Novel phenyl(thio)ureas bearing (thio)oxothiazoline group as potential BACE-1 inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.3109/14756366.2011.642375

We report the synthesis and the β-site amyloid precursor protein cleaving enzyme-1 inhibitory properties of novel phenyl(thio)ureas bearing 2-(thio)oxothiazoline derivatives. A library of analogues was prepared according to specific synthetic schemes and the inhibitory activity was monitored using a fluorescence resonance energy transfer assay. Several analogues show potent inhibitory activities ranging between 1 and 0.01 μM and the activity is related to the NH acidity of the (thio)urea motif. Our results illustrate once again the close relationship between molecular recognition, complexation of the active site in enzymatic system, and organocatalysis utilizing explicit hydrogen bonding.

Full text of DOI:10.3109/14756366.2011.642375

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(1): 153–162
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.642375
RESEARCH ARTICLE
Novel phenyl(thio)ureas bearing (thio)oxothiazoline group
as potential BACE-1 inhibitors: synthesis and biological
evaluation
Federico Andreoli1, Abdallah Larbi Doukara1, Mohammed Amine Mehdid1, Nicolas Vanthuyne1,
Christian Roussel1, Jean Dessolin2, and Jean-Louis Kraus3
1Chirosciences UMR 6263, University Paul-Cézanne, Aix-Marseille III, Marseille, France, 2UMR 5248 CBMN, CNRS-
Université Bordeaux IECB, Pessac, France, and 3Laboratoire de Chimie Biomoléculaire IBDML-UMR 6216-CNRS-
Université de la Méditerranée, Parc scientifique de Luminy, Marseille, France
Abstract
We report the synthesis and the β-site amyloid precursor protein cleaving enzyme-1 inhibitory properties of novel
phenyl(thio)ureas bearing 2-(thio)oxothiazoline derivatives. A library of analogues was prepared according to specific
synthetic schemes and the inhibitory activity was monitored using a fluorescence resonance energy transfer assay.
Several analogues show potent inhibitory activities ranging between 1 and 0.01 µM and the activity is related to
the NH acidity of the (thio)urea motif. Our results illustrate once again the close relationship between molecular
recognition, complexation of the active site in enzymatic system, and organocatalysis utilizing explicit hydrogen
bonding.
Keywords: BACE-1, N-aryl-thiazoline-2-(thi)ones, ureas, thioureas, Alzheimer disease
Introduction
hypothesized that aspartyl proteases are prone to be
A recent critical review outlined “the close relationship
and mutual interplay between molecular recognition,
active site considerations in enzyme catalysis involving
anions, and organocatalysis utilizing explicit hydrogen
bonding1”. Aryl(thio)ureas presenting two N-H groups
are well recognized in the very active field of non-cova-
lent organocatalysis for their ability to bind ketones, alde-
selectively complexed by the aryl(thio)urea scaffold. e
inhibition of BACE-1 (β-site amyloid precursor protein
(APP) cleaving enzyme), a key enzyme in the production
of α, β peptides involved in the formation of extracellular
senile plaques in Alzheimer’s disease21–23, was chosen to
monitor the binding capability (if any) of aryl(thio)ureas
to that important enzyme. e search of BACE-1 inhibi-
tors is a very active field of research24–31. Arylthioureas
have been recently described in several patents as poten-
tially active scaffolds for neurodegenerative diseases
hydes and nitro groups through hydrogen bonding2–8
.
In addition, they are outstanding neutral compounds
for the selective complexation of various anions9–14 and
they found application in the (enantio) selective extrac-
tion of carboxylate anions15–18. We have shown that some
ureas and thioureas derived from 3-(2-aminophenyl)-
4-methylthiazole-2(3H)-thione 1a enantioselectively
bind to carboxylate anion derived from amino-acids19
and act as organocatalysts for addition to aldehydes20.
In an extension of our interest in the binding capabili-
ties of aryl(thi)oureas through hydrogen bonding, we
including Alzheimer32,33
. Moreover, (thio)oxothiazol-
phenyl heterocycles represent a privileged moiety very
often encountered in bioactive molecules utilized for
the treatment of several pathologies including neurode-
generative diseases34,35. We report herein the synthesis of
focused library of heterocyclic aryl(thio)ureas. ey were
screened as BACE-1 inhibitors by the mean of a fluores-
cence resonance energy transfer (FRET) assay, which
Address for Correspondence: Dr. Jean-Louis Kraus, Department of Chemistry, université de la Méditerranée, faculté des sciences de Luminy,
chimie biomoléculaire, Marseille, 13288, France. Tel: (33) 04 91 82 91 41. E-mail: kraus@univmed.fr
(Received 24 October 2011; revised 15 November 2011; accepted 15 November 2011)
153

154 F. Andreoli et al.
uses purified baculovirus-expressed (BACE-1) and a
specific substrate (Rh-EVNLDAEFK-Quencher) based on
the Swedish mutation of the APP. is peptidic substrate
becomes highly fluorescent upon enzymatic cleavage.
pressure. iazoline 1b was then recovered by recrystal-
lization from EtOH: yield 73% (11 g, pale yellow crystals);
product description is given in Roussel et al.36 and Bellec
et al.37.
3-(2-Aminophenyl)-4-methylthiazol-2(3H)-one (1c)
Methods
In 50 mL of acetone solution, 2.5 g of 1a (11.2 mmol) were
solubilized, then iodomethane (10 eq, 70 mL) was added
and the solution stirred at r.t. After 24 h, the solvent and
the excess of reagent were evaporated and the resulting
thiazolium salt recovered quantitatively (4.1 g). en, a
solution of sodium methoxide (4 eq) in CH3OH was pre-
pared, 4.1 g (11.24 mmol) of thiazolium salt were added
and the solution stirred at r.t. After 48 h, the solvent was
evaporated, water (200 mL) was added and the mixture
extracted 3 × 100 mL of CH2Cl2. e organic layer was
then dried over MgSO4 and evaporated under reduced
pressure. e 1c was finally recovered by recrystalliza-
tion from EtOH: yield 74% (1.73 g, pale yellow crystals);
for product description see ref. Vanthuyne et al.38.
