Synthesis and biological evaluation of a new series of phenothiazine- containing protein farnesyltransferase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.11.008

Two new families of human farnesyltransferase inhibitors 13a-m and 14a-d, based on a phenothiazine scaffold, were synthesized. Compounds 14a and 14b were the most promising inhibitors of human farnesyltransferase with IC₅₀ values of 0.7 and 0.6 μM, respectively.

Full text of DOI:10.1016/j.ejmech.2012.11.008

European Journal of Medicinal Chemistry 59 (2013) 101e110
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of a new series of phenothiazine-containing
protein farnesyltransferase inhibitors
,
,
b
,
c
,
,
,c
Cristina-Maria Abuhaie a 1, Alina Ghinet a 1, Amaury Farce b d, Joëlle Dubois e, Philippe Gautret b
,
Benoît Rigo b c, Dalila Belei a, Elena Bîcu a
,
,
*
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 UCLille, EA 4481 (GRIIOT), Laboratoire de Pharmacochimie, HEI, 13 rue de Toul, F-59046 Lille, France
d Institut de Chimie Pharmaceutique Albert Lespagnol, EA GRIIOT (4481), IFR114, 3 Rue du Pr Laguesse, B.P. 83, F-59006 Lille, France
e Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, Centre de Recherche de Gif, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new families of human farnesyltransferase inhibitors 13aem and 14aed, based on a phenothiazine
scaffold, were synthesized. Compounds 14a and 14b were the most promising inhibitors of human
Received 26 July 2012
Received in revised form
6 November 2012
Accepted 7 November 2012
Available online 14 November 2012
farnesyltransferase with IC50 values of 0.7 and 0.6
m
M, respectively.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Phenothiazine
Fanesyltransferase inhibitors
Ylide
Pyridin-(1H)-ylidene
Anticancer agent
1. Introduction
occurring on several cell signalling proteins e.g. oncogenic Ras
proteins [3].
Since the discovery of protein farnesyltransferase (FTase) in the
late 1980s, its inhibition has generated much attention as an
important target for the conception of new anticancer agents with
reduced toxicity [1].
Activation of Ras proteins requires association with the plasma
membrane which is mainly dependent on farnesylation [4]. Inhi-
bition of farnesyltransferase prevents membrane localization of
Ras, and thus constitutes a valid target for the conception of new
cytostatic anticancer drugs [5]. Hence, farnesyltransferase inhibi-
tors (FTIs) have been developed as promising drugs for cancer
treatment, and diverse compounds (R-115777 [6,7], SCH-66336 [8],
BMS-214662 [9]) have emerged from these studies [10] before
undergoing clinical trials (Fig. 1). The approaches leading to new
FTIs included random screening of chemical libraries [11] or natural
as well as rational design of the CaaX peptidomimetics products
[12], have also been described.
We have recently discovered inhibitors, heretofore unencoun-
tered [13], such as compound 1 (Fig. 1), bearing a phenothiazine
unit. The investigation of the steric tolerance of the A2 binding site
of farnesyltransferase revealed that the ferrocene unit was a well-
tolerated bulky group (compound 2, Fig. 1) [14]. We then investi-
gated the possibility of obtaining phenothiazine-containing FTIs
able to coordinate with the zinc atom of the protein via chelating
groups and to interact with the A2 binding site via the
This heterodimeric metalloenzyme belongs to the prenyl-
transferase protein family. It catalyses the transfer of a farnesyl
moiety (C15) from farnesyl pyrophosphate (FPP) to the free thiol
group of the C cysteine found in the terminal CaaX motif (where
a are aliphatic amino acids and X a serine, a methionine, an alanine
or a glutamine) [2] of a group of membrane small G-proteins such
as lamin A and lamin B, RhoB or RhoE. This G-protein superfamily is
actively involved in many important cellular signalling pathways
and plays an important role in carcinogenesis. Indeed, protein far-
nesylation is an important post-translational modification
* Corresponding author.
E-mail address: elena@uaic.ro (E. Bîcu).
These authors have made equal contributions.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.008

102
C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
Cl
Cl
N
NH2
N
N
H
Br
N
O
N
S
N
NH2
O
N
SO2
N
N
O
N
NC
Cl
Tipifarnib
R-115777
BMS-214662
Lonafarnib
SCH-66336
Br
S
CN
CN
O
OMe
O
N
N N
N
OMe
OMe
N
N
O
-
S
Fe2+
O
O
-
Br
1
2
3
Fig. 1. Structure of some known FTIs and of compounds 1e3.
phenothiazine unit. Then, we decided to perform a preliminary SAR
study on the phenothiazine derivatives bearing an indolizine unit
with some structural modifications of the scaffold of compound 3
(Fig. 1).
ylides. Therefore, we decided to synthesize a series of compounds
with a ylide skeleton and to investigate the affinity of these new
compounds towards protein farnesyltransferase.
The obtention of carbanion disubstituted N-ylides could be due
to the two cyano and ester groups, linked to the carbanion of the
Michael addition intermediary, that prevent positioning near the
2. Results and discussion
carbocation in the
a-pyridinium position and/or at the existing
2.1. Chemistry
extended conjugation.
Note also that hydrolysis of trifluoromethyl group occurred
when the reaction was realized with salt 9a in presence of alkene 12
to afford ylide 13c (Table 1) in 57% yield. Such hydrolyses have been
reported in the literature [16].
3-Acylsubstituted tetrahydroindolizine derivatives 7 or dihy-
drobenzoindolizine can be obtained via a 1,3-dipole cycloaddition
reaction between N-ylides, generated in situ from pyridazinium or
benzopyridinium salts 4 in the presence of different bases, and
different substituted alkenes 5 or 6 (Scheme 1) [15], and we
thought that by using ethoxymethylene malononitrile instead of
alkenes 5 and 6, a double bond could be introduced in the pyrro-
lidine ring of compound 3.
We were interested in the synthesis of compounds bearing
complexing groups (CN, CO2R) that can bind the zinc atom of
protein farnesyltransferase. Different pyridinium salts 9aef were
reacted with trisubstituted alkenes (2-cyano-3-ethoxyacrylonitrile
10, ethyl 2-cyano-3-ethoxyacrylate 11 or (3E)-4-ethoxy-1,1,1-
trifluorobut-3-en-2-one 12). After the reactions were performed,
we found that the target compounds 3aem, analogues to 7, were
not obtained. In their stead, carbanion disubstituted N-ylides 13ae
m were isolated (Scheme 2 and Table 1).
As can be observed from Table 1, the reaction is quite general;
however, when the same sequence was realized with picoline salts
9g and 9h, unexpected red compounds 14aed were obtained as
unique reaction products, in place of ylides 13neq (Scheme 3). Note
that very few references have described the reaction of the methyl
group of 2- and 4-picolinium salts with dipolarophiles. Only five
allylidenedihydropyridine derivatives were isolated when ketene-
thioacetal (CMAN) was reacted with picolinium salts [17]; however,
no investigation on the generalization of the synthetic method has
been performed.
With this in mind, we furtherered the generalization of the
synthesis of such pyridineylidene derivatives. When the pheno-
thiazine unit was replaced by a phenyl moiety, the reaction
provided the corresponding ylide 15 in 78% yield (Table 2, entry
1). The replacement of the phenothiazine by a morpholine unit or
by a diethylamino group resulted in the synthesis of pyridin-
(1H)-ylidene derivatives 16 and 17 in 41% and 56% yields,
respectively; also, with morpholine salt 9j, a by-product 19 [18]
was isolated in 16% yield, which resulted from the cleavage of
the amide function, followed by the reaction of the morpholine
with alkene 11 (Table 2, entries 6 and 7). Finally, the same
sequence realized with pyridinium salt containing a carbazole
unit (9l) yielded only the cleavage product 9H-carbazole 18
(Table 2, entry 8).
To the best of our knowledge, cycloimmonium ylides have not
been encountered in the FTIs field and no biological properties have
been described in the literature for the phenothiazinepyridinium
R
Y
+
O
W1
N
-
W1
Cl
B:
- HCl
O
N
+
W2
W2
5: W = H, W = CN
S
N
Y
N
S
The study of the generalization of the synthesis of pyridin-(1H)-
ylidene derivatives starting from 2- and 4-picolinium salts showed
that an amide function was essential for reaction effectiveness; this
could perhaps result from stabilization of intermediates charged
species.
1
2
6: W = W = CO2Me
R
1
2
Y = H, N
4
7
Scheme 1. Synthesis of tetrahydroindolizines 7 [15].

