Orally active ghrelin receptor inverse agonists and their actions on a rat obesity model

Article abstract of DOI:10.1016/j.bmc.2015.05.047

A series of 2-alkylamino nicotinamide analogs was prepared as orally active ghrelin receptor (ghrelinR) inverse agonists. Starting from compound 1, oral bioavailability was improved by modifying metabolically unstable sites and reducing molecular weight. Brain-permeable compound 33 and compound 24 with low brain permeability were tested in rat models of obesity; 30 mg/kg of compound 33 suppressed weight gain. PK/PD analysis revealed that the anti-obesity effect of ghrelinR inverse agonists depends on their brain concentrations.

Full text of DOI:10.1016/j.bmc.2015.05.047

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Orally active ghrelin receptor inverse agonists and their actions
on a rat obesity model
⇑
Bitoku Takahashi , Hideaki Funami, Takehiko Iwaki, Hiroshi Maruoka, Makoto Shibata, Makoto Koyama,
Asako Nagahira, Yoshiyuki Kamiide, Satomi Kanki, Yoshiyuki Igawa, Tsuyoshi Muto
Asubio Pharma Co., Ltd, 6-4-3, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-alkylamino nicotinamide analogs was prepared as orally active ghrelin receptor (ghrelinR)
inverse agonists. Starting from compound 1, oral bioavailability was improved by modifying metaboli-
cally unstable sites and reducing molecular weight. Brain-permeable compound 33 and compound 24
with low brain permeability were tested in rat models of obesity; 30 mg/kg of compound 33 suppressed
weight gain. PK/PD analysis revealed that the anti-obesity effect of ghrelinR inverse agonists depends on
their brain concentrations.
Received 20 February 2015
Revised 20 May 2015
Accepted 22 May 2015
Available online xxxx
Keywords:
GhrelinR
Ó 2015 Elsevier Ltd. All rights reserved.
Inverse agonist
2
-Aminoalkyl nicotinamide
Obesity
1
. Introduction
pituitary gland, and the central nervous system (CNS). Although
how much each route works remains under dispute, some data
17,18
Ghrelin is a gut-derived 28-amino-acid peptide that has an
showed that the vagal afferent nerves route is dominant
whereas other data showed that the CNS effect is dominant.1
9–21
important role in food intake and energy homeostasis and is the
endogenous ligand for growth hormone secretagogue receptor 1a
While almost all anorectic drugs act through CNS targets, from
the viewpoint of drug discovery, a non-CNS anti-obesity drug is
attractive because of the lower risk of CNS-related adverse effects.
During the target validation, our in-house data reproduced the
result that ghrelin-induced food intake was canceled in vago-
1
(
GHS-R 1a), currently known as the ghrelin receptor (ghrelinR).
It has been suggested that ghrelin acts as a feeding signal as the
plasma ghrelin level increases before meals and declines after eat-
2
ing, and ghrelin is the only peripheral orexigenic hormone identi-
3
17
fied to date. Besides orexigenic action, ghrelin is known to
tomized rats, which encouraged us to seek peripheral ghrelinR
ligands.
regulate glucose homeostasis, gastric motility5 and addictive
4
6
behavior. Therefore, disruption of ghrelin signal is an attractive
Our first effort led to a non-CNS penetrable ghrelinR inverse
2
2
target for anti-obesity, anti-diabetes and anti-alcohol addiction
agonist (1) whose brain/plasma ratio (B/P ratio) was 0.005
(Fig. 1). This compound suppressed ghrelin-induced growth hor-
mone (GH) release in rats, ghrelin-induced gastric mobility in mice,
ghrelin-induced food intake in mice and cumulative food intake for
two weeks in high-fat diet-induced obesity (HFDIO) mice, which
further supports the idea that peripheral ghrelinR inhibition can
be sufficient for reducing food intake. Since compound 1 is not
orally available, we tried to optimize the structure to improve oral
bioavailability.
7
therapeutics.
In determining target profiles of small molecules that suppress
ghrelinR signaling, we considered two things: antagonists or
inverse agonists and peripheral or central. GhrelinR exhibits high
constitutive activity, and the constitutive signal reaches almost
8
half that of ghrelin-induced activity, which implies that ghrelinR
inverse agonists might suppress the signal more effectively than
ghrelinR antagonists. Nevertheless, ghrelinR inverse agonists are
relatively scarce and have been reported only recently by
1
4
15
16
Merck, AstraZeneca and Pfizer. Therefore, we decided to
search for inverse agonists. It is said that orexigenic action of ghre-
lin is caused mainly via three routes: the vagal afferent nerves, the
2
2
. Results and discussion
.1. Chemistry
⇑
Corresponding author. Tel.: +81 78 306 5309; fax: +81 78 306 5047.
E-mail address: takahashi.bitoku.cz@asubio.co.jp (B. Takahashi).
The 5-diazabicyclo pyridine analogs were prepared by the
method depicted in Scheme 1. Optically pure (S)-2-aminomethyl-
http://dx.doi.org/10.1016/j.bmc.2015.05.047
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
O
(13) proceeded smoothly, and following deprotection and acetyla-
N
tion yielded the final product 1. The yield of each reaction was
moderate to good, and the whole scheme was applicable to 10 g
scale synthesis without difficulty.
H
O
O
N
H
H
GhrelinR binding IC50 : 0.96 nM
GhrelinR inverse agonist IC50 : 47 nM
Oral bioavailability : 0.6%
N
NH
O
O
1
By analyzing the H NMR spectrum of compound 1, we found
that protons at the pyridine ring and the diazabicyclo moiety were
split into two peaks, and the integral ratio of the peaks was approx-
imately 0.45:0.55. Because compound 1 is a mixture of diastere-
omers, each diastereomer was separated through a chiral column
1
Brain / plasma ratio : 0.005
Figure 1. A previously reported ghrelinR inverse agonistic 2-aminoalkyl nicoti-
namide derivative.
1
and analyzed by H NMR spectroscopy. Interestingly, NMR spectra
1
,4-benzodioxane (5) was prepared using a chiral glycidol deriva-
of both isomers were almost identical, and the splits of the peaks
were also observed. We, therefore, concluded that the splits indi-
cate cis- and trans-rotamers of the amide bond on the bridge-head
nitrogen.
9
tive. Catechol and p-toluenesulfonic acid (2R)-(À)-glycidyl ester
were coupled to yield (S)-2-hydroxymethyl-1,4-benzodioxane
and the hydroxyl group was converted to an amino group via a
phthalimide intermediate (4). The dihydrobenzofuran ring of com-
Preparation of the alkynyl pyridine analogs is described in
Scheme 2. The key intermediate 20 was prepared from commer-
cially available 2-hydroxy nicotinic acid 18 through 5-iodination
using N-iodosuccinimide, chlorination using thionyl chloride, and
esterification. Compound 20 was coupled with 3,4-(methylene-
dioxy)benzylamine by heating without solvent, and an alkynyl
moiety was introduced by a Sonogashira reaction. No protection
of 2-methyl-3-butyl-2-amine was necessary in the reaction, and
compound 22 was obtained in good yield (90%). Acetylation, ester
hydrolysis, and amide coupling with 3-chloro-4-methoxybenzy-
lamine yielded compound 24. The amide part of compound 33
was prepared from compound 25 through di-fluorination using
N,N-diethylaminosulphur trifluoride (DAST), cuprate coupling to
introduce a cyano group, and reduction of the cyano group.
Starting from the 5-bromo intermediate 29, compound 33 was pre-
pared by applying the same procedure for compound 24. All the
1
0
pound 7 was constructed by intramolecular C–C bond formation,
and the bromo group of compound 7 was converted to a hydrox-
ymethyl group by lithiation, followed by paraformaldehyde treat-
ment. The same procedure for compound 5 was adopted for the
conversion of the hydroxymethyl group to the aminomethyl group
to yield 10. The diazabicyclo boronate intermediate (13) was pre-
pared by the method described in the literature11 with slight mod-
ification. Commercially available N-Boc-nortropine was converted
to vinyl triflate 12 and the triflate was coupled with bis(pinaco-
late)diboron to yield the boronate. Finally, these intermediates
(5, 10 and 13) were coupled with the nicotinic acid scaffold (14).
The amidation of compound 14 and compound 5 was performed
using BOP reagent and the amination at the 2-position of the pyr-
idine ring was accomplished by simply heating the mixture of
reactants. Suzuki coupling of the aryl bromide (16) and the borate
O
HO
HO
O
O
O
O
O
O
O
a,b,c
N
d
2
H N
S
+
O
O
2
3
4
5
O
Br
O
Br
e
Br
O
f
HO
O
H
g,h
i
O
O
N
2
H N
Br
O
6
7
8
9
10
Boc
Boc
Boc
N
N
N
H
j
H
k
O
O
OTf
B
H
H
H
O
11
12
13
O
O
O
Br
O
Br
N
OH
l
Br
O
O
m
n,o
N
H
H
N
NH
O
O
N
Cl
N
Cl
14
15
16
H
N
O
H
N
O
H
O
O
N
H
p
O
H
N
H
N
NH
O
O
H
N
NH
O
O
17
1
Scheme 1. Reagents and conditions: (a) K
8%; (e) n-BuLi, THF, paraformaldehyde, À74 °C, 74%; (f) t-BuLi, THF, paraformaldehyde, À74 °C, 74%; (g) SOCl
steps); (i) hydrazine hydrate, MeOH, 77 °C, 80%; (j) LDA, THF, À78 °C, then, PhNTf rt, 46%; (k) bis(pinacolato)diboron, PdCl
reagent, DIPEA, DMF, 0 °C–rt, 93%; (m) 10, 160 °C, 74%; (n) 13 Pd(pph , K PO , dioxane, H O, 100° C; (o) TFA, CH Cl , rt, 61% (2 steps); (p) AcCl, Et
2
CO
3
DMF, 60 °C; (b) TsCl, pyridine, rt; (c) phthalimide potassium, DMF, 90 °C, 57% (3 steps); (d) hydrazine hydrate, MeOH, reflux,
CH Cl , rt; (h) phthalimide potassium, DMF, 90 °C, 69% (2
(dppf), KOAc, dioxane, 80 °C, 67%; (l) 5, BOP
N, CH Cl , rt, 72%.
