Introduction of methionine mimics on 3-arylthiophene: Influence on protein farnesyltransferase inhibition and on antiparasitic activity

Article abstract of DOI:10.1039/c3md00065f

In the course of our search for new protein farnesyltransferase inhibitors with antiparasitic activity, we found that addition of a methionine residue at position 2 of 3-arylthiophenes greatly improved enzyme inhibition. To investigate the influence of this methionine residue on FTase and antiparasitic activities, 29 novel tetrasubstituted thiophenes bearing methionine or analogous moieties were synthesised. These new derivatives were evaluated on human and Trypanosoma brucei protein farnesyltransferases and on proliferation of 4 protozoan parasites: Plasmodium falciparum, Trypanosoma brucei brucei, Trypanosoma cruzi and Leishmania donovani. Some compounds showed promising low micromolar or submicromolar activities on T. b. brucei and L. donovani confirming the potential of this new class as antiparasitic agents.

Full text of DOI:10.1039/c3md00065f

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Introduction of methionine mimics on 3-arylthiophene:
influence on protein farnesyltransferase inhibition and
on antiparasitic activity†
Cite this: Med. Chem. Commun., 2013,
4, 1034
Damien Bosc,a Elisabeth Mouray,b Philippe Grellier,b Sandrine Cojean,c
c
a
*
¨
Philippe M. Loiseau and Joelle Dubois
In the course of our search for new protein farnesyltransferase inhibitors with antiparasitic activity, we
found that addition of a methionine residue at position 2 of 3-arylthiophenes greatly improved enzyme
inhibition. To investigate the influence of this methionine residue on FTase and antiparasitic activities,
29 novel tetrasubstituted thiophenes bearing methionine or analogous moieties were synthesised.
These new derivatives were evaluated on human and Trypanosoma brucei protein farnesyltransferases
and on proliferation of 4 protozoan parasites: Plasmodium falciparum, Trypanosoma brucei brucei,
Trypanosoma cruzi and Leishmania donovani. Some compounds showed promising low micromolar or
submicromolar activities on T. b. brucei and L. donovani confirming the potential of this new class as
antiparasitic agents.
Received 27th February 2013
Accepted 18th April 2013
DOI: 10.1039/c3md00065f
www.rsc.org/medchemcomm
interactions.7,8 Thus, FTase inhibition could lead to cellular
disorders and cell death.
Introduction
Parasitic diseases such as malaria, African sleeping sickness,
Chagas disease or Leishmaniasis affect each year 1 billion
people and are responsible for more than 1 million of deaths
according to WHO reports. To control parasite proliferation as
well as drug resistance development is crucial to ght against
these tropical diseases. The use of new targets to improve the
existing therapies could ll this unmet medical need. Among
them, protein farnesyltransferase (FTase), rst studied in anti-
cancer therapy,1 is considered nowadays a potential target in the
treatment of parasitic diseases.2,3 This key heterodimeric met-
alloenzyme belongs to the protein prenyl transferase family.
With the involvement of an essential zinc atom,4 FTase catalyses
the transfer of a farnesyl chain (C15) from farnesyl pyrophos-
phate (FPP) to the cysteine residue of some proteins. This
cysteine belongs to the C-terminal CaaX sequence where C is the
farnesylated cysteine, a is an aliphatic amino acid and X is a
serine, an alanine, a glutamine or a methionine.5,6 This farne-
sylation step allows CaaX proteins to be anchored to intracel-
lular membranes and could promote protein–protein
In the course of our search for a new class of antiparasitic
agents with FTase inhibitory activity, we have previously
reported on the discovery of tetrasubstituted 3-arylthiophenes.9
The rst modications around this original scaffold headed by
compound 1 revealed the importance of the presence of a nitrile
moiety at position 4 and a carboxylic acid at position 2 for FTase
inhibition (Fig. 1). Other structure–activity relationship (SAR)
studies on our hit compound 1 were also carried out. Position 5
was modied by SNAr or Liebeskind–Srogl coupling10 and
position
3
by solid- or liquid-phase Suzuki–Miyaura
couplings.11,12 These modications conrmed the potency of the
thio-isopropyl moiety at position 5 and of bulky or poly-
oxygenated aromatic groups at position 3 showing low micro-
molar activities on T. brucei FTase (TbFTase).
