Design, synthesis and biological evaluation of 1-phenanthryl-tetrahydroisoquinoline derivatives as novel p21-activated kinase 4 (PAK4) inhibitors

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DOI: 10.1039/C5OB00037H

Page 1 of 18

Organic & Biomolecular Chemistry

Design, synthesis and biological evaluation of 1-

phenanthryl-tetrahydroisoquinoline derivatives as

novel p21-activated kinase 4 (PAK4) inhibitors

Cite this: DOI: 10.1039/x0xx00000x

Received 00th January 2012,

Accepted 00th January 2012

Shuai Song^a, Xiaodong Li^b, Jing Guo^a, Chenzhou Hao^a, Yan Feng^a, Bingyu Guo^b,

Tongchao Liu^a, Qiaoling Zhang^a, Zhen Zhang^a, Ruijuan Li^a, Jian Wang^a, Bin Lin^a,

Feng Li,^b,*, Dongmei Zhao^a,*, Maosheng Cheng^a,*

DOI: 10.1039/x0xx00000x

www.rsc.org/

Functional versatility and elevated expression in cancers have promoted p21-activated kinase 4

(PAK4) as one of the first-in-class anti-cancer drug targets. In this study, a series of novel 1-

phenanthryl-tetrahydroisoquinoline analogues have been designed and synthesized as a novel

class of small-molecule PAK4 inhibitors to fit into the cavity of PAK4. All of the target

compounds were evaluated for their in vitro PAK4 inhibitory activities and antiproliferative

activities. Lead optimization identified all the derivatives with more potency than the lead

compound, especially compound 21a. Moreover, compound 21a significantly induced the cell

cycle in the G1/S phase, and inhibited migration and invasion of MCF-7 cells via the

regulation of the PAK4-LIMK1-cofilin signaling pathway. Molecular modeling study showed

possible novel binding modes between 21a and PAK4 and provided a structural basis for

further structure-guided design of PAK4 inhibitors.

A number of ATP-competitive PAK inhibitors have been

described in detail in patent applications. PF-3758309,

Introduction

PAKs (p21-activated kinases) comprise a family of Ser/Thr

kinases that are activated by Ras-related G proteins of 21 kDa

(p21) called Rac and CDC42.^1,2In 1977, the first member of

the PAK family was isolated at NIH from a soil amoeba as

Acanthamoeba myosin I heavy chain kinase (MIHCK).³They

are thought to play an important role in numerous cancer

cellular processes, including cytoskeletal signaling pathways,

cell cycle progression, cancer cell invasion and migration, cell

survival and apoptosis therefore have been extensively studied

since their discovery.^4-7

disclosed by Pfizer in 2010,^23,24is a potent, ATP-competitive,

pyrrolopyrazole inhibitor of PAK4. This compound progressed

into phase I clinical trials in patients with advanced/metastatic

solid tumors. Unfortunately, the phase I clinical trial of PF-

3758309 was prematurely terminated due to undesirable

pharmacokinetic characteristics and a lack of an observed dose–

response relationship; subsequent clinical investigation of PF-

3758309 has been placed on hold.²⁵Using a high throughput

screening and validation, researchers at Soongsil University and

Korea Research Institute of Chemical Technology identified

KY-04031 as a novel p21-activated kinase 4 inhibitor. The

crystal structure of PAK4 complexed with the compound

illustrated that both indole and indazole of KY-04031 are

responsible for PAK4 hinge interaction. Moreover, the triazine

molecular core of KY-04031 mimics the ribose of ATP,

distinguishing its unique scaffold from other known PAK4

inhibitors.²⁶Researchers from Genentech recently succeeded in

identifying a highly potent and highly selective group II PAK-

selective inhibitor, with a novel binding mode and exquisite

kinome selectivity. Hydrophobic interactions within a lipophilic

pocket past the methionine gatekeeper of group II PAKs

approached by these types I 1/2 binders were found to be

important for improving potency. A concentration-dependent

decrease in tumor cell migration and invasion in two triple-

Six different PAKs have been classified into two groups

(PAK1-3 of group I, and PAK4-6 of group II) based on the

domain architectures.^8,9The group I family has already been

studied intensively, and the members of the group II PAK

family are now emerging as more interesting targets for cancer

therapy.¹⁰PAK4 as the first identified and characterized

member of group II PAK, was widely studied with the most

cancer-related citations.^11-13Recently, a paired recombinant

protein assay identified LIM kinase 1 (LIMK1) as a substrate

for PAK4 in vitro^14,15which solidly confirmed the previous

works on LIMK1, as a substrate of PAK4 kinase, playing an

important role in actin filament dynamics of migrating cells.^16-

19

PAK4 is tumorogenic in vitro and in vivo^13,20and its

overexpressed or genetic amplification is found in numerous

cancer cell lines and tumours.^21,22Therefore, PAK4 is

considered as an attractive target for anti-cancer drug design.

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DOI: 10.1039/C5OB00037H

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Journal Name

negative breast cancer cell lines was observed with this

compound.²⁷

(According to the referee’s suggestion, we have removed the

Figure 1).

Herein we describe the identification of novel PAK4 inhibitors

with the scaffold of 1-phenanthryl-tetrahydroisoquinoline. The

lead compounds from which these compounds are derived were

(-)-β-Hydrastine (1, Fig. 1) and Noscapine (2, Fig. 1), identified

from our in-house natural products database, with moderate

inhibition in a PAK4 biochemical assay (IC₅₀= 29.8 μM, 28.5

μM) (manuscript to be submitted). (-)-β-Hydrastine, comprised

of tetrahydroisoquinoline and isobenzofuranone fragments, is a

natural product that was first extracted from the root of

Hydrastis canadensis in 1862 and used as a uterine hemostatic

agent, antibacterial agent, and anti-inflammatory agent.^28,29

Recently, (-)-β-Hydrastine and Noscapine were shown to be

microtubule-targeting molecules, then the two agents were

evaluated for activity in cell migration and invasion.³⁰Because

the PAK4 has been linked with survival, proliferation,

migration, invasion, and apoptosis in tumors, our group and

collaborator Prof. Li from China Medical University first

demonstrated that these two natural products mediated their

effects by modulating the PAK4 activation pathway for MCF-7

cells (manuscript to be submitted). However, these compounds

contain lactone in their structures therefore are prone to

hydrolysis. Our goals for lead optimization were to avoid the

stability problems from the isobenzofuranone fragment and

simplify the two chiral centers, in hopes of maintaining the

inhibition activity toward PAK4. Different replacements for the

isobenzofuranone fragment have been designed, synthesized,

and evaluated for their biological activities toward PAK4.

Phenanthryl ring as a replacement to afford 1-phenanthryl-

tetrahydroisoquinoline derivatives was found to result in the

best PAK4 inhibitions. Followed by lead optimization, a novel

Fig. 1 Structures of (-)-β-Hydrastine (1), Noscapine (2) and Compound 22

Results and discussion

Chemistry

The synthetic methods for the preparation of target compounds

are summarized as follows (Scheme 1). Overall, they were

prepared in 7 or 8 steps beginning with condensation of various

benzaldehydes with various phenyl acetic acids to obtain 13a-o

in a cis-trans-isomer ratio of 90:10 (isolated yield). The cis-

isomer, which was transformed into the trans isomer in the next

esterification reaction in MeOH/H₂SO_4,was not necessary to

isolate. Meanwhile, some references reported that both the cis

and trans esterifiable isomers could be coupled in the next

reaction.³¹The intramolecular coupling reaction is the key step

in the reaction of the stilbene with VOF₃, which was applied to

only substrates with at least three electrondonating groups

providing 15a-o ³²

. The methyl esters of 15a-q were hydrolyzed

to carboxylic acids with sodium hydroxide to afford the

phenanthrene-carboxylic acids 16a-o in quantitative yield.

Compounds 17a-o and 18a-o were synthesized by treating the

phenanthrenecarboxylic acids with HOBt and EDCI in DCM at

0

°C for 2.5 h, followed by homoveratrylamine or

homopiperonylamine to afford the amides after 15 min.³³

Bischler-Napieralski reaction of compounds 17a-o and 18a-o

using phosphorus oxychloride as a cyclization reagent under

neat conditions gave the intermediates dihydroisoquinolines in

refluxing dimethoxyethane (DME) after 3.5 h. These

intermediates were then directly used for the next step to

furnish the desired 19a-o and 20a-o from the reaction of

sodium borohydride in cooled MeOH overnight.³⁴

class

of

PAK4

inhibitors

with

1-phenanthryl-

tetrahydroisoquinoline scaffold was identified. Among them,

21a was discovered as unique small-molecule inhibitor of the

PAK4 interaction with good affinity (IC₅₀= 8.84 μM). Here, we

reported the synthesis, biological evaluation and the structure-

activity relationships (SARs) of this series of 1-phenanthryl-

tetrahydroisoquinoline derivatives. A molecular modeling study

of compound 21a was performed to elucidate its possible

binding modes and to provide a structural basis for the further

structure-guided design of PAK4 inhibitors.

Subsequently,

all

the

substituted

1-phenanthryl-

tetrahydroisoquinolines (19a-o and 20a-o), on treatment with

acetyl chloride in the presence of K₂CO₃as an acid-neutralizing

reagent, delivered the expected final products 21a-o and 22a-o

in quantitative yield. Finally, the deprotection of 21g-l and 22g-

l

in an ultrasonic reactor with 1,4-cyclohexadiene and 10%

Pd/C afforded 21p-u and 22p-u in quantitative yield.

2 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 2012

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DOI: 10.1039/C5OB00037H

Page 3 of 18

Organic & Biomolecular Chemistry

R₁

R₂

R₃

R₄

R₁

R₂

CHO

v

i

iii

COOH

ii

i

+

R₃

R₄

R₃

R₄

R₃

R₄

R₃

R₄

COOCH₃

COO

H

COOCH₃

COOH

R₅

:

3

:

4

:

5

:

6

:

7

:

8

=

OC

2

R₁

H

R

H

,

;

3

=

:

=

=-

=

,

R₁R₂

OC

H

9

R₃R₅

H

OC

R

H

OC

H

;

,

;

4

3

R₁

OC

H

R³

2

O

OC

-

B

H

10 R₃R₄

H

=

n

=

R

5

,

;

a-o

14

a-o

15

2

3

n ³

=

:

=

-

,

a-o

16

a-o

13

=

R₁

O

B

R

11 R₃R₄

H

R

H

;

,

3

2

5

=-

:

12

=

R₁R₂OCH₂

R₃R₄R₅OCH₃

;

=

OC

H

R₁

H

R

C

H

( )

3

2

,

2

O

C

O

NH

N

HN

O

a:

=

O

R₁R₃

H

R

,

;

:

=

=-

-

H

O

b

R₁R₃

R²

H

R

R₅

OC

;

2

R₅

R₄

R₅

R₄

R₅

R₄

c:

=

H

R₁R₂R₄OCH₃

A

:

=

NH₂

d

R₁R₂R₄R₅OCH₃

;

e:

=

R₁R₂R₃R₄R₅OCH₃

B

:

=

-

2

R₅

=

R

3

f

R₁R₂

OC

;

R₁R₄OCH₃R⁴OB

H

R₃

:

=

R₁

;

g

R₁

:

h

=

R

R₁R₄R₅OCH₃

R₂

a-o

:

k:

=

=-

-

i

j

R₁OCH₃

R₅OCH₂

=

H

;

,

;

4

-

21

l

g

a-o

19

=

n

=-

=

17

R₁OB

R

R₅

,

a-o

21

v

iii

2

-u

p

=

R₁OB , R₂R₄R₅OCH₃, R₃H;

v

i

v

ii

:

=

=-

l

R₁OB

R

R₅OCH₂

,

;

+

a-o

2

16

m

:

=

R₁R₂OCH₂

;

4

R₁R₂OCH₂R³

n

o:

:

=

;

O

C

O

=

R₁R₃

H

R

R₅OCH₃

,

;

4

:

=

R₁R₄OCH₃

H

;

H

;

p

HN

O

NH

N

:

=

O

R₁R₄R₅

q

3

r

=

=-

-

R₁OCH₃

R

=

R₅OCH₂

,

;

4

R₅

R₄

R₅

R₄

R₅

R₄

s:

=

R₁OH

R

O

,

R₁OH R²R₄R₅OCH₃

H

;

A

:

=

t

R₁OH R²OCH₃

R₅OCH₂

3

NH₂

u:

=

=-

-

2

B

R₃

R₁

R₂

a-o

R₂

a-o

R₂

-

22

l

g

a-o

18

20

22

v

iii

-u

p

22

Scheme1. Reagents and conditions: (i) (a) Ac₂O, Et₃N, reflux, 10 h; (b) NaOH (aq), 2 h; (ii) H₂SO₄, MeOH, reflux, 4 h; (iii) VOF₃, DCM, EtOAc, TFA, TFAA, ice-salt bath, 1.5

h; (iv) NaOH, MeOH-H₂O or THF-H₂O, reflux; (v) (a) EDCI, HOBt, DCM, 0 °C; (b) DIPEA, phenethylamine, rt; (vi) (a) POCl₃, DME, reflux; (b) NaBH₄, MeOH, 0 °C; (vii)

Acetyl chloride, TEA, DCM, 0 °C; (viii) 1,4-cyclohexadiene, DCM/MeOH, 10% Pd/C, ultrasound, 25 °C, 100 min.