8
2
2
2
2
2
)
3 4
3
4
2
2
2
3
2
2
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B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
O
O
O
I
O
I
I
OH
a
OH b,c
OH
O
d
e
N
NH
N
1
OH
N
N
Cl
O
O
8
19
21
20
O
O
NH
NH2
NH
O
O
O
Cl
O
O
f,g
OH
NH
h
N
H
N
NH
N
NH
N
O
O
O
O
O
24
22
23
O
O
F
F
F
Br
i
Br
j
NC
k
H
F
F
H2N
F
25
26
27
28
OH
O
O
O
Br
O
O
Br
l
m
O
n
N
NH
N
NH
N
Cl
O
O
O
O
29
30
31
OH
OH
O
O
F
o
OH
NH
N
H
F
N
N
NH
O
O
O
O
32
33
Scheme 2. Reagents and conditions: (a) N-iodosuccinimide, DMF, 50 °C, 76%; (b) SOCl
benzylamine, 110 °C, then, CH CN, H O, 80 °C–rt, 96%; (e) 2-methyl-3-butyn-2-amine, PdCl
MeOH, 50 °C, 95% (2 steps); (h) 3-chloro-4-methoxybenzylamine, BOP reagent, DIPEA, DMF, 0 °C, 85%; (i) DAST, CH
190 °C), 87%; (k) Pd–C, H , MeOH, rt, 97%; (l) 3,4-(methylenedioxy)-benzylamine, 110 °C, 45%; (m) 3-methyl-1-butyn-3-ol, PdCl
2
, DMF, 70 °C; (c) SOCl
2
, DMF, EtOH, 80 °C, 95% (2 steps); (d) 3,4-(methylenedioxy)-
CN, 60 °C, 90%; (f) AcCl, Et N, CH Cl , 0 °C; (g) 1 N NaOH,
Cl , 0 °C, 79%; (j) CuCN, pyridine, DMF, microwave
(PPh , CuI, Et N, 110 °C, 85%; (n) 1 N-
3
2
2
(PPh , CuI, Et N CH
)
3 2
3
3
3
3
3
2
2
(
2
2
)
3 2
3
NaOH, MeOH, 60 °C, 88%; (o) 28, BOP reagent, DIPEA, DMF, 0 °C–rt, 89%.
reactions proceeded in moderate to good yield and were no prob-
lem in 10 g scale synthesis.
beneficial because the molecular weight was decreased and the
chiral center was eliminated without loss of activity, the meta-
bolic stability was not improved (34a and 34b). Another substitu-
tion with potent activity was the 3,4-substituted benzyl group
such as in 34c and 34d. Metabolic stability was not improved
in combination with the dihydrobenzofuran moiety at the 2-po-
sition of the pyridine ring (34c). However, improvement of meta-
bolic stability was observed in combination with 3,4-
(methylenedioxy)benzylamine (34d). Although the metabolic rate
was improved, the pharmacokinetic study revealed that oral
bioavailability of compound 34d was not improved (as discussed
later). We then decided to put priority on seeking other substitu-
tions at the 5-position of the pyridine ring rather than further
analyzing the properties of diazabicyclo pyridine derivatives
because of the chirality of the diazabicyclo moiety and the rota-
tion barrier of amide bond discussed above, which can some-
2
.2. Structure–activity relationship
Because poor oral availability of compound 1 was considered
to be derived from a high metabolic rate, structural modification
was aimed at improving the metabolic stability while keeping the
main activity. GhrelinR binding affinity was evaluated by a radi-
olabeled ghrelin substitution assay, and inverse agonist activity
was evaluated by a reporter gene assay. Potencies of the com-
pounds were depicted as IC50 values. As in a previous structure
activity relationship (SAR) study, all compounds were pure
inverse agonists, which almost completely suppressed the consti-
tutive activity of ghrelinR at concentrations sufficiently higher
than the IC50. Metabolic sites were estimated by analyzing
metabolites in an in vitro liver microsome assay, which showed
that most metabolic reactions, mostly oxidations, occurred on
the 3- and 2-position moieties of the pyridine ring. Therefore,
we modified the structures of these parts (Table 1). Regarding
the main activity, structural tolerance at the 2-position of the
pyridine ring was very narrow, and we could only convert the
carbon atom to an oxygen atom on the dihydrobenzodioxane
ring. The 3-position of the pyridine ring was rather structurally
tolerable, and the benzodioxane ring could be substituted for a
simple phenoxy group. Although this modification seemed to be
1
2
times result in troublesome atropisomers.
We aimed for a simpler structure with reduced molecular
weight and found that the diazabicyclo ring can be replaced by
propargyl amine (24) or propargyl alcohol type substitutions with-
out loss of activity. These derivatives have lower molecular
weights, no chirality, and, surprisingly, good oral bioavailability.
The reason for the improvement of oral bioavailability remains to
be elucidated. Further optimization of the substitution at the 3-
and 5-position of the pyridine ring led to the highly potent com-
pound 33 whose molecular weight is 507, which is not so high
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4
B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
1
GhrelinR binding affinity, inverse agonist IC50, and metabolic rate for selected compounds varying R and X
O
N
H
O
R1
N
H
H
N
NH
O
X
a
Metabolic rateb (pmol min mg
À1
À1
Compd
R
1
X
GhrelinR binding IC50
Inverse agonist IC50 (nM)
)
(
nM)
Rat
Human
O
1
CH
2
2
0.96
0.39
47
302
O
O
3
4a
4b
CH
O
2.1
9.1
N.D.c
N.D.c
28
80
358
202
O
3
Cl
3
3
4c
CH
O
2
5.9
5.6
0.42
0.30
9.4
9.8
211
39
O
Cl
4d
O
All values indicate mean of at least two independent measurements.
a
All compounds showed the same efficacy (95% block).
Metabolic rate in human liver microsomes.
No data.
b
c
Table 2
GhrelinR binding affinity, inverse agonist IC50, and metabolic rate, total body clearance, oral bioavailability, and brain/plasma ratio for selected compounds
O
O
N
NH
OH
H
O
O
O
F
Cl
O
Cl
O
N
H
N
H
N
H
F
H
N
NH
N
NH
N
NH
O
O
O
O
O
O
24
33
3
4d
a
b
Metabolic ratec (pmol min mg
À1
À1
À1
À1
Oral BA (%) B/P ratiof
Compd
Binding IC50
nM)
Inverse agonist IC50 (nM)
Agonist IC50 (nM)
)
CLtot (L h kg
)
(
Rat
Human
Calcium Pituitary GH
3
2
3
4d
4
3
5.6
6.6
0.28 0.44
0.30
3.0
9.8
26
5.4
100
34
13
32.8
36.2
11.6
39
81
103
10.79d
7.2d
46.4
23.8
N.D.
0.07
e
e
e
e
1.67
e
0.21e
0.74
All values indicate mean of at least two independent measurements.
a
All compounds showed the same efficacy (>95% block).
All compounds blocked >95% of ghrelin-induced activity.
Metabolic rate in human liver microsomes.
Measured in male ICR mice.
Measured in male SD rats.
Determined at 2 h after intravenous administration.
b
c
d
e
f
among ghrelinR ligands.13 Antagonistic activity of the compounds
was tested in a calcium efflux assay (FLIPR) and a GH release assay
using primary rat pituitary cells. Because inverse agonist activity
cannot be evaluated in these assays, we adopted them only for
evaluating antagonist activity using ghrelin as an agonist. These
compounds blocked ghrelin-induced calcium efflux and GH release
almost completely at high concentrations, showing that they also
have antagonist activity. Antagonist potency of 34d was lower than
inverse agonist potency, and antagonist potencies of 24 and 33
were similar to inverse agonist potencies.
2.3. In vivo evaluation
2.3.1. Pharmacokinetics
Pharmacokinetic data were obtained in normal male ICR
mice (34d) or normal male SD rats (24 and 33) as depicted
in Table 2. In spite of a lower metabolic rate in human liver
microsomes (HLM), total body clearance (CLtot) of compound
34d was very high, which resulted in low oral bioavailability
(7%). CLtot of 24 and 33 were lower, and therefore, these com-
pounds showed good oral bioavailability (46.4% and 23.8%,
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B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
A
1
0
8
6
4
2
0
2
4
6
8
Vehicle
3
3
3(10mpk-BID)
3(30mpk-BID)
*
*
24(10mpk-BID)
4(30mpk-BID)
2
-
-
-
-
habituation (PO, BID)
-6 -4 -2
Administration (PO, BID)
-8
0
2
4
6
8
10
12
14
16
Days
B 1
0.0
C
1800
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
1
1
1
700
¶
600
500
1400
1300
*
*
*
1
1
200
100
1000
33
33
24
24
33
33
24
24
Normal Vehicle
PF
Normal Vehicle
PF
(
10,BID) (30,BID) (10,BID) (30,BID)
(10,BID) (30,BID) (10,BID) (30,BID)
Figure 2. Effect of twice daily oral treatment of compounds 24 or 33 on body weight (A and B) and food intake (C) in HFDIO rats. (A) Daily body weight change before and
*
after the treatment. (B) Body weight change on day 14 from day 0. (C) Total calorie intake between day 0 and day 14. PF indicates pair-fed group. Denotes p<0.05 versus
vehicle, **p<0.01 versus vehicle, and denotes p<0.001 versus normal. (Dunnet’s test) BW, body weight; BID, twice daily; mpk, mg/kg; PO, post oral; HFDIO, high-fat diet-
–
induced obesity.
Table 3
Plasma and brain concentrations and their ratios against binding IC50 for PK/PD analysis
Compound 33
Compound 24
1
0 mg/kg BIDd
30 mg/kg BIDd
10 mg/kg BIDd
30 mg/kg BIDd
Brain/plasma ratio
Plasma protein binding (%)
AUC0–24 (ng * h/mL)
0.21
99
0.07
99
1105
91
19
324
68
3.2
0.7
3314
272
57
972
203
97
24901
1890
132
286
19
74704
5670
370
859
58
a
Mean plasma concn (nM)
Mean brain concnb (nM)
Mean plasma concn/binding IC50
Mean brain concn/binding IC50
Mean plasma free conc /binding IC50
c
2.9
0.2
86
0.6
c
Mean brain free conc /binding IC50
2.0
a
b
c
Calculated by AUC0–24/24 (h).
Calculated by (mean plasma concn) * (B/P ratio).
Free concentration was calculated by multiplying the free fraction with total concentration.
Twice daily.
d
respectively). The B/P ratio of compound 24 was low (0.07) and
that of 33 was moderate (0.21), both of which were higher
than the lead compound 1. However, we thought that a B/P
ratio of 0.07 is acceptable for a peripheral drug if its pharma-
cological action depends on plasma concentration. Although
compound 33 penetrated the blood-brain barrier to some
extent, we evaluated this compound in comparison with com-
pound 24 in an in vivo pharmacology experiment in order to
prove our concept.
2.3.2. Pharmacology and PK/PD analysis
We chose HFDIO rats in this study because the roles of the vagal
afferent nerve in ghrelin signaling have been well characterized in
rats. Specifically, ghrelin-induced food intake and GH release were
1
7
canceled in vagotomized rats, and ghrelinR mRNA expression
was increased in high-fat diet induced obesity-prone rats but not
in low-fat diet rats or high-fat diet-induced obesity-resistant
2
3
rats, but these results were not reproduced in mice (in house
data).
