Synthesis, antioxidant properties and radioprotective effects of new benzothiazoles and thiadiazoles

Article abstract of DOI:10.1016/j.bmc.2009.05.039

In this work, we report the synthesis and characterization of new compounds derived from benzothiazoles and thiadiazoles. We observed that structural modifications on these skeletons affected the antioxidant activity. Thiol and aminothiol compounds derived from thiadiazoles and benzothiazoles showed an interesting antioxidant property. The radioprotective activity has also been evaluated in mice. Some of these compounds could be good radioprotectors.

Full text of DOI:10.1016/j.bmc.2009.05.039

Bioorganic & Medicinal Chemistry 17 (2009) 5275–5284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antioxidant properties and radioprotective effects of new
benzothiazoles and thiadiazoles
Damien Cressier a, Caroline Prouillac a, Pierre Hernandez a, Christine Amourette b, Michel Diserbo b,
Claude Lion c, Ghassoub Rima a,
*
a Laboratoire Hétérochimie Fondamentale et Appliquée, UMR 5069-CNRS/UPS, Université Paul Sabatier, 118, route de Narbonne, 31062 Toulouse Cedex 9, France
b Centre de Recherches du Service de Santé des Armées, 24, Avenue des Maquis du Grésivaudan, 38702 La Tronche Cedex, France
c ITODYS, Université Paris VII, 1, rue Guy de la Brosse, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, we report the synthesis and characterization of new compounds derived from benzothiaz-
oles and thiadiazoles. We observed that structural modifications on these skeletons affected the antiox-
idant activity. Thiol and aminothiol compounds derived from thiadiazoles and benzothiazoles showed an
interesting antioxidant property. The radioprotective activity has also been evaluated in mice. Some of
these compounds could be good radioprotectors.
Received 3 February 2009
Revised 24 April 2009
Accepted 14 May 2009
Available online 21 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antioxidant
Radioprotection
Phosphorothioates
Thiosulfonic acid
Benzothiazole
Thiadiazole
1. Introduction
It is also well known that the biological effects of ionizing
radiation are linked to the production of reactive oxygen species
The potential application of radioprotective chemicals in the
event of various human radiation exposures has been investigated
since nuclear exposure from accidents or warfare became possi-
ble.1 However, in recent years, another application of such radio-
protective compounds has been in the treatment of cancer
patients during radiotherapy in order to protect normal tissues
adjacent to the treated tumor.
In 1949, the ability of a chemical compound to protect from the
toxic effects of radiation was reported for the first time by Patt
et al.2 It was demonstrated that the administration of the sulfydryl
amino acid cysteine to rats before an 8 Gy whole-body X-irradia-
tion, significantly increased the animal’s resistance to cytotoxic
effects of the exposure. Since this initial finding, new agents have
been developed like mercaptans, di- and trisulfides, phosphoro-
thioates, alcohols, acid hydrazides, imidazoles, benzofurans, amine
oxides and thiazolidines.3 However, most of these compounds are
toxic and so there is still a need to develop new radioprotective
compounds that are more effective and less toxic.
in organisms which induce cellular DNA damage which leads to
mutations and chromosomal aberrations.4 Moreover, it is generally
accepted that endogenous antioxidants such as cellular non-pro-
tein thiols (glutathion. . .) and antioxidant enzymes provide some
degree of protection. Even if a variety of other radioprotective
mechanisms have been proposed to explain prophylactic and ther-
apeutic effects of a large number of agents, antioxidants are still
considered a main class of radioprotectors.3,5,6 We supposed that
a direct link between a thiol function that catches radicals and
an aromatic group that traps them by potential degradation of
the aromatic structure, and that this is the way the antioxidant
properties perform. Furthermore, the synthesis of N-alkylated aro-
matic heterocycles by well known antioxidant or radioprotective
functions can have an interesting effect on such properties. Since
the radioprotective activity of thiosulfonic acids and phosphoro-
thioates is well-known, we speculated that the introduction of
these important functional groups into new heterocycles like ben-
zothiazole and thiadiazole might provide potent activity.7–12 More
specifically, heterocycles such as benzimidazole, imidazole and
thiazolidines were studied in this area.13–15
So, in this work, we report the synthesis of N-alkylbenzothiaz-
ole and thiadiazole substituted by amide, thiosulfonic acid,
* Corresponding author. Tel.: +33 (0) 561556172; fax: +33 (0) 561558204.
E-mail address: rima@chimie.ups-tlse.fr (G. Rima).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.039

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D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
H
Therefore the synthesis of compounds which differ in the posi-
N
S
N
S
tion of the alkyl chain should be interesting. Indeed, by different
methods, the alkylation could occur either on the endocyclic (6–
9) or on the exocyclic nitrogen atom (2 and 5).16
Amides derivatives 2(a–e)—The reaction of propionyl chloride
with amines 1(a–e) in tetrahydrofurane (THF) in the presence of
triethylamine leads to amide derivatives (Scheme 2).
NH
NH2
NH2
R1
R1
H
N
N
N
N
NH
R2
R2
S
S
Thiols 4(a–e) and aminothiols derivatives 5(a–e)—They were
synthesized in two steps. Firstly, the amine function of aminoben-
zothiazole and aminothiadiazole derivatives 1(a–e) was substi-
tuted by chloride. The treatment of 1(a–e) with 37% hydrogen
chloride solution in presence of copper powder and sodium nitrite
leads to halogenated derivatives 3(a–e) (Scheme 3).
Thiol derivatives 4(a–e) were obtained in a second step by reac-
tion of thiourea in ethanol under reflux. This method has been pre-
viously reported in the literature.17 Compounds 3(a–b), 3d and 4a
have been already obtained by other methods described previ-
ously.18–20
1a: R1= H, 1b: R1 = CH3, 1c: R1 = CH3CH2O
1d: R2 = CH3CH2S, 1e: R2 = CH3CH2
Scheme 1. Structures of benzothiazole and thiadiazole derivatives 1(a–e).
thiobenzothiazole, thiothiadiazole and phosphorothioate groups.
We then present their free radical scavenging properties measured
in vitro and their radioprotective activity studied in mice. Finally,
we discuss about these in vitro and in vivo results.
2. Results and discussion
2.1. Synthesis
Finally, hydrochlorides of the aminothiols 5(a–e) were synthe-
sized under reflux in THF, by reaction of halogenated derivatives
3(a–e) with cysteamine (Scheme 3). Compound 5a has been al-
ready synthesized by another method.21
We have focused our work on the development of two new clas-
ses of radioprotector agents that are benzothiazole and thiadiazole
derivatives. Both types of molecules are characterized by their tau-
tomeric forms (Scheme 1).
Thiosulfonic acids 8(a–e) and phosphorothioates derivatives
9(a–e) were obtained by three steps. First, we synthesized alcohol
derivatives 6(a–e) by treatment of bromoethanol solution with
aminobenzothiazole and aminothiadiazole. In a second step, the
O
O
N
N
S
THF, Et3N
- Et3N, HCl
NH2
H3CH2C C Cl
+
NHCCH2CH3
S
R1
R1
2a-c
1a-c
a: R1 = H, b: R1 = CH3, c: R1 = CH3CH2O
O
N
N
N
N
O
THF, Et3N
+
NH2
H3CH2C
C Cl
NHCCH2CH3
- Et3N, HCl
R2
R2
S
S
2d-e
1d-e
d: R2 = CH3CH2S, e: R2 = CH3CH2
Scheme 2. Synthesis of amide derivatives 2(a–e).
S
H2NCNH2
EtOH/refluxed
- (H2N)2C=O
N
SH
S
4a-c
N
N
S
N
R1
HCl/H2O
Cu
NH2 NaNO2
+
Cl
S
3a-c
R1
R1
NHCH2CH2SH.HCl
1a-c
H2NCH2CH2SH
THF/refluxed
S
a: R1 = H, b: R1 = CH3, c: R1 = CH3CH2O
R1
5a-c
S
H2NCNH2
EtOH/refluxed
- (H2N)2C=O
N
N
R2
R2
SH
S
N
N
N
S
N
HCl/H2O
4d-e
NaNO2
+
NH2
Cu
R2
Cl
R2
S
N
N
3d-e
NHCH2CH2SH.HCl
H2NCH2CH2SH
THF/refluxed
1d-e
S
d: R2 = CH3CH2S, e: R2 = CH3CH2
5d-e
Scheme 3. Synthesis of thiol 4(a–e) and aminothiol 5(a–e) derivatives.

