Antiplatelet activity and TNF-α release inhibition of phthalimide derivatives useful to treat sickle cell anemia

Article abstract of DOI:10.1007/s00044-019-02371-z

Sickle Cell Anemia (SCA) is one of the most prevalent hereditary hematological diseases worldwide. The disease is characterized by chronic inflammation, hypercoagulable state, and pro-thrombotic profile, which lead the vaso-occlusive process. In this work

Full text of DOI:10.1007/s00044-019-02371-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02371-z
ORIGINAL RESEARCH
Antiplatelet activity and TNF-α release inhibition of phthalimide
derivatives useful to treat sickle cell anemia
Rafael C. Chelucci1 Isabela J. de Oliveira1 Karina P. Barbieri1 Maria E. Lopes-Pires2 Marisa C. Polesi1
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Diego E. Chiba1 Iracilda Z. Carlos1 Sisi Marcondes2 Jean L. Dos Santos1 ManChin Chung1
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Received: 22 February 2019 / Accepted: 22 May 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Sickle Cell Anemia (SCA) is one of the most prevalent hereditary hematological diseases worldwide. The disease is
characterized by chronic inflammation, hypercoagulable state, and pro-thrombotic profile, which lead the vaso-occlusive
process. In this work, we described the antiplatelet activity and the ability to reduce tumor necrosis factor-alpha (TNF-α)
levels of phthalimide derivatives. All compounds inhibited platelet aggregation induced by collagen and adenosine
diphosphate, at levels ranging from 26.0 to 74.2% and 30.7 to 79.6%, respectively. The compounds exhibited reduced
bleeding time compared to acetylsalicylic acid (ASA). Moreover, compounds 4c and 10c inhibited TNF-α levels at 73.5%
and 65.0%, respectively. These findings suggest that phthalimide derivatives 4c and 10c are promising lead compounds
useful to prevent vaso-occlusion and inflammation associated with the sickle cell anemia.
Graphical Abstract
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Keywords Sickle cell disease Vaso-occlusion Phthalimide Platelet aggregation inhibition TNF-α inhibition.
Introduction
Sickle Cell Anemia (SCA) is a chronic hereditary hemato-
logical disease caused by a single mutation in the HBB
gene, which encodes hemoglobin subunit beta. This muta-
* Jean L. Dos Santos
jean.santos@unesp.br
tion causes the replacement of glutamic acid (Glu) to valine
1
(Val) on the surface of the variant β-globin chain (βs-glo-
School of Pharmaceutical Sciences, São Paulo State University
(UNESP), Araraquara, São Paulo, Brazil
bin) of hemoglobin S (HbS) (Ingram 1957). Interactions
among βs-globin chains, at low oxygen tensions, lead to
polymers formation inside HbS and alter the red blood cell
2
Faculty of Medical Science, State University of Campinas
(Unicamp), Campinas, São Paulo, Brazil

Medicinal Chemistry Research
(RBC) cytoskeletal structure to sickle-shaped cells (Kato
et al. 2018). RBC containing HbS are prone to suffer
hemolysis, releasing hemoglobin and arginase into the
plasma. Arginase reduces the levels of arginine, a substrate
for endothelial nitric oxide synthase, and decreases the
levels of nitric oxide (NO). In addition, the released
hemoglobin kidnaps NO produced by endothelium con-
ferring a vaso-constrictor character to SCA patients. The
reduced bioavailability of NO also results in activation of
endothelial cells (increasing the expression of adhesion
molecules) and platelets (increasing the expression of P-
selectin and αIIbβ3 integrin; Kato et al. 2017).
Platelets have an important role in the vaso-occlusion
process of SCA (Davila et al. 2015). Several markers of
activated platelet are increased such as β-thromboglobulin,
thrombospondin and chemokine (C-X-C motif) ligand 4
(Davila et al. 2015; Sugiharam et al. 1992). Platelets are
also found in circulation as heterocellular aggregates and
contribute to the innate immune system through cytokines
releasing (Davila et al. 2015; Zhang et al. 2016). Therapies
containing antiplatelet drugs should be an alternative to
prevent cardiovascular diseases in SCA patients, once these
patients exhibit abnormal activation of the fibrinolytic sys-
tem, increased tissue factor expression and high risks of
thrombotic complications (Noubouossie et al. 2016).