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6
B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
HFDIO rats were treated with 10 mg/kg or 30 mg/kg of com-
4.1.1. (S)-2-((2,3-Dihydrobenzo[b][1,4]dioxin-2-yl)methyl)isoi-
ndoline-1,3-dione (4)
pound 33 or 24 through twice daily oral administration. Body
weight was increased constantly during the administration period
in the vehicle group the same as during the habituation period
To a solution of catechol (24.12 g, 219 mmol) and p-toluenesul-
fonic acid (2R)-(À)-glycidyl ester (50 g, 219 mmol) in DMF
(500 mL) was added potassium carbonate (45.4 g, 329 mmol) and
the mixture was stirred at 60 °C for 48 h. The mixture was concen-
trated in vacuo and quenched with water. After extraction with
[EtOAc/hexane = 1:1] solution, the organic phase was washed with
(
Fig. 2A). The daily weight increase was attenuated in treated
groups and statistically significant attenuation was observed on
day 14 only at 30 mg/kg of compound 33. A slight decrease in total
calorie intake for 14 days was observed in treated groups, but was
not statistically significant. Although a statistically significant
decrease in food intake was not observed, attenuation of body
weight gain of rats treated with compound 33 was conceivably
ascribed to the suppression of food intake because pair-fed animals
showed similar body weight changes. Plasma concentration in an
in vivo disease model was monitored at the first day of the admin-
istration and used for PK/PD analysis in combination with the B/P
ratio in normal rats and in vitro plasma protein binding. The
plasma and brain concentrations of the compounds and the ratio
of each free concentration versus binding IC50 are described in
Table 3. Considering the binding IC50 and B/P ratio, the difference
between compounds 33 and 24 can be reduced to the difference
in brain concentration. Mean plasma free concentration/binding
IC50 were similar (3.2 vs 2.9 and 97 vs 86 in 10 mg/kg and
2 4
water and brine, then dried over Na SO , filtered and concentrated
in vacuo to obtain (S)-(2,3-dihydrobenzo[b][1,4]dioxin-2-
yl)methanol as a pale red solid (36.4 g). The solid was dissolved
in pyridine (145 mL) and p-toluenesulfonyl chloride (50.1 g,
263 mmol) was added to the solution. The mixture was stirred at
room temperature for 2 h, then 3 N HCl (1000 mL) was slowly
added at 0 °C. After extraction with ether, the organic phase was
2 4
washed with water and brine, then dried over Na SO , filtered
and concentrated in vacuo to obtain (R)-(2,3-dihydrobenzo[b][1,4]-
dioxin-2-yl)methyl 4-methylbenzenesulfonate as a pale yellow oil
(60.45 g). The oil was dissolved in DMF (300 mL) and phthalimide
potassium salt (45.4 g, 245 mmol) was added to the mixture. The
mixture was stirred at 90 °C for 20 h and cooled to room tempera-
ture, then MeOH (1.5 L) was added with stirring. The resulting solid
was filtered, washed with MeOH and dried in vacuo to give 4 as a
3
0 mg/kg, respectively), and mean brain free concentration/bind-
ing IC50 was three times higher for compound 33 than 24 (0.7 vs
.2 and 2.0 vs 0.6 in 10 mg/kg and 30 mg/kg, respectively).
white solid (36.9 g, 57% from catechol).
1
0
3
H NMR (400 MHz, CDCl ) d = 7.88 (m, 2H), 7.75 (m, 2H), 6.86
Therefore, the anti-obesity activity of compound 33 seems to be
(m, 4H), 4.51 (dq, J = 2.4, 6.4, 1H), 4.31 (dd, J = 2.4, 12.0 Hz, 1H),
derived from suppression of CNS ghrelinR activity.
4.10 (dd, J = 6.4, 14.4 Hz, 1H), 4.05 (dd, J = 6.4, 12.0 Hz, 1H), 3.90
+
(
dd, J = 6.0, 14.4 Hz, 1H); MS (ESI+) 296.1 (M+H) .
3
. Conclusions
4
.1.2. (S)-(2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methanamine (5)
To a solution of 4 (15 g, 50.8 mmol) in MeOH (300 mL) was
Starting from compound 1, we improved oral bioavailability by
added hydrazine hydrate (12.7 g, 254 mmol) and the mixture
was stirred at reflux for 2 h. After cooling, chloroform (500 mL)
was added to the mixture and insolubles were removed by filtra-
tion. The filtrate was concentrated in vacuo and treated with
modifying the structures of metabolic sites and changing the substi-
tution at the 5-position of the pyridine ring. The B/P ratio increased
during the structural modification, and we chose two compounds
with low and moderate brain penetration. Pharmacological evalua-
tion in HFDIO rats revealed that the brain-permeable compound
attenuated weight gain effectively. Therefore, we concluded that
the anti-obesity activity of ghrelinR inverse agonists is attributed
to the suppression of brain ghrelinR activity.
0
.5 N NaOH (200 mL), then extracted with ether. The organic phase
was washed with 0.5 N NaOH, water and brine, then dried over
Na SO , filtered and concentrated in vacuo to obtain 5 as a color-
2
4
less oil (7.36 g, 88%).
1
3
H NMR (400 MHz, CDCl ) d = 6.86 (m, 4H), 4.27 (dd, J = 2.4,
1
1.4 Hz, 1H), 4.13 (m, 1H), 4.02 (dd, J = 7.8, 11.2 Hz, 1H), 2.99
+
4
4
. Experimental
.1. Chemistry
(dd, J = 3.2, 5.6 Hz, 2H); MS (ESI+) 166.1 (M+H) .
4.1.3. 6-Bromo-2,3-dihydrobenzofuran (7)
To
a
solution of 1,4-dibromo-2-(2-bromoethoxy)benzene
1
Proton nuclear magnetic resonance spectra ( H NMR) and carbon
(22.3 g, 62.2 mmol) in THF (180 mL) was added 1.57 M n-BuLi in
hexanes (40 mL, 62.8 mmol) under Ar at À76 °C. After stirring at
nuclear magnetic resonance spectra (13C NMR) were recorded on
Brucker ARX-400 or Brucker Avance III (400 MHz) spectrometer in
the indicated solvent. Chemical shifts (d) are reported in parts per
million relative to the internal standard tetramethylsilane. High-
resolution mass spectra (HRMS) and fast atom bombardment
À76 °C overnight, the mixture was quenched with sat. NH Cl aq
4
and extracted with AcOEt. The organic phase was washed with
brine and dried over Na SO . After filtration and evaporation, the
residue was recrystallized from ethanol (28 mL) and water (3 mL)
2
4
(
FAB) mass spectra were recorded on JEOL JMS-700 mass spectrom-
to obtain 7 as a white solid (9.4 g, 76%).
1
eter. Electro-spray ionization (ESI) mass spectra were recorded on
Agilent G1956A MSD spectrometer system. Purity was analyzed by
Agilent 1100 HPLC system. HPLC conditions utilized are as follows;
column: YMC-PackPro C18, 4.6 mm  100 mm, gradientand mobile
phase: 10% to 100% solvent B in solvent A for 8 min, solvent
H NMR (400 MHz, CDCl ) d = 7.03 (d, J = 7.6 Hz, 1H), 6.97 (d,
3
J = 1.6 Hz, 1H), 6.94 (dd, J = 1.6, 7.6 Hz, 1H), 4.59 (t, J = 8.8 Hz,
2H), 3.15 (t, J = 8.8 Hz, 1H); MS (ESI+) 200.1 (M+H) .
+
4.1.4. (2,3-Dihydrobenzofuran-6-yl)methanol (8)
A = MeCN/H
trifluoroacetic acid (100:0.09), UV detector: 210 nm, flow rate:
mL/min. Retention times (t ) are in minutes and purity is calcu-
2
O/trifluoroacetic acid (5:95:0.1), solvent B = MeCN/
To a solution of 7 (4.29 g, 21.6 mmol) in THF (43 mL) was added
1.76 M t-BuLi in pentane (25.7 mL, 45.2 mmol) under Ar at À74 °C.
Paraformaldehyde (0.97 g, 32.3 mmol) was added to the mixture at
À74 °C and the mixture was stirred at room temperature over-
1
R
lated as% total area. Other chemical reagents and solvents were pur-
chased from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical
Industries, Kanto Kagaku or Nacalai tesque and used without purifi-
cation. Flash column chromatography was performed using Merck
night. The mixture was quenched with satd NH Cl aq and extracted
4
with AcOEt. The organic layer was washed with brine and dried
over MgSO , then filtered. After removal of the solvent, the residue
was recrystallized from CH Cl (6.5 mL) and n-hexane (25.9 mL) to
obtain 8 as a pale red solid (2.39 g, 74%).
4
Silica gel 60 (230–400 mesh) or Purif-PackÒ SI 30
Shoko Scientific.
lm supplied by
2
2
Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047

B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
1H NMR (400 MHz, CDCl
J = 7.2 Hz, 1H), 6.81 (s, 1H), 4.63 (br s, 2H), 4.58 (t, J = 8.8 Hz, 2H),
) d = 7.16 (d, J = 7.2 Hz, 1H), 6.84 (d,
4.1.9. (S)-5-Bromo-2-chloro-N-((2,3-dihydrobenzo[b][1,4]diox-
in-2-yl)methyl)nicotinamide (15)
3
+
3
.20 (t, J = 8.8 Hz, 2H); MS (ESI+) 151.1 (M+H) .
To
a
solution of 5-bromo-2-chloronicotinic acid (5 g,
2
1.2 mmol) and 5 (4.19 g, 25.4 mmol) in DMF (50 mL) were added
4
.1.5. 2-((2,3-Dihydrobenzofuran-6-yl)methyl)isoindoline-1,3-
DIPEA (11.08 mL, 63.4 mmol) and BOP reagent (10.29 g,
23.3 mmol) at 0 °C. The mixture was stirred at room temperature
for 1 h, quenched with water (200 mL) and extracted with
[EtOAc/hexane = 1: 1] solution. The organic phase was washed
dione (9)
A solution of 8 (2.38 g, 15.8 mmol) in CH
cooled with ice bath and SOCl (9.44 g, 79 mmol) was added drop-
2
2
Cl (15.8 mL) was
2
wise. The mixture was stirred at room temperature for 1 h and
concentrated in vacuo. The residue was stirred with phthalimide
potassium salt (3.23 g, 17.4 mmol) and DMF (15.8 mL) at 90 °C
for 2 h and cooled, then partitioned between AcOEt (80 mL) and
water (60 mL). The aqueous phase was extracted with AcOEt and
the combined organic phase was washed with brine and 1 N
2 4
with brine, dried over Na SO and filtered. After removal of the sol-
vent in vacuo, the residue was recrystallized from AcOEt and n-
hexane to yield 15 as a white solid (4.82 g, 93%).
