Synthesis and antimicrobial activities of a novel series of heterocyclic α-aminophosphonates

Article abstract of DOI:10.1002/ardp.201200109

Two series of novel α-aminophosphonates having heterocyclic moieties were synthesized in high yields. The structures of the newly synthesized compounds were confirmed by their elemental analyses, IR, ¹H NMR and MS spectral data. These compounds

Full text of DOI:10.1002/ardp.201200109

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
1
Full Paper
Synthesis and Antimicrobial Activities of a Novel Series of
Heterocyclic a-Aminophosphonates
Mohamed F. Abdel-Megeed1, Badr E. Badr2, Mohamed M. Azaam1, and Gamal A. El-Hiti1,3
1 Faculty of Science, Department of Chemistry, Tanta University, Tanta, Egypt
2 Faculty of Science, Department of Botany, Tanta University, Tanta, Egypt
3 School of Chemistry, Cardiff University, Cardiff, UK
Two series of novel a-aminophosphonates having heterocyclic moieties were synthesized in high
yields. The structures of the newly synthesized compounds were confirmed by their elemental
analyses, IR, 1H NMR and MS spectral data. These compounds were screened for their
antibacterial activities against Escherichia coli (NCIM2065) as a Gram-negative bacterium, Bacillus
subtilis (PC1219) and Staphylococcus aureus (ATCC25292) as Gram-positive bacteria, and Candida
albicans and Saccharomyces cerevisiae as fungi. The minimum inhibitory concentrations (MICs) of the
synthesized compounds show high antibacterial and antifungal activities at low concentrations
(10–1000 mg/mL). Furthermore, their lethal doses indicated that such compounds are safe for use
as antimicrobial agents.
Keywords: a-Aminophosphonates / 2-Amino-4-chloro-6-methylpyrimidine / 3-Aminoquinazolin-4(3H)-one /
Antimicrobial activities / Lethal dose
Received: March 18, 2012; Revised: June 3, 2012; Accepted: June 19, 2012
DOI 10.1002/ardp.201200109
Introduction
have reported the successful synthesis, antimicrobial and
anticancer activities of a novel series of a-aminophospho-
nates [41]. The present work was aimed to synthesize novel
a-aminophosphonates containing quinazolin-4(3H)-one and
pyrimidine moieties with the hope that new antimicrobial
agents could be developed. We now report the successful
synthesis of a range of a-aminophosphonates and their anti-
microbial activities against Gram-negative and Gram-positive
bacteria and fungi.
a-Aminophosphonates are among the most common and
biologically active organophosphorus compounds [1–3].
a-Aminophosphonates and in particular the ones having
heterocyclic moieties show very interesting biological
activities and have been used as peptide mimics, inhibitors
of serine hydrolase, enzyme inhibitors, antibiotics, anti-
bacterial, antifungal, anticancer, anti-HIV and herbicidal
agents [4–20].
Various synthetic processes have been developed for the Results and discussion
production of a-aminophosphonates [21–27]. However, the
Chemistry
most efficient method involves the one-pot Mannich-type [28]
process of carbonyl compounds, amines and diphenyl
phosphite in the presence of a Lewis acid catalyst. Such
process is simple, general, high yielding and accommodates
various substituents into aminophosphonates [29–32].
We have previously reported efficient syntheses of various
biologically active heterocyclic compounds [33–40] as part of
our continuing interest in organic synthesis. Recently, we
Two series a-aminophosphonates were synthesized in which
heterocyclic amines namely 3-aminoquinazolin-4(3H)-one (1)
and 2-amino-4-chloro-6-methyl pyrimidine (2) were used.
Reactions of 3-aminoquinazolin-4(3H)-one (1; 4 molar equiva-
lents) with various aromatic aldehydes namely 2,4-dihydroxy-
benzaldehyde, 4-chlorobenzaldehyde and 4-nitrobenzaldehyde
(2 molar equivalents) and triphenylphosphite (3 molar equiv-
alents) in the presence of lithium perchlorate as a Lewis acid
catalyst in dichloromethane (DCM) at room temperature for
28–36 h gave the corresponding diphenyl (aryl)(4-oxoquina-
zolin-3(4H)-ylamino)methylphosphonates 3–5 (Scheme 1) in
70–83% yields after crystallization from ethanol.
