Synthesis and Kinetic Testing of Tetrahydropyrimidine-2-thione and Pyrrole Derivatives as Inhibitors of the Metallo-β-lactamase from Klebsiella pneumonia and Pseudomonas aeruginosa

Article abstract of DOI:10.1111/j.1747-0285.2012.01440.x

Metallo-β-lactamases (MBLs), produced by an increasing number of bacterial pathogens, facilitate the hydrolysis of many commonly used β-lactam antibiotics. There are no clinically useful antagonists against MBLs. Two sets of tetrahydropyrimidine-2-thione and pyrrole derivatives were synthesized and assayed for their inhibitory effects on the catalytic activity of the IMP-1 MBL from Pseudomonas aeruginosa and Klebsiella pneumoniae. Nine compounds tested (1a, 3b, 5c, 6b, 7a, 8a, 11c, 13a, and 16a) showed micromolar inhibition constants (K_i values range from ~20-80μm). Compounds 1c, 2b, and 15a showed only weak inhibition. In silico docking was employed to investigate the binding mode of each enantiomer of the strongest inhibitor, 5c (K_i=19±9μm), as well as 7a (K_i=21±10μm), the strongest inhibitor of the pyrrole series, in the active site of IMP-1.

Full text of DOI:10.1111/j.1747-0285.2012.01440.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01440.x
Chem Biol Drug Des 2012
Research Article
Synthesis and Kinetic Testing of
Tetrahydropyrimidine-2-thione and Pyrrole
Derivatives as Inhibitors of the Metallo-b-
lactamase from Klebsiella pneumonia and
Pseudomonas aeruginosa
Waleed M. Hussein1,2, Samar S. Fatahala1,
The b-lactam antibiotics are broad-spectrum agents against many
Zainab M. Mohamed1, Ross P. McGeary2,3
Gerhard Schenk2,4, David L. Ollis5 and
Mosaad S. Mohamed1,*
,
Gram-positive, Gram-negative, and anaerobic micro-organisms (1).
b-Lactam antibiotics interfere with the synthesis of the peptidogly-
can layer of bacterial cell walls (1), and b-lactamases are produced
by a growing number of bacteria, enabling resistance to the effects
of b-lactam antibiotics; their hydrolysis of the b-lactam ring renders
these antibiotics inactive. Penams, carbapenems, and cephalosporins
are examples of b-lactam antibiotics that are hydrolyzed by these
enzymes. There are two families of b-lactamases: the serine-b-lacta-
mases (SBLs), which operate via a nucleophilic serine residue in the
active site, and metallo-b-lactamases (MBLs), which employ a metal-
activated nucleophilic hydroxyl group (2,3). The development of inhib-
itors against SBLs and MBLs is urgent to retain the usefulness of
b-lactam antibiotics. Potent inhibitors for SBLs are available and are
in clinical use (e.g., clavulanic acid and its derivatives) (4), but no
clinically useful antagonist of MBL activity has been reported.
1Department of Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Helwan University, Ein Helwan, Helwan, Egypt
2School of Chemistry and Molecular Biosciences, The University of
Queensland, Brisbane, Qld 4072, Australia
3School of Pharmacy, The University of Queensland, Brisbane, Qld
4072, Australia
4Department of Chemistry, National University of Ireland-Maynooth,
Maynooth, Co. Kildare, Ireland
5The Australian National University Research School of Chemistry,
Canberra, ACT 0200, Australia
*Corresponding author: Mosaad S. Mohamed,
doctor.mosaadmohamed@yahoo.com
The MBLs are members of the binuclear metallohydrolase family (5). A
characteristic feature of the MBLs is the abba protein fold with either
one or two zinc (II) ions in their active sites. At least one of these zinc
(II) ions plays an essential role in the catalytic hydrolysis of the b-lac-
tam ring of the antibiotics (6–9). Of great concern for healthcare sys-
tems is that MBLs display a very broad range of utilizable substrates,
including penams (e.g., benzylpenicillin), cephalosporins (e.g., cephalo-
thin, cefoxitin), and carbapenems (e.g., meropenem and imipenem)
(2,7). These observations, together with the ease with which MBLs
may be spread between bacterial species through mobile genetic ele-
ments, make the development of new MBL inhibitors a very urgent
task (10–13). Imipenemase-1 (IMP-1) is one of the best characterized
MBLs and has been identified in, among others, the pathogenic
microbes Pseudomonas aeruginosa and Klebsiella pneumonia (6,7).
Metallo-b-lactamases (MBLs), produced by an
increasing number of bacterial pathogens, facili-
tate the hydrolysis of many commonly used b-lac-
tam antibiotics. There are no clinically useful
antagonists against MBLs. Two sets of tetrahy-
dropyrimidine-2-thione and pyrrole derivatives
were synthesized and assayed for their inhibitory
effects on the catalytic activity of the IMP-1 MBL
from Pseudomonas aeruginosa and Klebsiella
pneumoniae. Nine compounds tested (1a, 3b, 5c,
6b, 7a, 8a, 11c, 13a, and 16a) showed micromolar
inhibition constants (Ki values range from ꢀ20–
80 lM). Compounds 1c, 2b, and 15a showed only
weak inhibition. In silico docking was employed
to investigate the binding mode of each enantio-
mer of the strongest inhibitor, 5c (Ki = 19 9 lM),
as well as 7a (Ki = 21 10 lM), the strongest
inhibitor of the pyrrole series, in the active site
of IMP-1.
In our previous work (14), we designed and synthesized a set of thi-
osemicarbazide derivatives (Ki = 10–75 lM) as IMP-1 inhibitors
based on in silico docking and fragment-based screening of a 500-
compound Maybridgeꢀ library (15). In another report, we assayed
a series of pyrrole derivatives against IMP-1 that have Ki values
ranging from ꢀ10 to 30 lM (16).
Key words: inhibition assays, metallo-b-lactamases, pyrrole, tetra-
hydropyrimidine-2-thione
The aim of this work was to find new candidate IMP-1 lead inhibi-
tors with improved potency via the synthesis, evaluation, and in sil-
ico docking of two sets of compounds, pyrroles and thiopyrimidines.
Received 14 March 2012, revised 13 June 2012 and accepted for publi-
cation 19 June 2012
1

