Novel phthalimide derivatives with TNF-α and IL-1β expression inhibitory and apoptotic inducing properties

Article abstract of DOI:10.1039/c4md00070f

Modulation of the immune system is an emerging concept in the control of tumor growth. Bearing in mind the pharmacological properties of thalidomide and its phthalimide derivatives, we describe here the structural design, synthesis and pharmacological evaluation of N-acylhydrazones derived from phthalimide. The ability of these N-acylhydrazones in inhibiting the secretion of TNF-α in stimulated cells as well as in inhibiting the transcription of the TNF-α gene was evaluated. We identified N-acylhydrazones 6b and 9c, which substantially impaired TNF-α secretion, expression and reduced IL-1β production similar to thalidomide or Revlimid. N-Acylhydrazone 9c was also able to induce apoptosis in Jurkat cells, however it does not have either antiproliferative properties or cytotoxicity for mouse splenocytes. Beyond that, we have assayed the ability of these compounds to induce cell death and a number of them are able to induce apoptosis.

Full text of DOI:10.1039/c4md00070f

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Novel phthalimide derivatives with TNF-a and IL-1b
expression inhibitory and apoptotic inducing
properties
Cite this: Med. Chem. Commun., 2014,
5, 758
Lucas Cunha Duarte Coelho,a Marcos Verıssimo de Oliveira Cardoso,a
ˆ
´
Diogo Rodrigo Magalhaes Moreira,a Paulo Andre Teixeira de Moraes Gomes,a
˜
´
Suellen Melo Tiburcio Cavalcanti,a Arsenio Rodrigues Oliveira,a
´
Gevanio Bezerra de Oliveira Filho,a Lucianna Rabelo Pessoa de Siqueira,a
Miria de Oliveira Barbosa,a Elizabeth Fernanda de Oliveira Borba,b
Teresinha Gonçalves da Silva,b Belinda Kaskow,c Mahdad Karimi,c
d
a
*
Lawrence J. Abraham and Ana Cristina Lima Leite
Modulation of the immune system is an emerging concept in the control of tumor growth. Bearing in mind
the pharmacological properties of thalidomide and its phthalimide derivatives, we describe here the
structural design, synthesis and pharmacological evaluation of N-acylhydrazones derived from
phthalimide. The ability of these N-acylhydrazones in inhibiting the secretion of TNF-a in stimulated
cells as well as in inhibiting the transcription of the TNF-a gene was evaluated. We identified
N-acylhydrazones 6b and 9c, which substantially impaired TNF-a secretion, expression and reduced
IL-1b production similar to thalidomide or Revlimid. N-Acylhydrazone 9c was also able to induce
apoptosis in Jurkat cells, however it does not have either antiproliferative properties or cytotoxicity for
mouse splenocytes. Beyond that, we have assayed the ability of these compounds to induce cell death
and a number of them are able to induce apoptosis.
Received 19th February 2014
Accepted 28th March 2014
DOI: 10.1039/c4md00070f
www.rsc.org/medchemcomm
increases T-cell responses to mitogens (and indirectly activates
B cells), increases expression of vascular adhesion molecules,
Introduction
and induces a number of other proinammatory cytokines,
chemokines, and inammation-associated molecules that form
an amplifying cascade to stimulate an immune response.2
Tumor necrosis factor (TNF) can kill cells by apoptosis, and this
ability to induce apoptosis is presently the most intensely
studied area of TNF research.3
A series of recent papers has provided new hope that TNF
could yet be useful as an anti-tumor cytokine.4–8 The manipu-
lation of signaling pathways downstream of TNF receptor (TNF-
R) activation showed that TNF negatively regulates its own
ability to induce apoptosis. However, despite considerable
incentives, viable leads for analogous small-molecule inhibitors
of TNF have not been reported.8
Phthalimide derivatives have been attracting considerable
attention since 1979 when Chapman Jr et al. showed that
N-substituted phthalimides possess hypolipidemic activity.9
Two years later, Hall et al. examined 12 imide analogs and
proposed that phthalimides are able to inhibit acetyl CoA
carboxylase activity.10 The same group investigated 10 N-aryl-
phthalimides and found that o-(N-phthalimido)acetophenone
lowered both serum cholesterol and triglyceride levels by 57%
and 44% aer 16 and 14 days of treatment in Swiss white mice.11
This class of compounds is also endowed with anti-
The immune system's role in cancer involves many different
mechanisms, such as minimizing metastasis by attenuating the
expression of pro-angiogenic factors. Another aspect is the
enhancement of antitumor immunity facilitated by interferons
and interleukins. Overall, reducing inammation by modu-
lating the immune response is important over the course of
cancer. In light of these possibilities, the identication of
immunomodulating agents is a task that is receiving a great
deal of attention. In this way, a variety of cytokines, including
IL-1 and tumor necrosis factor (TNF), play different roles as
regulators of immune, inammatory, and growth responses.1
IL-1 ligands (IL-1a and IL-1b, collectively referred to as IL-1) are
pluripotent, proinammatory cytokines that orchestrate
inammatory and host defense responses in the body.2 IL-1
a
ˆ
ˆ
ˆ
´
Departamento de Ciencias Farmaceuticas, Centro de Ciencias da Saude, Universidade
Federal de Pernambuco, 50740-520, Recife, PE, Brazil. E-mail: acllb2003@yahoo.com.
br
b
´
ˆ
´
Departamento de Antibioticos, Centro de Ciencias Biologicas, Universidade Federal de
Pernambuco, 50740-520, Recife, PE, Brazil
cThe School of Chemistry & Biochemistry, The University of Western Australia,
Crawley, WA 6009, Australia
dSchool of Pathology & Laboratory Medicine, The University of Western Australia,
Crawley, WA 6009, Australia
758 | Med. Chem. Commun., 2014, 5, 758–765
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inammatory and immunomodulatory properties, with thalid-
omide as the best representative of bioactive phthalimides.
Furthermore, a biochemical strategy for derivatization of
carboxylic acid-containing non-steroidal anti-inammatory
drugs (NSAIDs) to esters or amides was able to produce mole-
cules capable of binding tightly to COX-2, but not COX-1. This
single chemical derivatization (amidation or esterication) of
the carboxylic acid moiety-containing NSAIDs generates an
impressive array of potent and highly selective COX-2 inhibi-
tors. Several of them exhibited anti-inammatory and anti-
angiogenic activities.12
Similarly, a number of hydrazones have been evaluated for
new drug development, and some of them have been shown to
exhibit anti-cancer properties.13 Some have demonstrated qual-
ities of an apoptotic inducer with anti-proliferative chemo-
preventive activity in tongue cancer cells, such as the compound
[bromomethyl(phenyl)methyl]-2-(2,4-dinitrophenyl)hydrazine.14
In fact, because of their functional properties, the attachment of
N-acylhydrazones has been employed in the design of new anti-
inammatory and immunomodulatory agents.