Chemistry general methods
1H NMR spectra were recorded at 500, 400, 300 or 200MHz
and 13C NMR spectra at 125, 100, 75 or 50 MHz on Bruker
Avance DRX-500 or 400, DPX-300 or 200 instruments,
respectively. Chemical shifts are reported in ppm with
the signal for residual solvent as internal standard. J val-
ues are reported in Hz. Elemental analyses were within
0.4% of theoretical values. High resolution mass spectra
were performed on Q-STAR Elite spectrometer. Melting
points were measured using a Büchi Melting Point B-545
apparatus or a Kofler hot stage apparatus and are not cor-
rected. Filtrations through silica gel were performed with
silica gel 60 (230–400 mesh). TLCs were carried out on
Merck 60F254 silica plates.
3-[2-Amino-5-(trifluoromethyl)phenyl]-4-methyl-1,3-
Thiazoline thiones and ones
thiazole-2(3H)-thione (1d)
3-(2-Aminophenyl)-4-methylthiazole-2(3H)-thione (1a)
Twenty-eight grams of 1,2-phenylenediamine (258
mmol) was suspended in CS2 (600 mL), then NEt3 (2 eq,
72 mL) was added dropwise and the mixture stirred at
r.t. for 3 h. e resulting dithiocarbamate salt (73 g, yel-
low powder, 99%) was filtered off, washed with Et2O and
used without further purification. Seventy-three grams
of dithiocarbamate salt (393 mmol) were suspended in
CH3CN (600 mL), then chloroacetone (1 eq, 21 mL) was
added dropwise and the mixture stirred at r.t. for 12h.
e solvent was then removed and HCl 37% (69 mL)
was added and the mixture vigorously stirred for 15 min.
Water (400 mL) was added and the mixture extracted
with 3 × 400 mL of CH2Cl2. e organic layer was then
washed with 3 × 500 mL of water, dried over MgSO4 and
evaporated under reduced pressure. iazoline 1a was
then recovered by recrystallization from EtOH: yield 70%
(41 g, pale yellow crystals); product description is given
in Roussel et al.36 and Bellec et al.37.
ree grams of 1,2-diamino-4-trifluoromethylbenzene
(17.03 mmol) were suspended in CS2 (40 mL), then NEt3
(2 eq, 5 mL) was added dropwise and the mixture stirred
at r.t. for 3 h. e resulting dithiocarbamate salt (5.98 g,
yellow powder, 99%) was filtered off, washed with Et2O
and used without further purification. In 50 mL of CH3CN
solution, 5.98 g of dithiocarbamate salt (16.92 mmol)
were suspended, then chloroacetone (1 eq, 1.36 mL)
was added dropwise and the mixture stirred at r.t. for
12 h. e solvent was then removed and HCl 37% (4 mL)
was added and the mixture vigorously stirred for 15 min.
Water (200 mL) was added and the mixture extracted
with 3 × 200 mL of CH2Cl2. e organic layer was then
washed with 3 × 400 mL of water, dried over MgSO4 and
evaporated under reduced pressure. iazoline 1d was
then recovered by recrystallization from EtOH: yield 68%
(3.32 g, pale yellow crystals); mp 188°C, Rf = 0.61 (CH2Cl2/
1
AcOEt 9:1), H NMR (200 MHz, CDCl3) δH 1.95 (d, J= 1.2
Hz, 3H, CH3), 4.04 (br s, 2H, NH2), 6.39 (q, J= 1.2 Hz, 1H,
H5), 6.91–6.99 (m, 1H, Ar), 7.27–7.31 (m, 1H, Ar), 7.47–
7.58 (m, 1H, Ar). 13C NMR (75 MHz, CDCl3) δ= 15.53,
107.00, 117.18, 121.31 (q, J= 34 Hz, 1C), 122.69, 123.96
(q, J= 271 Hz, 1C), 126.41 (q, J= 4 Hz, 1C), 127.98 (q, J= 4
Hz, 1C), 132.60, 146.90, 172.15. High resolution mass
spectrometry (HRMS) m/z C11H9N2F3S2 [M+H]+ calcd:
291.0231, found: 291.0231. e structure of 3-[2-amino-
5-(trifluoromethyl)phenyl]-4-methyl-1,3-thiazole-2-
(3H)-thione was fully established by X-ray single crystal
analysis.
3-(2-Aminophenyl)-4,5-dimethylthiazole-2(3H)-thione (1b)
Seven grams of 1,2-phenylenediamine (64.6 mmol) were
suspended in CS2 (150 mL), then NEt3 (2 eq, 18 mL) was
added dropwise and the mixture stirred at r.t. for 3 h.
e resulting dithiocarbamate salt (18 g, yellow powder,
99%) was filtered off, washed with Et2O and used without
further purification. Eighteen grams of dithiocarbamate
salt (93 mmol) were suspended in CH3CN (150 mL), then
3-chlorobutan-2-one (1 eq, 6.4 mL) was added dropwise
and the mixture stirred at r.t. for 24 h. e solvent was then
removed and H2SO4 (8 mL) was added and the mixture
vigorously stirred for 15 min. Water (100 mL) was added
and the mixture extracted with 3 × 100 mL of CH2Cl2.
e organic layer was then washed with 3 × 200 mL of
water, dried over MgSO4 and evaporated under reduced
3-(2-Amino-4-fluorophenyl)-4-methyl-1,3-thiazole-2(3H)-
thione (1e)
Ten grams (79 mmol) of 1,2-diamino-4-fluorobenzene
were suspended in CS2 (500mL), then NEt3 (2 eq, 22mL)
was added dropwise and the mixture stirred at r.t. for 2h.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenyl(thio)ureas bearing 2-(thio)oxothiazoline derivatives 155
e resulting dithiocarbamate salt was then filtered off,
washed with Et2O and used without further purifications
(24g,yellowpowder,99%).Twenty-fourgrams(79mmol)of
dithiocarbamate salt were suspended in CH3CN (500mL),
then chloroacetone (1.1 eq, 7.14mL) was added dropwise
and the mixture stirred at r.t. for 12h. e solvent was then
removed and HCl 37% (21mL) was added under vigorous
stirring. After 15min, water (150mL) was added and the
mixture extracted 3×150mL of CH2Cl2. e organic layer
was washed 3×400mL of water, dried with MgSO4 and
evaporated under reduced pressure. iazoline 1e was
then recovered by recrystallization from CH2Cl2: yield 68%
130.9, 131.0, 134.3, 135.2, 141.7, 188.6. NMR data are in
agreement with literature37.
