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ISSN: 1948-5948
Journal of Microbial & Biochemical Technology
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Synthesis and SAR Studies of New Oxadiazole-2-oxide Derivatives with Remarkable In Vitro Activity against Schistosoma japonicum

Gause M1, Wen-Hua F1, Li-Jun S2, Chuan-Xin Y2* and Bainian F1*

1School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China

2Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China

*Corresponding Author:
Chuan-Xin Y
School of Pharmaceutical Science
Jiangnan University, Wuxi 214122, China
E-mail: [email protected]
Bainian F
Key Laboratory on Technology for Parasitic Disease Prevention and Control
Ministry of Health Jiangsu Institute of Parasitic Diseases
Wuxi 214064, China
E-mail: [email protected]

 

Received date: January 16, 2017; Accepted date: February 01, 2017; Published date: February 08, 2017

Citation: Gause M, Wen-Hua F, Li-Jun S, Chuan-Xin Y, Bainian F (2017) Synthesis and SAR Studies of New Oxadiazole-2-oxide Derivatives with Remarkable In Vitro Activity against Schistosoma japonicum. J Microb Biochem Technol 9:017-025. doi: 10.4172/1948-5948.1000339

Copyright: © 2017 Gause M, 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

Schistosomiasis is the most neglected tropical disease in the world now where every day more and more people are being affected. The only approved drug Praziquantel (PZQ) that could not prevent re-infection but only effective against adult worms. Resistance of PZQ has been found in some areas, so an alternative drug would be needed as a substitute. Oxadiazole-2-oxide has been proven as potential anti schistosomal agents in which they kill both adult and juvenile worms. We synthesized 25 novel oxadiazole-2-oxide analogues and check their in vitro activity on adult Schistosoma japonicum. In here, all of them showed better in vitro activity than praziquantel and 1, 2, 5-oxadiazole- 2-oxide. Compounds 16, 17, 20, 23 and 24 showed excellent activity even in low concentration and short time period. There are no differences found while checking their in vitro activity against male and female worms. Some new functional groups would be synthesized here may lead us to the new treatment of schistosomiasis. The synthesis and structure activity relationship (SAR) studies of oxadiazole-2-oxide analogues with activity against S. japonicum can help us to design some more new derivatives to find out better activity and potential candidate. Some of these compounds could be used to develop new anti-schistosomal drugs while the mechanism of these compounds still needs to found.

Keywords

Schistosoma japonicum; Oxadiazole-2-oxide analogues; Antischistosomal activity; Neglected tropical disease.

Abbreviations

DIBAl-H: Diisobutylaluminium Hydride; TGR: Thioredoxin Glutathione Reductase; SjTGR: S. japonicum Thioredoxin Glutathione Reductase; THF: Tetrahydrofuran; n-BuLi: n-Butyllithium; PPh3: Triphenylphosphine; DMSO: Dimethyl Sulphoxide; PZQ: Praziquantel; NO: Nitric Oxide; SAR: Structure-activity Relationship.

Introduction

Schistosomiasis (also known as bilharzia) is a vector-borne parasitic disease caused by trematode flatworms of the genus Schistosoma. It has affected more than 250 million people all over the world [1,2]. It is still a neglected tropical disease because of various control effects did not work well. Mostly, three species are responsible for particular relevance; Schistosoma mansoni, which is endemic in Africa, the Arabian Peninsula, South America and the Caribbean, Schistosoma haematobium, which occurs in Africa and the Arabian Peninsula, and Schistosoma japonicum, which is only restricted to China, the Philippines and Indonesia [3,4]. The only World Health Organization (WHO) recognized drug Praziquantel (PZQ) cannot stop reinfection and doesn’t work against juvenile schistosomiasis [5]. However, the mechanism of action of PZQ is still unidentified although data shown it causes schistosomes to develop tetanic contractions and tegumental vacuoles; affected worms lose their hold on the vein wall, and then washed upstream to the liver and die [6-8]. Several PZQ-resistant isolates already have identified [9,10]. There have been several studies to modify PZQ, but have yet to reach clinical trials [11]. So, there are needs of new targets and drugs those can work both in adult and juvenile schistosomiasis. 1,2,5-Oxadiazole-2-oxide was identified by a group of scientist as new drug leads against Schistosoma japonica as well as other species, where thioredoxin glutathione reductase (TGR) showed new potential molecular target [5,12]. Before, it was believed that oxadiazole-2-oxide activity depends on NO production but later find out, there might be other targets and mechanisms than NO production or inhibition of SjTGR activity [12,13].

