = Pmc
Figure 1. Previously identified spermidine-based TR inhibitors 1–3 and norspermidine analogues 4–6.

M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
4515
O
H2N
H2N
NH
DdeHN
DdeHN
N
O
(i), (ii)
O
O
8
O
7
(iii), (iv)
O
4 (7%)
5 (5%)
6 (5%)
DdeHN
N
O
(v) - (ix)
H
N
DdeHN
O
O
9
(v), (viii), (x)
(v) - (vii), (ix)
10 (21%)
11 (7%)
12 (6%)
Scheme 1. Preparation of 4–6 and 10–12. Reagents and conditions: (i) 2-acetyldimedone, EtOH, reflux, 2 h, 99%; (ii) 40-nitrophenyl-(4-
hydroxymethylphenoxyallylacetate) carbonate, DMF, 16 h, 74%; (iii) Pd(PPh3)4, thiosalicylic acid, DCM, THF, 2 h, 70%; (iv) aminomethyl
polystyrene resin, HOBt, DIC, DCM, DMF, 16 h; (v) 5% H2NNH2/DMF, 30 min; (vi) Fmoc-Arg(Pmc)-OH, HOBt, DIC, DCM, DMF, 2 h; (vii)
20% piperidine/DMF, 2 · 10 min; (viii) Boc-Trp(Boc)-OH, HOBt, DIC, DCM, DMF, 2 h; (ix) TFA/thioanisole/DCM/water (10:2:7:1), 4 · 15 min;
(x) TFA/thioanisole/DCM/water (16:2:1:1), 2 h.
Table 1. Structures and results of kinetic analysis of 4–6, 10–12 and 17–31
R1HN
N
H
NHR2
Compound
R1
R2
Mode
Ki (lM)
4
Trp-Arg
Trp-Arg(Pmc)
Trp-Arg(Pmc)
Trp
Trp-Arg
Trp-Arg
Trp-Arg(Pmc)
Trp
Non-competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Nd
14 0.5
5
0.22 0.02
0.16 0.01
19 1.1
6
10
11
12
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Arg(Pmc)
Arg(Pmc)
Trp
Arg
Arg(Pmc)
H
11 0.5
0.3
3
>100
Trp
Trp
COCH3
CO(CH2)4CH3
H
Nd
Nd
>100
>100
Arg
Arg
Nd
Nd
>100
>100
COCH3
CO(CH2)4CH3
H
Arg
Nd
>100
131 7.2
69 3.2
Arg(Pmc)
Arg(Pmc)
Arg(Pmc)
Trp-Arg
Trp-Arg(Pmc)
Trp-Arg
Trp-Arg(Pmc)
Trp-Arg
Trp-Arg(Pmc)
Non-competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Competitive
Non-competitive
Competitive
Non-competitive
COCH3
CO(CH2)4CH3
H
7
0.2
83 4.4
1.1
H
9
COCH3
COCH3
CO(CH2)4CH3
CO(CH2)4CH3
69 5.8
12 0.4
24 2.0
6
0.2
4–6 exhibited non-competitive behaviour. Interestingly,
this was not the case for all the library members, with
several (10, 26, 28 and 30) exhibiting competitive
behaviour.
3). The low Km value of NADPH with TR (5 lM)30
meant that extremely low concentrations of NADPH
were required for this experiment, so reaction progres-
sion, as shown by NADPH consumption, was moni-
tored using fluorescence rather than UV absorbance.
Unfortunately, due to the difficult nature of these ki-
netic experiments, it was not possible to investigate all
library members in this way. An alternative approach
The mechanism of action of 4–6 was investigated in
more detail. Non-competitive behaviour of 5 was estab-
lished with respect to NADPH by kinetic analysis (Fig.

4516
M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
GDH. No inhibition was observed with 4–6 at a con-
centration of 100 lM.
DdeHN
O
N
O
H
N
TR is an a2-homodimer and the active site is composed
of residues from both subunits.32 As such, an alternative
possible mechanism of action involves binding of the
inhibitors in the dimer interface region of the protein
leading to the disruption of dimer formation, as has
been reported with inhibitors of HIV-1 protease.33–35
To investigate this possibility, analysis of TR by analyt-
ical ultracentrifugation (AUC) in the presence and
absence of 4–6 was undertaken (AUC is a powerful tech-
nique for the determination of the stoichiometry of mac-
romolecular complexes in their native, solution-phase
state).36 Analysis of TR in the absence of inhibitor gave
a mass in good agreement with that reported for the TR
dimer.4 Analysis of TR in the presence of 4–6
([I] > 5 · Ki) showed no significant change in the stoichi-
ometry of the enzyme. The AUC findings were con-
firmed by analysis of TR in the presence and absence
of 5 by gel filtration chromatography (Fig. 4).
O
O
TfaHN
13
(i) - (iii)
RHN
O
N
O
H
N
O
O
H2N
14 R = Boc
15 R = COCH3
16 R = CO(CH2)4CH3
(vi), (vii),
(iv), (viii)
(vi), (vii),
(v), or (viii)
(iv), (v)
3. Discussion
20 (59%)
21 (70%)
22 (73%)
23 (19%)
24 (21%)
25 (10%)
17 (16%)
18 (34%)
19 (21%)
26 (6%)
27 (6%)
28 (13%)
29 (12%)
30 (10%)
31 (7%)
The solid-phase synthesis of both the one and two
armed inhibitors was extremely clean as shown by the
HPLC trace of the crude cleavage mixture of 4–6 (Fig.
2). The poor isolated yields (Scheme 2) were the result
of instrumentally caused losses during semi-preparative
HPLC purification. The non-competitive behaviour
and Ki values obtained with 4–6 were in broad agree-
ment with the results reported for the spermidine
compounds 1–3 (Ki = 16 lM, 100 nM and 190 nM,
respectively) and confirmed that the substitution of nor-
spermidine for spermidine had minimal effect on the
inhibitory properties of the compounds.
Scheme 2. Preparation of 17–31. Reagents and conditions: (i) 5%
H2NNH2/DMF, 30 min; (ii) Boc2O, DIPEA, DCM, 2 h or acetic
anhydride/pyridine (1:1), 30 min or hexanoic anhydride/pyridine (1:1),
30 min; (iii) 1 M KOH/MeOH/THF (5:1:1), 2 · 2 h; (iv) Boc-Trp(Boc)-
OH, HOBt, DIC, DCM, DMF, 2 h; (v) TFA/thioanisole/DCM/water
(16:2:1:1), 2 h; (vi) Fmoc-Arg(Pmc)-OH, HOBt, DIC, DCM, DMF,
2 h; (vii) 20% piperidine/DMF, 2 · 10 min; (viii) TFA/thioanisole/
DCM/water (10:2:7:1), 4 · 15 min (overall isolated yields).
Kinetic analysis surprisingly showed both competitive
and non-competitive inhibitors within the library. The
competitive inhibitors were similar in structure to many
reported TR inhibitors, containing both a hydrophobic
group and a polyamine; however, this was also true of
other compounds in this series which displayed non-
competitive behaviour! No improvement in potency
compared to 5 and 6 was observed with any of the
was used to confirm these findings, whereby 4–6 were
screened against glutamate dehydrogenase (GDH), an
NADPH dependent enzyme that is involved in amino
acid metabolism.31 Although TR and GDH are unre-
lated enzymes, the NADPH binding sites are similar
in both and a tight binding ligand in the TR NADPH
pocket might be expected to show some activity with
Figure 2. Analytical HPLC trace of a crude cleavage mixture giving 4–6.

M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
4517
Figure 3. Eadie–Hofstee plots showing non-competitive behaviour with respect to disulfide substrate of (a) 5, (b) 6, (d) 12 and (f) 31 and competitive
behaviour of (c) 10 and (e) 26. Non-competitive behaviour of 5 with respect to NADPH is shown in (g). All assays were carried out in triplicate.
library members. Any change in structure was detrimen-
tal and resulted in an increase in Ki of at least an order
of magnitude. In general, activity was related to struc-
tural similarity to 5 and 6 with the most active deriva-
tive, 12, just lacking the tryptophan residues found in
6, while structural simplification led to a corresponding
loss of activity.
activity of 20–22 is not surprising as previously screened
polyamines show little activity against TR.18
3.2. Arginine residue
Removal of the arginine residue led to a change in
mechanism (compare 4 with 10) and a loss of potency
(compare 26, 28 and 30 with 17–19). The lack of activ-
ity of 17–19 may simply be due to the arginine residue
in 26, 28 and 30 acting as a spacer and placing the
polyamine and hydrophobic groups at an optimum
distance apart for binding. Alternatively, the arginine
side-chain in 26, 28 and 30 may be involved in a
3.1. Tryptophan residue
Removal of the tryptophan residue resulted in a loss of
potency (compare 5 and 6 with 11 and 12; 27, 29 and 31
with 23–25; and 26, 28 and 30 with 20–22. The lack of

4518
M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
25
Given the non-competitive nature of 5, the differences
in structure and especially the differences in charge, the
possibility of any of the non-competitive inhibitors bind-
ing at the NADPH pocket is small.
20
15
10
5
The possibility of inhibitor binding in the dimer interface
region of the enzyme, hence causing disruption of the TR
dimer, can be discounted as only dimeric enzyme was ob-
served by both AUC and gel filtration chromatography.