3-(2-Isothiocyanatophenyl)-4-methylthiazole-2(3H)-one (2c)
To a suspension of thiazoline 1c (600mg, 2.91 mmol) in
CS2 (25mL), NEt3 was added dropwise (10 eq, 4.0mL) and
the mixture stirred first 1h under reflux, then 5h at room
temperature. e resulting dithiocarbamate salt (880mg,
79%) was filtered off, washed with anhydrous Et2O and
used without further purification. en, to a suspension
of the dithiocarbamate salt (880mg, 2.29 mmol) in CH3CN
(25mL), DCC (1.05 eq, 493mg) was added and the mixture
stirred at room temperature. After 24h, the formed DCU
was filtered off and the pure isothiocyanate was recovered
by column chromatography (CH2Cl2): yield 80% referring
to dithiocarbamate salt (455mg, pale yellow powder); for
product description see ref. Vanthuyne et al.38.
1
(12.1g, yellow crystals); mp=138°C; H NMR (300MHz,
CDCl3) δH 1.95 (d, J=1.2 Hz, 3H, CH3), 3.80 (br s, 2H, NH2),
6.37 (q, J=1.2 Hz, 1H, H5), 6.57–6.99 (m, 3H, Ar). 13C NMR
(75MHz, CDCl3) δ=15.44, 103.9 (d, J=26 Hz, 1C), 106.4 (d,
J=24Hz, 1C), 106.6, 119.3(d, J=3Hz, 1C), 130.0(d, J=11Hz,
1C), 144.6 (d, J=12 Hz, 1C), 163.8 (d, J=248 Hz, 1C), 189.2,
HRMS m/z C10H10N2FS2 [M+H]+ calcd: 241.0264, found:
241.0262. e structure of 3-(2-amino-4-fluorophenyl)-4-
methyl-1,3-thiazole-2(3H)-thione was fully established by
X-ray single crystal analysis.
General procedure for the synthesis of thioureas and
ureas
To a solution of isothiocyanate (238 mg, 0.9 mmol) in
CH3CN (5 mL), the corresponding aniline was added (1.05
eq) and the solution stirred for 24 h at room temperature.
en, the solvent was partially evaporated and the result-
ing thiourea recovered by filtration. Modifications to the
general procedure are given when needed.
Isothiocyanates
3-(2-Isothiocyanatophenyl)-4-methylthiazole-2(3H)-thione (2a)
To a suspension of thiazoline 1a (1g, 4.5 mmol) in CS2
(40 mL), NEt3 was added dropwise (10 eq, 6.3mL) and
the mixture stirred first 1 h under reflux, then 5 h at room
temperature. e resulting dithiocarbamate salt (1.79g,
99%) was filtered off, washed with anhydrous Et2O and
used without further purification. en, to a suspen-
sion of the dithiocarbamate salt (916 mg, 2.29 mmol) in
CH3CN (30 mL), DiCyclohexylCarbodiimide (DCC) (1.05
eq, 475 mg) was added and the mixture stirred at room
temperature. After 24 h, the formed Dicyclohexylurea
(DCU) was filtered off and the pure isothiocyanate was
recoveredbycolumnchromatography(CH2Cl2):yield74%
referring to dithiocarbamate salt (448 mg, white powder);
for product description see ref. Vanthuyne et al.38.
1-(2-(4-Methyl-2-thioxothiazol-3(2H)-yl)phenyl)-
3-phenylthiourea (3a), 1-(2-(4-methyl-2-thioxothi-
azol-3(2H)-yl)phenyl)-3-phenylthiourea
(3b),
1-(2-(4-methyl-2-thioxothiazol-3(2H)-yl)phenyl)-3-
(pyridin-3-yl)thiourea(3c),1-(2-hydroxyphenyl)-3-(2-(4-
methyl-2-thioxothiazol-3(2H)-yl)phenyl)thiourea (3d),
1-(3,5-dimethylphenyl)-3-(2-(4-methyl-2-thioxothiazol-
3(2H)-yl)phenyl)thiourea (3e), 1-(2-methoxyphenyl)-3-
(2-(4-methyl-2-thioxothiazol-3(2H)-yl)phenyl)thiourea
(3f), 1-(2-(4-methyl-2-thioxothiazol-3(2H)-yl)phenyl)-
3-(3-(trifluoromethyl)-phenyl)thiourea (3g), 1-(3,5-
bis(trifluoromethyl)phenyl)-3-(2-(4-methyl-2-thioxothiazol
-3(2H)-yl)phenyl)thiourea
2-thioxothiazol-3(2H)-yl)phenyl)-3-phenylu-
rea (4a) were available from
previous study19.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(4-meth-
yl-2-thioxothiazol-3(2H)-yl)phenyl)urea (4c),
(3h),
1-(2-(4-methyl-
3-(2-Isothiocyanatophenyl)-4,5-dimethylthiazole-2(3H)-
thione (2b)
a
To a suspension of thiazoline 1b (1.09g, 4.61 mmol) in
CS2 (40 mL), NEt3 was added dropwise (10 eq, 6.5 mL) and
the mixture stirred first 1 h under reflux, then 4 h at room
temperature. e resulting dithiocarbamate salt (1.51g,
79%) was filtered off, washed with anhydrous Et2O and
used without further purification. en, to a suspension
of the dithiocarbamate salt (1.51 g, 3.65 mmol) in CH3CN
(40 mL), DCC (1.05 eq, 790 mg) was added and the mix-
ture stirred at room temperature. After 24 h, the formed
DCU was filtered off and the pure isothiocyanate was
recovered by column chromatography (CH2Cl2): yield
92% referring to the dithiocarbamate salt (931mg, pale
yellow powder); mp 140°C (Bellec et al. 37 138°C); Rf = 0.57
(CH2Cl2); 1H NMR (300 MHz, CDCl3): δH 1.85 (s, 3H,
CH3), 2.22 (s, 3H, CH3), 7.28–7.55 (m, 4H, Ar); 13C NMR
(75 MHz, CDCl3): δ= 11.9, 13.0, 118.6, 126.2, 128.2, 130.0,
1-(2-(4-methyl-2-thioxothiazol-3(2H)-yl)phenyl)-3-(4-
nitrophenyl)urea (4d), 1-(2-(4-methyl-2-thioxothiazol-
3(2H)-yl)phenyl)-3-p-tolylurea (4e) were available from
a previous study39.
