Synthesis and biological evaluation of some thio containing pyrrolo [2,3-d] Pyrimidine derivatives for their anti-inflammator

Article abstract of DOI:10.1016/j.ejmech.2010.03.028

The pyrroles Iaec were used as precursor for the preparation of pyrrolo [2, 3-d] pyrimidine-2 and/or 4 thione derivatives IIa-f. A series of 8-Aryl-pyrrolo [2, 3-d] thiazolo[3,2-a]pyrimidine VI and VII were prepared. Alkylation of the thione compounds in basic medium afforded the pyrrolo [2, 3-d] pyrimidine IV. Also, some 2-amino pyrrolo [2, 3-d]pyrimidines V were obtained. Some newly synthesizedcompounds were examined for their in vitro anti-inflammatory. Also, all new compounds were examined for their in vivo anti-microbial activity. Several derivatives, showed a promising anti-inflammatory activity in compared to ibuprofen. In this paper, we examine and discuss the structureeactivity relationships and anti-inflammatory activities of these compounds.

Full text of DOI:10.1016/j.ejmech.2010.03.028

European Journal of Medicinal Chemistry 45 (2010) 2994e3004
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of some thio containing pyrrolo [2,3-d]
Pyrimidine derivatives for their anti-inflammatory and anti-microbial activities
,
b
a
Mosaad S. Mohamed a , Rehab Kamel , Samar S. Fatahala
*
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Helwan, Egypt
b Toxicology and Pharmacology Department, Faculty of Pharmacy, Helwan University, Helwan, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The pyrroles Iaec were used as precursor for the preparation of pyrrolo [2, 3-d] pyrimidine-2 and/or 4
thione derivatives IIaef. A series of 8-Aryl-pyrrolo [2, 3-d] thiazolo[3,2-a]pyrimidine VI and VII were
prepared. Alkylation of the thione compounds in basic medium afforded the pyrrolo [2, 3-d] pyrimidine
Received 28 January 2010
Received in revised form
15 March 2010
Accepted 17 March 2010
Available online 25 March 2010
IV. Also, some 2-amino pyrrolo [2, 3-d]pyrimidines
V were obtained. Some newly synthesized
compounds were examined for their in vitro anti-inflammatory. Also, all new compounds were exam-
ined for their in vivo anti-microbial activity. Several derivatives, showed a promising anti-inflammatory
activity in compared to ibuprofen. In this paper, we examine and discuss the structureeactivity rela-
tionships and anti-inflammatory activities of these compounds.
Keywords:
Pyrrolo [2,3-d]pyrimidine
Anti-inflammatory activity
Anti-microbial activity
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
a well known non steroidal anti-inflammatory drugs (NSAIDs) [11]
which act by inhibition of prostaglandin synthesis constitutes the
The key roles purines and pyrimidines play in cellular processes
have made them valuable leads for drug discovery. One important
class of pyrimidine is 2-thiopyrimidine (2-TP) and its derivatives,
which are also well known as 2-mercaptopyrimidine compounds
[1]. In 2-TP ring sulfur atom serves as an interesting replacement
for the existing oxygen atom bonded to C-2 in uridine base [2].
Considering this assumption 2-TPs have attracted substantial
interest of synthetic-biochemists [3]. The European patent [4]
revealed the application of 2-TP derivatives in preparation of
cardiotonic drugs. Studies by Pathak et al. evaluated primary activity
of 2-TP derivatives against Mycobacterium tuberculosis (Mtb) [5].
6-Thiopurine (6 TP) [6] is a thio analog of hypoxanthine the
natural product of purine metabolism (Fig. 3).
Since the discovery of this antimetabolite more than half
a century ago, several thousand 6 TP derivatives have been synthe-
sized and characterized in biological experiments. Which proved to
be veryeffective for the treatment of leukemias [7] autoimmune and
rheumatic disorders, [7e10] and for immunosuppressant during
organ transplanta tion [7,8]. Pyrrolo[3,2-d]pyrimidines, a class of
7-deazapurine analogues, exhibit for instance interesting biological
activity in part due to their resemblance to pyrimidines and purines.
For example Tolmetin (RumatolÒ) and ketorolac (ketolacÒ) (Fig. 1),
primary mechanism of the anti-inflammatory action of these drugs.
PNU-142731A [12] (Fig. 1) is an anti-inflammatory, pyrrolopyr-
imidine that inhibits the production of cytokines in vivo. Tubercidin,
Toyocamycin and Sangivamycin (Fig. 3) are naturally occurring
pyrrolo [2,3-d]pyrimidine nucleoside antibiotics [13,14]. These
compounds (Fig. 2) have been observed to be active against the
growth of some micro-organisms (Fig. 4).
In our previous work; we have found [15e17] that the addition
of sulfur and/or ring-containing nitrogen allowed the activity to
increase in the direction of anti-microbial and anti-inflammatory.
Motivated by the abovementioned findings and in continuation of
our investigations in this field [15e17], we aimed to synthesize
novel derivatives of pyrrolopyrimidines and their thio related
compounds for study of the structure activity relationships. The
heterocyclic system pyrrole was selected on the basis of previous
reports [15e17].
2. Results and discussion
2.1. Chemistry
The synthetic strategy to synthesize the target sulfur
compounds II, III, IVeVII is depicted in Schemes 1e3. Compounds
[15] Iaec were used as key compound for this study and for further
syntheses due to the presence of the enaminonitrile moiety, which
is well known as a highly reactive and convenient reagent for the
* Corresponding author.
E-mail address: samarradwan1@yahoo.com (M.S. Mohamed).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.028

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
2995
NH2
R
N
N
HO
N
O
HO
Fig. 1. Anti-inflammatory drugs (NSAIDs).
OH
synthesis of nitrogen heterocycles [18,19]. Thus, the reaction of
Iaec with thiourea [20], and carbon disulfide [23,25,26] in DMF/
pyridine and/or NaOH/DMSO, afforded the corresponding pyrimi-
dine derivatives IIaef. Refluxing [20,22,27] of compounds Iaec
with phenyl isothiocyanate, in pyridine or DMF to give compound
IIgeh directly. Yet in stirring Iaec with phenyl isothiocyanate
without reflux, gave compounds IIIaec, which on heated at 80 ꢀC
afforded the expected compound IIgeh.
H
(Tubericidin)
R=
CN
(Toyocamycin)
CONH2 (Sangivamycin)
Fig. 3. Pyrrolopyrimidine nucleoside antibiotics.
than the reference drug. All the tested compounds have shown no
activity against fungi. All the other tested compounds showed no
anti-microbial activity. Test results of activity for compounds are
summarized in Tables 2, 3.
Alkylation [21e25] of Pyrrolopyrimidin-2-thiones IIaei with
a series of a-halocarbonyl compounds using 3 different methods,
gave the corresponding S-alkylated pyrimidine derivatives IVael.
The reaction [25,26] of S-alkylated compounds IVaef with certain
aromatic amine and/or hydrazine, independently, yielded the
2-amino derivative Vaef.
Using the same procedures as reported [23,25,26,28e30], that
heating under reflux a mixture of thio compounds IIdef, chloro-
acetic acid, aromatic aldehyde in acetic acid, and acetic anhydride
compounds VII were obtained in a good yield. When the reaction
was performed without aromatic aldehyde, the product was iden-
tified as thiazolopyrimidine derivative VI. Compound VII had been
independently synthesized through the interaction of VI with
aromatic aldehyde in the presence of a catalytic amount of piper-
idine or triethylamine.
2.2.3. Biological discussion
The potential therapeutic activity of these compounds was
assessed both for their anti-inflammatory activity. Ibuprofen was
used as reference compound for the in vivo assays.
The anti-inflammatory activity of these synthesized compounds
in vivo was evaluated using the carrageenan-induced rat paw
oedema assay. The potency and duration of action was compared
with those of the reference compound ibuprofen (Table 1).
Compounds IIb and Va have a stronger anti-inflammatory effect
in vivo than ibuprofen. In addition, the in vivo anti-inflammatory
activity was rapidly observed (2 h and 3 h) and sustained after 4 h,
whereas ibuprofen activity decreased quickly. Likewise,
compounds IIc, IIh and Vb, showed a higher inhibitory action at 2 h
and 3 h for the carrageenan-induced oedema than ibuprofen.
Compound Ic, showed the same behavior as the ibuprofen
(increased by time) and had same potency as it.
To analyze structureeactivity relationships, three structural
components were considered, the nature of the heterocycle
nucleus, the position of the side chain on the heterocycle system,
and the function of the side chain.
