Indian Journal of Chemistry Vol. 56B, January 2017, pp. 112-119 Synthesis and antischistosomal activity of new furoxan derivatives of praziquantel Singam Naveen Kumara, Partha Sarathi Sadhua, Kirti Kumari Sharmaa,b, Livia Pica-Mattocciac, Annalisa Bassoc, Donato Ciolic & Vaidya Jayathirtha Rao*a,b a Crop Protection Chemicals Division and b Academy of Scientific & Innovative Research (AcSIR) CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500 007, India c Institute of Cell Biology and Neurobiology, National Research Council, 00015 Monterotondo, Rome, Italy E-mail: vaidya.opv@gmail.com; dcioli@ibc.cnr.it Received 8 August 2016; accepted (revised) 19 December 2016 A series of new furoxan derivatives of praziquantel have been synthesized and evaluated for antischistosomal activity. The newly synthesized hybrid compounds have structural modifications at amide and aromatic rings and thus offer broad structure-activity variations. All the compounds have been tested against adult as well as immature Schistosoma mansoni. Compounds 15 and 18 show moderate activity against adult schistosomes. On immature worms, only compound 15 shows substantial activity whereas the standard drug PZQ is practically inactive at this stage. Keywords: Schistosomiasis, Praziquantel, furoxan, hybrid drug, Schistosoma mansoni, antischistosomal Schistosomiasis is a chronic infectious disease caused by trematode flatworms of the genus Schistosoma. It is the second most prevalent parasitic disease in the world after malaria1. WHO reports suggest that 240 million people are infected worldwide and 280,000 deaths/year were estimated in sub-Saharan Africa alone2. In this scenario, Praziquantel (PZQ) is the sole drug available for the treatment of schistosomiasis. It is highly effective against all schistosome species that cause human schistosomiasis (mainly S. mansoni, S. japonicum and S. haematobium)3,4. PZQ is administered to millions of people annually in mass chemotherapy programs. This may lead to the emergence of drug-resistant parasites. Although no conclusive report of clinically relevant drug resistance or tolerance has appeared5, various isolates of S. mansoni and S. haematobium have shown different levels of PZQ sensitivity6,7. The main limitation of PZQ is that it is inactive against juvenile schistosomes8, a fact that would require the administration of a second delayed dose to achieve complete cure. In search of new antischistosomal agents, synthesis of PZQ analogs has attracted the attention of various research groups worldwide. A number of structural modifications were introduced in the PZQ molecule to improve its antischistosomal activity9-13. Previously, our group also reported new PZQ analogs having some antischistosomal activity13. However, PZQ still remains the most potent drug among all reported analogs. Recently, novel oxadiazole-2-oxide analogs (furoxans) were reported as most promising antischistosomal agents other than PZQ14. Furoxans are well known to release nitric oxide (NO) in the presence of TGR (Thioredoxin Glutathione Reductase) and NADPH and have shown activity even against the juvenile stage of schistosomes, unlike PZQ which is fully active only against adult schistosomes14. Hybrid molecules are defined as chemical entities with two or more different pharmacophores. If pharmacophores have different biological mechanisms of action, the emergence of drug resistance is much less likely to occur. The hybrid drug concept has been introduced for diseases like cancer, heart conditions, malaria and HIV-AIDS15-18. Recently, a few hybrid molecules were reported to have moderate antischistosomal activity19-22. As a continuation of our work to unearth new antischistosomal agents13, in this paper we report the