1-(2,4-Dichlorophenyl)-3-(2-(4-methyl-2-thioxothiazol-
3(2H)-yl)phenyl)thiourea (3i)
According to the general procedure, the reaction of 2,4-
dichlorobenzenamine with isothiocyanate 2a afforded
thiourea 3i: yield 62% (238 mg, white solid); mp 180°C;
1H NMR (300 MHz, dimethyl sulfoxide (DMSO) d6): δH
1.99 (d, J= 1.0 Hz, 3H, CH3), 6.88 (q, J= 1.0 Hz, 1H, H5),
7.34–7.81 (m, 7H, Ar), 8.84 (s, 1H, NH), 9.95 (s, 1H, NH);
13C NMR (75 MHz, DMSO d6): δC 15.42, 107.50, 127.32,
127.67, 128.90, 128.93, 129.66, 130.58, 130.88, 130.91,
© 2013 Informa UK, Ltd.

156 F. Andreoli et al.
131.13, 133.12, 135.04, 135.92, 140.66, 181.88, 188.54;
HRMS: m/z [M+H]+ calcd. for C17H14N3S3Cl2+: 425.9721,
found: 425.9717.
2b (203 mg, 0.73 mmol) afforded thiourea 3m: yield 66%
(210 mg, white solid); mp 191°C; 1H NMR (300 MHz,
DMSO d6): δH 1.91 (s, 3H, CH3), 2.18 (s, 3H, CH3), 7.13–
7.23 (m, 2H, Ar), 7.31–7.62 (m, 5H, Ar), 7.72–7.80 (m, 1H,
Ar), 7.70–7.80 (m, 1H, Ar), 8.11 (br s, 1H, Ar), 8.76 (s, 1H,
NH), 10.51 (s, 1H, NH); 13C NMR (75 MHz, DMSO d6): δC
11.39, 13.23, 117.43, 118.94 (q, J= 4 Hz, 1C), 120.83 (q, J= 4
Hz, 1C), 124.01 (q, J= 272 Hz, 1C), 126.38, 127.66, 128.89,
129.03 (q, J= 32 Hz, 1C), 129.64, 129.72, 130.55, 133.77,
135.73, 135.85, 139.98, 180.41, 186.09; HRMS: m/z [M+H]+
calcd. for C19H17N3F3S3+: 440.0531, found: 440.0532.
1-(2-(4-methyl-2-thioxothiazol-3(2H)-yl)phenyl)-3-(pyrimidin-
2-yl)thiourea (3j)
According to the general procedure, the reaction of
2-aminopyrimidine with isothiocyanate 2aafforded thio-
urea 3j; in this case, CH3CN was evaporated and thiourea
3j recovered by column chromatography (CH2Cl2/AcOEt
9/1): yield 42% (136 mg, white solid); mp 242°C; Rf = 0.49
1
(CH2Cl2/AcOEt 9/1); H NMR (300 MHz, DMSO d6): δH
1.95 (d, J= 1.1 Hz, 3H, CH3), 6.78 (q, J= 1.1 Hz, 1H, H5),
7.22 (t, J= 4.9 Hz, 1H, Ar), 7.38–7.54 (m, 2H, Ar), 7.55–7.65
(m, 1H, Ar), 8.01–8.09 (m, 1H, Ar), 8.60 (d, J= 4.9 Hz,
2H, Ar), 11.26 (s, 1H, NH), 13.03 (s, 1H, NH). 13C NMR
(75 MHz, DMSO d6): δC 15.27, 107.63, 116.24, 127.61,
128.69, 129.03, 129.60, 132.12, 136.09, 140.13, 157.12,
158.01 (2C), 179.47, 189.19; HRMS: m/z [M+H]+ calcd. for
C15H14N5S3+: 360.0405, found: 360.0411.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(4,5-dimethyl-2-
thioxothiazol-3(2H)-yl)phenyl)-thiourea (3n)
According to the general procedure, the reaction of
4-fluorobenzenamine with isothiocyanate 2b (207 mg,
0.74 mmol) afforded thiourea 3n; in this case, the solvent
was evaporated and 3n recovered by column chromatog-
raphy (CH2Cl2): yield 78% (295 mg, pale yellow solid); mp
1
170°C, Rf = 0.34 (CH2Cl2), H NMR (300 MHz, DMSO d6):
δH 1.90 (s, 3H, CH3), 2.16 (s, 3H, CH3), 7.33–7.41 (m, 1H,
Ar), 7.43–7.63 (m, 2H, Ar), 7.70–7.77 (m, 1H, Ar), 7.81 (br
s, 1H, Ar), 8.33 (br s, 2H, Ar), 9.03 (s, 1H, NH), 10.73 (s,
1H, NH); 13C NMR (75 MHz, DMSO d6): δC 11.36, 13.21,
117.03 (m, 1C) 117.46, 122.42 (q, J= 3 Hz, 2C), 123.19 (q,
J= 273 Hz, 2C), 127.91, 129.10, 129.82, 130.12 (q, J= 33 Hz,
2C), 130.33, 133.71, 135.51, 135.67, 141.38, 180.38, 186.10;
4-(3-(2-(4-Methyl-2-thioxothiazol-3(2H)-yl)phenyl)thioureido)
benzoic acid (3k)
A different procedure was used: a solution of isothiocya-
nate 2a (238mg, 0.9 mmol) in CH3CN (3mL) and a Na2CO3
saturated aqueous solution containing 4-aminobenzoic
acid (1.1 eq) were mixed together and then the mixture put
under vigorous stirring at room temperature. After 24h, the
suspension was acidified with HCl 37% and the thiourea 3k
recovered by filtration: yield 84% (304mg, white solid); mp
146°C; 1H NMR (300MHz, DMSO d6): δH 2.00 (d, J=1.0 Hz,
3H, CH3), 6.87 (q, J=1.0 Hz, 1H, H5), 7.33–7.63 (m, 3H, Ar),
7.68–7.79 (m, 3H, Ar), 7.84–7.93 (m, 2H, Ar), 8.79 (s, 1H,
NH), 10.57 (s, 1H, NH), 12.74 (br s, 1H, COOH). 13C NMR
(75MHz, DMSO d6): δC 15.43, 107.60, 121.38 (2C), 126.04,
127.73, 128.94, 129.65, 129.97 (2C), 130.85, 133.27, 135.90,
140.57, 143.24, 166.85, 180.10, 188.61; HRMS: m/z [M+H]+
calcd. for C18H16N3O2S3+: 402.0399, found: 402.0398.
HRMS: m/z [M+H]+ calcd. for C20H16N3F6S3 : 508.0405,
+
found: 508.0409.
