Discovery of a new class of protein farnesyltransferase inhibitors in the arylthiophene series

Article abstract of DOI:10.1021/jm901280q

Screening of the ICSN chemical library led to the discovery of 3-(4-chlorophenyl)-4-cyano-5-thioalkylthiophene 2-carboxylic acids as potent farnesyltransferase inhibitors. Enzymatic kinetic studies showed that this original FTI series belongs to the CaaX

Full text of DOI:10.1021/jm901280q

J. Med. Chem. 2009, 52, 6205–6208 6205
DOI: 10.1021/jm901280q
Several biological activities have already been reported for
3-arylthiophene derivatives. Some of them display anthelmintic
activity against Haemonchus contortus,11 and others have anti-
leishmanial and antifungal activities12 or inhibit c-Jun-N-term-
inal kinases and other kinases.13 Certain arylthiophenes have
also been patented as herbicides14 or for fungicidal15 and HLA-
DM activities.16 Our compounds belong to the 3-aryl-4-cyano-
5-substituted thiophene 2-carboxylic acid family, among which
some are AMPA receptor allosteric modulators.17,18
In this paper we describe the determination of 1 binding
mode on the enzyme along with preliminary structure-acti-
vity relationship (SAR) studies on this new FTI series.
To understand the mode of action of our hit compound 1,
enzymatic kinetic experiments were carried out to determine
where it binds to FTase. These studies showed that 1 was a
CaaX competitive inhibitor with Ki = 105 nM and a FPP
noncompetitive inhibitor with Ki = 110 nM. Thus, this
compound occupies the CaaX binding site without interacting
with the FPP binding site. The CaaX peptide fills a large part
of the FTase active site cavity, and the zinc ion directly
coordinates the sulfur atom of the cysteine.4 Our kinetic
results suggest that these arylthiophene derivatives interact
with the zinc ion of the catalytic site probably by means of the
thioether function in conjunction with the sulfur atom of the
thiophene ring. Preliminary SAR studies were therefore un-
dertaken to determine the binding contribution of different
parts of the molecule.
We first looked at the importance of the thiophene central
core. We used a described protocol allowing variation of the
heteroaryl core of the molecule.18 Ketene dithioacetal 4 was
obtained by deprotonation of p-chlorobenzoylpropionitrile 3
followed by condensation with carbon disulfide and subse-
quent quenchingwithisopropyliodide. Compound1was then
obtained by cyclocondensation of 4 with ethyl thioglycolate
and subsequent basic hydrolysis (Scheme 1). This synthetic
pathway allowed us to prepare the corresponding furan 8 and
methylpyrrole9 by condensation of 4 with ethyl bromoacetate
or sarcosine ethyl ester, respectively.
Thiazole 15 was also considered as an alternative hetero-
cyclic derivative. Ethyl p-chlorobenzoylacetate 10, obtained
from p-chlorobenzoic chloride and ethyl acetate, was chlori-
nated and subjected to condensation with thiourea.19 Sand-
meyer substitution of the amino group by a bromide further
substituted by propane-2-thiol allowed introduction of the
thioether function. Saponification afforded thiazole 15
(Scheme 2).
Discovery of a New Class of Protein
Farnesyltransferase Inhibitors in the
Arylthiophene Series
ꢀ
Sebastien Lethu, Maryon Ginisty, Damien Bosc, and
€
Joelle Dubois*
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
Received August 26, 2009
Abstract: Screening of the ICSN chemical library led to the dis-
covery of 3-(4-chlorophenyl)-4-cyano-5-thioalkylthiophene 2-car-
boxylic acids as potent farnesyltransferase inhibitors. Enzymatic
kinetic studies showed that this original FTI series belongs to the
CaaX competitive inhibitor class. Preliminary SAR studies allowed
us to improve the IC50 from 110 to 7.5 nM.
Since its discovery in the late 1980s, protein farnesyltrans-
ferase (FTasea) has been the subject of numerous studies.1-3
This heterodimeric metalloenzyme belongs to the protein
prenyl transferase family. It catalyzes 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 motif of proteins where C is a cysteine, a an aliphatic
amino acid, and X a serine, a methionine, an alanine, or a
glutamine.4,5 Protein farnesylation is an important posttrans-
lational modification occurring on several cell signaling pro-
teins like small GTPases, including the oncogenic Ras pro-
teins. Therefore, farnesyltransferase inhibitors (FTIs) have
been extensivelydeveloped for anticancertherapy, anddiverse
compounds with druglike properties are available.6,7 Re-
cently, 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.8 Development of FTIs has been realized by
rational design focused on modifications of isoprenoid or
CaaX substrates or by library screening. These studies led to
the discovery of three potent FTIs that are currently under
clinical trials for anticancer therapy: BMS214662 (rational
design approach) and lonafarnib and tipifarnib (library
screening approach).9
In the course of our search for new FTIs, we screened our
Institut’s chemical and natural product libraries on our auto-
mated fluorescence-based FTase assay.10 Two arylthiophene
derivatives among the 4500 compounds in the ICSN library
held more specifically our attention because of their activity
and structure (Figure 1). Initial screening showed a submi-
cromolar activity for these compounds bearing an original
structure in the FTI’s field.
The importance of the cyano group in FTase binding
was evaluated by the synthesis of 3-(p-chlorophenyl)-
5-(isopropylthio)thiophene-2-carboxylic acid 16 using the
*To whom correspondence should be addressed. Phone: þ33 1 69 82
30 58. Fax: þ33 1 69 07 72 47. E-mail: joelle.dubois@icsn.cnrs-gif.fr.
a Abbreviations: FTase, protein farnesyltransferase; FPP, farnesyl
pyrophosphate; FTI, farnesyltransferase inhibitor; ICSN, Institut de
Chimie des Substances Naturelles; AMPA, R-amino-3-hydroxy-5-meth-
yl-4-isoxazolepropionic acid; SAR, structure-activity relationship; rt,
room temperature; IC50, inhibitor concentration that inhibits 50% of
protein farnesyltransferase enzyme activity.
Figure 1. Structure of new FTIs.
r
2009 American Chemical Society
Published on Web 09/22/2009
pubs.acs.org/jmc