First, regarding the side chain function, thino group confers
greater activity than the dithieno or alkyl thieno: while IIb showed
% inh z 95 (2 h and 3 h post-carrageenan) the dithieno IIdef
presented % inh z 3e4.8 (2 h and 3 h post-carrageenan) were
completely lack of anti-inflammatory activity. Hydrazino group in
Va showed % inh z 79 (4 h post-carrageenan) and in Vb showed %
inh z 95 (3 h post-carrageenan), over the thiazolo ring in VII which
was completely in active.
2.2. Biological results and discussion
2.2.1. In vivo anti-inflammatory assay (Table 1)
The newly obtained compounds were tested for their anti-
inflammatory activity using the carrageenan-induced rat paw
oedema assay. The potency and duration of action was compared
with those of the reference compound ibuprofen. The vivo anti-
inflammatory activity is shown in Table 1.
2.2.2. Anti-microbial assay
All of the newly obtained compounds were tested in vitro
against a number of bacteria (including Gram-positive and Gram-
negative) and Candida albicans as fungi. As shown in the result all
tested compound show very poor activity as anti-microbial agent.
Compound IIa (2-thio-pyrimidine) has shown the highest activity
against two types of bacteria (Staphylococcus aureus and Bacillus
subtilis). Compounds Ic and Va have shown the activity against
B. subtilis only. Only one of the tested compounds was more potent
100
80
R'
S
N
60
N
R
40
4hr
3hr
2hr
1hr
N
N
20
0
R''
R=R'=R''=H
R=NH2 R'=R''=H
(Mercaptopurine)
(thioguanine)
RF
Ic
Active compound compared to Ibuprophen
IIb
IIc
II h
Va
Vb
R=R''=H R'= (CH2)4COOH
(buthiopurine)
Fig. 4. Anti-inflammatory effect of tested compound, show that IIb, IIh, Va, and Vb
Fig. 2. 6-Thiopurine (6 TP).
have highest activity (more potent than the reference drug (RF)).

2996
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
Y
S
S
N
O
Ph
Ph
CN
Ph
H
N
Ph
NR
1-3
1
N
S
z
z
NH2
S
N
H
z
N
S
N
N
N
z
N
H
Ar
Ia-c
Ar
II a-i
Ar
Ar
II d-f
VI a-c
H3C
3 (Y: NH , R: Ph)
3
Anti=
N
Ph
CN
O
N
CH3
4
S
N
O
N
Ph
H
Ph
2
z
NHCSNHPh
N
z
Ph
Ar
III a-c
S
N
Ar= benzyl, 3,4-(Cl) C H , Anti
Ar
1= H2NCSNH2
2= CS2/NaOH
VII a-d
2
6
3
3= PhNCS/ Reflux 4=PhNCS/Stirring
R, Z= H, C H Y = S, NH
6
5
1= ClCH COOH/ Ac O/AcOH
2= ClCH COOH/Ar'CHO/ Ac O/AcOH
2
2
2
2
Scheme 1.
3= Ar'CHO/ EtOH
Scheme 3.
The influence of the nature of the aromatic heterocyclic system
easily observed as pyrrolopyrimidine (IIb, IIc, IIh, Va, Vb)
condensed ring showed the highest activity over open pyrrole Iaec
and thiazolo fused ring VIeVII. Also the 3, 4-dichlorophenyl (IIb,
IIh Vb) showed highest activity than benzyl (IIa, Va) and antipyrine
in IIc. Where antipyrine Ic increase the activity over the other 2
aromatic ring in open pyrrole form as shown in Ia, Ib.
The introduction of thino group at position 2 rather than 2 and 4
increase the activity (IIb over IIdef). Also N3-phenyl in compound
IIh showed the activity (% inh z 42 (1 h post-carrageenan)) from
first ably over all tested compounds.
recorded as potassium bromide pellets on a PerkineElmer 1650
spectrophotometer (USA), Faculty of Science, Cairo University,
Cairo, Egypt. 1H NMR spectra 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 apparatus (Shimadzu, Japan), Organic Microanal-
ysis Unit, Faculty of Science, Cairo University, Cairo, Egypt and the
results were within the accepted range (ꢁ0.40) of the calculated
values. Column Chromatography was performed on (Merck) Silica
gel 60 (particle size 0.06e0.20 mm).
2.2.4. Conclusion
We have synthesized and evaluated a series of heterocyclic
compounds as potential anti-inflammatory agents. Based on their
structure, we conclude that the best aromatic nucleus is the pyrrole
with an 3,4-dichlorophenyl ring substituent and thino or hydrazine
subunit on the C-2 such as in compound IIb and Va,b. From the
biological tests, this group of pyrrole was a promising as anti-
inflammatory agent while can say that they deprived from their
anti-microbial activity.
3.2. 4-Amino-7-(Aryl)-5,6-disubstitututed-1H-pyrrolo[2,3-d]
pyrim-idine-2(7H)-thione (IIaec)
A mixture of amino cyano I (0.01 mol) and thiourea (0.02 mol)
was refluxed in dry ethanol (20 mL) for 6 h. Then evaporated under
reduced pressure and the residues were recrystallized from
methanol to give IIaec.
3. Experimental
3.1. General methods
3.2.1. 4-Amino-7-benzyl-5, 6-diphenyl-1H-pyrrolo [2, 3-d]
pyrimidine-2(7H)-thione (Ia)
All melting points are uncorrected and measured using Electro-
thermal IA 9100 apparatus (Shimadzu, Japan). IR spectra were
Yield 76%, m.p. 188e190 ꢀC. IR (KBr) (cmꢂ1): 3430, 3330 (NH2),
y
1620 (C]S), absence of (C^N). m/z: 408 (Mþ, 30.0%), 409 (Mþ þ 1,
R'
R'
Y
Ph
H
N
Ph
N
Ph
2
N
1
R'=NH
2
NHNH2
SR
S
N
z
N
N
z
N
z
N
N
H
Ar
V a-c
Ar
IV a-l
Ar
II a-f
1= RX/ Base
R= CH , C H
2= NH NH /EtOH
2 2
R'= SCH ,SC H , NH
2
3
2
5
3
2
5
Scheme 2.

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
2997
9.3%), 410 (Mþ þ 2, 1.2%). 1H NMR (DMSO, 500 MHz)
d
(ppm): 5.22
35Cl, 100.0%), 403 (Mþ þ 1, 21.2%), 404 (Mþ þ 2, 37Cl, 73.0%). 1H NMR
(s, 2H, CH2), 6.0 (br.s, 2H, NH2, D2O exchangeable), 7.0e7.8 (m, 15H,
Ar-H), 9.20 (s,1H, NH, D2O exchangeable). Anal. Calcd for C25H20N4S
(408.52): C, 73.50; H, 4.93; N,13.71; S, 7.85. Found: C, 73.86; H, 4.99;
N, 13.98; S, 8.05
(DMSO, 500 MHz) d (ppm): 7.1e7.8 (m, 8H, Ar-H), 8.0 (s, 1H, C6eH),
9.11 (s, 1H, NH, N1, D2O exchangeable), 10.86 (s, 1H, NH, N3, D2O
exchangeable). Anal. Calcd for C18H11Cl2N3S2 (402.98): C, 53.47; H,
2.74; Cl, 17.54; N, 10.39; S, 15.86. Found: C, 53.58; H, 3.01; Cl, 17.68;
N, 10.45; S, 15.01.
3.2.2. 4-Amino-7-(3,4-dichlorophenyl)-5-phenyl-1H-pyrrolo[2,3-d]
pyram-idine-2(7H)-thione (IIb)
3.3.3. (5, 6-diphenyl-2, 4-dithioxo-3, 4-dihydro-1H-pyrrolo[2,3-d]
pyrimi-din-7(2H)-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-
one (IIf)
Yield 74%, m.p. 280e282 ꢀC. IR (KBr) (cmꢂ1): 3420, 3350 (NH2),
y
1610 (C]S), absence of (C^N), m/z: 386 (Mþ, 35Cl, 38.2%), 387
(Mþ þ 1, 20.4%), 388 (Mþ þ 2, 37Cl, 12.8%), 390 (Mþ þ 4,
Yield (A) 75%, (B) 85%, and (C) 76%, m.p. 135e138 ꢀC. IR (KBr)
y
2 ꢃ (37Cl),4.28%). 1H NMR (DMSO, 500 MHz)
d
(ppm): 6.1 (br.s, 2H,
(cmꢂ1): 3420, 3380 (NH), 1680 (C]O), 1620 (C]C), absence of
NH2, D2O exchangeable), 7.0e7.8 (m, 8H, Ar-H), 8.1 (s, 1H, C6eH),
9.20 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C18H12Cl2N4S
(386.29): C, 55.82; H, 3.12; Cl, 18.31; N, 14.47; S, 8.28. Found: C,
56.03; H, 3.42; Cl, 18.62; N, 14.73; S, 8.42.