D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
5277
O
S
CH2CH2Br
N
Br2S(O)
toluene or
acetonitrile
CH2CH2OH
N
CH2CH2S
N
OH
Na2S2O3
H2O/EtOH
O
NH.HBr
NH.HBr
NH.HBr
refluxed
S
refluxed
S
R1
S
R1
7a-c
6a-c
R1
8a-c
O
S
CH2CH2S
N
OH
refluxed
3 h
Na2S2O3
H2O/EtOH
1a-e
+
BrCH2CH2OH
O
N
NH.HBr
R2
refluxed
S
CH2CH2OH
CH2CH2Br
N N
Br2S(O)
toluene or
acetonitrile
8d-e
N
N
O
NH.HBr
NH.HBr
R2
R2
S
S
refluxed
CH2CH2SP
OH
6d-e
7d-e
Li3SPO3
H2O/DMF
N
N
OH
NH
- 2LiBr
R2
S
9d-e
Scheme 4. Synthesis of thiosulfonic acids 8(a–e) and phosphorothioates derivatives 9(a–e).
alcohol group was substituted by bromine by reaction with thionyl
bromide to give compounds 7(a–e).
3. Biological studies
In the last step, thiosulfate derivatives 8(a–e) were obtained by
treatment of an aqueous solution of sodium thiosulfate with corre-
sponding hydrobromides 7(a–e) in a mixture of water/ethanol, at
reflux (Scheme 4).
Finally, lithium hydrogen phosphorothioates 9(d–e) were pre-
pared from lithium thiophosphate and the appropriate hydrobro-
mide 7(d–e) by a standard reaction procedure (Scheme 4).22,23
Indeed, treatment of an aqueous solution of lithium thiophosphate
with hydrobromides followed by dilution of the aqueous reaction
mixture with dimethylformamide and through washing of the
resulting precipitate obtained after concentration with acetone
and methanol yielded a solid which gave the expected 31P NMR
resonance chemical shift.
3.1. Antioxidant properties
The majority of these compounds were subjected to antioxidant
activity screening by determining the DPPH or ABTS free radical
scavenging using simple UV spectroscopic methods.25,26 Precursor
compounds 1(a–e) have been also tested to demonstrate the effect
of the structural modifications on antioxidant activity in compari-
son to amifostine (WR-2721) or ascorbic acid.
Indeed, for many of them it was not possible to calculate an IC50
which was superior to 4 mM. As shown in Table 1, amines and
amides derivatives did not display free radical scavenging activity
towards DPPH. This low activity is most probably due to the dis-
ability of amine and amide to catch radicals.
The use of trilithium rather than trisodium phosphorothioate
makes the isolation of phosphorothioates from thiadiazole easier
9(d–e).
The aminoalcohols derivatives 6c, 6d and 6e, exhibit an IC50
lower than 4 mM but these values are still very high compared
to WR-2721 (0.1 mM) or thiols and aminothiols. This bears out
the higher efficiency of thiol and phosphorothioates over alcohols.
This in vitro evaluation has been previously reported for thiol
and aminothiol derivatives.27 It shows that thiol derivatives of ben-
zothiazole and thiadiazole are the best antioxidant compounds to-
ward the DPPH radical. Their radical scavenging is nearly the same
as WR-2721’s for compounds 4a, 4b, 4e. Compounds 4c
(IC50 = 0.046 mM) and 4d (IC50 = 0.053 mM) show a strong antiox-
idant property which is around half more than WR-2721. But,
ascorbic acid remains the best antioxidant compound.
The preparation of the corresponding phosphorothioate from
benzothiazole by a similar procedure leads to three compounds
(Scheme 5). One of these three compounds is an annulated deriva-
tive which was characterized by 31P NMR and mass spectrum. The
two other compounds correspond to O-alkylphosphorothioate
with a chemical shift at 37.23 ppm and S-alkylphosphorothioate
with a chemical shift at 15.85 ppm in 31P NMR.24 Only the annu-
lated (benzothiazol-2-yliden-amino) phosphorothioate was iso-
lated. O-alkylphosphorothioate and S-alkylphosphorothioate
seem to undergo a rearrangement to the annulated form which
is more stable in solution (Scheme 5).
This good activity of thiol derivatives of benzothiazole and thia-
diazole shows that the hypothesis of a direct link between thiol
O
OH
CH2CH2SP
OLi
N
NH
CH2CH2Br
N
S
O
OH
OH
Li3SPO3
N
S
+
NH.HBr
NH SP
S
H2O/DMF
S
OH
CH2CH2OP
OLi
N
NH
S
Scheme 5. Rearrangement of O- and S-alkylphosphorothioates.

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D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
Table 1
Table 2
Free radical scavenging activity of compounds for DPPH radical
Free radical scavenging activity of compounds for ABTS radical
a
a
Chemical family
Compound IC50 (mM)
Inhibitionb
(%)
Chemical family
Compound
IC50 (lM)
Ascorbic acid
46.46 0.33
79.84 3.55
76.66 5.97
88.35 3.28
23.08 2.17
21.26 1.41
WR-2721
Ascorbic
acid
0.100 0.005
0.020 0.004
—
—
Thiols derivatives of
Benzothiazole
4a
4b
4c
4d
4e
Trolox
0.034 0.006
Glutathion 0.096 0.008
—
—
Thiadiazole
Amine derivatives of
Amide derivatives of
Thiols derivatives of
Benzothiazole 1a
>4
>4
>4
>4
>4
6.03 0.57
5.57 0.98
9.03 1.01
9.57 0.68
4.8 0.78
Aminothiols derivatives of
Benzothiazole
5a
5b
5d
1520 90
4040 62
390 30
1b
1c
1d
1e
Thiadiazole
Thiadiazole
Thiadiazole
Thiosulfonic acids derivatives of
8d
8e
8.32 0.12
51.85 3.06
Benzothiazole 2a
>4
>4
>4
>4
>4
19.1 0.11
2.99 0.15
5.49 0.89
7.21 0.56
2.62 0.76
Phosphorothioates derivatives of
Thiadiazole
9d
9e
76.87 0.99
90.33 2.56
2b
Thiadiazole
2c
2d
2e
a
IC50: Concentration of test compounds needed to reduce ABTS absorption by
50% at 732 nm. Values are means of three independent determinations.
Benzothiazole 4a
0.092 0.006
0.091 0.004
0.046 0.005
0.053 0.006
0.084 0.005
—
—
—
—
—
These observations on thiols and aminothiols are corroborated
by the ABTS test. Toward the [ABTS]+Å radicals, thiols 4(a–e) are al-
ways good antioxidants with the same tendency for thiadiazole
ring which is more effective than benzothiazole ring. Futhermore,
aminothiols stay less efficient than thiols.
Under our experimental conditions, it was not possible to eval-
uate free radical scavenging activity towards the DPPH radical of
thiosulfonic acids and phosphorothioates because of the lack of
solubility of these compounds in ethanol. As a result, we used
the 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical
cation discoloration assay (ABTS test).
4b
4c
Thiadiazole
4d
4e
Aminothiols
derivatives of
Benzothiazole 5a
1.39 0.49
3.17 0.85
0.11 0.20
—
—
—
5b
Thiadiazole
5d
Aminoalcool
derivatives of
Benzothiazole 6a
>4
>4
1.4 0.5
3.5 0.8
2.4 0.6
—
—
—
—
—
6b
6c
Thiadiazole
6d
6e
As shown in Table 2, thiosulfonic acids and phosphorothioates
compounds displayed various degrees of free radical scavenging
activity towards the ABTS radical, with decreasing activity in the
following order: 8d > 8e > 9d > 9e. The most potent compound is
a
IC50: Concentration of test compounds needed to reduce DPPH absorption by
50% at 516 nm. Values are means of three independent determinations.
b
Inhibition (%) indicates the percent inhibition at 4 mM of test compounds.
the thiosulfonic acid 8d with an antiradical effect (IC50 = 8.32
higher than ascorbic acid (IC50 = 46.46 M). Other compounds
demonstrated potent antioxidant activity comparable to that of
ascorbic acid for 8e (IC50 = 51.85 M), or lower for compounds 9d
(IC50 = 76.87 M) and 9e (IC50 = 90.33 M). These results demon-
strate that thiosulfonic acids are more effective antioxidants than
the corresponding phosphorothioates. This could be explained by
the lack of alkaline phosphatase in the medium that is required
to convert phosphorothioate to the corresponding thiol active form
lM)
function and an aromatic ring was a good one. The thiol catches the
radical and after, the aromatic ring permits the trapping of this
radical. Moreover, aminothiol derivatives of benzothiazole 5a and
5b (IC50 = 1.39 mM and IC50 = 3.17 mM respectively) do not seem
to be strong radical scavengers but the aminothiol derivative of
thiadiazole 5d (IC50 = 0.11 mM) shows a better activity than WR-
2721 (0.1 mM). So, the aromatic ring interferes with the radical
scavenging: that thiadiazoles are usually better scavengers than
benzothiazoles may be due to the efficiency of the trapping.
l
l
l
l
Table 3
Radioprotective effect of some thiol and aminothiol derivatives
Compound
LD50tox/48H (mg kgꢀ1
>960
)
Posology (mg kgꢀ1
480
)
Animal model
Irr. dose (Gy)
Survival at day 30 (%) [t, min]a
4a
B6D2F1 mice
CD1 mice
8.8
8.2
10.2
8.8
100 [15]
60 [90]
0 [90]
240
260
B6D2F1 mice
B6D2F1 mice
90 [15]
4b
>520
8.8
10.8
8.2
100 [15]
0 [15]
80 [90]
0 [90]
CD1 mice
10.2
4d
4e
5a
5d
698
349
340
308
368
CD1 mice
CD1 mice
CD1 mice
CD1 mice
8.2
10.2
8.2
10.2
8.2
10.2
8.2
0 [90]
0 [90]
30 [90]
0 [90]
60 [90]
0 [90]
0 [90]
0 [90]
>680
>616
>736
10.2
WR-2721
800
400
B6D2F1 mice
CD1 mice
8.8
10.8
8.2
100 [15]
60 [15]
50 [90]
20 [90]
10.2
a
Time elapsed between injection of the drug and the beginning of the irradiation.

