Systemic optimization and structural evaluation of quinoline derivatives as transthyretin amyloidogenesis inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.003

Wild type transthyretin (TTR) and mutant TTR misfold and misassemble into a variety of extracellular insoluble amyloid fibril and/or amorphous aggregate, which are associated with a variety of human amyloid diseases. To develop potent TTR amyloidogenesis

Full text of DOI:10.1016/j.ejmech.2016.08.003

European Journal of Medicinal Chemistry 123 (2016) 777e787
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Systemic optimization and structural evaluation of quinoline
derivatives as transthyretin amyloidogenesis inhibitors
,
,
Boyoung Kim a 1, Hwanggue Park a 1, Seul Ki Lee a, Sung Jean Park b, Tae-Sung Koo a,
Nam Sook Kang a, Ki Bum Hong c **, Sungwook Choi a
,
, *
a Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro Yuseong-gu, Daejon, 305-764, Republic of Korea
b College of Pharmacy, Gachon University, 534-2 Yeonsu 3-dong, Yeonsu-gu, Incheon, 406-799, Republic of Korea
c New Drug Development Center (NDDC), Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu, 701-310, Republic
of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Wild type transthyretin (TTR) and mutant TTR misfold and misassemble into a variety of extracellular
insoluble amyloid fibril and/or amorphous aggregate, which are associated with a variety of human
amyloid diseases. To develop potent TTR amyloidogenesis inhibitors, we have designed and synthesized a
focused library of quinoline derivatives by Pd-catalyzed coupling reaction and by the Horner
eWadswortheEmmons reaction. The resulting 2-alkynylquinoline derivatives, (E)-2-alkenylquinoline
derivatives, and (E)-3-alkenylquinoline derivatives were evaluated to inhibit TTR amyloidogenesis by
utilizing the acid-mediated TTR fibril formation. Among these quinoline derivatives, compound 14c
exhibited the most potent anti-TTR fibril formation activity in the screening studies, with IC50 values of
Received 31 May 2016
Received in revised form
2 August 2016
Accepted 4 August 2016
Available online 6 August 2016
Keywords:
Amyloid
1.49 mM against WT-TTR and 1.63 mM against more amyloidogenic V30 M TTR mutant. That is comparable
Amyloidogenesis
Transthyretin
Quinoline
In silico docking study
Pharmacokinetic
to that of approved therapeutic drug, tafamidis, to ameliorate transthyretin-related amyloidosis.
Furthermore, rationalization of the increased efficacy of compound 14c bearing a hydrophobic substit-
uent, such as chloride, was carried out by utilizing in silico docking study that could focus on the region of
the thyroid hormone thyroxine (T4) binding sites. Additionally, the most potent compound 14c exhibited
good pharmacokinetics properties. Taken together, the novel quinoline derivatives could potentially be
explored as potential drug candidates to treat the human TTR amyloidosis.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
amyloidosis (SSA), familial amyloid polyneuropathy (FAP), familial
amyloid cardiomyopathy (FAC), or central nervous system selective
Human amyloid diseases such as Alzeheimer's disease, Parkin-
son's disease, type II diabetes, light chain amyloidosis, and trans-
thyretin amyloidosis are characterized by aberrant protein
amyloidosis (CNSA). SSA is associated with WT-TTR deposition in
the heart and a late onset disease that affects up to 20% of the aged
population [3], whereas tissue selective deposition of one of less
stable mutants of TTR and cell degeneration results in FAP (e.g.,
V30M-TTR), FAC (e.g., V122I-TTR), and CNSA (e.g., D18G and A25T-
TTR) [5e11].
misfolding, misassembly, and deposition in the fibrillary cross-b-
sheet amyloid fibril [1e3]. Transthyretin (TTR) is one of more than
30 nonhomologous human amyloidogenic proteins and peptides
[4e6]. Amyloidogenesis of wild type (WT) TTR or one of over 100
thermodynamically less stable mutants of TTR appears to elicit the
proteotoxicity and cell degeneration, which cause senile systemic
TTR is a homotetrameric protein composed of 127-amino-acid,
b-sheet-rich subunits [12]. The established physiological functions
of TTR are to bind to and transport the thyroid hormone thyroxine
(T4) and holo-retinol binding protein in the blood and cerebrospinal
fluid (CSF) [5,13]. TTR has two unique dimer-dimer interface,
creating two funnel-shaped thyroxine binding sites as shown in
Fig. 1A [14]. Because there are two major thyroxine carriers such as
albumin and thyroid-binding globulin in the blood, more than 99%
of thyroxine binding sites within TTR tetramer are unoccupied [5].
TTR fibril formation requires the rate-limiting tetramer dissociation
* Corresponding author.
** Corresponding author.
E-mail addresses: kbhong@dgmif.re.kr (K.B. Hong), swchoi2010@cnu.ac.kr
(S. Choi).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.08.003
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

778
B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
Fig. 1. (A) The X-ray crystal structure (PDB 2ROX) of WT-TTR with expanded view of one of thyroxine binding sites and proposed binding mode of small molecules. (B) Chemical
structure of thyroxine.
and misassembly of partially denatured monomeric subunits into
insoluble amyloid fibril and/or amorphous aggregate [5,15,16].
Reversible and irreversible small molecules that bind to one or two
of thyroxine binding sites stabilize the ground state of tetrameric
TTR and raise the kinetic barrier for dissociation, resulting in kinetic
stabilization on TTR and preventing aggregation [5,17e37].
Oral tafamidis (Vyndaqel®) is only one approved therapeutic
drug to ameliorate transthyretin-related amyloidosis. Tafamidis
was initially used in Europe in 2011 for adult patients with early
stage symptomatic polyneuropathy and has since been approved in
Argentina, Japan, and Mexico for the treatment of TTR familial
amyloid polyneuropathy (TTR-FAP). The drug selectively binds to
thyroxine binding sites within TTR tetramer with negative coop-
erativity and kinetically stabilize the WT-TTR and the variant TTR
tetramer through prevention of TTR dissociation, the rate-limiting
step of TTR amyloid fibril formation [38e40]. In this study,
numerous structure-activity relationship studies were carried out
to identify TTR amyloidogenesis inhibitors with desirable phar-
macological properties: aromatic X ring bearing polar substituents
for hydrogen bond interaction with the Ser-117 or Thr-119 hydroxyl
groups and van der Waals interaction in the inner binding cavity,
hydrophobic linker L, and aromatic Y ring bearing polar sub-
stituents for electrostatic and/or hydrogen bond interactions with
the Lys 15 and/or the Glu-54 in the outer binding cavity (Fig.1B). On
the basis of previous structural data [17,18,22e30,32,34,36], we
chose focused substituents on the two aromatic rings (X and Y) and
two kinds of linker to design potent WT- and V30M-TTR amyloi-
dogenesis inhibitors and compared their potency to that of tafa-
midis. We investigated the molecular mechanism for the anti-TTR
fibril formation activity of quinoline derivatives by performing in
silico docking studies and reported the PK evaluations of two potent
inhibitors in vivo.
the deprotection of MEM or TIPS groups and 2-alkynylquinoline
derivatives 7h was subjected to reduction with Sn powder in
AcOH and HCl to give 2-alkynylquinoline derivatives 7i.
The Heck coupling reaction of 2-chloroquinoline derivatives 5a
and 5d and terminal alkene 8 [34] in the presence of Pd2(dba)3, a
variety of ligands (P(o-Tol)3, PPh3, tButylXPhos), and a variety of
bases (Et3N, K2CO3, Cy2NMe) was utilized as shown in Scheme 2.
Among them, N,N-Dicyclohexylmethylamine (Cy2NMe) and tBu-
tylXPhos were found to be very effective additives to give (E)-2-
alkenylquinoline derivatives 9a and 9b. Deprotection of MEM
groups of 9a and 9b using concentrated HCl afforded the corre-
sponding (E)-2-alkenylquinoline derivatives 10a and 10b and sub-
sequently, the nitro compound 10b was treated with SnCl2$2H2O in
EtOH to produce the amine compound 10c.
Finally, the (E)-3-alkenylquinoline derivatives 14a and 14b were
prepared by the HornereWadswortheEmmons reactions between
substituted 2-chloroquinoline-carbaldehydes 11a and 11b and
diethyl phosphonate 12 [37] followed by the deprotection of MEM
groups using concentrated HCl as shown in Scheme 3. Subse-
quently, the demethylation of 14b with BBr3 provided (E)-3-
alkenylquinoline derivatives 14c.
2.2. Biological evaluation
2.2.1. Inhibition of WT- and V30M-TTR amyloidogenesis by
quinoline derivatives
The newly synthesized quinoline derivatives are evaluated their
ability to inhibit TTR amyloidogenesis using the previously estab-
lished acid-mediated TTR fibril formation assay [41]. Briefly, a
candidate inhibitor (3.6 or 7.2
mM) was preincubated with a phys-
iological concentration of WT-TTR or V30M-TTR (each 3.6
mM) and
amyloidogenesis was triggered by adjusting the pH to 4.4. The
turbidity of the sample solution was measured after 72 h incuba-
tion at 37 ꢀC to evaluate their ability to prevent WT-TTR or V30M-
TTR amyloidogenesis. The potency of candidate inhibitors was
expressed as the percentage of TTR fibril formation, compared to
TTR fibril formation in the absence of inhibitor, as shown in
Tables 1e3.
The first study was focused on the effect of a variety of hydro-
phobic substituents flanked by 4-hydroxyl group on an aryl-X ring.
