Synthesis, characterization and biological evaluation of benzothiazoles and tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and inhibitors of the insulin releasing process

Article abstract of DOI:10.1016/j.ejmech.2016.03.028

A series of 1,3-benzothiazoles (series I) and 4,5,6,7-tetrahydro-1,3-benzothiazoles (series II) bearing an urea or a thiourea moiety at the 2-position were synthesized and tested as myorelaxants and inhibitors of insulin secretion. Several compounds (i.e. 13u and 13v) from series I showed a marked myorelaxant activity. Benzothiazoles bearing a strong electron withdrawing group (NO₂, CN) at the 6-position and an alkyl group linked to the urea or the thiourea function at the 2-position were found to be the most potent compounds. The weak vasorelaxant activity of series II compounds evidenced the necessity of the presence of a complete aromatic heterocyclic system. The myorelaxant activity of some active compounds was reduced when measured on aorta rings precontracted by 80 mM KCl or by 30 mM KCl in the presence of 10 μM glibenclamide, suggesting the involvement of K_ATP channels in the vasorelaxant effect. Some compounds of series I tested on rat pancreatic islets provoked a marked inhibition of insulin secretion, among which 13a exhibited a clear tissue selectivity for pancreatic β-cells.

Full text of DOI:10.1016/j.ejmech.2016.03.028

Accepted Manuscript
Synthesis, characterization and biological evaluation of benzothiazoles and
tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and
inhibitors of the insulin releasing process
Kamel Harrouche, Jean-Francois Renard, Nafila Bouider, Pascal de Tullio, Eric
Goffin, Philippe Lebrun, Gilles Faury, Bernard Pirotte, Smail Khelili
PII:
S0223-5234(16)30202-1
10.1016/j.ejmech.2016.03.028
EJMECH 8453
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 November 2015
Revised Date: 7 March 2016
Accepted Date: 10 March 2016
Please cite this article as: K. Harrouche, J.-F. Renard, N. Bouider, P. de Tullio, E. Goffin, P. Lebrun, G.
Faury, B. Pirotte, S. Khelili, Synthesis, characterization and biological evaluation of benzothiazoles and
tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and inhibitors of the insulin
releasing process, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.028.
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ACCEPTED MANUSCRIPT
Synthesis, characterization and biological evaluation of benzothiazoles and
tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and
inhibitors of the insulin releasing process
Kamel Harrouche, Jean-Francois Renard, Nafila Bouider, Pascal de Tullio, Eric Goffin,
Philippe Lebrun, Gilles Faury, Bernard Pirotte, Smail Khelili
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis, characterization and biological evaluation of benzothiazoles and
tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and
inhibitors of the insulin releasing process
Kamel Harrouche a, Jean-Francois Renard b, Nafila Bouider a, Pascal de Tullio b, Eric
Goffin b, Philippe Lebrun c, Gilles Faury d, Bernard Pirotte b*, Smail Khelili a
a Laboratoire de Phytochimie et de Pharmacologie, Département de Chimie, Faculté des
Sciences Exactes et Informatique, Université Mohamed Seddik Ben Yahia Jijel, B.P. 98 Ouled
Aissa, 18000 Jijel, Algeria.
b Laboratoire de Chimie Pharmaceutique, Center for Interdisciplinary Research on Medicines
(CIRM), Université de Liège, 1, Avenue de l’Hôpital, B-4000 Liège, Belgium
c Laboratoire de Physiologie et Pharmacologie, Université Libre de Bruxelles, Faculté de
Médecine, 808, Route de Lennik, B-1070 Bruxelles, Belgium
d Laboratoire "Hypoxie : Physiopathologie Cardiovasculaire et Respiratoire" (HP2), INSERM
U1042-Université Grenoble Alpes, F-38042 La Tronche, France
* Corresponding author
Abstract
A series of 1,3-benzothiazoles (series I) and 4,5,6,7-tetrahydro-1,3-benzothiazoles
(series II) bearing an urea or a thiourea moiety at the 2-position were synthesized and
tested as myorelaxants and inhibitors of insulin secretion. Several compounds (i.e. 13u
and 13v) from series I showed a marked myorelaxant activity. Benzothiazoles bearing a
strong electron withdrawing group (NO2, CN) at the 6-position and an alkyl group
linked to the urea or the thiourea function at the 2-position were found to be the most
potent compounds. The weak vasorelaxant activity of series II compounds evidenced
the necessity of the presence of a complete aromatic heterocyclic system. The
myorelaxant activity of some active compounds was reduced when measured on aorta
rings precontracted by 80 mM KCl or by 30 mM KCl in the presence of 10 µM
glibenclamide, suggesting the involvement of KATP channels in the vasorelaxant effect.
Some compounds of series I tested on rat pancreatic islets provoked a marked inhibition
of insulin secretion, among which 13a exhibited a clear tissue selectivity for pancreatic
β-cells.
Keywords: diazoxide; pinacidil; benzothiazole; benzothiadiazine; KATP channel;
potassium channel openers.
1

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1. Introduction
ATP-sensitive potassium channels, usually named KATP channels, are expressed
in most excitable cells.1,2 By tuning the membrane permeability to potassium ions as a
function of intracellular ATP availability, they link membrane excitability to the
metabolic state of the cell.3,4 It is important to emphasize that KATP channels in different
tissues display various biophysical and pharmacological properties. Such a diversity
results from differences in the molecular composition of KATP channels. These channels
are involved in the response to cerebral and cardiac ischemia, the regulation of insulin
secretion by pancreatic β-cells, the control of vascular smooth muscle tone, the
epithelial K+ transport and the modulation of electrical activity in several types of
neurons. 5-7 KATP channels are blocked by high concentrations of intracellular ATP and
by several pharmacological substances, in particular by sulphonylurea drugs such as
8
glibenclamide, which are used to treat non-insulin-dependent diabetes mellitus.
Conversely, KATP channels are activated by a variety of potassium channel openers
(PCOs) such as cromakalim (1), pinacidil (2) and diazoxide (3) (Figure 1). 9
The main challenge in the development of new KATP channel modulators as
therapeutic agents is the discovery of compounds exhibiting the highest selectivity for a
single KATP channel subtype. The reference PCO diazoxide is known to be non-
selective; being equipotent on both vascular smooth muscle cells (vasorelaxant effect)
and on pancreatic β-cells (inhibition of insulin release).
Chemical optimizations of diazoxide resulted in compounds such as BPDZ 44
(4), BPDZ 73 (5), BPDZ 216 (6) and NN414 (7), which potently and selectively
activate the KATP channels of pancreatic β-cells.10–13 Recent work reported the apparent
high selectivity of some benzothiazole derivatives bearing a thioamide group (8) for the
vascular smooth muscle KATP channels (Figure 1). 14
Moreover, the introduction
of sulfonylurea moieties on the diazoxide skeleton at the 7-position (9) or at the 3-
position (10) of the benzothiadiazine ring gave rise to a series of compounds expressing
a vasodilatory activity similar to that of diazoxide. 15
In order to develop new tissue selective KATP channel activators, we designed
original compounds structurally related to compounds 8 and 10 (Figure 1). Starting from
compound 8, our strategy first involved the replacement of the carbon atom linked at the
2-position of the thiazole ring by a nitrogen atom providing urea and thiourea analogues
(Figure 2). The second modification was the introduction of a substituent at the 6-
position of the benzothiazole ring in order to better mimic benzothiadiazine dioxides
2

