5-Substituted [1]pyrindine derivatives with antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2009.11.028

We report herein the synthesis of 5-substituted [1]pyrindine derivatives and the evaluation of their antiproliferative properties on HeLa cells, a cervical carcinoma tumor cell line, and on the melanoma A2058 cell line. The most efficient compounds display cytotoxicity against tumor cells in the micromolar range but have interestingly no effect against the normal human fibroblasts CRL-2796. Generally, these pyrindines are active on both tumor cell lines. Compounds bearing large substituents with structural rigidity at position 5 such as phenyl-furyl show no inhibition of cell growth.

Full text of DOI:10.1016/j.ejmech.2009.11.028

European Journal of Medicinal Chemistry 45 (2010) 896–901
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
5-Substituted [1]pyrindine derivatives with antiproliferative activity
a
,
b
,
1
a
,
b
,
,
b
c,d
, Mary-Lorene Goddard 1, Ali Loukaci a , Odile Mondesert
,
´
`
´
Stephanie Kolb
Bernard Ducommun c e, Emmanuelle Braud a b, Christiane Garbay a
,
d
,
,
,b,
*
a Universite´ Paris Descartes, UFR Biome´dicale, Laboratoire de Pharmacochimie Mole´culaire et Cellulaire, 45 rue des Saints-Pe`res, Paris, F-75006, France
b INSERM U648, Paris, F-75006, France
c Universite´ de Toulouse, LBCMCP; 118 route de Narbonne, F-31062 Toulouse, France
d CNRS, LBCMCP-UMR5088, F-31062 Toulouse, France
e CHU Purpan, TSA 40031, F-31059 Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the synthesis of 5-substituted [1]pyrindine derivatives and the evaluation of their
antiproliferative properties on HeLa cells, a cervical carcinoma tumor cell line, and on the melanoma
A2058 cell line. The most efficient compounds display cytotoxicity against tumor cells in the micromolar
range but have interestingly no effect against the normal human fibroblasts CRL-2796. Generally, these
pyrindines are active on both tumor cell lines. Compounds bearing large substituents with structural
rigidity at position 5 such as phenyl-furyl show no inhibition of cell growth.
Received 2 December 2008
Received in revised form
6 November 2009
Accepted 12 November 2009
Available online 6 December 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Cancer
Cytotoxic agents
Pyrindine derivatives
1. Introduction
phosphatase activity of the recombinant MBP-CDC25B protein with
an IC50 value of 19.0 mM [6].
Pyrindine systems have been reported in a variety of natural
products displaying awide range of biological activities. For example,
louisianin C [1] and incarvilline [2] which possess the [2]pyrindine
core are alkaloid compounds with anti-angiogenic and potent
analgesic properties, respectively (Fig. 1). Molecules bearing the
[1]pyrindine moiety have also been described such as streptazolin
[3,4], a tricyclic compound displaying antibiotic and antifungal
properties and its by-product, the 5-O-(b-D-xylopyranosyl)-strepta-
zolin [5] which shows in vitro cytotoxic activity against several
human cancer cell lines (Fig. 1).
We recently reported in silico/in vitro screening experiments
targeting the dual-specificity phosphatases CDC25 to identify
original scaffolds with CDC25 inhibitory activity [6]. These phos-
phatases play critical roles in cell cycle regulation by activating
Cdk–cyclin complexes and are considered particularly attractive
targets for the treatment of cancer [7,8]. Among the most potent
compounds isolated from the screening, pyrindine 1 inhibited the
To date, a few compounds belonging to the family of [1]pyr-
indines of type I have been found to exhibit biological activity but
only as protein kinase inhibitors (Fig. 1). Using virtual screening
methods, Forino et al. identified compound 2 with Akt1 kinase
inhibitory activity [9,10]. Subsequent evaluation of analogues of 2
allowed for the discovery of two additional inhibitors, compounds
1 and 3 with IC50 of 60.2 and 126
patent related that compound 4 could inhibit B-Raf kinase activity
with an IC50 value superior to 20 M [11].
mM, respectively. Finally, a recent
m
Therefore, since the biological properties of these pyrindine
derivatives have been poorly studied, we decided to develop
a series of compounds of type I to improve the inhibitory activity
toward CDC25 phosphatases (Fig. 1). Since inhibitors of CDC25
activity are intended to present cytotoxic properties, these
compounds were also evaluated against two human cancer cell
lines as well as a normal human cell type.
2. Chemistry
The 5-substituted [1]pyrindines 8 and 11–29 were synthesized
in a two-step sequence starting with the preparation of the
common aminopyrindinecarbonitrile scaffold 5a,b obtained by
reacting two equivalents of malononitrile with 2,5-hexanedione in
the presence of piperidine in refluxing ethanol (Scheme 1) [12,13].