1
3
H NMR (400 MHz, CDCl ) d = 8.53 (d, J = 2.4 Hz, 1H), 8.21 (d,
J = 2.4 Hz, 1H), 6.96 (br s, 1H), 6.87 (m, 4H), 4.43 (m, 1H), 4.35
(dd, J = 2.2, 11.8 Hz, 1H), 4.05 (dd, J = 7.0, 11.8 Hz, 1H), 3.92 (ddd,
NaOH, then dried over MgSO
4
and filtered. After removal of the sol-
J = 4.0, 6.4, 14.4 Hz, 1H), 3.71 (ddd, J = 1.2, 6.4, 14.4 Hz, 1H); 13
C
vent in vacuo, the residue was recrystallized from MeOH (58 mL)
3
NMR (100 MHz, CDCl ) d = 163.6, 152.1, 145.6, 143.0, 142.5,
and AcOEt (27 mL) to obtain 9 as a red solid (3.07 g, 69%).
142.1, 131.7, 121.9, 119.6, 117.4, 117.3, 71.6, 65.6, 40.6; MS
(ESI+) 385.0 (M+H) .
1
+
3
H NMR (400 MHz, CDCl ) d = 7.84 (d, J = 8.4 Hz, 2H), 7.70 (d,
J = 8.4 Hz, 2H), 7.11 (d, J = 8.0 Hz, 1H), 6.91 (dd, J = 1.6, 7.6 Hz,
1
3
H), 6.84 (d, J = 1.6 Hz, 1H), 4.79 (s, 2H), 4.53 (t, J = 8.8 Hz, 2H),
.15 (t, J = 8.8 Hz, 2H); MS (ESI+) 280.2 (M+H) .
4
.1.10. (S)-5-Bromo-N-((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-
+
methyl)-2-(((2,3-dihydrobenzofuran-6-yl)methyl)amino)nico-
tinamide (16)
4
.1.6. (2,3-Dihydrobenzofuran-6-yl)methanamine (10)
A mixture of 15 (192 mg, 0.5 mmol) and 10 (90 mg, 0.6 mmol)
A mixture of 9 (3.07 g, 11 mmol) and hydrazine hydrate (2.75 g,
5 mmol) in MeOH (45 mL) was stirred at 77 °C for 3 h. The mix-
was stirred at 160 °C for 2 h. After cooling, the mixture was par-
5
titioned between AcOEt (6 mL) and satd NaHCO
the organic phase was washed with brine and dried over
Na SO . After filtration, the mixture was concentrated in vacuo
and the residue was purified by silica gel column chromatogra-
phy (Hexane/EtOAc; 60:40) to yield 16 as a yellow solid
3
aq (2 mL) and
ture was concentrated in vacuo and partitioned between AcOEt
60 mL) and water (58 mL). The organic layer was washed with
brine, dried over MgSO and evaporated to afford 10 as a red solid
1.31 g, 80%).
1H NMR (400 MHz, CDCl
J = 8.4 Hz, 1H), 6.75 (s, 1H), 4.56 (t, J = 8.8 Hz, 2H), 3.80 (s, 2H),
(
2
4
4
(
3
) d = 7.14 (d, J = 8.8 Hz, 1H), 6.78 (d,
(
184 mg, 74%).
1H NMR (400 MHz, CDCl
3
) d = 8.32 (t, J = 7.4 Hz, 1H), 8.24 (d,
+
3
.18 (t, J = 8.8 Hz, 2H); MS (ESI+) 150.1 (M+H) .
J = 2.4 Hz, 1H), 7.64 (d, J = 2.4 Ha, 1H), 7.11 (d, J = 7.6 Hz, 1H),
.85–6.95 (m, 4H), 6.82 (d, J = 7.6 Hz, 1H), 6.78 (s, 1H), 6.47 (t,
J = 7.6 Hz, 1H), 4.60 (d, J = 5.6 Hz, 1H), 4.54 (t, J = 8.8, 2H), 4.36
m, 1H), 4.30 (dd, J = 2.4, 11.4 Hz, 1H), 3.99 (dd, J = 6.8, 11.4 Hz,
6
4
.1.7. tert-Butyl 3-(((trifluoromethyl)sulfonyl)oxy)-8-azabicyc-
lo[3.2.1]oct-3-ene-8-carboxylate (12)
(
A solution of diisopropylamine (0.34 mL, 2.74 mmol) in THF
1
1
H), 3.80 (ddd, J = 4.0, 6.4, 14.4 Hz, 1H), 3.61 (ddd, J = 6.2, 6.6,
4.4 Hz, 1H), 3.17 (t, J = 8.8 Hz, 2H); C NMR (100 MHz, CDCl )
3
(
10 mL) was treated with 1.6 M n-BuLi in hexanes at À78 °C and
13
the mixture was stirred at À78 °C for 30 min. A solution of N-
Boc-nortropinone 11 (411 mg, 1.83 mmol) in THF was added to
the mixture and stirred at À78 °C for 3 h, then N,N-bis(trifluo-
romethanesulfonyl)aniline (717 mg, 2 mmol) in THF (5 mL) was
added. The mixture was stirred at room temperature overnight,
quenched with water and extracted with ether. The organic phase
d = 167.5, 160.5, 156.3, 152.7, 143.0, 142.6, 139.6, 137.2, 125.8,
1
7
24.8, 121.8, 121.8, 119.6, 117.3, 117.3, 110.7, 108.6, 104.2, 71.8,
1.3, 65.7, 44.9, 40.2, 29.5; MS (ESI+) 498.0 (M+H) .
+
4
.1.11. 5-(8-Azabicyclo[3.2.1]oct-3-en-3-yl)-N-(((S)-2,3-dihydr-
obenzo[b][1,4]dioxin-2-yl)methyl)-2-(((2,3-dihydrobenzofu-
ran-6-yl)methyl)amino)nicotinamide (17)
was washed with NaHCO
Na SO and filtered. After removal of the solvent in vacuo, the resi-
due was purified by silica gel column chromatography
3
aq, water and brine, then dried over
2
4
A mixture of 16 (3.24 g, 6.53 mmol), 13 (2.63 g, 7.83 mmol),
3 4 3 4
Pd(PPh ) (0.377 g, 0.326 mmol) and K PO (2.77 g, 13.1 mmol)
(
Hexane/EtOAc; 67/33) to yield 12 as a white solid (297 mg, 46%).
1
in 1,4-dioxane (40 mL) and water (20 mL) was stirred at 100 °C
for 7 h. After cooling, the mixture was quenched with water
and extracted with AcOEt. The organic phase was washed with
3
H NMR (400 MHz, CDCl ) d = 6.10 (s, 1H), 4.46 (m, 2H), 3.08 (m,
1
H), 2.24 (br s, 1H), 2.08 (m, 1H), 2.01 (m, 2H), 1.74 (br s, 1H), 1.45
+
(
s, 9H); MS (ESI+) 258.0 (MÀBoc+H)
brine, dried over Na
yield a pale yellow solid. The solid was dissolved in CH
50 mL) and treated with TFA (10 mL). The mixture was stirred
2
SO
4
, filtered and concentrated in vacuo to
2
Cl
2
4
8
.1.8. tert-Butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
-azabicyclo[3.2.1]oct-3-ene-8-carboxylate (13)
(
at room temperature for 1 h and concentrated in vacuo. The resi-
due was purified by column chromatography on N-H silica gel
A suspension of 12 (296 mg, 0.83 mmol), bis(pinacolato)di-
boron (252 mg, 0.99 mmol), PdCl
and potassium acetate (244 mg, 2.49 mmol) in 1,4-dioxane
5 mL) was stirred at 120 °C under microwave irradiation for
0 min. After cooling, the mixture was partitioned between water
and AcOEt. The organic phase was washed with water and brine,
dried over Na SO and filtered. The solvent was removed in vacuo
and the residue was purified by silica gel column chromatography
2
(dppf) (60.6 mg, 0.083 mmol)
(
6
MeOH/CH
1%).
1H NMR (400 MHz, CDCl
2 2
Cl ; 5:95) to yield 17 as a pale yellow solid (2.1 g,
(
2
3
) d = 8.55 (q, J = 5.6 Hz, 1H), 8.18 (d,
J = 1.2 Hz, 1H), 7.67 (t, J = 2.4 Hz, 1H), 7.56 (br s, 1H), 7.07 (d,
J = 7.6 Hz, 1H), 6.80–6.85 (m, 4H), 6.76 (s, 1H), 6.02 (t, J = 7.0 Hz,
2
4
1
H), 4.63 (d, J = 5.2 Hz, 2H), 4.49 (t, J = 8.8, 2H), 4.23–4.30 (m,
1
H), 4.20–4.23 (m, 1H), 4.05 (m, 1H), 3.93 (m, 1H), 3.90 (dd,
(
Hexane/EtOAc; 80/20) to yield 13 as a colorless oil (186 mg, 67%).
1
J = 7.2, 11.4 Hz, 1H), 3.57 (m, 1H), 3.11 (t, J = 8.8 Hz, 2H), 3.03 (m,
H), 2.20–2.32 (m, 2H), 2.07–2.15 (m, 2H), 1.71–1.79 (m, 1H);
3
H NMR (400 MHz, CDCl ) d = 6.76 (s, 1H), 4.30 (m, 2H), 3.08 (m,
1
1
1
H), 2.78 (m, 1H), 2.11 (m, 1H), 1.92 (m, 3H), 1.44 (s, 9H), 1.25 (s,
2H); MS (ESI+) 236.0 (MÀBoc+H)
+
+
MS (ESI+) 525.2 (M+H) .
Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047

8
B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4
.1.12. 5-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-en-3-yl)-N-(((S)-2,3-
1H NMR (400 MHz, CDCl
) d = 8.70 (d, J = 2.0 Hz, 1H), 8.41 (d,
3
dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-2-(((2,3-dihydrobenz-
ofuran-6-yl)methyl)amino)nicotinamide (1)
J = 2.0 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H); MS
(ESI+) 311.9 (M+H) .
