The in vivo antinociceptive and μ-opioid receptor activating effects of the combination of N-phenyl-2′,4′-dimethyl-4,5′-bi-1,3-thiazol-2-amines and naloxone

Article abstract of DOI:10.1016/j.ejmech.2019.01.063

Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the activation of μ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance, dependence, addiction, constipation, nau

Full text of DOI:10.1016/j.ejmech.2019.01.063

Accepted Manuscript
The in vivo antinociceptive and μ-opioid receptor activating effects of the combination
of N-phenyl-2′,4′-dimethyl-4,5′-bi-1,3-thiazol-2-amines and naloxone
Shu-Yu Lin, Yu-Hsien Kuo, Ya-Wen Tien, Yi-Yu Ke, Wan-Ting Chang, Hsiao-Fu
Chang, Li-Chin Ou, Ping-Yee Law, Jing-Hua Xi, Pao-Luh Tao, Horace H. Loh, Yu-
Sheng Chao, Chuan Shih, Chiung-Tong Chen, Shiu-Hwa Yeh, Shau-Hua Ueng
PII:
S0223-5234(19)30083-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.063
EJMECH 11070
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 November 2018
Revised Date: 17 January 2019
Accepted Date: 26 January 2019
Please cite this article as: S.-Y. Lin, Y.-H. Kuo, Y.-W. Tien, Y.-Y. Ke, W.-T. Chang, H.-F. Chang, L.-C.
Ou, P.-Y. Law, J.-H. Xi, P.-L. Tao, H.H. Loh, Y.-S. Chao, C. Shih, C.-T. Chen, S.-H. Yeh, S.-H. Ueng,
The in vivo antinociceptive and μ-opioid receptor activating effects of the combination of N-phenyl-2′,4′-
dimethyl-4,5′-bi-1,3-thiazol-2-amines and naloxone, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.01.063.
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ACCEPTED MANUSCRIPT
Graphical Abstract

The in vivo antinociceptiv
A
e
C
a
C
n
E
d
PT
µ
E
-
D
o
M
pi
A
o
N
idUSre
C
ceptor activating effects of
R
IP
T
the combination of N-phenyl-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amines
and naloxone
a
a
a
a
a
a
a
Shu-Yu Lin, Yu-Hsien Kuo, Ya-Wen Tien, Yi-Yu Ke, Wan-Ting Chang, Hsiao-Fu Chang, Li-Chin Ou,
b
b
e
b
a
a
Ping-Yee Law, Jing-Hua Xi, Pao-Luh Tao, Horace H. Loh, Yu-Sheng Chao, Chuan Shih, Chiung-Tong
a
a,d*
a,c*
Chen, Shiu-Hwa Yeh, Shau-Hua Ueng
a
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli
County 35053, Taiwan, R. O. C.
b
Department of Pharmacology, University of Minnesota, Medical School, Minneapolis, MN 55455, USA
c
School of Pharmacy, National Cheng Kung University, Tainan, Taiwan, R. O. C.
d
The PhD Program for Neural Regenerative Medicine, Taipei Medical University, Taipei, 110, Taiwan, R. O.
C.
e
Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Miaoli County, Taiwan,
R. O. C.
Corresponding Authors
*
Tel: +886-37-246166 ext. 35791. Fax: +886-37-586456. E-mail: shueng@nhri.org.tw
*
Tel: +886-37-246166 ext. 35759. Fax: +886-37-586456. E-mail: bau9763@nhri.org.tw
1

Abstract
ACCEPTED MANUSCRIPT
Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the
activation of µ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance,
dependence, addiction, constipation, nausea, sedation, and respiratory depression. To address this problem,
we sought alternative ways to activate MOR – either by use of novel ligands, or via a novel activation
mechanism. To this end, a series of compounds were screened using a sensitive CHO-K1/MOR/Gα15
®
cell-based FLIPR calcium high-throughput screening (HTS) assay, and the bithiazole compound 5a was
identified as being able activate MOR in combination with naloxone. Structural modifications of 5a resulted
in the discovery of lead compound 5j, which could effectively activate MOR in combination with the MOR
antagonist naloxone or naltrexone. In vivo, naloxone in combination with 100 mg/kg of compound 5j
elicited antinociception in a mouse tail-flick model with an ED of 17.5±4 mg/kg. These results strongly
50
suggest that the mechanism by which the 5j/naloxone combination activates MOR is worthy of further study,
as its discovery has the potential to yield an entirely novel class of analgesics.
Keywords
Opioid receptor activation, antagonist, antinociception, naloxone, naltrexone, high-throughput screen
2

1. Introduction
ACCEPTED MANUSCRIPT
Opioids have been used to treat pain for thousands of years.[1,2] Morphine is one of the best known,
and is widely used for treating acute and chronic pain, but is limited by severe side-effects, such as tolerance,
dependence, addiction, constipation, nausea, sedation, and respiratory depression. It has been proven that the
antinociception of morphine is mediated principally by the activation of µ-opioid receptor (MOR). However,
prolonged activation of MOR causes tolerance, dependence, and addiction.[3-5] Four potential strategies for
the activation of MOR have been proposed to relieve these side effects: (i) activation by multiple targeted,
opioid receptor ligands;[6-17] (ii) activation by beta-arrestin/c-AMP biased signaling pathway ligands;
[
18-25] ; (iii) activation by non-morphine like ligands; [26-28] and (iv) activation by way of a novel
mechanism.[29,30] The feasibility of strategy (iv) is supported by studies carried out since 1996 by the
groups of Prof. Horace H. Loh, Prof. Ping-Yee Law, and Prof. Pao-Luh Tao found that naloxone, an opioid
receptor antagonist, elicited antinociceptive effects by activating mutant MOR (Ser196→Ala196) in vivo,
without inducing tolerance, dependence, or rewarding responses. [31-33] Based on these significant and
unprecedented results, Prof. Loh postulated that the mutation caused conformational changes in the
endogenous MOR, converting naloxone from an antagonist to a MOR agonist. Accordingly, we sought a
compound binding at an allosteric site of MOR would be able to perturb the conformation of non-mutant
MOR in such a way that it could undergo agonistic activation by naloxone. A similar conceptual example in
another G-protein coupled receptor (GPCR), human A adenosine receptor, has been reported by Gao et al.
3
[
34]
A CHO-K1 cell line, expressing MOR and Gα15 (GenScript), was used to set up a sensitive
®
high-throughput screening (HTS) system. In a FLIPR calcium assay using CHO-K1/MOR/Gα15 cells,
activation of MOR elicits an intracellular calcium release leading to an increase in the relative fluorescence
2+
units (RFU). The relative amount of [Ca ] released by the cells at each concentration of test compound,
i
®
was determined by integrating the area under the real-time FLIPR calcium assay response curve (AUC). In
all, 140,000 compounds were screened; and lead compound 5a was identified. This compound exhibited
poor activation ability toward MOR in combination with a constant concentration of naloxone (30 nM), with
an EC value of 13.8 µM and a AUC value of 6510 (% of morphine AUC: 19800, 32.9 %). Subsequent lead
5
0
3

optimization resulted in compound 5j, which enabled activation of MOR by the combination of opioid
ACCEPTED MANUSCRIPT
antagonist naloxone or naltrexone, and elicited antinociceptive effects in the mouse tail-flick test with this
combination pair. The structure-activity relationships, in vitro activity, and in vivo mouse tail-flick test of 5j
are all reported.
2. Chemistry
The synthesis of the bithiazole analogues 5a-s was carried out by established methods [35,36] and is
depicted in scheme 1. α-Bromination of acetyl thiazoles 1a, 1b, 1d, 1e, propionyl thiazole 1c, and acetyl
oxazole 1f with phenyltrimethylammonium tribromide afforded the bromides 4a–f in 30–88% yield.
Anilines 2a–n were treated with ammonium thiocyanate in 1.0 N HCl(aq) to prepare the commercially
unavailable N-phenylthioureas 3a–n. Condensation of 3a–n with acetyl bromides and propionyl bromide
4
a–f gave the corresponding bithiazole analogues 5a–s. To study the structure activity relationships of 5a–s,
we modified the bithiazole moiety by replacing the outer thiazole ring with an oxazole ring.
-Bromo-1-(2,4-dimethyl-oxazol-5-yl)ethanone (4f) was synthesized and then coupled with thioureas 3d
2
and 3j to give 1,3-oxazol-5-yl-N-(2-ethylphenyl)-1,3-thiazol-2-amines 5u and 5t, respectively. The inner
thiazole modification was carried out by an azide reduction of 6, followed by condensation [37] with phenyl
isocyanate
7
in
the
presence
of
triphenylphosphine
to
give
1,3-thiazol-5-yl-N-(2-ethylphenyl)-1,3-oxazol-2-amine 8, scheme 2. To probe the pharmacophore of the
bithiazole compounds, thiazole analogues 10, 12, and 14 were synthesized through the condensation of
α-bromoketones 9, 11, and 4a with thioureas 3j and 13.
4