Scheme 1 Synthesis of the compounds. Reagents and conditions: (a)
Ala, Phe or Ile, 135 ꢀC, 30 min;15 (b) ethanol, H2SO4 (4 drops), magnetic
stirring, 60 ꢀC, 4 h; (c) THF, imidazole, magnetic stirring, 60 ꢀC, 4 h; (d)
ethanol, hydrazine hydrate (4 drops), magnetic stirring, 60 ꢀC, 4 h; and
(e) ethanol, aromatic aldehyde substituted, reflux, 4 h.
In view of this, we describe the synthesis of N-phthaloyl
amino acids 2a–c and their chemical derivatization to their
respective esters 3a–c, amides 4a–c, hydrazides 5a–c and
N-acylhydrazone derivatives 6a–c, 7a–c, 8a–c and 9a–c. All these
compounds are phthalimide derivatives designed to compare
S-enantiomers were obtained in all cases as veried by the
addition of the chiral shi reagent Eu(hfc)3 in 1H NMR spectra.
Biological activities
the bioisosteric relationship between a carboxylic acid and an To evaluate the levels of TNF and IL-1b, mouse macrophages
amide; moreover, we aimed to compare the bioisosteric inter- were incubated with phthalimide derivatives over a 24 h time
play between an amide and N-acylhydrazone. This examination course. These cells were then stimulated with lipopolysaccha-
also reports the pharmacological evaluation of their properties ride (LPS). The purpose of this test was to evaluate the biological
for inhibiting the secretion and expression of TNF and IL-1b in capacity of the molecules that were found to inhibit the
cells. Beyond that, we have assayed the ability of these synthesis of these extremely important cytokines in inamma-
compounds in inducing cell death and some of them are indeed tory processes and cancers. Data presented in Fig. 1 show that
apoptosis inducers.
twelve compounds were able to inhibit TNF production at
50 mM, better than Thl. Among all series of compounds, the
hydrazide and hydrazone series were the most promising. The
hydrazide derivative 5b, the phenyl-hydrazone derivative 6c and
the 4-methoxy-phenyl-hydrazone derivative 7c all inhibit secre-
Results and discussion
Chemistry
A single strategy for the production of new chemical entities is tion of TNF by macrophages at 50 mM. Concerning IL-1b, in
the chemical derivatization of acid or amine compounds to general, each series of compounds was able to inhibit IL-1b at
esters, amides or hydrazines. Indeed, N-phthaloyl amino acids the same concentration (50 mM). The ester derivative 3b was the
2a–c were synthesized following a known procedure15 that least potent. Compounds 5b, 6c, 7b and 8c were the most active
condenses phthalic anhydride 1 with the respective amino of the series. These preliminary results indicate that, in general,
acids. Then N-phthaloyl amino acids 2a–c were converted into the chemical derivatization to acylhydrazides and hydrazones
ethyl esters 3a–c and amides 4a–c (Scheme 1). The reactions of and apolar aminoacyl moieties produces new derivatives that
amidication to prepare amides 4a–c proceed well by using urea are most effective in modulating TNF and IL-1b expression.
upon reuxing with ethanol (24 h), but we adapted them under
To effectively measure the inhibition of the expression of
ultrasound conditions using NH4OH and K2CO3 as bases which TNF, the compounds were tested and evaluated for inhibition of
produced excellent yields in general (50–85%) and in shorter TNF transcriptional activity via the NFkB signaling pathway. For
time periods (30 min in most of the cases). The hydrazides 5a–c this purpose, a T cell line containing a transcriptional reporter
were prepared by reacting commercially available hydrazine was used (FRT-Jurkat TNF) as previously described.16,17 The
hydrate with the appropriate ethyl esters 3a–c under magnetic green uorescent protein (GFP) reporter gene is under the
stirring in EtOH with mild heating. Once hydrazides 5a–c were control of the TNF promoter sequence which contains an NFkB
isolated, we reacted them with aromatic aldehydes. These binding site. As a measure of TNF expression, the activity of GFP
reactions were carried out in anhydrous EtOH in reux under was quantied by ow cytometry as previously described.18
the presence of catalytic amounts of H2SO4, affording N-acyl- First, it was determined whether the compounds inhibited the
hydrazones 6a–c, 7a–c, 8a–c and 9a–c in low to moderate yields expression of TNF at 10 mM and 100 mM doses (Fig. 2). Thalid-
(38–84%). It is worth mentioning that only the desired omide was used as a standard for comparing the levels of
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Fig. 1 (a) Effect of phthalimides and Thl on the production of TNF-a in mouse macrophages (2 ꢁ 106) stimulated with LPS (2 mg mLꢂ1). TNF-a
was measured after 24 h of incubation with compounds (50 mM) by sandwich ELISA (eBioscience kit). Data are mean ꢃ S.D. obtained in triplicate.
(b) Effect of phthalimides and Thl on the production of interleukin-1b (IL-1b) in mouse macrophages (2 ꢁ 106) stimulated with LPS (2 mg mLꢂ1). IL-
1b was measured after 24 h of incubation with compounds (50 mM) by sandwich ELISA (eBioscience kit). Data are mean ꢃ S.D. (error bars)
obtained in triplicate. Thl, thalidomide; S.D., standard deviation.
