New farnesyltransferase inhibitors in the phenothiazine series

Article abstract of DOI:10.1016/j.bmcl.2012.06.007

The biological screening of the chemical library of our Organic Chemistry Department, carried out on an automated fluorescence-based FTase assay, allowed us to discover that a phenothiazine derivative (1d) was an inhibitor of farnesyltransferase. Three new series of human farnesyltransferase inhibitors, based on a phenothiazine scaffold, were synthesized with protein farnesyltransferase inhibition potencies in the low micromolar range. Ester derivative 9d was the most active compound in these series. Four synthesized compounds were evaluated for their antiproliferative activity on a NCI-60 cancer cell line panel. The modest results obtained in this preliminary investigation showed that mixing the phenothiazine and the 1,2,3-triazole motif in the structure of a single compound can lead to new scaffolds in the field of farnesyltransferase inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.06.007

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4517–4522
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New farnesyltransferase inhibitors in the phenothiazine series
Dalila Belei a, Carmen Dumea a, Alexandrina Samson a, Amaury Farce b,c, Joëlle Dubois d,
Elena Bîcu a, Alina Ghinet a,b,e,
⇑
a Department of Organic Chemistry, ‘Al. I. Cuza’ University of Iasi, Faculty of Chemistry, Bd. Carol I nr. 11, 700506 Iasi, Romania
b Univ Lille Nord de France, F-59000 Lille, France
c Institut de Chimie Pharmaceutique Albert Lespagnol, EA GRIIOT (4481), IFR114, 3 Rue du Pr Laguesse, B.P. 83, F-59006 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 UCLille, EA 4481 (GRIIOT), Laboratoire de Pharmacochimie, HEI, 13 rue de Toul, F-59046 Lille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The biological screening of the chemical library of our Organic Chemistry Department, carried out on an
automated fluorescence-based FTase assay, allowed us to discover that a phenothiazine derivative (1d)
was an inhibitor of farnesyltransferase. Three new series of human farnesyltransferase inhibitors, based
on a phenothiazine scaffold, were synthesized with protein farnesyltransferase inhibition potencies in
the low micromolar range. Ester derivative 9d was the most active compound in these series. Four syn-
thesized compounds were evaluated for their antiproliferative activity on a NCI-60 cancer cell line panel.
The modest results obtained in this preliminary investigation showed that mixing the phenothiazine and
the 1,2,3-triazole motif in the structure of a single compound can lead to new scaffolds in the field of
farnesyltransferase inhibitors.
Received 23 April 2012
Revised 1 June 2012
Accepted 3 June 2012
Available online 9 June 2012
Keywords:
Farnesyltransferase inhibitor
Anticancer agent
Phenothiazine
Triazole
Ó 2012 Elsevier Ltd. All rights reserved.
The G-protein superfamily is the most important category of
human CAAX proteins (A: aliphatic amino acids; X: methionine,
glutamine or serine for farnesyltransferase, leucine or isoleucine
for type I geranylgeranyltransferase). Many G-proteins, such as
Ras, Rho, Rac and CDC42, are located on the plasma membrane
or endomembranes. The G-protein superfamily is actively involved
in many important cellular signaling pathways, and plays an
important role in carcinogenesis. As one of the most important
G-proteins, Ras protein has a well-established role in oncogenesis.