C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
103
Scheme 2. Synthesis of ylides 13aem. Reagents and conditions: (i) pyridine derivative (2e5 equiv), EtOAc, reflux, 24 h; (ii) DBU (1 equiv), DMF or acetonitrile, alkene (2-cyano-3-
ethoxyacrylonitrile (10), ethyl 2-cyano-3-ethoxyacrylate (11) or (3E)-4-ethoxy-1,1,1-trifluorobut-3-en-2-one (12)) (2 equiv), 70 ꢁC, 24 h.
2.2. Biological evaluation
In the pyridinylidene series of products 14 (structures given in
Scheme 4 and Table 2), the p-substituted compounds showed
significant biological potential with protein farnesyltransferase
inhibition potencies in the submicromolar range (e.g. 14a:
The activity of the two new families of synthesized compounds
was evaluated on human FTase. Results are summarized in Tables 3
and 4. In the ylide series of compounds 13 (structures given in
Scheme 2 and Table 1), the 4-dimethylamino substitution of the
pyridinium ring proved to be favourable to bioactivity. Except the
3,5-dimethylpyridinium ylides 13l and 13m, that showed compa-
rable biological potency, all other structural modifications of the
pyridinium ring (H, 3-Me or 4-OMe) caused a slight reduction in
biological potential (compounds 13f, 13h or 13j vs 13a, Table 3).
Structural modifications of the chelating groups were also realized:
the replacement of one cyano group by an ethyl ester generally
conserved affinity towards FTase (compound 13a vs 13b, Table 3). In
addition, when only a carboxyl unit was kept to interact with the
zinc atom of the protein, a significant decrease in biological
IC50 ¼ 0.65 ꢀ 0.1
mM, Table 4). As seen in the previous ylide series,
the replacement of a cyano chelating unit with an ethyl ester, had
no effect on biological potential (compound 14a vs 14b, Table 4).
The o-substituted derivatives 14c and 14d, showed a slight reduc-
tion in inhibitory activity, compared to the p-substituted deriva-
tives 14a and 14b. Finally, the replacement of the phenothiazine
unit by a diethylamino group caused a significant decrease of the
inhibitory potential (compound 14b vs 17, Table 4), highlighting the
importance of the phenothiazine group in these series.
2.3. Molecular modelling
potential was observed (13a: IC50 ¼ 4.67 ꢀ 0.36
mM; 13c:
Farnesyltransferase structure was taken from the 1LD7 [19]
entry of the RCSB Protein Data Bank [20]. The crystallized inhib-
itor and water molecules were removed to permit docking of the
studied compounds, built from the standard fragments library of
Sybyl 6.9.1 [21] with GOLD 5.1 [22]. Thirty solutions were generated
and classed through an in-house scoring function based on Gold-
Score [22] and X-Score functions [23]. The consistency of the results
was assessed by visually examining the conformation cluster.
Compound 14b adopts a single conformation described in
Fig. 2(a), where the cyano and the ethyl ester groups are in front of
the zinc atom and the carbonyl moiety next to the nitrogen atom of
IC50 ¼ 86.49 ꢀ 16.96
m
M). Finally, the substitution of the pheno-
thiazine unit by a 2-chloro group conserved biological potency
(compound 13b vs 13e, Table 3).
Table 1
Structure of starting pyridinium salts 9aef and obtained ylides 13aem.
Salt Nꢁ
Starting salt
Product
Nꢁ
Ylide
R
R
R1
H
R1
W1
W2
9a
4-NMe2
13a
13b
13c
13d
13e
13f
13g
13h
13i
4-NMe2
4-NMe2
4-NMe2
4-NMe2
4-NMe2
H
H
3-Me
3-Me
4-OMe
4-OMe
3,5-diMe
3,5-diMe
H
H
H
CN
CN
H
CN
the phenothiazine forms a hydrogen bond with Arg 202b. The
CO2Et
CO2H
CN
CO2Et
CN
CO2Et
CN
CO2Et
CN
dinitrile 14a binds in a similar way to ester 14b and adopts a single
conformation, without forming hydrogen bonds (Fig. 2(b)).
The affinity of o-substituted compounds 14c and 14d is weaker
than that of p-substituted compounds 14a and 14b. They display
different conformations. However, one conformation of compound
14c fits into the binding site with the cyano units in front of the zinc
atom of the enzyme, while the phenothiazine ring establishes
9b
9c
9d
9e
9f
4-NMe2
H
2-Cl
H
2-Cl
2-Cl
H
H
H
H
H
H
H
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
3-Me
H
4-OMe
3,5-diMe
H
13j
13k
13l
CO2Et
CN
CO2Et
stacking interactions with Tyr361
possible hydrogen bonding could further anchor the compound 14c
with the hydroxyl Tyr 166 at the level of the cyano group
b. On other conformations,
H
13m
H
a

104
C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
R1
+
W
N
R2
O
-
..
CN
N
S
- EtOH
R1
(i)
W
13n: W = CN, R1 = Me, R2 = H
13o: W = CO2Et, R1 = Me, R2 = H
13p: W = CN, R1 = H, R2 = Me
13q: W = CO2Et, R1 = H, R2 = Me
+
+
R2
O
N
-
Cl
EtO
CN
10: W = CN
11: W = CO2Et
N
S
W
CN
N
9g: R1 = Me, R2 = H
9h: R1 = H, R2 = Me
O
N
S
14a: W = CN 55%
Position 4
Position 2
14b: W = CO2Et 66%
14c: W = CN 64%
14d: W = CO2Et 63%
Scheme 3. Products obtained in case of reaction between dipolarophiles and 2- and 4-picoline salts 9g and 9h. Conditions: (i) DBU (1 equiv), DMF, 70 ꢁC, 24 h.
(Fig. 2(d)). Compound 14d displays a similar behaviour, with the
cyano group in interaction with the zinc atom and stacking inter-
actions between the phenothiazine group and Tyr361b (Fig. 2(c)).
254 nm and 366 nm. Column chromatographies were performed
using a CombiFlash Rf Companion (Teledyne-Isco System) and
RediSep packed columns. IR spectra were recorded on a Bruker
Tensor 27. Elemental analysis (C, H, N, S) of new compounds was
determined on a Thermo Electron apparatus by ‘Pôle Chimie
Moléculaire’, Faculté de Sciences Mirande, Université de Bour-
gogne, Dijon, France.
3. Conclusions
The biological evaluation of the effect of synthesized products
on human farnesyltransferase, allowed us to discover that new
ylides with a phenothiazine scaffold possess protein farnesyl-
transferase inhibition potencies in the low micromolar range.
The nature of the substituent on the pyridine unit proved to be
important in determining inhibitory activity and replacement of
the cyanoacrylonitrile chelating function by a cyanoethylacrylate
group generally conserved biological activity.
Unexpected products were isolated from 2- and 4-picolinium
salts. Compounds 14a and 14b were the most promising inhibi-
tors of human farnesyltransferase in the present study, with IC50
values in the submicromolar range. The obtained results indicate
that phenothiazine derivatives deserve further investigation as
new cytotoxic agents of improved characteristics.
4.1.1. General procedure for the synthesis of pyridinium salts (9ael)
The pyridine derivative (2e5 equiv) was added to a solution of
halogenated compound (1 equiv) in ethyl acetate. The resulting
mixture was stirred at reflux for 24 h. After cooling the reaction
media to room temperature, the obtained precipitate was filtered,
washed with ethyl acetate or acetone and recrystallized from
ethanol to give the corresponding pure salt.
4.1.1.1. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-4-dimethylamino-
pyridinium chloride (9a). White solid, 93% yield; mp 228e230 ꢁC.
IR nmax/cmꢂ1: 1692, 1651, 1578, 1456, 1216, 1177, 775. 1H NMR
(DMSO-d6/D2O 1:1, 400 MHz)
d ppm: 3.20 (s, 6H, N(CH3)2), 5.55
(br s, 2H, COCH2N), 7.07 (d, J ¼ 7.8 Hz, 2H, PyH), 7.32e7.94 (m, 8H,
4. Experimental section
ArH), 8.30 (d, J ¼ 7.8 Hz, 2H, PyH). 13C NMR (DMSO-d6/D2O 1:1,
100 MHz)
d 40.0 (2CH3), 57.7 (CH2), 106.7 (2CH), 126.7e138.0
4.1. Materials and methods-general remarks
(8CH þ 4C), 143.4 (2CH), 155.9 (C), 165.4 (C). Anal. calcd for
C
21H20ClN3OS: C, 63.39; H, 5.07; N, 10.56; S, 8.06. Found: C, 63.04;
Starting materials are commercially available and were used
without further purification. Melting points were measured on
a Buchi 510 apparatus and are raw values. NMR spectra were
acquired at 400 MHz for 1H NMR and 100 MHz for 13C NMR on
a Bruker DRX 400 spectrometer or on a Varian 400 MHz Premium
ShieldedÒ spectrometer with tetramethylsilane as internal stan-
H, 4.97; N, 10.11%.
