New protein farnesyltransferase inhibitors in the 3-arylthiophene 2-carboxylic acid series: Diversification of the aryl moiety by solid-phase synthesis

Article abstract of DOI:10.3109/14756366.2011.643302

A new synthetic pathway was devised to reach tetrasubstituted 3-arylthiophene 2-carboxylic acids in a three-step solid-phase synthesis. This very efficient methodology provided more than 20 new compounds that were evaluated for their ability to inhibit protein farnesyltransferase from different species as well as Trypanosoma brucei and Plasmodium falciparum proliferation.

Full text of DOI:10.3109/14756366.2011.643302

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(1): 163–171
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.643302
RESEARCH ARTICLE
New protein farnesyltransferase inhibitors in the
3-arylthiophene 2-carboxylic acid series: diversification
of the aryl moiety by solid-phase synthesis
Sébastien Lethu1, Damien Bosc1, Elisabeth Mouray2, Philippe Grellier2, and Joëlle Dubois1
1Institut de Chimie des Substances Naturelles, CNRS, Centre de Recherche de Gif, Gif sur Yvette, France and 2UMR 7245
CNRS - MNHN, Paris, France
Abstract
A new synthetic pathway was devised to reach tetrasubstituted 3-arylthiophene 2-carboxylic acids in a three-step
solid-phase synthesis. This very efficient methodology provided more than 20 new compounds that were evaluated
for their ability to inhibit protein farnesyltransferase from different species as well as Trypanosoma brucei and
Plasmodium falciparum proliferation.
Keywords: Suzuki cross-coupling, heterocycles, structure–activity relationships, enzyme inhibition, antiparasitic
activity
Introduction
a new library of 3-arylthiophene derivatives and their
activity on FTase.
Protein farnesyltransferase (FTase) is a key enzyme
in protein post-translational lipidation1. Its inhibition
prevents correct protein localization in the cell and may
cause cellular disorders leading to cell death. ese
properties were first thought to be of interest to design
new anti-cancer agents2. Recently, FTase turned out to
be a potential target to fight against parasitic tropical
diseases3. In the course of our search for new protein
farnesyltransferase inhibitors (FTIs) with antiparasitic
activity, we discovered a new FTI family in the tetra-
substituted 3-arylthiophene series. Our preliminary
structure–activity relationship studies have enlight-
ened the importance of a carboxylic acid at position 2 as
well as a nitrile function at position 4 on the thiophene
ring4. To get more insight into this new FTI family, we
have modified the isopropyl ether at position 5 by SNAr
reactions5 and subsequently we intended to study the
role of the 3-aryl moiety. erefore, we elaborated a
different synthetic pathway, allowing rapid modifica-
tion at position 3 of the thiophene ring. In this article,
we describe a novel synthesis of 3-arylthiophenes 1, its
application on solid-phase synthesis, the production of
Experimental protocols
All solvents and reagents were commercially avail-
able and were used without further purification. Wang
resin was purchased from Novabiochem (1.3 mmol/g,
100–200 mesh). Solid-phase syntheses were realized on
Unimax 2010 (Heidolph) for the binding to Wang resin
and on synthesis 1 (Heidolph) for the Suzuki–Miyaura
coupling. Analytical thin-layer chromatography was
carried out on silica gel 60F254 aluminium sheets
(Merck). Column chromatography was performed with
silica gel with prepacked Redisep columns. Preparative
LC (PLC) was performed on silica gel 60F254 glass
1
plates (Merck). H and 13C NMR spectra were recorded
at Bruker Avance 300 (300 MHz) and Avance 500
1
(500 MHz). H chemical shifts (δ) are reported in parts
per million (ppm) relative to the singlet at 7.26 ppm
for residual CHCl3 and to the central line of residual
acetone at 2.05 ppm. 13C chemical shifts (δ are reported
in parts per million (ppm) relative to the central line
Address for Correspondence: Dr. Joelle Dubois, CNRS, Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, Avenue de
la Terrasse, 91198, Gif-sur-Yvette Cedex, France. Tel: +33 1 69 82 30 58. Fax: +33 1 69 07 72 47. E-mail: joelle.dubois@icsn.cnrs-gif.fr
(Received 27 September 2011; revised 16 November 2011; accepted 16 November 2011)
163

164 S. Lethu et al.
either of the triplet at 77.2 ppm for chloroform-d, or of
the massif at 29.9 ppm for acetone-d6. Splitting pat-
terns are designated as s, singlet; d, doublet; t, triplet;
q, quartet; qi, quintuplet; h: heptuplet; m, multiplet; b,
broad and combinations thereof. Coupling constants
J are reported in hertz (Hz). IR spectra were recorded
with an FTIR Perkin-Elmer Spectrum BX spectrometer.
Low and high resolutions mass spectra were recorded
by navigator LC/MS (source AQA) for electron spray
ionization. UHPLC analyses were realized on Waters
Acquity UPLC system. Elemental analyses were per-
formed by the ICSN Microanalytical Laboratory.
After stirring for 3 h under reflux, water was added to
the reaction mixture. e solution was filtered and the
precipitate was washed with water before purification by
flash chromatography on silica gel (heptane/EtOAc 10:0
to 9:1 (v/v) in 1 h) to afford 3 as a white amorphous solid
(4.52 g, 84%). Mp = 114°C; 1H NMR (500 MHz, CDCl3)
δ 1.32 (t, J= 7.0 Hz, 3H), 1.39 (d, J= 6.5 Hz, 6H), 3.54 (h,
J= 6.5 Hz, 1H), 4.27 (q, J= 7.0 Hz, 2H), 5.74 (bs, 2H). 13C
NMR (75 MHz, CDCl3) δ14.6, 23.3, 42.0, 60.9, 101.5, 103.8,
112.9, 153.2 153.6, 162.9. IR (film) 3424, 3332, 2982, 2928,
2218, 1668, 1622, 1537, 1296 cm−1. MS (ESI+, CH2Cl2/
MeOH) m/z 293 [M+Na]+. HRMS (ESI+, CH2Cl2/MeOH)
calcd for C11H14N2O2S2Na [M+Na]+ 293.0394, found
293.0401. Elemental analysis calcd (%) for C11H14N2O2S2:
C 48.97, H 5.22, N 10.36, O 11.84, S 23.72; found C 48.87,
H 5.23, N 10.35, O 11.91, S 23.94.
