New praziquantel derivatives containing NO-donor furoxans and related furazans as active agents against Schistosoma mansoni

Article abstract of DOI:10.1016/j.ejmech.2014.07.007

A series of NO-donor praziquantel hybrid compounds was obtained by combining praziquantel (PZQ) and furoxan moieties in a single entity. NO-donor properties of the furoxan derivatives were evaluated by detecting nitrite after incubation of the products in

Full text of DOI:10.1016/j.ejmech.2014.07.007

European Journal of Medicinal Chemistry 84 (2014) 135e145
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New praziquantel derivatives containing NO-donor furoxans and
related furazans as active agents against Schistosoma mansoni
,
Stefano Guglielmo a, Daniela Cortese a b, Francesca Vottero a, Barbara Rolando a,
,
,
Valerie P. Kommer b, David L. Williams b *, Roberta Fruttero a *, Alberto Gasco a
a
ꢀ
Dipartimento di Scienza e Tecnologia del Farmaco, Universita of Torino, Via P. Giuria 9, 10125 Torino, Italy
b Department of Immunology/Microbiology, Rush University Medical Center, 1735 West Harrison Street, Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of NO-donor praziquantel hybrid compounds was obtained by combining praziquantel (PZQ) and
furoxan moieties in a single entity. NO-donor properties of the furoxan derivatives were evaluated by
detecting nitrite after incubation of the products in 7.4 pH buffered solution in the presence of L-cysteine.
Structurally-related furazans, devoid of NO release capacity, were also synthesized for control purposes.
All products were studied for their ability to inhibit recombinant Schistosoma mansoni thioredoxin
glutathione reductase (TGR). Mobility and death of adult Schistosoma mansoni worms cultured in the
presence of the products were evaluated versus PZQ. Analysis of the results showed that some products
were endowed with both PZQ and NO-dependent antiparasitic properties. Compounds 6, 7, 18, and 24
emerged as the most interesting balanced hybrids, worthy of additional study on PZQ-resistant parasites.
© 2014 Elsevier Masson SAS. All rights reserved.
Received 5 April 2014
Received in revised form
20 May 2014
Accepted 3 July 2014
Available online 3 July 2014
Keywords:
Furoxans
Praziquantel
Schistosomiasis
Thioredoxin glutathione reductase
NO-donor praziquantel
1. Introduction
PZQ is active against the adult forms of all schistosome species, but
not against the juvenile forms [8]; its mechanism of action is still
Schistosomiasis, also known as Bilharzia, is one of the most
prominent neglected tropical diseases (NTDs). It is a parasitosis
caused by blood-dwelling flatworms of the genus Schistosoma; the
principal species parasitizing humans are Schistosoma mansoni,
Schistosoma japonicum, and Schistosoma haematobium [1]. Estimates
indicate that 600 million people are at risk of parasitosis and that
more than 200 million people suffer from schistosomiasis, resulting
in 200,000 deaths each year. The highest incidence of schistosomi-
asis is in sub-Saharan Africa, where the principal species responsible
are S. mansoni and S. haematobium [2e4]. Praziquantel (PZQ) is the
drug of choice for the treatment of schistosomiasis [4,5]. The
structure (2-(cyclohexylcarbonyl)-1,2,3,6,7,11b-hexahydro-4H-pyr-
azino[2,1-a]isoquinolin-4-one) (PZQ, Chart 1) assigned to the drug
was confirmed by X-ray analysis [6]. It is a fairly lipophilic product,
and is consequently endowed with low water solubility [7]. PZQ has
a stereogenic center and can thus exist as two optical stereoisomers:
the levo isomer ((ꢀ)PZQ) is more active than the dextro isomer ((þ)
PZQ), but since the dextro form has no side effects, for economic
reasons the drug is used as an orally-administered racemic mixture.
unclear. Calcium accumulation, alteration of schistosomal mem-
brane fluidity, reduction of glutathione concentration, destruction
of the tegument following binding to schistosomal actin, and
interference with components of the parasite's aerobic metabolism
have all been proposed as possible action mechanisms [4,9]. A
number of structural modifications of PZQ have been introduced
with the goal of improving its antihelmintic action [7,10]. Most of the
resulting products are devoid of substantial activity; others show
only moderate effects, and none is better than the lead.
One problem associated with the extensive use of PZQ is the risk
of the parasite's developing resistance [11,12]. To date, there is no
clear evidence for large-scale clinical resistance of schistosomes to
treatment with PZQ. Conversely, laboratory studies clearly indicate
that resistance to PZQ can be selected. In particular it has been
found that a schistosomal homologue of the mammalian P-glyco-
protein (P-gp) is upregulated in PZQ-treated worms and in juvenile
worms, which are resistant to PZQ activity [13]. For this reason
there is an urgent need to develop new chemical classes of drugs for
the treatment of schistosomiasis.
Recently, through a quantitative high-throughput screen, 1,2,5,-
oxadiazole 2-oxides (furoxans) have been identified as a new
chemical class for the control of schistosomiasis [14,15]. Furoxan (1,
Chart 1) is an old heterocyclic system, well known to chemists
* Corresponding authors.
E-mail addresses: david_l_williams@rush.edu (D.L. Williams), roberta.fruttero@
unito.it (R. Fruttero).
http://dx.doi.org/10.1016/j.ejmech.2014.07.007
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

136
S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
Chart 1. PZQ, Praziquantel; A, general structure of the furoxan hybrids 5e7, and of the related furazans 8e10; B, general structure of the furoxan hybrids 17, 18, 24, and of the related
furazans 20, 21, 26; 1, R ¼ R0 ¼ H, 1,2,5-oxadiazole 2-oxide (furoxan); 2, R ¼ C6H5, R0 ¼ CN, 4-phenylfuroxan-3-carbonitrile.
because of arguments over its structure [16]. In the recent past,
renewed interest has surrounded furoxan derivatives due to the
discovery that they can release nitric oxide (NO) under the action of
thiol cofactors [17,18]. The presence of electron withdrawing groups
at the ring, in particular at the 3-position, generally increases this
capacity.
It has been shown that furoxan derivatives are able to inhibit
thioredoxin glutathione reductase (TGR), a multifunctional parasite
protein that reduces both thioredoxin and glutathione disulfide and
provides deglutathionylation (glutaredoxin) activity in worms [19].
Specific reaction of furoxans with TGR results in localized NO pro-
duction and subsequent S- (or Se-) nitrosation and inactivation of
TGR. The exactnature of theresultingTGR modifications isnot known
[20]. Furoxan-3-carbonitrile derivatives are the most interesting
compounds studied thus far. The prototype of this series is 4-
phenylfuroxan-3-carbonitrile (2, Chart 1). The product was found to
pathway reported in Scheme 1. The known hexahydro-4H-pyr-
azinoisoquinoline derivative was coupled with 3-
3
methoxycarbonylfuroxan-4-carboxylic acid (4) in CH2Cl2/THF so-
lution, in the presence of N-hydroxysuccinimide, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), and a catalytic amount
of 4-(dimethylamino)pyridine (DMAP), to give the furoxan ester 5.
Treatment of this ester with methanol saturated with ammonia
afforded the related amide 6. Dehydration of this latter product
with trifluoroacetic anhydride in THF solution, in the presence of
pyridine, gave rise to the corresponding nitrile 7. The furazan target
product 8 was obtained by reduction with trimethylphosphite of
the related furoxan 5. Furazans 9 and 10 were prepared from 8,
following the same procedures used to obtain the corresponding
furoxans 6 and 7 from 5.
The second series of NO-donor PZQ hybrids (17, 18, 24) and of
the related des-NO furazans (20, 21, 26) was obtained through the
synthetic pathway reported in Scheme 2. The commercial 2-(4-
methoxyphenyl)ethanamine 11 was transformed into the related
isocyanide 12 by treatment with chloroform under basic condi-
tions, in the presence of a catalytic amount of triethylbenzy-
lammonium chloride (TEBAC). The 10-methoxy-substituted PZQ 14,
the common intermediate in preparing all target compounds, was
obtained from 12 by the same stepwise Ugi four-component reac-
tion and PictecteSpengler reaction used by Cao et al. to prepare
PZQ from 2-phenylethanamine [23]. Briefly, a mixture of para-
formaldehyde, dimethoxyethylamine and cyclohexyl carboxylic
acid in methanol was treated with 12 to give 13 (Ugi reaction). This
intermediate was added to methanesulfonic acid to afford 17
(PictecteSpengler reaction). Cleavage with BBr3 of the methoxy
group present in 14 gave rise to the phenol 15. Reaction of 15 with
the 4-(bromomethyl)furoxan-3-carboxamide (16), or with the
related furazan amide 19, in the presence of NaH yielded the ex-
pected final compounds 17 and 20, respectively. Dehydration of
these latter compounds with trifluoroacetic anhydride in pyridine
produced the final cyano-substituted related hybrids 18, 21. To
prepare the phenylsulfonyl-substituted target structures 24, 26, the
phenol derivative 15 was coupled with 3-bromopropan-1-ol in
CH3CN in the presence of K2CO3, to give the alcohol 22. This in-
termediate, treated either with 3,4-bisphenylsulfonylfuroxan (23)
or with the related furazan 25, gave the desired final products.
be an irreversible inhibitor of TGR (IC50 ¼ 6.3
mM). It was active
against all stages of S. mansoni and against cultured adult
S. japonicum, and S. haematobium worms. Intraperitoneal injection of
2 at 10 mg/kg in S. mansoni infected mice led to a marked reduction in
worm burden when treatment occurred 1 day after infection (skin-
stage parasites), 23 days after infection (juvenile, liver stage para-
sites), or 37 days after infection (adult, egg-laying parasites) [14,20].
