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Synthesis of Novel Apocynin

Dimer Derivatives

INTRODUCTION

Apocynin (4-hydroxyl-3-methoxyacetophenone, compound 1) was origi-nally isolated from the roots of Apocynum cannabinum L., and also found in Picrorhiza kurroa Royle ex Benth1. Apocynin is an efficient NADPH oxidase inhibitor2,3 with the function to decrease ROS production4 and was investi-gated as an anti-oxidative agent in treatment of different kinds of diseases5–7. It was also reported that apocynin could effectively reverse inflammatory injury5,8. The results indicated that apocynin had the potential to be used in the treatment of rheumatoid arthritis8, osteoarthritis9, asthma5, stroke10 and diabetes neuropathy11.

Apocynin was supposed to be activated by myeloperoxidase in phagocytes to form its active dimer, diapocynin, which inhibits NADPH oxidase complex assembly and activation12. Although apocynin is widely studied, there is only limited information about diapocynin, including structure modification. In this report, we prepared a diapocynin analogue

JJA-D0 (Fig. 1), which had more functional groups than diapocynin for structural modification. A series of derivatives of JJA-D0 were designed and synthesized for attaining lead compounds of better NADPH oxidase inhibition activity.

MATERIALS AND METHODS

Materials

All reagents were obtained from commercial suppliers as follows. Propionyl chloride, isobutyryl chloride, iodomethane, bromoethane, 2-bromopro-pane, 1-bromopro2-bromopro-pane, methylsulfonyl chloride, ethylsulfonyl chloride, tert-Butyldimethylsilyl chloride (TBDMS-Cl), tetrabutylammonium fluoride (TBAF), sodium cyanoborohydride, Di-tert-butyl dicarbonate ((Boc)2O) and trifluoroacetic acid (TFA) were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (Shanghai, China). Apocynin was purchased from Beijing J&K Scientific Co., LTD (Beijing, China). Solvents were dried according to standard procedures.

General separation and structure identification methods

Thin layer chromatography (TLC) analysis was performed on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Group Corp., Qingdao, China). Column chromatography was performed on Silica gel (200–300 mesh, Qingdao Haiyang Chemical Group Corp, Qingdao, China) column.

Jianbin Qi1, Yang Ou1, Mengying Guo1, Lingchao Zhu1, Sha Li1,2*, Jie Jiang1,3*

1 College of Pharmacy, Jinan University, Guangzhou 510632, China

2 Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Guangzhou 510632, China

3 Dongguan Institute of Jinan University, Dongguan 523808, China

n Address reprint requests to: *Dr Sha Li, College of Pharmacy, Jinan University, Guangzhou 510632, China, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Guangzhou 510632, China

E-mail: tlisha@jnu.edu.cn

*Dr Jie Jiang, College of Pharmacy, Jinan University, Guangzhou 510632, China, Dongguan Institute of Jinan Univer-sity, Dongguan 523808, China E-mail: jiejiang@jnu.edu.cn

n Article citation: Qi J, Ou Y, Guo M, Zhu L, Li S, Jiang J. Synthesis of novel apocynin dimer derivatives. J Pharm Biomed Sci 2017;07(9):330–337. Available at www.jpbms.info Statement of originality of work: The manuscript has been read and approved by all the authors, the requirements for authorship have been met, and that each author believes that the manuscript represents honest and original work.

Source of funding: This research was supported by project of National Natural Science Foundation of China (81441128) and Science and Technology Program of Guangzhou, China (201704020198).

Competing interest / Conflict of interest: The author(s) have no competing interests for financial support, publication of this research, patents, and royalties through this collaborative research. All authors were equally involved in discussed research work. There is no financial conflict with the subject matter discussed in the manuscript.

Disclaimer: Any views expressed in this paper are those of the authors and do not reflect the official policy or position of the Department of Defense.

NLM Title J Pharm Biomed Sci CODEN JPBSCT

2230-7885 ISSN No

DOI https://doi.org/10.20936/jpbms/170905

RESEARCH ARTICLE

ABSTRACT

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has been proved to be a target of anti-oxidation and anti-inflammation therapy. Apocynin dimer (diapocynin), converted from apocynin by peroxidase-mediated oxidation in vivo, was reported to confer stronger inhibition to NADPH oxidase than apocynin. However, there was limited information about the structural modification of diapocynin. In order to improve the bioac-tivity of dimeric apocynin, JJA-D0, an apocynin dimer analogue of more functional groups than diapocynin for structural modification, was synthesized in our lab. 14 apocynin dimer derivatives were designed and synthesized on the basis of chemical structure of JJA-D0, attempting to attain more efficient lead compounds for further development.