Hussein et al.
D2O exchangeable); Anal. Calcd for C15H16N2OS: C, 66.15; H, 5.92; N,
10.28; S, 11.77%. Found: C, 66.00; H, 5.73; N, 10.14; S, 11.49%.
Materials and Methods
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 apparatus (Shimadzu, Kyoto,
Japan); IR spectra were recorded as potassium bromide pellets on a
Ethyl 6-methyl-4-(4-methoxyphenyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (1d)
Yield: 87%; m.p. 144–146 ꢁC; IR (KBr) t (per cm): 3390 (NH), 1683
1
(C=O), 1231 (C=S); MS (EI) m ⁄ z: 306 (M+,18%). H NMR (DMSO-d6,
1
300 MHz) d (ppm): 1.20 (t, J = 7.1 Hz, 3H, CH3), 4.10 (q, 2H,
J = 7.1 Hz, CH2), 2.70 (s, 3H, CH3), 3.40 (s, 3H, OCH3), 5.10 (s, 1H,
pyrim), 7.40 (dd, J = 8.2, J = 2.2 Hz, 4H, Ar-H), 9.70, 10.40 (2xs, 2H,
2NH D2O exchangeable); Anal. Calcd for C15H18N2O3S: C, 58.80; H,
5.92; N, 9.14; S, 10.47%. Found: C, 58.47; H, 5.72; N, 9.42; S, 10.32%.
PerkinElmer 1650 spectrophotometer (Waltham, MA, USA). H NMR
spectra were recorded on a Varian Mercury (300 MHz) spectrometer
(Varian, Crawley, UK), and chemical shifts are expressed as ppm
against TMS as internal reference. Mass spectra were recorded at
70 eV (EI Ms-QP 1000 EX; Shimadzu). Microanalyses were operated
using Vario, Elementar apparatus (Shimadzu, Japan). Column chroma-
tography was performed on (Merck) Silica gel 60 (particle size 0.06–
0.20 mm). Compounds 7a–c, 10b, 13–16, and 19 were prepared
as reported in the literature (17–21). Compounds 8, 10a, 10c, and
11–12 were prepared as reported in the literature (22).
Ethyl 6-methyl-4-styryl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1e)
Yield: 85%; m.p. 205–207 ꢁC; IR (KBr) t (per cm): 3478 (NH), 1683
1
(C=O), 1220 (C=S); MS (EI) m ⁄ z: 302 (M+, 100%). H NMR (DMSO-
d6, 300 MHz) d (ppm): 1.10 (t, J = 7.5 Hz, 3H, CH3), 4.01 (q,
J = 7.5 Hz, 2H, CH2), 2.30 (s, 3H, CH3, pyrim), 5.10 (d, J = 8.2 Hz,
1H, pyrim), 6.30 (d, J = 8.2 Hz, 1H, CH=CH), 6.40 (t, J = 7.5 Hz, 1H,
CH=CH), 7.20–7.40 (m, 5H, Ar-H), 9.70, 10.40 (2xs, 2H, 2NH, D2O
exchangeable); Anal. Calcd for C16H18N2O2S: C, 63.55; H, 6.00; N,
9.26; S, 10.60%. Found: C, 63.21; H, 6.31; N, 9.00; S, 10.84%.
General procedure for the synthesis of
compounds 1a–e
A mixture of thiourea (0.76 g, 10 mmol), the appropriate aromatic
aldehyde (10 mmol), and either acetylacetone or ethyl acetoacetate
(10 mmol) in absolute ethanol (20 mL) containing 37% HCl (8 drops)
was heated under reflux for 8 h, and the reaction mixture was
allowed to cool and neutralized with 25% ammonia (0.5 mL). The
precipitate formed was filtered off, washed with 50% ethanol, and
recrystallized from ethanol to give compounds 1a–e.
General procedure for the synthesis of
compounds 2a–c
To a solution of chloroacetic acid (0.94 g, 10 mmol) and sodium
hydroxide (10 mmol) in water (5 mL), a solution of 1a–c (10 mmol)
and sodium hydroxide (10 mmol) in water (10 mL) was added. The
reaction mixture was stirred at room temperature for 3 h and then ren-
dered acidic with 37% HCl. The precipitate formed was filtered off,
washed with water and recrystallized from methanol to afford 2a–c.
1-(6-Methyl-4-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-yl)ethanone (1a)
Yield: 86%; m.p. 197–199 ꢁC; IR (KBr) t (per cm): 3366 (NH), 1680
1
(C=O); MS (EI) m ⁄ z: 246 (M+, 77%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.60 (s, 3H, CH3, pyrim), 2.70 (s, 3H, COCH3), 5.26 (s, 1H
pyrim), 7.23-7.33 (m, 5H, Ar-H), 9.60, 9.70 (2xs, 2H, 2NH, D2O
exchangeable); Anal. Calcd for C13H14N2OS: C, 63.39; H, 5.73; N,
11.37; S, 13.02%. Found: C, 63.15; H, 5.54; N, 11.24; S, 12.72%.
2-((5-Acetyl-6-methyl-4-phenyl-1,2,3,4-
tetrahydropyrimidine-2yl)sulfanyl)acetic acid (2a)
Yield: 80%; m.p. 195–197 ꢁC; IR (KBr) t (per cm): 3340 (NH), 1681
1
(C=O); MS (EI) m ⁄ z: 304 (M+, 5%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.21 (s, 3H, COCH3), 2.28 (s, 3H, CH3 pyrim), 4.21 (s, 2H,
CH2), 5.11 (s, 1H, pyrim), 6.98–7.21 (m, 5H, Ar-H), 9.82 (s, 1H, NH,
D2O exchangeable), 12.11 (s, 1H, CO2H, D2O exchangeable); Anal.
Calcd for C15H16N2O3S: C, 59.19; H, 5.30; N, 9.20; S, 10.54%.
Found: C, 59.43; H, 5.60; N, 9.51; S, 10.59%.
1-(6-Methyl-4-(4-methoxy-phenyl)-2-thioxo-
1,2,3,4-tetrahydro pyrimidine-5-yl)ethanone (1b)
Yield: 88%; m.p. 179–181 ꢁC; IR (KBr) t (per cm): 3336 (NH), 1675 (C=O);
1
MS (EI) m⁄z: 276 (M+, 100%). H NMR (DMSO-d6, 300 MHz) d (ppm):
2.25 (s, 3H, CH3, pyrim), 2.60 (s, 3H, COCH3), 3.83 (s, 3H, OMe), 5.12 (s,
1H pyrim), 7.20 (dd, J = 8.1, J = 2.3 Hz, 4H, Ph-OCH3), 9.69, 9.73 (2xs,
2H, 2NH, D2O exchangeable); Anal. Calcd for C14H16N2O2S: C, 60.85; H,
5.84; N, 10.14; S, 11.60%. Found: C, 60.59; H, 5.72; N, 9.98; S, 11.30%.
2-((5-Acetyl-6-methyl-4-(4-methoxyphenyl)-
1,2,3,4-tetrahydropyrimidine-2yl)sulfanyl)acetic
acid (2b)
1-(6-Methyl-4-styryl-2-thioxo-1,2,3,4-
Yield: 87%; m.p. 207–209 ꢁC; IR (KBr) t (per cm): 3333 (NH), 1678
1
(C=O); MS (EI) m ⁄ z: 334 (M+, 21%). H NMR (DMSO-d6, 300 MHz) d
tetrahydropyrimidine-5-yl)ethanone (1c)
Yield: 90%; m.p. 183–185 ꢁC; IR (KBr) t (per cm): 3279 (NH), 1686
(ppm): 2.26 (s, 3H, COCH3), 2.29 (s, 3H, CH3 pyrim), 3.73 (s, 3H, OCH3),
4.12 (s, 2H, CH2), 5.20 (s, 1H, pyrim), 7.90 (dd, J = 8.2, J = 2.3 Hz, 4H,
Ar-H), 9.84 (s, 1H, NH, D2O exchangeable), 11.90 (s, 1H, CO2H, D2O
exchangeable); Anal. Calcd for C16H18N2O4S: C, 57.47; H, 5.43; N,
8.38; S, 9.59%. Found: C, 57.86; H, 5.31; N, 8.54; S, 9.33%.
1
(C=O), 1620 (C=S); MS (EI) m⁄ z: 272 (M+, 21%). H NMR (DMSO-d6,
300 MHz) d (ppm): 2.26 (s, 3H, CH3, pyrim), 2.30 (s, 3H, COCH3), 5.00
(d, J = 8.1 Hz, 1H, pyrim), 6.20 (t, J = 7.1 Hz, 1H, CH=CH), 6.40 (d,
J = 8.1 Hz 1H, CH=CH), 7.20–7.80 (m, 5H, Ar-H), 10.00 (s, 2H, 2NH,
2
Chem Biol Drug Des 2012

