The importance of the 6- and 7-positions of tetrahydroisoquinolines as selective antagonists for the orexin 1 receptor

Article abstract of DOI:10.1016/j.bmc.2015.07.013

Selective antagonism of the orexin 1 (OX₁) receptor has been proposed as a potential mechanism for treatment of drug addiction. We have previously reported studies on the structure-activity relationships of tetrahydroisoquinoline-based antagonists. In this report, we elucidated the respective role of the 6- and 7-substitutions by preparation of a series of either 6-substituted tetrahydroisoquinolines (with no 7-substituents) or vice versa. We found that 7-substituted tetrahydroisoquinolines showed potent antagonism of OX₁, indicating that the 7-position is important for OX₁ antagonism (10c, K_e = 23.7 nM). While the 6-substituted analogs were generally inactive, several 6-amino compounds bearing ester groups showed reasonable potency (26a, K_e = 427 nM). Further, we show evidence that suggests several compounds initially displaying insurmountable antagonism at the OX₁ receptor are competitive antagonists with slow dissociation rates.

Full text of DOI:10.1016/j.bmc.2015.07.013

Bioorganic & Medicinal Chemistry 23 (2015) 5709–5724
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The importance of the 6- and 7-positions of tetrahydroisoquinolines
as selective antagonists for the orexin 1 receptor
David A. Perrey a, Ann M. Decker a, Jun-Xu Li b, Brian P. Gilmour a, Brian F. Thomas a, Danni L. Harris a,
Scott P. Runyon a, Yanan Zhang a,
⇑
a Research Triangle Institute, Research Triangle Park, NC 27709, United States
b Department of Pharmacology and Toxicology, University at Buffalo, The State University of New York, Buffalo, NY 14214, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective antagonism of the orexin 1 (OX1) receptor has been proposed as a potential mechanism for
treatment of drug addiction. We have previously reported studies on the structure–activity relationships
of tetrahydroisoquinoline-based antagonists. In this report, we elucidated the respective role of the
6- and 7-substitutions by preparation of a series of either 6-substituted tetrahydroisoquinolines (with
no 7-substituents) or vice versa. We found that 7-substituted tetrahydroisoquinolines showed potent
antagonism of OX1, indicating that the 7-position is important for OX1 antagonism (10c, Ke = 23.7 nM).
While the 6-substituted analogs were generally inactive, several 6-amino compounds bearing ester
groups showed reasonable potency (26a, Ke = 427 nM). Further, we show evidence that suggests
several compounds initially displaying insurmountable antagonism at the OX1 receptor are competitive
antagonists with slow dissociation rates.
Received 10 May 2015
Revised 1 July 2015
Accepted 9 July 2015
Available online 16 July 2015
Keywords:
Orexin
Antagonist
Selective
Tetrahydroisoquinoline
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
producing neurons in the ventral tegmental area (VTA) and their
afferents in the nucleus accumbens.7,8 Knockout mice lacking the
Addiction is a chronic, often relapsing brain disease that causes
compulsive drug seeking and use, despite harmful consequences.
Drug abuse and addiction represent a major public health problem
and a significant unmet medical need (www.drugabuse.gov).
Unfortunately, there are few effective pharmacotherapies for
addiction, and currently approved medications predominantly
cover alcohol, nicotine and opiates.1 The orexin system consists
of two neuropeptides, orexin-A and B (also known as the hypocre-
tins), and two G protein-coupled receptors, orexin 1 and 2 (OX1
and OX2).2,3 While the orexin system has been extensively impli-
cated in the regulation of arousal and sleep-wake cycles, OX1 in
particular has more recently been considered a candidate target
for addiction treatment.4–6 The orexins are produced in the
hypothalamus and the orexin neurons project to brain regions
implicated in incentive motivation, such as the dopamine
prepro-orexin gene demonstrate reduced morphine-induced con-
ditioned place preference, as well as reduced morphine- and
cocaine-induced dopamine release.9–12 Moreover, selective block-
ade of the OX1 receptor has been shown to attenuate addictive
behaviors such as cocaine-, alcohol- and morphine-seeking.13–16
Thus a selective OX1 antagonist would be a potentially useful ther-
apeutic agent for drug addiction.
It is generally accepted that arousal is most closely associated
with activation of the OX2 receptor and reward with OX1 receptor
activation, although modulation of both receptors simultaneously
may prove to be more effective than OX2 alone for sleep disor-
ders.13,17 A number of orexin receptor antagonists that inhibit both
receptors have been developed, including almorexant (1, Fig. 1)
and suvorexant, the latter of which was approved by the FDA for
the treatment of insomnia;18 however, much less has been done
on developing OX1 selective antagonists. While the availability of
the partially selective OX1 antagonist SB-334867 (ꢀ50 fold for
OX1 over OX2) allowed the initial elucidation of the function of
the OX1 receptor in drug abuse and addiction,19 recent studies sug-
gest that OX2 may also play a role in certain aspects of reward and
addiction. For instance, OX2 selective antagonists attenuated alco-
hol self-administration and conditioned place preference,20 as well
Abbreviations: BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-tria-
zolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HPLC, high performance liquid
chromatography; OX1, orexin 1 receptor; OX2, orexin 2 receptor; SAR, structure–
activity relationship; TLC, thin layer chromatography.
⇑
Corresponding author. Tel.: +1 919 541 1235; fax: +1 919 541 6499.
E-mail address: yzhang@rti.org (Y. Zhang).
http://dx.doi.org/10.1016/j.bmc.2015.07.013
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

5710
D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
O
O
O
N
O
O
N
H
N
N
H
O
N
2 (TCS-OX2-29)
CF3
1 (Almorexant)
7
O
O
O
O
N
N
F3C
O
O
N
H
N
O
O
N
H
6
1
O
O
4 (RTIOX-276)
3 (ACT-335827)
Figure 1. Tetrahydroisoquinoline-based orexin antagonists.
as cue-induced nicotine reinstatement.21 On the contrary, OX2
antagonist TCS-OX2-29 (2) showed no effect on cocaine self-ad-
ministration or cue-induced reinstatement.22 Together, these find-
ings underscore the importance of the development of OX1
antagonists with improved selectivity in order to further probe
the individual roles of the OX1 and OX2 receptors. Since the discov-
ery of SB334867, several other OX1 receptor selective antagonists
have been reported, but some OX2 activity still remains for most
of these compounds (e.g., 3, ACT-335827).23,24
on the THIQ core.30 Moreover, the 6- and 7-positions seem to be
highly sensitive to substitutions;26,31 therefore, the SARs around
these positions need to be further probed. In this study, we exam-
ined the importance of substitution at the 6- and 7-positions of the
THIQ core, respectively, and investigated the impact of removing
one of the methoxy groups on orexin receptor activity.
2. Methods
We previously reported our efforts in developing selective OX1
antagonists,25–27 in particular those based on the tetrahydroiso-
quinoline (THIQ) scaffold found in other orexin antagonists such
as dual antagonist almorexant (1) and the OX2 selective antagonist
TCS-OX2-29 (2). We have synthesized and pharmacologically eval-
uated a series of 6,7-disubsituted THIQs and identified several
highly potent and selective OX1 antagonists such as RTIOX-276
(4).26 However, the highly electron rich aromatic rings with poly-
methoxy groups present in these compounds may be susceptible
to oxidative metabolism as these methoxy groups could be
metabolically cleaved.28,29 While the dimethoxybenzyl group at
the 1-position can be replaced with other aromatic groups that
retain potency and OX1 selectivity,25 most orexin antagonists
based on this scaffold share the 6,7-dimethoxy substitution pattern
2.1. Chemistry
In the 6,7-dialkoxy series, analogs with electron deficient
groups at the 7-position demonstrated excellent OX1 potency and
selectivity.26 Therefore, we attempted to prepare 7-substituted
analogs with substituents of different nature. However, the syn-
thetic route previously used for 6,7-disubstituted analogs did not
afford any desired products in the Bischler–Napieralski cyclization
reaction on the appropriately 7-substituted amide 6 (R = H or
alkyl), even with increased heat and time (Scheme 1). After protec-
tion of the tyramine nitrogen as the methyl carbamate followed by
alkylation of the phenol with the desired substituents (7a–e), the
tetrahydroisoquinoline 8a–e (R = alkyl) was obtained in 20–27%
yield under modified Pictet–Spengler conditions in neat
H
N
c
R
N
O
d
O
NH2
O
MeO
OMe
R
OMe
O
HO
5
MeO
7a-e
8a-e
N
e
f
a
H
N
O
O
b
R
NH
R
O
R
O
N
O
MeO
H
MeO
MeO
MeO
OMe
MeO
6
9a-e
10a-e
Scheme 1. Synthesis of 7-alkoxytetrahydroisoquinoline derivatives. Reagents and conditions: (a) (i) 3,4-dimethoxyphenylacetic acid, BOP, iPr2EtN, DMF; (ii) R-I or R-Br,
K2CO3, DMF; (b) (i) POCl3, toluene; (ii) NaBH4, MeOH; (c) (i) MeOCOCl, iPr2EtN, CH2Cl2; (ii) R-I or R-Br, K2CO3, DMF; (d) 3,4-dimethoxyphenylacetaldehyde, TFA; (e) KOH,
NH2NH2ꢁH2O, (CH2OH)2; (f) BrCH2CONHBn, iPr2EtN, Bu4NI, DMF.

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5711
BnO
O
HN
O
NH2
BnO
NO2
HO
a
BnO
b
c
HO
OMe
OMe
12
11
13
14
O
BnO
O
O
R
N
d
N
e
f
NH
N
H
N
H
MeO
MeO
MeO
MeO
MeO
MeO
17a-f
16
15
g
h
O
O
O
O
O
O
O
S
R
N
N
R
N
H
N
H
MeO
MeO
MeO
MeO
19
18a-b
Scheme 2. Synthesis of 6-alkoxytetrahydroisoquinoline derivatives. Reagents and conditions: (a) (i) BnBr, K2CO3, DMF; (ii) MeNO2, NH4OAc, AcOH; (b) LiAlH4, Et2O, CH2Cl2;
(c) 3,4-dimethoxyphenylacetic acid, BOP, iPr2EtN, DMF; (d) (i) POCl3, toluene; (ii) NaBH4, MeOH; (e) (i) BrCH2CONHBn, iPr2EtN, Bu4NI, DMF; (ii) NH4HCOO, Pd/C, EtOH; (f) R-
Br, K2CO3, Bu4NI, DMF or R-I, K2CO3, DMF; (g) RSO2Cl, Et3N, CH2Cl2; (h) RCOCl, iPr2EtN, CH2Cl2.
trifluoroacetic acid at 70 °C. The aldehyde used in this step was
obtained via Dess–Martin oxidation from 2-(3,4-dimethoxypheny-
l)ethanol. However, neither conditions gave any cyclization pro-
duct with other electron-withdrawing substituents such as a
trifluoroethyl group or the free phenol (R = H). Several protecting
groups were investigated but none afforded any of the desired
compounds. Removal of the methyl carbamate protecting group
on 7a–e was also problematic. Several conditions were examined
and the best conditions found were potassium hydroxide and
hydrazine monohydrate in ethylene glycol at 120 °C (yields 32–
68%). Finally, N-alkylation of 9a–e with N-benzylbromoacetamide
gave the final compounds 10a–e.
6-Substituted tetrahydroisoquinoline derivatives were synthe-
sized by methods adapted from our earlier work,25,26 summarized
in Schemes 2 and 3. The 6-alkoxy series first required synthesis of
the phenylethylamines (Scheme 2). 3-Hydroxybenzaldehyde 11
was protected as the benzyl ether with benzyl bromide in the pres-
ence of potassium carbonate followed by a Henry reaction with
nitromethane to give the nitrostyrene 12, which upon reduction
with lithium aluminum hydride gave phenylethylamine 13.32
Coupling with 3,4-dimethoxyphenylacetic acid gave the amide
14, then Bischler–Napieralski cyclization followed by sodium boro-
hydride reduction gave the tetrahydroisoquinoline 15. N-alkyla-
tion with N-benzylbromoacetamide followed by benzyl ether
deprotection via transfer hydrogenation using palladium on carbon
in the presence of ammonium formate gave 16. The phenol 16 was
then elaborated via alkylation with alkyl halides in the presence of
potassium carbonate (17a–f), sulfonylation with sulfonyl chlorides
(18a–b) or acylation with acid chlorides (19).
protecting group was removed by basic hydrolysis. Hydrolysis
required extended heating time and an attempt to speed up this
process in ethanol resulted in an improved reaction rate but also
produced some transesterification product in the form of the ethyl
carbamate 23b. This aniline 24 could then be elaborated further via
reductive amination (25a–d, 25g–i, m, n, r–u) or base-catalyzed
alkylation (25e, f, j–l, o–q, 26a–h), sulfonylation (27), acylation
(28a–c) or urea formation (28d–e).
2.2. Biological assays
Activity of the target compounds at the OX1 and OX2 receptors
was evaluated in calcium mobilization based functional curve shift
assays as previously described.25–27,33 In these assays, EC50 curves
of the agonist orexin-A were obtained alone and together with the
test compound (15 min pre-incubation), respectively, and the
right-shift of the agonist curve was measured. The apparent disso-
ciation constant Ke was then calculated from compound-mediated
inhibition of orexin A activity as previously described.25,26 The EC50
values for orexin A at OX1 and OX2 were 0.13 0.02 nM and
4.2 0.2 nM, respectively. All the compounds that had OX1 Ke val-
ues <1 lM were also tested alone at 10 lM for agonist activity;
none of the compounds showed any agonist activity at either
receptor.
3. Results and discussion
Given the importance of the 7-position substitutions for activity
in the 6,7-disubstituted THIQs reported earlier, we first examined a
series of compounds that are only substituted at the 7-position
(Table 1). To this end, several 7-substituted alkoxy analogs were
synthesized and tested. Because of the synthetic difficulties
described above, other substitutions (e.g., electron withdrawing
groups) were not examined. Compared to the 6,7-disubstituted
analogs, the -7-substituted analogs showed similar activity at the
OX1 receptor and three (10b, 10c, 10d) showed excellent
For the 6-amino series (Scheme 3), 3-nitrophenylethylamine 20
was coupled with 3,4-dimethoxyphenylacetic acid to give amide
21. The nitro group was then reduced with Raney nickel and the
aniline was protected as the methyl carbamate (22) using methyl
chloroformate. Bischler–Napieralski reaction gave the dihydroiso-
quinoline which was readily reduced to the tetrahydroisoquinoline
using sodium borohydride. The amine was alkylated with the bro-
moacetamide in the presence of DIPEA to give 23a and then the

