The study of the effect of histidine derivatives as a novel antinociceptive and anti-inflammatory activity

Article abstract of DOI:10.1155/2011/173149

This study explains the biochemical activity of histidine derivatives The compounds were identified by CHN analysis, FT-IR and H1 NMR. The results certified that the chemical structures of the prepared compounds. The anti-inflammatory and antinociceptive activity was studied by two different tests; the hot plate test and writhing test for analgesic activity and two tests for anti-inflammatory activity they are formalin induced inflammation test and carrageen an induced inflammation test. The histidine derivatives were found to have potent activity as anti-inflammatory and antinociceptive. The active compounds were tested to acute toxicity. It was found that they are safety to the dose 5 g/kg orally in mice without any mortality. Copyright E-Journal of Chemistry 2004-2011.

Full text of DOI:10.1155/2011/173149

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2011, 8(4), 1832-1842
http://www.e-journals.net
The Study of the Effect of Histidine
Derivatives as a Novel Antinociceptive and
Anti-Inflammatory Activity
USAMA H. RAMADHAN* and NIRAN J. AL-SALIHI
*Department of Pharmaceutical Chemistry
College of Pharmacy, University of Basrah, Basrah, Iraq
Department of Chemistry
College of Science, University of Basrah, Basrah, Iraq
usama_ramadhan@yahoo.com
Received 8 September 2010; Revised 19 December 2010; Accepted 8 January 2011
Abstract: This study explains the biochemical activity of histidine derivatives
The compounds were identified by CHN analysis, FT-IR and H1 NMR. The
results certified that the chemical structures of the prepared compounds. The
anti-inflammatory and antinociceptive activity was studied by two different
tests; the hot plate test and writhing test for analgesic activity and two tests for
anti-inflammatory activity they are formalin induced inflammation test and
carrageen an induced inflammation test. The histidine derivatives were found to
have potent activity as anti-inflammatory and antinociceptive. The active
compounds were tested to acute toxicity. It was found that they are safety to the
dose 5 g/kg orally in mice without any mortality.
Keywords: Histidine derivative, Anti-inflammatory agent, Antinociceptive agent
Introduction
Aspirin was the first drug developed as a marketable drug in 1897 but other salicylates have
long been used in the treatment of pain. The ancient Greeks, for example, used the bark of
willow trees for pain relief. Aspirin blocks the production of some eicosanoids and thus relives
the symptoms of pain and reduces fever. The active ingredient of aspirin, acetylsalicylic acid,
irreversibly inhibits COX activity by transferring an acetyl group to an active site serine
residue of the bifunctional enzyme. By blocking the activity of COX, aspirin prevents the
formation of a variety of eicosanoids that were synthesized after the COX reaction1,2.