Scheme 1
ACCEPTED MANUSCRIPT
1
and 4
a
X = S; R1 = CH ; R2 = H
3
H
O
3
C
X = S; R1 = CH
; R2 = H
3
N
X
b
c
d
e
f
2
CH
; R2 = CH
R1
3 3 3
Ph(CH ) N Br
X = S; R1 = CH
3
3
HBr in HOAc
(30-88%)
X = S; R
1
= NH ; R = H
2
2
R2
X = S; R1 = Ph; R2 = H
X = O; R1 = CH ; R2 = H
1
3
S
R1
N
H C
NH
2
3
HN
NH2
R3
H
O
3
C
N
X
X
R6
R3
R4
R6
R1
R5
R4
R6
N
NH SCN
4
ethanol
r.t.
+
R2
1.0 N HCl(aq)
R4
N
H
S
R5
100 o
12-74%)
C
Br
R2
R5
(60-90%)
R3
(
2
3
4
5
2
and 3
5
R3 = CF ; R4 = H; R5 = Br; R6 = H
X = S; R1 = CH
X = S; R1 = CH ; R2 = H; R3 = CH ; R4 = H; R5 = Br; R6 = H
; R2 = H; R3 = CF ; R4 = H; R5 = Br; R6 = H
a
a
b
3 3
3
R3 = CH
; R4 = H; R5 = Br; R6 = H
b
c
3
3
3
R3 = CH
; R4 = H; R5 = CH
; R4 = H; R5 = CH
; R6 = H
X = S; R1 = CH ; R2 = H; R3 = CH ; R4 = H; R5 = CH ; R6 = H
3
3
3
3
c
d
e
f
3 3 3
d
R3 = CH
; R6 = CH
; R6 = H
; R4 = H; R5 = OCH ; R6 = H
g R3 = CH ; R4 = OCH ; R5 = H; R6 = H
3
X = S; R1 = CH
X = S; R1 = CH
X = S; R1 = CH
3
; R2 = H; R3 = CH
; R2 = H; R3 = CH
; R2 = H; R3 = OCH
; R2 = H; R3 = CH ; R4 = OCH
X = S; R1 = CH ; R2 = H; R3 = CH ; R4 = F; R5 = H; R6 = H
3
; R4 = H; R5 = CH
; R4 = H; R5 = OCH
; R4 = H; R5 = OCH
; R5 = H; R6 = H
3
; R6 = CH
; R6 = H
; R6 = H
3
e R3 = CH
R3 = OCH
; R4 = H; R5 = OCH
3
3
3
3
3
f
3
3
3
3
3
g X = S; R1 = CH
3
3
3
3
3
h R3 = CH
; R4 = F; R5 = H; R6 = H
R3 = CH ; R4 = H; R5 = H; R6 = F
h
i
3
3
3
X = S; R1 = CH ; R2 = H; R3 = CH ; R4 = H; R5 = H; R6 = F
i
3
3
3
R3 = CH CH ; R = H; R5 = H; R6 = H
4
X = S; R1 = CH
; R2 = H; R3 = CH
; R4 = H; R5 = H; R6 = H
j
j
3
2
CH
3
2
3
k R3 = CH
CH
CH
; R4 = H; R5 = Br; R6 = H
X = S; R1 = CH ; R2 = H; R3 = CH
; R4 = H; R5 = Br; R6 = H
4
k
l
CH
2
3
3 2 3
l
R3 = CH
2
3
; R4 = H; R5 = H; R6 = CH
4
3
X = S; R1 = CH ; R2 = H; R3 = CH CH ; R = H; R5 = H; R6 = CH3
3 2 3
m R3 = CH CF ; R = H; R5 = H; R6 = H
m
X = S; R1 = CH ; R2 = H; R3 = CH ; R4 = H; R5 = H; R6 = H
CF
2
3
3 2 3
n R3 = CH(CH ) ; R = H; R5 = H; R6 = CH(CH3)2
4
n X = S; R1 = CH ; R2 = H; R3 = CH(CH ) ; R4 = H; R5 = H; R6 = CH(CH3)2
3
2
3 3 2
o
X = S; R1 = CH ; R2 = H; R3 = CH ; R4 = H; R5 = H; R6 = H
CH CH
2 3 2 3
p X = S; R1 = NH
; R2 = H; R3 = CH ; R4 = H; R5 = H; R6 = H
CH
2
2 3
4
2
Ac O
q
X = S; R1 = NHAc; R2 = H; R3 = CH CH ; R = H; R5 = H; R6 = H
2 3
Et N
3
4
r
X = S; R1 = Ph; R2 = H; R3 = CH CH ; R = H; R5 = H; R6 = H
2
3
s
X = S; R1 = CH ; R2 = CH ; R3 = CH CH ; R = H; R5 = H; R6 = H
4
18%
3
3
2
3
t
X = O; R1 = CH
X = O; R1 = CH ; R2 = H; R3 = CH ; R4 = H; R5 = CH ; R6 = CH
3 3 3 3
3
; R2 = H; R3 = CH
2
CH
3
; R4 = H; R5 = H; R6 = H
u
5

ACCEPTED MANUSCRIPT
Scheme 2
H C
O
C
3
N
H C
H C
3
3
N
S
CH3
N
N
NaN
CH3
PPh3
3
N
O
S
O
+
DMF
r.t.
THF
r.t
3%
O
N
S
H
CH3
Br
9
3%
N3
4
a
7
8
6
S
N
S
S
HN
NH2
N
S
O
N
ethanol
r.t.
+
N
H
Br
7
7%
3
j
9
10
S
CH3
HN
NH2
N
OCH3
HOAc
S
Br
OCH3
N
H
+
reflux
26%
CH3
1
2
11
3
j
H C
N
CH
3
3
N
S
S
H C
3
CH3
ethanol
r.t.
S
+
O
H N
NH2
2
N
5
3%
1
3
Br
S
H N
2
4
a
14
3. Results and discussion
3.1. Identification and optimization of bithiazole compounds in combination with naloxone to activate MOR
using a calcium assay system, and structure-activity relationships (SAR)
To explore the pharmacophore and SAR of the 5a–s bithiazole series, several analogs of 5a were
®
synthesized and submitted to the FLIPR calcium assay for measurement of their EC and AUC values.
5
0
®
Table 1. FLIPR assay results of compounds 5a–5u, 8, 10, 12, and 14 in combination with naloxone (30
a
nM).
b
compounds
EC (µM)
AUC (% of morphine AUC: 19800)
5
0
5
a
13.8
> 30 µM
8.8
32.9
ND
57.2
52
5
b
5
c
5
d
6.1
5
e
14.0
32.6
5
f
19.0
45.2
6

5
g
3.3
27.2
ACCEPTED MANUSCRIPT
5
h
10.1
4.2
57.9
32
5
i
5
j
4.0
60.5
5.9
5
k
4.9
5
l
2.8
38.2
72.1
ND
32.5
ND
ND
ND
ND
76.2
43.2
34.4
ND
ND
ND
5
m
8.5
5
n
> 30 µM
8.6
5
o
p
q
5
> 30 µM
> 30 µM
> 30 µM
> 30 µM
3.3
5
5
r
s
5
5
t
5
u
3.9
8
13.3
10
12
14
> 30 µM
> 30 µM
> 30 µM
a
b
EC values are the means of at least three independent experiments. ND = Not detected.
5
0
Compounds 10, 12, and 14 were unable to activate MOR in combination with naloxone, suggesting
the thiazole and benzene moieties to be important for MOR activation, table 1. Replacement of the
trifluoromethyl group of 5a with a methyl group to give 5b reduced the activity (5a, EC = 13.8 µM, % of
5
0
morphine AUC = 32.9; 5b, EC > 30 µM). Replacement of the bromine in the phenyl ring with the larger
5
0
methyl group improved the activity (5c, EC = 8.8 µM, % of morphine AUC = 57.2; 5d, EC = 6.1 µM, %
5
0
50
of morphine AUC = 52). Introduction of methoxy and methyl groups at the ortho- or para-position of the
phenyl ring slightly improved the activity (5e, EC = 14.0 µM, % of morphine AUC = 32.6; 5f = EC =
50
50
19.0 µM, % of morphine AUC = 45.2). Methoxy substitution at the meta-position and methyl substitution at
the ortho-position improved the potency, but not the efficacy of activation of MOR (5g, EC = 3.3 µM; %
5
0
of morphine AUC = 27.2). The meta-fluoro, ortho-methyl variant (5h) gave a superior improvement of
efficacy than 5g (5h, EC = 10.1 µM, % of morphine AUC = 57.9); however, switching from a meta-fluoro
5
0
to a para-fluoro substituted phenyl ring improved potency but not efficacy (5i, EC50 = 4.2 µM, % of
morphine AUC = 32). Introduction of an ethyl group at the ortho- position, without any substitution at all at
7

the meta- and para-positions (compound 5j) resulted in a breakthrough: compound 5j exhibited lower EC50
ACCEPTED MANUSCRIPT
value and comparable AUC to 5c (5j, EC = 4.0 µM, % of morphine AUC = 60.5). The activity was
5
0
attenuated by a second substitution on the phenyl ring (5k, EC = 4.9 µM, % of morphine AUC = 5.9; 5l,
5
0
EC50 = 2.8 µM, % of morphine AUC = 38.2). Replacement of the ethyl group with the isosteric
1
,1,1-trifluoroethyl group gave compound 5m, which was less potent (EC = 8.5 µM, % of morphine AUC
50
=
72.1).
Further modification of the thiazole moiety by replacement of the 2-methyl group of the thiazole ring
with a NH , NHAc, or phenyl group, led to complete loss of activity (5j vs. 5p–r); and with an ethyl group,
2
decreased activity (5o, EC = 8.6 µM, % of morphine AUC = 32.5). Introduction of a methyl group into the
50
inner-thiazole ring also resulted in loss of activity (cf. 5j versus 5s). 1,3-Oxazol-5-yl-N-1,3-thiazole 5t and
5
5
u exhibited comparable activity to 5j and 5d, respectively (5t, EC = 3.3 µM, % of morphine AUC = 76.2;
50
u, EC = 3.9 µM, % of morphine AUC = 43.2). However, 5t and 5u were insufficiently stable in aqueous
50
solution for evaluation of in vivo activity to be possible. Replacement of the thiazole ring with an oxazole
gave compound 8, which exhibited a lower ability to activate MOR than 5j (8, EC = 13.3 µM, % of
50
morphine AUC = 34.4). Taken together, these results suggest that the potency of bithiazoles in combination
with naloxone to activate MOR is sensitive to the substituents on both of phenyl and thiazole moieties of the
molecule.
3.2. Activation of MOR by compound 5j and naloxone/naltrexone combination pairs
To determine the effect of 5j/naloxone combination on MOR activation, several in vitro assays were
®
performed using the FLIPR calcium assay platform. Concentration-response curves (CRCs) of naloxone in
the absence or presence of various concentrations of 5j were obtained and shown in figure 1, and the
5
j/naloxone combination modulates MOR-mediated calcium mobilization. Both the maximum efficacy
Emax) and the potency of naloxone were altered by varying the concentration of 5j. Similar to naloxone, the
j/naltrexone combination also activates MOR, with higher Emax values (14000–18000, % of morphine AUC
70.7–90.9) for various concentrations of 5j than the 5j/naloxone combination (Figure 2). A previous study
(
5
=
indicated that a potential allosteric modulator LUF6000 was able to convert A adenosine receptor
3
antagonist MRS542 to be an agonist. [34] The effect of 5j might be also due to the allosteric modulation of
8