More recently, a number of phenylsulfonyl-substituted furoxans have
been found to be endowed with potent antischistosomal activity [21].
This paper describes a new series of compounds with potential
dual antischistosomal action obtained by combining, in a single
entity, PZQ and NO-donor furoxan derivatives bearing at the
3 position CN, CONH2, COOMe, or SO2C6H5 moieties. In the first
group of hybrids, the furoxan substructures were substituted for the
cyclohexyl group of PZQ (Chart 1, general structure A; Scheme 1,
der.s 5e7). In the second group of hybrids the furoxan moiety was
linked to the 10-position of PZQ through appropriate bridges (Chart
1, general structure B; Scheme 2, der.s 17, 18, 24). The synthesis,
structural characterization, and preliminary in vitro and ex vivo
pharmacological profiles of all these products are reported and
discussed. Related furazan (1,2,5-oxadiazoles) derivatives, (Chart 1,
general structure A; Scheme 1, der.s 8e10; Chart 1, general structure
B; Scheme 2, der.s 20, 21, 26) were also considered for comparison,
since their structures are closely related to those of the corre-
sponding furoxans, but they do not release NO (des-NO furazans).
Therefore, if a given biological activity of a furoxan derivative is
similar to that of its furazan analogue, it will be assumed that NO is
not involved in that activity. The approach to compare the phar-
macological profile of an NO-donor with that of its analogue des-NO,
is frequently used in the study of hybrid NO-donor drugs [22,a,b,c].
2.1.2. NO release
The capacity of the final furoxan hybrids to release NO was
evaluated on the basis of the amount of nitrite produced following
incubation in buffered pH 7.4 solution in the presence of a
5:1 molar excess of L-cysteine. Nitrite was detected by the Griess
2. Results and discussion
reaction. The results expressed as percentages of NOꢀ2 are reported
in Table 1. NO production ranks the series 7 > 18 > 24 > 6 z 17 > 5.
This sequence should parallel the different capacities of the prod-
ucts to release NO under the action of free cellular thiols
(‘nonspecific’ release). Potentially, the NO formed can diffuse into
the worm resulting in antiparasitic action, following interaction
with a variety of cellular targets [24].
2.1. Chemistry
2.1.1. Synthesis
The first series of NO-donor PZQ hybrids (5e7) and of the
related des-NO furazans (8e10) was obtained through the synthetic

S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
137
Scheme 1. Reaction conditions: (a) N-hydroxysuccinimide, EDC$HCl, cat. DMAP, CH2Cl2/THF; (b) CH3OH(NH3); (c) TFAA, pyridine, THF, 0 ꢁC; (d) (CH3O)3P reflux.
Scheme 2. Reaction conditions: (a) NaOH/H2O, TEBAC, CHCl3, CH2Cl2, reflux; (b) paraformaldehyde, 2,2-dimethoxyethylamine, cyclohexyl carboxylic acid, CH3OH; (c) meth-
anesulfonic acid, 0 ꢁC then 70 ꢁC; (d) BBr3, CH2Cl2; (e) NaH 60% mineral oil disp., THF, reflux; (f) TFAA, pyridine, THF, 0 ꢁC; (g) 3-bromopropan-1-ol, K2CO3, CH3CN, reflux; (h) DBU,
CH2Cl2.
2.2. Biology
upon both binding affinity and appropriately aligned reactivity of
each product, and is a measure of each product's capacity to trigger
antiparasitic action following its ability to inactivate TGR. As ex-
pected, the des-NO furazan analogues did not display inhibitory
2.2.1. Inhibition of TGR
The inhibitory activity of all products described in this paper
was evaluated against recombinant S. mansoni TGR; the results,
expressed as IC50, are reported in Table 1. All the hybrid furoxan
products acted as potent TGR inhibitors; their inhibitory potency
follows the series 6 > 17 > 5 > 7 z 18 > 24. This sequence should
parallel the different capacities of the compounds to release NO
under action of the enzyme. This ‘specific’ NO release is dependent
activity when tested up to 50 mM concentration.
2.2.2. Action of compounds against ex vivo parasites
Adult S. mansoni worms were cultured in the presence of two
different concentrations of furoxan and furazan derivatives: 10 and
50
mM, in DMSO. The mobility and death of the parasites were

138
S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
Table 1
Activity of compounds against TGR and cultured adult Schistosoma mansoni worms and NO donation ability expressed as % of NO2ꢀ
.
Compound
TGR IC50
(
m
M)
Worm contraction
50 10
Worm-killinga
%NOꢀ2 (mol/mol) SEb
m
M
m
M
50
m
M
10 mM
PZQ
n.i.
Yes
Yes
40%c
15%c
e
2d
5.0
No
No
24 h
96 h
28.9 0.3
5
0.148
Yes
Yese
50%c
23%
<1
6
7
0.01
Yes
Yes
Yese
80%
30%
1.8 0.1
0.316
\ only
72 h
30%c
56.2 0.3
8
>50
Yese
Yese
18%
15%
e
9
>50
>50
Yese
Weake
23%
25%
23%
20%
e
10
Yese
Yese
e
17
18
0.083
0.35
No
No
No
No
No
1.1 0.2
Yesf
24 h
Noc
33.8 0.1
20
21
>50
>50
No
No
No
No
No
No
No
e
e
Yesf

S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
139
Table 1 (continued )
Compound
TGR IC50
(m
M)
Worm contraction
50 10
Worm-killinga
50 10 mM
%NOꢀ2 (mol/mol) SEb
m
M
m
M
m
M
24
26
8.5
_ only
Some, not all
24 h
Noc
48 h
No
15.7 0.1
>50
Yes
Yese
e
n.i. e no inhibition; n.d. e not determined.
a
Time (h) to 100% worm death or % dead worms at 144 h.
b
c
d
e
f
Nitrite was measured (Griess reaction) after 1 h incubation at 37 ꢁC of the product in buffered solution (pH ¼ 7.4) containing 5:1 excess of
L-cysteine.
Living worms moved very slowly.
Rai et al. (reference [20]).
Worms contracted immediately upon addition of compound, then relaxed over 24e48 h.
Worms contracted immediately upon addition of compound, then relaxed after <10 min.
monitored over 144 h. In Table 1, the results are compared with
those for PZQ and 2, taken as references. Contraction and paralysis
of the worms are the first observable effects of PZQ (Fig. 1). After
overnight culture with PZQ, these effects are reversible and the
worms return to normal length over several days (>2 days). At the
concentrations of PZQ tested, worms begin to die 4e5 days after
PZQ is removed from the culture media. The furoxan 2 displayed no
effect on worm length (i.e., it did not cause contractile paralysis),
but quickly killed 100% of worms at both the concentrations tested.
This indicates that NO is not involved in worm contraction, but is
responsible for the rapid death of the parasites.
Worm morphology was monitored after treatment with hybrid
compounds to indirectly assess their PZQ-like activity. Worms were
contracted and mobility impaired immediately after exposure to
the first group of products 5e10. In the case of furazans 8e10, the
subsequent recovery after their removal from the medium was
faster than that observed for PZQ, at both concentrations tested.