1833
U. H. RAMADHAN et al.
Experimental
The amino acid L-histidine (1.55 g, 0.01 mol) was dissolved in ethanol (10 mL) and a
solution of potassium hydroxide (0.56 g, 0.01 mol) in ethanol (10 mL) was added, the
reaction mixture were refluxed for 2 h, the solution were evaporated by rotary evaporator.
The residue was recrystallized from methanol, A white precipitate (compound 1) L-histidine
o
ethyl ester was formed (ethyl 2-amino-3-(1H-imidazole-4-yl)propanoate). M.p.198 C,
yield 72.1%.
Acetyl histidine derivatives4,5
L-Histidine (1.55 g, 0.01 mol) was dissolved in (15 mL) glacial acetic acid and was treated
with (1 mL, 0.01 mol) acetic anhydride. The mixture was boiled for 2 h. The solution was
cooled to room temperature and evaporated in rotary evaporator to dryness. The residue was
dissolved in 20 mL of water and extracted twice in a separating funnel with 15 mL carbon
tetrachloride. The aqueous layer was evaporated and the residue was taken up twice in about
20 mL of distilled water and the solution each time was evaporated in rotary evaporator to
dryness. The residue was dissolved in a few mL of distilled water then filtered. To the
filtrate 20 mL of acetone was added, the mixture was cooled to 5 ºC for several hours. The
precipitate were filtered under vacuum and washed with 70% acetone and then with 100%
acetone and finally with dry ether. The precipitate recrystallized with ethanol to afford
Nα-acetyl-L-histidine; 4-Imidazole-4yl-2-(N-acetyl)propanoic acid (Compound 2).
M.p.153 oC, yield 68.9%.
Compound 3 was prepared by reflux (1.83 g, 0.01 mol) of compound 1 and 10 mL
glacial acetic acid for 2 h. The solution were cooled to room temperature and evaporated in
rotary evaporator to dryness. The residue was dissolved in 20 mL distilled water and
extracted twice in separating funnel with 15 mL of carbon tetrachloride. The aqueous layer
was evaporated and the residue was taken up in about 20 mL of water and the solution were
evaporated in rotary evaporator to dryness. The residue was dissolved in a few mL of
distilled water then filtered. To the filtrate 20 mL of acetone was added and the mixture was
cooled to 5 ºC for several hours. The precipitate were filtered under vacuum and washed
with 70% acetone and then with 100% acetone, and finally with dry ether. The precipitate
recrystallized with ethanol to afford Nα-acetyl-L-histidine ethyl ester; Ethyl-2-acetamido-3-
(1H-imidazole-4-yl) propanoate (compound 3)6 M.p. 118 oC, yield 63.4%.
.
Propanoyl histidine derivative7
Dried L-histidine (1.55 g, 0.01 mol) was dissolved in 10 mL dried propanoic acid. The
mixture was refluxed for 2 h. The propanoic acid was evaporated in rotary evaporator to
dryness. The residue was dissolved in 20 mL of distilled water and extracted twice in
separating funnel with 15 mL of chloroform. The aqueous layer was evaporated and the
residue was taken up twice in about 20 mL of distilled water and the solution each time was
evaporated in rotary evaporator to dryness. The residue was dissolved in few mL of distilled
water and filtered. To the filtrate 20 mL of acetone was added and the mixture cooled to
o
5 C for several hours. The precipitate were filtered under vacuum and washed with 70%
acetone and then with 100% acetone and finally with dry ether. The precipitate
recrystallized with ethanol to afford Nα-propanoyl-L-histidine; 3-(1H-imidazole-4-yl)-2-
propanamido propanoic acid (compound 4). M.p. 191-192 oC, yield 61.6%.
In the same way the compound 4 was prepared by refluxed compound 1 (1.83 g, 0.01 mol)
with 10 mL of dried propanoic acid to give Nα-propanoyl-L-histidine ethyl ester; ethyl-3-
(1H-imidazole-4-yl)-propanamido propanoate (Compound 5). M.p. 234-236 oC, yield 43.16%.

The Study of the Effect of Histidine Derivatives
1834
Pharmacology
Animals
Male and female albino mice (20-30 g) were obtained from the animal house of university of
Basrah, college of sciences. Animals were housed in colony room (12/12 h) light/ dark cycle
at 23 2 ºC and had free access to food and water. Mice were divided into six groups in each
experiment (n=6, 3 male and 3 female).
Drugs preparation
The drugs used in the experiments were diluted in a way to obtained injection volume of 0.2 mL.
Each drug was dissolved in appropriate solvent as following: aspirin and diclofenac in water;
carrageenan 1% in normal saline; acetic acid 0.7% in water; formalin 1% in water.
Antinociceptive activity8
Hot plate test
A mouse was placed on a hot plate maintained at 56 2 ºC and the latency of its reaction to
this nociceptive stimulus (number of seconds before it licked its paw or jumping) was
quantified, with interruption time of 20 sec. The latency was measured just before
zero time and 1, 2, 3 and 4 h after injections. The hot plate test was carried out to assess the
effects of agent on the thermal nociceptive threshold. The compounds were given orally (50
mg/kg) and control group received (0.2 mL) distilled water orally. Aspirin (50 mg/kg) and
diclofenac (25 mg/kg) were used as test standards.
Writhing test9
The antinociceptive effect was evaluated by the writhing test, induced by acetic acid (0.7%) v/v
(0.2 mL) intrapretonial injected to mice. Then the mice placed in large glass beaker and intensity of
nociception was quantified by counting the total numbers of writhing occurring between zero and
30 min after stimulus injection. The writhing response consists of a contraction of the abdominal
muscles together with a stretching of the hind limbs. The compounds 50 mg/kg orally
administered 1 h before the nociceptive agent in treated group. Five minutes after the acetic acid
injection we observed the numbers of writhes for a period of 25 min. Control group was received (0.2
mL) distilled water orally. Aspirin (50 mg/kg) and diclofenac (25 mg/kg) were used as test standards.
Anti-Inflammatory activity
Formalin induced inflammation test10,11
The inflammation was produced by subaponeurotic injection of 20 µL of 1% formaldehyde in
the left hand paw of the mice on the first hour after the oral administration of the compounds
50 mg/kg orally. The control group was received distilled water (0.2 mL). Aspirin (50 mg/kg)
and diclofenac (25 mg/kg) were used as references drugs. The changes in paw size was
measured at zero-hour and then at 1 h intervals up to the 5 h with a micrometer device.
Carrageenan induced inflammation test 12
For the determination of the effects on acute inflammation, carrageenan induced
inflammation model was used. Inflammation was induced by sub-planter injection of a
homogenous suspension of 1% carrageenan in physiological saline 25 µL. Mice were orally
given compounds in a single dose 50 mg/kg orally one hour before the induction of paw
inflammation. The control group was given aspirin (50 mg/kg) and diclofenac (25 mg/kg)
orally one hour before the induction, while the blank group was received distilled water (0.2
mL) only. Paw size was measured in every mouse hourly during four hours after the
induction with a micrometer device12.

