Novel pyrimidoazepine analogs as serotonin 5-HT2A and 5-HT 2C receptor ligands for the treatment of obesity

Article abstract of DOI:10.1016/j.ejmech.2013.02.020

Obesity is one of the most serious public health problems worldwide in the 21st century. Current therapeutic treatment for obesity is mostly focused on preventive measures involving dietary control and physical exercises in combination with anti-obesity m

Full text of DOI:10.1016/j.ejmech.2013.02.020

European Journal of Medicinal Chemistry 63 (2013) 558e569
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel pyrimidoazepine analogs as serotonin 5-HT2A and 5-HT2C
receptor ligands for the treatment of obesity
,
,
,d
Ha Yun Yang a b, Jinsung Tae b, Yong Wan Seo a, Yoon Jung Kim a c, Hye Yeon Im a
,
Gil Don Choi e, Heeyeong Cho e, Woo-Kyu Park e, Oh Seung Kwon a, Yong Seo Cho a,
Minkyung Ko a, HyunSeo Jang a, Jaeick Lee f, Kihang Choi c, Chan-Hwa Kim d, Jiyoun Lee g
**
,
*
,
Ae Nim Pae a
,
a Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
b Department of Chemistry, Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea
c Department of Chemistry, Korea University, Seoul 136-701, Republic of Korea
d School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
e Pharmaceutical Screening Research Team, Korea Research Institute of Chemical Technology, Daejeon 305-343, Republic of Korea
f Doping Control Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
g Department of Global Medical Science, Sungshin Women’s University, Seoul 142-732, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Obesity is one of the most serious public health problems worldwide in the 21st century. Current
therapeutic treatment for obesity is mostly focused on preventive measures involving dietary control and
physical exercises in combination with anti-obesity medications. However, most of these anti-obesity
medications have little or no effect on weight loss, and some cases have demonstrated fatal side ef-
fects. Due to the urgent need for highly potent and selective anti-obesity agents, the serotonin receptors
(5-HTR) have been the focus of much interest as a novel therapeutic target. In this report, we have
developed pyrimidoazepine analogs targeting the 5-HT2A and 5-HT2C receptors and evaluated their
biological activity in vitro and in vivo as novel anti-obesity agents. We were able to identify 6p as the
most potent 5-HT2A and 5-HT2C ligand in vitro (IC50 ¼ 3 nM and 2.3 nM, respectively), and this com-
pound also demonstrated the greatest potency in vivo. In an acute obesity model, mice treated with 6p
showed significant decrease in body weight gain and food intake over approximately 77e94% compared
to a control group. In a chronic obesity model, mice treated with 6p also showed a marked decrease in
food intake and body weight gain.
Received 8 September 2012
Received in revised form
15 February 2013
Accepted 18 February 2013
Available online 27 February 2013
Keywords:
5-HT2A antagonist
5-HT2C antagonist
Obesity
Pyrimidoazepine
Serotonin receptor ligands
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
The main treatment for obesity is limiting food intake
with physical exercise in combination with anti-obesity medica-
Obesity is a chronic medical condition defined as abnormal or
excessive adipose tissue accumulation and is recognized as one of
the most serious public health problems worldwide. Excessive
food intake combined with lack of physical activity often lead to
obesity. In addition, endocrine disorders and psychiatric illnesses
can also cause obesity in some cases. Obesity becomes a serious
threat to public health worldwide because it is linked to a variety
of physical and mental disorders such as type II diabetes, ischemic
heart diseases, dyslipidemia, osteoarthritis, and depression [1].
tions. Currently there are three types of anti-obesity drugs in the
market; (1) dual-acting serotonin and norepinephrine reuptake
inhibitors such as sibutramine to suppress appetite; (2) thermo-
genesis inducers such as over-the-counter weight-loss pills to
increase energy consumption; (3) lipase inhibitors such as orlistat
to interfere with nutrient partitioning [2]. However, these drugs
have demonstrated little or no effect on weight loss, and appetite
suppressants, such as sibutramine, were found to have severe side
effects on cardiovascular system and withdrawn from the market
recently [3].
The serotonin receptors (5-HTR) are primarily found in the
gastrointestinal (GI), platelets, and in the central nervous system
(CNS) in human and play a major role in regulating mood, appetite,
sleep, and some cognitive functions including memory and
* Corresponding author. Tel.: þ82 2 920 7281; fax: þ82 2 920 2027.
** Corresponding author. Tel.: þ82 2 958 5185; fax: þ82 2 958 5189.
E-mail addresses: jlee@sungshin.ac.kr (J. Lee), anpae@kist.re.kr (A.N. Pae).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.020

H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
559
Scheme 1. Synthesis of pyrimidoazepine. Reagents: (a) ethyl diazoacetate, BF3eether complex, ether, ꢀ25 ꢁC; (b) 9 or 11, sodium ethoxide, ethanol, 0 ꢁC to rt; (c) triflic anhydride,
pyridine, CH2Cl2, 0 ꢁC to rt; (d) R2H, NaHCO3, MeCN, 24 h*; * Conditions used for 5a: Pd(OAc)2, 1,1’-bis(diphenylphosphino)ferrocene, triethylamine, formic acid, DMF, 50 ꢁC, 2 h; for
5h: Me3OBF4, DCM, 2 h (e) 30% trifluoroacetic acid, CH2Cl2; (f) 37% HCHO, NaBH(OAc)3, MeOH, 2 h.
learning [4]. The serotonin receptors are composed of 14 distinct
families that are G protein-coupled receptors with the exception of
the 5-HT3 receptor, a ligand-gated ion channel [5]. Among these
2. Chemistry
The syntheses of pyrimidoazepine derivatives started with
commercially available N-Boc piperidone as described in
Scheme 1. Lewis acid-catalyzed condensation of the piperidone
with ethyl diazoacetate yielded 2. Subsequent condensation re-
action with amidine fragments (9 and 11) produced the pyr-
imidoazepine core 3. After converting a tautomeric alcohol group
to a triflate, a library of substituted amines and alcohols were
added to yield the desired product 5 in 50e99% yield. Depro-
tection of a BOC group with 30% TFA afforded the free secondary
amine 6, and subsequent reductive amination of 6k yielded 6l.
Each final compound was produced in total five steps with an
overall yield of 10%.
The key amidine fragments 9 and 11 were synthesized from
commercially available starting material as shown in Scheme 2. The
piperazinyl fragment 9 was obtained by reacting commercially
available methyl piperazine (7) with 1H-pyrazole-1-carboxamidine
hydrochloride (8) in the presence of DIPEA. The benzyl fragment
11 was provided by employing Garigipati’s reaction with 2-
phenylacetonitrile (4) in 82% yield [15].
families, the 5-HT2 receptor has three subtypes, 5-HT2A, 5-HT2B
,
and 5-HT2C, which exhibit substantial sequence similarity. In
particular, the 5-HT2C receptors are present mostly in hypothala-
mus and are known to be involved in the regulation of appetite and
food intake. After the failure of the early anti-obesity agents due to
serious side effects, considerable efforts to find a new therapeutic
target were made toward the serotonin receptors. Extensive animal
studies and genetics analysis have proven that the 5-HT2C is a valid
target for the treatment of obesity. For example, mice lacking the
5-HT2C receptor display excessive food intake and severe obesity
[6]. A known 5-HT2C agonist, mCPP successfully showed reduced
food intake and weight loss when administered in mice model, and
a selective 5-HT2C agonist, locarserin [7], is approved by U.S. FDA in
2012 for the treatment of obesity in human.
Recently, the 5-HT2A receptor has also gained much attention
due to its similarity to the 5-HT2C. The 5-HT2A receptor regulates
adrenaline levels that are responsible for hepatic glucose produc-
tion, insulin secretion, and glucose uptake by tissues [8].
Up-regulation of the 5-HT2A receptor is often found in obesity and
diabetes, and leads to high blood sugar level [9]. Therefore, it is
suggested that selective blockade of the 5-HT2A in obese patients
can alleviate hyperglycemia. A number of 5-HT2A receptor antag-
onists have been developed and some of these antagonists are
currently undergoing preclinical and clinical trials for the treat-
ment of obesity [10e13].
Previously, we have identified a series of 5-HT2C ligands con-
taining an aminopyrimidine core based on a sequential virtual
screening with known 5-HT2C agonists [14]. We were also able to
find that these potent 5-HT2C ligands showed potent antagonistic
activity toward the 5-HT2A receptor (See supporting information,
Table S1). Because both the 5-HT2A and 5-HT2C receptors have been
the targets of interest in treating obesity, we believe that the
combined activity toward both receptors can be beneficial for
treating obesity. Therefore, we decided to further optimize these
compounds as dual acting anti-obesity agents. In this report, we
have designed and synthesized a library of 5-HT2A and 5-HT2C li-
gands containing pyrimidoazepine scaffold, and evaluated their
biological activities in vitro and in vivo.
Scheme 2. Synthesis of amidine fragments 9 and 11. Reagents: (a) DIPEA, DMF; (b)
Ammonium chloride, trimethylaluminium, toluene, 0e80 ꢁC.

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H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
3. Results and discussion
in C57BL/6 mice as shown in Table 4. After treating mice with
50 mg/kg of each compound, food intake was measured at an in-
terval of 2, 6,12 h and compared to vehicle control. Not surprisingly,
the compounds with a benzyl moiety on R1 position (Group II)
showed better in vivo activity, as it was observed from in vitro
activity. In general, all the tested compounds showed moderate to
significant effect in C57BL/6 mice. Among them, mice treated with
6p showed 77% of reduced food intake and 85% of reduced body
weight gain compared to a control group.
We further assessed anti-obesity activity of 6p by employing an
obesity-induced mice model, B6.V-Lepob/J (Table 5 and Fig. 1).
B6.V-Lepob/J is a homozygous strain for the obese spontaneous
mutation, and these mice can gain weight rapidly and may reach
three times the normal weight of wild-type control. With this mice
model, even further reduced food consumption and body weight gain
were observed upto 93.30% and 80.24% respectivelyafter 12 h (Fig.1).
