Novel indolizine derivatives with unprecedented inhibitory activity on human farnesyltransferase

Article abstract of DOI:10.1016/j.bmcl.2014.10.044

The rational structural modification of new substituted indolizin-3-yl(phenyl)methanones 1a-i, 2a-i and 3a-i has greatly improved human farnesyltransferase inhibition. The para-bromophenyl analog 2f bearing an ester unit on the indolizine ring demonstrates the highest inhibition potential, with IC₅₀ value of 1.3 ± 0.2 μM. The amidic series 1a-i proves to be the most promising for future modulations, particularly at the triple bond level.

Full text of DOI:10.1016/j.bmcl.2014.10.044

Accepted Manuscript
Novel indolizine derivatives with unprecedented inhibitory activity on human
farnesyltransferase
Carmen Dumea, Dalila Belei, Alina Ghinet, Joëlle Dubois, Amaury Farce, Elena
Bîcu
PII:
S0960-894X(14)01104-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.10.044
BMCL 22099
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 August 2014
13 October 2014
14 October 2014
Please cite this article as: Dumea, C., Belei, D., Ghinet, A., Dubois, J., Farce, A., Bîcu, E., Novel indolizine
derivatives with unprecedented inhibitory activity on human farnesyltransferase, Bioorganic & Medicinal Chemistry
Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.10.044
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Novel indolizine derivatives with unprecedented inhibitory activity on human
farnesyltransferase
Carmen Dumea,a Dalila Belei,a Alina Ghinet,a,b,c Joëlle Dubois,d Amaury Farce,b,e and Elena
Bîcua,*
a Department of Organic Chemistry, Faculty of Chemistry, ‘Al. I. Cuza’ University of Iasi, B-dul Carol I,
Nr. 11, Corp A, 700506 Iasi, Romania
b Univ Lille Nord de France, F-59000 Lille, France
c
UCLille, EA GRIIOT (4481), Laboratoire de pharmacochimie, Ecole des Hautes Etudes d’Ingénieur
(HEI), 13 rue de Toul, F-59046 Lille, France
d Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif, Avenue de
la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
e
Faculté des Sciences Pharmaceutiques et Biologiques, EA GRIIOT (4481), IFR114, 3 Rue du Pr
Laguesse, B.P. 83, F-59006 Lille, France
Graphical abstract
The rational structural modification of new substituted indolizin-3-yl(phenyl)methanones 1a-
i, 2a-i and 3a-i has greatly improved human farnesyltransferase inhibition. The para-
bromophenyl analogue 2f bearing an ester unit on the indolizine ring demonstrates the highest
inhibition potential, with IC50 value of 1.3 0.2 µM. The amidic series 1a-i proves to be the
most promising for future modulations, particularly at the triple bond level.
Indolizine derivatives display a broad spectrum of biological activity. Our interest in this field
has focused on the design and synthesis of substituted indolizin-3-yl(phenyl)methanones
which inhibit human farnesyltransferase. Similar indolizines have recently been patented to
treat cancer,1-5 cardiovascular disorders,1-5 degenerative joint diseases6 such as osteoarthritis
or spondyloses. Inhibition mechanisms involve basic fibroblast growth factors (b-FGF)
(compounds I and II, Figure 1) and leukotriene production7 (compound III, Figure 1). Other
substituted indolizin-3-yl(phenyl)methanones (e.g. compound IV, Figure 1) are known to
inhibit MPtpA/MPtpB phosphatases8 involved in infectious diseases. In addition, indolizines
substituted by a trimethoxyphenyl unit show potent anti-mitotic and anti-proliferative
activity.9,10
Our interest in anticancer therapy is to design, synthesize and evaluate new molecules
targeting protein farnesyltransferase (FTase).11-15 Protein farnesyltransferase (FTase) is a
heterodimeric metalloenzyme of the prenyltransferase family. Farnesylation is a
posttranslational modification of several cell signaling proteins such as small GTPases,
including the oncogenic Ras proteins.16 FTase catalyzes the process of protein prenylation,
controlling the activation of Ras proteins and thus affecting intracellular signal traduction, cell
growth and proliferation.17,18 Therefore, FTase inhibitors (FTIs) have been developed for
anticancer therapy,19-21 and diverse compounds with drug-like properties have been
synthesized. Many clinical trials have now been completed with FTIs, with disappointing
results, inconsistent with evidence for potential inhibition of FTase. In many clinical trials,
1

