Towards the synthesis of bisubstrate inhibitors of protein farnesyltransferase: Synthesis and biological evaluation of new farnesylpyrophosphate analogues

Article abstract of DOI:10.1016/j.bmc.2009.12.017

Protein farnesyltransferase (FTase) has recently appeared as a new target of parasitic diseases, a field poor in drugs in development. With the aim of creating new bisubstrate inhibitors of FTase, new farnesyl pyrophosphate analogues have been studied. Fa

Full text of DOI:10.1016/j.bmc.2009.12.017

Bioorganic & Medicinal Chemistry 18 (2010) 543–556
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Towards the synthesis of bisubstrate inhibitors of protein farnesyltransferase:
Synthesis and biological evaluation of new farnesylpyrophosphate analogues
Stéphanie Duez a, Laëtitia Coudray a, Elisabeth Mouray b, Philippe Grellier b, Joëlle Dubois a,
*
a Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif-sur-Yvette, Avenue de la Terrasse 91198 Gif-sur-Yvette Cedex, France
b Muséum National d’Histoire Naturelle, FRE 3206 CNRS/MNHN, Case 52, 57 Rue Cuvier 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein farnesyltransferase (FTase) has recently appeared as a new target of parasitic diseases, a field poor
in drugs in development. With the aim of creating new bisubstrate inhibitors of FTase, new farnesyl pyro-
phosphate analogues have been studied. Farnesyl analogues with a malonic acid function exhibited the
best inhibitory activity on FTase. This group was introduced into our imidazole-containing model leading
to new compounds with submicromolar activities. Kinetic experiments have been realized to determine
their binding mode to the enzyme.
Received 4 September 2009
Accepted 4 December 2009
Available online 11 December 2009
Keywords:
Farnesyltransferase inhibitors
Imidazole
Ó 2009 Elsevier Ltd. All rights reserved.
FPP analogues
Trypanosoma
Malaria
1. Introduction
teins where C is a cysteine, a an aliphatic amino acid, X a serine,
a methionine, an alanine or a glutamine (Scheme 1).
New drugs are desperately needed for diseases caused by proto-
zoan parasites, such as African sleeping sickness, Chagas disease
and leishmaniasis. These tropical diseases represent major health
problems. The World Health Organization estimates that 300,000
cases of African sleeping sickness occur annually in 36 countries
in sub-Saharan Africa, that 16–18 million people in Latin America
are chronically suffering from Chagas disease, and that 12 million
people worldwide are infected with Leishmania spp.1 Besides,
between one and two million people die annually from malaria
and a further 600 million cases occur each year.2 The currently
used drugs for these diseases are generally highly toxic and often
ineffective, and drug resistance has developed in some cases.
New and vulnerable protein targets within the cellular processes
of the parasite are needed in the search of novel therapeutics.
Farnesyltransferase inhibitors (FTIs) have been extensively
developed for anticancer therapy, and diverse compounds with
drug-like properties are available.9,10 Recently, FTase has also
appeared as a potential target for the treatment of parasitic
diseases as it has been shown that protein farnesylation occurs
in trypanosomatids and in the malaria parasite.11 A variety of FTIs
that are well tolerated in man have shown growth inhibition
activity and killing of Trypanosoma brucei and Plasmodium
falciparum.12–15 Most of FTIs are FPP or CaaX competitive inhibi-
tors.16–22 However, only a few bisubstrate compounds, that is, able
to bind both FPP and CaaX sites, have been reported. This kind of
compound is expected to exhibit better specificity and affinity for
the enzyme as it blocks both active sites.
In the course of our research on bisubstrate inhibitors, we have
designed new potential FTIs based on an imidazole ring known to
realize a strong interaction with the zinc atom in the FTase cata-
lytic binding site thanks to its basic nitrogen. Moreover, this im-
idazole ring is a common moiety of potent FTIs.23 We chose to
introduce an acidic function and a hydrophobic chain to mimic
Protein prenylation represents
a critical post-translational
modification as it plays a crucial role in intracellular signal trans-
duction, cell proliferation, and apoptosis. The process of prenyla-
tion includes the reaction of farnesylation. Since its identification,
the protein farnesyltransferase (FTase) has emerged as a promising
target in cancer therapy.3–7 FTase is an heterodimeric zinc metallo-
enzyme8 that catalyses the transfer of a farnesyl group (C15) from
farnesylpyrophosphate (FPP) to the free thiol group of a cysteine
residue embedded in the C-terminal CaaX sequence motif of pro-
SH
Protein-C-aaX
OPP
S
2
FTase
2
Protein-C-aaX
* Corresponding author. Tel.: +33 1 69 82 30 58; fax: +33 1 69 07 72 47.
E-mail address: joelle.dubois@icsn.cnrs-gif.fr (J. Dubois).
Scheme 1. Protein farnesylation.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.017

544
S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
Acid
X
n
acid
n
2
RN
N
3
2
aaX
Figure 3. Model of new FPP analogues X = C, O, N, carbonyl, amide.
Figure 1. General structure of potential bisubstrate analogues.
We began our investigation with monocarboxylic acid ana-
logues. In this series, derivatives where the carboxylic acid is
linked to the farnesyl moiety by a C–C bond has already been syn-
thesized.24 Therefore we intend to change this linkage to an ester
or a keto bond. Compounds with an ester bond were synthesized
by nucleophilic attack of trans–trans-farnesol 4 on Meldrum’s acid
(n = 0), succinic (n = 1) or glutaric anhydrides (n = 2) to give the
products 5a–c, respectively (Scheme 2).
As previously described for the synthesis of substituted FPP
analogues,32 the deprotonation with n-BuLi of sodium ethyloxala-
cetate followed by the addition of trans–trans-farnesyl bromide 6
led to the common ester intermediate 7 of the two keto analogues
11a and 11b. The attempt to hydrolyze the ester moiety of 7 under
different basic conditions did not allow us to form the keto
derivative 8. We isolated either the corresponding decarboxylated
product or the starting material depending on the reaction temper-
the FPP and a tripeptide in order to connect the CaaX-binding site
(Fig. 1).
The early first derivatives we synthesized were substituted
imidazole rings bearing an acidic moiety and either a hydrophobic
or a peptidic chain. Series of succinic acid–FPP analogues where the
lipophilic chain length has been modified 124 and of imidazole
derivatives functionalized by different tripeptides and acidic
chains 225 were synthesized and their inhibitory activity was eval-
uated on FTase (Fig. 2).
Among these first analogues, compound 1c bearing a farnesyl
chain was the most active derivative on human FTase (IC50
=
12 M) and proved to be competitive to FPP and uncompetitive
l
to CaaX motif. Compound 2c with the valine–phenylalanine–
methionine (VFM) tripeptide at the C-5 position of the imidazole26
and an acidic chain with n = 3 exhibited the best inhibitory activity
(IC50 = 60 lM) among the imidazole-containing derivatives. Con-
trary to compound 1c, this derivative is competitive to the CaaX
ature. The chain length was modified by
a-alkylation of 7 with
ethyl 2-bromoacetate 9a (n = 1) or ethyl 3-chloropropionate 9b
(n = 2) and gave, respectively, the keto diesters 10a and 10b.33
The one step saponification–decarboxylation of 10a–b at 70 °C pro-
vided the desired compounds 11a–b (Scheme 3).
motif and not to FPP.
Therefore our aim was to combine all these elements to synthe-
size more powerful FTIs. We present in this article the preparation
of new FPP analogues 3 (Fig. 3) with different acidic functions in or-
der to find a good mimic of the FPP pyrophosphate moiety as well
as the synthesis and biological activities of potential bisubstrate
inhibitors based on the general structure depicted in Figure 1.
In our previous work, we chose succinic acid as a FPP pyrophos-
phate mimic because it is present in several potent natural FPP
competitive inhibitors like chaetomellic acid A ( Fig. 4).34,35
A
stereoselective synthesis26 of these analogues has shown that the
(R) or (S) configuration of the succinic moiety have little influence
on the FTase inhibition. As malonic acid was already used as
replacement of the pyrophosphate moiety36 and to avoid any ster-
eoselective synthesis, we decided to build up malonic acid ana-
logues connected to the farnesyl moiety by a unsaturated or
saturated C–C bond, an ether or an amide linkage.
2. Results and discussion
2.1. Synthesis of new FPP analogues with various pyrophosphate
surrogates
trans–trans-Farnesal 12, easily obtained from trans–trans-farne-
sol 4,37 was submitted to Knoevenagel reaction using Yamamoto
and co-workers38 procedure and subsequent saponification affor-
ded compound 14 in low yield due to the instability of this
derivative. Starting from the commercially available dimethylmal-
onate, compound 16 was readily obtained in two steps by nucleo-
philic substitution of the trans–trans-farnesyl bromide 6 and ester
cleavage by lithium hydroxide. Finally, the nucleophilic attack of
hydroxymethylmalonate on trans–trans-farnesyl bromide 6 led to
malonic acid 18 after ester hydrolysis. The synthesis of the ami-
domalonic acid derivatives 21a–b (unsaturated and saturated
compounds, respectively) was realized by pseudopeptidic coupling
of diethylaminomalonate with two carboxylic acid analogues 19a–
b which have previously been described24 and by final deprotec-
tion of the diethylesters by lithium hydroxide (Scheme 4).
The FPP pyrophosphate moiety plays two roles: it participates
to FPP binding through its acidic functions and it is postulated to
be activated by Mg2+ to facilitate its departure during the enzy-
matic farnesylation.27,28 Therefore we chose to mimic pyrophos-
phate by other acidic functions or by groups able to bind Mg2+
.
Most of the FPP analogues described in the literature contained
various mono/di-acidic phosphoryl mimics like phosphonic acid
or phosphonate. The importance of this acidic part was demon-
strated as crucial for the inhibition mechanism.29–31 Because the
phosphoryl mimic has been largely studied and displayed some
bioavailability problems, we explored the synthesis and the biolog-
ical evaluation of FPP analogues with various other functions like
carboxylic, hydroxamic or sulfamic acids, amide, or sulfonamide.
Each analogue was designed to evaluate three parameters: the nat-
ure of the acidic moiety, the length and the nature of the chain
linking the farnesyl group to the acidic moiety (Fig. 3).
Hydroxamate derivatives have already been shown to bind to
Mg2+, for instance in yeast enolase.39 Therefore, in the series of me-
tal chelating FPP analogues, we decided to prepare hydroxamic
CO2H
CO2H
CO2H
CO2H
n
a or b
OH
O
n
N
N
CO2H
N
N
n
O
2
2
aaX
2a n = 1
2b n = 2
2c n = 3
1a n = 0
1b n =1
1c n = 2
4
5a n = 1
5b n = 2
5c n = 3
aaX = tripeptide
Scheme 2. Reagents and conditions: (a) Meldrum’s acid, toluene, reflux, 4 h (5a,
100%); (b) succinic or glutaric anhydride, DMAP, CH2Cl2, rt (5b, 90%; 5c, 70%).
Figure 2. Structure of previously synthesized imidazole-containing FTIs.

S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
545
a
b
Br
CO2Et
CO2H
O
O
2
2
2
6
7
8
X
CO2Et
n
c
9a n = 1, X = Br
9b n = 2, X = Cl
CO2Et
n
b
CO2H
CO2Et
n
O
O
2
2
11a n = 1
11b n = 2
10a n = 1
10b n = 2
Scheme 3. Reagents and conditions: (a) sodium oxalacetate, n-BuLi, THF, 0 °C (89%); (b) NaOH, EtOH, 70 °C, 1 h (11a, 100%; 11b, 85%); (c) KHMDS, THF, ꢀ78 °C, 1 h then 9a–b,
ꢀ78 °C to rt, 8 h (10a, 66%; 10b, 45%).
CO2H
a,b
NH2
19a
CO2H
O
2
22a
Figure 4. Chaetomellic acid A.
NH2
a,b
c
O
O
2
2
CO2R
CO2R
O
a
22b
23
19b
2
2
NHOH
12
13 R = Me
14 R = H
b
H
N
CO2R
CO2R
SO3H
d
e
O
O
2
2
24
25
2
2
c
19b
H
N
15 R =Me
16 R = H
b
SO2Ph
6
d
O
CO2R
CO2R
Scheme 5. Reagents and conditions: (a) ethylchloroformate, NEt3, THF, 0 °C,
30 min; (b) NH4Cl, KOH/MeOH, rt, 3 h (22a, 54%; 22b, 100%); (c) NH2OH, NMM,
EDCI, HOBt, CH2Cl2, rt, 3 h (23, 100%); (d) NH2SO3H, pivaloyl chloride, NEt3, CH2Cl2,
ꢀ78 °C then 0 °C, 1 h (24, 8%); (e) benzenesulfonamide, NMM, EDCI, HOBt, CH2Cl2,
rt, 18 h (25, 77%).
17 R = Et
18 R = H
b
The sulfamic moiety has been used as a surrogate of a phos-
phate moiety in the design of asparagine synthetase inhibitors.41,42
We first tried to connect the sulfamic moiety to the farnesyl group
through a sulfonate bond but the instability of the obtained com-
pound prompted us to change our strategy. We decided to synthe-
size the N-acyl sulfamic acid derivative 24 starting from the
carboxylic acid 19b. After different acid activation assays,
compound 24 was finally obtained with the mixed anhydride
methodology but in poor yield. These results led us to wonder if
the free sulfamic moiety was stable enough. We investigated the
synthesis of the phenyl analogue 25 because phenylsulfonamide
derivatives were efficient inhibitors of the zinc containing carbonic
anhydrase.43,44 Starting from carboxylic acid 19b and benzenesul-
fonamide, compound 25 was obtained in 77% yield (Scheme 5).
H
N
e
CO2R
CO2R
CO2H
2
O
2
b
20a-b R = Et
21a-b R = H
19a-b
Scheme 4. Reagents and conditions: (a) dimethylmalonate, KHMDS, THF, 0 °C then
rt, 5 h (13, 92%); (b) LiOH, THF/H2O 1:1, rt, 2 h (14, 22%; 16, 100%; 18, 100%; 21a,
100%; 21b, 37%); (c) dimethylmalonate, TiCl4, THF, 0 °C, 1 h then pyridine, rt, 2 h
(15, 63%) (d) diethylhydroxymalonate, NaH 60% in oil, THF, rt, 14 h (17, 42%); (e)
diethylaminomalonate, NMM, EDCI, HOBt, CH2Cl2, rt, 4 h/18 h (20a, 98%; 20b, 93%).
type analogues and their close amide derivatives. Compounds
22a–b were successfully obtained using an activation-nucleophilic
attack sequence40 on carboxylic acids 19a–b with ethyl chlorofor-
mate and ammoniac. Classical coupling conditions between
carboxylic acid 19b and hydroxylamine afforded 23 in a quantita-
tive yield (Scheme 5).
2.2. Synthesis of imidazole-containing analogues
Previous work in our group led to the synthesis of imidazole-
containing analogues with a succinic acid moiety and with either

