4-Bromo-2-(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b) inhibits Na+/K+-ATPase and Ras oncogene activity in cancer cells

Article abstract of DOI:10.1016/j.ejmech.2013.01.046

The in vitro growth inhibitory activity of 26 thiazoles (including 4-halogeno-2,5-disubtituted-1,3-thiazoles) and 5 thienothiazoles was assessed on a panel of 6 human cancer cell lines, including glioma cell lines. (4-Chloro-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12a) and (4-bromo-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12b) displayed ～10 times greater in vitro growth inhibitory activity than perillyl alcohol (POH), which therapeutically benefits glioma patients through the inhibition of both alpha-1 Na⁺/K⁺-ATPase (NAK) and Ras oncogene activity. The in vitro cytostatic activities (as revealed by quantitative videomicroscopy) displayed by 12a and 12b were independent of the intrinsic resistance to pro-apoptotic stimuli associated with cancer cells. Compounds 12a and 12b displayed relatively similar inhibitory activities on purified guinea pig brain preparations that mainly express NAK alpha-2 and alpha-3 subunits, whereas only compound 12b was efficacious against purified guinea pig kidney preparations that mainly express the NAK alpha-1 subunit, which is also expressed in gliomas, melanomas and non-small-cell lung cancers NSCLCs.

Full text of DOI:10.1016/j.ejmech.2013.01.046

European Journal of Medicinal Chemistry 63 (2013) 213e223
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Bromo-2-(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b)
inhibits Naþ/Kþ-ATPase and Ras oncogene activity in cancer cells
Florence Lefranc a 1, Zhanjie Xu e 1, Patricia Burth f, Véronique Mathieu b,
Germain Revelant e, Mauro Velho de Castro Faria g, Caroline Noyon c,
Diogo Gomes Garcia f, Damien Dufour c, Céline Bruyère b,
,
,
,
d
Cassiano Felippe Gonçalves-de-Albuquerque f, Pierre Van Antwerpen c
,
,
,
, ,1
*
Bernard Rogister h, Stéphanie Hesse e, Gilbert Kirsch e 1, Robert Kiss b
**
a Service de Neurochirurgie, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium
b Laboratoire de Toxicologie, Université Libre de Bruxelles, Brussels, Belgium
c Laboratoire de Chimie Pharmaceutique Organique, Université Libre de Bruxelles, Brussels, Belgium
d Plate-Forme Analytique, Faculté de Pharmacie, Université Libre de Bruxelles, Brussels, Belgium
e Université de Lorraine, Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Institut Jean Barriol, FR CNRS 2843,
1 Boulevard Arago, 57070 METZ, France
f Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Outeiro
de São João Batista, s/n Campus do Valonguinho, Centro-Niteroi, Rio de Janeiro, Brazil
g Departamento de Medicina Interna, Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
h Laboratoire de Neurobiologie du Développement, GIGA-Neurosciences, Université de Liège, Sart Tilman, B-4000 Liège, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The in vitro growth inhibitory activity of 26 thiazoles (including 4-halogeno-2,5-disubtituted-1,3-
thiazoles) and 5 thienothiazoles was assessed on a panel of 6 human cancer cell lines, including gli-
oma cell lines. (4-Chloro-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12a) and (4-bromo-2-
(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12b) displayed w10 times greater in vitro growth
inhibitory activity than perillyl alcohol (POH), which therapeutically benefits glioma patients through the
inhibition of both alpha-1 Naþ/Kþ-ATPase (NAK) and Ras oncogene activity. The in vitro cytostatic ac-
tivities (as revealed by quantitative videomicroscopy) displayed by 12a and 12b were independent of the
intrinsic resistance to pro-apoptotic stimuli associated with cancer cells. Compounds 12a and 12b dis-
played relatively similar inhibitory activities on purified guinea pig brain preparations that mainly ex-
press NAK alpha-2 and alpha-3 subunits, whereas only compound 12b was efficacious against purified
guinea pig kidney preparations that mainly express the NAK alpha-1 subunit, which is also expressed in
gliomas, melanomas and non-small-cell lung cancers NSCLCs.
Received 9 July 2012
Received in revised form
16 January 2013
Accepted 22 January 2013
Available online 16 February 2013
Keywords:
Thiazoles
Thienothiazoles
Perillyl alcohol
In vitro
Naþ/Kþ-ATPase
Ras oncogene
Apoptosis resistance
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
intrinsically resistant to pro-apoptotic stimuli [4e6]. Thus, these
cancers are also resistant to conventional radiotherapy and chemo-
Gliomas, especially glioblastomas [1], melanomas [2] and NSCLCs
[3] are associated with dismal prognoses because they are
therapy [7,8]. In addition, they develop the multidrug resistance
(MDR) phenotype during chronic chemotherapy [9e11]. Treatments
whose main effects do not relate to the activation of apoptosis are of
particular interest to combat these cancers. For example, the alky-
lating drug temozolomide, which contributes significant therapeutic
benefits to glioblastoma patients [12], induces sustained and irre-
versible pro-autophagic effects in glioma cells [13].