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J Pharm Biomed Sci JPBSCT

2230-7885

https://doi.org/10.20936/jpbms/170905

Melting points were measured using a Mel-Temp (X7L20, Beijing), and are uncorrected. NMR spectra were recorded at ambient temperature on a 300 MHz spectrometer (AV-400, Bruker) in CDCl3 or DMSO-d6. Electrospray ionization mass spectra (ESI-MS) were obtained in the positive ion detection mode on a Finnigan LCQ Advantage MAX mass spectrometer (Applied Biosystems, 4000 Q TRAP). HRMS was per-formed on a G2 Q-TOF mass spectrometer (Waters).

General process of synthesis of apocynin dimer

derivatives

The general structure of 14 novel JJA-D0 derivatives designed and synthesized in this work is shown in Fig. 1. Compound 2 was prepared by the Reimer-Tiemann reac-tion from apocynin (compound 1). Compound 4 was pre-pared by nitration of compound 1 followed by reduction of the nitro group (Scheme 1). Compound 2 and com-pound 4 made up motif 1 and 2 of JJA-D0, respectively, by producing Schiff base first and then reduced to give JJA-D0. The phenolic hydroxyls of JJA-D0 were protected by tert-butyldimethylsilyl (TBDMS) to obtain compound 6, and then the secondary amidogroup of JJA-D0 was acylated followed by TBDMS removal with tetrabutylam-monium fluoride (TBAF) to get compounds JJA-D12 and

JJA-D13 (Scheme 2).

The phenolic hydroxyl of compound 2 was sub-stituted by different haloalkanes and sulfonyl chlorides,

and the substituted compounds reacted with com-pound 4 to produce Schiff base, which was reduced to yield compounds JJA-D14, JJA-D15, JJA-D16,

JJA-D17, JJA-D25 and JJA-D26 (Scheme 3) using sodium cyanoborohydride. In order to selectively modify the phenolic hydroxyl of compound 4, its amino group was protected by di-tert-butyl dicar-bonate ((Boc)2O) in dichloromethane (DCM) to get compound 7. Then, the phenolic hydroxyl of com-pound 7 was substituted by different haloalkanes and sulfonyl chlorides, followed by removal of Boc with trifluoroacetic acid (TFA) in DCM to produce differ-ent substitutes of compound 4. The different substi-tutes reacted with compound 2 to afford compounds

JJA-D18, JJA-D19, JJA-D20, JJA-D21, JJA-D27 and

JJA-D28 (Scheme 4).

RESULTS AND DISCUSSION

According to the schemes mentioned above, 14 new apocynin dimer derivatives were yielded. The synthesis procedure of each compound and related intermediates were described as follows in detail as well as the spectral data for structure identification.

5-acetyl-2-hydroxy-3-methoxybenzaldehyde (2)

Compound 2 was prepared according to the procedure reported by Lu X et al.13

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Scheme 1. Reagents and conditions: (i) 50% NaOH aqua, ethanol, CHCl3, reflux, HCl; (ii) 65% HNO3, acetic acid, ice-bath to r.t.; (iii) H2, 10% Pd/C, ethanol.

Scheme 2. Reagents and conditions: (i) methanol; (ii) NaBH3CN, glacial acetic acid, r.t.; (iii) imidazole, tertButyldimethylsilyl chloride, DCM, r.t.; (iv) different acyl chloride, DCM, Et3N, N2 protection, ice-bath, 2 h; (v) TBAF, DCM, r.t. 2 h.

Scheme 3. Reagents and conditions: (i) different halohydrocarbon or sulfonyl chloride, K2CO3 or Et3N,DMF, 40°C or ice-bath, 3 h; (ii) methanol, r.t.; (iii) NaBH3CN, glacial acetic acid, r.t.

1-(3-amino-4-hydroxy-5-methoxyphenyl)

ethanone (4)

Compound 4 was prepared according to the procedure reported by Lu X et al.13

1-(3-(5-Acetyl-2-hydroxy-3-methoxybenzylamino)

-4-hydroxy-5-methoxyphenyl) ethanone (JJA-D0)

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1-(3-((5-acetyl-2-((tert-butyldimethylsilyl)

oxy)-3-methoxybenzyl)amino)-4-((tert-butyldimethylsilyl)oxy)-5-methoxyphenyl)

ethanone (6)

Compound JJA-D0 (3.59 g, 10 mmol), dissolved in anhydrous dichloromethane (DCM), was added imida-zole (4.10 g, 60 mmol), tert-Butyldimethylsilyl chloride (6.0 g, 40 mmol) successively with stirring for 5 h. After the reaction, the reaction mixture was washed with water, and purified by column chromatography, eluting with ethyl acetate/petroleum ether (1:5, v/v), to get purified compound 6. White solid. 1H-NMR (300 MHz, DMSO-d6) d 7.60 (s, 1H), 7.44 (s, 1H), 6.96 (s, 1H), 6.82 (s, 1H), 4.90 (s, 1H), 4.38 (d, J = 5.6 Hz, 2H), 3.87 (d, J = 5.7 Hz, 3H), 3.80 (s, 3H), 2.46 (s, 3H), 2.45 (s, 3H), 0.95 (s, 9H), 0.92 (s, 9H), 0.23 (d, J = 6.0 Hz, 6H), 0.16 (s, 6H). MS (ESI): m/z [M+H]+ 588.4.

