Accepted Manuscript New Praziquantel Derivatives Containing NO-donor Furoxans and Related Furazans as Active Agents against Schistosoma mansoni Stefano Guglielmo, Daniela Cortese, Francesca Vottero, Barbara Rolando, Valerie P. Kommer, David L. Williams, Roberta Fruttero, Alberto Gasco PII:

S0223-5234(14)00613-8

DOI:

10.1016/j.ejmech.2014.07.007

Reference:

EJMECH 7127

To appear in:

European Journal of Medicinal Chemistry

Received Date: 5 April 2014 Revised Date:

20 May 2014

Accepted Date: 3 July 2014

Please cite this article as: S. Guglielmo, D. Cortese, F. Vottero, B. Rolando, V.P. Kommer, D.L. Williams, R. Fruttero, A. Gasco, New Praziquantel Derivatives Containing NO-donor Furoxans and Related Furazans as Active Agents against Schistosoma mansoni, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

New Praziquantel Derivatives Containing NO-donor Furoxans and Related Furazans as Active Agents against Schistosoma mansoni.

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Graphical abstract

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New Praziquantel Derivatives Containing NO-donor Furoxans and Related

2

Furazans as Active Agents against Schistosoma mansoni.

3

Stefano Guglielmo1, Daniela Cortese1,2, Francesca Vottero1, Barbara Rolando1,

4

Valerie P. Kommer2, David L. Williams2*, Roberta Fruttero1*, Alberto Gasco1

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1

Dipartimento di Scienza e Tecnologia del Farmaco, Università of Torino, Via P.

Giuria 9, 10125 Torino, Italy 2

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Department of Immunology/Microbiology, Rush University Medical Center, 1735

West Harrison Street, Chicago, IL 60612 USA

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*Corresponding authors:

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DLW: Phone: 1-312-942-1375; Fax: 1-312-942-2808; Email:

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[email protected]

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RF: Phone: +39 011 6707850; Fax: +39 011 6707286; Email:

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[email protected].

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Abstract

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A series of NO-donor praziquantel hybrid compounds was obtained by combining

17

praziquantel (PZQ) and furoxan moieties in a single entity. NO-donor properties of

18

the furoxan derivatives were evaluated by detecting nitrite after incubation of the

19

products in 7.4 pH buffered solution in the presence of L-cysteine. Structurally-

20

related furazans, devoid of NO release capacity, were also synthesized for control

21

purposes. All products were studied for their ability to inhibit recombinant

22

Schistosoma mansoni thioredoxin glutathione reductase (TGR). Mobility and death of

23

adult Schistosoma mansoni worms cultured in the presence of the products were

24

evaluated versus PZQ. Analysis of the results showed that some products were

25

endowed with both PZQ and NO-dependent antiparasitic properties. Compounds 6, 7,

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18, and 24 emerged as the most interesting balanced hybrids, worthy of additional

27

study on PZQ-resistant parasites.

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KEYWORDS furoxans, praziquantel, schistosomiasis, thioredoxin glutathione

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reductase, NO-donor praziquantel.

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Introduction

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Schistosomiasis, also known as Bilharzia, is one of the most prominent neglected

33

tropical diseases (NTDs). It is a parasitosis caused by blood-dwelling flatworms of

34

the genus Schistosoma; the principal species parasitizing humans are S. mansoni, S.

35

japonicum, and S. haematobium.1 Estimates indicate that 600 million people are at

36

risk of parasitosis and that more than 200 million people suffer from schistosomiasis,

37

resulting in 200,000 deaths each year. The highest incidence of schistosomiasis is in

38

sub-Saharan Africa, where the principal species responsible are S. mansoni and S.

39

haematobium.2-4 Praziquantel (PZQ) is the drug of choice for the treatment of

40

schistosomiasis.4,5 The structure (2-(cyclohexylcarbonyl)-1,2,3,6,7,11b-hexahydro-

41

4H-pyrazino[2,1-a]isoquinolin-4-one) (PZQ, Chart 1) assigned to the drug was

42

confirmed by X-ray analysis.6 It is a fairly lipophilic product, and is consequently

43

endowed with low water solubility.7 PZQ has a stereogenic center and can thus exist

44

as two optical stereoisomers: the levo isomer ((-)PZQ) is more active than the dextro

45

isomer ((+)PZQ), but since the dextro form has no side effects, for economic reasons

46

the drug is used as an orally-administered racemic mixture. PZQ is active against the

47

adult forms of all schistosome species, but not against the juvenile forms;8 its

48

mechanism of action is still unclear. Calcium accumulation, alteration of

49

schistosomal membrane fluidity, reduction of glutathione concentration, destruction

50

of the tegument following binding to schistosomal actin, and interference with

51

components of the parasite’s aerobic metabolism have all been proposed as possible

52

action mechanisms.4,9 A number of structural modifications of PZQ have been

53

introduced with the goal of improving its antihelmintic action.7,10 Most of the

54

resulting products are devoid of substantial activity; others show only moderate

55

effects, and none is better than the lead.

56

One problem associated with the extensive use of PZQ is the risk of the parasite’s

57

developing resistance.11,12 To date, there is no clear evidence for large-scale clinical

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resistance of schistosomes to treatment with PZQ. Conversely, laboratory studies

59

clearly indicate that resistance to PZQ can be selected. In particular it has been found

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that a schistosomal homologue of the mammalian P-glycoprotein (P-gp) is

61

upregulated in PZQ-treated worms and in juvenile worms, which are resistant to PZQ

62

activity.13 For this reason there is an urgent need to develop new chemical classes of

63

drugs for the treatment of schistosomiasis.

64

Recently, through a quantitative high-throughput screen, 1,2,5,-oxadiazole 2-oxides

65

(furoxans) have been identified as a new chemical class for the control of

66

schistosomiasis.14,15 Furoxan (1, Chart 1) is an old heterocyclic system, well known to

67

chemists because of arguments over its structure.16 In the recent past, renewed

68

interest has surrounded furoxan derivatives due to the discovery that they can release

69

nitric oxide (NO) under the action of thiol cofactors.17,18 The presence of electron

70

withdrawing groups at the ring, in particular at the 3-position, generally increases this

71

capacity.

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It has been shown that furoxan derivatives are able to inhibit thioredoxin glutathione

73

reductase (TGR), a multifunctional parasite protein that reduces both thioredoxin and

74

glutathione disulfide and provides deglutathionylation (glutaredoxin) activity in

75

worms.19 Specific reaction of furoxans with TGR results in localized NO production

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and subsequent S- (or Se-) nitrosation and inactivation of TGR. The exact nature of

77

the resulting TGR modifications is not known.20 Furoxan-3-carbonitrile derivatives

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are the most interesting compounds studied thus far. The prototype of this series is 4-

79

phenylfuroxan-3-carbonitrile (2, Chart 1). The product was found to be an

80

irreversible inhibitor of TGR (IC50 = 6.3 µM). It was active against all stages of S.

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mansoni and against cultured adult S. japonicum, and S. haematobium worms.

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Intraperitoneal injection of 2 at 10 mg/Kg in S. mansoni infected mice led to a

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marked reduction in worm burden when treatment occurred 1 day after infection

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(skin-stage parasites), 23 days after infection (juvenile, liver stage parasites), or 37

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days after infection (adult, egg-laying parasites).14,20 More recently, a number of

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phenylsulfonyl substituted furoxans have been found to be endowed with potent

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antischistosomal activity.21

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This paper describes a new series of compounds with potential dual antischistosomal

89

action obtained by combining, in a single entity, PZQ and NO-donor furoxan

90

derivatives bearing at the 3- position CN, CONH2, COOMe, or SO2C6H5 moieties. In

91

the first group of hybrids, the furoxan substructures were substituted for the

92

cyclohexyl group of PZQ (Chart 1, general structure A; Scheme 1, der.s 5-7). In the

93

second group of hybrids the furoxan moiety was linked to the 10-position of PZQ

94

through appropriate bridges (Chart 1, general structure B; Scheme 2, der.s 17, 18,

95

24). The synthesis, structural characterization, and preliminary in vitro and ex vivo

96

pharmacological profiles of all these products are reported and discussed. Related

97

furazan (1,2,5-oxadiazoles) derivatives, (Chart 1, general structure A; Scheme 1,

98

der.s 8-10; Chart 1, general structure B; Scheme 2, der.s 20, 21, 26) were also

99

considered for comparison, since their structures are closely related to those of the

100

corresponding furoxans, but they do not release NO (des-NO furazans). Therefore, if

101

a given biological activity of a furoxan derivative is similar to that of its furazan

102

analogue, it will be assumed that NO is not involved in that activity. The approach to

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compare the pharmacological profile of a NO donor with that of its analogue des-NO,

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is frequently used in the study of hybrid NO-donor drugs.22,a,b,c

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Chart 1

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N

N

O

PZQ

O

N

Furoxan/furazan moieties

O

4

3

N

N O 2

5

N

N

N

O

R'

R

+

O 1

1, 2 O

Furoxan/furazan moieties

O B der.s 17, 18, 24; 20, 21, 26

A der.s 5-7; 8-10

109 110 111

PZQ, Praziquantel; A, general structure of the furoxan hybrids 5-7, and of the related

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furazans 8-10; B, general structure of the furoxan hybrids 17, 18, 24, and of the 5

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related furazans 20, 21, 26; 1, R=R’=H, 1,2,5-oxadiazole 2-oxide (furoxan); 2,

114

R=C6H5, R’=CN, 4-phenylfuroxan-3-carbonitrile.

