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Synthesis and In Vitro Anti-Influenza Evaluation of Rupestonic Acid Analogues: Effect of Configuration and Substitution at C (3) | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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Synthesis and In Vitro Anti-Influenza Evaluation of Rupestonic Acid Analogues: Effect of Configuration and Substitution at C (3)

Jiangyu Zhao1,2, Changzhi Dong3* and Haji Akber Aisa1,2*

1Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, China

2State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, China

3Equipe de Pharmacochimie, ITODYS, CNRS UMR7086, Université Paris Diderot, Paris, France

*Corresponding Author:
Changzhi Dong
Equipe de Pharmacochimie, ITODYS,
CNRS UMR7086, Université Paris Diderot, Paris, France
E-mail: [email protected]
 
Haji Akber Aisa
State Key Laboratory Basis of Xinjiang
Indigenous Medicinal Plants Resource Utilization
Xinjiang Technical Institute of Physics and Chemistry
Chinese Academy of Sciences, China
E-mail: [email protected]

Received date: April 18, 2016; Accepted date: May 03, 2016; Published date: May 10, 2016

Citation: Zhao J, Dong C, Aisa HA (2016) Synthesis and In Vitro Anti-Influenza Evaluation of Rupestonic Acid Analogues: Effect of Configuration and Substitution at C (3). Med chem (Los Angeles) 6:322-326. doi:10.4172/2161-0444.1000364

Copyright: © 2016 Zhao J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Rupestonic acid is the main active compound in Artemisia rupestris L, which mainly grows in Xinjiang of China. To find its active group, a series of novel rupestonic acid 3-carbonyl analogues were prepared. The structures of the compounds were confirmed by spectral and high resolution mass spectrum. All compounds were evaluated for antiviral activity against influenza A (H3N2 and H1N1) and B viruses in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. Compound 13 with allyl group was the most potent inhibitor against influenza A/H1N1 virus with an IC50 value of 4.27 μM and a SI value of 27.04. Dihydrogen amide 3 possesses the good potency against influenza B virus with an IC50 value of 5.5 μM and a SI value of 13.

Keywords

Rupestonic acid; Synthesis; Analogues; Anti-influenza activity

Anti-Influenza

Influenza is an infectious disease of respiratory tract caused by influenza viruses in humans throughout the world. Influenza viruses belongs to RNA viruses and composed of three general types: influenza A, B and C according to antigenicity of nucleoprotein [1,2]. Compared with influenza B and C, influenza A viruses are the most major pathogens. It exhibits 16 HA subtypes and 9 NA subtypes. Of them, H1N1, H3N2 and H1N2 subtypes cause acute respiratory disease in humans [3]. In the past for a long time, they had caused serious impact on global morbidity, mortality and economy. For example, Spanish flu (H1N1) in 1918, Asian influenza (H2N2) in 1957, Hong Kong influenza (H3N2) in 1968, avian influenza (H5N1) in 2007, swine influenza (H1N1) in 2009 and so on [4].

Currently, vaccination is the primary strategy for the prevention of influenza infections, but vaccines are ineffective against rapidly emerging mutant viral antigens [5]. In addition, the vaccine cannot effectively protect elderly people and children. Pharmacotherapy is another effective means of controlling the spread of influenza viruses. Until now, there are only four anti-influenza drugs approved for use by the United States FDA: M2 ion channel inhibitors (Amantadine and Rimantadine) and neuraminidase (NA) inhibitors (Oseltamivir and Zanamivir) [6]. The former are effective only against type A virus because of its self-structure and drug resistance has become widespread [7,8]. For the later, Oseltamivir phosphate (Tamiflu), an ethyl ester of GS4071, is an orally administered drug for the prophylaxis and treatment of human influenza A and B [9,10], whereas it has been reported that the H5N1 influenza virus has shown resistant to Oseltamivir [11,12]. Zanamivir is delivered by oral inhalation due to its poor oral bioavailability [13]. Thus, it is very urgent to search for new compounds with inhibitory activity against influenza.

2-(3,8-Dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl) acrylic acid (Rupestonic acid, 1), first isolated from the dichloromethane extract of leaves of Decachaeta scabrella and named pechueloic acid, was isolated from dry Artemisia rupestris L (Chinese name Yi Zhihao) and purified using silica gel chromatograph. It is a multi-functional sesquiterpenoid compound [14]. During our previous studies, more than two hundred amide and ester derivatives of rupestonic acid have been synthesized by modifying the carboxyl group (C13) and the in vitro activities of these compounds against influenza viruses A and B were assayed [15-19]. Mechanism of action study and in vivo activity of selected candidate compounds are under way.

