A new series of amodiaquine analogues modified in the basic side chain with in vitro antileishmanial and antiplasmodial activity

Article abstract of DOI:10.1016/j.ejmech.2009.09.012

The synthesis and the study of new amodiaquine derivatives bearing modified lateral basic chains as new agents with both antimalarial and antileishmanial activities are reported. The compounds were tested in vitro against Leishmania donovani MHOM/ET/67/HU3 and 2 strains of Plasmodium falciparum, 3D7 and K1. All the compounds show complex ionisation profiles. At physiological pH the ionised form(s) are in equilibrium with the uncharged form, while at acid pH all the products exist largely as protonated forms. The antiprotozoal profile indicates that all derivatives are endowed with both antimalarial and antileishmanial activity. Interestingly amodiaquine, together with some synthesised derivatives (11, 12, 15, 27, 34), displayed antileishmanial activity in the low micromolar range, although these compounds were also cytotoxic and have a narrow therapeutic window, most of the synthesised compounds proved to be potent antimalarials, a few of them showing a good activity against the chloroquine resistant K1 strain.

Full text of DOI:10.1016/j.ejmech.2009.09.012

European Journal of Medicinal Chemistry 44 (2009) 5071–5079
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new series of amodiaquine analogues modified in the basic side chain with in
vitro antileishmanial and antiplasmodial activity
,
a
a
a
Stefano Guglielmo a, Massimo Bertinaria a , Barbara Rolando , Marco Crosetti , Roberta Fruttero ,
*
Vanessa Yardley b, Simon L. Croft b, Alberto Gasco a
a Dipartimento di Scienza e Tecnologia del Farmaco, Via P. Giuria 9, I-10125 Torino, Italy
b Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and the study of new amodiaquine derivatives bearing modified lateral basic chains as new
agents with both antimalarial and antileishmanial activities are reported. The compounds were tested in
vitro against Leishmania donovani MHOM/ET/67/HU3 and 2 strains of Plasmodium falciparum, 3D7 and K1.
All the compounds show complex ionisation profiles. At physiological pH the ionised form(s) are in
equilibrium with the uncharged form, while at acid pH all the products exist largely as protonated forms.
The antiprotozoal profile indicates that all derivatives are endowed with both antimalarial and anti-
leishmanial activity. Interestingly amodiaquine, together with some synthesised derivatives (11, 12, 15,
27, 34), displayed antileishmanial activity in the low micromolar range, although these compounds were
also cytotoxic and have a narrow therapeutic window, most of the synthesised compounds proved to be
potent antimalarials, a few of them showing a good activity against the chloroquine resistant K1 strain.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 19 March 2009
Received in revised form
8 July 2009
Accepted 9 September 2009
Available online 15 September 2009
Keywords:
Amodiaquine analogues
Leishmania donovani
Plasmodium falciparum
Antiprotozoals
1. Introduction
derivatives bearing modified lateral basic chains. The synthesised
compounds can formally be grouped in two series. The first series
Infections caused by protozoa parasites are widespread in
developing regions of the world with an enormous impact on
public health. Generally speaking the development of agents useful
for the treatment of these diseases has been limited by the lack of
interest of the pharmaceutical sector to develop drugs without
a market. Amodiaquine (AQ, Chart 1) is an established antimalarial
drug recently reintroduced in the World Health Organisation Model
List of Essential Medicines [1,2]. Its clinical indication is the treat-
ment of chloroquine resistant P. falciparum infection in association
with artesunate [3]. In addition, it has been recently reported that
a series of pyrazolo[3,4-b]pyridine derivatives (general structure A,
Chart 1), structurally related to AQ and AQ itself are active against
consists of compounds 7–10, 13 and 16 bearing a third basic centre
in the side chain, and of compound 6, 11–12, 15 where the third
basic centre was replaced by bulky polar groups. It is generally
admitted that a stronger basicity of the molecule increases the
antimalarial activity due to a better uptake in the vacuole owing to
the pH gradient between the cytosol and the acidic vacuole, while
the use of bulky substituents was suggested as one possible factor
contributing to the improvement of the activity against CQ-resis-
tant P. falciparum strains [6]. The second series consists of
compounds 26–35 which were designed by joining the AQ scaffold
to a substituted piperazinyl moiety. The use of piperazine as a linker
proved of some success in antimalarial drug design [6,7]. Recent
work showed that compounds bearing a piperazinyl linker, a basic
centre and heteroaromatic rings were able to inhibit P. falciparum
promastigote forms of Leishmania amazonensis at mM concentration
[4]. These findings open new interesting potential perspectives for
AQ derivatives not only as antimalarial but also as antileishmanial
agents. A number of structural analogues of the lead AQ have been
described. They are principally connected with the modification of
the substitution pattern at the quinoline ring and with variations in
the Mannich side chain [5]. In this paper we report the synthesis
and the study of the dissociation constants (pKas) of some new AQ
growth at nM concentrations as well as to inhibit
b-hematin
formation at M concentrations [8]. Moreover the use of hetero-
m
aromatic rings as substituents in a series of 20-deoxyuridine
derivatives led to antileishmanial compounds with improved
potency [9]. According to these observations we decided to modify
the properties of this series of compounds introducing differently
substituted piperazine on the AQ scaffold. The synthesised
compounds were tested against two strains of Plasmodium falci-
parum 3D7, a clone of NF54 sensitive to all antimalarial drugs
(CQ-S) and K1, a strain originating from Thailand, resistant to
* Corresponding author. Tel.: þ39 011 6707737; fax: þ39 011 6707687.
E-mail address: massimo.bertinaria@unito.it (M. Bertinaria).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.012

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S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
chloroquine and pyrimethamine and against Leishmania donovani
MHOM/ET/67/HU3,
maniasis.
a reference strain causing visceral leish-
2. Results and discussion
2.1. Chemistry
The products we designed can be grouped into two different
classes on the basis of the synthetic pathway used for their prep-
aration. The former class includes compounds 3–4, 6–13, 15–16,
(Scheme 1) the latter includes compounds 26–35 (Scheme 2). The
synthesis of these products required the availability of the new
chloromethyl substituted intermediate 4 that is a very flexible
Chart 1.
Scheme 1. Preparation of key intermediate 4 and of final derivatives 3, 6–13, 15–16. Reagents and conditions: (a) EtOH, reflux, 2.5 h; (b) conc. HCl, reflux, 18 h; (c) Et3N, CH3CN, RT,
18 h; (d) CF3COOH, CH2Cl2, RT, 6 h; (e) DIPEA, HBTU, HOBt, N-Boc-AA(OH), RT, 16 h; (f) CF3COOH, CH2Cl2, RT, 6 h; (g) Et3N, PhNCO or PhNCS or PhC]NH(OCH3), CH3CN, RT or reflux;
(h) Et3N, 14, CH3CN, RT, 2 h; (i) PhNH2, CH3CN, reflux, 72 h; (l) 5 N HCl, reflux, 24 h.

S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
5073
Scheme 3. Preparation of intermediates 21–25. Reagents and conditions: (a) pipera-
zine, n-BuOH, reflux 18 h; (b) 17, toluene, reflux 1.5 h; (c) KOH, MeOH/H2O, 80 ꢀC, 12 h;
(d) piperazine, iPrOH or n-BuOH, base, 0 ꢀC-reflux.
Scheme 2. Preparation of final derivatives 26–35. Reagents and conditions: (a) 17–25,
CH3CN, RT, 18 h; (b) 5 N HCl, reflux, 18 h.
the basic ionisable centres of the products since the dissociation of
the acid phenol OH is undetectable by the potentiometric method
we used. Analysis of the data indicates that in many products there
is not a well separated ionisation of the basic centres following the
close pKa values. However, using AQ as reference compound, we are
induced to assign the pKa values in the range 7.36–7.79 (pKa2) to the
prevalent ionisation of the 4-amino substituted quinoline centre. In
the case of compounds 11, 12 the pKa1 and pKa2 values are too close
to do this attribution. At physiological pH the ionised form(s) are in
equilibrium with the uncharged form. In the sole products 7, 13, 16
the percentage of the uncharged form is <1. At acid pH of the food
vacuole (5.2) all the products exist largely as protonated forms and
this should assure a good trapping of the products in parasite-
infected erythrocytes, at least as far as the pH-driven mechanism is
concerned [10].
scaffold to prepare analogues of AQ. To prepare 4, 4-amino-2-
(hydroxymethyl)phenol (1) was treated in refluxing ethanol with
the chloro-substituted quinoline 2 to afford alcohol 3 in 95% yield.
This intermediate was then refluxed in concentrated HCl for 18 h
yielding desired 4 in high yield (91%). The intermediate 4 was
treated with tert-butylcarbamate 5 to give the protected amine 6
that was easily hydrolised in CF3COOH/CH2Cl2 to afford the amine
intermediate 7. O-Benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU)/1-hydroxybenzotriazole (HOBt)
mediated coupling of this compound with the appropriate N-Boc
protected aminoacid in DMF in the presence of di-isopropylethyl-
amine (DIPEA) yielded, after acid hydrolysis, the corresponding
amino acid derivatives 8–10. The target compounds 11–13 were
obtained by reaction of 7 in basic medium (Et3N) with phenyl-
isocyanate, phenylisothiocyanate and benzenecarboxyimidoate
respectively. Treatment of
7 with stoichiometric amount of
2.3. Biological activities
diphenyl benzoylimidocarbonate (14) gave rise to 15 that was
transformed into 16 by refluxing with aniline in CH3CN and
subsequent hydrolysis in boiling 5 N HCl.