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such as diazoxide or compounds 5, 6, 9 bearing a substituent at the 7-position. The
substituent on the second nitrogen atom of the urea or thiourea moiety was selected as a
short branched alkyl chain (as found in 8), an alicyclic nucleus or a diversely substituted
phenyl ring (figure 2, series I). A second series was obtained from the former by formal
saturation of the benzene ring of benzothiazole, in order to highlight the role of the
presence of an aromatic cycle. This led to tetrahydrobenzothiazole derivatives (Figure 2,
series II).
The biological effects of the new compounds (series I and II) were characterized
on rat pancreatic islets as well as on rat vascular smooth muscle in order to evaluate
their potential inhibitory effects on insulin secretion and putative vasorelaxant activity.
Further investigations were undertaken with a selection of compounds with the aim at
determining the mechanism of action.
2. Chemistry
The diversely 6-substituted 2-aminobenzothiazoles 12 were generally obtained by the
thiocyanation of the appropriate 4-substituted anilines (11), followed by ring closure in
the presence of bromine according to the reaction of Hugerscorff. 16,17 The 6-substituted
2-aminobenzothiazoles 12 and commercially available alkyl/cycloalkyl/aryl isocyanates
or isothiocyanates were refluxed in toluene to generate the urea and thiourea derivatives
13a-v (Scheme 1). The reaction rate and yields depended, firstly, on substituents
introduced on the benzothiazole ring and, secondly, on types of isocyanates and
isothiocyanates. We noticed that the reaction rate was greater when the substituent
beared on the benzothiazole ring was an electron-donating group such as C2H5 or CH3.
The rate became very low when an electron-withdrawing group, such as NO2, was
introduced. Isocyanates are knowm to be more reactive than isothiocyanates due to the
greater electronegativity of the oxygen atom compared to that of the sulfur atom.
The synthesis of 2-amino-4,5,6,7-tetrahydro-1,3-benzothiazole (15) was carried out
18,19
according to the Hantzsch's method described by King and Hlavacek.
The solvent
free reaction was performed by heating cyclohexanone (14) with thiourea in the
presence of iodine. The reaction of 15 with appropriate isocyanates and isothiocyanates
in dichloromethane successively provided the desired ureas and thioureas (16a-h)
(Scheme 2). In contrast to the 2-amino-1,3-benzothiazoles 12, 2-amino-4,5,6,7-
tetrahydro-1.3-benzothiazole 15 readily reacted with isocyanates and isothiocyanates,
clearly indicating that the aromaticity of the benzene cycle negatively influenced the
reactivity of the 2-amino-1,3-benzothiazoles.
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All newly synthesized benzothiazoles (13a-v) and tetrahydrobenzothiazoles (16a-h)
were purified by crystallization and characterized by IR, 1H NMR, 13C NMR (APT) and
elemental analyses.
3. Results and Discussion
The vasorelaxant activity of the newly synthesized compounds, 1,3-benzothiazoles 13a-
v and 4,5,6,7-tetrahydro-1,3-benzothiazoles 16a-h (series I and II, respectively), was
evaluated on isolated rat thoracic aorta rings precontracted with 30 mM KCl, by
comparison with diazoxide and pinacidil. The effects of the drugs were expressed as the
EC50 values, which corresponded to the drug concentrations giving 50% relaxation of
the precontracted smooth muscle preparation. 1,3-Benzothiazoles 13a-v (Table 1)
exhibited, in most cases, a more pronounced myorelaxant activity than the 4,5,6,7-
tetrahydrobenzothiazoles 16a-h (Table 2), indicating that the presence of the aromatic
ring was required for the biological activity. Compounds 13a, 13d, 13e, 13h-k, 13m,
13n, and 13q-s were less active than diazoxide and pinacidil (EC50 > 35 µM), while
compounds 13b, 13f, 13l, 13o, 13p and 13t were equipotent or slightly more potent than
diazoxide. Four compounds (13c, 13g, 13u and 13v) were found to be clearly more
active than diazoxide, especially 13u and 13v (EC50 equal to 2.78 and 2.11 µM,
respectively). It can be observed that the preferred R group for the myorelaxant ativity
was an alkyl chain, in particular a tert-butyl or a pentyl, while Y should be an electron-
withdrawing group such as CN or NO2. This observation could explain the weak
activity of 4,5,6,7-tetrahydro-1,3-benzothiazoles 16a-h (Table 2): the lack of aromatic
cycle, which would fail to transmit the electron-withdrawing effect of the substituent at
the 6-position to the 1,3-thiazole ring, seems to be required for a marked myorelaxant
activity.
In order to verify whether the vasorelaxant activity was related to smooth muscle cell
KATP channel activation, additional experiments were performed using the active
compounds 13c, 13f, 13g, 13u and 13v. These investigations included: i) two
experimental conditions into which the vasodilatory activity of KATP channel activators
is known to be suppressed or markedly decreased 20,21,25 [i.e. depolarization of rat aorta
rings by either 30 mM K+ in the continuous presence of the specific KATP channel
blocker glibenclamide (10 µM) or depolarization by 80 mM K+], and ii) experimental
conditions allowing to determine the involvement of endothelial cells in the compounds
effects (i.e. cell depolarization by 30 mM K+ on rat aorta rings with or without
endothelium).
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As shown in Table 3, the presence of glibenclamide in the bathing medium provoked a
substantial increase in the EC50 value of the selected compounds (about 2 fold for 13c
and 13g, about 6 fold for 13u, and about 9 fold for 13v), suggesting the involvement, at
least in part, of KATP channels in the vasodilatory activity of these molecules. Under
identical experimental conditions (Table 3), the EC50 value for diazoxide and pinacidil,
two known KATP channel activators, were increased by more than 4 fold and 20 fold;
respectively.
The same effects were observed with aorta rings precontracted by 80 mM KCl. The
EC50 values were increased about 2 fold for 13c and 13f, about 3 fold for 13g, about 5
fold for 13u, and about 7 fold for 13v, while those of diazoxide and pinacidil were
increased by more than 4 fold and 230 fold; respectively.
Such findings indicated that the vasodilatory action of the selected compounds was not
exclusively mediated by KATP channels activation.
The myorelaxant effects of the selected compounds were not significantly different in
the presence or absence of endothelium, indicating that the site of action was located on
the vascular smooth muscle cells and not on the endothelial cells (Table 3).
In the second set of pharmacological investigations, five newly synthesized compounds
(13a, 13c, 13e, 13f, 13g) were selected and tested (at 1, 10 and 50 µM) as inhibitors of
insulin secretion from glucose-stimulated (16.7 mM glucose) pancreatic β-cells (Table
4). Diazoxide, pinacidil and BPDZ 44 (4) were used as reference compounds.
The results clearly showed that all selected products provoked a marked inhibition of
insulin secretion at 50 µM, an effect more pronounced than that induced by pinacidil or
diazoxide. At 10 µM, 13g and 13a still exhibited a strong inhibitory activity on insulin
secretion. At 1µM, the effect of 13a was again more pronounced than that of diazoxide,
pinacidil or BPDZ 44 (4).
When the effects of the tested compounds were compared on vascular and pancreatic
cells (Table 4), it was observed that 13c, 13e, 13f and 13g did not disclose any marked
tissue-selectivity. Compound 13a, which clearly showed a preference for pancreatic β-
cells, expressed a tissue-selectivity roughly similar to that of BPDZ 44 (4). It could also
be noted that 13a was the only tested compound with an aromatic R group, while the
other molecules were bearing an alkyl chain (13c, 13f and 13g) or a benzyl group (13e).
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As previously observed for the antihypertensive drug diazoxide, the lack of obvious
vascular smooth muscle tissue selectivity requires to pay attention to the risk of
developing hyperglycemia when the compounds are administered in vivo. Compound
13a, with a clear selectivity for the pancreatic tissue, might be used in pathologies
characterized by the presence of a hyperinsulinism.
4. Conclusion
Starting from the structural modulation of compound 8, a benzothiazole recently
14
described as KATP channel opener , two series of compounds were prepared: series I
(13) and II (16). Some molecules from series I (13b, 13c, 13f, 13g, 13l, 13o, 13p, 13t,
and 13u-13v) exhibited a myorelaxant activity on 30 mM KCl-precontracted rat aorta
rings, especially 13u and 13v which were found to be more potent than diazoxide. The
myorelaxant activity of 13c, 13f, 13g, 13u and 13v was reduced by the presence of
glibenclamide (10 µM) in the experimental medium or when the contraction was
induced by 80 mM KCl. Such observations suggested an involvement, at least in part,
of KATP channels in the vasodilatory activity of these molecules. Further investigations
also indicated that the site of action was mainly located on the vascular smooth muscle
cells rather than on the endothelial cells. Five selected compounds (13a, 13c, 13e, 13f
and 13g) also provoked a marked inhibition of insulin secretion from rat pancreatic
islets, in particular 13a which exhibited a clear preference for pancreatic β-cells versus
vascular smooth muscle cells.
By contrast, most compounds of series II (16a-h) did not show any substantial
myorelaxant activity, indicating the potential role of an aromatic ring for inducing a
vasorelaxant effect.
Further work should be conducted with new representatives of the two series in order
to deepen the pharmacological profile of these compounds in terms of efficiency and
tissue selectivity.
5. Experimental section
5.1. Relaxation of rat aorta rings
Female Wistar rats weighing approximately 180–220 g were anesthetized by
intraperitoneal injection of 60 mg/kg pentobarbital, then sacrificed by exsanguination.
The thoracic aorta was isolated and excess fat and connective tissue were removed. The
vessels were cut into rings of about 2-3 mm length and mounted in an organ bath
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containing 10 mL of Krebs’ solution with the following composition (mM): NaCl 118,
KCl 5.6, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11 and CaCl2 2.4. The tissue
bath solution was maintained at 37 °C and bubbled with a 95% O2 – 5% CO2 mixture.
The pH of the saturated solution was 7.4. Two stainless steel hooks were inserted into
the aortic lumen; one was fixed while the other was connected to a force transducer
connected to an analysis software. The aorta rings were equilibrated in the medium for
75 min and maintained under an optimal tension of 1.8–2.0 g. KCl (30 mM) was then
added into the organ chamber to contract the rings. When the contraction reached a
stable plateau (usually 15 min), the test compounds were added in the organ bath to
assess their myorelaxant activity. Concentration–response curves were established by
cumulative addition at concentrations of 1, 10, 30, 50 and 100 µM or, for the active
compounds 13u and 13v, 0.1, 1, 5, 10, 30 and 100 µM. The same experiments were
repeated for some potent compounds by using aorta rings into which the endothelium
was removed. The endothelial removal was confirmed by the absence of vasodilator
effect of acetylcholine (10 ꢀM) added to vascular rings precontracted by 30 mM KCl.
Contractile responses were calculated as the difference between resting tension and
maximum tension developed in response to KCl stimulation. Data were expressed as
mean ± S.E.M. from 4–6 (n) experiments. All experiments were performed in
accordance with the ethical institutional guidelines.
5.2. Measurements of Insulin Release from Incubated Rat Pancreatic Islets
The method used for measuring insulin release from incubated pancreatic islets was
11,25
previously described.
Experiments were performed with pancreatic islets isolated
from adult fed Wistar rats. Groups of 10 islets, each derived from the same batch of
islets, were preincubated for 30 min at 37°C in 1 mL of a physiological salt medium
(in mM: NaCl 115, KCl 5, CaCl2 2.56, MgCl2 1, NaHCO3 24) supplemented with 2.8
mM glucose, 0.5% (w/v) dialysed albumin (fraction V, Sigma) and equilibrated against
a mixture of O2 (95%) and CO2 (5%). The islets were then incubated at 37°C for 90
min in 1 mL of the same medium containing 16.7 mM glucose and, in addition, the
reference or the required compound. The release of insulin was measured
radioimmunologically using rat insulin as a standard.
Residual insulin secretion was expressed as a percentage of the value recorded in
control experiments (100%); that is in the absence of drug and presence of 16.7 mM
glucose.
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Statistical analysis of the differences between the means of data was carried out
through a t-Student test. The biological results are considered statistically different
when p < 0.05.
5.3. Chemistry
Melting points were determined with a Büchi B-540 capillary apparatus and are
uncorrected. IR spectra were recorded as KBr pellets on a FTIR-8400 S, CE
(SHIMADSU) spectrophotometer. The NMR spectra were recorded on a Bruker
1
Avance (500 MHz for H NMR and 125 MHz for 13C NMR APT) instrument using
DMSO-d6 as the solvent with TMS as an internal standard; chemical shifts are reported
in δ values (ppm) relative to that of internal standard TMS.
The abbreviations s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, br =
broad and dd = doublet of doublet are used throughout. Elemental analyses (C, H, N, S)
were realized on a Thermo Scientific Flash EA 1112-elemental analyzer and were
within 0.4 % of the theoretical values. All reactions were routinely checked by TLC on
silica gel (Merck 60 F254).
5.3.1. General Method for the Synthesis of 1, 3-benzothiazoles (13)
5.3.1.1. 6-substituted 2-aminobenzothiazoles (12) 16,17
The appropriate 4-substituted aniline (11; 20 mmol), glacial acetic acid (20 mL) and
ammonium thiocyanate (40 mmol) were cooled in an ice bath and stirred for 10 min. A
solution of bromine (20 mmol) in AcOH (20 mL) was then added over 20 min. The
reaction mixture was stirred for 21 h at room temperature. The benzothiazole
hydrobromide salt was filtered, washed with acetic acid and dried. It was subsequently
dissolved in hot water and neutralized with aqueous ammonia solution (25%), filtered,
washed with water and dried under vacuum, recrystallized with aqueous ethanol to
obtain 6-substituted 1,3-benzothiazol-2-amine (12) in good yield.
5.3.1.2. 2-ureido (or thioureido)-susbstituted benzothiazoles
The solution of the appropriate 6-substituted 2-aminobenzothiazole (12) (1 mmol) in
anhydrous toluene (10 mL) was supplemented with the selected isocyanate or
isothiocyanate (1.1 mmol) and the reaction mixture was refluxed for 5 h. The solid
product was washed with water and purified by recrystallization from aqueous ethanol,
and air-dried to give the corresponding urea or thiourea compounds (13a-v).
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1-(6-bromo-1,3-benzothiazol-2-yl)-3-(4-cyanophenyl)urea (13a) : 55 % yield ; mp >
1
300°C. IR (KBr) (νmax/cm-1) : 3238, 3083, 2219, 1714, 1596, 1529, 815. H NMR
(DMSO) (δ/ppm) : 7.55 (m, 2H, CHar), 7.73 (m, 2H, CHar), 7.78 (m, 2H, CHar), 8.20
(s, 1H, CHar), 9.66 (s, 1H, NH), 11.16 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) :
(CHar) : 118.35, 118.75, 124.23, 129.07, 133.35 ; (Cq) : 112.08, 114.89, 119.07,
126.81, 134.51, 142.63, 143.65, 160.84. Anal. (C15H9BrN4OS) theoretical: 48.27% C,
2.43% H, 15.01% N, 8.59% S ; found: 48.38% C, 2.65% H, 15.13% N, 8.80% S.
1-(6-bromo-1,3-benzothiazol-2-yl)-3-isopropylurea (13b) : 50 % yield ; mp = 176-
1
178°C. IR (KBr) (νmax/cm-1) : 3296, 3060, 2974, 1672, 1533, 1269, 806. H NMR
(DMSO) (δ/ppm) : 1.14 (d, J = 6.7 Hz, 6H, CH(CH3)2), 3.80 (m, 1H, CH(CH3)2), 6.62
(d, J = 6.9 Hz,1H, NH), 7.49 (dd, J = 8.5, 2.0 Hz, 1H, CHar), 7.54 (d, J = 8.5 Hz,1H,
13
CHar), 8.13 (d, J = 1.7, 1H, CHar), 10.33 (s, 1H, NH). C NMR (DMSO) (δ/ppm) :
(CH3)2 : 22.60 ; (CH) 41.55 ; (CHar) : 121.22, 123.82, 128.65 ; (Cq) : 114.32, 133.30,
148.18, 152.71, 160.29. Anal. (C11H12BrN3OS) theoretical: 42.05% C, 3.85% H,
13.37% N, 10.21% S; found: 41.83% C, 3.84% H, 13.46% N, 10.40% S.
1-(6-bromo-1,3-benzothiazol-2-yl)-3-tert-butylurea (13c) : 40 % yield ; mp = 180-
182°C. IR (KBr) (νmax/cm-1) : 3255, 3076, 2970, 1683, 1531, 1448, 1272, 810. 1H NMR
(DMSO) (δ/ppm) : 1.32 (s, 9H, (CH3)3), 6.62 (s, 1H, NH), 7.49 (dd, J = 8.2, 1.6 Hz, 1H,
CHar), 7.53 (d, J = 8.2, 1H, CHar), 8.12 (d, J = 1.7 Hz, 1H, CHar), 10.36 (s, 1H, NH).
13C NMR (DMSO) (δ/ppm) : (CH3)3 : 28.65 ; (C(CH3)3) : 50.15 ; (CHar) : 121.20,
123.78, 128.63 ; (Cq) : 114.27, 133.66, 147.93, 152.04, 159.90. Anal. (C12H14BrN3OS)
theoretical: 43.91% C, 4.30% H, 12.80% N, 9.77% S; found: 43.84 % C, 4.26 % H,
13.08 % N, 10.07 % S.
1-(6-bromo-1,3-benzothiazol-2-yl)-3-[2-chloro-4-(trifluoromethyl)]phenylurea
(13d): 48 % yield ; mp > 300°C. IR (KBr) (νmax/cm-1) : 3379, 3112, 2972, 1704, 1598,
1
1523, 1315, 1116, 808. H NMR (DMSO) (δ/ppm) : 7.56 (dd, J = 8.6, 1.8 Hz, 1H,
CHar), 7.64 (d, J = 8.5 Hz, 1H, CHar), 7.76 (d, J = 8.5 Hz, 1H, CHar), 7.95 (s, 1H,
CHar), 8.23 (d, J = 1.4 Hz, 1H, CHar), 8.47 (d, J = 8.6 Hz, 1H, CHar), 9.43 (br s, 1H,
NH), 11.55 (br s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CHar) : 120.92, 121.77,
124.05, 125.06, 126.48, 129.09 ; (Cq) :112.09, 115.12, 117.33, 122.39, 124.05, 124.31,
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124.54 , 138.64, 151.10. Anal. (C15H8BrClF3N3OS) theoretical: 39.98% C, 1.79% H,
9.32% N, 7.12% S; found: 40.22% C, 1.87% H, 9.72% N, 6.95% S.
1-benzyl-3-(6-bromo-1,3-benzothiazol-2-yl)urea (13e) : 90 % yield ; mp = 222-224°C.
1
IR (KBr) (νmax/cm-1) : 3375, 3070, 2958, 1685, 1560, 1452, 1271, 696. H NMR
(DMSO) (δ/ppm) : 4.38 (d, J = 5.8 Hz, 2H, CH2), 7.27 (t, J = 5.5 Hz, 1H, NH), 7.30-
7.38 (m, 5H, CHar,), 7.50 (dd, J = 8.5, 1.9 Hz, 1H, CHar), 7.55 (d, J = 8.5 Hz, 1H,
CHar), 8.14 (s, 1H, CHar), 10.88 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH2) :
42.95 ; (CHar) : 121.27, 123.85, 126.98, 127.20, 128.40, 128.69 ; (Cq) : 114.40, 133.72,
139.35, 148.36, 153.79, 160.51. Anal. (C15H12BrN3OS) theoretical: 49.73% C, 3.34%
H, 11.60% N, 8.85% S ; found: 49.53% C, 3.48% H, 11.84% N, 8.80% S.
1-(6-nitro-1,3-benzothiazol-2-yl)-3-isopropylurea (13f) : 59 % yield ; mp = 252°C. IR
1
(KBr) (νmax/cm-1) : 3305, 3109, 2977, 1679, 1550, 1446, 1330, 1274, 1124. H NMR
(DMSO) (δ/ppm) : 1.15 (d, J = 6.6 Hz, 6H, CH(CH3)2), 3.84 (m, 1H, CH(CH3)2), 6.70
(d, J = 6.5 Hz,1H, NH), 7.74 (d, J = 8.9 Hz, 1H, CHar), 8.21 (dd, J = 8.9, 2.4 Hz,1H,
CHar), 8.94 (d, J = 2.2 Hz,1H, CHar), 10.89 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) :
(CH3)2 : 22.52 ; (C(CH3)2) : 41.71 ; (CHar) : 118.48, 119.44, 121.61 ; (Cq) : 132.22,
142.22, 152.22, 154.34, 165.20. Anal. (C11H12N4O3S) theoretical: 47.13% C, 4.32% H,
19.99% N, 11.44% S; found: 47.01% C, 4.28% H, 19.60% N, 11.17% S.
1-tert-butyl-3-(6-nitro-1,3-benzothiazol-2-yl)urea (13g) : 65 % yield ; mp >300°C. IR
1
(KBr) (νmax/cm-1) : 3402, 3255, 3105, 2979, 2364, 1686, 1517, 1340, 1278, 1203. H
NMR (DMSO) (δ/ppm) : 1.32 (s, 9H, (CH3)3), 6.69 (br s, 1H, NH), 7.74 (d, J = 8.9 Hz,
1H, CHar), 8.21 (dd, J = 8.9, 2.3 Hz, 1H, CHar), 8.93 (d, J = 2.1, 1H, CHar), 10.73 (br
13
s, 1H, NH). C NMR (DMSO) (δ/ppm) : (CH3)3 : 28.66 ; (C(CH3)3) : 50.25 ; (CHar) :
118.77, 119.99, 121.34 ; (Cq) : 132.26, 141.72, 153.23, 155.64, 165.29. Anal.
(C12H14N4O3S) theoretical: 48.42% C, 4.74% H, 18.91% N, 10.76% S; found: 48.30%
C, 4.51% H, 18.99% N, 11.12% S.
1-[2-chloro-4-(trifluoromethyl)]phenyl-3-(6-nitro-1,3-benzothiazol-2-yl)urea (13h) :
85 % yield ; mp >300°C. IR (KBr) (νmax/cm-1) : 3373, 3120, 2962, 1695, 1517, 1320,
1
744. H NMR (DMSO) (δ/ppm) : 7.76 (d, J = 8.7 Hz, 1H, CHar), 7.84 (d, J = 8.9 Hz,
1H, CHar), 7.96 (s, 1H, CHar), 8.26 (dd, J = 8.9, 2.4 Hz, 1H, CHar), 8.48 (d, J = 8.7
Hz, 1H, CHar), 9.02 (d, J = 2.3 Hz, 1H, CHar), 9.23 (br s, 1H, NH), 12.03 (br s, 1H,
10