To study the structure-activity relationship (SAR) of rupestonic acid derivatives comprehensively, effect of modification at C (3) will be discussed in this research. The synthetic routes of C (3) analogues were depicted in Schemes 1-6.

Scheme 1: Reagents and conditions: (a) Bop, HOBt, DIPEA, 60%; (b) H2, 10% Pd/C, EtOAc; (c) H2, 10% Pd/C, EtOH.

Scheme 2: 1S0%y Pndt/Ch, eEtsOHis, 70o%f. tetrahydrogen methyl rupestonate. Reagents and conditions: (a) K2CO3, CH2Cl2, 98%; (b) H2, 10% Pd/C, EtOAc, 83%; (c) H2, 10% Pd/C, EtOH, 70%.

Scheme 3: Synthesis of ketoximes ethers. Reagents and conditions: (a) NaOAc, MeOH, 98%; (b) appropriate alkyl iodide, NaH, THF, 48-89%.

Scheme 4: Reagents and conditions: (a) MeOH, 98%; (b) MsCl, Et3N, CH2Cl2.

Scheme 5: Synthesis of esters. Reagents and conditions: (a) appropriate acyl chlorides, pyridine, 32%-86%.

Scheme 6: Synthesis of ethers. Reagents and conditions: (a) appropriate alkyl iodine, Ag2O, CH3CN, 47%-71%.

As we know, carboxyl group possessed the high reactivity in organic chemical reaction and amide compounds were relatively more stable than carboxylic acid. In the beginning, amide was planned to prepare and protect carboxyl group. Amide 2 was formed from lead compound 1 and 4-chloroaniline using Bop (Benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate)) and N-Hydroxybenzotriazole (HOBt) as the coupling reagents,4-(dimethylamino) pyridine (DMAP) as the catalyst and N,N-Diisopropylethylamine (DIPEA) as the base [20]. Meanwhile, reaction activity of carbonyl in α, β-unsaturated ketone was relatively low, so the next step was the obtainment of saturated amide 2. It was carried out by hydrogenation over 10% Pd/C catalyst in EtOAC to afford dihydrogen amide 3 in 60% with high stereo selectivity, but it was found that C4=C5 in the five-membered ring did not hydrogenate. To finish the thorough hydrogenation of amide 2, EtOH was selected as the solvent, whose polarity was stronger than that of EtOAC [21]. Interestingly, dechlorination production compound 4 has been generated. Its structure was confirmed by NMR and HRMS, similar results have been reported previously [22-24].

We, therefore, decided to find out another method to protect the carboxyl group. Methylation of rupestonic acid 1 and dimethyl sulfate using K2CO3 as the base afforded methyl rupestonate 5 in 98% [25]. Compound 5 was carried out by hydrogenation over 10% Pd/C catalyst in EtOAC or EtOH to afford dihydrogen methyl rupestonate 6 or tetrahydrogen methyl rupestonate 7 in 83% or 70%, respectively [24]. As can be seen, the polarity of the solvent played an important role in the hydrogenation process.

Compound 7 served as the starting materials for the preparation of the C(3)-modified analogues 9-31. Various ketoximes ethers (9-13), esters (18-25) and ethers (27-31) analogues were prepared at this site and assayed in vitro anti-influenza activity.

O-ketone oxime 8 were obtained by starting with compound 7 and NH2OH·HCl using NaOAc as the base in near quantitative yield (98%). And then, it was treated with appreciate alkyl iodide to obtain ketoximes ethers 9-13 [26].

In Scheme 4, treatment of compound 7 with NaBH4 afforded two epimeric alcohols 14 (43%) and 15 (45%), which were easily separated by silica gel chromatography. Alcohol 14 with excess methane sulfonyl chloride/pyridine in CH2Cl2 at 0°C afforded alkene 16 in 70% yield; on the other hand, treatment of its epimer 15 by quick mesylation under the same condition afford the expected methane sulfonate 17. This was consistent with trans-elimination rule of alcohol. Unfortunately, the substitution reaction between compound 17 and sodium benzene thiolate with different substitution failed.

Standard acylation condition (appropriate acyl chlorides, pyridine) was then carried out and yielded the desired esters 18-22 from compound 14 and 23-26 from 15 with yields ranging from 32-73% (Scheme 5) [26]. As can be seen, the same condition was ineffective using compound 15 and tert-butyl formyl chloride as the starting materials. This may be attributed to the space steric hindrance of tertbutyl. Incorporation of ethers at C(3) was accomplished according to Scheme 6. Treatment of 14 with the appropriate alkyl iodide in the present of Ag2O created the corresponding ethers 27-31. Our attempts to prepare ethers failed using compound 15 as the starting material under the same conditions.