However, despite the efficacy of antiplatelet therapies to
reduce platelet aggregation in SCA patients, clinical trials
have failed to demonstrate the ability of these drugs to
prevent vaso-occlusion complications (Heeney et al. 2016).
This lack of effectiveness may be result from the com-
plexity of the disease, which involves other phenomenons
such as ischemia-reperfusion injury, chronic inflammation,
hemolysis, vaso-occlusion and NO deficiency (Noubouossie
et al. 2016).
Therefore, compounds designed to act through multiple
targets are promising strategies to find out an alternative to
hydroxyurea and glutamine – the only approved drugs to
treat SCA. Our research group has previously described that
compound 3-(1,3-dioxoisoindolin-2-yl)benzyl nitrate (4c)
exhibits multiple effects such as in vivo analgesic/anti-
inflammatory activities and NO-donor properties. Trans-
genic sickle mice treated with this compound for three
weeks have reduced priapism, reversed PDE5 protein
expression and reduced reactive-oxygen species markers in
the penises of SCA mice (Silva et al. 2016). In addition,
compound (4c) increased ~53% the fetal hemoglobin (HbF)
levels when compared to the vehicle, using a transgenic
SCA mouse model. This effect was superior to that of
hydroxyurea in the same assay (Lanaro et al. 2017).
In this study, we described the antiplatelet activity and
tumor necrosis factor-alpha (TNF-α) release from a culture
of mice monocytes of several phthalimide derivatives con-
taining a NO-donor subunit. Two different NO-donors
represented by organic nitrate esters and furoxan (1,2,5-
oxadiazole-2-N-oxide) were selected. In addition to anti-
platelet and vasodilatory effects, NO also contributes to
increase the levels of HbF. It has been reported that NO/
cGMP pathway plays an important role in γ-globin
expression by activating transcription factors, including c-
Jun and c-Fos (Perrine et al. 2014).
The phthalimide subunit, presented in the drug thalido-
mide, was selected due to its ability to reduce the levels of
TNF-α in culture of monocytes. The structure-activity
relationship shows that phthalimide subunit is the primary
pharmacophore responsible for reducing TNF-α levels,
being, therefore, selected in our studies (Miyachi et al.
1997; Muller et al. 1999). It was demonstrated that SCA
patients exhibit high levels of TNF-α in circulation aggra-
vating painful vaso-occlusive crisis and inflammatory epi-
sodes (Dworkis et al. 2011). Beyond the anti-inflammatory
effect, thalidomide also increases γ-globin mRNA expres-
sion through activation of p38 mitogen-activated protein
kinase (MAPK) signaling pathway along with histone H4
hyperacetylation (Aerbajinai et al. 2007).
Material and methods
General synthesis information
Reagents and solvents were purchased from commercial
suppliers at reagent purity grade and were used without any
further purification. Dry solvents used in the reactions were
obtained by distillation of technical grade materials over
appropriate dehydrating agents. Melting points were mea-
sured with an electrothermal melting-point apparatus
Stuart™ melting point apparatus SMP3 (Bibby Stuart Sci-
entific, Cole-Parmer, Staffordshire, UK) in open capillary
tubes and are uncorrected. Infrared spectra (range of
400–4000 cm−1) were obtained using KBr pellets in the
FTIR-8300 infrared spectrophotometer (Shimadzu Seisa-
kusho Co. Ltd., Kyoto, Japan). One-dimensional nuclear
1
magnetic resonance spectra of H and 13C were obtained
1
using the Fourier 300 spectrometer with Dual probe H/13C
(Bruker Corporation, Billerica, Massachusetts, EUA).