+
2 2
To a solution of 17 (2.2 g, 4.19 mmol) in CH Cl were added tri-
ethylamine (0.877 mL, 6.29 mmol) and acetyl chloride (0.358 mL,
4.1.15. Ethyl 2-((benzo[d][1,3]dioxol-5-ylmethyl)amino)-5-
iodonicotinate (21)
A mixture of 20 (26.2 g, 84 mmol) and 3,4-(methylenedioxy)-
benzylamine (25.4 g, 168 mmol) was stirred at 110 °C for 0.5 h
and stood at 110 °C for 3 h. The mixture was cooled to 80 °C and
5
0
.03 mmol), and the mixture was stirred at room temperature for
.5 h. The mixture was concentrated in vacuo and the residue
was
purified
by
silica
gel
column
chromatography
(
MeOH/AcOEt/n-hexane = 3:48.5:48.5) to yield 1 as a pale yellow
solid (1.72 g, 72%).
3
treated with CH CN (260 mL) and water (260 mL), then stirred at
80 °C for 1 h and cooled to room temperature. The mixture was
stirred at 0 °C for 1 h and the solid was filtered, washed with a
1
H NMR (400 MHz, CDCl
3
) d = 8.33 (m, 1H), 8.25 (s, 0.54H), 8.18
(
s, 0.46H), 7.61 (s, 0.46H), 7.53 (s, 0.54H), 7.11 (d, J = 7.6 Hz, 1H),
6.81–6.91 (m, 5H), 6.78 (s, 1H), 6.23 (d, J = 5.6 Hz, 1H), 4.94 (m,
0.46H), 4.87 (m, 0.54H), 4.64 (d, J = 5.2 Hz, 1H), 4.53 (t, J = 8.8 Hz,
2H), 4.34–4.41 (m, 2H), 4.32 (dd, J = 2.0, 12.0 Hz, 1H), 4.00 (dd,
1:1 mixture of CH
3
CN and water (30 mL) and dried in vacuo to
yield 21 as a pale gray solid (32.42 g, 96%).
1
3
H NMR (400 MHz, CDCl ) d = 8.41 (d, J = 2.4 Hz, 1H), 8.33 (d,
J = 7.6, 11.2 Hz, 1H), 3.75–3.84 (m, 1H), 3.60–3.68 (m, 1H), 3.15
t, J = 8.8 Hz, 2H), 3.06–3.13 (m, 0.54H), 2.86–2.91 (m, 0.46H),
J = 2.4 Hz, 1H), 8.25 (br s, 1H), 6.84 (d, J = 1.2 Hz, 1H), 6.80 (dd,
J = 1.2, 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H),
(
13
2
1
1
.27–2.37 (m, 1H), 2.07–2.17 (m, 2H), 2.10 (s, 1.38H), 2.07 (s,
.62H), 1.67–1.83 (m, 1H); 13C NMR (100 MHz, CDCl
) d = 168.7,
67.1 [166.2], 160.4, 157.0 [157.1], 148.7 [148.2], 143.1, 142.8
4.31 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H);
C NMR
3
(100 MHz, CDCl ) d = 166.4, 158.9, 157.0, 147.8, 146.9, 146.7,
3
133.0, 120.8, 108.5, 108.3, 100.9, 61.2, 44.6, 14.2; MS (ESI+) 427.0
+
[
[
[
142.8], 139.9, 132.1 [130.0], 131.8 [132.2], 125.6, 124.7, 124.5
126.0], 122.6 [122.3], 121.7, 121.6, 119.5, 117.3, 117.3, 109.0
109.4], 108.5, 72, 71.3, 65.9 [65.9], 55.0 [53.6], 50.0 [50.9], 44.9,
(M+H) .
4.1.16. Ethyl 5-(3-amino-3-methylbut-1-yn-1-yl)-2-((benzo[d]
[1,3]dioxol-5-ylmethyl)amino)nicotinate (22)
4
0.3, 35.9 [37.3], 35.3 [33.9], 29.5, 28.7 [30.5], 21.6 [20.8], the
minor counterparts of doubled signals due to rotamers are in the
A mixture of 21 (25 g, 58.7 mmol), 2-methyl-3-butyn-2-amine
+
brackets; MS (ESI+) 567.2 (M+H) ; HRMS (FAB) Calcd for
(6.34 g, 76 mmol), PdCl
2
(PPh
3
)
2
(1.24 g, 1.76 mmol), copper(I)
CN
+
C
33
H
35
N
4
O
5
: 567.2607; Found: 567.2574; HPLC purity 93.4%
iodide (0.335 g, 1.76 mmol) and triethylamine (125 mL) in CH
3
(
4.95 min).
(125 mL) was stirred at 60 °C for 3 h. The mixture was diluted with
AcOEt (500 mL) and water (500 mL) and filtered. The filtrate was
diluted with AcOEt (500 mL), then the organic phase was separated
4
.1.13. 2-Hydroxy-5-iodonicotinic acid (19)
A mixture of 2-hydroxynicotinic acid (24 g, 173 mmol), N-io-
and washed with 28% aqueous solution of NH
brine. The solution was dried over Na SO , filtered and concen-
trated, then the residue was recrystallized from CH CN (125 mL)
and water (125 mL) to give 22 as a pale gray solid (20.2 g, 90%).
3 4
, satd NH Cl aq and
dosuccinimide (50.5 g, 224 mmol) in DMF (300 mL) was stirred
under shading at room temperature for 1 h and at 50 °C for 20 h.
About 250 mL of DMF was removed in vacuo and water (400 mL)
was added to the mixture. The mixture was stirred at room tem-
perature for 1 h and at 0 °C for 1 h, and then the resulting solid
was filtered. The solid was suspended in MeOH (200 mL) and stir-
red at room temperature for 2 h, then the solid was filtered,
washed with MeOH (40 mL) and dried in vacuo to give 19 as a pale
2
4
3
1
3
H NMR (400 MHz, CDCl ) d = 8.35 (br s, 1H), 8.31 (d, J = 2.0 Hz,
1H), 8.13 (d, J = 2.4 Hz, 1H), 6.85 (d, J = 1.2 Hz, 1H), 6.81 (dd, J = 1.2,
8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 5.92 (s, 2H), 4.64 (d, J = 5.6 Hz,
2H), 4.31 (q, J = 7.2 Hz, 2H), 1.49 (s, 6H),1.38 (t, J = 7.2 Hz, 3H); 13
C
3
NMR (100 MHz, CDCl ) d = 167.0, 157.0, 156.1, 147.8, 146.7, 142.4,
yellow solid (38.4 g, 76%).
H NMR (400 MHz, CDCl
J = 2.4 Hz, 1H); MS (ESI+) 265.9 (M+H) .
133.1, 120.8, 108.3, 108.2, 107.4, 105.6, 100.9, 97.1, 77.0, 61.0, 45.8,
44.7, 32.0, 14.3; MS (ESI+) 382.1 (M+H) .
1
+
3
) d = 8.41 (d, J = 2.4 Hz, 1H), 8.23 (d,
+
4
.1.17. 5-(3-Acetamido-3-methylbut-1-yn-1-yl)-2-((benzo[d]
4
.1.14. Ethyl 2-chloro-5-iodonicotinate (20)
[1,3]dioxol-5-ylmethyl)amino)nicotinic acid (23)
The mixture of 19 (29.8 g, 107 mmol), DMF (1.5 mL) and SOCl
2
A solution of acetyl chloride (4.9 mL, 68.7 mmol) in CHCl
(10 mL) was added dropwise to a solution of 22 in CHCl
(190 mL) and triethylamine (29.5 mL) at 0 °C. The mixture was stir-
red at 0 °C for 15 min. and at room temperature for 2 h, then con-
centrated in vacuo. The residue was partitioned between AcOEt
(600 mL) and water (300 mL) and the organic phase was washed
3
(
78 mL, 10 eq.) was stirred at 70 °C for 4 h. The mixture was con-
centrated in vacuo and diluted with DMF (6 mL). Water (90 mL)
was slowly added to the mixture and then satd NaHCO aq
200 mL) was slowly added. The mixture was acidified to pH 4
3
3
(
by 1 N HCl (55 mL) and stirred at room temperature for 2 h and
at 0 °C overnight. After filtration, the solid was washed with water
and dried in vacuo to yield a crude mixture of 2-chloro-5-iodonico-
tinic acid as a pale yellow solid (27 g). The solid was suspended in
with satd NaHCO
organic solution was dried over Na
3
aq, half-satd NaHCO
3
and half-sat NaCl aq. The
4
, filtered and concentrated.
2
SO
The residue was treated with THF (100 mL) and concentrated in
vacuo again to yield a red oil (23.84 g). The oil was dissolved in
MeOH (106 mL) and THF (106 mL) and treated with 1 N NaOH
(106 mL). The mixture was stirred at 50 °C for 1 h and cooled to
0 °C. The mixture was neutralized by 1 N HCl (106 mL) at 0 °C
and concentrated in vacuo. AcOEt (660 mL) was added to the mix-
ture and the organic phase was washed with brine, dried over
CHCl
3
(135 mL) and SOCl
2
(13.9 mL) and the suspension was stir-
(27.8 mL) were
red at reflux for 0.5 h. DMF (0.5 mL) and SOCl
2
added to make a clear solution and the mixture was stirred at
reflux for 1.5 h, then cooled and concentrated in vacuo. EtOH
135 mL) was slowly added to the residue at 0 °C and the mixture
(
was stirred at 0 °C for 15 min. and at room temperature for 30 min.
The mixture was concentrated in vacuo and partitioned between
Na
pended in CH
room temperature. The suspension was further stirred at 0 °C for
1 h and the solid was filtered, washed with ice cooled CH CN and
dried in vacuo to yield 23 as a pale yellow solid (19.87 g, 95%).
2
SO
4
, filtered and concentrated in vacuo. The residue was sus-
AcOEt (270 mL) and satd NaHCO
was extracted with AcOEt (135 mL) and the combined organic
phase was washed with brine, dried over Na SO and filtered.
3
aq (135 mL). The aqueous phase
3
CN (210 mL), stirred at 80 °C for 1 h and cooled to
2
4
3
The mixture was concentrated in vacuo and the residue was puri-
fied by silica gel column chromatography (AcOEt/n-hexane; 0:100
to 20:80) to yield 20 as a white solid (27.25 g, 95%).
1
6
H NMR (400 MHz, DMSO-d ) d = 8.52 (t, J = 5.6 Hz, 1H), 8.24 (d,
J = 2.4 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.96 (s, 1H), 6.90 (d,
Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047

B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
J = 1.2 Hz, 1H), 6.4 (d, J = 8.0 Hz, 1H), 6.80 (dd, J = 1.6, 8.0 Hz, 1H),
4.1.21. (3-(Difluoromethyl)-4-methylphenyl)methanamine (28)
1
3
5
.97 (s, 2H), 4.59 (d, J = 4.8 Hz, 2H), 1.80 (s, 3H), 1.56 (s, 6H);
C
A mixture of 27 (1.92 g, 11.52 mmol), Pd–C (5%, 386 mg) and
NMR (100 MHz, CDCl
3
) d = 169.5, 168.1, 156.7, 155.0, 147.3,
4 N 1,4-dioxane solution of HCl in (7.7 mL) in MeOH (77 mL) was
1
7
46.1, 142.3, 133.4, 120.5, 108.1, 108.0, 106.6, 106.0, 100.8, 94.3,
6.5, 46.7, 43.6, 29.0, 23.3; MS (ESI+) 396.1 (M+H) .
