Design, synthesis, and pharmacological evaluation of novel hybrid compounds to treat sickle cell disease symptoms

Article abstract of DOI:10.1021/jm200531f

A novel series of thalidomide derivatives (4a-f) designed by molecular hybridization were synthesized and evaluated in vitro and in vivo for their potential use in the oral treatment of sickle cell disease symptoms. Compounds 4a-f demonstrated analgesic, anti-inflammatory, and NO-donor properties. Compounds 4c and 4d were considered promising candidate drugs and were further evaluated in transgenic sickle cell mice to determine their capacity to reduce the levels of the proinflammatory cytokine tumor necrosis factor α (TNFα). Unlike hydroxyurea, the compounds reduced the concentrations of TNFα to levels similar to those induced with the control dexamethasone (300 μMol/kg). These compounds are novel lead drug candidates with multiple beneficial actions in the treatment of sickle cell disease symptoms and offer an alternative to hydroxyurea treatment.

Full text of DOI:10.1021/jm200531f

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Pharmacological Evaluation of Novel Hybrid
Compounds To Treat Sickle Cell Disease Symptoms
Jean Leandro dos Santos,*,† Carolina Lanaro,‡ Lídia Moreira Lima,§ Sheley Gambero,‡
Carla Fernanda Franco-Penteado,‡ Magna Suzana Alexandre-Moreira,||
Marlene Wade,^ Shobha Yerigenahally,^ Abdullah Kutlar,# Steffen E. Meiler,^
Fernando Ferreira Costa,‡ and ManChin Chung†
†Laboratꢀorio de Pesquisa e Desenvolvimento de Fꢀarmacos (Lapdesf), Departamento de Fꢀarmacos e Medicamentos,
Faculdade de Ci^encias Farmac^euticas, Universidade Estadual Paulista (UNESP), Rodovia Araraquara Jaꢀu Km. 01, 14801-902,
Araraquara, SP, Brazil
‡The Haematology and Haemotherapy Centre, University of Campinas (UNICAMP), Hemocentro, Rua Carlos Chagas, 480,
Cidade Universitꢀaria, Bar~ao Geraldo, 13083-970, Campinas, SP, Brazil
§Laboratꢀorio de Avaliac-~ao e Síntese de Subst^ancias Bioativas (LASSBio, http://www.farmacia.ufrj.br/lassbio/), Faculdade de Farmꢀacia,
Universidade Federal do Rio de Janeiro, P.O. Box 68024, 21944-971, Rio de Janeiro, RJ, Brazil
Laboratꢀorio de Farmacologia e Imunidade (LaFI), Instituto de Ci^encias Biolꢀogicas e da Saꢀude, Universidade Federal de Alagoas,
Maceiꢀo, AL, Brazil
^Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Augusta, Georgia, United States
#Sickle Cell Center, Medical College of Georgia, Augusta, Georgia, United States
ABSTRACT: A novel series of thalidomide derivatives (4aꢀf) designed
by molecular hybridization were synthesized and evaluated in vitro and
in vivo for their potential use in the oral treatment of sickle cell disease
symptoms. Compounds 4aꢀf demonstrated analgesic, anti-inflamma-
tory, and NO-donor properties. Compounds 4c and 4d were considered
promising candidate drugs and were further evaluated in transgenic sickle
cell mice to determine their capacity to reduce the levels of the
proinflammatory cytokine tumor necrosis factor R (TNFR). Unlike
hydroxyurea, the compounds reduced the concentrations of TNFR to
levels similar to those induced with the control dexamethasone (300
μmol/kg). These compounds are novel lead drug candidates with multi-
ple beneficial actions in the treatment of sickle cell disease symptoms and offer an alternative to hydroxyurea treatment.
’ INTRODUCTION
(FDA) to treat SCD. HU is commonly used to treat a variety of
myeloproliferative disorders, and its effectiveness in SCD has
been attributed to its capacity to stimulate the production of fetal
hemoglobin (HbF).7,8 After its metabolism, HU can act as an exo-
genous nitric oxide (NO) source, which is responsible for numerous
HU benefits, including the induction of γ-globin gene expression,
vasodilatation, and the inhibition of platelet aggregation.9ꢀ11
Individuals with SCD have shown reduced physiological NO
levels in the vascular endothelium, resulting from the hemolytic
process.12 Much interest has centered on understanding the
positive benefits of NO in the treatment of SCD after some
studies demonstrated that NO inhalation reduced the severity
and duration of vaso-occlusive crises in children.10,13
Sickle cell disease (SCD) is one of the most common genetic
disorders worldwide, caused by a point mutation that changes
glutamic acid (Glu6) to valine (Val6) in the β chain of hemoglobin.
