Design and synthesis of 4-arylpiperidinyl amide and N-arylpiperdin-3-yl- cyclopropane carboxamide derivatives as novel melatonin receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2010.12.068

Two series of 4-arylpiperidinyl amide and N-arylpiperdin-3-yl-cyclopropane carboxamide derivatives exhibiting diverse functionality at rat MT₁ and MT₂ receptors are reported. Compounds 11f and 18b (MT ₁/MT₂ agonist) have human microsomal intrinsic clearance comparable to ramelteon.

Full text of DOI:10.1016/j.bmcl.2010.12.068

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1236–1242
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 4-arylpiperidinyl amide
and N-arylpiperdin-3-yl-cyclopropane carboxamide derivatives
as novel melatonin receptor ligands
Guiying Li a, , Hao Zhou a, Yu Jiang a, Holger Keim a, Sidney W. Topiol a, Suresh B. Poda b, Yong Ren b,
⇑
Gamini Chandrasena a, Darío Doller a
a Department of Chemical & Pharmacokinetic Sciences, Lundbeck Research USA, 215 College Rd., Paramus, NJ 07652, USA
b Department of Biological Research, Lundbeck Research USA, 215 College Rd., Paramus, NJ 07652, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of 4-arylpiperidinyl amide and N-arylpiperdin-3-yl-cyclopropane carboxamide derivatives
exhibiting diverse functionality at rat MT1 and MT2 receptors are reported. Compounds 11f and 18b
(MT1/MT2 agonist) have human microsomal intrinsic clearance comparable to ramelteon.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 October 2010
Revised 8 December 2010
Accepted 15 December 2010
Available online 19 December 2010
Keywords:
Melatonin
Melatonergic ligands
Melatonin (Fig. 1) is a pineal gland hormone, that is, secreted
during darkness and plays an important role in the synchroniza-
tion of circadian rhythms in both human and mammals.1,2 It is syn-
thesized from serotonin by the consecutive actions of two
enzymes, 5-HT-N-aceyltransferase and hydroxyindole-O-methyl-
transferase.3 In mammals, its biological actions are mediated via
two high-affinity (Ki values ca. 1 nM) G-protein coupled receptors,
MT1 and MT2.4–7 A third subtype, Mel1c, is expressed in avian spe-
cies but not in mammals.4 In addition, a melatonin-sensitive form
of the human enzyme Quinone Reductase 2, known as MT3 with
lower affinity for melatonin (Ki values in the range of 10–60 nM),
has been identified.8 Melatonin has been suggested as a therapeu-
tic agent for the treatment of delayed sleep phase syndrome,9 jet
lag,10 shift work disturbances,11 aging,12 affective disorders associ-
ated with biological rhythm disturbances,13,14 etc. However, its
pharmacological use is limited due to its short biological half-life,
poor bioavailability, and ubiquitous action.15 Thus, during the past
decade, considerable efforts have been devoted to developing mel-
atonergic ligands which are characterized by a better pharmacoki-
netic profile, and/or subtype selectivity. A variety of structurally
diverse melatonergic ligands, which range from simple indole
derivatives and their bioisosteres to phenylalkyl amides and con-
strained derivatives, have been reported and elegantly summa-
rized in several recent reviews.16–20 More specifically, several
melatonin ligands of existing chemical series related to our work,
such as phenylalkyl amides 121 and 2,22 N-(substituted-anilinoeth-
yl)amide 3,23 and aminopyrrolidine derivatives 4 and 524 are
shown in Figure 1. Two dual MT1/MT2 agonists, agomelatine and
ramelteon, are currently on the market, the former, also having
weak 5-HT2C antagonism activity.25,26 Upon embarking on our mel-
atonin receptor modulation exploratory program, one of our main
goals was to identify ligands with different subtype selectivity and
with reasonable pharmacokinetic profile for evaluating the patho-
physiological functions of MT1 and MT2 receptors in rat behavioral
models. Hence, we developed binding and high throughput
functional FLIPR assays against rat MT1 and MT2 receptors. We
report herein the design, synthesis and SAR of two chemotypes,
4-arylpiperidinyl amides and N-arylpiperidin-3-yl-carboxamides
exhibiting a diverse range of functional activities. In addition, we
present a comparison of rat pharmacokinetic properties of select
compounds with agomelatine and ramelteon.