N-(5-acetyl-2-hydroxy-3-methoxybenzyl)-N-(5-acetyl-2-hydroxy-3-methoxyphenyl)

propionamide (JJA-D12)

Compound 6 (5.87 g, 10 mmol), dissolved in anhydrous DCM, was added Et3N (2.02 g, 20 mmol) and propionyl chloride (1.85 g, 20 mmol) with N2 protection in ice bath for 2 h. After the reaction, the ice bath was removed, and the reaction mixture was washed with water and extracted with DCM. The organic layer was added TBAF dropwise with stirring. After 2 h, the reaction mixture was washed with water and the product was purified by column chromato-graphy, eluting with DCM/methanol (100:1, v/v) to afford purified compound JJA-D12.White solid (2.37 g, 57% yield). m.p. 184.2–185.5°C. 1H NMR (300 MHz, DMSO-d6) d 10.29 (s, 1H), 9.74 (s, 1H), 7.48 (d, J = 1.8 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 1.8 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 5.08 (d, J = 15.2 Hz, 1H), 4.45 (d, J =

15.3 Hz, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 2.45 (s, 3H), 2.39 (s, 3H), 2.03 (dt, J = 15.9, 7.0 Hz, 2H), 0.95 (dd, J = 9.6, 5.2 Hz, 3H). 13C NMR (75 MHz, CDCl

3) d 196.55, 195.70, 176.93, 150.65, 148.72, 147.70, 146.35, 129.62, 128.66, 127.03, 125.23, 123.47, 122.32, 110.99, 109.81, 56.55, 56.16, 49.03, 26.96, 26.09, 9.21, 1.03. MS (ESI): m/z [M+H]+ 416.4. ESI-HRMS m/z: 416.1703 [M+H]+, calcd for C22H25NO7 416.1704.

N-(5-acetyl-2-hydroxy-3-methoxybenzyl)-N-(5-acetyl-2-hydroxy-3-methoxyphenyl)

isobutyramide (JJA-D13)

Compound JJA-D13 was prepared from Compound

6 (5.87 g, 10 mmol) and isobutyryl chloride (2.12 g, 20 mmol) according to the procedure of JJA-D12. White solid (1.97 g, 46% yield). m.p. 187.3–188.9°C. 1H NMR (300 MHz, DMSO-d6) d 10.34 (s, 1H), 9.79 (s, 1H), 7.44 (d, J = 1.8 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 1.9 Hz, 1H), 7.26 (d, J = 1.9 Hz, 1H), 5.07 (d, J = 15.4 Hz, 1H), 4.40 (d, J = 15.3 Hz, 1H), 3.89 (d, J = 3.4 Hz, 3H), 3.82 (s, 3H), 2.73 (s, 1H), 2.45 (s, 3H), 2.40 (s, 3H), 1.15 – 0.90 (m, 6H). 13C NMR (75 MHz, CDCl

3)

d 196.56, 195.71, 180.60, 150.77, 148.72, 147.65, 146.36, 129.51, 128.57, 127.05, 125.26, 123.39, 122.21, 110.98, 109.85, 56.54, 56.15, 49.15, 31.69, 26.10, 19.85, 19.3. MS (ESI): m/z [M-H]+ 428.4. ESI-HRMS m/z: 430.1861 [M+H]+, calcd for C

23H27NO7 430.1860.

5-acetyl-2,3-dimethoxybenzaldehyde (14a)

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anhydrous sodium sulfate. Solvent was removed in vacuo. The product was purified by column chromatog-raphy, eluting with ethyl acetate/petroleum ether (1:8, v/v), to afford compound 14a. White solid. 1H-NMR (300 MHz, DMSO-d6) d 10.33 (s, 1H), 7.88 (d, J = 1.9 Hz, 1H), 7.79 (d, J = 1.8 Hz, 1H), 4.00 (s, 3H), 3.96 (s, 3H), 2.62 (s, 3H). MS (ESI): m/z [M+H]+ 209.4.