3.1. In vitro functional assays to measure receptor potency and
selectivity
To measure their binding affinity and effective concentrations of
all the synthesized ligands, in vitro biological activity was deter-
mined by competitive binding assays and calcium mobilization
assay as shown in Table 1. A 5-HT2A antagonist ketanserine, and a
5-HT2C agonist mCPP were included in all experiments as compar-
ative standards. Additionally metitepine, a receptor antagonist for
various dopamine and serotonin receptors is also added as a control.
Two groups of derivatives with pyrimidoazepine core were tested;
group I, compounds 6ael, have a methyl piperazine on R1 position
and group II, compounds 6mes, have a benzyl moiety on the same
position. In order to analyze structureeactivity relationship of the
pyrimidoazepine scaffold, in vitro activity of all compounds with
various R2 and R3-substituted analogs were tested.
The results from in vitro assays demonstrated that group II
compounds e the compounds with a benzyl group on R1 position
(6me6s) showed significantly higher binding affinity toward both
the 5-HT2C and 5-HT2A receptors compared to the compounds with a
piperazine group (group I, 6ae6l). Interestingly, most of group I
compounds with small aliphatic amine groups on R2 position (6ae
6g) seemed to be poor ligands, however, compounds with bulkier
aromatic substituents on R2 position (6ie6l) exhibited slightly
improved binding affinity with IC50 values in the low micromolar
range. Replacing electron-donating methoxyaniline (6j) to fluroani-
line (6k) further enhanced binding affinity against the 5-HT2C up to
10 fold.
Among the benzyl-substituted compounds (group II), the
aniline substituted analog, 6n, showed excellent binding affinity
toward both the 5-HT2A and 5-HT2C receptors (IC50 ¼ 5.2 and 14.8 nM
respectively). When an electron-withdrawing group such as fluorine
and chlorine was added to the aromatic ring, these compounds, for
example 6o, 6p, and 6r, showed higher binding affinity up to 50e100
fold compared to methoxyaniline substituted compounds (6q and
6s). Additionally, when m-methoxyaniline on R2 (6q) was replaced
with p-methoxyaniline (6s), IC50 against the 5-HT2C increased to 9-
fold, whereas IC50 against the 5-HT2A reduced by two thirds (from
84.4 nM to 57.4 nM) suggesting that the position of the substituents
on the aniline ring is likely to affect selectivity for binding. It is also
identified that aromatic amines on R2 position (6je6s) have antag-
onistic activity against the 5-HT2C whereas aliphatic amines (6ce6g)
have agonistic activity. Much to our surprise, all of the compounds
containing a substituted aniline group on R2 position displayed
3.3. Chronic in vivo assays to measure effects on rat food intake and
body weight
In order to evaluate chronic anti-obesity effect in animal model,
we performed a 14-day chronic study with compound 6p, and
measured food intake and body weight gain in mice. First, 6p was
injected at 5, 25, 50 mg/kg once daily in C57BL/6 mice over 14 days,
however only a reduced weight gain of 18.8% at the highest dose
(50 mg/kg) was observed (Fig. 2). Next, we decided to administer
6p once daily in obesity induced mice, B6.V-Lepob/J, however, again,
only a slight reduction in food intake and body weight gain at the
doses of 25 mg/kg and 50 mg/kg were observed, which was sta-
tistically insignificant (See supporting information, Fig. S1).
Finally, we treated obese mice with 6p twice daily and signifi-
cant decreases in food intake and body weight gain were observed
at the doses of 25 mg/kg and 50 mg/kg (Fig. 3). Unfortunately, at the
highest concentration, 50 mg/kg, some of the mice died during the
experiment due to possible toxicity. However, at the dose of 25 mg/
kg, significant reductions in food intake and body weight gain,
15.23% and 32.7% respectively, were observed. In addition, we
measured the weight of each organ harvested from the chronic
assays as shown in Table 6. Interestingly, mice treated with 25 mg/
kg of 6p showed a slightly lighter liver and gonadal adipose tissue
compared to the rest of mice suggesting that fat storage in these
mice might have been reduced.
3.4. Pharmacokinetics study of 6p
To evaluate the in vivo stability of 6p, we determined the
pharmacokinetic profile in rats and these parameters are shown in
Table 7. Compound 6p was absorbed rapidly and had high oral
bioavailability (55.1%); terminal half life (T1/2) was longer than 12 h,
and the mean B/P ratio (AUCbrain/AUCplasma) was high (9.33 in
intravenous and 0.66 in oral administration) indicating that 6p also
exhibited rapid brain uptake.
antagonistic activity toward the 5-HT2A as well as the 5-HT2C
.
To assess the selectivity of the synthesized ligands, IC50 values of
the compounds with higher binding affinity, 6ne6q, were
measured against various serotonergic, dopaminergic and adren-
ergic receptors as shown in Tables 2 and 3. When compared to IC50
values against the 5-HT2B receptors, compound 6n and 6p exhibited
some selectivity for the 5-HT2A and the 5-HT2C receptors having 4e
45-fold higher binding affinity (Table 2). All the selected compounds
showed little or no activity against the dopaminergic D3 and D4
receptors, and the adrenergic a1A and a2A receptors (Table 3).
Although these selected compounds also appear to have moderate
activity against the 5-HT6 and D2 receptors, again, they demon-
strated higher binding affinity toward the 5-HT2A and 5-HT2C
receptors.
4. Conclusion
In summary, we have developed a series of pyrimidoazepine
derivatives targeting the 5-HT2A and 5-HT2C receptors as novel anti-
obesity agents. Our structureeactivity relationship study revealed
that compounds with aliphatic amine substituents demonstrated
low binding affinity to both the receptors and exhibited agonistic
activity against the 5-HT2C. However, compounds with aniline
substituents demonstrated excellent binding affinity and showed
antagonistic activity to both the receptors. Since we found several
dual-targeting compounds showing high affinity for both the
5-HT2A and 5-HT2C receptors, we decided to move forward to
3.2. Acute in vivo assays to measure effects on mouse food intake
To determine effects on weight loss and food intake in vivo,
several compounds from both group I and group II were tested

H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
561
Table 1
Functional activity assays of 5-HT2C and 5-HT2A ligands.
R2
N
N
N R3
R1
a
Compound
R1
R2
R3
5-HT2C
5-HT2A
Binding affinity
IC50 (nM)
Functional assay
Binding affinity
IC50 (nM)
Functional assay
Type
EC50 (nM)
Type
EC50 (nM)
6a
6b
6c
6d
6e
Methyl piperazine
Methyl piperazine
Methyl piperazine
Methyl piperazine
Methyl piperazine
H
H
H
H
H
H
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
CH3NHe
CH3CH2NHe
(CH3)2Ne
(CH3CH2)2Ne
Antagonist
1200
2900
Agonist
Agonist
>10,000
1200
Antagonist
Antagonist
6f
Methyl piperazine
Methyl piperazine
Methyl piperazine
Methyl piperazine
H
H
H
H
>10,000
>10,000
>10,000
1262
Agonist
Agonist
9600
>10,000
>10,000
>10,000
2625
6g
6h
6i
>10,000
687
CH3Oe
6j
Methyl piperazine
H
H
2852
Antagonist
Antagonist
2300
1500
>10,000
6k
6l
Methyl piperazine
Methyl piperazine
337
779
215
684
Antagonist
153
CH3
6m
6n
Benzyl
Benzyl
H
H
333
5.2
1271
14.8
Antagonist
Antagonist
96.3
6.1
Antagonist
20.1
6o
6p
6q
Benzyl
Benzyl
Benzyl
H
H
H
13.3
2.3
4.92
3
Antagonist
Antagonist
Antagonist
27.9
6.4
Antagonist
Antagonist
Antagonist
25.1
9.2
37.3
84.4
77.6
6r
6s
Benzyl
Benzyl
H
H
8.5
2.6
1.95
57.4
269
Ketanserin
mCPP5
Antagonist
Agonist
1.04
7.2
Agonist
8.1
Metitepine
Antagonist
0.38
a
All compounds were tested as HCl salt form.
evaluate anti-obesity effects of these compounds in vivo. Based on
the in vitro functional activity and receptor selectivity, several
compounds were selected for further biological evaluation. Com-
pound 6p, which showed the highest in vitro potency, also
demonstrated the greatest effect on acute weight gain and food
consumption in mice. When administered twice daily at the dose of
25 mg/kg for 14 days in obese mice, 6p showed a marked decrease
in food intake and weight gain. In addition, we observed that mice
administered with 6p also showed a moderate decrease in the
weight of liver and gonadal adipose tissue. We believe that our
study provides a useful insight into dual-targeting 5-HTR ligands as
anti-obesity therapeutics. Based on the structure activity

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H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
Table 2
Table 4
In vitro selectivity and binding affinity of 6n and 6p against the 5-HT2A, 5-HT2B, and
Acute effects on food intake and body weight gain in C57BL/6 mice.
5-HT2C receptors.
Compound
(50 mg/kg)
Decreased
food intake %
Decreased body
weight %
Compounds
IC50 (nM)a
5-HT2A
Selectivityb
2B/2A
Positive control
Group I
mCPP (5.6 mg/kg)
13.12 ꢂ 3.27*
12.51 ꢂ 6.45*
4.17 ꢂ 3.36
5-HT2B
5-HT2C
2B/2C
6d
6e
6g
6k
6m
6n
6o
6p
6q
12.24 ꢂ 8.89*
9.01 ꢂ 5.75
6n
6p
2.69
7.53
89
32
1.98
3.94
33.1
4.2
44.9
8.1
2.10 ꢂ 3.74
ꢀ15.02 ꢂ 4.89
5.61 ꢂ 4.29
a
IC50 measurements were performed at 25 ꢁC (for 5-HT2A and 5-HT2C) and 37 ꢁ
C
10.98 ꢂ 2.91
18.44 ꢂ 5.39
64.24 ꢂ 8.50***
69.37 ꢂ 9.11***
76.59 ꢂ 7.46***
55.79 ꢂ 11.68***
(for 5-HT2B) by using radioligands of 0.5 nM [3H]Ketanserin (5-HT2A), 1.2 nM [3H]
Group II
11.97 ꢂ 4.71
Lysergic acid diethylamide (5-HT2B), and 1.0 nM [3H]Mesulergine (5-HT2C).