D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
5279
as is well known for WR-2721. IC50 of compounds 8(a–c) were not
determined because of lack of solubility.
and 100% in B6D2F1 treated 15 min before exposure. This good
radioprotective effect has been suggested by results of the
in vitro DPPH and ABTS studies (IC50 = 92 lM and 77 lM, respec-
3.2. In vivo evaluation
tively) or plasmide test.27 However no compound has a radiopro-
tective efficacy at LD99.9/30days-IRR + 2 Gy whatever the mice strain
and the delay of administration.
In vitro studies show that compounds 4(a–e), 5(a,b,d), 6(c,d,e)
and 8d exhibit an antioxidant effect. Compounds 6(c,d,e) are low
radical quenchers and this function usually not give good radiopro-
tection compared to thiols or phosphorothioates. Another test on
irradiated plasmids27 seems to show compounds 4(a–e) and 5d
(total DNA protection for concentration lower than 200
in a merest way 5a (more than 50% of DNA protection for concen-
tration lower than 200 M), as very good candidates for in vivo
4. Conclusion
In conclusion, we have synthesized new derivatives from ami-
nobenzothiazole and thiadiazole. Results of the antioxidant activ-
ity evaluation have demonstrated that only thiols, thiosulfonic
acids and phosphorothioates exhibit evident antioxidant activity.
The antioxidant properties have led us to expect that these com-
pounds could be potent radioprotective agents in comparison with
WR-2721.
lM) and,
l
studies. So, in order to complete the study of these compounds,
we tested the toxicity and the radioprotective effectiveness of
these compounds in vivo on two strains of mice.
In a preliminary study, we determined lethal doses for gamma
irradiations (60Co, 300 mGy minꢀ1) on 6 weeks old male mice: CD1
mice and B6D2F1 mice (Janvier, France). LD50/30days-IRR and
LD99.9/30days-IRR defined as irradiation doses which kill, respectively
50% and 99.9% of the animals 30 days after exposure, were deter-
mined by probit analysis. For these determinations, 20 mice of each
strain and for each irradiation dose were placed in a Plexiglas box
divided into 30 individual cells in an homogeneous field
28.5 cm ꢁ 28.5 cm in size, and exposed at different doses from 6.5
to 10 Gy by 0.5 Gy step. Dosimetry was checked with an ionization
chamber dosimeter (Dosimentor PTW, USA). In these conditions,
LD50/30days-IRR and LD99.9/30days-IRR on CD1 mice were estimated at
6.8 Gy and 8.2 Gy, respectively. The irradiation dose on B6D2F1 mice
was 7.4 and 8.8 Gy, respectively.
In vivo tests showed an efficient radioprotective effect at
LD99.9/30days-IRR for the compounds 4a and 5a and particularly for
2-mercapto-6-methylbenzothiazole 4b compared to WR-2721.
So, the new hypothesis of a direct link between the thiol func-
tion for catching the radical and the aromatic ring for the trapping
of this radical, gives very encouraging results for radioprotection.
Modifications of these structures are in progress in order to in-
crease the radioprotective effect of benzothiazole derivatives.
5. Experimental
Most reagents were purchased and all solvents were freshly dis-
tilled from sodium benzophenone or P2O5 before use. 1H NMR and
13C NMR spectra were recorded on Bruker AC 200 and 300 NMR
spectrometers operating at 200.1, 300.1 MHz and 75.4 MHz (chem-
ical shifts in ppm relative to TMS as internal standard), 31P NMR
spectra were recorded on an Bruker AC 300 spectrometer operating
at 121.5 MHz (chemical shifts in ppm relative to phosphorothioic
acid as internal standard). Mass spectra under electron impact
(EI) conditions were obtained on a Nermag R10-10H spectrometer.
Mass spectra under electrospray (ESI) and fast atom bombardment
(FAB) conditions were obtained on Qtrap and API365 (Applied Bio-
systems). IR spectra were recorded on Perkin–Elmer 1760 FT-IR.
Melting points were taken uncorrected on a Leitz Biomed hot-plate
microscope apparatus or, in capillary tubes, on a digital electro-
thermal apparatus. Elemental analyses (C, H and N) were per-
formed at the « Ecole Nationale Supérieure de Chimie », Toulouse,
France.
In a second experiment, for each of the tested molecules, the
acute (48 h) toxicity was evaluated in male CD1 mice. The mole-
cules were injected intraperitoneally at different concentrations
to determine the LD50tox/48H, defined as the concentration which
kills 50% of the animals at 48 h.
Then, compounds were injected intra peritoneally to 10 CD1
mice at the maximum tolerated dose (defined as 1/2LD50tox/48H
)
90 min before radiation exposure at doses LD99.9/30days-IRR (8.2 Gy)
and LD99.9/30days-IRR + 2 Gy (10.2 Gy). Survival rate was determined
30 days after exposure. WR 2721 (at 1/2 LD50tox/48H) was used as a
reference. To confirm results obtained in CD1 mice, compounds
4(a–b), and WR-2721 were tested in another experimental model:
they were administered 15 min before irradiation (instead of
90 min) in B6D2F1 mice exposed at doses of 8.8 Gy or 10.8 Gy.
The results are reported in Table 3.
The first comment is that our tested compounds are non toxic
(in most case, LD50 cannot be determined because of lack of solu-
bility) which indicates the possibility of development of radiopro-
tective drugs to prevent accidental irradiation.
5.1. Syntheses of the amides derivatives 2(a–e)
5.1.1. N-Benzothiazol-2-yl-propylamide (2a)
To a solution of 2-aminobenzothiazole (1.54 g, 10.26 mmol) and
freshly distilled triethylamine (2.02 g, 20 mmol) in tetrahydrofu-
rane (50 ml) was added a solution of 1.85 g of propionyl chloride
(20 mmol) in THF (20 ml). The reaction mixture was stirred at room
temperature for 3 h. The solvent was evaporated under reduced
pressure, afforded a solid residue which was washed with water
(30 ml) and hexane (30 ml). The solid obtained was crystallized in
methanol to give compound 2a (0.74, 36%). Mp: 198–190 °C. 1H
Considering thiol compounds 4(a,b,d,e), this study reveals that
benzothiazole derivatives are better radioprotectors than thiadia-
zole derivatives even if thiadiazole 4d presents the best antioxi-
dant activity measured in vitro. What’s more, the tendency
observed during the antioxidant screening in which thiadiazoles
are better than benzothiazoles is not observed in vivo. In fact, here,
that benzothiazoles are in a global way better than thiadiazoles
may be due to better vectorisation of the drug.
3
NMR (DMSO-d6, 300.1 MHz): 1.12 (3H, t, JH–H = 9.0 Hz, CH2CH3),
Comparing thiol and aminothiol derivatives of benzothiazole, in
3
2.50 (2H, q, JH–H = 9.0 Hz, CH2CH3), 7.27–7.98 (4H, m, C6H4). 13C
vitro tests show that thiol derivative 4a (IC50 DPPH = 92
ter antioxidant compound than aminothiol derivative 5a
(IC50 DPPH = 1390 M) but these two compounds present a similar
in vivo radioprotective effect in CD1 mice. Note that compound
4a is effective even at 1/4LD50tox/48H
lM) is a bet-
NMR (DMSO-d6, 75.4 MHz): 9.42 (CH2CH3), 28.93 (CH2CH3),
120.90 (C-aryl), 122.13 (C-aryl), 123.87 (C-aryl), 126.51 (C-aryl),
131.90 (C-aryl), 149.04 (C-aryl), 158.44 (C–NH), 173.47 (C@O). Mass
spectrum: m/z = 206 [Mꢀ56]+. Anal. Calcd for C10H10N2OS: C, 58.23;
H, 4.89; N, 13.58. Found: C, 58.02; H, 4.64; N, 14.04%.
Compounds 2(b–e) was also prepared using the general proce-
dure described for 2a.
l
.