The hybrid furoxans 5e7 retained PZQ's full worm-contraction
ability at the higher concentration tested, but mobility recovered
these compounds' PZQ-like activity was limited (Fig. 1). This is in
line with findings that modification of the PZQ aromatic ring
significantly reduces activity [7,10]. This might be due to decreased
interaction with parasite target protein(s), or to increased efflux of
the compounds. Furoxans 18 and 24 have better worm-killing ac-
tivity than the related furazans 21 and 26 suggesting that NO is
involved in this action. Since both furoxan derivatives are quite
efficient NO-donors under the action of cysteine, and quite potent
TGR inhibitors, we can infer that both specific (if no critical struc-
tural modifications of the products occur during their random walk
to the target) and nonspecific production of NO might be impli-
cated in the killing activity. Furoxan 17 and the related furazan 20
did not kill worms. This inability is surprising in the case of the
furoxan derivative since it is a quite potent TGR inhibitor, although
it is a weak NO-donor under the action of cysteine. Metabolic fate,
increased efflux, and lack of worm uptake could be responsible for
its inactivity. In light of these results, the specific NO release
following the direct interaction of furoxan derivatives with TGR
does not directly correlate with the ex vivo killing activity due to the
complexity of the biological system involved. In conclusion, the
only products of a certain interest among those belonging to this
group are the hybrids 18 and 24, which retain some PZQ-like ac-
tivity resulting in worm paralysis and also display NO-dependent
worm-killing capacity.
faster at the 10
effective worm killers than the furazan analogues (compare 5/8, 6/
9, 7/10), in particular at the 50 M concentration, indicating an
mM concentration. The hybrid furoxans were more
m
involvement of NO in this action, in keeping with what was
observed with 2. The furoxans 6 and 7 emerge as the most inter-
esting products, because of their balance between PZQ and NO-
dependent activities. Findings concerning TGR IC50 and cysteine-
induced NO release suggest that, in the case of compound 7 (high
NO release under the action of cysteine, high TGR inhibitory po-
tency) the worm-killing activity might be induced by a combination
of both specific and nonspecific NO production provided that the
compound's pharmacokinetic properties (e.g. worm uptake, meta-
bolic fate, increased efflux) do not limit its access to the TGR target.
In the case of 6 (low nonspecific release, high TGR inhibitory po-
tency) specific NO release and TGR inhibition appear to be princi-
pally involved. The modestly higher worm-killing activity of 5
compared to 8 is in keeping with the low cysteine-induced NO
release of product but not with its high TGR inhibitory potency. This
suggests that pharmacokinetic properties of 5 limit its access to this
target.
We recently demonstrated that furoxans can inhibit the activity
of P-gp, MRP1 and MRP3 transporters in different kinds of cells
[25,26]. Since multidrug resistance transporters (MDR) are
considered potentially attractive targets for the development of
new antischistosomal drugs [13], selected members of this new
class of PZQ/furoxan hybrid products are worthy of additional
investigation, in view of their potential activity against PZQ-
resistant schistosomes.
2.3. Conclusions
A new class of NO-donor PZQ hybrids was developed by joining
NO-donor furoxan moieties to different areas of the PZQ structure.
The inhibitory activity of these products, and that of the related des-
NO furazan derivatives, was evaluated against recombinant
S. mansoni TGR; their antiparasitic action against ex vivo adult S.
mansoni worms was likewise evaluated. Products 6, 7, 18, and 24
emerged as potent antischistosomal agents, endowed with both
PZQ-like and NO-dependent antiparasitic activity. These
The results obtained with the second group of compounds, 17,
18, 20, 21, 24, and 26, indicate that, although at the higher con-
centration tested several products induced worm contraction, this
effect was of a much shorter duration than that of PZQ (less than
10 min to ~24 h, depending on the compound), suggesting that

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S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
Fig. 1. Images of adult Schistosoma mansoni worms 24 h after addition of compounds.

S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
141
compounds are worthy of additional studies in view of their po-
tential activity against PZQ-resistant schistosomes.
13C NMR (DMSO-d6):
126.6, 127.1, 128.9, 132.7, 134.9, 151.9, 154.8, 155.7, 163.0.
MS CI (isobutane) (m/z): 358 [MHþ].
Anal. (C16H15N5O5) C, H, N.
d
28.0, 45.2, 48.6, 53.9, 110.3, 125.6, 126.3,
3. Experimental section
3.1. Chemistry
3.4. 4-[(4-Oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]
isoquinolin-2-yl)carbonyl]furoxan-3-carbonitrile (7)
1H and 13C NMR spectra were recorded on a Bruker Avance 300
at 300 and 75 MHz, respectively, using SiMe4 as internal standard.
Low resolution mass spectra were recorded with a Finnigan-Mat
TSQ-700. Melting points were determined with a capillary appa-
ratus (Büchi 540). Flash column chromatography was performed on
silica gel (Merck Kieselgel 60, 230e400 mesh ASTM). The progress
of the reactions was monitored by thin layer chromatography (TLC)
on 5 cm ꢂ 20 cm plates with a layer thickness of 0.2 mm. Organic
solvents were removed under vacuum at 30 ꢁC. Elemental analyses
(C, H, N) of the target compounds were performed by Section de
Pharmacie, Service de Microanalyse (Geneva), and the results are
within 0.4% of the theoretical values, unless otherwise stated.
Target compounds were prepared, as assessed using the afore-
mentioned standard spectroscopic techniques and elemental ana-
lyses, in ꢃ95% purity. Compounds 3 [27]; 4 [28]; 16 [29]; 19 [30]; 23
[31] and 25 [32] were obtained as described elsewhere.
A solution of 6 (130 mg, 0.36 mmol) in anhydrous THF (20 ml)
was cooled in an ice bath; anhydrous pyridine (4 eq.) and TFAA
(4 eq.) were added to the solution. The mixture was kept under
stirring at 0 ꢁC for 30 min, then the solvent was removed under
reduced pressure. The residue was taken up with 20 ml of CH2Cl2
and washed with H2SO4 0.5 M (2 ꢂ 10 ml), water (2 ꢂ 10 ml), brine,
dried over Na2SO4, filtered and evaporated under reduced pressure.
The yellow solid obtained was purified by flash chromatography on
silica gel (eluent CH2Cl2/EtOAc 90/10) to give the title product as
white solid yield 83%, mp 183e185 ꢁC dec. (CHCl3/n-Hex).
1H NMR (CDCl3):
d
2.80e3.02 (3H, m, H-6, 2 ꢂ H-7), 3.18 (0.5H,
dd, J ¼ 2.7, 13.5 Hz, H-1), 3.47 (0.5H, dd, J ¼ 3.0, 13.7 Hz, H-1), 4.09
(0.5H, d, J ¼ 18.6 Hz, H-1), 4.43 (0.5H, d, J ¼ 18.3 Hz, H-1), 4.82e5.22
(4H, m, 2 ꢂ H-3, H-6 and H-11b), 7.22e7.32 (4H, m, aromatic
protons).
13C NMR (CDCl3):
d 28.7, 39.0, 47.0, 50.0, 54.3, 55.7, 104.5, 125.4,
3.2. Methyl 4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]
isoquinolin-2-yl)carbonyl]furoxan-3-carboxylate (5)
127.2, 128.0, 129.7, 131.3, 135.0, 150.4, 153.4, 163.0.
MS CI (isobutane) (m/z): 340 [MHþ].
Anal. (C16H13N5O4) C, H, N.
3-methoxycarbonyl-4-furoxancarboxylic
acid
(300
mg,
1.59 mmol) was dissolved in anhydrous THF (7 ml) and CH2Cl2
(10 ml) and the solution obtained was cooled in an ice bath. N-
hydroxysuccinimide (1.1 eq.) and EDC$HCl (1.5 eq.) were added to
the solution, which was kept under stirring at room temperature
for one hour. A solution of 1,2,3,6,7,11b-hexahydro-pyrazino[2,1-a]
isoquinolin-4-one (1 eq.) in anhydrous CH2Cl2 (10 ml) was then
added and the resulting mixture was kept under stirring at room
temperature for 20 min. The solvent was removed under reduced
pressure, the residue was taken up with 30 ml of CH2Cl2 and
washed with water (2 ꢂ 20 ml), brine, dried over Na2SO4 and the
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel (eluent CH2Cl2/EtOAc
90/10) to give the title product as a white solid, yield 42%, mp
150e152 ꢁC dec (MeOH).
3.5. Methyl 4-[(4-oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]
isoquinolin-2-yl)carbonyl]furazan-3-carboxylate (8)
A solution of 5 (330 mg, 0.89 mmol) in (CH3O)3P (5 ml) was
refluxed for 4 h. The mixture was then poured into 20 ml of 2.5 M
H2SO4 and cooled in an ice bath. A white solid precipitated and was
collected by filtration. The filtrate was extracted with CH2Cl2
(2 ꢂ 10 ml), then the organic phase was washed with H2SO4 2.5 M
(3 ꢂ 10 ml), NaHCO3 saturated solution (3 ꢂ 10 ml), brine, dried
over Na2SO4, filtered and evaporated under reduced pressure. The
residue was combined with the filtered-out white solid and crys-
tallized from MeOH/H2O, to give the title product as a white solid,
yield 47%, mp 141e142 ꢁC.
