Synthesis and structure-activity relationships of tri-substituted thiazoles as RAGE antagonists for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2012.10.022

A series of thiazole derivatives were designed, and prepared to develop RAGE antagonist for the treatment of Alzheimer's disease (AD). SAR studies were performed to optimize inhibitory activity on Aβ-RAGE binding. SAR studies showed that introducing an amino group at part A was essential for inhibitory activity on Aβ-RAGE binding. Compounds selected from Aβ-RAGE binding screening displayed inhibitory activity on Aβ transport across BBB. They also showed inhibitory activity against Aβ-induced NF-κB activation. These results indicated that our derivatives had a potential as therapeutic agent for the treatment of AD.

Full text of DOI:10.1016/j.bmcl.2012.10.022

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7555–7561
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of tri-substituted thiazoles
as RAGE antagonists for the treatment of Alzheimer’s disease
Yun Suk Lee a,b, Hee Kim c, Young-Ho Kim c, Eun Joo Roh a, Hogyu Han b, Kye Jung Shin d,
⇑
a Korea Institute of Science and Technology, 39-1 Hawolgog-dong, Sungbuk-gu, Seoul 136-791, Republic of Korea
b Department of Chemistry, Korea University, 1 Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea
c Medifron DBT, Sandanro 349, Danwon-Gu, Ansan-City, Gyeonggi-do 425-839, Republic of Korea
d Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu,
Bucheon, Gyeonggi-do 420-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thiazole derivatives were designed, and prepared to develop RAGE antagonist for the treat-
ment of Alzheimer’s disease (AD). SAR studies were performed to optimize inhibitory activity on Ab-
RAGE binding. SAR studies showed that introducing an amino group at part A was essential for inhibitory
activity on Ab-RAGE binding. Compounds selected from Ab-RAGE binding screening displayed inhibitory
Received 13 August 2012
Revised 28 September 2012
Accepted 4 October 2012
Available online 13 October 2012
activity on Ab transport across BBB. They also showed inhibitory activity against Ab-induced NF-jB acti-
vation. These results indicated that our derivatives had a potential as therapeutic agent for the treatment
of AD.
Keywords:
Alzheimer’s disease
RAGE antagonist
Ó 2012 Elsevier Ltd. All rights reserved.
NF-jB
b-Amyloid
Alzheimer’s disease (AD) is a neurodegenerative disorder and is
Ab transport across BBB is mediated by the low-density lipopro-
tein receptor-related protein 1 (LRP-1) and the receptor for
advanced glycation end-products (RAGE). LRP-1 and RAGE play
contrary roles in Ab transport across BBB; efflux and influx of Ab.
RAGE is a multiligand cell surface receptor which is normally
expressed in endothelium, microglia and neurons. RAGE was origi-
nally identified as a receptor for the advanced glycation end prod-
ucts (AGEs) and amphoterin. Along with the involvement of RAGE
in disease and age-related manner, it was reported that expression
of RAGE was up-regulated in AD brain tissues and RAGE acted as a
receptor/transporter for Ab from blood into brain.7 Also, RAGE in
the most common cause of dementia. The pathological hallmarks
of AD include extracellular amyloid plaques that are consist of
aggregated Ab, cleaved peptides from amyloid precursor protein
(APP).1 Although pathogenic process of AD is not yet clear, the
beginning of AD is due to abnormal accumulation of Ab in the
brains causing neurodegeneration and finally the clinical symp-
toms of dementia.2 Therefore, Ab is considered to play a central
role in the pathogenesis of AD. In addition to accumulation of Ab,
evidences for the involvement of inflammatory responses in the
pathogenesis of AD have been documented. A number of inflam-
matory mediators with Ab-associated NF-
j
B activation have been
the surface of microglia and astrocyte activated NF-jB signaling
reported to be up-regulated in AD brain.3
pathway and triggered oxidative stress and inflammatory response,
causing cellular perturbation.7b,8 These studies suggested that RAGE
was a key target in the inflammatory and neurotoxic cascade.