More interestingly, although ester analogues 7–10 of
compounds 1–4 respectively showed micromolar inhibitory
activities on T. b. brucei proliferation, submicromolar activities
were obtained with esters 5 and 6 (Fig. 1). Furthermore, intro-
duction of methionine at position 2 of the thiophene ring
(compound 2) increased the activity on TbFTase inhibition by 2
orders of magnitude displaying submicromolar activities. This
activity improvement was not observed on T. b. brucei which led
us to suspect the cleavage of the amide bond between methio-
nine and thiophene ring in parasites.
In this article we report on two series of new 3-arylth-
iophenes. In the rst series, methionine was introduced on our
most active 3-arylthiophenes as mentioned above. The second
series deals with compound 2 analogues where the amide bond
was modied to be less sensitive to peptidase, or where
aCNRS, Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif,
Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. E-mail: joelle.dubois@.
cnrs.fr; Fax: +33 1 69 07 72 47; Tel: +33 1 69 82 30 58
b
´
Museum National d'Histoire Naturelle, UMR 7245 CNRS, Departement RDDM, CP52,
57 rue Cuvier, 75005 Paris, France
c
´
Univ Paris-Sud, CNRS, BioCIS-UMR 8076, Chimiotherapie Antiparasitaire, LabEx
´
´
ˆ
LERMIT, Faculte de Pharmacie, 5 rue J.-B. Clement, Chatenay-Malabry, F-92296,
France
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00065f
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Scheme 1 Synthesis of compounds 15–17 by methionine coupling followed by
saponification. Reagents and conditions: (a) EDCI, HOBt, NMM, CH2Cl2, rt, over-
night or HBTU, HOBt, DIEA, DMF, 24–36 h, 11: 64%, 12: 62%, 13: 33%, 14: 48%
and (b) NaOH, THF–H2O–EtOH (2 : 2 : 1), rt, 72 h or KF–Al2O3, THF, MW, 300 W,
5 ꢀ 30 min, 15: 62%, 16: 100%, 17: 23%.
In the CaaX motifs recognized by FTase, X is mainly a
methionine or a glutamine particularly for the parasitic
enzymes. Thus, 2-thio-sec-butylthiophene 18 having an inhibi-
tory activity close to that of thiophene 19 was coupled efficiently
with EDCI–HOBt to these two amino acids giving compounds 19
and 20 respectively (Scheme 2). Furthermore, in order to check
whether methionine in compound 2 mimics the X of the Ca1a2X
box or the a2 amino acid of this motif, the dipeptide Ile-Met-
OMe was classically coupled to acid 1 to afford compound 21
(Scheme 2). This sequence was chosen because it is oen
observed in the C-terminal part of FTase protein substrates for
example in K-Ras4B (CVIM) and B lamine (CAIM). Furthermore,
the isoleucine residue is the a2 part of the Rap2b protein (CVIL),
a GGTase I substrate.14
Fig. 1 Structures and inhibitory activities against TbFTase and T. b. brucei (Tbb)
proliferation of our most active thiophenes.
Moreover, one of the key interactions for FTI design is with
the hydrophobic cavity delimited by Trp102, Trp106 and Tyr361
called the a2 binding site of Ca1a2X box.15 This hydrophobic
pocket was considered essential for the recognition of the
inhibitor by FTase. Because of intermediate FTase inhibition
observed for 21 (see below) we wondered whether the arylth-
iophene core interacts in the a1 or a2 site. Therefore, methio-
nine was replaced by bulky hydrophobic moieties such as
adamantyl and naphthyl known to bind at the a2 site.16 These
groups were introduced with one or no methylene spacer by
formation of an amide bond between thiophene 1 and the
corresponding amines. As EDCI–HOBt coupling conditions
were ineffective with these groups, acylation reactions were
methionine was replaced by other amino derivatives. Synthesis
of these new thiophenes and their biological evaluation on
FTases and on P. falciparum, T. b. brucei, T. cruzi and L. donovani
proliferation are described.
Results and discussion
Chemistry
In the rst series, methionine was added to compounds 3 and 4
and to the acid derivatives of compounds 5 and 6. EDCI–HOBt
or HBTU–HOBt coupling agents proved to be efficient to afford
compounds 11–14 in moderate to good yields (Scheme 1).
