Synthesis of new (Nα-dipicolinoyl)-bis-L-valyl-L- phenylalanyl linear and macrocyclic bridged peptides as anti-inflammatory agents

Article abstract of DOI:10.1002/ardp.200600187

In continuation to our search for new chiral macrocyclic peptide-based anti-inflammatories, the suggestion, synthesis, structure elucidation of some N^α-bis-dipicolinoyl amino acids, linear, tetra and cyclic (penta and octa)-bridged peptides 3-1

Full text of DOI:10.1002/ardp.200600187

304
Arch. Pharm. Chem. Life Sci. 2007, 340, 304–309
Full Paper
a
Synthesis of New (N -Dipicolinoyl)-bis-L-valyl-L-phenylalanyl
Linear and Macrocyclic Bridged Peptides as Anti-Inflammatory
Agents
Abd El-Galil E. Amr1, Mohamed H. Abo-Ghalia2, and Mohamed M. Abdalah3
1 Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt
2 Peptide Chemistry Department, National Research Center, Dokki, Cairo, Egypt
3 Research Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
In continuation to our search for new chiral macrocyclic peptide-based anti-inflammatories, the
suggestion, synthesis, structure elucidation of some Na-bis-dipicolinoyl amino acids, linear, tetra
and cyclic (penta and octa)-bridged peptides 3–10, were realized herein. The newly synthesized
compounds showed potent anti-inflammatory activity with low toxicity (LD50) comparable to
indomethacin and diclofenac as reference anti-inflammatory drugs.
Keywords: Amino acids / Anti-inflammatory agents / Macrocyclic peptides /
Received: November 6, 2006; accepted: April 17, 2007
DOI 10.1002/ardp.200600187
enzyme cyclooxygenase-2 (COX-2) by the natriuretic pep-
Introduction
tides has been equally reported [11]. In this context, we
have previously approached the synthesis and investiga-
tion of some new conjugates of non-proteinogenic and
proteinogenic amino acid condensed with diclophenac.
The synthesized candidates proved to be considerably
potent and less toxic than the parent drugs [12–14].
Generally, inflammation can be defined as an abnormal,
but protective biological response to a tissue injury that
is induced by physical trauma, chemicals, microbial
agents, or even an autoimmune reaction. The inflamma-
tory reaction is initially activated by a release of chemical
mediators from the injured tissue and/or migrating cells.
The acting mediators may include amines (histamine),
lipids (prostaglandin), small or large peptides (brady-
kinin or interleukin-1). Inhibition of a released mediator
is, consequently, of a therapeutic impact [1]. Interest-
ingly, some individual amino acids exemplified by
valine, leucine, isoleucine, some glutamine, trypto-
phane, methionine, and phenylalanine were early
reported to have anti-inflammatory properties [2–7].
Similarly, some short peptides as valyl-alanine, valyl-typ-
tophan, and tyrosyl-valine [8] as well as, some longer pep-
tides [9, 10] showed anti-inflammatory properties. Addi-
tionally, the specific inhibition of the inflammatory
Results and discussions
Chemistry
Biochemically, dipicolinic acid (pyridine-2,6-dicarboxylic
acid, DPA, CAS, 499-83-2) is a naturally occurring dicar-
boxylic acid that is found mainly in bacterial endospores,
as recently indicated by spectrometric analysis of Bacillus
spores [15]. We have previously explored the analytical
and biological characteristics of some of rationalized bis-
amino acid and peptide conjugates of dipicolinic acid
[16]. Our studies of these compounds, exemplified by
compound A (Fig. 1), revealed an interesting anti-cancer
activity, probably via DNA intercalation, as well as out-
standing metal sensors properties, particularly, for the
serious pollutant, lead cations (Pb2+) [17–19]. Cyclization
of some of these bis-conjugates with the molecular bas-
kets calix[4]arene as presented by compound B (Fig. 1)
Correspondence: Abd El-Galil E. Amr, Applied Organic Chemistry De-
partment, National Research Center, Dokki, Cairo, Egypt.
E-mail: aamr1963@yahoo.com
Fax: +20 2337-0931
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Arch. Pharm. Chem. Life Sci. 2007, 340, 304–309
New Peptides as Anti-Inflammatory Agents
305
Figure 1. Chemical structures of cyclic and macrocyclic com-
pounds A and B.
provided new biologically and chemically interesting
molecular architectures [20].