1H NMR (CDCl3):
d
2.78e3.01 (3H, m, H-6, 2 ꢂ H-7), 3.16 (0.5H,
1H NMR (CDCl3):
d
2.78e3.06 (3H, m, H-6, 2 ꢂ H-7), 3.16 (0.5H,
dd, J ¼ 2.7, 13.4 Hz, H-1), 3.39 (0.5H, dd, J ¼ 3.0, 13.7 Hz, H-1), 4.03
(3H, d, J ¼ 3.9 Hz, eCOOCH3), 4.14 (1H, s, H-3), 4.27 (1H, m, H-3),
4.79e5.06 (2H, m, H-1 and H-6), 5.23e5.28 (1H, m, H-11b),
7.21e7.34 (4H, m, aromatic protons).
dd, J ¼ 2.4, 13.2 Hz, H-1), 3.43 (0.5H, dd, J ¼ 3.0, 13.8 Hz, H-1), 3.95
(3H, d, J ¼ 6.0 Hz, eCOOCH3), 4.05e4.41 (2H, m, 2 ꢂ H-3), 4.76e4.92
(1H, m, H-1), 4.98e5.22 (2H, m, H-6 and H-11b), 7.22e7.34 (4H, m,
aromatic protons).
13C NMR (CDCl3):
d 28.7, 39.0, 46.2, 50.2, 54.0, 54.5, 55.6, 125.3,
13C NMR (CDCl3):
d
28.7, 39.0, 46.1, 50.0, 53.9, 55.0, 106.5, 125.3,
127.5, 129.5, 131.6, 135.0, 147.0, 148.8, 155.7, 157.6, 163.1.
MS CI (isobutane) (m/z): 357 [MHþ].
Anal. (C17H16N4O5) C, H, N.
127.1, 127.9, 129.6, 131.6, 135.0, 150.3, 155.2, 155.9, 163.1.
MS CI (isobutane) (m/z): 372 [MHþ].
Anal. (C17H16N4O6) C, H, N.
3.6. 4-[(4-Oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]
3.3. 4-[(4-Oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]
isoquinolin-2-yl)carbonyl]furazan-3-carboxamide (9)
isoquinolin-2-yl)carbonyl]furoxan-3-carboxamide (6)
A solution of 8 (240 mg, 0.67 mmol) in MeOH saturated with
NH3 (9 ml) was stirred at room temperature for 10 min. A white
solid precipitated and was recovered by filtration. The solid ob-
tained was purified by flash chromatography on silica gel (CH2Cl2/
acetone 97/3) to give the product as a white solid, yield 83%, mp
138e139 ꢁC (iPrOH).
A solution of 5 (390 mg, 1.05 mmol) in MeOH saturated with
NH3 (15 ml) was kept under stirring at room temperature for
10 min. A white solid precipitated and was recovered by filtration.
The solid obtained was recrystallized from MeOH, to give the title
product, yield 53%, mp 206e208 ꢁC dec.
1H NMR (DMSO-d6):
d
2.74e2.95 (3H, m, H-6, 2 ꢂ H-7), 3.26
1H NMR (CDCl3):
d
2.79e3.04 (3H, m, H-6, 2 ꢂ H-7), 3.15 (0.5H,
(0.5H, dd, J ¼ 2.1, 13.9 Hz, H-1), 3.45 (0.5H, dd, J ¼ 3.0, 13.7 Hz, H-1),
3.96e4.15 (1H, m, H-1), 4.46e4.68 (3H, m, 2 ꢂ H-3 and H-6),
4.91e4.99 (1H, m, H-11b), 7.18e7.39 (4H, m, aromatic protons), 7.86
(1H, d, J ¼ 13.5 Hz, eNH2), 8.50 (1H, d, J ¼ 3.9 Hz, eNH2).
dd, J ¼ 2.4, 13.4 Hz, H-1), 3.39 (0.5H, dd, J ¼ 2.4, 13.5 Hz, H-1), 4.14
(1H, s, H-3), 4.23e4.29 (1H, m, H-3), 4.74e5.13 (2H, m, H-1 and H-
6), 5.22e5.28 (1H, m, H-11b), 6.61 (1H, br. s, eNH2), 6.76 (0.5H, br. s,
eNH2), 7.18e7.35 (4H, m, aromatic protons), 7.53 (0.5H, br. s, eNH2).

142
S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
13C NMR (CDCl3):
d
28.7, 39.0, 46.3, 49.6, 54.9, 125.4, 127.1, 127.8,
1H NMR (CDCl3):
d 1.22e1.25 (3H, m, cyclohexane protons), 1.45
129.5, 131.6, 135.0, 147.8, 148.3, 156.4, 157.0, 163.5.
MS CI (isobutane) (m/z): 342 [MHþ].
Anal. calc. for C16H15N5O4$0.7H2O: C, H, N 19.79%; found: C, H, N
19.19%.
(2H, m, cyclohexane protons), 1.60e1.76 (5H, m, cyclohexane pro-
tons), 2.24 (0.5H, t, J ¼ 11.1 Hz, cyclohexane protons), 2.59 (0.5H, t,
J
¼
11.4 Hz, cyclohexane protons), 2.70e2.78 (2H, m,
ePheCH2CH2NHe), 3.35 (3H, s, (CH3O)2CHCH2e), 3.38 (3H, s,
(CH3O)2CHCH2e), 3.43 (2H, t, 5.1 Hz, (CH3O)2CHCH2e),
3.48e3.55 (2H, m, ePheCH2CH2NHe), 3.78 (3H, s, CH3OePh), 3.99
(2H, d, 5.1 Hz, eCOCH2e), 4.40 (0.5H, t, 4.8 Hz,
J
¼
3.7. 4-[(4-Oxo-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]
isoquinolin-2-yl)carbonyl]furazan-3-carbonitrile (10)
J
¼
J
¼
(CH3O)2CHCH2e), 4.58 (0.5H, t, J ¼ 4.8 Hz, (CH3O)2CHCH2e), 6.50
(0.5H, br. s, eNH), 6.83 (2H, d, J ¼ 8.4 Hz, H-2 and H-6 aromatic
protons), 6.98 (0.5H, br. s, eNH), 7.10 (2H, d, J ¼ 8.1 Hz, H-3 and H-5
aromatic protons).
A solution of 9, (170 mg 0.50 mmol) in anhydrous THF (20 ml)
was cooled in an ice bath; anhydrous pyridine (4 eq.) and TFAA
(4 eq.) were added to the solution. The mixture was stirred at 0 ꢁC
for 30 min, then the solvent was removed under reduced pressure.
The residue was taken up with 20 ml of CH2Cl2 and washed with
H2SO4 0.5 M (2 ꢂ 10 ml), water (2 ꢂ 10 ml), brine, dried over
Na2SO4, filtered and evaporated under reduced pressure. The
resulting yellow solid was purified by flash chromatography on
silica gel (eluent CH2Cl2/EtOAc 95/5) and then crystallized from
CHCl3/n-hexane to give the product as a white solid, yield 83%, mp
149e150 ꢁC.
13C NMR (CDCl3):
d 25.5, 29.2, 34.7, 40.3, 41.2, 42.8, 50.3, 51.3,
52.3, 52.7, 54.9, 56.4, 102.9, 103.8, 114.0, 129.6, 130.6, 131.3, 158.5,
169.2, 169.6, 178.2.
MS CI (isobutane) (m/z): 377 [MHþ].
3.10. 2-(Cyclohexylcarbonyl)-10-methoxy-1,2,3,6,7,11b-hexahydro-
4H-pyrazino[2,1-a]isoquinolin-4-one (14)
1H NMR (CDCl3):
d
2.80e3.02 (3H, m, H-6, 2 ꢂ H-7), 3.19 (0.5H,
13 (19.0 g, 0.051 mol) was added portion-wise to meth-
anesulphonic acid (20 eq.) cooled in an ice bath. The mixture was
heated to 70 ꢁC for 5 h, then poured into an ice-water mixture. The
mixture was alkalinized to pH 8 with a 20% solution of NaOH, then
extracted with CH2Cl2 (4 ꢂ 50 ml). The organic phase was washed
with water (2 ꢂ 30 ml), brine, dried over Na2SO4 and evaporated
under reduced pressure. The resulting brown oil was purified by
flash chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) to
give a yellow solid, which was crystallized from EtOAc/n-hexane 1/
1 to give the product as a white solid, yield 40%, mp 145e146 ꢁC.
dd, J ¼ 2.4, 13.2 Hz, H-1), 3.49 (0.5H, dd, J ¼ 2.7, 14.3 Hz, H-1), 4.11
(0.5H, d, J ¼ 18.3 Hz, H-1), 4.42 (0.5H, d, J ¼ 17.7 Hz, H-1), 4.79e5.25
(4H, m, 2 ꢂ H-3, H-6 and H-11b), 7.18e7.34 (4H, m, aromatic
protons).