The 4-arylpiperidinyl amide template (e.g., compound 11) was
derived from reported phenylalkyl amides 1 and 2 by conforma-
tional constraint (Fig. 2). The general synthetic route for analogs
in this series is depicted in Scheme 1. Suzuki coupling of commer-
cially available boronic acid 7 with an appropriate aryl iodide or
bromide 6 produced compound 8. Catalytic hydrogenation of com-
pound 8 afforded compound 9. Removal of the N-Boc protecting
group from compound 9 was carried out using TFA, producing
the TFA salt of amine 10. Amidation of 10 with an appropriate acyl
chloride R2COCl afforded amides 11a to 11l, 11p–t, respectively. All
reactions proceeded in good to excellent yield. De-methylation of
compound 10 using BBr3 in CH2Cl2 produced phenol 12 in 15%
⇑
Corresponding author. Tel.: +1 201 350 0140.
E-mail address: guli@lundbeck.com (G. Li).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.068

G. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1236–1242
1237
O
OMe
NH-COR
HN
H
N
NHCOR
( )n
MeO
O
MeO
N
R2
R1
N
H
n = 1-4
1
2
3
Melatonin
O
O
HN
HN
O
O
O
O
MeO
MeO
N
N
N
H
N
Pr
H
4
Agomelatine
Ramelteon
5
Figure 1. Melatonin receptor ligands.
NH-COR
O
R2
O
N
R2
R1
R1
R1
R2
N
N
H
1
NHCOR
( )n
11
18
MeO
n = 1-4
2
Aromatic
Acceptor
Acceptor
Hydrophobic
HO
NH
O
NH
O
O
Cl
N
O
O
TAYWUL
EZUDUY
Figure 2. Design of novel melatonin ligands.
yield (unoptimized reaction conditions). Amidation of 12 with
cyclopropylcarbonyl chloride 13 gave compound 11o in 86% yield,
which upon alkylation with ethyl bromide or isopropyl bromide in
the presence of base K2CO3 in DMF at 90 °C afforded compound
11m and 11n, respectively, in about 50% yield. Experimental pro-
cedures, 1H NMR and/or LC–MS data are included as Supplemen-
tary data.
Amides 11a–t were evaluated in vitro for their binding and
functional activity at rat MT1 and MT2 receptors, and results are
summarized in Table 1. The SAR of this template is narrower when
compared to phenylalkylamides 121 and 2.22 On the piperidinyl
aryl ring, changing the position of the methoxy group from m- to
o- or p-, or increasing methoxy to ethoxy resulted in a dramatic
loss in potency. With respect to the amide, isopropyl and cyclopro-
pyl are optimal. The most promising compound in this series is
compound 11f, which has four-fold receptor selectivity toward
MT2 receptor (MT1 Ki/MT2 Ki = 4)
Using 3D pharmacophoric features coupled with X-ray crystal-
lography structures we identified other targets as follows. Upon
searching the Cambridge Crystallographic Database,28 we found
X-ray structures of both melatonin (CCDC ID: MELATN03) and com-
pounds with the core of 11. Using one of these structures (CCDC ID:
TAYWUL) we found that an overlay as depicted in Figure 2 sug-
gested a four-point pharmacophore as shown. Previous studies
using pharmacophore and homology models on other rigidified
analogs of melatonin29,30 proposed that the side chain of melatonin
and rigid analogs thereof are out-of-plane with respect to the indole
core. For those analogs, X-ray structures of the compounds and
their analogs were supportive of the models. The X-ray structure
of melatonin adopts a nearly in-plane conformation of the side

1238
G. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1236–1242
O
O
B
N Boc
R1
R1
R1
7
Pd/C, H2, EtOH
60-84%
N Boc
N Boc
Pd(PPh3)4, Na2CO3, LiCl
MeO(CH2)2OMe, reflux
50-80%
X
6
8
9
X = I or Br
ClCOR2
R1
CF3CO2H
CH2Cl2, 0oC
R1
O
NH
N
R2
Et3N, CH2Cl2
70-93%
90%
CF3CO2H salt
11a to 11l
11p to 11t
10
BBr3, CH2Cl2, 0oC
15% (R1 = m-OMe)
O
HO
O
HO
13
Cl
O
O
RBr, K2CO3
NH
N
N
DMF, 90oC
~ 50%
Et2NiPr, CH2Cl2
86%
12
11o
11m, R =Et
11n, R =iPr
Scheme 1. Synthesis of 4-arylpiperidinyl amides.
chain. For our series’, for example, compound 11, the melatonin X-
ray structure yields a better overlap of the key features and was
adopted herein as the template. We then considered a number of
variations of 11 with different rings, topologies with respect to het-
eroatom placement, etc. We searched the CCDC for examples of
each of the considered cores and found that in one such case, that
is, the core shown as 18, the related X-ray structure (CCDC ID: EZU-
DUY) allowed for a good overlap of three of the corresponding phar-
macophore features with obvious access to introduction of the
fourth via substitution at the meta position of the phenyl group.
