Tetrahydroprotoberberine alkaloids with dopamine and σ receptor affinity

Article abstract of DOI:10.1016/j.bmc.2016.03.037

Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that (a) contain various alkoxy substituents at the C10 position and, (b) were de-rigidified with respect to (±)-stepholidine, were synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2 receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3 receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the compounds.

Full text of DOI:10.1016/j.bmc.2016.03.037

Accepted Manuscript
TETRAHYDROPROTOBERBERINE ALKALOIDS WITH DOPAMINE
AND σ RECEPTOR AFFINITY
Satishkumar Gadhiya, Sudharshan Madapa, Thomas Kurtzman, Ian L. Alberts,
Steven Ramsey, Nagavara-Kishore Pillarsetty, Teja Kalidindi, Wayne W.
Harding
PII:
S0968-0896(16)30193-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.037
BMC 12886
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
14 February 2016
19 March 2016
Please cite this article as: Gadhiya, S., Madapa, S., Kurtzman, T., Alberts, I.L., Ramsey, S., Pillarsetty, N-K.,
Kalidindi, T., Harding, W.W., TETRAHYDROPROTOBERBERINE ALKALOIDS WITH DOPAMINE AND σ
RECEPTOR AFFINITY, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.03.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

TETRAHYDROPROTOBERBERINE ALKALOIDS WITH DOPAMINE AND σ RECEPTOR AFFINITY

Satishkumar Gadhiya,a,b Sudharshan Madapa,a Thomas Kurtzman,b,c,d Ian L. Alberts,e Steven Ramsey,c,d
Nagavara-Kishore Pillarsetty,f Teja Kalidindif and Wayne W. Harding *, a,b,c
a. Department of Chemistry, Hunter College, City University of New York, 695 Park Avenue, NY 10065, USA
b. Ph.D. Program in Chemistry, CUNY Graduate Center 365 5th Avenue, New York New York, United States
of America, 10016
c. Ph.D. Program in Biochemistry, CUNY Graduate Center 365 5th Avenue, New York New York, United
States of America, 10016
d. Department of Chemistry, Lehman College, The City University of New York, Bronx, NY 10468, USA.
e. Department of Natural Sciences, LaGuardia Community College, City University of New York, New York,
NY 11101, USA
f. Department of Radiology, Memorial Sloan Kettering Cancer Center , 1275 York Avenue, New York, NY
10065, USA
*Email: whardi@hunter.cuny.edu
Tel: (+)212-772-5359
Fax:(+)212-772-5332
1

ABSTRACT
Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that a) contain
various alkoxy substituents at the C10 position and, b) were de-rigidified with respect to (±)-stepholidine, were
synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2
receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all
compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity
for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-
rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB
framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3
receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond
interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as
well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the
compounds.
2

GRAPHICAL ABSTRACT
3

1. Introduction
The dopamine D3 receptor has received considerable attention, especially over the past two decades,
mainly due to its prominent role in mediating craving for addictive substances.1-3 In that regard, selective D3
antagonists have been sought after as potential therapeutics for cocaine addiction and the treatment of
cocaine relapse.4 Additionally, a growing body of evidence in the literature suggests that D3 antagonism may
be a valuable antipsychotic therapeutic strategy.5-7 Several selective D3 antagonists have been identified that
bear the key pharmacophoric elements of a phenylpiperazine motif connected to an aryl amide unit via a 4
carbon spacer.8,9,1 Other D3-preferring scaffolds are also known, although the phenypiperazine template has
been most prolific in terms of the number of compounds reported. Representative examples of selective D3
antagonists are depicted in Figure 1.
Figure 1. Structures of representative selective D3 antagonists
Despite advances in understanding the structure-activity relationships (SARs) of such D3 scaffolds, in
most cases, clinical translation of the resulting optimized compounds has been limited, primarily due to
pharmacokinetic issues.3 There is thus a constant search for new structural templates endowed with selective
D3 antagonist activity as such compounds could serve as useful leads for development as in vivo biological
tools and therapeutic leads, especially pertinent to the realms of neuropsychiatric disorders and drug abuse.
There are two types of σ receptors - σ1 and σ2, as borne out by differential binding of selective
radioligands as well as other biochemical and pharmacological studies.10-12 σ receptors are distributed in the
central nervous system (CNS) and in peripheral tissue.13,14 Within the CNS, σ1 receptors are concentrated in
the hippocampus and other limbic regions which control cognition and emotion.15,16 Thus these receptors have
been implicated in a number of neuropsychiatric disorders and have been targets for the development of
treatment interventions for such diseases.17-20 σ2 receptors are found predominantly in regions responsible for
4

motor functions in the CNS and are also highly expressed in the lung, liver and kidneys.15,16 The σ1 receptor
has been cloned but the σ2 receptor has not, and there is some debate about the identity of the σ2 receptor.21
Recent studies suggest that the σ2 receptor is Progesterone Receptor Membrane Component 1
(PGRMC1).22,23
Figure 2. Structural classes of σ2ligands and representative examples
σ2 receptors are overexpressed in tumor cells and are upregulated in rapidly proliferating cells and this
receptor has attracted interest as a biomarker for cancer proliferation.24,25 Some σ2 ligands (particularly σ2
receptor agonists) display cytotoxic activity.26 Thus, σ2 ligands have been of interest for the development of
"stand alone" antitumor drugs, targeted drug delivery agents and Positron Emission Tomography (PET) tools
for imaging the proliferative status of various cancers.27-30 σ2 receptor ligands have been predominantly from
four structural classes: 6,7-dimethoxytetrahydroisoquinolines, indoles, tropanes and cyclohexylpiperazines
(Figure 2). Many of these compounds lack sufficient selectivity or are otherwise hampered by unfavorable
pharmacokinetic drawbacks that limit their usefulness as drugs, anticancer drug delivery vehicles or tools.
New, potent and selective σ2 receptor ligands are sought after to fully exploit the potential of the σ2 receptor
as a target for cancer diagnosis and therapy.
The tetrahydroprotoberberine (THPB) alkaloid (-)-stepholidine (1, Figure 3) shows affinity for dopamine
D1 and D2 receptors and has been the focus of a number of structure-activity relationship (SAR) studies in this
regard.31,32 The antipsychotic and anti-drug abuse potential of (-)-stepholidine has been attributed to its
unusual D1 agonist/D2 antagonist profile. (-)-Stepholidine also shows good affinity for σ2 (Ki = 54 nM) and D3
receptors (Ki = 30 nM), as revealed by data from the Psychoactive Drug Screening Program (PDSP)
5

