Synthesis of Nitrate Derivatives of Apocynin Dimer Analogue 57

RESEARCH ARTICLE 56 Jie Jiang ISSN No 2230-7885 Synthesis of Nitrate CODEN JPBSCT NLM Title J Pharm Biomed Sci Derivatives of Apocynin DOI https://doi.org/10.20936/jpbms/08/04/01

Dimer Analogue Gaofang Wang1, 1 ABSTRACT Yang Ou , Zhijian Zhang1, Acute lung injury (ALI) is a mortal disease without satisfactory therapy and poor prognosis. 1,2* It is believed that lung injury relates much to excessive production of reactive oxygen Jie Jiang species (ROS) and a host of pro-inflammatory factors. Anti-oxidative and anti-inflammatory 1College of Pharmacy, Jinan University, agents may be potential for ALI treatment. Factors, such as ROS, TNF-α, and nitric oxide Guangzhou 510632, China (NO) play important role in oxidative stress and inflammation process of ALI. Apocynin, 2Dongguan Institute of Jinan University, a natural phenolic antioxidant, demonstrated activity against oxidative stress and inflam- Dongguan 523808, China mation. Apocynin was reported to turn into dimer or trimer derivatives to exert action. Our previous work showed that apocynin dimer analogue, JJA-D0, reduced ROS level n Address reprint requests to: *Jie Jiang, College of Pharmacy, Jinan and expression of TNF-α and NADPH oxidase. In this work, NO donor of nitrate was con- University, Guangzhou 510632, China; jugated with JJA-D0 by its phenolic groups to synthesize four nitrate derivatives, which Dongguan Institute of Jinan University, were expected to obtain enhanced anti-oxidative and anti-inflammatory activity through Dongguan 523808, China. increasing the action of modulating NO level. The four derivatives, JJA-D1–JJA-D4, were E-mail: [email protected] synthesized under moderate condition with a yield around 10–40% calculated by starting n Article citation: Wang G, Ou Y, Zhang Z, material of 5-acetyl-2-hydroxy-3-methoxybenzaldehyde (Apo-CHO) or 1-(3-amino-4- Jiang J. Synthesis of nitrate derivatives hydroxy-5-methoxyphenyl) ethanone (Apo-NH ). The docking of the derivatives with 1K4U 2 of apocynin dimer analogue. J Pharm subsection of human neutrophil NADPH oxidase system was preliminarily investigated BiomedSci 2018;8(4):56–62. using Goldscore function available in GOLD 5.0.1 program. The results demonstrated Available at www.jpbms.info that the derivatives got higher score (>44) than apocynin (25.8647), which indicated a possible stronger binding with NADPH oxidase. Statement of originality of work: The manuscript has been read and approved by KEYWORDS apocynin dimer analogue, nitrate derivative, synthesis all the authors, the requirements for authorship have been met, and that each author believes that the manuscript represents honest and original work. INTRODUCTION Source of funding: This research was Acute lung injury (ALI) is the early stage of acute respiratory distress syn- supported by project of National Natural Science drome (ARDS) and is a severe even mortal disease caused by various insults Foundation of China (81441128) and Science and Technology Program of Guangzhou, China inside and outside lung, leading acute progressive anoxic respiratory fail- (201704020198). ure, excessive amount of pro-inflammation cytokines release, and increased 1–4 Competing interest / Conflict of interest: pulmonary capillary permeability . From ARDS originally described by The author(s) have no competing interests Ashbaugh in 19675, the exact pathogenic mechanism was still not fully for financial support, publication of this understood over the past few decades. research, patents, and royalties through It is widely believed that excessive oxidative stress and inflammation is this collaborative research. All authors were related to the development of ALI closely. Excessive production of reactive equally involved in discussed research work. There is no financial conflict with the subject oxygen species (ROS) led to explosion of oxidative stress, and then acti- matter discussed in the manuscript. vated nuclear factor-kappa B (NF-kB)6. NF-κB is an important transcription Disclaimer: Any views expressed in this paper factor in inflammation development, its activation modulated the transcrip- are those of the authors and do not reflect the tion of various inflammatory factors, including TNF-α, inducible nitric official policy or position of the Department oxide synthase (iNOS), and nitric oxide (NO)6–8. TNF-α was a well-known of Defense. pro-inflammatory cytokine in inflammation process and up-regulated level of TNF-α was regarded to be crucial pathogenesis of ALI8. NO has been known as a famous gaseous signal molecule participating in kinds of biological function, including inflammation progression. NO derived from iNOS was reported to relate to pathophysiology of ALI and induce tissue damage through the formation of peroxynitrite and other inflammatory media- tors8–10. These factors interact mutually and cause cascade of ALI progression. Treatment against oxidative stress and inflammation may be potential method in ALI therapy. Apocynin, a phenolic chemical, is a natural antioxidant extracted from Picrorhiza kurroa plant and has been commonly used as an effective inhibi- tor of the Nox complex11,12. Apocynin was widely investigated as an anti- oxidative agent in many in vitro and in vivo models, and showed beneficial

