A simple O-sulfated thiohydroximate molecule to be the first micromolar range myrosinase inhibitor

Article abstract of DOI:10.1016/j.tetlet.2009.02.072

New non-hydrolyzable analogues of glucosinolates have been prepared. Myrosinase inhibition was observed with modified aglycon moieties, even bulky phenothiazine analogue 6 gave reasonable inhibition. The simplest structure 8 derived from dimethylaminoethanethiol has shown to be the most potent inhibitor with an IC₅₀ of 3.32 μM.

Full text of DOI:10.1016/j.tetlet.2009.02.072

Tetrahedron Letters 50 (2009) 3302–3305
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A simple O-sulfated thiohydroximate molecule to be the first micromolar
range myrosinase inhibitor
a,b
a
b
a
Deimante Cerniauskaite , Estelle Gallienne , Henreta Karciauskaite , Andrea S. F. Farinha ,
b
c
a,
b
a
*
Jolanta Rousseau , Sylvie Armand , Arnaud Tatibouët , Algirdas Sackus , Patrick Rollin
Institut de Chimie Organique et Analytique, Associé au CNRS, Université d’Orléans, B.P. 6759, F-45067 Orléans, France
Kaunas University of Technology, Radvilenu st.19, LT-50270 Kaunas, Lithuania
Centre de Recherche sur les Macromolécules Végétales (CERMAV–CNRS), Affiliated with Université Joseph Fourier, Grenoble, BP 53, F-38041 Grenoble Cedex 9, France
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
New non-hydrolyzable analogues of glucosinolates have been prepared. Myrosinase inhibition was
observed with modified aglycon moieties, even bulky phenothiazine analogue 6 gave reasonable inhibi-
tion. The simplest structure 8 derived from dimethylaminoethanethiol has shown to be the most potent
Received 20 January 2009
Revised 6 February 2009
Accepted 10 February 2009
Available online 14 February 2009
inhibitor with an IC50 of 3.32 lM.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Glucosinolate
Myrosinase inhibition
Thiohydroximate
Sulfation
Glucosinolates 1 are naturally occurring thiosugars mainly
found in the botanical order Brassicales. The structural framework
of glucosinolates invariably results from a combination of three
and the flexibility with regard to the aglycon moiety. More re-
cently, glucosinolates bearing modified aglycons have been syn-
thesized and studied as new pro-drugs able to deliver cytotoxic
7
segments: a
D
-glucopyrano unit, an O-sulfated anomeric thiohy-
isothiocyanates. So far, only 2-fluoro-2-deoxy-glucotropaeolin
droximate function and a broad library of aglycons, whose struc-
ture diversifies in the vegetal kingdom according to species.
Glucosinolates are substrates of a specific enzyme, myrosinase
2b was found as a good inhibitor acting through the formation of
a covalent glucosyl–enzyme intermediate. Glucotropaeolin (GTL)
2a was taken as a model because of its current use as a EU official
1
8
(
thioglucoside glucohydrolase EC 3.2.1.147), the only enzyme able
to hydrolyze those thiosaccharidic compounds. Via concomitant
expulsion of sulfate ion and -glucose release, glucosinolate hydro-
standard for glucosinolate analysis (Scheme 1).
GTL 2a appeared to be a good template to develop non-hydro-
lyzable substrates by keeping the benzyl thiohydroximate function
and modifying the osidic moiety. We have previously investigated
synthetic modifications involving carbasugars or more simple
alkyl derivatives, among which the most potent inhibitor was
D
lysis delivers a wide range of aglycon moieties modified into
isothiocyanates, nitriles, 1,3-oxazolidine-2-thiones, etc. which usu-
ally take part in the defense mechanism of plants and may have
2
9
some impact on human nutrition. The influence of glucosinolates
detected. The non-hydrolyzable GTL mimic 2c was shown to be
and their breakdown products on health and more specifically the
role they can play in cancer chemoprevention are still not perfectly
well understood. With a view to developing new chemical tools
the best inhibitor inducing 88% myrosinase inhibition at 1 mM.
