New condensed pyrroles of potential biological interest: Syntheses and structure-activity relationship studies

Article abstract of DOI:10.1016/j.ejmech.2011.04.034

The Pyrrole derivatives Ia-d were prepared and utilized for the preparation of pyrrolo[2,3-d]pyrimidine derivatives IIa-c, III, IVa-e, V and VIIIa-c; pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine VI and pyrrolo[4,3e][1,2,4]triazolo[1, 5-c]pyrimidine derivative

Full text of DOI:10.1016/j.ejmech.2011.04.034

European Journal of Medicinal Chemistry 46 (2011) 3022e3029
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New condensed pyrroles of potential biological interest
Syntheses and structureeactivity relationship studies
,
Mosaad S. Mohamed a, Rehab Kamel b, Samar S. Fatahala a
*
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, Helwan, Egypt
b Toxicology and Pharmacology Department, Faculty of Pharmacy, Helwan University, Ein Helwan, Helwan, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The Pyrrole derivatives Iaed were prepared and utilized for the preparation of pyrrolo[2,3-d]pyrimidine
derivatives IIaec, III, IVaee, V and VIIIaec; pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine VI and pyrrolo[4,3e]
[1,2,4]triazolo[1,5-c]pyrimidine derivative derivatives VIIaec. These some newly synthesized compounds
were examined for their in vitro antimicrobial activity and in vivo anti-inflammatory. Result indicated
that these compounds showed promising anti-inflammatory activity in comparison to ibuprofen (the
standard anti-inflammatory drug). The structureeactivity relationships (SAR) and anti-inflammatory
activities of these compounds are also discussed in this paper.
Received 20 November 2010
Received in revised form
5 April 2011
Accepted 12 April 2011
Available online 16 April 2011
Keywords:
Pyrrole
Ó 2011 Elsevier Masson SAS. All rights reserved.
Pyrrolo[2,3-d]pyrimidine
Structureeactivity-relationship
Anti-inflammatory activity
Antimicrobial
1. Introduction
antibiotics [5] that have been observed to inhibit the growth of
some microorganisms such as Candida albicans, Streptococcus neo-
The key roles played by purines and pyrimidines in cellular
processes have made them valuable leads for drug discovery. Pyr-
rolo[3,2-d]pyrimidines, a class of 7-deazapurine analogs, exhibit
interesting biological activity in part due to their resemblance to
pyrimidines and purines [1].
formans, and Mycobacterium tuberculosis.
In addition, toyocamycin has minor affects on some other
bacteria, fungi, or yeast microorganisms [6], tubercidin was known
to inhibit M. tuberculosis and S. faecalis [7].
Previous investigations have shown that the reactions of 4-
hydrazinopyrimidines with trimethylorthoformate (TMOF) and/or
trimethylorthoacetate (TMOA) (one carbon donor moieties) affor-
ded the crossponding isomeric triazolopyrimidines [8e19], which
are known to exhibit interesting pharmacological activities [20,21].
Motivated by these aforementioned findings, and in continu-
ance of our research efforts [22e25], we decided to prepare certain
novel pyrrole and pyrrolopyrimidine derivatives and evaluate them
for anti-inflammatory and antimicrobial activities.
For example Tolmetin (RumatolÒ) and Ketorolac (KetolacÒ)
(Fig. 2), nonsteroidal anti-inflammatory drugs (NSAIDs) [2] con-
taining pyrrole ring, are nonselective inhibitors of cyclooxygenase 1
and 2 enzymes that led to decrease prostaglandin synthesis.
Recently, a novel series of 3-amino-pyrrole derivatives (Fig. 1) were
synthesized [3] as lymphocyte-specific kinase (Lck) inhibitors,
a potential immunosuppressive agent for treatment of inflamma-
tory diseases such as rheumatoid arthritis, atopic dermatitis,
asthma, and organ transplant rejection.
PNU-142731A [4], (Fig. 1), is a potent and efficient pyrrolopyr-
imidine inhibitor of eosinophilic lung inflammation that is
currently in Phase II clinical evaluation for the potential treatment
of asthma.
2. Results and discussion
2.1. Chemistry
On the other hand, tubercidin, toyocamycin and sangivamycin
(Fig. 2) are naturally occurring pyrrolo[2,3-d]pyrimidine nucleoside
Compound Ia was prepared [25] by the reaction [26] of benzoin
with antipyrine amine (commercial available) and malononitrile in
non polar solvent and utilized for the preparation of pyrrole
derivatives Ibed using appropriate reagents and reaction condi-
tions: heating Ia with acetic anhydride afforded the corresponding
2-acetylamino Ib, while its reaction with triethyl orthoformate
* Corresponding author.
E-mail address: samarradwan1@yahoo.com (S.S. Fatahala).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.034

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
3023
to ibuprofen). Their activity profiles were the same as ibuprofen
(response increasing by time).
O
O
COOH
N
Compound IIa showed the opposite profile: it showed 63%
inhibition at 1hr post-carrageenan and 60% inhibition at 2 and 3 h
post-carrageenan then decreased to 27.5% inhibition at 4 h post-
carrageenan. Compound Ib exerted a stronger anti-inflammatory
effect than ibuprofen (66.5% inhibition at 3 h interval post-
carrageenan). Likewise, compounds IVa and VIIIa showed a signif-
icantly higher inhibitory action at the 3 h interval, especially,
compound VIIIa (90.8% inhibition). Compound VIIIa showed also
a stronger anti-inflammatory effect than ibuprofen, at 1st and 2nd
hours intervals (47.87% and 59.61% inhibition).
Compound V showed no activity at 1st and 2nd hours intervals
post-carrageenan injection. Yet, it exerted stronger action than
Ibuprofen at the 3rd hour post-carrageenan (73.34% inhibition).
Compounds IIb,c, IVb and VIIa showed weak activities 4 h after
carrageenan injection.
