Amodiaquine analogues containing NO-donor substructures: Synthesis and their preliminary evaluation as potential tools in the treatment of cerebral malaria

Article abstract of DOI:10.1016/j.ejmech.2011.02.029

The synthesis and physico-chemical properties of novel compounds obtained by conjugation of amodiaquine with moieties containing either furoxan or nitrooxy NO-donor substructures are described. The synthesised compounds were tested in vitro against both t

Full text of DOI:10.1016/j.ejmech.2011.02.029

European Journal of Medicinal Chemistry 46 (2011) 1757e1767
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Amodiaquine analogues containing NO-donor substructures: Synthesis and their
preliminary evaluation as potential tools in the treatment of cerebral malaria
,
,
Massimo Bertinaria a 1, Stefano Guglielmo a 1, Barbara Rolando a, Marta Giorgis a, Cristina Aragno a,
Roberta Fruttero a, Alberto Gasco a , Silvia Parapini , Donatella Taramelli , Yuri C. Martins ,
,
b
b
c
*
Leonardo J.M. Carvalho c
a Dipartimento di Scienza e Tecnologia del Farmaco, Via P. Giuria 9, I-10125 Torino, Italy
b Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy
c La Jolla Bioengineering Institute, 3535 General Atomics Court suite 210, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and physico-chemical properties of novel compounds obtained by conjugation of amo-
diaquine with moieties containing either furoxan or nitrooxy NO-donor substructures are described. The
synthesised compounds were tested in vitro against both the chloroquine sensitive, D10 and the chlo-
roquine resistant, W-2 strains of Plasmodium falciparum (P. falciparum). Most of the compounds showed
an antiplasmodial activity comparable to that of the parent drug. By comparing the activities of simple
related structures devoid of the ability to release NO, it appears that the contribution of NO to the
antiplasmodial action in vitro is marginal. All the compounds were able to relax rat aorta strips with
a NO-dependent mechanism, thus showing their capacity to release NO in the vessels. A preliminary in
vivo study using Plasmodium berghei ANKA-infected mice showed a trend for prolonged survival of mice
with cerebral malaria treated with compound 40, which is potent and fast amodiaquine-derived NO-
donor, when compared with amodiaquine alone or with compound 31, a milder NO-donor. The two
compounds showed in vivo antiplasmodial activity similar to that of amodiaquine.
Received 18 November 2010
Received in revised form
10 February 2011
Accepted 11 February 2011
Available online 22 February 2011
Keywords:
Amodiaquine
Cerebral malaria
Nitric oxide
Furoxans
Nitrooxy derivatives
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
learning, memory formation and neurotransmitters release, while
in the peripheral nervous system behaves as a neurotransmitter at
Malaria is one of the most important causes of parasitic infec-
tion and death in the world. A WHO report estimates in 189e327
millions the cases of malaria and in about 1 million the associated
deaths, the major part of them occurring in children living in sub-
Saharan Africa [1]. There is an increasing need both of new anti-
malarial drugs and of improvements of those already in use, in
particular of agents active against the infection due to Plasmodium
falciparum, which is the main agent of severe malaria.
Nitric oxide (NO) is a physiological messenger with multiple
actions. In the cardiovascular system it is involved in maintaining
the micro- and macro-vascular homeostasis [2]. In particular, it
induces vasodilation and inhibits platelet aggregation through
a cyclic GMP (cGMP) dependent mechanism and it modulates the
expression of the cell-adhesion molecules (CAMs) on endothelial
cells. In the central nervous system, it plays complex roles in
the ends of non-adrenergic, non-cholinergic nerves controlling
a number of gastrointestinal, respiratory and genitourinary func-
tions [3]. In the innate immune system NO is one of the final
effector molecules against pathogens of different origin. It regulates
other immunological functions, as well, including T- and B-cells
proliferation, leukocytes rolling and cytokines production [4].
Finally, NO seems to play an important role in the pathogenesis of
P. falciparum malaria both as antiplasmodial agent and regulator of
the immune response to the parasite. This suggested the hypothesis
that derivatives of NO could be effective antimalarial agents [5].
Indeed, it was found that some NO-donors display in vitro toxic
action against P. falciparum [6e8].
Severe falciparum malaria, including cerebral malaria (CM), is
associated with tissue ischemia related to cytoadherence of para-
sitized erythrocytes to microvascular endothelium. CM has been
reported to be associated to low NO bioavailability in the vascula-
ture [9,10]. This is due to both an increased scavenging of NO in the
blood, caused by the high concentration of free oxyhaemoglobin
(HbO2þ) from the haemolysis of parasitized red blood cells and to
* Corresponding author. Tel.: þ39 011 6707670; fax: þ39 011 6707286.
E-mail address: alberto.gasco@unito.it (A. Gasco).
1
Authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.029

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M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
Chart 1. Amodiaquine structure.
hypoargininemia. Administration of exogenous NO to the murine
model of CM induced by Plasmodium berghei ANKA (PbA) restored
signalling in the brain, decreased proinflammatory biomarkers in
the blood, and markedly reduced vascular leak and petechial
hemorrhages in the brain [10a]. Moreover, a significant improve-
ment of CM has been observed in patients after infusion of L-argi-
nine demonstrating that the NO pathway can be targeted for
adjunctive treatment in falciparum malaria [10b]. Amodiaquine
(AQ, Chart 1) is an established antimalarial drug recently reintro-
duced in the World Health Organisation Model List of Essential
Medicines [11,12].
As development of our previous work aimed at designing new
NO-donor multifunctional drugs [13], we now report a study con-
cerning the synthesis, the dissociation constants and the in vitro
anti P. falciparum activity of new NO-donor amodiaquine deriva-
tives (NO-AQ) containing either furoxan (1,2,5-oxadiazole 2-oxide)
or nitrooxy (-ONO2) NO-donor substructures. The capacity of NO-
AQ of relaxing rat aorta strips with a NO-dependent mechanism,
and in vivo preliminary data which seem to confirm their potential
utility in the treatment of CM, are also discussed.
Scheme 1. Preparation of derivatives 3, 4, 7, 12e15. Reagents and conditions: (a)
fuming HNO3, -15 ꢀC to RT, 24 h; (b) EtNH2, EtOH, RT, 48 h; (c) 1N HCl; (d) ion-
exchange chromatography, Amberlite IRA-400.
triethylamine, was alkylated with excess ethyl iodide in basic
medium to afford 22. Treatment of this product with thiophenol
and KOH under nitrogen atmosphere gave the expected secondary
amine 23. The dioxolane ring of this intermediate was cleaved by
treatment with refluxing 80% CF3COOH solution and subsequent
conversion of the obtained aminodiol trifluoroacetate 24 to the
corresponding free base by ion-exchange chromatography; 25 was
then treated with fuming nitric acid to afford the final double
nitrated aminodiol 26 as HNO3 salt.
2. Results and discussion
2.1. Chemistry
The synthesis of final AQ derivatives 28e31, 32e35 bearing
nitrooxy groups (Scheme 3) was carried out by nucleophilic
displacement of benzylic chlorine atom present on versatile inter-
mediate 27 we have previously described [15], by the appropriate
nitrooxy substituted primary and secondary amines. The reaction,
was performed in a 1:1 mixture of DMF and CH3CN, in the presence
of triethylamine. Compounds 28e30, 32e35 were converted to the
corresponding hydrochlorides by treatment of their methanolic
solution with HCl-saturated Et2O, while compound 31 was kept as
the free base.
The final AQ derivatives 37, 40 bearing furoxan moieties were
synthesised starting from 27 (Scheme 4). To obtain the phenyl-
furoxanyl substituted compound 37 the chloromethyl substituted
intermediate 27 was transformed into hydroxyamodiaquine 36
by treatment with 2-(ethylamino)ethanol in acetonitrile. This last
compound was treated with 3-phenyl-4-benzenesulfonyl furoxan
and 50% (w/w) NaOH, under nitrogen atmosphere in distilled THF/
DMF mixture, to give the desired product. Owing to extensive
decomposition of the reaction mixture, it was impossible to obtain
40 in the same way. This product was obtained by reacting 27 with
N-ethyl-2-((3-phenylsulfonyl furoxan-4-yl)oxy)ethanamine 39 that,
in its turn, was obtained by NaOH-mediated reaction of 2-(ethyl-
amino)ethanol with bis-benzenesulfonyl furoxan 38. The proposed
structure of 39 was confirmed by its 13C-NMR spectrum, which is
typical of a 4-alkoxy-3-phenylsulfonyl substituted furoxan [16].
Derivatives 3, 4, 7, 12e15, bearing nitrooxy functionalities and
a primary or a secondary amino group, used for building the final
nitrooxy substituted compounds, were synthesised following the
pathways described in Scheme 1. Compounds 3 and 4 were syn-
thesised by nitration with fuming nitric acid of the related
commercially available amino alcohols 1 and 2. For the synthesis of
6-(ethylamino)hexan-1-ol (6) the commercially available 6-chlor-
ohexanol (5) was treated with ethylamine and then with HCl to give
5a,HCl. The hydrochloride was converted into the corresponding
free base by ion-exchange chromatography on cationic resin
(Amberlite IRA-400). This base was transformed into 7,HNO3 by
treatment with fuming nitric acid in 34% overall yield. For the
synthesis of piperidino- and piperazino-based nitrooxy derivatives,
the commercially available piperidino or piperazino alcohols 8e11
were nitrated following the usual procedure to afford compounds
12e15 in 60e67% yields.
