Synthesis and pharmacological evaluation of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives as sigma-2 receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2017.11.016

Increasing evidences have implicated that sigma-2 receptor is a biomarker and significantly over-expressed in many proliferative cancer cells with no or low expression in normal cells. Sigma-2 receptor selective ligands have been successfully used as valuable tools to study its pharmacological functions, tumor imaging, and cancer therapeutics or adjuvants. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolinylalkyl benzamides are among a few categories of structures that have demonstrated high affinities and selectivities for sigma-2 receptor and been used extensively as study tools in various tumor imaging and therapy. As a continuous effort, we have synthesized a new series of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives and evaluated their affinities for both sigma-1 and sigma-2 receptors. Most of these newly developed analogs showed good to excellent binding affinities for sigma-2 receptor with no or low affinities for sigma-1 receptor. In particular, compounds 3b, 3e, 4b, and 4e demonstrated K_i values of 5–6 nM affinities and excellent selectivities for sigma-2 receptor. In addition, these analogs also demonstrated moderate anticancer activities against human liver Huh-7 tumor cells and human esophagus KYSE-140 cancer cells. But their cytotoxicities seem not to be correlated with their sigma-2 receptor affinities.

Full text of DOI:10.1016/j.ejmech.2017.11.016

Accepted Manuscript
Synthesis and pharmacological evaluation of 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline derivatives as sigma-2 receptor ligands
Yu-Tong Sun, Gui-Fei Wang, Yi-Qiu Yang, Fujun Jin, Yifei Wang, Xiao-Yang Xie,
Robert H. Mach, Yun-Sheng Huang
PII:
S0223-5234(17)30909-1
10.1016/j.ejmech.2017.11.016
EJMECH 9891
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2017
Revised Date: 13 October 2017
Accepted Date: 5 November 2017
Please cite this article as: Y.-T. Sun, G.-F. Wang, Y.-Q. Yang, F. Jin, Y. Wang, X.-Y. Xie, R.H. Mach, Y.-
S. Huang, Synthesis and pharmacological evaluation of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
derivatives as sigma-2 receptor ligands, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.016.
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ACCEPTED MANUSCRIPT
Graphical abstract
3b
R1 = Me, R2,R4,R5 = H, R3 = OMe, X = O;
3e R1,R3 = OMe, R2,R4,R5 = H, X = O;
4b
R1 = Me, R2,R4,R5 = H, R3 = OMe, X = OH;
4e R1,R3 = OMe, R2,R4,R5 = H, X = OH.
Analog
3b
K
> 1000
32.9 + 4.1
> 1000
> 1000
iσ1 ( nM)
K
iσ2 ( nM)
Huh-7 (µM)
KYSE-140 (µM)
5.1 + 1.5
5.8 + 0.8
5.2 + 1.0
6.2 + 1.6
3e
4b
4e
12.50 + 0.36
14.86 + 0.90

ACCEPTED MANUSCRIPT
Synthesis and pharmacological evaluation of
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives as
sigma-2 receptor ligands
Yu-Tong Suna,b, Gui-Fei Wanga, Yi-Qiu Yanga, Fujun Jinc, Yifei Wangc, Xiao-Yang Xiea.b, Robert H
Machd,* Yun-Sheng Huanga,b*
a School of Pharmacy, Guangdong Medical University, 1 Xincheng Ave, Songshan Lake Technology
Park, Dongguan, Guangdong 523808，China.
b
Dongguan Key Laboratory for Drug Design & Formulation, 1 Xincheng Ave, Songshan Lake
Technology Park, Dongguan, Guangdong 523808，China.
c Institute of Biomedicine, College of Life Science & Technology, Jinan University, Guangzhou 510632,
China.
d Department of Radiology, University of Pennsylvania, 231 S. 34th St, Philadelphia, PA 19104, USA
* Corresponding author: Yun-Sheng Huang, Tel: +86-0769-22896547, Email: yshuang@gdmu.edu.cn
* Corresponding author: Robert H Mach, Tel: 215-746-8233, Email: rmach@mail.med.upenn.edu
Abstract
Increasing evidences have implicated that sigma-2 receptor is a biomarker and
significantly over-expressed in many proliferative cancer cells with no or low
expression in normal cells. Sigma-2 receptor selective ligands have been successfully
used as valuable tools to study its pharmacological functions, tumor imaging, and
cancer therapeutics or adjuvants. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolinylalkyl
benzamides are among a few categories of structures that have demonstrated high
affinities and selectivities for sigma-2 receptor and been used extensively as study
tools in various tumor imaging and therapy. As a continuous effort, we have
synthesized a new series of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives
and evaluated their affinities for both sigma-1 and sigma-2 receptors. Most of these
newly developed analogs showed good to excellent binding affinities for sigma-2
receptor with no or low affinities for sigma-1 receptor. In particular, compounds 3b,
3e, 4b, and 4e demonstrated Ki values of 5-6 nM affinities and excellent selectivities
for sigma-2 receptor. In addition, these analogs also demonstrated moderate
anticancer activities against human liver Huh-7 tumor cells and human esophagus
KYSE-140 cancer cells. But their cytotoxicities seem not to be correlated with their
sigma-2 receptor affinities.
Keywords: Sigma receptor, ligands, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline,
affinity
1

