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“D”, “E” rings may not be indispensable for antofine: Discovery of phenanthrene and alkylamine chain containing antofine derivatives as novel antiviral agent against tobacco mosaic virus (TMV) based on interaction of antofine and TMV RNA Ziwen Wang, Peng Wei, Yuxiu Liu, and Qingmin Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5028894 • Publication Date (Web): 25 Aug 2014 Downloaded from http://pubs.acs.org on August 31, 2014

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Page 1 of 44

Journal of Agricultural and Food Chemistry

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“D”, “E” rings may not be indispensable for antofine: Discovery of

2

phenanthrene and alkylamine chain containing antofine derivatives

3

as novel antiviral agent against tobacco mosaic virus (TMV) based

4

on interaction of antofine and TMV RNA

5 6

Ziwen Wang,1 Peng Wei,1 Yuxiu Liu and Qingmin Wang*

7

State Key Laboratory of Elemento-Organic Chemistry, Research Institute of

8

Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and

9

Engineering(Tianjin), Nankai University, Tianjin 300071, China

10 11 12 13 14 15 16 17 18 19

* To whom correspondence should be addressed. E-mail: [email protected]; Tel.:

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0086-22-23503952; Fax: 0086-22-23503952. 1These authors contributed equally to this

21

paper.

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ACS Paragon Plus Environment


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Abstract

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Based on interaction of antofine and TMV RNA, a series of phenanthrene and alkylamine

4

chain containing antofine derivatives 1–41 were designed, synthesized and systematically

5

evaluated for their antiviral activity against TMV. The results showed that most of these

6

compounds exhibited good to excellent anti-TMV activity, which indicated that the “D”,

7

“E” rings of antofine may not be indispensable. Phenanthrene is important for these

8

compounds but not the more the better. Phenanthrene, benzene rings and alkylamine chain

9

containing compounds exhibited good antiviral activity. The optimum compounds 10, 18

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and 19 displayed higher activity than precursor antofine and commercial Ribavirin, thus

11

emerged as new lead compounds. The novel concise structure provides another new

12

template for antiviral studies.

13 14 15

Keywords: Phenanthrene; Alkylamine chain; Antofine derivatives; Antiviral activity;

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Tobacco mosaic virus; Structure activity relationship.

17 18 19 20 21 22

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Page 2 of 44

Page 3 of 44


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Introduction

2

Viral plant diseases are widespread in nature and cause heavy losses to modern

3

agriculture. As one of the most well-studied viruses, TMV is known to infect more than

4

198 individual species of 22 monocots families including many vegetables, tobaccos,

5

flowers, and weeds. The amount of loss can vary from 5 to 90% depending on the strain of

6

TMV, the total time of infection by TMV, the temperature during disease development,

7

and the presence of other diseases.1 Therefore, plant virus is difficult to control.2 As a

8

successfully registered plant viral inhibitor, Ribavirin (Figure 1) is widely used to prevent

9

TMV disease.3 However, the inhibitory effects of Ribavirin are less than 50% at 500

10

µg/mL. In fact, there are no super chemical pesticides that can absolutely inhibit TMV

11

once it has infected the plants.

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Because of the unsatisfactory cure rate (30–60%) by common antiviral agents and

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economic loss of tobacco, many efforts have been done to develop novel, potent and

14

structure concise antiviral agents. A series of chemicals have been found to possess

15

antiviral activity,4 such as thiadiazoles,5 pyrazole derivatives,6 α-aminophosphonate

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derivatives,7 and some natural products8–11. However, there are only a few reports on

17

economically viable antiviral chemicals available for application in agriculture,12 therefore,

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novel and more practical antiviral agents still need to be developed.

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Botanical agrochemicals offer many advantages that they can sometimes be speciﬁc to a

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target species and have unique mode of action with low toxicity to humans and non-target

21

organisms. Another benefit is their ability to decompose rapidly, thereby reducing their

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risk to the environment. So they are a potential alternative to chemical pesticides.13,14 In a

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1

program aimed at screening of plants for biologically active natural products as

2

alternatives to conventional synthetic agrochemicals, we first found that the extract from

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the aerial parts of Cyanchum komarovii showed excellent antiviral activity against TMV.

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The main active substances in C. komarovii were determined as tylophorine alkaloids, in

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which antofine (Figure 1) presents high level.15 The in vitro bioassay results showed that

6

antofine exhibited 63% inhibitory activity at the concentration of 1.0 µg/mL, which is

7

higher than that of commercial antiviral agents DHT (50%, Figure 1) and DADHT (50%,

8

Figure 1) at 500 µg/mL.16,17 Further structure-activity relationship studies showed that the

9

presence of nitrogen in tertiary amine and phenanthrene ring are essential for high

10

antiviral activity.18,19 The antiviral mechanism studies revealed that antofine-based

11

alkaloids exerted their virus inhibition by binding to oriRNA and interfering with virus

12

assembly initiation.20

13

Based on interaction of antofine and TMV RNA, a series of phenanthrene and

14

alkylamine chain containing antofine derivatives 1–41 (Figure 2) were designed,

15

synthesized and systematically evaluated for their antiviral activity against TMV.

16 17

Materials and methods

18

Synthetic Procedures. Reagents were purchased from commercial sources and were used

19

as received. All anhydrous solvent were dried and purified by standard techniques just

20

before use. Reaction progress was monitored by thin-layer chromatography on silica gel

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GF254 with detection by UV. Melting points were determined using an X-4 binocular

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microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China) and

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Page 5 of 44


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the thermometer was uncorrected. 1H NMR spectra were obtained by using Bruker AV 400

2

and a Varian Mercury plus 400 MHz spectrometer with CDCl3 or DMSO-d6 as a solvent.

3

Chemical shifts (δ) were given in parts per million (ppm) and were measured downfield

4

from internal tetramethylsilane. 13C NMR spectra were recorded by using Bruker AV 400

5

(100 MHz) with CDCl3 or DMSO-d6 as a solvent. Chemical shifts (δ) were reported in

6

parts per million using the solvent peak. High-resolution mass spectra were obtained with

7

an FT-ICR MS spectrometer (Ionspec, 7.0 T).

8

General Synthetic Procedure for Alcohols 45–47. To a mixture of LiAlH4 (0.03 mol) in

9

150 mL of THF at 0 °C was added corresponding acids 42–44 (0.02 mol) in portions. The

10

solution was refluxed for an additional 2 h, then brought back to 0 °C, at which point 10

11

mL of EtOAc was added dropwise, followed by 10 mL of 2 N HCl. The solution was

12

stirred and filtered, and the solvent was removed by rotary evaporation to give

13

corresponding alcohols 45–47.

14

(2,3,6,7-Tetramethoxyphenanthren-9-yl)methanol (45). Yield: 95%, white powder, mp:

15

183–185 °C, (lit.25, mp: 181–182 °C); 1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.78 (s,

16

1H), 7.59 (s, 1H), 7.55 (s, 1H), 7.21 (s, 1H), 5.13 (s, 2H), 4.13 (s, 3H), 4.13 (s, 3H), 4.07

17

(s, 3H), 4.03 (s, 3H), 1.75 (s, 1H); 13C NMR (100 MHz, CDCl3) 148.8, 148.6, 148.4, 148.3,

18

131.9, 125.6, 124.7, 124.3, 124.1, 123.3, 108.1, 104.5, 102.9, 102.4, 64.1, 55.8, 55.8, 55.7,

19

55.6.

20

(2,3,6-Trimethoxyphenanthren-9-yl)methanol (46). Yield: 93%, white powder, mp

21

183–184 °C (lit.26 mp 187 °C); 1H NMR (400 MHz, CDCl3) 8.05 (d, J = 9.0 Hz, 1H), 7.83

22

(s, 1H), 7.77 (s, 1H), 7.45 (s, 1H), 7.21 (d, J = 9.0 Hz, 1H), 7.09 (s, 1H), 5.07 (s, 2H), 4.06

5



1

(s, 3H), 4.00 (s, 3H), 3.96 (s, 3H), 2.02 (s, 1H); 13C NMR (100 MHz, CDCl3) 158.0, 149.4,

2

149.1, 132.8, 131.7, 127.0, 126.1, 124.3, 124.1, 123.1, 115.1, 108.3, 104.6, 103.3, 64.2,

3

56.0, 55.8, 55.5.

4

(3,6,7-Trimethoxyphenanthren-9-yl)methanol (47). Yield: 94%, white powder, mp

5

161–162 °C (lit.25 mp 157–158 °C); 1H NMR (400 MHz, CDCl3) 7.85 (s, 1H), 7.79 (s,

6

1H), 7.74 (d, J = 8.7 Hz, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 7.17 (d, J = 8.7 Hz, 1H), 5.06 (s,

7

2H), 4.09 (s, 3H), 4.04 (s, 3H), 4.00 (s, 3H), 1.78 (s, 1H);

8

158.4, 149.4, 148.8, 131.4, 131.2, 130.2, 125.8, 125.5, 124.8, 124.6, 115.4, 104.8, 103.9,

9

103.7, 64.7, 56.0, 55.9, 55.5.

13

C NMR (100 MHz, CDCl3)

10

Synthetic Procedure for 1. Alcohol 45 (10 mmol) was dissolved in 200 mL of CHCl3 and

11

cooled to 0 °C. A solution of PBr3 (15 mmol) in 40 mL of CHCl3 was added dropwise

12

under nitrogen. The solution was then stirred at room temperature for 4 h and poured over

13

ice, and the two layers were separated. The organic phase was dried over anhydrous

14

Na2SO4, filtered, and concentrated in vacuo to afford 48 as a white solid. Bromide 48 was

15

then redissolved in 120 mL of DMF.

16

To the solution of Me2NH (14.2 mmol) in DMF (80 mL) was added (i-Pr)2NEt (15.6

17

mmol), and the mixture was stirred for 10 min. Then the above solution of bromide 48 was

18

added. The mixture was stirred at 90 °C for 3 h, then cooled to room temperature. And

19

then 300 mL of H2O was added, followed by 300 mL of CH2Cl2. The product was

20

partitioned between CH2Cl2 and H2O. The organic layer was dried over anhydrous Na2SO4,

21

filtered, and concentrated to obtain a crude product. The crude product was purified by

22

flash column chromatography to give compound 1 (2.8 g, 80%) as a white powder, mp

6


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Page 7 of 44


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157–159 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.98 (s, 1H), 7.68 (s, 1H),

2

7.52 (s, 1H), 7.37 (s, 1H), 4.02 (s, 3H), 4.02 (s, 3H), 3.90 (s, 3H), 3.90 (s, 3H), 3.76 (s,

3

2H), 2.24 (s, 6H);

4

125.4, 125.2, 125.0, 124.6, 123.9, 108.4, 106.1, 103.9, 103.7, 62.7, 55.9, 55.8, 55.3, 55.1,

5

45.1; ESI-HRMS (m/z): calcd. for C21H26NO4 [M+H]+ 356.1856; found 356.1853.

6

General Synthetic Procedure for Compounds 24, 27, 30, 33, 36, 39 and 40.

7

Corresponding alcohols 45–47 (10 mmol) was dissolved in 200 mL of CHCl3 and cooled

8

to 0 °C. A solution of PBr3 (15 mmol) in 40 mL of CHCl3 was added dropwise under

9

nitrogen. The solution was then stirred at room temperature for 4 h and poured over ice,

10

and the two layers were separated. The organic phase was dried over anhydrous Na2SO4,

11

filtered, and concentrated in vacuo to afford corresponding bromides 48–50, which was

12

then redissolved in 120 mL of DMF.

