Direct synthesis of C3-mono-functionalized oxindoles from N-unprotected 2-oxindole and their antileishmanial activity

Article abstract of DOI:10.1016/j.bmc.2013.12.039

A novel approach for the synthesis of unprecedented C3-mono-functionalized indolin-2-ones is reported, starting from 2-oxindole and chalcones The reactions proceed regioselectively under mild conditions, without di- and tri-alkylated side products The new

Full text of DOI:10.1016/j.bmc.2013.12.039

Bioorganic & Medicinal Chemistry 22 (2014) 1063–1069
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Direct synthesis of C3-mono-functionalized oxindoles from
N-unprotected 2-oxindole and their antileishmanial activity
Angela Scala a,b, Massimiliano Cordaro a, Giovanni Grassi a, Anna Piperno a, Giuseppina Barberi c,
Antonio Cascio c, Francesco Risitano a,
⇑
a Dipartimento di Scienze Chimiche, Università di Messina, V.le F.Stagno d’Alcontres 31, Messina 98166, Italy
b CNR—ISMN Istituto per lo Studio dei Materiali Nanostrutturati, UOS Palermo, c/o Dip. Scienze Chimiche, Università di Messina, V.le F.Stagno d’Alcontres 31, Messina 98166, Italy
c Dipartimento di Patologia Umana, Università di Messina, Viale Consolare Valeria 1, Messina 98125, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel approach for the synthesis of unprecedented C3-mono-functionalized indolin-2-ones is reported,
starting from 2-oxindole and chalcones. The reactions proceed regioselectively under mild conditions,
without di- and tri-alkylated side products. The new compounds have been evaluated in vitro for their
antiproliferative effects against the protozoan Leishmania infantum. Interestingly, they appear able to kill
L. infantum promastigotes and amastigotes, without significant cytotoxic effects.
Received 5 September 2013
Revised 6 December 2013
Accepted 17 December 2013
Available online 25 December 2013
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Oxindoles
Michael addition
Cyclization
Diastereoselectivity
Leishmanicidal activity
Leishmania
1. Introduction
To date, no effective vaccination is available. The choice of drugs
(pentavalent antimonials, amphotericin B, liposomal amphotericin
Leishmaniasis comprises a group of infectious diseases caused
by several species of the genus Leishmania with three different clin-
ical forms, visceral, cutaneous, and mucocutaneous. It is associated
with significant rates of morbidity and mortality in many countries
around the world and affects ca. 15 million people.1
B, paromomycin, and miltefosine) has increased in the past decade,
but current treatments are limited, have the potential to develop
resistance, are expensive, and possess unacceptable toxicity.4 Thus,
there is a great necessity to develop new drugs that are efficacious,
safe, and more accessible.
The relevance of this parasitic disease is that the incidence of
new cases is increasing daily and currently is emerging as a com-
mon and serious opportunist infection in human immunodefi-
ciency virus (HIV)-infected patients and in organ transplant
patients.2
Infection takes place when a sandfly vector inoculates Leish-
mania promastigotes (extracellular flagellated forms of the para-
site) into the mammalian skin. Once in the host tissues, parasites
are phagocytosed by mononuclear blood cells, especially macro-
phages, where they differentiate into amastigotes (obligate intra-
cellular forms of the parasite). Amastigotes proliferate inside the
macrophages before inducing the bursting of the host cell and
being released into the bloodstream. This process occurs repeat-
edly, leading to tissue damage.3
The indole ring is the most common heterocycle present in nat-
ure and several indole-based biologically active natural products
and indole-derived drugs are known. For this reason, substituted
indoles have been referred to as ‘privileged structures’ owing to
their excellent binding ability for many receptors with high
affinity.5
In particular, the 2-oxindole scaffold constitutes an important
pharmacophoric moiety, encountered in natural products and
pharmaceutical compounds, with a wide spectrum of biological ef-
fects, such as antitumor, phosphodiesterase inhibitor activity and
tyrosine kinase inhibitor activity.6–8
Thus, the search of new methods for the preparation of oxin-
dole-containing heterocycles, especially those substituted at the
C-3 position, is of considerable interest.
The selective and direct monofunctionalization at C-3 of
N-unsubstituted 2-oxindole by alkyl, alkenyl, aryl, or heteroaryl
groups is a longstanding issue.9 Indeed, the conventional methods
(reactions with alkyl halides or Michael or Mannich reactions)
⇑
Corresponding author. Fax: +39 090 393897.
