Peptide chemistry applied to a new family of phenothiazine-containing inhibitors of human farnesyltransferase

Article abstract of DOI:10.1016/j.bmcl.2014.04.102

Novel phenothiazine derivatives bearing an amino acid residue were synthesized via peptide chemistry, and evaluated for their inhibitory potential on human farnesyltransferase. The phenothiazine unit proved to be an important bulky unit in the structure o

Full text of DOI:10.1016/j.bmcl.2014.04.102

Accepted Manuscript
Peptide chemistry applied to a new family of phenothiazine-containing inhibi-
tors of human farnesyltransferase
Gina-Mirabela Dumitriu, Alina Ghinet, Elena Bîcu, Benoît Rigo, Joëlle Dubois,
Amaury Farce, Dalila Belei
PII:
S0960-894X(14)00459-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.102
BMCL 21595
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 March 2014
23 April 2014
25 April 2014
Please cite this article as: Dumitriu, G-M., Ghinet, A., Bîcu, E., Rigo, B., Dubois, J., Farce, A., Belei, D., Peptide
chemistry applied to a new family of phenothiazine-containing inhibitors of human farnesyltransferase, Bioorganic
& Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.102
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Peptide chemistry applied to a new family of phenothiazine-containing
inhibitors of human farnesyltransferase
Gina-Mirabela Dumitriu,a Alina Ghinet,a,b,c Elena Bîcu,a Benoît Rigo,b,c Joëlle Dubois,d Amaury
Farce,b,e Dalila Beleia,*
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, 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
Institut de Chimie Pharmaceutique Albert Lespagnol, EA GRIIOT (4481), IFR114, 3 Rue du Pr
Laguesse, B.P. 83, F-59006 Lille, France
*Corresponding author: dalila@uaic.ro (D. Belei)
Abstract: Novel phenothiazine derivatives bearing an amino acid residue were synthesized via
peptide chemistry, and evaluated for their inhibitory potential on human farnesyltransferase. The
phenothiazine unit proved to be an important bulky unit in the structure of the synthesized
inhibitors. Propargyl ester 20 bearing a tyrosine residue exhibited the best biological potential in vitro
in the present study. Further syntheses and biological evaluation of phenothiazine derivatives are
necessary in order to gain a full view of SAR in this family of farnesyltransferase inhibitors.
Key-words: farnesyltransferase inhibitor, phenothiazine, peptide coupling, activated ester
Over the years, farnesyltransferase (FTase) has generated much attention as a major target
in the development of new anticancer agents.1 It could be interesting also against Progeria and
parasitic protozoa diseases such as malaria, Chagas diseases or Leishmanias.2 This heterodimeric zinc
metalloenzyme is one of the three prenyltransferases which catalyses covalent attachment of a
prenyl unit (C15) from a farnesylpyrophosphate (FPP) to the free thiol group of the C-cysteine found
in the terminal CAAX motif (where A are aliphatic amino acids, and X is a serine, a methionine or a
glutamine for farnesyltransferase, and
a
leucine or an isoleucine for type
I
geranylgeranyltransferase(GGTase))3 of a set of membrane small G-proteins. Many of these proteins,
such as lamin A and B, Rac, Ras, RhoB or RhoE actively involved in many important cellular signaling
pathways and in carcinogenesis. This protein farnesylation is critical for membrane binding and the
biological function of G-proteins.4 As one of the most important G-protein, Ras proteins have a well-
established role in oncogenesis, and function as switches that control growth signal from cell surface
receptors to nuclear transcription factors. It has been described that gene mutational activation of
the Ras occurs in about 20% of pancreatic and colorectal adenocarcinoma,5 as well as in many other
human cancers.6
Inhibition of protein farnesyltransferase prevents membrane localization of Ras, and so
constitutes a valid target for the conception of new cytostatic anticancer drugs,7 and recent reviews
report many data on SAR in these series.8 The main FTase inhibitors (FTIs) that have undergone
clinical development are non peptidic compounds such as Tipifarnib (R-115777),9,10 BMS-21466211 or
Lonafarnib (SCH-66336)10,12 (Figure 1).
1