Correspondence: Gamal A. El-Hiti, School of Chemistry, Cardiff
University, Main Building, Park Place, Cardiff CF10 3AT, UK.
E-mail: el-hitiga@cardiff.ac.uk
Fax: þ442920870600
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
M. F. Abdel-Megeed et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
O
O
N
O
H
N
OPh
NH2
N
P
P(OPh)3, LiClO4
N
OPh
Ar CHO
+
_
Scheme 1. Synthesis of diphenyl (aryl)(4-
oxoquinazolin-3(4H)-ylamino)methylphos-
phonates 3–5.
Ar
DCM, RT, 28 36 h
N
_
1
Ar = 2,4-di-HOC6H3, 4-ClC6H4, 3-NO2C6H4
3 5
The structures of a-aminophosphonates 3–5 were con-
Antimicrobial activities
firmed by various spectroscopic techniques including IR,
1H NMR and mass spectral data and their purities were
confirmed by elemental analyses. The IR spectra of 3–5 are
characterized by the presence of absorption bands within the
3471–3399 cmꢀ1 region corresponding to the stretching
vibrations of the NH and/or OH groups. The absorption bands
in the 1673–1659 cmꢀ1 region are due to the symmetric
The antimicrobial agents available on the market have
various drawbacks such as toxicity, narrow spectrum of
activity and some also exhibit drug–drug interactions. In
view of the high incidence of infections in immune com-
promised patients, demands for new antimicrobial agents
with a broad spectrum of activity and good pharmacokinetic
properties have increased [42].
stretching vibrations of the carbonyl groups, while the
ꢀ1
The bands in the 1396–1363 cmꢀ1 and 834–824 cmꢀ1 regions
a-Aminophosphonates 3–8 along with their starting
materials 1 and 2 were screened for their in vitro antibacterial
and antifungal activities against Escherichia coli (NCIM2065) as
a Gram-negative bacterium, Bacillus subtilis (PC1219) and
Staphylococcus aureus (ATCC25292) as Gram-positive bacteria
and Candida albicans and Saccharomyces cerevisiae as fungi.
The inhibition zones were measured in triplicates and the
results are reported in Table 1.
–
C N groups absorbed within the 1598–1515 cm region.
–
–
are due to the stretching vibrations of the P O and P–O–C
–
1
groups, respectively. The H NMR spectra showed a charac-
teristic CH doublet signal (J ¼ 16–18 Hz) within the
d ¼ 7.60–5.59 ppm region. They also showed an exchange-
able singlet signal that resonated in the d ¼ 10.56–9.45 ppm
region due to the NH protons. The structures of 3–5 were
confirmed further by mass spectral data. The electron impact
and the electrospray mass spectra of 3–5 indicated the
presence of molecular ion or pseudo molecular ion peaks
([MꢀH]þ). Moreover, the elemental analyses of 3–5 were con-
sistent with the suggested structures (see Experimental
Section for details).
The results show that compounds 3–5 showed moderate
antimicrobial activities except for B. subtilis. Compounds 6–8
showed high antimicrobial activities against all the tested
organisms. Also, it is clear that a-aminophosphonates 3–8
are more active than the corresponding starting materials
1 and 2.
Compounds 1–8 showed less antibacterial activities com-
pared to the standard drug (ciprofloxacin; MIC ¼ 5 mg/mL).
On the other hand, compounds 3, 6 and 8 showed high
antifungal activities against Candida albicans compared to
the standard drug (amphotericin B; MIC ¼ 15 mg/mL). Also,
compounds 4, 6 and 8 showed high antifungal activities
against S. cerevisiae compared to amphotericin B.
Similarly, it was found that reactions of 2-amino-4-chloro-6-
methyl pyrimidine (2) with aromatic aldehydes (2,4-dihydrox-
ybenzaldehyde, 4-chlorobenzaldehyde and 4-nitrobenzalde-
hyde) and triphenylphosphite in the presence of lithium
perchlorate in DCM under conditions similar to those
used in Scheme 1 gave the corresponding diphenyl (aryl)(4-
chloro-6-methylpyrimidin-2-ylamino)methylphosphonates
6–8 (Scheme 2) in 69–86% yield.