E-mail address: frisitano@unime.it (F. Risitano).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.039

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A. Scala et al. / Bioorg. Med. Chem. 22 (2014) 1063–1069
suffer of some limitations, such as lack of regioselectivity and dif-
ficulty in stopping the reaction at the monosubstitution,10,11 so
that di- and tri- alkylated products are obtained, as a consequence
of the close acidity (pKa 18.5) of the protons at the C-3 and N-1
positions.9
configuration was assigned on the basis of NOE experiments. In
particular, the irradiation of Ha (dd, 3.56 ppm, the upfield reso-
nance of methylene proton) resulted in a signal enhancement for
Hb (dd, 4.0 ppm, the downfield methylene proton), for Hc (ddd,
3.98 ppm) and for aromatic protons (d, 7.95 ppm, the orto protons
of the benzoyl group; m, 7.00–7.06 ppm, oxindole protons)
(Scheme 2). Furthermore, when Hd was irradiated (d, 3.80 ppm)
a positive NOE effect was observed for Hb and for aromatic protons
(d, 6.77 ppm, orto protons of the aryl substituent; m, 7.00–
7.06 ppm, oxindole protons). These results support an anti rela-
tionship between Hd and Hc consistent with 3b0 structure
Thereby, the reduction of 3-alkylidene- or 3-arylidene-oxin-
doles, prepared by condensation of oxindoles with aldehydes or
ketones, is generally preferred, although this method suffers of
limitations, such as the competitive aldol-type side reactions for
aliphatic aldehydes.12–14 Recently, this two-step procedure has
been converted into a more useful one-pot reaction, starting from
alcohol as aldehyde-precursor, in the presence of catalytic amount
of iridium,15 ruthenium complex,16 or Nichel-Raney.17 However,
the process is expensive and requires relatively long reaction time.
In view of these observations, in the framework of our studies
dealing with the design of poly functionalized N,O-heterocy-
cles,18–27 we propose a new, convenient and straightforward
approach for the synthesis of unprecedented C3-mono-functional-
ized N-unprotected indolin-2-ones, aiming at the identification of
novel small molecules with leishmanicidal effects.
(Scheme 2), having C3-R⁄, C -R⁄ configuration.
a
However, after one hour both 1H and 13C NMR spectra of 3b0 in
CDCl3 appeared complicated by the presence of three sets of sig-
nals. In particular, three methyl groups at 2.27, 2.20 and
2.18 ppm (14:2:1 ratio) are clearly observed.
One- and two-dimensional NMR data revealed that the signals
at 2.20 ppm (s, CH3), 3.50 ppm (dd, Ha), 3.82 ppm (d, Hd),
3.96 ppm (m, Hc) and 4.20 ppm (m, Hb) can be attributed to the
diastereomer 3b000 (Scheme 2). The positive NOE signals observed
within Hd–Hc–Ha of 3b000 fully support their syn relationship. The
formation of 3b000 can be rationalized by a keto–enol tautomerism
causing racemization at C-3.
In d6-DMSO/D2O a faster equilibrium was observed as indicated
by the presence of three singlets for three methyl groups at 2.05,
2.06 and 2.15 ppm respectively, with the relative ratio 4.5:1:4.5
(3b000/3b00/3b0). One- and two-dimensional NMR data indicated that
the signals at 2.15 ppm (s, CH3), 3.15 ppm (dd, Ha), 4.02 ppm (dd,
Hb) and 5.10 (dd, Hc) can be assigned to the enol form 3b00
(Scheme 2).
Finally, we further expanded the scope of the reaction to the
synthesis of spiro-oxindoles.
The cytotoxicity and the anti-proliferative effects against Leish-
mania infantum, the main causative agent of visceral leishmaniasis
in Mediterranean region, of all synthesized compounds were eval-
uated and compared with those of amphotericin B.
2. Results and discussion
2.1. Chemistry
Moreover, a very fast equilibrium between the two keto-forms
in CDCl3 was observed for halogenated derivatives 3d and 3e, lead-
ing to a 3:2 and a 3:1 mixture respectively, within a few minutes
(see ESM for NMR spectra).
In our previous work,28 we have described the AcOH-mediated
Michael addition of 4-unsubstituted azol-5-ones to
a,b-unsatu-
rated carbonyl compounds. This procedure extended to oxindole
Then, we turned our attention to testing the reactivity of benz-
alacetone 20 (R = Me). Under similar reaction conditions, the reac-
tion did not yield the expected Michael adduct.
1 as pronucleophilic agent and chalcone 2 (R = Ph) as selected
a,b-unsaturated carbonyl compound did not give the expected Mi-
chael adduct. Therefore, the development of an alternative method
applicable to this substrate has been studied. In particular, in
refluxing EtOH and in the presence of piperidine and 4 Å molecular
sieves, the reaction of oxindole 1 and chalcones 2a–e (R = Ph)
yielded the Michael adducts 3a–e (Scheme 1).
It was found that all chalcones 2a–e can tolerate the reaction
conditions with different yields (Scheme 1), in particular rising
from those m-substituted up to be excellent for p-substituted. Con-
versely, the o-substituted chalcones did not react.
In all cases, any bi- or tri-substituted adducts were isolated, nei-
ther intramolecular ring closure products, although the adduct 3
seem to possess the appropriate functionalities for the synthesis
of fused ring system.29
Piperidine was found the best choice, while the use of DBU pro-
vided an uninteresting mixture of di- and tri-alkylated derivatives.