It is clear that the hydrophobic group such as methyl group play a
critical role in enhancing binding potency (7a vs 7b). However, the
decrease in potency of compounds 7c-e with bigger hydrophobic
groups over compound 7b suggests that the small hydrophobic
group is essential for WT-TTR binding, due to spatial limitation in
the inner binding cavity. We then shift our attention to the effect of
substituents on a quinoline-Y ring, while holding the 3,5-dimethyl-
2. Results and discussion
2.1. Chemistry
The general procedure for the synthesis of 2-alkynylquinoline
derivatives 7a-i was outlined in Scheme 1. Intermediates 2a-e,
obtained by the protection of hydroxyl group with MEM group or
TIPS group, were treated with TMS-acetylene under Pd/Cu cata-
lyzed Sonogashira coupling conditions to afford TMS-protected
alkynes 3a-e in 40%e94% yield. TMS-deprotected alkynes 4a-e,
which were prepared from deprotection of TMS group with K2CO3
in MeOH, were coupled with 2-chloroquinoline derivatives 5a-d via
a second Pd/Cu catalyzed Sonogashira coupling reactions. The final
2-alkynylquinoline derivatives 7a-h were conveniently obtained by

B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
779
TMS
H
Br
Br
a
b
c
R1
R2
R1
R2
R1
R2
R1
R2
OPG
OH
OPG
2a-e
OPG
1a-e
3a-e
4a R1 = H, R2 = H, PG = MEM
4b R1 = Me, R2 = Me, PG = MEM
4c R1 = Et, R2 = Et, PG = TIPS
4d R1 = H, R2 = Et, PG = TIPS
4e R1 = H, R2 = i-Pr, PG = TIPS
R3
R3
H
R3
d
N
N
e
R2
R2
OPG
N
R1
R2
Cl
5a R3 = H
5b R3 = OMe
5c R3 = OH
5d R3 = NO2
OPG
4a-e
OH
R1
R1
7a R1 = H, R2 = H, R3 = H
6a-h
7b R1 = Me, R2 = Me, R3 = H
7c R1 = Et, R2 = Et, R3 = H
7d R1 = H, R2 = Et, R3 = H
7e R1 = H, R2 = i-Pr, R3 = H
7f R1 = Me, R2 = Me, R3 = OMe
7g R1 = Me, R2 = Me, R3 = OH
7h R1 = Me, R2 = Me, R3 = NO2
7i R1 = Me, R2 = Me, R3 = NH2
f)
Scheme 1. Reagents and conditions a) NaH, TIPSCl, 3e14 h for 2a and 2b or NaH, MEMCl, THF, 18 h for 4c-e; b) Ethynyltrimethylsilane, Pd(PPh3)2Cl2, CuI, Et3N, MeCN, 66 ꢀC, 3e17 h;
c) K2CO3, MeOH, 1.5e8 h; d) Pd(PPh3)2Cl2, CuI, Et3N 3e7 h, 80 ꢀC; e) conc.HCl, THF/MeOH ¼ 1:1 (v/v), r.t., 4e20 h for 7a, 7b, 7f, 7g or 1 M TBAF, THF, 1e2.5 h, for 7c-e, 7h ; f) Sn
powder, AcOH, HCl, r.t., 1 h for 7i.
R1
R1
R1
N
N
a
b
N
OMEM
Cl
OH
c
OMEM
10a R1 = H
10b R1 = NO2
10c R1 = NH2
5a R1 = H
5d R1 = NO2
9a R1 = H
9d R1 = NO2
8
Scheme 2. Reagents and conditions a) Pd2(dba)3, tButylXPhous, Cy2NMe, Dioxane, 120 ꢀC, 18e40 h; b) conc.HCl, THF/MeOH ¼ 1:1 (v/v), r.t.,6e20 h; c) SnCl2 2H2O, EtOH, r.t., 9 h.
R2
R2
O
P
N
N
OEt
OEt
Cl
Cl
O
a
b
H
R2
N
Cl
OMEM
OMEM
OH
11a R2 = H
11b R2 = OMe
12
13a R2 = H
13b R2 = OMe
14a R2 = H
14b R2 = OMe
14c R2 = OH
c
Scheme 3. Reagents and conditions a) tBuOK, DMF, 60 ꢀC, 1e6 h; b) conc.HCl, THF/MeOH ¼ 1:1 (v/v), r.t., 5e20 h; c) BBr3, DCM, ꢁ78 ꢀC to r.t., 22 h.
4-hydroxy-substituted aryl-X ring constant. This series with rela-
tive hydrophilic substituents displayed increased levels of potency
compared to the unsubstituted lead compound 7b, except
compound 7f with a hydrophobic methoxy group on the quinoline-
Y ring.
In order to induce further structural optimization, we asked

780
B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
Table 1
Table 3
Inhibition of WT-TTR amyloid fibril formation under acidic denaturation condition
in the presence of quinoline derivatives.
Inhibition of more amyloidogenic V30M-TTR amyloid fibril formation under acidic
denaturation condition in the presence of tafamidis and quinoline derivative 14c.4
R3
O
HO
OH
N
Cl
N
R2
N
O
OH
R1
Compound
R1
R2
R3
% Fibril Formation
(WT-TTR)a
IC50 (mM)
OH
Cl
Cl
Tafamidis
14c
7.2
m
M
3.6 mM
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
Me
Et
H
H
Me
Me
Me
Me
H
Me
Et
H
H
H
H
73.9%
10.2%
93.4%
99.0%
97.3%
22.1%
9.6%
e
e
25.6%
e
2.18
e
Compound
% Fibril Formation (V30M-
TTR)a
IC50 (mM)
Et
e
e
7.2
mM
3.6 mM
i-Pr
Me
Me
Me
Me
H
e
e
OMe
OH
NO2
NH2
e
e
Tafamidis
14c
3.2%
5.0%
17.0%
16.6%
1.62
1.63
32.1%
20.9%
17.9%
2.59
2.06
1.90
3.6%
4.8%
a
% Fibril formation represents the extent of more amyloidogenic V30M-TTR fibril
formation in the presence of inhibitors (7.2 mM or 3.6 mM inhibitor, 3.6 mM V30M-
TTR, pH 4.4, 37 ꢀC, 72 h), compared to untreated V30M-TTR (100% fibril formation).
a
% Fibril formation represents the extent of WT-TTR fibril formation in the
M or 3.6 M inhibitor, 3.6
M WT-TTR, pH 4.4, 37 ꢀC,
presence of inhibitors (7.2 M
m
m
m
72 h), compared to untreated WT-TTR (100% fibril formation).
halogen binding pockets (HBP) 1 and 1’.
Furthermore, the most potent compound 14c was reevaluated
against one of the most clinically significant mutant TTR (V30M-
TTR) as shown in Table 3. Importantly, compound 14c allowed 5% of
whether we could substitute the acetylene linker in the context of
an aryl-quinoline scaffold composed of 3,5-dimethyl-4-hydroxy-
substituted aromatic X ring and one aromatic Y ring bearing
different substituents. While a detrimental effect in potency was
observed with the simple replacement of the acetylene linker to the
ethylene linker (10a-c), addition of a hydrophobic substituent such
as a chloride group on the quinoline-Y ring dramatically increased
V30M-TTR fibril formation at a concentration of 7.2
V30M-TTR fibril formation at a concentration equal to the V30M-
TTR tetramer (3.6 M). Dose-dependent fibril formation inhibi-
tion demonstrated that compound 14c, with IC50 value of 1.63 M,
exhibited similar potency to tafamidis with IC50 value of 1.62 M.
mM and 16.6% of
m
m
m
the efficacy (14c, IC50 value of 1.49
mM), which is comparable to that
observed with tafamidis (IC50 value of 1.55
mM). It is likely that the
chloride substituents extend into the two symmetry-related
Table 2
Inhibition of WT-TTR amyloid fibril formation under acidic denaturation condition in the presence of tafamidis and quinoline derivatives.3
O
R1
R2
OH
N
Cl
N
N
O
OH
OH
Cl
Cl
Tafamidis
Compound
R1
R2
% Fibril Formation (WT-TTR)a
7.2
1.0%
26.3%
92.9%
26.8%
2.3%
IC50 (mM)
m
M
3.6 mM
Tafamidis
10a
10b
10c
14a
14.8%
e
1.55
e
H
e
e
e
H
NO2
NH2
e
e
e
51.3%
33.2%
17.7%
e
e
14c
e
OH
0.9%
1.49
a
% Fibril formation represents the extent of more amyloidogenic V30M-TTR fibril formation in the presence of inhibitors (7.2
4.4, 37 ꢀC, 72 h), compared to untreated WT-TTR (100% fibril formation).
mM or 3.6 mM inhibitor, 3.6 mM WT-TTR, pH

B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
781
Fig. 3. Docking mode of compound 14c on the TTR tetramer. The chloride atom of
quinoline ring forms the hydrophobic interactions with side chains of K15, T106 and
V121 shown in violet lines. As known in 2ROX structure bound with thyroxine, qui-
nolone ring shows the hydrophobic interaction with side chains of K15 and K015. The
hydroxyl group of quinolone ring electrostatically interacts with side chains of E54 and
E054. The hydrogen bond was described in red lines.
interaction with side chains of K15 and K015, as shown in 2ROX
complex structure. The chloride atom of quinolone-Y ring showed
the favorable interaction in the hydrophobic cavity formed by side
chains of K15, T106 and V121, and thus leading to the activity
improvement. Also, the hydroxyl group of quinoline-Y ring was
well positioned around the negative side chain of E54 and E054
showing the favorable electrostatic interaction. Through the hy-
drophobic channel surrounded by A108, L110, T119, L017, T0106 and
L0110, two methyl group on the aryl-X ring interacts with each
hydrophobic residue as shown in Fig. 3, indicated with violet lines.
