Design, synthesis and structure - Activity relationship of phthalimides endowed with dual antiproliferative and immunomodulatory activities

Article abstract of DOI:10.1016/j.ejmech.2015.04.041

Twenty-seven compounds showed dual anti-cancer and immunomodulatory activities.Series (6a-f) show perceptual inhibition range of 32.1-96.8 for SF-295 cell line.Compounds (6b) and (6f) show better inhibition profile of cancer cell lines than thl.The functionalization of series 3 to series 6 achieved better anti-cancer profile.

Full text of DOI:10.1016/j.ejmech.2015.04.041

European Journal of Medicinal Chemistry 96 (2015) 491e503
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureeactivity relationship of phthalimides
endowed with dual antiproliferative and immunomodulatory
activities
,
*
a
Marcos Veríssimo de Oliveira Cardoso a , Diogo Rodrigo Magalhaes Moreira ,
~
Gevanio Bezerra Oliveira Filho a, Suellen Melo Tiburcio Cavalcanti a,
a
a
a
ꢀ
Lucas Cunha Duarte Coelho , Jose Wanderlan Pontes Espíndola , Laura Rubio Gonzalez ,
Marcelo Montenegro Rabello a, Marcelo Zaldini Hernandes a,
Paulo Michel Pinheiro Ferreira , Claudia Pessoa d, Carlos Alberto de Simone e,
b
c,
ꢀ
Elisalva Teixeira Guimaraes g, Milena Botelho Pereira Soares g, Ana Cristina Lima Leite a
f,
~
a
^
^
^
Departamento de Ciencias Farmaceuticas, Centro de Ciencias da Saúde, Universidade Federal de Pernambuco, 50740-520, Recife, PE, Brazil
Departamento de Biofísica e Fisiologia, Programa de Pos-Graduaçao em Ciencias Farmaceuticas, Universidade Federal do Piauí, 64049-550, Teresina, PI,
b
ꢀ
~
^
^
Brazil
c
ꢀ
Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceara, 60430-270, Fortaleza, CE, Brazil
d
e
f
~
Fundaçao Oswaldo Cruz, 60180-900, Fortaleza, CE, Brasil
ꢀ
~
~
Departamento de Física e Informatica, Instituto de Física, Universidade de Sao Paulo, 13560-970, Sao Carlos, SP, Brazil
^
Departamento de Ciencias da Vida, Universidade do Estado da Bahia, 41150-000, Salvador, BA, Brazil
g
~
Hospital Sao Rafael, 41253-190, Salvador, BA, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The present work reports the synthesis and evaluation of the antitumour and immunomodulatory
properties of new phthalimides derivatives designed to explore molecular hybridization and bio-
isosterism approaches between thalidomide, thiosemicarbazone, thiazolidinone and thiazole series.
Twenty-seven new molecules were assessed for their immunosuppressive effect toward TNFa, IFNg, IL-2
and IL-6 production and antiproliferative activity. The best activity profile was observed for the (6aef)
series, which presents phthalyl and thiazolidinone groups.
Received 11 November 2014
Received in revised form
15 April 2015
Accepted 18 April 2015
Available online 20 April 2015
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Phthalimide
Thiosemicarbazone
Thiazole
Thiazolidinone
Antiproliferative
Immunosuppressive
1. Introduction
inflammatory cytokine tumour necrosis factor (TNFa) [2]. TNF is a
central regulator of the inflammatory cascade that controls many
effector pathways as anti-angiogenic, anti-inflammatory and
immunomodulatory molecule. The molecular mode of action of
thalidomide on TNFa expression is thought to involve the inflam-
matory NFjB signalling pathway, specifically inhibiting the activity
of the IjB kinase, IKKa [3].
Thalidomide appears to be a multi-target drug that impinges on
a number of seemingly distinct cellular processes, including
peptidase inhibition, glucosidase inhibition, androgen receptor
antagonism and (cyclooxygenase) COX inhibition [1].
One of the most studied biological activities influenced by
thalidomide is the inhibition of the expression of the pro-
Thalidomide is also known as an inhibitor of nuclear factor
kappa B (NF-
related transcription factors that play a major role in inflammation
and immune responses. Moreover, NF- B inhibits apoptosis, and
kB) activation [4e7]. NF-kB is a family of structurally
k
* Corresponding author.
E-mail address: marcosvocardoso@gmail.com (M.V.O. Cardoso).
http://dx.doi.org/10.1016/j.ejmech.2015.04.041
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

492
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
induces proliferation and angiogenesis, suggesting that NF-
pivotal role in oncogenesis and tumour progression [8,9].
kB has a
Immunomodulatory drugs (IMiDs) are thalidomide derivatives
with improved anti-tumour activity and safer toxicity profiles [10].
The two leading IMiD compounds, lenalidomide (CC-5013; IMiD3;
Revlimid) and pomalidomide (CC-4047; IMiD1; Actimid), were the
first drugs to enter into clinical trials for the treatment of multiple
myeloma in 1999 [11] and are the subject of clinical evaluation in
other haematological malignancies [12]. Studies on the structur-
eeactivity relationship (SAR) of the metabolites of thalidomide and
its analogues have revealed that the phthalimide ring system is an
essential pharmacophoric fragment [13].
In fact, substituted N-phenylphthalimides are of high interest
because they have been found to inhibit TNFa [1,14] and COX [1],
and have tubulin binding properties [15]. With these properties in
mind, phthalimide has usually been employed in the design of
potential antiinflammatory [16], immunomodulatory [17e19],
antiangiogenic [20e22] and antitumour [23e26] drug candidates.
In this promising scenario, the strategy of molecular hybridization
using phthalimide as a pharmacophoric fragment have figured
prominently and led to many successful cases [14]. On the other
hand, thiosemicarbazones are compounds of considerable interest
because of their important chemical properties and potentially
beneficial biological activities [27e30].
In general, the synthesis of thiosemicarbazone compounds
presents low cost and high atom economy because all the atoms
from the reagents (except the water liberated in the condensation)
are present in the final molecule.
4-N-substituted thiosemicarbazones show remarkable activity
in comparison with their unsubstituted counterparts. An enhanced
inhibitory effect may be attributed to the increased lipophilicity
that allows the molecules to easily cross the cell membrane. The 4-
N nitrogen of the thiosemicarbazone skeleton may contain: a) two
hydrogen atoms (unsubstituted thiosemicarbazones); b) one
hydrogen atom and one alkyl or aryl group and c) two alkyl or aryl
groups or may be a part of a cyclic ring [31].
Bearing in mind the molecular pharmacophores outlined above
and their structural requirements, some phthalimide derivatives
were designed after exploring molecular hybridization and bio-
isosterism approaches between thalidomide, thiosemicarbazone,
thiazolidinone and thiazole moieties (Fig. 1). These derivatives
were synthesized by our group and based on the obtained biolog-
ical data, and new SAR information was collected. Furthermore, a
number of the derivatives exhibited potent in vivo activity against
S-180 sarcoma cells that was comparable to that of the reference
drug, thalidomide [32].
Fig. 2. Design concept of target compounds.
In the design concept, the 2-N and 4-N nitrogen of the thio-
semicarbazone skeleton were then substituted by alkyl groups
(2aec) to improve the lipophilicity. A set of compounds (3aed,
4aef and 6aef) bearing thiazolidinones was then synthesized by
exploring bioisosteric relationship between thiazolidinones and
thiosemicarbazones. Our approach also investigated the homolo-
gation between the phthalyl system and thiazolidinones (3aed and
4aef) to investigate the influence of flexibility. Subsequently, a
bioisosteric exchange between the thiazolidinone and thiazole
nuclei was made, so that the 4-N nitrogen of the thiosemicarbazone
skeleton was then converted to a thiazole ring that contained alkyl
(7b) or phenyl groups (7a, 7ceh) (Fig. 2).
2. Results and discussion
2.1. Chemistry
The synthesis of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine
derivatives was adapted from the method described previously
[32,33] and is outlined in Scheme 1.
In a continuation of our work on the structureeactivity rela-
tionship, twenty-seven new phthalimide derivatives were pre-
pared to establish an appropriate SAR. Our design was based on the
molecular hybridization of the phthalimide ring system with a
thiosemicarbazone, thiazolidinone or thiazole subunit.