ACCEPTED MANUSCRIPT
NH). 13C NMR (DMSO) (δ/ppm) : (CHar) : 118.76, 119.98, 121.04, 121.78, 125.05,
126.50 ; (Cq) : 117.77, 122.37, 124.54, 132.22, 134.76, 138.54, 142.59, 147.29, 159.43.
Anal. (C15H8ClF3N4O3S) theoretical: 43.23% C, 1.93% H, 13.44% N, 7.69% S; found:
43.27% C, 2.01% H, 13.50% N, 7.63% S.
1-(1,3-benzothiazol-2-yl)-3-[2-chloro-4-(trifluoromethyl)]phenylurea (13i) : 64 %
yield ; mp = 162-163°C. IR (KBr) (νmax/cm-1) : 3373, 3311, 3110, 1726, 1595, 1541,
1
1307, 831. H NMR (DMSO) (δ/ppm) : 7.63 (s, 4H, CHar), 7.70 (d, J = 8.8 Hz, 1H,
CHar), 7.89 (s, 1H, CHar), 8.45 (d, J = 8.8 Hz, 1H, CHar), 8.69 (s, 1H, NH), 9.87 (s,
1H, NH). 13C NMR (DMSO) (δ/ppm) : (CHar) : 119.73, 120.46, 124.89, 126.34,
132.76 ; (Cq) : 112.03, 115.67, 121.58, 123.05, 123.29, 139.47, 140.97, 151.60. Anal.
(C15H9ClF3N3OS) theoretical: 48.46% C, 2.44% H, 11.30% N, 8.63% S; found: 48.48%
C, 2.64% H, 11.37% N, 8.79% S.
1-(1,3-benzothiazol-2-yl)-3-isopropylurea (13j) : 50 % yield ; mp = 175°C. IR (KBr)
1
(νmax/cm-1) : 3352, 3174, 3105, 2977, 1720, 1595, 1529, 1315, 1233, 837. H NMR
(DMSO) (δ/ppm) : 1.10 (d, J = 6.5 Hz, 6H, (CH3)2), 3.73 (m, 1H, CH), 6.13 (d, J = 7.5
Hz, 1H, NH), 7.53 (s, 2H, CHar), 7.61 (s, 2H, CHar), 9.14 (s, 1H, NH). 13C NMR
(DMSO) (δ/ppm) : (CH3)2 : 22.83; (CH) : 40.03 ; (CHar) : 118.81, 119.72, 132.69,
133.03 ; (Cq) : 141.45, 142.74, 151.97, 154.03. Anal. (C11H13N3OS) theoretical: 56.15%
C, 5.57% H, 17.86% N, 13.63% S; found: 55.86% C, 5.35% H, 17.51% N, 14.07% S.
1-cyclohexyl-3-(6-methyl-1,3-benzothiazol-2-yl)thiourea (13k) : 35 % yield ; mp =
226-227°C. IR (KBr) (νmax/cm-1) : 3458, 3170, 3033, 2923, 2853, 1571, 1527, 1213.
1H NMR (DMSO) (δ/ppm) : 1.31-1.95 (m, 10H, (CH2)5), 2.39 (s, 3H, CH3), 4.16 (m,
1H, CH), 7.22 (d, J = 8.0 Hz, 1H, CHar), 7.56 (br s, 1H, CHar), 7.67 (s, 1H, CHar),
10.21 (s, 1H, NH), 11.60 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH3) : 20.93 ;
(CH2) : 23.81, 25.06, 31.19 ; (CH) : 52.47; (CHar) : 119.59, 121.32, 127.44 ; (Cq) :
112.91, 126.75, 133.18, 160.17, 168.89. Anal. (C15H19N3S2) theoretical: 58.98% C,
6.27% H, 13.76% N, 20.99% S; found: 58.66% C, 6.11% H, 13.91% N, 20.64% S.
1-(6-methyl-1,3-benzothiazol-2-yl)-3-isopropylurea (13l) : 44 % yield ; mp = 177-
1
178°C. IR (KBr) (νmax/cm-1) : 3305, 3055, 2974, 1677, 1539, 1263. H NMR (DMSO)
(δ/ppm) : 1.13 (d, J = 6.6 Hz, 6H, CH(CH3)2) 2.37 (s, 3H, CH3), 3.81 (m, 1H,
CH(CH3)2), 6.61 (d, J = 6.9 Hz,1H, NH), 7.16 (dd, J = 8.2, 1.1 Hz, 1H, CHar), 7.48 (d, J
11