synthesis and antischistosomal activity of twenty new hybrid entities combining the NO donor furoxan moiety with PZQ. As both moieties have excellent activity against schistosome species, we thought that the hybrid entities might be worth a trial. KUMAR et al.: FUROXAN DERIVATIVES OF PRAZIQUANTEL Results and Discussion Chemistry The synthetic scheme was designed to prepare novel hybrid compounds with possible structural variations for a better understanding of structure– activity relationships. A total of twenty new furoxan derivatives of PZQ were prepared with structural variations at amide and aromatic moieties. Synthesis of vital intermediate furoxan compounds 5a-f, 6a-f is depicted in Scheme I (Ref 23). Synthesis starts with C2-Wittig homologation of commercially available substituted benzaldehydes 1a-f to give ethyl cinnamates 2a-f. Reduction of ester functionality of 2a-f with DIBAL-H yielded cinnamyl alcohols 3a-f. Further reaction of 3a-f with sodium nitrite in presence of glacial acetic acid afforded furoxan methanol derivatives 4a-f (Ref 23). Finally, furoxan intermediates i.e., furoxan acid derivatives 5a-f were synthesized from 4a-f using Jones oxidation protocol23, whereas synthesis of bromo derivatives 6a-f was accomplished by reaction of 4a-f with NBS in the presence of triphenylphosphine. Synthesis of hybrid compounds 8-15, 16-27 is depicted in Scheme II. Hexahydro-4H-pyrazinoisoquinoline derivatives 7a,b were synthesized according to the literature procedures24. The first series of hybrid compounds 8-15 were synthesized by reaction between furoxan acid compouds 5a-f 113 and amine compounds 7a,b under EDCI and HOBT conditions in 48-65% yields. The second series of hybrid compounds 16-27 were synthesized by the reaction of furoxan bromo compounds 6a-f with 7a,b in the presence of K 2CO3 as base. Biology In vitro anti-schistosomal test of Furoxan Derivatives of Praziquantel In a preliminary activity screening, we tested all compounds in vitro against adult schistosomes8, using a high concentration of each substance (100 µM) that was left continuously in the cultures for 3 days. Only two compounds showed lethal effects, while all the rest did not exert any adverse activity on the schistosomes. Compounds 15 and 18 rendered worms immobile and contracted. The two positive compounds were then tested at lower concentration with the standard protocol we use for PZQ8, namely leaving the substance in contact with the worms for 20 hr, washing and continuing culture for 7 days in drug-free medium. Results are shown in Table I. To put things in perspective, one should consider that PZQ under these conditions causes total contraction and death of adult schistosomes at concentrations between 1 µM and 3 µM. We also tested all compounds against immature schistosomes (4-week-old) that are known to be refractory to PZQ activity. Following the same procedure we had Scheme I — Reagents and conditions: (a) (OEt)2P(O)CH2COOEt, NaH, 0°C-RT, 1 h. (b) DIBAL-H, CH2Cl2, 0°C, 4 h. (c) Glacial acetic acid, NaNO2, RT, 4-6 h, 30-75%. (d) CrO3, H2SO4, Acetone. (e) PPh3, NBS, DCM, 2-3 h. INDIAN J. CHEM., SEC B, JANUARY 2017 114 Scheme II — Reagents and conditions: (a) 5a-f, EDC, HCl, HOBT, CH2Cl2, −20°C, 1 h., (b) 6a-f, K2CO3, Acetone, 4 h. Table I — In vitro activity after 20 h exposure followed by culture in drug-free medium. Observations on day 7 after drug wash. Each dish contained either 10 adults or ~20 four-week-old worms Table II — Effect of compounds 15 and 18 on adult worms upon in vivo treatment Compd Immature worms 50 µM Compd 75% immobile, contracted 6% immobile, contracted Slightly lower mobility, otherwise normal Untreated control 15 15 18 PZQ Adult worms 25 µM 50 µM Slow, slightly Immobile, slightly