Enzymatic Assay

C-2, 3-dimethoxy group, showed higher activities. The different

activities of the compounds with multiple substitution effects in

ring-B demonstrated that the modification of ring-B was

In an attempt to evaluate the ability of various 1-phenanthryl-

tetrahydroisoquinoline derivatives to inhibit PAK4, all the new

synthetic compounds and the reference compound (-)-β-

crucial. A comparison of compounds 21a

, 21c, 21h, and 21o

revealed a substitution effect at C-6 of ring-B. Compound 21o

,

Hydrastine (1) were initially assayed for their inhibitory effects

which contains an isopropoxy group, showed the most potent

activity against PAK4, with an IC₅₀value of 8.0μM. The

conversion of the C-6-OiPr to C-6-OMe (21a) resulted in a tiny

decrease in potency, with an IC₅₀value of 8.8 μM. The addition

of an extra benzyloxy group or methoxy group at C-7, as in

compound 21d or 21k, respectively, led to a decrease in

biological activity. When the C-6-OMe and C-7-OBn were

changed, the anti-kinase activities of compounds 21h and 21k

had no significant change. In the ring-B modification, the

deprotection of the benzyl to give an -OH group, accompanied

by a C-6-OMe or C-7-OMe, was found to decrease the

biological activity, indicating that the hydrogen bond donating

OH group is disfavoured. Consequently, this finding indicates

against PAK4 with Kinase Glo^®Luminescent Kinase Assays,

and compounds with high potency for PAK4 were selected for

further profiling (Table 1). The reported IC₅₀values are the

average of at least three independent experiments.

As shown in Table 1, most of the target compounds showed

moderate-to-good inhibitory effects against PAK4, with

potencies in the 5-20 μM range, indicating that the introduction

of the phenanthryl ring moiety as a suitable rigid scaffold to fit

into the large pocket of PAK4 increased the inhibition of kinase

activity. In the ring-C modification, similar activity was

observed for compounds 21a-o and 22a-o, which contain two

methoxy groups and a methylene-dioxy group, respectively. In

our previous docking study, it was shown that the

tetrahydroisoquinoline part faced the solvent distinct in an area

that was not sensitive to the alkoxy groups. When ring-A was

replaced with C-3-methoxy, C-2,3-dimethoxy, C-2,3,4-

trimethoxy, and C-2,3-methylene-dioxy substituents, the

that

the

combination

of

C-2,3-dimethoxy-6-

isopropoxyphenanthryl or C-2,3,6-trimethoxyphenanthryl is the

most favourable modification for the anti-proliferative activity

of 1-phenanthryl-tetrahydroisoquinoline analogues.

compounds 21a, 22h, 21k, 21n and 22o, which contained the

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Table 1. Inhibitory Effects of New Compounds against PAK4 kinase^a

compound

21a

PAK4(IC₅₀, μM) Purity%

compound

PAK4(IC₅₀, μM) Purity%

8.84 ± 0.09

10.17 ± 0.12

9.87 ± 0.47

13.44 ± 0.19

8.47 ± 0.15

14.47 ± 0.26

9.66 ± 0.11

11.70 ± 0.07

9.97 ± 0.03

10.17 ± 0.05

12.25 ± 0.06

8.61 ± 0.08

10.81 ± 0.14

9.90 ± 0.15

8.01 ± 0.06

16.58 ± 0.18

14.31 ± 0.12

10.36 ± 0.08

12.06 ± 0.06

11.28 ± 0.03

10.02 ± 0.05

0.028 ± 0.006

99.39

99.76

95.52

99.54

98.59

98.95

97.21

99.88

99.61

99.71

99.45

97.12

100.00

99.70

99.18

99.55

98.08

99.55

95.47

97.10

99.76

22a

22b

22c

22d

22e

22f

22g

22h

22i

22j

22k

22l

22m

22n

22o

22p

22q

22r

8.76 ± 0.04

10.74 ± 0.29

9.96 ± 0.46

17.67 ± 0.15

12.23 ± 0.33

16.51 ± 0.20

10.95 ± 0.17

12.49 ± 0.12

9.85 ± 0.13

10.48 ± 0.21

11.25 ± 0.10

9.52 ± 0.03

12.79 ± 0.03

15.86 ± 0.25

13.21 ± 0.18

15.44 ± 0.11

13.63 ± 0.22

11.27 ± 0.07

11.40 ± 0.12

11.89 ± 0.08

9.93 ± 0.04

0.011 ± 0.008

99.64

99.53

95.56

99.30

97.69

99.84

95.38

98.74

98.42

97.51

99.40

98.58

99.72

98.72

99.05

100.00

99.50

99.55

98.94

95.48

99.53

21b

21c

21d

21e

21f

21g

21h

21i

21j

21k

21l

21m

21n

21o

21p

21q

21r

21s

21t

22s

22t

21u

22u

PF-3758309^b

Staurosporine^b

^aIC₅₀s were calculated by the Logit method from the results of at least three independent tests with eleven concentrations each and expressed as the means ±

SD.

^bUsed as a positive control.

Cellular Inhibition Study

Table 2. Inhibitory Effects of Potent Compounds on Cell Proliferation

Based on the enzymatic assay, potent inhibitors were selected

for the cellular assay. The antiproliferative effects of the

IC₅₀(μM) ^a

compound

PAK4(IC₅₀, μM)

MCF-7

0.85

6.63

4.22

7.81

5.08

5.04

0.92

A549

1.35

5.28

>30

HT1080

>50

compounds are listed in Table 2 in the MCF-7 cell line and the

A549 cell line, in which PAK4 has been found to be

overexpressed. Meanwhile, the tumours cell line HT-1080

whose growth was not dependent on PAK4, was used to test the

potential off-target effects of the potent PAK4 inhibitors. In

parallel with the enzymatic results, all the selected compounds

displayed antiproliferative effects in the MCF-7 and A549 cell

lines. Compound 21a, bearing a C-6-OMe group in ring-B,

showed IC₅₀value of 0.85 μM and 1.35 μM in the MCF-7 and

the A549 cells respectively, and a 40-fold selectivity over

8.84

9.88

8.47

9.97

8.61

8.01

9.93

21a

21c

21e

21i

15.89

34.00

27.29

31.80

45.76

4.71

2.33

3.86

8.03

0.72

21l

21o

22u

^aIC₅₀: Concentration of the compound (μM) displaying 50% cell growth

inhibition after 24 h of drug exposure, as determined by the MTT assay. Each

experiment was carried out in triplicate.

HT1080 cells. Compounds 21i, 21l, and 21o were found to

decrease the biological activity. Compound 22u, bearing an -

OH group, showed high potency with IC₅₀values of 0.92 μM

and 0.72 μM in the MCF-7 and A549 cells respectively.

Meanwhile, compound 22u also showed high potency in the

HT1080 cells, indicating that general cytotoxicity might exist.

Quite disappointingly, compound 21o showed less potent

Cell cycle analysis

We next examined the effect of compound 21a on the

proliferation of human cancer cells. As shown in the MTT

assay, compound 21a treatment inhibited the proliferation of

MCF-7 and A549 cells at 1 μM. To investigate the mechanism

by which compound 21a inhibited the growth of human breast

cancer cells, we explored the effects of compound 21a on cell-

cycle distribution by flow cytometry. After 24 h of exposure,

compound 21a was found to induce an increase in the

percentage of cells in the G1 phase and decrease such in the S

phase compared with the vehicle control, indicating that

compound 21a arrests MCF-7 cells at the G1 phase of the cell

cycle (Fig. 2A). Since CyclinD1/3 and CDK2/4/6 are key

regulators in G1 phase of the cell cycle, we then examined the

expression level of these factors along with the level of

activated PAK4(phospho-Ser474) in compound 21a treated

activity in the MCF-7 cell line compared with 21a

.

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ARTICLE

cancer cells. Western blot analysis found that exposure of dependent manner (Fig. 2B). All these dates demonstrate that

MCF-7 to compound 21a (5, 10 μM) for 24h dramatically compound 21a suppresses the proliferation of breast cancer

decreased levels of phospho-PAK4, CDK2, CDK4, CDK6, cells and mediates cell cycle arrest via repression of cyclin D

cyclin D1 protein, and cyclin D3 protein expression in a dose- and CDKs.

Fig. 2 Compound 21a affects cell cycle distribution in MCF-7 cancer cells. (A) Untreated cells (Control) and cells treated with compound 21a at different

concentrations for 24 h; (B) 21a downregulates the expression level of cyclin D and CDKs.

(Fig. 3A). The results indicate that 21a can induce apoptosis in

breast cancer cells. To elucidate the molecular mechanisms

Compound 21a induces apoptosis in breast cancer cells

We further tested whether apoptosis induction could contribute involved in the observed apoptosis alterations, we detected the

to the growth inhibitory function of 21a in MCF-7 cells. The expression of several apoptosis regulators in 21a treated cancer

ability of 21a to induce apoptosis in MCF-7 cells was thus cells. Results showed that 21a decreased the levels of phospho-

evaluated by treatment of cells with 21a for 24 h and PAK4 and Bcl-2, increased BAX, caspase 3, and caspase 8

subsequently subjecting the cells to staining with annexin V expression in MCF-7 cells (Fig. 3B). Data above indicates that

and propidium iodide (PI), and flow cytometry analysis. As 21a induces apoptosis of breast cancer cells via affecting

shown in Fig 3, treatment with 21a (0 μM, 5 μM, 10 μM) for 24 indicated apoptosis regulators along with the inhibition of

h caused 13.3% annexin V-positive cells compared with PAK4 kinase activity.

controls, which showed only 5.7% annexin V-positive cells

Fig. 3 The effect of 21a on apoptosis of breast cancer cells. (A) Untreated cells (Control) and cells treated with compound 21a at different concentrations for 24 h; (B)

21a downregulates Bcl-2 expression and upregulates BAX, caspase 3, and caspase 8.

PAK4/SCG10 signaling pathways were examined by western

blot assay. signaling pathway by Western blot analysis. As we

expected, exposure of MCF-7 cells with different

concentrations of compound 21a for 24 h inhibited the levels of

activated PAK4, phosphor-LIMK1, phosphor-cofilin, and

phosphor-SCG10 phosphorylation in a dose-dependent manner

(Fig. 4B), indicating that the mechanism of 21a repressing

breast cancer cell migration and invasion correlates with 21a

inhibiting PAK4 kinase activity and its certain downstream

substrates.

Cell migration and invasion assay

PAK4 activity is involved in cytoskeleton dynamics and cancer

cell migration and invasion, therefor, inhibitory effect of 21a on

breast cancer cell migration and invasion were analysed. By

transwell assay, a dose-dependent decrease in cell migration

and invasion was observed following treatment with compound

21a (Fig. 4A). As reported, PAK4-regulated cell migration and

invasion via the PAK4/LIMK1/cofilin, PAK4/MMP2, or

PAK4/SCG10 signaling pathway. Here, the effects of

compound 21a on the PAK4/LIMK1/cofilin, PAK4/MMP2, or

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Fig. 4 21a suppresses the migration and invasion of human breast cancer cells and inhibits the PAK4/LIMK1/cofilin and PAK4/SCG10 signaling pathways (A) MCF-7

cells migratory and invasive capacities were evaluated by using a Boyden chamber matrigel invasion assay. After 24 h treatment with the indicated concentrations of

21a, the invaded cells were fixed and stained, and 10 random fields were counted. (B) Western blot analysis shows the protein level of the proteins involved in

migration and invasion.

Lentivirus production and infection

exhibited a significantly reduced the inhibition of the invasive

and migratory effects in PAK4 silenced cells, which were

transfected with sh-PAK4 recombinant lentivirus (Fig. 5B).

Further, we detected PAK4 activity and its substrates

phosphorylation level in PAK4WT overexpressed cells or PAK

silenced cells by western blot assay. Data showed the same

results with the migration and invasion assays (Fig. 5C). All

these experiments demonstrated that compound 21a represses

the invasive and migratory capacity of MCF-7 cells via

inhibiting PAK4 kinase activity.

To further clarify whether the regulation of 21a on MCF-7

migration and invasion depend on its inhibitory effect of PAK4

kinase activity, we stable transfected Flag-tagged PAK4WT

(wild type PAK4) or PAK4KM (kinase dead PAK4) into MCF-

7 cells and then performed transwell assay following 21a

treatment. As a result, PAK4WT-overexpressed cells showed

an enhanced-inhibitory effect by 21a on both migration and

invasion of MCF-7 cells comparing to vector control, while

PAK4KM did no. Furthermore, compound 21a treatment

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Fig. 5 21a suppresses migration and invasion of human breast cancer cells via PAK4 kinase activity. (A) The migratory and invasive capacities PAK4WT/KM stable

transfected MCF-7 cells of PAK4^Wtand PAK4^KMMCF-7 cells were evaluated by using a Boyden chamber matrigel invasion assay. (B) PAK4^SiMCF-7 cells migratory and

invasive capacities were evaluated. (C) Western blot analysis shows the protein levels involved in migration and invasion in PAK4 -overexpressed or PAK4-silenced

cells.