1-(2-(4-Methyl-2-oxothiazol-3(2H)-yl)phenyl)-3-
phenylthiourea (3o)
To a solution of isothiocyanate 2c (200 mg, 0.8 mmol)
in CH3CN (5 mL), aniline was added (1.05 eq) and the
solution stirred for 24 h at room temperature. en, the
solvent was partially evaporated and the resulting thio-
urea recovered by filtration: yield 80% (220 mg, white
solid); mp 183°C; 1H NMR (200 MHz, DMSO d6): δH
2.00 (d, J= 1.0 Hz, 3H, CH3), 6.28 (q, J= 1.0 Hz, 1H, H5),
7.06–7.21 (m, 1H, Ar), 7.24–7.60 (m, 7H, Ar), 7.63–7.76
(m, 1H, Ar), 8.87 (s, 1H, NH), 10.06 (s, 1H, NH); 13C NMR
(50 MHz, DMSO d6): δC 15.21, 96.45, 123.11 (2C), 124.67,
127.13, 128.56 (2C), 129.18, 129.26, 130.12, 131.28, 133.02,
136.79, 139.00, 171.43, 180.27; HRMS: m/z [M+H]+ calcd.
for C17H16N3OS2+: 342.0729, found: 342.0728.
1-(4-Fluorophenyl)-3-(2-(4-methyl-2-thioxothiazol-3(2H)-yl)
phenyl)thiourea (3l)
According to the general procedure, the reaction of
4-fluorobenzenamine with isothiocyanate 2a afforded
thiourea 3l: yield 86% (291 mg, white solid); mp 179°C;
1H NMR (300 MHz, DMSO d6): δH 1.99 (d, J= 1.0 Hz, 3H,
CH3), 6.87 (q, J= 1.0 Hz, 1H, H5), 7.13–7.23 (m, 2H, Ar),
7.32–7.60 (m, 5H, Ar), 7.72–7.80 (m, 1H, Ar), 8.44 (s, 1H,
NH), 10.21 (s, 1H, NH); 13C NMR (75 MHz, DMSO d6): δC
15.39, 107.55, 115.27 (d, J= 22 Hz, 2C), 125.70 (d, J= 8 Hz,
2C), 127.46, 128.71, 129.56, 130.63, 133.02, 134.96 (d, J= 3
Hz, 1C), 136.03, 140.56, 159.28 (d, J= 242 Hz, 1C), 180.56,
188.46; HRMS: m/z [M+H]+ calcd. for C17H15FN3S3+:
376.0406, found: 376.0404.
1-(2-(4-Methyl-2-thioxothiazol-3(2H)-yl)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (4b)
To a solution of thiazoline 1a (1g, 4.5 mmol) in CH3CN
(10mL),3-(trifluoromethyl)phenylisocyanate(1.05eq)was
added and the solution stirred at room temperature. After
24h, the solvent was evaporated and urea 4b recovered
by column chromatography (CH2Cl2/AcOEt 9.5/0.5): yield
85% (1.57g, white solid); mp 151°C, Rf =0.32 (CH2Cl2/AcOEt
9.5/0.5), 1H NMR (300MHz, DMSO d6): δH 1.85 (d, J=1.1 Hz,
3H, CH3), 6.93 (q, J=1.1 Hz, 1H, H5), 7.17–7.35 (m, 3H, Ar),
7.42–7.59 (m, 3H, Ar), 7.77 (s, 1H, NH), 7.98 (br s, 1H, Ar),
8.06–8.14 (m, 1H, Ar), 9.52 (s, 1H, NH). 13C NMR (75MHz,
1-(2-(4,5-Dimethyl-2-thioxothiazol-3(2H)-yl)phenyl)-3-(3-
(trifluoromethyl)phenyl)thiourea (3m)
According to the general procedure, the reaction of 3,5-
bis(trifluoromethyl)benzenamine with isothiocyanate
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenyl(thio)ureas bearing 2-(thio)oxothiazoline derivatives 157
136.83, 139.37, 152.26, 171.55; Anal. calcd. for C17H15N3O2S
(325.39): C, 62.75; H, 4.65; N, 12.91; S, 9.85%. Found: C,
62.76; H, 4.87; N, 13.09; S, 9.79%.
DMSO d6): δC 14.94, 107.86, 114.04 (q, J=4 Hz, 1C), 118.29
(d, J=4 Hz, 1C), 121.63, 123.19, 124.12 (q, J=272 Hz, 1C),
124.17, 128.01, 129.01, 129.56 (d, J=31 Hz, 1C), 130.01 (2C),
135.42, 139.97, 140.18, 152.29, 189.13; HRMS: m/z [M+H]+
calcd. for C18H15N3OF3S2+: 410.0603, found: 410.0611.
