Phthaloyl amino acids as anti-inflammatory and immunomodulatory prototypes

Article abstract of DOI:10.1007/s00044-013-0730-1

A series of phthalimide analogs were synthesized by derivatization of phthalic anhydride, a highly toxic substance, using a "one pot" condensation reaction to α-amino acids. All phthaloyl amino acid derivatives presented anti-oral inflammatory activity, but compounds 2e and 2g were found to possess the best activities comparable to thalidomide.Most of the compounds effectively suppressed nitric oxide production inmurine cells stimulatedwith lipopolysaccharide. N-phthaloyl amino acids did not exhibit any significant cytotoxicity in vitro when tested against tumor cells as well as a spleen cell culture of BALB/c mice. Compounds 2a, 2g, and 2h were able to inhibit TNF-α and IL-1β production by macrophages. At the same concentration, thalidomide did not exhibit significant inhibitory activity. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0730-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1701–1708
DOI 10.1007/s00044-013-0730-1
ORIGINAL RESEARCH
Phthaloyl amino acids as anti-inflammatory
and immunomodulatory prototypes
Ana Cristina Lima Leite
Diogo R. M. Moreira
Lucas Cunha D. Co eˆ lho
Gevanio Bezerra de Oliveira Filho
Vald eˆ nia Maria Oliveira de Souza
Val e´ ria R eˆ go A. Pereira
Luiza de C. Reis Paulo Michel Pinheiro Ferreira
Claudia Pessoa Almir Gon c¸ alves Wanderley
Fernanda Virg ´ı nia B. Mota
Teresinha G. da Silva
•
F a´ bio Fernandes Barbosa
•
Marcos Ver ´ı ssimo de Oliveira Cardoso
•
•
•
Elany Barbosa da Silva
•
•
•
•
•
•
•
•
•
Received: 7 May 2013 / Accepted: 15 August 2013 / Published online: 12 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of phthalimide analogs were synthesized
by derivatization of phthalic anhydride, a highly toxic sub-
stance, using a ‘‘one pot’’ condensation reaction to a-amino
acids. All phthaloyl amino acid derivatives presented anti-oral
inflammatory activity, but compounds 2e and 2g were found
to possess the best activities comparable to thalidomide. Most
of the compounds effectively suppressed nitric oxide pro-
ductionin murine cells stimulated with lipopolysaccharide. N-
phthaloyl amino acids did not exhibit any significant cyto-
toxicity in vitro when tested against tumor cells as well as a
spleen cell culture of BALB/c mice. Compounds 2a, 2g, and
Introduction
It is well known that the free radicals formed during
inflammation play an important role in killing the micro-
organism and activating leukocytes and macrophages.
Overproduction of these radicals is associated with a wide
range of pathological conditions (Ariel and Serhan, 2007;
Nathan, 2002). Thus, it is important to develop anti-
inflammatory and immunomodulatory drugs that could
regulate the overproduction of these undesirable species
(Moormann et al., 2001).
2
h were able to inhibit TNF-a and IL-1b production by
Phthalimides are well-known plant growth regulators
(Butula et al., 1975; Hoffmann and Smith, 1949; Koch,
1971), bacteriostatic agents (Kant and Saksena, 2003;
Midtvedt, 1963), and fungicides (Kennedy et al., 1975).
Thalidomide, a multi-target drug, is the best-known
phthalimide and is a hypnotic/sedative drug with teratogenic
effects. Despite these effects, thalidomide has never com-
pletely vanished as a therapeutic substance. It was found to
have a powerful anti-inflammatory effect due to its ability to
inhibit the production of the cytokine tumor necrosis factor
alpha (TNF-a), a potent stimulator of inflammation, cellular
macrophages. At the same concentration, thalidomide did not
exhibit significant inhibitory activity.