Chemical shifts are reported in parts per million (ppm - δ)
relative to tetramethylsilane, and the coupling constants (J)
are shown in Hertz (Hz). HPLC analysis was performed on
an LC-10AD chromatograph equipped with a model SPD-
10A UV–Vis detector (Shimadzu Seisakusho Co., Ltd.,
Kyoto, Japan). All compounds were analyzed by HPLC,
and their purity was confirmed to be >98.5%. The mass
spectra were obtained using an LCMS-8045 spectrometer
(Shimadzu Corporation, Kyoto, Japan), with triple quadru-
pole detector and electrospray ionization (ESI). Compounds
were separated on a reversed phase C18 column (5 µm

Medicinal Chemistry Research
particle, 250 mm × 4.6 mm I.D.) Shim-pack CLC-ODS(M)®
C18 (M). HPLC-grade solvents (acetonitrile, methanol,
acetic acid, and toluene) were used in the analyses. Thin
layer chromatography (TLC) analyses were performed on
TLC Silica gel 60 FSigma®, to monitor the reactions and
purifications of the synthesized compounds. Substances
were visualized in ultraviolet light (254 and 365 nm), and/or
by exposure to powdered iodine and/or functional group
developer when necessary. Merck silica gel (70–230 mesh)
was used for preparative column chromatography. The final
compounds 4a-e, 6a-b, and 10a-c and the intermediate
compounds 3a-e, 7 and 9a-c were previously obtained in
our laboratory (Dos Santos et al. 2011; Dos Santos et al.
2012) however, the synthetic methodology adopted in this
work to obtain 6b was altered and is shown below. Com-
pound 8 is unprecedented and its synthetic procedure and
structural characterization are demonstrated in this work and
in the supplemental material.
CH2-O); 7.67-7.62 (2 H; m; Ar-H); 7.36 (2 H; d; Jortho =
8,0 Hz; Ar-H); 2.45 (3 H; s; Ar-CH3).
4-(2-(1,3-dioxoisoindolin-2-yl) ethoxy)-3-(phenylsulfo-
nyl)-1,2,5-oxadiazole 2-N-oxide (6b): white powder, yield
73%; mp 150-155 °C. IR Vmax (cm−1; KBr pellets): 1780.3
and 1718.5 (C = O imide); 1620.2 and 1550.7 (C = N);
1363.6 and 1168.8 (O = S = O sulfone). 1H-NMR
(300 MHz, CDCl3), 7.78-7.76 (2 H; m; Ar-H); δ 7.91-7.89
(2 H; m; Ar-H); 4.20 (2 H; t; N-CH2-CH2-O); 4.69 (2 H; t;
N-CH2-CH2-O); 8.04-8.01 (2 H; m; Ar-H); 7.60-7.54 (2 H;
m; Ar-H); 7.74-7.69 (1 H; m; Ar-H). 13C-NMR (75 MHz,
CDCl3), 134.4 (CH, C-23, C-24); 123.7 (CH, C-22, C-25);
132.0 (C, C-21, C-26); 168.0 (C = O, C-20, C-18); 36.2 (N-
CH2-CH2-O, C-17); 68.0 (N-CH2-CH2-O, C-16); 158.6 (C,
C-4); 110.5 (C, C-3); 138.1 (SO2-C, C-9); 128.80 (CH, C-
10, C-14); 129.7 (CH, C-11, C-13); 135.6 (CH, C-14). MS-
ESI (m/z): C18H13N3O7S [M + H]+ 415.55.
General procedure for the synthesis of 4-(3-((4-(1,3-diox-
oisoindolin-2-yl)benzoyl)oxy) propoxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-N-oxide (8)
Synthetic procedures
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride (EDC) (2.7 mmol) and 4-dimethylaminopyridine
(DMAP) (0.2 mmol) was added in a solution containing 4-
(1,3-dioxoisoindolin-2-yl) benzoic acid (7) (1.5 mmol) in
dichloromethane (15.0 mL) at 0 °C. Then, it was slowly
added the intermediate 4-(3-hydroxypropoxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-N-oxide (11) (1.5 mmol)
(Cena et al. 2001). The reaction was stirring at room tem-
perature under nitrogen atmosphere for 4 h. The crude was
diluted with dichloromethane (50.0 mL) and it was washed
using brine and water (3 × 15.0 mL). The organic phase was
dried with sodium sulfate and dried under reduced pressure.