Ras proteins function as switches that control growth signals from
cell surface receptors to nuclear transcription factors. Human
cancer studies show that gene mutational activation of the Ras
subfamily (K-Ras, N-Ras and H-Ras) occurs in about 20% of human
pancreatic and colorectal adenocarcinoma.1–5 N-Ras mutation has
been reported in melanoma, hepatocellular cancer, myelodys-
plastics syndrome and acute myelogenous leukemia.6–8 Also,
several human cancers such as thyroid follicular and papillary
carcinoma, bladder cancer and renal cell cancer harbor H- or
N-Ras mutations.9,10
Inhibition of protein farnesyltransferase (FTase) prevents mem-
brane localization of Ras, and so constitutes a valid target for the
conception of new cytostatic anticancer drugs.12 Farnesyltransfer-
ase inhibitors (FTIs) have thus been developed as a new class of
promising drugs for cancer treatment.13 The main FTase inhibitors
that have undergone clinical development14 are non peptidic,
heterocyclic compounds such as tipifarnib (R-115777),15,16 L-
778123,17 BMS-214662,18 lonafarnib (SCH-66336)19 and SCH-
226374.20 Such FTase inhibitors have been discovered through
systematic screening approaches. X-ray diffraction studies of the
complex of BMS-214662 or tipifarnib with protein farnesyltrans-
ferase show that they bind to a hydrophobic cleft formed at the
interface of the alpha and beta subunits. The inhibitor forms a
ternary complex with the FPP substrate and the enzyme, and binds
to the catalytic zinc cation to the rim of the active site. Therefore,
they act within a peptide-competitive mechanism.21
In order to identify new FTIs hits, a screen of the chemical
library of our Organic Chemistry Department (Iasi) was carried
out on an automated fluorescence-based FTase assay.22 This led
to the discovery of a phenothiazine derivative 1d (Fig. 1), which
exhibits an inhibitory activity on human FTase in the micromolar
range (Table 1). Many biological properties have already been
described for phenothiazine derivatives;23 some of them display
anthelmintic activities,24 and others are (reversible) inhibitors of
trypanothione reductase,25 inhibit lipid peroxidation26 or tubulin
polymerization.27 To the best of our knowledge, phenothiazines
and 1,2,3-triazolyl ring have not encountered in the FTIs field. Thus
Activation of Ras proteins requires their localization on the
plasma membrane, which is mainly dependent on the attachment
of a farnesyl (15-carbon) or of a geranylgeranyl (20-carbon) group
to the cysteine residue located in the C-terminal tetrapeptidic
(CAAX) sequence at the carboxyl terminus of these proteins.11
⇑
Corresponding author. Tel.: +33 328 384 858; fax: +33 328 384 604.
E-mail address: alina.ghinet@hei.fr (A. Ghinet).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.007

4518
D. Belei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4517–4522
Cl
O
acid 11 with N-hydroxysuccinimide led to activated ester 12,34 and
then reaction with propargylamine gave acetylenic amide 13.
Then, click chemistry performed between 13 and -azidoketones
7 afforded triazoles 15. The same sequence realized with with
propargyl alcohol yielded ester 14 then triazoles 16.
Cl
NH2
N
N
a
N
N
O
N
N
N
To complete the study of compounds evaluated in this prelimin-
ary study, a second phenothiazine group was introduced. Thus,
products 22 and 23 were synthesized in a similar way by perform-
ing a click chemistry reaction between azidoketones 2035a and 2136
and amides 13 and 6 (Scheme 3).
NC
Cl
Tipifarnib
L-778123
S
The compound docking onto 1LD7 binding site37 was realized
with GOLD5.1 (CCDC 2011), using the standard parameters, and a
sphere of 10 Å around the extracted cocrystallized ligand. The best
solution of the docking run was defined as the most frequent con-
formation among the 30 conformations generated.
N
N
N N
N
N
N
H
H
N
O
O
S
N
SO2
Br
Compounds 1c and 1d can take a slightly more common confor-
mation described in Figure 2, where their tricyclic head lies in the
hydrophobic part of the pocket, in contact with Trp 602 and the
terminal aromatic in front of the zinc atom. In this conformation,
their amide analogs are not able to interact with any residue of
the binding site. They however also display a second conformation
where their tricycle moves away from the hydrophobic cleft, let-
ting the carbonyl of their amide to form a hydrogen bond with
Arg 702. Compared with the amides 1c and 1d, the esters 9c and
9d have a larger cluster of conformations; this may be related to
their higher potency (Fig. 2). However, all these compounds bind
in a mostly similar way, with the terminal phenyl in front of the
zinc ion, while the tricyclic head group is engaged in hydrophobic
interactions with Trp 602. Possible hydrogen bonds could further
anchor the compounds with the hydroxyls of both Tyr 166 and
861 at the level of the ester carbonyl and the triazole respectively.
The activity of all the synthesized derivatives was evaluated on
human FTase. Results are summarized in Table 1. The p-chloro and
the p-bromo substitutions of the phenyl ring proved to be favor-
able to bioactivity. We then investigated the influence of different
substituents (F, CN, OMe or Me) at the para-position of the phenyl
ring. These structural modifications caused a significant reduction
in biological activity (compound 1a vs 1c, Table 1). Next, the
replacement of the amide function by an ester enhanced the
inhibitory potency on FTase (compound 1c vs 9c, Table 1). In
addition, when the length of the carbon chain between the
phenothiazinic nitrogen and the amide group was shortened from
two (1d) to one atom (15d), FTase inhibition decreased (1d:
NC
BMS-214662
1d
Figure 1. Structure of FTIs.
we decided to perform a preliminary SAR study, and some struc-
tural modifications of the scaffold of compound 1d and we de-
scribed here the results of this preliminary investigation.