MOR.
ACCEPTED MANUSCRIPT
Figure 1. The effect of various concentrations of 5j and naloxone on the fluorescence response in the
®
FLIPR calcium assay. Concentration-response curves for 5j are shown to illustrate the potentiation of
naloxone. Data are expressed as mean ± SEM of triplicate tests, and curves were fitted using GraphPad
Prism 5. RFU: relative fluorescence units.
9

ACCEPTED MANUSCRIPT
Figure 2. The effect of various concentrations of 5j and naloxone on the fluorescence response in the
®
FLIPR calcium assay. Concentration-response curves for 5j are shown to illustrate the potentiation of
naltrexone in MOR expression cells. Data are expressed as mean ± SEM of triplicate tests, and curves were
fitted using GraphPad Prism 5. RFU: relative fluorescence units.
3.3. The combination of naloxone and 5j elicited an antinociceptive effect in mice
We investigated whether the 5j/naloxone combination could activate MOR and produce
antinociception in vivo. Naloxone, a MOR antagonist, was not found to elicit any measurable antinociceptive
effect in B6 mice at a dose of 100 mg/kg. However, significant inhibition of the tail-flick response was
observed in mice co-injected with naloxone (10 mg/kg) and 5j (25, 50, 100 mg/kg), with AUC values of
114.9 ± 24, 159.9 ± 55.8 and 384 ± 84, respectively (Figure 3), in a dose response manner. In mice injected
with 100 mg/kg of 5j, the ED of naloxone in inhibition of the tail-flick response was 17.5 ± 4 mg/kg. Mice
5
0
injected with 100 mg/kg of 5j alone or co-injected with an ultra-low-dose of naloxone (10 ng/kg) [38]
demonstrated no inhibition in tail-flick response at 30 min post-injection. No death of animal was observed
at a dose of 100 mg/kg of 5j, and the situation of animals were normal after the administration of 5j,
1
0

indicating that 100 mg/kg of 5j was a safe dosage for mice. [39] The antinociception of the 5j/naloxone
ACCEPTED MANUSCRIPT
combination and the mechanism by which they operate are both very interesting phenomena, worthy of
further study.
(b)
1
00 mg/kg 5j + 0.9% saline (n=12)
1
5
00 mg/kg 5j + 10 mg/kg naloxone (n=13)
0 mg/kg 5j + 0.9% saline (n=9)
***
1
000
#
##
8
6
4
2
00
00
00
00
0
50 mg/kg 5j + 10 mg/kg naloxone (n=10)
2
2
5 mg/kg 5j + 0.9% saline (n=7)
5 mg/kg 5j + 10 mg/kg naloxone (n=7)
Vehicle + 0.9% saline (n=6)
Vehicle + 10 mg/kg naloxone (n=8)
2
1
mg/kg morphine (n=6)
00 mg/kg 5j + 10 ng/kg naloxone (n=6)
Figure 3. Antinociceptive effects of 5j alone and 5j co-injected with naloxone in C57BL/6 (B6) mice. (a)
After detection of basal latencies, mice were injected with various doses of 5j (25, 50, or 100 mg/kg; i.v.) or
the 5j (25, 50, or 100 mg/kg; i.v.) / naloxone (10 mg/kg; s.c.) combination, and the radiant heat tail-flick
latencies measured at the indicated time points (30, 60, 90, 120, and 180 min after drug injection). *P < 0.05,
**P < 0.01 versus naloxone alone group, two-way ANOVA, Bonferroni’s post-hoc test. n = 7–13 per group.
The values indicate the mean ± SEM. (b) Antinociceptive effects of 5j alone and 5j co-injected with
naloxone in B6 mice in tail-flick test. Quantitative results from Figure 3 are represented as the area under the
1
1

curve (AUC: min × sec). *P < 0.05 versus 5j alone (100 mg/kg), one-way ANOVA with appropriate post hoc
ACCEPTED MANUSCRIPT
tests. n = 7–13 per group. The values indicate the mean ± SEM.
4. Conclusion
The HTS of 140,000 compounds led to the identification of 5a as a molecule able to activate MOR
when administered in combination with naloxone. After the SAR study in which 25 compounds were
synthesized and tested, N-(2-ethylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5j) was identified as a
lead compound. Compound 5j could activate MOR when combined with naloxone or naltrexone; the Emax
values of the 5j/naloxone and 5j/naltrexone combination were 10000–14000 (% of morphine AUC =
50.5–70.7) and 14000–18000 (% of morphine AUC = 70.7–90.9), respectively. Furthermore, the 5j/naloxone
combination elicited antinociception in the B6 mice tail-flick model; the ED value of naloxone as 17.5±4
5
0
mg/kg with 100 mg/kg co-injection of 5j. 5j/naloxone combination has the potential to open up a new area
in pain treatment, and the novel mechanism by which the 5j/naloxone combination elicited the observed
antinociception merits further study; for example, the conformational changes as well as signaling pathways
induced by 5j/naloxone combination.
5
5
5
. Experimental
.1. Chemistry
.1.1. General methods. All commercial chemicals and solvents were of analytical or reagent grade and used
without further purification, unless stated otherwise. 1-(4-Bromo-2-(trifluoromethyl)phenyl)thiourea (3a),
-(4-bromo-2-methyphenyl)thiourea (3b) and 1-(2,4-dimethoxyphenyl)thiourea (3f) were purchased from
Alfa Aesar, Sigma-Aldrich or Acros Organic Chemical Co.
-Bromo-1-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-ethanone (4e) was purchased from Maybridge-Fisher
1
2
Scientific. All reactions were carried out under an atmosphere of dry nitrogen or argon and were monitored
by thin layer chromatography (TLC), using Merck 60 F254 silica gel glass-backed plates; zones were
detected visually under UV irradiation (254 nm or 365 nm). Flash column chromatography was carried out
using silica gel (Merck Kieselgel 60, no. 9385, 230–400 mesh ASTM or SiliCycle, P60, R12030B, 230–400
mesh) or Teledyne Isco CombiFlash® Automated Flash Chromatography systems. Proton and carbon-13
NMR spectra were obtained with a Varian Mercury 300 or Varian Mercury 400 spectrometer using
1
2

chloroform-d, methanol-d (MeOD-d ), or dimethylsulfoxide-d (DMSO-d ) as the solvent. Chemical shifts
4
6
6
6
ACCEPTED MANUSCRIPT
(δ) were recorded in ppm and reported relative to the residual of solvent signals. Multiplicities were
recorded with the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Low-resolution mass spectra (LRMS) were measured with a HP 1100 Series LC/MSD System electrospray
ionization (ESI) and a Agilent 1200 Series LC/MSD VL atmospheric pressure chemical ionization (APCI).
High-resolution mass spectra (HRMS) were measured with a VARIAN 901-MS electrospray ionization (ESI)
or by using JEOL JMS-700 fast atom bombardment (FAB) mass spectrometer. Purity of the final compounds
was determined with an Hitachi 2000 series HPLC system using a C18 column (Agilent ZORBAX Eclipse
XDB-C18 5 mm, 4.6 mm × 150 mm), operating at 25 °C. Elution was carried out using CH CN as mobile
3
phase A and water containing 0.1% formic acid + 10 mmol NH OAc as mobile phase B. Elution conditions:
4
0
9
min, 10% phase A + 90% phase B; at 45 min, 90% phase A + 10% phase B; at 50 min, 10% phase A +
0% phase B; at 60 min, 10% phase A + 90% phase B. The flow rate of the mobile phase was 0.5 mL/min,
and the injection volume of the sample was 5 µL. Peaks were detected at 254 nm. The purity of all tested
compounds was > 95% except for compounds 5k (92.2%), 5p (91.1%), 5q (94.9%), and 5r (86.9%). The
chemical name of all compound was generated from their structures by ACD/Name software in accordance
IUPAC nomenclature rule.
5.1.2. Standard Procedure A for the Preparation of N-Phenylthioureas 3. Ammonium thiocyanate was
added to a solution of aniline in 1.0 N HCl(aq) . The reaction was stirred at 100 °C for 16 h to 20 h, cooled to
room temperature over 1 h, diluted with cold water (30 mL), and then neutralized with saturated NH OH(aq)
4
(pH > 7). The precipitate was collected by vacuum filtration and washed with water and diethyl ether :
n-hexanes (1 : 3) to give the desired product. The product was purified by Isco Combi-Flash Companion
column chromatography.
5.1.3. Standard Procedure B for the Preparation of Acetyl Bromide Derivatives 4.
Phenyltrimethylammonium tribromide was added to a solution of substituted thiazole or oxazole in 33%
HBr in acetic acid at room temperature, and the solution stirred overnight–36 h. The mixture was poured
into ice water, and extracted with CH Cl (3 × 50 mL). The combined organic layers were washed with
2
2
water (2 × 40 mL), dried over MgSO , filtered, and concentrated under reduced pressure to afford a
4
(s)
1
3