4.1.1.2. 1-[2-Oxo-2-(10H-2-chlorophenothiazin-10-yl)ethyl]-4-dimethy-
laminopyridinium chloride (9b). White solid, 92% yield; mp 225 ꢁC.
IR nmax/cmꢂ1: 1681, 1648, 1572, 1459, 1210, 1175, 775. 1H NMR
(DMSO-d6/D2O 1:1, 400 MHz) d ppm: 3.21 (s, 6H, N(CH3)2), 5.74 (br
dard, at room temperature. Chemical shifts (
d
) are expressed in
s, 2H, COCH2N), 7.10 (d, J ¼ 7.4 Hz, 2H, PyH), 7.36e7.55 (m, 4H, ArH),
ppm relative to TMS as internal standard. Thin layer chromatog-
raphy (TLC) was realized on Macherey Nagel silica gel plates with
fluorescent indicator and was visualized under a UV-lamp at
7.66 (d, J ¼ 8.0 Hz, 2H, ArH), 7.97 (br. s, 1H, ArH), 8.39 (d, J ¼ 7.4 Hz,
2H, PyH). 13C NMR (DMSO-d6/D2O 1:1, 100 MHz)
d 39.4 (2CH3), 57.6
(CH2), 106.9 (2CH), 126.0e138.0 (8CH þ 4C), 143.4 (2CH), 155.9 (C),

C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
105
Table 2
Study of the generalization of the synthesis of pyridin-(1H)-ylidene derivatives starting from 2- and 4-picolinium salts.
Entry
Salt no
Starting salt
Y
Product no
Structure final product
R
1
2
9i
C6H5
4-Me
15
9g
10H-phenothiazin-10-yl
4-Me
14a
3
4
14b
14c
14d
9h
2-Me
5
6
9j
Morpholin-4-yl
4-Me
16
19
7
8
9k
9l
NEt2
4-Me
4-Me
17
18
9H-carbazol-9-yl
165.4 (C). Anal. calcd for C21H19Cl2N3OS: C, 58.34; H, 4.43; N, 9.72; S,
7.42. Found: C, 58.74; H, 4.57; N, 9.91%.
1628, 1479, 1460, 1372, 1259, 1177, 760, 570. 1H NMR (DMSO-d6/D2O
1:1, 400 MHz) ppm: 5.35 (br s, 1H, COCH2N), 6.11 (br s, 1H,
d
COCH2N), 7.26e7.48 (m, 4H, ArH), 7.50e7.84 (m, 4H, ArH), 8.04 (t,
J ¼ 7.0 Hz, 2H, PyH), 8.54 (t, J ¼ 7.8 Hz, 1H, PyH), 8.77 (d, J ¼ 6.0 Hz,
4.1.1.3. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]pyridinium chlo-
ride (9c). White solid, 90% yield; mp 252e253 ꢁC. IR nmax/cmꢂ1
:
2H, PyH). 13C NMR (DMSO-d6/D2O 1:1, 100 MHz)
d 62.0 (CH2),
124.0e139.0 (10CH þ 4C), 146.3 (2CH), 146.9 (CH), 164.5 (C). Anal.
calcd for C19H15ClN2OS: C, 64.31; H, 4.26; N, 7.89; S, 9.04. Found: C,
64.38; H, 4.18; N, 7.90%.
Table 3
Inhibitory activities of ylides 13aem on human FTase.
Compound no.
IC50
(
m
M ꢀ SD)a
13a
13b
13c
13d
13e
13f
13g
13h
13i
4.67 ꢀ 0.36
5.33 ꢀ 0.55
86.49 ꢀ 16.96
3.18 ꢀ 0.23
4.92 ꢀ 0.45
13.10 ꢀ 1.02
14.98 ꢀ 2.31
10.98 ꢀ 1.65
7.01 ꢀ 0.93
10.29 ꢀ 1.48
13.74 ꢀ 0.99
6.36 ꢀ 0.74
7.27 ꢀ 0.73
Table 4
Inhibitory activities of compounds 14aed and 17 on human
FTase.
Compound no.
IC50
(
m
M ꢀ SD)a
14a
14b
14c
14d
15
0.65 ꢀ 0.10
0.63 ꢀ 0.06
1.20 ꢀ 0.29
1.07 ꢀ 0.17
n.t.b
13j
13k
13l
16
17
n.t.
43.09 ꢀ 9.81
13m
a
Values represent mean of two experiments.
not tested.
a
b
Values represent mean of two experiments.

106
C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
R
W
W
(i)
+
+
+
or
N
N
W
N
-
Cl
EtO
CN
-
CN
O
O
O
..
CN
Y
Y
Y
9g-l
10: W = CN
11: W = CO2Et
14a-d, 16, 17
15
Scheme 4. Study of the generalization of the synthesis of new unexpected compounds. Conditions: (i) DBU (1 equiv), DMF, 70 ꢁC, 24 h.
4.1.1.4. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-3-methylpyridi-
nium chloride (9d). White solid, 82% yield; mp 160e163 ꢁC.
IR nmax/cmꢂ1: 1677, 1461, 1372, 1303, 1262, 1166, 780, 748, 574.
(DMSO-d6/D2O 1:1, 100 MHz) d 58.4 (CH3), 60.1 (CH2), 113.2 (2CH),
124.0e139.0 (8CH þ 4C), 147.6 (2CH), 165.0 (C), 171.7 (C). Anal. calcd
for C20H17ClN2O2S: C, 62.41; H, 4.45; N, 7.28; S, 8.33. Found: C,
62.48; H, 5.15; N, 6.98%.
1H NMR (DMSO-d6/D2O 1:1, 400 MHz)
d ppm: 2.55 (s, 3H, CH3),
5.53 (br s, 1H, COCH2N), 6.54 (br s, 1H, COCH2N), 7.38e7.59 (m, 5H,
ArH), 7.64e7.80 (m, 3H, ArH), 8.17 (dd, J ¼ 8.0, 6.0 Hz, 1H, pyridine
ArH), 8.58 (d, J ¼ 8.0 Hz, 1H, pyridine ArH), 9.04 (d, J ¼ 6.0 Hz, 1H,
PyH), 9.12 (s, 1H, PyH). 13C NMR (DMSO-d6/D2O 1:1, 100 MHz)
4.1.1.6. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-3,5-dimethylpy-
ridinium chloride (9f). White solid, 88% yield; mp 262e264 ꢁC.
IR nmax/cmꢂ1: 1682, 1462, 1367, 1310, 1263, 1182, 755, 599. 1H
d
18.2 (CH3), 62.0 (CH2), 124.8e138.0 (8CH þ 5C), 138.4 (CH), 144.4
(CH), 146.3 (CH), 147.2 (CH), 164.7 (C). Anal. calcd for
20H17ClN2OS: C, 65.12; H, 4.65; N, 7.59; S, 8.69. Found: C, 64.90; H,
NMR (DMSO-d6/D2O 1:1, 400 MHz)
5.25 (br s, 1H, COCH2N), 5.97 (br s, 1H, COCH2N), 7.27e7.50 (m, 4H,
ArH), 7.53e7.83 (m, 4H, ArH), 8.20 (s, 1H, PyH), 8.43 (s, 2H, PyH). 13
NMR (DMSO-d6/D2O 1:1,100 MHz) 18.6 (2CH3), 62.4 (CH2),123.0e
d ppm: 2.37 (s, 6H, N(CH3)2),
C
C
5.06; N, 8.26%.
d
131.0 (10CH þ 2C), 139.2 (2C), 143.6 (2C), 148.5 (CH), 165.3 (C). Anal.
calcd for C21H19ClN2OS: C, 65.87; H, 5.00; N, 7.32; S, 8.37. Found: C,
65.86; H, 4.91; N, 7.76%.
4.1.1.5. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-4-methoxypyridi-
nium chloride (9e). White solid, 85% yield; mp 192e194 ꢁC. IR nmax
/
cmꢂ1: 1677, 1640, 1520, 1480, 1300, 1261, 1198, 999, 748, 536. 1H
NMR (DMSO-d6/D2O 1:1, 400 MHz) ppm: 4.02 (s, 3H, OCH3), 5.14
d
4.1.1.7. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-4-methylpyridi-
nium chloride (9g). White solid, 87% yield; mp 265e266 ꢁC.
IR nmax/cmꢂ1: 1677, 1635, 1462, 1374, 1261, 1132, 975, 785, 508.