As a preventive drug, praziquantel is not useful at all [14]. It also shows some side effects such as dizziness, rash, nausea, abdominal pain, pruritus, headache and drowsiness which could be connected with the result of worm death than the drug itself [15,16]. The shortcoming of PZQ is the circumstance that it is not active against juvenile schistosomes [17,18]. Oxamniquine is an aminoethyltetrahydroquinolone derivative, which is effective only against S. mansoni, generally the adult worms, and male worms are more sensitive to the drug [19]. However, a combination therapy for acute S. japonicum has failed to improved treatment efficacy compared with PZQ alone [20]. World Health Organization (WHO) approved six vaccine candidates until now which are still in trial phase [21]. There is an urgent need for integrated control programs and preventive chemotherapy. Therefore, we became interested in the synthesis of oxadiazole-2-oxide analogues to find out some new potential antischistosomal agents. We designed and synthesis some novel oxadiazole-2-oxide to target Schistosoma japonicum, where they have good killing activity. It did not inhibit the activity of SjTGR, so we found some new functional groups. Amine functional group could be a new lead of drug for treatment of Schistosoma japonicum, where the halogen contains oxadiazole-2-oxide showed good killing activity. These could be a new drug leads for the treatment and control of schistosomiasis. In here, we checked the in vitro activity against Schistosoma japonicum; maybe it will have effect other species too.

Materials and Methods

Experimental

Chemistry: All the chemicals were reagent grade and used as purchased. Some chemicals were stored in the laboratory as per chemical indication. 1H-NMR spectra (400 MHz) were recorded on a bruker AVII 400 MHz spectrometer. The chemical shifts were reported in (ppm) using the 7.26 signal of CDCl3 (1H-NMR) as internal standards. 13C-NMR spectra (100 MHz) were recorded on a bruker AVII 100 MHz spectrometer. The chemical shifts were reported in (ppm) using the 77.0 signal of CDCl3 (13C-NMR) as internal standards.

Procedure for the Preparation of Compound 2-10.

(E)-methyl 3-(4-nitrophenyl)acrylate (2): Compound 1 (25 g, 120 mmol) dissolved in 125 ml methanol, stirred until the material dissolved. Then H2SO4 (conc.) (2 g) was added slowly. Then reflux the solution overnight until the material consumed well. After check the TLC, filtered the solution to get the compound 2 (25 g, Yield=93%). 1H-NMR (400 MHz, CDCl3) δ: 3.84 (s, 3H), 6.57 (d, J=16.0 Hz, 1H), 7.66-7.71 (m, 2H), 7.23 (d, J=16.0 Hz, 1H), 8.24-8.27 (m, 2H).

(E)-methyl 3-(4-aminophenyl)acrylate (3): Compound 2 (20 g, 100 mmol) was dissolved in 160 ml of THF and 80 ml of methanol and stirred. NH4Cl (48 g, 900 mmol) dissolved in 80 ml of water and the solution was added. Then add Fe (21.5 g, 400 mmol) and stirred. The reaction mixture was refluxed about 5 h. Afterwards check the TLC, filtered the solution. The solvent was poured into NaHCO3 (aq.), to make pH-8, extracted with EtOAc, washed with water and brine/ dried over Na2SO4. Then concentrated the organic phase to develop compound 3 (5.25 g, Yield=30%). 1H-NMR (400 MHz, CDCl3) δ: 3.74 (s, 3H), 3.96 (d, J=3.2 Hz, 2H), 6.24 (d, J=16.0 Hz, 1H), 6.64-6.66 (m, 2H), 7.26-7.36 (m, 2H), 7.61 (d, J=16.0 Hz, 1H).

(E)-methyl 3-(4-(tert-butoxycarbonylamino)phenyl)acrylate (4): Compound 3 (5 g, 28 mmol) and Di-tert-butyl dicarbonate(Boc anhydride) (12.3 g, 56 mmol) dissolved in 50 ml dry THF and stirred. Then added 40 ml of Triethylamine and heated reflux overnight. After checking the TLC, concentrated to get the crude product. The crude product was then chromatographed (10:1- petroleum ether/EtOAc) to afforded compound 4 (6.8 g, Yield=95%). 1H-NMR (400 MHz, CDCl3) δ: 1.52 (s, 9H), 3.80 (s, 3H), 6.35 (d, J=16.0 Hz, 1H), 6.66(s, 1H), 7.38- 7.47 (m, 4H), 7.64 (d, J=16.0 Hz, 1H).

(E)-tert-butyl 4-(3-hydroxyprop-1-enyl)phenylcarbamate (5): Compound 4 (7 g, 25 mmol) dissolved in 70 ml DCM at -65ºC and stirred. Then DIBAl-H (8.97 g, 63 mmol) added drop wise. After finish the dropping, stirred the solution at RT for 3 h. Afterwards check the TLC, the reaction was quenched with KNaC4H4O6·4H2O, extracted with EtOAc, washed with water and brine/dried over Na2SO4. Then concentrated the organic phase to get compound 5 (4.25 g, Yield=68%). 1H-NMR (400 MHz, CDCl3) δ: 1.52 (s, 9H), 4.29-4.32 (m, 2H), 6.24- 6.31 (m, 1H), 6.51 (s, 1H), 6.53-6.58 (m, 1H), 7.32 (s, 4H).