Fig. 2 (a) Effect of phthalimides and Thl on the expression of TNF in the human T cell line. Jurkat cells containing GFP-gene report under control
of the TNF gene promoter (FRT-Jurkat TNF) were incubated for 24 h with the compounds (100 or 10 mM). Inhibition of TNF expression was
measured as the GFP expression. Data are mean ꢃ S.D. obtained in duplicate; one single experiment. (b) Dose–response curve of selected
phthalimides in the expression of TNF in the human T cell line (Jurkat); two independent experiments. Controls have only buffer and DMSO
(0.5%). Thl, thalidomide; Rev, Revlimid; S.D., standard deviation expressed in error bars.
inhibition. Revlimid (pomalidomide), a thalidomide derivative, compound. In this model, Revlimid shows only twice the
which has been clinically shown to be 1000 times more powerful potency of thalidomide, therefore it suggests that if
a
in relation to inhibition of TNF, was also used as a standard compound shows a level of inhibition in this model similar to
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Revlimid, in a clinical context, it will be a signicant improve- the 6, 8 and 9 series. These series have in common an N-phenyl-
ment over thalidomide. acyl-hydrazone moiety. In previous studies, it was observed that
The N-acyl-hydrazone 6a derivative showed a percentage improvements in thalidomide derivatives bearing hydrophobic
inhibition of TNF-a of 9.8% at both concentrations 100 mM and groups (phenyl or alkyl) have been found to provide highly
10 mM, which is about 3% more than thalidomide (around 5– potent inhibitors of TNF expression.19
6% at 100 mM and 10 mM). The 6b (phenyl-hydrazone derivative)
As shown in Fig. 3, we analyzed the induction of total cell
and 9c (3,4-dichloro-phenyl-hydrazone derivative) compounds death by ow cytometry. Observing the DMSO control it is clear
were the most effective with inhibition around 17% at 100 mM, that the solvent does not interfere with the test and does not
which is three times more effective than thalidomide and lead to cell death. Therefore, any toxicity observed would be due
equipotent to Revlimid (Fig. 2A). No signicant inhibitory solely to the presence of the compounds. By adding thalidomide
activity was veried at 10 mM. As we can see in Fig. 2B, (10 mM) it is possible to observe a rate of 2–3% of cell death,
compounds 6b and 9c inhibit TNF-a in a dose-dependent however Revlimid is the less toxic option. In other words,
manner. The TNF expression inhibition was more effective for Revlimid is the most effective in inhibiting TNF without killing
Fig. 3 Induction of total cell death. The effect of each compound on cellular viability was assessed using the FRT-Jurkat TNF reporter cell line
using a FACSCalibur 4 colour flow cytometer. Viable and non-viable cells present following either solvent (DMSO) alone or compound treatment
for 24 h were assayed. Cells were counted by gating on each cell population using a forward scatter (FSC) versus side scatter (SSC) plot. The
percentage of viable cells was determined as the percentage of cells inside the FSC/SSC gate that encompassed the major population in solvent-
only treated control cells. Viability was confirmed following propidium iodide staining and detection of fluorescence at 570 nm. Data are mean ꢃ
S.E. (standard error of the mean) obtained in triplicate. Thal, thalidomide, Rev, Revlimid.
Fig. 4 Induction of apoptosis. Treatments were carried out as in Fig. 3 using the parental Jurkat line. Apoptosis was measured following
treatment with a FITC-labelled caspase 3 substrate. The degree of apoptosis was assessed as the percentage of the cleaved caspase 3 substrate
measured as an increase in fluorescence at 515 nm due to FITC release. Data are mean ꢃ S.E. (standard error of the mean) obtained in triplicate.
Thal, thalidomide, Rev, Revlimid.
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cells. In general, all compounds showed acceptable levels of cell lines). 13C NMR spectra were recorded on a 75.5 MHz spec-
death comparable to thalidomide and even less than the trometer. All the experiments were monitored by analytical thin
Revlimid like acyl-hydrazones 6a, 6c and 9c (all at 10 mM). The layer chromatography (TLC) performed on silica gel GF254 pre-
compounds 6b, 9b, 8c and 9c have shown a high rate of cell coated plates. Aer elution, the plate was visualized under UV
death at 100 mM (Fig. 3). However, it was not previously possible illumination at 254 nm for UV active materials.
to say whether this was through programmed cell death.
It is interesting to note that the compound 6a (alanyl-
hydrazone derivative), at 10 mM, induced cell death at a lower
Chemistry
percentage than Revlimid, also at 10 mM, and showed a
General procedure for the synthesis of 3a–c. The respective
percentage of inhibition of TNF similar to Revlimid (9% and acid 2a–c (4.56 mmol), 15 mL of ethanol and H2SO4 (4 drops) were
14% respectively, both at 10 mM). To determine whether cell added to a 50 mL round bottom ask under magnetic stirring and
death was via apoptosis (programmed cell death), we used the warmed to 60 ꢀC for 4 h. Aer cooling back to rt, the precipitate
same T cell line, Jurkat, however without the GFP reporter was ltered off and the solvent was evaporated. A white solid was
(Fig. 4). Cells were treated with compounds and aer 24 hours, obtained, ltered in a Buchner funnel with a sintered disc lter,
the degree of caspase 3 cleavage was assessed as a measure of washed with cold water, and then dried over SiO2.
apoptosis. Cleavage could be measured as the release of the
added uorescein isothiocyanate (FITC)-labeled substrate formula: C13H13NO4. MW: 247.25 g. Yield: 70.09%. Rf: 0.6 (Hex/
against cleaved caspase 3.
AcEt: 8/2). M.p.: liquid product. IR (KBr, cmꢂ1): 3008 (C–H),
Ethyl 2-(1,3-dioxoisoindolin-2-yl)propanoate (3a). Chemical
In Fig. 4 it can be noted that the compound 6a in this trial 1711 (C]O) cmꢂ1. 1H NMR (300 MHz, DMSO-d6), d (ppm): 0.98
had a greater activity than the leading parent compound (M, 3H, CH3), 1.12 (d, 3H, CH3), 2.49 (m, 3H, CH and CH2), 7.85–
Revlimid. At both 100 mM and 10 mM concentrations, our 8.05 (m, 4H, Ar). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 10.8
synthesized compound 6a showed a lower apoptotic action (CH3), 15.4 (CH3), 20.8 (CH), 125.5 (Ar), 127.6 (Ar), 132.9 (Ar),
against the test cells. It is also observed that the compound 8c 152.2 (C]O), 170.1 (C]O). Elemental analysis: liquid product.
(4-Cl-phenyl-hydrazone derivative) has a high rate of apoptosis.
Ethyl 2-(1,3-dioxoisoindolin-2-yl)-3-phenylpropanoate (3b).
From the rst test (Fig. 2) it appears that this compound Chemical formula: C19H17NO4. MW: 323.34 g. Yield: 74.89%. Rf:
inhibits TNF-a around 13% while Revlimid inhibits 14% at 0.5 (Hex/AcEt: 8/2). M.p.: liquid product. IR (KBr, cmꢂ1): 3447
100 mM. Although the compound 8c does not show more effi- (NH2), 3018 (N–H), 1679 (C]O) cmꢂ1, 2918 (C–H), 1679 (C]O)
cient inhibition against TNF, it is equivalent to a drug for cmꢂ1
.