546
S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
a farnesyl or a peptidyl chain (compounds 1 and 2, Fig. 2). Based on
the biological evaluation of our FPP analogues (see below), we in-
tended to synthesize a new imidazole-containing analogue bearing
CO2Et
CO2Et
CO2Et
CO2Et
a
O
B
O
B
+
O
O
30
a malonic acid moiety and a farnesyl chain attached on the a-posi-
tion of the carboxylic acids. We chose this position for the farnesyl
moiety because previous results have shown that a close proximity
between the farnesyl chain and the imidazole ring was detrimental
to the activity.24 We planned the retrosynthetic route depicted in
Scheme 6. The peptidyl chain could be introduced at the end of
the synthesis by reductive amination of the aldehyde obtained
after deprotection and oxidation of the C-5 hydroxymethylimida-
zole derivative 27. The key step of the synthesis relies on a Suzuki
C–C cross coupling on the 2-iodoimidazole derivative 29.26
To apply this reaction, it was necessary to first synthesize the
aliphatic vinyl boronate 30 that was successfully obtained by
cross-coupling metathesis between diethylallylmalonate and
vinylpinacolborane (Scheme 7). After optimization of the Suzuki
cross-coupling reaction, compound 28a was obtained in 80% yield.
Hydrogenation of the double bond afforded the desired product
28b in 64% yield (Scheme 7).
CO2Et
N
CO2Et
EtO2C
EtO2C
c
I
b
N
N
N
N
N
OTBS
OTBS
OTBS
29
28a
28b
EtO2C
EtO2C
2
d
N
N
The first attempt to introduce the farnesyl chain on 28b in the
presence of sodium hydroxide led to the introduction of two far-
nesyl moieties together with the loss of the TBS group. Farnesyla-
tion occurred between the two carboxylic esters as expected and
on the second nitrogen of the imidazole to form the imidazolium
31 (Scheme 7). The attempts to avoid the formation of this
secondary product by using weak bases or no base at all were
unsuccessful. This peculiar reactivity of the imidazole ring is due
to the presence of two protonable nitrogens that stabilizes the po-
sitive charge by an equilibrated form. This reactivity has been
exploited for ionic liquids or as stable carben precursor in particu-
lar. To avoid this imidazolium formation, we changed our strategy
by carrying out first the oxidation of the alcohol in order to desta-
bilize the imidazolium (Scheme 8). Compound 32 was then submit-
ted to farnesylation in the presence of sodium hydride and provided
the expected compound 33 in a quantitative yield. The aldehyde 32
and VFM–OMe25 were coupled by a reductive amination followed
by saponification of the ester functions. Although the saponification
step was undertaken during a long reaction time, only two of the
three esters were hydrolyzed. Other conditions using stronger
bases or higher reaction temperature were tempted but none of
them were conclusive, leading to degradation or leaving starting
material unchanged (Scheme 8).
2
OH
31
Scheme 7. Reagents and conditions: (a) Grubbs I catalyst 10 mol %, CH2Cl2, reflux,
3 h (30, 92%); (b) 30, Pd(PPh3)4, Na2CO3, DMF, 130 °C, 5 h (28a, 80%); (c) H2, Pd/C,
AcOEt, rt, 18 h (28b, 64%); (d) NaH, 6, THF, 0 °C then rt, 3 h (31, 50%).
Compound 2c represents the derivative without the farnesyl group
and we synthesized compound 36 bearing no tripeptide.45 The syn-
thesis was carried out as for compound 35 starting from 2-iodo-1-
methylimidazole. The Suzuki cross-coupling with compound 30
afforded 37 in 85% yield that was further hydrogenated under Pd
catalyst. As for compound 28b farnesylation occurred both at the
imidazole ring and at the malonyl carbon. Therefore, to minimize
the imidazole ring farnesylation, the reaction with farnesyl bro-
mide 6 was carried out at low temperature leading to major forma-
tion of the desired compound 38. As previously observed in this
series, ester hydrolysis was difficult and the desired diacid 36
was obtained in only 27% yield (Scheme 9).
3. Biological evaluation
FPP analogues were evaluated for their inhibitory activity
against recombinant yeast and human FTases46 using a fluorescent
based assay47,48 and adapted to 96-well plate format. The IC50 were
determined and compared to the IC50 of chaetomellic acid A (Fig. 4)
measured under our conditions (Table 1).
To evaluate the actual influence of the peptidyl and farnesyl
chains, we needed compounds bearing only one of these chains.
HO2C
HO2C
R'O2C
R'O2C
For the monocarboxylic acids, the nature of the linkage does not
seem to be important. However the distance between the farnesyl
and acidic groups slightly modifies the activity. The best results
were obtained with short chains with C–C bond linkage (19a–b)
but only in the case of yeast FTase or with longer chain either with
an ester or a keto linkage (5c and 11b, respectively) for both en-
zymes. However all these activities are low compared to chaeto-
mellic acid A. As previously observed,24 the introduction of a
second carboxylic acid greatly enhances the activity whatever
the enzyme is concerned. On the contrary to C or O-linkage (16
and 18, respectively), the presence of an amide (21a–b) seems det-
rimental to the activity. The other non-carboxylic functions do not
lead to any advantage since, in almost all cases, compounds 22a–b,
2
2
N
N
N
N
VFM
OR
26
27
CO2R
RO2C
HO
I
OH
O
23–25 expressed an inhibitory activity superior to 200 lM. The
most active compounds 16 and 18 showed interesting activities
in the submicromolar range. When compared to its succinic ana-
N
N
N
N
O
OR
OR
HO
OH
29
28
logue (IC50 = 2.5 and 5.4
lM on yeast and human FTase, respec-
tively),24 compound 16 showed the same activity against yeast
Scheme 6. Retrosynthetic pathway.

S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
547
CO2Et
N
EtO2C
EtO2C
EtO2C
EtO2C
HO2C
EtO2C
EtO2C
2
2
d
2
a, b
c
e
N
N
N
N
28
N
N
N
O
VFM-OMe
34a-b
VFM-OH
35a-b
O
32a-b
33a-b
Scheme 8. Reagents and conditions: (a) TBAF, THF, rt, 2 h; (b) MnO2, CH2Cl2, 40 °C, 18 h (32a, 85%; 32b, 64%) (c) NaH, 6, THF, 0 °C, 2 h (33a, 100%; 33b, 77%) (d) (i) NH2–VFM–
OMe, 4 Å MS, CH2Cl2–MeOH, rt, 10 min then aldehyde 33a or 33b, (ii) NaBH3CN, MeOH/AcOH (10:1), rt, 18 h (34a, 34%; 34b, 46%) (e) LiOH, THF/MeOH/H2O (1:1:1), rt, seven
days (35a, 100%; 35b, 27%).
usually employed as prodrugs in cell-based assays.49 This was
verified with compounds 34a–b that were unable to inhibit human
FTase. It is to be noted that the presence of all chains greatly in-
creased the activity of our imidazole-containing inhibitors with
CO2Et
RO2C
RO2C
EtO2C
a
2
I
b, c
IC50 going from 60
l
M for 2c (no farnesyl chain) or 21
lM for 36
N
N
N
N
N
N
(no peptidyl chain) to 0.64
l
M and 0.24 M for 35a and 35b,
l
respectively. Compound 35b bearing the saturated acid chain re-
vealed to be the best inhibitor of our imidazole-containing inhibi-
tor series with a submicromolar range activity. To explain these
results and determine if our compounds were bisubstrate inhibi-
tors, kinetic studies were realized on both compounds 35a and
35b. These experiments showed that these derivatives were FPP
37
38 R = Et
36 R = H
d
Scheme 9. Reagents and conditions: (a) 30, Pd(PPh3)4, Na2CO3, DMF, 130 °C, 5 h
(85%); (b) H2, Pd/C, AcOEt, rt, 18 h (37, 93%); (c) NaH, 0 °C, 1 h then 6, THF, 0 °C, 1 h
30 min (38, 64%); (d) LiOH, THF/MeOH/H2O (1:1:1), rt, eight days (39, 27%).
competitive inhibitors with Kai pp = 0.5 and 0.23
l
M for 35a and
35b, respectively. However, these compounds were non-competi-
tive CaaX inhibitors with Kai pp = 17 and 7.7 M and Ki0app = 15 and
5.6
M, respectively.50 It is to be noted that Kai pp for FPP inhibition
are very similar for compounds 16, 35a and 35b (0.26, 0.5 and
0.23 M, respectively) whereas the CaaX inhibition goes from
l
FTase but a fivefold increase against the human enzyme. Before
introducing the malonic function into our potential bisubstrate
inhibitors, it was necessary to precise the binding mode of these
malonyl FPP analogues. As expected, the kinetic experiments,
realized on compound 16, showed that it binds to the FPP site
and not to the peptide one as it revealed to be a FPP competitive
l
l
uncompetitive to non-competitive inhibition. Therefore, the better
IC50 obtained for 35b compared to 16 and 35a must result from the
differences observed in their Kai pp for CaaX inhibition.
inhibitor (Kai pp = 0.26
lM) and to be uncompetitive toward the pep-
With the aim of finding new active compounds against some pro-
tozoan parasites, we evaluated the activity of our imidazole-con-
taining compounds against P. falciparum and T. brucei proliferation.
tidyl substrate Dns-GCVLS.
Generally, the esters of peptide-containing derivatives are much
less active than their acid analogues in the enzymatic assay and are
Table 1
Activity of FPP analogues on recombinant yeast and human FTases
X
acid
n
2
Compounds
n
X
Acid
IC50
(
lM)
IC50 (lM)
yeast FTase
human FTase
Chaetomellic acida
0.34
40
42
80
100
35
0.175
130
170
160
130
60
>200
60
Inactive
0.90
0.88
10
19ab
19bb
5a
2c
2
1
1
1
–CH–
–CH2–
–COOH
–COOH
–CO2H
–CO2H
–CO2H
–CO2H
–CO2H
HO2C–C–CO2H
HO2C–CH–CO2H
HO2C–CH–CO2H
HO2C–C–CO2H
HO2C–CH–CO2H
—
—
—
–SO3H
—
–OCO–CH2–
–OCO–(CH2)2–
–OCO–(CH2)3–
–CO–(CH2)2–
–CO–(CH2)3–
—
—
–O–
–CONH–
–CONH–
–CONH2
5b
5c
11a
11b
14
2
80
2
15
1c
1
ndd
4
16
18
1
5
nd
21a
21b
22a
22b
23
2c
2
200
Inactive
Inactive
Inactive
>200
>200
60
2c
2
Inactive
>200
nd
85
>200
–CONH2
2
2
2
–CONH–OH
–CONH–
–CONH–SO2Ph
24
25
a
b
c
IC50 measured under our conditions.
See Ref. 24.
Unsaturated derivative.
Not determined.
d