Copyright © 2018 J Pharm Biomed Sci | Vol. 08 No. 4 | 56–62 Received Date: 10 March 2018 – Accepted Date: 03 April 2018 – Published Online: 14 April 2018 Synthesis of nitrate derivatives of apocynin dimer analogue 57

2230-7885 effects in asthma, neurological diseases, atherosclero- Dimethylformamide (DMF), tetrahydrofuran (THF), JPBSCT sis, hypertension, hepatic dysfunction, and ALI12–14. and triethylamine (TEA) were purchased from AlfaAesar J Pharm Biomed Sci Apocynin appears to exert the action after forming dimer (China) Chemical Co., LTD and Shanghai Aladdin Bio- https://doi.org/10.20936/jpbms/08/04/01 and trimer derivatives13. Our previous research found Chem Technology Co., Ltd. (Shanghai, China). Apocynin dimer analogue of apocynin, JJA-D0, showed better was purchased from Beijing J&K Scientific Co., Ltd. activity in reducing ROS level and inhibiting expres- (Beijing, China). Solvents were dried according to stan- sion of NADPH oxidase and TNF-α15,16. Also, JJA-D0 dard procedures. was observed to slightly improve the lung histopatholo- gical damage in rats with ALI induced by General separation and structure identification lipopolysaccharide (LPS)17. Connelly et al.18 found that methods high concentration of exogenous NO depressed the activity of NF-κB and caused down-regulation of iNOS Thin layer chromatography (TLC) analysis was expression in RAW 264.7 macrophages treated with LPS. performed on pre-coated silica gel GF254 plates Thus, decreased level of iNOS may reduce the endoge- (Qingdao Haiyang Chemical Group Corp., Qingdao, nous output of NO, which is possible to ameliorate the China). Column chromatography was performed on pathophysiology of ALI. In order to explore more effec- silica gel (200–300 mesh, Qingdao Haiyang Chemical tive apocynin dimer analogue in treating ALI, NO donor Group Corp, Qingdao, China) column. Melting points groups were introduced to JJA-D0. Four new nitrate were measured using a Mel-Temp (X7L20, Beijing), derivatives of JJA-D0 were synthesized and identified. and are uncorrected. NMR spectra were recorded 1K4U sub-section of human neutrophil NADPH oxidase at ambient temperature on a 300 MHz spectrometer system was chosen for preliminary docking investiga- (AV-400, Bruker) in CDCl3. Electrospray ionization tion to predict the binding capacity of the derivatives mass spectra (ESI-MS) were obtained in the positive with NADPH oxidase. ion detection mode on a Finnigan LCQ Advantage MAX mass spectrometer (Applied Biosystems, 4000 Q TRAP). MATERIALS AND METHODS Materials General process of synthesis of nitrate derivatives of apocynin dimer analogue All reagents were obtained from commercial suppliers as follows: 1,2-dibromoethane, 1,4-dibromobutane, Four nitrate derivatives of apocynin dimer analogue

silver nitrate, di-tert-butyl dicarbonate ((Boc)2O), were designed based on JJA-D0 (Fig. 1). Apocynin was N, N-diisopropylethylamine (DIPEA), trifluoroacetic introduced with an aldehyde group and amino group

acid (TFA), sodium cyanoborohydride (NaBH3CN), to produce 5-acetyl-2-hydroxy-3-methoxybenzaldehyde

Figure 1. Nitrate derivatives of apocynin dimer analogue.

