Synthesis and anti-inflammatory activity of phthalimide derivatives, designed as new thalidomide analogues.

Article abstract of DOI:10.1016/S0968-0896(02)00152-9

This paper describes the synthesis and anti-inflammatory activity of new N-phenyl-phthalimide sulfonamides (3a-e) and the isosters N-phenyl-phthalimide amides (4a-e), designed as hybrids of thalidomide (1) and aryl sulfonamide phosphodiesterase inhibitor (2). In these series, compound 3e (LASSBio 468), having a sulfonyl-thiomorpholine moiety, showed potent inhibitory activity on LPS-induced neutrophil recruitment with ED(50)=2.5mg kg(-1), which was correlated with its inhibitory effect on TNF-alpha level.

Full text of DOI:10.1016/S0968-0896(02)00152-9

Bioorganic & Medicinal Chemistry 10 (2002) 3067–3073
Synthesis and Anti-Inflammatory Activity of Phthalimide
Derivatives, Designed as New Thalidomide Analogues
Lıdia M. Lima,a,b Paulo Castro,c Alexandre L. Machado,c Carlos Alberto M. Fraga,a,b
Claire Lugnier,d Vera Lucia Goncalves de Moraesc and Eliezer J. Barreiroa,b,
*
a
´
Laboratorio de Avaliac¸a˜o e Sıntese de Substancias Bioativas (LASSBio, http://www.farmacia.ufrj.br/lassbio),
´
ˆ
Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Brazil
´
Instituto de Quımica, Universidade Federal do Rio de Janeiro, Brazil
b
´
c
´
Departamento de Bioquımica Medica, Universidade Federal do Rio de Janeiro, Brazil
´
dCNRS URA 600, IllKirch, France
Received 3 December 2001; accepted 16 January 2002
Abstract—This paper describes the synthesis and anti-inflammatory activity of new N-phenyl-phthalimide sulfonamides (3a–e) and
the isosters N-phenyl-phthalimide amides (4a–e), designed as hybrids of thalidomide (1) and aryl sulfonamide phosphodiesterase
inhibitor (2). In these series, compound 3e (LASSBio 468), having a sulfonyl-thiomorpholine moiety, showed potent inhibitory
activity on LPS-induced neutrophil recruitment with ED50=2.5 mg kgꢀ1, which was correlated with its inhibitory effect on TNF-a
level. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
number of clinical trials with selective inhibitors of PDE
for different inflammatory disorders such as asthma.12,13
Trying to reduce side effects, much effort have been
expended to discover new substances that could selec-
tively block members of the PDEfamily, such as PDE-4,
the isoform found in all pro-inflammatory and immuno-
competent cells.14,15
Tumor necrosis factor-a (TNF-a), is a cytokine con-
sidered to be a primary mediator of the inflammatory
response.1 At lung level, it has been demonstrated that
the release of TNF-a favours the sequestration and
migration of neutrophils which play a critical role in the
pathogenesis of lung inflammation.2ꢀ4 Using different
models, it has been shown that adenosine cyclic mono-
phosphate (cAMP) elevating-agents can also regulate
the induction of TNF-a synthesis at the transcriptional
levels.5,6 Over production of TNF-a is associated with a
wide range of pathological conditions, this has led to
much recent effort in finding ways to downregulate its
production or inhibit its effects.7ꢀ9
It is well documented that elevated levels of cAMP
inhibit TNF-a production in activated monocytes and
peripheral blood mononuclear cells,22 and inhibition of
PDE-4 has been shown to be a successful method for
modulating TNF-a activity.
Thalidomide (1), a drug used to treat different inflam-
matory diseases is also an inhibitor of TNF-a
expression.16ꢀ21 It has been approved by the Food and
Drug Administration (FDA) in July 1998 for a treat-
ment of erythema nodosum leprosum. After that, the
interest in this drug has been intensified because of its
effective immunomodulatory and anti-inflammatory
properties.23 However, given the problems in admini-
stering an effective, nontoxic oral dose of thalidomide
(1), there is concern about the design of new compounds
that are based on the thalidomide structure, but which
have greater anti-TNF-a activity, less toxicity and
greater stability than the prototype (1).