548
S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
As expected, the ester derivatives were more active on parasites
than their acidic forms. We observed the same behavior on P. falcipa-
rum as on the free enzyme, that is, the activity goes from
5. Experimental
5.1. Chemistry
IC50 = 17.7
l
M (without the tripeptide) or 4
lM (without the far-
nesyl group) to 1
lM when all chains are present on the imidazole
5.1.1. General method
ring. Our derivatives 34a–b and 38 showed promising activity
against T. brucei, compound 34a being more active on the blood-
stream form than on the procyclic form. As such agents should not
be toxic for humans, we also evaluated their cytotoxicity on both
normal (MRC5) and cancer (KB) human cell lines. As shown in
Table 2 all our compounds are weakly or not cytotoxic, what is
encouraging for antiparasitic purpose.
Unless otherwise indicated, all reactions were carried out with
magnetic stirring and in case of air-sensitive compounds reactions
were carried out in oven-dried glassware under argon. Commercial
compounds were used without any further purification. Tetrahy-
drofuran (THF) was freshly distilled from sodium/benzophenone.
Dichloromethane (CH2Cl2), triethylamine (NEt3), diisopropylamine
and toluene were distilled over calcium hydride. N,N0-Dimethyl-
formamide (DMF) was dried over MgSO4 followed by distillation
under reduced pressure. Pyridine was stored over KOH and other
solvents over 4 Å molecular sieves. Analytical thin-layer chroma-
tography was carried out on precoated silica gel aluminum plates
(SDS TLC plates, Silica Gel 60F254). Column chromatography was
4. Conclusions
In a first part, we have synthesized sixteen new FPP analogues
with different functions introduced on the farnesyl chain in order
to find a good mimic of the FPP pyrophosphate moiety. This study
revealed that a malonic acid attached to the farnesyl group could
properly interact with FTase at the FPP binding site and was
accordingly chosen as the acidic part for the new FTase inhibitors
we aimed to synthesize. In a second part, we performed the syn-
thesis of new imidazole-containing derivatives composed by two
main moieties, a farnesyl chain with an acidic function and a pep-
tidyl chain linked by an imidazole ring in order to occupy both
FTase substrate binding sites. The key reaction of these syntheses
was a Suzuki cross coupling, a new C–C coupling methodology
on 2-iodoimidazole optimized in our laboratory with aliphatic vi-
nyl boronates.26 Biological profile evaluation of our imidazole ana-
logues has shown that the simultaneous presence of potential FPP
and CaaX mimics enhanced the inhibitory activity going from an
performed with silica gel SDS 60 A CC (40–63
packed RedisepÒ columns. Preparative TLC (PLC) was performed
on Merck TLC with Silica Gel 60F254
lm) or with pre-
.
NMR spectra (1H and 13C) were recorded on a Brucker Avance
300 (300 MHz) and Avance 500 (500 MHz). Chemical shifts (d)
are given in ppm relative to CDCl3 (7.27 ppm; 77.14 ppm), CD3OD
(3.34 ppm; 49.9 ppm). Splitting patterns are designed as: s, singlet;
d, doublet; t, triplet; q, quartet; qi, quintuplet; m, multiplet; br,
broad and combinations thereof. Coupling constants J are reported
in hertz (Hz). IR spectra were recorded on a Perkin–Elmer Spec-
trum BX. Mass spectra were recorded on Thermofinningan Auto-
mass with a quadripole detection (IE) and on Thermoquest AQA
Navigator with a TOF detection (ESI-HRMS).
5.1.2. General procedure A: saponification of FPP analogues
To a solution of ester 13, 15, 17 or 20 in THF/H2O (1:1), lithium
hydroxide (3.3 equiv) was added. After being stirred at room tem-
perature for 0.5–72 h, the reaction mixture was neutralized by HCl
10% and extracted by dichloromethane. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The
purification was realized when necessary by column chromatogra-
phy to afford the target compounds 14, 16, 18 or 21.
IC50 value around 60 lM to a submicromolar range. Kinetic
experiments have demonstrated that our compounds do not ex-
press bisubstrate properties; they have a good affinity for the FPP
but not for the CaaX-binding site. Our results also showed that
an increased inhibition of the CaaX motif greatly improved the
overall activity. These results as well as the encouraging activities
against P. falciparum and T. brucei and the weak cytotoxicity upon
mammalian cells prompt us to carry on our investigation on poten-
tial bisubstrate FTIs with an imidazole ring as central motif. The
relative position of the farnesyl and peptidyl chains in our imidaz-
ole-containing inhibitors is probably not appropriate enough to fit
both CaaX and FPP binding sites. Therefore, the synthesis of new
imidazole-containing analogues substituted on different positions
by the farnesyl and peptidyl chains is currently under
investigation.
5.1.3. General procedure B: pseudopeptidic coupling
To a solution of acid 19 in dichloromethane, N-methylmorpho-
line (2 equiv), EDCI (1.2 equiv), amine (1 equiv) and HOBt
(1.2 equiv) were added. After being stirred 4–18 h at room temper-
ature, the reaction mixture was neutralized by HCl 10% and ex-
tracted by dichloromethane. The organic layer was dried over
Table 2
Inhibition of recombinant human FTase and of proliferation of T. brucei, P. falciparum, KB and MRC5 cells by imidazole-containing analogues
Compound
IC50
hFTase
(
l
M)
% Inh. at 10
T. brucei brucei
l
M
IC50
P. falciparum
(
l
M)
% Inh. at 10
KB
lM
% Inh. at 10
MRC5
lM
Bloodstream
Procyclic
2ca
34a
35a
34b
35b
36
60
ndb
nd
17.7c
1.1
17.2
1.0
31.4
nd
4
nd
nd
Inactive
0.640
Inactive
0.240
21
92d
28e
60e
Inactive
Inactive
Inactive
Inactive
nd
Inactive
nd
Inactive
Inactive
54e
44e
Inactive
nd
Inactive
nd
nd
Inactive
nd
nd
38
nd
89f
nd
a
b
c
d
e
f
From Ref. 25.
Not determined.
Triester of compound 2c.
64% at 1 M.
Inactive at 1
24% at 1 M.
l
l
M.
l

S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
549
Na2SO4 and concentrated under reduced pressure. The crude prod-
uct was purified by column chromatography on silica gel to afford
5.1.8. General procedure G: saponification of imidazole-
containing analogues
desired compounds 20, 23 or 25.
Lithium hydroxide (20 equiv) was added to a solution 34 or 38
in THF/H2O/MeOH 1:1:1 (2 mL). The reaction mixture was stirred
at room temperature for 7–8 days then stopped by neutralization
with a solution of HCl 10% and the reaction mixture was extracted
with dichloromethane. The organic layer was dried over Na2SO4
and the solvent was removed under reduced pressure to afford
compound 35 or 36.
5.1.4. General procedure C: amine analogues formation
Ethyl chloroformate (1.1 equiv) and triethylamine (1.1 equiv)
were added to a solution of 19 in anhydrous THF at 0 °C under ar-
gon. After being stirred 30 min at 0 °C, the solution was filtered and
concentrated. A solution of KOH, 0.69 M in methanol, (1.4 equiv)
and ammonium chloride (1.4 equiv) was prepared at room temper-
ature under argon. After being stirred for 30 min, the reaction res-
idue in anhydrous THF was added to the ammonium hydroxide
suspension. After being stirred for 3 h at room temperature, the
solvent was removed under reduced pressure. The resulting white
powder was dissolved in CH2Cl2, filtered and concentrated. The
residue was purified by preparative TLC (CH2Cl2/MeOH 9:1) to af-
ford compound 22.
5.1.9. 3-Oxo-3((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl-
oxy)propanoic acid (5a)
To a solution of commercial Meldrum’s acid (67 mg, 0.47 mmol)
in anhydrous toluene (2.5 mL) under argon at room temperature
was added commercial trans–trans-farnesol 4 (113 lL, 0.45 mmol).
After being stirred 4 h at 110 °C, the solvent was removed under
reduced pressure to afford 5a (138 mg, 100%) as a yellow oil. 1H
NMR (300 MHz, CDCl3) d 1.60 (s, 3H), 1.60 and 1.68 (s, 6H), 1.72
(s, 3H), 1.92–2.18 (m, 8H), 3.44 (s, 2H), 4.70 (d, 2H, J = 7.0 Hz),
5.09 (m, 2H), 5.35 (t, 1H, J = 7.0 Hz), 9.56 (br s, 1H). 13C NMR
(75 MHz, CDCl3) d 16.3, 26.0, 16.8, 18.0, 26.5, 27.0, 39.8, 40.0,
41.0, 63.2, 117.7, 123.8, 124.6, 131.6, 135.9, 143.9, 167.4, 171.5.
IR (CH2Cl2) 2965, 2919, 2857, 1739, 1731, 1443, 1150 cmꢀ1. MS
(ESI+) m/z 331 [M+Na]+. HRMS (ESI+) calcd for C17H25O4 [M+Na]+:
331.1885, found 331.1877.
5.1.5. General procedure D: TBS deprotection–oxidation
Tetrabutylammonium fluoride in solution 1 M in THF (2 equiv)
was added to a solution of 28 in anhydrous THF at room tempera-
ture. After being stirred for 2–3 h at room temperature, the reac-
tion was allowed to cool at room temperature and some drops of
acetone were added. The reaction mixture was extracted with
ethyl acetate and washed with H2O. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography to afford the free
alcohol derivative. This latter was added to a suspension of acti-
vated manganese dioxide(IV) (6 equiv) in dichloromethane at
room temperature. The reaction mixture was stirred for 18 h at
40 °C, then, after cooling, filtered on a pad of Celite (EtOAc) and
concentrated.
5.1.10. 4-Oxo-4((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl-
oxy)butanoic acid (5b)
To a solution of DMAP (497 mg, 4.1 mmol) and succinic anhy-
dride (94 mg, 0.94 mmol) in anhydrous CH2Cl2 (2.5 mL) under
argon at room temperature was added commercial trans–trans-far-
nesol 4 (113 lL, 0.45 mmol). After being stirred for 2 h, the solvent
was evaporated. The reaction mixture was extracted with ethyl
acetate, washed with a 10% HCl solution and twice with brine.
The organic layer was dried over Na2SO4 and concentrated to af-
ford 5b (131 mg, 90%) as a yellowish oil. 1H NMR (300 MHz, CDCl3)
d 1.60 (s, 3H), 1.60 and 1.68 (s, 6H), 1.70 (s, 3H), 1.93–2.17 (m, 8H),
2.58–2.73 (m, 4H), 4.62 (d, 2H, J = 7.0 Hz), 5.09 (m, 2H), 5.34 (td,
1H, J = 7.0 Hz and J0 = 1.0 Hz). 13C NMR (75 MHz, CDCl3) d 16.3,
26.0, 16.8, 18.0, 26.0, 27.0, 29.2, 29.3, 39.8, 40.0, 62.1, 118.3,
123.9, 124.6, 131.6, 135.8, 142.9, 172.5, 178.5. MS (ESI+) m/z 345
[M+Na]+. HRMS (ESI+) calcd for C19H30O4Na [M+Na]+: 345.2042,
found 345.2054.
5.1.6. General procedure E: farnesylation
Sodium hydride, 95% (1 equiv) was added to a solution of 32 or
37 in anhydrous THF at 0 °C. After being stirred for 1 h at 0 °C,
trans–trans-farnesyl bromide 6 (1 equiv) was added at 0 °C. The
reaction mixture was stirred 1.5–2 h at 0 °C then the reaction
was stopped by addition of H2O and extracted with ethyl acetate.
The organic layer was dried over Na2SO4 and the solvent was re-
moved under reduced pressure. The crude product was purified if
necessary by column chromatography to afford compounds 33 or
38.
5.1.11. 5-Oxo-5-((2E,6E)-3,7,11-trimethyl-dodeca-2,6,10-trie-
nyl)pentanoic acid (5c)
5.1.7. General procedure F: reductive amination
A solution of trifluoroacetic acid (1.6 mL, 75%) in dichlorometh-
ane was added to the tripeptide (1.2 equiv) under anhydrous con-
ditions. After being stirred at 0 °C for 20 min, diethyl ether was
added to the reaction mixture and the solvent was removed under
reduced pressure. This process was repeated until a white solid is
obtained. To this solid in CH2Cl2/MeOH 8:2 (1.9 mL) were added
at room temperature molecular sieves and distilled triethylamine
(1 equiv). The reaction mixture was stirred for 10 min at room
temperature then aldehyde 33 (1 equiv) dissolved in CH2Cl2/MeOH
8:2 (1 mL) was added. The reaction mixture was stirred at room
temperature for 6 h then sodium cyanoborohydride (1.2 equiv) dis-
solved in a solution of 1% acetic acid in methanol (0.12 mL) was
added. After being stirred for 15 h at room temperature, the reac-
tion was stopped by addition of H2O and the reaction mixture
was extracted with dichloromethane. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography to afford com-
pound 34.
Commercial trans–trans-farnesol 4 (0.157 mL, 0.6 mmol) was
added to a solution of DMAP (667 mg, 5.5 mmol) and glutaric
anhydride (148 mg, 1.3 mmol) in dichloromethane (3.3 mL) at
room temperature. After being stirred for 1 h, the reaction mixture
was hydrolyzed by a saturated NaHCO3 solution. The organic layer
was neutralized with a 10% HCl solution then dried over Na2SO4
and concentrated under reduced pressure. The crude product was
purified by column chromatography on C18 silica (gradient H2O/
CH3CN 8:2 v/v to CH3CN in 20 min) to give pure compound 5c as
a colorless oil (137 mg, 70%). 1H NMR (500 MHz, CDCl3) d 1.60 (s,
6H), 1.68 (s, 3H), 1.71 (s, 3H), 1.93 (q, 2H, J = 4.0 Hz), 1.93–1.99
(m, 4H), 2.0–2.08 (m, 4H), 2.40–2.47 (m, 4H), 4.60 (d, 2H,
J = 7.0 Hz), 5.09 (m, 2H), 5.34 (td, 1H, J = 7.0 Hz and J0 = 2.0 Hz).
13C NMR (75 MHz, CDCl3) d 16.0, 16.5, 17.7, 20.2, 25.7, 33.0, 33.4,
39.5, 39.7, 61.5, 118.2, 123.6, 124.3, 131.3, 135.5, 142.5, 171.4,
178.6. MS (ESI+) m/z 359 [M+Na]+. HRMS (ESI+) calcd for
C20H32O4Na [M+Na]+: 359.2198, found 359.2196.