Innovative approaches to combat several types of cancers
associated with dismal prognoses involve the inhibition of various
types of ion channels, and we pay particular attention to the alpha-
1 subunit of the Naþ/Kþ-ATPase (NAK) in our group [14], which is
significantly overexpressed in gliomas [15], melanomas [16], and
Abbreviations: AECs, alveolar epithelial cells; ATCC, American Type Culture
Collection; DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen;
ECACC, European Collection of Cell Culture; FGF-10, fibroblast growth factor 10;
GBM, glioblastoma; GGR, global growth ratio; Kþ-pnppase, Kþ activated-p-nitro-
phenylphosphatase; MDR, multidrug resistance; MTT, 3-(4,5)-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; NAK, Naþ/Kþ-ATPase; NSCLC, non-small-cell
lung carcinoma; POH, perillyl alcohol; QVM, quantitative videomicroscopy.
* Corresponding author. Tel.: þ32 477622083.
** Corresponding author. Tel.: þ33 0387315295.
E-mail addresses: kirsch@univ-metz.fr (G. Kirsch), rkiss@ulb.ac.be (R. Kiss).
Authors contributed equally to this article.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.046

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F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
NSCLCs [17]. Impairing NAK alpha-1 subunit activity kills cancer
cells, and the effect is more detrimental to cancer cells than to
normal cells [15,16]. Such impairment induces irreversible auto-
phagy in glioma cells and lysosomal membrane permeabilization-
related cell death in NSCLC cells [14]. In addition, impairing NAK
alpha-1 activity represents an efficient approach to killing MDR
cancer cells [18].
we first tried these classical conditions on several 4-amino-1,3-
thiazoles, but the reactions either failed or the yields were rather
low. In some cases (e.g., when a cyano group was present at posi-
tion 5), we obtained some chlorothiazolotriazine derivatives, which
resulted from an intramolecular cyclization [31]. We then turned
our attention to Doyle’s method [32], which we have used suc-
cessfully to synthesize a thiophene series [33]. This method also
works on 2-amino-1,3-thiazoles [34]. 4-Chloro- and 4-bromo-1,3-
thiazoles were obtained via this substitutive deamination proto-
col with moderate to good yields (Scheme 1, Table 1). Generally,
bromo derivatives (1be13b) were obtained in higher yields than
chloro derivatives (1ae13a). This feature could relate to the greater
reactivity of CuBr2 compared to CuCl2 as emphasized in Ref. [32]
(and more precisely in Note 23 of Ref. [32]), in which the authors
report that in a competition experiment at 65 ꢀC between CuCl2
(7.0 mmol) and CuBr2 (7.0 mmol) that employed p-nitroaniline
(10 mmol) and tert-butyl nitrite (15 mmol), the recovered yield
of p-bromonitrobenzene (57%) was twice that of p-chloroni-
trobenzene (29%).
The proximity of the halogen and the electron-withdrawing
group then allowed several transformations, including access to
fused thiazoles. Thieno[2,3-d]thiazoles could be synthesized start-
ing either from thiophenes or from thiazoles. For example, 4-
bromo-5-nitrothiophene has been used as starting material and
converted via thiocyanate to the corresponding thienothiazoles
[35]. Thienothiazole-2-carboxamides have been synthesized start-
ing from aminothiophenes [36]. The construction of a thiophene
ring onto thiazole has also been described. Often, this ring forma-
tion involves a reaction on a chloroformylthiazole; these de-
rivatives have been prepared starting from thiohydantoin [37] or
thiazolidine-2,4-dione [38,39]. Here, we reported the condensation
of methylthioglycolate on 4-halogenothiazoles (1a,b and 2a,b) and
the subsequent cyclization with sodium methylate to afford the
corresponding thienothiazoles (14 and 15) in moderate to good
yields (Scheme 2). We then took advantage of the good leaving
group properties of the methylsulfanyl group and performed a
SNAr reaction with secondary amines. Compound 14 was refluxed
The most potent NAK ligands include cardiotonic steroids (car-
denolides and bufadienolides), which are useful to treat heart fail-
ure, but they cannot be used safely as anticancer agents due to their
narrow therapeutic indices [14]. In contrast, NAK inhibitors such as
perillyl alcohol (POH) [19] are much less potent than cardiotonic
steroids, but they nevertheless contribute significant therapeutic
benefits to glioma patients [20,21]. In our group, we have adopted a
pharmacological strategy that consists of analyzing the in vitro
growth inhibitory effects of original compounds in a panel of murine
and cancer cell lines that include apoptosis-sensitive versus
apoptosis-resistant models. Once we have identified compounds
that are active against apoptosis-resistant cancer cells, especially
glioma apoptosis-resistant ones, we investigate whether these
active compounds display anti-NAK activity. Adopting this strategy
in the current study, we identified compound 12b, which decreased
the growth of apoptosis-resistant glioma cells, a feature that seems
to partly relate to anti-NAK activity. Indeed, the present study de-
scribes the design and characterization of the in vitro growth
inhibitory activity (by means of an MTT colorimetric assay [15e17])
of 31 novel compounds; 26 thiazoles (including 4-halogeno-2,5-
disubstituted-1,3-thiazoles) and
5 thienothiazoles were tested
against a panel of 6 human cancer cell lines, which included 3 cell
lines that display sensitivity to pro-apoptotic stimuli (the MCF-7
breast [22], the PC-3 prostate [22], and the Hs683 oligoden-
droglioma [23] cancer cell lines) and 3 cell lines that display various
levels of resistance to pro-apoptotic stimuli (the U373 glioblastoma
[23], the SKMEL-28 melanoma [24] and the A549 NSCLC [25] cell
lines). Two compounds (12a and 12b) displayed IC50 in vitro growth
inhibitory concentrations < 100 mM against all 6 cancer cell lines,
and they were further analyzed in terms of anti-NAK activity. The
most potent NAK inhibitor, 12b, was then assayed for anti-Ras
oncogene activity because POH, in addition to its anti-NAK activity
[19], also inhibits Ras activity [26,27], and Ras represents a prom-
ising anticancer target for many cancer types [28], including gliomas
[29]. Lastly, computer-assisted phase-contrast microscopy (quanti-
tative videomicroscopy [15,17,23]) was used to determine whether
12b induced cytotoxic or cytostatic effects on cancer cells.