Because in most cases acids were most potent on FTase inhi-
bition, these compounds were saponied. Because of its low
availability compound 13 was not submitted to saponication.
Although compounds 11 and 12 were easily hydrolysed with
sodium hydroxide giving acids 15 and 16 respectively, ester 14
was resistant to these conditions. This lack of reactivity towards
sodium hydroxide has already been described for a few thio-
phenes bearing a hydroxyphenyl moiety at position 3 and ester
at position 2.12 Therefore, we adapted a hydrolysis procedure
using potassium uoride doped alumina under microwave
conditions13 giving acid 17 in a non-optimized 23% yield
(Scheme 1).
Scheme 2 Synthesis of compounds 19–21 and 26. Reagents and conditions: (a)
EDCI, HOBt, NMM, CH2Cl2, rt, 16–36 h, 19: 68%, 20: 69%, 21: 70%, and 26: 88%.
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performed between compound 1 acid chloride and the corre-
sponding amines to give analogues 22–25 in moderate to
excellent yields (Scheme 3).
To get more active compounds against parasites we designed
analogues of compound 2 less prone to peptidase cleavage. The
most common modications to prevent the peptide bond from
enzymatic hydrolysis are the inversion of the amino acid abso-
lute conguration, the methylation of the amide nitrogen or its
transformation to a thioamide. These moieties provide more
stable analogues with sometimes higher affinity towards bio-
logical receptors.17 Conguration of the methionine was rst
changed by the synthesis of (D)-methionine analogue 26
(Scheme 2). Thionation of the methionine amide bond was also
considered. Lawesson's reagent (LR) is the most used reagent
for amino acid thionation, thanks to its high selectivity based
on reaction temperatures and to its mild conditions.18 Choice of
the reaction solvent was crucial. In THF compound 8 conversion
was very low either at room temperature or at 70 ꢁC. However, in
toluene at 110 ꢁC, unselective thionation gave an inseparable
mixture of mono- and dithionated compounds whereas at 40 ꢁC
selective thioamidation afforded 27 in 55% yield (Scheme 4).
Several methods are available to carry out N-methylation of
amino acids.19,20 Benoiton's method, i.e. treatment of amide 8
with NaH and methyl iodide, afforded N-methylated methio-
nine thiophene 28 in a satisfactory yield (Scheme 4).
Aza-b3 amino acids are recent promising moieties which
could increase metabolic stability, biodisponibility and biolog-
ical absorption of drugs.21,22 As far as we know there is only one
article mentioning the formation of aza-b3 methionine as a
fmoc protected derivative.23 Therefore, to introduce this aza-b3
methionine bioisostere on the thiophene ring we envisioned
two possible pathways. Either (2-(methylthio)ethyl)hydrazine
would be added to thiophene 1 and the acetate moiety would
further be introduced or the aza-b3 methionine analogue would
be rst synthesized and then coupled to compound 1. Both ways
were undertaken. 1,1-Dimethoxy-2-(methylsulfanyl)ethane was
treated with HCl to afford the corresponding aldehyde which
was submitted without any purication to the classical condi-
tions of reductive amination with Boc-hydrazine to give
compound 29. Aer removal of the Boc group, the hydrazine
was coupled with compound 1 to afford compound 30 and
Scheme
4
Thionation and N-methylation of compound 8. Reagents and
conditions: (a) Lawesson's reagent, toluene, 40 ꢁC, 140 h, 55% and (b) NaH, MeI,
DMF, rt, 1 h, 53%.
unpredicted compound 31 both in poor yields, 10% and 17%
respectively. It could be hypothesized that 31 came from acyl-
ation of the secondary amine followed by N–N bond cleavage as
previously reported.24–26 Addition of the acetate moiety to
compound 30 was not possible because of the too small quan-
tity available. However addition of ethyl bromoacetate in the
presence of DIEA to compound 31 afforded compound 32
probably through the formation of a sulfonium ion (Scheme 5).