From the phthalic anhydride (1) obtained commercially, an
acetal intermediate was first synthesized by imidification reaction
with aminoacetaldehyde diethyl acetal reagent in the presence of
DMAP. Then, this intermediate underwent acid hydrolysis to obtain
the aldehyde intermediate, which was condensed with
Fig. 1. Bioisosteric relationship between thalidomide and the proposed thiosemicarbazones, thiazolidinones and thiazoles.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
493
Scheme 1. Synthesis of target compounds. Reagents and conditions: a) aminoacetaldehyde diethyl acetal, toluene, DMAP, reflux, 2 h; H2O, H2SO4, reflux, 2 h; thiosemicarbazide,
EtOH, H2SO4, reflux, 20 h b) BrCH2CO2CH3, AcONa, EtOH, reflux, 20 h; halogenated acids or esters, AcONa, EtOH, reflux, 20 h. c) W-ArCHO, AcOK, DMF, reflux, 12e20 h d) thio-
semicarbazide, DMAP, DMF, reflux, 1 h; BrCH2CO2CH3, AcONa, EtOH, reflux, 24 h. e) W-ArCHO, AcOK, DMF, reflux, 8e24 h.
thiosemicarbazide for the synthesis of phthalyl thiosemicarbazones
(2aec), producing crystals in good yields and in a short reaction
time. The thiazolidinone (3aed) was synthesized by the cyclization
cyclization reaction between the thiosemicarbazone (2a) and
different substituted 2-bromoacetophenone in NaOAc (Scheme 2).
The chemical structure of these products was established using
NMR (1H, 13C and DEPT), IR spectral and elemental analysis (for C, H,
N, S).
of the thiosemicarbazones obtained with a-halogenated acids or
esters in AcONa. Finally, through the reaction between the thiazo-
lidinone (3a) and different aryl-aldehydes in basic medium the
benzylidyl-phthalyl-4-thiazolidinones (4aef) were obtained via
Michael reaction.
2.2. X-ray analysis
In addition to the benzylidyl-phthalyl-4-thiazolidinones, (6aef),
a homologous series to the compounds (4aef), was also synthe-
sized. This synthesis occurred primarily through the reaction be-
tween the thiosemicarbazide and phthalic anhydride under acidic
conditions, followed by cyclization of the thiosemicarbazone with
methyl bromoacetate to obtain the intermediate (5). Then, this
intermediate was treated with the same aryl-aldehydes used in the
synthesis of compounds (4aef).
X-ray diffraction data collections were performed on an Enraf-
Nonius Kappa-CCD diffractometer (95 mm CCD camera on
goniostat) using graphite monochromated MoK radiation
(0.71073 Å), at room temperature. Data collections were carried out
using the COLLECT software [34] up to 50ꢀ in 2
. The final unit cell
k-
a
q
parameters were based on 6064 reflections for the (2a) compound
and 3662 for the (2b) compound. Integration and scaling of the
reflections, and correction for Lorentz and polarization effects were
performed with the HKL DENZO-SCALEPACK system of programs
The phthalyl-thiazoles (7aeh) were produced through
a
Scheme 2. Synthesis of target compounds. Reagents and conditions: a) substituted 2-bromoacetophenone, AcONa, ethanol, reflux, 4e24 h.

494
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
Table 1
[35]. The structures of compounds were solved by direct methods
with SHELXS-97 [36]. The models were refined by full-matrix least
squares on F2 using SHELXL-97 [36]. The program ORTEP-3 [37] was
used for graphic representation, and the program WINGX [38] was
used to prepare materials for publication. All H atoms were located
Main crystallographic parameters of compounds (2a) and (2b).
Compound (2a)
Compound (2b)
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C11 H10 N4 O2 S H2O
280.3
monoclinic
P21/c
5.4630(2)
10.1120(4)
23.9650(9)
90.0
94.790(2)
90.0
C12 H10 N4 O2
274.3
S
triclinic
P-1
by geometric considerations placed (CeH
¼
0.93e0.97 Å;
NeH ¼ 0.86 Å) and refined as riding with Uiso(H) ¼ 1.5Ueq(C-
methyl) or 1.2Ueq(other). An Ortep-3 diagram of the molecules is
shown in Fig. 3, and Table 1 shows the main crystallographic pa-
rameters. All bond distances and angles, fractional coordinates,
equivalent isotropic displacement parameters and other relevant
information can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif under deposit numbers CCDC 972715 and CCDC
972716, respectively.
10.1470(7)
9.9790(5)
14.0120(8)
77.804(3)
87.552(3)
67.989(3)
1284.67(2)
4
b (Å)
c (Å)
a
b
g
(Å)
(Å)
(Å)
V (Å3)
1319.25(2)
4
Z
Radiation (
l
, Å)
MoKa
0.255
none
(
l
¼ 0.71070 Å)
MoKa
0.255
none
(
l
¼ 0.71070 Å)
m
(mmꢁ1
)
Absorption correction
Temp. (K)
Bond lengths and angles are in good agreement with the ex-
pected values reported in the literature [39]. Compound (2a)
crystallized with one solvent water molecule in the packing form
OW-H1W/Si and OW-H2W/Sii [i ¼ x þ 1, y, z; ii ¼ -x þ 1, ꢁy, ꢁz],
and hydrogen-bonding interactions where: H1W/Si ¼ 2.387(2)Å;
295(2)
1.41
295(2)
1.42
Dcalc (Mg mꢁ3
)
Crystal dimensions (mm)
range (º)
0.32 ꢂ 0.23 ꢂ 0.07
0.20 ꢂ 0.19 ꢂ 0.07
q
3.3e27.5
3.1e27.3
Reflections collected(Rint
Independent reflections
Data/parameters
)
7398 [Rint ¼ 0.046]
2877
2254/184
13,033 [Rint ¼ 0.07]
5040
1880/343
OW-H1W/Si
¼
169ꢀ and H2W/Sii
¼
2.549(2) Å; OW-
H2W/Sii ¼ 158ꢀ.
Goodness-of-fit on F2
1.038
0.937
Final R indices (I > 2
s
(I)) R1 ¼ 0.041, wR2 ¼ 0.108 R1 ¼ 0.048,
wR2 ¼ 0.124
2.3. Pharmacological evaluation
R indices (all data)
R1 ¼ 0.055, wR2 ¼ 0.121 R1 ¼ 0.134,
wR2 ¼ 0.169
Once their structures were elucidated, all compounds were
tested as immunomodulatory and anticancer agents. First, the po-
tential immunological properties of the compounds were assessed
by measuring the secretion of cytokines from the animal macro-
protective immune response to bacterial and viral infections
[40,41]. However, it may also play a role in the pathogenesis of
phages (TNF
omide (Thl) and dexamethasone (Dex) were used as controls.
TNF , a highly pleiotropic cytokine produced primarily by
a and IL-6) and lymphocytes (IL-2 and IFNg). Thalid-
disease. Additionally, elevated levels of TNF
with fevers, malaise and weight loss that occur with chronic in-
fections [42]. Otherwise, reductions in TNF levels have been linked
a have been associated
a
a
monocytes and macrophages, plays a central role in the host's
with an improvement in clinical symptoms in a number of disease
states [43e45]. Immune stimulation with LPS was suitable for
analysing TNF
tested compounds, only four did not affect TNF
(compounds (4bed) and (7e)). The inhibition profiles were
observed for both concentrations tested (1 and 10 g/mL); however,
a better inhibition profile was observed at 10 g/mL. Compounds
a
production, and it was observed that among the 27
a
production at all
m
m
(3a), (4a), (4e), (4f), (6eef), (7a) and (7d) showed average % inhi-
bition cytokine between 52 and 73%. At the same concentration,
thalidomide did not have an inhibitory profile (Fig. 4).
When observing the thiazolidinone group, series 6, showed bet-
ter inhibition rates than did series 4, which contains a space group
between phthalyl and thiazolidinone ring. Phthalyl thio-
semicarbazones (series 2) showed only moderate inhibitory activity.
IL-2 is instrumental in the body's natural response to microbial
infection and is normally produced by TH1 cells [46]. Levels of this
cytokine were significantly inhibited by compound (2c), (4a), (6a)
and (6e) (45e72% range). We found that, among the twenty-seven
synthetic derivatives, compounds (4a) and (6e) displayed the
strongest ability to inhibit IL-2 secretion. Compounds (4bed), (4f),
(6bed), (7d) and (7f) showed only a modest inhibition of IL-2
(Fig. 5). It is worth mentioning that the inhibitory ability was
revealed only at a concentration of 1
For IL-2 cytokine, the thiazole nucleus (7aeh series) is inactive
for both concentrations. The activity was observed only at 1 g/mL,
and it seems that phthalyl thiazolidones at 1 g/mL (series 4aef
mg/mL.
m
m
and 6aef) are selective for the inhibition of IL-2. Among the thia-
zolidinone series, series 3aed was the only series that did not
produce inhibition. The main difference in this series is the ben-
zylidine substituent at C5 of series (4aef). Compounds (4a) and
(6e) were comparable to dexamethasone at the same concentra-
tion. Once again, Thl was inactive at the same concentration.