Inhibitors of metallo-beta-lactamases
General procedure for the synthesis of
2-((5-Acetyl-6-methyl-4-styryl-1,2,3,4-
tetrahydropyrimidine-2yl)sulfanyl)acetic acid (2c)
Yield: 77%; m.p. 204-206 ꢁC; IR (KBr) t (per cm): 3334 (NH), 1675
compounds 4a–c
A mixture of pyrimidine derivatives 1a–c (10 mmol) and the appro-
priate aromatic aldehyde (10 mmol) in 10% ethanolic sodium
hydroxide solution (50 mL) was stirred at room temperature for
24 h. The mixture was then heated under reflux for 1 h, cooled,
poured onto ice water (100 mL), and acidified with 37% HCl
(0.5 mL). The formed precipitate was filtered off, dried, and recrys-
tallized from aqueous DMF to give compounds 4a–c.
1
(C=O); MS (EI) m ⁄ z: 330 (M+, 8%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.27 (s, 3H, CH3, pyrim), 2.30 (s, 3H, COCH3), 4.00 (s, 2H,
CH2), 5.02 (d, J = 8.1 Hz, 1H, pyrim), 6.23 (d, J = 8.1 Hz, 1H,
CH=CH), 6.31 (t, J = 7.5 Hz, 1H, CH=CH), 7.35–8.21 (m, 5H, Ar-H),
9.70 (s, 1H, NH, D2O exchangeable), 12.01 (s, 1H, CO2H, D2O
exchangeable); Anal. Calcd for C17H18N2O3S: C, 61.80; H, 5.49; N,
8.48; S, 9.70%. Found: C, 61.49; H, 5.21; N, 8.54; S, 9.70%.
1-(6-Methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahy-
dropyrimidine-5yl)-3-(2-methoxyphenyl)-prop-
2en-1-one (4a)
General procedure for the synthesis of
compounds 3a–c
A solution of 1a–c (10 mmol) in benzene (50 mL) was treated
with malononitrile (10 mmol), ammonium acetate (10 g, 0.13 mol),
and glacial acetic acid (15 mL). The reaction mixture was heated
under reflux for 24 h using Dean-Starke water separator. On
cooling to room temperature, it was diluted with benzene
(100 mL) and washed successively with water (100 mL) and 5%
aq. sodium carbonate (200 mL). Then the solvent was removed
in vacuum on a steam bath and the residual oil crystallized out
upon standing at room temperature to afford dark brown crystals
of 3a–c.
Yield: 85%; m.p. 240–242 ꢁC; IR (KBr) t (per cm): 3403 (NH), 1749
1
(C=O); MS (EI) m ⁄ z: 364 (M+, 83%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.37 (s, 3H, CH3, pyrim), 3.75 (s, 3H, OMe), 4.99 (s, 1H,
pyrim), 5.50 (dd, J = 8.2, J = 2.3 Hz, 2H, CH=CH), 6.83–7.67 (m, 9H,
Ar-H), 9.65, 9.83 (2xs, 2H, 2NH, D2O exchangeable); Anal. Calcd for
C21H20N2O2S: C, 69.21; H, 5.53; N, 7.69; S, 8.80%. Found: C, 69.00;
H, 5.38; N, 7.45; S, 8.59%.
1-(6-Methyl-4-(4-methoxyphenyl)-2-thioxo-1,2,3,
4-tetrahydropyrimidine-5yl)-3-(2-methoxyphenyl)
-prop-2en-1-one (4b)
2-(1-(6-Methyl-4-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-yl)ethylidene)
malononitrile (3a)
Yield: 64%; m.p. 145–147 ꢁC; IR (KBr) t (per cm): 3357 (NH),
1696 (C=O); MS (EI) m ⁄ z: 394 (M+, 23%). 1H NMR (DMSO-d6,
300 MHz) d (ppm): 2.26 (s, 3H, CH3, pyrim), 3.63 (s, 3H, OMe),
3.73 (s, 3H, OMe), 5.52 (dd, J = 8.1, J = 5.2 Hz, 2H, CH=CH), 5.25
(s, 1H pyrim), 6.94–7.64 (m, 8H, Ar-H), 10.05, 10.13 (2xs, 2H, 2NH,
D2O exchangeable); Anal. Calcd for C22H22N2O3S: C, 66.98; H,
5.62; N, 7.10; S, 8.13%. Found: C, 66.94; H, 5.40; N, 7.31; S,
8.19%.
Yield: 45%; m.p. 220–222 ꢁC; IR (KBr) t (per cm): 2223 (CN), 3427
1
(NH); MS (EI) m ⁄ z: 294 (M+, 11%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.20 (s, 3H, CH3, pyrim), 2.40 (s, 3H, CH3), 5.21 (s, 1H, pyrim),
6.90–7.50 (m, 5H, Ar-H), 8.90, 10.40 (2xs, 2H, 2NH, D2O exchange-
able); Anal. Calcd for C16H14N4S: C, 65.28; H, 4.79; N, 19.03; S,
10.89%. Found: C, 65.12; H, 4.92; N, 19.25; S, 10.72%.
1-(6-Methyl-4-styryl-2-thioxo-1,2,3,4-tetrahy
dropyrimidine-5yl)-3-(2-methoxyphenyl)-prop-
2en-1-one (4c)
2-(1-(6-Methyl-4-(4-methoxyphenyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-yl)ethylidene)
malo- nonitrile (3b)
Yield: 75%; m.p. 148–150 ꢁC; IR (KBr) t (per cm): 3399 (NH),
1688 (C=O); MS (EI) m ⁄ z: 390 (M+, 5%). 1H NMR (DMSO-d6,
300 MHz) d (ppm): 2.46 (s, 3H, CH3, pyrim), 3.83 (s, 3H, OMe),
4.95 (d, J = 8.0 Hz, 1H pyrim), 5.09 (dd J = 8.0, J = 2.4 Hz, 2H,
CH=CH), 6.19 (t, J = 7.0 Hz, 1H, CH=CH), 6.50 (d, J = 8.0 Hz, 1H,
CH=CH), 6.94–7.69 (m, 9H, aromatic), 9.68, 9.73 (2xs, 2H, 2NH,
D2O exchangeable); Anal. Calcd for C23H22N2O2S: C, 70.74; H,
5.68; N, 7.17; S, 8.21%. Found: C, 70.44; H, 5.39; N, 7.49; S,
8.44%.
Yield: 49%; m.p. 210–212 ꢁC; IR (KBr) t (per cm): 2227 (CN), 3322
1
(NH); MS (EI) m ⁄ z: 324 (M+, 18%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.26 (s, 3H, CH3, pyrim), 2.41 (s, 3H, CH3), 3.81 (s, 3H, OMe),
5.11 (s, 1H, pyrim), 7.40 (dd, J = 8.2, J = 4.1 Hz, 4H, Ar-H), 9.30,
10.30 (2xs, 2H, 2NH, D2O exchangeable); Anal. Calcd for
C17H16N4OS: C, 62.94; H, 4.97; N, 17.27; S, 9.88%. Found: C, 63.00;
H, 5.20; N, 17.38; S, 10.10%.
2-(1-(6-Methyl-4-styryl-2-thioxo-1,2,3,4-tetrahyd
ropyrimidine-5-yl)ethylidene)malono-nitrile (3c)
Yield: 50%; m.p. 170–172 ꢁC; IR (KBr) t (per cm): 2220 (CN), 3279
General procedure for the synthesis of
compounds 5a–c
1
(NH); MS (EI) m ⁄ z: 320 (M+, 10%). H NMR (DMSO-d6, 300 MHz) d
To a solution of chalcone derivative 4a–c (10 mmol) in dry ethanol
(30 mL), phenyl hydrazine (4 mL) was first added, followed by gla-
cial acetic acid (5 mL). The reaction mixture was heated under
reflux for 5 h, cooled, and concentrated under reduced pressure;
the precipitate formed was filtered, dried, and recrystallized from
ethanol to give compounds 5a–c.
(ppm): 2.25 (s, 3H, CH3, pyrim), 2.42 (s, 3H, CH3), 5.22 (d, J = 8.2 Hz,
1H, pyrim), 6.30 (t, J = 7.2 Hz, 1H, CH=CH), 6.50 (d, J = 8.2 Hz, 1H,
CH=CH), 6.60–7.70 (m, 5H, Ar-H), 8.20, 10.40 (s, 2H, 2NH, D2O
exchangeable); Anal. Calcd for C18H16N4S: C, 67.47; H, 5.03; N, 17.49;
S, 10.01%. Found: C, 67.63; H, 4.99; N, 17.70; S, 10.21%.
Chem Biol Drug Des 2012
3

