Antiprotozoal and cysteine proteases inhibitory activity of dipeptidyl enoates

Article abstract of DOI:10.1016/j.bmc.2018.07.015

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k_2nd (M^?1s^?1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC₅₀ values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.

Full text of DOI:10.1016/j.bmc.2018.07.015

Accepted Manuscript
Antiprotozoal and Cysteine Proteases Inhibitory Activity of Dipeptidyl Enoates
Santiago Royo, Tanja Schirmeister, Marcel Kaiser, Sascha Jung, Santiago
Rodríguez, José Manuel Bautista, Florenci V. González
PII:
S0968-0896(18)30887-3
https://doi.org/10.1016/j.bmc.2018.07.015
BMC 14452
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
10 May 2018
2 July 2018
7 July 2018
Please cite this article as: Royo, S., Schirmeister, T., Kaiser, M., Jung, S., Rodríguez, S., Bautista, J.M., González,
F.V., Antiprotozoal and Cysteine Proteases Inhibitory Activity of Dipeptidyl Enoates, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.015
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Antiprotozoal and Cysteine Proteases
Inhibitory Activity of Dipeptidyl Enoates
Santiago Royoa, Tanja Schirmeisterb, Marcel Kaiserc,d, Sascha Jungb, Santiago Rodrígueza, José Manuel
Bautistae and Florenci V. Gonzáleza,*

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Antiprotozoal and Cysteine Proteases Inhibitory Activity of Dipeptidyl Enoates
Santiago Royoa, Tanja Schirmeisterb, Marcel Kaiserc,d, Sascha Jungb, Santiago Rodrígueza, José Manuel
Bautistae and Florenci V. Gonzáleza, ∗
aDepartament de Química Inorgànica i Orgànica, Universitat Jaume I, Avda. Sos Baynat s/n 12080 Castelló, Spain
bInstitute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55099 Mainz, Germany
cSwiss Tropical and Public Health Institute, Socinstrasse 57, 4051 Basel, Switzerland
dUniversity of Basel, Petersplatz 1, 4003 Basel, Switzerland
eDepartment of Biochemistry and Molecular Biology and Research Institute Hospital 12 de Octubre, Faculty of Veterinary Sciences, Complutense University of
Madrid, 28040 Madrid, Spain.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine
proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and
malaria, respectively. They have also been tested against human cathepsins B and L1 for
selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of
the members of the family are very potent inhibitors of parasitic cysteine proteases displaying
k2nd (M-1s-1) values of seven orders of magnitude. In vivo antiprotozoal testing was also
performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium
falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values
against Leishmania donovanii.
Available online
Keywords:
Inhibitors
Malaria
Sleeping sickness
Chagas disease
malaria
2009 Elsevier Ltd. All rights reserved
.
Cysteine proteases
∗ Corresponding author. Tel.: +34-964729156; fax: +34-964728214; e-mail: fgonzale@uji.es

1. Introduction
Figure 1. Schematic representation of binding of the dipeptidyl enoate
inhibitors into the active site and the binding pockets.
Malaria, sleeping sickness and Chagas disease are among the
most important tropical diseases, and the last two are considered
neglected.1,2 Human African trypanosomiasis (HAT) or sleeping
sickness, caused by the protozoan Trypanosoma brucei, is fatal
when untreated, and current treatments are ineffective and have
side effects. The cysteine protease rhodesain, being essential for
the development of T. brucei, has been identified as an
interesting target for the search of new drugs against this disease.
Chagas disease is caused by the protozoan Trypanosoma cruzi.
Approximately 7-8 million people are infected by T. cruzi in
Central and South America, with over 100 million people at risk
of infection.1 It has recently emerged in North America and
Europe as well. Benznidazole and nifurtimox are drugs currently
available but both have variable efficacy and side effects.3 The
cysteine protease cruzain has been identified as a target for the
search of new drugs against this disease.4 Malaria is the most
widespread and severe tropical infectious disease; in humans, it is
caused by several species of the Plasmodium genus, with
Plasmodium falciparum being the most dangerous and most
prevalent. The cysteine protease falcipain-2 has been recognized
as a potential drug target.
2.2. Synthesis of inhibitors
The designed inhibitors were initially prepared through a
synthetic route resulting from a combination of an asymmetric
Evans aldol reaction, followed by protection/deprotection steps
and then a Curtius reaction to afford the corresponding
isocyanate which upon coupling with corresponding N-protected
aminoacid yielded the hydroxylic enoates FGA40, FGA54,
FGA50, FGA67 and FGA58 (Scheme 1). Since the aldol reaction
affords a mixture of separable isomers, epimeric inhibitors
differing in the configuration of carbon-4 could also be prepared.
Then, the oxidation of FGA54 afforded the ketones FGA55 and
FGA56 (Scheme 1).
All three above mentioned parasitic cysteine proteases rhodesain,
cruzain and falcipain-2 belong to the papain superfamily. The
alignment of these three proteases with their homologous
cathepsin B and L shows striking similarities at three main
functional regions (see Supplementary Material)5 despite of some
other structural differences allowing the potential design of
inhibitors selectivity as antiparasitic compounds.6
Michael acceptors are among the most interesting inhibitors of
cysteine proteases. For example, K1777 a dipeptidyl vinyl
sulfone is a potent irreversible inhibitor.7
We previously reported dipeptidyl enoates as efficient
inhibitors against rhodesain,8 and we now report a structure-
activity study of dipeptidyl enoates as irreversible inhibitors
against the parasitic cysteine proteases falcipain-2, cruzain and
rhodesain as compared to their activity against human cathepsins
B and L. As it is reported herein in vitro and antiprotozoal
activity of these inhibitors which contain an enoate moiety at the
carboxyl terminus depended very much on the residues within
the peptidic framework and the protecting group of the amino
terminus.
Scheme 1. Preparation of inhibitors through an Evans aldol/Curtius
sequence.
Analogues displaying a reverse sequence were also prepared by
coupling the carboxylic acids resulting from chiral auxiliary
removal with an aminoester (FGA41, 42, 43, 45, 68) (Scheme 2).
Chemical derivatizations of these compounds afforded further
members of the family. FGA41 was oxidized to give the
corresponding ketone FGA73, and the ester FGA50 was
hydrolized into carboxylic acid FGA57 which was subsequently
derivatized into amide FGA71 (Scheme 2).
2. Results and discussion
2.1. Structure design of inhibitors
The structure of the inhibitors is a modified dipeptide having a
carbon-carbon double bond conjugated with an ester at the
carboxyl-terminus of the dipeptide and a protecting group at the
amino-terminus (Figure 1). The conjugated double bond at the
carboxyl-terminus is the warhead to be attacked by the thiolate of
the cysteine. Analogues with different substituens were prepared
in order to optimize the interactions at the sites S1, S2, S3 and S1´.
Scheme 2. Preparation of inhibitors displaying a reversed sequence.
In order to prepare more dipeptidyl enoates, the synthetic
strategy was changed. A straightforward approach was applied by
preparation of phosphonates derived from the corresponding

diprotected dipeptide. Then, Horner-Emmons reactions between
dipeptidyl phosphonate and ethyl glyoxalate afforded the
corresponding inhibitors in good yield (Scheme 3).
gave IC50 values between 1 and 4 µM. Interestingly, the
diastereomeric inhibitors FGA50 and FGA40 differing only in
the configuration of one stereocenter showed different activities.
FGA50, displaying S configuration at C-4, was more active than
FGA40, with R configured C-4 atom.
For all assayed cysteine proteases, FGA54 with an L-alanine
residue at the P1 site showed similar inhibitory (slightly lower)
activity than its L-homophenylalanine counterpart FGA50.
Inhibitor FGA57 having a carboxylic acid was slightly less
active than the ethyl ester FGA50, and the benzylamide FGA71
was not active.
FGA41, FGA42, FGA43 and FGA45 consisting of a reverse
amide bond as compared to the rest of inhibitors were not active
which demonstrates the importance of the correctly oriented
amide bond for these dipeptidic inhibitors.
Scheme 3. Preparation of inhibitors through phosphonates.
2.3. Inhibitory activity against cysteine proteases
In the alcohol series, in vitro testing against cysteine proteases
denote inhibitors to display a time-dependent inhibition with IC50
values in the micromolar range (Table 1). The most active
inhibitor of this series resulted to be FGA67 displaying a
morpholinyl carbonyl protecting group instead of the benzyloxy
carbonyl which is part of the other compounds. Inhibitor FGA67

Table 1. In vitro inhibitory activity of compounds against cysteine proteases
Cathepsin B
Cathepsin L
Falcipain-2
Rhodesain
Cruzain
b
c
b
c
b
c
b
c
b
c
Comp.
%Inh. IC50
k2nd
Inh.%
IC50
k2nd
%Inh. IC50
k2nd
Inh.%
IC50
k2nd
%Inh. IC50
k2nd
a
a
a
a
a
FGA40
FGA41
FGA42
FGA43
FGA45
FGA50
FGA54
FGA57
FGA58
FGA67
FGA68
FGA71
35
17
25
0
52.6
14
nd
58
0
47
77
nd
22
5
nd
nd
32
2
nd
nd
37
nd
nd
nd
nd
18
30
12
9
nd
0
nd
5
nd
6
nd
nd
0
nd
3
nd
1
nd
0
nd
0
nd
9
nd
4
nd
36
26
15
10
100
23.9
1.13
800
nd
57
80
47
14
98
13.1
14.3
20
1100
828
882
nd
41
41
24
11
34.4
26.8
75
557
2700
29
63
63
24
0
9.5
3200
11000
nd
nd
nd
15.1
nd
nd
nd
nd
nd
nd
4200
2.45
1900
99
1.13
36700
95
3.75
3600
9
nd
17
nd
1
nd
41
nd
aInh. % at 20 µM.
bIC50 in µM units
ck2nd in M-1s-1 units.
Inhibitors with a ketone group resulted to be more potent against
the tested cysteine proteases than the counterparts with hydroxyl
group (Table 2). The inhibitors FGA44, FGA55 and FGA75
containing a L-phenylalanine residue at P-2 site were more active
than FGA47, FGA56 and FGA77 having an L-leucine residue at
this position. For the P-1 site L-homophenylalanine (FGA44,
FGA47, FGA74, FGA75 and FGA77) or L-leucine (FGA69 and
FGA76) gave better results than L-alanine (FGA55 and FGA56).
Inhibitors FGA69 and FGA 70 differing in the configuration of
the leucine residue gave similar results with the L-leucine
derivative being slightly more active. A similar result was
observed when comparing inhibitors FGA74 and FGA75.
Interestingly, compound FGA78 having a dienoate warhead is
a reversible inhibitor as opposite to the ones having an enoate
moiety which are all irreversible. The (ir)reversibility of
inhibition was tested by dilution assays for compounds FGA74,
FGA75, and FGA78.
Tested compounds also inhibited human cathepsins B and L.
Further SAR studies will be carried out in the near future by
changing amino acids at P1 and P2 positions so as to improve
selectivity. The structures of human and parasite cathepsins
cocrystalized with inhibitors revealed sites for modification in the
inhibitor to enhance selectivity for the parasite protease over its
human ortholog.9
Inhibitors having a morpholine carbonyl group were less
potent than the ones with a benzyloxy carbonyl group. For
example, FGA44 displayed higher IC50 values than FGA75, and
the kinetic constants ratio k2nd was found to be higher against
rhodesain; similar results were observed when comparing FGA70
with FGA76 or FGA47 with FGA77.

