Synthesis and structure activity relationship of tetrahydroisoquinoline- based potentiators of GluN2C and GluN2D containing N-Methyl-D-aspartate receptors

Article abstract of DOI:10.1021/jm400177t

We describe here the synthesis and evaluation of a series of tetrahydroisoquinolines that show subunit-selective potentiation of NMDA receptors containing the GluN2C or GluN2D subunits. Bischler-Napieralski conditions were employed in the key step for the

Full text of DOI:10.1021/jm400177t

Subscriber access provided by BRIGHAM YOUNG UNIV
Article
Synthesis and Structure Activity Relationship of
Tetrahydroisoquinoline-based Potentiators of GluN2C
and GluN2D Containing N-Methyl-D-Aspartate Receptors
Rose M. Santangelo Freel, Kevin K. Ogden, Katie L. Strong, Alpa Khatri, Kathryn
M. Chepiga, Henrik S. Jensen, Stephen F. Traynelis, and Dennis C. Liotta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400177t • Publication Date (Web): 29 Apr 2013
Downloaded from http://pubs.acs.org on May 17, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesis and Structure Activity Relationship of Tetrahydroisoquinoline-based Potentiators of GluN2C and
GluN2D Containing N-Methyl-D-Aspartate Receptors
1
2
1
2
1
Rose M. Santangelo Freel , Kevin K. Ogden , Katie L. Strong , Alpa Khatri , Kathryn M. Chepiga , Henrik S.
3
2
1
Jensen , Stephen F. Traynelis , Dennis C. Liotta *
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta GA 30322
2
Department of Pharmacology, Emory University School of Medicine, 1510 Clifton Road, Atlanta GA 30322
3
Neuroscience Drug Discovery, H. Lundbeck A/S,Ottiliavej 9, DK-2500 Valby, Denmark
*
Corresponding Author
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
We describe here the synthesis and evaluation of a series of tetrahydroisoquinolines that show subunitꢀ
selective potentiation of NMDA receptors containing the GluN2C or GluN2D subunits. BischlerꢀNapieralski
conditions were employed in the key step for the conversion of acyclic amides to the corresponding
tetrahydroisoquinoline containing analogs. Compounds were evaluated using both two electrode voltage clamp
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
recordings from Xenopus laevis oocytes and imaging of mammalian BHK cells loaded with Ca ꢀsensitive dyes. The
most potent analogues had EC values of 300 nM and showed over 2ꢀfold potentiation of the response to maximally
50
effective concentrations of glutamate and glycine, but had no effect on responses from NMDA receptors containing
the GluN2A or GluN2B subunits, AMPA, kainate, GABA, or glycine receptors or a variety of other potential targets.
These compounds represent a potent class of small molecule subunitꢀselective potentiators of NMDA receptors.
Introduction
The ionotropic family of glutamate receptors comprises NꢀmethylꢀDꢀaspartate (NMDA), αꢀaminoꢀ3ꢀ
hydroxyꢀ5ꢀmethylꢀ4ꢀisoxazolepropionic acid (AMPA), and kainate receptor subtypes, subdivided on the basis of
amino acid sequence homology, structure homology, and pharmacology. The NMDA receptor mediates a slow,
2+
Ca ꢀpermeable component of excitatory synaptic transmission in the central nervous system, and plays a prominent
1
ꢀ8
role in normal brain processes such as learning, memory, synaptic plasticity, and neuronal development. In
addition, dysfunction of NMDA receptors, either overactivation or hypofunction, has been implicated as a
9
ꢀ11
12
contributing factor to a wide range of neurological disorders including schizophrenia , Alzheimer’s disease ,
1
3
14
15, 16
6
17
Parkinson’s disease , Huntington’s chorea , depression
, epilepsy , neuropathic pain , and acute brain injury
18ꢀ20
8, 21
following ischemia , hypoxia or trauma.
NMDA receptors are ligandꢀgated cation channels that are tetramers of two glycineꢀbinding GluN1 subunits
and two glutamateꢀbinding GluN2 subunits. There are four different GluN2 subunits (GluN2AꢀD), each of which
22, 23
endows the receptor with unique open probability, single channel conductance, and deactivation time course.
For
24ꢀ26
example, GluN2Cꢀ and GluN2Dꢀcontaining NMDA receptors have a lower open probability,
decreased
ACS Paragon Plus Environment

Page 3 of 82
Journal of Medicinal Chemistry
1
2
+ 27
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sensitivity to block by Mg , and can be activated by lower concentrations of glycine and glutamate than GluN2Aꢀ
28, 29
and GluN2Bꢀcontaining receptors.
The four different subunits are differentially expressed throughout the brain,
with particularly prominent GluN2C and GluN2D expression in the cerebellum, basal ganglia, and cortical and
30ꢀ32
hippocampal interneurons.
The distinct localized expression and the unique functional properties of each of the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
four subunits, along with the potential involvement of NMDA receptors in disease states and injuries, creates a
compelling rationale for development of subunitꢀselective modulators with potential use in a variety of
neuropathological conditions. Compounds that increase the strength of glutamatergic synapses have been
33ꢀ35
hypothesized to be therapeutically useful in treating schizophrenia.
For examples, agonists at the GluN1 subunit,
36
e.g. glycine and Dꢀserine, have received attention as potential therapies for schizophrenia ; however, these
molecules will increase the activity of all NMDA receptor subtypes to a similar degree and have activity at other cellꢀ
37
surface receptors expressed in the CNS. By contrast, small molecules directly potentiating the NMDA receptor at
regions other than the agonist binding site might exhibit advantageous subunitꢀselectivity and be more selective for
38
the NMDA receptor over other receptors. Moreover the GluN2C and GluN2D subunits are particularly interesting
27
targets in this context because they are expressed on hippocamapal and cortical interneurons, whose hypofunction is
35, 39, 40
thought to cause disinhibition of pyramidal cells leading to excessive drive of the dopaminergic system.
Each NMDA receptor subunit contains four semiꢀautonomous domains: an extracellular aminoꢀterminal
domain (ATD), an extracellular ligand binding domain (LBD), a transmembrane domain that contributes to the ion
conduction pore, and an intracellular carboxyꢀterminus. The binding sites for at least six classes of antagonists of the
NMDA receptor are known. Voltageꢀdependent channel blockers, typically rigid organic cations such as
4
1ꢀ43
phencyclidine (PCP), bind deep within the ion conduction pore in a voltageꢀdependent fashion.
Two additional
classes of NMDA receptor antagonists include competitive antagonists that bind with high affinity to either the
44ꢀ47
glycine site on the GluN1 subunit or the glutamate site on the GluN2 subunit.
A fourth class of nonꢀcompetitive
antagonists, which includes quinazolineꢀ4ꢀones and dihydroquinolineꢀpyrazolines, act at the membraneꢀproximal
4
8ꢀ50
portion of the ligand binding domain and are more potent after glutamate but not glycine binding.
A fifth class of
51
NMDA receptor antagonists binds to the weakly conserved ATD, and is highly selective for the GluN2B subunit.
Antagonists in this class, which include ifenprodil and a wide range of related structures, show well over 200ꢀfold
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
selectivity for GluN2B over GluN2A, GluN2C and GluN2D. Finally, a sixth class of antagonists selectively inhibits
GluN2Aꢀcontaining receptors through actions at the dimer interface between the GluN1 and GluN2 ligand binding
52, 53
domains. These compounds, typified by TCN201, are allosteric regulators of glycine binding.
In addition to
these six classes, several phenanthroic acid and napthoic acid analogues are noncompetitive antagonists that act
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
4, 55
outside the ATD.
In contrast to NMDA receptor antagonists, relatively few compounds have been shown to potentiate NMDA
56ꢀ58
59
60
receptor function. Polyamines (e.g. spermine) , aminoglycosides , and sulfated neurosteroids can enhance the
function of GluN2Bꢀcontaining NMDA receptors with EC values in range of 40ꢀ130 ꢁM. Phenanthroic acid and
5
0
napthoic acid derivatives increase the current response of GluN2Aꢀ and GluN2Bꢀcontaining receptors at
61, 62
concentrations around 100 ꢁM.
In addition, Dꢀcycloserine binds the GluN1 subunit and is a partial agonist at
GluN2A, GluN2B, or GluN2D receptors, but can activate GluN2Cꢀcontaining receptors to a greater extent than
63, 64
glycine.
Thus, the few potentiators that are known show strong GluN2 subunit dependence, suggesting
potentiation of NMDA receptors is mediated by less conserved portions of the receptor.
To identify small drugꢀlike subunitꢀselective modulators of NMDA receptors, we previously employed a
2
+
reverse chemical genetics approach utilizing a fluorescence Ca imagingꢀbased screen of a diversity library
65, 66
containing 100,000 compounds on BHK cell lines expressing GluN1/GluN2C or GluN1/GluN2D receptors.
We
identified an NMDA receptor potentiator with micromolar potency that selectively enhanced the function of both
GluN2Cꢀ and GluN2Dꢀcontaining NMDA receptors, but was without action on GluN2Aꢀ and GluN2Bꢀcontaining
receptors. This compound, (6,7ꢀdimethoxyꢀ1ꢀ((4ꢀmethoxyphenoxy)methyl)ꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀ
yl)(phenyl)methanone), had a tetrahydroisoquinoline backbone (1). Compound 1 enhanced the response of
GluN2C/Dꢀcontaining NMDA receptors by 1.5ꢀfold in the presence of maximally effective concentrations of coꢀ
agonists glutamate and glycine with an EC value estimated to be 12 ꢀM, but the solubility limit prevented accurate
50
determination of the maximal effect. Compound 1 and related analogues had no agonist activity on their own. We
recently described the mechanism and site of action of a related compound, 3ꢀchlorophenyl)(6,7ꢀdimethoxyꢀ1ꢀ((4ꢀ
methoxyphenoxy)methyl)ꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)methanone (2, also referred to as CIQ), which had a
6
6
reported EC value of 3 ꢀM and enhanced receptor responses less than 2ꢀfold.
50
ACS Paragon Plus Environment