ACCEPTED MANUSCRIPT
= 8.2 Hz, 1H, CHar), 7.65 (s, 1H, CHar), 10.32 (s, 1H, NH). 13C NMR (DMSO)
(δ/ppm) : (CH3) : 20.85, 22.66 ; (CH(CH3)2) : 41.45 ; (CHar) : 119.30, 121.00, 126.91 ;
(Cq) : 131.49, 131.85, 147.10, 152.86, 158.80. Anal. (C12H15N3OS) theoretical: 57.81%
C, 6.06% H, 16.85% N, 12.86% S; found: 57.54% C, 6.04% H, 16.90% N, 12.59% S.
1-benzyl-3-(6-methyl-1,3-benzothiazol-2-yl)thiourea (13m) : 50 % yield ; mp = 230°C.
1
IR (KBr) (νmax/cm-1) : 3419, 3188, 3040, 1562, 1527, 1444, 1271, 1207, 811. H NMR
(DMSO) (δ/ppm) : 2.38 (s, 3H, CH3), 4.85 (s, 2H, CH2), 7.20 (d, J = 8.1 Hz, 1H, CHar),
7.29 - 7.38 (m, 5H, CHar), 7.51 (m, 1H, CHar), 7.70 (m, 1H, CHar), 10.44 (s, 1H, NH),
11.84 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH3) : 15.88 ; (CH2) : 28.08 ; (CHar) :
127.18, 127.38, 127.46, 128.17, 128.46, 128.59; (Cq) : 132.91, 138.82, 148.79, 150.94,
159.71, 174.13. Anal. (C16H15N3S2) theoretical: 61.31% C, 4.82% H, 13.41% N,
20.46% S; found: 61.18% C, 4.80% H, 13.35% N, 20.22% S.
1-(3-nitrophenyl)-3-(6-ethyl-1,3-benzothiazol-2-yl)thiourea (13n) : 57 % yield ; mp =
204-205°C. IR (KBr) (νmax/cm-1) : 3180, 3026, 2964, 1647, 1521, 1338, 1186. 1H NMR
(DMSO) (δ/ppm) : 1.22 (t, J = 7.7 Hz, 3H, CH3), 2.70 (q, J = 7.6 Hz, 2H, CH2), 7.30 (d,
J = 8.1 Hz, 1H, CHar), 7.41 (s, 1H, CHar), 7.62 (t, J = 8.0 Hz, 1H, CHar), 7.72 (s, 1H,
CHar), 7.92 (m, 1H, CHar), 8.26 (m, 1H, CHar), 8.61 (m, 1H, CHar), 10.75 (s, 1H,
13
NH), 13.19 (s, 1H, NH). C NMR (DMSO) (δ/ppm) : (CH3) : 15.87 ; (CH2) : 28.05 ;
(CHar) : 112.73, 114.93, 117.69, 121.35, 124.63, 127.00, 129.66 ; (Cq) : 117.39,
129.45, 139.71, 141.03, 147.72, 169.66, 176.66. Anal. (C16H14N4O2S2) theoretical:
53.61% C, 3.94% H, 15.63% N, 17.89% S; found: 53.37% C, 3.79% H, 15.63% N,
17.67% S.
1-(6-ethyl-1,3-benzothiazol-2-yl)-3-isopropylurea (13o) : 70 % yield ; mp = 137-
1
138°C. IR (KBr) (νmax/cm-1) : 3309, 3058, 2964, 1674, 1544, 1265. H NMR (DMSO)
(δ/ppm) : 1.14 (d, J = 6.6 Hz, 6H, (CH3)2), 1.21 (t, J = 7.6 Hz, 3H, CH3), 2.67 (q, J = 7.6
Hz, 2H, CH2), 3.81 (m, 1H, CH), 6.61 (d, J = 6.9 Hz,1H, NH), 7.19 (dd, J = 8.2, 1.6 Hz,
13
1H, CHar), 7.51 (d, J = 8.2 Hz,1H, CHar), 7.68 (s, 1H, CHar), 10.33 (s, 1H, NH). C
NMR (DMSO) (δ/ppm) : (CH3) : 15.97, 22.67 ; (CH2) : 28.05 ; (C(CH3)2) : 40.78,
(CHar) : 119.38, 119.88, 125.82 ; (Cq) : 138.39, 141.42, 145.45, 147.28, 152.86. Anal.
(C13H17N3OS) theoretical: 59.29% C, 6.51% H, 15.96% N, 12.18% S; found
6.46% H, 15.78% N, 11.78% S.
:
59.17% C,
12