Then we carried out the second pathway starting with the
synthesis of the aza-b3 methionine analogue. Compound 33
was obtained by nucleophilic substitution between ethyl bro-
moacetate and Boc-hydrazine.27 Subsequent alkylation with
1-chloro-2-(methylsulfanyl)ethane proved to be difficult and
only high temperature and short time of microwave irradiation
allowed the formation of compound 34 though in a low yield.
Further Boc removal and coupling with thiophene 1 afforded
analogue 35 (Scheme 6).
For FTase inhibition evaluation, all the analogues were
hydrolyzed under appropriate conditions in good to excellent
yields (Table 1).
Scheme 5 Synthesis of compound 32. Reagents and conditions: (a) (i) HCl 1%,
reflux, 30 min, (ii) BocHN–NH2, NaBH(OAc)3, CH3COOH, DCE, rt, 16 h, 29%; (b) (i) 4
M HCl, dioxane, rt, 2 h, (ii) 1, HBTU, HOBt, DIEA, DMF, rt, 96 h, 10% (30), 17% (31);
(c) BrCH2CO2Et, DIEA, CH2Cl2, 45 ꢁC, 72 h, 52%.
Scheme 3 Synthesis of analogues 22–25. Reagents and conditions: (a) (i) SOCl2,
reflux, 4.5 h, and (ii) RNH2, pyridine, CH2Cl2, rt, 46 h, 22: 27%, 23: 66%, 24: 87%,
and 25: 91%.
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Table 2 Inhibitory activity of thiophenes 2, 8, 11–17 against recombinant
human FTase (hFTase) and T. brucei FTase (TbFTase)
Ar
Cpd
R
IC50 (mM) hFTase IC50 (mM) TbFTase
8
2
OMe Inactive
Inactive
0.12 ꢂ 0.02
OH
0.077 ꢂ 0.006
Scheme 6 Synthesis of aza-b3 analogue 35. Reagents and conditions: (a) Boc–
NH–NH2, DIEA, CH2Cl2, 45 ꢁC, 16 h, 54%; (b) 1-chloro-2-(methylsulfanyl)ethane,
DIEA, 150 ꢁC (MW), 2 ꢀ 30 min, 21%; (c) (i) TFA, CH2Cl2, rt, 1 h, (ii) 1, EDCI, HOBt,
NMM, CH2Cl2, rt, 6 h, 58%.
11
15
OMe 23 ꢂ 6
19 ꢂ 4
OH
0.042 ꢂ 0.003
0.042 ꢂ 0.001
12
16
OMe >50
>50
0.19 ꢂ 0.01
OH
0.14 ꢂ 0.009
Biological evaluation
All these analogues of 2-methionine thiophene 2 were evaluated
on recombinant human and T. brucei brucei FTases using a
uorescence-based assay28,29 adapted to a 96-well plate format.30
Results on the rst series of compound 2 analogues bearing a
methionine residue and variable 3-aryl groups are reported in
Table 2.
13
OMe 4.7 ꢂ 0.4
5.3 ꢂ 0.5
14
17
OMe 5.6 ꢂ 0.4
3.6 ꢂ 0.2
4.8 ꢂ 0.2
OH
8.7 ꢂ 1.5
Introduction of methionine at position 2 of our more active
3-arylthiophenes did not have the same effect as the initial
compound (Table 2). This transformation was favourable for
compounds bearing bulky m-biphenyl and dibenzofuranyl on hFTase and led to a 1.2 to 240-fold activity enhancement on
aromatics and improved their activities by 100-fold. Thus, TbFTase (compound 42 and compound 36 respectively).
interactions of analogues 15 and 16 in the FTase pocket seem to However none of these derivatives were more active than
be similar to compound 2.10 In contrast, addition of methionine compound 2 on both enzymes, except compound 36 which
to 3-polyhydroxyphenylthiophenes had no effect on their enzy- displayed slightly improved activity.
matic activity. These results support our former hypothesis12
Analogues 30–32 and 42 bearing long sulphur chains showed
that the binding mode or binding site of poly- micromolar activities, roughly in the same range as compound
hydroxyphenylthiophenes differs from that of our hit 1. Hydrophobic groups introduced to bind potentially on the
compound 1. This is also conrmed by the fact that esters 13 hydrophobic cavity of the a2 residue of the Ca1a2 box strongly
and 14 showed micromolar activities whereas ester 8 was decreased the inhibition activity (compounds 22–25). These
inactive.