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D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
H
O2N
O
N
c
a
HN
O
b
OMe
HN
O
O2N
NH2
OMe
OMe
OMe
OMe
20
22
21
R'
N
H
N
O
R
O
H2N
O
O
N
OR
N
N
H
d
N
e
N
H
N
H
MeO
MeO
MeO
MeO
MeO
MeO
25a-u
26a-h
24
23a-b
g
f
H
H
O
S
O
N
N
O
O
O
R
N
N
N
H
N
H
MeO
MeO
MeO
MeO
27
28a-e
Scheme 3. Synthesis of 6-aminotetrahydroisoquinoline derivatives. Reactions and conditions: (a) 3,4-dimethoxyphenylacetic acid, BOP, iPr2EtN, DMF; (b) (i) Raney Ni,
NH2NH2ꢁH2O, EtOH; (ii) MeOCOCl, iPr2EtN, CH2Cl2; (c) (i) POCl3, toluene; (ii) NaBH4, MeOH; (iii) BrCH2CONHBn, iPr2EtN, Bu4NI, DMF; (d) 2 N NaOH (aq), MeOH; (e) aldehyde,
Na(OAc)3BH, 1-2-DCE or alkyl halide, iPr2EtN, DMF; (f) (i) MsCl, Et3N, CH2Cl2; (ii) 2 N NaOH (aq); (g) RCO2H, BOP, iPr2EtN, CH2Cl2 or RCOCl/(RCO)2O, iPr2EtN, CH2Cl2 or
isocyanate, toluene.
Table 1
results clearly indicate that a substituent with appropriate size is
preferred for activity at the 7-position.
7-Alkoxy tetrahydroisoquinoline orexin antagonists
The importance of the 6-position had not been previously
examined. Therefore, we prepared a series of analogs with different
O
R
N
substituents to replace the methyl group at the 6-methoxy posi-
tion, including both electron rich and electron deficient alkyl and
aryl groups. As shown in Table 2, these compounds were mostly
inactive at the OX1 receptor, showing Ke values greater than
O
O
N
H
O
10 lM. Interestingly, the butanoate analog 17e showed a Ke of
a
a
No.
R
OX1 Ke (nM)
OX2 Ke (nM) or % inhibition at 10
lM
1000 nM, and was only slightly less potent than the 6,7-dimethoxy
analog 4, suggesting that the added ester group may have addi-
tional interaction with the receptor, or potentially occupy the same
space of the 7-methoxy as in compound 4. When screened at the
10a
10b
10c
10d
10e
Methyl
Ethyl
n-Propyl
Iso-propyl
n-Butyl
512 36
37.3 7.8
23.7 7.9
49.7 11
230 70
1680 330
b
2550 260
b
b
OX2 receptor for antagonist activity at 10
showed little inhibition.
lM, these compounds
a
Ke values are the mean SEM of three independent experiments performed in
duplicate. Results where Ke > 10 M are N = 2.
Less than 50% inhibition at 10 M.
We next prepared a series of 6-amino derivatives as shown in
Table 3. Compared to the alkoxy series, the 6-position nitrogen
can be di-substituted; one of those substituents may be able to
take up similar space as the 7-position substituents and therefore
make up for the potency lost by removal of the 7-substituent.
Given the difficulties faced in the synthesis of the 7-substituted
analogs, these 6-amino analogs may provide an alternative way
to improve the potency. The 6-alkylamino analogs showed greater
potency than the 6-alkoxy series, although mostly in the low
micromolar range. The potencies in general were improved by cap-
ping the nitrogen with a methyl group (25b vs 25c; 25f vs 25g),
though dialkylation gave similar potency to monoalkylation (25b
vs 25d). Larger alkyl groups (25r–u) gave no detectable potency,
as was the case with the cyclic piperidinyl group 25q, though the
slightly smaller pyrrolidinyl 25p showed some potency. The benzyl
substituent showed the opposite effect; the monobenzyl derivative
l
b
l
selectivity against the OX2 receptor (>100-fold). The n-propyl
derivative 10c was the most potent compound of the series, with
a Ke value of 23.7 nM and was 108-fold more selective for OX1 over
OX2. The ethyl analog 10b and isopropyl analog 10d were slightly
less potent than 10c, with Ke values of 37.3 nM and 49.7 nM,
respectively, but had higher OX1 selectivities (268- and 201-fold,
respectively). The 7-methoxy analog 10a had a Ke of 512 nM, and
was only two-fold less potent than compound 4. The previously
described trend of potency first increasing then decreasing as the
alkyl groups increase in size is also present in the 6,7-disubstituted
analogs.26 Both ethyl 10b and isopropyl 10d were almost as potent
as 10c, but potency decreased for the butyl analog 10e. These

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5713
Table 2
Table 3
6-Alkoxy tetrahydroisoquinoline orexin antagonists
6-Amino tetrahydroisoquinoline orexin antagonists
O
R'
N
R
O
N
R
O
N
H
N
O
O
N
H
O
O
a
a
No.
R
OX1 Ke
OX2 Ke (nM) or %
a
a
R0
OX1 Ke (nM) OX2 Ke (nM)
(nM)
inhibition at 10 lM
No.
R
17a n-Propyl
17b 2,2,2-Trifluoroethyl
17c Iso-propyl
>10,000
>10,000
>10,000
>10,000
b
b
b
b
25a
25b
25c
25d
25e
25f
25g
25h
25i
Methyl
Methyl
H
Methyl
n-Propyl 2350 490
H
1370 100
>10,000
619 96
b
b
b
b
b
b
b
b
b
n-Propyl
n-Propyl
n-Propyl
Iso-propyl
Cyclopropylmethyl
Cyclopropylmethyl
Benzyl
17d Benzyl
O
>10,000
>10,000
1400 240
1100 290
>10,000
17e
1000 190c
b
H
EtO
Methyl
H
Methyl
EtO
Benzyl
17f
>10,000
b
O
MeO
18a Methylsulfonyl
18b Benzenesulfonyl
>10,000
>10,000
>10,000
1330 260
b
1290 130
25j
H
2180 360c
b
MeO
3-Phenylpropyl
4-Phenoxybutyl
19
Propionoyl
25k
25l
H
H
H
H
1180 80
2080 20
>10,000
b
b
b
b
a
Ke values are the mean SEM of three independent experiments performed in
duplicate. Results where Ke > 10 M are N = 2.
Less than 50% inhibition at 10 M.
Pre-incubation of antagonist and test compound was 1 h.
l
25m 4-Pyridylmethyl
b
l
25n
3-Pyridylmethyl
1270 110
c
N
25o
H
>10,000
b
25h had a Ke of 1100 nM but this was lost with the methylated
analog 25i. Extending the linker to the phenethyl 25j and the
phenylpropyl 25k showed little change. Pyridylmethyl sub-
stituents 25m and 25n showed a decline in potency, indicating
that a basic nitrogen was not favorable but was tolerated for the
3-pyridyl, though making ethylpiperidinyl 25o caused a loss of
detectable potency. The alkyl ester series (26a–e) showed some
activity, with Ke values ranging from 427 to 949 nM, with the
exception of the branched analog 26b, suggesting that there is lim-
ited space. Reinforcing that idea is the drop in potency for the
longer alkyl esters 26f and 26g. Replacing the alkyl ester with an
alkyl amide 26h caused a large decline in potency, and all the
amide derivatives (28a–c) showed no detectable activity. The
methyl carbamate 23a showed slight potency, though the ethyl
carbamate 23b was not active, while the sulfonamide 27 and ureas
28d and 28e showed no significant potency. This may be due to a
lack of flexibility in the side chain, giving unfavorable interactions
with the binding site.
Interestingly, while most of the compounds tested in the cal-
cium mobilization curve shift assays displayed competitive antag-
onism, including the most potent compound 10c, several of the 6-
substituted analogs demonstrated insurmountable antagonism
under the testing conditions, such as 17e, 25j and 26e. They not
only shifted the orexin agonist curve to the right as expected with
antagonists, but also suppressed the maximal orexin response.
Almorexant has been known to behave as an insurmountable
antagonist at the OX2 receptor.34 While it is commonly observed
in allosteric modulation due to the nature of the indirect interac-
tion between allosteric ligands and orthosteric agonists, insur-
mountable antagonism can also be the result of competitive
orthosteric antagonists with slow dissociations rates. One way to
help distinguish between these two mechanisms involves perform-
ing curve shift assays as described above with longer antagonist-
receptor incubation periods, which allows for the system to reach
equilibrium.35 Steiner and co-workers used another method to dis-
tinguish between allosteric and competitive orthosteric antago-
nists by performing calcium mobilization curve shift experiments
25p
25q
25r
25s
25t
25u
Pyrrolidine
Piperidine
n-Pentyl
n-Pentyl
n-Hexyl
n-Hexyl
O
2480
6
b
b
b
b
b
b
>10,000
>10,000
>10,000
>10,000
>10,000
H
Methyl
H
Methyl
26a
26b
H
H
427 69
>10,000
b
b
EtO
O
EtO
EtO
26c
H
809 200
b
O
O
26d
26e
26f
H
H
H
591 67
>10,000
EtO
O
1010 200d
1600 190
b
b
O
MeO
O
EtO
26g
26h
H
>10,000
b
b
O
O
H
>10,000
N
27
Methylsulfonyl
Acetyl
Hexanoyl
H
H
H
H
H
H
H
H
>10,000
>10,000
>10,000
>10,000
2400 320
>10,000
>10,000
5230 680
b
b
28a
28b
28c
23a
23b
28d
28e
>10,000
b
>10,000
>10,000
b
b
Benzoyl
Methyl carbamoyl
Ethyl carbamoyl
PhNHCO
n-Hexyl-NHCO
a
Ke values are the mean SEM of three independent experiments performed in
duplicate. Results where Ke > 10 M are N = 2.
Less than 50% inhibition at 10 M.
l
b
l
c
Pre-incubation of antagonist and receptor was 45 min.
Pre-incubation of antagonist and receptor was 1 h.
d

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D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
15 minute pre-incubation of compound prior to Orexin A addition 45 minute pre-incubation of compound prior to Orexin A addition
8000
6000
4000
2000
0
Orexin A
Orexin A
Orexin A + 17e
Orexin A + 25j
Orexin A + 26e
Orexin A + 17e
Orexin A + 25j
Orexin A + 26e
20000
10000
0
-2
0
2
-2
0
2
log [nM]
log [nM]
1 hour pre-incubation of compound prior to Orexin A addition
4000
Simultaneous Addition of Orexin A and Compound
Orexin A
Orexin A
Orexin A + 17e
Orexin A + 25j
Orexin A + 26e
Orexin A + 17e
Orexin A + 25j
Orexin A + 26e
3000
2000
1000
0
10000
5000
0
-2
0
2
-2
0
2
log [nM]
log [nM]
Figure 2. Effect of incubation time on calcium mobilization of compounds 17e, 25j and 26e in OX1 cells. Each data point represents the composite of at least three individual
experiments performed in duplicate.
with simultaneous addition of antagonist and Orexin
A
as
Under increased pre-incubation time (45 min and 1 h) or simulta-
neous addition of antagonist and Orexin A, these compounds
appeared to be competitive antagonists, indicating that the insur-
mountable antagonism observed with a 15 min pre-incubation is
most likely the result of slow dissociation rates.
described in a recent publication.36 To help elucidate the mecha-
nism of antagonism for the insurmountable antagonists in our
study, we tested both methods with compounds 17e, 25j and
26e (Fig. 2). The 15 min pre-incubation clearly depicts insurmount-
able antagonism for all three compounds. As the pre-incubation
times were increased to 45 min and 60 min, the compounds
5. Experimental procedures
5.1. General
appeared
increasingly
to
be
competitive
antagonists.
Interestingly, all three compounds also appeared to be competitive
antagonists under the simultaneous addition. Thus, with our com-
pounds, both methods appear to distinguish between allosteric
and competitive orthosteric antagonists.
All solvents and chemicals were reagent grade. Unless other-
wise mentioned, all were purchased from commercial vendors
and used as received. Flash column chromatography was done on
a Teledyne ISCO CombiFlash Rf system using prepacked columns.
Solvents used were hexane, ethyl acetate (EtOAc), dichloro-
methane (DCM), methanol and chloroform/methanol/ammonium
hydroxide (80:18:2) (CMA-80). Purity and characterization of com-
pounds was established by a combination of high pressure liquid
chromatography (HPLC), thin layer chromatography (TLC), mass
spectrometry (MS) and nuclear magnetic resonance (NMR) analy-
sis. 1H and 13C NMR spectra were recorded on a Bruker Avance
DPX-300 (300 MHz) spectrometer and were determined in chloro-
form-d or methanol-d4 with tetramethylsilane (TMS) (0.00 ppm)
or solvent peaks as the internal reference. Chemical shifts are
reported in ppm relative to the reference signal, and coupling con-
stant (J) values are reported in Hz. TLC was done on EMD precoated
silica gel 60 F254 plates, and spots were visualized with UV light or
iodine staining. Low resolution mass spectra were obtained using a
Waters Alliance HT/Micromass ZQ system (ESI). High resolution
mass spectra were obtained using an Agilent 6230 time-of-flight
mass spectrometer. Melting points were determined using a Mel
Temp II melting point apparatus and are uncorrected. All test com-
pounds were greater than 95% pure as determined by HPLC on an
Agilent 1100 system using an Agilent Zorbax SB-Phenyl,
4. Conclusions
In conclusion, we prepared a series of 6- and 7-mono-substi-
tuted THIQ derivatives to investigate the contribution of the 6-
and 7-positions to OX1 potency. The 7-substituted analogs with
alkyl groups indeed showed potency at the OX1 receptor compara-
ble to the corresponding 6,7-disubstituted analogs, though
synthetic challenges made further SAR exploration difficult. The
6-substituted analogs were also examined and the observed Ke
values were only modest, in contrast to the much more active
6,7-disubstituted derivatives. However, several 6-position analogs
with terminal ester groups (26a, 26c, 26d) showed some activity at
the OX1 receptor, potentially introducing additional interactions
between the ester group and the receptor or alternatively, occupy-
ing the same space of the 7-methoxy as in compound 4. It is clear
that the 7-substituent is the dominant position for determining
potency in this series, though some contribution remains from
the 6-substituent, as a slight drop in potency is observed as com-
pared with the 6-methoxy analogs. Several 6-substituted com-
pounds displayed insurmountable antagonism at the OX1
receptor under the assay conditions (15 min pre-incubation).