6206 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lethu et al.
Scheme 1. Synthesis of Compound 1 and of Its Heteroaromatic
Scheme 3. Synthesis of Compound 16a
Analoguesa
a Reagents: (a) (i) NaH, DMF, rt; (ii) CS2, rt; (iii) i-PrI, TBAB, DMF,
60 °C (79%); (b) HSCH2CO2Et, K2CO3, EtOH, reflux (71%); (c)
NaOH, THF/EtOH/H2O (2:1:1), rt (64%).
Scheme 4. Modification of the Carboxylic Acid from
Compound 1a
a Reagents: (a) (i) NaH, DMF, rt; (ii) CS2, rt; (iii) i-PrI, 60 °C, (82%);
(b) HSCH2CO2Et, Et3N, EtOH, rt for 5 (92%), BrCH2CO2Et,
LiHMDS, THF, -78 °C to rt for 6 (34%), MeNHCH2CO2Et, Et3N,
EtOH, reflux for 7 (46%); (c) NaOH, THF/H2O/EtOH (4:3:2), rt for 1
(93%); NaOH, EtOH/H2O (1:1), rt for 8 (87%); LiOH, THF/MeOH/
H2O (2:1:1), 60 °C for 9 (89%).
a Reagents: (a) Cu, quinoline, 250 °C (81%); (b) (i) SOCl2, CH2Cl2, rt;
(ii) NH4OH, rt (86%); (c) Met-OMe, HOBt, Et3N, EDCI, CH2Cl2, rt
(90%); (d) LiOH, MeOH/THF/H2O (2:1:1), rt (50%).
Scheme 2. Synthesis of Thiazole 15a
Figure 2. Commercial amide derivative.
coupled with an amino acid under usual conditions. Because
our hit compound 1 is a CaaX competitive inhibitor, we
thought that introduction of an amino acid at this position
would improve binding affinity and solubility. In the CaaX
motifs recognized by FTase, X is often a methionine. There-
fore, this amino acid was chosen for this preliminary study.
Classical peptide formation with methionine methyl ester
afforded 25 that was further hydrolyzed by lithine to afford
the free acid 26 (Scheme 4).
a Reagents: (a) SO2Cl2, toluene, rt (85%); (b) thiourea, EtOH, reflux
(98%); (c) CuBr2, t-BuONO, CH3CN, 60 °C (89%); (d) i-PrSH,
t-BuOK, THF, 10 °C to rt (76%); (e) NaOH, EtOH/H2O (1:1), rt (95%).
To complete the 2-amide-3-(p-chlorophenyl)thiophene
series, the cyclopropylamide 27, commercially available as
the methyl thioether, was also evaluated (Figure 2).
Synthesis of thiophene 1 went through its ester 5, allowing
the evaluation of the importance of a free carboxylic acid in
this part of the molecule. Some modifications were realized on
this ester such as reduction by sodium borohydride yielding
the primary alcohol 28 followed by its acetylation (29).
Compound 5 was also transformed into the Weinreb amide
30 that was alkylated by methyllithium to afford the methyl
ketone 31 (Scheme 5).
same pathway as for thiophene 1 but starting from methyl
p-chlorophenyl ketone (Scheme 3).
Influence of the thioether group was studied with 3-(p-chloro-
phenyl)-4-cyano thiophene 2-carboxylic acids available in
commercial libraries. The alkyl group of the thioether varied
from one to six carbons (1, 2, and 17-21), and a N-morpholine
derivative 22 was also available to evaluate the importance of a
sulfur atom at this position.
Carboxylic acid was the most considered part of this
preliminary SAR study. The acid was decarboxylated at high
temperature (23), transformed into a primary amide (24), or