1-(3-((5-acetyl-2,3-dimethoxybenzyl)amino)-4-hydroxy-5-methoxyphenyl)ethanone (JJA-D14)

Compound 14a (2.08 g, 10 mmol) was dissolved in methanol and added compound 4 (1.99 g, 11 mmol) with stirring for 1 h. After precipitate appeared com-pletely, the mixture was filtered to obtain the precip-itate. Then the precipitate was dissolved in the ethyl acetate, appropriate amount of sodium cyanoborohy-dride and glacial acetic acid were added, reacting for 2 h. The reactant was purified by column chromatogra-phy, eluting with ethyl acetate/petroleum ether (1:4, v/v), to afford purified compound JJA-D14. Yellow solid (2.42 g, 65% yield). m.p. 182.2–183.9°C. 1H NMR (300 MHz, DMSO-d6) d 10.33 (s, 1H), 7.61 (d, J = 1.9 Hz, 1H), 7.44 (d, J = 1.9 Hz, 1H), 6.93 (d, J = 1.8 Hz, 1H), 6.83 (d, J = 1.8 Hz, 1H), 5.52 (s, 1H), 4.39 (s, 2H), 3.89 (s, 6H), 3.82 (s, 3H), 2.49 (s, 3H), 2.41 (s, 3H). 13C NMR (75 MHz, CDCl

3) d 197.42, 197.25, 152.60, 151.51, 145.73, 137.28, 135.57, 132.84, 132.26, 129.49, 122.69, 110.88, 105.70, 101.64, 61.00, 56.25, 55.93, 43.21, 26.44, 26.31. MS (ESI) m/z [M-H]+ 372.3. ESI-HRMS m/z: 374.1601 [M+H]+, calcd for C

20H23NO6 374.1598.

1-(3-((5-acetyl-2-ethoxy-3-methoxybenzyl)

amino)-4-hydroxy-5-methoxyphenyl) ethanone

(JJA-D15)

Compound JJA-D15 was prepared from compound 2

(1.94 g, 10 mmol) and bromoethane (2.16 g, 20 mmol) according to the procedure of JJA-D14. Yellow solid (2.39 g, 62% yield). m.p. 184.2–185.4°C. 1H NMR (300 MHz, DMSO-d6 ) d 9.45 (s, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 1.9 Hz, 1H), 6.93 (d, J = 1.8 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H), 5.53 (s, 1H), 4.40 (s, 2H), 4.15 (d, J = 7.0 Hz, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 2.49 (s, 3H), 2.41 (s, 3H), 1.34 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) d 197.24, 197.00, 152.51, 150.20, 146.70, 138.03, 137.53, 134.03, 132.68, 128.79, 121.79, 111.34, 104.91, 102.34, 68.90, 56.38, 56.30, 41.95, 26.91, 26.66, 16.10. MS (ESI) m/z [M-H]+ 386.3. ESI-HRMS m/z: 388.1760 [M+H]+, calcd for C

21H25NO6 388.1755.

1-(3-(((5-acetyl-2-hydroxy-3-methoxyphenyl)

amino)methyl)-4-isopropoxy-5-methoxyphenyl)

ethanone (JJA-D16)

Compound JJA-D16 was prepared from compound

2 (1.94 g, 10 mmol) and 2-bromopropane (2.44 g,

20 mmol) according to the procedure of JJA-D14. Yellow solid (2.12g, 53% yield). m.p. 185.2–187.9°C. 1H NMR (300 MHz, DMSO-d6) d 9.46 (s, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 6.99 (d, J = 2.0 Hz, 2H), 6.79 (d, J = 1.8 Hz, 1H), 5.60 (s, 1H), 4.70 (dt, J = 12.2, 6.1 Hz, 2H), 4.38 (s, 3H), 3.83 (m, 3H), 2.48 (m, 6H), 1.28 (d, J = 6.1 Hz, 6H). 13C NMR (75 MHz, CDCl

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d 197.47, 197.28, 152.58, 149.35, 145.69, 137.09, 132.89, 132.43, 129.57, 122.47, 110.47, 106.35, 101.27, 75.11, 56.21, 55.85, 43.31, 26.32, 22.71. MS (ESI) m/z [M+H]+ 402.1, [M+Na]+ 424.4. ESI-HRMS m/z: 402.1909 [M+H]+, calcd for C

22H27NO6 402.1911.

1-(3-(((5-acetyl-2-hydroxy-3-methoxyphenyl)

amino)methyl)-5-methoxy-4-propoxyphenyl)

ethanone (JJA-D17)

Compound JJA-D17 was prepared from compound

2 (1.94 g, 10 mmol) and 1-bromopropane (2.44 g, 20 mmol) according to the procedure of JJA-D14. Yellow solid (2.72 g, 68% yield). m.p. 185.5–187.3°C. 1H NMR (300 MHz, DMSO-d

6) d 9.46 (s, 1H), 7.61 (d, J = 1.7 Hz, 1H), 7.44 (d, J = 1.8 Hz, 1H), 6.93 (d, J = 1.6 Hz, 1H), 6.83 (s, 1H), 5.54 (s, 1H), 4.40 (s, 2H), 4.04 (t, J = 6.6 Hz, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 2.49 (s, 3H), 2.41 (s, 3H), 1.77 (d, J = 7.2 Hz, 2H), 1.01 (s, 3H). 13C NMR (75 MHz, DMSO-d