Our goal is to discover a potent antagonist that prevents Ab
transport across the BBB and decreases inflammatory response in
the brain. Initially, compounds known for RAGE antagonist were
considered as our strategy for molecular design of RAGE antagonist
(Fig. 1). Our newly designed molecule was divided into four parts;
parts A (aminoalkyl phenyl), part B (heterocyclic core), part C
(alkoxyphenyl), and part D (aliphatic chain). BBB permeation of
compounds was required for anti-inflammatory action of com-
pound in brain. Therefore, more lipophilic thiazole ring than benz-
imidazole was selected for structural modification of heterocyclic
Ab peptides were initially thought to derive solely from neurons
and glia.4 It is now believed that Ab originates from the entire tis-
sues and circulating Ab in plasma can contribute to accumulation
of neurotoxic Ab in the brain through transport of plasma Ab.5
The accumulation of Ab in AD brain was related with the balance
between production and clearance of Ab. Although there has been
a lot of reports regarding regulation of Ab level in the brain for the
treatment strategy of AD, Ab-lowering strategy by receptors that
mediate Ab transport across blood–brain barrier (BBB) recently be-
gin to receive attention.6
⇑
Corresponding author. Tel.: +82 2 2164 4060; fax: +82 2 2164 4059.
E-mail address: kyejung@catholic.ac.kr (K.J. Shin).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.022

7556
Y. S. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7555–7561
O
N
N
O
N
O
O
N
N
N
H
O
NHBoc
NHBoc
O
Cl
I
II
Part C
Alkoxy
phenyl
Part A
Aminoalkyl
phenyl
Part B
Heterocyclic
core
O
R3
R1
N
n
R2
X
core
R4
Part D
Aliphatic
chain
Figure 1. Compounds known for RAGE antagonist and synthetic strategy for new compounds.9
core at part B. In this study, we reported the synthesis of newly de-
signed compounds and established SAR studies for RAGE binding.
General synthesis for thiazole core ring was achieved by cycli-
then brominated with CuBr2 to give
Preparation of -chloroketones 16 and 20 was shown in Scheme 2.
Ullmann reaction of arylbromide 12 and phenol 7 provided diphe-
a
-bromoketones 5 and 11.12
a
zation reaction of thioamide and
a-haloketone. Synthesis of inter-
nyl ether 13. Ketones 15 and 19 were obtained from 14 and 18 by
mediates thioamide and -haloketone was shown in Scheme 1 and
a
Weinreb ketone synthesis strategy. Regiospecific a-chloroketones
Scheme 2. Treatment of benzonitriles (2 and 8) with NaSH and
MgCl2 in DMF provided desired thioamides 3 and 9.10 Benzonitriles
(2 and 8) were subject to reaction with pentylMgBr, followed by
acidic hydrolysis to afford ketones 4 and 10.11 These ketones were
16 and 20 were synthesized by treating 15 and 19 with sulfuryl
chloride, respectively.13
Thiazoles 21aÀ24a which had positional diversity were ob-
tained by cyclization reaction with intermediates thioamide and
BnO
HO
a
BnO
b
NH2
CN
CN
S
2
3
1
c
BnO
BnO
Br
d
Bu
Bu
O
O
4
5
S
Cl
NC
NC
Cl
Cl
e
f
H2N
+
HO
F
O
O
9
6
7
8
g
O
O
h
Bu
Cl
Bu
Cl
Br
O
O
10
11
Scheme 1. Reagents and conditions: (a) BnBr, K2CO3, acetone, reflux, 98%; (b) NaSH, MgCl2, DMF, rt, 95%; (c) (i) pentylMgBr, THF, 65 °C; (ii) aqueous H2SO4, 86%; (d) CuBr2,
EtOAc, 70 °C, 61%; (e) K2CO3, DMF, reflux, 96%; (f) NaSH, MgCl2, DMF, rt, 91%; (g) (i) pentylMgBr, THF, 65 °C; (ii) aqueous H2SO4, 53%; (h) CuBr2, EtOAc, 70 °C, 97%.