N
H3C
H3C
Tolmetin
Ketorolac
COOH
OMe
OPh
N
N
NC
N
O
N
N
N
N
H2N
COPh
3-aminopyrrole
PNU-142731A
To analyze structureeactivity relationships, three structural
components were considered: the nature of the heterocycle
nucleus, the nature of the side chain on the heterocycle system, and
the function of the side chain.
Fig. 1. Anti-inflammatory drugs (NSAIDs).
(TEOF) afforded the corresponding 2-ethoxy methylamino deriva-
tive Ic. On the other hand, careful hydrolysis [27] of Ia gave the
carboxamide Id, as revealed in Scheme A.
First, the influence of the nature of the aromatic heterocyclic
system was easily observed as pyrrolopyrimidine (IIa, IVa,V and
VIIIa) derivatives showed superior activity over pyrrole Iaed.
Regarding the side chain function, thione and hydrazino groups
conferred the greater activity over the amino or alkyl amino groups
and triazole ring system. While the thione group in V led to 73%
inhibition at 3rd hour and 64% inhibition at 4th hour post-
carrageenan injection, the amino group in IIc cause only 11%
inhibition at 3rd hour and 27.5% inhibition at 4th hour post-
carrageenan injection.
The derivative with the hydrazino group IVa showed 65% inhi-
bition at 3rd hour post-carrageenan injection. Also, cyano group
confers markedly but not significantly higher activity at the 4th
hour than the amide: while cyano compounds Ia showed 62%
inhibition 3rd hour and increased to 64% inhibition at 4th hour
post-carrageenan injection, the amide Id exerted 66.5% inhibition
at 3rd hour then decreased to 46.3% inhibition at 4th hour. The
presence of a carbonyl group produced an opposite profile to the
other groups: compound IIa showed 63e60% inhibition at 1ste3rd
hour post-carrageenan and reached 27.14% inhibition after 4 h
(activity decreased by time).
The pyrrole derivatives Iaed were converted to the corre-
sponding pyrrolo[2,3-d]pyrimidines IIaec via condensation with
formic acid, AcOH/HCl, NH4OH and/or formamide. Pyrrolo[2,3-d]
pyrimidine IIa was reacted with phosphorous oxychloride to
afford its corresponding 4-chloro derivative III which was subse-
quently reacted with hydrazine, aryl amines and thiourea to give
the corresponding 4-hydrazino IVa, 4-aryl amino IVbed and
pyrrolopyrimidin-4-thione V, respectively. It is worth mentioning
that compound IVa was also prepared via the condensation of
compound Ic with hydrazine, as revealed in Scheme B.
Both, 4-chloropyrrolopyrimidine III and its 4-hydrazino IVa
analog were used [28e30] to obtain the pyrrolo[3,2-e]tetrazolo[1,5-
c]pyrimidine VI, using sodium azide in acetic acid and/or sodium
nitrite in acetic acid, respectively, as revealed in Scheme C.
The condensation of 4-hydarzinopyrrolopyrimidine IVa with
formic acid or TEOF; acetic acid/acetic anhydride or triethyl
orthoacetate (TEOA) and CS2/KOH afforded pyrrolo[1,2,4]triazolo
[1,5-c]pyrimidines VIIaec, while reaction of compound IVa with
appropriate aldehydes in the presence of a catalytic amount of
glacial acetic acid afforded compounds VIIIaec [23,31,32], as
revealed in Scheme D.
Addition of the acetyl group in compound Ib leads to increased
activity (66% inhibition) at 3rd hour post-carrageenan injection
then to a decrease in activity (46.3% inhibition) at 4th hour post-
carrageenan injection. Also, addition of bulky groups (phenyl) to
hydrazine as in compound VIIIa contributes to produce the highest
activity (90.8% inhibition) at 3 h post-carrageenan overall tested
compounds (Fig. 3).
2.2. Biological results and discussion
2.2.1. Anti-inflammatory assay
The synthesized compounds were assessed for their anti-
inflammatory activity. As indicated in Table 1: compounds Ia and
Id induced a good anti-inflammatory activity, comparable with that
of ibuprofen (Ia was significantly difference at 3 h post-carrageenan
2.3. Antimicrobial assay
The synthesized compounds were tested in vitro against strains
of bacteria (Gram-positive and Gram-negative) and C. albicans as
fungi. Results indicated that compounds 1a, IIb and IIc were active
only against the Bacillus subtilis bacteria strain; while compound Ib
was active against the Staphylococcus aureus and B. subtilis strain.
However, all tested compounds were not as potent as the reference
drug (amoxicillin) and showed no activity against fungi (Table 2).
NH2
R
N
N
N
O
HO
R=
HO
2.4. Conclusion
O H
H
(Tubericidin)
CN
(Toyocamycin)
In the present study, we described a straightforward and effi-
cient synthesis of novel pyrrolo[2,3-d]pyrimidine and pyrrolo[1,2,4]
triazolo[1,5-c]pyrimidines asanti-inflammatory agents. The
CONH
(Sangivamycin)
2
Fig. 2. Pyrrolopyrimidine nucleoside antibiotics.

3024
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
R
CN
Ph
Ph
Ph
Ph
O
Ph
Ph
OH
2-4
1
Anti-NH2
+
Ph
R'
Ph
NH2
NH
OH
N
N
Anti
Anti
Anti
H3C
Anti=
Ia
I b-d
R
R'
b: CN
c: CN
d: CONH2 NH2
NHCOCH3
N=CHOEt
N
O
CH3
N
Ph
Reagents: 1=
CH2(CN)2
2= (CH3CO)2O 3= TEOF
4= NaOH/ EtOH
Scheme A. Synthesis of compounds Iaed.
structureeactivity relationship (SAR) results indicated that; the
pyrrole Ia containing amino and cyano groups displayed an anti-
inflammatory activity profile similar to that of ibuprofen. On
hydrolysis of the cyano group in Ia to carboxamide group in Id
didn’t enhance the activity. The introduction of hydrazine group to
IVa and thione group to V resulted to an increase in the anti-
inflammatory activity of the pyrrolopyrimidine analogs; and on
addition of phenyl group to compound VIIIa resulted in an
increased activity. These results and others demonstrated that the
synthesized pyrrole and pyrrolopyrimidine compounds are prom-
ising anti-inflammatory agents.