At low oxygen tensions, after constant cycles of oxygenationꢀ
deoxygenation, the sickle hemoglobin molecule (HbS) polymerizes
inside the red blood cells, altering the erythrocyte cytoskeletal
structure and leading to hemolysis or the formation of sickle-
shaped red blood cells. The abnormal adhesion of these blood
cells to the vascular endothelium contributes to a vaso-occlusive
process.1ꢀ3 Patients with SCD present a multifaceted pathophy-
siology characterized by painful vaso-occlusive crises, acute chest
syndrome, priapism, pulmonary hypertension, stroke, and many
other disorders that reduce their life expectancy.4ꢀ6
SCD patients demonstrate a chronic inflammatory response,
with significantly increased levels of circulating proinflammatory
cytokines, such as tumor necrosis factor R (TNFR).14ꢀ16 High
The cruelest characteristic of SCD is that there is no specific
drug for its treatment. Pharmacological therapies are based on
drugs that alleviate the main symptoms of the disease. Hydro-
xyurea (HU), a well-known ribonucleotide reductase inhibitor, is
the only drug approved by the U.S. Food and Drug Administration
Received: April 29, 2011
Published: July 18, 2011
r
2011 American Chemical Society
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Scheme 1. Structural Planning Using Molecular Hybridization
levels of TNFR increase the chemotactic properties of neutro-
phils and their adhesion to the vascular endothelium, contributing to
the vaso-occlusion process. It has been reported that HU treat-
ment increases TNFR levels, counteracting the beneficial effects
of the drug.17 TNFR also stimulates the production of free radicals
and the synthesis of other inflammatory mediators, such as inter-
leukin 1 (IL1) and prostaglandin E2, induces changes in coagulants
and anticoagulants properties, and sensitizes nociceptive terminals,
aggravating pain crises.18,19 Increased blood levels of TNFR in
patients with sickle cell anemia can aggravate painful vaso-occlusive
crises and lead to infection and inflammatory episodes.20,21
Thalidomide, first described as a hypnotic and sedative drug in
the late 1950s, presents a well-known TNFR-inhibitor property.
Nowadays the drug is used as an immunomodulatory agent to
treat some diseases, such as multiple myeloma and leprosy.
Thalidomide was also demonstrated to increase the production
of reactive oxygen species, inducing γ-globin mRNA expression
in a dose-dependent manner. Aerbajinai and co-workers (2007)
postulated that this effect could be mechanistically associated
with the p38 mitogen-activated protein kinase (MAPK) signaling
pathway and histone H4 acetylation.22 Other thalidomide deri-
vatives with TNFR-inhibitory properties, such as pomalidomide
and lenalidomide, induce HbF expression and modulate erythroid
differentiation during the specific commitment of CD34+ pro-
genitor cells.23 It has been hypothesized that pomalidomide regulates
HbF by modifying the chromatin structure, which results from
histone H3 acetylation. In vivo studies with transgenic sickle
mice treated with pomalidomide showed augmented HbF expres-
sion similar to that produced by treatment with HU.24
structural modification of the prototypes hydroxyurea (1) and
thalidomide (3) (Scheme 1).
The design concept was based on the molecular hybridization
of hydroxyurea (1) and thalidomide (3). Specifically, NO resulting
from the bioconversion of HU was inserted into nitrate ester
subunits to produce novel hybrid derivatives (4aꢀf) containing
both pharmacophoric elements: NO-donor properties and TNFR-
inhibitory properties. Wethuscombinedanalgesic, anti-inflammatory,
and vasodilatory activities in a single structure.
’ CHEMISTRY
The synthetic routes for the preparation of the phthalimide
derivatives (4aꢀf) are summarized in Schemes 2 and 3. The alkyl
and aryl derivatives (Scheme 2) were obtained in two or three
synthetic steps, with overall yields of 51%ꢀ80%. Compound 4f
was produced by the derivatization of the thalidomide molecule
(Scheme 3).
The initial stage of the reactions used to produce the phtha-
limide derivatives (6cꢀe) involved the coupling of functional-
ized amino derivatives with phthalic anhydride in pyridine, with
variable yields (68ꢀ89%). Compound 8a was produced with a
good yield (80%) by treating phthalimide with 37% formalde-
hyde under reflux conditions.25 The phthalimide derivative 8b
was acquired commercially. Compound 10 was synthesized by
the procedure described by Hess and co-workers (2001).26
The compounds containing alcohol functions (6cꢀe, 8a,b,
and 10) were then reacted in dichloromethane with a mixture of
glacial acetic acid and fuming nitric acid to produce the final
organic nitrate ester compounds 4aꢀf. The structures of all the
compounds were established by mass spectrometry, elemental
analysis, IR spectroscopy, and 1H and 13C NMR. All compounds
In a continuing effort to develop new candidate drugs to treat
SCD symptoms, we report here the design, synthesis, and pharma-
cological evaluation of thalidomide derivatives (4aꢀf) by the
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Scheme 2a
a Reagents and conditions: (a) pyridine, reflux, 2 h, 68ꢀ89%; (b) HNO3/Ac2O, CH2Cl2, room temp, overnight, 74ꢀ90%; (c) urea, 140ꢀC, 20 min, 80%;
(d) HCOH, H2O, reflux, 1 h, 93%.
Scheme 3a
a Reagents and conditions: (a) HCOH, H2O, reflux, 70%; (b) HNO3/Ac2O, CH2Cl2, room temp, overnight, 70%.
were analyzed by HPLC, and their purity was confirmed to be
over 98.5%.
of polymorphonuclear cells (PMNs) into the peritoneal cavity,
with maximal cell influx after 4 h. The exudates obtained from the
peritoneal cavities of rats 4 h after a thioglycollate injection were
confirmed to contain >98% neutrophils (data not shown). As demon-
strated in Figure 2, the accumulation of PMNs in the peritoneal
cavity in response to thioglycollate was delayed by derivatives
4aꢀf, indicating the ability of these compounds to inhibit leukocyte
migration.
Antinociceptive Activity. The antinociceptive profiles of com-
pounds 4aꢀf were evaluated using acetic acid induced abdominal
constrictions in mice.30 The compounds were administered orally at
100 μmol/kg. Compound 4d was the most active antinociceptive
compound, remarkably reducing the acetic acid induced abdom-
inal constrictions by 66%. Compounds 4a, 4c, 4e, and 4f showed
antinociceptive activities similar to that of the control, dypirone,
at the same dose. Compound 4b reduced the induced abdominal
constrictions by only 25% (Figure 3).