1H NMR (300 MHz, DMSO-d6), d (ppm): 1.09 (m, 3H,
clinical use. However, this compound is far more effective in CH3), 3.50 (m, 4H, 2 ꢁ CH2), 4.91 (m, 1H, CH), 7.08–7.14 (m, 5H,
promoting programmed cell death.
Ar), 7.12 (br, 2H, NH2), 7.78–7.91 (m, 4H, Ar). 13C NMR (75.5
MHz, DMSO-d6), d (ppm): 10.0 (CH3), 37.34 (CH2), 55.8 (CH),
123.6 (Ar), 126.8 (Ar), 128.6 (Ar), 129.1 (Ar), 134.9 (Ar), 168.1 (C]
O). Elemental analysis: liquid product.
Conclusion
In conclusion, new analogues of thalidomide and immuno-
Ethyl 2-(1,3-dioxoisoindolin-2-yl)-3-methylpentanoate (3c).
modulatory activity were obtained. Compounds inhibited the Chemical formula: C16H19NO4. MW: 289.33 g. Yield: 72.46%. Rf:
production of TNF-a and IL-1 also in vitro. The proles of 0.6 (Hex/AcEt: 8/2). M.p.: liquid product. IR (KBr, cmꢂ1): 3062
compounds 6a, 6b and 9c showed good inhibition of TNF-a as (C–H), 1776 (C]O) cmꢂ1
.
1H NMR (300 MHz, DMSO-d6), d
compared to the standards used (thalidomide and Revlimid). (ppm): 0.77 (m, 6H, 2 ꢁ CH3), 0.91 (m, 2H, CH2), 0.98 (m, 3H,
The compounds 6b, 8c, 9b and 9c showed high levels of cyto- CH3), 2.51 (m, 1H, CH), 4.34 (m, 1H, CH), 7.87–7.86 (m, 4H, Ar).
toxicity. In addition, compound 8c showed a high rate of 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 10.5, 11.2 (CH3), 17.1
induction of apoptosis. These results indicate that, in general (CH3), 25.8 (CH2), 33.3 (CH), 58.5 (CH), 123.6 (Ar), 135.0 (Ar),
the chemical derivatization to acylhydrazones and hydrazones, 168.1 (C]O), 170.1 (C]O). Elemental analysis: liquid product.
instead of esters or amides is most effective in modulating TNF
General procedure for the synthesis of 4a–c. The respective
and IL-1 expression and also is effective in promoting pro- acid (2a–c, 4.56 mmol), 15 mL of THF and imidazole (0.31 g,
grammed cell death. These select compounds are important 4.56 mmol) were added to a 50 mL round bottom ask under
lead candidates to act as immunomodulatory agents.
magnetic stirring and warmed to 60 ꢀC for 4 h. Aer cooling
back to rt, the precipitate was ltered off and the solvent was
evaporated. A white solid was obtained, ltered in a Buchner
funnel with a sintered disc lter, washed with cold water, and
then dried over SiO2.
Experimental
General
Melting points which were measured with a Fisatom (Mod.
2-(1,3-Dioxoisoindolin-2-yl)propanamide (4a). Chemical
430D, 60 Hz) melting point apparatus are uncorrected. 1H NMR formula: C11H10N2O3. MWꢀ: 218.21 g. Yield: 58.31%. Rf: 0.2 (Hex/
spectra were recorded on a 300 MHz spectrometer in appropriate AcEt: 3/7). M.p.: 187–189 C. IR (KBr, cmꢂ1): 3439 (NH); 1683
solvents using TMS as an internal standard or the solvent signals (C]O); 1467 (C–N–C); 1387 (C–N–C). 1H NMR (300 MHz,
as secondary standards, and the chemical shis are shown in the DMSO-d6), d (ppm): 1.12 (d, 3H, CH3), 2.49 (m, 1H, CH), 6.91 (br,
d (ppm) scale. Multiplicities of NMR signals are designated as s 2H, NH2), 7.85–7.91 (m, 2H, Ar), 8.05 (m, 2H, Ar). 13C NMR (75.5
(singlet), d (doublet), br (broad) and m (multiplet, for unresolved MHz, DMSO-d6), d (ppm): 15.4 (CH3), 20.9 (CH), 125.5 (Ar), 127.6
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(Ar), 132.9 (Ar), 152.2 (C]O), 170.1 (C]O). Elemental analysis – (m, 1H, CH), 7.12–7.49 (2 br, 2H, NH2), 7.87–7.86 (m, 4H, Ar),
EAtheor: N 12.84%; C 60.55%; H 4.62%; EAexp: N 12.74%; C 7.99 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 11.2
60.79%; H 4.71%.
(CH3), 17.1 (CH3), 25.8 (CH2), 33.3 (CH), 58.5 (CH), 123.6 (Ar),
2-(1,3-Dioxoisoindolin-2-yl)-3-phenylpropanamide
(4b). 135.0 (Ar), 168.1 (C]O), 170.1 (C]O). Elemental analysis –
Chemical formula: C17H14N2O3. MW: 294.30 g. Yield: 59.05%. EAtheor: N 15.26%; C 61.08%; H 6.22%; EAexp: N 15.13%; C
Rf: 0.5 (Hex/AcEt: 3/7). M.p.: 233–234 C. IR (KBr, cmꢂ1): 3387 61.21%; H 6.31%.
ꢀ
1
(NH); 1687 (C]O); 1468 (C–N–C); 1384 (C–N–C). H NMR (300
General procedure for the synthesis of 6a–9c. The respective
MHz, DMSO-d6), d (ppm): 3.50 (m, 2H, CH2), 4.91 (m, 1H, CH), hydrazide (5a–c, 4.22 mmol), 15 mL of ethanol and aromatic
7.08–7.14 (m, 5H, Ar), 7.12 (br, 2H, NH2), 7.78–7.91 (m, 4H, Ar). aldehyde p-substituted (4.22 mmol) were added to a 50 mL
13C NMR (75.5 MHz, DMSO-d6), d (ppm): 37.3 (CH2), 54.7 (CH), round bottom ask, under magnetic stirring and reux for 4 h.
123.6 (Ar), 126.8, 128.6, 129.1 (Ar), 134.9 (Ar), 168.1 (C]O), Aer cooling back to rt, the precipitate was ltered off and the
170.1 (C]O). Elemental analysis – EAtheor: N 9.52%; C 69.38%; solvent was evaporated. A white solid was obtained, ltered in a
H 4.79%; EAexp: N 9.36%; C 69.49%; H 4.87%.