Spectra data (1H NMR, 13C NMR and HRMS) consistent with the proposed structures were obtained for all the compounds prepared in this study. To determine the potential activity of compounds 2-31, they were preliminarily assayed in vitro against influenza A (H3N2, H1N1) and B viruses [27]. The inhibition and selectivity of these compounds were listed in Table 1.

Compd. R TC50
(µM)a
Against influenza
H3N2 virus
Against influenza
H1N1 virus
Against influenza
B virus
IC50
(µM)b
SIc IC50
(µM)b
SIc IC50
(µM)b
SIc
1d 4653 25.77 180.6 >1344 e >4032 e
Amides                
2 34.5 >11.5 e >11.5 e >11.5 e
3 71.4 >34.4 e 26.6 2.68 5.5 13.00
4 1776.5 1026.9 1.73 493.1 3.60 >32.5 e
Esters                
5 294.0 27.2 10.81 81.6 3.60 >141.4 e
6 542.3 108.9 4.98 81.0 6.69 60.3 9.00
7 417.7 >139.2 e 108.0 3.87 139.2 3.00
Ketoxime ethers              
8 H 395.4 >131.8 e 34.6 11.43 131.8 3.00
9 CH3 161.8 >41.86 e >41.86 e >41.9 e
10 CH3CH2 69.2 >13.33 e 13.33 5.19 >13.3 e
11 CH3CH2)2 66.2 >38.24 e 16.42 4.03 >38.2 e
12 CH3(CH2)3 329.7 >109.9 e 85.2 3.87 109.9 3.00
13 CH2=CH-CH2 115.4 >38.5 e 4.27 27.04 12.5 9.23
Alcohols                
14 287.5 >138.2 e >138.2 e >139.2 e
15 718.1 >414.6 e 414.6 e >414.6 e
Alkene                
16 256.6 >148.2 e 63.64 4.03 148.3 1.73
Methanesulfonate              
17 963.4 >321.2 e 198.6 4.85 321.1 3.00
Esters                
18 CH3 238.6 >72.3 e 46.8 5.10 >46.5 e
19 Ph 207.1 >99.6 e 69.03 3.00 >99.6 e
20 PhCH2CH2 1940.2 110.2 17.61 215.6 9.00 >277.8 e
21 CH3(CH2)3 1969.8 >946.7 e 83.15 23.69 947.0 2.08
22 (CH3)3C 182.2 >105.2 e 15.39 11.84 65.07 2.80
15 H 718.1 >414.6 e 414.6 e >414.6 e
23 CH3 90.77 >13.3 e 13.00 6.98 >13.3 e
24 Ph 298.68 >99.6 e 33.19 9.00 99.56 3.00
25 PhCH2CH2 481.1 >92.6 e >92.6 e >92.6 e
26 CH3(CH2)3 406.7 105.1 3.87 105.1 3.87 >105.2 e
Ethers                
27 CH3 131.3 >43.8 e 33.94 3.87 >43.8 e
28 CH3CH2 72.23 >41.7 e 32.39 2.23 >41.7 e
29 CH3(CH2)2 206.9 >119.5 e 92.80 2.23 >119.5 e
30 CH3(CH2)3 198.0 >114.3 e 88.79 2.23 88.8 e
31 CH2=CH-CH2 7.73 >4.45 e >4.45 e >4.45 e
Ribavirin 1278 3.9 329.4 1.9 665.6 14.7 86.9
Oseltamivir 1260 1.1 1135.1 15.5 81.3 >500 e

a50% cytotoxic concentration; b50% inhibitory concentration, determined by CPE inhibition assay; cSelectivity Index (TC50/IC50); dParent compound; eThe SI cannot be calculated, because the highest concentration tested was less than the IC50.

Table 1: Chemical structures and antiviral activity of Rupestonic acid analogues against influenza A (H3N2 and H1N1) and B viruses.

As can been seen from Table 1, most of the target compounds showed some potency against influenza H1N1 virus, but most did not show obvious activity against influenza H3N2 and B viruses. Among of them, parent compound 1 presented the best activity against influenza H3N2, with an IC50 value of 25.77 μM and a SI value of 180.6. Compound 13 with allyl group was the most potent analogy among the series against influenza H1N1 with an IC50 value of 4.27 μM and a SI value of 27.04, which was much better than that of Oseltamivir. Dihydrogen amide 3 showed the best activity against influenza B with an IC50 value of 5.5 μM and a SI value of 13, which was about 3 times higher than Ribavirin.