Moreover, the thiol derivative 4b presents a very interesting
radioprotective effect in mice exposed to LD99.9/30days-IRR with a
survival rate of 80% in CD1 mice treated 90 min before irradiation

5280
D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
5.1.2. N-(6-Methylbenzothiazol-2-yl)-propylamide (2b)
5.2.2. 2-Chloro-6-methylbenzothiazole (3b)
Yield 52%. Mp: 221–222 °C. 1H NMR (DMSO-d6, 300.1 MHz):
Yield 53%. Mp: 50–52 °C. 1H NMR (CDCl3, 300.1 MHz): 2.32 (3H,
s, CH3), 7.01–7.81 (4H, m, C6H3). 13C NMR (CDCl3, 75.4 MHz): 21.53
(CH3), 120.82 (C-aryl), 122.34 (C-aryl), 128.19 (C-aryl), 136.02 (C-
aryl), 149.03 (C-aryl), 152.04 (C-aryl), 172.04 (C–Cl). Mass spec-
trum: m/z = 184 [M+1]+. Anal. Calcd for C8H6ClNS: C, 52.32; H,
3.29; N, 7.63. Found: C, 52.04; H, 3.34; N, 7.58.
3
3
1.11 (3H, t, JH–H = 9.0 Hz, CH2CH3), 2.40 (3H, q, JH–H = 9.0 Hz,
CH2CH3 and CH3), 7.22–7.74 (3H, m, C6H3), 12.21 (1H, s, NH). 13C
NMR (DMSO-d6, 75.4 MHz): 9.46 (CH2CH3), 21.45 (CH3), 28.90
(CH2CH3), 120.55 (C-aryl), 121.74 (C-aryl), 127.82 (C-aryl), 132.05
(C-aryl), 133.30 (C-aryl), 147.01 (C-aryl), 157.57 (C–NH), 173.32
(C@O) ppm. Mass spectrum: m/z = 220 [Mꢀ56]+. Anal. Calcd for
C11H12N2OS: C, 59.97; H, 5.49; N, 12.72. Found: C, 59.59; H, 5.15;
N, 12.56.
5.2.3. 2-Chloro-6-ethoxybenzothiazole (3c)
Yield 64%. Mp: 54–56 °C. 1H NMR (CDCl3, 300.1 MHz): 1.35 (3H,
3
3
t, JH–H = 6.0 Hz, CH3), 4.07 (2H, q, JH–H = 6.0 Hz, OCH2), 7.09–7.85
(3H, m, C6H3). 13C NMR (CDCl3, 75.4 MHz): 15.00 (CH3), 64.23
(CH2), 106.05 (C-aryl), 116.78 (C-aryl), 123.43 (C-aryl), 137.57 (C-
aryl), 145.04 (C-aryl), 149.73 (C-aryl), 157.37 (C–Cl). Mass spec-
trum: m/z = 214 [M+1]+. Anal. Calcd for C9H8ClNOS: C, 50.59; H,
3.77; N, 6.55. Found: C, 49.17; H, 3.76; N, 6.80.
5.1.3. N-(6-Ethoxybenzothiazol-2-yl)-propylamide (2c)
Yield 53%. Mp: 206–208 °C. 1H NMR (DMSO-d6, 300.1 MHz):
3
1.11 (3H, t, JH–H = 9.0 Hz, –(CO)CH2CH3), 1.34 (3H, t, J = 9.0 Hz, –
3
O–CH2CH3), 2.45 (2H, q, JH–H = 9.0 Hz, –(CO)CH2CH3), 4.06 (2H, q,
3JH–H = 9.0 Hz, –O–CH2CH3), 6.98–7.61 (3H, m, C6H3), 12.14 (1H, s,
NH). 13C NMR (DMSO-d6, 75.4 MHz): 9.47 [(C@O)CH2CH3], 15.18
(OCH2CH3), 28.85 [(C@O)CH2CH3], 64.05 (OCH2CH3), 105.82 (C-
aryl), 115.62 (C-aryl), 121.47 (C-aryl), 133.18 (C-aryl), 143.05 (C-
aryl), 155.74 (C-aryl), 156.40 (C–NH), 173.15 (C@O). Mass spec-
trum: m/z = 250 [Mꢀ56]+. Anal. Calcd for C12H14N2O2S: C, 57.58;
H, 5.64; N, 11.19. Found: C, 57.31; H, 5.16; N, 11.07.
5.2.4. 2-Chloro-5-(ethylthio)-1,3,4-thiadiazole (3d)
3
Yield 56%. 1H NMR (CDCl3, 300.1 MHz): 1.37 (3H, t, JH–H
=
3
6.0 Hz, CH3), 3.24 (2H, q, JH–H = 6.0 Hz, OCH2). 13C NMR (CDCl3,
75.4 MHz): 14.44 (CH3), 28.38 (CH2CH3), 152.55 (C5), 168.15 (C2).
Mass spectrum: 181 [M+1]+. Anal. Calcd for C4H5ClN2S2: C, 26.59;
H, 2.79; N, 15.50. Found: C, 26.44; H, 2.57; N, 16.61.
5.1.4. N-[5-(Ethylthio)-1,3,4-thiadiazol-2-yl]-propylamide (2d)
Yield 96%. Mp: 163–165 °C. 1H NMR (DMSO-d6, 300.1 MHz):
5.2.5. 2-Chloro-5-ethyl-1,3,4-thiadiazole (3e)
3
3
3
1.09 (3H, t, JH–H = 9.0 Hz, –(O@C)CH2CH3), 1.33 (3H, t, JH–H
=
Yield 79%. 1H NMR (CDCl3, 300.1 MHz): 1.31 (3H, t, JH–H
=
3
3
9.0 Hz, –SCH2CH3), 2.47 (2H, q, JH–H = 9.0 Hz, –SCH2CH3), 3.21
6.0 Hz, CH3), 3.00 (2H, q, JH–H = 6.0 Hz, OCH2). 13C NMR (CDCl3,
75.4 MHz): 14.32 (CH3), 24.35 (CH2CH3), 153.85 (C5), 175.05 (C2).
Mass spectrum: m/z = 149 [M+1]+. Anal. Calcd for C4H5ClN2S: C,
32.33; H, 3.39; N, 18.85. Found: C, 32.56; H, 3.45; N, 18.56.
3
(2H, q, JH–H = 9.0 Hz, –(O@C)CH2CH3), 12.53 (1H, s, NH). 13C NMR
(DMSO-d6, 75.4 MHz): 9.42 [(C@O)CH2CH3], 15.22 (SCH2CH3),
28.52 [(C@O)CH2CH3], 28.57 (SCH2CH3), 158.76 (C5), 159.08 (C2),
172.79 (C@O). Mass spectrum: m/z = 217 [Mꢀ56]+. Anal. Calcd for
C7H11N3OS2: C, 38.69; H, 5.10; N, 19.34. Found: C, 38.70; H, 4.91;
N, 19.16.
5.2.6. 2-Mercaptobenzothiazole (4a)
A mixture of 3a (2.50 g, 14.79 mmol) and excess thiourea
(3.03 g, 47.33 mmol) in 36 ml of ethanol was refluxed for 3 h.
The mixture was cooled down to room temperature and a solution
of HCl 37% (5 ml) in 50 ml of water was added dropwise under stir-
ring, the solid formed was isolated by filtration and the aqueous
layer was extracted by CHCl3 (2 ꢁ 100 ml). The combined organic
layers were dried on Na2SO4. The solvent was removed in vacuo.
Recrystallization from ethanol gave 4a as a white solid (0.70 g,
5.1.5. N-(5-Ethyl-1,3,4-thiadiazol-2-yl)propylamide (2e)
Yield 96%. Mp: 224–226 °C. 1H NMR (DMSO-d6, 300.1 MHz):
3
3
1.09 (3H, t, JH–H = 9.0 Hz, (–(C@O)CH2CH3), 1.29 (3H, t, JH–H
=
3
9.0 Hz, CH3CH2), 2.47 (2H, q, JH–H = 9.0 Hz, CH3CH2), 2.97 (2H, q,
3JH–H = 9.0 Hz, –(C@O)CH2CH3), 12.31 (1H, s, NH). 13C NMR
(DMSO-d6, 75.4 MHz): 9.53 [(C@O)CH2CH3], 14.29 (CH2CH3),
23.10 [(C@O)CH2CH3], 28.66 (CH2CH3), 158.59 (C5), 165.64 (C2),
172.54 (C@O). Mass spectrum: m/z = 185 [Mꢀ56]+. Anal. Calcd for
C7H11N3OS: C, 45.39; H, 5.99; N, 22.68. Found: C, 45.10; H, 5.48;
N, 22.32.
28%). Mp 170–172 °C. IR (KBr): m .
SH = 2664 cmꢀ1 1H NMR (DMSO-
d6, 300.1 MHz): 3.49 (1H, s, SH), 7.15–7.48 (4H, m, C6H4). 13C
NMR (DMSO-d6, 75.4 MHz): 112.35 (C-aryl), 121.37 (C-aryl),
124.70 (C-aryl), 127.23 (C-aryl), 130.02 (C-aryl), 140.33 (C-aryl),
190.92 (C-SH). Mass spectrum: m/z = 167 [M]+Å. Anal. Calcd for
C7H5NS2: C, 50.27; H, 3.01; N, 8.37. Found: C, 50.55; H, 2.82; N,
8.86.
