A convenient synthesis of lubeluzole and its enantiomer: Evaluation as chemosensitizing agents on human ovarian adenocarcinoma and lung carcinoma cells

Article abstract of DOI:10.1016/j.bmcl.2013.06.077

Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a convenient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as assessed by HPLC analysis. The chemosensitizing e

Full text of DOI:10.1016/j.bmcl.2013.06.077

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4820–4823
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Bioorganic & Medicinal Chemistry Letters
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A convenient synthesis of lubeluzole and its enantiomer: Evaluation
as chemosensitizing agents on human ovarian adenocarcinoma and
lung carcinoma cells
Maria Maddalena Cavalluzzi a, Maurizio Viale b, Claudio Bruno a, Alessia Carocci a, Alessia Catalano a,
Antonio Carrieri a, Carlo Franchini a, Giovanni Lentini a,
⇑
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ‘Aldo Moro’, via E. Orabona 4, 70126 Bari, Italy
b IRCCS Azienda Ospedaliera Universitaria San Martino, IST Istituto Nazionale per la Ricerca sul Cancro (IRCCS SMIST), U.O.C. Terapia Immunologia,
L.go R. Benzi 10, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a conve-
nient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as
assessed by HPLC analysis. The chemosensitizing effects of both enantiomers were evaluated in combi-
nation with either doxorubicin (human ovarian adenocarcinoma A2780 cells) or paclitaxel (human lung
carcinoma A549 cells) by the MTT assay. At the lowest concentrations used, lubeluzole showed an overall
and remarkable tendency to synergize with both anticancer drugs. In ovarian cancer cells a clear preva-
lence of antagonistic effect was observed for the R-enantiomer. The synergistic effects of lubeluzole for
both drugs were observed over a wide concentration window (0.005–5 lM), the lowest limit being at
least 40 times lower than human plasma concentrations previously reported as causing serious side
effects.
Received 5 June 2013
Revised 26 June 2013
Accepted 27 June 2013
Available online 4 July 2013
Keywords:
Hydrolytic kinetic resolution
Enantiomeric excess
Antiproliferative activity
Synergism
Ó 2013 Elsevier Ltd. All rights reserved.
Drug repositioning
Lubeluzole [(S)-1a, Fig. 1] is a homochiral benzothiazole deriv-
ative that has shown neuroprotective properties in preclinical
models of ischemic stroke. Its beneficial effects might stem from
inhibition of glutamate release, inhibition of glutamate-activated
nitric oxide (NO) synthesis, and blockade of voltage-gated calcium
channels.1–4 Lubeluzole is also capable of blocking voltage-gated
sodium channels but it has been recently reported that its neuro-
protective action is likely not triggered by sodium channel inhibi-
tion.5 On the other hand, possible epigenetic control on NO
synthase (NOS) and calmodulin (CaM) activities has been hypoth-
esized2 to explain lubeluzole anti-ischemic activity. Thus, lubeluz-
ole neuroprotective properties might stem from NOS and CaM
pathways inhibition. However, the promise of anti-ischemic prop-
erties have failed to translate to the human paradigm and this is
why clinical stroke research on lubeluzole has now been aban-
doned.6 In fact, despite its efficacy in animal models, a number of
clinical trials did not show any beneficial effect in man.6,7 The
worst is that lubeluzole seems to be associated with significant in-
crease of heart-conduction disorders.7 In particular, concentrations
of lubeluzole which exerted neuroprotection in humans increased
the action potential duration thus leading to a prolongation of the
QT interval of the electrocardiogram in some of the treated
patients.7,8
A number of calcium channel blockers and CaM ligands act as
chemosensitizing agents,9,10 while several sodium channel block-
ers display anticancer potential.11,12 Indeed, calcium antagonist
sabeluzole [(RS)-1b, Fig. 1]—the monofluorinated analogue of lube-
luzole—has already been evaluated as chemosensitizing agent.13
Hence, being lubeluzole a sodium and calcium channel blocker,
we thought it was interesting to examine whether it is endowed
with chemosensitizing properties, at concentration significantly
lower than those inducing direct cytotoxicity. In the present study
lubeluzole and its enantiomer were prepared through a new con-
venient route employing a hydrolytic kinetic resolution method,
and their corresponding anticancer activities were evaluated on
human ovarian adenocarcinoma and lung carcinoma cells in com-
bination with either doxorubicin or paclitaxel, respectively.
Two methods have been reported for the preparation of lube-
luzole and its enantiomer as separated entities: a biocatalytic
method based on lipase-catalyzed kinetic resolution14 and a
stereospecific procedure using chiral pool starting material.15
Abbreviations: CaM, calmodulin; HKR, hydrolytic kinetic resolution; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide;
NOS, nitric oxide synthase; PgP, P-glycoprotein.
⇑
Corresponding author. Tel.: +39 080 5442744; fax: +39 080 5442050.
E-mail
addresses:
giovanni.lentini@uniba.it,
glentini@farmchim.uniba.it
(G. Lentini).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.077