71.81 ꢂ 9.91***
68.88 ꢂ 11.41**
84.75 ꢂ 7.98***
63.85 ꢂ 14.32***
b
Selectivity was calculated by dividing IC50 values against each 5-HT receptor.
relationship, further optimization to improve biological activity is
underway.
*P < 0.05, **P < 0.005, ***P < 0.0005.
The reaction was quenched with water, partitioned with ethyl acetate
and the phases separated. The combined organic layers were dried
over MgSO4, filtered and concentrated in vacuo to provide the crude
product as an orange gum. The residue was purified by flash column
chromatography to afford the title compound as a yellow solid
5. Experimental
5.1. Synthesis
(760 mg, 30%). 1H NMR (300 MHz, CDCl3)
d
ppm 3.73 (t, J ¼ 4.90 Hz,
5.1.1. General methods
All non aqueous reactions were carried out under an argon or
nitrogen atmosphere at room temperature, unless otherwise noted.
All reagents and solvents were purchased from commercial sources
and were used without further purification or distillation, unless
otherwise stated. Analytical thin-layer chromatography (TLC) was
performed on Merck KGaA silica gel 60 F254 (0.25 mm). Com-
pounds were visualized by UV light and/or stained with either
iodine or KMnO4 followed by heating. Flash column chromatog-
raphy was performed on Kanto chemical silica gel 60 (particle size
4H), 3.53 (d, J ¼ 35.68 Hz, 4H), 2.77 (d, J ¼ 10.46 Hz, 4H), 2.48 (t,
J ¼ 4.86 Hz, 4H), 2.35 (s, 3H), 1.50 (s, 9H).
5.1.1.3. tert-Butyl
2-benzyl-4-oxo-5,6,8,9-tetrahydro-3H-pyrimido
[4,5-d]azepine-7(4H)-carboxylate (3b). The title compound was
prepared by similar method to that of General Procedure A. The title
compound was obtained as a yellow solid (1.37 g, 73%). 1H NMR
(300 MHz, CDCl3)
d ppm 7.35 (m, 5H), 3.96 (s, 2H), 3.59 (d,
J ¼ 7.40 Hz, 4H), 2.98 (m, 2H), 2.90 (m, 2H), 1.49 (s, 9H).
of 63e210 m
m). 1H NMR and 13C NMR were carried out using Bruker
Avance 400 spectrometer at 400 MHz or Bruker Avance 300 spec-
trometer at 300 MHz. Chemical shift information for each signal is
given in part per million (ppm) relative to trimethylsilane (TMS).
High-resolution mass spectrometry (HRMS) was performed using
an LTQ Orbitrap (Thermo Electron Corporation).
5.1.1.4. General procedure B for the synthesis of tert-butyl 2-(4-methyl-
piperazin-1-yl)-4-(trifluoromethylsulfonyloxy)-5,6,8,9-tetrahydro-3H-
pyrimido[4,5-d]azepine-7(4H)-carboxylate (4a). Triflic anhydride
(1.20 ml, 3.6 mmol) was added dropwise to a solution of the tert-
butyl 2-(4-methylpiperazin-1-yl)-4-oxo-5,6,8,9-tetrahydro-3H-pyr-
imido[4,5-d]azepine-7(4H)-carboxylate 3a (1 g, 2.8 mmol) and pyr-
idine in CH2Cl2 at 0 ꢁC. The resulting solution was allowed to warm to
room temperature and stirred for 1 h. The reaction mixture was
quenched by addition of water then partitioned between 5% citric
acid and ethyl acetate. The organic layer was washed with sodium
bicarbonate (sat.), dried over MgSO4 and concentrated under vac-
uum to yield the crude product as a red gum (1.38 g, 90%). 1H NMR
5.1.1.1. tert-Butyl 4-oxo-5-propionylazepine-1-carboxyate (2). A so-
lution of N-Boc piperidone (3.5 g, 17.5 mmol) in dry ether (25 ml)
was treated with ethyl diazoacetate (3 g, 26.3 mol) and BF3eEther
complex (2.48 g, 17.5 mmol) dropwise simultaneously and stirred
1 h at ꢀ25 ꢁC. The product mixture was neutralized with saturated
potassium carbonate and extracted with EtOAc. The organic
phase was dried with MgSO4, and concentrated (5 g, 99%). 1H
(300 MHz, CDCl3)
d ppm 4.06 (s, 4H), 3.57 (m, 4H), 3.04 (m, 6H), 2.79
NMR (400 MHz, CDCl3)
d ppm 4.20 (m, 2H), 3.71 (m, 3H), 3.38
(s, 5H), 1.49 (s, 9H).
(m, 2H), 2.81 (m, 1H), 2.66 (m, 1H), 2.03 (m, 2H), 1.43 (s, 9H), 1.25
(t, J ¼ 12.9, 3H).
5.1.1.5. tert-Butyl 2-benzyl-4-(trifluoromethylsulfonyloxy)-8,9-
dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (4b). The ti-
tle compound was prepared by similar method to that of General
Procedure B. The title compound was obtained as a yellow solid
5.1.1.2. General procedure A for the synthesis of tert-butyl 2-(4-methyl-
piperazin-1-yl)-4-oxo-5,6,8,9-tetrahydro-3H-pyrimido[4,5-d]azepine-
7(4H)-carboxylate (3a). 4-Methyl-piperazine-1-carboximidamide 9
(1 g, 5.6 mmol) and tert-butyl 4-oxo-5-propionyl-azepine-1-
carboxyate 2 (1.08 g, 3.8 mmol) were added to a solution of NaOEt
in EtOH at 0 ꢁC and stirred at 0 ꢁC to room temperature overnight.
(1.37 g, 91%). 1H NMR (400 MHz, CDCl3)
d ppm 7.32 (m, 5H), 4.19 (s,
2H), 3.61 (m, 4H), 3.18 (m, 2H), 2.92 (m, 2H), 1.48 (s, 9H).
5.1.1.6. tert-Butyl 2-(4-methylpiperazin-1-yl)-8,9-dihydro-5H-pyrimido
[4,5-d]azepine-7(6H)-carboxylate (5a). Palladium acetate (7 mg,
0.03 mmol), (1,10-bis(diphenylphosphino)ferrocene) (33.3 mg,
Table 3
Binding affinity of 6n, 6o, 6p, 6q against serotonergic, dopaminergic, and adrenergic
receptors.
Table 5
Dose-dependent effects of 6p on food intake in B6.V-Lepob/J mice.
Compounds IC50 (nM)
5-HT1A 5-HT6 5-HT7 D2
D3
D4
a1A
a2A
12.5 mg/kg
25 mg/kg
50 mg/kg
6n
6o
6p
6q
394.8 36.8
1252.0 22.2
1057.0 21.8
822.8 30.4
1564 146.7 271.5 111.9 1390
>10,000
N/D
0e2 h (%)
0e6 h (%)
0e12 h (%)
98.56 ꢂ 0.85***
71.03 ꢂ 3.26 ***
47.76 ꢂ 6.23***
99.79 ꢂ 0.21***
90.15 ꢂ 2.87***
77.81 ꢂ 3.18***
99.82 ꢂ 0.18***
98.62 ꢂ 0.78***
93.30 ꢂ 2.84***
1059
1003
63.3 674.3 734.7 N/D
85.8 807.3 899.7 >10,000 >10,000
N/D
497 162.8 940.7 549.0 N/D
*P < 0.05, *P < 0.005, ***P < 0.0005.

H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
563
(300 MHz, CDCl3)
d ppm 3.76 (m, 4H), 3.57 (m, 4H), 2.95 (s, 3H),
2.85 (m, 2H), 2.46 (m, 6H), 2.43 (s, 3H), 1.45 (s, 9H).
5.1.1.8. tert-Butyl 2-(4-methylpiperazin-1-yl)-4-(propylamino)-8,9-
dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5c). The ti-
tle compound was prepared by similar method to that of General
Procedure C. The title compound was obtained as a white solid
(430 mg, 53%). 1H NMR (300 MHz, CDCl3)
d ppm 7.30 (m, 1H), 7.19
(t, J ¼ 8.14 Hz, 1H), 6.94 (d, J ¼ 2.53 Hz, 1H), 6.60 (d, J ¼ 5.27 Hz, 2H),
6.40 (s, 1H), 3.79 (m, 7H), 3.62 (m, 4H), 2.93 (t, J ¼ 5.26, 2H), 2.65
(t, J ¼ 4.89, 2H), 2.45 (t, J ¼ 4.95, 4H), 2.34 (s, 3H), 1.48 (s, 9H).
5.1.1.9. tert-Butyl 4-(dimethylamino)-2-(4-methylpiperazin-1-yl)-8,9-
dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5d). The title
compound was prepared by similar method to that of General
Procedure C. The title compound was obtained as a white solid
Fig. 1. Effects on body weight gain of acute administration of 6p in B6.V-Lepob/J mice
after 12 h (*P < 0.05, **P < 0.005, ***P < 0.0005).
(90 mg, 8%). 1H NMR (400 MHz, CDCl3)
d ppm 3.93 (m, 4H), 3.53 (m,
4H), 2.84 (m, 8H), 2.74 (s, 6H), 2.54 (s, 3H), 1.48 (s, 9H).
5.1.1.10. tert-Butyl 4-(diethylamino)-2-(4-methylpiperazin-1-yl)-8,9-
dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5e). The ti-
tle compound was prepared by similar method to that of General
Procedure C. The title compound was obtained as a yellow solid
d ppm 3.76 (m, 4H), 3.53
(m, 4H), 2.86 (m, 2H), 2.68 (m, 2H), 2.46 (m, 4H), 2.33 (s, 3H), 1.48
(s, 9H).