We have decided to follow two different approaches aimed at
3
for biological and pharmaceutical applications, we have explored
the enzyme-substrate recognition mechanism and developed vari-
ous classes of glucosinolate analogues. Previously, some substrate
mimics have been prepared with structural modifications on the
HO
HO
S
R
O
S
A
N
HO
KO SO
R
3
glycosidic moiety, on the aglycon chain5 or on the anionic site.
4
6
N
KO3SO
Most of these modifications proved useful in clearly demonstrating
the specificity of myrosinase towards the -glucopyrano moiety
general structure 1, R = OH
2c, R = H, IC50 = 0.6 mM
D
glucotropaeolin 2a, R = OH, A = Bn
2
-fluoro2-deoxyglucotropaeolin
2d, R = CH2OH, IC50 = 0.2 mM
2
b, R = F, A = Bn
*
Corresponding author. Tel.: +33 238494857; fax: +33 238417281.
E-mail address: arnaud.tatibouet@univ-orleans.fr (A. Tatibouët).
Scheme 1. Structure of O-sulfated thiohydroximates.
0
040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.02.072

D. Cerniauskaite et al. / Tetrahedron Letters 50 (2009) 3302–3305
3303
OH
S
N
O
KO SO
3
3
HO
S
S
HO
N
N
KO SO
KO SO
3
3
4
7
S
N
N
S
H
2c
O SO
N
3
KO SO
3
8
5
S
H N
3
n
N
S
S
N
O SO
3
9
1
,
n = 1
N
KO SO
0, n = 2
3
6
Scheme 2. Panel of myrosinase inhibitors.
improving myrosinase inhibition, either through modification of
the aglycon moiety (3–6) or by developing transition state
analogues (Scheme 2).
using standard conditions. After careful ionic exchange using
KHCO in excess, the O-sulfated thiohydroximates 3 and 4 were
obtained in similarly good yields.
3
We have synthesized a panel of glucosinolate-like molecules in
which aryl-type aglycons replace the benzyl group: 3 and 4 ana-
logues lack the benzylic methylene ‘knuckle’ while 5 and 6 intro-
duce bulky and fluorescent aromatic entities. On the other hand,
modifications of the carbohydrate in 2a afford a panel of geometric
mimics of the transition state formed during myrosinase catalysis.
These include the 2,5-anhydro-D-mannitol derivative 7 and the
oxonium mimics 8–10 which bear ammonium ions distal from
the benzylic moiety.
Hydroximoyl chlorides bearing a methylene ‘knuckle’ were pre-
pared following a different reaction sequence. A Henry-type proce-
dure was first applied to produce the nitroalkene derivatives
1
2
14b–c, which in turn were converted into chloro-oximes 15b–c
1
0
by using the protocol described by Kulkarni. S-Ethyl naphthyl-
acetothiohydroximate 16b was isolated in 67% yield, but the N-
methyl phenothiazinyl counterpart 16c was isolated in only 26%
1
3
yield. Finally, the derived O-sulfates 5 and 6 were obtained in
good yields.
The expected O-sulfated thiohydroximates were obtained by
standard sulfation of a precursor synthesized via a key reactive
species—a hydroximoyl chloride intermediate. This electrophilic
partner could be prepared either from nitroalkene derivatives fol-
All built on a common phenylacetothiohydroximate frame of
the GTL-type, other glucosinolate mimics 7–10 were obtained
through condensation of diverse mercaptans on a unique hydroxi-
moyl chloride 15a (Scheme 5) previously used to make 2a–d. The
10
lowing Kulkarni’s method (Scheme 4) or by substitutive chlorina-
anhydromannitol-derived thiol 20 was synthesized from D-gluco-
1
1
14
tion of aldoximes (Scheme 3).
samine 17 according to a previously described sequence. Under
nitrosation conditions, 17 underwent ring contraction leading to
Hydroximoyl chlorides devoid of a methylene ‘knuckle’ were
prepared from aldoximes 11a–b (Scheme 3) by reaction with
2,5-anhydro-
2,5-anhydro-
D
-mannose, which was reduced with NaBH
4
to yield
1
1
N-chlorosuccinimide: the unstable intermediates 12a–b were
engaged in the next step without further purification and reacted
with ethyl mercaptan. Thiohydroximates 13a and 13b obtained,
respectively, in 83% and 62% yields over two steps were sulfated
D-mannitol 18 (Scheme 5). Selective monotosylation
and further peracetylation gave a 35% yield of compound 19,
which was further converted into the corresponding thiol 20 in
three steps (64% overall yield): (1) substitutive iodination (2) for-
mation of the thiouronium salt (3) hydrolysis under reductive
conditions.