Minimum inhibitory concentrations
The structures of a-aminophosphonates 6–8 were con-
firmed by IR, 1H NMR and mass spectral data. The purity
of products was confirmed by their elemental analyses (see
Experimental Section for details).
The minimum inhibitory concentrations (MICs) of com-
pounds 1 and 2 and the newly synthesized a-aminophos-
phonates 3–8 were determined for each antimicrobial
agent by using the agar diffusion method. The inhibition
Ar
O
Cl
N
NH2
Cl
N
NH
P
P(OPh)3, LiClO4
OPh
PhO
Ar CHO
N
+
N
_
DCM, RT, 34 44 h
Scheme 2. Synthesis of diphenyl (aryl)(4-
chloro-6-methylpyrimidin-2-ylamino)methyl-
phosphonates 6–8.
Me
2
Me
6 8
_
Ar = 2,4-di-HOC6H3, 4-ClC6H4, 3-NO2C6H4
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Antimicrobial Activities of Heterocyclic a-Aminophosphonates
3
Table 1. Antimicrobial activities of compounds 1–8.a)
Compound
Inhibition zone diameter (mm)b)
LD50 (mg/mL)
Bacteria
Fungi
E. coli
S. aureus
B. subtilis
C. albicans
S. cerevisiae
1
2
3
4
5
6
7
8
6 ꢁ 1.1
8 ꢁ 0.5
10 ꢁ 1.0
10 ꢁ 1.0
21 ꢁ 3.0
20 ꢁ 1.9
18 ꢁ 2.5
25 ꢁ 4.0
17 ꢁ 2.0
18 ꢁ 1.6
–
8 ꢁ 0.5
ꢀ
8 ꢁ 0.8
7 ꢁ 1.0
10 ꢁ 1.0
10 ꢁ 1.0
16 ꢁ 0.2
17 ꢁ 1.0
14 ꢁ 2.3
22 ꢁ 2.2
14 ꢁ 1.5
20 ꢁ 1.9
38 ꢁ 3
175 ꢁ 12
2045 ꢁ 205
175 ꢁ 13
100 ꢁ 8
20 ꢁ 1.5
10 ꢁ 1.0
7 ꢁ 1.0
14 ꢁ 3.0
12 ꢁ 1.5
10 ꢁ 1.1
15 ꢁ 2.0
10 ꢁ 0.9
17 ꢁ 1.2
ꢀ
ꢀ
20 ꢁ 3.0
10 ꢁ 1.0
10 ꢁ 1.2
10 ꢁ 1.2
9 ꢁ 0.8
10 ꢁ 1.7
843 ꢁ 76
7841 ꢁ 405
4688 ꢁ 229
a)
The antimicrobial activities were measured at 10 mg/mL concentration.
DMSO was added to different organisms as control and showed no inhibition zone.
b)
Table 2. Minimum inhibitory concentrations of compounds 1–8.a)
Compd
Minimum inhibitory concentrations (MICs; mg/mL)
E. coli
S. aureus
B. subtilis
C. albicans
S. cerevisiae
Mean MICs (mg/mL)
1
2
3
4
5
6
6
8
1000 ꢁ 98
1000 ꢁ 101
10 ꢁ 1.2
1000 ꢁ 98
100 ꢁ 12
10 ꢁ 1.1
50 ꢁ 5.0
1000 ꢁ 101
10 ꢁ 1.0
50 ꢁ 4.0
10 ꢁ 0.7
1000 ꢁ 98
1000 ꢁ 101
1000 ꢁ 100
1000 ꢁ 100
1000 ꢁ 102
10 ꢁ 1.0
1000 ꢁ 100
1000 ꢁ 100
10 ꢁ 1.0
1000 ꢁ 100
100 ꢁ 14
50 ꢁ 4.5
10 ꢁ 1.0
1000 ꢁ 98
10 ꢁ 2.0
50 ꢁ 5.0
10 ꢁ 1.0
1000 ꢁ 98.8
640 ꢁ 65.6
216 ꢁ 21.5
252 ꢁ 23.8
820 ꢁ 80.6
10 ꢁ 1.3
100 ꢁ 11
1000 ꢁ 101
10 ꢁ 1.0
100 ꢁ 2.0
100 ꢁ 1.0
10 ꢁ 1.5
100 ꢁ 10
100 ꢁ 9.0
1000 ꢁ 97
100 ꢁ 0.9
100 ꢁ 9.0
10 ꢁ 1.2
260 ꢁ 25.0
46 ꢁ 2.6
a)
The standard antibiotics were ciprofloxacin for bacteria (MIC ¼ 5 mg/mL) and amphotericin B for fungi (MIC ¼ 15 mg/mL).