The synthesis of 6-(ethylamino)hexane-1,2-diyl dinitrate (26)
was achieved through the route depicted in Scheme 2. The
commercially available hex-5-en-1-ol (16) was converted into the
corresponding phtalimido derivative 17, by reaction of the inter-
mediate methansulphonate 16a with potassium phtalimide.
Double bond oxidation with KMnO4 in acetone/water afforded the
diol 18, which was then transformed into the dioxolane 19 with
acetone and catalytic pyridinium p-toluensulfonate (PPTS).
Cleavage of the phtalimide moiety by refluxing with hydrazine
hydrate in THF afforded the amine 20. The Fukuyama procedure
[14] was used in order to obtain the mono-alkylated amine 23.
2-nitro-benzenesulphonamide derivative 21, resulting from reac-
tion of 20 with 2-nitrobenzenesulfonyl chloride in the presence of
2.2. Dissociation constants determination
Potentiometric titrations of the final amodiaquine derivatives
were performed with a Sirius GLpKa automated potentiometric

M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
1759
Scheme 2. Preparation of 26. Reagents and conditions: (a) CH3SO2Cl, Et3N, CH2Cl2, RT; (b) potassium phtalimide, KI cat., CH3CN, reflux, 72 h (c) KMnO4, Acetone/H2O, 0 ꢀC to RT, 3 h;
(d) Acetone, PPTS cat, RT, 18 h; (e) N2H4 H2O, dist. THF, reflux, 24 h; (f) 2-nitrobenzensulfonyl chloride, Et3N, CH2Cl2, 0 ꢀC to RT, 30 min; (g) EtI, K2CO3, DMF, 60 ꢀC, 10 h; (h) PhSH,
KOH, CH3CN, 0 ꢀC to 50 ꢀC, 4 h, N2 atmosphere. (i) CF3COOH/H2O 8/2, reflux, 24 h; (j) ion-exchange chromatography, Amberlite IRA-400; (k) fuming HNO3, -15 ꢀC to RT, 40 h.
system. The titrations were carried out in water using methanol in
different ratios as co-solvent; the aqueous pKas were determined
by extrapolation to 0% methanol according to YasudaeShedlovsky
procedure (see experimental). The pKa values are listed in Table 1.
Using AQ as reference compound (pKa1 ¼ 8.47, lateral chain
nitrogen; pKa2 ¼ 7.42, 4-aminoquinoline center) [17] it is reason-
able to assign in the analogues 28e35, 37, 40, the higher pKa2 values
to the basic center of the lateral chains, and the lower pKa1 values to
the 4-aminoquinoline moiety. In the case of the piperazino
substituted compounds 34, 35, pKa3 values could be related with
aminoquinoline scaffold, while pKa1 and pKa2 values with the
piperazine substructure. From the data of the Table 1, it is imme-
diately deducible that at pH (5.2) of parasite food vacuole all the
products exist in the dicationic form in equilibrium with the
monocationic one, while at physiological pH (7.4) as complex
equilibrium between neutral, monocationic and dicationic species.
both strains in the low nM range. The most active compounds are the
piperidine and the piperazine derivatives 32, 33 and 35, which
display an antiplasmodial potency near to that of the lead. Also the
activity of this class against the W-2, CQ-R strain is similar to that of
AQ, within the limits of the experimental errors, with the only
exception of the furoxan and piperazine compounds 40 and 34,
respectively, which show signs of cross resistance with CQ.
No activity in the nM range was observed for the simple nitric
acid esters 3, 7, 12e15, and 26 used to prepare the nitrooxy
substituted NO-AQ, as well as for the two simple furoxans A, B
(Table 2) structurally similar to the furoxan moieties present in 37
and 40 respectively. These results indicate that the contribution of
NO to the antiplasmodial properties of NO-AQ is, if any, secondary
to that of the lead structure.
2.3.2. Vasodilator activity
All the NO-AQ products described in this work were able to relax
rat aorta strips pre-contracted with phenylephrine. The vasodilator
potencies, expressed as EC50, are reported in Table 2. Analysis of the
data indicates that the most potent compounds appear to be the
dinitrooxy- and the furoxan-substituted products 35 and 40,
respectively, which display their action in the nM range. The
remaining products show EC50 values all included in a very narrow
2.3. Biological activities
2.3.1. In vitro antimalarial activity against (CQS) D10 and (CQR) W-
2 strains of P. falciparum
All the final products were screened in vitro against the chloro-
quine sensitive (CQ-S) D10 and the chloroquine resistant (CQ-R) W-2
strains of P. falciparum. The results, expressed as 50% inhibitory
concentrations (IC50) are shown in Table 2. All tested compounds
including the NO-AQ derivatives retained significant activity against
range 0.10O1.2
presence of 1 M ODQ (1H-[1,2,4] oxadiazolo [4, 3-a]quinoxalin-1-
one), a well known inhibitor of the soluble guanylate cyclase (sGC),
mM. When the experiments were repeated in the
m

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M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
Scheme 3. Preparation of derivatives 28e35. Reagents and conditions: (a) 3, 4, 7, 26, Et3N, CH3CN/DMF 1/1, RT, 6 h; (b) HCl satd Et2O, MeOH; (c) 12-15, Et3N, CH3CN/DMF 1/1,
RT, 6 h.
a decrease in the potencies was observed. These results indicate the
ability of these products to release NO in the vessels with conse-
quent NO-dependent vasodilation, thus suggesting their potential
use as adjunct therapy for CM.
CM. Indeed, low NO bioavailability plays a role in the pathogenesis
of murine CM [10], which is associated with cerebral microcircu-
latory dysfunction and vasoconstriction [18]. Treatment with an
NO-donor, 1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-
1-ium-1,2-diolate (DPTA-NO) largely prevents the neurological
syndrome and this is associated with improved cerebral vascular
responses [19]. We asked whether a hybrid compound endowed
with antimalarial and NO activities could improve survival in mice
with late stage CM, in comparison with its lead compound pre-
senting antimalarial activity only. Our preliminary results with
compounds 40 and 31 in the murine model of CM by P. berghei
ANKA indeed showed a trend for increased survival of the animals
treated with compound 40, which is a potent and fast NO-donor. It
is remarkable that survival rates were increased three-fold, espe-
cially considering that mice left untreated at late stage of CM die in
a few hours, and AQ by itself was highly ineffective at this stage.
Earlier treatment and shorter intervals for drug administration may
help to further improve the efficacy. The same trend for increased
survival was not observed with compound 31, a milder NO-donor.
Further in vivo studies are in progress to disclose the interesting
potentialities of these products, and of other NO-donor antimalarial
drugs, in the treatment of CM.
2.3.3. Ability of compounds 31 and 40 to improve survival of mice
from late-stage cerebral malaria (CM)
To test this hypothesis, preliminary in vivo experiments were
conducted with compounds 31 and 40 using the murine model of
CM caused by P. berghei ANKA (PbA). Infected mice with late stage
CM (i.e., presenting neurological signs, hypothermia and low motor
scores) treated with compound 40 (1.4 mg per mouse) daily for 5
days presented a trend for delayed mortality and increased survival
as compared to AQ (1 mg per mouse) alone. Compound 40 slightly
improved the survival to 23% and had an efficacy in parasite
clearance (76% reduction of parasitemia in 24 h) similar to that of
AQ (8% survival, 72% of parasite clearance in 24 h) (Fig. 1A and C).
The same trend was not observed with compound 31 (1.15 mg per
mouse), a milder NO-donor (Fig. 1B and D). Total parasite clearance
with both compounds and AQ was achieved after 72 h and three
doses of treatment in mice that survived and no recrudescence was
observed after a 5-day treatment regimen (Fig. 1C and D).
3. Conclusions
4. Experimental
We have developed a new series of NO-AQ derivatives which are
able to retain a high degree of activity in vitro against both CQ-S and
CQ-R strains of P. falciparum. At least in vitro, the contribution of NO
to the antiplasmodial properties of these products seems to be
secondary to that of the lead. All the compounds are able, in different
degree, to dilate pre-contracted rat aorta strips with a NO-depen-
dent mechanism, and consequently they could be capable to help
restoring the vascular homeostasis which is deeply compromised in
4.1. Instrumentation and chemicals
Melting points were determined with a capillary apparatus
Büchi B-540 or Büchi B-530 (uncorrected). Melting point with
decomposition were determined after introduction of the sample at
a temperature 10 ꢀC lower than the melting point. A heating rate of
2
ꢀC minꢁ1 was used. 1H and 13C-NMR spectra were obtained on
a Bruker Avance 300, at 300 and 75 MHz respectively or Bruker

M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
1761
Scheme 4. Preparation of derivatives 37, 40. Reagents and conditions: (a) 2-(ethylamino)ethanol, CH3CN, RT, 20 h; (b) 3-phenyl-4-benzenesulfonyl furoxan, 50% aq NaOH, dist. THF/
DMF, RT, N2 atmosphere (c) 39, Et3N, iPrOH/DMF, RT, 24 h; (d) 2-(ethylamino)ethanol, 50% aq NaOH, dist. THF/DMF, RT, 20 h; N2 atmosphere.