ACCEPTED MANUSCRIPT
1. Introduction
Sigma receptors have attracted extensive interests in the scientific fields of
pharmacology, oncology, and medicinal chemistry due to their possible involvement
in diverse roles of physiological and neurological disorders as well as diseases such as
cancer, Alzheimer’s disease, schizophrenia, memory loss or damaged cognition [1-4].
Sigma receptors are currently classified into two subtypes, termed sigma-1 (σ1) and
sigma-2 (σ2) [5]. Their physiological functions have not yet been clearly elucidated
and their corresponding natural ligands are still not known, though some candidates
such as some neurosteroids and N,N-dimethyltryptamine (DMT) were indicated as
potential endogenous ligands for sigma-1 receptor [1,4]. Both receptors are widely
expressed in the central nervous system (CNS) as well as in many peripheral tissues
such as in the liver, kidney, prostate, etc [4,6]. Accumulating evidences indicate that
sigma-1 receptor is involved in CNS regulated cognition and movement, while the
sigma-2 receptor may play important roles in cell morphology, survival and
differentiation [1,4,7,8]. Sigma-1 receptor was cloned in 1996 and proposed to be a
transmembrane protein with a molecular weight of ~25.3 KDa [9]. Despite the
confirmed presence of a 21.5 kDa protein by photoaffinity labeling, the sigma-2
receptor was not able to be cloned for a very long time [10]. Due to its similarity to
progesterone receptor membrane component 1 (PGRMC1) protein in association with
tumorigenesis and coexistence in cellular environment [11,12], the sigma-2 receptor
was widely suggested to be or a component of the PGRMC1 protein complex [13].
But more recent studies have indicated that PGRMC1 protein and sigma-2 receptor
are two different bio-molecules [14-17]. Most recently, Alon and coworkers have been
able to purify the sigma-2 protein by an affinity column and identify the gene for the
sigma-2 receptor [18]. Therefore, they have provided more convincing evidence to
distinguish sigma-2 receptor from PGRMC1 protein. According to their study, the
sigma-2 receptor is the same as TMEM97 (an endoplasmic reticulum-resident
transmembrane protein that regulates the sterol transporter NPC1) and contains 4
transmembrane segments and 176 amino acids with Asp 29 and Asp 56 being the
essential ligand binding residues. The successful cloning and revealing of the sigma-2
receptor will accelerate the research toward better understanding and potential
application of this receptor in drug discovery.
An increasing interest in sigma-2 receptor is largely due to its involvement in
oncogenesis and cancer cell proliferation [19,20]. Sigma-2 receptor has been
identified to be a cancer cell proliferative biomarker that has almost 10 folds higher
densities in proliferative cancer cells than in quiescent tumor tissue [21-24]. Various
radio- or fluorescent labeled sigma-2 ligands have demonstrated to specifically bind
to the tumor tissues and thus are potentially useful for cancer diagnosis [25-34].
Another developing field is that sigma-2 ligands (agonists) bind to siamg-2 receptor
and inhibit cancer cell proliferation and trigger cancer cell death via apoptosis
pathway [1,4]. Many sigma-2 ligands showed strong inhibitory effects against various
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cancer cell lines both in vitro and in animal models [35-37]. In addition, sigma-2
ligands also demonstrated strong potentiation or synergistic effects when combined
with an anticancer agent [38-40]. Yet another newly developing field is to use sigma-2
receptor ligands as carriers that specifically deliver anticancer agents to the tumor
sites and thus to kill the cancer cells with no or minimum effects to normal cells
[41-45]. Though the exact mechanism of action of sigma-2 receptor and its ligands is
unknown, some evidence points to the coupling of sigma-2 receptor to cell signaling
pathways that may involve caspases, kinases, Ca2+ channels, K+ channels, reactive
oxygen species (ROS), etc [1,3,4].
The structural moiety of piperidine is the hallmark pharmacophore for both
sigma-1 and sigma-2 receptor binding pockets [1,46,47]. Though the sigma-1 receptor
has a wide variety of structural tolerance with various alkylamines binding to the
sigma-1 receptor with high affinities, the piperidine derived cycloalkylamine analogs
demonstrated extremely potent affinities for sigma-1 receptor [46-48]. On the other
hand, most reported sigma-2 ligands more or less contain the piperidine structure or
some modified form of it (Scheme 1). Among the four reported categories of sigma-2
receptor pharmacophores, compounds containing the
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety demonstrated the overall
highest selectivity for sigma-2 receptor over sigma-1 receptor [1,49-52]. Thus, in
order to be sigma-2 selective, the piperidine structure must be modified with more
bulky elements incorporated. We discovered, for the first time, that
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline was an ideal piperidine analog and that
compounds containing this moiety bound to sigma-2 receptor with high affinities and
selectivities [53]. Since our initial publication, many
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline containing benzamide analogs have
been synthesized and reported to have good to excellent affinities and selectivities for
sigma-2 receptor [1,54-59]. Recently, some hybrid analogs containing an indole
moiety and a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure linked with an
alkyl chain showed excellent affinities and selectivities for sigma-2 receptor
[50,51,60,61]. Some of these analogs demonstrated good anticancer activity against
multidrug resistant tumors [60,62]. In an effort to further explore the binding
requirement and discover novel and more selective sigma-2 ligands, we synthesized a
new series of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogs without the
benzamide moiety and evaluated their binding affinities for both sigma receptors.
Among them, four analogs demonstrated nanomolar affinities for sigma-2 receptor
and moderate or no significant affinities for sigma-1 receptor. These compounds are
among the most potent and selective sigma-2 ligands reported and thus potentially
useful as pharmacological tools to study the functions of sigma-2 receptor or can be
developed as cancer diagnostic agents. In addition, these analogs showed moderate
anticancer activities against human liver Huh-7 cancer cells and human esophagus
KYSE-140 cancer cells.
2. Results and discussion
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2.1. Chemistry
The benzamide structure was once believed to be involved in the binding to the
sigma-2 receptor, but later indicated not to be a required sigma-2 receptor
pharmacophore because a number of non-benzamide analogs, such as
indole-6,7-dimethoxy-1,2,3,4-tetrahydroisoquine hybrid analogs without the amide
moiety, were reported to have high affinities and selectivities for sigma-2 receptor
[50,60]. Efforts toward the modifications of the
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure did not improve the binding
affinity or selectivity for the sigma-2 receptor. For example, the removal, replacement,
or cyclolization of the 6,7-dimethoxy substituents resulted in reduced or loss of
activity; the opening or enlargement of the piperidine moiety, or changing the nitrogen
position on the piperidine ring of the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
moiety resulted in significantly decreased or loss of activity; the contraction of the
piperidine ring to form 5,6-dimethoxyindoline maintained binding activity for
sigma-2 receptor [54-56].
The strategy for this study was to synthesize a new series of hybrid compounds
containing 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline and an aromatic moiety of
electron rich or deficiency with an alkyl chain linkage between them. The syntheses
of these analogs were very straightforward and outlined in Schemes 2 and Scheme 3.
An appropriately substituted benzene was carried out Friedel-Crafts acylation with a
chloroalkylacyl chloride, followed by substitution of the chloro atom by
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline to give the phenyl ketone products
3a-3f, and subsequently undergone reduction with sodium borohydride to afford the
hydroxyl product 4a-4e (Scheme 2). A series of quinazoline substituted analogs were
prepared according to the well known established methods, substituted at the 3-N
position with a chloroalkyl bromide and followed by replacement of the chloro atom
by 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline to give the products 8a-8e (Scheme
3). All the novel intermediates were characterized by 1H NMR unless otherwise
notified and all the test analogs were confirmed by 1H NMR, 13C NMR, and elemental
analysis. Both the 1H NMR and 13C NMR results indicated that analog 3f lost one
methyl group of the tetramethoxyl substituted aromatic ring. This tetramethoxyl
substituted aromatic series of analogs are being further investigated and will be
reported elsewhere.
2.2. Pharmacology
Compounds 3a-3f, 4a-4e, and 8a-8e were evaluated for their binding affinities for
both sigma-1 and sigma-2 receptors according to methods as previously described
[48,58]. Sigma-1 receptor competitive binding assays were conducted in guinea pig
brain membrane homogenates with [3H](+)-pentazocine as the radioligand. Sigma-2
receptor binding assays were conducted in rat liver membrane homogenates with
[3H]di-o-tolylguanidine (DTG) as the radioligand and in the presence of 100 nM of
(+)-pentazocine (to mask the sigma-1 sites). All test compounds were dissolved in
ethanol, diluted to various concentrations ranging from 0.1 nM to 1000 nM in 50 mM
Tris-HCl (pH 8.0), added to the incubation at 25°C and terminated by the addition of
4