Cyclic-AMP or GMP are ubiquitous second messengers
involved in a vast array of cellular responses to different
inflammatory stimuli.10 Inside the cell, cyclic nucleotide
phosphodiesterase (PDE) represents a group of 11
distinct proteins, differentially expressed depending on
the tissue, whose function is to hydrolyse cyclic nucleo-
tides.10,11 Since the last decade, studies on the control of
cAMP or cGMP levels through the action of PDE
increased and its relevance can be demonstrated by the
*Corresponding author. e-mail: eliezer@ufrj.br
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00152-9

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L. M. Lima et al. / Bioorg. Med. Chem. 10 (2002) 3067–3073
Chart 1.
As part of a research program aiming at the synthesis
and pharmacological evaluation of novel anti-inflam-
matory lead-candidates, we describe in the present
paper the synthesis and anti-inflammatory profile of
new N-substituted phthalimides derivatives (3a–e) and
(4a–e), structurally designed as hybrids of thalidomide
(1) and aryl sulfonamides, described as selective PDE-4
inhibitors, like active compound (2)24 (Chart 1). The
rational basis to design the new derivatives 3 was foun-
ded to promote a dual anti-inflammatory profile acting
at TNF-a and phosphodiesterase levels. For instance,
this symbiotic activity25 was anticipated to be possible
in a compound having the phthalimide ring of 1 (A,
Chart 1) and the phenylsulfonamide moiety of 2 (B,
Chart 1), raising the new derivatives 3a–e.
yield. The synthetic route to the new derivatives 3a–e
was accomplished by classical functional group inter-
conversion. Thus, the target compounds 3a–e could be
prepared by condensation of 7 with corresponding
piperazine derivatives,28 as showed in Scheme 1.
Finally, the bioisosteric series 4a–e, possessing a car-
bonyl group instead of the sulfonyl unit of derivatives
3a–e (Chart 1), was synthesized by the route illustrated
in Scheme 1. The amide derivatives 4a–e were prepared
from functionalized phthalimide 9, in excellent yields,
exploring classical methodology, using thionyl chloride
to the more electrophilic acid chloride, followed by
treatment with functionalized piperazines in the pre-
sence of dichlorometane, at room temperature.29
In order to investigate the contribution of the C frame-
work of 2 (Chart 1) in the anti-inflammatory activity,
the isoindoline group was substituted by a hexahydro-
pyrazine moiety in the derivative 3a. Next, we decide to
vary the N-piperazinyl ring substituent as methyl (3b),
the large phenyl group (3c) and by isosteric morpholine
and thiomorpholine nucleous, providing derivatives 3d
and 3e, respectively (Chart 1).
Results and Discussion
Thalidomide analogues (3a–e) were screened for their
ability to inhibit the acute inflammatory response, mea-
sured by inhibition of LPS-induced TNF-a and neutro-
phil infiltration into mice lungs, with thalidomide as
standard. The results obtained are shown in Figure 1.
In the series of sulfonamide derivatives (3a–e), the most
active compound (3e), which possesses the thiomorpho-
line ring at the aryl moiety of the 4-sulfonylphenyl-
phthalimide framework, was selected to study the dose-
dependent response for this new bioactive compound.
Compound 3e (LASSBio 468) inhibited the neutrophil
Chemistry
Among several obvious synthetic routes to the new
thalidomide analogues 3a–e, we decided to explore in
our approach the sulfonylchloride derivative (7) as the
key intermediate. This compound could be easily
obtained, in high yield, by the synthetic sequence
depicted in Scheme 1. The condensation of phthalic
anhydride (5) with aniline at reflux, furnished the
N-phenylphthalimide (6) in 86% yield.26
infiltration induced by LPS with ED50 2.5 mg kgꢀ1
.
In order to investigate the importance of phthalimide
ring in the anti-inflammatory activity, compound 3e
(LASSBio 468) was hydrolised by treatment with
lithium hydroxide in a mixture of THF/MeOH/H2O,29
to furnish compound 8 in 77% yield (Scheme 1). The
pharmacological evaluation of this compound (8)
revealed a remarkable decrease in anti-inflammatory
activity (Table 1).
The second step in the synthesis of 7 was based on a
regioselectivy
employing a mixture of chlorosulfonic acid and phos-
eletrophilic
aromatic
substitution
phorous pentachloride,27 at 50 ꢁC, obtaining 7 in 70%

L. M. Lima et al. / Bioorg. Med. Chem. 10 (2002) 3067–3073
3069
Scheme 1. (a) C6H5NH2, reflux, 1 h; 86%; (b) ClSO3H, PCl5, 50 ꢁC, 30 min, 70%; (c) functionalized piperazines, CH2Cl2, rt, 30 min, 60–66%;
(d) LiOH, THF, MeOH, H2O, rt, 10 min, 77%; (e) 4-CO2HC6H4NH2, AcOH, reflux, 1 h, 91%; (f) (1) SO2Cl, DMF (cat), reflux, 1 h; (2) CH2Cl2,
functionalized piperazines, rt, 30 min, 81–97%.