Thus, the right side of 11 was kept constant as cyclopropyl, and
the core was modified to a 3-amino-piperdinyl (18, Fig. 2).
The general synthesis of N-arylpiperidin-3-yl-cyclopropane car-
boxamides (e.g., 18) is depicted in Scheme 2. Amidation of com-
mercially available 13 and 14 (racemate was used for the
synthesis of 18a; S-enantiomer was used for the synthesis of
18b, 18d, 18f, 18g and 18h; R-enantiomer was used for the synthe-
sis of 18c and 18e) produced compound 15. Removal of the N-Boc
protecting group of 15 yielded amine 16. Reaction yields were
excellent. N-arylation of 16 with commercially available aryl halide
17 afforded compounds 18a–g in 10–50% yield. The reaction condi-
tions were not optimized. De-methylation of compound 18b with
BBr3, followed by alkylation with Br(CH2)2OSi(Me)2tBu, and then
treatment with TBAF, afforded 18h in unoptimized 41% yield.
Experimental procedures, 1H NMR and LC–MS data are in Supple-
mentary data.
affinity to both MT1 and MT2 receptors. Both 18f and 18g were par-
tial MT2 agonists and weak MT1 antagonists in the FLIPR functional
assay. Chlorine on the para-position of the phenyl moiety was well
tolerated. The S-enantiomer 18d was a potent dual MT1/MT2 par-
tial agonist, while its R-enantiomer 18e was a MT1/MT2 full antag-
onist with about four-fold binding selectivity toward MT2 receptor.
The effect of chirality on receptor binding was also reported in the
literature for bicyclic aminopyrrolidine amide 5 (The S-enantiomer
of 5 was a non selective MT1/MT2 ligand: human MT1 Ki = 1.2 nM,
human MT2 Ki = 2.8 nM; while the R-enantiomer of 5 had about
nine-fold binding selectivity toward MT2 receptor: human MT1
Ki = 3.9 nM, human MT2 Ki = 35 nM).24 5-HEAT, a melatonin analog
with –O(CH2)2OH replacing methoxy, has been reported to be a full
agonist at human MT1 and an antagonist/weak partial agonist at
human MT2 with lower affinity than melatonin.31 In our case, the
–O(CH2)2OH analog 18h was a full antagonist at both rat MT1
and MT2 receptors.
Next, we explored the hypothesis that core modification of N-
arylpiperdin-3-yl-cyclopropane carboxamides could improve affin-
ity and/or binding selectivity. Results are summarized in Figure 3.
Replacement of the piperdin-3-yl moiety with pyridyl led to com-
pound 19, a very weak MT1 partial antagonist and MT2 partial ago-
nist. Incorporation of a phenyl on the piperidin-3-yl moiety led to
compound 20 (racemate), a MT1 full/ MT2 partial agonist with 11-
fold binding selectivity toward MT2 receptor. In comparison, the
non-constrained N-phenyl substituted anilinoethyl amide 3 (also
known as UCM765), a highly potent human MT2-selective partial
agonist (MT1 Ki = 4.2 nM, IAr = 0.79; MT2 Ki = 0.07 nM, IAr =
0.61),23 was also an MT2-selective partial agonist in our rat receptor
assays (rMT1 Ki = 290 nM, FLIPR EC50 = 78 nM, IA% = 81; rMT2
Ki = 8.7 nM, FLIPR EC50 = 2.3 nM, IA% = 57). Replacement of the pip-
erdin-3-yl moiety with pyrrolidin-2-yl-methyl was well tolerated.