* Corresponding author. Universite´ Paris Descartes, UFR Biome´dicale, Laboratoire
´
`
de Pharmacochimie Moleculaire et Cellulaire, 45 rue des Saints-Peres, Paris,
F-75006, France. Tel.: þ33 142 864 080; fax: þ33 142 864 082.
E-mail address: christiane.garbay@parisdescartes.fr (C. Garbay).
1
These authors contributed equally to the work.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.028

S. Kolb et al. / European Journal of Medicinal Chemistry 45 (2010) 896–901
897
H
O
OH
OH
N
N
O
O
H
N
louisianin C
(-)-incarvilline
CH3
(+)-streptazolin
R2
R1
OH
O
R
CH3
NC
CH3
N
NC
NC
CH3
CH3
CH3
H2N
N
H2N
N
H2N
CN
CN
CN
4
I
1 : R1= 3-CO2H R2 = H
2 : R1= 3-Cl
3 : R1= H
R2 = 4-CO2H
R2 = 4-CO2H
Fig. 1. Pyrindine system containing agents.
1H NMR spectrum obtained in DMSO revealed that an equilibrium
was observed between both tautomeric forms 5a and 5b in a ratio
of 32/68 respectively as previously reported [12].
glutathione-depleting compound menadione whose anti-
proliferative activity has already been demonstrated against some
cancer cell lines [14] was used as a control and displayed comparable
cytotoxicity against the tumor and normal cell types.
Interestingly, no pyrindine derivatives exhibited cytotoxic
activity against the normal CRL-2796 cells regardless the substi-
tution of the pyrindine system, except compound 22.
Concerning the cytotoxic effects on cancer cell lines, results in
Table 1 clearly showed that when the pyrindine core was substituted
by a phenyl-furylmethylene moiety, the corresponding compounds
1, 3, 6, 7, 9 and 10 did not display any relevant antiproliferative
The final compounds were synthesized using a Knoevenagel
condensation that was first described by Junek [13]. Thus, reaction of
the equilibrating scaffolds 5a, b with the appropriate aldehydes in
the presence of piperidine in methanol afforded the final compounds
8 and 11–29 with yields ranging from 23 to 62% (Scheme 1).
This synthetic path led to mixtures of Z/E isomer. NMR experi-
ments were conducted on compound 27 to fully assign each isomer
its own 1H NMR spectral data. Briefly, the HMBC spectrum showed
three-bond correlations from C3 to NH2 and 4-Me while comple-
mentary NOESY spectral data helped determine the correct
stereochemistry by correlating the vinylic H and 4- or 6-Me (Fig. 2).
Z/E ratio was subsequently determined for each compound from 1H
NMR spectra and Tables 1 and 2. Almost all pyrindines I showed
comparable proportions of each isomer except compounds 8, 14, 21
and 26 with 70, 68, 35 and 93% of the E form, respectively.
activity on both cancer cell lines at 100
mM. No effect was either
observed in case of compound 8 bearing the 5-bromofuryl moiety.
Pyridinyl derivatives 11–12 were found to be equally active on
both cancer cell lines, 12 being slightly more potent. In contrast,
2-pyridinyl 13 displayed no cytotoxic activity. Compound 14 was
the most efficient cytotoxic agent against HeLa cells with an IC50
value of 8.2 mM, ten-fold higher than that against A2058. No cyto-
toxic effect was observed for methylnaphthylderivative 15 whereas
derivatives 16 and 17 displayed comparable potency and appeared
more active toward A2058.
3. Results and discussion
Pyrindines 18–21 bearing a susbtituent at position 4 of the
phenyl ring displayed cytotoxicity. The 3-hydroxyl derivative 22
exhibited comparable IC50 values against both cancer cell lines
whereas its congener 23 revealed totally inactive. Bromo deriva-
tives 24–26 inhibited HeLa growth but only 25 was efficient against
The 27 pyrindine derivatives reported in Tables 1 and 2 were
first evaluated for their inhibitory activity on an enzymatic in vitro
assay using the recombinant MBP-CDC25B fused protein [14].
Unfortunately, none of the compounds was efficient at inhibiting
CDC25 phosphatase activity except compound 1 as previously
mentioned [6].
A2058 with an IC50 value of 4.2 mM. Potent cytotoxic activity was
observed in the case of nitro derivatives 27 and 28 against A2058
whereas no efficiency was found on HeLa cells. Finally compound
29 revealed selective toward HeLa cells whereas 30 displayed
comparable antiproliferative activities toward both cell lines.
In conclusion, [1]pyrindine derivatives substituted in the
5-position by a diversity of aromatic groups did not inhibit CDC25
Nonetheless, for a preliminary study, these molecules were
assessed as mixtures of both isomers for their antiproliferative
activity using a cell viability colorimetric assay against two human
cancer cell lines, cervical carcinoma HeLa and melanoma A2058.