microbial-biochemical-technology-Synthesis-compounds

Figure 1: Synthesis of compounds 2-10. Reagents and condition: (a) CH3OH, H2SO4, reflux, overnight, 93%; (b) NH4CL, THF, Fe, reflux, 5h, 30%; (c) (Boc)2O,THF, TEA, reflux, overnight, 95%; (d)DIBA1-H, DCM,-65°C, 3h, 68%; (e) CH3COOH, NaNo2, 40°C, 3h, 86%; (f) DMP, DCM, rt, overnight, 94%; (g) NH4OH.HCl, EtOH, Pyridine, 95°C, 1h, 80%; (h) SOCL2,DMF,0°C, 10min, rt, 1h, 80%; (1)TFA,DCM,0°C, 5min, rt,5h,82%.

4-(4-tert-butoxucarbonylamino)phenyl)-3-(hydroxymethyl)- 1,2,5-oxadiazole-2-oxide(6): Compound 5 (3 g, 12 mmol) was dissolved in 50 ml of acetic acid and stirred. Then a solution of NaNO2 (2.08 g, 30 mmol) was added drop wise in 20 ml of water, the reaction mixture was stirred at 40ºC for 3 h. After that check the TLC, the reaction residue was poured into NaHCO3(aq.), to make pH-7, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude product was then chromatographed (10:1- petroleum ether/EtOAc) to afforded compound 6 (3.2 g, Yield=86%). 1H-NMR (400 MHz, CDCl3) d: 1.54 (s, 9H), 4.75 (d, J=6.4 Hz, 2H), 7.53-7.56 (m, 2H), 7.74-7.77 (m, 2H); 13C-NMR (100 MHz, CDCl3) d: 28.5, 52.6, 80.1, 115.5, 118.7, 120, 128.8, 142.9, 153.1, 157.3.

microbial-biochemical-technology-Reagents-condition

Figure 2: Synthesis of compounds 11-17. Reagents and condition: (a) n-BuLi, THF, N2, -78°C, 15min, 0°C, 15min, -78°C, 18h, 69-70%; (b) NaNo2, H2So4, DME, -5C, rt, overnight, 29-30%; (c) NaNo2, H2So4, DCE, 50°C, 1h, 71%.

4-(4-(tert-butoxycarbonylamino)phenyl)-3-formyl-1,2,5- oxadiazole-2-oxide (7): Compound 6 (3.2 g, 10 mmol) dissolved in 50 ml of DCM and Dess Martin Periodinane (5.3 g, 12 mmol) was added. The solution was stirred overnight at RT. After checking the TLC, the solution was filtered, the filtered cake was washed with DCM twice. Combine the filtrate, washed with NaHCO3 (aq.), extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get compound 7 (3 g, Yield=94%). 1H-NMR (400 MHz, CDCl3) d: 1.47 (s, 9H), 4.05 (q, J=8.0 Hz, 1H), 7.43-7.48 (m, 2H), 7.80-7.83 (m, 2H).

( E ) - 4 - ( 4 - ( te r t - butoxyc arbony l amino)pheny l ) - 3 - ((hydroxyimino)methyl)-1,2,5-oxadiazole-2-oxide (8): Compound 7 (3 g, 9 mmol) was dissolved in 10 ml of Ethanol and stirred. Then NH4OH.HCl (857 mg, 12 mmol) and Pyridine (1.01 g, 12 mmol) was added. The solution was stirred at 95ºC for 1 h. TLC showed that the material was consumed. Then NH4OH.H2O was added to make the pH-7, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude product was then chromatographed (10:1- petroleum ether/EtOAc) to afforded compound 8 (2.5 g, Yield=80%). 1H-NMR (400 MHz, CDCl3) d: 1.53 (s, 9H), 7.49-7.54 (m, 2H), 7.66-7.84 (m, 2H), 8.05 (s, 1H).

microbial-biochemical-technology-aromatic-compounds

Figure 3: Synthesis of compounds 19-28. Reagents and condition: (a) DMP, DCM, rt, overnight, 90%; (b) PBr3, DCM, 0°C, 2h, 68%; (c) CH3OH, rt, 2h, 40-59%; (d) aromatic compounds, DCM, 0°C-rt, 2h-5h, 73-80%; (e) AgNO3, CH3CN, Light protected, 70°C, 5h, 64%.

