Anti-inflammatory and analgesic activities of newly synthesized chiral peptide derivatives using (3-benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)acetic acid ethylester as starting material

Article abstract of DOI:10.1002/ardp.200700158

A series of heterocyclic derivatives was synthesized using (3-benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)acetic acid ethyl ester 1 as starting material. Treatment of 1 with 1 N NaOH or hydrazine hydrate afford the corresponding acid 2 and acid hydrazides 4 and 5, which were reacted with several reagents to produce some new peptido hetero-organic derivatives 6-12. The pharmacological screening showed that many of these newly synthesized compounds have good antiinflammatory and analgesic activities comparable to diclofenac potassium and valdecoxib as reference drugs.

Full text of DOI:10.1002/ardp.200700158

174
Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
Full Paper
Anti-Inflammatory and Analgesic Activities of Newly
Synthesized Chiral Peptide Derivatives Using (3-Benzoyl-
4,5-dioxo-2-phenyl-pyrrolidin-1-yl)acetic Acid Ethylester as
Starting Material
Issa M. Fakhr1, Abdel-Galil E. Amr1, Nermien, M. Sabry1, and Mohamed M. Abdalah2
1 Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt
2
Research Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
A series of heterocyclic derivatives was synthesized using (3-benzoyl-4,5-dioxo-2-phenyl-pyrroli-
din-1-yl)acetic acid ethyl ester 1 as starting material. Treatment of 1 with 1 N NaOH or hydrazine
hydrate afford the corresponding acid 2 and acid hydrazides 4 and 5, which were reacted with
several reagents to produce some new peptido hetero-organic derivatives 6–12. The pharmaco-
logical screening showed that many of these newly synthesized compounds have good anti-
inflammatory and analgesic activities comparable to diclofenac potassium and valdecoxib as
reference drugs.
Keywords: Amino acids / Analgesic agents / Anti-inflammatory / Pyrrolidine-2,3-dione /
Received: August 1, 2007; accepted: November 8, 2007
DOI 10.1002/ardp.200700158
logical and pharmacological activities [23–25]. In view of
Introduction
these observations and as continuation of our previous
work in peptido-heterocyclic chemistry, we have synthe-
sized some new compounds containing amino acid and
hetero-organic moieties, and tested their selected biologi-
cal activities.
In previous works, Koskin and Merchant reported that
certain substituted heterocyclic systems were synthe-
sized via a,c-diketoesters using 2,3-pyrrolidinedione-N-
acetic acid ethyl ester as starting materials [1, 2]. In addi-
tion, the biological importance of 2,3-pyrrolidinediones
as potent and specific inhibitors of aldose reductase was
also reported [3, 4]. The peptide derivatives have antimi-
crobial and anti-inflammatory activities [5–10] and anti-
tumor properties [11–14]. We also demonstrated that
some peptido-heterocyclic derivatives exhibit a general
ionophoric potency for divalent cations [15] and are use-
ful for assembling novel thiocyanate-selective membrane
sensors [16]. Recently, some new heterocyclic derivatives
were synthesized which exhibit analgetic, anti-parkinso-
nian, androgenic anabolic, and anti-inflammatory activ-
ities [17–22]. On the other hand, semicarbazide, thiose-
micarbazide, and imide derivatives show promising bio-
Results and discussion
Chemistry
In the previous work, Koskin reported that the synthesis
and a preliminary biological activity screening of 2,3-pyr-
rolidinediones derivatives based on a,c-diketoesters [1].
The condensation of methyl benzoyl pyruvate (a,c-dike-
toester), benzaldehyde, and glycine ethyl ester hydro-
chloride in refluxing ethanol in the presence of triethyl-
amine afforded the corresponding (3-benzoyl-4,5-dioxo-2-
phenyl-pyrrolidin-1-yl)acetic acid ethyl ester 1 according
to the literature procedure [2]. Hydrolysis of the ethyl
ester 1 with 1 N NaOH [Method A] afforded the corre-
sponding acid 2, which could also get prepared directly
from condensation of the methyl benzoyl pyruvate, benz-
aldehyde, and glycine in refluxing ethanol [Method B;
Correspondence: Abdel-Galil E. Amr, Applied Organic Chemistry De-
partment, National Research Center, Dokki, Cairo, Egypt.
E-mail: aamr1963@yahoo.com
Fax: +20 2 3370-931
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
Anti-inflammatory / Analgesic Activities of Chiral Peptides
175
Scheme 1. Synthesis route of compounds 1–6.
Scheme 1). Reaction of L-leucine methyl ester hydrochlor-
ide with the acid derivative 2 and ethyl chloroformate in
the presence of triethylamine afforded the correspond-
ing peptide ester derivative 3; mixed anhydride method.