The hydroxyl part of aryl-X ring especially forms the hydrogen
bond with side chain of S117 and S0117 described in red lines. The
methyl substituents extend into the two symmetry-related halogen
binding pockets 3 and 30. The 4-OH substituent on the aryl-X ring
makes bridging hydrogen bonds with the Ser117 and Ser1170 side
chains of adjacent TTR subunits.
Fig. 2. (A) The time-course of WT-TTR (3.6
presence of quinoline derivatives and tafamidis (3.6
TTR (3.6 M) fibril formation in the absence and presence of quinoline derivative 14c
and tafamidis (3.6 M).
m
M) fibril formation in the absence and
m
M). (B) The time-course of V30M-
m
m
2.2.2. WT- and V30M-TTR aggregation time courses for quinoline
derivatives
To further evaluate the potency of selected quinoline derivatives
7b, 7g, 7h, 7i, and 14c, the extent of inhibition of acid-mediated
WT-TTR fibril formation (pH 4.4) by inhibitors at a concentration
2.4. In vivo pharmacokinetic profile
Plasma concentration profiles of compounds 7i and 14c in fasted
male SD rats are shown in the Supplementary Data section. Phar-
macokinetic parameters are listed in Table 4. Parameters were
calculated for each animal and averaged.
After a single IV injection of 2 mg/kg, the peak plasma concen-
trations (Cmax) of 7i and 14c in male rats were 3.107 and 2.381 mg/
mL, reached at 0.083 h after administration, respectively. The mean
systemic plasma clearance (CL) was 1505 and 440.2 mL/h/kg for 7i
and 14c, respectively. The mean steady state volume of distribution
(Vss) for 7i and 14c were 1479 and 1128 mL/kg, respectively. Ter-
minal half-life (T1/2) was 2.710 and 2.745 h for 7i and 14c, respec-
tively. Comparing to hepatic blood flow in rat (3312 mL/h/kg) [42],
the CL values of 7i and 14c were considered moderate and low,
respectively, indicating the elimination rate including metabolism
was not high. Meanwhile, Vss values of 7i and 14c were moderate,
suggesting that 7i and 14c were well distributed to tissue in rats.
After a single oral dosing of 5 mg/kg, Cmax of 7i and 14c in male
equal to the TTR tetramer (3.6 mM) were investigated over a 120 h
time course instead of at a fixed time (72 h). As shown in Fig. 2A, the
most potent inhibitor 14c inhibited 7e23% more WT-TTR fibril
formation than other inhibitors and significantly showed similar
potency to tafamidis. Furthermore, inhibitor 14c exhibited more
than 80% inhibition and significantly suppressed more amyloido-
genic V30M-TTR fibril formation (Fig. 2B). Under such an acidic
condition, the binding of 14c to only one T4 binding site within WT-
TTR tetramer was sufficient to kinetically stabilize and to prevent
the fibril formation of WT- and V30M-TTR tetramer.
2.3. In silico molecular docking studies
The expected binding mode of compound 14c with the best
activity was displayed in Fig. 3. Atomic coordinates for TTR (PDB ID:
2ROX) were obtained from RCSB protein databank (www.rcsb.org).
For compound 14c, the quinoline-Y ring forms a hydrophobic
rats was 0.138 and 0.665 mg/mL, reached at 2.450 and 0.598 h after

782
B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
Table 4
Pharmacokinetic parameters in rats after a single IV and oral administration of 7i and 14c.
PK Parameters
7i
14c
IV
Oral
IV
Oral
T
C
(h)
(mg/mL)
0.083 0.000
3.107 0.371
2.710 1.059
1.331 0.167
1.347 0.170
1505 211.5
1479 417
2.450 1.230
0.138 0.204
1.592 0.654
0.226 0.127
0.232 0.127
0.083 0.000
2.381 0.358
2.745 1.894
4.508 0.557
4.590 0.541
440.2 51.30
1128 431.2
0.598 0.318
0.665 0.247
3.364 1.428
2.845 0.639
3.122 0.698
max
max
T1/2 (h)
AUC0-t
AUC0-∞ (mg h/mL)
CL (mL/h/kg)
Vss (mL/kg)
BA (%)
(
m
g h/mL)
6.888 3.761
27.20 6.081
administration, respectively. T1/2 for oral dosing of 5 mg/kg was
1.592 and 3.364 h for 7i and 14c, respectively. Bioavailability (BA)
was estimated by comparing normalized areas under the plasma
performance liquid chromatography (RP-HPLC) performed on a
Waters Alience 2695 separation module, using a Waters 2487 dual
l
absorbance detector, equipped Alience 2695 autosampler and a
concentration-time curve (AUC0-∞) for oral and intravenous
Thermo Hypersil Keystone Betabasic-18 column (150 Å pore size,
administration was to be 6.880 and 27.20 for 7i and 14c, respec-
tively. Inhibitor 7i was absorbed slowly and showed low BA value
after oral administration. Considering moderate CL value, low BA
was not considered to be caused only by low metabolic stability but
also by the marginal absorption in intestine. Meanwhile, inhibitor
14c was absorbed rapidly and showed higher plasma exposure level
than 7i. With moderate BA and low clearance, 14c was also not
considered to be fully absorbed in the rat intestine.
3
m
m particle size, mobile phase A ¼ 0.1% TFA in 99.9% H2O, mobile
phase B ¼ 0.1% TFA in 99.9% MeCN). Linear gradients were run from
100:1 to 0:100 A:B over 25 min.
4.1.2. General procedure for MEM or TIPS protection for compound
2a-e
To a solution of compounds 3a-d (1.0 equiv.) and MEMCl or
TIPSCl (1.2 equiv.) in THF was slowly added sodium hydride (60% in
mineral oil,1.1 equiv.) in portion at 0 ꢀC. The solution was allowed to
r.t. and stirred for 3e18 h. The solution was quenched with water.
The solvent was evaporated and the residue was dissolved with
EtOAc. The organic layer was washed with water and brine, and
dried with Na2SO4. The solution was filtered and concentrated. The
crude material was purified by column chromatography (silica gel,
Hexane/EtOAc).
3. Conclusions
We have successfully carried out structure activity relationship
studies and identified potent quinoline derivatives, which has the
following characteristics as a TTR kinetic stabilizer; aromatic X ring
with polar substituents for hydrogen bond interaction and van der
Waals interaction, hydrophobic linker L, and aromatic Y ring with
polar substituents for electrostatic and/or hydrogen bond in-
teractions. In this study, the focused promising library of quinoline
derivatives have been designed by structural optimization by SAR
studies and their potency was evaluated by acid-mediated TTR fibril
formation. Among these compounds, compounds 7i and 14c
allowed less than 5% of WT-TTR fibril formation at a concentration
equal to the WT-TTR tetramer. The most potent inhibitor 14c
significantly exhibited 80% inhibition against more amyloidogenic
V30M-TTR at a concentration equal to the V30M-TTR tetramer over
a 120 h time course. In silico docking studies within the T4 binding
sites were carried out to rationalize the binding mode and molec-
ular mechanism for its anti-TTR fibril formation activity. From this
study, a new class of quinoline derivatives with high potency and
good PK properties that could potentially be applied for the treat-
ment of human TTR-related diseases.
4.1.2.1. 1-Bromo-4-((2-methoxyethoxy)methoxy)benzene
(2a).
Yield: 94%, 1H NMR (300 MHz, CDCl3)
d
3.38 (s, 3H), 3.55 (t,
J ¼ 4.5 Hz, 2H), 3.82 (t, J ¼ 4.5 Hz, 2H), 5.25 (s, 2H), 6.95 (dd,
J ¼ 12 Hz, 2H), 7.38 (dd, J ¼ 9.0 Hz, 2H); 13C NMR (150 MHz, CDCl3)
d
58.44, 67.28, 71.11, 93.04, 113.67, 117.68, 131.82, 155.98.
4.1. 2. 2. 5-Bromo-2-((2-methoxyethoxy)methoxy)-1, 3-
dimethylbenzene (2b). Yield: 94%, 1H NMR (300 MHz, CDCl3)
2.26
d
(s, 6H), 3.41 (s, 3H), 3.59e3.62 (m, 2H), 3.92e3.95 (m, 2H), 5.03 (s,
2H), 7.15 (s, 2H); 13C NMR (75 MHz, CDCl3)
71.75, 97.98, 116.69, 131.38, 133.23, 153.84.
d 16.71, 59.07, 69.18,
4.1.2.3. (4-Bromo-2,6-diethylphenoxy)triisopropylsilane
(2c).