residue, which was purified by column chromatography to give the desired product.
ACCEPTED MANUSCRIPT
5.1.4 Standard Procedure C for the Preparation of Thiazole Derivatives. N-Phenylthiourea 3 was added to
a solution of 2-bromo-1-(2,4-dimethyl-thiazol-5-yl)ethanone 4 in ethanol. The solution was then stirred at
room temperature for 30 min to 4.0 h and ethanol was removed under reduced pressure. The residue was
diluted with saturated aqueous NaHCO solution (15 mL) and extracted with CH Cl (2 × 15 mL). The
3
2
2
combined organic layers were washed with brine (10 mL), dried over MgSO , filtered, and concentrated
4
(s)
under reduced pressure to afford a residue, which was purified by Isco Combi-Flash Companion column
chromatography to give the desired product.
5.1.5. 1-(2,4-Dimethylphenyl)thiourea (3c). Following standard procedure A, 2,4-dimethylphenylaniline (2c,
0.720 g, 5.94 mmol), 1.0 N HCl(aq) (7.0 mL), and ammonium thiocyanate (0.540 g, 7.09 mmol) were used to
carry out the reaction. After the reaction was stirred for 20 h and worked-up, 3c (0.270 g, 25%) was
1
afforded as a white solid. H NMR (CDCl , 400 MHz) δ 7.52 (br s, 1H), 7.18–7.05 (m, 3H), 6.20–5.40 (br s,
3
2
5
0
H), 2.33 (s, 3H), 2.27 (s, 3H).
.1.6. 1-(2,4,6-Trimethylphenyl)thiourea (3d). Following standard procedure A, 2,4,6-trimethylaniline (2d,
.700 mL, 4.99 mmol), 1.0 N HCl(aq) (6.0 mL), and ammonium thiocyanate (0.418 g, 5.48 mmol) were used
to carry out the reaction. After the reaction was stirred for 20 h and worked-up, 3d (0.720 g, 74%) was
1
obtained as a white solid. H NMR (CDCl , 400 MHz) δ 7.40 (br s, 1H), 6.95 (s, 2H,), 6.00 (br s, 1H), 5.35
3
(br s, 1H), 2.29 (s, 3H), 2.25 (s, 6H).
5.1.7.
1-(4-Methoxy-2-methylphenyl)thiourea
(3e).
Following
standard
procedure
A,
4-methoxy-2-methylaniline (2e, 0.830 g, 6.05 mmol), 1.0 N HCl(aq) (7.0 mL), and ammonium thiocyanate
(0.510 g, 6.66 mmol) were used to carry out the reaction. After the reaction was stirred for 20 h and
1
worked-up, 3e (0.450 g, 38%) was afforded as a purple solid. H NMR (DMSO-d , 400 MHz) δ 9.06 (br s,
6
1
5
3
H), 7.02 (d, 1H), 6.81 (d, 1H), 6.73 (dd, 1H), 3.72 (s, 3H), 2.13 (s, 3H).
.1.8. 1-(3-Methoxy-2-methylphenyl)thiourea (3g). Following
-methoxy-2-methylphenylaniline (2g, 0.320 g, 2.60 mmol), 1.0 N HCl(aq) (4.5 mL), and ammonium
standard
procedure
A,
thiocyanate (0.240 g, 3.15 mmol) were used to carry out the reaction. After the reaction was stirred for 20
1
h and worked-up, 3g (0.133 g, 26%) was afforded as a purple solid. H NMR (CDCl + MeOD, 400 MHz) δ
3
1
4

7
.16 (dd, 1H), 6.81 (d, 1H), 6.78 (d, 1H), 3.81 (s, 3H), 2.08 (s, 3H).
ACCEPTED MANUSCRIPT
5.1.9. 1-(3-Fluoro-2-methylphenyl)thiourea (3h). Following standard procedure A, 3-fluoro-2-methylaniline
(2h, 0.520 g, 4.16 mmol), 1.0 N HCl(aq) (6.0 mL), and ammonium thiocyanate (0.350 g, 4.60 mmol) were
used to carry out the reaction. After the reaction was stirred for 18 h and worked-up, 3h (0.260 g, 34%)
1
was obtained as a white solid. H NMR (CDCl , 400 MHz) δ 7.94 (br s, 1H), 7.27–7.22 (m, 1H), 7.09–7.00
3
(
m, 2H), 5.98 (br s, 2H), 2.25 (s, 3H).
.1.10. 1-(2-Fluoro-6-methylphenyl)thiourea (3i). Following standard procedure A, 2-fluoro-6-methylaniline
2i, 0.440 g, 3.51 mmol), 1.0 N HCl(aq) (5.0 mL), and ammonium thiocyanate (0.294 g, 3.87 mmol) were
5
(
used to carry out the reaction. After the reaction was stirred for 16 h and worked-up, 3i (0.266 g, 41%) was
1
afforded as a white solid. H NMR (CDCl , 300 MHz) δ 7.52 (br s, 1H), 7.38–7.23 (m, 1H), 7.10 (d, 1H),
3
7.04 (t, 1H), 6.30–5.60 (br s, 1H), 2.35 (s, 3H).
5.1.11. 1-(2-Ethylphenyl)thiourea (3j). Following standard procedure A, 2-ethylaniline (2j, 1.00 mL, 8.10
mmol), 1.0 N HCl(aq) (8.0 mL), and ammonium thiocyanate (0.620 g, 8.15 mmol) were used to carry out the
reaction. After the reaction was stirred for 20 h and worked-up, the crude product was purified by Isco
Combi-Flash Companion column chromatography (10–60% EtOAc in n-hexane) to give 3j (0.600 g, 41%)
1
as a white solid. H NMR (CDCl , 400 MHz) δ 7.69 (br, 1H), 7.35–7.22 (m, 4H), 2.67 (q, 2H), 1.22 (t, 3H).
3
5.1.12. 1-(4-Bromo-2-ethylphenyl) thiourea (3k). Following standard procedure A, 4-bromo-2-ethylaniline
(2k, 0.840 g, 4.20 mmol), 1.0 N HCl(aq) (5.0 mL), and ammonium thiocyanate (0.380 g, 4.99 mmol) were
used to carry out the reaction. After the reaction was stirred for 20 h and worked-up, 3k (0.130 g, 12%)
1
was afforded as a white solid. H NMR (CDCl , 300 MHz) δ 7.54 (br s, 1H), 7.49 (d, 1H), 7.42 (dd, 1H),
3
7.12 (d, 1H), 6.10–5.60 (br s, 2H), 2.64 (q, 2H), 1.22 (t, 3H).
5.1.13. 1-(2-Ethyl-6-methylphenyl)thiourea (3l). Following standard procedure A, 2-ethyl-6-methylaniline
(2l, 0.860 g, 5.76 mmol), 1.0 N HCl(aq) (7.0 mL), and ammonium thiocyanate (0.530 g, 6.94 mmol) were
used to carry out the reaction. After the reaction was stirred for 20 h and worked-up, 3l (0.470 g, 39%) was
1
afforded as a white solid. H NMR (CDCl , 400 MHz) δ 7.78 (br s, 1H), 7.27–7.23 (m overlapped with s at
3
7
.26, 1H), 7.18–7.14 (m, 2H), 6.40–6.10 (br s, 1H), 5.45–5.20 (br s, 1H), 2.68–2.60 (m, 2H), 2.31 (s, 3H),
.21 (t, 3H).
1
1
5

5
.1.14. 1-(2-(2,2,2-trifluoroethyl)phenyl)thiourea (3m). A solution of 2-(2,2,2-trifluoroethyl)aniline (2m,
ACCEPTED MANUSCRIPT
48.9 mg, 0.279 mmol) and benzoyl isothiocyanate (41.2 µL, 0.308 mmol) in acetone (2.0 mL) was stirred at
room temperature for 5 h. The solution was concentrated under reduced pressure to give an intermediate.
The intermediate in methanol (2.0 mL) was treated with 5% NaOH (aq) (0.80 mL) and it was stirred at room
temperature for 1 h. The solution was quenched with 1.0 N HCl(aq) and extracted with ethyl acetate. The
combined organic layers were dried over anhydrous MgSO , filtered, and concentrated under reduced
4
1
pressure to give 3m as a light brown solid (56.5 mg, 86%). H NMR (CDCl , 400 MHz) δ 7.66 (br s, 1H),
3
7
5
0
.49–7.36 (m, 4H), 3.48 (q, 2H).
.1.15. 1-(2,6-Diisopropylphenyl)thiourea (3n). Following standard procedure A, 2,6-diisopropylaniline (2n,
.600 mL, 3.18 mmol), 1.0 N HCl(aq) (6.0 mL), and ammonium thiocyanate (0.270 g, 3.55 mmol) were used
to carry out the reaction. After the reaction was stirred for 20 h and worked-up, 3n (0.280 g, 37%) was
1
obtained as a brown solid. H NMR (CDCl , 300 MHz) δ 7.47 (br s, 1H), 7.38 (dd, 1H), 7.24 (d, 2H),
3
6
5
5
.20–5.80 (br s, 1H), 5.60–5.10 (br s, 1H), 3.16 (septet, 2H), 1.24 (d, 6H), 1.18 (d, 6H).
.1.16. 2-Bromo-1-(2,4-dimethyl-thiazol-5-yl)ethanone (4a). Following standard procedure B,
-acetyl-2,4-dimethylthiazole (1a, 2.59 g, 16.7 mmol), 33% HBr in acetic acid solution (18 mL), and
phenyltrimethylammonium tribromide (6.59 g, 17.5 mmol) were used to carry out the reaction. After the
solution was stirred overnight and worked-up, the residue was purified by Isco Combi-Flash Companion
1
column chromatography (0–2% EtOAc in CH Cl ) to give 4a (2.23 g, 57%) as a yellow oil. H NMR
2
2
(
CDCl , 400 MHz) δ 4.21 (s, 2H), 2.72 (s, 6H).
3
5
.1.17. 2-Bromo1-(2-ethyl-4-methyl-thiazol-5-yl)-ethanone (4b). Following standard procedure B,
-acetyl-2-ethyl-4-dimethylthiazole (1b, 0.801 g, 4.73 mmol), 33% HBr in acetic acid solution (6.0 mL),
5
and phenyltrimethylammonium tribromide (1.87 g, 4.97 mmol) were used to carry out the reaction. After the
solution was stirred overnight and worked-up, the residue was purified by Isco Combi-Flash Companion
1
column chromatography (0–5% EtOAc in CH Cl ) to give 4b (0.710 g, 61%) as a brown solid. H NMR
2
2
(
CDCl , 400 MHz) δ 4.23 (s, 2H), 3.03 (q, 2H), 2.73 (s, 3H), 1.41 (t, 3H).
3
5.1.18. 2-Bromo1-(2,4-dimethyl-thiazol-5-yl)propan-1-one (4c). Following standard procedure B,
1-(2.4-dimethylthiazol-5-yl)propan-1-one (1c, 0.101 g, 0.599 mmol), 33% HBr in acetic acid solution (0.80
1
6

