Design, synthesis, and biological evaluation of 1,3-diarylisoquinolines as novel topoisomerase I catalytic inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.11.011

With a goal of identifying potent topoisomerase (topo) inhibitor, the C4-aromatic ring of the anticancer agent, 3,4-diarylisoquinolone, was strategically shifted to design 1,3-diarylisoquinoline. Twenty-two target compounds were synthesized in three simple and efficient steps. The 1,3-diarylisoquinolines exhibited potent anti-proliferative effects on cancer cells but few compounds spared non-cancerous cells. Inhibition of topo I/IIα-mediated DNA relaxation by several derivatives was greater than that by camptothecin (CPT)/etoposide even at low concentration (20 μM). In addition, these compounds had little or no effect on polymerization of tubulin. A series of biological evaluations performed with the most potent derivative 4cc revealed that the compound is a non-intercalative topo I catalytic inhibitor interacting with free topo I. Collectively, the potent cytotoxic effect on cancer cells including the drug resistance ones, absence of lethal effect on normal cells, and different mechanism of action than topo I poisons suggest that the 1,3-diarylisoquinolines might be a promising class of anticancer agents worthy of further pursuit.

Full text of DOI:10.1016/j.ejmech.2017.11.011

Accepted Manuscript
Design, synthesis, and biological evaluation of 1,3-diarylisoquinolines as novel
topoisomerase I catalytic inhibitors
Daulat Bikram Khadka, Seojeong Park, Yifeng Jin, Jinhe Han, Youngjoo Kwon, Won-
Jea Cho
PII:
S0223-5234(17)30896-6
10.1016/j.ejmech.2017.11.011
EJMECH 9886
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 July 2017
Revised Date: 3 November 2017
Accepted Date: 4 November 2017
Please cite this article as: D.B. Khadka, S. Park, Y. Jin, J. Han, Y. Kwon, W.-J. Cho, Design, synthesis,
and biological evaluation of 1,3-diarylisoquinolines as novel topoisomerase I catalytic inhibitors,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.011.
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ACCEPTED MANUSCRIPT
Topo I
catalytic
inhibitor
IC50 (µM):
0
.74±0.05 (T47D)
5
.06±0.04 (HeLa)
.77±0.38 (HCT-15)
Topo I inhibition > CPT
2
4
cc

ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation of 1,3-diarylisoquinolines as novel
topoisomerase I catalytic inhibitors
a, 1
b, 1
a
a
b, **
Daulat Bikram Khadka , Seojeong Park , Yifeng Jin , Jinhe Han , Youngjoo Kwon
, Won-
a, *
Jea Cho
a
College of Pharmacy and Research Institute of Drug Development, Chonnam National University,
Gwangju 61186, Republic of Korea
b
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University,
Seoul 120-750, Republic of Korea
*
Corresponding author.
**
Corresponding author.
E-mail address: ykwon@ewha.ac.kr (Y. Kwon), wjcho@chonnam.ac.kr (W.-J. Cho).
1
Both authors contributed equally to this work.
1

ACCEPTED MANUSCRIPT
ABSTRACT
With a goal of identifying potent topoisomerase (topo) inhibitor, the C4-aromatic ring of the
anticancer agent, 3,4-diarylisoquinolone, was strategically shifted to design 1,3-diarylisoquinoline.
Twenty-two target compounds were synthesized in three simple and efficient steps. The 1,3-
diarylisoquinolines exhibited potent anti-proliferative effects on cancer cells but few compounds
spared non-cancerous cells. Inhibition of topo I/IIα-mediated DNA relaxation by several derivatives
was greater than that by camptothecin (CPT)/etoposide even at low concentration (20 µM). In
addition, these compounds had little or no effect on polymerization of tubulin. A series of biological
evaluations performed with the most potent derivative 4cc revealed that the compound is a non-
intercalative topo I catalytic inhibitor interacting with free topo I. Collectively, the potent cytotoxic
effect on cancer cells including the drug resistance ones, absence of lethal effect on normal cells, and
different mechanism of action than topo I poisons suggest that the 1,3-diarylisoquinolines might be a
promising class of anticancer agents worthy of further pursuit.
Keywords:
Antitubulin activity, Topoisomerase catalytic inhibitor, 1,3-Diarylisoquinoline, Suzuki coupling,
Topoisomerase
2

ACCEPTED MANUSCRIPT
1.
Introduction
Deregulated cell proliferation and suppressed cell death are two main characteristics of
cancer [1]. The factors that determine the efficacy of an anticancer therapy include identification and
targeting of the molecular anatomy responsible for the relentless proliferation of tumor cells and the
compensatory mutations necessary for their survival. The success of the anticancer drugs, etoposide,
doxorubicin (DOX), and the camptothecin (CPT) derivatives irinotecan and topotecan, is primarily
attributable to the exploitation of cancer cell mechanisms that differ from those of normal cells.
These anticancer agents interfere with the activity of a specialized class of enzymes called
topoisomerase (topo), which are overexpressed in cancer cells [2-6]. Topo is involved in resolving
the topological consequences of DNA like supercoils, knots, and catenation during cellular processes,
including transcription, recombination, and replication of rapidly proliferating cells [7].
Six topos are expressed in human cells [8-10]. On the basis of function they are classified as
type I and type II. Type I topos cleave single strand of DNA while type II affect both strands during
their enzymatic activities. Topos are further divided into A and B subtypes based on structure.
Among the type IA (topo IIIα and β), type IB (topo I and Imt), and type IIA (topo IIα and IIβ)
enzymes, topo I and II are established molecular targets of anticancer drugs. The catalytic function of
topo I(II) involves transient DNA strand(s) cleavage, DNA swivel (strands passage), and resealing of
broken strand(s) [11]. Topo cleaves phosphodiester backbone of DNA by nucleophilic attack from
catalytic tyrosine to form short-lived covalent topo-DNA complex, commonly referred as “topo-
DNA cleavage/cleavable complex” (topocc; Fig. 1). Topo I forms tyrosyl-DNA covalent link at the 3ʹ
end whereas topo II at the 5ʹ ends of broken strand(s) at the cleavage site. Rotation of broken strand
around its intact complement and passage of intact DNA duplex through double strand break of
another DNA duplex are the main events that relax, unknot, and decatenate DNA.
3

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Fig. 1. Schematic diagram showing different stages of topo I enzymatic processes and inhibitors
involved in each stage.
The enzymatic activities of topo can be obstructed at various stages. Inhibition of DNA
break resealing and stabilization of topocc are the most important mechanisms exploited for cancer
treatment. Topocc, when trapped during replication and transcription, causes permanent DNA
breakage due to collision with replication or transcription machineries, eventually leading to DNA
damage, cell cycle arrest, or apoptotic cell death [12]. Anticancer drugs like irinotecan and topotecan
can interact and freeze topo Icc. These topo inhibitors that permit topo I-mediated DNA cleavage but
prevent DNA resealing convert functional topo I into a lethal component and are called topo I
poisons (Fig. 1).
Apart from preventing DNA resealing, anticancer agents like the vanadium compound, Van-
7
[13], intercalates between DNA bases to distort and inhibit association of DNA with topo I. In
addition, betulinic acid [14] and isodiospyrin [15] bind with topo I and prevent interaction with
DNA; β-lapachone [16] interacts with topo I and allows assembly of topo I and DNA but inhibits the
formation of topo Icc. The latter types of topo I inhibitors are collectively known as topo I catalytic
inhibitors/suppressors [17, 18] (Fig. 1). Similarly, several topo II poisons and catalytic inhibitors act
at different points of the enzymatic reaction cycle [19, 20].
The topo I poisons prescribed clinically, irinotecan and topotecan, are chemically unstable
(the lactone ring of the drugs opens in physiological neutral pH into the inactive carboxylate form)
and are cross-resistant to cells expressing the drug efflux membrane transporters, ABCG2 and
4

ACCEPTED MANUSCRIPT
ABCB1 [8]. The topo II poison, etoposide, is associated with higher incidences of secondary
malignancies and most notably with the development of myeloid leukemia [21]. Etoposide-induced
carcinogenesis is linked with drug-related DNA double-strand breaks and DNA sequence
rearrangements. It may be possible to overcome the drawbacks and limitations of these drugs by
eliminating chemically liable groups, using a novel scaffold to avoid extrusion from the cell, and
developing drug candidates that affect a suitable target with different mode of activity to avoid the
development of secondary malignancies. We therefore report the design, synthesis, and biological
evaluation of cytotoxic 1,3-diarylisoquinolines, non-intercalative topo I catalytic inhibitors.
2.
Results and discussion
2.1.
Design of 1,3-diarylisoquinoline
Subtle changes in the bridge length and relative positions between aromatic rings of multi-
arylated compound comprise the general strategy utilized during drug design. The inhibition of
tubulin polymerization by 2,3-diarylpyridine 1 was restored with a comparable potency as that of
combretastatin A-4 (CA-4) when the bridge length between the two aromatic rings was increased to
3
atoms and the aromatic rings were placed between pyridine N, as in 2,6-diarylpyridine 2 (Fig. 2A)
[
22, 23]. Thus, it can be expected that the shift of the 4-aryl group of 3,4-diarylisoquinolone 3
inhibitor of topo activity) [24] to the C1 position to form the isomeric 1,3-diarylisoquinoline 4 may
(
be advantageous in terms of its pharmacological properties.
5

ACCEPTED MANUSCRIPT
Fig. 2. Design of 1,3-diarylisoquinoline. A) 2- and 3-atom linked diaryls. B) Synthetic drawbacks of
3,4-diarylisoquinolones. C) Retrosynthesis of 1,3-diarylisoquinolines. CA-4: combretastatin A-4.
Besides improving biological activity, synthesis of 1,3-diarylisoquinoline via an efficient
pathway is desirable; the synthetic pathway used to obtain 3,4-diarylisoquinolone has several
drawbacks and limitations (Fig. 2B). Free-radical halogenation of 3-arylisoquinolone 5 lacks
selectivity. Dihalogenation occurs when an activating group (e.g. -OMe) is present in the C3-aryl
ring (Scheme S2, Supplementary data) [24]. Moreover, the Suzuki reaction of 4-brominated
isoquinolin-1(2H)-one 6 with aryl boronic acid is always accompanied by a debrominated byproduct,
6