Yield: 77%, 1H NMR (300 MHz, CDCl3)
d
1.09 (d, J ¼ 7.2 Hz, 18H), 1.19
(t, J ¼ 7.5 Hz, 6H), 2.28 (m, J ¼ 7.2 Hz, 3H), 2.58 (q, J ¼ 7.5 Hz, 4H),
7.10 (s, 2H); 13C NMR (75 MHz, CDCl3)
113.71, 128.97, 136.27, 151.48.
d 14.16, 14.32, 18.04, 23.42,
4. Experimental section
4.1. Chemistry
4.1.2.4. (4-Bromo-2-ethylphenoxy)triisopropylsilane
78%, 1H NMR (300 MHz, CDCl3)
(2d). Yield:
1.10 (d, J ¼ 7.1 Hz, 18H), 1.19 (t,
d
4.1.1. General
J ¼ 7.6 Hz, 3H), 1.26e1.34 (m, 3H), 2.58 (q, J ¼ 7.5 Hz, 2H), 6.64 (d,
J ¼ 8.6 Hz, 1H), 7.12 (dd, J ¼ 2.6, 8.6 Hz, 1H), 7.24 (d, J ¼ 2.6 Hz, 1H);
Unless otherwise indicated, all reactions were run under nitro-
gen gas. The progress of the reactions was monitored by thin layer
chromatography (TLC). All products were determined by 1H, 13C
NMR spectra. 1H and 13C spectra were recorded on a BRUKER
300 MHz and 600 MHz spectrometer. Chemical shifts are reported
13C NMR (75 MHz, CDCl3)
d 13.00, 13.95, 18.02, 23.50, 112.77, 119.34,
129.15, 131.91, 136.71, 153.00.
relative to internal CDCl3 (Me4Si,
d
0.0) and DMSO-d6 (
d
2.50 for 1H
4.1.2.5. (4-Bromo-2-isopropylphenoxy)triisopropylsilane (2e) [43]..
and 39.52 for 13C). All mass spectrometry data were collected by
Waters Fraction Lynx MS Autopurification System at the Korea
Research Institute of Chemical Technology (KRICT). Final compound
purities were determined by analytical reverse phase high
Yield: 85%, 1H NMR (300 MHz, CDCl3)
d
1.10 (d, J ¼ 6.0 Hz, 18H), 1.18
(d, J ¼ 7.5 Hz, 6H), 1.27 (m, J ¼ 6.0 Hz, 3H), 3.30 (m, J ¼ 7.5 Hz, 1H),
6.63 (d, J ¼ 8.6 Hz, 1H), 7.10 (dd, J ¼ 2.6, 8.6 Hz, 1H), 7.27 (d,
J ¼ 2.6 Hz, 1H).

B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
783
4.1.3. General procedure of the Sonogashira coupling reaction for
58.98, 69.10, 71.69, 76.05, 83.48, 97.88, 117.60, 131.19, 132.57, 155.33.
alkynes 3a-e
To
a
solution of compounds 2a-e (1.0 equiv.),
4.1.4.3. (2,6-Diethyl-4-ethynylphenoxy)triisopropylsilane
Yield: 80%, 1H NMR (300 MHz, CDCl3)
1.09 (d, J ¼ 7.4 Hz, 18H), 1.17
(t, J ¼ 7.5 Hz, 6H), 1.27e1.34 (m, 3H), 2.58 (q, J ¼ 7.4 Hz, 4H), 3.00 (s,
1H), 7.18 (s, 2H); 13C NMR (75 MHz, CDCl3)
14.14, 14.33, 18.03,
(4c).
bis(triphenylphosphine)-palladium(II) dichloride (0.1 equiv.), cop-
per(I) iodide (0.1 equiv.) in degassed MeCN was added trime-
thylsilylacetylene (3e4 equiv.) and Et3N (3.0 equiv.) and stirred at
66 ꢀC for 3e17 h. The mixture was filtered through Celite and
washed with EtOAc. The organic solution was washed with water
and brine, and dried with Na2SO4. The solution was filtered and
concentrated. The crude material was purified by column chro-
matography (silica gel, Hexane/EtOAc).
d
d
23.30, 75.17, 84.53, 114.44, 130.39, 134.27, 153.28.
4.1.4.4. (2-Ethyl-4-ethynylphenoxy)triisopropylsilane
(4d).
Yield: 70%, 1H NMR (300 MHz, CDCl3)
d
1.10 (d, J ¼ 7.2 Hz, 18H), 1.17
(t, J ¼ 7.5 Hz, 3H), 1.25e1.35 (m, 3H), 2.59 (q, J ¼ 7.5 Hz, 2H), 2.99 (s,
1H), 6.70 (d, J ¼ 8.3 Hz, 1H), 7.19 (dd, J ¼ 2.2, 8.2 Hz, 1H), 7.30 (d,
4.1.3.1. (4-(2-Methoxy-ethoxymethoxy)-phenylethynyl)trimethylsi-
J ¼ 2.2 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 13.03, 18.01, 23.38,
lane (3a). Yield: 93%, 1H NMR (300 MHz, CDCl3)
d
0.24 (s, 9H), 3.37
75.30, 84.19, 99.97, 113.95, 117.79, 130.73, 133.18, 134.59, 154.63.
(s, 3H), 3.54 (t, J ¼ 4.5 Hz, 2H), 3.81 (t, J ¼ 4.5 Hz, 2H), 5.27 (s, 2H),
6.97 (d, J ¼ 9.0 Hz, 2H), 7.4 (d, J ¼ 9 Hz, 2H); 13C NMR (150 MHz,
4.1.4.5. (4-Ethynyl-2-isopropylphenoxy)triisopropylsilane
Yield: 69%, 1H NMR (300 MHz, CDCl3)
(4e).
CDCl3)
d
0.23, 59.20, 67.94, 71.75, 92.93, 93.43, 105.18,116.19, 116.67,
d
1.11 (d, J ¼ 7.2 Hz, 18H), 1.19
133.60, 157.53.
(d, J ¼ 6.9 Hz, 6H),1.28e1.35 (m, J ¼ 7.2 Hz, 3H), 2.99 (s,1H), 3.31 (m,
J ¼ 6.9 Hz, 1H), 6.70 (d, J ¼ 8.3 Hz, 1H), 7.17 (dd, J ¼ 2.1, 8.3 Hz, 1H),
4.1.3.2. 2((4-((2-methoxyethoxy)methoxy)-3,5-dimethylphenyl)ethy-
7.35 (d, J ¼ 2.1 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 13.08, 18.04,
nyl)trimethylsilane (3b) [34]. Yield: 99%, 1H NMR (400 MHz, CDCl3)
22.62, 26.58, 75.23, 84.38, 114.01, 117.81, 130.44, 138.88, 153.98.
d
0.23 (s, 9H), 2.24 (s, 6H), 3.40 (s, 3H), 3.56e3.62 (m, 2H),
3.90e3.94 (m, 2H), 5.04 (s, 2H), 7.15 (s, 2H).
4.1.5. General procedure of Sonogashira coupling reaction for
compounds 6a-h
4.1.3.3. (2,6-Diethyl-4-((trimethylsilyl)ethynyl)phenoxy)triisopro-
To a solution of substituted quinolines 5a-d (1.0 equiv.), ethy-
nylbenzene 4a-e (1.2e1.4 equiv.), bis(tri-phenylphosphine)
palladium(II) dichloride (0.1 equiv.), copper(I) iodide (0.1 equiv.)
in degassed Et3N. The solution was stirred under N2 at 80 ꢀC for
3e7 h. The mixture was filtered through Celite and washed with
EtOAc. The solution was washed with water and brine, and dried
with Na2SO4. The solution was filtered and concentrated. The crude
material was purified by column chromatography (silica gel, Hex-
ane/EtOAc).
pylsilane (3c). Yield: 54%, 1H NMR (300 MHz, CDCl3)
d 0.25 (s, 9H),
1.08 (d, J ¼ 7.3 Hz, 18H), 1.17 (t, J ¼ 7.5 Hz, 6H), 1.26 (m, 3H), 2.57 (q,
J ¼ 7.5 Hz, 4H), 7.16 (s, 2H); 13C NMR (75 MHz, CDCl3)
d 0.12, 14.17,
14.31, 18.02, 23.34, 91.76, 106.10, 115.44, 130.32, 134.18, 153.13.
4.1.3.4. (2-Ethyl-4-((trimethylsilyl)ethynyl)phenoxy)triisopropylsi-
lane (3d). Yield: 40%, 1H NMR (300 MHz, CDCl3)
d 0.24 (s, 9H), 1.09
(d, J ¼ 7.3 Hz, 18H), 1.16 (t, J ¼ 7.5 Hz, 3H), 1.26e1.33 (m, 3H), 2.60 (q,
J ¼ 7.5 Hz, 2H), 6.67 (d, J ¼ 8.3 Hz, 1H), 7.16 (dd, J ¼ 2.0, 8.4 Hz, 1H),
7.28 (d, J ¼ 2.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d
0.10, 13.04,
4.1.5.1. 2-((4-((2-Methoxyethoxy)methoxy)phenyl)ethynyl)quinoline
18.01, 23.39, 91.93, 105.75, 115.04, 117.78, 130.62, 133.10, 134.48,
154.47.
(6a). Yield: 97%, 1H NMR (300 MHz, CDCl3)
d 3.37 (s, 3H), 3.55e3.58
(m, 2H), 3.82e3.85 (m, 2H), 5.28 (s, 2H), 7.05 (d, J ¼ 8.8 Hz, 1H),
7.51e7.62 (m, 4H), 7.70e7.76 (m, 1H), 7.79e7.82 (m, 1H), 8.11e8.15
4.1.3.5. (3-Isopropyl-4-(2-methoxy-ethoxymethoxy)phenylethynyl)
(m, 1H); 13C NMR (75 MHz, CDCl3)
d 59.01, 67.83, 71.54, 88.55,
trimethylsilane (3e). Yield: 61%, 1H NMR (300 MHz, CDCl3)
d
0.25 (s,
90.08, 93.24, 115.24, 116.22, 124.30, 126.93, 126.99, 127.46, 129.26,
129.98, 133.77, 136.04, 143.86, 148.22, 157.94.