Table 1
Synthesis of 4-halogeno-2,5-disubstituted-1,3-thiazoles.
Structure
EWGa X ¼ Cl Yield (%) X ¼ Br Yield (%)
X
N
2. Chemistry
Ac
1a
80
78
31
83
1b
2b
3b
4b
85
87
68
81
CO2Et 2a
Bz
CN
S
EWG
X
3a
4a
S
Recently, we described a one-pot, four-step procedure to syn-
thesize 4-amino-2,5-disusbtituted-1,3-thiazoles (Scheme 1) [30].
Here, we report the transformation of these compounds to 4-
halogeno-2,5-disubstituted-1,3-thiazoles and the synthesis of
thieno[2,3-d]thiazoles.
N
Ac
5a
42
43
61
52
5b
6b
7b
8b
55
40
76
76
N
CO2Et 6a
Bz
CN
EWG
S
O
7a
8a
The Sandmeyer reaction is one of the most commonly used
procedures to convert a primary amine into a halogen. Therefore,
X
X
N
CO2Et 9a
Bz
CN
54
66
48
9b
10b
11b
64
71
91
10a
11a
N
EWG
EWG
S
N
Bz
CN
12a
13a
59
50
12b
13b
93
76
N
S
Scheme 1. Synthesis of 4-halogeno-2,5-disubstituted-1,3-thiazoles. Conditions and
reagents: (a) (i) Na2S$9H2O, DMF, 70 ꢀC, 2 h (ii) XeCH2eEWG, 70 ꢀC, 2 h (iii) K2CO3,
70 ꢀC, 1 h (iv) R1R2NH; (b) tBuONO, CuX2, CH3CN, reflux.
a
EWG ¼ electron withdrawing group.

F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
215
Scheme 2. Synthesis of thieno[2,3-d]thiazoles. Conditions and reagents: (a) HSCH2CO2Me, MeONa; (b) R1R2NH, reflux.
with excess amine overnight and led to compounds 16e18 in 39%e
68% yields (Scheme 2).
3.4. Evaluation of the cytotoxic versus cytostatic nature of the
compounds in this study
As indicated above, only 2 compounds, 12a and 12b, displayed
IC50 growth inhibitory concentrations < 100 M for each of the 6
3. Results
m
cancer cell lines analyzed (Table 2). These 2 compounds were thus
analyzed by quantitative videomicroscopy, which revealed that
their effect is cytostatic rather than cytotoxic in Hs683 glioma cells,
as illustrated by the morphological analyzes in Fig. 2. Indeed, Fig. 3
indicates that 12a (Fig. 3A) and 12b (Fig. 3B) delayed Hs683 pop-
ulation growth: calculations of the global growth ratio (GGR; see
3.1. Determination of the in vitro growth inhibitory IC50
concentration
Of the 31 compounds synthesized for this study, 16 displayed an
IC50 in vitro growth inhibitory concentration < 100
mM in at least
one of the 6 cancer cell lines analyzed (Table 2). The remaining 15
compounds displayed IC50 concentrations > 100 mM in each of the 6
cell lines analyzed (data not shown).
the legend to Fig. 3) revealed that at 40 mM 12a (Fig. 3C) and 12b
(Fig. 3D) decreased the growth of Hs683 cell populations by w40%.
This growth inhibition began on the 48th hour of cell culture and
remained until the 72nd hour of culture (Fig. 3C, D). Compounds
12a and 12b both exhibited GGR indices of 0.6 (see the legend to
POHdisplayedIC50 inhibitoryconcentrations> 1000
the 6 cell lines analyzed (Table 2). In fact, of the 31 compounds syn-
thesized, only 12a and 12b displayed IC50 concentrations < 100
mMineachof
mM
Fig. 3) at 40
mM; this value was in agreement with the IC50 con-
for each of the 6 cell lines analyzed (Table 2). In addition,12a and 12b
displayed similar in vitro growth inhibition toward not only those
cancer cell lines with various levels of resistance to pro-apoptotic
stimuli (i.e., U373 glioblastoma [23], SKMEL-28 melanoma [24] and
A549 NSCLC [25] cells) but also those lines that are sensitive to pro-
apoptotic stimuli (i.e., MCF-7 breast [22] and PC-3 prostate [22]
cancer, and Hs683 oligodendroglioma [23] cells; Table 2).
centrations (w40
mM) obtained from the MTT colorimetric assay on
Hs683 glioma cells (Table 2).
3.5. Inhibition of the Naþ/Kþ-ATPase activity
Compounds 12a and 12b displayed inhibitory effects on the
growth of various cancer cell lines when compared to POH
(Table 2). NAK inhibitory activity was analyzed at a concentration of
1 mM, i.e., the concentration used to demonstrate POH-related
anti-NAK activity [24]. Compounds 12a and 12b displayed inhibi-
tory activity > 50% at 1 mM on purified guinea pig brain NAK
(Fig. 4A), whereas only compound 12b displayed inhibitory
activity > 50% at 1 mM on purified guinea pig kidney NAK (Fig. 4B).