ACCEPTED MANUSCRIPT
which interferes with isolation procedures and requires additional protection and deprotection steps.
Meanwhile, 1,3-diarylisoquinoline 4 can be obtained in two reaction steps via Suzuki reaction of
monohalogenated isoquinoline 7, obtained simply by heating isoquinolone 5 with POCl (Fig. 2C).
3
Moreover, based on our previous experience, Suzuki coupling of 1-chloroisoquinoline and boronic
acid results in no detectable dehalogenated byproduct. The 1,3-diarylisoquinoline formed by the
reaction has a satisfactory solubility in organic solvents, which eases purification and isolation steps.
2.2.
Chemistry
Synthesis of 1,3-diarylisoquinolines 4 involved two steps, starting from 3-arylisoquinolones
5
(Scheme 1A; refer to Supplementary data for the synthesis of 3-arylisoquinolones, Scheme S1).
Isoquinolones 5 were converted to imine chlorides 7 on treatment with POCl . The isoquinolinyl
3
chlorides thus obtained were coupled with aryl boronic acids a-e under Suzuki reaction conditions to
form 1,3-diarylisoquinolines 4 with high yield (quantitative–62%). Phenol substituted 1,3-
diarylisoquinolines (4aa, ca, and dd) and alkyl halide with amino terminal were further reacted to
afford compounds 8 with an amino alkyl tail (Scheme 1B). Free amines 8 were converted to 8·HCl
salts with c-HCl.
7

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of 1,3-diarylisoquinolines 4 (A) and 1,3-diarylisoquinolines with amino alkyl
chain 8 (B). Reagents and conditions: (i) POCl , 75 °C; (ii) ArB(OH) , Pd(PPh ) (0), Na CO ,
3
2
3 4
2
3
(
MeOCH ) , 90 °C. (iii) RCl, K CO , DMF, 130 °C; (iv) c-HCl, acetone, room temperature (r.t.).
2 2 2 3
2.3.
Cytotoxicity
The in vitro cytotoxicity of the synthesized 1,3-diarylisoquinolines (4 and 8) was evaluated
®
using Adriamycin (DOX·HCl) as positive control. The assay was performed using non-cancerous
(human breast epithelial, MCF-10A) and tumor (human ductal breast epithelial tumor, T47D; human
prostate cancer, DU145; human colorectal adenocarcinoma, HCT-15; and human cervical cancer,
HeLa) cells [25]. Cytotoxicity results are summarized as IC (concentration that inhibits 50% of cell
5
0
proliferation) values (Tables 1 and 2).
Initially, eight 1,3-diarylisoquinolines were tested to assess their toxicity in non-cancerous
(MCF-10A) and cancerous (T47D, DU145, and HCT-15) cells (Table 1). Compounds 4ab, 4db, and
4de, with an anisole ring at C1, are significant, as they were non-toxic to normal MCF-10A cells but
had potent cytotoxicity against cancerous T47D cells (Fig. 3). Moreover, these compounds had no
8

ACCEPTED MANUSCRIPT
effect on prostate cancer (DU145) cells. The hydroxylated derivatives 4aa, 4da, 4dd and 4fa were,
however, toxic to both MCF-10A and DU145 cells. The phenolic compound 4ad exhibited a unique
cytotoxicity profile; it selectively suppressed the proliferation of T47D cells with no cytotoxic effect
on MCF-10A, DU145, and HCT-15 cells. Similarly, isoquinoline 4de had minimal anti-proliferative
effect on HCT-15 cells. Besides 4ad and 4de, the isoquinolines compiled in Table 1 exhibited more
potent antitumor activity than the standard, Adriamycin, in HCT-15 cells (IC : 0.37±0.02–2.52±0.1
5
0
µM).
Table 1. Cytotoxicity (IC ) of 1,3-diarylisoquinolines 4.
5
0
1
2
S.
Compound
R
R
R
IC (µM)
50
a
b
c
d
No.
MCF-10A
0.93±0.03
7.83±0.06
>50
T47D
DU145
HCT-15
1
2
3
4
5
6
7
8
9
.
.
.
.
.
.
.
.
.
Adriamycin
4aa
/
/
/
0.84±0.04
0.58±0.01
0.86±0.01
1.22±0.03
4.88±0.12
5.36±0.01
>50
3.76±0.12
1.2±0.07
1.3±0.01
>50
-H
4′-OMe
3ʺ-OH
3ʺ-OMe
4ʺ-OH
3ʺ-OH
3ʺ-OMe
4ʺ-OH
4ʺ-OMe
3ʺ-OH
4ab
-H
4′-OMe
4ad
-H
4′-OMe
>50
>50
4da
6-Me
6-Me
6-Me
6-Me
3′,4′-(OMe)2
3′,4′-(OMe)2
3′,4′-(OMe)2
3′,4′-(OMe)2
13.48±0.11 4.34±0.04
6.51±0.04
>50
0.37±0.02
1.84±0.05
2.52±0.1
28.69±0.35
0.54±0.03
4db
>50
7.86±0.15
4.42±0.05
3.34±0.18
3.92±0.16
4dd
18.1±0.24
>50
5.98±0.29
>50
4de
4fa
6,7-(OMe)2 3′,5′-(OMe)2
5.9±0.15
8.12±0.08
Each value represents the mean ± S.D. from three different experiments performed in triplicate.
a
MCF-10A: non-cancerous human breast epithelial cells.
b
T47D: human ductal breast epithelial tumor cells.
c
DU145: human prostate cancer cells.
d
HCT-15: human colorectal adenocarcinoma cells.
Fig. 3. Structure-activity relationship (SAR) of 1,3-diarylisoquinolines.
9

ACCEPTED MANUSCRIPT
The preliminary cytotoxicity profile of 1,3-diarylisoquinolines proves that this class of
compounds may be useful in the development of safer anticancer drugs. Encouraged by the initial
results, newer derivatives of 1,3-diarylisoquinolines were synthesized and further tested for anti-
proliferative activity against MCF-10A, T47D, HeLa, and HCT-15 cells (Table 2). In accordance
with the earlier result, anisoles 4ae and 4bb of the second batch of compounds were not toxic to non-
cancerous MCF-10A cells. Similarly, anilines 4bc and 4ec had no effect on these cells. However,
phenols and aminoalkyl substituted derivatives had detrimental effect on the proliferation of MCF-
10A cells at concentrations > 5 µM. On the other hand, 1,3-Diarylisoquinolines of Table 2 exhibited
potent antitumor activity against T47D cells at lower concentrations (IC < 1 µM). The notable
5
0
difference in cytotoxicity of 1,3-diarylisoquinolines between MCF-10A and T47D confers that they
are safer anticancer agents. C1-anisole substituted derivatives 4ae and 4bb were inactive against
HeLa cells, while the remaining compounds exhibited cytotoxicity at micromolar concentrations
(
IC : 3.06±0.01–13.19±0.85 µM). Importantly, the diarylisoquinolines listed in Table 2 exhibited
50
significant inhibitory effects on HCT-15 cell proliferation (IC : 0.84±0.01–4.08±0.11 µM). The
5
0
potent cytotoxicity of 1,3-diarylisoquinolines against HCT-15 indicates that their cellular
concentration is not affected by overexpressed multidrug resistance protein 1 (MDR1; also known as
P-glycoprotein, P-gp, or as ATP-binding cassette sub-family B member 1, ABCB1) [8, 26-28].
Table 2. Cytotoxicity (IC ) of 1,3-diarylisoquinolines 4 and 8.
5
0
1
2
S.
Compound
R
R
R
IC (µM)
50
a
b
c
d
No.
MCF-10A
T47D
HeLa
HCT-15
1
2
3
4
5
6
7
8
9
1
.
Adriamycin
4ac
4ae
4ba
4bb
4bc
4bd
4ca
4cc
/
/
/
0.72±0.06
1.15±0.01
1.51±0.22
8.61±0.68
>50
2.03±0.03
2.35±0.16
2.79±0.09
0.84±0.01
1.17±0.10
.
-H
4′-OMe
3ʺ-NH2
4ʺ-OMe
3ʺ-OH
18.76±1.12 0.95±0.04
.
-H
4′-OMe
>50
0.85±0.06
0.76±0.03
1.12±0.03
0.98±0.04
0.78±0.02
0.56±0.1
.
-H
3′,5′-(OMe)2
3′,5′-(OMe)2
3′,5′-(OMe)2
3′,5′-(OMe)2
2′-Me, 4′-OMe
2′-Me, 4′-OMe
3ʹ,4ʹ-(OMe)2
3ʹ,5ʹ-(OMe)2
4′-OMe
6.77±0.24
>50
3.87±0.01
>50
.
-H
3ʺ-OMe
3ʺ-NH2
4ʺ-OH
.
-H
>50
13.19±0.85 4.08±0.11
.
-H
7.28±0.1
5.05±0.06
5.74±0.15
5.6±0.1
4.58±0.09
5.51±0.06
5.06±0.04
4.19±0.03
7.33±0.34
6.86±0.46
4.92±0.11
3.06±0.01
3.48±0.09
2.78±0.00
1.75±0.06
2.77±0.38
2.70±0.24
2.53±0.02
1.75±0.09
2.2±0.07
.
6-Me
6-Me
6-Me
7-NMe2
-H
3ʺ-OH
.
3ʺ-NH2
3ʺ-NH2
3ʺ-NH2
3ʺ-O(CH ) NMe ·HCl
0.74±0.05
0.45±0.33
0.
4dc
4ec
11.
42.04±0.15 3.59±0.39
10.15±0.03 0.16±0.03
1
1
1
1
2.
3.
4.
5.
8a
2
3
2
8b
6-Me
6-Me
6-Me
2ʹ-Me, 4ʹ-OMe
2ʹ-Me, 4ʹ-OMe
3ʹ,4ʹ-(OMe)2
3ʺ-O(CH ) NMe ·HCl
2
2
2
6.35±0.07
6.07±0.04
5.31±0.04
0.6±0.18
0.29±0.02
2.33±0.04
8c
3ʺ-O(CH ) NMe ·HCl
2
3
2
0.87±0.02
0.97±0.04
8d
4ʺ-O(CH ) NMe ·HCl
2 2 2
1
0