In this work, the synthesis and investigation of anti-
inflammatory characteristics of some bis-Na-L-valine, and
Na-L-valyl-L-phenylalanine linear, tetra and cyclic (penta
and octa) bridged peptides of dipicolinic acid were under-
taken.
L-Valine methyl ester was initially coupled with dipico-
linic acid via the conventional acid chloride method [20].
The peptide linkage was then formed with L-phenylala-
nine methyl ester. While the synthesis of compounds 1
and 2 were previously reported [20], coupling of diacid 2
with L-phenylalanine methyl ester by mixed anhydride
method afforded the corresponding Na-dipicolinoyl-bis-[L-
valyl-L-phenylalanine methyl ester] 3. In this context, the
used mixed anhydride coupling method, low tempera-
ture, weak bases (TEA) proved generally more potent and
less racemizing than the alternatives methods, e.g. the
acid chloride or the azide coupling procedures. Hydrazo-
nolysis of 3 with hydrazine hydrate afforded the corre-
sponding bis-hydrazide 4 (Scheme 1).
a
Scheme 1. Synthesis of new (N -dipicolinoyl)-bis-L-valyl-L-phe-
nylalanyl linear tetra peptides 5 and 6.
is associated with moderate to low reaction yields. In
Compounds 5–8 are newly suggested and synthesized addition, the possibility of dimerization of compound 10
candidates obtained via simple condensation of the is chromatographically (TLC) not supported.
hydrazide 4 with tetrachlorophthalic anhydride thus
The possible modulation of the amino acid and peptide
affording the 2,6-bis-imide pyridine derivative 5, with ani- C-terminal ends as esters 1 and 3, acids 2 and 9, hydrazide
saldehyde in methanol giving the corresponding hydra- 4, tetrachlorophthalic hydrazine conjugate 5, and finally
zone derivative 6 and with 1,2,4,5-benzenetetra-carbox- hydrazone 6 permit the explorations of the correspond-
ylic dianhydride or 1,8,4,5-naphthaline-tetracarboxylic ing activity of these biologically significant functional
dianhydride affording the conjugates 7 and 8, respec- groups.
tively (Scheme 2).
Na-Dipicolinoyl-bis-[L-valyl-L-phenylalanine
In addition, bridging with benzenetetracarboxylic
methyl acid exemplified by 7 and naphthalene tetracarboxylic
ester] 3 was hydrolyzed with 1 N sodium hydroxide in acid affording compound 8 infer a planar molecular
methanol to give the corresponding bis-acid 9, which was geometry for the obtained octa peptides. These conforma-
cyclized with L-lysine methyl ester in the presence of tionally restricted structures offer the perspectives of
ethyl chloroformate/triethylamine (mixed anhydride investigating the biological responses of these planar
method) to afford the cyclopentapeptide derivative 10 macrocycles. Structural resemblance to the biologically
(45%), which was equally prepared by azide coupling attractive crown ethers may also be significantly consid-
with 35% yield (Scheme 3). Generally, peptide cyclization ered.
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Abd El-Galil E. Amr et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 304–309
a
Scheme 2. Synthesis of new (N -dipicolinoyl)-bis-L-valyl-L-phe-
nylalanyl cyclic octa-bridged peptides 7 and 8.
As expected, the mass spectral data (Scheme 2) con-
firmed that the bridged cyclic tetra peptides 79and 89
were not formed. This may indicate that such cyclic mol-
ecules, contrary to the corresponding octapeptides 7 and
8, are sterically hindered and consequently structurally
unfavorable.
a
Scheme 3. Synthesis of new (N -dipicolinoyl)-bis-L-valyl-L-phe-
nylalanyl cyclic penta-bridged peptides 10.
potent than the simple parent hydrazide 4. In all cases,
elongation with L-phenylalanine had a potentiating
effect. The general pronounced anti-inflammatory activ-
ity of the compounds may accept the hypothesis that the
cyclooxygenase (COX) may be the working inflammatory
receptor.
