Synthesis and structure anti-inflammatory activity relationships studies of andrographolide derivatives

Abstract Andrographolide is a main bioactive diterpene lactone in A. paniculata with anti-inflammatory activity. In this study, a series of andrographolide derivatives were synthesized and evaluated for their structure-anti-inflammatory activity relationships in vivo. Among all compounds, isoandrographolide and 14-deoxyandrographolide showed stronger anti-inflammatory activity than andrographolide. The results indicated that the introduction of tetrahydrofuran ring and cyclic olefinic bond plays an important role in enhancing the anti-inflammatory activity of andrographolide derivatives. Isoandrographolide and 14-deoxyandrographolide are potent inhibitor of inflammation.


Introduction
Andrographis paniculata (Burm.F.) Nees (Acanthaceae), a well known traditional medicinal plant in India, Thailand, and China, has attracted much attention recently because of its potential clinical application. A. paniculata contains diterpene lactones, flavonoids, and polyphenols. Many studies have shown that andrographolide possesses anti-inflammatory activity (Jiang et al. 2007;Al aa et al. 2009;Zhang et al. 2009), and the anti-inflammatory activity of andrographolide has been extensively studied in vivo and in vitro (Shen et al. 2002;Levita et al. 2010;Warisara et al. 2010;Lee et al. 2011;Du et al. 2012;Liu et al. 2013;Kou et al. 2014;Zhai et al. 2014.) In 1985, the anti-inflammatory activities of the water soluble derivatives of 1 had been investigated in vivo, the results show that the basic structure of diterpenoid bicyclic nuclus and five-membered c-lactone ring were the foundation of anti-inflammatory activity, the anti-inflammatory activity can fluctuate to varying degrees due to changes in hydroxyl (Deng et al. 1985). In 2009, 3, 19-dipalmitoryl-14-deoxy-11, 12-didehydroandrographolide was synthesized, testing of anti-inflammatory activity in the abdominal cavity were carried out in animal models. The results show that 3, 19-dipalmitoryl-14-deoxy-11, 12-didehydroandrographolide shows much greater anti-inflammatory activity than its parent compound (Suebsasana et al. 2009). We modified the structure of andrographolide by esterification with anhydride or acyl chloride at 3, 14, 19-OH and evaluated their anti-inflammatory activity in vivo 2. Results and discussion 1 was purchased from Jinxin Bioscience Co. Ltd. Compound 1a, 2, 3 & 4 were synthesized in our laboratory (Han et al. 2006) (Figure 1). Compound 1b, 2a-2c, 3a and 3c were synthesized according to the literature reported previously (Smith et al. 2000;Wang et al. 2005;Grigore et al. 2013;Luo et al. 2017). In the present study, A variety of acyl chloride and anhydride reacted with compounds 1~4 in anhydrous to form crude products 1a, 1c, 3b, 3d and 4a. The crude products were further purified by silica gel column chromatography (CC) to yield the corresponding andrographolide esterified derivatives ( Figure 1). Compounds 1c, 3b, 3d and 4a were characterized by MS and 1 H NMR.
The anti-inflammatory activity of all synthetic compounds was screened by the method of rat paw edema induced by egg white (Grigore et al. 2013). Compared with andrographolide group. The anti-inflammatory activities of compounds 1a-1c, 2, 2c, 3b and 3c were equal to andrographolide, and compounds 2a, 2b, 3a, 3d and 4a showed less activity. The anti-inflammatory activity of compound 2 was not enhanced compared with andrographolide. Therefore, the anti-inflammatory activity of the andrographolide derivatives can not be improved by formation of conjugated double bonds. The anti-inflammatory activity of compound 1a-1c, 2a-2c, 3a-3d, 4a were not enhanced compared with that of 1, 2, 3, 4. Therefore, the introduction of ester groups by modification of 3, 14, 19-hydroxyl does not enhance the anti-inflammatory activity of andrographolide derivatives. It may be that the conjugated double bonds react with the oxygen free radicals and the active molecules in the body, causing the structure of the compounds to change greatly. In addition, the formation of ester groups delayed the action of compounds and inflammatory molecules, resulting in less antiinflammatory activity. However, compounds 3 and 4 exhibited high anti-inflammatory activity ( Table 1). The newly formed tetrahydrofuran ring and cyclic olefine bond may help the compounds to interact with inflammatory molecules, thereby enhancing the anti-inflammatory activity of the compounds. Therefore, the newly formed tetrahydrofuran ring and cycloalkene bond are valuable in the structure of andrographolide.
Synthesis of isoandrographolide-3,19-bisuccinates monoester (3b) and isoandrographolide-3, 19-bioxalicacid monoester (3d): Succinic anhydride (20.0 g, 200 mmol) and compound 3 (8.0 g, 23.0 mmol) was stirred at 80 C under 0.02MPa vacuum. The progress of the reaction was monitored by using TLC. The reaction mixture was dissolved in ethyl acetate (300 mL), organic layer was separated and washed with brine, dried over anhydrous Na 2 SO 4 and concentrated under vacuum, vacuum drying at room temperature, milk white powder 3b (3.1 g) was obtained in 25% yield. And, compound 3c (5.0 g, 9.0 mmol) was dissolved in excessive sodium hydroxide solution with stirring, filtered, hydrochloric acid was added until the pH is 2, sealed and placed, pure white needle crystal 3d (2.8 g) was obtained in 60% yield.
Synthesis of 3, 19-diacetyl-14-deoxyandrographolide (4a): compound 1a (10.0 g. 21.0 mmol) was dissolved in 200 mL of methanol, with the NaBH 4 (4.0 g, 105 mmol) added, and the mixture was stirred at room temperature. The progress of the reaction was monitored by using TLC. The mixture was slowly poured into ice water with stirring and filtered. The residue was crystallized from ethyl acetate-petroleum ether (1:5) to afford white powder compound 4a (5.2g) in 59% yield.
The other compounds were also obtained according to the literature reported previously.

Conclusions
In summary, we have synthesized a series of andrographolide derivatives successfully. The anti-inflammatory activities of compounds 1a-1c, 2, 2c and 3c were equal to andrographolide, and compounds 1c, 3b, 3d and 4a were poor. Compounds 3 and 4 were the most potent anti-inflammatory activity compounds. The most promising derivative is 3, which exhibited stronger anti-inflammatory activity than andrographolide. Its anti-inflammatory activity and molecular mechanisms need to be further investigated. Study on the relationship between structure and anti-inflammatory activity indicated that the anti-inflammatory activity of the andrographolide derivatives can not be improved by introduction of exocyclic double bond and ester group by modification of 3, 14, 19-hydroxyl. The newly formed tetrahydrofuran ring and cycloalkene bond help to enhance the anti-inflammatory activity of andrographolide derivatives.