The structure of new compounds 3a–e was determined on the
basis of analytical and spectroscopic data.
Taking into account the experimental results, we suggest the
mechanism summarized in Scheme 3. The 1,4-addition of piperi-
dine to the
a
,b-unsaturated carbonyl compound30 leads to the
Mannich base 4 which could evolve according to two different
pathways. For chalcone 2 (R = Ph), the intermediate 4 is inter-
cepted by the oxindole 1, undergoing a rapid proton exchange
and a slow C-alkylation through SN2, leading to the expected ad-
duct 3. This proposed mechanism could explain the unsuccessful
reaction with the sterically hindered o-substituted chalcone and
the absence of bis-adducts as final products, because of the steric
hindrance of the chiral carbon at C-3.
Conversely, for benzalacetone 20 (R = Me), the intermediate 4 is
preferentially intercepted by the piperidine and converted into the
iminium ion 5. The latter readily stabilizes, providing the dien-
amine 6, after removal of the piperidinium ion 7. Obviously, the
electron-rich dienamine 6 is unable to be intercepted by the pro-
nucleophile oxindole 1 (Scheme 3).
Interestingly, this reaction creates two new stereocenters, but
the 1H NMR spectrum in CDCl3 of 3b, selected as model compound,
indicated the presence of only one diastereomer, and the relative
Based on recent reports,31 we further expanded the scope of the
reaction to dibenzalacetones 8 (Scheme 4), with the aim to
Scheme 1. Synthesis of compounds 3a–e and yields.
Scheme 2. Keto–enol tautomerism of compounds 3.

A. Scala et al. / Bioorg. Med. Chem. 22 (2014) 1063–1069
1065
Scheme 3. Proposed mechanism.
amastigotes.38 All tests were performed in triplicate and ampho-
tericin B was used as reference drug.39
Compounds 3a–e and 9a–d exhibit a good activity and toxicity
profile with respect to the precursors oxindole 1 and chalcone 2
(Tables 1 and 2). In particular, oxindole 1 results inactive at all
tested concentrations, while chalcone 2 show a good EC50/24 h va-
lue (5.7
significant toxicity (TC50/24 h: 28.1
After 24 h of incubation, 3a and 9b exhibit a EC50 value of
16.2 g/mL and 6.1 g/mL, respectively, with a quite high TC50
(71.6 g/mL and 62.8 g/mL, respectively) and a good TI (4.4 and
10.2, respectively).
After 48 h of incubation, 9b and 3a show high TI values for both
promastigotes (18.7 and 10.6, respectively) and amastigotes (20.3
and 11.2, respectively).
Interestingly, 3e presents a quite good therapeutic index TI for
both promastigotes and amastigotes (6.1 and 6.3, respectively)
after 24 h of incubation, suggesting that the presence of an elec-
tron-withdrawing group (Cl) at the meta position of the phenyl
ring maintains the biological activity, reducing the toxicity. Con-
versely, the presence of an electron-donor group in 3b and 3c
(Me, OMe) results in a decrease of antileishmanial activity.
A significant improvement of antiprotozoal activity has been
observed for compounds 9a–d comparing with 3a–e. Indeed, all
spiro[cyclohexanone-oxindoles] 9a–d are able to kill more than
74% of L. infantum promastigotes after 48 h of incubation, at all
tested concentrations (data not shown). In particular, 9b, although
less active than commercial drug Am B, results the most interest-
ing compound, with a TI more than two times higher than Am B,
for both promastigotes and amastigotes.
l
g/mL for promastigotes; 5.1
l
g/mL for amastigotes), but a
Scheme 4. Synthesis of compounds 9a–d and yields.
lg/mL; TC50/48 h: 30.7
lg/mL).
l
l
l
l
promote a double conjugate addition leading to spirocyclic com-
pounds, which are intriguing combinations of multistereogenic
cyclohexanone and oxindole motifs, with promising therapeutic
perspectives.32 Indeed, the spiro-oxindole is a privileged heterocy-
clic ring system, which constitutes the core of pharmaceutically
relevant natural and unnatural products, exhibiting a wide spec-
trum of bioactivities such as antitumor, antimycobacterial, antimi-
crobial and antitubercular properties.33
Initially, the above described procedure has been applied to the
reaction of oxindole 1 and dibenzalacetone 8, but in refluxing EtOH
and in the presence of piperidine and 4 Å molecular sieves, the
reaction did not yield the expected spirans. In this case, DBU was
found the best base-catalyst, in agreement with the literature,34
confirming the role of piperidine as nucleophile in the reaction of
chalcone 2, not as base catalyst.
Our strategy is outlined in Scheme 4. Oxindole 1 reacted with
dibenzalacetones 8a–d in CH2Cl2 under reflux conditions, affording
the spiro[cyclohexanone-oxindoles] 9a–d in good yields (60–84%)
and complete diastereoselectivity.
