Design and preparation of sterol mimetics as potential antiparasitics

Article abstract of DOI:10.1016/j.bmc.2010.08.007

We have previously shown that azasterols have activity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania species, which are the causative agents of various neglected tropical diseases. In this paper, we discuss the replacement of the sterol cor

Full text of DOI:10.1016/j.bmc.2010.08.007

Bioorganic & Medicinal Chemistry 18 (2010) 7291–7301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and preparation of sterol mimetics as potential antiparasitics
Federica Gigante a, Marcel Kaiser b, Reto Brun b, Ian H. Gilbert a,
*
a Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee, DD1 5EH, UK
b Swiss Tropical and Public Health Institute, Socinstrasse 57, Basel, Switzerland University of Basel, Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously shown that azasterols have activity against Trypanosoma brucei, Trypanosoma cruzi
and Leishmania species, which are the causative agents of various neglected tropical diseases. In this
paper, we discuss the replacement of the sterol core of the azasterols with sterol mimics. Various mimics
were designed, and the structures were minimised to see if they could adopt a similar conformation to
that of the azasterols. From this, two series of mimics were synthesised and then evaluated against
the parasites. Compounds showed moderate activity.
Received 6 April 2010
Revised 2 August 2010
Accepted 3 August 2010
Available online 9 August 2010
Keywords:
Trypanosoma
Leishmania
Azasterol
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
blood stream form T. brucei rhodesiense (Fig. 2).2,3 Interestingly,
we discovered that sterol 24-methyltransferase is expressed in
Parasitic infections caused by the trypanosomatid family, are a
major health problem in many parts of the world, particularly in
tropical countries. Protozoan parasites of this family give rise to
human African trypanosomiasis (Trypanosoma brucei spp.), Chagas
disease (Trypanosoma cruzi) and leishmaniasis (Leishmania spp.).1
There is a need for the development of new drugs to treat these
diseases, as the current drugs available are inadequate for a variety
of different reasons, including costs, efficacy and side effects.
We have reported on azasterols as having activity against the
parasites which cause these diseases.2–7 Examples of the class of
compounds that we have prepared are shown in Figure 1. The de-
sign of these molecules was predicated on the discovery that azas-
terols can inhibit the enzyme 24-sterol methyltransferase in yeast
and various plants.8–12 This enzyme is involved in the biosynthesis
of ergosterol and other related 24-alkylated sterols that are found
in yeast and plants and also in the trypanosomatid parasites. We
have further developed these initial leads in our laboratories.2–6
Subsequent work has shown that the mode of action of these com-
pounds is not (only) through inhibition of sterol 24-methyltrans-
ferase, but includes other modes of action. One particular case is
that blood stream form T. brucei is reported to scavenge cholesterol
from the host rather than biosynthesise ergosterol; hence inhibi-
tion of sterol 24-methyltransferase should not have any effect on
the growth of this life-stage form of T. brucei.13,14 However some
of the azasterols that we have investigated have a potent effect
on the growth of T. brucei.2–4 The most potent compound that
was discovered was compound A which had an EC50 of 12 nM for
blood stream form T. brucei, although these azasterols were not
inhibitors of this enzyme.2,3
The pharmacophore that we have discovered for the azasterols
is shown in Figure 2: there should be an acetate group at the 3b-
position and on the side chain, linked either via an amine and a
chain length of 3 carbon atoms, a carboxy ester. We were inter-
ested to investigate if the sterol molecule was a rigid spacer or
whether it had some particular role/interaction in the mode of ac-
tion of these compounds. Therefore, we decided to investigate
replacements of the sterol nucleus. By changing the sterol nucleus
to a mimic, it might be possible to manipulate the physicochemical
and pharmacokinetic properties of the molecules.
O
R
R
R'O
R'O
amide derivatives
amine derivatives
R = NHbutyl, piperidyl, NHCH2Ph, NH(CH2)nNH2 (n=2,4,6,8,10)
NH(CH2)nCOOMe, NH(CH2)nCOOH, (n= 1-7)
R' = H, Ac
* Corresponding author.
E-mail address: i.h.gilbert@dundee.ac.uk (I.H. Gilbert).
Figure 1. Examples of azasterols that have been prepared in our research group.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.007

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F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
to predict whether the conformation of the designed mimics could
ester
amine
be superimposed upon the conformation of the lead compound A.
This assumes that the active conformation of the azasterols such as
A is at an energetic minimum.
The lowest energy conformations were generated in vacuo using
Moloc,25 which uses the MAB force-field to calculate descriptions of
molecular geometries and energies. The search for the minimized
conformations was carried out using a stochastic run: with this
method, the atomic positions were randomly varied, starting from
the last minimized structure, in order to obtain a library of confor-
mations and identify that which corresponds to the energetic mini-
mum. The minimised structures obtained using Moloc were
imported into Sybyl 7.2 and further minimised, correcting for the
presence of water using a dielectric constant. The conformations
thus obtained were superimposed with the similarly obtained con-
formation for azasterol A. Atoms superimposed are shown in Figure
5.
O
acetate
N
H
3
O
C
D
O
O
A
B
optimum chain
length 3
A
N-23 azasterols
lead compound:
EC50 = 12nM T.b.rhodesiense
Figure 2. The pharmacophore for activity of the azasterols for anti-trypanosomal
and anti-leishmanial activity.
As expected, the urea derivatives 10 and 11 and the tricyclic mi-
mic 9 were predicted to be planar. These superimposed well with
the predicted low energy conformation of azasterol A (Fig. 6). One
of the benzanilide derivatives 7 also gave a good superimposition
with A, with the amide in the predicted trans (E) conformation
(Fig. 6C). In this minimised conformation, the ester is shown as
Development of sterol mimics is an attractive field, particularly
in the treatment of cancer and hormone regulation. A number of
different sterol mimics have been reported; a selection are refer-
enced.15–24 In this paper, we report the design, synthesis and eval-
uation of some sterol mimics for our azasterols. In the approach
that we have adopted, the B and C rings of the lead azasterol A
were removed (Fig. 2) and replaced by spacers intended to mimic
the planarity of the sterol nucleus and maintain the relative posi-
tions of the acetate in the 3b-position and the side chain at position
20 (Fig. 3).
p-stacking with one of the phenyl rings; this probably is a conse-
quence of the energy initially being calculated in vacuo. In an aque-
ous environment, the ester group would be solvated, which would
probably disrupt this conformation.
Interestingly the other benzanilides gave a cis (Z) conformation
about the amide bond (Fig. 6D). In this cis conformation the two phe-
nyl rings were twisted slightly out of plane, presumably to prevent
steric clashes between each other. The cis conformation was not pla-
nar and did not superimpose onto the planar structure A. The reason
for this cis conformation is unknown. It may be an artefact, as this
does not seem a reasonable conformation. One possibility is that a
2. Molecular modelling
In order to find a replacement for the sterol nucleus, that re-
tained the other functional groups in the correct relevant orienta-
tions, a modelling approach was adopted. This involved designing
new analogues, carrying out an energy minimisation and then
superimposing this on the low energy conformation of the most
potent azasterol A.
potential
p-cation stacking interaction between the protonated
amine and phenyl ring ‘A’ might be driving this conformation. To
investigate this, the substituents on the mimics were removed and
only the cores were minimised (Fig. 7). Similar results were ob-
tained, with the benzanilides giving a cis conformation.