Keywords N-phthaloyl amino acids ꢀ
Anti-inflammatory ꢀ Immunomodulatory
A. C. L. Leite (&) ꢀ F. F. Barbosa ꢀ M. V. O. Cardoso ꢀ
D. R. M. Moreira ꢀ L. C. D. Co eˆ lho ꢀ E. B. da Silva ꢀ
G. B. O. Filho ꢀ V. M. O. de Souza
C. Pessoa
Departamento de Fisiologia e Farmacologia, Centro de Ci eˆ ncias
da Sa u´ de, Universidade Federal do Cear a´ , Fortaleza,
CE 60430-270, Brazil
Departamento de Ci eˆ ncias Farmac eˆ uticas, Centro de Ci eˆ ncias da
Sa u´ de, Universidade Federal de Pernambuco, Recife,
PE 50740-520, Brazil
A. G. Wanderley
e-mail: acllb2003@yahoo.com.br
Departamento de Fisiologia e Farmacologia, Centro de Ci eˆ ncias
da Sa u´ de, Universidade Federal de Pernambuco, Recife,
PE 50740-520, Brazil
V. R. A. Pereira ꢀ L. de C. Reis
Departamento de Imunologia, Centro de Pesquisas Ageu
Magalh a˜ es-FIOCRUZ, Recife, PE, Brazil
F. V. B. Mota ꢀ T. G. da Silva
Departamento de Antibi o´ ticos, Universidade Federal de
Pernambuco, Recife, PE 50740-520, Brazil
P. M. P. Ferreira
Departamento de Ci eˆ ncias Biol o´ gicas, Universidade Federal do
Piau ´ı , Picos, Piau ´ı 64607-670, Brazil
123

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Med Chem Res (2014) 23:1701–1708
necrosis, and tissue damage in general (Fern a´ ndez-Mart ´ı nez
etal., 2001). Theanticanceractivityofthalidomideisbasedon
the inhibition of the growth of new vessels in the process of
angiogenesis (Lepper et al., 2004). Thalidomide is being
increasingly used in the clinical management of a wide
spectrum of immunologically mediated infectious diseases
and cancers such as erythema nodosum leprosum, multiple
myeloma, renal and intestinal carcinomas, Beh c¸ et’s, Crohn’s,
and a number of dermatologic diseases, as well as rheumatoid
arthritis and wasting syndrome in AIDS (Franks et al., 2004;
Gockel et al., 2004; Kerr and Ship, 2003; Lu et al., 2003;
Sayarlioglu et al., 2004; Srinivasan and Lichtenstein, 2004;
Zhu et al., 2003). It is also effective for mycobacterial infec-
tion in the central nervous system, for example, tuberculous
meningitiscausedbyMycobacteriumbovisorMycobacterium
bacillus (Tsenova et al., 1999).
Fig. 1 General synthesis of N-phthaloyl amino acid derivatives
Biological activities
Compounds 2a–g were screened for anti-inflammatory
activity in an air pouches model assay with a single dose of
Based on the broad spectra of thalidomide properties,
the phthalimide pharmacophore has been the target of
research with varying intentions. The method by which
phthalimide reduces TNF-a production is associated with
the induction of the degradation of TNF-a m-RNA (Kim
et al., 2004; Lentzsch et al., 2002; Orzeszko et al., 2003;
Yogeeswari et al., 2003). N-substituted phthalimides are of
high interest because they have been reported to possess
hypolipidemic activity (Srivastava et al., 2001) as well as
anti-inflammatory and immunomodulatory properties (Ha-
shimoto, 2002; Lima et al., 2002; Sena et al., 2003).
Phthalimides also have a number of applications in syn-
thetic chemistry (Casimir et al., 2000; Kukolja and
Lammert, 1975). In fact, a number of reports have
described the synthesis of N-phthaloyl amino acids fusing
free amino acids with phthalic anhydride (Billman and
Harting, 1948; Zeng et al., 2004). To the best of our
knowledge, no biological activity tests have been per-
formed for this structural core; therefore, we were inter-
ested in carrying out the pharmacological evaluations of
these small compounds. This communication reports the
results of the in vivo anti-inflammatory tests and immu-
nomodulatory profile. In addition, the compounds were
evaluated for their cytotoxicity against human tumor cell
lines and a spleen cell culture from BALB/c mice.
1
00 mg/kg orally administered to mice and evaluated for
their ability to inhibit leukocyte migration from the blood
circulation into air pouches (Table 1).
To determine the selectivity and immunomodulatory
action, the effect of the derivatives on the proliferation of
spleen cells from BALB/c mice (a method that is effective
for the evaluation of specific T lymphocyte cytotoxicity)
and their effects on NO-induced production in murine
spleen cells were tested using thalidomide (Thl) as a
reference.
Compounds 2a and 2b showed a weak anti-inflamma-
tory profile, whereas 2c, 2d, and 2h presented moderate
activity. The derivatives 2e, 2f, and 2g (an isoleucine,
glutamic acid, and phenylalanine derivative, respectively)
were the most potent anti-inflammatory agents. It is
Table 1 Anti-inflammatory activity orally of phthalimides 2a–h and
thalidomide (Thl)
6
No. of PMNL/mL (910 )
Treated (100 mg/kg)
Inhibition %
Vehicle
5.4 ± 0.1*
2.8 ± 0.2*
3.3 ± 0.2*
2.5 ± 0.1*
2.3 ± 0.3*
1.8 ± 0.2*
2.1 ± 0.4
–
2
2
2
2
a
b
c
47.7
38.4
54.2
57.8
67.5
61.7
64.2
43.8
72.1
d
Results and discussion
2e
2
f
Chemistry
2g
1.9 ± 0.1*
3.0 ± 0.2*
1.5 ± 0.2*
2
h
The compounds were synthesized according to described
procedures by fusing free amino acids with phthalic
anhydride (Fig. 1) (Zeng et al., 2004). It is worth men-
tioning that only the desired S-enantiomers were obtained
Thl
The data represent the mean ± the standard error of 6 animals
PMNL polymorphonuclear leukocytes
*
followed by Bonferroni’s post hoc test when compared with control
group
P \ 0.05. Significance was determined with one way ANOVA
in all cases, as verified by the addition of the chiral shift
1
reagent Eu(hfc) in the H-NMR spectra.