zone was measured in triplicates in four different concen-
trations (10–1000 mg/mL) and the mean value ꢁ standard
deviation (SD) [43] is recorded in Table 2.
brine shrimp lethality bioassay. The LD50 of compounds
1–8 are shown in Table 1.
It was found that the number of living Artemia was
decreased by increasing the concentration of 1–8. The lethal
doses of the starting materials 1 and 2 were low (38 and
175 mg/mL, respectively).
Table 2 shows that all compounds exhibited high antimi-
crobial activities at a concentration of 10–1000 mg/mL for all
microorganisms. Compound 3 was found to be the most
effective within the first series of a-aminophosphonates
3–5 in which the order of activity was as follows:
3 > 4 > 5. For the second series (i.e., compounds 6–8)
compound 6 was the most effective compound and the order
of activity within this series was as follows: 6 > 8 > 7.
Compound 3 has no effect on Artemia at low concentrations
(10 and 100 mg/mL) and killed up to 85% of Artemia at a
higher concentration (1000 mg/mL). Compounds 4 and 5
showed no effect on Artemia at lower concentration
(10 mg/mL) and the number of living Artemia was reduced
to 50–55% at a higher concentration (100 mg/mL). At an
even higher concentration (1000 mg/mL) all Artemia were
killed. Compound 3 was found to be the safest compound
within the first series of a-aminophosphonates 3–5 in which
the lethal dose was 2045 mg/mL. The lethal doses for
compounds 4 and 5 were found to be 175 and 100 mg/mL,
respectively.
The lethal dose
Cytotoxic anticancer substances have unique problems that
come primarily from the lack of safety and side effects.
Therefore, the cytotoxicity lethal doses (LD50) of compounds
1 and 2 and the newly synthesized a-aminophosphonates 3–8
were determined on the larvae of Artemia salina using the
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M. F. Abdel-Megeed et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
reduced pressure to give the crude product which was recrystal-
lized from ethanol to give the pure products 3–8 in high yields as
white solids.
Compounds 6–8 have no effect on Artemia at low concen-
trations (10 and 100 mg/mL). At a high concentration
(1000 mg/mL), compound 6 reduced the living Artemia to
25%, while compounds 7 and 8 reduced the living Artemia
to 35 and 15%, respectively. a-Aminophosphonates 6–8 were
found to be very safe to be used since their lethal doses were
high (843, 7841 and 4688 mg/mL, respectively).
Diphenyl (2,4-dihydroxyphenyl)(4-oxoquinazolin-3(4H)-
ylamino)methylphosphonate (3)
Reaction time: 28 h, yield: 83%; mp: 240–2428C; IR (KBr): 34ꢀ711
–
–
–
–
(NH/OH), 1659 (C O), 1515 (C N), 1363 (P O), 834 (P–O–C) cm
;
–
–
1H NMR (DMSO-d6): d 10.56 (br s, exch., 1H, NH), 9.14 (s, exch., 1H,
OH), 9.01 (s, exch., 1H, OH), 8.54–7.60 (m, 18 H, Ar–H), 6.41
(d, J ¼ 17 Hz, 1H, CH); EI-MS: m/z (%) 514 ([MꢀH]þ, 25), 430 (19),
146 (59), 76 (100); Anal. Calcd. for C27H22N3O6P (515.45): C, 62.91;
H, 4.30; N, 8.15; P, 6.01. Found: C, 62.92; H, 4.32; N, 8.19; P, 6.05.