Fig. 3. ORTEP-3 projections of the compounds: (a) 2a and (b) 2b, showing the atom-
numbering and displacement ellipsoids at the 50% probability level.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
495
Fig. 4. Effect of phthalimides on TNF production. Peritoneal macrophages were incubated in the presence of phthalimides (1 and 10
mg) or medium alone, and stimulated or not
with bacterial lipopolysaccharide (LPS; 500 ng/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs in the same concentrations. Cell free supernatants
were collected 4 h later for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean S.D. (error bars) obtained in duplicate; S.D., standard deviation.
Fig. 5. Effect of phthalimides on IL-2 production. Spleen cells were incubated in the presence of phthalimides (1 and 10
concanavalin A (ConA; 1 g/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs at the same concentrations. Cell-free supernatants were collected 24 h
later for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean S.D. (error bars) obtained in duplicate; S.D., standard deviation.
mg) or medium alone and were stimulated or not with
m
Interferon-
g
(IFN
g
) is a cytokine secreted by lymphocytes that
(93%). Both series representatives showed good inhibition activity
at 1 g/mL, but no trend can be identified with regard to spacing
promotes innate immunity, i.e., natural killer (NK) cells, and cells
m
that are components of the adaptive immune system (specific
groups or differences in ring structures (thiazolidinone versus
thiazole).
subsets of T cells) [47,48]. Furthermore, a role for IFN
tion against tumour development has recently been identified
[49]. The results have shown that endogenously produced IFN is
g in protec-
IL-6, a pro-inflammatory cytokine, is secreted by the TH1 cells
and macrophages and stimulates the immune response to trauma,
especially burns or other tissue damage leading to inflammation. In
terms of the host response to a foreign pathogen, IL-6 has been
shown to provide resistance to mice against the bacterium, Strep-
tococcus pneumoniae [51]. The effect of the tested compounds
g
critical not only for the rejection of transplantable tumours but
also to prevent primary tumour development [50]. The level of
IFN
(7h), 83% (2a), and 82% (3b) at 1 mg/mL (Fig. 6). Derivative (4b) is
the strongest inhibitor of IFN secretion among the twenty-seven
compounds and is comparable to dexamethasone (89%) and Thl
g secretion was reduced by 95% (4b), 93% (7b), 87% (7d), 85%
g
(10
mg/mL) on the secretion of this cytokine was only modest; the
inhibition percentage did not reach 50%. Compounds (2c), (6aed),
Fig. 6. Effect of phthalimides on IFN
concanavalin A (ConA; 1 g/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs at the same concentrations. Cell-free supernatants were collected 24 h
later for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean S.D. (error bars) obtained in duplicate; S.D., standard deviation.
g secretion. Spleen cells were incubated in the presence of phthalimides (1 and 10 mg) or medium alone, and stimulated or not with
m

496
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
Fig. 7. Effect of phthalimides on IL-6 secretion. Peritoneal macrophages were incubated in the presence of phthalimides (1 and 10 mg) or medium alone, and stimulated or not with
bacterial lipopolysaccharide (LPS; 500 ng/mL). Thalidomide (Thl) and dexamethasone (Dex) were used as reference drugs at the same concentrations. Cell-free supernatants were
collected 4 h later for cytokine analysis by sandwich ELISA (Duoset, R&D Systems kit). Data are the mean S.D. (error bars) obtained in duplicate; S.D., standard deviation.
(7a), (7c), (7d) and (7f) showed inhibition in the range of 31e39%
(Fig. 7).
To investigate the anticancer properties of these compounds,
these phthalimides were first evaluated against three tumour lines:
MDA/MB-435 (melanoma), HCT-8 (colon) and SF-295 (nervous
system).
Table 2 summarizes the cytotoxic action on tumour cells eval-
uated by MTT assay. Compounds from series (6aef) were the most
potent, especially compounds (6b) and (6f), which revealed cell
proliferation inhibition rates ranging from 87.0 11.1% (SF-295) to
as positive control, was active against all lines. On the other hand,
Thalidomide, the phthalimide of reference, was weakly cytotoxic on
tumour cells.
Likewise, the IC50 values for compounds (6b) and (6f) on human
lymphocytes were 9.4 and 7.7 mg/mL, respectively. As mentioned
before, the selectivity between normal and malignant cells is one of
the critical issues for the research and development of chemo-
therapeutic reagents.
In light of these findings, it is reasonable to draw some com-
ments about the dual behaviour of compounds (6b) and (6f). These
100 1.1% (MDA/MB-435) and IC50 values of 7.5 and 5.3
m
g/mL (SF-
compounds showed immunosuppressive activity toward TNF
10 g/mL and also showed anticancer properties against three
tumour cell lines.
a at
295) and 5.8 and 5.2
mg/mL (HCT-8), respectively. Doxorubicin, used
m
With regard to the structural features of the compounds and the
immunological profiles of all series of the tested compounds
(Fig. 8), the immunosuppressive and antiproliferative profile of the
(6aef) series of phthalimide derivatives were the most effective.
The main structural difference between the (6aef) and (4aef) se-
ries concerns the insertion of a flexible group (eCH2eCH]Ne)
between the phthalyl and thiazolidinone rings (in the 4aef series).
Another remark is the fact that series (7aeh) possesses a 4-phenyl-
thiazole nucleus instead of a thiazolidin-4-one nucleus such as that
present in the (4aef) and (6aef) series.
Table 2
Screening of the in vitro cytotoxicity of 27 phthalimides derivatives on cancer cells at
concentration of 50 g/mL and lymphocytes at 5 g/mL. The cytotoxicity on both
neoplasic and normal cells was determined by MTT assay.
m
m
Sample
SF-295
HCT-8
MDA/MB-435
LYMP (GI%)
SD
2.9%
4.4%
4.9%
8.8%
8.1%
16.1%
4.9%
1.9%
9.1%
3.3%
10.2%
42.3%
4.0%
12.4%
6.0%
2a
2b
2c
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
7g
7h
Dox
Thl
NT
NT
NT
NT
30.1 0.8
21.8 6.1
26.4 0.2
33.0 13.0
26 0.1
NT
17.8 4.1
28.5 10.2
22.0 6.1
8.0 1.2
NT
67.5%
13.2%
13.6%
9.4%
7.2 6.4
NT
2.4%
It is worth mentioning that our previous results showed that
phthalimide derivatives were inactive (IC50 > 300 mM) in in vitro
5.8 1.2
2.5 0.3
24.9 3.2
27.3 7.5
34.4 2.3
45.9 14.0
11.2 4.0
93.5 1.0
30.2 3.3
100.0 1.1
50.7 0.8
55.1 5.7
60.4 6.3
82.1 8.
35.5 7.8
28.4 3.0
24.8 3.3
22.8 1.9
16.3 13.5
NT
14.6%
35.7%
20.7%
NT
24.9%
16.7%
50.2%
32.1%
5.0%
1.4%
0.8%
NT
19.2%
35.6%
13.9%
59.9%
14.6%
4.0%
NT
NT
23.4 8.3
32.7 2.0
56.2 0.1
46.6 9.5
16.7 0.3
23.7 9.5
67.1 5.1
87.0 11.1
32.1 0.6
58.9 2.3
62.5 6.1
96.8 0.3
NT
64.8 1.0
NT
38.5 1.2
7.5 0.9
NT
58.7 3.5
11.1 4.0
100.00 0.7
10.4 4.7
33.3 6.5
33.7 0.4
50.2 13.1
47.5 0.3
42.7 2.0
42.2 15.1
65.3 2.7
97.1 3.2
41.0 0.1
56.5 5.5
62.8 7.9
91.3 4.4
25.3 9.8
45.8 7.8
NT
52.3 1.5
9.3 3.7
NT
72.5 3.9
24.1 1.1
83.62 2.9
35.7 3.2
tests against four tumour cells lines: MDA/MB-435 (breast), HCT-8
(colon), SF-295 (glioblastoma) and HL-60 (leukaemia). Likewise, in
general, these derivatives did not show immunosuppressive
properties, which is a characteristic that is highly desirable in new
immunomodulatory drug candidates [32].
2.6%
2.4. Docking studies
11.4%
10.7%
23.8%
3.4%
7.8%
14.2%
1.7%
4.4%
2.8%
11.4%
23.2%
2.9%
NF-
expression of various pro-survival genes. The multi-subunit protein
kinase, IKK, regulates NF- B activation in response to specific
external mediators, including tumour necrosis factor- (TNF ) and
interleukin-1 (IL-1) [52]. In the nucleus, NF- B binds to its cognate
kB is a significant transcription factor that regulates the
k
a
a
k
1.7%
7.9%
9.8%
17.0%
67.5%
13.2%
DNA site and enhances the expression of a number of genes related
to the immune response, cell proliferation and survival [53].