Cytotoxic activity was also evaluated on the non tumor human CRL-
2796 cell line. The results are reported in Tables 1 and 2. The
Scheme 1. Reagents and conditions: (i) piperidine, abs. EtOH, reflux, 1 h (45%); (ii) RCHO, piperidine, MeOH, rt, 24 h (23–92%).

898
S. Kolb et al. / European Journal of Medicinal Chemistry 45 (2010) 896–901
Fig. 2. Identification of Z- and E-isomers of compound 27.
phosphatase activity. Nevertheless, they provided antiproliferative
activities against HeLa and A2058 cell lines and induced no cyto-
toxicity toward normal CRL-2796 cells. Moreover, one can note that
some of these pyrindines were more active on A2058 cells, known
to be resistant to chemotherapy while others displayed cytotoxicity
on HeLa cells only. Further experiments will therefore be carried
out in order to identify the main biological targets of these prom-
ising candidates for the development of novel anticancer agents.
internal standard and coupling constants J are given in Hertz. Mass
spectra were determined on an LCQ Advantage spectrometer
(ThermoElectron, France) at the Laboratoire de Chimie et Biochimie
´
Pharmacologiques et Toxicologiques, Universite Paris Descartes,
Paris, France. Compounds 1, 3, 6, 7, 9,10 and 30 were purchased from
Akos, Germany.
4.2. 2-Amino-4,6-dimethyl-5H-cyclopenta[b]pyridine-3,7-
4. Materials and methods
dicarbonitrile (5)
4.1. General
To a solution of malononitrile (6.7 mL, 106 mol) in ethanol
(85 mL) cooled to 0 ꢁC were added piperidine (12.6 mL,127.8 mmol)
and 2,5-hexanedione (5.00 mL, 42.6 mmol). The reaction mixture
was heated under reflux for 1 h. The precipitate was filtered and
washed with ethanol to give 5 as a brown powder (4.03 g, 45%).
Kieselgel 60F254 plates (Merck) were used for analytical thin layer
chromatography and were visualized with ultraviolet light (254 nm).
All the commercial reagents and solvents were of analytical grade
and purchased from Sigma and Carlo Erba – SDS, respectively. NMR
spectra were performed on a Bruker WMFTꢀ250 MHz spectrometer
at the Laboratoire de Chimie et Biochimie Pharmacologiques et
Compound 5a: 1H NMR (DMSO–d6, 250 MHz)
d
2.34 (s, 3H, CH3),
2.42 (s, 3H, CH3), 3.63 (s, 2H, CH2), 6.85–6.88 (m, 2H, NH2).
Compound 5b: 1H NMR (DMSO–d6, 250 MHz)
2.25 (s, 3H, CH3),
d
´
Toxicologiques, Universite Paris Descartes, Paris, France. Chemical
2.50 (s, 3H, CH3), 6.03 (s, 1H, C–H), 6.85–6.88 (m, 2H, NH2), 12.75 (s,
1H, NH).
shifts are expressed in parts per million (ppm) with TMS used as an
Table 1
Antiproliferative activity against tumor and normal cell lines of compounds 1–17. IC50 values are expressed with standard error.
CH3
R
NC
CH3
H2N
N
CN
Entry
R
Yield (%)
–
Z/E
IC50 ꢂ SEM (
mM)
HeLa
A2058
CRL-2796
Menadione
–
–
–
–
24.7 ꢂ 2.5
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
13.4 ꢂ 1.8
n.d.
n.d.
21.0 ꢂ 2.0
n.d.
n.d.
n.a.
a
b
1
3
6
7
8
9
10
11
12
13
14
15
16
17
2-[5-(3-carboxyphenyl)]furyl
2-[5-(4-carboxyphenyl)]furyl
2-[5-(2-carboxyphenyl)]furyl
2-[5-(3-ethoxycarbonylphenyl)]furyl
2-(5-bromo)furyl
2-(5-phenyl)furyl
2-[5-(3-bromophenyl)]furyl
4-pyridinyl
3-pyridinyl
2-pyridinyl
4-quinolinyl
4-methylnaphthyl
1,3-benzodioxol-5-yl
Phenyl
–
–
–
–
a
b
a
b
n.a.
a
b
–
n.a.
n.a.
92
30/70
n.a.
n.a.
a
b
–
–
–
–
n.a.
n.a.
a
b
n.a.
n.a.
41
76
49
86
68
70
70
40/60
40/60
40/60
32/68
45/55
55/45
45/55
24.6 ꢂ 3.2
13.2 ꢂ 0.6
n.a.
37.3 ꢂ 1.2
12.2 ꢂ 0.4
n.a.
n.a.
n.a.
n.a.
8.2 ꢂ 0.2
79.9 ꢂ 2.1
n.a.
n.a.
n.a.
n.a.