[11]. On the other hand, when treating the ethyl ester 1
with hydrazine hydrate according to the published pro-
cedure [9] in refluxing ethanol, the corresponding hydra-
zino acid hydrazide 4 was isolated as main product along
with another acid hydrazide 5. The latter compound was
reacted with 1,2,4,5-benzenetetracarboxylic di-anhy-
dride in refluxing glacial acetic acid to afford the corre-
sponding bis-imide derivative 6 (Scheme 1).
In the presented work, we found it interesting to
extend this course of investigation by the synthesis of
some new peptido-heteroorganic compounds. Thus, con-
densation of hydrazino hydrazide 4 with aromatic alde-
hydes, namely, p-fluorobenzaldehyde or p-methyl-benzal-
dehyde was undertaken in refluxing ethanol to afford
the corresponding hydrazones 7a, b, respectively
(Scheme 2), while, it was treated with ethyl isothiocya-
nate in refluxing dioxane to afford the thiosemicarba-
zide derivative 8. Condensation of 4 with selected acid
anhydrides, namely phthalic or 1,2,4,5-tetrachlorophe-
thalic anhydrides, in refluxing glacial acetic acid
Scheme 2. Synthesis route of compounds 7–10.
afforded the corresponding imide derivatives 9 and 10,
respectively (Scheme 2).
Treatment of peptide ester 3 with hydrazine hydrate in
boiling ethanol gave the corresponding hydrazide 11,
which was reacted with 1,2,4,5-tetrachlorophthalic anhy-
dride in refluxing acetic acid to afford the corresponding
imide derivative 12 (Scheme 3).
Pharmacological screening
All animals were obtained from the Animal House Col-
ony, Research Institute of Ophthalmology, Giza, Egypt.
Initially, the acute toxicity of the compounds was assayed
determining their LD50. Interestingly, all the synthesized
compounds were less toxic than valdecoxib (Table 1).
Then, the newly synthesized compounds were screened
pharmacologically for their analgesic and anti-inflamma-
tory activities using male albino rats (Tables 2 and 3).
Anti-inflammatory activity
Purpose and rational
For the determination of the antiphlogistic potency of
the synthesized compounds, two standard tests were
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I. M. Fakhr et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
Table 2. Anti-inflammatory activities of some new synthesized
compounds.
Groups
Dose
% Protection
% Inhibition of
[mg/kg]
against edema plasma PGE2
1
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
5.0
2.5
78.88 l 0.091
92.00 l 0.062
97.45 l 0.053
98.89 l 0.044
90.26 l 0.035
99.08 l 0.036
99.12 l 0.078
99.09 l 0.064
86.14 l 0.053
98.16 l 0.052
91.16 l 0.065
95.12 l 0.076
79.28 l 0.081
92.90 l 0.098
93.44 l 0.074
98.56 l 0.063
90.46 l 0.092
99.34 l 0.081
81.00 l 0.075
95.80 l 0.063
85.16 l 0.056
96.19 l 0.067
82.16 l 0.078
93.15 l 0.061
70.14 l 0.061
57.57 l 0.051
76.00 l 0.041
81.27 l 0.041
82.50 l 0.053
62.12 l 0.051
83.00 l 0.049
84.15 l 0.041
86.00 l 0.031
59.43 l 0.051
81.09 l 0.055
73.44 l 0.049
78.56 l 0.041
57.90 l 0.041
77.11 l 0.042
75.71 l 0.041
80.00 l 0.061
63.16 l 0.031
86.16 l 0.071
63.11 l 0.041
79.88 l 0.031
61.14 l 0.041
77.11 l 0.041
58.99 l 0.031
74.56 l 0.041
54.00 l 0.041
2
3
4
6
7a
7b
8
9
Scheme 3. Synthesis route of compounds 11 and 12.
10
11
12
Table 1. Acute toxicity (LD50) of the synthesized compounds.
Compound
LD50 [mg/kg]
1
2
3
4
6
7a
7b
8
9
10
11
12
1620.14 l 0.24
2213.78 l 0.19
2213.87 l 0.18
2100.89 l 0.17
1272.87 l 0.17
2612.56 l 0.16
1345.98 l 0.14
1456.09 l 0.12
2112.55 l 0.11
2134.89 l 0.10
1720.00 l 0.11
1249.87 l 0.12
1180.01 l 0.23
Diclofenac
potassium
5.0
75.23 l 0.083
70.00 l 0.051
All results were significantly different from the standard and
normal control value at P f 0.05.
Analgesic activity
All tested compounds exhibited analgesic activity in a
hot-plate assay (Table 3). Interestingly, the analgesic
activities of all the compounds 1–12 were more potent
than valdecoxib as a reference drug (Table 3) and, com-
pared to valdecoxib after 120 min these analgesic activ-
ities were increased. Compounds 9, 3, 4, 2, 8, 6, 11, 10, 7a,
12, 7b, and 1 are arranged in descending order of analge-
sic potency. Compound 9 showed more than three times
the activity of valdecoxib, while compounds 2, 3, 4, and 8
showed double activity as compared to valdecoxib after
two hours.