Molecular modelling

structure of PAK4 in complex with PF3758309 (PDB entry

23a

code: 2X4Z)

was used as the receptor. Molecular docking

To investigate the mechanism by which compound 21a

interacts with PAK4, we performed docking study between 21a

and PAK4. Both isomers of 21a were studied. The crystal

was carried out using Glide XP (Schrodinger 2014). The ligand

poses that Glide generated passed through a series of

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ARTICLE

hierarchical filters that evaluated the ligand’s interaction with resolution accurate mass determinations (HRMS) for all final

the receptor. The final binding model was shown in Fig. 6. target compounds were obtained on a Bruker Micromass Time

In 2014, Li et al³⁵constructed a ligand-based pharmacophore of Flight mass spectrometer equipped with electrospray

model for PAK4 inhibitors consisting of five chemical features: ionisation (ESI). Column chromatography was performed with

a positive ionizable center, two hydrophobic groups, a silica gel (200-300 mesh) purchased from Qingdao Haiyang

hydrogen donor, and a hydrogen acceptor. It was demonstrated Chemical Co. Ltd. The purities of compounds used for

that the crucial anchoring points inside the binding site include biological evaluation (> 95%) were determined on a Waters

Glu396, Phe397, and Leu398 which play important roles in the 2000 HPLC system: column, Unitary C18, 4.6 mm × 250 mm,

affinity of PAK4 inhibitors. The other anchoring point is the 5 μm; solvent A = H₂O, solvent B = MeOH; flow rate, 1.0

conserved water molecule W2142 including Asp458 and mL/min. Samples were eluted with a gradient of 70%

Lys350 which influences PAK4 selectivity. In Fig. 6, both MeOH/H₂O to 95% MeOH/H₂O over 15 min and detected at

isomers of compound 21a bind to the PAK4 catalytic domain 254 nm.

fairly well. The bulky and lipophilic phenanthrene group makes

GENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

effective hydrophobic interactions with the highly hydrophobic 13

A-O. A solution of substituted phenylacetic acid (7.5 mmol),

ATP binding site. ^36,37In particular, compound (R)-21a makes substituted benzene formaldehyde (7.5 mmol), acetic anhydride

hydrogen bonds with conserved water molecule and protein (3 mL), and triethylamine (1.5 mL) was refluxed with stirring

residue Glu329 while compound (S)-21a makes hydrogen under N₂for 10 h. The resulting solution was cooled to room

bonds with protein residues (Glu396, Ile327, Ala402). This temperature, 10% NaOH solution (25 mL) was added, and then

model suggested that hydrophobic interactions play an the resulting mixture was stirred for 2 h at 90°C. The reaction

important role between the compound and PAK4 and they mixture was cooled to room temperature and neutralized with

offered a structural explanation for the activity of the concentrated HCl (pH 3). The yellow stilbene acid obtained

compound.

was collected by filtration and recrystallized from acetonitrile

to yield pure 13a-o as yellow needles. For example:

(E/Z)-2-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-acrylic

acid (13a). Yellow needle, yield: 73%. mp 214.1-215.5 °C. H

1

NMR (400MHz, DMSO-d₆) δ 12.43 (s, 1H), 7.67 (s, 1H), 7.06-

7.04 (d, J = 8.84 Hz, 2H), 6.98-6.96 (d, J = 8.24 Hz, 1H), 6.80-

6.78 (d, J = 8.88 Hz, 2H), 6.74-6.73 (d, J = 1.88 Hz, 1H), 6.68-

6.66 (dd, J = 8.12, 1.88Hz, 1H), 3.78 (s, 1H), 3.71 (s, 1H), 3.66

(s, 1H). ¹³C NMR (100MHz, DMSO-d₆) δ 169.22, 160.32,

149.26, 148.67, 138.98, 132.42(×2), 131.00, 129.40, 127.47,

122.20, 114.29(×2), 113.63, 112.40, 56.00, 55.90, 55.63. MS

(ESI) m/z 315.2[M+H]⁺, 337.2[M+Na]⁺, 353.1[M+K]⁺.

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

14A-O. Stilbene acid 13a-o (5 mmol) was dissolved in MeOH

(15 mL) containing concentrated H₂SO₄(0.1 mL) and the

resulting solution was refluxed for 6 h and then cooled to room

temperature overnight. The mixture was filtered and the solid

was washed with cooled MeOH twice. The solid was dried in

vacuum to give 14a-o. The filtrate was evaporated under

reduced pressure; water was added. The mixture was extracted

with CH₂Cl₂and brine. The organic phase was dried over

Na2SO4 overnight and the solvent was removed in a vacuum.

The oil was purified by silica gel column chromatography (n-

hexane: EtOAc as eluent) to afford an addition of 14a-o as off-

yellow crystals. For example:

Fig. 6 Predicted binding models of PAK4 in complex with the most active

compound 21a. PAK4 is shown in a cartoon representation with the bound

inhibitor in a stick representation. The gray dots indicate potential hydrogen

bonds between the compound and PAK4. (A) Space matching of compound (R)-

21a. (B) Space matching compound (S)-21a. (C) Best docking pose of compound

(R)-21a. (D) Best docking pose of compound (S)-21a

.

Experimental

Z-2-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-acrylic acid

methyl ester (14a). Off-yellow needle, yield: 74%. mp 110.6-

111.7 °C. ¹H NMR (400MHz, CDCl₃) δ 7.78 (s, 1H), 7.03-7.01

(d, J = 8.88 Hz, 1H), 6.91-6.89 (d, J = 8.20 Hz, 1H), 6.80-6.77

(dd, J = 8.16, 1.88 Hz, 1H), 6.74-6.73 (d, J = 1.88 Hz, 1H),

6.71-6.68 (d, J = 8.88 Hz, 2H), 3.93 (s, 1H), 3.80 (s, 1H), 3.79

(s, 1H), 3.76 (s, 1H). ¹³C NMR (100MHz, CDCl₃) δ 168.74,

160.29, 149.14, 148.58, 140.17, 132.40(×2), 129.62, 128.66,

127.30, 122.12, 113.72(×2), 112.86, 111.45, 55.89, 55.82,

Chemistry

G

ENERAL CHEMISTRY METHODS. Unless otherwise noted, all

materials were obtained from commercially available sources

and were used without purification. TLC was performed on

silica gel plates (thickness 250 mm, Indicator F-254) plates and

visualized by UV-light. H NMR and ¹³C NMR spectra were

recorded on a Bruker 400 MHz NMR spectrometer in DMSO-

d₆and CDCl₃, respectively and the chemical shifts were

recorded in parts per million downfield from TMS. High

1

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Journal Name

55.22, 52.29. MS (ESI) m/z 329.2[M+H]⁺, 351.2[M+Na]⁺, 7.21-7.18 (dd, J= 8.76, 2.28 Hz, 1H), 7.60 (s, 1H), 6.83 (s, 2H),

367.1[M+K]⁺.

6,82 (s, 1H), 6.17-6.14 (t, J = 5.68 Hz, 1H), 4.11 (s, 3H), 4.03

(s, 3H), 4.02 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.78-3.38 (q, J

= 13.16, 6.90 Hz, 2H), 3.00-2.96 (t, J = 6.80 Hz, 2H). ¹³C-NMR

(100MHz, CDCl₃) δ 170.05, 159.30, 149.73, 149.29, 149.13,

132.02, 131.35, 130.68, 129.82, 124.88, 124.52, 124.38,

124.25, 120.77, 115.98, 112.06, 111.45, 106.49, 103.84,

103.30, 55.99, 55.94(×2), 55.88, 55.54, 41.22, 35.30. MS (ESI)

m/z 475.2[M+H]⁺.

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

15A-O. A solution composed of stilbene ester 14a-o (2.5 mmol)

was dissolved in dry CH₂Cl₂(8 mL) containing trifluoroacetic

acid (0.2 mL) and trifluoroacetic anhydride (0.04 mL). A

solution of vanadium oxytrifluoride (5 mmol) in CH₂Cl₂(6.5

mL) and EtOAc (3.5 mL) was added dropwise. During the

addition (90 min), the reaction mixture was cooled in an ice-salt

bath. The dark brown mixture was stirred for an additional 0.5

h in an ice-salt bath, then poured onto crushed ice, and was

extracted with CH₂Cl₂. The organic layer was washed with

brine, dried over Na₂SO₄, and concentrated under reduced

pressure. The residue was purified by silica gel column

chromatography (CHCl₃as eluent) to afford 15a-o as white

solids. For example:

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

19

A

-

O AND 20A-O. A solution of 17a-o or 18a-o (1 mmol) in

dimethoxymethane (5 mL) in a round-bottomed flask was

added phosphorus oxychloride (2 mL) and the resulting mixture

was refluxed for 3.5h. The reaction was then cooled to 0 °C for

1

h

and the solution was filtered to afford the

dihydroisoquinoline. These compounds were found to be

unstable and decompose on standing or exposure to silica gel.

They were therefore reacted without further purification

immediately. To a solution of the dihydroisoquinoline in

MeOH (5 mL) at 0 °C, sodium borohydride (37.8 mg) was

added slowly. The resulting mixture was stirred for 1.5 h at 0

°C a brine solution was added and then extracted with DCM.

The amine was then purified by column chromatography (TEA

neutralized silica gel, CH₂Cl₂: MeOH 20:1 as eluent) to furnish

19a-o or 20a-o as white solids. For example:

3,6,7-trimethoxyphenanthrene-9-carboxylic acid methyl ester

1

(

15a). White solid, yield: 87%. mp 151.2-152.0 °C. H NMR

(400MHz, CDCl₃) δ 8.65 (s, 1H), 8.44 (s, 1H), 7.87 (s, 1H),

7.86-7.84 (d, J = 8.80 Hz, 1H), 7.81-7.80 (d, J = 2.24 Hz, 1H),

7.23-7.20 (dd, J = 8.76, 2.40 Hz, 1H), 4.11 (s, 1H), 4.09 (s,

1H), 4.03 (s, 1H), 4.01 (s, 1H). ¹³C NMR (100MHz, CDCl₃) δ

168.24, 160.20, 149.94, 148.91, 133.43, 131.87, 131.29,

125.19, 124.99, 124.27, 121.68, 116.03, 106.97, 103.75,

103.25, 55.92, 55.88, 55.55, 52.02. MS (ESI) m/z

327.2[M+H]⁺, 349.2[M+Na]⁺, 325.1[M-H]^-.

6,7-dimethoxy-1-(3,6,7-trimethoxyphenanthren-9-yl)-1,2,3,4-

tetrahydroisoquinoline (19a). White solid, yield: 63% (for 2

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

1

16A-O. 15a-o (3 mmol), NaOH (18 mmol) were dissolved in

steps). mp 270.5-272.8 °C. H NMR (400MHz, CDCl₃) δ 8.06

MeOH (9 mL) and water (9 mL) and refluxed for 4h. On

cooling, the solvent was neutralized with concentrated HCl (pH

2). The crude solids were filtered, washed with water, and dried

under vacuum for 12h to give 16a-o as white solids. For

example:

(s, 1H), 8.06-8.05 (d, J = 2.24 Hz, 1H), 7.79-7.77 (d, J = 8.72

Hz, 1H), 7.71 (s, 1H), 7.44 (s, 1H), 7.20-7.17 (dd, J = 8.72,

2.24 Hz, 1H), 6,80 (s, 1H), 6.20 (s, 1H), 5.58 (s, 1H), 4.13 (s,

3H), 4.13-4.09 (q, 1H), 4.00 (s, 3H), 3.99 (m, 3H), 3.74 (s, 3H),

3.68 (s, 3H), 3.17-3.15 (m, 4H), 3.00-2.97 (m, 1H), 2.79-2.75

(m, 1H). ¹³C NMR (100MHz, CDCl₃) δ 158.53, 148.76, 148.57,

147.82, 147.36, 131.13, 130.45(×2), 127.97, 126.80, 126.00,

125.50, 125.37(×2), 116.26(×2), 112.47, 111.00, 104.74,

104.53, 56.26, 55.99(×2), 55.93, 55.86, 55.37, 49.06, 29.39. MS

(ESI) m/z 460.3[M+H]⁺, 482.3[M+Na]⁺.

3,6,7-trimethoxyphenanthrene-9-carboxylic acid (16a). White

solid, yield: 98%. mp 213.0-214.6 °C. ¹H NMR (400MHz,

DMSO-d₆) δ 12.94 (s, 1H), 8.58 (s, 1H), 8.45 (s, 1H), 8.14 (s,

1H), 8.11-8.10 (d, J = 2.16 Hz, 1H), 8.05-8.03 (d, J = 8.84 Hz,

1H), 7.30-7.27 (dd, J = 8.76, 2.32 Hz, 1H), 4.06 (s, 3H), 4.04

(s, 3H), 3.92 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 169.40,

160.41, 149.98, 149.30, 133.30, 132.16, 130.78, 125.19,

124.98, 124.26, 117.03, 107.29, 104.86, 104.55, 56.32, 56.15,

55.71. MS (ESI) m/z 310.9[M-H]^-

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

21

A

-

O AND 22

A-O. A solution of 19a-o or 20a-o (0.5mmol) in

dry acetone (5mL) in a round-bottomed flask was cooled to 0°C

then added acetyl chloride (35μL).The mixture was stirred for

3.5 h at 0 °C and then water was added. The organic layer was

washed with water and brine and dried over Na₂SO₄overnight.

Removal of the solvent gave a residue that was purified by

column chromatography (silica gel, CH₂Cl₂: acetone 20:1 as

eluent) to furnish 21a-o or 22a-o as white solids. For example:

1-(6,7-dimethoxy-1-(3,6,7-trimethoxyphenanthren-9-yl)-3,4-

dihydroisoquino-in-2-(1H)-yl) ethanone (21a) White solid,

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

17

A

-

O AND 18

A-O. A solution of 16a-o (2 mmol), EDCI (3

mmol) and HOBt (3 mmol) in dry CH₂Cl₂(6 equiv) were

stirred at 25°C for 3.5h. Then substituted phenethylamine (2

mmol) and DIPEA (4 mmol) were added and the reaction was

stirred at the same temperature for 1.5 h. The organic layer was

washed with water and brine and dried over Na₂SO₄. Removal

of the solvent gave a residue that was purified by column

chromatography (silica gel, CH₂Cl₂: acetone 20:1 as eluent) to

furnish 17a-o or 18a-o as white solids. For example:

1

221mg, yield: 96%. H NMR (400MHz, CDCl₃) δ 8.24 (s, 1H),

7.95 (s, 1H), 7.87-7.86 (d, J = 2.28 Hz, 1H), 7.60-7.58 (d, J =

8.76 Hz, 1H), 7.47 (s, 1H), 7.16-7.13 (dd, J = 8.76, 2.28 Hz,

1H), 6.91 (s, 1H), 6.75 (s, 1H), 6.61 (s, 1H), 4.15 (s, 3H), 4.14

(s, 3H), 4.03 (s, 3H), 3.97 (s, 3H), 3.76 (s, 3H), 3.69-3.63 (m,

1H), 3.55-3.47 (m, 1H), 3.13-3.04 (m, 1H), 2.86-2.81 (m, 1H),

2.21 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ 169.15, 158.57,

N-(3,4-dimethoxyphenethyl)-2,6,7-trimethoxyphenanthrene-9-

carboxamide (17a). White solid, yield: 82%. mp 149.8-151.5

1

°C. H NMR (400MHz, CDCl₃) δ 7.89 (s, 1H), 7.86 (s, 1H),

7.81-7.80 (d, J = 2.28 Hz, 1H), 7.73-7.71 (d, J = 8.76 Hz, 1H),

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ARTICLE

149.88, 148.90, 147.67, 133.25, 131.02, 148.26, 130.51, 127.58, 125.74, 124.32, 119.07, 108.84, 108.34(×2), 107.42,

127.88, 126.68, 128.46, 126.41, 125.03, 124.56, 115.19, 102.59, 100.95, 61.26, 60.49, 56.60, 55.77, 55.71, 53.05, 39.45,

111.49, 111.20, 106.17, 103.85, 103.56, 56.71, 56.03, 55.95, 28.77, 21.47. HRMS calcd for C₃₂H₃₅NO₈Na, [M+Na]⁺,

55.85, 55.58, 52.81, 39.46, 28.37, 21.53. HRMS calcd for 584.2225; found 584.2229.