13

C NMR (100 MHz, DMSO-d6) δ 149.0, 148.7, 148.6, 148.0, 130.1,

13

To the solution of corresponding alkylamines (3 mmol) in DMF (80 mL) was added

14

(i-Pr)2NEt (12 mmol), and the mixture was stirred for 10 min. Then the above solution of

15

bromide 48 was added. The mixture was stirred at 90 °C for 3 h, then cooled to room

16

temperature. And then 300 mL of H2O was added, followed by 300 mL of CH2Cl2. The

17

product was partitioned between CH2Cl2 and H2O. The organic layer was dried over

18

anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product

19

was purified by flash column chromatography to give compounds 24, 27, 30, 33, 36, 39

20

and 40.

21

Methyl 6-(bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate (24).

22

Yield: 60%, white powder, mp: 191–192 °C; 1H NMR (400 MHz, CDCl3) δ 7.74 (s, 2H),

7



Page 8 of 44

1

7.74 (s, 2H), 7.56 (s, 2H), 7.48 (s, 2H), 7.17 (s, 2H), 4.11 (s, 6H), 4.07 (s, 6H), 4.06 (s,

2

4H), 4.03 (s, 6H), 3.58 (s, 3H), 3.49 (s, 6H), 2.61 (t, J = 8.0 Hz ,2H), 2.10 (t, J = 8.0 Hz,

3

2H), 1.78–1.69 (m, 2H), 1.48–1.39 (m, 2H), 1.22–1.16 (m, 2H);

4

CDCl3) δ 174.0, 149.1, 148.9, 148.8, 148.4, 130.6, 126.9, 126.1, 125.9, 124.9, 124.3,

5

108.1, 106.1, 103.0, 102.8, 59.6, 56.1, 56.0, 55.9, 55.4, 54.5, 51.4, 33.9, 27.1, 27.0, 24.7;

6

ESI-HRMS (m/z): calcd. for C45H51NO10Na [M+Na]+ 788.3405; found 788.3405.

7

Methyl 5-(bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)pentanoate (27).

8


9

7.53 (s, 2H), 7.44 (s, 2H), 7.15 (s, 2H), 4.09 (s, 6H), 4.06 (s, 6H), 4.02 (s, 10H), 3.57 (s,

10

3H), 3.48 (s, 6H), 2.59 (t, J = 8.0 Hz, 2H), 2.10 (t, J = 8.0 Hz, 2H), 1.75–1.68 (m, 2H),

11

1.58–1.48 (m, 2H);

12

130.5, 126.8, 126.1, 125.9, 125.0, 124.3, 108.2, 106.1, 103.0, 102.8, 59.5, 56.1, 56.0, 55.9,

13

55.5, 54.4, 51.4, 33.7, 26.7, 22.9; ESI-HRMS (m/z): calcd. for C44H49NO10Na [M+Na]+

14

744.3249; found 744.3246.

15

Methyl 4-(bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)butanoate (30).

16


17

7.74 (s, 2H), 7.57 (s, 2H), 7.46 (s, 2H), 7.17 (s, 2H), 4.11 (s, 6H), 4.08 (s, 10H), 4.03 (s,

18

6H), 3.49 (s, 6H), 3.45 (s, 3H), 2.65 (t, J = 8.0 Hz, 2H), 2.17 (t, J = 8.0 Hz, 2H), 2.07–2.02

19

(m, 2H); 13C NMR (100 MHz, CDCl3) δ 172.6, 148.2, 147.9, 147.8, 147.5, 129.3, 126.05,

20

124.9, 124.8, 123.9, 123.3, 107.2, 104.9, 102.0, 101.8, 58.4, 55.1, 55.0, 54.8, 54.4, 52.8,

21

50.3, 30.9, 21.6; ESI-HRMS (m/z): calcd. for C43H47NO10Na [M+Na]+ 760.3092; found

22

760.3099.

13

13

C NMR (100 MHz,

C NMR (100 MHz, CDCl3) δ 173.8, 149.2, 148.9, 148.8, 148.5,

8


Page 9 of 44


1

Methyl 3-(bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)propanoate (33).

2


3

7.73 (s, 2H), 7.57 (s, 2H), 7.43 (s, 2H), 7.16 (s, 2H), 4.10 (s, 6H), 4.09 (s, 4H), 4.07 (s,

4

6H), 4.03 (s, 6H), 3.53 (s, 6H), 3.41 (s, 3H), 3.05 (t, J = 8.0 Hz, 2H), 2.62 (t, J = 8.0 Hz,

5

2H); 13C NMR (100 MHz, CDCl3) δ 172.9, 149.3, 148.9, 148.8, 148.6, 130.0, 126.9, 126.0,

6

125.8, 125.0, 124.4, 108.2, 106.0, 103.1, 102.8, 59.2, 56.1, 56.0, 55.9, 55.5, 51.4, 50.4,

7

32.1; ESI-HRMS (m/z): calcd. for C42H45NO10Na [M+Na]+ 746.2936; found, 746.2935.

8

Methyl

9

Yield: 68%, white powder, mp: 126–128 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.96 (s,

10

2H), 7.95 (s, 2H), 7.63 (s, 2H), 7.52 (s, 2H), 7.34 (s, 2H), 4.20 (s, 4H), 4.01 (s, 6H), 3.99

11

(s, 6H), 3.90 (s, 6H), 3.57 (s, 3H), 3.43 (s, 2H), 3.38 (s, 6H); 13C NMR (100 MHz, CDCl3)

12

δ 171.8, 149.3, 149.0, 148.8, 148.7, 129.8, 127.0, 126.0, 125.8, 125.0, 124.5, 108.2, 106.4,

13

103.0, 102.9, 77.5, 77.1, 76.8, 58.8, 56.1, 56.0, 55.9, 55.7, 54.8, 51.2; ESI-HRMS (m/z):

14

calcd. for C41H43NO10Na [M+Na]+ 732.2779; found 732.2774.

15

Methyl 6-(bis((2,3,6-trimethoxyphenanthren-9-yl)methyl)amino)hexanoate (39). Yield:

16

66%, white powder, mp: 189–190 °C; 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 8.0 Hz,

17

2H), 7.82 (d, J = 4.0 Hz, 4H), 7.47 (s, 2H), 7.16 (s, 2H), 6.92 (dd, J = 12.0, 4.0 Hz, 2H),

18

4.09 (s, 6H), 4.03 (s, 6H), 4.00 (s, 10H), 3.59 (s, 3H), 2.56 (t, J = 8.0 Hz, 2H), 2.09 (t, J =

19

8.0 Hz, 2H), 1.68–1.61 (m, 2H), 1.46–1.39 (m, 2H), 1.18–1.10 (m, 2H);

20

MHz, CDCl3) δ 173.1, 156.7, 148.3, 147.8, 130.7, 130.5, 127.0, 126.0, 124.7, 124.4, 123.2,

21

113.4, 107.1, 102.8, 102.3, 57.7, 55.0, 54.8, 54.4, 53.0, 50.3, 32.9, 26.0, 25.0, 23.6;

22


2-(bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)acetate

9


(36).

13

C NMR (100


Page 10 of 44

1

Methyl 6-(bis((3,6,7-trimethoxyphenanthren-9-yl)methyl)amino)hexanoate (40). Yield:

2

63%, white powder, mp: 111–113 °C; 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 2H), 7.82 (s,

3

2H), 7.75 (d, J = 8.8 Hz, 2H), 7.61 (s, 2H), 7.48 (s, 2H), 7.18 (d, J = 8.8 Hz, 2H), 4.07 (s,

4

6H), 4.05 (s, 4H), 4.01 (s, 6H), 3.58 (s, 3H), 3.47 (s, 6H), 2.60 (t, J = 8.0 Hz, 2H), 2.09 (t,

5

J = 8.0 Hz, 2H), 1.77–1.69 (m, 2H), 1.46–1.38 (m, 2H), 1.22–1.13 (m, 2H); 13C NMR (100

6

MHz, CDCl3) δ 173.9, 158.2, 149.2, 148.7, 131.0, 129.9, 127.4, 127.3, 125.7, 124.8, 115.4,

7

106.2, 103. 9, 103.5, 59.4, 56.0, 55.6, 55.4, 54.4, 51.4, 33.9, 27.1, 26.9, 24.7; ESI-HRMS

8

(m/z): calcd. for C43H47NO8Na [M+Na]+ 728.3194; found 728.3192.

9

General Synthetic Procedure for Compounds 25, 28, 31, 34 and 37. The mixture of

10

corresponding esters 24, 27, 30, 33 and 36 (0.34 mmol), 4 M NaOH (15 mL) and

11

1,4-dioxane (15 mL) was refluxed for 4 h, and concentrated to 15 mL. The mixture was

12

acidified (pH = 6) with diluted hydrochloric acid, and filtrated to afford corresponding

13

acids 25, 28, 31, 34 and 37.

14

6-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoic acid (25). Yield:

15

92%, white powder, mp: 235 °C (dec.); 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 2H),

16

7.94 (s, 2H), 7.62 (s, 2H), 7.39 (s, 2H), 7.32 (s, 2H), 4.00 (s, 6H), 3.98 (s, 10H), 3.89 (s,

17

6H), 3.33 (s, 6H), 2.45 (t, J = 8.0 Hz , 2H), 1.82 (t, J = 8.0 Hz, 2H), 1.72–1.63 (m, 2H),

18

1.34–1.25 (m, 2H), 1.15–1.05 (m, 2H);

19

148.7, 148.5, 148.0, 129.9, 126.5, 125.3, 125.2, 124.7, 123.9, 108.2, 105.8, 103.9, 103.6,

20

58.7, 55.8, 55.8, 55.3, 54.7, 54.3, 36.5, 27.3, 26.8, 25.6; ESI-HRMS (m/z): calcd. for

21

C44H49NO10Na [M+Na]+ 774.3249; found 774.3247.

22

5-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)pentanoic

13

C NMR (100 MHz, DMSO-d6) δ 177.0, 149.0,

10


acid

(28).

Page 11 of 44


1

Yield: 82%, white powder, mp: >300 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.91 (s, 4H),

2

7.53 (s, 2H), 7.33 (s, 2H), 7.29 (s, 2H), 3.98 (s, 6H), 3.96 (s, 6H), 3.88 (s, 10H), 3.28 (s,

3

6H), 2.44 (t, J = 8.0 Hz, 2H), 1.84 (t, J = 8.0 Hz, 2H), 1.71–1.57 (m, 2H), 1.37–1.25 (m,

4

2H);

5

125.3, 125.3, 124.6, 123.9, 108.2, 105.7, 103.9, 103.6, 58.5, 55.8, 55.7, 55.3, 54.7, 54.4,

6

36.7, 26.8, 24.0; ESI-HRMS (m/z): calcd. for C43H47NO10Na [M+Na]+ 760.3092; found

7

760.3088.