3
1
23

Med Chem Res (2014) 23:1701–1708
1703
interesting to note that the amino acid derivative 2f (a
synthetic precursor of Thl) presented a 15 % decrease in
anti-inflammatory activity relative to Thl.
10 mg/kg, it displayed a 64.7 % inhibition of inflamma-
tion, which is comparable to thalidomide (76.3 % inhibi-
tion). Compound 2h (a tryptophyl derivative), which
presented poor anti-inflammatory activity orally, was very
potent in inhibiting the production of NO, and it did not
show cytotoxicity for mammalian cells at a high dose
(50 lg/mL) (Fig. 2).
Furthermore, the compounds were evaluated in vitro
against a 4-cell-line panel consisting of MDA/MB-435
(human breast), HCT-8 (human colon carcinoma), and SF-
295 (human glioblastoma) using a previously described
MTT assay (Mosmann, 1983). In this protocol, each cell
line is inoculated and preincubated on a microtiter plate.
Test agents were then added at a single concentration
(25 lg/mL), and the culture was incubated for 48 h. End-
point determinations are made with MTT. The results for
each test agent were reported as the percent of growth of
the treated cells compared to the untreated control cells.
Compounds that reduce the growth of any one of the cell
lines to 32 % or less are considered cytotoxic (de Moreira
et al., 2007). According to these criteria, the derivatives
were considered non-cytotoxic (Table 3).
Previous work has shown that the inflammatory effects
of carrageenan in the air pouch are due to an influx of
predominantly neutrophilic leukocytes (mainly PMNL)
from the blood circulation, and the reduction in PMNL
suggests the inhibition of prostaglandin (PG) production
(
Jacobs et al., 1981). Therefore, the air pouch model is an
attractive method for the direct measurement of PG, TNF-
a, and NO, among other inflammatory mediators.
The data shown in Table 2 provide evidence that these
phthalimides are efficient at reducing lipopolysaccharide
(
LPS)-induced nitric oxide production at non-cytotoxic
levels and at stimulating the proliferation of spleen cells
without affecting cell viability at the dose assayed. These
results indicate that these compounds are promising anti-
inflammatory agents, especially compounds 2e and 2g.
Compound 2b (an alanine derivative) did not provide sig-
nificant anti-inflammatory activity, but it was able to
reduce the production of NO at non-cytotoxic levels.
To better understand the inhibitory effects on NO for the
amino acid 2h, the in vivo biological aspects were inves-
tigated using the air pouches model and the NO content of
the fluid was measured by the Griess reaction. In the fluid
samples from the air pouch, 2h and Thl (at a dose of
To evaluate the levels of TNF-a and IL-1b, human
macrophages were incubated with 50 lmol of compounds
2a–h over a 24-h time course. These cells were then
stimulated with LPS. The purpose of this test was to
evaluate the biological capacity of the molecules found to
inhibit the synthesis of these extremely important cytokines
in inflammatory processes and cancers. The data presented
in Fig. 3a show that compounds 2a and 2h both inhibit the
secretion of TNF-a by macrophages at 50 lmol/mL. In
addition, as shown in Fig. 3b, compounds 2a, 2g, and 2h
inhibit IL-1b production at the same concentration
1
0 mg/kg) significantly decreased the concentration of
LPS-induced nitric oxide production compared to the
control group (Table 2).
