Thalidomide analogues: Tumor necrosis factor-alpha inhibitors and their evaluation as anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejps.2015.12.017

A series of related thalidomide derivatives (2-9) were synthesized by microwave irradiation and evaluated for anti-inflammatory activity. Such activity was assessed in vivo and ex vivo. Compounds 2, 8 and 9 showed the highest levels of inhibition of TNF-α production. On rat paw edema and hyperalgesia assays, compound 9, (1,4-phthalazinedione) demonstrated the highest in vivo anti-inflammatory activity. Thus, compound 9 can be considered as a promising compound to be subjected to further modification to obtain new agents for the treatment of inflammatory diseases.

Full text of DOI:10.1016/j.ejps.2015.12.017

Thalidomide analogues: Tumor necrosis factor-alpha inhibitors and their
evaluation as anti-inflammatory agents
Juan Jose´ Casal, Mariela Bollini, Mar´ıa Elisa Lombardo, Ana Mar´ıa
Bruno
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DOI:
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S0928-0987(15)30091-9
doi: 10.1016/j.ejps.2015.12.017
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28 November 2015
11 December 2015
Please cite this article as: Casal, Juan Jos´e, Bollini, Mariela, Lombardo, Mar´ıa Elisa,
Bruno, Ana Mar´ıa, Thalidomide analogues: Tumor necrosis factor-alpha inhibitors and
their evaluation as anti-inflammatory agents, (2015), doi: 10.1016/j.ejps.2015.12.017
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Thalidomide analogues: tumor necrosis factor-alpha inhibitors and their evaluation as
anti-inflammatory agents
Juan José Casala,§, Mariela Bollinia,§, María Elisa Lombardob,*, Ana María Brunoa,*.
aDepartamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, Junín 956, 1113. Ciudad Autónoma de Buenos Aires, Argentina.
bCentro de Investigaciones sobre Porfirias y Porfirinas (CIPYP, UBA-CONICET), Hospital de
Clínicas José de San Martín, Avenida Córdoba 2351, 1120. Ciudad Autónoma de Buenos Aires,
Argentina.
*Corresponding authors. Ana María Bruno: Telephone/Fax: 54-11-4964-8251
anabruno@ffyb.uba.ar
E-mail:
María Elisa Lombardo: Telephone/Fax: 54-11-5950-8347 E-mail: elombardo@qb.fcen.uba.ar
§: Both authors contributed equally to this work.

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ABSTRACT
A series of related thalidomide derivatives (2-9) were synthesized by microwave irradiation and evaluated
for anti-inflammatory activity. Such activity was assessed in vivo and ex vivo. Compounds 2, 8 and 9
showed the highest levels of inhibition of TNF-α production. On rat paw edema and hyperalgesia assays,
compound 9, (1,4-phthalazinedione) demonstrated the highest in vivo anti-inflammatory activity. Thus,
compound 9 can be considered as a promising compound to be subjected to further modification to obtain
new agents for the treatment of inflammatory diseases.
Keywords: Phtalimides, Phtalazinone derivate, TNF-α, Anti-inflammatory effect

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1. Introduction
Thalidomide (2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione), compound 1, is a synthetic
glutamic acid derivative developed as a sedative hypnotic agent to treat emesis during pregnancy
(Fig. 1). Despite its early successful clinical results, this compound had to be withdrawn from the
market due to its teratogenic effects (Ito et al., 2011). Nevertheless, over the past years, the
interest in this drug has resurged due to its potential usefulness in the treatment of erythema
nodosum leprosum (Sampaio et al., 1993; Haslett et al., 2005), multiple myeloma (Singhal et al.,
1999), AIDS as well as various cancers (Jacobson et al., 1999; Singhal et al., 2001). Thalidomide
has been found to have several biological activities, including, the inhibition of tumor necrosis
factor- (TNF-) production (Sampaio et al., 1991, Moreira et al., 1993), as well as anti-
inflammatory, anti-angiogenic (D’Amato et al., 1994), and cycloxygenase inhibitory activities
(Sano et al., 2005).
TNF- was originally described as a circulating factor that can cause necrosis of tumors. Later, it
has been identified as a key regulator of the inflammatory response. The central role of TNF- in
inflammation has been demonstrated through the ability of TNF-α blocking agents to treat a wide
range of inflammatory conditions.
TNF- production inhibitory activity was initially considered to be one of the key thalidomide
action mechanisms. This cytokine plays a critical role in several physiological immunological
processes, causing severe damage when produced in excess.
A number of phthalimide derivatives have been previously synthesized employing different
strategies in order to obtain molecules that can act as modulators of the over production of TNF-
(Niwayama et al.,1998; Muller et al.,1998; Lima et al., 2002; Cupertino Da Silva et al., 2010;
Stewart et al., 2007; Zhu et al., 2003; Machado et al., 2005; Chaulet et al., 2011; Tweedie et al.,