As a consequence of the modelling and the potential complexity
of the synthetic chemistry, it was decided to proceed with synthe-
sis of the benzanilide 7 and the phenyl-urea-pyrrolidine 11. These
were predicted to mimic the conformation of the sterol nucleus
and the directionality of the side chains from the sterol nucleus,
although not perfectly the conformations of the side chains.
A selection of the new analogues designed is shown in Figure 4.
These analogues were designed as follows:
ꢀ The ‘A’ ring was replaced by a phenyl ring. The acetate in the 3b-
position was either attached directly to the sterol nucleus, or via
methylene linker. The latter should have greater chemical stabil-
ity, as phenoxide is a good leaving group.
ꢀ The ‘B’ and ‘C’ rings were replaced by amide or urea linkages.
3. Chemistry
ꢀ The ‘normal’ and ‘inverse’ amides were used.
ꢀ The ‘D’ ring was either retained or replaced by a phenyl ring.
ꢀ A slight variation in the linker to the side chain was allowed.
ꢀ In one case the ‘B’ ring was retained.
The synthesis of benzanilide 7 and phenyl-urea-pyrrolidine 11
compounds was carried out. In accordance with previous studies,
where the best activity was found for analogues with a side chain
between 3 and 5 carbons, and to keep the main features of lead A,
initially only derivatives with 3 or 5 carbons side chain were
planned.
2.1. Conformational analysis and superimposition
Studies of conformational analysis were carried out on the
structure of A and on those of the proposed derivatives, in order
3.1. Benzanilide derivatives
The synthetic pathway followed for the synthesis of benzanilide
derivatives is shown in Scheme 1. 4-(Hydroxymethyl)-benzoic acid
was acetylated with acetic anhydride, introducing the acetyl group.
This was then coupled to 4-aminophenyl ethanol. The hydroxyl
group was silyl protected prior to the coupling reaction, to ensure
coupling to the acid 15 was through the amine rather than the hy-
droxyl group. The coupling of acid 15 and amine 16 was carried out
using TBTU and HOBT,26 to give the required benzanilide 17. The
yield of this reaction was lowered by concomitant hydrolysis of
N
O
H
n
O
O
O
sterol mimics
Figure 3. New analogues to mimic the azasterol compounds.

F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
7293
R
H
N
R
R
O
N
H
O
N
N
H
H
H
H
H
N
H
N
H
AcO
AcO
AcO
AcO
AcO
1
2
3
H
R
R
N
O
R
N
H
N
H
H
H
H
H
N
N
H
O
AcO
O
4
5
6
H
H
H
H
R
N
N
R
N
O
R
H
N
H
H
N
H
O
AcO
AcO
AcO
7
8
9
R
N
H
H
H
H
N
H
N
N
N
O
10
AcO
O
AcO
R
= (CH2)3COOMe
11
Figure 4. Key analogues minimised.
O
N
H
3
O
O
O
lead A
R1
R1
R
H
O
R1
N
N
N
H
O
R
R
Figure 5. Points over-layed in the superimposition analysis.
the silyl group during coupling. The silyl group was then removed
using TBAF.
ported scission of the C–C bond during oxidation of a series of
homobenzylic alcohols. We carried out reductive amination with
this aldehyde and methyl 4-aminobutyric acid 20 to give the target
molecule 3.
In order to investigate the conformation of the product 3, a
NOESY spectrum was taken. This showed that compound 3 main-
tains a planar conformation, with the phenyl rings trans to each
other. This was proved by lack of coupling between 8-H and 17-
H, as it would happen in a cis conformation (Fig. 8); in this spec-
trum only the coupling of the amide proton with the 13-H/17-H
was detected (see Supplementary data). The trans-conformation
was not what was predicted from the modelling for this com-
The alcohol 18 was then oxidized to aldehyde, in order to attach
the side chain through a reductive amination. Oxidation with the
Dess–Martin periodinane, gave the best results for the reagents
investigated. The aldehyde 19 was not purified, to avoid possible
decomposition of the aldehyde, and was used crude for next step.
The target molecule 7 was then prepared by reductive amination
with methyl 4-aminobutyric acid 20.
Interestingly, when the alcohol 18 was oxidised with PCC, the
product recovered was the aldehyde 21, in which loss of a methy-
lene group was observed. Fernandes and Kumar27 have also re-

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F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
Figure 6. Minimised structures: (A) Compound 9 (yellow) superimposed with compound A (blue); (B) compound 11 (orange) over-layed with compound A (blue); (C)
compound 7; (D) compound 2.
31, which occurred by lactamisation of the side chain. A similar
reaction was observed previously for azasterol derivatives with a
O
H
3 carbon chain between the N and the ester group.
N
N
NH
For these analogues, the NOESY experiment (see Supplementary
data) confirmed what had been suggested by the modelling stud-
ies: no enhancement seemed to occur between the 8-H of the phe-
nyl and the 13-H or 16-H of the pyrrolidine (Fig. 9), which supports
the hypothesis that the ureas should retain a ‘trans’ planar confor-
mation, and hence be a good mimic of the sterol nucleus.
For derivatives 11, 30 and 31 a characteristic doubling of peaks
was observed in the 13C NMR spectra for the carbons of the pyrrol-
idine. This was due to restricted rotation around the pyrrolidine
ring. In order to prove this, the NMR experiment was performed
at 25 °C and at 50 °C: the signals for the carbons belonging to the
pyrrolidine ring and the side chain, which were split in two at
room temperature, merged into one when the experiment was car-
ried out at 50 °C, thus confirming the presence of rotational con-
formers for this kind of molecules.
O
12
13
14
cis-folded
trans-extended
trans-extended
Figure 7. Core structures minimised.
pound, rather a cis conformation; however the trans-conformation
was observed experimentally.
3.2. Phenyl-urea-pyrrolidine derivatives
The successful route for the preparation of the phenyl-urea-pyr-
rolidine analogues is outlined in Scheme 2. 4-Aminobenzyl alcohol
22 was acetylated with acetic anhydride to give the mono-acety-
lated derivative 23; by careful control of conditions, it was possible
to get selective acetylation of the hydroxyl group rather than the
aniline. The urea 27 was then formed in two steps. Firstly the acet-
ylated 4-aminobenzyl alcohol 23 was treated with p-nitrophenyl
chloroformate to form the intermediate p-nitrophenyl carbamate
24.16,20 This was immediately reacted with pyrrolidine 26 (which
was obtained by deprotection of the commercially available Boc-
hydroxymethylpyrrolidine 25) to give the required urea 27 in a
yield of 57%.