13C NMR (CDCl3):
d 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.
MS CI (isobutane) (m/z): 324 [MHþ].
Anal.(C16H13N5O3) C, H, N.
3.8. 1-(2-Isocyanoethyl)-4-methoxybenzene (12)
1H NMR (CDCl3):
d 1.26e1.32 (3H, m, cyclohexane protons),
1.49e1.61 (2H, m, cyclohexane protons), 1.73e1.81 (5H, m, cyclo-
hexane protons), 2.47 (1H, t, J ¼ 11.1 Hz, cyclohexane proton),
2.69e2.91 (4H, m, 2 ꢂ H-1 and 2 ꢂ H-7), 3.79 (3H, s, eOCH3), 4.08
(1H, d, J ¼ 17.4 Hz, H-3), 4.47 (1H, d, J ¼ 17.4 Hz, H-3), 4.75e4.82 (2H,
m, 2 ꢂ H-6), 5.14 (1H, dd, J ¼ 13.2 Hz, H-11b), 6.80 (2H, d, J ¼ 9.6 Hz,
H-9 and H-11 aromatic protons), 7.09 (1H, d, J ¼ 8.1 Hz, H-8 aro-
matic proton).
A
solution of 2-(4-methoxyphenyl)ethanamine 11 (15 ml,
100 mmol), TEBAC (0.01 eq.) and CHCl3 (1 eq.) in CH2Cl2 (32 ml) was
added dropwise over 10 min to an aqueous solution of NaOH (8 eq.,
32 ml). The mixture was refluxed for 4 h, and then a mixture of ice
and water (50 ml) was added. The organic phase was collected and
the aqueous phase was extracted with CH2Cl2 (2 ꢂ 50 ml). The
organic phases were then washed with H2SO4 0.5 M (3 ꢂ 100 ml),
water (100 ml), brine, dried over Na2SO4 and evaporated under
reduced pressure. The residue was purified by flash chromatog-
raphy on silica gel (eluent CH2Cl2) to give the product as a yellow
oil, yield 57%.
13C NMR (CDCl3):
d 25.7, 27.9, 29.0, 29.2, 39.4, 40.8, 45.2, 49.0,
55.1, 55.4, 110.1, 114.1, 126.7, 130.3, 133.7, 158.5, 164.4, 174.8.
MS CI (isobutane) (m/z): 343 [MHþ].
1H NMR (CDCl3):
d
2.91 (2H, t, J ¼ 6.9 Hz, eCH2CH2eNC), 3.55
3.11. 2-(Cyclohexylcarbonyl)-10-hydroxy-1,2,3,6,7,11b-hexahydro-
4H-pyrazino[2,1-a]isoquinolin-4-one (15)
(2H, t, J ¼ 6.9 Hz, eCH2CH2eNC), 3.79 (3H, s, eOCH3), 6.87 (2H, d,
J ¼ 8.4 Hz, H-2 and H-6 aromatic protons), 7.14 (2H, d, J ¼ 8.4 Hz, H-
3 and H-5 aromatic protons).
BBr3 (1 M solution in CH2Cl2) (3.5 eq.) was added dropwise to a
solution of 14 (6.4 g, 0.019 mol) in CH2Cl2 (300 ml) cooled in an ice
bath. The mixture was stirred at room temperature for 3 h, and then
cooled to 0 ꢁC. An aqueous solution of KHCO3 (10 eq.) was added
dropwise to the reaction mixture, and the resulting suspension was
stirred for 1 h. The organic phase was collected and washed with
water (100 ml), brine, dried over Na2SO4 and evaporated under
reduced pressure. The residue was crystallized from CHCl3/n-hex-
ane to give the product as a white solid, yield 81%, mp 238e239 ꢁC.
13C NMR (CDCl3):
d 34.8, 43.2, 52.3, 114.2, 128.7, 129.4, 129.7,
156.4.
MS CI (isobutane) (m/z): 162 [MHþ].
3.9. N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(2-(4-methoxy)
phenethylamino)-ethyl)cyclohexanecarboxamide (13)
1-(2-isocyanoethyl)-4-methoxybenzene 12 (9.2 g, 0.057 mol)
was added portion-wise to a suspension of paraformaldehyde
(1 eq.), 2,2-dimethoxyethylamine (1 eq.), cyclohexanecarboxylic
acid (1 eq.) in MeOH (60 ml) cooled in an ice bath, and the mixture
was stirred at room temperature for 48 h. The solvent was then
evaporated under reduced pressure and the residue was dissolved
in diethyl ether (150 ml). The organic phase was washed with
H2SO4 0.5 M (50 ml), NaOH 0.5 M (50 ml), water, brine, dried over
Na2SO4 and evaporated under reduced pressure. The yellow oil
solidifies to give the product as a pale yellow solid pure enough for
the next step, yield 89%.
1H NMR (DMSO-d6):
d 1.18e1.39 (5H, m, cyclohexane protons),
1.64e1.72 (5H, m, cyclohexane protons), 2.65e2.86 (5H, m, 1
cyclohexane proton, 2 ꢂ H-1 and 2 ꢂ H-7), 3.72 (0.5H, d, J ¼ 17.7 Hz,
H-3), 4.08 (0.5H, d, J ¼ 17.1 Hz, H-3), 4.39 (1H, s, H-3), 4.45e4.51
(2H, m, 2 ꢂ H-6), 4.66e4.78 (1H, m, H-11b), 6.64 (2H, d, J ¼ 9.3 Hz,
H-9 and H-11 aromatic protons), 6.99 (1H, d, J ¼ 8.1 Hz, H-8 aro-
matic proton), 9.38 (1H, br. s, eOH).
13C NMR (DMSO-d6):
d, 24.9, 25.5, 27.3, 28.9, 30.9, 45.5, 48.1,
48.3, 54.7, 112.1, 114.4, 124.9, 129.8, 134.0, 155.8, 164.6, 173.6.
MS CI (isobutane) (m/z): 329 [MHþ].

S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
143
3.12. 4-({[2-(Cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-
2H-pyrazino[2,1-a]isoquinolin-10-yl]oxy}methyl)furoxan-3-
carboxamide (17)
room temperature. 4-Bromomethylfurazan-3-carboxamide (19)
(1 eq.) was added to the mixture which was stirred at 60 ꢁC for 2 h.
The mixture was then cooled in an ice bath and water was added.
THF was evaporated under reduced pressure and the aqueous
phase was extracted with EtOAc (4 ꢂ 20 ml). The organic phase was
washed with water (2 ꢂ 20 ml), brine, dried over Na2SO4 and
evaporated under reduced pressure. The residue was purified by
flash chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) to
give a white solid which was crystallized from EtOAc, yield 42%, mp
203e204 ꢁC.
NaH (60% suspension in mineral oil, 4 eq.) was added to a sus-
pension of 15 (1.0 g, 3.1 mmol) in anhydrous THF (100 ml) cooled in
an ice bath. The mixture was refluxed for 1.5 h, and then cooled to
room temperature. 4-Bromomethylfuroxan-3-carboxamide (16)
(1 eq.) was added to the mixture, which was then stirred at 60 ꢁC
for 2 h. The mixture was then cooled in an ice bath and water was
added. THF was evaporated under reduced pressure and the
aqueous phase was extracted with EtOAc (5 ꢂ 20 ml). The organic
phase was washed with water (20 ml), brine, dried over Na2SO4 and
evaporated under reduced pressure. The residue was purified by
flash chromatography on silica gel (eluent CH2Cl2/MeOH 98/2) to
give a yellow solid which was crystallized from iPrOH, to give the
product as a white solid, yield 42%, mp 200e202 ꢁC (dec.).
1H NMR (DMF-d7):
d 1.11e1.43 (5H, m, cyclohexane protons),
1.64e1.79 (5H, m, cyclohexane protons), 2.74e2.92 (5H, m, 1
cyclohexane proton, 2 ꢂ H-1 and 2 ꢂ H-7), 4.16e4.21 (1H, m, H-3),
4.47e4.55 (1H, m, H-3), 4.71e4.92 (2H, m, 2 ꢂ H-6), 4.96e5.04 (1H,
m, H-11b), 5.66 (2H, s, eCH2Oe), 7.03 (2H, d, J ¼ 7.8 Hz, H-9 and H-
11 aromatic protons), 7.21 (1H, d, J ¼ 8.4 Hz, H-8 aromatic proton),
8.30 (1H, br. s, eCONH2), 8.67 (1H, br. s, eCONH2).