Y. S. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7555–7561
7557
Me
N
Cl
Cl
Cl
a
b
MeO2C
MeO
MeO2C
+
O
O
O
Br
HO
12
7
13
Cl
14
Bu
Cl
Bu
Cl
c
d
O
O
O
O
15
16
BnO
BnO
BnO
O
BnO
g
O
e
f
O
OMe
N
Me
CN
Bu
Bu
Cl
17
18
19
20
Scheme 2. Reagents and conditions: (a) Cu2O, Cs2CO3, N,N-dimethylglycine, molecular sieve, DMF, 110 °C, 42%; (b) (i) 1 N NaOH, MeOH, reflux, then aqueous HCl, 95%; (ii)
SOCl2, reflux; (iii) N,O-dimethylhydroxylamine, NEt3, CH2Cl2, 0 °C, 55%; (c) n-BuLi, THF, À78 °C, 52%; (d) SO2Cl2, CH2Cl2, À10 °C, 75%; (e) (i) 30% NaOH, reflux, then aqueous
HCl, 88%; (ii) CDI, THF, then N,O-dimethylhydroxylamine, 32%; (f) n-BuLi, THF, À78 °C, 86%; (g) SO2Cl2, CH2Cl2, À10 °C, 42%.
a
-haloketone in EtOH (Scheme 3). After debenzylation, Mitsunobu
We introduced thiazole ring at heterocyclic core of molecules
and attempted to investigate the effect on substitution site of
thiazole ring. Their inhibitory activity on Ab-RAGE binding was
evaluated using in vitro enzyme-linked immunosorbent assay
(ELISA).14 The results of Ab-RAGE binding study were summarized
in Table 1 and 2. It showed that there was no difference in
inhibitory activity between 2,4-phenyl-substituted (Table 1) and
2,5-phenyl-substituted (Table 2) derivatives. Also, the shifting for
R1 (part A) and R2 (part B) had no effect on inhibitory activity.
Inhibitory activity depended rather on amino group of part A.
Compounds 21aÀ24b that did not contain amino group showed
no activity, whereas compounds 21cÀ24d with amino group
reaction with appropriate aminoalcohols provided desired com-
pounds 21c–24d.
Alternatively, for modification of alkylamino group, 22b reacted
with dibromoalkane followed by the amination using the corre-
sponding amines to afford compounds 22gÀq (Scheme 4).
Compound 27a was prepared from 26b by amide coupling reac-
tion (Scheme 5). Compound 27b which is reverse amide form of
27a was prepared by condensation reaction with thiourea and
11, followed by amidation, debenzylation, and Mitsunobu reaction
(Scheme 6). Compound 27c was obtained by cyclization reaction
with 28 and 11 (Scheme 7).
Cl
Cl
RO
RO
BnO
S
Br
a
c
Cl
+
H2N
N
S
N
Bu
O
O
O
O
S
Bu
Bu
5
9
21a
. R = Bn
. R = H
21c
. R = (1-ethylpiperidin-3-yl)methyl
b
21d
. R = 3-(diethylamino)propyl
21b
Cl
Cl
O
RO
RO
BnO
Bu
Cl
c
a
+
N
N
NH2
Br
O
O
O
S
S
S
11
3
Bu
22a.
Bu
R = Bn
22c. R = (1-ethylpiperidin-3-yl)methyl
22d. R = 3-(diethylamino)propyl
b
22b. R = H
Cl
Cl
RO
RO
S
BnO
O
a
c
Cl
+
H2N
S
S
N
Bu
O
O
O
O
Cl
N
Bu
Bu
20
9
23a. R = Bn
23c
23d
. R = (1-ethylpiperidin-3-yl)methyl
. R = 3-(diethylamino)propyl
b
23b
. R = H
Cl
Cl
RO
RO
Cl
BnO
a
c
Bu
Cl
+
NH2
S
S
O
O
O
N
N
S
3
16
Bu
24a. R = Bn
Bu
24c
24d
. R = (1-ethylpiperidin-3-yl)methyl
. R = 3-(diethylamino)propyl
b
24b
. R = H
Scheme 3. Reagents and conditions: (a) EtOH, reflux, 38À79%; (b) BBr3, CH2Cl2, À78 °C, 72À92%; (c) aminoalcohols, PPh3, DIAD, THF, 0 °C ? rt, 18À47%.