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2011). In an attempt to maintain the beneficial properties while avoiding its side effects, new
achiral synthetic thalidomide analogues have been designed.
In this work we optimized the synthesis process and evaluated the anti-inflammatory activity of a
series of phthalimides that are, structurally related to thalidomide (Fig. 1). To test the anti-
inflammatory activity in vivo (rat paw edema, hyperalgesia and myeloperoxidase activity) and ex
vivo (TNF-α production) assays were employed. A phthalazinone derivative was synthesized and
its anti-inflammatory activity was also evaluated.
2. Materials and methods
Melting points (uncorrected) were determined in a capillary with an Electrothermal 9100
SERIES-Digital apparatus. The microwave-assisted reaction was carried out in Monowave 300
Anton Paar. Reactions were monitored by thin-layer chromatography (TLC) in silica gel plates
(F245 Merck) and the products visualized under ultraviolet light (254 and 365 nm).
IR spectra were recorded with a FT Perkin Elmer Spectrum One employing KBr discs. 1H- and 13
C-NMR spectra were determined in DMSO-d6 and CDCl3 solutions using a Bruker 300 MHz and
Bruker Biospin 600 MHz AVIII600 spectrometers at room temperature with tetramethylsilane as
internal standard. Chemical shifts () are reported in ppm and coupling constants (J) in Hertz.
2.1. Chemistry
2.1.1. General procedure for the synthesis of phthalimide derivatives
A mixture of phthalic anhydride (2.5 mmol, 0.37 g), 2.5 mmol of the corresponding amine and
0.1 mL of DMF were subjected to microwave irradiation (Fig. 1). Reaction time and temperature
varies for each derivative being synthesized. The reaction was monitored by TLC employing
Cl2CH2: MeOH (8:2) as mobile phase. After complete conversion, the reaction mixture was

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triturated with ethanol and the solid product was recrystallized from a solvent mixture (EtOH:
H2O, 2:1).
2.1.1.1 (E)-2-((4-nitrobenzylidene)amino)isoindoline-1,3-dione (2)
Reaction temperature: 170°C, reaction time: 2 min. A pale yellow solid was obtained after
recrystallization. Yield: 0.45 g (62%). Mp: 260-264 °C. Mp Lit. (Hearn et al., 1982, Salman et
al., 1981): 301-305 °C and 290°C. IR (KBr, cm-1): 1724 (C=O), 1597 (C=N), 1513 and 1345
(NO2), 837 (C-H). 1H NMR (600 MHz, DMSO-d6): δ 8.48 (s, 1H, HC=N), 8.28 (d, 2H, J=8.2
Hz, ArH), 7.76 -7.84 (m, 3H, ArH), 7.50 (dd, 3H, J=7.8 1.6 Hz, ArH).
13C NMR (151 MHz, DMSO-d6): 163.9, 148.5, 136.7, 135.6, 129.5, 129.3, 125.3, 124.7, 124.1.
2.1.1.2 2-(4H-1,2,4-triazol-4-yl)isoindoline-1,3-dione (3)
Reaction temperature: 170 °C, reaction time: 4:00 min. A white solid was obtained after
recrystallization. Yield: 0.52 g (87%). Mp: 266-269°C. Mp Lit. (Sena et al., 2003): 269.7–270.4
°C. 1H NMR (300 MHz, DMSO-d6):  8.44 (s, 2H, H-3’, H-5’), 7.84 (d, 2H, J = 7.5 Hz, ArH),
7.74 (d, 2H, J = 7.5 Hz, ArH). 13C NMR (75 MHz, DMSO-d6): δ 166.9, 141.2, 135.5, 133.8,
132.9, 128.9, 119.4, 118.7.
2.1.1.3 2-(4-methylpirimidin-2-yl)isoindolin-1,3-dione (4)
Reaction temperature: 160°C, reaction time: 4:00 min. A white solid was obtained after
recrystallization. Yield: 0.13 g (22%). Mp: 178-180 °C. Mp Lit. (Cingolani et al., 1976): 188-190
°C. 1H NMR (300 MHz, DMSO-d6):  8.78 (d, 1H, J = 6.0 Hz, H-6’), 7.99-7.97 (m, 2H, ArH),