115

Results and Discussion

117

Chemistry.

118

Synthesis. The first series of NO-donor PZQ hybrids (5-7) and of the related des-NO

119

furazans (8-10) was obtained through the synthetic pathway reported in Scheme 1.

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The known hexahydro-4H-pyrazinoisoquinoline derivative 3 was coupled with 3-

121

methoxycarbonylfuroxan-4-carboxylic acid (4) in CH2Cl2/THF solution, in the

122

presence of N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

123

(EDC), and a catalytic amount of 4-(dimethylamino)pyridine (DMAP), to give the

124

furoxan ester 5. Treatment of this ester with methanol saturated with ammonia

125

afforded the related amide 6. Dehydration of this latter product with trifluoroacetic

126

anhydride in THF solution, in the presence of pyridine, gave rise to the corresponding

127

nitrile 7. The furazan target product 8 was obtained by reduction with

128

trimethylphosphite of the related furoxan 5. Furazans 9 and 10 were prepared from 8,

129

following the same procedures used to obtain the corresponding furoxans 6 and 7

130

from 5.

131

Scheme 1

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N N H 3

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HOOC O + N

COOMe

N

a

O

+

N O O 4

O

N

N

b OMe

O

N

+

5

N N

NH2 O

+

+

N O

N O N O

6

O

c

O

O

N O N O

N

O

N O

7

d

N

O

N

O

b N

O

OMe

O

N

N

9

8

6

O

O

N O

133

N

c

N

NH2

N O

N N O

O

10

N N O

ACCEPTED MANUSCRIPT

conditions:

(a)

N-hydroxysuccinimide,

EDC.HCl,

134

Reaction

cat.

DMAP,

135

CH2Cl2/THF; (b) CH3OH(NH3); (c) TFAA, pyridine, THF, 0°C; (d) (CH3O)3P reflux.

136

The second series of NO-donor PZQ hybrids (17, 18, 24) and of the related des-NO

138

furazans (20, 21, 26) was obtained through the synthetic pathway reported in Scheme

139

2. The commercial 2-(4-methoxyphenyl)ethanamine 11 was transformed into the

140

related isocyanide 12 by treatment with chloroform under basic conditions, in the

141

presence of a catalytic amount of triethylbenzylammonium chloride (TEBAC). The

142

10-methoxy-substituted PZQ 14, the common intermediate in preparing all target

143

compounds, was obtained from 12 by the same stepwise Ugi four-component

144

reaction and Pictect–Spengler reaction used by Cao et al. to prepare PZQ from 2-

145

phenylethanamine.23 Briefly, a mixture of paraformaldehyde, dimethoxyethylamine

146

and cyclohexylcarboxylic acid in methanol was treated with 12 to give 13 (Ugi

147

reaction). This intermediate was added to methanesulfonic acid to afford 17 (Pictect-

148

Spengler reaction). Cleavage with BBr3 of the methoxy group present in 14 gave rise

149

to the phenol 15. Reaction of 15 with the 4-(bromomethyl)furoxan-3-carboxamide

150

(16), or with the related furazan amide 19, in the presence of NaH yielded the

151

expected final compounds 17 and 20, respectively. Dehydration of these latter

152

compounds with trifluoroacetic anhydride in pyridine produced the final cyano-

153

substituted related hybrids 18, 21. To prepare the phenylsulfonyl-substituted target

154

structures 24, 26, the phenol derivative 15 was coupled with 3-bromopropan-1-ol in

155

CH3CN in the presence of K2CO3, to give the alcohol 22. This intermediate, treated

156

either with 3,4-bisphenylsulfonylfuroxan (23) or with the related furazan 25, gave the

157

desired final products.

158

Scheme 2

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7

ACCEPTED MANUSCRIPT MeO NH2

NC

a

MeO

MeO

N

b 13

N

MeO

c

N O

MeO

12

11

OMe O d N

O O 14

CONH 2

H2N

+

N

N O O

+

N

e

N

O

O N O N

16

BrCH 2

e

N

N

O

18

N O

H2N N

N

O

O

N O N

f

O N

N

15

HO

O

N

SO2Ph +

N O O

23

N

O

PhO2S

h

O

N

O

PhO2S N

h

O

24

SO2Ph

O

25

O

N

N + N O

O

22

N

O

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PhO2S

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N

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O

21

20

O

N

O

O

O

N

O

N

N

19

g

N

O

O 17

CONH 2 O

+

O N O N

f

O 15

O

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HO

O

SC

BrCH 2

N

N

O

N N O

N

O

O

N O 26

Reaction conditions: (a) NaOH/H2O, TEBAC, CHCl3, CH2Cl2, reflux; (b)

162

paraformaldehyde, 2,2-dimethoxyethylamine, cyclohexyl carboxylic acid, CH3OH;

163

(c) methanesulfonic acid, 0 °C then 70 °C; (d) BBr3, CH2Cl2; (e) NaH 60% mineral

164

oil disp., THF, reflux; (f) TFAA, pyridine, THF, 0°C; (g) 3-bromopropan-1-ol,

165

K2CO3, CH3CN, reflux; (h) DBU, CH2Cl2.

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NO-release. The capacity of the final furoxan hybrids to release NO was evaluated on

168

the basis of the amount of nitrite produced following incubation in buffered pH 7.4

169

solution in the presence of a 5:1 molar excess of L-cysteine. Nitrite was detected by

170

the Griess reaction. The results expressed as percentages of NO2- are reported in

171

Table 1. NO production ranks the series 7 > 18 > 24 > 6 ≈ 17 > 5. This sequence

172

should parallel the different capacities of the products to release NO under the action 8

ACCEPTED MANUSCRIPT 173

of free cellular thiols (‘nonspecific’ release). Potentially, the NO formed can diffuse

174

into the worm resulting in antiparasitic action, following interaction with a variety of

175

cellular targets.24

176

Biology

178

Inhibition of TGR. The inhibitory activity of all products described in this paper was

179

evaluated against recombinant S. mansoni TGR; the results, expressed as IC50, are

180

reported in Table 1. All the hybrid furoxan products acted as potent TGR inhibitors;

181

their inhibitory potency follows the series 6 > 17 > 5 > 7 ≈ 18 > 24. This sequence

182

should parallel the different capacities of the compounds to release NO under action

183

of the enzyme. This ‘specific’ NO release is dependent upon both binding affinity

184

and appropriately aligned reactivity of each product, and is a measure of each

185

product’s capacity to trigger antiparasitic action following its ability to inactivate

186

TGR. As expected, the des-NO furazan analogues did not display inhibitory activity

187

when tested up to 50 µM concentration.

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Action of compounds against ex vivo parasites. Adult S. mansoni worms were

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cultured in the presence of two different concentrations of furoxan and furazan

191

derivatives: 10 and 50 µM, in DMSO. The mobility and death of the parasites were

192

monitored over 144 hrs. In Table 1, the results are compared with those for PZQ and

193

2, taken as references. Contraction and paralysis of the worms are the first observable

194

effects of PZQ (Figure 1). After overnight culture with PZQ, these effects are

195

reversible and the worms return to normal length over several days (>2 days). At the

196

concentrations of PZQ tested, worms begin to die 4-5 days after PZQ is removed

197

from the culture media. The furoxan 2 displayed no effect on worm length (i.e., it did

198

not cause contractile paralysis), but quickly killed 100% of worms at both the

199

concentrations tested. This indicates that NO is not involved in worm contraction, but

200

is responsible for the rapid death of the parasites.

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Worm morphology was monitored after treatment with hybrid compounds to

202

indirectly assess their PZQ-like activity. Worms were contracted and mobility

203

impaired immediately after exposure to the first group of products 5-10. In the case of

204

furazans 8-10, the subsequent recovery after their removal from the medium was

205

faster than that observed for PZQ, at both concentrations tested. The hybrid furoxans

206

5-7 retained PZQ’s full worm-contraction ability at the higher concentration tested,

207

but mobility recovered faster at the 10 µM concentration. The hybrid furoxans were

208

more effective worm killers than the furazan analogues (compare 5/8, 6/9, 7/10), in

209

particular at the 50 µM concentration, indicating an involvement of NO in this action,

210

in keeping with what was observed with 2. The furoxans 6 and 7 emerge as the most

211

interesting products, because of their balance between PZQ and NO-dependent

212

activities. Findings concerning TGR IC50 and cysteine-induced NO release suggest

213

that, in the case of compound 7 (high NO release under the action of cysteine, high

214

TGR-inhibitory potency) the worm-killing activity might be induced by a

215

combination of both specific and nonspecific NO production provided that the

216

compound’s pharmacokinetic properties (e.g. worm uptake, metabolic fate, increased

217

efflux) do not limit its access to the TGR target. In the case of 6 (low nonspecific

218

release, high TGR inhibitory potency) specific NO release and TGR inhibition appear

219

to be principally involved. The modestly higher worm-killing activity of 5 compared

220

to 8 is in keeping with the low cysteine-induced NO release of product but not with

221

its high TGR inhibitory potency. This suggests that pharmacokinetic properties of 5

222

limit its access to this target.