Thereafter, compounds 2e and 2h were selected to test
the dose-dependent response of the anti-inflammatory
activity. When compound 2e was given at an oral dose of
Table 2 Inhibitory effects on LPS-induced nitric oxide production
and cytotoxic effect on spleen cell BALB/c mice
a
Treated ? LPS NO-levels (lg/mL) ± SD* Cytotoxic effects
b
c
at 25 lg/mL at 50 lg/mL Conc. (lg/mL) % Inhibition
Thl
3.6 ± 0.0* 3.6 ± 0.0* [100
4.2 ± 0.9 3.6 ± 2.3 50
2.1 ± 0.3* 1.5 ± 0.6* [100
None
25.60
None
22.20
24.69
26.06
08.75
21.52
27.62
2
2
2
2
2
2
2
2
a
b
c
d
e
f
3.6 ± 0.0* 4.4 ± 1.2
6.4 ± 0.0 2.7 ± 0.6*
50
25
3.1 ± 0.0* 2.3 ± 0.0*
2.9 ± 0.9* 3.4 ± 0.3*
2.3 ± 0.0* 0.7 ± 0.0*
1.3 ± 0.9* 2.1 ± 1.4*
12.5
50
g
h
25
50
NO-induced production by stimulating with lipopolysaccharide (LPS-1.0 lg/
mL) and measured by Griess’s method [36]
*
a
P \ 0.05 compared with control (only LPS stimulus)
NO-induced levels induced by LPS 7.5 ± 1.2 lg/mL
The highest nontoxic concentration on spleen cell of BALB/c mice
Fig. 2 Anti-inflammatory effects of the compounds 2e, 2h, and Thl.
The results are expressed as the percentage of inhibition for n = 6
animals. The percent of inhibition was obtained by comparison with a
vehicle control group (data not shown) after 6 h (p.o.) of treatment
b
c
%
of viable cells
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Med Chem Res (2014) 23:1701–1708
Table 3 Antiproliferative activity against human cancer cells
Treated Inhibition of cell proliferation (% ± SD) at 25 lg/mL
Conclusion
a
In summary, N-phthaloyl amino acids were readily syn-
thesized and evaluated for their anti-inflammatory activity
and cytotoxicity, as well as their NO, TNF-a, and IL-1b
production inhibition. These amino acids possess simple
structures and could be easily structurally improved. In this
series, derivatives from the amino acids, isoleucine and
phenylalanine, showed the best oral anti-inflammatory
activity in an air pouch model. Moreover, the stimulation
of lymphocyte proliferation and the inhibition of NO pro-
duction suggest that the compounds could be considered as
potential immunomodulatory agents. The tryptophyl
derivative presented the best immunomodulatory activity
of the series. Investigation of immunomodulation and
toxicological profiles are subjects for further studies.
MDA/MB-435
HCT-8
SF-295
2
2
2
2
2
2
2
2
a
b
c
d
e
f
-4.72 ± 0.25
5.01 ± 22.38
-3.71 ± 0.29
7.82 ± 2.87
-0.61 ± 2.30
6.11 ± 2.88
18.94 ± 12.53
4.28 ± 1.87
15.02 ± 3.67
6.52 ± 2.88
-7.23 ± 2.30
10.44 ± 3.67
97.07 ± 4.15
3.98 ± 15.39
12.21 ± 4.26
12.57 ± 7.01
2.74 ± 5.88
14.95 ± 1.32
12.92 ± 5.96
11.72 ± 1.03
13.75 ± 2.43
94.64 ± 0.93
20.00 ± 6.01
14.42 ± 7.63
13.54 ± 5.38
24.51 ± 8.39
81.36 ± 0.80
g
h
Dox
a
Standard deviation is given in parentheses
(
50 lmol/mL). Thalidomide did not provide significant
inhibitory activity.
Experimental
Compound 2a (a phthaloyl-glycine derivative) had
presented weak anti-inflammatory activity, but it exhibited
an immunomodulatory profile when tested for NO, TNF-a,
and IL-1b production. Compound 2g (a phthaloyl-phenyl-
alanine derivative) showed oral anti-inflammatory activity
in the air pouch model, and its immunomodulatory profile
showed moderate activity for NO production and strong
inhibitory activity for IL-1b production. The phthaloyl-
tryptophyl derivative, 2h, exhibited immunomodulatory
activity toward NO, TNF-a, and IL-1b production but poor
anti-inflammatory activity with oral administration.
General
Melting points were determined on an electrothermal
1
capillary melting point apparatus and were uncorrected. H
1
3
and C NMR spectra were recorded on a Varian Unit Plus
instrument (300 MHz for protons and 75.5 MHz for car-
bon). All chemical shifts are reported in ppm relative to
TMS as an internal standard. IR spectra were recorded with
a Brucker model IFS66 FT-IR spectrophotometer using
KBr pellets. All chemicals were purchased from Aldrich,
Vetec, or Fluka and were used without additional
purification.
Chemistry
All reagents were used as purchased from commercial
sources (Sigma-Aldrich, Acros Organics, Vetec, or Fluka).