1835
U. H. RAMADHAN et al.
Statistical analysis13,14
All values in the tables and the figures are expressed as the mean standard error. The
comparisons between groups were made by the analysis of variant student’s t-test (2-tailed,
3-typed) two-tailed distribution, two-sample unequal variance, with microsoft excel software.
Differences with P<0.05 between experimental groups were considered statistically significant.
Acute toxicity15
In order to verify the LD50, mice (n=10; 5 male and 5 female) in each group administered
orally following single dose of (1, 3 and 5 gm/kg) to each compound dissolved in (0.5 mL)
distilled water, control group received 0.5 mL distilled water only. The mortality was
observed during 24 h.
Result and Discussion16,17
Thin layer chromatography (TLC) of starting materials and products were preformed using
suitable solvents. The spots visualized by using ultraviolet light lamp at 254 nm instrument,
iodine vapour, 10% sulphuric acid and ninhydrin.
The mobile phase of TLC was propanol: water (1:1). Compounds (1, 3 and 5) give positive to
ester test and compounds (2-5) give positive to secondary amine test with two test benzene
sulfonyl chloride and diazonium ion. The esterification of carboxylic acids with alcohols is a
reversible reaction and it can be accomplished only if a means is available to drive the equilibrium
to the right. There are many ways of doing this and in this case by adding excess of alcohol to
driving the equilibrium to the products. Table 1 shows the physical properties of the prepared
compounds while Table 2 indicates the CHN chemical analysis of compounds 3 and 5. Elemental
analyses were performed for the final compounds to confirm their basic chemical compositions.
The measured percentages show a reasonable good agreement with calculated results.
Table 3 shows the major bands of the histidine derivatives. The NH of imidazole and
α-NH bond stretching was between (3444-3282 cm-1), methylene hydrogen bond C=CH
between (3168-3016 cm-1) and the aliphatic hydrogen attributed between (2980-2820 cm-1).
The carbonyl bond was absorbed between (1714-1587 cm-1), the C=C bond was stretching
between (1400-1433 cm-1) and the NH bending between (1587-1535 cm-1)18. Figures 1 to 4
show the FT-IR spectrums of compounds.
Table 1. Characterizations and physical data of histidine derivatives
Comp
Compound name
L-histidine ethyl ester
Nα-acetyl-L-histidine
M.wt m.p.oC
Colour
Rf
%Yield
72.1
68.9
1
2
3
183.2
197.2
198
153
118
White crystals 0.61
White powder 0.67
White powder 0.65
Nα-acetyl-L-histidine ethyl ester 225.3
63.4
Pale yellow
0.53
4
5
Nα-propanoyl-L-histidine
211.2 191-192
61.6
powder
Nα-propanoyl-L-histidine
ethyl ester
239.3 234-236 Beige powder 0.49
43.16
Table 2. Elemental analyses of compounds
Elemental analysis %
C
Compound Molecular formula M.wt
H
N
Cal.
Found. 52.74 6.63 18.28
Cal. 55.16 7.1 17.55
Found. 55.95 7.38 17.72
53.3 6.65 18.64
3
5
C10H15N3O3
C11H17N3O3
225.3
239.3

