Clofazimine analogs with antileishmanial and antiplasmodial activity

Article abstract of DOI:10.1016/j.bmc.2014.11.028

A set of novel riminophenazine derivatives has been synthesized and evaluated for in vitro activity against chloroquine-sensitive (CQ-S) and chloroquine-resistant (CQ-R) strains of Plasmodium falciparum and against different species of Leishmania promasti

Full text of DOI:10.1016/j.bmc.2014.11.028

Bioorganic & Medicinal Chemistry 23 (2015) 55–65
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Clofazimine analogs with antileishmanial and antiplasmodial
activity
Anna Barteselli a, Manolo Casagrande a, Nicoletta Basilico b, Silvia Parapini c, Chiara M. Rusconi a,
Michele Tonelli d, Vito Boido d, Donatella Taramelli c, Fabio Sparatore d, Anna Sparatore a,
⇑
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b Dipartimento di Scienze Biomediche Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Via C. Pascal 36, 20133 Milano, Italy
c Dipartimento di Scienze Farmacologiche e Biomolecolari—DiSFeb, Università degli Studi di Milano, via C. Pascal 36, 20133 Milano, Italy
d Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of novel riminophenazine derivatives has been synthesized and evaluated for in vitro activity
against chloroquine–sensitive (CQ-S) and chloroquine–resistant (CQ-R) strains of Plasmodium falciparum
and against different species of Leishmania promastigotes. Most of the new compounds inhibited the
growth of Leishmania promastigotes as well as CQ-S and CQ-R strains of P. falciparum with IC50 in submi-
cromolar range, resulting in the best cases 1–2 orders of magnitude more potent than the parent com-
pound clofazimine.
Received 26 September 2014
Revised 17 November 2014
Accepted 19 November 2014
Available online 26 November 2014
Keywords:
Quinolizidinyl-alkyliminophenazine
derivatives
Ó 2014 Elsevier Ltd. All rights reserved.
Pyrrolizidinyl-alkyliminophenazine
derivatives
Clofazimine
Clofazimine analogs
Antiplasmodial activity
Antileishmanial activity
1. Introduction
In most of the developing countries the therapy is still based on
pentavalent antimonials as first choice drug, whereas amphoteri-
Malaria is a major life-threatening parasitic disease in humans
worldwide. Despite all the efforts and investments done over the
last decade to roll back the disease, malaria was still responsible
for 207 million clinical cases and 627,000 deaths in 2012, most
of them children living in sub-Saharan Africa.1 The emergence of
resistant strains of Plasmodium falciparum (P.f.) even to the most
recent combinations of antimalarial drugs,2 including the artemis-
inin derivatives,3 makes urgent the development of new anti-plas-
modial molecules using different strategies.
After malaria, leishmaniasis is the second most important para-
sitic infection worldwide for mortality in humans. It is transmitted
by the bite of a sand-fly infected by a flagellate protozoan of the
genus Leishmania. Three different forms of the disease are
described: visceral, cutaneous and muco-cutaneous leishmaniasis.
The disease is endemic in 89 countries, leading to 500,000 new
cases and 50,000 annual deaths, mostly due to the visceral form
caused by Leishmania donovani.4
cin B, miltefosine, paromomycine and pentamidine are considered
second-line drugs. However, all these drugs may cause several side
effects, are expensive and toxic; the treatment is prolonged, and
antimonial-resistant parasites have emerged in several endemic
areas.5
Clofazimine (Fig. 1) is a fat-soluble riminophenazine dye used in
combination with rifampicin and dapsone as multidrug therapy
(MDT) for the treatment of leprosy. It is included in the World
Health Organization’s List of Essential Medicines, a list of the most
important medications needed in any basic health system.6
Recently, the WHO guidelines included clofazimine within a group
of 5 drugs for the treatment of Multidrug Resistant Tuberculosis
(MDR-TB), especially of cases that are extensively drug-resistant
(XDR-TB).7
Consequently, several analogs of clofazimine with reduced lipo-
philicity, obtained through the introduction of a basic moiety in
the structure, have been recently synthesized and studied as
MDR-TB agents,8–10 achieving encouraging results. Clofazimine is
also described to possess antiprotozoal activity, exhibiting a mod-
erate antimalarial activity in murine models11 and antileishmanial
⇑
Corresponding author. Tel.: +39 02 50319365; fax: +39 02 50319359.
E-mail address: anna.sparatore@unimi.it (A. Sparatore).
http://dx.doi.org/10.1016/j.bmc.2014.11.028
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

56
A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
effects both in vitro and in vivo;12,13 however, only few studies are
available on this topic. Moreover, tetramethylpiperidine-substi-
tuted phenazines (Fig. 1), structurally related to clofazimine, have
been described14 to be endowed with activity against multidrug
resistant strains of P.f.
As reported by Zhang et al.9 and by Liu B. et al.22 phenylpyrid-
inyliminophenazine compounds (27–29) have been prepared in
five steps, as indicated in Scheme 3. The suitable substituted
N1-phenylbenzene-1,2-diamines, previously synthesized or com-
mercially available, were condensed with 1,5-difluoro-2,4-dinitro-
benzene in the presence of triethylamine (TEA). Nitrocompounds
(30 and 31) were reacted with pyridin-3-amine or 6-methoxypyri-
din-3-amine to afford compounds 32–34, which after reduction
with metallic zinc and acetic acid to the corresponding diamino
derivatives, underwent a spontaneous oxidative cyclization reac-
tion. The obtained imino compounds (27–29) were then reacted
with 4-(octahydro-1H-quinolizin-9a-yl)butan-1-amine or with
N1,N1-dimethylpropane-1,3-diamine to afford the final products
(18–21).
In the search for more effective alternatives to the presently
used antileishmanial drugs and with the aim to study more thor-
oughly the antimalarial potentialities of this kind of structures,
we synthesized and tested a set of novel riminophenazines bearing
a bicyclic basic head, as a quinolizidine or a pyrrolizidine, linked
through an alkyl chain to the imino nitrogen in position 3 on
the phenazine nucleus (Fig. 2, series A). The choice of this kind of
basic heads is related to our previous successful experience with
1-quinolizidinylmethyl and 7-pyrrolizidinymethyl derivatives of
7-chloro-4-aminoquinoline, named AM-1 and MG-3, respectively
(Fig. 3), which exhibited an excellent in vitro activity against chlo-
roquine-sensitive (CQ-S) and chloroquine resistant (CQ-S) strains
of P. falciparum and good in vivo efficacy in murine models of
malaria.15–18 In addition to the riminophenazine derivatives bear-
The required
x-(octahydro-2H-quinolizin-1-yl)alkanamines
were prepared as previously described by some of us23–25 and the
(hexahydro-1H-pyrrolizin-7a-yl)methanamine and 2-(hexahydro-
1H-pyrrolizin-1-yl)ethylamine have been obtained as described
by Miyano et al.26,27
ing a
x
-(quinolizidin-1
a
/1b-yl)alkylamino or a pyrrolizidinyleth-
Finally,
x-(octahydro-1H-quinolizin-9a-yl)alkan-1-amines
ylamino group on position 3, which have been previously
prepared and studied by some of us for antitubercular activity,19
we have now synthesized new achiral quinolizidine derivatives
connected to the imino group in position 3 of the phenazine
through an alkyl chain attached to the bridgehead carbon 9a, thus
avoiding any stereochemical issue. To evaluate the role of the steric
hindrance of the bicyclic basic heads, the smaller pyrrolidinyl
derivative 5 was also studied.