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5715
2.1 mm ꢂ 150 mm, 5
l
m column with gradient elution using the
4.68 (br s, 1H), 4.51 (td, J = 6.03, 12.06 Hz, 1H), 3.66 (s, 3H), 3.34–
3.45 (m, 2H), 2.74 (t, J = 6.97 Hz, 2H), 1.33 (d, J = 6.03 Hz, 6H).
mobile phases (A) H2O containing 0.1% CF3COOH and (B) MeCN,
with a flow rate of 1.0 mL/min.
5.7. Methyl N-[2-(4-butoxyphenyl)ethyl]carbamate (7e)
5.2. Methyl N-[2-(4-hydroxyphenyl)ethyl]carbamate
This was prepared as per 7b but using 1-bromobutane to get the
desired butyl ether as a white solid. Yield 81%. 1H NMR (300 MHz,
CHLOROFORM-d) d 7.09 (d, J = 8.48 Hz, 2H), 6.80–6.88 (m, 2H), 4.67
(br s, 1H), 3.94 (t, J = 6.50 Hz, 2H), 3.66 (s, 3H), 3.34–3.45 (m, 2H),
2.74 (t, J = 6.97 Hz, 2H), 1.70–1.82 (m, 2H), 1.42–1.56 (m, 2H), 0.97
(t, J = 7.35 Hz, 3H).
To a suspension of tyramine (1.0 g, 7.29 mmol) in DCM (35 mL)
cooled in an ice bath under N2 was added DIPEA (1.41 g, 1.9 mL,
10.94 mmol); then methyl chloroformate (0.86 g, 0.7 mL,
9.11 mmol) was slowly added. A homogeneous solution formed
and the reaction was allowed to warm slowly to RT overnight.
The reaction was washed with NaHCO3 solution and brine, and
dried over MgSO4 and the solvent was removed under reduced
pressure. The crude material was purified by chromatography on
silica (0–40% EtOAc/hexane) to give the desire carbamate as a clear
oil (0.74 g, 52%). 1H NMR (300 MHz, CHLOROFORM-d) d 7.04 (d,
J = 8.48 Hz, 2H), 6.75–6.82 (m, 2H), 5.49 (br s, 1H), 4.71 (br s,
1H), 3.66 (s, 3H), 3.35–3.45 (m, 2H), 2.73 (t, J = 6.97 Hz, 2H).
5.8. Methyl 1-[(3,4-dimethoxyphenyl)methyl]-7-methoxy-1,2,3,-
4-tetrahydroisoquinoline-2-carboxylate (8a)
Carbamate 7a (209 mg, 1.0 mmol) and 3,4-dimethoxyphenylac-
etaldehyde (216 mg, 1.2 mmol) were combined in trifluoroacetic
acid (3 mL) and heated to 70 °C overnight. The reaction was care-
fully quenched by pouring into saturated NaHCO3 solution then
extracted with EtOAc, The organic fraction was washed with brine,
and dried over MgSO4 and the solvent was removed under reduced
pressure. The crude was purified by chromatography on silica (0–
20% EtOAc/hexane) to give the tetrahydroisoquinoline 8a (89 mg,
24%). 1H NMR (300 MHz, CHLOROFORM-d) d 7.02 (dd, J = 8.48,
11.68 Hz, 1H), 6.69–6.81 (m, 2H), 6.61 (dd, J = 8.19, 14.03 Hz, 1H),
6.32–6.56 (m, 2H), 5.14–5.33 (m, 1H), 3.48–3.89 (m, 13H), 3.20–
3.38 (m, 1H), 2.93–3.17 (m, 2H), 2.46–2.90 (m, 2H).
5.3. Methyl N-[2-(4-methoxyphenyl)ethyl]carbamate (7a)
This was prepared as per methyl N-[2-(4-hydrox-
yphenyl)ethyl]carbamate but using 4-methoxyphenylethylamine.
Yield quantitative. 1H NMR (300 MHz, CHLOROFORM-d) d 7.11
(d, J = 8.48 Hz, 2H), 6.85 (d, J = 8.67 Hz, 2H), 4.68 (br s, 1H), 3.79
(d, J = 0.57 Hz, 3H), 3.66 (s, 3H), 3.35–3.45 (m, 2H), 2.75
(t, J = 6.88 Hz, 2H).
5.9. Methyl 1-[(3,4-dimethoxyphenyl)methyl]-7-ethoxy-1,2,3,4-
tetrahydroisoquinoline-2-carboxylate (8b)
5.4. Methyl N-[2-(4-ethoxyphenyl)ethyl]carbamate (7b)
This was prepared as per 8a using 7b. Yield 21%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.00 (dd, J = 8.38, 11.77 Hz, 1H),
6.69–6.80 (m, 2H), 6.61 (dd, J = 8.10, 15.07 Hz, 1H), 6.35–6.56 (m,
2H), 5.13–5.33 (m, 1H), 3.87–4.00 (m, 2H), 3.46–3.87 (m, 11H),
3.18–3.36 (m, 1H), 2.93–3.14 (m, 2H), 2.44–2.89 (m, 2H), 1.30–
1.43 (m, 2H).
Methyl
N-[2-(4-hydroxyphenyl)ethyl]carbamate
(0.4 g,
2.05 mmol) and potassium carbonate (0.85 g, 6.15 mmol) were
combined in dry DMF (10 mL) and iodoethane (0.48 g, 0.25 mL,
3.07 mmol) was added and the reaction was stirred at RT over-
night. The reaction was diluted with EtOAc, then washed with
NaHCO3 solution, water and brine, and dried over MgSO4 and the
solvent was removed under reduced pressure. The crude material
was purified by chromatography on silica (0–30% EtOAc/hexane)
5.10. Methyl 1-[(3,4-dimethoxyphenyl)methyl]-7-propoxy-1,
2,3,4-tetrahydroisoquinoline-2-carboxylate (8c)
to give the ether as
a
white solid (0.36 g, 78%). 1H NMR
(300 MHz, CHLOROFORM-d) d 7.09 (d, J = 8.67 Hz, 2H), 6.80–6.87
(m, 2H), 4.67 (br s, 1H), 4.01 (q, J = 7.03 Hz, 2H), 3.66 (s, 3H),
3.35–3.46 (m, 2H), 2.74 (t, J = 6.97 Hz, 2H), 1.41 (t, J = 7.06 Hz, 3H).
This was prepared as per 8a using 7c. Yield 27%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.00 (dd, J = 8.48, 11.49 Hz, 1H),
6.69–6.80 (m, 2H), 6.61 (dd, J = 8.95, 13.09 Hz, 1H), 6.33–6.56 (m,
2H), 5.14–5.32 (m, 1H), 3.93–4.05 (m, 2H), 3.46–3.89 (m, 10H),
3.19–3.36 (m, 1H), 2.92–3.15 (m, 2H), 2.66–2.89 (m, 1H), 2.45–
2.64 (m, 1H), 1.75 (dt, J = 6.97, 13.56 Hz, 2H), 0.94–1.08 (m, 3H).
5.5. Methyl N-[2-(4-propoxyphenyl)ethyl]carbamate (7c)
This was prepared as per 7b but using 1-iodopropane to get the
desired ether as a white solid (0.56 g, 76%). 1H NMR (300 MHz,
CHLOROFORM-d) d 7.05–7.17 (m, 2H), 6.80–6.91 (m, 2H), 4.68
(br s, 1H), 3.86–3.98 (m, 2H), 3.67 (s, 3H), 3.34–3.49 (m, 2H),
2.70–2.82 (m, J = 3.60 Hz, 2H), 1.73–1.88 (m, 2H), 0.99–1.11 (m,
3H).
5.11. Methyl 1-[(3,4-dimethoxyphenyl)methyl]-7-(propan-2-yloxy)-
1,2,3,4-tetrahydroisoquinoline-2-carboxylate (8d)
This was prepared as per 8a using 7d. Yield 20%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.00 (dd, J = 8.48, 11.30 Hz, 1H),
6.68–6.79 (m, 2H), 6.61 (dd, J = 9.04, 14.13 Hz, 1H), 6.35–6.55 (m,
2H), 5.13–5.32 (m, 1H), 4.27–4.47 (m, 1H), 3.47–3.88 (m, 10H),
3.18–3.36 (m, 1H), 2.93–3.15 (m, 2H), 2.45–2.88 (m, 2H), 1.20–
1.33 (m, 6H).
5.6. Methyl N-{2-[4-(propan-2-yloxy)phenyl]ethyl}carbamate (7d)
Methyl
N-[2-(4-hydroxyphenyl)ethyl]carbamate
(0.4 g,
2.05 mmol), potassium carbonate (0.85 g, 6.15 mmol) and tetra-
butylammonium iodide (0.15 g, 0.41 mmol) were combined in
dry DMF (10 mL) and 2-bromopropane (0.38 g, 0.29 lL, 3.07 mmol)
5.12. Methyl 7-butoxy-1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,
4-tetrahydroisoquinoline-2-carboxylate (8e)
was added and the reaction was stirred at 50 °C overnight. The
reaction was diluted with EtOAc, then washed with NaHCO3 solu-
tion, water and brine, and dried over MgSO4 and the solvent was
removed under reduced pressure. The crude material was purified
by chromatography on silica (0–30% EtOAc/hexane) to give the
This was prepared as per 8a using 7e. Yield 24%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.00 (dd, J = 8.38, 11.96 Hz, 1H),
6.69–6.80 (m, 2H), 6.34–6.67 (m, 3H), 5.13–5.33 (m, 1H), 3.46–
3.92 (m, 12H), 3.19–3.36 (m, 1H), 2.93–3.15 (m, 2H), 2.45–2.88
(m, 2H), 1.63–1.79 (m, 2H), 1.38–1.55 (m, 2H), 0.92–1.01 (m, 3H).
ether as
a
clear oil (0.31 g, 63%). 1H NMR (300 MHz,
CHLOROFORM-d) d 7.08 (d, J = 8.48 Hz, 2H), 6.79–6.86 (m, 2H),