ACCEPTED MANUSCRIPT
Each value represents the mean ± S.D. from three different experiments performed in triplicate.
a
MCF-10A: non-cancerous human breast epithelial cells.
b
T47D: human ductal breast epithelial tumor cells.
c
HeLa: human cervical cancer cells.
d
HCT-15: human colorectal adenocarcinoma cells.
2.4.
Inhibition of topo-mediated DNA relaxation
Inhibition of topo activity is a principle mechanism of cell cytotoxicity of a huge library of
3-arylisoquinoline derivatives [24, 29-31], analogs [32-34], and compounds bearing the 3-
arylisoquinoline skeleton [10]. The inhibition of topo activity by the 1,3-diarylisoquinolines 4 and 8
was determined using the “DNA relaxation assay” (Fig. 4) [25]. In the DNA relaxation assay, topo
relaxes supercoiled plasmid, which has a different electrophoretic mobility from that of completely
relaxed DNA. Inhibition of topo activity by a chemical compound in the assay system is measured
by determining the extent of supercoiled plasmid that was prevented from relaxing. The DNA
relaxation assay was conducted using human topo I/IIα, pBR322 with CPT (as topo I positive
control), and etoposide (as topo IIα positive control). The assay was performed with 100 and 20 µM
concentrations of positive controls and test compounds. The inhibition of topo activity by the test
compounds has been represented as % inhibition (Tables S1–S4). Furthermore, to ease the structure-
activity relationship (SAR) study, the relative topo inhibition potency of various diarylisoquinolines
was determined as the ratio of topo inhibition by individual compounds to that by CPT/etoposide
(Table 3).
11

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Fig. 4. Inhibition of topo I (A) and topo IIα (B) activity by 1,3-diarylisoquinolines 4 and 8 at 100 µM
and 20 µM (DNA relaxation assay). Lane D: pBR322 only, lane T: pBR322 + topo I/IIα, lane C:
pBR322 + topo I + camptothecin (CPT), lane E: pBR322 + topo IIα + etoposide, remaining lanes:
pBR322 + topo I/IIα + the indicated 1,3-diarylisoquinolines. Rel: relaxed, Sc: supercoiled.
Table 3. Relative inhibition of topo activity by 1,3-diarylisoquinolines 4 and 8.
1
2
S.
Compound
R
R
R
Topo I
Topo IIα
No.
100 µM 20 µM
100 µM 20 µM
a
1
2
3
.
.
.
4aa
4ab
4ac
-H
-H
-H
4′-OMe
4′-OMe
4′-OMe
3ʺ-OH
0.12
0.58
0.0
-
1.07
0.0
0.0
3ʺ-OMe
3ʺ-NH2
0.0
-
-
-
0.12
1
2

ACCEPTED MANUSCRIPT
4
5
6
7
8
9
1
.
4ad
4ae
4ba
4bb
4bc
4bd
4ca
4cc
4da
4db
4dc
4dd
4de
4ec
4fa
8a
-H
4′-OMe
4ʺ-OH
4ʺ-OMe
3ʺ-OH
3ʺ-OMe
3ʺ-NH2
4ʺ-OH
0.09
0.0
-
0.0
-
.
-H
4′-OMe
-
0.11
0.59
0.26
0.37
0.82
0.2
-
.
-H
3′,5′-(OMe)2
3′,5′-(OMe)2
3′,5′-(OMe)2
3′,5′-(OMe)2
0.0
-
0.9
.
-H
0.0
-
-
.
-H
0.0
-
-
.
-H
0.0
-
1.17
0.
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
7-NMe2
2′-Me, 4′-OMe 3ʺ-OH
2′-Me, 4′-OMe 3ʺ-NH2
1.13
1.22
0.03
0.05
0.11
0.005
0.04
1.12
0.06
0.81
0.05
0.6
2.46
-
11.
2.53
0.06
0.38
0.0
-
1
1
1
1
1
1
1
1
2
2
2
2.
3.
4.
5.
6.
7.
8.
9.
0.
1.
2.
3′,4′-(OMe)2
3′,4′-(OMe)2
3ʹ,4ʹ-(OMe)2
3′,4′-(OMe)2
3′,4′-(OMe)2
3ʹ,5ʹ-(OMe)2
3ʺ-OH
-
0.01
3ʺ-OMe
-
-
3ʺ-NH2
-
0.11
0.4
-
4ʺ-OH
-
0.0
4ʺ-OMe
-
0.0
-
3ʺ-NH2
2.5
-
0.04
0.0
-
6,7-(OMe)2 3′,5′-(OMe)2
3ʺ-OH
-
-H
4′-OMe
3ʺ-O(CH ) NMe ·HCl
0.38
-
0.32
0.81
0.67
1.24
-
2
3
2
8b
6-Me
6-Me
6-Me
2ʹ-Me, 4ʹ-OMe 3ʺ-O(CH ) NMe ·HCl
2
2
2
0.35
0.28
0.41
8c
2ʹ-Me, 4ʹ-OMe 3ʺ-O(CH ) NMe ·HCl
2
3
2
0.0
0.0
8d
3ʹ,4ʹ-(OMe)2
4ʺ-O(CH ) NMe ·HCl
2 2
2
1.22
Relative inhibition of topo activity = inhibition of topo activity by compound/inhibition of topo
activity by camptothecin (CPT) or etoposide.
a
Not determined.
Semi quantitative representation: 0.0–0.25 (no potency), 0.25–0.5 (low potency), 0.5–0.8 (moderate
potency), 0.8-1.0 (potency similar to CPT/etoposide) and >1.0 (greater potency than CPT/etoposide).
The inhibition of topo I activity by 1,3-diarylisoquinoline 4ec was slightly and about 2.5-
fold greater than that by CPT at 100 and 20 µM, respectively (Table 3). The potency was lost when
7
-Me was removed as in 4bc (Fig. 3). Similar to 4ec, diarylisoquinolines 4ca and 4cc also had
2
strong interference on topo I activity. The efficacy of the derivatives was lost when 2ʹ-Me, 4ʹ-OMe
groups were replaced by 3ʹ,4ʹ-(OMe) (4da and 4dc). The potency of phenolic compound 4dd at 100
2
µM increased after substitution with amino alkyl chain (8d). In addition, amino alkyl substituted
compounds 8a and 8c exhibited significant potency at 100 µM.
The phenolic derivative 4aa had similar topo IIα inhibition potency as that of etoposide at
100 µM (Table 3). Likewise, phenols 4ba and 4bd had a considerable inhibitory effect on topo IIα
activity at both 100 and 20 µM. Conversion of 3ʺ-OH (4aa and 4ba) to 3ʺ-OMe (4ab and 4bb) had
detrimental effect on potency to inhibit topo II activity (Fig. 3). On the other hand, the potency of
phenol 4ca was elevated by aminoalkyl substitution (8b and 8c). Importantly, compound 8d inhibited
1
3

ACCEPTED MANUSCRIPT
DNA relaxation by both topo I and IIα.
A positive correlation was found between inhibition of topo I/IIα activity and cytotoxicity
for 8 1,3-diarylisoquinolines 4aa, 4bd, 4ca, 4cc, 4ec, 8a, 8b, and 8d. Furthermore, it can be
concluded that the cytotoxic effect of 3 compounds 4ab, 4ba, and 8c is partially due to inhibition of
topos. However, no obvious relationship between inhibition of enzyme activity and cytotoxicity was
observed with remaining diarylisoquinolines. The potent antiproliferative effect of these compounds
on cancer cells might be due to interference with function of other biological targets.
2.5.
Inhibition of tubulin polymerization
,3-Diaryliosquinolines 4 are structurally similar to 2,6-diarylpyridine 2, inhibitors of
1
tubulin polymerization. Thus, to investigate the effect of compounds 4 on microtubule dynamics the
tubulin polymerization assay was performed using porcine brain tubulin. 1,3-Diarylisoquinolines 4ba,
4bd, 4ca, 4cc and 4ec that showed potent inhibition of topo activity even at low concentration (20
µM) were considered for the test (Table 3). Paclitaxel (PTX) and DMSO were used as positive
control and vehicle, respectively. PTX has been known to enhance tubulin polymerization and inhibit
dissociation of microtubules [35]. As shown in Figure 5A, diarylisoquinolines 4ba, 4bd, 4ca, 4cc
and 4ec at 10 µM did not increase the absorbance unlike PTX, which reflects that compounds neither
enhanced nor stabilized tubulin polymerization. However, compared to vehicle, 4ca appeared to
slightly inhibit tubulin polymerization. Similarly, compound 4cc exhibited low potency at even
higher concertation (15 µM, Fig. 5B). The inhibition of tubulin polymerization by 4ac and 4cc is
very weak in comparison to CA-4 which nearly completely blocks tubulin polymerization at 10 µM
[
23].
Fig. 5. Effect of 3,4-diarylisoquinolines on microtubule dynamics (tubulin polymerization assay).
1
4

ACCEPTED MANUSCRIPT
(A) The absorbance of diarylisoquinolines 4ba, 4bd, 4ca, 4cc, and 4ec- (10 µM), PTX- (10 µM) and
DMSO-treated tubulin solution at 340 nm. (B) The absorbance of diarylisoquinoline 4cc- (5, 10, and
15 µM) and PTX-treated solution (5 and 10 µM) at 340 nm. OD: optical density.
Among the tested 1,3-diarylisoquinolines, compound 4cc had the most potent inhibitory
effect on topo I activity, was cytotoxic to cancer cells at low micromolar concentration, and had no
significant inhibitory effect on tubulin polymerization. Thus, further tests were performed to
determine the mode of inhibition of topo I activity by the compound.
2.6.
Topo I-DNA cleavage complex assay
The well-known mechanism of topo I inhibition is stabilization of topo Icc and prevention of
DNA break resealing by topo I poison. This mechanism of topo I inhibition can be investigated via
the topo I-DNA cleavage complex assay. In the cleavage complex assay, supercoiled plasmid
pBR322 was incubated with topo I and the test compounds, followed by treatment with sarcosyl and
proteinase K to remove any covalently bound topo I and electrophoresis in a system containing
ethidium bromide (DNA intercalator). Ethidium bromide inserts between DNA bases of relaxed
closed circular DNA, unwinds double-helical structure, and increases its electrophoretic mobility
[
36]. In contrast, nicked-DNA is partially unaffected. Nicked-DNA is a consequence of inhibition of
DNA break resealing by the topo I poison, CPT. In fact, CPT increased nicked-DNA compared to the
negative control (treated with only DMSO, Fig. 6A). Upon treatment with the isoquinoline 4cc,
however, there was no change in the level of nicked-DNA, even at a higher concentration (500 µM).
This observation indicates that compound 4cc does not act as a topo I poison.
1
5