A summaryof theactivityof thecompoundsisreported inTable2.
The compounds 26, 28–35, bearing a piperazinyl moiety, were
synthesised in good yields according to the procedure reported in
Scheme 2. The intermediate 4 was treated with an excess of the
appropriate piperazines 17–25 at room temperature in CH3CN to
afford the expected target compounds, which were characterised as
hydrochloride salts with the exception of 32, kept as free base. The
monosubstituted piperazino-derivative 27 was obtained by acid
hydrolysis of the protected precursor 26.
The heteroaryl piperazines 21, 23–25 were prepared by reaction
of the suitable halo-substituted heterocycles with an excess of
piperazine following, with marginal modifications, the procedures
described in literature (see Section 4.1). The 1-thiophen-2-ylpi-
perazine (22) was synthesised by reaction of commercially avail-
able 2-mercaptothiophene with 17 followed by basic hydrolysis of
the ethoxycarbonyl protection (Scheme 3).
2.3.1. Leishmania donovani
Generally speaking compounds 6–35 proved to be active against
L. donovani intracellular amastigotes, their IC50 values lying in the
1.83–17.7
active than AQ (IC50 value of 1.4
value of 1.83 M, showed the best activity comparable to that of the
lead. The standard drug, sodium stibogluconate (NaSbV) had an IC50
mM range. All of the synthesised compounds were less
m
M). Compound 11, with an IC50
m
value of 188 m
M SbV. However, at the higher concentrations used in
Table 1
Dissociations constants of the synthesised compounds and reference AQ.
No. Dissociation
constantsa
No. Dissociation
constantsa
No. Dissociation
constantsa
pKa1 pKa2 pKa3
pKa1
pKa2
pKa3
pKa1 pKa2 pKa3
AQ 8.50 7.07
–
–
–
–
11
12
13
15
7.65b 7.79b
7.53b 7.62b
–
–
29
30
6.49 7.59 5.54
6.38 7.60 2.83
5.56 7.52 2.86
6.42 7.58 2.66
6.11 7.45 5.09
6.72 7.62 5.12
6.25 7.60 5.14
2.2. Dissociation constants (pKas)
3
4
–
–
7.71
7.78
5.85
6.66
5.94
6.08
3.87
6.34
7.62
7.62
7.61
7.40
7.50
7.68
10.0 31
32
10.1 33
34
6
7.30 7.57
–
Potentiometric titrations of the final target compounds were
performed out with a Sirius GLpKa automated potentiometric
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 Yasuda–Shedlovsky
procedure. The pKa values are listed in Table 1 and are referred to
7
8
9
10
5.87 7.55 9.05 16
6.25 7.59 7.99 26
5.98 7.36 8.05 27
6.11 7.42 8.05 28
–
8.88 35
2.86
a
Determined by potentiometry; S.D. ꢁ 0.07.
b
Uncertain attribution.

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S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
Table 2
potent against both Plasmodium strains, the remainder displaying
strain sensitivity.
Antiparasitic activity of synthesised compounds and AQ against L. donovani, P. fal-
ciparum 3D7 and K1 strains and cytotoxicity against KB cells.
No.
L. donovani
P. falciparum
P. falciparum
Toxicity IC50
(m
M)b
3. Conclusion
MHOM/ET/67/
3D7 IC50 (nM)a
K1 IC50 (nM)a
HU3 IC50 (m
M)a
The introduction on AQ scaffold of lateral chains containing
basic centres and/or hydrogen bonding moieties has produced
derivatives that exhibit both antimalarial and antileishmanial
actions. This has been reported previously for other quinoline-
based compounds, for example chloroquine derivatives and propyl-
quinoline derivatives [11]. A few compounds show highly potent
antimalarial activity against the chloroquine resistant K1 strain.
This activity is comparable to that showed by isoquine, an
improved amodiaquine analogues recently reported [12]. To the
best of our knowledge, this is the first report where the anti-
leishmanial action of AQ and of some newly synthesised analogues
against intracellular amastigote L. donovani is described. Although
these compounds show a narrow therapeutic margin against
L. donovani, there is chemical space to explore the design of more
selective compounds. Further studies are necessary to address
toxicity issues, in particular the synthesis of isoquine-like models
with improved metabolic profile is being considered.
AQ
3
4
6
7
1.4
2.15
712
695
8.61
1420
557
2.12
5.47
14100
14100
14300
238
6.50
290
10.1
12100
7.04
225
1.80
90.0
198
>89
>83.5
8.77
5.65
5.15
15.3
4.69
1.83
2.05
7.10
2.86
11.7
14.9
2.29
9.68
6.71
7.21
17.7
9.14
25.6
8.81
25.6
10.4
54.4
22.3
5.33
8.46
5.34
10.2
9.03
11.4
11.8
8.63
16.1
8.03
5.02
50.4
2.12
5.47
12.2
47.0
22.6
8.22
1.63
48.4
6.73
126
5.28
18.8
3.52
0.323
3.48
0.670
2.22
8
9
10
11
12
13
15
16
26
27
28
29
30
31
32
1.40
10.4
0.420
Unable to
fit curve
1480
1.54
33
34
35
12.9
1.87
14.6
14.6
3.08
1.68
45.2
2.44
8.02
4. Experimental
387
4.1. Instrumentation and chemicals
a
The IC50 represents the concentrations of the test compounds required to inhibit
parasite growth by 50%; IC50 values are the mean of at least three determinations;
standard errors were all within 10% of the mean; IC50 values of control drugs: SbV
All compounds were purified by recrystallisation before char-
acterisation. Melting points were determined with a capillary
apparatus Bu¨chi B-540. Melting points with decomposition were
determined after introduction of the sample at a temperaturꢂe110 ꢀC
lower than the melting point. A heating rate of 2 ꢀC min was
used. Compounds 7–10 were highly hygroscopic amorphous foams,
the determination of their melting point was affected by the
complex thermal behaviour of these compounds; consequently the
melting point was not reported. 1H and 13C NMR spectra were
obtained on a Bruker Avance 300, at 300 and 75 MHz respectively;
188
m
M; Chloroquine (3D7) 7.75 nM; Artesunate (K1) 2.46 nM.
Toxicity determined on KB cells; The IC50 represents the concentrations of test
compounds required to kill 50% of the test cells.
b
the assay, namely 30 and 10 mg/mL, toxicity to the host cell
macrophage was seen for the majority of compounds as reflected in
the cytotoxicity IC50 values. Sodium stibogluconate showed no
toxicity at the top concentrations tested. The highest Therapeutic
Index (TI), the IC50 cytotoxicity/IC50 antileishmanial activity,
showed a narrow margin between in vitro efficacy and cytotoxicity
(Table 2). At this stage the structural basis for selective toxicity
against L. donovani amastigotes is difficult to determine.
d
in ppm rel. to SiMe4 as 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, AQ: amodiaquine
structure, pip: piperazine, pyr: pyridine, pyrim: pyrimidine, Tz:
thiazole, Tph: thiophene, Bzt: benzothiazole, Bzm: benzoimidazole,
Bzo: benzooxazole. Mass spectra were recorded on a Finnigan-Mat
TSQ-700. Flash chromatography (FC) was performed on BDH silica
2.3.2. Plasmodium falciparum 3D7 and K1
As expected compounds 3 and 4, lacking the basic side chain,
showed no activity against both Plasmodium strains. When the
tertiary amine in benzylic position was re-introduced (compound
6) the antiplasmodial activity was restored (IC50 2.12 nM against
both CQ-S and CQ-R strains). Addition of a primary amino group as
the third basic centre (compound 7) maintained the antiplasmodial
activity with a slight decrease in the toxicity against KB cells. When
the amino group was incorporated into an aminoacidic moiety
(compounds 8–10) or a stronger basic functionality was used
(compounds 13, 16), the antiplasmodial activity against the CQ-S
strain was lowered and that against CQ-R strain was completely
abolished. The replacement of the third basic centre by non-basic
bulky polar residues as in compounds 11–12, 15 led to a drop in the
antiplasmodial activity with the exception of derivative 12 which
maintained the same activity with respect to the lead compound.
In the piperazine series the best results were obtained with
2-pyridyl, 2-thiazolyl, 2-benzoimidazolyl N4-substituted pipera-
zine derivatives 29, 31 and 34 which showed IC50 values within the
0.32–3.08 nM range against CQ-S strain and 0.42–1.54 nM range
against CQ-R strain, this might be attributable to an additional
effect exerted by the heteroaromatic ring used. In general the
compounds 4, 6, 7, 12, 15, 26, 28, 29, 30, 31 and 34, were equally
gel (particle size 40–63 mm). HPLC measurements were carried out
on a Varian Pro-Star 210 chromatograph equipped with a variable
wavelength detector (iPro-Star 325). The chromatography was
performed using a 5
250–25 C18 with a flow rate of 39 mL/min,
m
m particle size prepacked column Licrospher
254 nm. When not
l
otherwise specified, anhydrous magnesium sulphate (MgSO4) was
used as the drying agent of organic phases. Analysis (C, H, N) of the
new compounds was performed by REDOX (Monza); the analytical
results were within ꢃ0.4% of the theoretical value. Structures 1 [13],
5 [14], 14 [15], 21 [16], 23 [17], 24 [18], 25 [19] were synthesised
according to reported methods.