The crude was purified by column chromatography using
silica gel (60 Å pore size; 40–60 μm particle size) and
mobile phase hexane: ethyl acetate (60:40; v/v) in
isocratic mode.
General procedure for the synthesis of 4-(2-(1,3-dioxoi-
soindolin-2-yl)
ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadia-
zole 2-oxide (6b)
The synthesis of compound (6b) involves two steps.
First, it was synthesized the furoxan derivative 3-(phe-
nylsulfonyl)-4-(2-(tosyloxy)ethoxy)-1,2,5-oxadiazole 2-N-
oxide. A mixture of 4-(2-hydroxyethoxy)-3-(phenylsulfo-
nyl)-1,2,5-oxadiazole 2-N-oxide (1.5 mmol) and triethyla-
mine (4.5 mmol) was added in dry dichloromethane
(10.0 mL) at 0 °C. Then, using addition funnel, p-toluene-
sulfonyl chloride (2.5 mmol) previously solubilized in dry
dichloromethane (10.0 mL) was added slowly to the med-
ium. The reaction was stirring at room temperature under
nitrogen atmosphere for 3 h. The solvent was dried under
reduced pressure to provide a crude, which was purified by
column chromatography (5.0 cm diameter column; sta-
tionary phase silica: 60 Å pore size; 40–60 μm particle size;
mobile phase: hexane: ethyl acetate (80:20 → 60:40, v/v) in
gradient mode).
In the second step for the synthesis of compound (6b), a
mixture of 3-(phenylsulfonyl)-4-(2-(tosyloxy)ethoxy)-1,2,5-
oxadiazole 2-N-oxide (7.5 mmol) and potassium phthali-
mide (5) (7.5 mmol) was added in N,N-dimethylformamide
(DMF) (15.0 mL). The reaction was stirring at room tem-
perature under nitrogen atmosphere for 24 h. After, ice-cold
water (10.0 mL) was added to precipitate the product, which
was filtrated and washed with distilled water.
4-(3-((4-(1,3-dioxoisoindolin-2-yl)benzoyl)oxy)pro-
poxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-N-oxide (8):
White powder, yield 41%; mp 176-180 °C. IR Vmax (cm-1;
KBr pellets): 1.716 (C = O imide); 1.650 (C = O ester);
1
1.384 (O = S = O sulfone). H-NMR (300 MHz, CDCl3);
7.84-7.81 (m; 2 H; Ar-H); 7.99-7.96 (m; 2 H; Ar-H); 7.65-
7.60 (m; 4 H; Ar-H); 8.18 (dt; Jortho = 8.8 Hz/Jmeta = 2.2 Hz;
2 H; Ar-H); 4.61 (t; 2 H; CH2); 2.38 (q; 2 H; CH2); 4.55 (t;
2 H; CH2); 8.08-8.05 (m; 2 H; Ar-H); 7.75 (tt; Jortho
=
7.5 Hz/Jmeta = 1.3 Hz; 1 H; Ar-H). 13C-NMR (75 MHz,
CDCl3), 134.8 (CH, C-33, C-34); 124.1 (CH, C-32, C-35);
131.6 (C, C-31, C-36); 166.9 (C = O, C-28, C-30); 136.3
(N-C, C-24); 126.2 (CH, C-23, C-25); 130.6 (CH, C-22, C-
26); 129.0 (C-C = O, C-21); 165.6 (C = O, C-20); 61.1 (O-
CH2-CH2-CH2-O, C-18); 28.2 (O-CH2-CH2-CH2-O, C-17);
68.1 (O-CH2-CH2-CH2-O, C-16); 159.0 (C, C-4); 110.6 (C,
C-3); 138.1 (SO2-C, C-9); 128.6 (CH, C-10, C-14); 129.8
3-(phenylsulfonyl)-4-(2-(tosyloxy)ethoxy)-1,2,5-oxadia-
zole 2-N-oxide: white powder, yield 78%; mp 117-120 °C.