Conclusion
We developed a convenient process for the synthesis of
various a-aminophosphonates having quinazolin-4(3H)-one
and pyrimidine moieties in high yields. The antimicrobial
activities of the newly synthesized compounds show high
activities against Gram-positive, Gram-negative bacteria and
fungi at low concentrations. Their lethal doses indicated that
the synthesized compounds are safe and are promising for
their use as in vivo antimicrobial agents.
Diphenyl (4-chlorophenyl)(4-oxoquinazolin-3(4H)-
ylamino)methylphosphonate (4)
Reaction time: 30 h, yield: 75%; mp: 170–1728C; IR (KBr): 3424
(NH), 1672 (C O), 1595 (C N), 1396 (P O), 825 (P–O–C) cmꢀ1; 1H
–
–
–
–
–
–
NMR (DMSO-d6): d 9.45 (br s, exch., 1H, NH), 8.65–7.74 (m, 19H,
Ar–H), 7.60 (d, J ¼ 16 Hz, 1H, CH); ESþ–MS: m/z (%) 519 ([M37Cl]þ,
2), 517 ([M35Cl]þ, 6), 376 (100), 339 (16), 249 (7), 237 (26), 232 (13),
229 (11); Anal. Calcd. for C27H21ClN3O4P (517.90): C, 62.62; H, 5.09;
N, 8.11; P, 5.98. Found: C, 62.72; H, 4.87; N, 8.15; P, 5.95.
Experimental
General experimental
Diphenyl (3-nitrophenyl)(4-oxoquinazolin-3(4H)-
ylamino)methylphosphonate (5)
Melting point determinations were performed by the open capil-
lary method using an Electrothermal MEL-TEMP II apparatus and
are reported uncorrected. IR spectra were recorded on a Perkin-
Elmer 1430 spectrophotometer using the KBr disc technique.
1H NMR spectra were recorded on a Bruker AC400 spectrometer
operating at 400 MHz. The spectra were recorded in DMSO-d6.
Chemical shifts d are reported in parts per million (ppm) relative
to TMS. Assignments of signals are based on integration values
and expected chemical shift values and have not been rigorously
confirmed. EI mass spectra were recorded at energy 70 eV with
Reaction time: 36 h, yield: 70%; mp: 190–1928C; IR (KBr): 33ꢀ919
–
–
–
–
(NH), 1673 (C O), 1526 (C N), 1393 (P O), 824 (P–O–C) cm
;
–
–
1H NMR (DMSO-d6): d 9.61 (br s, exch., 1H, NH), 8.65–7.53 (m, 19H,
Ar–H), 5.92 (d, J ¼ 16 Hz, 1H, CH); EI–MS: m/z (%) 528 (Mþ, 2), 493
(11), 419 (23), 345 (72), 270 (88), 197 (100) and 150 (92); ESþ–MS:
m/z (%) 528 (Mþ, 52), 474 (12), 415 (16), 403 (13), 295 (9), 243 (15),
202 (100), 188 (17), 147 (16); Anal. Calcd. for C27H21N4O6P
(528.45): C, 61.37; H, 4.01; N, 10.60; P, 5.86. Found: C, 61.35;
H, 4.03; N, 10.63; P, 5.85.
a
7070 EQ mass spectrometer. Electrospray (ES) analyses
were performed on a ZQ4000 spectrometer in both positive
and negative ionization modes. Microanalysis was performed
by analytical service at both the Universities of Tanta and
Cairo, Egypt. Analytical thin layer chromatography (TLC) was
performed on EM silica gel F254 sheets (0.2 mm) with petroleum
ether (40–608C)/acetone (5:2 by volume) as a developing eluent.
The spots were detected with a UV lamp model UV GL-58.
Reagents and solvents were obtained from commercial sources
and used without purification.
3-Aminoquinazoline-4(3H)-one (1) was prepared according to
the literature procedure, mp 208–2108C (lit. 209–2108C) [44].
2-Amino-4-chloro-6-methylpyrimidine (2) was purchased from
Aldrich Chemical Company; mp of 184–1868C.