34.5 ꢂ 3.2
38.9 ꢂ 0.6
10.0 ꢂ 0.1
8.5 ꢂ 0.3
n.a.
n.a.
a
Compound purchased from Akos, Germany.
Quantity not sufficient to establish Z/E ratio. n.d.: not determined. n.a.: no activity at 100 mM.
b

S. Kolb et al. / European Journal of Medicinal Chemistry 45 (2010) 896–901
899
following the general procedure as an orange solid (216 mg, 76%). 1H
NMR (DMSO–d6, 250 MHz) 1.66 (s, 3H, CH3), 2.02 (s, 3H, CH3), 2.58
(s, 3H, CH3), 2.72 (s, 3H, CH3), 7.24 (s, 2H, NH2), 7.32 (s, 2H, NH2), 7.49–
7.56 (m, 2H, Har), 7.78–7.83 (m, 2H, Har and C–H), 7.93–7.96 (m, 1H,
Har), 8.08 (s, 1H, C–H), 8.59–8.69 (m, 4H, Har); MS (ESI) m/z 298.3
[M ꢀ H]ꢀ.
Table 2
Antiproliferative activity against tumor and normal cell lines of compounds 18–30.
IC50 values are expressed with standard error.
d
R2
CH3
R1
NC
CH3
4.7. (Z/E)-2-Amino-4,6-dimethyl-5-(pyridin-2-ylmethylene)-5H-
H2N
N
cyclopenta[b]pyridine-3,7-dicarbonitrile (13)
CN
Compound 13 was obtained from compound
0.57 mmol) and 2–pyridinecarboxaldehyde (108 L, 1.14 mmol)
following the general procedure as an orange solid (84 mg, 49%). 1H
NMR (DMSO–d6, 250 MHz) 1.63 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.57
(s, 3H, CH3), 2.72 (s, 3H, CH3), 7.25 (s, 2H, NH2), 7.31 (s, 2H, NH2),
7.417.46 (m, 2H, Har), 7.55 (d, 1H, J ¼ 7.5, Har), 7.66–7.72 (m, 3H, Har
and C–H), 7.90–7.99 (m, 4H, Har and C–H), 8.72 (s, 2H, Har); MS (ESI)
m/z 300.3 [M þ H]þ.
5 (120 mg,
m
Entry R1
R2
Yield Z/E
(%)
IC50 ꢂ SEM (
HeLa
50/50 17.6 ꢂ 0.3 7.9 ꢂ 0.2
m
M)
A2058
CRL-2796
n.a.
d
18
19
20
21
22
23
24
25
26
27
28
29
4-Me
4-Ph
H
H
H
H
H
H
H
H
H
H
H
H
31
82
77
87
69
86
44
23
39
66
67
68
53/47 17.0 ꢂ 2.3 13.3 ꢂ 0.2 n.a.
55/45 39.4 ꢂ 1.2 34.5 ꢂ 3.8 n.a.
65/35 11.3 ꢂ 0.7 28.5 ꢂ 1.3 n.a.
44/56 25.8 ꢂ 0.9 17.6 ꢂ 1.1 54.7 ꢂ 1.3
4-SMe
4-N(Me)2
3-OH
2-OH
4-Br
3-Br
2-Br
4-NO2
3-NO2
4-(4-Cl-
Ph)O
52/48 n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
47/53 27.1 ꢂ 0.8 n.a.
4.8. (Z/E)-2-Amino-4,6-dimethyl-5-(quinolin-4-ylmethylene)-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (14)
40/60 12.5 ꢂ 0.9 4.2 ꢂ 0.7
7/93
36.6 ꢂ 0.9 n.a.
46/54 99.0 ꢂ 1.0 12.2 ꢂ 0.6 n.a.
44/56 97.2 ꢂ 6.6 19.8 ꢂ 1.9 n.a.
Compound 14 was obtained from compound
5 (200 mg,
53/47 15.2 ꢂ 1.1 n.a.
n.a.
0.95 mmol) and 4-quinolinecarboxaldehyde (299 mg, 1.9 mmol)
following the general procedure as a red solid (285 mg, 86%). 1H
a
b
30
4-[(2-CN- 3-Br
Ph)CH2]O
–
–
15.8 ꢂ 2.4 13.1 ꢂ 1.0 n.a.
NMR (DMSO–d6, 250 MHz) d 1.29 (s, 3H, CH3), 1.75 (s, 3H, CH3), 2.69
a
(s, 3H, CH3), 2.80 (s, 3H, CH3), 7.28–7.36 (m, 4H, 2 ꢃ NH2), 7.59–7.74
(m, 4H, Har), 7.83–7.90 (m, 3H, Har and C–H), 8.04 (d, 1H, J ¼ 7.9,
Har), 8.12–8.15 (m, 3H, Har), 8.33 (s, 1H, C–H), 8.95–8.99 (m, 2H,
Har); MS (ESI) m/z 348.7 [M ꢀ H]ꢀ.