Table 1. Inhibitory effects of compounds 1, 3e, 4e, and 8 on neutro-
plihs recruitment induced by LPSa
Compd
% Inhibition
DMSO (vehicle)
Thalidomide (1)
LASSBio 468 (3e)
LASSBio 595 (4e)
LASSBio 596 (8)
0.0ꢂ0.0
50.0ꢂ7.3
72.0ꢂ8.6
48.0ꢂ11.7
50.0ꢂ7.3
aNeutrophils number was determined by BALF CTR=animals
pretreated with vehicle. Results are expressed as meanꢂSEM of five
animals.4
Figure 1. Effect of compounds 3a–e and thalidomide on neutrophil
influx induced by LPS into BALF of mice lungs.
In order to study the role of the sulfonyl group present
in the aryl-sulfonamide moiety found in the prototype
(2), we were able to replace this group by an isosteric
carbonyl unit, as in compounds 4a–e. The pharmacolo-
gical evaluation of amide compound 4e (LASSBio 596),
designed upon the bioassays results from 3e, revealed a
significant loss of activity when compared with the ori-
ginal compound 3e (Table 1), suggesting the crucial role
of the phenyl-sulfonyl-piperazine framework in the
investigated bioactivity.
Figure 2. Effect of compound 3e (LASSBio 468) on TNF-a levels and
neutrophil influx into the BALF of mice lungs.
It is well-known that elevation of intracellular levels of
cAMP in leukocytes is accompanied by significant inhi-
bition of the production of TNF-a and is associated
with inhibition of PDE-4 activity.6,30,31 Therefore, the
new thalidomide analogues 3e, 4e and 8 were also eval-
uated, in vitro, as PDE-4 and PDE-3 inhibitors. The
results obtained with PDE’s from bovine aorta assay,32
To correlate the anti-inflammatory activity of com-
pound 3e (LASSBio 468) with a possible effect on TNF-a
production, the cytokine levels were also evaluated as
shown in Figure 2. This result revealed the ability of
compound 3e (LASSBio 468) to inhibit TNF-a levels in
BALF of mice lungs treated with LPS.

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L. M. Lima et al. / Bioorg. Med. Chem. 10 (2002) 3067–3073
indicated that none of the compounds assayed pre-
sented a significative inhibitory effect on PDE-4 and
PDE-3 activity. For instance, 3e (LASSBio 468) pre-
sented only 40% of PDE-4 inhibition at 300 mM con-
centration and was ineffective on PDE-3. Compound 8
(LASSBio 596) and 4e (LASSBio 595) presented the
same poor profile of bioactivity (data not shown).
CDCl3) d 123.91 (C4 and C7), 126.74 (C20 and C60),
128.26 (C40), 129.28 (C30 and C50), 131.86 (C10), 131.94
(C3a–C7a), 134.56 (C5–C6), 167.44 (C1 and C3).
4-(1,3-Dioxo-2,3-dihydro-1H-2-isoindolyl)-1-benzene-
sulfonyl chloride (7).27 To a solution of 0.16 g (0.09 mL,
1,35 mmol) of chlorosulfonic acid and 0.14 g
(0.67 mmol) of phosphorus pentachloride that had been
stirred for 10 min, was added 0.15 g (0.67 mmol) of
N-phenyl-phthalimide (6) in small portion. The result-
ing mixture was stirred and heated at 50 ꢁC for 30 min.
The reaction mixture was poured onto ice and extracted
with chloroform. The organic phase was separated,
washed with brine, dried over anhyd Na2SO4 and eva-
porated at reduced pressure to furnish compound (7) in
In conclusion, in an effort to discover new thalidomide
analogues with anti-inflammatory activity we found a
novel lead-compound candidate 3e (LASSBio 468). The
preliminary SAR with this compound revealed the
importance of the sulfonyl group, the nature of the
N-terminal piperazine ring, and also indicated the role
of the phthalimide ring in the anti-inflammatory activity
of this compound (3e). Further, 3e presented a great
inhibitory activity on neutophil recruitment and on
TNF-a level in BALF of mice lungs treated with LPS.