A recent biological screening of the chemical library of our Organic Chemistry Department
(Iasi) allowed us to discover new FTIs hits, such as compounds IV and V heretofore unencountered,
bearing a bulky phenothiazine group to be placed in the A2 binding site.13 Interestingly, precursors 1a
and 2a also present low inhibition of human farnesyltransferase (Figure 1). Thus, it was decided to
explore the FTase inhibition potential of tricyclic scaffolds bearing a chain with a terminal propargylic
amide 1a and ester 2a. These compounds VI were mainly obtained either by exchanging the
phenothiazine ring by a carbazole, or by increasing the length of the chain by the incorporation of
glycine or tyrosine unit, able to form new hydrogen bonds or stacking interactions (Figure 1).
Cl
Cl
N
NH2
N
N
H
Br
N
O
N
S
N
NH2
O
N
SO2
N
N
O
N
NC
Cl
Tipifarnib
I
BMS-214662
III
Lonafarnib
II
Br
IC50 (bovine FTase) = 8.3 nM10
IC50 (bovine FTase) = 0.65 nM10
IC50 (human FTase) = 1.4 nM11
Br
O
N
O
O
O
X
Spacer
X
( )n
X
H
H
N
N
N
N
N
S
S
A
bulky unit
IV X = NH IC50 = 8.3 µM
V X = O IC50 = 1.3 µM
1a X = NH 59%
2a X = O 46%
A = bond, S
Spacer = bond, (Gly)x(Tyr)y
x = 0,1,2
y = 0,1
IC50 (human FTase) 13
%inhibition (human FTase) at 100 µM
X = O, NH
n = 1,2
VI Target compounds
Figure 1. Structure of known FTIs I-III,10,11 of FTIs synthesized by our team IV, V,13 and of target
compounds VI.
We first studied the nature of the heterocycle (carbazole versus phenothiazine) while the
number of methylene units X was varied from n = 1 to n = 2; the general synthesis of these
compounds is outlined in Scheme 1. Starting acid 3 was obtained by condensation of phenothiazine 4
with acrylonitrile in the presence of Triton B14 then by hydrolysis of nitrile 5 to acid 3 with aqueous
sodium hydroxide in methanol.14 EDCI [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride] coupling15 of carboxylic acid 3 with N-hydroxysuccinimide led to activated ester 6,13,16
and then reaction with propargylamine or propargyl alcohol in presence of triethylamine gave
propargylic amide 1a and ester 2a respectively.13,17 The same sequence allow to obtain the ester 2b
(n = 2) in 69% yield, and applied to carbazole 718 led to 85% of hydroxysuccinyl ester 10, and then to
the corresponding amide 11a (n = 1, 83%) and esters 12a (n = 1, 80%) and 12b (n = 2, 67%) (Scheme
1).
2

O
O
X
O
O N
( )n
H
z
O
H
N
HX
N
A
N
A
N
A
(i)
(iii)
( )n
(iv)
H
A
11a A = bond, n = 1, X = NH 83%
12a A = bond, n = 1, X = O 80%
12b A = bond, n = 2, X = O 67%
1a A = S, n = 1, X = NH 87% Lit. 13
8 A = bond, Z = CN Lit. 17
9 A = bond, Z = CO2H Lit. 17
(ii)
(ii)
10 A = bond 85%
6 A = S 83%
7 A = bond
4 A = S
Lit. 13
5 A = S, Z = CN
3 A = S, Z = CO2H
Lit. 14
Lit. 14
Lit. 13
2a A = S, n = 1, X = O 78%
2b A = S, n = 2, X = O 69%
Scheme 1. Reagents and conditions: (i) Triton B, 0 °C to reflux, 2 h; (ii) aqueous NaOH, MeOH, reflux, 15 h; (iii)
1.2 equiv N-hydroxysuccinimide, 1.2 equiv EDCI, CH2Cl2, rt, 24 h; (iv) 1.7 equiv propargylamine, propynol or
butynol, 2.2 equiv triethylamine, CH2Cl2, rt, 24 h.
The bioactivities of compounds possessing a carbazole sub-unit proved to be inferior to that
of corresponding phenothiazine derivatives (see later), thus the new syntheses were performed in
these series. Activated ester 6 was condensed with glycinyl or tyrosyl esters to give acids 13 and 14
after saponification of the esters 15 and 16. Acids 13 and 14 were directly condensed with propargyl
amine or alcohol in the presence of EDCI, thus furnishing the compounds 17-20 with a Gly (x = 1, y =
0) or Tyr (x = 0, y = 1) linker, in yields from 65% to 91%. Again, acid 13 was utilized in a new sequence
of activation, reaction with ethyl glycinate, saponification, and then EDCI condensation with
propargyl amine or alcohol. Propargylic amide 24 and ester 25, with a glycylglycyl (x = 2, y = 0) (Gly-
Gly) linker were thus obtained in cumulated 44% and 28% yields respectively (Scheme 2). One-pot
reactions were also utilized starting from activated esters 6 and 21 whose the sequential
condensation firstly with GlyTyrOH (Et3N) then with propargyl amine or alcohol (EDCI) in
dimethylformamide allowed obtaining propargylic products 28-31 with a Gly-Tyr (x = 1, y = 1) or Gly-
Gly-Tyr (x = 2, y = 1) respectively, in 40% to 65% yield (Scheme 2).
3