To complete the study and to evaluate the contribution of the
increment of polarity on the antiprotozoal activity, we also pre-
pared compounds characterized by the replacement of the aniline
moiety in position 2 with an aminopyridine (Fig. 2, series B) or by
the quaternarization of the basic nitrogen in the side chain with a
methyl group (compound 17).
were obtained in six steps, as indicated in Scheme 4. d-Valerolac-
tam was reacted firstly with sodium hydride and then with ethyl
5-bromovalerate to generate the corresponding tertiary amide
35. After treatment with soda lime and distillation, the obtained
2,3,4,6,7,8-hexahydro-1H-quinolizine was converted into the cor-
responding perchlorate 36.28,29 Compound 36 was treated with
the proper Grignard reagent to generate the olefinic quinolizidines
(37 and 38),29,30 which underwent a hydroboration reaction to
obtain the alcoholic derivatives (39 and 40).29 After a Mitsunobu
reaction in the presence of phthalimide, DEAD and triphenylphos-
phine and the following acidic hydrolysis,31 the desired (octahy-
dro-1H-quinolizin-9a-yl)alkan-1-amine dihydrochloride salts (43
and 44) were obtained.
2.2. Biological activity
2. Results and discussion
2.1. Chemistry
The 21 compounds of Figure 2 were tested in vitro against D10
(CQ-S) and W2 (CQ-R) strains of P. falciparum. Their antiplasmodial
activity was quantified as inhibition of parasite growth, measured
as the activity of parasite lactate dehydrogenase (pLDH)32 and the
Compounds of Figure 2 were synthesized by reacting, in dioxane
solution, the 2-[(4-R-phenyl)amino]-10-(4-R-phenyl)-2,10-dihy-
dro-3-iminophenazines hydrochloride (22–26) with the suitable
cyclic alkylamines, ((1S,9aR)- or (1R,9aR)-(octahydro-2H-quinoli-
zin-1yl)alkylamines, (hexahydro-1H-pyrrolizin-1-yl)alkylamines,
(octahydro-1H-quinolizin-9a-yl)alkan-1-amines and 3-(pyrroli-
din-1-yl)propylamine in analogy to the previously described
riminophenazines8,20 (Scheme 1).
The quaternary ammonium salt 17 was obtained by reacting
compound 12 with iodomethane, as shown in Scheme 2.
The required iminophenazines hydrochloride 22–26 were
prepared by oxidation of the corresponding N1-(substituted-
phenyl)benzene-1,2-diamine (in acid solution) with ferric
chloride.21
results, expressed as IC50 SD (lM), are reported in Table 1. The
ratios between the IC50 of each compound against the CQ-R and
CQ-S strains of P. falciparum are also indicated. This value
(resistance index, R.I.) is suggestive of cross resistance between
the compound and chloroquine. Cytotoxicity on a human endothe-
lial cell line (HMEC-1) was assayed using the MTT test33 and the
selectivity index (S.I.; IC50 HMEC-1/IC50 on different strains of P.
falciparum) was calculated (Table 1).
Seventeen of these compounds were also tested in vitro against
different species of Leishmania promastigotes, using the MTT
assay34,35 and the results, expressed as IC50 SD (
lM), are reported
in Table 1 together with their corresponding selectivity indexes (IC50
HMEC-1/IC50 on different species of Leishmania promastigotes).
All the tested compounds inhibited the growth of different spe-
cies of Leishmania promastigotes as well as CQ-S and CQ-R strains
of P.f. Most of them exhibited an IC50 in the nanomolar range, with
a clear improvement of potency compared to clofazimine, which
was, generally, from one to two orders of magnitude less potent.
These results confirm the importance of the introduction of a basic
head on the imino nitrogen in position 3 on the phenazine.
Concerning the antiplasmodial activity, the tested compounds
exhibited similar activity against CQ-S and CQ-R strains of P.f., with
very low R.I. (0.4–2.4), thus demonstrating that this kind of com-
pounds do not share the same resistance mechanisms of CQ.
The most active compound on both P.f. strains was the quinoliz-
idinylpropyl derivative 9, which inhibited the D10 (CQ-S) and W2
Cl
R
NH
N
N
N
N
N
N
N
H
N
H
Cl
R
Clofazimine
TMP-substituted phenazines
Figure 1. Structures of clofazimine and tetramethylpiperidine-substituted
phenazines.

A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
57
R1
R1
R2
R3
n
R3
N
N
N
n
N
N
N
N
H
R1
N
H
R2
N
R2
A
B
H2C
H2C
H2C
N
H
N
H
N
N
H2C
;
N
CH3
;
H2C
;
;
;
H2C
N
;
N(CH3)2
I
g
a
b
c
e
f
d
Series
A
R1
R2
H
R3
n
Compound
H
Cl
H
a
a
a
a
b
c
0
0
1
1
1
0
0
1
2
0
0
3
5
3
5
5
3
3
3
3
3
1
2
A
H
A
H
3
A
Cl
H
H
4
A
H
5
A
H
H
6
A
Cl
H
H
c
7
A
H
c
8
A
H
H
c
9
A
H
H
d
c
10
11
12
13
14
15
16
17
18
19
20
21
A
CH3
H
H
A
H
e
e
e
e
e
f
A
H
H
A
F
H
A
F
H
A
F
Cl
H
A
H
B
H
H
e
e
e
g
B
H
OCH3
H
B
Cl
Cl
B
H
Figure 2. Structures of the riminophenazines under investigation.
resistance index improved with the elongation of the alkyl spacer,
whereas the change of its attachment, from trans to cis, to the quin-
olizidine nucleus (compound 6 vs compound 10) produced a slight
improvement in the activity of the latter.
In the series of symmetrical quinolizidinylalkyl derivatives
(12–16), the different length of the alkyl chain (4 or 6 CH2) did
not influence the antiplasmodial activity.
Interestingly, the quaternarization of the quinolizidine nitrogen
with a methyl group (compound 17) increased significantly the
selectivity index against P.f., showing reduced toxicity against the
tested human cell line, while leaving unchanged the antiplasmo-
dial activity. Thus the effect of the quaternarization of the basic
head is surely worth of a more extensive investigation.
N
HN
N
NH
N
H
N
Cl
Cl
(_)-AM1
MG3
Figure 3. Structures of AM1 and MG3.
The introduction on the phenyl rings of a substituent like Cl, F or
CH3 did not affect significantly the antiplasmodial activity or
toxicity on the human cell line. The replacement of the aniline moi-
ety in position 2 with a differently substituted aminopyridine
(compounds of series B) slightly increased the selectivity indexes
(CQ-R) strains with IC50 = 123 and 63 nM, respectively, thus show-
ing a R.I. = 0.5 and a limited toxicity on HMEC-1 cell line with a
quite good S.I. (37 and 71, respectively).
In the series of 1-quinolizidinylalkylderivatives (compounds 6,
8 and 9, with R1 = R2 = H) the antiplasmodial activity and the

58
A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
R1
R1
R2
R2
R3
n
N
N
NH HCl
NH
N
N
N
a
N
H
R1
R2
A
R2
R1
H2C
H2C
R1=R2= H
22
23
24
25
26
R =
3
H2C
N
H C
2
N
H
N
H
N
;
;
N
;
;
H C
2
R1= F, R2= H
R1= F, R2= Cl
R1= Cl, R2= H
R1= CH3, R2= H
e
a
b
c
d
n
R2 R3
Compound
R1
0
0
1
1
1
0
0
1
2
0
0
3
5
3
5
5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
a
a
a
a
b
c
c
c
c
d
c
e
e
e
e
e
1
2
H
Cl
H
Cl
H
H
Cl
H
H
H
3
4
5
6
7
8
9
10
11
12
13
14
15
16
CH3
H
H
F
F
F
Scheme 1. Reagents and conditions: (a) R3-(CH2)n-NH2, 1,4-dioxane, reflux.
N
N
N
N
N
N
a
N
N
I
NH
NH
12
17
Scheme 2. Reagents and conditions: (a) CH3I, dry THF, rt.
on P.f. and the resistance indexes (R.I. = 0.4–0.8). Indeed com-
pounds of this series were always more active on W2 (CQ-R) strain
of P.f. than on D10 (CQ-S) strain. Finally, the nature and the size of
the basic head did not influence significantly the antiplasmodial
activity.
the substitution with halogen atoms on the phenyl rings, did not
affect significantly the antileishmanial activity whereas, when the
aniline moiety in position 2 was replaced by an aminopyridine
(compounds of series B), the presence of a chlorine atom on the phe-
nyl ring was clearly favourable (compare compound 20 with 18).