The proposed reaction mechanism consists in a double Michael
addition of an enolate to the dibenzalacetone 8, leading to 9 in a
diastereoselective fashion.
The structure of compounds 9a–d has been determined on the
basis of analytical and spectroscopic data and by comparison with
authentic samples.32 According to literature data,33 only the more
stable trans isomer has been detected in the crude reaction
mixture.
It is noteworthy that several advantages have been achieved
with respect to previous methods,32–36 such as quicker reaction
time, mild conditions, and simple workup. Furthermore, the
protection of the oxindole-NH37 and the use of additives32–36 are
not required. However, the reaction proceeds with high
diastereoselectivity.
3. Conclusions
In summary, an efficient approach for the synthesis of
C3-mono-functionalized N-unprotected oxindoles has been
achieved. The procedure offers several advantages, including mild
conditions, high product yield, and simple workup, and provides
straightforward access to important synthetic intermediates. Fur-
thermore, spiro[cyclohexanone-oxindoles] have been synthesized
in a more convenient fashion with respect to previous reports.
The obtained compounds were evaluated in vitro for leishmanicid-
al activity against L. infantum. The results showed for both series 3
and 9 lack of cytotoxicity and significant dose-dependent antipro-
liferative activity. The overall activity profile demonstrated that
the modification of the oxindole nucleus is a convenient way for
the identification of new antiparasitic drugs. Further studies are
currently in progress to explore the mechanism of action and more
extensive details will be reported in future publications.
2.2. Biology
All new synthesized compounds were evaluated in vitro for
antileishmanial activity. The screening for leishmanicidal com-
pounds was carried out with Leishmania promastigotes and axenic

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A. Scala et al. / Bioorg. Med. Chem. 22 (2014) 1063–1069
Table 1
Toxic concentration on RAW 264.7 cells, antileishmanial activity (vs promastigotes and axenic amastigotes) and therapeutic index of the tested compounds after 24 h of
incubation
RAW 264.7
Promastigotes
Amastigotes
TC50 SD (lg/mL)
EC50 SD (
lg/mL)
EC95 SD (
lg/mL)
TI (TC50/EC50
)
EC50 SD (lg/mL)
EC95 SD (lg/mL)
TI (TC50/EC50)
3a
3b
3c
3d
3e
9a
9b
9c
9d
1
71.6 4.5
93.3 6.4
41 2.3
36.9 3.3
193.1 12.4
88.8 7.3
62.8 3.5
80.5 6.4
44 3.5
16.2 3.5
62.0 2.7
29.7 0.3
19.7 1.1
31.6 0.3
29.8 2.2
6.1 1.4
23.9 2.2
23.4 1.3
232.4 19
5.7 1.2
77.8 6.7
110.9 4.6
78.5 1.2
76.5 1.3
85.5 0.5
86.1 2.6
72.1 0.3
82.4 3.7
4.4
1.5
1.3
1.8
6.1
2.9
10.2
3.3
1.8
0.5
15.3 2.1
60.1 1.8
24.2 0.2
18.5 0.9
30.5 0.2
28.5 1.7
5.9 1.2
21 1.6
75.6 4.2
99.5 3.4
75.4 0.9
74 1.1
80.2 0.3
78.3 1.5
69.4 0.2
79.3 2.5
75.2 0.7
4.7
1.6
1.7
2
6.3
3.1
10.6
3.8
2.1
0.6
5.5
39.1
77.1
1
21 1.1
125.8 11.3
28.1 2.3
19.6 1.4
447.4 36
83.5 1.3
3.9 0.02
199 11
5.1 0.9
0.5 0.05
430.5 15.5
79.5 1.1
3.9 0.02
2
AmB
4.9
35.1
0.55 0.08
Table 2
Toxic concentration on RAW 264.7 cells, antileishmanial activity (vs promastigotes and axenic amastigotes) and therapeutic index of the tested compounds after 48 h of
incubation
RAW 264.7
Promastigotes
Amastigotes
TC50 SD (
lg/mL)
EC50 SD (
6.3 1.2
lg/mL)
EC95 SD (
lg/mL)
TI (TC50/EC50
)
EC50 SD (
lg/mL)
EC95 SD (
lg/mL)