Anti-inflammatory activity
Table 1 resumes the anti-inflammatory activity and LD50
of the candidates. Comparable to the two reference anti-
inflammatory drugs namely, indomethacin and diclo-
phenac (100%), the determined anti-inflammatory activ-
ity of the candidates (carrageenan induced paw edema in
rats) revealed a general significant activity (64–84%). In
particular, the potency of the (Na-dipicolinoyl)-bis-L-valyl-
L-phenylalanyl-p-methoxybenzaldehyde hydrazone 6 was
of 72% and 84% relative to the reference drugs, respec-
tively. Additionally, an acceptable acute toxicity was
observed (LD50: 2980 mg/kg comparable to 2700 and 2850
for indomethacin and diclophenac, respectively).
Experimental
Chemistry
Melting points were determined on open glass capillaries using
an Electrothermal IA 9000 SERIES digital melting point appara-
tus (Electrothermal, Essex, UK) 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
Other significant conclusions can be outlined in the
following: The cyclic compound 7 proved to be more
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Arch. Pharm. Chem. Life Sci. 2007, 340, 304–309
New Peptides as Anti-Inflammatory Agents
307
Table 1. Anti-inflammatory potency and toxicity (LD50) of the synthesized compounds.
Compound
% Increase in the weight
of rat paw edema (g)
% Protection
Aa)
Bb)
Cc)
LD50 (mg/Kg)
Diclofenac
Indomethacin
10.318 l 0.321
13.865 l 0.828
17.286 l 0.726
20.666 l 0.823
27.649 l 0.768
23.539 l 0.468
41.718 l 1.178
16.351 l 1.108
16.847 l 0.891
27.814 l 0.763
21.613 l 0.591
82.70
77.11
71.47
64.24
54.36
61.06
31.15
72.20
72.19
54.09
64.33
107.24
100
100
93.24
2850
2700
2090
2500
2433
2520
2315
2980
2741
2713
2870
1
2
3
4
5
6
7
8
9
80.20
70.90
50.14
58.90
33.23
84.79
82.29
49.84
64.15
59.68
49.92
37.31
43.83
24.73
63.10
61.24
37.09
47.73
a)
A: absolute figure.
B: % relative to Indomethacin.
C: % relative to Diclophenac.
b)
c)
Table 2. Physicochemical data of newly synthesized compounds.
Comp. No
Yield (%)
M.p. (8C)
[a]D25 (c 1, MeOH)
Color and solvent
for cryst.
Mol. Formula (Mol. wt)
3
4
5
6
7
8
9
10
76
70
80
82
68
64
72
165–168
120–122
A250
+ 82
+ 71
+ 95
+ 74
+ 84
+ 92
+ 78
+ 68
White MeOH
White EtOAc
White EtOH
Orange MeOH
Yellow MeOH
Brown MeOH
White MeOH
Pale yellow EtOAc
C37H45N5O8 (687.79)
C35H45N9O6 (687.79)
C51H41Cl8N9O10 (1223.56)
C51H57N9O8 (924.06)
C90H86N18O20 (1739.78)
C98H90N18O20 (1839.90)
C35H41N5O8 (659.73)
C42H53N7O8 (783.92)
217–219
160–162
A250
115–117
Oil
45 [A] 35 [B]
(KBr) were recorded on a FT IR-8201 PC Spectrophotometer (Shi-
madzu, Tokyo, Japan). The 1H 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, Santa Fe, NM, USA)
using EI and the values of m/z are indicated in Dalton. The start-
ing materials 1 and 2 were prepared according to published pro-
cedure [17]. The reactions were followed by TLC (Silica gel, alumi-
num sheets 60 F254, Merck, Darmstadt, Germany).
IR (KBr, cm– 1): 3392 (NH), 1739 (CO, ester), 1660 (CO, amide).
1H-NMR (DMSO-d6) d: 0.90–1.00 (m, 12H, 46 CH3), 2.28–2.34 (m,
2H, 26CH), 3.35 (d, 4H, 26CH2Ph), 3.58 (s, 6H, 26OCH3), 4.18–
4.25 (m, 2H, 26 CH-NH), 4.55–4.65 (m, 2H, 26 CH-NH), 6.92–
7.24 (m, 10H, 26Ph-H), 8.15–8.35 (m, 3H, pyr-H) and 8.45, 8.55
(2s, 4H, 46NH, exchangeable with D2O). 13C-NMR (DMSO-d6) d:
17.35, 18.66 (CH3), 33.76 (CH), 42.12 (CH2-Ph), 52.57, 52.80 (26
CH-NH), 55.66 (OCH3-ester), 124.12, 128.34, 129.46, 138.64 (Ar-C),
125.42, 140.00, 148.43 (pyr-C), 163.73, 168.96 (CO-amide), 172.44
(CO-ester). MS m/z (%): 687 [M+] (25), corresponding to the molecu-
lar formula C37H45N5O8 and at 331 (100), base peak.