Table 2. In vitro inhibitory activity of ketone compounds against cysteine proteases
Cathepsin B
Cathepsin L
Falcipain-2
Rhodesain
Cruzain
b
c
b
c
b
c
b
c
b
c
Comp.
%Inh.a IC50
k2nd
Inh.% a
IC50
k2nd
%Inh.a IC50
k2nd
%Inh.a
IC50
k2nd
%Inh.
IC50
k2nd
a
FGA44
FGA47
FGA55
100
100
100
0.07
0.05
0.14
45500
42400
47000
100
100
100
0.05
0.26
0.08
45300
29900
100
98
0.06
0.58
0.19
17100
6000
100
98
0.03
0.145
0.59
1274500
116200
6500
99
100
100
0.08
0.3
60800
19730
0
99
28300
100
13960
0
FGA56
FGA69
100
100
0.07
0.05
85500
99
0.5
9800
98
nd
1.2
nd
12000
nd
99
0.8
14200
99
0.63
0.02
14400
138500
100
0.01
75860
100
0.02
1611100
100
51670
0
31180
0
0
70450
0
FGA70
nd
nd
nd
100
0.06
116700
100
0.01
100
0.03
1533300
100
0.02
FGA74
FGA75
FGA76
FGA77
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
99
0.08
0.07
0.09
0.14
108400
97000
49500
31400
100
100
100
100
> 2
nd
99
0.15
0.15
0.11
0.14
62400
142000
324500
120000
99
0.33
0.30
0.20
0.34
22000
100
100
100
0.18
0.09
0.21
28500
52200
19600
100
100
100
100
100
23400
10600
5600
Ki =
0.044
µM
Ki=1.4
µM
Ki=0,7
8 mM
FGA78
K1777
90
2.25
99
1.49
nd
nd
nd
98
0.56
> 2
0.56
95000
0
552300d
23900
aInh. % at 20 µM.
bIC50 in µM units
ck2nd in M-1s-1 units
d ref. value 555000
2.4. Docking studies
Molecular modeling studies were performed in order to get
an idea of the reasons for the remarkable differences in
inhibition potency of keto compounds compared to the allylic
alcohol compounds. Therefore, FGA50 (Ki = 9.5 µM at
rhodesain) and FGA44 (Ki = 0.3 µM at rhodesain) were
selected as representative examples. Docking was performed
using the crystal structure of rhodesain bound to K11777 (pdb
2p7u).10 First, non-covalent docking was carried out with
FlexX/LeadIT to simulate the initial non-covalent enzyme-
inhibitor complex (Figure 2).11 The affinity of the keto
compound is significantly higher (FlexX score: FGA44 –25.7,
FGA50 –16.7) and the Michael system is closer to the

nucleophilic cysteine (FGA44: 3.38 Å, FGA50: 3.85 Å). The
results suggest that the keto compound is attacked at the
carbon next to the keto group. Furthermore, covalent docking
was performed with DOCKTITE to simulate the final protein
bound state (Figure 3).12 The scores obtained do not differ
significantly (Table 3), indicating no major differences once
the covalent complex is formed. The main reason for the
higher potency of the keto compounds may therefore be
attributed to the higher non-covalent affinity resulting in a
more stable non-covalent complex in which the Michael
system comes closer to the nucleophilic cysteine for
subsequent reaction.
Table 3. Docking results between rhodesain and compounds FGA44 and FGA50
Compound
Non-covalent docking
Covalent docking
Distance Cys-
Michael system (Å)
London dG
score
FlexX score
DSX score
Affinity dG score
DSX score
FGA44
FGA50
– 25.7
– 16.8
– 168.9
– 133.7
3.38
3.85
– 6.2
– 6.4
– 10.6
– 11.3
– 130.0
– 132.9
Figure 3. Covalent complex of FGA44.
Figure 2. Overlay of non-covalent docking solutions. FGA44: cyan,
FGA50: magenta.
2.5. Activity against Protozoa
Selected inhibitors were tested against the parasites
inhibitory activity against T. brucei (IC50=0.93 µM). FGA75
was also active against P. falciparum (IC50=6.81 µM).
Inhibitor FGA47 resulted to be very active with an IC50 value
of 1.6 µM against P. falciparum, and even more interestingly
inhibited L. donovanii with an IC50 value of 90 nM. Such
higher activity of FGA47 against Leishmania donovanii as
compared to other tested parasites, might indicate the existence
of other cysteine proteases in L. donovanii as potential targets.
Trypanosoma
brucei
causing
Human
African
Trypanosomiasis, Trypanosoma cruzi which causes Chagas
disease, and Plasmodium falciparum causing malaria. Some
inhibitors were also tested against Leishmania donovanii
which causes kala-azar, also called visceral leishmaniasis. As
shown in Table 4, IC50 values ranged from nanomolar to
micromolar, depending on the organism and the chemical
structure of the inhibitor. Thus, FGA44 showed the best
Table 4. Antiprotozoal activity of dipeptidyl enoates (IC50 µM )
inhibitor
Plasmodium
falciparum
Trypanosoma
brucei
Trypanosoma
cruzi
Leishmania
donovanii
12.57
0.09
nd
FGA-44
FGA-47
FGA-69
FGA-75
FGA-77
16.08
1.61
0.93
2.32
nda
nd
20.14
7.14
nd
14.94
6.81
nd
nd
17.74
nd
nd
nd
and= non-determined.
vitro culture. As seen in Figure 4, all assayed compounds allowed
a 100% viability at 1µM. Thus, all these compounds performed
with low cytotoxicity at low micromolar range.
In addition, selectivity of the inhibitors was evaluated by
assaying selected compounds for viability of human cells on in

Figure 4. Cytotoxicity in human hepatoma cells HEP-G2 of selected
inhibitors. HEPG2 human hepatoma cells were incubated with the inhibitors
at three different concentrations (1, 50 and 100 µM). Viability of the HEPG2
human hepatoma cells after 24 h is plotted in comparison with 100% viability
of control cultures in the absence of inhibitors.
Figure 5. Optimization process of inhibitors.
4. Experimental Section
4.1. Chemistry
General. 1H NMR spectra and 13C NMR spectra were
measured in CDCl3 (1H, 7.24 ppm; 13C 77.0 ppm) solution at 30
°C on a 300 MHz or a 500 MHz NMR spectrometer. Mass
spectra were measured in a QTOF I (quadrupole-hexapole-TOF)
mass spectrometer with an orthogonal Z-spray-electrospray
interface. IR spectra were recorded as oily films on NaCl plates
on a FT-IR spectrometer. EM Science Silica Gel 60 was used for
column chromatography while TLC was performed precoated
plates (0.25 mm). Unless otherwise specified, all reactions were
carried out under nitrogen atmosphere with magnetic stirring.
3. Conclusions
An optimization study of the dipeptidyl enoates was performed
by chemical structure modifications (Figure 5). Compound
FGA50 displaying R configuration at C-3 was more active than
its epimer FGA40. Two modifications of the chemical structure
of FGA50 were done: the hydroxyl group of the warhead was
interchanged by a ketone group resulting into FGA44, and the
CBZ group at the P3 site was replaced by a morpholine carbonyl
group yielding FGA67. The ketone group remarkably increased
the potency, while the morpholine group lowered the calculated
lipophilicity. Hence, FGA75 was prepared by combining both
moieties: a ketone group at the warhead for having high activity
and a morpholine carbonyl group at P3 site for a balanced
lipophilicity. The resulting inhibitor FGA75 showed good
activity against protozoa Plasmodium falciparum and low
lipophilicity.
1-[(4S)-4-Benzyl-2-thioxo-thiazolidin-3-yl]-4-phenyl-
butan-1-one 1. To an ice-bath cold solution of (S)-4-
benzylthiazolidine-2-thione13 (18.37 g, 87.87 mmol) and Et3N
(26.66 mL, 175.74 mmol) in CH2Cl2 (266 mL) was added 4-
phenylbutanoyl chloride (freshly prepared from 4-phenylbutyric
acid (17.31g, 105.44 mmol)). The resulting mixture was stirred at
room temperature for 24 hours. Then brine was added and
extracted with CH2Cl2 (3 x 40 mL), the organic layers were
washed (brine), dried (Na2SO4) and concentrated. The yellow
crude oil was purified through chromatography (silica-gel,
hexanes/EtOAc (98:2) and (95:5)) to afford 24.76 mg (77%);
[αD25]= +157.80 (c= 2.3, CHCl3). IR (NaCl) ν 3025, 2935, 2849,
1603, 1695, 1496, 1454, 1342, 1394, 1359, 1342, 1293, 1264,
1192, 1157, 1135, 1040, 893, 746, 701 cm-1. 1H NMR (500MHz,
CDCl3) δ 7.16-7.34 (10H, m), 5.33 (1H, ddd, J= 4.0, 7.5 and 11.5
Hz), 3.38 (1H, ddd, J= 6.0, 9.0 and 17.0Hz), 3.33 (1H, dd, J= 7.5
and 11.5Hz), 3.13-3.20 (2H, m), 3.01 (1H, dd, J= 10.5 and 13.0
Hz), 2.84 (1H, d, J= 11.5 Hz), 2.68 (2H, t, J= 7.0 Hz). 13C NMR
(125MHz, CDCl3) δ 201.06, 173.73, 141.53, 136.56, 129.44,
128.88, 128.53, 128.36, 127.19, 125.96, 68.53, 37.93, 36.79,
35.11, 31.90, 26.43 ppm.. HRMS m/z calcd. for C20H21ONS2Na
[M+Na+]: 378.0962, found: 378.0970; m/z calcd. for
C20H21ONS2K [M+K+]: 394.0702, found: 394.0724.
Further SAR studies will be carried out to first optimize efficacy
in vitro, and then to provide proof of concept activity in vivo. For
in vitro activity against Leishmania, parasites within
macrophages will be used to ensure that compounds can enter the
parasitophorous vacuoles within which parasites reside. This in
vitro SAR study will then be followed by a microsomal stability
study and “snapshot” pharmacokinetics prior to testing in animal
models of infection. Also a preliminary toxicology study in
animal models will be carried out to ensure that the compounds
are safe.