1H-NMR (300 MHz, CDCl3), 7.77-7.75 (1 H; m; Ar-H);
7.83-7.80 (2 H; m; Ar-H); 8.08-8.05 (2 H; m; Ar-H); 4.62-
4.59 (2 H; m; O-CH2-CH2-O); 4.43-4.40 (2 H; m; O-CH2-

Medicinal Chemistry Research
(CH, C-11, C-13); 135.8 (CH, C-14). MS-ESI (m/z):
C26H19N3O9S [M + H]+ 550.20.
results were presented as mean standard error of the
mean (SEM).
Animals
Bleeding time assay
Adult males of Mus muscules (Swiss albino), weighing
~25–30 g, were obtained from the central laboratory of São
Paulo State University (UNESP). The animals were main-
tained under controlled conditions of temperature (22 2 °
C), humidity and light (dark cycle/light 12 h/12 h). The
animals had access to food and water ad libitum. The
experiment was previously approved by the Institutional
Ethics Committee of the Faculty of Pharmaceutical Sci-
ences (FCFAr) as described below in the section “com-
pliance with ethical standards”.
Campus Araraquara (CEUA / FCFAr) under No. 37/
2013, 38/2013 and 09/2014. All experiments were per-
formed in accordance with current guidelines for laboratory
animal care and ethical guidelines for the investigation of
experimental pain in conscious animals.
The bleeding time assay was performed as described pre-
viously (Dejana et al. 1979). Mus musculus mice (Swiss
albino) (n = 6) received the compounds (4a-e; 6a-b; 8 and
10a-c), ASA (positive control) at concentration of 100 µM
or carboxymethylcellulose (CMC) 0.5% (negative control)
orally. After 60 min, the animals were anesthetized with
ketamine hydrochloride (100 mg/Kg) by intraperitoneal (IP)
route. An incision of 2.0 mm counted from the end of the
tail was performed and the bleeding time registered from
that moment. It was determinate 15 min as the maximum
time if bleeding remains continuously. Statistical analysis
was performed using the statistical program GraphPad
Prism version 6.01 for Windows (GraphPad Software, San
Diego, California, USA) applying ANOVA with a sig-
nificance level as a p-value <0.05 (p < 0.05), followed by
Tukey’s multiple comparisons test. The results were
expressed as mean SEM.
Antiplatelet activity assay
The antiplatelet activity assay was performed according to
the method described previously (Born and Cross, 1963).
Blood from mice was collected and added to 3.8% triso-
dium citrate (9:1 v/v). The platelet-rich plasma (PRP) was
obtained by centrifugation of whole blood (200 × g) at room
temperature for 15 min. Washing buffer (140.0 mM NaCl,
0.5 mM KCl, 12.0 mM trisodium citrate, 10.0 mM glucose
and 12.5 mM sucrose, pH 6.0) in the ratio 7:5 (buffer/
plasma) was added to PRP and centrifuged for 13 minutes
(800 × g), twice. Platelets were suspended in Krebs-Ringer
solution devoid of calcium ions and the platelet count was
adjusted to 1.2 × 10-8 platelets/mL by manual counting
using the Neubauer chamber. Calcium chloride (CaCl2) was
added to the platelet suspension to a final concentration of
1.0 mM.
The platelet solution (400.0 μL) was transferred to the
aggregation cuvette and taken to the two-channel aggreg-
ometer (Chrono-log Lumi-Aggregometer model 560-Ca,
Harvertown, PA, USA). Platelets were incubated in pre-
sence of compounds (4a-e; 6a-b; 8 and 10a-c) at con-
centration of 150 µM, dissolved in 0.1% DMSO, for
3 minutes prior to the addition of ADP (10.0 µM) or col-
lagen (5.0 µg/mL). The aggregation was monitored for
10 minutes. Acetylsalicylic acid (ASA) at 150.0 μM was
used as positive control.
TNF-α cytokine inhibition assay
The determination of cell viability and the detection of
TNF-α of the compounds (4a-e; 6a-b; 8 and 10a-c) were
performed using Mus musculus mice (Swiss albino). The
animals were previously inoculated by IP route with 3.0 mL
of a 3% sodium thioglycolate solution. After 72 h of sti-
mulation, the animals were euthanized in a CO2 chamber.