No monomeric enzyme was observed as would be ex-
pected in the case of dimer disruption. Clearly, inhibitor
binding does not cause disruption to the dimeric form of
the enzyme and therefore inhibitor binding presumably
occurs at a site distinct from the dimer interface region.
0
-5
0
10
20
30
40
50
60
Retention time (min)
Figure 4. Gel filtration analysis of TR in absence (red) and presence
(blue) of inhibitor 5 (10 lM).
A recent report described the action of a class of com-
pounds termed aggregation inhibitors.37 These inhibi-
tors possess an unusual mechanism of action, whereby
aggregates of many individual molecules form in solu-
tion and inactivate the target enzyme by absorption or
adsorption. These inhibitors are typically highly sensi-
tive to enzyme concentration and show little selectivity.
This mechanism of action was discounted for the inhib-
itors in the present study as there was no evidence of
aggregation by either AUC or gel filtration analysis,
while activity was not enzyme-concentration dependent,
nor were the compounds active against other enzymes.
binding interaction, increasing the potency of these
compounds.
3.3. Pmc group
The Pmc group was clearly important for potency, as
the eight top inhibitors all contained this structural fea-
ture. Removal of the group led to an increase in Ki
(compare 5 and 6 with 4; 27, 29 and 31 with 26, 28
and 30; and 23–25 with 20–22). A change in mechanism
also resulted (compare 27, 29 and 31 with 26, 28 and 30).
Similar compounds containing other hydrophobic argi-
nine protecting groups have been studied in our group
and found to be potent TR inhibitors.24
4. Conclusions
In summary, multiple parallel solid-phase chemistry was
utilised in the synthesis of a library of compounds that
were screened against TR. No improvement in potency
from the lead compounds was achieved, but SAR data
were developed and both competitive and non-competi-
tive behaviour was observed. The mechanism of action
of the non-competitive inhibitors was investigated using
a variety of techniques, and evidence for an allosteric
mechanism involving inhibitor binding at a site distinct
from the disulfide substrate active site, the NADPH ac-
tive site, and the dimer interface region was obtained.
The site of binding of the non-competitive inhibitors
and specific interactions of the competitive inhibitors
in the active site are currently under investigation by
protein crystallography. The results presented strongly
suggest that caution is needed when studying this pro-
tein in that simple assumptions about binding sites
and mode of action may not always be justified as
shown here for the clear distinction and change of mech-
anism from competitive to non-competitive with just
small changes in structure.
3.4. One- and two-armed structures
The most potent compounds were derivatised on both
arms of norspermidine, although significant activity
was also observed with one-armed compounds. No cor-
relation between the mode of action and either one- or
two-armed structures was observed. In the one-armed
series, the more hydrophobic hexanoyl capping group
generally gave the best inhibition and the primary amino
group the worst.
3.5. Norspermidine
The lack of activity of the dipeptides 32 and 33 demon-
strated the importance of the polyamine portion of the
inhibitors. The presence of the carboxylate moiety is
almost certainly having a detrimental effect on the
binding, as the TR active site is known to disfavour
the binding of negatively charged over positively
charged compounds.
Kinetic analysis of 5 showed non-competitive behaviour
with respect to NADPH and the lack of activity against
GDH of 4–6 supported this finding. Although NADPH
and 4–6 share some structural similarities, such as
hydrophobic portions (the adenine and nicotinamide
in NADPH, the indole and PMC group in 4–6), overall
there is little similarity between the structures. At physio-
logical pH, NADPH is negatively charged and the
binding pocket in TR has a corresponding positive
charge, whereas the inhibitors are positively charged.
5. Experimental
5.1. General information
NMR spectra were recorded on a Bruker DPX-400
spectrometer operating at 400 MHz for 1H and
100 MHz for 13C. All coupling constants (J values) were
measured in Hertz. ES mass spectra were recorded on a
VG Platform Quadrupole Electrospray Ionisation mass

M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
4519
spectrometer. High resolution ES mass spectra were re-
corded on a Bruker Apex III FT-ICR mass spectrome-
ter. Infra-red spectra were recorded on a BioRad FTS
135 Paragon 1000 spectrometer with a Golden Gate
ATR accessory. Melting points were recorded on a Gal-
lenkamp melting point apparatus and are uncorrected.
Analytical RP-HPLC was performed on a Hewlett
Packard HP 1100 chemstation equipped with a Pheno-
menex Prodigy 5 lm C18 (150 · 4.6 mm) column with
a flow rate of 0.5 mL/min. Semi-preparative RP-HPLC
was performed on a Hewlett Packard HP 1100 chemsta-
tion equipped with a Phenomenex Prodigy 5 lm C18
(250 · 10 mm) column with a flow rate of 2.5 mL/min.
Mobile phase A was 0.1% TFA in water, mobile phase
B was 0.042% TFA in acetonitrile. Gradient 1 was T = 0
min, B = 10%, T = 10 min, B = 90%, T = 15 min, B =
90%. Gradient 2 was T = 0 min, B = 0%, T = 5 min,
B = 0%, T = 15 min, B = 60%, T = 16 min, B = 100%,
T = 20 min, B = 100%. Gradient 3 was T = 0 min,
B = 0%, T = 40 min, B = 50%, T = 45 min, B = 90%,
T = 50 min, B = 90%. Thin layer chromatography
(TLC) was performed on Alugram Sil G/UV/254
precoated plates and visualised under UV light. Column
chromatography was performed using Sorbsil C60, 40–
60 mesh silica. All reactions were performed at room
temperature unless otherwise stated. UV/vis spectra
and kinetics were recorded on a Hewlett Packard
HP8452A diode array spectrophotometer. Fluorescence
spectra were recorded on a Hitachi F2000 fluorescence
spectrophotometer.
DMF (1 mL per 100 mg of resin) for 2 · 10 min. The re-
sin was filtered and washed sequentially with DMF,
DCM, MeOH and Et2O (3 mL per 100 mg of resin, five
times with each).
5.6. Boc protection
The resin was swollen in DCM (1 mL per 100 mg of re-
sin) for 10 min, filtered and treated with a solution of di-
tert-butyl dicarbonate (5 equiv relative to resin-bound
amine) and DIPEA (2 equiv) in DCM (1 mL per
100 mg of resin) for 2 h. The resin was filtered and
washed sequentially with DMF, DCM, MeOH and
Et2O (3 mL per 100 mg of resin, five times with each).
5.7. N-Capping with acetic anhydride
The resin was swollen in DCM (1 mL per 100 mg of re-
sin) for 10 min, filtered and treated with a mixture of
acetic anhydride and pyridine (1:1, 1 mL per 100 mg of
resin) for 30 min. The resin was filtered and washed
sequentially with DMF, DCM, MeOH and Et2O
(3 mL per 100 mg of resin, five times with each).
5.8. N-Capping with hexanoic anhydride
The resin was swollen in DCM (1 mL per 100 mg of resin)
for 10 min, filtered and treated with a mixture of hexanoic
anhydride and pyridine (1:1, 1 mL per 100 mg of resin) for
30 min. The resin was filtered and washed sequentially
with DMF, DCM, MeOH and Et2O (3 mL per 100 mg
of resin, five times with each).
5.2. General procedures for solid-phase chemistry
Reactions were monitored by the use of a qualitative
Kaiser test.38
5.9. Tfa deprotection
The resin was swollen in DCM (1 mL per 100 mg of re-
sin) for 10 min, filtered and treated with a solution of
1 M KOH/THF/MeOH (5:1:1, 1 mL per 100 mg of re-
sin) for 2 · 2 h. The resin was filtered and washed
sequentially with DMF/water (1:1), MeOH/water (1:1),
DMF, DCM, MeOH and Et2O (3 mL per 100 mg of re-
sin, five times with each).
5.3. Coupling of carboxylic acids
Acid (3 equiv relative to resin-bound amine) and N-
hydroxybenzotriazole (3 equiv) were dissolved in
DCM/DMF (4:1, 1 mL per 100 mg of resin) and the
resultant solution shaken for 10 min. N,N0-Diisopropyl-
carbodiimide (3 equiv) was added and the resultant solu-
tion shaken for a further 10 min. The solution was
added to the resin (pre-swollen in DCM (1 mL per
100 mg of resin) for 10 min) and the mixture shaken
for 2 h. The resin was filtered and washed sequentially
with DMF, DCM, MeOH and Et2O (3 mL per 100
mg of resin, five times with each).
5.10. Cleavage from the resin with TFA
The resin was swollen in DCM (1 mL per 100 mg of resin)
for 10 min, filtered and treated with a mixture of TFA/
thioanisole/DCM/water (16:2:1:1, 1 mL per 100 mg of
resin) for 2 h or treated with a mixture of TFA/thioani-
sole/DCM/water (10:2:7:1, 1 mL per 100 mg of resin)
for 4 · 15 min. The resin was filtered and washed with
MeOH (3 mL per 100 mg of resin, five times), the filtrate
and washings combined and concentrated in vacuo. The
crude product was then dissolved in TFA, precipitated
from ice-cold Et2O, the precipitate collected by centrifu-
gation and lyophilised.
5.4. Dde deprotection
The resin was swollen in DCM (1 mL per 100 mg of resin)
for 10 min, filtered and treated with 5% v/v hydrazine in
DMF (1 mL per 100 mg of resin) for 30 min. The resin
was filtered and washed sequentially with DMF, DCM,
MeOH and Et2O (3 mL per 100 mg of resin, five times
with each).