General experimental procedure for the aldol reaction.14
through chromatography (silica-gel, hexane/EtOAc (8:2) to
afford a yellow oil.
To MgBr2·Et2O (weighed in a dry-box) (230 mg, 0.89 mmol)
were added sequentially a solution of compound 3 (2.43 g, 6.85
mmol) in EtOAc (mL), a solution of (E)-ethyl 3-formylacrylate
(1.75 g, 13.70 mmol) in EtOAc (mL), and then freshly distilled
triethylamine (2.1 ml, 15.07 mmol) and trimethylsilyl chloride
(1.47 ml, 11.65 mmol). The resulting yellowish suspension was
stirred under nitrogen atmosphere at room temperature for 12
days. Then the reaction mixture was filtered through a pad of
silica-gel using ethyl ether and then concentrated under vacuum.
Then the resulting orange oil was solved in THF (180 mL) and
treated with hydrochloric acid (1M aqueous solution) (4 mL).
Then solid NaHCO3 was added until no more gas was evolved.
Then extracted with CH2Cl2 (3 x 40 mL), dried (Na2SO4) and
concentrated. The yellow crude oil was purified through
chromatography (silica-gel, CH2Cl2/Et2O (20:1)) to afford 2.15 g
of a yellow oil (yield 65%, over consumed starting material).
Spectroscopical data for anti silylated aldol: (2E,4S,5S)-5-
[(4S)-4-Benzyl-2-thioxo-thiazolidine-3-carbonyl]-4-(tert-butyl-
dimethyl-silanyloxy)-7-phenyl-hept-2-enoic acid ethyl ester 4.
(yield= 98%). [αD21] = +92.65 (c= 5.8, CHCl3). IR (NaCl) ν
3063, 3027, 2930, 2857, 1716, 1659, 1604, 1496, 1455, 1367,
1
1341, 1230, 1159, 1038, 984, 837, 779, 746, 701, 667 cm-1. H
NMR ( 500MHz, CDCl3 ) δ 7.15-7.37 (10H, m), 6.93 (1H, dd, J =
7.0 and 15.5 Hz), 6.01 (1H, d, J = 15.5 Hz), 5.37 (1H, ddd, J =
3.5, 7.0 and 11.0 Hz), 5.21 (1H, m), 4.58 (1H, t, J= 6.5 Hz), 4.22
(2H, t, J= 7.0 Hz), 3.31 (1H, dd, J= 7.5 and 11.5 Hz), 3.20 (1H,
dd, J= 3.5 and 13.5 Hz), 3.04 (1H, dd, J= 11.0 and 13.5 Hz), 2.85
(1H, d, J= 11.0 Hz), 2.76 (1H, m), 2.64 (1H, m), 2,10 (1H, m),
1.90 (1H, m), 1.31 (3H, t, J = 7.0 Hz), 0.92 (9H, m), 0.08 (3H, s),
0.04 (3H, s). 13C NMR ( 125 MHz, CDCl3) δ 201.44, 174.18,
165.76, 147.52, 141.60, 136.39, 129.35, 128.80, 128.29, 128.26,
127.12, 125.86, 122.40, 74.27, 68.93, 60.47, 49.32, 36.81, 33.04,
31.55, 30.67, 25.61, 17.86, 14.11, -4.27, -5.05 ppm. HRMS m/z
calcd. for C32H43O4NS2SiNa [M+Na+]: 620.2301, found:
620.2283.
Spectroscopical data for anti aldol: (2E,4S,5S)-5-[(4S)-4-
Benzyl-2-thioxo-thiazolidine-3-carbonyl]-4-hydroxy-7-
phenyl-hept-2-enoic acid ethyl ester 2.
[αD29] = +113.31(c= 3.7, CHCl3). IR (NaCl) ν 3483, 3027,
2980, 1716, 1603, 1496, 1455, 1368, 1342, 1263, 1164, 1137,
1038, 982, 912, 865, 747, 701 cm-1.1H NMR (500 MHz, CDCl3)
δ 7.05-7.25 (10H, m), 6.84 (1H, dd, J = 15.5 and 4.0 Hz), 6.03
(1H, dd, J= 15.5, 1.0 Hz), 5.14 (1H, ddd, J= 4.5, 7.5 and 11.0
Hz), 4.72 (1H, m), 4.46 (1H, m), 4.10 (2H, q, J= 7.5 Hz), 3.25
(1H, dd, J = 7.5 and 11.0 Hz, ), 3.04 (1H, dd, J = 4.5 and 13.5
Hz), 2.91 (1H, dd, J = 10.0 and 13.5 Hz), 2.76 (2H, d, J = 11.5
Hz), 2.57 (1H, m), 2.12 (1H, m), 2.03 (1H, m), 1.18 (3H, t, J= 7.0
Hz). 13C NMR (125 MHz, CDCl3) δ 201.85, 175.77, 166.09,
147.74, 141.15, 136.14, 129.39, 128.88, 128.43, 128.40, 127.27,
126.10, 121.89, 71.25, 68.44, 60.59, 48.01, 36.71, 32.87, 32.40,
30.45, 14.16 ppm. HRMS m/z calcd. for C26H29O4NS2Na
[M+Na+]: 506.1436, found: 506.1429; m/z calcd. for
C26H29O4NS2K [M+K+]: 522.1175, found: 522.1180.
Spectroscopical data for syn silylated aldol: (2E,4R,5S)-5-
[(4S)-4-Benzyl-2-thioxo-thiazolidine-3-carbonyl]-4-(tert-butyl-
dimethyl-silanyloxy)-7-phenyl-hept-2-enoic acid ethyl ester 5.
(yield= 94%). [αD20] = +112.1 (c= 1.5, CHCl3). IR (NaCl) ν
3063, 3027, 2954, 2930, 2857, 1718, 1659, 1604, 1496, 1471,
1455, 1365, 1341, 1262, 1193, 1161, 1135, 1083, 1038, 983, 939,
883, 838, 779, 747, 701, 667 cm-1. 1H NMR ( 500MHz, CDCl3 ) δ
7.15-7.36 (10H, m), 6.94 (1H, dd, J= 5.5 and 15.5 Hz), 5.93 (1H,
d, J= 15.5 Hz), 5.26 (1H, ddd, J = 4.0, 7.0 and 11.0 Hz), 5.07
(1H, dq, J = 5.0 Hz), 4.55 (1H, t, J = 5.0 Hz), 4.20 (2H, q, J= 6.5
Hz), 3.30 (1H, dd, J= 7.0 and 11.5 Hz), 3.17 (1H, dd, J= 4.0 and
13.0 Hz), 3.01 (1H, dd, J= 11.0 and 13.0 Hz), 2.84 (1H, d, J=
11.5 Hz), 2.61-2.73 (2H, m), 2.24 (1H, m), 1.87 (1H, m), 1.30
(3H, t, J= 7.0 Hz), 0.89 (9H, s), 0.01 (3H, s), 0.00 (3H, s). 13C
NMR ( 125 MHz, CDCl3) δ 201.57, 173.62, 166.04, 147.67,
141.61, 136.45, 129.45, 129.42, 129.36, 129.10, 128.40, 128.37,
128.35, 128.32, 127.18, 125.91, 121.72, 73.46, 69.23, 60.47,
49.68, 36.67, 33.52, 31.78, 29.07, 25.73, 25.67, 18.03, 14.20, -
4.61, -5.21 ppm. HRMS m/z calcd. for C32H43O4NS2SiNa
Spectroscopical data for syn aldol: (2E,4R,5S)-5-[(4S)-4-
Benzyl-2-thioxo-thiazolidine-3-carbonyl]-4-hydroxy-7-
phenyl-hept-2-enoic acid ethyl ester 3.
[M+Na+]:
C32H43O4NS2SiK [M+K+]: 636.2040, found: 636.2079.
620.2301,
found:
620.2310;
calcd.
for
[αD29] = +55.0 (c= 1.9, CHCl3). IR (NaCl) ν 3468, 3027, 2929,
1714, 1604, 1496, 1455, 1368, 1342, 1264, 1162, 1038, 911, 745,
701 cm-1.1H NMR ( 500 MHz, CDCl3) δ 7.10-7.28 (10H, m),
6.83 (1H, dd, J= 15.5 and 4.5 Hz), 6.03 (1H, dd, J= 15.5, 1.0 Hz),
5.30 (1H, ddd, J= 4.0, 7.5 and 11.5 Hz), 5.05 (1H, dq, J= 4.5 Hz),
4.58 (1H, m), 4.13 (2H, q, J= 7.5 Hz), 3.31 (1H, dd, J= 7.5 and
12.0 Hz), 3.08 (1H, dd, J= 3.5 and 13.5 Hz), 2.94 (1H, dd, J =
10.5 and 13.5 Hz), 2.85 (2H, d, J= 11.5 Hz), 2.54-2.67 (2H, m),
2.46 (1H, d, J= 4.0 Hz), 2.12-2.18 (m, 2H), 1.82-1.89 (m, 2H),
1.22 (3H, t, J= 7.0 Hz). 13C NMR ( 125 MHz, CCl3) δ 202.06,
175.03, 166.14, 146.61, 141.59, 136.32, 129.48, 129.42, 129.29,
129.00, 128.49, 128.47, 127.37, 126.11, 122.12, 72.02, 69.05,
60.63, 48.21, 36.98, 33.69, 32.00, 28.84, 14.26 ppm. HRMS m/z
calcd. for C26H29O4NS2Na [M+Na+]: 506.1436, found: 506.1441.
General experimental procedure for the removal of the
chiral auxiliary.
To an ice-bath cold solution of protected aldol ( 2.24 g, 3.75
mmol) in THF-H2O (3:1) (67.5 mL) was added H2O2 (30% v/v)
(2.32 mL, 22.5 mmol) and then a solution of LiOH (180 mg, 7.50
mmol) in H2O (1.8 mL). The resulting mixture was stirred cold
with an ice-bath for 2 h. Then quenched with a saturated aqueous
solution of Na2SO3 and NaHCO3 and stirred for 30 min. Then
extracted with CH2Cl2 (3 x 40 mL), dried (Na2SO4) and
concentrated. The crude oil was purified through chromatography
(acidified silica-gel, hexanes/EtOAc (7:3)) to afford an oil.
General experimental procedure for the protection of
aldols.
(2E,4S,5S)-4-(tert-Butyl-dimethyl-silanyloxy)-5-phenethyl-
hex-2-enedioic acid 1-ethyl ester 6.
To a -70ºC cold solution of aldol (1.81 g, 3.75 mmol) in
CH2Cl2 (38 mL) was added 2,6-lutidine (1.31 mL, 11.25 mmol)
and then TBSOTf (2.15 mL, 9.37 mmol). The resulting mixture
was stirred at –70 ºC for 6h. Then quenched with saturated
aqueous NaHCO3 solution, and extracted with CH2Cl2 (3 x 40
mL), dried (Na2SO4) and concentrated. The crude was purified
(yield= 88%). [αD21] = -9.88 (c= 8.6, CHCl3). IR (NaCl) ν
3028, 2930, 2858, 2649, 1944, 1710, 1660, 1605, 1497, 1472,
1455, 1391, 1369, 1260, 1163, 1092, 1035, 1006, 982, 938, 837,
1
778, 748, 700, 670 cm-1. H NMR ( 500 MHz, CDCl3) δ 10.58
(1H, s), 7.14-7.27 (5H, m), 6.82 (1H, dd, J= 6.0 and 15.5 Hz),
5.96 (1H, d, J= 1.5 and 15.5 Hz), 4.50 (1H, t, J = 1.0 and 7.0 Hz),
4.18 (2H, m), 2.71 (1H, m), 2.61 (2H, m), 1.87 (1H, m), 1.76