ACCEPTED MANUSCRIPT
Fig. 6. 1,3-diarylisoquinoline 4cc is not a topo I poison. A) Topo I-DNA cleavage complex assay. B
&
C) Assessment of DNA damage/repair by comet assay. CPT: camptothecin, Nck: nicked, Sc:
supercoiled, Rel: relaxed.
2.7.
Comet assay
The comet assay confirms the degree of DNA damage induced by a chemical compound.
Debris (fragments) of broken or damaged DNA form comet tails (tail DNA) [37]. DNA damage and
comet formation are characteristic properties of topo I poison. The assay is therefore useful to study
1
6

ACCEPTED MANUSCRIPT
the mode of topo I inhibition. In this assay, CPT-treated T47D cells showed severe DNA damage
with 8 times more tail DNA than untreated control, even at a low concentration of CPT (25.0±4.53%
at 5 µM, Fig. 6B, C). However, isoquinoline 4cc induced less tail DNA (5.82±1.63% at 5 µM and
9.43±1.74% at 10 µM), somewhat comparable to that of the untreated control (2.99±1.27%). This
result is consistent with that of cleavage complex assay and confirms that the 1,3-diarylisoquinoline
4cc is not a topo I poison.
2.8.
DNA unwinding assay
Besides topo Icc stabilization and prevention of DNA break resealing, DNA intercalation is
also a mode of inhibition of topo I activity. A DNA intercalator unwinds relaxed plasmid DNA into a
compact form. The DNA unwinding assay, performed with amsacrine (m-AMSA), one of the
eminent intercalative topo II poisons [38] and compound 4cc, shows that m-AMSA recovered the
fully relaxed pHOT1 DNA to its supercoiled form, while isoquinoline 4cc failed to do so even at a
higher concentration (1 mM, Fig. 7A).
1
7

ACCEPTED MANUSCRIPT
Fig. 7. A) DNA unwinding assay. B) Hoechst assay. Fluorescence spectra of Hoechst-ctDNA in
absence and presence of 4cc. Free Hoechst 33342 (1 µg/mL) in 10 mM Tris-HCl (pH 7.2) showed
very weak emission intensity, while when Hoechst interacted with ctDNA, the fluorescence intensity
was strong. The addition of various concentrations of 4cc (0–100 µM) to the pre-reacted Hoechst-
ctDNA did not cause change in the florescence intensity. C) Band depletion assay. Rel: relaxed, Sc:
supercoiled, CPT: camptothecin.
2
.9. Hoechst Assay
Hoechst assay was further performed to investigate the effect of compound 4cc on DNA (Fig.
B). Hoechst 33342 is a well-known DNA minor groove binder and the Hoechst–DNA complex is
7
known to be excited at 361 nm [39, 40]. To determine whether compound 4cc is a DNA minor
groove binder, Hoechst and circulating tumor DNA (ctDNA) were pre-reacted and then treated with
4cc in a series of concentrations (0–100 µM). It was observed that fluorescence intensity did not
decrease even when 100 µM of 4cc was treated. The results of DNA-related experiments confirmed
that the compound 4cc is neither a DNA intercalator nor a DNA ancillary groove binder but may act
as topo I catalytic inhibitor (i.e. it may interact with free topo I, prevent association with supercoiled
DNA and stop the consequent processes or may stabilize non-covalent topo I-DNA complex and
prevent DNA break).
2.10. Band depletion assay
To further decipher the mechanism of topo I inhibition by diarylisoquinoline 4cc, a band
depletion assay was performed. Following treatment with CPT (50 µM) for 2 h at 37 °C, free topo I
in T47D cells decreased, indicating the formation of topo Icc (Fig. 7C). In contrast, free topo I
remained unaffected in presence of compound 4cc. This result infers that the isoquinoline 4cc binds
with topo I and inhibits the harbor of DNA. Furthermore, treatment with compound 4cc before CPT
treatments restored free topo I in T47D cells. This indicates that compound 4cc binds with free topo I
and can competitively sequester topo I from being incorporated into the cleavage complex by co-
treated topo I poison. Taken together, 1,3-diarylisoquinoline 4cc functions as a non-intercalative topo
I catalytic inhibitor, interacting with free topo I.
3.
Conclusion
,4-Diarylisoquinolone (with 2 Cs spacer between aryl rings) was modified to 1,3-
diarylisoquinoline (separated by two covalent bonds) and synthesized in three simple steps with high
3
1
8

ACCEPTED MANUSCRIPT
yield. It was found that most of the 1,3-diarylisoquinolines showed potent anti-proliferative effects
against cancer cells. The potent cytotoxic effects of 1,3-diarylisoquinoline against HCT-15 cells
suggest decreased risk of drug resistance due to overexpressed drug efflux membrane transporters.
Several of the compounds tested had no effect on normal (non-cancerous) cells. Furthermore, 1,3-
diarylisoquinolines inhibited topo I/IIα-mediated DNA relaxation. The inhibition of topo I/IIα
activity by several compounds including the most potent derivative, 4cc, at 20 µM was greater than
that of CPT and had weak or no antitubulin activity. Topo I-DNA cleavage complex, comet, DNA
unwinding, Hoechst, and band depletion assays revealed that the diarylisoquinoline 4cc acts as a
non-intercalative topo I catalytic inhibitor that binds with free topo I to stop the downward steps and
prevent DNA damage. Altogether, this study might provide valuable information for the further
research related to isoquinoline and topo I catalytic inhibitor.
4.
Experimental section
4.1.
Chemistry
Solvents were distilled prior to use; THF was distilled from sodium/benzophenone. The
reaction temperature mentioned, except for the lithiated toluamide-benzonitrile cycloaddition
reaction, is the set temperature of an oil bath. Column chromatography and medium performance
liquid chromatography (MPLC, flow rate: 10 mL/min, make: Yamazen) were performed with Merck
silica gel 60 (70–230 mesh). Thin-layer chromatography (TLC) was performed using plates coated
with silica gel 60 F254 (Merck). Melting points were determined by the capillary method with a
®
MEL-TEMP capillary melting point apparatus and were uncorrected. IR spectra were obtained on a
1
13
JASCO FT/IR 300E Fourier transform infrared spectrometer using KBr pellets. H NMR, and C
NMR spectra were obtained on Varian Unity Plus 300 MHz, Varian Unity Inova 500 MHz and
Bruker Ascend 400 spectrometers at the Korea Basic Science Institute. The chemical shift (δ) is
reported in parts per million (ppm) downfield to tetramethylsilane (δ = 0). The coupling constant, J,
is given in Hertz (Hz). The data are reported in the following order: chemical shift, multiplicity,
coupling constant, number of protons, and proton assignment where applicable. The multiplicity of
proton signals is reported as s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, b: broad, bs:
broad singlet, and dd: doublet of doublets, dt: doublet of triplets, ddd: doublet of doublet of doublets.
Mass spectra were obtained on a Shimadzu LCMS-2010 EV liquid chromatograph mass
spectrometer and Quattro micro API Tandem Quadrupole System (Waters) using the electron spray
ionization (ESI) method.
4.1.1. 1-Chloro-3-(4-methoxyphenyl)isoquinoline (7a)
1
9

ACCEPTED MANUSCRIPT
A solution of compound 5a (1.46 g, 5.83 mmol) and POCl (20 mL) was heated using an oil
3
bath set at 75 °C. Upon completion of the reaction, excess POCl and volatile substances were
3
evaporated by vacuum distillation. Saturated (sat.) NaHCO solution (100 mL) was added to the
3
residue obtained and was extracted with CH Cl . The organic extract was further washed with water,
2
2
dried over anhydrous Na CO and concentrated under reduced pressure. The residue was then
2
3
purified by column chromatography (n-hexane:EtOAc = 5:1, 3:1) to obtain compound 7a as a white
1
floppy solid (1.54 g, 98%). R = 0.45 (n-hexane:EtOAc = 3:1). H NMR (300 MHz, DMSO-d6): δ
f
8
.41 (s, 1H, 4-H), 8.25 (dd, J = 8.4, 0.9 Hz, 8-H), 8.15–8.07 (m, 3H, 2′,6′-(H) , Ar-H), 7.90–7.85 (m,
2
1
H, Ar-H), 7.78–7.73 (m, 1H, Ar-H), 7.12–7.07 (m, 2H, 3′,5′-(H) ), 3.32 (s, 3H, OCH ).
2
3
4.1.2. 1-Chloro-3-(3,5-dimethoxyphenyl)isoquinoline (7b)
The procedures described for 7a were used with 5b (894 mg, 3.17 mmol) and POCl (20
3
mL), followed by column chromatography (n-hexane:EtOAc = 5:1, EtOAc) to obtain compound 7b
1
as a white solid (925 mg, 97%). R = 0.32 (n-hexane:EtOAc = 4:1). H NMR (300 MHz, CDCl ): δ
f
3
8.36–8.32 (m, 1H, 8-H), 7.97 (d, J = 0.3 Hz, 1H, 4-H), 7.90–7.87 (m, 1H, 5-H), 7.77–7.72 (m, 1H,
Ar-H), 7.69–7.63 (m, 1H, Ar-H), 7.27 (d, J = 2.1 Hz, 2H, 2′,6′-(H) ), 6.54 (t, J = 2.4 Hz, 1H, 4′-H),
2
+
3
.90 (s, 6H, 3′,5′-(OCH ) ). MS (ESI): m/z 300 (M+H) .
3
2
4.1.3. 1-Chloro-3-(4-methoxy-2-methylphenyl)-6-methylisoquinoline (7c)
The procedures described for 7a were used with a mixture of 5c, S3 and polar byproducts
(
798 mg) and POCl (20 mL), followed by column chromatography (n-hexane:EtOAc = 5:1) to
3
1
obtain compound 7c as a white solid (810 mg). R = 0.65 (n-hexane:EtOAc = 4:1). H NMR (300
f
MHz, DMSO-d6): δ 8.16 (d, J = 8.4 Hz, 1H, Ar-H), 7.86 (s, 2H, Ar-H), 7.63 (dd, J = 8.7, 1.5 Hz, 1H,
Ar-H), 7.47–7.44 (m, 1H, Ar-H), 6.91–6.87 (m, 2H, Ar-H), 3.80 (s, 3H, OCH ), 2.54 (s, 3H, Ar-CH ),
3
3
2.39 (s, 3H, Ar-CH3).
4.1.4. 1-Chloro-3-(3,4-dimethoxyphenyl)-6-methylisoquinoline (7d)
The procedures described for 7a were used with 5d (1.41 g, 4.8 mmol) and POCl (20 mL),
3
followed by column chromatography (n-hexane:EtOAc = 5:1, 3:1) to obtain compound 7d as an off-
1
white solid (1.45 g, 96%). R = 0.38 (n-hexane:EtOAc = 3:1). H NMR (500 MHz, DMSO-d6): δ
f
8.33 (s, 1H, Ar-H), 8.13 (d, J = 8.5 Hz, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 7.75–7.71 (m, 2H, Ar-H), 7.58
(
dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.10 (d, J = 9.0 Hz, 1H, Ar-H), 3.89 (s, 3H, OCH ), 3.83 (s, 3H,
3
OCH ), 2.53 (s, 3H, 6-CH ).
3
3
2
0