UHPLC analysis
e purity for all target compounds was measured using
reversed-phase UHPLC (HSS C-18 1.8 µm, 2.1 × 50 mm
column) with two different solvent systems: compounds
were eluted with 95:5 A/B for 0.5 min then with a gradient
of 5–100% B/A for 3.5 min followed by 0:100 isocratic for
1 min at a flow rate of 0.6 mL/min, where solvent A was
0.1% formic acid in H2O and solvent B was 0.1% formic
acid in MeCN (system 1) or 0.1% formic acid in MeOH
(system 2). Purity was determined on TAC (total absor-
bance current from 200 to 400 nm).
Ethyl 3-bromo-4-cyano-5-(isopropylthio)thiophene-2-
carboxylate (4)
To a solution (CH3CN, 50 mL) of tBuONO (2.9 mL, 25
mmol, 1.5 equiv.) and CuBr2 (4.85 g, 21.7 mmol, 1.3
equiv.) a solution (CH3CN, 50 mL) of 3 (4.5 g, 16.7 mmol,
1 equiv.) was added through cannula. After completion of
the addition, the reaction mixture was stirred for 10 min
at room temperature, poured on a HCl solution (20%,
100 mL) and extracted four times with diethyl ether.
Combined organic layers were washed with brine, dried
over MgSO4 and concentrated under reduced pressure.
e crude product was purified by flash chromatog-
raphy on silica gel (heptane/EtOAc 10:0 to 7:3 (v/v) in
1 h) to afford 4 as a white amorphous solid (5.25 g, 94%).
Mp = 59°C; 1H NMR (500 MHz, CDCl3) δ 1.39 (t, J= 7.0 Hz,
3H), 1.43 (d, J= 6.5 Hz, 6H), 3.61 (h, J= 6.5 Hz, 1H), 4.38
(q, J= 7.0 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ14.4, 23.3,
42.7, 62.4, 112.8, 117.9, 118.2, 129.1, 154.1, 159.2. IR (film)
2983, 2224, 1722, 1226, 1099, 750 cm−1. MS (ESI+, CH2Cl2/
MeOH) m/z 356 and 358 [M+Na]+. HRMS (ESI+, CH2Cl2/
MeOH) calcd for C11H1279BrNO2S2Na [M+Na]+ 355.9391,
found 355.9384; calcd for C11H1281BrNO2S2Na [M+Na]+
357.9370, found 357.9380. Elemental analysis calcd (%)
for C11H12BrNO2S2: C 39.53, H 3.62, N 4.19, O 9.57, S 19.19;
found C 39.45, H 3.51, N 4.01, O 9.52, S 18.82.
Chemistry
2-(Bis(isopropylthio)methylene)malononitrile (2)
To a solution (DMF, 350 mL) of malononitrile (10.0 g,
151 mmol, 1.0 equiv.), K2CO3 (22.0 g, 159 mmol, 1.05
equiv.) was added. After stirring for 30 min at room
temperature, carbon disulfide (9.13 mL, 151 mmol, 1.0
equiv.) was added dropwise and the reaction mixture
was stirred at room temperature for 30 min before
i-propyl iodide (30.3 mL, 302 mmol, 2.0 equiv.) and
tetrabutylammonium bromide (9.75 g, 30.2 mmol, 0.2
equiv.) were then added. After being stirred for 70 h
at 60°C, the reaction mixture was cooled, diluted with
water and extracted four times with diethyl ether. The
combined organic layers were washed with sodium
thiosulfate 5% and brine, dried over MgSO4 and con-
centrated under reduced pressure. The crude product
was purified by flash chromatography on silica gel
(heptane/EtOAc 98:2 (v/v)) to afford 2 as a white amor-
phous solid (24.9 g, 73%). Mp = 68°C; 1H NMR (500 MHz,
CDCl3) δ 1.41 (d, J = 6.5 Hz, 12H), 3.99 (h, J = 6.5 Hz,
2H). 13C NMR (75 MHz, CDCl3) δ 23.2, 42.7, 80.6, 113.1,
180.1. IR (film) 2970, 2928, 2867, 2219, 1479 cm−1. MS
(ESI+, MeOH) m/z 227 [M+H]+. HRMS (ESI+, MeOH)
calcd for C10H15N2S2 [M+H]+ 227.0677, found 227.0670.
Elemental analysis calcd (%) for C10H14N2S2: C 53.06, H
6.23, N 12.38, S 28.33; found C 53.05, H 6.45, N 12.23,
S 28.46.
Ethyl 3-(4-chlorophenyl)-4-cyano-5-(isopropylthio)
thiophene-2-carboxylate (5a)
To a solution (toluene/EtOH 9:1 (v/v), 100 mL) of 4 (5.0 g,
15.0 mmol, 1.0 equiv.), K2CO3 (2 M in water, 18.8 mL, 37.5
mmol, 2.5 equiv.), Pd(PPh3)4 (1.74 g, 1.5 mmol, 10% mol)
and 4-chlorophenyl boronic acid (4.69 g, 30 mmol, 2.0
equiv.) were added. After stirring for 4 h under reflux,
water was added to the reaction mixture which was
extracted three times with EtOAc. Combined organic
layers were washed with brine, dried over MgSO4 and
concentrated under reduced pressure. e crude prod-
uct was purified by flash chromatography on silica gel
(heptane/EtOAc 10:0 to 5:5 (v/v) in 40 min) to afford 5a
as a white amorphous solid (4.99 g, 91%). e analytical
data were identical to those previously reported4.