Page 5 of 82
Journal of Medicinal Chemistry
1
2
3
4
In this study, we report synthetic efforts towards the optimization of substituents on the core structure of 2
67
that have led to the development of a structureꢀactivity relationship (SAR) for this class of compounds. The active
compounds in this class selectively potentiated current response from GluN2Cꢀ and GluN2Dꢀcontaining receptors.
While the core backbone of these compounds is similar to that of the previously identified GluN2Bꢀselective
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
68
antagonist, HON0001 (3, Figure 1) , none of the compounds studied here inhibit GluN2B at relevant concentrations.
Moreover, this class of compounds did not affect responses from GluN2Aꢀcontaining receptors nor from GluA1
AMPA receptors and GluK2 kainate receptors, suggesting this class of compounds shows strong subunit selectivity.
Results
Chemistry. To explore the SAR of the tetrahydroisoquinoline compound class as posititve allosteric modulators of
GluN2C/2Dꢀcontaining NMDA receptors, a series of derviatives were synthesized. For SAR development, the core
tetrahydroisoquinoline was divided into 3 modifiable rings (A, B, and C) and 2 modifiable linker regions (LinkerꢀA
and LinkerꢀB) (Figure 2). Compounds 5, 6, and 7 were synthesized from 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline hydrochloride (4) and the corresponding benzoyl chloride (Scheme 1). All other synthetically
derived analogues were generated via one of three pathways, each starting with substituted phenethylamines. 2ꢀ(3ꢀ
(
benzyloxy)phenyl)ethanamine (12) was prepared from 3ꢀmethoxyphenethylamine (8) in the sequence shown in
Scheme 2. The starting material (8) was demethylated in a refluxing mixture of acetic acid and hydrobromic acid to
form the phenol 9. The primary amine was then Bocꢀprotected to form 10 followed by an alkylation of the phenol
with benzyl bromide to give 11. The Boc group was removed under acidic conditions to give the phenethylamine 12.
The synthetic scheme employed for the preparation of each analogue was dependent upon the identity of the
linker between the tetrahydroisoquinoline core and the B ring (Figure 2). For compounds containing an ether or
thioether linker, an appropriately substituted phenethylamine (8, 12-18) was acylated with chloroacetyl chloride to
afford the αꢀchloroamideꢀcontaining compounds 19-26. Displacement of the chlorine with a phenol or thiophenol in
the presence of base afforded 27-42 and 43, respectively. The amides 27-43 were then subjected to Bischlerꢀ
Napieralski conditions to form the dihydroisoquinolines (45-62). The nitroꢀcontaining amide compound 42 was
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
subjected to hydrogenolysis, followed by a reductive amination to afford the dimethylaminoꢀcontaining intermediate
which was then cyclized using BischlerꢀNapieralski conditions to give the dihydroisoquinoline 44. The
dihydroisoqunolines were subsequently reduced with sodium borohydride (NaBH ) to afford the racemic
4
tetrahydroisoquinolines 63-80. Acylation of 63-80 with a substituted benzoyl chloride afforded analogues 2 and 81-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
27 (Scheme 3). Nitroꢀcontaining analogues 128-130 were reduced to the corresponding anilines (131-133) with tin
69
(II) chloride dehydrate. The anilines (131-133) were then subjected to reductive amination conditions to give the
6
9, 70
methylaminoꢀcontaining analogues 134 and 135.
Although the Aꢀring in each of the compounds described above
is a substituted phenyl ring, a number of compounds were synthesized with various aromatic or heterocyclic moieties
in place of the phenyl. These compounds were synthesized in one step from tetrahydroisoquinoline 63 in an
acylation reaction with an aryl chloride or 4ꢀmorpholinyl chloride (Scheme 4). In addition, tetrahydroisoquinoline 63
was acylated with chloroacetyl chloride followed by chlorine displacement with morpholine to afford 23, with a
modified Aꢀring and linker A. Tetrahydroisoquinoline 63 was also subjected to alkylation conditions to form the
tertiary amine 141 (Scheme 4).
Additionally, compounds with either a saturated or an unsaturated carbon linker, a substituted cinnamic acid
or phenylpropionic acid underwent standard EDCꢀcoupling conditions with a substituted phenethylamine (8 or 13) to
afford amides 142-145. Amides 142-145 were then subjected to BischlerꢀNapieralski conditions in refluxing POCl3
to afford the corresponding dihydroisoquinolines (146-149), which were subsequently reduced with NaBH to afford
4
the tetrahydroisoquinolines 150-153. Acylation of 150-153 with a series of substituted benzoyl chlorides afforded
the final analogues 154-162 (Scheme 5). Compounds with a shorter linker than those described above were
synthesized according to the procedure shown in Scheme 6. The commercially available phenylacetic acids (163 and
1
64) were each treated with thionyl chloride to form the corresponding acid chlorides, which were subsequently
reacted with 3,4ꢀdimethoxyphenethylamine (13) to give amides 165 and 166. The amides were then subjected to
BischlerꢀNapieralski reaction conditions to form the dihydroisoquinoline intermediates (167 and 168), which were
further reduced to the tetrahydroisoquinolines (169 and 170) with sodium borohydride. The core was then acylated
with an appropriately substituted benzoyl chloride to afford final compounds 171-175.
Stereoselective synthesis of compound 2
ACS Paragon Plus Environment

Page 7 of 82
Journal of Medicinal Chemistry
1
2
3
4
The stereoisomers for 2 were synthesized according to the procedure shown in Scheme 7. Either enantiomer
of the commercially available chiral amine (177) was subjected to acylation conditions with 3,4ꢀ
dimethoxyphenylacetyl chloride (176), to give the secondary amide 178. Reduction of the amide with borane
dimethylsulfide complex and boron trifluoride etherate gave the corresponding amine (179) in excellent yields.
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
71
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Acylation with chloroacetyl chloride followed by halogen displacement with 4ꢀmethoxyphenol afforded the tertiary
amide (181). Treatment of amide 181 with phosphorus oxychloride afforded the chiral iminium intermediate, which
was subsequently reduced to the tetrahydroisoquinoline (182) with sodium borohydride. Although crystallographic
data will be needed to confirm the assignment, the stereochemistry of the reduction has been assigned based on the
72ꢀ
model shown in Figure 3 and on literature precedent for the use of this chiral directing group in similar reductions
7
4
.
Tetrahydroisoquinoline 182 was subjected to hydrogenolysis to remove the chiral directing group, followed by
acylation with 3ꢀchlorobenzoyl chloride, to give the assigned Rꢀ(+) and S-(-) enantiomers of 2.
Potentiation of NMDA Receptors
All of the compounds synthesized in addition to 33 commercially available analogues (184-217) were
evaluated using two electrode voltage clamp recordings of currents from four recombinant NMDA receptor subtypes:
GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, and GluN1/GluN2D. We initially evaluated the conditions under
which to best assess compound activity. At low concentrations of agonist (0.3 ꢀM glutamate, 0.1 ꢀM glycine),
compound 2 produced strong potentiation (353+36% of control at 10 ꢀM for GluN2D ), but full concentrationꢀeffect
curves could not be fitted with the Hill equation, limiting our ability to obtain EC values with which to compare
50
analogues. By contrast, concentrationꢀeffect curves for compound 2 recorded in response to maximallyꢀeffective
concentrations of glutamate (100 ꢀM) and glycine (30 ꢀM) were wellꢀfitted by the Hill equation (maximum
2
28+8.8% of control for GluN2D). For all subsequent experiments, concentrationꢀresponse curves for each
compound were generated by coꢀapplying increasing concentrations of compound with maximally effective
concentrations of glutamate and glycine. Each compound was tested in at least 5 oocytes from two different frogs.
The mean degree of potentiation measured at 10 ꢂM for each compound at GluN1/GluN2C and GluN1/GluN2D is
presented in Tables 1ꢀ9. For compounds exhibiting more than 115% potentiation at 10 ꢂM, the EC values were
5
0
determined by fitting composite concentrationꢀresponse curves to equation 1 (Table 1ꢀ9). None of the compounds
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tested potentiated responses from receptors comprised of GluN1/GluN2A or GluN1/GluN2B, and thus these data are
not shown. A small subset of compounds produced weak inhibition (70% of the control response at 100 ꢂM) of
GluN1/GluN2A (compounds 7, 84, 110, 186) or GluN1/GluN2B (compounds 1, 96, 100, 101, 109, 139, 192, 206);
these inhibitory effects were not studied further.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In addition, several active compounds were tested against a representative member of the AMPAꢀselective
class of glutamate receptors (GluA1) and of the kainateꢀselective glutamate receptor class (GluK2). Compounds 1, 2,
5, 87, 96, 98, 111, 117, 118, 138, 140, 155, 157, 158, 159, 161, 162, 190, 197 coꢀapplied with 100 ꢂM glutamate had
no effects on homomeric GluA1 responses recorded under two electrode voltage clamp. Compounds 1, 2, 190, and
1
97 were coꢀapplied with 100 ꢂM glutamate to GluK2 expressing oocytes and had no effect on GluK2ꢀmediated
currents. Compound 83 was tested on an additional 19 receptors (e.g., AMPA, GABA, glycine, nicotinic
acetylcholine, purinergic) using the oocyte expression system (see below). Together, these results suggest strong
GluN2C/Dꢀselectivity for this series of compounds.
Effect of removing Linker-B and the B-ring.
In order to determine the importance of each portion of the compound for potency, fragments of the lead
compound 1 and two lead compounds 2, 192 were synthesized and tested. Compounds 5, 6, and 7 correspond to
compounds 1, 2, and 190 without the LinkerꢀB and the Bꢀring (Figure 4). All three of these compounds were
inactive against all recombinant NMDA receptors (n=6ꢀ10 oocytes for GluN1/GluN2A, GluN1/GluN2B,
GluN1/GluN2C, GluN1/GluN2D, data not shown). Reducing the length of the linker to a single methylene also
removed all activity (171-175 in Figure 4; data not shown). Thus, the Bꢀring and the two atom linker are essential for
activity in this series.
Optimization of A- and B-Ring Substituent Position.
We evaluated the optimal placement of substituents on the Aꢀring (Table 1). Substituents in the 3ꢀposition,
(
R in Table 1) showed the strongest potentiation compared to substitution in the 2ꢀ (R ) or 4ꢀ (R ) position. This is
3 4 2
evident when comparing potentiation associated with the methylꢀcontaining analogues 184, 185, and 186. For this
subset of compounds, 185, with a methyl in the 3ꢀposition (R , Table 1), is the most effective and potentiates
3
ACS Paragon Plus Environment