Diphenyl (4-chloro-6-methylpyrimidin-2-ylamino)-
(2,4-dihydroxyphenyl)methylphosphonate (6)
Reaction time: 34 h, yield: 86%; mp: 280–2828C; IRꢀ(1KBr): 3369
(NH/OH), 1596 (C N), 1397 (P O), 761 (P–O–C) cm
;
1H NMR
–
–
–
–
(DMSO-d6): d 9.92 (s, exch., 1H, OH), 9.59 (s, exch., 1H, OH),
7.46–6.13 (m, 14H, Ar–H), 5.91 (br s, 1H, NH), 5.59 (d,
J ¼ 18 Hz, 1H, CH), 2.23 (s, 3H, CH3); EI–MS: m/z (%) 498
([M37ClꢀH]þ, 2), 496 ([M35ClꢀH]þ, 6), 247 (6), 211 (46), 119 (31),
94 (43), 76 (100); Anal. Calcd. for C24H21ClN3O5P (497.87): C, 57.90;
H, 4.25; N, 8.44; P, 6.22. Found: C, 57.92; H, 4.22; N, 8.45; P, 6.24.
Diphenyl (4-chloro-6-methylpyrimidin-2-ylamino)-
(4-chlorophenyl)methylphosphonate (7)
Chemistry
General procedure for the synthesis of
a-aminophosphonates (3–8)
A mixture of 1 or 2 (4.0 mmol) and a DCM solution of LiClO4
(3 mL, 5.0 M; 15.0 mmol) in DCM (10 mL) was stirred for 2 min
and the aromatic aldehyde (2.0 mmol) was then added. After
10 min, triphenylphosphite (0.93 g, 3.0 mmol) was added and
the mixture was stirred at room temperature for 28–44 h. Water
(10 mL) was added and the organic phase was separated and
dried over anhydrous Na2SO4. The solvent was removed under
Reaction time: 38 h, yield: 78%; mp: 155–1578C; IR (KBr): 3369
(NH), 1596 (C N), 1397 (P O), 827 (P–O–C) cmꢀ1; 1H NMR (DMSO-
–
–
–
–
d6): d 7.82–7.03 (m, 15H, Ar–H), 6.84 (br s, exch., 1H, NH), 6.24
(d, J ¼ 16 Hz, 1H, CH), 2.32 (s, 3H, CH3); ESþ–MS: m/z (%) 567
([M37Cl2þMeCNNa]þ, 23), 565 ([M37Cl35ClþMeCNNa]þ, 70), 563
([M35Cl2þMeCNNa]þ, 100), 526 ([M37Cl2þNa]þ, 18), 524
([M37Cl35ClþNa]þ, 35), 522 ([M35Cl2þNa]þ, 20), 504 ([M37Cl2]þ,
5), 502 ([M37Cl35Cl]þ, 12), 500 ([M35Cl2]þ, 15), 160 (35), 145 (37),
112 (7); Anal. Calcd. for C24H20Cl2N3O3P (500.31): C, 57.62;
H, 4.03; N, 8.40; P, 6.19. Found: C, 57.63; H, 4.06; N, 8.42; P, 6.22.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Antimicrobial Activities of Heterocyclic a-Aminophosphonates
5
Diphenyl (4-chloro-6-methylpyrimidin-2-ylamino)-
(3-nitrophenyl)methylphosphonate (8)
Determination of minimum inhibitory concentrations (MICs)
The MIC was determined by agar diffusion assay using the
filter paper disc method. The MICs were determined for the
synthesized compounds against E. coli, S. aureus and B. subtilis
as bacteria and C. albicans and S. cerevisiae as yeasts. It was carried
out by impregnation of different concentrations of synthesized
compounds (0, 10, 50, 100 and 1000 mg/mL) in DMSO as a solvent
and then placed on filter paper discs of the same diameter
(5 mm). The agar plate dilution method was used to inoculate
the bacteria and yeasts used in the plate. Nutrient agar medium
was seeded with 100 mL of inoculum size (5 ꢂ 105 for bacteria
and 4 ꢂ 104 for yeasts). The impregnated discs containing the
tested samples of different concentrations were placed on the
agar medium seeded with tested microorganisms. Standard anti-
biotic discs (ciproflaxcicin, 5 mg/mL for bacteria and 15 mg/mL
amphotericin B for yeasts) and blank discs (impregnated with
DMSO) were used as positive and negative control. The plates
were then incubated at 378C for 24–48 h to allow maximum
growth of the microorganisms.