Ethyl 3-amino-4-cyano-5-(isopropylthio) thiophene-2-
carboxylate (3)
To a solution (EtOH, 60 mL) of 2 (4.5 g, 19.9 mmol, 1.0
equiv.), K2CO3 (3.07 g, 21.9 mmol, 1.1 equiv.) and ethyl
thioglycolate (2.4 mL, 21.9 mmol, 1.1 equiv.) were added.
Journal of Enzyme Inhibition and Medicinal Chemistry

Diversification of 3-arylthiophene FTIs by solid-phase synthesis 165
THF/MeOH 1:1 (3 × 20 mL), MeOH (3 × 20 mL) and Et2O
(2 × 20 mL), respectively to afford 7 (1.33 g, 97%, 0.94
mmol/g)6.
3-(4-Chlorophenyl)-4-cyano-5-(isopropylthio)thiophene-2-
carboxylic acid (1a)
is compound was obtained according to previously
reported procedure4.
Polymer-bond 3-aryl-4-cyano-5-(isopropylthio)thiophene-2-
carboxylate (8) Resin 7 (130 mg, 0.75 mmol/g, 1 equiv.)
was loaded on a teflon reactor for solid-phase synthesis
and suspended in toluene/tBuOH (4 mL, 9:1 v/v). e
resin was swollen for 6 h at room temperature. en,
Pd(PPh3)4 (11.3 mg, 9.8 µmol, 10% mol), K3PO4 (104 mg,
0.49 mmol, 5 equiv.) and boronic acid (0.49 mmol, 5
equiv.) were added to the solution and the mixture was
shaken for 65 h at 110°C. e resin was collected by
filtration, washed with THF (2 × 10 mL), THF/H2O 1:1
(2 × 10 mL), MeOH (2 × 10 mL) CH2Cl2 (2 × 10 mL), DMF
(2 × 10 mL), THF (2 × 10 mL), MeOH (2 × 10 mL) and
CH2Cl2 (2 × 10 mL), respectively to afford 8.
3-Bromo-4-cyano-5-(isopropylthio)thiophene-2-carboxylic
acid (6)
To a solution (THF/EtOH 2:1 (v/v), 20mL) of 4 (2.58 g,
7.74 mmol, 1.0 equiv.), aqueous NaOH (2 M, 20 mL) was
added. After stirring overnight at room temperature, the
mixture was neutralized with aqueous HCl (1 M, 40 mL).
e precipitated product was filtered, washed with water
anddriedundervacuumtoaffordacid6asawhitepowder
(2.17 g, quant.). 1H NMR (500 MHz, acetone-d6) δ 1.46 (d,
J= 6.5 Hz, 6H), 3.75 (h, J= 6.5 Hz, 1H). 13C NMR (75 MHz,
acetone-d6) δ 23.3, 43.3, 114.3, 119.2, 119.3, 131.6, 155.5,
161.0. IR (film) 2969, 2221, 1689, 1650, 1498, 1241 cm−1.
MS (ESI−, CH2Cl2/MeOH) m/z 304 and 306 [M-H]−. HRMS
(ESI−, CH2Cl2/MeOH) calcd for C9H779BrNO2S2 [M-H]−
303.9102, found 303.9102; calcd for C9H781BrNO2S2 [M-H]−
305.9078, found 305.9081. Elemental analysis calcd (%)
for C9H8BrNO2S2: C 35.30, H 2.63, N 4.57, O 10.45, S 20.94;
found C 35.35, H 2.62, N 4.51, O 10.58, S 20.78.
3-aryl-4-cyano-5-(isopropylthio)thiophene-2-carboxylicacid
(1) To the swelled resin 8, CH2Cl2/TFA (1:1 v/v, 5 mL)
was added and the mixture was shaken for 5 min. e
solution was filtered, then CH2Cl2/TFA (1:1 v/v, 5 mL)
was added again on the resin that was shaken 40min. e
resin was filtered and washed with CH2Cl2. e filtrates
were pooled and concentrated under vacuum. e crude
residue was diluted to 1 mg/mL and analysed by UHPLC.
Whenneeded, theproductwaspurifiedbyPLC(Heptane/
EtOAc 5:5) to afford pure desired compounds.
Ethyl 3-(4-methoxyphenyl)-4-cyano-5-(isopropylthio)
thiophene-2-carboxylate (5j)
To a solution (toluene/EtOH 9:1 (v/v), 20 mL) of 4 (0.71 g,
2.12 mmol, 1.0 equiv.), K2CO3 (2 M in water, 2.65 mL,
5.3 mmol, 2.5 equiv.), Pd(PPh3)4 (0.245 g, 0.21 mmol, 10
% mol) and 4-methoxyphenyl boronic acid (0.645 g, 4.2
mmol, 2.0 equiv.) were added. After stirring for 20 h under
reflux, water was added to the reaction mixture which
was extracted three times with EtOAc. Combined organic
layers were washed with brine, dried over MgSO4 and
concentrated under reduced pressure. e crude prod-
uct was purified by flash chromatography on silica gel
(heptane/EtOAc 100:0 to 85:15 (v/v) in 35 min) to afford
5j as a white amorphous solid (0.302 g, 50%). Mp = 90°C;
1H NMR (300 MHz, CDCl3) δ 1.23 (t, J= 7.0 Hz, 3H), 1.46
(d, J= 6.5 Hz, 6H), 3.63 (h, J= 6.5 Hz, 1H), 3.85 (s, 3H), 4.22
(q, J= 7.0 Hz, 2H), 6.97 (d, J= 8.5 Hz, 2H), 7.38 (d, J= 8.5
Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 14.0, 23.2, 42.1,
55.3, 61.6, 113.5, 113.8, 116.0, 124.2, 128.6, 130.8, 149.1,
152.7, 160.3. IR (film) 2971, 2918, 2837, 2216, 1713, 1610,
1530, 1496, 1458, 1365, 1358, 1264, 1180, 1083, 1024, 828,
759 cm−1. MS (ESI+, CH2Cl2/MeOH) m/z 384 [M+Na]+.