AC-200, at 200 and 50 MHz respectively;
d
in ppm rel. to SiMe4 as
4.2. Chemistry
the internal standard; coupling constants J in Hz. 13C-NMR spectra
were fully decoupled. The following abbreviations are used: s:
singlet, d: doublet, dd: doublet doublet, t: triplet, qt: quartet, m:
multiplet, br: broad, Fx: furoxan, Q: quinoline ring, AQ: amodia-
quine. Mass spectra were recorded on a Finnigan-Mat TSQ-700.
Flash chromatography (FC) was performed on BDH silica gel
4.2.1. 6-(ethylamino)hexan-1-ol (6)
6-chlorohexan-1-ol (5) (3.5 g; 26 mmol) was added to 70 ml of
EtNH2 70% solution in water. The solution was kept under stirring at
room temperature for 48 h. The solution was then evaporated
under reduced pressure and the residue was taken up with 1N HCl
and washed three times with CH2Cl2; the aqueous phase was
filtered and dried under reduced pressure. The residue was eluted
through an ion-exchange column over Amberlite IRA-400 resin to
afford 1.76 g of the title product as a colourless oil (52% free base).
(particle size 40e63 mm). When not otherwise specified, anhydrous
magnesium sulphate (MgSO4) was used as the drying agent of
organic phases. Analysis (C, H, N) of the target compounds was
performed by Service de Microanalyse, Université de Genève,
Genève (CH) and REDOX (Monza) and the results were within
ꢂ0.4% of the theoretical. 3-phenyl-4-benzenesulfonyl furoxan [20],
3 [21], 27 [15], 38 [22], A, B [23], were synthesised according to
reported methods.
1H-NMR (CD3OD):
CH2NHCH2); 1.68e1.41 (m, 6H, 3 CH2); 1.28 (t, J ¼ 7.2 Hz, 3H, CH3).
d, 3.58e3.53 (m, 2H, CH2O); 3.02e2.89 (m, 4H,
13C-NMR (CD3OD):
d, 61.3; .47.4; 42.8; 32.0; 26.5; 26.2; 25.2; 10.8.
MS CI (isobutane) (m/z): 146 [MHþ].
Table 1
4.2.2. N-Ethyl-6-(nitrooxy)hexan-1-ammonium nitrate (7)
Dissociation constants of the final compounds.
To 10 ml of fuming nitric acid at e 15 ꢀC, 0.55 g (3.8 mmol) of 6
were added dropwise. The mixture was allowed to warm at room
temperature and was kept under stirring for 24 h. The solution was
then evaporated under reduced pressure and the residue was
dropped in 100 ml of anhydrous Et2O at ꢁ15 ꢀC. After 2 h at 0 ꢀC the
solvent was decanted off and the semisolid residue was triturated
several times with fresh diethyl ether and was used without further
purification. The product was characterized as free base which was
obtained by extraction of a slurry of the semisolid with Na2CO3,
with EtOAc. The organic phase was then washed with brine, dried
over Na2SO4 and the solvent was evaporated under reduced pres-
sure. The residue was purified by flash chromatography on silica gel
a
a
a
Compound
pKa1
pKa2
pKa3
28
29
30
31
32
33
34
35
37
40
7.42
7.06
7.08
6.99
7.08
7.05
3.00
3.06
6.18
6.12
8.47
8.14
8.56
8.09
7.86
7.98
6.46
6.71
7.66
7.68
e
e
e
e
e
e
7.59
7.69
e
e
a
Determined by potentiometry; S.D. ꢃ 0.07.

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M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
Table 2
Antiplasmodial action and vasodilating ability of the synthesised compounds and reference derivatives.
Compound
D10 (CQ-S) IC50
(
m
M)a
W2 (CQ-R) IC50
(m
M)a
EC50
(m
M)b
EC50
(
m
M)b þ ODQ 1
mM
28
29
30
31
32
33
34
35
37
40
3
0.027 ꢂ 0.012
0.027 ꢂ 0.016
0.031 ꢂ 0.012
0.038 ꢂ 0.007
0.019 ꢂ 0.007
0.019 ꢂ 0.001
0.055 ꢂ 0.007
0.018 ꢂ 0.003
0.023 ꢂ 0.005
0.046 ꢂ 0.029
> 5
> 5
> 5
> 5
> 5
> 5
> 5
0.062 ꢂ 0.027
0.031 ꢂ 0.013
0.028 ꢂ 0.013
0.021 ꢂ 0.004
0.028 ꢂ 0.008
0.025 ꢂ 0.012
0.138 ꢂ 0.028
0.034 ꢂ 0.017
0.024 ꢂ 0.003
0.152 ꢂ 0.075
> 5
> 5
> 5
> 5
> 5
> 5
> 5
0.42 ꢂ 0.09
11 ꢂ 1
0.45 ꢂ 0.12
3.3 ꢂ 0.8
> 30c
> 30c
> 30c
> 30c
6.5 ꢂ 0.8
3.6 ꢂ 0.4
> 10c
1.0 ꢂ 0.1
e
0.45 ꢂ 0.05
0.22 ꢂ 0.04
0.10 ꢂ 0.04
0.10 ꢂ 0.02
0.12 ꢂ 0.02
0.079 ꢂ 0.012
1.2 ꢂ 0.2
0.017 ꢂ 0.003
e
e
e
e
e
e
e
7
e
12
13
14
15
26
e
e
e
e
e
A
B
> 5
> 5
> 5
14.3 ꢂ 3.8
> 10c
4.94 ꢂ 2.03
0.030 ꢂ 0.004
e
AQ
0.012 ꢂ 0.001
0.025 ꢂ 0.007
0.016 ꢂ 0.004
0.632 ꢂ 0.209
Inactive
e
Inactive
e
Chloroquine
e ¼ not tested.
a
The IC50 represents the
m
m
M equivalents of test compounds required to inhibit parasite growth by 50% (data are expressed as mean ꢂ SD).
M equivalents of test compounds required to relax the pre-contracted rat aorta strips by 50% (data are expressed as means ꢂ SE).
not able to reach 50% tissue relaxation at the maximum concentration tested.
b
c
The EC50 represents the
Fig. 1. Efficacy of amodiaquine (AQ), compound 40 and compound 31 in rescuing mice with late stage CM. A and B: survival curves of of PbA-infected mice with late stage CM
following treatment with: (A) AQ (solid circle) 1 mg per mouse (n ¼ 12) or compound 40 (open square, dashed line) at an equivalent molar dose (1.4 mg per mouse, n ¼ 12) and; (B)
AQ (solid circle) 1 mg per mouse (n ¼ 29) or compound 31 (open triangle, dashed line) also at an equivalent molar dose (1.15 mg per mouse, n ¼ 19). C and D: parasitemia curves
showing the profile of parasite clearance of the same treatment groups showed in A and B, respectively. Results in C and D are the mean ꢂ SEM.

M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
1763
eluting with DCM/MeOH 5% to afford 0.62 g of the desired product
as a colourless oil (65%). 1H-NMR (DMSO-d6):
4.2.5. 2-(5,6-dihydroxyhexyl)-1H-isoindole-1,3(2H)-dione (18)
d, 4.51 (t, J ¼ 6.1 Hz,
To a solution of 5 g of 17 (22 mmol) in 200 ml of acetone cooled
in an ice bath, a solution of KMnO4 in 200 ml of water was added
dropwise. After the addition was over the mixture was allowed to
raise to room temperature and kept under stirring for further 3 h;
the brownish solid was filtered off on a sintered-glass funnel and
the filtrate was concentrated under reduced pressure. The residue
was taken up with 100 ml of water and extracted with EtOAc
(6 ꢄ 50 ml). The organic extracts were washed with brine
(1 ꢄ 100 ml), dried and the solvent was evaporated under reduced
pressure. The crude product was purified by flash chromatography
on silica gel eluting with CH2Cl2/CH3OH 5% to afford 3.16 g (56%) of
the desired product as white solid. Spectral data were consistent
2H, CH2ONO2); .2.55e2.43 (m, 4H, CH2NCH2); 1.70e1.61 (m, 2H,
CH2); 1.44e1.31 (m, 6H, 3CH2); 0.99 (t, J ¼ 7.1 Hz, 3H, CH3). 13C-NMR
(DMSO-d6): d, 74.2; 49.2; 43.8; 29.3; 26.7; 26.4; 25.4; 15.1.