The lower activity of the 3-(quinolizidin-1-yl)propyl derivative 9
compared to compound 12 could be due to the different disposition
in the space of the basic head, which could influence the biological
activity, since in both cases five carbon atoms are interposed
between the two nitrogen atoms. Differently from antiplasmodial
activity, the quaternarization of the quinolizidine ring with a methyl
group was detrimental for the antileishmanial activity (compare
compound 17 with compound 12). The most potent compound 13
showed activity against different Leishmania promastigotes
comparable with that of the reference drug Amphotericin B and
activity against the W2 (CQ-R) strain of P.f. higher than that of
chloroquine and a selectivity index more favorable than that of
clofazimine.
Concerning the antileishmanial activity, all the 17 tested
compounds inhibited the growth of L. infantum and L. tropica
promastigotes with IC50 in the range 0.12–6.02
lM and seven of
them showed IC50 60.5 M. The different activity of these com-
l
pounds seems to depend, at least in part, on their lipophilicity.
In the series of the pyrrolizidinyl derivatives (1–4), the length of
the alkyl spacer and the presence of a chlorine substituent on the
phenyl rings clearly influenced the antileishmanial activity, with
the most lipophilic compounds (3 and 4) exhibiting the best
activity. Coherently with this, the less lipophilic pyrrolidinylethyl
derivative 5 showed the lowest activity.
9a-Quinolizidinyl derivatives (12–16) were the most active and
in this group the different length of the chain spacer, as well as

A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
59
NO2
NO2
H
H
N
N
NH2
H
N
O2N
F
NO2
NH
NO2
N
NH
NO2
+
a
b
HN
F
R1
F
R2
R1 = H, Cl
R1
R1
32
33
34
R1= H
30
R1= H, R2= H
R1= Cl, R2= H
R1= H, R2= OCH3
R1= Cl 31
R1
R1
NH2
H
N
N
N
NH
NH
N
N
NR3
NH
NH
NH2
N
d
e
c
HN
R2
B
N
N
R1
R2
R2
18
19
20
21
R1= H, R2= H, R3= a
R1= H, R2= OCH3, R3= a
R1= Cl, R2= H, R3= a
R1= Cl, R2= H, R3= b
27
28
29
R1= H, R2= H
R1= Cl, R2= H
R1= H, R2= OCH3
N
H2C
;
N
H2C
R3 =
a
b
Scheme 3. Reagents and conditions: (a) TEA, EtOH, rt; (b) 3-aminopyridine or 6-methoxypyridin-3-amine, TEA, dry THF, reflux; (c) Zn, AcOH, rt; (d) air, MeOH, rt; (e) 4-
(octahydro-1H-quinolizin-9a-yl)butan-1-amine or N1,N1-dimethylpropane-1,3-diamine, 1,4-dioxane, reflux.
O
O
COOEt
c, d
a, b
e
NH
N
N
+
BrMg
n
ClO4
35
36
O
NH2
2HCl
N
OH
n
n
n
n
f, g
h
O
i
N
N
N
N
n= 2 39
n= 4 40
n= 2 41
n= 4 42
n= 2 43
n= 4 44
n= 2 37
n= 4 38
Scheme 4. Reagents and conditions: (a) NaH 60%, dry THF from 0 °C to rt; (b) ethyl 5-bromovalerate, reflux; (c) Soda lime, distillation (160–170 °C); (d) HClO4 70%, EtOH; (e)
ZnBr2, dry THF, from 50 °C to reflux; (f) 1 N BH3 in THF, 2-methyl-2-butene, dry THF, rt; (g) H2O, 6 N NaOH, H2O2 35%, rt; h) phthalimide, Ph3P, DEAD, dry THF, rt; (i) 6 N HCl,
reflux.

Table 1
In vitro data on antileishmanial activity against different species of Leishmania promastigotes, antiplasmodial activity against D10 and W2 strains of P. falciparum and cytotoxicity on the human endothelial cell line (HMEC-1) of the
compounds under study
Compoundsa
L. infantum IC50
(l
M)
S.I.c
L. tropica IC50
(
lM)
S.I.c
L. brasiliensis IC50
(l
M)
S.I.c
D-10 (CQ-S) IC50
(l
M)
S.I.c
W-2 (CQ-R) IC50
(lM)
R.I.d
S.I.c
HMEC-1 IC50 (lM)
b
a
b
b
b
b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
n.t.
n.t.
0.91 0.26
0.30 0.03
n.t.
n.t.
n.t.
n.t.
1.59 0.94
n.t.
0.46 0.20
0.37 0.11
0.23 0.05
0.48 0.26
0.43 0.17
0.35 0.08
2.67 1.53
6.02 2.70
4.44 0.21
0.34 0.07
0.41 0.21
4.48 1.06
0.08 0.02
n.t.
—
—
2.1
3.3
—
—
—
—
2.8
—
3.4
3.3
5.4
4.1
4.3
3.9
4.1
0.6
1.4
6.9
11.4
4.2
321.2
—
9.85 0.25
7.44 1.55
1.10 0.33
0.54 0.30
12.28 5.01
n.t.
n.t.
n.t.
2.70 0.65
n.t.
0.47 0.23
0.22 0.08
0.12 0.03
0.84 0.53
0.33 0.07
0.24 0.07
1.09 0.34
2.01 0.51
2.32 0.24
0.34 0.11
0.77 0.31
2.96 1.25
0.09 0.04
n.t.
0.3
0.5
1.7
1.8
0.1
—
—
—
1.7
—
3.3
5.6
10.3
2.3
5.5
5.5
10.1
1.9
2.7
6.9
6.1
6.3
285.6
—
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
0.17 0.08
0.14 0.03
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
0.09 0.02
n.t.
—
—
—
—
—
—
—
—
—
—
—
7.2
8.9
—
—
—
—
—
—
—
—
—
285.6
—
0.64 0.16
0.62 0.07
0.34 0.02
0.19 0.04
0.18 0.04
0.37 0.10
0.45 0.14
0.18 0.05
0.12 0.03
0.14 0.07
0.21 0.04
0.21 0.03
0.17 0.05
0.24 0.02
0.28 0.08
0.31 0.06
0.19 0.03
0.28 0.05
0.81 0.25
0.22 0.03
0.40 0.03
4.82 0.82
n.t.
4.1
5.8
5.5
5.2
7.7
—
—
—
36.6
—
7.5
5.9
7.3
8.2
6.7
4.4
58.2
13.9
7.8
10.6
11.7
3.9
—
0.44 0.06
0.75 0.10
0.29 0.03
0.45 0.10
0.11 0.02
0.81 0.25
0.96 0.21
0.27 0.08
0.063 0.004
0.24 0.14
0.40 0.11
0.27 0.03
0.21 0.07
0.39 0.05
0.34 0.07
0.57 0.03
0.27 0.03
0.21 0.06
0.35 0.12
0.18 0.05
0.28 0.04
7.02 1.10
n.t.
0.7
1.2
0.9
2.4
0.6
2.2
2.1
1.5
0.5
1.7
1.9
1.3
1.2
1.6
1.2
1.9
1.4
0.8
0.4
0.8
0.7
1.5
—
6.0
4.8
6.5
2.2
12.6
—
—
—
71.4
—
3.9
4.5
5.9
5.0
5.5
2.4
41.0
18.5
18.1
12.9
16.6
2.6
—
2.65 0.41
3.57 0.82
1.90 0.37
0.98 0.10
1.39 0.34
n.t
n.t.
n.t.
4.50 0.30
n.t.
1.57 0.34
1.23 0.03
1.24 0.12
1.96 0.25
1.87 0.10
1.35 0.17
11.06 3.70
3.89 0.73
6.32 1.64
2.33 1.03
4.66 1.61
18.59e
17
18
19
20
21
Clofazimine
Amph B
CQ
25.7 1.90
>38
0.02 0.005f
>1900
0.36 0.06g
18.0
>119
n.t.: not tested.
a
Compounds were tested as corresponding hydrochlorides with the exception of compounds 6–10 and 21 which were tested as free bases and 17 which is a quaternary iodide.