C₃₀H₃₁NO₆Na, [M+Na]⁺, 524.2044; found 524.2063.

1-(6,7-dimethoxy-1-(2-methoxyphenanthro[3,2-d][1,3]dioxol-

1-(1-(2,3-dimethoxyphenanthro[3,2-d][1,3]dioxol-6-yl)-6,7-

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone 21f)

(

6-yl)-3,4-dihydroisoquinolin-2(1H)-yl)ethanone(21b

)

White White solid, 193mg, yield: 91%. ¹H NMR (400MHz, CDCl₃) δ

solid, 206mg, yield 92%. ¹H NMR (600MHz, CDCl₃) δ 8.22 (s, 8.18 (s, 1H), 7.90 (s, 1H), 7.73 (s, 1H), 7.38 (s, 1H), 6.95 (s,

1H), 7.99 (s, 1H), 7.80-7.79 (d, J = 2.04 Hz, 1H), 7.56-7.55 (d, 1H), 6.84 (s, 1H), 6.73 (s, 1H), 6.55 (s, 1H), 6.11-6.09 (d, J =

J = 8.70 Hz, 1H), 7.38 (s, 1H), 7.14-7.11 (dd, J = 8.70, 2.22 Hz, 7.69 Hz, 2H), 4.08 (s, 3H), 3.96 (s, 3H), 3.95 (s, 3H), 3.73 (s,

1H), 6.91 (s, 1H), 6.73 (s, 1H), 6.55 (s, 1H), 6.14-6.12 (d, J = 3H), 3.67-3.61 (m, 1H), 3.52-3.44 (m, 1H), 3.10-3.01 (m, 1H),

12.90 Hz, 2H), 4.00 (s, 3H), 3.95 (s, 1H), 3.73 (s, 3H), 3.66- 2.84-2.79 (m, 1H), 2.20 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ

3.63 (m, 1H), 3.52-3.47 (m, 1H), 3.09-3.03 (m, 1H), 2.84-2.80 169.20, 149.64, 148.92, 148.28, 147.86, 147.71, 134.41,

(m, 1H), 2.21 (s, 3H). ¹³C NMR (150MHz, CDCl₃) δ 169.11, 128.12, 127.97, 126.80, 126.48, 125.29, 124.81, 111.58,

158.51, 148.36, 148.14, 147.63, 147.56, 133.68, 131.21, 111.25, 108.48, 103.41, 102.73, 101.37, 100.35, 56.03, 55.97,

130.32, 128.50, 127.97, 127.87, 126.34, 126.25, 124.93, 55.94, 55.88, 52.99, 39.62, 28.33, 21.60. HRMS calcd for

116.18, 111.32, 111.08, 103.41, 103.10, 101.39, 100.82, 55.86, C₃₀H₂₉NO₇Na, [M+Na]⁺, 538.1836; found 538.1829.

55.75, 55.41, 52.79, 39.48, 28.21, 21.64. HRMS calcd for 1-(1-(3-(benzyloxy)-2,6-dimethoxyphenanthren-9-yl)-6,7-

C₂₉H₂₇NO₆Na [M+Na]⁺, 508.1731; found 508.1732.

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

White solid, 261mg, yield: 92%. H NMR (400MHz, CDCl₃) δ

(

21g

)

1

1-(6,7-dimethoxy-1-(2,3,6-trimethoxyphenanthren-9-yl)-3,4-

dihydroisoquinolin-2(1H)-yl)ethanone

(

21c

)

White solid, ¹H NMR (400MHz, CDCl₃) δ 8.63-8.61 (d, J = 9.20 Hz, 1H),

209mg, yield: 95%. ¹H NMR (400MHz, CDCl₃) δ 8.68-8.65 (d, 7.89 (s, 1H), 7.71-7.70 (d, J = 2.44 Hz, 1H), 7.56-7.54 (d, J =

J = 9.20 Hz, 1H), 7.93-7.92 (d, J = 2.40 Hz, 1H), 7.86 (s, 1H), 7.40 Hz, 2H), 7.47 (s, 1H), 7.41-7.37 (m, 2H), 7.33-7.27 (m,

7.49 (s, 1H), 7.34-7.31 (dd, J = 9.16, 2.52 Hz, 1H), 6.98 (s, 2H), 6.99 (s, 1H), 6.78 (s, 1H), 6.73 (s, 1H), 6.56 (s, 1H), 5.38

1H), 6.79 (s, 1H), 6.73 (s, 1H), 6.58 (s, 1H), 4.10 (s, 3H), 4.03 (s, 2H), 3.98 (s, 6H), 3.95 (s, 3H), 3.73 (s, 3H), 3.65-3.59 (m,

(s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 3.73 (s, 3H), 3.66-3.61 (s, 1H), 3.52-3.44 (m, 1H), 3.09-3.00 (m, 1H), 2.83-2.78 (m, 1H),

1H), 3.53-3.45 (s, 1H), 3.10-3.01 (m, 1H), 2.84-2.79 (m, 1H), 2.18 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ 169.09, 150.08,

2.19 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ 169.09, 158.15, 148.40, 148.25, 147.68, 137.08, 135.00, 131.61, 128.70(×2),

149.47, 149.39, 148.20, 147.63, 134.85, 131.65, 128.03, 128.05(×2), 127.36(×2), 127.28, 127.14, 126.69, 126.47,

127.35, 127.22, 126.51, 126.38, 125.00, 124.19, 115.24, 124.91, 124.08, 115.74, 111.58, 111.24, 108.98, 106.55,

111.54, 111.21, 108.71, 104.77, 103.24, 56.10, 55.94(×2), 104.21, 71.50, 55.99, 55.97, 55.87, 55.42, 52.59, 39.63, 28.37,

55.86, 55.55, 52.56, 39.63, 28.35, 21.67. HRMS calcd for 21.62. HRMS calcd for C₃₆H₃₅NO₆Na, [M+Na]⁺, 600.2357;

C₃₀H₃₁NO₆Na, [M+Na]⁺, 524.2044; found 524.2048.

found 600.2349.

1-(6,7-dimethoxy-1-(2,3,6,7-tetramethoxyphenanthren-9-yl)-

1-(1-(3-(benzyloxy)-2,6,7-trimethoxyphenanthren-9-yl)-6,7-

3,4-dihydroisoquinolin-2(1H)-yl)ethanone (21d) White solid, dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

(

21h)

237mg, yield: 96%. H NMR (400MHz, CDCl₃) δ 8.19 (s, 1H), White solid, 273mg, yield: 95%. H NMR (400MHz, CDCl₃) δ

7.84 (s, 1H), 7.77 (s, 1H), 7.47 (s, 1H), 6.98 (s, 1H), 6.83 (s, 8.17 (s, 1H) 7.78 (s, 1H), 7.60 (s, 1H), 7.57 (s, 1H), 7.55 ( s,

1

，

1H), 6.75 (s, 1H), 6.61 (s, 1H), 4.14 (s, 3H), 4.11 (s, 6H), 3.97 1H), 7.44 (s, 1H), 7.41-7.37 (m, 2H), 7.33-7.30 (m, 1H), 6.99

(s, 3H), 3.96 (s, 3H), 3.75 (s, 3H), 3.68-3.62 (m, 1H), 3.53-3.45 (s, 1H), 6.81 (s, 1H), 6.74 (s, 1H), 6.59 (s, 1H), 5.39 (s, 2H),

(m, 1H), 3.12-3.03 (m, 1H), 2.86-2.80 (m, 1H), 2.21 (s, 3H). 4.09 (s, 3H), 4.06 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.74 (s,

¹³C NMR (100MHz, CDCl₃) δ 169.30, 149.63, 149.24, 149.12, 3H), 3.65-3.61 (m, 1H), 3.50-3.42 (m, 1H), 3.10-3.01 (m, 1H),

148.81, 148.30, 147.72, 133.80, 127.96, 126.40, 125.53, 2.83-2.78 (m, 1H), 2.18 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ

125.28, 124.78, 124.40, 111.59, 111.24, 108.77, 106.08, 169.06, 149.35, 149.11, 149.03, 148.54, 148.26, 147.68,

103.12, 102.68, 56.68, 56.15, 56.09, 55.97, 55.90, 53.01, 39.73, 137.29, 134.15, 128.70(×2), 128.03(×2), 127.76, 127.30(×2),

28.40, 21.33. HRMS calcd for C₃₁H₃₃NO₇Na, [M+Na]⁺, 126.49, 125.56, 125.39, 124.72, 124.19, 111.61, 111.26,

554.2149; found 554.2164.

108.97, 106.27, 106.07, 102.93, 71.70, 56.63, 55.97(×2),

55.87(×2), 52.81, 39.64, 28.39, 21.47. HRMS calcd for

1-(6,7-dimethoxy-1-(2,3,5,6,7-pentamethoxyphenanthren-9-yl)-

3,4-dihydroisoquinolin-2(1H)-yl)ethanone (21e) White solid, C₃₇H₃₇NO₇Na, [M+Na]⁺, 630.2462; found 630.2461.

1

233mg, yield: 94%. H NMR (400MHz, CDCl₃) δ 9.09 (s, 1H), 1-(1-(2-(benzyloxy)-3-methoxyphenanthro[3,2-d][1,3]dioxol-6-

8.02 (s, 1H), 7.39 (s, 1H), 6.99 (s, 1H), 6.90(s, 1H), 6.71 (s, yl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

1H), 6.55 (s, 1H), 6.00-5.99 (d, J = 0.90 Hz, 1H), 5.93-5.92 (d,

(

21i) White solid, 239mg, yield: 91%. ¹H NMR (400MHz,

J = 0.96 Hz, 1H), 4.08 (s, 6H), 4.05 (s, 3H), 4.00 (s, 3H), 3.97 CDCl₃) δ 8.15 (s, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.54 (s, 1H),

(s, 3H), 3.90 (s, 3H), 3.61-3.60 (s, 1H), 3.48-3.42 (m, 1H), 7.52 (s, 1H), 7.41-7.36 (m, 3H), 7.33-7.29 (m, 1H), 6.97 (s,

3.06-3.00 (m, 1H), 2.81-2.78 (m, 1H), 2.17 (s, 3H). ¹³C NMR 1H), 6.83 (s, 1H), 6.72 (s, 1H), 6.54 (s, 1H), 6.09 (s, 1H), 6.07

(100MHz, CDCl₃) δ 169.08, 152.14, 151.52, 148.99, 147.94, (s, 1H), 5.35 (s, 2H), 3.96 (s, 3H), 3.94 (s, 3H), 3.72 (s, 3H),

146.96, 146.26, 142.42, 133.40, 130.01, 129.10, 128.22, 3.65-3.60 (m, 1H), 3.51-3.43 (m, 1H), 3.09-3.01 (m, 1H), 2.83-

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ARTICLE

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2.78 (m, 1H), 2.20 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ 3.94 (s, 3H), 3.72 (s, 3H), 3.66-3.61 (m, 1H), 3.53-3.45 (m,

169.35, 149.47, 148.66, 148.29, 147.82, 147.72, 147.66, 1H), 3.09-3.01 (m, 1H), 2.84-2.79 (m, 1H), 2.20 (s, 3H). ¹³C

136.83, 134.38, 128.68(×2), 128.04, 127.98, 127.38(×2), NMR (100MHz, CDCl₃) δ 169.69, 158.25, 148.41, 148.27,

126.66, 126.47, 126.36, 125.55, 124.67, 111.51, 111.22, 147.81, 147.67, 134.40, 131.96, 127.87, 127.64, 127.54,

108.76, 105.59, 103.29, 101.34, 100.34, 77.24, 71.17, 126.91, 126.14, 125.93, 124.91, 116.33, 111.38, 111.20,

55.97(×2), 55.86, 53.08, 39.64, 28.32, 21.46. HRMS calcd for 106.06, 104.31, 101.37, 100.73, 55.96, 55.87, 55.47, 53.02,

C₃₆H₃₃NO₇Na, [M+Na]⁺, 614.2150; found 614.2148.

1-(1-(2-(benzyloxy)-3,6-dimethoxyphenanthren-9-yl)-6,7-

39.79, 28.34, 21.13. HRMS calcd for C₂₉H₂₇NO₆Na, [M+Na]⁺,

508.1731; found 508.1724.