HRMS (ESI+, CH2Cl2/MeOH) calcd for C18H19NO3S2Na
[M+Na]+ 384.0704, found 384.0722.
Biological assays
Yeast FTase assay
Assays were realized on 96-well plates, prepared with
Biomek NKMC and Biomek 3000 from Beckman Coulter
and read on Wallac Victor fluorimeter from Perkin-
Elmer. Per well, 20 µL of farnesyl pyrophosphate (10
µM) was added to 180 µL of a solution containing 2 µL of
varied concentrations of potential inhibitors (dissolved
in DMSO) and 178 µL of a solution composed by 0.1 mL
of partially purified recombinant yeast FTase (2.2 mg/
mL) and 7.0 mL of dansyl-GCVLS peptide (in the fol-
lowing buffer: 5.8 mM DTT, 6 mM MgCl2, 12 µM ZnCl2
and 0.09% (w/v) CHAPS, 53 mM Tris/HCl, pH 7.5). en
the fluorescence development was recorded for 15 min
(0.7 second per well, 20 repeats) at 30°C with an excita-
tion filter at 340 nm and an emission filter at 486 nm.
Each measurement was realized twice as duplicate or
triplicate.
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 µL
of partially purified human FTase (1.5 mg/mL)7 in 1 mL
peptide solution.
Solid-phase synthesis
Polymer-bond
3-bromo-4-cyano-5-(isopropylthio)thio-
phene-2-carboxylate (7) To a solution (THF, 8 mL) of
swelled Wang resin (1.3 mmol/g, 1.0 g, 1.3 mmol, 1
equiv.), bromothiophene 6 (793 mg, 2.6 mmol, 2 equiv.),
DIAD (1.02 mL, 5.2 mmol, 4 equiv.) and PPh3 (1.36 g, 5.2
mmol, 4 equiv.) were added. e mixture was stirred on
an orbital shaker for 6 h at room temperature. e resin
was collected by filtration, washed with THF (3× 20 mL),
Cloning and expression of recombinant protein Tb-FTase
e cloning of the αβ-Tb-FTase from the Trypanosoma
brucei brucei strain WT 2913 was realized according to
© 2013 Informa UK, Ltd.

166 S. Lethu et al.
Buckner et al.8. Briefly, procyclic forms of T. brucei brucei
were maintained in semi-defined medium 79 contain-
ing 10% (v/v) heat-inactivated fetal calf serum (FCS) at
27°C. Cells were collected by centrifugation from loga-
rithmic cultures and DNA extracted. e α subunit was
amplified from genomic DNA by PCR using the sense
(5′-CCGGATCCCATGAATAAGAGCGCGGTGCGTAGT
GAAGAAAGCCGC-3′) and antisense oligonucleotides
(5′-GAAAGCTTCATATGTTAAAACTCCTCAACATAG
TCCCGGTTCATAACG-3′), respectively. e 1800 bp PCR
fragment was purified (DNA clean up-GEHealthcare)
and ligated into a TOPO 2.1 vector (Clontech). e β
subunit was similarly amplified using the sense (5′-
ATACTTCCATATGTCTTTTGTACCACCTCCAGC-3′)
and antisense oligonucleotides (5′-CTATCCTAGGAG
100 µL culture medium in 96-well plates. Asynchronous
parasite cultures (100 µL, 1% parasitaemia and 1%
final hematocrite) were then added to each well and
incubated for 24 h at 37°C prior to the addition of 0.5µ
Ci of [3H]-hypoxanthine (GE Healthcare, France, 1 to 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. e growth inhibition for each
drug concentration was determined by comparison of
the radioactivity incorporated in the treated culture with
that in the control culture maintained on the same plate.
e concentration causing 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 indepen-
dent experiments.
ATCTTCAGCACATGAAAGTCTTTGCGC-3′),
respec-
tivelyandalsoclonedintotheTOPO2.1vector(Clontech).
e α-subunit gene was excised from its vector by BamHI
and NdeI digestion, gel purified and cloned into the
BamHI/NdeI sites immediately upstream the β-subunit
gene into the vector TOPO 2.1-β previously digested with
Bam HI and NdeI. e resulting TOPO 2.1-α−β plasmid
was digested with BamHI and BglII to release a 3600 bp
fragment corresponding to the α and β subunits in tan-
dem of the Tb-FTase gene. e α–β tandem was cloned
into the BamHI site of the expression plasmid pRSF-1b
(Novagen). e recombinant plasmid was used to trans-
form top 10 E. coli and the clones with proper orientation
were designated pRSF1-TbPFT. For the Tb-FTase protein
expression, the E. coli strain BL21(DE3) was transformed
with pRSF1-TbPFT, grown in LB + kanamycine until DO
(A600) = 0.6. e culture was then cooled down to 24°C
before induction by addition of 0.4 mM IPTG (isopropyl-
ß-D-thiogalactopyranoside) and grown 3 h at room tem-
perature. e bacteria are harvested by centrifugation
(12 min, 8000 rpm) and the pellet frozen at −20°C. To
purify the soluble recombinant protein, bacteria were
lysed using Dyno Mill (0.2 mm) and purified according to
our reported procedure for recombinant human FTase7
and stored with 5% glycerol at 4°C.