8

4-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)butanoic acid (31). Yield:

9

95%, white powder, mp: 224–226 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 2H), 7.94

10

(s, 2H), 7.63 (s, 2H), 7.43 (s, 2H), 7.32 (s, 2H), 4.00 (s, 10H), 3.98 (s, 6H), 3.89 (s, 6H),

11

3.35 (s, 6H), 2.55 (t, J = 4.0 Hz, 2H), 1.95–1.88 (m, 2H), 1.86–1.79 (m, 2H); 13C NMR

12

(100 MHz, DMSO-d6) δ 176.7, 149.0, 148.6, 148.1, 130.0, 126.5, 125.4, 125.3, 124.7,

13

123.9, 108.3, 105.8, 104.0, 103.7, 58.7, 55.9, 55.8, 55.3, 54.9, 54.6, 35.3, 23.8;

14


15

3-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)propanoic

16

Yield: 97%, yellow powder, mp: 234–236 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s,

17

2H), 7.94 (s, 2H), 7.63 (s, 2H), 7.43 (s, 2H), 7.33 (s, 2H), 4.00 (s, 6H), 3.98 (s, 10H), 3.89

18

(s, 6H), 3.35 (s, 6H), 2.76 (t, J = 8.0, 2H), 2.40–2.34 (t, J = 8.0, 2H); 13C NMR (100 MHz,

19

DMSO-d6) δ 176.4, 149.0, 148.7, 148.5, 148.0, 130.1, 126.4, 125.4, 125.3, 124.7, 123.8,

20

108.2, 106.0, 103.9, 103.7, 58.5, 55.9, 55.8, 55.3, 54.8, 51.8, 35.6; ESI-HRMS (m/z):

21


22

2-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)acetic acid (37). Yield:

13

C NMR (100 MHz, DMSO-d6) δ 178.1, 149.0, 148.6, 148.5, 148.0, 129.9, 126.5,

11


acid

(34).


1

96%, white powder, mp: >300 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.97 (s, 2H), 7.95 (s,

2

2H), 7.94 (s, 2H), 7.62 (s, 2H), 7.32 (s, 2H), 4.09 (s, 4H), 4.00 (s, 6H), 3.98 (s, 6H), 3.90

3

(s, 6H), 3.41 (s, 6H), 2.99 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 174.7, 148.9, 148.8,

4

148.5, 148.3, 130.7, 126.4, 125.8, 125.4, 124.6, 123.9, 108.3, 107.2, 103.8, 103.7, 58.7,

5

55.9, 55.8, 55.3, 55.3; ESI-HRMS (m/z): calcd. for C40H41NO10Na [M+Na]+ 718.2623;

6

found 718.2616.

7

General Synthetic Procedure for Compounds 26, 29, 32, 35 and 38. To a mixture of

8

LiAlH4 (1.17 mmol) in 30 mL of THF at 0 °C was added corresponding esters 24, 27, 30,

9

33 and 36 (0.39 mmol) in portions. The solution was stirred for 8 h at room temperature,

10

then brought back to 0 °C, at which point 30 mL of CH2Cl2 was added, followed by 30 mL

11

of 2 M NaOH. The product was partitioned between CH2Cl2 and H2O. The organic layer

12

was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product.

13

The crude product was purified by flash column chromatography to give compounds 26,

14

29, 32, 35 and 38.

15

6-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexan-1-ol (26). Yield:

16


17

2H), 7.50 (s, 2H), 7.17 (s, 2H), 4.11 (s, 6H), 4.08 (s, 6H), 4.06 (s, 4H), 4.03 (s, 6H), 3.72

18

(s, 1H), 3.48 (s, 6H), 3.44–3.39 (m, 2H), 2.60 (t, J = 8.0 Hz , 2H), 1.77–1.68 (m, 2H),

19

1.36–1.29 (m, 2H), 1.23–1.17 (m, 2H), 1.14–1.09 (m, 2H); 13C NMR (100 MHz, CDCl3) δ

20

148.1, 147.8, 147.8, 147.4, 129.6, 125.9, 125.1, 124.8, 123.9, 123.2, 107.1, 105.2, 101.9,

21

101.7, 61.7, 58.6, 55.1, 55.0, 54.8, 54.4, 53.4, 31.6, 26.2, 24.4; ESI-HRMS (m/z): calcd.

22

for C44H51NO9Na [M+Na]+ 760.3456; found 760.3449.

12


Page 12 of 44

Page 13 of 44


1

5-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)pentan-1-ol (29). Yield:

2


3

2H), 7.57 (s, 2H), 7.48 (s, 2H), 7.17 (s, 2H), 4.11 (s, 6H), 4.08 (s, 6H), 4.06 (s, 4H), 4.03

4

(s, 6H), 3.47 (s, 6H), 3.38 (t, J = 4.0 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.79–1.72 (m, 2H),

5

1.53 (s, 1H), 1.32–1.20 (m, 4H);

6

148.5, 130.7, 126.9, 126.1, 125.9, 125.0, 124.3, 108.2, 106.2, 103.0, 102.9, 62.8, 59.7,

7

56.1, 56.1, 55.9, 55.5, 54.6, 32.5, 27.2, 23.7; ESI-HRMS (m/z): calcd. for C43H49NO9Na

8

[M+Na]+ 746.3300; found 746.3297.

9

4-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)butan-1-ol (32). Yield:

10


11

2H), 7.42 (s, 2H), 7.14 (s, 2H), 4.09 (s, 6H), 4.05 (s, 6H), 4.01 (s, 10H), 3.46 (s, 6H), 3.38

12

(t, J = 4.0 Hz, 2H), 2.60 (t, J = 4.0 Hz, 2H), 1.80–1.71 (m, 2H), 1.47–1.38 (m, 2H); 13C

13

NMR (100 MHz, CDCl3) δ 148.1, 147.8, 147.7, 147.4, 129.4, 125.8, 125.0, 124.8, 123.9,

14

123.2, 107.1, 105.1, 101.9, 101.7, 61.3, 58.4, 55.0, 55.0, 54.8, 54.4, 53.5, 29.6, 22.5;

15


16

3-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)propan-1-ol (35). Yield:

17


18

2H), 7.35 (s, 2H), 7.17 (s, 2H), 4.11 (s, 6H), 4.09 (s, 4H), 4.06 (s, 6H), 4.02 (s, 6H), 3.48

19

(s, 6H), 3.42 (t, J = 4.0, 2H), 2.89–2.81 (m, 2H), 1.94–1.86 (m, 2H), 1.43 (s, 1H); 13C

20

NMR (100 MHz, CDCl3) δ 148.3, 147.9, 147.8, 147.7, 128.6, 126.5, 124.7, 123.9, 123.4,

21

107.1, 104.5, 102.1, 101.8, 61.7, 58.4, 55.1, 54.9, 54.9, 54.3, 52.5, 28.1; ESI-HRMS (m/z):

22


13

C NMR (100 MHz, CDCl3) δ 149.2, 148.9, 148.9,

13



Page 14 of 44

1

2-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)ethanol (38). Yield: 58%,

2

white powder, mp: 246–248 °C; 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 2H), 7.74 (s, 2H),

3

7.58 (s, 2H), 7.29 (s, 2H), 7.18 (s, 2H), 4.16 (s, 4H), 4.11 (s, 6H), 4.06 (s, 6H), 4.03 (s,

4

6H), 3.64 (s, 2H), 3.41 (s, 6H), 2.86 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 148.4, 148.0,

5

147.9, 147.9, 128.5, 126.7, 124.7, 124.1, 123.6, 107.2, 104.0, 102.3, 101.8, 58.6, 57.9,

6

55.1, 55.0, 54.9, 54.6, 54.2; ESI-HRMS (m/z): calcd. for C40H43NO9Na [M+Na]+

7

704.2830; found 704.2823.

8

Synthetic Procedure for 2,3,6,7-Tetramethoxyphenanthrene-9-carbaldehyde (51). The

9

mixture of alcohol 45 (5 g, 15.23 mmol), silica (10 g) and pyridinium chlorochromate

10

(PCC, 9.85 g, 45.69 mmol) in CH2Cl2 (150 mL) was stirred at room temperature for 10 h,

11

and then filtered. The filtrate was concentrated by rotary evaporation and purified by flash

12

column chromatography to give compound 51 (4.02 g, 80%) as a yellow powder: mp

13

227–228 °C (lit.27 210–214 °C); 1H NMR (400 MHz, CDCl3) δ 10.27 (s, 1H), 8.98 (s, 1H),

14

8.07 (s, 1H), 7.80 (s, 1H), 7.78 (s, 1H), 7.35 (s, 1H), 4.17 (s, 3H), 4.14 (s, 3H), 4.11 (s,

15

3H), 4.07 (s, 3H).

16

Synthetic

17

6-(((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate (2). The mixture

18

of aldehyde 51 (1.2 g, 3.68 mmol), Et3N (0.42 g, 4.12 mmol), AcOH (5 d) and methyl

19

6-aminohexanoate hydrochloride (0.74 g, 4.05 mmol) in CH2Cl2 (40 mL) was refluxed for

20

6 h. The solution was cooled to room temperature and concentrated in vacuo. The result

21

solid was dissolved in MeOH (30 mL), followed addition of AcOH (5 d) and NaBH3CN

22

(0.47 g, 7.40 mmol). The solution was stirred at room temperature for 3 h, and

Procedure

for

14


Methyl

Page 15 of 44


1

concentrated in vacuo. To the result mixture was added CH2Cl2 (30 mL), H2O (20 mL) and

2

saturated NaHCO3 solution (10 mL), and the two layers were separated. The organic phase

3

was dried over anhydrous Na2SO4, filtered, concentrated and purified by flash column

4

chromatography to give compound 2 (1.14 g, 68%) as a yellow powder: mp: 104–106 °C;

5

1

6

1H), 4.16 (s, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 4.06 (s, 3H), 4.02 (s, 3H), 3.64 (s, 3H), 2.79 (t,

7

J = 8.0 Hz, 2H), 2.28 (t, J = 8.0 Hz, 2H), 1.71–1.65 (m, 2H), 1.64–1.57 (m, 2H),

8

1.40–1.33 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 174.1, 149.3, 149.0, 148.9, 148.8,

9

130.1, 126.0, 125.1, 125.0, 124.8, 124.3, 108.3, 104.7, 103.4, 102.8, 56.1, 56.0, 56.0, 55.9,

10

51.7, 51.5, 49.2, 33.9, 29.2, 26.8, 24.7; ESI-HRMS (m/z): calcd. for C26H33NO6Na

11

[M+Na]+ 478.2200; found 478.2208.

12

Synthetic

13

6-(((2,3,6,7-Tetramethoxyphenanthren-9-yl)methyl)amino)hexan-1-ol (3). The similar

14

procedure for the preparation of compounds 26, 29, 32, 35 and 38 was used. Yield: 89%,

15

white powder, mp: 83–85 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.98 (s, 1H),

16

7.60 (s, 1H), 7.58 (s, 1H), 7.34 (s, 1H), 4.34 (s, 1H), 4.13 (s, 2H), 4.03 (s, 3H), 4.02 (s,

17

3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.40–3.36 (m, 2H), 2.67 (t, J = 6.8 Hz, 2H), 1.56–1.47 (m,

18

2H), 1.44–1.39 (m, 2H), 1.35–1.28 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 149.1, 148.9,

19

148.8, 148.6, 130.8, 126.0, 125.1, 125.0, 124.3, 124.1, 108.2, 104.6, 103.3, 102.7, 62.7,

20

56.0, 56.0, 55.9, 52.1, 49.7, 32.7, 29.8, 27.1, 25.7; ESI-HRMS (m/z): calcd. for

21

C25H34NO5 [M+H]+ 428.2431; found 428.2440.