The peritoneum was exposed in laminar flow for removal of
the peritoneal exudate. Cells removed from the peritoneal
exudate were washed three times with 5.0 mL of phosphate
buffered saline (PBS) (pH 7.2; 4 °C) and centrifuged (400 ×
g/5 min) at room temperature. The pelleted cells were then
suspended in complete RPMI-1640-C culture medium. The
number of cells was determined using Neubauer’s chamber,
and trypan blue. The cell concentration was then diluted to
5 × 106 cell/mL. Cells were plated and incubated at 37 °C
for 60 min in an atmosphere containing 5% of CO2 for
adherence of macrophages.
The cell viability was determinate by a colorimetric
method in 96-well microplate using MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mos-
mann 1983). The cell and all compounds (4a-e; 6a-b; 8 and
10a-c) were incubated for 24 h at 37 °C in an atmosphere
containing 5% CO2, in presence of lipopolysaccharide LPS
1.0 μg/mL. After the contents of the plates were discarded
and 100.0 μL of MTT solution (1.0 g/mL) was added to
each well. The plate was incubated for 3 hours at 37 °C.
After this time, the contents of the plate were discarded
once again and 100.0 μL of isopropyl alcohol was added to
Statistical analysis was performed using GraphPad Prism
version 6.01 for Windows (GraphPad Software, San Diego,
California, USA), by analysis of variance (ANOVA) with
the determination of significance levels as p-value <0.05
(p < 0.05) from Tukey’s multiple comparisons test. The

Medicinal Chemistry Research
Scheme 1 The synthesis scheme
of phthalimides containing
organic nitrate ester subunit
(4a-e)
Scheme 2 The synthesis scheme of phthalimides containing furoxan subunit (6a-b; 8 and 10a-c)
each well. Absorbance reading was performed using UV/
Vis Spectrophotometer BioTeck® plate reader with wave-
length (λ) of 540 and 620 nm. The results of the absorbance
values were converted into a percentage of cell viability,
where the negative control corresponded to 100% of via-
bility. For quantification of the TNF-α levels, concentra-
tions with cell viability >70% were considered.
The levels of cytokine TNF-α were quantified in the
culture supernatants by enzyme-linked immunosorbent
assay ELISA (OptEIA; BD Biosciences, San Diego, CA)
according to the manufacturer’s instructions. The cytokine
concentrations were calculated from a standard curve of
known cytokine concentrations and expressed in pg/mL. In
all of the experiments, the results were expressed as the

Medicinal Chemistry Research
Table 1 Inhibition of platelet aggregation induced by collagen (5 µg/
mL), ADP (10 µM) and bleeding time for phthalimide derivatives
et al. 2012), compound (8) is novel and its synthetic pro-
cedure is described in the previous section. All furoxan
derivatives were coupled with phthalimides (7 and 9a-c),
Compounds
Inhibition of platelet
aggregation (%)
Bleeding
time (sec)
using
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(EDC) and dimethylaminopiridine (DMAP) as coupling
reactive, to obtain the final compounds (8 and 10a-c) at
yields ranging from 39 to 55% (Scheme 2). The alkyl
chloride or tosyl containing furoxan derivatives were pre-
pared through a reaction involving with the respective
alcohol (Cena et al. 2001; Dos Santos et al. 2012) with
thionyl chloride or p-toluenesulfonyl chloride. After, com-
pounds (6a-b) were obtained by nucleophilic substitution
using potassium phthalimide (2), with yields of 73–91%,
respectively (Scheme 2). The structures of all compounds
Collagen
(5 µg/mL)
ADP
(10 µM)
Negative control 27.2 5.4b
26.9 6.6b
26.6 11.4
58.3 7.3
41.3 8.3
52.3 5.9
48.0 7.1
79.7 3.3ab
58.2 4.7ab
52.3 6.9
30.8 5.6
40.4 9.6
47.5 7.0
55.0 8.0
369.0 23.0d
828.0 100.3c
448.2 37.3d
344.6 24.0d
276.0 16.3d
–
ASA
4a
64.4 4.8a
39.5 9.4
26.0 6.4
58.5 13.9
74.3 12.6a
72.5 14.8a
38.6 7.8
48.5 5.6
61.6 5.8
48.8 1.3
59.4 6.1
45.8 6.4
4b
4c
4d
4e
258.0 28.4d
998.8 183.4c
288.5 91.9d
276.0 45.9d
678,5 56.8
263.0 40.2d
398.6 48.0d
6a
1
were established by infrared spectroscopy, and H and 13C
nuclear magnetic resonance (1H and 13C NMR). All com-
pounds were analyzed by high-performance liquid chro-
matography and their purities were >98.5%.