(C^N). m/z: 521 (Mþ, 15%), 522 (Mþ þ 1, 4.7%), 523 (Mþ þ 2, 1.51%).
1H NMR (DMSO, 500 MHz)
d
(ppm): 1H NMR (DMSO, 500 MHz)
d
(ppm): 2.43 (s, 3H, CH3), 3.24 (s, 3H, NeCH3), 6.7e7.8 (m, 15H,
Ar-H), 9.52 (s, 1H, NH, N1, D2O exchangeable), 11.80 (s, 1H, NH, N3,
D2O exchangeable). Anal. Calcd for C29H23N5OS2 (521.66): C, 66.77;
H, 4.44; N,13.43; O, 3.07; S,12.29. Found: C, 66.59; H, 4.28; N,13.58;
O, 2.98; S, 12.00.
3.2.3. 5-(4-Amino-5,6-diphenyl-2-thioxo-1H-pyrrolo[2,3-d]
pyrimidin-7(2H)-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(IIc)
Yield 74%, m.p. 170e172 ꢀC. IR (KBr)
y
(cmꢂ1): 3410, 3330 (NH2),
3.4. General method for preparation of II gei
1630 (C]S), absence of (C^N). m/z: 504 (Mþ, 70.0%), 505 (Mþ þ 1,
22.17%), 506 (Mþ þ 2, 3.73%).1H NMR (DMSO, 500 MHz)
d
(ppm): 2.43
Method A: A mixture of I (0.01 mol) and phenyl isothiocyanate
(0.01 mol) was dissolved in pyridine. The reaction mixture was
refluxed in an oil bath for 12 h, then cooled and poured into ice
bath. The product was precipitated, filtered, washed with acidi-
fied water, dried and recrystallized from ethanol/H2O to give II
gei.
(s, 3H, CH3), 3.12 (s, 3H, NeCH3), 6.3 (br.s, 2H, NH2, D2Oexchangeable),
7.0e7.8 (m,15H, Ar-H), 9.35 (s,1H, NH, D2O exchangeable). Anal. Calcd
for C29H24N6OS (504.61): C, 69.03; H, 4.79; N, 16.65; O, 3.17; S, 6.35,
Found: C, 69.40; H, 4.92; N, 16.81; O, 3.25; S, 6.59.
3.3. General method for preparation of IIdef
Method B: A mixture of I or III (0.01 mol) and phenyl iso-
thiocyanate (0.01 mol) was dissolved in dimethyl formamide
(25 mL) and 10% aqueous KOH (5.6 mL, 0.01 mol) was added over
period of time not exceed 30 min with stirring at 90 ꢀC for 12 h and
then diluted with water. The precipitate formed was filtered off,
was washed with acidified water, dried, and recrystallized from
ethanol/H2O to give II gei. Compound II hei prepared by Methods
A and B have the identical m.p. and mixed m.p.
Method A: A mixture of I (0.02 mol) and carbon disulfide (5 mL)
in dry pyridine (15 mL) was heated under reflux on a steam bath for
24 h. The solid product that separated after cooling was collected
and recrystallized from ethanol/H2O to give IIdef as orange yellow
needles.
Method B: A mixture of I (0.001 mol), carbon disulfide (6 mL), in
dimethyl sulphoxide(25 mL)and 10%aqueous KOH(5.6mL, 0.01 mol)
was added over period of time not exceed 30 min with stirring at ice
bath, was stirred at r.t. for 24 h and then diluted with water. The
precipitate formed was filtered off, was washed with water,acidified
with HCl, dried, and recrystallized from ethanol/H2O to give IIdef.
Method C: A mixture of I (0.01 mol) and excess carbon disulfide
(7 mL) in ethanolic sodium hydroxide solution (0.01 mol in 20 mL of
ethanol) was heated at 80 ꢀC with stirring for 12 h. The reaction
mixture was allowed to cool to r.t., stand allover the night, poured
into water, and neutralized by diluted HCl. The solid product
obtained was filtered off, dried and crystallized from ethanol/H2O
to give IIdef.
3.4.1. 7-Benzyl-4-imino-3,5,6-triphenyl-3, 4-dihydro-1H-pyrrolo
[2,3-d]pyrimidine-2(7H)-thione (II g)
Yield (A) 70% and (B) 75%, m.p. 112e115 ꢀC. IR (KBr)
y
(cmꢂ1):
3380 (NH), 1620(C]NH), absence of (C^N). m/z: 484 (Mþ, 21%),
485 (Mþ þ 1, 7.8%), 486 (Mþ þ 2, 2.5%), 1H NMR (DMSO,
500 MHz)
d (ppm): 5.92 (s, 2H, CH2), 6.8e7.7 (m, 20H, Ar-H), 9.2
(s, 1H, NH (pyrimidine), D2O exchangeable), 10.85 (s, 1H, NH,
C]NH, D2O exchangeable). Anal. Calcd for C31H24N4S (484.61): C,
76.83; H, 4.99; N, 11.56; S, 6.62. Found: C, 77.04; H, 5.12; N, 11.98;
S, 6.82
Compounds IIdef prepared by Methods A, B and C have the
identical m.p. and mixed m.p.
3.4.2. 7-(3,4-dichlorophenyl)-4-imino-3,5-diphenyl-3,4-dihydro-
1H-pyrrolo [2,3-d]pyrimidine-2(7H)-thione (II h)
Yield (A) 71% and (B) 74%, m.p. 260e262 ꢀC. IR (KBr)
y
(cmꢂ1):
3.3.1. 7-Benzyl-5,6-diphenyl-1H-pyrrolo [2, 3-d] pyrimidine-2,4
(3H, 7H)-dithione (IId)
3390 (NH), 1630(C]N), absence of (C^N). m/z: 462.05 (Mþ, 35Cl,
100.0%), 463 (Mþ þ 1, 26.9%), 464 (Mþ þ 2, 37Cl, 68.5%), 466
Yield (A) 70%, (B) 75%, and (C) 70%, m.p. 98e102 ꢀC. IR (KBr)
y
(Mþ þ 4, 2(37Cl), 13.6%). 1H NMR (DMSO, 500 MHz)
d (ppm):
(cmꢂ1): 3380 (NH), 1620(C]S), absence of (C^N). m/z: 425 (Mþ,
6.8e7.8 (m, 13H, Ar-H), 8.0 (s, 1H, C6eH), 9.21 (s, 1H, NH (pyrim-
idine), D2O exchangeable), 10.80 (s, 1H, NH, C]NH, D2O
exchangeable). Anal. Calcd for C24H16Cl2N4S (462.38): C, 62.21; H,
3.48; Cl, 15.30; N, 12.09; S, 6.92. Found: C, 62.52; H, 3.59; Cl, 15.68;
N, 12.28; S, 7.01.
12%), 426 (Mþ þ 1, 3.5%), 427 (Mþ þ 2, 2.1%).1H NMR (DMSO,
500 MHz)
d (ppm): 5.90 (s, 2H, CH2), 7.1e7.7 (m, 15H, Ar-H), 9.2
(s, 1H, NH, N1, D2O exchangeable), 10.85 (s, 1H, NH, N3, D2O
exchangeable). Anal. Calcd for C25H19N3S2 (425.57): C, 70.56; H,
4.50; N, 9.87; S, 15.07. Found: C, 70.64; H, 4.82; N, 9.98; S, 15.35.
3.4.3. 5-(4-Imino-3,5,6-triphenyl-2-thioxo-3,4-dihydro-1H-pyrrolo
[2,3-d] pyr-imidin-7(2H)-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3
(2H)-one (II i)
3.3.2. 7-(3, 4-dichlorophenyl)-5-phenyl-1H-pyrrolo [2,3-d]
pyrimidine-2,4 (3H, 7H)-dithione (IIe)
Yield (A) 70%, (B) 80%, and (C) 74%, m.p. 280e282 ꢀC. IR (KBr)
y
Yield (A) 71%, (B) 81%, m.p. 92e94 ꢀC. IR (KBr) (cmꢂ1): 3420,
y
(cmꢂ1): 3390 (NH), 1630(C]S), absence of (C^N). m/z: 402 (Mþ,
3380 (NH2), 3360 (NH), 1680 (C]O), 1620(C]C), absence of (C^N).