0.06 mmol), triethylamine (0.63 ml, 4.5 mmol) and formic acid
(0.11 ml, 3.0 mmol) were added to a solution of tert-butyl 2-(4-
methylpiperazin-1-yl)-4-(trifluoromethylsulfonyloxy)-5,6,8,9-tetrah-
ydro-3H-pyrimido[4,5-d]azepine-7(4H)-carboxylate (4a) in DMF
(1 ml) and the reaction mixture was warm to 50 ꢁC for 2 h with
stirring, to give a mixture of the corresponding pyrimidinone. Water
(4 ml) was added and products extracted with diethyl ether. The
combined organics were washed with brine, dried over MgSO4,
filtered and the solvent evaporated in vacuo to product residue as a
gum. The crude material was purified by flash column chromatog-
raphy using a CH2Cl2:EtOAc (7:3) mixture to provide the title com-
pound as a brown solid (110 mg, 65%). 1H NMR (300 MHz, CDCl3)
(180 mg, 14%). 1H NMR (300 MHz, CDCl3)
5.1.1.11. General procedure D for the synthesis of tert-butyl 2-(4-
methylpiperazin-1-yl)-4-morpholino-8,9-dihydro-5H-pyrimido[4,5-d]
azepine-7(6H)-carboxylate (5f). A solution of morpholine (0.9 ml,
9 mmol) was added to a solution of tert-butyl 2-(4-methylpiperazin-
1-yl)-4-(trifluoromethylsulfonyloxy)-5,6,8,9-tetrahydro-3H-pyrimi-
do[4,5-d]azepine-7(4H)-carboxylate (4a, 600 mg, 0.3 mmol) in
MeCN (9 ml) at room temperature with stirring. The mixture was
stirred at room temperature for 24 h. The solvent was evaporated in
vacuo to provide the crude title compound as a yellow gum. The
crude material was purified by flash column chromatography to
yield the desired product as a white crystalline solid (240 mg, 18%).
d
ppm 7.96 (s, 1H), 3.77 (m, 4H), 3.49 (m, 4H), 2.87 (m, 2H), 2.65 (m,
2H), 2.41 (m, 4H), 2.28 (s, 3H), 1.48 (s, 9H).
5.1.1.7. General procedure C for the synthesis of tert-butyl 4-(meth-
ylamino)-2-(4-methylpiperazin-1-yl)-8,9-dihydro-5H-pyrimido[4,5-
d]azepine-7(6H)-carboxylate (5b). A solution of methylamine
(0.62 ml, 1.0 mmol) and sodium bicarbonate (50 mg, 0.6 mmol) was
added to a solution of tert-butyl 2-(4-methylpiperazin-1-yl)-4-
(trifluoromethyl-sulfonyloxy)-5, 6, 8, 9-tetrahydro-3H-pyrimido
[4,5-d]azepine-7(4H)-carboxylate (4a, 100 mg, 0.2 mmol) in MeCN
(5 ml) at room temperature with stirring. The mixture was stirred
at room temperature for 24 h. The solvent was evaporated in vacuo
to provide the crude title compound as a yellow gum. The crude
material was purified by flash column chromatography to yield the
desired product as a white crystalline solid (88 mg, 95%). 1H NMR
1H NMR (300 MHz, CDCl3)
d ppm 3.97 (m, 4H), 3.80 (m, 4H), 3.58 (m,
2H), 3.50 (m, 2H), 3.17 (m, 4H), 2.97 (m, 6H), 2.67 (m, 5H), 1.49
(s, 9H).
5.1.1.12. tert-Butyl 2-(4-methylpiperazin-1-yl)-4-thiomorpholino-8,9-
dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5g). The ti-
tle compound was prepared by similar method to that of General
Procedure D. The title compound was obtained as a pale yellow solid
(470 mg, 27%). 1H NMR (300 MHz, CDCl3)
d ppm 3.97 (m, 4H), 3.80
Fig. 2. Effects on body weight gain (a) and food intake (b) of chronic administration of 6p (once daily) in C57BL/6 mice.

564
H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
Fig. 3. Effects on body weight gain (a) and food intake (b) of chronic administration of 6p (twice daily) in B6.V-Lepob/J mice.
(m, 4H), 3.55 (m, 4H), 3.17 (m, 4H), 2.86 (m, 6H), 2.67 (m, 2H), 2.66
(m, 3H), 1.49 (s, 9H).
of General Procedure D. The title compound was obtained as a
white solid (400 mg, 49%). 1H NMR (300 MHz, CDCl3)
ppm 7.43 (q,
d
J ¼ 6.85 Hz, 2H), 7.03 (t, J ¼ 8.72 Hz, 2H), 6.26 (s, 1H), 3.70 (m, 8H),
2.94 (m, J ¼ 5.27 Hz, 2H), 2.66 (m, 2H), 2.45 (t, J ¼ 4.95 Hz, 3H), 1.49
(s, 9H).
5.1.1.13. tert-Butyl 4-methoxy-2-(4-methylpiperazin-1-yl)-8,9-dihydro-
5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5h). To a solution of
tert-butyl 2-(4-methylpiperazin-1-yl)-4-(trifluoromethylsulfonyloxy)-
5,6,8,9-tetrahydro-3H-pyrimido[4,5-d]azepine-7(4H)-carboxylate (4a,
300 mg, 0.83 mmol) in DCM (15 ml) was added trimethyloxonium
tetrafluoroborate (367 mg, 2.5 mmol) and the mixture was stirred for
2 h. The organic layers were combined, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by column chroma-
tography eluting with EtOAc:pentane ¼ 1:1. The relevant fractions
were combined and concentrated in vacuo to afford the title com-
5.1.1.17. tert-Butyl 2-benzyl-4-thiomorpholino-8,9-dihydro-5H-pyr-
imido[4,5-d]azepine-7(6H)-carboxylate (5m). The title compound
was prepared by similar method to that of General Procedure D. The
title compound was obtained as a yellow solid (220 mg, 61%). 1H
NMR (300 MHz, CDCl3)
7.23 (m, 1H), 4.08 (s, 2H), 3.60 (m, 2H), 3.49 (m, 6H), 3.00 (m, 2H),
2.72 (m, 6H), 1.48 (s, 9H).
d
ppm 7.41 (d, J ¼ 4.21, 2H), 7.31 (m, 2H),
pound as a yellow solid (10 mg, 3%). 1H NMR (300 MHz, CDCl3)
d ppm
4.06 (s, 3H), 3.29 (m, 4H), 3.15 (m, 4H), 2.81 (m, 4H), 2.36 (m, 4H), 2.26
(s, 3H), 1.38 (s, 9H).
5.1.1.18. tert-Butyl 2-benzyl-4-(phenylamino)-8,9-dihydro-5H-pyr-
imido[4,5-d]azepine-7(6H)-carboxylate (5n). The title compound
was prepared by similar method to that of General Procedure D. The
title compound was obtained as a white solid (288 mg, 82%). 1H
5.1.1.14. tert-Butyl 4-(furan-3-ylmethylamino)-2-(4-methylpiperazin-
1-yl)-8,9-dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5i).
The title compound was prepared by similar method to that of
General Procedure D. The title compound was obtained as a yellow
NMR (300 MHz, CDCl3)
d
ppm 7.41 (m, 4H), 7.36 (t, J ¼ 1.62, 2H), 7.29
(m, 3H), 7.07 (t, J ¼ 3.83, 1H), 6.44 (s, 1H), 4.10 (s, 2H), 3.72 (d,
J ¼ 11.12, 4H), 3.09 (m, 2H), 2.74 (m, 2H), 1.47 (s, 9H).
solid (89 mg, 99%). 1H NMR (300 MHz, CDCl3)
1H), 6.33 (m, 1H), 6.23 (d, J ¼ 1.51, 1H), 4.61 (d, J ¼ 2.55, 2H), 3.82 (m,
4H), 3.58 (m, 4H), 2.87 (t, J ¼ 4.98, 2H), 2.50 (m, 6H), 2.37 (s, 3H),1.46
(s, 9H).
d
ppm 7.36 (d, J ¼ 0.80,
5.1.1.19. tert-Butyl 2-benzyl-4-(3-fluorophenylamino)-8,9-dihydro-
5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5o). The title com-
pound was prepared by similar method to that of General Pro-
cedure D. The title compound was obtained as a white solid
5.1.1.15. tert-Butyl 4-(3-methoxyphenylamino)-2-(4-methylpiper-
azin-1-yl)-8,9-dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carbox-
ylate (5j). The title compound was prepared by similar method to
that of General Procedure D. The title compound was obtained as a
(327 mg, 71%). 1H NMR (300 MHz, CDCl3)
d
ppm 7.43 (d, J ¼ 0.7,
1H), 7.38 (d, J ¼ 5.48, 2H), 7.33 (t, J ¼ 5.15, 2H), 7.20 (m, 2H), 7.03
(d, J ¼ 0.65, 1H), 7.00 (t, J ¼ 0.63, 1H), 6.72 (s, 1H), 4.11 (s, 2H),
3.67 (m, 4H), 3.09 (t, J ¼ 5.55, 2H) 2.72 (t, J ¼ 5.08, 2H), 1.47
(s, 9H).
white solid (79 mg, 14%). 1H NMR (300 MHz, CDCl3)
d ppm 7.33
(t, J ¼ 2.1 Hz, 1H), 7.20 (t, J ¼ 8.1 Hz, 1H), 6.93 (d, J ¼ 1.2 Hz, 1H), 6.63
(m, 1H), 6.40 (s, 1H), 3.80 (s, 8H), 2.95 (m, 7H), 2.67 (m, 2H), 1.39
(s, 9H).