Page 9 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
GluN2Cꢀ and GluN2Dꢀcontaining receptors to a greater extent. When a chlorine substituent was placed on the Aꢀ
ring, as in compounds 187, 2, and 188, the 3ꢀchloro analogue (2) showed greater potentiation compared to the 2ꢀ and
4
ꢀchloro derivatives (187, 188; Table 1). Analogues with dichloro (86, 85, 194, 87) or 3ꢀchloroꢀ4ꢀfluoro (88)
substitutions on the Aꢀring showed EC values (when active) ranging from 1.4ꢀ21 ꢀM on GluN2C and 1.9ꢀ24 ꢀM
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
on GluN2D (Table 1).
We also evaluated the positional effects of Bꢀring substitutions. Data in Table 2 show that the paraꢀposition
(
R , Table 2) on the Bꢀring is the only position that affords any detectable activity for methoxy substituents. When a
2
methoxy substituent is placed in either the orthoꢀ or metaꢀposition, all activity is lost (Table 2).
Optimization of Substituent Identity on A- and B-Rings.
Since substitution in the metaꢀposition on the Aꢀring yielded the most potent and effective potentiators, a
number of modifications were made to this position (R in Table 3). Substitution on the Aꢀring with 3ꢀbromo (190),
1
6
6
3
ꢀiodo (93), and 3ꢀtrifluoromethyl (198) gave strong potentiation with potencies similar to compound 2 . All other
substitutions on the 3ꢀposition on the Aꢀring showed either decreased potency or decreased maximum degree of
potentiation. These data suggest that the binding pocket has both size and electronic requirements. Since 2 and 190
show similar potencies and maximal effects, and they are more active than the 3ꢀfluoro (197) analogue, it was
expected that the 3ꢀiodo (93) analogue would be active. While not as potent as the 3ꢀbromo (190) derivative, the 3ꢀ
iodo (93) analogue showed enhanced maximal potentiation. Interestingly, addition of a similarly large phenyl (94)
ring shows reasonable potency but a diminished degree of potentiation. In addition, when R is an electronꢀ
3
withdrawing group, such as nitro (84), the compounds are less potent but more efficacious (Table 3). Figure 5A
summarizes optimal substitutions on the A ring. We subsequently evaluated a range of different ring systems in
place of RingꢀA (Table 4). Only 2ꢀthiophene (203) showed weak activity at GluN2Cꢀcontaining receptors with no or
minimal potentiating effects at GluN2Dꢀcontaining receptors. The weak selectivity of this ring system for GluN2C
suggests a possible starting point from which to develop future compounds with selectivity for GluN2C over
GluN2D.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As shown in Table 2, only compounds with a paraꢀsubstitution pattern around the Bꢀring have detectable
potentiation on recombinant GluN2C/GluN2D receptors. Several modifications were made to the paraꢀposition
substituent on the Bꢀring to test for the optimal functionality in this position (R in Table 5). When a thiomethyl is
2
placed in the 4ꢀposition, there is potentiation if the analogue also contains a 3ꢀchloro on the Aꢀring (104), but not a
hydrogen (103). The potency of 104 is similar to the 4ꢀmethoxy containing analogue (2), but the degree of
potentiation is reduced. The 4ꢀethyl analogue is weakly active at GluN2C (207; Table 5). A 4ꢀdimethylamino
substituent shows potentiation preferentially of GluN2C if the analogue also contains a 3ꢀchloro or 3ꢀbromo on Ringꢀ
A (107 and 108, respectively), but not a hydrogen (106). Substitution with ethoxy (100, 101), benzyloxy (109),
amino (133, 132, 131), or nitro (129, 130, 128) led to compounds with no detectable activity on recombinant
GluN2Cꢀ or GluN2Dꢀcontaining receptors. These results suggest that the methyl ether is important for binding.
Moreover, steric effects may explain the lack of efficacy for the larger substituents such as benzyloxy (109; Table 5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Optimization of C-Ring Substitutions
All of the compounds described above contain a dimethoxy substitution pattern on the Cꢀring (R and R ,
3
4
Table 6). Substitution of methyl groups in place of both methoxy substituents results in modestly enhanced potency
~ 1 ꢀM EC ) and increased maximal potentiation (compare 122 and 123 to 2 and 190). When a dioxolane is fused
(
50
at these two positions (110, 111, 112), the potency is decreased when compared to the dimethoxy analogues (1, 2,
90). When the R ꢀmethoxy is removed and a R ꢀmethoxy is installed (Table 6), the resulting compounds show
1
3
2
modest changes in either potency or maximal effect. Compounds with dimethoxy substitutions at R and R (120 and
4
5
1
21; Table 6) are inactive. This suggests that substitution at the R position introduces unfavorable steric interactions
5
within the binding pocket. In addition, the decrease in potency associated with a nonꢀhydrogen substituent at R may
2
be explained by steric interactions between linkerꢀB and the substituent at R on the Cꢀring.
2
Interestingly, when a single methoxy is placed at R on the Cꢀring, as in compounds 116 and 117, potency is
4
increased when compared to the dimethoxy counterparts (Table 6). While compound 118 does not show increased
potency compared to its dimethoxy analog, it does exhibit a greater maximum potentiation, further suggesting that a
single substitution at R on the Cꢀring is optimal for activity. This trend of increased effectiveness also appears to
4
hold for the methylꢀsubstituted C ring. Compounds 122 and 123 show increased potency compared to 2 and 190, and
ACS Paragon Plus Environment

Page 11 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
when a single methyl is placed at R on the Cꢀring as in compounds 124, 125, and 126 the maximal effect is slightly
4
increased. Interestingly, introduction of Oꢀbenzyl substitutions at the R position results in the most potent
4
compounds tested. Compounds 82 and 83 show submicromolar EC values (0.3ꢀ0.4 ꢀM) with strong potentiation of
5
0
2
54ꢀ257% at GluN2C and 198ꢀ219% at GluN2D, suggesting that space exists within the binding pocket that can be
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
exploited to enhance potency. Figure 5B summarizes optimal substitutions on the C ring.
Optimization of Linker-A and Linker-B.
There are two regions on the backbone that link the 3 rings together: linkerꢀA and linkerꢀB (Figure 2). In
both the original screening hit (1) and 2, linkerꢀA is an amide linkage. As shown in Table 7, extension of this linker
into a urea (211, 212) or thiourea (211, 212, 213, 214, and 215) leads to a loss of activity for all but one analogue
(
215), which retained only weak potentiating activity (Table 7). Substitution of the amide with a sulfonamide (216,
217) eliminated all activity on GluN1/GluN2C and GluN1/GluN2D receptors (Table 8), as did reduction of the amide
linkage to a tertiary amine (141; Table 8). These data suggest that both the potency and maximal degree of
potentiation of this series is dependent on the amide linkage.
All of the compounds described thus far contained an ether linkage in linkerꢀB. Table 9 shows modifications
to this linker region. When the linker is aliphatic, as in 154, 160, 161, 162, the compounds either show a significant
reduction in potency or are inactive. Similarly, replacing the linkage with a thioether (127) eliminated activity of the
compounds. When the compounds contain unsaturation within linkerꢀB, the compounds appear less soluble and show
varying effects. For example, 156, 158, and 159 all showed increased potency compared to the related compounds
containing an ether linkage (2, 117, and 118, respectively). By contrast, 155 shows decreased potency compared to
1
. Analogues with a single methoxy on the Cꢀring (158 and 159) were similarly potent as analogs with a dimethoxy
(
156 and 157), indicating two different changes (single methoxy, removal of ether) did not have additive effects.
These data suggest modest enhancements in potency can be gained through alterations in the composition of this
linker.
Off-Target Testing
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We subsequently tested the most potent analogue (83) against a variety of other targets. We used two
electrode voltage clamp recordings to assess the ability of 3 ꢀM 83 (10 times the EC ) to inhibit GABA, glycine,
5
0
serotonin, nicotinic acetylcholine, AMPA, kainate, and purinergic receptors. Although compound 83 had a
statistically significant effect on seven of the receptors tested (Table 10), it showed less than 10% inhibition or 5%
potentiation, suggesting compound 83 shows approximately 100ꢀfold selectivity for GluN2C or GluN2D compared
to these receptors. Compound 83 was also submitted to the National Institute of Mental Health Psychoactive Drug
Screening Program (PDSP, http://pdsp.med.unc.edu/) for further offꢀtarget profiling (Supplementary Table S1). In
the PDSP assays less than 50% inhibition of ligand binding was observed with 5 ꢁM compound 83 at serotonin
receptors (5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT , 5ꢀHT ), adrenergic receptors
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
A
1B
1D
1E
2A
2C
3
5A
6
7
(
α , α , α , α , α , α , β , β ), brain and peripheral benzodiazepine sites, dopamine receptors (D , D , D , D ,
1A 1B 1D 2A 2B 2C 2 3 1 2 3 4
D ), opioid receptors (δ and ꢂ) , histamine receptors (H , H , H ), acetylcholine muscarinic receptors (M , M , M ,
5
1
2
3
1
2
3
M , M ), sigma receptors (σ , σ ), as well as serotonin, dopamine, and norepinephrine transporters (n=4 for each).
4
5
1
2
The initial screen determined significant inhibition at the κ opioid receptors, which was studied further. Compound
3 inhibited antagonist binding to kappa opioid with a Ki greater than 5 ꢂM, 15ꢀfold higher than the EC for NMDA
8
5
0
receptor potentiation. The combined data from these offꢀtarget screens highlights the remarkable selectivity of this
series for GluN2Cꢀ and GluN2Dꢀcontaining receptors.
Potency of Stereoisomers.
All compounds in this series thus far have been synthesized and tested as racemic mixtures. We studied the
stereoisomers of compound 2 by first separating the two enantiomers using chiral supercritical fluid chromatography
(
SFC; see Methods). We assessed the activity of each enantiomer in BHK cells that stably express GluN1/GluN2C
2+
or GluN1/GluN2D using the Ca sensitive dye Fluo4 and in oocytes expressing GluN1/GluN2C or GluN1/GluN2D
using two electrode voltage clamp recordings. The activity determined in these two assays suggested that one
enantiomer potentiates the response of GluN1/GluN2C and GluN1/GluN2D receptors to a far greater extent than the
2+
other. In Ca ꢀimaging experiments with BHK cells, the active SFC peak potentiated GluN2C and GluN2D
responses with EC values of 7.1 and 6.6 ꢀM, respectively (N=4). We therefore carried out stereoselectiveꢀsynthesis
50
of the two enantiomers of compound 2 and directly compared the activity of these two enantiomers using two
ACS Paragon Plus Environment

Page 13 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
electrode voltage clamp recordings. The results suggested that the Sꢀ(ꢀ)ꢀenantiomer (>98% ee) of compound 2 (with
72ꢀ74
stereochemistry assigned based on literature precedent
) potentiates the response of GluN2C and GluN2D by
3
04% and 294% with estimated EC values of 9.0 and 8.0 ꢀM, respectively. Like the racemic form, 2-Sꢀ(ꢀ) had no
50
effect on GluN2Aꢀ or GluN2Bꢀcontaining receptors. By contrast, the Rꢀ(+)ꢀenantiomer (90% ee) possessed minimal
activity, which was indistinguishable from that predicted for the 5% residual Sꢀ(ꢀ)ꢀenantiomer (Fig 5C). These data
suggest that the binding pocket preferentially accommodates one enantiomer, and this stereoselectivity may provide a
means to further enhance the selectivity of this compound class over other central nervous system targets. Given the
limited solubility of compound 2, we attribute the slightly reduced potency of the purified enantiomer (8ꢀ9 ꢀM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compared to the racemic mixture (4.6ꢀ5.0 ꢀM) to the greater amount of active species in solution for the purified
enantiomer compared to racemic mixture at concentrations near the limit of solubility. The larger amount of the
active enantiomer in solution before the solubility limit allows a better estimate of the maximum potentiation, which
was higher for the Sꢀ(ꢀ)ꢀisomer (304% and 294% for GluN2C and GluN2D) than for racemic 2 (233% and 215% for
GluN2C and GluN2D). The higher potentiation led to increases in EC values compared to racemic mixture.
5
0
Conclusion
Introduction of a single of Oꢀbenzyl substitution at the R position of the tetrahydroisoquinoline of
4
compound 2 provided compound 83, a GluN2Cꢀ and GluN2Dꢀselective potentiator with 300 nM potency. Moreover,
the majority of the activity attributed to the compounds in this class of tetrahydroisoquinoline potentiators appears to
arise from a single enantiomer, tentatively assigned with Sꢀstereochemistry. This class of potentiators also
demonstrates strong selectivity for GluN2C and GluN2D receptors with the most potent analogue showing no
activity at over 65 receptors, channels, and transporters expressed in the central nervous system. Additionally,
compounds 107, 108, 205 and 203 may represent potential starting point for developing GluN2Cꢀselective
modulators.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biology Experimentals.
Twoꢀelectrode voltageꢀclamp recordings were performed on Xenopus laevis oocytes expressing recombinant
rat GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, GluN1/GluN2D, GluA1, or GluK2 receptors. cDNAs for rat
GluN1ꢀ1a (GenBank accession numbers U11418 and U08261; hereafter GluN1), GluN2A (D13211), GluN2B
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
U11419), GluN2C (M91563), GluN2D (L31611), GluA1 (X17184), GluK2 (Z11548) were provided by Drs. S.
Heinemann (Salk Institute), S. Nakanishi (Kyoto University), and P. Seeburg (University of Heidelberg). Oocyte
7
5
isolation, RNA synthesis, and RNA injection were completed as described in detail elsewhere ; all protocols
involving Xenopus laevis were approved by the Emory University Institutional Animal Care and Use Committee.
During twoꢀelectrode voltageꢀclamp recordings, oocytes were placed into a perfusion chamber and continually
washed with recording solution containing (in mM) 90 NaCl, 1.0 KCl, 0.5 BaCl , 0.005 EDTA, and 10 HEPES at pH
2
o
7
.4 (23 C). Glass electrodes with a tip resistance of 0.3ꢀ1.0 Mꢃ were pulled from thinꢀwalled glass capillary tubes
and filled with 3.0 M KCl. An OCꢀ725C amplifier (Warner Instruments) was used to hold the membrane potential of
the oocytes at ꢀ40 mV during current recording. All compounds were made as 20 mM stock solutions in DMSO, and
dissolved to reach the desired final concentration in recording solution containing 100 ꢂM glutamate and 30 ꢂM
glycine for use on oocytes expressing NMDA receptors. Final DMSO content was 0.05ꢀ0.5% (vol/vol). Oocytes
expressing GluK2 receptors were preꢀtreated with 10 ꢀM concanavalin A for 10 minutes. Recombinant GluA1 and
GluK2 receptors were activated by 100 ꢀM glutamate. In order to prevent a gradual increase in current response
over the course of the experiment, which is a common feature of GluN1/GluN2A receptor responses in oocytes, some
oocytes expressing GluN1/GluN2A were either pretreated for 10 minutes with 50 ꢀM BAPTAꢀAM (1,2ꢀbis(2ꢀ
aminophenoxy)ethaneꢀN,N,N',N'ꢀtetraacetic acid tetrakis(acetoxymethyl ester)) or injected with 50 nl of 2 mM Kꢀ
BAPTA (potassium 1,2ꢀbis(2ꢀaminophenoxy)ethaneꢀN,N,N',N'ꢀtetraacetic acid). For every test compound, we
recorded 5ꢀ7 concentrations in at least 5 oocytes obtained from two different frogs. We subsequently determined
mean potentiation at 10 ꢀM (+SEM), and when potentiation exceeded 115% of control we determined the EC (halfꢀ
50
maximally effective concentration of potentiator) by fitting the equation
N
Response = maximum + (100 – maximum) / (1 + ( [concentration] / EC ) )
(1)
5
0
ACS Paragon Plus Environment