5716
D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5.13. 1-[(3,4-Dimethoxyphenyl)methyl]-7-methoxy-1,2,3,4-tetra-
hydroisoquinoline (9a)
14.98 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.74 (s, 3H), 3.58–3.72
(m, 2H), 3.37–3.48 (m, 1H), 3.11–3.34 (m, 2H), 2.82–2.99 (m,
4H), 2.46–2.56 (m, 1H). m/z 461 (M+H).
Carbamate 8a (84 mg, 0.226 mmol), potassium hydroxide
(89 mg, 1.583 mmol) and hydrazine monohydrate (79 mg, 77
lL,
5.19. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-7-ethoxy-
1.583 mmol) were combined in ethylene glycol (1 mL) and heated
to 120 °C for 24 h. the reaction was cooled, diluted with water and
extracted 3 times with DCM. The combined extracts were dried
over MgSO4 and the solvent was removed under reduced pressure.
The crude was purified by chromatography on silica (0–5%
MeOH/DCM) to give the amine 9a (37 mg, 52%). 1H NMR
(300 MHz, CHLOROFORM-d) d 7.03 (d, J = 8.29 Hz, 1H), 6.71–6.86
(m, 5H), 4.17 (dd, J = 3.86, 9.14 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 3H),
3.79 (s, 3H), 3.15–3.25 (m, 2H), 2.64–2.94 (m, 4H).
1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (10b)
This was prepared as per 10a from 9b. Yield quantitative. 1H
NMR (300 MHz, CHLOROFORM-d) d 7.19–7.33 (m, 3H), 7.06–7.12
(m, 2H), 7.02 (d, J = 8.48 Hz, 1H), 6.93 (br s, 1H), 6.59–6.78 (m,
5H), 4.48 (dd, J = 8.10, 15.07 Hz, 1H), 4.00 (q, J = 7.10 Hz, 2H),
3.80 (s, 3H), 3.74 (s, 3H), 3.56–3.72 (m, 2H), 3.36–3.48 (m, 1H),
3.11–3.33 (m, 2H), 2.82–2.99 (m, 4H), 2.45–2.55 (m, 1H), 1.41 (t,
J = 6.97 Hz, 3H). m/z 475 (M+H).
5.14. 1-[(3,4-Dimethoxyphenyl)methyl]-7-ethoxy-1,2,3,4-tetra-
hydroisoquinoline (9b)
5.20. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-7-propoxy-1,-
2,3,4-tetrahydroisoquinolin-2-yl}acetamide (10c)
This was prepared as per 9a using 8b. Yield 67%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.02 (d, J = 8.48 Hz, 1H), 6.76–6.85
(m, 3H), 6.71–6.76 (m, 2H), 4.16 (dd, J = 3.77, 9.42 Hz, 1H), 4.01
(q, J = 6.97 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.15–3.24 (m, 2H),
2.64–2.95 (m, 4H), 1.41 (t, J = 6.97 Hz, 3H).
This was prepared as per 10a from 9c. Yield 90%. 1H NMR
(300 MHz, CHLOROFORM-d)
d 7.18–7.38 (m, 3H), 7.09 (d,
J = 6.78 Hz, 2H), 7.02 (d, J = 8.48 Hz, 1H), 6.89–6.97 (m, 1H), 6.66–
6.79 (m, 3H), 6.56–6.66 (m, 2H), 4.48 (dd, J = 8.29, 15.07 Hz, 1H),
3.88 (t, J = 6.50 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 3.55–3.71 (m,
2H), 3.35–3.51 (m, 1H), 3.09–3.34 (m, 2H), 2.81–3.00 (m, 4H),
2.44–2.57 (m, 1H), 1.73–1.87 (m, 2H), 1.04 (t, J = 7.44 Hz, 3H).
m/z 489 (M+H).
5.15. 1-[(3,4-Dimethoxyphenyl)methyl]-7-propoxy-1,2,3,4-tetra-
hydroisoquinoline (9c)
This was prepared as per 9a using 8c. Yield 32%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.02 (d, J = 8.29 Hz, 1H), 6.71–6.86
(m, 5H), 4.17 (dd, J = 3.39, 9.23 Hz, 1H), 3.88–3.93 (m, 2H), 3.86–
3.88 (m, 3H), 3.85 (s, 3H), 3.16–3.26 (m, 2H), 2.64–2.95 (m, 4H),
1.74–1.87 (m, 2H), 1.04 (t, J = 7.44 Hz, 3H).
5.21. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-7-(propan-
2-yloxy)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (10d)
This was prepared as per 10a from 9d. Yield 98%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.19–7.32 (m, 4H), 7.06–7.12 (m,
1H), 7.02 (d, J = 8.48 Hz, 1H), 6.90–6.97 (m, 1H), 6.66–6.77 (m,
3H), 6.63 (s, 1H), 6.61 (s, 1H), 4.43–4.54 (m, 2H), 3.80 (s, 3H),
3.74 (s, 3H), 3.56–3.72 (m, 2H), 3.36–3.48 (m, 1H), 3.12–3.33 (m,
2H), 2.82–2.98 (m, 4H), 2.45–2.54 (m, 1H), 1.33 (d, J = 4.71 Hz,
3H), 1.31 (d, J = 4.52 Hz, 3H). m/z 489 (M+H).
5.16. 1-[(3,4-Dimethoxyphenyl)methyl]-7-(propan-2-yloxy)-1,2,
3,4-tetrahydroisoquinoline (9d)
This was prepared as per 9a using 8d. Yield 65%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.01 (d, J = 8.29 Hz, 1H), 6.77–6.85
(m, 3H), 6.70–6.76 (m, 2H), 4.44–4.57 (m, 1H), 4.14 (dd, J = 3.77,
9.42 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.15–3.24 (m, 2H), 2.63–
2.94 (m, 4H), 1.33 (dd, J = 2.17, 6.12 Hz, 6H).
5.22. N-Benzyl-2-{7-butoxy-1-[(3,4-dimethoxyphenyl)methyl]-
1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (10e)
This was prepared as per 10a from 9e. Yield quantitative. 1H
NMR (300 MHz, CHLOROFORM-d) d 7.19–7.33 (m, 3H), 7.06–7.11
(m, 2H), 7.02 (d, J = 8.48 Hz, 1H), 6.89–6.96 (m, 1H), 6.67–6.78
(m, 3H), 6.64 (s, 1H), 6.59–6.61 (m, 1H), 4.48 (dd, J = 8.10,
15.07 Hz, 1H), 3.92 (t, J = 6.50 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 3H),
3.55–3.72 (m, 2H), 3.36–3.48 (m, 1H), 3.11–3.33 (m, 2H), 2.82–
2.99 (m, 4H), 2.45–2.55 (m, 1H), 1.71–1.81 (m, 2H), 1.43–1.56
(m, 2H), 0.98 (t, J = 7.44 Hz, 3H). m/z 503 (M+H).
5.17. 7-Butoxy-1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetra-
hydroisoquinoline (9e)
This was prepared as per 9a using 8e. Yield 68%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.01 (d, J = 8.29 Hz, 1H), 6.77–6.86
(m, 3H), 6.70–6.77 (m, 2H), 4.15 (dd, J = 3.67, 9.51 Hz, 1H), 3.94
(t, J = 6.50 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.15–3.25 (m, 2H),
2.63–2.94 (m, 4H), 1.70–1.81 (m, 2H), 1.43–1.56 (m, 2H), 0.98 (t,
J = 7.35 Hz, 3H).
5.23. 2-[3-(Benzyloxy)phenyl]ethan-1-amine (13)
5.18. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-7-methoxy-
1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (10a)
This was prepared in 3 steps from 3-hydroxybenzaldehyde
according to literature procedure.32
9a (37 mg, 0.118 mmol), N-benzyl-2-bromoacetamide (40 mg,
5.24. N-{2-[3-(Benzyloxy)phenyl]ethyl}-2-(3,4-dimethoxyphenyl)-
0.177 mmol) and tetrabutylammonium iodide (9 mg, 0.024 mmol)
acetamide (14)
were combined in dry DMF (1 mL) and DIPEA (38 mg, 51 lL,
0.295 mmol) was added. The reaction was stirred at RT overnight
under N2. The reaction was diluted with EtOAc, washed with
NaHCO3 solution, water and brine, and then dried over MgSO4
and the solvent was removed under reduced pressure. The crude
was purified by chromatography on silica (0–60% EtOAc in hexane)
to give the desired product (54 mg, quantitative). 1H NMR
(300 MHz, CHLOROFORM-d) d 7.19–7.34 (m, 3H), 7.01–7.13 (m,
3H), 6.89–6.97 (m, 1H), 6.57–6.79 (m, 5H), 4.49 (dd, J = 8.01,
Phenylethylamine 13 (1.0 g, 4.40 mmol), 3,4-dimethoxypheny-
lacetic acid (0.95 g, 4.84 mmol) and BOP (2.53 g, 5.72 mmol) were
combined in dry DMF (40 mL). DIPEA (2.27 g, 3.1 mL, 17.6 mmol)
was added and the reaction was stirred under N2 at RT overnight.
The reaction was diluted with EtOAc, washed with 2 N HCl,
NaHCO3 solution and brine, and dried over MgSO4 and the solvent
was removed under reduced pressure to give the amide (1.78 g,
quantitative). 1H NMR (300 MHz, CHLOROFORM-d) d 7.29–7.46

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5717
(m, 5H), 7.14 (t, J = 7.86 Hz, 1H), 6.69–6.88 (m, 4H), 6.67 (s, 1H),
6.62 (d, J = 7.54 Hz, 1H), 5.41 (br s, 1H), 5.02 (s, 2H), 3.84 (s, 3H),
3.81 (s, 3H), 3.41–3.51 (m, 4H), 2.71 (t, J = 6.83 Hz, 2H).
was removed under reduced pressure. The crude was purified by
chromatography on silica (0–50% EtOAc/hexane) to give the ether
(12 mg, 44%). 1H NMR (300 MHz, CHLOROFORM-d) d 7.19–7.36
(m, 3H), 7.09 (d, J = 7.91 Hz, 2H), 6.90–7.02 (m, 2H), 6.58–6.79
(m, 5H), 4.49 (dd, J = 8.19, 14.98 Hz, 1H), 3.90 (t, J = 6.55 Hz, 2H),
3.79 (s, 3H), 3.74 (s, 3H), 3.56–3.72 (m, 2H), 3.35–3.47 (m, 1H),
3.12–3.32 (m, 2H), 2.79–3.03 (m, 4H), 2.48–2.58 (m, 1H), 1.82 (s,
2H), 1.04 (t, J = 7.39 Hz, 3H). m/z 489 (M+H).
5.25. 6-(Benzyloxy)-1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-
tetrahydroisoquinoline (15)
Amide 14 (1.78 g, 4.40 mmol) was dissolved with warming in
toluene (25 mL) then phosphorus oxychloride (3.55 g, 2.2 mL,
23.18 mmol) added. The solution was heated to 90 °C for 2 h. The
reaction was cooled, poured into water and stirred for 10 min.
Layers were separated, and the aqueous portion was treated with
2 N NaOH solution until a pH of 8–9 was reached; then it was
extracted 3 times with DCM. Combined DCM fractions were dried
over MgSO4, and the solvent was removed under reduced pressure.
The crude dihydroisoquinoline was redissolved in methanol
(20 mL) and cooled in ice. Sodium borohydride (0.75 g,
19.87 mmol) was added portionwise. After initial reaction had sub-
sided, the ice bath was removed and the reaction was stirred at RT
for 30 min. The reaction was quenched with water and then the
methanol was removed under reduce pressure. The aqueous solu-
tion was extracted 3 times with DCM, the combined extracts were
dried over MgSO4 and the solvent was removed under reduced
pressure to give the tetrahydroisoquinoline 15 (1.42 g, 92%). 1H
NMR (300 MHz, CHLOROFORM-d) d 7.28–7.47 (m, 5H), 7.16 (d,
J = 8.57 Hz, 1H), 6.67–6.89 (m, 5H), 5.05 (s, 2H), 4.15 (dd, J = 3.53,
9.28 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.80–3.86 (m, 1H), 3.16–
3.25 (m, 1H), 2.66–3.00 (m, 4H).
5.29. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(2,2,2-tri-
fluoroethoxy)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (17b)
Phenol 16 (25 mg, 0.056 mmol) was combined with cesium car-
bonate (55 mg, 0.168 mmol) in DMF (0.5 mL) and 2,2,2-trifluoroio-
doethane (24 mg, 11 lL, 0.112 mmol) was added. The reaction was
stirred at 50 °C overnight. The reaction was cooled, diluted with
EtOAc, washed with NaHCO3 solution and brine, dried over
MgSO4 and the solvent was removed under reduced pressure.
The crude was purified by chromatography on silica (0–50%
EtOAc/hexane) to give the ether (10 mg, 33%). 1H NMR (300 MHz,
CHLOROFORM-d) d 7.20–7.34 (m, 3H), 7.09 (d, J = 7.91 Hz, 2H),
7.01 (d, J = 8.48 Hz, 1H), 6.87–6.96 (m, 1H), 6.78 (dd, J = 2.26,
8.48 Hz, 1H), 6.60–6.73 (m, 4H), 4.49 (dd, J = 8.19, 14.98 Hz, 1H),
4.34 (q, J = 8.23 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 3.57–3.73 (m,
2H), 3.36–3.48 (m, 1H), 3.11–3.33 (m, 2H), 2.79–3.03 (m, 4H),
2.51–2.60 (m, 1H). 19F NMR (282 MHz, CHLOROFORM-d)
ꢃ73.96. m/z 529 (M+H).
d
5.30. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(propan-
2-yloxy)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (17c)
5.26. N-Benzyl-2-[6-(benzyloxy)-1-[(3,4-dimethoxyphenyl)met-
hyl]-1,2,3,4-tetrahydroisoquinolin-2-yl]acetamide (17d)
This was prepared as for 17a but using 2-bromopropane at
50 °C. Yield 48%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–
7.33 (m, 3H), 7.06–7.13 (m, 2H), 6.90–7.00 (m, 2H), 6.68–6.76
(m, 2H), 6.59–6.67 (m, 3H), 4.43–4.59 (m, 2H), 3.79 (s, 3H), 3.74
(s, 3H), 3.57–3.72 (m, 2H), 3.34–3.47 (m, 1H), 3.12–3.32 (m, 2H),
2.79–3.01 (m, 4H), 2.51 (dd, J = 4.33, 16.58 Hz, 1H), 1.33 (d,
J = 6.03 Hz, 6H). m/z 489 (M+H).
This was prepared from 14 using the method for 10a. Purified
by chromatography on silica (0–60% EtOAc/hexane). Yield 35%.
1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.47 (m, 8H), 7.05–
7.13 (m, 2H), 6.99 (d, J = 8.57 Hz, 1H), 6.89–6.97 (m, 1H), 6.82
(dd, J = 2.59, 8.43 Hz, 1H), 6.68–6.75 (m, 2H), 6.66 (d, J = 1.79 Hz,
1H), 6.59–6.64 (m, 1H), 5.05 (s, 2H), 4.48 (dd, J = 8.10, 14.98 Hz,
1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.57–3.72 (m, 2H), 3.35–3.47 (m,
1H), 3.12–3.33 (m, 2H), 2.79–3.03 (m, 4H), 2.53 (dd, J = 4.29,
16.62 Hz, 1H). m/z 537 (M+H).
5.31. Ethyl 4-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethox-
yphenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-
yl}oxy)butanoate (17e)
5.27. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-hydro-
xy-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (16)
This was prepared as for 17a but using ethyl 4-bromobutyrate.
Yield 23%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.17–7.36 (m,
3H), 7.05–7.12 (m, 2H), 6.89–7.01 (m, 2H), 6.59–6.78 (m, 5H),
4.48 (dd, J = 8.10, 14.88 Hz, 1H), 4.15 (q, J = 7.16 Hz, 2H), 3.99 (t,
J = 6.03 Hz, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 3.54–3.72 (m, 2H),
3.34–3.49 (m, 1H), 3.10–3.33 (m, 2H), 2.78–3.03 (m, 4H), 2.44–
2.59 (m, 3H), 2.10 (quin, J = 6.59 Hz, 2H), 1.27 (t, J = 7.16 Hz, 3H).
m/z 561 (M+H).
Benzyl ether 15 (90 mg, 0.168 mmol), ammonium formate
(32 mg, 0.503 mmol) and palladium on carbon (10%, 45 mg, 50%
w/w) were combined in ethanol (2 mL) and heated to reflux for
2 h. The reaction was cooled, filtered through Celite, rinsed thor-
oughly with ethanol, and then the solvent was removed under
reduced pressure to give the desired phenol (75 mg, quantitative).
1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.37 (m, 3H), 7.08 (d,
J = 7.44 Hz, 2H), 6.95–7.02 (m, 1H), 6.89 (d, J = 8.19 Hz, 1H), 6.60–
6.73 (m, 4H), 6.55 (s, 1H), 4.48 (dd, J = 8.19, 14.79 Hz, 1H), 3.80
(s, 3H), 3.72–3.75 (m, 3H), 3.54–3.65 (m, 2H), 3.32–3.47 (m, 1H),
3.05–3.30 (m, 2H), 2.71–2.94 (m, 4H), 2.37–2.49 (m, 1H).
5.32. Ethyl 7-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethox-
yphenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}oxy)hepta-
noate (17f)
This was prepared as for 17a but using ethyl 7-bromohep-
5.28. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-propoxy-1,-
2,3,4-tetrahydroisoquinolin-2-yl}acetamide (17a)
tanoate. Yield 65%. 1H NMR (300 MHz, CHLOROFORM-d)
d
7.18–7.34 (m, 3H), 7.06–7.13 (m, 2H), 6.97 (d, J = 8.48 Hz, 2H),
6.58–6.78 (m, 5H), 4.48 (dd, J = 8.19, 14.98 Hz, 1H), 4.13 (q,
J = 7.16 Hz, 2H), 3.93 (t, J = 6.40 Hz, 2H), 3.79 (s, 3H), 3.74 (s, 3H),
3.55–3.72 (m, 2H), 3.35–3.49 (m, 1H), 3.11–3.33 (m, 2H), 2.78–
3.03 (m, 4H), 2.53 (dd, J = 4.14, 16.58 Hz, 1H), 2.31 (t, J = 7.44 Hz,
2H), 1.72–1.84 (m, 2H), 1.59–1.72 (m, 2H), 1.34–1.55 (m, 4H),
1.26 (t, J = 7.16 Hz, 3H). m/z 603 (M+H).
Phenol 16 (25 mg, 0.056 mmol) was combined with potassium
carbonate (23 mg, 0.168 mmol) and tetrabutylammonium iodide
(4 mg, 0.011 mmol) in DMF (0.5 mL) and 1-bromopropane
(10 mg, 8 lL, 0.084 mmol) was added. The reaction was stirred at
RT overnight. The reaction was diluted with EtOAc, washed with
NaHCO3 solution and brine, and dried over MgSO4 and the solvent