J Pharm Biomed Sci | Vol. 08 No. 4 | 56–62 Received Date: 10 March 2018 – Accepted Date: 03 April 2018 – Published Online: 14 April 2018 58 Jie Jiang

(Apo-CHO) and 1-(3-amino-4-hydroxy-5-methoxy- were synthesized. Briefly, dibromo-alkane combined phenyl) ethanone (Apo-NH2), respectively. Apo-CHO with the phenolic group of Apo-CHO or Apo-NH2 first and Apo-NH2 reacted to form Schiff base, which was by ether bond, respectively, and then silver nitrate was then reduced to yield 1-(3-((5-Acetyl-2-hydroxy-3- used to substitute the other bromine and form nitrate methoxybenzyl)amino)-4-hydroxy-5-methoxyphenyl) bond (Schemes 1–4). For preparing nitrate derivatives of 15,19 ethanone (JJA-D0) . Apo-CHO and Apo-NH2 made Apo-NH2, the amino group of Apo-NH2 was protected up motifs 1 and 2 of JJA-D0, respectively. by di-tert-butyl dicarbonate ((Boc)2O) before modifi- Two kinds of NO donor groups of nitrate were intro- cation of phenolic group, and the protective group was duced to the phenolic group of motif 1 or 2 of JJA-D0, removed after nitration step in dichloromethane (DCM) respectively, and JJA-D1, D2, D3, and D4 were yielded. with TFA. The obtained nitrate derivatives of Apo-CHO

First, the nitrate derivatives of Apo-CHO or Apo-NH2 reacted with Apo-NH2 to generate JJA-D1 and D2,

Scheme. 1 Reagents and conditions: (a) 1,2-dibromoethane/DMF/K2CO3, 80°C; (b) silver nitrate/acetonitrile, 80°C; (c) NaBH3CN/ CH3OH, glacial acetic acid, r.t.

Scheme. 2 Reagents and conditions: (a) 1,4-dibromobutane/DMF/K2CO3, 80°C; (b) silver nitrate/acetonitrile, 80°C; (c) NaBH3CN/ CH3OH, glacial acetic acid, r.t.

Scheme. 3 Reagents and conditions: (a) (Boc)2O/TEA, r.t.; (b) 1,2-dibromoethane/DMF/DIPEA, r.t.; (c) silver nitrate/acetonitrile, 80°C; (d) DCM/TFA; (e) NaBH3CN/CH3OH, glacial acetic acid, r.t.

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Scheme. 4 Reagents and conditions: (a) (Boc)2O/TEA, r.t.; (b) 1,4-dibromobutane/DMF/K2CO3, r.t.; (c) silver nitrate/acetonitrile, 80°C; (d) DCM/TFA; (e) NaBH3CN/CH3OH, glacial acetic acid, r.t.

while the nitrate derivatives of Apo-NH2 reacted with this work, we introduced NO donor groups of nitrate Apo-CHO to generate JJA-D3 and D4. with different carbon chain length to different phenolic group of JJA-D0 to synthesize four nitrate derivatives. The synthesis procedure of each compound and related Preliminary investigation of docking of nitrate intermediates were described as follows in detail, as well derivatives of JJA-D0 with 1K4U subsection as the spectra data for structure identification. of NADPH oxidase It is generally believed that apocynin reduced the 2-(4-acetyl-2-(((5-acetyl-2-hydroxyphenyl) ROS production through inhibiting cytosolic sub- amino)methyl)-6-methoxy phenoxy)ethyl unit of NADPH oxidase to translocate to membrane. nitrate (JJA-D1) p67phox and p47phox are two important subunits of NADPH oxidase existing in cytoplasm. The 1K4U Compound Apo-CHO (1.94 g, 10 mmol) was dissolved subsection of human neutrophil NADPH oxi- in DMF, 1,2-dibromoethane (2.23 g, 12 mmol) and dase system consists of parts of the C-terminal SH3 K2CO3 (2.76 g, 20 mmol) were added to react overnight domain of p67phox complexed with the C-terminal at 80°C. At the end of reaction, DCM was added and tail region of p47phox20. In order to predict the bind- the organic layer was then washed by water. After eva- ing capacity of the nitrate derivatives of JJA-D0 to poration of solvent, the residue was separated by NADPH oxidase, we performed docking of JJA-D1, silica column chromatography, eluting with ethyl D2, D3, and D4 with 1K4U subsection of NADPH acetate/petroleum ether (1:6, v/v) to afford inter- oxidase system using GOLD 5.0.1 program (CCDC), mediate Apo-CHO-(CH2)2Br. The intermediate was and the binding action was scored using Goldscore dissolved in acetonitrile, and silver nitrate was added function available in GOLD 5.0.1 program. The study to react in darkness for 8 h at 80°C. The reaction also included docking of apocynin for comparison. product was purified by column chromatography, eluting with ethyl acetate/petroleum ether (1:5, v/v)