dose limiting toxicities have been due to myelosuppression.22,23 This has been seen with
several peptidomimetic and non-peptidomimetic FTI compounds.22 Research work continues
to resolve these problems for anticancer therapy and other pathologies. One case is the in vitro
and in vivo pharmacological modulation of FTase in treatment of opioid withdrawal
syndrome.24
We have previously synthetized a series of FTIs bearing a triazole unit.11 In this series, the
para-chloro or bromo-phenyl substitution (e.g. compounds Va,b, Figure 1) enhanced the
biological activity on FTase. It is important to note that (1) these moieties are conserved in the
structure of the current target indolizines and that (2) the replacement of the ester group in the
structure of compounds Va,b with an amidic function, diminishes inhibitory activity.11 These
modulations have been retained in order to improve our functional knowledge of the ester
function (compounds 1a-i, 2a-i and 3a-i, Figure 1). We have already observed that propargyl
groups sometimes lead to improved FTI properties.15. As such indolizine-based FTIs have yet
to be described. We thus carried out a study of a new series of indolizines and the inhibitory
potential on human FTase (Figure 1).
Me N
O
2
N
O
O
O
O
O
N
N
N
N
Cl
O
N
H
O
H2N
Me
Me
OMe
OMe
t-Bu
Me
.
.
Na
CO H
2
Me CO
H
2
H O
2
I
II
3
inhibitor of fibroblast growth factors1-5
inhibitor of basic fibroblast growth factors6 inhibitor of leukotriene production7
B
N
B
N
N
O
R
H
N
O
A
A
C
O
N
O
CO2H
O
O
D
X
O
X
C
D
Y
N
N
O
n
S
Va X=Cl
Vb X=Br
IV
CN
1a-i Y=NH, n=1, X=H, Cl, Br, R=H, Me
2a-i Y=O, n=1, X=H, Cl, Br, R=H, Me
3a-i Y=O, n=2, X=H, Cl, Br, R=H, Me
phosphatase inhibitor8
inhibitors of human farnesyltransferase11
target compounds
Figure 1. Structure of some biologically active indolizin-3-yl(phenyl)methanones and of
target compounds
Here we endeavor to highlight the nature and the position of substituents on the indolizine
ring, those that may play an important role in the biological activity on protein
farnesyltransferase. A [3+2] cycloaddition synthetic pathway was thus privileged to achieve
different substitutions on the indolizine ring. The starting bromoacetyl derivatives 4a-c are
commercially available and were reacted with pyridines 5a-c using the same conditions as
reported in the literature25-32 to give pyridinium salts 6a-i in very good yields (84-93%) (see
2

‘supplementary data’ section) (Scheme 1). The construction of the indolizine unit in products
7a-i was then achieved by [3+2] cycloaddition reaction of the corresponding ylide (not
isolated), generated in situ by triethylamine treatment of pyridinium salts 6a-i, with ethyl
propiolate (Scheme 1).10,33 Saponification of indolizines 7a-i, followed by citric acid
acidification of corresponding sodium carboxylates yielded carboxylic acids 8a-i.34 Activated
esters 9a-i and 10d-i were then prepared by reaction of carboxylic acids 8a-i with N-
hydroxysuccinimide or benzotriazole. The synthetic strategy for the target compounds was
then accomplished by coupling reactions of activated esters 9a-i and 10d-i with
propargylamine, propyn-1-ol or but-3-yn-1-ol to obtain the corresponding amides 1a-i and
esters 2a-i and 3a-i (Scheme 1 and Table 1). The reaction between esters activated with N-
hydroxysuccinimide 9d-i and propyn-1-ol or but-3-yn-1-ol did not yield the expected target
compounds 2d-i or 3d-i. In order to obtain these compounds, esters 10d-i, activated with
benzotriazole, were synthesized and reacted with propyn-1-ol or but-3-yn-1-ol to provide the
desired compounds in 21-73% yield (Table 1).
1
2
2
1
2
X=H, R1, R2=H
X=Cl, R1, R2=H
X=Br, R1, R2=H
X=H, R1=Me, R2=H
X=Cl, R1=Me, R2=H
X=Br, R1=Me, R2=H
X=H, R1=H, R2=Me
X=Cl, R1=H, R2=Me
X=Br, R1=H, R2=Me
7a
7b
7c
7d
7e
7f
(ii)
(i)
-
1
7g
7h
7i
R1, R2=H
5a
6a-i
7a-i
5a-c
4a-c
X=H
X=Cl
X=Br
4a
4b
4c
R1=Me, R2=H
R1=H, R2=Me
5b
5c
(iii)
1
1
1
2
2
2
(v)
(iv)
8a-i
W:
9a-i
Y=NH, n=1
Y=O, n=1
Y=O, n=2
1a-i
2a-i
3a-i
W:
10d-i
Scheme 1. Reagents and conditions: (i) EtOAc or acetone, reflux, 4-24 h; (ii) ethyl propiolate (2.0 equiv), TEA (1.2
equiv), CH2Cl2, rt, 4-12 h; (iii) 2N NaOHaq:MeOH 1:1, reflux, 3-5 h, then aqueous citric acid; (iv) N-
hydroxysuccinimide (1.2 equiv) or HOBT (1.2 equiv), EDCI (1.2 equiv), CH2Cl2, rt, 24 h; (v) propargylamine (1.7
equiv) or propyn-1-ol (1.7 equiv) or but-3-yn-1-ol (2.0 equiv), TEA (2.2 equiv) or DBU (1.2 equiv), DMF, dioxane or
CH2Cl2, rt, 24-48 h.
3

