Synthesis and kinetic testing of new inhibitors for a metallo-β- lactamase from Klebsiella pneumonia and Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.ejmech.2011.10.030

There are currently no clinically useful inhibitors against metallo-β-lactamases (MBLs), enzymes that confer resistance against a broad spectrum of commonly used antibiotics and that are produced by an increasing number of bacterial pathogens. New pyrrole

Full text of DOI:10.1016/j.ejmech.2011.10.030

European Journal of Medicinal Chemistry 46 (2011) 6075e6082
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and kinetic testing of new inhibitors for a metallo-b-lactamase from
Klebsiella pneumonia and Pseudomonas aeruginosa
,
,
,d
Mosaad S. Mohamed a, Waleed M. Hussein a b, Ross P. McGeary b c, Peter Vella b, Gerhard Schenk b
,
,
Rania H. Abd El-hameed a
*
a Helwan University, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Ein Helwan, Helwan, Egypt
b The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, QLD 4072, Australia
c The University of Queensland, School of Pharmacy, Brisbane, Qld 4072, Australia
d National University of Ireland- Maynooth, Department of Chemistry, Maynooth, Co. Kildare, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
There are currently no clinically useful inhibitors against metallo-b-lactamases (MBLs), enzymes that
Received 21 July 2011
Received in revised form
11 October 2011
Accepted 13 October 2011
Available online 21 October 2011
confer resistance against a broad spectrum of commonly used antibiotics and that are produced by an
increasing number of bacterial pathogens. New pyrrole derivatives were synthesized and assayed for their
inhibitory effect on the catalytic activity of the IMP-1 MBL from Pseudomonas aeruginosa and Klebsiella
pneumoniae. Six compounds tested (3a-3c, 5, 7 and 8) show micromolar inhibition constants (Ki values
range from w10 to 30 mM). In silico docking was employed to investigate the binding mode of the strongest
inhibitor, 3b, in the active site of IMP-1. Implications for further improvements of binding efficiency and
specificity are discussed.
Keywords:
Pyrrole
Pyrrolo[2,3-d]pyrimidine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Metallo-b-lactamase inhibitors
Structureeactivity relationship
1. Introduction
may be spread between bacterial species on plasmids, the devel-
opment of new MBL inhibitors is very urgent [9,10].
The
treatment of bacterial infections. In bacteria, the production of
enzymes such as the serine- -lactamases (SBLs) and metallo-
lactamases (MBLs) increasingly confers resistance to an ever
broader range of common -lactam antibiotics such as penams,
b
-lactam antibiotics are crucial chemotherapeutics for the
It is observed from the literature that the pyrrole nucleus and
pyrrolo[2,3-d]pyrimidines [11e16] have a variety of biological
applications due to their antibacterial [14,17e25], antiviral [26e28],
anticancer [29e35], anti-inflammatory [36,37] and anti-
hyperglycemic [38,39] activities.
b
b-
b
carbapenems and cephalosporins [1,2]. Thus, the development of
an inhibitor for SBLs and MBLs is an attractive approach to maintain
the usefulness of existing antibiotics. While there are efficient
inhibitors for SBL activity available, e.g. clavulanic acid derivatives
[3], no clinically useful antagonist of MBL activity has yet been re-
ported. The MBLs are characterized by their abba protein fold and
the presence of one or two zinc (II) atoms in their active sites (at
least one of these metal ions is required for catalysis [4e7]). The
MBLs belong to the large family of binuclear metallohydrolases [8]
and generally display a broad substrate profile, hydrolysing penams
(e.g. benzylpenicillin), cephalosporins (e.g. cephalothin, cefoxitin)
and carbapenems (e.g. meropenem and imipenem) [1,5]. Given this
broad range of utilizable substrates, and the ease with which MBLs
The aim of this work was to develop candidate MBL lead
inhibitors through the synthesis, testing and in silico docking of
compounds based on pyrrole. An IMP-1 MBL which occurs in both
Pseudomonas aeroginosa and Klebsiella pneumoniae [40e43] was
selected as a target since these pathogens are well known for
recorded outbreaks of antibiotics-resistant bacterial infections in
medical settings [44,45], and since this enzyme is relatively well
studied (e.g. crystal structures have been reported [46]).