5.2. Syntheses of the thiols and aminothiols derivatives 4(a–e)
and 5(a–e)
Compound 4(b-e) was obtained by using the same procedure
described for 4a.
5.2.1. 2-Chlorobenzothiazole (3a)
To a stirred solution of hydrogen chloride (105 ml, 1.06 mol)
with copper powder (1.70 g, 26.75 mmol) in 45 ml of water, a mix-
ture of 2-aminobenzothiazole (5 g, 33.3 mmol) and an excess of
NaNO2 (6.83 g, 100 mmol), was added slowly at ꢀ5 °C. The reaction
mixture was stirred for 2 h at room temperature and heated at
55 °C until the evolution of gas ceased. The reaction mixture was
extracted by CHCl3 (3 ꢁ 200 ml), the combined organic extracts
were washed with sulfuric acid (10%, 100 ml), dried over Na2SO4,
filtered and concentrated in vacuo. Compound 3a is a red oil
(5.15 g, 94%). 1H NMR (CDCl3, 300.1 MHz): 7.34–7.95 (4H, m,
C6H4). 13C NMR (CDCl3, 75.4 MHz): 121.84 (C-aryl), 123.55 (C-aryl),
126.50 (C-aryl), 127.42 (C-aryl), 150.71 (C-aryl), 153.56 (C-aryl),
172.75 (C–Cl). Mass spectrum: m/z = 169 [M]+Å. Anal. Calcd for
C7H4ClNS: C, 49.56; H, 2.38; N, 8.26. Found: C, 49.08; H, 2.11; N,
9.01.
5.2.7. 2-Mercapto-6-methylbenzothiazole (4b)
Yield 87%. Mp: 150–152 °C. IR (KBr): m .
SH = 2509 cmꢀ1 1H NMR
(DMSO-d6, 300.1 MHz): 2.33 (3H, s, CH3), 3.59 (1H, s, SH), 7.16–
8.05 (4H, m, C6H3). 13C NMR (DMSO-d6, 75.4 MHz): 21.19 (CH3),
112.68 (C-aryl), 123.74 (C-aryl), 128.55 (C-aryl), 130.56 (C-aryl),
134.46 (C-aryl), 151.73 (C-aryl), 184.33 (C-SH). Mass spectrum:
m/z = 182 [M+1]+. Anal. Calcd for C8H7NS2: C, 53.00; H, 3.89; N,
7.73. Found: C, 52.60; H, 4.03; N, 7.99.
5.2.8. 2-Mercapto-6-ethoxybenzothiazole (4c)
Yield 63%. Mp: 180–182 °C. IR (KBr): m .
SH = 2509 cmꢀ1 1H NMR
3
(DMSO-d6, 300.1 MHz): 1.32 (3H, t, JH–H = 6.0 Hz, CH3), 3.56 (1H,
3
s, SH), 4.0 (2H, q, JH–H = 6.0 Hz, CH2), 6.94–7.31 (4H, m, C6H4).
13C NMR (DMSO-d6, 75.4 MHz): d = 15.05 (CH3), 64.19 (CH2),
General procedure described for 3a was used for the synthesis
of compounds 3(b–e).
106.93 (C-aryl), 113.55 (C-aryl), 115.63 (C-aryl), 131.14 (C-aryl),

D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
5281
3
3
135.58 (C-aryl), 156.31 (C-aryl), 188.75 (C-SH). Mass spectrum: m/
z = 212 [M+1]+. Anal. Calcd for C9H9NOS2: C, 51.16; H, 4.29; N, 6.63.
Found: C, 51.03; H, 3.85; N, 7.20.
(2H, t, JH–H = 6.0 Hz, NHCH2), 3.29 (2H, q, JH–H = 6.0 Hz, CH2CH3),
3
3.36 (1H, s, SH), 3.55 (2H, t, JH–H = 6.0 Hz, NHCH2), 8.32 (1H, s,
NH). 13C NMR (DMSO-d6, 75.4 MHz): 14.97 (CH3), 31.21 (CH2S),
38.55 (NHCH2), 39.07 (CH2SH), 164.10 (C5), 166.23 (C2). Mass spec-
trum: m/z = 222 [M+1]+. Anal. Calcd for C6H12ClN3S3: C, 27.95; H,
4.69; N, 16.30. Found: C, 27.78; H, 4.75; N, 16.41.
5.2.9. 2-Mercapto-5-(ethylthio)-1,3,4-thiadiazole (4d)
Yield 73%. Mp: 102–104 °C. IR (KBr): m .
SH = 2528 cmꢀ1 1H NMR
3
(DMSO-d6, 300.1 MHz): 1.31 (3H, t, JH–H = 9.0 Hz, CH3), 3.16 (2H,
3
q, JH–H = 9.0 Hz, CH2), 7.03 (1H, s, SH). 13C NMR (DMSO-d6,
5.2.15. 2-[5-(Ethyl)-1,3,4-thiadiazol-2-ylamino]ethanethiol
hydrochloride (5e)
Yield 44%. IR. (KBr):
NMR (DMSO-d6, 300.1 MHz): 1.37 (3H, t, JH–H = 6.0 Hz, CH2SH),
75.4 MHz,): 14.94 (CH3), 28.17 (CH2), 162.13 (C5), 188.32 (C2). Mass
spectrum: m/z = 179 [M+1]+. Anal. Calcd for C4H6N2S3: C, 26.95; H,
3.39; N, 15.71. Found: C, 25.73; H, 3.56; N, 16.77.
m . m .
SH = 2036 cmꢀ1 NH = 3372 cmꢀ1 1H
3
3
3
3.06 (2H, t, JH–H = 6.0 Hz, NHCH2), 3.43 (2H, q, JH–H = 6.0 Hz,
3
5.2.10. 2-Mercapto-5-ethyl-1,3,4-thiadiazole (4e)
CH2CH3), 3.61 (1H, s, SH), 3.80 (2H, t, JH–H = 6.0 Hz, NHCH2),
Yield 34%. Mp: 68–70 °C. IR (KBr):
(DMSO-d6, 300.1 MHz): 1.19 (3H, t, JH–H = 9.0 Hz, CH3), 2.00 (2H,
m
SH = 2523 cmꢀ1
.
1H NMR
8.62 (1H, s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 13.99 (CH3),
23.82 (CH2S), 32.92 (NHCH2), 39.78 (NHCH2), 164.29 (C5),
172.78 (C2). Mass spectrum: m/z = 188 [M+1]+. Anal. Calcd for
C6H12ClN3S2: C, 31.92; H, 5.36; N, 18.61. Found: C, 31.68; H,
5.55; N, 18.66.
3
3
q, JH–H = 9.0 Hz, CH2), 7.03 (1H, s, SH). 13C NMR (DMSO-d6,
75.4 MHz): 12.69 (CH3), 24.40 (CH2), 165.98 (C5), 189.35 (C2). Mass
spectrum: m/z = 146 [M+1]+. Anal. Calcd for C4H6N2S2: C, 32.85; H,
4.14; N, 19.16. Found: C, 32.59; H, 4.04; N, 19.52.
5.3. Syntheses of the thiosulfonic acids 8(a–e) and
phosphorothioates derivatives 9(d–e)
5.2.11. 2-(Benzothiazol-2-ylamino)ethanethiol hydrochloride
(5a)
Solution of cysteamine (0.7 g, 9.00 mmol) in THF (15 ml) was
added to a stirred suspension of 3a (1.5 g, 8.87 mmol) in THF
(30 ml). After heating under argon for 3 h, the solvent was evapo-
rated in vacuo and the yellow solid residue was washed with THF/
pentane (1/3; 30 ml). Drying in vacuo afforded pure 5a (0.65 g,
5.3.1. 2-Iminobenzothiazol-3-yl-ethanol hydrobromide (6a)
A mixture of 2-aminobenzothiazole (10.19 g, 65.89 mmol) and
2-bromoethanol (45.76 g, 366.16 mmol) was heated under reflux
for 3 h. After one more hour stirring at room temperature 200 ml
of diethyl ether were added. The solid formed was isolated by fil-
tration, washed with 50 ml of acetone and 100 ml of diethyl ether.
Recrystallization in ethanol gave compound 6a as a white solid
(11.47 g, 90%). Mp: 200–202 °C (dec). 1H NMR (DMSO-d6,
300.1 MHz): 3.79 (2H, t, JH–H = 6.0 Hz, NCH2), 4.41 (2H, t, JH–
H = 6.0 Hz, CH2OH), 4.71 (1H, s, OH), 7.23–8.03 (4H, m, C4H6),
10.13 (1H, s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 48.13 (NH-
CH2), 58.41 (CH2-OH), 114.18 (C-aryl), 122.74 (C-aryl), 123.54 (C-
aryl), 125.40 (C-aryl), 127.86 (C-aryl), 139.12 (C-aryl), 168.86
(C@NH). Mass spectrum: m/z = 195 [M+1]+. Anal. Calcd for
C9H11BrN2OS: C, 39.28; H, 4.03; N, 10.18. Found: C, 39.43; H,
4.10; N, 9.96.