We first modified the nature of the aromatic ring substituent;
the general synthesis of 1,2,3-triazole derivatives 1 is outlined in
Scheme 1. Starting acid was obtained by condensation of phenothi-
azine 2 with acrylonitrile in the presence of Triton B28 then by
hydrolysis of nitrile 3 to acid 4 with aqueous sodium hydroxide
in methanol.28 Coupling of carboxylic acid 4 with N-hydroxy-
succinimide led to activated ester 5,29 and then reaction with prop-
argylamine gave acetylenic amide 6.30 The expected triazoles 1
were obtained by click chemistry between 6 and
a-azidoketones
7 that have already been described31 but were synthesized by
using a new biphasic (CHCl3–H2O) condition with a phase transfer
agent. The same sequence realized with propargyl alcohol yielded
ester 8 then triazoles 9, allowing the evaluation of the importance
of the amide group in that position.
The importance of the length between the phenothiazine group
and the amido function was considered (Scheme 2). Starting acid
was provided from phenothiazine 2 by reacting its sodium salt
with ethyl bromoacetate in DMF32 followed by saponification of
ester 10 with aqueous sodium hydroxide.33 Coupling of carboxylic
Table 1
Inhibitory activities of 1,2,3-triazoles 1a–f, 9a–e, 15a–f, 16a–c, 22a–b and 23a on human FTase
O
N
N
X
N
n
N
R
S
O
38
Compound
n
X
R
IC50
7.7
(
l
M)
0.8
Compound
n
X
R
Inhibition ratioa (%)
Compound
n
X
R
Inhibition ratioa (%)
1c
1d
1f
9c
9d
15d
2
2
2
2
2
1
NH
NH
NH
O
O
NH
Cl
1a
1b
1e
9a
9b
9e
9f
15a
15b
2
2
2
2
2
2
2
1
1
NH
NH
NH
O
O
O
O
NH
NH
Me
OMe
F
Me
OMe
F
CN
Me
OMe
44.2
29.3
52.6
10.7
22.3
35.2
55.1
19.0
24.4
15c
15e
15f
16a
16b
16c
22a
22b
23a
1
1
1
1
1
1
1
1
2
NH
NH
NH
O
O
O
NH
NH
NH
Cl
F
CN
Br
F
CN
—
—
—
40.0
0
Br
CN
Cl
Br
Br
8.3 0.9
5.2 1.3
1.5 0.3
1.3 0.3
24.3 5.0
24.9
35.5
31.9
20.7
0
b
0
0
a
Inhibition ratio at a 100
The terminal phenyl is changed to a phenothiazine unit.
l
M concentration.
b

D. Belei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4517–4522
4519
O
(i)
(ii)
(iii)
HO
O
N
O
O
HN
N
N
N
S
S
S
S
S
N
CH=CH2
NC
O
2
3 87%
4 56%
5 83%
N
N
N
(iv)
(v)
X
X
N
N
S
N3
O
O
R
O
O
R
6 X = NH 87%
8 X = O 78%
7
9a X = O R = Me 77%
9b X = O R = OMe 80%
9c X = O R = Cl 78%
9d X = O R = Br 70%
9e X = O R = F 67%
9f X = O R = CN 58%
1a X = NH R = Me 64%
1b X = NH R = OMe 74%
1c X = NH R = Cl 79%
1d X = NH R = Br 72%
1e X = NH R = F 76%
1f X = NH R = CN 60%
Scheme 1. Reagents and conditions: (i) Triton B, 0 °C ? reflux, 2 h, 87% (lit. 92%)28; (ii) aqueous NaOH, MeOH, reflux, 15 h, 56% (lit. 60%)28; (iii) 1.2 equiv N-
hydroxysuccinimide, 1.2 equiv EDC, CH2Cl2, rt, 24 h, 83%; (iv) 1.7 equiv propargylamine or propargyl alcohol, 2.2 equiv triethylamine, DMF, rt, 24 h; (v) 0.1 equiv CuSO4.5H2O,
0.2 equiv sodium ascorbate, t-BuOH:MeOH:MeCN 10:2:1, H2O, rt, 24 h.