4.2. Chemistry
4.2.1. 4-[(7-Chloroquinolin-4-yl)amino]-2-(hydroxymethyl)phenol
hydrochloride (3)
To a solution of 4-amino-2-(hydroxymethyl)phenol hydrochlo-
ride 1 (11.4 g; 64.9 mmol) in EtOH (540 mL) 4,7-dichloroquinoline 2
(12.8 g; 64.9 mmol) was added and the mixture was refluxed for
2.5 h. The reaction was cooled and the solid collected on a buchner

S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
5075
funnel and dried in a dessiccator (over P2O5) to give the desired
product (20.8 g; 95%) as yellow solid. An analytical sample was
obtained by recrystallisation from MeOH/H2O. Mp: 323 ꢀC (dec.) 1H
procedure 7 (1.48 g; 98%) was obtained as a white amorphous solid
which dissolves if left in open air. 1H NMR (DMSO-d6):
, 10.95
d
(s, 1H,exch. sign.); 8.85 (d, 1H, J ¼ 9.1 Hz, AQ-H5); 8.52 (d, 1H,
J ¼ 7.0 Hz, AQ-H2); 8.25 (s, br, 2H, exch. sign.); 8.10 (d, 1H, J ¼ 2.0 Hz,
AQ-H8); 7.89 (dd, 1H, J1 ¼9.1 Hz, J2 ¼ 2.0 Hz, AQ-H6); 7.52 (d, 1H,
NMR (DMSO-d6): d, 11.07 (s, 1H, exch. signal); 10.12 (s, 1H, exch.
signal); 8.83 (d, 1H, J ¼ 9 Hz, AQ-H5); 8.46 (d, 1H, J ¼ 6.9 Hz, AQ-H2);
0
0
0
8.17 (s, 1H, AQ-H8); 7.84 (d, 1H, J ¼ 9 Hz, AQ-H6), 7.35 (s, 1H, AQ-H3 );
J ¼ 2.5, AQ-H3 ); 7.39 (dd, 1H, J1 ¼8.6 Hz, J2 ¼ 2.5 Hz AQ-H5 ); 7.18
0
0
0
0
7.14 (d, 1H, J ¼ 8.4 Hz, AQ-H5 ); 6.99 (d, 1H, J ¼ 8.4 Hz, AQ-H6 ); 6.65
(d, 1H, J ¼ 6.9 Hz, AQ-H3); 5.21 (br s, 1H, exch. signal); 4.53 (s, 2H,
(d, 1H, J ¼ 8.6 Hz, AQ-H6 ); 6.72 (d, 1H, J ¼ 7.0 Hz, AQ-H3 ); 4.21
(s, 2H, PhCH2N); 3.28 (m, 6H, J ¼ 5.8 Hz, CH2NCH2CH2N); 1.27 (t, 3H,
CH2OH). 13C NMR (DMSO-d6):
d
, 155.3; 153.6; 143.1; 139.2; 138.3;
J ¼ 6.8 Hz, CH3). 13C NMR (DMSO-d6):
d, 156.3; 155.3; 143.4; 139.0;
130.6; 127.7; 127.2; 126.1; 124.7; 124.3; 119.3; 115.7; 115.6; 100.0;
57.9. Anal. Calc. For C16H13ClN2O2$HCl C% 56.99, H% 4.18, N% 8.31;
found C% 56.85, H% 4.23, N% 8.36.
138.5; 129.9; 128.8; 127.9; 127.4; 125.6; 122.5; 119.34; 118.6; 117.6;
116.9; 115.6; 114.6; 110.7; 100.2; 51.0; 47.9; 47.7; 33.4; 8.4. Anal.
Calc. For C20H23ClN4O$3 CF3COOH$H2O C% 42.72, H% 3.86, N% 7.66;
found C% 42.51, H% 3.81, N% 7.40.
4.2.2. 2-Chloromethyl-4-[(7-chloroquinolin-4-yl)amino]phenol
hydrochloride (4)
4.2.5. General procedure for the synthesis of derivatives 8–10
To a solution of 7 (0.4 g; 0.85 mmol) in DMF (10 mL), DIPEA
(0.74 mL; 4.24 mmol), HBTU (0.64 g; 1.70 mmol), HOBt (0.23 g;
1.70 mmol) and the appropriate Boc-protected aminoacid
(1.70 mmol) were added. After 16 h the reaction mixture was
evaporated, the residue was dissolved in CH2Cl2 (25 mL) and
washed with 10% NaHCO3 (3 ꢄ 10 mL), brine (10 mL), dried and
evaporated to yield a yellow oil. The oil was purified by flash
chromatography eluting with EtOAc to obtain a yellow solid
(94–96% yield). The product was dissolved in 30% CF3COOH in
CH2Cl2 (15 mL); after 6 h under stirring the mixture was evaporated
and the residue was recrystallised from MeOH/Et2O, then purified
by RP18-HPLC (eluent: H2O/gradient from 5 to 40% of CH3CN).
Derivative 3 (7.5 g; 22.2 mmol) was suspended in conc. HCl
(400 mL) and the mixture was refluxed for 18 h. The solvent was
evaporated under reduced pressure and the residue was triturated
with Et2O and filtered to give the desired product (7.2 g; 91%) as
yellow solid. The product was used without further purification.
Mp: 239.3–241.1 ꢀC (dec.) 1H NMR (DMSO-d6):
d, 11.12 (s, 1H, exch.
signal), 10.56 (s, 1H, exch. signal); 8.87 (d, 1H, J ¼ 9 Hz, AQ-H5); 8.50
(d, 1H, J ¼ 6.9 Hz, AQ-H2); 8.20 (s, 1H, AQ-H8); 7.85 (d, 1H, J ¼ 9 Hz,
0
0
AQ-H6); 7.36 (s, 1H, AQ-H3 ); 7.13 (d, 1H, J ¼ 8.5 Hz, AQ-H5 ); 7.01
0
(d, 1H, J ¼ 8.5 Hz, AQ-H6 ); 6.64 (d, 1H, J ¼ 6.9 Hz, AQ-H3); 4.77
(s, 2H, CH2); 3.62 (br s, 1H, exch. signal). 13C NMR (DMSO-d6):
d,
156.2; 156.1; 143.9; 139.8; 139.1; 129.0; 128.5; 128.4; 128.1; 126.9;
125.9; 119.9; 117.5; 116.5; 100.8; 42.3. Anal. Calc. For
C16H12Cl2N2O$HCl$0.2 H2O C% 53.49, H% 3.77, N% 7.80; found C%
53.42, H% 3.71, N% 7.44.
4.2.5.1. N1-{2-[{5-[(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
(ethyl)amino]ethyl} alaninamide (8). After evaporation and freeze-
drying the product was obtained as a yellow solid. 1H NMR (DMSO-
4.2.3. (2-{[5-(7-Chloroquinolin-4-ylamino)2-hydroxybenzyl]-
ethylamino}ethyl)carbamic acid, tert-butyl ester (6)
d6): d, 10.89 (s, 1H), 9.72 (s br, 1H), 8.80 (m, 1H, AQ-H5), 8.71 (d, 1H,
J ¼ 9.2 Hz, CONH), 8.53 (d, 1H, J ¼ 7.0 Hz, AQ-H2), 8.19 (s br, 2H), 8.08
To a stirred suspension of 4 (1.25 g; 2.81 mmol) in CH3CN
(10 mL) triethylamine (1.56 mL; 1.12 mmol) was added. After
10 min (2-ethylamino-ethyl)carbamic acid tert-butyl ester (5)
(1.59 g; 8.43 mmol) was added and the reaction mixture was stirred
at room temperature (r.t.) for 18 h. The solvent was removed under
reduced pressure and the residue taken up with water (20 mL) and
extracted with EtOAc (3 ꢄ 25 mL). The organic phase was dried
(Na2SO4), the solvent removed under reduced pressure to leave
a dark oil. The crude product was purified by flash chromatography
eluting with CH2Cl2/MeOH 5% to obtain 7 (0.64 g; 48) as a light
brown oil that became solid on standing. The product was recrys-
tallized from iPr2O/EtOAc to obtain a white amorphous solid. Mp:
(d,1H, J ¼ 1.9 Hz, AQ-H8), 7.89 (dd,1H, J ¼ 9.0 Hz,1.9 Hz, AQ-H6), 7.51
0
0
(d, 1H, J ¼ 2.2 Hz, AQ-H3 ), 7.39 (dd, 1H, J ¼ 8.6 Hz, 2.2 Hz, AQ-H5 ),
0
7.16 (d, 1H, J ¼ 8.6 Hz, AQ-H6 ), 6.71 (d, 1H, J ¼ 7.0 Hz, AQ-H3), 4.33
(s, 2H, PhCH2N), 3.85 (m, 1H, CHAla), 3.57 (m, 2H, NHCH2CH2), 3.17
(s br, 4H, CH2NCH2),1.35–1.26 (m, 6H, 2 CH3). 13C NMR (DMSO-d6):
d,
170.2, 156.3, 155.3, 143.8, 139.4, 138.5, 129.8, 128.8, 128.2, 127.5,
125.7, 119.7, 118.0, 117.0, 115.8, 100.3, 53.6, 51.2, 50.4, 47.6, 34.0, 17.0,
8.6. Anal. Calc. For C23H28ClN5O2$3 CF3COOH$2 H2O C% 42.47, H%
4.30, N% 8.54; found C% 42.72, H% 4.12, N% 8.64.