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

White solid, 240mg, yield: 93%. H NMR (400MHz, CDCl₃) δ dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

(

21j

)

1-(1-(2,3-dimethoxyphenanthro[2,3-d][1,3]dioxol-5-yl)-6,7-

1

(21n)

1

8.67-8.65 (d, J = 9.20 Hz, 1H), 7.94-7.93 (d, J = 2.44 Hz, 1H), White solid, 201mg, yield: 90%. H NMR (400MHz, CDCl₃) δ

7.89 (s, 1H), 7.49-7.47 (m, 3H), 7.40-7.36 (m, 2H), 7.34-7.30 8.19 (s, 1H), 7.84 (s, 1H), 7.80 (s, 1H), 7.45 (s, 1H), 6.97 (s,

(m, 2H), 7.07 (s, 1H), 6.77 (s, 1H), 6.72 (s, 1H), 6.57 (s, 1H), 1H), 6.81 (s, 1H), 6.72 (s, 1H), 6.58 (s, 1H), 6.06-6.05 (m, 2H),

5.21 (s, 2H), 4.03 (s, 3H), 3.94 (s, 3H), 3.73 (s, 3H), 3.65-3.60 4.11 (s, 6H), 3.94 (s, 3H), 3.73 (s, 3H), 3.67-3.61 (m, 1H), 3.51-

(m, 1H), 3.53-3.45 (m, 1H), 3.09-3.00 (m, 1H), 2.83-2.78 (m, 3.43 (m, 1H), 3.11-3.02 (m, 1H), 2.84-2.79 (m, 1H), 2.19 (s,

1H), 2.19 (s, 3H). ¹³C-NMR (100MHz, CDCl₃) δ 169.10, 3H). ¹³C NMR (100MHz, CDCl₃) δ 169.11, 149.29, 149.12,

158.16, 149.92, 148.89, 148.25, 147.69, 136.70, 134.82, 148.32, 148.27, 147.73, 146.84, 134.00, 128.20, 127.86,

131.64, 128.57(×2), 128.05, 127.95, 127.43(×2), 127.33, 126.46, 126.44, 125.94, 125.61, 125.18, 111.53, 111.29,

127.23, 126.49, 126.39, 125.07, 124.54, 115.28, 111.61, 105.98(×2), 103.12, 101.22, 100.07, 56.64, 55.94(×2), 55.86,

111.30, 110.67, 104.88, 103.91, 70.74, 56.27, 55.96, 55.87, 52.75, 39.65, 28.34, 21.45 HRMS calcd for C₃₀H₂₉NO₇Na,

55.56, 52.61, 39.65, 28.36, 21.64. HRMS calcd for [M+Na]⁺, 538.1836; found 538.1829.

C₃₆H₃₅NO₆Na, [M+Na]⁺, 600.2357; found 600.2359.

1-(1-(3-isopropoxy-6,7-dimethoxyphenanthren-9-yl)-6,7-

1-(1-(2-(benzyloxy)-3,6,7-trimethoxyphenanthren-9-yl)-6,7-

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

White solid, 196mg, yield: 92%. H NMR (400MHz, CDCl₃) δ

(21o)

1

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

(

21k

)

1

White solid, 199mg, yield: 90%. H NMR(400MHz, CDCl₃) δ 8.20 (s, 1H), 7.92 (s, 1H), 7.90-7.89 (d, J = 2.12 Hz, 1H), 7.57-

8.19 (s, 1H), 7.85 (s, 1H), 7.81 (s, 1H), 7.40-7.37 (m, 2H), 7.34- 7.55 (d, J = 8.80 Hz, 1H), 7.44 (s, 1H), 7.14-7.11 (dd, J = 8.76,

7.30 (m, 1H), 7.07 (s, 1H), 6.81 (s, 1H), 6.73 (s, 1H), 6.60 (s, 2.28 Hz, 1H), 6.87 (s, 1H), 6.73 (s, 1H), 6.59 (s, 1H), 4.81-4.75

1H), 5.20 (s, 2H), 4.13 (s, 3H), 4.11 (s, 3H), 4.10 (s, 3H), 3.95 (m, 1H), 4.11 (s, 6H), 3.95 (s, 3H), 3.74 (s, 3H), 3.66-3.61 (m,

(s, 3H), 3.74 (s, 3H), 3.66-3.61 (m, 1H), 3.52-3.45 (m, 1H), 1H), 3.53-3.45 (m, 1H), 3.11-2.92 (m, 1H), 2.84-2.79 (m, 1H),

3.11-3.02 (m, 1H), 2.85-2.80 (m, 1H), 2.21 (s, 3H). ¹³C NMR 2.20 (s, 3H), 1.44-1.42 (dd, J = 6.00, 0.76 Hz, 6H). ¹³C NMR

(100MHz, CDCl₃) δ 169.10, 150.10, 149.23, 149.09, 148.25, (100MHz, CDCl₃) δ 169.10, 156.71, 149.80, 148.86, 147.01,

148.19, 147.68, 136.78, 133.97, 128.57(×2), 128.02, 127.93, 146.28, 133.16, 131.17, 130.38(×2), 129.00, 128.41, 127.61,

127.87, 127.44(×2), 126.50, 125.64, 125.31, 124.74, 124.70, 126.55, 124.96, 124.58, 116.12, 108.88, 108.35, 107.35,

111.62, 111.29, 110.76, 106.18, 103.38, 103.15, 70.75, 56.65, 106.09, 103.60, 100.98, 70.41, 56.67, 56.01, 53.02, 39.46,

56.36, 56.09, 55.96, 55.89, 52.84, 39.67, 28.38, 21.49. HRMS 28.85, 22.17(×2), 21.46. HRMS calcd for C₃₂H₃₅NO₆Na,

calcd for C₃₇H₃₇NO₇Na, [M+Na]⁺, 630.2462; found 630.2463.

1-(1-(3-(benzyloxy)-2-methoxyphenanthro[3,2-d][1,3]dioxol-6- 1-(5-(3,6,7-trimethoxyphenanthren-9-yl)-7,8-dihydro-

yl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone [1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone (22a) White

21l) White solid, 228mg, yield: 96%. ¹H NMR (400MHz, solid, 204mg, yield: 96%. H NMR (400MHz, CDCl₃) δ 8.17

[M+Na]⁺, 552.2357; found 552.2369.

1

(

CDCl₃) δ 8.21 (s, 1H), 7.93 (s, 1H), 7.79 (s, 1H), 7.51-7.49 (m, (s, 1H), 7.92 (s, 1H), 7.84-7.83 (d, J = 2.20 Hz, 1H), 7.62 (s,

2H), 7.42-7.32 (m, 4H), 7.07 (s, 1H), 6.84 (s, 1H), 6.74 (s, 1H), 1H), 7.60-7.58 (d, J = 8.76 Hz, 1H), 7.41 (s, 1H), 7.14-7.11 (dd,

6.57 (s, 1H), 6.14-6.11 (d, J = 7.44 Hz, 2H), 5.22 (s, 2H), 4.10 J = 8.72, 2.36 Hz, 1H), 6.92 (s, 1H), 6.70 (s, 1H), 6.56 (s, 1H),

(s, 3H), 3.97 (s, 3H), 3.74 (s, 3H), 3.68-3.63 (m, 1H), 3.54-3.46 5.99-5.98 (d, J = 1.08 Hz, 1H), 5.94-5.93 (d, J = 1.12 Hz, 1H),

(s, 1H), 3.12-3.03 (s, 1H), 2.86-2.80 (m, 1H), 2.22 (s, 3H). ¹³C 4.12 (s, 1H), 4.11 (s, 1H), 4.01 (s, 1H), 3.63-3.60 (m, 1H), 3.49-

NMR (100MHz, CDCl₃) δ 169.15, 150.07, 148.21(×2), 147.81, 3.42 (m, 1H), 3.07-2.98 (m, 1H), 2.81-2.76 (m, 1H), 2.18 (s,

147.70, 147.65, 136.73, 134.41, 128.58(×2), 128.07, 1H). ¹³C NMR (100MHz, CDCl₃) δ 169.04, 158.59, 149.90,

127.93(×2), 127.42(×2), 126.85, 126.42(×2), 125.22, 125.07, 148.93, 147.04, 146.31, 133.21, 131.06, 130.47, 129.03,

111.52, 111.23, 110.37, 103.42, 103.16, 101.37, 100.37, 70.70, 128.41, 127.65, 126.69, 125.02, 124.63, 115.24, 108.89,

56.12, 55.95, 55.86, 52.92, 39.60, 28.31, 21.67. HRMS calcd 108.37, 106.20, 103.84, 103.64, 100.98, 56.68, 56.04, 55.58,

for C₃₆H₃₃NO₇Na, [M+Na]⁺, 614.2149; found 614.2141.

1-(6,7-dimethoxy-1-(2-methoxyphenanthro[2,3-d][1,3]dioxol-

53.02, 39.47, 28.86, 21.48. HRMS calcd for C₂₉H₂₇NO₆Na,

[M+Na]⁺, 508.1731; found 508.1736.

5-yl)-3,4-dihydroisoquinolin-2(1H)-yl)ethanone (21m) White 1-(5-(2-methoxyphenanthro[3,2-d][1,3]dioxol-6-yl)-7,8-

1

solid, 187mg, yield: 89%. H NMR (400MHz, CDCl₃) δ 8.65- dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

8.63 (d, J = 9.16 Hz, 1H), 7.91 (s, 1H), 7.86-7.85 (d, J = 2.44

(

22b) White solid, 213mg, yield: 95%. ¹H NMR (600MHz,

Hz, 1H), 7.48 (s, 1H), 7.33-7.30 (m, 1H), 6.98 (s, 1H), 6.78 (s, CDCl₃) δ 8.18 (s, 1H), 7.89 (s, 1H), 7.79-7.78 (d, J = 2.16 Hz,

1H), 6.71 (s, 1H), 6.55 (s, 1H), 6.07-6.06 (s, 2H), 4.01 (s, 3H), 1H), 7.59-7.57 (d, J = 8.76 Hz, 1H), 7.34 (s, 1H), 7.14-7.11 (dd,

12 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

ARTICLE

J = 8.70, 2.28 Hz, 1H), 6.94 (s, 1H), 6.70 (s, 1H), 6.52 (s, 1H), 6.99 (s, 1H), 6.89 (s, 1H), 6.70 (s, 1H), 6.52 (s, 1H), 6.11-6.09

6.14-6.12 (d, J = 11.46 Hz, 2H), 5.99 (s, 1H), 5.95-5.94 (d, J = (d, J = 6.80 Hz, 1H), 5.98 (s, 1H), 5.93-5.92 (d, J = 1.00 Hz,

1.14 Hz,1H), 4.01 (s, 3H), 3.64-3.61 (m, 1H), 3.50-3.45 (m, 1H), 4.08 (s, 3H), 3.97 (s, 3H), 3.64-3.59 (m, 1H), 3.50-3.43

1H), 3.06-3.00 (m, 1H), 2.81-2.78 (m, 1H), 2.20 (s, 3H). ¹³C (m, 1H), 3.07-2.98 (m, 1H), 2.82-2.77 (m, 1H), 2.19 (s, 3H).

NMR (150MHz, CDCl₃) δ 160.11, 158.59, 148.41, 147.71, ¹³C NMR(100MHz, CDCl₃) δ 169.19, 149.61, 148.89, 147.85,

146.99, 146.29, 133.66, 131.31, 130.33, 129.05, 128.51, 147.68, 147.02, 146.34, 134.23, 129.14, 127.86, 127.57,

127.99, 127.59, 126.36, 124.98, 116.26, 108.79, 108.33, 126.51, 125.23, 124.84, 108.84, 108.43, 108.32, 103.32,

103.44, 103.16, 101.45, 100.97, 100.89, 55.46, 53.09, 39.41, 102.74, 101.38, 100.99, 100.35, 56.00, 55.92, 53.22, 39.46,

21.66, 28.80. HRMS calcd for C₂₈H₂₄NO₆[M+H]⁺, 470.1598; 28.83, 21.55. HRMS calcd for C₂₉H₂₆NO₇, [M+H]⁺, 500.1704;

found 470.1615.

found 500.1703.

1-(5-(2,3,6-trimethoxyphenanthren-9-yl)-7,8-dihydro-

1-(5-(3-(benzyloxy)-2,6-dimethoxyphenanthren-9-yl)-7,8-

[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone (22c) White dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

1

solid, 203mg, yield: 95%. H NMR (400MHz, CDCl₃) δ 8.64-

(

22g) White solid, 251mg, yield: 88%. ¹H NMR (400MHz,

8.61 (d, J = 9.16 Hz, 1H), 7.92-7.91 (d, J = 2.44 Hz, 1H), 7.86 CDCl₃) δ 8.59-8.57 (d, J = 9.20 Hz, 1H), 7.89 (s, 1H), 7.71-

(s, 1H), 7.46 (s, 1H), 7.32-7.29 (m, 1H), 7.02 (s, 1H), 6.84 (s, 7.70 (d, J = 2.40 Hz, 1H), 7.43-7.37 (m, 3H), 7.33-7.28 (m,

1H), 6.71 (s, 1H), 6.55 (s, 1H), 5.99 (s, 1H), 5.92 (s, 1H), 4.10 2H), 7.03 (s, 1H), 6.83 (s, 1H), 6.70 (s, 1H), 6.53 (s, 1H), 5.98

(s, 3H), 4.02 (s, 3H), 3.98 (s, 3H), 3.63-3.58 (m, 1H), 3.51-3.43 (s, 1H), 5.92-5.91 (d, J = 1.12Hz, 1H), 5.37 (s, 2H), 3.98 (s,

(m, 1H), 3.07-2.98 (m, 1H), 2.82-2.76 (m, 1H), 2.18 (s, 3H). 3H), 3.97 (s, 3H), 3.61-3.56 (m, 1H), 3.49-3.41 (m, 1H), 3.06-

¹³C NMR (100MHz, CDCl₃) δ 169.10, 158.14, 149.40, 149.39, 2.97 (m, 1H), 2.80-2.75 (m, 1H), 2.17 (s, 3H). ¹³C NMR

147.00, 146.30, 134.65, 131.67, 129.07, 127.62, 127.29, (100MHz, CDCl₃) δ 169.03, 158.14, 150.07, 148.48, 147.00,

127.15, 126.32, 124.93, 124.25, 115.21, 108.87, 108.67, 146.31, 137.07, 134.87, 131.64, 129.11, 128.69(×2), 128.04,

108.32, 104.83, 103.25, 100.98, 56.08, 55.94, 55.55, 52.85, 127.64, 127.38(×2), 127.18, 127.09, 126.64, 124.85, 124.13,

39.48, 28.85, 21.57. HRMS calcd for C₂₉H₂₇NO₆Na, [M+Na]⁺, 115.73, 108.96, 108.87, 108.33, 106.56, 104.22, 100.97, 71.49,

508.1731; found 508.1726.