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6207
Scheme 5. Modification of the Carboxylic Acid from Ester 5a
Table 2. Inhibitory Activities of 5-Modified 3-(p-Chlorophenyl)-
thiophenes
compd
R
IC50 (μM)
1
S-i-Pr
S-Et
S-Me
0.110 ( 0.008
0.325 ( 0.01
0.61 ( 0.03
1.24 ( 0.1
0.89 ( 0.06
0.345 ( 0.02
1.45 ( 0.15
28 ( 2.4
2
17
18
19
20
21
22
S-n-Bu
S-i-Bu
S-s-Bu
S-c-Hex
N-morpholine
a Reagents: (a) NaBH4, LiCl, THF/EtOH (1:2), rt (78%); (b) Ac2O,
DMAP, CH2Cl2, rt (76%); (c) NHMe(OMe), i-PrMgCl, THF, -15 °C
(80%); (d) MeLi, THF, -78 °C (74%).
Table 3. Inhibitory Activities of 2-Modified 3-(p-Chlorophenyl)-
thiophenes
Table 1. Inhibitory Activities of Various Heterocyclesa
compd
Het
IC50 (μM)
1
thiophene
furan
0.11 ( 0.008
2.0 ( 0.17
47 ( 3
8
9
15
NMe-pyrrole
thiazole
compd
R
IC50 (μM)
51 ( 5.5
1
CO2H
CO2Et
H
0.11 ( 0.008
3.8 ( 0.25
inactive
a IC50(FTase inhibitor I) = 0.032 μM in our assay conditions.
5
23
24
25
26
27a
28
29
30
31
All these derivatives were evaluated on FTase and com-
pared with commercial FTase inhibitor I.20 Results are
summarized in Tables 1-3.
CONH2
1.1 ( 0.05
5.7 ( 0.4
0.0075 ( 0.0007
22 ( 2.4
inactive
45 ( 5
inactive
CO-Met-OMe
CO-Met-OH
CONH-c-Pr
CH2OH
The thiophene ring is the best heterocycle in this series.
Furan 8 retains a relatively good inhibitory activity, 20-fold
less than that of thiophene 1. NMe-pyrrole 9 and thiazole 15
are weak inhibitors probably because 9 bears a methyl group
at position 1 that can hamper binding to FTase and 15 lacks
thecyanogroupatposition 4(Table 1). Theimportance ofthis
latter group is confirmed by the weak activity of 16 where the
cyano moiety has been replaced by hydrogen. Though 16 is
more active than thiazole 15, it exhibited IC50 = 15 ( 1 μM, 2
orders of magnitude lower than 1.
All the thioether derivatives show close activities within the
micromolar range (Table 2). Hit compound 1 is the most
active derivative. Inhibitory activity decreases when the alkyl
group is smaller (2 and 17) or larger (18-21) than isopropyl
and varies with the alkyl chain length. Branched alkyl groups
are better than linear ones especially when the branched
carbon is adjacent to the sulfur atom (20 vs 19). The thioether
at the C-5 position may contribute to FTase binding, since
CH2OAc
CON(Me)OMe
COMe
0.79 ( 0.08
a Methylthioether derivative.
ketone 31 retains a good activity, though 8-fold lower than
that of thiophene 1, like the amide derivative 24. The inhibi-
tory activity drops when the amide is substituted by an alkyl
group (27) and disappears when two substituents are present
on the amide as in the Weinreb amide 30. However, when
methionine is added to 1, the activity remains the same for the
ester derivative 25 as for 5 but greatly increases for the acid
form 26 with an IC50 in the low nM range. This is in good
agreement with our general observation that an acidic deri-
vative is a better inhibitor than its corresponding ester in the
enzymatic assay. This result also supports our kinetic findings
that these arylthiophene derivatives occupy the CaaX binding
site.
In conclusion, this new FTI series belongs to the nonpepti-
dic CaaX inhibitors, the most active FTI class. Our prelimi-
nary SAR study has shown that the presence of the two sulfur
atoms and of the cyano function contributes to the good
affinity of these derivatives. A carbonyl group at the
2-position is necessary to inhibit FTase, and compounds
bearing a carboxylic acid or an amino acid are the best
inhibitors of this series. Through the addition of a methionine
on the acidic function we have succeeded in increasing the hit
compound 1 activity by 1.5 orders of magnitude. Variations
around this amino acid are currently under investigation.
Many other modifications are also conceivable such as the
the N-morpholine analogue 22 is much less active with IC50 =
28 μM, compared to IC50 = 1.45 μM for cyclohexyl derivative
21.
In general, removal or modification of the carboxylic
function is detrimental to the activity (Table 3). When the
carboxylic acid is removed (23), no activity is seen any more.
Its reduction (28) abolishes the inhibition though acetylation
of the primary alcohol (29) restores it slightly. Therefore, it
appears that the presence of a carbonyl group is important for
binding to FTase. The absence of a free carboxyl group lowers
the activity as seen by the 30-fold decrease for ester derivative
5. Predictably, furan, pyrrole, and thiazole esters 6, 7, and 14
are completely inactive (data not shown). When the carbonyl
group is still present, some inhibition is retained. Methyl