In Vitro TNFr Measurements. Levels of the cytokine TNFR
were measured in the supernatants of monocyte cultures of adult
knockoutꢀtransgenic sickle cell mice. The purity of the monocytes
was >90%, as determined with MayꢀGiemsa staining. Compounds
4c and 4d were added to the monocyte cultures at different
concentrations (100 or 300 μM). Dexamethasone (Dex) was
used as the positive anti-inflammatory control, and HU was used
as the SCD drug control. After treatment with 100 or 300 μM
HU, the levels of TNFR were 463.3 ( 130.9 and 579.8 (
60.2 pg/mL, respectively. Dexamethasone (1 μM) inhibited 83%
of TNFR. Only compound 4c reduced the in vitro level of TNFR.
Compound 4c (100 and300μM) inhibited 45% and 91% of TNFR
production, respectively, and compound 4d (100 and 300 μM)
’ RESULTS
Detection of Nitrite (Griess Reaction). The in vitro ability of
compounds 4aꢀf to act as NO donors was measured using the
Griess reaction by quantifying the nitrite produced from the
oxidative reaction of nitric oxide, oxygen, and water.27 The extent
of thiol-induced NO generation was determined similarly after
incubation for 1 h in the presence of a large excess of cysteine
(1:50).Theresults, expressedaspercentagenitrite(NO2ꢀ, mol/mol),
are summarized in Table 1.
All organic nitrate ester compounds (4aꢀf) were capable of
inducing nitrite formation at concentrations between 5.3% and
6.2%. Isosorbide dinitrate (DNS), used as the control, induced
11.7% nitrite formation. However, DNS has two organic nitrate
ester groups (ONO2) that can release NO, whereas compounds
4aꢀf have only one group that releases NO.
Capsaicin-Induced Ear Edema. The anti-inflammatory activity
of compounds 4aꢀf was evaluated using the in vivo capsaicin-
induced ear edema model,28 and they were administered orally to
mice at doses of 300 μmol/kg. Compounds 4aꢀf all inhibited ear
edema by about 43ꢀ65%, producing a significant anti-inflammatory
effect similar to that produced by indomethacin, which was used
as the positive control (Figure 1).
Thioglycollate-Induced Peritonitis in Mice. The anti-
inflammatory effects of compounds 4aꢀf were confirmed by testing
their effects on thioglycollate-induced peritonitis in mice.29 The
thioglycollate stimulus induces a time-dependent accumulation
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Table 1. NO Release Data
% NO2ꢀ (mol/mol),a
compd
50 ꢁ 10ꢀ4 M L-Cys
DNSb
4a
11.7 ( 0.3
5.4 ( 0.1
6.2 ( 0.2
6.0 ( 0.05
5.5 ( 0.08
6.1 ( 0.06
5.8 ( 0.15
4b
4c
4d
4e
4f
a All values are the mean ( SEM. Determined by Griess reaction, after
incubation for 1 h at 37 ꢀC in pH 7.4 buffered water, in the presence of
1:50 molar excess of L-cysteine. b DNS: isosorbide dinitrate. DNS
possesses two ONO2 groups that may release NO, whereas compounds
4aꢀf possess only one group that may release NO.
Figure 1. Effects of compounds 4aꢀf and indometacin (Ind) on edema
ear capsaicin-induced in mice. All compounds were administered po at a
dose of 300 μmol/kg. Results are expressed as the mean ( SEM for n = 8
animals (triplicate). The % of inhibition was obtained by comparison
with vehicle control group (data not shown): /, P < 0.01 (ANOVA
followed by Dunnett’s test).
inhibited 67% and 84% of TNFR production, respectively
(Figure 4).
’ DISCUSSION
Several strategies have been developed to identify compounds
useful in the treatment of SCD.31 These strategies involve (a)
agents that reduce iron overload (chelating agents),32 (b) agents
that induce γ-globin expression and HbF synthesis (i.e., cytidine,
azacytidine, short-chain fatty acids),33 (c) agents that prevent
hemoglobin dehydration (i.e., Gardos channel inhibitors),34 (d)
agents that bind covalently to hemoglobin (i.e., aldehyde
derivatives),3,35 (e) agents that modify rheological blood properties
(i.e., poloxamer-188),36 and (f) agents that increase NO avail-
ability.37 In general, the compounds used in these strategies are
available for therapeutic uses other than the treatment of SCD. In
fact, the only drug approved by the regulatory agency U.S. FDA
for the treatment of SCD is HU. Despite the beneficial effects of
HU, long-term treatment with it is associated with several deleter-
ious effects, which limit its life-long utility for SCD patients.38ꢀ40
Therefore, to find new therapeutic alternatives with which to
treat SCD symptoms, we have designed and synthesized a series
of thalidomide derivatives containing an organic nitrate ester
function as an NO-donor subunit (Scheme 1). The NO-donor
properties of the organic nitrate ester are well established in the
literature. Therefore, because the most beneficial effect of HU is
its ability to generate NO, we evaluated this effect for all the
synthesized compounds (4aꢀf). All compounds (4aꢀf) demon-
strated a similar ability to generate nitrite in the cell culture
medium in the presence of cysteine, measured with the Griess
reaction. Interestingly, it has been reported that NO donors such
as S-nitrosocysteine and deta-NONOate can increase γ-globin
mRNA in K562 cells.41 The same kind of property was observed
in these compounds, which induced the expression of γ-globin
mRNA in K562 cells at lower concentrations than were used for
HU (unpublished results).
cardiovascular system, compounds with anti-inflammatory activ-
ity should contribute to the control of some disease symptoms.