1-(2-(4-Methyl-2-oxothiazol-3(2H)-yl)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (4i)
To a solution of thiazoline 1c (236 mg, 1.17 mmol) in
CH3CN (5 mL), 3-(trifluoromethyl)phenyl isocyanate
(1.05 eq) was added and the solution stirred at room tem-
perature for 24 h. en, the solvent was partially evapo-
rated and the resulting urea recovered by filtration: yield
79% (355 mg, white solid); mp 234°C, 1H NMR (300 MHz,
DMSO d6): δH 1.78 (d, J= 1.2 Hz, 3H, CH3), 6.36 (q, J= 1.2
Hz, 1H, H5), 7.12–7.37 (m, 3H, Ar), 7.40–7.58 (m, 3H, Ar),
7.94–8.16 (m, 3H, Ar and NH), 9.48 (s, 1H, NH). 13C NMR
(75 MHz, DMSO d6): δC 14.79, 97.02, 114.09 (q, J= 4 Hz,
1C), 118.29 (d, J= 4 Hz, 1C), 121.69, 122.45, 123.68, 124.14
(q, J= 272 Hz, 1C), 125.73, 129.55, 129.57 (d, J= 31 Hz,
1C), 129.76, 130.01, 132.54, 136.40, 140.21, 152.27, 171.52;
Anal. calcd. for C18H14F3N3O2S (393.39): C, 54.96; H, 3.59;
N, 10.68; S, 8.15%. Found: C, 55.03; H, 3.55; N, 10.78; S,
8.13%.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(4-methyl-2-
thioxothiazol-3(2H)-yl)-4-(trifluoro-methyl)phenyl)urea (4f)
To a solution of thiazoline 2d (100mg, 0.34 mmol) in
CH3CN (10 mL), 3,5-bis(trifluoromethyl)phenyl isocya-
nate (1.05 eq) was added and the solution stirred at room
temperature. After 24 h, the stirring was stopped and 4f
recovered by crystallization: yield 70% (131 mg, colour-
1
less crystals); mp 268°C, H NMR (300 MHz, DMSO d6):
δH 1.88 (d, J= 1.0 Hz, 3H, CH3), 6.99 (q, J= 1.0 Hz, 1H, H5),
7.69 (br s, 1H, Ar), 7.74–7.79 (m, 1H, Ar), 7.84–7.91 (m,
1H, Ar), 8.07 (br s, 2H, Ar), 8.19 (s, 1H, NH), 8.43–8.51 (m,
1H, Ar), 9.96 (s, 1H, NH). 13C NMR (75 MHz, DMSO d6):
δC 14.92, 108.42, 115.19 (sept, J= 4 Hz, 1C), 118.22 (d, J= 3
Hz, 2C), 122.22, 123.22 (q, J= 272 Hz, 2C), 123.72 (q, J= 271
Hz, 1C), 123.98 (q, J= 33 Hz, 1C), 127.07 (q, J= 4 Hz, 1C),
127.12, 127.31 (q, J= 4 Hz, 1C), 130.86 (q, J= 33 Hz, 2C),
139.29, 139.76, 140.92, 151.87, 189.63; HRMS: m/z [M+H]+
calcd. for C20H13N3OF9S2+: 546.0350, found: 546.0349.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(2-(4-methyl-2-
oxothiazol-3(2H)-yl)phenyl)urea (4j)
To a solution of thiazoline 1c (236 mg, 1.17 mmol) in
CH3CN (5 mL), 3,5-bis(trifluoromethyl)phenyl isocya-
nate (1.05 eq) was added and the solution stirred at room
temperature for 24 h. en, the solvent was partially
evaporated and the resulting urea recovered by filtra-
tion: yield 75% (396 mg, white solid); mp 267°C, 1H NMR
(300 MHz, DMSO d6): δH 1.79 (d, J= 1.2 Hz, 3H, CH3), 6.36
(q, J= 1.0 Hz, 1H, H5), 7.17–7.30 (m, 2H, Ar), 7.42–7.53 (m,
1H, Ar), 7.65 (br s, 1H, Ar), 7.93–8.20 (m, 4H, Ar and NH),
9.81 (s, 1H, NH). 13C NMR (75 MHz, DMSO d6): δC 14.81,
97.04, 114.62 (sept, J= 4 Hz, 1C), 117.86 (d, J= 3 Hz, 2C),
122.75, 123.24 (q, J= 272 Hz, 2C), 124.12, 126.09, 129.60,
129.80, 130.78 (q, J= 33 Hz, 2C), 132.54, 136.02, 141.41,
152.22, 171.51; Anal. calcd. for C19H13F6N3O2S (461.29): C,
49.46; H, 2.84; N, 9.11; S, 6.95 %. Found: C, 49.73; H, 2.78;
N, 9.23; S, 7.11%.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(5-fluoro-2-(4-methyl-2-
thioxothiazol-3(2H)-yl)-phenyl)urea (4g)
To a solution of thiazoline 1e (300 mg, 1.25 mmol) in
CH3CN (10 mL), 3,5-bis(trifluoromethyl)phenyl isocya-
nate (1.05 eq) was added and the solution stirred at room
temperature. After 24 h, the stirring was stopped and 4g
recovered by crystallization: yield 72% (445 mg, colour-
1
less crystals); mp 250°C, H NMR (300 MHz, DMSO d6):
δH 1.87 (d, J= 1.0 Hz, 3H, CH3), 6.96 (q, J= 1.0 Hz, 1H, H5),
7.05–7.15 (m, 1H, Ar), 7.25–7.36 (m, 1H, Ar), 7.68 (br s,
1H, Ar), 7.98–8.11 (m, 4H, Ar and NH), 9.92 (s, 1H, NH).
13C NMR (75 MHz, DMSO d6): δC 14.97, 108.00, 108.74
(d, J= 28 Hz, 1C), 110.82 (d, J= 23 Hz, 1C), 115.04 (sept,
J= 4 Hz, 1C), 118.10 (d, J= 3 Hz, 2C), 123.22 (q, J= 273
Hz, 2C), 123.40 (d, J= 3 Hz, 1C), 130.83 (q, J= 33 Hz, 2C),
131.22 (d, J= 10 Hz, 1C), 137.10 (d, J= 13 Hz, 1C), 140.05,
141.00, 151.97, 162.16 (d, J= 244 Hz, 1C), 189.60; HRMS:
m/z [M+H]+ calcd. for C19H13N3OF7S2+: 496.0383, found:
496.0382.
1,3-Bis(3,5-bis(trifluoromethyl)phenyl)urea (4k)
To
a
solution of 3,5-bis(trifluoromethyl)benze-
namine (200 mg, 0.87 mmol) in CH3CN (5 mL), 3,5-
bis(trifluoromethyl)phenyl isocyanate (1.05 eq) was
added and the solution stirred at room temperature for
1 h. en, the solvent was partially evaporated and the
resulting urea recovered by filtration: yield 70% (296 mg,
white solid); mp 244°C (40 240–242°C), 1H NMR (300 MHz,
DMSO d6): δH 7.16 (br s, 2H), 7.60 (br s, 2H), 7.94 (br s,
4H). NMR data are in agreement with literature40.
1-(2-(4-Methyl-2-oxothiazol-3(2H)-yl)phenyl)-3-phenylurea (4h)
To a solution of thiazoline 1c (185 mg, 0.90 mmol) in
CH3CN (5 mL), phenyl isocyanate (1.05 eq) was added
and the solution stirred at room temperature. After 24 h,
the solvent was evaporated and replaced with CH2Cl2
(10 mL); then the solution was washed with HCl 5%
(3 × 10 mL), and the organic phase evaporated to dry-
ness: yield 76% (221 mg, white solid); mp 146°C, 1H NMR
(300 MHz, DMSO d6): δH 1.78 (d, J= 1.2 Hz, 3H, CH3),
6.36 (q, J= 1.2 Hz, 1H, H5), 6.95–7.50 (m, 8H, Ar), 7.79
(s, 1H, 1NH), 8.14 (m, 1H, Ar), 9.14 (s, 1H, NH). 13C NMR
(75 MHz, DMSO d6): δC 14.78, 96.98, 118.17 (2C), 122.05,
122.14, 123.23, 125.40, 128.85 (2C), 129.52, 129.72, 132.57,
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3,5-dimethylphenyl)
urea (4l)
To a solution of 3,5-dimethylbenzenamine (200 mg,
1.65 mmol) in CH3CN (5 mL), 3,5-bis(trifluoromethyl)
phenyl isocyanate (1.05 eq) was added and the solution
© 2013 Informa UK, Ltd.