223

The results obtained with the second group of compounds, 17, 18, 20, 21, 24, and 26,

224

indicate that, although at the higher concentration tested several products induced

225

worm contraction, this effect was of a much shorter duration than that of PZQ (less

226

than 10 minutes to ~24 hrs, depending on the compound), suggesting that these

227

compounds’ PZQ-like activity was limited (Figure 1). This is in line with findings

228

that modification of the PZQ aromatic ring significantly reduces activity.7,10 This

229

might be due to decreased interaction with parasite target protein(s), or to increased

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efflux of the compounds. Furoxans 18 and 24 have better worm killing activity than

231

the related furazans 21 and 26 suggesting that NO is involved in this action. Since

232

both furoxan derivatives are quite efficient NO-donors under the action of cysteine,

233

and quite potent TGR inhibitors, we can infer that both specific (if no critical

234

structural modifications of the products occur during their random walk to the target)

235

and nonspecific production of NO might be implicated in the killing activity. Furoxan

236

17 and the related furazan 20 did not kill worms. This inability is surprising in the

237

case of the furoxan derivative since it is a quite potent TGR inhibitor, although it is a

238

weak NO-donor under the action of cysteine. Metabolic fate, increased efflux, and

239

lack of worm uptake could be responsible for its inactivity. In light of these results,

240

the specific NO release following the direct interaction of furoxan derivatives with

241

TGR does not directly correlate with the ex-vivo killing activity due to the

242

complexity of the biological system involved. In conclusion, the only products of a

243

certain interest among those belonging to this group are the hybrids 18 and 24, which

244

retain some PZQ-like activity resulting in worm paralysis and also display NO-

245

dependent worm-killing capacity.

246

We recently demonstrated that furoxans can inhibit the activity of P-gp, MRP1 and

247

MRP3 transporters in different kinds of cells.25,26 Since multidrug resistance

248

transporters (MDR) are considered potentially attractive targets for the development

249

of new antischistosomal drugs,13 selected members of this new class of PZQ/furoxan

250

hybrid products are worthy of additional investigation, in view of their potential

251

activity against PZQ-resistant schistosomes.

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Table 1. Activity of compounds against TGR and cultured adult Schistosoma

254

mansoni worms and NO donation ability expressed as % of NO2-. TGR Compound

(µM)

IC50

%NO2Worm contraction

Worm killinga

(mol/mol) SEb

50 µM 11

10 µM

50 µM

10 µM

±

ACCEPTED MANUSCRIPT PZQ

CN

2d

N N

5.0

No

No

24 hrs

96 hrs

28.9 ± 0.3

O

OMe

0.148

Yes

Yese

50%c

0.01

Yes

Yese

80%

30%

1.8 ± 0.1

O

Yes

♀ only

72 hrs

30%c

56.2 ± 0.3

Yese

Yese

18%

15%

---

> 50

Yese

Weake

23%

23%

---

> 50

Yese

Yese

25%

20%

---

O O

NH2 +

N O N O

N

O N

0.316

N O

23%

SC

N O

M AN U

N

50

EP

N

O

TE D

+

N O N O

N

N

O N

10

---

+

N

9

15%c

O

N O

8

40%c

N O

O

7

Yes

RI PT

O

6

Yes

+

N

5

n.i.

N O

N N O

12

ACCEPTED MANUSCRIPT O

H2N +

17

N

O

O N O N

O

N

0.083

No

No

No

No

1.1 ± 0.2

0.35

Yesf

No

24 hrs

Noc

33.8 ± 0.1

> 50

No

No

No

No

---

No

No

---

24 hrs

48 hrs

15.7 ±0.1

Noc

No

---

O

N

18

N

O

O

N

RI PT

+

O N O N

O

O

N

20

N

O O N

O

N

N

21

N

O

N O N

O

> 50

N O

N

N + N O

O

O

O

26

PhO2S

N N O

O

N

O

N

O

8.5

♂ only

> 50

Yes

TE D

24

N

O

Yesf

No

Some,

not all

Yese

EP

O PhO2S

M AN U

O

SC

H2N

O

n.i. – no inhibition; n.d. – not determined; atime (hrs) to 100% worm death or % dead

256

worms at 144 hrs; bnitrite was measured (Griess reaction) after 1 hr incubation at 37

257

°C of the product in buffered solution (pH = 7.4) containing 5:1 excess of L-cysteine;

258

c

AC C

255

living worms moved very slowly; dRai et al. (reference 20); eworms contracted

259

immediately upon addition of compound, then relaxed over 24-48 hrs; fworms

260

contracted immediately upon addition of compound, then relaxed after < 10 min.

261

Figure 1. Images of adult Schistosoma mansoni worms 24 hrs after addition of

262

compounds. 13

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ACCEPTED MANUSCRIPT

263

14

ACCEPTED MANUSCRIPT

Conclusions. A new class of NO-donor PZQ hybrids was developed by joining NO-

265

donor furoxan moieties to different areas of the PZQ structure. The inhibitory activity

266

of these products, and that of the related des-NO furazan derivatives, was evaluated

267

against recombinant S. mansoni TGR; their antiparasitic action against ex vivo adult

268

S. mansoni worms was likewise evaluated. Products 6, 7, 18, and 24 emerged as

269

potent antischistosomal agents, endowed with both PZQ-like and NO-dependent

270

antiparasitic activity. These compounds are worthy of additional studies in view of

271

their potential activity against PZQ-resistant schistosomes.

SC

272

RI PT

264

Experimental section

274

Chemistry. 1H and 13C NMR spectra were recorded on a Bruker Avance 300 at 300

275

and 75 MHz, respectively, using SiMe4 as internal standard. Low resolution mass

276

spectra were recorded with a Finnigan-Mat TSQ-700. Melting points were

277

determined with a capillary apparatus (Büchi 540). Flash column chromatography

278

was performed on silica gel (Merck Kieselgel 60, 230-400 mesh ASTM). The

279

progress of the reactions was monitored by thin layer chromatography (TLC) on 5 cm

280

x 20 cm plates with a layer thickness of 0.2 mm. Organic solvents were removed

281

under vacuum at 30 °C. Elemental analyses (C, H, N) of the target compounds were

282

performed by Section de Pharmacie, Service de Microanalyse (Geneva), and the

283

results are within 0.4% of the theoretical values, unless otherwise stated. Target

284

compounds were prepared, as assessed using the aforementioned standard

285

spectroscopic techniques and elemental analyses, in ≥95% purity. Compounds 3;27

286

4;28 16;29 19;30 2331 and 2532 were obtained as described elsewhere.

AC C

EP

TE D

M AN U

273

287 288

Methyl

4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinolin-2-

289

yl)carbonyl]furoxan-3-carboxylate (5).

290

3-methoxycarbonyl-4-furoxancarboxylic acid (300 mg, 1.59 mmol) was dissolved in

291

anhydrous THF (7 ml) and CH2Cl2 (10 ml) and the solution obtained was cooled in

292

an ice bath. N-hydroxysuccinimide (1.1 eq.) and EDC.HCl (1.5 eq.) were added to the 15

ACCEPTED MANUSCRIPT

solution, which was kept under stirring at room temperature for one hour. A solution

294

of 1,2,3,6,7,11b-hexahydro-pyrazino[2,1-a]isoquinolin-4-one (1 eq.) in anhydrous

295

CH2Cl2 (10 ml) was then added and the resulting mixture was kept under stirring at

296

room temperature for 20 minutes. The solvent was removed under reduced pressure,

297

the residue was taken up with 30 ml of CH2Cl2 and washed with water (2 x 20 ml),

298

brine, dried over Na2SO4 and the solvent was evaporated under reduced pressure. The

299

residue was purified by flash chromatography on silica gel (eluent CH2Cl2/EtOAc

300

90/10) to give the title product as a white solid, yield 42%, mp 150-152 °C dec

301

(MeOH). 1

SC

302

RI PT

293

H-NMR (CDCl3): δ 2.78-3.06 (3H, m, H-6, 2 x H-7), 3.16 (0.5H, dd, J = 2.4, 13.2

Hz, H-1), 3.43 (0.5H, dd, J = 3.0, 13.8 Hz, H-1), 3.95 (3H, d, J = 6.0 Hz, -COOCH3),

304

4.05-4.41 (2H, m, 2 x H-3), 4.76-4.92 (1H, m, H-1), 4.98-5.22 (2H, m, H-6 and H-

305

11b), 7.22-7.34 (4H, m, aromatic protons).

306

13

M AN U

303

C-NMR (CDCl3): δ 28.7, 39.0, 46.1, 50.0, 53.9, 55.0, 106.5, 125.3, 127.1, 127.9,

129.6, 131.6, 135.0, 150.3, 155.2, 155.9, 163.1.

308

MS CI (isobutane) (m/z): 372 [MH+].

309

Anal. (C17H16N4O6) C, H, N.

TE D

307

310

4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinolin-2-

312

yl)carbonyl]furoxan-3-carboxamide (6).

313

A solution of 5 (390 mg, 1.05 mmol) in MeOH saturated with NH3 (15 ml) was kept

314

under stirring at room temperature for 10 minutes. A white solid precipitated and was

315

recovered by filtration. The solid obtained was recrystallized from MeOH, to give the

316

title product, yield 53%, mp 206-208 °C dec.

317

AC C

EP

311

1

H-NMR (DMSO-d6): δ 2.74-2.95 (3H, m, H-6, 2 x H-7), 3.26 (0.5H, dd, J = 2.1,

318

13.9 Hz, H-1), 3.45 (0.5H, dd, J = 3.0, 13.7 Hz, H-1), 3.96-4.15 (1H, m, H-1), 4.46-

319

4.68 (3H, m, 2 x H-3 and H-6), 4.91-4.99 (1H, m, H-11b), 7.18-7.39 (4H, m,

320

aromatic protons), 7.86 (1H, d, J = 13.5 Hz, –NH2), 8.50 (1H, d, J = 3.9 Hz, –NH2).