58.5 1.8
NT
96.7 4.5
40.5 7.9
Thus, the inhibition of IKK
b on the NF-kB pathway could be
involved the anti-inflammatory and anti-cancer mechanism of the
molecules reported in this work.
4.4%
Data are presented as inhibition perceptual of the antiproliferative rate obtained
from at least three independent experiments for human tumor lines (SF-295, ner-
vous system; HCT-8, colon; MDA/MB-435, melanoma) and normal human lym-
phocytes (LYMP). Doxorubicin was used as positive control (Dox); Thalidomide as
drug of reference (Thl). NT: non toxic.
To understand a possible correlation between cell proliferation
inhibition and IKK
reported in this work with IKK
docking studies. The binding mode for these ligands was
b
, we investigated the interaction of compounds
b
(PDB ID: 4KIK) by conducting

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
497
Fig. 8. Effect of substitutions in the thiazole ring on antiproliferative activity.
determined by the highest (most positive) score among the
possible solutions for each ligand. These calculations were gener-
ated according to the ChemPLP Fitness Score [54].
Fig. 9 shows the trend observed between the in silico docking
scores and the cytotoxic activity on tumour cells evaluated by MTT
assay. The assay indicates that the compounds with higher cyto-
toxic action on tumour cells are usually those with the higher
docking scores, i.e., the molecules with high cytotoxic action also
docking score values for the (6b) and (3d) molecules, which are
78.07 and 62.15, respectively. Due to the trend observed between
the in silico docking scores (ChemPLP scores) and the in vitro
cytotoxic activity on tumour cells, the molecules with high affinity
(in silico) for the IKKb target seem to prevent more cell proliferation
(in vitro), at least for the three tumour lines tested in this work (SF-
295, HCT-8 and MDA/MB-435).
have a high affinity for the IKKb target, as revealed by the docking
3. Conclusions
score values. It is important to highlight the large variation of
docking scores (range: from 61.22 to 81.04), and the percentage of
inhibition (range: from zero to 100.0%), which contributes to the
reliability of this in silico-in vitro trend.
To identify the molecular reasons for the two extremes of cell
proliferation inhibition (highest and lowest percentage inhibition
for (6b) and (3d), respectively), we performed a detailed analysis of
The current investigation has described the facile synthesis of
anti-cancer compounds, which showed significant cytotoxic activ-
ity toward three human cancer cell lines and immunosuppressive
activity over cytokines TNF
studies have shown that the molecules with more stable or positive
docking scores (i.e., greater in silico affinity for the IKK target) are
also the most cytotoxic in human cancer cell lines. In summary,
compounds (6b) and (6f) hold potential as immunosuppressive
agents with anticancer properties. The described findings may
open up new possibilities for developing a new class of drugs with
immunosuppressive and cytotoxic activity.
a, IFNg, IL-2 and IL-6. In silico docking
b
the intermolecular interactions between the target (IKKb) and the
docked molecules. The superimposition of molecules (6b), (3d) and
co-crystallized ligand (named “K252A”) can be seen in Fig. 10.
The difference between the binding modes of the (6b) and (3d)
molecules is shown in Fig. 11 and Table 3. The residues of the IKK
target that participate in hydrophobic interactions, hydrogen bonds
and interactions are highlighted in Fig. 11. It seems that the
b
pep
4. Experimental methods
additional hydrophobicity of the Cl-phenyl group in molecule (6b)
provides a greater contact surface for interactions with hydropho-
bic residues of the target in comparison with molecule (3d), which
4.1. General
ensures more stability in the complex formed with IKK
b.
Melting points were measured with a Fisatom (Mod. 430D,
60 Hz) melting point apparatus and are uncorrected. 1H NMR
This stability difference is also revealed when we analyse the
Fig. 9. Trends observed between the cell proliferation inhibition and in silico (docking ChemPLP score) results. The blue rhombus shows trends for the SF-295 tumour line, the red
square for the HCT-8 tumour line and the green triangle for the MDA/MB-435 tumour line. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

498
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
Fig. 10. Superimposition of the docking solutions for compounds (6b) (blue stick), (3d) (red stick) and the crystallographic structure of the “K252A” co-crystallized ligand (gray
line). (A) Full view and (B) active site view. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 11. Detailed view of the docking solutions for (A) compound (6b) and (B) compound (3d). Residues involved in hydrophobic interactions (green), hydrogen bonds (cyan), and
pe
p
T-shaped interactions (orange) are highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
spectra were recorded on a 300 MHz spectrometer in appropriate
solvents using TMS as an internal standard or the solvent signals as
secondary standards. The chemical shifts are shown in
scale. Multiplicities of NMR signals are designated as s (singlet),
d (doublet), br (broad) and m (multiplet, for unresolved lines). 13
NMR spectra were recorded on a 75.5 MHz spectrometer. All the
experiments were monitored by analytical thin layer chromatog-
raphy (TLC) performed on silica gel GF254 pre-coated plates. After
elution, plates were visualized under UV illumination at 254 nm for
UV active materials.
d (ppm)
Table 3
Molecular interaction of IKK
b
with molecules (6b) and (3d).
C
Residues
Molecules
6b
3d
LEU21
GLY22
THR23
VAL29
ARG31
ALA42
LYS44
VAL74
MET96
GLU97
TYR98
CYS99
ASP103
ILE165
HC
e
HC
HC
3.0
e
e
HC
HC
HC
HC
HC
HC
2.9
HC
3.3
e
e
4.2. General procedure for the synthesis of thiazolidinones (3aed).
Example for compound (3a)
HC
e
HC
HC
e
Thiosemicarbazone (2a) (0.4 g, 1.52 mmol), anhydrous sodium
acetate (0.5 g, 6.08 mmol), and 50 mL ethanol were added to a
100 mL round bottom flask under magnetic stirring and slightly
warmed for 10e15 min. Then, ethyl 2-bromoacetate (0.26 g,
1.52 mmol) was added, and the colourless reaction was kept under
reflux heating for 18 h. After cooling the solution back to room
temperature (r.t.), the precipitate was filtered off and the solvent
was evaporated for half of its volume and then cooled to 0 ꢀC. A
white solid was obtained, filtered in a Büchner funnel with a
PI
HC
3.3
e
HC
Docking score
78.07
62.15
HC means hydrophobic contacts, PI means
the hydrogen bond distances between donor and acceptor, in Ångstroms. (See
Fig. 11).
pep interaction, and the numbers are

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
499
sintered disc filter, washed with cold water and then dried in SiO2.
Products were purified by recrystallization using the solvent sys-
tem detailed below for each compound.
water was added to the flask and a yellow precipitate was formed.
The precipitate was filtered off and the solvent was discarded. A
yellow solid is obtained, filtered in Büchner funnel with sintered
disc filter, washed with cold water, and then dried in SiO2. Products
are purified by column chromatography using the solvent system
detailed below for each compound.
4.2.1. 2-(4-Oxothiazolidin-2-ylidene)hydrazono)ethyl)isoindoline-
1,3-dione (3a)
After crystallization with toluene, colourless crystals were ob-
tained; yield ¼ 74%; M.p. (ꢀC): 262e263; IR (KBr) 3075 (NeH), 2968
(CeH), 1771 and 1714 (C]O), 1641 (C]N) cmꢁ1. 1H NMR (300 MHz,
4.3.1. 2-(2-((-5-(4-Fluorobenzylidene)-4-oxothiazolidin-2-ylidene)
hydrazono)ethyl)isoindoline-1,3-dione (4a)
DMSO-d6):
1H, CH); 7.82e7.92 (m, 4H, Ar); 11.84 (s, 1H, NH). 13C NMR
(75.5 MHz, DMSO-d6): 32.8 (CH2 heterocycle); 37.6 (CH2); 123.1
(CH Ar); 131.73 (CH Ar); 134.5 (Ar); 153.5 (HC]N); 165.7 (SeC]N);
167.5 (C]O); 167.6 (C]O); 174.4 (C]O heterocycle). Anal. Calcd.
For (3a): C, 51.65; H, 3.33; N, 18.53; S, 10.61; Found: C, 51.42; H,
3.40; N, 18.13; S, 10.36. Rf: 0.35.
d
3.68 (s, 2H, CH2 heterocycle); 4.47 (d, 2H, CH2); 7.71 (t,
After elution with hexane/acetate (8:2), yellow crystals were
obtained; yield ¼ 38%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
3471 (NeH), 2935 (CeH), 1716 (C]O), 1634 and 1600 (C]N), 1233
d
(CeF) cmꢁ1. 1H NMR (300 MHz, DMSO-d6):
d 4.32 (d, 2H, CH2); 7.22
(d, 2H, Ar); 7.48 (t, 1H, CH); 7.69 (s, 1H, CH); 7.72 (d, 2H, Ar);
7.79e7.96 (m, 4H, Ar); 8.52 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6):
d 37.6 (CH2); 115.1 (SeC]C); 120.4 (CH Ar); 130.8 (CH Ar);
123.6 (CH Ar); 131.8 (CH Ar); 132.2 (Ar); 132.7 (Ar); 142 (HC]C);
162.1 (CeF); 163 (C]N); 163.7 (HC]N); 168.2 (C]O); 170.2 (C]O).