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Y. S. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7555–7561
Cl
Cl
Cl
Br
O
R
HO
O
n
n
a and b
c
N
N
N
O
O
O
S
S
S
Bu
22b
Bu
Bu
22g-22q
22e
. n = 2
22f. n = 3
Scheme 4. Reagents and conditions: (a) dibromoethane, K2CO3, MeCN, reflux, 67%; (b) dibromopropane, K2CO3, MeCN, reflux, 80%; (c) amines, K2CO3, MeCN, reflux, 48À94%.
Cl
Cl
Et2N
O
3
O
NH2
S
O
a
c
Bu
Cl
O
RO2C
N
N
N
H
+
O
Br
O
b
OEt
S
O
S
25
11
Bu
Bu
26a. R = Et
26b
27a
. R = H
Scheme 5. Reagents and conditions: (a) EtOH, reflux, 44%; (b) 1 N NaOH, EtOH, reflux, then aqueous, HCl 84%; (c) (i) SOCl2, reflux; (ii) 4-(3-diethylaminopropoxy)aniline,
Et3N, CH2Cl2, 0 °C, 21%.
Cl
Cl
RO
O
H
N
a
b
NH2
S
Bu
Cl
H2N
N
N
+
O
H2N
Br
O
O
S
O
S
28
11
Bu
26d. R = Bn
26e
Bu
26c
c
. R = H
Cl
Et2N
O
3
H
N
d
N
O
O
S
Bu
27b
Scheme 6. Reagents and conditions: (a) EtOH, reflux, 94%; (b) 4-benzyloxybenzoyl chloride, Et3N, CH2Cl2, 0 °C, 50%; (c) BBr3, CH2Cl2, À78 °C, 77%; (d) 3-diethylamino-1-
propanol, PPh3, DIAD, THF, 0 °C ? rt, 25%.
Cl
O
Et2N
O
3
a
H
N
S
Bu
Cl
+
N
O
N
H
NH2
Br
Et2N
S
O
O
Bu
28
11
27c
Scheme 7. Reagents and conditions: (a) EtOH, reflux, 40%.
ncreased inhibitory activity for Ab-RAGE binding (% inhibition,
47.7À58.9% at 10 M).
compared with thiazole derivatives in Table 1 and Table 2 (% inhi-
M). The result confirmed that modifica-
tion of heterocyclic core did not have a significant effect on
increase of inhibitory activity.
Based on in vitro SAR study above, we selected compounds 22d,
which showed high binding activity (IC50 = 0.66 lM), to explore ef-
fect of aminoalkyl group at part A. With fixed diphenyl ether and
butyl groups in part C and D, various amino groups with change
of carbon chain length were substituted at part A. As shown in Ta-
ble 4, most of compounds kept its inhibitory activity on Ab-RAGE
l
bition, 47.7À58.9% at 10
l
As thiazole ring of part B with positional diversity was not re-
lated to inhibition of Ab-RAGE binding, we further continued to
identify influence of part B in binding assay by modifying thiazole
ring. Heterocyclic core at part B was converted into modified thia-
zole (compounds 27a, 27b, and 27c). We inserted amide and amino
groups between phenyl ring of part A and thiazole ring of part B, to
change bond length between part A and part B, and logP value of
compounds. Their inhibitory activity was kept when inserted
amide and amine groups (Table 3). However, striking increase of
binding and exhibited good activity (IC50 = 0.91À3.18
lM).
activity was not observed (% inhibition, 48.1À60.9% at 10
lM), as
Although there was not remarkable difference in inhibitory activity

Y. S. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7555–7561
7559
Table 1
In vitro inhibitory activity of 2,4-phenyl-substituted thiazole derivatives on Ab-RAGE binding
R1
N
R2
S
Bu
Compd.