a
N -Dipicolinoyl-bis-[L-valyl-L-phenylalanine methyl ester]
3
a
N -dipicolinoyl-bis-[L-valyl-L-phenylalanine hydrazide] 4
To a cold and stirred dry dichloromethane solution (25 mL, –
208C) of diacid 2 (0.36 g, 1 mmol), ethyl chloroformate (0.2 g,
2 mmol) and triethylamine (0.2 g, 2 mmol) were successively
added. Ten minutes later, a cold methylene chloride solution
(10 mL, –208C) of L-phenylalanine methyl ester (0.35 g, 2 mmol)
was added. Stirring of the cold reaction mixture (–208C) was
continued for 3 h and then at room temperature overnight. The
solution was washed with water, 1 N hydrochloric acid, 1 N
sodium bicarbonate, and finally with water (263 mL). The dried
solution (anhydrous CaCl2) was evaporated and the obtained oily
residue was solidified by dry ether trituration, filtered off, dried
under vacuum, and crystallized from methanol / ether to afford
3 (Table 2).
Hydrazine hydrate (90%, 0.5 g, 10 mmol) was added to a metha-
nolic solution (10 mL) of 3 (0.68 g, 1 mmol). The reaction mix-
ture was refluxed for 5 h after which the solvent was evaporated.
The obtained residue was triturated with ether, filtered off, and
crystallized from methanol/ether to afford 4 (Table 2).
IR (KBr, cm– 1): 3460–3350 (br, NH2, NH), 1666 (CO, amide). 1H-
NMR (DMSO-d6) d: 0.86–0.98 (m, 12H, 46CH3), 2.18–2.28 (m, 2H,
26CH), 3.42 (d, 4H, 26 CH2Ph), 3.48 (brs, 4H, 26 NH2, exchange-
able with D2O), 4.22–4.28 (m, 2H, 26CH-NH), 4.54–4.68 (m, 2H,
26CH-NH), 6.95–7.15 (m, 10H, 26Ph-H), 8.18–8.25 (m, 3H, pyr-
H), 8.60, 8.74, 9.10 (3s, 6H, 66NH, exchangeable with D2O). 13C-
NMR (DMSO-d6): d 17.32, 18.58 (CH3), 33.72 (CH), 42.22 (CH2-Ph),
52.44, 53.00 (26CH-NH), 124.10, 128.22, 129.38, 138.68 (Ar-C),
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125.34, 139.92, 148.36 (pyr-C), 163.73, 169.05 (CO-amide), 170.44
(CO-hydrazide). MS m/z (%):687 [M+] (15), corresponding to the
molecular formula C35H45N9O6 and at 331 (100), base peak.
NH), 4.55–4.60 (m, 4H, 46CH-NH), 6.95–7.30 (m, 20H, 46Ph-H),
7.40 (s, 4H, Ar-H), 8.10–8.28 (m, 6H, 26pyr-H), 8.88, 9.68, 9.72
(3s, 12H, 126NH, exchangeable with D2O). 13C-NMR (DMSO-d6) d:
17.69, 18.55 (CH3), 33.86 (CH), 41.95, 42.12 (CH2Ph), 51.50, 52.46
(CH-NH), 125.32, 140.15, 148.57 (pyr-C), 124.45, 128.43, 129.34,
134.50, 138.28, 152.46 (Ar-C), 164.34, 170.86, 172.10 (CO, amide),
174.18 (CO, imide). MS m/z (%): 1739 [M+] (8), corresponding to
the molecular formula C90H86N18O20 and at 331 (100), base peak.
a
N -dipicolonoyl-bis-[L-valyl-L-phenylalanyl]-
tetrachlorophthalic-1,2-hydrazine conjugate 5
A stirred glacial acetic acid suspension (50 mL) of Na-dipicolonyl-
bis-[L-valyl-L-phenylalanine hydrazide] (4, 0.68 g, 1 mmol) and tet-
rachlorophthalic anhydride (0.57 g, 2 mmol) was heated (808C)
for 6 hrs. The reaction mixture was concentrated under reduced
pressure, cooled and the separated solid was collected by filtra-
tion, dried and crystallized from acetic acid/ether to yield the
corresponding compound 5 (Table 2).