Moreover, the ability of compound 3e (LASSBio 468)
to inhibit TNF-a production seems to not correlate with
activity awards PDE-4 or PDE-3, major isoforms found
in all pro-inflammatory and immunocompetent cells.
ꢁ
1
70% yield, as a white solid mp 180–182 C. H NMR
(200 MHz, DMSO-d6) d 7.41 (d, J=8.25 Hz, H2–H60),
7.74 (d, J=8.23 Hz, H30 and H50), 7.91 (m, H4–H7); 13
C
NMR (50 MHz, DMSO-d6) d 124.05 (C4 and C7),
126.66 (C20 and C60), 127.34 (C30 and C50), 132.08
(C3a–C7a), 132.67 (C10), 135.37 (C5–C6), 147.97 (C40),
167.50 (C1 and C3).
General procedures for the preparation of sulfonamides
3a–e28
Experimental
Chemistry
To a solution of sulfonyl chloride derivative (7) (0.5 g;
1.56 mmol) in 50 mL of methylene chloride, were added
the functionalized piperazine derivatives (3.12 mmol).
The reaction mixture was stirred for about 30 min, at
room temperature, when the end of reaction was
observed by TLC. Next, the phthalimide derivatives 3a–
e were isolated by addition of 50 mL of methylene
chloride and extraction with 10% aq HCl and brine.
The organic layer was dried over anhyd Na2SO4 and
evaporated at reduced pressure to give the sulfonamide
derivatives 3a–e in good yields.
Melting points were determined with a Buchi 540 appa-
ratus and are uncorrected. Proton magnetic resonance
(1H NMR), unless otherwise stated, was determined in
deuterated dimethylsulfoxide containing ca. 1% tetra-
methylsilane as an internal standard with Bruker AC
200, Bruker DRX 300 spectrometers at 200 and
300 MHz, respectively. Splitting patterns are as follows:
s, singlet; d, doublet; t, triplet; q, quartet; qt, quintet;
dd, double doublet; br, broad; m, multiplet. Carbon
magnetic resonance (13C NMR) was determined in the
same spectrometers described above at 50 and 75 MHz,
respectively, using deuterated dimethylsulfoxide as
internal standard. Infrared (IR) spectra were obtained
with Nicolet-550 Magna spectrophotometer using
potassium bromide plates. The mass spectra (MS) were
obtained by electron impact (70 eV) with a GC/VG
Micromass 12 spectrometer.
2-(4-Hexahydro-1-pyrazinylsulfonylphenyl)-1,3-isoindo-
linedione (3a). The title compound was obtained as a
white powder, in 60% yield, by condeꢁnsing 7 with
1
piperazine, as described above, mp >250 C. H NMR
(200 MHz, DMSO-d6) d 3.21 (sl, H100 and H200 and H300
and H400), 7.82 (d, J=8.58 Hz, H20 and H60), 7.97 (d,
J=8.62 Hz, H30 and 50), 7.99 (s, H5–H6 and H4–H7).
13C NMR (50 MHz, DMSO-d6) d 42.59 (C300 and C400),
43.30 (C100 and C200), 124.13 (C7 and C4), 128.18 (C20
and C60), 128.89 (C30 and C50), 131.93 (C7a and C3a),
133.73 (C6 and C5), 135.45 (C10), 137.04 (C40), 166.96
(C1 and C3). IR (KBr) 3495 and 3363 (n NH), 1787 and
1766 (n C¼O), 1747 and 1720 (n C¼O), 1381 (n C–N–
C), 1343 (n SO2) cmꢀ1. Anal. calcd for C18H17N3O4S: C,
58.21; H, 4.61; N, 11.31; O, 17.23; S, 8.63. Found: C,
58.19; H, 4.60; N, 11.33; O, 17.22; S, 8.62.
The progress of all reactions was monitored by tlc per-
formed on 2.0 ꢃ 6.0 cm aluminum sheets precoated with
silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm.
The developed chromatograms were viewed under
ultraviolet light at 254 nm. For column chromatography
Merck silica gel (70–230 mesh) was used.