Compound Structure Conc. (µm) Number of Worms Killing Activity*
24 h 48 h 72 h
1640 - - 8 0.00% 0.00% 0.00%
1% DMSO - - 9 0.00% 0.00% 0.00%
PZQ equation 10 8 0.00% 0.00% 0.00%
25 7 14.3% 28.6% 42.9%
50 9 22.2% 22.2% 44.4%
100 7 28.6% 42.9% 71.4%
1,2,5-Oxadiazole-2-oxide equation 10 9 0.00% 0.00% 11.1%
25 8 75.0% 100% 100%
50 8 100% 100% 100%
100 8 100% 100% 100%
06 equation 10 8 0.00% 0.00% 0.00%
25 8 0.00% 0.00% 25%
50 10 0.00% 40.0% 40.0%
100 8 0.00% 75.0% 100%
09 equation 10 8 0.00% 0.00% 0.00%
25 8 0.00% 50.0% 75.0%
50 8 50.0% 100% 100%
100 8 50.0% 100% 100%
10 equation 10 8 0.00% 0.00% 50.0%
25 10 0.00% 40.0% 60.0%
50 8 75.0% 100% 100%
100 8 100% 100% 100%
14 equation 10 10 0.00% 0.00% 0.00%
25 8 0.00% 50.0% 50.0%
50 8 0.00% 100% 100%
100 8 75.0% 100% 100%
15 equation 10 8 0.00% 0.00% 0.00%
25 8 0.00% 0.00% 0.00%
50 8 0.00% 0.00% 0.00%
100 8 75.0% 100% 100%
16 equation 10 8 0.00% 75.0% 100%
25 8 100% 100% 100%
50 8 100% 100% 100%
100 8 100% 100% 100%
17 equation 10 10 0.00% 40.0% 40.0%
25 8 50.0% 100% 100%
50 10 100% 100% 100%
100 8 100% 100% 100%
20 equation 10 8 0.00% 75.0% 100%
25 9 66.7% 100% 100%
50 10 100% 100% 100%
100 8 100% 100% 100%
21 equation 10 8 0.00% 0.00% 0.00%
25 10 0.00% 0.00% 0.00%
50 8 0.00% 0.00% 50.0%
100 8 100% 100% 100%
22 equation 10 8 0.00% 0.00% 25.0%
25 8 0.00% 0.00% 25.0%
50 8 0.00% 0.00% 25.0%
100 8 25.0% 25.0% 100%
23 equation 10 9 0.00% 66.7% 77.8%
25 9 44.4% 100% 100%
50 8 100% 100% 100%
100 8 100% 100% 100%
24 equation 10 8 0.00% 0.00% 0.00%
25 8 100% 100% 100%
50 10 100% 100% 100%
100 8 100% 100% 100%
25 equation 10 8 0.00% 0.00% 0.00%
25 8 0.00% 0.00% 0.00%
50 8 0.00% 0.00% 0.00%
100 8 0.00% 50.0% 50.0%
26 equation 10 8 0.00% 0.00% 0.00%
25 8 0.00% 0.00% 66.7%
50 12 0.00% 0.00% 50.0%
100 8 0.00% 0.00% 50.0%
27 equation 10 10 0.00% 0.00% 0.00%
25 8 0.00% 0.00% 0.00%
50 10 0.00% 0.00% 0.00%
100 8 0.00% 0.00% 25.0%
28 equation 10 8 0.00% 0.00% 0.00%
25 8 75.0% 100% 100%
50 10 100% 100% 100%
100 8 100% 100% 100%

Table 1: Worm-killing activity on S. japonicum adult worms in vitro (Only the active compound) 1640 without any drugs in the negative control; 1% DMSO to join the maximum drug concentration of DMSO concentration; PZQ for praziquantel at different concentrations on adults killing effect, as a positive control.

4-(4-(tert-butoxycarbonylamino)phenyl)-3-cyano-1,2,5- oxadiazole-2-oxide (9): Compound 8 (2 g, 6 mmol) was dissolved in 30 ml of Dimethylformamide at 0ºC and stirred. Then SOCl2 (1.8 ml, 24 mmol) was added slowly at 0ºC. Stirred at 0ºC for 10 min. Then stirred at RT for 1 h. After checking the TLC, slowly added NH3 solution then filter and concentrated to get the compound 9 (1.5 g, Yield=80%). 1H-NMR (400 MHz, CDCl3) δ: 1.54 (s, 9H), 7.58 (d, J=8.0 Hz, 2H), 7.86 (d, J=8.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ: 28.5, 80.4, 108, 117.5, 118.9, 128.2, 144, 153, 155.1.

4-(4-aminophenyl)-3-cyano-1,2,5-oxadiazole-2-oxide (10): Compound 9 (1 g, 3 mmol) was dissolved in DCM and stirred at 0ºC. Then add TFA (1.51 g, 13 mmol) was added at 0ºC and stirred for 5 min. The solution then stirred at RT for 5 h. After checking the TLC, the reaction residue was poured into NaHCO3 (aq.), to make pH-7, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (10:1- petroleum ether/EtOAc) to afford compound 10 (550 mg, Yield=82%). 1H-NMR (400 MHz, CDCl3) δ: 4.16 (s, 2H), 6.76-6.79 (m, 2H), 7.71-7.75 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ: 98, 108.4, 110.1, 114.2, 128.6, 153.4, 155.5.