1
-Thio-3,4,6-tri-O-acetyl-2,5-anhydro-D-mannitol 20 was con-
NH2OH,
pyridine
A
NCS, pyridine
CHCl3
A
Cl
densed with 15a to give thiohydroximate 21 in 50% yield. A final
two-step sulfation/de-O-acetylation sequence gave the glucosino-
late analogue 7 in 73% yield.
A
O
N
N
OH
11a 90%
1
OH
EtOH
12a
12b
The unprecedented ammonium analogues 8–10 were prepared
following a similar sequence involving stereospecific addition of
the corresponding aminothiols to the transient nitrile oxide gener-
1b 99%
Et3N, EtSH
DCM
15
9
ated from 15a, followed by standard sulfation. A slight modifica-
tion was needed for the synthesis of 9, where the starting
a = Ph
b = naphthyl
1
6
aminothiol was used in the N-tBoc protected form. Sulfate 23 ob-
tained was deprotected in the last step to generate zwitterionic 9
(Scheme 6).
a-pyridine.SO3,
DCM
A
S
N
A
S
N
This amino acid 9 was easily isolated, thanks to its precipitation
in the aqueous reaction medium but unfortunately on re-solubili-
OSO K
OH
3
b- KHCO3, H O
2
1
7
zation in DMSO, a spontaneous decomposition occurred: intra-
molecular nucleophilic addition of the amine onto the O-sulfated
thiohydroximate resulted in the formation of thiazoline 24 through
1
1
3a 83% (after two steps)
3b 62% (after two steps)
3
4
87%
86%
1
8
Scheme 3. Synthesis of thiohydroximates via oxime chlorination.
sulfamic acid elimination. Preparation of the amino acid 10 was

3
304
D. Cerniauskaite et al. / Tetrahedron Letters 50 (2009) 3302–3305
CH3NO2,
NH OAc
TiCl4, Et SiH
4
3
Cl Et3N, EtSH
S
A
O
NO2
A
A
A
DCM
OH
N
N
DCM
OH
1
4b 61%
1
1
5b
5c
16b
1
1
4c 97%
67% (after two steps)
6c 26% (after two steps)
a-pyridine.SO3,
DCM
a
∗
b
∗
c
b- KHCO3, H O
2
S
∗
A =
S
N
A
CH3
N
5
6
91%
73%
OSO K
3
Scheme 4. Synthesis of thiohydroximates from a nitroalkene precursor.
HO
HO
HO
OH
a) NaNO2, AcOH, H O
2
O
OH
O
NH3
Cl
b) NaBH4, EtOH
OH
HO
OH
18
D-glucosamine 17
a) TsCl, pyridine, DMAP
b) Ac O, pyridine
2
OAc
O
OAc
a) NaI, acetone, Δ
b) thiourea, butanone,Δ
O
SH c) Na S O H O-CHCl3
AcO
OTs
OAc
AcO
2
2
5,
2
OAc
20
19
1
5a, Et N, DCM
3
OAc
OH
a) pyr.SO3, CH CN
b) MeOH, MeOK
3
O
O
Bn
N
Bn
N
S
AcO
S
HO
OAc
OH
HO
K
O SO
3
21
7
Scheme 5. Synthesis of the anhydromannitol-derived thiohydroximate 7.
attempted from the corresponding azidopropyl derivative 26,
obtained in two steps and 61% yield from thiohydroximate 25.