2. Results and discussion
2.1. Synthesis of lead compounds
The synthetic route [47,48] to compounds 1ae1d is shown in
Scheme 1. Condensation of benzoin with the appropriate primary
aromatic amine in refluxing toluene gave the corresponding
* Corresponding author. Tel.: þ20 125517104; fax: þ20 225541601.
E-mail address: zeiadomar@yahoo.com (R.H. Abd El-hameed).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.030

6076
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 6075e6082
Ph
Ph
O
+
Ph
Ph
O
i
Ph
CN
4-R- C H -NH
2
6
4
OH
NHC H R
6
4
X
NH
2
N
1 a-d
R
Ph
O
Ph
O
i
4-R-
+
C H -NH
6 4 2
Br
NHC H R
6
4
R
X
CH
a
Ph
3
OMe
Ph
H
b
CH
c
d
3
CH (CN)
2
Reagents: i=
2
OMe
H
Scheme 1. Synthesis of compounds 1ae1d.
a
-aminoketone intermediates which were subsequently condensed,
with bezaldehyde in absolute ethanol. Finally, pyrrolo[3,2-e][1,2,4]
triazolo[4,3-c]pyrimidines 8, 9 and 10 were obtained from 6 by
reaction with carbon disulfide, formic acid and acetic anhydride,
respectively, [48,54] as revealed in Scheme 3.
in situ, with malononitrile to afford compounds 1a and 1b.
Alternatively, refluxing a mixture of phenacyl bromide and
primary aromatic amines in dry ethanol gave the respective
a
-aminoketone intermediates which were condensed with malo-
nonitrile to afford compounds 1c and 1d.
2.2. Enzymatic inhibition assays
Pyrroles 1ae1d reacted with formic acid [49] to yield pyrrolo
[2,3-d]pyrimidin-4(3H)-ones 2ae2d, and these reacted with
phosphorus oxychloride [50] to afford 4-chloro-pyrrolo[2,3-d]
pyrimidines 3ae3d which were converted to pyrrolo[2,3-d]pyr-
imidin-4(3H)-thiones 4ae4d by reaction with thiourea in refluxing
ethanol [51]. Preparation of pyrrolo[2,3-d]pyrimidin-4-ylidene-
malononitrile 5 was accomplished by the reaction of 4b with
malononitrile in absolute ethanol [52] as revealed in Scheme 2.
On the other hand, synthesis of 4-hydrazino-pyrrolo[2,3-d]
pyrimidines 6 was achieved by the reaction of 3a with hydrazine
hydrate [53] in absolute ethanol. N-Benzylidene-N0-pyrrolo[2,3-d]
pyrimidin-4-yl-hydrazine 7 was obtained by the reaction [54] of 6
The inhibitory effects of the 11 compounds 1c, 1d, 2c, 3ae3c, 5,
7, 8, 9 and 10 (Table 1) on the catalytic activity of IMP-1 were
initially assessed by measuring enzyme activity in the absence and
presence of 10
mM of compound. A previously developed assay
based on the chromogenic cephalosporin substrate CENTA (70
mM)
was used [43,55]. The hydrolysis of CENTA by IMP-1 releases the
chromophore 4-nitrophenolate, the formation of which can be
measured spectrophotometrically with a plate reader at
l
¼ 405 nm
(at pH 7.0, ε ¼ 6400 Mꢀ1 cmꢀ1). The results are presented in Table 1.
Six compounds (3ae3c, 5b, 7 and 8) were of interest as they
showed promising inhibition effects at a concentration of 10 mM.
Cl
O
H
N
Ph
Ph
N
Ph
CN
NH
i
ii
N
X
N
X
N
X
N
N
2
3 a-d
1 a-d
2 a-d
Ph
S
R
CN
R
R
H
N
NC
H
N
Ph
iii
iv
4b
N
X
N
N
Ph
N
4 a-d
5
R
R
X
OMe
CH
a
Ph
Ph
3
OMe
b
ii =
Reagents:
POCl
i = HCOOH
3
CH
c
H
H
3
d
OMe
iii= CS(NH)
CH (CN)
2
iv =
2
2
Scheme 2. Synthesis of compounds 2e5.