5.11. Preparation of N1,N9-bis-1-(4,4-dimethyl-2,6-dioxo-
cyclohexylidene)ethyl-norspermidine
5.5. Fmoc deprotection
Norspermidine (0.96 mL, 6.9 mmol) and 2-acetyl dime-
done (2.50 g, 13.7 mmol) were dissolved in ethanol
(47 mL) and heated at reflux for 2 h. The solvent was
The resin was swollen in DCM (1 mL per 100 mg of re-
sin) for 10 min and treated with 20% v/v piperidine in

4520
M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
removed in vacuo and the resulting yellow oil dissolved
in EtOAc (50 mL), washed with water (3 · 50 mL), dried
(MgSO4) and concentrated in vacuo to give the title
compound as a colourless oil (3.13 g, 99%).
were added and the reaction mixture stirred for 2 h. Sol-
vent was removed in vacuo and the resulting oily residue
purified by column chromatography (eluting with
EtOAc, then EtOAc/MeOH, 3:2) to yield the title com-
pound as a colourless oil (1.59 g, 70%).
Rf (DCM/MeOH, 17:3) 0.39. RP-HPLC (analytical, gra-
dient 1, k = 220 nm): Rt = 6.3 min. dH (400 MHz,
CDCl3): 13.40 (br s, 2H, 2 · C@C(CH3)NH), 3.49 (m,
4H, 2 · C@C(CH3)NHCH2), 2.74 (t, 4H, J = 7, CH2-
NHCH2), 2.56 (s, 6H, 2 · C@C(CH3)NH), 2.35 (s, 8H,
4 · COCH2), 1.83 (tt, 4H, J = 7, 7, 2 · CH2CH2CH2),
1.50 (br s, 1H, CH2NHCH2), 1.02 (s, 12H, 2 ·
C(CH3)2). dC (100 MHz, CDCl3): 197.8 (C), 173.8 (C),
108.2 (C), 53.2 (CH2), 47.1 (CH2), 41.6 (CH2), 30.4 (C),
29.7 (CH2), 28.5 (CH3), 18.2 (CH3). IR m cmꢀ1: 2954,
2867, 2958, 1633, 1567. MS (ES+) m/z: 460.4 (100%)
[M+H]+, 919.8 (18%) [2M+H]+. HRMS (ES+)
C26H42N3O4 calcd: 460.3170. Found: 460.3167 [M+H]+.
Rf (EtOAc/MeOH, 2:1) 0.18. RP-HPLC (analytical, gra-
dient 1, k = 220 nm): Rt = 8.5 min. dH (400 MHz,
CDCl3): 13.40 (br s, 2H, 2 · NH), 7.30 (d, 2H, J = 8.5,
2 · ArCH), 6.88 (d, 2H, J = 8.5, 2 · ArCH), 5.08 (s,
2H, OCH2), 4.67 (s, 2H, OCH2), 3.43–25 (m, 8H, 2 ·
CH2CH2CH2), 2.52–2.33 (m, 14H, 2 · C@C(CH3)NH,
4 · CH2C(CH3)2), 1.96–1.80 (m, 4H, 2 · CH2CH2CH2),
1.03 (s, 12H, 2 · C(CH3)2). dC (100 MHz, CDCl3): 197.3
(C), 172.8 (C), 169.8 (C), 156.9 (C), 155.8 (C), 129.3
(CH), 128.7 (C), 113.6 (CH), 107.0 (C), 66.0 (CH2),
65.2 (CH2), 51.7 (CH2), 42.0 (CH2), 40.1 (CH2), 29.1
(C), 27.2 (CH3), 25.2 (CH2), 16.9 (CH3). IR m cmꢀ1
:
2950, 1692, 1561. MS (ES+) m/z: 668.5 (100%)
[M+H]+, 690.5 (40%) [M+Na]+. HRMS (ES+)
C36H50N3O9 calcd: 668.3542. Found: 668.3563 [M+H]+.
5.12. Preparation of N1,N9-bis-1-(4,4-dimethyl-2,6-dioxo-
cyclohexylidene)ethyl-N5-(4-(allyloxycarbonylmethoxy)-
phenyl-methoxycarbonyl)-norspermidine (8)
5.14. Preparation of polymer supported N1,N9-bis-1-(4,4-
N1,N9-bis-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-
norspermidine (3.00 g, 6.5 mmol) and 40-nitrophenyl-(4-
hydroxymethylphenoxy-allylacetate) carbonate (3.03 g,
7.8 mmol) were dissolved in DMF (20 mL) and stirred
for 16 h. The reaction mixture was poured into 1 M
KHSO4 (100 mL) and extracted with EtOAc (3 ·
100 mL). The combined organic extracts were washed
with water (5 · 100 mL), dried (MgSO4) and concen-
trated in vacuo. The resulting oily residue was purified
by column chromatography (eluting with EtOAc, then
EtOAc/MeOH, 49:1) to give the title compound as a col-
ourless oil (2.61 g, 74%).
dimethyl-2,6-dioxocyclohexylidene)ethyl-norspermidine
(9)
N1,N9-bis-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-
N5-4-((carboxy-methyl)phenyl-methoxy-carbonyl)-nor-
spermidine (1.50 g, 2.3 mmol) was coupled to amino-
methyl-polystyrene resin (1% DVB cross-linked, 1.11
mmol/g, 1.35 g, 1.5 mmol) according to the general pro-
cedure for carboxylic acid coupling with the exceptions
that 1.5 equiv of reagents was used and the reaction
was shaken for 16 h. The Dde groups of a small portion
of resin were removed according to the general proce-
dure and loading, measured by a quantitative Kaiser
test, determined to be 1.32 mmol/g of amine (equivalent
to 0.66 mmol/g of norspermidine).
Rf (EtOAc/MeOH, 49:1) 0.20. RP-HPLC (analytical,
gradient 1, k = 220 nm): Rt = 10.0 min. dH (400 MHz,
CDCl3): 13.50 (br s, 2H, 2 · NH), 7.28 (d, 2H, J = 9,
2 · ArCH), 6.86 (d, 2H, J = 9, 2 · ArCH), 5.91 (ddt,
1H, J = 17, 10.5, 5.5, CH@CH2), 5.33 (dd, 1H, J = 17, 1,
CH@CHH), 5.26 (dd, 1H, J = 10.5, 1, CH@CHH), 5.06
(s, 2H, OCH2), 4.69 (d, 2H, J = 5.5, CH2CH@CH2),
4.65 (s, 2H, OCH2), 3.43–3.30 (m, 8H, 2 · CH2CH2CH2),
2.55–2.45 (m, 6H, 2 · C@C(CH3)NH), 2.34 (s, 8H,
4 · CH2C(CH3)2), 1.93–1.82 (m, 4H, 2 · CH2CH2CH2),
1.01 (s, 12H, 2 · C(CH3)2). dC (100 MHz, CDCl3): 197.3
(C),173.6 (C), 168.5 (C), 157.8 (C), 156.2 (C), 131.5
(CH), 130.2 (CH), 129.7 (C), 119.3 (CH2), 114.7 (CH),
108.0 (C), 67.2 (CH2), 66.0 (CH2), 65.3 (CH2), 53.6
(CH2), 52.3 (CH2), 45.1 (CH2), 44.4 (CH2), 40.7
IR m cmꢀ1: 2928, 1697, 1637, 1573, 1452.
5.15. Preparation of N1,N9-bis-(L-tryptophanyl-L-argin-
yl)-norspermidine (4), N1-L-tryptophanyl-L-arginyl-N9-L-
tryptophanyl-L-arginyl(2,2,5,7,8-pentamethylchroman-6-
sulfonamide)-norspermidine (5), N1,N9-bis-(L-tryptophan-
yl-L-arginyl(2,2,5,7,8-pentamethylchroman-6-sulfon-
amide))-norspermidine (6)
Compounds 4–6 were synthesised from 9 (0.60 g,
0.4 mmol) as outlined in Scheme 1 using the general
procedures for solid-phase chemistry. The desired com-
pounds were purified as their TFA salts by semi-prep
HPLC (gradient 3). The desired compounds eluted at
22.4 min (4, 25 mg, 7%), 33.8 min (5, 23 mg, 5%) and
43.5 min (6, 28 mg, 5%).
(CH2), 30.2 (C), 28.3 (CH3), 17.9 (CH3). IR m cmꢀ1
:
2955, 2859, 2958, 1758, 1636, 1570. MS (ES+) m/z:
708.2 (100%) [M+H]+. HRMS (ES+) C39H54N3O9
calcd: 708.3855. Found: 708.3865 [M+H]+.