Buchner funnel with a sintered disc lter, washed with cold
(4c). water, and then dried over SiO2.
2-(1,3-Dioxoisoindolin-2-yl)-3-methylpentanamide
Chemical formula: C14H16N2O3. MW: ꢀ260.29 g. Yield: 56.39%.
(Z)-N0-Benzylidene-2-(1,3-dioxoisoindolin-2-yl)propanehydra-
Rf: 0.6 (Hex/AcEt: 3/7). M.p.: 218–219 C. IR (KBr, cmꢂ1): 3402 zide (6a). Chemical formula: C18H15N3O3. MW: 321.33 g. Yield:
(NH); 1650 (C]O); 1458 (C–N–C); 1389 (C–N–C). H NMR (300 44.77%. Rf: 0.6 (Hex/AcEt: 9/1). M.p.: 86–88 C. IR (KBr, cmꢂ1):
1
ꢀ
MHz, DMSO-d6), d (ppm): 0.77 (m, 3H, CH3), 0.91 (m, 2H, CH2), 3018 (NH2), 2897 (C–H), 1661 (C]O) cmꢂ1. 1H NMR (300 MHz,
0.98 (m, 2H, CH3), 2.51 (m, 1H, CH), 4.34 (m, 1H, CH), 7.12 and DMSO-d6), d: 1.11 (d, 3H, CH3), 2.49 (m, 1H, CH), 4.15 (s, 3H,
7.49 (2 br, 2H, NH2), 7.87–7.86 (m, 4H, Ar). 13C NMR (75.5 MHz, CH3), 7.51–7.72 (m, 4H, Ar), 7.85–8.05 (m, 4H, Ar), 8.09 (s, 1H,
DMSO-d6), d (ppm): 11.2 (CH3), 17.1 (CH3), 25.8 (CH2), 33.3 CH]N). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 15.4 (CH3),
(CH), 58.5 (CH), 123.6 (Ar), 135.0 (Ar), 168.1 (C]O), 170.1 (C] 20.8 (CH), 123.6 (Ar), 125.5 (Ar), 128.6 (Ar), 129.1 (Ar), 132.9 (Ar),
O). Elemental analysis – EAtheor: N 10.76%; C 64.60%; H 6.20%; 134.9 (Ar), 152.2 (C]O), 159.5 (CH]N), 170.1 (C]O).
EAexp: N 10.57%; C: 64.74%; H: 6.36%.
Elemental analysis – EAtheor: N 13.08%; C 67.28%; H 4.71%;
General procedure for the synthesis of 5a–c. The respective EAexp: N 12.98%; C 67.17%; H 4.76%.
ester (3a–c, 4.04 mmol), 15 mL of ethanol and hydrazine hydrate
(Z)-N0-Benzylidene-2-(1,3-dioxoisoindolin-2-yl)-3-phenyl-
(4 drops) were added to a 50 mL round bottom ask under propanehydrazide (6b). Chemical formula: C24H19N3O3. MW:
ꢀ
magnetic stirring and warmed to 60 ꢀC for 4 h. Aer cooling 397.43 g. Yield: 42.22%. Rf: 0.6 (Hex/AcEt: 9/1). M.p.: 90–92 C.
1
back to rt, the precipitate was ltered off and the solvent was IR (KBr, cmꢂ1): 1664 (C]N); 1624 (C]O). H NMR (300 MHz,
evaporated. A white solid was obtained, ltered in a Buchner DMSO-d6), d (ppm): 0.77 (m, 3H, CH3), 0.91 (m, 2H, CH2), 0.98
funnel with a sintered disc lter, washed with cold water, and (m, 2H, CH3), 2.51 (m, 1H, CH), 4.34 (m, 1H, CH), 7.10–7.52
then dried over SiO2.
(m, 8H, Ar), 7.87–7.86 (m, 4H, Ar), 7.99 (s, 1H, NH), 8.01 (s, 1H,
2-(1,3-Dioxoisoindolin-2-yl)propanehydrazide (5a). Chemical CH]N). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 25.8 (CH2),
formula: C11H11N3O3. MW: 233.22 g. Yield: 61.45%. IR (KBr, 33.3 (CH), 58.5 (CH), 123.6 (Ar), 125.0 (Ar), 126.5 (Ar), 131.0 (Ar),
cmꢂ1): 3018 (NH); 1661 (C]O); 1458 (C–N–C); 1377 (C–N–C). 1H 135.0 (Ar), 168.1 (C]O), 170.1 (C]O). Elemental analysis –
NMR (300 MHz, DMSO-d6), d (ppm): 1.12 (d, 3H, CH3), 2.49 (m, EAtheor: N 10.57%; C 72.53%; H 4.82%; EAexp: N 10.52%; C
1H, CH), 4.11 (br, 2H, NH2), 7.85–8.05 (m, 4H, Ar). The signal in 72.38%; H 4.77%.
4.11 disappears aer the addition of 3 drops of D2O. 13C NMR
(Z)-N0-Benzylidene-2-(1,3-dioxoisoindolin-2-yl)-3-methyl-
(75.5 MHz, DMSO-d6), d (ppm): 15.4 (CH3), 20.8 (CH), 125.5 (Ar), pentanehydrazide (6c). Chemical formula: C21H21N3O3. MW:
ꢀ
127.6 (Ar), 132.9 (Ar), 152.2 (C]O), 170.1 (C]O). EAtheor: N 363.41 g. Yield: 42.84%. Rf: 0.6 (Hex/AcEt: 9/1). M.p.: 93–95 C.
1
18.02%; C 56.65%; H 4.75%. Elemental analysis – EAexp: N IR (KBr, cmꢂ1): 1664 (C]N); 1623 (C]O). H NMR (300 MHz,
17.99%; C 56.68%; H 4.68%.