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ice-cold 10 mM Tris-HCl (pH 8.0). Initially, all the test compounds were subjected to
a displacement assay at 1 µM concentration and only those with significant
displacement of the [3H](+)-pentazocine (sigma-1) or [3H]-DTG (sigma-2) were
identified as “hits” (greater than 50% displacement). The hits were then undergone
full assays and were measured for their binding Ki values for sigma-1 and sigma-2
receptors. Nonspecific bindings were determined in the presence of 10 µM
haloperidol for sigma-1 receptor and 5 µM DTG for sigma-2 receptor. All binding
experiments were repeated at least three times and the binding results are shown in
Table 1.
The binding results in Table 1 indicated that all the tested compounds, except for
3f, 8c, 8d, and 8e, showed moderate to excellent affinities for sigma-2 receptor. Only
two analogs, 3a and 3e, showed moderate binding affinities for sigma-1 receptor with
Ki(σ1) values of 13.3 and 32.9 nM, respectively, and the rest of the analogs had no
measurable affinities for sigma-1 receptor. In particular, analogs 3b, 3e, 4b and 4e
demonstrated the highest binding affinities with Ki(σ2) values of 5.1, 5.8, 5.2 and 6.2
nM, respectively, for sigma-2 receptor. Since analogs 3b, 4b and 4e had no
measurable affinities for sigma-1 receptor, these three analogs displayed the highest
selectivities of >196, >192, and >161, respectively, for sigma-2 over sigma-1 receptor.
Analogs 3a, 3c, 3d, 4a, 4c, and 4d also demonstrated moderate affinities with Ki(σ2)
values of 23.7, 53.8, 68.1, 18.2, 62.0, and 30.2 nM, respectively, for sigma-2 receptor.
It is interesting to note that all the hydroxyl series of analogs (4a-4e) did not show any
measurable affinities for sigma-1 receptor, whereas two of the corresponding ketone
analogs, 3a and 3e, showed moderate affinities for sigma-1 receptor. Therefore, the
reduction of the ketone moiety (3a-3e) to form the corresponding hydroxyl group
(4a-4e) has no harmful effect or slightly positive effect for sigma-2 receptor affinity
and has a negative effect for sigma-1 receptor binding. This may attribute to the
conjugation between the carbonyl group and the aromatic moiety and the electron
withdrawing effect of the carbonyl group, resulting in a reduced electron density on
the aromatic moiety that is projected to bind to the primary hydrophobic binding
pocket of the sigma-2 receptor [1,58]. Analog 3f was designed as a tetramethoxyl
substituted aromatic ketone and expected to show at least some sigma-2 receptor
affinity. As a result, one of the methoxyl group (most likely the one adjacent to the
carbonyl group or its para- position) was demethylated (very likely during the
Friedel-Crafts reaction) and the resultant trimethoxyphenol ketone did not show any
measurable affinity for both sigma receptors. The exact reason for the lack of affinity
of 3f toward either sigma receptor is not clear. However, it is well known that both
sigma-1 and sigma-2 receptors’ binding pocket consist at least one primary
hydrophobic binding site, one ionizable nitrogen binding site, and maybe an
additional hydrophobic binding site (even more true for sigma-2 receptor) [1]. Thus
the lack of binding affinity of 3f for both sigma receptors is probably due to an
increased hydrophilicity that may have a detrimental effect toward the binding of the
phenol aromatic moiety to the primary hydrophobic binding pocket of the receptor
protein.
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A number of quinazoline linked 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
analogs 8a-8e were also synthesized and measured their affinities for both sigma
receptors. Only 8a and 8b showed moderate affinities with Ki values of 13.9 and 43.8
nM, respectively, for sigma-2 receptor and all these quinazoline analogs did not
display any affinities for sigma-1 receptor. Quinazoline is an electron deficient
aromatic moiety and therefore does not favorably bind to the primary hydrophobic
binding pocket of the sigma receptor. However, when the quinazoline ring is
substituted with two methoxyl groups, the resultant analogs 8a and 8b bind to the
sigma-2 receptor with moderate affinities, which may be explained that the electron
donating methoxyl groups reverse the electron deficiency and help the substituted
quinazoline moiety bind to the sigma-2 receptor’s primary hydrophobic binding site.
On the other hand, 6-nitro-7-chloro substituted quinazoline moiety is even more
electron deficient than the quinazoline ring itself. As expected, the corresponding
analogs 8c-8e did not show any binding affinities for either receptor.
These analogs were tested their anticancer activities against human liver Huh-7
cancer cells and human esophagus KYSE-140 cancer cells. Several of them showed
moderate cytotoxicities agsinst either or both cell lines. Particularly, analog 4b
displayed IC50 values of 12.5 µM and 14.86 µM against the growth of Huh-7 and
KYSE-140 cells, respectively, which are comparable to 15.31 µM and 21.34 µM
against Huh-7 and KYSE-140 cells, respectively, for cisplatin in the same assay.
Analogs 3a, 3d, 3f, and 4d demonstrated moderate anticancer activities with IC50
values of 42.83, 75.33, 81.63, and 45.89 µM, respectively, against Huh-7 cell growth.
Analogs 3a, 3e, 3f, and 4d also demonstrated moderate activities with IC50 values of
60.11, 88.32, 93.73, 26.10 µM, respectively, against KYSE-140 cell growth. These
analogs’s anticancer activities are comparable to the IC50 values for several recently
published sigma ligands (though against different cancer cell lines) [63]. Analog 4b
was one of the best sigma-2 ligands in this study and also had the most potent
anticancer activity against both Huh-7 and KYSE-140 cells, but the other two potent
and selective sigma-2 ligands 3b and 3e did not show similar potent anticancer
activities against Huh-7 cells or KYSE-140 cells (Note: analog 4e was not available at
the time of anticancer screening and not tested in this assay). Therefore, the anticancer
activities of these analogs may not attribute to their sigma-2 receptor interactions.
Another possibility is that analog 4b is an agonist for sigma-2 receptor, whereas,
analogs 3b and 3e are antagonists for sigma-2 receptor. Thus, it is necessary to further
investigate the agnostic/antagonistic profile and mechanism of action for these
analogs in future studies.
3. Conclusions
A
series of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogs were
synthesized and evaluated for both sigma-1 and sigma-2 receptor affinities. Many of
these analogs showed good to excellent affinities for sigma-2 receptor and no or
moderate affinities for sigma-1 receptor. Among them, analogs 3b, 3e, 4b and 4e
showed the highest affinities and analogs 3b, 4b and 4e had the highest selectivities
6

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for sigma-2 receptor. The common structural feature among these four analogs is that
all
of
them
have
5
carbon
units
between
the
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety and the aromatic moiety. These
four analogs are among the most potent and selective sigma-2 ligands reported and
thus are potentially useful for the development of cancer diagnostic agents, functional
study tools, and anticancer adjuvants. Analog 4b has cytotoxicities that are
comparable to those of cisplatin against both liver Huh-7 and esophagus KYSE-140
cancer cells and thus may be worth further investigation of its mechanism of
anticancer action and potentially useful for drug development against liver or
esophageal cancer.
4. Experimental Section
4.1. Chemistry
All reagents or solvents purchased commercially were used directly without
further purification. Reactions were monitored by analytical thin-layer
chromatography (TLC) on silica gel F254 glass plates and visualized under UV light
(254 and 365 nm) and temperatures were recorded using regular thermometer without
correction. Flash column chromatography was performed on silica gel (200–300
1
mesh). Melting points were measured using capillary method without correction. H
NMR spectra were recorded with a Bruker Avance III 400 MHz NMR spectrometer at
room temperature. Chemical shifts were recorded as parts per million (ppm)
downfield to tetramethylsilane (TMS). The following abbreviations are used for
multiplicity of NMR signals: s) singlet, d) doublet, t) triplet, q) quartet, m) multiplet,
dd) doublet of doublets, td) triplet of doublets; dt) doublet of triplets, dq) doublet of
quartets, b) broad, 13C NMR or DEPT-13C (distortionless enhancement by polarization
transfer 13C-NMR) spectra were recorded with Bruker Avance III 400 MHz NMR
spectrometer (100 MHz).
4.1.1. 3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(4-methoxy-2-methy
lphenyl)propan-1-one (3a)
To a 100 mL flask was added dichloromethane (50 mL), 3-methylanisole (5 g,
40.9 mmol), AlCl3 (6.5 g, 48.7 mmol). The mixture was stirred at stirred at 0 oC for 30
min and added 3-chloropropinoyl chloride (5.2 g, 40.9 mml). The reaction was stirred
o
at 0 C for 6 hr and then was poured into ice-water, extrcted with dichloromethane,
washed with brine, and dried over anhydrous Na2SO4. The solvent was removed and
the product was recrystallized from ethyl acetate and petroleum to give 2a as a white
solid (in freezer) that quickly became an oil in room temperature (7.6 g, yield 87%).
1H NMR (DMSO-d6, ppm): δ 7.68-7.70 (d, 1H), 6.73-6.80 (m, 2H), 3.81-3.84 (m, 5H),
3.31-3.34 (t, 2H), 2,53 (s, 3H).
The above intermediate 2a (5 g, 23.5 mmol)was dissolved in dichloromethane (25
mL) and was added 6,7-dimethoxy1,2,3,4-tetrahydroisoquinoline hydrochloride (5.4 g,
23.5 mmol) and triethylamine (4 mL, 28.8 mmol). The reaction mixture was stirred at
rt overnight and then poured into ice-water, extracted with dichloromethane, washed
7