4.2.3. General procedure for the synthesis of derivatives 4, 12e15
The appropriate alcohol was added dropwise to 15 ml of fuming
nitric acid at e 15 ꢀC. The mixture was allowed to warm at room
temperature and was kept under stirring for 24 h. The solution was
then evaporated under reduced pressure and the residue was
dropped in 100 ml of anhydrous Et2O at ꢁ15 ꢀC. After 2 h at 0 ꢀC the
solvent was decanted off and the semisolid residue was triturated
several times with fresh diethyl ether to obtain an off-white solid
(60e93% yields).
with the reported ones [28]. 1H-NMR (CD3OD):
4Harom); 3.70e3.30 (m, 5H, CH2N, CHOH,CH2OH); 1.74e1.27 (m,
6H, CH2CH2CH2). 13C-NMR (CD3OD):
, 169.8; 135.3; 133.4; 124.0;
d, 7.85e7.76 (m,
d
4.2.3.1. N-Ethyl-3-(nitrooxy)propan-1-ammonium nitrate (4). 1H-
73.0; 67.3; 38.8; 33.9; 29.6; 24.0; 23.3þ.
NMR (DMSO-d6):
CH2ONO2); 2.99 (m, 2H, CH2CH2CH2); 1.17 (t, J ¼ 5.5 Hz, 3H, CH3).
13C-NMR (DMSO-d6):
, 70.8; 43.2; 42.2; 23.2; 11.0.
d
, 8.45 (s, 2H, exch. sign.); 4.60 (t, J ¼ 4.5 Hz, 2H,
MS (CI) (isobutane) (m/z): 264 [MH ]. M.p. (iPr2O/iPrOH): 65 ꢀC.
d
4.2.6. 2-[4-(2,2-dimethyl-1,3-dioxolan-4-yl)butyl]-1H-isoindole-
1,3(2H)-dione (19)
4.2.3.2. 4-[(nitrooxy)methyl]piperidinium nitrate (12). 1H-NMR
(DMSO-d6):
, 8.68 (br s, 1H, NHþ); 8.34 (br s, 1H, NHþ); 4.45 (d,
To a solution of 18 (3.16 g; 12 mmol) in 200 ml of acetone,
pyridinium p-toluenesulfonate (0.3 g; 1 mmol) was added. The
mixture was stirred at room temperature for 18 h, then evaporated
under reduced pressure and purified by flash chromatography on
silica gel to afford 3.03 g (80%) of title product as a white solid.
Spectral data were consistent with the reported ones [28]. 1H-NMR
d
J ¼ 6.4 Hz, 2H, CH2ONO2); 3.34e3.30 (m, 2H, CH2-piperidine);
2.98e2.86 (m, 2H, CH2-piperidine); 2.10e2.04 (m, 1H, CH-piperi-
dine); 1.87e1.82 (m, 2H, CH2-piperidine); 1.51e1.38 (m, 2H, CH2-
piperidine). 13C-NMR (DMSO-d6):
d, 76.6; 42.8; 31.4; 25.1. Spectral
data were consistent with the reported ones [22].
(CDCl3): d, 7.86e7.80 (m, 2H arom); 7.74e7.68 (m, 2H arom);
4.11e4.00 (m, 2H, 2CHO); 3.70 (t, J ¼ 7.2 Hz, 2H, CH2N); 3.54e3.44
4.2.3.3. 4-[2-(nitrooxy)ethyl]piperidinium nitrate (13). 1H-NMR
(m,1H, CHO); 1.77e1.46 (m, 6H, 3CH2); 1.40 (s, 3H, CH3); 1.34 (s, 3H,
(CD3OD):
d
, 4.45 (t, J ¼ 6.3 Hz, 2H, CH2ONO2); 3.30e3.25 (m, 2H,
CH3). 13C-NMR (CDCl3):
d, 168.4; 165.7; 133.9; 132.1; 123.2; 108.72;
CH2-piperidine) 2.91e2.83 (m, 2H, CH2-piperidine); 1.88e1.83
(m, 2H, CH2-piperidine); 1.64e1.58 (m, 3H, CH2, CH); 1.37e1.30
75.8; 71.9; 69.4; 37.8; 33.1; 28.6; 26.9; 25.7; 23.1. MS CI (isobutane)
(m/z): 304 [MHþ]. M.p. (n-hexane): 121 ꢀC.
(m, 2H, CH2-piperidine). 13C-NMR (CD3OD):
d, 71.5; 55.2; 53.3;
44.5; 33.0; 31.3; 29.0. Spectral data were consistent with the
reported ones [24].
4.2.7. 4-(2,2-Dimethyl-1,3-dioxolan-4-yl)butan-1-amine (20)
To a stirred solution of 19 (3 g; 10 mmol) in 100 ml of distilled
THF, hydrazine monohydrate (3.9 ml; 8 eq.) was added and the
mixture was stirred under reflux for 24 h. After cooling the mixture
was filtered on a sintered-glass funnel and the filtrate was evapo-
rated under reduced pressure. The residue was purified by flash
chromatography on silica gel eluting with DCM/MeOH 5%, DCM/
MeOH 10% NH3 soln 1% affording the desired product as a colourless
oil (0.9 g 52%). Spectral data were consistent with the reported ones
4.2.3.4. 1-[2-(nitrooxy)ethyl]piperazin-4-ium nitrate (14). 1H-NMR
(DMSO-d6):
d
, 9.09 (br s, 2H, 2NHþ); 4.87 (t, J ¼ 4.6 Hz, 2H,
CH2ONO2); 3.62 (m, 2H, CH2-lateral chain); 3.41e3.32 (m, 8H,
4CH2-piperazine). 13C-NMR (DMSO-d6):
d, 67.3; 52.6; 48.6; 40.4.
Spectral data were consistent with the reported ones [25].
4.2.3.5. 1-[3-(nitrooxy)propyl]piperazin-4-ium nitrate (15). 1H-NMR
[29].1H-NMR (CDCl3):
CH); 2,7 (t, 2H, J ¼ 6.9 Hz, CH2N); 1.95 (s, 3H, CH2, CH); 1.40 (s, 3H,
d, 4.13e4.01 (m, 2H, CH2O); 3.54e3.47 (m,1H,
(DMSO-d6):
d
, 9.10 (br s, 2H, 2NHþ); 4.61 (t, J ¼ 6.0 Hz, 2H,
CH2ONO2); 4.0e3.28 (m, 10H, 5 CH2); 2.14e2.05 (m, 2H, CCH2C-
CH3); 1.35 (s, 3H, CH3). 13C-NMR (CDCl3):
d, 108.6; 77.2; 75.9; 69.4;
lateral chain). 13C-NMR (DMSO-d6):
d, 70.8; 52.7; 48.5; 40.6; 21.4.
41.8; 33.3; 26.9; 25.7; 23.2. MS CI (isobutane) (m/z): 174 [MHþ].
Spectral data were consistent with the reported ones [25].
4.2.8. N-[4-(2,2-dimethyl-1,3-dioxolan-4-yl)butyl]-2-
4.2.4. 2-Hex-5-enyl-1H-isoindole-1,3(2H)-dione (17)
nitrobenzenesulfonamide (21)
5-hexen-1-ol 16 (10 g, 10 mmol) was converted to the corre-
sponding methanesulfonate according to a reported procedure
[26]. The crude product was then suspended in CH3CN with
potassium phtalimide (1.5 eq) and a catalytic amount of potassium
iodide. The mixture was refluxed for 72 h, then cooled and filtered
on a sintered-glass funnel. The filtrate was purified by flash chro-
matography eluting with PE/EtOAc 10% to afford the desired
product as a white solid (20 g, 86%). Spectral data were consistent
To a solution of 0.9 g (5 mmol) of 20 and Et3N (1.1 eq) in 20 ml of
DCM cooled in an ice bath, 2-nitrobenzensulfonilchloride (1 eq)
was added portionwise. The mixture was stirred at RT for 30 min,
taken up with DCM and washed with 20 ml of 1N HCl; the aqueous
phase was extracted with DCM (2 ꢄ 10 ml) and the organic phase
was then washed with water and brine, dried over Na2SO4, filtered
and evaporated under reduced pressure. The residue was purified
by flash chromatography on silica gel, eluting with PE/EtOAc 30% to
afford the desire product as a colourless oil (1.55 g, 83%). 1H-NMR
with the reported ones [27]. 1H-NMR (CDCl3):
d, 7.86e7.80 (m, 2H
arom); 7.64e7.68 (m, 2H arom); 5.85e5.71 (m, 1H, CH]);
5.04e4.92 (m, 2H, CH2]); 3.69 (t, 2H, J ¼ 7.2 Hz, CH2N); 2.13e2.05
(m, 2H, CH2CH); 1.72e1.67 (m, 2H, CH2); 1.50e1.40 (m, 2H, CH2).
(CDCl3): d, 8.13e8.10 (m, 1H arom); 7.86e7.77 (m, 3H arom);
5.76e5.73 (m, 1H, CH); 4.14e3.98 (m, 2H, CH2O); 3.38e3.44 (m, 1H,
NH); 3.13 (qt, 2H, J ¼ 6.3 Hz, CH2N); 1.60e1.36 (m, 6H, CH2CH2CH2);
13C-NMR (CDCl3):
d
, 168.4; 138.2; 133.8; 132.1; 123.1; 114.9; 37.8;
1.32 (s, 3H, CH3); 1.26 (s, 3H, CH3). 13C-NMR (CDCl3):
d, 148.2; 135.6;
33.2; 28.0; 26.1; 21.0. MS CI (isobutane) (m/z): 230 [MHþ]. M.p. (n-
134.2; 133.6; 133.2; 131.1; 125.5; 108.8; 76.0; 69.5; 43.9; 33.2; 29.8;
hexane): 52.5 ꢀC.