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 6075e6082
NHN=CH-Ph
6077
NHNH
2
Cl
Ph
N
Ph
Ph
Ph
Ph
N
N
ii
i
N
Ph
N
N
N
N
N
7
6
CH
3a
3
CH
CH
3
3
iii
v
H
N
N
N
iv
N
N
S
CH
3
Ph
Ph
Ph
Ph
N
N
N
N
N
N
N
N
Ph
Ph
N
8
N
10
CH
CH
3
3
9
CH
3
iii = CS
ii =PhCHO
Reagents:
i =
NH -NH
2
2
2
v = (CH CO) O
3
iv =HCOOH
2
Scheme 3. Synthesis of compounds 6e10.
Therefore, they were selected for further analysis to determine
their mode of inhibition and the magnitude of the corresponding
inhibition constant(s) (Ki values).
8 results in activity derivative with considerable inhibitory effect.
Finally, the benzylidene-pyrrolo[2,3-d]pyramidineehydrazine
7
also inhibits MBL activity. The mode of inhibition is generally
competitive and the flexible loop in the vicinity of the active site
may play an important role in binding these lead compounds. In
summary, pyrrole and pyrrolopyrimidine compounds are prom-
ising leads in the development of novel MBL inhibitors.
As shown in Table 2, compound 3b is the strongest inhibitor for
IMP-1, acting in essence competitively with a Kic of 12
mM. However,
the remaining compounds are also good inhibitors with inhibition
constants ranging from 15
the known MBL inhibitor
those of compounds discovered from a fragment-based screening
approach range from w500 M to w1600 M [43]. To investigate
m
M to 33
m
M. For comparison, the Kic of
L-captopril is 12.5
mM ꢁ 2.4 [43], and
m
m
4. Experimental
the potential binding mode of 3b to IMP-1, in silico docking was
employed (Fig. 1). The method employed was described in our
previous work [43] with the exception that structures here were
drawn in JME editor with hydrogen atoms added using the Dundee
PRODRG 2 server (http://davapc1.bioch.dundee.ac.uk/prodrg/) [56].
Compound 3b was docked into the active site of IMP-1 (Fig. 1); the
model illustrates that 3b binds within the active site occupying the
groove parallel to the flexible loop and close to the two zinc atoms.
The oxygen atom of the methoxy group of 3b is located close to
4.1. Synthesis of lead compounds
All commercial chemicals used as starting materials and
reagents in this study were purchased from Merck (Darmstadt,
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
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 Chromatography
was performed on (Merck) Silica gel 60 (particle size
0.06e0.20 mm). All compounds prepared in this paper are new and
confirmed with spectral data except 1b, 2b, 3b and 4b [47].
ꢀ
ꢀ
both metal ions in the active site (Zn1: 3.4 A; Zn: 2.7 A). Apart from
these interactions with the metal ions hydrophobic interactions
with residues in the flexible loop, notably residue Trp 64, may play
an important role in enhancing the binding affinity of the inhibitor.
The N3 atom of the pyrimidine ring in 3b is oriented approximately
ꢀ
3.5 A towards Glu 59 and binds to the oxygen atom of Glu 59
through a hydrogen bond.
3. Conclusion
In the present study, we describe a straightforward and efficient
synthesis of novel pyrrole and pyrrolo[2,3-d]pyrimidine derivatives
as potential MBL inhibitors. The structure activity relationship
(SAR) results indicate that the pyrrole derivatives 1c, 1d and the
pyrrolo[2,3-d]pyrimidine 2c have no activity. However, the intro-
duction of a chloro group in 3ae3c and the formation of the ary-
lidine malononitrile 5 leads to pyrrolopyrimidine analogs that are
rather potent inhibitors of MBL activity. Similarly, while the pyrrolo
[3,2-e][1,2,4]triazolo[4,3-c]pyrimidines 9 and 10 have no effect on
MBL activity, the addition of a thione group in position 3 of the
pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine moiety in compound
4.1.1. General procedure for the synthesis of compounds 1a and 1b
A mixture of benzoin (2 g, 0.01 mol), aryl amine (0.01 mol) and
conc. HCl (6e8 drops) in toluene (50 mL) was heated under reflux
for 6 h and cooled. Malononitrile (0.66 g, 0.01 mol) was added,
followed by a catalytic amount (1.5 mL) of pyridine portion wise
and left to reflux until a solid was formed. The solvent was evap-
orated under reduced pressure and the residue was recrystallized
from methanol to give compounds 1ae1b.

6078
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 6075e6082
Table 1
Percentage inhibition of IMP-1 MBL (2 nM) at pH 7.0 by 11 pyrrole derivatives using CENTA (70
m
M) as substrate.