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7.84-7.80 (m, 2H, ArH, H-5’), 7.25 (dt, 1H, J = 6.0, 0.51 Hz, ArH), 2.89 (s, 3H, CH3). 13C NMR
(75 MHz, DMSO-d6): δ 168.8, 167.6, 158.5, 135.2, 133.9, 132.9, 128.9, 126.1, 21.1.
2.1.1.4 2-(4-nitrophenyl)isoindolin-1,3-dione (5)
Reaction temperature: 160°C, reaction time: 2:00 min. A yellow solid was obtained after
recrystallization. Yield 0.54 g (82%). Mp: 273-275 °C. Mp. Lit. (Aliabadi et al., 2014): 265°C.
IR (KBr, cm-1): 1738 (C=O), 1523 and 1346 (NO2), 837 (C-H). 1H NMR (600 MHz, DMSO-
d6): δ 8.42 (d, 2H, J = 8.6 Hz, ArH), 8.02 (dt, 2H, J = 7.6, 3.9 Hz, ArH), 7.95 (dd, 2H, J = 5.5,
3.1 Hz, ArH), 7.81 (d, 2H, J = 8.6 Hz, ArH).13C NMR (151 MHz, DMSO-d6): δ 166.8, 146.6,
138.2, 135.4, 131.9, 128.2, 124.6, 124.2.
2.1.1.5 2-(4-methoxy-2-nitrophenyl)isoindolin-1,3-dione (6)
Reaction temperature: 160 °C, reaction time: 4:00 min. A yellow solid was obtained after
recrystallization. Yield: 0.65 g (88%). Mp: 132-134 °C. Mp Lit. (Davood et al., 2013): 148-153
°C. 1H NMR (600 MHz, DMSO-d6): δ 8.03 (dd, 2H, J = 5.5, 3.0 Hz, ArH), 7.97 (td, 2H, J = 5.2,
2.1 Hz, ArH), 7.76 (d, 1H, J = 2.9 Hz, ArH), 7.70 (d, 1H, J = 8.8 Hz, ArH), 7.53 (dd, 1H, J =
8.8, 2.9 Hz, ArH), 3.94 (s, 3H, OCH3). 13C NMR (151 MHz, DMSO-d6): δ 166.9, 160.2, 146.8,
135.7, 132.8, 131.8, 124.3, 120.8, 117.5, 110.9, 56.8.
2.1.1.5 2-(pyridin-2-yl)isoindolin-1,3-dione (7)
Reaction temperature: 170 °C, Reaction time: 4:00 min. A white solid was obtained after
recrystallization. Yield 0.49 g (88%). Mp: 227-230 °C. Mp Lit. (Guirado et al., 1997): 225-226
°C. IR (KBr, cm-1): 1713 (C=O), 1586 and 1571 (C=C and C=N), 717 (C-H).1H NMR (600