Compound purchased from Akos, Germany.
Quantity not sufficient to establish Z/E ratio. n.a.: no activity at 100 mM.
b
4.3. General procedure for the synthesis of pyrindine derivatives
4.9. (Z/E)-2-Amino-4,6-dimethyl-5-[(4-methyl-1-
naphthyl)methylene]-5H-cyclopenta[b]pyridine-3,7-dicarbonitrile
(15)
To a solution of compound 5 (1 eq) and the appropriate alde-
hyde (2 eq) in ethanol were added two drops of piperidine. The
reaction mixture was stirred at room temperature for 24 h. The
precipitate was filtered and washed with ethanol.
Compound 15 was obtained from compound
5 (200 mg,
0.95 mmol) and 4-methyl-1-naphthaldehyde (324 mg, 1.9 mmol)
following the general procedure as a brown solid (234 mg, 68%). 1H
4.4. (Z/E)-2-Amino-5-[(5-bromo-2-furyl)methylene]-4,6-dimethyl-
5H-cyclopenta[b]pyridine-3,7-dicarbonitrile (8)
NMR (DMSO–d6, 250 MHz) d 1.37 (s, 3H, CH3), 1.84 (s, 3H, CH3), 2.69
(s, 3H, CH3), 2.74 (s, 6H, 2 ꢃ CH3), 2.81 (s, 3H, CH3), 7.19–7.22 (m, 4H,
2 ꢃ NH2), 7.43–7.47 (m, 3H, Har), 7.60–7.71 (m, 4H, Har), 8.04 (d, 2H,
J ¼ 8.5, Har), 8.13–8.19 (m, 3H, Har), 8.24 (s, 1H, C–H), 8.45 (s, 1H, C–
H); MS (ESI) m/z 361.2 [M ꢀ H]ꢀ.
Compound
8 was obtained from compound 5 (900 mg,
4.28 mmol) and 5-bromo-2-furaldehyde (1.5 g, 8.56 mmol)
following the general procedure as a red solid (1.45 g, 92%). 1H NMR
(DMSO–d6, 250 MHz) d 2.13 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.64 (s, 3H,
CH3), 2.72 (s, 3H, CH3), 6.93 (d, 1H, J ¼ 3.7, Har), 6.97 (d, 1H, J ¼ 3.7,
Har), 7.10 (d, 1H, J ¼ 3.7, Har), 7.14 (s, 2H, NH2), 7.29 (s, 2H, NH2), 7.36
(d, 1H, J ¼ 3.7, Har), 7.40 (s, 1H, C–H), 7.57 (s, 1H, C–H); MS (ESI) m/z
368.0 [M þ H]þ.
4.10. (Z/E)-2-Amino-5-(1,3-benzodioxol-5-ylmethylene)-4,6-
dimethyl-5H-cyclopenta[b]pyridine-3,7-dicarbonitrile (16)
Compound 16 was obtained from compound
5 (105 mg,
0.5 mmol) and piperonal (150 mg, 1 mmol) following the general
procedure as a brown solid (120 mg, 70%). 1H NMR (DMSO–d6,
4.5. (Z/E)-2-Amino-4,6-dimethyl-5-(pyridin-4-ylmethylene)-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (11)
250 MHz) d 1.84 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.55 (s, 3H, CH3), 2.69
(s, 3H, CH3), 6.13 (s, 2H, CH2), 6.15 (s, 2H, CH2), 6.85–7.20 (m, 10H,
Har and 2 ꢃ NH2), 7.70 (s, 1H, C–H), 8.02 (s, 1H, C–H); MS (ESI) m/z
341.4 [M ꢀ H]ꢀ.
Compound 11 was obtained from compound
0.57 mmol) and 4-pyridinecarboxaldehyde (109 L, 1.14 mmol)
following the general procedure as a brown solid (70 mg, 41%). 1H
NMR (DMSO–d6, 250 MHz) 1.64 (s, 3H, CH3), 1.99 (s, 3H, CH3), 2.56
5 (120 mg,
m
d
4.11. (Z/E)-2-Amino-5-benzylidene-4,6-dimethyl-5H-
(s, 3H, CH3), 2.70 (s, 3H, CH3), 7.28 (s, 2H, NH2), 7.33–7.38 (m, 4H, NH2
and Har), 7.48–7.50 (m, 2H, Har), 7.70 (s, 1H, C–H), 8.00 (s, 1H, C–H),
8.67–8.68 (m, 4H, Har); MS (ESI) m/z 297.5 [M ꢀ H]ꢀ.
cyclopenta[b]pyridine-3,7-dicarbonitrile (17)
Compound 17 was obtained from compound
5 (105 mg,
0.5 mmol) and benzaldehyde (0.1 mL,1 mmol) following the general
4.6. (Z/E)-2-Amino-4,6-dimethyl-5-(pyridin-3-ylmethylene)-5H-
procedure as a yellow solid (149 mg, 70%). 1H NMR (DMSO–d6,
cyclopenta[b]pyridine-3,7-dicarbonitrile (12)
250 MHz) d 1.68 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.58 (s, 3H, CH3), 2.72
(s, 3H, CH3), 7.18 (s, 2H, NH2), 7.25 (s, 2H, NH2), 7.40–7.49 (m, 10H,
Har), 7.81 (s, 1H, H, C–H), 8.14 (s, 1H, C–H); MS (ESI) m/z 297.3
[M ꢀ H]ꢀ.