Compound
Structure
Inhibition % (10
m
M)
Compound
Structure
Inhibition % (10 mM)
1c
0
5
17
1d
0
7
13
2c
0
8
14
3a
10
9
4
3b
15
10
7
3c
9
4.1.1.1. 2-Amino-1-(4-methylphenyl)-4,5-diphenyl-1H-pyrrole-3-
4.1.1.2. 2-Amino-1-(4-methoxyphenyl)-4,5-diphenyl-1H-pyrrole-3-
carbonitrile (1b). Yield: 93%; m.p.: 204e206 ꢂC; IR (KBr) (cmꢀ1):
3526, 3659 (NH2), 2205 (C^N); 1246 (CeO); MS (EI) m/z: 365 (Mþ,
5.6%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 3.79 (s, 3H, OCH3), 5.1
carbonitrile (1a). Yield: 81%; m.p.: 193e194 ꢂC; IR (KBr)
y
y
(cmꢀ1): 3567, 3526 (NH2), 2205 (C^N); MS (EI) m/z: 349 (Mþ,
74.5%); 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 2.27 (s, 3H, CH3),
d
5.09 (br s, 2H, NH2, D2O exchangeable), 6.5e7.9 (m, 14H, AreH);
Anal. calcd. for C24H19N3 (349.44): C, 82.52; H, 5.44; N, 12.03%.
Found: C, 82.16; H, 5.81; N, 11.84%.
(br s, 2H, NH2, D2O exchangeable), 6.6e7.8 (m, 14H, AreH); Anal.
calcd. for C24H19N3O (365.44): C, 78.90; H, 5.20; N, 11.51; O, 4.38%.
Found: C, 78.58; H, 5.39; N, 11.22; O, 4.61%.

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 6075e6082
6079
Table 2
4.1.2.2. 2-Amino-1-(4-methoxyphenyl)-4-phenyl-1H-pyrrole-3-
carbonitrile (1d). Yield: 73%; m.p.: 164e166 ꢂC; IR (KBr)
Inhibition constants for the inhibibition of IMP-1 MBL by compounds 3ae3c, 5, 7 and
8.
y
(cmꢀ1): 3332, 3259 (NH2), 2210 (C^N); 1250 (CeO); MS (EI) m/z:
289 (Mþ, 18.2%); 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 3.41
Compound
Kic
(
m
M)
Kiuc (mM)
3a
3b
3c
5
7
8
19 ꢁ 7
12 ꢁ 4
33 ꢁ 16
29 ꢁ 7
18 ꢁ 9
15 ꢁ 5
112 ꢁ 74
182 ꢁ 184
106 ꢁ 60
225 ꢁ 139
e
(s, 3H, OCH3), 4.7 (br s, 2H, NH2, D2O exchangeable), 6.8e7.9
(m, 10H, AreH); Anal. calcd. for C18H15N3O (289.34): C, 74.73;
H, 6.27; N, 13.76; O, 5.53%. Found: C, 75.02; H, 6.39; N, 13.42;
O, 5.69%.
e
4.1.3. General procedure for the synthesis of compounds 2ae2d
Compounds 1aed (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 2ae2d.
4.1.2. General procedure for the synthesis of compounds 1c and 1d
To a solution of phenacyl bromide (2 g, 0.01 mol) and aryl amine
(0.01 mol) in ethanol (20 mL) was added a saturated solution of
sodium bicarbonate (5 mL). The reaction mixture was kept at 70 ꢂC
for 1 h, cooled, thenpouredintocold water, filtered off and dried. The
obtained product was dissolved in an appropriate amount of ethanol
(20 mL) and then malononitrile (0.66 g, 0.01 mol) was added portion
wise, followed by sodium ethoxide (0.01 mol) and left to reflux until
a solid formed. The solvent was removed under reduced pressure
and the residue was recrystallized from methanol to give 1c and 1d.
4.1.3.1. 7-(4-Methylphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyr-
imidin-4(3H)-one (2a). Yield: 82%; m.p.: 236e238 ꢂC; IR (KBr)
y
(cmꢀ1): 3182 (NeH), 1675 (C]O), 1595 (C]N) and disappearance
of the CN group; MS (EI) m/z: 377 (Mþ, 10%); 1H NMR (DMSO-d6,
300 MHz)
d (ppm): 2.4 (s, 3H, CH3), 6.9e7.5 (m, 14H, AreH), 8.1 (s,
1H, C2eH), 8.3 (s, 1H, NH, D2O exchangeable); Anal. calcd. for
C25H19N3O (377.45): C, 79.58; H, 5.04; N, 11.14; O, 4.24%. Found: C,
79.38; H, 5.25; N, 10.86; O, 4.61%.
