N-Acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline: The first orexin-2 receptor selective non-peptidic antagonist

Article abstract of DOI:10.1016/j.bmcl.2003.08.038

The identification of potent and selective orexin-2 receptor (OX ₂R) antagonists is described based on the modification of N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogue 1, recently discovered during high throughput screening (HTS). Substitution of an acyl group in 1 with tert-Leucine (tert-Leu), and introduction of a 4-pyridylmethyl substituent onto the amino function of tert-Leu improved compound potency, selectivity, and water solubility. Thus, compound 29 is a promising tool to investigate the role of orexin-2 receptors.

Full text of DOI:10.1016/j.bmcl.2003.08.038

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4497–4499
N-Acyl 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline: The First
Orexin-2 Receptor Selective Non-peptidic Antagonist
Masaaki Hirose*, Shin-ichiro Egashira, Yasuhiro Goto, Takashi Hashihayata,
Norikazu Ohtake, Hisashi Iwaasa, Mikiko Hata, Takehiro Fukami, Akio Kanatani
and Koji Yamada
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, Okubo 3, Tsukuba 300-2611, Ibaraki, Japan
Received 16 June 2003; accepted 15 August 2003
Abstract—The identification of potent and selective orexin-2 receptor (OX2R) antagonists is described based on the modification of
N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogue 1, recently discovered during high throughput screening (HTS).
Substitution of an acyl group in 1 with tert-Leucine (tert-Leu), and introduction of a 4-pyridylmethyl substituent onto the amino
function of tert-Leu improved compound potency, selectivity, and water solubility. Thus, compound 29 is a promising tool to
investigate the role of orexin-2 receptors.
# 2003 Elsevier Ltd. All rights reserved.
Orexin-Aand -B are the novel neuropeptides that have
been identified as ligands of orphan G protein coupled
receptors (GPCR) from rat brain extracts.1 Evidence
continues to indicate that they are involved in the reg-
ulation of many neuronal functions, including feeding,1
sleep/wake cycles,2,3 neuroendocrine activity,4,5 stress
reactions,6 and activation of the sympathetic nerve sys-
tem.7 The innervation of orexin neurons in the brain
supports diversity in function of the orexins.8 The
physiological functions of orexins are evoked by two
receptor subtypes, orexin-1R (OX1R) and orexin-2R
(OX2R) which belong to a family of GPCR. Orexin-Ais
a 33-residue peptide with two intra-molecular disulfide
bridges, which possessed potent agonistic activity
toward both subtypes of orexin receptors. In contrast,
orexin-B, which consists of 28-amino acids, possessed
greater affinity for OX2R than for OX1R.1 Moreover,
the expression patterns of the two orexin receptors in
the brain are different.9 The orexin receptor subtypes
are likely to be involved in different pharmacological
functions. Recently, a small molecular OX1R selective
antagonist was developed as a potential tool to elucidate
the functions of OX1R.10 Selective agonist of OX2R,
which can address the pharmacological functions of
OX2R, were recently identified;11 however, OX2R selec-
tive antagonists remain critical to understanding the
physiological and pathophysiological roles of OX2R.
Therefore, the development of small molecular OX2R
selective antagonists for use as pharmacological tools is
important for clarification of the functions of OX2R.
This communication describes the development of N-
acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline ana-
logues as the first OX2R selective non-peptidic antago-
nist and reports progress in optimizing this structural
class, leading to compound 29, which possesses potency,
selectivity, and water solubility. As a part of our pro-
gram to develop selective OX2R antagonists, we dis-
covered the non-selective orexin antagonist 1 (human
OX1R (hOX1R) IC50: 7 mM; human OX2R (hOX2R)
IC50: 2 mM) through HTS screening.12
Substitution and modification of the 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline skeleton in 1 with other
acyclic and cyclic amine moieties resulted in loss of
potency against both hOX1R and hOX2R (data not
shown). Thus, the 6,7-dimethoxy-1,2,3,4-tetrahydro-
isoquinoline skeleton seems to be an essential core for
retaining biological activity. Based on these results,
our attention focused on modification of the acyl
portion of 1.
*Corresponding author. Tel.: +81-298-77-2000; fax: +81-298-77-
2029; e-mail: hirosems@banyu.co.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.038