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MHz, DMSO-d6): δ 8.66 (dd, 1H, J = 7.1, 3.1 Hz, ArH), 8.06 (t, 1H, J = 7.0 Hz, ArH), 8.01 (dd,
2H, J = 7.1, 3.1 Hz, ArH), 7.95 (dd, 2H, J = 7.3, 3.0 Hz, ArH), 7.58 – 7.53 (m, 2H, ArH). 13C
NMR (151 MHz, DMSO-d6): δ 166.9, 149.8, 146.3, 139.1, 135.4, 131.8, 124.5, 124.1, 123.5.
2.1.1.6 2-(benzo[d]tiazol-2-yl)isoindolin-1,3-dione (8)
Reaction temperature: 170°C, reaction time: 4:00 min. A white solid was obtained after
recrystallization. Yield: 0.64 g (92%). Mp: 147-148°C. Mp Lit. (El Sadek et al., 1989): 143-147
°C. IR (KBr, cm-1): 1725 (C=O), 1594 and 1512 (C=C and C=N), 838 (C-H). 1H NMR (600
MHz, DMSO-d6): δ 8.16 (dd, 1H, J = 7.4, 3.1 Hz, ArH), 8.07 – 8.03 (m, 3H, ArH), 7.98 (d, 2H, J
= 7.4 Hz, ArH), 7.57 (t, 1H, J = 7.4 Hz, ArH), 7.48 (t, 1H, J = 7.4 Hz, ArH). 13C NMR (151
MHz, DMSO-d6): δ 165.0, 152.5, 149.4, 135.7, 133.1, 131.5, 126.9, 125.7, 124.6, 122.8, 122.4.
2.1.1.7 2-(4-chlorophenyl)2,3-dihydrophthalazin-1,4-dione (9)
Reaction temperature: 180°C, reaction time: 4:00 min. A white solid was obtained after
recrystallization. Yield: 0.49 g (74%). Mp: 182-184°C. (Prime et al., 2011). IR (KBr, cm-1):
3165 (NH), 1663 (C=O), 1496 (NH) and 790 (C-Cl). 1H NMR (600 MHz, DMSO-d6): δ 8.54 (s,
1H, NH), 7.98 (dt, 2H, J = 7.4, 3.7 Hz, ArH), 7.93 – 7.92 (m, 2H), 7.62 (d, J = 8.6 Hz, 2H), 7.51
(d, J = 8.6 Hz, 2H). 13C NMR (151 MHz, DMSO-d6):  161.0, 160.6, 135.2, 133.0, 132.3, 131.3,
129.6, 129.4, 124.0.
2.2. Biology
2.2.1. Animals

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Male BALB/c mice (25-30 g) and male Spraguee-Dawley rats (240-270 g) were obtained from
the LASSBio breeding unit (Faculdade de Farmácia, Universidade Federal do Rio de Janeiro,
Brazil). Animals were maintained in a temperature-controlled room and received water and food
ad libitum. Animals received human care and were treated according to the “Principles of
Laboratory Animal Care and Use in Research” (Colégio Brasileiro de Experimentacão Animal -
COBEA/Instituto Brasileiro Carlos Chagas Filho - IBCCF, Brazil) that is based upon the
International Guidelines for the Care and Use of Laboratory Animals and Ethical Guidelines for
the Investigation of Experimental Pain in Conscious Animals (Zimmerman M., 1983).
2.2.2. Ex vivo anti-inflammatory activity assay: TNF-α production
Male BALB/c mice were used throughout this study. Animals were pretreated with saline
solution (vehicle) ip. 45 min before LPS inhalation. The inhalation procedure was performed as
previously described (Corti et al., 1994). After aspiration, airspaces were washed with saline to
obtain 4 ml of bronchoalveolar lavage fluid (BALF). Aliquots of BALF were used for TNF-α
assay after incubation with either drugs (10 - 100 µM) or with thalidomide (100 - 300 µM). The
concentration of TNF-α was determined by ELISA according to the manufacturer´s instructions.
The assay had a detection limit of 250 pg/ml (Genzyme, Cambridge, MA, USA).
2.2.3. In vivo anti-inflammatory activity assays
2.2.3.1. Rat paw edema
Male Spraguee-Dawley rats were divided in different groups which received an ip. injection of
test compound (10mg/kg as a suspension in 1ml of 0.5% methylcellulose). Control animals
received an equal volume of vehicle. An hour later each group of animals received a subplantar