Compound 12 was obtained from compound
0.95 mmol) and 3-pyridinecarboxaldehyde (179
5
(200 mg,
m
L, 1.9 mmol)

900
S. Kolb et al. / European Journal of Medicinal Chemistry 45 (2010) 896–901
4.12. (Z/E)-2-Amino-4,6-dimethyl-5-(4-methylbenzylidene)-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (18)
2.56 (s, 3H, CH3), 2.70 (s, 3H, CH3), 6.87–7.04 (m, 6H, Har), 7.13–7.18
(m, 3H, Har and NH2), 7.28–7.34 (m, 3H, Har and NH2), 7.69 (s, 1H,
C–H), 7.99 (s, 1H, C–H), 10.10 (s, 1H, OH), 10.27 (s, 1H, OH); MS (ESI)
m/z 315.4 [M þ H]þ.
Compound 18 was obtained from compound
0.95 mmol) and p-tolualdehyde following the general procedure as
a brown solid (92 mg, 31%). 1H NMR (DMSO–d6, 250 MHz)
1.73 (s,
5 (200 mg,
d
4.18. (Z/E)-2-Amino-5-(4-bromobenzylidene)-4,6-dimethyl-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (24)
3H, CH3), 2.09 (s, 3H, CH3), 2.41 (s, 6H, 2 ꢃ CH3), 2.56 (s, 3H, CH3),
2.70 (s, 3H, CH3), 7.15 (s, 2H, NH2), 7.22 (s, 2H, NH2), 7.28–7.32 (m,
5H, Har), 7.40–7.43 (m, 3H, Har), 7.76 (s, 1H, C–H), 8.10 (s, 1H, C–H);
MS (ESI) m/z 313.4 [M þ H]þ.
Compound 24 was obtained from compound
5 (120 mg,
0.57 mmol) and 4-bromobenzaldehyde (211 mg, 1.14 mmol)
following the general procedure as a brown solid (93 mg, 44%). 1H
4.13. (Z/E)-2-Amino-5-(biphenyl-4-ylmethylene)-4,6-dimethyl-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (19)
NMR (DMSO–d6, 250 MHz) d 1.73 (s, 3H, CH3), 2.05 (s, 3H, CH3), 2.56
(s, 3H, CH3), 2.70 (s, 3H, CH3), 7.21 (s, 2H, NH2), 7.29 (s, 2H, NH2),
7.35 (d, 2H, J ¼ 8.1, Har), 7.47 (d, 2H, J ¼ 8.1, Har), 7.67–7.72 (m, 5H,
Har and C–H), 8.04 (s, 1H, C–H); MS (ESI) m/z 378.1 [M þ H]þ.
Compound 19 was obtained from compound
5 (200 mg,
0.95 mmol) and 4-biphenylcarboxaldehyde (347 mg, 1.9 mmol)
following the general procedure as an orange solid (292 mg, 82%).1H
4.19. (Z/E)-2-Amino-5-(3-bromobenzylidene)-4,6-dimethyl-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (25)
NMR (DMSO–d6, 250 MHz) d 1.80 (s, 3H, CH3), 2.15 (s, 3H, CH3), 2.59
(s, 3H, CH3), 2.73 (s, 3H, CH3), 7.19 (s, 2H, NH2), 7.27 (s, 2H, NH2),
7.39–7.53 (m, 8H, Har), 7.62 (d, 2H, J ¼ 8.3, Har), 7.77–7.86 (m, 9H, Har
and C–H), 8.16 (s, 1H, C–H); MS (ESI) m/z 373.2 [M ꢀ H]ꢀ.
Compound 25 was obtained from compound
0.57 mmol) and 3-bromobenzaldehyde (133 L, 1.14 mmol)
following the general procedure as a yellow solid (48 mg, 23%). 1H
NMR (DMSO–d6, 250 MHz) 1.70 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.55
5 (120 mg,
m
4.14. (Z/E)-2-Amino-4,6-dimethyl-5-[4-
(methylsulfanyl)benzylidene]-5H-cyclopenta[b]pyridine-3,7-
dicarbonitrile (20)
d
(s, 3H, CH3), 2.70 (s, 3H, CH3), 7.22 (s, 2H, NH2), 7.32–7.50 (m, 6H, NH2
and Har), 7.64–7.67 (m, 3H, Har), 7.75–7.76 (s, 2H, Har and C–H), 8.06
(m, 1H, C–H); MS (ESI) m/z 378.1 [M þ H]þ.