As a benchmark, Fig. 4A, B also include the inhibition obtained with
1 mM POH.
As shown in Fig. 4C, compound 12b inhibited purified kidney
and brain NAK with some differences. The NAK IC50 concentration
for 12b was approximately 0.25 mM for the purified brain enzyme
(including mainly a2 and a3 NAK isoforms) and approximately
0.35 mM for the whole brain homogenate (Fig. 4C). However, 12b
displayed a lower inhibitory potency for the kidney a1 isoform. In
this case, the 12b-related anti-NAK IC50 concentration was
approximately 0.55 mM for both the purified and the homogenate
kidney enzymes (Fig. 4D). Fig. 4C and D also show that the ouabain-
insensitive MgþþATPases in the brain and kidney tissue homoge-
nates were not affected within the range of drug concentrations
tested.
3.2. Evaluation of the in vitro growth inhibitory IC50 concentration
of 12a and 12b in normal astrocytes
Primocultures of mouse normal astrocytes were established as
validated previously [40] and detailed in the Materials and
Methods. Compounds 12a and 12b were thus assayed by means of
the MTT colorimetric assay on normal mouse astrocytes at 0 (con-
trol), 30, 60 and 300
50% (data not shown). At 300
m
M without reaching growth inhibition below
M, the in vitro growth inhibition
M, respectively.
m
induced by 12a and 12b were 86 ꢁ 4 and 68 ꢁ 6
m
Compound 12a and 12b thus displayed bioselectivity indices > 5 in
terms of in vitro growth inhibition between normal and tumor
astrocytes.
3.3. In vitro stability determination for compounds 12a and 12b
The stability of compounds 12a and 12b was determined as
detailed in the Materials and Methods. Fig. 1 shows that compound
12a remained stable over time (72 h) in both water and RPMI cell
culture media, while compound 12b degraded over time. A com-
parison of the data in Table 1 with those in Fig. 1 suggests that the
degradation process observed for compound 12b compared to
compound 12a did not impair its in vitro growth inhibitory activity
in various cancer cell lines.
Kinetic studies were performed to evaluate the influence of a
fixed concentration of compound 12b on the activation of brain- or
kidney-purified NAK by Naþ, Kþ and ATP (Fig. 5); the results
revealed that 12b interacts by non-competitive inhibition with
both ions. Double reciprocal plots also demonstrated that

216
F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
Table 2
Characterization of the in vitro growth inhibitory activity of 4-halogeno-2,5-disubstituted-1,3-thiazoles and thieno[2,3-d]thiazoles on a panel of six human cancer cell lines.
Compounda
Chemical structure
Growth inhibitory IC50
interestb
(mM) concentration after culturing the cancer cells for 72 h with the compound of
MCF-7
A549
PC-3
*
U373
*
Hs683
*
SKMEL-28
*
Mean ꢁ SEM
>99
3a
3b
91
*
67
88
97
*
*
89
83
*
*
*
*
>92
>95
5a
*
7a
7b
70
51
78
84
*
*
92
*
*
*
>90
>88
*
93
9a
79
63
61
50
78
*
*
54
44
82
98
>76
>73
10a
82
10b
69
97
*
*
*
*
>94
11a
12a
99
56
*
*
*
*
*
>99
41
69
96
36
91
65 ꢁ 10
12b
35
48
62
78
38
91
59 ꢁ 9
13b
14
77
69
93
78
*
*
84
79
*
*
>92
90
92
**

F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
217
Table 2 (continued )
Compounda
Chemical structure
Growth inhibitory IC50
interestb
(mM) concentration after culturing the cancer cells for 72 h with the compound of
MCF-7
A549
PC-3
*
U373
*
Hs683
92
SKMEL-28
Mean ꢁ SEM
16
17
78
96
*
**
**
56
86
*
89
91
*
18
90
**
*
93
**
*
*
*
**
POH
**
**
**
**
>1000
*>100
m
M; **>1000
Only those compounds with an IC50 concentration < 100
concentrations > 100 M on each of the six cell lines analyzed are not reported in Table 2. The IC50 concentrations of perillyl alcohol (POH), a reference compound in the
mM.
a
m
M on at least one of the six cancer cell lines are reported in Table 2. Thus, those compounds that displayed IC50
m
current study, are reported in Table 2.
b
The in vitro growth inhibitory IC50 concentrations were determined using the MTT colorimetric assay. The human cell lines include the MCF-7 breast cancer (DSMZ code
ACC115), the A549 NSCLC (DSMZ code ACC107), the PC-3 prostate cancer (DSMZ code ACC465), the U373 glioblastoma (ECACC code 08061901), the Hs683 oligodendroglioma
(ATCC code HTB-138) and the SKMEL-28 melanoma (ATCC code HTB-72) cell lines.
compound 12b acted on purified NAK through an uncompetitive
inhibition toward the substrate ATP, regardless of the enzyme
origin.
activity on the a1 NAK subunit (Fig. 4B). As detailed in the
Discussion, NAK and Ras interplay to cross-regulate each other’s
activity. We therefore analyzed the anti-Ras activity of 12b and
used POH as a dual reference compound for anti-Ras activity
Kþ-activated-p-nitrophenylphosphatase (Kþ-pnppase) activity,
which measures phase II of the catalytic NAK cycle, was not
inhibited by 12b in the kidney purified preparations, whereas the
brain enzyme was affected (Fig. 6).