27.2; 26.0; 25.5; 23.0; 14.5. MS CI (isobutane) (m/z): 359 [MHþ].

1764
M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
4.2.9. N-[4-(2,2-dimethyl-1,3-dioxolan-4-yl)butyl]-N-ethyl-2-
nitrobenzenesulfonamide (22)
4.2.13. General procedure for the synthesis of the derivatives 28e35
The appropriate nitric ester and 27 (1 eq.) were suspended in
a mixture of CH3CN/DMF 1/1. To the suspension, cooled at 0 ꢀC, 3.5
eq. of Et3N were added dropwise. The mixture was kept under
stirring at room temperature for 6 h. After that period the solvent
was distilled under reduced pressure and the residue was taken up
with water and extracted with EtOAc. The organic phase was
washed with brine, dried over Na2SO4, filtered and evaporated
under reduced pressure. The residue was purified by flash chro-
matography on silica gel eluting with mixture of DCM/MeOH to
afford the desired products as yellow solids in 35e56% yields.
To a stirred solution of 21 (1.55 g; 4.3 mmol) in 50 ml of DMF,
K2CO3 (10 eq) and EtI (1.5 eq) were added. The solution was stirred
at 60 ꢀC for 10 h, taken up with 100 ml of Et2O and washed with
water (3 ꢄ 50 ml) and brine; the organic phase was dried over
Na2SO4, filtered and dried under reduced pressure. The residue was
purified by flash chromatography on silica gel eluting with PE/
EtOAc 30%, to afford the desired product as a pale yellow oil (1.63 g
98%). 1H-NMR (CDCl3):
d, 8.02e7.99 (m, 1H arom); 7.70e7.67 (m, 2H
arom); 7.65e7.60 (m, 1H, arom); 4.07e3.99 (m, 2H, CH2O);
3.50e3.46 (m, 1H, CH); 3.40e3.28 (m, 4H, NCH2CH3, NCH2);
1.66e1.29 (m, 12H, 3 CH2, CH3CCH3); 1.12 (t, J ¼ 7.2 Hz, 3H, CH3CH2).
4.2.13.1. 7-Chloro-4-{[4-hydroxy-3-({[3-(nitrooxy)propyl]ammonio}
methyl)phenyl]amino}quinolinium dihydrchloride (28). The free
base was taken up with MeOH and HCl saturated Et2O was added at
0 ꢀC; the solution was kept under stirring at room temperature for
2 h, then the solvent was evaporated under reduced pressure and
the solid was freeze dried to give the product as the hydrochloride.
13C-NMR (CDCl3):
d, 133.8, 133.4, 130.6127.0, 75.8, 69.3, 46.7, 42.0,
36.5, 33.0, 31.4, 28.3, 26.9, 25.7, 22.8, 13.7. MS CI (isobutane) (m/z):
387 [MHþ].
4.2.10. N-[4-(2,2-dimethyl-1,3-dioxolan-4-yl)butyl]-N-ethylamine
(23)
1H-NMR (DMSO-d6):
d, 11.20 (br s, 1H, exch. signal); 10.79 (br s, 1H,
A solution of 1.08 ml (2.5 eq) of thiophenol in 15 ml of CH3CN kept
under N2 was cooled in an ice bath. To this solution 0.60 g of KOH (2.5
eq) dissolved in 2 ml of water were added dropwise. After 10 min
1.63 g of 22 (4.0 mmol) dissolved in 10 ml of CH3CN were added
dropwise over a period of 20 min. The mixture was stirred at 50 ꢀC
for 4 h, then it was filtered and the filtrate was evaporated under
reduced pressure. The residue was purified by flash chromatography
on silica gel, eluting with DCM, DCM/MeOH5%, DCM/MeOH10% NH3
1% to afford the title product as a colourless oil (0.65 g 77%). 1H-NMR
exch. signal); 9.50 (br s, 2H, exch. signal) 8.95 (d, J ¼ 9.0 Hz, 1H
arom.); 8.46 (d, J ¼ 7.0 Hz, 1H arom.); 8.19 (d, J ¼ 1.9 Hz, 1H arom.);
7.82 (dd, J1 ¼ 9.0 Hz, J2 ¼ 1.9 Hz, 1H arom.); 7.60 (d, J ¼ 2.3 Hz, 1H
arom.); 7.34 (d, J1 ¼ 8.6 Hz, J2 ¼ 2.3 Hz, 1H arom.); 7.19 (d, J ¼ 8.6 Hz,
1H arom.); 6.86 (dd, J ¼ 7.0 Hz, 1H arom.); 4.66 (t, J ¼ 6.0 Hz, 2H,
CH2ONO2); 4.13 (s, 2H, CH2Ph); 3.06 (m, 2H, CH2N); 2.17 (m, 2H,
CCH2C). 13C-NMR (DMSO-d6):
d, 155.4; 155.3; 154.9; 142.9; 138.8;
138.2; 128.7; 127.8; 127.2; 125.9; 119.2; 119.0; 116.4; 115.5; 100.2;
70.7; 44.6; 43.2; 22.8. M. p.: 177e179 ꢀC (dec.). Anal. Calc. for:
C19H19ClN4O4∙2HCl∙1.7H2O C% 45.07, H% 4.86, N% 11.06; Found
45.16, H% 4.49, N% 10.69.
(CDCl3):
d
, 4.10e4.01 (m, 2H, CH2O); 3.50 (t, 1H, J ¼ 7.09 Hz, CH);
2.70e2.60 (m, 4H, CH2NCH2); 1.66e1.43 (m, 6H, CH2CH2CH2); 1.41
(s, 3H, CH3);1.37(s, 3H, CH3); 1.12(t, 3H, J ¼ 7.2Hz, CH3CH2).13C-NMR
(CDCl3):
d, 108.7; 69.5; 49.5; 44.1; 33.5; 29.9; 27.0; 25.7; 23.6; 15.1.
4.2.13.2. 7-Chloro-4-{[3-({ethyl[3-(nitrooxy)propyl]ammonio}methyl)-
4-hydroxyphenyl]amino}quinolinium dihydrochloride (29). The free
base was taken up with MeOH and HCl saturated Et2O was added
at 0 ꢀC; the solution was kept under stirring at room temperature
for 2 h, then the solvent was evaporated under reduced pressure
and the solid was freeze dried to give the product as the hydro-
MS CI (isobutane) (m/z): 202 [MHþ].
4.2.11. 6-(ethylamino)hexane-1,2-diol (25)
Of compound 23 (0.64 g; 3.2 mmol) was dissolved in 20 ml of 80%
TFA in water. The mixture was refluxed for 24 h, then it was evapo-
rated under reduced pressure. The residuewas taken up with 20 ml of
water and activated carbon was added; the suspension was heated to
boiling andfilteredthroughabed ofcelite. The filtratewasevaporated
under reduced pressure and the residue was eluted through an ion-
exchange column over Amberlite IRA-400 resin to afford 0.42 g of the
chloride. 1H-NMR (free base) (CDCl3):
d
, 8.42 (d, J ¼ 5.6 Hz, 1H
arom.); 7.97e7.93 (m, 2H arom.); 7.41 (dd, J1 ¼ 9.0 Hz, J2 ¼ 2.5 Hz,
1H arom.); 7.14 (dd, J1 ¼ 8.2 Hz, J2 ¼ 2.5 Hz, 1H arom.); 6.96
(d, J ¼ 2.5 Hz, 1H arom.); 6.87 (d, J ¼ 8.2 Hz, 1H arom.); 6.63 (d,
J ¼ 5.6 Hz, 1H arom.); 4.49 (t, J ¼ 6.2 Hz, 2H, CH2ONO2); 3.77 (s, 2H,
CH2Ph); 2.67 (m, 4H, CH2NCH2); 1.99 (m, 2H, CCH2C); 1.13 (t,
title product as a colourless oil (82% free base). 1H-NMR (CD3OD):
d,
3.61e3.59 (m, 1H, CHOH); 3.47e3.45 (m, 2H, CH2OH); 3.10e2.97 (m,
4H, CH2NHCH2); 1.73e1.40 (m, 6H, CH2CH2CH2); 1.31 (t, 3H, J ¼ 7.4 Hz,
J ¼ 7.2 Hz, 3H, CH3). 13C-NMR (free base) (CDCl3):
d, 156.1; 150.7;
149.7; 148.3; 135.5; 129.9; 127.7; 125.8; 125.7; 125.2; 125.1; 117.1;
101.1; 70.8; 57.2; 49.1; 47.1; 24.0; 10.9. MS CI (free base) (isobu-
tane) (m/z): 431/433 [MHþ]. M. p.: 182e183 ꢀC (dec.). Anal. Calc.
for: C21H23ClN4O4∙2HCl∙1.5H2O C% 47.51, H% 5.31, N% 10.55; Found
47.62, H% 5.02, N% 10.27.
CH3).13C-NMR (CD3OD):
d, 72.8;67.2; 48.4;44.0;33.7;27.3;23.6;11.6.
MS CI (isobutane) (m/z): 162 [MHþ].