4.2.5.2. N1-{2-[{5-[(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
(ethyl)amino]ethyl} threoninamide (9). After evaporation and
freeze-drying the product was obtained as a yellow solid. 1H NMR
161.4–161.8 ꢀC (dec). 1H NMR (DMSO-d6):
d, 10.73 (s, 1H, exch.
signal), 8.91 (s, 1H, exch. signal); 8.43 (d, 1H, J ¼ 9 Hz, AQ-H5); 8.38
(DMSO-d6): d, 10.97 (s, 1H), 9.83 (s br, 1H), 9.02 (m, 1H, AQ-H5), 8.73
(d, 1H, J ¼ 5.4 Hz, AQ-H2); 7.86 (d, 1H, J ¼ 2 Hz, AQ-H8); 7.53 (dd, 1H,
(d, 1H, J ¼ 9.1 Hz, CONH), 8.53 (d, 1H, J ¼ 7.0 Hz, AQ-H2), 8.22 (s br,
0
0
J ¼ 9, 2 Hz, AQ-H6); 7.11 (m, 2H, AQ-H3 , AQ-H5 ); 6.81 (m, 2H,
3H), 8.11 (d,1H, J ¼ 1.3 Hz, AQ-H8), 7.88 (d, 1H, J ¼ 8.9 Hz, AQ-H6),
0
0
0
NHBoc, AQ-H6 ); 6.60 (d, 1H, J ¼ 5.4 Hz, AQ-H3); 3.75 (s, 2H, CH2Ph);
7.54 (s, 1H, AQ-H3 ), 7.40 (dd, 1H, J ¼ 8.6 Hz, 1.9 Hz, AQ-H5 ), 7.19
0
3.09 (m, 2H, CH2NH); 2.60 (m, 4H, CH2NCH2); 1.37 (s, 9H, t-Bu); 1.04
(d, 1H, J ¼ 8.6 Hz, AQ-H6 ), 6.72 (d, 1H, J ¼ 7.0 Hz, AQ-H3), 4.37 (s, 2H,
(t, 3H, J ¼ 6.9 Hz, CH3). 13C NMR (DMSO-d6):
d
, 155.5; 154.5; 152.7;
PhCH2N), 3.76 (s br, 1H, CHNH2), 3.60–3.58 (m, 2H, AQ-CH2), 3.23–
3.21 (m, 4H, 2 CH2), 1.67–1.55 (m, 3H, CH2 þ CH-Leu) 1.31 (t, 3H,
J ¼ 6.9 Hz CH3CH2), 0.88 (d, 6H, J ¼ 3.1 Hz, 2 CH3 Leu); 13C NMR
151.8; 149.4; 133.6; 130.4; 127.5; 125.4, 124.5; 124.4; 124.2; 124.1;
117.6; 115.9; 100.4; 77.5; 54.7; 51.9; 46.4; 37.3; 28.1; 10.8. MS (CI/
isobutane) m/z: 471/473 [MHþ]. Anal. Calc. For C25H31ClN4O3 C%
63.75, H% 6.63, N% 11.90; found C% 63.60, H% 6.62, N% 12.01.
(DMSO-d6): d, 167.61, 156.2, 155.2, 143.5, 139.1, 138.4, 129.8, 128.7,
127.9, 127.3, 125.5, 119.4, 117.8, 116.9, 115.6, 100.2, 65.4, 58.1, 50.8,
50.0, 47.5, 33.8, 19.7, 8.4. Anal. Calc. For C24H30ClN5O3$3 CF3COOH$2
H2O C% 42.39, H% 4.39, N% 8.24; found C% 42.49, H% 4.10, N% 8.35.
4.2.4. 2-{[(2-Aminoethyl)(ethyl)amino]methyl}-4-[(7-chloro-
quinolin-4-yl)amino]phenol trifluoroacetate (7)
To a stirred solution of 6 (1.00 g; 2.12 mmol) in CH2Cl2 (10 mL),
CF3COOH 2.38 mL was added, the mixture was stirred at r.t. for
6 h, then the solvent was evaporated under reduced pressure.
The obtained residue was triturated with dry Et2O (2 x 25 mL) at
ꢂ15 ꢀC until it was solid enough to be filtered and immediately
stored in a dessiccator over P2O5/paraffin under vacuum. With this
4.2.5.3. N1-{2-[{5-[(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
(ethyl)amino]ethyl} leucinamide (10). After evaporation and
freeze-drying the product was obtained as a yellow solid. 1H NMR
(DMSO-d6): d, 10.97 (s, 1H), 9.83 (s, 1H), 9.02 (m, 1H), 8.73 (d, 1H,
J ¼ 9.1 Hz), 8.53 (d, 1H, J ¼ 7.0 Hz), 8.28 (br s, 2H), 8.11 (d,
1H, J ¼ 1.3 Hz), 7.88 (d, 1H, J ¼ 8.9 Hz), 7.54 (s, 1H), 7.40 (dd, 1H,

5076
S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
J1 ¼8.6 Hz, J2 ¼1.9 Hz), 7.19 (d,1H, J ¼ 8.6 Hz), 6.72 (d,1H, J ¼ 7.0 Hz),
4.37 (s, 2H), 3.76 (br s, 1H), 3.60–3.58 (m, 2H), 3.23–3.21 (m, 4H),
1.67–1.55 (m, 3H), 1.31 (t, 3H, J ¼ 6.9 Hz), 0.88 (d, 3H, J ¼ 3.1 Hz); 13C
4.2.8. N-{2-[{5-[(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
ethylamino}ethyl]-benzencarboximidamide trihydrochloride (13)
To a solution of 7 (0.40 g; 0.56 mmol) and Et3N (0.23 mL;
NMR (DMSO-d6):
d
, 169.7, 156.3, 155.3, 143.4, 139.0, 138.4, 129.9,
1.68 mmol) in CH3CN (10 mL),
a
solution of methyl-
128.8, 127.9, 127.4, 125.6, 122.6, 119.3, 118.6, 117.8, 116.9, 115.6, 114.7,
110.8, 100.2, 50.9, 49.9, 47.5, 33.7, 23.5, 22.4, 21.7, 8.4. Anal. Calc. For
C26H34ClN5O2$3.2 CF3COOH$2 H2O C% 43.98, H% 4.69, N% 7.91;
found C% 43.95, H% 4.37, N% 7.80.
benzenecarboximidoate [20] (0.08 g; 0.56 mmol) in CH3CN (5 mL)
was added dropwise under stirring. After the addition was over the
mixture was stirred under reflux for 72 h. After the mixture reached
r.t. the solvent was evaporated under reduced pressure. The crude
product was purified by flash chromatography eluting with CH2Cl2/
MeOH 5% affording a pale yellow oil. The pure product (0.28 g, 80%)
was dissolved in MeOH and converted into the corresponding
hydrochloride by treatment with HCl–saturated Et2O. The solvent
was decanted off and the yellow semisolid material was recrystal-
lized from dry MeOH/Et2O. The yellow solid thus obtained was
dissolved in water and freeze-dried to afford the desired product.
4.2.6. N-[2-({[5-(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
ethylamino)ethyl]-N0-phenyl-urea dihydrochloride (11)
To a solution of 7 (0.40 g; 0.56 mmol) and Et3N (0.23 mL;
1.68 mmol) in CH3CN (10 ml), a solution of phenylisocyanate
(0.07 g; 1.68 mmol) in CH3CN (5 ml) was added dropwise under
stirring. After the addition was over the mixture was stirred at r.t.
for 24 h. The solvent was evaporated under reduced pressure. The
crude product was purified by flash chromatography eluting with
CH2Cl2/MeOH 5% affording a pale yellow oil. The pure product
(0.28 g; 84%) was dissolved in MeOH and converted into the
corresponding hydrochloride by treatment with HCl–saturated
Et2O. The solvent was decanted off and the yellow semisolid
material was recrystallized from dry MeOH/Et2O. The yellow solid
thus obtained was dissolved in water and freeze-dried to afford
Mp: 201.3 ꢀC (dec.)1H NMR (CD3OD):
d
, 8.64 (d, 1H, J ¼ 9.1 Hz, AQ-
H5); 8.36 (d, 1H, J ¼ 7.1 Hz, AQ-H2); 7.96 (d, 1H, J ¼ 2.0 Hz, AQ-H8);
0
7.84–7.57 (m, 7H, 5PhH, AQ-H3 , AQ-H6); 7.44 (dd, 1H, J1 ¼8.7 Hz,
0
0
J2 ¼ 2.3 Hz, AQ-H5 ); 7.14 (d, 1H, J ¼ 8.7 Hz, AQ-H6 ); 6.93 (d, 1H,
J ¼ 7.1 Hz, AQ-H3); 4.58 (s, 2H, PhCH2); 4.10 (m, 2H, CH2NH); 3.71 (m,
2H, CH2N); 3.30 (m, 2H, NCH2CH3); 1.52 (t, 3H, J ¼ 7.11 Hz, CH3). 13
C
NMR (CD3OD): d, 166.5; 157.8; 157.3; 144.2; 141.4; 140.4; 135.1;
131.6; 130.6; 130.5; 130.0; 129.3: 129.1; 126.5; 120.4; 118.7; 118.2;
117.2; 101.9; 52.9; 51.1; 50.4; 39.4; 25.3; 9.3. Anal. Calc. For
C27H28ClN5O$3 HCl$2 H2O C% 52.36, H% 5.70, N% 11.31; found C%
52.41, H% 5.41, N% 11.05.