(1H, m), 1.27 (3H, t, J= 7.0 Hz), 0.86 (9H, s), 0.03 (3H, s), 0.00
(3H, s). 13C NMR ( 125MHz, CDCl3) δ 178.51, 166.00, 146.81,
141.04, 128.53, 128.50, 126.17, 122.83, 73.01, 60.65, 52.03,
33.68, 29.31, 25.71, 18.06, 14.24, -4.37, -4.50 ppm. HRMS m/z
calcd. for C22H34O5SiNa [M+Na+]: 429.2073, found: 429.2099.
Ethyl
(4S,5S,E)-5-(((S)-1-(benzyloxy)-1-oxo-3-
phenylpropan-2-yl)carbamoyl)-4-hydroxy-7-phenylhept-2-
enoate (FGA42).
(yield = 61%). 1H NMR ( 300 MHz, CDCl3) δ 7.43-7.12 (11H,
m), 7.01-6.98 (4H, m), 6.80 (1H, dd, J= 4.5 and 16.1 Hz), 6.21
(1H, d, J = 8.3 Hz), 6.06 (1H, dd, J= 16.1 and 1.7 Hz), 5.20 (1H,
d, J = 12.0 Hz), 5.12 (1H, d, J = 12.0 Hz), 4.92 (1H, m), 4.32
(1H, m), 4.12 (2H, q, J = 7.1 Hz), 3.70 (1H, m), 3.10 (dd, J =
13.5, 6.5 Hz, 1H), 3.00 (dd, J = 13.5, 8.5 Hz, 1H), 2.52-2.32 (2H,
m), 2.22-2.24 (1H, m), 2.12-2.20 (1H, m), 1.80-1.87 (1H, m),
1.26 (3H, t, J= 7.1 Hz) ; 13C NMR ( 75 MHz, CDCl3) δ 173.8,
171.5, 166.2, 147.1, 140.9, 135.6, 129.1, 128.8, 128.7, 128.6,
128.5, 128.4, 127.3, 126.2, 122.2, 72.0, 67.6, 60.5, 53.2, 51.0,
37.5, 33.1, 30.8, 14.2 ppm
(2E,4R,5S)-4-(tert-Butyl-dimethyl-silanyloxy)-5-phenethyl-
hex-2-enedioic acid 1-ethyl ester 7.
(yield= 83%). [αD20] = -10.35 (c= 1.3, CHCl3). IR (NaCl) ν
3028, 2957, 2931, 2859, 1712, 1660, 1604, 1497, 1472, 1455,
1391, 1369, 1260, 1165, 1125, 1084, 1037, 1006, 979, 939, 837,
1
778, 748, 699, 671 cm-1. H NMR ( 500MHz, CDCl3 ) δ 10.58
(1H, s), 7.15-7.28 (5H, m), 6.87 (1H, dd, J= 5.5 and 15.5 Hz),
5.97 (1H, d, J= 15.5 Hz), 4.60 (1H, t, J= 4.5 Hz), 4.19 (2H, m),
2.77 (1H, m), 2.55 (2H, m), 2.05 (1H, m), 1.82 (1H, m), 1.28
(3H, t, J= 7.0 Hz), 0.88 (9H, s), 0.03 (3H, s), 0.00 (3H, s). 13C
NMR ( 125 MHz, CDCl3) δ 178.48, 166.12, 147.41, 141.22,
128.48, 128.46, 126.10, 122.21, 72.68, 60.58, 51.02, 33.74,
28.10, 25.75, 18.10, 14.23, -4.47, -4.52 ppm. HRMS m/z calcd.
for C22H34O5SiNa [M+Na+]: 429.2072, found: 429.2108; m/z
calcd. for C22H34O5SiK [M+K+]: 445.1813, found: 445.1866.
Ethyl
(4S,5S,E)-5-(((S)-1-(methyloxy)-4-methyl-1-
oxopentan-2-yl)carbamoyl)-4-hydroxy-7-phenylhept-2-enoate
(FGA43).
1
(yield = 51%). H NMR ( 500 MHz, CDCl3) δ 7.30-7.15 (5H,
m), 6.81 (1H, dd, J= 4.4 and 15.6 Hz), 6.10 (1H, dd, J= 15.6 and
1.6 Hz), 4.60 (1H, m), 4.35 (1H, m), 4.21 (1H, q, J = 7.0 Hz),
3.65 (3H, s), 2.72 (1H, m), 2.61 (1H, m), 2.25 (1H, m), 2.15 (1H,
m), 1.80 (1H, m), 1.55 (1H, m), 1.49 (1H, m), 1.10 (3H, t, J= 7.0
Hz), 0.70 (6H, d, J = 4.4 Hz).
General experimental procedure for the conversion of the
carboxylic acid into isocianate.
To a solution of carboxylic acid ( 660 mg, 1.63 mmol) in
toluene ( 13 mL) was added 4 molecular sieves ( 196 mg), Et3N (
249 µL, 1.79 mmol) and then diphenylphosphorylazide ( 401 µL,
1.79 mmol). The resulting mixture was stirred at room
temperature for 30 min. Then was quenched with a saturated
aqueous solution of hydrogen carbonate and extracted with Et2O
(3 x 30 mL ), dried (Na2SO4) and concentrated. The crude oil was
purified through chromatography (silica-gel, hexanes/EtOAc
(8:2) and (7:3)) to afford an oil.
Ethyl
(4S,5S,E)-5-(((S)-1-(methyloxy)-1-oxo-3-
phenylpropan-2-yl)carbamoyl)-4-hydroxy-7-phenylhept-2-
enoate (FGA45).
(yield = 50%). 1H NMR ( 300 MHz, CDCl3) δ 7.35-6.89 (10H,
m), 6.82 (1H, dd, J= 4.5 and 16.1 Hz), 6.10 (1H, m), 6.08 (1H, d,
J= 16.1 Hz), 4.85 (1H, m), 4.30 (1H, m), 4.10 (2H, q, J = 7.1 Hz),
3.75 (1H, m), 3.70 (s, 3H), 3.10 (dd, J = 13.5, 6.5 Hz, 1H), 3.00
(dd, J = 13.5, 8.5 Hz, 1H),2.55-2.34 (2H, m), 2.21 (1H, m), 2.05
(1H, m), 1.75 (1H, m), 1.25 (3H, t, J= 7.1 Hz) ; 13C NMR ( 75
MHz, CDCl3) δ 173.8, 172.0, 166.2, 147.2, 140.9, 135.7, 129.0,
128.8, 128.5, 128.4, 127.4, 126.2, 122.2, 72.1, 60.5, 53.1, 52.6,
50.8, 37.6, 33.1, 30.9, 14.2 ppm.
(2E,4S,5S)-4-(tert-Butyl-dimethyl-silanyloxy)-5-isocyanato-
7-phenyl-hept-2-enoic acid ethyl ester 8.
(yield= 54%). [αD20] = -16.05 (c= 4.5, CHCl3). IR (NaCl) ν
3028, 2956, 2931, 2859, 2253, 1783, 1722, 1660, 1604, 1497,
1472, 1367, 1260, 1175, 1128, 1038, 982, 838, 778, 700 cm-1. 1H
NMR ( 500 MHz, CDCl3) δ 7.13-7.27 (5H, m), 6.84 (1H, dd, J=
5.0 and 15.5 Hz), 6.00 (1H, dd, J= 15.5 and 1.5 Hz), 4.23 (1H,
m), 4.19 (2H, m), 3.22 (1H, m), 2.83 (1H, m), 2.62 (1H, m), 1.82
(2H, q, J= 7.5 Hz), 1.27 (3H, t, J= 7.0 Hz), 0.88 (9H, s), 0.03
(3H, s), 0.00 (3H, s). 13C NMR ( 75 MHz, CDCl3) δ 165.89,
146.34, 140.51, 128.58, 128.46, 126.24, 122.95, 73.86, 60.59,
59.21, 34.55, 32.34, 25.69, 18.07, 14.19, -4.40, -4.99 ppm.
HRMS m/z calcd. for C22H33O4NSiNa [M+Na+]: 426.2077,
found: 426.2071.
Ethyl (E)-5-(((S)-1-(benzyloxy)-4-methyl-1-oxopentan-2-
yl)carbamoyl)-4-oxo-7-phenylhept-2-enoate (FGA73).
To an ice-bath cold solution of ethyl (4S,5S,E)-5-(((S)-1-
(benzyloxy)-4-methyl-1-oxopentan-2-yl)carbamoyl)-4-hydroxy-
7-phenylhept-2-enoate (FGA41) ( 310 mg, 0.63 mmol) in
dichloromethane (6.25 mL) was added Dess-Martin periodinane
(517 mg, 1.22 mmol). The resulting mixture was stirred at room
temperature for 1h. Then saturated aqueous solution of sodium
bicarbonate with some sodium thiosulfate crystals (10 mL) was
added and stirred for additional 15 min. The mixture was
extracted with Et2O (3 x 20 mL), dried over MgSO4 and
concentrated under reduce pressure. The product was purified
through chromatography (silica-gel, hexane/ethyl acetate (8:2)) to
afford a white solid.
Ethyl
(4S,5S,E)-5-(((S)-1-(benzyloxy)-4-methyl-1-
oxopentan-2-yl)carbamoyl)-4-hydroxy-7-phenylhept-2-enoate
(FGA41).
(yield = 62%). 1H NMR ( 500 MHz, CDCl3) δ 7.13-6.89 (10H,
m), 6.60 (1H, dd, J= 4.4 and 15.6 Hz), 6.12 (1H, d, J = 8.3 Hz),
5.86 (1H, dd, J= 15.6 and 1.6 Hz), 4.94 (1H, d, J = 12.3 Hz), 4.90
(1H, d, J = 12.3 Hz), 4.44 (1H, td, J = 9.0 and 4.6 Hz), 4.25 (1H,
m), 3.93 (2H, q, J = 7.1 Hz), 2.49-2.43 (1H, m), 2.25-2.31 (1H,
m), 2.16-2.20 (1H, m), 1.80-1.88 (1H, m), 1.51-1.58 (1H, m),
1.46-1.40 (1H, m), 1.35-1.30 (1H, m) 1.03 (3H, t, J= 7.1 Hz),
0.70 (6H, d, J = 4.4 Hz); 13C NMR ( 75 MHz, CDCl3) δ 173.8,
173.0, 166.2, 147.1, 141.0, 135.3, 128.6, 128.5, 128.4, 128.3,
126.2, 122.2, 72.2, 67.3, 60.5, 51.4, 51.2, 40.7, 33.4, 30.7, 25.0,
22.8, 21.6, 14.2 ppm.
(yield = 55%). 1H NMR ( 300 MHz, CDCl3) δ 7.45-7.11 (10H,
m), 6.72 (1H, d, J= 4.5 Hz), 6.60 (1H, d, J= 4.5 Hz), 6.45 (1H,
m), 6.10 (1H, m), 5.72 (1H, d, J= 16.1 Hz), 5.66 (1H, d, J= 16.1
Hz), 4.70 (1H, m), 4.25 (2H, m), 3.70 (1H, m), 2.55-3.25 (3H,
m), 2.20 (1H, m), 1.47-1.75 (3H, m), 1.26 (3H, t, J= 7.1 Hz) 0.82
(6H, m); 13C NMR ( 75 MHz, CDCl3) δ 197.1, 196.7, 172.5,
172.2, 171.8, 167.7, 167.6, 166.4, 165.0, 162.4, 140.2, 137.6,
137.3, 134.3, 132.8, 128.8, 128.7, 128.6, 128.4, 128.3, 126.5
126.4, 125.4, 105.1, 67.3, 67.2, 61.5, 60.8, 58.3, 51.1, 51.0, 41.6,
36.9, 33.2, 28.3, 25.0, 22.8, 22.7, 22.1, 21.8, 14.3, 14.1 ppm.