ACCEPTED MANUSCRIPT
1-tert-butyl-3-(6-ethyl-1,3-benzothiazol-2-yl)urea (13p) : 85 % yield ; mp = 164-
1
166°C. IR (KBr) (νmax/cm-1) : 3411, 3238, 3097, 2964, 1681, 1546, 1272. H NMR
(DMSO) (δ/ppm) : 1.21 (t, J = 7.6 Hz, 3H, CH3), 1.32 (s, 9H, (CH3)3), 2.67 (q, J = 7.6
Hz, 2H, CH2), 6.64 (s, 1H, NH), 7.19 (dd, J = 8.2, 1.6 Hz, 1H, CHar), 7.50 (d, J = 8.2
13
Hz, 1H, CHar), 7.67 (s, 1H, CHar), 10.19 (s, 1H, NH). C NMR (DMSO) (δ/ppm) :
(CH3) : 15.96, 28.38 ; (CH2) : 28.04 ; (C(CH3)3) : 50.03 ; (CHar) : 119.34, 119.85,
125.80 ; (Cq) : 131.46, 138.34, 147.22, 152.54, 158.72. Anal. (C14H19N3OS) theoretical:
60.62% C, 6.90% H, 15.15% N, 11.56% S; found: 60.72% C, 6.75% H, 15.15% N,
11.50% S.
1-benzyl-3-(6-ethyl-1,3-benzothiazol-2-yl)urea (13q) : 70 % yield ; mp = 183-185°C.
IR (KBr) (νmax/cm-1) : 3340, 3053, 2966, 1706, 1533, 1258. 1H NMR (DMSO) (δ/ppm) :
1.21 (t, J = 7.6 Hz, 3H, CH3), 2.68 (q, J = 7.6 Hz, 2H, CH2), 4.38 (d, J = 5.9 Hz, 2H,
CH2-ar), 7.20 (dd, J = 8.2, 1.2 Hz, 1H, CHar), 7.24-7.34 (m, 5H, CHar), 7.37 (m,1H,
NH), 7.53 (d, J = 8.2 Hz, 1H, CHar), 7.69 (s, 1H, CHar), 10.69 (s, 1H, NH). 13C NMR
(DMSO) (δ/ppm) : (CH3) : 15.98 ; (CH2) : 28.05 ; (CH2-Ar) : 42.98, (CHar) : 119.42,
119.90, 125.87, 126.94, 127.19, 128.39, (Cq) : 133.60, 138.48, 139.38, 144.59, 153.34,
155.72. Anal. (C17H17N3SO) theoretical: 65.57% C, 5.50% H, 13.49% N, 10.30% S ;
found: 65.37% C, 5.52% H, 13.65% N, 10.09% S.
1-[2-chloro-4-(trifluoromethyl)]phenyl-3-(6-ethyl-1,3-benzothiazol-2-yl)urea (13r) :
50 % yield ; mp > 300°C. IR (KBr) (νmax/cm-1) : 3384, 3134, 2970, 1701, 1599, 1535,
1327, 1271, 1116, 752. 1H NMR (DMSO) (δ/ppm) : 1.23 (t, J = 7.7 Hz, 3H, CH3), 2.71
(q, J = 7.6 Hz, 2H, CH2), 7.27 (dd, J = 8.3, 1.6 Hz, 1H, CHar), 7.61 (d, J = 8.2 Hz, 1H,
CHar), 7.75 (dd, J = 8.8, 1.6 Hz, 1H, CHar), 7.78 (s, 1H, CHar), 7.94 (d, J = 1.5 Hz, 1H,
CHar), 8.50 (d, J = 8.7 Hz, 1H, CHar), 9.43 (s, 1H, NH), 11.55 (s, 1H, NH). 13C NMR
(DMSO) (δ/ppm) : (CH3) : 15.92 ; (CH2) : 28.08 ; (CHar) : 120.16, 120.80, 125.00,
125.03, 126.27, 126.48 ; (Cq) :122.30, 123.57, 123.83, 124.09, 124.58, 138.86, 139.19,
143.62, 152.03. Anal. (C17H13ClF3N3OS) theoretical: 51.07% C, 3.28% H, 10.51% N,
8.02% S; found: 50.76% C, 3.30% H, 10.56% N, 7.98% S.
1-benzyl-3-(6-ethyl-1,3-benzothiazol-2-yl)thiourea (13s) : 25 % yield ; mp > 300°C. IR
1
(KBr) (νmax/cm-1) : 3398, 3174, 3046, 1568, 1529, 1464, 1276, 1220, 689. H NMR
(DMSO) (δ/ppm) : 1.21 (t, J = 7.6 Hz, 3H, CH3), 2.68 (q, 2H, CH2), 4.86 (s, 2H, CH2),
7.23 (d, J = 8.3 Hz, 1H, CHar), 7.29-7.38 (m, 5H, CHar), 7.53 (m, 1H, CHar), 7.73 (s,
13