O
O
H
N
H
N
X
O
O
H
N
H
H
X
O
R
N
S
N
S
24 X = NH 70%
25 X = O 45%
17 R = H X = NH 91%
18 R = CH2C6H4OH(p) X = NH 79%
19 R = H X = O 74%
20 R = CH2C6H4OH(p) X = O 65%
HX
HX
(iv)
(iv)
H
H
O
O
N
O
O
O
H
N
H
N
H
N
O
O
N
CO2R1
(iii)
O
O
N
O
O
N
H
CO2R1
R
R
O
N
S
N
S
N
S
(i)
(i)
S
22 R1 = Et 77%
23 R1 = H 97%
21 R = H 84%
6
15 R = H, R1 = Et 79%
13 R = H, R1 = H 95%
16 R = CH2C6H4OH(p), R1 = Me 73%
(ii)
(ii)
(ii)
14 R = CH2C6H4OH(p), R1 = H 95%
O
CO2H
R
CO2H
R
O
(v)
(v)
H2N
H2N
N
H
N
H
O
O
H
N
H
N
H
N
O
CO2H
O
N
H
NH
O
R
R
CO2H
N
S
N
S
26 R = CH2C6H4OH(p)
27 R = CH2C6H4OH(p)
HX
HX
(iv)
(iv)
H
H
O
R
O
O
X
H
N
H
N
H
N
O
O
H
NH
N
H
X
O
R
H
O
N
S
N
S
28 R = CH2C6H4OH(p), X = NH 65%
29 R = CH2C6H4OH(p), X = O 45%
30 R = CH2C6H4OH(p), X = NH 62%
31 R = CH2C6H4OH(p), X = O 40%
Scheme 2. Reagents and conditions: (i) 1.2 equiv L-glycine ethyl ester hydrochloride or L-tyrosine methyl ester
hydrochloride, 1.2 equiv triethylamine, CH2Cl2, rt, 24 h; (ii) NaOH 2N, 80 °C, 2-5 h; (iii) 1.2 equiv N-
hydroxysuccinimide, 1.2 equiv EDCI, CH2Cl2, rt, 24 h; (iv) 1.7 equiv propargylamine or propargyl alcohol, 1.2
equiv EDCI, DMF, rt, 24 h; (v) 1.0 equiv glycine-L-tyrosine, 2.2 equiv triethylamine, DMF, rt, 5 h.
The activity of all the synthesized phenothiazines was evaluated on human FTase.19 Results are
presented in Tables 1-3 and in Figure 2. The importance of the nature of the tricycle was considered firstly.
The replacement of the phenothiazine unit by a carbazole motif was detrimental to the affinity for the
protein (e. g.: compound 2a vs 12a (Table 1)). The influence of the length between the amide or ester
function and the acetylene group was also studied. A methylene linkage (n = 1) led to compounds with
better inhibitory activity than an ethylene one (n = 2) (e. g.: compound 2a vs 2b (Table 1)). The methylene
4