Page 15 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to the average composite concentrationꢀresponse data normalized to the current in the absence of potentiator (100%).
N is the Hill slope, which ranged between 1 and 2 and is not reported; maximum is the maximal response predicted
for saturating concentration of potentiator. A few compounds produced modest inhibition (70% of controls at 100
ꢂM); these inhibitory actions were not studied further.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For offꢀtarget data, GABA (ρ1), glycine (α1), serotonin (5ꢀHT ), nicotinic acetylcholine (nAChR, α1β1δγ,
C
3A
α4β2, α3β4, α9α10, α7), and purinergic (P2 rat, P2 human) receptors were expressed in Xenopus oocytes.
X2
X2
cRNA encoding the receptor subunits was synthesized from linear template cDNA using the mMessage mMachine
kit (Ambion). Oocytes were injected with 1ꢀ5 ng cRNA in a 50 nL volume and incubated in Barth's solution at 15°C
for 2ꢀ5 days prior to recording. α1β1δγ nAChR subunits were injected at a 1:1:1:1 ratio while α4β2, α3β4, and
α9α10 nAChR subunits were injected at a 1:1 ratio. The cDNAs encoding GABA , glycine, and serotonin subunits
C
were provided by Dr. D. Weiss (University of Texas Health Science Center at San Antonio). cDNAs encoding
nicotinic acetylcholine receptor subunits were provided by Drs. R. Papke (University of Florida) and S. Heinemann
(
Salk Institute). cDNAs encoding the purinergic receptors were provided by Dr. R. Hume (University of Michigan).
GABA receptors were activated by 2 ꢀM GABA. Acetylcholine was used at the indicated concentrations (in ꢂM) to
C
activate the nicotinic acetylcholine receptors: α1β1δγ (1), α4β2 (10), α3β4 (10), α9α10 (1), α7(300). 50 ꢀM glycine
was used to activate the glycine receptor, 100 ꢀM serotonin was used to activate 5ꢀHT3A receptor, and 9 ꢀM ATP
was used to activate the purinergic receptors. K determinations and receptor binding profiles were generously
i
provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSNꢀ271ꢀ
2008ꢀ00025ꢀC (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North
Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA. For experimental details
please refer to the PDSP web site http://pdsp.med.unc.edu/
2+
76
Ca imaging experiments were performed as described previously with the following modifications. One
day prior to the experiment, the cells were seeded in 20 ꢁl media at 600,000 cells/ml in black clear bottom 384 well
plates (Corning CellBind). On the day of the experiment, the media was aspirated and replaced with Flouꢀ4 NW
(Invitrogen) dissolved in HEPESꢀbuffered saline (HBSS, Gibco #14175ꢀ053) comprised of (in mM) 5.33 KCl, 0.441
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
KH PO , 4.17 NaHCO , 137.9 NaCl, 0.338 Na HPO , 5.56 Dꢀglucose, 2 CaCl , and 20 HEPES (pH 7.4), with 2.5
2
4
3
2
4
2
mM (1%) Probenecid, and 30 ꢁM 7ꢀchlorokynurenic acid for 60 minutes at 37° C in the dark. Cells were then gently
washed with 30 ꢁl/well using the same buffer without the Flouꢀ4 dye, and placed in 20 ꢁl/well buffer. Using a
FDSS7000 instrument (Hamamatsu) real time recordings of changes in Fluoꢀ4 emission was performed (excitation
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
80 nm and emission 540 nm) at room temperature (20ꢀ22° C). After 10 seconds of baseline recordings, 10 ꢂl/well of
3x concentrated test compound, controls, or assay buffer in HBSS (pH 7.4) and 1 mM glycine (final concentration)
were added. After 2 minutes, an additional 10 ꢂl/well were added containing a 4x concentrated solutions of NMDA
(
GluN2C: 1000 ꢂM and GluN2D: 300 ꢂM). Changes in fluorescence were recorded for subsequent 2 minutes. For
determination of the concentrationꢀresponse relationships, test compounds were serial diluted 3ꢀfold over 10
concentration steps. Responses (fluorescence units, FU) were normalized to the first recording and expressed as
percent of maximally effective concentration of NMDA (see above). The EC value was determined by nonꢀlinear
50
least squares fitting of equation 1 to the data.
The maximum solubility (20 ꢀM) was determined for compound 2 using a BMG Labtech Nephelostar
nephelometer (Offenburg, Germany), according to manufacturer’s instructions. Only responses for concentrations
below the experimentally determined limit of solubility were measured; whenever visual evidence of precipitation at
higher concentrations was observed, experiments were repeated with 1ꢀ10 mM 2ꢀhydroxypropylꢀβꢀcyclodextrin
added to the recording solution to ensure that the compounds remained in solution. 2ꢀhydroxypropylꢀβꢀcyclodextrin
had no detectable effect on NMDA receptor response amplitude (data not shown). We also evaluated the stability of
potentiator activity by making a solution of 10 ꢂM 2 and continuously agitating it. After increasing periods of time,
some of the solution was filtered (0.2 ꢂm nylon filter) and used to measure potentiation of GluN1/GluN2D receptors
expressed in X. laevis oocytes. Similarly, we tested the concentrationꢀresponse effects of 2 on maximal responses
induced by NMDA on GluN1/GluN2C in BHK cells by measuring changes in fluorescence of Fluoꢀ4 at different
time points after making the compound solutions. Activity (potency and efficacy) was retained without loss until 6
hours, whereas no activity was seen after 24 hours. Results from these two experiments suggested that compound 2
remains active in solution up to 2ꢀ3 hours, with activity decreasing thereafter until by 24 hours there is no detectable
activity.
ACS Paragon Plus Environment

Page 17 of 82
Journal of Medicinal Chemistry
1
2
3
4
The enantiomers of 2 were obtained by supercritical fluid chromatography on a Berger Multigram II
operating at 50 ml/min at 35 °C and 100 bar backpressure. The column was a Phenomenex Lux 5u Celluloseꢀ1 (250
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
x 21.2 mm). The eluent was CO (60 %) and methanol + 0.1 % diethylamine (40%). 15 mg were dissolved in
2
methanol and 200 stacked injections of 0.4 ml were performed. Enantiomeric excess was determined on an Aurora
Fusion A5 SFC system operating at 3 ml/min at 40 °C and 100 bar backpressure. The column was a Phenomenex
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Lux 3u Celluloseꢀ1 (150 x 4.6 mm). The eluent was CO (70 %) and ethanol + 0.1 % diethylamine (30%). EE of
2
peak 1 (rt 1.584 min) determined at 220 nM 98.3 %. Peak 2 (rt 2.708 min) 98.2 %.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Chemistry Experimentals. Compounds not described were purchased from either Life Chemicals (Compounds
1
1
84, 187, 189-194, 196-197, 200-201, 203-206, 208, 210-217) or Chem Div (Compounds 2, 185-186, 188, 195,
98, 127, 207, 209)). All purchased compounds were greater than 90% pure, as determined by the suppliers, by
HPLC or NMR.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All reagents for synthesis were obtained from commercial suppliers and used without further purification.
Reaction progress was monitored by thin layer chromatography (TLC) on precoated aluminum plates (silica gel 60
F254, 0.25 mm). Proton and carbon NMR spectra were recorded on an INOVAꢀ400 (400 MHz) or VNMRS 400
(
400 MHz). The spectra obtained were referenced to the residual solvent peak. Mass spectra were performed by the
Emory University Mass Spectroscopy Center on either a VG 70ꢀS Nier Johnson or JEOL instrument. Elemental
analyses were performed by Atlantic Microlab Inc. Purity was established using HPLC, unless indicated by
combustion analysis, and were found to be ≥ 95%. C, H, N agreed with proposed structures ±0.4% of theoretical
o
values unless indicated. Optical activity was measured at 20 C with a PerkinꢀElmer model 341 polarimeter. Flash
chromatography was performed on a Teldyne ISCO CombiFlash Companion System with prepackaged Teledyne
RediSep or Silicycle normal phase columns with silica gel.
General preparation for 1,2,3,4-tetrahydroisoquinoline compounds (Procedure I). 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline hydrochloride (1.0 g, 4.4 mmol) was dissolved in DCM (20 mL) and saturated aqueous
o
sodium bicarbonate solution (20 mL). The biphasic reaction mixture was cooled to 0 C in an ice bath and benzoyl
chloride (3 equiv) was added dropwise. After complete addition, the reaction was warmed to room temperature and
stirred for an additional 2 hours, when TLC indicated complete conversion. The organics were separated and the
aqueous phase was extracted with DCM (2x). The organics were combined, washed with brine and water, dried over
MgSO4, filtered and concentrated in vacuo. The resulting residue was subjected to flash column chromatography.
General preparation for 2-chloro-N-phenethylacetamide compounds (Procedure II). A solution of a
o
phenethylamine (1.0 equiv) in DCM was cooled to 0 C in an ice bath. To this solution, Et N (3.0 equiv) was added
3
followed by dropwise addition of an acid chloride (1.2 equiv). Upon complete addition, the reaction was warmed to
room temperature and stirred for 2 hours. When TLC indicated complete conversion, the reaction mixture was
ACS Paragon Plus Environment