ACCEPTED MANUSCRIPT
1H, CHar), 10.45 (s, 1H, NH), 11.85 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH3) :
15.88 ; (CH2) : 28.08, 47.61 ; (CHar) : 126.27, 126.36, 127.07, 127.37, 128.20, 128.45 ;
(Cq) : 136.31, 139.59, 143.97, 153.03, 168.37, 170.75. Anal. (C17H17N3S2) theoretical:
62.35% C, 5.23% H, 12.83% N, 19.58% S; found: 62.07% C, 5.27% H, 12.75% N,
18.98% S.
1-(6-cyano-1,3-benzothiazol-2-yl)-3-pentylurea (13t) : 61 % yield ; mp = 320-322 °C.
1
IR (KBr) (νmax/cm-1) : 3430, 2960, 2225, 1670, 1600, 1550, 1280. H NMR (DMSO)
(δ/ppm) : 0.88 (t, 3H, CH3), 1.31 (m, 4H, (CH2)2), 1.49 (m , 2H, CH2), 3.17 (q, J = 6.0
Hz, 2H, CH2-N), 6.79 (s, 1H, NH), 7.75 (dd, J = 8.39, 1.43 Hz, 1H, CHar), 7.82 (d, J =
8.41 Hz, 1H, CHar), 8.46 (s, 1H, CHar), 11.01 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm)
: (CH3) : 13.92 ; (CH2) : 21.78, 28.44, 28.95 ; (CHar) : 120.14, 126.37, 129.43 ; (Cq) :
104.17, 119.35, 132.26, 142.39, 152.49, 163.64. Anal. (C14H16N4OS) theoretical:
55.61% C, 4.67% H, 18.53% N, 10.61% S; found: 55.63% C, 4.43% H, 18.49% N,
10.73% S.
1-tert-butyl-3-(6-cyano-1,3-benzothiazol-2-yl)urea (13u) : 56 % yield ; mp =390-
1
392°C. IR (KBr) (νmax/cm-1) : 3420, 2960, 2225, 1700, 1645, 1540, 1260. H NMR
(DMSO) (δ/ppm) : 1.33 (s, 9H, (CH3)3), 6.67 (s, 1H, NH), 7.72 (d, J = 8.43 Hz, 1H,
CHar), 7.75 (dd, J = 8.43, 1.54 Hz, 1H, CHar), 8.45 (d, J = 1 Hz, 1H, CHar), 10.63 (s,
1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH3) : 28.58 ; (C(CH3)3) : 50.30 ; (CHar) :
120.17, 126.37, 129.44 ; (Cqar) : 104.15, 119.35, 131.95, 139.99, 151.95, 163.24. Anal.
(C14H16N4OS) theoretical: 53.41% C, 4.86% H, 19.16% N, 10.97% S; found: 53.42% C,
4.92% H, 19.36% N, 11.39% S.
1-(6-nitro-1,3-benzothiazol-2-yl)-3-pentylurea (13v) : 58 % yield ; mp = 195-197 °C.
1
IR (KBr) (νmax/cm-1) : 3440, 2920, 1680, 1600, 1510, 1340, 1275. H NMR (DMSO)
(δ/ppm) : 0.89 (t, 3H, CH3), 1.30 (m, 4H, (CH2)2), 1.48 (m, 2H, CH2), 3.17 (q, J = 6.3
Hz, 2H, CH2-N), 6.81 (s, 1H, NH) 7.76 (d, J = 8.41 Hz, 1H, CHar), 8.23 (dd, J = 8.39,
1.43 Hz, 1H, CHar), 8.95 (d, J = 1.02 Hz, 1H, CHar). 11.13 (s, 1H, NH). 13C NMR
(DMSO) (δ/ppm) : (CH3) : 13.92 ; (CH2) : 21.79, 28.44, 28.94 ; (CHar) : 118.51,
119.48, 121.63 ; (Cq) : 132.23, 142.21, 153.46, 161.22, 165.29. Anal. (C13H16N4O3S)
theoretical: 50.64% C, 5.23% H, 18.17% N, 10.40% S; found: 50.48% C, 4.96% H,
17.95% N, 10.18% S.
14

ACCEPTED MANUSCRIPT
5.3.2. General Method for the Synthesis of 4,5,6,7-tetrahydrobenzothiazoles (16)
5.3.2.1. 2-amino-4,5,6,7-tetrahydrobenzothiazoles (15) 18,19
Cyclohexanone (2.07 mL, 20 mmol) was added to a mixture of thiourea (3.04 g, 40
mmol) and iodine (5.07 g, 20 mmol) and heated without solvent at 110°C for 12 hrs.
The reaction mixture was then cooled to 20°C and quenched in water (20 mL) and
extracted by diethylether. The organic layer was washed with saturated sodium
bicarbonate solution and then filtered to remove insoluble solids (2-amino-4,5,6,7-
tetrahydrobenzothiazole hydroiodide salt).
The 2-amino-4,5,6,7-tetrahydrobenzothiazole hydroiodide salt was solubilized in
saturated sodium carbonate solution and heated, then the aqueous solution was extracted
with dichloromethane, dried over MgSO4 and evaporated. The residue was precipitated
in diethylether to lead to 2-amino-4,5,6,7-tetrahydrobenzothiazole (15).
5.3.2.2. 2-ureido (or thioureido)-4,5,6,7-tetrahydrobenzothiazoles (16a-h)
The solution of (15) (1 mmol) in anhydrous dichloromethane (10 mL) was
supplemented with the appropriate isocyanate or isothiocyanate (1.1 mmol) and
refluxed for 5 h. The solid product was washed with water and purified by re-
crystallization from aqueous ethanol, and air-dried to give the corresponding urea or
thiourea compounds (16a-h).
1-(3-nitrophenyl)-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)thiourea (16a) : 70 %
yield ; mp = 203°C. IR (KBr) (νmax/cm-1) : 3473, 3182, 3029, 2939, 1585, 1521, 1342,
1197, 686. 1H NMR (DMSO) (δ/ppm) : 1.79 (br s, 4H, CH2), 2.54 (br s, 4H, CH2), 7.55
(t, J = 8.2 Hz, 1H, CHar), 7.83 (d, J = 7.9 Hz, 1H, CHar), 8.28 (d, J = 7.6 Hz, 1H,
CHar), 8.58 (s, 1H, CHar), 10.34 (br s, 1H, NH), 12.73 (br s, 1H, NH). 13C NMR
(DMSO) (δ/ppm) : (CH2) : 21.60, 22.28, 22,43 ; (CHar) : 115.15, 116.66, 127.17,
129.51 ; (Cq) : 135.58, 141.43, 147.77, 159.50, 166.17, 183.83. Anal. (C14H14N4O2S2)
theoretical: 50.28% C, 4.22% H, 16.75% N, 19.18% S; found: 50.53% C, 4.36% H,
16.55% N, 18.91% S.
1-isopropyl-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)urea (16b) : 60 % yield ; mp =
92-94°C. IR (KBr) (νmax/cm-1) : 3294, 3070, 2927, 1679, 1569, 1234. 1H NMR (DMSO)
(δ/ppm) : 1.09 (d, J = 6.6 Hz, 6H, (CH3)2), 1.73 (br s, 4H, CH2), 2.47 (br s, 2H, CH2),
2.56 (br s, 2H, CH2), 3.75 (m, 1H, CH), 6.41 (d, J = 7.3 Hz, 1H, NH), 9.83 (br s, 1H,
15