results may have two reasons: either methionine of compound 2
The second series of compound 2 analogues was also eval- did not occupy the a2 binding site, or the other substituents
uated on FTase inhibition (Table 3). In most cases, acidic around the thiophene hampered an optimal conguration
derivatives are more active than their ester analogues as previ- allowing adamantyl and naphthyl groups to interact with this
ously described.31 Modication at position 2 of our hit 1 with pocket. Similarly, introduction of another amino acid between
methionine derivatives improved IC50 to a 4 to 240-fold extent methionine and thiophene such as isoleucine which could
mimic the a2 part of the Ca1a2X box decreased the activity.
Taken together, these results suggest that the 3-arylthiophene
motif occupies the a2 binding site in the same manner as
Table 1 Hydrolysis of estersa
phenylalanine in the complex CVFM-FPP-rat FTase (PDB I.D.
code 1JCR).32
Entry
Ester
Acid
Method
Yieldb (%)
Adding a methionine residue was the optimal trans-
formation affording submicromolar activities. As previously
described with analogues bearing a carboxylic acid at position
2,9 the thioalkyl group at position 5 had little inuence on the
inhibitory activity (analogues 2 vs. 36). The analogues designed
to minimize peptidase sensitivity (acids 26–28 and 35 and esters
39–41 and 43) were all less active than acid 2 but more efficient
than compound 1. Submicromolar inhibitory activities were
obtained for the thioamide derivative 40 and for the aza-b3
1
2
3
4
5
6
7
8
19
20
21
26
27
28
32
35
36
37
38
39
40
41
42
43
A
C
B
A
B
B
B
B
94
55
100
61
100
95
100
100
a
Hydrolysis with NaOH 2 M (Method A) or LiOH 2 M (Method B) in
THF–EtOH or TFA, Et3SiH, CH2Cl2 (Method C). b Isolated yields.
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Table 3 Inhibitory activity of novel tetrasubstituted thiophenes against recombinant human FTase (hFTase) and Trypanosoma brucei FTase (TbFTase)
IC50 (mM)
hFTase
IC50 (mM)
TbFTase
IC50 (mM)
TbFTase
R
Cpd
R0
R
Cpd
R0
IC50 (mM) hFTase
1.9 ꢂ 0.1
7
1
OEt
OH
Inactive
16 ꢂ 2
Inactive
11 ꢂ 2
30
—
>50
>50
8
2
OMe
OH
Inactive
0.077 ꢂ 0.006
Inactive
0.12 ꢂ 0.02
31
—
24 ꢂ 6
19a
36a
OMe
OH
>50
0.067 ꢂ 0.005
>50
0.079 ꢂ 0.003
32
42
OEt
OH
9.3 ꢂ 0.6
3.7 ꢂ 0.3
22 ꢂ 2
8.9 ꢂ 0.8
20a
37a
OtBu
OH
10.4 ꢂ 0.6
29 ꢂ 13
35
43
OEt
OH
>50
1.1 ꢂ 0.1
>50
0.53 ꢂ 0.03
0.076 ꢂ 0.006
1.2 ꢂ 0.07
21
38
OMe
OH
>50
1.9 ꢂ 0.3
>50
2.2 ꢂ 0.60
22
—
>50
>50
26
39
OMe
OH
>50
0.45 ꢂ 0.02
2.8 ꢂ 0.5
1.4 ꢂ 0.1
23
24
25
—
—
—
21.6 ꢂ 1.6
Inactive
Inactive
>50
>50
>50
27
40
OMe
OH
0.46 ꢂ 0.08
1.7 ꢂ 0.1
8.2 ꢂ 1
0.23 ꢂ 0.02
28
41
OMe
OH
Inactive
1.8 ꢂ 0.05
Inactive
3.3 ꢂ 1
a
SsBu instead of SiPr at position 5.
analogue 43 on TbFTase and for the D-methionine bearing groups.12 Thus for these two compounds, the ester moiety should
compound 39 on the human enzyme. induce a conformational or position change allowing a better
This interesting discrepancy between two FTases from interaction with the FTase binding site.