Oxidation of hydroxyl 27 with Dess–Martin periodinane gave
the required aldehyde. This was then reductively aminated with
either methyl 4-aminobutyric acid 20 to give the target molecule
11 with a 3 carbon linker, or with methyl 6-aminohexanoic acid
29 to give the target molecule 30 with a 5 carbon linker. In the case
of 11, the yield was lowered due to formation of the side product
4. Results and discussion
New benzanilide and phenyl-urea-pyrrolidine derivatives were
synthesised as potential mimics of azasterol compounds which had
showed activity against trypanosomes and Leishmania species.2,3,7
For analogues 7 and 11 the modelling studies had suggested a
‘trans-extended’ conformation, which should allow them to repro-
duce the structure and the features of the lead compound A. This
was confirmed by NOESY spectra (see Supplementary data). Also
for compound 30, the homologue of 11, the NOESY spectra sug-
gested a trans-extended conformation, and no coupling was ob-
served between the two rings. For analogue 3 the conformational
analysis had suggested a folded conformation with the two phen-
yls cis to each other; however the NOESY experiment indicated
that this compound had a planar conformation, with the two phe-

F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
7295
O
OH
OH
HO
H2N
4-aminophenyl ethanol
hydroxymethyl benzoic acid
TBDMSCl
Ac2O
78%
39%
imidazole, DMF
pyridine
0o
r.t.
O
OTBS
DIPEA, HOBt,
TBTU, DMF
O
OTBS
OH
+
N
H
O
H2N
O
27%
15
O
16
17
O
H
OH
O
O
O
N
H
TBAF, THF
79%
N
H
Dess-Martin reagent
DCM
O
O
18
19
O
O
HCl . H2N
COOMe
3
PCC
DCM
37%
O
20
NaCNBH3, TEA, MeOH
24%
O
H
O
H
N
N
H
O
O
O
21
N
H
7
O
O
O
HCl . H2N
COOMe
3
NaCNBH3, TEA, MeOH
22%
O
O
O
N
H
N
H
O
3
O
Scheme 1. Synthesis of the benzanilde analogues 3 and 7.
O
14
6
9
H
O
13
5
N
O
N
H
3
6
8
O
O
13
14
5
17
16
N
H
17
O
O
16
8
O
9
O
trans
cis
N
3
O
H
Figure 8. trans and cis conformation for benzanilide derivative 3.
nyl rings trans to each other, giving a promising mimic of the lead
A. This indicates that the modelling studies only give an indication
of the conformations.
Compounds 3, 27 and 31 were evaluated to gain further infor-
mation on the SAR. The new derivatives were evaluated as growth
inhibitors of the clinically relevant forms of the kinetoplastids: T.

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F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
NH2
Ac2O pyridine
0 o C
ClCO2C6H4NO2, TEA
NH2
O
DCM, 0oC
91%
HO
O
23
44%
22
OH
N
Boc
25
TFA 5%
DCM
100%
OH
H
N
OH
N
H
O
26 H.TFA
N
N
O
O
Dess-Martinreagent
DCM
TEA, MeOH
m.w. 30', 100oC
57%
O
O
O
NO2
24
O
27
O
O
3
N
H
20, NaCNBH3,
O
H
N
H
TEA, MeOH
H
N
N
+
N
O
O
11%
O
O
28
O
11
O
O
N
5
NaCNBH3
TEA
MeOH
H
N
HCl. H2N
COOMe
N
29
O
O
5
O
O
31
N
H
O
H
N
N
n= 5
O
O
O
30 (10%)
Scheme 2. Synthesis of phenyl-urea-pyrrolidine analogues.
O
NH
H
N
6
9
O
11
14 17
7
5
13
10
19
22
O
21
12
H
N
15
6
9
2
N
O
11
7
N
8
5
13
1
4
10
12 16
16
15
3
O
H
O
2
O
O
14
8
N
1
4
3
17
O
22
O
trans
cis
Figure 9. trans and cis conformation for the urea derivatives.
brucei rhodesiense blood stream forms; T. cruzi intracellular
amastigotes; and Leishmania donovani axenic amastigotes. Com-
pounds were also screened against L6-cells, which are rat myo-
blasts, as a measure of toxicity against mammalian cells. The
data for this is shown in Table 1.
Unfortunately neither of the benzanilide compounds 3 or 7
showed potent antiparasitic activity. The only compound showing
marginal activity against L. donovani was 3. Similarly, the phenyl-
urea-pyrrolidine compounds failed to give significant growth inhi-
bition of the parasites.

F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
7297
M)
Table 1
Biological results for benzanilide and phenyl-urea-pyrrolidine derivatives
T. b. rhodesiense EC50
(lM)
T. cruzi EC50
(l
M)
L. donovani EC50
(
lM)
L6-cells TD50 (l
Control drugs
Melarsoprol 0.01
Benznidazole 1.6
Miltefosine 0.31
Podophyl. 0.012
O
O
N
H
N
H
O
3
7
78
207
2.6
73
161
O
O
O
O
O
H
N
O
O
N
11.5
141
>218
>308
H
OH
H
N
N
27
11
52
40
>308
26
80
O
O
O
O
N
H
O
H
N
N
>230
>230
O
O
O
O
N
H
N
31
43
181
30
>250
N
O
O
O
O
O
N
O
H
H
N
N
30
76
>215
41
>214
O
O
L6-cells are rat skeletal myoblasts and are used as an indication of toxicity to mammalian cells. Data for A, as reported by Gros et al.:2 T.b. rhodesiense, EC50 0.012
donovani, EC50 3.2 M; Toxicity, L-6 cells, 19.2 M. Controls are: T.b. rhodesiense, melarsoprol; L. donovani, miltefosine; L6-cells, podophyllotoxin.
lM; L.
l
l
Therefore neither the benzanilide nor the urea derivatives
showed significant antiproliferative activity against the parasites,
with general EC50 values higher than those of the lead compound
A. This might be due to poor mimetic of the lead compound,
although the modelling studies had suggested a planar conforma-
tion for these derivatives, which matched the sterol nucleus. Alter-
natively this might also suggest that the sterol nucleus has an
important interaction with the molecular target, which does not
take place with these analogues.
activity of these type of compounds, and that it is difficult to
replace.
6. Experimental
6.1. General experimental details
When applicable, all glassware were oven-dried overnight and
all reactions were carried out under Argon atmosphere. Sensitive
liquids and solutions were transferred via syringe and were intro-
duced into reaction vessels through rubber septa. Reagents were
purchased from Aldrich or Fluka. All the reactions were carried
out using dry solvents unless otherwise stated. All dry solvents,
ethanol, methanol, dichloromethane, tetrahydrofuran, dimethyl-
formamide were purchased from Aldrich or Fluka in sure sealed
bottles. Analytical TLCs were performed on Silica Gel 60 F254
plates (Merck). Visualisation of spots was effected by one of the
following techniques: (a) UV illumination, (b) immersion of the
plate in a 3% solution of ninhydrin in ethanol followed by heating
and (c) immersion of the plate in a 48 g Lꢁ1 solution of phospho-
5. Conclusions
Using a combination of background literature, molecule model-
ling and chemical intuition, we developed two classes of sterol
mimics. These were successfully synthesized, and NOESY spectra
indicated that these mimics had similar steric properties to sterols.