Table 1. Nature of substituents and isolated yields for intermediate compounds 8a-i, 9a-i, 10d-i and for target
compounds 1a-i, 2a-i and 3a-i
R1
H
R2
H
X
H
Cpd no.
8a
Yield(%)a
84
Cpd no.
9a
Yield(%)a
63
Cpd no.
-
Yield(%)a
Cpd no.
1a
Yield(%)a
70
Cpd no.
2a
Yield(%)a
54
Cpd no.
3a
Yield(%)a
32
-
H
H
Cl
Br
H
8b
84
9b
-
-
1b
68
2b
41
3b
31
75
H
H
8c
91
9c
-
-
1c
75
2c
45
3c
34
79
CH3
CH3
CH3
H
H
8d
99
9d
10d
10e
10f
10g
10h
10i
1d
75
2d
38
3d
25
97
72
91
86
77
88
90
H
Cl
Br
H
8e
95
9e
1e
70
2e
45
3e
22
93
H
8f
92
9f
1f
83
2f
45
3f
30
90
CH3
CH3
8g
94
9g
1g
83
2g
73
3g
30
70
H
Cl
8h
92
9h
1h
84
2h
70
3h
30
85
H
CH3 Br
8i
93
9i
1i
77
2i
68
3i
21
79
a Isolated yield.
The biological potentials of newly synthesized indolizines 1a-i, 2a-i and 3a-i were
evaluated on human farnesyltransferase by fluorescence measurements.35 Chaetomellic acid
A, a known FTI isolated along with chaetomellic acid B from Chætomella acutiseta,36,37 was
used as positive control in the biological assay. The results are described in Table 2. The
esters are generally hydrolyzed to carboxylic acids in vivo under the action of esterases. For
this reason and for their great solubility and permeability, they are often used as pro-drugs of
carboxylic acids.38 Consequently, intermediary carboxylic acids 8a-i were tested in vitro by
fluorescence measurements on farnesyltransferase. Furthermore, the COOH group could
chelate the zinc atom of the protein.
The analysis of the four structural regions modified in the current study A-D (Figure
1) shows that: 1) in the A region, a para-chloro or a para-bromophenyl substitution is the
most tolerated and proves to be favorable to bioactivity while a hydrogen substituent in this
position resulted in a significant reduction in biological activity; 2) the replacement of the
triazole unit from region B11 with an indolizine ring is successful since six new indolizine-
based FTIs show inhibitory potential in the low micromolar range (compounds 1b,c, 2e,f, 3d
and 3f, Table 2); 3) as observed previously for our series of FTIs bearing a triazole unit,11 the
amidic function from region C of compounds 1a-i slightly decreased the affinity toward
FTase (e.g. 1f: IC50 (FTase) = 11.4 1.3 µM vs 2f: IC50 (FTase) = 1.3 0.2 µM, Table 2), the
ester function being more favorable; the biological evaluation of carboxylic acids 8a-i shows
a diminished activity on the protein; 4) finally, the investigation of the chain length between
the amide/ester group and the triple bond in zone D (n=1, 2) shows that, except compounds
2d and 3d, molecules with n = 2 are worse inhibitors compared to those with n = 1.
The most active compounds in the current study 2f and 3f have been placed in the binding site
of the farnesyltransferase cocrystallized with an inhibitor (PDB entry 1LD7) using GOLD
(Figure 2). 30 conformations were generated and the stability of the solution assessed
visually. The most representative conformation was taken as the final docking output,
considering both sheer number of conformations with the same placement and their relative
score.
Compound 2f (Figure 2, left panel) has roughly a single conformation, with only 5
conformations differing either by the rotation of the bromophenyl group or the orientation of
the propynic moiety. It is characterized by the placement of this last in close proximity with
the zinc atom, but is pointing toward the other side of the pocket. The carbonyl between the
indolizine and the phenyl group is in a favorable position to form a hydrogen bond with Tyr
4