6208 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lethu et al.
p-chlorophenyl moiety that has not been modified in this
preliminary study. Aswell, otherZn-chelating functionscould
be introduced in the C-5 position of the thiophene ring to
improve the activity and solubility of these derivatives. The
aim of our research is to find new antiparasitic derivatives
through FTase inhibition. Therefore, we will focus our efforts
to modulate these arylthiophenes to make them selectively
active on parasitic FTase.
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(10) Our enzymatic assay is the adaptation to 96-well plate format of the
fluorescence-based assay described in the following references: (a)
Pompliano, D. L.; Gomez, R. P.; Anthony, N. J. Intramolecular
fluorescence enhancement: a continuous assay of Ras farnesyl:
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ꢀ
Acknowledgment. The authors thank J.-B. Crechet for the
generous gift of the E. coli strain expressing the yeast FTase,
Dr. J. Ouazzani and P. Lopes for the production and pur-
ification of recombinant yeast FTase, N. Maroteau for his
ꢀ
(11) Gonzalez, I. C.; Davis, L. N.; Smith, C. K., II. Novel thiophenes
and analogues with anthelmintic activity against Haemonchus
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ꢀ
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ICSNcompound library, andE. Galmiche andO. Thoisonfor
HPLC analyses. Pr. J. Y. Lallemand is acknowledged for his
interest in this work, and ICSN and CNRS are acknowledged
for providing financial support.
Supporting Information Available: Synthesis details and
spectral data for all new compounds, experimental details for
FTase inhibition, Lineweaver-Burk plots of the kinetic data,
and Ki determination. This material is available free of charge
via the Internet at http://pubs.acs.org.
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