contracted contracted Slow, Immobile, contracted contracted Immobile, Immobile, contracted contracted adopted for the adults, all compounds were left for 3 days in the cultures at the concentration of 100 µM. As with adults, only compounds 15 and 18 showed activity. When tested at lower concentration and following the pulse-and-wash procedure, only compound 15 showed a substantial activity, while PZQ, as expected8, was essentially ineffective (Table I). Since compounds 15 and 18 appear to possess some antischistosomal activity, we also carried out preliminary in vitro tests of toxicity using HepG2 cells. Compound 15 did not show toxic effects at the highest concentration tested (25 µM), while cells treated with compound 18 were free of toxic effects at 5 µM, but gave signs of toxicity at 25 µM. The two compounds showing some in vitro activity against adult worms, were also tested in vivo25. Results are shown in Table II. As a term of comparison, PZQ at 500 mg/kg causes >80% worm 18 Dose (mg/kg) No. of Worms recovered (mg/kg) mice (mean ± S.D.) P (t-test) – 6 16.0±11.3 – 400 800 400 800 6 6 5 6 16.8±8.6 14.0±2.6 10.4±2.7 17.2±5.4 0,8888 n.s. 0,6820 n.s. 0,3114 n.s. 0,8242 n.s. reduction. None of the animals showed overt signs of toxicity after treatment. Experimental Section All reagents (highest grade) were commercially available and were used without further purification unless otherwise noted. All dry reactions were carried out under an inert atmosphere unless mentioned otherwise, and standard syringe-septa techniques were followed. Solvents were freshly dried and purified by conventional methods prior to use. The progress of all reactions was monitored by TLC, using TLC aluminium-backed sheets precoated with silica gel 60 F254 to a thickness of 0.25 mm (Merck). Column chromatography was performed on silica gel (60–120 mesh and 100-200 mesh), and EtOAc, hexane were used as eluents. Melting points were determined using the Barnstead Electrothermal digital KUMAR et al.: FUROXAN DERIVATIVES OF PRAZIQUANTEL melting point apparatus and are uncorrected. IR spectra were recorded with a Perkin-Elmer FT-IR spectro-photometer. 1H and 13C NMR spectra were recorded with Varian Gemini 200 MHz, Bruker Avance 300 MHz, Varian Unity 400 MHz or Varian Inova 500 MHz spectrometers. TMS was used as an internal standard in CDCl3. Mass spectra were recorded with a VG Micromass 7070H (EI), QSTAR XL high resolution mass spectrometer, and a Thermo Finnigan ESI ion trap mass spectrometer. General procedure for the synthesis of final compounds 8-15 To a solution of acid compound 5a-f (0.10 mmol) in DCM (25 mL) at −20°C, EDCI (0.2 mmol), HOBT (0.2 mmol) were added simultaneously. After 15 min of stirring at the same temperature, the amine compound 7a/7b (0.15 mmol) and triethylamine (0.30 mmol) were added and the reaction mixture was stirred for one additional hour. After completion of the reaction, the mixture was quenched by addition of water and extracted twice with chloroform (2×10 mL). The combined organic layer was washed with dil. HCl and sat. NaHCO3, then dried (Na2SO4) and concentrated under reduced pressure to give the crude product. The crude residue was purified by silica gel column chromatography to yield pure compound 8-15. 3-(4-Oxo-2,3,4,6,7,11b-hexahydro-1H-pyrazino [2,1-a]isoquinoline-2-carbonyl)-4-phenyl-1,2,5oxadiazole 2-oxide, 8: Yield 55%. Light brown solid. m.p.160-63°C; IR (neat): 3012, 2928, 2855, 1656, 1597, 1019, 755 cm−1; 1H NMR (300 MHz, CDCl3): 7.79-7.72 (m, 2H), 7.57-7.43 (m, 3H), 7.33-7.09 (m, 4H), 5.10-5.00 (m, 1H), 4.98-4.72 (m, 2H), 4.16-4.12 (m, 1H), 4.09-3.96 (m, 1H), 3.41-3.20 (m, 1H), 3.05-2.74 (m, 3H); 13C NMR (75 MHz, CDCl3): 163.4, 162.9, 155.1, 135.1, 134.8, 131.8, 131.7, 131.1, 129.5, 129.2, 127.8, 127.7, 127.6, 127.1, 125.6, 125.3, 55.6, 54.5, 50.3, 48.5, 46.7, 45.9, 39.3, 38.8, 28.8, 28.5; ESI-MS: m/z 391 [M+H]+; ESI-HRMS: m/z Calcd for C21H19N4O4: 391.1406 [M+H]+. Found: 391.1400. 