The Study of the Effect of Histidine Derivatives
1836
Table 3. Characteristic FT-IR absorptions (cm-1) of histidine derivatives
Stretching
υN-H
3444, 3419 3091, 3016
Stretching
υC=C-H
Stretching
υC-H
2820
Stretching
υC=O
1640
Stretching
υC=C
1410
Bending
Comp.
δN-H
1587
1540
1542
1535
1
2
3
4
3282,
3413
3338
3140, 3020 2935, 2870 1714, 1654
1716, 1639
1633
1400
1433
1403
3147, 3031
3099, 3016 2980, 2863
2893
2975,
3168, 3109
5
3338
1641, 1587
1404
1535
2940, 2860
O
O
NH
N H
O
O
CCHH3
3
N
N
N H
NH2
2
Figure 1. The FT-IR spectrum of compound 1
O
O
NH
NH
O
CH
3
CH
O
N
NH
NH
CH
3
3
CH
N
3
O
O
Figure 2. The FT-IR spectrum of compound 3

1837
U. H. RAMADHAN et al.
O
NH
N
OH
O
NH
CH
2
CH
3
Figure 3. The FT-IR spectrum of compound 4
O
NH
O
CH
3
N
NH
CH
3
O
Figure 4. The FT-IR spectrum of compound 5
The 1H NMR spectra of compounds 1 and 4 shows the NH peak at 7.7 ppm and 8.02 ppm
respectively, the hydrogen in imidazole ring peak between 8.33-6.8 ppm, CH 4.03 and
4.39 ppm respectively, CH2 between 3.19- 2.8 ppm and CH3 1.1 and 0.97 ppm respectively.
The Table 4 and the Figure 5 and 6 show the characteristics and NMR spectra of
compounds 1 and 4.

The Study of the Effect of Histidine Derivatives
1838
Table 4. Characteristics H1 NMR of compounds 1 and 4
imidazole
Comp
δ NH
7.7 s
δ CH-CH2
δ CH2-CH3
3.74 d CH2,
1.1 t CH3
2.11 q CH2,
0.97 t CH3
δ N=CH-NH
δ C=CH-NH
4.03 t CH,
3.19,3.03 m CH2
4.39 q CH,
1
4
8.33 s
7.22 s
8.02 d
7.63 s
6.8 s
2.92, 2.8 m CH2
s = Singlet, d = doublet, t = triplet, q = quartet, m = multi
Figure 5. The H1 NMR spectrum of compound 1
Figure 6. The H1 NMR spectrum of compound 4

1839
U. H. RAMADHAN et al.
Table 5 and Figure 7 shows the results of hot plate test. All derivatives were found
to have analgesic effect in hot plate test, compound 3 (Nα-acetyl-L-histidine ethyl ester)
was more effective than other histidine derivatives. The substitution on both amino and
carboxyl groups of histidine increases the activity of drug. Compound 5 (Nα-propanoyl-
L-histidine ethyl ester) was less effective than compound 3 but compound 4 (Nα-
propanoyl-L-histidine) still more effective than the other derivatives thus the
substitution on amino group must not be more than three carbon groups (C3) and it was
found that two carbon groups (C2) are more effective than three carbon groups if
carboxyl group was substituted. This may be due to the size of the small molecule to be
sitting in an active site of enzyme19.
Table 5. Hot plate tests of histidine derivatives
Latency time in sec
2 h
Comp.
0 h
1 h
3 h
4 h
Control
3.4 1.37
2 1.0
3.7 1.5
4.4 1.75
9 1.5 *
5.6 2.5
4 0.5
9.11 1.0*
Aspirin
4.8 1.25
7.8 3.25
12.6 2.8 *
13.4 3.3 *
17.5 2.5 *
17.2 1.6 *
7.66 1.55
7.2 2.75
14.1 3.5 *
15.5 4.1 *
16 1.7 *
28.2 2.83*
18.6 2.42*
22.3 2.782*
Diclofenac
3 0.5
14 2.5 *
8.33 1.1
17.6 1.5 *
31 3.13*
18.4 2.32*
7.66 2.88
9.6 3.5
1
2
3
4
5
4.6 2.11
6 2.4
24 4.2 2*
20.4 2.2 2*
28 3.3 3*
13.5 1.3 *
18.3 2.222*
5.2 2
4.5 2.2
5.33 1.1
* =P<0.05; 2* =P<0.01; 3* =P<0.005
35
control
aspirin
30
25
20
15
10
diclofenac
comp.1
comp.2
comp.3
comp.4
comp.
5
5
0
0
1
2
3
4
Time, h
Figure 7. Hot plate tests of histidine derivatives
Table 6 and Figure 8 shows the result of writhing test of histidine derivatives. Compound 1
(L-histidine ethyl ester) and compound 4 (Nα-propanoyl-L-histidine) show significant effect
(comp1 p<0.05, comp4 p<0.05). The activity increased when carboxyl group was
substituted, but decreased when amino group was substituted except compound 4 which is
substituted with three carbons (C3) group and carboxyl group was free.
Table 6. Writhing tests of histidine derivatives
control aspirin
43 2
6.3 25*
* =P<0.05; 2* =P<0.01; 3* =P<0.005; 4* = P<0.001; 5* =P<0.0005
diclofenac comp.1 comp.2 comp.3 comp.4
comp.5
44 2.5
10.6 25* 27 3* 28 2*
36 2.8 42 3