the desired product. Mp: 183.8–184.1 ꢀC. 1H NMR (DMSO-d6):
d,
11.09 (s, 1H, exch. sign.); 11.00 (s, br, 1H, exch. sign.); 10.11 (s, br,
1H, exch. sign.); 9.22 (s, 1H, exch. sign); 8.85 (d, 1H, J ¼ 9.1 Hz, AQ-
H5); 8.50 (d, 1H, J ¼ 7.0 Hz, AQ-H2); 8.13 (d, 1H, J ¼ 2.0 Hz, AQ-H8);
7.84 (dd, 1H, J1 ¼9.1 Hz, J2 ¼ 2.0 Hz, AQ-H6); 7.65 (d, 1H, J ¼ 2.5,
4.2.9. N0-benzoyl-N-{2-[({5-[(7-Chloroquinolin-4-yl)amino]-2-
hydroxybenzyl}ethylamino]ethyl)carbamimidicacid, phenyl ester (15)
To a solution of 7 (0.40 g; 0.56 mmol) and Et3N (0.23 mL;
1.68 mmol) in CH3CN (15 ml), a solution of 14 (0.18 g; 0.56 mmol)
in CH3CN (5 ml) was added dropwise under stirring. After the
addition was over the mixture was stirred at r.t. for 2 h. The
solvent was evaporated under reduced pressure and the crude
product was purified by flash chromatography eluting with
CH2Cl2/MeOH 5% affording a pale yellow oil. The pure product
(0.27 g, 80%) was recrystallized from dichloromethane/n-hexane
0
0
AQ-H3 ); 7.35–7.11 (m, 6H, 5PhH, AQ-H5 ); 6.81–6.76 (m, 2H,
0
CH2NHCO, AQ-H6 ); 6.75 (d, 2H, J ¼ 7.0 Hz AQ-H3); 4.35 (s, 2H,
PhCH2N); 3.52 (d, 2H, J ¼ 5.8 Hz, CH2NH); 3.29–3.18 (m, 4H,
CH2NCH2); 1.36 (t, 3H, J ¼ 7.1 Hz, CH3). 13C NMR (DMSO-d6):
d,
169.2; 156.0; 155.9; 155.0; 143.1; 140.0; 138.9; 138.1; 130.0;
128.6; 128.3; 127.9; 127.1; 125.9; 121.0; 119.1; 117.6; 116.8; 115.5;
100.2; 51.7; 50.4; 47.9; 34.4; 8.6. Anal. Calc. For C27H28ClN5O2$ 2
HCl$2.5 H2O C% 53.34, H% 5.80, N% 11.52; found C% 53.18, H% 5.54,
N% 11.66.
to afford a yellow solid. Mp: 144.5–144.9 ꢀC. 1H NMR (CDCl3):
d,
4.2.7. N-[2-({[5-(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
ethylamino)ethyl]-N0-phenyl-thiourea dihydrochloride (12)
10.21 (t, 1H, exch. sign.) 8.43 (d, 1H, J ¼ 5.4 Hz, AQ-H2); 7.97 (d, 1H,
J ¼ 1.5 Hz, AQ-H8); 7.89(m, 2H, COPhHo); 7.78 (d, 1H, J ¼ 8.9 Hz,
0
To a solution of 7 (0.40 g; 0.56 mmol) and Et3N (0.23 mL;
1.68 mmol) in CH3CN (15 ml), a solution of phenylisothiocyanate
(0.08 g; 0.56 mmol) in CH3CN (5 ml) was added dropwise under
stirring. After the addition was over the mixture was stirred at r.t.
for 24 h. The solvent was evaporated under reduced pressure. The
crude product was purified by flash chromatography eluting with
CH2Cl2/MeOH 5% affording a pale yellow oil. The pure product
(0.31 g, 91%) was dissolved in MeOH and converted into the cor-
responding hydrochloride by treatment with HCl–saturated Et2O.
The solvent was decanted off and the yellow semisolid material was
recrystallized from dry MeOH/Et2O. The yellow solid thus obtained
was dissolved in water and freeze-dried to afford the desired
AQ-H5); 7.44–7.11 (m, 10H, 7PhH, AQ-H6, AQ-H5 , NHCON); 6.94 (s,
0
0
1H, AQ-H3 ); 6.84 (d, 1H, J ¼ 8.5 Hz, AQ-H6 ); 6.77 (s, 1H, OPhHp);
6.61 (d, 1H, J ¼ 5.4 Hz, AQ-H3); 3.85 (s, 2H, PhCH2); 3.68 (m, 2H,
CH2CH3); 2.89 (t, 2H, J ¼ 6.6 Hz, NCH2); 2.77 (m, 2H, HNCH2); 1.82
(t, 3H, J ¼ 7.1 Hz, CH3). 13C NMR (CDCl3):
d, 177.9; 162.6; 155.9;
151.8; 151.6, 149.4; 149.2; 137.0; 135.1; 131.9; 130.4; 129.3; 129.1;
128.7; 127.9; 125.8; 125.7; 125.6; 125.4; 122.9; 122.0; 121.3; 117.5;
117.4; 101.3; 57.4; 52.1; 47.9; 38.9; 11.4. Anal. Calc. For
C34H32ClN5O3 C% 68.74, H% 5.43, N% 11.79; found C% 68.35, H%
5.55, N% 11.61.
4.2.10. N-[2-({5-[(7-Chloroquinolin-4-yl)amino]-2-
hydroxybenzyl}ethylamino)ethyl]-N0-phenylguanidine
product. Mp: 167.4–167.7 ꢀC. 1H NMR (DMSO-d6):
d, 11.18 (s, 1H,
exch. sign.); 10.96 (s, br,1H, exch. sign.); 10.27 (s, br, 2H, exch. sign.);
8.91 (d, 1H, J ¼ 9.1 Hz, AQ-H5); 8.47 (d, 1H, J ¼ 7.1 Hz, AQ-H2); 8.39
(m, 1H, NHCS); 8.18 (d, 1H, J ¼ 2.0 Hz, AQ-H8); 7.84 (dd, 1H,
trihydrochloride (16)
To a solution of 15 (0.89 g, 1.5 mmol) in CH3CN (15 ml) aniline
(1.4 g; 15 mmol) was added under stirring. The mixture was
refluxed for 72 h and evaporated under reduced pressure. The
residue was taken up with water (100 ml) and extracted with
EtOAc (3 ꢄ 30 ml). The organic phase was washed with brine
(100 mL), dried over anhydrous Na2SO4 and evaporated under
reduced pressure to leave a dark-yellow semisolid material. The
crude product was partially purified by flash chromatography
eluting with CH2Cl2/MeOH 5% and immediately dissolved in 5 N
HCl (30 ml) and refluxed for 24 h. After cooling the mixture was
evaporated under reduced pressure to leave a yellow oil which
0
J1 ¼9.1 Hz, J2 ¼ 2.0 Hz, AQ-H6); 7.70 (d, 1H, J ¼ 2.3 Hz, AQ-H3 ); 7.42–
0
0
7.20 (m, 6H, 4PhH, AQ-H5 , AQ-H6 ); 7.08 (m, 1H, PhHp); 6.83 (d, 1H,
J ¼ 7.1 Hz, AQ-H3); 4.35 (s, 2H, PhCH2N); 3.96 (d, 2H, J ¼ 5.0 Hz,
CH2NH); 3.27 (m, 4H, CH2NCH2); 1.37 (t, 3H, J ¼ 7.0 Hz, CH3). 13C
NMR (DMSO-d6): d, 180.9; 156.3; 155.1; 143.2; 139.1; 139.0; 138.4;
130.3; 128.8; 128.6; 128.0; 127.4; 126.2; 124.4; 123.2; 119.2; 117.6;
117.0; 115.8; 100.6; 51.7; 50.0; 48.0; 38.6; 8.7. Anal. Calc. For
C27H28ClN5OS$2 HCl$2 H2O C% 52.73, H% 5.57, N% 11.39; found C%
52.62, H% 5.22, N% 11.49.