3. Experimental
3.1. Chemistry
All commercial chemicals used as starting materials and
reagents in this study were purchased from Merck (Darm-
stadt,Germany) and were of reagent grade. All melting points were
uncorrected and measured using Electro-thermal IA 9100 appa-
ratus (Shimadzu, Japan); IR spectra were recorded as potassium
bromide pellets on a PerkineElmer 1650 spectrophotometer (USA),
Faculty of Science, Cairo University, Cairo, Egypt. 1H- NMR spectra
X
H
R
Ph
Ph
N
1-3
R''
Ph
R'
N
N
Ph
N
a: X= O R''=H
b: X= O R''=CH3
c: X= NH R''= H
Anti
Ia-d
Anti
IIa-c
Cl
NHR
Ph
Ph
N
N
6
5
4
IIa
Ic
N
N
Ph
Ph
N
N
Anti
Anti
IV a-d
III
a: R= NH2
b: R= Ph
c: R= o-tolyl
d: R= 4-Cl-phenyl
7
S
H
N
Ph
H3C
N
Ph
N
Anti=
Anti
N
O
CH3
V
N
Ph
1 =
HCOOH
2 = AcOH/ HCl
5 = RNH2/EtOH
3 = NH3 or HCONH2
6 = N2H2 7 = NH2CSNH2
Reagents:
4 = POCl3
Scheme B. Synthesis of compounds IIeV.

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
3025
5.3 (br.s, 2H, NH2, D2O exchangeable), 7.0e7.8 (m, 15H, AreH); Anal.
Calcd for C28H23N5O (445.52): C, 75.49; H, 5.20; N, 15.72; O, 3.59%.
Found: C, 75.55; H, 5.22; N, 15.79, O, 3.65%.
N
N
N
1
III
Ph
N
3.1.2. N-(3-cyano-1-(2,4-dimethyl-5-oxo-1-phenyl-2,5-dihydro-
1H–pyrazol-3-yl)-4,5-diphenyl-1H-pyrrol-2-yl)-acetamide (Ib)
Compound Ia (4.45 g, 0.01 mol) in acetic anhydride (40 mL) was
refluxed for 2 h, cooled, poured onto ice water, neutralized with
ammonia to give a precipitate which was filtered off, dried, and
recrystallized from methanol, to give compound 1b. Yield: 76%;
N
Ph
N
Anti
2
IVa
VI
NaNO / AcOH
2=
Reagents:
NaN3/ AcOH
1=
2
M.P. 135e138 ꢀC; IR (KBr)
y
(cmꢁ1): 3330 (NH), 2230 (C^N), 1720,
1703 (C]O); MS (EI) m/z: 487 (Mþ, 33.1%), 488 (Mþþ1, 6.2%),410
Scheme C. Synthesis of compounds VI.
(Mþ, 100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.23 (s, 3H,
COeCH3), 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 7.0e7.8 (m, 15H,
AreH), 9.5 (s, 1H, NH, D2O exchangeable); Anal. Calcd. for
C30H25N5O2 (487.55): C, 73.90; H, 5.17; N, 14.36; O, 6.56. Found: C,
73.95; H, 5.20; N, 14.41, O, 6.70%.
were determined on a Varian Mercury (300 MHz) spectrometer
(Varian UK) and chemical shifts were expressed as ppm against
TMS as internal reference (Faculty of Science, Cairo University,
Cairo, Egypt). Mass spectra were recorded on 70 eV (EI Ms-QP 1000
EX, Shimadzu, Japan), Faculty of Science, Cairo University, Cairo,
Egypt. Microanalyses were operated using Vario, Elmentar appa-
ratus (Shimadzu, Japan), Organic Microanalysis Unit, Faculty
of Science, Cairo University, Cairo, Egypt. Column Chrom-
atography was performed on (Merck) Silica gel 60 (particle size
0.06e0.20 mm). All compounds prepared in this paper (Except; Ia
[25]) are new and confirmed with spectral data.
3.1.3. Ethyl N-3-cyano-1-(2,4-dimethyl-5-oxo-1-phenyl-2,5-
dihydro-1H–pyrazol-3-yl)-4,5-diphenyl-1H-pyrrol-2-yl
formimidate (Ic)
Compound Ia (4.45 g, 0.01 mol) in triethyl orthoformate
(30 mL) were refluxed for 6 h. The solvent was removed under
reduced pressure and the residue was recrystallized from meth-
anol/water to afford Ic. Yield: 66%; M.P. 120e122 ꢀC; IR (KBr)
y
(cmꢁ1): 3070, 2900(CH), 2310(CN), 1560(C]N), 1620 (C]C); MS
(EI) m/z: 501 (Mþ, 21.5%), 502 (Mþþ1, 3.81%), 314 (Mþ,100%); 1H
3.1.1. 2-Amino-1-(2,4-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H–
pyrazol-3-yl)-4,5-diphenyl-1H-pyrrole-3-carbonitrile (Ia)
NMR (DMSO-d6, 300 MHz)
d
(ppm): 1.30 (t, 3H, J ¼ 7.1 Hz, CH3),
A mixture of benzoin (2 g, 0.01 mol), antipyrine amine (2.03 g,
0.01 mol) in dry benzene (50 mL) was kept at 80 ꢀC for 6 h. The
reaction mixture was cooled, then malononitrile (0.66 mg,
0.01mol) was added, followed by catalytic amount of pyridine
portion wise and left to reflex till solid formed. The solvent was
evaporated under reduced pressure and the residue was recrys-
tallized from methanol to give of Ia. Yield: 36%; M.P. 145e148 ꢀC; IR
2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 4.2 (q, 2H, J ¼ 7.1 Hz, CH2),
6.9e7.8 (m, 16H, AreH and N]CH); Anal. Calcd. for C31H-27N5O2
(501.578): C, 74.23; H, 5.43; N, 13.96; O, 6.38%, Found: C, 74.52; H,
5.55; N, 14.11; O, 6.52%.