158 F. Andreoli et al.
stirred at room temperature for 1 h. en, the solvent
was partially evaporated and the resulting urea recov-
ered by filtration: yield 76% (472 mg, white solid); mp
was submitted to a quick minimization protocol to
ensure correct angles and atom distances (500 steps
Steepest Descent algorithm, 0.01 gradient, then 2000
steps Conjugate Gradient algorithm, 0.01 gradient). All
ligands used in the study were prepared in the mol2
format with DS 2.1, and were considered as fully flex-
ible during the docking process.
e docking software used in this study was CDocker
(J Comp Chem 2003, 24, 1549–1562) as implemented in
Discovery Studio 2.1. Briefly, the grid based algorithm
used a simulated annealing process to account for the
flexibility of the ligand into a rigid receptor, the obtained
complex energy was considered to evaluate the affinity
of the docked ligand. e ligands were docked into the
active site using a 10 Å sphere centered on a point situ-
ated at equal distance between Asp 228 and Tyr 71 Cα.
Pymol software was used to prepare Figure 1 (DeLano,
W.L. e PyMOL Molecular Graphics System. (2008)
DeLano Scientific LLC, Palo Alto, CA, USA. http://www.
pymol.org).
1
235°C, H NMR (300 MHz, DMSO d6): δH 2.24 (s, 6H,
2CH3), 6.66 (br s, 1H, Ar), 7.10 (br s, 2H, Ar), 7.62 (br
s, 1H, Ar), 8.12 (br s, 2H, Ar), 8.78 (s, 1H, NH), 9.32 (s,
1H, NH). 13C NMR (75 MHz, DMSO d6): δC 21.07 (2C),
114.25 (sept, J = 4 Hz, 1C), 116.58 (2C), 117.88 (d, J = 4
Hz, 2C), 123.32 (q, J = 272 Hz, 2C), 124.13, 130.69 (q,
J = 33 Hz, 2C), 137.81 (2C), 138.82, 141.91, 152.36;
HRMS: m/z [M+H]+ calcd. for C17H15N2OF6+: 377.1083,
found: 377.1084.
Biology
BACE-1 enzymatic assay
ese experiments have been performed using
BACE-1 (β-secretase) FRET Kit assay, from PanVera
Corporation (Madison, WI, USA), according to the
described protocol and using a multiwell spectrofluo-
rometer instrument capable of 530–545 nm excitation
and 570–590 nm emission wavelengths (Wallac Victor2–8
1420, Perkin Elmer, Turku, Finland). e procedure is
as follow: the substrate and enzyme are diluted accord-
ing to the described protocol into the provided assay
buffer (50 mM Tris, pH 7.5, 10% glycerol). Each inhibi-
tor is diluted into DMSO at the desired concentration.
e substrate (Rh-EVNLDAEFK-Quencher; 10 µL of
the main solution) and each inhibitor (1 µL of the cor-
responding solution) are introduced in the 96-well flat
bottom black polystyrene plate (Corning, NY, USA).
e resulting mixtures are gently mixed and 10 µL of
the enzyme solution are then added to each well to
start the reaction. e reaction mixtures are incubated
at 25°C for 90 min and the spectrofluorescence is
monitored at 530–545 nm (excitation wavelength) and
570–590 nm (emission wavelength). e kinetic assays
are performed in sixplicate for each inhibitor, using
BACE-1 inhibitor (H-Lys-r-Glu-Glu-Ile-Ser-Glu-Val-
Results
A library of thioureas 3a–o was prepared according to
Scheme 1. N-(2-aminophenyl)-thiazoline-2-thiones
1a,b and N-(2-aminophenyl)-thiazolin-2-one 1c were
transformed into the corresponding isothiocyanates
2a–c by the CS2, DCC route under very mild conditions.
ese isothiocyanates are stable, they can be chromatog-
raphied using alcohol-free mobile phases and stored in
the fridge for a long period of time. Intermediates 2a–c
afforded a platform from which the thioureas 3a–o were
easily prepared in good yield by reaction with the corre-
sponding aniline, aminopyridine or aminopyrimidine at
room temperature in CH3CN.
Asn-(3S,4S)-Stat-Val-Ala-Glu-Phe-OH,
IC50 = 30 nM;
Calbiochem, Beeston, UK) as reference (negative test,
no clivage), the provided BACE-1 Product Standard
(Rh-EVNL) as positive test (100% clivage) and a con-
trol test using only the enzyme and the substrate in the
same conditions to allow a 15% clivage of the substrate
after 90 minutes.
Molecular docking
BACE-1 protein coordinates were obtained from the
Research Collaboratory for Structural Bioinformatics
Protein Data Bank, code 1SGZ and modified with
Discovery Studio 2.1 (Accelrys, San Diego, USA).
Chains were separated and considered individually,
water molecules were removed, incomplete residues
were modified with the “Clean Protein” command
from the Protein Reports and Utilities module. Finally,
hydrogen atoms were added, charges were calculated
with CHARMm forcefield and the resulting structures
were saved in the mol2 format. e obtained receptor
Figure 1. Molecular docking for 4c with BACE-1.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenyl(thio)ureas bearing 2-(thio)oxothiazoline derivatives 159
Scheme 1. Synthesis of thioureas 3.
Scheme 2. Synthesis of ureas 4.
In contrast to isothiocyanates 2, the isocyanates of
N-(2-aminophenyl)-thiazoline-2-(thi)ones 1 were not
stable. Another route was used to prepare ureas 4a–j in
good yields. N-(2-aminophenyl)-thiazoline-2-thiones
1a,b and N-(2-aminophenyl)-thiazolin-2-ones 1c–e were
reacted with commercially available arylisocyanates
under very mild conditions (Scheme 2).
All the compounds are displayed at the same time in
Schemes 1 and 2 for convenience. Of course the library
was elaborated step by step and the logic behind the
design was strongly connected with the arriving inhibi-
tion results.