2
stirred under H atmosphere at room temperature for 6 h. The mix-
+
ture was filtered and the filtrate was concentrated in vacuo. The
residue was suspended in ether (26 mL) and stirred at room tem-
perature overnight. The solid was filtered, washed with ether and
dried in vacuo to yield 28 as a white solid (2.33 g, 97%).
4
.1.18. 5-(3-Acetamido-3-methylbut-1-yn-1-yl)-2-((benzo[d]-
[
1,3]dioxol-5-ylmethyl)amino)-N-(3-chloro-4-methoxybenzyl)-
1
nicotinamide (24)
H NMR (400 MHz, MeOD) d = 7.63 (s, 1H), 7.48 (d, J = 8.0 Hz,
A solution of 23 (12.7 g, 32.1 mmol) and 3-chloro-4-methoxy-
benzylamine hydrochloride (8.02 g, 38.5 mmol) in DMF (127 mL)
was cooled to 0 °C and treated with DIPEA (16.8 mL) and BOP
reagent (15.63 g, 35.3 mmol). The mixture was stirred at 0 °C for
1H), 7.37 (d, J = 8.0 Hz, 1H), 6.95 (t, J = 55.0 Hz, 1H), 4.13 (s, 2H),
2.50 (s, 3H); MS (ESI+) 172.1 (M+H) .
+
4.1.22. Ethyl 2-((benzo[d][1,3]dioxol-5-ylmethyl)amino)-5-
2
(
h and diluted with a solution of AcOEt (900 mL) and n-hexane
450 mL). The organic solution was washed with water (600 mL),
half-satd NaHCO aq (300 mL), water (600 mL) and brine
600 mL), then dried over Na SO , filtered and concentrated in
vacuo. The residue was suspended in CH CN (88 mL) with heating
bromonicotinate (30)
A mixture of ethyl 5-bromo-2-chloronicotinate 29 (27 g,
3
102 mmol) and 3,4-(methylenedioxy)-benzylamine (30.9 g,
204 mmol) was stirred at 110 °C for 15 min. During the reaction
a pale brown solution changed to a black solid. The black solid
was pound with a mortar and suspended in AcOEt (500 mL). The
suspension was stirred for 30 min. and the solid was filtered off.
(
2
4
3
and water (88 mL) was added to the suspension. The suspension
was stirred at 80 °C for 1 h and slowly cooled to 0 °C with stirring.
The solid was filtered, washed with an ice-cooled mixture of
The filtrate was washed with water and brine, dried over Na
2 4
SO ,
[
3
CH CN/water = 1:1] and dried under vacuum to yield 24 as a pale
filtered and concentrated in vacuo. The residue was purified by sil-
yellow solid (16 g, 91%).
3
ica gel column chromatography (CHCl ) to yield 30 as a white solid
1
H NMR (400 MHz, CDCl
3
) d = 8.60 (br s, 1H), 8.25 (d, J = 2.0 Hz,
(17.34 g, 45%).
1
1
8
H), 7.62 (d, J = 2.0 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.19 (dd, J = 2.4,
.4 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 6.80 (dd,
3
H NMR (400 MHz, CDCl ) d = 8.30 (d, J = 2.8 Hz, 1H), 8.24 (br s,
1H), 8.21 (d, J = 2.8 Hz, 1H), 6.84 (d, J = 1.2 Hz, 1H), 6.81 (dd, J = 1.6,
8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 5.93 (s, 2H), 4.61 (d, J = 5.6 Hz,
2H), 4.32 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H); MS (ESI+) 381.0
J = 1.6, 8.0 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.53 (br s, 1H), 5.92 (s,
2
2
H), 5.65 (br s, 1H), 4.60 (d, J = 5.6 Hz, 2H), 4.45 (d, J = 5.6 Hz,
H), 3.89 (s, 3H), 1.96 (s, 3H), 1.69 (s, 6H); 13C NMR (100 MHz,
(M+H) .
+
3
CDCl ) d = 169.7, 167.6, 156.5, 154.8, 154.5, 147.7, 146.6, 137.8,
1
1
33.2, 131.3, 129.9, 127.4, 122.6, 120.8, 112.2, 109.1, 108.3,
08.2, 106.2, 100.9, 93.0, 78.2, 56.2, 48.2, 44.7, 42.8, 29.4, 24.2;
4.1.23. Ethyl 2-((benzo[d][1,3]dioxol-5-ylmethyl)amino)-5-(3-
hydroxy-3-methylbut-1-yn-1-yl)nicotinate (31)
To a mixture of 30 (18.2 g, 48 mmol), 3-methyl-1-butyn-3-ol
(12.11 g, 144 mmol) in triethylamine (182 mL) were added
+
+
MS (ESI+) 549.1 (M+H) ; HRMS (FAB) Calcd for C20
4
H30ClN O
5
:
5
4
5
49.1905; Found: 549.1935; HPLC purity 97.2% (5.84 min).
2 3 2
PdCl (PPh ) (1.68 g, 2.4 mmol) and cupper(I) iodide (0.914 g,
.1.19. 4-Bromo-2-(difluoromethyl)-1-methylbenzene (26)
DAST (6 mL, 45.4 mmol) was added dropwise to a solution of
-bromo-2-methylbenzaldehyde (3.0 g, 15.1 mmol) in CH Cl
4.8 mmol) and the mixture was stirred at reflux for 2 h under Ar
atmosphere. The mixture was cooled and concentrated in vacuo.
The residue was suspended in AcOEt (364 mL) at 40 °C and filtered
2
2
(
60 mL) at 0 °C and the mixture was stirred at 0 °C for 1 h and
at room temperature for 2 h. The mixture was diluted with
CH Cl (60 mL), cooled to 0 °C and quenched with water
30 mL). The organic phase was separated, washed with brine,
dried over Na SO and filtered. After removal of the solvent in
through celite. The filtrate was washed with satd NH
and brine, dried over Na SO and filtered. The solution was concen-
trated in vacuo and the residue was recrystallized from CH CN
(91 mL) and water (46 mL) to yield 31 as a white solid (15.56 g, 85%).
4
Cl aq, water
2
4
2
2
3
(
1
2
4
3
H NMR (400 MHz, CDCl ) d = 8.40 (t, J = 5.0 Hz, 1H), 8.38 (s,
vacuo, the residue was purified by silica gel column chromatog-
raphy (AcOEt/n-hexane; 0:100 to 4:96) to yield 26 as a colorless
1H), 8.33 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 6.85 (d,
J = 1.6 Hz, 1H), 6.81 (dd, J = 1.6, 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz,
oil (2.62 g, 79%).
1H), 5.93 (s, 2H), 4.64 (d, J = 5.6 Hz, 2H), 4.31 (q, J = 7.2 Hz, 2H),
1
13
H NMR (400 MHz, CDCl
3
) d = 7.64 (d, J = 0.8 Hz, 1H), 7.47 (dd,
2.01 (s, 1H), 1.62 (s, 6H), 1.38 (t, J = 7.2 Hz, 3H);
C NMR
J = 0.8, 8.0 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.70 (t, J = 55.0 Hz,
H), 2.37 (s, 3H); MS (ESI+): compound did not ionize under the
condition.
(100 MHz, CDCl ) d = 166.9, 157.2, 156.2, 147.8, 146.8, 142.6,
3
1
133.0, 120.8, 108.3, 108.3, 106.8, 105.7, 100.9, 94.1, 79.2, 65.7,
+
61.1, 44.7, 31.6, 14.2; MS (ESI+) 383.1 (M+H) .
4
.1.20. 3-(Difluoromethyl)-4-methylbenzonitrile (27)
4.1.24. 2-((Benzo[d][1,3]dioxol-5-ylmethyl)amino)-5-(3-
hydroxy-3-methylbut-1-yn-1-yl)nicotinic acid (32)
A mixture of 31 (15.54 g, 40.6 mmol) and 1 N aqueous NaOH
(81 mL, 81 mmol) in MeOH (22.4 mL) was stirred at 60 °C for 2 h.
The organic solvent was removed in vacuo and the mixture was
neutralized with 1 N HCl (81 mL) and extracted with AcOEt. The
A mixture of 26 (2.62 g, 11.85 mmol) and CuCN (2.12 g,
3.7 mmol) in pyridine (1.3 mL) and DMF (1.3 mL) was stirred at
90 °C under microwave irradiation for 1.5 h. The mixture was
2
1
cooled to 0 °C and insolubles were filtered off and washed with
AcOEt (200 mL). The filtrate was diluted with n-hexane (65 mL),
washed with water and brine, dried over Na
The mixture was concentrated in vacuo and the residue was puri-
fied by silica gel column chromatography (AcOEt/n-hexane; 0:100
2
SO
4
and filtered.
organic phase was washed with brine, dried over Na
and concentrated in vacuo. The residue was recrystallized from
CH CN to yield 32 as a pale yellow solid (12.7 g, 88%).
2 4
SO , filtered
3
1
to 27:73) to give 27 as a white solid (1.73 g, 87%).
6
H NMR (400 MHz, DMSO-d ) d = 13.4 (br s, 1H), 8.62 (br s, 1H),
1
3
H NMR (400 MHz, CDCl ) d = 7.81 (s, 1H), 7.65 (d, J = 8.0 Hz,
8.26 (d, J = 2.4 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 6.90 (s, 1H), 6.84 (d,
J = 8.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 5.97 (s, 2H), 4.58 (d,
1
H), 7.36 (d, J = 8.0 Hz, 1H), 6.76 (t, J = 55.0 Hz, 1H), 2.51 (s, 3H);
+
+
MS (ESI+) 186.1 (M+H
2
O+H) .
J = 5.2 Hz, 2H), 1.44 (s, 6H); MS (ESI+) 355.1 (M+H) .
Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047

1
0
B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4
.1.25. 3-(Difluoromethyl)-4-methylbenzyl 2-((benzo[d][1,3]-
0.55H), 2.88 (d, J = 16.0 Hz, 0.45H), 2.25–2.37 (m, 1H), 2.08–
dioxol-5-ylmethyl)amino)-5-(3-hydroxy-3-methylbut-1-yn-1-
yl)nicotinate (33)
2.19 (m, 2H), 2.09 (s, 1.35H), 2.06 (s, 1.65H), 1.89–1.97 (m,
1H), 1.70–1.85 (m, 1H); C NMR (100 MHz, CDCl ) d = 169.4,
3
1
3
To a mixture of 32 (12.65 g, 35.7 mmol), 28 (8.9 g, 42.8 mmol)
and DIPEA (18.7 mL, 107 mmol) in DMF (126 mL) was added BOP
reagent (17.37 g, 39.3 mmol) over the period of 10 min at 0 °C.