Reaction time: 44 h, yield: 69%; mp: 121–1238C; IRꢀ(1KBr): 3271
(NH), 1568 (C N), 1352 (P O) and 825 (P–O–C) cm
;
1H NMR
–
–
–
–
(DMSO-d6): d 7.75–6.98 (m, 15H, Ar–H), 6.74 (br s, exch., 1H, NH),
6.04 (d, J ¼ 15 Hz, 1H, CH), 2.49 (s, 3H, CH3); ESꢀ–MS: m/z (%) 549
([M37Clþ37Cl]ꢀ, 11), 547 ([M37Clþ35Cl or [M35Clþ37Cl]ꢀ, 52), 545
([M35Clþ35Cl]ꢀ, 75), 511 ([M37ClꢀH]ꢀ, 5), 509 ([M35ClꢀH]ꢀ, 18),
491 (21), 433 (37), 397 (53), 339 (19), 257 (100), 233 (40); Anal.
Calcd. for C24H20ClN4O5P (510.87): C, 56.43; H, 3.95; N, 10.97;
P, 6.06. Found: C, 55.91; H, 3.67; N, 10.67; P, 6.05.
Antimicrobial activities
Tested microorganisms
Gram-negative bacteria
After the Gram-staining procedure, Gram-negative cells appear
pink. The Gram-negative bacterium used in this study was E. coli
which is known as the backbone example for Gram-negative
bacteria and to cause urinary infection, wound infection and
gastroenteritis.
The lethal dose
Brine shrimps lethality bioassay is a very simple bench-top
assay used to measure the cytotoxicity of plant extracts as well
as of synthesized compounds. Brine shrimp eggs are available
commercially and are used as fish food. Different concentrations
of each compound (10, 100 and 1000 mg/mL) were suspended in
5 mL vials containing saline solution and 20 shrimps. Three
replicates were used for each concentration and living larvae were
counted after 72 h. All data were expressed as mean ꢁ SD [46].
Gram-positive bacteria
The thick cell wall of a Gram-positive organism retains the
crystal violet dye used in the Gram-staining procedure, so the
stained cells appear purple under magnification. Gram-positive
bacteria used in this study were B. subtilis and S. aureus. B. subtilis
are mostly involved in urinary infection, wound, ulceration and
septicemia. S. aureus is the milestone of Gram-positive bacteria
and it is a causative agent of pneumonia, meningitis and food
poisoning.
The authors have declared no conflict of interest.
References
Fungi
Pathogenic fungi especially yeasts are responsible for a number
of diseases in human and animals. A number of pathogenic
strains of fungi are represented in C. albicans and S. cerevisiae.
The tested organisms were obtained from the culture collection
of Bacteriology Unit, Department of Botany, Faculty of Science,
Tanta University, Egypt.
[1] F. Palacios, C. Alonso, J. M. de los Santos, Curr. Org. Chem.
2004, 8, 1481–1496.
[2] K. Moonen, I. Laureyn, C. V. Stevens, Chem. Rev. 2004, 104,
6177–6216.
[3] K. A. Schug, W. Lindner, Chem. Rev. 2005, 105, 67–114.
[4] C. H. Hassal, E. F. Hahn, Antibiotics, Vol VI, Springer, Berlin
1983, pp. 1–11.
Media used and antimicrobial assay
[5] M. C. Allen, W. Fuhrer, B. Tuck, R. Wade, J. M. Wood, J. Med.
Chem. 1989, 32, 1652–1661.
Nutrient and Sabouraud’s broths, nutrient and Sabouraud’s
agar were used for growing and maintaining the tested
bacteria and yeast, respectively. The antimicrobial spectrum of
the synthesized compounds was determined as powdered
samples by the cut-plug method on plates seeded with the tested
bacteria (E. coli, B. subtilis and S. aureus) on nutrient agar, which
contained per liter: peptone (3 g), beef extract (5 g), NaCl (5 g)
and agar (20 g) at pH 7. The test was also performed on
plates seeded with C. albicans and S. cerevisiae on Sabaraud’s agar
that contained per liter: glucose (40 g), peptone (10 g) and
agar (20 g) at pH 6.0. After solidification, the wells were
made and each was filled with powdery compounds (10 mg).