O
(i)
(ii)
(iii)
HN
N
N
N
S
S
S
S
Br
EtO
HO
N O
EtO
(iv)
O
O
O
O
O
2
10 58%
11 70%
12 81%
N
N
N
(v)
N
O
N
S
S
N3
X
X
R
O
O
O
R
7
13 X = NH 80%
14 X = O 63%
15a X = NH R = Me 72%
16a X = O R = Br 66%
16b X = O R = F 47%
16c X = O R = CN 54%
15b X = NH R = OMe 68%
15c X = NH R = Cl 65%
15d X = NH R = Br 68%
15e X = NH R = F 43%
15f X = NH R = CN 60%
Scheme 2. Reagents and conditions: (i) 1 equiv NaH, DMF, 0 °C ? rt, 12 h; 58% (lit. 67%)32; (ii) aqueous NaOH, EtOH, MeOH, reflux, 1 h, 70%; (iii) 1.2 equiv N-
hydroxysuccinimide, 1.2 equiv EDC, CH2Cl2, rt, 24 h, 81%; (iv) 1.67 equiv propargylamine, 2.2 equiv triethylamine, DMF, rt, 24 h, 80%; (v) 0.1 equiv CuSO4.5H2O, 0.2 equiv
sodium ascorbate, t-BuOH:MeOH:MeCN 10:2:1, H2O, 50 °C, 24 h.
IC50 = 8.3 0.9
also conserved in the series of esters (e.g., compound 9c vs 16a,
Table 1; 9c: IC50 = 1.5 0.3 M; 16a: 36% inhibition at a 100 lM
concentration). Finally, the introduction of a second phenothiazine
unit in place of the benzene ring abolished affinity toward FTase
(compound 1c vs 23a).
The four phenothiazine derivatives 1c–e and 15d were selected
by NCI for screening against 60 human tumor cell lines. The
representative results are summarized in Table 2. Compound 1c
exhibited the most interesting cell growth inhibitory activity
among the tested compounds (68% inhibition on HL-60(TB), 40%
inhibition on MOLT-4 and 38% inhibition on PC-3 cell lines at
l
M; 15d: IC50 = 24.3 5.0
l
M). This tendency is
inhibition on MOLT-4 and 26% inhibition on PC-3). Compound
15d showed very modest cell growth inhibition, conserving
antiproliferative potential only on MOLT-4 cell lines (42% inhibi-
tion), compared to compound 1c.
l
The biological screening of the chemical library of our Organic
Chemistry Department, carried out on an automated fluores-
cence-based FTase assay, allowed us to discover that a compound
with a phenothiazine scaffold (1d) was an inhibitor of farnesyl-
transferase. Structural modifications of this compound were envis-
aged in order to enhance inhibitory activity. Three families of new
phenothiazine derivatives were synthesized and evaluated for
their ability to inhibit human farnesyltransferase in vitro. The
length of the chain between the phenothiazine group and the
amido function proved to be very important in determining
inhibitory activity, as shown for compounds 1d and 15d (Table
1). Replacement of the amidic function by an ester group enhanced
biological activity (e.g., compound 9d vs 1d, Table 1). The introduc-
tion of a second phenothiazine motif in place of the p-substituted
10 lM concentration). As observed in the FTase assay (Table 1),
nearly identical activities were observed on most cell lines for
compound 1d (41% inhibition on HL-60(TB), 34% inhibition on
MOLT-4 and 42% on PC-3 cell lines). Replacement of the p-chloro
group of 1c with a p-fluoro moiety (1e) caused a reduction in
antiproliferative activity (only 20% inhibition on HL-60(TB), 38%

4520
D. Belei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4517–4522
R
R
N
R
N
O
O
(i)
(ii)
NH
S
S
S
ClCH2COCl
Cl
N3
2 R = H
18 R = H 75%
20 R = H 85%
21 R = Cl 85%
17 R = Cl
19 R = Cl 65%
N
N
N
R
H
N
H
N
n
(iii)
O
N
N
O
20, 21
N
+
n
S
S
O
S
22a n = 1 R = H 85%
22b n = 1 R = Cl 60%
23a n = 2 R = H 81%
23b n = 2 R = Cl 85%
13 n = 1
6 n = 2
Scheme 3. Reagents and conditions: (i) toluene, reflux, 5 h, 75% for compound 18 (lit. 76%)35b and 65% for compound 19 (lit. 65%)36; (ii) 1.1 equiv NaN3, 0.012 equiv TBAB,
CHCl3, H2O, rt, 48 h, 85% for compounds 20 and 2135; (iii) 0.1 equiv CuSO4.5H2O, 0.2 equiv sodium ascorbate, t-BuOH:MeOH:MeCN 10:2:1, H2O, 50 °C, 24 h.