3-(9,10-Dimethoxy-4-oxo-2,3,4,6,7,11b-hexahydro -1H-pyrazino[2,1-a]isoquinoline-2-carbonyl)-4-phenyl -1,2,5-oxadiazole 2-oxide, 9: Yield 48%. Brown solid. m.p.198-200°C; IR (neat): 2930, 1656, 1598, 1253, 1016, 783 cm−1; 1H NMR (300 MHz, CDCl3): 7.78-7.70 (m, 2H), 7.57-7.45 (m, 3H), 6.75-6.61 (m, 2H), 5.03-4.76 (m, 3H), 4.17-4.08 (m, 2H), 3.88 115 (s, 3H), 3.85 (s, 3H), 3.35-3.27 (m, 1H), 2.97-2.79 (m, 2H), 2.74-2.66 (m, 1H). 13C NMR (75 MHz, CDCl3): 163.4, 162.8, 155.1, 154.8, 154.3, 148.6, 148.2, 131.8, 131.7, 129.2, 127.7, 127.5, 127.2, 125.3, 125.2, 123.4, 122.6, 111.9, 108.0, 56.2, 55.9, 55.4, 54.5, 50.9, 48.6, 46.8, 45.9, 39.5, 38.8, 28.1, 28.4; ESI-MS: m/z 451 [M+H]+; ESI-HRMS: m/z Calcd for C23H23N4O6: 451.1617 [M+H]+. Found: 451.1609. 4-(2-Methoxyphenyl)-3-(4-oxo-2,3,4,6,7,11bhexahydro-1H-pyrazino[2,1-a]isoquinoline-2-carbonyl) -1,2,5-oxadiazole 2-oxide, 10: Yield 48%. White solid. m.p.115-17°C; IR (neat): 3010, 2925, 2854, 1654, 1596, 1218, 771 cm−1; 1H NMR (300 MHz, CDCl3): 7.77-7.71 (m, 1H), 7.54-7.49 (m, 1H), 7.31-7.25 (m, 3H), 7.23-7.20 (m, 1H), 7.13 (dt, 1H, J = 7.6, 0.7 Hz), 6.95 (dd, 1H, J = 10.5, 8.3 Hz), 5.24-4.91 (m, 2H), 4.89-4.74 (m, 1H), 4.35-4.24 (m, 1H), 4.20-3.97 (m, 1H), 3.72-3.68 (m, 3H), 3.33-3.09 (m, 1H), 3.84-2.87 (m, 2H), 2.85-2.80 (m, 1H); 13C NMR (75 MHz, CDCl3): 163.6, 163.6, 156.4, 156.2, 155.9, 155.1, 154.5, 135.0, 134.7, 133.2, 133.1, 131.8, 131.3, 130.0, 129.9, 129.4, 129.4, 127.7, 127.7, 127.1, 125.8, 125.3, 121.7, 114.6, 114.4, 112.1, 111.9, 111.2, 55.6, 55.4, 55.3, 54.4, 50.4, 48.3, 46.5, 45.9, 39.0, 38.6, 28.8, 28.6; ESI-MS: m/z 421 [M+H]+; ESI-HRMS: m/z Calcd for C22H21N4O5: 421.1511 [M+Na]+. Found: 421.1498. 3-(9,10-Dimethoxy-4-oxo-2,3,4,6,7,11b-hexahydro 1H-pyrazino[2,1-a]isoquinoline-2-carbonyl)-4-(2methoxyphenyl)-1,2,5-oxadiazole 2-oxide, 11: Yield 47%. Brown solid. m.p.188-90°C; IR (neat): 3013, 2924, 2853, 1654, 1596, 1218, 771 cm−1; 1H NMR (300 MHz, CDCl3): 7.76-7.68 (m, 1H), 7.53-7.49 (m, 1H), 7.14-7.10 (m, 1H), 6.95 (dd, 1H, J = 13.7, 8.2 Hz), 6.78-6.65 (m, 2H), 5.14-4.70 (m, 3H), 4.35-4.23 (m, 1H), 4.13-3.99 (m, 1H), 3.89-3.70 (m, 6H), 3.71 (s, 1.5H), 3.65 (s, 1.5H), 3.23-3.13 (m, 1H), 2.98-2.82 (m, 2H), 2.75-2.69 (m, 1H); 13 C NMR (75 MHz, CDCl3): 163.6, 163.4, 156.3, 156.2, 155.7, 154.8, 154.5, 153.8, 148.5, 148.4, 148.1, 148.1, 133.1, 133.0, 129.8, 129.7, 127.3, 126.9, 123.5, 122.8, 121.5, 114.5, 114.3, 112.2, 111.8, 111.6, 111.1, 108.8, 107.9, 56.1, 56.0, 55.8, 55.4, 55.2, 55.2, 54.1, 50.7, 48.2, 46.5, 45.8, 39.0, 38.5, 28.3, 28.0; ESI-MS: m/z 481 [M+H]+; ESI-HRMS: m/z Calcd for C24H25N4O7: 481.1723 [M+H]+. Found: 481.1731. 4-(4-Methoxyphenyl)-3-(4-oxo-2,3,4,6,7,11b-hexahydro -1H-pyrazino[2,1-a]isoquinoline-2-carbonyl)-1,2,5oxadiazole 2-oxide, 12: Yield 52%. Whitish solid. m.p.184-86°C; IR (neat): 3002, 2925, 2854, 1632, 116 INDIAN J. CHEM., SEC B, JANUARY 2017 1575, 1021, 763 cm−1; 1H NMR (300 MHz, CDCl3): 7.73-7.68 (m, 2H), 7.33-7.10 (m, 4H), 7.01-6.95 (m, 2H), 5.11-5.00 (m, 1H), 4.96-4.73 (m, 2H), 4.15-4.13 (m, 1H), 4.11-3.98 (m, 1H), 3.85 (s, 3H), 3.39-3.19 (m, 1H), 3.00-2.75 (m, 3H); 13C NMR (75 MHz, CDCl3): 163.4, 162.9, 162.2, 162.1, 155.3, 155.2, 154.6, 154.4, 135.0, 134.8, 131.7, 131.0, 129.4, 129.2, 129.1, 127.8, 127.7, 127.0, 125.5, 125.2, 117.5, 117.3, 114.6, 114.6, 110.4, 110.1, 55.5, 55.4, 55.4, 54.5, 50.7, 50.3, 48.5, 46.6, 45.8, 39.2, 38.7, 28.7, 28.5; ESI-MS: m/z 421 [M+H]+; ESI-HRMS: m/z Calcd for C22H21N4O5: 421.1511 [M+H]+. Found: 421.1500. 