1-(5-(2,3,6,7-tetramethoxyphenanthren-9-yl)-7,8-dihydro-

55.98, 55.41, 52.81, 39.46, 28.86, 21.59. HRMS calcd for

C₃₅H₃₁NO₆Na, [M+Na]⁺, 584.2044; found 584.2022.

[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone (22d) White 1-(5-(3-(benzyloxy)-2,6,7-trimethoxyphenanthren-9-yl)-7,8-

1

solid, 229mg, yield: 97%. H NMR (400MHz, CDCl₃) δ 8.17 dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(s, 1H), 7.85 (s, 1H), 7.79 (s, 1H), 7.45 (s, 1H), 7.04 (s, 1H),

(

22h) White solid, 258mg, yield: 91%. ¹H NMR (400MHz,

6.91 (s, 1H), 6.75 (s, 1H), 6.60 (s, 1H), 6.03-6.02 (d, J = 0.76 CDCl₃) δ 8.12 (s, 1H), 7.78 (s, 1H), 7.59 (s, 1H), 7.57 (s, 1H),

Hz, 1H), 5.97 (s, 1H), 4.16 (s, 3H), 4.14 (s, 3H), 4.13 (s, 3H), 7.55 (s, 1H), 7.41-7.40 (d, J = 1.28 Hz, 1H), 7.39 (s, 1H), 7.37

4.00 (s, 3H), 3.67-3.62 (m, 1H), 3.53-3.47 (m, 1H), 3.12-3.05 (s, 1H), 7.33-7.30 (m, 1H), 7.02 (s, 1H), 6.86 (s, 1H), 6.71 (s,

(m, H), 2.86-2.82 (m, 1H), 2.24 (s, 3H). ¹³C NMR (100MHz, 1H), 6.55 (s, 1H), 6.00-5.99 (d, J = 0.96 Hz, 1H), 5.93-5.92 (d,

CDCl₃) δ 169.08, 149.59, 149.19, 149.10, 148.77, 147.04, J = 1.08 Hz, 1H), 5.39 (s, 2H), 4.08 (s, 3H), 4.06 (s, 3H), 3.98

146.33, 133.80, 129.10, 127.83, 127.64, 125.52, 125.23, (s, 3H), 3.62-3.57 (m, 1H), 3.47-3.40 (m, 1H), 3.07-2.98 (m,

124.80, 124.43, 108.94, 108.73, 108.37, 106.11, 103.10, 1H), 2.81-2.76 (m, 1H), 2.17 (s, 3H). ¹³C NMR (100MHz,

102.70, 101.01, 56.66, 56.13, 56.08, 55.93, 53.10, 39.51, 28.91, CDCl₃) δ 169.14, 149.32, 149.09, 149.01, 148.55, 147.05,

21.47. HRMS calcd for C₃₀H₂₉NO₇Na, [M+Na]⁺, 538.1836; 146.33, 137.25, 133.87, 129.00, 128.70(×2), 128.04, 127.72,

found 538.1835.

1-(5-(2,3,5,6,7-pentamethoxyphenanthren-9-yl)-7,8-dihydro-

127.58, 127.30(×2), 125.47, 125.29, 124.75, 124.25, 108.91,

108.35, 106.23, 105.94, 102.91, 101.01, 71.68, 56.64, 55.97,

[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone (22e) White 55.85, 53.13, 39.50, 28.88, 21.33. HRMS calcd for

1

solid, 221mg, yield: 90%. H NMR (400MHz, CDCl₃) δ 9.09 C₃₆H₃₃NO₇Na, [M+Na]⁺, 614.2149; found 614.2160.

(s, 1H), 8.02 (s, 1H), 7.39 (s, 1H), 6.99 (s, 1H), 6.90 (s, 1H), 1-(5-(2-(benzyloxy)-3-methoxyphenanthro[3,2-d][1,3]dioxol-6-

6.71 (s, 1H), 6.55 (s, 1H), 6.00-5.99 (d, J = 0.90 Hz, 1H), 5.93- yl)-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-

1

5.92 (d, J = 0.96 Hz, 1H), 4.08 (s, 6H), 4.05 (s, 3H), 4.00 (s, yl)ethanone (22i) White solid, 230mg, yield: 90%. H NMR

3H), 3.97 (s, 3H), 3.90 (s, 3H), 3.61-3.60 (m, 1H), 3.48-3.42 (400MHz, CDCl₃) δ 8.30 (s, 1H), 8.12 (s, 1H), 8.03 (s, 1H),

(m, 1H), 3.06-3.00 (m, 1H), 2.81-2.78 (m, 1H), 2.19 (s, 3H). 7.55 (s, 1H), 7.53 (s, 1H), 7.43-7.40 (m, 2H), 7.36-7.32 (m,

¹³C-NMR (400MHz, CDCl₃) δ 169.08, 152.14, 151.52, 148.99, 1H), 7.19 (s, 1H), 7.17 (s, 1H), 6.92 (s, 1H), 6.85 (s, 1H), 6.64

147.94, 146.96, 146.26, 142.42, 133.40, 130.01, 129.10, (s, 1H), 6.19 (s, 1H), 6.16 (s, 1H), 6.03 (s, 1H), 5.97 (s, 1H),

128.22, 127.58, 125.74, 124.32, 119.07, 108.84, 108.34(×2), 5.35 (s, 2H), 3.84 (s, 3H), 3.72-3.67 (m, 1H), 3.29-3.21 (m,

107.42, 102.59, 100.95, 61.26, 60.49, 56.60, 55.77, 55.71, 1H), 3.04-2.95 (m, 1H), 2.79-2.74 (m, 1H), 2.12 (s, 3H). ¹³C

53.05, 39.45, 28.77, 21.47. HRMS calcd for C₃₉H₄₁NO₉Na, NMR (100MHz, CDCl₃) δ 169.06, 149.45, 148.63, 147.79,

[M+Na]⁺, 568.2111; found 568.2109.

147.61, 146.99, 146.31, 136.84, 134.46, 129.17, 128.67(×2),

128.03, 127.80, 127.62, 127.40(×2), 126.67, 126.49, 125.53,

124.69, 108.83, 108.74, 108.32, 105.64, 103.33, 101.31,

1-(5-(2,3-dimethoxyphenanthro[3,2-d][1,3]dioxol-6-yl)-7,8-

dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(

22f) 179mg, White solid, yield: 86%. ¹H NMR (400MHz, 100.97, 100.33, 71.17, 55.97, 53.10, 39.42, 28.82, 21.62.

CDCl₃) δ 8.14 (s, 1H), 7.89 (s, 1H), 7.73 (s, 1H), 7.34 (s, 1H),

J. Name., 2012, 00, 1-3 | 13

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Organic & Biomolecular Chemistry

Page 14 of 18

ARTICLE

Journal Name

HRMS calcd for C₃₆H₃₃NO₇Na, [M+Na]⁺, 598.1836; found 4.00 (s, 3H), 3.63-3.57 (m, 1H), 3.49-3.41 (m, 1H), 3.05-3.00

598.1854.

(m, 1H), 2.80-2.75 (m, 1H), 2.17 (s, 3H). ¹³C NMR (100MHz,

CDCl₃) δ 169.13, 158.19, 148.17, 147.61, 147.03, 146.32,

134.76, 131.96, 128.91, 127.64(×2), 127.57, 127.05, 125.88,

1-(5-(2-(benzyloxy)-3,6-dimethoxyphenanthren-9-yl)-7,8-

dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(

22j) White solid, 229mg, yield: 89%. ¹H-NMR(400MHz, 124.99, 116.25, 108.77, 108.34, 106.00, 104.21, 101.33,

CDCl₃) δ 8.64-8.61 (d, J = 9.20 Hz, 1H), 7.93-7.92 (d, J = 2.48 100.98, 100.75, 55.44, 52.79, 39.50, 28.84, 21.53. HRMS calcd

Hz, 1H), 7.89 (s, 1H), 7.49-7.45 (m, 3H), 7.41-7.37 (m, 2H), for C₂₈H₂₃NO₆Na, [M+Na]⁺, 492.1418; found 492.1389.

7.34-7.29 (m, 2H), 7.11 (s, 1H), 6.82 (s, 1H), 6.70 (s, 1H), 6.54 1-(5-(2,3-dimethoxyphenanthro[2,3-d][1,3]dioxol-5-yl)-7,8-

(s, 1H), 5.99-5.98 (d, J = 0.88 Hz, 2H), 5.93-5.92 (d, J = 1.24 dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

1

Hz, 2H), 5.21 (s, 2H), 4.09 (s, 3H), 4.03 (s, 3H), 3.63-3.57 (m,

(

22n) White solid, 197mg, yield: 89%. H NMR (400MHz,

1H), 3.51-3.43 (m, 1H), 3.06-2.97 (m, 1H), 2.81-2.76 (m, 1H), CDCl₃) δ 8.15 (s, 1H), 7.83 (s, 1H), 7.79 (s, 1H), 7.41 (s, 1H),

2.17 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.03, 158.15, 7.00 (s, 1H), 6.84 (s, 1H), 6.69 (s, 1H), 6.55 (s, 1H), 6.05 (s,

149.92, 148.90, 146.98, 146.29, 136.73, 134.72, 131.66, 2H), 5.98-5.97 (d, J = 1.24 Hz, 1H), 5.93-5.92 (d, J = 1.28 Hz,

129.12, 128.58(×2), 127.96, 127.64, 127.45(×2), 127.23(×2), 1H), 4.10 (s, 3H), 4.09 (s, 3H), 3.63-3.58 (m, 1H), 3.48-3.40

126.34, 125.03, 124.59, 115.26, 110.68, 108.89, 108.34, (m, 1H), 3.07-2.98 (m, 1H), 2.80-2.75 (m, 1H), 2.17 (s, 3H).

104.89, 103.93, 100.95, 70.79, 56.25, 55.55, 52.80, 39.48, ¹³C NMR (100MHz, CDCl₃) δ 169.01, 149.26, 149.10, 148.25,

28.85, 21.62. HRMS calcd for C₃₆H₃₅NO₆Na, [M+Na]⁺, 147.04, 146.82, 146.31, 133.92, 128.95, 128.16, 127.62,

584.2225; found 584.2229.

126.40, 125.97, 125.56, 125.21, 108.37, 105.95(×2), 103.12,

108.82, 101.22, 100.98, 100.10, 56.63, 55.92, 52.97, 39.49,

28.85, 21.44. HRMS calcd for C₂₉H₂₆NO₇, [M+H]⁺, 500.1704;

1-(5-(2-(benzyloxy)-3,6,7-trimethoxyphenanthren-9-yl)-7,8-

dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(

22k) White solid, 201mg, yield: 92%. ¹H NMR (400MHz, found 500.1703.

CDCl₃) δ 8.16 (s, 1H), 7.84 (s, 1H), 7.80 (s, 1H), 7.42 (s, 1H), 1-(5-(3-isopropoxy-6,7-dimethoxyphenanthren-9-yl)-7,8-

7.39-7.37 (m, 2H), 7.34-7.30 (m, 1H), 7.11 (s, 1H), 6.86 (s, dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

1H), 6.71 (s, 1H), 6.56(s, 1H), 5.99 (s, 1H), 5.93-5.92 (d, J =

(

22o) White solid, 193mg, yield: 92%.¹H NMR (400MHz,

0.84 Hz, 1H), 5.20 (s, 2H), 4.13 (s, 3H), 4.10(s, 6H), 3.63-3.58 CDCl₃) δ 8.16 (s, 1H), 7.92(s, 1H), 7.89-7.88 (d, J = 1.88 Hz,

(m, 1H), 3.50-3.42 (m, 1H), 3.08-2.99 (m, 1H), 2.82-2.77 (m, 1H), 7.59-7.57 (d, J = 8.76 Hz, 1H), 7.40 (s, 1H), 7.13-7.10 (dd,

1H), 2.19 (s, 3H). ¹³C NMR (100MHz, CDCl₃) δ 169.03, J = 8.72, 2.28 Hz, 1H), 6.90 (s, 1H), 6.70 (s, 1H), 6.56 (s, 1H),

150.09, 149.21, 149.08, 148.20, 147.01, 146.30, 136.80, 5.98-5.93 (m, 2H), 4.81-4.75 (m, 1H), 4.11 (s, 3H), 4.10 (s,

133.86, 129.09, 128.57(×2), 127.95, 127.79, 127.67, 3H), 3.63-3.58 (m, 1H), 3.50-3.43 (m, 1H), 3.07-2.98 (m, 1H),

127.46(×2), 125.61, 125.25, 124.76, 110.76, 108.93, 108.39, 2.81-2.76 (m, 1H), 2.18 (s, 3H),1.44-1.42 (dd, J = 6.00, 1.36

106.15, 103.38, 103.16, 100.98, 70.80, 56.63, 56.32, 56.07, Hz, 6H). ¹³C NMR (100MHz, CDCl₃) δ 169.12, 156.73, 149.83,

53.04, 39.49, 28.86, 21.47. HRMS calcd for C₃₆H₃₃NO₇Na, 148.88, 147.02, 146.29, 133.16, 131.18, 130.38, 129.01,

[M+Na]⁺, 614.2149; found 614.2149.