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with brine and dried over Na2SO4. The solvent was removed and the crude product
was purified by silica gel colum to afford 3a as a light yellow oil (6.8 g, yield 78%,
1
HPLC 97.9%). H NMR (CDCl3, ppm): δ 7.75-7.78 (t, 1H), 6.74-6.77 (m, 2H), 6.59
(s, 1H), 6.52 (s, 1H), 3.82-3.83 (m, 9H), 3.61 (s, 2H), 3.18-3.22 (t, 2H), 2.93-2.97 (t,
2H), 2.80-2.82 (t, 2H), 2.75-2.77 (t, 2H), 2.54 (s, 3H). 13C NMR (CDCl3, ppm): δ
200.89, 161.82, 147.51, 147.18, 142.04, 131.54, 129.99, 126.34, 125.98, 117.53,
111.31, 110.61, 109.41, 55.90, 55.87, 55.70, 55.30, 53.39, 51.11, 38.83, 28.67, 22.38.
Anal: C 71.28, H 7.23, N 3.66 (Calcd: C 71.52, H 7.37, N 3.79).
4.1.2. 3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(4-methoxy-2-methy
lphenyl)propan-1-ol (4a)
A solution of 3a (4.5 g, 12.2 mmol) in methanol (15 mL) and NaBH4 (0.5 g, 13.2
mmol) was stirred at rt for 6 hr. The mixture was poured into water and extracted with
ethyl acetate, washed with brine and dried over Na2SO4. The solvent was removed in
vacuum and the product was purified by silica gel column to give 4a as a white solid
1
product (3.8 g, yield 84%, HPLC 98.0%). mp 113-115℃; H NMR (CDCl3, ppm): δ
7.48-7.50 (d, 1H), 6.78-6.81 (dd, 1H), 6.70-6.71 (d, 1H), 6.63 (s,1H), 6.57 (s, 1H),
5.10-5.13 (m, 1H), 3.87 (6H, 2OMe), 3.78-3.83 (4H, CH-O + OMe), 3.62-3.78 (m,
13
2H), 2.74-2.94 (m, 6H), 2.32 (s, 3H), 1.88-1.93 (m, 2H); C NMR (CDCl3, ppm): δ
158.24, 147.67, 147.31, 135.44, 135.19, 126.77, 125.98, 125.87, 115.77, 111.30,
111.07, 109.41, 72.06, 56.95, 55.97, 55.94, 55.87, 55.19, 50.81, 32.70, 28.56, 19.20;
Anal: C 70.91, H 7.68, N 3.59 (Calcd: C 71.13, H 7.87, N 3.77).
4.1.3. 5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(4-methoxy-2-methy
lphenyl)pentan-1-one (3b)
5-Chloro-1-(4-methoxy-2-methylphenyl)pentan-1-one (2b) was prepared
according to the procedure of 2a and abtained as a white solid in 80% yield. mp
97-98℃; 1H NMR (CDCl3, ppm): δ 7.71-7.74 (d, 1H), 6.75-6.78(m, 2H), 3.85 (s, 3H),
3.56-3.59 (m, 2H), 2.88-2.96 (m, 2H), 2.55 (s, 3H), 1.84-1.87 (m, 4H).
5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(4-methoxy-2-methylphen
yl)pentan-1-one (3b) was prepared according to the procedure of 3a and obtained as
1
an oil in 78% yield (HPLC 98.0%). H NMR (CDCl3, ppm): δ 7.72-7.74 (d, 1H),
6.68-6.74 (m, 2H), 6.58 (s, 1H), 6.51 (s, 1H). 3.81-3.83 (t, 9H), 3.54 (s, 2H),
2.90-2.94 (t, 2H), 2.79-2.82 (t, 2H), 2.68-2.71 (t, 2H), 2.51-2.55 (m, 5H), 1.73-1.79
(m, 2H), 1.64-1.70 (m, 2H); 13C NMR (CDCl3, ppm): δ 202.12, 161.86, 147.43,
147.13, 141.86, 131.61, 130.08, 126.70, 126.22, 117.47, 111.31, 110.50, 109.45, 58.06,
55.90, 55.87, 55.83, 55.27, 51.03, 40.58, 28.71, 26.86, 22.79, 22.42; Anal: C 72.56, H
7.77, N 3.61 (Calcd: C 72.52, H 7.86, N 3.52).
4.1.4. 5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(4-methoxy-2-methy
lphenyl)pentan-1-ol (4b)
5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(4-methoxy-2-methylphen
yl)pentan-1-ol (4b) was prepared according to the procedure of 4a to give a white
1
solid in 86% yield (HPLC 98.8%). mp 131-132℃; H NMR (CDCl3, ppm): δ
8

ACCEPTED MANUSCRIPT
7.35-7.37 (d, 1H), 6.72-6.75 (dd, 1H), 6.66-6.67 (d, 1H), 6.58 (s, 1H), 6.49 (s, 1H),
4.81-4.84 (m, 1H), 3.79-3.85 (m, 10H, CH-O + 3OMe), 3.53 (s, 2H), 2.80-2.83 (t,
13
2H), 2.70-2.73 (t, 2H), 2.49-2.53 (t, 2H), 2.29 (s, 3H), 1.54-1.77 (m, 6H); C NMR
(CDCl3, ppm): δ 158.34, 147.50, 147.17, 135.95, 135.58, 126.51, 126.19, 126.01,
115.76, 111.27, 111.23, 109.40, 70.03, 58.03, 55.89 (2C), 55.59, 55.16, 50.96, 37.95,
28.34, 26.80, 23.94, 19.30; Anal: C 71.92, H 8.19, N 3.66 (Calcd: C 72.15, H 8.33, N
3.45).
4.1.5. 2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphen
yl)ethanone (3c)
2-Chloro-1-(2,4-dimethoxyphenyl)ethanone (2c) was prepared according to the
o
1
procedure of 2a and obtained as a white solid in 87.5% yield. mp 116-118 C; H
NMR (DMSO-d6, ppm): δ 7.95-7.98 (d, 1H), 6.57-6.60 (dd, 1H), 6.47-6.48 (d, 1H),
4.77 (s, 2H), 3.94 (s, 3H), 3.88 (s, 3H).
2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphenyl)eth
anone (3c) was prepared according to the procedure of 3a and was obtained as a light
yellow solid in 79% yield (HPLC 98.2%). mp 117-119 oC; 1H NMR (CDCl3, ppm): δ
7.90-7.93 (d, 1H), 6.46-6.60 (m, 4H), 3.76-3.99 (m, 16H), 2.86-2.92 (m, 4H); 13C
NMR-DEPT135 (CDCl3, ppm): δ 132.86, 111.33, 109.37, 105.32, 98.19, 67.88, 55.88,
55.58, 55.50, 55.38, 51.18, 28.51; Anal: C 68.06, H 6.65, N 3.71 (Calcd: C 67.91, H
6.78, N 3.77).
4.1.6. 2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphen
yl)ethanol (4c)
2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphenyl)eth
anol (4c) was prepared according to the procedure of 4a to give a white solid in 92%
o
1
yield (HPLC 99.1%). mp 119-120 C; H NMR (CDCl3, ppm): δ 7.46-7.49 (d, 1H),
6.64 (s, 1H), 6.53-6.57 (m, 2H), 6.46-6.47 (d, 1H), 5.18-5.21 (m, 1H), 3.83-3.88
(4OMe+CH2 + CH-O, 15H), 3.63-3.66 (d, 1H), 3.01-3.07 (m, 1H), 2.74-2.95 (m, 4H),
2.57-2.62 (m, 1H); 13C NMR-DEPT135 (CDCl3, ppm): δ 127.10, 111.36, 109.38,
104.26, 98.31, 64.27, 63.89, 55.97, 55.94, 55.49, 55.40, 55.37, 51.02, 28.62; Anal: C
64.37, H 7.31, N 3.54 (Calcd (+H2O): C 64.43, H 7.47, N 3.58).
4.1.7. 3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphen
yl)propan-1-one (3d)
3-Chloro-1-(2,4-dimethoxyphenyl)propan-1-one (2d) was prepared according to
o
1
the procedure of 2a and obtained as a white solid in 92% yield. mp 98-99 C; H
NMR (DMSO-d6, ppm): δ 7.69-7.72 (d, 1H), 6.60-6.65 (m, 2H), 3.84-3.90 (m, 8H),
3.36-3.39 (t, 2H).
3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphenyl)pro
pan-1-one (3d) was prepared according to the procedure of 3a and obtained as a light
yellow oil in 97.7% yield (HPLC 97.8%). 1H NMR (DMSO-d6, ppm): δ 7.73-7.75 (d,
1H), 6.56-6.62 (m, 4H), 3.90 (s, 3H), 3.83 (s, 3H), 3.72 (s, 6H), 3.50 (s, 2H), 3.19 (b,
13
2H), 2.83 (b, 2H), 2.71 (b, 2H), 2.65 (b, 2H); C NMR (DMSO-d6, ppm): δ 198.54,
9