database.33 Given the comparatively high σ2 and D3 affinity of 1, we considered that the THPB template may
be exploitable towards the development of novel potent and selective σ2 receptor or D3 receptor ligands.
However, no SAR studies have been performed on THPBs in relation to their σ2 or D3 receptor affinity.
Furthermore, computational evaluations on THPBs particularly at the D3 receptor (for which a crystal structure
is available)34 are lacking in the literature. Such studies could propel our understanding of the potential utility
of this scaffold towards obtaining novel, selective σ2 or D3 receptor ligands. Our explorations in this regard
are described herein.
Figure 3. Structure of (-)-stepholidine (1)
2. Results and discussion
Stepholidine was used as the lead molecule for our SAR evaluations. To initiate this study, we decided
to prepare a series of racemic C10 stepholidine analogues in order to probe the tolerance of this position for
larger substituent groups at both receptors. THPBs are structurally similar to the 6,7-
dimethoxytetrahydroisoquinoline class of σ2 ligands in that both possess a tetrahydroisoquinoline motif.
Tetrahydroisoquinoline sub-structures are also found in a number of D3 antagonist ligands (eg. SB277011A,
Figure 1).1 However, known 6,7-dimethoxytetrahydroisoquioline σ2 ligands, like the known D3-preferring
tetrahydroisoquinolines are flexible, extended molecules whereas the THPB scaffold is obviously a more rigid
structure. To elucidate the importance of structural rigidity of the THPB core on σ2 and D3 receptor affinity,
less rigid stepholidine analogues were prepared and evaluated. Given that THPBs have been known to show
affinity for D1 and D2 receptors, the analogues were also assessed at D1 and D2 receptors to further gauge
selectivity. Computational studies were also performed to help to rationalize observed affinities.
2.1 Chemistry
We engaged a route that we have recently reported for the synthesis of the THPB 10 for the synthesis
of the C10 alkoxy derivatives.35 This route is depicted in Scheme 1. Commercially available phenol 2 was
reacted with ethoxymethyl chloride. The protected aryl bromide (3) thus formed was then treated with LDA
(generated from reaction of n-BuLi with diisopropylamine) to afford diester 4. Selective hydrolysis of the
aliphatic ester in 4 gave acid 5. Compound 5 was coupled to amine 6 and the resulting amide (7) was
subjected to cyclization under Bischler-Napieralski conditions. Cyclization was accompanied by removal of the
ethoxymethyl protecting group. The imine which formed was not isolated, but was treated thereafter with
sodium borohydride, whereupon cyclization to afford the 8-oxoprotoberberine core ensued (compound 8).
6

Reduction of the 8-oxo group of 8 with lithium aluminum hydride gave the THPB 9 containing the unprotected
C10 phenol group. Williamson ether alkylation of phenol 9 with various alkyl halides afforded the C9 alkoxy/C2
benzyl protected derivatives. Finally, the benzyl group of these intermediates was removed by treatment with
HCl in refluxing methanol to obtain the racemic C10 analogues 10-17. Compound (±)-1 was obtained by
deprotection of the benzyl group of phenol 9 in similar fashion as for other C10 analogues.
Scheme 1. Synthesis of C10 analogues. Reagents and conditions: (a) DIPEA, (chloromethoxy)ethane, DCM, 98%; (b)
diisopropylamine, n-BuLi, DMM, THF, -78 °C, 54%; (c) K2CO3, 1:1 H2O/MeOH, reflux, 95%; (d) CDI, THF, 78%; (e) i)
POCl3, CH3CN, reflux; ii) NaBH4, MeOH, 79% over 2 steps; (f) LAH, THF, reflux, 84%; (g) i) appropriate alkyl halide, DMF,
CH3CN, rt to reflux; ii) conc. HCl, MeOH, reflux; 64-77% over 2 steps; (h) conc. HCl, MeOH, reflux, 81 %
The route for preparation of the flexible analogues is shown in Scheme 2. Here, primary amine 6 was
converted to the secondary amine 18 via reductive amination with 3-benzyloxy-2-methoxybezaldehyde. Pictet-
Spengler cyclization followed by acidic debenzylation were implemented to convert 18 to the
tetrahydroisoquinoline analogue 19. A sequence of reductive amination and benzyl group hydrolysis reactions
gave tertiary amines 20 and 21 from the secondary amine 18.
7

Scheme 2. Synthesis of flexible analogues. Reagents and conditions: a) Na(OAc)3BH, DCM, rt, appropriate aldehyde; b)
i) CF3COOH, 1,1-diethoxyethane, DCM, rt; ii) Conc. HCl, MeOH, reflux, 60% over two steps; c) Conc. HCl, MeOH, reflux.
2.2 Biological evaluations
2.2.1 SAR on C10 analogues
Compounds (±)-1, 10-17 and 19-21 were assayed for binding affinity to human dopamine D1, D2 and
D3 receptors and σ1 and σ2 receptors by the Psychoactive Drug Screening Program (PDSP). Details of the
screening protocol may be found online at the PDSP website (https://pdspdb.unc.edu/pdspWeb/). In brief, the
compounds were initially assayed in quadruplicate at a 10 µM concentration at D1, D2, D3, σ1 and σ2
receptors. Compounds that showed an inhibition of binding of >50% were progressed to secondary assays to
measure Ki. The Ki determinations were performed via 12-point concentration-response curves in triplicate
(unless noted otherwise). This data is presented in Tables 1 (for C10 analogues) and 2 (for de-rigidified
analogues).
(±)-1 displayed approximately 20-fold stronger affinity for the D1 receptor as compared to the D2 and
D3 receptors (Ki of 5.6, 115.5 and 101 nM at D1, D2 and D3 respectively). For C10 alkoxy derivatives
alkylation tends to reduce D1 affinity, with the analogues showing a 1.2 to 7-fold decrease in affinity as
compared to (±)-1. The C10 phenol plays role but is not absolutely required for D1 affinity, given the relatively
small drop in affinity seen for some analogues (for example 12 and 16). This result is in accordance with
observations from previous SAR work on the THPB framework by others.32,31 Methyl and ethyl substituents
resulted in an approximately 2-fold decrease in affinity (compounds 10 and 11 respectively). In the n-propyl
(12) to n-hexyl (15) homologous series, a progressive decrease in affinity was observed. The D1 receptor
affinity seen for the hydroxypropyl analogue 16 was comparable to that observed for (±)-1 and the n-
8

Table 1. Affinitydata (Ki in nM) for C10 analogues at dopamine and σ receptors
Ki (nM)a
D3d
Selectivity
D2/D3
Compound
R
D1b
D2c
D2/D1
D3/D1
1e
269
106
124
57
2f
9.0
1.0
1.3
6.7
49
1/2
30
(±)-1
10
H
5.6g
115.5g
102
159
99
101g
20.6
6.8
18.0
2.5
3.5
6.8
5.9
5.3
5.5
16.4
4.5
1.1
2.8
3.6
1.9
1.9
2.8
NDi
1.9
1
Me
Et
15
37
106
95
11
12.4
7.8
11
44
12.8
12.7
11
12
n-Pr
53
8.5
13
n-Bu
121
283
NAh
211
54
65
13
0.27
1.22
0.35
13.9
5
14
n-Pent
19
101
220
110
54
119
37
97
14.9
NDi
31.5
4.5
15
n-Hex
40
107
3.8
2.4
16
Hydroxypropyl
Cyclopropylmethyl
6.7
53
17
12
12
a
b
c
Experiments carried out in triplicate - SEM values are within 13% of reported Ki; [3H]SCH2390 used as radioligand/(+)-butaclamol used as reference compound - Ki = 2.3 nM; [3H]N-
methylspiperone used as radioligand/haloperidol used as reference compound - Ki = 9.6 nM; d [3H]N-methylspiperone used as radioligand/chlorpromazine used as reference compound - Ki =
11.0 nM; e [3H](+)-pentazocine used as radioligand/haloperidol used as reference compound - Ki = 1.1 nM; f [3H]DTG used as radioligand/haloperidol used as reference compound - Ki = 10.0 nM;
g experiments carried out in duplicate; hNA- not active (<50% inhibition in primary assay therefore no secondary assay was performed); iND - not determined
9