35%). Mp: 174–176 °C. IR (KBr): m , m .
SH = 2446 cmꢀ1 NH = 3444 cmꢀ1
1H NMR (DMSO-d6, 300.1 MHz): 3.18 (2H, t, 3JH–H = 6.0 Hz, CH2SH),
3
3.36 (1H, s, SH) 3.58 (2H, t, JH–H = 6.0 Hz, NHCH2), 7.29–7.98 (4H,
m, C6H4), 8.49 (1H, s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 29.46
(NHCH2), 38.84 (CH2SH), 120.9 (C-aryl), 125.09 (C-aryl), 125.75
(C-aryl), 134.58 (C-aryl), 152.22 (C-aryl), 164.54 (C–NH). Mass spec-
trum: m/z = 211 [M+1]+. Anal. Calcd for C9H11ClN2S2: C, 43.80;
H, 4.49; N, 11.35. Found: C, 43.67; H, 4.75; N, 11.22.
3
3
Using the same procedure described for 5a, compounds 5(b–e)
were obtained.
5.2.12. 2-(6-Methylbenzothiazol-2-ylamino)ethanethiol
hydrochloride (5b)
Using the same operating conditions described for 6a com-
pounds 6(b–e) were obtained.
Yield 16%. mp 170–172 °C. IR (KBr):
m ,
SH = 2580 cmꢀ1
m
NH = 3434 cmꢀ1
.
1H NMR (DMSO-d6, 300.1 MHz): 2.43 (3H, s,
5.3.2. (2-Imino-6-methylbenzothiazol-3-yl)ethanol
hydrobromide (6b)
3
CH3), 3.26 (2H, t, JH–H = 6.0 Hz, NHCH2), 3.36 (1H, s, SH), 3.64
3
(2H, t, JH–H = 6.0 Hz, CH2SH), 7.30–7.83 (4H, m, C6H3), 8.47 (1H,
Yield 76%. Mp: 232–234 °C (dec). 1H NMR (DMSO-d6,
s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 21.40 (CH3), 30.36 (NHCH2),
38.64 (CH2SH), 121.25 (C-aryl), 121.98 (C-aryl), 128.24 (C-aryl),
134.88 (C-aryl), 135.32 (C-aryl), 151.12 (C-aryl), 164.35 (C–NH).
Mass spectrum: m/z = 225 [M+1]+. Anal. Calcd for C10H13ClN2S2:
C, 46.05; H, 5.02; N, 10.74. Found: C, 45.90; H, 4.95; N, 10.96.
3
300.1 MHz,): 2.39 (3H, s, CH3), 3.77 (2H, t, JH–H = 6.0 Hz, NHCH2),
3
4.39 (2H, t, JH–H = 6.0 Hz, CH2OH), 4.69 (1H, s, OH), 7.24–7.81
(3H, m, C6H3), 10.07 (1H, s, NH). 13C NMR (75.4 MHz, DMSO-d6):
21.16 (CH3), 48.14 (NCH2), 58.42 (CH2OH), 113.89 (C-aryl),
122.67 (C-aryl), 123.33 (C-aryl), 128.74 (C-aryl), 135.22(C-aryl),
137.00 (C-aryl), 168.18 (C@NH). Mass spectrum: m/z = 209
[M+1]+. Anal. Calcd for C10H13BrN2OS: C, 41.53; H, 4.53; N, 9.69.
Found: C, 41.22; H, 4.74; N, 9.79.
5.2.13. 2-(6-Ethoxybenzothiazol-2-ylamino)ethanethiol
hydrochloride (5c)
Yield 33%. 1H NMR (DMSO-d6, 300.1 MHz): 1.34 (3H, t, CH3),
3
3.19 (2H, t, JH–H = 6.0 Hz, CH2SH), 3.34 (1H, s, SH), 3.56 (2H, t,
3JH–H = 6.0 Hz, NHCH2), 4.05 (2H, q, CH3CH2), 7.02–7.84 (4H, m,
C6H3), 8.42 (1H, s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 15.07
(CH3), 31.24 (NHCH2), 38.44 (CH2SH), 64.18 (CH3CH2), 106.01 (C-
aryl), 115.98 (C-aryl), 122.17 (C-aryl), 136.66 (C-aryl), 147.38 (C-
aryl), 156.52 (C-aryl), 162.36 (C2–NH). Mass spectrum: m/z = 225
[M+1]+. Anal. Calcd for C11H15ClN2OS2: C, 45.43; H, 5.20; N, 9.63.
Found: C, 45.29; H, 5.30; N, 9.67.
5.3.3. (2-Imino-6-ethoxybenzothiazol-3-yl)ethanol
hydrobromide (6c)
Yield 86%. Mp: 225–227 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz): 1.35 (3H, t, JH–H = 6.0 Hz, CH2CH3), 3.76 (2H, t,
3
3JH–H = 6.00 Hz, NHCH2), 4.06 (2H, q, JH–H = 6.0 Hz, CH2CH3), 4.37
3
(2H, t, JH–H = 6.0 Hz, CH2OH), 4.91 (1H, s, OH), 7.07–7.67 (3H, m,
C6H3), 9.98 (1H, s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 15.01
(CH3), 48.15 (NHCH2), 62.01 (CH2OH), 64.40 (CH2–O), 108.89 (C-
aryl), 115.14 (C-aryl), 115.47 (C-aryl), 123.98 (C-aryl), 132.99 (C-
aryl), 156.52 (C-aryl), 167.79 (C@NH). Mass spectrum: m/z = 239
[M+1]+. Anal. Calcd for C11H15BrN2O2S: C, 41.39; H, 4.74; N, 8.78.
Found: C, 41.19; H, 4.80; N, 8.92.
5.2.14. 2-[5-(Ethylthio)-1,3,4-thiadiazol-2-ylamino]ethanethiol
hydrochloride (5d)
Yield 16%. IR. (KBr):
(DMSO-d6, 300.1 MHz): 1.38 (3H, t, JH–H = 6.0 Hz, CH2SH), 3.19
m
SH = 2539 cmꢀ1
,
m
NH = 3375 cmꢀ1. 1H NMR
3

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D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
5.3.4. (2-Imino-5-ethylthio-1,3,4-thiadiazol-3-yl)ethanol
hydrobromide (6d)
168.10 (C@NH). Mass spectrum: m/z = 303 [M+1]+. Anal. Calcd for
C11H14Br2N2OS: C, 34.58; H, 3.69; N, 7.33. Found: C, 34.62; H,
3.87; N, 7.11.
Yield 59%. Mp: 136–138 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz,): 1.36 (3H, t, JH–H = 6.0 Hz, CH2CH3), 3.22 (2H, q,
3
3JH–H = 6.0 Hz, CH2CH3), 3.77 (2H, t, JH–H = 6.0 Hz, NHCH2), 4.25
5.3.9. 3-Bromoethyl-5-ethylthio-1,3,4-thiadiazole-2-imine
hydrobromide (7d)
3
(2H, t, JH–H = 6.0 Hz, CH2OH), 4.90 (1H, s, OH), 9.96 (1H, s, NH).
13C NMR (DMSO-d6, 75.4 MHz): 14.78 (CH3), 28.39 (CH2CH3),
53.44 (NHCH2), 58.47 (CH2OH), 154.11 (C5), 167.97 (C2). Mass
spectrum: m/z = 206 [M+1]+. Anal. Calcd for C6H12BrN3OS2: C,
25.18; H, 4.23; N, 14.68. Found: C, 25.25; H, 4.12; N, 14.72.
Yield 63%. Mp: 140–142 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz): 1.35 (3H, t, JH–H = 6.0 Hz, CH2CH3), 3.22 (2H, q,
3
3JH–H = 6.0 Hz, CH2CH3), 3.88 (2H, t, JH–H = 6.0 Hz, CH2Br), 4.61
3
(2H, t, JH–H = 6.0 Hz, NHCH2), 10.18 (1H, s, NH). 13C NMR
(DMSO-d6, 75.4 MHz): 14.72 (CH3), 28.34 (CH2S), 29.70 (CH2Br),
51.63 (CH2NH), 154.77 (C5), 168.01 (C2). Mass spectrum: m/
z = 269 [M]+Å. Anal. Calcd for C6H11Br2N3S2: C, 20.64; H, 3.18; N,
12.04. Found: C, 20.52; H, 3.23; N, 12.11.
5.3.5. (2-Imino-5-ethyl-1,3,4-thiadiazol-2-yl)ethanol
hydrobromide (6e)
Yield 56%. Mp: 150–152 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz): 1.22 (3H, t, JH–H = 6.0 Hz, CH2CH3), 2.90 (2H, q,
3
3JH–H = 6.0 Hz, CH2CH3), 3.74 (2H, t, JH–H = 6.0 Hz, NHCH2), 4.23
5.3.10. 3-Bromoethyl-5-ethyl-1,3,4-thiadiazole-2-imine
hydrobromide (7e)
3
(2H, t, JH–H = 6.0 Hz, CH2OH), 5.45 (1H, s, OH), 9.81 (1H, s, NH).