TI (TC50/EC50)
3a
3b
3c
3d
3e
9a
9b
9c
9d
1
66.6 5.4
67.6 4.1
35.4 4.2
72.1 0.3
96 1.6
74 0.4
73.6 0.4
74 0.7
73.2 0.9
71.4 0.7
75.2 0.2
72.1 0.8
10.6
1.3
2.5
3
8.8
9.4
18.7
3.1
4.2
2.1
20.1
8.2
5.9 1.1
69.4 0.2
94.3 1.1
71.2 0.3
70.2 0.2
71.5 0.5
74.1 0.7
69.9 0.5
74.8 0.4
69.9 0.7
11.2
1.3
2.7
3.5
9.3
11.4
20.3
3.4
4.4
2.2
51.6
1
48.4
1
14.2 1.1
11.5 1.1
13.2 1.3
6.9 1.7
13.1 1.1
10.1 0.9
12.5 1.1
5.7 0.9
1.9 0.8
35
116.3 9.9
65.2
5
7
38.6 4.7
51 5.9
21.3 1.9
154.5 20.6
30.7 3.4
3.1 0.2
2
0.9
16.2 1.2
5.1 1.7
15.1
1
4.9 0.9
69.5 4.1
1.3 0.2
72.4
6
146.4 13.5
71.1 0.1
3.9 0.03
135.5 12.1
69.9 0.7
3.4 0.02
2
AmB
1.5 0.3
0.4 0.07
23.7
8.2
0.4 0.07
1704, 1682; 1H NMR (500 MHz, CDCl3): d = 3.58 (dd, J = 6.6,
16.3 Hz, 1H), 3.81 (d, J = 7.1 Hz, 1H), 3.96–3.99 (m, 1H), 3.99–4.04
(m, 1H), 6.70 (t, J = 8.4 Hz, 2H Ar), 6.87 (t, J = 7.5 Hz, 1H Ar),
7.14–7.21 (m, 6H, Ar), 7.44 (t, J = 7.0 Hz, 2H Ar), 7.55 (t, J = 7.0 Hz,
1H Ar), 7.94 (d, J = 7.0 Hz, 2H Ar); 13C NMR (125 MHz, CDCl3):
d = 41.9, 42.7, 49.7, 109.4, 122.0, 125.4, 127.1, 127.9, 128.0, 128.1,
128.2, 128.3, 128.5, 128.6, 133.1, 137.0, 141.0, 178.2, 198.3; ESI-
MS (m/z) = 342.14 [M+1]+; Anal. Calcd for C23H19NO2: C, 80.95; H,
5.68; N, 4.20.
4. Experimental
4.1. General
Melting points were determined on a Kofler melting apparatus
and are uncorrected. IR spectra were recorded in Nujol with a Nico-
let Impact 410D spectrometer. 1H and 13C NMR spectra were ob-
tained on a Varian 500 MHz spectrometer. The chemical shifts (d)
and coupling constants (J) are expressed in ppm and hertz respec-
tively. Microanalyses and mass spectrometry analyses were carried
out on a Carlo Erba EA 1102 and on a 3200 QTRAP (Applied Biosys-
tem SCIEX) respectively. All solvents and reagents were obtained
from commercial sources and purified before use if necessary.
Merck Kieselgel 60F254 plates were used for TLC, and Merck Silica
gel 60 (0.063–0.100 mm) for column chromatography.
4.1.1.2. (C3-R⁄,C
lin-2-one (3b).
a
-R⁄)-3-(3-oxo-3-phenyl-1-p-tolylpropyl)indo-
White solid; yield: 77%; mp: 158–160 °C; IR
(Nujol): 1702, 1677; 1H NMR (500 MHz, CDCl3): d = 2.28 (s, 3H),
3.56 (dd, J = 6.1, 15.5 Hz, 1H), 3.80 (d, J = 6.6 Hz, 1H), 3.98 (ddd,
J = 6.1, 6.6, 7.0 Hz, 1H), 4.0 (dd, J = 7.0, 15.5 Hz 1H), 6.73 (d,
J = 8.0 Hz, 1H Ar), 6.77 (d, J = 7.1 Hz, 1H Ar), 6.89 (t, J = 7.5 Hz, 1H
Ar), 7.0–7.06 (m, 4H Ar), 7.16 (t, J = 8.0 Hz, 1H Ar), 7.45 (t,
J = 8.4 Hz, 2H Ar), 7.54 (t, J = 8.4 Hz, 1H Ar), 7.95 (d, J = 8.4 Hz, 2H
Ar); 13C NMR (125 MHz, CDCl3): d = 21.0, 42.0, 42.4, 49.8, 109.4,
121.9, 125.4, 128.1, 128.2, 128.6, 129.0, 133.0, 136.6, 137.0,
137.9, 141.4, 178.1, 198.2; ESI-MS (m/z) = 356.14 [M+1]+; Anal.
Calcd for C24H21NO2: C, 81.15; H, 5.99; N, 3.98.
4.1.1. Typical procedure for the preparation of compounds 3a–e
To a stirred solution of 1 (0.5 g, 7.5 mmol) in abs EtOH (20 mL)
chalcone 2a–e (7.5 mmol) and piperidine (7.5 mmol) were added.
The mixture was stirred and heated at reflux for 2 h. After cooling,
the reaction mixture was evaporated, affording compounds 3a–e.
For compound 3a, the resulting residue was washed with cool
H2O (20 mL) and the aqueous suspension was then extracted with
Et2O (3 ꢀ 30 mL). The combined organic layers were dried over
anhydrous Na2SO4, filtered and evaporated under reduced pres-
sure. The residue was recrystallized from MeOH leading to 3a as
white crystals.