16

ACCEPTED MANUSCRIPT 321

13

C-NMR (DMSO-d6): δ 28.0, 45.2, 48.6, 53.9, 110.3, 125.6, 126.3, 126.6, 127.1,

322

128.9, 132.7, 134.9, 151.9, 154.8, 155.7, 163.0.

323

MS CI (isobutane) (m/z): 358 [MH+].

324

Anal. (C16H15N5O5) C, H, N.

RI PT

325

4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinolin-2-

327

yl)carbonyl]furoxan-3-carbonitrile (7).

328

A solution of 6 (130 mg, 0.36 mmol) in anhydrous THF (20 ml) was cooled in an ice

329

bath; anhydrous pyridine (4 eq.) and TFAA (4 eq.) were added to the solution. The

330

mixture was kept under stirring at 0°C for 30 minutes, then the solvent was removed

331

under reduced pressure. The residue was taken up with 20 ml of CH2Cl2 and washed

332

with H2SO4 0.5 M (2 x 10 ml), water (2 x 10 ml), brine, dried over Na2SO4, filtered

333

and evaporated under reduced pressure. The yellow solid obtained was purified by

334

flash chromatography on silica gel (eluent CH2Cl2/EtOAc 90/10) to give the title

335

product as white solid yield 83%, mp 183-185 °C dec. (CHCl3/n-Hex).

M AN U

1

H-NMR (CDCl3): δ 2.80-3.02 (3H, m, H-6, 2 x H-7), 3.18 (0.5H, dd, J = 2.7, 13.5

TE D

336

SC

326

Hz, H-1), 3.47 (0.5H, dd, J = 3.0, 13.7 Hz, H-1), 4.09 (0.5H, d, J = 18.6 Hz, H-1),

338

4.43 (0.5H, d, J = 18.3 Hz, H-1), 4.82-5.22 (4H, m, 2 x H-3, H-6 and H-11b), 7.22-

339

7.32 (4H, m, aromatic protons).

340

13

EP

337

C-NMR (CDCl3): δ 28.7, 39.0, 47.0, 50.0, 54.3, 55.7, 104.5, 125.4, 127.2, 128.0,

129.7, 131.3, 135.0, 150.4, 153.4, 163.0.

342

MS CI (isobutane) (m/z): 340 [MH+].

343

Anal. (C16H13N5O4) C, H, N.

AC C

341

344 345

Methyl

4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinolin-2-

346

yl)carbonyl]furazan-3-carboxylate (8).

347

A solution of 5 (330 mg, 0.89 mmol) in (CH3O)3P (5 ml) was refluxed for 4 hours.

348

The mixture was then poured into 20 ml of 2.5 M H2SO4 and cooled in an ice bath. A

349

white solid precipitated and was collected by filtration. The filtrate was extracted 17

ACCEPTED MANUSCRIPT

with CH2Cl2 (2 x 10 ml), then the organic phase was washed with H2SO4 2.5 M (3 x

351

10 ml), NaHCO3 saturated solution (3 x 10 ml), brine, dried over Na2SO4, filtered and

352

evaporated under reduced pressure. The residue was combined with the filtered-out

353

white solid and crystallized from MeOH/H2O, to give the title product as a white

354

solid, yield 47%, mp 141-142 °C.

355

1

RI PT

350

H-NMR (CDCl3): δ 2.78-3.01 (3H, m, H-6, 2 x H-7), 3.16 (0.5H, dd, J = 2.7, 13.4

Hz, H-1), 3.39 (0.5H, dd, J = 3.0, 13.7 Hz, H-1), 4.03 (3H, d, J = 3.9 Hz, -COOCH3),

357

4.14 (1H, s, H-3), 4.27 (1H, m, H-3), 4.79-5.06 (2H, m, H-1 and H-6), 5.23-5.28 (1H,

358

m, H-11b), 7.21-7.34 (4H, m, aromatic protons).

359

13

SC

356

C-NMR (CDCl3): δ 28.7, 39.0, 46.2, 50.2, 54.0, 54.5, 55.6, 125.3, 127.5, 129.5,

131.6, 135.0, 147.0, 148.8, 155.7, 157.6, 163.1.

361

MS CI (isobutane) (m/z): 357 [MH+].

362

Anal. (C17H16N4O5) C, H, N.

363

M AN U

360

4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinolin-2-

365

yl)carbonyl]furazan-3-carboxamide (9).

366

A solution of 8 (240 mg, 0.67 mmol) in MeOH saturated with NH3 (9 ml) was stirred

367

at room temperature for 10 minutes. A white solid precipitated and was recovered by

368

filtration. The solid obtained was purified by flash chromatography on silica gel

369

(CH2Cl2/acetone 97/3) to give the product as a white solid, yield 83%, mp 138-139

370

°C (iPrOH). 1

EP

AC C

371

TE D

364

H-NMR (CDCl3): δ 2.79-3.04 (3H, m, H-6, 2 x H-7), 3.15 (0.5H, dd, J = 2.4, 13.4

372

Hz, H-1), 3.39 (0.5H, dd, J = 2.4, 13.5 Hz, H-1), 4.14 (1H, s, H-3), 4.23-4.29 (1H, m,

373

H-3), 4.74-5.13 (2H, m, H-1 and H-6), 5.22-5.28 (1H, m, H-11b), 6.61 (1H, br. s, -

374

NH2), 6.76 (0.5H, br. s, -NH2), 7.18-7.35 (4H, m, aromatic protons), 7.53 (0.5H, br. s,

375

-NH2).

376

13

C-NMR (CDCl3): δ 28.7, 39.0, 46.3, 49.6, 54.9, 125.4, 127.1, 127.8, 129.5, 131.6,

377

135.0, 147.8, 148.3, 156.4, 157.0, 163.5.

378

MS CI (isobutane) (m/z): 342 [MH+]. 18

ACCEPTED MANUSCRIPT 379

Anal. calc. for C16H15N5O4.0.7H2O: C, H, N 19.79%; found: C, H, N 19.19%.

380

4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinolin-2-

382

yl)carbonyl]furazan-3-carbonitrile (10).

383

A solution of 9, (170 mg 0.50 mmol) in anhydrous THF (20 ml) was cooled in an ice

384

bath; anhydrous pyridine (4 eq.) and TFAA (4 eq.) were added to the solution. The

385

mixture was stirred at 0°C for 30 minutes, then the solvent was removed under

386

reduced pressure. The residue was taken up with 20 ml of CH2Cl2 and washed with

387

H2SO4 0.5 M (2 x 10 ml), water (2 x 10 ml), brine, dried over Na2SO4, filtered and

388

evaporated under reduced pressure. The resulting yellow solid was purified by flash

389

chromatography on silica gel (eluent CH2Cl2/EtOAc 95/5) and then crystallized from

390

CHCl3/n-hexane to give the product as a white solid, yield 83%, mp 149-150 °C.

SC

1

M AN U

391

RI PT

381

H-NMR (CDCl3): δ 2.80-3.02 (3H, m, H-6, 2 x H-7), 3.19 (0.5H, dd, J = 2.4, 13.2

Hz, H-1), 3.49 (0.5H, dd, J = 2.7, 14.3 Hz, H-1), 4.11 (0.5H, d, J = 18.3 Hz, H-1),

393

4.42 (0.5H, d, J = 17.7 Hz, H-1), 4.79-5.25 (4H, m, 2 x H-3, H-6 and H-11b), 7.18-

394

7.34 (4H, m, aromatic protons).

395

13

TE D

392

C-NMR (CDCl3): δ 28.7, 39.0, 46.9, 50.3, 54.3, 55.7, 106.2, 125.4, 127.6, 129.7,

131.4, 134.3, 135.0, 149.8, 153.5, 163.0.

397

MS CI (isobutane) (m/z): 324 [MH+].

398

Anal.(C16H13N5O3) C, H, N.

AC C

399

EP

396

400

1-(2-isocyanoethyl)-4-methoxybenzene (12).

401

A solution of 2-(4-methoxyphenyl)ethanamine 11 (15 ml, 100 mmol), TEBAC (0.01

402

eq.) and CHCl3 (1 eq.) in CH2Cl2 (32 ml) was added dropwise over 10 minutes to an

403

aqueous solution of NaOH (8 eq., 32 ml). The mixture was refluxed for 4 hours, and

404

then a mixture of ice and water (50 ml) was added. The organic phase was collected

405

and the aqueous phase was extracted with CH2Cl2 (2 x 50 ml). The organic phases

406

were then washed with H2SO4 0.5 M (3 x 100 ml), water (100 ml), brine, dried over

407

Na2SO4 and evaporated under reduced pressure. The residue was purified by flash 19

ACCEPTED MANUSCRIPT 408

chromatography on silica gel (eluent CH2Cl2) to give the product as a yellow oil,

409

yield 57%.

410

1

H-NMR (CDCl3): δ 2.91 (2H, t, J = 6.9 Hz, -CH2CH2-NC), 3.55 (2H, t, J = 6.9 Hz, -

CH2CH2-NC), 3.79 (3H, s, -OCH3), 6.87 (2H, d, J = 8.4 Hz, H-2 and H-6 aromatic

412

protons), 7.14 (2H, d, J = 8.4 Hz, H-3 and H-5 aromatic protons).

413 414

13

RI PT

411

C-NMR (CDCl3): δ 34.8, 43.2, 52.3, 114.2, 128.7, 129.4, 129.7, 156.4.

MS CI (isobutane) (m/z): 162 [MH+].