Unfortunately when derivatised in a similar pattern to the lead
azasterol, none of these compounds gave antiparasitic activity of
the same order as the lead compound A. This may indicate that
the sterol nucleus itself may be important for the antiparasitic

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F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
molibdic acid in methanol followed by heating. Column chroma-
tography was carried out on Silica Gel 60 (40–60 micron) pur-
chased from Fluka.
in DMF (12 mL), DIPEA (2.1 mL, 12.24 mmol, 2.5 equiv) was added
and the reaction stirred and monitored by TLC. When the forma-
tion of the activated ester with HOBt was complete (after ꢃ1 h),
the amine 16 was added (1.418 g, 5.63 mmol, 1.15 equiv) and the
reaction stirred overnight at room temperature. The mixture was
then extracted with CHCl3 (30 mL) and washed with H2O
(2 ꢂ 30 mL), the organic layer was dried over MgSO4 and then con-
centrated under reduced pressure. After purification by column
chromatography (CHCl3/MeOH 100:0?90:10), the product was
collected as a yellow solid (0.473 g, 27% yield); Rf = 0.94 (CHCl3/
MeOH: 90:10); LRMS, m/z (ES+ mode): 428.25 ([M+H]+, 100%);
Anal. Calcd for C24H33NO4Si (Theory): C, 67.41; H, 7.78; N, 3.28.
Found: C, 67.36; H, 7.41; N, 4.23; mp = 83–86 °C; 1H NMR
(500 MHz, CDCl3): d 0.01 6Y, s, 20-CH3 and 21-CH3), 0.88 (9H, s,
23-CH3, 24-CH3 and 25-CH3), 2.15 (3H, s, 1-CH3), 2.82 (2H, t,
J = 7.0, 18-CH2), 3.81 (2H, t, J = 7.0, 19-CH2), 5.18 (2H, s, 3-CH2),
7.22 (2H, d, J = 8.5, 14-CH and 16-CH), 7.48 (2H, d, J = 8.3, 5-CH
and 9-CH), 7.55 (2H, d, J = 8.1, 13-CH and 17-CH), 7.72 (1H, br s,
11-NH), 7.87 (2H, d, J = 8.3, 6-CH and 8-CH); 13C NMR (125 MHz,
CDCl3): d ꢁ5.4 20-CH3 and 21-CH3), 18.4 (22-C), 21.0 (1-CH3),
26.0 (23-CH3, 24-CH3 and 25-CH3), 39.0 (18-CH2), 64.5 (19-CH2),
65.5 (3-CH2), 120.1 (13-CH and 17-CH), 127.3 (5-CH and 9-CH),
128.3 (6-CH and 8-CH), 129.8 (14-CH and 16-CH), 134.8 (7-C),
135.7 (12-C), 135.9 (15-C), 139.9 (4-C), 165.1 (10-C@O), 170.8
(2-C@O).
NMR spectra were recorded on a Bruker Avance DPX 300 MHz
spectrometer at 300 MHz for 1H and 75 MHz for 13C or a Bruker
Avance DPX 500 MHz spectrometer at 500 MHz for 1H, 125 MHz
for 13C. Chemical shifts are reported downfield in parts per million
and coupling constants (J values) are in Hertz. Melting points were
determined with a GallenKamp melting point apparatus. Low-res-
olution mass measurements were performed on Applied Biosystem
Mariner API-TOF. LC–MS and accurate mass measurements were
performed in house with Agilent 1100 HPLC in series with Bruker
MicroTof spectrometer or at EPSRC National Mass Spectrometry
Service Centre in the Chemistry Department, University of Wales
Swansea, Swansea, Wales, UK.
6.2. Biological in vitro assays
The in vitro assays against T. b. rhodesiense was carried out as
described by Wenzler et al.28 The assays for T. cruzi, L. donovani
and L6-cells were performed following the procedure reported by
Ganapaty et al.29
6.2.1. 4-(Acetoxymethyl) benzoic acid (15)
Ac2O (1.02 mL, 10.84, 1.1 equiv) was added to a solution of com-
mercially available hydroxymethyl benzoic acid (1.497 g,
9.84 mmol) in pyridine (10 mL), and the reaction was stirred over-
night at room temperature. The mixture was then diluted with H2O
(20 mL) and extracted with EtOAc (20 mL); the organic layer was
washed with HCl 1 M (10 mL), dried over MgSO4 and concentrated
under reduced pressure, to yield pure 15, as a white solid (1.500 g,
78% yield); Rf = 0.53 (CHCl3/MeOH: 80:20); LRMS, m/z (ES+ mode):
217.1 ([M+Na]+, 100%); Anal. Calcd for C10H10O4 (Theory): C, 61.85;
H, 5.19. Found: C, 61.62; H, 5.11; mp = 124 °C; 1H NMR (500 MHz,
CDCl3): d 2.18 (3H, s, 1-CH3), 5.22 (2H, s, 3-CH2), 7.49 (2H, d, J = 8.4,
5-CH and 9-CH), 8.14 (2H, d, J = 8.3, 6-CH and 8-CH); 13C NMR
(125 MHz, CDCl3): d 21.0 (1-CH3), 65.5 (3-CH2), 127.8 (5-CH and
9-CH), 128.9 (7-C), 130.5 (6-CH and 8-CH), 142.0 (4-C), 170.8
(10-C@O), 171.0 (2-C@O).
6.2.4. 4-4-(2-Hydroxyethyl)phenylcarbamoylbenzyl acetate (18)
To a solution of 17 (0.255 g, 0.60 mmol) in THF (8 mL) TBAF
(0.59 mL, 0.60 mmol, 1 equiv) was added and the reaction was stir-
red at room temperature until the TLC showed complete disap-
pearance of the starting material. The solvent was then removed
under reduced pressure and the crude was purified by column
chromatography (DCM/MeOH 100:0?85:15) to afford 18 as a
white solid (0.148 g, 79% yield); Rf = 0.35 (CHCl3/MeOH: 90:10);
LRMS, m/z (ES+ mode): 314.14 ([M+H]+, 100%); Anal. Calcd for
C18H19NO4 (Theory): C, 68.99; H, 6.11; N, 4.47. Found: C, 68.80;
H, 6.08; N, 4.82; mp = 163–164 °C; 1H NMR (500 MHz, CDCl3): d
2.14 (3H, s, 1-CH3), 2.85 (2H, t, J = 7.1, 18-CH2), 3.77 (2H, t,
J = 7.1, 19-CH2), 5.21 (2H, s, 3-CH2), 7.26 (2H, d, J = 8.7, 14-CH
and 16-CH), 7.53 (2H, d, J = 8.1, 5-CH and 9-CH), 7.63 (2H, d,
J = 8.4, 13-CH and 17-CH), 7.95 (2H, d, J = 8.4, 6-CH and 8-CH);
13C NMR (125 MHz, CDCl3): 21.0 (1-CH3), 38.7 (18-CH2), 63.7
(19-CH2), 65.5 (3-CH2), 120.5 (13-CH and 17-CH), 127. 3 (5-CH
and 9-CH), 128.3 (6-CH and 8-CH), 129.7 (14-CH and 16-CH),
134.7 (7-C), 134.9 (12-C), 136.3 (15-C), 140.0 (4-C), 165.2 (10-
C@O), 170.8 (2-C@O).