2-Phenyl-1,3-isoindolinedione (6).26 A mixture of 0.5 g
(3.38 mmol) of phthalic anhydride in 0.4 mL
(4.06 mmol) of aniline was refluxed for 30 min. The
N-phenyl phthalimide (6) separates out on cooling. A
nearly white powder appeared which was filtered
through a Buchner funnel and washed twice with 20 mL
of water, to give the desired 2-phenyl-1,3-iso-
indolinedione (6) in 86% yield, mp 204–205 ꢁC. 1H
NMR (200 MHz, CDCl3) d 7.46 (m, H20-H60), 7.80 (m,
H5 and H6), 7.96 (m, H4 and H7). 13C NMR (50 MHz,
2 - [4 - (4 - Methylhexahydro - 1 - pyrazinylsulfonyl)phenyl]-
1,3-isoindolinedione (3b). The title compound was
obtained as a white powder, in 63% yield, by conden-
sing 7 with 1-methylpiperazine, as described above, mp
210–213 ꢁC. H NMR (200 MHz, DMSO-d6) d 3.21 (sl,
1
H100 and H200 and H300 and H400), 7.82 (d, J=8.58 Hz,
H20 and H60), 7.97 (d, J=8.62 Hz, H30 and 50), 7.99 (s,
H5–H6 and H4–H7). 13C NMR (50 MHz, DMSO-d6) d

L. M. Lima et al. / Bioorg. Med. Chem. 10 (2002) 3067–3073
3071
42.59 (C300 and C400), 43.30 (C100 and C200), 124.13 (C7
and C4), 128.18 (C20 and C60), 128.89 (C30 and C50),
131.93 (C7a and C3a), 133.73 (C6 and C5), 135.45 (C10),
137.04 (C40), 166.96 (C1 and C3). IR (KBr) 3495 and
3363 (n NH), 1787 and 1766 (n C¼O), 1747 and 1720 (n
C¼O), 1381 (n C–N–C), 1343 (n SO2) cmꢀ1. Anal. calcd
for C19H19N3O4S: C, 59.21; H, 4.97; N, 10.90; O, 16.60; S,
8.32. Found: C, 59.20; H, 4.96; N, 10.88; O, 16.59; S, 8.31.
2-[4-(1,4-Thiazinan-4-ylsulfonyl)phenylcarbamoyl]benzoic
acid (8).29 To a solution of 0.2 g (0.51 mmol) of the
phthalimide derivative (3e) in a solution of 6 mL of tet-
rahydrofuran/methanol/water (3:1:1) was added 0.1 g
(4.12 mmol) LiOH 98% and the resulting solution was
stirred at room temperature for 2 h. The reaction mix-
ture was diluted with ethyl ether (20 mL) and carefully
acidified with 2 N aq HCl. The organic layer was
washed with brine, dried over anhyd Na2SO4 and eva-
porated at reduced pressure to give the acid derivative
2-[4-(4-Phenylhexahydro-1-pyrazinylsulfonyl)phenyl]-1,3-
isoindolinedione (3c). The title compound was obtained
as a white powder, in 65% yield, by condensing 7 with
1-phenylpiperazine, as described above, mp 231–233 ꢁC.
1H NMR (200 MHz, CDCl3) d 3.26 (s, H100 and H200
and H300 and H400), 6.90 (m, H3000-H5000), 7.27 (dd,
J=7.58 and 8.25 Hz, H2000 and H6000), 7.76 (d,
J=8.74 Hz, H20 and H60), 7.86 (m, H5 and H6), 7.94 (d,
J=8.77 Hz, H30 and 50), 8.00 (m, H4 and H7). 13C
NMR (50 MHz, CDCl3) d 46.10 (C300 and C400), 49.28
(C100 and C200), 117.05 (C2000 and C5000), 120.95 (C4000),
124.09 (C7 and C4), 126.31 (C20 and C60), 128.66 (C30
and C50), 129.24 (C3000 and C5000), 131.41 (C7a and C3a),
134.44 (C6 and C5), 134.88 (C10), 136.12 (C40), 150.68
(C1000), 166.54 (C1 and C3). IR (KBr) 1789 and 1745 (n
C¼O), 1714 (n C¼O), 1384 (n C–N–C), 1348 (n SO2)
cmꢀ1. Anal. calcd for C24H21N3O4S: C, 64.42; H, 4.73;
N, 9.39; O, 14.30; S, 7.16. Found: C 64.43; H 4.72; N,
9.40; O, 14.29; S, 7.17.