6) d 197.23, 196.97, 152.46, 150.40, 146.67, 138.05, 137.52, 133.87, 132.64, 128.77, 121.78, 111.40, 104.85, 102.37, 74.76, 56.36, 56.29, 41.89, 26.90, 26.63, 23.67, 10.90. MS (ESI) m/z [M-H]+ 400.5. ESI-HRMS m/z: 402.1912 [M+H]+, calcd for C22H27NO6 402.1911.

tert-Butyl

(5-acetyl-2-hydroxy-3-methoxyphenyl)carbamate (7)

Compound 4 (1.81 g, 10 mmol), dissolved in anhydrous DCM, was added (Boc)2O (4.36 g, 20 mmol) and Et3N (1.01 g, 10 mmol) and stirred overnight. The reaction mixture was washed with water and extracted with DCM. The product was purified by column chromatography, eluting with ethyl acetate/petroleum ether (1:8, v/v) to afford compound 7. White solid. 1H NMR (300 MHz, DMSO-d6) d 10.02 (s, 1H), 8.06 (d, J = 1.1 Hz, 1H), 7.89 (s, 1H), 7.26 (d, J = 1.9 Hz, 1H), 3.87 (s, 3H), 2.50 (s, 3H), 1.47 (s, 9H). MS (ESI) m/z [M-H]+ 280.5.

1-(3-amino-4,5-dimethoxyphenyl)ethanone (18c)

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DCM/methanol (100:1, v/v) to afford compound 18c. White solid. 1H NMR (300 MHz, DMSO-d

6) d 6.99 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 5.13 (s, 2H), 3.80 (s, 3H), 3.69 (s, 3H), 2.47 (s, 3H). MS (ESI) m/z [M+H]+ 196.3.

1-(3-(((5-acetyl-2,3-dimethoxyphenyl)amino)

methyl)-4-hydroxy-5-methoxyphenyl) ethanone

(JJA-D18)

Compound 2 (1.94 g, 10 mmol), dissolved in meth-anol, was added compound 18c (2.15 g, 11 mmol) with stirring for 1 h. The precipitate was collected and dissolved in ethyl acetate, and then added appropriate amount of sodium cyanoborohydride and glacial acetic acid. After 2 h reaction, the reaction mixture was washed with water and the solvent was removed in vacuo. Then the mixture was purified by column chromatography, eluting with ethyl acetate/petroleum ether (1:4, v/v), to afford purified compound JJA-D18. Yellow solid (2.05 g, 55% yield). m.p. 181.2–182.8°C. 1H NMR (300 MHz, DMSO-d6) d 9.87 (s, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.37 (d, J = 1.9 Hz, 1H), 6.85 (s, 2H), 5.95 (s, 1H), 4.36 (d, J = 6.7 Hz, 2H), 3.87 (s, 3H), 3.81 (s, 3H), 3.75 (s, 3H), 2.44 (d, J = 2.9 Hz, 6H). 13C NMR (75 MHz, CDCl3) d 197.75, 196.89, 152.05, 148.64, 146.72, 141.59, 140.14, 134.08, 129.44, 124.01, 123.54, 108.98, 105.70, 102.26, 60.14, 56.22, 55.92, 43.00, 26.44, 26.17. MS (ESI) m/z [M+H]+ 374.6, [M+Na]+ 396.5. ESI-HRMS m/z: 374.1599 [M+H]+, calcd for C20H23NO6 374.1598.

1-(3-(((5-acetyl-2-ethoxy-3-methoxyphenyl)

amino)methyl)-4-hydroxy-5-methoxyphenyl)

ethanone (JJA-D19)

Compound JJA-D19 was prepared from compound

7 (2.81 g, 10 mmol) and bromoethane (2.16 g, 20 mmol) according to the procedure of JJA-D18. Yellow solid (1.70 g, 44% yield). m.p. 182.4–184.5°C. 1H NMR (300 MHz, DMSO-d

6) d 9.89 (s, 1H), 7.59 (d, J = 1.8 Hz, 1H), 7.37 (d, J = 1.9 Hz, 1H), 6.85 (d, J = 2.4 Hz, 2H), 5.85 (s, 1H), 4.37 (d, J = 4.0 Hz, 2H), 3.99 (d, J = 7.0 Hz, 2H), 3.87 (s, 3H), 3.80 (s, 3H), 2.45 (s, 3H), 2.43 (s, 3H), 1.31 (s, 3H). 13C NMR (75 MHz, DMSO-d6) d 197.70, 196.55, 152.32, 149.55, 147.46, 142.54, 138.16, 133.07, 128.45, 126.31, 122.80, 110.10, 104.89, 101.60, 67.99, 56.40, 56.12, 41.52, 26.97, 26.56, 15.95. MS (ESI) m/z [M-H]+ 386.1. ESI-HRMS m/z: 388.1756 [M+H]+, calcd for C

21H25NO6 388.1755.