6b
8
10a
10b
10c
Antiplatelet activity
Results are expressed as the mean SEM. All compounds were
evaluated at 150 μM. The statistical analysis used was one-way
ANOVA followed by Tukey’s multiple comparisons test
ap < 0.05 phthalimide derivatives vs. Negative control group
bp < 0.05 phthalimide derivatives vs. ASA group
cp < 0.05 phthalimide derivatives vs. Negative control group
The inhibition of platelet aggregation for all compounds
was evaluated in vitro using platelet-rich plasma in the
presence of ADP (10 μM) and collagen (5 μg/mL; Table 1)
(Born and Cross, 1963). All compounds, tested at 150 μM,
were able to inhibit the platelet aggregation induced by both
of these agonists. For collagen-platelets stimulated, the
percentage of inhibition ranged from 26.0 to 74.2%. The
most active compounds found were 4d and 4e, which
inhibited the aggregation platelet at 72.5 and 74.2%,
respectively. For ADP-platelet stimulated, the percentage of
inhibition ranged from 30.7 to 79.6%. The most active
compound found was 4e, which inhibited 79.6% of platelet
aggregation induced by ADP. Despite the differences of
NO-release levels among organic nitrate esters and furoxans
(Dos Santos et al. 2011; Dos Santos et al. 2012), it was not
found differences among these NO-donors to inhibit platelet
aggregation induced by both agonists. Only compound 4e
demonstrated activity superior to acetylsalicylic acid (ASA)
(Table 1), one of the most used antiplatelet drug in cardi-
ovascular diseases.
(bleeding time)
dp < 0.05 phthalimide derivatives vs. ASA group (bleeding time)
mean SEM. Each experiment was performed in triplicate
at least three times. One-way ANOVA with Tukey’s Mul-
tiple Comparison Test as a post hoc test was performed
using GraphPad Prism version 6.01 for Windows (Graph-
Pad Software, San Diego, California, USA). The statistical
significance was defined as p-value <0.05.
Results and discussion
Synthesis
Compounds (4a-e) were prepared in three followed steps
(Scheme 1). Intermediates (3a-e) were obtained by con-
densation of phthalic anhydride and phthalimide with
functionalized amines in yields ranging from 93 to 65%.
The last step, involving the nucleophilic substitution of
alcohol function to organic nitrate esters (4a-e), in reaction
medium containing acetic acid and nitric acid (99.5%), with
yields of 90-74% (Dos Santos et al. 2011).
Bleeding time
Bleeding is a serious adverse effect and one of the current
limitations of antiplatelet therapy. Assays aiming to char-
acterize the bleeding time are useful to evaluate platelet
function and the ability of the body to form clots. In this
work, we found for all compounds values of bleeding time
ranging from 258.0–998.7 s. Except for compound 6a, all
derivatives demonstrated reduced bleeding time compared
to ASA, which exhibits bleeding time of 828.0 s (Table 1).