Hussein et al.
5-(5-(2-Methoxyphenyl)-1-phenyl-1H-pyrazol-3-
yl)-6-methyl-4-phenyl-1,2,3,4-tetrahydropyr
imidine-2(1H)-thione (5a)
(ppm): 2.33 (s, 3H, CH3, pyrim), 5.01 (d, J = 8.1 Hz, 1H, pyrim), 6.70
(d, J = 8.1 Hz, 1H, CH=CH), 6.90 (t, J = 7.1 Hz, 1H, CH=CH), 7.04–
8.30 (m, 12H, Ar-H), 9.50, 10.20, 10.40, 11.23 (4xs, 4H, 4NH, D2O
exchangeable); Anal. Calcd for C24H22N6O3S2: C, 56.90; H, 4.38; N,
16.59; S, 12.66%. Found: C, 57.10; H, 4.21; N, 16.64; S, 12.57%.
Yield: 60%; m.p. 185–187 ꢁC; IR (KBr) t (per cm): 3393 (NH), 1622
1
(C=N); MS (EI) m ⁄ z: 452 (M+, 59%). H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.30 (s, 3H, CH3, pyrim), 3.80 (s, 3H, OCH3), 5.09 (s, 1H
pyrim), 7.07–8.22 (m, 15H, Ar-H), 9.73, 9.82 (2xs, 2H, 2NH, D2O
exchangeable); Anal. Calcd for C27H24N4OS: C, 71.66; H, 5.35; N,
12.38; S, 7.08%. Found: C, 71.83; H, 5.00; N, 12.00; S, 7.32%.
General procedure for the preparation of
compounds 8a–c
A mixture of 7 (10 mmol) in 37% HCl (10 mL) was cooled with stir-
ring to 0–5 ꢁC in ice, and cooled sodium nitrite solution (2.50 g,
36 mmol) in water (10 mL) was added to it dropwise during 30 min.
The reaction mixture was then stirred for 30 min. Without separa-
tion, an ice-cold mixture of malononitrile (1.00 g, 15 mmol) and
sodium acetate (4.10 g, 50 mmol) in ethanol (50 mL) was added
dropwise with stirring during 15 min. Stirring was continued for
30 min on ice, and the reaction mixture was then left for 12 h at
room temperature. The precipitate was filtered off and recrystallized
from ethanol ⁄ H2O to give 8.
5-(5-(2-Methoxyphenyl)-1-phenyl-1H-pyrazol-3-
yl)-6-methyl-4-(4-methoxyphenyl)-1,2,3,4-
tetrahydropyrimidine-2(1H)-thione (5b)
Yield: 92%; m.p. 130–132 ꢁC; IR (KBr) t (per cm): 3479 (NH), 1599
1
(C=N); MS (EI) m ⁄ z: 482 (M+, 86%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.26 (s, 3H, CH3, pyrim), 3.63 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 5.02 (s, 1H pyrim), 7.07–8.32 (m, 14H, Ar-H), 10.02, 10.31
(2xs, 2H, 2NH, D2O exchangeable); Anal. Calcd for C28H26N4O2S: C,
69.69; H, 5.43; N, 11.61; S, 6.64%. Found: C, 69.39; H, 5.70; N,
11.84, S, 6.34%.
(1-Benzyl-3-cyano-4,5-diphenyl-1H-pyrrol-2-
yl)carbonohydrazonoyl dicyanide (8a)
5-(5-(2-Methoxyphenyl)-1-phenyl-1H-pyrazol-3-
yl)-6-methyl-4-styryl-1,2,3,4-tetrahydropyr-
imidine-2(1H)-thione (5c)
Yield 58%, m.p. 115–120 ꢁC. IR (KBr) t (per cm): 3280 (NH), 2330
(C”N), 1585 (C=N). MS (EI) m ⁄ z(%): 426 (M+, 18%), 427 (M++1,
5.5%), 428 (M++2, 1.5%). 1H NMR (DMSO-d6, 300MHz) d (ppm):
5.62 (s, 2H, CH2), 6.90 (s, 1H, NH, hydrazone), 7.20–7.80 (m, 15H,
Ar-H). Anal. Calcd for C27H18N6 (426.47): C, 76.04; H, 4.25; N, 19.71.
Found: C, 76.25; H, 4.41; N, 19.86.
Yield: 70%; m.p. 120–122 ꢁC; IR (KBr) t (per cm): 3462 (NH), 1622
1
(C=N); MS (EI) m ⁄ z: 478 (M+, 65%). H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.33 (s, 3H, CH3, pyrim), 3.84 (s, 3H, OCH3), 5.23 (d, J = 8.3 Hz,
1H pyrim), 6.21 (t, J = 7.2 Hz, 1H, CH=CH), 6.82 (d, J = 8.3 Hz, 1H,
CH=CH), 7.05–8.32 (m, 15H, Ar-H), 9.60, 10.40 (2xs, 2H, 2NH, D2O
exchangeable); Anal. Calcd for C29H26N4OS: C, 72.78; H, 5.48; N,
11.71; S, 6.70%. Found: C, 72.54; H, 5.52; N, 11.93; S, 6.88%.
(3-Cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-4,5-diphenyl-1H-pyrrol-
2-yl)carbonohydrazonoyl dicyanide (8b)
Yield 48%, m.p. 153–157 ꢁC. IR (KBr) t (per cm): 3290 (NH), 2320
(C”N), 1695(C=O), 1585 (C=N). MS (EI) m ⁄ z(%): 522 (M+, 18%), 523
(M++1, 8.5%), 524 (M++2, 0.8%). 1H NMR (DMSO-d6, 300MHz) d
(ppm): 2.20 (s, 2H, CH2-C=O), 2.33 (s, 3H, CH3), 3.12 (s, 3H, N-CH3),
6.90 (s, 1H, NH, hydrazone), 7.30–7.90 (m, 15H, Ar-H). Anal. Calcd
for C31H22N8O (522.559): C, 71.25; H, 4.24; N, 21.44; O, 3.06. Found:
C, 71.48; H, 4.62; N, 21.55; O, 3.17.
General procedure for the synthesis of
compounds 6a–b
A mixture of pyrimidine derivative 1d–e (10 mmol) and sulfadiazine
(2.50 g, 10 mmol) in DMF (30 mL) was heated under reflux for 8 h.
The solution was poured onto ice water, and the precipitate was
filtered off and washed with water to give 6a–b.
4-(4-Methoxyphenyl)-5-(sulfadiazinyl-amido)-6-
methyl-1,2,3,4-tetrahydropyrimidine-2-thiones
(6a)
(3-Cyano-1-(3,4-dichlorophenyl)-4-phenyl-1H-
pyrrol-2-yl)carbon hydrazonoyl dicyanide (8c)
Yield 45%, m.p. 218–222 ꢁC. IR (KBr) t (per cm): 3300 (NH), 2310
(C”N), 1565 (C=N). MS (EI) m ⁄ z(%): 405 (M+, 35Cl, 19%), 407
Yield: 79%; m.p. 195–197 ꢁC; IR (KBr) t (per cm): 3380 (NH), 1720
1
1
(C=O); MS (EI) m ⁄ z: 510 (M+, 10%). H NMR (DMSO-d6, 300 MHz) d
(M++2, 37Cl, 12.2%), 409 (M++4, 2*(37Cl), 2.1%). H NMR (DMSO-d6,
(ppm): 2.35 (s, 3H, CH3, pyrim), 3.75 (s, 3H, OCH3), 5.23 (s, 1H, pyrim),
7.33–8.62 (m, 11H, Ar-H), 9.70, 10.80, 11.00, 11.23 (4xs, 4H, 4NH, D2O
exchangeable); Anal. Calcd for C23H22N6O4S2: C, 54.10; H, 4.34; N,
16.46; S, 12.56%. Found: C, 53.86; H, 4.51; N, 17.00; S, 12.72%.
300MHz) d (ppm): 6.90 (s, 1H, NH, hydrazone), 7.00 (s, 1H, C6-H),
7.20–7.80 (m, 8H, Ar-H). Anal. Calcd for C20H10Cl2N6 (405.24): C,
59.28; H, 2.49; Cl, 17.50; N, 20.74. Found: C, 59.41; H, 2.62; Cl,
17.65; N, 20.86.
4-Styryl-5-(sulfadiazinyl-amido)-6-methyl-
1,2,3,4-tetrahydropyrimidine-2-thiones (6b)
Yield: 65%; m.p. 105–107 ꢁC; IR (KBr) t (per cm): 3390 (NH), 1655.90
2-Amino-1-(aryl)-4-phenyl-5-substituted-1H-
pyrrole-3-carboxamide (10)
To a suspension of compound 7 (10 mmol) in a mixture of 37% HCl
(5 mL) and glacial acetic acid (10 mL) at 0–5 ꢁC, a solution of
1
(C=O); MS (EI) m ⁄ z: 506 (M+, 2%). H NMR (DMSO-d6, 300 MHz) d
4
Chem Biol Drug Des 2012