ACCEPTED MANUSCRIPT
164.60, 160.98, 147.72, 147.47, 132.38, 126.98, 126.27, 120.88, 112.02, 110.27,
106.10, 98.67, 55.97, 55.77 (2Cs), 55.71, 55.52, 53.62, 51.09, 41.41, 28.86; Anal: C
68.42, H 6.94, N 3.49 (Calcd: C 68.55, H 7.06, N 3.63).
4.1.8. 3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphen
yl)propan-1-ol (4d)
3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphenyl)pro
pan-1-ol (4d) was prepared according to the procedure of 4a to give a white solid in
93% yield (HPLC 99.2%). mp 114-116 oC; 1H NMR (DMSO-d6, ppm): δ 7.31-7.33 (d,
1H), 6.63-6.65 (d, 2H), 6.51-6.52 (m, 2H), 4.91-4.94 (m, 1H), 3.69-3.77 (m, 13H,
CH-O + 4OMe), 3.44-3.46 (m, 2H), 2.70-2.72 (m, 2H), 2.55-2.63 (m, 4H), 1.63-1.89
(m, 2H); 13C NMR (DMSO-d6, ppm): δ 159.62, 156.78, 147.60, 147.34, 127.10,
127.07, 126.98, 126.31, 112.15, 110.39, 104.83, 98.38, 65.71, 55.91, 55.88, 55.77,
55.68, 55.54, 55.45, 51.19, 35.42, 28.78; Anal: C 68.07, H 7.28, N 3.55 (Calcd: C
68.20, H 7.54, N 3.61).
4.1.9. 5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphen
yl)pentan-1-one (3e)
5-Chloro-1-(2,4-dimethoxyphenyl)pentan-1-one (2e) was prepared according to
o
1
the procedure of 2a and obtained as a white solid in 89% yield. mp 58-59 C; H
NMR (CDCl3, ppm): δ 7.80-7.22 (d, 1H), 5.52-5.55 (dd, 2H), 5.46-5.47 (d, 1H), 3.90
(s, 3H), 3.87 (s, 3H), 3.56-3.59 (m, 2H), 2.97-3.00 (m, 2H), 1.83-1.85 (m, 4H).
5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphenyl)pen
tan-1-one (3e) prepared according to the procedure of 3a and obtained as a white solid
o
1
in 73% yield (HPLC 97.7%). mp 71-72 C; H NMR (CDCl3, ppm): δ 7.78-7.80 (d,
1H), 6.58 (s, 1H), 6.51-6.52 (m, 2H), 6.44-6.45 (d, 1H), 3.82-3.87 (4OMe, 12H), 3.54
(s, 2H), 2.97-3.01 (t, 2H), 2.79-2.82 (t, 2H), 2.68-2.71 (t, 2H), 2.51-2.54 (t, 2H),
1.63-1.76 (m, 4H); 13C NMR (CDCl3, ppm): δ 200.50, 164.24, 160.65, 147.44, 147.14,
132.64, 126.55, 126.16, 121.24, 111.29, 109.44, 105.03, 98.35, 58.24, 55.84, 55.80,
13
55.77, 55.45, 55.41, 50.99, 43.45, 28.61, 26.97, 22.59; C NMR-DEPT135 (CDCl3,
ppm): δ 132.64, 111.28, 109.43, 105.03, 98.34, 58.32, 55.90, 55.87, 55.82, 55.51,
55.46, 51.04, 43.47, 28.72, 27.03, 22.62;. Anal: C 69.59, H 7.45, N 3.26 (Calcd: C
69.71, H 7.56, N 3.39).
4.1.10. 5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphen
yl)pentan-1-ol (4e)
5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2,4-dimethoxyphenyl)pen
tan-1-ol (4e) was prepared according to the procedure of 4a to give a white solid in
o
1
93% yield (HPLC 99.0%). mp 91-92 C; H NMR (CDCl3, ppm): δ 7.21-7.23 (dd,
1H), 6.59 (s, 1H), 6.51 (s, 1H), 6.45-6.47 (m, 2H), 4.81-4.85 (m, 1H), 3.81-3.85
(4OMe, 12H), 3.54 (s, 2H), 2.95 (b, 1H), 2.81-2.84 (t, 2H), 2.68-2.71 (t, 2H),
2.48-2.52 (t, 2H), 1.77-1.84 (m, 2H), 1.61-1.68 (m, 2H), 1.49-1.56 (m, 1H), 1.37-1.45
(m, 1H); 13C NMR-DEPT135 (CDCl3, ppm): δ 127.46, 111.28, 109.42, 104.00, 98.58,
69.98, 58.27, 55.90, 55.88, 55.78, 55.37, 55.28, 51.07, 37.10, 28.60, 26.98, 24.03;
10

ACCEPTED MANUSCRIPT
Anal: C 69.59, H 7.45, N 3.26 (Calcd: C 69.71, H 7.56, N 3.39).
4.1.11. 3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-1-(2-hydroxy-3,4,5-tri
methoxy-6-methylphenyl)propan-1-one (3f)
A solution of 1,2,3,4-tetramethoxy-5-methylbenzene (1.0 g, 4.7 mmol) in
dichloromethane (25 mL) was added with AlCl3 (0.75 g, 5.6 mmol) and stirred at 0 oC.
A solution of 3-chloropropanoyl chloride (0.65 g, 5.1 mmol) was added dropwise at 0
oC and then the reaction was stirred at rt overnight. The reaction mixture was poured
onto 10% HCl ice-water, extracted with dichloromethane, washed with brine and
dried, solvent removed to give 2f (1.05 g, crude yield 74%) as an oil product that
decomposed quickly upon standing or column purification, therefore was used
directly in the next reaction.
To a solution of 2f (0.4 g, 1.3 mmol) in dichloromethane (10 mL) and was added
6,7-dimethoxy1,2,3,4-tetrahydroisoquinoline hydrochloride (0.3 g, 1.3 mmol) and
triethylamine (0.4 g, 4.0 mmol). The reaction was stirred at rt for 6 hr and extrated
with dichloromethane, eashed with brine, dried over anhydrious sodium sulfate. The
crude product was purified by silica gel column to give 3f as a light yellow solid (0.21
o
1
g, 36%). mp 87-90 C; H NMR (CDCl3, ppm): δ 6.58 (s, 1H), 6.51 (s, 1H), 3.96 (s,
3H), 3.82-3.85 (m, 9H), 3.75 (s, 3H), 3.69 (s, 2H), 3.05-3.10 (m, 4H), 2.86 (s, 4H),
2.17 (s, 3H); 13C NMR (CDCl3, ppm): δ 206.89, 148.48, 147.75, 147.38, 145.73,
144.51, 139.67, 125.50, 125.10, 123.68, 123.63, 111.29, 109.40, 61.04, 61.03, 60.82,
55.92, 55.91, 55.12, 53.70, 50.88, 42.55, 27.49, 12.76; Anal: C 64.73, H 6.97, N 3.08
(Calcd: C 64.70, H 7.01, N 3.14).
4.1.12. 3-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-6,7-dimetho
xyquinazolin-4(3H)-one (8a)
To a 100 mL flask was added 3,4-dimethoxybenzoic acid (10g, 54.9 mmol) and
nitric acid (50 mL, 20%) in an ice-bath. The reaction mixture was then stirred at 60 oC
for 6 hr. After cooling to rt, the mixture was poured onto ice-water. The solid was
filtered, washed with water, dried. The crude was purified by a silica gel column to
afford 2-nitro-4,5-dimethoxybenaoic acid as a light yellow solid (9.6 g, yield 77%).
o
1
mp 195-197 C; H NMR (CDCl3, ppm): δ 7.42 (s, 1H), 7.26 (s, 1H), 4.03 (s, 3H),
4.02 (s, 3H).
A solution of 2-nitro-4,5-dimethoxybenaoic acid (8 g, 35.2 mmol) in EtOH ( 50
mL) was added with ammonium formate (8.8 g, 139.7 mmol) and 5% Pd-C (0.19 g)
and wad heated to reflux for 6 hr. The reaction was cooled to rt and filtered off the
solid. The solution was concentrated to dryness that was then purified by silica gel
column to give 2-amino-4,5-dimethoxy benzoic acid (5a) as a white solid (6 g, yield
o
1
65%). mp 156-157 C; H NMR (CDCl3, ppm): δ 7.36 (s, 1H), 6.17 (s, 1H), 3.91 (s,
3H), 3.86 (s, 3H).
6,7-dimethoxyquinazolin-4(3H)-one (6a) was prepared according to a similar
procedure of Luth and Lowe [62], with formamide in replace of formamidine acetate.
Briefly, a solution of 5a (5 g, 25.4 mmol) in formamide (30 mL) was heated to reflux
for 4 hr, cooled to rt and poured onto ice-water, extracted with ethyl acetate, washed
11