Anal. Calcd. For (4a): C, 58.82; H, 3.21; N, 13.72; S, 7.85; Found: C,
58.74; H, 3.48; N, 13.81; S, 8.03. Rf: 0.631.
4.2.2. 2-(5-Methyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)
isoindoline-1,3-dione (3b)
After crystallization with toluene, colourless crystals were ob-
tained; yield ¼ 70%; M.p. (ꢀC): 227; IR (KBr) 3461 (NeH), 3034
(CeH), 1772 and 1718 (C]O), 1648 (C]N) cmꢁ1. 1H NMR (300 MHz.
4.3.2. 2-(2-((-5-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylidene)
hydrazono)ethyl)isoindoline-1,3-dione (4b)
DMSO-d6):
7.71 (t, 1H, CH); 7.84e7.93 (m, 4H, Ar); 11.82 (s, 1H, NH). 13C NMR
(75.5 MHz, DMSO-d6): 18.7 (CH3); 37.4 (CH2); 42.1 (CH hetero-
d 1.35 (d, 3H, CH3); 4.00 (q, 1H, CH); 4.47 (d, 2H, CH2);
After elution with hexane/acetate (8:2), yellow crystals were
d
obtained; yield ¼ 46%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
cycle); 123.2 (CH Ar); 131.7 (CH Ar); 134.6 (Ar); 153.8 (HC]N);
163.9 (SeC]N); 167.5 (C]O); 177.4 (C]O heterocycle). Anal. Calcd.
For (3b): C, 53.16; H, 3.82; N, 17.71; S, 10.14; Found: C, 53.55; H,
3.90; N, 17.43; S, 10.08. Rf: 0.526.
2925 (CeH), 1724 (C]O), 1596 (C]N), 821 (CeCl) cmꢁ1 1H NMR
.
(300 MHz, DMSO-d6):
2H, Ar); 7.63 (s, 1H, CH); 7.67 (d, 2H, Ar); 7.82e7.99 (m, 4H, Ar); 8.54
(s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
37.6 (CH2); 116.0
d 4.35 (d, 2H, CH2); 7.55 (t, 1H, CH); 7.60 (d,
d
(SeC]C); 123.3 (CH Ar); 129.5 (CH Ar); 129.7 (CH Ar); 131.9 (CH
Ar); 133.3 (Ar); 133.5 (Ar); 134.7 (Ar); 142 (HC]C); 163.7 (HC]N);
165.0 (C]N); 168.2 (C]O); 170.0 (C]O); 170.2 (C]O). Anal. Calcd.
For (4b): C, 56.54; H, 3.08; N, 13.19; S, 7.55; Found: C, 56.37; H, 3.07;
N, 13.49; S, 7.77. Rf: 0.635.
4.2.3. 2-(5-Ethyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)
isoindoline-1,3-dione (3c)
After crystallization with toluene, colourless crystals were ob-
tained; yield ¼ 79%; M.p. (ꢀC): 191e192; IR (KBr) 3397 (NeH), 2963
(CeH), 1770 and 1714 (C]O), 1651 (C]N) cmꢁ1. 1H NMR (300 MHz,
DMSO-d6):
CH2); 7.43 (t,1H, CH heterocycle); 7.68 (t,1H, CH); 7.83e7.91 (m, 4H,
Ar); 11.23 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
10.3 (CH3);
25.4 (CH2); 37.6 (CH2); 49.3 (CH heterocycle); 123.0 (Ar); 131.5 (Ar);
134.2 (Ar); 140.3 (HC]N); 152.6 (C]N); 167.3 (C]O); 177.2 (C]O);
178.0 (C]O). Anal. Calcd. For (3c): C, 54.53; H, 4.27; N, 16.96; S,
9.71; Found: C, 54.37; H, 4.42; N, 17.16; S, 10.05. Rf: 0.5.
d
0.80 (t, 3H, CH3); 1.59e1.98 (m, 2H, CH2); 4.36 (d, 2H,
4.3.3. 2-(2-((-5-(4-Bromobenzylidene)-4-oxothiazolidin-2-ylidene)
hydrazono)ethyl)isoindoline-1,3-dione (4c)
d
After elution with hexane/acetate (8:2), yellow crystals were
obtained; yield ¼ 60%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
2933 (CeH), 1716 (C]O), 1607 (C]N), 558 (CeBr) cmꢁ1 1H NMR
.
(300 MHz, DMSO-d6):
2H, Ar); 7.64 (d, 2H, Ar); 7.72 (s,1H, CH); 7.80e8.06 (m, 4H, Ar); 8.55
(s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
37.6 (CH2); 116.3
d 4.37 (d, 2H, CH2); 7.50 (t, 1H, CH); 7.55 (d,
d
4.2.4. 2-(3-Methyl-4-oxothiazolidin-2-ylidene)hydrazono)ethyl)
isoindoline-1,3-dione (3d)
After crystallization with toluene, colourless crystals were ob-
tained; yield ¼ 65%; M.p. (ꢀC): 180e181; IR (KBr) 2979 (CeH), 1770
and 1718 (C]O), 1637 (C]N) cmꢁ1. 1H NMR (300 MHz. DMSO-d6):
(SeC]C); 122.3 (CH Ar); 122.9 (CH Ar); 128.6 (CH Ar); 131.5 (CH
Ar); 132.3 (Ar); 132.7 (Ar); 134.2 (Ar); 142 (HC]C); 163.7 (HC]N);
164 (C]N); 168.4 (C]O); 173.1 (C]O). Anal. Calcd. For (4c): C,
51.18; H, 2.79; N, 11.94; S, 6.83; Found: C, 50.99; H, 2.76; N, 11.80; S,
7.11. Rf: 0.625.
d
3.04 (s, 3H, CH3); 3.74 (s, 2H, CH2 heterocycle); 4.49 (d, 2H, CH2);
7.40 (t, 1H, CH); 7.83e7.90 (m, 4H, Ar). 13C NMR (75.5 MHz. DMSO-
d6): 29.1 (CH2 heterocycle); 30.6 (CH2); 31.81 (CH3); 123.0 (CH Ar);
4.3.4. 2-(2-((-5-Benzylidene-4-oxothiazolidin-2-ylidene)
hydrazono)ethyl)isoindoline-1,3-dione (4d)
d
131.6 (CH Ar); 134.4 (Ar); 154.8 (HC]N); 164.8 (SeC]N); 167.4
(C]O); 172.1 (C]O); 178.0 (C]O heterocycle). Anal. Calcd. For
(3d): C, 51.16; H, 3.82; N, 17.71; S, 10.14; Found: C, 51.40; H, 4.02; N,
17.42; S, 9.89. Rf: 0.562.
After elution with hexane/acetate (8:2), yellow crystals were
obtained; yield ¼ 56%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
3026 (CeH), 1715 (C]O), 1608 (C]N) cmꢁ1 1H NMR (300 MHz,
.
DMSO-d6):
d
3.95 (d, 2H, CH2); 7.53 (t, 1H, CH); 7.41e7.89 (m, 10H;
40.3
9H Ar and 1H CH); 8.50 (NH). 13C NMR (75.5 MHz, DMSO-d6):
d
4.3. General procedure for the synthesis of benzylidenes (4aef).
Example for benzylidene (4a)
(CH2); 116.0 (SeC]C); 122.9 (CH Ar); 127.3 (CH Ar); 127.9 (CH Ar);
128.8 (CH Ar); 132.3 (CH Ar); 133.7 (Ar); 135.2 (Ar); 142.5 (HC]C);
158.4 (HC]N); 159.9 (C]N); 164.6 (C]O); 168 (C]O). Anal. Calcd.
For (4d): C, 61.53; H, 3.61; N, 14.35; S, 8.21; Found: C, 61.47; H, 3.77;
N, 14.77; S, 8.49. Rf: 0.66.