Inhibitors of metallo-beta-lactamases
sodium nitrite (4 g, 58 mmol) in water (10 mL) was added dropwise
with continuous stirring during 30 min. After 8 h of stirring at room
temperature, the foamy mixture was diluted with water and neu-
tralized with aqueous ammonia. The solid was filtered off and
recrystallized from EtOH ⁄ H2O to give compound 10. Compound
10c prepared with this method is identical in all respects (physical
and spectral data) as reported (18).
7-(3,4-Dichlorophenyl)-5-phenyl-2-thioxo-2,3-
dihydro-1H-pyrrolo[2,3-d]pyrimidine-4(7H)-one
(11c)
Yield 75%, m.p. 138–140 ꢁC. IR (KBr) t (per cm): 3360 (NH), 1690
(C=O), 1605 (C=S). MS (EI) m ⁄ z: 387 (M+, 35Cl, 20%), 389 (M++2,
37Cl, 8.3%), 391 (M++4, 2*(37Cl), 7.5%). 1H NMR (DMSO-d6,
300MHz) d (ppm): 7.00 (s, 1H, C6-H), 7.30–8.00 (m, 8H, Ar-H), 12.20
(s, 1H, NH), 13.30 (s, 1H, NH). Anal. Calcd for C18H11Cl2N3OS
(388.270): C, 55.68; H, 2.86; Cl, 18.26; N, 10.82; O, 4.12; S, 8.26.
Found: C, 55.89; H, 2.97; Cl, 18.58; N, 10.99; O, 4.23; S, 8.49.
2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-
carboxamide (10a)
Yield 60%, m.p. 98–102 ꢁC. IR (KBr) t (per cm): 3350-3300 (NH2),
1680 (C=O). MS (EI) m ⁄ z(%): 367 (M+, 27%), 368 (M++1, 8.5%), 369
(M++2, 1.3%). 1H NMR (DMSO-d6, 300MHz) d (ppm): 5.62 (s, 2H,
CH2), 6.50 (s, 2H, NH2), 7.20–8.00 (m, 15H, Ar-H and 2H, CONH2).
Anal. Calcd for C24H21N3O (367.443): C, 78.45; H, 5.76; N, 11.44; O,
4.35. Found: C, 78.26; H, 5.59; N, 11.32; O, 4.61.
7-Aryl-5,6-disubstituted-2-(ethylthio)-3H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (12)
Compound 11 (10 mmol) was added to a warmed alcoholic solution
of sodium (0.56 g, 10 mmol) in ethanol (50 mL) and heating was
continued for 20 min. The mixture was allowed to cool to room
temperature, and alkyl halide (20 mmol) was added dropwise with
continuous stirring during 30 min. The mixture was stirred under
reflux for 5 h, monitored by TLC, allowed to cool to room tempera-
ture, and finally poured into cold water. The solid product was fil-
tered off and washed with water. The residue was dried and
recrystallized from methanol to give 12.
2-Amino-1-(3,4-dichlorophenyl)-4-phenyl-1H-
pyrrole-3-carboxamide (10c)
Yield 58%, m.p. 178–181 ꢁC. IR (KBr) t (per cm): 3330-3280 (NH2),
1670 (C=O). MS (EI) m ⁄ z(%): 346 (M+, 35Cl, 32%), 348 (M++2, 37Cl,
19%), 350 (M++4, 2*(37Cl), 5.6%). 1H NMR (DMSO-d6, 300MHz) d
(ppm): 6.60 (s, 2H, NH2), 7.00 (s, 1H, C6-H), 7.30–8.00 (m, 8H, Ar-H
and 2H, CONH2). Anal. Calcd for C17H13Cl2N3O (346.211): C, 58.98;
H, 3.78; Cl, 20.48; N, 12.14; O, 4.62. Found: C, 59.04; H, 3.96; Cl,
20.71; N, 12.47; O, 4.85.
7-Benzyl-2-(ethylthio)-5,6-diphenyl-3H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (12a)
Yield 80%, m.p. 84–86 ꢁC. IR (KBr) t (cm-1): 3330 (NH), 1680(C=O).
MS (EI) m ⁄ z(%): 437 (M+, 34%), 438 (M++1, 10.28%), 439 (M++2,
1.4%). 1H NMR (DMSO-d6, 300MHz)
d (ppm): 1.32 (t, 3H,
7-Aryl-5,6-disubstituted-2-thioxo-2,3-dihydro-
1H-pyrrolo[2,3-d]pyrimidine-4(7H)-one (11)
A mixture of compound 10 (10 mol) and thiourea (1.20 g, 20 mmol)
was refluxed in dry ethanol (20 mL) for 10 h. The reaction mixture
was evaporated under reduced pressure and the residue was
recrystallized from methanol to give 11.
J = 7.2 Hz, CH3), 3.53 (q, 2H, J = 7.2 Hz, CH2), 5.98 (s, 2H, CH2),
7.10–7.80 (m, 15H, Ar-H), 12.59 (s, 1H, NH). Anal. Calcd for
C27H23N3OS (437.556): C, 74.11; H, 5.30; N, 9.60; O, 3.66; S, 7.33.
Found: C, 74.41; H, 5.63; N, 9.80; O, 3.79; S, 7.56.
7-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-2-(ethylthio)-5,6-diphenyl-3H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (12b)
7-Benzyl-5,6-diphenyl-2-thioxo-2,3-dihydro-1H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (11a)
Yield 64%, m.p. 145–147 ꢁC. IR (KBr) t (per cm): 3450 (NH), 1700
(C=O), 1675 (C=O). MS (EI) m ⁄ z(%): 533 (M+, 31.15%), 534 (M++1,
11.2%), 535 (M++2, 2.3%). 1H NMR (DMSO-d6, 300MHz) d (ppm):
1H NMR (DMSO-d6, 300MHz) d (ppm): 1.46 (t, 3H, J = 7.5, CH3),
2.43 (s, 3H, CH3), 3.01 (q, 3H, J = 7.5, CH3), 3.21 (s, 3H, N-CH3),
7.00–7.80 (m, 15H, Ar-H), 12.49 (s, 1H, NH). Anal. Calcd for
C31H27N5O2S (533.643): C, 69.77; H, 5.10; N, 13.12; O, 6.00; S, 6.01.
Found: C, 70.01; H, 5.38; N, 16.01; O, 6.25; S, 6.35.
Yield: 80%, m.p. 92–94 ꢁC. IR (KBr) t (per cm): 3350 (NH), 1680 (C=O),
1615 (C=S). MS (EI) m ⁄ z(%): 409 (M+, 45%), 410 (M++1, 13.5%), 411
(M++2, 1.1%). 1H NMR (DMSO-d6, 300MHz) d (ppm): 5.56 (s, 2H,
CH2), 7.10–7.80 (m, 15H, Ar-H), 12.20 (s, 1H, NH), 13.30 (s, 1H, NH).
Anal. Calcd for C25H19N3OS (409.503): C, 73.32; H, 4.68; N, 10.26; O,
3.91; S, 7.83. Found: C, 73.49; H, 4.90; N, 10.42; O, 3.63; S, 7.86.
7-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-5,6-diphenyl-2-thioxo-2,3-dihydro-
1H-pyrrolo[2,3-d]pyrimidine-4(7H)-one (11b)
Yield 74%, m.p. 132–137 ꢁC. IR (KBr) t (per cm): 3390 (NH), 1690
(C=O), 1630 (C=S). MS (EI) m ⁄ z(%): 505 (M+, 70.0%), 506 (M++1,
7-(3,4-Dichlorophenyl)-2-(ethylthio)-5-phenyl-3H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (12c)
Yield 76%, m.p. 125–128 ꢁC. IR (KBr) t (cm-1): 3410 (NH), 1680 (C=O).
MS (EI) m ⁄ z (%): 416 (M+, 35Cl, 43.2%), 418 (M++2, 37Cl, 8.9%), 420
(M++4, 2*(37Cl), 2.1%). 1H NMR (DMSO-d6, 300MHz) d (ppm): 1.43 (t,
3H, J = 6.8,CH3), 3.23 (q, 3H, J = 6.8, CH3), 6.90 (s, 1H, C6-H), 7.20–
7.80 (m, 8H, Ar-H), 12.61 (s, 1H, NH). Anal. Calcd for C20H15Cl2N3OS
(416.324): C, 57.70; H, 3.63; Cl, 17.03; N, 10.09; O, 3.84; S, 7.70.
Found: C, 57.93; H, 3.89; Cl, 17.26; N, 10.18; O, 3.96; S, 7.75.
1
22.17%), 507 (M++2, 3.73%). H NMR (DMSO-d6, 300MHz) d (ppm):
2.43 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 7.20–7.80 (m, 15H, Ar-H),
12.32 (s, 1H, NH), 13.14 (s, 1H, NH). Anal. Calcd for C29H23N5O2S
(505.590): C, 68.89; H, 4.59; N, 13.85; O, 6.33; S, 6.34. Found: C,
68.89; H, 4.59; N, 13.85; O, 6.33; S, 6.34.
Chem Biol Drug Des 2012
5