(2E,4R,5S)-4-(tert-Butyl-dimethyl-silanyloxy)-5-
isocyanato-7-phenyl-hept-2-enoic acid ethyl ester 9.
ammonium fluoride (1M in THF) ( 2.70 mL, 2.70 mmol ). The
resulting mixture was stirred cold with an ice-bath for 5.5h. Then
was concentrated and the resulting crude was purified by
chromatography (Hexanes/AcOEt, (1:1) and (1:2)) to afford a
white solid.
(yield= 60%). [αD20] = -79.56 (c= 10.5, CHCl3). IR (NaCl) ν
3028, 2956, 2931, 2859, 2254, 1817, 1783, 1722, 1660, 1604,
1497, 1472, 1367, 1260, 1175, 1126, 1095, 1036, 1007, 982, 838,
1
779, 749, 700, 671 cm-1. H NMR ( 500 MHz, CDCl3 ) δ 7.13-
(2E,4S,5S)-5-[(2S)-2-Benzyloxycarbonylamino-3-phenyl-
propionylamino]-4-hydroxy-7-phenyl-hept-2-enoic acid ethyl
ester (FGA40).
7.27 (5H, m), 6.86 (1H, dd, J= 5.0 and 15.5 Hz), 5.97 (1H, d, J=
15.5 Hz), 4.54 (1H, t, J= 5.5 Hz), 4.16 (2H, m), 2.75 (1H, m),
2.51-2.60 (2H, m), 2.07 (1H, m), 1.88 (1H, m), 1.26 (3H, t, J=
7.0 Hz), 0.88 (9H, s), 0.03 (3H, s), 0.00 (3H, s). 13C NMR ( 125
MHz, CDCl3) δ 165.89, 146.98, 140.88, 128.55, 128.42, 126.22,
122.59, 72.25, 60.62, 52.56, 33.46, 28.56, 25.77, 18.12, 14.22, -
4.30, -4.50 ppm. HRMS m/z calcd. for C22H33O4NSiNa [M+Na+]:
426.2077, found: 426.1997; m/z calcd. for C22H33O4NSiK
[M+K+]: 442.1816, found: 442.1780.
(yield= 77%). mp= 141-143 ºC. [αD20] = -16.27 (c= 2.7,
CHCl3). IR (NaCl) ν 3438, 3309, 3029, 2927, 1690, 1642, 1532,
1495, 1455, 1385, 1369, 1302, 1258, 1219, 1190, 1135, 1115,
1
1041, 971, 911, 874, 750, 700, 673 cm-1. H NMR ( 300 MHz,
CDCl3 ) δ 7.09-7.35 (15H, m), 6.74 (1H, dd, J= 4.5 and 15.5 Hz),
6.31 (1H, s), 6.04 (1H, dd, J= 2.0 and 15.5 Hz), 5.42 (1H, s), 4.38
(1H, q, J= 7.2Hz), 4.23 (1H, td, J= 1.5 and 4.2 Hz), 4.17 (2H, q,
J= 6.9Hz), 3.89 (1H, m), 3.01 (2H, m), 2.57 (2H, m), 1.93 (1H,
m), 1.75 (1H, m), 1.26 (3H, t, J= 7.0Hz) ppm. 13C NMR (
75MHz, CDCl3 ) δ 171.82, 166.22, 156.10, 146.72, 141.18,
136.26, 136.05, 129.26, 129.04, 128.96, 128.82, 128.56, 128.53,
128.37, 128.27, 128.12, 127.20, 126.10, 122.19, 72.35, 67.25,
60.53, 56.57, 54.08, 38.29, 32.32, 32.25, 14.23 ppm. HRMS m/z
calcd. for C32H36O6N2Na [M+Na+]: 567.2471, found: 567.2482;
m/z calcd. for C32H36O6N2K [M+K+]: 583.2210, found: 583.2203.
General experimental procedure for the peptidic coupling.
To an ice-bath cold solution of the isocianate ( 394 mg, 0.98
mmol ) in CH2Cl2 (6 mL) was added a solution of Cbz-L-Phe (
352 mg, 1.18 mmol) in CH2Cl2 ( 2 mL) and then a solution of 4-
DMAP ( 16 mg, 0.13 mmol) in CH2Cl2 ( 2 mL ). The resulting
mixture was stirred cold with an ice-bath for 1.5 h and then at
room temperature for 6 h. Then quenched with brine, extracted
with CH2Cl2 (3 x 30mL), dried (Na2SO4) and concentrated. The
crude was purified through chromatography (silica-gel,
hexane/EtOAc, (7:3) to afford an orange oil
(2E,4R,5S)-5-[(2S)-2-Benzyloxycarbonylamino-3-phenyl-
propionylamino]-4-hydroxy-7-phenyl-hept-2-enoic acid ethyl
ester (FGA50).
(2E,4S,5S)-5-[(2S)-2-Benzyloxycarbonylamino-3-phenyl-
propionylamino]-4-(tert-butyl-dimethyl-silanyloxy)-7-phenyl-
hept-2-enoic acid ethyl ester 10.
(yield= 78%). 1H NMR ( 500 MHz, CDCl3 ) δ 7.04-7.36
(15H, m), 6.75 (1H, dd, J = 3.5 and 15.5 Hz), 6.00 (1H, dd, J =
2.0 and 15.5 Hz), 5.84 (1H, s), 5.28 (2H, s), 5.07 (2H, s), 4.32
(1H, m), 4.16 (1H, q, J = 7.5 Hz), 4.01 (1H, m), 3.07 (1H, dd, J =
6.0 and 14.0 Hz), 2.99 (1H, dd, J = 8.0 and 13.5 Hz), 2.51 (2H,
m), 1.72 (1H, m), 1.60 (1H, m), 1.24 (1H, t, J = 7.0 Hz). 13C
NMR ( 125 MHz, CDCl3 ) δ 171.7, 166.2, 156.1, 145.6, 141.1,
136.4, 136.0, 129.3, 128.9, 128.6, 128.5, 128.4, 128.2, 127.3,
126.1, 122.5, 73.1, 67.4, 60.6, 56.8, 53.9, 38.5, 32.4, 30.5, 14.2
ppm.
(yield= 79%). [αD20] = -14.31 (c= 1.7, CHCl3). IR (NaCl) ν
3322, 2929, 2858, 1719, 1659, 1535, 1497, 1455, 1366, 1260,
1
1174, 1130, 1031, 837, 778, 746, 698 cm-1. H NMR ( 500MHz,
CDCl3 ) δ 7.07-7.35 (15H, m), 6.53 (1H, dd, J = 4.5 and 15.5 Hz),
5.83 (1H, d, J = 15.5 Hz), 5.21 (1H, s), 5.07 (2H, s), 4.31 (1H, q,
J = 7.0 Hz), 4.17 (3H, m), 3.94 (1H, m), 3.04 (2H, d, J= 7.5 Hz),
2.52 (2H, m), 1.86 (1H, m), 1.45 (1H, m), 1.26 (3H, t, J= 7.0 Hz,
0.86 (9H, s), 0.03 (3H, s), 0.00 (3H, s) ppm. 13C NMR ( 125MHz,
CDCl3 ) δ 170.74, 166.04, 155.94, 146.69, 141.32, 136.36,
136.13, 129.28, 128.84, 128.56, 128.49, 128.44, 128.25, 128.11,
127.25, 126.03, 122.12, 72.22, 67.20, 60.47, 56.62, 53.36, 38.36,
32.49, 31.70, 25.82, 18.08, 14.27, -4.92, -5.15 ppm. HRMS m/z
calcd. for C38H50O6N2SiNa [M+Na+]: 681.3336, found:681.3384.
Ethyl (4R,5S,E)-5-((S)-2-(((benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-4-hydroxyhex-2-enoate (FGA54).
(yield= 69%). 1H NMR ( 500 MHz, CDCl3 ) δ 7.15-7.40
(10H, m), 6.65 (1H, dd, J = 3.5 and 15.5 Hz), 6.48 (1H, m), 6.05
(1H, dd, J = 2.0 and 15.5 Hz), 5.75 (1H, s), 5.12 (1H, d, J = 16.0
Hz), 5.05 (1H, d, J = 16.0 Hz), 4.30 (1H, m), 4.12 (1H, q, J = 7.5
Hz), 4.01 (1H, m), 3.01 (2H, m), 1.21 (1H, t, J = 7.0 Hz), 0.95
(3H, d = 6.5 Hz). 13C NMR ( 125 MHz, CDCl3 ) δ 171.2, 166.2,
156.1, 146.0, 136.4, 136.0, 129.3, 128.7, 128.5, 128.2, 128.0,
127.1, 122.1, 72.8, 67.1, 60.5, 56.6, 49.5, 38.9, 21.0, 14.2 ppm.
HRMS m/z calcd. for C25H31O6N2 [M+H+]: 455.2182, found:
455.2179.
(2E,4R,5S)-5-[(2S)-2-Benzyloxycarbonylamino-3-phenyl-
propionylamino]-4-(tert-butyl-dimethyl-silanyloxy)-7-phenyl-
hept-2-enoic acid ethyl ester 11.
(yield= 63%). [αD20] = -6.3 (c= 10.5, CHCl3). IR (NaCl) ν
3303, 3064, 3030, 2955, 2930, 2858, 1720, 1659, 1538, 1497,
1472, 1455, 1390, 1368, 1260, 1174, 1132, 1107, 1040, 981, 838,
1
778, 747, 699, 671 cm-1. H NMR ( 500 MHz, CDCl3 ) δ 7.08-
7.41 (15H, m), 6.87 (1H, dd, J = 4.0 and 16.0 Hz ), 6.19 (1H, dd,
J = 1.5 and 16.0 Hz ), 5.24 (1H, s), 5.03 (2H, s), 4.36 (1H, m),
4.20 (2H, q, J = 7.0 Hz), 4.07 (1H, m), 3.13 (1H, dd, J = 6.0 and
14.0 Hz), 3.04 (1H, m), 2.55 (2H, m), 1.82 (1H, m), 1.70 (1H,
m), 1.27 (3H, t, J = 7.0 Hz), 0.89 (9H, s), 0.06 (3H, s), 0.01 (3H,
s). 13C NMR ( 125 MHz, CDCl3 ) δ 170.48, 166.00, 156.10,
146.97, 141.64, 136.43, 135.94, 129.43, 128.73, 128.47, 128.41,
128.31, 128.27, 128.21, 128.10, 127.05, 125.99, 122.38, 73.80,
67.21, 60.52, 56.30, 53.67, 37.49, 32.39, 30.08, 25.78, 18.08,
14.21, -4.57, -5.13 ppm
Ethyl
(4R,5S,E)-4-hydroxy-5-((S)-2-(morpholine-4-
carboxamido)-3-phenylpropanamido)-7-phenylhept-2-enoate
(FGA 67).
(yield= 99%). 1H NMR ( 500 MHz, CDCl3 ) δ 7.05-7.45
(10H, m), 6.80 (1H, dd, J = 3.5 and 15.5 Hz), 6.10 (1H, dd, J =
2.0 and 15.5 Hz), 5.68 (1H, s), 4.72 (1H, m), 4.27 (1H, s), 4.30
(2H, q, J = 7.5 Hz), 4.01 (1H, m), 3.10-3.40 (6H, m), 2.55 (1H,
m), 2.47 (1H, m), 1.70-1.55 (2H, m), 1.25 (1H, t, J = 7.0 Hz). 13
C
NMR ( 75 MHz, CDCl3 ) δ 173.1, 166.2, 157.2, 146.3, 141.2,
137.0, 129.3, 128.4, 128.2, 128.2, 126.8, 125.8, 121.9, 73.0, 66.2,
60.3, 56.1, 53.8, 43.9, 38.6, 32.3, 30.5, 14.1 ppm.
General experimental procedure for the deprotection.
To an ice-bath cold solution of silylated peptide ( 419 mg,
0.77 mmol ) in THF ( 8 mL ), was added tetra-n-butyl