Unfortunately on catalytic hydrogenation of 26 and similarly to
moiety (compound 6, entry 6) did not significantly hamper the
inhibition capacity. Neither the transition state mimic 7, bearing
the 2,5-anhydro-D-mannitol moiety (Table 1, entry 7), nor the
9
, rapid degradation occurred. On the contrary, the N,N-dimethyl-
azide 26 (Table 1, entry 9) displayed a significant ability to inhibit
myrosinase. Surprisingly, the most potent myrosinase inhibitor is
the most simple constructed one. The N,N-dimethylammonium-
ammonium derivative 8, which could be smoothly prepared in
only two steps and 47% yield, proved fairly stable in solution and
could therefore be tested.
based transition state mimic 8 displayed an IC50 of 3.32 lM and
The activity of Sinapis alba myrosinase towards the different
glucosinolate analogues was determined by titration of released
glucose from sinigrin (Table 1).8 From this small panel of mole-
cules, a major information related to the inhibition of myrosinase
could be put into light. Comparison with both standard compounds
at 1 mM it completely ablated myrosinase activity. The potency
shown by 8 is approximately 100-fold greater than shown by the
previous millimolar inhibitors developed in our laboratories.
Major features to create new potent inhibitors could thus be ex-
tracted from the above results: the presence of a methylene
knuckle, the possibility of bulky fluorescent moieties and, more
importantly, the introduction of a dialkylamino group to mimic
the transition state and mostly to introduce stability. Further
exploration on designing and studying new non-hydrolyzable
inhibitors of myrosinase is ongoing and will be published in due
course.
2
c and 2d demonstrated the importance of the methylene knuckle:
only a small 20.8% inhibition was observed at 1 mM with the phe-
nyl group (compound 3, entry 3) while no activity was detected
with a more bulky naphthyl group (compound 4, entry 4). Intro-
ducing back a methylene knuckle restored good inhibitory proper-
ties in compound 5 (entry 5), while even a bulkier phenothiazinyl

D. Cerniauskaite et al. / Tetrahedron Letters 50 (2009) 3302–3305
3305
S
S
TFA-H O (2-1)
2
H3N
BocHN
K
N
N
0°C
O SO
O3SO
3
9
23
resolubilisation
in DMSO
pyr.SO3, CH Cl
then KHCO3, H2O
2
2
S
BocHN
N
N
HO
24
S
2
2
1
2
) NaI, NaN3, DMF
80°C, 26h
N3
S
Cl
S
N
N
) pyr.SO3, CH CN
O SO
3
HO
3
then KHCO3, H O
2
K
25
2
6
Scheme 6. Synthesis of the ammonium-type thiohydroximates.
Table 1
Myrosinase inhibition results
8. Official method ISO-9167-1 (a) EEC Regulation No. 1864/90, Enclosure VIII
Offic. J. Eur. Commun. 1990, L170, 27–34; (b) Wathelet, J.-P.; Iori, R.; Leoni, O.;
Rollin, P.; Quinsac, A.; Palmieri, S. Agroindustria 2004, 3, 257–266.
9. Bourderioux, A.; Lefoix, M.; Gueyrard, D.; Tatibouët, A.; Cottaz, S.; Arzt, S.;
Burmeister, W. P.; Rollin, P. Org. Biomol. Chem 2005, 3, 1872–1879.
Entry
Molecules
% Inhibition at 1 mM
IC50 (mM)
1
2
3
4
5
6
7
8
9
2d
2c
3
4
5
6
7
8
26
88
67
20.8
0
77.3
44
20.9
100
26.1
0.2
1
0. (a) Kumaran, G.; Kulkarni, G. H. Tetrahedron Lett. 1994, 35, 9099–9100; (b)
Kumaran, G.; Kulkarni, G. H. J. Org. Chem. 1997, 62, 1516–1520.
1. Liu, K. C.; Shelton, B. R.; Howe, R. K. J. Org. Chem. 1980, 45, 3916.
2. (a) Fujiwara, A. N.; Acton, E. M.; Goodman, L. J. Heterocycl. Chem. 1967, 10, 126–
127; (b) Winchester, M. J.; Popp, F. D. J. Heterocycl. Chem. 1975, 12, 547–549; (c)
Luzzio, F. A. Tetrahedron 2001, 57, 915–945.