[26,27] and anti-
show that at 40
a
m
1 NAK subunit activity [19]. The data in Fig. 7
M (the MTT colorimetric assay IC50 concentra-
tion; Table 2) 12b reduced the Ras activity of Hs683 glioma cells
by > 50%; this effect was more rapid and efficient than the anti-Ras
activity of 2 mM POH.
3.6. Compound 12b inhibits Ras oncogene activity
Compound 12b displays in vitro growth inhibitory activity with
an IC50 < 100 mM on each of the 6 cancer cell lines tested, an effect
4. Discussion
that was independent of the cell lines’ resistance to pro-apoptotic
stimuli (Table 2). In addition, 12b also displayed inhibitory
Targeting the ion channels overexpressed in chemoresistant
cancers is a new anti-cancer strategy [50]. One promising target is
the
[15], melanomas [16], NSCLCs [17] and other cancer types [14]. The
most potent NAK subunit inhibitors are related to cardiotonic
a1 NAK channel subunit [14], which is overexpressed in gliomas
a
steroids, which are useful for treating heart failure but have narrow
therapeutic indices [14]. Therefore, these compounds cannot be
used safely as anticancer agents, even if the potential of these
compounds as anticancer drugs [41e43], including drugs for MDR
cancers [18], are heavily emphasized. Non-cardiotonic, steroid-
related compounds also inhibit NAK activity, although the effect is
less pronounced than for cardiotonic steroids [14]. Non-cardiotonic,
steroid-related drugs include coumestans, iantherans, gossypol,
aplysiallene, kaur-16-en-19-oic acid derivatives and dienome A.
Among these drugs, in vitro anticancer activity has already been
demonstrated for gossypol, kaur-16-en-19-oic acid derivatives and
dienome A (see Ref. [14] for review). More recently, anti-NAK ac-
tivity has been reported for POH [19], a monoterpene found in the
essential oils of lavender, peppermint, spearmint, sage, cherries,
cranberries, lemongrass, and wild bergamot [44]. POH also displays
Fig. 1. Stability analyzes of compounds 12a and 12b over 72 h in water versus RPMI
cell culture medium.

218
F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
Fig. 2. Morphological illustrations (200ꢂ magnification) of the growth patterns of human Hs683 glioma cells cultured for 72 h in the absence (control) or the presence of either
40 M 12a or 40 M 12b. The morphological images were recorded by computer-assisted phase-contrast microscopy (quantitative videomicroscopy).
m
m
Fig. 3. Based on the quantitative videomicroscopy-related morphological images (see Fig. 2), we calculated the global growth rates of Hs683 glioma cells left untreated (control) or
treated with either 40 M 12a (A) or 40 M 12b (B) for 72 h. One image was digitized every 4 min, so a total of 1080 digitized images were recorded for each experimental condition;
m
m
each condition was performed in triplicate. All videomicroscopy experiments were performed in parallel; thus the controls in Fig. 3A and B are identical. The global growth rates
therefore correspond to the ratio of the mean number of cells present in the 1080th image to the number of cells present in the first image (at 0 h). We divided the ratios obtained
from the 12a- (C) or 12b- (D) treated experiments by the ratio obtained in the control, enabling the global growth ratio (GGR) index to be calculated. The GGR value of 0.6 in Figs C
and D means that 60% of Hs683 glioma cells grew in the treated experiment compared to the control over a 72-hr observation period.

F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
219
Fig. 4. The inhibition of purified brain (A) and kidney (B) Naþ/Kþ-ATPase by 1 mM of compounds 12a, 12b and POH. Each bar indicates the mean ꢁ SEM of 5 different experiments.
(C) Purified brain Naþ/Kþ-ATPase, brain homogenate Naþ/Kþ-ATPase and brain homogenate Mgþþ ATPase inhibition by 12b. (D) Purified kidney Naþ/Kþ-ATPase, kidney homogenate
Naþ/Kþ-ATPase and kidney homogenate Mgþþ ATPase inhibition by 12b. Each point in C and D is the mean ꢁ SEM of 3 different experiments.
anti-Ras activity [26,27], and, as explained below, NAK and Ras
activity are closely connected.
(Table 2). Compounds 12a and 12b displayed relatively similar
inhibitory activities on purified guinea pig brain preparations
In the present study, we have focused on distinct types of thi-
azoles. Among those tested, (4-chloro-2-(piperidin-1-yl)thiazol-5-
yl)(phenyl)methanone (12a) and (4-bromo-2-(piperidin-1-yl)thia-
zol-5-yl)(phenyl)methanone (12b) displayed higher than a 10-fold
increase in growth inhibition in vitro on a panel of 6 human cancer
cell lines than POH (Table 2). In addition, the in vitro growth
inhibitory activity displayed by 12a and 12b was independent of the
cancer cell lines’ resistance (or sensitivity) to pro-apoptotic stimuli
(Fig. 4), which overexpressed the a2 and a3 NAK subunits [19]. In
contrast, only compound 12b was efficacious against purified
guinea pig kidney preparations, which, like glioma cells [15], mel-
anoma cells [16] and NSCLC cells [17], contain only the
subunit [19].
a1 NAK
Compound 12b also displayed anti-Ras activity that was
approximately 40 times more pronounced than the anti-Ras ac-
tivity of POH (Fig. 7).