The mixture was stirred at 0 °C for 1 h and at room temperature
for 1 h. The mixture was diluted with a solution of [AcOEt/n-hex-
167.1 [166.2], 158.5, 157.0 [156.9], 148.5 [148.2], 147.7, 146.6,
133.5, 131.7 [132.1], 129.6 [129.6], 129.6 [130.9], 124.6
[126.3], 122.7 [122.5], 121.3 [121.3], 120.7, 114.6, 109.4
[109.7], 108.3, 108.2, 100.9, 66.6 [66.6], 55.0 [50.8], 49.9 [53.6],
44.8, 39.5, 35.9 [37.4], 35.3 [33.8], 28.7 [30.5], 21.7 [20.8], the
minor counterparts of doubled signals due to rotamers are in
ane = 2:1] and washed with satd NaHCO
The solution was dried over Na SO , filtered and concentrated in
vacuo. The residue was recrystallized from CH CN (65 mL) to yield
3 as a white solid (16.08 g, 89%).
3
aq, water and brine.
+
2
4
the brackets; MS (ESI+) 541.2 (M+H) ; HPLC purity 99.1%
(4.85 min).
3
3
1
3
H NMR (400 MHz, CDCl ) d = 8.60 (t, J = 5.4 Hz, 1H), 8.28 (d,
4.1.28. 5-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-en-3-yl)-N-(3-chlo-
ro-4-methoxybenzyl)-2-(((2,3-dihydrobenzofuran-6-yl)methyl)
amino)nicotinamide (34c)
J = 2.0 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.45 (s, 1H), 7.32 (dd,
J = 3.2, 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 0.8 Hz, 1H),
6
.81 (dd, J = 0.8, 8.0 Hz, 1H), 6.76 (t, J = 55.4 Hz, 1H), 6.74 (d,
Starting form compound 14, the title compound was prepared
by the method described in the preparation of compound 1 with
J = 4.0 Hz, 1H), 6.31 (t, J = 5.2 Hz, 1H), 5.93 (s, 2H), 4.61 (d,
J = 5.6 Hz, 2H), 4.55 (d, J = 5.6 Hz, 2H), 2.43 (s, 3H), 1.59 (s, 3H),
compound 5 replaced by 3-chloro-4-methoxybenzylamine.
1
3
1
1
.56 (s, 3H); C NMR (100 MHz, CDCl
3
) d = 167.4, 156.6, 154.8,
3
H NMR (400 MHz, CDCl ) d = 8.33 (q, J = 5.2 Hz, 1H), 8.22 (d,
1
47.8, 146.7, 137.7, 135.9 (t, 3
J
C–F = 4.4 Hz), 135.7, 133.1, 132.8 (t,
J = 2.0 Hz, 0.55H), 8.13 (d, J = 2.0 Hz, 0.45H), 7.59 (d, J = 2.0 Hz,
0.45H), 7.50 (d, J = 2.0 Hz, 0.55H), 7.34 (s, 1H), 7.20 (dt, J = 2.0,
8.8 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.86 (t,
J = 5.4 Hz, 0.45H), 6.82 (dd, J = 1.2, 8.0 Hz, 1H), 6.79 (s, 1H), 6.68
(t, J = 5.4 Hz, 0.55H), 6.20 (dd, J = 5.2, 12.0 Hz, 1H), 4.86 (t,
J = 5.6 Hz, 0.45H), 4.80 (t, J = 5.2 Hz, 0.55H), 4.64 (t, J = 5.2 Hz,
2
3
J
C–F = 20.8 Hz), 131.7, 130.1, 125.3 (t,
J
C–F = 7.4 Hz), 120.9,
1
1
7
14.1(t,
9.2, 65.7, 44.8, 43.4, 31.5, 18.2; MS (ESI+) 508.1 (M+H) ; HRMS
JC–F = 238 Hz), 108.9, 108.4, 108.2, 106.1, 100.9, 94.4,
+
+
(
FAB) Calcd for C28
28 2 3 4
H F N O : 508.2048; Found: 508.2013; HPLC
purity 99.6% (6.30 min).
2
H), 4.53 (t, J = 8.8 Hz, 2H), 4.47 (d, J = 5.6 Hz, 2H), 4.25–4.35 (m,
4
.1.26. 5-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-en-3-yl)-2-(((2,3-di-
1H), 3.89 (s, 3H), 3.16 (t, J = 8.8 Hz, 2H), 3.03 (d, J = 16.0 Hz,
0.55H), 2.85 (d, J = 16.0 Hz, 0.45H), 2.25–2.37 (m, 1H), 2.09–2.16
(m, 1H), 2.02–2.09 (m, 1H), 2.06 (s, 1.35H), 2.03 (s, 1.65H), 1.89–
hydrobenzofuran-6-yl)methyl)amino)-N-(2-phenoxyethyl)nic-
otinamide (34a)
Starting form compound 14, the title compound was prepared
by the method described in the preparation of compound 1 with
1
3
3
1.97 (m, 1H), 1.70–1.85 (m, 1H); C NMR (100 MHz, CDCl )
d = 167.9, 167.1 [166.3], 160.4, 154.6, 139.9 (br), 132.1 (br), 131.8
[132.5], 131.2, 129.9 [129.9], 127.4 [127.4], 125.6, 124.7, 124.7
[126.5], 122.8, 122.6 [122.4], 119.6, 112.4, 108.6, 71.3, 56.2, 55.0
[50.8], 50.0 [53.6], 44.9, 42.9, 36.0 [37.4], 35.3 [33.8], 29.5, 28.7
[30.5], 21.6 [20.8], the minor counterparts of doubled signals due
to rotamers are in the brackets (3 signals corresponding to pyridine
ring were not observed due to line broadening most possibly
compound 5 replaced by 2-phenoxyethanamine.
1
3
H NMR (400 MHz, CDCl ) d = 8.34 (m, 1H), 8.24 (d, J = 2.4 Hz,
0
.55H), 8.17 (d, J = 2.4 Hz, 0.45H), 7.58 (d, J = 2.4 Hz, 0.45H), 7.51
(
d, J = 2.4 Hz, 0.55H), 7.28–7.35 (m, 2H), 7.11 (d, J = 7.2 Hz, 1H),
6.95–7.00 (m, 1H), 6.92 (dd, J = 1.2, 8.8 Hz, 2H), 6.83 (d, J = 8.4 Hz,
1H), 6.79 (s, 1H), 6.74 (t, J = 6.0 Hz, 0.45H), 6.64 (t, J = 6.0 Hz,
0.55H), 6.22 (dd, J = 1.2, 5.6 Hz, 1H), 4.94 (t, J = 4.8 Hz, 0.45H),
4.87 (t, J = 4.8 Hz, 0.55H), 4.64 (d, J = 5.6 Hz, 2H), 4.54 (t,
+
caused by amide rotation), MS (ESI+) 573.1 (M+H) ; HPLC purity
92.8% (5.03 min).
J = 8.8 Hz, 2H), 4.33–4.37 (m, 1H), 4.14 (t, J = 5.2 Hz, 2H), 3.81 (q,
J = 5.2 Hz, 2H), 3.16 (t, J = 8.8 Hz, 2H), 3.11 (dd, J = 4.0, 17.2 Hz,
4.1.29. 5-((1R,5S)-8-Acetyl-8-azabicyclo[3.2.1]oct-3-en-3-yl)-2-
((benzo[d][1,3]dioxol-5-ylmethyl)amino)-N-(3-chloro-4-meth-
oxybenzyl)nicotinamide (34d)
Starting form compound 14, the title compound was prepared
by the method described in the preparation of compound 1 with
compound 5 replaced by 3-chloro-4-methoxybenzylamine and
0
2
1
.55H), 2.86 (dd, J = 4.0, 16.8 Hz, 0.45H), 2.25–2.37 (m, 1H), 2.11–
.19 (m, 2H), 2.10 (s, 1.35H), 2.07 (s, 1.65H), 1.89–1.97 (m, 1H),
1
3
.70–1.85 (m, 1H); C NMR (100 MHz, CDCl
3
) d = 168.4, 167.1
[
[
166.2], 160.4, 158.5, 157.0 [157.1], 148.6 [148.3], 140, 132.1
130.1], 131.5 [131.9], 129.6 [129.6], 125.6, 124.7, 124.6 [126.3],
1
7
3
22.6 [122.5], 121.3 [121.3], 119.6, 114.6, 109.4 [109.7], 108.6,
1.3, 66.6 [66.6], 55.0 [50.8], 49.9 [53.6], 44.9, 39.5, 36.0 [37.4],
5.3 [33.9], 29.5, 28.7 [30.6], 21.7 [20.8], the minor counterparts
compound 10 replaced by 3,4-(methylenedioxy)benzylamine.
1
3
H NMR (400 MHz, CDCl ) d = 8.46 (q, J = 5.6 Hz, 1H), 8.22 (d,
J = 2.4 Hz, 0.55H), 8.13 (d, J = 2.4 Hz, 0.45H), 7.65 (d, J = 2.4 Hz,
0.45H), 7.54 (d, J = 2.4 Hz, 0.55H), 7.35 (t, J = 2.0 Hz, 1H), 7.20 (dt,
J = 2.0, 8.8 Hz, 1H), 7.15 (m, 0.45H), 6.95 (m, 0.55H), 6.88 (d,
J = 8.0 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 6.80 (dd, J = 1.6, 8.0 Hz,
1H), 6.73 (d, J = 7.6 Hz, 1H), 6.21 (d, J = 5.2 Hz, 0.55H), 6.17 (d,
J = 5.2 Hz, 0.45H), 5.91 (s, 2H), 4.82 (t, J = 5.2 Hz, 0.45H), 4.77 (t,
J = 5.2 Hz, 0.55H), 4.60 (d, J = 5.6 Hz, 2H), 4.70 (d, J = 5.6 Hz, 2H),
4.29–4.36 (m, 1H), 3.88 (s, 3H), 2.99 (d, J = 16.8 Hz, 0.55H), 2.85
(d, J = 16.8 Hz, 0.45H), 2.07–2.35 (m, 2H), 2.02–2.07 (m, 1H), 2.04
of doubled signals due to rotamers are in the brackets; MS (ESI+)
5
+
39.2 (M+H) ; HPLC purity 92.2% (4.87 min).