5718
D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5.33. 2-[(Benzylcarbamoyl)methyl]-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl methanesulfonate (18a)
(d, J = 7.54 Hz, 1H), 6.33 (s, 1H), 5.43 (br s, 1H), 3.89 (s, 3H), 3.84
(s, 3H), 3.46–3.49 (m, 2H), 3.39–3.46 (m, 2H), 2.58–2.65 (m, 2H).
To phenol 16 (25 mg, 0.056 mmol) and TEA (17 mg, 23
0.168 mmol) in DCM (0.5 mL) was added methanesulfonyl chloride
(13 mg, 9 L, 0.112 mmol) and the reaction was stirred at RT over-
l
L,
5.38. Methyl N-(3-{2-[2-(3,4-dimethoxyphenyl)acetamido]ethyl}-
phenyl)carbamate (22)
l
night. The reaction was taken directly into purification by chro-
matography on silica (0–80% EtOAc/hexane) to give the sulfonate
(17 mg, 59%). 1H NMR (300 MHz, CHLOROFORM-d) d 7.19–7.34
(m, 3H), 7.09 (t, J = 4.71 Hz, 5H), 6.83–6.93 (m, 1H), 6.60–6.75
(m, 3H), 4.49 (dd, J = 8.10, 14.88 Hz, 1H), 3.80 (s, 3H), 3.75 (s,
3H), 3.58–3.78 (m, 2H), 3.36–3.49 (m, 1H), 3.16 (s, 3H), 3.10–
3.35 (m, 2H), 2.80–3.06 (m, 4H), 2.61 (dd, J = 4.33, 16.77 Hz, 1H).
m/z 525 (M+H).
To the aniline (1.55 g, 4.84 mmol) in DCM (30 mL) cooled in an
ice bath under N2 was added DIPEA (1.23 g, 1.65 mL, 9.50 mmol)
and then dropwise addition of methyl chloroformate (0.75 g,
0.61 mL, 7.91 mmol). The reaction was stirred in ice for 15 min
then at RT overnight. The reaction completion was checked by
TLC (5% MeOH/DCM). The reaction was washed with water and
the aqueous fraction was extracted once with DCM. The combined
organic fractions were washed with 2 N HCl solution, then NaHCO3
solution, and dried over MgSO4 and the solvent was removed
under reduced pressure. The crude mixture was purified by chro-
matography on silica (0–5% MeOH in DCM) to give the carbamate
5.34. 2-[(Benzylcarbamoyl)methyl]-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl benzenesulfonate (18b)
as
a
yellow viscous oil (1.53 g, 85%). 1H NMR (300 MHz,
CHLOROFORM-d) d 7.13–7.24 (m, 2H), 7.10 (s, 1H), 6.77–6.82 (m,
1H), 6.68–6.75 (m, 3H), 6.65 (br s, 1H), 5.41 (br s, 1H), 3.88 (s,
3H), 3.82 (s, 3H), 3.75–3.79 (m, 3H), 3.41–3.50 (m, 4H), 2.71 (t,
J = 6.73 Hz, 2H).
This was prepared as per 18a using benzenesulfonyl chloride.
Yield 67%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.83–7.91 (m,
2H), 7.64–7.73 (m, 1H), 7.50–7.60 (m, 2H), 7.19–7.34 (m, 3H),
7.04–7.13 (m, 2H), 6.94 (d, J = 8.48 Hz, 1H), 6.80–6.89 (m, 2H),
6.59–6.74 (m, 4H), 4.48 (dd, J = 8.10, 14.88 Hz, 1H), 3.78 (s, 3H),
3.74 (s, 3H), 3.57–3.72 (m, 2H), 3.34–3.46 (m, 1H), 3.07–3.31 (m,
2H), 2.75–2.97 (m, 4H), 2.45–2.58 (m, 1H). m/z 587 (M+H).
5.39. Methyl N-{1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetra-
hydroisoquinolin-6-yl}carbamate
Amide 22 (1.53 g, 4.11 mmol) was dissolved with warming in
toluene (20 mL) and then phosphorus oxychloride (3.15 g, 1.9 mL,
20.54 mmol) was added. The reaction was heated to 90 °C for
2 h. The reaction was cooled, poured into water and stirred for
10 min. Layers were separated, and the aqueous portion was trea-
ted with 2 N NaOH solution until a pH of 8–9 was reached and then
it was extracted 3 times with DCM. Combined DCM fractions were
dried over MgSO4 and the solvent was removed under reduced
pressure. The crude dihydroisoquinoline was redissolved in metha-
nol (40 mL) and cooled in ice. Sodium borohydride (0.77 g,
20.5 mmol) was added portionwise. After initial reaction had sub-
sided, the ice bath was removed and the reaction was stirred at RT
for 30 min. The reaction was quenched with water then the metha-
nol was removed under reduce pressure. The aqueous solution was
extracted 3 times with DCM, the combined extracts were dried
over MgSO4 and the solvent was removed under reduced pressure
to give the tetrahydroisoquinoline (1.17 g, 80%). 1H NMR
(300 MHz, CHLOROFORM-d) d 7.11–7.21 (m, 3H), 6.73–6.86 (m,
3H), 6.55 (s, 1H), 4.14 (dd, J = 3.81, 9.65 Hz, 1H), 3.87 (s, 3H), 3.85
(s, 3H), 3.77 (s, 3H), 3.16–3.25 (m, 2H), 2.68–2.95 (m, 4H).
5.35. 2-[(Benzylcarbamoyl)methyl]-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl propanoate (19)
This was prepared as per 18a using propionyl chloride. Yield
89%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.34 (m, 3H),
7.03–7.13 (m, 3H), 6.83–6.96 (m, 3H), 6.67–6.74 (m, 1H), 6.59–
6.67 (m, 2H), 4.48 (dd, J = 8.10, 15.07 Hz, 1H), 3.79 (s, 3H), 3.74
(s, 3H), 3.58–3.73 (m, 2H), 3.35–3.47 (m, 1H), 3.11–3.34 (m, 2H),
2.81–3.04 (m, 4H), 2.52–2.65 (m, 3H), 1.27 (t, J = 7.54 Hz, 3H).
m/z 503 (M+H).
5.36. 2-(3,4-Dimethoxyphenyl)-N-[2-(3-nitrophenyl)ethyl]aceta-
mide (21)
3-Nitrophenylethylamine hydrochloride (1.0 g, 4.94 mmol),
3,4-dimethoxyphenylacetic acid (1.07 g, 5.43 mmol) and BOP
(2.84 g, 6.42 mmol) were combined in dry DMF (50 mL). DIPEA
(2.55 g, 3.4 mL, 19.74 mmol) was added and the reaction was stir-
red under N2 at RT overnight. The reaction was diluted with EtOAc,
washed with 2 N HCl, NaHCO3 solution and brine, dried over
MgSO4 and the solvent was removed under reduced pressure to
give the amide (1.7 g, quantitative). 1H NMR (300 MHz,
METHANOL-d4) d 7.93–8.08 (m, 2H), 7.37–7.56 (m, 2H), 6.78–
6.87 (m, 2H), 6.67–6.74 (m, 1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.48 (t,
J = 6.78 Hz, 2H), 3.35 (s, 2H), 2.86–2.94 (m, 2H).
5.40. Methyl N-{2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}carbamate
(23a)
Tetrahydroisoquinoline (1.17 g, 3.28 mmol), N-benzyl-2-bro-
moacetamide (1.12 g, 4.92 mmol) and tetrabutylammonium iodide
(0.24 g, 0.66 mmol) were combined in dry DMF (30 mL) and DIPEA
(1.06 g, 1.4 mL, 8.21 mmol) was added. The reaction was stirred at
RT overnight under N2. The reaction was diluted with EtOAc,
washed with NaHCO3 solution, water and brine, and then dried
over MgSO4 and the solvent removed under reduced pressure.
The crude was purified by chromatography on silica (0–70%
EtOAc in hexane) to give the desired product (0.85 g, 52%). 1H
NMR (300 MHz, CHLOROFORM-d) d 7.19–7.32 (m, 4H), 7.06–7.15
(m, 2H), 7.00 (d, J = 8.29 Hz, 1H), 6.92 (dd, J = 4.76, 7.49 Hz, 1H),
6.59–6.74 (m, 4H), 6.57 (s, 1H), 4.48 (dd, J = 8.10, 14.98 Hz, 1H),
3.80 (s, 3H), 3.78 (s, 3H), 3.74 (s, 3H), 3.57–3.73 (m, 2H), 3.36–
3.47 (m, 1H), 3.11–3.32 (m, 2H), 2.80–3.02 (m, 4H), 2.56 (dd,
J = 4.57, 16.44 Hz, 1H). m/z 504 (M+H).
5.37. N-[2-(3-Aminophenyl)ethyl]-2-(3,4-dimethoxyphenyl)ace-
tamide
To the amide 21 (1.70 g, 4.94 mmol) in ethanol (65 mL) was
added hydrazine monohydrate (3.26 g, 3.2 mL, 65.1 mmol) and
the reaction was heated to 50 °C. Raney nickel (2880 type as slurry
in water, 0.45 g) was added and the reaction was stirred at 50 °C
for 1 h. The reaction was cooled, filtered through Celite, rinsed
thoroughly with ethanol. The solvent was removed under reduced
pressure to give the desired aniline (1.55 g, 98%). 1H NMR
(300 MHz, CHLOROFORM-d) d 7.00 (t, J = 7.68 Hz, 1H), 6.79–6.86
(m, 2H), 6.66–6.76 (m, 2H), 6.51 (dd, J = 2.26, 8.01 Hz, 1H), 6.40