The Study of the Effect of Histidine Derivatives
1840
50
40
30
20
10
0
l
o
c
a
n
i
1
2
3
4
5
.
p
.
p
.
p
.
p
.
p
r
r
i
p
t
n
n
o
e
m
o
m
o
m
o
m
o
m
o
s
a
f
o
c
l
c
c
c
c
c
c
i
d
Figure 8. Writhing tests of histidine derivatives
Table 7 and Figure 9 shows the result of formalin induced inflammation test. The
substitution on both carboxyl and amino groups of histidine increased anti-inflammatory
activity and substitution on carboxyl group (Compound 1) was more effective than
substituted on amino group (Compound 2 and 4).
Table 7. Formalin induced inflammation tests of histidine derivatives
change of paw size, mm×10 -2
comp.
0 h
0 2
1 h
77 2.5
52 16
2 h
96 9
3 h
84 2
4 h
5 h
87 14
38 11.5*
43 24
control
aspirin
diclofenac 0 23
91 14
0 10
77 21
52.5 25
48 22
65 11
61 13
57.5 25
31 162*
59.5 13
40 12 *
22.5 202*
29 92*
61 15
33.5 212*
43 10 *
64 15
1
2
0 9
0 15
25 212*
72 23
1 35*
3
0 17
37 182*
25 153*
30 142*
3 84*
4
5
0 13
0 8
54 21
32 14*
62 14
17 7 2*
71 18
20 6 3*
32 162*
21 6.22*
46 19
18 4.4 3*
* =P<0.05; 2* =P<0.01; 3* =P<0.005; 4* = P<0.001; 5* =P<0.0005
120
control
aspirin
diclofenac
100
80
comp.1
comp.2
comp.3
comp.4
comp.5
60
40
20
0
1
2
3
4
5
Time, h
Figure 9. Formalin induced inflammation tests of histidine derivatives
Table 8 and Figure 10 shows the result of carrageenan induced inflammation test of
histidine derivatives. All derivatives were given significant effects. Compound 3 was more
effective than the other compounds because it is substituted on positions amine and carboxyl
group, then compound 2 because it substituted on amino group.

1841
U. H. RAMADHAN et al.
Table 8. Carrageenan induced inflammation tests of histidine derivatives.
change of paw size, mm×10-2
Comp.
0 h
0 5 140 15 111 21 112.5 12 120 15 95 25
1 h
2 h
3 h
4 h
5 h
Control
Aspirin
0 22 118 1
85 15
0 12 82 20
0 7 52 23
90 2
85 29
60 25
64 4
58.5 3
69 10
80 2
72 22
59 9
77 5
62 7
75 20
Diclofenac 0 1
1
2
3
4
5
43 21 32.5 15* 34 17* 29 12*
0 16 82 18
0 19 52 16
0 11 90 24
19 63*
41 12
59 17
29 14*
36 16*
58 13
39 8*
51 6
36 5*
50 18
53.5 14 60 12
* =P<0.05; 2* =P<0.01; 3* =P<0.005
160
140
120
100
control
aspirin
diclofenac
comp.1
comp.2
comp.3
comp.4
comp.5
80
60
40
20
0
1
2
3
4
5
Time, h
Figure 10. Carrageenan induced inflammation test of histidine derivatives
The injection of carrageenan increased in leukocyte infiltration and accumulation of
PGE2, LTB4 and nitrate. Carrageenan also induced elevated expression of
cyclooxygenase-1 and -2, inducible nitric oxide synthase and 5-lipoxygenase in the
inflammatory exudates20.
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Volume 2014
Volume 2014
Volume 2014
Volume 2014
Volume 2014