3.1.4. 2-Amino-1-(2,4-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-
pyrazol-3-yl)-4,5-diphenyl-1H-pyrrole-3-carboxamide (Id)
Compound Ia (4.45 g, 0.01 mol) was heated at reflux tempera-
ture in 20 ml of alcoholic sodium hydroxide solution (sodium
hydroxide 1.68 g, 7 ml water) for 4 h, the solvent was evaporated
(KBr)
y
(cmꢁ1): 3430, 3330 (NH2), 2230 (C^N),1700 (C]O); MS (EI)
m/z: 445 (Mþ, 25%), 446 (Mþþ1, 7.5%), 368 (Mþ, 100%); 1H NMR
(DMSO-d6, 300 MHz) d (ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3),
Scheme D. Synthesis of compounds VIIeVIII.

3026
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
Table 1
In vivo anti-inflammatory activity.
Compounds
Oedema induced by carrageenan (% oedema inhibition relative to control)
1 h
2 h
3 h
4 h
Swel ꢂSE
% inh
Swel ꢂSE
% inh
Swel ꢂSE
% inh
Swel ꢂSE
% inh
Ia
Ib
Id
IIa
IIb
IIc
IVa
IVb
IVc
0.208 ꢂ 0.034
0.226 ꢂ 0.035
0.182 ꢂ 0.044
0.086 ꢂ 0.003a,b
0.223 ꢂ 0.022
0.229 ꢂ 0.029
0.13 ꢂ 0.043
0.225 ꢂ 0.02
0.2238 ꢂ 0.023
0.324 ꢂ 0.014
0.2255 ꢂ 0.02
0.096 ꢂ 0.03
0.216 ꢂ 0.034
0.23 ꢂ 0.033
9.5
1.73
19.13
63.04
2.16
0.89
43.74
1.99
1.95
0
0.165 ꢂ 0.048
0.252 ꢂ 0.0415
0.192 ꢂ 0.0403
0.098 ꢂ 0.013a,b
0.245 ꢂ 0.029
0.254 ꢂ 0.037
0.14 ꢂ 0.051
36.53
3.07
26.15
60.57
5.37
2.35
46.15
5.11
5.17
0
0.122 ꢂ 0.04a,b
0.182 ꢂ 0.019a,b
0.28 ꢂ 0.08
62.62
66.54
47.8
60.01
13.22
11.4
65.07
13.03
13.33
73.34
9.9
0.178 ꢂ 0.076a
0.338 ꢂ 0.055
0.23 ꢂ 0.062a
0.468 ꢂ 0.062
0.458 ꢂ 0.093
0.466 ꢂ 0.016
0.245 ꢂ 0.0122
0.446 ꢂ 0.09
0.450 ꢂ 0.02
0.18 ꢂ 0.078a
0.432 ꢂ 0.019
0.332 ꢂ 0.071
0.192 ꢂ 0.012a
0.63 ꢂ 0.037
64.68
46.35
62.55
27.14
27.65
27.51
53.97
27.85
27.99
64.28
28.67
48.41
69.52
0.174 ꢂ 0.061a,b
0.471 ꢂ 0.065
0.482 ꢂ 0.072
0.14 ꢂ 0.051a,b
0.474 ꢂ 0.098
0.470 ꢂ 0.099
0.116 ꢂ 0.056a,b
0.487 ꢂ 0.092
0.04 ꢂ 0.028a,b
0.214 ꢂ 0.024a
0.544 ꢂ 0.081
0.250 ꢂ 0.0252
0.2489 ꢂ 0.0257
0.32 ꢂ 0.013
V
VIIa
VIIIa
Ibuprofen
Control
1.73
47.82
6.08
0.2407 ꢂ 0.024
0.04 ꢂ 0.02a,b
0.1425 ꢂ 0.031
0.26 ꢂ 0.049
4.92
59.61
45
90.8
60.66
Swel ¼ mean difference in rat paw volume between right and left paw. ꢂ S.E.
%inhibition ¼(1 ꢁ rt/rc) ꢃ 100 [rt ¼ swel. of tested group; rc ¼ swel. of control group].
a: Significantly different from control at the same time interval at p < 0.05.
b: Significantly different from ibuprofen at the same time interval at p < 0.05.
Swel ¼ swelling, SE ¼ Standard Error, %inh ¼ % inhibition, Anti ¼ Antipyrine.
under reduced pressure, and the residue was poured onto acidified
ice water to give a precipitate which was filtered, dried, and
recrystallized from ethanol to afford Id. Yield: 46%; M.P.
d (ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.9e7.8 (m, 15H,
AreH), 8.4 (s, 1H, C2eH), 12.40 (s, 1H, NH, D2O exchangeable).Anal.
Calcd. for C29H23N5O2 (473.19): C, 73.56; H, 4.90; N, 14.79; O, 6.56.
Found: C, 73.71; H, 4.98; N, 14.90; O, 6.85.
164e168 ꢀC; IR (KBr) (cmꢁ1): 3450, 3380 (NH2), 1710, 1690 (C]O),
y
and disappearance of the CN group; MS (EI) m/z: 463 (Mþ, 32.2%),
464 (Mþþ1, 5.1%), 386 (Mþ, 100%);1H NMR (DMSO-d6, 300 MHz)
3.1.6. 7-(2,4-Dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H–pyrazol-3-
yl)-2-methyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one
(IIb)
d
(ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.4 (s, 2H, NH2, D2O
exchangeable), 7.5 (s, 2H, CONH2, D2O exchangeable), 6.9e7.8 (m,
15H, Ar-H); Anal. Calcd. for C28H25N5O2 (463.53): C, 72.55; H, 5.44;
N, 15.11; O, 6.90%, Found: C, 72.62; H, 5.56; N, 15.18; O, 6.97%.