13C NMR (DMSO-d6, 75.4 MHz): 12.80 (CH2CH3), 23.86 (CH2CH3),
53.00 (NHCH2), 58.47 (CH2OH), 122.67 (C5), 159.58 (C2). Mass
spectrum: m/z = 174 [M+1]+. Anal. Calcd for C6H12BrN3OS: C,
28.36; H, 4.76; N, 16.53. Found: C, 28.42; H, 4.55; N, 16.68.
Yield 30%. Mp: 206–208 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz): 1.23 (3H, t, JH–H = 6.0 Hz, CH2CH3), 2.92 (2H, q,
3
3JH–H = 6.0 Hz, CH2CH3), 3.86 (2H, t, JH–H = 6.0 Hz, CH2Br), 4.60
3
(2H, t, JH–H = 6.0 Hz, NHCH2), 10.11 (1H, s, NH). 13C NMR
(DMSO-d6, 75.4 MHz): 12.76 (CH3), 23.91 (CH2CH3), 29.74 (CH2Br),
51.27 (CH2NH), 160.14 (C5), 168.01 (C2). Mass spectrum: m/z = 237
[M+1]+. Anal. Calcd for C6H11Br2N3S: C, 22.73; H, 3.50; N, 13.25.
Found: C, 22.46; H, 3.71; N, 13.31.
5.3.6. 3-Bromoethylbenzothiazole-2-imine hydrobromide (7a)
Thionyl bromide (1.2 ml, 15.45 mmol) was added to a stirred
suspension of 6a (1 g, 5.15 mmol) in 80 ml of anhydrous toluene.
The reaction mixture was heated under reflux until the evolution
of gas ceased. After cooling down to room temperature, a solid
was isolated by filtration and washed with 50 ml of acetone. The
product was recrystallized from methanol and provided (0.96 g,
72%) of 7a. Mp: 205–207 °C (dec). 1H NMR (DMSO-d6,
5.3.11. S-2-(2-iminobenzothiazol-3-yl-ethyl)thiosulfonic acid
hydrobromide (8a)
A solution of 3-bromoethyl- benzothiazole-2-imine (1.00 g,
3.89 mmol) in ethanol/water (20/10) was added dropwise to a stir-
red solution of sodium thiosulfate (1.02 g, 4.13 mmol) in water
(42 ml). The mixture was heated under reflux for 1 h 30 min and
than cooled down to 4 °C. The solid residue formed gave after fil-
tration and recrystallization from ethanol/water 8a (0.21 g, 19%).
Mp: 230–232 °C (dec). 1H NMR (DMSO-d6, 300.1 MHz): 3.24 (2H,
3
300.1 MHz): 3.87 (2H, t, JH–H = 6.0 Hz, NHCH2), 4.78 (2H, t,
3JH–H = 6.0 Hz, CH2Br), 7.11–8.05 (4H, m, C6H4), 10.41 (1H, s, NH).
13C NMR (DMSO-d6, 75.4 MHz): 28.63 (CH2Br), 46.00 (NHCH2),
114.20 (C-aryl), 122.60 (C-aryl), 124.13 (C-aryl), 125.80 (C-aryl),
128.22 (C-aryl), 138.42 (C-aryl), 168.91 (C@NH). Mass spectrum:
m/z = 259 [M+1]+. Anal. Calcd for C9H10Br2N2S: C, 31.98; H, 2.98;
N, 8.29. Found: C, 31.79; H, 3.04; N, 8.42.
3
3
t, JH–H = 6.0 Hz, CH2S), 4.56 (2H, t, JH–H = 6.0 Hz, NCH2), 7.34–
7.94 (4H, m, C6H4), 10.01 (1H, s, NH). 13C NMR (DMSO-d6:
75.4 MHz): 31.39 (CH2–S), 45.13 (CH2–N), 113.99 (C-aryl), 122.74
(C-aryl), 123.96 (C-aryl), 125.74 (C-aryl), 128.36 (C-aryl), 138.87
(C-aryl), 168.97 (C@NH). Mass spectrum: m/z = 291 [M+1]+. Anal.
Calcd for C9H11BrN2O3S3: C, 29.11; H, 2.99; N, 7.54. Found: C,
29.29; H, 3.05; N, 7.30.
5.3.7. 3-Bromoethyl-6-methylbenzothiazole-2-imine
hydrobromide (7b)
Thionyl bromide (1.6 ml, 20.61 mmol) was added to a stirred
suspension of 6a (1.5 g, 7.21 mmol) in 80 ml of anhydrous acetoni-
trile. The solution was heated until the evolution of gas ceased. The
reaction mixture was cooled down to room temperature and the
solid formed was filtered and washed with 50 ml of diethyl ether.
Recrystallization from methanol gave 7b as a white solid (1.5 g,
80%). Mp: 219–221 °C (dec). 1H NMR (DMSO-d6, 300.1 MHz):
Following the same procedure as the preceding preparation
8(b–e) were obtained.
5.3.12. S-2-(2-Imino-6-methylbenzothiazol-3-yl)-ethyl
thiosulfonic acid hydrobromide (8b)
3
2.38 (3H, s, CH3), 3.86 (2H, t, JH–H = 6.0 Hz, CH2Br), 4.77 (2H, t,
Yield 27%. Mp: 233–235 °C (dec). 1H NMR (DMSO-d6,
3
3JH–H = 6.0 Hz, NHCH2), 7.34–7.82 (4H, m, C6H3), 10.37 (1H, s,
NH). 13C NMR (75.4 MHz, DMSO-d6): 21.27 (CH3), 28.71 (CH2Br),
46.02 (NHCH2), 113.91 (C-aryl), 123.88 (C-aryl), 125.09 (C-aryl),
129.00 (C-aryl), 135.52 (C-aryl), 136.31 (C-aryl), 168.49 (C@NH).
Mass spectrum: m/z = 273 [M]+Å. Anal. Calcd for C10H12Br2N2S: C,
34.11; H, 3.44; N, 7.96. Found: C, 34.46; H, 3.36; N, 7.69.
Using the same operating conditions described for 7b com-
pounds 7c–e were obtained.
300.1 MHz): 2.49 (3H, s, CH3), 3.27 (2H, t, JH–H = 6.0 Hz, CH2S),
3
4.57 (2H, t, JH–H = 6.0 Hz, N–CH2), 7.37–7.77 (3H, m, C6H3), 9.99
(1H, s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 20.68 (CH3), 30.98
(CH2-S), 44.63 (CH2-N), 113.24 (C8-aryl), 123.25 (C5-aryl), 124.51
(C-aryl), 128.72 (C-aryl), 135.04 (C-aryl), 136.29 (C-aryl), 168.10
(C@NH). Mass spectrum: m/z = 305 [M+1]+. Anal. Calcd for
C10H13BrN2O3S3: C, 31.17; H, 3.40; N, 7.27. Found: C, 30.99; H,
3.59; N, 7.26.
5.3.8. 3-Bromoethyl-6-ethoxybenzothiazole-2-imine
hydrobromide (7c)
5.3.13. S-2-(2-Imino-6-ethoxybenzothiazol-3-yl)-ethyl
thiosulfonic acid hydrobromide (8c)
Yield 73%. Mp: 225–227 °C (dec). 1H NMR (DMSO-d6,
Yield 34%. Mp: 256–258 °C (dec). 1H NMR (DMSO-d6,
3
3
300.1 MHz): 1.34 (3H, t, JH–H = 6.0 Hz, CH2CH3), 3.86 (2H, t,
300.1 MHz): 1.36 (3H, t, JH–H = 6.0 Hz, CH3), 3.27 (2H, t,
3
3
3JH–H = 6.0 Hz, CH2Br), 4.05 (2H, q, JH–H = 6.0 Hz, CH2CH3), 4.74
3JH–H = 6.0 Hz, CH2S), 4.08 (2H, q, JH–H = 6.0 Hz, CH2CH3), 4.56
3
3
(2H, t, JH–H = 6.0 Hz, NHCH2), 7.67–7.09 (4H, m, C6H3), 10.22 (1H,
(2H, t, JH–H = 6.0 Hz, N–CH2), 7.12–7.76 (3H, m, C6H3), 9.91 (1H,
s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 15.01 (CH3), 28.86 (CH2Br),
46.06 (NHCH2), 64.42 (CH2CH3), 109.11 (C-aryl), 114.99 (C-aryl),
115.61 (C-aryl), 123.79 (C-aryl), 132.06 (C-aryl), 156.70 (C-aryl),
s, NH). 13C NMR (DMSO-d6, 75.4 MHz): 14.98 (CH3), 31.46 (CH2-
S), 45.12 (CH2-N), 64.43 (CH2CH3), 109.01 (C-aryl), 114.70 (C-aryl),
115.71 (C-aryl), 123.89 (C-aryl), 132.50 (C-aryl), 156.70 (C-aryl),

D. Cressier et al. / Bioorg. Med. Chem. 17 (2009) 5275–5284
5283
167.99 (C@NH). Mass spectrum: m/z = 334 [M+1]+. Anal. Calcd for
C11H17BrN2O4S3: C, 31.81; H, 3.64; N, 6.74. Found: C, 31.70; H,
3.84; N, 6.65.