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M. Hirose et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4497–4499
Table 2. Antagonistic activity of 1-(tert-leucyl)-tetrahydroisoquino-
line analogues against hOX1R and hOX2R)a
SARof 3-Phenylpropanoyl Amides
To understand the structure–activity relationship (SAR)
of 1, initially we prepared N-(3-phenylpropanoyl) 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline
analogues.
Removal of the bromine from the benzene ring of 1
resulted in a 30-fold increase in potency toward hOX2R
(2). Removal of the tert-butyl group from 2 afforded 3,
which did not possess antagonistic activity against
either orexin receptor. The results of functional group
substitutions for the tert-butyl group of 2 are listed in
Table 1. Adimethylamino or benzylamino group at this
position was not allowed (5 and 6), suggesting that neutral
bulky substituents at this position are indispensable for
antagonistic activity toward hOX2R. Although replace-
ment of the tert-butyl moiety with a benzoylamino group
(7,8) led to significant reduction of activity toward hOX2R
compared to 2, the (S)-isomer (8) showed relatively high
potency than did its racemate (7). Introduction of neutral
bulky substituent(s) on the benzoyl group in 8 improved
potency toward hOX2R; thus, the (S)-3,5-dichloro-
benzoylamino analogue (10) brought about a 6-fold
improvement in hOX2R potency relative to 8.
No.
R
hOX1R
hOX2R
(IC50, nM)b
12
13
14
15
16
17
18
19
20
H
Phenyl
Benzoyl
Benzyl
Benzylc
Benzyld
>10,000
>10,000
>10,000
>10,000
3850
>10,000
>10,000
>10,000
>10,000
>10,000
2100
>10,000
910
130
2900
2-Phenylethyl
3-Phenylpropyl
Cyclohexylmethyl
>10,000
>10,000
>10,000
aAll compounds were synthesized as racemic mixture otherwise noted.
bValues are the mean of more than two independent experiments per-
formed in duplicate.
c(S)-Isomer.
d(R)-Isomer.
loss of potency (12, 13, 18 and 19). Substitution of a
benzyl group in 15 for a cyclohexylmethyl group also
resulted in complete loss of potency (20). Thus, aro-
matic functionality in the N-benzyl group seemed to be
essential. Regarding to the stereochemistry of the tert-
Leu portion, (S)-configuration is more preferable than
(R)-configuration (16 vs 17).
SARof N-Substituted tert-Leu Amides
To improve water solubility and develop useful phar-
macological tools, we modified the benzyl group in 1.
Introduction of hydrophilic substituents such as a
dimethyl amino group on the benzene ring of 1 resulted
in loss of potency toward both orexin receptors (data
not shown). In some cases, substitution of the benzyl
group with a substituted amino group afforded tert-Leu
amide analogues and resulted in greater water solubility
without loss of potency or selectivity (Table 2), espe-
cially in the case of the N-benzyl substituent (15).
Reduction or elongation of the carbon chain between
the nitrogen atom and phenyl group in 15 resulted in
None of the other N-benzyl amino acid analogues, such
as (N-benzyl)phenylalanine 6, N-benzyl-valine 21 or (N-
benzyl)phenylglycine 22, possessed greater potency
toward hOX2R than the tert-Leu analogue 15 (Table 3).
Only the N-benzylvaline 21 analogue showed weak
activity against hOX2R (IC50: 3300 nM).
Substitution of the benzyl group in 16 with other aryl-
methyl groups improved potency (Table 4). Replace-
ment of the benzene ring in 16 with five-membered
heteroaromatic rings generally improved hOX2R
potency. In particular, the 2-thienyl analogue 25 led to a
Table 1. Antagonistic activity of 1-(3-phenylpropanoyl)-tetra-
hydriisoquinoline analogues against hOX1R and hOX2Ra
Table 3. Antagonistic activity of N-benzyl a-aminoacyl tetra-
hydroisoquinoline analogues against hOX1R and hOX2Ra
No
R1
R1
hOX1R
hOX2R
(IC50, nM)b
1
2
3
4
5
6
7
8
Br
H
H
H
H
H
H
H
H
H
H
tert-Butyl
tert-Butyl
H
Phenyl
7000
5500
2000
56
>10,000
395
>10,000
>10,000
900
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
5300
No.
R
hOX1R
hOX2R
(IC50, nM)b
Dimethylamino
Benzylaminoc
Benzoylamino
Benzoylaminoc
6
Benzylc
tert-Butyl
tert-Butylc
i-Propyl
>10,000
>10,000
3850
>10,000
>10,000
>10,000
910
130
3300
>10,000
15
16
21
22
195
36
30
70
9
10
11
3,5-Dichlorobenzoylamino
3,5-Dichlorobenzoylaminoc
3-Bromo-4-fluorobenzoylamino
2300
5900
Phenylc
aAll compounds were synthesized as racemic mixture otherwise noted.
bValues are the mean of more than two independent experiments per-
formed in duplicate.
aAll compounds were synthesized as racemic mixture otherwise noted.
bValues are the mean of more than two independent experiments per-
formed in duplicate.
c(S)-Isomer.
c(S)-Isomer.