DMSO-d6), d (ppm): 0.77 (m, 3H, CH3), 0.91 (m, 2H, CH2), 0.98
2-(1,3-Dioxoisoindolin-2-yl)-3-phenylpropanehydrazide (5b). (m, 2H, CH3), 2.51 (m, 1H, CH), 4.34 (m, 1H, CH), 7.19–7.32 (m,
Chemical formula: C17H15N3O3. MW: 309.32 g. Yield: 69.96%. 5H, Ar), 7.87–7.86 (m, 4H, Ar), 7.99 (s, 1H, NH), 8.01 (s, 1H,
IR (KBr, cmꢂ1): 3026 (NH); 1662 (C]O); 1493 (C–N–C); 1378 (C– CH]N). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 11.2 (CH3),
N–C). 1H NMR (300 MHz, DMSO-d6), d (ppm): 3.50 (m, 2H, CH2), 17.1 (CH3), 25.8 (CH2), 33.3 (CH), 58.5 (CH), 123.6 (Ar), 135.0
4.91 (m, 1H, CH), 7.08–7.14 (m, 5H, Ar), 7.12 (br, 2H, NH), 7.46 (Ar), 168.1 (C]O), 170.1 (C]O). Elemental analysis – EAtheor: N
(NH2), 7.78–7.91 (m, 4H, Ar). 13C NMR (75.5 MHz, DMSO-d6), d 11.56%; C 69.41%; H 5.82%; EAexp: N 11.49%; C 69.58%; H
(ppm): 37.3 (CH2), 54.7 (CH), 126.8 (Ar), 128.6 (Ar), 129.1 (Ar), 5.73%.
123.6 (Ar), 134.9 (Ar), 168.1 (C]O), 170.1 (C]O). EAtheor: N
13.58%; C 66.01%; H 4.89%; elemental analysis – EAexp: N propanehydrazide) (7a). Chemical formula: C19H17N3O4. MW:
13.43%; C 66.17%; H 5.06%. 351.36 g. Yield: 47.95%. Rf: 0 : 3 (Hex/AcEt: 9/1). M.p.: 169–170
2-(1,3-Dioxoisoindolin-2-yl)-3-methylpentanehydrazide (5c). ꢀC. IR (KBr, cmꢂ1): 3018 (NH2), 2897 (C–H), 1661 (C]O) cmꢂ1
(Z)-2-(1,3-Dioxoisoindolin-2-yl)-N0-(4-methoxybenzylidene-
.
Chemical formula: C14H17N3O3. MW: 275.30 g. Yield: 75.17%. 1H NMR (300 MHz, DMSO-d6), d (ppm): 1.12 (d, 3H, CH3), 2.49
IR (KBr, cmꢂ1): 3018 (NH); 1662 (C]O); 1494 (C–N–C); 1378 (C– (m, 1H, CH), 4.11 (s, 3H, CH3), 7.21–7.31 (m, 4H, Ar), 7.85–8.05
N–C). 1H NMR (300 MHz, DMSO-d6), d (ppm): 0.77 (m, 3H, CH3), (m, 4H, Ar), 8.09 (s, 1H, CH]N). 13C NMR (75.5 MHz, DMSO-
0.91 (m, 2H, CH2), 0.98 (m, 2H, CH3), 2.51 (m, 1H, CH), 4.34 d6), d (ppm): 15.4 (CH3), 20.8 (CH), 123.6 (Ar), 125.5 (Ar), 128.6
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(Ar), 129.1 (Ar), 132.9 (Ar), 134.9 (Ar), 152.2 (C]O), 159.5 (CH] NH), 8.01 (s, 1H, CH]N). 13C NMR (75.5 MHz, DMSO-d6), d
N), 170.1 (C]O). Elemental analysis – EAtheor: N 11.96%; C (ppm): 11.2 (CH3), 17.1 (CH3), 25.8 (CH2), 33.3 (CH), 45.9 (CH3O),
64.95%; H 4.88%; EAexp: N 11.83%; C 64.90%; H 5.04%.
58.5 (CH), 135.0 (Ar), 123.6 (Ar), 168.1 (C]O), 170.1 (C]O).
(Z)-2-(1,3-Dioxoisoindolin-2-yl)-N0-(4-methoxybenzylidene)-3- Elemental analysis – EAtheor: N 10.56%; C 63.40%; H 5.07%;
phenylpropanehydrazide (7b). Chemical formula: C25H21N3O4. EAexp: N 10.72%; C 63.51%; H 5.11%.
MW: 427.45 g. Yield: 49.12%. Rf: 0.3 (Hex/AcEt: 9/1). M.p.: 169–
(Z)-N0-(3,4-Dichlorobenzylidene)-2-(1,3-dioxoisoindolin-2-yl)-
170 ꢀC. IR (KBr, cmꢂ1): 1620 (C]N); 1602 (C]O). 1H NMR (300 propanehydrazide (9a). Chemical formula: C18H13Cl2N3O3. MW:
MHz, DMSO-d6), d (ppm): 2.61 (m, 1H, CH), 4.11 (s, 3H, OCH3), 390.22 g. Yield: 45.76%. Rf: 0.7 (Hex/AcEt: 9/1). M.p.: 170–172 ꢀC.
4.34 (m, 1H, CH), 7.12 and 7.49 (2 br, 2H, NH2), 7.10–7.52 IR (KBr, cmꢂ1): 3442 (NH2), 2997 (C–H), 1710 (C]O), 1627 (C]N)
(m, 8H, Ar), 7.86–7.87 (m, 4H, Ar), 7.99 (s, 1H, NH), 8.01 (s, 1H, cmꢂ1. 1H NMR (300 MHz, DMSO-d6), d (ppm): 1.11 (d, 3H, CH3),
CH]N). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 25.8 (CH2), 2.49 (m, 1H, CH), 4.11 (s, 3H, CH3), 7.51–7.63 (4H, Ar), 7.85–8.05
33.3 (CH), 45.9 (CH3O), 58.5 (CH), 123.6 (Ar), 125.0 (Ar), 126.5 (m, 4H, Ar), 8.19 (s, 1H, CH]N). 13C NMR (75.5 MHz, DMSO-d6),
(Ar), 131.0 (Ar), 135.0 (Ar), 168.1 (C]O), 170.1 (C]O). 7b – d (ppm): 15.4 (CH3), 20.8 (CH), 125.5 (Ar), 128.6 (Ar), 129.1 (Ar),
elemental analysis – EAtheor: N 9.83%; C 70.25%; H 4.95%; 132.9 (Ar), 134.9 (Ar), 147.9 (Ar, C–Cl), 152.2 (C]O), 159.5 (CH]
EAexp: N 10.02%; C 70.13%; H 4.98%.
(Z)-2-(1,3-Dioxoisoindolin-2-yl)-N0-(4-methoxybenzylidene)-3- 55.40%; H 3.36%; EAexp: N 10.67%; C 55.46%; H 3.25%.
methylpentanehydrazide (7c). Chemical formula: C22H23N3O4.