to afford intermediate Apo-CHO-(CH2)2O-NO2. RESULTS AND DISCUSSION Apo-CHO-(CH2)2O-NO2 (2.83 g, 10 mmol) was dissolved in methanol, and Apo-NH (1.81 g, 10 Synthesis of nitrate derivatives of apocynin 2 dimer analogue mmol) was added under stirring. After precipitate formation completed, the mixture was filtered Nitrogen oxide donor compound can slowly release to obtain precipitate. Then, the precipitate was dis- NO gas in cells and animals, producing corres- solved in ethyl acetate, appropriate amount of sodium ponding pharmacological activities. Organic nitrate cyanoborohydride and glacial acetic acid were added, ester is a group of important NO donor compound. In reacting for 2 h. The reaction product was purified

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by column chromatography, eluting with ethyl ace- Apo-NH2-(CH2)2O-NO2 (2.70 g, 10 mmol) was dis- tate/petroleum ether (1:4, v/v) to afford JJA-D1. solved in methanol, and Apo-CHO (1.94 g, 10mmol) White solid (2.55 g, 57% yield). m.p.: 161–163°C. was added under stirring. After precipitate formation ESI-MS: m/z [M+H]+ 449.3, [M-H]+ 447.3. 1H-NMR completed, the mixture was filtered to obtain precip

(300 MHz, CDCl3) d:9.46 (s, 1H), 7.66 (d, 1H), 7.50 itate. Then, the precipitate was dissolved in ethyl acetate, (d, 1H), 7.12 (d, 1H), 7.07 (d, 1H), 5.52 (s, 1H), 4.79– appropriate amount of sodium cyanoborohydride 4.82 (m, 2H), 4.51 (s, 2H), 4.45–4.48(m, 2H), 3.93 and glacial acetic acid were added, reacting for 3 h. (s, 3H), 3.91 (s, 3H), 2.56 (s, 3H), 2.52 (s, 3H). 13C- The reaction product was purified by column chroma-