5.13. Preparation of N1,N9-bis-1-(4,4-dimethyl-2,6-dioxo-
cyclohexylidene)ethyl-N5-4-((carboxy-methyl)phenyl-
methoxy-carbonyl)-norspermidine
Compound 4: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 11.8 min. dH (400 MHz, CD3OD):
7.71 (d, 2H, J = 7.5, 2 · ArCH), 7.44 (d, 2H, J = 7.5,
2 · ArCH), 7.30 (s, 2H, 2 · ArCH), 7.19 (t, 2H,
J = 7.5, 2 · ArCH), 7.11 (t, 2H, J = 7.5, 2 · ArCH),
4.36–4.27 (m, 4H, 2 · Arg aCH, 2 · Trp aCH), 3.50
(dd, 2H, J = 15, 6, 2 · Trp bCHH), 3.40–3.17 (m, 10H,
A solution of 8 (2.40 g, 3.4 mmol) in DCM/THF (1:1,
21 mL) was purged with N2 for 1 h. Thiosalicylic acid
(1.05 g, 6.8 mmol) and Pd(PPh3)4 (0.39 g, 0.3 mmol)

M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
4521
2 · Trp bCHH, 2 · Arg dCH2, 2 · CONHCH2), 2.99–
2.93 (m, 4H, CH2NH CH2), 1.94–1.59 (m, 12H, 2 ·
Arg bCH2, 2 · Arg cCH2, 2 · NHCH2CH2CH2NH).
dC (100 MHz, CD3OD): 173.9 (C), 170.2 (C), 158.5
(C), 138.0 (C), 128.1 (C), 125.5 (CH), 122.8 (CH),
120.2 (CH), 119.0 (CH), 112.5 (CH), 107.7 (C), 54.6
(CH), 46.2 (CH2), 41.7 (CH2), 36.9 (CH2), 30.0 (CH2),
28.5 (CH2), 27.2 (CH2), 26.1 (CH2). MS (ES+) m/z:
816.5 (100%) [M+H]+. HRMS (ES+) C40H62N15O4
calcd: 816.5103. Found: 816.5103 [M+H]+.
(approximately 40%) of library compounds was also
characterised by 13C NMR.
5.17. Preparation of N1,N9-bis-(L-tryptophanyl)-norsper-
midine (10)
Compound 10 was synthesised from
9
(0.20 g,
0.13 mmol) as outlined in Scheme 1 using the general
procedures for solid-phase chemistry. The desired com-
pound was purified as the TFA salt by semi-prep HPLC
(gradient 3), elution time = 21.4 min (17 mg, 21%).
Compound 5: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 14.2 min. dH (400 MHz, CD3OD):
7.67 (d, 2H, J = 7.5, 2 · ArCH), 7.40 (d, 2H, J = 7.5,
2 · ArCH), 7.26 (s, 2H, 2 · ArCH), 7.15 (t, 2H,
J = 7.5, 2 · ArCH), 7.06 (t, 2H, J = 7.5, 2 · ArCH),
4.40 (br s, 1H, Arg aCH), 4.33–4.22 (m, 3H, Arg
aCH, 2 · Trp aCH), 3.50–3.42 (m, 2H, 2 · Trp bCHH),
3.31–3.11 (m, 10H, 2 · CONHCH2, 2 · Trp bCHH,
2 · Arg dCH2), 2.98–2.92 (m, 4H, CH2NHCH2), 2.65
(t, 2H, J = 6.5, ArCH2), 2.59 (s, 3H, ArCH3), 2.57 (s,
3H, ArCH3), 2.11 (s, 3H, ArCH3), 1.90–1.54 (m, 14H,
2 · Arg bCH2, 2 · Arg cCH2, 2 · NHCH2CH2CH2NH,
ArCH2CH2), 1.31 (s, 6H, C(CH3)2). dC (100 MHz,
CD3OD): 174.5 (C), 174.2 (C), 170.4 (C), 170.1 (C),
158.7 (C), 154.9 (C), 138.2 (C), 136.6 (C), 136.2 (C),
134.3 (C), 128.3 (C), 125.7 (CH), 125.1 (C), 123.0
(CH), 120.4 (CH), 119.5 (C), 119.2 (CH), 112.6 (CH),
107.9 (C), 74.9 (C), 54.8 (CH), 54.5 (CH), 46.5 (CH2),
46.5 (CH2), 41.9 (CH2), 37.1 (CH2), 33.7 (CH2), 30.3
(CH2), 30.2 (CH2), 28.7 (CH2), 27.4 (CH2), 27.4
(CH2), 26.9 (CH3), 26.3 (CH2), 22.3 (CH2), 19.0
(CH3), 17.9 (CH3), 12.3 (CH3). MS (ES+) m/z: 542.2
(100%) [M+2H]2+, 1082.9 (13%) [M+H]+.
RP-HPLC (analytical, gradient 2, k = 220 nm):
Rt = 12.3 min. dH (400 MHz, CD3OD): 7.62 (d, 2H,
J = 7.5, 2 · ArCH), 7.40 (d, 2H, J = 7.5, 2 · ArCH),
7.22 (s, 2H, 2 · ArCH), 7.16 (t, 2H, J = 7.5, 2 · ArCH),
7.08 (t, 2H, J = 7.5, 2 · ArCH), 4.11 (t, 2H, J = 7.5,
2 · Trp aCH), 3.42–3.16 (m, 8H, 2 · Trp bCH2,
2 · CONHCH2), 2.65 (t, 4H, J = 7, CH2NH CH2),
1.71 (tt, 4H, J = 7, 7, 2 · CH2CH2CH2). dC (100 MHz,
CD3OD): 170.9 (C), 138.2 (C), 128.4 (CH), 125.5
(CH), 123.0 (CH), 120.3 (CH), 119.1 (CH), 112.7 (C),
108.1 (C), 55.3 (CH), 46.2 (CH2), 37.4 (CH2), 28.8
(CH2), 27.1 (CH2). MS (ES+) m/z: 504.4 (100%)
[M+H]+, 526.4 (11%) [M+Na]+. HRMS (ES+)
C28H38N7O2 calcd: 504.3082. Found: 504.3087 [M+H]+.
5.18. Preparation of N1-L-arginyl(2,2,5,7,8-pentamethyl-
chroman-6-sulfonamide)-N9-L-arginyl-norspermidine (11)
and N1,N9-bis-(L-arginyl(2,2,5,7,8-pentamethylchroman-
6-sulfonamide))-norspermidine (12)
Compounds 11 and 12 were synthesised from 9 (0.40 g,
0.26 mmol) as outlined in Scheme 1 using the general
procedures for solid-phase chemistry. The desired com-
pounds were purified as the TFA salts by semi-prep
HPLC (gradient 3), elution time = 31.0 min (11, 14 mg,
7%) and 40.7 min (12, 18 mg, 6%).
Compound 6: RP-HPLC (analytical, gradient 2, k =
220 nm): Rt = 16.2 min. dH (400 MHz, CD3OD): 7.63
(d, 2H, J = 8, 2 · ArCH), 7.36 (d, 2H, J = 8, 2 · ArCH),
7.23 (s, 2H, 2 · ArCH), 7.11 (t, 2H, J = 8, 2 · ArCH),
7.03 (t, 2H, J = 8, 2 · ArCH), 4.39 (br s, 2H, 2 · Arg
aCH), 4.22 (m, 2H, 2 · Trp aCH), 3.43 (dd, 2H, J =
15, 6, 2 · Trp bCHH), 3.33–3.16 (m, 8H, 2 · Trp
bCHH, 2 · Arg dCHH, 2 · CONHCH2), 3.13–3.07 (m,
2H, 2 · Arg dCHH), 2.98–2.94 (m, 4H, CH2NHCH2),
2.62 (t, 4H, J = 6.5, 2 · ArCH2CH2), 2.56 (s, 6H, 2 ·
ArCH3), 2.54 (s, 6H, 2 · ArCH3), 2.08 (s, 6H,
2 · ArCH3), 1.90–1.75 (m, 10H, 2 · NHCH2CH2CH2-
NH, 2 · ArCH2CH2, 2 · Arg bCHH), 1.74–1.63 (m,
2H, 2 · Arg bCHH), 1.60–1.51 (m, 4H, 2 · Arg cCH2),
1.29 (s, 12H, 2 · C(CH3)2). dC (100 MHz, CD3OD):
174.4 (C), 170.2 (C),158.2 (C), 155.0 (C), 138.2 (C),
136.5 (C), 136.1 (C), 134.3 (C), 128.4 (C), 125.7 (CH),
125.2 (C), 122.9 (CH), 120.4 (CH), 119.5 (C), 119.2
(CH), 112.6 (CH), 107.9 (C), 75.0 (C), 54.8 (CH), 54.5
(CH), 46.6 (CH2), 37.1 (CH2), 33.7 (CH2), 30.3 (CH2),
28.8 (CH2), 27.5 (CH2), 26.9 (CH3), 22.4 (CH2), 19.1
(CH3), 18.0 (CH3), 12.3 (CH3). MS (ES+) m/z: 675.4
(100%) [M+2H]2+, 1349.0 (22%) [M+H]+.
Compound 11: RP-HPLC (analytical, gradient 2, k =
220 nm): Rt = 13.3 min. dH (400 MHz, CD3OD): 3.97–
3.87 (m, 2H, 2 · Arg aCH), 3.50–3.16 (m, 8H, 2 · Arg
dCH2, 2 · CONHCH2), 3.10 (t, 4H, J = 7.5, CH2NHCH2),
2.69 (t, 2H, J = 7, ArCH2CH2), 2.59 (s, 3H, ArCH3),
2.58 (s, 3H, ArCH3), 2.12 (s, 3H, ArCH3), 2.03–
1.83 (m, 10H, 2 · Arg bCH2, 2 · NHCH2CH2CH2NH,
ArCH2CH2), 1.74–1.61 (m, 4H, 2 · Arg cCH2), 1.33 (s,
6H, C(CH3)2). MS (ES+) m/z: 356.1 (100%) [M+2H]2+
,
710.7 (23%) [M+H]+. HRMS (ES+) C32H60N11O5S
calcd: 710.4494. Found: 710.4500 [M+H]+.