1-(3-((5-acetyl-2-hydroxy-3-methoxybenzyl)

amino)-4-isopropoxy-5-methoxyphenyl)

ethanone (JJA-D20)

Compound JJA-D20 was prepared from compound 7

(2.81 g, 10 mmol) and 2-bromopropane (2.46 g,

20 mmol) according to the procedure of JJA-D18. Yellow solid (1.76 g, 44% yield). m.p. 186.2–187.5°C. 1H NMR (300 MHz, DMSO-d

6) d 9.88 (s, 1H), 7.59 (d, J = 1.7 Hz, 1H), 7.37 (d, J = 1.8 Hz, 1H), 6.93 – 6.79 (m, 2H), 5.73 (t, J = 6.6 Hz, 1H), 4.55 (dt, J = 12.2, 6.0 Hz, 1H), 4.38 (d, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.79 (s, 3H), 2.45 (s, 3H), 2.42 (s, 3H), 1.25 (s, 3H), 1.23 (s, 3H). 13C NMR (75 MHz, CDCl

3) d 197.71, 196.85, 152.08, 148.62, 146.74, 142.36, 137.82, 132.76, 129.39, 124.07, 123.27, 108.85, 105.71, 102.32, 75.00, 56.18, 55.84, 43.10, 26.39, 26.14, 22.69. MS (ESI) m/z [M+H]+ 402.2, [M+Na]+ 424.3. ESI-HRMS m/z: 402.1915 [M+H]+, calcd for C

22H27NO6 402.1911.

1-(3-((5-acetyl-2-hydroxy-3-methoxybenzyl)

amino)-5-methoxy-4-propoxyphenyl) ethanone

(JJA-D21)

Compound JJA-D21 was prepared from compound 7

(2.81 g, 10 mmol) and 1-bromopropane (2.46 g, 20 mmol) according to the procedure of JJA-D18. Yellow solid (2.08 g, 52% yield). m.p. 185.2–187.4°C. 1H NMR (300 MHz, DMSO-d6) d 9.88 (s, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H), 6.87 (s, 2H), 5.71 (s, 1H), 4.39 (d, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.90 (t, J = 5.2 Hz, 2H), 3.80 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.73 (dd, J = 14.4, 7.1 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d

6) d 197.72, 196.57, 152.20, 149.57, 147.47, 142.32, 138.47, 132.99, 128.42, 126.24, 122.87, 110.11, 104.97, 101.75, 74.07, 56.40, 56.15, 41.62, 26.99, 26.56, 23.44, 10.79. MS (ESI) m/z [M+H]+ 402.3, [M+Na]+ 424.5. ESI-HRMS m/z: 402.1908 [M+H]+, calcd for C

22H27NO6 402.1911.

4-acetyl-2-formyl-6-methoxyphenyl

methanesulfonate (25a)

Compound 2 (1.94 g, 10 mmol), dissolved in DMF, was added methylsulfonyl chloride (2.28 g, 20 mmol) and Et3N (2.02 g, 20 mmol) in ice bath. After 3 h reac-tion, the reaction mixture was washed with water and extracted with DCM. Then the mixture was purified by column chromatography, eluting with ethyl acetate/ petroleum ether (1:8, v/v), to afford purified com-pound 25a. White solid, 1H NMR (300 MHz, DMSO-d

6)

d 10.19 (d, J = 5.0 Hz, 1H), 7.97 – 7.92 (m, 2H), 4.01 (s, 3H), 2.68 (m, 3H), 1.49 – 1.44 (m, 3H). MS (ESI) m/z [M+H]+ 273.2.

4-acetyl-2-(((5-acetyl-2-hydroxy-3-

methoxyphenyl)amino)methyl)-6-methoxyphenyl methanesulfonate (JJA-D25)

(7)

cyanoborohydride and glacial acetic acid. After 2 h reac-tion, the reaction mixture was washed with water and the solvent was removed in vacuo. The mixture was purified by column chromatography, eluting with ethyl acetate/ petroleum ether (1:4, v/v), to afford purified compound

JJA-D25. Yellow solid (2.79 g, 64% yield). m.p. 172.4– 173.6°C. 1H NMR (300 MHz, DMSO-d

6) d 9.54 (s, 1H), 7.62 (s, 1H), 7.55 (s, 1H), 6.94 (d, J = 1.0 Hz, 1H), 6.70 (s, 1H), 5.82 (s, 1H), 4.49 (d, J = 4.8 Hz, 2H), 3.96 (s, 3H), 3.83 (s, 3H), 3.59 (s, 3H), 2.53 (s, 3H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO-d

6) d 197.44, 196.91, 152.10, 146.77, 140.30, 138.08, 137.12, 136.41, 136.12, 128.77, 120.51, 111.66, 104.74, 102.35, 56.76, 56.37, 41.98, 27.19, 26.64. MS (ESI) m/z [M+H]+ 438.3. ESI-HRMS m/z: 438.1213 [M+H]+, calcd for C

20H23NO8S 438.1217.