Naphthalene tetracarboxamide bis-[L-valyl-L-
phenylalanyl] cyclic octa-bridged peptide 8
IR (KBr, cm– 1): 3560–3258 (NH), 1734 (CO), 1656 (CO, amide). 1H-
NMR (DMSO-d6): d = 0.85–1.00 (m, 24H, 86CH3), 2.19–2.26 (m,
4H, 46CH), 3.38–3.44 (m, 8H, 46CH2-Ph), 4.18–4.36 (m, 4H,
46CH-NH), 4.50–4.65 (m, 4H, 46CH-NH), 7.15–7.60 (m, 28H, Ar-
H), 8.05–8.25 (m, 6H, 26pyr-H), 8.95, 9.64, 10.15 (3s, 12H,
126NH, exchangeable with D2O). MS m/z (%): 1840 [M+] (5), corre-
sponding to the molecular formula C98H90N18O20 and at 625
(100), base peak.
IR (KBr, cm– 1): 3345, 3276 (NH), 1728 (C=O), 1656 (CO, amide).
1H-NMR (DMSO-d6) d: 0.88–1.05 (m, 12H, 46CH3), 2.20–2.27 (m,
2H, 26CH), 3.40 (d, 4H, 26CH2Ph), 4.18–4.30 (m, 2H, 26CHN),
4.35–4.42 (m, 2H, 26CH-NH), 6.92–7.24 (m, 10H, 26Ph-H),
8.10–8.22 (m, 3H, py-H), 8.45–9.15 (brs, 6H, 66NH, exchange-
able with D2O). MS m/z (%): 1223 [M+] (24), corresponding to the
molecular formula C51H41Cl8N9O10 and at 625 (100), base peak.
a
N -dipicolinoyl-bis-[L-valyl-L-phenylalanine] 9
a
N -dipicolonoyl-bis[L-valyl-L-phenylalanyl]-p-
Sodium hydroxide (1 N, 25 mL) was added dropwise to a cold
and stirred methanolic solution (0.68 g, 1 mmol, –58C) of 3. Stir-
ring 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, and crystallized from ethanol/ether
mixture to afford 9 (Table 2).
methoxybenzaldehyde hydrazone 6
A stirred solution of Na-dipicolonyl-bis-[L-valyl-L-phenylalanine
hydrazide] (4, 0.68 g, 1 mmol) and p-methoxy benzaldehyde
(0.27 g, 2 mmol) in absolute methanol (50 mL) was refluxed
reflux for 6 h. The reaction mixture was allowed to stand at
room temperature overnight, then it was evaporated under
reduced pressure. The obtained oily product was solidified by
trituration with benzene/petrolether (40–608C), the remaining
solid was filtered off, dried, and crystallized from methanol to
give the corresponding hydrazone 6 (Table 2).
IR (KBr, cm– 1): 3655–3540 (OH), 3325 (NH), 1725 (CO, acid),
1656 (C=O, amide). 1H-NMR (DMSO-d6) d: 0.85–0.95 (m, 12H,
46CH3), 2.16–2.33 (m, 2H, 26CH), 3.38 (d, 4H, 26CH2Ph), 4.22–
4.26 (m, 2H, 26CH-NH), 4.35–4.50 (m, 2H, 26 CH-NH), 7.00–
7.25 (m, 10H, 26Ph-H), 8.10–8.45 (m, 3H, pyr-H), 8.65, 8.82 (2s,
4H, 46NH, exchangeable with D2O), 11.98 (s, 2H, 26OH,
exchangeable with D2O). 13C-NMR (DMSO-d6) d: 17.98, 18.56 (CH3),
33.68 (CH), 41.98 (CH2-Ph), 52.54 (CH-NH), 124.18, 128.22, 129.40,
138.64 (Ar-C), 125.36, 139.89, 148.42 (pyr-C), 164.05, 169.10 (CO-
amide), 171.40 (CO-acid). MS m/z (%): 659 [M+] (65), corresponding
to the molecular formula C35H41N5O8 and at 625 (100), base peak.