Compound 21 (racemate) was an MT1/MT2 full agonist with six-fold
selectivity toward rat MT2 receptor. Synthesis of compound 19 is
illustrated in Scheme 3. Compounds 20 and 21 were made using
the same route described in Scheme 2 for the synthesis of 18, from
cyclopropanecarboxylic acid (1,2,3,4-tetrahydro-quinolin-3-yl)-
amide, which was made from the amidation of 3-aminoquinoline
and cyclopropanecarbonyl chloride followed by hydrogenation,
and tert-butyl 2-(aminomethyl)pyrrolidine-1-carboxylate, respec-
The in vitro functional and binding activity of compounds 18a–
h were evaluated, and results are summarized in Table 2. It appears
that chirality affects the affinity and the nature of the functional re-
sponse. For example, the S-enantiomer 3-methoxy analog 18b was
a potent MT1/MT2 partial to full agonist, which is similar to the lit-
erature compound S-aminopyrrolidine amide
4 (human MT1
Ki = 3.7 nM, MT2 Ki = 2.6 nM);24 while its R-enantiomer 18c was a
MT2 antagonist with weak partial agonist activity toward the rat
MT1 receptor. The corresponding racemic compound 18a showed
full MT1/MT2 agonist activity in the FLIPR functional assay with
about six-fold selectivity toward MT2 receptor in the binding assay
(MT1 Ki/MT2 Ki = 5.6). Reduced electron density appears to have
negative impact on binding affinity. For example, replacing –OMe
with –OCHF2 (18f) or –OCF3 (18g) resulted in significantly loss of

G. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1236–1242
1239
Table 1
Binding affinity and functional activity of 4-arylpiperidinyl amides27
Compd
R1
R2
EC50 (nM) (IA%)
rMT2
Ki (nM)
rMT1
rMT1
rMT2
Melatonin
Agomelatine
Ramelteon
11a
0.56 0.1
(97 1)
1.1 0.4
(97 3)
1.4 0.2
(100 2)
0.89 0.02
(95 4)
>10,000
(19 1)
0.99 0.01
0.58 0.07
0.14 .0.01
0.03 0.007
n.d.
2.1 0.1
(94 10)
3.1 0.6
(100 4)
>10,000
(6 1)
1.2 0.1
2.0 0.1
n.d.
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
Me
11b
Et
1300 275
(31 4)
42
(66 14)
19
9
810 270
160 62
n.d.
150 37
31 3.6
n.d.
11c
iPr
98
6
9
(87 10)
2300 380
(32 3)
1700 300
(39 1)
(76 3)
790 62
(35 2)
1800 200
(35 2)
11d
1-Ethylpropyl
iBu
11e
n.d.
n.d.
11f
Cyclopropyl
65
1
9.5
2
40
4
10 0.8
(41 5)
610 190
(19 0.5)
640 158
(33 3)
380 29
(52 0.1)
210 20
(67 0.5)
7500 620
(42 13)
>10,000
(12 1)
n.d.
(74 4)
140 16
(65 2)
147 16
(30 2)
11g
(1S,2S)-2-Methyl-cyclopropyl
2,2-Dimethyl-cyclopropyl
Cyclobutyl
150 25
n.d.
35
4
11h
n.d.
11i
200
(62 3)
110
9
140 42
n.d.
67 23
n.d.
11j
Cyclopentyl
7
(74 10)
2800 200
(36 17)
4400 140
(60 5)
n.d.
>10,000
(10 2)
11k
Cyclohexyl
n.d.
n.d.
11l
Ph
650 80
110
7
11m
11n
m-OEt
m-OiPr
Cyclopropyl
Cyclopropyl
5700 370
n.d.
4100 34
n.d.
>10,000
(9 1)
11o
11p
11q
11r
11s
11t
m-OH
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Me
>10,000
(96 8)
>10,000
(0.4 0.1)
>10,000
(2 0.1)
>10,000
(58 3)
1700 208
(34 3)
>10,000
(9 1)
3600 135
(23 2)
>10,000
(15 1)
>10,000
(3 1)
5000 100
(94 9)
3400 190
(140 17)
1000 280
(54 4)
n.d.
n.d.
m-Me
n.d.
n.d.
o-OMe
p-OMe
p-OMe
m-OMe
n.d.
n.d.
1100 127
4700 260
2000 410
870 78
360 60
860 400
OMe
tively; and using an Ullmann coupling for the N-arylation step.
Experimental procedures, 1H NMR and LC–MS data are in Supple-
mentary data.