Assay for in vitro inhibition of T. brucei brucei growth
Bloodstream forms of T. brucei brucei strain 90–13 were
cultured in HMI9 medium supplemented with 10% FCS
at 37°C under an atmosphere of 5% CO211. In all experi-
ments, log-phage cell cultures were harvested by centrif-
ugation at 3000g and immediately used. Drug assays were
based on the conversion of a redox-sensitive dye (resa-
zurin) to a fluorescent product by viable cells12 and were
performed according to the manufacturer recommenda-
tions (AlamarBlue® Assay, Invitrogen Corporation). Drug
stock solutions were prepared in pure DMSO. T. brucei
brucei bloodstream forms (3 × 104 cells/mL) were cul-
tured as described above in 24-well plates (1 mL per well)
either in the absence or in the presence of different con-
centrations of inhibitors (0, 0.1, 1, 10 and 100 µM) with a
final DMSO concentration of 1%. After a 72-h incubation,
AlamarBlue® solution (100 µL) was added in each well
and fluorescence was measured at 530 nm excitation and
590 nm emission wavelengths after a further 4-h incuba-
tion. Each inhibitor concentration was tested in triplicate
and the experiment repeated twice. e percentage of
inhibition of parasite growth rate was calculated by com-
paring the fluorescence of parasites maintained in the
presence of drug to that of in the absence of drug.
T. brucei FTase assay
Assays were realized on 96-well plates, as described
for human FTase with the dansylated peptide Dansyl-
GCAIM and the solution contains 15 µL of partially puri-
fied Tb-FTase (1.0 mg/mL) in 1 mL peptide solution.
Results and discussion
Novel synthetic pathway to 3-arylthiophene
2-carboxylic acids
Assay for in vitro inhibition of Plasmodium falciparum growth
e chloroquine-resistant strain FcB1/Colombia of P. f al-
ciparum was maintained in vitro on human erythrocytes
in RPMI 1640 medium 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
assays was measured by [3H]-hypoxanthine incorpora-
tion as described9 using a modification of the semi-au-
tomated microdilution technique of Desjardins et al.10.
Drugs were prepared in DMSO at a 10mM concentra-
tion. Compounds were serially diluted two-fold with
According to our strategy to develop new 3-modified
derivatives, it seemed reasonable to carry out the intro-
duction of the phenyl ring at the end of the synthesis
by a general and efficient method allowing the forma-
tion of the biaromatic unit with various substituants.
Suzuki–Miyaura cross-coupling13,14 fulfilled our require-
ments because of its common use in arylation of halog-
enothiophene15–19, though less frequently described in
solid phase than in homogenous synthesis20–22. is
palladium-catalyzed reaction could afford the desired
compounds from the corresponding 3-bromothiophene
Journal of Enzyme Inhibition and Medicinal Chemistry

Diversification of 3-arylthiophene FTIs by solid-phase synthesis 167
derivative obtained by a Sandmeyer reaction on a
3-amino thiophene23 itself synthesized according to pre-
viously described Fiesselmann-type reaction on a ketene
dithioacetal prepared from commercially available
malononitrile (Scheme 1)24. is pathway was applied to
the synthesis of our hit compound, 3-(4-chlorophenyl)-4-
-cyano-5-(isopropylthio)thiophene-2-carboxylic acid 1a
and proceeded in a very efficient 48% overall yield from
malononitrile (Scheme 2). Ketene dithioacetal 2 was
obtained by deprotonation of malononitrile followed
by condensation with carbon disulfide and subsequent
quenching with isopropyl iodide. Cyclocondensation of
2 with ethyl thioglycolate afforded the amino derivative 3
that was converted to bromothiophene 4 by a Sandmeyer
reaction.
e crucial step for our methodology, Suzuki–Miyaura
palladium-catalyzed coupling of compound 4 with
4-chlorophenylboronicacid,hadtobeoptimized.Among
many assayed conditions, tetrakis(triphenylphosphine)
palladium was found to be the best catalyst for this reac-
tion, potassium carbonate proved to be the most efficient
base, the mixture of toluene/ethanol (9:1, v/v) was the
best solvent and running the reaction at 110°C afforded
the ester 5a in excellent yield. Compound 1a was finally
obtained after basic hydrolysis. e efficiency of this new
pathway where the aryl moiety is introduced at the end of
the synthesis allowed us to plan the generation of diverse
3-arylthiophene analogues.
Solid-phase synthesis
To rapidly obtain a 3-arylthiophene-2-carboxylic acid
library, we decided to realize the synthesis on solid sup-
port. e thiophene could be linked to the resin by an
ester function after reaction with the 2-carboxylic moi-
ety. We previously noted that acid derivatives were bet-
ter FTIs than other 2-carbonyl-containing compounds
such as amides or esters4. erefore we chose the Wang
resin as solid support that could provide directly the
expected acid by cleavage of the resin. Our thiophene
derivative was linked to the Wang resin just before the
Suzuki–Miyaura cross-coupling step (Scheme 3). us,
compound 4 had to be saponified to allow subsequent
linkage to the resin. Under the conditions used to obtain
1a, compound 4 hydrolysis afforded the acid derivative 6
in quantitative yield. To attach compound 6 to the poly-
mer, we first investigated the EDC/HOBt method involv-
ing the acid activation. Variations in coupling agent, base
or acid amounts did not allow to get more than 60% of
polymer-bond thiophene. is was observed whatever
was the polymer substitution rate we used25. Increasing
the reaction time did not improve the attachment yield
either.Becauseouracid6mightbestericallytoohindered,
we rather thought to activate the polymer-bond hydroxyl
function to achieve linkage to the resin. As expected, such
coupling was successfully achieved under Mitsunobu
conditions26, leading to a linkage rate up to 97% (calcu-
lated by elemental analysis from the sulfur ratio in the
polymer). Before performing the Suzuki–Miyaura cross-
coupling, we checked whether the classical cleavage
method using trifluoroacetic acid (TFA) in CH2Cl2 would
afford the expected thiophene-2-carboxylic acid. Under
these conditions, no degradation or secondary products
were observed and the bromo acid 6 was recovered in
quantitative yield.