Hussein et al.
7-(Aryl)-2-(chloromethyl)-5,6-disubstituted-3H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (17)
423 (M++2, 1.5%). 1H NMR (DMSO-d6, 500MHz) d (ppm): 3.02 (s,
2H, CH2-NH), 4.92 (s, 2H, NH2), 5.85 (s, 2H, CH2), 6.80 (s, 1H, NH),
7.00–7.70 (m, 15H, Ar-H), 12.45 (s, 1H, NH pyrimidine). Anal. Calcd
for C26H23N5O (421.494): C, 74.09; H, 5.50; N, 16.62; O, 3.80. Found:
C, 74.16; H, 5.63; N, 16.71; O, 3.96.
A mixture of 7 (10 mmol) and chloroacetyl chloride (5.65 g,
50 mmol) in dry dioxane (30 mL) was allowed to stir at room tem-
perature overnight and then under reflux for 5 h. The solvent was
evaporated, and the remaining mixture was triturated with ethanol.
The solid product that formed was collected by filtration and recrys-
tallized from ethanol ⁄ H2O to give 17.
7-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-2-(hydrazinylmethyl)-5,6-diphenyl-
3H-pyrrolo[2,3-d]pyrimidine-4(7H)-one (18b)
Yield 55%, m.p. 130–132 ꢁC. IR (KBr) t (per cm): 3380-3320 (NH,
NH2), 1690, 1665 (C=O). MS (EI) m ⁄ z: 517 (M+, 35%), 518 (M++1,
12%), 519 (M++2, 2.8%). 1H NMR (DMSO-d6, 500MHz) d (ppm):
2.59 (s, 3H, CH3), 3.21–3.25 (s, 5H, N-CH3 and CH2-NH), 4.90 (s, 2H,
NH2), 6.80 (s, 1H, NH), 7.20–7.80 (m, 15 H, Ar-H), 12.44 (s, 1H, NH
pyrimidine). Anal. Calcd for C30H27N7O2 (517.581): C, 69.62; H, 5.26;
N, 18.94; O, 6.18. Found: C, 69.79; H, 5.42; N, 19.08; O, 6.21.
1-(4-Amino-1-benzyl-6-methyl-2,3-diphenyl-1H-
pyrrolo[2,3-b]pyridin-5-yl) ethanone (17a)
Yield 64%, m.p. 130–135 ꢁC. IR (KBr) t (per cm): 3320 (NH), 1670
(C=O). MS (EI) m ⁄ z: 425 (M+, 65%), 426 (M++1, 15%), 427 (M++2, 37Cl,
1
23.5%). H NMR (DMSO-d6, 500MHz) d (ppm): 3.60 (s, 2H, CH2-Cl),
5.85 (s, 2H, CH2), 7.00–7.60 (m, 15H, Ar-H), 11.14 (s, 1H, NH pyrimi-
dine). Anal. Calcd for C26H2ꢁClN3O (425.909): C, 73.32; H, 4.73; Cl, 8.32;
N, 9.87; O, 3.76. Found: C, 73.43; H, 4.86; Cl, 8.41; N, 9.93; O, 3.85.
7-(Aryl)-8,9-disubstituted-3,7-dihydro-2H-
imidazo[1,2-c]pyrrolo[3,2-e]pyrimidine-2-one
(20)
A mixture of 19 (10 mmol) and ethyl bromoacetate (50 mmol) in
absolute ethanol (25 mL) containing pyridine (1–2 mL) was heated
on a water bath for 15 h. The solid product obtained after cooling
was collected by filtration and recrystallized from ethanol to give
20.
2-(Chloromethyl)-7-(1,5-dimethyl-3-oxo-2-
phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5,6-
diphenyl-3H-pyrrolo[2,3-d]pyrimidine-4(7H)-one
(17b)
Yield 61%, m.p. 110–112 ꢁC. IR (KBr) t (per cm): 3335 (NH), 1700,
1680 (C=O). MS (EI) m ⁄ z: 521 (M+, 44%), 522 (M++1, 12%), 523
1
(M++2, 37Cl, 15.5%). H NMR (DMSO-d6, 500MHz) d (ppm): 2.39 (s,
3H, CH3), 3.21 (s, 3H, N-CH3), 3.62 (s, 2H, CH2-Cl), 7.00–7.60 (m,
15H, Ar-H), 12.24 (s, 1H, NH pyrimidine). Anal. Calcd for
C30H24ClN5O2 (521.997): C, 69.03; H, 4.63; Cl, 6.79; N, 13.42; O,
6.13. Found: C, 69.10; H, 4.84; Cl, 6.88; N, 13.15; O, 5.99.
7-Benzyl-8,9-diphenyl-3,7-dihydro-2H-
imidazo[1,2-c]pyrrolo[3,2-e]pyrimidine-2-one
(20a)
Yield 52%, m.p. 128–130 ꢁC. IR (KBr) t (per cm): 1695 (C=O). MS
1
2-(Chloromethyl)-7-(3,4-dichlorophenyl)-5-phenyl-
3H-pyrrolo[2,3-d]pyrimidine-4(7H)-one (17c)
(EI) m ⁄ z: 416 (M+, 18%), 417 (M++1, 5.5%), 418 (M++2, 2.25%). H
NMR (DMSO-d6, 500MHz) d (ppm): 3.68 (s, 2H, CO-CH2), 5.88 (s,
2H, CH2), 7.00–7.70 (m, 15H, Ar-H), 8.44 (s, 1H, NH pyrimidine).
Anal. Calcd for C27H20N4O (416.474): C, 77.87; H, 4.84; N, 13.45; O,
3.84. Found: C, 77.97; H, 4.99; N, 13.63; O, 3.95.
Yield 63%, m.p. 103–105 ꢁC. IR (KBr) t (per cm): 3330 (NH), 1680
(C=O). MS (EI) m ⁄ z: 404 (M+, 35Cl, 75%), 406 (M++2, 37Cl, 51%),
408 (M++4, 2*(37Cl), 17.5%), 410 (M++6, 3*(37Cl), 4.5%). 1H NMR
(DMSO-d6, 500MHz) d (ppm): 3.46 (s, 2H, CH2-Cl), 6.70 (s, 1H, C6-H),
7.00–7.50 (m, 8H, Ar-H), 12.04 (s, 1H, NH pyrimidine). Anal. Calcd
for C19H12Cl3N3O (404.677): C, 56.39; H, 2.99; Cl, 26.28; N, 10.38;
O, 3.95. Found: C, 56.12; H, 2.63; Cl, 26.09; N, 10.25; O, 3.72.
7-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-8,9-diphenyl-3,7-dihydro-2H-
imidazo[1,2-c]pyrrolo[3,2-e]pyrimidine-2-one
(20b)
7-(Aryl)-2,5,6-trisubstituted-3H-pyrrolo[2,3-
d]pyrimidine-4(7H)-one (18)
Yield 51%, m.p. 142–144 ꢁC. IR (KBr) t (per cm): 1690, 1680 (C=O).
MS (EI) m ⁄ z: 512 (M+, 19%), 513 (M++1, 5.6%), 514 (M++2, 1.05%).
1H NMR (DMSO-d6, 500MHz) d (ppm): 2.32 (s, 3H, CH3), 3.21 (s, 3H,
N-CH3), 3.79 (s, 2H, CO-CH2), 7.00–7.60 (m, 15 H, Ar-H), 8.26 (s, 1H,
NH pyrimidine). Anal. Calcd for C31H24N6O2 (512.561): C, 72.64; H,
4.72; N, 16.40; O, 6.24. Found: C, 72.77; H, 4.89; N, 16.63; O, 6.41.
To a solution of 17 (10 mmol), hydrazine hydrate (5 mL) and pyri-
dine (2 mL) in ethanol (30 mL) were added. The reaction mixture
was refluxed for 8 h (monitored by TLC). The mixture was then
cooled to room temperature, poured onto crushed ice (25 g), and
neutralized with HCl. The precipitate was filtered off and recrystal-
lized from ethanol ⁄ H2O to give 18.
7-(3,4-Dichlorophenyl)-9-phenyl-3,7-dihydro-2H-
imidazo[1,2-c]pyrrolo[3,2-e]pyrimidine-2-one
(20c)
Yield 48%, m.p. 280–282 ꢁC. IR (KBr) t (per cm): 1680 (C=O). MS
(EI) m ⁄ z: 395 (M+, 35Cl, 22%), 397 (M++2, 37Cl, 6.5%), 399 (M++4,
2*(37Cl), 1.7%), 410 (M++6, 3*(37Cl), 4.5%). 1H NMR (DMSO-d6,
7-Benzyl-2-(hydrazinylmethyl)-5,6-diphenyl-3H-
pyrrolo[2,3-d]pyrimidine-4(7H)-one (18a)
Yield 61%, m.p. 117–120 ꢁC. IR (KBr) t (per cm): 3350-3290 (NH,
NH2), 1670 (C=O). MS (EI) m ⁄ z: 421 (M+, 32%), 422 (M++1, 11%),
6
Chem Biol Drug Des 2012

Inhibitors of metallo-beta-lactamases
500MHz) d (ppm): 3.65 (s, 2H, CO-CH2), 6.80 (s, 1H, C6-H), 7.00–
7.70 (m, 8H, Ar-H), 8.26 (s, 1H, NH pyrimidine). Anal. Calcd for
C20H12Cl2N4O (395.241): C, 60.78; H, 3.06; Cl, 17.94; N, 14.18; O,
4.05. Found: C, 60.88; H, 3.28; Cl, 17.69; N, 14.05; O, 4.36.
Vmax½Sꢁ
ꢀ
ꢁ
ꢀ
ꢁ
m ¼
ð1Þ
½Iꢁ
½Iꢁ
ic
½Sꢁ 1 þ K þ KM 1 þ K
iuc
Results and Discussion
Enzyme expression and purification
The IMP-1 enzyme, lacking the first 21 signal peptide amino acid
residues, was expressed and purified using a previously published
procedure (15,23).
Synthesis of lead compounds
The syntheses of compounds 1a–e (26–30) were achieved by con-
densing thiourea, 1,3-dicarbonyl derivatives, and an aromatic alde-
hyde in refluxing ethanol containing a few drops of 37% HCl, to
give the 5-acetyl tetrahydrothiopyrimidine-2-thione derivatives 1a–
1c, or the 5-ethoxycarbonyl derivatives 1d–e, in very good yields,
as shown in Figure 3.
Kinetic assays
Inhibition assays were performed in 96-well 400-lL multi-titer
plates using a UV ⁄ Vis multi-plate spectrophotometer. The assay
was performed in HEPES buffer (50 mM HEPES, 0.1 M NaCl,
100 lM ZnCl2, pH 7.0). The substrate CENTA (Figure 1) was syn-
thesized according to the previously published work (15,24) with
final substrate concentrations ranging between 5–70 lM. The final
concentration of IMP-1 in the assay was 2 nM. A final concentra-
tion of 20 lg ⁄ mL of bovine serum albumin was added to the
assay. Three different concentrations of the compounds 1a, 1c,
2b, 3b, 5c, 6b, 7a, 8a, 11c, 13a, 15a, and 16a were used
as illustrated in Figure 2 for compound 5c. The inhibition data
were analyzed by nonlinear regression using WinCurveFit (Kevin
Raner Software) and eqn 1, where Vmax is the maximum rate, KM
the Michaelis constant, and Kic and Kiuc the inhibition constants
for the competitive and uncompetitive inhibition modes, respec-
tively (25).
As shown in Figure 4, tetrahydropyrimidine-2-thiones 1a–c reacted
with chloroacetic acid in aqueous sodium hydroxide to afford acetic
acid derivatives 2a–c. On the other hand, when 1a–c were con-
densed with malononitrile in glacial acetic acid and ammonium ace-
tate, they give ethylidene malononitrile derivatives 3a–c. The
reactions of 1a–c with 2-methoxybenzaldehyde in ethanolic sodium
hydroxide afforded chalcone derivatives 4a–c, which were con-
verted to pyrazole derivatives 5a–c by their reactions with phenyl
hydrazine.
The ethoxycarbonyl tetrahydropyrimidine-2-thiones (1d–e) reacted
with sulfadiazine in DMF to afford amide derivatives 6a–b, as
shown in Figure 5.
The 2-amino-3-cyanopyrrole derivatives (7a–c) were prepared as
reported in our previous works (17–21). These compounds were
used for the preparation of pyrrole derivatives 8a–c through the
formation of diazonium chloride salt intermediates. Diazotization of
2-amino-pyrroles have been reported several times (31–34). Diazoti-
zation of 7a–c using a mixture of sodium nitrite and concentrated
HCl at 0–5 ꢁC followed by the addition of an ethanolic solution of
malononitrile in the presence of sodium acetate afforded the corre-
sponding hydrazono derivatives 8a–c (Figure 6). On the other hand,
diazotization of 7a–c using sodium nitrite in a mixture of acetic
acid and concentrated HCl (2:1) at 0–5 ꢁC afforded the carboxamide
derivatives 10a–c. This was unexpected, as there are many reports
(31–37) that diazotization of compounds similar to 7 gives the cor-
responding triazine derivatives (9). The formation of compounds 10
can be explained by the hydrolysis of the cyano group in acidic
medium into the amide without formation of the unstable diazonium
salt from the amino group. The pyrrole derivatives 10a–10c were
converted to the corresponding pyrrolo[2,3-d]pyrimidine-2-thiones
11a–c by heating (38) with thiourea in ethanol. Ethylation (39–41)
of 11a–c with ethyl iodide in the presence of base gave the corre-
sponding S-ethyl pyrimidine derivatives 12a–12c (Figure 6).
Figure 1: Chemical structure of CENTA.
The 2-amino-3-cyanopyrrole derivatives (7a–c) were also used for
the preparation of pyrrolopyrimidine derivatives 13–20. Condensa-
tion of 7a–c with formic acid afforded the corresponding pyrrolo-
pyrimidine-4-ones (13a–c) (Figure 7) (17–21,42). Reacting 13a–c
(17–21,42) with phosphorous oxychloride gave their corresponding
4-chloro derivatives 14a–c, which were subsequently reacted (17–
21,42) with thiourea and hydrazine hydrate, separately, to give the
Figure 2: Determination of the inhibition constants for com-
pound 5c against IMP-1 MBL (2 nM) using CENTA as the substrate.
Inhibitor concentrations are indicated on the right side. The experi-
mental data were fit to eqn 1; error bars indicate standard errors.
Chem Biol Drug Des 2012
7