[brain] = 15 ng/mL; rat 2: [plasma] = 64 ng/mL, [brain] = 176 ng/
mL), 11f and 18b had extremely low plasma and brain expo-
sures(<5 ng/mL), which is presumably caused by high first pass
metabolism, and perhaps solubility limited absorption. It was
noted that agomelatine, 11f and 18b were dosed as suspensions
rather than solutions in the vehicle used. Different vehicles were
screened to make possible solution-dosing of these compounds,
and their exposures were enhanced (Table 3). One hour after IP
dosing, agomelatine, ramelteon and 11f showed good ability to
cross the blood–brain barrier; albeit compound 18b less so. The
brain-to-plasma ratios of compounds 11f and agomelatine were
similar, and higher than that of ramelteon. The brain-to-plasma ra-
tio of compound 18b was about 15% of agomelatine and 30% of
ramelteon, respectively. The calculated physicochemical properties
of these compounds (MW 243–310, cLogP 1.9–2.8, tPSA 30–42 Å2,
H-bond donor 0–1, H-bond acceptor 2) are all within the range
targeted for CNS drugs, and they all showed good membrane
permeability (19.8–25.0 ꢀ 10ꢁ6 cm/sec) in a PAMPA assay.33 The
observed low B/P ratio of 18b may be caused by transporter efflux,
which needs to be further investigated.
The most promising compounds, 11f (4-arylpiperidinyl amide
series), 18b, 18d, and 18f (N-arylpiperidine-3-yl-cyclopropane car-
boxamide series), were further evaluated for in vitro human and
rat microsomal stability (Table 3). All showed high human micro-
somal intrinsic clearance (CLINT), comparable to that of ramelteon
but lower than agomelatine. Compound 11f had comparable rat
in vitro microsomal clearance to that of agomelatine and ramelt-
eon, while compounds 18b, 18d and 18f had higher rat in vitro
microsomal clearance than agomelatine and ramelteon. Reducing
the electron density of the oxygen in the methoxyphenyl moiety
of 18b with –OCHF2 (18f) or blocking one of the potential meta-
bolic sites with a chlorine atom on the phenyl ring (18d) resulted
in no improvements of microsomal stability. Compounds 11f and
18b were tested in vivo in order to compare rat plasma and brain
levels with agomelatine and ramelteon. When the compounds
were dosed via intraperitoneal (IP) injection and 20% 2-hydroxy-
propyl-b-cyclodextrin (HP-bCD) was used as dosing vehicle, only
ramelteon showed reasonable exposure (Table 3), while agomela-
tine had highly variable exposure (rat 1: [plasma] = 9.6 ng/mL,
In summary, two novel series (4-arylpiperdinyl amide and N-aryl-
piperdin-3-yl-cyclopropane carboxamide derivatives) are reported

1240
G. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1236–1242
O
O
O
Cl
13
HN
HN
NH2
1) 4 M HCl /dioxane, EtOAc
Boc
N
HN
N
2) a) Si-Tosic acid resin, CH2Cl2
b) 2M NH3/MeOH
CH2Cl2, Et3N
Crude 100%
Boc
14
Crude 98%
16
15
R1
R2
I (or Br)
17
O
R1
HN
18a to 18g
R2
N
Dave-phos, Pd2(dba)3, NaOtBu, Toluene
or K2CO3, CuI, L-proline, DMSO
10-50%
O
O
MeO
HN
O
HN
1) BBr3, CH2Cl2
HO
2) Br(CH2)2OSi(Me)2tBu, Cs2CO3, DMF, 60oC
N
N
3) 1M TBAF/THF
41%
18a
18h
Scheme 2. Synthesis of N-arylpiperdin-3-yl-cyclopropane carboxamides.
Table 2
Binding affinity and functional activity of N-aryl piperidin-3-yl-cyclopropane carboxamides27
Compd
Chirality
R1
R2
EC50 (nM) (IA%)
IC50 (nM) (Inh. %)
Ki (nM)
rMT1
rMT2
rMT1
rMT2
rMT1
rMT2
18a
18b
18c
18d
18e
18f
( )
S
OMe
H
H
H
Cl
Cl
H
H
H
9.7 1.7
(85 2.8)
3.9 0.6
(77 3)
3500 1000
(30 1)
0.74 0.1
(73 3)
>10,000
(19 5)
390 50
(21 5)
>10,000
(1 0.3)
>10,000
(3 0.5)
2.9 0.6
(99 0.8)
6.2 0.2
(74 7)
>10,000
>10,000
n.d.
15 0.3
2.7 0.1
OMe
n.d.
n.d.
n.d.
9.9 0.9
1000 27
1.2 0.7
2.7 0.4
95 32
2.1 0.6
R
S
OMe
93
9
10
1
(23 10)
2.3 0.5
(65 4)
>10,000
(23 5)
4.2 0.7
(57 5)
(97 2)
n.d.