butyl analogue 13 (6.7, 5.6 and 11 nM respectively). This suggests that the hydroxyl group in 16 plays a
relatively minor role in binding to the D1 receptor. The presence of the hydroxypropyl substituent in 16
restores the D1 affinity of stepholidine, improves the selectivity versus the D2 receptor (20.6 versus 31.5 for
D2/D1 selectivity for stepholidine and 16 respectively) and retains the selectivity versus the D3 receptor (18
versus 16-fold for D3/D1 selectivity). Among the alkylated analogues, a 3 carbon chain length seems to be
optimal for D1 affinity (consider 12 and 16). The cyclopropylmethyl analogue (17) had similar affinity to the n-
butyl analogue 13 and ethyl analogue 11. Thus some degree of branching on the alkyl chain may be tolerated
for D1 affinity.
On a whole, the D2 affinity of the analogues was lower than their D1 affinities. Thus the compounds
retained selective affinity for the D1 receptor, as was observed in the lead molecule (±)-1. However, most
compounds had lower than the 20-fold D1 selectivity seen for (±)-1 (except for compounds 15 and 16).
Compounds in the homologous series from a methyl group to an n-butyl chain (10-13), had D2 affinities that
are similar to (±)-1, ranging from 99 to 159 nM. However, compounds with the larger n-pentyl and n-hexyl
chains had reduced affinities; in fact, the n-hexyl analogue (15) did not show any appreciable affinity for the D2
receptor (<50% inhibition) in the primary assay. The cyclopropylmethyl analogue (17) was the only compound
in this series that displayed a significantly higher affinity for the D2 receptor than (±)-1.
There was a clear trend in the methyl to n-hexyl series at the D3 receptor, where an increase in the
alkyl chain length was associated with a progressive decrease in affinity for the receptor. The hydroxypropyl
analogue had similar affinity for the D3 receptor as compared to stepholidine. In the case of the
cyclopropylmethyl analogue, D3 receptor affinity was improved two-fold as compared to stepholidine (54 nM vs
101 nM).
At the σ1 receptor, all analogues tested showed increased affinity as compared to (±)-1 (ranging from
approximately 2-fold to 22-fold increase in σ1affinity). Therefore, the C10 phenol functionality is not required
for σ1 receptor affinity. No clear SAR trend could be discerned in the homologous series 10-15. The n-butyl
analogue 13 had the highest affinity of the compounds in this series [Ki = 13 nM for 13 vs 269 nM for (±)-1].
The hydroxypropyl analogue 16 had lower affinity than 13 (53 vs 13 nM) which suggests that the hydroxyl
group in 16 does not have any significant binding interactions with the σ1 receptor (akin to observations at the
D1 receptor). The cyclopropylmethyl and n-butyl analogues had similar σ1 affinities- approximately 12 nM (a
situation that mirrors results for 17 at the D1 receptor). It thus appears that the cyclopropylmethyl group may
function as a less hydrophobic bioisostere of the n-butyl group in this series at σ1 and D1 receptors.
A clear trend was seen at the σ2 receptor for analogues 10-15, wherein there was a progressive
decrease in σ2 receptor affinity as the alkyl chain was extended. Compounds 10 and 11 had the highest
affinity of all compounds assayed (1 nM and 1.3 nM respectively), representing an approximately 7- to 9-fold
higher σ2 receptor affinity than (±)-1. With respect to binding at σ receptors, both 10 and 11 exhibited higher
σ2 receptor selectivity than compound (±)-1 [approximately 100-fold for 10 and 11 versus 30-fold for compound
10

(±)-1]. All other analogues had decreased selective affinity for the σ2 receptor as compared to the σ1 receptor.
In fact, this σ receptor selectivity was reversed for compounds 13 and 15 which both showed approximately 3-
fold higher selectivity for σ1 versus σ2 receptors. Analogues 10-12 had higher affinity than (±)-1, indicating
that small C10 alkoxy groups are better tolerated than the C10 phenolic group at the σ2 receptor. Compounds
16 and 17 displayed very high σ2 receptor affinity, comparable to 10 and 11; thus there is some tolerance for
small polar or hydrophobic groups at C10 for σ2 affinity.
2.2.2 SAR on flexible analogues
All de-rigidified analogues showed significantly lower affinity than (±)-1 for dopamine D1, D2 and D3
receptors. Thus, the intact THPB core seems to be very important for affinity to dopamine receptors.
Compounds 20 and 21 which lacked a tetrahydroisoquinoline motif had lower affinities for the D1, D2 and D3
receptor than compound 19 which possesses such a motif. Therefore it would appear that the presence of an
intact tetrahydroisoquinoline moiety contributes significantly to dopamine receptor affinity.
De-rigidification did not significantly improve σ1 receptor affinity. Only a 2-fold increase in σ1 affinity
was seen for compounds 19 and 20 as compared to (±)-1; a decrease in σ1 affinity was observed with
compound 21. All flexible analogues showed a deterioration in σ2 receptor affinity as compared to the lead
(227 - 788 nM vs 9 nM). However, in general the de-rigidified analogues showed higher selectivity for σ
receptors as compared to dopamine receptors suggesting that molecular flexibility is better tolerated at σ
receptors within this series.
Table 2. Ki data for flexible analogues
Ki (nM)a
D1b
D2c
D3d
1e
130
122
350
2f
301
227
788
Compound
19
20
21
833
3111
1095
1367
2680
1811
>10,000
>10000
>10000
a
Experiments carried out in triplicate - SEM values are within 13% of reported Ki; b [3H]SCH2390 used as radioligand/(+)-butaclamol used as reference
c
d
compound - Ki = 2.3 nM; [3H]N-methylspiperone used as radioligand/ haloperidol used as reference compound - Ki = 9.6 nM; [3H]N-methylspiperone
e
used as radioligand/chlorpromazine used as reference compound - Ki = 11.0 nM; [3H](+)-pentazocine used as radioligand/ haloperidol used as
reference compound - Ki = 1.1 nM; f [3H]DTG used as radioligand/ haloperidol used as reference compound - Ki = 10.0 nM
2.2.3 Cytotoxicity evaluation
11

As alluded to before, some compounds have shown cytotoxic actions that are attributable to their
interaction with σ receptors. We were curious to determine the extent to which our newly identified high affinity
σ2 receptor ligands displayed cytotoxicity. Therefore, isocorypalmine (10), the most potent and selective σ2
ligand identified in this study, was selected for cytotoxicity evaluation in an Alamar Blue assay.36 Compound
10 lacked significant cytotoxic activity in this assay, with an IC50 of 178 µM. Since σ2 receptor agonists are
generally cytotoxic,37-39 this result tends to imply that compound 10 is a σ2 receptor antagonist (although this
needs to be ratified with further assays).
2.2.4 Receptor docking studies at the D3 receptor
The phenolic group at C10 of stepholidine does not seem to play a major role in its binding to dopamine
D1 and D2 receptors (given the similarity in D1 and D2 affinities of the analogues as compared to
stepholidine). In the case of the D3 receptor, a number of analogues had moderately improved affinity as
compared to stepholidine. Therefore it seems that the C10 phenolic group is not an absolute necessity for
affinity of stepholidine to the D3 receptor.
Table 3. Predicted binding affinity from Glidescore and ligand strain energy for C10 and flexible analogues at
the D3 receptor prepared from the crystal structure with PDB code 3PBL
Compound
R
Glidescore
-7.9
Ki at D3 (nM)
101
(±)-1
10
11
12
13
14
15
16
17
19
20
21
H
Me
Et
-7.5
37
-7.4
44
n-Pr
-7.5
53
n-Bu
-7.4
65
n-Pent
-7.5
101
n-Hex
-7.3
220
Hydroxypropyl
Cyclopropylmethyl
-8.1
110
-7.3
54
-6.2
1367
2680
1811
-6.3
-6.4
Based on the SAR trend observed (increasing alkyl chain length decreases D3 affinity), it is apparent
that the C10 alkoxy substituents of the analogues might project into a shallow hydrophobic pocket in the
receptor. To get further clarity into this possibility, and to aid in rationalizing the D3 affinity trend observed,
docking evaluations were performed on analogues (±)-1, 10-17 and 19-21 at the D3 receptor.
12