The progress of the reactions was monitored by thin-layer
chromatography (TLC) analysis (Merck, silica gel 60 F254
in aluminum foil). The chemical identity was confirmed by
NMR and IR spectroscopy and accurate mass (HRMS). IR
was performed in KBr pellets. For NMR spectroscopy, we
used either a Varian UnityPlus 300 MHz 1H (300 MHz)
and 13C (75.5 MHz) or a Bruker AMX-300 MHz 1H
(300 MHz) and 13C (75.5 MHz) instrument. DMSO-d6 and
Fig. 3 a Effect of the amino acyl phthalimides and Thl in on the
6
D O were purchased from CIL or Sigma-Aldrich. Chemi-
2
production of TNF-a in mouse macrophages (2 9 10 cells/mL)
cal shifts are reported in ppm, and multiplicities are given
as s (singlet), d (doublet), t (triplet), q (quartet), and m
stimulated with LPS (2 lg/mL). TNF-a was measured after 24 h of
incubation with compounds (50 lM) by sandwich ELISA (eBio-
science kit). Data are the mean ± S.D obtained in triplicate. b Effect
of phthalimides and Thl on the production of interleukin-1b (IL-1b)
(multiplet). NH signals were localized in each spectrum
after the addition of a few drops of D O. Mass spectrom-
2
6
-1
in mouse macrophages (2 9 10 ) stimulated with LPS (2 lg/mL ).
IL-1b was measured after 24 h of incubation with compounds
etry experiments were performed on an LC–IT–TOF
(Shimadzu). Unless otherwise specified, ESI was carried
(50 lM) by sandwich ELISA (eBioscience kit). Data are the
mean ± SD (standard deviation) obtained in triplicate. Thl,
thalidomide
out in the positive ion mode. Typical conditions were as
follows: capillary voltage of 3 kV, cone voltage of 30 V,
1
23

Med Chem Res (2014) 23:1701–1708
1705
and a peak scan between 50 and 1,000 m/z. The com-
pounds were previously synthesized by Zeng et al. (Zeng
et al., 2004).
(300 MHz, ppm):d 0.87 (d, J = 7.2 Hz, 3H); 1.22 (t,
J = 6.3 Hz, 3H); 1.50–1.49 (m, 2H); 2.54–2.52 (m, 1H);
4.70 (d, J = 8.4 Hz, 1H); 7.87–7.72 (m, 4H); 9.45 (s, 1H,
1
3
OH). C-NMR (75.5 MHz, ppm):d 10.6 (CH ); 16.6
3
Synthesis
(CH ); 25.4 (CH ); 33.9 (CH); 56.5 (CHa); 122.9 (C4 and
3
2
C9); 131.3 (C5 and C8); 133.8 (C6 and C7); 167.5
-
1
A mixture of 0.5 g (3.38 mmol) of phthalic anhydride 1
and the respective L-amino acid (3.38 mmol) in TEA or
(C = O); 170.3 (CO H). IR (KBr, cm ):3250 (mOH);
2
1763 (mC = O); 1703 (mC = O); 1460 (mC–N–C); 1394
(mC–N–C).
(S)-N-Phthaloylglutamine (2f) yield = 31.9 %. M.p.
4
-DMAP (0.5 mL) and DMF (three drops) was heated by
microwave irradiation for 2 min. The crude mixture was
1
then washed with n-hexane (30 mL), filtered through a
(°C):204–209. Rf = 0.58 (8:2-chloroform:methanol). H-
Buchner funnel and recrystallized with 0.1 M KHSO aq.
4
NMR (300 MHz, ppm):d 2.48–2.63 (m, 1H, CH2);
2.11–2.05 (m, 1H, CH ); 2.96–2.84 (m, 2H, CH );
or chloroform (40 mL), and dried over anhydrous Na SO .
2
4
2
2
The compounds 2f and 2h were obtained by heating at 180
C in an oil bath for 10 min.
S)-N-Phthaloylglycine (2a) yield: 96 %. M.p.
5.13–5.19 (m, 1H, CHa); 7.87–7.90 (m, 4H, Ar); 7.80 (m,
4H, Ar); 11.15 (s, 1H, CO H); 11.33 (s, 1H, CO H); The
8
2
2
1
11.15 and 11.33 signals disappear after adding D O. C-
3
(
2
1
(
(
°C):187–191. Rf:0.5 (8:2-chloroform/methanol). H-NMR
NMR (75.5 MHz, ppm):d 22.044 (CH ); 30.995 (CH );
2
2
300 MHz, ppm):d 4.15 (s, 2H, CH ); 7.50–7.70 (m, 4H,
49.025 (CHa); 122.967 (C2 and C5); 131.261 (C1 and C6);
134.927 (C3 and C4); 167.217 (C = O); 169.960 (CO H);
2
1
3
Ar); 10.58 (s, 1H, CO H). C-NMR (75.5 MHz, ppm):d
2
2
-
1
3
9.1 (CH ); 122.4 (C2 and C5); 132.6 (C1 and C6); 135.8
2
172.873 (CO H). IR (KBr, cm ): 3201 (mOH); 1775
2
(
C3 and C4); 165.9 (C = O); 174.9 (CO H). IR (KBr,
2
(mC = O); 1740 (mC = O); 1465 (mC–N–C).