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injection of 0.2 ml of either 1% carrageenan solution in saline (w/v) or sterile saline (0.9% NaCl)
on the right and left hind paws respectively. Paw volumes were measured with a plethysmometer
(UGO Basile, Italy) immediately before the subplantar injection (0 h) and 3 h after. The edema
(µl) was calculated by subtracting the difference between the carrageenan- and saline- treated
paws evaluated at 0 h to the same difference measured at 3 h.
2.2.3.2. Hyperalgesia
Carrageenan-induced hyperalgesia was quantified in separate groups of animals as a measure of
the nociceptive response to a thermal stimulus using the hot-plate test with the temperature
adjusted to 51 ± 2°C (Bannon, 2001). For this study male Spraguee-Dawley rats were used and
test drugs were administered orally at a dose of 10 mg/kg, 1 h before carrageenan administration.
The control group received only vehicle (10 ml/kg). Animals were then injected subplantarly
with 0.2 ml of either 1% carrageenan solution in saline (w/v) or sterile saline (0.9% NaCl) on the
right and left hind paws respectively. Each hind paw was placed on the heated surface at
intervals of 1 h for 4 h after carrageenan injection and the time between placement and the first
sign of paw licking or withdrawal was recorded as latency. The latency time (withdrawal time)
of each hind paw was determined and the anti-inflammatory activity defined as a decrease in the
Δ latency (s) calculated as difference between carrageenan and saline paw latency times. A cut-
off time of 40 s was established to prevent injury to the paws.
2.2.3.3. Myeloperoxidase activity
Animals were treated as described above for the rat paw edema test. The methodology used to
measure MPO activity was adapted from the one previously described by Posadas et al., 2004.

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Briefly, rats were killed 6 h after carrageenan administration and inflamed paws were weighed,
cut and suspended in hexadecyl trimethylammonium bromide (HTBA) buffer (containing 5g
HTBA in 1 l potassium phosphate buffer 50 mM pH 6.0) in ratio of 50:1 (mg tissue:ml buffer).
Resuspended tissues were homogenized at 4°C with a Turrax ® at 13,000 rpm for 3 min. After
centrifugation at 13,000 rpm for 2 min, supernatants were assayed for MPO activity. Samples
(100 µl) were mixed with 50 mM potassium phosphate buffer pH 6.0 (100 µl) containing 2 mM
o-dianisidine dihydrochloride and 0.002% hydrogen peroxide in a microtiter plate. The
absorbance at 450 nm was measured after incubation at 25 ºC with constant stirring for 15
minutes. One unit of MPO is defined as the amount of enzyme converting 1 µmol of hydrogen
peroxide to water in 1 min at 25° C. MPO activity was expressed as Units/mg fresh inflamed tissue.
2.2.4. Data analysis
Results are presented as means ±n S.E.M. of measurements made on at least four to six animals in
each group. Differences between responses were evaluated by the Student t-test and two ways
analysis of variance (ANOVA). Statistical differences were considered significant from P<0.05.
To calculate physicochemical descriptors PaDEL-descriptor software were performed. (Yap,
2011).
3. Results and Discussion
3.1. Chemistry
The general method for the synthesis of N-substituted (alkyl, aryl, heteroaryl) phthalimides
employing the corresponding amines and phthalic anhydride requires long reflux reaction times.
The synthesis methods using microwave irradiation shortens reaction times and provides higher