6.2.2. (2-p-Aminophenylethoxy)-tert-butyldimethylsilane (16)
A
solution of tert-butyldimethylsilyl chloride (2.703 g,
17.93 mmol, 1.2 equiv) and imidazole (1.267 g, 18.61 mmol,
1.3 equiv) in DMF (8 mL) was cooled at 0 °C under Argon, then a
solution of commercially available 4-aminophenyl ethanol
(2.000 g, 14.58 mmol) in DMF (8 mL) was slowly added. The reac-
tion was allowed to warm to room temperature and stirred over-
night. The mixture was then diluted with H2O (20 mL) and
extracted with EtOAc (20 mL), the organic layer was washed with
NaHCO3 (3 ꢂ 20 ml), dried over MgSO4 and concentrated under re-
duced pressure. The crude was purified by column chromatogra-
phy (Hexane/EtOAc 100:0?85:15) to afford 16 as a yellow oil
(1.418 g, 39% yield); Rf = 0.33 (Hexane/EtOAc: 80:20); LRMS, m/z
(ES+ mode): 252.18 ([M+H]+, 100%); 1H NMR (500 MHz, CDCl3): d
0.01 6Y, s, 10-CH3 and 11-CH3), 0.88 (9H, s, 13-CH3, 14-CH3 and
15-CH3), 2.72 (2H, t, J = 7.3, 8-CH2), 3.56 (1H, br s, 2-NH), 3.74
(2H, t, J = 7.3, 9-CH2), 6.62 (2H, d, J = 8.3, 3-CH and 7-CH), 6.99
(2H, d, J = 8.1, 4-CH and 6-CH); 13C NMR (125 MHz, CDCl3): d
ꢁ5.3 10-CH3 and 11-CH3), 18.4 (12-C), 26.0 (13-CH3, 14-CH3 and
15-CH3), 38.8 (8-CH2), 65.0 (9-CH2), 115.2 (3-CH and 7-CH),
129.1 (5-C), 129.9 (4-CH and 6-CH), 144.5 (2-C).
6.2.5. 4-4-(Formylmethyl)phenylcarbamoylbenzyl acetate (19)
The Dess–Martin periodinane (0.406 g, 0.96 mmol, 2 equiv) and
18 (0.150 g, 0.48 mmol) were dissolved in DCM (15 mL) and the
reaction was stirred overnight at room temperature. The mixture
was then extracted with EtOAc (20 mL) and washed with NaHCO3
(20 mL), Na2S2O3 (25 mL) and brine (20 ml) and dried over MgSO4.
The solvent was removed under reduced pressure to afford 19 as a
brown sticky solid. The product was not purified but used as a
crude for next step; Rf = 0.63 (CHCl3/MeOH: 90:10); LRMS, m/z
(ES+ mode): 312.15 ([M+H]+, 100%); mp = 126–127 °C; 1H NMR
(500 MHz, CDCl3): d 2.12 (3H, s, 1-CH3), 3.68 (2H, t, J = 2.3, 18-
CH2), 5.16 (2H, s, 3-CH2), 7.22 (2H, d, J = 8.4, 14-CH and 16-CH),
7.46 (2H, d, J = 8.2, 5-CH and 9-CH), 7.64 (2H, d, J = 8.4, 13-CH
and 17-CH), 7.86 (2H, d, J = 8.2, 6-CH and 8-CH), 9.69 (1H, t,
J = 2.3, 19-CH); 13C NMR (125 MHz, CDCl3): 21.0 (1-CH3), 50.0
(18-CH2), 65.5 (3-CH2), 120.7 (13-CH and 17-CH), 127. 3 (5-CH
and 9-CH), 128.0 (6-CH and 8-CH), 128.3 (14-CH and 16-CH),
130.4 (15-C), 134.6 (7-C), 137.2 (12-C), 140.1 (4-C), 166.2 (10-
C@O), 165.3 (2-C@O), 199.3 (19-C@O).
6.2.3. 4-4-2-(tert-Butyldimethylsilyloxy)ethylphenylcarbamoylb
enzyl acetate (17)
To
a solution of 15 (0.950 g, 4.89 mmol), HOBt (1.059 g,
0.78 mmol, 1.6 equiv) and TBTU (2.516 g, 7.83 mmol, 1.6 equiv)

F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
7299
6.2.6. 4-(4-Formylphenylcarbamoyl)benzyl acetate (21)
(0.033 g, 22% yield); Rf = 0.6 (CHCl3/MeOH: 80:20); LRMS, m/z
(ES+ mode): 399.19 ([M+H]+, 100%); HRMS (ES+ mode), Calculated
for C22H27N2O5 (M+H+): 399.1914. Found: 399.1920; 1H NMR
(500 MHz, CDCl3): d 1.87 2Y, m, 21-CH2), 2.16 (3H, s, 1-CH3), 2.43
(2H, t, J = 7.3, 22-CH2), 2.71 (2H, t, J = 7.3, 20-CH2), 3.69 (3H, s,
24-CH3), 3.80 (2H, s, 18-CH3), 5.20 (2H, s, 3-CH2), 7.35 (2H, d,
J = 8.3, 14-CH and 16-CH), 7.50 (2H, d, J = 8.2, 5-CH and 9-CH),
7.62 (2H, d, J = 8.3, 13-CH and 17-CH), 7.87 (1H, br s, 11-NH),
7.90 (2H, d, J = 8.2, 6-CH and 8-CH); 13C NMR (125 MHz, CDCl3):
21.0 (1-CH3), 29.7 (21-CH2), 31.9 (22-CH2), 46.1 (18-CH2), 46.6
(20-CH2), 51.7 (24-CH3), 65.5 (3-CH2), 120.5 (13-CH and 17-CH),
127. 3 (5-CH and 9-CH), 128.3 (6-CH and 8-CH), 129.0 (14-CH
and 16-CH), 133.0 (15-C), 134.9 (7-C), 136.3 (12-C), 140.0 (4-C),
165.0 (10-C@O), 170.8 (2-C@O, 174.1(23-C@O).
Pyridinium chlorochromate (0.460 g, 2.13 mmol, 2.1 equiv) and
18 (0.323 g, 1.03 mmol) were stirred in DCM (20 mL) overnight at
room temperature, in presence of molecular sieves. The mixture
was then poured onto a silica pad and eluted with 1.5 L of DCM, then
with 1.5 L DCM/MeOH (90:10). The filtrate collected was concen-
trated under reduced pressure and the crude was purified by column
chromatography (CHCl3/MeOH in presence of TEA, 100:0?95:5) to
obtain 21 as a yellow powder (0.113 g, 37% yield); Rf = 0.86 (CHCl3/
MeOH: 90:10); LRMS, m/z (ES+ mode): 298.11 ([M+H]+, 100%); Anal.
Calcd for C17H15NO4ꢄ0.15H2O (Theory): C, 68.06; H, 5.14; N, 4.67.