Zn
Zn
FPP
FPP
Tyr 861
Tyr 861
Trp 606
Trp 606
Tyr 166
Tyr 166
Trp 602
Leu 596
Trp 602
Arg 702
Arg 702
Cys 595
Cys 595
Leu 596
(b) amide 1c
(a) amide 1d
FPP
FPP
Zn
Zn
Tyr 861
Tyr 861
Tyr 166
Trp 606
Tyr 166
Trp 606
Arg 702
Arg 702
Trp 602
Leu 596
Trp 602
Leu 596
Cys 595
Cys 595
(d) ester 9c
(c) ester 9d
Figure 2. Docking of compounds 1c–d and 9c–d in the active site of FTase.

D. Belei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4517–4522
4521
Table 2
Results of the in vitro human cancer cell growth inhibition39
Cell type
Cell line
Inhibition ratio (%) at 10 l
Ma
1c
1d
1e
15d
Leukemia
CCRF-CEM
HL-60 (TB)
MOLT-4
RPMI-8226
SNB-75
19
68
40
19
20
8
18
41
34
15
18
7
24
17
24
29
na
42
37
42
14
29
20
17
13
42
22
18
16
na
na
5
25
6
13
13
ndc
9
20
38
3
CNS cancer
Colon cancer
Non-small cell lung cancer
melanoma
19
nab
22
12
14
14
na
18
17
26
3
HCT-15
EKVX
30
9
LOX IMVI
SK-MEL-5
UACC-62
OVCAR-8
UO-31
CAKI-1
PC-3
MDA-MB-231/ATCC
T-47D
13
26
17
36
32
38
4
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
22
7
12
a
b
c
Data obtained from NCI’s in vitro 60 cell single dose screen (10 lM).
Not active.
Not determined.
13. Ohkanda, J.; Knowles, D. B.; Blaskovich, M. A.; Sebti, S. M.; Hamilton, A. D. Curr.
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pound 23a vs 1d, Table 1). The modest results obtained in this pre-
liminary investigation showed that mixing the phenothiazine and
the 1,2,3-triazole motif in a single compound structure can lead
to new scaffolds in the field of farnesyltransferase inhibitors.
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Acknowledgments
The authors gratefully acknowledge the CNCSIS (project PN-II-
ID-PCE2008, code ID_2643) and CommScie (project POSDRU/89/
1.5/S/63663) for financial support, and the NCI (National Cancer
Institute) for the biological evaluation of compounds on their 60-
cell panel. We also give special thanks to Professor Benoît RIGO
for his interest in this work.
Supplementary data
21. Reid, T. S.; Beese, L. S. Biochemistry 2004, 43, 6877.
Supplementary data (synthesis details and physico-chemical
characterization for all new compounds) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2012.06.007.
22. This assay represents 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. J. Am. Chem. Soc. 1992, 114,
7945; (b) Cassidy, P. B.; Dolence, J. M.; Poulter, C. D. Methods Enzymol. 1995,
250, 30. All compounds were initially evaluated at a single high dose (100 lM
concentration); only compounds which satisfied pre-determined threshold
inhibition criteria (more than 50% FTase inhibition) progressed to the 8-dose
screen and were retained for the IC50 calculation..
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lM
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i
constant concentration of Dns-GCVLS of 2.5
l
M, or with Dns-GCVLS as varied
M. Non linear
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38. Farnesyltransferase assay: Assays were realized in 96-well plates, prepared with
a Biomek NKMC and a Biomek 3000 from Beckman Coulter and read on a
Wallac Victor fluorimeter from Perkin–Elmer. Per well, 20
pyrophosphate (10 M) was added to 180 L of a solution containing 2
varied concentrations of potential inhibitors (dissolved in DMSO) and 178
a solution composed by 10 L of partially purified recombinant human FTase
lL
of farnesyl
L of
L of
l
l
l
l
l