4.1.2.1. 2-Amino-1-(4-methylphenyl)-4-phenyl-1H-pyrrole-3-carbo-
nitrile (1c). Yield: 66%; m.p.: 173e175 ꢂC; IR (KBr)
y
(cmꢀ1): 3342,
3226 (NH2), 2212 (C^N); MS (EI) m/z: 273 (Mþ, 23%); 1H NMR
(DMSO-d6, 300 MHz) (ppm): 2.24 (s, 3H, CH3),4.3 (br s, 2H, NH2,
4.1.3.2. 7-(4-Methoxyphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyr-
d
imidin-4(3H)-one (2b). Yield: 79%; m.p.: 261e263 ꢂC; IR (KBr)
y
D2O exchangeable), 6.7e7.8 (m, 10H, AreH); Anal. calcd. for
C18H15N3 (273.34): C, 79.10; H, 5.53; N, 15.37%. Found: C, 81.03; H,
5.61; N, 15.65%.
(cmꢀ1): 3130 (NeH), 1682 (C]O), 1587 (C]N), 1510 (CeO) and
disappearance of the CN group; MS (EI) m/z: 393 (Mþ, 12.9%); 1H
NMR (DMSO-d6, 300 MHz) d (ppm): 3.78 (s, 3H, OCH3), 6.7e8.3 (m,
14H, AreH), 8.8 (s, 1H, C2eH), 8.9 (s, 1H, NH, D2O exchangeable);
Anal. calcd. for C25H19N3O2 (393.45): C, 76.34; H, 4.83; N, 10.69; O,
8.13%. Found: C, 76.65; H, 5.21; N, 10.45; O, 7.85%.
4.1.3.3. 7-(4-Methylphenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-
4(3H)-one (2c). Yield: 78%; m.p.: 182e184 ꢂC; IR (KBr)
y
(cmꢀ1):
3280 (NeH), 1671 (C]O), 1594 (C]N) and disappearance of the CN
group; MS (EI) m/z: 301 (Mþ, 36%); 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 2.37 (s, 3H, CH3), 6.9e7.8 (m, 10H, AreH), 8.31 (s, 1H,
C2eH), 8.5 (s, 1H, NH, D2O exchangeable); Anal. calcd. for
C19H15N3O (301.35): C, 75.73; H, 5.02; N, 13.94; O, 5.31%. Found: C,
75.41; H, 4.85; N, 14.22; O, 4.99%.
4.1.3.4. 7-(4-Methoxyphenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-
4(3H)-one (2d). Yield: 82%; m.p.: 189e191 ꢂC; IR (KBr)
y
(cmꢀ1):
3202 (NeH), 1687 (C]O), 1599 (C]N), 1503 (CeO) and disap-
pearance of the CN group; MS (EI) m/z: 317 (Mþ, 25%); 1H NMR
(DMSO-d6, 300 MHz)
d (ppm): 3.5 (s, 3H, OCH3), 6.8e7.9 (m, 10H,
AreH), 8.2 (s, 1H, C2eH), 8.3 (s, 1H, NH, D2O exchangeable); Anal.
calcd. for C19H15N3O2 (317.35): C, 71.91; H, 4.76; N, 13.24; O, 10.08%.
Found: C, 72.28; H, 5.01; N, 13.39; O, 10.35%.
4.1.4. General procedure for the synthesis of compounds 3ae3d
Compounds 2aed (0.01 mol) were refluxed in phosphorus
oxychloride (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 3ae3d.