IR (KBr, cm– 1): 3500–3250 (NH), 1660 (C=O, amid). 1H-NMR
(DMSO-d6) d 0.9–1.2 (m, 12H, 46CH3), 2.22–2.28 (m, 2H, 26CH),
3.36 (d, 4H, 26CH2Ph), 3.42 (s, 6H, 2 x OCH3), 4.24–4.45 (m, 4H,
46CH-NH), 4.52 (s, 2H, 26CH=N), 6.95–7.44 (m, 18H, Ar-H),
8.12–8.32 (m, 3H, py-H), 8.60, 9.86, 10.00 (36s, 6H, 66NH,
exchangeable with D2O). 13C-NMR (DMSO-d6) d: 17.49, 18.65 (CH3),
33.86 (CH), 41.98 (CH2-Ph), 52.09, 54.74 (CH-NH), 124.26, 127.26,
128.16, 128.68, 129.44, 130.25, 133,89, 138.66 (Ar-C), 147.84
(CH=N), 124.98, 140.00, 148.25 (pyr-C), 163.62, 169.40, 172.38
(C=O). MS m/z (%): 924 [M+] (12), corresponding to the molecular
formula C51H57N9O8 and at 331 (100), base peak.
a
Cyclo-(N -dipicolinoyl)-bis-[L-valyl-L-phenylalanyl]-L-
lysine methyl ester 10
Method A: Mixed anhydride method
Bis-[L-valyl-L-phenylalanyl] cyclic octa-bridged peptides 7
Triethylamine (0.2 g, 2 mmol) was added to a cold and stirred
dichloromethane (25 mL, –208C) suspension of diacid 9 (0.65 g,
1 mmol), and ethyl chloroformate (0.22 g, 2 mmol). Stirring was
continued for 20 min after which L-lysine methyl ester (0.16 g,
1 mmol) was added. The reaction mixture was stirred at (–208C)
for 3 h and then for 12 h at room temperature. The reaction mix-
ture was washed with water, 1 N hydrochloric acid, 1 N sodium
bicarbonate and water, then dried over anhydrous calcium
chloride. The solvent was evaporated and the crude product was
purified by preparative thin layer chromatography using metha-
nol/benzene mixture (1 : 9 (vol/vol) as an eluent to give the corre-
sponding cyclic peptide ester 10 (Table 2).
and 8
A stirred suspension of a mixture of Na-dipicolonyl-bis-[L-valyl-L-
phenylalanene hydrazide] (4; 0.68 g, 1 mmol) and 1,2,4,5-benze-
netetracarboxylic acid dianhydride or 1,8,4,5-naphthaline-tetra-
carboxylic acid dianhydride (1 mmol) in glacial acetic acid
(50 mL) was heated (808C) for 7 h. The reaction mixture was con-
centrated under reduced pressure. The obtained solid was col-
lected by filtration, dried, and crystallized from DMF/H2O to give
7 and 8, respectively (Table 2).
Benzene tetracarboxamide bis-[L-valyl-L-
IR (KBr, cm– 1): 3350 (NH), 1745 (C=O, ester), 1656 (C=O, amide).
1H-NMR (DMSO-d6) d: 0.90–0.80 (m, 12H, 46 CH3), 1.24–1.46 (m,
4H, 26CH2), 1.60–1.75 (m, 2H, CH2), 2.30–2.35 (m, 2H,
26CH(CH3)2), 3.00–3.20 (m, 2H, CH2), 3.32 (d, 4H, 26CH2Ph), 3.55
phenylalanyl]cyclic octa-bridged peptide 7
IR (KBr, cm– 1): 3550-3300 (NH), 1726 (C=O), 1655 (C=O, amid). 1H-
NMR (DMSO-d6) d: 0.82–0.90 (m, 24H, 86CH3), 2.26–2.34 (m, 4H,
46CH), 3.36–3.40 (m, 8H, 46CH2Ph), 4.28–4.37 (m, 4H, 46CH-
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Arch. Pharm. Chem. Life Sci. 2007, 340, 304–309
New Peptides as Anti-Inflammatory Agents
309
(s, 3H, OCH3), 3.90–4.05 (m, 4H, 46CH-NH), 4.38–4.44 (m, 1H,
CH-NH), 7.12–7.30 (m, 10H, 26Ph-H), 8.05–8.18 (m, 3H, pyr-H),
8.88, 8.96, 9.15 (3s, 6H, 6 x NH, exchangeable with D2O). 13C-NMR
(DMSO-d6) d: 17.65, 18.97 (CH3), 22.56, 28.30, 30.35, 38.00
(46CH2), 33.85 (CH), 41.99, 42.05 (2 CH2-Ph), 52.36, 52.80 (46CH–
NH), 54.48 (OCH3-ester), 60.50 (CH-ester), 124.32, 128.30, 129.42,
138.74 (Ar-C), 125.23, 139.37, 149.57 (pyr-C), 163.81, 169.15,
170.64 (CO, amide), 173.89 (CO, ester). MS m/z (%): 784 [M+] (16),
corresponding to the molecular formula C42H53N7O8 and at 331
(100), base peak.