Phe106
Asp110
Cys181
Ser192
Phe345
a)
b)
Figure 4. a) Docked poses of the lead molecule (±)-1 and the C10 analogues 10-15 and 17. Key hydrogen bonding
interactions are given by the yellow dashed lines and hydrophobic interactions by the turquoise dashed lines. b)
compound 10 docked in binding pocket
Investigation of the docked ligand poses and recognition of key protein-ligand interactions provides
insights into the SAR outcomes of the series of C10 analogues and the de-rigidified compounds with respect to
D3. In this study, ligand docking was performed retrospectively in order to elucidate and rationalize the
measured D3 affinity data. Overall, the calculated binding energies for stepholidine and the C10 analogues are
13

in reasonable agreement with activities derived from the experimental data as shown in Table 3. Compounds
(±)-1, 10-17 and 19-21 were docked into a pre-prepared 3-dimensional structure of the D3 receptor derived
from the crystal structure with PDB code 3PBL.34 A representation of the docked poses for the ligands (±)-1,
10-15 and 17 within the D3 receptor binding pocket is depicted in Figure 4. Each ligand generated multiple
binding modes and the structures shown display the top ranked poses according to Glidescore (+ligand strain).
These poses maintain key receptor-ligand interactions, including the critical protonated tertiary N – Asp110 salt
bridge motif, H-bonds to Ser192 and Cys181 and hydrophobic interactions to Phe106 and Phe345 (Figure 4a).
The analogues are positioned such that the aryl substituents project into hydrophobic cavities in the receptor
(depicted in Figure 4b for compound 10). The estimated binding energies according to Glidescore are very
similar for this series of C10 analogue ligands, covering a narrow range from -7.9 to -7.3 kcal/mol. Overall, the
relatively small decrease in the predicted binding energy as the length of the alkyl chain increases for
compounds 10-15 qualitatively matches the decrease in the measured affinity for these ligands.
Asp110
Glu90
Ser192
Phe345
Figure 5. Docked pose of the hydroxypropyl analogue, compound 16.
Quantitatively, the binding energy differences are very small compared to the wider, yet still relatively
low, variation in measured affinities. The Glidescore value for stepholidine (-7.9 kcal/mol) was found to be
lower than that for the other C10 analogues, including the cyclopropylmethyl analogue (-7.3 kcal/mol), in
contrast to the experimental measurements. However, in a similar manner to the measured affinities, the
hydroxypropyl analogue, with docked pose illustrated in Figure 5, had a predicted binding energy that is close
to that of stepholidine (-8.1 kcal/mol vs -7.9 kcal/mol, respectively). Examination of the D3 receptor binding site
shows that the alkyl, cyclopropylmethyl and hydroxypropyl groups of the C10 analogues fit into the extracellular
hydrophobic region of the binding pocket, consisting of the extracellular loops and the junction of several
14

helices, without incurring any significant clashes with the protein structure. Hence, the small difference in
predicted binding energies for these systems is unsurprising. The hydroxyl group in compound 16 also forms
an additional H-bond to Glu90.
In agreement with the experimental affinity measurements at the D3 receptor, the de-rigidified, flexible
compounds 19-21 are predicted to have significantly lower binding energies than the above more rigid C10
analogues according to the in silico docking outputs. The Glidescore values for the three compounds are very
similar, ranging only from -6.4 kcal/mol to -6.2 kcal/mol. Analysis of the generated poses and the key protein-
ligand interactions (depicted in Figures 6 and 7) suggests that the lowered affinity and predicted binding energy
for these de-rigidified analogues is a consequence of (i) extra ligand flexibility, which leads to a small amount
of strain for the ligand to fit in the site compared to the nearest minimized ligand conformation, and (ii) a lower
lipophilic interaction score with the binding site residues due to lack of the tetrahydroisoquinoline motif (20, 21)
and the THBP core (19). Each of these effects is relatively small (< 1 kcal/mol), however, the overall
consequence is to reduce the binding affinity of the flexible analogues.
Asp110
Ser192
Figure 6. Docked pose of the flexible analogue, compound 19.
Others have investigated docking of stepholidine at the D3 receptor computationally.40 As compared to
prior work, we found similarities in the key residues that are important for affinity of stepholidine (and the C10
analogues), particularly Asp110, Ser192 and Phe345. Apart from the key protonated nitrogen-Asp110 salt
bridge interaction, in both studies Ser192 interacts with an aromatic hydroxyl group via a hydrogen bond and
Phe345 interacts with an aromatic ring via aromatic stacking (Fig 4a). The main difference is that the
orientation of stepholidine in our Glide docking study is reversed compared to the AutoDock derived pose in
the previous work. Thus in our case Ser192 is H-bonded to the phenolic hydroxyl in ring A and Phe345
interacts with ring A, whereas in prior work Ser192 and Phe345 have interactions with the ring D hydroxyl and
15

ring D respectively. Given the close to symmetric structure of the molecule and its high rigidity, this reversal
has little impact on the interactions with the receptor pocket, except for the formation of an additional H-
bonding interaction between the phenolic group in ring D of stepholidine and the backbone O of Cys181 in our
docked pose.
In order to assess the binding stability of flexible compounds 19-21 the ligands were simulated in
complex with D3 (crystal structure 3pbl).34 Ligand parameters were generated in Generalized Amber Force
Field (GAFF),41 protein parameters were assigned from AMBERFF14SB,42 and the system was solvated in a
10 Å buffer of Tip4pew (~12000 water molecules). Ligands were allowed to move freely, however, protein
heavy atoms were restrained with 2.5 kcal/Å2 harmonic restraints to maintain model accuracy (in the absence
of lipids). The starting configuration utilized for these simulations was the top ranked docked poses discussed
above. These configurations were minimized and heated to 300 K, then equilibrated for 20 ns in NPT, and
finally run in a 100 ns production NVT molecular dynamics simulation sampling every 1 ps.
Tyr373
His349
Figure 7. Docked poses of the flexible analogues, compounds 20 and 21.
The results of these simulations show that the docking poses are stable within this D3 configuration.
Two metrics were utilized to assess ligand stability throughout the production simulations: ligand RMSD and
the distance between the ligand’s center of mass and Asp110 (critical for binding). Ligand RMSD was
computed for each frame of the production trajectories compared to the ligand configuration in the original
docked pose. This analysis shows that while ligand positions fluctuated over the course of the simulation,
however, this did not differ greatly from their starting poses (Figure 8). The distance between ligand center of
mass and Asp110 was utilized to determine whether the ligand maintained this key contact over the course of
16

the simulation. Though the distance does change by a small amount in compounds 19 and 21, all three
ligands maintain a bound state during the simulation (Figure 9). These data suggest that compounds 19-21
maintain a configuration similar to the predicted docked pose during a molecular dynamics simulation and that
these predicted poses are stable with respect to the crystal D3 configuration. This supports the assertion that
the difference in binding affinity is not likely due to destabilized binding, but rather the added ligand flexibility
and decreased lipophilic interactivity.
Figure 8. RMSD of flexible compounds 19 (left), 20 (middle) and 21 (right) between all atom coordinates at each frame,
compared to the coordinates of the docked pose (starting configuration). All three compounds display limited motion within
the binding site, fluctuating about their original docked pose.
Figure 9. Distance between flexible compounds 19 (left), 20 (middle), and 21 (right) center of mass and the key aspartate
Asp110 over the course of a 100 ns simulation. All three ligands maintain this contact throughout the sampled time.
3. Conclusions
The purpose of this study was to shed light on how substituent groups at the C10 position of THPBs as
well as molecular rigidity impact σ2 receptor and D3 receptor affinity and selectivity (as compared to σ1, D1
and D2 receptors). Among the 5 receptors evaluated, the C10 THPB analogues seem to exhibit a general
preference for the D1 receptor. The lowest D1 affinity seen for any analogue was 40 nM (for compound 15).
17