ACCEPTED MANUSCRIPT
4.1.5. 1-Chloro-3-(3,5-dimethoxyphenyl)-N,N-dimethylisoquinolin-7-amine (7e)
The procedures described for 7a were used with 5e (800 mg, 2.46 mmol) and POCl (20 mL),
3
followed by column chromatography (n-hexane:EtOAc = 4:1, 2:1, EtOAc) to obtain compound 7e as
1
a yellow solid (243 mg, 28%). R = 0.59 (n-hexane:EtOAc = 2:1). H NMR (300 MHz, DMSO-d6): δ
f
8
2
3
.33 (s, 1H, Ar-H), 7.93 (d, J = 9.3 Hz, 1H, Ar-H), 7.57 (dd, J = 9.3, 2.7 Hz, 1H, Ar-H), 7.25 (d, J =
.1 Hz, 2H, 2′,6′-(H) ), 7.04 (d, J = 2.4 Hz, 1H, Ar-H), 6.52 (t, J = 2.4 Hz, 1H, 4′-H), 3.83 (s, 6H,
2
+
′,5′-(OCH ) ), 3.11 (s, 6H, N(CH ) ). MS (ESI): m/z 343 (M+H) .
3
2
3 2
4.1.6. 1-Chloro-3-(3,5-dimethoxyphenyl)-6,7-dimethoxyisoquinoline (7f)
The procedures described for 7a were used with 5f (607 mg, 1.77 mmol) and POCl (20 mL),
3
followed by column chromatography (n-hexane:EtOAc = 3:1, EtOAc) to obtain compound 7f as a
1
light yellow solid (627 mg, 98%). R = 0.23 (n-hexane:EtOAc = 3:1). H NMR (300 MHz, DMSO-
f
d6): δ 8.36 (s, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.45 (s, 1H, Ar-H), 7.27 (d, J = 2.1 Hz, 2H, 2′,6′-(H)2),
6
.56 (t, J = 2.4 Hz, 1H, 4′-H), 3.98 (s, 3H, OCH ), 3.97 (s 3H, OCH ), 3.33 (s, 6H, (OCH ) ).
3
3
3 2
4.1.7. 3-(3-(4-Methoxyphenyl)isoquinolin-1-yl)phenol (4aa)
A solution of 3-hydroxyphenylboronic acid (143 mg, 1.03 mmol) in MeOH (5 mL) was
added to a solution of compound 7a (200 mg, 0.74 mmol) in 1,2-dimethoxyethane (5 mL) followed
by Pd(PPh ) (0) (43 mg, 0.03 mmol) and sat. Na CO solution (2 mL). The reaction mixture was
3
4
2
3
heated using an oil bath set at 90 °C. Upon completion of the reaction, water (150 mL) was added.
The resulting mixture was extracted with CH Cl (100 mL x 8); the organic extract was washed with
2
2
water (100 mL) and concentrated under reduced pressure. The residue thus obtained was purified by
column chromatography (n-hexane:EtOAc = 5:1) to obtain compound 4aa as an off-white solid (220
-1 1
mg, 90%). R = 0.27 (n-hexane:EtOAc = 3:1). Melting point (mp): 146–148 °C. IR: 3236 cm . H
f
NMR (300 MHz, DMSO-d6): δ 9.68 (bs, 1H, OH), 8.32 (s, 1H, Ar-H), 8.21–8.18 (m, 2H, 2′,6′-(H)2),
8.07–8.02 (m, 2H, Ar-H), 7.79–7.74 (m, 1H, Ar-H), 7.61–7.55 (m, 1H, Ar-H), 7.41–7.35 (m, 1H, Ar-
H), 7.15–7.12 (m, 2H, Ar-H), 7.10–7.06 (m, 2H, 3′, 5′-(H) ), 6.96–6.93 (m, 1H, 2″-H), 3.83 (s, 3H,
2
13
OCH ). C NMR (100 MHz, CDCl ): δ 160.4, 160.1, 155.8, 150.1, 140.5, 137.8, 132.1, 130.3, 129.3,
3
3
128.5, 127.7, 127.1, 126.7, 125.5, 122.4, 117.6, 116.1, 115.5, 114.1, 55.3. MS (ESI): m/z 328.12
+
+
+
-
(
M+H) , 329.50 (M+2H) , 327.33 (M) , 325.97 (M-H) .
4.1.8. 1-(3-Methoxyphenyl)-3-(4-methoxyphenyl)isoquinoline (4ab)
The procedures described for 4aa were used with 7a (200 mg, 0.74 mmol), a solution of 3-
methoxyphenylboronic acid (146 mg, 0.96 mmol) in 3 mL MeOH, Pd(PPh ) (0) (43 mg, 0.03 mmol),
3
4
2
1

ACCEPTED MANUSCRIPT
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(
CH Cl , n-hexane:EtOAc = 3:1) to obtain compound 4ab as a transparent semi-solid, which
2 2
solidified upon storage (260 mg, quantitative). R = 0.33 (n-hexane:EtOAc = 5:1). Mp: 117–120 °C.
f
1
H NMR (300 MHz, DMSO-d6): δ 8.34 (s, 1H, Ar-H), 8.22–8.19 (m, 2H, 2′,6′-(H) ), 8.07 (d, J = 8.1
2
Hz, 1H, Ar-H), 8.01 (d, J = 8.4 Hz, 1H, Ar-H), 7.08–7.74 (m, 1H, Ar-H), 7.61–7.55 (m, 1H, Ar-H),
7
3
1
.51 (t, J = 7.8 Hz, 1H, Ar-H), 7.30–7.26 (m, 1H, Ar-H), 7.15–7.11 (m, 1H, 2″-H), 7.10–7.05 (m, 2H,
1
3
′,5′-(H) ), 3.85 (s, 3H, OCH ), 3.83 (s, 3H, OCH ). C NMR (100 MHz, CDCl ): δ 160.1, 160.0,
2
3
3
3
59.5, 149.8, 141.2, 137.9, 132.2, 129.9, 129.2, 128.3, 127.5, 127.2, 126.5, 125.4, 122.7, 115.6,
+
+
1
14.6, 114.3, 114.0, 55.4, 55.3. MS (ESI): m/z 343.37 (M+2H) , 341.39 (M) .
4.1.9. 3-(3-(4-Methoxyphenyl)isoquinolin-1-yl)aniline (4ac)
The procedures described for 4aa were used with 7a (200 mg, 0.74 mmol), a solution of 3-
aminophenylboronic acid monohydrate (161 mg, 1.03 mmol) in 3 mL MeOH, Pd(PPh ) (0) (43 mg,
3
4
0
.03 mmol), sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (8 mL), followed by column
2 3
chromatography (n-hexane:EtOAc = 4:1) to obtain compound 4ac as a light yellow semi-solid,
which solidified upon prolonged heating and vacuum pumping (193 mg, 79%). R = 0.61 (n-
f
1
hexane:EtOAc = 1:1). Mp: 119–123 °C. H NMR (300 MHz, CDCl ): δ 8.18 (m, 3H, Ar-H), 7.97 (d,
3
J = 0.9 Hz, 1H, 4-H), 7.88 (d, J = 8.4 Hz, 1H, Ar-H), 7.67–7.61 (m, 1H, Ar-H), 7.48–7.43 (m, 1H,
Ar-H), 7.34–7.29 (m, 1H, Ar-H), 7.16–7.11 (m, 2H, Ar-H), 7.04–6.99 (m, 2H, 3′, 5′-(H) ), 6.85–6.81
2
1
3
(
m, 1H, 2″-H), 3.87 (s, 3H, OCH ), 3.81 (bs, 2H, NH ). C NMR (100 MHz, CDCl ): δ 160.4, 160.0,
3 2 3
149.8, 146.4, 140.9, 137.8, 132.3, 129.9, 129.0, 128.3, 127.7, 127.1, 126.3, 125.4, 120.7, 116.8,
+
.
+
1
15.3, 114.5, 114.0, 55.3. MS (ESI): m/z 327 (M+H) MS (ESI): m/z 327.03 (M+H) , 328.44
+
+
(
M+2H) , 326.41 (M) .
4.1.10. 4-(3-(4-Methoxyphenyl)isoquinolin-1-yl)phenol (4ad)
The procedures described for 4aa were used with 7a (200 mg, 0.74 mmol), a solution of 4-
hydroxyphenylboronic acid (143 mg, 1.03 mmol) in 3 mL MeOH, Pd(PPh ) (0) (43 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(
n-hexane:EtOAc = 7:1) to obtain compound 4ad as a white solid (151 mg, 62%). R = 0.48 (n-
f
1
hexane:EtOAc = 3:1). Mp: 160 °C. H NMR (300 MHz, DMSO-d6): δ 9.82 (bs, 1H, OH), 8.26 (s,
1H, 4-H), 8.22–8.18 (m, 2H, Ar-H), 8.09–8.02 (m, 2H, Ar-H), 7.77–7.72 (m, 1H, Ar-H), 7.62–7.54
13
(
m, 3H, Ar-H), 7.10–7.06 (m, 2H, Ar-H), 6.99–6.96 (m, 2H, Ar-H), 3.83 (s, 3H, OCH ). C NMR
3
(100 MHz, DMSO-d6): δ 160.3, 159.7, 158.5, 149.0, 138.1, 131.8, 130.67, 130.64, 128.3, 127.9,
+
+
1
27.44, 127.41, 125.0, 115.6, 114.6, 114.0, 55.7. MS (ESI): m/z 327.98 (M+H) , 329.50 (M+2H) ,
2
2