1H NMR (DMSO-d6):
d
1.03 (6H, d, J ¼ 6 Hz, 2 ꢂ CH3 iPrOH)
13C NMR (DMF-d7):
d 25.9, 26.5, 28.3, 30.0, 39.3, 39.8, 46.5, 49.0,
1.25e1.31 (5H, m, cyclohexane protons), 1.52e1.80 (5H, m, cyclo-
hexane protons), 2.38e1.61 (1H, m, cyclohexane proton), 2.71e2.89
(4H, m, 2 ꢂ H-1 and 2 ꢂ H-7), 4.06e4.12 (2H, m, CH iPrOH, H-3),
4.47 (1H, d, J ¼ 17.4 Hz, H-3), 4.75e4.82 (2H, m, 2 ꢂ H-6), 5.08 (1H,
m, H-11b), 5.44 (2H, s, eCH2Oe), 6.4 (1H, br. s, eCONH2), 6.90e6.96
(2H, m, H-9 and H-11 aromatic protons), 7.12 (1H, d, J ¼ 8.4 Hz, H-8
aromatic proton), 7.55 (1H, br. s, eCONH2).
56.0, 60.4, 113.1, 114.5, 129.2, 130.8, 149.7, 153.2, 157.4, 159.2, 162.9,
165.7, 174.9.
MS CI (isobutane) (m/z): 454 [MHþ].
Anal. (C23H27N5O5) C, H, N.
3.15. 4-({[2-(Cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-
2H-pyrazino[2,1-a]isoquinolin-10-yl]oxy}methyl)furazan-3-
carbonitrile (21)
13C NMR (DMSO-d6):
d 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.
Anhydrous pyridine (4 eq.) and TFAA(4 eq.) were added to a
solution of 20 (550 mg, 1.21 mmol) in anhydrous THF (70 ml). The
mixture was stirred at room temperature for 45 min. The solvent
was then evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (50 ml). The organic phase was washed with
H2SO4 0.5 M (2 ꢂ 30 ml), water, brine, dried over Na2SO4, filtered
and evaporated under reduced pressure. The resulting yellow solid
was purified by flash chromatography on silica gel (eluent CH2Cl2/
MeOH 98/2), to give the product as a white solid, yield 28%, mp
69e70 ꢁC (iPr2O/EtOAc).
MS CI (isobutane) (m/z): 470 [MHþ].
Anal. calc. for C23H27N5O6$iPrOH: C, H, N.
3.13. 4-({[2-(Cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-
2H-pyrazino[2,1-a]isoquinolin-10-yl]oxy}methyl)furoxan-3-
carbonitrile (18)
Anhydrous pyridine (4 eq.) and TFAA (4 eq.) were added to a
solution of 17 (200 mg, 0.43 mmol) in anhydrous THF (25 ml). The
mixture was stirred at room temperature for 45 min. The solvent
was then evaporated under reduced pressure and the residue was
dissolved in CH2Cl2 (50 ml). The organic phase was washed with
H2SO4 0.5 M (2 ꢂ 30 ml), water, brine, dried over Na2SO4, filtered
and evaporated under reduced pressure. The resulting yellow solid
was purified by flash chromatography on silica gel (eluent CH2Cl2/
EtOAc 95/5) to give the product as a white solid, yield 41%, mp
95e97 ꢁC (iPr2O/EtOAc).
1H NMR (CDCl3):
d 1.27e1.57 (5H, m, cyclohexane protons),
1.72e1.81 (5H, m, cyclohexane protons), 2.46 (1H, m, cyclohexane
proton), 2.73e2.96 (4H, m, 2 ꢂ H-1 and 2 ꢂ H-7), 4.10 (1H, d,
J ¼ 17.4 Hz, H-3), 4.46 (1H, d, J ¼ 17.7 Hz, H-3), 4.76e4.83 (2H, m,
2 ꢂ H-6), 5.04e5.09 (1H, m, H-11b), 5.40 (2H, s, eCH2Oe), 6.92 (2H,
d, J ¼ 6.6 Hz, H-9 and H-11 aromatic protons), 7.16 (1H, d, J ¼ 8.7 Hz,
H-8 aromatic proton).
13C NMR (CDCl3):
d, 21.0, 25.7, 27.9, 29.1, 39.3, 40.8, 44.9, 49.0,
1H NMR (CDCl3):
d
1.26e1.31 (5H, m, cyclohexane protons),
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.
1.48e1.81 (5H, m, cyclohexane protons), 2.46 (1H, m, cyclohexane
proton), 2.73e2.98 (4H, m, 2 ꢂ H-1 and 2 ꢂ H-7), 4.10 (1H, d,
J ¼ 17.7 Hz, H-3), 4.46 (1H, d, J ¼ 17.7 Hz, H-3), 4.76e4.83 (2H, m,
2 ꢂ H-6), 5.04 (1H, dd, J ¼ 2.4,10.8 Hz, H-11b), 5.24 (2H, s, eCH2Oe),
6.91e6.94 (2H, m, H-9 and H-11 aromatic protons), 7.16 (1H, d,
J ¼ 8.3 Hz, H-8 aromatic proton).
MS CI (isobutane) (m/z): 436 [MHþ].
Anal. (C23H25N5O4 H2O) C, H, N.
3.16. 2-(Cyclohexylcarbonyl)-10-(3-hydroxypropoxy)-1,2,3,6,7,11b-
hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one (22)
13C NMR (CDCl3):
d 25.7, 27.9, 29.0, 29.2, 39.3, 40.8, 44.8, 49.1,
53.5, 60.9, 96.0, 104.9, 109.7, 114.9, 129.3, 130.8, 134.5, 153.4, 155.8,
164.4, 174.8.
3-Bromopropan-1-ol (1 eq.) and K2CO3 (2 eq.) were added to a
suspension of 15 (1.0 g, 3.2 mmol) in CH3CN. The mixture was
refluxed for 24 h, and then cooled to room temperature. The solvent
was evaporated under reduced pressure; the residue was dissolved
in EtOAc (50 ml) and washed with 0.5 M NaOH solution (4 ꢂ 30 ml),
water (30 ml), brine, dried over Na2SO4 and evaporated under
reduced pressure.
MS CI (isobutane) (m/z): 452 [MHþ].
Anal. (C23H25N5O5$0.5H2O) C, H, N.
3.14. 4-({[2-(Cyclohexylcarbonyl)-4-oxo-1,3,4,6,7,11b-hexahydro-
2H-pyrazino[2,1-a]isoquinolin-10-yl]oxy}methyl)furazan-3-
carboxamide (20)
The yellow solid was purified by flash chromatography on silica
gel (eluent CH2Cl2/MeOH 98/2) to give the product as a white solid,
yield 66%, mp 187e188 ꢁC (MeOH/H2O).
NaH (60% suspension in mineral oil, 4 eq.) was added to a sus-
pension of 15 (1.0 g, 3.1 mmol) in anhydrous THF (100 ml) cooled in
an ice bath. The mixture was refluxed for 1.5 h, and then cooled to
1H NMR (CDCl3):
d
1.18e1.39 (3H, m, cyclohexane protons),
1.52e1.60 (2H, m, cyclohexane protons), 1.73e1.80 (5H, m,

144
S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
cyclohexane protons), 2.02e2.17 (2H, m, cyclohexane proton and
H-1), 2.43e2.50 (1H, m, H-1), 2.69e2.76 (2H, m, 2 ꢂ H-7),
2.81e2.95 (2H, m, HOeCH2CH2CH2Oe), 3.84e3.87 (2H, m, 2 ꢂ H-3),
4.05e4.25 (3H, m, eOH, HOeCH2CH2CH2Oe), 4.28e4.36 (1H, m, H-
6), 4.43e4.49 (1H, m, H-6), 4.73e4.82 (2H, m, HOeCH2CH2CH2Oe),
5.06e5.10 (1H, m, H-11b), 6.81 (2H, d, J ¼ 8.1 Hz, H-9 and H-11
aromatic protons), 7.08 (1H, d, J ¼ 7.8 Hz, H-8 aromatic protons).
protons), 7.73 (1H, t, J ¼ 7.2 Hz, aromatic protons), 8.07 (2H, d,
J ¼ 7.8 Hz, aromatic protons).