(Z)-N0-(3,4-Dichlorobenzylidene)-2-(1,3-dioxoisoindolin-2-yl)-
MW: 393.44 g. Yield: 48.75%. Rf: 0.2 (Hex/AcEt: 9/1). M.p.: 169– 3-phenylpropanehydrazide (9b). Chemical formula:
171 ꢀC. IR (KBr, cmꢂ1): 1661 (C]N); 1623 (C]O). 1H NMR (300
24H17Cl2N3O3. MW: 466.32 g. Yield: 46.82%. Rf: 0.7 (Hex/AcEt:
N), 170.1 (C]O). Elemental analysis – EAtheor: N 10.77%; C
C
MHz, DMSO-d6), d (ppm): 0.77 (m, 3H, CH3), 0.91 (m, 2H, CH2), 9/1). M.p.: 171–173 ꢀC. IR (KBr, cmꢂ1): 3442 (NH2), 2997 (C–H),
0.98 (m, 2H, CH3), 2.51 (m, 1H, CH), 4.11 (s, 3H, OCH3), 4.34 1700 (C]O), 1687 (C]N) cmꢂ1. 1H NMR (300 MHz, DMSO-d6),
(m, 1H, CH), 7.12 and 7.49 (2 br, 2H, NH2), 7.19–7.32 (m, 4H, d (ppm): 2.49 (m, 1H, CH), 4.11 (s, 3H, CH3), 7.10–7.52 (m, 8H,
Ar), 7.87–7.86 (m, 4H, Ar), 7.99 (s, 1H, NH), 8.01 (s, 1H, CH]N). Ar), 7.85–8.05 (m, 4H, Ar), 8.19 (s, 1H, CH]N). 13C NMR (75.5
13C NMR (75.5 MHz, DMSO-d6), d (ppm): 11.2 (CH3), 17.1 (CH3), MHz, DMSO-d6), d (ppm): 25.8 (CH2), 33.3 (CH), 58.5 (CH), 125.0
25.8 (CH2), 33.3 (CH), 45.9 (CH3O), 58.5 (CH), 123.6 (Ar), 135.0 (Ar), 126.5 (Ar), 131.0 (Ar), 135.0 (Ar), 143.6 (Ar), 168.1 (C]O),
(Ar), 168.1 (C]O), 170.1 (C]O). Elemental analysis – EAtheor: N 170.1 (C]O). Elemental analysis – EAtheor: N 9.01%; C 61.82%;
10.68%; C 67.16%; H 5.89%. EAexp: N 10.85%; C 67.30%; H H 3.67%; EAexp: N 9.24%; C 61.74%; H 3.46%.
5.69%.
(Z)-N0-(3,4-Dichlorobenzylidene)-2-(1,3-dioxoisoindolin-2-yl)-
(Z)-N0-(4-Chlorobenzylidene)-2-(1,3-dioxoisoindolin-2-yl)pro- propanehydrazide (9c). Chemical formula: C21H19Cl2N3O3. MW:
panehydrazide (8a). Chemical formula: C18H14ClN3O3. MW: 432.30 g. Yield: 50.02%. Rf: 0.7 (Hex/AcEt: 9/1). M.p.: 178–
355.78 g. Yield: 60.04%. Rf: 0.7 (Hex/AcEt: 9/1). M.p.: 209– 180 C. IR (KBr, cmꢂ1): 3442 (NH2), 2997 (C–H), 1700 (C]O),
ꢀ
210 ꢀC. IR (KBr, cmꢂ1): 1660 (C]N); 1624 (C]O). 1H NMR (300 1687 (C]N) cmꢂ1. 1H NMR (300 MHz, DMSO-d6), d (ppm): 1.11
MHz, DMSO-d6), d (ppm): 1.11 (d, 3H, CH3), 2.49 (m, 1H, CH), (d, 3H, CH3), 2.49 (m, 1H, CH), 4.11 (s, 3H, CH3), 7.51–7.63
4.11 (s, 3H, CH3), 7.51–7.72 (m, 4H, Ar), 7.85–8.05 (m, 4H, Ar), (m, 2H, Ar), 7.85–8.05 (m, 4H, Ar), 8.19 (s, 1H, CH]N). 13C NMR
8.09 (s, 1H, CH]N). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): (75.5 MHz, DMSO-d6), d (ppm): 15.4 (CH3), 20.8 (CH), 125.5 (Ar),
15.4 (CH3), 20.8 (CH), 123.6 (Ar), 125.5 (Ar), 128.6, 129.1 (Ar), 128.6 (Ar), 129.1 (Ar), 132.9 (Ar), 134.9 (Ar), 147.9 (Ar, C–Cl),
132.9 (Ar), 134.9 (Ar), 147.9 (Ar, C–Cl), 152.2 (C]O), 159.5 (CH] 152.2 (C]O), 159.5 (CH]N), 170.1 (C]O). Elemental analysis –
N), 170.1 (C]O). Elemental analysis – EAtheor: N 11.81%; C EAtheor: N 9.72%; C 58.35%; H 4.43%; EAexp: N 9.60%; C 58.27%;
60.77%; H 3.97%; EAexp: N 11.72%; C 60.68%; H 3.89%.
(Z)-N0-(4-Chlorobenzylidene)-2-(1,3-dioxoisoindolin-2-yl)-3-phe-
nylpropanehydrazide (8b). Chemical formula: C24H18ClN3O3.
H 4.67%.
Biological in vitro evaluation
MW: 431.87 g. Yield: 50.25%. Rf: 0.7 (Hex/AcEt: 9/1). M.p.: 210–
1
211 C. IR (KBr, cmꢂ1): 1658 (C]N); 1624 (C]O). H NMR (300
TNF-a and IL-1 levels. Mice peritoneal macrophages were
ꢀ
MHz, DMSO-d6), d (ppm): 2.51 (m, 2H, CH2), 4.34 (m, 1H, CH), placed into 96 well plates at a cell density of 2 ꢁ 106 cells per mL
6
ꢀ
7.10–7.52 (m, 8H, Ar), 7.87–7.86 (m, 4H, Ar), 7.99 (s, 1H, NH), 8.01 and incubated for 2 h at 37 C and 5% CO2. Cells at 2 ꢁ 10
(s, 1H, CH]N). 13C NMR (75.5 MHz, DMSO-d6), d (ppm): 25.8 concentration were suspended in RPMI 1640 with 5% FBS, 100
(CH2), 33.3 (CH), 45.9 (CH3O), 58.5 (CH), 123.6 (Ar), 125.0 (Ar), UI mLꢂ1 of penicillin, 100 mg mLꢂ1 of streptomycin and 50 mM
126.5 (Ar), 131.0 (Ar), 135.0 (Ar), 168.1 (C]O), 170.1 (C]O). 2-mercaptoethanol. 100 mL of suspension and 100 mL of samples
Elemental analysis – AEteor: N 9.73%; C 66.75%; H 4.20%; AEexp: N were incubated with LPS 2 mg mLꢂ1 (positive control) or with the
9.92%; C 66.83%; H 4.47%.