415

N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(4-methoxy)phenethylamino)-

417

ethyl)cyclohexanecarboxamide (13).

418

1-(2-isocyanoethyl)-4-methoxybenzene 12 (9.2 g, 0.057 mol) was added portion-wise

419

to a suspension of paraformaldehyde (1 eq.), 2,2-dimethoxyethylamine (1 eq.),

420

cyclohexanecarboxylic acid (1 eq.) in MeOH (60 ml) cooled in an ice bath, and the

421

mixture was stirred at room temperature for 48 hours. The solvent was then

422

evaporated under reduced pressure and the residue was dissolved in diethyl ether

423

(150 ml). The organic phase was washed with H2SO4 0.5 M (50 ml), NaOH 0.5 M

424

(50 ml), water, brine, dried over Na2SO4 and evaporated under reduced pressure. The

425

yellow oil solidifies to give the product as a pale yellow solid pure enough for the

426

next step, yield 89%.

TE D

M AN U

1

EP

427

SC

416

H-NMR (CDCl3): δ 1.22-1.25 (3H, m, cyclohexane protons), 1.45 (2H, m,

cyclohexane protons), 1.60-1.76 (5H, m, cyclohexane protons), 2.24 (0.5H, t, J = 11.1

429

Hz, cyclohexane protons), 2.59 (0.5H, t, J = 11.4 Hz, cyclohexane protons), 2.70-2.78

430

(2H, m, -Ph-CH2CH2NH-), 3.35 (3H, s, (CH3O)2CHCH2-), 3.38 (3H, s,

431

(CH3O)2CHCH2-), 3.43 (2H, t, J = 5.1 Hz, (CH3O)2CHCH2-), 3.48-3.55 (2H, m, -Ph-

432

CH2CH2NH-), 3.78 (3H, s, CH3O-Ph), 3.99 (2H, d, J = 5.1 Hz, -COCH2-), 4.40

433

(0.5H, t, J = 4.8 Hz, (CH3O)2CHCH2-), 4.58 (0.5H, t, J = 4.8 Hz, (CH3O)2CHCH2-),

434

6.50 (0.5H, br. s, -NH), 6.83 (2H, d, J = 8.4 Hz, H-2 and H-6 aromatic protons), 6.98

435

(0.5H, br. s, -NH), 7.10 (2H, d, J = 8.1 Hz, H-3 and H-5 aromatic protons).

AC C

428

20

ACCEPTED MANUSCRIPT 436

13

C-NMR (CDCl3): δ 25.5, 29.2, 34.7, 40.3, 41.2, 42.8, 50.3, 51.3, 52.3, 52.7, 54.9,

437

56.4, 102.9, 103.8, 114.0, 129.6, 130.6, 131.3, 158.5, 169.2, 169.6, 178.2.

438

MS CI (isobutane) (m/z): 377 [MH+].

439

2-(cyclohexylcarbonyl)-10-methoxy-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-

441

a]isoquinolin-4-one (14).

442

13 (19.0 g, 0.051 mol) was added portion-wise to methanesulphonic acid (20 eq.)

443

cooled in an ice bath. The mixture was heated to 70°C for 5 hours, then poured into

444

an ice-water mixture. The mixture was alkalinized to pH 8 with a 20% solution of

445

NaOH, then extracted with CH2Cl2 (4 x 50 ml). The organic phase was washed with

446

water (2 x 30 ml), brine, dried over Na2SO4 and evaporated under reduced pressure.

447

The resulting brown oil was purified by flash chromatography on silica gel (eluent

448

CH2Cl2/MeOH 98/2) to give a yellow solid, which was crystallized from EtOAc/n-

449

hexane 1/1 to give the product as a white solid, yield 40%, mp 145-146 °C.

SC

1

M AN U

450

RI PT

440

H-NMR (CDCl3): δ 1.26-1.32 (3H, m, cyclohexane protons), 1.49-1.61 (2H, m,

cyclohexane protons), 1.73-1.81 (5H, m, cyclohexane protons), 2.47 (1H, t, J = 11.1

452

Hz, cyclohexane proton), 2.69-2.91 (4H, m, 2 x H-1 and 2 x H-7), 3.79 (3H, s, -

453

OCH3), 4.08 (1H, d, J = 17.4 Hz, H-3), 4.47 (1H, d, J = 17.4 Hz, H-3), 4.75-4.82 (2H,

454

m, 2 x H-6), 5.14 (1H, dd, J = 13.2 Hz, H-11b), 6.80 (2H, d, J = 9.6 Hz, H-9 and H-

455

11 aromatic protons), 7.09 (1H, d, J = 8.1 Hz, H-8 aromatic proton).

EP

13

C-NMR (CDCl3): δ 25.7, 27.9, 29.0, 29.2, 39.4, 40.8, 45.2, 49.0, 55.1, 55.4, 110.1,

AC C

456

TE D

451

457

114.1, 126.7, 130.3, 133.7, 158.5, 164.4, 174.8.

458

MS CI (isobutane) (m/z): 343 [MH+].

459 460

2-(cyclohexylcarbonyl)-10-hydroxy-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-

461

a]isoquinolin-4-one (15).

462

BBr3 (1 M solution in CH2Cl2) (3.5 eq.) was added dropwise to a solution of 14 (6.4

463

g, 0.019 mol) in CH2Cl2 (300 ml) cooled in an ice bath. The mixture was stirred at

464

room temperature for 3 hours, and then cooled to 0°C. An aqueous solution of 21

ACCEPTED MANUSCRIPT

KHCO3 (10 eq.) was added dropwise to the reaction mixture, and the resulting

466

suspension was stirred for 1 hour. The organic phase was collected and washed with

467

water (100 ml), brine, dried over Na2SO4 and evaporated under reduced pressure. The

468

residue was crystallized from CHCl3/n-hexane to give the product as a white solid,

469

yield 81%, mp 238-239 °C.

470

1

RI PT

465

H-NMR (DMSO-d6): δ 1.18-1.39 (5H, m, cyclohexane protons), 1.64-1.72 (5H, m,

cyclohexane protons), 2.65-2.86 (5H, m, 1 cyclohexane proton, 2 x H-1 and 2 x H-7),

472

3.72 (0.5H, d, J = 17.7 Hz, H-3), 4.08 (0.5H, d, J = 17.1 Hz, H-3), 4.39 (1H, s, H-3),

473

4.45-4.51 (2H, m, 2 x H-6), 4.66-4.78 (1H, m, H-11b), 6.64 (2H, d, J = 9.3 Hz, H-9

474

and H-11 aromatic protons), 6.99 (1H, d, J = 8.1 Hz, H-8 aromatic proton), 9.38 (1H,

475

br. s, -OH). 13

M AN U

476

SC

471

C-NMR (DMSO-d6): δ, 24.9, 25.5, 27.3, 28.9, 30.9, 45.5, 48.1, 48.3, 54.7, 112.1,

477

114.4, 124.9, 129.8, 134.0, 155.8, 164.6, 173.6.

478

MS CI (isobutane) (m/z): 329 [MH+].

479

4-({[2-(cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-

481

a]isoquinolin-10-yl]oxy}methyl)furoxan-3-carboxamide (17).

482

NaH (60% suspension in mineral oil, 4 eq.) was added to a suspension of 15 (1.0 g,

483

3.1 mmol) in anhydrous THF (100 ml) cooled in an ice bath. The mixture was

484

refluxed for 1.5 hours, and then cooled to room temperature. 4-Bromomethylfuroxan-

485

3-carboxamide (16) (1 eq.) was added to the mixture, which was then stirred at 60°C

486

for 2 hours. The mixture was then cooled in an ice bath and water was added. THF

487

was evaporated under reduced pressure and the aqueous phase was extracted with

488

EtOAc (5 x 20 ml). The organic phase was washed with water (20 ml), brine, dried

489

over Na2SO4 and evaporated under reduced pressure. The residue was purified by

490

flash chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) to give a yellow solid

491

which was crystallized from iPrOH, to give the product as a white solid, yield 42%,

492

mp 200-202 °C (dec.).

AC C

EP

TE D

480

22

ACCEPTED MANUSCRIPT 493

1

H-NMR (DMSO-d6): δ 1.03 (6H, d, J = 6 Hz, 2 x CH3 iPrOH) 1.25-1.31 (5H, m,

cyclohexane protons), 1.52-1.80 (5H, m, cyclohexane protons), 2.38-1.61 (1H, m,

495

cyclohexane proton), 2.71-2.89 (4H, m, 2 x H-1 and 2 x H-7), 4.06-4.12 (2H, m, CH

496

iPrOH, H-3), 4.47 (1H, d, J = 17.4 Hz, H-3), 4.75-4.82 (2H, m, 2 x H-6), 5.08 (1H,

497

m, H-11b), 5.44 (2H, s,-CH2O-), 6.4 (1H, br. s, -CONH2), 6.90-6.96 (2H, m, H-9 and

498

H-11 aromatic protons), 7.12 (1H, d, J = 8.4 Hz, H-8 aromatic proton), 7.55 (1H, br.

499

s, -CONH2).

502 503

C-NMR (DMSO-d6): δ 22.7, 25.4, 25.7, 27.95, 28.01, 39.4, 40.8, 45.0, 49.0, 54.9,

61.3, 64.9 110.4, 111.8, 114.9, 126.0, 128.4, 130.4, 134.1, 154.7, 156.6, 164.4, 174.9. MS CI (isobutane) (m/z): 470 [MH+]. Anal. calc. for C23H27N5O6.iPrOH: C, H, N.