13C NMR (CDCl3):
d 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, 157.4, 161.2, 164.1, 164.4, 174.8.
MS CI (isobutane) (m/z): 595 [MHþ].
Anal. (C30H34N4O7S): C, H, N.
13C NMR (CDCl3):
d 25.7, 28.6, 29.0, 29.5, 31.0, 39.4, 40.8, 45.2,
49.0, 55.1, 60.0, 65.8, 110.8, 114.6, 126.9, 130.2, 133.7, 157.7, 164.4,
3.19. Biology
174.9.
MS CI (isobutane) (m/z): 387 [MHþ].
Female Swiss-Webster mice infected with S. mansoni cercariae
by percutaneous tail exposure were obtained from the Biomedical
Research Institute, Rockville, MD. Parasites were obtained from
mice 7 weeks after infection by perfusion with RPMI 1640 medium
(Cellgro, Fisher) [33]. Worms were washed thoroughly several
times in RPMI 1640 in a laminar flow hood; they were then washed
several times in complete RPMI 1640 (cRPMI) containing 25 mM
3.17. 2-(Cyclohexylcarbonyl)-10-(3-{[3-(phenylsulfonyl)furoxan-4-
yl]oxy}propoxy)-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]
isoquinolin-4-one (24)
DBU (2 eq.) was added to a solution of 23 (1.2 eq.) in CH2Cl2
(20 ml). The solution was cooled in an ice bath and a solution of 22
(140 mg, 0.36 mmol) in CH2Cl2 (10 ml) was added. The mixture was
stirred at room temperature for 1 h, washed with a 0.5 M H2SO4
solution (2 ꢂ 20 ml), water (2 ꢂ 20 ml), brine, dried over Na2SO4
and evaporated under reduced pressure. The resulting yellow oil
was partially purified by flash chromatography on silica gel (eluent
CH2Cl2/MeOH 98/2) and then purified by HPLC (eluent CH3CN/H2O
0.1% CF3COOH 70/30), to give the product as a white solid, yield 14%,
mp 82e84 ꢁC (iPrOH).
HEPES (Cellgro, Fisher), 150 units/mL penicillin, 125 mg/mL strep-
tomycin, and 10% fetal bovine serum (Atlanta Biologicals). Worms
were then distributed into 6-well tissue culture plates with 15e20
worms per well and cultured overnight in 5 ml cRPMI in 5% CO2 at
37 ꢁC. The following day, media were removed from each well and
replaced with 5 ml fresh cRPMI. Test compounds were dissolved in
DMSO at 10 mM and added to the wells at the indicated concen-
trations. The same volume of DMSO was added to each well.
Negative control worms were treated with an equal volume of
DMSO alone. After overnight culture, the media with compounds
were removed and replaced with fresh media alone. Worm
mobility and survival were observed under a stereomicroscope
(Zeiss Stemi 2000-C) for 10 s per worm at the indicated time points.
Media were removed and replaced with fresh media every 48 h.
This study was approved by the Institutional Animal Care and Use
Committee at Rush University Medical Center (IACUC number
08e058; DHHS animal welfare assurance number A3120-01).
TGR preparation and IC50 determination were performed as
1H NMR (CDCl3):
d 1.26e1.32 (3H, m, cyclohexane protons),
1.49e1.57 (2H, m, cyclohexane protons), 1.72e1,80 (5H, m, cyclo-
hexane protons), 2.34e2.47 (3H, m, cyclohexane proton and 2 ꢂ H-
1), 2.71e2.92 (4H, m, 2 ꢂ H-7 and HOeCH2CH2CH2Oe), 4.06e4.50
(4H, m, 2 ꢂ H-3 and HOeCH2CH2CH2Oe), 4.62e4.66 (2H, m, 2 ꢂ H-
6), 4.75e4.83 (2H, m, HOeCH2CH2CH2Oe), 5.08e5.17 (1H, m, H-
11b), 6.83 (2H, d, J ¼ 7.5 Hz, H-9 and H-11 aromatic protons), 7.11
(1H, d, J ¼ 7.8 Hz, H-8 aromatic protons), 7.54e7.59 (2H, m, aromatic
protons), 7.74 (1H, t, J ¼ 7.5 Hz, aromatic protons), 8.02 (2H, d,
J ¼ 7.5 Hz, aromatic protons).
described [34]. Briefly, assays were run in 96-well plates in 200
mL
13C NMR (CDCl3):
d 25.7, 27.9, 28.5, 29.0, 29.2, 39.4, 40.8, 45.2,
with 20 nM enzyme and 100 M NADPH in 0.1 M potassium
m
phosphate (pH 7.4), 10 mM EDTA. Test compounds were dissolved
in DMSO. The enzyme was incubated for 10 min at room temper-
ature with NADPH and compounds at 50 mM, 20 mM, 10 mM, 1 mM,
49.0, 55.0, 63.7, 68.1, 110.5, 110.8, 111.2, 114.3, 127.2, 128.5, 129.6,
130.4, 133.9, 135.6, 138.0, 157.5, 158.9, 164.4, 174.8, 177.2.
MS CI (isobutane) (m/z): 611 [MHþ].
500 nM, 100 nM, 10 nM, or 5 nM before the addition of 3 mM 5, 50-
dithio-bis(2-nitrobenzoic acid). Enzyme activity was monitored by
observing the change in A412 due to the formation of 5-thio-2-
nitrobenzoic acid during the first 3 min. Residual TGR activity
was compared to controls, incubated with equal volumes of DMSO.
All assays were done in triplicate.
Anal. (C30H34N4O8S): C, H, N.
3.18. 2-(Cyclohexylcarbonyl)-10-(3-{[4-(phenylsulfonyl)furazan-3-
yl]oxy}propoxy)-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]
isoquinolin-4-one (26)
DBU (2 eq.) was added to a solution of 25 (1.2 eq.) in CH2Cl2
(20 ml). The solution was cooled in an ice bath and a solution of 22
(150 mg, 0.39 mmol) in CH2Cl2 (10 ml) was added to it. The mixture
was stirred at room temperature for 1 h, washed with H2SO4 0.5 M
solution (2 ꢂ 20 ml), water (2 ꢂ 20 ml), brine, dried over Na2SO4
and evaporated under reduced pressure. The resulting yellow oil
was partially purified by flash chromatography on silica gel (eluent
CH2Cl2/MeOH 98/2) and then purified by HPLC (eluent CH3CN/H2O
0.1% CF3COOH 75/25), to give the product as a white solid, yield
35%, mp 70e71 ꢁC (iPrOH).
Acknowledgements
We would like to thank Dr. Matthew S. Tucker, Biomedical
Research Institute, Rockville, MD, for parasite material through
NIH-NIAID contract HHSN272201000005I. This work was sup-
ported in part by the NIH/National Institute of Allergy and Infec-
tious Diseases (NIAID) grant R01AI065622 (DLW) and by Ministero
ꢀ
dell'Istruzione, dell'Universita e della Ricerca grant (PRIN 2009
prot. 20097FJHPZ_002).
1H NMR (CDCl3):
d 1.28e1.31 (3H, m, cyclohexane protons),
References
1.49e1.57 (2H, m, cyclohexane protons), 1.72e1,80 (5H, m, cyclo-
hexane protons), 2.20e2.34 (3H, m, cyclohexane proton and 2 ꢂ H-
1), 2.71e2.95 (4H, m, 2 ꢂ H-7 and HOeCH2CH2CH2Oe), 4.06e4.50
(4H, m, 2 ꢂ H-3 and HOeCH2CH2CH2Oe), 4.57e4.61 (2H, m, 2 ꢂ H-
6), 4.74e4.83 (2H, m, HOeCH2CH2CH2Oe), 5.09e5.14 (1H, m, H-
11b), 6.73e6.81 (2H, m, H-9 and H-11 aromatic protons), 7.10 (1H, d,
J ¼ 8.7 Hz, H-8 aromatic protons), 7.56e7.66 (2H, m, aromatic
[1] B. Gryseels, Schistosomiasis, Infect. Dis. Clin. N. Am. 26 (2012) 383e397.
[2] P. Steinmann, J. Keiser, R. Bos, M. Tanner, J. Utzinger, Schistosomiasis and
water resources development: systematic review, meta-analysis, and esti-
mates of people at risk, Lancet Infect. Dis. 6 (2006) 411e425.
[3] M.J. Van der Werf, S.J. de Vlas, S. Brooker, C.W. Looman, N.J. Nagelkerke,
J.D. Habbema, D. Engels, Quantification of clinical morbidity associated with
schistosoma infection in Sub-Saharan Africa, Acta Trop. 86 (2003) 125e139.