4.1.4.1. 4-Chloro-7-(4-methylphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidine (3a). Yield: 75%; m.p.: 236e238 ꢂC; IR (KBr)
y
(cmꢀ1):
1605 (C]N); MS (EI) m/z: 395 (Mþ, 35Cl, 77%), 397 (Mþþ2, 37Cl,
Fig. 1. Surface view of the IMP-1 active site for the highest Autodock Vina score
conformationof IMP-1 with3bdockedintotheactive site. Forclarity, Trp64 of theflexible
loop adjacent tothe active site has been given a ‘stick’ representation. Atom colours are as
follows: blue, nitrogen; red, oxygen; white, carbon (on IMP-1); green, carbon (on
inhibitor); magenta, zinc active site metals. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
31%) 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.45 (s, 3H, CH3),
6.9e7.5 (m, 14H, AreH), 8.16 (s, 1H, C2eH); Anal. calcd. for
C25H18ClN3 (395.90): C, 75.95; H, 4.56; Cl, 8.86; N, 10.63%. Found: C,
75.61; H, 4.12; Cl, 8.49; N, 10.81%.

6080
M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 6075e6082
4.1.4.2. 4-Chloro-7-(4-methoxyphenyl)-5,6-diphenyl-7H-pyrrolo
4.1.6. 2-(7-(4-Methoxyphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-4-ylidene)-malononitrile (5)
[2,3-d]pyrimidine (3b). Yield: 87%; m.p.: 209e211 ꢂC; IR (KBr)
y
(cmꢀ1): 1605 (C]N), 1226 (CeO); MS (EI) m/z: 411 (Mþ, 35Cl,
Compound 4b (4.09 g, 0.01 mol) and malononitrile (0.66 g,
0.01 mol) was heated under reflux in dry ethanol (30 mL) for 8 h,
cooled, poured onto ice-water to give precipitate which was filtered
off, dried, and recrystallized from methanol to give 5. Yield: 67%;
40%), 413 (Mþþ2, 37Cl, 14.5%) 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 3.65 (s, 3H, OCH3), 7.1e7.9 (m, 14H, AreH), 8.6 (s, 1H,
C2eH); Anal. calcd. for C25H18ClN3O (411.89): C, 72.99; H, 4.38; Cl,
8.52; N, 10.22; O, 3.88%. Found: C, 73.24; H, 4.21; Cl, 8.48; N, 9.85;
O, 3.62%.
m.p.: 212e214 ꢂC; IR (KBr) (cmꢀ1): 3325 (NeH), 2210 (C^N), 1546
y
(C]N), 1249 (CeO); MS (EI) m/z:441 (Mþ, 21%), 1H NMR (DMSO-d6,
300 MHz) (ppm): 3.66 (s, 3H, OCH3), 6.6e7.9 (m, 14H, AreH), 8.65
d
4.1.4.3. 4-Chloro-7-(4-methylphenyl)-5-phenyl-7H-pyrrolo[2,3-d]
(s, 1H, C2eH), 8.74 (s, 1H, NH, D2O exchangeable); Anal. calcd. for
C28H19N5O (441.50): C, 76.18; H, 4.34; N, 15.86; O, 3.62%. Found: C,
76.35; H, 4.26; N, 15.98; O, 3.77%.
pyrimidine (3c). Yield: 65%; m.p.: 193e195 ꢂC; IR (KBr)
y
(cmꢀ1):
1612 (C]N); MS (EI) m/z: 319 (Mþ, 35Cl, 6%), 321 (Mþþ2, 37Cl, 2.2%)
1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.27 (s, 3H, CH3), 6.9e7.8
(m, 10H, AreH), 8.23 (s, 1H, C2eH); Anal. calcd. for C19H14ClN3
(319.80): C, 71.36; H, 4.41; Cl, 11.09; N, 13.14%. Found: C, 71.61; H,
4.72; Cl, 10.82; N, 12.99%.
4.1.7. (7-(4-Methylphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)-hydrazine (6)
Compound 3a (3.95 g, 0.01 mol) and hydrazine hydrate (5 mL,
0.015 mol, 98%) was heated under reflux in dry ethanol (30 mL)
for 4 h, cooled, poured onto ice-water to give precipitate which
was filtered off, dried, and recrystallized from methanol to give 6.