unit was thus conserved in the skeleton of amides and esters bearing an amino acid residue (Table 2). In
this series, compounds with a glycyl spacer were inactive. A tyrosyl spacer provides the best
pharmacomodulation in the present work (compound 20, Table 2). The replacement of the mono amino
acid spacer by a di- or tri- amino acid led to a slight loss of activity. The target compounds 18, 20, 24 and 25
were synthesized starting from intermediary carboxylic acids 14 and 23 that were also evaluated for their
ability to inhibit human FTase (Tables 2 and 3). Interestingly, they exhibited similar potency as
corresponding final products (e. g.: acid 14: IC50 (FTase) = 30.3 ± 2.6 µM vs propargyl derivative 20: IC50
(FTase) = 18.0 ± 2.4 µM, and acid 23: IC50 (FTase) = 80.5 ± 6.7 µM vs propargyl derivative 25: IC50 (FTase) =
78.7 ± 5.5 µM, Table 2). This comparison was not possible for the intermediary acids for the synthesis of
compounds 28-31 since they were obtained via a one-pot reaction, without isolating the acid precursors.
However, starting esters 15, 16 and 22 were also tested and did not show noticeable inhibitory activity
(Table 3).
Table 1. Inhibitory activities of propargyl derivatives 1a, 2a-b, 11a, and 12a-b on human farnesyltransferase
Compound
A
S
S
S
X
NH
O
N
1
1
2
1
1
2
% Inh (FTase)a,b
O
X
1a
2a
2b
11a
12a
12b
59
46
9
40
13
0
n
O
bond NH
bond
bond
N
A
O
O
a Inhibition ratio of protein farnesyltransferase at a 100 µM concentration.
b Values represent mean of two experiments.
Table 2. Inhibitory activities of propargyl derivatives bearing amino acid(s) residue 17-20, 24-25, and 28-31 on
human farnesyltransferase
Compound
Spacer
Gly
Gly
Tyr
Tyr
Gly-Gly
Gly-Gly
Gly-Tyr
Gly-Tyr
Gly-Gly-Tyr
Gly-Gly-Tyr
X
NH
O
NH
O
NH
O
NH
O
% Inh (FTase)a,b
IC50 (µM ± SDc)b
n. d. e
n. d.
n. d.
18.0 ± 2.4
n. d.
78.7 ± 5.5
24.4 ± 3.3
36.3 ± 0.9
39.7 ± 1.0
30.1 ± 2.7
R2d
-
-
-
0.935
-
0.939
0.898
0.994
0.994
0.909
17
19
18
20
24
25
28
29
30
31
42
25
43
90
42
59
68
69
82
70
X
O
spacer
N
S
NH
O
a Inhibition ratio of protein farnesyltransferase at a 100 µM concentration.
b Values represent mean of two experiments.
c SD: standard deviation.
d R2: regression factor.
e Not determined.
Table 3. Inhibitory activities of some intermediary esters (15, 16, 22) and carboxylic acids (13, 14, 23) on human
farnesyltransferase
Compound
% Inh (FTase)a,b
IC50 (µM ± SDc)
n.d.d
30.3 ± 2.6
n.d.
n.d.
13
14
15
16
22
23
48
74
41
33
42
60
n.d.
80.5 ± 6.7
a Inhibition ratio of protein farnesyltransferase at a 100 µM concentration.
5

b Values represent mean of two experiments.
c SD: Standard deviation.
d Not determined.
90
90
80
70
60
50
40
30
20
10
0
82
74
70
69
68
60
59
Acid precursor series*
Amide series
43
42
Ester series
14 18 20 23 24 25
28 29
30 31
Compound number
Figure 2. Comparative inhibitory activity on human FTase of amides (in red), esters (in green) and their acid precursors
(in blue) at a 100 µM concentration. *Esters and amides 28-31 were synthesized via a one-pot procedure, without
isolating the corresponding acid precursors.
To further expand the understanding of the experimental results, molecular modeling studies for
esters 20, 29 and 31 were carried out in the active site of protein FTase. Docking results indicated that
indeed they can be placed in this site (Figure 3a-c).
Farnesyltransferase structure was taken from the 1LD720 entry of the RCSB Protein Data Bank21 due
to the structural proximity of the co-crystallized ligand with our own molecules. Both the ligand and the
water molecules were removed from the enzyme, before validating the protocol by docking the
cocrystallized ligand back into the binding site, defined as a 10Å sphere around the position of the ligand in
the complex. A formal charge of +2 was assigned to the farnesyltransferase zinc cation to ensure it would
be correctly handled for the docking. The molecules under investigation were built from the standard
fragments library of Sybyl 6.9.122 and their geometry optimized using Gasteiger-Hückel partial charges and
a dielectric constant set to 4.30 solutions were generated for each compound using GOLD 5.1,23 then
ranked by PLP fitness score after assessing the backward compatibility versus the previous in house
GoldScore/X-Score consensus scoring scheme. The consistency of the results was checked visually and in
case of several clusters of solutions, the most representative solution of each was selected as the results of
the docking run.
The best conformation obtained for compound 20 (Figure 3(a)) is different from those of
compounds 29 and 31 (Figure 3(b) and (c)). However, compound 20 has a larger cluster of conformations;
this may be related to its slightly higher potency. No hydrogen bonds are established, only stacking
interactions are observed.
Esters 29 and 31 occupy roughly the same place of the pocket with the bulky tricycle in front of the
FPP chain. The two nitrogen atoms from the two amide functions of ester 29 establish two hydrogen bonds
with His 201 and Lys 164. However, these interactions are represented on a secondary potential
conformation. In the same way, ester 31 bearing a longer chain presents another conformation (with only
three solutions) where the phenol group forms a hydrogen bond with Asn 127 and the two nitrogen atoms
from the two last amide functions form hydrogen bonds with Lys 164.
Also, the docking studies showed that the presence of the propargylic ester was not needed for the
biological activity. This is in accordance with our biological assay results since carboxylic acids 14 and 23
have the same inhibitory potential as final corresponding esters 20 and 25 (Figure 2). However, it is well
known that carboxylic acids are generally more active than corresponding esters on the protein
6