5.1.1.20. tert-Butyl 2-benzyl-4-(3-chlorophenylamino)-8,9-dihydro-
5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5p). The title com-
pound was prepared by similar method to that of General Proce-
dure D. The title compound was obtained as a white solid (193 mg,
5.1.1.16. tert-Butyl 4-(4-fluorophenylamino)-2-(4-methylpiperazin-
1-yl)-8,9-dihydro-5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate
(5k). The title compound was prepared by similar method to that
51%). 1H NMR (300 MHz, CDCl3)
d
ppm 7.71 (br s, 1H), 7.45 (d,
J ¼ 0.73, 2H), 7.37 (t, J ¼ 15.91, 2H), 7.16 (m, 3H), 7.04 (d, J ¼ 3.24,
Table 6
Dose-dependent effects on organ weight after chronic treatment of 6p in B6.V-Lepob/J mice.a
Dose
Brain
Liver
Spleen
Thymus
Heart
Kidney
Lung
Gonadal adipose tissue
Control
0.340 ꢂ 0.017
0.344 ꢂ 0.013
0.369 ꢂ 0.006
0.360 ꢂ 0.010
3.396 ꢂ 0.162
3.394 ꢂ 0.295
3.173 ꢂ 0.207
3.870 ꢂ 0.020
0.097 ꢂ 0.052
0.057 ꢂ 0.011
0.111 ꢂ 0.073
0.075 ꢂ 0.025
0.029 ꢂ 0.006
0.033 ꢂ 0.003
0.034 ꢂ 0.002
0.040 ꢂ 0.010
0.150 ꢂ 0.003
0.163 ꢂ 0.008
0.143 ꢂ 0.005
0.170 ꢂ 0.010
0.339 ꢂ 0.009
0.329 ꢂ 0.020
0.329 ꢂ 0.010
0.350 ꢂ 0.010
0.143 ꢂ 0.013
0.124 ꢂ 0.005
0.154 ꢂ 0.020
0.140 ꢂ 0.010
3.599 ꢂ 0.155
3.776 ꢂ 0.211
3.127 ꢂ 0.148
3.475 ꢂ 0.245
5 mg/kg
25 mg/kg
50 mg/kg
a
All values are reported in grams.

H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
565
Table 7
(300 MHz, CDCl3)
d ppm 4.52 (m, 1H), 3.79 (m, 4H), 2.95 (m, 4H),
2.85 (m, 4H), 2.46 (m, 7H), 2.32 (s, 3H); 13C NMR (75 MHz, CDCl3)
Pharmacokinetic parameters after intravenous (n ¼ 4) and oral (n ¼ 4) adminis-
tration (10 mg/kg) of 6p in male SpragueeDawley rats.a
d
ppm 167.5, 161.0, 159.9, 105.0, 55.2, 48.0, 46.6, 46.3, 43.8, 42.1, 28.3;
HR-MS m/z [M þ H]þ calcd. for C13H22N5 ¼ 248.18748, found
Plasma
Intravenous
Oral
AUC0eN
AUClast
(
m
m
g min/ml)
114.35 ꢂ 44.06
103.60 ꢂ 41.07
135.06 ꢂ 13.10
e
63.02 ꢂ 50.93
22.50 ꢂ 5.90
761.24 ꢂ 1044.3
0.07 ꢂ 0.02
210 (120e240)b
e
248.18967.
(
g min/ml)
Terminal half-life (min)
Cmax g/ml)
5.1.1.25. N-Methyl-2-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepin-4-amine (6b). A similar method to that
of General Procedure E, by using 5b as starting material provided
39 mg of the title compound as a pale yellow solid in 38% yield. 1H
(
m
Tmax (min)
CL (ml/mim/kg)
MRT (min)
Vss (ml/kg)
Ae (%)
Plasma concentration (
Plasma concentration (
Brain concentration (
Brain-to-plasma ratio (B/P) at 2 h
F (%)
e
97.21 ꢂ 35.35
112.86 ꢂ 41.09
16,992.95 ꢂ 9984.04
0.03
e
e
NMR (300 MHz, CDCl3)
6H), 2.69 (m, 2H), 2.46 (m, 4H), 2.33 (s, 3H), 2.19 (s, 1H); 13C NMR
(75 MHz, CDCl3) ppm 170.9, 160.7, 156.6, 122.3, 55.0, 49.2, 46.9,
d ppm 7.99 (s, 1H), 3.81 (m, 4H), 2.94 (m,
0.03
m
m
g/ml) at 2 h 0.19 ꢂ 0.08
0.05 ꢂ 0.02
0.03 ꢂ 0.01
0.0292 ꢂ 0.0123
0.66
d
g/ml) at 8 h 0.06 ꢂ 0.02
H]þ calcd. for
m
g/ml) at 2 h
1.4825 ꢂ 0.1443
46.3, 43.8, 43.3, 34.3; HR-MS m/z [M
C14H25N6 ¼ 277.21384, found 277.21352.
þ
9.33
55.1
Abbreviations AUC0eN, total area under the plasma concentrationetime curve from
time zero to time infinity; AUClast, total area under the plasma concentrationetime
5.1.1.26. 2-(4-Methylpiperazin-1-yl)-N-propyl-6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepin-4-amine (6c). A similar method to that of
General Procedure E, by using 5c as starting material provided
30 mg of the title compound as a yellow solid in 20% yield. 1H NMR
curve from time zero to last measured time; Cmax, peak plasma concentration; Tmax
,
time to reach Cmax; CL, time-averaged total body clearance; MRT, mean residence
time; Vss, apparent volume of distribution at steady state; Ae, Excreted amount; F,
bioavailability.
(300 MHz, CD3OD)
d ppm 3.74 (m, 4H), 3.33 (m, 4H), 3.12 (t,
a
Data are the mean ꢂ SD
J ¼ 14.6 Hz, 2H), 2.94 (d, J ¼ 10.3 Hz, 2H), 2.79 (t, J ¼ 10.2 Hz, 2H),
b
Median (range) for Tmax
.
2.45 (m, 2H), 2.35 (s, 3H), 1.62 (d, J ¼ 7.2 Hz, 2H), 0.94 (t, J ¼ 7.4 Hz,
3H); 13C NMR (75 MHz, CDCl3)
d ppm 169.6, 158.4, 133.7, 109.8, 54.7,
53.5, 50.0, 47.5, 45.5, 34.9, 27.2, 23.2, 11.5.
1H), 6.44 (br s, 1H), 4.10 (s, 2H), 3.70 (m, 4H), 3.10 (m, 2H), 2.72
(m, 2H), 1.47 (s, 9H).
5.1.1.27. N,N-Dimethyl-2-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepin-4-amine (6d). A similar method to that of
General Procedure E, by using 5d as starting material provided 39 mg
of the title compound as a yellow solid in 38% yield. 1H NMR
5.1.1.21. tert-Butyl 2-benzyl-4-(3-methoxyphenylamino)-8,9-dihydro-
5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5q). The title com-
pound was prepared by similar method to that of General Procedure
D. The title compound was obtained as a yellow solid (450 mg, 58%).
(300 MHz, CDCl3) d ppm 3.75 (m, 4H), 2.89 (m, 7H), 2.81 (s, 6H), 2.78
(m, 2H), 2.70 (m, 2H), 2.41 (t, J ¼ 10.2 Hz, 4H), 2.30 (s, 3H); 13C NMR
1H NMR (300 MHz, CDCl3)
d
ppm 7.39 (d, J ¼ 3.79, 2H), 7.23 (m, 5H),
(75 MHz, CDCl3) d ppm 170.6,167.2,159.2,109.6, 55.2, 53.4, 48.7, 46.5,
6.95 (d, J ¼ 3.85, 1H), 6.62 (d, J ¼ 4.07, 1H), 6.52 (s, 1H), 4.10 (s, 2H),
46.3, 43.8, 42.5, 41.5, 31.0; HR-MS m/z [M þ H]þ calcd. for
C17H31N6 ¼ 319.26047, found 319.26080.
3.73 (m, 5H), 3.68 (m, 2H), 3.10 (m, 2H), 2.73 (m, 2H), 1.47 (s, 9H).
5.1.1.22. tert-Butyl 2-benzyl-4-(4-fluorophenylamino)-8,9-dihydro-
5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5r). The title com-
pound was prepared by similar method to that of General Proce-
dure D. The title compound was obtained as a yellow solid (88 mg,
5.1.1.28. N,N-Diethyl-2-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepin-4-amine (6e). A similar method to that of
General Procedure E, by using 5e as starting material provided
90 mg of the title compound as a yellow solid in 66% yield. 1H NMR
95%). 1H NMR (400 MHz, CDCl3)
d
ppm 7.31 (m, 7H), 6.93 (t, J ¼ 8.69,
(300 MHz, CDCl3) d ppm 3.72 (m, 4H), 3.39 (s, 1H), 3.15 (t,
2H), 6.40 (s, 1H), 4.07 (s, 2H), 3.70 (d, J ¼ 29.79, 4H), 3.09 (t, J ¼ 5.57,
J ¼ 20.94 Hz, 4H), 2.94 (t, J ¼ 19.75 Hz, 2H), 2.87 (m, 4H), 2.66 (m,
2H), 2.71 (m, 2H), 1.46 (s, 9H).
2H), 2.39 (m, 4H), 2.27 (s, 3H), 1.09 (t, J ¼ 13.95 Hz, 6H); 13C NMR
(75 MHz, CDCl3)
d ppm 170.2, 166.3, 159.2, 110.4, 55.1, 53.4, 48.4,
5.1.1.23. tert-Butyl 2-benzyl-4-(4-methoxyphenylamino)-8,9-dihydro-
5H-pyrimido[4,5-d]azepine-7(6H)-carboxylate (5s). The title com-
pound was prepared by similar method to that of General Procedure
D. The title compound was obtained as a white solid (324 mg, 69%).
46.4, 46.3, 44.4, 43.8, 42.1, 30.5, 13.1, 13.07; HR-MS m/z [M þ H]þ
calcd. for C17H31N6 ¼ 319.26047, found 319.26047.