All the compounds (4aꢀf) demonstrated anti-inflammatory
activity, with compound 4c demonstrating activity higher than
that of the positive control indomethacin. All the compounds
(4aꢀf) inhibited leukocyte migration in a similar manner. It is
well established that stimulated neutrophils produce super-
oxide and other highly reactive oxygen products capable of
inducing cellular injury, so the inhibition of leukocyte migra-
tion is important in the prevention of the damage associated
with the inflammatory processes. Both experiments showed that
the synthesized compounds have important anti-inflammatory
activities.
The inflammatory response also involves an increase in
proinflammatory cytokines. It has been reported that patients
with SCD present with high levels of TNFR, IL1, and IL6.14
Malavꢀe and co-workers (1993) reported an inverse correlation
between the percentage of HbF and levels of TNFR in humans,
demonstrating the importance of controlling cytokine levels.16
Furthermore, Laurance and co-workers (2010) demonstrated
that HU therapy stimulates the production of proinflammatory
gene expression and levels of cytokines, such as TNFR, IL1A,
IL1B, IL6, and IL8.17 These cytokines increase the expression of
vascular cell adhesion molecules on leukocytes, encouraging the
initiation of the vaso-occlusion process.
To characterize the analgesic activity of compounds 4aꢀf, we
examined their effects after their oral administration in a mouse
model of acetic acid induced abdominal constriction.30 All the
thalidomide derivatives (4aꢀf) showed analgesic activity, but the
most active compound was 4d, which reduced the acetic acid
induced constrictions by 66%. Ribeiro and co-workers (2002)
demonstrated that TNFR also plays an important role in the
nociceptive writhing response in mice and that the analgesic
effect of thalidomide results from the inhibition of this
cytokine.19 It has been observed that therapies that inhibit TNFR
production have an important role in pain management.
The genotoxicity of the compounds was evaluated in vitro
using a Salmonella/microsome assay and in vivo using the micro-
nucleus test.46 An interesting mutagenic relationship was observed
The inflammatory pathway in SCD is an important therapeutic
target. Recurrent inflammation causes the increased expression
of adhesion molecules by activated leukocytes and vascular endo-
thelial cells. Several studies have associated high levels of
leukocyte adhesion molecules with the clinical severity of the
disease.42 The vaso-occlusion process is also frequently triggered
by inflammatory processes.43ꢀ45 Because inflammation plays
a deleterious role in SCD, resulting in progressive damage
to most organs including the lungs, brain, kidneys, bones, and
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Figure 4. Level of proinflammatory mediator TNF-R, determined by
ELISA in the supernatant of mononuclear culture treated with LPS and
co-incubated with test drugs (4c and 4d; 100 and 300 μM, 24 h), was
used a positive anti-inflammatory control. Dexamethasone (Dex) at 1
μM was used a positive anti-inflammatory control. Hydroxyurea was
used to determine its anti-inflammatory capacity (100 and 300 μM).
Data are represented as the mean ( SEM; P < 0.05, compared to LPS
(ANOVA followed by Dunnett’s test); N g 4 experiments; /, 100 μM;
//, 300 μM.
Figure 2. Effect of compounds 4aꢀf and indomethacin (Ind) on cell
migration in peritonite 3% thioglycollate-induced in mice. All com-
pounds were administered po at a dose of 300 μmol/kg. Mice were killed
at the time-point of 4 h after % thioglycollate-induced peritonitis. Total
cell migration was counted using a Neubauer chamber. Data represent
the mean ( SEM from at least six animals: /, P < 0.01 (ANOVA
followed by Dunnett’s test).
derivatives are related to their inhibition of TNFR.48 HU showed
no anti-inflammatory effect but an increase in the production of
the inflammatory mediator TNFR at a concentration similar to
that of the positive control (LPS). Lanaro and co-workers
reported similar data, indicating that HU has proinflammatory
properties.14
These results together demonstrate that combining the prop-
erties of thalidomide derivatives with NO-donor capacity repre-
sents a successful new approach to the treatment of some sickle
cell disease symptoms and could be a therapeutic alternative to
HU treatment.
’ CONCLUSIONS
Figure 3. Antinociceptive effect of dypirone (Dyp) and compounds
4aꢀf (all in doses of 100 μmol/kg, po) in the 0.6% acetic acid (AcOH)
induced abdominal constrictions observed for 20 min after the admin-
istration of acetic acid. Data are expressed as the inhibition percentage of
total writhings calculated from eight animals: /, P < 0.01 (ANOVA
followed by Dunnett’s test).