Method A: Compound Ia (4.45 g, 0.01 mol) in acetic acid/HCl
(3:1) (40 mL) was refluxed for 6 h. The reaction mixture was cooled,
poured onto ice water, neutralized with ammonia to give a precip-
itate which was filtered, dried, and recrystallized from methanol, to
3.1.5. 7-(2,4-Dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-3-
yl)-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (IIa)
Compound Ia or Id (0.01 mol) in formic acid (20 mL, 85%) was
refluxed for 8 h. The reaction mixture was cooled, poured onto ice
water to give a precipitate which was filtered, dried, and recrys-
tallized from ethanol to afford IIa. Yield: 76%, M.P. 172e176 ꢀC; IR
give compound IIb. Yield: 67%; M.P. 200e202 ꢀC; IR (KBr) (cmꢁ1):
y
3330 (NH),1730,1700 (C]O),1560 (C]N) and disappearance of the
CN group; MS (EI) m/z: 487 (Mþ, 45%), 488 (Mþþ1, 15.2%), 300 (Mþ,
100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.35 (s, 3H, C2eCH3),
2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.9e7.8 (m, 15H, AreH), 12.40
(s, 1H, NH, D2O exchangeable); Anal. Calcd. for C30H25N5O2
(487.55): C, 73.90; H, 5.17; N, 14.36; O, 6.56%. Found: C, 73.91; H,
5.22; N, 14.40, O, 6.65%.
(KBr)
y
(cmꢁ1): 3330 (NH), 1720, 1690 (C]O), 1560 (C]N) and
disappearance of the CN group; MS (EI) m/z: 473 (Mþ, 23.5%), 474
(Mþþ1, 7.1%), 286 (Mþ, 100%); 1H NMR (DMSO-d6, 300 MHz)
100
80
60
40
20
0
4 Hr
3 Hr
2 Hr
1 Hr
Tested Compounds
Fig. 3. Anti-inflammatory effect (% Inhibition) of the tested compounds compared to the reference drug (ibuprofen).

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
3027
Table 2
for 6 h. The solvent was removed under reduced pressure and the
Antimicrobial activity results (MIC
standard drugs.
mg/ml) of newly synthesized compounds with the
residues were recrystallized from methanol to give IVa. Yield: 76%;
M.P. 162e166 ꢀC; IR (KBr)
y
(cmꢁ1): 3430, 3330 (NH2), 3210 (NH),
1560 (C]N),1620 (C]C); MS (EI) m/z: 487 (Mþ, 42.5%), 488 (Mþþ1,
Compds &
Stander
MIC of the tested compounds (
m
g/ml) against
9.5%), 300 (Mþ, 100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.43
B. subtilis
S. aureus
E. coli
P. aeruginosa
C. albicans
(s, 3H, CH3), 3.12 (s, 3H, NeCH3), 4.9e5 (brs, 2H, NH2, D2O
exchangeable), 6.9e7.4 (m, 16H, AreH, NH, D2O exchangeable), 8.1
(s, 1H, C2eH); Anal. Calcd. for C29H25N7O (487.56): C, 71.44; H, 5.17;
N, 20.11; O, 3.28%, Found: C, 71.52; H, 5.24; N, 20.23; O, 3.38%.
Method B: Compound Ic (5.01 g, 0.01 mol) in dry benzene
(20 mL), hydrazine hydrate (5 mL, 0.015 mol, 98%) was added with
stirring at room temperature for 4 h. The solvent was removed
under reduced pressure, and the residue was recrystallized from
methanol to give IVa, Yield: 56%. Compound IVa prepared by this
method is identical in all respects (physical and spectral data) to
that prepared from Method A.
ATCC
6633
ATCC
29213
ATCC
25922
ATCC
278533
ATCC
10231
Ia
Ib
IIa
IIc
512
256
256
512
64
NT
256
NT
NT
1
NT
NT
NT
NT
256
NT
NT
NT
NT
NT
64
NT
NT
NT
NT
NT
512
Amoxacillin
Fluconazol
NT
NT
NT
All tested compound ¼ 20
m
g/disc ꢃ 30
mg/disc.
A (amoxicillin) ¼ 25
m
g mLꢁ1B (fluconaze) ¼ 4
m
g mLꢁ1NT ¼ Not tested.
N.B: Ic,d*, IIa,d*, III*, IV*, V*,VI*,VII* and VIII*, were inactive toward all tested
organisms in both used concentration.
Method B: Compound Ib (4.87 g, 0.01 mol) in acetic acid/HCl
(3:1) (40 mL) was refluxed for 5 h. The reaction mixture was cooled,
poured onto ice water, neutralized with ammonia to give a precip-
itate which were filtered, dried, and recrystallized from methanol,
to give compound IIb. Yield: 60%. Compound IIb prepared by this
method is identical in all respects (physical and spectral data) to
that prepared from Method A.
3.1.10. General procedure for the synthesis of compounds (IVbed)
A mixture of 4-chloro III (4.91 g, 0.01 mol), aryl amines
(0.01 mol) and triethylamine in a catalytic amount were refluxed in
dry ethanol (30 mL) for 8 h. The solvent was removed under
reduced pressure and the residue was washed with water, acidified
with conc. HCl, filtered, dried and recrystallized from methanol to
give IVbed.
3.1.7. 5-(4-Amino-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (IIc)
3.1.11. 5-(5,6-Diphenyl-4-(phenylamino)-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(IVb)
Method A: Compound Ia (4.45 g, 0.01 mol) and formamide
(30 mL, 0.066 mol) was heated at 110 ꢀC for 8 h. The reaction
mixture was cooled, poured onto ice water to give a precipitate,
which were filtered, dried, and recrystallized, from Methanol to
Yield: 75%; M.P. 162e166 ꢀC; IR (KBr)
y
(cmꢁ1): 3330 (NH), 1580
(C]N), 1630 (C]C); MS (EI) m/z: 548 (Mþ, 38.8%), 549 (Mþþ1,
6.65%), 471 (Mþ,100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.43
afford IIc. Yield: 70%; M.P. 152e156 ꢀC; IR (KBr)
y
(cmꢁ1): 3430,
(s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.4 (brs, 1H-, NH1, D2O
exchangeable), 6.9e7.6 (m, 20H, AreH) 8.2 (s, 1H, C2eH); Anal.