Thus the C10 position seems to be fairly tolerant of alkoxy substituent groups with respect to D1 affinity. Of all
C10 analogues tested, the lowest affinities were seen for D2 and σ1 receptors. Among the 3 dopamine
receptors, affinity was generally highest for D1 receptor and lowest for the D2 receptor. Overall, the general
order of affinities for C10 analogues at the 5 receptors evaluated is D1>σ-2>D3>D2≈σ1. However, individual
compounds may exhibit high σ2 receptor affinity and selectivity. This is the situation with compounds 10 and
11. Compound 10 is 15-fold more selective for the σ2 receptor as compared to the D1 receptor, thirty-seven-
fold more selective as compared to the D3 receptor and approximately 100-fold more selective as compared to
the σ1 receptor. A similar selectivity profile was obtained for compound 11. The data suggests that relatively
small n-alkoxy groups can be accommodated at C10 for high σ2 affinity and σ2 versus σ1 selectivity. It
appears that high σ2 affinity may be obtained (perhaps to the detriment of selectivity) with small branched
alkoxy groups (as is seen in for compound 17). However the generality of this premise will be need to be
tested via the evaluation of a larger set of analogues. Compound 10 is the racemic form of the known natural
product isocorypalmine which has been reported to possess anti-cocaine effects in animals.43 It is not clear at
this time to what extent σ2 receptor affinity may play in this activity of naturally-occurring isocorypalmine.
Rigidity of the THPB core seems to be important to impart high σ2 receptor affinity but de-ridification of the
THPB core negatively impacts dopamine receptor affinity to a greater extent than σ receptor affinity.
The ligand binding energies from the D3 receptor docking studies show some similarities and minor
discrepancies compared to the experimentally derived affinities. This is not surprising as empirical scoring
functions, such as Glidescore used in this study, cannot rank order correctly and quantitatively discriminate all
protein-ligand complexes based on the predicted scores. Such scoring functions work best in terms of
distinguishing ligands as active or inactive, in contrast to quantitative rank ordering. In the current work, the
relatively small series of compounds analyzed in the biological assays and in silico simulations, the relatively
small range of affinities observed and the formation of multiple ligand binding modes in the D3 receptor binding
pocket represent additional causes for the absence of a clear correlation between the docking outcomes and
the experimentally derived affinities. Further exploration of a significantly larger and structurally more diverse
ligand library would be required to provide deeper understanding and quantitative evaluation of the
effectiveness of our in silico modeling process. Nevertheless, despite these issues, the predictive model
successfully differentiated the flexible analogues from the more rigid systems and allowed for the identification
of important protein-ligand interactions within the D3 receptor binding site that should potentially be conserved
in the search for more active and selective compounds. The lack of crystal structures for σ receptors
precluded similar studies at these receptors; this would have been similarly insightful, had it been possible.
This study is the first to investigate structural requirements of stepholidine analogues for affinity to σ
receptors and the dopamine D3 receptor, via the utility of a combination of SAR and computational techniques.
Our work has provided important revelations into the future design of THPB-based D3 and σ2 ligands and has
uncovered compounds with higher D3 and σ2 receptor affinity than the lead, such as compound 10 and the
18

new THPBs 11, 12 and 17. These compounds may be useful as leads for further SAR explorations to optimize
σ2 and D3 affinity and selectivity. In closing, we posit that the THPB scaffold represents a good starting point
from which to mine ligands with interesting mono- and multi-receptor profiles, particularly at dopamine D3 and
σ2 receptors.
4. Experimental
4. 1
General methods
All moisture-sensitive and oxygen-sensitive reactions were carried out in flame-dried glassware under a
nitrogen atmosphere. Solvents and all other reagents were purchased at the highest commercial quality from
Aldrich and Fisher Scientific USA and used without further purification. Anhydrous sodium sulfate was used as
drying agent for work-up of reactions. HRESIMS spectra were obtained using an Agilent 6520 QTOF
1
instrument. H NMR and 13C NMR spectra were recorded using Bruker DPX-500 spectrometer (operating at
500 MHz for 1H; 125 MHz, for 13C) or Bruker Avance III spectrometer (operating at 400 MHz for 1H; 100 MHz,
for 13C) using CDCl3 as solvent, unless stated otherwise. Tetramethylsilane  0.00 ppm) served as an internal
standard in 1H NMR and CDCl3  77.0 ppm) in 13C NMR unless stated otherwise. Chemical shift  0.00 ppm)
values are reported in parts per million and coupling constants in Hertz (Hz). Splitting patterns are described as
singlet (s), doublet (d), triplet (t), and multiplet (m). Reactions were monitored by TLC with Whatman Flexible
TLC silica gel G/UV 254 precoated plates (0.25 mm). TLC plates were visualized by UV (254 nm) and by
staining in an iodine chamber. Flash column chromatography was performed with silica gel 60 (EMD
Chemicals, 230-400 mesh, 0.063 µm particle size).
4.2
Chemistry
General procedure for synthesis of compounds 10-17 (as described for compound 10):
3,9,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (10)
Compound 9 (0.1 g, 0.24 mmol) was dissolved in DMF. Potassium carbonate (0.07 g, 0.48 mmol) and methyl
iodide (0.02 mL, 0.36 mmol) were added and the reaction mixture was stirred overnight at rt. The crude
reaction solution was filtered and the filtrate was evaporated to dryness. The residue was refluxed in a mixture
of MeOH (5 mL) and concentrated HCl (3 mL) for 3 h. The MeOH was evaporated and the crude mixture was
basified with ammonia solution, and extracted with DCM (3 × 15 mL). The combined organic layer was dried
over anhydrous Na2SO4 and concentrated to give the crude product, which was purified by flash column
chromatography on silica gel (2% MeOH/DCM) to give compound 10 (0.052 g, 64%) as an off-white solid; mp
1
208-210 °C; H NMR (CDCl3, 400 MHz) δ 6.87-6.77 (m, 3H), 6.60 (s, 1H), 5.56 (s, 1H), 4.24 (d, J = 15.7 Hz,
1H), 3.87 (s, 3H), 3.85 (s, 6H), 3.54-3.50 (m, 2H), 3.26-3.11 (m, 3H), 2.81 (dd, J = 16, 11.5 Hz, 1H), 2.68-2.61
(m, 2H); 13C NMR (CDCl3, 100 MHz) δ 150.2, 145.1, 143.9, 143.9, 130.6, 128.7, 127.9, 126.1, 124.0, 111.3,
19