3-(9,10-Dimethoxy-4-oxo-2,3,4,6,7,11b-hexahydro -1Hpyrazino[2,1-a]isoquinoline-2-carbonyl)-4-(4methoxyphenyl)-1,2,5-oxadiazole 2-oxide, 13: Yield 46%. Yellow solid. m.p.96-98°C; IR (neat): 3012, 2924, 2853, 1653, 1575, 1255, 771 cm−1; 1H NMR (300 MHz, CDCl3): 7.70 (d, 1H, J = 9.0 Hz), 7.65 (d, 1H, J = 9.0 Hz), 6.97 (dd, 2H, J = 12.8, 9.0 Hz), 6.76-6.60 (m, 2H), 5.04-4.77 (m, 3H), 4.19-4.09 (m, 2H), 3.90-3.83 (m, 9H), 3.36-3.26 (m, 1H), 2.96-2.80 (m, 2H), 2.72-2.66 (m, 1H); 13C NMR (75 MHz, CDCl3): 163.5, 162.8, 162.1, 155.3, 154.5, 154.4, 148.6, 148.5, 148.1, 148.1, 129.2, 129.0, 128.8, 127.5, 127.1, 123.4, 122.5, 117.4, 117.3, 114.6, 114.4, 114.2, 111.8, 111.7, 110.6, 110.0, 108.5, 107.9, 56.1, 56.1, 55.8, 55.4, 54.4, 50.8, 48.5, 46.7, 45.7, 39.5, 38.8, 28.3, 28.0; ESI-MS: m/z 503 [M+Na]+; ESI-HRMS: m/z Calcd for C24H24N4O7Na: 503.15372 [M+Na]+. Found: 503.15582. 4-(3-Fluorophenyl)-3-(4-oxo-2,3,4,6,7,11bhexahydro-1H-pyrazino[2,1-a]isoquinoline-2carbonyl)-1,2,5-oxadiazole 2-oxide, 14: Yield 29%. Yellow solid. m.p.150-52°C; IR (neat): 3018, 2923, 2853, 1655, 1589, 1218, 770 cm−1; 1H NMR (300 MHz, CDCl3): 7.66-7.41 (m, 3H), 7.36-7.11 (m, 5H), 5.19-4.69 (m, 3H), 4.20-4.13 (m, 1H), 4.20-3.98 (m, 1H), 3.41-3.20 (m, 1H), 3.05-2.77 (m, 3H); ESI-MS: m/z 409 [M+H]+; ESI-HRMS: m/z Calcd for C21H18FN4O4: 409.1306 [M+H]+. Found: 409.1303. 4-(2-Fluorophenyl)-3-(4-oxo-2,3,4,6,7,11bhexahydro-1H-pyrazino[2,1-a]isoquinoline2-carbonyl)-1,2,5-oxadiazole 2-oxide, 15: Yield 31%. White solid. m.p.148-50°C; IR (neat): 3011, 2926, 2857, 1726, 1657, 1602, 1459, 1294, 771 cm−1; 1 H NMR (300 MHz, CDCl3): 7.88-7.69 (m, 1H), 7.59-7.51 (m, 1H), 7.38-7.27 (m, 3H), 7.23-7.10 (m, 3H), 5.20-4.75 (m, 3H), 4.26-4.00 (m, 2H), 3.43-3.12 (m, 1H), 3.02-2.88 (m, 2H), 2.85-2.78 (m, 1H); ESI-MS: m/z 409 [M+H]+; ESI-HRMS: m/z Calcd for C21H18FN4O4: 409.1304 [M+H]+. Found: 409.1303. General procedure for the synthesis of final compounds 16-27 To a solution of bromo compound 6a-f (0.10 mmol) and amine 7a/7b (0.12 mmol) in CH2Cl2, was added K2CO3 (0.5 mmol) and the reaction mixture was stirred for 4 h. After completion of reaction, the reaction mixture was diluted with water, the organic layer was separated and the aqueous layer was extracted using chloroform. The combined organic layer was dried (Na2SO4), concentrated under reduced pressure and purified using silica gel column chromatography to yield pure compound. 3-((4-Oxo-3,4,6,7-tetrahydro-1H-pyrazino[2,1a]isoquinolin-2(11bH)-yl)methyl)-4- phenyl-1,2,5oxadiazole 2-oxide, 16: Yield 92%. White solid. m.p.98-101°C; IR (neat): 3017, 2932, 1645, 1599, 1457, 1027, 757 cm−1; 1H NMR (300 MHz, CDCl3): 7.92 (d, 1H, J = 1.51 Hz), 7.89 (d, 1H, J = 1.5 Hz), 7.58-7.52 (m, 3H), 7.24-7.15 (m, 3H), 7.08-7.04 (m, 1H), 4.86-4.73 (m, 2H), 3.69 (dd, 2H, J = 18.4, 13.5 Hz), 3.58 (dd, 1H, J = 16.0, 1.3 Hz), 3.48 (ddd, 1H, J = 12.0, 4.7, 1.8 Hz), 3.19 (d, 1H, J = 16.05 Hz), 3.04-2.88 (m, 2H), 2.81-2.74 (m, 1H), 2.64-2.57 (m, 1H); 13C NMR (75 MHz, CDCl3): 165.2, 157.4, 134.8, 133.5, 131.3, 129.3, 129.27, 127.8, 127.2, 126.7, 126.4, 124.5, 112.0, 56.7, 55.5, 55.1, 48.6, 38.9, 28.5; ESI-MS: m/z 377 [M+H]+; ESI-HRMS: m/z Calcd for C21H21O3N4: 377.1608 [M+H]+. Found: 377.1607. 3-((9,10-Dimethoxy-4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)4-phenyl-1,2,5-oxadiazole 2-oxide, 17: Yield 93%. White solid. m.p.120-22°C; IR (neat): 3014, 2933, 1645, 1598, 1454, 1027, 748 cm−1; 1H NMR (300 MHz, CDCl3): 7.90 (d, 1H, J = 1.8 Hz), 7.88 (d, 1H, J = 1.8 Hz), 7.58-7.51 (m, 3H), 6.62 (s, 1H), 6.49 (s, 1H), 4.86-4.77 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.69 (dd, 2H, J = 17.56, 13.59 Hz), 3.56 (dd, 1H, J = 16.0, 1.3 Hz), 3.44 (ddd, 1H, J = 11.8, 4.3, 1.8 Hz), 3.19 (d, 1H, J = 16.05 Hz), 2.94-2.81 (m, 2H), 2.71-2.63 (m, 1H), 2.58 (dd, 1H, J = 11.89, 10.19 Hz); 13C NMR (75 MHz, CDCl3): 