2998
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
Table 1
In vivo anti-inflammatory activity
Compounds
Oedema induced by carrageenan (% oedema inh. relative to control)
1 h
2 h
3 h
4 h
Swel
% inh
Swel
% inh
Swel
% inh
12.59
Swel
% inh
23.8
CN
Ph
0.223
3.04
0.249
4.23
0.472
0.48
NH2
Ph
N
Ia
Bez
CN
Ph
NH
0.225
0.208
0.226
2.17
0.228
0.038*
0.25
12.3
36.53
3.84
0.465
13.88
62.62
13.97
0.51
19.04
64.68
24.76
2
N
Ib
Ar
CN
Ph
NH2
9.5
0.122**
0.178*
Ph
N
Ic
Anti
H2N
Ph
N
S
1.73
0.468
0.474
N
Ph
N
H
Bez
IIa
H2N
Ph
N
S
0.216
6.08
0.012**
95.38
0.024***
95.95
0.202*
76.82
N
N
H
Ar
II b
NH2
Ph
N
S
0.32
0
0.32
0
0.116***
73.34
0.18*
64.28
N
H
Ph
N
II c
Anti
S
H
N
Ph
S
0.224
2.608
0.246
5.38
0.472
13.23
0.456
27.61
N
Ph
N
H
IId
Bez

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
2999
Table 1 (continued)
Compounds
Oedema induced by carrageenan (% oedema inh. relative to control)
1 h
2 h
3 h
4 h
Swel
% inh
Swel
% inh
Swel
% inh
12.53
Swel
% inh
S
Ph
NH
S
0.226
1.73
0.26
0.28
0.249
4.48
0.5
0.52
26
N
N
H
IIe
Ar
S
Ph
NH
S
0.23
0
3.94
0.451
14.1
0.501
28.8
N
Ph
N
H
IIf
Anti
HN
Ph
S
Ph
N
0.224
1.98
5.08
0.470
13.03
0.441
27.98
N
H
IIi
Ph
N
Bez
HN
Ph
Ph
N
S
0.172
42
0.135
48.07
0.022**
76.83
0.198*
68.57
N
H
N
IIh
Ar
H2N
Ph
N
NHNH2
0.206
10.43
0.101
61.15
0.142***
73.9
0.132**
79.04
N
Ph
N
Va
Bez
H2N
Ph
N
NHNH2
0.196
14.78
0.182
30
0.022***
95.58
0.282
67.43
N
N
Vb
Ar
O
S
CHPh
Ph
N
S
0.222
3.498
0.241
7.31
0.49
9.92
0.55
12.69
N
H
Ph
N
Bez
VIIa
(continued on next page)

3000
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
Table 1 (continued)
Compounds
Oedema induced by carrageenan (% oedema inh. relative to control)
1 h
2 h
3 h
4 h
Swel
% inh
Swel
% inh
Swel
% inh
17.1
Swel
% inh
20.47
O
S
CHPh
Ph
N
S
0.229
0
0.239
8.07
0.451
0.501
N
N
H
Ar
VIIb
O
S
CHPh
Ph
N
S
0.226
1.74
6.08
0.249
4.23
0.49
9.92
0.441
28.57
69.52
N
H
Ph
N
Anti
VIIc
Ibuprophen
Control
0.216
0.23
0.14
0.26
45
0.214**
0.544
60.66
0.192*
0.63
Swel ¼ the mean difference of tested compound and control.
% inhibition ¼ (1 ꢂ rt/rc) ꢃ 100 [rt ¼ result of tested group; rc ¼ result of control group].
* indicate compound is significantly difference compared to the tested drug. As indicated: *P < 0.05; **P < 0.01; ***P < 0.001.
Swel ¼ swelling; % inh ¼ % inhibition; Bez ¼ CH2Ph; Ar ¼ 3,4-Cl2C6H3; Anti ¼ Antipyrine.
m/z: 580 (Mþ, 15%), 581 (Mþ þ 1, 5.2%), 582 (Mþ þ 2, 1.1%). 1H NMR
3.5.1. 1-(1-Benzyl-3-cyano-4,5-diphenyl-1H-pyrrol-2-yl)-3-
phenylthiourea (IIIa)
(DMSO, 500 MHz)
d (ppm): 2.43 (s, 3H, CH3), 3.24 (s, 3H, NeCH3),
6.7e7.8 (m, 20H, Ar-H), 9.21 (s, 1H, NH (pyrimidine), D2O
exchangeable), 10.80 (s, 1H, NH, C]NH, D2O exchangeable). Anal.
Calcd for C35H28N6OS (580.70): C, 72.39; H, 4.86; N,14.47; O, 2.76; S,
5.52. Found: C, 72.65; H, 5.08; N, 14.78; O, 2.98; S, 5.78.
Yield 60%, m.p. 96e100 ꢀC. IR (KBr) (cmꢂ1): 3390 (NH), 3350
y
(NH), 2250 (C^N), 1620(C]N). m/z: 484 (Mþ, 10%), 485 (Mþ þ 1,
3.8%), 486 (Mþ þ 2, 1.2%). 1H NMR (DMSO, 500 MHz)
d (ppm): 5.92
(s, 2H, CH2), 6.8e7.7 (m, 20H, Ar-H), 11.56 (s, 1H, NH, D2O
exchangeable), 12.25 (s, 1H, NH, S¼CeNH, D2O exchangeable).Anal.
Calcd for C31H24N4S (484.61): C, 76.83; H, 4.99; N, 11.56; S, 6.62.
Found: C, 77.06; H, 5.22; N, 11.78; S, 6.80.
3.5. General method for preparation of III aec
3.5.2. 1-(3-Cyano-1-(3,4-dichlorophenyl)-4-phenyl-1H-pyrrol-2-
yl)-3-phenyl thiourea (IIIb)
A mixture of I (0.01 mol) and phenyl isothiocyanate (0.01 mol)
was dissolved in dimethyl formamide (25 mL) and 10% aqueous
KOH (5.6 mL, 0.01 mol) was added over period of time not exceed
30 min with stirring at 50 ꢀC for 24 h and then diluted with
water. The precipitate formed was filtered off, was washed with
acidified water, dried, and recrystallized from ethanol to give III
aec.
Yield 60%, m.p. 92e96 ꢀC. IR (KBr)
y
(cmꢂ1): 3390 (NH), 3350
(NH), 2230 (C^N), 1620(C]N). m/z: 463 (Mþ, 35Cl, 100.0%), 463
(Mþ þ 1, 26.9%), 465 (Mþ þ 2, 37Cl, 68.5%). 1H NMR (DMSO,
500 MHz) d (ppm): 6.8e7.8 (m, 13H, Ar-H), 8.02 (s, 1H, C6eH), 11.52
(s, 1H, NH, D2O exchangeable), 12.45 (s, 1H, NH, S¼CeNH, D2O
exchangeable). Anal. Calcd for C24H16Cl2N4S (463.38): C, 62.21; H,
3.48; Cl, 15.30; N, 12.09; S, 6.92. Found: C, 62.58; H, 3.69; Cl, 15.55;
N, 12.42; S, 7.0.
Table 2
Antibacterial and antifungal in vitro activity expressed as diameter of growth
inhibitory area.
Strain
Disc diffusion test (mm)
Table 3
Compounds and reference drugs
Anti-microbial activity results (MIC
the standard drugs.
mg/ml) of newly synthesized compounds with
Ic
IIa
IId
A
B
C. albicans ATCC 10231
P. aeruginosa ATCC 278533
M. phlei ATCC 10142
E. col ATCC 25922
S. aureus ATCC 29213
B. subtilis ATCC 6633
e
e
e
e
e
e
e
e
e
e
8
e
20
e
e
e
e
e
Compds & MIC of the tested compounds (
m
g/ml) against
e
12
e
Stander
B. subtilis S. aureus
E. coli P. aeruginosa C. albicans
e
ATCC 6633 ATCC 29213 ATCC 25922 ATCC 278533 ATCC 10231
e
13
13
12
16
25
Ic
IIa
IId
512
512
512
NT
256
NT
16
NT
NT
NT
256
NT
NT
NT
NT
64
NT
NT
NT
NT
NT
512
10
All tested compound ¼ 20
m
g/disc ꢃ 30
mg/disc.
Amoxacillin 64
Fluconazol NT
A (amoxicillin) ¼ 25
m
g mLꢂ1; B (fluconaze) ¼ 4
m
g mLꢂ1 e inactive compound.
NT
N.B: Ia,b, IIaec, IIeej*, II, IV*, V*, and VI*, were inactive towards all tested organisms
in both used concentration.
NT [ Not tested.