166α. Compound 3f behaves quite similarly (Figure 2, right panel), with a reduced number of
conformations in the larger cluster. This may be relevant to its slightly lesser affinity. The
only real difference is the position of the butynic group, taking a more upright conformation
and lying squarely in front of the zinc atom in about three fourth of the solutions, rather than
folded toward the inside of the pocket as for compound 2f. The remaining part of the
compound is superimposed with its congener.
Zn
Zn
FPP
FPP
Tyr361β
Tyr361β
Trp106β
Trp106β
Tyr166α
Tyr166α
Trp102β
Arg202β
Arg202β
Trp102β
Figure 2. Docking of indolizines 2f (left panel) and 3f (right panel) in the active site of FTase.
The investigation of substituted indolizin-3-yl(phenyl)methanones allowed us to
demonstrate that six new indolizines present good biological activity. The para-bromophenyl
analogue 2f bearing a propargylic ester at position 1 of the indolizine unit proved to be the
most active FTI in the current study with IC50 value of 1.3 0.2 µM (Table 2). However, new
FTIs leads must have specific pharmacokinetic parameters to envisage future biological and
chemical investigation. For instance, a log P value between 2 and 4 is preferred.
Consequently, the amidic series 1a-i proved to be the most promising (series 1a-i: mean log P
= 3.6 vs series 2a-i: mean log P = 5.0, Table 2) for future modulations, particularly at the
triple bond level. The synthesis of indolizine-like analogues, taking into account the
imperatives of bioavailability, will allow improvement in the inhibition properties on FTase
and thus new lead compounds for the development of chemical entities with improved anti-
cancer potential.
5