165.0, 157.4, 148.1, 147.8, 131.3, 129.2, 127.8, 127.1, 126.3, 125.2, 112.0, 111.7, 107.5, 56.7, 56.1, 55.8, 55.8, 54.8, 48.5, 38.9, 28.1; ESI-MS: m/z 437 [M+H]+; ESI-HRMS: m/z Calcd for C23H25O5N4: 437.18195 [M+H]+. Found: 437.18097. KUMAR et al.: FUROXAN DERIVATIVES OF PRAZIQUANTEL 4-(2-Methoxyphenyl)-3-((4-oxo-3,4,6,7-tetrahydro -1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl) -1,2,5-oxadiazole 2-oxide, 18: Yield 91%. Light brown solid. m.p.140-42°C; IR (neat): 3015, 2929, 2843, 1642, 1598, 1217, 749 cm−1; 1H NMR (300 MHz, CDCl3): 7.56-7.49 (m, 2H), 7.19-7.16 (m, 2H), 7.12-7.09 (m, 3H), 6.88-6.85 (m, 1H), 4.70-4.64 (m, 1H), 4.44 (dd, 1H, J = 9.82, 4.5 Hz), 3.86 (s, 3H), 3.75 (dd, 2H, J = 27.1, 14.3 Hz), 3.32 (dd, 1H, J = 15.8, 1.5 Hz), 3.23 (ddd, 1H, J = 12.0, 4.5, 2.2 Hz), 2.92 (d, 1H, J = 15.8 Hz), 2.82-2.65 (m, 3H), 2.30 (dd, 1H, J = 12.0, 10.5 Hz); 13C NMR (75 MHz, CDCl3): 165.2, 157.1, 155.8, 134.7, 133.5, 132.8, 130.6, 129.2, 127.1, 126.5, 124.5, 121.2, 115.6, 113.3, 111.2, 56.4, 55.6, 55.6, 55.0, 48.9, 38.6, 28.5; ESI-MS: m/z 407 [M+H]+; ESI-HRMS: m/z Calcd for C22H23N4O4: 407.17193 [M+H]+. Found: 407.17077. 3-((9,10-Dimethoxy-4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)4-(2-methoxyphenyl)-1,2,5-oxadiazole 2-oxide, 19: Yield 92%. Brownish solid. m.p.148-50°C; IR (neat): 3014, 2929, 2843, 1644, 1598, 1217, 768 cm−1; 1H NMR (300 MHz, CDCl3): 7.57-7.49 (m, 2H), 7.11-7.01 (m, 2H), 6.58 (s, 1H), 6.38 (s, 1H), 4.78-4.71 (m, 1H), 4.45 (dd, 1H, J = 10.5, 5.2 Hz), 3.86 (s, 3H), 3.85 (drs, 6H), 3.74 (dd, 1H, J = 18.8, 14.3 Hz), 3.31 (dd, 1H, J = 15.8, 1.5 Hz), 3.24 (ddd, 1H, J = 12.0, 4.5, 1.5 Hz), 2.92 (d, 1H, J =15.8 Hz), 2.82-2.55 (m, 3H), 2.31 (dd, 1H, J = 12.0 10.57 Hz); 13C NMR (75 MHz, CDCl3): 165.1, 156.9, 155.8, 147.9, 147.6, 132.7, 130.4, 127.0, 125.1, 121.1, 115.4, 113.2, 111.5, 111.1, 107.6, 56.2, 56.0, 55.8, 55.7, 55.5, 54.8, 48.7, 38.5, 28.0; ESI-MS: m/z 467 [M+H]+; ESI-HRMS: m/z Calcd for C24H27N4O6: 467.1924 [M+H]+. Found: 467.19214. 4-(4-Methoxyphenyl)-3-((4-oxo-3,4,6,7-tetrahydro 1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)1,2,5-oxadiazole 2-oxide, 20: Yield 92%. Light yellow solid. m.p.150-52°C; IR (neat): 3004, 2928, 2837, 1646, 1594, 1016, 762 cm−1; 1H NMR (300 MHz, CDCl3): 7.83 (d, 1H, J = 8.35 Hz), 7.22-7.20 (m, 2H), 7.16-7.14 (m,1H), 7.07-7.04 (m, 1H), 7.02 (d, 1H, J = 8.35 Hz), 4.83 (dd, 1H, J = 9.4, 4.1 Hz), 4.77-4.74 (m, 1H), 3.86 (s, 3H), 3.66 (dd, 2H, J = 22.9, 13.5 Hz), 3.56 (dd, 1H, J = 15.6, 1.0 Hz), 3.46 (ddd, 1H, J = 12.5, 5.2, 2.0 Hz), 3.17 (d, 1H, J = 15.6 Hz), 3.89-3.00 (m, 2H), 2.77-2.73 (m, 1H), 2.60 (m, 1H); 13C NMR (75 MHz, CDCl3): 165.1, 161.8, 157.1, 134.7, 133.5, 129.3, 129.2, 127.1, 126.6, 124.5, 118.6, 114.7, 111.9, 56.7, 55.4, 55.3, 55.0, 117 48.6, 38.8, 28.5; ESI-MS: m/z 407 [M+H]+; ESI-HRMS: m/z Calcd for C22H23N4O4: 407.1719 [M+H]+. Found: 407.1707. 3-((9,10-Dimethoxy-4-oxo-3,4,6,7-tetrahydro-1H -pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)-4(4-methoxyphenyl)-1,2,5-oxadiazole 2-oxide, 21: Yield 91%. Light brown solid. m.p.170-72°C; IR (neat): 3010, 2928, 2838, 1645, 1594, 1014, 749 cm−1; 1 H NMR (300 MHz, CDCl3): 7.82 (d, 1H, J = 9.4 Hz), 7.02 (d, 1H, J = 9.4 Hz), 6.61 (s, 1H), 6.49 (s, 1H), 4.83-4.76 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.66 (dd, 2H, J = 22.9, 13.5 Hz), 3.55 (dd, 1H, J = 16.7, 1.0 Hz), 3.42 (ddd, 1H, J = 11.4, 4.1, 2.0 Hz), 3.18 (d, 1H, J = 16.7 Hz), 2.94-2.82 (m, 2H), 2.67-2.63 (m, 1H), 2.59 (d, 1H, J = 12.5, 10.4 Hz); 13 C NMR (75 MHz, CDCl3): 165.0, 161.7, 157.0, 147.9, 147.6, 129.2, 126.9, 125.2, 118.4, 114.5, 111.9, 111.5, 107.4, 56.5, 55.9, 55.6, 55.6, 55.2, 54.7, 48.4, 38.8, 28.0; ESI-MS: m/z 467 [M+H]+; ESI-HRMS: m/z Calcd for C24H27N4O6: 467.1930 [M+H]+. Found: 467.1921. 