ACCEPTED MANUSCRIPT
13
NH). C NMR (DMSO) (δ/ppm) : (CH3) : 22.76 ; (CH2) : 22.04, 22.63, 22.98, 25.94 ;
(CH) : 41.23 ; (Cq) : 141.45, 142.74 , 151.97, 167.83. Anal. (C11H17N3OS) theoretical:
55.20% C, 7.16% H, 17.56% N, 13.39% S; found: 55.55% C, 7.21% H, 17.30% N,
12.99% S.
1-isopropyl-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)thiourea (16c) : 50 % yield ;
mp = 103-105°C. IR (KBr) (νmax/cm-1) : 3326, 3224, 2926, 1670, 1560, 1442, 1232,
675. 1H NMR (DMSO) (δ/ppm) : 1.10 (d, J = 6.5 Hz, 6H, (CH3)2), 1.73 (br s, 4H, CH2),
2.47 (br s, 2H, CH2), 2.56 (br s, 2H, CH2), 3.75 (m, 1H, CH), 6.41 (d, J = 6.7 Hz, 1H,
13
NH), 9.82 (br s, 1H, NH). C NMR (DMSO) (δ/ppm) : (CH3) : 22.76 ; (CH2) : 22.04,
22.63, 22.98, 25.94 ; (CH) : 41.23 ; (Cq) : 143.45, 152.97, 156.76, 167.92. Anal.
(C11H17N3S2) theoretical: 51.73% C, 6.71% H, 16.45% N, 25.11% S; found: 52.12% C,
7.01% H, 16.52% N, 24.11% S.
1-tert-butyl-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)urea (16d) : 63 % yield ; mp =
178 -180°C. IR (KBr) (νmax/cm-1) : 3375, 3068, 2939, 1701, 1676, 1556, 1261. 1H NMR
(DMSO) (δ/ppm) : 1.28 (s, 9H, (CH3)3), 1.73 (br s, 4H, CH2), 2.46 (br s, 2H, CH2), 2.56
(br s, 2H, CH2), 6.47 (br s, 1H, NH), 9.70 (br s, 1H, NH). 13C NMR (DMSO) (δ/ppm) :
(CH2) : 22.19, 22.64, 22.98, 26.00 ; (CH3) : 28.79 ; (CH(CH3)3) : 49.75 ; (Cq) : 119.08,
143.39, 152.66, 156.64. Anal. (C12H19N3OS) theoretical: 56.89% C, 7.56% H, 16.58%
N, 12.66% S; found: 56.53% C, 7.40% H, 16.33% N, 12.63% S.
1-(4-cyanophenyl)-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)urea (16e): 70% yield ;
mp = 247-248°C. IR (KBr) (νmax/cm-1) : 3371, 3109, 2935, 2216, 1706, 1647, 1581,
1523, 1434, 1307, 1220. 1H NMR (DMSO) (δ/ppm) : 1.76 (br s, 4H, CH2), 2.59 (br s,
4H, CH2), 7.69 (d, J = 8.0 Hz, 2H, CHar), 7.73 (d, 2H, CHar), 9.53 (br s, 1H, NH),
10.80 (br s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH2) : 22.24, 22.32, 22.78, 25.06 ;
(CHar) : 118.25, 133.22 ; (Cq) : 103.64, 118.60, 133.31, 142.11, 143.32, 143.76,
159.76. Anal. (C15H14N4OS) theoretical: 60.38% C, 4.74% H, 18.78% N, 10.74% S;
found: 60.26% C, 4.69% H, 18.80% N, 10.83% S.
1-(4-cyanophenyl)-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)thiourea (16f) : 65 %
yield ; mp = 206°C. IR (KBr) (νmax/cm-1) : 3423, 3201, 3024, 2941, 2223, 1604, 1529,
1442, 1372, 1308, 1173. 1H NMR (DMSO) (δ/ppm) : 1.78 (br s, 4H, CH2), 2.53 (s, 4H,
CH2), 7.69 (d, J = 7.9 Hz, 2H, CHar), 8.03 (d, J = 8.2 Hz, 2H, CHar), 10.31 (s, 1H, NH),
16

ACCEPTED MANUSCRIPT
12.92 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH2) : 21.51, 22.12, 22.25, 22.36 ;
(CHar) : 120.17, 132.68 ; (Cq) : 99.49, 103.13, 117.07, 119.50, 144.42, 155.02, 182.51.
Anal. (C15H14N4S2) theoretical: 57.30% C, 4.49% H, 17.82% N, 20.40% S; found:
57.18% C, 4.43% H, 18.02% N, 20.08% S.
1-[4-chloro-3-(trifluoromethyl)]phenyl-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)
urea (16g) : 70 % yield ; mp = 196-197°C. IR (KBr) (νmax/cm-1) : 3413, 3101, 2950,
1
1699, 1605, 1416, 1264, 1134, 738. H NMR (DMSO) (δ/ppm) : 1.77 (m, 4H, CH2),
2.58 (m, 4H, CH2), 7.62 (d, J = 8.5 Hz, 1H, CHar), 7.75 (d, J = 8.5 Hz, 1H, CHar), 8.14
(s, 1H, CHar), 9.48 (s, 1H, NH), 10.85 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm) : (CH2)
: 22.22, 22.25, 22;74 ; (CHar) : 116.86, 123.18, 131.95 ; (Cq) : 119.52, 121.61, 122.52,
125.85, 126.50, 126.74, 139.06, 150.96. Anal. (C15H13ClF3N3OS) theoretical: 47.94%
C, 3.49% H, 11.18% N, 8.53% S; found: 48.17% C, 3.60% H, 11.18% N, 8.88% S.
1-(4-chlorophenyl)-3-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)urea (16h): 70 % yield
; mp = 265°C. IR (KBr) (νmax/cm-1) : 3357, 3294, 3074, 2935, 1674, 1552, 1296, 821. 1H
NMR (DMSO) (δ/ppm) : 1.76 (m, 4H, CH2), 2.60 (m, 4H, CH2), 7.34 (d, J = 7.6 Hz,
13
2H, CHar), 7.51 (d, J = 7.9 Hz, 2H, CHar), 9.14 (s, 1H, NH), 10.38 (s, 1H, NH). C
NMR (DMSO) (δ/ppm) : (CH2) : 22.22, 22.45, 22.85 ; (CHar) : 119.93, 128.66 ; (Cq) :
114.53, 125.41, 125.88, 138.00, 138.52, 152.30. Anal. (C14H14ClN3OS) theoretical:
54.63% C, 4.58% H, 13.65% N, 10.42% S; found: 54.31% C, 4.65% H, 13.22% N,
9.90% S.
6. References
[1] L. Aguilar-Bryan, J. Bryan, Molecular biology of adenosine triphosphate-sensitive
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17

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alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives potently and
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thiadiazine 1,1-dioxides as powerful inhibitors of insulin release from rat pancreatic β-
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benzothiazole-based thioamides and their evaluation as KATP channel openers,
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[15] S. Khelili, G. Leclerc, G. Faury, J. Verdetti, Synthesis and vasodilator effects of 3-
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Mechanical and ionic response of rat aorta to diazoxide, Eur. J. Pharmacol. 216 (1992)
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19

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Legends for figures
Figure 1: Chemical structure of reference potassium channel openers.
Figure 2 : Modulation of compound 8 leading to benzothiazoles and
tetrahydrobenzothiazoles.
20

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Figures
O
N
O
NC
N
N
NC
OH
N
H
N
H
Cromakalim (1)
Pinacidil (2)
O
O
O
O
S
Cl
S
N
N
N
N
H
N
N
H
H
Diazoxide (3)
BPDZ 44 (4)
O
S
O
O
O
O
Cl
S
N
N
N
H
N
N
H
N
H
H
BPDZ 73 (5)
BPDZ 216 (6)
O
O
S
S
S
N
NH
S
Cl
N
H
N
N
H
NN414 (7)
(8)
O
O
O
O
O
O
O
R
S
S
S
N
H
N
H
N
N
O
O
O
S
N
H
N
H
N
N
R
H
H
(9)
(10)
Figure 1
21