4.1.4.4. 4-Chloro-7-(4-methoxyphenyl)-5-phenyl-7H-pyrrolo[2,3-d]
pyrimidine (3d). Yield: 69%; m.p.: 198e200 ꢂC; IR (KBr)
y
(cmꢀ1):
1596 (C]N), 1217 (CeO); MS (EI) m/z: 335 (Mþ, 35Cl, 35%), 337
Yield: 82%; m.p.: 204e206 ꢂC; IR (KBr) (cmꢀ1): 3412, 3320
y
(Mþþ2, 37Cl, 11.8%) 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 3.5 (s,
(NH2), 3237 (NeH), 1533 (C]N); MS (EI) m/z: 391 (Mþ, 34%), 1H
NMR (DMSO-d6, 300 MHz) (ppm): 2.46 (s, 3H, CH3), 4.9 (br s, 2H,
3H, OCH3), 6.7e8.0 (m, 10H, AreH), 8.4 (s, 1H, C2eH); Anal. calcd.
for C19H14ClN3O (335.80): C, 68.28; H, 5.16; Cl, 10.08; N, 11.94; O,
4.76% Found: C, 68.49; H, 4.88; Cl, 10.39; N, 12.21; O, 4.48%.
d
NH2, D2O exchangeable), 6.7e7.9 (m, 15H, AreH, NH, D2O
exchangeable), 8.3 (s, 1H, C2eH); Anal. calcd. for C25H21N5
(391.48): C, 76.70; H, 5.41; N, 17.89%. Found: C, 76.93; H, 5.12; N,
17.56%.
4.1.5. General procedure for the synthesis of compounds 4ae4d
Compound 3aed (0.01 mol) and thiourea (1.5 g, 0.02 mol) was
heated under reflux in dry ethanol (30 mL) for 4 h, cooled, poured
onto ice-water, to give precipitate which was filtered off, dried, and
recrystallized from methanol to give 4ae4d.
4.1.8. N-Benzylidene-N0-(7-(4-methylphenyl)-5,6-diphenyl-7H-
pyrrolo[2,3-d]pyramidin-4-yl)-hydrazine (7)
Compound 6 (3.91 g, 0.01 mol) and benzaldehyde (3 mL) was
heated under reflux in dry ethanol (30 mL) for 8 h, cooled, poured
onto ice-water to give precipitate which was filtered off, dried, and
recrystallized from methanol to give 7. Yield: 32%; m.p.:
4.1.5.1. 7-(4-Methylphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-
4(3H)-thione (4a). Yield: 67%; m.p.: 214e216 ꢂC; IR (KBr)
y
(cmꢀ1):
3326 (NeH), 1645 (C]S), 1609 (C]N); MS (EI) m/z: 393 (Mþ, 14%),
187e189 ꢂC; IR (KBr)
y
(cmꢀ1): 3323 (NeH),1608 (C]N); MS (EI) m/
(ppm): 2.54 (s,
1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 2,4 (s, 3H, CH3), 6.6e7.9 (m,
z: 479 (Mþ, 37.9%), 1H NMR (DMSO-d6, 300 MHz)
d
14H, AreH), 8.1 (s, 1H, C2eH), 9.3 (d, 1H, NH, D2O exchangeable);
Anal. calcd. for C25H19N3S (393.51): C, 76.34; H, 4.83; S, 8.14; N,
10.69%. Found: C, 76.10; H, 5.12; S, 7.88; N, 10.47%.
3H, CH3), 5.12 (br s,1H, NH, D2O exchangeable), 7.0e7.9 (m, 19H,
AreH), 8.26 (s, 1H, C2eH), 8.32 (s, 1H, CH); Anal. calcd. for C32H25N5
(479.59): C, 80.14; H, 5.25; N, 14.60%. Found: C, 79.82; H, 5.61; N,
14.36%.
4.1.5.2. 7-(4-Methoxyphenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyr-
imidin-4(3H)-thione (4b). Yield: 89%; m.p.: 205e207 ꢂC; IR (KBr)
y
4.1.9. 7-(4eMethylphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]
triazolo[4,3-c]pyrimidine-3-thione (8)
(cmꢀ1): 3368 (NeH), 1631 (C]S), 1586 (C]N), 1241 (CeO); MS (EI)
m/z: 409 (Mþ, 6.5%), 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 3.7 (s,
Compound 6 (3.91 g, 0.01 mol) and carbon disulfide (10 mL)
in absolute ethanol (30 mL) was heated under reflux for 3 h,
cooled, poured onto ice-water to give a precipitate which was
filtered off, dried, and recrystallized from ethanol to give 8.
3H, OCH3), 6.8e7.8 (m, 14H, AreH), 8.0 (s, 1H, C2eH), 9.5 (d, 1H, NH,
D2O exchangeable); Anal. calcd. for C25H19N3SO (409.51): C, 73.35;
H, 4.65; S, 7.82; N, 10.27; O, 3.91%. Found: C, 73.41; H, 4.59; S, 7.59;
N, 10.39; O, 4.22%.