As previously mentioned, the Suzuki–Miyaura cross-
coupling has not often been used for thiophene arylation
on solid support20–22 and, to our knowledge, it has never
been described for tetrasubstitued resin-bond deriva-
tives. erefore the reaction of 7 with 4-chlorophenyl
boronic acid had to be optimized. Pd(PPh3)4 was chosen
ascatalystaccordingtoourpreviousstudyinhomogenous
Scheme 1. Retrosynthetic pathway.
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, RT, 30min, then CS2, RT, 30min, then iPrI, TBAB, 60°C, 70h, 73%; (b) HSCH2CO2Et,
K2CO3, EtOH, reflux, 3h, 84%; (c) tBuONO, CuBr2, CH3CN, RT, 10min, 94%; (d) Pd(PPh3)4, K2CO3, 4-ClPhB(OH)2, toluene/EtOH (9:1), reflux,
4h, 91%; (e) NaOH 2M, EtOH, RT, 16h, 92%.
© 2013 Informa UK, Ltd.

168 S. Lethu et al.
Scheme 3. Reagents and conditions: (a) NaOH 2M, EtOH, RT, 16h, quant.; (b) PPh3, DIAD, THF, RT, 6h, 97%; (c) Pd(PPh3)4, ArB(OH)2,
110°C, 65h; (d) TFA/CH2Cl2 (1:1, v/v), RT, 45min.
medium and the palladium-catalyzed reaction was car-
ried out with an excess of boronic acid and base. Several
bases, solvents and temperatures were assayed for opti-
mization (Table 1). e conversion rate was determined
by 1H NMR of the crude filtrate after TFA cleavage.
Table 1. Optimization of Suzuki–Miyaura cross-coupling
conditions for 1a synthesis*.
Entry Base
Solvent
Temperature Conversion
†
†
1
2
3
4
5
6
7
8
Toluene/EtOH 10:1
90°C
90°C
90°C
90°C
90°C
90°C
90°C
110°C
26%
8%
K2CO3
K2CO3
DME
Temperature was an important factor for the reac-
tion and below 100°C, conversion rate was very low
(entries 1–7), whereas increasing temperature to reflux
improved it significantly (entries 1 vs 8 and 7 vs 17).
Among the solvents we used, the toluene/ethanol
mixture as well as dioxane gave the best results. e
amount of ethanol was varied to find the best solvent
conditions. Toluene alone afforded the desired cou-
pling compound in very low yield (data not shown).
Up to 20% EtOH (entry 13), an excellent reaction rate
was observed but dropped dramatically as the amount
of alcohol increased (entries 14 and 15). e base effi-
ciency was dependant on solvent as experienced with
cesium salts that gave good results in dioxane (entry 19)
but were ineffective in toluene/ethanol (entries 9 and
10). As well, aqueous potassium carbonate was found
to be the best base both in dioxane (entry 17) and in tol-
uene/ethanol (entry 8) but in the latter mixture, aque-
ous sodium carbonate and potassium phosphate were
equally efficient (entries 8, 11 and 12). At that stage of
our study, we found difficulties to reproduce our cross-
coupling reaction in similar yields whereas conversion
was complete. In such basic media, we suspected etha-
nol to realize transesterification on the polymer-bound
compound resulting in loss of substitution rate on the
resin. erefore we changed ethanol to tert-butanol
less prone to induce transesterification. As expected,
excellent and very reproducible yields were observed
in such conditions. Potassium phosphate giving the
best conversion in toluene/tBuOH, the chosen condi-
tions were Pd(PPh3)4 (10 mol%), boronic acid (5 equiv.),
K3PO4 (5 equiv.) in toluene/tBuOH (9:1, v/v) at 110°C
and the reaction time was increased from 24 h to 65 h
to achieve complete conversion in most cases. ese
conditions were applied on a variety of commercially
available arylboronic acids (Table 2). Conversion rates
†
†
†
†
†
THF
7%
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
DME
7%
Xylene/EtOH 25:1
DMF
15%
0%
Dioxane
15%
100%
†
Toluene/EtOH 10:1
Toluene/EtOH 10:1
K2CO3
9
10
11
CsF
110°C
110°C
110°C
0%
0%
Cs2CO3 Toluene/EtOH 10:1
†
Toluene/EtOH 10:1
Toluene/EtOH 10:1
Toluene/EtOH 4:1
Toluene/EtOH 3:1
Toluene/EtOH 2:1
Dioxane
100%
Na2CO3
K3PO4
12
13
14
15
16
17
18
19
20
21
110°C
110°C
110°C
110°C
100°C
100°C
100°C
100°C
110°C
110°C
100%
100%
85%
0%
†
K2CO3
†
K2CO3
†
K2CO3
†
100%
73%
77%
75%
75%
100%
K2CO3
†
Dioxane
Na2CO3
K3PO4
Dioxane
Cs2CO3 Dioxane
†
Toluene/tBuOH 9:1
Toluene/tBuOH 9:1
K2CO3
K3PO4
*Reaction conditions: Pd(PPh3)4 10% mol, 4-ClPhB(OH)2 5 equiv.,
base 5 equiv., 24h, then CH2Cl2/TFA (1:1, v/v).
†2M in water.
were determined by UHPLC dosing after acidic release
from the resin, referring to bromo acid 6 as standard.