its radical form, DPPH. has an absorption band at 516 nm which
disappears upon reduction by an antiradical compound. The reac-
tion mixture contained in 3 ml of ethanol, 80 l
M DPPH. and test
compounds at different concentrations. After 30 min at room tem-
perature, the absorbance was recorded at 516 nm on an Agilent
8453. All experiments were carried out in triplicate. The percent-
age of remaining DPPH. (DPPH. REM) was calculated as follows:
5.3.14. S-2-(2-Imino-5-ethylthio)-1,3,4-thiadiazol-ethyl
thiosulfonic acid hydrobromide (8d)
Yield 11%. Mp: 180–182 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz): 1.33 (3H, t, JH–H = 6.0 Hz, CH2CH3), 3.17 (2H, q,
%DPPHÅREM ¼ ½DPPHÅꢃt=½DPPHÅꢃ0 ꢁ 100
For all derivatives, IC50 that is, the concentration of test com-
pound needed to reduce DPPH. absorption by 50% at 516 nm was
determined.
3
3JH–H = 6.0 Hz, CH2CH3), 3.32 (2H, t, JH–H = 6.0 Hz, CH2-S), 4.44
3
(2H, t, JH–H = 6.0 Hz, N–CH2), 9.90 (1H, s, NH). 13C NMR (DMSO-
d6, 75.4 MHz): 13.75 (CH3), 28.59 (CH2CH3), 32.54 (CH2-S), 49.81
(CH2-N), 132.60 (C5), 173.68 (C2). Mass spectrum: m/z = 300
[Mꢀ1]+. Anal. Calcd for C6H12BrN3O3S4: C, 18.85; H, 3.16; N,
10.99. Found: C, 18.63; H, 3.32; N, 11.05.
5.4.2. ABTS test
Secondly, 2,20-azinobis(3-ethylbenz-thiazoline-6-sulfonic acid)
radical cation discoloration assay (ABTS test) was used. The ABTS+Å
radical cation scavenging activity of thiols (4–5), thiosulfonic acids
and phosphorothioates (8–9), and ascorbic acid was determined
according to Re et al. (1999).18 Briefly, to an aqueous solution of
ABTS 7.0 mM, potassium persulfate 140 mM was added to obtain
a final solution of 3.5 mM potassium persulfate. The mixture was
kept overnight in the dark to yield the ABTS+Å radical cation. Prior
to use in the assay, the ABTS+Å radical cation was diluted with eth-
anol to give an initial absorbance of 0.70 at 734 nm, at a controlled
temperature of 30 °C. Free radical scavenging activity was assessed
5.3.15. S-2-(2-Imino-5-ethyl)-1,3,4-thiadiazol-3-yl-ethyl
thiosulfonic acid hydrobromide (8e)
Yield 14%. Mp: 212–214 °C (dec). 1H NMR (DMSO-d6,
3
300.1 MHz): 1.23 (3H, t, JH–H = 6.0 Hz, CH2CH3), 2.95 (2H, q,
3
3JH–H = 6.0 Hz, CH2CH3), 3.07 (2H, t, JH–H = 6.0 Hz, CH2-S), 4.0 2
3
(2H, t, JH–H = 6.0 Hz, N-CH2), 9.90 (1H, s, NH). 13C NMR (DMSO-
d6, 75.4 MHz): 12.79 (CH3), 24.10 (CH2CH3), 35.39 (CH2-S), 49.29
(CH2-N), 159.72 (C5), 168.00 (C2). Mass spectrum: m/z = 174
[M+1]+. Anal. Calcd for C6H12BrN3O3S3: C, 20.57; H, 3.45; N,
12.00. Found: C, 20.34; H, 3.65; N, 12.03.
by mixing 1.5 ml diluted ABTS+Å radical cation with 10
lL of test
5.3.16. S-2-(2-Imino-5-ethylthio)-1,3,4-thiadiazol-ethyl lithium
hydrogen phosphorothioate (9d)
compound at different concentrations and monitoring the change
in absorbance at 0, 0.5, 1 min after the addition and again at
5 min intervals until a steady state was achieved. The antioxidant
capacity of test compounds was expressed as IC50, the concentra-
tion necessary to obtain 50% reduction of ABTS+Å radical cation.
3-Bromoethyl-5-ethylthio-1,3,4-thiadiazol-2-imine 7d (1.92 g,
5.50 mmol) was added in portions to a stirred solution of Li3S-
PO3ꢂ5H2O (0.99 g, 5.32 mmol) in water (24 ml). 12 ml of DMF were
added and the solution was stirred at room temperature for 3 h.
The solvent was removed in vacuo. The residual was washed in
methanol and dried under reduced pressure to give 9d (0.32 g,
20%). Mp: 250–252 °C (dec). 1H NMR (D2O, 300.1 MHz): 1.31 (3H,
5.5. In vivo radioprotective effectiveness
It is essential to establish certain criteria in order to evaluate
radioprotective effectiveness and toxicity of compounds. For each
of the molecules we tested in mice, first we evaluated its acute
(48 h) toxicity in male six weeks old Swiss CD1 mouse (Janvier,
France). The molecule was injected intraperitonally at different
concentrations to determine the LD50tox/48H, defined as the concen-
tration which kills 50% of the animals at 48 h.
Then radioprotective effectiveness was determined in the same
animal model, then in male six week old B6D2F1 mice (Janvier,
France). Compounds were dissolved in Mygliol 812 and adminis-
tered (ip, 20 ml/kg) at the maximum tolerated dose (defined as
1/2 LD50tox/48H). In Swiss CD1 mice, compounds were injected
90 min before radiation; in B6D2F1 mice they were injected
15 min before exposure. Gamma irradiation (60Co) was delivered
at doses LD100irr/30days and LD100irr/30days + 2 Gy. LD100irr/30days is de-
fined as the irradiation dose which kills 100% of the animals
30 days after exposure.
3
t, JH–H = 9.0 Hz, CH2CH3), 3.00 (4H, m, CH3CH2–S and CH2SP),
3
4.28 (2H, t, JH–H = 9.0 Hz, N-CH2), 7.80 (1H, s, NH). 13C NMR
(D2O, 75.4 MHz): 11.78 (CH3), 27.76 (CH2CH3), 28.54 (CH2–S),
50.68 (CH2–N), 158.73 (C5), 168.45 (C2). 31P NMR (D2O,
121.5 MHz): 15.80. Mass spectrum: m/z = 308 [M+1]+. Anal. Calcd
for C6H11LiN3O3PS3: C, 23.45; H, 3.61. N, 13.67. Found: C, 23.06;
H, 3.89; N, 13.78.
5.3.17. S-2-(2-Imino-5-ethyl)-1,3,4-thiadiazol-ethyl lithium
hydrogen phosphorothioate (9e)
N-3-Bromoethyl-5-ethylthio-1,3,4-thiadiazol-2-imine 7e (2.13 g,
6.72 mmol) was added in portions to a stirred solution of Li3S-
PO3ꢂ5H2O (1.20 g, 6.45 mmol) in water (30 ml). 15 ml of DMF were
added and the solution was stirred at room temperature for 3 h. The
solvents were removed in vacuo. The residual was washed in metha-
nol (20 ml) and dried under reduced pressure to give 9e (0.6 g, 32%).
Mp: 250–252 °C (dec) 1H NMR (D2O, 300.1 MHz): 1.25 (3H, t,
3
3JH–H = 9.0 Hz, CH2CH3), 2.81 (2H, q, JH–H = 9.0 Hz, CH2CH3), 3.06
Acknowledgements
3
3
(2H, m, JH–H = 9.0 Hz, CH2-S), 4,32 (2H, t, JH–H = 9.0 Hz, N-CH2),
7.80 (1H, s, NH). 13C NMR (D2O, 75.4 MHz): 11.83 (CH3), 23.74
(CH2CH3), 27.90 (CH2–S), 49.59 (CH2–N), 158.73 (C), 167.82 (C)
ppm. 31P NMR (D2O, 121.5 MHz): 15.80. Mass spectrum: m/z = 275
[M+1]+. Anal. Calcd for C6H11LiN3O3PS2: C, 26.18; H, 4.03; N, 15.27.
Found: C, 25.99; H, 4.43; N, 15.06.
We thank the ‘Délégation Générale pour l’Armement’ (DGA/
DSP/STTC/DT/DH), Ministère de la Défense Nationale, France, for
their financial support and their interest in this research. We are
very grateful to Dr. William Fauquette and Dr Patrick Martigne
for their technical help in the in vivo tests. The authors want to
dedicate this work to deceased Dr. Célariès.
5.4. In vitro antioxidant activity
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