5.1.1.29. 4-(2-(4-Methylpiperazin-1-yl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepin-4-yl)morpholine (6f). A similar method to that
of General Procedure E, by using 5f as starting material provided
50 mg of the title compound as a white solid in 14% yield. 1H NMR
1H NMR (300 MHz, CDCl3)
6.33 (s, 1H), 4.06 (s, 2H), 3.82 (s, 3H), 3.71 (d, J ¼ 13.22, 4H), 3.07 (t,
J ¼ 3.57, 2H), 2.71 (m, 2H), 1.47 (s, 9H).
d
ppm 7.32 (m, 7H), 6.81 (dd, J ¼ 4.51, 2H),
(300 MHz, CDCl3)
d
ppm 3.74 (s, 8H), 3.37 (d, J ¼ 4.9 Hz, 1H), 3.11 (t,
5.1.1.24. General procedure E for the synthesis of 2-(4-methylpiperazin-
1-yl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepine (6a). A solution
of trifluoroacetic acid (TFA, 1.1 ml) in CH2Cl2 (3.3 ml) was added to a
solution of tert-butyl 2-(4-methylpiperazin-1-yl)-8,9-dihydro-5H-
pyrimido[4,5-d]azepine-7(6H)-carboxylate 5a (110 mg, 0.49 mmol)
in CH2Cl2 (5 ml) by using a dropping funnel. The mixture was stirred
at room temperature for 10 h. The reaction was quenched by addi-
tion of sat. sodium bicarbonate and extracted into CH2Cl2. The
combined organics were dried over MgSO4, filtered and concen-
trated in vacuo providing the crude product as a brown gum. The
title compound was purified by flash column chromatography
eluting with (CHCl3:MeOH:NH4Cl:H2O ¼ 80:20:1:1) to yield the
desired product as a white crystalline solid (70 mg, 89%). 1H NMR
J ¼ 4.5 Hz, 4H), 2.88 (m, 5H), 2.66 (t, J ¼ 13.9 Hz, 4H), 2.40 (t,
J ¼ 4.9 Hz, 4H), 2.27 (s, 3H); 13C NMR (75 MHz, CDCl3)
d ppm 171.12,
166.72, 159.41, 111.16, 77.55, 77.33, 77.13, 76.71, 66.70, 54.99, 50.09,
48.67, 46.47, 46.23, 43.67, 42.66, 30.60, 29.63.
5.1.1.30. 4-(2-(4-Methylpiperazin-1-yl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepin-4-yl)thiomorpholine (6g). A similar method to
that of General Procedure E, by using 5g as starting material pro-
vided 76 mg of the title compound as a beige solid in 28% yield. 1H
NMR (300 MHz, CDCl3)
d
ppm 5.27 (s, 1H), 3.73 (t, J ¼ 4.9 Hz, 4H),
3.41 (t, J ¼ 4.5 Hz, 4H), 2.96 (m, 2H), 2.90 (m, 4H), 2.68 (m, 6H), 2.41
(t, J ¼ 4.9 Hz, 4H), 2.30 (s, 3H); 13C NMR (75 MHz, CDCl3)
d ppm
170.73, 167.35, 159.38, 110.72, 60.36, 55.04, 52.01, 48.36, 46.32,

566
H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
46.14, 43.72, 41.81, 29.92, 27.27; HR-MS m/z [M þ H]þ calcd. for
C17H29N6S ¼ 349.21724, found 349.21689.
46.29, 43.81, 38.95, 25.52; HR-MS m/z [M þ H]þ calcd. for
C20H28N6F ¼ 371.23569, found 371.23540.
5.1.1.31. 4-Methoxy-2-(4-methylpiperazin-1-yl)-6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepine (6h). A similar method to that of Gen-
eral Procedure E, by using 5h as starting material provided 10 mg of
the title compound as a white solid in 68% yield. 1H NMR (300 MHz,
5.1.1.36. 4-(2-Benzyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepin-
4-yl)thiomorpholine (6m). A similar method to that of General
Procedure E, by using 5m as starting material provided 75 mg of the
title compound as a yellow solid in 44% yield. 1H NMR (300 MHz,
CDCl3)
(m, 2H), 2.47 (t, J ¼ 4.9 Hz, 4H), 2.45 (s, 3H), 2.35 (s, 3H), 2.02 (s,1H);
13C NMR (75 MHz, CDCl3)
ppm 170.91,167.27,159.61,108.16, 55.08,
d
ppm 3.88 (s, 3H), 3.81 (t, J ¼ 4.9 Hz, 4H), 2.91 (m, 6H), 2.76
CDCl3)
4.08 (s, 2H), 3.47 (m, 4H), 2.98 (q, J ¼ 6.82, 4H), 2.90 (m, 2H), 2.71
(m, 6H), 2.09 (s, 1H); 13C NMR (75 MHz, CDCl3)
ppm 170.70,
d
ppm 7.40 (d, J ¼ 4.10, 2H), 7.29 (t, J ¼ 4.00, 2H), 7.19 (m, 1H),
d
d
53.19, 48.64, 46.80, 46.32, 43.80, 42.73, 30.92, 27.26.
166.57, 165.22, 138.94, 129.16, 126.18, 119.89, 52.01, 48.10, 46.41,
45.48, 42.65, 31.52, 27.23; HR-MS m/z [M þ H]þ calcd. for
C19H25N4S ¼ 341.18066, found 341.17944.
5.1.1.32. N-(Furan-2-ylmethyl)-2-(4-methylpiperazin-1-yl)-6,7,8,9-
tetrahydro-5H-pyrimido[4,5-d]azepin-4-amine (6i). A similar method
to that of General Procedure E, by using 5i as starting material pro-
vided 15 mg of the title compound as a yellow solid in 20% yield. 1H
5.1.1.37. 2-Benzyl-N-phenyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]
azepine-4-amine (6n). A similar method to that of General Proce-
dure E, by using 5n as starting material provide 50 mg of the title
compound as a white solid in 22% yield. 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3)
d
ppm 7.37 (s, 1H), 6.34 (d, J ¼ 0.88, 1H), 6.24
(s, 1H), 4.85 (m, 1H), 4.62 (d, J ¼ 2.40 Hz, 2H), 3.83 (m, 4H), 3.18 (m,
2H), 3.13 (m, 2H), 3.04 (m, 2H), 2.73 (m, 2H), 2.50 (m, 4H), 2.37 (m,
d
ppm 7.42 (t, J ¼ 4.24, 4H), 7.34 (t, J ¼ 3.47, 2H), 7.26 (t, J ¼ 12.93,
3H), 7.03 (t, J ¼ 7.33, 1H), 6.62 (s, 1H), 4.11 (s, 2H), 3.06 (m, 4H), 2.99
(m, 2H), 2.75 (m, 2H); 13C NMR (75 MHz, CDCl3)
ppm 168.33,
3H); 13C NMR (75 MHz, CD3OD)
d ppm 165.89,160.48,159.66,153.53,
141.15, 109.82, 105.98, 104.18, 54.43, 46.23, 44.78, 44.55, 43.16, 37.42,
36.77, 23.52.
d
166.27, 157.75, 139.40, 138.84, 129.65, 128.70, 128.27, 126.27, 122.98,
120.44, 114.13, 46.84, 45.82, 45.59, 41.08, 28.11; HR-MS m/z
[M þ H]þ calcd. for C21H23N4 ¼ 331.19172, found 331.19173.
5.1.1.33. N-(3-Methoxyphenyl)-2-(4-methylpiperazin-1-yl)-6,7,8,9-
tetrahydro-5H-pyrimido[4,5-d]azepin-4-amine (6j). A similar method
to that of General Procedure E, by using 5j as starting material pro-
vided 19 mg of the title compound as a white solid in 31% yield. 1H
5.1.1.38. 2-Benzyl-N-(3-fluorophenyl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepine-4-amine (6o). A similar method to that of
General Procedure E, by using 5o as starting material provided
71 mg of the title compound as a pale yellow solid in 28% yield. 1H
NMR (300 MHz, CDCl3)
J ¼ 8.1 Hz, 1H), 6.93 (d, J ¼ 1.2 Hz, 1H), 6.63 (m, 1H), 6.40 (s, 1H), 3.80
(s, 8H), 2.95 (m, 7H), 2.67 (m, 2H); 13C NMR (75 MHz, CDCl3)
ppm
d
ppm 7.33 (t, J ¼ 2.1 Hz, 1H), 7.20 (t,
d
NMR (300 MHz, CDCl3)
d
ppm 7.50 (d, J ¼ 5.80, 1H), 7.41 (d, J ¼ 3.63,
169.76, 160.42, 159.53, 157.76, 141.18, 129.31, 112.55, 108.61, 105.94,
77.47, 77.25, 77.04, 76.62, 55.20, 55.10, 53.42, 48.10, 46.48, 43.88,
42.52, 28.85.
2H), 7.33 (t, J ¼ 7.44, 2H), 7.25 (m, 1H), 7.14 (m, 1H), 7.01 (d, J ¼ 4.02,
1H), 6.79 (s, 1H), 6.72 (t, J ¼ 3.12, 1H), 4.11 (s, 2H), 2.98 (m, 6H), 2.70
(m, 2H), 2.48 (s,1H); 13C NMR (75 MHz, CDCl3)
d ppm 169.09,166.17,
164.57, 161.35, 157.38, 141.19, 141.04, 138.68, 129.68, 129.55, 128.39,
126.38, 115.35, 115.31, 114.74, 109.49, 109.20, 107.76, 107.37, 47.13,
46.10, 45.63, 41.93, 28.82; HR-MS m/z [M þ H]þ calcd. for
C21H22N4F ¼ 349.18230, found 349.18198.
5.1.1.34. N-(4-Fluorophenyl)-2-(4-methylpiperazin-1-yl)-6,7,8,9-
tetrahydro-5H-pyrimido[4,5-d]azepin-4-amine (6k). A similar method
to that of General Procedure E, by using 5k as starting material
provided 600 mg of the title compound as a white solid in 77% yield.