General procedure for the preparation of compound 11-13

Phenylacetylene (6 g, 51 mmol) was dissolved in 100 ml of THF under N2 and cooled to -78ºC. n-BuLi (44 ml, 1.5 M, 66 mmol) was added drop wise. The reaction mixture was stirred at -78ºC for 15 min and for further 15 min at 0ºC. Then the suspension was cooled to - 78ºC. For compound 11; NCS (66mmol), compound 12; NBS (66 mmol) and compound 13; a solution of I2 in THF were added slowly. The reaction mixture was stirred for 1 h at -78ºC and for another 18 h at RT. After checking the TLC, add NH4OH.H2O was added to make the pH-7, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the compound.

(Chloroethynyl)benzene (11): Yield=70%; 1H-NMR (400 MHz, CDCl3) δ: 7.28-7.36 (m, 3H), 7.43-7.51 (m, 2H).

(Bromoethynyl)benzene (12): Yield=77%; 1H-NMR (400 MHz, CDCl3) δ: 7.24-7.36 (m, 3H), 7.43-7.50 (m, 2H).

(Iodoethynyl)benzene (13): Yield=69%; 1H-NMR (400 MHz, CDCl3) δ: 7.31-7.37 (m, 3H), 7.50-7.54 (m, 2H).

General procedure for the preparation of compound 14-16

Compound 11~13 (1 g, 7 mmol) dissolved in 20 ml of DME at -5ºC and added H2SO4 (14 mmol) and NaNO2 (14 mmol). The solution was stirred at RT for overnight. After checking the TLC, the reaction residue was poured into NaHCO3(aq.), extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (30:1~10:1- petroleum ether/EtOAc) to afford compound 14-16.

3-chloro-4-phenyl-1,2,5-oxadiazole 2-oxide (14): Yield=30%; 1H-NMR (400 MHz, CDCl3) δ: 7.47-7.74 (m, 3H), 7.82-7.88 (m,1H), 8.16-8.22 (m, 1H).

3-bromo-4-phenyl-1,2,5-oxadiazole 2-oxide (15): Yield=29%; 1H-NMR (400 MHz, CDCl3)δ: 7.46-7.55 (m, 3H), 7.82-7.86 (m, 1H), 8.16-8.18 (m, 1H).

3-iodo-4-phenyl-1,2,5-oxadiazole 2-oxide (16): Yield=29%; 1H-NMR (400 MHz, CDCl3) δ: 7.50-7.63 (m, 3H), 7.67-7.74 (m, 1H), 8.85-8.87 (m, 1H).

Procedure for the Preparation of Compound 17 (3-nitro-4- phenyl-1,2,5-oxadiazole-2-oxide)

2-Phenylacrylic acid (1 g, 6 mmol) was dissolved in 10 ml of DCE and H2SO4 (conc.) (9 mmol) was added. Then NaNO2 (1.4 g, 21 mmol) was added slowly into the solution. The solution was stirred for 1 h at 50ºC. After checking the TLC, the reaction residue was poured into Na2CO3 (aq.), extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (15:1- petroleum ether/EtOAc) to afford compound 17 (1 g, Yield=71%). 1H-NMR (400 MHz, CDCl3) δ: 7.55-7.69 (m, 5H).

Procedure for the preparation of compound 18-28

3-formyl-4-p-tolyl-1,2,5-oxadiazole 2-oxide (19): Compound 18 (500 mg, 2.5 mmol) dissolved in 20 ml of DCM and Dess Martin Periodinane (1.23 gm, 3 mmol) was added. The solution was stirred overnight at RT. After checking the TLC, the solution was filtered, the filtered cake was washed with DCM twice. Combine the filtrate, washed with NaHCO3(aq.), extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get compound 19 (450 mg, Yield=90%). 1H-NMR (400 MHz, CDCl3) δ: 2.38(s, 3H), 7.28 (d, J=8.0 Hz, 2H), 7.73 (d, J=8.0 Hz, 2H), 9.91 (s, 1 H).

3-(bromomethyl)-4-p-tolyl-1,2,5-oxadiazole 2-oxide (20): Compound 18 (1 g, 4 mmol) dissolved in 10 ml of DCM and cooled to 0ºC. Then PBr3 (0.5 ml, 5 mmol) was added drop wise. The solution was stirred at this temperature for 2 h. After checking the TLC, the reaction residue was poured into NaHCO3(aq.) to make pH-7, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get compound 20 (884 mg, Yield=68%). 1H-NMR (400 MHz, CDCl3) δ: 2.46 (s, 3H), 4.40 (s, 2H), 7.38 (d, J=8.0 Hz, 2H), 7.83 (d, J=8.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ: 17.5, 21.5, 113.3, 122.9, 127.4, 130.3, 142.1, 155.7.