Yield: 52%; m.p.: 190e192 ꢂC; IR (KBr)
y
(cmꢀ1): 3314 (NeH),
1652 (C]S), 1567 (C]N); MS (EI) m/z: 433 (Mþ, 13%), 1H NMR
(DMSO-d6, 300 MHz) (ppm): 2.36 (s, 3H, CH3), 6.8e7.5 (m,
4.1.5.3. 7-(4-Methylphenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-
d
4(3H)-thione (4c). Yield: 63%; m.p.: 189e191 ꢂC; IR (KBr)
y
(cmꢀ1):
14H, AreH), 8.89 (s, 1H, C5eH), 8.98 (br s,1H, NH, D2O
exchangeable); Anal. calcd. for C26H19N5S (433.54): C, 72.03; H,
4.42; N, 16.15; S, 7.40%. Found: C, 71.85; H, 4.78; N, 15.83; S,
7.14%.
3293 (NeH), 1636 (C]S), 1595 (C]N); MS (EI) m/z: 317 (Mþ, 9%),
1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 2.75 (s, 3H, CH3), 6.9e8.0
(m, 10H, AreH), 8.3 (s, 1H, C2eH), 9.2 (d, 1H, NH, D2O
exchangeable); Anal. calcd. for C19H15N3S (317.42): C, 71.92; H,
4.73; S, 10.09; N, 13.25%. Found: C, 72.21; H, 4.36; S, 10.34; N,
13.57%.
4.1.10. 7-(4-Methylphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-e][1,2,4]
triazolo[4,3-c] pyrimidine (9)
Compound 6 (3.91 g, 0.01 mol) was heated under reflux for 8 h
in formic acid (20 ml, 85%), cooled, poured onto ice-water to give
a precipitate which was filtered off, dried, and recrystallized from
4.1.5.4. 7-(4-Methoxyphenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-
4(3H)-thione (4d). Yield: 77%; m.p.: 176e178 ꢂC; IR (KBr) (cmꢀ1):
y
3345 (NeH), 1652 (C]S), 1617 (C]N),1238 (CeO); MS (EI) m/z: 333
ethanol to give 9. Yield: 64%; m.p.: 176e178 ꢂC; IR (KBr) (cmꢀ1):
y
(Mþ, 24%), 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 3.54 (s, 3H,
1613 (C]N); MS (EI) m/z: 401 (Mþ, 100%), 1H NMR (DMSO-d6,
300 MHz) (ppm): 2.34 (s, 3H, CH3), 6.94e7.96 (m, 14H, AreH),
OCH3), 7.0e7.9 (m, 10H, AreH), 8.3 (s, 1H, C2eH), 8.8 (d,1H, NH, D2O
exchangeable); Anal. calcd. for C19H15N3SO (333.41): C, 68.46; H,
4.50; S, 9.60; N, 12.61; O, 4.80%. Found: C, 68.75; H, 4.26; S, 9.82; N,
12.98; O, 4.54%.
d
8.0 (s, 1H, C3eH), 8.9 (s, 1H, C5eH); Anal. calcd. for C26H19N5
(401.47): C, 77.79; H, 4.77; N, 17.44%. Found: C, 78.03; H, 5.02; N,
17.81%.

M.S. Mohamed et al. / European Journal of Medicinal Chemistry 46 (2011) 6075e6082
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4.1.11. 3-Methyl-7-(4-methylphenyl)-8,9-diphenyl-7H-pyrrolo[3,2-
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m.p.: 185e187 ꢂC; IR (KBr)
y
(cmꢀ1): 1598 (C]N); MS (EI) m/z: 415
(ppm): 2.21 (s, 3H,
(Mþ, 6.49%), 1H NMR (DMSO-d6, 300 MHz)
d
C3eCH3), 2.32 (s, 3H, AreCH3), 6.98e7.6 (m, 14H, AreH), 8.2 (s,1H,
C5eH); Anal. calcd. for C27H21N5 (415.50): C, 78.05; H, 5.09; N,
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4.3. Kinetic assays
Inhibition assays were performed in 96 well 400
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mL multi-titre
mM
ZnCl2, pH 7.0). The substrate CENTA was synthesized according to
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mM. The final concentration of IMP-
1 in the assay was 2 nM. A final concentration of 20
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