22

General Synthetic Procedure for Compounds 6, 9, 10 and 13–21. The mixture of

H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.67 (s, 1H), 7.58 (s, 1H), 7.42 (s, 1H), 7.18 (s,

Procedure

15


for


1

compound 3 (0.18 g, 0.40 mmol) and NaH (0.03 g, 1.20 mmol) was stirred at room

2

temperature for 30 min, then corresponding bromides or iodides (0.60 mmol) was added.

3

The solution was stirred for further 3 h, followed addition of H2O (150 mL) and CH2Cl2

4

(100 mL), and the two layers were separated. The organic phase was dried over anhydrous

5

Na2SO4, filtered, concentrated and purified by flash column chromatography to give

6

corresponding compounds 6, 9, 10 and 13–21.

7

Methyl

8

(6). Reaction with CH3I, yield: 89%, yellow powder, mp: 128–129 °C; 1H NMR (400

9

MHz, DMSO-d6) δ 8.00 (s, 1H), 7.98 (s, 1H), 7.77 (s, 1H), 7.51 (s, 1H), 7.36 (s, 1H), 4.02

10

(s, 3H), 4.02 (s, 3H), 3.90 (s, 6H), 3.81 (s, 2H), 3.55 (s, 3H), 2.43 (t, J = 8.0 Hz, 2H), 2.22

11

(t, J = 8.0 Hz, 2H), 2.13 (s, 3H), 1.56–1.52 (m, 2H), 1.48–1.41 (m, 2H), 1.31-1.26 (m, 2H);

12

13

13

107.2, 104.9, 102.0, 101.8, 56.7, 55.1, 55.0, 54.9, 54.8, 50.5, 40.9, 32.9, 26.1, 26.0, 23.8;

14

ESI-HRMS (m/z): calcd. for C27H35NO6 [M+Na]+ 492.2357; found 492.2361.

15

Methyl 6-(butyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate (9).

16

Reaction with BuI, yield: 80%, yellow powder, mp: 96–98 °C; 1H NMR (400 MHz,

17

CDCl3) δ 7.97 (s, 1H), 7.81 (s, 1H), 7.79 (s, 1H), 7.50 (s, 1H), 7.20 (s, 1H), 4.13 (s, 6H),

18

4.04 (s, 6H), 3.94 (s, 2H), 3.62 (s, 3H), 2.52–2.42 (m, 4H), 2.18 (t, J = 7.2 Hz, 2H),

19

1.56–1.46 (m, 6H), 1.31–1.20 (m, 4H), 0.84 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,

20

CDCl3) δ 174.1, 149.0, 148.8, 148.7, 148.2, 131.4, 126.1, 126.1, 125.7, 124.9, 124.2,

21

108.2, 106.5, 102.9, 102.8, 59.3, 56.1, 56.0, 55.9, 53.9, 53.5, 51.4, 34.0, 29.4, 27.0, 26.7,

22

24.8, 20.7, 14.1; ESI-HRMS (m/z): calcd. for C30H42NO6 [M+H]+ 512.3007; found

6-(methyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate

C NMR (100 MHz, CDCl3) δ 173.1, 148.1, 147.9, 147.8, 147.4, 124.9, 124.0, 123.3,

16


Page 16 of 44

Page 17 of 44


1

512.3012.

2

Methyl

3

(10). Reaction with BnBr, yield: 95%, yellow powder, mp: 90–92 °C; 1H NMR (400 MHz,

4

DMSO-d6) δ 7.99 (s, 1H), 7.97 (s, 1H), 7.74 (s, 1H), 7.60 (s, 1H), 7.36 (s, 1H), 7.33–7.27

5

(m, 4H), 7.23–7.20 (m, 1H), 4.02 (s, 3H), 4.01 (s, 3H), 3.93 (s, 2H), 3.90 (s, 3H), 3.83 (s,

6


7

(m, 2H), 1.34–1.25 (m, 2H), 1.17–1.09 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 173.0,

8

148.1, 147.8, 147.8, 147.2, 138.9, 129.9, 128.1, 127.1, 125.8, 125.2, 125.0, 124.0, 123.3,

9

107.2, 105.5, 101.9, 101.9, 58.2, 58.0, 55.1, 55.0, 55.0, 54.9, 52.6, 50.4, 32.9, 25.9, 25.6,

6-(benzyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate

10

23.7; ESI-HRMS (m/z): calcd. for C33H39NO6Na [M+Na]+ 568.2670; found 568.2674.

11

Methyl

12

6-((4-methylbenzyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate

13

(13). Reaction with p-methylbenzyl bromide, yield: 70%, white powder, mp: 88–90 °C; 1H

14

NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.80 (s, 1H), 7.78 (s, 1H), 7.53 (s, 1H), 7.20–7.18

15

(m, 3H), 7.07 (d, J = 7.6 Hz, 2H), 4.12 (s, 3H), 4.11 (s, 3H), 4.04 (s, 3H), 3.96 (s, 5H),

16

3.61 (s, 3H), 3.54 (s, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H), 2.15 (t, J = 7.6 Hz, 2H),

17


18

174.2, 149.2, 149.0, 148.9, 148.4, 136.9, 136.5, 131.2, 129.3, 128.9, 126.3, 126.2, 126.2,

19

125.1, 124.4, 108.3, 106.7, 103.1, 103.0, 59.3, 58.8, 56.3, 56.1, 56.0, 53.6, 51.5, 34.1, 27.1,

20

26.8, 24.9, 21.2; ESI-HRMS (m/z): calcd. for C34H42NO6 [M+H]+ 560.3007; found

21

560.3010.

22

Methyl

17



Page 18 of 44

1

6-((4-(tert-butyl)benzyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexan

2

oate (14). Reaction with 4-tert-butylbenzyl bromide, yield: 92%, yellow powder, mp:

3

84–86 °C; 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.79 (s, 1H), 7.77 (s, 1H), 7.54 (s,

4

1H), 7.28 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (s, 1H), 4.12 (s, 3H), 4.11 (s,

5

3H), 4.04 (s, 3H), 3.96 (s, 2H), 3.93 (s, 3H), 3.61 (s, 3H), 3.56 (s, 2H), 2.46 (t, J = 7.2 Hz,

6

2H), 2.15 (t, J = 7.6 Hz, 2H), 1.61–1.56 (m, 2H), 1.48–1.42 (m, 2H), 1.29 (s, 9H),

7

1.24–1.19 (m, 2H);

8

148.2, 136.8, 131.1, 128.9, 126.9, 126.2, 126.0, 125.0, 125.0, 124.3, 108.2, 106.6, 102.9,

9

59.2, 58.7, 56.1, 56.1, 56.0, 55.9, 53.7, 51.4, 34.4, 34.0, 31.4, 26.9, 26.7, 24.7; ESI-HRMS

13

C NMR (100 MHz, CDCl3) δ 174.1, 149.7, 149.1, 148.8, 148.8,

10

(m/z): calcd. for C37H48NO6 [M+H]+ 602.3476; found 602.3481.

11

Methyl

12

6-((4-fluorobenzyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate

13

(15). Reaction with 4-fluorobenzyl bromide, yield: 94%, white powder, mp: 116–118 °C;

14

1

15

7.23–7.20 (m, 3H), 6.93 (t, J = 8.8 Hz, 2H), 4.13 (s, 3H), 4.12 (s, 3H), 4.04 (s, 3H), 3.98 (s,

16

3H), 3.98 (s, 2H), 3.62 (s, 3H), 3.52 (s, 2H), 2.45 (t, J = 7.6 Hz, 2H), 2.18 (t, J = 7.6 Hz,

17

2H), 1.64–1.57 (m, 2H), 1.53–1.45 (m, 2H), 1.27–1.20 (m, 2H);

18

CDCl3) δ 174.0, 149.2, 148.9, 148.8, 148.3, 144.3, 130.5, 129.4, 129.1, 128.8, 128.5,

19

128.3, 126.3, 125.9, 125.8, 125.6, 125.1, 125.0, 125.0, 124.9, 124.9, 124.4, 122.9, 120.2,

20

108.1, 106.3, 102.9, 102.8, 59.3, 58.3, 56.1, 56.0, 55.9, 55.9, 54.3, 51.5, 33.9, 27.0, 26.8,

21

24.7.

22

Methyl

H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.81 (s, 1H), 7.78 (s, 1H), 7.54 (s, 1H),

18


13

C NMR (100 MHz,

Page 19 of 44


1

6-(((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)(4-(trifluoromethyl)benzyl)amino)

2

hexanoate (16). Reaction with 4-(trifluoromethyl)benzyl bromide, yield: 88%, white

3

powder, mp: 127–129 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.96 (s, 1H),

4

7.71 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.61 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.36 (s, 1H),

5

4.02 (s, 3H), 4.01 (s, 3H), 3.97 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.64 (s, 2H), 3.52 (s,

6

3H), 2.41 (t, J = 7.2 Hz, 2H), 2.12 (t, J = 7.2 Hz, 2H), 1.58–1.51 (m, 2H), 1.35–1.28 (m,

7

2H), 1.18–1.13 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 174.0, 149.2, 148.9, 148.8, 148.3,

8

144.3, 130.5, 129.1, 128.9 (q, J = 31.9 Hz, 1C), 126.3, 125.9, 125.8, 125.1, 124.9 (q, J =

9

3.6 Hz, 1C), 124.4, 124.2 (q, J = 270.1 Hz, 1C), 120.2, 108.1, 106.3, 102.9, 102.8, 59.3,

10

58.3, 56.1, 56.0, 55.9, 55.9, 54.3, 51.5, 33.9, 27.0, 26.8, 24.7; ESI-HRMS (m/z): calcd. for

11

C34H39F3NO6 [M+H]+ 614.2724; found 614.2725.

12

Methyl

13

6-((4-nitrobenzyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoate

14

(17). Reaction with 4-nitrobenzyl bromide, yield: 96%, yellow powder, mp: 132–134 °C;

15

1

16

1H), 7.54 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.19 (s, 1H), 4.14 (s, 3H), 4.12 (s, 3H), 4.04 (s,

17


18

(m, 2H), 1.59–1.53 (m, 2H), 1.33–1.25 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 174.0,

19

149.3, 149.0, 148.9, 148.3, 148.2, 146.8, 130.2, 129.3, 126.4, 125.9, 125.7, 125.1, 124.5,

20

123.2, 108.1, 106.2, 103.0, 102.8, 59.5, 58.0, 56.1, 56.0, 55.9, 54.9, 51.5, 33.9, 27.0, 27.0,

21

24.8; ESI-HRMS (m/z): calcd. for C33H39N2O8 [M+H]+ 591.2701; found 591.2711.