mL), and phenyltrimethylammonium tribromide (0.246 g, 0.658 mmol) were used to carry out the reaction.
ACCEPTED MANUSCRIPT
After the solution was stirred for 36 h and worked-up, the residue was purified by column chromatography
1
(
5% EtOAc in CH Cl ) to give 4c (130 mg, 88%) as a yellow liquid. H NMR (CDCl , 400 MHz) δ 4.81 (q,
2
2
3
1
5
H), 2.73 (s, 3H), 2.72 (s, 3H), 1.86 (d, 3H).
.1.19. 1-(2-Amino-4-methyl-1,3-thiazol-5-yl)-2-bromoethanone hydrobromide (4d). Following standard
procedure B, 5-acetyl-2-amino-4-methylthiazole (1d, 0.711 g, 4.55 mmol), 33% HBr in acetic acid solution
8.0 mL), and phenyltrimethylammonium tribromide (1.88 g, 4.99 mmol) were used to carry out the reaction.
(
After the solution was stirred overnight and worked-up, the undissolved solid was collected from the organic
1
layer and washed with diethyl ether to give 4d (0.680 g, 47%) as a white solid. H NMR (DMSO-d , 300
6
MHz) δ 8.90–8.40 (br, 2H), 4.53 (s, 2H), 2.46 (s, 3H).
5.1.20. 2-Bromo-1-(2,4-dimethyl-oxazol-5-yl)ethanone (4f). Following standard procedure B,
5-acetyl-2,4-dimethyloxazole (1f, 0.360 g, 2.59 mmol), 33% HBr in acetic acid solution (4.0 mL), and
phenyltrimethylammonium tribromide (0.971 g, 2.59 mmol) were used to carry out the reaction. After the
solution was stirred overnight and worked-up, the residue was purified by Isco Combi-Flash Companion
1
column chromatography (0–3% EtOAc in CH Cl ) to give 4f (0.160 g, 30%) as a brown oil. H NMR
2
2
(
CDCl , 400 MHz) δ 4.27 (s, 2H), 2.54 (s, 3H), 2.49 (s, 3H).
3
5.1.21. N-[4-Bromo-2-(trifluoromethyl)phenyl]-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5a). Following
standard procedure C, 4a (69.2 mg, 0.296 mmol), ethanol (3.0 mL), and 3a (88.4 mg, 0.296 mmol) were
used to carry out the reaction. After the solution was stirred for 2 h and worked-up, the residue was purified
by Isco Combi-Flash Companion column chromatography (10–50% EtOAc in n-hexane) to give 5a (104 mg,
1
8
1
1
1%) as a yellow solid. H NMR (CDCl , 400 MHz) δ 8.20 (d, 1H), 7.74 (d, 1H), 7.68 (dd, 1H), 7.25 (br s,
3
1
3
H), 6.68 (s, 1H), 2.67 (s, 3H), 2.60 (s, 3H); C NMR (CDCl , 75 MHz) δ 165.65, 162.51, 148.01, 143.78,
3
3
1
2
37.18, 136.11, 129.50 ( JC-F = 5.7 Hz), 126.34, 123.20 ( J = 271 Hz), 122.24, 119.98 ( J = 29.8 Hz),
C-F
C-F
+
1
15.11, 104.57, 18.94, 16.99; HPLC Purity = 99.6%, t = 44.07 min; LC-MS (APCI) m/z: 434.0 [M + H] ;
R
+
HRMS (ESI): m/z calculated for C H BrF N S [M + H] : 433.9608 Found: 433.9608.
1
5
12
3
3 2
5.1.22. N-(4-Bromo-2-methylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5b). Following standard
procedure C, 4a (130 mg, 0.556 mmol), ethanol (3.0 mL), and 3b (136 mg, 0.556 mmol) were used to carry
1
7

out the reaction. After the solution was stirred for 3 h and worked-up, the residue was purified by Isco
ACCEPTED MANUSCRIPT
Combi-Flash Companion column chromatography (10–40% EtOAc in n-hexane) to give 5b (191 mg, 90%)
1
as a beige solid. H NMR (CDCl , 400 MHz) δ 7.61 (d, 1H), 7.42–7.37 (m, 2H), 6.91 (br s, 1H), 6.58 (s, 1H),
3
1
3
2
.67 (s, 3H), 2.58 (s, 3H), 2.31 (s, 3H); C NMR (CDCl , 100 MHz) δ 165.62, 163.40, 147.87, 143.27,
3
137.60, 133.68, 131.75, 130.08, 126.35, 122.52, 117.41, 103.22, 18.89, 17.64, 16.91; HPLC Purity = 100%,
+
+
t = 41.47 min; LC-MS (ESI) m/z: 380.0 [M + H] ; HRMS (ESI): m/z calculated for C H BrN S [M + H] :
R
15 15
3 2
379.9891 Found: 379.9890.
5.1.23. N-(2,4-Dimethylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5c). Following standard
procedure C, 4a (106 mg, 0.452 mmol), ethanol (3.0 mL), and 3c (81.4 mg, 0.452 mmol) were used to carry
out the reaction. After the solution was stirred for 2 h and worked-up, the residue was purified by column
1
chromatography (40% EtOAc in n-hexane) to give 5c (126 mg, 88%) as a red brown solid. H NMR (CDCl3,
300 MHz) δ 7.44 (d, 1H), 7.08–7.05 (m, 2H), 6.90 (br s, 1H), 6.50 (s, 1H), 2.66 (s, 3H), 2.57 (s, 3H), 2.33 (s,
1
3
3
1
H), 2.29 (s, 3H); C NMR (CDCl , 100 MHz) δ 167.24, 163.12, 147.81, 143.36, 135.87, 135.34, 131.88,
3
30.83, 127.74, 126.62, 122.34, 102.64, 20.89, 18.99, 17.73, 17.04; HPLC Purity = 100%, t = 38.29 min;
R
+
+
LC-MS (APCI) m/z: 316.1 [M + H] ; HRMS (ESI): m/z calculated for C H N S [M + H] : 316.0942
16
18
3 2
Found: 316.0937.
.1.24. 2',4'-Dimethyl-N-(2,4,6-trimethylphenyl)-4,5'-bi-1,3-thiazol-2-amine (5d). Following standard
5
procedure C, 4a (96.9 mg, 0.414 mmol), ethanol (3.0 mL), and 3d (80.5 mg, 0.414 mmol) were used to carry
out the reaction. After the solution was stirred for 2 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (15–45% EtOAc in n-hexane) to give 5d (113 mg, 83%)
1
as a white solid. H NMR (CDCl , 400 MHz) δ 6.97 (s, 2H), 6.65 (br s, 1H), 6.41 (s, 1H), 2.65 (s, 3H), 2.56
3
1
3
(
s, 3H), 2.32 (s, 3H), 2.27 (s, 6H); C NMR (CDCl , 100 MHz) δ 170.27, 162.85, 147.59, 143.49, 137.87,
3
1
36.79, 134.70, 129.52, 126.71, 102.46, 21.03, 18.82, 18.02, 16.93; HPLC Purity = 99.8%, t = 38.61 min;
R
+
+
LC-MS (ESI) m/z: 330.1 [M + H] ; HRMS (ESI): m/z calculated for C H N S [M + H] : 330.1099 Found:
1
7
20
3 2
330.1090.
5.1.25. N-(4-Methoxy-2-methylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5e). Following standard
procedure C, 4a (101 mg, 0.431 mmol), ethanol (3.0 mL), and 3e (84.5 mg, 0.431 mmol) were used to carry
1
8

out the reaction. After the solution was stirred for 1 h and worked-up, the residue was purified by Isco
ACCEPTED MANUSCRIPT
Combi-Flash Companion column chromatography (10–50% EtOAc in n-hexane) to give 5e (117 mg, 82%)
1
as a brown solid. H NMR (CDCl , 400 MHz) δ 7.39 (d, 1H), 6.83–6.77 (m, 2H), 6.72 (br s, 1H), 6.47 (s,
3
1
3
1
H), 3.82 (s, 3H), 2.66 (s, 3H), 2.57 (s, 3H), 2.31 (s, 3H); C NMR (CDCl , 75 MHz) δ 169.09, 163.01,
3
158.02, 147.72, 143.50, 135.07, 131.59, 126.69, 126.43, 116.45, 112.13, 102.59, 55.38, 18.94, 18.07, 16.99;
+
HPLC Purity = 99.8%, t = 33.41 min; LC-MS (APCI) m/z: 332.1 [M + H] ; HRMS (ESI): m/z calculated
R
+
for C H N OS [M + H] : 332.0891 Found: 332.0890.
1
6
18
3
2
5.1.26. N-(2,4-Dimethoxyphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5f). Following standard
procedure C, 4a (109 mg, 0.466 mmol), ethanol (3.0 mL), and 3f (98.9 mg, 0.466 mmol) were used to carry
out the reaction. After the solution was stirred for 1.5 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (5–60% EtOAc in n-hexane) to give 5f (85.3 mg, 53%) as
1
a white solid. H NMR (CDCl , 400 MHz) δ 7.87 (d, 1H), 7.41 (br s, 1H), 6.58–6.53 (m, 3H), 3.88 (s, 3H),
3
1
3
3
1
1
.82 (s, 3H), 2.67 (s, 3H), 2.60 (s, 3H); C NMR (CDCl , 100 MHz) δ 164.54, 163.07, 155.95, 149.51,
3
47.69, 143.50, 126.94, 123.44, 118.62, 103.69, 102.28, 99.11, 55.72, 55.60, 19.01, 17.09; HPLC Purity =
+
00%, t = 34.39 min; LC-MS (ESI) m/z: 348.1 [M + H] ; HRMS (ESI): m/z calculated for C H N O S
R
16 18
3
2
2
+
[
M + H] : 348.0840 Found: 348.0836.
5.1.27. N-(3-Methoxy-2-methylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5g). Following standard
procedure C, 4a (116 mg, 0.494 mmol), ethanol (3.0 mL), and 3g (80.8 mg, 0.412 mmol) were used to carry
out the reaction. After the solution was stirred for 4 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (0–50% EtOAc in n-hexane) to give 5g (105 mg, 77%) as
1
a solid. H NMR (CDCl , 300 MHz) δ 7.25–7.20 (m, 2H), 6.97 (br s, 1H), 6.73 (d, 1H), 6.53 (s, 1H), 3.85 (s,
3
1
3
3
1
9
H), 2.66 (s, 3H), 2.58 (s, 3H), 2.19 (s, 3H); C NMR (CDCl , 75 MHz) δ 166.54, 163.13, 158.36, 147.82,
3
43.35, 139.26, 126.97, 126.63, 118.82, 113.93, 107.18, 102.94, 55.70, 18.99, 17.03, 9.96; HPLC Purity =
+
6.5%, t = 35.47 min; LC-MS (ESI) m/z: 332.1 [M + H] ; HRMS (ESI): m/z calculated for C H N OS
R
16 18
3
2
+
[
M + H] : 332.0891 Found: 332.0894.
5.1.28. N-(3-Fluoro-2-methylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5h). Following standard
procedure C, 4a (109 mg, 0.464 mmol), ethanol (3.0 mL), and 3h (109 mg, 0.464 mmol) were used to carry
1
9