111.0, 110.6, 60.2, 59.3, 59.3, 55.9, 54.0, 51.7, 36.3, 29.2; HRMS (ESI) m/z calcd. for C20H23NO4 [M+H]+,
342.1700, found 342.1704.
10-Ethoxy-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (11)
1
Yield 67%; off-white powder, mp 186-188 °C ; H NMR (CDCl3, 400 MHz) δ 6.85-6.77 (m, 3H), 6.60 (s, 1H),
4.24 (d, J = 15.8 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 3.52 (d, J = 15.3 Hz, 2H), 3.27-
3.10 (m, 3H), 2.84-2.61 (m, 3H), 1.44 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 147.5, 143.5 143.2,
142.0, 128.7, 126.7, 125.9, 124.2, 122.0, 110.5, 109.5, 108.7, 62.5, 58.2, 57.4, 54.0, 52.2, 49.8, 34.4, 27.3,
13.1; HRMS (ESI) m/z calcd. for C21H25NO4 [M+H]+, 356.1856, found 356.1861.
3,9-Dimethoxy-10-propoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (12)
Yield 68%; brown solid, mp 87-90 °C; 1H NMR (CDCl3, 500 MHz) δ 6.84-6.78 (m, 3H), 6.60 (s, 1H), 4.23 (d, J =
15.8 Hz, 1H), 3.94 (dt, J = 6.7, 1.8 Hz, 2H), 3.87 (s, 6H), 3.51 (d, J = 15.3 Hz, 2H), 3.25-3.10 (m, 3H), 2.80 (dd,
J = 15.6, 11.4 Hz, 1H), 2.68-2.60 (m, 2H), 1.84 (sext, J = 7.1 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3,
125 MHz) δ 149.6, 145.3, 145.1, 144.0, 130.6, 128.6, 127.8, 126.0, 123.8 112.3, 111.3, 110.6, 70.4, 60.2,
59.2, 55.9, 54.1, 51.7, 36.3, 29.2, 22.8, 10.7; HRMS (ESI) m/z calcd. for C22H27NO4 [M+H]+, 370.2013, found
370.2020.
10-Butoxy-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (13)
1
Yield 73%; orange oil; H NMR (CDCl3, 400 MHz) δ 6.84-6.77 (m, 3H), 6.60 (s, 1H), 4.24 (d, J = 15.7 Hz, 1H)
3.98 (dt, J =1.0, 6.45 Hz, 2H), 3.86 (s, 6H), 3.52 (d J = 15.0 Hz, 2H), 3.25-3.10 (m, 3H), 2.80 (dd, J = 15.2,
11.3 Hz, 1H), 2.67-2.60 (m, 2H), 1.80 (quint, J = 6.9 Hz, 2H), 1.52 (sex, J = 7.6 Hz, 2H), 0.98 (t, J = 7.4 Hz,
3H); 13C NMR (CDCl3, 100 MHz) δ 149.6, 145.3, 145.1, 143.9, 130.5, 128.5, 127.7, 126.0, 123.8, 112.2, 111.3,
110.6, 68.5, 60.1, 59.2, 55.9, 54.0, 51.6, 36.2, 31.5, 29.2, 19.4, 13.9; HRMS (ESI) m/z calcd. for C23H29NO4
[M+H]+, 384.2169, found 384.2173.
3,9-Dimethoxy-10-(pentyloxy)-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (14)
1
Yield 65%; brown oil; H NMR (CDCl3, 400 MHz ) δ 6.84-6.76 (m, 3H), 6.60 (s, 1H), 4.23 (d, J = 15.8 Hz, 1H),
3.97 (dt, J =1.5, 6.55 Hz, 2H), 3.87 (s, 6H), 3.51 (d, J = 15.2 Hz, 2H), 3.26-3.10 (m, 3H), 2.80 (dd, J = 15.7,
13
11.5 Hz, 1H), 2.68-2.60 (m, 2H), 1.82 (quint, J = 6.7 Hz, 2H), 1.49-1.36 (m, 4H), 0.93 (t, J = 7.2 Hz, 3H); C
NMR (CDCl3, 100 MHz) δ 149.6, 145.3, 145.0, 143.9, 130.6, 128.6, 127.7, 126.1, 123.8, 112.3, 111.3, 110.6,
68.9, 60.2, 59.2, 55.9, 54.6, 51.6, 36.3, 29.2, 29.1, 28.3, 22.5, 14.0; HRMS (ESI) m/z calcd. for C24H31NO4
[M+H]+, 398.2326, found 398.2326.
10-(Hexyloxy)-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (15)
20

1
Yield 70%; dark brown oil; H NMR (CDCl3, 500MHz ) δ 6.84-6.76 (m, 3H), 6.60 (s, 1H), 4.23 (d, J = 15.7 Hz,
1H), 3.97 (t, J = 6.6 Hz , 2H), 3.86 (s, 6H), 3.53-3.50 (m, 2H), 3.26-3.10 (m, 3H), 2.80 (dd, J = 15.8, 11.6 Hz,
1H), 2.68-2.60 (m, 2H), 1.81 (quin, J = 6.7 Hz, 2H), 1.48 (quint, J= 7.2, 2H), 1.34 (sex, J = 3.8 Hz, 4H), 0.91 (t,
J= 7.0 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ 149.6, 145.3, 145.0, 143.9, 130.6, 128.6, 127.7, 126.1, 123.8,
112.3, 111.4, 110.6, 68.9, 60.2, 59.2, 55.9, 54.1, 51.7, 36.3, 31.6, 29.4, 29.2, 25.8, 22.6, 14.0; HRMS (ESI) m/z
calcd. for C25H33NO4 [M+H]+, 412.2482, found 412.2429.
10-(3-Hydroxypropoxy)-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (16)
Yield 76%; reddish-brown solid, mp 73-78 °C; 1H NMR (CDCl3, 500MHz ) 6.87-6.80 (m, 3H), 6.60 (s, 1H), 4.22
(d, J = 15.7 Hz, 1H), 4.16 (td, J = 6.1, 1.5 Hz, 2H), 3.91-3.84 (m, 8H), 3.52 (d, J = 15.4 Hz, 2H), 3.27-3.10 (m,
13
3H), 2.80 (dd, J = 15.7, 11.6 Hz, 1H), 2.68-2.61 (m, 2H), 2.07 (quint, J = 5.8 Hz, 2H); C NMR (CDCl3, 125
MHz) δ 149.4, 145.4, 145.1, 143.9, 130.5, 128.8, 128.4, 126.1, 124.1, 112.5, 111.3, 110.6, 67.5, 61.0, 60.3,
59.2, 55.9, 54.0, 51.6, 36.3, 32.1, 29.2; HRMS (ESI) m/z calcd. for C22H27NO5 [M+H]+, 386.1884, found
386.1897.
10-(Cyclopropylmethoxy)-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol
(17)
Yield 81%; yellow oil; 1H NMR (CDCl3, 500 MHz ) δ 6.83-6.76 (m,3H), 6.60 (s, 1H), 4.23 (d, J = 15.7 Hz, 1H),
3.90- 3.83 (m, 8H), 3.51 (d, J = 15.1 Hz, 2H), 3.26-3.11 (m, 3H), 2.80 (dd, J = 15.0, 12.0 Hz, 1H), 2.68-2.62 (m,
2H), 1.31-1.27 (m, 1H), 0.61 (q, J = 5.7 Hz, 2H), 0.36 (q, J = 4.85 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 149.5,
145.7, 145.0, 143.9, 130.6, 128.6, 128.1, 126.1, 123.8, 113.1, 111.3, 110.6, 73.9, 60.2, 59.2, 55.9, 54.1, 51.6,
36.3, 29.2, 10.5, 3.2, 3.2; HRMS (ESI) m/z calcd. for C23H27NO4 [M+H]+, 382.2013, found 382.2019.
N-(3-(Benzyloxy)-2-methoxybenzyl)-2-(4-(benzyloxy)-3-methoxyphenyl)ethan-1-amine (18)
Amine 6 (0.25g, 0.97 mmol) and 3-(benzyloxy)-2-methoxybenzaldehyde (0.24 g ,0.97 mmol) were dissolved in
DCM and the solution stirred at 0 ºC for 10 min. Sodium triacetoxyborohydride (0.31 g, 1.5 mmol) was added
to the reaction mixture at 0 ºC and allowed to stir for 3 h at rt. The reaction mixture was washed with saturated
NaHCO3 and the organic layer was dried over sodium sulfate and concentrated to give the compound 18 (0.4
1
g, 83%) as a brown oil; H NMR (CDCl3, 500MHz ) δ 7.40-7.27 (m, 10H), 6.98-6.95 (m, 1H), 6.89-6.6.85 (m,
2H), 6.79-6.74 (m, 2H), 6.66 (dd, J = 1.6, 8.1 Hz, 1H), 5.12 (s, 2H), 5.10 (s, 2H), 3.85-3.79 (m, 8H), 2.86 (t, J =
7.3, 2H), 2.77 (t, J = 7.3 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 151.6, 149.5, 147.7, 146.5, 137.3, 137.0,
133.5, 133.0, 128.6, 128.5, 128.0, 127.7, 127.3, 127.2, 123.8, 122.2, 122.1, 121.3, 120.6, 114.7, 114.1, 113.3,
112.4, 111.9, 71.1, 70.1, 60.7, 55.9, 50.3, 48.8, 35.7 ; HRMS (ESI) m/z calcd. for C31H33NO4 [M+H]+, 484.2488,
found 484.2475.
21