4.2.12. N-Ethyl-5,6-bis(nitrooxy)hexan-1-ammonium nitrate (26)
To 20 ml of fuming nitric acid cooled at ꢁ15 ꢀC, 25 (1.5 g;
9.3 mmol) was added dropwise over a period of 10 min The mixture
was then allowed to warm to room temperature and was kept
under stirring for 40 h. After that period the solvent was distilled
under reduced pressure and the residue was dropped and tritu-
rated in 150 ml of dry diethyl ether at ꢁ15 ꢀC. The white solid thus
obtained was collected through filtration and washed thoroughly
4.2.13.3. 7-Chloro-4-{[3-({ethyl[6-(nitrooxy)hexyl]ammonio}methyl)-
4-hydroxyphenyl]amino}quinolinium dihydrochloride (30). The free
base was taken up with MeOH and HCl saturated Et2O was added at
0
ꢀC; the solution was kept under stirring at room temperature for
2 h, then the solvent was evaporated under reduced pressure and
the solid was freeze dried to give the product as the hydrochloride.
with dry diethyl ether to afford 1.97 g of the title compounds (67%
1H-NMR (CD3OD):
d
, 8.65 (d, J ¼ 9.1 Hz,1H arom.); 8.39 (d, J ¼ 7.1 Hz,
as nitric acid salt). 1H-NMR (DMSO-d6):
d, 8.21 (br s, 2H NH2
)
1H arom.); 7.76 (dd, J1 ¼ 9.1 Hz, J2 ¼ 1.9 Hz, 1H arom.); 7.61 (d,
J ¼ 2.5 Hz, 1H arom.); 7.45 (dd, J1 ¼ 8.7, Hz, J2 ¼ 2.5 Hz, 1H arom.);
7.15 (d, J ¼ 8.7 Hz, 1H arom.); 6.86 (d, J ¼ 7.1 Hz, 1H arom.); 4.49
(t, J ¼ 6.5 Hz, 2H, CH2ONO2); 4.23 (s, 2H, CH2Ph); 3.30e3.20 (m, 4H,
CH2NCH2); 1.83 (m, 2H, CH2); 1.74 (m, 2H, CH2) 1.42 (m, 7H, 2CH2,
þ
5.46e5.39 (m,1H, CHONO2); 4.97e4.92 (dd, J1 ¼ 2.4 Hz, J2 ¼ 12.9 Hz,
1H, CH’H00ONO2); 4.74e4.68 (dd, J1 ¼ 6.2 Hz, J2 ¼ 12.8 Hz, 1H,
CH’H’’ONO2); 2.99e2.85 (m, 4H, CH2NH2þCH2) 1.78e1.70 (m, 2H,
CH2); 1.63e1.37 (m, 6H, 2CH2) 1.16 (t, 3H, J ¼ 7.2 Hz, CH3). 13C-NMR
(DMSO-d6):
d
, 80.0; 71.8; 45.8; 41.8; 27.6; 25.1; 21.3; 10.9. M. p.:
CH3). 13C-NMR (CD3OD):
d, 157.8; 157.5; 144.2; 141.4; 140.5; 131.4;
64e65 ꢀC. Anal. Calc. for: C8H17N3O6∙HNO3∙0.5H2O C% 29.72, H%
130.5; 129.9; 129.1; 126.5; 120.4; 119.3; 118.0; 117.2; 101.7; 74.5;
5.92, N% 17.33; Found 29.61, H% 5.65, N% 17.53.
53.7; 52.6; 49.8; 27.6; 27.3; 26.3; 24.6; 9.2. M. p.: 191e193 ꢀC (dec.).

M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
1765
Anal. Calc. for: C24H29ClN4O4∙2HCl∙H2O C% 51.31, H% 5.77, N% 9.90;
Found 51.12, H% 5.90, N% 9.94.
J1 ¼ 6.9 Hz, J2 ¼ 2.1 Hz, 1H arom.); 7.15e6.90 (m, 2H arom.); 6.84
(d, J ¼ 8.5, Hz, 1H arom.); 6.62 (d, J ¼ 5.6 Hz, 1H arom.); 4.63 (t,
J ¼ 6.5 Hz, 2H, CH2ONO2); 3.74 (s, 2H, CH2Ph); 2.76 (t, J ¼ 5.4 Hz,
2H, CH2CH2ONO2) 2.57 (br s 8H, 4CH2-piperazine). 13C-NMR (free
4.2.13.4. 6-[{5-[(7-chloroquinolin-4-yl)amino]-2-hydroxybenzyl}(ethyl)
amino]hexan-1,2-diyl dinitrate (31). 1H-NMR (CD3OD):
d, 8.32e8.24 (m,
base) (CD3OD): d, 156.7; 152.6; 152.2; 149.9; 136.7; 132.1; 127.6;
2H arom.); 7.85 (s,1H arom.); 7.49e7.46 (m,1H arom.); 7.16 (d, J¼ 4.5 Hz,
1H arom); 7.08 (s, 1H arom.); 6.88e6.84 (m, 1H arom.); 6.66e6.64 (m,
1H arom.); 5.43e5.39 (m, 1H, CHONO2); 4.96e4.88 (m, 1H, CH2ONO2);
4.63e4.56 (m, 1H, CH2ONO2); 3.84 (s, 2H, PhCH2N); 2.73e2.61 (m, 4H,
CH2NCH2); 1.82e1.49 (m, 6H, CH2CH2CH2); 1.19e1.15 (m, 3H, CH3). 13C-
127.5; 126.9; 126.5; 124.6; 124.1; 119.0; 117.6; 101.8; 71.4; 60.9;
59.8; 55.9; 54.2; 53.8; 53.3. MS CI (free base) (isobutane) (m/z):
458 [MHþ]. M. p.: 153e154 ꢀC (dec.). Anal. Calc. for:
C22H24ClN5O4∙3HCl∙2.5H2O C% 43.15, H% 5.27 N% 11.44; Found
42.87, H% 4.93, N% 11.20.
NMR (DMSO-d6): d, 154.8; 151.8; 149.44; 149.38; 133.7; 130.4; 127.5;
125.4; 124.6; 124.5; 124.2; 124.0; 117.6; 116.0; 100.4; 80.2; 71.8; 55.0;
51.9; 46.4; 28.0; 25.5; 22.1; 10.8. M. p.: 120e122 ꢀC (dec.). Anal. Calc. for
C24H28ClN5O7 C% 53.09, H% 5.38, N% 12.90; Found C% 52.79, H% 5.12,
N% 12.61.
4.2.13.8. 7-Chloro-4-{[4-hydroxy-3-({4-[3-(nitrooxy)propyl]piperazine
diium-1-yl}methyl)phenyl]amino}quinolinium
trihydrochloride
(35). The free base was taken up with MeOH and HCl saturated
Et2O was added at 0 ꢀC; the solution was kept under stirring at
room temperature for 2 h, then the solvent was evaporated under
reduced pressure and the solid was freeze dried to give the product
4.2.13.5. 7-Chloro-4-{[4-hydroxy-3-({4-[(nitrooxy)methyl]piperi-
dinium-1-yl}methyl)phenyl]amino}quinolinium
dihydrochloride
as the hydrochloride. 1H-NMR (free base) (DMSO-d6):
d, 8.91 (br s,
(32). The free base was taken up with MeOH and HCl saturated
Et2O was added at 0 ꢀC; the solution was kept under stirring at
room temperature for 2 h, then the solvent was evaporated under
reduced pressure and the solid was freeze dried to give the product
exch. sign. 1H) 8.43e8.36 (m, 2H arom.); 7.85 (s, 1H arom.);
7.54e7.51 (m, 1H arom.); 7.11e7.09 (m, 2H arom.); 6.84 (d, J ¼ 8.9,
Hz, 1H arom.); 6.60e6.59 (m, 1H arom.); 4.55 (t, J ¼ 6.2 Hz, 2H,
CH2ONO2); 3.64 (s, 2H, CH2Ph); 2.51e2.38 (m, 10H, 5CH2) 1.85e1.81
as the hydrochloride. 1H-NMR (free base) (DMSO-d6):
d
, 8.90 (s, 1H
(m 2H, CH2). 13C-NMR (free base) (DMSO-d6):
d, 155.7; 154.8; 153.3;
arom.); 8.43e8.36 (m, 2H arom.); 7.85 (d, J ¼ 1.8 Hz,1H arom.); 7.52
(dd, J1 ¼ 7.2, Hz, J2 ¼ 1.8 Hz, 1H arom.); 7.10e7.08 (m, 2H arom.);
6.82 (d, J ¼ 9.2 Hz, 1H arom.); 6.57 (d, J ¼ 5.4 Hz, 1H arom.); 4.42 (d,
J ¼ 6.3 Hz, 2H, CH2ONO2); 3.64 (s, 2H, CH2Ph); 2.91e2.51 (m, 2H,
CH2-piperidine); 2.11e2.04 (m, 2H, CH2-piperidine); 1.79e1.70 (m,
3H, CH-piperidine, CH2-piperidine) 1.32e1.28 (m, 2H, CH2-piper-
150.9; 135.4; 132.0; 129.0; 127.9; 127.3; 126.1; 125.8; 124.9; 119.2;
117.5; 102.0; 73.9; 59.8; 56.8; 55.2; 53.8; 48.3; 25.1; 14.9. M. p.:
167e168 ꢀC (dec.). Anal. Calc. for: C22H24ClN5O4∙3HCl∙2.5H2O C%
44.10, H% 5.47 N% 11.18; Found 43.81, H% 4.99, N% 10.88.