(br s, 1H, COCH2N), 5.93 (br s, 1H, COCH2N), 7.27e7.80 (m, 10H, 8H
phenoth. þ 2H pyridine ArH), 8.54 (d, J ¼ 7.4 Hz, 2H, PyH). 13C NMR
Fig. 2. Docking of compounds 14aed in the active site of FTase: (a) compound 14b, (b) compound 14a, (c) compound 14d, (d) compound 14c.

C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
107
1H NMR (DMSO-d6/D2O 1:1, 400 MHz)
d
ppm: 2.56 (s, 3H, CH3),
precipitate was filtered, washed with cold distilled water and
recrystallized from absolute ethanol. Some ylures necessitated
supplementary chromatographic purification using ethanol as
eluent.
5.31 (br s, 1H, COCH2N), 6.11 (br s, 1H, COCH2N), 7.27e7.64 (m, 8H,
ArH), 7.88 (d, J ¼ 6.8 Hz, 2H, PyH), 8.66 (d, J ¼ 6.8 Hz, 2H, PyH). 13
C
NMR (DMSO-d6/D2O 1:1, 100 MHz) d 22.0 (CH3), 61.2 (CH2), 124.0e
140.0 (8CH þ 4C phenoth. þ 2CH pyridine), 145.4 (2CH pyridine),
160.9 (C), 164.7 (C). Anal. calcd for C20H17ClN2OS: C, 65.12; H, 4.65;
N, 7.59; S, 8.69. Found: C, 64.90; H, 5.06; N, 8.26%.
4.1.2.1. 3-(2-Cyanoacrylonitrile)-10H-phenothiazin-10-yl-carbonyl-
4-dimethylaminopyridinium methylide (13a). Yellow solid; 51%
yield; mp 281e283 ꢁC. IR nmax/cmꢂ1: 2189, 2168, 1634,1567, 1540,
4.1.1.8. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-2-methylpyridi-
nium chloride (9h). White solid, 81% yield; mp 256e258 ꢁC. IR
nmax/cmꢂ1: 2918, 1675, 1629, 1462, 1346, 1263, 1181, 1033, 759, 547.
1279, 1259, 1169, 751, 595. 1H NMR (DMSO-d6, 400 MHz)
d ppm:
3.19 (s, 6H, N(CH3)2), 6.71 (s, 1H, CH]), 6.86 (d, J ¼ 7.2 Hz, 2H, PyH),
7.21 (t, J ¼ 7.6 Hz, 2H, ArH), 7.34 (t, J ¼ 7.6 Hz, 2H, ArH), 7.43 (d,
J ¼ 7.6 Hz, 2H, ArH), 7.80 (d, J ¼ 7.6 Hz, 2H, ArH), 8.00 (d, J ¼ 7.2 Hz,
1H NMR (DMSO-d6/D2O 1:1, 400 MHz)
d ppm: 2.55 (s, 3H, CH3),
5.31 (br s, 1H, COCH2N), 6.05 (br s, 1H, COCH2N), 7.25e7.75 (m, 8H,
ArH), 7.89e7.97 (m, 2H, PyH), 8.44 (dt, J ¼ 7.8, 1.1 Hz, 1H, PyH), 8.82
(d, J ¼ 6.0 Hz, 1H, PyH). 13C NMR (DMSO-d6/D2O 1:1, 100 MHz)
2H, PyH). 13C NMR (DMSO-d6, 100 MHz)
d 39.7 (2CH3), 40.1 (C),
106.9 (2CH), 112.5 (C), 118.0 (C), 121.2 (C), 124.9 (2CH), 126.0 (2CH),
127.6 (2CH), 127.7 (2CH), 130.0 (2C) 140.6 (2C), 143.3 (CH), 146.0
(2CH), 155.8 (C), 163.6 (C). Anal. calcd for C25H19N5OS: C, 68.56; H,
4.34; N, 15.99. Found: C, 68.58; H, 4.26; N, 15.29%.
d
19.7 (CH3), 59.5 (CH2), 124.0e140.0 (10CH þ 4C), 147.1 (CH), 146.9
(CH), 156.2 (C), 164.0 (C). Anal. calcd for C20H17ClN2OS: C, 65.12; H,
4.65; N, 7.59; S, 8.69. Found: C, 64.92; H, 4.98; N, 7.96%.
4.1.2.2. 3-(2-Cyanoethylacrylate)-10H-phenothiazin-10-yl-carbonyl-
4.1.1.9. 1-[2-Oxo-2(phenyl)ethyl]-4-methylpyridinium chloride (9i).
White solid with the same physico-chemical properties as
described in the literature [24]; 85% yield.
4-dimethylaminopyridinium
methylide
(13b). Yellow-greenish
solid; 57% yield; mp 287e289 ꢁC. IR nmax/cmꢂ1: 3539, 3456, 2193,
1645, 1618, 1541, 1197, 1097, 757. 1H NMR (DMSO-d6, 400 MHz)
d
ppm: 1.10 (t, J ¼ 7.2 Hz, 3H, CO2CH2CH3), 3.19 (s, 6H, N(CH3)2), 3.91
4.1.1.10. 1-(2-Morpholin-4-yl-2-oxoethyl)-4-methylpyridinium chlo-
(q, J ¼ 7.2 Hz, 2H, CO2CH2CH3), 6.85 (d, J ¼ 7.2 Hz, 2H, PyH), 7.16 (t,
J ¼ 7.6 Hz, 2H, ArH), 7.28 (t, J ¼ 7.6 Hz, 2H, ArH), 7.38 (d, J ¼ 7.6 Hz,
2H, ArH), 7.61 (s, 1H, CH]), 7.75 (d, J ¼ 7.6 Hz, 2H, ArH), 7.98 (d,
ride (9j). Off-white solid; 48% yield. 1H NMR (D2O, 400 MHz)
d
ppm: 2.56 (s, 3H, CH3), 3.31 (t, J ¼ 4.6 Hz, 2H, NCH2CH2O), 3.46 (t,
J ¼ 4.7 Hz, 2H, NCH2CH2O), 3.66 (t, J ¼ 4.7 Hz, 2H, NCH2CH2O), 3.72
(sym m, J ¼ 4.6 Hz, 2H, NCH2CH2O), 5.56 (s, 2H, COCH2N), 7.80 (d,
J ¼ 6.9 Hz, 2H, ArH), 8.40 (d, J ¼ 6.9 Hz, 2H, ArH). 13C NMR (D2O,
J ¼ 7.2 Hz, 2H, PyH). 13C NMR (DMSO-d6, 100 MHz)
d 14.6 (CH3), 39.7
(2CH3), 58.6 (CH2), 65.4 (C), 106.8 (2CH), 110.9 (C), 119.4 (C), 124.1
(2CH), 125.5 (2CH), 127.5 (4CH), 129.4 (2C), 141.2 (2C), 142.5 (CH),
146.2 (2CH), 155.7 (C), 165.0 (C), 167.0 (C). Anal. calcd for
100 MHz) d 21.5 (CH3), 44.9 (CH2), 59.5 (CH2), 63.5 (CH2), 65.9 (CH2),
66.1 (CH2), 128.3 (2CH), 144.6 (2CH), 161.5 (C), 164.7 (C). Anal. calcd
for C12H17ClN2O2: C, 56.14; H, 6.67; N, 10.91. Found: C, 56.37; H,
6.46; N, 11.07%.
C27H24N4O3S: C, 66.86; H, 4.95; N, 11.55. Found: C, 66.74; H, 5.15; N,
11.41%.
4.1.2.3. 3-(Prop-2-enoic acid)-10H-phenothiazin-10-yl-carbonyl-4-
dimethylaminopyridinium methylide (13c). Yellow solid; 57% yield;
mp 207e210 ꢁC. IR nmax/cmꢂ1: 1630, 1564, 1516, 1440, 1182, 1150,
4.1.1.11. 1-[2-(Diethylamino)-2-oxoethyl]-4-methylpyridinium chlo-
ride (9k). Very hygroscopic white solid; 95% yield. IR nmax/cmꢂ1
:
3379 (hydrated form),1641,1521,1472,1433,1385,1308,1273,1203,
754, 714. 1H NMR (DMSO-d6, 400 MHz)
d ppm: 3.19 (s, 6H, N(CH3)2),
1147, 951. 1H NMR (DMSO-d6, 400 MHz)
d
ppm: 1.06 (t, J ¼ 7.1 Hz,
4.55 (d, J ¼ 12.8 Hz, 1H, CH]), 6.84 (d, J ¼ 7.2 Hz, 2H, pyridine ArH),
7.16 (t, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.29 (t, J ¼ 7.6 Hz, 2H, phenoth.