2-(3-Hydroxy-2-methoxybenzyl)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol (19)
Compound 18 (0.3 g, 0.6 mmol) was dissolved in DCM. 1,1 diethoxyethane (0.13 mL, 0.9 mmol) and TFA
(0.14 mL, 1.8 mmol) were added to the reaction mixture and allowed to stir o.n. The reaction mixture was
washed with saturated NaHCO3 and organic layer was dried over sodium sulfate and evaporated to dryness.
The residues were refluxed in a mixture of MeOH (5 mL) and concentrated HCl (3 mL) for 3 h. The methanol
was evaporated and the crude mixture was basified with ammonia solution, and extracted with
dichloromethane (3 × 10 mL). The combined organic layer was dried over sodium sulfate and concentrated to
give the crude product, which was purified by flash column chromatography on silica gel (3% MeOH/DCM) to
1
give the compound 19 (0.12 g, 60%) as a yellow powder, mp 56-58 °C; H NMR (CDCl3, 500 MHz) δ 7.00-6.98
(m, 2H), 6.88 (dd, J =6.85, 2.8 Hz, 1H), 6.64 (s, 1H), 6.55 (s, 1H), 3.85-3.79 (m, 8H), 3.67 (d, J = 13.6 Hz, 1H),
3.07-3.02 (m, 1H), 2.83-2.77 (m, 1H), 2.68 (dt, J = 12.5, 4.5 Hz, 1H), 2.58 (dt, J = 13, 4.5 Hz, 1H), 1.37 (d, J =
6.6 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ 149.0, 145.9, 144.9, 143.6, 133.0, 132.7, 125.6, 124.6, 122.1,
114.3, 113.0, 110.6, 61.8, 56.3, 55.9, 52.2, 44.0, 27.0, 20.0; HRMS (ESI) m/z calcd. for C19H23NO4 [M+H]+,
330.1700, found 330.1707.
General procedures for synthesis of compounds 20 and 21 (as described for compound 20):
3-(((4-Hydroxy-3-methoxyphenethyl)(methyl)amino)methyl)-2-methoxyphenol (20)
Amine 18 (0.2g, 0.41 mmol) and formaldehyde (0.06 mL, 1.64 mmol) were dissolved in DCM and allowed to
stir the solution at 0 ºC for 10 minutes. Sodium triacetoxy borohydride (0.13g, 0.62 mmol) was added in to the
reaction mixture at 0 ºC and allowed it to stir for 3 h at room temperature. Reaction mixture was washed with
saturated NaHCO3 and organic layer was dried over sodium sulfate and concentrated. The residues were
refluxed in to the mixture of methanol (5 mL) and concentrated HCl (3 mL) for 3 h. Methanol was evaporated
and the crude mixture was basified with ammonia solution, and extracted with dichloromethane (10 mL × 3).
The combined organic layers were dried over sodium sulfate and concentrated to give the crude product,
which was purified by flash column chromatography on silica gel (3% methanol/dichloromethane) to give the
1
compound 20 (0.1g, 74%) as a yellow oil; H NMR (CDCl3, 500 MHz) δ 7.0-6.89 (m, 3H), 6.82 (d, J = 8.1 Hz,
1H), 6.68-6.66 (m, 2H), 3.86 (s, 3H), 3.80 (s, 3H), 3.64 (s, 2H), 2.79 (s, 2H), 2.70 (s, 2H), 2.33 (s, 3H); 13C
NMR (CDCl3, 125 MHz) δ 149.0, 146.3, 145.9, 143.8, 124.7, 122.5, 121.2, 121.2, 114.2, 114.1, 114.0, 111.2,
61.7, 55.9, 55.7, 52.2, 42.0, 33.3; HRMS (ESI) m/z calcd. for C18H23NO4 [M+H]+, 318.1700, found 318.1693
3-((Ethyl(4-hydroxy-3-methoxyphenethyl)amino)methyl)-2-methoxyphenol (21)
Yield 68%, brown oil; 1H NMR (CDCl3, 500MHz ) δ 6.98-6.93 (m, 2H), 6.87 (dd, J = 2.0, 7.5 Hz, 1H), 6.80 (d, J
= 8.4 Hz, 1H), 6.65-6.64 (m, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.67 (s, 2H), 2.71 (s, 4H), 2.62 (q, J = 6.8 Hz, 2H),
13
1.1 (t, J = 7.0 Hz, 3H); C NMR (CDCl3, 125 MHz) δ 149.0, 146.3, 145.7, 143.7, 124.6, 122.3, 121.3, 121.3,
22