M. M. Cavalluzzi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4820–4823
4821
presence of several both commercially available or homemade19,20
mandelic acid derivatives as chiral solvating agents failed. By
applying a previously reported HPLC method,15 higher ee (>99%)
than the ones previously reported (97%15 and 98.916) was demon-
strated. Subsequently, (R)-4 was converted into the corresponding
tosyl derivative (S)-5 which underwent nucleophilic substitution
with 9 affording the R-enantiomer of lubeluzole [(R)-1a]. Although
its ee was relatively low (84%), recrystallization from abs EtOH/
hexane gave (R)-1a in acceptable enantiomeric purity (96%). 2-
Aminobenzothiazole derivative 9 was prepared from ethyl 4-
oxopiperidine-1-carboxylate (6) as previously described,15 while
shortening the reaction times by microwave irradiation (Scheme 2).
Moreover, microwave-assisted synthesis of 9 resulted in increased
yield with respect to the conventional synthetic procedure.15 As
concerning the overall yields, considering the maximum amount
of the two products which can be stechiometrically obtained in
the HKR step, we may assume that lubeluzole and its R-enantiomer
[(R)-1a] were obtained with yields at least similar to those previ-
ously reported.14,15 Compared with the most convenient route to
lubeluzole, which gave (S)-1a in 61% overall yield and 98.9% ee,16
this route seems to be more convenient since both lubeluzole
and its enantiomer can be easily obtained with higher enantiopu-
rity and acceptable overall yield from inexpensive racemic starting
material. Moreover, the novelty of this synthetic route stems from
S
N
OH
N
R
F
O
N
1a,b
Figure 1. Structures of lubeluzole [(S)-1a, R = F], and sabeluzole [(RS)-1b, R = H].
However, both methods have some drawbacks such as the need of
relative high numbers of synthetic steps14 or the need of an expen-
sive chiral starting material.15 Very recently a three-step version of
the chiral pool approach gave (S)-1a in good yield.16 With the aim
of overcoming these drawbacks, we thought to obtain highly enan-
tioenriched chiral building blocks from racemic materials accord-
ing to the Jacobsen’s hydrolytic kinetic resolution (HKR)
strategy,17 as outlined in Scheme 1. (RS)-2, obtained as reported
in the literature,15 was subjected to HKR18 in order to provide both
the oxirane (S)-3 and the diol (R)-4 intermediates in their corre-
sponding enantiomerically enriched forms. (R,R)-(salen)CoIII(OAc)
was prepared according to Jacobsen’s A procedure,17 and used as
the catalyst. (S)-3 was then subjected to a ring-opening reaction
with 9, obtained as reported in Scheme 2, to provide lubeluzole
[(S)-1a]. Attempts to evaluate ee by 1H NMR esperiments in the
O
O
F
F
2
(RS)-
i
O
OH
O
F
F
F
F
O
OH
a
3
)
(R)-4 (33%, 66%a)
(S)- (30%, 60%
ii
iii
S
N
OH
N
F
F
O
N
OH
O
O
F
F
O
S
O
1a
(S)-
(71%)
5
(S)- (52%)
iv
S
N
OH
N
F
F
O
N
1a
(R)-
(79%)
Scheme 1. Reagents and conditions: (i) (R,R)-(salen)CoIII(OAc), H2O, room temperature; (ii) compd 9, Yb(OTf)3, dry CH2Cl2, room temperature; (iii) tosyl chloride, dry
pyridine, dry CH2Cl2, 0 °C, then room temperature; (iv) compd 9, K2CO3, i-PrOH; abased on the maximum amount which can be stechiometrically obtained, that is, on a half
amount of (RS)-2 used.