9H), 1.10 (d, J ¼ 7.2 Hz, 18H), 1.19 (d, J ¼ 6.9 Hz, 6H), 1.27 (m,
J ¼ 6.9 Hz, 3H), 3.33 (m, 1H), 6.67 (d, J ¼ 8.4 Hz, 1H), 7.15 (dd, J ¼ 1.8,
8.4 Hz, 1H), 7.32 (d, J ¼ 1.8 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
4.1.5.2. 2-((4-((2-Methoxyethoxy)methoxy)3,5-dimethylphenyl)ethy-
d
0.11, 13.08, 18.03, 22.62, 26.58, 115.08, 117.78, 117.80, 130.28,
nyl)quinoline (6b). Yield: 92%, 1H NMR (300 MHz, CDCl3)
d 2.26 (s,
130.39, 130.44, 138.74, 153.82.
6H), 3.40 (s, 3H), 3.60e3.63 (m, 2H), 3.94e3.97 (m, 2H), 5.09 (s, 2H),
7.35 (s, 2H), 7.51e7.59 (m, 2H), 7.70e7.75 (m, 1H), 7.79 (d, J ¼ 8.0 Hz,
4.1.4. General procedure for the synthesis of terminal alkynes 4a-4e
To a solution of compounds 3a-e (1.0 equiv.) in MeOH was added
K2CO3 (1.0 equiv.) at 0 ꢀC and stirred for 1 h. The reaction mixture
was stirred for additional 2.5e5 h at room temperature. The solu-
tion was diluted with EtOAc. The solution was washed with water
and brine, and dried with Na2SO4 and concentrated. The crude
material was purified by column chromatography (silica gel, Hex-
ane/EtOAc or Hexane only).
1H), 8.11 (d, J ¼ 8.4 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 16.82, 59.10,
69.21, 71.77, 88.61, 90.08, 97.98, 117.67, 124.33, 126.95, 127.02,
127.47, 129.30, 129.97, 131.41, 132.86, 136.04, 143.83, 148.24, 155.83.
4.1.5.3. 2-((3,5-Diethyl-4-(triisopropylsilyloxy)phenyl)ethynyl)quino-
line (6c). Yield: 60%, 1H NMR (300 MHz, CDCl3)
d
1.11 (d, J ¼ 7.3 Hz,
18H),1.21 (t, J ¼ 7.5 Hz, 6H),1.31 (m, 3H), 2.63 (q, J ¼ 7.5 Hz, 4H), 7.37
(s, 2H), 7.54 (dd, J ¼ 8.1 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.70 (dd,
J ¼ 8.4 Hz, 1H), 7.79 (d, J ¼ 8.1 Hz, 1H), 8.12 (dd, J ¼ 3.3, 8.4 Hz, 2H);
4.1.4.1. 1-Ethynyl-4-(2-methoxy-ethoxymethoxy)-benzene
(4a).
13C NMR (75 MHz, CDCl3)
d 14.09, 14.37, 18.05, 23.30, 88.17, 91.35,
Yield: 74%, 1H NMR (300 MHz, CDCl3)
d
3.01 (s, 1H), 3.38 (s, 3H),
114.56, 124.32, 126.76, 126.98, 127.45, 129.34, 129.87, 130.65, 134.40,
135.90, 144.24, 148.37, 153.81.
3.54e3.57 (m, 2H), 3.81e3.84 (m, 2H), 6.99 (d, J ¼ 8.9 Hz, 2H), 7.41
(d, J ¼ 8.8 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 58.93, 67.72, 71.50,
76.00, 83.44, 93.20, 115.33, 116.08, 133.48, 157.52.
4.1.5.4. 2-((3-Ethyl-4-(triisopropylsilyloxy)phenyl)ethynyl)quinoline
(6d). Yield: 78%, 1H NMR (300 MHz, CDCl3)
d
1.13 (d, J ¼ 7.2 Hz,
4.1.4.2. 5-Ethynyl-2-((2-methoxyethoxy)methoxy)-1,3-
18H), 1.21 (t, J ¼ 7.5 Hz, 3H), 1.30 (m, 3H), 2.63 (q, J ¼ 7.5 Hz, 2H),
6.77 (d, J ¼ 8.4 Hz, 1H), 7.37 (dd, J ¼ 2.2, 8.2 Hz, 1H), 7.5e7.56 (m,
2H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.73 (dd, J ¼ 7.8 Hz, 1H), 7.79 (d,
J ¼ 8.2 Hz, 1H), 8.12 (d, J ¼ 8.4 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
dimethylbenzene (4b). Yield: 72%, 1H NMR (300 MHz, CDCl3)
d
2.27
(s, 6H), 2.99 (s, 1H), 3.40 (s, 3H), 3.59e3.62 (m, 2H), 3.93e3.96 (m,
2H), 5.05 (s, 2H), 7.17 (s, 2 H); 13C NMR (75 MHz, CDCl3)
16.64,
d

784
B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
d
13.10, 13.93, 18.03, 23.37, 88.26, 91.06, 114.10, 118.00, 124.37,
d
6.84 (d, J ¼ 8.8 Hz, 2H), 7.50 (d, J ¼ 8.7 Hz, 2H), 7.60e7.69 (m, 2H),
126.78, 126.99, 127.46, 129.33, 1219.89, 131.01, 133.47, 134.77,
135.92, 144.22, 148.34, 155.17.
7.77e7.83 (m, 1H), 7.98 (d, J ¼ 9.5 Hz, 2H), 8.38 (d, J ¼ 8.4 Hz, 1H),
10.10 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d 87.65, 91.39, 111.07,
116.02, 124.25, 126.72, 127.28, 127.85, 128.01, 130.64, 133.86, 137.21,
142.79, 146.98, 159.11; HRMS (ESþ) m/z calcd for C17H12NO (MHþ)
246.0919, found 246.0908; Purity: 94%.
4.1.5.5. 2-((3-Isopropyl-4-(triisopropylsilyloxy)phenyl)ethynyl)quin-
oline (6e). Yield: 60%, 1H NMR (300 MHz, CDCl3)
d
1.13 (d, J ¼ 7.3 Hz,
18H), 1.23 (d, J ¼ 6.9 Hz, 6H), 1.33 (m, 3H), 3.37 (q, J ¼ 6.9 Hz, 1H),
6.76 (d, J ¼ 8.3 Hz, 1H), 7.35 (dd, J ¼ 2.2, 8.2 Hz, 1H), 7.55 (m, 2H),
7.59 (d, J ¼ 8.5 Hz, 1H), 7.73 (dd, J ¼ 8.0 Hz, 1H), 7.79 (d, J ¼ 7.9 Hz,
4.1.6.2. 2,6-Dimethyl-4-(quinolin-2-ylethynyl)phenol
(7b).
Compound 7b was prepared according to the general procedure for
MEM deprotection reaction. The residue was subjected to reverse
column chromatography over C18 (mobile phase A ¼ 0.1% TFA in
H2O, mobile phase B ¼ 0.1% TFA in MeCN). Linear gradients were
run from 65%: 35%e30%: 70% (A:B) to give 7b (97%). 1H NMR
1H), 8.12 (d, J ¼ 8.4 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 13.08,18.05,
22.69, 26.64, 88.12, 91.20, 114.01, 117.96, 124.35, 126.79, 126.94,
127.46, 129.26, 129.92, 130.69, 130.89, 135.97, 139.05, 144.15, 148.25,
154.48.
(300 MHz, DMSO-d6)
7.78e7.83 (m, 1H), 7.98 (d, J ¼ 8.6 Hz, 2H), 8.38 (d, J ¼ 8.4 Hz, 1H),
8.94 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
16.33, 87.82, 90.73,
d 2.20 (s, 6H), 7.28 (s, 2H), 7.60e7.67 (m, 2H),
4.1.5.6. 6-Methoxy-2-((4-((2-methoxyethoxy)methoxy)-3,5-
dimethylphenyl)ethynyl)quinoline (6f). Yield: 98%, 1H NMR
d
(300 MHz, CDCl3)
d
2.29 (s, 6H), 3.40 (s, 3H), 3.59e3.62 (m, 2H),
111.25, 124.13, 124.92, 126.61, 127.03, 127.89, 128.38, 130.25, 132.13,
136.54, 143.22, 147.60, 155.10; HRMS (ESþ) m/z calcd for C19H16NO
(MHþ) 274.1232, found 274.1220; Purity: 99%.
3.93e3.96 (m, 5H), 5.08 (s, 2H), 7.04 (d, J ¼ 2.8 Hz, 1H), 7.33 (s, 2H),
7.35e7.39 (dd, J ¼ 9.3, 2.8 Hz, 1H), 7.51 (d, J ¼ 8.5 Hz, 1H), 7.98 (dd,
J ¼ 8.4, 3.0 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 16.78, 53.37, 55.53,
59.06, 69.17, 71.75, 88.63, 89.22, 97.94, 104.98, 117.88, 122.72,
124.63, 128.12, 130.75, 131.32, 132.71, 134.67, 141.18, 144.37, 155.62,
158.16.
4.1.6.3. 2,6-Diethyl-4-(quinolin-2-ylethynyl)phenol
(7c).