22

Methyl

H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 8.4 Hz, 2H), 7.85 (s, 1H), 7.79 (s, 1H), 7.76 (s,

19



1

6-((4-(dimethylamino)benzyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)

2

hexanoate (18). Reaction with 4-(dimethylamino)benzyl bromide, yield: 37%, yellow

3

powder, mp: 44–46 °C; 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.79 (s, 1H), 7.78 (s,

4

1H), 7.53 (s, 1H), 7.20 (s, 1H), 7.18 (d, J = 8.0 Hz, 2H), 6.66 (d, J = 8.0 Hz, 2H), 4.12 (s,

5

6H), 4.04 (s, 3H), 3.95 (s, 3H), 3.94 (s, 2H), 3.61 (s, 3H), 3.50 (s, 2H), 2.91 (s, 6H), 2.43 (t,

6

J = 6.8 Hz, 2H), 2.15 (t, J = 7.6 Hz, 2H), 1.60–1.52 (m, 2H), 1.47–1.42 (m, 2H),

7

1.23–1.17 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 174.1, 149.7, 149.0, 148.8, 148.7,

8

148.2, 131.3, 130.1, 127.6, 126.1, 126.0, 124.9, 124.2, 112.5, 108.2, 106.7, 102.9, 102.8,

9

77.4, 77.3, 77.1, 76.7, 59.0, 58.3, 56.1, 56.1, 56.0, 55.9, 53.2, 51.4, 40.8, 34.0, 26.9, 26.6,

10

24.7; ESI-HRMS (m/z): calcd. for C35H45N2O6 [M+H]+ 589.3272; found 589.3278.

11

Methyl

12

6-((4-methoxybenzyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoa

13

te (19). Reaction with 4-methoxybenzyl bromide, yield: 83%, white powder, mp:

14

92–94 °C; 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.80 (s, 1H), 7.78 (s, 1H), 7.53 (s,

15

1H), 7.21–7.20 (m, 3H), 6.80 (d, J = 8.0 Hz, 2H), 4.12 (s, 3H), 4.12 (s, 3H), 4.04 (s, 3H),

16

3.97 (s, 3H), 3.96 (s, 2H), 3.77 (s, 3H), 3.61 (s, 3H), 3.51 (s, 2H), 2.44 (t, J = 7.2 Hz, 2H),

17

2.16 (t, J = 7.2 Hz, 2H), 1.63–1.56 (m, 2H), 1.49–1.43 (m, 2H), 1.26–1.19 (m, 2H); 13C

18

NMR (100 MHz, CDCl3) δ 174.1, 158.5, 149.1, 148.8, 148.7, 148.2, 131.9, 131.1, 130.3,

19

128.7, 126.2, 126.0, 125.0, 124.3, 113.5, 108.2, 106.6, 102.9, 102.8, 59.1, 58.2, 56.1, 56.0,

20

56.0, 55.9, 55.3, 53.4, 51.4, 34.0, 26.9, 26.7, 24.7; ESI-HRMS (m/z): calcd. for

21

C34H42NO7 [M+H]+ 576.2956; found 576.2952.

22

Methyl

20


Page 20 of 44

Page 21 of 44


1

6-(((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)(3,4,5-trimethoxybenzyl)amino)he

2

xanoate (20). Reaction with 4-(3,4,5-trimethoxy)benzyl bromide, yield: 54%, white

3

powder, mp: 129–131 °C; 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.81 (s, 1H), 7.77 (s,

4

1H), 7.55 (s, 1H), 7.19 (s, 1H), 6.42 (s, 2H), 4.12 (s, 3H), 4.11 (s, 3H), 4.03 (s, 5H), 3.99

5

(s, 3H), 3.76 (s, 3H), 3.71 (s, 6H), 3.63 (s, 3H), 3.48 (s, 2H), 2.56 (t, J = 7.6 Hz, 2H), 2.22

6

(t, J = 7.2 Hz, 2H), 1.70–1.63 (m, 2H), 1.59-1.52 (m, 2H), 1.34-1.28 (m, 2H); 13C NMR

7

(100 MHz, CDCl3) δ 174.0, 152.8, 149.2, 148.9, 148.8, 148.3, 136.5, 136.0, 130.9, 126.2,

8

126.0, 125.9, 125.1, 124.3, 108.1, 106.6, 105.5, 103.0, 102.9, 60.8, 59.1, 58.7, 56.1, 56.0,

9

55.9, 54.6, 51.5, 34.0, 27.1, 26.9, 24.8; ESI-HRMS (m/z): calcd. for C36H46NO9 [M+H]+

10

636.3167; found 636.3172.

11

Methyl

12

6-((naphthalen-2-ylmethyl)((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)he

13

xanoate (21). Reaction with 2-(bromomethyl)naphthalene, yield: 69%, yellow viscous oil;

14

1

15

1H), 7.63 (s, 1H), 7.50–7.37 (m, 3H), 7.37 (s, 1H), 4.02 (s, 3H), 4.01 (s, 5H), 3.91 (s, 3H),

16

3.81 (s, 3H), 3.72 (s, 2H), 3.50 (s, 3H), 2.44 (t, J = 8.0 Hz, 2H), 2.09 (t, J = 8.0 Hz, 2H),

17


18

174.0, 149.2, 148.9, 148.8, 148.3, 137.5, 133.3, 132.6, 130.9, 127.7, 127.6, 127.6, 126.3,

19

126.0, 125.9, 125.5, 125.0, 124.4, 108.2, 106.6, 102.9, 59.4, 59.1, 56.1, 56.0, 55.9, 53.8,

20

51.4, 33.9, 27.0, 26.7, 24.8; ESI-HRMS (m/z): calcd. for C37H41NO6Na [M+Na]+

21

618.2826; found 618.2832.

22

6-(Methyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoic acid (7).

H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.97 (s, 1H), 7.88–7.82 (m, 4H), 7.79 (s,

21



1

The similar procedure for the preparation of compound 25 was used. Yield: 60%, yellow

2

powder, mp: 168–170 °C; 1H NMR (400 MHz, MeOD) δ 7.84 (s, 1H), 7.77 (s, 1H), 7.59

3

(s, 1H), 7.29 (s, 1H), 7.18 (s, 1H), 4.50 (s, 2H), 3.89 (s, 6H), 3.86 (s, 3H), 3.79 (s, 3H),

4

3.05–2.97 (m, 2H), 2.57 (s, 3H), 2.05 (t, J = 6.4 Hz, 2H), 1.70–1.62 (m, 2H), 1.48–1.40 (m,

5

2H), 1.26–1.19 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 174.4, 149.3, 148.8, 148.6,

6

148.1, 125.2, 125.0, 124.6, 124.1, 108.4, 106.0, 104.0, 103.7, 62.8, 56.8, 55.9, 55.9, 55.4,

7

55.3, 40.9, 33.6, 26.3, 24.3; ESI-HRMS (m/z): calcd. for C26H34NO6 [M+H]+ 456.2381;

8

found 456.2387.

9

6-(Benzyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexanoic acid (11).

10

The similar procedure for the preparation of compound 25 was used. Yield: 73%, white

11

powder, mp: 148–150 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 7.99 (s, 1H),

12

7.97 (s, 1H), 7.76 (s, 1H), 7.60 (s, 1H), 7.36 (s, 1H), 7.32–7.25 (m, 4H), 7.22–7.19 (m,

13

1H), 4.02 (s, 3H), 4.01 (s, 3H), 3.94 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.54 (s, 2H), 2.38 (t,

14

J = 6.8 Hz, 2H), 2.05 (t, J = 7.2 Hz, 2H), 1.58–1.50 (m, 2H), 1.35–1.27 (m, 2H),

15

1.18–1.10 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 174.4, 149.0, 148.7, 148.6, 147.9,

16

139.6, 130.1, 128.7, 128.1, 126.7, 125.9, 125.4, 125.2, 124.7, 123.9, 108.3, 106.1, 103.8,

17

103.7, 58.4, 58.0, 55.9, 55.7, 55.4, 55.3, 53.1, 33.5, 26.4, 25.9, 24.2; ESI-HRMS (m/z):

18

calcd. for C32H38NO6 [M+H]+ 532.2694; found 532.2699.

19

6-(Methyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexan-1-ol (8). The

20

similar procedure for the preparation of compound 26 was used. Yield: 90%, white powder,

21

mp: 101–103 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.98 (s, 1H), 7.78 (s, 1H),

22

7.51 (s, 1H), 7.36 (s, 1H), 4.31 (t, J = 4.8 Hz, 1H), 4.02 (s, 6H), 3.90 (s, 6H), 3.80 (s, 2H),

22


Page 22 of 44

Page 23 of 44


1

3.33–3.27 (m, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.12 (s, 3H), 1.57–1.49 (m, 2H), 1.35–1.33 (m,

2

2H), 1.30–1.25 (m, 2H), 1.23–1.18 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 149.1, 148.8,

3

148.7, 148.3, 130.7, 126.0, 126.0, 125.7, 125.0, 124.3, 108.2, 106.2, 102.9, 102.8, 62.9,

4

62.3, 57.8, 56.1, 56.0, 55.9, 55.8, 42.1, 32.7, 30.3, 29.7, 27.5, 27.3, 25.6; ESI-HRMS (m/z):

5


6

6-(Benzyl((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)hexan-1-ol (12). The

7

similar procedure for the preparation of compound 26 was used. Yield: 86%, yellow

8

powder, mp: 99–101 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.97 (s, 1H), 7.77

9

(s, 1H), 7.59 (s, 1H), 7.36 (s, 1H), 7.30–7.27 (m, 4H), 7.22–7.17 (m, 1H), 4.25 (s, 1H),

10

4.02 (s, 3H), 4.01 (s, 3H), 3.94 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.54 (s, 2H), 3.27–3.20

11

(m, 2H), 2.38 (t, J = 6.4 Hz, 2H), 1.59–1.52 (m, 2H), 1.26–1.23 (m, 2H), 1.17–1.12 (m,

12

2H), 1.09–1.03 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 148.1, 147.8, 147.7, 147.1, 138.9,

13

130.0, 128.2, 127.1, 125.8, 125.2, 125.0, 125.0, 123.9, 123.3, 107.1, 105.6, 101.8, 101.7,

14

61.8, 58.3, 58.0, 55.1, 55.0, 54.9, 54.9, 52.5, 31.7, 29.3, 26.0, 25.9, 24.4; ESI-HRMS (m/z):

15


16

Synthetic

17

2-Methyl-2-(((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)propane-1,3-diol

18

(4). The mixture of 2-amino-2-methyl-1,3-propanediol (0.98 mmol), Et3N (1.34 mmol)

19

and bromide 48 (0.89 mmol) in DMF (20 mL) was stirred at room temperature for 4 h,

20

followed addition of H2O (150 mL) and CH2Cl2 (100 mL), and the two layers were

21

separated. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated and

22

purified by flash column chromatography to give compound 4 (0.24 g, 64%) as a white

Procedure

23


for


Page 24 of 44

1

powder, mp 108–110 °C; 1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.72 (s, 1H), 7.70 (s,

2

1H), 7.58 (s, 1H), 7.20 (s, 1H), 4.23 (s, 2H), 4.09 (s, 6H), 4.04 (s, 3H), 4.02 (s, 3H),

3

3.59–3.50 (m, 4H), 1.16 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.30, 148.96, 148.93,

4

148.87, 130.59, 126.05, 125.48, 125.11, 125.02, 124.38, 108.23, 104.75, 103.37, 102.74,

5

67.22, 57.68, 56.04, 56.02, 55.99, 55.90, 44.48, 18.20; ESI-HRMS (m/z): calcd. for

6

C23H29NO6Na [M+Na]+ 438.1887; found 438.1884.