Compound 12: RP-HPLC (analytical, gradient 2, k =
220 nm): Rt = 15.5 min. dH (400 MHz, CD3OD): 3.96
(t, 2H, J = 6.5, 2 · Arg aCH), 3.50–3.08 (m, 12H, 2 ·
NHCH2CH2CH2NH, 2 · Arg dCH2), 2.67 (t, 4H, J = 7,
2 · ArCH2CH2), 2.58 (s, 6H, 2 · ArCH3), 2.56 (s, 6H,
2 · ArCH3), 2.11 (s, 6H, 2 · ArCH3), 2.03–1.81 (m,
12H, 2 · Arg bCH2, 2 · ArCH2CH2, 2 · NHCH2CH2-
CH2NH), 1.64 (tt, 4H, J = 7, 7, 2 · Arg cCH2), 1.32
(s, 12H, 2 · C(CH3)2). MS (ES+) m/z: 489.1 (100%)
5.16. Library characterisation
[M+2H]2+
,
998.6 (12%) [M+Na]+. HRMS (ES+)
All compounds were characterised by 1H NMR, LRMS,
C46H78N11O8S2 calcd: 976.5471. Found: 976.5475
[M+H]+.
HRMS and RP-HPLC.
A representative sample

4522
M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
5.19. Preparation of 17–25
J = 7, CH3). MS (ES+) m/z: 416.3 (100%) [M+H]+.
HRMS (ES+) C23H38N5O2 calcd: 416.3020. Found:
Compounds 17–25 were synthesised from 13 (0.20 g,
0.1 mmol) as outlined in Scheme 2 using the general pro-
cedures for solid-phase chemistry.
416.3025 [M+H]+.
5.23. N1-L-Arginyl-norspermidine (20) (17 mg, 59%)
5.20. N1-L-Tryptophanyl-norspermidine (17)
dH (400 MHz, CD3OD): 4.11 (q, 1H, J = 6.5, Arg aCH),
3.57–3.14 (m, 10H, Arg dCH2, CH2CH2CH2NHCH2-
CH2CH2), 2.29–1.99 (m, 6H, Arg bCH2, CH2CH2-
CH2NHCH2CH2CH2), 1.84–1.74 (m, 2H, Arg cCH2).
dC (100 MHz, CD3OD): 170.7 (C), 158.1 (C), 53.9
(CH), 46.3 (CH2), 45.7 (CH2), 41.4 (CH2), 37.6 (CH2),
37.4 (CH2), 29.1 (CH2), 26.6 (CH2), 26.3 (CH2), 24.9
(CH2). MS (ES+) m/z: 288.4 (100%) [M+H]+. HRMS
(ES+) calcd: 288.2506. Found: 288.2506 [M+H]+.
The desired compound was isolated as the TFA salt by
semi-prep HPLC (gradient 3), elution time = 20.8 min
(7 mg, 16%).
RP-HPLC (analytical, gradient 2, k = 220 nm): Rt =
10.8 min. dH (400 MHz, CD3OD): 7.63 (d, 1H, J = 8,
ArCH), 7.41 (d, 1H, J = 8, ArCH), 7.23 (s, 1H, ArCH),
7.17 (t, 1H, J = 8, ArCH), 7.09 (t, 1H, J = 8, ArCH),
4.11 (t, 1H, J = 7.5, Trp aCH), 3.44–3.17 (m, 4H, Trp
bCH2, CONHCH2), 3.10–2.98 (m, 4H, CH2CH2CH2-
NH2), 2.81 (t, 2H, J = 7.5, CONHCH2CH2CH2), 2.13–
2.02 (m, 2H, CH2CH2NH2), 1.84–1.73 (m, 2H, CON-
HCH2CH2). MS (ES+) m/z: 318.2 (100%) [M+H]+.
HRMS (ES+) C17H28N5O calcd: 318.2288; (found)
318.2287 [M+H]+.
5.24. N1-L-Arginyl-N9-acetyl-norspermidine (21) (23 mg,
70%)
dH (400 MHz, CD3OD): 3.97 (br s, 1H, Arg aCH), 3.46–
3.23 (m, 6H, Arg dCH2, 2 · CONHCH2), 3.11–3.01 (m,
4H, CH2NHCH2), 2.05–1.87 (m, 9H, Arg bCH2,
2 · NHCH2CH2CH2NH, CH3), 1.77–1.67 (m, 2H, Arg
cCH2). dC (100 MHz, CD3OD): 174.2 (C), 170.7 (C),
158.7 (C), 54.0 (CH), 46.6 (CH2), 41.5 (CH2), 37.4
(CH2), 37.1 (CH2), 29.6 (CH2), 27.5 (CH2), 27.2
(CH2), 25.3 (CH2), 22.5 (CH3). MS (ES+) m/z: 330.4
(100%) [M+H]+, 352.4 (8%) [M+Na]+. HRMS (ES+)
C14H32N7O2 calcd: 330.2612. Found: 330.2605 [M+H]+.
5.21. N1-L-Tryptophanyl-N9-acetyl-norspermidine (18)
The desired compound was isolated as the TFA salt by
semi-prep HPLC (gradient 3), elution time = 22.4 min
(16 mg, 34%).
RP-HPLC (analytical, gradient 2, k = 220 nm): Rt =
11.2 min. dH (400 MHz, CD3OD): 7.63 (d, 1H, J = 8,
ArCH), 7.42 (d, 1H, J = 8, ArCH), 7.24 (s, 1H, ArCH),
7.15 (t, 1H, J = 8, ArCH), 7.09 (t, 1H, J = 8, ArCH),
4.13 (t, 1H, J = 7.5, Trp aCH), 3.39 (dd, 1H, J = 14.5,
7.5, Trp bCHH), 3.35–3.21 (m, 5H, Trp bCHH,
2 · CONHCH2), 2.88 (t, 2H, J = 7, CH2NHCH2), 2.69
(t, 2H, J = 7, CH2NHCH2), 1.99 (s, 3H, CH3), 1.85
(tt, 2H, J = 7, 7, CH2CH2CH2), 1.75 (tt, 2H, J = 7, 7,
CH2CH2CH2). dC (100 MHz, CD3OD): 174.4 (C),
170.8 (C), 138.2 (C), 128.4 (C), 125.5 (CH), 122.9
(CH), 119.2 (CH), 112.7 (CH), 108.2 (C), 55.3 (CH),
46.4 (CH2), 46.1 (CH2), 37.3 (CH2), 36.9 (CH2), 28.7
(CH2), 27.6 (CH2), 27.1 (CH2), 22.5 (CH3). MS (ES+)
m/z: 360.2 (100%) [M+H]+, 382.2 (25%) [M+Na]+.
HRMS (ES+) C19H30N5O2 calcd: 360.2394. Found:
360.2392 [M+H]+.
5.25. N1-L-Arginyl-N9-hexanoyl-norspermidine (22)
(28 mg, 73%)
dH (400 MHz, CD3OD): 3.98 (br s, 1H, Arg aCH), 3.48–
3.24 (m, 6H, Arg dCH2, 2 · CONHCH2), 3.12–3.05 (m,
4H, CH2NHCH2), 2.25 (t, 2H, J = 7.5, COCH2), 2.08–
1.89 (m, 6H, Arg bCH2, 2 · NHCH2CH2CH2NH),
1.79–1.58 (m, 4H, Arg cCH2, COCH2CH2), 1.42–1.29
(m, 4H, CH3CH2CH2), 0.95 (t, 3H, J = 7, CH3). dC
(100 MHz, CD3OD): 177.3 (C), 170.7 (C), 158.8 (C),
54.0 (CH), 46.6 (CH2), 46.5 (CH2), 41.4 (CH2), 37.4
(CH2), 37.0 (CH2), 32.6 (CH2), 29.6 (CH2), 27.6 (CH2),
27.2 (CH2), 26.7 (CH2), 25.3 (CH2), 23.4 (CH2), 14.3
(CH3). MS (ES+) m/z: 386.5 (100%) [M+H]+.
5.26. N1-L-Arginyl(2,2,5,7,8-pentamethylchroman-6-sul-
fonamide)-norspermidine (23)
5.22. N1-L-Tryptophanyl-N9-hexanoyl-norspermidine (19)
The desired compound was purified as the TFA salt by
semi-prep HPLC (gradient 3), elution time = 32.4 min
(13 mg, 19%).
The desired compound was purified as the TFA salt by
semi-prep HPLC (gradient 3), elution time = 28.9 min
(11 mg, 21%).
RP-HPLC (analytical, gradient 2, k = 220 nm): Rt =
13.7 min. dH (400 MHz, CD3OD): 3.98 (t, 1H, J = 6.5,
Arg aCH), 3.52–3.32 (m, 4H, Arg dCH2, CONHCH2),
3.23–3.08 (m, 6H, H2NCH2CH2CH2NHCH2), 2.72 (t,
2H, J = 6.5, ArCH2), 2.63 (s, 3H, ArCH3), 2.61 (s, 3H,
ArCH3), 2.18–2.09 (m, 5H, ArCH3, NHCH2CH2CH2NH),
2.08–1.86 (m, 6H, Arg bCH2, NHCH2CH2CH2NH,
ArCH2CH2), 1.67 (tt, 2H, J = 7, 7, Arg cCH2), 1.36 (s,
6H, C(CH3)2). MS (ES+) m/z: 554.4 (100%) [M+H]+,
576.4 (70%) [M+Na]+. HRMS (ES+) C26H48N7O4S
calcd: 554.3483. Found: 554.3498 [M+H]+.