Compound 7c was prepared according to the general procedure for
TIPS deprotection reaction. The residue was subjected to chroma-
tography (silica gel, Hexane/EtOAc ¼ 5:1) to provide 7c (64%). 1H
4.1.5.7. 2-((4-((2-Methoxyethoxy)methoxy)-3,5-dimethylphenyl)
NMR (300 MHz, CDCl3)
d
1.29 (t, J ¼ 7.5 Hz, 6H), 2.61 (q, J ¼ 7.5 Hz,
ethynyl)quinolin-6-ol (6g). Yield: 49%, 1H NMR (300 MHz, CDCl3)
4H), 5.02 (s, 1H), 7.37 (s, 2H), 7.52 (dd, J ¼ 7.5 Hz, 1H), 7.58 (d,
J ¼ 8.4 Hz, 1H), 7.71 (dd, J ¼ 7.6 Hz, 1H), 7.80 (d, J ¼ 7.9 Hz, 1H), 8.12
d
2.22 (s, 6H), 3.40 (s, 3H), 3.60e3.62 (m, 2H), 3.92e3.95 (m, 2H),
5.05 (s, 2H), 7.18 (s, 3H), 7.38 (dd, J ¼ 9.1, 2.7 Hz, 1H), 7.49 (d,
(d, J ¼ 8.5 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
d 14.10, 22.68,
J ¼ 8.4 Hz, 1H), 7.92 (d, J ¼ 8.5 Hz, 1H), 8.02 (d, J ¼ 9.1 Hz, 1H); 13
C
87.91, 90.85, 111.68, 124.16, 126.65, 127.05, 127.92, 128.45, 130.27,
130.49, 131.12, 136.51, 143.30, 147.70, 154.07; HRMS (ESþ) m/z calcd
for C21H20NO (MHþ) 302.1545, found 302.1531; Purity: 99%.
NMR (75 MHz, CDCl3)
d 16.72, 59.09, 69.15, 71.74, 87.74, 88.23,
90.15, 97, 91, 109.13, 115.84, 117.57, 122.93, 124.57, 128.56, 130.15,
131.31, 132.66, 134.97, 143.40, 155.66.
4.1.6.4. 2-Ethyl-4-(quinolin-2-ylethynyl)phenol (7d).
4.1.5.8. 2-((3,5-Dimethyl-4-(triisopropylsilyloxy)phenyl)ethynyl)-6-
Compound 7d was prepared according to the general procedure for
TIPS deprotection reaction. The residue was subjected to chroma-
tography (silica gel, Hexane/EtOAc ¼ 3:1) to provide 7d (40%). 1H
nitroquinoline (6h). Yield: 40%, 1H NMR (300 MHz, CDCl3)
d 1.13 (d,
J ¼ 7.2 Hz, 18H), 1.32 (m, 3H), 2.32 (s, 6H), 7.36 (s, 2H), 7.63 (d,
J ¼ 4.7 Hz, 1H), 8.22 (dd, J ¼ 9.1, 2.2 Hz, 1H), 8.49 (d, J ¼ 9.2 Hz, 1H),
9.01 (dd, J ¼ 4.5, 2.5 Hz, 1H), 9.31 (s, 1H); 13C NMR (75 MHz, CDCl3)
NMR (300 MHz, CDCl3)
d
1.13 (t, J ¼ 7.5 Hz, 3H), 2.55 (m, 2H), 6.85
(d, J ¼ 8.3 Hz, 1H), 7.33e7.40 (m, 2H), 7.60e7.68 (m, 2H), 7.77 (dd,
d
14.24, 17.75, 17.96, 82.73, 102.62, 113.40, 123.25, 123.27, 124.44,
J ¼ 8.4 Hz, 1H), 7.98 (d, J ¼ 8.3 Hz, 1H), 8.97 (d, J ¼ 8.3 Hz, 2H); 13
C
126.87, 129.06, 131.66, 132.79, 132.95, 145.79, 150.16, 153.15, 155.78.
NMR (75 MHz, DMSO-d6) d 13.76, 22.30, 89.84, 90.64, 111.17, 115.25,
124.05, 126.56, 126.92, 127.81, 128.37, 130.13, 130.75 130.93, 132.76,
136.37, 143.23, 147.63, 156.67; HRMS (ESþ) m/z calcd for C19H16NO
(MHþ) 274.1232, found 274.1219; Purity: 99%.
4.1.6. General procedure of MEM or TIPS deprotection
MEM deprotection: To a solution of MEM protected compound
(1.0 equiv.) in MeOH and THF (1:1) was slowly added a catalytic
amount of concentrated HCl. The solution was stirred for 4e20 h at
room temperature. The solution was diluted with EtOAc. The
organic layer was washed with water and brine. The solution was
dried with Na2SO4. The crude material was purified by reverse
column chromatography over C 18 (mobile phase A ¼ 0.1% TFA in
H2O, mobile phase B ¼ 0.1% TFA in MeCN).
4.1.6.5. 2-Isopropyl-4-(quinolin-2-ylethynyl)phenol
(7e).
Compound 7e was prepared according to the general procedure for
TIPS deprotection reaction. The residue was subjected to chroma-
tography (silica gel, Hexane/EtOAc ¼ 3:1) to provide 7e (97%). 1
H
NMR (300 MHz, CDCl3)
d
1.18 (d, J ¼ 6.9 Hz, 6H), 3.19 (q, J ¼ 6.8 Hz,
1H), 6.86 (d, J ¼ 8.3 Hz, 1H), 7.32 (dd, J ¼ 2.1, 8.2 Hz, 1H), 7.41 (d,
J ¼ 2.1 Hz, 1H), 7.62 (dd, J ¼ 6.8 Hz, 1H), 7.67 (d, J ¼ 8.5 Hz, 1H),
7.77e7.83 (m, 1H), 7.98 (d, J ¼ 8.4 Hz, 2H), 8.38 (d, J ¼ 8.5 Hz, 1H);
TIPS Deprotection: To a solution of TIPS protected compound (1.0
equiv.) in THF was added TBAF (tetra-n-butylammonium fluoride,
1 M in THF, 1.7 equiv.).The solution was stirred for 1e2.5 h. The
solution was quenched with sat. NH4Cl. The solution was diluted
with EtOAc and washed with water and brine. The solution was
dried with Na2SO4. The solution was filtered and concentrated. The
crude material was purified by column chromatography (silica gel,
Hexane/EtOAc).
13C NMR (75 MHz, DMSO-d6)
d 22.16, 26.20, 89.81, 90.80, 111.26,
115.45, 124.07, 126.57, 126.93, 127.81, 128.36, 130.05, 130.14, 130.68,
135.09, 136.3.7, 143.24, 147.64, 156.10; HRMS (ESþ) m/z calcd for
C
20H18NO (MHþ) 288.1388, found 288.1376; Purity: 99%.
4.1.6.6. 4-((6-Methoxyquinolin-2-yl)ethynyl)-2,6-dimethylphenol
(7f). Compound 7f was prepared according to the general pro-
cedure for MEM deprotection reaction. The residue was subjected
to reverse column chromatography over C18 (mobile phase
A ¼ 0.1% TFA in H2O, mobile phase B ¼ 0.1% TFA in MeCN). Linear
gradients were run from 85%: 15%e45%: 55% (A:B) to give 7f (86%).
4.1.6.1. 4-(Quinolin-2-ylethynyl)phenol (7a). Compound 7a was
prepared according to the general procedure for MEM deprotection
reaction. The residue was subjected to reverse column chroma-
tography over C18 (mobile phase A ¼ 0.1% TFA in H2O, mobile phase
B ¼ 0.1% TFA in MeCN). Linear gradients were run from 95%: 5%e
60%: 40% (A:B) to give 11a (95%). 1H NMR (300 MHz, DMSO-d6)
1H NMR (300 MHz, DMSO-d6)
2H), 7.42 (d, J ¼ 2.6 Hz, 1H), 7.45 (dd, J ¼ 9.2, 2.8 Hz, 1H), 7.62 (d,
d 2.20 (s, 6H), 3.92 (s, 3H), 7.26 (s,

B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
785
J ¼ 8.5 Hz,1H), 7.90 (d, J ¼ 9.0 Hz,1H), 8.31 (d, J ¼ 8.5 Hz,1H), 8.93 (s,
1H); 13C NMR (75 MHz, DMSO-d6)
16.38, 55.70, 87.04, 91.68,
4.1.7.2. (E)-2-(4-((2-Methoxyethoxy)methoxy)-3,5-dimethylstyryl)-
d
6-nitro quinoline (9b). Yield: 64%, 1H NMR (300 MHz, DMSO-d6)
105.89, 111.19, 123.41, 124.54, 124.99, 128.08, 128.87, 132.13, 136.37,
139.73, 142.44, 155.22, 157.94; LC/MS (ESþ) m/z 303.6 (Mþ); Purity:
98%.
d 2.31 (s, 6H), 3.27 (s, 3H), 3.50e3.53 (m, 2H), 3.84e3.87 (m, 2H),
5.08 (s, 2H), 7.56e7.61 (m, 3H), 7.99 (d, J ¼ 4.7 Hz, 1H), 8.05 (d,
J ¼ 16 Hz, 1H), 8.21 (d, J ¼ 9.3 Hz, 1H), 8.48 (dd, J ¼ 2.5, 9.3 Hz, 2H),
9.05 (d, J ¼ 4.7 Hz, 1H), 9.36 (d, J ¼ 2.4, 1H); 13C NMR (75 MHz,
4.1.6.7. 2-((4-Hydroxy-3,5-dimethylphenyl)ethynyl)quinolin-6-ol
(7g). Compound 7g was prepared according to the general pro-
cedure for MEM deprotection reaction. The residue was subjected
to reverse column chromatography over C18 (mobile phase
A ¼ 0.1% TFA in H2O, mobile phase B ¼ 0.1% TFA in MeCN). Linear
gradients were run from 95%: 5%e20%: 80% (A:B) to give 7g (83%).