Page 19 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrated in vacuo. The resulting residue was taken up into DCM and washed with a saturated solution of NH Cl
4
and brine. The aqueous layer was then extracted with DCM (2x). The organic layers were combined, washed with
water, dried over MgSO and filtered. The solvent was removed in vacuo. The resulting solid was subjected to flash
4
column chromatography.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General preparation for N-(phenethyl)-2-(phenoxy)acetamide and N-(phenethyl)-2-(phenyl)thio)acetamide
compounds (Procedure III). To a solution of phenol or thiophenol (7.3 mmol) in MeCN (15 mL) was added
Cs CO ( 3.0 equiv.) at room temperature and the reaction mixture was allowed to stir for 2 hours. A solution of the
2
3
αꢀchloroamide (1.2 equiv.) dissolved in dry MeCN (15 mL) was added and the resulting reaction mixture was stirred
for 18 h under an argon atmosphere. After TLC indicated complete conversion, the volatiles were removed in vacuo
and the resulting residue was treated with NH Cl and extracted into EtOAc (2x). The resulting organic layer was
4
washed with brine and water, dried over MgSO , filtered, and concentrated in vacuo. The resulting solid was
4
subjected to flash column chromatography.
General preparation for 3,4-dihydroisoquinoline compounds (Procedure IV). To a solution of 3 (3.5 mmol)
suspended in toluene (40 mL) was added POCl (3.0 equiv.), dropwise. The reaction mixture was brought to reflux
3
and allowed to stir for 90 minutes before cooling to room temperature. The resulting precipitate was filtered and
carried on without further purification.
General preparation for 3,4-dihydroisoquinoline compounds (Procedure V). The amide (1.0 equiv) was
dissolved in dry toluene (30 mL) and brought to reflux. Phosphorus pentoxide (7.0 equiv) was added to the refluxing
solution over about 15 minutes. The reaction was refluxed for an additional 30 minutes. The toluene was decanted
and the remaining residue was dissolved in water and washed with ether (2x). The aqueous solution was treated with
NH OH and extracted into DCM (3x). The organic layer was separated, dried over MgSO , filtered and concentrated
4
4
in vacuo. The crude product was taken on without further purification.
General preparation for 1,2,3,4-tetrahydroisoquinoline compounds (Procedure VI). The dihydroisoquinoline 4
o
(
4.0 mmol) was suspended in anhydrous MeOH (40 mL). The reaction mixture was cooled to 0 C in an ice bath.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NaBH (3.0ꢀ4.0 equiv.) was added slowly to the reaction mixture under an argon atmosphere. It was then allowed to
4
warm to room temperature and stirred for 6 hours to overnight, until TLC indicated complete conversion. Volatiles
were removed in vacuo. The resulting residue was dissolved in DCM and washed with 1 N HCl, water, and brine.
The aqueous layer was extracted with DCM (2x). The organic layers were combined, dried over MgSO and filtered.
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Volatiles were removed in vacuo. The crude residue was subjected to flash column chromatography.
General preparation for 3,4-dihydroisoquinolin-2(1H)-yl)methanone compounds (Procedure VII). The
o
tetrahydroisoquinoline 5 (1 mmol) was dissolved in anhydrous DCM (10 mL). The solution was cooled to 0 C in an
ice bath. Triethylamine (3.0 equiv.) was added to the cooled solution followed by dropwise addition of a benzoyl
chloride (1.2 equiv.). The reaction mixture was allowed to warm to room temperature and stirred for an additional 90
minutes. Volatiles were removed in vacuo. The resulting residue was treated with NH Cl saturated solution
4
followed by extraction with DCM (2x). The organic phase was washed with brine and water, dried over MgSO4,
filtered, and concentrated to yield a residue. The resulting residue was subjected to column chromatography to afford
the final products as a mixture of two rotamers.
General preparation for 3,4-dihydroisoquinolin-2(1H)-yl)methanone compounds (Procedure VIII). Benzoic
o
acid (1.0ꢀ1.3 equiv) was dissolved in dry DCM (15 mL) and cooled to 0 C in an ice bath. DMAP (1.2 equiv) and
o
EDC (1.2 equiv) were added and the reaction was stirred at 0 C for 2 hours. Tetrahydroisoquinoline (1.0 equiv)
dissolved in DCM (10 mL) was added to the cooled reaction and it was warmed to room temperature and stirred for
an additional 18 hours. When TLC indicated complete conversion, 1M HCl was added and the aqueous phase was
extracted with DCM (2x). The organics were combined and washed with brine and water, dried over MgSO4,
filtered and concentrated in vacuo. The resulting residue was subjected to column chromatography to afford the final
products as a mixture of two rotamers.
General preparation of N-(phenethyl)-3-phenylpropanamide and (E)-N-(phenethyl)-3-(phenyl)acrylamide
compounds (Procedure IX). Phenylpropionic acid (6.7 mmol, 1.0 equiv) or cinnamic acid (5.6 mmol, 1.0 equiv)
o
was dissolved in dry DCM (20 mL) and DMF (10 mL) and the reaction mixture was brought to 0 C in an ice bath.
o
EDC (1.3 equiv) and DMAP (1.1 equiv) were added and the reaction mixture was stirred at 0 C for 2 hours. 3,4ꢀ
ACS Paragon Plus Environment

Page 21 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dimethoxyphenethylamine (6.7 mmol, 1.0 equiv) was added dropwise. The solution was warmed to room
temperature and stirred overnight. After TLC indicated complete conversion, 1M HCl was added and the reaction
mixture was extracted into DCM (3x). The organics were combined, washed with brine and water, dried over
MgSO , filtered and concentrated in vacuo. The resulting solid was subjected to flash column chromatography.
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)(phenyl)methanone (5). Compound 5 was prepared according to
Procedure I using benzoyl chloride (1.5 mL, 13 mmol, 3.0 equiv). The crude residue was purified by silica gel
chromatography (ISCO, Redisep 12 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a
1
white solid (mixture of rotamers, 0.9 g, 67%). H NMR (CDCl , 400 MHz) δ: 7.45ꢀ7.39 (m, 7H), 6.62ꢀ6.38 (m, 1H),
3
1
3
4
.81 (m, 1H), 3.97 (m, 1H), 3.86 (s, 6H), 3.76 (m, 1H), 3.60 (m, 1H), 2.81 (m, 1H). C NMR (CDCl , 100 MHz) δ:
3
1
71.1, 170.6, 147.9, 136.4, 129.9, 128.7, 127.4, 127.0, 125.8, 125.0, 111.5, 109.6, 108.8, 56.2, 56.1, 49.8, 45.6, 44.7,
+
+
29.3, 28.1. HRMS calcd. for C H NO , 298.14377 [M + H] ; found, 298.14362 [M + H] .
18
20
3
(3-chlorophenyl)(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone (6). Compound 6 was prepared
according to Procedure I using 3ꢀchlorobenzoyl chloride (1.7 mL, 13 mmol, 3.0 equiv). The crude residue was
purified by silica gel chromatography (ISCO, Redisep 24 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the
1
title compound as a white solid (mixture of rotamers, 1.1 g, 76%). H NMR (CDCl , 400 MHz) δ: 7.51ꢀ7.31 (m, 4H),
3
1
3
6
.65ꢀ6.39 (m, 2H), 4.80 (s, 1H), 3.96 (m, 1H), 3.84 (s, 6H), 3.77 (m, 1H), 3.60 (m, 1H), 2.88 (m, 1H). C NMR
(CDCl , 100 MHz) δ: 169.6, 169.2, 148.2, 137.9, 134.8, 133.2, 130.5, 129.9, 127.3, 125.6, 125.2, 124.7, 111.6,
3
+
1
09.5, 108.8, 56.2, 56.1, 49.7, 45.6, 44.7, 29.3, 27.9. HRMS calcd. for C H NO Cl , 332.10480 [M + H] ; found,
1
8
19
3
+
3
32.10483 [M + H] .
(
3-bromophenyl)(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone (7). Compound 7 was prepared
according to Procedure I using 3ꢀbromobenzoyl chloride (1.7 mL, 13 mmol, 3.0 equiv). The crude residue was
purified by silica gel chromatography (ISCO, Redisep 24 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the
1
title compound as a white solid (1.4 g, 85%). H NMR (CDCl , 400 MHz) δ: 7.56ꢀ7.52 (m, 2H), 7.30 (d, J= 7.6 Hz,
3
1
H), 7.29ꢀ7.24 (m, 1H), 6.63ꢀ6.68 (m, 2H), 4.76 (m, 1H), 4.45 (m, 1H), 3.93 (m, 1H), 3.82 (s, 6H), 3.75 (m, 1H),
13
3
.57 (m, 1H), 2.81 (m, 1H). C NMR (CDCl , 100 MHz) δ:169.4, 148.2, 138.2, 133.1, 130.4, 130.2, 126.6, 125.9,
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
25.6, 124.7, 124.4, 122.9, 111.7, 109.5, 56.2, 56.1, 54.1, 49.7, 45.6, 44.7, 29.2, 28.1. HRMS calcd. for
+
+
C H NO Br , 376.05428 [M + H] ; found, 376.05471 [M + H] .
18
19
3
(
6-(benzyloxy)-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(phenyl)methanone (81).
Compound 81 was prepared according to Procedure VII using tetrahydroisoquinoline 63 (0.2 g, 0.53 mmol) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzoyl chloride (0.09 g, 0.07 mL, 0.64 mmol, 1.2 equiv). The crude residue was purifed by silica gel
chromatography (ISCO, Redisep 4 g column, 0ꢀ60% EtOAc/hexanes gradient) to give the title compound as an offꢀ
1
white amorphous solid (mixture of rotamers, 0.18 g, 71%). H NMR (CDCl , 400 MHz) δ: 7.52ꢀ7.22 (m, 10H), 6.93ꢀ
3
6
.71 (m, 7H), 1H [6.01, 5.19 (t, J = 4.4 Hz; dd, J = 3.8 Hz, J = 8.6 Hz)], 5.05 (s, 2H), 1H [4.88, 4.10 (dd, J = 5.4
1 2 1
13
Hz, J = 13 Hz; t, J = 9.6 Hz), 4.36 (d, J = 4.4 Hz, 1H), 3.93ꢀ3.66 (m, 4H), 3.27ꢀ3.15 (m, 1H), 3.93ꢀ2.70 (m, 2H).
C
2
NMR (CDCl , 100 MHz) δ: 172.0, 171.3, 158.4, 157.9, 154.2, 153.1, 152.7, 137.0, 136.8, 136.7, 136.6, 135.9, 129.8,
3
129.7, 128.9, 128.6, 128.3, 127.7, 126.8, 126.5, 126.1, 125.1, 115.9, 115.6, 115.4, 115.2, 114.9, 113.8, 113.6, 96.1,
+
7
1.4, 70.4, 70.2, 57.2, 56.0, 51.8, 42.9, 35.4. 30.1, 28.7. HRMS calcd. for C H NO , 480.21694 [M + H] ;
3
1
30
4
+
found,480.21792 [M + H] .
(
(
6-(benzyloxy)-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-chlorophenyl)methanone
82). Compound 82 was prepared according to Procedure VII using tetrahydroisoquinoline 63 (0.2 g, 0.53 mmol)
and 3ꢀchlorobenzoyl chloride (0.1 g, 0.08 mL, 0.64 mmol, 1.2 equiv). The crude residue was purified by silica gel
chromatography (ISCO, Redisep 4 g column, 0ꢀ60% EtOAc/hexanes gradient) to give the title compound as an off
1
white amorphous solid (mixture of rotamers, 0.20 g, 70%). H NMR (CDCl , 400 MHz) δ: 7.59ꢀ7.21 (m, 9H), 6.93ꢀ
3
6
.76 (m, 7H), 1H [5.98, 5.14 (t, J = 4.8 Hz; dd, J = 4.4 Hz, J = 9.2 Hz)], 5.04 (s, 2H), 1H [4.85, 4.13 (dd, J = 5.2
1 2 1
1
3
Hz, J = 13 Hz; m)], 4.35 (m, 1H), 3.94ꢀ3.67 (m, 4H), 3.26ꢀ2.73 (m, 3H). C NMR (CDCl , 100 MHz) δ: 170.5,
2
3
1
69.7, 168.5, 158.5, 158.0, 154.4, 153.0, 152.6, 138.3, 137.1, 137.0, 136.7, 135.7, 134.9, 134.6, 133.4, 130.3, 129.9,
128.9, 128.4, 127.7, 127.1, 125.8, 125.7, 124.9, 124.7, 115.9, 115.5, 115.3, 114.9, 113.9, 113.7, 71.3, 70.2, 57.3,
+
+
5
6.0, 52.0, 42.9, 35.5, 30.0, 28.6. HRMS calcd. for C H NO Cl, 514.17796 [M + H] ; found, 514.17923 [M + H] .
3
1
29
4
(
(
6-(benzyloxy)-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-bromophenyl)methanone
83). Compound 83 was prepared according to Procedure VII using tetrahydroisoquinoline 63 (0.2 g, 0.53 mmol)
ACS Paragon Plus Environment