Hussein et al.
Figure 3: Synthesis of com-
pounds 1a–e.
Figure 4: Synthesis of com-
pounds 2–5.
afforded the corresponding pyrrolopyrimidines 19a–c. Heating
19a–c with ethyl bromoacetate and pyridine in ethanol solution
yielded the corresponding imidazopyrrolopyrimidine-2-ones 20a–c
(Figure 7).
Enzymatic inhibition assays
Based on the compounds' solubility under the assay conditions, the
inhibitory effects of 10 thiopyrimidine derivatives 1a–e, 2b, 3b,
4c, 5c, and 6b (Table 1) and 17 pyrrole derivatives 7a–c, 8a–b,
10c, 11c, 13a–b, 15a–c, 16a–c, 18a, and 20b (Table 2) on
the catalytic activity of IMP-1 were initially assessed by measuring
enzyme activity in the absence and presence of 10 lM of each
compound. A previously developed assay based on the chromogenic
cephalosporin substrate CENTA was used (15,24). 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 k = 405 nm (at pH 7.0, e = 6400 ⁄ M ⁄ cm). The results
are presented in Tables 1 and 2.
Figure 5: Synthesis of compounds 6a–b.
corresponding pyrrolopyrimidine-4-thione (15a–c) and 4-hydrazino
derivatives (16a–c), respectively.
Heating (43,44) 7a–c with chloroacetyl chloride in refluxing dioxane
afforded the pyrrolopyrimidine-4-one derivatives 17a–c, which on
reaction with hydrazine hydrate in refluxing ethanol gave the hydra-
zinomethyl derivatives 18a–c. The enaminonitrile moiety in com-
pound 7a–c proved to be highly reactive toward other nitrogen
nucleophiles. Thus, the reaction of 7a–c with formamide (17–21)
Twelve compounds, six thiopyrimidine derivatives (1a, 1c, 2b, 3b,
5c, and 6b), and six pyrrole derivatives (7a, 8a, 11c, 13a, 15a,
and 16a) showed promising inhibition at a concentration of 10 lM.
8
Chem Biol Drug Des 2012

Inhibitors of metallo-beta-lactamases
Figure 6: Synthesis of com-
pounds 7–12.
Figure 7: Synthesis of compounds 13–20.
Therefore, they were selected for further analysis to determine their
mode of inhibition and the magnitude of the corresponding inhibi-
tion constant(s) (Ki values).
thus implying alternative possible binding interactions with the
enzyme. Compound 5c is the most potent inhibitor of IMP-1, with a
Kic of 19 lM. For comparison, the Kic of the known MBL inhibitor
L-captopril is 12.5 € 2.4 lM (15), and those of compounds discov-
As shown in Table 3, compounds 1a, 5c, 7a, 8a, and 11c are the
strongest inhibitors for IMP-1; all have inhibition constants of
ꢀ20 lM. The remaining compounds in Table 3 are also good inhibi-
tors with inhibition constants ranging from 36 to 148 lM. Com-
pounds 1a, 5c, 6b, and 7a are purely competitive inhibitors in
this group with all the others displaying a mixed mode of inhibition,
ered from a fragment-based screening approach range from
ꢀ500 lM to ꢀ1600 lM (15). The inhibition constants for the com-
pounds reported here are also of comparable efficiency to a range
of pyrrole derivatives reported previously (44). It is noted that, in
general, the weaker the inhibitory effect is, the larger is the error
associated with the corresponding inhibition constants (Table 3).
Chem Biol Drug Des 2012
9

Hussein et al.
Table 1: Percentage inhibition of IMP-1 MBL (2 nM) at pH 7.0 by thiopyrimidine derivatives using CENTA (70 lM) as substrate
Inhibition
Inhibition
Compound
Structure
% (10 lM)
Compound
Structure
% (10 lM)
OCH3
1a
29
2b
12
20
0
O
O
HN
CH3
CH3
HN
CH3
HO
O
S
N
H
S
N
CH3
OCH3
O
OCH3
NC
1b
1c
1d
1e
0
27
0
3b
4c
5c
6b
CN
CH3
HN
HN
CH3
S
N
H
CH3
S
N
H
CH3
H3CO
O
O
HN
CH3
HN
S
N
H
CH3
S
N
CH3
H
OCH3
O
17
H3CO
N
OEt
HN
HN
N
S
N
H
CH3
S
N
H
CH3
N
N
O
0
12
NH
O
S
O
O
N
H
OEt
HN
HN
S
N
H
S
N
CH3
CH3
H
MBL, metallo-b-lactamase.
To investigate the possible binding modes of the most potent
inhibitor 5c to IMP-1, in silico docking was employed using Mo-
legro Virtual Docker (45). The chemical structures of the two e-
nantiomers of 5c were drawn in ChemBioOffice, and their
energies were minimized, and the resulting structures were saved
in SYBL format (46,47). Molegro Virtual Docker was also used to
modify the charge of the sulfur atom of the thiopyrimidine ring
in 5c to be negative (48), and the three bonds in the thiourea
linkage in the same ring were modified to be delocalized bonds.
The IMP-1 crystal structure (PDB Code: 1JJT) (47) was used in
these docking studies. The crystal structure was modified for
docking simulations by allowing residues Glu60, Val61, Asn62,
Gly63, and Trp64 in a loop close to the active site to be flexible
(49).
10
Chem Biol Drug Des 2012

Inhibitors of metallo-beta-lactamases
Table 2: Percentage inhibition of IMP-1 MBL (2 nM) at pH 7.0 by pyrrole derivatives using CENTA (70 lM) as substrate
Inhibition
Inhibition
Compound
Structure
% (10 lM)
Compound
Structure
% (10 lM)
7a
17
15a
11
S
Ph
Ph
CN
NH2
Ph
NH
N
N
Ph
N
CN
N
S
N
Ph
7b
0
15b
15c
16a
16b
16c
0
Ph
Ph
NH
NH2
CH3
Ph
N
N
N
O
O
CH3
N
N
CH3
Ph
CH3
Ph
S
Ph
CN
7c
7
0
Ph
NH
NH2
N
N
N
Cl
Cl
Cl
Cl
H2NHN
N
8a
57
17
Ph
Ph
CN
CN
CN
N
Ph
N
N
N
H
N
Ph
NHNH2
Ph
N
Ph
N
8b
6
5
CN
CN
N
N
Ph
Ph
O
N
CN
N
N
N
H
CH3
O
CH3
N
N
Ph
Ph
CH3
CH3
O
H2NHN
Ph
Ph
10c
0
11
NH2
NH2
N
N
N
N
Cl
Cl
Cl
Cl
Chem Biol Drug Des 2012
11