ACCEPTED MANUSCRIPT
with brine and dried over Na2SO4, After removal of the solvent, the residue was
purified by silica gel column to give 6,7-dimethoxyquinazolin-4(3H)-one (6a) as a
white solid (4.5 g, yield 86%). mp 310-312 oC (reference mp 300 oC [64]).
To a 100 mL flask was added 6a (2 g, 9.7 mmol), DMF (20 mL) and
1-bromo-3-chloropentane (3 g, 19 mmol). The solution was stirred at rt and added
o
NaH (0.47 g, 19.6 mmol) and then heated to 80 C for 6 hr. After cooled to rt, the
solution was poured onto ice-water, extracted with dichloromethane, washed with
brine and dried over Na2SO4. The crude product was purified by silica gel column to
afford 3-(3-chloropropyl)-6,7-dimethoxyquinazolin-4(3H)-one (7a) as an oil (1.7 g,
yield 62%). 1H NMR (CDCl3, ppm): δ 8.28 (s, 1H), 6.83-6.85 (m, 1H), 6.68-6.71 (m,
1H), 3.83-3.88 (m, 8H), 3.48-3.52 (t, 2H). 1.97-2.07 (m, 2H).
A solution of 7a (1.0 g, 3.5 mmol), DMF (10 mL), K2CO3 (1 g, 7.2 mmol), and
o
1,2,3,4-tetrahydroisoquinoline hydrochloride (0.9 g, 3.9 mmol) was stirred at 85 C
for 8 hr. After cooled to rt, the mixture was poured onto ice-water, extracted with
dichloromethane, washed with brine, and dried over Na2SO4. The crude product was
purified by a silica gel column to afford 8a as an oil (1.1 g, yield 71%, HPLC 97.1%).
1H NMR (CDCl3, ppm): δ 8.30 (s, 1H), 6.82-6.86 (m, 1H), 6.69-6.74 (m, 1H), 6.56 (s,
1H), 6.46 (s, 1H), 3.81-3.88 (m, 14H), 3.47-3.48 (s, 2H), 2.75-2.79 (m, 2H), 2.62-2.65
(t, 2H), 2.49-2.53 (t, 2H), 1.81-1.85 (m, 2H); 13C NMR (CDCl3, ppm): δ 162.49,
149.60, 148.20, 147.48, 147.16, 134.06, 126.30, 126.01, 117.39, 111.39, 111.27,
109.36, 108.70, 56.10, 56.04, 55.89, 55.88, 55.62, 55.33, 50.97, 43.85, 28.56, 25.36;
Anal: C 62.87, H 6.67, N 9.07 (Calcd (+H2O): C 63.00, H 6.83, N 9.18).
4.1.13. 3-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-6,7-dimethox
yquinazolin-4(3H)-one (8b)
3-(4-chlorobutyl)-6,7-dimethoxyquinazolin-4(3H)-one (7b) was prepared
according to the procedure of 7a and obtained as an light yellow oil in 63% yield. 1H
NMR (CDCl3, ppm): δ 8.28-8.30 (d, 1H), 6.86-6.88 (m, 1H), 6.67-6.68 (d, 1H),
3.87-3.89 (m, 6H), 3.77-3.80 (t, 2H), 3.52-3.55 (t, 1H), 3.39-3.42 (t, 1H), 1.72-1.92
(m, 4H).
3-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-6,7-dimethoxyquin
azolin-4(3H)-one (8b) was prepared according to the procedure of 8a and obtained as
an oil in 72% yield (HPLC 97.3%). 1H NMR (CDCl3, ppm): δ 8.30 (s, 1H), 6.83-6.86
(m, 1H), 6.73-6.75 (m, 1H), 6.58 (s, 1H), 6.51 (s, 1H), 3.79-3.92 (m, 14H), 3.56 (s,
1H), 2.80-2.83 (t, 2H), 2.70-2.73 (t, 2H), 2.51-2.55 (t, 2H), 1.61-1.63 (m, 4H); 13C
NMR (CDCl3, ppm): δ 162.55, 149.60, 148.24, 147.57, 147.24, 133.87, 125.88,
117.49, 111.39, 111.28, 109.40, 108.77, 57.44, 56.10, 55.92, 55.89, 55.70, 55.50,
50.79, 45.07, 28.28, 25.47, 24.10; Anal: C 64.90, H 6.81, N 8.94 (Calcd (+1/2H2O): C
64.92, H 6.97, N 9.08).
4.1.14. 7-Chloro-3-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-6-nit
roquinazolin-4(3H)-one (8c)
A solution of 2-amino-4-chlorobenaoic acid (20 g, 116.5 mmol) in formamide (50
o
mL) was heated to 130 oC under stirring for 30 min and then at 160 C for 4 hr. after
12