S. Guglielmo et al. / European Journal of Medicinal Chemistry 84 (2014) 135e145
145
[4] S.A.L. Thetiot-Laurent, J. Boissier, A. Robert, B. Meunier, Schistosomiasis
chemotherapy, Angew. Chem. Int. Ed. 53 (2013) 17936e17956.
[5] R. Gonnert, P. Andrews, Praziquantel, a new broad-spectrum antischistosomal
agent, Z. Parasitenk 52 (1977) 129e150.
[6] Y. Liu, X. Wang, J.K. Wang, C.B. Ching, Investigation of the phase diagrams of
chiral praziquantel, Chirality 18 (2006) 259e264.
[22] a) R. Fruttero, D. Boschi, A. Di Stilo, A. Gasco, The furoxan system as a useful
tool for balancing "Hybrids" with mixed alpha-1-Antagonist and NO-like
vasodilator activities, J. Med. Chem. 38 (1995) 4944;
b) M.F. Buonsanti, M. Bertinaria, A. Di Stilo, C. Cena, R. Fruttero, A. Gasco, Nitric
oxide donor beta(2)-agonists: furoxan derivatives containing the fenoterol
moiety and related furazans, J. Med. Chem. 50 (2007) 5003e5011;
c) T. Dunlap, S.O. Abdul-Hay, R.E.P. Chandrasena, G.K. Hagos, V. Sinha,
Z. Wang, H. Wang, G.R.J. Thatcher, Nitrates and NO-NSAIDs in cancer che-
moprevention and therapy: in vitro evidence querying the NO donor func-
tionality, Nitric Oxide-Biol. Chem. 19 (2008) 115e124.
[7] A. Doemling, K. Khoury, Praziquantel and schistosomiasis, ChemMedChem 5
(2010) 1420e1434.
[8] A. Fenwick, J.P. Webster, Schistosomiasis: challenges for control, treatment
and drug resistance, Curr. Opin. Infect. Dis. 19 (2006) 577e582.
[9] A.D. Aragon, R.A. Imani, V.R. Blackburn, P.M. Cupit, S.D. Melman, T. Goronga,
T. Webb, E.S. Loker, C. Cunningham, Towards an understanding of the
mechanism of action of praziquantel, Mol. Biochem. Parasitol 164 (2009)
57e65.
[10] P.S. Sadhu, S.N. Kumar, M. Chandrasekharam, L. Pica-Mattoccia, D. Cioli,
V.J. Rao, Synthesis of new praziquantel analogues: potential candidates for the
treatment of schistosomiasis, Bioorg. Med. Chem. Lett. 22 (2012) 1103e1106.
[11] E. van den Enden, Pharmacotherapy of helminth infection, Exp. Opin. Phar-
macother. 10 (2009) 435e451.
[23] H. Cao, H. Liu, A. Doemling, Efficient multicomponent reaction synthesis of the
schistosomiasis drug praziquantel, Chem. Eur. J. 16 (2010) 12296e12298.
[24] M. Colasanti, L. Gradoni, M. Mattu, T. Persichini, L. Salvati, G. Venturini,
P. Ascenzi, Molecular bases for the anti-parasitic effect of NO (Review), Int. J.
Mol. Med. 9 (2002) 131e134.
[25] R. Fruttero, M. Crosetti, K. Chegaev, S. Guglielmo, A. Gasco, F. Berardi, M. Niso,
R. Perrone, M.A. Panaro, N.A. Colabufo, Phenylsulfonylfuroxans as modulators
of multidrug-resistance associated protein-1 and P-glycoprotein, J. Med.
Chem. 53 (2010) 5467e5475.
[12] W. Wang, L. Wang, Y.S. Liang, Susceptibility or resistance of praziquantel in
human schistosomiasis: a review, Parasitol. Res. 111 (2011) 1871e1877.
[13] R.S. Kasinathan, W.M. Morgan, R.M. Greenberg, Schistosoma mansoni express
higher levels of multidrug resistance-associated protein 1 (SmMRP1) juvenile
worms and in response to praziquantel, Mol. Biochem. Parasitol 173 (2010)
25e31.
[14] A.A. Sayed, A. Simeonov, C.J. Thomas, J. Inglese, C.P. Austin, D.L. Williams,
Identification of oxadiazoles as new drug leads for the control of schistoso-
miasis, Nat. Med. 14 (2008) 407e412.
[15] G. Rai, C.J. Thomas, W. Leister, D.J. Maloney, Synthesis of oxadiazole-2-oxide
analogues as potential antischistosomal agents, Tetrahedron Lett. 50 (2009)
1710e1713.
[16] A. Gasco, A.J. Boulton, Furoxans and benzofuroxans, Adv. Heterocycl. Chem. 29
(1981) 251.
[26] K. Chegaev, C. Riganti, L. Lazzarato, B. Rolando, S. Guglielmo, I. Campia,
R. Fruttero, A. Bosia, A. Gasco, Nitric oxide donor doxorubicins accumulate
into doxorubicin-resistant human colon cancer cells inducing cytotoxicity,
Med. Chem. Lett. 2 (2011) 494e497.
[27] J.H. Kim, Y.S. Lee, H. Park, C.S. Kim, Formation of pyrazinoisoquinoline ring
system by the tandem amidoalkylation and N-acyliminium ion cyclization: an
efficient synthesis of praziquantel, Tetrahedron 54 (1998) 7395e7400.
[28] C. Grundmann, G.W. Nickel, R.K. Bansal, Das Tetramere der Knallsaure (Iso-
cyanilsaure) und seine Derivate (Nitrile oxides. XVIII. Tetramer of fulminic acid
(isocyanilic acid) and its derivatives), Liebigs Ann. Chem. (1976) 1029e1050.
[29] A. Di Stilo, S. Visentin, C. Cena, A.M. Gasco, G. Ermondi, A. Gasco, New 1,4-
dihydropyridines conjugated to furoxanyl moieties, endowed with both ni-
tric oxide-like and calcium channel antagonist vasodilator activities, J. Med.
Chem 41 (1998) 5393e5401.
[17] M. Feelisch, K. Schonafinger, E. Noack, Thiol-mediated generation of nitric-
oxide accounts for the vasodilator action of furoxans, Biochem. Pharmacol.
44 (1992) 1149e1157.
[18] A. Gasco, K. Schoenafinger, The NO-releasing heterocycles, in: P.G. Wang,
T.B. Cai, N. Taniguchi (Eds.), Nitric Oxide Donors, WILEY-VCH Verlag GmbH &
Co KGaA, Weinheim, 2005, pp. 131e175.
[19] H.M. Alger, A.A. Sayed, M.S. Stadecker, D.L. Williams, Molecular and enzimatic
characterization of Schistosoma mansoni thioredoxin, Int. J. Parasitol. 32
(2002) 1285e1292.
[20] G. Rai, A.A. Sayed, W.A. Lea, H.F. Luecke, H. Chakrapani, S. Prast-Nielsen,
A. Jadhav, W. Leister, M. Shen, J. Inglese, C.P. Austin, L. Keefer, E.S.J. Arner,
A. Simeonov, D.J. Maloney, D.L. Williams, C.J. Thomas, Structure mechanism
insights and the role of nitric oxide donation guide the development of
oxadiazole-2-oxides as therapeutic agents against schistosomiasis, J. Med.
Chem. 52 (2009) 6474e6483.
[30] M. Bertinaria, U. Galli, G. Sorba, R. Fruttero, A. Gasco, M.I. Brenciaglia,
M.M. Scaltrito, F. Dubini, Synthesis and anti-Helicobacter pylori properties of
NO-donor/metronidazole hybrids and related compounds, Drug. Dev. Res. 60
(2003) 225e239.
[31] W.V. Farrar, The 3,4-bisarenesulphonylfuroxans, J. Chem. Soc. (1964) 904e906.
[32] D. Boschi, A. Di Stilo, C. Cena, M. Lolli, R. Fruttero, A. Gasco, Studies on agents
with mixed NO-dependent vasodilating and beta-blocking activities, Pharm.
Res. 14 (1997) 1750e1758.
[33] F. Lewis, Schistosomiasis, in: J.E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W. Strober (Eds.), Current Protocols in Immunology, edition ed.,
John Wiley & Sons, New York, 1998, pp. 19.11.11e19.11.28.
ꢁ
[34] A.N. Kuntz, E. Davioud-Charvet, A.A. Sayed, L.L. Califf, J. Dessolin, E.S.J. Arner,
D.L. Williams, Thioredoxin glutathione reductase from Schistosoma mansoni:
an essential parasite enzyme and a key drug target, PLoS Med. 4 (2007)
1071e1086.
[21] To be submitted for publication.