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5719
5.41. 2-{6-Amino-1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetra-
hydroisoquinolin-2-yl}-N-benzylacetamide (24)
2H), 6.96–7.02 (m, 1H), 6.92 (d, J = 8.57 Hz, 1H), 6.68–6.75 (m,
1H), 6.65 (d, J = 1.79 Hz, 1H), 6.61 (d, J = 8.10 Hz, 1H), 6.54–6.59
(m, 1H), 6.38 (d, J = 2.54 Hz, 1H), 4.48 (dd, J = 8.15, 15.02 Hz, 1H),
3.78 (s, 3H), 3.73 (s, 3H), 3.56–3.69 (m, 2H), 3.33–3.45 (m, 1H),
3.13–3.32 (m, 4H), 2.91 (s, 3H), 2.76–3.01 (m, 4H), 2.49 (dd,
J = 3.96, 16.48 Hz, 1H), 1.59–1.67 (m, 2H), 0.93 (t, J = 7.39 Hz, 3H).
m/z 502 (M+H).
To a solution of the carbamate 23a (0.85 g, 1.69 mmol) in
methanol (10 mL) was added 2 N sodium hydroxide solution
(1.69 mL, 3.38 mmol) and the reaction was heated to 50 °C over-
night. Two additional aliquots of 2 N NaOH solution (1.7 mL) was
added after 24 h and 48 h, respectively, and heating continued at
50 °C until all starting material was gone by TLC (4:1
EtOAc/hexane). Methanol was removed under reduced pressure
and the aqueous solution diluted with water. The solution was
extracted 3 times with EtOAc and the combined extracts were
dried over MgSO4 and the solvent was removed under reduced
pressure to give the free amine as a white solid (0.63 g, 84%). 1H
NMR (300 MHz, CHLOROFORM-d) d 7.19–7.34 (m, 3H), 7.09 (d,
J = 7.82 Hz, 2H), 6.95 (br s, 1H), 6.86 (d, J = 8.19 Hz, 1H), 6.58–
6.73 (m, 3H), 6.53 (dd, J = 1.84, 8.15 Hz, 1H), 6.44 (s, 1H), 4.48
(dd, J = 8.15, 15.02 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.56–3.68
(m, 2H), 3.33–3.47 (m, 1H), 3.22 (q, J = 17.02 Hz, 2H), 2.77–2.96
(m, 4H), 2.39–2.51 (m, 1H).
5.45. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(dipropyl-
amino)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25d)
This was prepared as per 25b but with 2 equiv of propionalde-
hyde and 2.5 equiv of sodium triacetoxyborohydride. Yield 94%. 1H
NMR (300 MHz, CHLOROFORM-d) d 7.17–7.32 (m, 3H), 7.05–7.12
(m, 2H), 6.98 (dd, J = 5.04, 7.58 Hz, 1H), 6.90 (d, J = 8.48 Hz, 1H),
6.67–6.73 (m, 1H), 6.63 (d, J = 1.79 Hz, 1H), 6.56–6.61 (m, 1H),
6.50 (dd, J = 2.64, 8.57 Hz, 1H), 6.31 (d, J = 2.45 Hz, 1H), 4.47 (dd,
J = 8.05, 15.02 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 3H), 3.56–3.68 (m,
2H), 3.36 (d, J = 3.11 Hz, 1H), 3.13–3.27 (m, 6H), 2.75–2.98 (m,
4H), 2.45 (dd, J = 3.67, 16.39 Hz, 1H), 1.51–1.66 (m, 4H), 0.88–
0.97 (m, 6H). m/z 530 (M+H).
5.42. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(dimethyl-
amino)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25a)
5.46. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(propan-
2-yl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25e)
To a solution of aniline 24 (25 mg, 0.056 mmol) and formaldehyde
(37% aqueous, 23 mg, 21 lL, 0.281 mmol) in 1,2-dichloroethane
Aniline 24 (25 mg, 0.056 mmol), 2-bromopropane (7 mg, 5
0.056 mmol) and tetrabutylammonium iodide (4 mg, 0.011 mmol)
were combined in anhydrous DMF (0.5 mL). DIPEA (18 mg, 24 L,
lL,
(0.5 mL) was added portionwise sodium triacetoxyborohydride
(59 mg, 0.281 mmol) and the reaction was stirred at RT overnight.
The solvent was removed under reduced pressure, and the crude
was redissolved in EtOAc, washed with NaHCO3 solution and brine,
and dried over MgSO4 and the solvent was removed under reduced
pressure. The crude sample was purified by chromatography on silica
(0–100% EtOAc/hexane) to give the desired dimethylamine (23 mg,
l
0.140 mmol) was added and the reaction was heated at 50 °C over-
night. The reaction was cooled, diluted with EtOAc and washed
with NaHCO3 solution and brine, and then dried over MgSO4 and
the solvent removed under reduced pressure. The crude was puri-
fied by chromatography on silica (0–60% EtOAc/hexane) to give the
desired amine (5 mg, 19%). 1H NMR (300 MHz, CHLOROFORM-d) d
7.18–7.34 (m, 3H), 7.09 (d, J = 7.91 Hz, 2H), 6.97 (br s, 1H), 6.87 (d,
J = 8.29 Hz, 1H), 6.58–6.74 (m, 3H), 6.44 (dd, J = 2.17, 8.19 Hz, 1H),
6.31 (s, 1H), 4.48 (dd, J = 8.19, 14.98 Hz, 1H), 3.79 (s, 3H), 3.73 (s,
3H), 3.53–3.68 (m, 3H), 3.33–3.46 (m, 1H), 3.13–3.32 (m, 2H),
2.76–2.97 (m, 4H), 2.41–2.52 (m, 1H), 1.21 (d, J = 6.22 Hz, 6H).
m/z 488 (M+H).
88%). 1H NMR (300 MHz, CHLOROFORM-d)
d ppm 2.50 (dd,
J = 16.58, 4.24 Hz, 1H), 2.77–3.01 (m, 4H), 2.92 (s, 6H), 3.11–3.32
(m, 2H), 3.33–3.46 (m, 1H), 3.55–3.69 (m, 2H), 3.73 (s, 3H), 3.78 (s,
3H), 4.47 (dd, J = 14.98, 8.19 Hz, 1H), 6.44 (d, J = 2.54 Hz, 1H), 6.57–
6.73 (m, 4H), 6.94 (d, J = 8.48 Hz, 2H), 7.05–7.11 (m, 1H), 7.17–7.32
(m, 4H). m/z 474 (M+H).
5.43. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(propyl-
amino)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25b)
5.47. N-Benzyl-2-{6-[(cyclopropylmethyl)amino]-1-[(3,4-dimeth-
oxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25f)
To a solution of aniline 24 (50 mg, 0.112 mmol) and propi-
onaldehyde (7 mg,
8 lL, 0.112 mmol) in 1,2-dichloroethane
(1 mL) was added portionwise sodium triacetoxyborohydride
(36 mg, 0.168 mmol) and the reaction was stirred at RT overnight.
The solvent was removed under reduced pressure, and the crude
was redissolved in EtOAc, washed with NaHCO3 solution and brine,
and dried over MgSO4 and the solvent was removed under reduced
pressure. The crude sample was purified by chromatography on sil-
ica (0–60% EtOAc/hexane) to give the desired amine (37 mg, 67%).
1H NMR (300 MHz, CHLOROFORM-d) d 7.17–7.34 (m, 3H), 7.05–
7.12 (m, 2H), 6.92–7.02 (m, 1H), 6.88 (d, J = 8.29 Hz, 1H), 6.68–
6.74 (m, 1H), 6.67 (s, 1H), 6.58–6.64 (m, 1H), 6.43–6.51 (m, 1H),
6.31–6.36 (m, J = 2.00 Hz, 1H), 4.48 (dd, J = 8.01, 14.98 Hz, 1H),
3.79 (s, 3H), 3.73 (s, 3H), 3.53–3.69 (m, 3H), 3.33–3.45 (m, 1H),
3.13–3.32 (m, 2H), 3.07 (t, J = 7.11 Hz, 2H), 2.77–2.98 (m, 4H),
2.41–2.52 (m, 1H), 1.60–1.71 (m, 2H), 0.96–1.05 (m, 3H). m/z 488
(M+H).
This was prepared as per 25e using bromomethylcyclopropane.
After stirring overnight at 50 °C, the reaction was stirred an addi-
tional 6 h at 70 °C. Yield 43%. 1H NMR (300 MHz, CHLOROFORM-
d) d 7.18–7.34 (m, 4H), 7.05–7.12 (m, 2H), 6.96 (dd, J = 4.90,
7.72 Hz, 1H), 6.88 (d, J = 8.29 Hz, 1H), 6.65–6.74 (m, 2H), 6.58–
6.63 (m, 1H), 6.48 (dd, J = 2.45, 8.29 Hz, 1H), 6.34 (d, J = 2.07 Hz,
1H), 4.48 (dd, J = 8.19, 14.98 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H),
3.54–3.68 (m, 2H), 3.33–3.45 (m, 1H), 3.22 (q, J = 16.95 Hz, 2H),
2.94 (d, J = 6.97 Hz, 2H), 2.77–2.92 (m, 4H), 2.42–2.51 (m, 1H),
1.02–1.17 (m, 1H), 0.51–0.60 (m, 2H), 0.20–0.27 (m, 2H). m/z 500
(M+H).
5.48. N-Benzyl-2-{6-[(cyclopropylmethyl)(methyl)amino]-1-[(3,-
4-dimethoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl}-
acetamide (25g)
5.44. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[methyl-
This was prepared as per 25a from 25f. Yield 94%. 1H NMR
(propyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25c)
(300 MHz, CHLOROFORM-d) d 7.18–7.33 (m, 3H), 7.09 (d,
J = 6.97 Hz, 2H), 6.89–7.04 (m, 2H), 6.58–6.76 (m, 4H), 6.47 (s,
1H), 4.48 (dd, J = 8.01, 14.98 Hz, 1H), 3.78 (s, 3H), 3.74 (s, 3H),
3.57–3.70 (m, 2H), 3.34–3.46 (m, 1H), 3.12–3.33 (m, 4H), 2.96 (s,
This was prepared as per 25a from 25b. Yield 100%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.18–7.34 (m, 3H), 7.06–7.12 (m,

5720
D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
3H), 2.78–3.01 (m, 4H), 2.50 (dd, J = 3.67, 16.67 Hz, 1H), 0.97–1.09
(m, 1H), 0.47–0.56 (m, 2H), 0.17–0.25 (m, 2H). m/z 514 (M+H).
CHLOROFORM-d) d 7.18–7.36 (m, 6H), 7.04–7.12 (m, 2H), 6.84–
7.01 (m, 5H), 6.64–6.74 (m, 2H), 6.60 (s, 1H), 6.47 (dd, J = 2.26,
8.29 Hz, 1H), 6.33 (d, J = 2.07 Hz, 1H), 4.48 (dd, J = 7.91, 15.07 Hz,
1H), 4.02 (t, J = 5.93 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.53–3.69
(m, 2H), 3.33–3.46 (m, 1H), 3.10–3.32 (m, 4H), 2.76–2.99 (m,
4H), 2.41–2.53 (m, 1H), 1.75–1.98 (m, 4H). m/z 594 (M+H).
5.49. N-Benzyl-2-[6-(benzylamino)-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl]acetamide (25h)
Aniline 23 (30 mg, 0.067 mmol), sodium bicarbonate (2 mg,
0.027 mmol) and benzaldehyde (8 mg, 8
l
L, 0.074 mmol) were
5.54. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(pyridin-
4-ylmethyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25m)
combined in anhydrous methanol (0.5 mL) and heated to 40 °C
for 1 h The reaction was then cooled in an ice bath and sodium
borohydride was added. The reaction was allowed to warm slowly
to RT overnight. The solvent was removed under reduced pressure
and the crude purified by chromatography on silica (0–100%
EtOAc/hexane) to obtain the desired amine (19 mg, 53%). 1H
NMR (300 MHz, CHLOROFORM-d) d 7.36–7.42 (m, 3H), 7.20–7.35
(m, 4H), 7.10 (d, J = 7.63 Hz, 2H), 6.97 (br s, 1H), 6.90 (d,
J = 8.19 Hz, 1H), 6.57–6.76 (m, 4H), 6.51 (dd, J = 2.17, 8.19 Hz,
1H), 6.39 (d, J = 2.07 Hz, 1H), 4.49 (dd, J = 8.15, 15.02 Hz, 1H),
4.32 (s, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 3.54–3.70 (m, 2H), 3.34–
3.49 (m, 1H), 3.13–3.33 (m, 2H), 2.77–3.00 (m, 4H), 2.41–2.54
(m, 1H). m/z 536 (M+H).
This was prepared as per 25b from 24 using 4-pyridine carbox-
aldehyde. Yield 33%. 1H NMR (300 MHz, CHLOROFORM-d) d 8.57
(d, J = 5.75 Hz, 2H), 7.19–7.37 (m, 6H), 7.04–7.12 (m, 2H), 6.94 (s,
1H), 6.88 (d, J = 8.29 Hz, 1H), 6.57–6.75 (m, 3H), 6.45 (dd, J = 2.45,
8.29 Hz, 1H), 6.30 (d, J = 2.26 Hz, 1H), 4.47 (dd, J = 8.15, 15.02 Hz,
1H), 4.37 (s, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.53–3.67 (m, 2H),
3.33–3.46 (m, 1H), 3.21 (q, J = 16.95 Hz, 2H), 2.76–2.95 (m, 4H),
2.36–2.49 (m, 1H). m/z 537 (M+H).
5.55. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(pyridin-3-
ylmethyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25n)
5.50. N-Benzyl-2-{6-[benzyl(methyl)amino]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25i)
This was prepared as per 25b from 24 using 3-pyridine carbox-
aldehyde. Yield 53%. 1H NMR (300 MHz, CHLOROFORM-d) d 8.64 (s,
1H), 8.54 (d, J = 4.71 Hz, 1H), 7.71 (d, J = 7.72 Hz, 1H), 7.19–7.35 (m,
4H), 7.09 (d, J = 7.72 Hz, 2H), 6.91–6.99 (m, 1H), 6.89 (d, J = 8.29 Hz,
1H), 6.58–6.75 (m, 3H), 6.50 (d, J = 8.29 Hz, 1H), 6.36 (s, 1H), 4.48
(dd, J = 7.96, 15.02 Hz, 1H), 4.36 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H),
3.54–3.68 (m, 2H), 3.33–3.47 (m, 1H), 3.21 (q, J = 16.99 Hz, 2H),
2.77–2.97 (m, 4H), 2.38–2.53 (m, 1H). m/z 537 (M+H).
This was prepared as per 25a from 25h. Yield 67%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.18–7.39 (m, 8H), 7.05–7.12 (m,
2H), 6.88–7.01 (m, 2H), 6.67–6.75 (m, 1H), 6.57–6.67 (m, 3H),
6.47 (d, J = 2.07 Hz, 1H), 4.51 (s, 2H), 4.42–4.49 (m, 1H), 3.78 (s,
3H), 3.73 (s, 3H), 3.55–3.69 (m, 2H), 3.33–3.46 (m, 1H), 3.13–
3.32 (m, 2H), 2.99 (s, 3H), 2.76–2.97 (m, 4H), 2.42–2.54 (m, 1H).
m/z 550 (M+H).
5.56. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-{[2-(pip-
eridin-1-yl)ethyl]amino}-1,2,3,4-tetrahydroisoquinolin-2-yl}
acetamide (25o)
5.51. N-Benzyl-2-(6-{[2-(3,4-dimethoxyphenyl)ethyl]amino}-1-
[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-
2-yl)acetamide (25j)
This was prepared as per 25e using 1-(2-chloroethyl)piperidine
hydrochloride. Yield 16%. 1H NMR (300 MHz, CHLOROFORM-d) d
7.17–7.34 (m, 3H), 7.08 (d, J = 6.59 Hz, 2H), 6.92–7.01 (m, 1H),
6.89 (d, J = 8.29 Hz, 1H), 6.64–6.75 (m, 2H), 6.57–6.63 (m, 1H),
6.51 (dd, J = 2.26, 8.29 Hz, 1H), 6.36 (d, J = 2.07 Hz, 1H), 4.48 (dd,
J = 8.19, 15.16 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.53–3.68 (m,
2H), 3.33–3.48 (m, 1H), 3.09–3.32 (m, 4H), 2.75–2.99 (m, 4H),
2.58 (t, J = 6.03 Hz, 2H), 2.47–2.54 (m, 1H), 2.35–2.45 (m, 3H),
1.59 (td, J = 5.30, 10.69 Hz, 4H), 1.46 (d, J = 5.09 Hz, 2H). m/z 558
(M+H).
This was prepared as per 25e using 3,4-dimethoxyphenethyl
bromide, stirring at RT overnight. Yield 12%. 1H NMR (300 MHz,
CHLOROFORM-d) d 7.18–7.34 (m, 3H), 7.08 (d, J = 6.78 Hz, 2H),
6.96 (br s, 1H), 6.65–6.91 (m, 6H), 6.58–6.64 (m, 1H), 6.47 (dd,
J = 2.26, 8.29 Hz, 1H), 6.34 (d, J = 1.70 Hz, 1H), 4.48 (dd, J = 8.19,
14.98 Hz, 1H), 3.88 (s, 6H), 3.79 (s, 3H), 3.73 (s, 3H), 3.50–3.68
(m, 2H), 3.32–3.46 (m, 3H), 3.22 (q, J = 16.95 Hz, 2H), 2.77–2.98
(m, 6H), 2.41–2.51 (m, 1H). m/z 611 (M+H).
5.52. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(3-phe-
nylpropyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25k)
5.57. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(pyrro-
lidin-1-yl)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25p)
This was prepared as per 25e using 3-phenyl-1-bromopropane,
stirring at RT overnight. Yield 41%. 1H NMR (300 MHz,
CHLOROFORM-d) d 7.16–7.36 (m, 8H), 7.04–7.13 (m, 2H), 6.92–
7.01 (m, 1H), 6.87 (d, J = 8.29 Hz, 1H), 6.64–6.74 (m, 2H), 6.58–
6.63 (m, 1H), 6.44 (dd, J = 2.26, 8.29 Hz, 1H), 6.29 (d, J = 2.26 Hz,
1H), 4.48 (dd, J = 8.19, 14.98 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H),
3.52–3.69 (m, 2H), 3.32–3.47 (m, 1H), 3.11 (s, 2H), 3.05–3.32 (m,
2H), 2.78–2.98 (m, 4H), 2.74 (t, J = 7.63 Hz, 2H), 2.38–2.50 (m,
1H), 1.95 (quin, J = 7.30 Hz, 2H). m/z 564 (M+H).
This was prepared as per 25e using 1-bromo-4-chlorobutane.
Yield 16%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.33 (m,
5H), 7.06–7.12 (m, 1H), 6.97–7.02 (m, 1H), 6.94 (d, J = 8.48 Hz,
1H), 6.66–6.76 (m, 1H), 6.58–6.65 (m, 1H), 6.45 (dd, J = 2.45,
8.48 Hz, 1H), 6.28 (d, J = 2.26 Hz, 1H), 4.48 (dd, J = 8.19, 14.79 Hz,
1H), 3.80 (s, 3H), 3.73 (s, 3H), 3.53–3.69 (m, 2H), 3.36–3.48 (m,
1H), 3.13–3.33 (m, 6H), 2.79–3.02 (m, 4H), 2.51 (dd, J = 4.05,
16.67 Hz, 1H), 1.99 (td, J = 3.34, 6.50 Hz, 4H). m/z 500 (M+H).
5.58. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(piperi-
5.53. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(4-phenoxy-
din-1-yl)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25q)
butyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25l)
Aniline 24 (25 mg, 0.056 mmol), potassium carbonate (9 mg,
This was prepared as per 25e using 3-phenyl-1-bromopropane,
0.062 mmol) and 1,5-dibromopentane (14 mg, 8
were combined in DI water and heated to 120 °C for 20 min in
lL, 0.062 mmol)
stirring at RT overnight. Yield 41%. 1H NMR (300 MHz,