out the reaction. After the solution was stirred for 2 h and worked-up, the residue was purified by Isco
ACCEPTED MANUSCRIPT
Combi-Flash Companion column chromatography (20–40% EtOAc in n-hexane) to give 5h (127 mg, 86%)
1
as a yellow solid. H NMR (CDCl , 400 MHz) δ 7.49 (d, 1H), 7.24–7.18 (m, 1H), 6.99 (br s, 1H), 6.87 (dd,
3
1
3
1
H), 6.59 (s, 1H), 2.67 (s, 3H), 2.59 (s, 3H), 2.24 (s, 3H); C NMR (CDCl , 100 MHz) δ 165.33, 163.34,
3
1
3
3
1
61.52 ( J = 243 Hz), 148.00, 143.42, 139.94 ( JC-F = 6.1 Hz), 127.40 ( J = 9.9 Hz), 126.41, 116.57
C-F
C-F
2
4
2
3
(
JC-F = 18.3 Hz), 115.84 ( J = 2.3 Hz), 111.35 ( J = 22.9 Hz), 103.34, 19.00, 17.02, 9.20 ( J = 5.3
C-F C-F C-F
+
Hz); HPLC Purity = 99.6%, t = 35.97 min; LC-MS (ESI) m/z: 320.2 [M + H] ; HRMS (ESI): m/z
R
+
calculated for C H FN S [M + H] : 320.0691 Found: 320.0684.
1
5
15
3 2
5.1.29. N-(2-Fluoro-6-methylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5i). Following standard
procedure C, 4a (105 mg, 0.447 mmol), ethanol (3.0 mL), and 3i (82.4 mg, 0.447 mmol) were used to carry
out the reaction. After the solution was stirred for 3 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (10–35% EtOAc in n-hexane) to give 5i (105 mg, 74%)
1
as a beige solid. H NMR (CDCl , 400 MHz) δ 7.24–7.19 (m, 1H), 7.09 (d, 1H), 7.03 (dd, 1H), 6.68 (br s,
3
1
3
1
1
1
3
3
5
4
H), 6.52 (s, 1H), 2.65 (s, 3H), 2.56 (s, 3H), 2.37 (s, 3H); C NMR (CDCl , 100 MHz) δ 168.70, 163.09,
3
1
3
2
58.56 ( J = 247 Hz), 147.85, 143.46, 138.54, 128.32 ( J = 8.4 Hz), 126.53, 126.51 ( J = 13.0 Hz),
C-F
C-F
C-F
3
2
26.38 ( J = 3.8 Hz), 113.90 ( J = 20.6 Hz), 103.64, 18.93, 17.74, 16.91; HPLC Purity = 99.3%, t =
C-F
C-F
R
+
+
2.91 min; LC-MS (APCI) m/z: 320.1 [M + H] ; HRMS (ESI): m/z calculated for C H FN S [M + H] :
1
5
15
3 2
20.0691 Found: 320.0701.
.1.30. N-(2-Ethylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5j). Following standard procedure C,
a (72.1 mg, 0.308 mmol), ethanol (3.0 mL), and 3j (50.5 mg, 0.280 mmol) were used to carry out the
reaction. After the solution was stirred for 30 min and then worked up, the residue was purified by Isco
Combi-Flash Companion column chromatography (20–40% EtOAc in n-hexane) to give 5j (59.2 mg, 67%)
1
as a yellow solid. H NMR (CDCl , 300 MHz) δ 7.61 (d, 1H), 7.29–7.25 (m, 2H), 7.17 (dd, 1H), 6.98 (br s,
3
1
3
1
1
1
H), 6.52 (s, 1H), 2.72–2.64 (m, 5H), 2.58 (s, 3H), 1.25 (t, 3H); C NMR (CDCl , 100 MHz) δ 167.23,
3
63.06, 147.81, 143.41, 137.97, 136.70, 129.24, 127.08, 126.56, 125.70, 122.63, 102.71, 24.22, 18.92, 16.95,
+
4.24; HPLC Purity = 95.6%, t = 37.76 min; LC-MS (ESI) m/z: 316.1 [M + H] ; HRMS (ESI): m/z
R
+
calculated for C H N S [M + H] : 316.0942 Found: 316.0945.
1
6
18
3 2
2
0

5
.1.31. N-(4-Bromo-2-ethylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5k). Following standard
ACCEPTED MANUSCRIPT
procedure C, 4a (72.4 mg, 0.309 mmol), ethanol (3.0 mL), and 3k (80.1 mg, 0.309 mmol) were used to carry
out the reaction. After the solution was stirred for 2 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (10–40% EtOAc in n-hexane) to give 5k (99.8 mg, 82%)
1
as a brown solid. H NMR (CDCl , 400 MHz) δ 7.57 (d, 1H), 7.42–7.36 (m, 2H), 6.93 (br s, 1H), 6.55 (s,
3
1H), 2.66 (s overlapped with q at 2.64, 3H), 2.64 (q overlapped with s at 2.66, 2H), 2.58 (s, 3H), 1.25 (t, 3H);
1
3
C NMR (CDCl , 100 MHz) δ 166.59, 163.41, 147.88, 143.24, 138.54, 137.05, 132.04, 130.04, 126.30,
3
1
23.88, 118.44, 103.12, 24.08, 18.87, 16.87, 13.87; HPLC Purity = 92.2%, t = 43.59 min; LC-MS (APCI)
R
+
+
m/z: 394.1 [M + H] ; HRMS (ESI): m/z calculated for C H BrN S [M + H] : 394.0047 Found: 394.0045.
1
6
17
3 2
5.1.32. N-(2-Ethyl-6-methylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5l). Following standard
procedure C, 4a (109 mg, 0.466 mmol), ethanol (3.0 mL), and 3l (90.6 mg, 0.466 mmol) were used to carry
out the reaction. After the solution was stirred for 3 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (10–40% EtOAc in n-hexane) to give 5l (92.6 mg, 60%)
1
as a yellow solid. H NMR (CDCl , 400 MHz) δ 7.25–7.16 (m, 3H), 6.82 (br s, 1H), 6.41 (s, 1H), 2.68 (q,
3
1
3
2
1
1
H), 2.64 (s, 3H), 2.55 (s, 3H), 2.32 (s, 3H), 1.19 (t, 3H); C NMR (CDCl , 75 MHz) δ 170.08, 163.03,
3
47.73, 143.40, 142.85, 137.17, 136.45, 128.95, 128.48, 127.17, 126.60, 102.69, 24.56, 18.93, 18.24, 17.00,
+
4.97; HPLC Purity = 99.7%, t = 37.46 min; LC-MS (APCI) m/z: 330.1 [M + H] ; HRMS (ESI): m/z
R
+
calculated for C H N S [M + H] : 330.1099 Found: 330.1094.
1
7
20
3 2
5.1.33. N-[2-(2,2,2-trifluoroethyl)phenyl]-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5m). Following
standard procedure C, 4a (62.1 mg, 0.241 mmol), ethanol (3.0 mL), and 3m (56.5 mg, 0.265 mmol) were
used to carry out the reaction. After the solution was stirred for 8 h and worked-up, the residue was purified
by preparative thin layer chromatography plates (1.0 mm of silica gel on glass support, 30% ethyl acetate in
1
n-hexane) to give 5m (68.5 mg, 77%) as a lightly brown solid. H NMR (CDCl , 400 MHz) δ 7.67 (d, 1H),
3
1
3
7
.44–7.39 (m, 2H), 7.29 (d, 1H), 6.53 (s, 1H), 3.50 (q, 2H), 2.69 (s, 3H), 2.57 (s, 3H); C NMR (CDCl3,
1
1
00 MHz) δ 166.96, 163.34, 148.01, 143.67, 139.48, 132.51, 129.91, 126.62, 126.43, 125.84 ( J = 276
C-F
2
Hz), 124.93, 124.76, 103.37, 35.79 ( J = 29.8 Hz), 19.01, 17.02; HPLC Purity = 97.2%, t = 25.61 min;
C-F
R
+
+
LC-MS (ESI) m/z: 370.1 [M + H] ; HRMS (ESI): m/z calculated for C H F N S [M + H] : 370.0659
16
15
3
3 2
2
1