ArH), 7.37 (d, J ¼ 7.6 Hz, 2H, phenoth.), 7.72e7.78 (m, 3H, 2H phe-
noth. ArH þ CH]), 7.93 (d, J ¼ 7.2 Hz, 2H, pyridine ArH). 13C NMR
3H, NCH2CH3), 1.25 (t, J ¼ 7.1 Hz, 3H, NCH2CH3), 2.64 (s, 3H, CH3),
3.33 (q, J ¼ 7.1 Hz, 2H, NCH2CH3), 3.39 (q, J ¼ 7.1 Hz, 2H, NCH2CH3),
5.77 (s, 2H, COCH2N), 8.01 (d, J ¼ 6.6 Hz, 2H, ArH), 8.86 (d, J ¼ 6.6 Hz,
2H, ArH). 13C NMR (DMSO-d6, 100 MHz)
d
12.7 (CH3), 13.6 (CH3),
(DMSO-d6, 100 MHz) d 39.7 (2CH3), 85.8 (CH), 107.3 (2CH), 115.5 (C),
21.4 (CH3), 40.1 (CH2), 40.7 (CH2), 60.0 (CH2), 127.6 (2CH), 145.3
(2CH), 159.4 (C), 163.6 (C). Anal. calcd for C12H19ClN2O 5H2O: C,
43.31; H, 8.78; N, 8.42. Found: C, 44.04; H, 9.16; N, 8.87%.
124.5 (2CH), 126.0 (2CH), 127.5 (4CH), 129.7 (2C), 140.5 (2C), 140.7
(CH), 144.6 (2CH), 155.3 (C), 164.0 (C), 168.9 (C). Anal. calcd for
C24H21N3O3S: C, 66.58; H, 4.85; N, 9.71; S, 7.39. Found: C, 66.31; H,
4.86; N, 9.61%.
4.1.1.12. 1-[2-Oxo-2-(9H-carbazol-9-yl)ethyl]-4-methylpyridinium
chloride (9l). White solid, 78% yield; mp 294e296 ꢁC 1H NMR
4.1.2.4. 3-(2-Cyanoacrylonitrile)-10H-(2-chlorophenothiazin)-10-yl-
carbonyl-4-dimethylaminopyridinium methylide (13d). Yellow solid;
41% yield; mp 271e273 ꢁC. IR nmax/cmꢂ1: 2193, 2173, 1630, 1537,
(DMSO-d6, 400 MHz)
d ppm: 2.72 (s, 3H, ArCH3), 6.67 (s, 2H,
COCH2N), 7.53 (t, J ¼ 7.6 Hz, 2H, ArH), 7.61 (t, J ¼ 7.6 Hz, 2H, ArH),
8.15 (d, J ¼ 6.4 Hz, 2H, PyH), 8.26e8.31 (m, 4H, ArH), 9.02 (d,
1278, 1171, 743, 599. 1H NMR (CDCl3, 400 MHz)
d ppm: 3.22 (s, 6H,
J ¼ 6.4 Hz, 2H, PyH). 13C NMR (DMSO-d6/D2O 1:1, 100 MHz)
d
21.8
N(CH3)2), 6.63 (d, J ¼ 7.6 Hz, 2H, PyH), 7.02 (s, 1H, CH]), 7.10e7.17
(m, 2H, ArH), 7.21 (d, J ¼ 7.6 Hz, 1H, ArH), 7.25e7.30 (m, 2H, ArH),
7.49 (d, J ¼ 7.6 Hz, 1H, ArH), 7.64 (d, J ¼ 7.6 Hz, 2H, PyH), 7.88 (s, 1H,
(CH3), 64.3 (CH2), 116.5 (2CH), 120.6 (2CH), 124.7 (2CH), 126.1 (2C),
127.9 (2CH),128.2 (2CH),137.5 (2C),145.5 (2CH),160.5 (C),166.5 (C).
ArH). 13C NMR (CDCl3, 100 MHz)
d 40.3 (2CH3), 43.3 (C), 107.0 (2CH),
4.1.2. General procedure for the preparation of ylides (13aem)
DBU (1 equiv) was added to a solution of pyridinium salt (1
equiv) in a minimum volume of solvent (DMF or acetonitrile). The
reaction media became yellow. This corresponded to the in situ
formation of the ylure. The trisubstituted alkene (2-cyano-3-
ethoxyacrylonitrile (10), ethyl 2-cyano-3-ethoxyacrylate (11) or
(3E)-4-ethoxy-1,1,1-trifluorobut-3-en-2-one (12)) (2 equiv) was
then added. The reaction mixture became red-brown and was
heated at 70 ꢁC for 24 h. After cooling to room temperature, cold
distilled water was added to the reaction media. The obtained
111.6 (C), 118.3 (C), 120.6 (C), 124.4 (CH), 123.7 (CH), 125.8 (CH),
126.3 (CH), 127.8 (CH), 128.0 (CH), 128.2 (CH), 129.1 (C), 130.6 (C),
133.1 (C),140.9 (C),141.7 (C),145.6 (CH),146.3 (2CH),156.1 (C),164.3
(C). Anal. calcd for C25H18ClN5OS: C, 63.56; H, 3.81; N, 14.83; S, 6.78.
Found: C, 63.18; H, 4.16; N, 15.02%.
4.1.2.5. 3-(2-Cyanoethylacrylate)-10H-(2-chlorophenothiazin)-10-yl-
carbonyl-4-dimethylaminopyridinium methylide (13e). Yellow solid;
38% yield; mp 250e253 ꢁC. IR nmax/cmꢂ1: 2178, 1644, 1630, 1557,
1365, 1191, 1098, 746. 1H NMR (CDCl3, 400 MHz)
d ppm: 1.21 (t,

108
C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
J ¼ 7.2 Hz, 3H, CO2CH2CH3), 3.21 (s, 6H, N(CH3)2), 4.08 (q, J ¼ 7.2 Hz,
2H, CO2CH2CH3), 6.66 (d, J ¼ 7.2 Hz, 2H, PyH) 7.07e7.13 (m, 2H,
ArH), 7.21 (d, J ¼ 8.0 Hz, 2H, ArH), 7.31 (d, J ¼ 7.2 Hz, 1H, ArH), 7.50
(d, J ¼ 8.0 Hz, 1H, ArH), 7.71 (d, J ¼ 7.2 Hz, 2H, PyH), 7.80 (s, 1H,
142.7 (CH), 144.0 (CH), 145.6 (CH), 147.9 (CH), 164.2 (C), 167.0 (C).
Anal. calcd for C26H21N3O3S: C, 68.49; H, 4.60; N, 9.21; S, 7.02.
Found: C, 68.55; H, 4.82; N, 9.20%.
CH]), 7.87 (s,1H, ArH). 13C NMR (CDCl3,100 MHz)
d
14.6 (CH3), 40.2
4.1.2.10. 3-(2-Cyanoacrylonitrile)-10H-phenothiazin-10-yl-carbonyl-
4-methoxypyridinium methylide (13j). Yellow solid; 50% yield; mp
242e245 ꢁC. IR nmax/cmꢂ1: 2181, 2170, 1625, 1511, 1238, 1167, 1124,
(2CH3), 59.8 (CH2), 68.1 (C), 106.9 (2CH), 110.3 (C), 120.2 (C), 124.0
(CH), 124.3 (CH), 125.4 (CH), 125.8 (CH), 127.6 (CH), 127.9 (CH), 128.1
(CH), 129.0 (C), 130.3 (C), 133.0 (C), 141.6 (C), 142.3 (C), 144.2 (CH),
146.5 (2CH), 156.2 (C), 165.7 (C), 167.1 (C). Anal. calcd for
938, 746, 595. 1H NMR (CDCl3, 400 MHz)
d ppm: 4.06 (s, 3H, OCH3),
7.07 (d, J ¼ 7.2 Hz, 2H, pyridine ArH), 7.08 (s, 1H, CH]), 7.15 (t,
J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.27e7.32 (m, 4H, phenoth. ArH), 7.71
(d, J ¼ 7.6 Hz, 2H, phenoth. ArH), 8.03 (d, J ¼ 7.2 Hz, 2H, pyridine
C
27H23ClN4O3S: C, 62.41; H, 4.43; N, 10.78; S, 6.16. Found: C, 62.08;
H, 4.26; N, 10.39%.