4-acetyl-2-(((5-acetyl-2-hydroxy-3-

methoxyphenyl)amino)methyl)-6-methoxyphenyl ethanesulfonate (JJA-D26)

Compound JJA-D26 was prepared from compound 2

(1.94 g, 10 mmol) and ethylsulfonyl chloride (2.56 g, 20 mmol) according to the procedure of JJA-D25. Yellow solid (3.06 g, 68% yield). m.p. 174.2–176.5°C. 1H-NMR (300 MHz, DMSO-d6) d 9.53 (s, 1H), 7.62 (d, J = 1.9 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 6.93 (d, J = 1.8 Hz, 1H), 6.69 (d, J = 1.7 Hz, 1H), 5.83 (s, 1H), 4.48 (d, J = 5.8 Hz, 2H), 3.94 (s, 3H), 3.82 (d, J = 4.7 Hz, 3H), 3.71 (q, J = 7.3 Hz, 2H), 2.53 (s, 3H), 2.37 (s, 3H), 1.48 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl

3) d 197.72, 197.03, 152.05, 145.74, 140.45, 136.93, 136.17, 135.75, 135.13, 129.69, 121.62, 110.42, 106.66, 101.25, 56.25, 47.76, 43.06, 26.60, 26.36, 8.39. MS (ESI) m/z [M+H]+ 452.3, [M+Na]+ 474.3. ESI-HRMS m/z: 452.1369 [M+H]+, calcd for C

21H25NO8S 452.1374.

4-acetyl-2-amino-6-methoxyphenyl

methanesulfonate (27c)

Compound 7 (2.81 g, 10 mmol), dissolved in anhydrous DMF, was added methylsulfonyl chloride (1.36 g, 12 mmol) and Et3N (2.02 g, 20 mmol) in ice bath. After 2 h reaction, the reaction mixture was washed with water and extracted with DCM, then TFA (5 mL) was added drop-wise. After 1 h reaction, NaHCO3 was used to neutralize the mixture, the organic layer was concentrated to get compound 27c. White solid. 1H NMR (300 MHz, DMSO-d6) d 7.07 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 5.39 (s, 2H), 3.84 (s, 3H), 3.46 (d, J = 26.4 Hz, 3H), 2.52 (m, 3H). MS (ESI) m/z [M+H]+ 260.4.

4-acetyl-2-((5-acetyl-2-hydroxy-3-methoxybenzyl)amino)-6-methoxyphenyl

methanesulfonate (JJA-D27)

Compound 27c (2.80 g, 11 mmol) was dissolved in methanol and was added compound 2 (1.94 g, 10 mmol). After precipitate formed completely, appropriate amount

of sodium cyanoborohydride and glacial acetic acid were added, reacting for 2 h. The reaction mixture was purified by column chromatography, eluting with ethyl acetate/petroleum ether (1:3, v/v), to afford purified compound JJA-D27. Yellow solid (2.57 g, 59% yield). m.p. 179.2–181.4°C. 1H NMR (300 MHz, DMSO-d

6) d 9.95 (s, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 6.90 (s, 2H), 5.95 (s, 1H), 4.41 (t, J = 10.0 Hz, 2H), 3.87 (d, J = 6.8 Hz, 6H), 3.52 (s, 3H), 2.50 (s, 3H), 2.44 (s, 3H). 13C NMR (75 MHz, DMSO-d6) d 197.86, 196.64, 152.79, 149.48, 147.54, 142.63, 136.31, 129.05, 128.57, 125.53, 122.95, 109.78, 105.21, 100.72, 56.48, 56.38, 41.13, 27.19, 26.59. MS (ESI) m/z [M+H]+ 438.3, [M+Na]+ 460.3. ESI-HRMS m/z: 438.1214 [M+H]+, calcd for C20H23NO8S 438.1217.

4-acetyl-2-((5-acetyl-2-hydroxy-3-methoxybenzyl)amino)-6-methoxyphenyl

ethanesulfonate (JJA-D28)

Compound JJA-D28 was prepared from compound 7

(2.81 g, 10 mmol) and ethylsulfonyl chloride (1.53 g, 12 mmol) according to the procedure of JJA-D27. Yellow solid (2.84 g, 63% yield). m.p. 177.2–180.1°C. 1H NMR (300 MHz, DMSO-d

6) d 9.96 (s, 1H), 7.66 (s, 1H), 7.38 (d, J = 1.3 Hz, 1H), 6.91 (s, 2H), 5.85 (t, J = 6.0 Hz, 1H), 4.42 (d, J = 5.9 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.66 (q, J = 7.3 Hz, 2H), 2.50 (s, 3H), 2.44 (s, 3H), 1.42 (dd, J = 15.3, 5.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) d 197.84, 196.63, 152.66, 149.49, 147.55, 142.79, 136.26, 129.01, 128.56, 125.48, 123.02, 109.77, 105.26, 100.75, 56.48, 56.38, 47.66, 41.23, 27.20, 26.58, 8.65. MS (ESI) m/z [M+H]+ 452.2, [M+Na]+ 474.4. ESI-HRMS m/z: 452.1369 [M+H]+, calcd for C

21H25NO8S 452.1374.