4.1.1.3. (C3-R⁄,C
propyl)indolin-2-one (3c).
a
-R⁄)-3-(1-(4-methoxyphenyl)-3-oxo-3-phenyl-
white solid; yield: 72%; mp: 145–
146 °C; IR (Nujol): 1703, 1678; 1H NMR (500 MHz, CDCl3): d = 3.57
(dd, J = 6.5, 16.2 Hz, 1H), 3.75 (s, 3H), 3.78 (dd, J = 6.9 Hz, 1H), 3.94–
3.97 (m, 2H), 6.73 (d, J = 8.8 Hz, 2H Ar), 6.80 (d, J = 7.3 Hz, 1H Ar),
6.90 (t, J = 7.2 Hz, 1H Ar), 7.06 (d, J = 8.8 Hz, 2H Ar), 7.16 (t,
J = 7.2 Hz, 1H Ar), 7.43–7.46 (m, 3H Ar), 7.55 (t, J = 7.3 Hz, 1H Ar),
4.1.1.1.
(3a).
(C3-R⁄,C
a
-R⁄)-3-(1,3-diphenylpropyl)indolin-2-one
White solid; yield: 80%; mp: 150–152 °C; IR (Nujol):

A. Scala et al. / Bioorg. Med. Chem. 22 (2014) 1063–1069
1067
7.95 (d, J = 7.3 Hz, 2H Ar); 13C NMR (125 MHz, CDCl3): d = 42.0,
42.1, 50.0, 55.1, 109.4, 113.6, 122.0, 125.4, 128.0, 128.1, 128.2,
128.6, 129.5, 132.9, 133.1, 137.0, 178.3, 198.2; ESI-MS (m/
z) = 372.16 [M+1]+; Anal. Calcd for C24H21NO3: C, 77.69; H, 5.75;
N, 3.71.
4.1.3.1. (2S,6S)-2,6-Diphenylspiro[cyclohexane-1,30-indoline]-
20,4-dione (9a). Lit.32
4.1.3.2.
20,4-dione (9b).
(2S,6S)-2,6-Dip-tolylspiro[cyclohexane-1,30-indoline]-
Lit.32
4.1.1.4. 3-(1-(4-Chlorophenyl)-3-oxo-3-phenylpropyl)indolin-2-
4.1.3.3.
(2S,6S)-2,6-Bis(4-methoxyphenyl)spiro[cyclohexane-
Lit.32
Inseparable 3:2 mixture of (C3-R⁄,C -R⁄) and (C3-
a
1,30-indoline]-20,4-dione (9c).
one (3d).
S⁄,C -R⁄) white solid; yield: 65%; mp: 160–162 °C; IR (Nujol):
1704, 1675; (C3-R⁄,C -R⁄) isomer 1H NMR (500 MHz, CDCl3):
a
a
4.1.3.4. (2S,6S)-2,6-Bis(4-chlorophenyl)spiro[cyclohexane-1,30-
indoline]-20,4-dione (9d). Lit.32
d = 3.60 (m, 1H), 3.83 (d, J = 0.4 Hz, 1H), 4.05 (dd, J = 7.0,
14.5 Hz, 1H), 4.19–4.23 (m, 1H), 6.64 (d, J = 8.0 Hz, 1H Ar), 6.87
(s, 1H Ar), 7.02 (t, J = 7.9 Hz, 1H Ar), 7.04–7.18 (m, 5H Ar),
7.44–7.46 (m, 3H Ar), 7.47–7.57 (m, 1H Ar), 8.03 (d, J = 7.1 Hz,
4.2. Biological materials and methods
1H Ar); (C3-R⁄,C -R⁄) isomer 13C NMR (125 MHz, CDCl3):
a
4.2.1. Activity assay against promastigotes and axenic
amastigotes of L. infantum
d = 41.6, 42.0, 49.8, 109.3, 122.4, 124.5, 127.9, 128.0, 128.2,
128.6, 129.7, 133.2, 133.3, 137.0, 140.8, 178.0, 198.5; (C3-
The in vitro efficacies of the synthesized compounds were per-
formed on promastigotes and on axenic amastigotes of L. infantum
(zymodeme MON-1). Promastigotes were cultured at 26 °C in
Schneider’s Drosophila Medium supplemented with 10% (v/v)
heat-inactivated fetal calf serum (FCS) at pH 7.2. Parasites were
harvested from the medium, in the logarithmic phase; were
counted in a Neubauer’s chamber and adjusted to a concentration
of 5 ꢀ 106 parasites/mLꢁ1, for the drug assay.40–43
S⁄,C -R⁄) isomer 1H NMR (500 MHz, CDCl3): d = 3.58 (dd, J = 7.4,
a
7.3 Hz, 1H), 3.79 (d, J = 7.0 Hz, 1H), 4.19–4.23 (m, 2H), 6.75 (d,
J = 7.5 Hz, 1H Ar), 6.85 (s, 1H Ar), 6.93 (t, J = 7.3 Hz, 1H Ar),
7.04–7.18 (m, 5H Ar), 7.33 (d, J = 7.5 Hz, 1H Ar), 7.44–7.46 (m,
2H Ar), 7.47–7.57 (m, 1H Ar), 7.93 (d, J = 7.0 Hz, 1H Ar); (C3-
S⁄,C -R⁄) isomer 13C NMR (125 MHz, CDCl3): d = 39.7, 41.3,
a
49.8, 109.6, 122.1, 125.2, 127.4, 128.0, 128.4, 128.7, 129.9,
132.5, 137.0, 138.0, 140.8, 177.8, 198.0; ESI-MS (m/z) = 376.16
[M+1]+; Anal. Calcd for C23H18ClNO2: C, 73.55; H, 4.88; Cl,
9.47; N, 3.74.