OMe
R
S
OMe
440 59
(94 3)
870 11 9
(96 4)
2100 78
(95 3)
930 52
(91 3)
24
5
130 24
31
44
4
6
(98 2)
n.d.
OCHF2
OCF3
410 129
190 61
610 122
18g
18h
S
12
1
n.d.
66 11
(53 2)
>10,000
(12 1)
S
O(CH2)2OH
31
5
150 97
(94 4)
O
O
O
N
O
N
N
N
H
H
O
19
20
O
N
N
H
rMT1 EC50 >10,000 nM (IA 15%)
rMT2 EC50 1200 nM (IA 57%)
rMT1 IC50 3800 nM (41% inh.)
rMT1 Ki 1500 nM
rMT1 EC50 170 nM (IA 84%)
rMT2 EC50 50 nM (IA 35%)
rMT1 Ki 75 nM
rMT2 Ki 6.7 nM
rMT2 Ki 83 nM
rMT1 EC50 18 nM (IA 89%)
rMT2 EC50 2.5 nM (IA 98%)
rMT1 Ki 21 nM
O
N
O
N
rMT2 Ki 3.3 nM
H
21
Figure 3. Core modifications of N-arylpiperdin-3-yl-cyclopropane carboxamide.

G. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1236–1242
1241
O
Br
NH2
O
O
O
Cl
O
23
13
N
NH2
NH
Pd(PPh3)4, K2CO3, Toluene, MeOH, H 2O
91%
Et3N, CH2Cl2
78%
B(OH)2
19
24
22
N
N
Scheme 3. Synthesis of compound 19.
Table 3
In vitro microsomal CLINT and brain/plasma ratios of select compounds
Compd
Microsomal CLINT
Human
(l
L/min/mg)32
Rat
Exposure in Rat (1 h, 10 mpk, IP)
Brain (nM)
Plasma (nM)
B/P
Vehicle
Agomelatine
Ramelteon
11f
18b
18d
361
47
29
28
45
48
144
60
91
616
199
485
1030
390
540
180
—
700
580
390
730
—
1.5
0.7
1.4
0.2
—
15% Solutol
20% HP-bCD
15% Solutol
10% DMSO, 2.5% PEG400 in 15% solutol
18f
—
—
—
18. Rivara, S.; Mor, M.; Bedini, A.; Spadoni, G.; Tarzia, G. Curr. Top. Med. Chem. 2008,
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Acknowledgements
The authors acknowledge the contributions of colleagues in the
DMPK and Analytical Chemistry Groups of the Chemical & Pharma-
cokinetic Sciences Department, Lundbeck Research USA, for in vitro
and in vivo pharmacokinetic data, PAMPA results and HPLC purifi-
cations, and scientists in the Global HTS group of Biological Re-
search Department, Lundbeck Research USA, for in vitro binding
and functional data.
26. Kupfer, D. J. Eur. Neuropsychopharmacol. 2006, 16, S639.
27. Values were determined in triplicate and expressed as average SEM
(a) Functional assay: Melatonin functional assay
Supplementary data
CHO-G
384-well poly-
a
16 cell line expressing rat MT1 and rat MT2 receptor were plated in
D-lysine-coated black FLIPR plates (Corning) 24 h before the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.068. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
assay. Cell plates were washed once with Hanks’ balanced salt solution buffer
(HBSS buffer) supplemented with 20 mM HEPES + 0.1% BSA pH 7.4 (Wisent
Inc.) containing 2.5 mM Probenecid (Sigma). The plates were loaded with 50
HBSS containing the calcium-sensitive dye Fluo4-AM (Invitrogen) at 1.5
lL
lM
final concentration and pluronic acid (2% final concentration, Molecular
Probes) and were incubated at 37 °C for 1 h in a humidified chamber (5%
CO2/95% air). Following the incubation step, cells were washed three times in
References and notes
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lL
l
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1242
G. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1236–1242
C ¼ C0 ꢂ eꢁkt; LnC ¼ LnC0 ꢁ kt; k ¼ ꢁslope;
C ¼ ð1=2Þ C0 ¼ T1=2 ¼ 0:693=k
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0:693
incubation volume ðmLÞ
CLINT
¼
ꢀ
T1=2 ðminÞ microsomal protein ðmgÞ
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quenched incubations were analyzed by LC/MS to determine the remained
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