A novel series of compounds (4aꢀf), designed by molecular
hybridization and containing organic nitrate ester functions, was
synthesized and characterized by mass spectrometry, elemental
1
analysis, IR spectroscopy, and H and 13C NMR. Compounds
4aꢀf inhibited leukocyte migration in a similar manner in a
thioglycollate-induced model of peritonitis. All compounds
demonstrated analgesic and anti-inflammatory activities and
NO-donor properties. Compound 4d was the most active
antinociceptive compound, reducing acetic acid induced abdom-
inal constrictions by 66%. Compound 4c was more active than
indomethacin, inhibiting 65% of ear edema. Compounds 4c and
4d, given at 300 μmol/kg, reduced TNFR levels in the super-
natants of monocyte cultures from transgenic sickle cell mice,
whereas HU showed no activity. Compounds 4c and 4d have
emerged from these in vivo and in vitro studies as novel lead drug
candidates for the treatment of SCD symptoms.
and allowed us to characterize compounds 4c and 4d as less
mutagenic in the TA100 and TA102 strains of Salmonella
typhimurium than the other compounds.46 An in vivo micro-
nucleus test using mouse peripheral blood demonstrated that all
the compounds were less genotoxic than HU. All the compounds
induced an average frequency of less than six micronucleated
reticulocytes (MNRET) after their oral administration at 100
mg/kg, whereas HU induced an average frequency of 33.7
MNRET at the same concentration.47
Considering all these results, we selected compounds 4c and
4d for further evaluation of their TNFR inhibitory activity in
adult knockoutꢀtransgenic sickle cell mice. Compounds 4c and
4d reduced the level of TNFR relative to that in the lipopoly-
saccharide (LPS) treated control mice. Both 100 and 300 μM
showed dose-dependent effects. At 300 μM, compound 4c
decreased TNFR levels more than compound 4d at the same
concentration. Dexamethasone, the positive control, reduced
TNFR but not to the same degree as compound 4c. These
results are consistent with the literature, which has demonstrated
that the analgesic and anti-inflammatory activities of phthalimide
’ EXPERIMENTAL SECTION
General. Melting points were measured with an electrothermal
melting-point apparatus (SMP3, Bibby Stuart Scientific) in open
capillary tubes and are uncorrected. Infrared spectra (KBr disk) were
produced on an FTIR-8300 Shimadzu and the frequencies expressed
1
in cmꢀ1. H NMR and 13C NMR spectra were scanned on a Bruker
DRX-400 (400 MHz) NMR spectrometer using DMSO-d6 as the
solvent. Chemical shifts were expressed in parts per million (ppm)
relative to tetramethylsilane. Elemental analyses (C, H, and N) were
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performed on a Perkin-Elmer model 240C analyzer, and the data were
within (0.4% of the theoretical values. HPLC analysis was performed on
a Shimadzu LC-10AD chromatograph equipped with a model SPD-10A
UVꢀvisible detector (Shimadzu). All compounds were analyzed by
HPLC, and their purity was confirmed to be greater than 98.5%. The
compounds were separated on a reversed phase C18 Shimadzu Shim-
pack CLC-ODS (M) column (5 μm particle, 250 mm ꢁ 4.6 mm i.d.).
HPLC-grade solvents (acetonitrile, methanol, acetic acid, and toluene)
were used in the analyses and were bought from a local supplier. The
progress of all reactions was monitored by TLC, which was performed
on 2.0 ꢁ 6.0 cm2 aluminum sheets precoated with silica gel 60 (HF-254,
Merck) to a thickness of 0.25 mm. The developed chromatograms were
viewed under UV light (254ꢀ265 nm) and treated with iodine vapor.
Merck silica gel (70ꢀ230 mesh) was used for preparative column
chromatography. Reagents and solvents were purchased from commer-
cial suppliers and used as received.
temperature under nitrogen atmosphere. The compounds were isolated
by the addition of 40 mL of dichloromethane and 30 mL of iceꢀwater.
The organic phase was isolated and washed three more times with 20 mL
of saturated sodium bicarbonate solution. The organic phase was
evaporated at reduced pressure to obtain compounds 4aꢀe with variable
yields (65ꢀ85%).
If necessary, the compounds were further purified with silica gel
column chromatography using hexane/ethyl acetate (6:4) as the
mobile phase.
(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl Nitrate (4a). Yield:
74%. Mp: 75ꢀ77 ꢀC. IR νmax (cmꢀ1, KBr pellets): 2929 (CꢀH),
1768 and 1701 (CdO imide), 1622 and 1278 (O-NO2). 1H NMR (400
MHz, CDCl3) δ: 7.93 (2H; m, H6 and H9); δ 7.80 (2H; m, H7 and H8; J
= 8,1 Hz); 6.08 (2H; s, H10) ppm. 13C NMR (400 MHz, CDCl3) δ:
166.0; 135.0; 131.5; 124.3; 66,7 ppm. Anal. Calcd for C9H6N2O5: C,
48.6; H, 2.7; N, 12.6. Found: C, 48.4; H, 2.5; N, 12.5. MS/ESI m/z: [M]+
222.0, [M ꢀ ONO2]+ 161.05 (100%).
Compounds 7, 8a, and 10 were synthesized according to a previously
described methodology.25,26 Compound 8b was purchased commercially.
General Procedure for the Synthesis of Phthalimide Deri-
vatives (6cꢀe). A mixture of the selected amine (2.02 mmol), phthalic
anhydride (5) (0.3 g, 2.02 mmol), and 10 mL of pyridine was stirred
under nitrogen at 140 ꢀC for 3 h. The mixture was then cooled and
50 mL of dichloromethane added; successive washes with a 10% copper
sulfate solution were performed until a blue color was produced in
aqueous phase. The organic phase was washed twice with 30 mL of water
and dried with sodium sulfate or magnesium sulfate. After filtration, the
organic phase was concentrated under reduced pressure to produce
compounds with variable yields (70%ꢀ90%).