Fig. 5. The influence of compound 12b on the activation of purified brain Naþ/Kþ-ATPase by Naþ (A), Kþ (B) and ATP (C). The influence of 12b on the activation of purified kidney
Naþ/Kþ-ATPase by Naþ (D), Kþ (E) and ATP (F). Each point is the mean ꢁ SEM of 3e6 different experiments.

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F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
Activity differences between purified and whole homogenate
NAK can be used to indicate selectivity because nonspecific binding
in crude homogenates decreases the compound’s inhibitory effect
on NAK [19]. Based on this criterion, the data shown in Fig. 4
indicate that 12b was somewhat less selective toward the brain
enzyme (mainly a2 and a3 isoforms) than the kidney enzyme (a1
isoform), which showed the same IC50 values for purified and ho-
mogenate NAK activities. Other Mgþþ-dependent ATPases in crude
tissue homogenates were not affected by this compound at the
concentrations used (0.1e1.0 mM). NAK inhibition by 12b was thus
uncompetitive with the substrate ATP. In this type of inhibition, the
inhibitor does not bind to the enzyme alone. Rather, the inhibitor
binds to the enzymeesubstrate complex, which is, in this case,
the ATPeNAK complex. Considering the two phases of the NAK
catalytic cycle (I and II), these data indicate that compound 12b acts
on phase I of the NAK cycle. ATP first binds to the enzyme and then
is the substrate for phosphorylation of the Naþ activated-enzyme.
POH displayed a similar behavior [19], but the inhibitory capacity
of 12b for the kidney isoform is approximately 3 times higher than
the activity measured for POH. Interestingly, Kþ-pnppase activity,
which measures phase II of the NAK cycle, was not affected by 12b
in purified kidney preparations, whereas some degree of inhibition
was noted for the purified brain enzyme. The brain enzyme was
also less selective to this compound described above. Therefore, at
least for the kidney enzyme, 12b seems to act primarily in phase I of
the NAK cycle.
Fig. 6. The influence of compound 12b on the inhibition of Kþ-pnppase activity from
purified brain and kidney NaK preparations. Each point is the mean ꢁ SEM of 3
different experiments.
As emphasized by Li et al. [45], NAK was originally discovered as
an ion pump that is essential for cell viability and provides a means
for the epithelium to secrete and/or absorb solutes and nutrients.
However, as elegantly demonstrated by Xie and Askari [46] and
Liang et al. [47], in addition to pumping ions across the cell mem-
brane, the
transduction molecule. In glioma cells, inhibiting NAK activity does
not modify [Ca2þ and [Naþ]i but does massively induce non-
a1 subunit of NAK also functions as a scaffold and signal
]
i
apoptotic cell death [15]. Recently, a
has been identified as one of the central components of
a
1 NAK/Src receptor complex
1 NAK-
a
Despite these findings, it is still possible that compound 12b
directly binds to some NAK sites, and the possibility that it interacts
with the lipid microenvironment surrounding the enzyme cannot
be discharged. Notably, the lipid composition of brain and kidney
cells is different.
mediated signal transduction [48]. Furthermore, the Xie group
[45] demonstrated the in vivo effectiveness in human tumor xe-
nografts targeting NAK/Src-mediated signal transduction to
develop new anticancer therapeutics.
Another signaling pathway controlled by NAK is the Ras/Raf/
MEK/ERK1/2 pathway [49]. For example, translationally controlled
tumor protein (TCTP) is implicated in cell growth and malignant
transformation, and TCTP interacts directly with the third cyto-
plasmic domain of the alpha NAK subunits [50]. Jung et al. [50] have
demonstrated that TCTP induces Src release from the alpha NAK
subunit, Src activation and activation of the Ras/Raf/MEK/ERK1/2
pathway. However, Ras also controls NaK activity. For example, over
a short time scale, fibroblast growth factor-10 (FGF-10) up-
regulates NAK activity in alveolar epithelial cells (AECs) via the
Grb-SOS/Ras/MAPK pathway [51]. AECs that express dominant
negative Ras protein prevent FGF-10-mediated NAK recruitment to
the AEC plasma membrane [51].
In conclusion, the current study reveals that 4-bromo-2-
(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b) displays
in vitro growth inhibitory activity that partially relates to the inhi-
bition of the
observed some specificity of the compound toward the
a
subunits of the Naþ/Kþ-ATpase. Furthermore, we
a1 subunit,
which is overexpressed in gliomas, melanomas and NSCLCs. The
most potent anti-NAK inhibitors are cardiotonic steroids, which
exhibit cardiotoxicity and have very narrow therapeutic indices as
potential anticancer drugs. It is possible that 12b will not display
cardiotoxic effects as pronounced as the cardiotonic steroids; we
are currently performing cardiotoxicity tests. Another non-
cardiotonic steroid-related compound, POH, has already been
demonstrated to contribute significant therapeutic benefits to gli-
oma patients. POH exerts its anti-glioma effects at least partly
through both
12b appears to be approximately 3 times more potent than POH in
inhibiting 1 NAK subunit activity and 40 times more potent than
a1 NAK subunit and Ras oncogene inhibition. Notably,
a
POH in inhibiting Ras activity.