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
3001
3.5.3. 1-(3-Cyano-1-(2,4-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-
pyrazol-3-yl)-4,5-diphenyl-1H-pyrrol-2-yl)-3-phenylthiourea (IIIc)
3.6.4. 7-Benzyl-2-(ethylthio)-5, 6-diphenyl-7H-pyrrolo [2, 3-d]
pyrimidin-4-amine (IVd)
Yield 81%, m.p. 75e78 ꢀC. IR (KBr)
y
(cmꢂ1): 3380 (NH), 2235
Yield 80%, m.p. 172e176 ꢀC. IR (KBr)
y
(cmꢂ1): 3350 (NH), 1625
(C^N), 1690 (C]O), 1620(C]N). m/z: 580 (Mþ, 20%), 581
(Mþ þ 1, 7.64%), 582 (Mþ þ 2, 1.8%). 1H NMR (DMSO, 500 MHz)
(C]C). m/z: 436 (Mþ, 44%), 437 (Mþ þ 1, 12.98%), 438 (Mþ þ 2,
2.28%).1H NMR (DMSO, 500 MHz)
d
(ppm): 1.53 (t, 3H, J ¼ 7.2 Hz,
d
(ppm): 1H NMR (DMSO, 500 MHz)
d
(ppm): 2.43 (s, 3H, CH3),
CH3), 2.43 (q, 2H, J ¼ 7.2 Hz, CH2), 5.98 (s, 2H, CH2), 6.58 (s, 2H, NH2,
D2O exchangeable), 6.9e7.8 (m, 15H, Ar-H). Anal. Calcd for
C27H24N4S (436.57): C, 74.28; H, 5.54; N, 12.83; S, 7.3; Found: C,
74.51; H, 5.68; N, 13.10; S, 7.56.
3.24 (s, 3H, NeCH3), 6.7e7.8 (m, 20H, Ar-H), 11.21 (s, 1H, NH, D2O
exchangeable), 12.28 (s, 1H, NH, S¼CeNH, D2O exchangeable).
Anal. Calcd for C35H28N6OS (580.70): C, 72.39; H, 4.86; N,
14.47; O, 2.76; S, 5.52. Found: C, 72.65; H, 5.08; N, 14.78; O, 2.98;
S, 5.78.
3.6.5. 7-(3,4-dichlorophenyl)-2-(ethylthio)-5-phenyl-7H-pyrrolo
[2,3-d] pyr- -imidin-4-amine (IVe)
3.6. 4-Amino-7-(Aryl)-5,6-disubstitututed-2-(alkylthio)-1Hpyrrolo
[2,3-d]pyrimidine-2(7H)-thione (IV)
Yield 80%, m.p. 128e130 ꢀC. IR (KBr) (cmꢂ1): 3420, 3350 (NH2),
y
1620 (C]C), absence of (C^N). m/z: 414 (Mþ, 35Cl, 43.2%), 415
(Mþ þ 1, 8.9%), 416 (Mþ þ 2, 37Cl, 28.9%). 1H NMR (DMSO, 500 MHz)
Method A: To a warmed ethanolic potassium hydroxide solution
(prepared by dissolving 0.56 g, 0.01 mol of potassium hydroxide in
50 mL of ethanol) compound IIaef (0.01 mol) was added, and
heating was continued for 30 min. The mixture was allowed to cool
to r.t., and alkyl halide (0.02 mol) was added. The mixture was
stirred under reflux for 5 h, allowed to cool to r.t., and finally poured
into cold water (100 mL). The solid product precipitated was
filtered off and washed with 100 mL water, residue was dried off
and recrystallized from methanol
Method B: A mixture of IIaef (0.01 mol), alkyl halide (0.05 mol),
and anhydrous sodium acetate (2 g) in ethanol (30 mL) was heated
under reflux for 6 h, and then left to cool. The product that formed
was filtered off and recrystallized from methanol
Method C: A mixture of IIaef (0.01 mol) and anhydrous
potassium carbonate (2.07 g, 0.015 mol) were dissolved in dry
acetone (30 ml) and heated at 80 ꢀC under for 30 min. Methyl
iodide (0.61 ml, 0.01 mol) was added to the refluxing solution and
the mixture heated for 5 h on the water bath. The excess solvent
was removed and the residue was recrystallized ethanol.
Compound IV prepared by Methods A, B and C has the identical
m.p. and mixed m.p.
d
(ppm): 1.43 (t, 3H, J ¼ 6.8,CH3), 2.23 (q, 3H, J ¼ 6.8, CH3), 6.1 (br.s,
2H, NH2, D2O exchangeable), 7.0e7.8 (m, 8H, Ar-H), 8.1 (s, 1H,
C6eH). Anal. Calcd for C20H16Cl2N4S (414.34): C, 57.84; H, 3.88; Cl,
17.07; N,13.49; S, 7.72. Found: C, 58.03; H, 3.98; Cl,17.41; N,13.65; S,
7.89.
3.6.6. 5-(4-Amino-2-(ethylthio)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (IVf)
Yield 84%, m.p. 145e148 ꢀC. IR (KBr) (cmꢂ1): 3450, 3330 (NH2),
y
1710 (C]O), 1630 (C]C). m/z: 532 (Mþ, 35.5%), 533 (Mþ þ 1,
12.12%), 534.21 (Mþ þ 2, 2.35%). 1H NMR (DMSO, 500 MHz)
d
(ppm): 1H NMR (DMSO, 500 MHz)
d
(ppm): 1.43 (t, 3H, J ¼ 7.5,
CH3), 2.43 (s, 3H, CH3), 3.01 (q, 3H, J ¼ 7.5, CH3), 3.21 (s, 3H, NeCH3),
6.41 (br.s, 2H, NH2, D2O exchangeable), 7.0e7.8 (m, 15H, Ar-H). Anal.
Calcd for C31H28N6OS (532.66): C, 69.90; H, 5.30; N, 15.78; O, 3.00;
S, 6.02, Found: C, 70.01; H, 5.38; N, 16.01; O, 3.25; S, 6.35.
3.6.7. 7-Benzyl-2, 4-bis(methylthio)-5,6-diphenyl-7H-pyrrolo[2,3-
d]pyrimidine (IVg)
Yield 75%, m.p. 125e128 ꢀC. IR (KBr)
y
(cmꢂ1): 2925 (CH aliph.),
1620(C]S), absence of (NH). m/z: 453 (Mþ, 25%), 454 (Mþ þ 1,
7.5%), 455 (Mþ þ 2, 2.4%), 1H NMR (DMSO, 500 MHz)
d (ppm): 2.6
3.6.1. 7-Benzyl-2-(methylthio)-5, 6-diphenyl-7H-pyrrolo [2,3-d]
pyrimidin-4-amine (IVa)
(s, 6H, CH3), 5.92 (s, 2H, CH2), 7.1e7.7 (m, 15H, Ar-H). Anal. Calcd for
C27H23N3S2 (453.62): C, 71.49; H, 5.11; N, 9.26; S, 14.14. Found: C,
70.98; H, 4.89; N, 9.48; S, 14.25.
Yield 80%, m.p. 140e142 ꢀC. IR (KBr)
y
(cmꢂ1): 3350 (NH), 1625
(C]C). m/z: 422 (Mþ, 60%), 423 (Mþ þ 1, 17.52%), 424 (Mþ þ 2,
2.7%). 1H NMR (DMSO, 500 MHz)
d
(ppm): 2.43 (s, 3H, CH3), 6.02 (s,
3.6.8. 7-(3,4-dichlorophenyl)-2,4-bis(methylthio)-5-phenyl-7H-
pyrrolo[2,3-d]pyrimidine (IVh)
2H, CH2), 6.58 (s, 2H, NH2, D2O exchangeable), 6.9e7.8 (m, 15H, Ar-
H). Anal. Calcd for C26H22N4S (422.54): C, 73.90; H, 5.25; N, 13.26; S,
7.59; Found: C, 74.01; H, 5.48; N, 13.50; S, 7.86.
Yield 80%, m.p. 185e188 ꢀC. IR (KBr)
y
(cmꢂ1): 2930 (CH aliph.),
1630(C]S), absence of (NH). m/z: 431 (Mþ, 35Cl,100%), 432 (Mþ þ 1,
23.5%), 433 (Mþ þ 2, 37Cl, 64.5%). 1H NMR (DMSO, 500 MHz)
3.6.2. 7-(3,4-dichlorophenyl)-2-(methylthio)-5-phenyl-7H-pyrrolo
[2,3-d] pyrimidin-4-amine (IVb)
d (ppm): 2.45 (s, 6H, CH3), 7.1e7.8 (m, 8H, Ar-H), 8.0 (s, 1H, C6eH).
Anal. Calcd for C20H15Cl2N3S2 (432.39): C, 55.56; H, 3.50; Cl, 16.40;
N, 9.72; S, 14.83. Found: C, 55.58; H, 3.42; Cl, 16.69; N, 10.15; S, 15.0.