Valdecoxib
All results were significantly different from the normal control
value at P f 0.05.
realized at a dose level 2.5 and 5 mg/kg body weight of
the rats, namely, the protection against carrageenan-
induced edema according to Winter et al. [26] and the
inhibition of plasma PGE2. The latter is known as a good
confirming indicator for the carrageenan-induced rat
paw edema [27]. Regarding the protection against carra-
geenan-induced edema, all tested compounds, were
found to be more potent than diclofenac potassium. For
these compounds, a similar activity profile was realized
for the inhibition of plasma PGE2. Concerning the anti-
inflammatory activities, the descending order of activity
is 9, 4, 3, 2, 8, 6, 11, 10, 7a, 12, 7b, and 1. Compounds 9, 4,
3, 2, and 8 are the most active products.
Structure activity relationship (SAR)
The phenylpyrrolidinone nucleus is essential for both
the anti-inflammatory and analgesic activities. The
nucleophilicity of the substituents at positions 1 and 4
increases the activities. As the aromaticity increases with
minimal steric hindrance, the activities increase. But an
increase in steric hindrance alongside with an increase
in molecular weight decreases the activities.
The authors have declared no conflict of interest.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
Anti-inflammatory / Analgesic Activities of Chiral Peptides
177
Table 3. Analgesic activities of some new synthesized compounds.
Compound
Comparative analgesic potency to Valdecoxib after time [in min]
10 min
20 min
30 min
60 min
90 min
120 min
1
2
3
4
6
7a
7b
8
9
10
11
12
0.40 l 0.01
0.61 l 0.02
0.65 l 0.01
0.64 l 0.02
0.51 l 0.01
0.47 l 0.01
0.41 l 0.01
0.55 l 0.01
0.72 l 0.03
0.45 l 0.02
0.45 l 0.01
0.45 l 0.01
1.00
0.40 l 0.03
0.61 l 0.05
0.63 l 0.06
0.76 l 0.07
0.53 l 0.05
0.47 l 0.03
0.40 l 0.04
0.56 l 0.03
0.83 l 0.07
0.47 l 0.03
0.48 l 0.04
0.45 l 0.03
1.00
0.45 l 0.04
0.90 l 0.03
0.97 l 0.08
0.81 l 0.07
0.81 l 0.07
0.55 l 0.05
0.50 l 0.05
0.85 l 0.05
0.98 l 0.09
0.59 l 0.05
0.61 l 0.06
0.50 l 0.04
1.00
0.58 l 0.05
1.00 l 0.01
1.00 l 0.01
1.15 l 0.15
0.85 l 0.08
0.67 l 0.09
0.61 l 0.06
0.89 l 0.08
1.22 l 0.12
0.74 l 0.07
0.76 l 0.07
0.65 l 0.06
1.00
0.89 l 0.08
1.18 l 0.10
1.24 l 0.12
1.43 l 0.12
1.11 l 0.11
0.87 l 0.09
0.97 l 0.09
1.12 l 0.12
1.63 l 0.16
0.99 l 0.09
0.99 l 0.09
0.80 l 0.08
1.00
1.11 l 0.07
2.34 l 0.12
2.56 l 0.11
2.45 l 0.14
1.89 l 0.08
1.47 l 0.14
1.28 l 0.16
2.22 l 0.21
3.55 l 0.13
1.58 l 0.04
1.63 l 0.06
1.30 l 0.08
1.00
Valdecoxib
All results were significantly different from the standard and normal control value at P=0.05.
1. Stirring was continued at that temperature for 2 h and then
for 12 h at room temperature followed by evaporation of the sol-
vent. The cold reaction mixture was acidified with 1 N hydro-
chloric acid to pH l 3, and the obtained solid was filtered off,
washed with cold water, dried, and crystallized to afford the
acid 2. Yield 63%, m.p. 240–2428C (EtOH/ether); IR (KBr, cm– 1):
3530–3225 (OH), 1723 (C=O, acid), 1692–1678 (3 C=O); 1H-NMR
(DMSO-d6) d: 3.62 (d, 1H, CH), 4.42 (d, 1H, CH), 5.47 (s, 2H, CH2),
7.10–7.86 (m, 10H, Ar-H), 10.65 (s, 1H, OH, exchangeable with
D2O); MS m/z (%): 337 [M+] (26), corresponding to the molecular
formula C19H15NO5 and base peak at 243 (100).