Found: C, 68.13; H, 5.21; N, 4.71; mp = 137 °C; 1H NMR (500 MHz,
CDCl3): d 2.17 (3H, s, 1-CH3), 5.20 (2H, s, 3-CH2), 7.53 (2H, d, J = 8.4,
5-CH and 9-CH), 7.93 (6H, m, 6-CH, 8-CH, 13-CH, 14-CH, 16-CH
and 17-CH), 9.97(1H, s, 18-CH); 13C NMR (125 MHz, CDCl3): 21.0
(1-CH3), 65.4 (3-CH2), 119.6 (13-CH and 17-CH), 127. 4 (5-CH and
9-CH), 128.4 (6-CH and 8-CH), 131.3 (14-CH and 16-CH), 134.8
(15-C), 135.2 (7-C), 139.4 (12-C), 142.4 (4-C), 165.2 (10-C@O),
170.8 (2-C@O), 191.0 (18-C@O).
6.2.9. 4-Aminobenzyl acetate (23)
Ac2O (1.28 mL, 13.63 mmol, 1 equiv) was added to a solution of
commercially
available
4-aminobenzyl
alcohol
(2.015 g,
16.36 mmol, 1.2 equiv) in pyridine (10 mL) and the reaction was
stirred at 0°C and monitored by TLC. When all the starting material
had disappeared (26 h) the mixture was extracted with EtOAc
(20 mL) and washed with H2O (2 ꢂ 20 mL), then with HCl
(15 mL) and then dried over MgSO. The solvent was removed un-
6.2.7. Methyl N-4-(4-acetoxymethylbenzamidyl)phenethyl-4-
aminobutanoate (7)
TEA (0.078 ml, 0.56 mmol, 2.3 equiv) was added to a solution of
methyl 4-aminobutanoate hydrochloride 20 H2N(CH2)3COOMeHCl
(0.079 g, 0.51 mmol, 2.1 equiv) in MeOH (8 mL) and the solution
stirred at room temperature. After 30 min, a solution of 19
(0.076 g, 0.24 mmol) in MeOH (2 mL) was added and the reaction
stirred for a further 30 min. Finally NaCNBH3 (0.44 mL of 1 M solu-
tion in THF, 0.44 mmol, 1.8 equiv) was added and the reaction stir-
red overnight at room temperature. The mixture was then diluted
with H2O (10 ml), extracted with CHCl3 (15 mL), the organic layer
was washed with a saturated solution of NaCl (10 mL), dried over
MgSO4 and concentrated under reduced pressure. The crude was
purified by column chromatography (CHCl3/MeOH 100:0?90:10)
to afford the product 7 as a white sticky compound (0.024 g, 24%
yield); Rf = 0.53 (CH2Cl2/MeOH: 90:10); LC–MS, m/z (ES+ mode):
413.18 ([M+H]+, M = 9, 75%), 381.16 ([M+H]+, M = lactam deriva-
tive, MW = 380.17, 16%); HRMS (ES+ mode), Calcd for C23H29N2O5
(M+H+): 413.2071. Found: 413.2061; 1H NMR (500 MHz, CDCl3):
d 2.02 2Y, m, 22-CH2), 2.07 (3H, s, 1-CH3), 2.43 (2H, m, 18-CH2),
2.48 (2H, m, 23-CH2), 3.02 (2H, m, 21-CH2), 3.26 (2H, m, 19-CH2),
3.64 (3H, s, 25-CH3), 5.09 (2H, s, 3-CH2), 7.15 (2H, m, 14-CH and
16-CH), 7.44 (4H, m, 5-CH, 9-CH, 13-CH and17-CH), 7.82 (2H, d,
J = 8.3, 6-CH and 8-CH), 8.21 (1H, br s, 11-NH); 13C NMR
(125 MHz, CDCl3): 19.9 (1-CH3), 28.7 (22-CH2), 30.5 (23-CH2),
43.6 (18-CH2), 50.0 (19-CH2), 51.2 (21-CH2), 53.3 (25-CH3), 64.4
(3-CH2), 120.7 (13-CH and 17-CH), 126.6 (5-CH and 9-CH), 127.1
(6-CH and 8-CH), 128.5 (14-CH and 16-CH), 131.3 (7-C), 133.0
(15-C), 135.8 (12-C), 139.2 (4-C), 164.8 (10-C@O), 169.8 (2-C@O,
173.1 (24-C@O).
der reduced pressure, to recover pure 23 as
a white solid
(1.182 g, 44% yield); Rf = 0.22 (CHCl3/MeOH: 90:10); LRMS, m/z
(ES+ mode): 166.09 ([M+H]+, 100%); Anal. Calcd for C9H11NO4 (The-
ory): C, 65.44; H, 6.71; N, 8.48. Found: C, 66.33; H, 6.88; N, 8.77;
mp = 144; 1H NMR (500 MHz, CDCl3): d 2.21 (3H, s, 1-CH3), 4.68
(2H, s, 3-CH2), 7.20 (2H, br s, 10-NH2), 7.35 (2H, d, J = 8.3, 6-CH
and 8-CH), 7.52 (2H, d, J = 8.3, 6-CH and 8-CH); 13C NMR
(125 MHz, CDCl3): d 24.7 (1-CH3), 65.0 (3-CH2), 120.0 (6-CH and
8-CH), 127.9 (5-CH and 9-CH), 136.8 (4-C), 137.3 (7-C), 168.2 (2-
C@O).
6.2.10. 4-(4-Nitrophenyloxycarbonylamino)benzyl acetate (24)
Commercially available p-nitrophenylchloroformate (0.648 g,
3.22 mmol, 1 equiv) and 23 (0.532 g, 3.22 mmol) were dissolved
in CH2Cl2 (4 mL) at 0°C, then TEA (0.45 mL, 3.22 mmol, 1 equiv)
was added and the reaction was allowed to warm to room temper-
ature and stirred until the complete disappearance of starting
material was observed by TLC and LC–MS. The precipitate formed
was then filtered and the filtrate was extracted with CH2Cl2
(10 mL), washed with H2O (2 ꢂ 10 mL) and the solvent was re-
moved under reduced pressure to afford the product as a yellow
solid (0.965 g, 91% yield). The product was not purified and only
identified by 1H NMR and LC–MS, then used as a crude for next
step; Rf = 0.86 (CH2Cl2/MeOH: 90:10); LRMS, m/z (ES+ mode):
331.12 ([M+H]+, 100%); Anal. Calcd for C16H14N2O6 (Theory): C,
58.18; H, 4.27; N, 8.48. Found: C, 57.71; H, 4.45; N, 8.65; 1H
NMR (500 MHz, CDCl3): d 2.12 (3H, s, 1-CH3), 5.18 (2H, s, 3-CH2),
7.11 (1H, br s, 10-NH), 7.31 (2H, m, 13-CH and 17-CH), 7.34 (2H,
d, J = 8.5, 5-CH and 9-CH), 7.48 (2H, d, J = 8.3, 6-CH and 8-CH),
8.20 (2H, dd, J1 = 2.2, J2 = 7.0, 14-CH and 16-CH).