DMSO-d6) d 16.52, 58.04, 68.51, 71.11, 97.42, 117.99, 120.15, 121.23,
122.58, 124.70, 128.25, 130.94, 131.40, 131.73, 136.58, 144.95, 145.07,
150.29, 153.59, 155.36.
4.1.7.3. (E)-2,6-Dimethyl-4-(2-(quinolin-2-yl)vinyl)phenol
(10a).
Compound 10a was prepared according to the general procedure
1H NMR (300 MHz, DMSO-d6)
d
2.19 (s, 6H), 7.22e7.25 (m, 3H), 7.40
for MEM deprotection reaction. Yield: 73%. 1H NMR (300 MHz,
(dd, J ¼ 9.1, 2.5 Hz, 1H), 7.61 (d, J ¼ 8.6 Hz, 1H), 7.87 (d, J ¼ 9.1 Hz,
DMSO-d6)
d
2.22 (s, 6H), 7.21 (d, J ¼ 16 Hz, 1H), 7.31 (s, 2H), 7.49 (dd,
1H), 8.30 (d, J ¼ 8.7 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d
16.40,
J ¼ 7.4 Hz, 1H), 7.64 (d, J ¼ 16 Hz, 1H), 7.72e7.79 (m, 2H), 7.89e7.96
86.57, 92.55, 108.63, 111.07, 123.82, 124.33, 125.03, 128.27, 128.48,
132.18,136.56,138.36,140.73,155.34,156.67; HRMS (ESþ) m/z calcd
for C19H16NO2 (MHþ) 290.1181, found 290.1169; Purity: 99%.
(m, 2H), 8.27 (d, J ¼ 8.7 Hz, 1H), 8.60 (s, 1H). 13C NMR (75 MHz,
DMSO-d6)
d 17.07, 120.18, 125.08, 125.82, 126.23, 127.29, 127.83,
128.10, 128.20, 129.00, 130.13, 134.96, 136.71, 148.22, 154.84, 156.67;
HRMS (ESþ) m/z calcd for C19H18NO (MHþ) 276.1388, found
276.1377; Purity: 98%.
4.1.6.8. 2,6-Dimethyl-4-((6-nitroquinolin-2-yl)ethynyl)phenol (7h).
Compound 7h was prepared according to the general procedure for
MEM deprotection reaction. The residue was subjected to chro-
matography over silica gel (Hexane/EtOAc ¼ 3:1) to give 7h (75%).
4.1.7.4. (E)-2,6-Dimethyl-4-(2-(6-nitroquinolin-2-yl)vinyl)phenol
(10b). Compound 10b was prepared according to the general pro-
cedure for MEM deprotection reaction. Yield: 63%. 1H NMR
1H NMR (300 MHz, DMSO-d6)
d 2.23 (s, 6H), 7.36 (s, 2H), 7.85 (m,
1H), 8.28 (d, J ¼ 8.9 Hz, 1H), 8.53 (d, J ¼ 9.2 Hz, 1H), 9.11 (s, 2H); 13
C
(300 MHz, DMSO-d6)
d
2.24 (s, 6H), 7.48 (s, 2H), 7.52 (d, J ¼ 16 Hz,
NMR (75 MHz, DMSO-d6)
d
16.37, 82.19, 102.31, 110.71, 122.07,
1H), 7.90 (m, 2H), 8.19 (d, J ¼ 9.3 Hz, 1H), 8.45 (dd, J ¼ 2.4, 9.3 Hz,
123.31, 124.56, 125.08, 125.73, 131.19, 131.59, 132.20, 145.43, 149.43,
153.77, 155.73; HRMS (ESþ) m/z calcd for C19H15N2O3 (MHþ)
319.1083, found 319.1070; Purity: 95%.
1H), 9.01 (d, J ¼ 4.8 Hz, 1H), 9.35 (d, J ¼ 2.3 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6)
d 16.40, 117.40, 117.49, 121.17, 122.44, 124.47,
124.65, 127.19, 128.18, 131.33, 137.35, 144.94, 145.33, 150.35, 153.45,
154.70; HRMS (ESþ) m/z calcd for C19H16N2O3 (MHþ) 321.1239,
found 321.1226; Purity: 98%.
4.1.6.9. 4-((6-Aminoquinolin-2-yl)ethynyl)-2,6-dimethylphenol (7i).
To a suspension of 7h (38 mg, 0.119 mmol) in AcOH and HCl (1/
0.1 mL, v/v) was added Sn powder (56.7 mg, 0.477 mmol) and
stirred for 1 h. The mixture was diluted with EtOAc and neutralized
by addition of saturated NaHCO3. The solution was washed with
saturated brine and dried with Na2SO4. The solution was filtered
and concentrated. The residue was subjected to chromatography
over silica gel (Hexane/EtOAc ¼ 1:2) to give 7i (67%). 1H NMR
4.1.8. (E)-4-(2-(6-Aminoquinolin-2-yl)vinyl)-2,6-dimethyl phenol
(10c)
To a solution of 10b (150 mg, 0.468 mmol) in EtOH (0.1 M) was
added Tin (II) chloride dehydrate (SnCl2$2H2O) (518 mg,
2.295 mmol) and stirred for 9 h at room temperature. The mixture
was concentrated and diluted with EtOAc. The organic layer was
washed water and brine, and dried with Na2SO4. The solution was
filtered and concentrated. The crude material was purified by col-
umn chromatography (silica gel, Hexane:EtOAc ¼ 1:1) to provide
(300 MHz, DMSO-d6)
d 2.21 (s, 3H), 5.89 (s, 2H), 7.18e7.24 (m, 2H),
7.30 (s, 2H), 7.37 (dd, J ¼ 4.5, 1.6 Hz, 1H), 7.71 (d, J ¼ 8.9 Hz, 1H), 8.42
(dd, J ¼ 4.4, 1.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d 16.41, 83.93,
98.65, 102.64, 111.93, 121.95, 122.85, 124.92, 125.21, 129.17, 130.35,
10c (83.4 mg, 61%). 1H NMR (300 MHz, DMSO-d6)
d 2.22 (s, 6H), 5.61
131.94, 141.86, 144.13, 148.04, 154.94; HRMS (ESþ) m/z calcd for
(s, 2H), 7.14 (dd, J ¼ 2.4, 8.9 Hz, 2H), 7.23e7.31 (m, 4H), 7.49e7.55
C
19H17N2O (MHþ) 289.1341, found 289.1328; Purity: 96%.
(m, 2H), 7.68 (d, J ¼ 8.9 Hz, 1H), 8.42 (d, J ¼ 4.7 Hz, 1H), 8.55 (s, 1H);
13C NMR (75 MHz, DMSO-d6)
d 16.63, 101.14, 116.12, 119.51, 121.21,
4.1.7. General procedure of the Heck coupling reaction for
compounds 9a-b
124.47, 127.29, 127.67, 127.80, 130.24, 133.43, 139.13, 142.58, 144.67,
147.03, 153.98; HRMS (ESþ) m/z calcd for C19H19N2O (MHþ)
291.1497, found 291.1484; Purity: 96%.
To a solution of substituted quinolines (5a-b) (1.0e1.5 equiv.),
compound 8 (1.0 equiv.) in degassed 1,4-dioxane were added
Pd2(dba)3 (0.1e0.2 equiv.), t-butylXphos (0.1e0.2 equiv.), and
Cy2NMe (2.0 equiv.). The solution was stirred under N2 at 120 ꢀC for
18e40 h. The mixture was filtered through Celite and washed with
EtOAc. The solution was washed with water and brine, and dried
with Na2SO4. The crude material was purified by column chroma-
tography (silica gel, Hexane/EtOAc).
4.1.9. General procedure of Horner-Wadsworth-Emmons reaction
for compounds 13a-b
To a solution of compound 12 (1.5e2.0 equiv.) in dry DMF
(0.1e0.2 M) was added t-BuOK (1.5e2.0 equiv.) at 0 ꢀC. The reaction
solution was stirred for 10 min. Compound 11a-b was added to the
reaction solution at 0 ꢀC. The solution was stirred for 1e6 h at 60 ꢀC.
The solution was diluted with EtOAc. The organic layer was washed
with water and brine. The solution was filtered and concentrated.
The crude material was purified by column chromatography (silica
gel, Hexane:EtOAc).
4.1.7.1. (E)-2-(4-((2-Methoxyethoxy)methoxy)-3,5-dimethylstyryl)
quinoline (9a). Yield: 24%, 1H NMR (300 MHz, DMSO-d6)
d 2.28 (s,
6H), 3.26 (s, 3H), 3.50e3.53 (m, 2H), 3.84e3.87 (m, 2H), 5.04 (s, 2H),
7.35 (d, J ¼ 16.3 Hz,1H), 7.44 (s, 2H), 7.52 (t, J ¼ 7.8 Hz,1H), 7.71e7.76
(m, 2H), 7.82 (d, J ¼ 8.7 Hz, 1H), 7.92 (t, J ¼ 9 Hz, 2H), 8.32 (d,
4.1.9.1. (E)-2-Chloro-3-(4-((2-methoxyethoxy)methoxy)-3,5-
J ¼ 8.5 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d
13.96, 16.53, 20.62,
dimethylstyryl)quinoline (13a). Yield:, 43%, 1H NMR (300 MHz,
58.04, 68.47, 71.12, 97.38, 99.40, 119.74, 125.91, 126.85, 127.62,
127.65, 128.52, 129.64, 130.95, 131.86, 133.56, 136.29, 147.59, 154.96,
155.66.