ArH). 13C NMR (CDCl3, 100 MHz)
d: 43.0 (C), 57.7 (CH3), 112.4 (2CH),
4.1.2.6. 3-(2-Cyanoacrylonitrile)-10H-phenothiazin-10-yl-carbonyl-
pyridinium methylide (13f). Orange solid; 36% yield; mp 292e
294 ꢁC. IR nmax/cmꢂ1: 2193, 2172, 1631, 1542, 1458, 1358, 1284,
118.0 (2C), 120.2 (C), 125.0 (2CH), 126.3 (2CH), 127.6 (2CH), 127.9
(2CH),131.4 (2C),140.4 (2C), 144.7 (CH),149.9 (2CH),163.5 (C), 170.7
(C). Anal. calcd for C24H16N4O2S: C, 67.84; H, 3.76; N, 13.19; S, 7.53;
Found: C, 67.41; H, 3.66; N, 12.90%.
1256, 1174, 746, 589. 1H NMR (DMSO-d6, 400 MHz)
d ppm: 6.84 (s,
1H, CH]), 7.22 (t, J ¼ 7.6 Hz, 2H, ArH), 7.35 (t, J ¼ 7.6 Hz, 2H, ArH),
7.40 (d, J ¼ 7.6 Hz, 2H, ArH), 7.86 (d, J ¼ 8.0 Hz, 2H, ArH), 8.01 (t,
J ¼ 6.8 Hz, 2H, PyH), 8.50 (t, J ¼ 7.6 Hz, 1H, PyH), 8.93 (d, J ¼ 6.0 Hz,
4.1.2.11. 3-(2-Cyanoethylacrylate)-10H-phenothiazin-10-yl-carbonyl-
4-methoxypyridinium methylide (13k). Yellow solid; 41% yield; mp
211e213 ꢁC. IR nmax/cmꢂ1: 2181, 1674, 1624, 1512, 1455, 1312, 1185,
2H, PyH). 13C NMR (DMSO-d6, 100 MHz)
d 40.2 (C), 114.0 (C), 116.9
(C) 120.4 (C), 125.1 (2CH), 126.3 (2CH), 127.5 (2CH), 127.7 (2CH),
127.8 (2CH), 130.3 (2C), 140.0 (2C), 142.5 (CH), 145.6 (CH), 149.1
(2CH), 162.6 (C). Anal. calcd for C23H14N4OS: C, 69.96; H, 3.54; N,
14.19. Found: C, 69.58; H, 3.97; N, 13.90%.
1103, 748, 593. 1H NMR (CDCl3, 400 MHz)
d
ppm: 1.22 (t, J ¼ 7.2 Hz,
3H, CO2CH2CH3), 4.02 (s, 3H, OCH3), 4.09 (q, J ¼ 7.2 Hz, 2H,
CO2CH2CH3), 7.05e7.19 (m, 4H, 2H pyridine and 2H phenoth. ArH),
7.24 (t, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.30 (d, J ¼ 7.6 Hz, 2H, phenoth.
ArH), 7.71 (d, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.82 (s, 1H, CH]), 8.07
4.1.2.7. 3-(2-Cyanoethylacrylate)-10H-phenothiazin-10-yl-carbonyl-
pyridinium methylide (13g). Orange solid; 42% yield; mp 259e
261 ꢁC. IR nmax/cmꢂ1: 2181, 1644, 1615, 1560, 1334, 1232, 1195,
(d, J ¼ 6.8 Hz, 2H, pyridine ArH). 13C NMR (CDCl3, 100 MHz)
d 14.6
(CH3), 57.5 (CH2), 59.8 (CH3), 67.4 (C), 111.0 (C), 112.3 (2CH), 120.0
(C), 124.7 (2CH), 125.8 (2CH), 127.4 (2CH), 127.7 (2CH), 131.2 (2C),
140.9 (2C), 143.1 (CH), 149.9 (2CH), 164.7 (C), 167.0 (C), 170.4 (C).
Anal. calcd for C26H21N3O4S: C, 66.16; H, 4.45; N, 8.90. Found: C,
66.41; H, 4.28; N, 8.91%.
1098, 754, 596. 1H NMR (CDCl3, 400 MHz)
d
ppm: 1.23 (t, J ¼ 7.0 Hz,
3H, CO2CH2CH3), 4.11 (q, J ¼ 7.0 Hz, 2H, CO2CH2CH3), 7.12 (t,
J ¼ 7.6 Hz, 2H, PyH) 7.20e7.35 (m, 4H, ArH), 7.70e7.80 (m, 4H, ArH),
7.92 (s, 1H, CH]), 8.20 (t, J ¼ 7.6 Hz, 1H, PyH), 8.32 (d, J ¼ 6.0 Hz, 2H,
PyH). 13C NMR (CDCl3, 100 MHz)
d
14.6 (CH3), 60.0 (CH2), 68.1 (C),
4.1.2.12. 3-(2-Cyanoacrylonitrile)-10H-phenothiazin-10-yl-carbonyl-
3,5-dimethylpyridinium methylide (13l). Yellow solid; 57% yield; mp
266e269 ꢁC. IR nmax/cmꢂ1: 2194, 2171, 1639, 1536, 1456, 1255, 1029,
112.3 (C), 119.4 (C), 124.8 (2CH), 126.0 (2CH), 126.9 (2CH), 127.5
(2CH), 127.8 (2CH), 131.3 (2C), 140.7 (2C), 142.7 (CH), 143.5 (CH),
148.5 (2CH), 164.2 (C), 166.9 (C). Anal. calcd for C25H19N3O3S: C,
67.94; H, 4.30; N, 9.51; S, 7.24. Found: C, 67.6; H, 4.54; N, 9.48%.
759, 594. 1H NMR (DMSO-d6, 400 MHz)
d ppm: 2.30 (s, 6H, 2CH3),
7.13 (s, 1H, CH]), 7.20 (t, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.32e7.36
(m, 4H, phenoth. ArH), 7.85 (d, J ¼ 7.6 Hz, 2H, phenoth. ArH), 8.06
(s, 1H, pyridine ArH), 8.53 (s, 2H, pyridine ArH). 13C NMR (DMSO-d6,
4.1.2.8. 3-(2-Cyanoacrylonitrile)-10H-phenothiazin-10-yl-carbonyl-
3-methylpyridinium methylide (13h). Orange solid; 49% yield; mp
170e174 ꢁC. IR nmax/cmꢂ1: 2193, 2171, 1632, 1537, 1455, 1353, 1280,
100 MHz) d 17.5 (2CH3), 40.7 (C), 114.4 (C), 117.1 (C), 120.7 (C), 125.1
(2CH), 126.2 (2CH), 127.5 (2CH), 127.7 (2CH), 130.5 (2C), 137.1 (2C),
139.6 (2C), 142.2 (CH), 145.2 (2CH), 145.8 (CH), 162.4 (C). Anal. calcd
for C25H18N4OS: C, 71.00; H, 4.26; N, 13.25; S, 7.57. Found: C, 70.77;
H, 4.31; N, 13.25%.
1250, 1170, 754, 589. 1H NMR (DMSO-d6, 400 MHz)
d ppm: 2.39 (s,
3H, CH3), 6.92 (s, 1H, CH]), 7.20 (dt, J ¼ 7.6, 0.8 Hz, 2H, phenoth.
ArH), 7.31e7.38 (m, 4H, phenoth. ArH), 7.78e7.87 (m, 3H, 1H pyri-
dine and 2H phenoth. ArH), 8.27 (d, J ¼ 8.0 Hz, 1H, H-4 pyridine
ArH), 8.69 (d, J ¼ 6.0 Hz, 1H, H-6 pyridine ArH), 8.75 (s, 1H, H-2
4.1.2.13. 3-(2-Cyanoethylacrylate)-10H-phenothiazin-10-yl-carbonyl-
3,5-dimethylpyridinium methylide (13m). Orange solid; 61% yield;
mp 245e246 ꢁC. IR nmax/cmꢂ1: 2190, 1672, 1628, 1522, 1457, 1188,
pyridine ArH). 13C NMR (DMSO-d6, 100 MHz)
d 17.6 (CH3), 40.5 (C),
114.2 (C), 117.0 (C), 120.5 (C), 125.1 (2CH), 126.2 (2CH), 126.7 (CH),
127.6 (2CH),127.8 (2CH),130.4 (2C),138.0 (C),139.8 (2C),142.4 (CH),
145.6 (CH), 146.2 (CH), 147.9 (CH), 162.5 (C). Anal. calcd for
1021, 755, 598. 1H NMR (CDCl3, 400 MHz)
d
ppm: 1.25 (t, J ¼ 6.8 Hz,
3H, CO2CH2CH3), 2.38 (s, 6H, 2CH3), 4.14 (q, J ¼ 6.8 Hz, 2H,
CO2CH2CH3), 7.10 (t, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.21 (d, J ¼ 7.6 Hz,
2H, phenoth. ArH), 7.26 (t, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.69 (s, 1H,
CH]), 7.81 (d, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.87 (s, 2H, pyridine
C
24H16N4OS: C, 70.50; H, 3.91; N, 13.70; S, 7.83. Found: C, 70.40; H,
4.39; N, 13.28%.