4.2.14. 4-[(7-chloroquinolin-4-yl)amino]-2-{[ethyl-(2-
hydroxyethyl)amino]methyl}phenol dihydrochloride (36)
idine). 13C-NMR (free base) (DMSO-d6):
d
, 154.0; 151.8; 149.4;
149.0; 133.7; 131.2; 129.4; 127.8; 127.5; 124.7; 124.1; 117.7; 116.8;
116.4; 100.8; 77.2; 65.3; 59.3; 54.0; 34.7; 28.2. M. p.: 170e172 ꢀC
(dec.). Anal. Calc. for: C22H25ClN4O4∙2HCl∙2H2O C% 47.88, H%
5.30 N% 10.15; Found 47.60, H% 4.87, N% 10.07.
To a stirred solution of 2-(ethylamino)ethanol (0.82 mL;
8.43 mmol) in CH3CN (20 mL), kept under nitrogen, 27 (1 g;
2.81 mmol) was added. The yellow suspension was stirred for 18 h.
The solvent was evaporated and the yellow residue was treated with
water (15 mL) and stirred for 15 min. The solid was collected on
a buchner funnel and washed with water (50 mL). After drying over
P2O5 the product was purified by flash chromatography eluting with
CH2Cl2/MeOH20%togive 36(0.72g;69%)asyellowsolid. Theproduct
was converted into the corresponding hydrochloride by treatment
with HCl saturated MeOH and recrystallised from dry MeOH/Et2O to
obtain an analytical sample. Mp: 228e229.5 ꢀC (dec.) (230 ꢀC dec.
4.2.13.6. 7-Chloro-4-{[4-hydroxy-3-({4-[2-(nitrooxy)ethyl]piperi-
dinium-1-yl}methyl)phenyl]amino}quinolinium
dihydrochloride
(33). The free base was taken up with MeOH and HCl saturated
Et2O was added at 0 ꢀC; the solution was kept under stirring at
room temperature for 2 h, then the solvent was evaporated under
reduced pressure and the solid was freeze dried to give the product
as the hydrochloride. 1H-NMR (free base) (CD3OD þ CDCl3):
d
, 8.28
[30]).1H-NMR (DMSO-d6þD2O):
, 8.33 (d,1H, J ¼ 9.1 Hz, AQ-H5);8.21
d
(d, J ¼ 5.6 Hz, 1H arom.); 8.21 (d, J ¼ 9.0 Hz, 1H arom.); 7.83 (d,
J ¼ 2.0 Hz, 1H arom.); 7.44 (dd, J1 ¼ 6.9, Hz, J2 ¼ 2.1 Hz, 1H arom.);
7.13 (dd, J1 ¼ 6.0, Hz, J2 ¼ 2.5 Hz, 1H arom.); 7.02 (d, J ¼ 2.4 Hz, 1H
arom.); 6.84 (d, J ¼ 8.5 Hz, 1H arom.); 6.62 (d, J ¼ 5.6 Hz, 1H arom.)
4.54 (t, J ¼ 6.5 Hz, 2H, CH2ONO2); 3.73 (s, 2H, CH2Ph); 3.07e3.03 (m,
2H, CH2-piperidine) 2.21e2.14 (m, 2H, CH2-piperidine); 1.85e1.81
(m, 2H, CH2-piperidine); 1.74e1.68 (m, 2H, CH2-lateral chain);
1.63e1.55 (m, 1H, CH-piperidine); 1.46e1.33 (m, 2H, CH2-piperi-
(d, 1H, J ¼ 7.1 Hz, AQ-H2); 7.82 (d, 1H, J ¼ 2 Hz, AQ-H08); 7.65 (dd, 1H,
J ¼ 2, 9.1 Hz, AQ-H6); 7.38 (d, 1H, J ¼ 2.5 Hz, AQ-H3 )0; 7.34 (dd, 1H,
0
J ¼ 2.5, 8.6 Hz AQ-H5 ); 7.06 (d, 1H, J ¼ 8.6 Hz, AQ-H6 ); 6.72 (d, 1H,
J ¼ 7.1 Hz, AQ-H3); 4.32 (s, 2H, CH2Ph); 3.79 (t, 2H, J ¼ 5.1 Hz, CH2O);
3.2 (m, 4H, 2(CH2N)); 1.26 (t, 3H, J ¼ 7.2 Hz, CH3). 13C-NMR (DMSO-
d6þD2O):
d,154.0 (two overlapping peaks); 141.2; 138.1; 136.9; 128.1;
127.5; 126.9; 126.5; 123.3; 117.6; 116.2; 115.5; 113.9; 98.7; 53.6; 52.3;
50.6; 47.0; 6.9. MS (free base) (EI) m/z : 371 (52), 340 (35), 283 (100).
Anal. Calc. For C20H22ClN3O2 $ 2 HCl $ 0.1H2O C% 53.78, H% 5.46, N%
9.40; found C% 53.58, H% 5.39, N% 9.33.
dine). 13C-NMR (free base) (CD3OD þ CDCl3):
d, 157.2; 156.3; 143.1;
141.1; 140.8; 139.7; 131.0; 129.3; 128.8; 125.8; 119.8; 117.5; 117.4;
116.5; 101.2; 71.1; 57.9; 55.3; 53.0; 49.7þ; 32.9; 31.2; 29.5. MS CI (free
base) (isobutane) (m/z): 457/459 [MH ]. M. p.: 163e164 ꢀC (dec.).
Anal. Calc. for: C23H25ClN4O4∙2HCl C% 52.14, H% 5.14 N% 10.57;
Found 51.79, H% 5.34, N% 10.17.
4.2.15. 4-[(7-chloroquinolin-4-yl)amino]-2-[({ethyl-[2-(3-
phenylfuroxan-4-yl)oxy]ethyl}amino)methyl]phenol
dihydrochloride (37)
To a suspension of 36 (1.3 g; 2.92 mmol) in distilled THF/DMF
14/1 (27.5 mL), stirred under nitrogen at RT, 50% NaOH aqueous
solution (14.6 mmol) was added dropwise followed by a solution of
3-phenyl-4-benzenesulfonyl furoxan (1.24 g; 4.1 mmol) in distilled
THF. The reaction mixture was stirred for 5 h, then the mixture was
concentrated under reduced pressure to leave a brown oil. This
residue was taken up with water (20 mL) and extracted with EtOAc
(4 ꢄ 30 mL). The organic phase was washed with brine (40 mL),
dried and evaporated under reduced pressure to afford the crude
4.2.13.7. 7-Chloro-4-{[4-hydroxy-3-({4-[2-(nitrooxy)ethyl]piperazine
diium-1-yl}methyl)phenyl]amino}quinolinium
trihydrochloride
(34). The free base was taken up with MeOH and HCl saturated
Et2O was added at 0 ꢀC; the solution was kept under stirring at
room temperature for 2 h, then the solvent was evaporated under
reduced pressure and the solid was freeze dried to give the
product as the hydrochloride. 1H-NMR (free base) (CD3OD):
d,
8.30e8.23 (m, 2H arom.); 7.82 (d, J ¼ 2.1 Hz, 1H arom.); 7.50 (dd,

1766
M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
product as brown solid. The solid was purified by flash chroma-
tography eluting with CH2Cl2/MeOH 3%. The fractions containing
the desired product were collected in tubes filled with 0.2 mL of
HCl saturated MeOH to readily convert the product into the cor-
responding dihydrochloride. After evaporation under reduced
pressure 37 was obtained in 50% yield as bright-yellow solid. The
product was recrystallised from MeOH/Et2O. Mp: 229e231 ꢀC
(CD3OD þ D2O): , 8.54 (d, 1H, J ¼ 9.1 Hz, AQ-H5); 8.3 (d, 1H,
d
J ¼ 7.1 Hz, AQ-H2); 7.98 (d, 2H, J ¼ 7.4 Hz, FxPh-Ho); 7.94 (d, 1H,
J ¼ 2 Hz, AQ-H8); 7.81e7.74 (m, 2H, AQ-H6, FxPh-Hp); 7.69e7.62
0
0
(m, 3H, AQ-H3 , F0xPh-Hm); 7.48e7.45 (m, 1H, AQ-H5 ); 7.17 (d, 1H,
J ¼ 8.7 Hz, AQ-H6 ); 6.83 (d, 1H, J ¼ 7.1 Hz, AQ-H3); 4.95 (br m, 2H,
CH2O); 4.64 (s, 2H, PhCH2N); 3.83 (br m, 2H, OCH2CH2N); 3.49 (q,
2H, J ¼ 7.2 Hz, CH3CH2N); 1.38 (t, 3H, J ¼ 7.2 Hz CH3). 13C-NMR
(dec.) 1H-NMR (DMSO-d6):
d
, 11.24 (s, 1H, exch. signal); 11.15 (br s,
(CD3OD þ D2O):
d, 159.6; 157.7; 157.6; 143.9; 141.4; 140.3; 138.2;
1H, exch. signal); 10.98 (br s, 1H, exch. signal); 8.94 (d, 1H,
J ¼ 8.8 Hz, AQ-H5); 8.4 (d, 1H, J ¼ 6.5 Hz, AQ-H2); 8.18 (s, 1H,
AQ-H8); 8.02 (d, 2H, J ¼ 6.8 Hz, FxPh-Ho); 7.81 (d, 1H, J ¼ 8.8 Hz,
137.3; 131.3; 131.0; 130.6; 129.9; 129.6; 129.2; 126.2; 120.4; 119.2;
118.2; 117.0; 111.9; 101.6; 67.1; 54.1; 52.0; 51.0; 9.6. Anal. Calc. For
C28H26ClN5O6S $ 2 HCl C% 50.27, H% 4.22, N% 10.47; found C%
50.29, H% 4.23, N% 10.27.