S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
5077
was purified by flash chromatography with gradient elution
4.2.13. 4-[(7-Chloroquinolin-4-yl)amino]-2-[(piperazin-1-
yl)methyl]-phenol trihydrochloride (27)
CH2Cl2 with 10 to 30% NH3-saturated MeOH. The pure product
(0.40 g, 55%) was dissolved in MeOH/conc. HCl 1/1 (20 ml) and
the mixture was stirred at r.t. for 3 h. The solvent was evaporated
under reduced pressure and the yellow semisolid material was
recrystallized from iPrOH/iPr2O. The yellow solid obtained was
freeze-dried to afford the desired product. Mp: 180.4–180.7 ꢀC
A solution of 26 (1.27 g; 2.47 mmol) in 6 N HCl (20 mL) was
refluxed for 18 h, after evaporation of the solvent under reduced
pressure the obtained product was recrystallized from iPrOH to
yield 27 (1.1 g; 94%) as a yellow solid that was freeze-dried. Mp:
271.6–272.8 ꢀC. 1H NMR (DMSO-d6): , 12.22 (s, br, 1H, NHþ); 11.25
d
(dec.). 1H NMR (CD3OD):
d
, 8.30 (d, 1H, J ¼ 9.4 Hz, AQ-H5); 8.26
(s. br, 1H, AQ-OH); 10.94 (s, br, 1H, NHþ); 10.09 (s, br, 2H, NH2þ); 8.96
(d, 1H, J ¼ 9.0 Hz, AQ-H5); 8.46 (d, 1H, J ¼ 6.9 Hz, AQ-H2); 8.19 (s, 1H,
(d, 1H, J ¼ 5.5 Hz, AQ-H2); 7.79 (d, 1H, J ¼ 1.8 Hz, AQ-H8); 7.44–
0
0
0
7.12 (m, 8H, 5PhH, AQ-H3 , AQ-H6, AQ-H5 ); 6.87 (d, 1H, J ¼ 9.6 Hz,
AQ-H8); 7.82 (d, 1H, J ¼ 9.0 Hz, AQ-H6); 7.65 (s, 1H, AQ-H3 ); 7.40
0
0
0
AQ-H6 ); 6.58 (d, 1H, J ¼ 5.5 Hz, AQ-H3); 3.75 (s, 2H, PhCH2); 3.47
(d, 1H, J ¼ 8.5 Hz, AQ-H5 ); 7.25 (d, 1H, J ¼ 8.5 Hz, AQ-H6 ); 7.04
(d, 1H, J ¼ 6.9 Hz, AQ-H3); 4.35 (s, 2H, CH2-Ph); 3.51 (s, br, 8H, 4CH2-
(m, 2H, CH2CH2NH); 2.79 (m, 2H, NCH2CH2); 2.69 (m, 2H,
CH2CH3); 1.14 (t, 3H, J ¼ 7.0 Hz, CH3). 13C NMR (CD3OD):
d
, 156.4;
Pip). 13C NMR (DMSO-d6):
d, 156.5; 155.0; 154.8; 143.1; 139.0;
155.2; 151.5; 151.1; 148.7; 135.8; 135.5; 131.1; 130.0; 127.4, 127.3;
126.4; 125.9; 125.6; 125.3; 124.7; 124.0; 118.0; 116.6; 100.7;
54.7; 49.0; 48.0; 40.4; 10.7. Anal. Calc. For C27H29ClN6O$3 HCl$2.5
H2O C% 50.33, H% 5.80, N% 13.04; found C% 50.01, H% 5.45, N%
12.60.
138.3; 130.5; 128.6; 127.9; 127.2; 119.1; 117.1; 116.7; 115.8; 101.0;
61.4; 47.1. Anal. Calc. For C20H21ClN4O3$3 HCl$3 H2O C% 45.13, H%
5.60, N% 10.52; found C% 44.81, H% 5.23, N% 10.32.
4.2.14. General procedure for the synthesis of derivatives 28–35
To a stirred solution of 4 (0.5 g; 1.4 mmol) in CH3CN (15 mL) the
appropriate piperazine 18–25 (6.3 mmol) was added and the
mixture was stirred at r.t. for 18 h. The solvent was evaporated
under reduced pressure, the residue treated with water (40 mL)
and stirred for 20 min until a yellow precipitate was formed. The
solid, consisting of crude product, was collected, washed with
water, dried and then purified by flash chromatography eluting
with CH2Cl2/MeOH 5–10% to yield the desired product.
4.2.11. 1-(Thiophen-2-yl)piperazine (22)
To a stirred solution of 2-mercaptothiophene (2.0 g; 17.5 mmol)
in toluene (14 mL) ethoxycarbonyl piperazine (2.7 g; 17.5 mmol)
was added and the mixture was refluxed for 1.5 h. The reaction
mixture was purified by flash chromatography eluting with PE/
CH2Cl2 50% then CH2Cl2 100% to yield 2.7 g of a yellow oil. The
obtained product was dissolved in MeOH/H2O 4/1 (110 mL) and
KOH (7.45 g; 133 mmol) was slowly added under stirring at r.t. After
the addition was over the mixture was heated at 80 ꢀC for 12 h. The
solvent was evaporated under reduced pressure and the residue
taken up with water (30 mL) and extracted with CH2Cl2
(3 ꢄ 25 mL). The organic phase was dried and evaporated under
reduced pressure to leave an oil which was purified by flash
chromatography eluting with CH2Cl2/MeOH 10% to give 1.45 g
(49%) of a colorless oil, pure enough to be used in the next step. 1H
4.2.14.1. 4-[(7-Chloroquinolin-4-yl)amino]-2-[(4-phenyl-piperazin-
1-yl)methyl]-phenol trihydrochloride (28). The product was con-
verted into the corresponding hydrochloride by treatment with HCl
saturated MeOH, recrystallized from MeOH/Et2O and freeze-dried
to afford a yellow solid (66%). Mp: 256.5–258.3 1H NMR (DMSO-d6):
d
, 11.39 (s, br, 1H, NHþ); 11.24 (s, br, 1H, AQ-OH); 10.90 (s, br, 1H,
NHþ); 8.95 (d, 1H, J ¼ 9.2 Hz, AQ-H5); 8.47 (d, 1H, J ¼ 6.8 Hz, AQ-H2);
8.20 (d, 1H, J ¼ 2.0 Hz, AQ-H8); 7.84 (d, 1H, J ¼ 9.2 Hz, AQ-H6); 7.73
NMR (DMSO-d6):
d, 6.77–6.74 (m, 1H, H4); 6.69 (m, 1H, H5); 6.12
(m,1H, H3); 2.96 (m, 4H, 2CH2-pip); 2.82 (m, 4H, 2CH2-pip). 13C NMR
(d,1H, J ¼ 2.4 Hz, AQ-H3 ); 7.42–7.38 (dd,1H, J ¼ 2.4, 8.7 Hz, AQ-H5 );
0
0
0
(DMSO-d6):
d
, 139.7; 126.3; 111.6; 104.5; 52.1; 45.1. MS (CI/isobu-
7.28–7.23 (m, 3H, AQ-H6 , Ph-H3,5); 7.0–6.95 (m, 3H, AQ-H3, Ph-
tane) (m/z): 169 [MHþ]. Spectral data are in keeping with those of
H2,6); 6.87 (t, 1H, Ph-H4); 4.37 (s, 2H, ArCH2-N); 4.28 (s, br, 2H,
an authentic sample [21].
2NHþ-Pip); 3.82 (m, 2H, CH2-Pip); 3.49 (m, 2H, CH2-Pip); 3.30–3.23
(m, 4H, 2CH2-Pip). 13C NMR (DMSO-d6):
d, 157.2; 155.7; 150.4;
4.2.12. 4-{5-[(7-Chloroquinolin-4-yl)amino]-2-hydroxybenzyl}-
piperazine-1-carboxylic acid, ethyl ester dihydrochloride (26)
143.9; 139.8; 139.2; 131.5; 130.0; 129.5; 128.8; 128.1; 127.1; 120.9;
119.9; 117.9; 117.6; 116.8; 116.6; 101.5; 53.7; 51.2; 46.1. MS (CI/
isobutane) (m/z): 445/447 [MHþ]. Anal. Calc. For C26H25ClN4O$3
HCl$0.75 H2O C% 54.99, H% 5.24, N% 9.87; found C% 55.02, H% 5.27,
N% 9.89.
To a stirred solution of 4 (1.2 g; 3.38 mmol) in CH3CN (30 mL),
1-ethoxycarbonylpiperazine 17 (2.2 mL; 15 mmol) was added and
the mixture was stirred at r.t. for 3 h. The solvent was evaporated
under reduced pressure, the residue treated with water (40 mL)
and stirred for 20 min until a pale yellow precipitate was formed.