0.6
n.d.b
1
1
b
n.d.
n.d.b
b
n.d.
n.d.b
13. Davidson, N. E.; Rutherford, T. J.; Botting, N. P. Carbohydr. Res. 2001, 330, 295–
307.
14. Cassel, S.; Debaig, C.; Benvegnu, T.; Chaimbault, P.; Lafosse, M.; Plusquellec, D.;
Rollin, P. Eur. J. Org. Chem. 2001, 875–896. and references cited therein.
15. In a representative synthetic sequence, the crude phenylacetohydroximoyl
chloride 15a (0.67 mmol)9 under argon was dissolved in dichloromethane
À3a
3.32 Â 10
n.d.b
a
IC50 = 3.32 ± 0.13
n.d.: not determined.
lM.
b
(
10 mL) and NEt
3
(280
lL, 2 mmol) just before commercial N-tBoc-amino-
ethanethiol (135
l
L, 0.8 mmol) was added. After stirring for 5 h, the mixture
Acknowledgements
was evaporated in vacuo. Flash chromatography (petroleum ether/AcOEt, 7:3)
purification of the residue gave compound 22 (0.14 g, 0.45 mmol, 67%) as a
white solid, mp: 143–145 °C. IR (KBr):
m
3343, 2980, 1672, 1524, 1286,
): d 11.15 (s, 1H, OH), 7.35–7.20 (m, 5H, Harom),
.05 (t, 1H, J = 5.6, NH), 3.83 (s, 2H, CH Ph), 3.06–2.98 (m, 2H, CH N), 2.70 (t,
). C NMR (DMSO-d ): d 155.5 (C@O), 150.8
C–), 40.7 (CH N),
We are grateful to the ANR and the CNRS and to La Ligue contre
le Cancer for financial support. We would also like to thank Dr. R.
Iori (CRA-CIN Bologna) for a gift of myrosinase, the Kaunas Techno-
logical University, the Université d’Orléans and the GILIBERT pro-
gram for multiform support.
À1
1
1163 cm . H NMR (DMSO-d
6
7
2
2
13
2H, J = 7.2, CH
2
S), 1.39 (s, 9H, CH
3
6
(
C@N), 137.1 (Carom), 128.4, 128.1, 126.5 (CHarom), 77.9 (Me
3
2
+
37.2 (CH
2
Ph), 28.1 (CH
3
), 27.9 (CH
2
S). ESI-HRMS calcd for C15
H
22
2
N O
3
S [M+H] :
311.1429, found: 311.1454.
9
1
6. Compound 22 was O-sulfated according to a previously described protocol.
Flash chromatography (AcOEt/MeOH, 9:1) purification of the residue gave
References and notes
compound 23 (91%) as a white solid, mp: 151–155 °C (dec). IR (KBr):
m
3357,
): d 7.33–7.23 (m,
Ph), 3.05–2.97 (m, 2H,
). C NMR (DMSO-d ): d 155.5
C@O or C@N), 155.2 (C@N or C@O), 136.4 (Carom), 128.5, 127.9, 126.8 (CHarom),
8.0 (Me C–), 40.5 (CH N), 37.1 (CH Ph), 28.3 (CH S), 28.1 (CH ). ESI-HRMS
calcd for C15 [M] : 389.0841, found: 389.0826.
7. To the protected compound 23 (0.3 g, 0.7 mmol) dissolved in water (2 mL) at
°C was added TFA (4 mL). After stirring at room temperature for 4 h, the
formed white precipitate of zwitterionic 9 was filtered and rinsed with water
À1
1
2977, 1700, 1574, 1409, 1294, 1070 cm
. H NMR (DMSO-d
6
1.
2.
3.
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(
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3
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N and
7
CH
2
2
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2
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2
654.
1
2
3
1
4
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36.3 (Carom), 128.9, 128.3, 126.7 (CHarom), 64.3 (C-4), 39.6 (CH
6
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.
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13
2
2 3
S). C NMR (CDCl
2
+
(
2
CH S). MS (IS+): m/z: 178 [M+H] .