1H NMR (300 MHz, CD3OD)
d
ppm 7.49 (m, 2H), 7.03 (t, J ¼ 3.5 Hz,
5.1.1.39. 2-Benzyl-N-(3-chlorophenyl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepine-4-amine (6p). A similar method to that of
General Procedure E, by using 5p as starting material provided
44 mg of the title compound as a white solid in 29% yield. 1H NMR
2H), 3.73 (t, J ¼ 4.5 Hz, 4H), 3.30 (m, 5H), 3.03 (m, 4H), 2.51 (t,
J ¼ 5.0 Hz, 4H), 2.36 (s, 3H); 13C NMR (75 MHz, CD3OD)
d ppm 166.74,
160.42, 159.50, 158.96, 157.23, 136.12, 136.08, 123.72, 123.62, 114.48,
114.18, 104.11, 54.24, 53.41, 48.48, 48.19, 47.91, 47.63, 47.34, 47.06,
46.78, 45.84, 44.60, 43.99, 42.95, 35.34, 22.22; HR-MS m/z [M þ H]þ
calcd. for C19H26N6F ¼ 357.21975, found 357.21934.
(300 MHz, CD3OD)
3.51 (m, 2H), 3.47 (m, 2H), 3.36 (m, 2H), 3.22 (m, 2H); 13C NMR
(75 MHz, CDCl3) ppm 169.19, 166.16, 157.33, 140.63, 138.77, 134.27,
d ppm 7.56 (m, 1H), 7.26 (m, 8H), 4.10 (s, 2H),
d
129.65, 129.55, 128.44, 126.36, 122.77, 120.26, 118.18, 114.77, 47.18,
46.16, 45.75, 42.04, 28.92; HR-MS m/z [M þ H]þ calcd. for
C21H21ClN4 ¼ 364.1455, found 364.1454.
5.1.1.35. N-(4-Fluorophenyl)-7-methyl-2-(4-methylpiperazin-1-yl)-
6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepin-4-amine (6l). A stir-
ring solution of N-(4-fluorophenyl)-2-(4-methylpiperazin-1-yl)-
6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepin-4-amine (6k, 69 mg,
0.19 mmol) in MeOH (2 ml) was added 37% HCHO (0.1 ml,
3.2 mmol) at room temperature. The reaction was stirred for 2 h at
room temperature. The mixture was added NaBH(OAc)3 (282 mg,
1.3 mmol) and then stirred for 3 h. The reaction mixture was
evaporated and extracted with CH2Cl2 and sat. sodium bicarbonate.
The combined organic layers were dried over MgSO4, filtered and
concentrated in vacuo providing the crude product as a brown gum.
The title compound was purified by flash column chromatography
eluting with (CHCl3:MeOH:NH4Cl:H2O ¼ 80:20:1:1) to yield the
desired product as a white crystalline solid (30 mg, 42%). 1H NMR
5.1.1.40. 2-Benzyl-N-(3-methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepine-4-amine (6q). A similar method to that of
General Procedure E, by using 5q as starting material provided
25 mg of the title compound as a yellow solid in 30% yield. 1H NMR
(400 MHz, CDCl3)
d
ppm 7.39 (d, J ¼ 3.53, 2H), 7.23 (m, 5H), 6.95 (d,
J ¼ 0.92, 1H), 6.61 (d, J ¼ 1.19, 1H), 6.51 (s, 1H), 4.26 (s, 2H), 3.73 (s,
3H), 3.04 (m, 6H), 2.77 (m, 2H), 2.10 (s, 1H); 13C NMR (75 MHz,
CDCl3)
d ppm 168.77, 166.01, 160.05, 157.73, 140.70, 138.93, 129.39,
128.29, 126.23, 114.61, 112.61, 108.68, 106.12, 55.23, 47.07, 46.07
45.71, 41.75, 28.78; HR-MS m/z [M
C22H25ON4 ¼ 361.20333, found 361.20229.
þ
H]þ calcd. for
(300 MHz, CDCl3)
1H), 3.74 (s, 4H), 2.91 (s, 2H), 2.64 (m, 7H), 2.43 (t, J ¼ 3.9 Hz, 4H),
2.39 (s, 3H), 2.31 (s, 3H); 13C NMR (75 MHz, CDCl3)
ppm 169.28,
d
ppm 7.43 (m, 2H), 7.01 (t, J ¼ 8.5 Hz, 2H), 6.31 (s,
5.1.1.41. 2-Benzyl-N-(4-fluorophenyl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepine-4-amine (6r). A similar method to that of
General Procedure E, by using 5r as starting material provided
27.6 mg of the title compound as a yellow solid in 32% yield. 1H
d
160.23, 159.57, 157.70, 157.02, 135.76, 135.73, 122.65, 122.55, 115.33,
115.04, 105.28, 77.47, 77.04, 76.62, 56.84, 56.91, 55.24, 55.08, 47.57,

H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
567
NMR (300 MHz, CDCl3)
6.49 (s, 1H), 4.09 (s, 2H), 3.02 (m, 6H), 2.73 (t, J ¼ 4.68, 2H), 2.17 (s,
1H); 13C NMR (75 MHz, CDCl3)
ppm 168.80, 166.11, 160.20, 157.55,
157.00, 138.83, 135.27, 129.68, 128.25, 126.24, 122.20, 122.09, 115.34,
115.04, 114.14, 47.23, 46.22, 45.50, 42.06, 28.91; HR-MS m/z
[M þ H]þ calcd. for C21H22N4F ¼ 349.18230, found 349.18228.
d
ppm 7.33 (m, 7H), 6.92 (t, J ¼ 8.45, 2H),
saponin. Then, the incubations were terminated by rapid filtration
using an Innotech cell harvester (Innotech Biosystems, Switzerland)
through Whatman GF/C glass fiber filter presoaked in 0.05% Brij. The
filter was covered with MeltiLex, sealed in a sample bag followed by
drying in the microwave oven, and counted by MicroBeta Plus
(Wallac, Finland). Nonspecific binding was determined in the
d
presence of mianserin (0.5 mM). Competition binding studies were
5.1.1.42. 2-Benzyl-N-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyr-
imido[4,5-d]azepine-4-amine (6s). A similar method to that of
General Procedure E, by using 5s as starting material provided
10 mg of the title compound as a yellow solid in 4% yield. 1H NMR
carried out with 7e8 varied concentrations of the test compounds
run in duplicate tubes, and isotherms from three assays were
calculated by computerized nonlinear regression analysis (Graph-
Pad Prism Program, San Diego, USA) to yield IC50 values.
(300 MHz, CDCl3)
J ¼ 4.45, 2H), 6.38 (s,1H), 4.07 (s, 2H), 3.82 (s, 3H), 3.03 (m, 4H), 2.99
(m, 2H), 2.71 (m, 2H), 1.92 (s, 1H); 13C NMR (75 MHz, CDCl3)
ppm
168.40, 166.09, 157.86, 155.59, 139.00, 132.44, 129.64, 128.21, 126.16,
122.44, 113.84, 55.52, 47.19, 46.21, 45.59, 41.93, 28.86.
d
ppm 7.40 (d, J ¼ 3.62, 2H), 7.29 (m, 5H), 6.81 (d,
5.2.1.2. [3H]Mesulergine binding to serotonin 5-HT2C receptor.
Frozen membranes from stable CHO-K1 cell line expressing the
human recombinant 5-HT2C receptor were used. For the binding
d
assay, [3H]Mesulergine (1 nM), receptor membrane (4
m
g/well) and
test compounds were added into 50 mM TriseHCl (pH 7.4) buffer
containing 0.1% ascorbic acid and 10 M pargyline. Nonspecific
binding was determined using 0.5 M mianserin. The incubations
5.1.1.43. 4-Methyl piperazine-1-carboximidamide (9). To a stirring
solution of methyl piperazine (500 mg, 4.5 mmol) in DMF (7.5 ml)
was added amidinopyrazole hydrochloride (666 mg, 4.5 mmol) and
DIPEA (0.8 ml, 4.5 mmol). The reaction was heated at 80 ꢁC for 10 h.
The mixture was concentrated in vacuo. The residue was diluted
with CH2Cl2 and recrystallized with ether. The solid is filtered and
dried in vacuo to afford a pale yellow solid (580 mg, 65%). 1H NMR
m
m
were performed for 30 min at 37 ꢁC, and these were terminated by
rapid filtration through Whatman GF/C glass fiber filters presoaked
in 1% BSA (bovine serum albumin).
5.2.1.3. [3H]8-OH-DPAT binding to serotonin 5-HT1A receptor.
Membranes from stable HEK293-EBNA cell line expressing the
human recombinant 5-HT1A serotonin receptor (PerkinElmer) were
used. For the binding assay, aliquots of receptor membranes, 2 nM
[3H]8-OH-DPAT and appropriate concentrations of test compounds
were added to 0.25 ml of 50 mM TriseHCl (pH 7.4) buffer con-
taining 5 mM MgSO4. Nonspecific binding was determined using
(300 MHz, CDCl3)
J ¼ 5.06 Hz, 4H), 2.37 (s, 3H).
d
ppm 4.87 (s, 5H), 3.53 (t, J ¼ 5.19 Hz, 4H), 2.55 (t,
5.1.1.44. 2-Phenylacetimidamide (11). A stirring solution of ammo-
nium chloride (2.32 g, 43.4 mmol) in toluene (20 ml) was added
trimethylaluminium (21.65 ml, 43.4 mmol) dropwise at 0 ꢁC. The
reaction was stirred for 2 h at room temperature. The mixture was
added 2-phenylacetonitrile (5 ml, 43.3 mmol) and then heated to
80 ꢁC and stirred for 16 h. The reaction mixture was cooled and
slowly poured into a slurry of silica gel in CH2Cl2 and stirred for
10 min. The silica was filtered and washed with MeOH. The filtrate
and wash were combined and concentrated in vacuo to obtain a
yellow gum-like solid (1.15 g, 82%). 1H NMR (300 MHz, DMSO-d6)
10 mM serotonin (5-HT). Incubations were carried out for 120 min at
37 ꢁC, and these were terminated by rapid filtration using an Ino-
tech cell harvester through Filtermat A glass fiber filter presoaked
in 0.3% PEI (polyethyleneimine).