References
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Method B: Azide method
An aqueous solution of sodium nitrite (10%, 0.13 g, 2 mmol) was
added to a cold (–58C) and stirred solution of the dihydrazide 4
(0.68 g, 1 mmol) in 5 N HCl (3 mL) and acetic acid (3 mL). Stirring
was continued for 30 min after which the reaction mixture was
extracted with ether, washed with water, NaHCO3, and water;
then dried over anhydrous sodium sulfate. The cold ethereal sol-
ution (–58C) was then added to a cold (–58C) dichloromethane
solution of L-lysine methyl ester (0.16 g, 1 mmol, 10 mL). Stirring
was continued for 5 h and at room temperature for 2 h. The reac-
tion mixture was washed with 1 N hydrochloric acid, water and
then dried (anhydrous sodium sulfate). Evaporation of the sol-
vent afforded crude 10 (Table 2), which upon preparative TLC
was found identical with that obtained via the mixed anhydride
method.
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Anti-inflammatory assay
[12] M. H. Abo-Ghalia, A. M. Shalaby, W. I. El-Eraqui, H. M.
Animals: Adult male albino rats weighing l120–200 g were
obtained from the animal house colony, Research Institute for
Ophthalmology, Cairo, Egypt and fed on standard laboratory
diet. Drugs and chemicals: Indomethacin (Sigma, USA) and diclo-
fenac (Novartis, Switzerland) were used as reference drugs. Car-
rageenan (BDH, England); other used chemicals are of analytical
grade (Sigma-Aldrich, USA).
Awad, Amino Acids 1999, 16, 425–440.
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Methodology: Three groups of rats were classified as followed:
The first group containing six rats received a vehicle of 0.2 mL of
7% aqueous solution of Tween 80m and served as control group.
The second group was divided into two subgroups of six rats,
each received either indomethacin or diclofenac (7 mg/kg). The
third group was divided into nine subgroups of six rats each; the
rats received a candidate compound (7 mg/kg) orally. As for the
first group, identical conditions also were followed for the sec-
ond and third group. The anti-inflammatory activity (Table 1)
was determined principally according to Winter et al. [21]. The
rates were dosed with a candidate or a reference drug. One hour
later, a foot-paw edema was induced by sub-planter injection of
0.05 mL of 1% suspension of carrageenan in saline into the
planter tissue of the rat hind paw. Identical experimental condi-
tions, except for dosing, were followed for the reference group.
Three hours after carrageenan injection the average weight of
edema in the rapidly excised paws of the decapitated rats was
determined. For each group, the mean value of the obtained
results was then considered. Statistical analysis of data was com-
puted via the Student`s t-test [22] A 0.05 level of probability was
regarded as significant according to Sendecor and Cochran [23].
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1962, 111, 541–545.
[22] J. T. jr Litchfield, F. Wilcoxon, Pharmacol. Ther. 1949, 96,
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[23] W. G. Senecor, G. W. Cochran (Eds), Statistical Methods,
Iowa State University, Ames, Iowa, 1971.
[24] K. F. Austen, W. E. Brocklehurst, J. Exp. Med. 1961, 113,
521–524.
Toxicity
The LD50 was determined by using rats and inject different
increasing doses and calculate the dose that kill 50% from the
animal, according to Austen et al. [24].
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