Yield 80%, m.p. 140e145 ꢀC. IR (KBr) (cmꢂ1): 3420, 3350 (NH2),
y
1620 (C]C), absence of (C^N). m/z: 401 (Mþ, 35Cl, 71.3%), 401
(Mþ þ 1, 16.8%), 402 (Mþ þ 2, 37Cl, 46.87%). 1H NMR (DMSO,
3.6.9. 5-(2,4-bis(methylthio)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)ꢂ1,4-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (IVi)
500 MHz)
d (ppm): 2.23 (s, 3H, CH3), 6.1 (br.s, 2H, NH2, D2O
exchangeable), 7.0e7.8 (m, 8H, Ar-H), 8.1 (s, 1H, C6eH). Anal. Calcd
for C19H14Cl2N4S (401.31): C, 56.86; H, 3.52; Cl, 17.67; N, 13.96; S,
7.99. Found: C, 57.03; H, 3.68; Cl, 17.00; N, 14.13; S, 6.89.
Yield 76%, m.p. 158e160 ꢀC. IR (KBr) (cmꢂ1): 2920 (CH aliph.),
y
1690 (C]O), 1630(C]S), absence of (NH). m/z: 549 (Mþ, 42%), 550
(Mþ þ 1, 15%), 551 (Mþ þ 2, 4.1%). 1H NMR (DMSO, 500 MHz)
3.6.3. 5-(4-Amino-2-(methylthio)-5, 6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (IVc)
d d (ppm): 2.43 (s, 3H, CH3), 2.58
(ppm): 1H NMR (DMSO, 500 MHz)
(s, 6H, CH3), 3.24 (s, 3H, NeCH3), 6.7e7.8 (m, 15H, Ar-H). Anal. Calcd
for C31H27N5OS2 (549.71): C, 67.73; H, 4.95; N, 12.74; O, 2.91; S,
11.67. Found: C, 67.59; H, 4.68; N, 13.0; O, 2.98; S, 11.60.
Yield 84%, m.p. 135e137 ꢀC. IR (KBr) (cmꢂ1): 3410, 3330 (NH2),
y
1698 (C]O), 1640 (C]C). m/z: 518 (Mþ, 52%), 519 (Mþ þ 1, 18.5%),
520 (Mþ þ 2, 2.82%). 1H NMR (DMSO, 500 MHz)
d (ppm): 2.43
(s, 3H, CH3), 2.58 (s, 3H, SeCH3), 3.21 (s, 3H, NeCH3), 6.41 (br.s, 2H,
NH2, D2O exchangeable), 7.0e7.8 (m, 15H, Ar-H). Anal. Calcd for
C30H26N6OS (518.63): C, 69.48; H, 5.05; N, 16.20; O, 3.08; S, 6.18,
Found: C, 69.59; H, 5.32; N, 16.57; O, 3.27; S, 6.58.
3.6.10. 7-Benzyl-2,4-bis(ethylthio)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidine (IVj)
Yield 78%, m.p. 115e120 ꢀC. IR (KBr)
y
(cmꢂ1): 2925 (CH aliph.),
1640(C]N), absence of (NH). m/z: 481 (Mþ, 36%), 482 (Mþ þ 1,

3002
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
11.7%), 483 (Mþ þ 2, 3.51%). 1H NMR (DMSO, 500 MHz)
d
(ppm):
(Mþ, 65%), 503 (Mþ þ 1, 20.1%), 1H NMR (DMSO, 500 MHz)
1.6e2.0 (t, 6H, CH3), 3.1e3.2 (q, 4H, CH2), 5.91 (s, 2H, CH2), 7.0e7.7
(m, 15H, Ar-H). Anal. Calcd for C29H27N3S2 (481.67): C, 72.31; H,
5.65; N, 8.72; S, 13.31. Found: C, 72.58; H, 5.59; N, 8.49; S, 13.25.
d d (ppm): 2.43 (s, 3H, CH3),
(ppm): 1H NMR (DMSO, 500 MHz)
3.21 (s, 3H, NeCH3), 4.78 (s, 2H, NH2, D2O exchangeable), 6.70
(s, 2H, NH2, D2O exchangeable), 7.0e7.8 (m, 15H, Ar-H), 9.30 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C29H26N8O (502.57): C,
69.31; H, 5.21; N, 22.30; O, 3.18, Found: C, 69.61; H, 5.39; N, 22.78;
O, 3.28.
3.6.11. 7-(3,4-dichlorophenyl)-2,4-bis(ethylthio)-5-phenyl-7H-
pyrrolo[2,3-d]pyrimidine (IVk)
Yield 85%, m.p. 170e174 ꢀC. IR (KBr)
y
(cmꢂ1): 2930 (CH aliph.),
1625(C]N), absence of (NH). m/z: 460 (Mþ, 35Cl, 100%), 461
(Mþ þ 1, 25.4%), 462 (Mþ þ 2, 37Cl, 73.6%). 1H NMR (DMSO,
3.8. General method to prepare VI
500 MHz)
d (ppm): 1.7e2.0 (t, 6H, CH3), 3.0e3.1 (q, 4H, CH2),
A mixture of IIdef (0.01 mol), chloroacetic acid (0.03 mol), and
fused sodium acetate (2 g) in a mixture of acetic acid/acetic anhy-
dride (30 mL, 1:1) was heated under reflux for 9 h and left to cool.
The reaction mixture was then diluted with water, shaken well, and
allowed to stand 6 h. The residue was triturate with ethanol; solid
product was filtered off and recrystallized from the ethanol/H2O to
give VI.
6.9e7.8 (m, 8H, Ar-H), 8.1 (s, 1H, C6eH). Anal. Calcd for
C22H19Cl2N3S2 (460.44): C, 57.39; H, 4.16; Cl, 15.40; N, 9.13; S, 13.93.
Found: C, 57.58; H, 4.42; Cl, 15.69; N, 9.25; S, 14.01.
3.6.12. 5-(2,4-bis(ethylthio)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-7-yl) ꢂ1,4-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (IVl)
Yield 74%, m.p. 162e166 ꢀC. IR (KBr)
y
(cmꢂ1): 2920 (CH aliph.),
3.8.1. 8-Benzyl-6,7-diphenyl-5-thioxo-5,8-dihydropyrrolo[2,3-d]
thiazolo[3,2-a]pyrimidin-3(2H)-one (VIa)
1690 (C]O), 1630(C]N), absence of (NH). m/z: 577 (Mþ, 22%), 578
(Mþ þ 1, 8.37%), 579 (Mþ þ 2, 2.1%). 1H NMR (DMSO, 500 MHz)
Yield 55%, m.p. 114e117 ꢀC. IR (KBr) (cmꢂ1): 1720 (C]O), 1620
y
d
(ppm): 1H NMR (DMSO, 500 MHz)
d (ppm): 1.6e2.0 (t, 6H, CH3),
(C]S). m/z: 465 (Mþ, 10%), 466 (Mþ þ 1, 3.52%). 1H NMR (DMSO,
2.43 (s, 3H, CH3), 3.0e3.1 (q, 4H, CH2), 3.24 (s, 3H, NeCH3), 6.7e7.8
(m, 15H, Ar-H). Anal. Calcd for C33H31N5OS2 (577.76): C, 68.60; H,
5.41; N, 12.12; O, 2.77; S, 11.10. Found: C, 68.79; H, 5.58; N, 12.01; O,
2.98; S, 11.35.
500 MHz) d (ppm): 3.8 (s, 2H, COeCH2eS), 5.70 (s, 2H, CH2), 7.0e7.7
(m, 15H, Ar-H). Anal. Calcd for C27H19N3OS2 (465.59): C, 69.65; H,
4.11; N, 9.03; O, 3.44; S, 13.77. Found: C, 70.01; H, 4.02; N, 9.38; O,
3.24; S, 14.05.
3.7. General method to prepare V
3.8.2. 8-(3,4-dichlorophenyl)-6-phenyl-5-thioxo-5,8-
dihydropyrrolo[2,3-d]thia-zolo[3,2-a]pyrimidin-3(2H)-one (VIb)
Method A: A mixture of IV aef (0.01 mol) and hydrazine hydrate
(99%, 0.03 mol) was heated under reflux in pyridine (30 mL) for 6 h
and then left to cool. Pour to ice water/acidified with HCl. The solid
product was filtered off, and recrystallized from ethanol to give V.
Method B. A mixture of IV aef (0.01 mol) and hydrazine hydrate
(99%, 0.03 mol) in absolute ethanol (20 mL) was refluxed for 10 h.
The reaction mixture was poured onto ice. The product was isolated
and recrystallized from methanol to give V as buff colored crystals.
Compound V prepared by Methods A and B have the identical m.p.
and mixed m.p.
Yield 54%, m.p. 210e215 ꢀC. IR (KBr) (cmꢂ1): 1710 (C]O), 1620
y
(C]S). m/z: 444 (Mþ, 35Cl, 73.6%), 443 (Mþ þ 1,21.8%), 446 (Mþ þ 2,
37Cl, 46.0%). 1H NMR (DMSO, 500 MHz)
d (ppm): 3.9 (s, 2H,
COeCH2eS), 7.2e7.8 (m, 8H, Ar-H), 8.0 (s, 1H, C6eH). Anal. Calcd for
C20H11Cl2N3OS2 (444.36): C, 54.06; H, 2.50; Cl, 15.96; N, 9.46; O,
3.60; S, 14.43. Found: C, 54.38; H, 2.42; Cl, 15.69; N, 9.15; O, 3.40; S,
14.21.