Experimental
Material and methods
Melting points were determined in open glass capillaries using
an Electrothermal IA 9000 SERIES digital melting point appara-
tus (Electrothermal, Essex, U.K.) and are uncorrected. Elemental
analyses were performed in the Microanalytical Unit, National
Research Centre, Cairo, Egypt and were found within l 0.4% of
the theoretical values. Analytical data were obtained from the
Microanalytical Unit, Cairo University, Egypt. The IR spectra
(KBr) were recorded on a FT IR-8201 PC spectrophotometer (Shi-
madzu, Tokyo, Japan). The 1 H-NMR spectra were measured with
Jeol 270 MHz (JEOL, Tokyo, Japan) in DMSO-d6. The chemical
shifts were recorded (d, ppm) relative to TMS. The Mass spectra
were run at 70 eV with a Finnigan SSQ 7000 spectrometer
(Thermo-Instrument system incorporation, USA) using EI, and
the values of m/z are indicated in Dalton.
Method B
A mixture of methyl benzoyl pyruvate (0.4 g, 2 mmol), benzalde-
hyde (0.2 mL, 2 mmol) and glycine (0.15 g, 2 mmol) in ethanol
30 mL was refluxed for 15 h, the unreacted glycine was filtered
off, the filtrate was leave overnight at room temperature, the
obtained solid was filtered off, crystallized from ethanol/ether
to give the corresponding acid 2. The product was identified by
their m.p. and Rf-values in comparison with authentic samples
previously obtained by method A.
All animals were obtained from the Animal House Colony,
Research Institute of Ophthalmology, Giza, Egypt.
Chemistry
(3-Benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)acetic acid
ethyl ester 1
2-[2-(3-Benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)-
The stirred, cold solution of glycine ethyl ester hydrochloride
(2.1 g, 15 mmol) and triethylamine (1.4 mL, 15 mmol) in ethanol
5 mL was stirred for 5 min, then poured into the solution of
methyl benzoyl pyruvate (3.0 g, 15 mmol) and benzaldehyde
(2.4 mL, 15 mmol) in 30 mL ethanol. The reaction mixture was
refluxed for 15 h, left 2 days, and filtered off, crystallized to give
the ester derivative 1. Yield 76%, m.p. 195–1988C (EtOH / petrol
ether 40–608C); IR (KBr, cm– 1): 1747 (C=O, ester), 1695–1675
(C=O); 1H-NMR (DMSO-d6) d: 1.28 (t, 3H, CH3), 3.64 (d, 1H, CH), 4.15
(q, 2H, CH2), 4.38 (d, 1H, CH), 5.45 (s, 2H, CH2), 7.15–7.82 (m, 10H,
Ar-H); MS m/z (%): 365 [M+] (9), corresponding to the molecular
formula C21H19NO5 and base peak at 243 (100).
acetyamino]-4-methyl-pentanoic acid methyl ester 3
To a cold and stirred dry dichloromethane solution (25 mL, –
208C) of the acid 2 (0.34 g, 1 mmol), ethyl chloroformate (0.1 g,
1 mmol) and triethylamine (0.1 g, 1 mmol) were successively
added. Ten minutes later, a cold methylene chloride solution
(10 mL, –208C) of L-leucine methyl ester (0.15 g, 1 mmol) was
added. Stirring of the cold reaction mixture (–208C) was contin-
ued for 3 h and at room temperature for overnight. The solution
was then washed with water, 1 N hydrochloric acid, 1 N sodium
bicarbonate, and finally with water (263 mL). The dried solu-
tion (anhydrous CaCl2) was evaporated and the obtained oily res-
idue was solidified by dry ether trituration, filtered off, dried
under vacuum, and crystallized to afford the ester 3. Yield 66%,
m.p. 99–1008C(EtOH/ether); [a]2D5 = + 65 (MeOH); IR (KBr, cm– 1):
1745 (C=O, ester), 1698–1674 (C=O); 1H-NMR (DMSO-d6) d: 1.34–
1.65 (m, 6H, 26CH3), 1.98–2.20 (m, 2H, CH2CH), 2.24–2.32 (m,
1H, CH(CH3)2), 3.56 (s, 3H, OCH3), 3.65 (d, 1H, CH), 4.22–4.32 (m,
(3-Benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)acetic acid 2
Method A
Sodium hydroxide (1 N, 25 mL) was added dropwise to a cold
and stirred ethanolic solution (0.36 g, 1 mmol, –58C) of the ester
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Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
1H, CHNH), 4.43 (d, 1H, CH), 5.42 (s, 2H, CH2), 7.24–7.94 (m, 10H,
Ar-H), 8.90 (s, 1H, NH, exchangeable with D2O); MS m/z (%): 464
[M+] (32), corresponding to the molecular formula C26H28N2O6
and base peak at 244 (100).
sure. The obtained oily product was solidified by trituration
with ethanol/petrol ether (40–608C), the remaining solid was fil-
tered off, dried, and crystallized to give the corresponding
hydrazones 7a, b, respectively.
(3-{[(4-Fluorobenzylidene)-hydrazono]-phenylmethyl}-
4,5-dioxo-2-phenyl-pyrrolidin-1-yl)-acetic acid (4-
Synthesis of acid hydrazide derivatives 4 and 5
Hydrazine hydrate (0.8 mL, 16 mmol) was added to a ethanolic
solution (50 mL) of the ester 1 (0.36 g, 1 mmol). The reaction mix-
ture was refluxed for 5 h after which the solvent was evaporated.