Compound 20 was obtained from compound
0.95 mmol) and 4-(methylthio)benzaldehyde (253 L, 1.90 mmol)
following the general procedure as a red solid (252 mg, 77%). 1H
NMR (DMSO–d6, 250 MHz) 1.80 (s, 3H, CH3), 2.13 (s, 3H, CH3),
5 (200 mg,
m
4.20. (E)-2-Amino-5-(2-bromobenzylidene)-4,6-dimethyl-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (26)
d
2.51–2.56 (m, 9H, 3x CH3), 2.71 (s, 3H, CH3), 7.15 (s, 2H, NH2), 7.23
(s, 2H, NH2), 7.33–7.37 (m, 6H, Har), 7.48 (d, 2H, J ¼ 8.2, Har), 7.74
(s, 1H, C–H), 8.07 (s, 1H, C–H); MS (ESI) m/z 343.7 [M ꢀ H]ꢀ.
Compound 26 was obtained from compound
0.57 mmol) and 2-bromobenzaldehyde (133 L, 1.14 mmol)
following the general procedure as a yellow solid (85 mg, 39%). 1H
NMR (DMSO–d6, 250 MHz) 1.94 (s, 3H, CH3), 2.71 (s, 3H, CH3), 7.27
5 (120 mg,
m
4.15. (Z/E)-2-Amino-5-[4-(dimethylamino)benzylidene]-4,6-
dimethyl-5H-cyclopenta[b]pyridine-3,7-dicarbonitrile (21)
d
(s, 2H, NH2), 7.42–7.53 (m, 4H, Har), 7.79–7.82 (m, 1H, Har), 7.89
(s, 1H, C–H); MS (ESI) m/z 378.1 [M þ H]þ.
Compound 21 was obtained from compound
5 (200 mg,
0.95 mmol) and 4-dimethylaminobenzaldehyde (284 mg, 1.9 mmol)
4.21. (Z/E)-2-Amino-4,6-dimethyl-5-(4-nitrobenzylidene)-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (27)
following the general procedure as a dark red solid (282 mg, 87%).1H
NMR (DMSO–d6, 250 MHz) d 2.09 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.64
(s, 3H, CH3), 2.80 (s, 3H, CH3), 3.15 (s,12H, 4 ꢃ CH3), 6.89–6.93 (m, 4H,
Har), 7.02 (s, 2H, NH2), 7.11 (s, 2H, NH2), 7.41–7.45 (d, 2H, J ¼ 10, Har),
7.53–7.57 (d, 2H, J ¼ 10, Har), 7.80 (s, 1H, C–H), 8.16 (s, 1H, C–H); MS
(ESI) m/z 340.4 [M ꢀ H]ꢀ.
Compound 27 was obtained from compound
5 (200 mg,
0.95 mmol) and 4-nitrobenzaldehyde (287 mg, 1.9 mmol) following
the general procedure as a red solid (217 mg, 66%). (E)-2-Amino-4,6-
dimethyl-5-(4-nitrobenzylidene)-5H-cyclopenta[b]pyridine-
3,7-dicarbonitrile (27-E). 1H NMR (DMSO–d6, 250 MHz)
d 2.00
4.16. (Z/E)-2-Amino-5-(3-hydroxybenzylidene)-4,6-dimethyl-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (22)
(s, 3H, CH3), 2.71 (s, 3H, CH3), 7.40 (s, 2H, NH2), 7.79–7.82 (m, 3H, Har
and C–H), 8.33 (d, 2H, J ¼ 10, Har). (Z)-2-Amino-4,6-dimethyl-5-
(4-nitrobenzylidene)-5H-cyclopenta[b]pyridine-3,7-dicarbonitrile
Compound 22 was obtained from compound
5 (120 mg,
(27-Z). 1H NMR (DMSO–d6, 250 MHz)
d 1.67 (s, 3H, CH3), 2.58 (s, 3H,
0.57 mmol) and 3-hydroxybenzaldehyde (139 mg, 1.14 mmol)
CH3), 7.29 (s, 2H, NH2), 7.66 (d, 2H, J ¼ 10, Har), 8.11 (s,1H, C–H), 8.33
following the general procedure as a yellow solid (123 mg, 69%). 1H
(d, 2H, J ¼ 10, Har); MS (ESI) m/z 342.1 [M ꢀ H]ꢀ.