Calcd. for C35H28N6O (548.64): C, 76.62; H, 5.14; N, 15.32; O, 2.92,
Found: C, 76.75; H, 5.21; N, 15.61; O, 3.04.
3330 (NH2), 1560 (C]N), MS (EI) m/z: 472 (Mþ, 33.7%), 473 (Mþþ1,
4.91%), 455 (Mþ,100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.43
(s, 3H, CH3), 3.12 (s, 3H, NeCH3), 5.6 (brs, 2H, NH2, D2O
exchangeable), 6.9e7.4 (m, 15H, AreH,), 8.2 (s, 1H, C2eH); Anal.
Calcd for C29H24N6O (472.54): C, 73.71; H, 5.12; N, 17.78; O, 3.39%,
Found: C, 73.85; H, 5.20; N, 17.90; O, 3.41%.
Method B: Compound Ic (5.01 g, 0.01 mol) in dry ethanol
(20 mL), ammonium hydroxide solution (5 mL, 25%) was added
with stirring at 0 ꢀC for 30 min, the reaction mixture was left at
room temperature for 6 h. The solvent was removed under reduced
pressure and the residue was recrystallized from methanol to
afford IIc. Yield: 65%. Compound IIc prepared by this method is
identical in all respects (physical and spectral data) to that prepared
from Method A.
3.1.12. 5-(5,6-Diphenyl-4-(o-tolylamino)-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (IVc)
Yield: 68%; M.P. 162e168 ꢀC; IR (KBr) (cmꢁ1): 3330 (NH),
y
1560(C]N), 1610 (C]C); MS (EI) m/z: 562 (Mþ, 41.9%), 563 (Mþþ1,
9.5%), 471 (Mþ, 100%);1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.1 (s,
3H, aromatic CH3), 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.3 (brs,
1H-, NH1, D2O exchangeable), 6.9e7.6 (m, 19H, AreH,), 8.2 (s, 1H,
C2eH); Anal. Calcd for C36H30N6O (562.66): C, 76.85; H, 5.37; N,
14.94; O, 2.84%, Found: C, 76.08; H, 5.49; N, 15.06; O, 3.02%.
3.1.13. 5-(4-(4-Chlorophenylamino)-5,6-diphenyl-7H-pyrrolo[2,3-
d]pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(IVd)
3.1.8. 5-(4-Chloro-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (III)
Compound IIa (0.01 mol) was refluxed in phosphorus oxy-
chloride (30 mL) for 6 h. The solution was cooled and poured with
stirring onto ice and the formed precipitated was filtered, washed
several times with water, dried and recrystallized from ethanol to
afford III. Yield: 76%; orange brown solid; M.P. 184e188 ꢀC; IR (KBr)
Yield: 74%; M.P. 198e200 ꢀC; IR (KBr) (cmꢁ1): 3350 (NH), 1550
y
(C]N), 1620 (C]C); MS (EI) m/z: 583.20 (Mþ, 35Cl, 38.2%), 585.19
(Mþþ2, 37Cl,12.8%), 456 (Mþ, 100%);1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.3 (brs, 1H-, NH1,
D2O exchangeable), 6.9e7.8 (m,19H, AreH,), 8.3 (s,1H, C2eH); Anal.
Calcd. for C35H27ClN6O (583.08): C, 72.10; H, 4.67; Cl, 6.08; N, 14.41;
O, 2.74, Found: C, 72.38; H, 4.92; Cl, 6.18; N, 14.92; O, 2.89.
y
(cmꢁ1): 3080, 2840(CH), 1730 (C]O), 1612 (C]C), 1580 (C]N);
MS (EI) m/z: 491 (Mþ, 35Cl, 39.98), 493 (Mþþ2, 37Cl, 10%), 456 (Mþ,
100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.43 (s, 3H, CH3),
3.12 (s, 3H, NeCH3), 6.9e7.8 (m, 15H, AreH), 8.5 (s, 1H, C2eH).Anal.
Calcd. for C29H22ClN5O (491.15): C, 70.80; H, 4.51; Cl, 7.21; N, 14.24;
O, 3.25%, Found: C, 70.88; H, 4.56; Cl, 7.25; N, 14.30; O, 3.30%.
3.1.14. 5-(5,6-Diphenyl-4-thioxo-3H-pyrrolo[2,3-d]pyrimidin-
7(4H)-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (V)
Compound III (4.91g, 0.01 mol) and thiourea (1.2g, 0.02 mol)
was refluxed in dry ethanol (20 mL) for 8 h. The reaction mixture
was evaporated under reduced pressure and the residues were
recrystallized from methanol to give V. Yield: 80%; M.P. 170e174 ꢀC;
3.1.9. 5-(4-Hydrazinyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-7-
yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (IVa)
Method A: Compound III (4.91 g, 0.01 mol) and hydrazine
hydrate (8 mL, 0.015 mol, 98%) was refluxed in dry ethanol (30 mL)
IR (KBr) y
(cmꢁ1): 3350 (NH), 1625 (C]S); MS (EI) m/z: 489 (Mþ,
25%), 490 (Mþþ1, 8.91%), 430 (Mþ, 100%); 1H NMR (DMSO-d6,

3028
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
300 MHz)
d
(ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.9e7.8
3.1.18. 5-(2-Mercapto-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]
triazolo[1,5-c]pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (VIIc)
(m, 15H, AreH), 9.02 (s, 1H, C2eH). 12.20 (s, 1H, NH, D2O
exchangeable). Anal. Calcd. for C29H23N5S (489.59): C, 71.14; H,
4.74; N, 14.30; O, 3.27; S, 6.55, Found: C, 71.35; H, 4.90; N, 14.62; O,
3.55; S, 6.86.