(E)-3-((phenylimino)methyl)-4-p-tolyl-1,2,5-oxadiazole 2-oxide (21): Compound 19 (100 mg, 0.5 mmol) and Phenylamine (45 mg, 0.5 mmol) dissolved in 2 ml of methanol and stirred at RT for 2 h. After checking the TLC, concentrated the solution and chromatographed (30:1- petroleum ether/EtOAc) to afford compound 21 (80 gm, Yield=59%). 1H-NMR (400 MHz, CDCl3) δ: 2.44 (s, 3H), 7.20-7.22 (m, 2H), 7.30-7.33 (m, 3H), 7.38-7.41 (m, 2H), 7.89-7.91 (m, 2H), 8.43 (s, 1H); 13C-NMR (100 MHz, CDCl3) δ: 20.5, 113.2, 120.1, 121.8, 122.1, 127, 128.2, 128.3, 140.7, 149, 155.

(E)-3-((2-(6-chloropyridin-3-yl)hydrazono)methyl)-4-p-tolyl- 1,2,5-oxadiazole 2-oxide (22): Compound 19 (100 mg, 0.5 mmol) and 2-chloro-5-hydrazinylpyridine (70 mg, 0.5 mmol) dissolved in 3 ml of methanol and stirred at RT for 2 h. After checking the TLC, filtered the solution. Filtered cake(solid) is compound 22 (64 mg, Yield=40%). 1H-NMR (400 MHz, CDCl3) δ: 2.49 (s, 3H), 7.13-7.19 (m, 2H), 7.35 (d, J=7.6 Hz, 2H), 7.71 (d, J=8.0 Hz, 2H), 8.01 (d, J=2.4 Hz, 1H), 8.13 (s, 1H)); 13C-NMR (100 MHz, CDCl3) δ: 21.6, 113.4, 123.3, 124, 124.5, 129, 129.3, 134.7, 138.7, 141.8, 143.5, 155.6.

3-(chloromethyl)-4-p-tolyl-1,2,5-thiadiazole 2-oxide (23): Compound 18 (50 mg, 0.24 mmol) dissolved in 5 ml of DCM at 0ºC. Then SOCl2 (85 mg, 0.6 mmol) was added slowly at the same temperature. Then stirred at RT for 5 h. After checking the TLC, the reaction residue was poured into NaHCO3(aq.) to make pH-7, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get compound 23 (40 mg, Yield=74%). 1H-NMR (400 MHz, CDCl3) δ: 2.46 (s, 3H), 4.59 (s, 2H), 7.38 (d, J=8.0 Hz, 2H), 7.67 (d, J=8.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ: 21.5, 32.8, 113, 123, 127.8, 130.3, 142.1, 156.

3-(iodomethyl)-4-p-tolyl-1,2,5-thiadiazole 2-oxide (24): Compound 18 (50 mg, 0.24 mmol) dissolved in 5 ml of DCM and added PPh3 (94 mg, 0.35 mmol) and stirred for 5 min. Then imidazole (21 mg, 0.3 mmol) was added and stirred another 5 min and cooled it down to 0ºC. Then add I2 (182 mg, 0.7 mmol) was added slowly and stirred at RT for 2 h. After checking the TLC, the reaction residues was poured into water, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (5:1- petroleum ether/EtOAc) to afford compound 24 (60 mg, Yield=79%). 1H-NMR (400 MHz, CDCl3) δ: 2.46 (s, 3H), 4.25 (s, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.65 (d, J=8.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ: 13.6, 21.6, 114.7, 123.2, 127.4, 130.3, 142, 155.2.

3-(fluoromethyl)-4-p-tolyl-1,2,5-thiadiazole 2-oxide (25): Compound 18 (100 mg, 0.5 mmol) was dissolved in 5 ml of DCM at 0ºC and 1 drop of methanol was added and stirred. Then DAST (619 mg, 4 mmol) was added slowly at this temperature. The solution was stirred at 0ºC for 1 h. After checking the TLC, the reaction residues was poured into water, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (10:1- petroleum ether/EtOAc) to afford compound 25 (80 mg, Yield=79%). 1H-NMR (400 MHz, CDCl3) δ: 2.45 (s, 3H), 5.40 (d, J=48.0 Hz, 2H), 7.36-7.39 (m, 2H), 7.65-7.69 (m, 2H); 13C-NMR (100 MHz,CDCl3) δ: 21.5, 72.7, 111.2, 123, 127.5, 130.2, 142.2, 156.9.

3-(benzoyloxymethyl)-4-p-tolyl-1,2,5-oxadiazole 2-oxide (26): Compound 18 (50 mg, 0.24 mmol) and Benzoyl Chloride (40 mg, 0.3 mmol) was dissolved in 3 ml of DCM and stirred. Then add TEA (36 mg, 0.35 mmol) was added and stirred at RT for 5 h. After checking the TLC, the reaction residues was poured into water, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (10:1- petroleum ether/EtOAc) to afford compound 26 (60 mg, Yield=80%). 1H-NMR (400 MHz, CDCl3) δ: 2.42 (s, 3H), 5.38 (s, 2H), 7.32-7.34 (m, 2H), 7.43-7.47 (m, 2H), 7.54-7.66 (m, 3H), 7.99-8.00 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ: 21.5, 54.8, 111.3, 123.2, 127.5, 128.6, 128.7, 129.9, 130.1, 133.8, 142, 156.8, 165.7.