Axenic amastigotes were grown at 37 °C with 5% CO2 in a cell
free medium called MAA/20 (medium for axenically grown
amastigotes).44 MAA/20 consisted of modified medium 199 (Gibco)
with Hank’s balanced salt solution supplemented with 0.5% trypto-
casein (Oxoid), 15 mM d8-glucose (Sigma), 5 mM glutamine (Gib-
co), 4 mM NaHCO3 (Sigma), 0.023 mM bovine hemin (Fluka), and
25 mM HEPES to a final pH of 6.5 and supplemented with 20% heat
inactivated fetal calf serum FBS (Gibco). The growth of parasites
was determined by Image stream multispectral imaging flow
cytometer.
4.1.1.5. 3-(1-(3-Chlorophenyl)-3-oxo-3-phenylpropyl)indolin-2-
one (3e).
S⁄,C -R⁄); white solid; yield: 37%; mp: 145–146 °C; IR (Nujol):
1704, 1675; (C3-R⁄,C -R⁄) isomer 1H NMR (500 MHz, CDCl3):
Inseparable 3:1 mixture of (C3-R⁄,C -R⁄) and (C3-
a
a
a
d = 3.54–3.58 (m, 1H), 3.85 (d, J = 0.2 Hz, 1H), 3.94–3.98 (m, 1H),
4.15–4.17 (m, 1H), 6.67 (d, J = 7.5 Hz, 1H Ar), 7.01–7.19 (m, 5H
Ar), 7.29 (d, J = 7.1 Hz, 1H Ar), 7.41–7.48 (m, 4H Ar), 7.53–7.58
Tested compounds, solubilized in DMSO (the highest concentra-
tion used was 1% v/v, not hazardous to the parasites), were added
to the above parasite culture for screening, using a 96-well micro-
(m, 1H Ar), 8.02 (d, J = 7.5 Hz, 1H Ar), 8.03 (br s, NH); (C3-R⁄,C
a-
R⁄) isomer 13C NMR (125 MHz, CDCl3): d = 41.5, 42.3, 49.9, 109.6,
122.4, 124.6, 126.6, 127.1, 127.7, 128.1, 128.2, 128.4, 128.6,
titre plate, in a concentration range of 100–10
or axenic amastigote cultures were distributed in 96-well flat-
bottom microplates (195 L/well); 5 L of each concentration of
products were added to the culture in a range from 10 to 100 g/
mL, with a final volume of 200 L/well and incubated at 26 °C or
lg/mL. Promastigote
129.5, 133.2, 133.9, 136.9, 141.1, 141.8, 178.5, 197.6; (C3-S⁄,C
a-
R⁄) isomer 1H NMR (500 MHz, CDCl3): d = 3.54–3.58 (m, 1H), 3.80
(d, J = 0.4 Hz, 1H), 4.15–4.17 (m, 2H), 6.68 (d, J = 7.0 Hz, 1H Ar),
6.91 (t, J = 7.5 Hz, 1H Ar), 7.01–7.19 (m, 6H Ar), 7.41–7.48 (m, 4H
l
l
l
l
Ar), 7.93 (d, J = 7.5 Hz, 1H Ar), 8.42 (br s, NH); (C3-S⁄,C -R⁄) isomer
a
37 °C with 5% CO2, respectively for promastigotes and axenic
amastigotes, for 24/48 h.
13C NMR (125 MHz, CDCl3): d = 39.5, 41.6, 49.8, 109.8, 122.1, 125.1,
126.9, 127.3, 127.6, 128.0, 128.4, 128.5, 129.3, 129.5, 133.2, 134.1,
136.8, 141.6, 143.2, 178.3, 198.4; ESI-MS (m/z) = 376.18 [M+1]+;
Anal. Calcd for C23H18ClNO2: C, 73.56; H, 4.89; Cl, 9.44; N, 3.76.
After 24/48 h of incubation at specific temperatures (26 °C for
promastigotes and 37 °C with 5% CO2 for axenic amastigotes), par-
asite’s survival was evaluated. The number of surviving promastig-
otes was counted in Neubauer’s chamber and compared to the
controls (parasites without the drugs).