The furoxan derivatives (10a-c) and the synthetic inter-
mediates (7 and 9a-c) were prepared according to proce-
dures previously described (Cena et al. 2001; Dos Santos

Medicinal Chemistry Research
Table 2 Reduction of TNF-α
levels in murine monocytes
cultures stimulated with LPS
induced by phthalimide
derivatives
Compounds
Reduction of TNF-α levels (%)
100.0 µM
50.0 µM
25.0 µM
12.5 µM
6.2 µM
3.1 µM
1.5 µM
Thalidomide
–
28.4 2.3
–
21.1 4.4
–
14.9 3.7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4a
4b
–
–
–
–
–
–
–
–
–
7.0 4.5
6.1 3.1
14.0 6.2
4c 73.5 5.9a 65.2 2.9a 38.3 8.4
4d 21.2 11.6
3.2 1.6
4.3 2.5
–
7.6 4.4
6.6 4.8
–
4e
6a
0.3 0.1
–
33.1 5.3 19.4 5.1 14.0 5.8
6b 19.6 5.4
23.8 7.1
13.8 5.8
22.1 10.6
5.7 3.9
17.0 6.7
37.5 10.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8
10a
10b
30.7 12.0 20.3 8.0
–
1.4 0.7
7.8 4
–
23.2 5.9
29.0 9.5
10c 65.0 9.3a 53.3 8.8
–
Results are expressed as the mean SEM of Inhibition of TNF-α (%). The statistical analysis used was one-
way ANOVA followed by Tukey’s multiple comparisons test
ap < 0.05 vs. thalidomide (50.0 µM) group
Quantification of TNF-α levels in the macrophage
supernatant
findings demonstrated that all compounds exhibit anti-
platelet effect. In addition, the compounds were able to
reduce TNF-α levels in murine monocytes cultures stimu-
lated with LPS. Compounds 4c and 10c were the most
potent to reduce TNF-α levels. Although compounds 4c and
10c were not the most active antiplatelet compounds, it was
found for these compounds an appropriate balance between
all investigated effects. Moreover, compounds 4c and 10c
have shown reduced bleeding time compared to ASA.
Considering the current limitations for SCA treatment,
compounds 4c and 10c have emerged as promising lead
compounds useful to prevent vaso-occlusion and inflam-
mation associated with the disease.
The ability of all compounds to decrease TNF-α was
characterized in murine monocytes cultures stimulated with
lipopolysaccharide (LPS). Initially, the cellular viability was
determined for all compounds in order to obtain the suitable
dose that could be used in the assay to quantify TNF-α
levels (Mosmann 1983). For this experiment, we have
considered only those concentrations with cellular viability
equal to or >95%. The results were expressed as a per-
centage of inhibition, comparing the values obtained with
those of positive control - thalidomide. Thalidomide was
selected with control once it has well-described TNF-α
inhibitory activity (Sampaio et al. 1991; Moreira et al.
1993). Also due to the fact that the structural planning for
the molecules evaluated in this work contains the phthali-
mide subunit present in thalidomide and responsible for its
inhibitory action of TNF-α. The reduction of TNF-α levels
ranged from 0.3 to 73.5%. The most active organic nitrate
ester and furoxan were 4c and 10c. At 100 μM, these
compounds inhibited TNF-α levels at 73.5 and 65.0%,
respectively (Table 2). At 50 μM, compounds 4c and 10c
were more active than thalidomide, inhibiting TNF-α at
65.2 and 53.3%, respectively.
Acknowledgements This study was supported by Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Ref. Process:
2013/23461-3; 2015/19531-1) and Conselho Nacional de Desenvol-
vimento Científico e Tecnológico (CNPq) (Ref. Process: 135821/
2011-8).
Compliance with ethical standards
Ethical approval The experiment was previously approved by the
Institutional Ethics Committee of the Faculty of Pharmaceutical Sci-
ences (FCFAr), Campus Araraquara (CEUA/FCFAr) under No. 37/
2013, 38/2013, and 09/2014. All experiments were performed in
accordance with current guidelines for laboratory animal care and
ethical guidelines for the investigation of experimental pain in con-
scious animals.
Conclusions
Conflict of interest The authors declare that they have no conflict of
interest.
In summary, we synthesized phthalimide derivatives (4a-e;
6a-b; 8 and 10a-c) containing different NO-donor subunit
represented by organic nitrate ester and furoxan. Our
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Medicinal Chemistry Research
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