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5721
the microwave at 50 W power. The aqueous was removed via pip-
ette and the residue was dissolved in EtOAc, then washed with
NaHCO3 solution and brine, and dried over MgSO4 and the solvent
was removed under reduced pressure. The crude was purified by
chromatography on silica (0–50% EtOAc/hexane) to give the piper-
idine (17 mg, 59%). 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–
7.34 (m, 3H), 7.09 (d, J = 7.91 Hz, 2H), 6.95 (d, J = 8.29 Hz, 2H),
6.80 (dd, J = 2.26, 8.48 Hz, 1H), 6.68–6.74 (m, 1H), 6.57–6.67 (m,
3H), 4.48 (dd, J = 8.10, 15.07 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H),
3.55–3.71 (m, 2H), 3.33–3.45 (m, 1H), 3.08–3.32 (m, 6H), 2.78–
3.01 (m, 4H), 2.45–2.55 (m, 1H), 1.66–1.76 (m, 4H), 1.58 (d,
J = 5.09 Hz, 2H). m/z 514 (M+H).
5.63. Ethyl 2-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)acetate
(26a)
This was prepared as per 25e from 24 using ethyl bromoacetate.
Yield 90%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.34 (m,
3H), 7.09 (d, J = 7.91 Hz, 2H), 6.92–7.00 (m, 1H), 6.90 (d,
J = 8.29 Hz, 1H), 6.58–6.74 (m, 3H), 6.48 (dd, J = 2.12, 8.24 Hz,
1H), 6.33 (s, 1H), 4.47 (dd, J = 8.10, 15.07 Hz, 1H), 4.25 (q,
J = 7.16 Hz, 2H), 3.89 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.57–3.69
(m, 2H), 3.33–3.47 (m, 1H), 3.11–3.32 (m, 2H), 2.76–2.98 (m,
4H), 2.42–2.53 (m, 1H), 1.31 (t, J = 7.11 Hz, 3H). m/z 532 (M+H).
5.59. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(pentyl-
amino)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25r)
5.64. Ethyl 2-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)propa-
noate (26b)
This was prepared as per 25b from 24 using valeraldehyde.
Yield 46%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.34 (m,
3H), 7.05–7.13 (m, 2H), 6.93–7.01 (m, 1H), 6.88 (d, J = 8.29 Hz,
1H), 6.65–6.74 (m, 2H), 6.58–6.64 (m, 1H), 6.46 (dd, J = 2.26,
8.29 Hz, 1H), 6.33 (s, 1H), 4.48 (dd, J = 8.29, 15.07 Hz, 1H), 3.79
(s, 3H), 3.73 (s, 3H), 3.51–3.68 (m, 3H), 3.33–3.46 (m, 1H), 3.13–
3.32 (m, 2H), 3.08 (t, J = 7.06 Hz, 2H), 2.77–2.99 (m, 4H), 2.42–
2.52 (m, 1H), 1.58–1.68 (m, 2H), 1.32–1.44 (m, 4H), 0.88–0.97
(m, 3H). m/z 516 (M+H).
This was prepared as per 25e from 24 using ethyl 2-bromopro-
pionate. Yield 65%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–
7.32 (m, 3H), 7.08 (d, J = 6.78 Hz, 2H), 6.91–6.99 (m, 1H), 6.88 (d,
J = 8.29 Hz, 1H), 6.58–6.73 (m, 3H), 6.47 (dd, J = 2.07, 8.10 Hz,
1H), 6.31–6.35 (m, 1H), 4.47 (dd, J = 8.01, 14.98 Hz, 1H), 4.20 (q,
J = 7.10 Hz, 2H), 4.06–4.14 (m, 1H), 3.79 (s, 3H), 3.73 (s, 3H),
3.55–3.67 (m, 2H), 3.32–3.45 (m, 1H), 3.21 (q, J = 17.08 Hz, 2H),
2.76–2.97 (m, 4H), 2.38–2.51 (m, 1H), 1.47 (d, J = 5.46 Hz, 3H),
1.27 (dt, J = 2.26, 7.16 Hz, 3H). m/z 546 (M+H).
5.60. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[methyl-
(pentyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25s)
5.65. Ethyl 3-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)propa-
noate (26c)
This was prepared as per 25a from 25r. Yield 82%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.18–7.34 (m, 3H), 7.06–7.13 (m,
2H), 6.96–7.02 (m, 1H), 6.93 (d, J = 8.48 Hz, 1H), 6.69–6.75 (m,
1H), 6.54–6.68 (m, 3H), 6.39 (s, 1H), 4.48 (dd, J = 8.10, 15.07 Hz,
1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.56–3.70 (m, 2H), 3.39 (dt,
J = 4.71, 12.15 Hz, 1H), 3.14–3.33 (m, 4H), 2.91 (s, 3H), 2.77–3.01
(m, 4H), 2.49 (dd, J = 3.77, 16.58 Hz, 1H), 1.57 (td, J = 7.23,
14.36 Hz, 2H), 1.23–1.42 (m, 4H), 0.91 (t, J = 6.88 Hz, 3H). m/z
530 (M+H).
This was prepared as per 25e from 24 using ethyl 3-bromopro-
pionoate. Yield 12%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–
7.34 (m, 3H), 7.08 (d, J = 6.97 Hz, 2H), 6.96 (t, J = 6.31 Hz, 1H),
6.89 (d, J = 8.29 Hz, 1H), 6.58–6.74 (m, 3H), 6.48 (dd, J = 2.26,
8.29 Hz, 1H), 6.36 (d, J = 1.88 Hz, 1H), 4.48 (dd, J = 8.19, 14.98 Hz,
1H), 4.16 (q, J = 7.16 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.54–3.67
(m, 2H), 3.44 (t, J = 6.30 Hz, 2H), 3.36–3.49 (m, 1H), 3.22 (q,
J = 17.08 Hz, 2H), 2.77–2.97 (m, 4H), 2.61 (t, J = 6.22 Hz, 2H),
2.42–2.53 (m, 1H), 1.22–1.31 (m, 3H). m/z 546 (M+H).
5.61. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-(hexyl-
amino)-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (25t)
5.66. Ethyl 4-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)buta-
noate (26d)
This was prepared as per 25b from 24 using 1-bromohexane.
Yield 33%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.34 (m,
3H), 7.09 (d, J = 6.59 Hz, 2H), 6.96 (dd, J = 4.80, 7.82 Hz, 1H), 6.88
(d, J = 8.29 Hz, 1H), 6.65–6.74 (m, 2H), 6.57–6.64 (m, 1H), 6.46
(dd, J = 2.26, 8.29 Hz, 1H), 6.33 (d, J = 1.88 Hz, 1H), 4.48 (dd,
J = 8.10, 15.07 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.52–3.69 (m,
3H), 3.33–3.46 (m, 1H), 3.13–3.32 (m, 2H), 3.08 (t, J = 7.06 Hz,
2H), 2.77–2.98 (m, 4H), 2.42–2.51 (m, 1H), 1.53–1.67 (m, 2H),
1.27–1.47 (m, 6H), 0.86–0.94 (m, 3H). m/z 530 (M+H).
This was prepared as per 25e from 24 using ethyl 4-bromobu-
tanoate. Yield 35%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–
7.34 (m, 3H), 7.05–7.12 (m, 2H), 6.96 (dd, J = 4.71, 7.72 Hz, 1H),
6.88 (d, J = 8.29 Hz, 1H), 6.65–6.75 (m, 2H), 6.59–6.64 (m, 1H),
6.47 (dd, J = 2.26, 8.29 Hz, 1H), 6.33 (d, J = 2.26 Hz, 1H), 4.48 (dd,
J = 8.19, 14.98 Hz, 1H), 4.15 (q, J = 7.16 Hz, 2H), 3.79 (s, 3H), 3.73
(s, 3H), 3.54–3.67 (m, 2H), 3.34–3.45 (m, 1H), 3.11–3.32 (m, 4H),
2.77–2.99 (m, 4H), 2.49 (d, J = 5.27 Hz, 1H), 2.43 (t, J = 7.16 Hz,
2H), 1.95 (quin, J = 6.97 Hz, 2H), 1.26 (t, J = 7.16 Hz, 3H). m/z 560
(M+H).
5.62. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[hexyl-
(methyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(25u)
5.67. tert-Butyl 4-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimeth-
oxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)buta-
noate (26e)
This was prepared as per 25a from 25t. Yield 90%. 1H NMR
(300 MHz, CHLOROFORM-d)
d 7.18–7.34 (m, 3H), 7.09 (d,
J = 7.16 Hz, 2H), 6.99 (dd, J = 5.27, 7.72 Hz, 1H), 6.93 (d,
J = 8.48 Hz, 1H), 6.68–6.75 (m, 1H), 6.53–6.67 (m, 3H), 6.38 (d,
J = 2.07 Hz, 1H), 4.48 (dd, J = 8.10, 15.07 Hz, 1H), 3.78 (s, 3H), 3.74
(s, 3H), 3.55–3.70 (m, 2H), 3.39 (dt, J = 4.62, 12.20 Hz, 1H), 3.13–
3.32 (m, 4H), 2.90 (s, 3H), 2.77–3.01 (m, 4H), 2.49 (dd, J = 3.96,
16.58 Hz, 1H), 1.50–1.62 (m, 2H), 1.31 (br s, 6H), 0.86–0.94 (m,
3H). m/z 544 (M+H).
This was prepared as per 25e from 24 using t-butyl 4-bromobu-
tanoate. Yield 21%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–
7.33 (m, 3H), 7.09 (d, J = 6.78 Hz, 2H), 6.98 (br s, 1H), 6.87 (d,
J = 8.29 Hz, 1H), 6.65–6.73 (m, 2H), 6.58–6.64 (m, 1H), 6.46 (dd,
J = 2.17, 8.19 Hz, 1H), 6.33 (d, J = 2.07 Hz, 1H), 4.47 (dd, J = 8.01,
14.98 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.54–3.69 (m, 2H), 3.32–