1-(5-(3-(benzyloxy)-2-methoxyphenanthro[3,2-d][1,3]dioxol-6- 108.35, 107.36, 106.12, 103.65, 100.97, 70.43, 56.66, 56.02,

yl)-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)- 53.03, 39.46, 28.85, 22.18(×2), 21.45. HRMS calcd for

yl)ethanone (22l) White solid, 222mg, yield: 94%. H NMR C₃₁H₃₁NO₆Na, [M+Na]⁺, 536.2044; found 536.2048.

(400MHz, CDCl₃) δ 8.14 (s, 1H), 7.90 (s, 1H), 7.76 (s, 1H),

7.49 (s, 1H), 7.47 (s, 1H), 7.40-7.36 (m, 2H), 7.33-7.30 (m, 21

128.41, 127.62, 126.57, 124.97, 124.59, 116.16, 108.87,

1

G

ENERAL SYNTHETIC PROCEDURE FOR THE PREPARATION OF

U AND 22P-U. To a solution of 21g-l or 22g-l (0.17 mmol)

P

-

2H), 7.08 (s, 1H), 6.86 (s, 1H), 6.69 (s, 1H), 6.50 (s, 1H), 6.11 in absolution MeOH (30 mL) was added 1,4-cyclohexadiene

(s, 1H), 6.09 (s, 1H), 5.98 (s, 1H), 5.92-5.91 (d, J = 0.96 Hz, (3.5 mL) and Pd/C (10%, 50mg) in an ultrasonic reactor for

1H), 5.20 (s, 2H), 4.07 (s, 3H), 3.63-3.58 (m, 1H), 3.49-3.43 100min at 25°C. The solution was filtered and dried under

(m, 1H), 3.06-2.97 (m, 1H), 2.81-2.76 (m, 1H), 2.19 (s, 3H). reduced pressure to give off-white solids which was purified by

¹³C NMR (100MHz, CDCl₃) δ 169.08, 150.11, 148.26, 147.82, recrystallization with MeOH to furnish 21p-u or 22p-u as white

147.70, 146.98, 146.31, 136.77, 134.32, 129.16, 128.57(×2), solids. For example:

127.94, 127.83, 127.61, 127.44(×2), 126.82, 126.47, 125.20, 1-(1-(3-hydroxy-2,6-dimethoxyphenanthren-9-yl)-6,7-

125.15, 110.44, 108.85, 108.35, 103.38, 103.24, 101.37, dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

(21p).

100.96, 100.39, 70.79, 56.11, 53.14, 39.44, 28.82, 21.64. White solid, 87mg, yield: 97%. ¹H NMR (400MHz, DMSO-d₆)

HRMS calcd for C₃₅H₂₉NO₇Na, [M+Na]⁺, 598.1836; found δ 9.44 (s, 1H), 8.50-8.48 (d, J = 9.24 Hz, 1H), 8.01 (s, 1H),

598.1854.

7.90-7.88 (d, J = 2.20 Hz, 1H), 7.29 (s, 1H), 7.22-7.19 (dd, J =

9.12, 2.24 Hz, 1H), 7.14 (s, 1H), 6.86 (s, 1H), 6.82 (s, 1H), 6.65

(s, 1H), 3.97 (s, 3H), 3.87 (s, 3H), 3.80 (s, 1H), 3.73-3.68 (m,

1-(5-(2-methoxyphenanthro[2,3-d][1,3]dioxol-5-yl)-7,8-

dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

1

(22m) White solid, 184mg, yield: 88%. H NMR (400MHz, 1H), 3.59 (s, 1H), 3.32-3.24 (m, 1H), 3.05-2.96 (s, 1H), 2.81-

CDCl₃) δ 8.61-8.59 (d, J = 9.16 Hz, 1H), 7.90 (s, 1H), 7.85- 2.76 (m, 1H), 2.11 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ

7.84 (d, J = 2.48 Hz, 1H), 7.44 (s, 1H), 7.31-7.26 (m, 1H), 7.30- 169.09, 158.18, 149.26, 148.35, 147.63, 147.54, 134.10,

7.28 (m, 1H), 7.00 (s, 1H), 6.81 (s, 1H), 6.68 (s, 1H), 6.52 (s, 131.57, 128.06, 127.41, 127.21, 126.94, 125.73, 124.87,

1H), 6.06 (s, 2H), 5.97 (s, 1H), 5.93-5.92 (d, J = 1.20 Hz, 1H), 124.49, 116.16, 112.28, 112.00, 109.33, 107.74, 104.31, 56.05,

14 | J. Name., 2012, 00, 1-3

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DOI: 10.1039/C5OB00037H

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Journal Name

Organic & Biomolecular Chemistry

ARTICLE

55.89(×2), 55.88, 55.76, 52.24, 28.16, 21.85. HRMS calcd for 1-(1-(3-hydroxy-2-methoxyphenanthro[3,2-d][1,3]dioxol-6-yl)-

C₂₉H₃₀NO₆, [M+H]⁺, 488.2068; found 488.2066.

1-(1-(3-hydroxy-2,6,7-trimethoxyphenanthren-9-yl)-6,7-

6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanon (21u)

White solid, 80mg, yield: 91%. H NMR (400MHz, DMSO-d₆)

δ 9.38 (s, 1H), 8.30 (s, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 7.18 (s,

1

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

(21q)

1

White solid, 84mg, yield: 96%. H NMR (400MHz, DMSO-d₆) 1H), 6.89 (s, 1H), 6.85 (s, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 6.19-

δ 9.33 (s, 1H), 8.06 (s, 1H), 7.94 (s, 1H), 7.85 (s, 1H), 7.27 (s, 6.16 (d, J = 13.76 Hz, 2H), 4.00 (s, 3H), 3.81 (s, 3H), 3.73-3.68

1H), 7.09 (s, 1H), 6.86 (s, 1H), 6.81 (s, 1H), 6.66 (s, 1H), 3.98 (m, 1H), 3.59 (s, 3H), 3.31-3.23 (s, 1H), 3.05-2.96 (s, 1H),

(s, 3H), 3.89 (s, 3H), 3.85 (s, 3H), 3.81 (s, 3H), 3.73-3.68 (m, 2.82-2.77 (m, 1H), 2.13 (s, 3H). ¹³C NMR (100MHz, DMSO-

1H), 3.60 (s, 3H), 3.28-3.21 (m, 1H), 3.05-2.97 (m, 1H), 2.80- d₆) δ 169.25, 149.57, 148.39, 147.83, 147.65, 147.20, 147.11,

2.75 (m, 1H), 2.13 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 134.37, 128.02, 127.39, 127.33, 126.22, 125.55, 126.97,

168.79, 148.67, 148.47, 147.96, 147.88, 147.13, 147.09, 124.25, 112.28, 112.03(×2), 104.28, 102.96, 101.69(×2),

127.52, 132.82, 127.05, 126.93, 124.72, 124.26, 124.21, 56.40(×2), 55.92(×2), 52.58, 39.60, 28.04, 21.90. HRMS calcd

124.12, 111.83, 111.59, 108.75, 106.76, 105.87, 103.53, 55.62, for C₂₉H₂₈NO₇, [M+H]⁺, 524.1680; found 524.1676.

55.52, 55.42, 55.40, 52.03, 27.72, 21.20. HRMS calcd for 1-(5-(3-hydroxy-2,6-dimethoxyphenanthren-9-yl)-7,8-dihydro-

C₃₀H₃₂NO₇, [M+H]⁺, 518.2173; found 518.2184.

1-(1-(2-hydroxy-3-methoxyphenanthro[3,2-d][1,3]dioxol-6-yl)- solid, 83mg, yield: 93%. H NMR (400MHz, DMSO-d) δ 9.45

6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone (21r (s, 1H), 8.46-8.44 (d, J = 8.12 Hz, 1H), 8.00 (s, 1H), 7.89-7.88

[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone (22p) White

1

)

1

White solid, 79mg, yield: 95%. H NMR (400MHz, DMSO-d₆) (d, J = 2.12 Hz, 1H), 7.25 (s, 1H), 7.21-7.20 (d, J = 2.04 Hz,

δ 9.39 (s, 1H), 8.05 (s, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 7.18 (s, 1H), 7.18 (s, 1H), 6.85 (s, 1H), 6.84 (s, 1H), 6.65 (s, 1H), 6.03

1H), 7.10 (s, 1H), 6.86 (s, 2H), 6.64 (s, 1H), 6.18 (s, 1H), 6.14 (s, 1H), 5.97 (s, 1H), 3.88 (s, 3H), 3.72-3.67 (m, 1H), 3.30-

(s, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.73-3.68 (m, 1H), 3.59 (s, 3.22(m, 1H), 3.97 (s, 3H), 3.03-2.94 (s, 1H), 2.79-2.74 (m, 1H),

3H), 3.31-3.24 (m, 1H), 3.06-2.97 (m, 1H), 2.81-2.76 (m, 1H), 2.09 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 169.11, 158.18,

2.12 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 169.25, 148.72, 149.26, 147.55, 146.88, 146.07, 134.01, 131.57, 129.26,

148.40, 147.75, 147.73, 147.68, 147.45, 133.72, 128.07, 128.76, 127.25, 126.92, 125.68, 124.78, 124.49, 116.16,

127.96, 127.38, 126.58, 126.29, 125.18, 124.75, 112.30, 109.39, 109.01, 108.73, 107.73, 104.32, 101.25, 56.05,

112.04, 109.08, 107.25, 103.02, 101.71, 101.11, 56.04(×2), 55.76(×2), 52.47, 28.67, 21.85. HRMS calcd for C₂₈H₂₆NO₆,

55.89(×2), 52.63, 28.12, 21.88. HRMS calcd for C₂₉H₂₈NO₇, [M+ H]⁺, 472.1755; found 472.1753.

[M+H]⁺, 502.1860; found 502.1847.

1-(1-(2-hydroxy-3,6-dimethoxyphenanthren-9-yl)-6,7-

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

1-(5-(3-hydroxy-2,6,7-trimethoxyphenanthren-9-yl)-7,8-

dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(

22q) White solid, 83mg, yield: 97%. ¹H NMR (400MHz,

(

21s

)

White solid, 75mg, yield: 92%. ¹H NMR (400MHz, DMSO-d₆) DMSO-d₆) δ 9.35 (s, 1H), 8.03 (s, 1H), 7.94 (s, 1H), 7.86 (s,

δ 9.48 (s, 1H), 8.50-8.48 (d, J = 9.24 Hz, 1H), 8.09-8.08 (d, J = 1H), 7.25 (s, 1H), 7.14 (s, 1H), 6.86 (s, 1H), 6.84 (s, 1H), 6.68

2.36 Hz, 1H), 8.04 (s, 1H), 7.28 (s, 1H), 7.23-7.20 (m, 1H), (s, 1H), 6.05 (s ,1H), 5.99 (s, 1H), 3.99 (s, 3H), 3.89 (s, 3H),

6.91 (s, 1H), 6.85 (s, 1H), 6.64 (s, 2H), 4.02-3.99 (d, J = 12.72 3.87 (s, 3H), 3.73-3.68 (m, 1H), 3.26-3.18 (m ,1H), 3.04-2.96

Hz, 6H), 3.81 (s, 3H), 3.73-3.68 (s, 1H), 3.59 (s, 3H), 3.28-3.23 (m, 1H), 2.79-2.74 (m, 1H), 2.13 (s, 3H). ¹³C NMR (100MHz,

(m, 1H), 3.04-2.95 (m, 1H), 2.82-2.76 (m, 1H), 2.11 (s, 3H). DMSO-d₆) δ 169.31, 149.18, 148.96, 148.46, 147.61, 146.91,

¹³C NMR (100MHz, DMSO-d₆) δ 168.95, 158.27, 149.24, 146.07, 133.22, 129.22, 128.78, 127.62, 125.14, 124.77,

148.39, 147.78, 147.67, 134.74, 132.08, 128.07, 127.34, 124.74, 124.57, 109.31, 109.03, 108.79, 107.27, 106.37,

126.93, 126.71, 124.63, 123.43, 115.40, 112.46, 112.27, 104.06, 101.26, 56.12, 56.03(×2), 52.76, 39.26, 28.75, 21.69.

111.96, 105.00, 104.58, 56.57, 56.38, 55.92, 55.85, 52.26, HRMS calcd for C₂₉H₂₈NO₇, [M+H]⁺, 502.1860; found

31.06, 28.15, 21.83, 18.95. HRMS calcd for C₂₉H₃₀NO₆, 502.1862.

[M+H]⁺, 488.2068; found 488.2075.