4-(3-Bromophenyl)-3-((4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)1,2,5-oxadiazole 2-oxide, 22: Yield 94%. White color solid. m.p.151-53°C; IR (neat): 3016, 2934, 2844, 1643, 1599, 1217, 750 cm−1; 1H NMR (300 MHz, CDCl3): 8.24-8.23 (m, 1H), 7.88 (dd, 1H, J = 6.7, 1.5 Hz), 7.22-7.68 (m, 1H), 7.42 (t, 1H, J = 8.3 Hz), 7.25-7.16 (m, 3H), 7.13-7.09 (m, 1H), 4.95 (dd, 1H, J = 10.5, 4.5 Hz), 4.84-4.78 (m, 1H), 3.69 (s, 2H), 3.57 (dd, 1H, J = 15.8, 1.5 Hz), 3.51 (ddd, 1H, J = 12.0, 4.5, 2.2 Hz), 3.21 (d, 1H, J = 15.8 Hz), 3.05-2.88 (m, 2H), 2.81-2.75 (m, 1H), 2.66 (dd, 1H, J = 12.0, 9.8 Hz); 13C NMR (75 MHz, CDCl3): 164.8, 155.9, 134.7, 134.0, 133.3, 131.0, 130.7, 129.2, 128.2, 127.1, 126.6, 126.1, 124.5, 123.0, 111.5, 56.2, 55.7, 55.1, 48.7, 38.7, 28.5; ESI-MS: m/z 455 [M+H]+; ESI-HRMS: m/z Calcd for C21H20O3N4Br: 455.07133 [M+H]+. Found: 455.07050. 4-(3-Bromophenyl)-3-((9,10-dimethoxy-4-oxo3,4,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin2(11bH)-yl)methyl)-1,2,5-oxadiazole 2-oxide, 23: Yield 92%. Light brown solid. m.p.161-63°C; IR (neat): 3017, 2937, 2837, 1643, 1601, 1217, 743 cm−1; 1 H NMR (300 MHz, CDCl3): 8.23 (brs, 1H), 7.87 (d, 1H, J = 8.3 Hz), 7.70 (d, 1H, J = 8.3 Hz), 7.42 (t, 1H, J = 8.3 Hz), 6.64 (s, 1H), 6.56 (s, 1H), 6.56 (s, 1H), 4.93-4.83 (m, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.69 (s, 2H), 3.56 (d, 1H, J = 15.8 Hz), 3.48 (dd, 1H, J = 12.0, 3.0 Hz), 3.22 (d, 1H, J = 15.8 Hz), 118 INDIAN J. CHEM., SEC B, JANUARY 2017 2.93-2.80 (m, 2H), 2.68-2.60 (m, 2H); 13C NMR (75 MHz, CDCl3): 164.7, 155.9, 147.9, 147.6, 134.0, 131.0, 130.7, 128.2, 127.0, 126.1, 124.9, 123.0, 111.6, 107.4, 56.2, 56.1, 55.9, 55.7, 54.9, 48.7, 38.7, 28.1; ESI-MS: m/z 515 [M+H]+; ESI-HRMS: m/z Calcd. for C23H24O5N4Br: 515.09246 [M+H]+. Found: 515.09094. 4-(3-Fluorophenyl)-3-((4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)1,2,5-oxadiazole 2-oxide, 24: Yield 91%. White solid. m.p.145-47°C; IR (neat): 3010, 2931, 2845, 1643, 1599, 1217, 749 cm−1; 1H NMR (300 MHz, CDCl3): 7.74-7.70 (m, 2H), 7.56-7.48 (m, 1H), 7.31-7.17 (m, 4H), 7.10-7.05 (m, 1H), 4.48 (dd, 1H, J = 10.0, 4.5 Hz), 4.81-4.74 (m, 1H), 3.69 (dd, 2H, J = 16.8, 13.7 Hz), 3.58 (dd, 1H, J = 16.0, 1.5 Hz), 3.48 (ddd, 1H, J = 11.8, 6.4, 1.8 Hz), 3.20 (d, 1H, J = 16.2 Hz), 3.04-2.89 (m, 2H), 2.82-2.74 (m, 1H), 2.64 (dd, 1H, J = 11.8, 10.1 Hz); 13C NMR (75 MHz, CDCl3): 164.9, 163.8, 161.9, 156.3, 134.8, 133.4, 131.1, 131.1, 129.3, 128.3, 128.3, 127.2, 126.7, 124.5, 123.6, 123.5, 118.5, 118.3, 115.1, 114.9, 111.7, 56.6, 55.6, 55.1, 48.7, 38.9, 28.6; ESI-MS: m/z 395 [M+H]+; ESI-HRMS: m/z Calcd for C21H20O3N4F: 395.15140 [M+H]+. Found: 395.15057. 3-((9,10-Dimethoxy-4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)4-(3-fluorophenyl)-1,2,5-oxadiazole 2-oxide, 25: Yield 89%. Light brown solid. m.p.154-56°C; IR (neat): 3019, 1644, 1601, 1474, 1215, 742 cm−1; 1 H NMR (300 MHz, CDCl3): 7.73-7.69 (m, 2H), 7.56-7.49 (m, 1H), 7.31-7.28 (m, 1H), 6.63 (s, 1H), 6.5 (s, 1H), 4.87-4.79 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.69 (dd, 2H, J = 15.8, 13.5 Hz), 3.56 (dd, 1H, J = 15.8, 1.5 Hz), 3.45 (ddd, 1H, J = 12.0, 4.5, 2.2 Hz), 3.20 (d, 1H, J = 15.8 Hz), 2.90-2.81 (m, 2H), 2.71-2.66 (m, 1H), 2.61 (dd, 1H, J = 12.0, 10.5 Hz); 13 C NMR (75 MHz, CDCl3): 165.0, 164.5, 161.2, 156.4, 148.2, 147.8, 131.2, 131.1, 128.4, 128.2, 127.2, 125.1, 123.6, 123.6, 118.6, 118.3, 115.2, 114.9, 111.8, 111.8, 107.5, 56.5, 56.0, 55.9, 54.9, 48.7, 39.0, 28.2, 25.3; ESI-MS: m/z 455 [M+H]+; ESI-HRMS: m/z Calcd for C23H24O5N4F: 455.1726 [M+H]+. Found: 455.17087. 