4.1.2.9. 3-(2-Cyanoethylacrylate)-10H-phenothiazin-10-yl-carbonyl-
3-methylpyridinium methylide (13i). Red solid; 43% yield; mp 220e
224 ꢁC. IR nmax/cmꢂ1: 2181, 1658, 1635, 1527, 1475, 1231, 1196, 764,
ArH), 8.09 (s, 1H, pyridine ArH). 13C NMR (100 MHz, CDCl3)
d 14.6
(CH3), 18.5 (2CH3), 59.9 (CH2), 68.0 (C), 112.3 (C), 119.4 (C), 124.9
(2CH), 125.8 (2CH), 127.4 (2CH), 127.5 (2CH), 131.0 (2C), 137.2 (2C),
140.5 (2C), 142.4 (CH), 144.7 (CH), 144.9 (2CH), 164.1 (C), 167.2 (C).
Anal. calcd for C27H23N3O3S: C, 69.00; H, 4.89; N, 8.94. Found: C,
68.87; H, 4.64; N, 8.75%.
597. 1H NMR (CDCl3, 400 MHz)
CO2CH2CH3), 2.46 (s, 3H, ArCH3), 4.09 (q, 7.2 Hz, 2H,
d
ppm: 1.23 (t, J ¼ 7.2 Hz, 3H,
J
¼
CO2CH2CH3), 7.11 (t, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.20e7.30 (m, 4H,
phenoth. ArH), 7.59 (t, J ¼ 6.8 Hz, 1H, pyridine ArH), 7.78 (d,
J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.94 (d, J ¼ 6.8 Hz, 1H, pyridine ArH),
8.00 (s, 1H, CH]), 8.07 (s, 1H, pyridine ArH), 8.12 (d, J ¼ 6.8 Hz, 1H,
4.1.3. General procedure for the synthesis of compounds 14aed, 15,
16 and 17
DBU (1 equiv) was added to a solution of cycloimmonium salt
(1 equiv) in a minimum volume of DMF. The reaction media
become yellow. This corresponded to the formation in situ of the
pyridine ArH). 13C NMR (CDCl3, 100 MHz)
d
14.6 (CH3), 18.7 (CH3),
60.0 (CH2), 68.2 (C), 112.3 (C), 119.4 (C), 124.8 (2CH), 125.9 (2CH),
126.2 (CH), 127.5 (2CH), 127.7 (2CH), 131.2 (2C), 138.1 (C), 140.7 (2C),

C.-M. Abuhaie et al. / European Journal of Medicinal Chemistry 59 (2013) 101e110
109
intermediate ylure. The trisubstituted alchene (2-cyano-3-
ethoxyacrylonitrile 10 or ethyl 2-cyano-3-ethoxyacrylate 11) (2
equiv) was then added. The reaction mixture become red and was
heated at 70 ꢁC for 24 h. A precipitate was formed during the
reaction. After cooling to room temperature, the obtained precipi-
tate was filtered, washed with absolute ethanol and recrystallized
from absolute ethanol. Compound 16 necessitated a supplementary
chromatographic purification using AcOEt/n-heptane 8/2 as eluent.
129.0 (8CH), 134.0 (2C), 136.7 (CH), 141.8 (CH), 143.0 (2C), 148.2
(CH), 153.9 (C), 164.2 (C), 165.7 (C). Anal. calcd for C26H21N3O3S: C,
68.55; H, 4.65; N, 9.22; S, 7.04. Found: C, 68.44; H, 4.71; N, 9.22%.
4.1.3.5. 3-(2-Cyanoethylacrylate)-benzoyl-4-methylpyridinium
methylide (15). Yellow solid, 78% yield; mp 252e254 ꢁC. IR nmax
/
cmꢂ1: 2185, 1697, 1640, 1516, 1233, 1172, 1115, 736, 662. 1H NMR
(CDCl3, 400 MHz)
d
ppm: 1.22 (t, J ¼ 7.2 Hz, 3H, CO2CH2CH3), 2.70 (s,
3H, ArCH3), 4.14 (q, J ¼ 7.2 Hz, 2H, CO2CH2CH3), 7.41e7.52 (m, 3H,
phenyl ArH), 7.62 (d, J ¼ 6.8 Hz, 2H, phenyl ArH), 7.74 (d, J ¼ 5.6 Hz,
2H, phenyl ArH), 8.17 (s, 1H, CH]), 8.31 (d, J ¼ 5.6 Hz, 2H, pyridine
4.1.3.1. {2-[1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]pyridin-
4(1H)-ylidene]ethylidene}malononitrile (14a). Orange red solid; 55%
yield; mp 265e267 ꢁC. IR nmax/cmꢂ1: 2187, 1680, 1652, 1543, 1461,
ArH). 13C NMR (CDCl3, 100 MHz)
d 14.5 (CH3), 22.5 (CH3), 60.3 (CH2),
1261, 1176, 754. 1H NMR ((CD3)2CO, 400 MHz)
d
ppm: 5.20 (br s, 2H,
70.2 (C), 118.8 (C), 121.1 (C), 127.9 (2CH), 128.4 (2CH), 128.5 (2CH),
130.7 (CH), 138.6 (C), 145.2 (CH), 147.7 (2CH), 158.7 (C), 166.8 (C),
185.4 (C). Anal. calcd for C20H17N2O3: C, 71.99; H, 5.09; N, 8.39.
Found: C, 71.57; H, 4.88; N, 8.31%.
NCH2CO), 5.61 (d, J ¼ 14.0 Hz, 1H, CH]), 6.70 (d, J ¼ 6.4 Hz, 1H,
pyridine ArH), 7.25e7.32 (m, 3H, 2H phenoth. and 1H pyridine ArH),
7.35 (dt, J ¼ 8.0, 1.2 Hz, 2H, phenoth. ArH), 7.50 (dd, J ¼ 8.0, 1.2 Hz,
2H, phenoth. ArH), 7.57 (d, J ¼ 7.9 Hz, 2H, phenoth. ArH), 7.59 (d,
J ¼ 14.0 Hz,1H, CH]), 7.66 (d, J ¼ 6.4 Hz, 2H, pyridine ArH). 13C NMR
4.1.3.6. Ethyl (2Z)-2-cyano-4-[1-(2-morpholin-4-yl-2-oxoethyl)pyr-
(DMSO-d6, 100 MHz)
d
53.1 (C), 59.5 (CH2), 102.2 (CH), 112.8 (CH),
idin-4(1H)-ylidene]but-2-enoate (16). Red oil; 41% yield. 1H NMR
117.4 (C), 117.7 (CH), 119.6 (C), 127.1 (2CH), 127.5 (2CH), 127.7 (2CH),
128.1 (2CH),132.8 (2C),137.6 (2C),140.2 (CH),141.2 (CH),149.3 (CH),
152.5 (C), 165.2 (C). Anal. calcd for C24H16N4OS: C, 70.57; H, 3.95; N,
13.72. Found: C, 69.99; H, 3.62; N, 12.35%.
(CDCl3, 400 MHz)
d
ppm: 1.27 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3), 3.32 (t,
J ¼ 4.6 Hz, 2H, NCH2CH2O), 3.49 (t, J ¼ 4.7 Hz, 2H, NCH2CH2O), 3.69 (t,
J ¼ 4.7 Hz, 2H, NCH2CH2O), 3.76 (sym m, J ¼ 4.6 Hz, 2H, NCH2CH2O),
4.21 (q, J ¼ 7.2 Hz, 2H, CO2CH2CH3), 4.55 (s, 2H, COCH2N), 5.76 (d,
J ¼ 13.9 Hz,1H, CH]), 6.44 (sym m,1H, ArH), 6.93e6.99 (m, 3H, ArH),
8.08 (d, J ¼ 13.9 Hz,1H, CH]). Anal. calcd for C18H21N3O4: C, 62.96; H,
6.16; N, 12.24. Found: C, 62.79; H, 6.43; N, 12.51%.