SC

501

13

M AN U

500

RI PT

494

504

4-({[2-(cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-

506

a]isoquinolin-10-yl]oxy}methyl)furoxan-3-carbonitrile (18).

507

Anhydrous pyridine (4 eq.) and TFAA (4 eq.) were added to a solution of 17 (200

508

mg, 0.43 mmol) in anhydrous THF (25 ml). The mixture was stirred at room

509

temperature for 45 minutes. The solvent was then evaporated under reduced pressure

510

and the residue was dissolved in CH2Cl2 (50 ml). The organic phase was washed with

511

H2SO4 0.5 M (2 x 30 ml), water, brine, dried over Na2SO4, filtered and evaporated

512

under reduced pressure. The resulting yellow solid was purified by flash

513

chromatography on silica gel (eluent CH2Cl2/EtOAc 95/5) to give the product as a

514

white solid, yield 41%, mp 95-97 °C (iPr2O/EtOAc).

EP

1

AC C

515

TE D

505

H-NMR (CDCl3): δ 1.26-1.31 (5H, m, cyclohexane protons), 1.48-1.81 (5H, m,

516

cyclohexane protons), 2.46 (1H, m, cyclohexane proton), 2.73-2.98 (4H, m, 2 x H-1

517

and 2 x H-7), 4.10 (1H, d, J = 17.7 Hz, H-3), 4.46 (1H, d, J = 17.7 Hz, H-3), 4.76-

518

4.83 (2H, m, 2 x H-6), 5.04 (1H, dd, J = 2.4, 10.8 Hz, H-11b), 5.24 (2H, s, -CH2O-),

519

6.91-6.94 (2H, m, H-9 and H-11 aromatic protons), 7.16 (1H, d, J = 8.3 Hz, H-8

520

aromatic proton).

23

ACCEPTED MANUSCRIPT 521

13

C-NMR (CDCl3): δ 25.7, 27.9, 29.0, 29.2, 39.3, 40.8, 44.8, 49.1, 53.5, 60.9, 96.0,

522

104.9, 109.7, 114.9, 129.3, 130.8, 134.5, 153.4, 155.8, 164.4, 174.8.

523

MS CI (isobutane) (m/z): 452 [MH+].

524

Anal. (C23H25N5O5.0.5H2O) C, H, N.

RI PT

525

4-({[2-(cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-

527

a]isoquinolin-10-yl]oxy}methyl)furazan-3-carboxamide (20).

528

NaH (60% suspension in mineral oil, 4 eq.) was added to a suspension of 15 (1.0 g,

529

3.1 mmol) in anhydrous THF (100 ml) cooled in an ice bath. The mixture was

530

refluxed for 1.5 hours, and then cooled to room temperature. 4-Bromomethylfurazan-

531

3-carboxamide (19) (1 eq.) was added to the mixture which was stirred at 60°C for 2

532

hours. The mixture was then cooled in an ice bath and water was added. THF was

533

evaporated under reduced pressure and the aqueous phase was extracted with EtOAc

534

(4 x 20 ml). The organic phase was washed with water (2 x 20 ml), brine, dried over

535

Na2SO4 and evaporated under reduced pressure. The residue was purified by flash

536

chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) to give a white solid which

537

was crystallized from EtOAc, yield 42%, mp 203-204 °C.

M AN U

1

TE D

538

SC

526

H-NMR (DMF-d7): δ 1.11-1.43 (5H, m, cyclohexane protons), 1.64-1.79 (5H, m,

cyclohexane protons), 2.74-2.92 (5H, m, 1 cyclohexane proton, 2 x H-1 and 2 x H-7),

540

4.16-4.21 (1H, m, H-3), 4.47-4.55 (1H, m, H-3), 4.71-4.92 (2H, m, 2 x H-6), 4.96-

541

5.04 (1H, m, H-11b), 5.66 (2H, s, -CH2O-), 7.03 (2H, d, J= 7.8 Hz,

542

H-9 and H-11 aromatic protons), 7.21 (1H, d, J= 8.4 Hz, H-8 aromatic proton), 8.30

543

(1H, br. s, -CONH2), 8.67 (1H, br. s, -CONH2). 13

AC C

544

EP

539

C-NMR (DMF-d7): δ 25.9, 26.5, 28.3, 30.0, 39.3, 39.8, 46.5, 49.0, 56.0, 60.4,

545

113.1, 114.5, 129.2, 130.8, 149.7, 153.2, 157.4, 159.2, 162.9, 165.7, 174.9.

546

MS CI (isobutane) (m/z): 454 [MH+].

547

Anal. (C23H27N5O5) C, H, N.

548

24

ACCEPTED MANUSCRIPT

4-({[2-(cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-

550

a]isoquinolin-10-yl]oxy}methyl)furazan-3-carbonitrile (21).

551

Anhydrous pyridine (4 eq.) and TFAA(4 eq.) were added to a solution of 20 (550 mg,

552

1.21 mmol) in anhydrous THF (70 ml). The mixture was stirred at room temperature

553

for 45 minutes. The solvent was then evaporated under reduced pressure, and the

554

residue was dissolved in CH2Cl2 (50 ml). The organic phase was washed with H2SO4

555

0.5 M (2 x 30 ml), water, brine, dried over Na2SO4, filtered and evaporated under

556

reduced pressure. The resulting yellow solid was purified by flash chromatography

557

on silica gel (eluent CH2Cl2/MeOH 98/2), to give the product as a white solid, yield

558

28%, mp 69-70 °C (iPr2O/EtOAc).

SC

1

H-NMR (CDCl3): δ 1.27-1.57 (5H, m, cyclohexane protons), 1.72-1.81 (5H, m,

M AN U

559

RI PT

549

cyclohexane protons), 2.46 (1H, m, cyclohexane proton), 2.73-2.96 (4H, m, 2 x H-1

561

and 2 x H-7), 4.10 (1H, d, J = 17.4 Hz, H-3), 4.46 (1H, d, J = 17.7 Hz, H-3), 4.76-

562

4.83 (2H, m, 2 x H-6), 5.04-5.09 (1H, m, H-11b), 5.40 (2H, s,

563

-CH2O-), 6.92 (2H, d, J = 6.6 Hz, H-9 and H-11 aromatic protons), 7.16 (1H, d, J =

564

8.7 Hz, H-8 aromatic proton).

565

13

TE D

560

C-NMR (CDCl3): δ, 21.0, 25.7, 27.9, 29.1, 39.3, 40.8, 44.9, 49.0, 55.0, 60.4, 111.3,

114.8, 129.2, 130.8, 132.5, 134.4, 153.0, 155.9, 164.4, 171.2, 174.8.

567

MS CI (isobutane) (m/z): 436 [MH+].

568

Anal. (C23H25N5O4 H2O) C, H, N.

AC C

569

EP

566

570

2-(cyclohexylcarbonyl)-10-(3-hydroxypropoxy)-1,2,3,6,7,11b-hexahydro-4H-

571

pyrazino[2,1-a]isoquinolin-4-one (22).

572

3-Bromopropan-1-ol (1 eq.) and K2CO3 (2 eq.) were added to a suspension of 15 (1.0

573

g, 3.2 mmol) in CH3CN. The mixture was refluxed for 24 hours, and then cooled to

574

room temperature. The solvent was evaporated under reduced pressure; the residue

575

was dissolved in EtOAc (50 ml) and washed with 0.5 M NaOH solution (4 x 30 ml),

576

water (30 ml), brine, dried over Na2SO4 and evaporated under reduced pressure.

25

ACCEPTED MANUSCRIPT 577

The yellow solid was purified by flash chromatography on silica gel (eluent

578

CH2Cl2/MeOH 98/2) to give the product as a white solid, yield 66%, mp 187-188 °C

579

(MeOH/H2O).

580

1

H-NMR (CDCl3): δ 1.18-1.39 (3H, m, cyclohexane protons), 1.52-1.60 (2H, m,

cyclohexane protons), 1.73-1.80 (5H, m, cyclohexane protons), 2.02-2.17 (2H, m,

582

cyclohexane proton and H-1), 2.43-2.50 (1H, m, H-1), 2.69-2.76 (2H, m, 2 x H-7),

583

2.81-2.95 (2H, m, HO-CH2CH2CH2O-), 3.84-3.87 (2H, m, 2 x H-3), 4.05-4.25 (3H,

584

m, -OH, HO-CH2CH2CH2O-), 4.28-4.36 (1H, m, H-6), 4.43-4.49 (1H, m, H-6),

585

4.73-4.82 (2H, m, HO-CH2CH2CH2O-), 5.06-5.10 (1H, m, H-11b), 6.81 (2H, d, J =

586

8.1 Hz, H-9 and H-11 aromatic protons), 7.08 (1H, d, J = 7.8 Hz, H-8 aromatic

587

protons). 13

SC

M AN U

588

RI PT

581

C-NMR (CDCl3): δ 25.7, 28.6, 29.0, 29.5, 31.0, 39.4, 40.8, 45.2, 49.0, 55.1, 60.0,

589

65.8, 110.8, 114.6, 126.9, 130.2, 133.7, 157.7, 164.4, 174.9.

590

MS CI (isobutane) (m/z): 387 [MH+].

591

2-(cyclohexylcarbonyl)-10-(3-{[3-(phenylsulfonyl)furoxan-4-yl]oxy}propoxy)-

593

1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one (24).