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yields. Therefore, the target compounds were obtained within minutes by a microwave-assisted
method. With the exception of compound 4, good yields and excellent purities were obtained
(Fig. 1). Reaction conditions were optimized by using different solvents (dioxane, acetic acid and
DMF) observing that DMF as an aprotic reaction solvent at 160-170 °C gave the best yields
(compounds 2-8).
By applying microwave radiation, compound 9 was synthesized in one step obtaining a very
good yield.
The synthesis of these compounds involving the reaction of phthalic anhydride with electron-rich
phenylhydrazines affords the corresponding phthalazine-1,4-diones in one step, though with
moderate yields (30-50%), whereas electron poor phenylhydrazines require an additional ring-
expansion step under harsh conditions after the formation of the initial isoindole-1,3-diones to
give the target phthalazine-1,4-diones in good yields (70-80%) (Prime et al., 2011).
The structures of compounds 2-9 were established on the basis of spectral (1H NMR, 13C NMR,
FTIR) analyses and they were identical to those found in literature.
All synthesized compounds meet the rule of Oprea (Oprea et al., 2001) which considers the
physicochemical descriptors required to generate leadlike compounds (Table 1).
3.2. Anti-inflammatory activity
TNF-α is a cytokine considered to be a primary mediator of the inflammatory response. To
evaluate if the compounds 2-9 had anti-inflammatory activity, its effect on TNF-α production
was investigated. The results (Fig. 2A) revealed that all compounds tested at10 and 100 µM had
higher anti-inflammatory activity than thalidomide at 100 and 300 µM (in our experiments
thalidomide were employed for comparison purposes rather than as a positive control).

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Compound 2 was not tested at 100 µM due to its low solubility in DMSO. Compound 5 showed,
independently of the concentration, equal inhibitory activity. A different behaviour was observed
for compound 3, which showed a lower inhibitory activity at the highest concentration.
Fig. 2B shows the IC50 (50% inhibitory concentration) values obtained for the different
thalidomide derivatives. Compounds 2, 8 y 9 with IC50 values of 8.4, 1.2 and 2.1 M
respectively, were selected for further testing since they were the ones displaying the highest
inhibitory activities on TNF-α production.
Finally, we investigated the possible correlation between the IC50 values and the
physicochemical properties (Table 1). A good inverse correlation (r = -0.8738, P<0.05) was
observed between inhibitory activity on TNF-α production and CLogP, indicating that higher
ClogP values (high lipophilicity and ability to cross membranes) correlated with low IC50 values
(higher anti-inflammatory activity). However, no relationship was found with the other
physicochemical descriptors.
Results for the determination of anti-inflammatory activity of compounds 2, 8 and 9 on the rat
paw edema are illustrated in Fig. 3. At the concentration tested (10 mg/kg), compound 9 was the
only one which exerted inhibition of the paw edema (36%). The inhibition produced by the
compounds 2, 8 and thalidomide showed no statistically significant differences as compared to
control animals (P<0.05).
Fig. 4 depicts the effect of the treatment with compounds 2, 8 and 9 (10 mg/kg) on carrageenan-
induced hyperalgesia (compound 1 was used for comparison). During the period spanning from 0
to 4, the injection of carrageenan induced a substantial increase in Δ latency, which was
significantly lower (about 45%) in rats treated with compound 9. No significant differences with
the control group were found for animals treated with compounds 1, 2, or 8.

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Finally, the values of MPO activity, a neutrophils sequestration marker enzyme, were determined
(Fig. 5). All the three compounds tested reduced MPO activity significantly as compared to the
control group (between 48-60%, P = 0.05). As expected, inhibition of this enzyme was correlated
with decreased production of TNF-α. For animals treated with thalidomide, the MPO activity
values were similar (P = 0.05) to those corresponding to the control group.
Our results suggested that replacement of the glutarimide ring of thalidomide by N- phenyl
functionalized and N-heteroaromatic derivatives improved the inhibitory activity on TNF-α
production. Compounds 2 and 8 exhibited remarkable anti-inflammatory activity and were 30-
and 200- fold more potent than thalidomide (IC50 = 8.4 and 1.2 M vs 239.1 M) respectively.
Differently, compounds 3 and 5 did not show inhibitory activities on TNF-α production.
When the phthalimide ring was replaced by a phthalazinone structure (compound 9) afforded the
most potent in vivo anti-inflammatory agent of all synthesized compounds. It can be considered
to be scaffold for the future design and development of anti-inflammatory agents.
4. Conclusion
In this work, we report the use of microwave irradiation to carry out the synthesis of compounds
2-9. These compounds were screened for their anti-inflammatory activity (inhibition of TNF-α
production, rat paw edema, hyperalgesia and myeloperoxidase activity). Compound 9, a
phthalazin-1,4-dione, may be considered as a new prototype molecule with excellent anti-
inflammatory features that manifest both in vivo and ex vivo.
The importance of this compound cannot be ignored, as it is an intermediary in the lead
optimization process.
ACKNOWLEDGMENTS