RP-HPLC (analytical, gradient 2, k = 220 nm): Rt =
13.0 min. dH (400 MHz, CD3OD): 7.51 (d, 1H, J = 8,
ArCH), 7.29 (d, 1H, J = 8, ArCH), 7.12 (s, 1H, ArCH),
7.03 (t, 1H, J = 8, ArCH), 6.97 (t, 1H, J = 8, ArCH),
3.99 (t, 1H, J = 7.5, Trp aCH), 3.32–3.08 (m, 6H, Trp
bCH2, 2 · CONHCH2), 2.76 (t, 2H, J = 7, CH2NHCH2),
2.59 (t, 2H, J = 7, CH2NHCH2), 2.11 (t, 2H, J = 7.5,
COCH2), 1.78–1.47 (m, 6H, COCH2CH2, 2 · NHCH2-
CH2), 1.29–1.14 (m, 4H, CH2CH2CH3), 0.81 (t, 3H,

M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
4523
5.27. N1-L-Arginyl(2,2,5,7,8-pentamethylchroman-6-sul-
fonamide)-N9-acetyl-norspermidine (24)
cCH2, NHCH2CH2CH2NH). MS (ES+) m/z: 474.4
(100%) [M+H]+. HRMS (ES+) C23H40N9O2 calcd:
474.3299. Found: 474.3291 [M+H]+.
The desired compound was purified as the TFA salt by
semi-prep HPLC (gradient 3), elution time = 34.3 min
(15 mg, 21%).
Compound 27: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 14.2 min. dH (400 MHz, CD3OD):
7.67 (d, 1H, J = 8, ArCH), 7.39 (d, 1H, J = 8, ArCH),
7.25 (s, 1H, ArCH), 7.15 (t, 1H, J = 8, ArCH), 7.07 (t,
1H, J = 8, ArCH), 4.44 (br s, 1H, Arg aCH), 4.23 (dd,
1H, J = 8, 6, Trp aCH), 3.46 (dd, 1H, J = 15, 6, Trp
bCHH), 3.38–3.19 (m, 3H, Trp bCHH, CONHCH2),
3.17–3.00 (m, 8H, Arg cCH2, CH2NHCH2CH2CH2),
2.68 (t, 2H, J = 6.5, ArCH2), 2.59 (s, 3H, ArCH3),
2.57 (s, 3H, ArCH3), 2.13–2.04 (m, 5H, ArCH3,
CH2CH2NH2), 1.95–1.79 (m, 5H, Arg bCHH,
ArCH2CH2, NHCH2CH2CH2NH), 1.77–1.55 (m, 3H,
Arg bCHH, Arg cCH2), 1.31 (s, 6H, C(CH3)2). MS
(ES+) m/z: 740.5 (100%) [M+H]+, 370.9 (90%)
RP-HPLC (analytical, gradient 2, k = 220 nm): Rt =
14.1 min. dH (400 MHz, CD3OD): 3.99 (t, 1H, J = 6.5,
Arg aCH), 3.50–3.19 (m, 6H, 2 · CONHCH2, Arg
dCH2), 3.12–2.95 (m, 4H, CH2NHCH2), 2.73 (t, 2H, J =
7, ArCH2), 2.62 (s, 3H, ArCH3), 2.60 (s, 3H, ArCH3),
2.15 (s, 3H, ArCH3), 2.04–1.86 (m, 11H, COCH3,
ArCH2CH2, Arg bCH2, 2 · NHCH2CH2CH2NH), 1.68
(t, 2H, J = 7, Arg cCH2), 1.36 (s, 6H, C(CH3)2). MS
(ES+) m/z: 596.5 (100%) [M+H]+, 618.5 (35%)
[M+Na]+. HRMS (ES+) C28H50N7O5S calcd:
596.3589. Found: 596.3581 [M+H]+.
[M+2H]2+
.
HRMS (ES+) C37H58N9O5S calcd:
5.28. N1-L-Arginyl(2,2,5,7,8-pentamethylchroman-6-sul-
fonamide)-N9-hexanoyl-norspermidine (25)
740.4276. Found: 740.4285 [M+H]+.
5.31. N1-L-Tryptophanyl-L-arginyl-N9-acetyl-norspermi-
dine (28) and N1-L-tryptophanyl-L-arginyl(2,2,5,7,8-pen-
tamethylchroman-6-sulfonamide)-N9-acetyl-norspermi-
dine (29)
The desired compound was purified as the TFA salt by
semi-prep HPLC (gradient 3), elution time = 39.3 min
(8 mg, 10%).
RP-HPLC (analytical, gradient 2, k = 220 nm): Rt =
15.0 min. dH (400 MHz, CD3OD): 4.01 (t, 1H, J = 6.5,
Arg aCH), 3.54–3.18 (m, 6H, Arg dCH2, 2 ·
CONHCH2), 3.13–3.05 (m, 4H, CH2NHCH2), 2.72 (t,
2H, J = 6.5, ArCH2), 2.62 (s, 3H, ArCH3), 2.61 (s, 3H,
ArCH3), 2.24 (t, 2H, J = 7.5, COCH2), 2.15 (s, 3H,
ArCH3), 2.07–1.86 (m, 8H, Arg bCH2, ArCH2CH2, 2 ·
NHCH2CH2CH2NH), 1.73–1.59 (m, 4H, COCH2CH2,
Arg cCH2), 1.40–1.30 (m, 10H, CH3CH2CH2, C(CH3)2),
0.95 (t, 3H, J = 7, CH3CH2). MS (ES+) m/z: 652.5
(100%) [M+H]+, 674.5 (90%) [M+Na]+, 326.9 (70%)
The desired compounds were purified as the TFA salts
by semi-prep HPLC (gradient 3), elution time = 19.4
min (28, 16 mg, 13%) and 37.0 min (29, 21 mg, 12%).
Compound 28: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 11.1 min. dH (400 MHz, CD3OD):
7.66 (d, 1H, J = 8, ArCH), 7.39 (d, 1H, J = 8, ArCH),
7.26 (s, 1H, ArCH), 7.15 (t, 1H, J = 8, ArCH), 7.06 (t,
1H, J = 8, ArCH), 4.30–4.24 (m, 2H, Arg aCH, Trp
aCH), 3.45 (dd, 1H, J = 15, 6.5, Trp bCHH), 3.38–
3.16 (m, 7H, Trp bCHH, 2 · CONHCH2, Arg dCH2),
2.96–2.88 (m, 4H, CH2NHCH2), 1.95 (s, 3H, CH3CO),
1.90–1.80 (m, 4H, 2 · NHCH2CH2CH2NH), 1.78–1.57
(m, 4H, Arg bCH2, Arg cCH2). dC (100 MHz, CD3OD):
174.3 (C), 174.2 (C), 170.4 (C), 158.7 (C), 138.1 (C),
128.4 (C), 125.8 (CH), 122.9 (CH), 120.4 (CH), 119.2
(CH), 112.6 (CH), 107.9 (C), 54.9 (CH), 54.8 (CH),
46.5 (CH2), 46.3 (CH2), 41.9 (CH2), 37.0 (CH2), 36.9
(CH2), 30.1 (CH2), 28.7 (CH2), 27.6 (CH2), 27.4
(CH2), 26.3 (CH2), 22.5 (CH3). MS (ES+) m/z:
258.9 (100%) [M+2H]2+, 516.5 (46%) [M+H]+. HRMS
(ES+) C25H42N9O3 calcd: 516.3405. Found: 516.3410
[M+H]+.
[M+2H]2+
.
HRMS (ES+) C32H58N7O5S calcd:
652.4215. Found: 652.4213 [M+H]+.
5.29. Preparation of 26–31
Compounds 26–31 were synthesised from 13 (0.40 g,
0.2 mmol) as outlined in Scheme 2 using the general
procedures for solid-phase chemistry.
5.30. N1-L-Tryptophanyl-L-arginyl-norspermidine (26)
and N1-L-tryptophanyl-L-arginyl(2,2,5,7,8-pentameth-
ylchroman-6-sulfonamide)-norspermidine (27)
The desired compounds were purified as the TFA salts
by semi-prep HPLC (gradient 3), elution time = 18.2
min (26, 7 mg, 6%) and 35.6 min (27, 11 mg, 6%).