5722
D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
3.47 (m, 1H), 3.14 (t, J = 6.88 Hz, 2H), 3.07–3.32 (m, 2H), 2.76–2.99
(m, 4H), 2.41–2.54 (m, 1H), 2.34 (t, J = 7.16 Hz, 2H), 1.90 (quin,
J = 7.02 Hz, 2H), 1.46 (s, 9H). m/z 588 (M+H).
(31 mg, 21 lL, 0.269 mmol) and the reaction was stirred at RT
overnight. The solution was taken directly into purification by
chromatography on silica (0–70% EtOAc/hexane) to give the bis-
sulfonamide (30 mg, 56%).
5.68. Methyl 5-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)penta-
noate (26f)
The bis-sulfonamide was suspended in 2 N NaOH solution
(2 mL) and heated to 80 °C overnight. The reaction was cooled
and acidified with 2 N HCl solution. The precipitate formed was
collected by filtration to give the sulfonamide as a white solid
(19 mg, 73%). 1H NMR (300 MHz, DMSO-d6) d 7.18–7.33 (m, 3H),
6.97–7.13 (m, 4H), 6.81–6.92 (m, 3H), 6.75 (s, 2H), 4.29 (dd,
J = 7.72, 15.07 Hz, 1H), 3.70–3.74 (m, 1H), 3.68 (s, 3H), 3.61 (s,
3H), 3.53–3.60 (m, 1H), 3.21–3.31 (m, 2H), 2.85–2.98 (m, 3H),
2.82 (s, 3H), 2.70–2.80 (m, 3H), 2.39–2.46 (m, 1H). m/z 524 (M+H).
This was prepared as per 25e from 24 using methyl 5-bro-
mopentanoate, stirring overnight at RT. Yield 19%. 1H NMR
(300 MHz, CHLOROFORM-d) d 7.18–7.35 (m, 3H), 7.05–7.12 (m,
2H), 6.93–7.01 (m, J = 4.30 Hz, 1H), 6.88 (d, J = 8.29 Hz, 1H), 6.65–
6.74 (m, 2H), 6.57–6.64 (m, 1H), 6.46 (dd, J = 2.26, 8.29 Hz, 1H),
6.32 (d, J = 2.07 Hz, 1H), 4.48 (dd, J = 8.10, 15.07 Hz, 1H), 3.79 (s,
3H), 3.73 (s, 3H), 3.68 (s, 3H), 3.53–3.66 (m, 2H), 3.33–3.45 (m,
1H), 3.06–3.32 (m, 4H), 2.76–2.98 (m, 4H), 2.42–2.53 (m, 1H),
2.33–2.41 (m, 2H), 1.56–1.83 (m, 4H). m/z 560 (M+H).
5.72. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-acetamido-
1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (28a)
To a solution of aniline 24 (25 mg, 0.056 mmol) in DCM (0.5 mL)
5.69. Ethyl 7-({2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)hepta-
noate (26g)
was added DIPEA (22 mg, 29
lL, 0.168 mmol) and then acetic
anhydride (11 mg, 11 L, 0.112 mmol) and the resulting solution
l
was stirred at RT overnight. The solution was applied directly to
column for chromatography on silica (0–100% EtOAc/hexane) to
give the amide (27 mg, quantitative). 1H NMR (300 MHz,
CHLOROFORM-d) d 7.37 (s, 1H), 7.17–7.33 (m, 4H), 7.14 (s, 1H),
7.09 (d, J = 8.01 Hz, 2H), 7.01 (d, J = 8.29 Hz, 1H), 6.92 (s, 1H),
6.66–6.74 (m, 2H), 6.60–6.66 (m, 1H), 4.49 (dd, J = 8.15, 14.93 Hz,
1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.56–3.72 (m, 2H), 3.36–3.49 (m,
1H), 3.10–3.33 (m, 2H), 2.79–3.03 (m, 4H), 2.52–2.63 (m, 1H),
2.17 (s, 3H). m/z 488 (M+H).
This was prepared as per 25e from 24 using ethyl 5-bromohep-
tanoate, stirring overnight at RT. Yield 18%. 1H NMR (300 MHz,
CHLOROFORM-d) d 7.17–7.36 (m, 3H), 7.03–7.15 (m, 2H), 6.97 (dd,
J = 4.62, 7.63 Hz, 1H), 6.88 (d, J = 8.29 Hz, 1H), 6.65–6.74 (m, 2H),
6.58–6.64 (m, 1H), 6.46 (dd, J = 2.35, 8.19 Hz, 1H), 6.32 (d,
J = 2.07 Hz, 1H), 4.48 (dd, J = 8.01, 14.98 Hz, 1H), 4.13 (q, J = 7.16 Hz,
2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.52–3.70 (m, 2H), 3.34–3.46 (m, 1H),
3.13–3.32 (m, 2H), 3.09 (t, J = 7.06 Hz, 2H), 2.76–2.99 (m, 4H), 2.41–
2.52 (m, 1H), 2.31 (t, J = 7.44 Hz, 2H), 1.55–1.71 (m, 4H), 1.33–1.49
(m, 4H), 1.22–1.30 (m, 3H). m/z 602 (M+H).
5.73. N-{2-[(Benzylcarbamoyl)methyl]-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}hexanamide (28b)
5.70. 2-({2-[(Benzylcarbamoyl)methyl]-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}amino)-N,N-dimethyl-
acetamide (26h)
Aniline 24 (30 mg, 0.067 mmol) and BOP (30 mg, 0.067 mmol)
were combined in DCM (1 mL) and hexanoic acid (8 mg, 8.5
0.067 mmol) was added, followed by DIPEA (17 mg, 24
l
l
L,
L,
0.135 mmol) and the reaction was stirred at RT overnight. The
reaction was diluted with EtOAc, washed with NaHCO3 solution
and brine, and dried over MgSO4 and the solvent was removed
under reduced pressure. The crude was purified by chromatogra-
phy on silica (0–75% EtOAc/hexane) to give the desired amide
(34 mg, 97%). 1H NMR (300 MHz, CHLOROFORM-d) d 7.42 (s, 1H),
7.17–7.35 (m, 4H), 7.05–7.14 (m, 3H), 7.01 (d, J = 8.29 Hz, 1H),
6.87–6.96 (m, 1H), 6.59–6.74 (m, 3H), 4.48 (dd, J = 8.15, 15.02 Hz,
1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.60 (dd, J = 4.71, 14.98 Hz, 2H),
3.35–3.51 (m, 1H), 3.08–3.33 (m, 2H), 2.78–3.03 (m, 4H), 2.51–
2.63 (m, 1H), 2.34 (t, J = 7.49 Hz, 2H), 1.66–1.79 (m, 2H), 1.31–
1.41 (m, 4H), 0.87–0.95 (m, 3H). m/z 544 (M+H).
Ester 25q (18 mg, 0.034 mmol) was dissolved in ethanol
(0.5 mL) and 2 N sodium hydroxide solution (0.1 mL, 0.2 mmol)
was added. The reaction was stirred at RT overnight. The ethanol
was removed under reduced pressure, the reaction was diluted
with water and the pH was adjusted to 7 with 2 N HCl. All solvents
were removed under reduced pressure, and the crude was dis-
solved as far as possible in methanol. The solution was filtered to
remove solids and the solvent removed under reduced pressure
to give the acid.
The acid was combined with dimethylamine hydrochloride
(7 mg, 0.079 mmol) and HATU (23 mg, 0.060 mmol) in DMF
(0.5 mL) and DIPEA (26 mg, 35 lL, 0.199 mmol) was added. The
reaction was stirred at RT overnight, diluted with EtOAc, washed
with NaHCO3 solution and brine, and dried over MgSO4 and the
solvent was removed under reduced pressure. The crude was puri-
fied by chromatography on silica (0–10% CMA-80/EtOAc) to give
the amide (9 mg, 43%). 1H NMR (300 MHz, CHLOROFORM-d) d
7.19–7.35 (m, 3H), 7.09 (d, J = 7.72 Hz, 2H), 6.94–7.02 (m, 1H),
6.81–6.93 (m, 2H), 6.59–6.75 (m, 3H), 6.52 (d, J = 8.29 Hz, 1H),
6.34 (s, 1H), 4.48 (dd, J = 8.19, 14.98 Hz, 1H), 3.85 (s, 2H), 3.79 (s,
3H), 3.74 (s, 3H), 3.55–3.69 (m, 2H), 3.33–3.47 (m, 1H), 3.13–
3.32 (m, 2H), 3.01–3.08 (m, 4H), 2.83–2.99 (m, 4H), 2.81 (s, 2H),
2.43–2.55 (m, 1H). m/z 531 (M+H).
5.74. N-{2-[(Benzylcarbamoyl)methyl]-1-[(3,4-dimethoxyphenyl)-
methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}benzamide (28c)
This was prepared as per 27a except using benzoyl chloride.
Yield 81%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.84–7.90 (m,
2H), 7.82 (s, 1H), 7.45–7.60 (m, 4H), 7.36 (d, J = 8.48 Hz, 1H),
7.19–7.33 (m, 3H), 7.03–7.12 (m, 3H), 6.88–6.96 (m, 1H), 6.67–
6.75 (m, 2H), 6.59–6.66 (m, 1H), 4.49 (dd, J = 8.15, 15.02 Hz, 1H),
3.81 (d, J = 0.94 Hz, 3H), 3.73–3.76 (m, 3H), 3.55–3.73 (m, 2H),
3.38–3.51 (m, 1H), 3.11–3.34 (m, 2H), 2.81–3.08 (m, 4H), 2.61
(dd, J = 4.57, 16.81 Hz, 1H). m/z 550 (M+H).
5.71. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-methane-
5.75. Ethyl N-{2-[(benzylcarbamoyl)methyl]-1-[(3,4-dimethoxy-
sulfonamido-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide (27)
phenyl)methyl]-1,2,3,4-tetrahydroisoquinolin-6-yl}carbamate (23b)
To aniline 24 (40 mg, 0.090 mmol) and TEA (36 mg, 50
0.359 mmol) in DCM (0.5 mL) was added methanesulfonyl chloride
l
L,
This was isolated from the hydrolysis of carbamate 23a in etha-
nol. Yield 15%. 1H NMR (300 MHz, CHLOROFORM-d) d 7.18–7.37

D. A. Perrey et al. / Bioorg. Med. Chem. 23 (2015) 5709–5724
5723
(m, 4H), 7.05–7.16 (m, 3H), 7.00 (d, J = 8.38 Hz, 1H), 6.92 (dd,
J = 4.85, 7.77 Hz, 1H), 6.65–6.74 (m, 2H), 6.64 (s, 1H), 6.61 (s, 1H),
4.49 (dd, J = 8.10, 14.98 Hz, 1H), 4.22 (q, J = 7.16 Hz, 2H), 3.79 (s,
3H), 3.74 (s, 3H), 3.56–3.72 (m, 2H), 3.35–3.50 (m, 1H), 3.09–3.34
(m, 2H), 2.78–3.03 (m, 4H), 2.48–2.61 (m, 1H), 1.27–1.35 (m,
3H). m/z 518 (M+H).
plotted against the log of compound concentration. Data were fit to
a three-parameter logistic curve to generate EC50 values (Prism,
version 6.0, GraphPad Software, Inc., San Diego, CA). Apparent Ke
values were calculated using the equation Ke = [L]/((EC+50/EC5ꢃ0) ꢃ 1)
where [L] is the concentration of test compound, EC+50 is the EC50 of
orexin A with test compound, and ECꢃ50 is the EC50 of orexin A. Ke val-
ues were considered valid when the EC+50/EC5ꢃ0 ratio was at least 4.
5.76. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(phenyl-
carbamoyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}acetamide
(28d)
Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Aniline 24 (25 mg, 0.056 mmol) and phenyl isocyanate (7 mg,
7 lL, 0.062 mmol) were combined in toluene (0.5 mL) and heated
to 75 °C for 4 h The reaction was cooled and the solvent was
removed under reduced pressure. The crude was purified by chro-
matography on silica (0–80% EtOAc/hexane) to give the urea
(32 mg, quantitative). 1H NMR (300 MHz, CHLOROFORM-d) d 7.82
(s, 1H), 7.46 (s, 1H), 7.17–7.35 (m, 8H), 6.95–7.12 (m, 6H), 6.70–
6.78 (m, 2H), 6.60–6.68 (m, 1H), 4.48 (dd, J = 8.19, 15.07 Hz, 1H),
3.81 (s, 3H), 3.72 (s, 3H), 3.56–3.68 (m, 2H), 3.35–3.49 (m, 1H),
3.05–3.34 (m, 2H), 2.70–2.91 (m, 4H), 2.40–2.53 (m, 1H). m/z 565
(M+H).
Acknowledgments
We are grateful to National Institute on Drug Abuse, National
Institutes of Health, U.S. (Grants DA032837 and DA026582 to
Y.Z.) for the financial support of this research. We thank Tiffany
Langston, Keith Warner and Dr. Angela Giddings for their valuable
technical assistance. We also thank Dr. Gerald Pollard for critical
reading of the manuscript.
References and notes
5.77. N-Benzyl-2-{1-[(3,4-dimethoxyphenyl)methyl]-6-[(hexyl-
carbamoyl)amino]-1,2,3,4-tetrahydroisoquinolin-2-yl}aceta-
mide (28e)
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Two individual stable cell lines were created by over-expressing
human OX1 and OX2 receptors in CHO-RD-HGA16 (Molecular
Devices) cells. The day before the assay, cells were plated into
96-well black-walled assay plates at 25,000 cells/well in Ham’s
F12 supplemented with 10% fetal bovine serum, 100 units of peni-
cillin and streptomycin, and 100 lg/mL normocin™. The cells were
incubated overnight at 37 °C, 5% CO2. Prior to the assay, Calcium 5
dye (Molecular Devices) was reconstituted according to the manu-
facturer instructions. The reconstituted dye was diluted 1:40 in
pre-warmed (37 °C) assay buffer (1ꢂ HBSS, 20 mM HEPES,
2.5 mM probenecid, pH 7.4 at 37 °C). Growth medium was
removed and the cells were gently washed with 100
pre-warmed (37 °C) assay buffer. The cells were incubated for 45
minutes at 37 °C, 5% CO2 in 200 L of the diluted Calcium 5 dye.
lL of
l
A single concentration of each test compound was prepared at
10ꢂ the desired final concentration in 2.5% BSA/8% DMSO/assay
buffer. Serial dilutions of orexin A were prepared at 10ꢂ the
desired final concentration in 0.25% BSA/1% DMSO/assay buffer,
aliquoted into 96-well polypropylene plates, and warmed to
37 °C. After the dye-loading incubation period, the cells were
pre-treated with 25 lL of the test compounds and incubated for
25. Perrey, D. A.; German, N. A.; Decker, A. M.; Thorn, D.; Li, J. X.; Gilmour, B. P.;
Thomas, B. F.; Harris, D. L.; Runyon, S. P.; Zhang, Y. ACS Chem. Neurosci. 2015, 6,
599.
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