Phenylboronic acids bearing ortho substituants
were less reactive than their meta or para substi-
tuted analogues (entries 3, 6 and 12) or completely
unreactive (entries 9 and 18) probably because of
steric hindrance. Electron withdrawing groups were
also detrimental to cross-coupling (entries 14–17
and 28). Thus, p- and m-carbomethoxyphenyl were
successfully introduced on the thiophene ring but
in moderate to low yields. Amino containing phenyl
Journal of Enzyme Inhibition and Medicinal Chemistry

Diversification of 3-arylthiophene FTIs by solid-phase synthesis 169
that the ester forms of our FTIs were more active on
parasites than their acidic forms34. erefore, to evaluate
the antiparasitic activity of our series, we synthesized the
corresponding esters of the selected active thiophene
2-carboxylic acids 1j, 1aa and 1gg (see below), according
to Scheme 2. e esters 5j, 5aa and 5gg were obtained
from compound 4 and the corresponding boronic acids
in 50%, 61% and 74% yields, respectively.
Table 2. 3-arylthiophenes 1 prepared from Wang resin*.
Entry Ar Conversion Product Yield†
1
2
4-chlorophenyl
100%
100%
66%
100%
100%
36%
100%
99%
3%
1a
1b
1c
1d
1e
1f
1g
1h
1i
78%
69%
66%
94%
94%
33%
69%
86%
0%
3-chlorophenyl
2-chlorophenyl
4-fluorophenyl
3-fluorophenyl
2-fluorophenyl
4-(trifluoromethyl)phenyl
3-(trifluoromethyl)phenyl
2-(trifluoromethyl)phenyl
4-methoxyphenyl
3- methoxyphenyl
2- methoxyphenyl
4- hydroxyphenyl
4-formylphenyl
4-acetylphenyl
4-(methoxycarbonyl)phenyl
3-(methoxycarbonyl)phenyl
2-(methoxycarbonyl)phenyl
phenyl
3
4
5
6
7
Biological evaluation
8
Compounds 1a-h, 1j-l, 1p-q, 1s-v, 1x-aa, and 1ff-gg were
evaluatedfortheirinhibitoryactivityagainstrecombinant
yeast35, human7 and T. brucei FTases using a fluorescent-
based assay36,37 adapted to 96-well plate format. Results
are reported in Table 3.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
78%
70%
72%
7%
1j
1k
1l
73%
68%
49%
0%
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
1x
1y
1z
8%
0%
efirstobservationisthedifferenceofinhibitoryactiv-
ity according to FTase species. In general, arylthiophenes
are much more active on yeast FTase (IC50 = 0.085–10 µM)
than on human or trypanosomal enzymes (IC50 = 3–>50
µM). is was also observed with the reference com-
pound FTI-27638, evaluated under our assay conditions,
which showed a 20-fold increased inhibitory activity
on yeast enzyme relative to the human one. Ortho sub-
stitution is detrimental for activity in all cases (1c, 1f, 1l
and 1v) but the presence of a para-substituent generally
improves the inhibitory activity (compare 1s with 1a, 1d,
1p and 1t). e nature of the substituent seems to have
little influence on the activity that depends on the FTase
species. Bulky aromatic rings like naphtyl (1x-y), biphe-
nyl (1z-aa) or dibenzofuranyl (1gg) moieties increase or
at least retain the inhibitory activity against human and
Tb-FTases with a different exception for each enzyme:
the 4-biphenyl derivative 1z for the trypanosomal
protein and the 1-naphtyl analogue 1x for the human
species. On the contrary, there is a 3 to 30-fold drop in
activity against the yeast enzyme for these analogues. e
most active compound against yFTase in this series is the
4-carbomethoxyphenylthiophene (1p) which displays
a slight increased activity (1.3 fold) compared to par-
ent compound 1a. For hFTase the 3-biphenyl derivative
1aa exhibits the best activity together with 2-naphtyl 1y
and 4-biphenyl 1z with a 5 and 3-fold increased activ-
ity, respectively. e gain in inhibition is less important
for Tb-FTase with a 3-fold and 2-fold increase for 1gg
and 1aa, respectively. However, as far as our search for
antiparasitic agents is concerned, compounds 1j and 1gg
displayed a better selectivity against the parasitic enzyme
with a 4.5 ratio compared to the human one. erefore,
9%
0%
45%
54%
0%
28%
49%
0%
100%
90%
100%
99%
5%
87%
76%
86%
86%
0%
4-tolyl
3-tolyl
2-tolyl
4-vinylphenyl
1-naphtyl
94%
100%
100%
100%
13%
10%
6%
74%
80%
78%
2-naphtyl
4-biphenyl
3-biphenyl
1aa 76%
4-cyanophenyl
4-acetamidophenyl
4-(dimethylamino)phenyl
3-pyridyl
1bb
1cc
1dd
1ee
1ff
0%
0%
0%
4%
0%
3-thienyl
70%
66%
44%
1-dibenzofuranyl
1gg 64%
*Reaction conditions: Pd(PPh3)4 (10% mol), ArB(OH)2 (5 equiv.),
K3PO4 (5 equiv.), in toluene/tBuOH (9:1, v/v) at 110°C, 65h, then
CH2Cl2/TFA (1:1, v/v).
†Isolated yield.
boronic acids gave no reaction with bromothiophene
7 as well as 4-hydroxyphenyl boronic acid (entries
13, 15, 28-31). We did not succeed in introducing a
4-vinylphenyl group by Suzuki–Miyaura coupling
either (entry 23). Such phenylboronic acids bearing
electron withdrawing or amino groups have already
been coupled on thiophene or benzothiophene rings
following this methodology. However, these aromat-
ics were seldom tetrasubstituted or the thiophene
ring was not as electrodeficient as our substrate27–31
Interestingly, there is no report on palladium-cata-
lyzed thiophene arylation with 4-vinyl phenylboronic
acid. Either the vinyl moiety was introduced on the
4-iodo derivative after Suzuki coupling32 or a rhodium
catalyst was used33.