2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl Nitrate (4b). Yield:
80%. Mp: 87ꢀ89 ꢀC. IR νmax (cmꢀ1, KBr pellets): 2925 (CꢀH), 1762
and 1716(CdO imide), 1622 and 1278 (OꢀNO2). 1H NMR (400 MHz,
CDCl3) δ: 7.85 (2H; m, H6 and H9); 7.73 (2H; m, H7 and H8; J = 7.9
Hz); 4.66 (2H; t, H11); 4.05 (2H; t, H10) ppm. 13C NMR (400 MHz,
CDCl3) δ: 167.82; 134.3; 131.8; 123.6; 69.52; 35.22 ppm. Anal. Calcd for
C10H8N2O5: C, 50.85;H, 3.41; N, 11.86. Found: C, 50.92;H, 3.5; N, 11.9.
MS/ESI m/z: [M]+ 236.0, [M ꢀ ONO2]+ 175 (100%).
3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl Nitrate (4c). Yield:
90%. Mp: 126ꢀ128 ꢀC. IR νmax (cmꢀ1, KBr pellets): 2926 (CꢀH),
1
1774 and 1722 (CdO imide), 1620 and 1279 (OꢀNO2). H NMR
If necessary, the samples were further purified with silica gel column
chromatography, using hexane/ethyl acetate (6:4) as the mobile phase.
2-(3-(Hydroxymethyl)phenyl)isoindoline-1,3-dione (6c). Yield: 82%.
Mp: 149ꢀ151 ꢀC. IR νmax (cmꢀ1, KBr pellets): 3541 (OꢀH), 2933
(CꢀH), 1772 and 1716 (CdO imide). 1H NMR (400 MHz, CDCl3) δ:
(400 MHz, CDCl3) δ: 7.98 (2H; dd, H6 and H9; J = 8.1 Hz); 7.82 (2H;
0
0
dd, H7 and H8; J = 8.1 Hz); 7.57 (1H; d, H2 ); 7.53 (1H; dd, H5 J = 7.8
Hz); 7.55 (1H; d, H6 ); 7.45 (1H; d, H4 ); 5.5 (2H; s, H1 ) ppm. 13C
NMR (400 MHz, CDCl3) δ: 167; 124.1; 133.7; 131.5; 127.6; 127.0;
128.9; 134.8; 132.5; 129.8; 74.2 ppm. Anal. Calcd for C15H10N2O5: C,
60.4; H, 3.38; N, 9.39. Found: C, 60.1; H, 3.5; N, 9.45. MS/ESI m/z:
[M]+ 298, [M ꢀ ONO2]+ 237 (54%).
0
0
00
7.94 (2H; dd, H6 and H9; J = 8.4 Hz); 7.78 (2H; dd, H7 and H8; Jorto
=
0
0
0
8.4 Hz); 7.49 (1H; d, H2 ); 7.41 (2H; dd, H6 ; J = 7.6 Hz); 7.37 (H4 and
H5 ); 4.76 (2H; s, H1 ) ppm. 13C NMR (400 MHz, CDCl3) δ: 166.9;
141.9; 123.4; 131.5; 125.4; 116.8; 126.3; 134.1; 132.0; 128.9; 64.4 ppm.
Anal. Calcd for C15H11NO3: C, 71.1; H, 4.38; N, 5.53. Found: C, 71.4;
H, 4.11; N, 5.7.
0
00
4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl Nitrate (4d). Yield:
88%. Mp: 159ꢀ161 ꢀC. IR νmax (cmꢀ1, KBr pellets): 2924 (CꢀH),
1
1786 and 1718 (CdO imide), 1620 and 1279 (OꢀNO2). H NMR
(400 MHz, CDCl3) δ: 7.98 (2H; dd; H6 and H9; J = 8.4 Hz); 7.81 (2H;
2-(4-(Hydroxymethyl)phenyl)isoindoline-1,3-dione (6d). Yield: 63%.
Mp: 155ꢀ158 ꢀC. IR νmax (cmꢀ1, KBr pellets): 3516 (OꢀH), 2916
(CꢀH), 1768 and 1710 (CdO imide). 1H NMR (400 MHz, CDCl3) δ:
7.99 (2H; dd, H6 and H9; J = 8.5 Hz); 7.80 (2H; dd, H7 and H8; J = 8.5
0
0
dd, H7 and H8; J = 8.4 Hz); 7.5 (2H; d, H3 and H5 ; J = 8.4 Hz); 7.53
(2H; d, H2 and H6 ; J = 8.4 Hz); 5.48 (2H; s, H1 ) ppm. 13C NMR
(400 MHz, CDCl3) δ: 167.2; 124.1; 133.0; 132.3; 131.9; 137.8; 126.9;
129.9; 74.1 ppm. Anal. Calcd for C15H10N2O5: C, 60.4; H, 3.38; N,
9.39. Found: C, 60.6; H, 3.22; N, 9.53. MS/ESI m/z: [M]+ 298,
[M ꢀ ONO2]+ 237 (65%).
0
0
00
0
0
0
0
Hz); 7.52 (2H; d, H2 and H6 ; J = 8.5 Hz); 7.44 (2H; d, H3 and H5 ;
J = 8.5 Hz); 4.77 (2H; s, H1 ) ppm. 13C NMR (400 MHz, CDCl3) δ:
00
2-[4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate (4e).
Yield: 76%. Mp: 141ꢀ144 ꢀC. IR νmax (cmꢀ1, KBr pellets): 2924
(CꢀH), 1786 and 1718 (CdO imide), 1610 and 1283 (OꢀNO2). 1H
NMR (400 MHz, CDCl3) δ: 7.95 (2H; m, H6 and H9; J = 8.4 Hz); 7.79
167.2; 140.9; 134.4; 128.2; 131.7; 127.5; 123.7; 126.7; 64.8 ppm. Anal.