ACCEPTED MANUSCRIPT
+
-
3
27.37 (M) , 326.00 (M-H) .
4.1.11. 1,3-Bis(4-methoxyphenyl)isoquinoline (4ae)
The procedures described for 4aa were used with 7a (200 mg, 0.74 mmol), a solution of 4-
methoxyphenylboronic acid (146 mg, 0.96 mmol) in 3 mL MeOH, Pd(PPh ) (0) (43 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by MPLC (n-
2
3
hexane:CH Cl = 3:1, 1:1) to obtain compound 4ae as a white solid (208 mg, 82%). R = 0.5 (n-
2
2
f
1
hexane:EtOAc = 4:1). Mp: 131–133 °C. H NMR (300 MHz, CDCl ): δ 8.19–8.12 (m, 3H, Ar-H),
3
7.96 (d, J = 0.9 Hz, 1H, 4-H), 7.88 (d, J = 8.4 Hz, 1H, Ar-H), 7.80–7.75 (m, 2H, Ar-H), 7.67–7.62 (m,
1H, Ar-H), 7.50–7.44 (m, 1H, Ar-H), 7.11–7.06 (m, 2H, Ar-H), 7.04–6.99 (m, 2H, Ar-H), 3.91 (s, 3H,
13
OCH ), 3.87 (s, 3H, OCH ). C NMR (100 MHz, CDCl ): δ 160.1, 160.0, 159.8, 149.8, 138.0, 132.5,
3
3
3
132.3, 131.6, 129.9, 128.3, 127.6, 127.3, 126.4, 125.4, 114.2, 114.0, 113.7, 55.42, 55.36. MS (ESI):
+
+
+
m/z 343.41 (M+2H) , 341.40 (M) , 327.79 (M-CH +2H) .
3
4.1.12. 3-(3-(3,5-Dimethoxyphenyl)isoquinolin-1-yl)phenol (4ba)
The procedures described for 4aa were used with 7b (200 mg, 0.66 mmol), a solution of 3-
hydroxyphenylboronic acid (129 mg, 0.93 mmol) in 3 mL MeOH, Pd(PPh ) (0) (38 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by MPLC (n-
2
3
hexane:EtOAc = 7:1) to obtain compound 4ba as a transparent semi-solid, which solidified into a
white solid upon prolonged heating (243 mg, quantitative). R = 0.68 (n-hexane:EtOAc = 2:1). Mp:
f
-1 1
1
40–142 °C. IR: 3265 cm . H NMR (300 MHz, DMSO-d6): δ 9.71 (bs, 1H, OH), 8.45 (s, 1H, 4-H),
8.11–8.05 (m, 2H, Ar-H), 7.82–7.77 (m, 1H, Ar-H), 7.66–7.60 (m, 1H, Ar-H), 7.41–7.36 (m, 3H, Ar-
H), 7.17–7.14 (m, 2H, Ar), 6.97–6.93 (m, 1H, 2″-H), 6.58 (t, J = 2.2 Hz, 1H, 4′-H), 3.84 (s, 6H, 3′,5′-
1
3
(
OCH ) ). C NMR (100 MHz, CDCl ): δ 161.0, 160.6, 156.0, 150.1, 141.6, 140.1, 137.6, 130.4,
3 2 3
129.2, 127.7, 127.3, 127.1, 126.0, 122.1, 117.5, 117.0, 116.0, 105.4, 100.9 55.5. MS (ESI): m/z 358
+
-
-
(
M+H) , 356 (M-H) . MS (ESI): m/z 355.91 (M-H) .
4.1.13. 3-(3,5-Dimethoxyphenyl)-1-(3-methoxyphenyl)isoquinoline (4bb)
The procedures described for 4aa were used with 7b (200 mg, 0.66 mmol), a solution of 3-
methoxyphenylboronic acid (142 mg, 0.93 mmol) in 3 mL MeOH, Pd(PPh ) (0) (38 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL), and 1,2-dimethoxy ethane (5 mL), followed by MPLC (n-
2
3
hexane:EtOAc = 7:1) to obtain compound 4bb as a transparent semi-solid, which solidified upon
1
storage (186 mg, 75%). R = 0.61 (n-hexane:EtOAc = 3:1). Mp: 95–98 °C. H NMR (300 MHz,
f
CDCl ): δ 8.14 (dd, J = 8.4, 0.9 Hz, 1H, Ar-H), 8.04 (s, 1H, 4-H), 7.93 (d, J = 8.1 Hz, 1H, Ar-H),
3
2
3

ACCEPTED MANUSCRIPT
7.71–7.65 (m, 1H, Ar-H), 7.54–7.48 (m, 1H, Ar-H), 7.46–7.43 (m, 1H, Ar-H), 7.38–7.34 (m, 4H, Ar-
13
H), 7.08–7.04 (m, 1H, 2″-H), 6.53(t, J = 2.2 Hz, 1H, 4′-H), 3.89 (s, 9H, 3′,5′, 3″-(OCH ) ). C NMR
3
3
(
100 MHz, CDCl ): δ 161.1, 160.0, 159.5, 149.8, 141.7, 141.1, 137.7, 130.1, 129.2, 127.5, 127.4,
3
+
1
27.0, 125.9, 122.7, 116.1, 115.6, 114.3, 105.2, 100.7, 55.5, 55.4. MS (ESI): m/z 372 (M+H) . MS
+
+
+
(
ESI): m/z 373.26 (M+2H) , 371.38 (M) , 357.93 (M-CH +2H) .
3
4.1.14. 3-(3-(3,5-Dimethoxyphenyl)isoquinolin-1-yl)aniline (4bc)
The procedures described for 4aa were used with 7b (200 mg, 0.66 mmol), a solution of 3-
aminophenylboronic acid monohydrate (145 mg, 0.93 mmol) in 3 mL MeOH, Pd(PPh ) (0) (38 mg,
3
4
0
.03 mmol), sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (10 mL), followed by column
2 3
chromatography (n-hexane:EtOAc = 5:1, 3:1) to obtain compound 4bc as a light yellow transparent
semi-solid, which solidified upon storage (212 mg, 89%). R = 0.22 (n-hexane:EtOAc = 2:1). Mp:
f
1
5
8–62 °C. H NMR (300 MHz, CDCl ): δ 8.16 (d, J = 8.4 Hz, 1H, Ar-H), 8.01 (s, 1H, 4-H), 7.91 (d,
3
J = 8.4 Hz, 1H, Ar-H), 7.69–7.64 (m, 1H, Ar-H), 7.52–7.47 (m, 1H, Ar-H), 7.36–7.29 (m, 3H, Ar-H),
7.16–7.11 (m, 2H, Ar-H), 6.85–6.81 (m, 1H, 2″-H), 6.52 (t, J = 2.2 Hz, 1H, 4′-H), 3.89 (s, 6H, 3′,5′-
1
3
(
OCH ) ), 3.81 (s, 2H, NH ). C NMR (100 MHz, CDCl ): δ 161.1, 160.5, 149.8, 146.4, 141.9,
3 2 2 3
140.8, 137.6, 130.0, 129.0, 127.8, 127.4, 126.9, 126.0, 120.7, 116.9, 116.0, 115.3, 105.3, 100.6, 55.5.
+
+
MS (ESI): m/z 357 (M+H) . MS (ESI): m/z 356.93 (M+H) .
4.1.15. 4-(3-(3,5-Dimethoxyphenyl)isoquinolin-1-yl)phenol (4bd)
The procedures described for 4aa were used with 7b (200 mg, 0.66 mmol), a solution of 4-
hydroxyphenylboronic acid (129 mg, 0.93 mmol) in 3 mL MeOH, Pd(PPh ) (0) (38 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (7 mL), followed by MPLC (n-
2
3
hexane:EtOAc = 7:1) to obtain compound 4bd as a transparent semi-solid, which solidified into a
white solid upon prolonged heating and vacuum pumping (69 mg, 28%). R = 0.62 (n-hexane:EtOAc
f
1
=
2:1). Mp: 185–188 °C. H NMR (300 MHz, CDCl ): δ 8.14 (dd, J = 8.4, 0.9 Hz, 1H, Ar-H), 8.00 (s,
3
1
H, 4-H), 7.91 (d, J = 8.1 Hz, 1H, Ar-H), 7.72–7.65 (m, 3H, 2″,6″-(H) , Ar-H), 7.54–7.48 (m, 1H,
2
Ar-H), 7.36 (d, J = 2.4 Hz, 2H, 2′,6′-(H) ), 6.99–6.95 (m, 2H, 3″,5″-(H) ), 6.53 (t, J = 2.2 Hz, 1H, 4′-
2
2
1
3
H), 5.34 (s, 1H, OH), 3.89 (s, 6H, 3′,5′-(OCH ) ). C NMR (100 MHz, DMSO-d6): δ 161.3, 159.7,
3
2
158.6, 148.7, 141.5, 138.0, 131.8, 130.7, 130.5, 128.2, 127.9, 127.4, 125.6, 115.68, 115.64, 105.1,
+
-
+
-
1
00.9, 55.8. MS (ESI): m/z 358 (M+H) , 356 (M-H) . MS (ESI): m/z 357.98 (M+H) , 355.95 (M-H) .
4.1.16. 3-(3-(4-Methoxy-2-methylphenyl)-6-methylisoquinolin-1-yl)phenol (4ca)
The procedures described for 4aa were used with 7c (350 mg, 1.17 mmol), a solution of 3-
2
4