The obtained residue was dissolved in a small amount of ethanol
and the product was precipitated with ether. The obtained solid
was filtered off and crystallized to afford the hydrazino-hydra-
zide derivative 4. While the filtrate was evaporated under
reduced pressure, the obtained oil was solidified with n-hexane
to afford another acid hydrazide 5.
fluorobenzylidine)-hydrazide 7a
Yield 86%, m.p. 156–1588C (AcOH); IR (KBr, cm– 1): 3354–3215
(NH), 1698–1678 (3 C=O), 1665 (C=N); 1H-NMR (DMSO-d6) d: 3.63
(d, 1H, CH), 4.46 (d, 1H, CH), 4.86 (s, 2H, 2CH=N), 5.47 (s, 2H, CH2),
6.90–7.96 (m, 18H, 4 Ph-H), 9.95 (s, 1H, NH, exchangeable with
D2O); MS m/z (%): 577 [M+] (18), corresponding to the molecular
formula C35H25F2N5O3 and base peak at 345 (100).
(3-{[(4-Methylbenzylidene)-hydrazono]-phenylmethyl}-
4,5-dioxo-2-phenyl-pyrrolidin-1-yl)-acetic acid (4-
methylbenzylidine)-hydrazide 7b
[3-(Hydrazinophenylmethyl)-4,5-dioxo-2-phenyl-
pyrrolidin-1-yl]-acetic acid hydrazide 4
Yield 45%, m.p. 202–2048C(EtOH/ether); IR (KBr, cm– 1): 3518–
3350 (NH, NH2), 1692-1679 (3 C=O); 1H-NMR (DMSO-d6) d: 3.65 (d,
1H, CH), 4.10 (s, 2H, NH2, exchangeable with D2O), 4.41 (d, 1H,
CH), 5.44 (s, 2H, CH2), 6.42 (s, 2H, NH2, exchangeable with D2O),
7.24–7.84 (m, 10H, Ar-H), 9.72 (s, 1H, NH, exchangeable with
D2O); MS m/z (%): 365 [M+] (32), corresponding to the molecular
formula C19H19N5O3 and base peak at 362 (100).
Yield 82%, m.p. 110–1128C (EtOH); IR (KBr, cm– 1): 3360-3200
(NH), 1695–1682 (3 C=O), 1662 (C=N); 1H-NMR (DMSO-d6) d: 1.05
(s, 6H, 2 CH3), 3.65 (d, 1H, CH), 4.45 (d, 1H, CH), 4.82 (s, 2H,
2CH=N), 5.54 (s, 2H, CH2), 7.16–7.98 (m, 18H, 4 Ph-H), 9.90 (s, 1H,
NH, exchangeable with D2O); MS m/z (%): 569 [M+] (8), correspond-
ing to the molecular formula C35H31N5O3 and base peak at 118
(100).
(3-Benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)acetic acid
hydrazide 5
(3-(Ethylthiosemihydrazino-phenylmethyl-4,5-dioxo-2-
phenyl-pyrrolidin-1-yl)-acetic acid N9-thiopropionyl
hydrazide 8
Yield 30%, m.p. 178–1808C(EtOH/n-hexane); IR (KBr, cm– 1):
3495–3315 (NH, NH2), 1696–1680 (4 C=O); 1H-NMR (DMSO-d6) d:
3.62 (d, 1H, CH), 4.43 (d, 1H, CH), 5.45 (s, 2H, CH2), 6.47 (s, 2H,
NH2, exchangeable with D2O), 7.18–7.95 (m, 10H, Ar-H), 9.78 (s,
1H, NH, exchangeable with D2O); MS m/z (%): 351 [M+] (12), corre-
sponding to the molecular formula C19H17N3O4 and base peak at
166 (100).
A mixture of hydrazide 4 (0.36 g, 1 mmol) and ethyl isothiocya-
nate (l0.2 g, 2 mmol) in 50 mL dry dioxane containing 2 mL of
triethylamine was heated under reflux for 10 h. After cooling,
the obtained solid was filtered off, washed with diethyl ether,
dried, and crystallized to give thiosemicarbazide derivative 8.
Yield 62%, m.p. 1358C (dioxane/ether); IR (KBr, cm– 1): 3460–3225
1
(br, NH), 1698–1680 (3 C=O), 1235 (C=S); H-NMR (DMSO-d6) d:
1.10 (t, 6H, 2 CH3), 3.56 (m, 4H, 2 CH2), 3.61 (d, 1H, CH), 4.40 (d,
1H, CH), 4.65 (brs, 2H, 2 NH, exchangeable with D2O), 5.52 (s, 2H,
CH2), 7.05–7.90 (m, 10H, 2 Ph-H), 8.45, 8.62, 8.85 (3s, 3H, 3 NH,
exchangeable with D2O); MS m/z (%): 539 [M+] (42), corresponding
to the molecular formula C25H29N7O3S2 and base peak at 451
(100).