Calcd for C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.2; H,
4.21; N, 5.7.
2-(4-(2-Hydroxyethyl)phenyl)isoindoline-1,3-dione (6e). Yield: 74%.
Mp: 172ꢀ175 ꢀC. IR νmax (cmꢀ1, KBr pellets): 3516 (OꢀH), 2916
(CꢀH), 1768 and 1701 (CdO imide). 1H NMR (400 MHz, CDCl3) δ:
7.95 (2H; dd, H6 and H9; J = 8,5 Hz); 7.81 (2H; dd, H7 and H8); 7.38
0
0
0
(2H; m, H7 and H8); 7.41 (2H; d, H2 and H6 ); 7.37 (2H; d, H3
and H5 ; J = 8.5 Hz); 4.66 (2H; t, H2 ); 3.08 (2H; t, H1 ) ppm. 13C
NMR (400 MHz, CDCl3) δ: 167.7; 132.1; 134.8; 131.1; 130.1; 127.3;
124.2; 117.6; 73.4; 33.4 ppm. Anal. Calcd for C16H12N2O5: C, 61.54; H,
3.87; N, 8.97. Found: C, 61.6; H, 3.72; N, 8.8. MS/ESI m/z: [M]+ 312,
[M ꢀ ONO2]+ 251 (47%).
0
00
00
0
0
0
0
00
00
(4H; m, H2 , H3 , H5 and H6 ); 3.9 (2H; t, H2 ); 2.93 (2H; t, H1 ) ppm.
13C NMR (400 MHz, CDCl3) δ: 167.7; 139.1; 134.8; 132.1; 130.2;
127.1; 124.1; 117.6; 63.9; 39.3 ppm. Anal. Calcd for C16H13NO3: C,
71.9; H, 4.9; N, 5.24. Found: C, 71.8; H, 4.8; N, 5.01.
[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopiperidin-1-
yl]methyl nitrate (4f). Yield: 70%. Mp: 162ꢀ165 ꢀC. IR νmax (cmꢀ1
,
General Procedure for the Synthesis of Phthalimide Deri-
vatives (4aꢀe). A mixture of 2.61 mmol of properly functionalized
phthalimide derivative was added to 17 mL of dichloromethane. After
dissolution, 0.0062 g (0.103 mmol) of urea was added. The reaction
mixture was cooled to 0 ꢀC in an ice bath and was added dropwise to a
previously prepared mixture of 3 mL of dichloromethane containing
0.42 mL of HNO3 (9.56 mmol) and 1.16 mL (10.44 mmol) of acetic
anhydride. The mixture was stirred continuously for 24 h at room
KBr pellets): 2930 (CꢀH), 1785 and 1715 (CdO imide), 1613 and
1281 (OꢀNO2). 1H NMR (400 MHz, CDCl3) δ: 8.0 (4H; m, H6, H7,
0
0
H8, H9); 6.2 (2H; s, H1”); 5.3 (1H; t, H1 ); 2.8 (2H; q, H2 ); 2.2 (2H; t,
H3 ) ppm. 13C NMR (400 MHz, CDCl3) δ: 171.5, 170.3, 168.4; 134.8;
0
131.1; 124.2; 69.5; 49; 30.2, 19.1 ppm. Anal. Calcd for C14H11N3O7:
C, 50.46; H, 3.33; N, 12.61. Found: C, 50.67; H, 3.11; N, 12.8. MS/ESI
m/z: [M]+ 333, [M ꢀ ONO2]+ 272 (29%).
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Journal of Medicinal Chemistry
ARTICLE
Pharmacology. Drugs and Reagents. The agents used here
were acetic acid and indomethacin (Merck), arabic gum, and dipyrone
(Sigma Chemical). A solution of 2.5% formalin was prepared with
formaldehyde (Merck) in saline (NaCl 0.9%). The compounds and
standards were used as suspensions in arabic gum in all experiments.
Detection of Nitrite (Griess Reaction). A solution of the
appropriate compound (20 μL) in DMSO was added to 2 mL of a
mixture of 50 mM phosphate buffer (pH 7.4) and methanol (1:1, v:v),
containing 5 mM L-cysteine. The final concentration of the compound
was 10ꢀ4 M. After 1 h at 37 ꢀC, 1 mL of the reaction mixture was treated
with 250 μL of Griess reagent (4 g of sulfanilamide, 0.2 g of
N-naphthylethylenediamine dihydrochloride, 85% phosphoric acid
[10 mL] in distilled water [final volume, 100 mL]). After 10 min at
room temperature, the absorbance was measured at 540 nm using a
Shimadzu UV-2501PC spectrophotometer. Standard sodium nitrite
solutions (10ꢀ80 nmol/mL) were used to construct the calibration
production of nitrite was observed in the absence of L-cysteine.27
Animals. Adult male and female Swiss albino mice (20ꢀ35 g) were
used in the experiments. They were housed in single-sex cages under a 12 h
light/12 h dark cycle (lights on at 0600) in a controlled-temperature
room (22 ( 2 ꢀC). The mice had free access to food and water. Groups
of six animals were used in each test group, and the control animals
received vehicle only. The experiments were performed after the protocol
was approved by the local Institutional Ethics Committee (No. 006443/
2005/78). All experiments were performed in accordance with the current
guidelines for the care of laboratory animals and the ethical guidelines for
the investigation of experimental pain in conscious animals.