5.2.1.4. [3H]LSD binding to serotonin 5-HT6 and 5-HT7 receptor.
For receptor binding assays, human 5-HT6 and 5-HT7 serotonin
receptors expressed in CHO-K1 cells (PerkinElmer) were used. For
5-HT6 receptor binding, frozen membrane, 1 nM [3H]LSD and
appropriate concentrations of test compounds were added to
0.25 ml of assay buffer. Incubations were carried out for 60 min at
27 ꢁC, and these were terminated by rapid filtration through Fil-
termat A glass fiber filter presoaked in 0.5% PEI. Methiothepin
d
ppm 9.31 (br s, 1H), 8.83 (br s, 2H), 7.47 (d, J ¼ 6.83, 2H), 7.35 (m,
3H), 3.73 (s, 2H).
5.2. Biological evaluations
5.2.1. In vitro functional assays to measure receptor potency and
selectivity
(10 mM) and 50 mM TriseHCl (pH 7.4) containing 4 mM CaCl2 and
Radioligands, [3H]Ketanserin and [3H]Imipramine, were pur-
chased from PerkinElmer (PerkinElmer Life and Analytical Sciences,
Boston, USA), whereas [3H]Mesulergine was obtained from Amer-
sham Biosciences (Buckinghamshire, UK). Cloned human recom-
binant serotonin 5-HT2A and 5-HT2C receptors were obtained from
Euroscreen (Brussels, Belgium). Receptor binding assays for the
adrenergic a1A/2A (Table 3) and the 5-HT2A/2B/2C receptors (Table 2)
were performed by Eurofins Panlabs, Inc. (Taipei, Taiwan). Receptor
binding assays for the dopaminergic D2, D3 and the 5-HT receptors
were performed by following the procedures described in the
literature [16].
0.1% ascorbic acid were used as the nonspecific ligand or assay
buffer, respectively.
For 5-HT7 receptor binding assay, cell membrane, 3 nM [3H]LSD
and appropriate concentrations of test compounds were added to
0.25 ml of assay buffer. The mixture was incubated for 120 min at
27 ꢁC, and the reaction was terminated by rapid filtration through
Filtermat A glass fiber filter presoaked in 0.3% PEI. Methiothepin
(10 mM) and 50 mM TriseHCl (pH 7.4) containing 10 mM MgSO4
and 0.5 mM EDTA, were used as the nonspecific ligand or assay
buffer, respectively.
5.2.1.5. [3H]Spiperone binding to dopamine D2L and D3 receptors.
Competition binding assays at dopamine D2L and D3 receptors were
performed using 0.2 nM and 0.25 nM of [3H]Methylspiperone,
respectively, by the protocols provided by the supplier of mem-
branes (PerkinElmer Life and Analytical Sciences) with minor
modifications. Briefly, receptor membranes were incubated at 27 ꢁC
for 60 min (30 min for D3 receptor) in a final volume of 0.25 ml
reaction mixture containing [3H]Methylspiperone (PerkinElmer)
and various concentrations of the drug in 50 mM TriseHCl (pH 7.4)
buffer containing 10 mM MgCl2, 1 mM EDTA and 120 mM NaCl
5.2.1.1. [3H]Ketanserin binding to serotonin 5-HT2A receptor.
Competition binding assays of serotonin 5-HT2A receptor were
performed using 1 nM of [3H]Ketanserin, based on the protocol
provided by the supplier of CHO-K1 membranes (Euroscreen,
Brussels, Belgium) with minor modifications. Briefly, receptor
membranes (15 mg/well) were incubated at 25 ꢁC for 60 min in a
final volume of 0.25 ml reaction mixture containing [3H]Ketanserin
and various concentrations of the drug in 50 mM TriseHCl (pH 7.4)
buffer containing 5 mM CaCl2, 0.1% ascorbic acid and 10 ug/ml

568
H.Y. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 558e569
(5 mM MgCl2 for D3 receptor). Then, the incubations were termi-
nated by rapid filtration using an Innotech cell harvester (Innotech
Biosystems, Switzerland) through Filtermat A glass fiber filter
presoaked in 0.5% polyethylenimine (PEI). The filter was covered
with MeltiLex, sealed in a sample bag followed by drying in the
microwave oven, and counted by MicroBeta Plus (Wallac, Finland).
Nonspecific binding was determined in the presence of 10 uM
containing 9 animals per group and provided free access to water
and food. Treatment groups included vehicle control were orally
administered 6p for 14 days, twice daily in C57BL/6 and B6.V-Lepob/J
mice and once daily in B6.V-Lepob/J mice. Dose was 5, 25, 50 mg/kg.
C57BL/6 and B6.V-Lepob/J mice were dosed 2 times at 9:30 and
19:00 each day. Body weight and food intake were measured at
19:00 every day.
haloperidol or 20 mM GR103691 for D2 and D3 receptors, respec-
tively. IC50 was calculated by computerized nonlinear regression
analysis (GraphPad Prism Program, CA, USA).
5.2.4. Pharmacokinetics
Seven-week-old male SD rat were purchased from Nara-biotech
and maintained for a week. Animal care and handling followed
institutional guidelines (Korea Institute of Science and Technology).
Animals were maintained with free access to food and water under
a 12 h light/dark cycle. They were starved in cage for 12 h prior to
drug administration. Compound 6p was dissolved completely at
5 mg/ml of N-methyl-2-pyrrolidone, tween80, and D.W. (1:2:7 by
volume) by sonication for 5 min. This preparation was orally
administered at 2 ml/kg with a blunt needle via the esophagus into
the stomach and via bolus tail vein injection at 2 ml/kg. Samples
were collected at various time points (n ¼ 5) after drug adminis-
tration and were stored at ꢀ70 ꢁC until LC-MS/MS analysis. To
5.2.1.6. [3H]YM-09151-2 binding to dopamine D4.2 receptors. In [3H]
YM-09151-2 receptor binding assays, cloned dopamine D4.2 re-
ceptor membranes prepared from Sf9 cells (PerkinElmer) and
0.2 nM of [3H]YM-09151-2 (PerkinElmer) were incubated in the
buffer containing 50 mM TriseHCl (pH 7.4), 5 mM MgCl2, 5 mM
EDTA, 5 mM KCl and 1.5 mM CaCl2. Nonspecific binding was defined
with clozapine (10
mM) for D4.2 receptors. After incubation for
60 min at 27 ꢁC, assay was terminated by rapid filtration through
Filtermat A glass fiber filter presoaked in 0.3% PEI.
5.2.1.7. Calcium mobilization assay. FlexStation (Molecular Devices,
USA) was used to measure the intracellular calcium concentration
in HEK293 cells stably expressing human 5-HT2A and 5-HT2C re-
ceptors. The cells were seeded at 10,000 cells/well in a 384-well,
black-wall, clear-bottom, tissue culture treated plate and grown at
37 ꢁC in an incubator with 5% CO2 for 24 h. After removing the
culture media, the cells were washed once with Hank’s balanced
salt solution (HBSS) containing 20 mM HEPES buffer. Fluo-4 NW
calcium dye (Invitrogen, USA) dissolved in HBSS containing 2.5 mM
probenecid were added to the cells and incubated for 50 min.
Subsequently, the cells were treated with various concentrations of
compounds and further incubated for 5 min at 37 ꢁC. Activation of
human 5-HT2A and 5-HT2C receptors was achieved by the addition
of serotonin to the cells and the changes in relative fluorescence
unit due to the increase in the intracellular calcium concentration
were monitored for 1 min. All the assays were duplicated or
quadruplicated and repeated several times. The data were calcu-
lated by the nonlinear regression analysis (GraphPad Prism Pro-
gram) to yield 50% inhibition values (IC50).
determine the drug concentration in the plasma, a 0.1
bamazepine in acetonitrile (an internal standard) was added to
50 l plasma samples to deproteinize. After vortex-mixing, the
mixture was centrifuged for 10 min at 10,000 ꢃ g. 100 l of the
mg/ml car-
m
m
supernatant was analyzed by LC-MS/MS. The Agilent 1100 Series
(Agilent Technologies, Waldbronn, Germany) was used. Mass
spectrometer API 3200 (Applied Biosystems Sciex, Rotterdam, The
Netherlands) with electrospray-positive ionization was used. The
multiple reaction monitor was set at 854.20e266.20 m/z for com-
pound and 237.4e194.4 m/z for the internal standard. The analytical
column was Waters XTrerraÒ MS C18 (3.5
mm, 2.1 mm
i.d. ꢃ 50 mm). The mobile phase of paclitaxel consisted of (A) water
containing 0.1% formic acid and (B) 90% acetonitrile containing 0.1%
formic acid. 5 ml aliquots were injected, and the flow rate was set at
0.35 ml/min. The initial composition was 10% (B), programmed
linearly to 90% (B) after 1 min, and held for 1 min. The column was
then re-equilibrated at 10% for 3 min.
Acknowledgments
5.2.2. Acute in vivo assays to measure effects on mouse food intake
Compounds were screened for the ability to reduce food intake
in male C57BL/6 and B6.V-Lepob/J mice. C57BL/6 is the most widely
used genetic background for genetically modified mice for use as
models of human diseases. And B6.V-Lepob/J is a strain of mice ho-
mozygous for the obese spontaneous mutation (Lepob; commonly
referred to as ob or ob/ob). Homozygous mutant mice gain weight
rapidly and may reach three times the normal weight of wild-type
controls. In addition to obesity, mutant mice exhibit hyperphagia,
a diabetes-like syndrome of hyperglycemia. The mice were accli-
mated for 1 week under the control of constant temperature
(23 ꢂ 3 ꢁC) and humidity (50 ꢂ 20%). The experimental and control
group were fasted for 24 h before experiment and then were
injected intraperitoneally with vehicle, 50 mg/kg in male C57BL/6
mouse (each compounds) and 12.5, 25, 50 mg/kg in male B6.V-
Lepob/J mice (only 6p). Food intake was measured an interval of 2, 6,
12 h and compared to vehicle control.
This work was supported by Korea Institute of Science and
Technology.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.02.020.
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