To an alcoholic NaOH solution of compound IVa (4.87 g,
0.01 mol) in ethanol (20 mL), carbon disulfide (10 mL) was added
then the reaction mixtures were refluxed on a water-bath for 5 h.
The reaction mixture cooled, poured onto ice water, neutralized
with 2e3 drops of hydrochloric acid (35%), filtered, dried and
recrystallized from methanol to give VIIc. Yield: 66%, M.P.
3.1.15. 5-(8,9-Diphenyl-7H-pyrrolo[3,2-e]tetrazolo[1,5-c]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (VI)
Method A: Compound III (4.91g, 0.01 mol) and sodium azide
(0.23 gm, 0.02 mol) were stirred in glacial acetic acid (20 mL) at 70ꢀ
C for 4 h. The reaction mixture was cooled, poured onto ice water to
give precipitates which was filtered, dried, and recrystallized from
146e148 ꢀC; IR (KBr)
y
(cmꢁ1): 3090, 2890 (CH), 1580 (C]N), 1620
(C]C), 3340, 3460 (SH). MS (EI) m/z: 529 (Mþ, 13.1%), 530 (Mþþ1,
3.85%), 484 (Mþ,100%); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.36
ethanol to afford VI. Yield: 70%, M.P. 230e235 ꢀC; IR (KBr)
y
(cmꢁ1):
(s, 3H, C2eCH3), 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 4.5e4.8 (brs,
1H-, SH, D2O exchangeable), 6.9e7.6 (m, 15H, AreH), 9.50 (s, 1H,
C2eH); Anal. Calcd. for C30H23N7OS (529.61): C, 68.03; H, 4.38; N,
18.51; O, 3.02; S, 6.05, Found: C, 68.40; H, 4.54; N, 18.78; O, 3.27.
1520 (C]N), 1630 (C]C); MS (EI) m/z: 498 (Mþ, 41%), 499 (Mþþ1,
12.3%), 311 (Mþ,100%);1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.43
(s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.9e7.6 (m, 15H, AreH,), 9.56 (s,
1H, C2eH); Anal. Calcd. for C29H22N8O (498.19): C, 69.87; H, 4.45; N,
22.48; O, 3.21, Found: C, 69.99; H, 4.81; N, 22.85; O, 3.45.
Method B: Compound IVa (4.87g, 0.01 mol) and sodium nitrite
(0.69 gm, 0.01 mol dissolve in 5 ml H2O) were stirred in glacial acetic
acid (20 mL) at 40ꢀ C for 8 h. The reaction mixture was cooled,
poured onto ice water to give precipitates which were filtered,
dried, and recrystallized from ethanol to afford VI. Yield: 60%.
Compound 8 prepared by this method is identical in all respects
(physical and spectral data) to that prepared from Method A.
3.1.19. General procedure for the synthesis of compounds (VIIIaec)
Compound IVa (4.87 g, 0.01 mol), aldehyde (0.01 mol) in ethanol
(30 mL), and a catalytic amount of acetic acid (10 drops) were
heated under reflux for 6 h. The precipitate was filtered while hot,
washed with water several times and dried to give compounds
VIIIaec.
3.1.20. 5-(4-(2-Benzylidenehydrazinyl)-5,6-diphenyl-7H-pyrrolo
[2,3-d]pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-
one (VIIIa)
3.1.16. 5-(8,9-Diphenyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(VIIa)
Yield: 86%; M.P. 142e145 ꢀC; IR (KBr)
y
(cmꢁ1): 3350e3220
(broad NH), 1595 (C]N), 1620 (C]C); MS (EI) m/z: 575 (Mþ, 51.2%),
576 (Mþþ1, 20.8%), 387 (Mþ, 100%); 1H NMR (DMSO-d6, 300 MHz)
Method A: Compound IVa (4.87g, 0.01 mol) and triethyl
orthoformate (20 mL) was refluxed for 10 h. The solvent was
removed under reduced pressure and the residue was recrystal-
lized from methanol to give VIIa. Yield: 85%, M.P. 200e205 ꢀC; IR
d
(ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.70 (s, 1H, N]CH),
7.1e7.8 (m, 21H, AreH and NH, D2O exchangeable), 8.3 (s, 1H,
C2eH); Anal. Calcd. for C36H29N7O (573.65): C, 75.11; H, 5.08; N,
17.03; O, 2.78%, Found: C, 75.45; H, 5.29; N, 17.42; O, 2.86%.
(KBr)
y
(cmꢁ1): 3090, 2890 (CH), 1580 (C]N), 1630 (C]C); MS (EI)
m/z: 497 (Mþ, 32.2%), 498 (Mþþ1, 6.14%), 310 (Mþ, 100%);1H NMR
(DMSO-d6, 300 MHz)
d
(ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3),
3.1.21. 5-(4-(2-(4-Chlorobenzylidene)hydrazinyl)-5,6-diphenyl-7H-
pyrrolo[2,3-d]pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (VIIIb)
6.9e7.6 (m, 15H, AreH), 8.3 (s, 1H, C5eH), 9.26 (s, 1H, C2eH); Anal.
Calcd. for C30H23N7O (497.55): C, 72.42; H, 4.66; N, 19.71; O, 3.22,
Found: C, 72.95; H, 4.83; N, 19.96; O, 3.26.
Method B: Compound IVa (4.87g, 0.01 mol) was refluxed in
formic acid (20 mL, 85%) for 8 h. The reaction mixture was cooled,
poured onto ice water, filtered, dried, and recrystallized from
ethanol to afford to give VIIa. Yield: 66% Compound VIIa prepared
by this method is identical in all respects (physical and spectral
data) to that prepared from Method A.