2-(3-Benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)-N-{6-
[2(3-benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)-
acetylamino]-1,3,5,7-tetraoxo-3,5,6,7-tetrahydro-1H-
pyrrolo[3,4-f]isoindol-2-yl}-acetamide 6
A stirred suspension of a mixture of hydrazide 5 (0.35 g, 1 mmol)
and 1,2,4,5-benzenetetracarboxylic acid di-anhydride (0.11 g,
0.5 mmol) in glacial acetic acid (50 mL) was heated (808C) for 7 h.
The reaction mixture was concentrated under reduced pressure;
the obtained solid was collected by filtration, dried, and purified
by crystallization to give bis-imide 6. Yield 45%, m.p. 1848C
(AcOH/ether); IR (KBr, cm– 1): 3500–3310 (OH, NH), 1720 (4 C=O),
1690–1682 (6 C=O); 1H-NMR (DMSO-d6) d: 3.62 (d, 2H, 2CH), 4.38
(d, 2H, 2 CH), 5.50 (s, 4H, 2CH2), 7.16–7.95 (m, 22H, 4 Ar-H), 9.82
(s, 2H, 2NH, exchangeable with D2O); MS m/z (%): 882 [M+-2] (16),
corresponding to the molecular formula C48H32N6O12 and base
peak at 300 (100).
Synthesis of imide derivatives 9 and 10
A stirred glacial acetic acid suspension (50 mL) of hydrazide 4
(0.36 g, 1 mmol) and acid anhydride derivatives, namely
phthalic or 1,2,4,5-tetrachlorophthalic anhydride (2 mmol), was
heated (808C) for 6 h. The reaction mixture was concentrated
under reduced pressure, cooled, and the separated solid was col-
lected by filtration, dried, and crystallized to yield the corre-
sponding imide derivatives 9 and 10, respectively.
N-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-2-{3-[(1,3-dioxo-
1,3-dihydro-isoindol-2-ylimino)-phenylmethyl]-4,5-dioxo-
Synthesis of hydrazone derivatives 7a, b
2-phenyl-pyrrolidin-1-yl)-acetamide 9
A stirred solution of hydrazide 4 (0.36 g, 1 mmol) and aromatic
aldehyde, namely, p-flouro-benzaldehyde or p-methyl benzalde-
hyde (2 mmol) in absolute methanol (50 mL) was refluxed for
6 h. The reaction mixture was allowed to stand at room temper-
ature overnight, then, it was evaporated under reduced pres-
Yield 78%, m.p. 150–1528C (AcOH/H2O); IR (KBr, cm– 1): 3410–
3252 (NH), 1731, 1728 (2 C=O), 1695–1678 (3 C=O); 1H-NMR
(DMSO-d6) d: 3.63 (d, 1H, CH), 4.42 (d, 1H, CH), 5.49 (s, 2H, CH2),
7.10–8.05 (m, 18H, 4 Ph-H), 10.48 (s, 1H, NH, exchangeable with
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Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
Anti-inflammatory / Analgesic Activities of Chiral Peptides
179
D2O); MS m/z (%): 625 [M+] (100, as base peak), corresponding to
the molecular formula C35H23N5O7.
Anti-inflammatory activity
Carrageenan-induced edema (rats paw test)
Groups of adult male albino rats (150–180 g), each of eight ani-
mals were orally dosed with tested compounds at a dose level of
2.5–5 mg/kg one hour before the carrageenan challenge. Foot-
paw edema was induced by subplantar injection of 0.05 cm3 of a
1% suspension of carrageenan in saline into the plantar tissue of
one hind paw. An equal volume of saline was injected to the
other hind paw and served as control. Four hours after drug
administration, the animals were decapitated, blood was col-
lected, and the paws were rapidly excised. The average weight of
edema was examined for the treated as well as for the control
group, and the percentage inhibition of weight of edema was
evaluated. Diclofenac potassium (5 mg/kg) was employed as
standard reference to which the tested compounds were com-
pared (Table 2).
2-{2,3-Dioxo-5-phenyl-4-[phenyl-(4,5,6,7-tetrachloro-1,3-
dioxo-1,3-dihydro-isoindol-2-yl imino)-methyl]pyrrolidin-1-
yl}-N-(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-isoindol-
2-yl)-acetamide 10
Yield 88%, m.p. A3008C (AcOH/H2O); IR (KBr, cm– 1): 3412-3256
(NH), 1728, 1726 (2 C=O), 1697–1675 (3 C=O); 1H-NMR (DMSO-d6)
d: 3.62 (d, 1H, CH), 4.43 (d, 1H, CH), 5.52 (s, 2H, CH2), 7.05-7.98 (m,
10H, 2 Ph-H), 10.45 (s, 1H, NH, exchangeable with D2O); MS m/z
(%): 902 [M++1] (12), corresponding to the molecular formula
C35H15Cl8N5O7 and base peak at 318 (100).