(S)-N-Phthaloylphenylalanine (2 g) yield = 87.4 %.
-
1
cm ):3212 (mOH); 1775 (mC = O); 1715 (mC = O); 1401
(
mC–N–C); 1374 (mC–N–C).
S)-N-Phthaloylalanine (2b) yield: 70 %. M.p.
M.p. (°C):167–170. Rf:0.54 (8:2-chloroform:methanol).
H-NMR (300 MHz, ppm):d 3.60 (d, J = 9.0 Hz, 2H,
1
(
1
(
(
°C):135. Rf:0.46 (8:2-chloroform:methanol). H-NMR
CH ); 5.258 (t, J = 7.0 Hz, 1H, CHa); 7.15–7.196 (m, 5H,
2
300 MHz, ppm):d 1.70 (d, J = 7.5 Hz, 3H, CH ); 5.02 (q,
Ar); 7.65–7.68 (m, 2H, Ar); 7.76–7.790 (m, 2H, Ar); 9.01
1
3
3
J = 7.5 Hz, 1H, CH); 7.70–7.85 (m, 4H, Ar); 10.02 (s, 1H,
3
CO H). C-NMR (75.5 MHz, ppm):d 16.9 (CH ); 51.7
(CHa); 122.4 (C2 and C5); 132.7 (C1 and C6); 135.8 (C3
and C4); 165.6 (C = O); 171.9 (CO H). IR (KBr,
(s, 1H, OH). C-NMR (75.5 MHz, ppm):d 34.0 (CH );
2
1
0 0
52.9 (CHa); 123.4 (C2 and C5 ); 126.6 (C5 and C9); 128.3
2
3
0 0 0
(C6); 128.7 (C8); 130.7 (C7 ); 135.0 (C3 and C4 ); 137.3
-
(C4); 167.2 (C = O); 170.2 (CO H). IR (KBr, cm ):3274
1
2
2
-
1
cm ):3205 (mOH); 1752 (mC = O); 1740 (mC = O); 1469
(mCO H); 1752 (mC = O); 1704 (mC = O); 1450 (mC–N–C);
2
(
mC–N–C); 1397 (mC–N–C).
S)-N-Phthaloylbetaalanine (2c) yield: 90 %. M. p.
1398 (mC–N–C).
(S)-N-Phthaloyltryptophan (2 h) yield = 96 %. M.p.
(
1
1
(
(
°C):133–135. Rf:0.5(8:2-chloroform:methanol). H-NMR
(°C):94–96. Rf = 0.48 (8:2-chloroform:methanol). H-
300 MHz, ppm):d 2.50 (t, J = 15.0 Hz, 2H, CH ); 3.77 (t,
NMR (300 MHz, ppm):d 3.58 (d, J = 7.00 Hz, 2H, CH2);
5.15 (t, J = 7.00 Hz, 1H, CHa); 6.89 (t, J = 8.00 Hz, 4H,
Ar); 7.03 (m, 4H, Ar); 7.27 (d, J = 9.00 Hz, 4H, Ar); 7.49
(d, J = 9.00 Hz, 4H, Ar); 7.79 (s, 4H, Ar); 10.762 (s, 1H,
2
J = 15.0 Hz, 2H, CH ), 7.67–7.51 (m, 4H, Ar), 8.07 (s,
2
1
3
1
H, CO H). C-NMR (75.5 MHz, ppm):d 29.5 (CH2);
2
3
3.1 (CHa); 122.4 (C2 and C5); 132.6 (C1 and C6); 135.8
C3 and C4); 165.9 (C = O); 175.9 (CO H). IR (KBr,
1
3
(
NH); The 10.762 signal disappear after adding D O. C-
2
2
-
1
cm ):3170 (mOH); 1771 (mC = O); 1727 (mC = O); 1466
NMR (75.5 MHz, ppm):d 24.140 (CH ); 52.680 (CHa);
2
(
mC–N–C); 1411 (mC–N–C).
S)-N-Phthaloylvaline (2d) yield: 50 %. M.p.
109.758 (C7); 111.517 (C4); 117.960 (C10); 118.475 (C9);
0 0
(
121.049 (C8); 123.399 (C2 and C5 ); 126.964 (C11);
0 0 0 0
130.920 (C1 and C6 ); 134.923 (C3 and C4 ); 168.545
1
(
(
°C):79–81. Rf:0.60 (8:2-chloroform:methanol). H-NMR
300 MHz, ppm):d 1.9 (d, J = 4.0 Hz, 6H, CH3);
-
1
(C = O); 170.461 (CO H). IR (KBr, cm ): 3406 (mNH);
2
2
8
1
.70–2.79 (m, 1H, CH); 4.98 (d, J = 5.0 Hz, 1H, CHa);
3258 (mOH); 1771 (mC = O); 1715 (mC = O); 1464 (mC–
N–C).