(9) in 77% yield, as a white solid, mp 187–189 ꢁC. H
1
NMR (200 MHz, DMSO-d6) d 2.68 (sl, H300 and H400),
3.18 (sl, H100 and H200), 7.61 (d, J=8.14 Hz, H20 and
H60), 7.67 (d, J=8.10 Hz, H30 and 50), 7.96 (m, H4 and
H5), 7.95 (m, H3 and H6), 10.80 (s, CONHAr), 12.41
(sl, ArCO2H). 13C NMR (50 MHz, DMSO-d6) d 26.91
(C300 and C400), 48.28 (C100 and C200), 119.77 (C20 and
C60), 124.13 (C3), 128.42 (C6), 128.94 (C30 and C50),
130.15 (C5), 130.34 (C1), 130.94 (C2), 132.39 (C4),
138.89 (C40), 144.20 (C10), 167.71 (ArCONHR), 168.55
(ArCO2H).
4-(1,3-Dioxo-2,3-dihydro-1 H-2-isoindolyl)benzoic acid
(9).26 Dissolve 1.0 g (7.29 mmol) of 4-aminobenzoic
acid and 1.0 g (6.75 mmol) of phthalic anhydride in
10 mL of glacial acetic acid and reflux for 1 h. The
N-substituted phthalimide (9) separates out on cooling.
A nearly white powder appeared that was filtered
through a Buchner funnel and washed twice with 30 mL
of water, to give the desired 4-(1,3-dioxo-2,3-dihydro-
1H-2-isoindolyl)benzoic cid (9) in 91% yield, as white
2-[4-(1,4-Oxazinan-4-ylsulfonyl)phenyl]-1,3-isoindoline-
dione (3d). The title compound was obtained as a white
powder, in 66% yield, by condensing 7 with morpho-
line, as described above, mp 218–220 ꢁC. 1H NMR
(300 MHz, CDCl3) d 3.05 (t, J=4.70 Hz, H100 and H200),
3.77 (t, J=4.71 Hz, H300 and H400), 7.76 (d, J=8.76 Hz,
H20 and H60), 7.85 (m, H5 and H6), 7.90 (d, J=8.77 Hz,
H30 and 50), 8.00 (m, H4 and H7) ppm. 13C NMR
(75 MHz, CDCl3) d: 46.04 (C100 and C200), 66.16 (C300
and C400), 124.19 (C7 and C4), 126.42 (C20 and C60),
128.75 (C30 and C50), 131.43 (C7a and C3a), 134.08 (C6
and C5), 135.01 (C10), 136.22 (C40), 166.65 (C1 and C3)
ppm. IR (KBr): 1743 and 1716 (n C¼O), 1595 (n C–O–
C), 1373 (n C–N–C), 1352 (n SO2) cmꢀ1. Anal. calcd for
C18H16N2O5S: C, 58.06; H 4.33; N, 7.52; O, 21.48; S,
8.61. Found: C, 58.05; H 4.31; N, 7.53; O, 21.49; S, 8.60.
powder, mp >250 ꢁC. H NMR (200 MHz, DMSO-d6/
1
40 ꢁC) d 7.70 (d, J=8.70 Hz, H20–H60), 7.90 (m, H5–
H6), 7.99 (m, H4–H7), 8.08 (d, J=8.70 Hz, H30-H50),
13.02 (sl, RCO2H). 13C NMR (50 MHz, DMSO-d6/
40 ꢁC) d 123.42 (C4 and C7), 126.78 (C20 and C60),
129.75 (C30 and C50), 129.98 (C40), 131.36 (C3a and
C7a), 134.71 (C5 and C6), 135.73 (C10), 166.51
(ArCO2H), 166.62 (C1 and C3). IR (KBr) 3547 (n OH),
1782 and 1748 (n C¼O), 1746 (n C¼O), 1700 (n C¼O),
1428 (n N–C–O), 1380 (n C–N–C) cmꢀ1
.
General procedures for the preparation of amides 4a–e29
A solution of 0.2 g (0.75 mmol) of the acid (9) in 3 mL of
freshly distilled thionyl chloride and catalytic amount of
dimethylformamide was vigorously stirred under reflux
for 1 h. After this time, the solvent was carefully evapo-
rated at reduced pressure and a solution of 0.97 mmol of
the respective amine in 10 mL of methylene chloride was
added. The reaction mixture was stirred for 30 min at
room temperature, then poured in 20 mL of water and
extracted with methylene chloride (3 ꢃ 15 mL). The
organic layer was dried over anhydrous Na2SO4 and
evaporated at reduced pressure to give the amides deri-
vatives (4a–e) in excellent yields.