ACCEPTED MANUSCRIPT
cooling to rt, the mixture was poured onto ice-water and the solid wad filtered,
washed and dried, and recrystallized from ethyl acetate to give
1
7-chloroquinazolin-4(3H)-one (6b) as a white solid (19.1 g, yield 91%). H NMR
(DMSO-d6, ppm): δ 12.41 (b, 1H), 8.11-8.15 (m, 2H), 7.73 (s, 1H), 7.55-7.57 (m,
1H).
To a 100-mL flask was added concentrated sulfuric acid (10 mL), concentrated
o
nitric acid (5 mL) and 6b (10 g, 55.4 mmol) under stirring at 0 C. The mixture was
o
heated and stirred at 90 C for 3 hr. After cooling to rt, the mixture was poured onto
ice-water, filtered, washed with water, dried. The crude product was recrystallized
from acetic acid to afford 7-chloro-6-nitroquinazolin-4(3H)-one (6c) as a light yellow
o
1
solid (9 g, yield 72%). mp 315-316 C; H NMR (DMSO-d6, ppm): δ 12.79 (b, 1H),
8.67 (s, 1H), 8.31 (s, 1H), 8.01 (s, 1H).
A solution of 6c (2 g, 8.8 mmol) in DMF (20 mL), 1-bromo-2-chloroethane (1.9 g,
13.2 mmol) and NaH (0.4 g, 17.9 mmol) was stirred at 80 oC for 6 hr, cooled, poured
onto ice-water. The solid was filtered, washed with water, dried, to give
7-chloro-3-(2-chloroethyl)-6-nitroquinazolin-4(3H)-one (7c) as a light yellow solid
o
1
(2.5 g, yield 98%). mp 186-187 C; H NMR (CDCl3, ppm): δ 8.83 (s, 1H), 8.21 (s,
1H), 7.93 (s, 1H), 4.41-4.44 (m, 1H), 4.34-4.37 (m, 1H), 3.93-3.96 (m, 1H), 3.79-3.82
(m, 1H).
A solution of 7c (1 g, 3.47 mmol), 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0,88 g, 3.8 mmol) and K2CO3 (1 g, 7.2 mmol) in DMF (20 mL) was
o
stirred at 80 C for 6 hr. After cooling to rt, the mixture was poured onto ice-water,
filtered, washed and dried. The crude product was purified by a silica gel column to
o
1
afford 8c as a white solid (0.96 g, yield 62%, HPLC 98.6%). mp 179-180 C; H
NMR (CDCl3, ppm): δ 8.69 (s, 1H), 8.13 (s, 1H), 7.30 (s, 1H), 6.67 (s, 1H), 6.62 (s,
1H), 4.34 (s, 2H), 4.28-4.31 (t, 2H), 3.90-3.93 (t, 2H), 3.87-3.88 (d, 6H), 3.52-3.55 (t,
2H), 2.95-2.98 (t, 2H); 13C NMR (CDCl3, ppm): δ 159.55, 151.00, 149.61, 148.76,
148.04, 147.80, 139.75, 126.88, 126.16, 124.48, 114.11, 112.51, 111.30, 109.03, 56.04,
55.99, 51.66, 49.16, 48.99, 41.96, 27.82; Anal: C 56.54, H 4.62, N 12.37 (Calcd: C
56.70, H 4.76, N 12.59).
4.1.15. 7-Chloro-3-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-6-
nitroquinazolin-4(3H)-one (8d)
7-Chloro-3-(3-chloropropyl)-6-nitroquinazolin-4(3H)-one (7d) was prepared
according to the procedure of 7a to afford a light yellow solid in 95% yield, mp
o
1
150-151 C. H NMR (CDCl3, ppm): δ 8.81 (s, 1H), 8.22-8.24 (d, 1H), 7.91 (s, 1H),
4.21-4.25 (m, 2H), 3.61-3.64 (t, 1H), 3.45-3.48 (t, 1H), 2.38-2.44 (m, 1H), 2.30-2.36
(m, 1H).
7-Chloro-3-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-6-nitroqu
inazolin-4(3H)-one (8d) was prepared according to the procedure of 8c as a light
yellow solid in 63% yield (HPLC 98.2%). mp 177-179 oC; 1H NMR (CDCl3, ppm): δ
8.67 (s, 1H), 8.08 (s, 1H), 7.26 (s, 1H), 6.67 (s, 1H), 6.61 (s, 1H), 4.32 (s, 2H),
4.14-4.17 (t, 2H), 3.87-3.88 (d, 6H), 3.59-3.62 (t, 2H), 3.51-3.54 (t, 2H), 2.94-2.97 (t,
13
2H), 2.26-2.32 (m, 2H); C NMR (CDCl3, ppm): δ 159.82, 151.43, 149.37, 148.62,
13

ACCEPTED MANUSCRIPT
148.02, 147.78, 139.80, 126.75, 126.17, 124.54, 114.36, 112.94, 111.31, 109.03, 56.03,
55.98, 51.65, 49.20, 44.52, 41.51, 30.98, 27.83; Anal: C 57.41, H 4.89, N 12.03
(Calcd: C 57.58, H, 5.05, N 12.21).
4.1.16. 7-Chloro-3-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-6-ni
troquinazolin-4(3H)-one (8e)
7-Chloro-3-(4-chlorobutyl)-6-nitroquinazolin-4(3H)-one (7e) was prepared
according to the procedure of 7a to afford a light yellow solid in 91% yield, mp
o
1
153-154 C. H NMR (CDCl3, ppm): δ 8.82 (s, 1H), 8.17 (s, 1H), 7.91 (s, 1H),
4.07-4.11 (m, 2H), 3.47-3.64 (m, 2H), 1.88-2.05 (m, 4H).
7-Chloro-3-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-6-nitroqui
nazolin-4(3H)-one (8e) was prepared according to the procedure of 8c as a light
yellow solid in 65% yield (HPLC 98.8%). mp 175-176 oC; 1H NMR (CDCl3, ppm): δ
8.66 (s, 1H), 8.02 (s, 1H), 7.24 (s, 1H), 6.66 (s, 1H), 6.61 (s, 1H), 4.32 (s, 2H),
3.98-4.02 (t, 2H), 3.86-3.87 (d, 6H), 3.57-3.60 (t, 2H), 3.49-3.52 (t, 2H), 2.93-2.96 (t,
2H), 1.92-2.00 (m, 2H), 1.82-1,89 (m, 2H); 13C NMR (CDCl3, ppm): δ 159.72, 151.39,
149.11, 148.57, 148.00, 147.76, 139.79, 126.81, 126.17, 124.56, 114.35, 112.99,
111.30, 109.04, 56.03, 55.97, 51.63, 49.24, 46.08, 44.15, 29.40, 27.84, 26.92; Anal: C
58.24, H 5.26, N 11.69 (Calcd: C 58.41, H 5.33, N 11.85).
4.2. Biological Assays
4.2.1. Tissue Source and Radioligands
Sigma-1 receptor binding was labeled with [3H]-(+)-pentazocine (DuPont-NEN,
Billerica, MA) in guinea pig brain membranes (Rockland Biological, Gilbertsville, PA)
according to published procedures [10]. Sigma-2 receptor binding was labeled with
[3H]-DTG (DuPont-NEN, Boston, MA) in rat liver membranes in the presence of
(+)-pentazocine.
4.2.2. Membrane Preparation
Quinea pig brain membrane homogenates were prepared from frozen guinea pig
brains without cerebellum. Liver membrane homogenates were prepared from the
livers of male Sprague-Dawley rats (300-350 g) according to reported procedures
[47].
4.2.3. Sigma-1 Binding Assay
Guinea pig brain membrane homogenates (100 µL, 300 µg) were incubated with
50 µL of 6 nM [3H]-(+)-pentazocine (~42 Ci/mmol) and 50 µL of each test compound
solution in 50 mM Tris-HCl (pH 8.0) for a total volume of 200 µL at 25 °C for 120
min. Test compounds were dissolved in ethanol and then diluted in buffer and added
in concentrations ranging from 0.1 to 1000 nM. Incubations were terminated by the
addition of ice-cold 10 mM Tris-HCl (pH 8.0) and followed by rapid filtration through
Whatman GF/B glass fiber filters (presoaked in 0.5% polyethylenimine) using a
Brandel cell harvester (Gaithersburg, MD). Filters were washed 3 times with 5 mL of
ice cold buffer. Each filter was removed with forceps, added into 4 mL vials, added 3
14