550
S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
5.1.12. (6E,10E)-Ethyl 7,11,15-trimethyl-3-oxohexadeca-6,10,
14-trienoate (7)
122.1, 124.0, 124.4, 131.4, 135.1, 137.0, 170.0, 173.0, 204.5. MS
(ESI+) m/z 457 [M+Na]+.
n-BuLi, 1,6 M in hexane, (2 mL, 3.2 mmol) was added to a solu-
tion of commercial sodium ethyl acetoacetate (480 mg, 3.10 mmol)
in anhydrous THF (14.5 mL) at 0 °C under argon. After being stirred
5.1.15. (7E,11E)-8,12,16-Trimethyl-4-oxoheptadeca-7,11,15-tri-
enoic acid (11a)
45 min at 0 °C, commercial trans–trans-farnesyl bromide 6 (271
l
L,
An aqueous solution of 2 M NaOH (0.35 mL, 0.70 mmol) was
added to a solution of 10a (90.4 mg, 0.23 mmol) in ethanol at room
temperature. After being stirred 1 h at 70 °C, the reaction was
cooled to room temperature then hydrolyzed by 1 M HCl. The reac-
tion mixture was extracted with ethyl acetate. The organic layer
was washed with brine and dried over Na2SO4. The expected com-
pound 11a was obtained after solvent removal (73.0 mg, 100%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) d 1.62 (3s, 9H), 1.65 (s, 3H),
1.96–2.16 (m, 8H), 2.24–2.34 (m, 2H), 2.54 (m, 4H), 2.75 (m, 2H),
5.13 (m, 3H). 13C NMR (75 MHz, CDCl3) d 16.9, 18.8, 26.8, 24.3,
28.4, 28.7, 29.6, 39.0, 41.7, 44.4, 125.0, 126.3, 133.0, 136.9, 138.1,
177.4, 212.4. IR (CH2Cl2) 2916, 1712 cmꢀ1. MS (ESIꢀ) m/z 319
[MꢀH]ꢀ. HRMS (ESIꢀ) calcd for C20H31O3 [MꢀH]ꢀ: 319.2273, found
319.2275.
1 mmol) was added dropwise and the reaction mixture was stirred
30 min at 0 °C. The mixture was poured into a solution of saturated
K2HPO4 and extracted three times with CH2Cl2. The organic layer
was dried over Na2SO4 and concentrated. The crude product was
then purified by column chromatography (heptane/CH2Cl2 1:1) to
afford 7 (300 mg, 89%) as a yellow oil. 1H NMR (300 MHz, CDCl3)
d 1.27 (t, 3H, J = 7.0 Hz), 1.59 and 1.67 (s, 6H), 1.59 (s, 3H), 1.61
(s, 3H), 1.92–2.11 (m, 8H), 2.30 (t, 2H, J = 7.5 Hz), 2.56 (t, 2H,
J = 7.5 Hz), 3.42 (s, 2H), 4.18 (q, 2H, J = 7.0 Hz), 5.03–5.14 (m, 3H).
13C NMR (75 MHz, CDCl3) d 14.4, 16.3, 17.9, 26.0, 22.5, 26.8, 27.0,
39.9, 40.0, 43.3, 49.7, 61.6, 122.4, 124.3, 124.7, 131.5, 135.3,
137.0, 167.5, 202.8. MS (ESI+) m/z 357 [M+Na]+. HRMS (ESI+) calcd
for C21H34O3Na [M+Na]+: 357.2406, found 357.2361.
5.1.16. (8E, 12E)-9,13,17-Trimethyl-5-oxo-octadeca-8,12,16-tri-
enoic acid (11b)
5.1.13. Diethyl 2-((4E,8E)-5,9,13-trimethyltetradeca-4,8,12-trie-
noyl)succinate (10a)
An aqueous solution of 2 M NaOH (0.612 mL, 1.22 mmol) was
added to a solution of 10b (177.3 mg, 0.41 mmol) in ethanol
(3.2 mL) at room temperature. After being stirred for 1 h at 70 °C,
the reaction was cooled to room temperature and acidified by
1 M HCl. The reaction mixture was extracted by ethyl acetate.
The organic layer was washed by brine, dried over Na2SO4 and con-
centrated under reduced pressure. The crude product was purified
by recrystallisation of the calcium salt in dichloromethane to give
pure compound 11b (116.4 mg, 85%) 1H NMR (500 MHz, CDCl3) d
1.60 and 1.62 (s, 6H), 1.63 (s, 3H), 1.69 (s, 3H), 1.93 (qi, 2H,
J = 7.0 Hz), 1.96–2.0 (m, 4H), 2.06–2.1 (m, 4H), 2.29 (q, 2H,
J = 7.0 Hz), 2.40 (t, 2H, J = 7.0 Hz), 2.45 (t, 2H, J = 8.0 Hz), 2.51
(t, 2H, J = 7.0 Hz), 5.08 (m, 3H). 13C NMR (75 MHz, CD3OD) d 16.0,
17.7, 23.4, 25.7, 26.8, 29.7, 39.7, 39.73, 42.9, 116.4, 122.6, 124.4,
133.0, 136.9, 141.1, 173.4, 212.4. MS (ESI+) m/z 357 [M+Na]+. HRMS
(ESI+) calcd for C21H34O3Na [M+Na]+: 357.2406, found 357.2424.
To a solution of KHMDS (181.8 mg, 0.91 mmol) in anhydrous THF
(0.9 mL) was added 7 (201 mg, 0.6 mmol) in anhydrous THF (2.5 mL)
at ꢀ78 °C under argon. After being stirred for 1 h, commercial ethyl
bromoacetate was added dropwise at ꢀ78 °C. The reaction mixture
was stirred 35 min at ꢀ78 °C and then allowed to warm to room
temperature. After being stirred for 7 h at room temperature, the
reaction was quenched by water and a 10% HCl solution and ex-
tracted with ethyl acetate. The organic layer was dried over Na2SO4
and concentrated. The residue was purified by column chromatogra-
phy (heptane/AcOEt 95:5) to afford 10a (166 mg, 66%) as a colorless
oil. 1H NMR (500 MHz, CDCl3) d 1.25 (t, 3H, J = 7.0 Hz); 1.27 (t, 3H,
J = 7.0 Hz); 1.60 and 1.69 (s, 6H); 1.61 (s, 3H); 1.62 (s, 3H); 1.95–
2.01 (m, 4H); 2.03–2.10 (m, 4H); 2.30 (q, 2H, J = 7.5 Hz); 2.65 (dt,
1H, J = 7.5 Hz and J0 = 17.5 Hz); 2.76 (dt, 1H, J = 7.5 Hz and
J0 = 17.5 Hz); 2.83 (dd, 1H, J = 6.5 Hz and J0 = 17.5 Hz); 2. 96 (dd,
1H, J = 8.0 Hz and J0 = 17.5 Hz); 3.97 (t, 1H, J = 7.0 Hz); 4.13 (q, 2H,
J = 7.0 Hz); 4.20 (q, 2H, J = 7.0 Hz); 5.09 (m, 3H). 13C NMR (75 MHz,
CDCl3) d 14.4, 16.3, 18.0, 26.0, 22.4, 26.9, 27.1, 32.7, 40.0, 43.1,
54.4, 61.2, 62.0, 122.6, 124.4, 124.7, 131.6, 135.4, 136.9, 168.8,
171.7, 204.0. IR (CH2Cl2) 2979, 2933, 1730, 1715, 1160 cmꢀ1. MS
(ESI+) m/z 443 [M+Na]+. HRMS (ESI+) calcd for C25H40O5Na
[M+Na]+: 443.2773, found 443.2787.
5.1.17. Dimethyl 3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-tri-
enylidene)malonate (13)
To a solution titanium tetrachloride(IV) (0.299 mL, 2.72 mmol)
in anhydrous THF (10 mL) were added dimethylmalonate
(0.234 mL, 2.04 mmol) and farnesal 12 (0.300 g, 1.36 mmol) in
THF (2 mL) at 0 °C under argon. The reaction mixture was stirred
1 h at 0 °C then pyridine (0.440 mL, 5.44 mmol) was added. After
being stirred 2 h at room temperature, H2O was added and the
reaction was extracted by ethyl acetate. The organic layer was
dried over Na2SO4 then the solvent was removed under reduced
pressure. The crude product was purified by preparative TLC
(CH2Cl2) to afford pure compound 13 as a colorless oil (288 mg,
63%). 1H NMR (500 MHz, CDCl3) d 1.60 (s, 6H), 1.68 (s, 3H), 1.94
(s, 3H), 1.97–2.40 (m, 8H), 3.79 and 3.85 (s, 6H), 5.08 (m, 2H),
6.28 (d, 1H, J = 13.0 Hz), 7.69 (dd, 1H, J = 13.0 Hz and J0 = 6.0 Hz).
13C NMR (75 MHz, CDCl3) d 16.1, 17.7, 25.7, 26.2, 26.7, 39.6, 40.9,
52.2, 120.5, 122.9, 124.3, 131.6, 136.4, 141.3, 156.2, 167.3. IR (neat)
5.1.14. Diethyl 2-((4E,8E)-5,9,13-trimethyl-tetradeca-4,8,12-trie-
noyl)pentanedioate (10b)
Compound 7 (0.300 g, 0.90 mmol) in anhydrous THF (3.2 mL)
was added to a solution of KHMDS (0.268 g, 1.34 mmol) in THF
(1.3 mL) at ꢀ78 °C. After being stirred for 1 h at ꢀ78 °C, ethyl-3-
chloropropionate (0.1335 mL, 1.08 mmol) was added dropwise.
The reaction mixture was stirred for 35 min at ꢀ78 °C then allowed
to warm to room temperature. After being stirred for 8 h, the reac-
tion mixture was neutralized by 10% HCl and extracted by ethyl
acetate and H2O. The organic layer was dried over Na2SO4 and
the solvent was removed under reduced pressure. The crude prod-
uct was purified by column chromatography (heptane then hep-
tane/AcOEt 9:1) to give pure compound 10b as a colorless oil
(177.3 mg, 45%) 1H NMR (500 MHz, CDCl3) d 1.28 (t, 6H, J =
7.0 Hz), 1.60 and 1.61 (s, 6H), 1.62 (s, 3H), 1.69 (s, 3H), 1.95–2.01
(m, 4H), 2.03–2.10 (m, 4H), 2.17 (t, 2H, J = 7.0 Hz), 2.30 (q, 2H,
J = 7.5 Hz), 2.36 (t, 2H, J = 7.5 Hz), 2.55 and 2.62 (t, 1H, J = 7.0 Hz),
3.58 (t, 1H, J = 7.0 Hz), 4.13 (q, 2H, J = 6.0 Hz), 4.20 (q, 2H, J =
7.0 Hz), 5.10 (m, 3H). 13C NMR (75 MHz, CDCl3) d 14.2, 16.0, 17.7,
22.1, 23.0, 25.7, 26.6, 26.7, 31.6, 39.7, 42.2, 57.6, 60.5, 61.4,
1740 cmꢀ1
.
5.1.18. 3-((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trienylidene)-
malonic acid (14)
Prepared according to general procedure A on 13 (0.233 g,
0.698 mmol) in 3 mL solvent with lithium hydroxide (0.097 g,
2.30 mmol) for 72 h. The crude product was purified by HPLC on
C18 silica gel to afford pure compound 14 as a white solid
(213 mg, 100%). 1H NMR (500 MHz, CDCl3) d 1.60 (s, 6H), 1.68 (s,
3H), 1.89 (s, 3H), 1.99–2.54 (m, 8H), 5.09 (m, 2H), 7.50 (d, 1H,