2-[4-(1,4-Thiazinan-4-ylsulfonyl)phenyl]-1,3-isoindoline-
dione (3e). The title compound was obtained as a white
powder, in 60% yield, by condensing 7 with thiomor-
pholine, as described above, mp 190–192 ꢁC. H NMR
1
(200 MHz, CDCl3) d 2.73 (t, J=5.22 Hz, H30 and H40),
3.40 (t, J=5.06 Hz, H10 and H20), 7.74 (d, J=8.58 Hz,
H20 and H60), 7.85 (m, H5 and H6), 7.88 (d, J=8.65 Hz,
H30 and 50), 7.99 (m, H4 and H7). 13C NMR (50 MHz,
CDCl3) d 26.95 (C300 and C400), 47.48 (C100 and C200),
123.65 (C7 and C4), 126.07 (C20 and C60), 127.79 (C30
and C50), 130.93 (C7a and C3a), 134.55 (C6 and C5),
135.25 (C10), 135.63 (C40), 166.55 (C1 and C3). IR
(KBr) 1786 and 1766 (n C¼O), 1745 and 1716 (n C¼O),
1379 (n C–N–C), 1337 (n SO2) cmꢀ1. Anal. calcd for
C18H16N2O4S2: C, 55.66; H 4.15; N, 7.21; O, 16.47; S,
16.51. Found: C, 55.67; H 4.15; N, 7.22; O, 16.48; S,
16.50.
2-(4-Hexahydro-1-pyrazinylcarbonylphenyl)-1,3-isoindo-
linedione (4a). The title compound was obtained as a
white powder, in 82% yield, by condensing 9 with
piperazine, as described above, mp >250 ꢁC. H NMR
1
(200 MHz, CDCl3) d 1.60 (sl, H100 and H200), 3.72 (sl,
H300 and H400), 7.59 (m, H20-60 and H30-H50), 7.84 (m,

3072
L. M. Lima et al. / Bioorg. Med. Chem. 10 (2002) 3067–3073
H5 and H6), 7.97 (m, H4 and H7). IR (KBr): 3470 (n
NH), 1739 and 1713 (n C¼O), 1621 (n C¼O), 1466 (n
N–C–O), 1389 (n C–N–C) cmꢀ1. Anal. calcd for
C19H17N3O3: C, 68.05; H 5.11; N, 12.53; O, 14.31;
Found: C, 68.06; H 5.10; N, 2.56; O, 14.32.
morpholine, as described above, mp 197–199 ꢁC. 1H
NMR (200 MHz, CDCl3) d 2.64 (sl, H300–H400), 3.85 (sl,
H100–H200), 7.54 (s, H20–H60 and H30–H50), 7.81 (m, H5
and H6), 7.96 (m, H4 and H7); 13C NMR (50 MHz,
CDCl3) d 27.40 (C300 and C400), 44.24 (C100), 49.70 (C200),
123.52 (C4 and C7), 126.10 (C20 and C60), 127.33 (C30
and C50), 131.20 (C3a and C7a), 132.71 (C40), 134.30
(C5 and C6), 134.80 (C10), 166.55 (C1 and C3), 169.43
(ArCONR2). IR (KBr) 1780 and 1761 (n C¼O), 1739
and 1715 (n C¼O), 1635 (n C¼O), 1459 (n N–C–O),
1379 (n C–N–C) cm ꢀ1. Anal. calcd for C19H16N2O3S:
C, 64.76; H 4.58; N, 7.95; O, 13.62; S, 9.10; Found: C,
64.77; H 4.59; N, 7.97; O, 13.61; S, 9.09.
2-[4-(4-Methylhexahydro-1-pyrazinylcarbonyl)phenyl]-
1,3-isoindolinedione (4b). The title compound was
obtained as a white powder, in 96% yield, by condensing 9
with 1-methylpiperazine, as described above, mp 140–
142 ꢁC. H NMR (200MHz, CDCl3) d 1.26 (R2NCH3),
1
3.05 (sl,0H100-H200, 3.14 (sl, H300-H400), 7.55 (s, H20-H60 and
H30-H5 ), 7.82 (m, H5 and H6), 7.98 (m, H4 and H7); 13
C
NMR (50 MHz, CDCl3) d 29.87 (R2NCH3), 35.59 (C100
and C200), 39.85 (C300 and C400), 124.05 (C4 and C7), 126.37
(C20 and C60), 128.14 (C30 and C50), 131.80 (C3a and C7a),
133.03 (C10), 134.76 (C5 and C6), 135.82 (C40), 167.15 (C1
and C3), 170.95 (ArCONR2). IR (KBr) 2923 (n CH3),
1788 and 1712 (n C¼O), 1617 (n C¼O), 1491 (n N–C–O),
1388 (n C–N–C) cmꢀ1. Anal. calcd for C20H19N3O3: C,
68.75; H 5.48; N, 12.03; O, 13.74. Found: C, 68.76; H 5.49;
N, 12.05; O, 13.75.