5. Experimental section
5.1. Chemistry
5.1.1. General methods
All solvents were of reagent grade and were purified and dried
by standard methods when necessary. All chemicals were pur-
chased from Acros or SigmaeAldrich. All reactions were routinely
checked by TLC analysis on an Alugram SIL G/UV254 (Macherey-
Nagel) with spots visualized by UV light. The concentration of so-
lutions after reactions and extractions involved the use of a rotary
evaporator operating at reduced pressure. Organic solutions were
dried over anhydrous magnesium sulfate, and column chroma-
Fig. 7. Characterization of the Ras oncogene inhibitory profiles of POH (positive con-
trol) and compound 12b in human Hs683 glioma cells. Each experimental condition
was performed in triplicate. Data are presented as the mean ꢁ SEM.
tography was performed using silica gel 60 (50e200
mm diameter).
Melting points were determined in open capillaries on a Stuart

F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
221
SMP3 apparatus and are uncorrected. The 1H and 13C NMR spectra
were measured on an AC Bruker 250 MHz spectrometer in DMSO-
d6; chemical shifts are reported in parts per million (ppm). All
coupling constants (J) are given in Hz. MS spectra were recorded on
an Agilent Technologies GCeMS instrument equipped with a 7683
injector, 6890 N gas chromatograph and a 5973 mass selective
detector. The mass spectrometer was operated in EI mode at 70 eV,
and MS spectra were recorded from m/z 50 to 650. Elemental an-
alyzes (C, H, N, S) were used to confirm the purity of all compounds
(>95%) and were performed on a CHN ThermoScientific Flash 2000
apparatus.
into water (80 mL) while stirring. The precipitate was filtered,
washed with water, and dried at room temperature overnight. The
isolated solid was purified by recrystallization in isopropanol.
All compounds under study are described in the Supplemental
Information section.
5.2. Stability test
5.2.1. Solvents
Formic acid (FA) and high-purity LC-MS methanol (MeOH) were
purchased from Merck (Darmstadt, Germany). Water was purified
usingaMilli-Qpurificationsystem fromMillipore(Bedford,MA,USA).
5.1.1.1. Synthesis of 2,5-substitued-4-halogeno-1,3-thiazole by
Doyle’s method. Twelve millimoles of either anhydrous copper (II)
chloride or anhydrous copper (II) bromide were added to a solution
of tert-butyl nitrite (15 mmol) in acetonitrile anhydrous (40 mL).
The mixture was stirred at 50 ꢀC for 1 h, and then 2,5-substituted 4-
amino-1,3-thiazole was added slowly. The resulting mixture was
stirred at room temperature overnight. The reaction mixture was
quenched in water (100 mL). When the precipitate appeared, it was
filtered, washed twice with 20 mL of water, and dried under room
temperature until a constant weight was reached. The isolated solid
was purified by recrystallization in isopropanol. When a viscous
mixture appeared, the solution was extracted with ethyl acetate
(3 ꢂ 25 mL). The organic layers were dried with MgSO4, filtered,
and evaporated. The oily solid was then purified by column chro-
matography on silica gel.
5.2.2. Infusion-MS analysis
Compounds 12a and 12b were dissolved in DMSO (5 mM).
Before injection, these solutions were diluted 100-fold in water or
in RPMI. Analyzes were performed using a Rapid Resolution Liquid
Chromatography (RRLC) 1200 series from Agilent Technologies
(Santa Clara, CA, USA) coupled to a 6520 series electrospray ion
source (ESI) e quadrupole time-of-flight (QTOF) from Agilent
Technologies. One microliter of samples was infused using a by-
pass and FA 0.2%-MeOH 50e50 (v/v) as solvent. The stability test
has been performed at 37 ꢀC for 72 h for each compound. The ESI-
QTOF parameters were: positive mode, extended dynamic range
mode (2 GHz), gas temperature of 300 ꢀC, drying gas of 9 l/min,
nebulizer pressure of 40 psi, capillary voltage of 3000 V, fragmentor
90 V and MS scan range of 110e950 m/z at 6 spectra/s. Data were
acquired by the Mass Hunter AcquisitionÒ software from Agilent
Technologies and analyzed by the Mass Hunter Qualitative Ana-
lysisÒ software (AgilentTechnologies). Compounds were identified
by their molecular peak and their adducts merged into one chro-
We detail here below the characteristics of 12a and 12b. All
remaining compounds under study are described in the
Supplemental Information section.
5.1.1.2. (4-Chloro-2-(piperidin-1-yl)-1,3-thiazol-5-yl)(phenyl)meth-
matogram for each compound: 307.0666 m/z [M
þ
H]þ,
anone (12a). Yield, 59%; yellow so1id; mp 145 ꢀC; 1H NMR
329.0486 m/z [M þ Na]þ and 345.0225 m/z [M þ K]þ for compound
12a and 351.0161 m/z [M þ H]þ, 372.9981 m/z [M þ Na]þ and
388.9720 m/z [M þ K]þ for compound 12b.