Compound 29: RP-HPLC (analytical, gradient 2, k =
220 nm): Rt = 14.7 min. dH (400 MHz, CD3OD): 7.70
(d, 1H, J = 8, ArCH), 7.42 (d, 1H, J = 8, ArCH), 7.29
(s, 1H, ArCH), 7.18 (t, 1H, J = 8, ArCH), 7.10 (t, 1H,
J = 8, ArCH), 4.46 (br s, 1H, Arg aCH), 4.26 (t, 1H,
J = 7, Trp aCH), 3.49 (dd, 1H, J = 15, 6, Trp bCHH),
3.37–3.23 (m, 5H, Trp bCHH, 2 · CONHCH2), 3.20–
3.12 (m, 2H, Arg dCH2), 3.05–2.97 (m, 4H,
CH2NHCH2), 2.71 (t, 2H, J = 6.5, ArCH2CH2), 2.62
(s, 3H, ArCH3), 2.61 (s, 3H, ArCH3), 2.14 (s, 3H,
ArCH3), 1.98 (s, 3H, COCH3), 1.92–1.82 (m, 6H,
2 · NHCH2 CH2CH2NH, ArCH2CH2), 1.80–1.69 (m,
2H, Arg bCH2), 1.67–1.58 (m, 2H, Arg cCH2), 1.35 (s,
Compound 26: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 10.8 min. dH (400 MHz, CD3OD):
7.69 (d, 1H, J = 7.5, ArCH), 7.41 (d, 1H, J = 7.5,
ArCH), 7.27 (s, 1H, ArCH), 7.17 (t, 1H, J = 7.5, ArCH),
7.09 (t, 1H, J = 7.5, ArCH), 4.36–4.23 (m, 2H, Arg
aCH, Trp aCH), 3.49 (dd, 1H, J = 15, 6, Trp bCHH),
3.38–2.97 (m, 11H, Trp bCHH, Arg dCH2,
NHCH2CH2CH2NHCH2 CH2CH2NH2), 2.13–2.05 (m,
2H, CH2CH2NH2), 1.93–1.56 (m, 6H, Arg bCH2, Arg

4524
M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
6H, C(CH3)2). dC (100 MHz, CD3OD): 174.5 (C), 174.3
(C), 170.2 (C), 158.6 (C), 154.9 (C), 138.3 (C), 136.5 (C),
136.1 (C), 134.4 (C), 128.4 (C), 125.7 (CH), 125.1 (C),
123.0 (CH), 119.5 (CH), 119.2 (C), 119.0 (CH), 112.6
(CH), 107.9 (C), 75.0 (C), 54.8 (CH), 54.4 (CH), 46.6
(CH2), 46.5 (CH2), 37.0 (CH2), 33.8 (CH2), 30.3
(CH2), 28.8 (CH2), 27.7 (CH2), 27.5 (CH2), 26.9
(CH3), 22.5 (CH2), 22.4 (CH3), 19.0 (CH3), 17.9
(CH3), 12.3 (CH3). MS (ES+) m/z: 782.7 (100%)
[M+H]+, 804.7 (55%) [M+Na]+. HRMS (ES+)
C39H60N9O6S calcd: 782.4382. Found: 782.4357
[M+H]+.
cyclohexylcarbodiimide (0.13 g, 0.6 mmol). After 2 h,
the reaction mixture was filtered and the filtrate concen-
trated in vacuo to yield a white foam. The crude product
was dissolved in DMF (5 mL) and a solution of H-
Arg(Pmc)-OH (0.27 g, 0.6 mmol) and NEt3 (86 lL,
0.6 mmol) in DMF (2 mL) was added. The reaction mix-
ture was stirred for 16 h and concentrated in vacuo. The
residue was taken into water (50 mL), extracted with
EtOAc (4 · 50 mL), the organic extracts combined,
dried (MgSO4) and concentrated in vacuo to yield the
title compound as a white solid (0.39 g, 77%).
Rf (EtOAc/hexane, 1:1) 0.37. Mp 203–206 ꢁC. RP-
5.32. N1-L-Tryptophanyl-L-arginyl-N9-hexanoyl-norsper-
midine (30) and N1-L-tryptophanyl-L-arginyl(2,2,5,7,8-
pentamethylchroman-6-sulfonamide)-N9-hexanoyl-nor-
spermidine (31)
HPLC
(analytical,
gradient
1,
k = 220 nm):
Rt = 12.0 min. dH (400 MHz, CD3OD): 8.05 (d, 1H,
J = 7.5, ArCH), 7.59 (d, 1H, J = 7.5, ArCH), 7.47 (s,
1H, ArCH), 7.24 (t, 1H, J = 7.5, ArCH), 7.17 (t, 1H,
J = 7.5, ArCH), 4.41–4.32 (m, 2H, Arg aCH, Trp
aCH), 3.22–3.10 (m, 3H, Trp bCHH, Arg dCH2), 2.92
(dd, 1H, J = 14.5, 9, Trp bCHH), 2.61 (t, 2H, J = 7,
ArCH2), 2.53 (s, 3H, ArCH3), 2.51 (s, 3H, ArCH3),
2.06 (s, 3H, ArCH3), 1.90–1.73 (m, 3H, Arg bCHH,
ArCH2CH2), 1.70–1.58 (m, 10H, Arg bCHH,
CO2C(CH3)3), 1.55–1.45 (m, 2H, Arg cCH2), 1.34–1.23
(m, 15H, CO2C(CH3)3, C(CH3)2). dC (100 MHz,
CD3OD): 174.7 (C), 174.3 (C), 157.6 (C), 154.7 (C),
151.0 (C), 136.8 (C), 136.5 (C), 136.1 (C), 134.7
(C),131.8 (C), 125.4 (CH), 125.0 (C), 123.6 (CH), 120.1
(CH), 119.3 (C), 117.5 (C), 116.0 (CH), 84.6 (C), 80.7
(C), 74.8 (C), 55.9 (CH), 53.2 (CH), 41.5 (CH2), 34.7
(CH2), 33.8 (CH2), 30.1 (CH2), 28.7 (CH3), 28.4
(CH3), 27.0 (CH3), 22.3 (CH2), 19.0 (CH3), 17.9
(CH3), 12.3 (CH3). IR m cmꢀ1: 3300, 1728, 1547, 1453,
1368, 1252, 1157. MS (ES+) m/z: 827.5 (100%)
[M+H]+, 849.5 (92%) [M+Na]+. HRMS (ES+)
C41H58N6O10SNa calcd: 849.4008. Found: 849.3797
[M+Na]+.
The desired compounds were purified as the TFA salts
by semi-prep HPLC (gradient 3), elution time = 26.6
min (30, 14 mg, 10%) and 41.7 min (31, 14 mg, 7%).
Compound 30: RP-HPLC (analytical, gradient 2, k =
220 nm): Rt = 12.8 min. dH (400 MHz, CD3OD): 7.70
(d, 1H, J = 8, ArCH), 7.42 (d, 1H, J = 8, ArCH), 7.28
(s, 1H, ArCH), 7.18 (t, 1H, J = 8, ArCH), 7.10 (t, 1H,
J = 8, ArCH), 4.33 (dd, 1H, J = 8, 6, Arg aCH), 4.27
(dd, 1H, J = 8, 6, Trp aCH), 3.49 (dd, 1H, J = 15, 6,
Trp bCHH), 3.38–3.20 (m, 7H, Trp bCHH, Arg dCH2,
2 · CONHCH2), 3.03–2.90 (m, 4H, CH2NHCH2), 2.22
(t, 2H, J = 7.5, COCH2), 1.90–1.59 (m, 10H, Arg
bCH2, Arg cCH2, 2 · NHCH2CH2CH2 NH, CH2-
CH2CO), 1.39–1.27 (m, 4H, CH3CH2CH2), 0.92 (t,
3H, J = 7, CH3CH2). MS (ES+) m/z: 286.9 (100%)
[M+2H]2+
,
572.4 (70%) [M+H]+, 594.4 (15%)
[M+Na]+. HRMS (ES+) C29H50N9O3 calcd: 572.4031.
Found: 572.4041 [M+H]+.
Compound 31: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 15.5 min. dH (400 MHz, CD3OD):
7.71 (d, 1H, J = 8, ArCH), 7.44 (d, 1H, J = 8, ArCH),
7.29 (s, 1H, ArCH), 7.19 (t, 1H, J = 8, ArCH), 7.11 (t,
1H, J = 8, ArCH), 4.50 (br s, 1H, Arg aCH), 4.26 (t,
1H, J = 7, Trp aCH), 3.49 (dd, 1H, J = 15, 6, Trp
bCHH), 3.39–3.14 (m, 7H, Trp bCHH, Arg dCH2,
2 · CONHCH2), 3.09–2.97 (m, 4H, CH2NHCH2), 2.72
(t, 2H, J = 7, ArCH2CH2), 2.63 (s, 3H, ArCH3), 2.61
(s, 3H, ArCH3), 2.22 (t, 2H, J = 7.5, COCH2), 2.15 (s,
3H, ArCH3), 1.97–1.83 (m, 6H, ArCH2CH2,
2 · NHCH2CH2CH2NH), 1.82–1.71 (m, 2H, Arg
bCH2), 1.70–1.57 (m, 4H, CH2CH2CO, Arg cCH2),
1.39–1.28 (m, 10H, C(CH3)2, CH3CH2CH2), 0.94 (t,
3H, J = 7, CH3CH2). MS (ES+) m/z: 420.0 (100%)
5.34. Preparation of L-tryptophanyl-L-arginine (32) and
L-tryptophanyl-NG-2,2,5,7,8-pentamethylchroman-6-sul-
fonamide-L-arginine (33)
Boc-L-tryptophanyl-(Boc)-NG-2,2,5,7,8-pentamethylchro-
man-6-sulfonamide-L-arginine (0.10 g, 0.1 mmol) was
treated with a solution of TFA/thioanisole/DCM/water
(10:2:7:1, 5 mL) at room temperature for 30 min. The
reaction mixture was concentrated in vacuo, the result-
ing oily residue dissolved in TFA, precipitated from
ice-cold Et2O, collected by centrifugation and lyophi-
lised. The desired compounds were purified as the
TFA salts by semi-prep HPLC (gradient 3), elution
time = 22.0 min (32, 12 mg, 33%) and 42.6 min (33,
15 mg, 24%).
[M+2H]2+
,
860.7 (50%) [M+Na]+, 838.7 (45%)
[M+H]+. HRMS (ES+) C43H68N9O6S calcd: 838.5008.
Found: 838.5002 [M+H]+.