test compounds in different concentrations. Aer 24 h, the
(Z)-N0-(4-Chlorobenzylidene)-2-(1,3-dioxoisoindolin-2-yl)-3- supernatants were removed and kept at ꢂ80 C until the eval-
methylpentanehydrazide (8c). Chemical formula: C21H20ClN3O3. uation of cytokine levels (TNF and IL-1). The doses of cytokines
MW: 397.86 g. Yield: 55.54%. Rf: 0.7 (Hex/AcEt: 9/1). M.p.: 210– in the exudates were assayed by sandwich ELISA, using mono-
211 ꢀC. IR (KBr, cmꢂ1): 1660 (C]N); 1624 (C]O). 1H NMR (300 clonal antibodies specic to the detection of cytokines.
ꢀ
MHz, DMSO-d6), d (ppm): 0.77 (m, 3H, CH3), 0.91 (m, 2H, CH2),
TNF-a, cell death and apoptosis inhibition. The effect of
0.98 (m, 2H, CH3), 2.51 (m, 1H, CH), 4.11 (s, 3H, OCH3), 4.34 (m, each compound on inhibiting TNF expression and decreasing
1H, CH), 7.19–7.32 (m, 4H, Ar), 7.87–7.86 (m, 4H, Ar), 7.99 (s, 1H, cell viability was determined using the cell line FRT-Jurkat TNF.
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The analysis was carried out using a ow cytometer FACSCali-
bur 4-Colour (Becton, Dickinson and Company, New Jersey,
USA). The data analysis was obtained with FlowJo soware
(Tree-Star, Ashland, OR, USA). The study of cell viability with
DMSO and with the compounds, for 24 h, was carried out by
ow cytometry. The compounds were suspended and diluted to
100 mM and 10 mM in dimethyl sulfoxide. TNF inhibition was
determined as a decrease in GFP uorescence, which was
detected at a wavelength of 515 nm, in channel FL3. Viable and
non-viable cells, present following either the solvent alone or
compound treatment, were counted by gating on each cell
population using a forward scatter (FSC) versus side scatter
(SSC) plot. Viability was also assessed following propidium
iodide staining and detection of uorescence at 570 nm in the
3 J. Drewe, S. Kasibhatla, B. Tseng, E. Shelton, D. Sperandio,
R. M. Yee, J. Litvak, M. Sendzik, J. R. Spencer and S. X. Cai,
Bioorg. Med. Chem. Lett., 2007, 17, 4987–4990.
4 A. A. Beg and D. Baltimore, Science, 1996, 274, 782–784.
5 Z. G. Liu, H. Hsu, D. V. Goeddel and M. Karin, Cell, 1996, 87,
565–576.
6 D. J. Van Antwerp, S. J. Martin, T. Kafri, D. R. Green and
I. M. Verma, Science, 1996, 274, 787–789.
7 C. Y. Wang, M. W. Mayo and A. S. Baldwin, Science, 1996, 274,
784–787.
8 M. A. Palladino, F. R. Bahjat, E. A. Theodorakis and
L. L. Moldawer, Nat. Rev. Drug Discovery, 2003, 2, 736–746.
9 J. M. Chapman, G. H. Cocolas and I. H. Hall, J. Med. Chem.,
1979, 22, 1399–1402.
FL2 channel. For detection of apoptosis following treatment 10 I. H. Hall, J. M. Chapman and G. H. Cocolas, J. Pharm. Sci.,
with each compound, parental Jurkat cells were treated with the 1981, 70, 326–328.
substrate, labelled with uorescein isothiocyanate (FITC), 11 J. M. Chapman, P. J. Voorstad, G. H. Cocolas and I. H. Hall,
against cleaved caspase 3 by the Nucview-488 caspase 3 assay J. Med. Chem., 1983, 26, 237–243.
(Biotium Inc., Hayward, USA). The resulting uorescence was 12 T. O. Daniel, H. Liu, J. D. Morrow, B. C. Crews and
quantitated in channel FL3.
L. J. Marnett, Cancer Res., 1999, 59, 4574–4577.
¨ ¨
¨
13 S. Rollas and S. G. Kuçukguzel, Molecules, 2007, 12, 1910–
1939.
Acknowledgements
14 N. A. Zulkepli, K. V. K. Rou, W. N. H. W. Sulaiman, A. Salhin,
B. Saad and A. Seeni, Asian Pac. J. Cancer Prev., 2011, 12, 259–
263.
15 Q. Zeng, Z. Liu, B. Li and F. Wang, Amino Acids, 2004, 27,
183–186.
16 M. Karimi, L. C. Goldie, D. Ulgiati and L. J. Abraham,
BioTechniques, 2007, 42, 217–224.
17 K. M. Kroeger, K. S. Carville and L. J. Abraham, Mol.
Immunol., 1997, 34, 391–399.
We would like to thank the Brazilian National Research Council
(CNPq), the Research Foundation of Pernambuco State
(FACEPE) and the Cancer Council, Western Australia for
nancial support. M.V.O.C. is a recipient of the FACEPE
scholarship. Our thanks are also due to the Department of
Chemistry at the Federal University of Pernambuco (UFPE), for
1
recording the H NMR and IR spectra of all compounds.
18 S. G. Stewart, C. J. Braun, S.-L. Ng, M. E. Polomska, M. Karimi
and L. J. Abraham, Bioorg. Med. Chem., 2010, 18, 650–662.
19 S. Y. Yeung, S. Kampmann, K. A. Stubbs, B. W. Skelton,
B. J. Kaskow, L. J. Abraham and S. G. Stewart,
MedChemComm, 2011, 2, 1073.
References
1 A. L. Akeson, C. W. Woods, L. B. Mosher, C. E. Thomas and
R. L. Jackson, Atherosclerosis, 1991, 86, 261–270.
2 K. D. Nam, M. Han, J. Yoon, E.-A. Kim, S.-W. Cho and
H.-G. Hahn, Bull. Korean Chem. Soc., 2013, 34, 271–274.
This journal is © The Royal Society of Chemistry 2014
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