6.2.8. Methyl N-4-(4-acetoxymethylbenzamidyl)benzyl-4-amino
butanoate (3)
TEA (0.122 ml, 0.88 mmol, 2.3 equiv) was added to a solution of
methyl 4-aminobutanoate hydrochloride 20 H2N(CH2)3COOMeꢄHCl
(0.123 g, 0.80 mmol, 2.1 equiv) in MeOH (10 mL) and the solution
stirred at room temperature. After 30 min, a solution of 21
(0.114 g, 0.38 mmol) in MeOH (2 mL) was added and the reaction
stirred for a further 30 min. Finally NaCNBH3 (0.69 mL of 1 M solu-
tion in THF, 0.69 mmol, 1.8 equiv) was added and the reaction stir-
red overnight at room temperature. The mixture was then diluted
with H2O (25 mL) and extracted with CHCl3 (30 mL), the organic
layer was washed with a saturated solution of NaCl (25 ml), dried
over MgSO4 and the solvent concentrated under reduced pressure.
The crude was purified by column chromatography (CHCl3/MeOH
100:0?90:10) to afford product 3 as a white oily compound
6.2.11. (Pyrrolidin-3-yl)methanol trifluoroacetate (26)
TFA (0.5 mL, 1.6 equiv) was added to a solution of N-Boc-hydrox-
ymethylpyrrolidine commercially available (0.852 g, 4.23 mmol) in
DCM (4.5 mL) and the reaction stirred and monitored by TLC and LC–
MS. When the removal of the Boc group was completed, all the sol-
vent was removed under reduced pressure, leaving pure 26 as a pink
oil (quantitative); Rf = 0.36 (CHCl3/MeOH: 90:10); LRMS, m/z (ES+
mode): 102.10 ([MꢁCF3COO]+); 1H NMR (500 MHz, CDCl3): d 1.96
(1H, m, 4a-CH2), 2.36 (1H, m, 3-CH), 2.93 (1H, m, 4b-CH2), 3.22 (1H,
m, 2a-CH2), 3.43 (1H, m, 2b-CH2), 3.54 (1H, m, 5a-CH2), 3.60 (1H, m,
5b-CH2), 4.41 (1H, dd, J1 = 6.9, J2 = 11.2, 6a-CH2), 4.49 (1H, dd,
J1 = 5.5, J2 = 11.3, 6b-CH2); 13C NMR (125 MHz, CDCl3): d 27.0

7300
F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
(4-CH2), 36.6 (3-CH), 45.5 (5-CH2), 47.5 (2-CH2), 67.1 (6-CH2); 19F
NMR (470 MHz, CDCl3): d ꢁ75.9 3F, CF3COOH).
1.95 (1H, m, 15b-CH2), 2.11 (3H, s, 1-CH3), 2.31 (2H, m, 21-CH2),
2.52 (1H, m, 14-CH), 2.58 (3H, m, 17a-CH2 and 19-CH2), 2.99 (1H,
m, 17b-CH2), 3.28 (1H, m, 16a-CH2), 3.48 (3H, m, 13-CH2 and 16b-
CH2), 3.60 (3H, s, 23-CH3), 5.00 (2H, s, 3-CH2), 7.25 (2H, d,
J = 8.1 Hz, 5-CH and 9-CH), 7.29 (1H, br s, 10-NH), 7.41 (2H, d,
J = 8.4, 6-CH and 8-CH); 13C NMR (125 MHz, CDCl3): 24.7 (1-CH3),
24.9 (20-CH2), 29.2 and 29.9 (15-CH2), 31.8 (21-CH2), 41.2 (14-CH),
49.2 (16-CH2), 50.3 (19-CH2), 50.3 (17-CH2), 51.7 (23-CH3), 52.3
and 52.4 (13-CH2), 66.3 (3-CH2), 119.9 (6-CH and 8-CH), 129.0 (5-
CH and 9-CH), 132.9 (7-C), 137.6 (4-C), 154.9 (11-C@O), 158.1 (2-
C@O), 174.1 (22-C@O).
6.2.12. N-(4-Acetoxymethylphenyl)-3-hydroxymethylpyrroli
dine-1-carboxamide (27)
TEA (0.613 mL, 4.42 mmol, 1.6 equiv) was added to 24 (0.935 g,
2.83 mmol) in MeOH (18 mL), then 26 (0.950 g, 4.41 mmol,
1.5 equiv) was added and the reaction stirred in the microwave for
30 min at 100°C. The precipitate formed was filtered and the filtrate
was concentrated under reduced pressure. The crude was then ex-
tracted with CH2Cl2 (15 mL) and washed with H2O (3 ꢂ 10 mL), then
dried over MgSO4 and concentrated under reduced pressure. Purifi-
cation by column chromatography (CH2Cl2/MeOH 100:0?90:10)
afforded 27 as light yellow oil (0.471 g, 57% yield); Rf = 0.30
(CH2Cl2/MeOH: 90:10); LRMS, m/z (ES+ mode): 310.18 ([M+NH4]+,
100%); HRMS (ES+ mode), Calcd for C15H21N2O4 (M+H+): 293.1496.
Found: 293.1505; 1H NMR (500 MHz, MeOD): d 1.70 (1H, m, 15a-
CH2), 2.00 (1H, m, 14-CH), 2.12 (3H, s, 1-CH3), 2.39 (1H, m, 15b-
CH2), 3.17 (2H, m, 16-CH2), 3.52 (4H, m, 13-CH2 and 17-CH2), 5.06
(2H, s, 3-CH2), 7.32 (2H, d, J = 7.8, 5-CH and 9-CH), 7.54 (2H, d,
J = 8.5, 6-CH and 8-CH); 13C NMR (125 MHz, MeOD): d 23.8 (1-
CH3), 28.3 and 29.1 (15-CH2), 41.8 (14-CH), 42.6 (16-CH2), 46.5 and
46.8 (13-CH2), 64.3 (17-CH2), 67.7 (3-CH2), 121.0 (6-CH and 8-CH),
129.7 (5-CH and 9-CH), 133.9 (7-C), 139.8 (4-C), 156.8 (11-C@O),
171.7 (2-C@O).
The side product of this reaction, 31, was also isolated pure as a
white sticky solid (0.026 g, 10% yield).
6.2.15. N-(4-Acetoxymethylphenyl)-3-(2-oxopyrrolidin-1-yl)
methylpyrrolidine-1-carboxamide (31)
Rf = 0.76 (CHCl3/MeOH: 90:10); LRMS, m/z (ES+ mode): 377.23
([M+NH4]+, 100%; HRMS (ES+ mode), Calcd for C19H26N3O4
(M+H+): 360.1918. Found: 360.1924; 1H NMR (500 MHz, CDCl3):
d 1.55 (1H, m, 15a-CH2), 1.89 (1H, m 15b-CH2), 1.96 (2H, m, 20-
CH2), 2.09 (3H, s, 1-CH3), 2.32 (2H, t, J = 8.1, 21-CH2), 2.32 (1H,
m, 14-CH), 3.04 (1H, m, 17a-CH2), 3.22 (1H, m, 17b-CH2), 3.43
(6H, 13-CH2, 16-CH2 and 19-CH2), 4.99 (2H, s, 3-CH2), 7.22 (2H,
d, J = 8.3, 5-CH and 9-CH), 7.42 (2H, d, J = 7.5, 6-CH and 8-CH),
7.89 (1H, br s, 10-NH); 13C NMR (125 MHz, CDCl3): d 18.1 (20-
CH2), 24.5 (1-CH3), 28.9 and 29.4 (15-CH2), 30.9 (21-CH2), 36.7
and 37.7 (14-CH), 45.1 and 45.2 (17-CH2), 45.6 (19-CH2), 47.9
and 47.9 (16-CH2), 49.5 and 49.9 (13-CH2), 66.5 (3-CH2), 119.9
(6-CH and 8-CH), 128.8 and 128.9 (5-CH and 9-CH), 132.6 (7-C),
137.9 (4-C), 154.9 (11-C@O), 168.7 (2-C@O), 175.4 (22-C@O).