NMR (DMSO–d6, 250 MHz) d 1.75 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.56
(s, 3H, CH3), 2.70 (s, 3H, CH3), 6.77 (s,1H, Har), 6.84–6.91 (m, 5H, Har),
7.16 (s, 2H, NH2), 7.24–7.33 (m, 4H, Har and NH2), 7.72 (s, 1H, C–H),
8.06 (s,1H, C–H), 9.67 (s,1H, OH), 9.70 (s,1H, OH); MS (ESI) m/z 315.4
[M þ H]þ.
4.22. (Z/E)-2-Amino-4,6-dimethyl-5-(3-nitrobenzylidene)-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (28)
Compound 28 was obtained from compound
5 (200 mg,
4.17. (Z/E)-2-Amino-5-(2-hydroxybenzylidene)-4,6-dimethyl-5H-
cyclopenta[b]pyridine-3,7-dicarbonitrile (23)
0.95 mmol) and 3-nitrobenzaldehyde (287 mg,1.9 mmol) following
the general procedure as a red solid (218 mg, 67%). 1H NMR (DMSO–
d6, 250 MHz)
d 1.61 (s, 3H, CH3), 1.93 (s, 3H, CH3), 2.50 (s, 3H, CH3),
Compound 23 was obtained from compound
0.57 mmol) and 2-hydroxybenzaldehyde (122 L, 1.14 mmol)
following the general procedure as an orange solid (154 mg, 86%).
1H NMR (DMSO–d6, 250 MHz)
1.77 (s, 3H, CH3), 2.15 (s, 3H, CH3),
5
(120 mg,
2.65 (s, 3H, CH3), 7.19 (s, 2H, NH2), 7.30 (s, 2H, NH2), 7.66–7.73 (m,
3H, Har), 7.78 (s, 1H, C–H), 7.89 (d, 1H, J ¼ 7.5, Har), 8.07 (s, 1H, C–H),
8.18–8.25 (m, 3H, Har), 8.31 (s, 1H, Har); MS (ESI) m/z 342.0
[M ꢀ H]ꢀ.
m
d

S. Kolb et al. / European Journal of Medicinal Chemistry 45 (2010) 896–901
901
4.23. (Z/E)-2-Amino-5-[4-(4-chlorophenoxy)benzylidene]-4,6–
dimethyl-5H-cyclopenta[b]pyridine-3,7-dicarbonitrile (29)
430 nm with a Bio-Rad microplate reader. The results are expressed
as the mean of three independent experiments with three deter-
minations per tested concentration and per experiment. For each
compound, the IC50 value was determined from a sigmoidal dose-
response using GraphPad Prism (GraphPad Software, San
Diego, CA).
Compound 29 was obtained from compound
5 (200 mg,
0.95 mmol) and 4-(4-chlorophenoxy)benzaldehyde (443 mg,
1.9 mmol) following the general procedure as a red solid (274 mg,
68%). 1H NMR (DMSO–d6, 250 MHz)
d 1.82 (s, 3H, CH3), 2.14 (s, 3H,
CH3), 2.57 (s, 3H, CH3), 2.71 (s, 3H, CH3), 7.09–7.23 (m, 12H, Har and
2 ꢃ NH2), 7.44–7.58 (m, 8H, Har), 7.76 (s, 1H, C–H), 8.10 (s, 1H, C–H);
MS (ESI) m/z 423.9 [M ꢀ H]ꢀ.
Acknowledgments
We are grateful to Dr Wang-Qing Liu for mass spectra recording.
This research is supported by La Ligue Nationale contre le Cancer:
4.24. Cell culture and chemicals
´
´
Equipe Labellisee 2006 (U648) and Equipe Labellisee 2008
(UMR 5088).
Human cancer cell line HeLa was obtained from Aptanomics
(Lyon, France). A2058 cells and CRL-2796 cells were obtained from
ATCC (Rockville, MD, USA). Cells were cultured at 37 ꢁC in Dulbecco’s
minimum essential medium complemented with 10% fetal bovine
serum and 100 units/ml penicillin/streptomycin in a humidified
atmosphere of 5% CO2. All compounds were solubilized in dimethyl
sulfoxide so that the DMSO final concentration was <1%. The tetra-
zolium salt WST-1 was purchased from Roche Diagnostics (Man-
nheim, Germany).
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4.25. Cytotoxic assay
The inhibition of cell proliferation was determined using
a colorimetric assay based on the cleavage of the WST-1 tetrazo-
lium salt by mitochondrial dehydrogenases in viable cells, leading
to formazan formation. At day 0, HeLa cells, A2058 cells and CRL-
2796 cells were plated in 96-well culture plates with 95
medium/well at 3000, 4000 and 5000 cells/well respectively. At
day 1, cells were treated with 5 L of increasing concentrations of
drug. At day 6 (HeLa), 5 (A2058) or 4 (CRL-2796), after addition of
10
L of WST-1 per well, cells were incubated 2 h at 37 ꢁC in
a humidified atmosphere of 5% CO2. Absorbance was measured at
mL of
m
m