Yield: 81%; M.P. 205e208 ꢀC; IR (KBr)
y
(cmꢁ1): 3350e3220
(broad NH), 1595 (C]N), 1620(C]C); MS (EI) m/z: 610.21 (Mþ, 35Cl,
39.3%), 612.20 (Mþ, 37Cl,13.3%), 497 (Mþ,100%); 1H NMR (DMSO-d6,
300 MHz) d (ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.70 (s,1H,
N]CH), 7.1e7.8 (m, 20H, AreH and NH, D2O exchangeable), 8.3 (s,
1H, C2eH); Anal. Calcd. for C36H28ClN7O (610.11): C, 70.87; H, 4.63;
Cl, 5.81; N, 16.07; O, 2.62%, Found: C, 70.99; H, 4.99; Cl, 5.93; N,
16.42; O, 2.87%.
3.1.17. 1,4-Dimethyl-5-(2-methyl-8,9-diphenyl-7H-pyrrolo[3,2-e]
[1,2,4]triazolo[1,5-c]pyrimidin-7-yl)-2-phenyl-1H-pyrazol-3(2H)-
one (VIIb)
3.1.22. 5-(4-(2-(3,4-Dimethylbenzylidene)hydrazinyl)-5,6-
diphenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-
1H-pyrazol-3(2H)-one (VIIIc)
Method A: Compound IVa (4.87g, 0.01 mol) and triethyl
orthoacetate (20 mL) was refluxed for 12 h. The solvent was
removed under reduced pressure and the residue was recrystal-
lized from methanol to give VIIb. Yield: 70%, M.P. 210e215 ꢀC; IR
Yield: 81%; M.P. 196e198 ꢀC; IR (KBr)
y
(cmꢁ1): 3350e3220
(broad NH), 1595 (C]N), 1620(C]C); MS (EI) m/z: 603 (Mþ, 42%),
604 (Mþþ1, 10.15%), 471 (Mþ, 100%); 1H NMR (DMSO-d6, 300 MHz)
(KBr)
y
(cmꢁ1): 3090, 2890 (CH), 1580 (C]N), 1630 (C]C); MS (EI)
d (ppm): 2.34e2.38 (s, 6H, CH3), 2.43 (s, 3H, CH3), 3.12 (s, 3H,
m/z: 511 (Mþ, 22%), 512 (Mþþ1, 7.7%), 324 (Mþ, 100%);1H NMR
NeCH3), 6.70 (s, 1H, N]CH), 7.1e7.8 (m, 19H, AreH and NH, D2O
exchangeable), 8.3 (s, 1H, C2eH); Anal. Calcd. for C38H33N7O
(603.71): C, 75.60; H, 5.51; N, 16.24; O, 2.65%, Found: C, 75.78; H,
5.56; N, 16.45; O, 2.86%.
(DMSO-d6, 300 MHz)
d (ppm): 2.36 (s, 3H, C2eCH3), 2.43 (s, 3H,
CH3), 3.12 (s, 3H, NeCH3), 6.9e7.6 (m, 15H, AreH), 9.54 (s, 1H,
C2eH); Anal. Calcd. for C31He25N7O (511.58): C, 72.78; H, 4.93; N,
19.17; O, 3.13, Found: C, 72.98; H, 4.99; N, 19.51; O, 3.26.
Method B: Compound IVa (4.87 g, 0.01 mol), acetic anhydride
(20 mL), and acetic acid (10 mL) were refluxed for 8 h. The reaction
mixture was cooled, poured onto ice water, filtered, dried, and
recrystallized from ethanol to afford VIIb. Yield: 80%. Compound
VIIb prepared by this method is identical in all respects (physical
and spectral data) to that prepared from Method A.
3.2. Anti-inflammatory activity
3.2.1. Animals
Seventy adult male SpragueeDawley rats (5 rats per group),
weighing 150e170 g, were housed at cages in a temperature-
controlled (25 ꢂ 1 ꢀC) environment and provided free access to

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 3022e3029
3029
pelleted food and purified drinking water ad libitum. The animal
experiments described below comply with the ethical principles
and guidelines for the care and use of laboratory animals adopted
by the National Egyptian Community.
The lowest concentration of the compound that completely
inhibits macroscopic growth was determined and minimum inhib-
itory concentrations (MICs) were reported. DMSO (SigmaeAldrich,
80%), pure microorganisms, and pure media were used as control
wells.
3.2.2. Assessment of anti-inflammatory activity
Rat paw edema assay was carried out according to Winter et al.
[33] Prepared compounds (equimolar to the reference drug) were
dissolved in DMSO and administrated subcutaneously. One hour
later, paw edema was induced by subplantar injection of 0.1 mL of
1% carrageenan (SigmaeAldrich, St. Louis, USA) into the right hind
paw. Paw volume was measured using a water plethysmometer
(Basile, Comerio, Italy). The difference between the right and left
paw volume was measured at 1, 2, 3 and 4 h after induction of
inflammation. Control group (five rats per group) received DMSO
subcutaneously and carrageenan in subplantar region. Results were
expressed as percentage inhibition of inflammation. Ibuprofen
(70 mg/kg) was used as the reference drug [34]
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Media for disc sensitivity tests were the nutrient agar and
MullereHinton agar (MHA) purchased from Difco (USA). Non-sterile
powder of the tested compound was dissolved in sterile DMSO to
Yield: 20 and 30
mg/mL, and passed through0.2 mm membrane filters
(Millipore Corp, USA). The filtrates were dispensed as 2 mL samples
into sterile, small screw-capped vials, frozen and kept stored
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reference substances.
MIC (minimum inhibitory concentration) for each tested
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micro dilution technique according to NCCLS guidelines 1997 [37].
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overnight at 37 ꢀC and all Candida isolates were sub cultured in SDA
plates at 35 ꢀC for 24e48 h. The microorganisms were passage at
least twice to ensure purity and viability. The solution of the newly
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512, 256, 128, 64, 32 and 16 mg/mL concentrations using serial two-
folds dilutions in DMSO (dimethyl sulphoxide), each concentration
was mixed with sterile nutrient agar (SigmaeAldrich) in sterile
plate, bacteria inoculums were added to each well of the micro
dilution trays. The trays were incubated at 37 ꢀC in a humid chamber
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