Ethyl
(4R,5S,E)-5-((S)-2-(((benzyloxy)carbonyl)amino)-4-
Benzyl ((S)-1-(((3S,4R,E)-7-(benzylamino)-4-hydroxy-7-oxo-
1-phenylhept-5-en-3-yl)amino)-1-oxo-3-phenylpropan-2-
yl)carbamate (FGA71)
methylpentanamido)-4-hydroxy-7-phenylhept-2-enoate
(FGA58).
(yield = 84%). 1H NMR (CDCl3, 500 MHz) δ 7.21-7.45 (m,
10H), 6.90 (1H, dd, J = 3.0 and 15.5 Hz), 6.65 (1H, m), 6.15 (1H,
dd, J = 2.5 and 15.5 Hz), 5.32 (m, 1H), 5.10 (m, 2H), 4.30 (m,
1H), 4.21 (2H, q, J = 7.5 Hz), 3.75 (m, 1H), 2.60 (m, 2H), 1.75
(m, 1H), 1.60 (m, 1H), 1.45 (m, 1H), 1.25 (t, J = 7.5 Hz, 3H),
0.90 (m, 6H) ppm; 13C NMR (CDCl3, 125 MHz) δ 173.5, 166.4,
161.4, 156.5, 146.0, 141.1, 136.2, 128.6, 128.1, 126.1, 122.5,
73.7, 67.2, 60.5, 54.3, 53.9, 41.2, 32.3, 31.3, 25.6, 24.5, 22.7,
14.2 ppm. HRMS m/z calcd. for C29H39O6N2 [M+H+]: 511.2803,
found: 511.2801.
To an ice-bath cold solution of the (4R,5S,E)-5-((S)-2-
(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-4-
hydroxy-7-phenylhept-2-enoic acid (FGA57) ( 49 mg, 0.095
mmol ) in a mixture of dry CH2Cl2 (2 mL) and DMF ( 1 mL) was
added subsequently HOBT (13 mg, 1eq), EDCI hydrochloride
(18 mg, 1 eq), NMM (47 µL, 4.5 eq) and benzylamine (12 µL,
1.2 eq). The resulting mixture was stirred cold with an ice-bath
for 1.5 h and then at room temperature for 6 h. Then quenched
with brine, extracted with EtOAc(3 x 30mL), dried (Na2SO4) and
concentrated to afford the product.
Ethyl (S,E)-5-((S)-2-(((benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-4-oxohex-2-enoate (FGA55).
(yield= 75%). 1H NMR ( 300 MHz, CDCl3 ) δ 8.48 (1H, t, J =
4 Hz), 7.90 (1H, d, J = 8 Hz), 7.50 (1H, d, J = 7.9 Hz), 7.10-7.35
(20H, m), 6.68 (1H, dd, J = 3.5 and 15.5 Hz), 6.12 (1H, dd, J =
2.0 and 15.5 Hz), 5.20 (1H, m), 4.95 (2H, s), 4.32 (3H, m), 4.12
(1H, m), 3.75 (1H, m), 2.45-2.77 (2H, m), 1.82 (1H, m), 1.65
(1H, m). 13C NMR ( 75 MHz, CDCl3 ) δ 172.0, 165.1, 156.4,
143.6, 142.6, 139.9, 138.8, 137.5, 129.7, 128.8, 128.7, 128.4,
128.1, 127.9, 127.6, 127.1, 126.6, 126.1, 124.7, 72.8, 65.6, 53.0,
42.5, 37.9, 32.3, 32.0 ppm. HRMS m/z calcd. for C37H39O5N3Na
[M+Na+]: 628.2787, found: 628.2775.
1
(yield= 43%). H NMR ( 300 MHz, CDCl3 ) δ 7.20-7.45 (10H,
m), 7.20 (1H, d, J = 15.5 Hz), 6.80 (1H, d, J = 15.5 Hz), 6.65
(1H, m), 5.35 (1H, m), 5.15 (2H, s), 4.74 (1H, m), 4.48 (1H, m),
4.25 (2H, q, J = 7.5 Hz), 3.05 (3H, m), 1.22 (1H, t, J = 7.0 Hz),
1.20 (3H, d = 6.5 Hz). 13C NMR ( 75 MHz, CDCl3 ) δ 196.7,
170.6, 165.0, 155.9, 136.1, 135.6, 133.0, 129.6, 129.3, 129.2,
128.9, 128.8, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.4,
127.2, 76.6, 67.2, 61.6, 56.2, 53.3, 45.6, 38.6, 17.3, 14.1 ppm.
HRMS m/z calcd. for C25H29O6N2 [M+H+]: 453.2026, found:
453.2025.
General experimental procedure for the preparation of
phosphonates
Ethyl (S,E)-5-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-oxohex-2-enoate (FGA56).
To a -70 ºC cold solution of dimethyl methylphosphonate
(2.08 mL, 19.17 mmol) in THF (20 mL) was added n-butyl
lithium (1.6 M in hexanes) (1.98 mL, 19.17 mmol). The resulting
mixture was stirred cooled at -70 ºC for 15 min and then a
solution of the corresponding diprotected dipeptide (already
prepared by standard procedures) (2.40 mmol) in THF (20 mL)
was added dropwise. The resulting mixture was stirred at -70 ºC
for 2 h and then was quenched with 10% acetic acid (20 mL) and
extracted with AcOEt (3 x 30 mL), the organic layers were
washed (saturated sodium bicarbonate solution), dried (Na2SO4)
and concentrated. The crude oil was directed submitted to the
next step without any further purification.
1
(yield= 87%). H NMR ( 300 MHz, CDCl3 ) δ 7.30-7.45 (5H,
m), 7.12 (1H, d, J = 15.5 Hz), 6.80 (1H, d, J = 15.5 Hz), 6.70
(1H, m), 5.20 (2H, s), 4.25 (2H, q, J = 7.5 Hz), 4.20 (1H. m),
1.35-1.60 (3H, m), 1.25 (1H, t, J = 7.5 Hz), 0.90 (6H, m). 13C
NMR ( 75 MHz, CDCl3 ) δ 197.2, 171.8, 165.0, 156.4, 136.2,
135.7, 133.0, 128.6, 128.3, 128.1, 76.6, 67.2, 61.6, 53.4, 41.5,
24.7, 22.9, 22.0, 17.3, 14.2 ppm. HRMS m/z calcd. for
C22H31O6N2 [M+H+]: 419.2177, found: 419.2180.
(4R,5S,E)-5-((S)-2-(((benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-4-hydroxy-7-phenylhept-2-enoic acid
(FGA57)
Spectroscopic data for phosphonates:
To an ice-bath cold solution of (2E,4R,5S)-5-[(2S)-2-
benzyloxycarbonylamino-3-phenyl-propionylamino]-4-hydroxy-
7-phenyl-hept-2-enoic acid ethyl ester (FGA50) (65 mg, 0.12
mmol) in a mixture of THF:H2O (5 mL) was added KOH 1M in
EtOH (240 µL, 0.24 mmol). The mixture was stirred for 2h at
0ºC and overnight at room temperature. The THF was removed
under reduced pressure and 5 mL of cold water were added.
KHSO4 1M aqueous solution was added until pH = 1 and the
mixture was extracted using EtAcO. The organic phase was dried
over MgSO4 and concentrated. The product was recrystallized
from a mixture of EtAcO-hexane.
Benzyl ((S)-1-(((S)-1-(dimethoxyphosphoryl)-2-oxo-5-
phenylpentan-3-yl)amino)-1-oxo-3-phenylpropan-2-
1
yl)carbamate 12. (yield = 98%). H NMR (CDCl3, 300 MHz) δ
7.48 (d, J = 7.8 Hz, 1H), 7.27-7.08 (m, 15H), 5.68 (d, J = 7.2 Hz,
1H), 5.07 (d, J = 12.6 Hz, 1H), 5.01 (d, J = 12.3 Hz, 1H), 4.63-
4.53 (m, 2H), 3.71 (s, 3H), 3.67 (s, 3H), 3.18-2.91 (m, 2H), 2.53
(t, J = 7.8 Hz, 2H), 2.24-2.13 (m, 1H), 1.89-1.80 (m, 1H) ppm;
13C NMR (CDCl3, 75 MHz) δ 200.3, 170.9, 156.1, 140.7, 136.2,
129.2, 128.6, 128.4, 128.0, 127.9, 126.9, 126.0, 66.9, 58.7, 56.2,
53.1, 53.0, 38.6, 36.9, 31.9, 31.5 ppm.
1
(yield= 92%). H NMR ( 500 MHz, CDCl3 ) δ 7.12-7.27
Benzyl ((S)-1-(((S)-1-(dimethoxyphosphoryl)-2-oxo-5-
phenylpentan-3-yl)amino)-4-methyl-1-oxopentan-2-
(15H, m), 6.81 (1H, dd, J = 3.5 and 15.5 Hz), 6.05 (1H, dd, J =
2.0 and 15.5 Hz), 5.12 (2H, s), 4.45 (1H, m), 4.12 (1H, m), 3.85
(1H, m), 3.13 (1H, dd, J = 6.0 and 14.0 Hz), 2.92 (1H, dd, J = 8.0
and 13.5 Hz), 2.65 (1H, m), 2.47 (1H, m), 1.83 (1H, m), 1.75
(1H, m). 13C NMR ( 125 MHz, CDCl3 ) δ 172.8, 172.4, 168.2,
156.8, 147.9, 141.8, 137.2, 136.8, 128.9, 128.2, 128.1, 128.0,
127.9, 127.8, 127.5, 127.3, 126.4, 125.4, 121.8, 72.7, 66.1, 56.6,
53.0, 37.7, 31.7, 31.1 ppm. HRMS m/z calcd. for C30H33O6N2
[M+H+]: 517.2339, found: 517.2338.
1
yl)carbamate 13. (yield = 89%). H NMR (CDCl3, 500 MHz) δ
7.38-7.13 (m, 10H), 5.47 (d, J = 7.0 Hz, 1H), 5.11 (d, J = 12.0
Hz, 1H), 5.07 (d, J = 12.5 Hz, 1H), 4.61 (dq, J = 8.5, 4.5 Hz, 1H),
4.25 (m, 1H), 3.71 (s, 3H), 3.69 (s, 3H), 3.28 (dd, J = 22.6, 14.1
Hz, 1H), 3.05 (dd, J = 21.9, 14.1 Hz, 1H), 2.60 (m, 2H), 2.23 (m,
1H), 1.92 (m, 1H), 1.69 (m, 2H), 1.52 (m, 1H), 0.93 (m, 6H)
ppm; 13C NMR (CDCl3, 125 MHz) δ 200.8, 172.9, 156.0, 140.6,
136.1, 128.4, 128.0, 127.9, 126.0, 66.8, 58.5, 53.8, 52.1, 52.0,
41.3, 38.6, 36.9, 31.8, 31.6, 24.7, 23.1 ppm.