4.1.3.2. Ethyl (2Z)-2-cyano-4-[1-[2-oxo-2-(10H-phenothiazin-10-yl)
ethyl]pyridin-4(1H)-ylidene]but-2-enoate (14b). Red solid; 66%
yield; mp 268e271 ꢁC. IR nmax/cmꢂ1: 2183, 1675, 1652, 1553, 1256,
1197, 1093, 852, 749. 1H NMR (DMSO-d6, 400 MHz)
d
ppm: 1.20 (t,
4.1.3.7. Ethyl 2-cyano-3-morpholin-4-yl acrylate (19) [18]. By-
product from the synthesis of compound 16. White solid; 16%
yield. IR nmax/cmꢂ1: 2206, 1682, 1608, 1439, 1368, 1285, 1260, 1211,
J ¼ 7.2 Hz, 3H, CO2CH2CH3), 4.08 (q, J ¼ 7.2 Hz, 2H, CO2CH2CH3), 5.20
(br s, 2H, NCH2CO), 5.65 (d, J ¼ 14.0 Hz, 1H, CH]), 6.85 (br s, 1H,
pyridine ArH), 7.25e7.50 (m, 5H, 4H phenoth. and 1H pyridine ArH),
7.64 (d, J ¼ 7.6 Hz, 2H, phenoth. ArH), 7.70 (d, J ¼ 7.6 Hz, 2H, phe-
noth. ArH), 7.75 (br s, 2H, pyridine ArH), 8.05 (d, J ¼ 14.0 Hz, 1H,
1163, 1099, 1028, 1004, 855, 762. 1H NMR (CDCl3, 400 MHz)
d (ppm)
1.31 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3), 3.50 (large s, 2H, CH2), 3.79 (t,
J ¼ 4.8 Hz, 4H, 2CH2), 4.05 (large s, 2H, CH2), 4.24 (q, J ¼ 7.1 Hz, 2H,
CH]). 13C NMR (DMSO-d6, 100 MHz)
d
14.6 (CH3), 57.7 (CH2), 59.0
CO2CH2CH3), 7.70 (s, 1H, CH). 13C NMR (CDCl3, 100 MHz)
d (ppm)
(CH2), 75.7 (C), 101.8 (CH), 112.8 (CH), 118.0 (CH), 120.0 (C), 126.9
(2CH), 127.6 (4CH), 128.2 (2CH), 132.0 (2C), 137.0 (C), 140.7 (CH),
141.4 (CH), 146.3 (CH), 152.7 (C), 165.5 (C), 166.0 (C). Anal. calcd for
14.4 (CH3), 46.7 (CH2), 55.2 (CH2), 61.1 (CH2), 66.0 (CH2), 66.9 (CH2),
70.6 (C), 117.8 (C), 155.6 (CH), 166.5 (C).
C
26H21N3O3S: C, 68.55; H, 4.65; N, 9.22. Found: C, 68.04; H, 4.59; N,
4.1.3.8. Ethyl (2Z)-2-cyano-4-[1-[2-(diethylamino)-2-oxoethyl]pyr-
9.35%.
idin-4(1H)-ylidene]but-2-enoate (17). Red solid; 56% yield. IR nmax
cmꢂ1: 2972, 2183, 1648, 1519, 1480, 1183, 1135, 1072, 1019, 948, 860,
815. 1H NMR (CDCl3, 400 MHz)
/
4.1.3.3. {(2E)-2-[1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]pyridin-
2(1H)-ylidene]ethylidene}malononitrile (14c). Yellow solid; 64%
yield; mp 270e271 ꢁC. IR nmax/cmꢂ1: 2196, 2188, 1680, 1545, 1283,
d
ppm: 1.16 (t, J ¼ 7.1 Hz, 3H,
NCH2CH3), 1.28 (t, J ¼ 7.2 Hz, 3H, CO2CH2CH3), 1.31 (t, J ¼ 7.1 Hz, 3H,
NCH2CH3), 3.33 (q, J ¼ 7.1 Hz, 2H, NCH2CH3), 3.42 (q, J ¼ 7.1 Hz, 2H,
NCH2CH3), 4.23 (q, J ¼ 7.2 Hz, 2H, CO2CH2CH3), 4.55 (s, 2H, COCH2N),
5.77 (d, J ¼ 13.9 Hz, 1H, CH]), 6.44 (sm, 1H, ArH), 6.93e6.99 (m, 3H,
ArH), 8.08 (d, J ¼ 13.9 Hz, 1H, CH]). 13C NMR (CDCl3, 100 MHz)
1176, 772, 576. 1H NMR (DMSO-d6, 400 MHz)
d ppm: 4.96 (br s, 1H,
NCH2CO), 5.16 (d, J ¼ 13.6 Hz, 1H, CH]), 5.50 (br s, 1H, NCH2CO),
6.85 (t, J ¼ 6.8 Hz, 1H, pyridine ArH), 7.38e7.58 (m, 4H, phenoth.
ArH), 7.63e7.80 (m, 5H, 4H phenoth. þ 1H pyridine ArH), 7.83 (d,
J ¼ 13.6 Hz, 1H, CH]), 7.87 (d, J ¼ 6.8 Hz, 1H, pyridine ArH), 7.91 (d,
d
12.9 (CH3), 14.4 (CH3), 14.6 (CH3), 41.1 (CH2), 56.8 (CH2), 60.3
(CH2), 81.8 (C), 103.7 (CH), 112.5 (CH), 117.9 (CH), 119.8 (C), 137.9
(CH), 138.6 (CH), 148.2 (CH), 151.3 (C), 163.9 (C), 166.8 (C). Anal.
calcd for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76. Found: C, 65.29; H,
7.33; N, 12.75%.
J ¼ 6.8 Hz, 1H, pyridine ArH). 13C NMR (DMSO-d6, 100 MHz)
d 50.2
(CH2), 56.2 (C), 92.7 (CH), 114.3 (CH), 118.1 (C), 120.3 (C), 120.6 (CH),
121.0e138.0 (8CH phenoth. þ 4C), 138.3 (CH), 142.3 (CH), 149.6
(CH), 153.1 (C), 164.2 (C). Anal. calcd for C24H16N4OS: C, 70.57; H,
3.95; N, 13.72; S, 7.85. Found: C, 70.35; H, 4.42; N, 12.72%.
4.2. Molecular modelling
4.1.3.4. Ethyl (2Z,4E)-2-cyano-4-[1-[2-oxo-2-(10H-phenothiazin-10-
yl)ethyl]pyridin-2(1H)-ylidene]but-2-enoate (14d). Red solid; 63%
yield; mp 135e137 ꢁC. IR nmax/cmꢂ1: 2184, 1684, 1521, 1412, 1224,
Based on previous studies [25], 1LD7 was chosen due to the
structural similarity of the cocrystallized ligand with the
compounds related in this work. The reference ligand and water
molecules were removed, then the cocrystallized inhibitor was
1156, 1082, 748. 1H NMR ((CD3)2CO, 400 MHz)
d ppm: 1,24 (t,
ꢀ
J ¼ 7.2 Hz, 3H, CO2CH2CH3), 4.14 (q, J ¼ 7.2 Hz, 2H, COCH2CH3), 5.19
(br s, 2H, NCH2CO), 5.42 (d, J ¼ 13.6 Hz, 1H, CH]), 6.62 (t, J ¼ 6.8 Hz,
1H, H pyridine, ArH), 7.33e7.50 (m, 5H, 4H phenoth. þ 1H pyridine
ArH), 7.55e7.70 (m, 3H, 2H phenoth. þ 1H pyridine ArH), 7.71e7.85
(m, 3H, 2H phenoth. þ 1H pyridine ArH), 8.17 (d, J ¼ 13.6 Hz, 1H,
repositioned into the binding site defined as a 10A sphere around
its position in the crystal, needing a distance constraint to maintain
proximity to the zinc. This constraint was later withdrawn as metal
atoms were directly taken into account by newer versions docking
software and the studied compounds became more remote from
the cocrystallized inhibitor. The protonation state of the protein
and ligands was found to be compatible with a pH of 7.5 and the
CH]). 13C NMR ((CD3)2CO, 100 MHz)
d 14.1 (CH3), 56.1 (CH2), 59.2
(CH2), 79.7 (C), 91.5 (CH), 112.1 (CH), 119.0 (C), 120.1 (CH), 123.0e

110
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geometry of the ligands optimized using GasteigereHückel partial
charges, with a formal charge of þ2 applied to iron atoms and
a dielectric constant set to 4. A careful inspection of the geometry
and charges of the ferrocene moiety was done to verify the distance
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consistency of the best ranking conformation and select the most
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Acknowledgements
The authors gratefully acknowledge the “Programul Operational
Sectorial Dezvoltarea Resurselor Umane 2007e2013” (project
POSDRU/88/1.5/S/47646), the CommScie (project POSDRU/89/1.5/S/
63663) and the PRES (Pôle de Recherche et d’Enseignement
Supérieur, Université Lille Nord de France) for financial support.
Thanks to Dr. David F. Perry for reviewing this manuscript.
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