ACCEPTED MANUSCRIPT
hydroxyphenylboronic acid (227 mg, 1.64 mmol) in 5 mL MeOH, Pd(PPh ) (0) (68 mg, 0.05 mmol),
3
4
sat. Na CO solution (3 mL) and 1,2-dimethoxy ethane (10 mL), followed by column
2
3
chromatography (n-hexane:EtOAc = 5:1) to obtain compound 4ca as a transparent semi-solid, which
solidified into a white solid upon prolonged heating and vacuum pumping (394 mg, 94%). R = 0.28
f
1
(
n-hexane:EtOAc = 3:1). Mp: 104–107 °C. H NMR (300 MHz, DMSO-d6): δ 9.65 (s, 1H, OH),
7.97 (d, J = 8.7 Hz, 1H, Ar-H), 7.82 (s, 1H, Ar-H), 7.79 (s, 1H, Ar-H), 7.50–7.44 (m, 2H, Ar-H), 7.35
(t, J = 8.1 Hz, 1H, Ar-H), 7.10–7.08 (m, 2H, Ar-H), 6.93–6.86 (m, 3H, Ar-H), 3.80 (s, 3H, OCH3),
1
2
.51 (≈3H, Ar-CH , overlapped with DMSO peaks), 2.42 (s, 3H, Ar-CH ). H NMR (300 MHz,
3
3
CDCl ): δ 7.99 (d, J = 8.7 Hz, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.48–7.45 (m, 1H,
3
Ar), 7.35–7.28 (m, 2H, Ar-H), 7.22–7.19 (m, 1H, Ar-H), 7.12–7.11 (m, 1H, Ar-H), 6.86–6.80 (m, 3H,
1
3
Ar-H), 6.08 (bs, 1H, OH), 3.83 (s, 3H, OCH ), 2.55 (s, 3H, Ar-CH ), 2.44 (s, 3H, Ar-CH ). C NMR
3
3
3
(
100 MHz, CDCl ): δ 159.9, 159.3, 156.1, 152.2, 140.8, 139.9, 137.7, 133.1, 131.3, 129.2, 129.0,
3
127.7, 125.8, 123.7, 121.8, 119.4, 117.8, 116.4, 116.1, 111.2, 55.2, 21.9, 20.8. MS (ESI): m/z 356
+
-
+
-
(
M+H) , 354 (M-H) . MS (ESI): m/z 356.04 (M+H) , 353.95 (M-H) .
4.1.17. 3-(3-(4-Methoxy-2-methylphenyl)-6-methylisoquinolin-1-yl)aniline (4cc)
The procedures described for 4aa were used with 7c (395 mg, 1.32 mmol), a solution of 3-
aminophenylboronic acid monohydrate (288 mg, 1.85 mmol) in 5 mL MeOH, Pd(PPh ) (0) (77 mg,
3
4
0
.06 mmol), sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (8 mL), followed by column
2 3
chromatography (n-hexane:EtOAc = 7:1) to obtain compound 4cc as a brown semi-solid (368 mg,
1
7
8%). R = 0.28 (n-hexane:EtOAc = 3:1). H NMR (400 MHz, CDCl ): δ 8.06 (d, J = 8.4 Hz, 1H,
f
3
Ar-H), 7.63 (s, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.50–7.47 (m, 1H, Ar-H), 7.34–7.31 (m, 1H, Ar-H),
.29–7.25 (m, ≈1H. Ar-H, partially overlapped with CHCl peak), 7.09 (dt, J = 7.6, 1.2 Hz, 1H, Ar-
7
3
H), 7.05 (t, J = 1.8 Hz, 1H, Ar-H), 6.83–6.81 (m, 2H, Ar-H), 6.77 (ddd, J = 8, 2.4, 0.8 Hz, 1H, Ar-H),
1
3
3
.83 (s, 3H, OCH ), 3.30 (bs, 2H, NH ), 2.54 (s, 3H, Ar-CH ), 2.46 (s, 3H, Ar-CH ). C NMR (100
3 2 3 3
MHz, CDCl ): δ 159.7, 159.3, 152.4, 146.3, 140.7, 140.4, 137.8, 137.7, 133.5, 131.3, 129.0, 127.6,
3
+
1
25.9, 123.5, 120.7, 118.9, 116.8, 116.1, 115.3, 111.2, 55.3, 21.8, 21.0. MS (ESI): m/z 355 (M+H) .
+
+
+
MS (ESI): m/z 354.99 (M+H) , 356.54 (M+2H) , 354.39 (M) .
4.1.18. 3-(3-(3,4-Dimethoxyphenyl)-6-methylisoquinolin-1-yl)phenol (4da)
The procedures described for 4aa were used with 7d (200 mg, 0.63 mmol), solution of 3-
hydroxyphenylboronic acid (132 mg, 0.95 mmol) in 3 mL MeOH, Pd(PPh ) (0) (37 mg, 0.03 mmol),
3
4
sat. Na CO solution (3 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(n-hexane:EtOAc = 3:1, 1:1) to obtain compound 4da as a light green semi-solid, which solidified
2
5

ACCEPTED MANUSCRIPT
upon prolonged heating and vacuum pumping (260 mg, quantitative). R = 0.55 (n-hexane:EtOAc =
f
-1 1
1
1
7
:1). Mp: 96–99 °C. IR: 3411 cm . H NMR (300 MHz, DMSO-d6): δ 9.66 (bs, 1H, OH), 8.25 (s,
H, Ar-H), 7.93 (b, J = 8.7 Hz, 1H, Ar-H), 7.82–7.79 (m, 3H, Ar-H), 7.44–7.35 (m, 2H, Ar-H), 7.14–
.07 (m, 3H, Ar), 6.96–6.92 (m, 1H, Ar-H), 3.87 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 2.52 (s, 3H, 6-
3
3
13
CH ). C NMR (100 MHz, CDCl ): δ 160.4, 156.1, 150.3, 149.4, 149.0, 140.9, 140.0, 138.0, 132.6,
3
3
1
3
29.2, 127.6, 125.9, 124.1, 121.8, 119.9, 117.6, 116.1, 115.8, 111.1, 110.5, 55.9, 21.9. MS (ESI): m/z
+
71.93 (M+H) .
4.1.19. 3-(3,4-Dimethoxyphenyl)-1-(3-methoxyphenyl)-6-methylisoquinoline (4db)
The procedures described for 4aa were used with 7d (200 mg, 0.63 mmol), a solution of 3-
methoxyphenylboronic acid (145 mg, 0.95 mmol) in 3 mL MeOH, Pd(PPh ) (0) (37 mg, 0.03 mmol),
3
4
sat. Na CO solution (3 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(
CH Cl , n-hexane:EtOAc = 5:1, 3:1) to obtain compound 4db as a transparent semi-solid, which
2 2
solidified into a white solid upon storage (248 mg, quantitative). R = 0.23 (n-hexane:EtOAc = 3:1).
f
1
Mp: 58–61 °C. H NMR (300 MHz, DMSO-d6): δ 8.28 (s, 1H, Ar-H), 7.92 (d, J = 8.7 Hz, 1H, Ar-H),
7.83–7.79 (m, 3H, Ar-H), 7.53–7.48 (m, 1H, Ar-H), 7.42 (dd, J = 8.7, 1.8 Hz, 1H, Ar-H), 7.30–7.26
(
m, 2H, Ar-H), 7.14–7.07 (m, 2H, Ar-H), 3.87 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), 3.82 (s, 3H,
3
3
13
OCH ), 2.52 (s, 3H, 6-CH ). C NMR (100 MHz, CDCl ): δ 159.6, 159.5, 149.9, 149.5, 149.1,
3
3
3
141.4, 140.3, 138.2, 132.7, 129.1, 128.9, 127.3, 126.1, 123.9, 122.7, 119.5, 115.6, 114.5, 114.2, 111.1,
+
+
+
1
10.2, 56.0, 55.9, 55.4. MS (ESI): m/z 385.94 (M+H) , 387.58 (M+2H) , 385.37 (M) .
4.1.20. 3-(3-(3,4-Dimethoxyphenyl)-6-methylisoquinolin-1-yl)aniline (4dc)
The procedures described for 4aa were used with 7d (200 mg, 0.63 mmol), a solution of 3-
aminophenylboronic acid monohydrate (148 mg, 0.95 mmol) in 3 mL MeOH, Pd(PPh ) (0) (37 mg,
3
4
0
.03 mmol), sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by MPLC (n-
2 3
hexane:EtOAc = 5:1) to obtain compound 4dc as a yellow semi-solid, which solidified upon
prolonged heating and vacuum pumping (202 mg, 85%). R = 0.51 (n-hexane:EtOAc = 1:1). Mp:
f
1
1
22–126 °C. H NMR (300 MHz, DMSO-d6): δ 8.22 (s, 1H, Ar-H), 7.94 (d, J = 9 Hz, 1H, Ar-H),
.82–7.79 (m, 3H, Ar-H), 7.42–7.38 (m, 1H, Ar-H), 7.23–7.18 (m, 1H, Ar-H), 7.08 (d, J = 9.3 Hz, 1H,
7
Ar-H), 6.92–6.91 (m, 1H, Ar-H), 6.83–6.79 (m, 1H, Ar-H), 6.74–6.70 (m, 1H, 2ʺ-H), 5.27 (s, 2H,
NH ), 3.87 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 2.51 (≈3H, Ar-CH , overlapped with DMSO peaks).
2
3
3
3
1
H NMR (300 MHz, CDCl ): δ 8.02 (d, J = 8.7 Hz, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 7.78–7.72 (m, 2H,
3
Ar-H), 7.65 (s, 1H, Ar-H), 7.34–7.26 (m, 2H, Ar-H), 7.16–7.10 (m, 2H, Ar-H), 6.98 (d, J = 8.4 Hz,
H, Ar-H), 6.85–6.81 (m, 1H, 2″-H), 4.01 (s, 3H, OCH ), 3.94 (s, 3H, OCH ), 3.79 (bs, 2H, NH ),
1
3
3
2
2
6