3-((2-chloroacetoxy)methyl)-4-p-tolyl-1,2,5-oxadiazole 2-oxide (27): Compound 18 (50 mg, 0.24 mmol) and Chloroacetyl chloride (40 mg, 0.3 mmol) was dissolved in 3 ml of DCM and stirred. Then add TEA (36 mg, 0.35 mmol) and stirred at RT for 5 h. After checking the TLC, the reaction residues was poured into water, extracted with EtOAc, washed with water and brine/dried over Na2SO4 and concentrated to get the crude product. The crude products were then chromatographed (10:1- petroleum ether/EtOAc) to afford compound 27 (50 mg, Yield=73%). 1H-NMR (400 MHz, CDCl3) δ: 2.45 (s, 3H), 4.11 (s, 2H), 5.25 (s, 2H), 7.35-7.36 (m, 2H), 7.57-7.59 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ: 21.5, 40.2, 55.6, 110.7, 123, 127.5, 130.2, 142.1, 156.7, 166.6.

3-(nitrooxymethyl)-4-p-tolyl-1,2,5-thiadiazole 2-oxide (28): Compound 20 (50 mg, 0.19 mmol) was dissolved in 2 ml of Acetonitrile and AgNO3 (75 mg, 0.7 mmol) was added into a flask protected from light. The suspension was stirred for 5 h at 70ºC. After cooling to room temperature, the solution was filtered, the solvent was concentrated, then extracted with EtOAc, the water phase washed again with two times, then combine the organic phase and brine/dried over Na2SO4 and concentrated to get compound 28 (30 mg, Yield=64%). 1H-NMR (400 MHz, CDCl3) δ:: 2.46 (s, 3H), 5.49 (s, 2H), 7.37 (d, J=8.0 Hz, 2H), 7.57 (d, J=8.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ: 21.5, 61.8, 108.9, 122.6, 127.5, 130.3, 142.4, 156.7.

Parasites killing activity of compounds 2-28 on schistosoma paponicum adult worms in vitro: Compounds 2-28 solutions and praziquantel were prepared by dissolving 1 mg of the drugs in 0.4 ml dimethyl sulfoxide (DMSO) and by adding 0.6 ml RPMI 1640 medium. S. japonicum worms obtained from mice (C57BL/6, female, 22–24 g, each of them was infected with 50 cercariae) were washed in RPMI 1640 medium, kept at pH 7.5 with HEPES 20 mM and supplemented with penicillin (100 UI/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, Gibco). Centrifugation was performed for 1 min at 1500 rpm. After centrifugation, the supernatant solution was removed. The cercariae at the bottom were washed twice and resuspended with RPMI 1640. Approximately 8-20 cercariae (1 ml of the resuspension medium) were added to one well in a 24 well culture plate. They were incubated overnight at 37°C with 5% CO2 and then different concentrations of compounds 2-28 (10, 25, 50, 100 μM) diluted with RPMI 1640 medium were added. Control worms were treated with equal volumes of RPMI 1640 or DMSO, and worms treated with 10, 25, 50, 100 μM praziquantel were also observed. The worm mobility, tegumental alterations and parasite survival were monitored under an inverted microscope (Leica, Wetzlar, Germany) at 24, 48 and 72 H. Parasite death was defined as having no motor activity during 2 min of continuous observation as well as morphological and tegumental alterations. The tests were repeated two times when compounds showed worm killing activity below the concentration of 100 μM.

Results

Chemistry

As shown in Figure 1, we designed compound 10 with adding amine functional group to position 1 substitution and examined the worm killing activities against Schistosoma japonicum. During the chain reaction, we found compound 6 and compound 9 also have worm killing activities against Schistosoma japonicum.

From the Figure 1, compound 1 was esterified with methyl to develop compound 2, where it was reduced by Fe to get the compound 3. The amino was protected with (Boc)2O at 80-90ºC to afford the compound 4, followed by closing the ring with NaNO2. The hydroxyl was oxidized with Dess-Martin Periodinane at room temperature with yield 94% to get the aldehyde. To extend compound 8 reacted with NH2OH.HCl to get the oxime, then hydrolyzed to get cyano compound 9. Finally it was deprotection with TFA to get the compound 10 with a yield of 82%.

As the earlier reports, we found that 3-(bromomethyl)-4-phenyl- 1,2,5-oxadiazole 2-oxide exhibited activity against Schistosoma japonicum. So, we designed some new analogues changing its position 13 substitutions with different atoms. The halogenated group includes chloride (-Cl), iodide (-I), bromide (-Br), nitrogen dioxide (-NO2). In, phenylacetylene react with a thiolate salt presence of an amine mediator with excellent yields ranging from 65-80% (Compound 11-13). Then it was thermal isomerization with NaNO2 and H2SO4 (conc.) to afford the compound 14-16. Compound 17 obtained by domino reaction of acrylic acids with action of sodium nitrite NaNO2.