Page 23 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 3ꢀbromobenzoyl chloride (0.1 g, 0.08 mL, 0.64 mmol, 1.2 equiv). The crude residue was purified by silica gel
chromatography (ISCO, Redisep 4 g column, 0ꢀ60% EtOAc/hexanes gradient) to give the title compound as an offꢀ
1
white amorphous solid (mixture of rotamers, 0.21 g, 71%). H NMR (CDCl , 400 MHz) δ: 7.56ꢀ7.21 (m, 9H), 6.94ꢀ
3
6
.76 (m, 7H), 1H [5.98, 5.13 (t, J = 4.7 Hz; dd, J = 2.6 Hz, J = 9.4 Hz)], 5.05 (s, 2H), 1H [4.85, 4.13 (m, m)], 4.34
1 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
(m, 1H), 3.93ꢀ3.67 (m, 4H), 3.26ꢀ2.72 (m, 3H). C NMR (CDCl , 100 MHz) δ: 170.3, 169.6, 158.5, 158.0, 154.4,
3
1
1
4
53.1, 152.6, 138.6, 138.4, 136.9, 136.7, 135.7, 132.9, 132.8, 131.1, 130.5, 130.2, 129.9, 129.0, 128.9, 128.3, 127.7,
26.3, 125.7, 125.3, 124.7, 123.0, 122.7, 115.9, 115.5, 115.3, 115.0, 114.9, 114.0, 113.7, 71.3, 70.2, 57.3, 56.0, 52.0,
+
+
2.9, 35.5, 30.0, 29.0. HRMS calcd. for C H NO Br, 558.12745 [M + H] ; found, 558.12878 [M + H] .
31
29
4
(
6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-nitrophenyl)methanone
(
84). Compound 84 was prepared according to Procedure VII using 63 (0.2 g, 0.6 mmol, 1.0 equiv) and 3ꢀ
nitrobenzoyl chloride (0.1 g, 0.6 mmol, 1.0 equiv). The crude material was purified by silica gel chromatography
(
ISCO, Redisep 4 g column, 0ꢀ60% EtOAc/hexanes) to afford the title compound as a white amorphous solid (0.1,
1
3
3%, mixture of rotamers). H NMR (CDCl , 400 MHz) δ: 1H [8.57, 8.30 (s, s)], 8.28 (d, J = 7.6 Hz, 1H), 1H [7.86,
3
7
.74 (d, J = 8.0 Hz; d, J = 7.2 Hz), 7.64ꢀ7.57 (m, 1H), 6.89 (d, J = 8.8 Hz, 1H), 6.86ꢀ6.81 (m, 3H), 1H [ 6.70, 6.65 (s,
s)], 1H [6.46, 5.98 (s, m)], 1H [5.04, 4.90 (dd, J = 3.4 Hz, J = 9.8 Hz; dd, J = 5.6 Hz, J = 13.2 Hz)], 4.39 (d, J =
1
2
1
2
13
5.2 Hz, 1H), 4.23ꢀ3.97 (m, 1H), 3.88ꢀ3.74 (m, 9H), 3.74ꢀ3.72 (m, 1H), 3.30ꢀ3.12 (m, 1H), 2.91ꢀ2.71 (m, 2H).
C
NMR (CDCl , 100 MHz) δ: 169.3,168.5, 154.5, 152.9, 152.3, 149.0, 148.6, 148.2, 147.9, 138.1, 133.9, 133.0, 130.2,
3
1
5
29.8, 127.2, 126.0, 124.9, 124.5, 123.5, 122.1, 115.9, 115.4, 115.0, 112.1, 111.5, 110.3, 109.8, 71.0, 69.9, 57.7,
+
6.3, 56.2, 55.9, 52.2, 43.1, 35.7, 29.2, 27.7. HRMS calcd. for C H N O , 479.18128 [M + H] ; found, 479.18062
2
6
27
2
7
+
[
M + H] .
(
3,4-dichlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (85). Compound 85 was prepared according to Procedure VII using 64 (0.2 g, 0.6 mmol) and 3,4ꢀ
dichlorobenzoyl chloride (0.2 g, 0.7 mmol, 1.2 equiv). The crude material was purified by silica gel chromatography
(
(
ISCO, Redisep 4g column, 0ꢀ70% EtOAc/hexanes) to afford the title compound as a white amorphous solid
1
mixture of two rotamers, 0.2 g, 67%). H NMR (CDCl , 400 MHz) δ: 7.74ꢀ7.44 (m, 2H), 7.37ꢀ7.20 (m, 1H), 6.86ꢀ
3
6
.77 (m, 4H), 1H [6.66, 6.60 (s, s)], 1H [6.47, 5.93 (s, m)], 1H [5.07, 5.05 (dd, J = 3.6 Hz, J = 9.2 Hz; dd, J = 6.0
1 2 1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hz, J = 13 Hz)], 4.34 (d, J = 4.0 Hz, 1H), 1H [4.16, 3.96 (t, J = 10.0 Hz; dd, J = 4.0 Hz, J = 10.0 Hz)], 3.86ꢀ3.74
2
1
2
13
(
m, 10H), 1H [td, J = 4.0 Hz, J = 13 Hz; td, J = 5.2 Hz, J = 10.0 Hz), 2.92ꢀ2.66 (m, 2H). C NMR (CDCl , 400
1 2 1 2 3
MHz) δ: 169.6, 154.5, 154.3, 152.9, 152.4, 149.0, 148.5, 148.1, 136.3, 134.0, 132.9, 131.0, 130.7, 130.2, 127.2,
26.1, 125.0, 123.8, 115.9, 115.5, 114.9, 112.0, 111.5, 109.8, 96.8, 57.6, 56.3, 56.2, 56.0, 43.0, 35.8, 29.2, 27.7.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
HRMS calcd. for C H NO Cl , 502.11826 [M + H] ; found, 502.11841 [M + H] . Anal. (C H NO Cl ): C, H, N.
2
6
26
5
2
26 25
5
2
(2,3-dichlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (86). Compound 86 was prepared according to Procedure VIII using 64 (0.2 g, 0.6 mmol, 1.0 equiv)
and 2,3ꢀdichlorobenzoic acid (0.1 g, 0.6 mmol, 1.0 equiv). The crude material was purified by silica gel
chromatography (ISCO, Redisep 4g column, 0ꢀ70% EtOAc/hexanes) to afford the title compound as a white
1
amorphous solid (mixture of two rotamers, 0.1 g, 33%). H NMR (CDCl , 400 MHz) δ: 7.50ꢀ7.45 (m, 1H), 7.30ꢀ7.22
3
(m, 1H), 1H [7.19, 7.12 (dd, J = 1.6 Hz, J = 5.2 Hz; dd, J = 1.6 Hz, J = 5.2 Hz)], 6.87ꢀ6.83 (m, 1H), 6.83ꢀ6.70 (m,
1
2
1
2
3
H), 6.67ꢀ6.60 (m, 1H), 6.50ꢀ5.92 (m, 1H), 1H [4.93, 4.76 (m, m)], 4.41ꢀ4.32 (m, 1H), 1H [4.08, 3.93 (t, J =10.0 Hz;
1
13
dd, J = 4.2 Hz, J = 10.0 Hz), 3.86ꢀ3.72 (m,9H), 3.60ꢀ3.44 (m, 1H), 3.27ꢀ2.96 (m, 1H), 2.84ꢀ2.64 (m, 2H).
C
1
2
NMR (CDCl , 100 MHz) δ: 174.1, 167.2, 166.8, 154.2, 152.8, 152.6, 148.4, 148.0, 147.8, 138.2, 133.8, 133.2, 130.9,
3
1
28.2, 127.2, 126.4, 126.2, 125.7, 125.1, 124.8, 124.0, 115.8, 115.4, 114.8, 111.9, 111.5, 109.9, 81.3, 70.9, 70.5,
+
56.3, 56.1, 55.9, 51.8, 42.4, 29.0, 28.0. HRMS calcd. for C H NO Cl , 502.11826 [M + H] ; found, 502.11832[M
2
6
26
5
2
+
+
H] . Anal. (C H NO Cl ): C, H, N.
26 25 5 2
(
3,5-dichlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (87). Compound 87 was prepared according to Procedure VII using 64 (0.11 g, 0.33 mmol) and 3,5ꢀ
dichlorobenzoyl chloride (0.08 g, 0.37 mmol. 1.1 equiv). The crude material was purified by silica gel
chromatography (ISCO, Silicycle 4 g column, 0ꢀ60% EtOAc/hexanes gradient) to afford the title compound as a
1
white amorphous solid (mixture of rotamers, 0.06 g, 36%). H NMR (CDCl , 400 MHz) δ: 7.51 (s, 1H), 7.42 (s, 1H),
3
7
.25 (m, 1H), 6.9ꢀ6.79 (m, 4H), 1H [6.68, 6.64 (s, s)], 1H [6.51, 5.94 (s, m)], 1H [5.06, 4.85 (dd, J = 2.8 Hz, J = 9.6
1 2
Hz; dd, J = 5.6 Hz, J = 13.2 Hz)], 4.36 (d, J = 4.4 Hz, 1H), 1H [4.21, 3.99 (t, J = 10.0 Hz; dd, J = 3.6 Hz, J = 10.0
1
2
1
2
13
Hz), 3.89ꢀ3.73 (m, 9H), 3.72ꢀ3.68 (m, 1H), 3.27ꢀ3.08 (m, 1H), 2.94ꢀ2.70 (m, 2H). C NMR (CDCl , 100 MHz) δ:
3
1
68.2, 154.5, 154.4, 149.0, 148.5, 148.1, 147.9, 139.2, 135.7, 135.3, 129.9, 129.8, 127.2, 126.5, 126.1, 125.3, 124.9,
ACS Paragon Plus Environment