Thiazolidinone (3a) (0.4 g, 1.32 mmol), anhydrous potassium
acetate (0.39 g, 3.96 mmol), and 5 mL dimethylformamide were
added to a 100 mL round bottom flask under magnetic stirring and
slightly warmed for 10e15 min. Then 4-fluorobenzaldehyde (0.16 g,
1.32 mmol) was added and the reaction acquired yellow colour was
kept under heating under reflux for 24 h. After cooling back to r.t.,
4.3.5. 2-(2-((-5-(3-Methoxybenzylidene)-4-oxothiazolidin-2-
ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4e)
After elution with hexane/acetate (8:2), yellow crystals were

500
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
obtained; yield ¼ 57%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
3402 (NeH), 2952 (CeH), 1718 (C]O), 1642 and 1594 (C]N), 1265
4.4.3. 2-((-5-(4-Bromobenzylidene)-4-oxothiazolidin-2-ylidene)
amino)isoindoline-1,3-dione (6c)
(CeO) cmꢁ1. 1H NMR (300 MHz, DMSO-d6):
d
3.80 (s, 1H, CH3); 4.53
(d, 2H, CH2); 6.89e7.85 (m, 10H; 8H Ar and 2H CH); 8.55 (NH). 13
NMR (75.5 MHz, DMSO-d6): 43.1 (CH2); 56.8 (CH3); 113.5 (CH Ar);
After elution with hexane/acetate (6:4), yellow crystals were
obtained; yield ¼ 33%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
3434 (NH), 2937 (CeH), 1715 (C]O), 1645 and 1583 (C]N), 558
C
d
116.0 (SeC]C); 120.9 (CH Ar); 124.2 (CH Ar); 127.9 (CH Ar); 130.8
(Ar); 134.0 (Ar); 136.2 (HC]C); 158.3 (HC]N); 159.9 (CeO Ar);
168.0 (C]O); 168.2 (C]O). Anal. Calcd. For (4e): C, 59.99; H, 3.84;
N, 13.33; S, 7.63; Found: C, 59.56; H, 3.78; N, 12.94; S, 7.28. Rf: 0.58.
(CeBr) cmꢁ1. 1H NMR (300 MHz, DMSO-d6):
d
7.53 (d, 2H, Ar); 7.61
(d, 2H, Ar); 7.73 (s, 1H, CH); 7.86e7.90 (m, 4H, Ar); 8.59 (s, 1H, NH).
13C NMR (75.5 MHz, DMSO-d6):
116.0 (SeC]C); 122.5 (CH Ar);
d
128.7 (CH Ar); 124.0 (CH Ar). 131.3 (CH Ar); 132.0 (Ar); 132.3 (CH
Ar); 142.0 (HC]C); 144.6 (C]N); 159.9 (C]O); 165.4 (C]O). Anal.
Calcd. For (6c): C, 50.48; H, 2.35; N, 9.81; S, 7.49; Found: C, 50.22; H,
2.58; N, 9.62; S, 7.20. Rf: 0.552.
4.3.6. 2-(2-((-5-(4-Methoxybenzylidene)-4-oxothiazolidin-2-
ylidene)hydrazono)ethyl)isoindoline-1,3-dione (4f)
After elution with hexane/acetate (8:2), yellow crystals were
obtained; yield ¼ 35%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
2932 (CeH), 1717 (C]O), 1597 (C]N), 1256 (CeO) cmꢁ1. 1H NMR
4.4.4. 2-((-5-Benzylidene-4-oxothiazolidin-2-ylidene)amino)
isoindoline-1,3-dione (6d)
(300 MHz, DMSO-d6):
2H, Ar); 7.51 (t, 1H, CH); 7.63 (d, 2H, Ar); 7.73 (s, 1H, CH); 7.83e7.88
(m, 4H, Ar); 8.55 (NH). 13C NMR (75.5 MHz, DMSO-d6):
40.1 (CH2);
d
3.85 (s, 1H, CH3); 4.56 (d, 2H, CH2); 6.99 (d,
After elution with hexane/acetate (6:4), yellow crystals were
obtained; yield ¼ 40%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
3023 and 2951 (CeH), 1712 (C]O), 1646 and 1593 (C]N) cmꢁ1. 1H
d
57.1 (CH3); 114.6 (CH Ar); 116.0 (SeC]C); 123.3 (CH Ar); 128.4 (CH
Ar); 130.7 (CH Ar); 132.0 (Ar); 142.0 (HC]C); 157.9 (CeO Ar); 159.7
(HC]N); 160 (C]N); 163.3 (C]O); 168.2 (C]O). Anal. Calcd. For
(4f): C, 59.99; H, 3.84; N, 13.33; S, 7.63; Found: C, 59.67; H, 3.62; N,
13.47; S, 7.57. Rf: 0.632.
NMR (300 MHz, DMSO-d6):
d
7.45e7.85 (m, 9H, Ar); 8.40 (s, 1H,
115.6 (SeC]
CH); 8.51 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d
C); 128.6 (CH Ar); 128.8 (CH Ar); 129.8 (CH Ar); 129.9 (CH Ar); 130.2
(CH Ar); 130.6 (Ar); 130.8 (Ar); 131.8 (HC]C); 134.8 (C]N); 157.2
(C]O); 158.0 (C]O). Anal. Calcd. For (6d): C, 61.88; H, 3.17; N,
12.03; S, 9.18; Found: C, 61.50; H, 3,28; N, 12.39; S, 9.46. Rf: 0.592.
4.4. General procedure for the synthesis of benzylidenes (6aef).
Example for benzylidene (6a)
4.4.5. 2-((-5-(3-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)
amino)isoindoline-1,3-dione (6e)
Thiazolidinone (5) (0.4 g, 1.52 mmol), anhydrous potassium
acetate (0.45 g, 4.56 mmol), and 5 mL of dimethylformamide were
added to a 100 mL round bottom flask under magnetic stirring and
slightly warmed for 10e15 min. Next, 4-fluorobenzaldehyde (0.19 g,
1.52 mmol) was added, and the reaction acquired a yellow colour
and was kept under heating under reflux for 24 h. After cooling
back to r.t., water was added to the flask and a yellow precipitate
was formed. The precipitate was filtered off and the solvent was
discarded. A yellow solid was obtained, filtered in Büchner funnel
with a sintered disc filter, washed with cold water, and then dried
in SiO2. Products were purified by column chromatography using
the solvent system detailed below for each compound.
After elution with hexane/acetate (6:4), yellow crystals were
obtained; yield ¼ 22%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
3460 (NeH), 2936 (CeH), 1711 (C]O), 1641 and 1594 (C]N), 1266
(CeO) cmꢁ1
.
1H NMR (300 MHz, DMSO-d6):
d
3.99 (s, 3H, CH3);
6.89e7.46 (m, 4H, Ar). 7.72 (s, 1H, CH); 7.85e7.88 (m, 4H, Ar); 8.51
(NH). 13C NMR (75.5 MHz, DMSO-d6):
57.2 (CH3); 114.3 (CH Ar);
d
116.0 (SeC]C); 120.1 (CH Ar); 122.6 (CH Ar); 128.2 (CH Ar); 132.5
(CH Ar). 132.8 (Ar); 136.8 (Ar); 139.1 (HC]C); 142.0 (C]N); 157.4
(CeO Ar); 159.2 (C]O); 163.6 (C]O). Anal. Calcd. For (6e): C, 60.15;
H, 3.45; N, 11.08; S, 8.45; Found: C, 60.01; H, 3.25; N, 11.20; S, 8.47.
Rf: 0.578.
4.4.6. 2-((-5-(4-Methoxybenzylidene)-4-oxothiazolidin-2-ylidene)
amino)isoindoline-1,3-dione (6f)
4.4.1. 2-((-5-(4-Fluorobenzylidene)-4-oxothiazolidin-2-ylidene)
amino)isoindoline-1,3-dione (6a)
After elution with hexane/acetate (6:4), yellow crystals were
After elution with hexane/acetate (6:4), yellow crystals were
obtained; yield ¼ 46%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
obtained; yield ¼ 36%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
2952 (CeH), 1710 (C]O), 1651 and 1598 (C]N), 1255 (CeO) cmꢁ1
.
2950 (CeH), 1709 (C]O), 1651 and 1598 (C]N), 1231 (CeF) cmꢁ1
.
1H NMR (300 MHz, DMSO-d6):
7.65 (d, 2H, Ar); 7.84e7.89 (m, 4H, Ar); 8.38 (s, 1H, CH); 8.49 (s, 1H,
NH). 13C NMR (75.5 MHz, DMSO-d6):
55.9 (CH3); 115.2 (CH Ar);
d 4.01 (s, 3H, CH3); 6.69 (d, 2H, Ar);
1H NMR (300 MHz, DMSO-d6):
7.72 (s, 1H, CH); 7.84e7.88 (m, 4H, Ar); 8.40 (s, 1H, NH). 13C NMR
(75.5 MHz, DMSO-d6): 115.4 (CH Ar); 116.0 (SeC]C); 122.8 (CH
d 7.21 (d, 2H, Ar); 7.61 (d, 2H, Ar);
d
d
116.0 (SeC]C); 124.4 (CH Ar); 128.3 (CH Ar); 132.1 (CH Ar); 133.6
(Ar); 133.9 (Ar); 142.8 (HC]C); 144.3 (C]N); 148.3 (CeO Ar); 160.3
(C]O); 163.2 (C]O). Anal. Calcd. For (6f): C, 60.15; H, 3.45; N,11.08;
S, 8.45; Found: C, 60.48; H, 3.74; N, 11.05; S, 8.23. Rf: 0.605.