6.2.13. N-(4-Acetoxymethylphenyl)-pyrrolidine-3-carbaldehyde
-1-carboxamide (28)
The Dess–Martin periodinane (1.086 g, 2.56 mmol, 2 equiv) and
27 (0.374 g, 1.28 mmol) were dissolved in CH2Cl2 (30 mL) under
Argon and the reaction was stirred overnight at room temperature.
The mixture was then diluted with EtOAc (30 mL) and washed
with NaHCO3 (20 mL), Na2S2O3 (20 mL) and brine (20 mL), dried
over MgSO4 and the solvent was removed under reduced pressure
to afford 28 as a brown sticky solid (0.370 g, quantitative). The
product was not purified but used as a crude for next step;
Rf = 0.63 (CH2Cl2/MeOH: 90:10); LRMS, m/z (ES+ mode): 318.17
([M+NH4]+, 100%); 1H NMR (500 MHz, CDCl3): d 2.06 (1H, m, 15a-
CH2), 2.11 (3H, s, 1-CH3), 2.18 (1H, m, 15b-CH2), 2.99 (1H, m, 14-
CH), 3.37 (2H, m, 16-CH2), 3.50 (1H, m, 13a-CH2), 3.72 (1H, m,
13b-CH2), 5.02 (2H, s, 3-CH2), 7.26 (2H, d, J = 6.3, 5-CH and 9-CH),
7.41 (2H, d, J = 7.4, 6-CH and 8-CH), 9.62 (1H, d, J = 2.9, 17-CH);
13C NMR (125 MHz, CDCl3): d 24.7 (1-CH3), 29.7 (15-CH2), 44.9
(13-CH), 45.3 (16-CH2), 49.6 (14-CH2), 66.6 (3-CH2), 119.9 (6-CH
and 8-CH), 129.0 (5-CH and 9-CH), 133.1 (7-C), 137.7 (4-C), 158.1
(11-C@O), 168.5 (2-C@O), 196.8 (17-C@O).
6.2.16. N-(4-Acetoxymethylphenyl)-3-N0-5-methoxycarbon
ylpent-1-yl)-aminomethyl-pyrrolidine-1-carboxamide (30)
TEA (0.22 mL, 1.58 mmol, 2.3 equiv) was added to a solution of
methyl 6-aminohexanoate hydrochloride 29 (0.262 g, 1.44 mmol,
2.1 equiv) in methanol (15 mL) and the solution stirred at room tem-
perature. After 30 min a solution of 28 (0.200 g, 0.69 mmol) in MeOH
(5 mL) was added and the reaction stirred for a further 30 min. Final-
ly NaCNBH3 (1.24 mL of 1.0 M solution in THF, 1.24 mmol, 1.8 equiv)
was added and the reaction stirred overnight at room temperature.
The mixture was then diluted with H2O (20 mL) and extracted with
CHCl3 (20 mL), the organic layer was dried over MgSO4, then concen-
trated under reduced pressure. The crude was purified by column
chromatography (CHCl3/MeOH 100:0?90:10) to afford product 30
as a white oily compound (0.029 g, 10% yield); Rf = 0.56 (CHCl3/
MeOH:80:20); LRMS, m/z (ES+ mode):420.24 ([M+H]+, 100%);HRMS
(ES+ mode), Calcd for C22H34N3O5 (M+H+): 420.2493. Found:
420.2496; 1H NMR (500 MHz, CDCl3): d 1.28 (2H, m, 21-CH2), 1.55
(5H, m, 15a-CH2, 20-CH2 and 22-CH2), 1.97 (1H, m, 15b-CH2), 2.10
(3H, s, 1-CH3), 2.25 (2H, t, J = 7.4, 23-CH2), 2.37 (1H, m, 14-CH),
2.62 (3H, m, 17a-CH2 and 19-CH2), 3.01 (1H, m, 17b-CH2), 3.28 (1H,
m, 16a-CH2), 3.49 (3H, m, 13-CH2 and 16b-CH2), 3.59 (3H, s, 25-
CH3), 4.99 (2H, s, 3-CH2), 7.24 (2H, m, 5-CH and 9-CH), 7.34 (1H, br
s, 10-NH), 7.40 (2H, m, 6-CH and 8-CH); 13C NMR (125 MHz, CDCl3):
23.5 (1-CH3), 25.5 (22-CH2), 27.9 (21-CH2), 28.7 and 28.9 (20-CH2),
29.4 (15-CH2), 32.8 (23-CH2), 37.4 (14-CH), 44.2 and 44.6 (16-CH2),
48.3 and 48.4 (19-CH2), 48.9 (17-CH2), 49.3 (13-CH2), 50.5 (25-
CH3), 65.4 (3-CH2), 119.0 (6-CH and 8-CH), 127.8 and 127.9 (5-CH
and 9-CH), 131.9 (7-C), 136.6 (4-C), 153.8 (11-C@O), 166.7 (2-
C@O), 173.1 (24-C@O).
6.2.14. N-(4-Acetoxymethylphenyl)-3-(N0-3-
methoxycarbonylpropyl)-aminomethylpyrrolidine-1-
carboxamide (11)
TEA (0.22 mL, 1.58 mmol, 2.3 equiv) was added to a solution of
methyl 4-aminobutanoate hydrochloride 20 (0.222 g, 1.45 mmol,
2.1 equiv) in methanol (15 mL) and the solution was stirred at room
temperature. After 30 min, a solution of 28 (0.200 g, 0.69 mmol) in
MeOH (5 mL) was added and the reaction stirred for a further
30 min. Finally NaCNBH3 (1.24 mL of 1.0 M solution in THF,
1.24 mmol, 1.8 equiv) was added and the reaction stirred overnight
at room temperature. The mixture was then diluted with H2O
(20 mL) and extracted with CHCl3 (20 mL), the organic layer was
dried over MgSO4, then concentrated under reduced pressure. The
crude was purified by column chromatography (CHCl3/MeOH
100:0?95:5) to afford product 11 as a white oily compound
(0.029 g, 11% yield); Rf = 0.42 (CHCl3/MeOH: 80:20); LRMS, m/z
(ES+ mode): 392.25 ([M+H]+, 100%); HRMS (ES+ mode), Calcd for
Acknowledgements
We would like to acknowledge the College of Life Sciences, Uni-
versity of Dundee and the Welsh School of Pharmacy for funding
(FG) and also the UNDP/World Bank/WHO Programme for Re-
C
20H30N3O5 (M+H+): 392.2180. Found: 392.2186; 1H NMR
(500 MHz, CDCl3): d 1.52 (1H, m, 15a-CH2), 1.74 (2H, m, 20-CH2),

F. Gigante et al. / Bioorg. Med. Chem. 18 (2010) 7291–7301
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.007.
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