7

2-(((2,3,6,7-Tetramethoxyphenanthren-9-yl)methyl)amino)propane-1,3-diol (5). The

8

similar procedure for the preparation of compound 4 was used. Yield: 51%, white powder,

9

mp: 182–184 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.98 (s, 1H), 7.62 (s, 2H),

10

7.35 (s, 1H), 4.52 (s, 2H), 4.20 (s, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 3.93 (s, 3H), 3.90 (s,

11

3H), 3.56–3.52 (m, 2H), 3.48–3.44 (m, 2H), 3.32 (s, 1H), 2.81–2.76 (m, 1H);

12

(100 MHz, DMSO-d6) δ 148.9, 148.7, 148.6, 148.3, 125.6, 124.8, 124.5, 123.9, 123.7,

13

108.4, 105.5, 104.1, 103.8, 61.2, 61.1, 55.9, 55.9, 55.4, 55.3, 49.9; ESI-HRMS (m/z):

14


15

2-Methyl-3-((2,3,6,7-tetramethoxyphenanthren-9-yl)methoxy)-2-(((2,3,6,7-tetrametho

16

xyphenanthren-9-yl)methyl)amino)propan-1-ol (22). The similar procedure for the

17

preparation of compound 24 was used. Yield: 31%, white powder, mp: 161–163 °C; 1H

18

NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.99 (s, 1H), 7.97 (s, 1H), 7.95 (s, 1H), 7.65 (s,

19

1H), 7.55 (s, 2H), 7.35 (s, 2H), 7.26 (s, 1H), 4.96 (dd, J = 28.0, 12.0 Hz, 2H), 4.64–4.61

20

(m, 1H), 4.02–4.01 (m, 14H), 3.89 (s, 3H), 3.88 (s, 3H), 3.81 (s, 3H), 3.74 (s, 3H), 3.54 (s,

21

2H), 3.47–3.44 (m, 2H), 1.15 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 149.2, 148.8,

22

148.8, 148.6, 148.5, 148.4, 148.3, 148.0, 132.4, 129.3, 125.6, 125.2, 125.0, 124.9, 124.6,

24


13

C NMR

Page 25 of 44


1

124.5, 124.2, 123.7, 123.6, 108.5, 108.2, 105.5, 105.2, 104.0, 103.9, 103.7, 103.6, 72.8,

2

72.1, 65.0, 56.6, 55.9, 55.8, 55.8, 55.3, 55.3, 55.1, 55.0, 44.4, 19.2; ESI-HRMS (m/z):

3


4

2-(Bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)amino)propane-1,3-diol

5

The similar procedure for the preparation of compound 24 was used. Yield: 69%, yellow

6

powder, mp: 152–154°C; 1H NMR (400 MHz, CDCl3) δ 7.75 (s, 2H), 7.73 (s, 2H), 7.59 (s,

7

2H), 7.23 (s, 2H), 7.16 (s, 2H), 4.40 (s, 4H), 4.12 (s, 4H), 4.11 (s, 6H), 4.06 (s, 6H), 4.02

8

(s, 6H), 3.42 (s, 6H), 3.30–3.23 (m, 1H), 2.36 (s, 2H); 13C NMR (100 MHz, CDCl3) δ

9

149.4, 149.0, 149.0, 148.9, 129.5, 128.1, 125.7, 125.7, 125.14, 124.5, 108.2, 104.8, 103.4,

10

102.8, 59.7, 56.1, 56.0, 55.9, 55.1, 54.6, 45.7; ESI-HRMS (m/z): calcd. for C41H45NO8Na

11

[M+Na]+ 734.2936; found 734.2941.

12

Synthetic

13

N1,N1-Dimethyl-N2,N2-bis((2,3,6,7-tetramethoxyphenanthren-9-yl)methyl)ethane-1,2-

14

diamine

15

N,N-dimethylethylenediamine (0.049 g, 0.55 mmol), NaBH3CN (0.11 g, 1.65 mmol),

16

AcOH (5 d) in MeOH (30 mL) and CH2Cl2 (15 mL) was refluxed for 6 h. Then the

17

mixture was cooled to room temperature, concentrated and purified by flash column

18

chromatography to give compound 41 (0.15 g, 39%) as a yellow powder, mp: 138–140 °C;

19

1

20

2H), 4.10 (s, 6H), 4.07 (s, 10H), 4.03 (s, 6H), 3.52 (s, 6H), 2.85 (t, J = 6.8 Hz, 2H), 2.62 (t,

21

J = 6.8 Hz, 2H), 2.13 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 148.2, 147.9, 147.8, 147.5,

22

129.1, 125.9, 124.9, 124.8, 123.9, 123.3, 107.1, 105.0, 102.0, 101.7, 58.9, 56.2, 55.1, 55.0,

Procedure

(41).

The

mixture

of

aldehyde

(23).

for

51

(0.38

g,

1.17

mmol),

H NMR (400 MHz, CDCl3) δ 7.73 (s, 2H), 7.72 (s, 2H), 7.58 (s, 2H), 7.45 (s, 2H), 7.17 (s,

25



1

54.9, 54.5, 51.2, 44.3; ESI-HRMS (m/z): calcd. for C42H49N2O8 [M+H]+ 709.3483; found

2

709.3482.

3

Antiviral Biological Assay.

4

Phytotoxic Activity.

5

The growing 5–6 leaf stage tobaccos (Nicotiana tabacum var Xanthi nc) were selected.

6

The compound solution (500 µg/mL) was smeared on the leaves. The local lesion numbers

7

were then counted and recorded 3–4 days.

8

Purification of Tobacco Mosaic Virus.

9

Using Gooding’s method,21 the upper leaves of Nicotiana tabacum L. inoculated with

10

TMV were selected and ground in phosphate buffer and then filtered through double-layer

11

pledget. The filtrate was centrifuged at 10000g for 15 min, treated with polyethyleneglycol

12

(PEG) twice, and centrifuged again. The whole experiment was processed at 4 °C.

13

Absorbance value was estimated at 260 nm by ultraviolet spectrophotometer.

14 15

Virus concn = ( A 260 × dilution

. 1 %, 260 nm ratio ) E / 10cm

Antiviral activity of compounds against TMV in vitro.

16

In vitro activity of the synthesized compounds against TMV was performed by the

17

conventional half-leaf method.22 Fresh leaf of the growing 5–6 leaf stage tobacco

18

(Nicotiana tabacum var Xanthi nc) mechanically inoculated (concentration of TMV is 10

19

µg/mL) was cut into halves along the main vein. The halves were immersed into the

20

solution of 500 µg/mL of the compounds and double distilled water for 20 min,

21

respectively, and then kept still at 25 °C for 72 h. Each compound was replicated at least

22

three times.

26


Page 26 of 44

Page 27 of 44


1

Protective effect of compounds against TMV in vivo.23

2


3

The compound solution was smeared on the left side and the solvent serving as control on

4

the right side of growing tobacco (Nicotiana tabacum var Xanthi nc) leaves of the same

5

ages. The leaves were then inoculated with the virus after 12 h. A brush was dipped in

6

TMV of 10 µg/mL to inoculate the leaves, which were previously scattered with silicon

7

carbide. The leaves were then washed with water and rubbed softly along the nervature

8

once or twice. The local lesion numbers appearing 3–4 days after inoculation were

9

counted. There are three replicates for each compound.

10

Inactivation effect of compounds against TMV in vivo.23

11


12

The virus of 10 µg/mL was inhibited by mixing with the compound solution at the same

13

volume for 30 min. The mixture was then inoculated on the left side of the leaves, whereas

14

the right side of the leaves was inoculated with the mixture of solvent and the virus for

15

control. The local lesion numbers were recorded 3–4 days after inoculation. There are

16

three replicates for each compound.

17

Curative effect of compounds against TMV in vivo.23

18

Growing 5–6 leaf stage tobaccos (Nicotiana tabacum var Xanthi nc) were selected.

19

TMV (concentration of 10 µg/mL) was dipped and inoculated on the whole leaves. Then

20

the leaves were washed with water and dried. The compound solution was smeared on the

21

left side, and the solvent was smeared on the right side for control. The local lesion

22

numbers were then counted and recorded 3–4 days after inoculation. There are three

27



1

replicates for each compound.

2

The in vitro and in vivo inhibition rates of the compound were then calculated according

3

to the following formula (“av” means average, and controls were not treated with

4

compound).

5 6

Inhibition rate (%) = [(av local lesion no. of control − av local lesion no. of drug-treated)/av local lesion no. of control] × 100%

7 8

Results and discussion

9

Chemistry.

10

Reduction of acids 42–4424 with LiAlH4 gave alcohols 45–47, which were treated with

11

PBr3 to afford bromides 48–50. Coupling of 48–50 with corresponding amines gave the

12

antofine derivatives 1, 24, 27, 30, 33, 36, 39, and 40. Treatment of 24, 27, 30, 33 and 36

13

with 4 M NaOH solution gave acids 25, 28, 31, 34 and 37. Reduction of 24, 27, 30, 33 and

14

36 with LiAlH4 afforded alcohols 26, 28, 32, 35 and 38 (Scheme 1).

15

As shown in Scheme 2, oxidation of alcohol 45 with pyridiniumchlorochromate (PCC)

16

gave aldehyde 51, which was coupled with methyl 6-aminohexanoate hydrochloride to

17

give compound 2. Reduction of 2 with LiAlH4 afforded alcohol 3. Coupling of 2 with

18

corresponding bromides or iodides gave the antofine derivatives 6, 9, 10 and 13–21.

19

Treatment of 6 and 10 with 4 M NaOH solution gave acids 7 and 11. Reduction of 6 and

20

10 with LiAlH4 afforded alcohol 8 and 12.

21

Coupling of bromide 48 with corresponding alkamines gave the antofine derivatives 4,

22

5, 22 and 23 (Scheme 3). It is worth mentioning that reaction of 48 with excess alkamine

28


Page 28 of 44

Page 29 of 44


1

only gave N,O-dialkylation compound 22. Using the similar procedure for preparation of

2

derivatives 1, 24, 27, 30, 33, 36, 39, and 40 couldn’t afford 41 due to the presence of

3

N,N-dimethyl. As an alterative method, compound 41 was prepared by coupling of 51 with

4

excess amine (Scheme 4).

5

Bioactivities and the structure-activity relationship.

6

Phytotoxic activity. The antofine derivatives 1–41 were first tested for their phytotoxic

7

activity against Nicotiana tabacum var Xanthi nc, and the results indicated that these

8

natural product-based compounds showed no phytotoxic activity at 500 µg/mL.

9

Antiviral activity in vitro and in vivo. To make a judgment of the antiviral potency of

10

the antofine derivatives 1–41, the precursor antofine and commercial plant virucide

11

Ribavirin were used as the controls.

12

The first in vitro anti-TMV bioassay indicated that all of the tested compounds

13

displayed good antiviral activity, of which compounds 1, 2, 6, 10, 18, 19, 33, 38 and 40

14

exhibited higher inhibition than Ribavirin (Table 1). Therefore, these compounds were

15

bioassayed further to investigate their antiviral activity in vivo.

16

As shown in Table 1, the antofine derivatives 1–41 also exhibited good to excellent in

17

vivo anti-TMV activity, especially for compounds 10, 18 and 19 displayed higher antiviral

18

activity than the precursor antofine and commercial plant virucide Ribavirin, thus

19

emerging as new lead compounds.