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mL microscintillation 20, and counted in microbeta counter with 45% efficiency.
Nonspecific binding was determined in the presence of 10 µM (+)-pentazocine.
4.2.4. Sigma-2 Binding Assay
Rat liver membrane homogenates (100 µL, 300 µg) were incubated with 50 µL of
5.65 nM [3H]DTG (~82 Ci/ mmol), 50 µL of each test compound solution, and 50 µL
of 100 nM (+)-pentazocine in 50 mM Tris-HCl (pH 8.0) for a total volume of 250 µL
at 25 °C for 120 min. Test compounds were dissolved in ethanol and then diluted in
buffer and added in concentrations ranging from 0.1 to 1000 nM. Incubations were
terminated by the addition of ice-cold 10 mM Tris-HCl (pH 8.0) and followed by
rapid filtration through Whatman GF/B glass fiber filters (presoaked in 0.5%
polyethylenimine) using a Brandel cell harvester (Gaithersburg, MD). Filters were
washed 3 times with 5 mL of ice-cold buffer. Each filter was removed with forceps,
added into 4 mL vials, added 3 mL microscintillation 20, and counted in microbeta
counter with 45% efficiency. Nonspecific binding was determined in the presence of 5
µM DTG.
4.2.5. Data Analysis
The IC50 values for sigma receptors were generally determined in triplicate from
nonlinear regression of binding data as analyzed by Prism (GraphPad Software, Inc.,
La Jolla, CA), using -10 concentrations of each compound. Ki values were calculated
using the method of Cheng-Prusoff and represent mean values.
4.2.6. Cytotoxicity Assay
CCK-8 assays were carried out on Huh7 hepatocellular carcinoma cells and
KYSE-140 esophageal squamous carcinoma cells to primarily determine the
anti-cancer activities of these drugs. Briefly, Huh7 and KYSE-140 cells with
exponential growth were seeded into 96-well culture plates at a density of 4,000
cells/well and allowed to adhere overnight. The cells were then treated with a series of
6 concentrations of the test compounds for 48 h. Cells were treated with an equal
volume of DMSO as the control. Thereafter, 10 µL CCK-8 solutions (Dojindo
Laboratories) were added to each well and the plates were incubated at 37 °C for 2 h
in the dark. The UV absorbance was measured at 450nm with an enzyme
immunoassay reader (Bio-Rad). Cell viability was calculated as (optical density of
experimental sample/optical density of control) ×100%. The IC50 values were
calculated using the Prism 6.0 program (GraphPad).
Supporting Information
13
Spectroscopic data (1H and C NMR) for the intermediates and the test compounds
are attached at the end of this manuscript.
Acknowledgments
This research was supported by Guangdong Science Foundation (Grant No.
2016A030313676).
15

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Representative tropane and granatane sigma-2 analogs
Me
CH3
CH3
N
O
N
H
O
H
N
O
NH2
N
N
H
OMe
NH
6
O
Ki ( 1): 1418 + 439 nM
Ki ( 2): 5.19 + 0.8 nM
SV119
Ki ( 1): 6300 nM
Ki ( 2): 32 nM
BIMU-1
Representative cyclohexylpiperazine sigma-2 analogs
N
N
N
N
OMe
N
Ki ( 1): 3450 + 1660 nM
Ki ( 2): 12.6 + 3.2 nM
PB28
Ki ( 1): 13.6 + 1.9 nM
Ki ( 2): 0.34 + 0.02 nM
F281
Representative 6,7-dimethoxytetrahydroisoquinoline benzamide sigma-2 analogs
OMe
OMe
F
OMe
OMe
OMe O
O
O
N
MeO
N
N
N
H
H
Me
I
Ki ( 1): 3078 + 86.89 nM
Ki ( 2): 10.33 + 1.46 nM
Ki ( 1): 2150 + 410 nM
Ki ( 2): 0.26 + 0.07 nM
[18F]ISO-1
RMH-1
Representative indole sigma-2 analogs
MeO
N
O
N
N
N
H
4
F
Ki ( 1): 9310 + 630 nM
Ki ( 2): 90.4 + 10.1 nM
Ibogaine
Ki ( 1): 17 nM
Ki ( 2): 0.12 nM
Siramesine
Lu28-179
Scheme 1. Representative sigma-2 selective ligands from 4 major structural classes.
Binding data were extracted from reference [1].
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Table 1. Sigma receptor binding profiles in cell membranes and cytotoxicities against
liver Huh-7 and KYSE-140 cancer cells for analogs 3a-3f, 4a-4e, 8a-8e.
Ki
(nM + SEM) a
IC50 (µM + SEM)e
b
c
d
Analog
3a
σ1
σ2
σ1/σ2
Huh-7
KYSE-140
60.11 + 2.61
NA
13.3 + 1.6
> 1000
> 1000
> 1000
32.9 + 4.1
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
58.5 + 5.8
23.7 + 7.4
0.5
42.83 + 4.37
NA
3b
3c
5.1 + 1.5
53.8 + 1.9
68.1 + 21.2
5.8 + 0.8
> 1000
> 196
> 18
> 14
5.6
NA
NA
3d
3e
75.33 + 5.81
NA
NA
88.32 + 1.80
93.73 + 5.53
NA
3f
81.63 + 2.85
NAf
4a
18.2 + 3.9
5.2 + 1.0
62.0 + 9.3
30.2 + 3.9
6.2 + 1.6
13.9 + 2.9
43.8 + 0.3
> 1000
> 55
> 192
> 16
4b
4c
12.50 + 0.36
NA
14.86 + 0.90
NA
4d
4e
> 33
45.89 + 5.36
NTg
26.10 + 3.22
NT
> 161
> 71.9
> 22.8
8a
NA
NA
8b
8c
NA
NA
NA
NA
8d
8e
> 1000
NA
NA
> 1000
NA
NA
DTG
19.4 + 1.2
Cisplatin
15.31 + 0.17
21.34 + 1.08
a
Mean (SEM), Ki values were determined by at least three experiments. Each inhibition curve consisted of 10
b
points from each binding assay. Ki values for σ1 receptor were measured on guinea pig brain membranes using
c
[3H](+)-pentazocine as the radioligand. Ki values for σ2 receptor were measured on rat liver membranes using
d
[3H]-DTG as the radioligand in the presence of (+)-pentazocine. Ki value for σ1 receptor/Ki value for σ2 receptor.
e
IC50 values were determined by at least three experiments. Each inhibition curve consisted of7 points from each
inhibition assay against the growth of Huh-7 liver cancer cells. f NA indicates cytotoxicity IC50 > 100 nM or
not accurately determined. g NT indicates not tested.
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Scheme 2. Synthesis of analogs 3a-3f and 4a-4e.
Reagents and conditions: a) chloroalkyl chloride/AlCl3, 0 oC; b) 6,7-dimethoxy-1,2,3,4-tetrahydro
–isoquinoline HCl/Et3N, rt; c) NaBH4, rt.
O
N
O
R1
R2
COOH
NH2
a
R1
R2
R1
R2
Cl
n
b
c
NH
N
N
5a R1,R2 = OMe
5b R1 = H, R2 = Cl
7a R1,R2 = OMe, n = 2
7b R1,R2 = OMe, n = 3
7c R1 = NO2, R2 = Cl, n = 1
7d R1 = NO2, R2 = Cl, n = 2
7e R1 = NO2, R2 = Cl, n = 3
6a R1,R2 = OMe
6b R1 = H, R2 = Cl
6c R1 = NO2, R2 = Cl
OMe
OMe
O
R1
R2
N
n
N
N
8a R1,R2 = OMe, n = 2
8b R1,R2 = OMe, n = 3
8c R1 = NO2, R2 = Cl, n = 1
8d R1 = NO2, R2 = Cl, n = 2
8e R1 = NO2, R2 = Cl, n = 3
Scheme 3. Synthesis of analogs 8a-8e.
Reagents and conditions: a) formamide, reflux; b) bromoalkyl chloride, 80 oC; c)
6,7-dimethoxy-1,2,3,4-tetrahydro –isoquinoline HCl/Et3N, 85 oC.
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Highlights
ò A total of 16 of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogs were
1
synthesized and their structures were confirmed by H NMR, 13C NMR,
elemental analysis.
ò These analogs were assayed for sigma-1 and sigma-2 receptor binding with 4 of
them showing excellent affinities and selectivities for sigma-2 receptor.
ò These four analogs are among the most potent sigma-2 ligands and are potentially
useful for further development as cancer diagnostic agents, anticancer adjuvants,
or pharmacological tools for the study of sigma-2 receptor.
ò These analogs were also screened for their cytotoxicities against liver cancer
Huh-7 and esophagus KYSE-140 cancer cells and analog 4b showed promising
anticancer activities against both Huh-7 and esophagus KYSE-140 cancer cells,
which are comparable to those of cisplatin in the same assay.