2-[2-(3-Benzoyl-4,5-dioxo-2-phenyl-pyrrolidin-1-yl)-
acetyamino]-4-methyl-pentanoic acid hydrazide 11
Hydrazine hydrate (0.8 mL, 16 mmol) was added to a methanolic
solution (10 mL) of 3 (0.46 g, 1 mmol). The reaction mixture was
refluxed for 5 h, after which the solvent was evaporated under
reduced pressure. The obtained residue was triturated with
ether, filtered off, and crystallized to afford the corresponding
hydrazide 11. Yield 68%, m.p. 120–1248C (EtOH/ether); [a]2D5 = +
45 (MeOH); IR (KBr, cm– 1): 3444–3310 (NH, NH2), 1698–1674 (4
C=O); 1H-NMR (DMSO-d6) d: 1.30–1.62 (m, 6H, 26CH3), 1.95–2.10
(m, 2H, CH2CH), 2.28–2.34 (m, 1H, CH(CH3)2), 3.62 (d, 1H, CH),
4.15 (s, 2H, NH2, exchangeable with D2O), 4.25–4.35 (m, 1H,
CHNH), 4.42 (d, 1H, CH), 5.40 (s, 2H, CH2), 6.45 (s, 2H, NH2,
exchangeable with D2O), 7.20–7.96 (m, 10H, Ar-H), 8.85, 9.15 (2s,
2H, 2 NH, exchangeable with D2O); MS m/z (%): 478 [M+] (12), cor-
responding to the molecular formula C25H30N6O4 and base peak
at 245 (100).
Estimation of plasma prostaglandin E2 (PGE2)
Heparinized blood samples were collected from rats (n = 8),
plasma was separated by centrifugation at 12000g for 2 min at
408C, immediately frozen, and stored at 208C until use. The
design correlate EIA prostaglandin E2 (PGE2) kit (Aldrich, Stein-
heim, Germany) is a competitive immuno assay for the quantita-
tive determination of PGE2 in biological fluids. The kit uses a
monoclonal antibody to PGE2 to bind, in a competitive manner,
the PGE2 in the sample after a simultaneous incubation at room
temperature. The excess reagents were washed away and the
substrate was added. After a short incubation time, the enzyme
reaction was stopped, and the yellow color generated was read
on a microplate reader DYNATech, MR 5000 at 405 nm (Dyna-
tech Industries Inc., McLean, VA, USA). The intensity of the
bound yellow color is inversely proportional to the concentra-
tion of PGE2 in either standard or samples.
2-(2-{2,3-Dioxo-5-phenyl-4-[phenyl-(4,5,6,7-tetrachloro-
1,3-dioxo-1,3-dihydro-isoindol-2-yl imino)-
methyl]pyrrolidin-1-yl}-acetylamino)-4-methyl-pentanoic
acid (4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-isoindol-2-
Analgesic activity
Sixty Webster mice of both sexes weighting 20–25 g were div-
ided into ten groups. One group was kept as control (received sal-
ine), the second group received vehicle (gum acacia), and the
third one received valdecoxib as a reference drug, whereas the
other groups received the test compounds (s.c. administration).
Mice were dropped gently in a dry glass beaker of 1 dm3 capacity
maintained at 55–55.58C. Normal reaction time in seconds for
all animals was determined at time intervals of 10, 20, 30, 45,
60, 90, and 120 minutes. This is the interval extending from the
instant the mouse reaches the hot beaker till the animals licks
its feet or jump out of the beaker (dose 5 mg/kg) [29]. The relative
potencies to valdecoxib were determined (Table 3).
yl)-amide 12
A stirred glacial acetic acid suspension (50 mL) of hydrazine –
acid hydrazide 11 (0.47 g, 1 mmol) and 1,2,4,5-tetrachloroph-
thalic anhydride (0.6 g, 2 mmol) was heated (808C) for 6 h. The
reaction mixture was concentrated under reduced pressure,
cooled and the separated solid was collected by filtration, dried,
and crystallized to yield the corresponding bis-imide 12. Yield
74%, m.p. A3008C (AcOH/ether); [a]2D5 = + 72 (MeOH); IR (KBr, cm–
1): 3455-3325 (NH), 1705 (C=O), 1695–1684 (4 C=O); 1H-NMR
(DMSO-d6) d: 1.34–1.60 (m, 6H, 26CH3), 1.98–2.15 (m, 2H,
CH2CH), 2.30–2.38 (m, 1H, CH(CH3)2), 3.64 (d, 1H, CH), 4.22–4.35
(m, 1H, CHNH), 4.40 (d, 1H, CH), 5.42 (s, 2H, CH2), 7.18–7.98 (m,
10H, Ar-H), 8.98, 9.25 (2s, 2H, 2 NH, exchangeable with D2O); MS
m/z (%): 1014 [M+] (16), corresponding to the molecular formula
C41H26Cl8N6O8 and base peak at 198 (100).