594

DBU (2 eq.) was added to a solution of 23 (1.2 eq.) in CH2Cl2 (20 ml). The solution

595

was cooled in an ice bath and a solution of 22 (140 mg, 0.36 mmol) in CH2Cl2 (10

596

ml) was added. The mixture was stirred at room temperature for 1 hour, washed with

597

a 0.5 M H2SO4 solution (2 x 20 ml), water (2 x 20 ml), brine, dried over Na2SO4 and

598

evaporated under reduced pressure. The resulting yellow oil was partially purified by

599

flash chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) and then purified by

600

HPLC (eluent CH3CN/H2O 0.1% CF3COOH 70/30), to give the product as a white

601

solid, yield 14%, mp 82-84 °C (iPrOH).

602

AC C

EP

TE D

592

1

H-NMR (CDCl3): δ 1.26-1.32 (3H, m, cyclohexane protons), 1.49-1.57 (2H, m,

603

cyclohexane protons), 1.72-1,80 (5H, m, cyclohexane protons), 2.34-2.47 (3H, m,

604

cyclohexane proton and 2 x H-1), 2.71-2.92 (4H, m, 2 x H-7 and HO-CH2CH2CH2O-

605

), 4.06-4.50 (4H, m, 2 x H-3 and HO-CH2CH2CH2O-), 4.62-4.66 (2H, m, 2 x H-6), 26

ACCEPTED MANUSCRIPT 606

4.75-4.83 (2H, m, HO-CH2CH2CH2O-), 5.08-5.17 (1H, m, H-11b), 6.83 (2H, d, J =

607

7.5 Hz, H-9 and H-11 aromatic protons), 7.11 (1H, d, J = 7.8 Hz, H-8 aromatic

608

protons), 7.54-7.59 (2H, m, aromatic protons), 7.74 (1H, t, J = 7.5 Hz, aromatic

609

protons), 8.02 (2H, d, J = 7.5 Hz, aromatic protons). 13

C-NMR (CDCl3): δ 25.7, 27.9, 28.5, 29.0, 29.2, 39.4, 40.8, 45.2, 49.0, 55.0, 63.7,

RI PT

610

68.1, 110.5, 110.8, 111.2, 114.3, 127.2, 128.5, 129.6, 130.4, 133.9, 135.6, 138.0,

612

157.5, 158.9, 164.4, 174.8, 177.2.

613

MS CI (isobutane) (m/z): 611 [MH+].

614

Anal. (C30H34N4O8S): C, H, N.

SC

611

615

2-(cyclohexylcarbonyl)-10-(3-{[4-(phenylsulfonyl)furazan-3-yl]oxy}propoxy)-

617

1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one (26).

618

DBU (2 eq.) was added to a solution of 25 (1.2 eq.) in CH2Cl2 (20 ml). The solution

619

was cooled in an ice bath and a solution of 22 (150 mg, 0.39 mmol) in CH2Cl2 (10

620

ml) was added to it. The mixture was stirred at room temperature for 1 hour, washed

621

with H2SO4 0.5 M solution (2 x 20 ml), water (2 x 20 ml), brine, dried over Na2SO4

622

and evaporated under reduced pressure. The resulting yellow oil was partially

623

purified by flash chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) and then

624

purified by HPLC (eluent CH3CN/H2O 0.1% CF3COOH 75/25), to give the product

625

as a white solid, yield 35%, mp 70-71 °C (iPrOH).

TE D

EP

1

H-NMR (CDCl3): δ 1.28-1.31 (3H, m, cyclohexane protons), 1.49-1.57 (2H, m,

AC C

626

M AN U

616

627

cyclohexane protons), 1.72-1,80 (5H, m, cyclohexane protons), 2.20-2.34 (3H, m,

628

cyclohexane proton and 2 x H-1), 2.71-2.95 (4H, m, 2 x H-7 and HO-CH2CH2CH2O-

629

), 4.06-4.50 (4H, m, 2 x H-3 and HO-CH2CH2CH2O-), 4.57-4.61 (2H, m, 2 x H-6),

630

4.74-4.83 (2H, m, HO-CH2CH2CH2O-), 5.09-5.14 (1H, m, H-11b), 6.73-6.81 (2H, m,

631

H-9 and H-11 aromatic protons), 7.10 (1H, d, J = 8.7 Hz, H-8 aromatic protons),

632

7.56-7.66 (2H, m, aromatic protons), 7.73 (1H, t, J = 7.2 Hz, aromatic protons), 8.07

633

(2H, d, J = 7.8 Hz, aromatic protons).

27

ACCEPTED MANUSCRIPT 634

13

C-NMR (CDCl3): δ 25.7, 28.0, 28.6, 29.2, 29.6, 39.4, 40.8, 45.2, 49.0, 55.0, 63.7,

70.5, 111.1, 114.3, 127.2, 129.0, 129.6, 129.7, 130.4, 133.8, 135.4, 137.9, 148.8,

636

157.4, 161.2, 164.1, 164.4, 174.8.

637

MS CI (isobutane) (m/z): 595 [MH+].

638

Anal. (C30H34N4O7S): C, H, N.

RI PT

635

639

Biology. Female Swiss-Webster mice infected with S. mansoni cercariae by

641

percutaneous tail exposure were obtained from the Biomedical Research Institute,

642

Rockville, MD. Parasites were obtained from mice 7 weeks after infection by

643

perfusion with RPMI 1640 medium (Cellgro, Fisher).33 Worms were washed

644

thoroughly several times in RPMI 1640 in a laminar flow hood; they were then

645

washed several times in complete RPMI 1640 (cRPMI) containing 25 mM HEPES

646

(Cellgro, Fisher), 150 units/mL penicillin, 125 µg/mL streptomycin, and 10% fetal

647

bovine serum (Atlanta Biologicals). Worms were then distributed into 6-well tissue

648

culture plates with 15-20 worms per well and cultured overnight in 5 mL cRPMI in

649

5% CO2 at 37°C. The following day, media were removed from each well and

650

replaced with 5 mL fresh cRPMI. Test compounds were dissolved in DMSO at 10

651

mM and added to the wells at the indicated concentrations. The same volume of

652

DMSO was added to each well. Negative control worms were treated with an equal

653

volume of DMSO alone. After overnight culture, the media with compounds were

654

removed and replaced with fresh media alone. Worm mobility and survival were

655

observed under a stereomicroscope (Zeiss Stemi 2000-C) for 10 seconds per worm at

656

the indicated time points. Media were removed and replaced with fresh media every

657

48 hours. This study was approved by the Institutional Animal Care and Use

658

Committee at Rush University Medical Center (IACUC number 08–058; DHHS

659

animal welfare assurance number A3120-01).

660

TGR preparation and IC50 determination were performed as described.34 Briefly,

661

assays were run in 96-well plates in 200 µL with 20 nM enzyme and 100 µM

662

NADPH in 0.1 M potassium phosphate (pH 7.4), 10 mM EDTA. Test compounds

AC C

EP

TE D

M AN U

SC

640

28

ACCEPTED MANUSCRIPT

were dissolved in DMSO. The enzyme was incubated for 10 minutes at room

664

temperature with NADPH and compounds at 50 µM, 20 µM, 10 µM, 1 µM, 500 nM,

665

100 nM, 10 nM, or 5 nM before the addition of 3 mM 5, 5'-dithio-bis(2-nitrobenzoic

666

acid). Enzyme activity was monitored by observing the change in A412 due to the

667

formation of 5-thio-2-nitrobenzoic acid during the first 3 minutes. Residual TGR

668

activity was compared to controls, incubated with equal volumes of DMSO. All

669

assays were done in triplicate.

RI PT

663

670

Acknowledgements

672

We would like to thank Dr. Matthew S. Tucker, Biomedical Research Institute,

673

Rockville,

674

HHSN272201000005I. This work was supported in part by the NIH/National

675

Institute of Allergy and Infectious Diseases (NIAID) grant R01AI065622 (DLW) and

676

by MIUR grant (PRIN 2009 prot. 20097FJHPZ_002).

for

parasite

material

through

NIH-NIAID

contract

M AN U

MD,

SC

671

677

References

679

1)

Gryseels, B., Schistosomiasis. Infect. Dis. Clin. of N. Am. 2012, 26, 383-397.

680

2)

Steinmann, P.; Keiser, J.; Bos, R.; Tanner, M.; Utzinger, J. Schistosomiasis and

TE D

678

water resources development: systematic review, meta-analysis, and estimates of

682

people at risk. Lancet Infect. Dis. 2006, 6, 411-425. 3)

Van der Werf, M.J.; de Vlas, S.J.; Brooker, S.; Looman, C.W.; Nagelkerke, N.J.;

AC C

683

EP

681

684

Habbema, J.D.; Engels, D. Quantification of clinical morbidity associated with

685

schistosoma infection in sub-Saharan Africa. Acta Trop. 2003, 86, 125-139.

686

4)

Chemotherapy. Angewandte Chemie-International Edition 2013, 53, 17936-7956

687 688

5)

691

Gonnert, R.; Andrews, P. Praziquantel, a new broad-spectrum antischistosomal agent. Z. Parasitenk. 1977, 52, 129-50.

689 690

Thetiot-Laurent, S. A. L. ; Boissier, J.; Robert, A.; Meunier, B. Schistosomiasis

6)

Liu, Y.; Wang, X.; Wang, J. K.; Ching, C. B. Investigation of the phase diagrams of chiral praziquantel. Chirality 2006, 18, 259-264. 29

ACCEPTED MANUSCRIPT 7)

2010, 5, 1420-1434, and references therein.