The solid, consisting of crude product, was collected, washed with
water, dried and then purified by flash chromatography eluting
with CH2Cl2/MeOH 5% to yield the desired product as a yellow
solid (1.27 g; 86%). The product was converted into the corre-
sponding hydrochloride by treatment with HCl–saturated MeOH,
recrystallized from MeOH/Et2O and freeze-dried to afford a yellow
4.2.14.2. 4-[(7-Chloroquinolin-4-yl)amino]-2-{[4-(pyridin-2-yl)pi-
perazin-1-yl]methyl}-phenol trihydrochloride (29). The product was
converted into the corresponding hydrochloride by treatment with
HCl–saturated MeOH, recrystallized from MeOH/Et2O and freeze-
dried to afford a yellow solid (85%). M.p.: 243.1–245.3 ꢀC. 1H NMR
(DMSO-d6): d
, 11.68 (s, br, 1H, NHþ); 11.21 (s, br, 1H, AQ-OH); 10.89
(s, br, 1H, NHþ); 8.94 (d, 1H, J ¼ 9.2 Hz, AQ-H5); 8.50 (d, 1H,
J ¼ 7.0 Hz, AQ-H2); 8.20 (d, 1H, J ¼ 2.0 Hz, AQ-H8); 8.11 (dd, 1H,
J ¼ 1.4, 5.7 Hz, Pyr-H6); 7.97 (t, 1H, Pyr-H4); 7.85 (dd, 1H, J ¼ 2.0,
solid. Mp: 242.4–244.7 ꢀC. 1H NMR (DMSO-d6):
d, 11.21 (s, br, 1H,
AQ-OH); 8.95 (d, 1H, J ¼ 9.0 Hz, AQ-H5); 8.48 (d, 1H, J ¼ 7.0 Hz. AQ-
0
H2); 8.20 (d, 1H, J ¼ 2.0 Hz, AQ-H8); 7.85 (dd, 1H, J ¼ 2.0, 9.0 Hz, AQ-
9.1 Hz, AQ-H6); 7.71 (d, 1H, J ¼ 2.5 Hz, AQ-H3 ); 7.4 (d, 2H, J ¼ 2.5,
0
0
0
H6); 7.70 (d, 1H, J ¼ 2.4 Hz, AQ-H3 ); 7.40 (dd, 1H, J ¼ 2.5, 8.7 Hz,
8.7 Hz, AQ-H5 ); 7.35 (d, 1H, Pyr-H3); 7.25 (d, 1H, J ¼ 8.7 Hz, AQ-H6 );
7.02–6.94 (m, 2H, AQ-H3, Pyr-H5); 4.36 (s, 2H, PhCH2); 3.70 (m, 2H,
CH2-Pip); 3.52 (d, 2H, CH2-Pip); 3.30 (s, 2H, CH2-Pip).
0
0
AQ-H5 ); 7.23 (d, 1H, J ¼ 8.7 Hz, AQ-H6 ); 7.01 (d, 1H, J ¼ 7.0 Hz, AQ-
H3); 4.31 (s, 2H, CH2-Ph); 4.10 (q, 2H, J ¼ 7.0 Hz, O–CH2); 3.40 (s, 8H,
4CH2-Pip); 1.21 (t, 3H, J ¼ 7.0 Hz, CH3). 13C NMR (DMSO-d6):
d
,
13C NMR (Free base) (DMSO-d6):
d,159.2; 154.3; 152.2; 149.8;
156.3; 154.9; 154.4; 143.2; 139.0; 138.4; 130.5; 128.6; 127.9; 127.3;
119.2; 116.9; 116.8; 115.7; 100.7; 66.7; 61.4; 53.3; 50.3; 14.6. MS
(CI/isobutane) (m/z): 441/443 [MHþ]. Anal. Calc. For
C23H25ClN4O3$2 HCl$3 H2O C% 48.64, H% 5.86, N% 9.86; found C%
48.69, H% 5.48, N% 9.98.
149.8; 147.9; 137.9; 134.1; 131.0; 127.9; 126.4; 125.0; 124.9; 124.6;
123.9; 118.1; 116.4; 113.4; 107.4; 100.9; 58.2; 52.4; 44.9.
MS (CI/isobutane) (m/z): 446/448 [MHþ]. Anal. Calc. For.
C25H24ClN5O$3 HCl$3.5 H2O C% 48.56, H% 5.54, N% 11.32; found C%
48.33, H% 5.14, N% 11.38.

5078
S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
4.2.14.3. 4-[(7-Chloroquinolin-4-yl)amino]-2-{[4-(pyrimidin-2-yl)pi-
perazin-1-yl]methyl}-phenol trihydrochloride (30). The product was
converted into the corresponding hydrochloride by treatment with
HCl–saturated MeOH, recrystallized from MeOH and freeze-dried
to afford a yellow solid (86%). Mp: 255.4–256.7 ꢀC. 1H NMR
Pip); 3.82 (m, 2H, CH2-Pip); 3.36 (m, 2H, CH2-Pip); 2.51 (m, 2H,
CH2-Pip). 13C NMR (DMSO-d6):
, 167.6; 162.2; 154.6; 142.9; 138.8;
d
138.1; 130.3; 129.8; 128.5; 127.8; 127.1; 126.2; 126.1; 122.1; 121.5;
118.9; 118.4; 116.8; 116.4; 115.6; 100.6; 52.9; 49.4; 44.9. MS (CI/
isobutane) (m/z): 502/504 [MHþ]. Anal. Calc. For C27H24ClN5OS$3
HCl$4 H2O C% 47.45, H% 5.16, N% 10.25; found C% 47.65, H% 4.77,
N% 10.41.
(DMSO-d6): d
, 11.52 (s, br, 1H, NHþ); 11.26 (s, br, 1H, AQ-OH); 10.9
(s, br, 1H, NHþ); 8.97 (d, 1H, J ¼ 9.1 Hz, AQ-H5); 8.46 (m, 3H, AQ-H2,
Pyrim-H4/6); 8.20 (d, 1H, J ¼ 2.0 Hz, AQ-H8); 7.82 (dd, 1H, J ¼ 2.0,
0
9.1 Hz, AQ-H6); 7.73 (d, 1H, J ¼ 2.5 Hz, AQ-H3 ); 7.39 (dd, 1H, J ¼ 2.5,
4.2.14.7. 2-{[4-(1H-Benzoimidazol-2-yl)piperazin-1-yl]methyl}-4-
[(7-Chloroquinolin-4-yl)amino]-phenol trihydrochloride (34). The
product was converted into the corresponding hydrochloride by
treatment with conc. HCl (15 mL) in MeOH (20 mL), treated with
abs. EtOH (3 ꢄ 40 mL), recrystallised from MeOH and freeze-dried
to afford a yellow solid (75%). Mp: 279.1–281 ꢀC. 1H NMR (DMSO-
0
0
8.7 Hz, AQ-H5 ); 7.25 (d, 1H, J ¼ 8.7 Hz, AQ-H6 ); 6.99 (d, 1H,
J ¼ 7.0 Hz, AQ-H3); 6.78 (t, 1H, J ¼ 4.8 Hz, Pyrim-H5); 4.68 (m, 2H,
CH2-Pip); 4.39 (s, 2H, CH2-Ph); 3-58-3.47 (m, 4H, 2CH2-Pip); 3.17
(m, 2H, CH2-Pip). 13C NMR (DMSO-d6):
d, 160.7; 158.5; 156.7; 155.1;
143.4; 139.3; 138.6; 130.9; 128.9; 128.3; 127.6; 126.9; 126.6; 119.4;
117.3; 117.1; 116.0; 115.4; 111.6; 101.1; 100.9; 98.7; 53.5; 50.6; 40.7.
MS (CI/isobutane) (m/z): 447/449 [MHþ]. Anal. Calc. For
C24H23ClN6O$3HCl$H2O C% 50.19, H% 4.91, N% 14.63; found C%
50.09, H% 5.04, N% 14.67.
d6):
d
, 11.23 (s, br, 1H, AQ-OH); 8.93 (d, 1H, J ¼ 9.1 Hz, AQ-H5); 8.54
(d, 1H, J ¼ 7.0 Hz, AQ-H2); 8.19 (d, 1H, J ¼ 2.0 Hz, AQ-H8); 7.82 (dd,
0
1H, J ¼ 2.0, 9.1 Hz, AQ-H6); 7.69 (s, 1H, AQ-H3 ); 7.46–7.38 (m, 3H,
0
0
Bzm-H4, 7, AQ-H6 ); 7.28–7.23 (m, 3H, AQ-H5 , Bzm-H5, 6); 7.0
(d, 1H, J ¼ 7.0 Hz, AQ-H3); 4.34 (s, 2H, CH2-Ph); 3.49 (m, 8H, 4 CH2-
4.2.14.4. 4-[(7-Chloroquinolin-4-yl)amino]-2-{[4-(1,3-thiazol-2-
yl)piperazin-1-yl]methyl}-phenol trihydrochloride (31). The product
was converted into the corresponding hydrochloride by treatment
with HCl–saturated MeOH, recrystallized from MeOH/Et2O and
freeze-dried to afford a yellow solid (85%). Mp: 255.5–257.3 ꢀC. 1H
Pip). 13C NMR (DMSO-d6):
d, 156.2; 154.7; 149.7; 143.0; 138.8;
138.1; 130.2; 130.1; 128.5; 127.8; 127.1; 126.0; 123.2; 118.9; 116.8;
115.5; 111.3; 111.2; 100.6; 93.2; 59.7; 53.0; 49.0; 43.9. MS
(CI/isobutane) (m/z): 485/487 [MHþ]. Anal. Calc. For.
C27H25ClN6O$3 HCl$3 H2O C% 50.01, H% 5.28, N% 12.96; found C%
50.18, H% 4.97, N% 13.06.