Found: 370.0653.
.1.34. N-(2,6-Diisopropylphenyl)-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amine (5n). Following standard
ACCEPTED MANUSCRIPT
5
procedure C, 4a (79.6 mg, 0.340 mmol), ethanol (3.0 mL), and 3n (80.3 mg, 0.340 mmol) were used to carry
out the reaction. After the solution was stirred for 4 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (10–40% EtOAc in n-hexane) to give 5n (106 mg, 84%)
1
as a yellow solid. H NMR (CDCl , 400 MHz) δ 7.38 (dd, 1H), 7.25 (d overlapped with solvent peak, 2H),
3
1
3
6
1
1
.73 (br s, 1H), 6.41 (s, 1H), 3.27 (septet, 2H), 2.72 (s, 3H), 2.57 (s, 3H), 1.20 (d, 12H); C NMR (CDCl3,
00 MHz) δ 171.24, 163.17, 147.69, 142.87, 134.09, 129.28, 126.32, 124.37, 102.65, 28.31, 23.97, 18.83,
+
6.94; HPLC Purity = 95.6%, t = 44.83 min; LC-MS (ESI) m/z: 372.1 [M + H] ; HRMS (ESI): m/z
R
+
calculated for C H N S [M + H] : 372.1568 Found: 372.1562.
2
0
26
3 2
5.1.35. 2'-Ethyl-N-(2-ethylphenyl)-4'-methyl-4,5'-bi-1,3-thiazol-2-amine (5o). Following standard procedure
C, 4b (110 mg, 0.445 mmol), ethanol (3.0 mL), and 3j (80.3 mg, 0.445 mmol) were used to carry out the
reaction. After the solution was stirred for 2 h and worked-up, the residue was purified by Isco Combi-Flash
Companion column chromatography (0–30% EtOAc in n-hexane) to give 5o (90.6 mg, 63%) as a beige solid.
1
H NMR (CDCl , 400 MHz) δ 7.60 (d, 1H), 7.30–7.25 (m, 2H), 7.18 (dd overlapped with br s at 7.16, 1H),
3
7.16 (br s overlapped with dd at 7.18, 1H), 6.53 (s, 1H), 3.00 (q, 2H), 2.68 (q, 2H), 2.59 (s, 3H), 1.40 (t, 3H),
1
3
1
.25 (t, 3H); C NMR (CDCl , 100 MHz) δ 170.12, 166.97, 147.76, 143.55, 137.88, 136.38, 129.32, 127.16,
3
1
26.10, 125.63, 122.24, 102.72, 26.82, 24.27, 17.08, 14.28, 14.24; HPLC Purity = 100%, t = 41.07 min;
R
+
+
LC-MS (ESI) m/z: 330.1 [M + H] ; HRMS (ESI): m/z calculated for C H N S [M + H] : 330.1099 Found:
1
7
20
3 2
3
5
4
30.1092.
2
.1.36. N -(2-ethylphenyl)-4'-methyl-4,5'-bi-1,3-thiazole-2,2'-diamine (5p). Following standard procedure C,
d (122 mg, 0.387 mmol), ethanol (3.0 mL), and 3j (69.8 mg, 0.387 mmol) were used to carry out the
reaction. After the solution was stirred overnight and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (0–90% EtOAc in n-hexane) to give 5p (88.6 mg, 72%)
1
as a red solid. H NMR (CDCl , 400 MHz) δ 7.59 (d, 1H), 7.29–7.24 (m, 2H), 7.16 (dd, 1H), 7.15–7.00 (br s,
3
1
3
1
H), 6.38 (s, 1H), 5.40–5.10 (br s, 2H), 2.67 (q, 2H), 2.53 (s, 3H), 1.24 (t, 3H); C NMR (CDCl , 100 MHz)
3
δ 167.13, 165.69, 143.48, 138.00, 136.70, 129.29, 127.13, 125.66, 122.55, 119.47, 116.62, 101.09, 24.28,
2
2

+
1
6.54, 14.33; HPLC Purity = 91.1%, t = 24.99 min; LC-MS (ESI) m/z: 317.0 [M + H] ; HRMS (ESI): m/z
R
ACCEPTED MANUSCRIPT
+
calculated for C H N S [M + H] : 317.0895 Found: 317.0892.
1
5
17
4 2
5.1.37. N-[2-(2-Ethylphenylamino)-4'-methyl-[4,5']bithiazolyl-2'-yl]-acetamide (5q). To a solution of 5p
(
(
69.2 mg, 0.219 mmol) in CH Cl was added acetic anhydrous (0.021 mL, 0.222 mmol) and triethylamine
2
2
0.031 mL, 0.222 mmol) at 0 °C. The reaction was stirred for 20 h and diluted with CH Cl . The solution
2
2
was washed with water and brine , dried over MgSO , filtered, and concentrated under reduced pressure
(aq)
4(s)
to afford a residue. The residue was purified by Isco Combi-Flash Companion column chromatography
1
(
0–55% EtOAc in CH Cl ) to give 5q (13.9 mg, 18%) as an orange solid. H NMR (CDCl , 400 MHz) δ
2
2
3
7.59 (d, 1H), 7.29–7.24 (m, 2H), 7.19 (dd, 1H), 6.52 (s, 1H), 2.70 (q, 2H), 2.56 (s, 3H), 2.33 (s, 3H), 1.26 (t,
1
3
3
H); C NMR (CDCl , 100 MHz) δ 167.65, 167.11, 156.25, 143.16, 141.82, 137.82, 136.31, 129.33, 127.20,
3
1
25.62, 122.19, 121.29, 101.09, 24.30, 23.15, 16.63, 14.29; HPLC Purity = 94.9%, t = 32.5 min; LC-MS
R
+
+
(
ESI) m/z: 359.2 [M + H] ; HRMS (ESI): m/z calculated for C H N OS [M + H] : 359.1000 Found:
17 19 4 2
3
5
59.0997.
.1.38. N-(2-Ethylphenyl)-4'-methyl-2'-phenyl-4,5'-bi-1,3-thiazol-2-amine (5r). Following standard
procedure C, 4e (73.4 mg, 0.248 mmol), ethanol (2.0 mL), and 3j (44.7 mg, 0.248 mmol) were used to carry
out the reaction. After the solution was stirred overnight and worked-up, the residue was purified by column
1
chromatography (20% EtOAc in n-hexane) to give 5r (81.4 mg, 87%) as a brown gum. H NMR (CDCl3,
400 MHz) δ 7.95 (d, 2H), 7.61 (d, 1H), 7.50–7.27 (m, 5H), 7.22 (dd, 1H), 6.61 (s, 1H), 2.78–2.68 (m, 5H),
1
3
1
.26 (t, 3H); C NMR (CDCl , 100 MHz) δ 167.41, 164.62, 149.48, 143.09, 137.79, 136.76, 133.56, 129.79,
3
129.38, 128.86, 127.22, 127.18, 126.26, 125.89, 122.54, 103.11, 24.28, 17.40, 14.32; HPLC Purity = 86.9%,
+
+
t = 48.49 min; LC-MS (APCI) m/z: 378.2 [M + H] ; HRMS (ESI): m/z calculated for C H N S [M + H] :
R
21 20
3 2
3
5
4
78.1099 Found: 378.1093.
.1.39. N-(2-Ethylphenyl)-2',4',5-trimethyl-4,5'-bi-1,3-thiazol-2-amine (5s). Following standard procedure C,
c (126 mg, 0.506 mmol), ethanol (3.0 mL), and 3j (91.2 mg, 0.506 mmol) were used to carry out the
reaction. After the solution was stirred overnight and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (0–30% EtOAc in n-hexane) to give 5s (120 mg, 72%) as
1
a yellow solid. H NMR (CDCl , 400 MHz) δ 7.55 (d, 1H), 7.26–7.20 (m, 2H), 7.13 (dd, 1H), 2.78–2.60 (m,
3
2
3

1
3
5
H), 2.36 (s, 3H), 2.22 (s, 3H), 1.23 (t, 3H); C NMR (CDCl , 100 MHz) δ 164.71, 164.43, 149.61, 138.22,
3
ACCEPTED MANUSCRIPT
137.79, 136.57, 129.17, 126.99, 125.38, 124.34, 122.27, 119.07, 24.27, 19.10, 16.22, 14.25, 12.19; HPLC
+
Purity = 95.0%, t = 38.3 min; LC-MS (ESI) m/z: 330.1 [M + H] ; HRMS (ESI): m/z calculated for
R
+
C H N S [M + H] : 330.1099 Found: 330.1098.
17
20
3 2
5.1.40. 4-(2,4-Dimethyl-1,3-oxazol-5-yl)-N-(2-ethylphenyl)-1,3-thiazol-2-amine (5t). Following standard
procedure C, 4f (99.6 mg, 0.457 mmol), ethanol (3.0 mL), and 3j (73.9 mg, 0.410 mmol) were used to carry
out the reaction. After the solution was stirred for 4 h and worked-up, the residue was purified by Isco
Combi-Flash Companion column chromatography (5–40% EtOAc in n-hexane) to give 5t (74.2 mg, 60%) as
1
an orange solid. H NMR (CDCl , 300 MHz) δ 7.62 (d, 1H), 7.30–7.24 (m, 2H), 7.16 (dd, 1H) 7.01 (br s,
3
1
3
1
1
2
H), 6.61 (s, 1H), 2.68 (q, 2H), 2.48 (s, 3H), 2.43 (s, 3H), 1.24 (t, 3H); C NMR (CDCl , 100 MHz) δ
3
67.61, 159.38, 141.66, 141.03, 137.88, 136.12, 132.78, 129.31, 127.14, 125.53, 122.05, 102.13, 26.82,
+
4.27, 14.26, 13.93, 12.68; HPLC Purity = 98.5%, t = 36.1 min; LC-MS (ESI) m/z: 300.1 [M + H] ; HRMS
R
+
(
ESI): m/z calculated for C H N OS [M + H] : 300.1171 Found: 300.1174.
16
18
3
5.1.41. 4-(2,4-Dimethyl-1,3-oxazol-5-yl)-N-(2,4,6-trimethylphenyl)-1,3-thiazol-2-amine (5u). Following
standard procedure C, 4f (78.3 mg, 0.359 mmol), ethanol (3.0 mL), and 3d (69.8 mg, 0.359 mmol) were
used to carry out the reaction. After the solution was stirred for 3 h and worked-up, the residue was purified
by Isco Combi-Flash Companion column chromatography (0–20% EtOAc in n-hexane) to give 5u (82.7 mg,
1
7
2
1
4%) as a beige solid. H NMR (CDCl , 300 MHz) δ 6.97 (s, 2H), 6.49 (s, 1H), 2.46 (s, 3H), 2.41 (s, 3H),
3
1
3
.32 (s, 3H), 2.27 (s, 6H); C NMR (CDCl , 100 MHz) δ 170.78, 159.26, 141.76, 141.10, 138.02, 136.68,
3
34.45, 132.57, 129.65, 102.06, 21.03, 18.02, 13.86, 12.72; HPLC Purity = 99.0%, t = 36.65 min; LC-MS
R
+
+
(
ESI) m/z: 314.1 [M + H] ; HRMS (ESI): m/z calculated for C H N OS [M + H] : 314.1327 Found:
17 20 3
3
5
14.1324.
.1.42. 2-Azido-1-(2,4-dimethyl-thiazol-5-yl)ethanone (6). Sodium azide (100 mg, 1.54 mmol) was added to
a solution of 4a (301 mg, 1.28 mmol) in dimethylformamide (3.0 mL) at room temperatura. The mixture
was stirred for 40 min, then diluted with waterand extracted with EtOAc. The combined organic layers were
dried over MgSO , filtered, concentrated, and dried under high vacuum to give 6 (238 mg, 93%) as a
4
(s)
1
brown solid. H NMR (CDCl , 400 MHz) δ 4.26 (s, 2H), 2.73 (s, 6H).
3
2
4