The results are expressed as IC50 SD of at least three different experiments each performed in duplicate or triplicate.
Selectivity Index: IC50 HMEC/IC50 different species of Leishmania or different strains of P. falciparum.
Resistance Index: Ratio IC50 CQ-R/CQ-S strains of P. falciparum.
b
c
d
e
f
Mean of two experiments, each performed in duplicate.
Mean value from many different experiments; range: 0.014–0.030
Mean value from many different experiments; range: 0.247–0.508
l
l
M.
M.
g

A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
61
In series B, the replacement of the bulky bicyclic basic head
(quinolizidine) of compound 20 with the small dimethylamino
group (compound 21) led to a compound with somewhat reduced
activity against both the Leishmania promastigotes and P.f. strains,
but also with reduced toxicity on the endothelial cell line HMEC-1.
7.18–7.09 (m, 2H); 6.86 (s, 1H); 6.41 (d, J = 7.98 Hz, 1H); 5.28 (s,
1H); 3.20–3.09 (m, 4H); 2.69 (s, 2H); 1.97–1.67 (m, 6H); 1.65–
1.61 (m, 2H). HRMS (ESI) m/z calcd for C32H30N5Cl2 [M+H]+:
554.18783; found: 554.18695. The obtained solid was converted
into the corresponding dihydrochloride salt with 1 N HCl in etha-
nol. Mp 199.0–202.0 °C (dec).
3. Conclusions
4.2.3. 3-[4-(Octahydro-1H-quinolizin-9a-yl)butylimino]-N,5-
diphenyl-3,5-dihydrophenazin-2-amine (12)
The in vitro biological results confirm the good potential of the
iminophenazine chemical scaffold on the antiprotozoal activity
and new potent antiplasmodial (e.g. 9 and 17) or antileishmanial
(e.g. 13) compounds have been identified. They can be considered
interesting ‘lead compounds’ worthy of more investigations. Fur-
ther studies will be focused on structural optimization, to improve
efficacy and selectivity on protozoa and safety versus human cells.
The possibility to identify a single molecule targeting different spe-
cies and different genera of protozoa (Plasmodium and Leishmania)
is attractive and may lead to innovative therapies for multiple
diseases.
Solid rinsed with a mixture of diethyl ether/petroleum ether
(1:1) to give dark red crystals. Yield: 37%. Mp 157.9–158.7 °C. 1H
NMR (CDCl3) d: 7.75–7.63 (m, 4H); 7.37–7.33 (m, 6H); 7.18–7.09
(m, 3H); 6.93 (s, 1H); 6.50–6.47 (m, 1H); 5.27 (s, 1H); 3.16–3.11
(m, 2H); 2.59–2.54 (m, 2H); 2.43–2.40 (m, 2H); 1.67–1.14 (m,
18H). The exchangeable proton is not visible, probably for the
interaction with the solvent. HRMS (ESI) m/z Calcd for C37H42N5
[M+H]+: 556.34402; found: 556.34283.
The product was converted into the corresponding dihydrochlo-
ride salt with 1 N HCl in ethanol. Mp 260–261 °C (dec). 1H NMR
(DMSO-d6) d: 10.28 (br s, 1H, collapsed with D2O); 10.15–10.07
(m, 2H, collapsed with D2O); 8.18 (d, 1H, J = 8.25 Hz); 7.92–7.84
(m, 3H); 7.82–7.72 (m, 4H); 7.54–7.45 (m, 4H); 7.31 (s, 1H);
7.28–7.23 (m, 1H); 7.05 (d, 1H, J = 8.25 Hz); 5.73 (s, 1H); 3.20–
3.07 (m, 4H); 2.92–2.89 (m, 2H); 1.88–1.13 (m, 18H).
4. Experimental
4.1. General
All commercially available solvents and reagents were used
without further purification, unless otherwise stated. CC = flash
column chromatography. Mps: Büchi apparatus, uncorrected. 1H
NMR and 13C NMR spectra: Varian Mercury 300VX spectrometer;
CDCl3, DMSO-d6, or TFA-d; d in ppm, J in Hertz. High-resolution
mass spectra (HRMS) on FT-Orbitrap mass spectrometer in positive
electro spray ionization (ESI).
4.2.4. 3-[6-(Octahydro-1H-quinolizin-9a-yl)hexylimino]-N,5-
diphenyl-3,5-dihydrophenazin-2-amine (13)
Solid rinsed with petroleum ether and few drops of diethyl
ether to give dark red crystals. Yield: 52%. Mp 129.2–130.6 °C. 1H
NMR (CDCl3) d: 7.75–7.70 (m, 4H); 7.66.7.33 (m, 6H); 7.19–7.06
(m, 3H); 6.92 (s, 1H); 6.48 (d, 1H, J = 7.98 Hz); 5.28 (s, 1H); 3.15–
3.11 (m, 2H); 2.64–2.60 (m, 2H); 2.57–2.44 (m, 2H); 1.61–1.11
(m, 22H). The exchangeable proton is not visible, probably for
the interaction with the solvent. HRMS (ESI) m/z Calcd for
4.2. 3-[x-(Azacycloalkyl)alkyl]imino)-N,5-diphenyl-3,5-dihydro
phenazin-2-amine (series A: 1–16)
C
39H46N5 [M+H]+: 584.37532; found: 584.37421.
The product was converted into the corresponding dihydrochlo-
ride salt with 1 N HCl in ethanol. Mp 207.3–209.0 °C (dec). 1H NMR
(DMSO-d6) d: 10.26–10.03 (m, 3H, collapsed with D2O); 8.19 (d, 1H,
J = 8.53 Hz); 7.90–7.84 (m, 3H); 7.75–7.73 (m, 4H); 7.54–7.45 (m,
4H); 7.31–7.25 (m, 2H); 7.05 (d, 1H, J = 8.53 Hz); 5.72 (s, 1H);
4.98–3.08 (m, 4H); 2.90–2.72 (m, 2H); 1.90–1.10 (m, 22H). 13C
NMR (DMSO-d6) d: 152.2; 146.2; 141.4; 140.0; 139.3; 136.8;
135.9; 132.5; 132.0 (2C); 130.6; 130.4 (4C); 130.2; 128.3 (2C);
128.1; 125.6; 123.4 (2C); 117.8; 90.4; 63.1 (2C); 48.7; 32.9 (2C);
29.6 (2C); 27.1; 26.9; 22.9; 22.7; 22.6; 22.4; 17.9 (2C).
General procedure: Differently substituted phenazine (22–26;
1.6 mmol) and the proper amine (1.6 mmol) were dissolved in
7 ml of 1,4-dioxane and refluxed for 5 h. After cooling, the solvent
was evaporated and the obtained mixture diluted with H2O and
aqueous 6 N NaOH and extracted several times with diethyl ether.
The organic layer was dried with anhydrous Na2SO4 and evapo-
rated to dryness. The crude product was purified by CC (CH2Cl2/
MeOH/conc. NH3 in gradient) and the purified fraction was treated
with the indicated solvent. The synthesis and characterization of
compounds 3, 4 and 6–11 has been recently described by some
of us;19 compound 5 has been described by O’Sullivan et al.8
4.2.5. N,5-Bis(4-fluorophenyl)-3-[4-(octahydro-1H-quinolizin-
9a-yl)butylimino]-3,5-dihydrophenazin-2-amine (14)
Solid recrystallized with a mixture of diethyl ether/petroleum
ether (1:1) and rinsed with diethyl ether to give dark brown crys-
tals. Yield: 35%. Mp 135.3–137.0 °C. 1H NMR (CDCl3) d: 7.70 (m,
1H); 7.42–7.05 (m, 8H); 6.75 (s, 1H); 6.48 (m, 1H); 5.26 (s, 1H);
3.16 (m, 2H); 2.68–2.47 (m, 4H); 1.61–1.19 (m, 18H). The
exchangeable proton is not visible, probably for the interaction
with the solvent. HRMS (ESI) m/z Calcd for C37H40N5F2 [M+H]+:
592.32518; found: 592.32410.