Ar); 124.9 (CH Ar); 127.4 (CH Ar); 131.6 (Ar); 132.2 (Ar); 133.0 (HC]
C); 141.6 (C]N); 144.7 (CeF Ar); 167.7 (C]O); 169.8 (C]O); 175.1
(C]O). Anal. Calcd. For (6a): C, 58.85; H, 2.74; N, 11.44; S, 8.73;
Found: C, 58.83; H, 2.58; N, 11.51; S, 8.56. Rf: 0.697.
4.5. General procedure for the synthesis of 1,3-thiazoles (7aeh).
Example for thiazole (7a)
4.4.2. 2-((-5-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylidene)
amino)isoindoline-1,3-dione (6b)
After elution with hexane/acetate (6:4), yellow crystals were
Thiosemicarbazone (2a) (0.15 g, 0.57 mmol), anhydrous sodium
acetate (0.18 g, 2.28 mmol), and 50 mL ethanol were added to a
100 mL round bottom flask under magnetic stirring and slightly
warmed for 10e15 min. Then, 2-bromoacetophenone (0.11 g,
0.57 mmol) was added, and the reaction acquired purple colour and
was kept under heating under reflux for 4 h. After cooling back to
r.t., the precipitate was filtered off and the solvent was evaporated
for half of its volume and then cooled to 0 ꢀC. A purple solid was
obtained, filtered in Büchner funnel with a sintered disc filter,
washed with cold water, and then dried in SiO2. Products were
obtained; yield ¼ 51%; M.p. (ꢀC): unidentified to 300 ꢀC; IR (KBr)
2938 (CeH), 1708 (C]O), 1647 (C]N), 745 (CeCl) cmꢁ1 1H NMR
.
(300 MHz, DMSO-d6):
CH); 7.85e7.89 (m, 4H, Ar); 8.57 (s, 1H, NH). 13C NMR (75.5 MHz,
DMSO-d6): 115.0 (SeC]C); 125.3 (CH Ar); 128.8 (CH Ar); 129.0
d 7.40 (d, 2H, Ar); 7.62 (d, 2H, Ar); 7.71 (s, 1H,
d
(CH Ar); 131.9 (CH Ar). 132.2 (Ar); 133.5 (Ar); 133.7 (CeCl Ar); 140.5
(HC]C); 144.1 (C]N); 159.8 (C]O); 161.3 (C]O). Anal. Calcd. For
(6b): C, 56.33; H, 2.63; N, 10.95; S, 8.35; Found: C, 56.20; H, 2.32; N,
10.96; S, 8.17. Rf: 0.526.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
501
purified by recrystallization using the solvent system detailed
below for each compound.
(m, 4H, CH Ar); 11.99 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d 37.4 (CH2); 55.1 (CH3); 101.1 (CH heterocycle); 113.9 (CH Ar); 123.1
(CH Ar); 126.7 (CH Ar); 127.5 (CH Ar); 131.7 (Ar); 134.5 (Ar); 138.0
(Ar); 150.2 (NeC]C); 154.1 (HC]N); 158.7 (C]N); 167.6 (C]O);
168.1 (C]O). Anal. Calcd. For (7e): C, 61.21; H, 4.11; N, 14.28; S, 8.17;
Found: C, 61.03; H, 4.28; N, 13.91; S, 7.87. Rf: 0.63.
4.5.1. 2-(4-Phenylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-
dione (7a)
After crystallization with water, purple crystals were obtained;
yield ¼ 59%; M.p. (ꢀC): 207e209; IR (KBr) 3262 (NeH), 3039
(CeH), 1767 and 1712 (C]O), 1561 (C]N) cmꢁ1
.
1H NMR
4.5.6. 2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazono)ethyl)
isoindoline-1,3-dione (7f)
(300 MHz, DMSO-d6):
d 4.44 (d, 2H, CH2); 7.16 (s, 1H, CH hetero-
cycle); 7.26 (t, 1H, CH); 7.36 (t, 2H, Ar, 1H, CeH); 7.77 (d, 2H, Ar);
After crystallization with water, white crystals were obtained;
7.91 (m, 4H, Ar); 11.88 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
yield ¼ 72%; M.p. (ꢀC): 190; IR (KBr) 3471 and 3403 (NeH), 2955
d
37.4 (CH2); 104.4 (SeCH heterocycle); 124.2 (CH Ar); 126.4 (CH
(CeH), 1770 and 1716 (C]O), 1560 (C]N), 719 (CeBr) cmꢁ1 1H
.
Ar); 128.5 (CH Ar); 129.6 (CH Ar); 132.7 (CH Ar); 135.6 (Ar); 139.1
(HC]N); 151.3 (C]N); 168.6 (C]O); 169.2 (C]O). Anal. Calcd. For
(7a): C, 62.97; H, 3.89; N, 15.46; S, 8.85; Found: C, 62.69; H, 4.19; N,
15.68; S, 8.98. Rf: 0.625.
NMR (300 MHz, DMSO-d6): d 4.40 (d, 2H, CH2); 7.28 (s, 1H, CH
heterocycle); 7.31 (t, 1H, CH); 7.49 (d, 2H, Ar); 7.57 (d, 2H, Ar);
7.85e7.94 (m, 4H, Ar); 11.73 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6):
d 37.4 (CH2); 105.0 (CH heterocycle); 120.0 (CeBr); 123.2 (CH
Ar); 129.7 (CH Ar); 131.2 (CH Ar); 131.7 (CH Ar); 134.3 (Ar); 134.5
(Ar); 138.5 (NeC]C); 155.7 (HC]N); 164.4 (C]N); 167.6 (C]O).
Anal. Calcd. For (7f): C, 51.71; H, 2.97; N, 12.70; S, 7.27; Found: C,
51.42; H, 3.13; N, 12.93; S, 7.17. Rf: 0.73.
4.5.2. 2-(4-Methylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-
dione (7b)
After crystallization with toluene, brown crystals were ob-
tained; yield ¼ 55%; M.p. (ꢀC): 209e210; IR (KBr) 3410 (NeH), 2934
(CeH), 1772 and 1715 (C]O), 1639 (C]N) cmꢁ1. 1H NMR (300 MHz,
4.5.7. 2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazono)ethyl)
isoindoline-1,3-dione (7g)
After crystallization with water, white crystals were obtained;
yield ¼ 73%; M.p. (ꢀC): 216; IR (KBr) 3465 (NeH), 3049 (CeH), 1774
and 1719 (C]O), 1550 (C]N), 720 (CeCl) cmꢁ1. 1H NMR (300 MHz,
DMSO-d6):
heterocycle); 7.30 (t, 1H, CH); 7.84e7.92 (m, 4H, Ar); 11.51 (s, 1H,
NH). 13C NMR (75.5 MHz, DMSO-d6):
18.1 (CH3); 37.4 (CH2); 103.0
d 2.08 (s, 3H, CH3); 4.39 (d, 2H, CH2); 6.20 (s, 1H, CH
d
(SeCH heterocycle); 124.1 (CH Ar); 132.7 (CH Ar); 135.5 (Ar); 138.9
(NeC]C); 148.3 (HC]N); 155.0 (C]N); 168.6 (C]O); 168.9 (C]
O). Anal. Calcd. For (7b): C, 55.99; H, 4.03; N, 18.65; S, 10.68; Found:
C, 55.76; H, 4.37; N, 18.48; S, 10.87. Rf: 0.3.
DMSO-d6):
Ar); 7.44 (d, 2H, Ar); 7.53 (t, 1H, CH); 7.84e7.95 (m, 4H, Ar); 11.85 (s,
1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
37.4 (CH2); 104.5 (CH
d 4.25 (d, 2H, CH2); 7.28 (CH heterocycle); 7.32 (d, 2H,
d
heterocycle); 123.1 (CH Ar); 127.1 (CH Ar); 128.5 (CH Ar); 131.6 (CH
Ar); 131.8 (Ar); 134.5 (Ar); 138.3 (Ar); 150.2 (NeC]C); 155.7 (HC]
N); 159.3 (C]N); 167.5 (C]O); 168.2 (C]O). Anal. Calcd. For (7g):
C, 57.50; H, 3.30; N, 14.12; S, 8.08; Found: C, 57.41; H, 3.44; N, 14.02;
S, 7.88. Rf: 0.75.