Page 25 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
23.7, 115.9, 115.4, 115.0, 114.9, 112.0, 111.5, 110.3, 109.8, 71.0, 70.1, 57.6, 56.3, 56.2, 55.9, 52.1, 42.9, 35.8, 29.2,
+
+
7.7. HRMS calcd. for C H NO Cl , 502.11826 [M + H] ; found, 502.11748 [M + H] .
26
26
5
2
(
3-chloro-4-fluorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (88). Compound 88 was prepared according to Procedure VIII using 64 (0.1 g, 0.3 mmol) and 3ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chloroꢀ4ꢀfluorobenzoic acid (0.06 g, 0.4 mmol, 1.2 equiv). The crude material was purified by silica gel
chromatography (ISCO, Redisep 4 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a
1
white amorphous solid (mixture of rotamers, 0.10 g, 68%). H NMR (CDCl , 400 MHz) δ: 8.08ꢀ7.74 (m, 1H), 7.46ꢀ
3
7
.13 (m, 2H), 6.88ꢀ6.80 (m, 4H), 6.72ꢀ6.63 (m, 1H) 1H [6.50, 5.95 (s, m)], 1H [5.10, 4.84 (m, dd, J = 5.2 Hz, J = 12
1 2
Hz)], 4.36 (m, 1H), 4.22ꢀ3.97 (m, 1H), 3.87ꢀ3.76 (m, 9H), 3.73ꢀ3.66 (m, 1H), 3.30ꢀ3.07 (m, 1H), 2.88ꢀ2.70 (m, 2H).
1
3
C NMR (CDCl , 400 MHz) δ: 169.7, 154.5, 152.9, 152.5, 148.9, 148.5, 148.1, 147.8, 133.5, 130.8, 127.3, 126.1,
3
125.1, 123.8, 115.9, 115.5, 114.9, 112.0, 109.8, 70.3, 57.7, 56.2, 56.1,55.9, 43.0, 35.8, 29.9, 29.2, 27.7, 15.5. HRMS
+
(
m/z): [M] calcd. for C H NO ClF , 486.14781; found, 486.14826. Anal. (C H NO ClF ) C, H, N.
26
26
5
26 25
5
6
,7-dimethoxy-1-(phenoxymethyl)-3,4-dihydroisoquinolin-2(1H)-yl(phenyl)methanone (89). Compound 89 was
prepared according to Procedure VII using 79 (0.6 g, 2 mmol) and benzoyl chloride (0.4 g, 0.3 mL, 3 mmol, 1.3
equiv). The crude residue was purified by silica gel chromatography (ISCO, Redisep 12 g column, gradient 0ꢀ60%
EtOAc/hexanes) to afford the title compound as a white amorphous solid (mixture of two rotamers, 0.8 g, 99%
1
yield). H NMR (CDCl , 400 MHz) δ: 7.50 (m, 1H), 7.38 (m, 4H), 7.23 (m, 2H), 6.94ꢀ6.89 (m, 2H), 1H [6.81, 6.60
3
(
s, s)], 1H [6.78, 6.66 (s, s)], 1H [6.47, 5.93 (s, m)], 1H [5.16, 4.84 (dd, J = 4.0 Hz, J = 9.0 Hz; dd, J = 6.0 Hz, J
1 2 1 2
=
9.4 Hz), 4.39 (m, 1H), 1H [4.18, 3.99 (m, m)], 3.84ꢀ3.75 (m, 6H), 1H [3.78, 3.63 (m, m)], 3.28ꢀ3.02 (m, 1H), 2.91ꢀ
13
2
.61 (m, 2H). C NMR (CDCl , 100 MHz) δ: 171.9, 171.2, 158.9, 158.5, 148.8, 148.4, 148.0, 147.7, 136.6, 129.8,
3
1
28.8, 128.6, 127.7, 127.5, 126.8, 126.5, 125.4, 124.3, 121.5, 121.3, 114.9, 114.6, 112.1, 111.6, 110.5, 110.0, 70.3,
+
69.6, 57.2, 56.2, 56.1, 51.7, 43.0, 35.7, 29.3, 27.9. HRMS calcd. for C H NO , 404.18564 [M + H] ; found,
2
5
25
4
+
4
04.18538 [M + H] . Anal. (C H NO ): C, H, N.
25 24 4
(
6,7-dimethoxy-1-((3-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(phenyl)methanone (90).
Compound 90 was prepared according to Procedure VII using 65 (0.2 g, 0.6 mmol, 1.0 equiv) and benzoyl chloride
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
0.1 g, 0.7 mmol, 1.2 equiv, 0.08 mL). The crude residue was purified by silica gel chromatography (ISCO, Redisep
4
g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a white amorphous solid (mixture of
1
rotamers, 0.18 g, 60%). H NMR (CDCl , 400 MHz) δ: 8.06ꢀ7.12 (m, 6H), 6.80ꢀ5.98 (m, 4H), 1H [5.16, 4.89 (dd, J
3
1
13
=
4.0 Hz, J = 8.8 Hz; dd, J = 6.0 Hz, J = 12 Hz)], 4.40ꢀ3.96 (m, 2H), 3.85ꢀ3.75 (m, 10H), 3.66ꢀ2.64 (m, 3H).
C
2
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (CDCl , 100 MHz) δ: 171.9, 171.3, 161.1, 160.1, 159.7, 148.8, 148.4, 147.9, 147.7, 136.5, 133.5, 130.3, 129.9,
3
1
5
4
28.8, 128.6, 127.7, 127.5, 126.9, 126.4, 125.3, 124.2, 112.0, 111.5, 110.4, 109.9, 107.0, 106.8, 101.3, 70.4, 69.7,
+
7.2, 56.2, 56.1, 55.6, 51.6, 43.0, 35.7, 29.3, 27.9. HRMS calcd. for C H NO , 434.19620 [M + H] ; found,
2
6
28
5
+
34.19602 [M + H] . Anal. (C H NO ): C, H, N.
26
27
5
(
(
3-chlorophenyl)(6,7-dimethoxy-1-((3-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone
91). Compound 91 was prepared according to Procedure VII using 65 (0.2 g, 0.6 mmol, 1.0 equiv) and 3ꢀ
chlorobenzoyl chloride (0.1 g, 0.7 mmol, 1.2 equiv, 0.07 mL). The crude residue was purified by silica gel
chromatography (ISCO, Redisep 4 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a
1
white amorphous solid (mixture of rotamers, 0.20 g, 67%). H NMR (CDCl , 400 MHz) δ: 8.03ꢀ7.46 (m, 1H), 7.39ꢀ
3
7
.29 (m, 3H), 7.16 (t, J = 8.4 Hz, 1H), 1H [6.78,6.66 (s, s)], 1H [6.61, 6.47 (s,s)], 6.52ꢀ5.95 (m, 3H), 1H [5.10, 4.86
(
dd, J = 3.0 Hz J = 9.0 Hz; dd, J = 5.6 Hz, J = 13 Hz)], 4.38 (m, 1H), 1H [4.20, 3.98 (m, m)], 3.85ꢀ3.75 (m, 9H),
1
2
1
2
1
3
3.75ꢀ3.45 (m, 1H), 3.28ꢀ3.04 (m, 1H), 2.92ꢀ2.66 (m, 2H). C NMR (CDCl , 100 MHz) δ: 170.4, 169.6, 161.1,
3
1
60.0, 148.0, 147.8, 138.2, 134.6, 132.3, 130.3,130.0, 128.2, 128.0, 127.3, 127.1, 125.7, 125.0, 123.8, 111.5, 110.3,
09.9, 107.3, 107.0, 106.5, 101.3, 101.0, 70.3, 57.4, 56.2, 56.1, 55.6, 42.9, 35.7, 29.2, 27.8. HRMS calcd. for
1
+
+
C H NO Cl, 468.15723 [M + H] ; found, 468.15689 [M + H] . Anal. (C H NO Cl): C, H, N.
26
27
5
26 26
5
(
(
3-chlorophenyl)(6,7-dimethoxy-1-((2-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone
92). Compound 92 was prepared according to Procedure VII using 66 (0.5 g, 2 mmol, 1.0 equiv) and 3ꢀ
chlorobenzoyl chloride (0.3 g, 2 mmol, 1.2 equiv, 0.22 mL). The crude residue was purified by silica gel
chromatography (ISCO, Redisep 12 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a
1
white amorphous solid (mixture of rotamers, 0.5 g, 70%). H NMR (CDCl , 400 MHz) δ: 7.81ꢀ7.23 (m, 4H), 6.98ꢀ
3
6
.72 (m, 5H), 1H [6.64, 6.59 (s, s)], 1H [6.48, 5.98 (s, m)], 1H [5.12, 4.84 (dd, J = 4.0 Hz, J = 9.6 Hz; dd, J = 5.8
1 2 1
Hz, J =13 Hz)], 4.42ꢀ4.38 (m, 1H), 4.05ꢀ4.01 (m, 1H), 3.89ꢀ3.66 (m, 10H), 3.30ꢀ3.04 (m, 1H), 2.93ꢀ2.60 (m, 2H).
2
ACS Paragon Plus Environment