The product was converted into the corresponding dihydrochlo-
ride salt with 1 N HCl in ethanol. Mp 214–215 °C (dec). 1H NMR
(DMSO-d6) d: 10.23–10.05 (m, 3H, collapsed with D2O); 8.19 (m,
1H); 7.79–7.10 (m, 12H); 5.76 (s, 1H); 3.27–2.94 (m, 6H); 1.72–
1.22 (m, 18H).
4.2.1. 3-[(Hexahydro-1H-pyrrolizin-7a-yl)methylimino]-N,5-
diphenyl-3,5-dihydrophenazin-2-amine (1)
Solid rinsed with petroleum ether to obtain brick-red crystals.
Yield: 45%. Mp 160.0–162.0 °C (dec). 1H NMR (CDCl3) d: 7.75–
7.63 (m, 4H); 7.54 (d, J = 7.7 Hz, 2H); 7.37–7.30 (m, 4H); 7.23–
7.02 (m, 4H); 6.51 (d, J = 7.7 Hz, 1H); 5.32 (s, 1H); 3.60 (s, 2H);
3.27 (s, 2H); 2.84–2.80 (m, 2H); 2.07–1.91 (m, 5H); 1.79–1.73 (m,
3H). The exchangeable proton is not visible, probably for the inter-
action with the solvent. HRMS (ESI) m/z calcd for C32H32N5 [M+H]+:
486.26577; found: 486.26483. The obtained solid was converted
into the corresponding dihydrochloride salt with 1 N HCl in etha-
nol. Mp 189.0–191.5 °C (dec).
4.2.2. N,5-Bis(4-Chlorophenyl)-3-[(hexahydro-1H-pyrrolizin-7a-
yl)methylimino]-3,5-dihydrophenazin-2-amine (2)
Solid rinsed with diethyl ether to give brick-red crystals. Yield:
11%. Mp 168.4–169.4 °C (dec). 1H NMR (CDCl3) d: 8.62 (br s, 1H,
collapsed with D2O); 7.68 (d, J = 7.7 Hz, 3H); 7.32–7.20 (m, 6H);
4.2.6. N,5-Bis(4-fluorophenyl)-3-[6-(octahydro-1H-quinolizin-
9a-yl)hexylimino]-3,5-dihydrophenazin-2-amine (15)
Solid rinsed with a mixture of diethyl ether/petroleum ether
(1:1) to give dark red crystals. Yield: 23%. Mp 128.5–130.0 °C. 1H

62
A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
NMR (CDCl3) d: 7.69 (m, 1H); 7.46–6.91 (m, 10H); 6.73 (s, 1H); 6.48
(m, 1H); 5.26 (s, 1H); 3.44–3.13 (m, 2H); 2.63–2.46 (m, 4H); 1.62–
1.12 (m, 22H). The exchangeable proton is not visible, probably for
the interaction with the solvent. HRMS (ESI) m/z Calcd for
4.4.2. N1-(4-Chlorophenyl)-N2-(5-fluoro-2,4-
dinitrophenyl)benzene-1,2-diamine (31)
Yield: 94%. Mp 211.7–214.0 °C (lit.36 209–211 °C; lit.22 216–
217 °C). 1H NMR (CDCl3) d: 9.6 (br s, 1H, collapsed with D2O);
9.14 (d, 1H, J = 7.70 Hz); 7.36–7.21 (m, 5H); 7.10–7.04 (m, 1H);
6.97.6.93 (m, 2H); 6.67–6.63 (m, 1H); 5.63 (br s, 1H, collapsed with
D2O).
C
39H44N5F2 [M+H]+: 620.35648; found: 620.35535.
The product was converted into the corresponding dihydrochlo-
ride salt with 1 N HCl in ethanol. Mp 213–214 °C (dec). 1H NMR
(DMSO-d6) d: 10.18 (br s, 1H, collapsed with D2O); 9.96 (m, 2H, col-
lapsed with D2O); 8.18 (d, 1H, J = 7.70 Hz); 7.82–7.74 (m, 6H);
7.50–7.45 (m, 2H); 7.39–7.33 (m, 2H); 7.19 (s, 1H); 7.10 (d, 1H,
J = 7.70 Hz); 5.72 (s, 1H); 3.41–3.14 (m, 4H); 3.10–2.94 (m, 2H);
1.79–1.01 (m, 22H).
4.5. N1-(Substituted phenyl)-N2-[2,4-dinitro-5-(substituted
pyridin-3-ylamino)phenyl]benzene-1,2-diamine (32–34)
General procedure: According to Zhang et al.9 and Liu et al.22
pyridine-3-amine (5.38 mmol) and TEA (4.89 mmol) were added
to a solution of the proper N1-(5-fluoro-2,4-dinitrophenyl)-N2-
((4-substitutedphenyl)benzene)-1,2-diamine 30–31 (4.89 mmol)
in 18 ml of THF and the mixture was refluxed for 20 h. The formed
precipitate was filtered and washed with THF and CH2Cl2.
4.2.7. N,5-Bis(3-chloro-4-fluorophenyl)-3-[6-(octahydro-1H-
quinolizin-9a-yl)hexylimino]-3,5-dihydrophenazin-2-amine
(16)
Solid rinsed with diethyl ether to give brick-red crystals. Yield:
25%. Mp 108.2–109.4 °C (dec). 1H NMR (DMSO-d6) d: 8.76 (br s, 1H,
collapsed with D2O); 7.99–7.88 (m, 1H); 7.85–7.82 (m, 1H); 7.63–
7.59 (m, 3H); 7.45–7.42 (m, 2H); 7.22 (m, 2H); 6.61 (s, 1H); 6.53
(m, 1H); 5.18 (s, 1H); 3.50–3.32 (m, 2H); 3.18–2.72 (m, 2H); 2.32
(m, 2H); 1.50–1.05 (m, 22H). HRMS (ESI) m/z Calcd for
4.5.1. N1-[2,4-Dinitro-5-(pyridin-3-ylamino)phenyl]-N2-phenyl
benzene-1,2-diamine (32)
Yield: 88%. Mp 213.4–215.2 °C. (Lit.9 212–213 °C). 1H NMR
(DMSO-d6) d: 9.70 (br s, 1H, collapsed with D2O); 9.48 (br s, 1H,
collapsed with D2O); 8.99 (s, 1H); 8.40–8.37 (m, 2H); 7.62–7.60
(m, 2H); 7.36–7.32 (m, 1H); 7.20–7.10 (m, 5H); 6.85–6.82 (m,
4H); 5.96 (s, 1H).
C
39H42N5Cl2F2 [M+H]+: 688.27853; found: 688.27771.
The product was converted into the corresponding dihydrochlo-
ride salt with 1 N HCl in ethanol. Mp 245–247 °C (dec). 1H NMR
(DMSO-d6) d: 10.23–9.97 (m, 3H, collapsed with D2O); 8.21–8.01
(m, 2H); 7.97 (d, 1H, J = 8.80 Hz); 7.84–7.75 (m, 3H); 7.64–7.48
(m, 3H); 7.31–7.21 (m, 1H); 7.19 (d, 1H, J = 8.80 Hz); 5.77 (s, 1H);
3.58–3.42 (m, 4H); 3.38–2.94 (m, 4H); 1.84–1.02 (m, 20H).
4.5.2. N1-(4-Chlorophenyl)-N2-[2,4-dinitro-5-(pyridin-3-ylam
ino)phenyl]benzene-1,2-diamine (33)
Yield: 94%. Mp 211.7–214.0 °C. 1H NMR (CDCl3) d: 9.72 (s, 1H,
collapsed with D2O); 9.49 (s, 1H, collapsed with D2O); 9.01 (s,
1H); 8.41 (m, 2H); 7.77 (s, 1H, collapsed with D2O); 7.38–7.15
(m, 6 H); 7.61 (m, 1H); 6.90–6.80 (m, 3H); 5.94 (s, 1H).