4.5.3. 2-(4-(4-Fluorophenyl)thiazol-2-yl)hydrazono)ethyl)
isoindoline-1,3-dione (7c)
After crystallization with water, white crystals were obtained;
yield ¼ 69%; M.p. (ꢀC): 214e126; IR (KBr) 3460 (NeH), 3102 (CeH),
1772 and 1718 (C]O), 1571 (C]N), 1394 (CeF) cmꢁ1 1H NMR
.
(300 MHz, DMSO-d6):
cycle); 7.35 (t,1H, CH); 7.77e7.82 (m, 4H, Ar); 7.85e7.94 (m, 4H, Ar);
11.88 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
37.4 (CH2); 104.1
(SeCH heterocycle); 116.6 (CH Ar); 124.2 (CH Ar); 128.4 (CH Ar);
132.3 (CH Ar); 132.7 (Ar); 135.6 (Ar); 139.3 (CeF Ar); 150.2 (NeC]
C); 150.3 (HC]N); 160.9 (C]N); 168.7 (C]O); 169.4 (C]O). Anal.
Calcd. For (7c): C, 59.99; H, 3.44; N, 14.73; S, 8.43; Found: C, 60.08;
H, 3.61; N, 15.01; S, 8.24. Rf: 0.625.
d
4.42 (d, 2H, CH2); 7.12 (s, 1H, CH hetero-
4.5.8. 2-(4-p-Tolylthiazol-2-yl)hydrazono)ethyl)isoindoline-1,3-
dione (7h)
d
After crystallization with water, white crystals were obtained;
yield ¼ 53%; M.p. (ꢀC): 209e210; IR (KBr) 3281 (NeH), 3029 (CeH),
1775 and 1717 (C]O), 1552 (C]N) cmꢁ1 1H NMR (300 MHz,
.
DMSO-d6):
d 2.34 (s, 3H, CH3); 4.13 (d, 2H, CH2); 6.98 (s, 1H, CH
heterocycle); 7.38 (t, 1H, CH); 7.52 (d, 2H, Ar); 7.65 (d, 2H, Ar);
7.87e7.92 (m, 4H, Ar); 11.80 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6): d 21.3 (CH3); 37.4 (CH2); 102.8 (CH heterocycle); 123.5 (CH Ar);
4.5.4. 2-(4-(4-Nitrophenyl)thiazol-2-yl)hydrazono)ethyl)
isoindoline-1,3-dione (7d)
After crystallization with water, yellow crystals were obtained;
yield ¼ 72%; M.p. (ꢀC): 240; IR (KBr) 3229 (NeH), 3110 (CeH), 1768
and 1705 (C]O), 1572 (C]N), 1510 and 1345 (NO2) cmꢁ1. 1H NMR
125.8 (CH Ar); 129.5 (CH Ar); 132.1 (CH Ar); 132.4 (Ar); 134.9 (Ar);
137.1 (Ar); 138.5 (NeC]C); 154.7 (HC]N); 151.8 (C]N); 168.0 (C]
O); 168.6 (C]O). Anal. Calcd. For (7h): C, 63.81; H, 4.28; N, 14.88; S,
8.52; Found: C, 63.77; H, 4.26; N, 14.69; S, 8.29. Rf: 0.684.
(300 MHz, DMSO-d6):
1H, CH); 7.86e7.98 (m, 4H, Ar); 8.05 (d, 2H, Ar); 8.22 (d, 2H, Ar);
11.96 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
37.4 (CH2); 108.3
(CH heterocycle); 123.2 (CH Ar); 124.1 (CH Ar); 126.3 (CH Ar); 131.7
(CH Ar); 134.6 (Ar); 140.6 (Ar); 146.1 (Ar); 150.2 (NeC]C); 154.7
(HC]N); 160.9 (C]N); 167.6 (C]O); 168.8 (C]O). Anal. Calcd. For
(7d): C, 56.01; H, 3.22; N, 17.19; S, 7.87; Found: C, 55.78; H, 3.27; N,
16.95; S, 8.15. Rf: 0.71.
d
4.42 (d, 2H, CH2); 7.39 (t, 1H, CH); 7.57 (s,
4.6. Molecular modelling
d
The structures of all compounds were obtained by applying the
RM1 method [55], which is available as part of the SPARTAN'08
program [56] by using internal default settings for convergence
criteria. Docking calculations and analyses were carried out using
the structure of human IkB Kinase b e IKKb e (PDB ID code: 4KIK)
as the target, which is composed of a co-crystallized complex with
the inhibitor, referred as “K252A” [57]. The active site was defined
as all atoms within a radius of 6.0 Å from the co-crystallized ligand.
The residues LEU21, THR23, LYS44, MET96, GLU97, TYR98, CYS99,
ASP103, ILE165 and ASP166 were treated as flexible during the
calculations. The GOLD 5.2 program [58] was used for docking
calculations. Next, the Binana program [59] was used to analyse the
molecular interactions present in the best docking solutions, using
default settings except for the hydrogen bond distance, which was
4.5.5. 2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazono)ethyl)
isoindoline-1,3-dione (7e)
After crystallization with water, white crystals were obtained;
yield ¼ 84%; M.p. (ꢀC): 225; IR (KBr) 3273 (NeH), 3115 (CeH), 1766
and 1711 (C]O), 1563 (C]N), 1248 (CeO) cmꢁ1. 1H NMR (300 MHz,
DMSO-d6):
d 3.7 (d, 2H, CH2); 3.85 (s, 3H, CH3); 7.05 (d, 2H, Ar); 7.28
(s, 1H, CH heterocycle); 7.50 (t, 1H, CH2); 7.55 (d, 2H, Ar); 7.85e7.88

502
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 96 (2015) 491e503
changed to a maximum of 3.5 Å. Figures were generated with
Pymol [60].
4.7.5. Cytokine determinations
Cytokine concentrations were determined in cell-free culture
supernatants using specific sandwich ELISA kits for each cytokine,
following the manufacturer's instructions (Duoset, R&D Systems,
Minneapolis, MN, EUA).
4.7. Biological in vitro evaluation
4.7.1. Measuring cytotoxicity against tumour cell lines by MTT assay
The cytotoxicity of the compounds was evaluated by MTT assay
against three human cancer cell lines: SF-295 (nervous system),
HCT-8, (colon) and MDA/MB-435 (melanoma), all of which were
obtained from the National Cancer Institute (NCI, Bethesda, MD,
USA). Cell lines were maintained in RPMI 1640 medium supple-
mented with 10% foetal bovine serum, 2 mM glutamine, 100 U/mL
penicillin and 100 mg/mL streptomycin, at 37 ꢀC with 5% CO2.
Tumour cell proliferation was quantified indirectly through the
ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to form a
purple formazan product [61]. Briefly, cells were plated in 96-well
Acknowledgements
We would like to thank the Brazilian National Research Council
(CNPq) and the Research Foundation of Pernambuco State (FACEPE)
for financial support. M.V.O.C. is recipient of a FACEPE scholarship
(BFP-0107-4.03/12). A.C.L.L. is recipient of a CNPq fellowship
(308806/2013-1). C.P. is grateful to FUNCAP (Fundaçao Cearense de
Apoio ao Desenvolvimento Científico de Tecnologico). P.M.P.F. is
also grateful to FAPEPI (Fundaçao de Amparo a Pesquisa do Estado
do Piauí) for financial support. Our thanks are also due to the
Department of Chemistry at the Federal University of Pernambuco
(UFPE) for recording the NMR (1H and 13C), IR spectra and the
elemental analysis of all compounds.
~
ꢀ
~
ꢁ
plates and the compounds (50 mg/mL) were added to wells. After
69 h of incubation, the supernatant was replaced with fresh me-
dium containing 10% MTT. Three hours later, the MTT formazan
product was dissolved in 150 mL DMSO, and the absorbance was
measured at 595 nm (DTX-880, Beckman Coulter). Doxorubicin
Appendix A. Supplementary data
(Dox, Sigma Aldrich) was used as a positive control (0.3 mg/mL). To
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.041.
avoid false proliferation data, the experiments were performed in
triplicate and the proliferation rate was always compared to
negative controls. All replicates had similar inhibition rates, and cell
proliferation rates in negative control wells were higher than those
in the treated wells.
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Design, synthesis and antiinflammatory activity of novel phthalimide
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final volume of 0.2 mL. Thalidomide and dexamethasone were used
as reference drugs. Cell supernatants were collected at 24 h of in-
cubation to determine IFN-
g and IL-2 levels.

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