CDCl3) d 2.36 (s, 6H), 3.43 (s, 3H), 3.62e3.65 (m, 2H), 3.96e4.00 (m,
2H), 5.10 (s, 2H), 7.09 (d, J ¼ 16 Hz, 1H), 7.40 (d, J ¼ 16 Hz, 1H),
7.54e7.59 (m, 1H), 7.68e7.73 (m, 1H), 7.84 (d, J ¼ 8.1 Hz,1H), 7.99 (d,

786
B. Kim et al. / European Journal of Medicinal Chemistry 123 (2016) 777e787
J ¼ 8.2 Hz, 1H), 8.36 (s, 1H); 13C NMR (75 MHz, CDCl3)
d
17.02, 59.12,
CHARMm force field [45]. The designed molecules were docked
into the binding site of the TTR by the LibDock [46] protocol of
Discovery Studio 4.5.
89.18, 71.80, 77.20, 89.01, 122.46, 127.21, 127.47, 127.55, 127.57,
128.29, 130.06, 130.53, 131.49, 132.48, 132.85, 133.41, 146.70, 155.23.
4.1.9.2. (E)-2-Chloro-7-methoxy-3-(4-((2-methoxyethoxy)methoxy)-
4.3. Rat pharmacokinetics
3,5-dimethylstyryl)quinoline (13b). Yield: 55%, 1H NMR (300 MHz,,
DMSO-d6) d 2.27 (s, 6H), 3.26 (s, 3H), 3.48e3.52 (m, 2H), 3.83e3.86
Eighteen male SD rats (Orient Bio Inc., Seongnam, Korea)
weighing 164e181 g for 7i and 174e194 g for 14c were used. Animal
room was controlled for illumination (12-h light/dark cycle), tem-
perature (20e25 ꢀC) and relative humidity (>40%). The Institutional
Animal Care and Use Committee of the Chungnam National Uni-
versity (Korea) approved the study protocol (license number: CNU-
00576).
(m,2H), 3.93 (s, 3H), 5.04 (s, 2H), 7.25e7.38 (m, 6H), 7.03 (d,
J ¼ 9.0 Hz,1H), 8.73 (s, 1H); 13C NMR (75 MHz, CDCl3)
d 16.63, 55.68,
58.13, 68.54, 71.19, 97.46, 106.33, 120.22, 121.67, 122.51, 127.17,
127.20, 129.21, 131.09, 132.04, 132.19, 134.13, 147.92, 149.35, 154.81,
161.14.
4.1.9.3. (E)-4-(2-(2-Chloroquinolin-3-yl)vinyl)-2,6-dimethyl phenol
(14a). Compound 14a was prepared according to the general pro-
cedure for MEM deprotection reaction. The residue was subjected
to chromatography (silica gel, Hexane/EtOAc ¼ 5:1) to provide 14a
After overnight fasting, four rats were received an intravenous
(IV) dose via tail vein at 2 mg/kg of 7i or 14c and five rats were
received an oral using gavage needle at 5 mg/kg of 7i or 14c. Dosing
solution was prepared by dissolving 7i or 14c in mixture of 10%
DMSO, 40% PEG400 and 50% saline at a concentration of 1 and
2.5 mg/mL for IV and oral, respectively. Blood samples (0.3 mL)
were collected from the jugular vein at 0.083 (IV only), 0.25, 0.5, 1,
3, 7, 10 and 24 h after administration.
(68%). 1H NMR (300 MHz, MDSO-d6)
d 2.21 (s, 6H), 7.22e7.36 (m,
4H), 7.62e7.68 (m, 1H), 7.74e7.79 (m, 1H), 7.91 (d, J ¼ 8.3 Hz, 1H),
8.02 (d, J ¼ 7.3 Hz, 1H), 8.79 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
16.64, 118.91, 124.62, 127.29, 127.44, 127.48, 127.49, 127.53, 127.96,
130.20, 130.28, 133.71, 134.01, 145.78, 149.18, 154.26; HRMS (ESþ)
m/z calcd for C19H16ClNO (MHþ) 310.0999, found 310.0987; Purity:
99%.
An aliquot (50
pine and 600 ng/mL chlorzoxazone in acetonitrile for 7i and 14c,
respectively) and 400 L of acetonitrile were added to an aliquot
(50 L) of plasma to induce precipitation of plasma proteins. This
solution was vigorously mixed for 10 min, followed by centrifuga-
tion at 12,000 g for 10 min. An aliquot (5 L) of supernatant was
mL) of internal standard (100 ng/mL carbamaze-
m
m
4.1.9.4. (E)-4-(2-(2-Chloro-7-methoxyquinolin-3-yl)vinyl)-2,6-
dimethylphenol (14b). Compound 14b was prepared according to
the general procedure for MEM deprotection reaction. The residue
was subjected to chromatography (silica gel, Hexane/EtOAc ¼ 4:1)
m
injected into the HPLC/MS/MS system (Agilent 1200 HPLC, Agilent
Technologies) which had an API 4000 tandem quadrupole mass
spectrometer (Applied Biosystems).
to provide 14b (84%). 1H NMR (300 MHz, DMSO-d6)
d 2.21 (s, 6H),
Compounds were separated on Agilent Eclipse Plus® C18 col-
3.92 (s, 3H), 7.22 (s, 3H), 7.27e7.33 (m, 2H), 7.91 (d, J ¼ 8.9 Hz, 1H),
8.69 (s, 1H); 13C NMR (75 MHz, CDCl3)
d
16.61, 55.64, 106.29, 119.10,
umn (3.5
m
m, 2.1 ꢂ 50 mm) with a gradient of 0.1% formic acid (A)
120.13, 122.59, 124.56, 127.06, 127.57, 127.59, 129.08, 132.73, 133.55,
147.65, 149.32, 154.00, 160.93; LC/MS (ESþ) m/z 339.6 (Mþ); Purity:
94%.
and 0.1% formic acid in acetonitrile (B). For 7i, the portion of (B) was
maintained 50% until 5.0 min. For 14c, the portion of (B) was
maintained 5% until 1.0 min, increased linearly to 95% from 1.0 to
1.5 min, and maintained until 2.5 min. After that, the portion of (B)
decreased linearly to 5% from 2.5 to 3.0 min, and maintained until
5 min. A constant flow rate was 0.3 mL/min. A positive multiple
reaction monitoring (MRM) mode was used for quantification at m/
z 289.236 to 273.300 and 237.198 to 194.200 for 7i and carba-
mazepine, respectively. A negative MRM mode was used for
quantification at m/z 324.115 to 272.900 and 167.958 to 131.900 for
14c and chlorzoxazone, respectively. The quantifiable range was
from 3 to 3000 ng/mL for 7i and 14c. The retention times of 7i,
carbamazepine, 14c and chlorzoxazone were 3.27, 3.29, 1.84 and
0.77 min, respectively.
4.1.10. (E)-2-Chloro-3-(4-hydroxy-3,5-dimethylstyryl) quinolin-7-
ol (14c)
To solution of 14b (20 mg, 0.588 mmol) in DCM was added
dropwise BBr3 (20
m
L, 0.206 mmol) at ꢁ78 ꢀC. The solution was
stirred for 1 h, allowed to r.t. and stirred for 26 h. The solution was
quenched with water and diluted with EtOAc. The organic layer was
washed with water and brine, and dried with Na2SO4. The crude
material was purified by column chromatography (silica gel, Hex-
ane/EtOAc ¼ 4:1) to provide 14c (10.3 mg, 54%). 1H NMR (300 MHz,
DMSO-d6)
d
2.20 (s, 6H), 7.12e7.21 (m, 6H), 7.85 (d, J ¼ 8.9 Hz, 1H),
8.52 (s, 1H), 8.63 (s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
16.49,
The plasma concentration-time profiles were analyzed using
moment analysis with WinNonlin 4.1 (Pharsight Corporation).
108.84, 119.30, 120.04, 121.70, 124.53, 126.66, 126.91, 127.67, 129.27,
132.16, 133.51, 147.72, 149.21, 153.82, 159.40; HRMS (ESþ) m/z calcd
for C19H17ClNO2 (MHþ) 326.0948, found 326.0934; Purity: 99%.
Acknowledgements
4.2. In silico docking study
We are grateful for support from the DRC program funded by the
National Research Council of Science & Technology (DRC-15-01-
KRICT) and Basic Science Research Program through the National
Research Foundation of Korea funded by the Ministry of Education,
Science and Technology (2015R1D1A3A01020384).
We carried out a docking study of selected molecules using the
released crystal structure complex (PDB ID: 2ROX) [44] of TTR from
the RCSB protein databank (www.rcsb.org). The protein was sub-
jected to the “Prepare protein” [45] process using the CHARMm
force field and default conditions. Also we built homo-dimer TTR by
the homology modeling method of Discovery Studio 4.5 using the
symmetry matrix to add other subunits. The prepared homo-dimer
structure was then defined as a receptor for the docking calculation.
The binding site of TTR was defined as a 15.0 Å sphere around the
original ligand of the 2ROX structure. In order to prepare the input
molecules, each conformation of the designed molecules was ob-
tained through energy minimization calculations under the
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.003.
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