4.3. 5-Methyl-9a-{4-[10-phenyl-3-(phenylamino)phenazin-
2(10H)-ylideneamino]butyl}-decahydroquinolizinium iodide
(17)
4.5.3. N1-[5-(6-Methoxypyridin-3-ylamino)-2,4-dinitrophenyl]-
N2-phenylbenzene-1,2-diamine (34)
4 ll (0.05 mmol) of iodomethane were added to a solution of
Yield: 85%. Mp 189.9–191.3 °C. 1H NMR (DMSO-d6) d: 9.59 (s,
1H, collapsed with D2O); 9.43 (s, 1H, collapsed with D2O); 8.98
(s, 1H); 7.95 (d, 1H, J = 2.47 Hz); 7.61 (s, 1H); 7.51 (dd; 1H,
J = 2.47; 8.80 Hz); 7.20–7.10 (m, 5H); 6.85–6.76 (m, 5H); 5.77 (s,
1H); 3.34 (s, 3H).
30 mg (0.05 mmol) of 3-(4-(octahydro-1H-quinolizin-9a-yl)butyli-
mino)-N,5-diphenyl-3,5-dihydrophenazin-2-amine (12) in 1.5 ml
of anhydrous THF and the mixture was stirred at rt for 72 h. The
obtained precipitate was filtered and washed with a mixture of
THF/diethyl ether (1:1) to provide a dark red solid. Yield: 65%.
Mp 191.0–192.5 °C. 1H NMR (CDCl3) d: 7.77–7.69 (m, 4H); 7.36–
7.25 (m, 4H); 7.11–6.99 (m, 6H); 6.56–6.4 (m, 1H); 5.3 (s, 1H);
4.02–3.47 (m, 4H); 3.27 (s, 3H); 3.15–3.07 (m, 2H); 2.1–1.2 (m,
18H). 1H is not visible, probably for the interaction with the sol-
vent. 13C NMR (CDCl3) d: 153.2; 143.8; 140.0; 137.6; 135.3; 131.7
(4C); 130.3; 129.7 (4C); 128.8 (3C); 128.5; 128.1; 124.0; 122.1
(2C); 114.8; 89.1; 71.3 (2C); 60.9; 57.4; 49.1; 46.9; 32.8; 30.8;
27.3; 22.4; 20.3; 19.0; 17.9; 16.7. HRMS (ESI) m/z Calcd for
4.6. 3-Imino-N-(substituted pyridin-3-yl)-5-(substituted
phenyl)-3,5-dihydrophenazin-2-amine (27–29)
General procedure: According to Zhang et al.9 zinc powder
(36.16 mmol) was added portion wise into
a suspension of
the proper N1-(substituted phenyl)-N2-[2,4-dinitro-5-(substituted
pyridin-3-ylamino)phenyl]benzene-1,2-diamine 32–34 (1.81 mmol)
in 10 ml of glacial acetic acid cooled with an ice-water bath. The
mixture was stirred at rt until the color turned to light green and
then filtered and washed with glacial acetic acid and methanol.
The filtrate was concentrated and the residue was treated with
water and alkalized with conc. NH3. The formed precipitate was fil-
tered, washed with water and then dissolved in methanol. The
solution was stirred under air overnight. The formed solid was fil-
tered and washed with methanol and diethyl ether. The crude
product was directly used for the next step.
C
38H44N5 [M+H]+: 570.35967; found: 570.35858.
4.4. N1-(5-Fluoro-2,4-dinitrophenyl)-N2-(4-substitutedphenyl)
benzene-1,2-diamine (30, 31)
General procedure: 1,5-Difluoro-2,4-dinitrobenzene (10.86
mmol) and TEA (10.94 mmol) were added to a solution of the
proper
N1-((4-substituted)phenyl)benzene-1,2-diamine
(10.86 mmol) in 40 ml of ethanol. The mixture was stirred at rt
for 1.5 h. The formed suspension was filtered and the resulting
solid washed with ethanol.
4.6.1. 3-Imino-5-phenyl-N-(pyridin-3-yl)-3,5-dihydrophenazin-
2-amine (27)
4.4.1. N1-(5-Fluoro-2,4-dinitrophenyl)-N2-phenylbenzene-1,2-
diamine (30)
Yield: 84%. Mp 206.3–209.5 °C (dec). (lit.9 216–218 °C) 1H NMR
(DMSO-d6) d: 9.90 (br s, 2H, collapsed with D2O); 8.55 (s, 1H); 8.28
(d, 1H, J = 2.80 Hz); 7.80–7.63 (m, 5H); 7.57 (d, 2H, J = 7.15 Hz);
7.42–7.38 (m, 1H); 7.25–7.18 (m, 2H); 6.65 (s, 1H); 6.42 (d, 1H,
J = 8.80 Hz); 5.42 (s, 1H).
Yield: 93%. Mp 163.6–164.7 °C (lit.9 165–167 °C). 1H NMR
(CDCl3) d: 9.60 (br s, 1H, collapsed with D2O); 9.13 (d, 1H,
J = 7.70 Hz); 7.36–7.24 (m, 5H); 7.07–6.99 (m, 4H); 6.65 (d, 1H,
J = 13.20 Hz); 5.65 (br s, 1H, collapsed with D2O).

A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
63
4.6.2. 5-(4-Chlorophenyl)-3-imino-N-(pyridin-3-yl)-3,5-dihydro
phenazin-2-amine (28)
5.79–5.77 (m, 1H); 3.44–3.39 (m, 2H); 3.28–3.07 (m, 2H); 2.91–
2.87 (m, 2H); 1.91–1.01 (m, 18 H). 1H is not visible, probably for
the interaction with the solvent. HRMS (ESI) m/z Calcd for C36H40-
N6Cl [M+H]+: 591.30030; found: 591.29919.
Yield: 42%. Mp 190.0–194.0 °C. 1H NMR (DMSO-d6) d: 9.91 (br s,
2H, collapsed with D2O); 8.54 (s, 1H); 2.28 (d, 1H, J = 2.80 Hz);
7.89–7.78 (m, 3H); 7.62–7.58 (m, 3H); 7.41–7.39 (m, 1H); 7.22–
7.20 (m, 2H); 6.64 (s, 1H); 6.51–6.43 (m, 1H); 5.42 (s, 1H).
4.7.4. N1-[10-(4-Chlorophenyl)-3-(pyridin-3-ylamino)phenazin-
2(10H)-ylidene]-N3,N3-dimethylpropane-1,3-diamine (21)
The amorphous residue was purified by CC (silica gel; CH2Cl2/
MeOH in gradient). The obtained dark red oil was crystallized with
a mixture of petroleum ether/diethyl ether (95:5) to give a dark red
solid. Yield: 45%. Mp 115.5–117.5 °C. 1H NMR (DMSO-d6) d: 8.60–
8.58 (m, 1H); 8.35–8.33 (m, 1H); 7.80–7.60 (m, 4H); 7.32–7.28 (m,
3H); 7.23–7.15 (m, 2H); 6.86 (s, 1H); 6.49–6.46 (m, 1H); 5.29 (s,
1H); 3.20 (t, 2H, J = 6.83 Hz); 2.36 (t, 2H, J = 6.83 Hz); 2.22 (s, 6
H); 1.82 (q, 2H, J = 7.15 Hz). The exchangeable proton is not visible,
probably for the interaction with the solvent. HRMS (ESI) m/z Calcd
for C28H28N6Cl [M+H]+: 483.20640; found: 483.20581.