4
.1.27. 5-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-en-3-yl)-2-((benzo-
[
d][1,3]dioxol-5-ylmethyl)amino)-N-(2-phenoxyethyl)nicotina-
mide (34b)
Starting form compound 14, the title compound was prepared
by the method described in the preparation of compound 1 with
compound 5 replaced by 2-phenoxyethanamine and compound
1
3
(s, 1.35H), 2.02 (s, 1.65H), 1.65–1.92 (m, 2H); C NMR (100 MHz,
CDCl ) d = 168.0, 167.1 [166.3], 156.9 (br), 154.6, 148.5 (br),
1
0 replaced by 3,4-(methylenedioxy)benzylamine.
3
1
H NMR (400 MHz, CDCl
3
) d = 8.33 (m, 1H), 8.24 (s, 0.55H),
147.8, 146.6, 133.4, 132.0 [130.1], 131.8 [132.6] (br), 131.2, 129.9
[129.9], 127.4 [127.4], 124.8 [126.5] (br), 122.7 [122.4], 122.7,
120.7 [120.8], 112.3, 109.3 (br), 108.3, 108.2, 100.8, 56.2, 55.0
[50.9], 50.0 [53.6], 44.8, 42.9, 36.0 [37.3], 35.3 [33.8], 28.7 [30.5],
21.6 [20.8], the minor counterparts of doubled signals due to rota-
8.17 (s, 0.45H), 7.61 (s, 0.45H), 7.53 (s, 0.55H), 7.26–7.32 (m,
2H), 6.95–7.00 (m, 1H), 6.91 (d, J = 8.0 Hz, 2H), 6.85 (s, 1H),
6.80 (d, J = 8.4 Hz, 1H), 6.72–6.82 (m, 1H), 6.73 (d, J = 8.0 Hz,
1H), 6.22 (br s, 1H), 5.91 (s, 2H), 4.92 (t, J = 4.8 Hz, 0.45H),
4.85 (t, J = 4.8 Hz, 0.55H), 4.60 (d, J = 6.0 Hz, 2H), 4.33–4.37 (m,
1H), 4.09–4.16 (m, 2H), 3.75–3.83 (m, 2H), 3.06 (d, J = 16.0 Hz,
+
mers are in the brackets; MS (ESI+) 575.2 (M+H) ; HPLC purity
97.4% (4.97 min).
Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047

B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
4
4
.2. In vitro assays
Modified Eagle’s Medium (DMEM), supplemented with penicillin
50 U/ml), streptomycin (50 g/ml), HEPES (25 mM), 2.5% fetal
(
l
.2.1. Radioligand binding assay
bovine serum, and 10% horse serum. Cells were plated into 24-well
5
Binding affinity of the compounds was determined based on the
plate at densities of 1.5 Â 10 cells per well.
1
25
displacement of [ I] human ghrelin from its receptors in the cell
membrane fraction obtained from rat or human ghrelinR stably
4.2.4.2. Growth hormone secretion assay.
Pituitary cells
overexpressed in Chinese hamster ovary (CHO) cells. 3.34
l
g pro-
were cultured for 2 days, and then were pre-incubated with the
reaction buffer (Hanks’s balanced salt solution, containing 1%
bovine serum albumin, 0.25% glucose, and 25 mM HEPES) 1.5 h
before tested compounds were applied. The reaction buffer was
exchanged with a new one 15 min before the application of com-
pounds. Cells were treated with compounds at 37 °C for 15 min,
and then the supernatants were collected into the tubes containing
2% EDTA and 2000 U/ml Aprotinin. After centrifugation (2000g for
15 min at 4 °C), GH in the supernatants was measured with Growth
hormone ELISA kit (LINCO Research). The percentage of inhibition
caused by each compound was calculated from the formula
(C À E)/C Â 100, where C stands for the GH concentration released
with Ghrelin (1 nM) and E stand for that released with each
compound.
tein of the membrane fraction, various concentrations of com-
pounds and 0.04 nM [1 I] human ghrelin (PerkinElmer) were
incubated at room temperature for 30 min in 50 mM Hepes (pH
25
7
2 2
.5), 1 mM CaCl , 5 mM MgCl , 75 mM NaCl, and 0.5% w/v BSA in
MultiScreen HTS, Durapore, HV, 96-well filter plates (Millipore)
pretreated with 0.01% polyethyleneimine. Membranes were har-
vested and washed with 50 mM Hepes (pH 7.5), 1 mM CaCl
mM MgCl and 75 mM NaCl using vacuum manifold
Millipore). After addition of Microscint™-O, radioactivity was
2
,
5
2
,
a
(
counted in the TopCount™ NXT HTS Microplate Scintillation and
Luminescence Counter (Perkin Elmer).
4
.2.2. Inverse agonist assay
HEK293 (human embryonic kidney) cells were seeded at 15,000
cells/well into a 96-well collagen-coated plate in RPMI1640 con-
taining 10% FBS and cultured overnight at 37 °C. Growth medium
was removed and prewarmed Opti-MEM (Life Technologies) was
added to the plates. For transfection of cells in each well, 10 ng
rat ghrelinR expression vector, 100 ng PathDetect pNFAT-Luc plas-
4.2.5. Metabolic stability in liver microsomes
For the microsomal stability assay, 1 lM of each compound was
incubated with human liver microsomes (0.5 mg protein/mL) in
50 mM phosphate buffer (pH 7.4) containing 1 mM NADPH (the
reduced form of nicotinamide adenine dinucleotide phosphate) at
37 °C for 30 min. After the enzyme reaction was terminated by
the addition of a 5-fold volume of acetonitrile, the reaction mixture
was centrifuged at 4000 rpm for 5 min. The resultant supernatant
was used as a test sample to measure the stability in human liver
microsomes by quantitating the compound in the sample using
LC/MS/MS (liquid chromatography tandem mass spectrometry).
Metabolites were analyzed in the same sample.
mid (Agilent Technologies) and 0.3 lL Lipofectamine 2000 (Life
Technologies) were used. Plasmids and Lipofectamine were diluted
separately and preincubated for 5 min with Opti-MEM before they
were mixed together and incubated for 20 min. Then, they were
added to the plates. After incubation for about 5 h at 37 °C, the
plasmids-Lipofectamine solution was removed, compound solu-
tions diluted to the desired final concentrations in RPMI1640 con-
taining 0.1% dialyzed FBS were added, and then plates were
incubated overnight at 37 °C. Luciferase activity was detected
using the Steadylite HTS™ assay (PerkinElemer) and luminescence
was measured with the EnVision multilabel reader (PerkinElmer).
AlamarBlue (Life Technologies) was used to measure cell viability.
4.2.6. Equilibrium dialysis to determine plasma protein binding
Compounds (10 lM) were mixed with human plasma. The mix-
tures then were subjected to equilibrium dialysis against
Dulbecco’s phosphate-buffered saline (pH 7.4) at 37 °C for 4 h
using a Rapid Equilibrium Dialysis (Thermo Fisher Scientific K.K.).
On completion of the dialysis period, both the plasma and buffer
fractions were removed from the device. The plasma and buffer
fractions were mixed with a 5-fold volume of acetonitrile and then
the reaction mixture was centrifuged at 4000 rpm for 5 min. The
resultant supernatant was used as a test sample to measure the
protein binding in human plasma by quantitating the compound
in the sample using LC/MS/MS.
4
.2.3. Intracellular Ca2+ mobilization assay
The FLIPR Calcium 3 Assay Kit (Molecular Devices) was used for
the assay. CHO cells stably overexpressing rat ghrelinR were
seeded at 20,000 cells/well into 96-well black-wall, clear-bottom
plates in F-12 medium containing 10% FBS and were cultured over-
night at 37 °C. Growth medium was removed, and plates were
washed with calcium buffer (1 Hank’s Balanced Salt solution with
2
0 mM Hepes, 2.5 mM probenecid and 1 mg/mL BSA). Calcium 3
reagent reconstituted with calcium buffer was added to the plates
and cells were incubated for 2.5 h at room temperature.
FLEXstation (Molecular Devices) was used for monitoring the fluo-
rescence, adding compounds, and subsequently adding 1 nM
human ghrelin. The values of the fluorescence increase were calcu-
lated as the peak fluorescence value minus the basal level.
4.3. In vivo assays
4.3.1. Pharmacokinetics
Non-fasted SD rats (n = 3 per treatment group) were adminis-
tered compounds orally at a dose of 10 mg/kg or by the intra-
venous route (iv) at a dose of 1 mg/kg. Compounds were
dissolved in a vehicle of 5% DMSO and 10% 2-hydroxypropyl-b-cy-
clodextrin (HPCD) in saline. Blood samples were collected with
heparin as an anticoagulant at 0.08, 0.25,0.5 1, 2, 4, 6, 8, and 24 h
following iv dosing and at 0.25, 0.50, 1, 2, 4, 6, 8, and 24 h following
oral dosing. Samples were centrifuged, and the plasma was col-
lected and stored at À80 °C before analysis. Samples were analyzed
by LC/MS/MS. The pharmacokinetic parameters were calculated by
noncompartmental analysis.
4
4
.2.4. Growth hormone secretion in rat primary pituitary cells
.2.4.1. Rat pituitary cell culture24
Anterior pituitary glands
.
were obtained from male Sprague-Dawley (SD) rats at 6–8 weeks
old (Charles River Laboratories), and were minced into small frag-
ments. The fragments were dispersed to single cells with two incu-
bations of 3 mg/ml collagenase (Wako Pure Chemical Industries) at
3
7 °C for 30 min and Papain Dissociation System (Worthington
Biochemical Corporation) at 37 °C for 30 min. The resultant cell
suspension was isolated by gentle pipetting and passed through
4.3.2. HFDIO rat model
4
0
l
m filter. Cells were centrifuged at 300Âg for 5 min, and the
Male SD rats were fed with a high-fat diet (containing 60% fat)
for 10 weeks before the start of the study. All animals were habit-
uated to oral administration with vehicle for three weeks and
pelleted cells were subjected to a discontinuous density gradient
to remove cell debris. Cells were suspended in the Dulbecco’s
Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047

1
2
B. Takahashi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
animals that showed abnormal behavior during the habituation
were excluded from the study. Animals with mean ± 2SD body
weight were included in the study and were assigned randomly
to vehicle, treatment, or pair-fed groups. Compound or vehicle
was administered orally as a suspension in 0.5% hydroxypropyl cel-
lulose (w/v) at 9:30 am and 6:00 pm. Body weight and daily food
consumption were measured before the morning administration.
In the pair-fed group, the amount of food was limited to that of
the average food intake of compound-treated group the day before,
and animals were treated with vehicle twice daily.
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Please cite this article in press as: Takahashi, B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.047