NMR (75 MHz, CDCl3) d:198.69, 198.40, 153.59, tography, eluting with ethyl acetate/petroleum ether 150.79, 147.08, 138.43, 137.09, 134.79, 133.99, (1:2, v/v) to afford JJA-D3. Yellow-brown solid (2.15 131.11, 124.00, 112.26, 107.57, 102.96, 73.40, g, 48% yield). m.p.: 138–139°C. ESI-MS: m/z [M+H]+ 1 70.25, 57.68, 57.36, 44.50, 27.82, 27.64. 449.4. H-NMR (300 MHz, CDCl3) d: 9.87 (s, 1H), 7.59 (d, 1H), 7.44 (d, 1H), 7.05 (d, 1H), 6.96 (d, 1H), 5.95 4-(4-acetyl-2-(((5-acetyl-2-hydroxyphenyl) (s, 1H), 4.72–4.75(m, 2H), 4.46 (s, 2H), 4.33–4.36 amino)methyl)-6-methoxyphenoxy)butyl nitrate (m, 2H), 3.92 (s, 3H), 3.87 (s, 3H), 2.53 (s, 3H), 2.51 13 d (JJA-D2) (s, 3H). C-NMR (75 MHz, CDCl3) : 197.61, 196.91, Compound JJA-D2 was prepared from Apo-CHO 151.89, 148.62, 146.68, 141.35, 137.51, 133.67, (1.94 g, 10 mmol) and 1,4-dibromobutane (2.57 g, 129.49, 123.72, 123.55, 109.01, 106.02, 101.88, 12 mmol) according to the procedure of JJA-D1. Light 71.96, 68.20, 56.23, 55.88, 43.00, 26.48, 26.18. yellow solid (2.81 g, 59% yield). m.p.: 151–152°C. ESI- + 1 MS: m/z [M+H] 477.4. H-NMR (300 MHz, CDCl3) 4-(4-acetyl-2-((5-acetyl-2-hydroxy- d: 9.46 (s, 1H), 7.61(d, 1H), 7.47(d, 1H), 7.05(d, 1H), 3-methoxybenzyl)amino)phenoxy)butyl 7.00(d, 1H), 5.52 (s, 1H), 4.48(t, 2H), 4.43(s, 2H), nitrate (JJA-D4) 4.14(t, 2H), 3.88(s, 3H), 3.85(s, 3H), 2.52(s, 3H), Compound JJA-D4 was prepared from Apo-NH-Boc 2.50(s, 3H), 1.87–1.94(m, 4H).13C-NMR (75 MHz, (2.82 g, 10 mmol) and 1,4-dibromobutane (2.57 g, CDCl3) d: 197.37, 197.22, 152.49, 150.42, 145.73, 12 mmol) according to the procedure of JJA-D3. White 137.22, 135.45, 132.85, 132.11, 129.48, 122.84, solid (2.09 g, 44% yield). m.p.: 160–162°C. ESI-MS: 110.84, 106.18, 101.94, 73.00, 72.24, 56.20, 55.85, + 1 m/z [M+H] 477.6. H-NMR (300 MHz, CDCl3) d: 43.47, 26.52, 26.42, 26.25, 23.53. 9.87 (s, 1H), 7.60 (d, 1H), 7.45 (d, 1H), 7.08 (d, 1H), 7.00 (d, 1H), 5.95 (s, 1H), 4.53 (t, 2H), 4.48 (s, 2H), 2-(4-acetyl-2-((5-acetyl-2-hydroxy- 4.07 (t, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 2.53 (s, 3H), 3-methoxybenzyl)amino)phenoxy)ethyl 2.52 (s, 3H), 1.85–1.97(m, 4H). 13C-NMR (75 MHz, nitrate (JJA-D3) CDCl3) d: 197.58, 196.78, 151.96, 148.60, 146.66, With reference to previous work19, the amino group 141.05, 138.97, 133.13, 129.48, 123.63, 123.59, of compound Apo-NH2 was protected using (Boc)2O 109.10, 106.25, 102.58, 72.96, 71.49, 56.22, 55.86, to afford intermediate Apo-NH-Boc. Apo-NH-Boc 43.53, 26.46, 26.20, 23.61. (2.82 g, 10 mmol) was then dissolved in DMF, In synthesis of intermediate Apo-CHO-(CH2)2Br 1,2-dibromoethane (2.23 g, 12 mmol) and DIPEA and Apo-CHO-(CH2)4Br, K2CO3 was used as acid-bind- were added to react at room temperature for 8 h. ing reagent to create a moderate alkaline condition; thus, At the end of the reaction, DCM was added and the to absorb HBr generated during reaction, and the reac- organic layer was then washed with water. After evap- tion was performed at a somewhat high temperature of oration of solvent, the residue was separated by silica 80°C. While in synthesis of intermediate Apo-NH-Boc- column chromatography, eluting with ethyl acetate/ (CH2)2Br and Apo-NH-Boc-(CH2)4Br, the reaction petroleum ether (1:8, v/v) to afford intermediate condition was changed to be milder due to the active

Apo-NH-Boc-(CH2)2Br. The intermediate was dis- reactivity of the hydrogen of amino group in Apo-NH2 solved in acetonitrile, and silver nitrate was added to motif. Although the amino group was protected with react in darkness for 8 h at 80°C. The reaction prod- (Boc)2O, the rest hydrogen in amino group was suscep- uct was purified by column chromatography, eluting tible to react with the end bromine of the ether deriva- with ethyl acetate/petroleum ether (1:6, v/v) to afford tive of Apo-NH-Boc-R-Br to form a cyclic compound intermediate Apo-NH-Boc-(CH2)2O-NO2. Apo-NH- as illustrated in Scheme 5, when K2CO3 was used and Boc-(CH2)2O-NO2 was dissolved in DCM, then, TFA the reaction temperature was 80°C. Therefore, when was added dropwise. After 1 h reaction, NaHCO3 preparing Apo-NH-Boc-(CH2)4Br, reaction tempera- saturated solution was added to neutralize the reac- ture was changed to room temperature and K2CO3 was tion mixture. The mixture was extracted with DCM, still used. In synthesis of Apo-NH-Boc-(CH2)2Br, the and the organic layer was collected and evaporated reaction condition was required to be even milder with to produce intermediate Apo-NH2-(CH2)2O-NO2. K2CO3 substituted by DIPEA, a weaker organic alkali.

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docking demonstrated higher score was achieved by the nitrate derivatives when compared with apocynin. It predicted a possible stronger action on NADPH oxidase, and further studies of biological activity are expected.

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