S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
551
J = 14.0 Hz), 8.48 (m, 1H). 13C NMR (75 MHz, CDCl3) d 16.5, 17.9,
25.7, 27.3, 40.9, 121.9, 123.3, 124.2, 131.6, 135.7, 137.9, 141.4. IR
(neat) 2920, 1740, 1700 cmꢀ1. Mp 139 °C. MS (ESI+) m/z 329
[M+Na]+. HRMS (ESI+) calcd for C18H26O4Na [M+Na]+: 329.1728,
found 329.1728.
2H, J = 8.0 Hz), 3.51 (s, 1H), 5.12 (m, 3H). 13C NMR (75 MHz, CDCl3)
d 16.2, 17.3, 25.5, 27.2, 27.4, 39.9, 63.5, 79.7, 118.5, 124.0, 124.2,
131.6, 134.5, 138.4, 174.2. IR (neat) 1730 cmꢀ1. MS (ESIꢀ) m/z
323 [MꢀH]ꢀ. HRMS (ESIꢀ) calcd for C18H28O5 [MꢀH]ꢀ: 323.1858,
found 323.1873.
5.1.19. Dimethyl 2-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)malo-
nate (15)
5.1.23. Diethyl 2-(5,9,13-trimethyltetradeca-2,4,8,12-tetraenoyl-
amino)-malonate (20a)
To a mixture of KHMDS (2.14 mL, 2.16 mmol) and dimethylmal-
onate (0.248 mL, 2.16 mmol) in anhydrous THF (6 mL), a solution
of trans–trans-farnesyl bromide 6 (0.391 mL, 1.44 mmol) in THF
(3 mL) was added at 0 °C. After being stirred 5 h at room tempera-
ture, the reaction mixture was poured into H2O then extracted by
ethyl acetate. The organic layer was dried over Na2SO4 and concen-
trated under reduced pressure. The crude product was purified by
column chromatography (heptane then heptane/AcOEt 9:1) to give
pure compound 15 as colorless oil (0.462 g, 92%). 1H NMR
(500 MHz, CDCl3) d 1.61 (s, 3H), 1.62 (s, 3H), 1.65 (s, 3H), 1.70 (s,
3H), 1.97–2.01 (m, 4H), 2.02–2.10 (m, 4H), 2.63 (t, 2H, J = 8.0 Hz),
3.39 (t, 1H, J = 8.0 Hz), 3.74 (s, 6H), 5.09 (m, 3H). 13C NMR
(75 MHz, CDCl3) d 16.0, 17.7, 25.7, 26.5, 26.8, 27.6, 39.7, 51.9,
52.4, 119.4, 123.9, 124.4, 131.3, 135.2, 138.8, 169.6. IR (neat)
1740 cmꢀ1. MS (ESI+) m/z 359 [M+Na]+ HRMS (ESI+) calcd for
C20H32O4Na [M+Na]+: 359.2198, found 359.2192.
Prepared according to general procedure B on 19a (0.060 g,
0.229 mmol) 2 mL solvent for 18 h with N-methylmorpholine
(0.050 mL, 0.457 mmol), EDCI (0.053 g, 0.274 mmol), diethylami-
nomalonate (0.048 g, 0.229 mmol) and HOBt (0.037 g, 0.274
mmol). After work up, the purification was realized by column
chromatography (CH2Cl2 then CH2Cl2/MeOH 9:1) to afford pure
compound 20b as a colorless oil (89.6 mg, 93%). 1H NMR (500
MHz, CDCl3) d 1.30 (t, 6H, J = 7 Hz), 1.59 (s, 6H), 1.66 (s, 3H), 1.87
(s, 3H), 1.95–2.15 (m, 8H), 4.23–4.33 (m, 4H), 5.08 (m, 2H), 5.23
(d, 1H, J = 8.0 Hz), 5.84 (d, 1H, J = 15.0 Hz), 5.97 (d, 1H,
J = 12.0 Hz), 6.42 (d, 1H, J = 6.0 Hz), 7.56 (dd, 1H, J = 15.0 Hz and
J0 = 12.0 Hz). 13C NMR (75 MHz, CDCl3) d 14.0, 16.0, 17.4, 17.7,
25.7, 26.2, 26.7, 39.7, 40.3, 56.6, 62.6, 119.8, 123.1, 124.2, 131.5,
135.9, 139.0, 149.9, 166.1, 166.5. IR (neat) 3350, 1740, 1670,
1520 cmꢀ1. MS (ESI+) m/z 442 [M+Na]+. HRMS (ESI+) calcd for
C24H37NO5Na [M+Na]+: 442.2569, found 442.2563.
5.1.20. 2-(3,7,11-Trimethyl-dodeca-2,6,10-trienyl)malonic acid
(16)
5.1.24. 2-(5,9,13-Trimethyltetradeca-2,4,8,12-tetraenoylamino)-
malonic acid (21a)
Prepared according to general procedure A on 15 (0.100 g,
0.30 mmol) in 2.7 mL solvent with lithium hydroxide (0.041 g,
0.98 mmol) for 2 h. The crude product was purified by column
chromatography on C18 silica (gradient H2O/CH3CN 7:3 to CH3CN
in 15 min) to afford 16 as a colorless oil (0.020 g, 22%). 1H NMR
(500 MHz, CDCl3) d 1.61 (s, 3H), 1.62 (s, 3H), 1.66 (s, 3H), 1.70 (s,
3H), 1.97–2.01 (m, 4H), 2.02–2.10 (m, 4H), 2.69 (t, 2H, J = 7.0 Hz),
3.46 (t, 1H, J = 7.0 Hz), 5.11 (m, 3H). 13C NMR (75 MHz, CDCl3) d
16.1, 17.7, 25.7, 26.5, 26.8, 27.6, 39.7, 51.4, 118.7, 123.8, 124.4,
131.3, 135.3, 139.6, 173.9. MS (ESI+) m/z 331 [M+Na]+. HRMS
(ESI+) calcd for C18H28O4Na [M+Na]+: 331.1885, found 331.1896.
Prepared according to general procedure A on 20a (0.096 g,
0.23 mmol) in 6 mL solvent with lithium hydroxide (0.032 g,
0.76 mmol) for 30 min. After work up, the crude product was puri-
fied by column chromatography (CH2Cl2/MeOH 8:2) to afford pure
compound 21a as a colorless oil (30.3 mg, 37%). 1H NMR (500 MHz,
CD3OD) d 1.61 and 1.63 (s, 6H), 1.67 (s, 3H), 1.89 (s, 3H), 1.91–2.20
(m, 8H), 3. 75 (s, 1H), 5.11 (m, 2H), 6.07 (m, 2H), 7.55 (dd, 1H,
J = 17.0 Hz and J0 = 11.0 Hz). 13C NMR (75 MHz, CD3OD) d 16.2,
17.3, 17.8, 25.9, 27.2, 27.9, 40.8, 41.3, 68.9, 121.8, 124.7, 125.4,
131.9, 138.8, 139.4, 150.2, 169.9. IR (neat) 3340, 2920, 1750,
1640, 1520 cmꢀ1. MS (ESIꢀ) m/z 362 [MꢀH]ꢀ. HRMS (ESIꢀ) calcd
for C20H28NO5 [MꢀH]ꢀ: 362.1952, found 362.1967.
5.1.21. Diethyl 2-(3,7,11-trimethyldodeca-2,6,10-trienyloxy)malo-
nate (17)
5.1.25. Diethyl2-(5,9,13-trimethyltetradeca-4,8,12-trienoylamino)-
malonate (20b)
Diethylhydroxy malonate (0.062 g, 0.35 mmol) was added to a
suspension of sodium hydride (60% in oil, 0.017 g, 0.42 mmol), in
anhydrous THF (10 mL) under argon then trans–trans-farnesyl bro-
mide 6 (0.095 mL, 0.35 mmol) was added. After being stirred 14 h
at room temperature, the reaction mixture was neutralized by 10%
HCl and extracted with dichloromethane. The organic layer was
washed with brine then dried over Na2SO4 and the solvent was re-
moved under reduced pressure. The purification of the crude prod-
uct was realized by column chromatography (heptane/AcOEt 95:5)
to afford pure compound 17 as a colorless oil (55.5 mg, 42%). 1H
NMR (500 MHz, CDCl3) d 1.28 (t, 6H, J = 7.0 Hz), 1.58 (s, 3H), 1.60
(s, 3H), 1.65 (s, 3H), 1.68 (s, 3H), 1.96–2.07 (m, 8H), 2.76 (d, 2H,
J = 8.0 Hz), 3.71 (s, 1H), 4.24 (q, 4H, J = 7.0 Hz), 5.09 (m, 3H). 13C
NMR (75 MHz, CDCl3) d 14.0, 16.2, 16.7, 25.9, 26.5, 26.7, 39.9,
51.4, 65.4, 95.3, 116.1, 123.9, 124.4, 131.5, 139.5, 140.7, 172.8. IR
(neat) 1730 cmꢀ1. MS (ESI+) m/z 403 [M+Na]+. HRMS (ESI+) calcd
for C22H36O5Na [M+Na]+: 403.2460, found 403.2450.
Prepared according to general procedure B on 19b (0.100 g,
0.39 mmol) 5 mL solvent for 4 h with N-methylmorpholine
(0.085 mL, 0.77 mmol), EDCI (0.0887 g, 0.46 mmol), diethylamino-
malonate (0.0816 g, 0.39 mmol) and HOBt (0.0625 g, 0.46 mmol).
After work up and purification by column chromatography (hep-
tane/AcOEt 8:2) pure compound 20b was obtained as a colorless
oil (157.9 mg, 98%). 1H NMR (500 MHz, CDCl3) d 1.32 (t, 6H,
J = 7.0 Hz), 1.61 (s, 6H), 1.65 (s, 3H), 1.70 (s, 3H), 1.90–2.03 (m,
4H), 2.06–2.12 (m, 4H), 2.31–2.39 (m, 4H), 4.23–4.34 (m, 4H),
5.12 (m, 4H), 6.48 (d, 1H, J = 6.5 Hz). 13C NMR (75 MHz, CDCl3) d
14.2, 16.1, 17.9, 23.9, 25.5, 26.9, 36.1, 40.1, 56.4, 62.8, 122.3,
124.5, 128.6, 137.2, 144.3, 166.5, 172.7. IR 1730, 1640,
1540 cmꢀ1. MS (ESI+) m/z 444 [M+Na]+. HRMS (ESI+) calcd for
C24H39NO5Na [M+Na]+: 444.2726, found 444.2728.
5.1.26. 2-(5,9,13-Trimethyltetradeca-4,8,12-trienoylamino)mal-
onic acid (21b)
5.1.22. 2-(3,7,11-Trimethyldodeca-2,6,10-trienyloxy)malonic
acid (18)
Prepared according to general procedure A on 20b (0.1579 g,
0.38 mmol) in 4.5 mL solvent with lithium hydroxide (0.0521 g,
1.24 mmol) for 1 h. Compound 21a was obtained as a pure white
solid (0.137 g, 100%). 1H NMR (500 MHz, CDCl3) d 1.60 (s, 9H),
1.67 (s, 3H), 1.95–2.07 (m, 8H), 2.32–2.40 (m, 4H), 5.10 (m, 3H),
5.17 (d, 1H, J = 7.0 Hz), 7.22 (d, 1H, J = 7.0 Hz), 9.17 (br s, 2H). 13C
NMR (75 MHz, CDCl3) d 16.0, 16.1, 17.7, 23.8, 25.7, 26.7, 26.8, 35.
Prepared according to general procedure A on 17 (55.5 mg,
0.146 mmol) in 3 mL solvent with lithium hydroxide (20.2 mg,
0.482 mmol) for 4 h. The product 18 was obtained as a pure color-
less oil (47 mg, 100%). 1H NMR (500 MHz, CDCl3) d 1.60 (s, 3H),
1.61 (s, 3H), 1.66 (s, 3H), 1.69 (s, 3H), 1.97–2.08 (m, 8H), 2.79 (d,