1-(1-(2-hydroxy-3,6,7-trimethoxyphenanthren-9-yl)-6,7-

1-(5-(2-hydroxy-3-methoxyphenanthro[3,2-d][1,3]dioxol-6-yl)-

7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(

22r) White solid, 75mg, yield: 90%. ¹H NMR (400MHz,

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone

(

21t)

1

White solid, 79mg, yield: 93%. H NMR (400MHz, DMSO-d₆) DMSO-d₆) δ 9.39 (s, 1H), 8.00 (s, 1H), 7.95 (s, 1H), 7.85 (s,

δ 9.33 (s, 1H), 8.06 (s, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 7.27 (s, 1H), 7.14 (s, 2H), 6.86 (s, 1H), 6.85 (s, 1H), 6.63 (s, 1H), 6.17

1H), 6.88 (s, 1H), 6.86 (s, 1H), 6.66 (s, 1H), 6.65 (s, 1H), 4.03 (s, 1H), 6.14 (s, 1H), 6.03 (s, 1H), 5.97 (s, 1H), 3.86 (s, 3H),

(s, 6H), 3.90 (s, 3H), 3.81 (s, 3H), 3.73-3.68 (m, 1H), 3.60 (s, 3.72-3.67 (m, 1H), 3.28-3.21 (m, 1H), 3.04-2.95 (m, 1H), 2.79-

1H), 3.28-3.20 (m, 1H), 3.05-2.96 (m, 1H), 2.81-2.76 (m, 1H), 2.74 (m, 1H), 2.11 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ

2.13 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 169.32, 149.46, 169.25, 148.71, 147.75, 147.43, 146.91, 146.09, 133.61,

149.30, 148.86, 148.40, 147.64, 146.96, 134.05, 128.00, 129.25, 128.72, 127.99, 126.48, 126.28, 125.16, 124.69,

127.46, 127.02, 125.56, 125.28, 125.01, 123.71, 112.37(×2), 109.14, 109.00, 108.72, 107.23, 103.00, 101.71, 101.26,

112.08, 106.42, 104.54, 104.28, 56.48, 56.40, 56.09, 55.94(×2), 101.10, 56.05, 52.85, 39.21, 28.64, 21.88. C₂₈H₂₃NO₇Na,

55.37, 52.51, 28.16, 21.71. HRMS calcd for C₃₀H₃₂NO₇, [M+Na]⁺, 508.1367; found 508.1353.

[M+H]⁺, 518.2173; found 518.2177.

1-(5-(2-hydroxy-3,6-dimethoxyphenanthren-9-yl)-7,8-dihydro-

[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone (22s) White

J. Name., 2012, 00, 1-3 | 15

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DOI: 10.1039/C5OB00037H

Organic & Biomolecular Chemistry

Page 16 of 18

ARTICLE

Journal Name

1

solid, 71mg, yield: 87%. H NMR (400MHz, DMSO-d₆) δ 9.47 The in vitro anti-proliferative activities for some of the target

(s, 1H), 8.46-8.44 (d, J = 9.24 Hz, 1H), 8.09-8.08 (d, J = 2.36 compounds were determined by MTT (Sigma) assay. MCF-7,

Hz, 1H), 8.04 (s, 1H), 7.24-7.19 (m, 1H), 6.94 (s, 1H), 6.84 (s, A549, and HT1080 (1×10⁴/well) were plated in 0.1 mL of the

1H), 6.66 (s, 1H), 6.64 (s, 1H), 6.01-5.98 (d, J = 11.68 Hz, 2H), medium containing 10% FBS in 96-well Corning plates; 24 h

4.03 (s, 3H), 4.00 (s, 3H), 3.72-3.66 (m, 1H), 3.29-3.21 (m, later, the medium was removed and replaced with 0.1 mL

1H), 3.02-2.93 (m, 1H), 2.80-2.75 (m, 1H), 2.10 (s, 3H). ¹³C medium containing the indicated concentrations of PF-3758309

NMR (100MHz, DMSO-d₆) δ 169.11, 158.33, 149.33, 147.85, and the target compounds for 24 h. At the end of the incubation,

146.88, 146.07, 134.77, 132.12, 129.24, 128.77, 126.92, the capability of cellular proliferation was measured by the

126.66, 124.56, 123.44, 115.50, 112.47, 108.95, 108.66, modified tetrazolium salt-3-(4,5-dimethylthiozol-2yl)-2,5-

105.22, 104.74, 101.25, 56.49, 55.98, 56.45, 52.45, 28.60, biphenyl-tetrazolium bromide (MTT) assay. For this, 0.01 mL

21.85, 19.02. HRMS calcd for C₂₈H₂₆NO₆, [M+H]⁺, 472.1755; of MTT solution (5 mg/mL in PBS) was added to each well.

found 472.1754.

After a 4h incubation at 37 °C, medium was replaced by 0.15

mL DMSO. After 15 min incubation at 37 °C, the optical

densities at 595 nm were measured using a Microplate Reader

1-(5-(2-hydroxy-3,6,7-trimethoxyphenanthren-9-yl)-7,8-

dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone

(

22t) White solid, 77mg, yield: 91%. ¹H NMR (400MHz, (BIO-RAD). The data were calculated and plotted as the

DMSO-d₆) δ 9.32 (s, 1H), 8.02 (s, 1H), 8.01 (s, 1H), 7.96 (s, percent viability compared to the control. The 50% inhibitory

1H), 7.23 (s, 1H), 6.91 (s, 1H), 6.84 (s, 1H), 6.66 (s, 2H), 6.02 concentration (IC₅₀) was defined as the concentration that

(s, 1H), 5.99 (s, 1H), 4.02 (s, 3H), 3.88 (s, 3H), 3.72-3.67 (m, reduced the absorbance of the untreated wells by 50% of the

1H), 3.25-3.17 (m, 1H), 3.02-2.93 (m, 1H), 2.79-2.74 (m, 1H), vehicle in the MTT assay.

2.12 (s, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 169.32, 149.46,

C

ELL-CYCLE ANALYSIS BY FLOW CYTOMETRY

149.31, 148.85, 146.95, 146.90, 146.07, 133.96, 129.18, MCF-7 cells (8×10⁴cells) were incubated with the indicated

128.79, 127.07, 125.50, 125.29, 124.94, 123.71, 112.39, concentrations of 21a for the indicated time. After incubation,

108.97, 108.73, 106.42, 104.57, 104.24, 101.26, 56.46, 56.41, cells were collected, washed with PBS and then suspended in a

56.09, 52.72, 39.28, 28.66, 21.70. HRMS calcd for C₂₉H₂₈NO₇, staining buffer (10 μg/mL propidium iodide, 0.5% Tween-20,

[M+H]⁺, 502.1680; found 502.1682.

1-(5-(3-hydroxy-2-methoxyphenanthro[2,3-d][1,3]dioxol-6-yl)- FACSVantage flow cytometer with the Cell Quest acquisition

7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)ethanone and analysis software program (Becton Dickinson and Co., San

22u) White solid, 78mg, yield: 90%. ¹H NMR (400MHz, Jose, CA). Gating was set to exclude cell debris, doublets and

DMSO-d₆) δ 9.37 (s, 1H), 8.28 (s, 1H), 8.00 (s, 1H), 7.96 (s, clumps.

0.1% RNase in PBS). The cells were analyzed using a

(

A

NALYSIS OF CELL APOPTOSIS

1H), 7.13 (s, 1H), 6.91 (s, 1H), 6.84 (s, 1H), 6.70 (s, 1H), 6.62

(s, 1H), 6.18 (s, 1H), 6.15 (s, 1H), 6.01 (s, 1H), 5.98 (s, 1H), The ability of 21a to induce cell apoptosis of A549 cells was

4.00 (s, 3H), 3.70-3.68 (m, 1H), 3.27-3.21 (m, 1H ), 3.01-2.98 quantified by annexin V and PI staining and flow cytometry, as

(m, 1H), 2.80-2.76 (m, 1H), 2.27-2.24 (m, 1H), 2.12 (s, 3H). described previously. Briefly, after treatment with 21a for 24 h,

¹³C NMR (100MHz, DMSO-d₆) δ 169.23, 149.58, 147.84, cells were collected and washed with PBS twice, and subjected

147.18, 147.10, 146.89, 146.08, 134.30, 129.21, 128.72, to annexin V and propidium iodide staining using annexin-V

127.35, 126.97, 126.14, 125.49, 124.25, 112.06, 108.93, FITC apoptosis kit following the step-by-step protocol provided

108.64, 104.24, 102.94, 101.69(×2), 101.25, 56.38, 52.79, by the manufacturer. After staining, flow cytometry was

39.22, 28.56, 21.88. HRMS calcd for C₂₈H₂₃NO₇Na, [M+Na]⁺, performed for the quantification of apoptotic cells.

C

ELL INVASION ASSAY

508.1367; found 508.1377.

Matrigel invasion assays were performed using modified

Boyden chambers with polycarbonate Nucleopore membrane.

Precoated filters (6.5 mm in diameter, 8μm pore size, and

Matrigel 100 μg/cm²) were rehydrated with 100 μL medium.

Then, 1×10⁵cells in 100 μL serum-free DMEM supplemented

with 0.1% bovine serumalbumin were placed in the upper part

of each chamber, whereas the lower compartments were filled

with 600 μL DMEM containing 10% serum. After incubating

for 18 h at 37 °C, non-invaded cells were removed from the

upper surface of the filter with a cotton swab, and the invaded

cells on the lower surface of the filter were fixed, stained,

Biology

K

INASE

G

LO® LUMINESCENT

K

INASE ASSAYS

.

PAK4 was preincubated with PF-3758309(

1

) and all the target

molecules for 30 minutes, and then the kinase assay was

performed. Reactions were stopped by the addition of an equal

volume of Kinase-Glo reagent (Promega) and incubated for 10

min at room temperature. Chemiluminescence was measured at

555 nm (20 nm emission slit) using a Cary Eclipse fluorescence

spectrophotometer (Tecan) equipped with a microplate carrier.

MTT ASSAYS

.

The human breast cell line MCF-7, the human pulmonary photographed and counted under high-power magnification.

L

ENTIVIRAL PRODUCTION AND INFECTION

carcinoma cell line A-549 and the human fibrosarcoma cell line

HT-1080 were cultured in RPMI-1640 medium containing 10%

FBS, 100 U/mL streptomycin and 100 U/mL penicillin at 37 °C

in a humidified atmosphere containing 5% CO₂.

Recombinant lentiviruses including PAK4-Lentivirus, PAK4-

RNAi-Lentivirus and pGC FU-GFP-LV, NC-GFP-LV vectors

were purchased from Shanghai GeneChem Company. To stably

overexpress PAK4, cells were infected with lentivirus carrying

16 | J. Name., 2012, 00, 1-3

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DOI: 10.1039/C5OB00037H

Page 17 of 18

Journal Name

Organic & Biomolecular Chemistry

ARTICLE

PAK4, and then selected with puromycin (1.5 μg/mL). The Technology Research and Development Program of China

pGC FU-GFP-LV vector was used as control.

(Grant 2007AA02Z305) and Program for Innovative Research

Team of the Ministry of Education, and Program for Liaoning

W

ESTERN BLOT ANALYSIS

To determine the expression of protein, whole cell extracts Innovative Research Team in University.

were prepared from 1×10⁶cells in RIPA lysis buffer (50 mM

Tris/HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% Na-

deoxycholate, 1 mM EDTA and protease inhibitor cocktail).

Notes and references

^aKey Laboratory of Structure-Based Drug Design & Discovery of

Equal amounts of denatured protein were separated by SDS-

PAGE and transferred to a PVDF membrane (Millipore). The

membrane was blocked with 5% nonfat dry milk in TBS-T (20

mM Tris, pH 7.4, 137 mM NaCl, 0.05% Tween-20) for 3 h at

room temperature, and the proteins were probed with specific

antibodies: CDK2, CDK4, CDK6, cyclinD1 (Neomarker),

cyclinD3, cyclinB1, Bcl-2, BAX, caspase 3, caspase 8, LIMK1,

phospho-LIMK1, cofilin, phospho-cofilin, PAK4, phospho-

PAK4/Ser474, SCG-10, phospho-SCG10/Ser50. All PVDF

membranes were detected by chemiluminescence (ECL, Pierce

Technology). To assure equal loading, membranes were

stripped and reprobed with antibody against GAPDH and

MMP2 (Shang Hai Kangchen).

Ministry of Education, Shenyang Pharmaceutical University,

Shenyang 110016, P. R. China. Email:mscheng@263.net, dongmeiz-

67@163.com; Fax: +86-24-23995043; Tel: +86-24-23986413.

^bDepartment of Cell Biology, Key Laboratory of Cell Biology,

Ministry of Public Health, and Key Laboratory of Medical Cell

Biology, Ministry of Education, China Medical University, Shenyang

110001, P. R. China. Email: fli@mail.cmu.edu.cn; Fax: +86-24-

23261056; Tel: +86-24-23256666.

†Electronic Supplementary Information (ESI) available: [Details of

experimental procedure, spectral data of all novel compounds.]. See

DOI: 10.1039/b000000x/

Conclusions

1

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350-355.

Following the identification of (-)-β-Hydrastine as a PAK4

inhibitor from our in-house natural product database, we

undertook a detailed SAR investigation aimed at improving

potency as well as increasing metabolic stability of this

compound. A combination of traditional and modern medicinal

chemistry principles led to the identification of a novel, potent,

ATP-competitive PAK4 inhibitor (21a), which demonstrated

target modulation in PAK4 driven cancer cells (MCF-7). It was

shown that compound 21a suppressed the expression of cyclin

D (cyclinD1, D3) and CDKs (CDK2, 4, 6), in turn inhibiting

the transition of cells from G1 phase to S phase, and resulted in

the anti-proliferative effect on MCF-7 cells together with the

induction of apoptosis. Furthermore, Transwell and Western

blot studies suggested that compound 21a caused significant

inhibition of migration and invasion in breast cancer cells via

blockage of the PAK4/LIMK1/Cofilin, PAK4/MMP2, or

PAK4/SCG10 signaling pathways. Molecular docking study

demonstrated possible novel binding modes for the interactions

between compound 21a and PAK4. In summary, this work not

only broadened the scope of searching for PAK4 kinase

inhibitors with novel scaffolds, but also laid a solid foundation

for looking for more potent PAK4 inhibitors in the future.

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We gratefully acknowledge the financial support from the

National Natural Science Foundation of China (Grant 1230077,

81102379, 81001092, and J1210029), the National High

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DOI: 10.1039/C5OB00037H

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18 | J. Name., 2012, 00, 1-3

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Design, synthesis and biological evaluation of 1-phenanthryl-tetrahydroisoquinoline derivatives as novel p21-activated kinase 4 (PAK4) inhibitors

DOI: 10.1039/c5ob00037h

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