Benzyl ((S)-1-(((S)-1-(dimethoxyphosphoryl)-5-methyl-2-
oxohexan-3-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate
14. (yield = 92%). H NMR (CDCl3, 500 MHz) δ 7.35-7.17 (m,
HRMS m/z calcd. for C28H35O6N2Na [M+Na+]: 517.2315, found:
517.2317.
1
(S,E)-Ethyl
5-((S)-2-(((benzyloxy)carbonyl)amino)-3-
10H), 5.46 (d, J = 7.5 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 5.04 (d,
J = 12.5 Hz, 1H), 4.62 (dt, J = 10.0, 4.0 Hz, 1H), 4.50 (m, 1H),
3.74 (d, J = 6.5 Hz, 3H), 3.72 (d, J = 6.5 Hz, 3H), 3.22-3.06 (m,
3H), 2.97 (dd, J = 22.0, 14.0 Hz, 1H), 1.62 (ddd, J = 13.6, 9.4, 4.1
Hz, 1H), 1.38-1.53 (m, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.85 (d, J =
6.5 Hz, 3H) ppm; 13C NMR (CDCl3, 125 MHz) δ 201.4, 171.0,
155.8, 136.1, 129.4, 128.7, 128.6, 128.4, 128.0, 127.1, 67.0, 57.5,
56.2, 53.1, 52.9, 39.6, 38.6, 38.0, 24.6, 23.1, 21.3 ppm.
phenylpropanamido)-7-methyl-4-oxooct-2-enoate (FGA69).
Yellow oil. [α]D = -28.1 (c= 1.2, CHCl3). IR (NaCl) ν 3417,
20
3020, 2961, 1720, 1498, 1386, 1302, 1216, 1027, 910, 771, 762,
738, 669 cm-1. 1H NMR (CDCl3, 500 MHz) δ 7.39-7.20 (m,
10H), 6.43 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 12.0 Hz, 1H), 6.00 (d,
J = 12.0 Hz, 2H), 5.19 (br s, 1H), 5.10 (s, 2H), 4.77 (td, J = 10.0,
4.0 Hz, 1H), 4.47 (q, J = 6.5 Hz, 1H), 4.20 (m, 2H), 3.12 (m,
2H), 1.76 (m, 1H), 1.55 (m, 1H), 1.46 (m, 1H), 1.29 (t, J = 7.5
Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H) ppm;
13C NMR (CDCl3, 125 MHz) δ 197.2, 170.5, 165.1, 155.6, 139.5,
136.0, 132.5, 129.4, 129.2, 128.9, 128.5, 128.2, 128.0, 126.8,
67.3, 61.6, 56.7, 56.1, 40.4, 38.3, 24.9, 23.2, 21.8, 14.1 ppm.
HRMS m/z calcd. for C28H35O6N2Na [M+Na+]: 517.2315, found:
517.2317.
General experimental procedure for the preparation of
dipeptidyl enoates
To
a stirred solution of the corresponding dipeptidyl
phosphonate (1 mmol) in ethanol (7.5 mL) was added potassium
carbonate (1 mmol) and ethyl glyoxalate (1 mmol). The resulting
mixture was stirred at room temperature for 2 h and then was
filtered off, neutralized using acetic acid and concentrated. The
crude oil was purified through silica gel chromatography
(hexanes/ethyl acetate, 7/3).
Ethyl (R,E)-5-((S)-2-(morpholine-4-carboxamido)-3-
phenylpropanamido)-4-oxo-7-phenylhept-2-enoate (FGA 74).
(yield= 34%). 1H NMR ( 500 MHz, CDCl3 ) δ 7.00-7.41
(10H, m), 6.88 (1H, d, J = 16.0 Hz), 6.00 (1H, d, J = 16.0 Hz),
5.20 (1H, m), 4.70-4.81 (2H, m), 4.51 (1H, m), 4.40 (2H, q, J =
7.5 Hz), 3.70 (5H, m), 3.30 (5H, m), 3.20 (2H, m), 2.56 (2H, m),
2.32 (1H, m), 1.80 (1H, m), 1.26 (3H, t, J = 7.0 Hz). 13C NMR (
125 MHz, CDCl3 ) δ 200.8, 172.2, 165.6, 157.1, 141.2, 136.6,
129.8, 128.4, 127.1, 126.3, 66.5, 61.4, 57.8, 55.6, 44.0, 38.5,
32.6, 31.4, 14.0 ppm. HRMS m/z calcd. for C29H35O6N3Na
[M+Na+]: 544.2424, found: 544.2425.
Spectroscopic data for dipeptidyl enoates:
(S,E)-ethyl
5-((S)-2-(((benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-4-oxo-7-phenylhept-2-enoate (FGA44).
(yield = 76%). White solid (mp 115-119 ºC). [α]D20 -12.0 (c= 0.1,
CHCl3). IR (NaCl) ν 3619, 3019, 2896, 2399, 1716, 1518, 1385,
1213, 1046, 928, 746, 734, 669, 627 cm-1. 1H NMR (CDCl3,
500MHz) δ 7.35-7.06 (m, 16H), 6.72 (d, J = 16.0 Hz, 1H), 6.50
(br s, 1H), 5.21 (br s, 1H), 5.13 (d, J = 12.5 Hz, 2H), 5.09 (d, J =
12.0 Hz, 2H), 4.84 (q, J = 7.0 Hz, 1H), 4.44 (m, 1H), 4.28 (q, J =
7.5 Hz, 2H), 3.07 (m, 2H), 2.45-2.58 (m, 2H), 2.18-2.25 (m, 1H),
1.83-1.90 (m, 1H), 1.34 (t, J = 7.5 Hz, 3H) ppm; 13C NMR
(CDCl3, 125 MHz) δ 196.3, 170.7, 165.0, 155.9, 140.3, 136.2,
135.8, 132.7, 129.3, 128.7, 128.6, 128.5, 128.4, 128.2, 128.0,
127.1, 126.4, 67.2, 61.7, 56.9, 56.2, 38.4, 32.7, 31.2, 14.1 ppm.
HRMS m/z calcd. for C32H35O6N2 [M+H+]: 543.2495, found:
543.2496.
Ethyl (S,E)-5-((S)-2-(morpholine-4-carboxamido)-3-
phenylpropanamido)-4-oxo-7-phenylhept-2-enoate (FGA75).
1
(yield= 78%). H NMR ( 500 MHz, CDCl3 ) δ 7.12-7.37
(10H, m), 7.26 (1H, d, J = 16.0 Hz), 6.45 (1H, d, J = 16.0 Hz),
5.20 (1H, m), 4.70-4.81 (2H, m), 4.51 (1H, m), 4.50 (2H, q, J =
7.5 Hz), 3.65 (5H, m), 3.30 (5H, m), 3.20 (2H, m), 2.62 (2H, m),
2.20 (1H, m), 1.80 (1H, m), 1.27 (3H, t, J = 7.0 Hz). 13C NMR (
125 MHz, CDCl3 ) δ 209.5, 196.5, 171.9, 164.8, 156.9, 141.8,
140.4, 136.7, 136.0, 132.5, 129.3, 128.7, 128.6, 128.5, 128.4,
127.0, 126.3, 66.3, 61.2, 57.0, 55.8, 43.7, 38.7, 33.0, 31.5, 13.8
ppm. HRMS m/z calcd. for C29H35O6N3Na [M+Na+]: 544.2424,
found: 544.2426.
(S,E)-ethyl
5-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-oxo-7-phenylhept-2-enoate (FGA47).
(yield = 80%). Colourless oil. [α]D20 = -14.5 (c= 1.1, CHCl3). IR
(NaCl) ν 3404, 3064, 2961, 1736, 1679, 1518, 1455, 1369, 1247,
1188, 1029, 979, 764, 726, 717 cm-1. 1H NMR (CDCl3, 300
MHz) δ 7.33-7.07 (m, 11H), 6.81 (m, 1H), 6.73 (d, J = 15.9 Hz,
1H), 5.13 (s, 2H), 4.84-4.90 (m, 1H), 4.26 (q, J = 6.6 Hz, 2H),
4.18 (m, 1H), 2.60 (m, 1H), 2.24 (m, 1H), 1.92 (m, 1H), 1.68 (m,
2H), 1.51 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 0.94 (m, 6H) ppm;
13C NMR (CDCl3, 75 MHz) δ 196.9, 172.0, 165.2, 156.2, 140.4,
135.8, 132.8, 128.6, 128.6, 128.5, 128.3, 128.1, 128.1, 126.4,
67.2, 61.6, 57.1, 53.6, 41.2, 32.8, 31.3, 24.7, 22.9, 22.0, 14.1
ppm. HRMS m/z calcd. for C29H37O6N2 [M+H+]: 509.2652,
found: 509.2657.
Ethyl (S,E)-7-methyl-5-((S)-4-methyl-2-(morpholine-4-
carboxamido)pentanamido)-4-oxooct-2-enoate (FGA76).
(yield= 43%). 1H NMR ( 500 MHz, CDCl3 ) δ 7.24 (1H, d, J
= 16.0 Hz), 6.90 (1H, m), 6.80 (1H, d, J = 16.0 Hz), 5.05 (1H,
m), 4.78 (1H, m), 4.45 (1H, m), 4.54 (2H, q, J = 7.5 Hz), 4.48
(1H, m), 3.72 (5H, m), 3.32 (5H, m), 1.30-1.75 (7H, m), 1.30
(3H, t, J = 7.0 Hz), 1.25 (2H, m), 0.75 (12H, m). 13C NMR ( 125
MHz, CDCl3 ) δ 198.0, 173.2, 165.3, 157.8, 136.3, 132.1, 66.4,
61.6, 56.1, 52.6, 44.1, 42.0, 40.4, 24.9, 23.2, 22.8, 22.4, 21.9,
14.2 ppm. HRMS m/z calcd. for C22H37O6N3Na [M+Na+]:
462.2580, found: 462.2574.
(R,E)-Ethyl
5-((S)-2-(((benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-7-methyl-4-oxooct-2-enoate (FGA70).
20
(yield = 84 % for 6 and 7) White solid (mp 71-75 ºC). [α]D
=
Ethyl
(S,E)-5-((S)-4-methyl-2-(morpholine-4-
+8.0 (c= 1.0, CHCl3). IR (NaCl) ν 3618, 3019, 2896, 2436, 2399,
carboxamido)pentanamido)-4-oxo-7-phenylhept-2-enoate
(FGA77).
1717, 1519, 1369, 1211, 1046, 929, 793, 765, 755, 747, 731, 719,
1
625 cm-1. H NMR (CDCl3, 500 MHz) δ 7.36-7.12 (m, 10H),
(yield= 78%). 1H NMR ( 500 MHz, CDCl3 ) δ 7.25-7.40 (6H,
m), 6.70 (1H, d, J = 16.0 Hz), 5.20 (1H, m), 4.75 (1H, m), 4.40
(1H, m), 4.20 (2H, m), 3.72 (5H, m), 3.30 (5H, m), 2.60 (2H, m),
2.20 (1H, m), 1.71 (1H, m), 1.60 (3H, m), 1.32 (3H, t, J = 7.0
Hz), 0.75 (6H, m). 13C NMR ( 125 MHz, CDCl3 ) δ 197.1, 173.8,
173.7, 165.1, 165.0, 157.5, 140.4, 136.1, 132.3, 132.2, 128.5,
128.4, 126.3, 66.4, 61.4, 57.1, 53.0, 44.1, 41.3, 32.5, 31.4, 24.8,
6.80 (d, J = 16.0 Hz, 1H), 6.61 (br s, 1H), 5.49 (d, J = 8.0 Hz,
1H), 5.08 (s, 2H), 4.83 (td, J = 9.0, 3.5 Hz, 1H), 4.52 (m, 1H),
4.28 (q, J = 7.0 Hz, 2H), 3.07 (m, 2H), 1.55 (m, 2H), 1.34 (t, J =
7.0 Hz, 3H), 1.30 (m, 1H), 0.93 (d, J = 5.5 Hz, 3H), 0.86 (d, J =
6.0 Hz, 3H) ppm; 13C NMR (CDCl3, 125 MHz) δ 197.1, 170.6,
165.0, 155.8, 136.2, 136.1, 132.5, 129.3, 128.6, 128.1, 127.1,
67.1, 61.5, 56.9, 56.7, 40.2, 38.2, 24.7, 23.0, 21.7, 14.1 ppm.