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4499
Table 4. Antagonisitc activity of N-arylmethyl (S)-tert-leucyl tetra-
hydroisoquinoline analogues against hOX1R and hOX2R
borohydride.14 Other compounds described here were
synthesized by similar methods.
In summary, in this communication, the structure–
activity relationship of the tetrahydroisoquinoline 1
toward hOX2R was outlined. 6,7-Dimethoxy-1,2,3,4-tet-
rahydroisoquinoline was the essential core skeleton for
hOX1R and hOX2R potency. N-Arylmethyl tert-leucyl
6,7-dimethoxy-1,2,3,4-tetrahydroiso-quinoline analogues
generally showed high potency for the hOX2R. Their
selectivity could be enhanced by modifying the arylmethyl
motif of the amino functionality. Nitrogen-containing
hetero-aromatics were superior for improving both
potency and selectivity toward hOX2R. Finally, introduc-
tion of a 4-pyridylmethyl group on the amino function of
the tert-Leu moiety yielded a potent hOX2R antagonist 29
with high selectivity and high water solubility.
No.
Ar
hOX1R
hOX2R
(IC50, nM)a
16
23
24
25
26
27
28
29
Phenyl
2-(N-Methyl)pyrrolyl
2-Thiazolyl
2-Thienyl
3850
3600
>10,000
1130
3670
>10,000
>10,000
>10,000
130
28
59
25
3-Thienyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
35
1400
240
40
aValues are the mean of more than two independent experiments per-
formed in duplicate.
References and Notes
5-fold improvement in the hOX2R potency; however, its
selectivity against hOX2R over the hOX1R was
decreased compared to 16. Nitrogen containing hetero-
aromatic analogues were generally showed high OX1R/
OX2R selectivity. For the pyridine analogues, hOX2R
potency depended heavily on the position of the nitro-
gen. 4-Pyridyl methyl analogue 29 possessed the most
potent activity for hOX2R compared to other regio iso-
mers (27 and 28). Furthermore, 29 showed over 250-
fold selectivity for hOX2R compared with hOX1R
(Table 4), as well as over 50 receptors, ion channels, and
transporters (<30% inhibition at 10 mM), which
includes G-protein coupled receptors associated with
food intake including galanin and neuripeptide Y (data
not shown). The high water solubility of 29 was another
benefit (0.81 mg/mL at pH 7) to use pharmacological
experiment. Full details of additional pharmacological
testing of 29 will be described elsewhere.
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Synthesis of Compound 29
Compound 29 was prepared according to Scheme 1.
Commercially available 6,7-dimethoxy-1,2,3,4-tetra-
hydroisoquinoline hydrogen chloride was condensed
with N-Boc tert-leucine using PyBrop13 in pyridine in
the presence of DMAP followed by treatment of HCl/
AcOEt to yield the amine hydrogen chloride 30. Con-
version of 30 to the 4-pyridylmethyl analogue 29 was
accomplished by the standard reductive amination pro-
cedure using isonicotinaldehyde and sodium triacetoxy-
11. Asahi, S.; Egashira, S.; Matsuda, M.; Iwaasa, H.; Kana-
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12. Brief screening protocol: CHO-K1 cells stably expressing
hOX1R or hOX2R were seeded into 96-well plates and incu-
bated with a cytoplasmic calcium indicator, Fluo-3 AM. After
the cells were washed four times, the intracellular Ca2+ mobi-
lization evoked by 0.3 nM of orexins-Awas monitored as a
change in cell fluorescence intensity by FLIPR (Molecular
Devices). Varying concentration of orexin antagonists were
added to the plate 5 min prior to the addition of orexin-A. The
antagonistic activities were calculated as IC50 values.
13. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett.
1990, 31, 205.
14. Characterization of 29: 1H NMR (300 MHz, CDCl3) d
0.97 and 1.02 (s each, 9H), 2.61–2.90 (m, 2H), 3.20–3.51 (m,
3H), 3.62–4.13 (m, 8H), 4.31–4.99 (m, 2H), 6.40, 6.60, 6.60
and 6.65 (s each, 2H), 7.09–7.32 (m, 2H), 8.40–8.56 (m, 2H).
Scheme 1. Synthesis of compound 29.