R1
R2
% inhibition (10
2.4
l
M)
IC50 (lM)
BnO
HO
Cl
Cl
Cl
Cl
21a
—
O
O
O
O
21b
21c
0.2
—
Et2N
O
57.0
1.12
1.91
O
N
21d
55.9
Cl
Cl
Cl
Cl
BnO
HO
22a
22b
22c
—
—
O
O
O
O
—
—
Et2N
O
58.9
1.72
O
N
22d
58.0
0.66
Table 2
In vitro inhibitory activity of 2,5-phenyl-substituted thiazole derivatives on Ab-RAGE binding
R1
S
R2
N
Bu
Compd.
R1
R2
% inhibition (10
—
lM)
IC50 (lM)
BnO
HO
Cl
Cl
Cl
Cl
23a
—
O
O
O
O
23b
23c
—
—
Et2N
O
57.1
2.01
1.14
O
N
23d
53.9
Cl
Cl
Cl
Cl
BnO
HO
24a
24b
24c
—
—
O
O
O
—
—
—
Et2N
O
49.6
O
N
24d
47.7
—

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Y. S. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7555–7561
Table 3
In vitro inhibitory activity of modified thiazole derivatives on Ab-RAGE binding
Compd.
Structure
% inhibition (10
lM)
IC50 (lM)
HCl
Et2N
Cl
O
O
O
27a
48.1
—
N
N
H
O
O
S
S
Bu
HCl
Et2N
Cl
H
N
27b
60.2
60.9
5.59
0.55
N
O
Bu
Cl
HCl
H
N
27c
N
O
Et2N
HCl
S
O
Bu
Table 4
In vitro inhibitory activity of thiazole derivatives on Ab-RAGE binding
Table 5
Cl
In vitro inhibitory activity of compounds on Ab transport across BBB and NF-jB
activation
RO
Compd.
BBB permeation of Ab
Luciferase assay
% inhibition (1 lM)
N
% inhibition (10
lM)
O
S
21c
21d
22c
22d
23c
23d
22h
22i
71.7
68.3
73.3
77.5
63.5
61.5
64.3
64.3
39.5
55.3
68.6
66.6
NT
—
—
—
Bu
Compd.
R
% inhibition (10
47.8
lM)
IC50 (lM)
3.0
10.3
4.9
11.7
3.1
2.5
13.5
17.2
2.1
11.3
22g
—
N
N
22h
22i
59.2
59.2
1.84
1.24
22l
22m
22n
22p
22q
N
N
N
O
22j
22k
22l
40.6
46.2
64.2
—
N
—
using BBB and reporter gene assay, respectively (Table 5)15,16 In
BBB assay, RAGE-overexpressing bEND.3 cells with tight junction
were used for artificial BBB system. The result of BBB assay was con-
sistent with binding assay. Most of compounds with over 50% inhib-
itory activity in binding assay inhibited Ab transport across BBB (%
0.91
N
N
22m
22n
60.1
54.7
1.04
1.21
N
N
inhibition = 39.5À77.5% at 10
was efficient for inhibition of Ab-RAGE binding, with IC50 value of
0.66 M, showed the highest activity in the BBB assay (77.5% at
10 M). On the other hand, active compounds in binding assay were
less active in luciferase reporter assay. Inhibitory activity of com-
pounds on Ab-induced NF- B activation had relatively weak po-
tency (% inhibition, 2.1À17.2% at 1 M). It appeared that these
series of compounds had partial antagonistic effect on Ab-associated
NF- B activation.
lM). Particularly, compound 22d that
N
l
O
22o
22p
22q
22.9
51.8
55.1
—
l
N
N
3.18
1.58
j
l
j
In summary, thiazole derivatives were designed, synthesized,
and evaluated for RAGE antagonist. In SAR studies, alkylamino
group of part A was essential for inhibitory activity of Ab-RAGE
binding, and compounds that can bind to RAGE inhibited transport
of Ab. In particular, we identified that compound 22n inhibited not
according to amine substituents, morpholine seemed to be not
good substituent for inhibition of Ab-RAGE binding (% inhibition,
22j = 40.6%, 22o = 22.9%). These results of binding assay suggested
that amino group of part A was essential for inhibitory activity on
Ab-RAGE binding, and inhibitory activity depended more on amino
group of part A than part B.
only transport of Ab across BBB but also Ab-associated NF-jB acti-
vation by blocking Ab-RAGE binding. This study indicated that our
synthetic compounds had a potential as promising agents for RAGE
antagonist.