20

Among the phenanthrenyl methyl amines 2–5, long-chain containing compounds 2 and

21

3 exhibited better antiviral activity, and the ester group is favorable for activity (antiviral

22

effect: 2 > 3). It is worth mentioning that dimethyl amine compound 1 showed higher

29



1

antiviral effect than compounds 2–5, and compound 6 displayed better activity than 2,

2

which indicated that the hydrogen on the N-atom is unfavorable for antiviral activity. In

3

order to further investigate the substituents on the long-chain, compounds 6–8 and 10–12

4

were prepared. The bioassay results indicated that the ester group is more suitable than

5

acid and hydroxy groups (antiviral effect: 6 > 7, 8; antiviral effect: 10 > 11, 12). The

6

mainly difference between compounds 6, 9 and 10 lies in the substituents on the N-atom.

7

Compounds 6 and 9 displayed about similar good antiviral activity, which is lower than

8

that of compound 10. The results indicated that the replacement of methyl with n-butyl is

9

tolerant, and benzyl is more suitable for antiviral activity than alkyl group. In order to

10

investigate the substituents on benzyl, compounds 13–21 were prepared. Only compounds

11

18 and 19 maintained good antiviral activity (antiviral effect: 10 ≈ 18 ≈ 19). The results

12

indicated that the benzyl lies in a sensitive area for antiviral activity.

13

In previous work, we have found that the presence of nitrogen in tertiary amine and

14

phenanthrene ring are essential for high antiviral activity.18,19 The increase of

15

phenanthrene ring may raise the interaction between small molecular compound and TMV

16

RNA. Based on the above hypothesis, a series of two phenanthrene rings containing

17

compounds 22–41 were synthesized and evaluated for their antiviral activity.

18

Unfortunately, most of these compounds only exhibited moderate antiviral activity. Only

19

compounds 22, 38 and 40 maintained good antiviral activity (antiviral effect: 22 ≈ 38 ≈ 40

20

≈ 6). The results indicated that phenanthrene is important for these compounds but not the

21

more the better.

22

In summary, based on our previous structure activity relationship and antiviral

30


Page 30 of 44

Page 31 of 44


1

mechanism studies, a series of phenanthrene and alkylamine chain containing antofine

2

derivatives 1–41 were prepared and systematically evaluated for their antiviral activity

3

against TMV. The bioassay results indicated that most of these compounds exhibited good

4

to excellent anti-TMV activity, of which compounds 10, 18 and 19 displayed higher

5

activity than the precursor antofine and commercial Ribavirin, thus emerging as potential

6

inhibitors of plant virus. The “D”, “E” rings of antofine were proved to be not

7

indispensable. Phenanthrene is important for these compounds but not the more the better.

8

The novel concise structure provides another new template for antiviral studies, which

9

may have different mechanisms of action. Further studies on structural optimization and

10

mode of action are currently underway in our laboratories.

11 12

ACKNOWLEDGEMENTS

13

This work was supported by the National Key Project for Basic Research

14

(2010CB126106), the National Natural Science Foundation of China (21132003,

15

21121002, 21372131), and the Specialized Research Fund for the Doctoral Program of

16

Higher Education (20130031110017).

17 1

H NMR,

13

C NMR and HRMS spectra of

18

Supporting Information Available:

19

compounds 1–41. This material is available free of charge via the Internet at

20

http://pubs.acs.org.

21 22

Literature cited

31



Page 32 of 44

1

(1) Hari, V.; Das, P. Ultra microscopic detection of plant viruses and their gene products.

2

In Plant Disease Virus Control; Hadidi, A., Khetarpal, R. K., Koganezawa, H., Eds.;

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APS Press: St. Paul, 1998; pp 417–427.

4 5

(2) Ritzenthaler, C. Resistance to plant viruses: old issue, new answer. Curr. Opin. Biotech. 2005, 16, 118–122.

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(3) Wei, L. Z.; Meng, F. S. Antiviral composition containing fucoidan and ribavirin for

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preventing and treating plant viral diseases. 2010, CN 101869111A, 20101027, CAN

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153:573294.

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(4) Song, B. A.; Yang, S.; Jin, L. H.; Bhadury, P. S. Environment-friendly anti-plant viral agents, Chemical Industry Press (Beijing) & Springer Press, 2009, 1–305. (5) Xue, W.; Song, B. A.; Wang, H.; He, W.; Yang, S.; Jin, L. H.; Hu, D. Y.; Liu, G.; Lu, P.

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Synthesis

and

Anti-TMV

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of

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2-[5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazole-2-ylthiomethyl]-1-(2,3,4-trimethoxy)p

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henyl ketoxime ether derivativies. Chinese J. Org. Chem. 2006, 26, 702–706.

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(6) Ouyang, G. P.; Chen, Z.; Cai, X. J.; Song, B. A.; Bhadury, P. S.; Yang, S.; Jin, L. H.;

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Xue, W.; Hu, D. Y.; Zeng, S. Synthesis and antiviral activity of novel pyrazole

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derivatives containing oxime esters group. Bioorg. Med. Chem. 2008, 16, 9699–9707.

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(7) Chen, M. H.; Chen, Z.; Song, B. A.; Bhadury, P. S.; Yang, S.; Cai, X. J.; Hu, D. Y.;

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Xue, W.; Zeng, S. Synthesis and antiviral activities of chiral thiourea derivatives

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containing an α-aminophosphonate moiety. J. Agric. Food Chem. 2009, 57,

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1383–1388.

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(8) Ouyang, M. A.; Wein, Y. S.; Zhang, Z. K.; Kuo, Y. H. Inhibitory activity against

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tobacco mosaic virus (TMV) replication of pinoresinol and syringaresinol lignans and

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their glycosides from the root of rhus javanica var. roxburghiana. J. Agric. Food Chem.

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2007, 55, 6460–6465.

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(9) Chen, J.; Yan, X. H.; Dong, J. H.; Sang, P.; Fang, X.; Di, Y. T.; Zhang, Z. K.; Hao, X. J.

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Tobacco mosaic virus (TMV) inhibitors from picrasma quassioides benn. J. Agric.

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Food Chem. 2009, 57, 6590–6595.

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(10) Lu, M.; Han, Z. Q.; Xu, Y.; Yao, L. In vitro and in vivo anti-tobacco mosaic virus

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activities of essential oils and individual compounds. J. Microbiol. Biotechnol. 2013,

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23, 771–778.

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(11) Hu, Q. F.; Zhou, B.; Huang, J. M.; Gao, X. M.; Shu, L. D.; Yang, G. Y.; Che, C. T.

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Antiviral phenolic compounds from Arundina gramnifolia. J. Nat. Prod. 2013, 76,

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292−296.

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(12) Hansen, A. J. Antiviral chemicals for plant disease control. Crit. Rev. Plant Sci. 1989, 8, 45–88. (13) Qian, X. H.; Lee, P. W.; Cao, S. China: Forward to the green pesticides via a basic research program. J. Agric. Food Chem. 2010, 58, 2613–2623. (14) Seiber, J. N. Sustainability and agricultural and food chemistry. J. Agric. Food Chem. 2011, 59, 1–21.

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(15) An, T. Y.; Huang, R. Q.; Yang, Z.; Zhang, D. K.; Li, G. R.; Yao, Y. C.; Gao, J.

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Alkaloids from Cyanachum komarovii with inhibitory activity against the tobacco

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mosaic virus. Phytochemistry 2001, 58, 1267–1269.

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(16) Huang, Z. Q.; Liu, Y. X.; Fan, Z. J.; Wang, Q. M.; Li, G. R.; Yao, Y. C.; Yu, X. S.;

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Huang, R. Q. Antiviral activity of alkaloids from Cynanchum komarovii. Fine

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Chemical Intermediates 2007, 37, 20–24, 46.

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(17) Wang, Q. M.; Yao, Y. C.; Huang, R. Q.; Fan, Z. J.; Li, G. R.; Yu, X. S. Antiviral

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activity of antofine from Cynanchum komarovii. Agrochemicals 2007, 46, 425–427.

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(18) Wang, Q. M.; Wang, K. L.; Huang, Z. Q.; Liu, Y. X.; Li, H.; Hu, T. S.; Jin, Z.; Fan, Z.

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J.; Huang, R. Q. Derivatives and salts of phenanthro indolizidine and phenanthro

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quinolizidine, and their application as agricultural virucides. 2008, CN 101189968A,

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20080604, CAN 149:97630.

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(19) Cui, M. B.; Wang, K. L.; Wang, Q. M.; Huang, R. Q. Concise synthesis of

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benzoindolizidine derivatives and bioactivity evaluation. Lett. Org. Chem. 2008, 5,

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98–102.

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(20) Gao, S.; Zhang, R. Y.; Yu, Z. H.; Xi, Z. Antofine analogues can inhibit tobacco mosaic

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virus assembly through small-molecule–RNA interactions. Chembiochem 2012, 13,

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1622–1627.

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(21) Gooding, G. V. J.; Hebert, T. T. A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 1967, 57, 1285–1290. (22) Chen, N. C. Pesticide’s Bioassay Technique; Beijing Agriculture University: Beijing, People's Republic of China, 1990, p 194. (23) Li, S. Z.; Wang, D. M.; Jiao, S. M. In Pesticide Experiment Methods-Fungicide Sector, Li, S. Z., Ed., Agriculture Press of China: Beijing, China, 1991, pp 93–94. (24) Wang, K. L.; Lü, M. Y.; Wang, Q. M.; Huang, R. Q. Iron(III) chloride-based mild synthesis

of

phenanthrene

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application

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to

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phenanthroindolizidine alkaloids. Tetrahedron, 2008, 64, 7504–7510. (25) Yamashita, S.; Kurono, N.; Senboku, H.; Tokuda, M.; Orito, K. Synthesis of

3

phenanthro[9,10-b]indolizidin-9-ones,

phenanthro[9,10-b]quinolizidin-9-one,

and

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related benzolactams by Pd(OAc)2-catalyzed direct aromatic carbonylation. Eur. J.

5

Org. Chem., 2009, 8, 1173–1180.

6

(26) Buckley III, T. F.; Rapoport, H. Amino acids as chiral educts for asymmetric products.

7

Chirally specific syntheses of tylophorine and cryptopleurine. J. Org. Chem. 1983, 48,

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4222–4232.

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(27) Rozwadowska, M. D.; Tomczak, A. The synthesis of (4S, 5S)-(–)-isocytoxazone. Tetrahedron: Asymmetry, 2009, 20, 2048–2051.

11 12 13 14 15 16 17 18 19 20 21 22

35



1 2

Figure Captions

3

Figure 1.

4

Figure 2. Chemical Structures of Antofine Derivatives 1–41.