552
S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
8, 39.7, 39.7, 56.6, 121.7, 124.0, 124.4, 131.3, 135.1, 137.7, 168.3,
175.4. IR (neat) 3320, 1730, 1640, 1540 cmꢀ1. MS (ESI+) m/z 388
[M+Na]+. HRMS (ESI+) calcd for C20H31NO5Na [M+Na]+: 388.2100,
found 388.2137.
98:2 then 95:5) to afford pure compound 24 as a colorless oil
(10.5 mg, 8%). 1H NMR (500 MHz, CDCl3) d 1.62 (s, 6H), 1.66 and
1.70 (s, 6H), 1.98–2.12 (m, 8H), 2.38 (m, 2H), 2.50 (m, 2H), 5.13
(m, 3H). 13C NMR (75 MHz, CDCl3) d 16.4, 18.1, 23.2, 26.1, 26.9,
27.2, 35.9, 40.0, 121.7, 124.4, 124.8, 131.7, 135.6, 137.9, 169.5. IR
(neat) 1709, 1438 cmꢀ1. MS (ESIꢀ) m/z 342 [MꢀH]ꢀ. HRMS (ESIꢀ)
calcd for C17H28NO4S [MꢀH]ꢀ: 342.1739, found 342.1737.
5.1.27. (2E,4E,8E) 5,9,13-Trimethyltetradeca-2,4,8,12-tetraen-
amide (22a)
Prepared according to general procedure C on 19a (50 mg,
0.19 mmol) with triethylamine (31
l
L, 0.21 mmol) and ethyl chlo-
5.1.31. (4E,8E)-5,9,13-Trimethyl-N-(phenylsulfonyl)tetradeca-
4,8,12-trienamide (25)
roformate (21 L, 0.21 mmol) in 0.4 mL solvent. The ammonium
l
hydroxide solution was prepared with KOH, 0.69 M in methanol
(0.4 mL, 0.27 mmol) and ammonium chloride (14 mg, 0.27 mmol).
After work up, the residue was purified by preparative TLC (CH2Cl2/
MeOH 9:1) to afford 22a as a white powder (26.4 mg, 50%). 1H
NMR (300 MHz, CD3OD) d 1.62, 1.64 and 1.69 (s, 9H), 1.93 (s,
3H), 1.97–2.16 (m, 4H), 2.19–2.24 (m, 4H), 5.07–5.18 (m, 2H),
5.95 (dl, 1H, J = 14.5 Hz), 6.04 (d, 1H, J = 11.5 Hz), 7.53 (dd, 1H,
J = 11.5 Hz and J0 = 14.5 Hz). 13C NMR (75 MHz, CD3OD) d 17.0,
18.1, 18.6, 26.8, 28.1, 28.6, 41.7, 42.1, 123.2, 125.4, 125.6, 126.2,
133.0, 137.6, 140.1, 150.5, 172.7. IR 3332, 3189, 2968, 2930,
1660, 1593, 1397. MS (ESI+) m/z 284 [M+Na]+.
Prepared according to general procedure B on 19a (0.035 g,
0.134 mmol) 2 mL solvent with N-methylmorpholine (0.029 mL,
0.268 mmol), EDCI (0.031 g, 0.161 mmol), benzenesulfonamide
(0.021 g, 0.134 mmol) and HOBt (0.022 g, 0.161 mmol) for 18 h.
The purification of the crude product was realized by preparative
TLC (CH2Cl2/MeOH 95:5) to afford pure compound 25 as a colorless
oil (38 mg, 77%). 1H NMR (500 MHz, CDCl3) d 1.58 and 1.61 (s, 6H),
1.62 and 1.70 (s, 6H), 1.96–2.10 (m, 8H), 2.30 (m, 4H), 5.04 (m, 1H),
5.09 (m, 2H), 7.57 (t, 2H, J = 7.0 Hz), 7.57 (t, 1H, J = 8.0 Hz), 8.08 (t,
2H, J = 8.0 Hz). 13C NMR (75 MHz, CDCl3) d 16.0, 16.1, 17.7, 23.0,
25.7, 26.5, 26.7, 36.5, 39.6, 39.7, 121.2, 123.8, 124.3, 128.4, 128.9,
131.7, 133.9, 135.3, 138.4, 138.9, 170.2. MS (ESI+) m/z 426
[M+Na]+. HRMS (ESI+) calcd for C23H33NO3SNa [M+Na]+: 426.2079,
found 426.2091.
5.1.28. (4E,8E)-5,9,13-Trimethyltetradeca-4,8,12-trienamide (22b)
Prepared according to general procedure C on 19b (52 mg,
0.19 mmol) with triethylamine (31
lL, 0.21 mmol) and ethyl chlo-
roformate (21 L, 0.21 mmol) in 0.4 mL solvent. The ammonium
l
5.1.32. (E)-Diethyl 2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)allylmalonate (30)
hydroxide solution was prepared with KOH, 0.69 M in methanol
(0.4 mL, 0.27 mmol) and ammonium chloride (14 mg, 0.27 mmol).
After work up, the residue was purified by preparative TLC (CH2Cl2/
MeOH 95:5) to afford 22b as a white powder (30 mg, 54%). 1H NMR
(300 MHz, CDCl3) d 1.60, 1.61, 1.64 and 1.69 (s, 12H), 1.95–2.12 (m,
8H), 2.26 (t, 2H, J = 7.0 Hz), 2.34 (td, 2H, J = 6.5 Hz and J0 = 7.0 Hz),
5.05 (m, 3H). 13C NMR (75 MHz, CDCl3) d 16.4, 18.0, 24.3, 26.0,
26.8, 27.1, 36.3, 40.0, 40.1, 122.8, 124.3, 131.7, 135.5, 137.5,
175.7. IR (neat) 3339, 3189, 2966, 2919, 2853, 1659, 1445,
1380. MS (ESI+) m/z 286 [M+Na]+.
Pinacol vinyl boronate (3.123 mL, 18.42 mmol) was added to a
solution of diethylallylmalonate (1.218 mL, 6.14 mmol) in dichlo-
romethane (60 mL). The reaction mixture was warmed to 40 °C.
Grubbs I catalyst (0.758 g, 0.91 mmol) was added in three times
every hour. After being stirred 3 h at 40 °C, the reaction mixture
was cooled to room temperature and the catalyst was removed
by filtration on silica gel. The solvent was removed under reduced
pressure and the crude product was purified by column chroma-
tography (heptane/AcOEt 9:1) to afford pure compound 30 as an
oil (1.8367 g, 92%). 1H NMR (500 MHz, CDCl3) d 1.24 (m, 12H),
2.73 (t, 2H, J = 7.5 Hz), 3.46 (t, 1H, J = 7.0 Hz), 4.18 (q, 4H,
J = 7.5 Hz), 5.50 (d, 1H, J = 18.0 Hz), 6.52 and 6.57 (td, 1H,
J = 18.0 Hz and J0 = 7.0 Hz). 13C NMR (75 MHz, CDCl3) d 14.2, 24.7,
34.6, 51.0, 61.6, 83.2, 121.0, 148.8, 168.8. IR (neat) 1731,
1361 cmꢀ1. MS (ESI+) m/z 349 [M+Na]+. HRMS (ESI+) calcd for
C16H27O6BNa [M+Na]+: 349.1798, found 349.1814.
5.1.29. (4E,8E)-N-Hydroxy-5,9,13-trimethyltetradeca-4,8,12-tri-
enamide (23)
To a solution of 19a (0.064 g, 0.24 mmol) in dichloromethane
(1 mL), N-methylmorpholine (0.058 mL, 0.53 mmol), EDCI
(0.056 g, 0.29 mmol), hydroxylamine (0.017 g, 0.24 mmol) and
HOBt (0.039 g, 0.29 mmol) were added. After being stirred for 3 h
at room temperature, the reaction mixture was hydrolyzed by
H2O then extracted by dichloromethane. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. La
purification of the crude product was realized by column chroma-
tography (heptane/AcOEt 9:1) to give pure compound 33 as color-
less oil (67 mg, 100%). 1H NMR (500 MHz, CDCl3) d 1.61 (s, 6H),
1.65 (s, 3H), 1.70 (s, 3H), 1.90–2.10 (m, 8H), 2.29–2.54 (m, 4H),
5.12–5.16 (m, 4H), 8.87 (br s, 1H). MS (ESI+) m/z 302 [M+Na]+.
5.1.33. (E)-Diethyl 2-(3-(5-((tert-butyldimethylsilyloxy)methyl)-
1-methyl-1H-imidazol-2-yl)allylmalonate (28a)
Compound 30 (1.202 g, 3.08 mmol), sodium bicarbonate
(0.358 g, 3.38 mmol) and tetrakis(triphenylphosphine) palladium
(0.178 g, 0.154 mmol) were added to
a solution of 29 (1 g,
3.08 mmol) in DMF (6 mL). The reaction mixture was warmed to
130 °C and stirred for 4 h. The reaction was allowed to cool to room
temperature and was extracted with ethyl acetate and H2O. The
crude product was purified by column chromatography (gradient
from heptane/AcOEt 7:3 to heptane/AcOEt 2:8) to afford pure com-
pound 28a as a yellow oil (1.05 g, 80%). 1H NMR (500 MHz, CDCl3) d
0.08 (s, 6H), 0.91 (s, 9H), 1.29 (t, 6H, J = 7.0 Hz), 2.86 (t, 2H,
J = 7.0 Hz), 3.53 (t, 1H, J = 7.0 Hz), 3.61 (s, 3H), 4.20 (m, 4H), 4.69
(s, 2H), 6.38 (d, 1H, J = 15.5 Hz), 6.65 (m, 1H), 6.89 (s, 1H). 13C
NMR (75 MHz, CDCl3) d ꢀ5.0, 14.4, 26.1, 30.5, 32.6, 51.9, 55.7,
61.9, 119.1, 127.7, 130.8, 131.8, 146.3, 169.0. IR (neat) 1730,
5.1.30. (4E, 8E)-5,9,13-Trimethyltetradeca-4,8,12-trienoylsulfamic
acid (24)
To a solution of 19a (0.100 g, 0.38 mmol) in dichloromethane
(1 mL) at ꢀ78 °C, pivaloyl chloride (0.047 mL, 0.38 mmol) was
added and the reaction mixture was stirred for 1 h at ꢀ78 °C. In an-
other flask, acetic acid (0.002 mL, 0.034 mmol) and distilled trieth-
ylamine (0.057 mL, 0.40 mmol) were introduced successively to a
solution of sulfamic acid (0.033 g, 0.34 mmol) in dichloromethane
(1 mL). The mixture was warmed to 30 °C for 10 min then cooled to
0 °C. The first solution was added to the second one. After being
stirred for 1 h at 0 °C, H2O was added and the reaction mixture
was extracted by dichloromethane. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography (heptane/AcOEt
1050 cmꢀ1
.
5.1.34. Diethyl 2-(3-(5-((tert-butyldimethylsilyloxy)methyl)-1-
methyl-1H-imidazol-2-yl)propyl) malonate (28b)
Palladium on activated carbon Pd 10% (0.300 g, 88% w/w) was
added to 28a (0.340 g, 0.802 mmol) in ethyl acetate (8 mL). The