By this method, 22 new arylthiophenes 1 were
obtained in sufficient amount to evaluate their activity
on protein farnesyltransferase. We previously observed
.
we decided to evaluate the antiparasitic activities of the
two most active compounds on Tb-FTase together with
the 4-methoxy derivative which display a good selectiv-
ity for the parasitic enzyme. e evaluation was realized
on their ester forms 5j, 5aa and 5gg against P. falciparum
(intraerythrocytic forms) and T. brucei (bloodstream
form) proliferation. For comparison, we also evaluated
our hit compound 1a and its ester derivative 5a. Results
are reported in Table 4.
© 2013 Informa UK, Ltd.

170 S. Lethu et al.
Table 3. Inhibition of FTases by 3-arylthiophene-2-carboxylic
acids 1.
Conclusion
We designed an original method to rapidly obtain a small
library of 3-aryl-4-cyano-5-(isopropylthio)thiophen-
2-carboxylic acids. is method using Suzuki–Miyaura
cross-coupling has successfully been employed for the
first time on resin-bond tetrasubstitued thiophenes
to afford 22 new arylthiophenes 1 in sufficient yield to
evaluate their activity on FTase. However it is not appli-
cable to arylthiophene derivatives bearing electron
withdrawing groups, hydroxyl or amino moieties or
hindered ortho substituants. For these derivatives, the
synthesis via arylpropionitrile4 was more appropriate
and will be reported soon. Evaluation of the inhibitory
activity of these new 3-arylthiophenes has shown that
it is greatly dependent on the FTase species, showing
a 2-order of magnitude difference between yeast and
human or trypanosomal enzymes. For them all, para
or meta substitution is preferred to nude phenyl group,
substitution at the ortho position being a less favorable
pattern for FTase inhibition. Finally, we succeeded in
improving the FTase inhibitory activity of our 3-arylthio-
phene derivatives by introducing either a carbomethoxy
group in the para position of the phenyl ring for the yeast
enzyme (allowing IC50 to go below 100 nM), or a phenyl
in the meta or para position for the human protein and
by replacing the p-chlorophenyl group by a m-biphenyl
or benzofuranyl moiety for Tb-FTase. As we are aim-
ing to find selective inhibitors of the parasitic enzyme,
the para-methoxyphenyl and benzofuranyl derivatives
seemed promising because of their 4.5 ratio between
human and trypanosomal FTase inhibitory activity.
ese compounds are also more active on T. brucei pro-
liferation, though to a small extent. Because of the high
arylthiophene hydrophobicity, cell penetration may be
a problem for these compounds. erefore, we thought
to add oxygen atoms on these molecules to increase cel-
lular activity what is under current investigation and will
be reported soon.
IC50 (µM)
yFTase
IC50 (µM)
hFTase
IC50 (µM)
Tb-FTase
Compound
1a
1b
1c
1d
1e
1f
1g
1h
1j
1k
1l
1p
1q
1s
0.11 0.008
1.3 0.1
16
48
2
9
10.6
2
14.5 1.5
1.4 0.3
> 50
20
37
29
32
8
8
5
0.27 0.02
0.46 0.08
1.3 0.2
2
4
23
> 50
nd
34
46 13
0.58 0.13
1.6 0.15
0.25 0.03
1.6 0.2
15% inhat 10 µM
5
4
4
19
4
39
8.6 1.1
29
18
37
14
4
7
2
9.7 2.8
> 50
0.085 0.016
0.62 0.07
0.61 0.04
0.19 0.04
1.4 0.2
12.8 1.4
7.0 0.7
6.9 0.6
34
21
5
2
3
42
17
9
4
8
1t
1u
1v
1x
1y
16
34
3.1 0.8
> 50
32
> 50
10.5
1.1 0.2
6
2
0.32 0.05
2.7 0.5
4.8 0.15
5.5 0.6
3.3 0.2
9.7 1.7
32
5.4 0.9
26
1z
6
1aa
1ff
1gg
FTI-276
0.64 0.06
0.35 0.07
1.0 0.2
31
5
7
15 1.3
3.3 0.4
0.00078 0.00008 0.015 0.004
0.010 0.002
Table 4. Inhibition of P. falciparum and T. brucei proliferation
with selected 3-arylthiophenes*.
Compound
IC50 P. falciparum (µM) IC50 T. brucei (µM)
1a
Inactive at 10 µM
14 0.9
71†
40†
5a
5j
5aa
5gg
Pentamidine
Chloroquine
27 0.7
30 0.9
15 0.9
–
7.0 0.2
15 0.3
11 0.9
0.011 0.0017
–
0.072 0.0074
*Otherwise indicated IC50 were expressed as the mean values
standard deviations determined from at least three independent
experiments.
Acknowledgements
e 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 FTase, Dr P. Durand for fruitful discus-
sions and his help for solid-phase synthesis, O. oison
and her coworkers for UHPLC analyses, M-F. Bricot for
elemental analysis, ICSN and CNRS for financial support.
†Determined from two experiments.
As predicted, ester 5a is more active than the free
carboxylic acid 1a. e improved enzyme inhibition
led only to a slight augmentation of the antiparasitic
activity. It is to note that these arylthiophenes are more
active against T. brucei than against P. falciparum but
their IC50 remains in the same micromolar range what-
ever the aryl moiety may be. However, the methoxy
derivative 5j is the most active compound against T.
brucei although its Tb-FTase inhibition is equivalent to
that of our hit compound. It can be assumed that the
presence of an additional oxygen atom on these hydro-
phobic molecules could improve cell penetration and/
or aqueous solubility.
Declaration of interest
is work was financially supported by ICSN and CNRS.
e authors report no conflicts of interest.
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