.11 (d, J = 7 Hz, Ar); 8.16 (d, J = 7.0 Hz, Ar); 9.14 (s,
1
3
H, OH). C-NMR (75.5 MHz, ppm): d 18.9 (CH ); 29.5
3
(
CH); 56.5 (CHa); 122.4 (C2 and C5); 130.7 (C1 and C6);
35.9 (C3 and C4); 165.5 (C = O); 172.9 (CO H). IR
Biological evaluation
1
2
-
1
(
(
KBr, cm ):3234 (mCO H); 1763 (mC = O); 1691
Culture of normal, tumor cells, and cytotoxicity assays
2
mC = O); 1469 (mC–N–C); 1401 (mC–N–C).
(
S)-N-Phthaloylisoleusine (2e) yield: 74 %. M.p.
1
Heparinized human blood (from healthy, nonsmoker
donors who had not taken any drugs for at least 15 days
(
°C):110–112. Rf:0.5 (8:2-chloroform:methanol). H-NMR
123

1
706
Med Chem Res (2014) 23:1701–1708
prior to sampling, aged between 18 and 35 years old) was
collected, and peripheral blood mononuclear cells
In vitro cytotoxicity assays
(
PBMCs) were isolated by a standard method of density
The cytotoxicity of compounds 2a–h was determined using
5
BALB/c mice splenocytes (6 9 10 cells/well) cultured in
gradient centrifugation over Ficoll-Hypaque. All studies
were performed in accordance with Brazilian research
guidelines (Law 196/96, National Council of Health) and
with the Declaration of Helsinki.
96-well plates in RPMI 1640 media (Sigma Chemical Co.,
St. Louis, MO) supplemented with 10 % fetal bovine
serum (FCS; Cultilab, Campinas, SP, Brazil) and 50 lg/
mL of gentamycin (Novafarma, An a´ polis, GO, Brazil).
Each compound was evaluated at six concentrations (100,
Culture of colon (HCT-8)
5
0, 25, 10, 5, and 1 lg/mL) in triplicate in two independent
3
assays. Cultures were incubated in the presence of [ H]-
Glioblastoma (SF-295) and melanoma (MDA/MB-435)
cancer lines and PBMC were grown in RPMI 1640 medium
supplemented with 20 % fetal bovine serum, 2 mM glu-
tamine, 100 U/mL penicillin, and 100 lg/mL streptomycin
thymidine (Amersham Biosciences) (1 lCi/well) for 24 h
at 37 8C and 5 % CO . After this period, the content of the
2
3
plate was harvested to determine the [ H]-thymidine
3
at 37 °C with 5 % CO . The cytotoxicity of the compounds
([ H]TdR) incorporation using a beta-radiation counter (b-
matrix 9600, Packard). The toxicity of compounds 2a–h
2
against human cancer cells was determined by the MTT
assay (Mosmann, 1983), which analyzes the ability of
living cells to reduce the yellow dye 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to
3
was determined by comparing the percentage of [ H]-thy-
midine incorporation (as an indicator of cell viability) of
lectin-treated wells compared to untreated wells. Saponine
(0.05 %), concanavalin A (Con A), and phytohemaggluti-
nin (PHA) were used as positive controls. Non-cytotoxic
concentrations were defined as those causing a reduction in
a purple formazan product. Briefly, cells were plated in
5
6-well plates (0.3-0.7 9 10 cells/well) and incubated to
9
allow cell adhesion. Twenty-four hours later, extracts were
added to each well (0.04–100 lg/mL). After 72 h of
incubation, the supernatant was replaced by fresh medium
containing 10 % MTT, the formazan product was dissolved
in DMSO, and the absorbance was measured at 595 nm.
Quantification of cell proliferation was determined spec-
trophotometrically using a multiplate reader (DTX 880
Multimode Detector, Beckman Coulter). Control groups
3
[ H]-thymidine incorporation below 30 % in relation to
untreated controls.
Carrageenan-induced air pouch
The anti-inflammatory activity of the compounds was tes-
ted by the formation of air pouches on the dorsal cervical
region of mice (25–30 g) by subcutaneous injection of
2.5 mL of sterile air on day 0 followed by a second
injection of 2.5 mL of sterile air 3 days later. On the 6th
day, the mice received vehicle or the test compounds
orally. One hour after drug administration, inflammation
was induced by injecting 1 mL of carrageenan suspension
(1 % in saline solution) into the air pouch. After 6 h, the
mice were killed and the lumen of the air pouch was
lavaged by intrapouch injection of 3 mL of PBS containing
50 ng/mL heparin and subsequent aspiration of the fluid.