4.6.3. 3-Imino-N-(6-methoxypyridin-3-yl)-5-phenyl-3,5-dihydr
ophenazin-2-amine (29)
Yield: 49%. Mp 191.2–193.0 °C. 1H NMR (DMSO-d6) d: 9.12 (br s,
2H, collapsed with D2O); 8.15 (d, 1H, J = 2.75 Hz); 7.79–7.66 (m,
4H); 7.59–7.56 (m, 1H); 7.47 (d, 2H, J = 7.15 Hz); 7.18–7.15 (m,
2H); 6.89 (d, 1H, J = 8.53 Hz); 6.39–6.37 (m, 1H); 6.36 (s, 1H);
5.31 (s, 1H); 3.86 (s, 3H).
4.7. N-(Substituted pyridin-3-yl)-3-(aminoalkyl)-5-substituted
phenyl-3,5-dihydrophenazin-2-amine (series B: 18–21)
General procedure: The proper phenazine 27–29 (0.56 mmol)
and 4-(octahydro-1H-quinolizin-9a-yl)butan-1-amine or N0,N0-
dimethylpropane-1,3-diamine (1.12 mmol) were dissolved in
5 ml of 1,4-dioxane and refluxed for 24 h (compounds 18 and 19)
or 4 h (compounds 20 and 21). After cooling, the solvent was evap-
orated and the obtained mixture diluted with CH2Cl2 and washed
three times with water. The organic layer was dried with anhy-
drous Na2SO4 and evaporated to dryness. The crude product was
purified as indicated.
4.8. 9a-(Alkylenyl)-octahydro-1H-quinolizine (37, 38)
General procedure: A solution of 4-bromo-1-butene or 6-bromo-
1-hexene (17.3 mmol) in 5 ml of anhydrous THF was added drop-
wise to a mixture of granular magnesium (17.3 mmol) and a cata-
lytic amount of zinc bromide in 9 ml of anhydrous THF. After the
addition of a iodine crystal to trigger the reaction, the mixture
was stirred for 4 h at 50 °C under nitrogen. When the Grignard
reagent was completely formed, 30 ml of anhydrous THF and
1,2,3,4,5,6,7,8,9-octahydroquinolizinium perchlorate 3629 (8.65
mmol) were charged and the resulting mixture was refluxed for
20 h. After cooling, the mixture was diluted with 10 ml of a satu-
rated aqueous solution of NH4Cl and acidified with 6 N HCl. The
aqueous layer was separated and extracted three times with
diethyl ether, then alkalized and saturated with K2CO3 and
extracted six times with diethyl ether. The collected organic layers
were dried with anhydrous Na2SO4 and evaporated to dryness to
give an orange oil, which was directly used for the next step.
4.7.1. 3-{[4-(Octahydro-1H-quinolizin-9a-yl)butyl]imino}-5-phe
nyl-N-(pyridin-3-yl)-3,5-dihydrophenazin-2-amine dihydro
chloride (18)
The amorphous residue was digested with diethyl ether. The fil-
trate was evaporated and washed with a mixture of petroleum
ether/diethyl ether (1:1). The solid was converted into the corre-
sponding dihydrochloride salt with 1 N HCl in ethanol. Yield:
38%. Mp 218.0–219.8 °C (dec). 1H NMR (DMSO-d6) d: 10.75 (s,
1H, collapsed with D2O); 10.22 (s, 1H, collapsed with D2O); 10.05
(s, 1H, collapsed with D2O); 8.53 (d, 1H, J = 5.28 Hz); 8.35–8.26
(m, 2H); 7.93–7.67 (m, 9H); 7.12–7.10 (m, 1H); 5.76 (s, 1H);
3.19–3.06 (m, 4H); 2.91–2.88 (m, 2H); 1.89–1.56 (m, 14H); 1.22–
4.8.1. 9a-(But-3-enyl)-octahydro-1H-quinolizine (37)
Yield: 89%. 1H NMR (CDCl3) d: 5.92–5.78 (m, 1H); 5.00–4.91 (m,
2H); 2.63–2.55 (m, 2H); 2.44–2.26 (m, 2H); 1.92–1.23 (m, 13H);
1.23–1.11 (m, 3H).
1.18 (m, 4H). HRMS (ESI) m/z Calcd for
C
36H41N6 [M+H]+:
557.33927; found: 557.33838.
4.8.2. 9a-(Hex-5-enyl)-octahydro-1H-quinolizine (38)
Yield: 63%. 1H NMR (CDCl3) d: 5.92–5.78 (m, 1H); 5.02–4.91 (m,
2H); 2.63–2.55 (m, 2H); 2.44–2.30 (m, 2H); 2.18–2.01 (m, 2H);
1.92–1.11 (m, 18H).
4.7.2. N-(6-Methoxypyridin-3-yl)-3-{[4-(octahydro-1H-quino
lizin-9a-yl)butyl]imino}-5-phenyl-3,5-dihydrophenazin-2-
amine dihydrochloride (19)
The amorphous residue was purified by CC (neutral alumina,
grade IV; CH2Cl2/MeOH in gradient). The obtained solid was con-
verted into the corresponding dihydrochloride salt with 1 N HCl
in ethanol. Yield: 24%. Mp 217.6–219.0 °C (dec). 1H NMR (DMSO-
d6) d: 10.28–10.22 (m, 3H, collapsed with D2O); 8.24–8.18 (m,
2H); 7.92–7.71 (m, 6H); 7.21–6.92 (m, 5H); 5.72 (s, 1H); 3.91 (s,
3H); 3.19–2.88 (m, 6H); 1.89–1.55 (m, 14H); 1.34–1.22 (m, 4H).
HRMS (ESI) m/z Calcd for C37H43N6O [M+H]+: 587.34984; found:
587.34906.
4.9.
x-(Octahydro-1H-quinolizin-9a-yl)alkan-1-ol (39, 40)
General procedure: A solution of 9a-(alkylenyl)-octahydro-1H-
quinolizine (9.3 mmol) in 40 ml of anhydrous THF was added
dropwise during 30 minutes to an ice-cooled mixture of 1 M
borane tetrahydrofuran complex solution (37.2 mmol) and 2-
methyl-2-butene (74.5 mmol) diluted with 7 ml of anhydrous
THF. The mixture was stirred at rt for 4.5 h and then diluted with
11 ml of H2O, 37 ml of aqueous 6 N NaOH and 9.2 ml of aqueous
H2O2 (35%). The resulting mixture was stirred at rt for 1 h and
the aqueous layer was saturated with Na2CO3, separated from
the organic solution and extracted three times with diethyl ether.
The collected organic layers were evaporated and the resulting
oil was diluted with diethyl ether and extracted three times with
4 N HCl. The aqueous layer was alkalized and saturated with
Na2CO3 and extracted several times with diethyl ether. The organic
layer, dried with anhydrous Na2SO4 and evaporated to dryness,
gave a crude oil which was purified by CC (neutral alumina, grade
IV; petroleum ether/diethyl ether in gradient).
4.7.3. 5-(4-Chlorophenyl)-3-{[4-(octahydro-1H-quinolizin-9a-yl)
butyl]imino}-N-(pyridin-3-yl)-3,5-dihydrophenazin-2-amine
trihydrochloride (20)
The amorphous residue was purified by CC (silica gel; CH2Cl2/
MeOH in gradient). The obtained solid was converted into the cor-
responding trihydrochloride salt with 1 N HCl in ethanol. Yield:
86%. Mp 228.0–231.0 °C (dec). 1H NMR (DMSO-d6) d: 10.74 (s,
1H, collapsed with D2O); 10.22–10.13 (m, 2H, collapsed with
D2O); 8.83 (s, 1H); 8.53–8.51 (m, 1H); 2.27–2.24 (m, 2H); 8.05–
7.97 (m, 3H); 7.85–7.77 (m, 4H); 7.69 (s, 1H); 7.14–7.11 (m, 1H);

64
A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
4.9.1. 4-(Octahydro-1H-quinolizin-9a-yl)butan-1-ol (39)
The obtained oil crystallized spontaneously into a white solid,
which was rinsed with petroleum ether/diethyl ether (1:1). Yield:
80%. Mp 92.4–93.2 °C. 1H NMR (DMSO-d6) d: 4.34–4.31 (br s, 1H,
collapsed with D2O); 3.4–3.3 (m, 2H); 2.5–2.44 (m, 2H); 2.28–
2.24 (m, 2H); 1.52–1.32 (m, 14H); 1.12–0.96 (m, 4H).