4-(4-Fluorophenyl)-3-((4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)1,2,5-oxadiazole 2-oxide, 26: Yield 91%. White solid. m.p.145-47°C; IR (neat): 3007, 2931, 2847, 1644, 1597, 1297, 771 cm−1; 1H NMR (300 MHz, CDCl3): 7.75-7.71 (m, 2H), 7.56-7.49 (m, 1H), 7.30-7.15 (m, 5H), 7.11-7.06 (m, 1H), 4.88 (dd, 1H, J = 9.8, 3.7 Hz), 4.81-4.76 (m, 1H), 3.69 (dd, 2H, J = 16.6, 13.5 Hz), 3.58 (dd, 1H, J = 15.8 Hz, 1.51 Hz), 3.48 (ddd, 1H, J = 12.0, 4.5, 2.2 Hz), 3.21 (d, 1H, J = 15.8 Hz), 3.04-2.89 (m, 2H), 2.82-2.74 (m, 1H), 2.64 (dd, 1H, J = 11.3, 9.8 Hz); 13C NMR (75 MHz, CDCl3): 164.9, 163.8, 161.9, 156.3, 156.3, 134.8, 133.4, 131.1, 131.1, 129.3, 128.3, 128.3, 127.2, 126.7, 124.5, 123.6, 123.5, 118.5, 118.3, 115.1, 114.9, 111.7, 56.6, 55.6, 55.1, 48.7, 38.8, 28.6; ESI-MS: m/z 395 [M+H]+; ESI-HRMS: m/z Calcd for C21H20O3N4F: 395.15140 [M+H]+. Found: 395.15063. 3-((9,10-Dimethoxy-4-oxo-3,4,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)methyl)4-(4-fluorophenyl)-1,2,5-oxadiazole 2-oxide, 27: Yield 90%. Light brown solid. m.p.161-63°C; IR (neat): 3009, 2934, 2836, 1645, 1598, 1221, 771 cm−1; 1 H NMR (300 MHz, CDCl3): 7.74-7.69 (m, 2H), 7.57-7.49 (m, 1H), 7.32-7.28 (m, 1H), 6.63 (s, 1H), 6.51 (s, 1H), 4.87-4.80 (m, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.69 (dd, 2H, J = 15.8, 13.5 Hz), 3.56 (dd, 1H, J = 16.6, 1.5 Hz), 3.45 (ddd, 1H, J = 12.0, 4.5, 2.2 Hz), 3.20 (d, 1H, J = 16.6 Hz), 2.94-2.82 (m, 2H), 2.71-2.65 (m, 1H), 2.62 (dd, 1H, J = 8.3, 6.79 Hz); 13 C NMR (75 MHz, CDCl3): 164.8, 163.8, 161.8, 156.3, 148.1, 147.8, 131.1, 131.0, 128.3, 128.2, 127.1, 125.1, 123.5, 123.5, 118.4, 118.2, 115.1, 114.9, 111.7, 107.5, 56.5, 56.0, 55.9, 55.8, 54.8, 48.6, 38.8, 28.1; ESI-MS: m/z 455 [M+H]+; ESI-HRMS: m/z Calcd for C23H24O5N4F: 455.17252 [M+H]+. Found: 455.17087. Conclusions The widespread use of a single drug (PZQ) for treatment of schistosomiasis may lead to the emergence of resistant parasites. Therefore, identify-cation of antischistosomal drugs other then PZQ, or to be used in combination with PZQ, are in high demand. In this work we successfully synthesized the furoxan derivatives of praziquantel and evaluated their antischistosomal activity against adult S. mansoni. Among all the synthesized compounds, compounds 15 and 18 showed unequivocal signs of worm damage in vitro, but not in vivo. This discrepancy can be most likely explained in terms of host metabolism. Also, compound 15 showed substantial activity against immature worms, while PZQ is inactive at this stage. As the preliminary study on these hybrid molecules has provided some hints at possible PZQ modifications leading to activity against immature parasites, an attempt is being made to synthesize KUMAR et al.: FUROXAN DERIVATIVES OF PRAZIQUANTEL additional hybrid molecules that enhanced antischistosomal activity. can show Supplementary Information Supplementary information is available in the website http://nopr.niscair.res.in/handle/123456789/60. Acknowledgements The IICT authors thank the Director IICT for support. SNK and PSS thank CSIR, New Delhi for the fellowships. VJR acknowledges ORIGIN-CSC-0108 for financial assistance. The authors from ICBN thank Giuliana Papoff of the Rome lab, for performing toxicity tests and Giovina Ruberti for logistic support. 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On week 6 after infection, mice were randomly divided into 5 groups: group 1 (untreated controls) received only the vehicle used to re-suspend the compounds; groups 2 and 3 received compound 15 at 400 and 800 mg/kg, respectively; groups 4 and 5 received compound 18 at 400 and 800 mg/kg, respectively. Compounds were initially suspended in DMSO and subsequently diluted with 4 parts of 2% Cremophor EL, to be administered to mice by oral gavage. Three weeks after treatment with a single dose, all mice were subjected to portal perfusion and the average number of schistosomes recovered reported in Table II.