3.8.3. 8-(2,4-Dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-3-
yl)-6,7-di phenyl-5-thioxo-5,8-dihydropyrrolo[2,3-d]thiazolo[3,2-a]
pyrimidin-3(2H)-one (VIc)
3.7.1. 7-Benzyl-2-hydrazinyl-5, 6-diphenyl-7H-pyrrolo [2,3-d]
pyrimidin-4-amine (Va)
Yield 54%, m.p. 180e183 ꢀC. IR (KBr)
y
(cmꢂ1): 1710e1690 (C]
O), 1630 (C]S). m/z: 561 (Mþ, 25.0%), 562 (Mþ þ 1, 9.21%), 563
Yield (A) 60% and (B) 85%, m.p. 100e102 ꢀC. IR (KBr)
y
(cmꢂ1):
(Mþ þ 2, 2.68%). 1H NMR (DMSO, 500 MHz)
d
(ppm): 1H NMR
3460e3380 (NH2), 3350 (NH), 1630(C]C). m/z: 406 (Mþ, 10%), 407
(DMSO, 500 MHz)
d (ppm): 2.43 (s, 3H, CH3), 3.24 (s, 3H, NeCH3),
(Mþ þ 1, 3.1%). 1H NMR (DMSO, 500 MHz)
d (ppm): 4.85 (s, 2H, NH2,
3.9 (s, 2H, COeCH2eS), 6.7e7.8 (m, 15H, Ar-H). Anal. Calcd for
C31H23N5O2S2 (561.68): C, 66.29; H, 4.13; N, 12.47; O, 5.70; S, 11.42.
Found: C, 66.49; H, 4.48; N, 12.12; O, 5.98; S, 11.25.
D2O exchangeable), 5.92 (s, 2H, CH2), 6.75 (s, 2H, NH2, D2O
exchangeable), 6.9e7.8 (m, 15H, Ar-H), 9.2 (s, 1H, NH, D2O
exchangeable). Anal. Calcd for C25H22N6 (406.48): C, 73.87; H, 5.46;
N, 20.677. Found: C, 74.01; H, 5.49; N, 21.00.
3.9. General method to prepare VII
3.7.2. 7-(3,4-dichlorophenyl)-2-hydrazinyl-5-phenyl-7H-pyrrolo
[2,3-d] pyrimidin-4-amine (Vb)
Method A: A mixture of IIdef (0.01 mol), chloroacetic acid
(0.08 mol), appropriate aldehyde (0.05 mol), and fused sodium
acetate (2.0 g) in a mixture of acetic acid/acetic anhydride (30 mL,
1:1) was heated under reflux for 8 h and left to cool. The reaction
mixture was diluted with water. The residue was triturate with
ethanol; solid product was filtered off and recrystallized from the
ethanol/H2O to give VII.
Method B: To a mixture of VI (0.01 mol) and appropriate alde-
hyde (0.01 mol) in absolute ethanol (30 mL) was added piperidine
(5 drops). The mixture was heated under reflux for 8 h. The crys-
talline product thus obtained after cooling was collected and
recrystallized from ethanol to give VII as orange crystals.
Compound VII prepared by methods A and B have the same m.p.
and mixed m.p.
Yield (A) 52% and (B) 80%, m.p. 166e168 ꢀC. IR (KBr)
y
(cmꢂ1):
3480e3400 (NH2), 3380 (NH), 1640(C]C). m/z: 384 (Mþ, 35Cl,
100%), 385 (Mþ þ 1, 19.6%), 386 (Mþ þ 2, 37Cl, 63.9%), 388 (Mþ þ 4, 2
(
37Cl), 9.6%). 1H NMR (DMSO, 500 MHz)
d (ppm): 4.68 (s, 2H, NH2,
D2O exchangeable), 6.70 (s, 2H, NH2, D2O exchangeable), 7.0e7.8
(m, 8H, Ar-H), 8.0 (s, 1H, C6eH). 9.36 (s, 1H, NH, D2O exchangeable).
Anal. Calcd for C18H14Cl2N6 (385.25): C, 56.12; H, 3.66; Cl, 18.41; N,
21.81. Found: C, 56.48; H, 3.98; Cl, 18.78; N, 22.01.
3.7.3. 5-(4-Amino-2-hydrazinyl-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)-1,4-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (Vc)
Yield (A) 56% and (B) 84%, m.p. 165e168 ꢀC. IR (KBr)
y
(cmꢂ1):
3420, 3390 (NH2), 3340 (NH), 1710 (C]O), 1640(C]C). m/z: 502

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
3003
3.9.1. 8-Benzyl-2-benzylidene-6,7-diphenyl-5-thioxo-5,8-
Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 278533), and (C. albicans ATCC 10231) as a repre-
sentative for fungi. All micro-organisms used were obtained from
the culture collection of the Department of Microbiology and
Immunology, Faculty of Pharmacy, Helwan University, Cairo, Egypt.
Media for disc sensitivity tests were the nutrient agar and
MullereHinton agar (MHA) purchased from Difco (USA). Nonsterile
powder of the tested compound was dissolved in sterile DMSO to
dihydropyrrolo[2, 3-d] thiazolo[3,2-a]pyrimidin-3(2H)-one(VII a)
Yield (A) 65% and (B) 85%, m.p. 140e143 ꢀC. IR (KBr)
y
(cmꢂ1):
1720 (C]O), 1620(C]S). m/z: 553 (Mþ, 11.89%), 554 (Mþ þ 1,
4.81%), 555 (Mþ þ 2, 1.12%). 1H NMR (DMSO, 500 MHz)
d (ppm): 4.9
(s, 1H, C]CH), 5.75 (s, 2H, CH2), 6.8e7.7 (m, 20H, Ar-H). Anal. Calcd
for C34H23N3OS2 (553.69): C, 73.75; H, 4.19; N, 7.59; O, 2.89; S, 11.58.
Found: C, 73.91; H, 4.35; N, 7.92; O, 3.04; S, 11.85.
yield 20 and 30 m mm membrane
g mLꢂ1, and passed through 0.2
3.9.2. 2-Benzylidene-8-(3,4-dichlorophenyl)-6-phenyl-5-thioxo-
5,8-dihydro pyrrolo[2,3-d] thiazolo[3,2-a]pyrimidin-
3(2H)-one (VII b)
filters (Millipore Corp, USA). The filtrates were dispensed as 2 mL
samples into sterile, small screw-capped vials, frozen and kept
stored at ꢂ15 ꢀC. The vials were refrozen after thawing. Disc
diffusion sensitivity test was done in the manner identical to that of
Bauer et al. [34]. DMSO showed no inhibition zones. Amoxicillin
(Bioanalyse, Turkey) and Fluconazol (SigmaeAldrich, USA) were
used as reference substances.
MIC (minimum inhibitory concentration) for each tested
compound was determined on Muller-Hinton agar (MHA), by micro
dilution technique according to NCCLS guidelines 1997 [35]. All
bacterial isolates were sub cultured in MHA plates and incubated
overnight at 37 ꢀC and all Candida isolates were sub cultured in SDA
plates at 35 ꢀC for 24e48 h. The micro-organisms were passage at
least twice to ensure purity and viability. The solution of the newly
synthesized compounds and standard drugs were prepared at 1024,
Yield (A) 55% and (B) 86%, m.p. 260e265 ꢀC. IR (KBr)
y
(cmꢂ1):
1710 (C]O), 1620 (C]S). m/z: 531 (Mþ, 35Cl, 33.0%),532 (Mþ þ 1,
9.1%) 533 (Mþ þ 2, 37Cl, 23.4%). 1H NMR (DMSO, 500 MHz)
d (ppm):
2.3 (s, 1H, C]CH), 6.8e7.8 (m, 13H, Ar-H), 8.0 (s, 1H, C6eH). Anal.
Calcd for C27H15Cl2N3OS2 (532.464): C, 60.90; H, 2.84; Cl, 13.32; N,
7.89; O, 3.00; S, 12.04. Found: C, 61.18; H, 3.02; Cl, 12.79; N, 7.93; O,
3.20; S, 11.86.