Compound 32: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 11.6 min. dH (400 MHz, CD3OD):
7.76 (d, 1H, J = 7.5, ArCH), 7.56 (d, 1H, J = 7.5,
ArCH), 7.42 (s, 1H, ArCH), 7.30 (t, 1H, J = 7.5, ArCH),
7.21 (t, 1H, J = 7.5, ArCH), 4.52–4.37 (m, 2H, Arg
aCH, Trp aCH), 3.60–3.42 (m, 2H, Trp bCH2), 3.34–
3.25 (m, 2H, Arg dCH2), 2.16–1.64 (m, 4H, Arg bCH2,
Arg cCH2). dC (100 MHz, CD3OD): 175.0 (C), 170.9
(C), 158.1 (C), 137.7 (C), 128.1 (C), 126.0 (CH), 122.9
5.33. Preparation of Boc-L-tryptophanyl-(Boc)-NG-
2,2,5,7,8-pentamethylchroman-6-sulfonamide-L-arginine
To a stirred solution of Boc-Trp(Boc)-OH (0.25 g,
0.6 mmol) and N-hydroxy succinimide (0.71 g,
0.6 mmol) in THF (5 mL) at 0 ꢁC was added N,N0-di-

M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
4525
(CH), 120.3 (CH), 119.0 (CH), 112.7 (CH), 107.4 (C),
5.37. Glutamate dehydrogenase assay
54.7 (CH), 53.6 (CH), 41.7 (CH2), 29.5 (CH2), 28.1
(CH2), 25.5 (CH2). MS (ES+) m/z: 361.1 (100%)
[M+H]+. HRMS (ES+) C17H25N6O3 calcd: 361.1982.
Found: 361.1978 [M+H]+.
Kinetic analyses were performed in assays of a total
volume of 1 mL, containing sodium phosphate buffer
(100 mM NaH2PO4, pH 7.5), EDTA (1 mM), NADPH
(100 lM), a-ketoglutarate (250 lM), NH4Cl (50 mM)
and glutamate dehydrogenase (10 lM). Initial rates were
recorded at 340 nm at an inhibitor concentration of
100 lM. Data were collected in triplicate and compared
to a control containing no inhibitor.
Compound 33: RP-HPLC (analytical, gradient 2,
k = 220 nm): Rt = 15.8 min. dH (400 MHz, CD3OD):
7.73 (d, 1H, J = 7.5, ArCH), 7.41 (d, 1H, J = 7.5,
ArCH), 7.27 (s, 1H, ArCH), 7.17 (t, 1H, J = 7.5, ArCH),
7.09 (t, 1H, J = 7.5, ArCH), 4.46 (dd, 1H, J = 8.5, 4.5,
Arg aCH), 4.24 (dd, 1H, J = 9, 5.5, Trp aCH), 3.50
(dd, 1H, J = 15, 5.5, Trp bCHH), 3.29–3.15 (m, 3H,
Trp bCHH, Arg dCH2), 2.70 (t, 2H, J = 7, ArCH2),
2.59 (s, 3H, ArCH3), 2.57 (s, 3H, ArCH3), 2.13 (s, 3H,
ArCH3), 2.02–1.91 (m, 1H, Arg bCHH), 1.86 (t, 2H,
J = 7.0, ArCH2CH2), 1.82–1.71 (m, 1H, Arg bCHH),
1.69–1.59 (m, 2H, Arg cCH2), 1.34 (s, 6H, C(CH3)2).
dC (100 MHz, CD3OD): 174.4 (C), 170.2 (C), 158.0
(C), 154.8 (C), 138.3 (C), 136.6 (C), 136.2 (C), 134.3
(C), 128.3 (C), 125.8 (CH), 125.1 (C), 122.9 (CH),
120.3 (CH), 119.5 (C), 119.1 (CH), 112.6 (CH), 107.8
(C), 75.0 (C), 54.8 (CH), 53.6 (CH), 41.5 (CH2), 33.8
(CH2), 29.9 (CH2), 28.8 (CH2), 26.9 (CH3), 22.3
(CH2), 18.9 (CH3), 17.8 (CH3), 12.2 (CH3). MS (ES+)
m/z: 627.4 (100%) [M+H]+, 649.4 (48%) [M+Na]+.
HRMS (ES+) C31H43N6O6S calcd: 627.2960. Found:
627.2972 [M+H]+.
5.38. Analytical ultracentrifugation
Sedimentation equilibrium experiments were conducted
on a Beckman Optima XL/A analytical ultracentrifuge,
using 12 mm path length Beckman cells with six channel
charcoal-filled Epon centrepieces and quartz windows,
in an AN-50Ti rotor (three cells plus counterbalance)
at 20 ꢁC. Reference buffer (120 lL) and sample
(110 lL) were loaded into the cells and absorbance scans
(k = 280 nm) recorded at 3000 rpm to check loading
concentrations and uniform distribution of cell contents.
The rotor speed was increased to 8000 rpm and absor-
bance scans recorded approximately every 4 h until sed-
imentation equilibrium was achieved (as judged by
absence of change in overlays or subtractions of succes-
sive scans). Scans were conducted in step mode with 10
replicates per data point. The rotor speed was increased
to 13,000 rpm and the analysis repeated. Samples were
prepared in ammonium acetate buffer (50 mM
NH4OAc, 1 mM EDTA, pH 7.5) and consisted of TR
(0.5 mg/mL) alone, TR (0.5 mg/mL) with 4 (70 lM),
TR (0.5 mg/mL) with 5 (15 lM) and TR (0.5 mg/mL)
with 6 (70 lM). Data from all samples were very similar
and no change in the protein oligomerisation state was
observed. Molecular weight estimates were generated
by fitting the data from each scan using Beckman Origin
software to a one-species model. The estimated molecu-
lar mass obtained for each sample were TR alone
(114 kDa, 109 kDa), TR+4 (106 kDa, 103 kDa), TR+5
(114 kDa, 109 kDa) and TR+6 (110 kDa, 105 kDa) at
8,000 and 13,000 rpm, respectively, in good agreement
with the reported mass of TR dimer (107.8 kDa).4
5.35. Trypanothione reductase enzymatic assays
Kinetic analysis was performed in assays of a total
volume of 1 mL which contained ammonium acetate
(50 mM NH4OAc, 1 mM EDTA, pH 7.5), NADPH
(100 lM), T. cruzi TR (9.3 nM) and various known con-
centrations of nortrypanothione disulfide. Initial rates
were recorded at 340 nm at inhibitor concentrations esti-
mated to be 0.5, 1 and 2 Ki. NADPH kinetic analysis
was performed in assays of a total volume of 1 mL
which contained ammonium acetate buffer (50 mM
NH4OAc, 1 mM EDTA, pH 7.5), N-benzyloxycar-
bonyl-L-cysteinylglycine-3-dimethylaminopropylamide
disulfide39 (300 lM), TR (9.3 nM) and various known
concentrations of NADPH. Initial rates were recorded
by monitoring the fluorescence of NADPH (kex
340 nm, em = 460 nm) at inhibitor concentrations
=
5.39. Gel filtration chromatography
k
estimated to be 0.5, 1 and 2 Ki. Data were collected in
triplicate and the type of inhibition deduced from
Eadie–Hofstee and Lineweaver–Burke plots. Ki values
were generated using the appropriate model with the
commercial program GraFit (Erithacus Software Ltd).
Brief sonication was necessary to effect dissolution of
some of the inhibitors in the assay buffer.
Gel filtration chromatography was performed on a Gil-
son HPLC system (234 autoinjector, 321 pump, 155 UV/
vis detector) equipped with a Sephadex G-200 column
(Amersham Pharmacia Biotech), eluted with phosphate
buffer (50 mM Na3PO4, 150 mM NaCl, pH 7.0) at a
flow rate of 0.5 mL/min with UV detection at 220 and
280 nm. Column calibration was achieved by plotting
Log10Mr against Rt for six protein standards (4 mg/
mL, 20 lL): horse myoglobin (17 kDa, 32.3 min),
carbonic anhydrase (29 kDa, 30.8 min), chicken egg
albumin (43 kDa, 28.2 min), bovine serum albumin
(66 kDa, 25.2 min), b-amylase (200 kDa, 21.9 min) and
blue dextran (2000 kDa, 14.6 min). TR (1 mg/mL,
20 lL) was then analysed and a retention time of
25.0 min, equating to a Mr of 103.0 kDa, recorded.
The column was then equilibrated with buffer containing
5 (10 lM) and column calibration repeated. Analysis of
5.36. Single-point library screening
Kinetic analyses were performed in assays of a total
volume of 1 mL, containing ammonium acetate buffer
(50 mM NH4OAc, 1 mM EDTA, pH 7.5), NADPH
(100 lM), nortrypanothione (30 lM), TR (9.3 nM) and
inhibitor (100 lM). Initial rates were recorded at
340 nm, data were collected in triplicate and compared
to a control containing no inhibitor.

4526
M. J. Dixon et al. / Bioorg. Med. Chem. 13 (2005) 4513–4526
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TR (1 mg/mL) incubated for 30 min with 5 (10 lM) gave
a Rt of 24.7 min, equating to a Mr of 105.3 kDa.
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Acknowledgments
We thank Prof. M. G. Gore (Department of Biosciences,
University of Southampton) for helpful discussions and
the use of his fluorescence spectrophotometer, Dr. A.
Leech (Molecular Interactions Lab, University of York)
for AUC analysis and the EPSRC (MD), BBSRC (RM)
and a Marie Curie postdoctoral fellowship from the Euro-
pean Community (JO) for funding.
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