farnesyltransferase, esters being more appropriate for the cellular evaluation. We thus synthesized them in
this perspective.
(a)
(b)
(c)
Figure 3. Docking of esters bearing a phenothiazine moiety in the active site of FTase: (a) compound 20, (b) compound
29, and (c) compound 31.
In this study, we have synthesized and evaluated new phenothiazine derivatives bearing a glycine
(Gly), a tyrosine (Tyr), a glycylglycine (Gly-Gly), a glycyltyrosine (Gly-Tyr) or a glycylglygyltyrosine (Gly-Gly-
Tyr) unit as human farnesyltransferase inhibitors. These structural modifications furnished compounds that
showed decreased inhibitory potency relative to our previously described phenothiazines.13,24 However,
most of these compounds possess in vitro inhibition activities in the micromolar range. Propargyl ester 20
bearing a tyrosine residue exhibited the most potent in vitro biological potential in the present study.
Nevertheless, given these modest results obtained, non-specific interactions are not excluded.
The phenothiazine unit is an important bulky unit that can be placed in the A2 binding site of the
farnesyltransferase.25 This fact was supported once again since the replacement of the phenothiazine by a
carbazole unit was unfavorable to the affinity for the protein.
We are continuing synthesis and biological evaluation of additional phenothiazine analogs by
focusing on other points of the molecule in order to gain a full view of the SAR in this family of
farnesyltransferase inhibitors.
Acknowledgement
The authors gratefully acknowledge the "Ministerul Educaţiei, Cercetării, Tineretului şi Sportului" (Romania)
for a PhD scholarship (G. -M. D.).
Supplementary data
Supplementary data (synthesis details, physico-chemical characterization for all new compounds)
associated with this article can be found in this section.
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9

Peptide chemistry applied to a new family of phenothiazine-containing
inhibitors of human farnesyltransferase
Gina-Mirabela Dumitriu,a Alina Ghinet,a,b,c Elena Bîcu,a Benoît Rigo,b,c Joëlle Dubois,d Amaury
Farce,b,e Dalila Beleia,*
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, 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
Institut de Chimie Pharmaceutique Albert Lespagnol, EA GRIIOT (4481), IFR114, 3 Rue du Pr
Laguesse, B.P. 83, F-59006 Lille, France
*Corresponding author: dalila@uaic.ro (D. Belei)
Abstract: Novel phenothiazine derivatives bearing an amino acid residue were synthesized via
peptide chemistry, and evaluated for their inhibitory potential on human farnesyltransferase. The
phenothiazine unit proved to be an important bulky unit in the structure of the synthesized
inhibitors. Propargyl ester 20 bearing a tyrosine residue exhibited the best biological potential in vitro
in the present study. Further syntheses and biological evaluation of phenothiazine derivatives are
necessary in order to gain a full view of SAR in this family of farnesyltransferase inhibitors.
X
O
HN
Gly
X
OH
O
O
O
NH
Tyr
HN
O
S
N
HN
O
N
S
Gly
OH
Tyr
18: X = NH, % Inh (human FTase) at 100μM: 43
30: X = NH, % Inh (human FTase) at 100μM: 82
IC50 (μM ± SD) = 39.7 ± 1.0
20: X = O, % Inh (human FTase) at 100μM: 90
IC50 (μM ± SD) = 18.0 ± 2.4
31: X = O, % Inh (human FTase) at 100μM: 70
IC50 (μM ± SD) = 30.1 ± 2.7
10