Hussein et al.
Table 2: Continued
Inhibition
Inhibition
Compound
Structure
% (10 lM)
Compound
Structure
% (10 lM)
11c
15
15
0
18a
6
O
O
Ph
Ph
Ph
NH
NH
S
CH2NHNH2
N
N
N
H
N
Cl
Cl
O
N
O
Ph
13a
20b
0
Ph
NH
N
Ph
O
N
Ph
N
N
N
CH3
N
N
CH3
Ph
O
Ph
13b
NH
Ph
N
N
O
CH3
N
N
Ph
CH3
MBL, Metallo-b-lactamase.
The lowest energy-binding orientation of (R)-5c in the active site of
IMP-1 is shown in Figure 8. The modeling indicates that (R)-5c
binds to the two zinc ions via the thiolate anion with sulfur–metal
distances of 2.2 ꢀ (Zn1) and 2.0 ꢀ (Zn2). Apart from these interac-
tions with the metal ions, one of the nitrogen atoms of the thiopyr-
imidine ring forms hydrogen bond to the nitrogen atom of the side
chain of Lys224. Furthermore, there are hydrophobic interactions
between the isopropyl group of Val67 and the methoxybenzene moi-
ety. Furthermore, the interactions between the benzene ring of
Phe87 and the isopropyl group of Val61 with the benzene ring of
the styryl group also contribute to inhibitor binding (Figure 9).
docking score between the two enantiomers of 5c, which indicates
the more favorable binding energy for the (R) isomer.
To investigate the binding mode of the pyrrole series, the most potent
inhibitor of pyrrole derivatives against IMP-1, 7a with a Kic of 21 lM,
was docked into the active site of IMP-1 by using Molegro Virtual
Docker as discussed before. The modeling illustrates that 7a binds
within the active site, close to the two zinc atoms. The nitrogen atom
of the amino group of 7a is located close to both metal ions in the
active site (Zn1: 4.0 ꢀ; Zn2: 3.3 ꢀ) (Figure 12).
One of the protons on the amino group of 7a forms a hydrogen
bond with the nitrogen atom on the imidazole ring of His197 (N–N
distance 3.0 ꢀ) (Figure 13). Apart from this interaction, two hydro-
phobic interactions between 7a and Val67 and Phe87 of IMP-1
may play an important role in enhancing the binding affinity of the
inhibitor (Figure 13). The binding mode shown in Figures 10 and 11
represents competitive inhibition, as the close proximity of com-
pound 7a to the metal ions implies that the access of a substrate
to the catalytically relevant binuclear metal center is blocked.
In contrast, the lowest energy conformation of (S)-5c occupies the
active site, close to the two zinc ions, but without any significant
binding between the inhibitor and either of the two zinc ions (Fig-
ure 10). However, two hydrogen bonds are observed between the
inhibitor and the enzyme-one between the oxygen of the methoxy
group and the backbone N-H of Asn233, and the second between one
of the N atoms of the thiopyrimidine ring of 5c and the nitrogen atom
of the side chain of Lys224. Furthermore, there are hydrophobic inter-
actions between both the isopropyl group of Val61 and the benzene
ring of residue Phe87 in IMP-1 and the benzene ring attached to the
nitrogen atom of the pyrazole moiety in 5c, between the isopropyl
group of Val67 and the benzene ring of the styryl moiety, and also
between the imidazole ring of His118 and the benzene ring attached
to the methoxy group (Figure 11).There is a large difference in the
Conclusion
We have described a straightforward and efficient synthesis of thio-
pyrimidine and pyrrole derivatives that act as inhibitors of the MBL
12
Chem Biol Drug Des 2012

Inhibitors of metallo-beta-lactamases
Figure 10: Surface view of the IMP-1 active site with the low-
est energy conformation of (S)-5c docked into the active site. Atom
colors are as follows: blue – nitrogen, red – oxygen, white – car-
bon (on IMP-1); green – carbon, yellow – sulfur (on inhibitor);
magenta – zinc active site metals. The flexible loop consisting of
residues Asn62, Gly63, Trp64, and Asn233 has been omitted for
clarity.
Figure 8: Surface view of the IMP-1 active site with the lowest
energy conformation of (R)-5c docked into the active site. Atom col-
ors are as follows: blue – nitrogen, red – oxygen, white – carbon
(on IMP-1); green – carbon, yellow – sulfur (on inhibitor); magenta
– zinc active site metals. The flexible loop consisting of residues
Asn62, Gly63, Trp64, and Asn233 has been omitted for clarity.
Figure 11: Surface view of the IMP-1 active site for the high-
est docking score conformation of IMP-1 with (S)-5c docked into
the active site shows (i) the hydrogen bond (3.1 ꢀ) between the
oxygen of the methoxy group of (S)-5c and the backbone N-H of
Asn233, (ii) the hydrogen bond (2.6 ꢀ) between one of the N atoms
of the thiopyrimidine ring of (S)-5c and the nitrogen atom of the
side chain of Lys224, (iii) the hydrophobic interactions between both
the isopropyl group of Val61 and the benzene ring of Phe87 in IMP-
1 and the benzene ring attached to the nitrogen atom of the pyraz-
ole moiety in (S)-5c, (iv) the hydrophobic interaction between the
isopropyl group of Val67 and the benzene ring of the styryl moiety
of (S)-5c, and (v) the hydrophobic interaction between the imidazole
ring of His118 and the benzene ring attached to the methoxy group
of (S)-5c. Atom colors are as follows: blue – nitrogen, red – oxy-
gen, white – carbon (on IMP-1), green – carbon (on inhibitor),
magenta – zinc ions.
Figure 9: Surface view of the IMP-1 active site for the highest
docking score conformation of IMP-1 with (R)-5c docked into the
active site shows (i) the hydrogen bond (3.2 ꢀ) between the amino
group of (R)-5c and the nitrogen atom of the side chain of Lys224,
(ii) the hydrophobic interaction between the isopropyl group of Val67
and the methoxybenzene moiety, and (iii) the hydrophobic interaction
between the benzene ring of Phe87 and the isopropyl group of
Val61 with the benzene ring of the styryl group of (R)-5c. Atom col-
ors are as follows: blue – nitrogen, red – oxygen, white – carbon
(on IMP-1), green – carbon (on inhibitor), magenta – zinc ions.
IMP-1. The inhibitory effects of 27 compounds 1a–e, 2b, 3b, 4c,
5c, 6b, 7a–c, 8a–b, 10c, 11c, 13a–b, 15a–c, 16a–c, 18a,
and 20b were tested against IMP-1. The thiopyrimidine derivatives
1b, 1d, 1e, and 4c and the pyrrole derivatives 7b, 10c, 13b,
15b, 15c, and 20b had no activity. In thiopyrimidine derivatives,
compound 5c showed the most potent inhibitory effect
(Kic = 19 € 9 lM) with a purely competitive mode of inhibition. The
inhibitory potency of 5c is similar to the best known MBL inhibi-
tors, L-captopril (Kic = 12.5 € 2.4 lM) and a pyrrole compound we
recently reported (Kic ꢀ 10 lM) (16). In silico docking of the (R) and
Chem Biol Drug Des 2012
13

Hussein et al.
Table 3: Competitive (Kic) and uncompetitive (Kiuc) inhibition con-
stants for the inhibition of IMP-1 MBL by the most potent inhibitors
Compound Kic (lM)
Kiuc (lM)
Compound Kic (lM)
Kiuc (lM)
1a
1c
29 € 16
176 € 332 110 € 58
–
7a
8a
21 € 10
50 € 28
23 € 8
58 € 64
148 € 248 39 € 10
36 € 20 50 € 26
–
21 € 4
47 € 12
58 € 25
2b
3b
235 € 665
38 € 59
19 € 9
74 € 38
12 € 2.4
70 € 25
82 € 141 13a
–
–
–
11c
5c
6b
15a
16a
L-Captopril
MBL, metallo-b-lactamase.
Experiments were carried out in a duplicate mode.
Figure 12: Surface view of the IMP-1 active site for the high-
est docking score conformation of IMP-1 with 7a docked into the
active site. For clarity, atom colors are as follows: blue – nitrogen,
red – oxygen, white – carbon (on IMP-1), green-carbon (on inhibi-
tor), magenta-zinc active site metals.
1b dramatically decreases the inhibitory effect. The introduction of
the dicyanide moiety (i.e., as in 3b) results in mixed-mode inhibi-
tion kinetics (3b: Kic = 38 € 59 lM and Kiuc = 82 € 141 lM). The
introduction of a sulfadiazine group in 6b also results in consider-
able improvement in the inhibitory effect while maintaining a com-
petitive mode of inhibition (6b: Kic = 38 € 59 lM). In contrast, the
introduction of an acetic acid moiety to the sulfur atom in 2b
improves the inhibitory effect, but the uncompetitive mode of inhibi-
tor binding becomes dominant (2b: Kic = 235 € 665 lM and
Kiuc=70 € 25 lM). Resolution of the two enantiomers of 5c and
their individual testing in these assays will allow us to validate the
modeling described here, with our prediction that the (R)-enantio-
mer will be a much more potent MBL inhibitor than the (S)-enantio-
mer.
On the other hand, in the pyrrole series, compound 7a showed the
most potent inhibition against IMP-1 (Kic 21 € 10 lM). The analysis
of the highest docking score conformation of 7a, in the active site
of IMP-1, shows an interaction between the nitrogen atom of the
amino group to the two zinc ions, along with further contributions
involving hydrogen bonds and hydrophobic interactions between the
inhibitor and various amino acid side chains in the active site.
Based on Ki values, in silico docking, and SAR, we propose that
the presence of 2-amino-3-cyanopyrrole nucleus, illustrated by 7a,
is crucial for achieving tight inhibitor binding. Introducing further
substituents to the benzyl group and the two phenyl groups, the
potency of 7a against IMP-1 may be significantly enhanced. Steps
toward substantiating these hypotheses are currently in progress.
In summary, thiopyrimidine and pyrrole compounds have been
shown to be promising leads in the development of novel MBL
inhibitors.
Figure 13: Surface view of the IMP-1 active site for the high-
est docking score conformation of IMP-1 with 7a docked into the
active site shows (i) the hydrogen bond (3.0 ꢀ) between the amino
group of 7a and the nitrogen atom on the imidazole ring of His197,
(ii) the hydrophobic interaction between the benzene ring attached
to position 4 of pyrrole and the isopropyl group on Val67, and (ii)
the hydrophobic interaction between the benzene ring attached to
position 5 of the pyrrole moiety and both the isopropyl group on
Val67 and the benzene ring of Phe87. Atom colors are as follows:
blue – nitrogen, red – oxygen, white – carbon (on IMP-1), green –
carbon (on inhibitor), magenta – zinc ions.
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