114.3, 114.1, 114.1, 111.2, 61.7, 55.8, 55.3, 51.9, 47.5, 33.0, 11.8; HRMS (ESI) m/z calcd. for C19H25NO4
[M+H]+, 332.1856, found 332.1851.
5. Acknowledgements
This publication was made possible by Grant Numbers 1SC1GM092282 (WWH), G12MD007599 (WWH), and
5SC3GM095417 (TK) from the National Institutes of Health. Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of the NIH or its divisions. Ki determinations, and
receptor binding profiles were generously provided by the National Institute of Mental Health's Psychoactive
Drug Screening Program, Contract # HHSN-271-2008-00025-C (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 Driscol at
NIMH, Bethesda MD, USA. For experimental details please refer to the PDSP website
http://pdsp.med.unc.edu/ and click on "Binding Assay" or "Functional Assay" on the menu bar.
References
1.
2.
3.
4.
5.
Micheli, F.; Heidbreder, C. Expert Opin. Ther. Pat .2013, 23, 363.
Heidbreder, C. CNS Neurol. Disord. Drug Targets 2008, 7, 410.
Keck, T. M.; John, W. S.; Czoty, P. W.; Nader, M. A.; Newman, A. H. J Med Chem 2015, 58, 5361.
Heidbreder, C. Naunyn Schmiedebergs Arch. Pharmacol. 2013, 386, 167.
Luedtke, R. R.; Rangel-Barajas, C.; Malik, M.; Reichert, D. E.; Mach, R. H. Curr. Pharm. Des. 2015, 21,
3700
6.
7.
8.
Pich, E. M.; Collo, G. Eur Neuropsychopharmacol. 2015, 25, 1437.
Gross, G.; Wicke, K.; Drescher, K. U. Naunyn Schmiedebergs Arch. Pharmacol. 2013, 386, 155.
Hackling, A. E.; Stark, H. Chembiochem 2002, 3, 946.
9.
10.
11.
Newman, A. H.; Grundt, P.; Nader, M. A. J. Med. Chem. 2005, 48, 3663.
Bouchard, P.; Quirion, R. Neuroscience 1997, 76, 467.
Quirion, R.; Bowen, W. D.; Itzhak, Y.; Junien, J. L.; Musacchio, J. M.; Rothman, R. B.; Su, T. P.; Tam,
S. W.; Taylor, D. P. Trends Pharmacol. Sci. 1992, 13, 85.
12.
13.
McCann, D. J.; Weissman, A. D.; Su, T. P. Synapse 1994, 17, 182.
Hellewell, S. B.; Bruce, A.; Feinstein, G.; Orringer, J.; Williams, W.; Bowen, W. D. Eur. J. Pharmacol.
1994, 268, 9.
14.
15.
16.
McCann, D. J.; Su, T. P. Eur. J. Pharmacol.1990, 188, 211.
Guitart, X.; Codony, X.; Monroy, X. Psychopharmacology (Berl) 2004, 174, 301.
Walker, J. M.; Bowen, W. D.; Walker, F. O.; Matsumoto, R. R.; De Costa, B.; Rice, K. C. Pharmacol.
Rev. 1990, 42, 355.
17.
18.
19.
20.
21.
22.
23.
Matsumoto, R. R.; Nguyen, L.; Kaushal, N.; Robson, M. J. Adv. Pharmacol.2014, 69, 323.
Robson, M. J.; Noorbakhsh, B.; Seminerio, M. J.; Matsumoto, R. R. Curr. Pharm. Des. 2012, 18, 902.
Fishback, J. A.; Robson, M. J.; Xu, Y. T.; Matsumoto, R. R. Pharmacol. Ther. 2010, 127, 271.
Matsumoto, R. R. Expert Rev. Clin. Pharmacol. 2009, 2, 351.
Abate, C.; Niso, M.; Infantino, V.; Menga, A.; Berardi, F. Eur. J. Pharmacol. 2015, 758, 16.
Zeng, C.; Garg, N.; Mach, R. H. Mol. Imaging Biol. 2015.
Xu, J.; Zeng, C.; Chu, W.; Pan, F.; Rothfuss, J. M.; Zhang, F.; Tu, Z.; Zhou, D.; Zeng, D.;
Vangveravong, S.; Johnston, F.; Spitzer, D.; Chang, K. C.; Hotchkiss, R. S.; Hawkins, W. G.; Wheeler,
K. T.; Mach, R. H. Nat. Commun. 2011, 2, 380.
24.
Mach, R. H.; Smith, C. R.; al-Nabulsi, I.; Whirrett, B. R.; Childers, S. R.; Wheeler, K. T. Cancer Res.
1997, 57, 156.
23

25.
26.
27.
Wheeler, K. T.; Wang, L. M.; Wallen, C. A.; Childers, S. R.; Cline, J. M.; Keng, P. C.; Mach, R. H. Br. J.
Cancer 2000, 82, 1223.
Zeng, C.; Rothfuss, J.; Zhang, J.; Chu, W.; Vangveravong, S.; Tu, Z.; Pan, F.; Chang, K. C.; Hotchkiss,
R.; Mach, R. H. Br. J. Cancer 2012, 106, 693.
van Waarde, A.; Rybczynska, A. A.; Ramakrishnan, N. K.; Ishiwata, K.; Elsinga, P. H.; Dierckx, R. A.
Biochim. Biophys. Acta. 2015, 1848, 2703.
28.
29.
30.
Mach, R. H.; Zeng, C.; Hawkins, W. G. J. Med. Chem. 2013, 56, 7137.
Hornick, J. R.; Spitzer, D.; Goedegebuure, P.; Mach, R. H.; Hawkins, W. G. Surgery 2012, 152, S152.
Makvandi, M.; Tilahun, E. D.; Lieberman, B. P.; Anderson, R. C.; Zeng, C.; Xu, K.; Hou, C.; McDonald,
E. S.; Pryma, D. A.; Mach, R. H. Biochem. Biophys. Res. Commun. 2015, 467, 1070.
Sun, H.; Zhu, L.; Yang, H.; Qian, W.; Guo, L.; Zhou, S.; Gao, B.; Li, Z.; Zhou, Y.; Jiang, H.; Chen, K.;
Zhen, X.; Liu, H. Bioorg. Med. Chem. 2013, 21, 856.
31.
32.
Qian, W.; Lu, W.; Sun, H.; Li, Z.; Zhu, L.; Zhao, R.; Zhang, L.; Zhou, S.; Zhou, Y.; Jiang, H.; Zhen, X.;
Liu, H. Bioorg. Med. Chem. 2012, 20, 4862.
33.
34.
Roth, B. L.; Lopez, E.; Beischel, S.; Westkaemper, R. B.; Evans, J. M. Pharmacol. Ther. 2004, 102, 99.
Chien, E. Y.; Liu, W.; Zhao, Q.; Katritch, V.; Han, G. W.; Hanson, M. A.; Shi, L.; Newman, A. H.;
Javitch, J. A.; Cherezov, V.; Stevens, R. C. Science 2010, 330, 1091.
35.
36.
37.
Gadhiya, S.; Ponnala, S.; Harding, W. W. Tetrahedron 2015, 71, 1227.
Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. J. Immunol. Methods 1997, 204, 205.
Behensky, A. A.; Yasny, I. E.; Shuster, A. M.; Seredenin, S. B.; Petrov, A. V.; Cuevas, J. J. Pharmacol.
Exp. Ther. 2013, 347, 458.
38.
Rybczynska, A. A.; Dierckx, R. A.; Ishiwata, K.; Elsinga, P. H.; van Waarde, A. J. Nucl. Med. 2008, 49,
2049.
39.
40.
41.
42.
Korpis, K.; Weber, F.; Wunsch, B.; Bednarski, P. J. Pharmazie 2014, 69, 917.
Li, B.; Li, W.; Du, P.; Yu, K. Q.; Fu, W. J. Phys. Chem. B 2012, 116, 8121.
Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. J. Comput. Chem. 2004, 25, 1157.
Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. J. Chem.
Theory Comput. 2015, 11, 3696.
43.
Xu, W.; Wang, Y.; Ma, Z.; Chiu, Y. T.; Huang, P.; Rasakham, K.; Unterwald, E.; Lee, D. Y.; Liu-Chen, L.
Y. Drug Alcohol Depend. 2013, 133, 693.
24