0
AQ-H6); 7.71 (s, 1H, AQ-H3 ); 7.52 (m, 3H, FxPh-Hm; FxPh0-Hp ); 7.38
0
(d, 1H, J ¼ 8.4 Hz, AQ-H5 ); 7.22 (d, 1H, J ¼ 8.4 Hz, AQ-H6 ); 6.89 (d,
1H, J ¼ 6.5 Hz, AQ-H3); 4.97 (br m, 2H, CH2O); 4.26 (s, 2H, PhCH2N);
4.3. Dissociation constants determination
3.74 (br m, 2H, CH2N); 3.29 (br m, 2H, CH3CH2N ); 1.36 (br m, 3H,
CH3). 13C-NMR (DMSO-d6):
d
, 161.6; 155.9; 154.6; 142.8; 138.7;
The ionisation constants of compounds were determined by
potentiometric titration with the GLpKa apparatus (Sirius Analytical
Instruments Ltd, Forest Row, East Sussex, UK). Apparent ionisation
constants (psKa) were obtained in co-solvent mixtures because of
the low aqueous solubility of compounds according to the following
procedure. At least five separate 20 mL semiaqueous solutions of the
compounds (about 1 mM in 17e65 Wt% methanol for chloroquine
derivatives and 40-65 Wt% methanol for amodiaquine derivatives)
were initially acidified to pH 1.8 with 0.5 N HCl. The solutions were
then titrated with standardised 0.5 N KOH to pH 10.5. The titrations
were performed under argon at 25.0 ꢂ 0.1 ꢀC. The initial estimates
of the apparent ionisation constants (psKa) were obtained by
Bjerrum plots; these values were finally refined by a weighted
non-linear least-squares procedure. Aqueous pKa values were
obtained by extrapolation using the Yasuda-Shedlovsky procedure
[31]. The molar % of species were calculated using the experimental
pKa values from the HendersoneHasselbalch equation.
138.1; 130.5; 130.1; 128.7; 128.4; 127.8; 127.0; 126.3; 126.2; 121.5;
118.9; 117.0; 116.7; 115.5; 107.6; 100.4; 65.2; 50.2; 49.6; 47.9; 8.5.
Anal. Calc. For C28H26ClN5O4 $ 2 HCl C% 55.59, H% 4.66, N% 11.58;
found C% 55.29, H% 4.64, N% 11.44.
4.2.16. 2-{[(3-phenylsulfonylfuroxan-4-yl)oxy]ethyl}ethylamine
hydrochloride (39)
To a stirred solution of 3,4-bis(phenylsulfonyl)furoxan (38) (1 g;
2.72 mmol) in dist. THF (14 mL) kept under nitrogen, 2-(ethylamino)
ethanol (0.54 mL; 5.52 mmol) was added. The obtained mixture was
cooled to 15 ꢀC and 50% NaOH aqueous solution (0.64 g; 8.16 mmol)
was added dropwise. The reaction mixture was stirred for further
40 min and the solvent evaporated under reduced pressure to give
a yellow solid. The solid was taken up with water (30 mL) and
extracted with CH2Cl2 (4 ꢄ 30 mL), dried (Na2SO4) and evaporated
to give the crude product as an oil. The product was purified by FC
eluting with CH2Cl2/MeOH 5% to obtain the title product (0.4 g;
48%) as colourless oil. The product was either used immediately in
the next step or stored at ꢁ21 ꢀC overnight. An analytical sample
was obtained as the hydrochloride by treatment with HCl satu-
rated MeOH, precipitation with dry Et2O and recrystallisation
4.3.1. Parasite cultures
P. falciparum cultures were carried out according to Trager and
Jensen’s with slight modifications [32]. The CQ-sensitive, strain D10
and the CQ-resistant, strain W2 were maintained at 5% hematocrit
(human type A-positive red blood cells) in RPMI 1640 (EuroClone,
Celbio) medium with the addition of 1% AlbuMax (Invitrogen,
Milan, Italy), 0.01% hypoxantine, 20 mM Hepes, and 2 mM gluta-
mmine. All the cultures were maintained at 37 ꢀC in a standard gas
mixture consisting of 1% O2, 5% CO2, 94% N2.
from dry MeOH/Et2O. Mp: 136.8e138. 1H-NMR (DMSO-d6):
d, 9.49
(s, 2H, exch. signal); 8.08 (d, 2H, J ¼ 7.5 Hz, Ph-Ho); 7.91 (t, 1H,
J ¼ 7.5 Hz, Ph-Hp); 7.76 (t, 2H, J ¼ 7.7 Hz, Ph-Hm); 4.78 (t, 2H,
J ¼ 4.9 Hz, CH2O); 3.35 (m, 2H, CH2N); 3.08 (q, 2H, J ¼ 7.2 Hz,
NCH2CH3); 1.27 (t, 3H, J ¼ 7.2, CH3). 13C-NMR (DMSO-d6):
d, 158.5;
136.8; 136.1; 129.9; 128.5; 110.8; 67.0; 44.2; 42.3; 11.0. Anal. Calc.
For C12H15N3O5S $ HCl C% 41.21, H% 4.61, N% 12.01; found C% 41.51,
H% 4.81, N% 11.88.
4.3.2. Parasite growth and drug susceptibility assay
Compounds were dissolved in either water (chloroquine) or
DMSO and then diluted with medium to achieve the required
concentrations (final DMSO concentration <1%, which is non-
toxic to the parasite). Drugs were placed in 96 wells flat-bottom
microplates (COSTAR) and serial dilutions made. Asynchronous
cultures with parasitemia of 1e1.5% and 1% final hematocrit
were aliquoted into the plates and incubated for 72 h at 37 ꢀC.
4.2.17. 4-[(7-chloroquinolin-4-yl)amino]- 2-{[({[2-(3-phenyl-
sulfonylfuroxan-4-yl)oxy]ethyl}ethyl)amino]methyl}phenol dihydro-
chloride (40)
To a suspension of 27 (0.9 g; 2.53 mmol) in iPrOH/DMF 10/1
(16.5 mL), stirred under nitrogen at RT, triethylamine (10.1 mmol)
was added. After 15 min of stirring a solution of 39 (0.7 g;
3.54 mmol) in iPrOH/DMF 10/1 (10 mL) was added dropwise at RT.
The reaction mixture was stirred for 24 h, the solvent evaporated
under reduced pressure to leave an oily residue which was taken
up with water (20 mL) and extracted with EtOAc (4 ꢄ 30 mL). The
organic phase was washed with brine (40 mL), dried and evapo-
rated under reduced pressure to give the crude product as light-
brown oil. The product was purified by flash chromatography
eluting with CH2Cl2/MeOH 3%. The fractions containing the
desired product were collected in tubes filled with 0.2 mL of HCl
saturated MeOH to readily convert the product into the corre-
sponding dihydrochloride. After evaporation under reduced
pressure 40 was obtained in 40% yield as white solid. The product
was recrystallised from MeOH. Mp: 214e215 ꢀC (dec.) 1H-NMR
Parasite growth was determined spectrophotometrically (OD650
)
by measuring the activity of the parasite lactate dehydrogenase
(pLDH), according to a modified version of Makler’s method in
control and drug-treated cultures [33,34]. Antimalarial activity
is expressed as the 50% inhibitory concentrations (IC50); each
IC50 value is the mean and standard deviation of at least three
separate experiments.
4.3.3. Vasodilator activity assay
Thoracic aortas were isolated from male Wistar rats weighing
180e200 g. The endothelium was removed and the vessels were
elically cut: three strips were obtained from each aorta. The tissues
were mounted in organ baths containing 30 mL of Krebs-bicar-
bonate buffer of the following composition (mM): NaCl 111.2, KCl

M. Bertinaria et al. / European Journal of Medicinal Chemistry 46 (2011) 1757e1767
1767
5.0, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.0, NaHCO3 12, glucose 11.1
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Acknowledgments
This work was supported bya grant from Università degli Studi di
Torino, Progetti locali di ricerca 2008, by NIH grants R01-HL087290
and R01-AI082610 and by PRIN 2009 to DT & SP. Thanks are also due
to the Associazione Volontari Italiani Sangue (AVIS Comunale
Milano) for providing fresh blood for parasite cultures.
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522 (2002) 3e5.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.029.