Biological assays
Male BALB/c mice weighing 25–30 g (UFF, RJ, BR.)
were used throughout this study. Animals were main-
tained in a temperature-controlled room and received
water and food ad libitum. During all experiments mice
were care in accordance with published guidelines for
animal use in laboratory.33 Mice were pretreated with
saline (vehicle) or 10 mg/kg of each compound ip,
45 min before LPS inhalation. The inhalation procedure
has been done as previously described.4 After different
time intervals, airspaces were washed with saline to
provide 4 mL of bronchoalveolar lavage fluid (BALF).
Aliquots were used for evaluate the neutrophil number
and for TNF-a assay. TNF-a levels were determined
by a highly specific ELISA with a detection limit of
50 pg/mL. Results are expressed as meanꢂSEM of five
animals.4
2-[4-(4-Phenylhexahydro-1-pyrazinylcarbonyl)phenyl]-
1,3-isoindolinedione (4c). The title compound was
obtained as a yellow powder, in 97% yield, by conden-
sing 9 with 1-phenylpiperazine, as described above, mp
242–244 ꢁC. 1H NMR (200 MHz, CDCl3) d 3.21 (sl,
H100–H200), 3.80 (m, H300–H400), 6.95 (d, J=8.58 Hz,
H2000 and H6000), 7.44 (m, H3000, H4000 and H5000), 7.58 (s,
H20–H60 and H30–H50), 7.82 (m, H5 and H6), 7.99 (m,
H4 and H7); 13C NMR (50 MHz, CDCl3) d 29.83 (C100
and C200), 49.93 (C300 and C400), 116.92 (C2000 and C6000),
120.82 (C4000), 124.04 (C4 and C7), 126.52 (C20 and C60),
128.17 (C30 and C50), 129.41 (C3000 and C5000), 131.74
(C3a and C7a), 133.26 (C40), 134.77 (C5 and C6), 135.13
(C10), 151. 06 (C1000), 167.09 (C1 and C3), 169. 61
(ArCONR2). IR (KBr) 1780 and 1760 (n C¼O), 1736
and 1711 (n C¼O), 1633 (n C¼O), 1461 (n N–C–O),
1379 (n C–N–C) cmꢀ1. Anal. calcd for C25H21N3O3: C,
72.98; H 5.14; N, 10.21; O, 11.67. Found: C, 72.97; H
5.12; N, 10.22; O, 11.65.
Acknowledgements
Thanks are due to CNPq (#52.0033/98–5), FAPERJ
(E-26/151.868/2000) and FUJB-UFRJ (BR.) for the
financial support of this work and to CNPq and CAPES
(BR.) for fellowships (LML, PC, ALM, CAMF, VLGM
and EJB). We are grateful to Analytical Center of NPPN
(UFRJ-BR) and Instituto de Quımica (UFRJ-BR).
2-[4-(1,4-Oxazinan-4-ylcarbonyl)phenyl]-1,3-isoindoline-
dione (4d). The title compound was obtained as a white
powder, in 87% yield, by condensing 9 with morpho-
line, as described above, mp 158–160 ꢁC. 1H NMR
(200 MHz, DMSO-d6) d 3.62 (sl, H100–H200 and H300–
H400), 7.56 (s, H20–H60 and H30–H50), 7.95 (m, H4–H7
and H5–H6); 13C NMR (50 MHz, DMSO-d6) d 66.38
(C100 and C200), 66.56 (C300 and C400), 123.98 (C4 and
C7), 127.67 (C20 and C60), 128.13 (C30 and C50), 132.00
(C3a and C7a), 133.37 (C40), 135.26 (C5 and C6), 135.54
(C10), 167.29 (C1 and C3), 168.90 (ArCONR2). IR
(KBr) 1786 and 1764 (n C¼O), 1740 and 1708 (n C¼O),
1625 (n C¼O), 1518 (n C–O–C), 1440 (n N–C–O), 1380
(n C–N–C) cmꢀ1. Anal. calcd for C19H16N2O4: C, 67.85;
H 4.79; N, 8.33; O, 19.03; Found: C, 67.86; H 4.79; N,
8.31; O, 19.04.
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