After getting the activity result of Figures 1 and 2, we resynthesized and design more analogues by changing the same position 13 with halide and other atoms. The title 1,2,5-oxadiazole-2-oxide derivatives shown in Figure 3, wherein, R includes methylene chloride, methylene iodide, methylene fluoride, n-methyleneaniline, 2-chloro- 5-(2-methylenehydrazinyl) pyridine, methyl benzoate, methyl 2-chloroacetate, methyl nitrate which all show worm killing activity against Schistosoma japonicum.

Biological activities

We checked the biological activities from compound 2-28, among them most of the compound showed good worm killing activities. Table 1 contains the compound which is only showing activities.

Discussion

On the basis of Figures 1-3, we checked their worm killing activity. Among the 28 compounds, 16 compounds showed worm killing activity. Table 1 shows the list of that compound. Compound 6 containing a hydroxyl group killed 100.0 ± 0.0% of worms at 100 μM in 72 h. Continuation the chain reaction compound 7 and 8 have not shown any activity. Compound 9 with a cyano group and position 1 with Boc anhydride has also shown good worm killing activity, concentration of 50 μM in 48 h. Compound 10 by changing the 1 position with an amine functional group also exhibits same activity. So, we believe cyano group and hydroxyl group is important factor against the killing activity.

Comparing with earlier compounds, compounds 14-16 changed the cyano group with halogen atom showed good worm killing activity ever. Compound 14 containing a chloro atom killed 100.0 ± 0.0% of worms at 50 μM in 48 h, while compound 15 containing bromo atom killed 100.0 ± 0.0% of worms at 100 μM in 48 h. Compound 16 showed excellent activity which contains an iodine atom that killed 100.0 ± 0.0% of worms at 10 μM in 72 h. Compound 17 containing a nitro group in position 3, also have better activity than other compounds. In here, there are no cyano groups, but still they have shown good activity against the Schistosoma japonicum. Halogen atom could be a new kind to treat schistosomiasis, where it killed all worms at very low concentration with very short period of time.

By increasing the ring size of oxadiazole-2-oxide analogues, Compound 21 and compound 22 contains a phenylamino and chloropyridin atom also showed worm killing activity. Following the compound 20, 23, 24 also showed best result. These compounds containing a CH2 with Bromo, chloro, Iodine atom killed 100.0 ± 0.0% of worms. Compound 24 killed 100.0 ± 0.0% of worms at 25 μM in 24 h. If we see the compound 16 and 24, both containing an iodine atom but compound 24 contains an additional CH2 bond. We believe, iodine could have an important role in killing worms because of its low concentration of killing activity. Also, if we compare compound 15 and compound 20, bromide makes an excellent activity in which we believe its electron withdrawn group. Compound 26 and 27 have also showed better activity than PZQ. Compound 28 containing nitro-oxymethyl group killed 100.0 ± 0.0% of worms at 25 μM in 48 h. If we check the structure of 17 and 28 they are similar and their activity is almost same. So, nitro group could be a new way of treatment. Evaluation of their cytotoxity is in progress. This structure activity relationship (SAR) could have important implications for further drug development of the oxadiazole-2-oxides against schistosomiasis. The oxadiazole-2-oxide core is a class of NO donating compounds [22]. These compounds show us that schistosomiasis may have other targets, while different compounds work on different target group. The killing activities were done against the adult worms. This study helps us to synthesize some novel oxadiazole-2-oxide analogues to kill the worm also their structure activity relationship with it. There is a great chance to combined therapy with these compounds to control the schistosomiasis.

Conclusion

The designs and synthesizes of novel 1,2,5-oxadiazole-2-oxide analogues are relatively simple in structure and easy to prepare and could be used for preparation of medicaments for treatment of schistosomiasis, and have the same effect as commercial drugs and can overcome the poor efficacy of the existing anti-schistosomiasis drug-induced drug resistance or ineffective treatment. The only chemotherapy PZQ has the potential of risk to prevent widespread of schistosomiasis all over the world. There is an urgent need of new agent, which can work against both juvenile and adult worms. We should select one suitable and appropriate candidate to developed and employed. In this work, we studied about 1,2,5-oxadiazole-2-oxide and its related previous published journal. We believe its analogue can be a potential candidate for treatment of schistosomiasis. Some of these were shown very good result during the in-vitro process. The progress regarding in vivo activity and toxicity will be reported later on.

Acknowledgement

This work was supported by grants from the National Natural Science Foundation of China (Nos. 30972581, 8120316), the Natural Science Foundation of Jiangsu Province, China (Nos. BK2012544, BK20151120), the Laboratory Research of Parasitic Disease Prevention and Control Platform (No. wk014-002), and Jiangsu Science and Technology Department (No. BM2015024).

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