4.12. P. falciparum cultures and drug susceptibility assay
P.f. cultures were carried out according to Trager and Jensen
with slight modifications.37 The CQ-susceptible strains 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% hypoxanthine, 20 mM Hepes, and 2 mM gluta-
mine. All the cultures were maintained at 37 °C in a standard gas
mixture consisting of 1% O2, 5% CO2, and 94% N2.
All compounds were tested as the corresponding hydrochlo-
rides with the exception of compounds 6–10 and 21, which were
tested as free bases and compound 17 which is a quaternary
iodide. Compounds were dissolved in either water 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-well flat-bottomed microplates (COSTAR)
and serial dilutions made. Asynchronous cultures with parasita-
emia of 1–1.5% and 1% final hematocrit were aliquoted into the
plates and incubated for 72 h at 37 °C. Parasite growth was deter-
mined spectrophotometrically (OD650) by measuring the activity
of the parasite lactate dehydrogenase (pLDH), according to a mod-
ified version of the method of Makler in control and drug-treated
cultures.32 The antimalarial activity is expressed as 50% inhibitory
concentrations (IC50); each IC50 value is the mean and standard
deviation of at least three separate experiments performed in
duplicate.15
4.9.2. 6-(Octahydro-1H-quinolizin-9a-yl)hexan-1-ol (40)
A light yellow oil was obtained. Yield: 74%. 1H NMR (DMSO-d6)
d: 4.32–4.29 (br s, 1H, collapsed with D2O); 3.39–3.30 (m, 3H);
2.51–2.44 (m, 3H); 2.28–2.25 (m, 2H); 1.50–1.38 (m, 16H); 1.27–
1.00 (m, 4H).
4.10. 2-[x-(Octahydro-1H-quinolizin-9a-yl)alkyl]isoindoline-
1,3-dione (41, 42)
General procedure: Diethyl azodicarboxylate (16.7 mmol) diluted
with 6.5 ml of anhydrous THF was added dropwise to an ice-cooled
mixture of 8-(octahydro-1H-quinolizin-9a-yl)alkan-1-ol 39 or 40
(8.0 mmol), phthalimide (16.0 mmol), triphenylphosphine (16.0
mmol) and 24 ml of anhydrous THF. The resulting mixture was
stirred at rt for 20 h. The solvent was evaporated and the crude
orange oil was purified by CC (silica gel; CH2Cl2/MeOH/conc. NH3
in gradient).
4.10.1. 2-[4-(Octahydro-1H-quinolizin-9a-yl)butyl]isoindoline-
1,3-dione (41)
A yellow oil was obtained. Yield: 95%. 1H NMR (CDCl3) d: 7.83–
7.71 (m, 4H); 3.69–3.47 (m, 2H); 2.58–2.42 (m, 4H); 1.59–1.17
(m, 18H).
4.13. Promastigote stage of Leishmania spp cultures and
antileishmanial activity
4.10.2. 2-[6-(Octahydro-1H-quinolizin-9a-yl)hexyl]isoindoline-
1,3-dione (42)
Promastigote stage of L. infantum strain MHOM/TN/80/IPT1
(kindly provided by Dr. M. Gramiccia and Dr. T. Di Muccio, ISS,
Roma), L. tropica (MHOM/IT/2012/ISS3130) and L. braziliensis
(MHOM/IT/2006/ISS2848) were cultured in RPMI 1640 medium
(EuroClone) supplemented with 15% heat-inactivated fetal calf
An orange oil was obtained. Yield: 98%. 1H NMR (CDCl3) d: 7.85–
7.80 (m, 2H); 7.73–7.69 (m, 2H); 3.70–3.66 (t, 2H, J = 7.42 Hz);
2.64–2.59 (m, 2H); 2.56–2.40 (m, 2H); 1.68–1.00 (m, 22H).
serum (EuroClone), 20 mM Hepes, and 2 mM -glutamine at 22 °C.
L
To estimate the 50% inhibitory concentration (IC50), the
MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bro-
mide) method34,35 was used with modifications. Compounds were
dissolved in DMSO and then diluted with medium to achieve the
required concentrations. Drugs were placed in 96 wells round-bot-
tom microplates and seven serial dilutions made. Amphotericin B
was used as the reference antileishmanial drug. Parasites were
4.11.
x-(Octahydro-1H-quinolizin-9a-yl)alkan-1-amine
dihydrochloride (43, 44)
General procedure: 2-(x-(Octahydro-1H-quinolizin-9a-yl)alkyl)
isoindoline-1,3-dione 41 or 42 (5.9 mmol) was dissolved in 20 ml
of aqueous 6 N HCl and refluxed for 20 h. The solution was stored
in the freezer overnight and the precipitated phthalic acid was fil-
tered and washed with a small amount of cold water. The filtered
aqueous solution was evaporated under vacuum and the resulting
amorphous solid was crystallized and rinsed with different
solvents.
diluted in complete medium to 5 ꢀ 106 parasites/mL and 100
ll
of the suspension was seeded into the plates, incubated at 22 °C
for 72 h and then 20
each well for 3 h. The plates were then centrifuged, the superna-
tants discarded and the resulting pellets dissolved in 100 l of lys-
ll of MTT solution (5 mg/mL) was added into
l
ing buffer consisting of 20% (w/v) of a solution of SDS (Sigma), 40%
of N,N-dimethylformamide (Merck) in H2O. The absorbance was
measured spectrophotometrically at a test wavelength of 550 nm
and a reference wavelength of 650 nm. The results are expressed
as IC50 which is the dose of compound necessary to inhibit cell
growth by 50%; each IC50 value is the mean standard deviation
of at least three separate experiments performed in duplicate.
4.11.1. 4-(Octahydro-1H-quinolizin-9a-yl)butan-1-amine
dihydrochloride (43)
Solid was crystallized with a mixture of ethanol/diethyl ether
(9.5:0.5) and rinsed with diethyl ether to give a white solid. Yield:
88%. Mp 221.0–222.7 °C. 1H NMR (DMSO-d6) d: 10.45 (br s, 1H, col-
lapsed with D2O); 8.08 (br s, 3H, collapsed with D2O); 3.10–3.02
(m, 2H); 2.89–2.78 (m, 4H); 1.95–1.03 (m, 18H).
4.14. Cytotoxicity assay
4.11.2. 6-(Octahydro-1H-quinolizin-9a-yl)hexan-1-amine
dihydrochloride (44)
The long-term human microvascular endothelial cell line
(HMEC-1) immortalized by SV 40 large T antigen38 was maintained
in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with
10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/ml of epi-
Solid was crystallized with a mixture of ethanol/diethyl ether
(1:1) and rinsed with diethyl ether to give a white solid. Yield:
59%. Mp 184–185 °C. 1H NMR (DMSO-d6) d: 10.56 (br s, 1H, col-
lapsed with D2O); 8.10 (br s, 3H, collapsed with D2O); 3.32–3.10
(m, 2H); 2.88–2.85 (m, 2H); 2.72–2.49 (m, 2H); 1.97–1.12
(m, 22H).
dermal growth factor (Chemicon), 1
2 mM glutamine, 100 U/ml of penicillin, 100
cin, and 20 mM Hepes buffer (EuroClone). Unless stated otherwise,
l
g/ml of hydrocortisone,
lg/ml of streptomy-

A. Barteselli et al. / Bioorg. Med. Chem. 23 (2015) 55–65
65
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pounds for 72 h and cell proliferation evaluated using the MTT
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is the dose of compound necessary to inhibit cell growth by 50%.
Each IC50 value is the mean and standard deviation of at least three
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part supported by Università degli Studi di Milano-‘‘Piano sviluppo
linea B’’.
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