The viability of axenic amastigotes was evaluated by Image-
Stream Multispectral Imaging Cytometer (Ideas v4.0, Amnis Corp.,
Seattle, WA) using the IDEAS data analysis software (Inspire v4.0,
Amnis Corp.).
4.1.2. Detection of compound 3b00
Five milligrams of compound 3b was dissolved in 0.7 mL of d6-
DMSO. The 1H NMR spectra were recorded at different times
(1 min, 10 min). One drop of D2O was then added and the relative
1H NMR spectrum was registered (see ESM).
Axenic amastigotes treated and untreated (control) were
stained with molecular probe TO-PRO-3 (Invitrogen) for nucleic
acid labeling (absorbance at 642 nm and emission at 661 nm),
maintained for 10 min at room temperature before analyses.
Conventional flow cytometric methods do not provide direct
morphologic evidence of cell death. Imagestream system, with its
technology can provide the statistical power offered by flow
cytometry, coupled with the critical assessment capabilities associ-
ated with microscopic analysis.45
4.1.3. Typical procedure for the preparation of compounds 9a–d
To a stirred solution of 1 (0.5 g, 7.5 mmol) in CH2Cl2 (20 mL)
dibenzalacetone 8a–d (7.5 mmol) and DBU (7.5 mmol) were
added. The mixture was stirred and heated at reflux for 24 h.
After cooling, the reaction mixture was evaporated. A 10% solu-
tion of HCl and 5 mL of MeOH were added to the residue and
the product was then extracted with Et2O (3 ꢀ 30 mL). The com-
bined organic layers were dried over anhydrous Na2SO4, filtered
and evaporated under reduced pressure, leading to compounds
9a–d.
Briefly, images acquired on the Imagestream imaging cytometer
(at least 10,000 events) were collected for each sample. Labeled
parasites were excited using a 642 nm laser. Brightfield, side

1068
A. Scala et al. / Bioorg. Med. Chem. 22 (2014) 1063–1069
each well and incubated for 4 h at 37 °C, followed by low centrifu-
gation at 400 rpm for 10 min. Then, the 200 L of supernatant
culture medium were carefully aspirated and the reaction was
interrupted with 100 L of DMSO to dissolve the formation of
crystals. The culture plate was placed on a microplate reader and
the absorbance was measured at 540 nm.38,40,41 The amount of
color produced is directly proportional to the number of viable cell.
All assays were performed in triplicate. Cell viability rate was
calculated as the percentage of MTT absorption as follows:
A
58
l
BrightField
BrightField
SSC
SSC
Topro3
Topro3
l
B
215
%survival = (mean
experimental
absorbance/mean
control
absorbance) ꢀ 100.
Figure 1. ImageStream multispectra analysis of axenic amastigotes. Before acqui-
sition, amastigotes were labeled with ToPro-3, allowing differentiation of viable (A)
from non-viable parasites (B).
Toxicity of the compounds were assessed by their half maximal
toxic concentration (TC50) (dosage causing 50% macrophage death)
values at 24 and 48 h in comparison to identical cultures without
the compound.
scatter, fluorescent parasite image were acquired at 40ꢀ magnifi-
cation (Fig. 1).
Post-acquisition data analysis was performed using IDEAS im-
age analysis software package, analyzing the morphological char-
acteristics, size, and vitality (Fig. 2).46
Antileishmanial activity of the compounds were estimated by
their EC50 and EC95 values (dosage causing 50% and 95% parasites
death, respectively) at 24 and 48 h in comparison to identical cul-
tures without the compounds.
An Excel add-in ED50V10 was used for calculating EC50, EC95
and TC50. Therapeutic index (TI) (TC50/EC50) was computed in order
to find compounds with large therapeutic window.
Acknowledgments
This work was partially supported by MIUR (project PRIN
2010-2011).
We thank Dr. Gregorio Costa (University of Messina, Italy) for
his assistance at the earliest stage of the recourse to the Image-
stream apparatus (Amnis).
4.2.2. Cytotoxicity assay
The cytotoxicity effect of tested compounds was assayed on
RAW 264.7 cells grown in RPMI 1640 medium, supplemented with
Supplementary data
1 mmol L-1 L-glutamine, 10% (v/v) fetal bovine serum (FBS), Peni-
cillin/Streptomycin. The 1 ꢀ 105 cells/well were cultivated on
microplates and incubated at 37 °C overnight in a humidified 5%
CO2 atmosphere to ensure the adherence of the cells. After 24 h
of incubation, the compounds were added to the cell culture at
the respective concentration already assayed for L. infantum (10,
Supplementary data (1H and 13C NMR spectra of new
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.12.039.
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50 and 100
After incubation, 20
l
g/mL) and re-incubated at 37 °C for 24 h and 48 h.
L aliquots of (3-[4,5-dimethylthiazol-2-yl]-
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