For study regarding RAGE antagonist, we further identified inhib-
itory activity on Ab transport and Ab-induced NF-jB activation

Y. S. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7555–7561
7561
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References and notes
14. Ab-RAGE binding assay: 1
10 M Ab solution and 10
l
l
g of purified biotinylated-human-RAGE, 1
lL of
l
M of the compound in 100 L of TBS-T with 2.5%
l
1. Esler, W. P.; Wolfe, M. S. Science 2001, 293, 1449.
BSA were incubated on a streptavidin-coated plate for 60 min at ambient
temperature. After washing the plate with TBS-T, the horseradish-peroxidase
conjugated 4G8 antibody (4G8-HRP, 1:1000 dilution) in 100 lL of TBS-T with
2.5% BSA was added into each well to detect the bound Ab. The plate was
incubated for 60 min at ambient temperature. After washing with TBS-T, the
plate was developed with TMB substrate: the reaction was stopped with
sulfuric acid. The absorbance was read on a Sunrise plate reader (TECHAN) at
450 nm.
2. (a) Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.; Freed, R.; Liosatos,
M.; Morgan, T. E.; Rozovsky, I.; Trommer, B.; Viola, K. L.; Wals, P.; Zhang, C.;
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15. Measurement of BBB-permeated Ab: Collagen-coated transwell-Col inserts
(Corning) were washed with Krebs-ringer bicarbonate buffer (Sigma). bEND.3
cells were grown for 3–4 days to confluence on inserts, and the resistance of
inserts was measured using the Millicell ERS Voltohmmeter (Millipore,
Billerica, MA). The transendothelial electrical resistance (TEER) of the inserts
was calculated by subtracting the resistance of blank inserts from that of the
inserts with bEND.3 cells and multiplying the subtracted values by the area of
the insert. The TEER was used as an index of the barrier property of the bEND.3
4. Masters, C. L.; Multhaup, G.; Simms, G.; Pottgiesser, J.; Martins, R. N.;
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monolayer. For the efficacy of RAGE antagonists, 5
lM of Ab42-FITC (BACHEM)
with indicated compound (10 M) was treated in the inserts with bEND.3 cells.
l
To measure transported Ab42-FITC, the fluorescence level of the media from
donor chamber was measured using SAPIRE (TECHAN) at 520 nm.
8. (a) Fang, F.; Lue, L. F.; Yan, S.; Xu, H.; Luddy, J. S.; Chen, D.; Walker, D. G.; Stern,
D. M.; Schmidt, A. M.; Chen, J. X.; Yan, S. S. FASEB J. 2010, 24(4), 1043; (b)
Marshak, D. R.; Pesce, S. A.; Stanley, L. C.; Griffin, W. S. Neurobiol. Aging 1992, 13,
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16. Luciferase reporter gene assay: For the measurement of RAGE-NF-
the pNF- B-Luciferase vector was transfected on CHO cell line and stable cell
line was generated following manufacture’s protocol (Exp. Mol. Med. 2006, 38,
445). CHO-NF- B-Luciferase cells were plated at a density of 20,000 cells/well
in 96-well plates in 100 L of Opti-MEM (Invitrogen, Carlsbad, CA). After
overnight incubation, compounds (1 M) were added to the cultures for 18 h at
37 °C with or without 5 M of Ab42. For measure luciferase level, the cells
jB signaling,
j
j
l
l
l
were lysis with Passive lysis buffer (Promega). Cell lysates were transferred to
new 96 well plate and mixed with Luciferase assay substrate (Promega). The
values of relative light units (RLU) were measured using luminometer (Turner
Designs, Sunnyvale, CA).