N-H of the thiourea and the carbonyl group of the het-
erocycle in 3o.
Compound 4c gave the best binding of the whole
library: 30% inhibition at 100 nM. e binding difference
with thiourea 3h and urea 4c deserves a brief comment.
It is well documented that thioureas are more acidic
than the corresponding ureas and one should expect a
better binding through H-bonding for the thioureas. We
have previously shown that the thioureas of the 3 series
adopted a Z,E conformation while the urea analogue
adopted a Z,Z conformation19. e preorganization in the
urea 4 was accounting for a better binding to carboxylate
anion than the thiourea 3 which required a costly confor-
mational change for the same binding. We believe that
the same consideration is operating for the better bind-
ing observed in 4c. Similar observations were recently
reported for the binding abilities of urea and thiourea in
1,3-disubstituted thia[4]calixarenes and corresponding
monofunctional receptors for their anion recognition
properties41.
e two atropisomers of 4c showed no significant
difference in binding and they gave binding values very
close the one obtained for the racemate.
Figure 1 reported molecular docking studies with
analogue 4c showed that Asp 228 from BACE-1 inter-
acts with the first NH donor of the urea while the sec-
ond NH interacts with Gly 34. A close proximity was
observed between the ligand CF3 and residues Trp 76
and Arg 128.
Enzymatic assays were conducted as reported in the
experimental part. e percent inhibition values were
determined at least six times. Selected values are sum-
marized in Table 1.
Results in Table 1 show that the inhibition range
covered by the library goes from no inhibition at 30 µM
(3k and 4e) to 30% inhibition at 100 nM (4c). Taking
3a which produced 30% inhibition at 3 µM as a refer-
ence, the introduction of electron donating groups on
the N-aryl group drastically decreased the binding.
For instance, compounds 3d, 3e and 3f in which the
aryl group is equipped with OH, di-Me or MeO groups
are inactive at 3 µM. On the other hand, a ten-fold
increase in the binding (230 nM) is observed for 3g
and 3h which present one or two CF3 group on the aryl.
Interestingly, compounds 3i, 3k and 3l which present
electron attracting groups weaker than CF3 are inac-
tive. Compounds 3b, 3c and 3j (Scheme 1) in which
the aryl group was replaced by a pyridine or a pyrimi-
dine as electro-deficient groups were found inactive at
30 µM. Comparison of 3g vs 3m and 3h vs 3n indicates
that the introduction of a methyl group in position 5 of
the thiazoline ring significantly decreases the binding.
e smaller binding for 3o vs 3a is probably reflecting a
detrimental intramolecular hydrogen bond between a
e discussion of the data reported in Table 1 led
to a coherent view of the important feature to achieve
a good binding in series 3 and 4. Ureas are better
than thioureas and the east phenyl group should be
equipped with two CF3 in 3,5 positions. Based on these
remarks, two very simple ureas 4k and 4l were pre-
pared and assayed as BACE-1 inhibitors. 1,3-Bis(3,5-
Bis(trifluoromethyl)phenyl)urea, 4k is well known in
© 2013 Informa UK, Ltd.

160 F. Andreoli et al.
Table 1. BACE-1 inhibition according to a FRET assay.
Cpd
3a
3d
3e
3f
X1
S
X2
S
R1
H
R2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
R4
H
R5
H
R6
H
R7
H
% Inh (µM)*
30 (3)
S
S
H
OH
H
H
H
H
Inac (3)
Inac (3)
Inac (3)
35 (0.23)
35 (0.23)
Inac (3)
Inac (30)
Inac (3)
40 (3)
S
S
H
CH3
H
H
CH3
H
S
S
H
OCH3
H
H
3g
3h
3i
3k
3l
3m
3n
3o
4a
4b
4c
4d
4e
4f
S
S
H
CF3
CF3
H
H
H
S
S
H
H
H
CF3
H
S
S
H
Cl
H
Cl
CO2H
F
S
S
H
H
H
S
S
H
H
H
H
S
S
CH3
CH3
H
H
CF3
CF3
H
H
H
S
S
H
H
CF3
H
40 (1)
O
S
S
H
H
Inac (10)
Inac (3)
Inac (3)
30 (0.1)
Inac (3)
Inac (30)
Inac (3)
Inac (3)
30 (3)
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
S
H
H
CF3
CF3
H
H
H
S
H
H
H
CF3
H
S
H
H
NO2
CH3
H
S
H
H
H
H
S
H
H
CF3
CF3
H
CF3
CF3
H
4g
4h
4i
S
H
H
H
O
O
O
H
H
H
H
H
H
H
H
CF3
CF3
H
H
50 (10)
4j
H
H
H
CF3
30 (1)
*Standard deviation on the inhibition percentages are 5% of indicated value.
the field of organocatalysis42–50. e two N-H are highly
Table 2. Comparison of % of inhibition for 3h, 4c, 4k and 4l at
various concentrations.
acidic and equivalent in 4k. 1-(3,5-Bis(trifluoromethyl)-
phenyl)-3-(3,5-dimethylphenyl)urea 4l is a new com-
pound in which the two N-H groups should present
marked difference in acidity.
Entry
3h
4c
4k
4l
10 µM
100%
100%
100%
100%
1 µM
80%
0.1 µM
25 5% (%)
50 5%
100
0.01 µM
Inactive
Inactive
15 5%
Inactive
100%
100%
100%
Urea 4k produced 15% inhibition at 10 nM level,
while urea 4l gave 80% inhibition at 100 nM level. For the
most active BACE-1 inhibitors (Table 2) 3h, 4c, 4k and
4l, curves plotting drug concentrations versus inhibition
percentage were established allowing determination
of the IC50 values (values which correspond to the drug
concentration leading to 50% enzyme inhibition). e
most potent BACE-1 inhibitor is analogue 4k (IC50 value
ranging between 0.01 and 0.1 µM), while IC50 values for
analogues 4c and 4l were respectively 0.1 0.03 µM and
0.08 0.03 µM; in contrast IC50 value for analogue 3h
ranged between 0.1 and 1 µM.
80 5%
Declaration of interest
OSEO − ANVAR: grant A06 07 008 UAT and URMA
(Unis pour la Recherche sur la Maladie d’Alzheimer)
association are greatly acknowledged for financial
support. e authors report no conflicts of interest.
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Journal of Enzyme Inhibition and Medicinal Chemistry

Novel phenyl(thio)ureas bearing 2-(thio)oxothiazoline derivatives 161
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