Table 2. Inhibitory activities of compounds 1a-i, 2a-i, 3a-i and 8a-i on human farnesyltransferase
% FTIa,b
Cpd no.
IC50 (µM ± SD)b
R2c
Log Pd ± SD
Cpd no.
% FTIa,b
IC50 (µM ± SD)b
R2
Log P ± SD
8a
8b
8c
8d
100
100
100
48
19.7 ± 0.4
20.7 ± 9.1
19.4 ± 12.8
N.D.e
0.8888
0.7975
0.7622
-
3.4 ± 1.1
4.1 ± 1.1
4.3 ± 1.1
3.9 ± 1.1
4.6 ± 1.1
4.8 ± 1.1
3.9 ± 1.1
4.6 ± 1.1
4.8 ± 1.1
2.8 ± 1.1
3.5 ± 1.1
3.7 ± 1.2
3.2 ± 1.1
3.9 ± 1.1
4.2 ± 1.2
3.2 ± 1.1
3.9 ± 1.1
4.2 ± 1.2
8.0 ± 0.4
2a
2b
2c
2d
2e
2f
100
54
--f
14.4 ± 5.9
N.D.
N.D.
0.8299
-
-
0.9175
4.1 ± 1.1
4.8 ± 1.1
5.0 ± 1.1
4.6 ± 1.1
5.3 ± 1.1
5.5 ± 1.1
4.6 ± 1.1
5.3 ± 1.1
5.5 ± 1.1
4.4 ± 1.1
5.1 ± 1.1
5.4 ± 1.2
4.9 ± 1.1
5.6 ± 1.1
5.8 ± 1.2
4.9 ± 1.1
5.6 ± 1.1
5.8 ± 1.2
93
100
100
72
0
20.8 ± 3.3
2.6 ± 0.4
1.3 ± 0.2
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
7.3 ± 0.9
20.7 ± 3.4
2.2 ± 0.4
N.D.
N.D.
N.D.
8e
8f
8g
8h
8i
1a
79
91
32.2 ± 3.6
14.8 ± 1.6
72.3 ± 33.6
N.D.
0.8823
0.9689
0.6836
-
0.9453
0.8561
100
0
0
100
94
100
74
2g
2h
2i
3a
3b
3c
3d
3e
3f
-
-
-
-
N.D.
-
--f
61
0
22.1 ± 7.3
5.9 ± 1.6
4.4 ± 1.2
57.8 ± 3.2
14.9 ± 2.5
11.4 ± 1.3
15.3 ± 9.1
N.D.
0.8240
0.9301
0.9178
0.9790
0.9020
0.9683
0.6951
-
1b
1c
1d
-
0
-
0.9582
94
97
100
0
0
68
1e
1f
1g
1h
97
90
0.8602
0.8826
-
-
-
100
--f
3g
3h
3i
1i
Chaetomellic
--f
N.D.
0.18 ± 0.01
-
0.9890
100
acid Ag
a Inhibition of human farnesyltransferase at a 100 µM concentration.
b Values represent mean of two experiments.
c Regression factor.
d Predicted value with ACD software.
e Not determined.
f Not quantifiable; competition with the enzyme fluorescence.
Acknowledgements
This work was supported by the strategic grant POSDRU/159/1.5/S/137750, ”Project
Doctoral and Postdoctoral programs support for increased competitiveness in Exact Sciences
research” cofinanced by the European Social Found within the Sectorial Operational
Program Human Resources Development 2007 – 2013 (PhD scholarship C.D.) and the
Romanian Ministry of Education, CNCS-UEFISCDI, project number PN-II-RU-PD-2012-3-0426.
We also give special thanks to Professor Benoît Rigo for his interest in this work and Dr. D.
Perry for reviewing this manuscript.
Supplementary data
Synthesis details and physico–chemical characterization of all new compounds are available
in the ‘supplementary data’ section.
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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 10 µL
of partially purified recombinant human FTase (1.5 mg/mL) and 1.0 mL of Dansyl-
GCVLS peptide (in the following buffer: 5.6 mM DTT, 5.6 mM MgCl2, 12 µM ZnCl2 and
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development was recorded for 15 min (0.7 sec per well, 20 repeats) at 30 °C with an
excitation filter to 340 nm and an emission filter of 486 nm. Each measurement was
realized twice, in duplicate or in triplicate. The kinetic experiments were realized
under the same conditions, either with FPP as varied substrate with a constant
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8

Novel indolizine derivatives with unprecedented inhibitory activity on human
farnesyltransferase
Carmen Dumea,a Dalila Belei,a Alina Ghinet,a,b,c Joëlle Dubois,d Amaury Farce,b,e and Elena
Bîcu,a,*
a Department of Organic Chemistry, Faculty of Chemistry, ‘Al. I. Cuza’ University of Iasi, B-dul Carol I,
Nr. 11, Corp A, 700506 Iasi, Romania
b Univ Lille Nord de France, F-59000 Lille, France
c
UCLille, EA GRIIOT (4481), Laboratoire de pharmacochimie, Ecole des Hautes Etudes d’Ingénieur
(HEI), 13 rue de Toul, F-59046 Lille, France
d Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif, Avenue de
la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
e
Faculté des Sciences Pharmaceutiques et Biologiques, EA GRIIOT (4481), IFR114, 3 Rue du Pr
Laguesse, B.P. 83, F-59006 Lille, France
* Corresponding author: elena@uaic.ro
Graphical abstract
The rational structural modification of new substituted indolizin-3-yl(phenyl)methanones 1a-
i, 2a-i and 3a-i has greatly improved human farnesyltransferase inhibition. The para-
bromophenyl analogue 2f bearing an ester unit on the indolizine ring demonstrates the highest
inhibition potential, with IC50 value of 1.3 0.2 µM. The amidic series 1a-i proves to be the
most promising for future modulations, particularly at the triple bond level.
Keywords: indolizine, farnesyltransferase inhibitor, activated ester, propargyl ester, butynyl
ester, propargyl amide
B
R
A
C
D
1a-i
2a-i
3a-i
2f
Y=NH, n=1, X=H, Cl, Br, R=H, Me
Y=O, n=1, X=H, Cl, Br, R=H, Me
Y=O, n=2, X=H, Cl, Br, R=H, Me
IC50 (FTase) = 1.3 µM
target compounds
9