ACCEPTED MANUSCRIPT
1
3
2
1
5
.54 (s, 3H, 6-CH ). C NMR (100 MHz, CDCl ): δ 160.1, 149.9, 149.5, 149.1, 146.3, 141.0, 140.2,
3
3
38.1, 132.8, 129.0, 128.8, 127.6, 126.0, 123.9, 120.7, 119.6, 116.8, 115.2, 114.5, 111.1, 110.2, 56.0,
+
+
+
5.9, 21.8. MS (ESI): m/z 371 (M+H) . MS (ESI): m/z 371.01 (M+H) , 372.42 (M+2H) , 370.39
+
(
M) .
4.1.21. 4-(3-(3,4-Dimethoxyphenyl)-6-methylisoquinolin-1-yl)phenol (4dd)
The procedures described for 4aa were used with 7d (200 mg, 0.63 mmol), a solution of 4-
hydroxyphenylboronic acid (114 mg, 0.82 mmol) in 3 mL MeOH, Pd(PPh ) (0) (37 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(
CH Cl , n-hexane:EtOAc = 4:1) to obtain compound 4dd as a light hay-colored semi-solid, which
2 2
1
solidified upon storage (236 mg, 99%). R = 0.22 (n-hexane:EtOAc = 2:1). Mp: 190–193 °C. H
f
NMR (300 MHz, DMSO-d6): δ 9.79 (bs, 1H, OH), 8.19 (s, 1H, Ar-H), 7.97 (d, J = 8.7 Hz, 1H, Ar-H),
7
1
6
.81–7.78 (m, 3H, Ar-H), 7.61–7.58 (m, 2H, 2″,6″-(H) ), 7.42–7.39 (m, 1H, Ar-H), 7.08 (d, J = 9 Hz,
2
H, Ar-H), 6.98–6.95 (m, 2H, 3″,5″-(H) ), 3.87 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 2.51–2.48 (3H,
2
3
3
1
-CH , overlapped with DMSO peaks). H NMR (400 MHz, CDCl ): δ 8.00 (d, J = 8.4 Hz, 1H, Ar-
3
3
H), 7.88 (s, 1H, Ar-H), 7.78 (d, J = 2 Hz, 1H, Ar-H), 7.72 (dd, J = 8.4, 2 Hz, 1H, Ar-H), 7.69–7.66
m, 3H, 2ʺ,6ʺ-(H) , Ar-H, overlapped), 7.32–7.29 (m, 1H, Ar-H), 7.00–6.95 (m, 3H, Ar-H, 3ʺ,5ʺ-(H) ,
(
2
2
1
3
overlapped), 5.35 (bs, 1H, OH), 4.01 (s, 3H, OCH ), 3.95 (s, 3H, OCH ), 2.55 (s, 3H, 6-CH ). C
3
3
3
NMR (100 MHz, DMSO-d6): δ 159.3, 158.5, 149.9, 149.3, 149.2, 140.4, 138.4, 132.2, 131.8, 130.7,
1
3
29.5, 127.3, 126.7, 123.5, 119.7, 115.5, 113.8, 112.2, 110.5, 56.06, 56.02, 21.8. MS (ESI): m/z
+
-
71.92 (M+H) , 369.93 (M-H) .
4.1.22. 3-(3,4-Dimethoxyphenyl)-1-(4-methoxyphenyl)-6-methylisoquinoline (4de)
The procedures described for 4aa were used with 7d (200 mg, 0.63 mmol), a solution of 4-
methoxyphenylboronic acid (126 mg, 0.82 mmol) in 3 mL MeOH, Pd(PPh ) (0) (37 mg, 0.03 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(
CH Cl , n-hexane:EtOAc = 5:1) to obtain compound 4de as a transparent semi-solid, which
2 2
solidified into an off-white solid upon storage (231 mg, 94%). R = 0.32 (n-hexane:EtOAc = 3:1).
f
1
Mp: 84–88 °C. H NMR (300 MHz, DMSO-d6): δ 8.22 (s, 1H, Ar-H), 7.95 (d, J = 8.7 Hz, 1H, Ar-H),
7
7
2
1
2
.82–7.79 (m, 3H, Ar-H), 7.73–7.69 (m, 2H, 2″,6″-(H) ), 7.42 (dd, J = 8.4, 1.5 Hz, 1H, Ar-H), 7.17–
2
.13 (m, 2H, 3″,5″-(H) ), 7.09 (d, J = 9.3 Hz, 1H, Ar-H), 3.87 (s, 6H, (OCH ) ), 3.82 (s, 3H, OCH ),
2
3 2
3
13
.52 (s, 3H, 6-CH ). C NMR (100 MHz, CDCl ): δ 160.0, 159.4, 149.9, 149.5, 149.1, 140.1, 138.3,
3
3
32.9, 132.6, 131.5, 128.8, 127.4, 126.3, 123.9, 119.5, 114.0, 113.7, 111.2, 110.2, 56.03, 56.0, 55.4,
+
+
+
1.8. MS (ESI): m/z 386.08 (M+H) , 387.62 (M+2H) , 385.34 (M) .
2
7

ACCEPTED MANUSCRIPT
4.1.23. 1-(3-Aminophenyl)-3-(3,5-dimethoxyphenyl)-N,N-dimethylisoquinolin-7-amine (4ec)
The procedures described for 4aa were used with 7e (197 mg, 0.57 mmol), a solution of 3-
aminophenylboronic acid monohydrate (125 mg, 0.80 mmol) in 3 mL MeOH, Pd(PPh ) (0) (33 mg,
3
4
0
.02 mmol), sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (10 mL), followed by MPLC (n-
2 3
hexane:EtOAc = 5:1) to obtain compound 4ec as a yellow semi-solid, which solidified upon
-1 1
prolonged heating (157 mg, 68%). Mp: 101–103 °C. IR: 3473 cm . H NMR (300 MHz, DMSO-
d6): δ 8.21 (s, 1H, Ar-H), 7.91 (d, J = 9 Hz, 1H, Ar-H), 7.51–7.47 (m, 1H, Ar-H), 7.34 (d, J = 2.4 Hz,
2
H, 2′, 6′-(H) ), 7.19 (t, J = 7.8 Hz, 1H, Ar-H), 7.13 (d, J = 2.4 Hz, 1H, Ar-H), 6.99 (t, J = 1.9 Hz, 1H,
2
Ar-H), 6.90–6.87 (m, 1H, Ar-H), 6.70–6.66 (m, 1H, 2″-H), 6.51 (t, J = 2.2 Hz, 1H, 4′-H), 5.25 (s, 2H,
13
NH ), 3.82 (s, 6H, 3′,5′-(OCH ) ), 2.97 (s, 6H, N(CH ) ). C NMR (100 MHz, CDCl ): δ 161.0,
2
3 2
3 2
3
1
1
58.0, 149.1, 146.4, 146.3, 142.3, 141.5, 130.5, 128.8, 128.2, 127.5, 120.4, 119.9, 116.8, 115.9, 115.0,
+
+
05.4, 104.8, 99.9, 55.5, 40.6. MS (ESI): m/z 400 (M+H) . MS (ESI): m/z 399.97 (M+H) .
4.1.24. 3-(3-(3,5-Dimethoxyphenyl)-6,7-dimethoxyisoquinolin-1-yl)phenol (4fa)
The procedures described for 4aa were used with 7f (200 mg, 0.55 mmol), a solution of 3-
hydroxyphenylboronic acid (100 mg, 0.72 mmol) in 3 mL MeOH, Pd(PPh ) (0) (32 mg, 0.02 mmol),
3
4
sat. Na CO solution (2 mL) and 1,2-dimethoxy ethane (5 mL), followed by column chromatography
2
3
(n-hexane:EtOAc = 5:1, 3:1) to obtain compound 4fa as a semi-solid, which solidified into a white
solid upon prolonged heating and vacuum pumping (147 mg, 63%). R = 0.46 (n-hexane:EtOAc =
f
-
1 1
1
1
2
:1). Mp: 174–176 °C. IR: 3302 cm . H NMR (300 MHz, DMSO-d6): δ 9.65 (bs, 1H, OH), 8.28 (s,
H, Ar-H), 7.49 (s, 1H, Ar-H), 7.40–7.35 (m, 4H, Ar-H), 7.21–7.19 (m, 2H, Ar-H), 6.93–6.89 (m, 1H,
1
3
″-H), 6.55 (t, J = 2.1 Hz, 4′-H), 3.97 (s, 3H, OCH ), 3.83 (s, 6H, (OCH ) ), 3.79 (s, 3H, OCH ). C
3
3 2
3
NMR (100 MHz, CDCl ): δ 160.9, 158.4, 156.6, 153.1, 150.1, 149.4, 141.9, 140.2, 134.5, 129.2,
3
121.9, 121.3, 117.7, 116.3, 116.1, 105.7, 105.3, 105.2, 100.8, 56.1, 55.9, 55.4. MS (ESI): m/z 417.97
+
+
+
-
(
M+H) , 419.38 (M+H) , 417.33 (M) , 415.88 (M-H) .
4.1.25. 3-(3-(3-(4-Methoxyphenyl)isoquinolin-1-yl)phenoxy)-N,N-dimethylpropan-1-amine (8a)
Compound 4aa (149 mg, 0.45 mmol) was dissolved in N,N-dimethylformamide (DMF, 7
mL). Potassium carbonate (378 mg, 2.73 mmol) and 96% 3-dimethylamino-1-propylchloride
hydrochloride (216 mg, 1.31 mmol) were added to the solution. The reaction mixture was heated
using an oil bath set at 100 °C. Water (100 mL) was poured into the reaction mixture; the product
was extracted with CH Cl (100 mL x 9), washed with water (100 mL) and concentrated under
2
2
reduced pressure. The residue thus obtained was purified by MPLC (n-hexane:EtOAc = 3:1, EtOAc,
2
8