Page 27 of 82
Journal of Medicinal Chemistry
1
1
3
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C NMR (CDCl , 100 MHz) δ: 170.5, 169.6, 149.9, 149.7, 147.9, 147.7, 138.3, 138.2, 134.8, 134.6, 132.0, 130.2,
3
1
1
29.9, 128.3, 127.3, 127.1, 126.2, 126.1, 125.8, 125.2, 124.9, 124.1, 122.1, 121.1, 120.8, 114.2, 113.4, 112.3, 112.0,
11.8, 111.5, 110.6, 110.0, 71.1, 70.1, 57.5, 56.1, 56.0, 55.8, 51.7, 42.8, 35.6, 29.2, 27.7. HRMS calcd. for
+
+
C H NO Cl, 468.15723 [M + H] ; found, 468.15685 [M + H] . Anal. (C H NO Cl): C, H, N.
26 27
5
26 26
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-iodophenyl)methanone
(93). Compound 93 was prepared according to Procedure VIII using 64 (0.2 g, 0.6 mmol, 1.0 equiv) and 3ꢀ
iodobenzoic acid (0.2 g, 0.6 mmol, 1.0 equiv). The crude material was subjected to silica gel chromatography
(
ISCO, Redisep 4 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a white amorphous
1
solid (mixture of rotamers, 0.2 g, 59%). H NMR (CDCl , 400 MHz) δ: 1H [7.94, 7.70 (s, s)], 7.74 (d, J = 7.6 Hz,
3
1
H), 1H [7.46, 7.33 (d, J = 8.0 Hz; d, J = 7.6 Hz)], 7.13 (q, J = 8.0 Hz, 1H), 6.86 (d, J = 9.6 Hz, 1H), 6.80 (m, 3H),
1H [ 6.66, 6.61 (s, s)], 1H [6.47, 5.92 (s, m)], 1H [5.07, 4.83 (dd, J = 3.8 Hz, J = 9.8 Hz; dd, J = 6.8 Hz, J = 13
1
2
1
2
Hz)], 4.33 (m, 1H), 4.17ꢀ4.01 (m, 1H), 3.97ꢀ3.60 (m, 1H), 3.86ꢀ3.74 (m, 9H), 3.27ꢀ3.04 (m, 1H), 2.90ꢀ2.64 (m, 2H).
13
C NMR (CDCl , 100 MHz) δ: 170.0, 169.3, 154.4, 154.3, 153.0, 152.5, 148.9, 148.4, 148.0, 147.8, 138.8, 138.6,
3
1
38.5, 136.7, 135.6, 130.3, 127.3, 126.8, 126.2, 125.2, 124.0, 115.9, 115.5, 114.9, 112.0, 111.5, 109.9, 94.5, 94.4,
+
7
1.1, 70.2, 57.5, 56.3, 56.1, 55.9, 43.0, 35.7, 29.2. HRMS calcd. for C H NO I, 560.09285 [M + H] ; found,
26 27
5
+
560.09224 [M + H] . Anal. (C H NO I): C, H, N.
26
26
5
[1,1'-biphenyl]-3-yl(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (94). Compound 94 was prepared according to Procedure VIII using 64 (0.5 g, 1.5 mmol) and 3ꢀ
phenylbenzoic acid (0.3 g, 1.5 mmol, 1.0 equiv) in DCM (10 mL) and DMF (2.0 mL). The crude yellow residue was
purified by silica gel chromatography (ISCO, Silicycle 12 g column, 0ꢀ70% EtOAc/hexanes gradient) to afford the
1
title compound as a white amorphous solid (0.26 g, 34%, mixture of rotamers). H NMR (CDCl , 400 MHz) δ: 7.83ꢀ
3
7.30 (m, 8H), 6.92ꢀ6.74 (m, 5H), 1H [6.68, 6.62 (s, s)], 1H [6.49, 6.01 (s, m)], 1H [ 5.21, 4.91 (dd, J = 3.8 Hz, J =
1
2
9
.0 Hz; dd, J = 5.6 Hz, J = 12.8 Hz), 4.43ꢀ4.35 (m, 1H), 1H [4.22, 4.00 (m, m)], 3.90ꢀ3.72 (m, 9H), 1H [3.67, 3.26
1 2
13
(
m, m)], 1H [3.15, 2.89 (m, m)], 2.78ꢀ2.65 (m, 2H). C NMR (CDCl , 100 MHz) δ: 171.8, 171.1, 154.4, 154.3,
3
1
53.1, 152.6, 148.8, 148.4, 148.0, 147.7, 141.8, 141.5, 140.6, 140.4, 137.2, 129.3, 129.1, 128.5, 128.4, 128.0, 127.9,
27.4, 126.6, 126.5 (2), 125.6, 125.5, 124.5, 115.9, 115.6, 114.9, 112.1, 111.6, 110.5, 110.0, 71.2, 70.5, 57.5, 56.3,
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 82
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
5
6.1, 55.9, 51.8, 43.0, 35.8, 29.3, 27.8. HRMS calcd. for C H NO , 510.22750 [M + H] ; found, 510.22802 [M +
32 32 5
+
H] . Anal. (C H NO ): C, H, N.
32
31
5
(
(
6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-hydroxyphenyl)methanone
95). Compound 95 was prepared according to Procedure VIII using 64 (0.16 g, 0.5 mmol) and 3ꢀhydroxybenzoic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
acid (0.07 g, 0.5 mmol). The crude material was purified by silica gel chromatography (ISCO, Redisep 4 g column,
0
ꢀ60% EtOAc/hexanes gradient) to afford the title compound as a white amorphous solid (0.09 g, 41%, mixture of
1
rotamers). H NMR (CDCl , 400 MHz) δ: 8.14ꢀ8.02 (m, 1H), 7.72ꢀ7.40 (m, 3H), 7.34ꢀ7.04 (m, 2H), 6.84ꢀ6.74 (m,
3
2
1
1
H), 6.65ꢀ5.98 (m, 2H), 1H [5.20, 4.83 (m, m)], 4.32 (m, 1H), 4.18ꢀ3.90 (m, 1H), 3.84ꢀ3.70 (m, 9H), 3.70ꢀ3.68 (m,
13
H), 3.30ꢀ3.02 (m, 1H), 2.84ꢀ2.59 (m, 2H). C NMR (CDCl , 100 MHz) δ: 171.7, 157.1, 154.3, 153.0, 152.6, 148.4,
3
47.9, 137.0, 130.0, 126.4, 125.1, 117.9, 117.5, 117.1, 115.9, 115.6, 115.4, 114.9, 114.3, 112.0, 111.5, 110.4, 71.1,
+
67.7, 63.2, 56.2, 56.1, 55.9, 52.0, 42.9, 29.2, 27.9. HRMS calcd. for C H NO , 450.19111 [M + H] ; found,
2
6
28
6
+
4
59.19206 [M + H] . Anal. (C H NO ) C, H, N.
26 27 6
(
3-(chloromethyl)phenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (96). Compound 96 was prepared according to Procedure VII using 64 (0.8 g, 2.4 mmol) and 3ꢀ
chloromethyl)benzoyl chloride (0.5 g, 2.9 mmol, 1.2 equiv). The crude material was purified by silica gel
(
chromatography (ISCO, Silicycle 12 g column, 0ꢀ70% EtOAc/hexanes) to afford the title compound as a white
1
amorphous solid (mixture of rotamers, 0.30 g, 26%). H NMR (CDCl , 400 MHz) δ: 7.57ꢀ7.34 (m, 3H), 6.92ꢀ6.77
3
(
m, 4H), 6.69ꢀ6.62 (m, 2H), 1H [6.50, 5.98 (s, m)], 1H [5.15, 4.90 (m, dd, J = 5.6 Hz, J = 13.2 Hz)], 4.60 (m, 2H),
1 2
13
4
1
1
.43ꢀ4.31 (m, 1H), 4.19ꢀ4.11 (m, 1H), 3.88ꢀ3.67 (m, 10H), 3.32ꢀ3.08 (m, 1H), 2.96ꢀ2.66 (m, 2H). C NMR (CDCl ,
3
00 MHz) δ: 170.7, 169.6, 154.4, 154.3, 153.0, 152.6, 148.8, 148.4, 148.0, 138.3, 138.0, 137.0, 133.5, 130.8, 130.3,
30.2, 130.0, 129.3, 129.1, 128.1, 127.7, 127.3, 127.1, 126.8, 126.3, 125.3, 124.2, 115.9, 115.6, 114.9, 112.0, 111.5,
110.4, 109.9, 71.1, 70.3, 57.5, 56.2, 56.1, 55.9, 51.9, 46.0, 45.7, 43.0, 35.7, 29.2, 27.9. HRMS calcd. for
+
+
C H NO Cl, 482.17288 [M + H] ; found, 482.17250 [M + H] . Anal. (C H NO Cl): C, H, N.
27
29
5
27 28
5
(
3-(dichloromethyl)phenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-
yl)methanone (97). Compound 97 was prepared according to Procedure VII using 64 (0.2 g, 0.6 mmol) and 3ꢀ
ACS Paragon Plus Environment

Page 29 of 82
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
dichloromethyl)benzoyl chloride (0.16 g, 0.72 mmol, 1.2 equiv). The crude material was purified by silica gel
chromatography (ISCO, Silicycle 4 g column, 0ꢀ60% EtOAc/hexanes) to afford the title compound as a white
1
amorphous solid (0.07 g, 21%). H NMR (CDCl , 400 MHz) δ: 7.76ꢀ7.44 (m, 4H), 6.91ꢀ6.64 (m, 6H), 1H [6.50,
3
5
.99 (s, m)], 1H [5.13, 4.90 (m, m)], 4.39 (m, 1H), 4.20ꢀ3.94 (m, 1H), 3.88ꢀ3.69 (m, 10H), 3.29ꢀ3.13 (m, 1H), 2.98ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
2.70 (m, 2H). C NMR (CDCl , 100 MHz) δ: 170.8, 170.1, 154.3, 153.0, 152.5, 148.9, 147.8, 141.1, 137.1, 129.4,
3
1
29.3, 128.3, 127.5, 125.8, 125.2, 124.8, 124.0, 115.5, 114.9, 71.2, 64.3, 57.5, 56.3, 56.2, 56.1, 55.9, 52.0, 43.0, 27.2.
+
+
HRMS calcd. for C H NO Cl , 516.13391 [M + H] ; found, 516.13457 [M + H] . Anal. (C H NO Cl ): C, H, N.
2
7
28
5
2
27 27
5
2
3
-(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzonitrile (98).
Compound 98 was prepared according to Procedure VII using 64 (0.21 g, 0.63 mmol) and 3ꢀcyanobenzoyl chloride
0.13 g, 0.76 mmol, 1.2 equiv). The crude material was purified by silica gel chromatography (ISCO, Redisep 4 g
(
1
column, 0ꢀ70% EtOAc/hexanes gradient) to afford the title compound as a white amorphous solid (0.28 g, 99%). H
NMR (CDCl , 400 MHz) δ: 8.00ꢀ7.44 (m, 4H), 6.98ꢀ6.75 (m, 4H), 1H [6.70, 6.62 (s, s)], 1H [6.48, 6.00 (s, m)], 1H
3
13
[
(
5.02, 4.88 (m, m)], 4.38 (m, 1H), 4.06ꢀ3.71 (m, 10H), 3.28 (m, 1H), 3.14 (m, 1H), 2.96ꢀ2.69 (m, 2H). C NMR
CDCl , 100 MHz) δ: 169.4, 168.6, 154.4, 154.2, 152.7, 152.2, 148.9, 148.4, 147.9, 147.7, 137.6, 134.5, 134.0,
3
133.1, 133.0, 132.0, 131.5, 131.0, 130.3, 129.7, 129.3, 127.1, 125.9, 124.7, 123.4, 118.2, 118.0, 115.7, 115.3, 114.9,
114.7, 113.0, 112.6, 111.4, 110.2, 109.7, 70.8, 70.0, 57.5, 56.1, 55.9, 55.7, 51.9, 43.0, 35.6, 29.0, 27.6. HRMS calcd.
+
+
for C H N O , 459.19145 [M + H] ; found, 459.19087 [M + H] . Anal. (C H N O ): C, H, N.
27
27
2
5
27 27
2
5
1
-(3-(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-
carbonyl)phenyl)ethanone (99). Compound 99 was prepared according to Procedure VIII using 64 (1.0 g, 3.0
mmol) and 3ꢀacetylbenzoic acid (0.5 g, 3.04 mmol, 1.0 equiv) in DCM (10 mL) and DMF (2.0 mL). The crude
residue was purified by silica gel chromatography (ISCO, Silicycle 12 g column, 0ꢀ70% EtOAc/hexanes gradient) to
1
afford the title compound as a white amorphous solid (0.32 g, 22%, mixture of rotamers). H NMR (CDCl , 400
3
MHz) δ: 8.15ꢀ7.97 (m, 2H), 7.72ꢀ7.46 (m, 2H), 6.87ꢀ6.78 (m, 4H), 1H [6.66, 6.61 (s, s)], 1H [6.43, 5.95 (s, m)], 1H
[
5.06, 4.87 (dd, J = 3.6 Hz, J = 9.2 Hz; dd, J = 5.8 Hz, J = 13 Hz)], 4.39ꢀ4.31 (m, 1H), 1H [4.14, 3.96 (m, m)],
1 2 1 2
13
3
.84ꢀ3.72 (m, 9H), 1H [3.66, 3.25 (m, m)], 1H [3.12, 2.86 (m, m)], 2.77ꢀ2.61 (2H), 3H [2.58, 2.56 (s, s)]. C NMR
(
CDCl , 100 MHz) δ: 197.8, 197.6, 170.9, 170.2, 154.5, 154.3, 153.0, 152.5, 148.9, 148.4, 148.0, 137.6, 137.4,
3
ACS Paragon Plus Environment