4822
M. M. Cavalluzzi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4820–4823
O
O
O
S
N
N
i
ii
iii
O
N
O
O
N
N
N
N
S
O
NH
N
H
7
8
9
6
(90%)
(76%)
(85%)
Scheme 2. Reagents and conditions: (i) NaBH3CN, CH3NH2ÁHCl, MeOH, MW, 110 °C, 11 min; (ii) 2-chloro-1,3-benzothiazole, TEA, (CH2OH)2, MW, 180 °C, 20 min; (iii) 48%
HBr, MW, 140 °C, 15 min.
Figure 2. D values obtained for the combined treatment of A2780 cells with doxorubicin and lubeluzole or its enantiomer. The experimental D value for additivity was
calculated using combinations of two serial dilutions of all drugs. Lubeluzole and R-enantiomer [(R)-1a] concentrations: j, 50
0.005 M. For the sake of clarity, the standard deviation bars were omitted. Full data (means SD for each D value) are given in Tables 1 and 2, pages S2 and S3 of
Supplementary data. The statistical analysis was performed by means of the Mann–Whitney test for non-parametric data (n = 4–5, sham value n = 33).
lM; h, 5 lM; d, 0.5 lM; s, 0.05 lM; N,
l
Figure 3. D values obtained for the combined treatment of A549 cells with paclitaxel and lubeluzole or its enantiomer. The experimental D value for additivity was calculated
using combinations of two serial dilutions of all drugs. Lubeluzole and R-enantiomer [(R)-1a] concentrations: j, 50 M; h, 5 M; d, 0.5 M; s, 0.05 M. For the
l
l
l
lM; N, 0.005 l
sake of clarity, the standard deviation bars were omitted. Full data (means SD for each D value) are given in Tables 3 and 4, page S4 and S5 of Supplementary data. The
statistical analysis was performed by means of the Mann–Whitney test for non-parametric data (n = 4–5, sham value n = 24).
the possibility to use both products of HKR strategy. In fact, this
procedure is usually aimed at the synthesis of just one product:
either the enantioenriched epoxide of S configuration, directly
exploitable as a chiral buildingblock,17 or the R-diol which is in
turn converted into the corresponding R-epoxide.21 The synthetic
strategy herein proposed guarantees that the starting material is
not wasted, taking advantage of both (S)-3 and (R)-4 to obtain
lubeluzole and its enantiomer, respectively.
(A549) cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay. (S)-1a and its enantiomer (R)-1a
showed discrete antiproliferative activities in both A2780 and
A549 human cell lines with IC50s in the micromolar range of con-
centrations. Nevertheless, while some stereoselectivity was ob-
served in A2780 cells, (S)-1a being significantly more active than
its enantiomer (5.7 2.7 vs 14.0 3.0
ative activities of both enantiomers on A549 cells were almost
comparable (14 6 vs 18 M). For the anticancer drugs doxoru-
lM, p = 0.01), the antipolifer-
Antiproliferative effects of (S)-1a and (R)-1a, alone or in combi-
nation with doxorubicin or paclitaxel, were evaluated in human
ovarian carcinoma (A2780) cells and human lung carcinoma
4 l
bicin and paclitaxel the IC50s on A2780 and A549 cells were 23
8
and 14 4 nM, respectively.

M. M. Cavalluzzi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4820–4823
4823
When administered in association with the anticancer drugs
doxorubicin and paclitaxel, (S)-1a and (R)-1a displayed different
behaviors (Figs. 2 and 3). In fact, (S)-1a showed an overall and
remarkable tendency to synergize with both anticancer drugs at
sub-micromolar concentrations, this effect being more evident in
ovarian cancer cells. On the contrary, in both experimental condi-
tions, when the drug combination contained at least one of the
drugs at its higher concentration(s), the effect was additive or even
antagonistic (Figs. 2 and 3). This observation is intriguing since it
suggests the possibility that lubeluzole may potentiate in vivo pac-
litaxel or doxorubicin anticancer activity, at concentrations signif-
icantly lower than those inducing direct cytotoxicity. To this
regard, it is worth noting that lubeluzole, given alone at its lower
Rebesco and the staff of the Unit Anticancer Drugs (IRCCS SMIST)
for kindly providing paclitaxel and doxorubicin. The authors are in-
debted to Dr. Capodiferro for technical help. Thanks go also to the
anonymous reviewers of this Journal for their critical review,
which helped to improve the manuscript significantly.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.
077.
References and notes
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a more narrow concentration window (1.2–0.012
playing direct cytotoxicity at 1.2
M concentration.13 Considering
that the lowest lubeluzole concentration displaying synergistic ef-
lM) while dis-
l
fect (0.005 lM) is about 50 times lower than human plasma con-
centration related to QTc interval prolongation (>100 ng/mL),22
lubeluzole may be considered as a candidate for possible reposi-
tioning23 as an adjuvant for anticancer agents.24
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M. S. Tetrahedron: Asymmetry 2003, 14, 1363.
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Acknowledgments
23. Sleigh, S. H.; Barton, C. L. Pharm. Med. 2010, 24, 151.
24. Synthetic procedures and characterization for all the new compounds, and
general procedures for the biological activity are provided in the
Supplementary data.
This work was accomplished thanks to financial support from
the Ministero dell’Istruzione, dell’Università e della Ricerca (Grants
MIUR 2009 EL5WBP_003 and 2010FPTBSH_002). We thank Dr.