References
[1] H. Koskin, Rev. Faculte Sci. Univ. Istanboul, 11A, 1 (1946);
C.A. 40, 5427 (1946).
[2] J. R. Merchant, R. M. Bhandarkar, J. Ind. Chem. Soc. 1963,
40, 353–360.
Pharmacological screening
Determination of acute toxicity (LD50)
[3] P. L. Southwick, R. T. Crouch, J. Am. Chem. Soc. 1953, 75,
The LD50 was determined by using rats. They were injected with
different increasing doses of the synthesized compounds. The
dose that killed 50% of the animals was calculated according to
Austen et al. [28] (Table 1).
3413–3417.
[4] E. Dagne, N. Castagnoli Jr., J. Med. Chem. 1972, 15, 356–
360.
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

180
I. M. Fakhr et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 174–180
[5] A. E. Amr, A. M. Mohamed, A. A. Ibrahim, Z. Naturforsch.
2003, 58b, 861–868.
[17] A. E. Amr, M. I. Hegab, A. A. Ibrahim, M. M. Abdalah, Mon-
atsh. Chem. 2003, 134, 1395–1409.
[18] A. E. Amr, M. M. Abdalla, Arch. Pharm. Chem. Life Sci. 2005,
338, 433–440.
[6] M. H. Abou-Ghalia, A. E. Amr, M. M. Abdalah, Z. Natur-
forsch. 2003, 58b, 903–910.
[19] A. E. Amr, M. M. Abdalla, Arch. Pharm. Chem. Life Sci.,
2006, 339, 88–95.
[7] M. I. Hegab, A. E. Amr, F. M. E. Abdel-Megeid, Z. Natur-
forsch. 2002, 57b, 922–927.
[20] A. E. Amr, M. M. Abdulla, Ind J. Heterocycl. Chem. 2002, 12,
[8] A. E. Amr, Ind J. Heterocycl Chem. 2000, 10, 49–58.
129–134.
[9] A. E. Amr, O. I. Abdel-Salam, A. Attia, I. Stibor, Collect.
Czech Chem. Commun. 1999, 64, 288–298.
[21] A. E. Amr, N. A. Abdel–Latif, M. M. Abdalla, Bioorg. Med.
Chem. 2006, 14, 373–384.
[10] A. Attia, O. I. Abdel-Salam, A. E. Amr, I. Stibor, M. Bude-
[22] A. E. Amr, M. M. Abdalla, Bioorg. Med. Chem. 2006, 14,
sinsky, Egypt. J. Chem. 2000, 43, 187–201.
4341–4352.
[11] M. H. Abou–Ghalia, A. E. Amr, Amino Acids 2004, 26,
[23] A. E. Amr, Z. Naturforsch. 2005, 60b, 990998.
283–289.
[24] A. E. Amr, M. H. Abo-Ghalia, M. M. Abdalla, Z. Naturforsch.
2006, 61b, 1335–1345.
[12] A. G. Hammam, A. F. M. Fahmy, A. E. Amr, A. M.
Mohamed, Ind. J. Chem. 2003, 42B, 1985–1993.
[25] N. A. Abdel-Latif, A. E. Amr, A. A. Ibrahim, Monatsh. Chem.
2007, 138, 559–567.
[13] M. F. Brana, J. M. Castellano, M. Moran, M. J. Perez de
Vega, et al., Eur. J. Med. Chem. 1995, 30, 235–240.
[26] C. A. Winter, E. A. Risely, G. W. Nuss, Proc. Soc. Exp. Bio.
Med. 1962, 111, 541–545.
[14] A. E. Amr, A. M. Mohamed, S. F. Mohamed, N. A. Abdel-
Hafez, A. G. Hammam, Bioorg. Med. Chem. 2006, 14,
5481–5488.
[27] F. Herrmann, A. Lindemann, J. Gamss, R. Mertelsmann,
Eur J Immunol 1999, 20, 2513–2517.
[15] S. S. M. Hassan, M. H. Abou-Ghalia, A. E. Amr, A. H. K.
[28] K. F. Austen, W. E. Brocklehurst, J. Exp. Med. 1961, 113,
Mohamed, Talanta 2003, 60, 81–91.
521–524.
[16] S. S. M. Hassan, M. H. Abou-Ghalia, A. E. Amr, A. H. K.
[29] A. Tgolsen, G. H. Rofland, O. G. Berge, K. Hole, J. Pharma-
col. Ther. 1991, 25, 241–246.
Mohamed, Anal Chim Acta 2003, 482, 9–18.
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www.archpharm.com