A series of apocynin dimer derivatives were prepared according to Schemes 2, 3 and 4. In Scheme 2, the phe-nolic hydroxyl group were protected by tert-Butyldi-methylsilyl (TBDMS) first, then the secondary amido group of JJA-D0 was amidated to give JJA-D12 and JJA-D13, otherwise, the phenolic hydroxyl groups would be esterified simultaneously. In Schemes 3 and 4, the pheno-lic hydroxyl group in motif 1 or motif 2 was substituted, respectively, to produce two groups of dimeric deriva-tives for further investigation of the influence of pheno-lic hydroxyl groups in two motifs on pharmacological efficacy. In Scheme 4, the amino group of compound

4 was protected by ((Boc)2O) before the substitution of phenolic hydroxyl group to avoid the production of impurity. In summary, the designed scheme was simple and feasible, and all of the 14 apocynin dimer derivatives were produced under moderate reaction condition with easy post-processing and satisfactory yield.

CONCLUSION

(8)

alkylation or sulfonylation, and 14 novel apocynin dimer derivatives were attained, respectively. The new compounds will be further investigated for biological activities against oxidation injury and inflammation to seek for promising lead compounds, which can be fur-ther developed as fur-therapeutics for oxidative stress and inflammation related diseases like acute lung injury, asthma, diabetes mellitus, cardiovascular and cerebrova-sular diseases.

REFERENCES

1. Francis S, Laurieri N, Nwokocha C, Delgoda R. Treatment of rats with apocynin has considerable inhibitory effects on arylamine N-acetyltransferase activity in the liver. Sci Rep. 2016;6:26906. 2. Seo JE, Hasan M, Rahaman KA, Kang MJ, Jung BH, Kwon OS.

A leading role for NADPH oxidase in an in-vitro study of experimental autoimmune encephalomyelitis. Mol Immunol. 2016;72:19–27.

3. Sharma M, Kaur T, Singla SK. Role of mitochondria and NADPH oxidase derived reactive oxygen species in hyperox-aluria induced nephrolithiasis: therapeutic intervention with combinatorial therapy of N-acetyl cysteine and apocynin. Mitochondrion. 2016;27:15–24.

4. Lin CC, Yang CC, Cho RL, Wang CY, Hsiao LD, Yang CM. Sphingosine 1-phosphate-induced ICAM-1 expression via NADPH oxidase/ROS-dependent NF-κB cascade on human pulmonary alveolar epithelial cells. Front Pharmacol. 2016;7:80. 5. Kim SY, Moon KA, Jo HY, Jeong S, Seon SH, Jung E, et al. Anti-inflammatory effects of apocynin, an inhibitor of NADPH oxidase, in airway inflammation. Immunol Cell Biol. 2012;90:441–448.

6. Wang Q, Tompkins KD, Simonyi A, Korthuis RJ, Sun AY, Sun GY. Apocynin protects against global cerebral ischemia-reperfusion- induced oxidative stress and injury in the gerbil hippocampus. Brain Res. 2006;1090:182–189.

7. Hamilton CA, Brosnan MJ, Al-Benna S, Berg G, Dominiczak AF. NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels. Hypertension. 2002;40:755–762.

8. Hougee S, Hartog A, Sanders A, Graus YM, Hoijer MA, Garssen J, et al. Oral administration of the NADPH-oxidase inhibitor apocynin partially restores diminished cartilage proteoglycan synthesis and reduces inflammation in mice. Eur J Pharmacol. 2006;531:264–269.

9. Hsu HC, Chang WM, Wu JY, Huang CC, Lu FJ, Chuang YW, et al. Folate deficiency triggered apoptosis of synoviocytes: role of overproduction of reactive oxygen species generated via NADPH oxidase/mitochondrial complex II and calcium pertur-bation. PLos One. 2016;11:e0146440.

10. Tang LL, Ye K, Yang XF, Zheng JS. Apocynin attenuates cerebral infarction after transient focal ischaemia in rats. J Int Med Res. 2007;35:517–522.

11. Cotter MA, Cameron NE. Effect of the NAD(P)H oxidase inhib-itor, apocynin, on peripheral nerve perfusion and function in diabetic rats. Life Sci. 2003;73:1813–1824.

12. Kanegae MP, Condino-Neto A, Pedroza LA, de Almeida AC, Rehder J, da Fonseca LM, et al. Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells. Biochem Biophys Res Commun. 2010; 393:551–554.

Figure

Fig. 1 The novel apocynin dimer derivatives designed and synthesized.

References

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