(250 MHz, DMSO-d6):
d
1.55 (m, 6H, 3ꢂ CH2), 3.55 (m, 4H, 2ꢂ CH2),
7.54 (m, 2H), 7.61 (m, 1H), 7.67 (m, 2H); 13C NMR (62.5 MHz, DMSO-
d6):
d 23.0, 24.6, 48.6, 116.9, 128.0, 128.3, 131.7, 138.8, 142.1, 169.4,
185.4; HRMS calcd for C15H16ClN2OS [M þ H]þ 307.0666, found
307.0650. Anal. Calcd for C15H15ClN2OS: C, 58.72; H, 4.93; N, 9.13; S,
10.45. Found: C, 58.32; H, 5.22; N, 9.32; S, 10.14.
5.3. Pharmacology
5.3.1. Determination of in vitro anticancer activity
The histological types and origins of the six cancer cell lines that
were used for the MTT colorimetric assay are detailed in the legend
of Table 2. The cells were cultured in RPMI (Lonza, Verviers,
Belgium) medium supplemented with 10% heat inactivated fetal
calf serum (Lonza). All culture media were supplemented with
5.1.1.3. (4-Bromo-2-(piperidin-1-yl)-1,3-thiazol-5-yl)(phenyl)meth-
anone (12b). Yield, 93%; yellow so1id; mp 123 ꢀC; 1H NMR
(250 MHz, DMSO-d6):
d
1.60 (m, 6H, 3ꢂ CH2), 3.52 (m, 4H, 2ꢂ CH2),
7.51 (m, 2H), 7.58 (m, 1H), 7.63 (m, 2H); 13C NMR (62.5 MHz, DMSO-
d6):
d
23.1, 24.6, 48.7, 118.5, 128.1, 128.3, 131.2, 131.8, 138.8, 170.5,
4 mM glutamine, 100
mg/mL gentamicin, 200 U/mL penicillin and
185.7; HRMS calcd for C15H16BrN2OS [M þ H]þ 351.0161, found
351.0170. Anal. Calcd for C15H15BrN2OS: C, 51.29; H, 4.30; N, 7.98; S,
9.13. Found: C, 51.18; H, 4.40; N, 7.98; S, 9.30.
200 g/mL streptomycin (Lonza).
m
Normal astrocytes were obtained as detailed previously [40].
Briefly, neonatal NMRI mouse cortices were freed of meninges,
minced into small pieces of tissue with microscissors and then sus-
pended in MEM (Invitrogen) supplemented with glucose 6 g/L and
10% fetal calf serum (FCS; Invitrogen). The cell suspension was then
5.1.1.4. Synthesis of methyl 6-substitued-2-(methylsulfanyl)thieno
[2,3-d]thiazole-5-carboxylate. Methylthioglycolate (5 mmol) was
dissolved in methanol (30 mL). Sodium methylate (5 mmol) was
added, and the solution was stirred for 15 min at 60 ꢀC. 5-substituted
4-halogeno-2-methylsulfanyl-1,3-thiazole was added, and the
mixture was refluxed for 5 h. Then, fresh sodium methylate (6 mmol)
was added and heated at 50ꢀ for 1 h. The mixture was poured into
water (100 mL) with stirring. The precipitate was filtered, washed
with water, and dried at room temperature overnight. The isolated
solid was purified by recrystallization in isopropanol.
filtered through a 225 mm-pore and a 25 mm-pore filter. The filtered
cell suspension was plated on an uncoated T25 flask for 72 h. The
mediumwasthenchanged, andthecellsweregrownuntilconfluence.
The overall growth level of the human cancer cell lines was
determined using a colorimetric MTT (3-[4,5-dimethylthiazol-2yl]-
diphenyl tetrazolium bromide, Sigma, Belgium) assay as detailed
previously [15e17]. Each experimental condition was performed in
six replicates.
All compounds under study are described in the Supplemental
Information section.
5.3.2. Computer-assisted phase contrast microscopy (quantitative
videomicroscopy)
5.1.1.5. Synthesis of methyl 6-methyl-2-(substituted)thieno[2,3-d]
[1,3]thiazole-5-carboxylate 14. (3 mmol) and the corresponding
amine (10 mL) were refluxed overnight. The mixture was poured
The direct visualization of compound-induced effects, in terms
of cytotoxicity versus cytostaticity, in human Hs683 glioma cells
was performed as detailed elsewhere [15,17,23].

222
F. Lefranc et al. / European Journal of Medicinal Chemistry 63 (2013) 213e223
5.3.3. Biochemical characterization of anti-Naþ/Kþ-ATPase activity
Guinea pig brains and kidneys were used in the preparation of
homogenates and Naþ/Kþ-ATPase-enriched fractions. Guinea pigs
were kept in compliance with the Guide for the Care and Use of
Laboratory Animals (US Department of Health and Human Services,
N.I.H.) and in accordance with regulations of our institution.
Lyophilized Naþ/Kþ-ATPase enriched fractions were prepared
according to a modification of the JØrgensen protocol [52] as
detailed previously [53]. The specific activities of the purified en-
the chemiluminescence was read within 15 min after the addition
of the Chemiluminescent working solution using a luminometer
(ActiveGlo LR-100 luminometer, DSLabs, USA).
Acknowledgments
R.K. is a Director of Research with the Fonds National de la
Recherche Scientifique (FNRS, Belgium). G.K. thanks Alzchem
GmbH (Trostberg, Germany) for a gift of cyanamide. We also
warmly thank Thierry Gras for his excellent technical assistance.
zymes were 106 mmol Pi/h/mg of protein (brain) and 100 mmol Pi/h/
mg of protein (kidney). Protein assays were performed by Peter-
son’s method [54].
Appendix A. Supplementary data
Naþ/Kþ-ATPase activity was determined in microtiter plates as
described previously [53]. The final reaction volume (120 mL) con-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.046.
tained 110 mM NaCl, 20 mMKCl, 5 mM MgCl2, 50 mM TriseHCl pH
7.6, inhibitor solutions (absent in controls), an enzyme preparation
and 5 mM ATP. The amount of the enzyme preparation used in all
experiments (including tissue homogenates) corresponded to an
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ꢀC. The supernatant was collected, and the protein content was
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