When rupestonic acid was converted into amide 2, 3 and 4, it was found that the activity against influenza H3N2 decreased obviously compared with parent compound 1. For compound 4, it can be seen that removal of the chlorine group by hydrogenation reduced the toxicity. For methyl ester compound 5, we can see the activity was very close to that of compound 1, but its toxicity increased dramatically (TC50=294 μM). But when C4=C5 or C11=C13 hydrogenated, dihydrogen methyl rupestonate 6 (TC50=542.3 μM) or tetrahydrogen methyl rupestonate 7 (TC50=417.7 μM) possessed lower toxicity than methyl ester compound 5 (TC50=294.0 μM).

It can be seen that the size of the alkyl substituents in the ketoximes ethers (9-13) was critical for the activity. It was noted that the activity against influenza H1N1 virus was in the order: CH2=CH-CH2(13)> CH3CH2(10)> CH3(CH2)2(11)> H(8)> CH3(9)> CH3(CH2)3(12). Of these, compound 13 linked to allyl group showed the best activity against influenza H1N1 and B viruses with an IC50 value of 4.27 μM and 12.5 μM respectively. As we know, oxime ether structure was often selected as the active group in drug research and discovery. So, the C=C of CH2=CH-CH2 and C=N of oxime ether may strong the interaction of compound 13 with the neuraminidase of the influenza virus. All of these derivatives did not show the activity against influenza H3N2. For epimeric alcohol 14 and 15, it was observed that 14 (TC50=287.5 μM) was more toxic than 15 (TC50=718.1 μM).

Similar SAR patterns were observed in the esters (18-25). There were obvious activity difference between enantiomers, for example, compound 18 and 23, 19 and 24, 20 and 25, 21 and 26. Compound 23 with methyl remained the best activity against influenza H1N1 with an IC50 value of 13.00 μM. Compound 22 with tertiary butyl possessed the best activity against influenza B with an IC50 value of 65.07 μM. It can be exemplified by the relative contribution of related R groups to anti-influenza H1N1 activity between 18-22 as follows: (CH3)3C(22)> CH3(18)> Ph(19)> CH3(CH2)3 (21)>PhCH2CH2(20). For ethers 27-31, unfortunately, they did not show obvious activity against influenza A and B viruses, although their structures were very similar to that of ketoxime ethers 8-13. And we can see, the structures of compound 27- 31 were very close to the saturated molecules. So due to the lack of active functional group in ether, they showed very weak anti-influenza virus activity. Meanwhile, it can conclude that the functional groupketoxime played a very important role against influenza virus.

In summary, a series of novel rupestonic acid analogues were designed, synthesized and evaluated for their in vitro anti-influenza activity. Several compounds were found to possess moderate influenza inhibitory activity, although in all cases, measured activities were lower than that of Oseltamivir and Ribavirin. The most active compound 13 (IC50 = 4.27 μM) has good anti-influenza H1N1 activity which is fourfold more potent than positive drug Oseltamivir (IC50 = 15.5 μM) and it also showed better anti-influenza B activity than positive drug Ribavirin (IC50 = 14.7 μM). Our study indicated that rupestonic acid analogues could show potent inhibitory activity and the research finding will be used to design novel influenza inhibitors.

The Procedure for the Anti-Influenza Virus Assay

Each compound was dissolved in DMSO at an initial concentration of 1000 μg/mL and then diluted 3-fold successively to obtain 8 different concentrations (333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46 and 0.15, 0.05 μg/mL, respectively) as stock solutions for the following experiments. (2) Madin-Darby canine kidney cells (MDCK) were seeded in 96-well trays and cultured at 37°C in a humidified CO2 incubator (95% air, 5% CO2) for 24 h. Then, the cells were infected with influenza A virus with 10-4 [316 times the 50% tissue culture infective dose (TCID50)] and with influenza B virus with 1/210-2 [158 times of the 50% tissue culture infective dose (TCID50)], respectively. All of infected tissue culture plates (96 wells) were incubated at 37°C for 2 h, and then the medium was removed. Subsequently, 100 μL aliquots of the solutions with different concentrations of each compound were added to the wells (one per well), and the plates were incubated again for 36 h at 37°C. Then, the inhibition of the virus-induced cytopathic effect (CPE) for each sample was recorded relative to the cell control and the virus control. The 50% cell-inhibitory concentration (IC50) values of active compounds were calculated accordingly. The inhibitory potentials of rupestonic acid derivatives were comparable to those of the parent compound and the commercial drugs Ribavirin (RBV) and Oseltamivir.

Acknowledgements

This work was financially supported by West Light Foundation of the Chinese Academy of Sciences (No. XBBS201218), the National Natural Science Foundation of China (No. 81402808), Natural Science Foundation of Xinjiang (No. 2014211A070) and Youth Innovation Promotion Association, CAS. We would like to thank the members of Institute of Medicinal Biotechnology, Academy of Medical Sciences and Peking Union Medical College, for testing the anti-influenza activity of the synthesized compounds.

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