S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
553
reaction mixture was stirred under hydrogen atmosphere at room
temperature for 18 h. The mixture was filtered over silica gel and
washed with ethyl acetate, methanol and dichloromethane. The
solvent was removed under reduced pressure to afford pure com-
pound 28b as a colorless oil (341 mg, 100%). 1H NMR (500 MHz,
CDCl3) d 0.00 (s, 6H), 0.84 (s, 9H), 1.22 (t, 6H, J = 7.0 Hz), 1.78 (m,
2H), 1.95 (m, 2H), 2.65 (t, 2H, J = 7.0 Hz), 3.34 (t, 1H, J = 7.0 Hz),
3.50 (s, 3H), 4.14 (dd, 4H, J = 7.0 Hz, J0 = 2.0 Hz), 4.57 (s, 2H), 6.76
(s, 1H). 13C NMR (75 MHz, CDCl3) d ꢀ5.3, 14.1, 25.3, 25.8, 26.7,
28.4, 30.2, 51.8, 55.5, 61.4, 125.8, 129.0, 146.3, 169.3. MS (ESI+)
m/z 427 [M+H]+. HRMS (ESI+) calcd for C21H39N2O5Si [M+H]+:
427.2628, found 427.2607.
1.95 (q, 2H, J = 7.5 Hz), 2.70 (t, 2H, J = 7.5 Hz), 3.34 (t, 1H,
J = 7.5 Hz), 3.59 (s, 3H), 4.14 (m, 4H), 4.57 (s, 2H), 6.77 (s, 1H).
13C NMR (75 MHz, CDCl3) d 14.0, 25.2, 26.6, 28.3, 30.2, 51.7, 54.4,
61.4, 125.7, 131.3, 148.8, 169.3. MS (ESI+) m/z 313 [M+H]+. HRMS
(ESI+) calcd for C15H25N2O5 [M+H]+: 313.1363, found 313.1753.
Activated manganese dioxide(IV) (0.272 g, 3.12 mmol) was then
added to the colorless oil (0.150 g, 0.48 mmol) in 5 mL solvent to
afford 32b (95 mg, 64%) as a colorless oil. 1H NMR (500 MHz,
CDCl3) d 1.29 (t, 6H, J = 9.0 Hz), 1.79 (qi, 2H, J = 7.0 Hz), 2.01 (q,
2H, J = 7.0 Hz), 2.80 (t, 2H, J = 7.0 Hz), 3.41 (t, 1H, J = 7.0 Hz), 3.88
(s, 3H), 4,20 (m, 4H), 7.71 (s, 1H), 9.69 (s, 1H).
5.1.38. Diethyl 2-((E)-3-(5-formyl-1-methyl-1H-imidazol-2-yl)-
allyl)-2-((2E,6E)-3,7,11-trimethyl dodeca-2,6,10-trienyl)malon-
ate (33a)
5.1.35. 2-((6E,10E)-4,4-Bis(ethoxycarbonyl)-7,11,15-trimethylhex-
adeca-6,10,14-trienyl)-5-(hydroxymethyl)-1-methyl-3-((2E,6E)-
3,7,11-trimethyldodeca-2,6,10-trienyl)-1H-imidazol-3-ium (31)
Prepared according to general procedure E on 28b (0.267 g,
Prepared according to general procedure E on 32a (0.073 g,
0.237 mmol) with NaH (0.057 g, 0.237 mmol) and 6 (0.064 mL,
0.237 mmol) in 2 mL solvent. Compound 33a was obtained after
work up as gray oil (0.123 g, 100%). 1H NMR (500 MHz, CDCl3) d
1.26 (t, 6H, J = 8.0 Hz), 1.62 (s, 6H), 1.64 (s, 3H), 1.70 (s, 3H),
1.99–2.09 (m, 8H), 2.69 (d, 2H, J = 6.0 Hz), 2.89 (t, 2H, J = 6.0 Hz),
3.92 (s, 3H), 4.21 (q, 4H, J = 7.0 Hz), 5.03 (t, 1H, J = 8.0 Hz), 5.11
(t, 2H, J = 7.0 Hz), 6.35 (d, 1H, J = 16.0 Hz), 6.86 (m, 1H), 7.75 (s,
1H), 9.69 (s, 1H). 13C NMR (75 MHz, CDCl3) d 14.2, 16.0, 16.5,
17.7, 25.7, 26.5, 26.7, 30.9, 31.3, 36.4, 39.7, 40.0, 57.7, 61.4,
117.3, 117.9, 123.8, 124.3, 131.9, 132.4, 135.3, 136.4, 139.6,
144.0, 151.3, 170.7, 178.9. IR (neat) 1727, 1669 cmꢀ1. MS (ESI+)
m/z 535 [M+Na]+. HRMS (ESI+) calcd for C30H44N2O5Na [M+Na]+:
535.3148, found 535.3132.
0.96 mmol) with NaH (0.046 g, 1.15 mmol) and
6 (0.26 mL,
0.96 mmol) in 1 mL solvent for 3 h. After work up, the crude prod-
uct was purified by column chromatography (gradient CH2Cl2 to
CH2Cl2/MeOH 9:1 in 15 min then CH2Cl2/MeOH 9:1 for 10 min)
to afford compound 33b as a colorless oil (0.345 g, 50%). 1H NMR
(500 MHz, CDCl3) d 1.26 (t, 6H, J = 7.5 Hz), 1.61 (m, 17H), 1.69
(m, 6H), 1.82 (s, 3H), 1.90 (m, 2H), 1.96–2.13 (m, 16H), 2.61 (d,
2H, J = 8.0 Hz), 3.03 (t, 2H, J = 8.0 Hz), 3.90 (s, 3H), 4.18 (q, 4H,
J = 7.5 Hz), 4.69 (m, 4H), 4.95 (t, 1H, J = 8.0 Hz), 5.09 (m, 4H), 5.28
(t, 1H, J = 8.0 Hz), 7.17 (s, 1H), 7,72 (s, 1H). 13C NMR (75 MHz,
CDCl3) d 14.1, 16.0, 16.4,16.9, 17.7, 22.5, 24.5, 25.7, 26.1, 26.6,
26.7, 30.9, 32.4, 32.6, 33.1, 39.4, 39.7, 40.0, 46.4, 52.7, 57.3, 61.5,
115.2, 117.0, 119.2, 123.0, 123.7, 124.1, 124.2, 131.4, 131.5,
134.5, 135.4, 136.1, 139.8, 145.8, 145.9, 171.0. MS (ESI+) m/z 721
[M]+. HRMS (ESI+) calcd for C45H73N2O5 [M]+: 721.5519, found
721.5542.
5.1.39. Dimethyl 2-(3-(5-formyl-1-methyl-1H-imidazol-2-ylpro-
pyl)-2-((2E,6E)-3,7,11-trimethyl dodeca-2,6,10-trienyl)malonate
(33b)
Prepared according to general procedure E on 32b (0.095 g,
0.306 mmol) with NaH (0.008 g, 0.337 mmol) and 6 (0.083 mL,
0.306 mmol) in 5 mL solvent. Compound 33b was obtained after
work up as a colorless oil (121 mg, 77%). 1H NMR (500 MHz, CDCl3)
d 1.25 (t, 6H, J = 7.0 Hz), 1.61 (m, 9H), 1.72 (s, 3H), 1.72–1.77 (m,
2H), 1.97–2.10 (m, 12H), 2.66 (d, 2H, J = 8.0 Hz), 2.76 (t, 2H,
J = 8.0 Hz), 3.89 (s, 3H), 4,18 (q, 4H, J = 7.0 Hz), 4.98 (t, 1H,
J = 8.0 Hz), 5.11 (m, 2H), 7.71 (s, 1H), 9.68 (s, 1H). 13C NMR
(75 MHz, CDCl3) d 14.1, 16.0, 16.4, 17.7, 22.2, 25.7, 26.6, 26.8,
30.0, 31.9, 32.3, 39.7, 57.4, 61.3, 117.5, 123.9, 124.5, 131.7, 135.4,
136.3, 139.8, 150.5, 158.2, 171.6, 179.1. IR (neat) 1727,
1669 cmꢀ1. MS (ESI+) m/z 515 [M+H]+. HRMS (ESI+) calcd for
C30H47N2O5 [M+H]+: 515.3485, found 515.3478.
5.1.36. (E)-Diethyl 2-(3-(5-formyl-1-methyl-1H-imidazol-2-yl)-
allyl)malonate (32a)
Prepared according to general procedure D on 28a (0.300 g,
0.707 mmol) in 4 mL solvent with TBAF (1.41 mL, 1.41 mmol) for
2 h. After work up, the crude product was purified by column chro-
matography (gradient CH2Cl2 to CH2Cl2/MeOH 9:1 in 15 min) to af-
ford pure alcohol as a yellow oil (115 mg, 52%). 1H NMR (500 MHz,
CDCl3) d 1.28 (t, 6H, J = 8.0 Hz), 2.86 (t, 2H, J = 7.0 Hz), 3.53 (t, 1H,
J = 8.0 Hz), 3.65 (s, 3H), 4.21 (m, 4H), 4.61 (s, 2H), 6.38 (d, 1H,
J = 15.0 Hz), 6.63 (m, 1H), 6.90 (s, 1H). 13C NMR (75 MHz, CDCl3)
d 14.1, 30.2, 32.2, 51.5, 54.6, 61.6, 118.6, 127.9, 131.2, 131.4,
146.7, 168.7. MS (ESI+) m/z 333 [M+Na]+. HRMS (ESI+) calcd for
C15H22N2O5Na [M+Na]+: 333.1426, found 333.1435. Activated
manganese dioxide(IV) (0.208 g, 2.39 mmol) was then added to
the yellow oil (0.124 g, 0.399 mmol) in 3 mL solvent to afford
32a (104 mg, 85%) as a colorless oil. 1H NMR (500 MHz, CD3OD) d
1.28 (t, 6H, J = 8.0 Hz), 2.91 (t, 2H, J = 7.0 Hz), 3.54 (t, 1H,
J = 8.0 Hz), 3.93 (s, 3H), 4.22 (m, 4H), 6.44 (d, 1H, J = 15.0 Hz),
6.92 (m, 1H), 7.72 (s, 1H), 9.68 (s, 1H). 13C NMR (75 MHz, CDCl3)
d 14.1, 31.9, 32.2, 51.1, 61.7, 117.1, 131.8, 137.0, 143.9, 151.3,
168.4, 178.8. IR (neat) 1726, 1660 cmꢀ1. MS (ESI+) m/z 333
[M+Na]+. HRMS (ESI+) calcd for C15H22N2O5Na [M+Na]+:
333.1426, found 333.1435.
5.1.40. Methyl N-((2-((1E,6E,10E)-4,4-bis(ethoxycarbonyl)-7,11,15-
trimethylhexadeca-1,6,10,14-tetraen-1-yl)-1-methyl-1H-imidazol-
5-yl)methyl)-L-valyl-L-phenylalanyl-L-methioninate (34a)
According to general procedure F on 33a (0.112 g, 0.22 mmol)
with tripeptide (0.135 g, 0.26 mmol) deprotected with TFA
(2.7 mL, 75%), triethylamine (0.030 mL, 0.22 mmol) and NaBH3CN
(0.016 g, 0.26 mmol). The crude product was purified by column
chromatography (gradient from CH2Cl2 to CH2Cl2/MeOH 9:1 in
15 min) to afford pure compound 34a as a colorless oil (67 mg,
34%). 1H NMR (500 MHz, CDCl3)
d 0.74 and 0.82 (d, 6H,
J = 7.0 Hz), 1.27 (t, 6H, J = 7.0 Hz), 1.61 (s, 9H), 1.66 (s, 3H), 1.69
(s, 3H), 1.76–1.92 (m, 3H), 1.94- 2.14 (m, 11H), 2.43 (t, 2H,
J = 7.0 Hz), 2.69 (t, 2H, J = 7.0 Hz), 2.87 (m, 3H), 3.14 (m, 2H), 3.49
(s, 3H), 3.72 (s, 3H), 3.84 (s, 2H), 4.19 (m, 4H), 4.64 (q, 1H,
J = 7.0 Hz), 4.75 (q, 1H, J = 7.0 Hz), 5.06 (m, 1H), 5.20 (m, 2H),
6.35 (d, 1H, J = 15.0 Hz), 6.70 (m, 1H), 7.22–7.33 (m, 6H). 13C
NMR (75 MHz, CDCl3) d 14.1, 15.4, 15.5, 16.0, 17.9, 17.9, 19.4,
25.7, 26.6, 26.7, 29.7, 30.2, 31.0, 36.2, 38.0, 39.7, 40.0, 42.8, 51.6,
52.5, 54.0, 57.9, 61.3, 67.9, 117.5, 119.9, 123.9, 124.3, 126.9,
5.1.37. Diethyl 2-(3-(5-formyl-1-méthyl-1H-imidazol-2-yl)propyl)-
malonate (32b)
Prepared according to general procedure D on 28b (0.342 g,
0.803 mmol) in 4 mL solvent with TBAF (1.60 mL, 1.60 mmol) for
3 h. After work up, the crude product was purified by column chro-
matography (gradient CH2Cl2 to CH2Cl2/MeOH 9:1 in 15 min) to af-
ford pure compound 32b as a colorless oil (70.5 mg, 29%). 1H NMR
(500 MHz, CDCl3) d 1.27 (t, 6H, J = 7.0 Hz), 1.78 (qi, 2H, J = 7.5 Hz),