Total numbers of PMNL cell infiltration were determined
with a Neubauer hemocytometer.
(
negative and positive) received the same amount of
DMSO (0.1 %). Doxorubicin (Dox) (0.02–8.6 lM) was
used as a positive control.
Preparation of splenocytes
Mouse splenocytes were obtained by Pereira et al. (Her-
nandes et al., 2010). After killing the animal with CO gas,
2
the spleen of each mouse was removed aseptically and
placed in a Falcon tube containing RPMI 1640 with fetal
calf serum (complete medium). In a vertical flow, each
spleen was transferred to a Petri dish where they were
soaked. The cell suspensions obtained were transferred to
Falcon tubes containing approximately 10 mL of incom-
plete medium by spleen. Spleen homogenates were over-
TM
laid onto a Ficoll-Paque
PLUS layer with the density
In vitro nitrite analysis
adjusted to 1.076 g/mL and centrifuged at 1,0009g at
room temperature for 25 min. The interface cell layer
containing immune cells was recovered by a Pasteur pip-
ette, washed twice in PBS, and centrifuged two times at
The mice spleen cells were used to evaluate the concen-
tration of nitrite after treatment with LPS and the com-
pounds 2a–h (at 25 and 50 lg/mL), and after 72 h of
incubation, the media were carefully collected for mea-
surement by the colorimetric Griess method (Ding et al.,
1988). The NO concentration was estimated by a standard
curve of sodium nitrite (3.12-50 lmol/mL).
5
009g for 10 min. Cells were counted in a Neubauer
chamber, and cell viability was determined by the trypan
blue exclusion method. Cells were used only when viability
was greater than 98 %.
1
23

Med Chem Res (2014) 23:1701–1708
1707
TNF-a and IL-1b levels
Hernandes MZ, Rabello MM, Leite ACL et al (2010) Studies toward
the structural optimization of novel thiazolylhydrazone-based
potent antitrypanosomal agents. Bioorg Med Chem
Mice peritoneal macrophages were placed into 96-well
6
plates at a cell density of 2 9 10 cells/mL and incubated for
1
8:7826–7835. doi:10.1016/j.bmc.2010.09.056
Hoffmann OL, Smith AE (1949) A new group of plant growth
regulators. Science (New York, NY) 109:588. doi: 10.1126/
science.109.2841.588
Jacobs RS, White S, Wilson L (1981) Selective compounds derived
from marine organisms: effects on cell division in fertilized sea
urchin eggs. Fed Proc 40:26–29
2
2
h at 37 °C and 5 % CO . Cells at a concentration of
2
6
9 10 were suspended in RPMI 1640 with 5 % FBS, 100
UI/mL of penicillin, 100 lg/mL of streptomycin, and
0 mM 2-mercaptoethanol. One hundred microliters of the
suspension and 100 lL of the samples were incubated with
lg/mL LPS (positive control) or with the test compounds
5
Kant P, Saksena RK (2003) Synthesis and antimicrobial activity of
0
0
0
0
some new 2-phenyl-3-p-(2 -methyl-3 -aryl-4 -oxo-thiazolin-2 -
0
2
yl) phenyl quinazolin-4-ones and 2-phenyl-3-p-(1 -aryl-3-phtha-
0
in different concentrations. After 24 h, the supernatants were
removed and kept at -80 °C until the evaluation of cytokine
levels (TNF-a and IL-1b). The doses of cytokines in the
exudates were assayed by sandwich ELISA using mono-
clonal antibodies specific to the detection of the cytokine.
0
0
limido-4 -methylazetidin-2 -one-2 -yl)
ones. Indian J Heterocycl Chem 12:315–318
phenyl-quinazolin-4-
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Acknowledgments We would like to thank the Brazilian National
Research Council (CNPq), FACEPE, and FIOCRUZ for financial
support. MVOC received a CNPq doctoral scholarship and currently
receives a FACEPE researcher scholarship. We also thank the
Department of Fundamental Chemistry-UFPE for recording the H-
NMR and IR spectra of all compounds.
2
165/00128071-200304100-00002
Kim YS, Kim JS, Jung HC, Song IS (2004) The effects of thalidomide
on the stimulation of NF-kappaB activity and TNF-alpha
production by lipopolysaccharide in a human colonic epithelial
cell line. Mol Cells 17:210–216
Koch H (1971) Phytopharmakologische Untersuchung von Thalido-
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bindungen. Sci Pharm 39:209–247
1
Conflict of interest The authors state no conflict of interest.
Kukolja S, Lammert SR (1975) Azetidinone antibiotics. XIV.
Removal of a phthaloyl protective group from acid and base
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