different species was also noticed with other derivatives. Though
In our aim to develop antiparasitic agents, our ester deriva-
methionine and glutamine are generally described as possible X tives were evaluated on the intraerythrocytic stages of Plasmo-
residues of the CaaX moiety for both human and T. brucei FTases, dium falciparum,33,34 responsible for malaria, the bloodstream
compound 37 displayed a 15-fold lower inhibition of TbFTase forms of Trypanosoma brucei brucei, the pathogenic agent of
than the corresponding methionine analogue 36. On the other African sleeping sickness in cattle, the intracellular develop-
hand, IC50 values were very close for both compounds on the ment of Trypanosoma cruzi responsible for Chagas disease and
human enzyme. In contrast, analogue 40 with thio-methionine Leishmania donovani, the causative agent of visceral Leish-
was seven times more active on TbFTase. Surprisingly, this maniasis (Table 4).35–37 In most cases, our thiophenes are poorly
selectivity changed with ester analogue 27 displaying a sub- active on P. falciparum and on T. cruzi. Our best compound was
micromolar activity. The spatial disposition of the J[CSNH]Met- thiophene 35 with the aza-b3 moiety giving an activity of 10 mM
OMe chain should be different with the ester and would allow on P. falciparum. For most of the compounds evaluated on
supplementary favourable interactions in the FTase pocket. T. cruzi, IC50 values were around 15 mM, the best result being
Moreover, activities of esters 20 and 27 on TbFTase have a similar observed for compound 12 with IC50 ¼ 7.6 mM. Concerning
behaviour to ester analogues bearing polyhydroxyphenyl T. b. brucei proliferation inhibition by the rst series of
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methionine containing thiophenes, only the biphenyl derivative observed for the same compounds according to the considered
(9) showed a 2-fold better activity (IC50 ¼ 5.8 mM). It is notice- parasite. However compound 35 bearing an aza-b3 moiety was a
able that addition of a methionine on polyhydroxythiophene good TbFTase inhibitor and was among the most active deriv-
analogues lowered the inhibition rate. In the second series, atives against P. falciparum, T. b. brucei and L. donovani. Finally,
analogues designed to be resistant to carboxypeptidases three compounds displayed encouraging submicromolar activ-
(compounds 39–41) did not display better activities than ities on L. donovani. These promising molecules could be the
compound 7. Therefore, the hypothesis of a rapid hydrolysis in starting point for further modulations, in particular at the not-
parasites is not attested. Thus, to explain the difference between well-explored position 5 of the thiophene ring, to complete our
in vitro enzymatic activity and in cellulo proliferation inhibition, structure–activity relationship studies on this original class of
problems of cell penetration and/or aqueous solubility could be FTIs and antiparasitic agents.
suspected. It is noteworthy that compounds 22, 23, and 25 with
adamantyl or naphthyl amide inhibited parasite proliferation in
the micromolar range though they were inactive on the isolated
Acknowledgements
enzyme. Thus, for these derivatives, a target other than FTase The authors thank Dr J. Ouazzani and P. Lopes for the
should be responsible for this activity and would deserve to be production and purication of FTases, O. Thoison and her
identied. The best results were obtained for compounds 30–32 coworkers for UHPLC analyses, M.-F. Bricot for elemental
and 35 exhibiting low micromolar or submicromolar inhibitory analysis, ICSN and CNRS for nancial support.
activities on T. b. brucei (IC50 from 0.8 to 3.4 mM). This
improvement could be due to the higher lipophilicity of these
analogues bearing an aliphatic chain that may allow a better cell
Notes and references
permeability. Most of the derivatives evaluated on L. donovani
displayed toxicity against infected macrophage, precluding
inhibitory activity measurement. Three derivatives revealed
promising submicromolar activities. These compounds were
substituted at position 2 of the thiophene ring by one or two
amino acids (compounds 12 and 21 respectively) or by an aza-b3
moiety (compound 35). In this in vitro model, the compounds
should cross-over three membrane barriers (macrophage,
phagolysosome and amastigote). Interestingly, no problem of
compound uptake was observed with L. donovani. Since the
protein farnesyltransferase systems also exist in Leishmania
parasites and are sensitive to FTI,38 it is probable that the
compounds act on this target. Anyway, the level of activity
monitored justies further in vivo evaluation on the L. donovani/
Balb/C mice model.
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