3.9.3. 2-Benzylidene-8-(2,4-dimethyl-5-oxo-1-phenyl-2,5-dihydro-
1H-pyrazol-3-yl)-6,7-diphenyl-5-thioxo-5,8-dihydropyrrolo[2,3-d]
thiazolo[3,2-a]py- -rimidin-3(2H)-one (VII c)
Yield (A) 52% and (B) 80%, m.p. 150e155 ꢀC. IR (KBr)
y
(cmꢂ1):
1710e1680 (C]O), 1620 (C]S). m/z: 649 (Mþ,12.40%), 650
(Mþ þ 1,5.6%), 651 (Mþ þ 2, 1.25%). 1H NMR (DMSO, 500 MHz)
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 inoculum was added to each well of the micro dilution trays.
d
(ppm): 1H NMR (DMSO, 500 MHz)
d (ppm): 2.3 (s, 1H, C]CH),
2.43 (s, 3H, CH3), 3.24 (s, 3H, NeCH3), 6.9e7.8 (m, 20H, Ar-H). Anal.
Calcd for C38H27N5O2S2 (649.783): C, 70.24; H, 4.19; N, 10.78; O,
4.92; S, 9.87. Found: C, 70.49; H, 4.28; N, 10.52; O, 4.98; S, 9.55.
ꢀC
The trays were incubated at 37 in a humid chamber and MIC end
points were read after 24 h of incubation.
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 micro-organisms, and pure media were used as control
wells.
3.10. Biological assay
3.10.1. Anti-inflammatory activity [31]
In vivo assay: The carrageenan-induced rat paw oedema assay
was carried out using a modified winter’s method as a preliminary
screening test [32]. The rats (in groups of five animals weighing
120e170 g, young adult male SpragueeDawley) were housed in
a controlled environment and provided with standard rodent chow
and water for 24 h before a dose of the test compounds (50 and
70 mg/kg sc) was administered. One hour later, the volume of the
right hind paw was measured, and 0.05 mL of a 1% suspension of
carrageenan in sterile pyrogen-free 0.9% NaCl solution was injected
subcutaneously into planter aponeurosis of the hind paw. One hour
after the injection of carrageenan, the paw volume was again
measured by a water pletysmometer. The mean increase of paw
volume at each time interval was compared with that of control
group (five rats treated with carrageenan, but without test
compounds) at the same time intervals. The percentage inhibition
values were calculated according to the formula: % Anti-inflam-
matory activity ¼ (1 ꢂ Rt/Rc) ꢃ 100 (Rt ¼ result of tested group;
Rc ¼ result of control group). All experiments involving animals
were carried out using protocols approved by the Animal
Committee, University of Barcelona (Spain). Animal care was in
compliance with Generalitat of Catalunya regulations on protection
of animals used for experimental and other scientific purposes.
References
[1] S.M. Sondhi, R.N. Goyal, A.M. Lahoti, Bioorg. Med. Chem. 13 (2005) 3185.
[2] (a) H. Sierzputowska-Gracz, E. Sochacka, A. Malkiewicz, K. Kuo, C. Gehrke,
P.F. Agris, J. Am. Chem. Soc. 109 (1987) 7171;
(b) E. Sochacka, I. Fratczak, Tetrahedron Lett. 45 (2004) 6729.
[3] (a) S. Stoyanov, I. Petkov, L. Antonov, T. Stoyanova, P. Karagiannides,
P. Aslanidis, Can. J. Chem. 68 (1990) 1482;
(b) J.C. Hazelton, B. Iddon, H. Suschitzky, L.H.J. Woolley, Chem. Soc. Perkin
Trans. 1 6 (1992) 685.
[4] Z.G. Hajos, R.M. Kanojia, Chem. Abstr. 116 (1991) 83701.
[5] A.K. Pathak, V. Pathak, L.E. Seit, W.J. Sulng, R.C.J. Reynolds, Med. Chem. 41
(2004) 273.
[6] E.V. Aleksandrova1, Pharm. Chem. J. 37 (2003) 645.
[7] M.D. Mashkovskii, Drugs. Moscow 1e2 (2000) (in Russian).
[8] G.N. Pershin, Z.h. Vses, o-va im, Mendeleev Communications 15 (2) (1970)
216.
[9] E. Berger, L. Rumbach, La Revue de médecine interne 20 (1999) 3465.
[10] (a) T. Munster, D.E. Furst, Clin. Exp. Rheumatol. 17 (1999) 529;
(b) R.P. Baughman, E.E. Lower, Clin. Immunother. 6 (1996) 431;
(c) C.G. Groth, L. Backman, I.M. Morales, Transplantation 67 (1999) 1036;
(d) N. Perico, G. Remuzzi, Drugs 54 (4) (1999) 533.
[11] J.L.S. Banitt, G.L. Bundy, I.M. Richards, J. Pharmacol. Exp. Ther. 290 (1999)
188.
[12] B. Howard Cottam, Z. Kazimierczuk, S. Geary, P. McKernan, J. Med. Chem. 28
(1985) 1461.
[13] M.J. Motsuoka, Antibiotics (Tokyo) 13A (1960) 121.
[14] K. Anzai, S.J. Marumo, Antibiotics (Tokyo) 10A (1957) 20.
[15] M.A. Mohamed, A.E. Rashad, M.E.A. Zaki, S.S. Fatahala, Acta Pharm. 55 (2005)
237.
[16] M.A. Mohamed, A.E. Rashad, M.E.A. Zaki, S.S. Fatahala, Arch. Pharm. Chem. Life
Sci. (2006) 339.
[17] M.A. Mohamed, A.E. Rashad, M.E.A. Zaki, S.S. Fatahala, M.Z. Monem, Natur-
forsch C (2007) 62.
[18] M.T. Cocco, C. Congiu, A. Maccioni, V. Onnis, J. Heterocycl. 30 (1993) 253.
[19] A.Z.A. Hassanien, I.S. Abdel Hafiz, M.H. Elnagdi, J. Chem. Res. (S) (1999) 8.
[20] A.A. Aly, Phosphorus, Sulfur, and Silicon 181 (2006) 2395.
3.10.2. Anti-microbial activity
3.10.2.1. Materials and methods. Antibacterial activity was exam-
ined by the disc-diffusion method and the MIC method under
standard conditions of the National Committee for Clinical
Laboratory Standards [33]. The in vitro anti-microbial activity of the
synthesized compounds was investigated against several patho-
genic representative Gram-positive bacteria (S. aureus ATCC 29213,
B. subtilis ATCC 6633, Mycobacterium phlei ATCC 10142) and

3004
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 45 (2010) 2994e3004
[21] Z.H. Ismail, S.M. Abdel-Gawad, M.M. Ghorab, Phosphorus, Sulfur, and Silicon
178 (2003) 1795.
[30] S.M. Hussain, A.M.H. El-Reedy, A.M. Rezk, Kh.A Sife-Eldien, J. Heterocyclic.
Chem. 24 (1987) 1605.
[22] A.M.K. El-Dean, M.E. Abdel-Moneam, Phosphorus, Sulfur, and Silicon 177
(2002) 2745.
[23] A.E. Abdel-Rahman, I.M.A. Awad, E.A. Bakhete, Phosphorus, Sulfur, and Silicon
60 (1991) 189.
[24] M.I.A. Moneam, Phosphorus, Sulfur, and Silicon 180 (2005) 633.
[25] A.B.A. El-Gazzar, H.A.R. Hussein, A.S. Aly, Phosphorus, Sulfur, and Silicon 181
(2006) 2771.
[26] T.I. El-Emary, Sh.A. Abdel-Mohsen, Phosphorus, Sulfur, and Silicon 181 (2006)
2459.
[27] K.M. Hassan, M.S.K. Youssef, M.F. El-zohry, R. Abo El-wafa, Phosphorus, Sulfur,
and Silicon 40 (1988) 237.
[28] F. Abou-El, G. Hamman’, M.I. All, J. Chem. Eng. Data 26 (1981) 99.
[29] M.B. Devani, C.J. Shishoo, U.S. Pathak, S.H. Parikh, G.F. Shah, A.C. Padhya,
J. Pharm. Sci. 65 (1976) 660.
[31] Y. Harrak, G. Rosell, G. Daidone, Bioorg. Med. Chem. 15 (2007) 4876.
[32] C.A. Winter, E.A. Risley, G.W.J. Nuss, Pharmacol. Exp. Ther. 141 (1963) 369.
[33] National Committee for Clinical Laboratory Standards, Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically
Approved Standard NCCLS Document M7-A4, fourth ed., vol. 17, no. 2. NCCLS,
1997;
Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts; Approved Standard. NCCLS Document M27-A2, second ed., vol. 22, no.
15; National Committee for Clinical Laboratory Standards: 940 West Valley
Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, (2002).
[34] A.W. Bauer, M.M. Kirby, J.C. Sherris, Am. J. Clin. Pathol. 45 (1996) 493.
[35] A.C. Gales, H.S. Sader, R.N. Jones, Urinary tract infection trends in Latin
American hospitals: report from the SENTRY antimicrobial surveillance
program. Diagn. Microbial. Infect. Dis. 44 (1997e2000) 289.