693 694

8)

Fenwick, A.; Webster, J. P. Schistosomiasis: challenges for control, treatment and drug resistance. Curr. Opin. Infect. Dis. 2006, 19, 577-582.

695 696

Doemling, A.; Khoury, K. Praziquantel and Schistosomiasis. ChemMedChem

9)

Aragon, A. D.; Imani, R. A.; Blackburn, V. R.; Cupit, P. M.; Melman, S. D.;

RI PT

692

697

Goronga, T.; Webb, T.; Loker, E. S.; Cunningham, C. Towards an understanding

698

of the mechanism of action of praziquantel. Mol. Biochem. Parasit. 2009, 164, 57-

699

65.

700

10) Sadhu,

SC

P. S.; Kumar, S. N.; Chandrasekharam, M.; Pica-Mattoccia, L.; Cioli, D.;

Rao, V. J. Synthesis of new praziquantel analogues: Potential candidates for the

702

treatment of schistosomiasis. Bioorg. Med. Chem. Lett. 2012, 22, 1103-1106.

704 705 706 707

11) van

den Enden, E. Pharmacotherapy of helminth infection. Expert Opinion on

Pharmacotherapy 2009, 10, 435-451. 12) Wang,

W.; Wang, L.; Liang, Y.S. Susceptibility or Resistance of Praziquantel in

Human Schistosomiasis: A Review. Parasitol. Res. 2011, 111, 1871-1877. 13) Kasinathan,

R.S.; Morgan, W.M.; Greenberg,R.M. Schistosoma mansoni express

TE D

703

M AN U

701

higher levels of multidrug resistance-associated protein 1 (SmMRP1) juvenile

709

worms and in response to praziquantel. Molecular & Biochemical Parasitology

710

2010, 173, 25-31.

711

14) Sayed,

EP

708

A. A.; Simeonov, A.; Thomas, C. J.; Inglese, J.; Austin, C. P.; Williams,

D. L. Identification of oxadiazoles as new drug leads for the control of

713

schistosomiasis. Nat. Med. 2008, 14, 407-412.

714

15) Rai,

AC C

712

G.; Thomas, C. J.; Leister, W.; Maloney, D. J. Synthesis of oxadiazole-2-

715

oxide analogues as potential antischistosomal agents. Tetrahedron Lett. 2009, 50,

716

1710-1713.

717 718

16) Gasco,

A.; Boulton, A. J. Furoxans and Benzofuroxans. Adv. Heterocycl. Chem.

1981, 29, 251.

30

ACCEPTED MANUSCRIPT 719

17) Feelisch,

M.; Schonafinger, K.; Noack, E. Thiol-Mediated Generation of Nitric-

720

Oxide Accounts For The Vasodilator Action of Furoxans. Biochem. Pharmacol.

721

1992, 44, 1149-1157.

722

18) Gasco,

A., Schoenafinger, K. The NO-releasing Heterocycles. In Nitric Oxide

Donors, Wang, P. G., Cai, T.B., Taniguchi, N., Ed. WILEY-VCH Verlag GmbH

724

& Co KGaA Weinheim, 2005; pp 131-175.

725

19) Alger,

RI PT

723

H. M.; Sayed, A. A.; Stadecker, M. S.; Williams, D. L. Molecular and

enzimatic characterization of Schistosoma mansoni thioredoxin. Int. J. Parasitol.

727

2002, 32, 1285-1292.

728

20) Rai,

SC

726

G.; Sayed, A. A.; Lea, W. A.; Luecke, H. F.; Chakrapani, H.; Prast-Nielsen,

S.; Jadhav, A.; Leister, W.; Shen, M.; Inglese, J.; Austin, C. P.; Keefer, L.; Arner,

730

E. S. J.; Simeonov, A.; Maloney, D. J.; Williams, D. L.; Thomas, C. J. Structure

731

Mechanism Insights and the Role of Nitric Oxide Donation Guide the

732

Development

733

Schistosomiasis. J. Med. Chem. 2009, 52, 6474-6483.

of

M AN U

729

Oxadiazole-2-Oxides

as

Therapeutic

Agents

against

21) To

be submitted for publication.

735

22) a)

Fruttero, R.; Boschi, D.; Di Stilo, A.; Gasco, A. The Furoxan System as a

TE D

734

Useful Tool for Balancing "Hybrids" with Mixed alpha-1-Antagonist and NO-

737

Like Vasodilator Activities" J. Med. Chem. 1995, 38, 4944. b) Buonsanti, M.F.;

738

Bertinaria, M., Di Stilo, A.; Cena, C.; Fruttero, R.; Gasco, A. Nitric Oxide Donor

739

beta(2)-Agonists: Furoxan Derivatives Containing the Fenoterol Moiety and

740

Related Furazans. J. Med. Chem. 2007, 50, 5003-5011. c) Dunlap, T.; Abdul-Hay,

741

S. O.; Chandrasena, R. E. P.; Hagos, G. K.; Sinha, V.; Wang, Z.; Wang, H.;

742

Thatcher, G. R. J. Nitrates and NO-NSAIDs in cancer chemoprevention and

743

therapy: In vitro evidence querying the NO donor functionality. Nitric Oxide-

744

Biology and Chemistry 2008, 19, 115-124.

745 746

AC C

EP

736

23)

Cao, H.; Liu, H.; Doemling, A. Efficient Multicomponent Reaction Synthesis of the Schistosomiasis Drug Praziquantel. Chem. Eur. J. 2010, 16, 12296-12298.

31

ACCEPTED MANUSCRIPT 747

24) Colasanti,

M.; Gradoni, L.; Mattu, M.; Persichini, T.; Salvati, L.; Venturini, G.;

748

Ascenzi, P. Molecular bases for the anti-parasitic effect of NO (Review).

749

International Journal of Molecular Medicine 2002, 9, 131-134.

750

25) Fruttero,

R.; Crosetti, M.; Chegaev, K.; Guglielmo, S.; Gasco, A.; Berardi, F.;

Niso, M.; Perrone, R.; Panaro, M. A.; Colabufo, N. A. Phenylsulfonylfuroxans as

752

modulators of multidrug-resistance associated protein-1 and P-glycoprotein. J.

753

Med. Chem. 2010, 53, 5467-5475.

754

26) Chegaev,

RI PT

751

K.; Riganti, C.; Lazzarato, L. Rolando, B.; Guglielmo, S.; Campia, I.;

Fruttero, R.; Bosia, A.; Gasco, A. Nitric Oxide Donor Doxorubicins Accumulate

756

into Doxorubicin-Resistant Human Colon Cancer Cells Inducing Cytotoxicity

757

Medicinal Chemistry Letters 2011, 2, 494-497. 27) Kim,

M AN U

758

SC

755

J. H.; Lee, Y. S.; Park, H.; Kim, C. S. Formation of Pyrazinoisoquinoline

759

Ring System by the Tandem Amidoalkylation and N-Acyliminium ion

760

Cyclization: An Efficient Synthesis of Praziquantel. Tetrahedron 1998, 54, 7395-

761

7400. 28) Grundmann,

C.; Nickel, G.W.; Bansal, R.K. Das Tetramere der Knallsaure

TE D

762 763

(Isocyanilsaure) und seine Derivate (Nitrile oxides. XVIII. Tetramer of fulminic

764

acid (isocyanilic acid) and its derivatives). Liebigs Ann. Chem. 1976, 1029-1050. 29) Di

Stilo, A.; Visentin, S.; Cena, C.; Gasco, A. M.; Ermondi, G.; Gasco, A. New

EP

765

1,4-Dihydropyridines Conjugated to Furoxanyl Moieties, Endowed with Both

767

Nitric Oxide-like and Calcium Channel Antagonist Vasodilator Activities. J.

768

Med.Chem. 1998, 41, 5393-5401.

769

AC C

766

30) Bertinaria,

M.; Galli, U.; Sorba, G.; Fruttero, R.; Gasco, A.; Brenciaglia, M. I.;

770

Scaltrito, M. M.; Dubini, F. Synthesis and anti-Helicobacter pylori properties of

771

NO-donor/metronidazole hybrids and related compounds. Drug. Dev. Res. 2003,

772

60, 225-239.

773

31) Farrar,

W.V. The 3,4-bisarenesulphonylfuroxans. J .Chem. Soc. 1964, 904-906.

32

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32) Boschi,

D.; Di Stilo, A.; Cena, C.; Lolli, M.; Fruttero, R.; Gasco, A. Studies on

775

agents with mixed NO-dependent vasodilating and beta-blocking activities.

776

Pharm. Res. 1997, 14, 1750-1758.

777

33) Lewis,

F. Schistosomiasis. in Current Protocols in Immunology, eds. J. E.

Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John

779

Wiley & Sons, New York, Edition edn., 1998, 19.11.11-19.11.28.

780

34)

RI PT

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Kuntz, A.N.; Davioud-Charvet, E.; Sayed, A.A.; Califf, L.L.; Dessolin, J.; Arnér, E.S.J; Williams, D.L. Thioredoxin glutathione reductase from Schistosoma

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mansoni: an essential parasite enzyme and a key drug target. PLoS Med. 2007, 4,

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1071-1086.

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New Praziquantel Derivatives Containing NO-donor Furoxans and Related Furazans as Active Agents against Schistosoma mansoni.

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1. New NO-donor derivatives of praziquantel (PZQ) have been synthesized and studied. 2. The compounds are potential therapeutic tools against schistosomiasis. 3. Several compounds showed interesting TGR inhibiting and anti-parasitic activities. 4. The compounds could be useful in case of infection by PZQ-resistant schistosomes.