lyse the erythrocytes and release the malaria parasites. To
eliminate all traces of hemoglobin, the pellet was washed by the
addition of 200 µL of PBS followed by centrifugation at 600 × g.
The washing step was repeated twice to ensure complete removal
of hemoglobin. Finally, pellets were resuspended in 100 µL of
PBS. A 100-µL volume of PicoGreen diluted in TE was added to
each well. Plates were incubated for 30 to 60 min in the dark, and
the fluorescence intensity was measured at 485-nm excitation and
528-nm emission. Growth inhibition defining the half maximal
inhibitory concentration (IC50) was calculated as previously
described.23 Upon treatment, the parasite morphology was
evaluated by microscopic analysis of thin blood smears stained
with Wright's stain. Smears from drug-free cultures were used as
a control.
22.8, 22.3, 14.1 ppm. HRMS m/z calcd. for C26H37O6N2Na
[M+Na+]: 510.2581, found: 510.2580.
Ethyl (S,2E,4E)-7-((S)-2-(morpholine-4-carboxamido)-3-
phenylpropanamido)-6-oxo-9-phenylnona-2,4-dienoate
(FGA78).
To a stirred solution of the corresponding dimethyl ((3R,6S)-
6-(morpholine-4-carboxamido)-2,5-dioxo-3-phenethyl-7-
phenylheptyl)phosphonate (0.67 mmol) in ethanol (7.5 mL) was
added potassium carbonate (0.67 mmol) and ethyl (E)-4-oxobut-
2-enoate (341 mg, 2.66 mmol) solved in EtOH (10 mL). The
resulting mixture was stirred at room temperature for 2 h and
then was filtered off, neutralized using acetic acid and
concentrated. The crude oil was purified through silica gel
chromatography using ethyl acetate to afford 151 mg of the
desired product.
In vitro cell viability assay
HEPG2 human hepatoma cells were cultured 24 h in triplicate in
96-well flat-bottomed microplates in DMEM Glutamax (GIBCO)
growth medium in a final volume of 100 µL per well. Cells
grown in the presence of medium alone were used as controls. To
evaluate the potential cytotoxic effect of the G6PD inhibitors,
one set of plates received serial dilutions from 0.2 to 2 mM of
each inhibitor. Cells were seeded during 24 h and 48 h in
triplicate in 96-well flat-bottomed microplates at a density of 5 ×
103 cells per well in 100 µL growth medium. Treatment and
control in growth medium was performed after 24 h and during
further 24 h incubation in a final volume of 200 µL (100 µL cells
+ 100 µL medium with or without treatment). Following
treatment, cell viability was determined by means of the
colorimetric EZ4U (Biomedica, Vienna, Austria) according to the
manufacturer instructions, and absorbance at 490/610 nm was
measured spectrophotometrically in a Cary 50 BIO Microplate
Reader 50MPR (Varian).
1
(yield= 41%). H NMR ( 500 MHz, CDCl3 ) δ 7.12-7.37
(12H, m), 7.26 (1H, dd, J = 16.0, 15.0 Hz), 6.45 (1H, dd, J =
16.0, 15.0 Hz), 5.20 (1H, m), 4.60-4.76 (2H, m), 4.40 (1H, m),
4.24 (2H, q, J = 7.5 Hz), 3.82 (4H, m), 3.30 (4H, m), 3.22 (2H,
m), 2.50 (2H, m), 2.25 (2H, m), 1.27 (3H, t, J = 7.0 Hz). 13C
NMR ( 125 MHz, CDCl3 ) δ 196.7, 180.3, 171.7, 165.6, 157.1,
140.8, 140.6, 140.3, 136.9, 136.7, 131.8, 130.0, 129.3, 128.7,
128.6, 128.5, 128.4, 127.0, 126.2, 66.3, 60.9, 56.4, 55.4, 38.6,
33.1, 14.1 ppm. HRMS m/z calcd. for C31H36O6N3Na [M+Na+]:
570.2580, found: 570.2579.
4.2. Biological evaluation
4.2.1 Enzyme assays
Activity against Trypanosoma brucei rhodesiense STIB900.
This stock was isolated in 1982 from a human patient in
Tanzania and after several mouse passages cloned and adapted to
axenic culture conditions28 Minimum Essential Medium (50 µl)
supplemented with 25 mM HEPES, 1g/l additional glucose, 1%
MEM non-essential amino acids (100x), 0.2 mM 2-
mercaptoethanol, 1mM Na-pyruvate and 15% heat inactivated
horse serum was added to each well of a 96-well microtiter plate.
Serial drug dilutions of eleven 3-fold dilution steps covering a
range from 100 to 0.002 µg/ml were prepared. Then 4x103
bloodstream forms of T. b. rhodesiense STIB 900 in 50 µl was
added to each well and the plate incubated at 37 °C under a 5 %
CO2 atmosphere for 70 h. 10 µl Alamar Blue (resazurin, 12.5 mg
in 100 ml double-distilled water) was then added to each well and
incubation continued for a further 2–4 h.29 Then the plates were
read with a Spectramax Gemini XS microplate fluorometer
(Molecular Devices Cooperation, Sunnyvale, CA, USA) using an
excitation wave length of 536 nm and an emission wave length of
588 nm. The IC50 values were calculated by linear regression30
from the sigmoidal dose inhibition curves using SoftmaxPro
software (Molecular Devices Cooperation, Sunnyvale, CA,
USA). Melarsoprol (Arsobal Sanofi-Aventis, received from
WHO) is used as control.
The inhibitory activity was tested as reported previously.8,15-24
Dilution assays were performed as described previously.18
4.2.2 Protozoa assays
Antimalarial drug activity assay
P. falciparum strain Dd2 (clone MRA-150; Malaria Research and
Reference Reagent Resource Center: http://www.mr4.org) was
maintained in continuous culture following the protocol
previously described.22 The culture media consisted of standard
RPMI 1640 (Sigma-Aldrich) supplemented with 0.5 % Albumax
I (Gibco), 100 µM hypoxanthine (Sigma-Aldrich), 25 mM
HEPES (Sigma-Aldrich), 12.5 µg/mL gentamicine (Sigma-
Aldrich), and 25 mM NaHCO3 (Sigma-Aldrich), and incubated in
5% CO2 at 37 ºC in tissue culture flasks (Iwaki). Growth progress
was monitored by microscopy in thin blood smears stained with
Wright's eosin methylene blue solution (Merck), using the freely
available Plasmoscore software26 to determine parasitaemia
values. A detailed description of P. falciparum culture and
synchronization methods used in this work has been reported
previously.25
Drug activity was assayed by the PicoGreen microfluorimetric
DNA-based assay monitoring parasite growth inhibition at
different drug concentrations.27 PicoGreen (P7589) was
purchased from Invitrogen and diluted as indicated by the
manufacturer in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH
7.5). Synchronized rings from stock cultures were used to test
serial dilutions of the inhibitors in 96-well culture microplates.
Thus, 150 µL of parasites at 2% hematocrit and 1% parasitemia
were allowed to grow for 48 h in 5% CO2 at 37 ºC. The parasites
were then centrifuged at 600×g for 10 min and resuspended in
saponin (0.15%, wt/vol, in phosphate-buffered saline (PBS)) to
Activity against T. cruzi.
Rat skeletal myoblasts (L-6 cells) were seeded in 96-well
microtitre plates at 2000 cells/well in 100 µL RPMI 1640
medium with 10% FBS and 2 mM l-glutamine. After 24 h the
medium was removed and replaced by 100 µl per well containing
5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4
containing the β-galactosidase (Lac Z) gene.31 After 48 h the
medium was removed from the wells and replaced by 100 µl

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fresh medium with or without a serial drug dilution of eleven 3-
fold dilution steps covering a range from 100 to 0.002 µg/ml.
After 96 h of incubation the plates were inspected under an
inverted microscope to assure growth of the controls and sterility.
Then the substrate CPRG/Nonidet (50 µl) was added to all wells.
A color reaction developed within 2–6 h and could be read
photometrically at 540 nm. Data were analyzed with the graphic
programme Softmax Pro (Molecular Devices), which calculated
IC50 values by linear regression30 from the sigmoidal dose
inhibition curves. Benznidazole is used as control (IC50
0.5 0.2µg/ml).
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Amastigotes of L. donovani strain MHOM/ET/67/L82 were
grown in axenic culture at 37 °C in SM medium32 at pH 5.4
supplemented with 10% heat-inactivated fetal bovine serum
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of culture medium with 105 amastigotes from axenic culture with
or without a serial drug dilution were seeded in 96-well
microtitre plates. Serial drug dilutions of eleven 3-fold dilution
steps covering a range from 100 to 0.002 µg/ml were prepared.
After 70 h of incubation the plates were inspected under an
inverted microscope to assure growth of the controls and sterile
conditions. 10 µl of Alamar Blue (12.5 mg resazurin dissolved in
100 ml distilled water)33 were then added to each well and the
plates incubated for another 2 h. Then the plates were read with a
Spectramax Gemini XS microplate fluorometer (Molecular
Devices Cooperation, Sunnyvale, CA, USA) using an excitation
wave length of 536 nm and an emission wave length of 588 nm.
Data were analyzed using the software Softmax Pro (Molecular
Devices Cooperation, Sunnyvale, CA, USA). Decrease of
fluorescence (= inhibition) was expressed as percentage of the
fluorescence of control cultures and plotted against the drug
concentrations. From the sigmoidal inhibition curves the IC50
values were calculated.
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Acknowledgments
This work was financed by Generalitat Valenciana
(AICO/2016/32) and MINECO (BIO2016-77430R). We also
thank Serveis Centrals d’Instrumentació Científica from
Universitat Jaume I for technical support. The authors thank
Sabine Maehrlein, Nicole Denk and Ulrike Nowe for
performing the enzyme assays, Susana Pérez-Benavente for
technical assistance in cytotoxicity and antimalarial assays,
Dr. Jochen Kesselring for performing the dialysis and dilution
assays, and Patrick Johé for expression and purification of
enzymes.
S.R. thanks the Generalitat Valenciana for a postdoctoral
research grant under theVALi + d Program and the UJI for a
postdoctoral researcher position.
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Supplementary Material