NMR (DMSO-d6): d
, 11.76 (s, br, 1H, NHþ); 11.25 (s, br, 1H, AQ-OH);
10.92 (s, br, 1H, NHþ); 8.96 (d, 1H, J ¼ 9.1 Hz, AQ-H5); 8.45 (d, 1H,
J ¼ 6.9 Hz, AQ-H2); 8.19 (d, 1H, J ¼ 1.2 Hz, AQ-H8); 7.8 (d, 1H,
4.2.14.8. 2-{[4-(Benzooxazol-2-yl)piperazin-1-yl]methyl}-4-[(7-
0
J ¼ 9.1 Hz, AQ-H6); 7.7 (d,1H, J ¼ 1.9 Hz, AQ-H3 ); 7.38 (dd,1H, J ¼ 1.9,
Chloroquinolin-4-yl)amino]-phenol
dihydrochloride
(35). The
0
8.7 Hz, AQ-H6 ); 7.32 (d,1H, J ¼ 3.8 Hz, Tz-H4); 7.24 (d,1H, J ¼ 8.7 Hz,
product was converted into the corresponding hydrochloride by
treatment with conc. HCl (15 mL) in MeOH (20 mL), treated with
abs. EtOH (3 ꢄ 40 mL), recrystallised from MeOH and freeze-dried
to afford a yellow solid (94%). Mp: 253.5–255.2 ꢀC. 1H NMR (DMSO-
0
AQ-H5 ); 7.05 (d, 1H, J ¼ 3.8 Hz, Tz-H5); 7.0 (d, 1H, J ¼ 6.9 Hz, AQ-H3);
4.35 (s, 2H, CH2-Ph); 4.13 (m, 2H, CH2-Pip); 3.76 (m, 2H, CH2-Pip);
3.5 (m, 2H, CH2-Pip); 3.34 (m, 2H, CH2-Pip). 13C NMR (DMSO-d6):
d,
170.2; 156.5; 154.9; 143.2; 139.1; 138.5; 134.8; 130.7; 128.9; 128.2;
127.4; 126.5; 119.3; 117.2; 116.6; 115.9; 110; 100.9; 53.1; 49.5; 48.9.
MS (CI/isobutane) (m/z): 452/454 [MHþ]. Anal. Calc. For
C23H22ClN5O$3 HCl$2 H2O C% 46.24, H% 4.89, N% 11.72; found C%
45.86, H% 4.86, N% 11.63.
d6): d
, 11.61 (s, br, 1H, NHþ); 11.20 (s, 1H, AQ-OH); 10.88 (s, br, 1H,
NH\); 8.94 (d, 1H, J ¼ 9.0 Hz, AQ-H5); 8.48 (d, 1H, J ¼ 6.9 Hz, AQ-H2);
8.20 (d, 1H, J ¼ 2.0, AQ-H8); 7.84 (d, 1H, J ¼ 2.0, 9.0 Hz, AQ-H6); 7.73
0
0
(d, 1H, J ¼ 2.4 Hz, AQ-H3 ); 7.47 (d, 1H, J ¼ 7.8 Hz, AQ-H6 ), 7.39–7.35
0
(m, 2H, Bzo-H4, 7); 7.26–7.21 (m, 2H, AQ-H5 , Bzo-H5/6); 7.10 (t, 1H,
J ¼ 7.8 Hz, Bzo-H5/6); 7.02 (d, 1H, J ¼ 6.9 Hz, AQ-H3); 4.36 (s, 2H,
PhCH2); 4.30–4.25 (m, 2H, CH2-Pip); 3.82–3.74 (m, 2H, CH2-Pip);
3.54–3.50 (m, 2H, CH2-Pip); 3.37–3.20 (m, 2H, CH2-Pip). 13C NMR
4.2.14.5. 4-[(7-Chloroquinolin-4-yl)amino]-2-{[4-(thiophen-2-yl)pi-
perazin-1-yl]methyl}-phenol (32). The product was recrystallised
from EtOH and freeze-dried to afford a yellow solid (83%). Mp:
(DMSO-d6): d, 160.9; 156.2; 154.7; 148.3; 143.1; 141.9; 138.9; 138.2;
181.9–182.7 ꢀC. 1H NMR (CDCl3 þ CD3OD):
d
, 8.31 (d, 1H, J ¼ 5.4 Hz,
130.4; 128.5; 127.8; 127.2; 126.0; 124.1; 121.2; 119.0; 116.8; 116.5;
116.0; 115.6; 109.2; 100.6; 53.0; 49.4; 42.4. Anal. Calc. For
C27H24ClN5O2$2 HCl$2 H2O C% 54.51, H% 5.08, N% 11.77; found C%
54.55, H% 4.83, N% 11.60.
AQ-H2); 8.12 (d, 1H, J ¼ 8.0 Hz, AQ-H5); 7.87 (s, 1H, AQ-H8); 7.48–
0
0
7.42 (m, 1H, AQ-H6); 7.16 (m, 1H, AQ-H6 ); 7.4 (s, 1H, AQ-H3 ); 6.90
0
(m, 1H, AQ-H3); 6.79 (m, 2H, AQ-H5 , Tph-H4); 6.68–6.64 (m, 1H,
Tph-H5); 6.20 (d, J ¼ 2.5, 1H, Tph-H3); 3.81 (s, 2H, CH2-Ph); 3.25
(s, 4H,
2
CH2-Pip); 2.78 (s, 4H,
2
CH2-Pip). 13C NMR
4.3. Dissociation constants (pKas)
(CDCl3 þ CD3OD):
d, 158.3; 155.4; 151.4; 151.0; 149.1; 138.4; 135.9;
127.3; 126.3; 126.1; 123.1; 122.5; 118.1; 117.3; 117.2; 116.8; 113.7;
107.1; 101.2; 61.5; 52.6; 52.5. MS (CI/isobutane) (m/z): 451/453
[MHþ]. Anal. Calc. For C24H23ClN4OS C% 63.92, H% 5.14, N% 12.42;
found C% 63.67, H% 5.14, N% 12.42.
The ionisation constants of compounds were determined by
potentiometric titration with the GLpKa apparatus (Sirius Analyt-
ical Instruments Ltd, Forest Row, East Sussex, UK). Apparent ion-
isation 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 40–65 wt% methanol)
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 12.2. The titra-
tions were performed under argon at 25.0 ꢃ 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–Shed-
lovsky procedure [22]. The molar % of species was calculated using
the experimental pKa values from the Henderson–Hasselbalch
equation.
4.2.14.6. 2-{[4-(Benzothiazol-2-yl)piperazin-1-yl]methyl}-4-[(7-
Chloroquinolin-4-yl)amino]-phenol
trihydrochloride
(33). The
product was converted into the corresponding hydrochloride by
treatment with HCl–saturated MeOH, recrystallised from MeOH
and freeze-dried to afford a yellow solid (86%). Mp: 250.4–
252.2 ꢀC. 1H NMR (DMSO-d6): , 1.72 (s, br, 1H, NHþ); 11.23 (s, 1H,
d
AQ-OH); 10.91 (s, br, 1H, NHþ); 8.96 (d, 1H, J ¼ 9.27 Hz, AQ-H5);
8.47 (d, 1H, J ¼ 6.9 Hz, AQ-H2); 8.21 (d, 1H, J ¼ 2.0 Hz, AQ-H8);
0
7.88–7.81 (m, 2H, AQ-H6, Bzt-H4); 7.73 (d, 1H, J ¼ 2.3 z, AQ-H3 );
0
7.55 (d, 1H, J ¼ 8.0 Hz, Bzt-H4); 7.39–7.35 (m, 2H, AQ-H5 , Bzt-H5/6);
0
7.25 (d, 1H, J ¼ 8.7 Hz, AQ-H6 ); 7.17 (t, 1H, J ¼ 8.0 Hz, Bzt-H5/6); 7.02
(d, 1H, J ¼ 7.0 Hz, AQ-H3); 4.39 (s, 2H, CH2-Ph); 4.23 (m, 2H, CH2-

S. Guglielmo et al. / European Journal of Medicinal Chemistry 44 (2009) 5071–5079
5079
4.4. Biological studies
Special Programme for Research and Training in Tropical Diseases
(TDR).
4.4.1. Leishmania donovani
Briefly, peritoneal exudate macrophages were harvested from
CD1 mice (Charles Rivers Ltd, UK), 24 h after starch induction. After
washing the macrophages were dispensed into Lab-tekÔ16-well
tissue culture slides and maintained in RPMI 1640 (Sigma,
UK) þ 10% heat-inactivated foetal calf serum (HIFCS, Harlan, UK) at
37 ꢀC, 5% CO2/air mixture for 24 h. Leishmania donovani MHOM/ET/
67/HU3 amastigotes were harvested from an infected golden
hamster spleen and were used to infect the macrophages at a ratio
of 5 parasites:1 macrophage. Infected cells were left for a further
24 h and then exposed to drug for a total of 5 days, with the overlay
being replaced on day 3 [23]. The top concentration for the test
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and then incubated for 48 h. The plates were freeze/thawed rapidly,
harvested and dried. Radioactive hypoxanthine uptake was
measured by scintillation counter. IC50 values were calculated
(Sigmoidal regression, PrismÔ).
4.4.3. Toxicity on KB cells
KB cells, derived from a human carcinoma of the nasopharynx
and typically used in assays for antineoplastic agents, were main-
tained in RPMI 1640 medium (Sigma, UK), 10% heat-inactivated
foetal calf serum, 37 ꢀC, 5% CO2. KB cell monolayers, prepared in 96-
well plates, were exposed to the test compounds for 72 h [26].
Podophyllotoxin was used as a positive control. 20 ml of Alamar
BlueÔ was added to each well. After a further 2–4 h incubation the
plates were read (Molecular Devices GeminiÔ) at EX/EX 530/580,
cut-off 550 nm. The IC50 values were calculated by sigmoidal
regression analysis (PrismÔ)
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`
This investigation received financial support from Universita
degli Studi di Torino and from the UNDP/World Bank/WHO