554
S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
127.1, 128.6, 129.2, 136.2, 131.3, 135.2, 136.4, 139.3, 146.0, 170.9,
171.8, 173.8. IR (neat) 3295, 1730, 1644, 1442 cmꢀ1. MS (ESI+) m/z
906 [M+H]+. HRMS (ESI+) calcd for C50H76N5O8S [M+H]+: 906.5415,
found 906.5396.
130.7, 132.2, 136.3, 138.8, 140.5, 150.0, 172.9, 173.0, 174.2,
174.7. IR (neat) 3300, 1730, 1650 cmꢀ1 MS (ESI+) m/z 866
.
[M+H]+. HRMS (ESI+) calcd for C47H72N5O8S [M+H]+: 866.5102
found 866.5071.
5.1.41. Diethyl 2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl)-
5.1.44. (E)-Diethyl-2-(3-(1-methyl-1H-imidazol-2-yl)allyl)mal-
onate (37)
malonate-1-methyl-1H-imidazol-5-yl)methyl)-L-valyl-L-phenylal-
anyl- -methioninate (34b)
L
2-Iodo-1-(1H)-methyl-imidazole (0.435 g, 2.09 mmol), sodium
bicarbonate (0.244 g, 2.30 mmol) and tetrakis(triphenylphos-
phine)palladium (0.121 g, 0.10 mmol) were added to a solution
of 30 (3.818 g, 2.51 mmol) in DMF (6 mL). The reaction mixture
was warmed to 130 °C and stirred for 3 h. The reaction was
stopped by filtration on silica gel and extracted with ethyl acetate
and H2O. The organic layer was dried over Na2SO4. The crude prod-
uct was purified by chromatography on silica gel (CH2Cl2/MeOH
98:2) to afford pure compound 37 as a brown oil (497.4 mg,
85%). 1H NMR (500 MHz, CDCl3) d 1.27 (m, 6H), 2.86 (td, 2H,
J = 7.5 Hz, J0 = 2.0 Hz), 3.53 (t, 1H, J = 7.5 Hz), 3.63 (s, 3H), 4.22 (m,
4H), 6.38 (d, 1H, J = 15.5 Hz), 6.59 (m, 1H), 6.82 (s, 1H), 7.00 (s,
1H). 13C NMR (75 MHz, CDCl3) d 14.1, 32.2, 32.7, 51.6, 61.6,
118.4, 121.1, 128.3, 130.6, 144.9, 168.7. MS (ESI+) m/z 281
[M+H]+. HRMS (ESI+) calcd for C14H21N2O4 [M+H]+: 281.1501,
found 281.1507.
According to general procedure F on 33b (0.066 g, 0.128 mmol)
with tripeptide (0.052 g, 0.128 mmol) deprotected with TFA
(1.6 mL, 75%), triethylamine (0.018 mL, 0.128 mmol) and NaBH3CN
(0.010 g, 0.154 mmol). The crude product was purified by column
chromatography (gradient from CH2Cl2 to CH2Cl2/MeOH 9:1 in
15 min) to afford pure product 34b as a colorless oil (67 mg,
34%). 1H NMR (500 MHz, CDCl3)
d 0.82 and 0.85 (d, 6H,
J = 7.0 Hz), 1.26 (t, 6H, J = 7.0 Hz), 1.60, 1.62 and 1.63 (s, 9H), 1.69
(s, 3H), 1.87–2.14 (m, 17H), 2.46 (t, 2H, J = 7.0 Hz), 2.64 (d, 2H,
J = 8 Hz), 2.79 (m, 2H), 2.83 (d, 2H, J = 7.0 Hz), 3.04–3.20 (m, 2H),
3.45–3.60 (m, 2H), 3.57 (s, 3H), 3.74 (s, 3H), 4.18 (q, 4H,
J = 7.0 Hz), 4.64 (t, 1H, J = 8.0 Hz), 4.79 (q, 1H, J = 8.0 Hz), 4.99 (t,
1H, J = 8.0 Hz), 5.08 (m, 2H), 6.73 (s, 1H), 6.80 (d, 1H, J = 7.0 Hz),
7.20–7.32 (m, 5H), 7.41 (d, 1H, J = 8.0 Hz). 13C NMR (75 MHz,
CDCl3) d 14.1, 15.4, 16.0, 16.4, 17.7, 18.2, 19.4, 25.7, 26.7, 29.7,
30.8, 31.6, 39.7, 40.0, 46.3, 51.6, 52.5, 54.1, 57.5, 61.4, 67.7,
117.4, 123.9, 124.3, 127.1, 128.7, 129.3, 135.2, 136.6, 139.1,
148.2, 171.0, 171.4, 171.8, 173.8. IR (neat) 3295, 1730, 1644,
1442 cmꢀ1. MS (ESI+) m/z 908 [M+H]+. HRMS (ESI+) calcd for
C50H78N5O8S [M+H]+: 908.5571, found 908.5546.
5.1.45. Diethyl 2-(3-(1-methyl-1H-imidazol-2-yl)propyl)-2-
((1E,5E)-2,6,10-trimethyldodeca-1,5,9-trienyl)malonate (38)
Palladium on activated carbon Pd 10% (0.140 g, 14% w/w) was
added to 37 (0.100 g, 0.357 mmol) in ethyl acetate (4 mL). The
reaction mixture was stirred under hydrogen atmosphere at room
temperature for 4 h at room temperature. The mixture was filtered
over silica gel and washed with ethyl acetate, methanol and dichlo-
romethane. The solvent was removed under reduced pressure to
afford pure compound 38 as an orange oil (94.1 mg, 93%). 1H
NMR (500 MHz, CDCl3) d 1.27 (m, 6H), 1.84 (q, 2H, J = 7.0 Hz),
2.01 (q, 2H, J = 7.0 Hz), 2.73 (t, 2H, J = 8.0 Hz), 3.39 (t, 1H,
J = 8.0 Hz), 3.60 (s, 3H), 4.21 (m, 4H), 6.81 (s, 1H), 6.95 (s, 1H).
13C NMR (75 MHz, CDCl3) d 14.5, 25.8, 26.6, 28.7, 37.7, 52.0, 61.8,
120.9, 127.2, 147.5, 169.7. MS (ESI+) m/z 283 [M+H]+. HRMS
(ESI+) calcd for C14H23N2O4 [M+H]+: 283.1658, found 283.1652.
The orange oil (0.0764 g, 0.271 mmol) was added to a suspension
of NaH, 60% in oil (0.011 g, 0.271 mmol) in anhydrous THF (2 mL)
at 0 °C. The reaction mixture was stirred 1 h at 0 °C then trans–
trans-farnesyl bromide 6 (0.073 mL, 0. 271 mmol) was added. After
being stirred 1 h 30 min at 0 °C, the reaction mixture was extracted
with ethyl acetate and washed with H2O. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The
crude product was purified by column chromatography (gradient
from CH2Cl2 to CH2Cl2/MeOH 9:1 in 30 min) to afford pure product
38 as a colorless oil (85.1 mg, 64%). 1H NMR (500 MHz, CDCl3) d
1.25 (t, 6H, J = 7.0 Hz), 1.60 and 1.62 (s, 9H), 1.69 (m, 5H), 1.97–
2.08 (m, 10H), 2.64 (d, 2H, J = 7.0 Hz), 2.75 (t, 2H, J = 8.0 Hz), 3.61
(s, 3H), 4.17 (qi, 4H, J = 8.0 Hz), 4.98 (t, 1H, J = 8.0 Hz), 5.11 (m,
2H), 6.81 (s, 1H), 6.99 (s, 1H). 13C NMR (75 MHz, CDCl3) d 14.1,
16.0, 16.3, 17.7, 22.8, 24.9, 25.7, 26.6, 31.0, 32.0, 32.8, 39.7, 40.0,
57.5, 61.2, 117.6, 120.5, 123.9, 124.3, 128.6, 131.3, 135.2, 139.1,
147.7, 171.4. IR (neat) 1727 cmꢀ1. MS (ESI+) m/z 487 [M+H]+. HRMS
(ESI+) calcd for C29H47N2O4 [M+H]+: 487.3536, found 487.3519.
5.1.42. N-((2-((1E,6E,10E)-4-Carboxy-4-(ethoxycarbonyl)-7,11,15-
trimethylhexadeca-1,6,10,14-tetraen-1-yl)-1-methyl-1H-imid-
azol-5-yl)methyl)-L-valyl-L-phenylalanyl-L-methionine (35a)
Prepared according to general procedure G on 34a (0.046 g,
0.051 mmol) with LiOH (0.021 g, 0.51 mmol) in 2 mL solvent for
seven days. After work up, compound 35a was obtained as a color-
less oil (42 mg, 100%). 1H NMR (500 MHz, CD3OD) d 1.00 and 1.09
(d, 6H, J = 7.0 Hz), 1.29 (t, 3H, J = 6.0 Hz), 1.63 (s, 6H), 1.70 (s, 6H),
2.00–2.20 (m, 14H), 2.56 (m, 2H), 2.62 (m, 2H), 2.95 (m, 3H), 3.27–
3.41 (m, 2H), 3.60 (m, 1H), 3.68 (s, 3H), 4.10 (m, 1H), 4.24 (m, 2H),
4.60 (m, 1H), 4.91 (m, 1H), 5.11 (m, 3H), 6.75 (d, 1H, J = 16.0 Hz),
6.94 (m, 1H), 7.17 (t, 1H, J = 7.0 Hz), 7.32 (t, 2H, J = 7.0 Hz), 7.40
(d, 2H, J = 8.0 Hz), 7.58 (s, 1H). 13C NMR (75 MHz, CDCl3) d 4.4,
15.2, 16.2, 16.7, 17.8, 18.6, 19.3, 25.9, 27.5, 27.8, 29.8, 31.2, 31.8,
32.2, 33.1, 38.1, 39.2, 40.2, 40.9, 52.6, 55.8, 59.1, 62.7, 67.8,
115.0, 118.7, 122.7, 125.1, 125.4, 128.0, 129.7, 130.7, 138.7,
132.2, 136.4, 143.4, 145.4, 145.8, 172.2, 172.9, 173.5, 174.7. MS
(ESI+) m/z 864 [M+H]+. HRMS (ESI+) calcd for C47H70N5O8S
[M+H]+: 864.4945 found 864.4958.
5.1.43. N-((2-((4E,8E)-4-Carboxy-4-(ethoxycarbonyl)-7,11,15-tri-
methylhexadeca-1,6,10,14-tetraen-1-yl)-1-methyl-1H-imidazol-
5-yl)methyl)-L-valyl-L-phenylalanyl-L-methionine (35b)
Prepared according to general procedure G on 34b (0.025 g,
0.027 mmol) with LiOH (0.023 g, 0.55 mmol) in 2 mL solvent for
eight days. After work up, compound 35b was obtained as a color-
less oil (6 mg, 27%). 1H NMR (500 MHz, CD3OD) d 1.02 and 1.11 (d,
6H, J = 7.0 Hz), 1.29 (m, 3H), 1.63 (s, 6H), 1.70 (s, 6H), 1.79 (m, 2H),
1.89 (m, 2H), 2.00–2.11 (m, 16H), 2.53 (m, 2H), 2.62 (m, 2H), 2.52–
2.74 (m, 4H), 2.95 (m, 1H), 3.05–3.08 (m, 2H), 3.28 (m, 2H), 3.63 (s,
3H), 3.74 (m, 3H), 4.14–4.24 (m, 2H), 4.61 (m, 1H), 4.98 (m, 1H),
5.12 (m, 3H), 7.15 (t, 1H, J = 7.0 Hz), 7.32 (t, 2H, J = 7.0 Hz), 7.41
(d, 2H, J = 8.0 Hz), 7.62 (s, 1H), 8.55 (d, 1H, J = 7.0 Hz), 8.85 (d,
1H, J = 7.0 Hz). 13C NMR (75 MHz, CDCl3) d 14.4, 15.2, 16.2, 16.6,
17.8, 18.6, 19.3, 23.1, 25.9, 26.2, 27.1, 27.6, 27.8, 31.2, 31.8, 32.2,
32.7, 32.8, 33.1, 39.2, 40.4, 40.9, 41.1, 48.4, 48.6, 48.9, 49.0, 49.6,
49.8, 52.6, 55.8, 58.6, 62.5, 67.9, 119.0, 125.2, 125.4, 128.0, 129.7,
5.1.46. 2-(3-(1-Methyl-1H-imidazol-2-yl)propyl)-2-((2E,6E)-3,7,
11-trimethyldodeca-2,6,10-trienyl)malonic acid (36)
Lithium hydroxide (0.147 g, 3.5 mmol) was added to a solution
of 38 (0.085 g, 0.175 mmol) in THF/H2O 1:1 (1 mL). The reaction
mixture was stirred at room temperature for eight days then
stopped by neutralization with a solution of HCl 10% and the reac-
tion mixture was extracted with dichloromethane. The organic
layer was dried over Na2SO4 and the solvent was removed in

S. Duez et al. / Bioorg. Med. Chem. 18 (2010) 543–556
555
reduced pressure to afford pure compound 36 as colorless oil
(21 mg, 27%). 1H NMR (CDCl3) d 1.60, 1.61 and 1.62 (s, 9H), 1.68
(m, 5H), 1.90–2.08 (m, 10H), 2.53 (d, 2H, J = 8 Hz), 2.91 (t, 2H,
J = 8 Hz), 3.78 (s, 3H), 5.10 (m, 3H), 7.28 (s, 1H), 7.33 (s, 1H). 13C
NMR (CDCl3) d 16.8, 17.2, 18.5, 25.0, 26.0, 25.5, 28.3, 35.0, 38.5,
39.7, 41.5, 41.8, 58.9, 120.8, 121.4, 124.7, 126.0, 126.2, 132.7,
136.7, 140.3, 147.7, 166.3. MS (ESI+) m/z 453 [M+Na]+. HRMS
(ESI+) calcd for C25H38N2O4Na [M+Na]+: 453.2729, found 453.2721.
heat-inactivated fetal calf serum (FCS) at 27 °C.52 Bloodstream
forms of T. brucei brucei strain 93 were cultured in HMI9 medium
supplemented with 10% FCS at 37 °C under an atmosphere of 5%
CO2.53 In all experiments, log-phage cell cultures were harvested
by centrifugation at 3000g and immediately used. Drug assays
were based on the conversion of a redox-sensitive dye (resazurin)
to a fluorescent product by viable cells54 and were performed
according to the manufacturer recommendations (Alamar-
BlueÒAssay, Invitrogen Corporation). Drug stock solutions were
prepared in pure DMSO. T. brucei procyclic (1 ꢁ 106 cells/mL) or
bloodstream forms (3 ꢁ 104 cells/mL) were cultured as described
above in 24-well plates (1 mL per well) either in the absence or
in the presence of different concentrations of inhibitors (0, 0.1, 1,
5.2. Biological assays
5.2.1. Yeast FTase assay
Assays were realized on 96-well plates, prepared with Biomek
NKMC and Biomek 3000 from Beckman Coulter and read on Wallac
10 and 100
l
M) with a final DMSO concentration of 1%. After a
Victor fluorimeter from Perkin–Elmer. Per well 20
pyrophosphate (10 M) was added to 180 L of a solution contain-
ing 2 L of varied concentrations of potential inhibitors (dissolved
in DMSO) and 178 L of a solution composed by 0.1 mL of partially
lL of farnesyl
72-h incubation, AlamarBlueÒ solution (100
lL) was added in each
l
l
well and fluorescence was measured at 530 nm excitation and
590 nm emission wavelengths after a further 4-h incubation. Each
inhibitor concentration was tested in triplicate and the experiment
repeated twice. The percentage of inhibition of parasite growth
rate was calculated by comparing the fluorescence of parasites
maintained in the presence of drug to that of in the absence of
drug.
l
l
purified recombinant yeast FTase (2.2 mg/mL) and 7.0 mL of Dan-
syl-GCVLS peptide (in the following buffer: 5.8 mM DTT, 6 mM
MgCl2, 12 lM ZnCl2 and 0.09% (w/v) CHAPS, 53 mM Tris/HCl, pH
7.5). Then the fluorescence development was recorded for 15 min
(0.7 s per well, 20 repeats) at 30 °C with an excitation filter at
340 nm and an emission filter at 486 nm. Each measurement was
realized twice as duplicate or triplicate.
5.2.5. Cytotoxicity55
Human mouth epidermal carcinoma (KB) or human diploid
embryonic lung (MRC-5) cells were seeded into 96-well micro-
plates at 2000 cells per well. The cell lines were incubated for
72 h with different concentrations of drugs. The final volume in
each experiment was made up with the media containing 1%
DMSO final volume. Docetaxel was used as positive control. The
experiments were performed in triplicate. Cell growth inhibition
was determined by the MTS assay according to the recommenda-
tions of the manufacturer [Promega]. The optical density was
measured at 490 nm. The number of viable cells was proportional
to the extent of formazan production. The percent cytotoxicity
index [(OD490 treated/OD490 control) ꢁ 100] was calculated from
three experiments. For IC50 determinations, the cytotoxicity
index was plotted against the drug concentration ranged over
10–0.5 nM and the value resulting in 50% cytotoxicity was
determined.
5.2.2. Human FTase assay
Assays were realized on 96-well plates, as described for yeast
FTase but octyl-D-glucopyranoside (0.18%) was used instead of
CHAPS and the solution contains 5 lL of partially purified human
FTase (1.5 mg/mL)24 in 1 mL peptide solution.
The kinetic experiments were realized under the same condi-
tions either with FPP as varied substrate with constant concentra-
tion of Dns-GCVLS of 2.5 lM or with Dns-GCVLS as varied
substrate with constant concentration of FPP of 10 lM. Non-linear
regressions were made by KALEIDAGRAPH 4.03 software. Kmapp and Vapp
max
were obtained from Michaelis–Menten fits of fluorescent data and
Ki and Ki0 were deduced from the linear regression of Kapp/Vapp and
m
max
app
max
1/V
as a function of the inhibitor concentration, respectively.
5.2.3. Assay for in vitro inhibition of P. falciparum growth
The chloroquine-resistant strain FcB1/Colombia of P. falciparum
was maintained in vitro on human erythrocytes in RPMI 1640 med-
ium supplemented by 8% (v/v) heat-inactivated human serum, at
37 °C, under an atmosphere of 3% CO2, 6% O2, 91% N2. In vitro drug
susceptibility was measured by [3H]-hypoxanthine incorporation
as described.51 Drugs were prepared in DMSO at a 10 mM concentra-
Acknowledgments
The authors thank J.-B. Créchet for the generous gift of the E. coli
strain expressing the yeast FTase, Dr. J. Ouazzani and P. Lopez for
the production and purification of recombinant yeast and human
FTases, G. Aubert for cytotoxicity assays and Research Minister
and CNRS for financial support.
tion. Compounds were serially diluted twofold with 100
medium in 96-well plates. Asynchronous parasite cultures (100
1% parasitemia and 1% final hematocrit) were then added to each
well and incubated for 24 h at 37 °C prior to the addition of 0.5 Ci
lL culture
lL,
Supplementary data
l
of [3H]-hypoxanthine (GE Healthcare, France, 1–5 Ci mmol/mL)
per well. After a further incubation of 24 h, plates were frozen and
thawed. Cell lysates were then collected onto glass-fiber filters and
counted in a liquid scintillation spectrometer. The growth inhibition
for each drug concentration was determined by comparison of the
radioactivityincorporatedin thetreatedculturewiththatinthecon-
trol culture maintained on the same plate. The concentration caus-
ing 50% growth inhibition (IC50) was obtained from the drug
concentration–response curve and the results were expressed as
the mean values standard deviations determined from several
independent experiments.
Supplementary data (Lineweaver–Burk plots of the fluorescent
data and Ki determination for compounds 16, 35a and 35b) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.12.017.
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