Capsaicin-Induced Ear Edema. Ear edema was induced in
female Swiss mice (8 weeks old) as previously described.28 The mice
were divided into groups of six and had access to food and water ad
libitum. Each mouse received 12.5 μg/mL capsaicin in acetone on the
right ear. The phlogistic agent was applied with an automatic pipet in 10
μL volumes to both the inner and outer surfaces of the ear. The test
compounds (100 μmol/kg) and indomethacin, which was used as the
standard reference drug (100 μmol/kg), were administrated po in arabic
gum 1 h before treatment. The control group received vehicle only
(10 mL/kg, po). The left ear was treated with acetone, delivered in the
same manner. Thirty minutes after the capsaicin application, the mice
were killed and both ears were removed. Circular sections were taken,
using a cork borer with a diameter of 7 mm, and weighed. The increase in
weight caused by the irritant was measured by subtracting the weight of
the untreated left ear section from that of the treated right ear sections.
Ear edema was measured as the differences in weight between the
challenged and unchallenged ears.
Antinociceptive Activity. Analgesic activity was determined
in vivo with the acetic acid induced (0.6%, 0.1 mL/10 g) abdominal
constriction test in mice.30 Swiss mice of both sexes (18ꢀ23 g) were
used. The compounds were administered orally (100 μmol/kg) as a
suspension in 5% arabic gum in saline (vehicle). Dypirone (100 μmol/
kg) was used as the standard drug, administered under the same
conditions. Acetic acid solution was administered ip 1 h after the
administration of the compounds. Ten minutes after the ip acetic acid
injection, the number of constrictions per animal was recorded for 20
min. The control animals received an equal volume of vehicle. Anti-
nociceptive activity was expressed as percentage inhibition of the
constrictions compared with those in the vehicle-treated control group.
The results are expressed as the mean ( SEM of 10 animals per group.
The data were analyzed statistically with Student’s t test at a significance
level of P < 0.05.
Monocyte Stimulation and in Vitro TNFr Measurement.
Adult knockoutꢀtransgenic sickle cell mice were anesthetized with
ketamine/xylazine, and their blood was collected by intracardiac punc-
ture. The mononuclear cells were purified from the peripheral blood
using Ficoll gradient separation. The mononuclear cells were placed in
plastic dishes with Dulbecco’s modified Eagle’s medium and 10% calf
serum and incubated for 2 h in humidified air (5% CO2 at 37 ꢀC). The
purity of the monocytes was >90%, as determined by MayꢀGiemsa
staining. Compounds 4c and 4d in the same medium containing calf
serum were added to the monocyte cultures at different concentrations
(100 or 300 μM) 30 min before the addition of LPS (1 μg/mL).
Dexamethasone (1 μM) was used as the positive anti-inflammatory
control. The possible anti-inflammatory effect of HU was evaluated at
two different concentrations (100 and 300 μM). After incubation for 20
h, the supernatants were collected and stored at ꢀ80 ꢀC. The levels of
the cytokine TNFR were measured in the supernatants of the monocyte
cultures. The concentration of TNFR was determined by enzyme-linked
immunosorbent assay (ELISA), with a commercially available ELISA kit
(R&D Systems, Minneapolis, MN, U.S.).
ꢀ
curve. The yields of nitrite are expressed as % NO2 (mol/mol). No
Statistical Analysis. Data were analyzed statistically using the
INSTAT software, version 3.0. The results were compared before and
after treatment with the new compounds using ANOVA followed by
Dunnett’s test. A P value of less than 0.05 was considered to be
statistically significant.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +55-16-3301-6972. Fax: +55-16-3301-6960. E-mail:
santosjl@fcfar.unesp.br.
Percentage inhibition was calculated using the formula [(Cꢀ T)/(3C)] ꢁ
100, where C and T indicate the untreated (vehicle) control edema and
drug-treated edema, respectively. The significance of the differences
between the experimental groups and the control group was determined
using ANOVA in the program Prisma. The differences were considered
significant when P < 0.05. The results are expressed as the mean ( SEM,
as indicated in the captions to the figures.
’ ACKNOWLEDGMENT
This study was supported by Fundac-~ao de Amparo ꢁa Pesquisa
do Estado de S~ao Paulo (FAPESP Ref. Process: 10/12495-6 and
07/56115-0). The study protocol was approved by the local
Institutional Ethics Committee (No. 006443/2005/78).
Thioglycollate-Induced Peritonitis in Mice. This series of
experiments was performed according to a previously described method.29
The animals were treated with the thalidomide derivatives or indomethacin
(300 μmol/kg, po in arabic gum) and 1 h afterward were administered ip
1 mL of 3% thioglycollate (Difco-BD Biosciences). After 4 h, the animals
were killed by cervical dislocation and their peritoneal cavities were washed
with 1.5 mL of cold PBS and gentle manual massage. The cells were
retrieved from the peritoneal exudates and their volumes measured. The
significance of the differences between the experimental groups and the
control group was calculated with ANOVA in the program Prisma.
Differences were considered significant when P < 0.05. The results are
expressed as the mean ( SEM, as indicated in the captions to the figures.
’ ABBREVIATIONS USED
SCD, sickle cell disease; Hb, hemoglobin; HU, hydroxyurea;
FDA, Food and Drug Administration; NO, nitric oxide;
TNFR, tumor necrosis factor R; DNS, isosorbide dinitrate;
IL, interleukin; LPS, lipopolysaccharides
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