ACCEPTED MANUSCRIPT
Figure 2
22

ACCEPTED MANUSCRIPT
Schemes
NH2
Y
Y
S
x
i
S
ii
NH2
NH C NH
R
N
N
(Y = H, CH , C H , Br, CN, NO )
13a-v
12
Y
3
2
5
2
11
Scheme1. Reagents and conditions: (i) NH4SCN, Br2, CH3COOH; (ii) RNCX, toluene.
O
X
NH C NH
S
S
i
ii
R
NH2
N
N
16a-h
14
15
Scheme 2. Reagents and conditions: (i) 1) thiourea, I2, 110°C, 2) Na2CO3, H2O; (ii)
RNCX, CH2Cl2.
23

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Tables
Table 1. Effects of benzothiazoles 13a–v on the contractile activity of K+-depolarized
rat aorta rings.
Y
x
S
NH
C
NH
R
N
13a-v
R
EC50 (µM)a
(mean ± SEM (n))
Compound
Y
X
Br
Br
O
O
O
O
O
O
O
O
O
O
S
4-CN-phenyl
isopropyl
> 100
13a
13b
22.79 ± 4.42 (5)
13.03 ± 2.35 (5)
>100
Br
tert-butyl
13c
Br
2-Cl,4-CF3-phenyl
benzyl
13d
Br
64.20 ± 7.39 (4)
16.16 ± 4.77 (6)
10.76 ± 2.52 (6)
54.32 ± 8.18 (4)
> 100
13e
NO2
NO2
NO2
H
isopropyl
tert-butyl
2-Cl,4-CF3-phenyl
2-Cl,4-CF3-phenyl
isopropyl
cyclohexyl
isopropyl
benzyl
13f
13g
13h
13i
H
35.49 ± 4.93 (4)
52.06 ± 8.35 (4)
18.47 ± 3.37 (6)
>100
13j
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CN
CN
NO2
---
13k
O
S
13l
13m
13n
S
3-NO2-phenyl
isopropyl
tert-butyl
benzyl
>100
O
O
O
O
S
24.40 ± 1.15 (6)
25.27 ± 0.96 (5)
>100
13o
13p
13q
2-Cl,4-CF3-phenyl
benzyl
> 100
13r
> 100
13s
O
O
O
---
---
pentyl
14.76 ± 6.47 (5)
2.78 ± 0.94 (6)
2.11 ± 1.12 (5)
23.68 ± 3.31 (6)
0.39 ± 0.07 (4)
13t
tert-butyl
pentyl
13u
13v
---
Diazoxide
Pinacidil
---
---
a
EC50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aortic
rings. Results are expressed as mean ± sem. Numbers in parentheses refer to the number (n) of individual
experiments performed in each group.
24

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Table 2. Effects of tetrahydrobenzothiazoles 16a-h on the contractile activity of K+-
depolarized rat aorta rings.
X
S
NH
C
NH
R
N
16a-h
R
EC50 (µM) a
(mean ± SEM (n))
> 100
Compound
X
S
O
S
3-NO2-phenyl
isopropyl
16a
16b
47.63 ± 7.62 (6)
> 100
isopropyl
16c
O
O
S
tert-butyl
75.24 ± 4.34 (5)
> 100
16d
4-CN-phenyl
4-CN-phenyl
4-Cl,3-CF3-phenyl
4-Cl-phenyl
---
16e
> 100
16f
O
O
---
---
> 100
16g
> 100
16h
23.68 ± 3.31 (6)
0.39 ± 0.07 (4)
Diazoxide
Pinacidil
---
a
EC50: drug concentration giving 50% relaxation of the 30 mM KCl-induced contraction of rat aortic
rings. Results are expressed as mean ± sem. Numbers in parentheses refer to the number (n) of individual
experiments performed in each group.
25

ACCEPTED MANUSCRIPT
Table 3. Myorelaxant effect of selected compounds on 30 mM KCl-precontracted rat
aorta rings in the absence or presence of 10 µM glibenclamide, as well as on 80 mM
KCl-precontracted rat aorta rings.
rat aorta rings 30 mM KCl
EC50 (µM) a (mean ± SEM (n))
absence of
rat aorta rings
80 mM KCl
Compound
EC50 (µM) a
presence of
10 µM
endothelium
endothelium
glibenclamide b
23.30 ± 5.14 (4)
mean ± SEM (n)
26.87 ± 4.13 (4)
13.03 ± 2.35 (5)
12.12 ± 2.10 (4)
13c
13f
16.16 ± 4.77 (6)
10.76 ± 2.52 (6)
2.78 ± 0.94 (6)
2.11 ± 1.12 (5)
23.68 ± 3.31 (6)
0.39 ± 0.07 (4)
17.82 ± 2.88 (4)
14.79 ± 5.09 (5)
4.66 ± 1.27 (5)
4.75 ± 1.04 (4)
23.17 ± 3.57 (4)
nd
nd
36.39 ± 2.14 (5)
31.00 ± 4.15 (5)
15.02 ± 5.49 (4)
14.19 ± 3.38 (5)
>100
19.46 ± 2.82 (5)
15.61 ± 3.62 (4)
19.57 ± 4.42 (3)
>100
13g
13u
13v
Diazoxide
Pinacidil
8.02 ± 2.50 (5)
92.06 ± 17.06 (5)
a
EC50 is the drug concentration (µM) giving 50% relaxation of the 30 or 80 mM KCl-induced
contraction (mean ± s.e.m.). Numbers in parentheses refer to the number (n) of individual experiments
b
performed in each group. EC50 values obtained when glibenclamide (10 µM) was added to the bath
medium.
26

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Table 4. Effects of 13a, 13c, 13e, 13f and 13g on insulin secretion from rat pancreatic
islets and on contractile activity of K+-depolarized rat aorta rings.
Myorelaxant
Activity EC50
(µM) b
Rat pancreatic β-cells
% RIS a (mean ± SEM (n))
10 ꢀM
Compound
50 ꢀM
1 ꢀM
Mean ± SEM (n)
10.68 ± 1.16 (20) 11.03 ± 0.98 (15) 60.50± 2.27 (24)
> 100
13a
13c
15.89 ± 0.83 (22) 77.97 ± 3.65 (15)
17.85 ± 1.11 (21) 82.97 ± 5.40 (24)
nd
nd
nd
13.05 ± 4.40 (5)
81.91 ± 7.39 (4)
15.70 ± 3.00 (6)
13e
8.52 ± 0.69 (19)
62.03 ± 2.16 (23)
13f
11.77 ± 0.55 (22) 30.98 ± 2.06 (23) 83.52 ±5.87 (15) 11.09 ± 1.69 (6)
13g
26.7 ± 1.6 (16) c
88.8 ± 4.5 (13) c
8.6 ± 0.9 (23) d
73.9 ± 4.4 (16) c
92.1 ± 3.9 (13) c
13.7 ± 1.2 (23) d
87.5 ± 5.0 (15) c 23.68 ± 3.31 (6)
97.7 ± 6.7 (19) c
0.39 ± 0.07 (4)
82.9 ± 3.6 (22) 154.4 ± 14.5 (8) d
Diazoxide
Pinacidil
BPDZ 44
a % RIS: percentage of residual insulin release from rat pancreatic islets incubated in the presence of 16.7
mM glucose. Effects of the drugs at 1, 10 and 50 µM. b EC50: drug concentration giving 50% relaxation of
the 30 mM KCl-induced contraction of rat aortic rings. Numbers in parentheses refer to the number (n) of
individual experiments performed in each group. Published results : c ref 23, d ref 24.
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Synthesis, characterization and biological evaluation of benzothiazoles and
tetrahydrobenzothiazoles bearing urea or thiourea moieties as vasorelaxants and
inhibitors of the insulin releasing process
Kamel Harrouche, Jean-Francois Renard, Nafila Bouider, Pascal de Tullio, Eric Goffin,
Philippe Lebrun, Gilles Faury, Bernard Pirotte, Smail Khelili
Highlights
A series of 1,3-benzothiazoles and 4,5,6,7-tetrahydro-1,3-benzothiazoles were synthesized.
Several 1,3-benzothiazole derivatives showed a myorelaxant activity higher than diazoxide.
Their myorelaxant activity was explained, at least in part, by the opening of KATP channels.
Some 1,3-benzothiazole derivatives also provoked a marked inhibition of insulin secretion.