5.1.43.
5-(2,4-Dimethyl-1,3-thiazol-5-yl)-N-(2-ethylphenyl)-1,3-oxazol-2-amine
(8).
2-Ethylphenyl
ACCEPTED MANUSCRIPT
isocyanate (7, 86.0 µL, 0.610 mmol) was added to a solution of 6 (108 mg, 0.552 mmol) and
triphenylphosphine (217 mg, 0.828 mmol) in THF (5.0 mL) at room temperature and stirred for 4 h. The
mixture was quenched by the addition of saturated NaHCO3(aq), and extracted with CH Cl . The combined
2
2
organic layers were dried over MgSO , filtered, and concentrated to afford a residue, which was purified
4
(s)
by Isco Combi-Flash Companion column chromatography (5–60% EtOAc in n-hexane) to give an oil. This
1
was recrystallized from a mixture of CH Cl and ether to give 8 (5.1 mg, 3%). H NMR (CDCl , 400 MHz)
2
2
3
δ 7.96 (d, 1H), 7.29–7.22 (m, 2H), 7.06 (dd, 1H), 6.90 (s, 1H), 6.67 (br s, 1H), 2.69 (s overlapped with q at
1
3
2
1
1
.68, 3H), 2.68 (q overlapped with s at 2.69, 2H), 2.49 (s, 3H), 1.30 (t, 3H); C NMR (CDCl , 100 MHz) δ
3
63.32, 156.80, 147.97, 138.09, 135.70, 132.13, 131.89, 128.61, 127.05, 123.69, 123.56, 119.04, 24.10,
+
9.07, 16.61, 13.74; HPLC Purity = 95.1%, t = 33.36 min; LC-MS (ESI) m/z: 300.1 [M + H] ; HRMS
R
+
(
ESI): m/z calculated for C H N OS [M + H] : 300.1171 Found: 300.1174.
16
18
3
5.1.44. N-(2-Ethylphenyl)-2,4'-bi-1,3-thiazol-2'-amine (10). Synthesized according to standard procedure C
using 2-bromo-1-(1,3-thiazo-2-yl)ethanone (9, 78.6 mg, 0.381 mmol), ethanol (3.0 mL), and 3j (68.7 mg,
.381 mmol). After the solution was stirred for 3 h and worked-up, the residue was purified by Isco
0
Combi-Flash Companion column chromatography (0–30% EtOAc in n-hexane) to give 10 (84.8 mg, 77%)
1
as a yellow solid. H NMR (CDCl , 400 MHz) δ 7.83 (d, 1H), 7.60 (d, 1H), 7.34–7.26 (m, 3H), 7.20 (dd,
3
1
3
1
H), 7.08 (br s, 1H), 2.70 (q, 2H), 1.26 (t, 3H); C NMR (CDCl , 100 MHz) δ 167.61, 163.05, 145.27,
3
143.57, 137.68, 136.52, 129.41, 127.24, 125.85, 122.33, 118.98, 105.02, 24.30, 14.32; HPLC Purity = 100%,
+
+
t = 36.19 min; LC-MS (ESI) m/z: 288.0 [M + H] ; HRMS (ESI): m/z calculated for C H N S [M + H] :
R
14 14
3 2
2
5
1
3
88.0629 Found: 288.0624.
.1.45. N-(2-Ethylphenyl)-4-methyl-1,3-thiazol-2-amine (12). A solution of 3j (0.360 g, 2.00 mmol) and
-bromo-2,4-dimethoxypropane (11, 0.330 mL, 2.44 mmol) in acetic acid (4.0 mL) was stirred at reflux for
h. After the solution was cooled to room temperature and concentrated, it was diluted with EtOAc and
washed with water and saturated NaHCO3(aq). The organic layer was dried over MgSO , filtered, and
4(s)
concentrated to afford a residue, which was purified by Isco Combi-Flash Companion column
1
chromatography (0–20% EtOAc in n-hexane) to give 12 (0.114 g, 26%) as a yellow solid. H NMR (CDCl3,
2
5

3
00 MHz) δ 7.57 (d, 1H), 7.27–7.20 (m, 2H), 7.11 (dd, 1H), 7.05 (br s, 1H), 6.13 (s, 1H), 2.66 (q, 2H), 2.28
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1
3
(
s, 3H), 1.23 (t, 3H); C NMR (CDCl , 75 MHz) δ 167.32, 148.57, 138.25, 136.07, 129.23, 127.04, 125.08,
3
1
21.85, 101.53, 24.32, 17.23, 14.25; HPLC Purity = 100%, t = 29.20 min; LC-MS (ESI) m/z: 219.0 [M +
R
+
+
H] ; HRMS (ESI): m/z calculated for C H N S [M + H] : 219.0956 Found: 219.0950.
1
2
15
2
5.1.46. 2',4'-Dimethyl-4,5'-bi-1,3-thiazol-2-amine (14). Following standard procedure C, 4a (92.4 mg, 0.395
mmol), ethanol (3.0 mL), and thiourea (13, 30.1 mg, 0.395 mmol) were used to carry out the reaction. After
the solution was stirred for 4 h and worked-up, the crude product was washed with a solution of 10% diethyl
1
ether in n-hexane to give 14 (43.9 mg, 53%) as a yellow solid. H NMR (DMSO-d , 400 MHz) δ 7.15 (br s,
6
1
3
2
1
2
5
5
H), 6.62 (s, 1H), 2.54 (s, 3H), 2.44 (s, 3H); C NMR (CDCl +CD OD, 100 MHz) δ 168.22, 163.54,
3 3
47.13, 142.03, 126.46, 103.49, 18.35, 16.34; HPLC Purity = 99.8%, t = 14.46 min; LC-MS (ESI) m/z:
R
+
+
12.0 [M + H] ; HRMS (ESI): m/z calculated for C H N S [M + H] : 212.0316 Found: 212.0312.
8
10
3 2
.2. Biology
.2.1. Calcium assay. One day before the assay, CORNING® black with clear flat bottom 96-well assay
plates were coated with a 0.1 mg/mL Poly-L-Lysine solution. Chinese hamster ovary (CHO)-K1 cells, stably
expressing hMOR and Gα15 (CHO-K1/OPRM1/Gα15), were suspended in the F12 medium and plated at a
4
density of ~ 8 × 10 cells/well in 200 µl of medium. Cells were then incubated in a humidified atmosphere of
1
0% CO at 37 °C overnight. At the day of assay, 50 µL FLIPR® calcium assay reagent dissolved in 1x
2
assay buffer (HBSS: KCl 5 mM, KH PO 0.3 mM, NaCl 138 mM, NaHCO 4 mM, Na HPO 0.3 mM,
2
4
3
2
4
D-glucose 5.6 mM, with additional 20 mM HEPES and 13 mM CaCl , pH 7.4), with 2.5 mM probenecid was
2
added to the plate and incubated at 37 °C for 1 h. Compounds and other reagents were dissolved in the assay
2
+
buffer. Using a FlexStationIII (Molecular Devices Corp.), the [Ca ]i fluorescence increases after robotic
injections of compounds or other reagents are monitored every 1.52 s interval with excitation wavelength at
2
+
4
85 nm and with emission wavelength at 525 nm. The [Ca ]i fluorescence is measured up to 90 s after
compounds injection. The fluorescence intensity from 6 to 12 wells of cells are averaged and the relative
2
+
2+
amount of [Ca ]i release is determined by integrating the AUC of the [Ca ]i fluorescence averages. Results
were exported and analysed in GraphPad Prism.
5.2.2. Animals. Male wild-type C57BL/6 (B6) mice (25–30 g) and mu-opioid receptor-knockout mice [40]
2
6

(
kindly provided by Dr. Pao-Luh Tao, National Health Research Institutes, Taiwan) were kept in a
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temperature-controlled animal room with a 12:12 h light-dark cycle. The protocol was approved by the
Institutional Animal Care and Use Committee of the National Health Research Institutes, Taiwan. Animal
experiments were conducted in accordance with the Policies on the Use of Animals in Neuroscience
Research and the ethical guidelines for investigations of experimental pain in conscious animals established
by the International Association for the Study of Pain. In each experiment, animals were randomized to an
experimental group.
5.2.3. Tail-flick test. To test drug-induced antinociception, thermal pain hypersensitivity was determined
using the Tail-Flick Analgesia Meter (Columbia Instruments). The basal latency was recorded before
treatment and test latencies were recorded 30, 60, 90, 120, and 180 min after administration of drugs. The i.v.
dosing solution of 5j was prepared in 5% dimethyl sulfoxide, 5% cremophor, and 90% saline. At each time
point, the antinociceptive effect was defined as the difference between the test latency and the basal latency.
A tail-flick cut-off time of 10 s was set to avoid tissue damage. The ED50 was determined by the
up-and-down method. [41] Briefly, a series of test levels is chosen with equal spacing between each log dose
of compound. Then, a series of trials (n ≧ 6) is performed following the rule that the compound dose is
reduced after inhibition of the tail-flick response and the compound dose is increased after no inhibition of
the tail-flick response. Each mouse underwent only one trial. The ED value is derived from the equation
5
0
ED = X + k × d, where X is the last dose administered, k is the tabular value, and d is the interval between
5
0
f
f
doses. The AUC value was obtained by calculating the area under the time-response curve of the
antinociceptive effect after treatment with the drugs. The values indicate the mean ± SEM.
Author Information
Corresponding Authors
*
S.-H.Y.: phone, +886-37-246-166, extension 35759; fax, +886-37-586-456; e-mail, bau9763@nhri.org.tw.
S.-H.U.: phone, +886-37-246-166, extension 35791; fax, +886-37-586-456; e-mail, shueng@nhri.org.tw.
*
Note
The disclosed compounds of this paper are covered in issued patents: US9,827,228 and TWI625120.
Acknowledgments
2
7

This work was supported by grants 100-2325-B-400-006, 101-2325-B-400-005, 102-2325-B-400-005,
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1
1
03-2325-B-400-018, 104-2325-B-400-008, 105-2325-B-400-007, 107-2320-B-400-007 and
07-2635-B-400-001 from the Ministry of Science and Technology, and Intramural Research Program of the
National Health Research Institutes (04A1-BPPP03-005, BP-103-PP-05), Taiwan.
Abbreviations Used
MOR, µ-opioid receptor; HTS, high-throughput screening; AUC, area under curve; SAR, structure-activity
relationship; CRCs, concentration-response curves; RFU, relative fluorescence units.
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