Chemical Structures of Ribavirin, Antofine, DHT and DADHT.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

36


Page 36 of 44

Page 37 of 44


1 2

Table 1. Antiviral Activity of Compounds 1–41 against TMV.a Compd. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Conc. (µg/mL)

In vitro inhibition rate (%)

500 100 500 100 500 100 500 500 500 100 500 500 100 500 100 500 100 500 500 500 100 500 100 500 100 500 500 500 100 500 100 500 100 500 100 500 100

43.0±1.8 22.3±2.0 43.6±1.6 18.1±1.3 32.6±1.4 0 9.8±1.5 12.7±2.0 51.3±1.2 20.0±2.1 35.4±1.3 24.3±1.7 0 40.6±1.5 25.0±2.1 57.6±1.1 19.5±1.6 27.7±2.3 31.1±1.8 30.0±1.3 0 40.7±1.1 12.5±1.2 42.2±1.9 0 29.6±1.6 27.8±3.0 55.3±2.3 23.9±1.1 50.0±1.2 27.6±1.7 38.2±1.2 0 32.2±2.1 0 42.5±2.1 19.3±2.2

In vivo Inactivation effect (%)

Curative effect (%)

Protection effect (%)

47.8±2.3 9.7±3.4 37.5±2.4 20.1±2.3 35.4±2.2 5.9±2.3 0 15.7±3.1 42.2±1.6 26.4±2.3 18.9±2.8 29.5±1.9 0 49.3±3.6 29.6±3.0 52.6±1.3 29.8±1.2 18.3±1.9 21.4±1.1 32.4±1.2 0 38.4±1.5 10.6±1.5 39.5±1.2 10.0±1.6 28.5±1.3 30.0±4.1 45.8±3.1 20.5±2.3 45.8±1.4 21.4±1.6 43.2±2.1 17.3±1.3 30.3±2.6 0 44.3±2.0 18.9±1.7

46.9±1.5 14.2±2.1 40.3±3.5 16.5±2.1 38.7±1.8 10.3±2.2 10.4±3.1 26.3±4.0 43.4±1.3 21.5±2.5 32.0±2.3 36.7±3.2 0 45.8±3.2 20.0±1.7 49.4±1.7 20.2±1.6 23.6±2.8 28.5±1.2 34.1±1.4 0 38.3±1.8 15.4±1.3 33.5±1.2 7.9±1.2 34.6±1.3 32.2±3.2 50.9±2.1 26.5±1.8 46.9±1.2 23.1±1.6 40.0±2.4 10.6±1.9 40.9±2.3 12.7±1.6 45.8±2.3 18.4±2.1

42.6±3.1 18.3±2.7 39.2±2.1 15.8±3.0 28.9±1.7 0 15.2±3.3 23.4±3.8 50.8±2.0 18.2±1.9 28.2±2.7 34.1±2.0 0 44.5±2.5 17.6±1.2 55.3±1.4 27.1±1.7 17.4±2.1 26.9±1.9 31.8±1.5 0 30.2±1.8 0 37.6±1.7 12.4±1.4 31.8±1.8 23.6±4.7 46.3±1.9 21.7±1.5 46.2±2.1 24.7±2.0 38.9±1.5 11.1±1.4 37.1±2.5 8.2±1.5 52.1±1.3 22.7±2.1

37



23

500

0

28.5±1.9

17.7±1.9

22.7±2.1

24

500 500 100 500 100 500 500 100 500 500 100 500 500 500 100 500 500 100 500 500 500 100 500 500 100 500 500 100 500 100

30.0±2.4 38.6±3.1 17.2±1.3 32.7±2.1 0 27.5±3.4 36.1±2.3 10.9±3.1 30.6±1.3 38.3±2.1 18.2±2.5 28.8±2.1 33.3±3.2 47.3±3.1 12.8±1.8 30.0±2.1 39.8±1.3 19.8±2.5 25.1±3.1 15.9±1.4 47.8±2.1 22.5±2.7 20.7±1.3 50.1±3.0 23.7±3.1 30.0±1.4 52.8±1.3 27.4±1.5 40.4±2.0 12.5±2.1

24.6±2.4 40.3±3.2 12.2±1.9 36.3±3.1 0 20.0±2.1 30.0±2.8 0 28.3±3.2 39.5±2.0 11.5±2.6 30.5±2.0 21.5±2.6 44.7±2.3 23.2±3.2 27.2±1.4 37.4±1.5 16.2±1.6 35.2±1.7 23.4±3.2 45.6±2.6 18.6±2.1 31.5±4.2 41.9±2.8 18.3±2.7 22.2±1.6 42.7±1.8 15.3±1.8 34.7±1.8 14.4±2.5

22.3±3.2 32.0±1.7 8.3±1.5 28.6±2.5 0 26.7±2.7 27.5±2.6 0 25.9±1.7 32.9±1.8 12.4±2.3 24.1±2.0 27.4±2.1 37.6±2.6 14.3±1.7 20.9±2.6 33.2±2.1 10.3±2.4 29.4±2.3 23.6±2.8 41.7±2.2 12.0±1.7 24.2±2.1 44.1±2.3 13.2±2.3 20.9±1.8 46.0±2.1 19.4±1.8 35.9±1.2 10.5±3.1

28.9±2.6 37.5±2.3 10.6±1.2 30.5±2.1 5.4±1.4 23.6±2.3 34.1±2.8 11.8±1.9 21.7±1.6 34.2±1.7 15.8±2.7 28.2±2.4 31.3±2.4 42.5±4.2 19.5±2.1 27.4±3.2 36.0±2.4 17.1±1.7 27.9±2.7 18.8±1.6 49.3±1.7 20.6±1.2 27.0±2.5 44.9±2.2 17.0±1.3 28.5±1.6 45.5±1.5 20.4±1.5 38.2±1.9 15.2±2.7

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Antofine Ribavirin 1

a

Page 38 of 44

All results are expressed as mean ± SD.

2 3 4 5 6 7 8 9 10 11 12 38


Page 39 of 44


1 2 3

4 5

Figure 1. Chemical Structures of Ribavirin, Antofine, DHT and DADHT.

6

39



OMe

OMe

MeO

O

H N

H N

O

MeO OMe

OMe

OMe

2

3

O N MeO

OMe

OMe

6

7

OMe

OMe 10

O N

OH

OMe

OMe

OMe

O

OMe

N

OMe

N

O

OMe

N MeO

OMe 19

OMe 20

OMe 21 OMe

OMe

MeO

MeO

MeO

OH N H CH3

OH

O N 2

OMe

OMe 22 OMe

MeO

OMe

23

24

25 OMe

OMe MeO

N(CH2)5CH2OH 2

N(CH2)4CO2CH3 2

MeO

2

MeO

2

2

MeO

MeO OMe 38

N(CH2)5CO2CH3

N(CH2)5CO2CH3

2

MeO OMe 37

MeO

MeO

2

MeO OMe 36 OMe

OMe 35 OMe

34

NCH2CH2OH

NCH2CO2H

2

MeO OMe

MeO

MeO NCH2CO2CH3

N(CH2)2CH2OH

2

MeO OMe 33 OMe

OMe 32 OMe

OMe 31 OMe

N(CH2)2CO2H

N(CH2)2CO2CH3

MeO

MeO

MeO

OMe 30 OMe MeO

2

2

N(CH2)3CO2CH3

2

MeO OMe 29 OMe

MeO

N(CH2)3CH2OH

N(CH2)3CO2H

2

N(CH2)4CH2OH 2

MeO

MeO

MeO

MeO

N(CH2)4CO2H 2

OMe 28 OMe

OMe 27 OMe

MeO

OMe MeO

MeO

MeO OMe 26 OMe

MeO OMe

MeO

MeO

N(CH2)5CO2H

2

OMe

OMe

MeO

N(CH2)5CO2CH3

2

OH

MeO

OMe

MeO

OMe 39

MeO N(CH2)2N(CH3)2

2

MeO

1

O

MeO

OMe

OMe

OMe

O N

O

O

MeO

MeO

OMe O

O N

O

OMe 17 OMe

OMe

MeO

OMe

O

MeO OMe 16 OMe

O

OMe

O N

O

MeO

MeO

NO2

O

OMe 15 OMe

14 OMe

MeO

CF3

O

MeO

MeO

OMe 13

MeO

F

O

MeO OMe 12

MeO

O

N

OH

MeO OMe 11

OMe 18

MeO

O N MeO

MeO

OMe

MeO

O

N

9

OMe

O

N

OMe

8

MeO

O

MeO OMe

OMe

MeO

N

OH

OH

MeO

MeO

MeO

MeO

O N

MeO

5 OMe

MeO

O

N

OMe

4

O

MeO

OH

OMe

OMe

MeO

NH

MeO

OMe

MeO

N

OH

OH CH3 OH

NH MeO

1 OMe

N

MeO

OH

MeO

MeO

OMe

MeO

MeO

N MeO

OMe

OMe

MeO

Page 40 of 44

OMe 41

40


40

Page 41 of 44


1 2 3

Figure 2. Chemical Structures of Antofine Derivatives 1–41.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 41



R1 MeO

R1

R1

MeO

O OH

MeO

LiAlH4

OH

(i-Pr)2NEt DMF 90 °C

0 °C to rt

MeO

R2 1 42: R = R2 = CH3O 43: R1 = H, R2 = CH3O 44: RI = CH3O, R2 = H

Me2NH, or Br H2N(CH2)nCO2CH3•HCl

PBr3 CH2Cl2

THF 0 °C to reflux

MeO

Page 42 of 44

MeO

R2 1 45: R = R2 = CH3O 46: R1 = H, R2 = CH3O 47: RI = CH3O, R2 = H

R2 48: R1 = R2 = CH3O 49: R1 = H, R2 = CH3O 50: RI = CH3O, R2 = H R1 MeO N(CH2)nCO2H

4M NaOH

R1

OMe

dioxane reflux MeO

MeO

MeO N

N(CH2)nCO2CH3

2

or

2

25, 28, 31, 34, 37 R2 R1

MeO MeO

MeO OMe 1

LiAlH4

R2 24, 27, 30, 33, 36, 39, 40

26, 29, 32, 35, 38 R2

1 2

N(CH2)nCH2OH

2

THF 0 °C to rt MeO

Scheme 1. Synthesis of Compounds 1, 24–40.

3

42


Page 43 of 44


OMe

OMe

MeO

MeO H2N(CH2)5CO2CH3•HCl

PCC OH MeO

O CH2Cl2 rt

MeO

OMe 45

OMe 51

OMe

Et3N/AcOH (5d) CH2Cl2, reflux, then NaBH3CN/AcOH (5d) MeOH, rt

OMe

MeO

OMe

MeO

MeO R

RCH2Br or RCH2I NH(CH2)5CO2CH3 MeO

N(CH2)5CO2CH3 THF

NaH or Et3N DMF 0 °C to rt MeO

OMe 2

0 °C

OMe 6, 9, 10, 13-21

0 °C

OMe

4M NaOH dioxane reflux

OMe MeO

R N(CH2)5CO2H

NH(CH2)5CH2OH MeO

MeO

1 2

OMe

OMe 3

7, 11

Scheme 2. Synthesis of Compounds 2, 3, 6–21.

3

4 5

Scheme 3. Synthesis of Compounds 4, 5, 22, 23.

6 OMe

OMe

MeO

MeO O

H2N(CH2)2N(CH3)2 2

MeO OMe

7 8

Et3N/AcOH (5d) CH2Cl2, reflux, then NaBH3CN/AcOH (5d) MeOH, rt

N(CH2)2N(CH3)2

MeO

51

N(CH2)5CH2OH MeO OMe 8, 12

LiAlH4 THF MeO

R

LiAlH4

OMe 41

Scheme 4. Synthesis of Compound 41. 43



1 2 3

TOC graphic

4 5 6 7 8

“D”, “E” rings may not be indispensable for antofine: Discovery of phenanthrene and alkylamine chain containing antofine derivatives as novel antiviral agent against tobacco mosaic virus (TMV) based on interaction of antofine and TMV RNA Ziwen Wang, Peng Wei, Yuxiu Liu and Qingmin Wang*

9

Agrochemical Bioregulators

10

11 12

44


Page 44 of 44

D and E Rings May Not Be Indispensable for ... - ACS Publications

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