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Authors wish to thank Dr A. L. Palhares de Miranda and Dr L. Moreira Lima for hosting us in
your laboratory. JJC is also thankfuls to the PROSUL-CNPq (Programa Sul Americano de Apoio
às Atividades de Cooperação em Ciência e Tecnología- Conselho Nacional de Desenvolvimento
Científico e Tecnológico-Brazil), the grant received to carry out anti-inflammatory tests at
Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Departamento de
Fármacos, Faculdade de Farmácia, UFRJ, Brazil of E. J. Barreiro. This work was supported by a
grant from University of Buenos Aires UBACyT 20020100100770BA.
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Legends to Figures
Fig. 1. Structure of thalidomide (compound 1). Synthesis of phthalimide derivatives (compounds
2-9) from phthalic anhydride and the corresponding amines.
Fig. 2. A: Effect of the thalidomide (compound 1) and a series of phthalimides (compounds 2-9)
on TNF-α production in BALF from mice pre-treated with LPS. (++) P<0.01: significant
difference between LPS-induced TNF-α levels and basal level. (*) P< 0.05 and (**) P<0.01
significant difference between TNF-α production in BALF from mice pre-treated with LPS in
presence and absence of test compounds. B: IC50 values for tested compounds. IC50:
concentration that inhibits 50 % of TNF- α production.
Fig. 3. Effect of the thalidomide (compound 1) and the most active compounds (2, 8, and 9)
administered intraperitoneally on the carrageenan-induced rat paw edema. (++) P<0.01:
significant difference between carrageenan-induced group and group treated with the vehicle. (*)
P< 0.05 significant difference between carrageenan-induced group in presence and absence of
test compounds.
Fig. 4. Time course effect of thalidomide (compound 1) and selected phtalimides (2, 8, and 9)
administered orally on carrageenan-induced rat hyperalgesia. (+) P<0.05 and (++) P<0.01:
significant difference between carrageenan-induced group and the group treated with the vehicle.
(*) P<0.05 and (**) P<0.01 significant difference between carrageenan-induced group in
presence and absence of test compounds.
Fig. 5. Effect of pretreatment with compounds 1, 2, 8 and 9 on MPO in carrageenan-induced
inflammation of rat paws. (++) P<0.01: significant difference between carrageenan-induced

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group and the group treated with the vehicle. (**) P<0.01 significant difference between
carrageenan-induced group in presence and absence of test compounds.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Table 1. Calculated Physicochemical Properties
CMRg
66.36
83.93
56.36
68.01
76.00
82.55
65.26
81.69
75.62
Compound CLogPa nRBb nHBAc nHBDd
MWe
TopoPSAf
83.55
92.88
68.09
63.16
80.52
89.75
50.27
78.51
49.41
0.11
2.45
0.18
2.26
2.75
2.76
2.38
4.00
3.14
1
3
1
2
2
3
1
1
2
6
7
6
5
6
7
4
4
4
1
0
0
0
0
0
0
0
1
258.232
295.059
214.049
239.069
268.048
298.059
224.059
280.031
272.035
1
2
3
4
5
6
7
8
9
aCLogP: Crippen's logP, bnRB: number rotatable bonds, cnHBA: number hydrogen bond acceptors,
dnHBD: number of hydrogen bond donors, eMW: molecular weight (Dalton), fTopoPSA: Topological
polar surface area, gCMR: Crippen's molar refractivity.

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Graphical abstract