The plates were then incubated at 308C for 24–48 h, after which
the diameters of the inhibition zones were measured.
Compounds which produced the highest inhibition zones
were selected and assayed further at different concentrations
in suspensions to quantify their inhibitory effects. Nutrient
and Sabouraud’s broths were used in activation of organisms
[45].
[6] E. Alonso, E. Alonso, A. Solis, C. Del Pozo, Synlett 2000, 698–
700.
[7] P. Kafarski, B. Lejczak, Curr. Med. Chem. Anti-Cancer Agents
2001, 1, 301–312.
[8] L. Jin, B. Song, G. Zhang, R. Xu, S. Zhang, X. Gao, D. Hu, S.
Yang, Bioorg. Med. Chem. Lett. 2006, 16, 1537–1543.
[9] G. S. Prasad, J. R. Krishna, M. Manjunath, O. V. S. Reddy, M.
Krishnaiah, C. S. Reddy, V. G. Puranikd, Arkivoc 2007, xiii,
133–141.
[10] E. D. Naydenova, P. T. Todorov, M. N. Topashka-Ancheva, G.
Ts. Momekov, T. Z. Yordanova, S. M. Konstantinov, K. D.
Troev, Eur. J. Med. Chem. 2008, 43, 1199–1205.
ˇ
ˇi´
ˇ ´
[11] L. Tusek-Boz c, M. Juribasic, P. Traldi, V. Scarcia, A. Furlani,
Polyhedron 2008, 27, 1317–1328.
[12] B. Wang, Z. W. Miao, J. Wang, R. Y. Chen, X. D. Zhang, Amino
Acids 2008, 35, 463–468.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
M. F. Abdel-Megeed et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
´
[13] Z. Rezaei, H. Firouzabadi, N. Iranpoor, A. Ghaderi, M. R.
Jafari, A. A. Jafari, H. R. Zare, J. Med. Chem. 2009, 44, 4266–
4275.
[30] C. Caldes, B. Vilanova, M. Adrover, F. Mun˜oz, J. Donoso,
Bioorg. Med. Chem. 2011, 19, 4536–4543.
[31] L. Ning, W. Wang, Y. Liang, H. Peng, L. Fu, H. He, J. Med. Chem.
2012, 47, 379–384.
[14] N. Oni¸ta, I. Sisu, M. Penescu, V. L. Purcarea, L. Kurunczi,
Farmacia 2010, 58, 531–545.
[32] C. B. Reddy, K. S. Kumar, M. A. Kumar, M. V. N. Reddy,
B. S. Krishna, M. Naveen, M. K. Arunasree, C. S. Reddy,
C. N. Raju, C. D. Reddy, J. Med. Chem. 2012, 47, 553–559.
[15] J. Liu, S. Yang, X. Li, H. Fan, P. Bhadury, W. Xu, J. Wu, Z.
Wang, Molecules 2010, 15, 5112–5123.
[16] D. Y. Hu, Q. Q. Wan, S. Yang, B. A. Song, P. S. Bhadury, L. H.
Jin, K. Yan, F. Liu, Z. Chen, W. Xue, J. Agric. Food Chem. 2008,
56, 998–1001.
[33] K. Smith, G. A. El-Hiti, M. A. Abdo, M. F. Abdel-Megeed, J.
Chem. Soc. Perkin Trans. 1995, 1, 1029–1033.
[34] K. Smith, G. A. El-Hiti, M. F. Abdel-Megeed, M. A. Abdo, J. Org.
Chem. 1996, 61, 647–655.
[17] X. Zhang, Y. Qu, X. Fan, C. Bores, D. Feng, G. Andrei, R.
Snoeck, E. De Clercq, P. M. Loiseau, Nucleosides Nucleotides
Nucleic Acids 2010, 29, 616–627.
[35] G. A. El-Hiti, Synthesis 2003, 2799–2804.
[36] G. A. El-Hiti, Synthesis 2004, 363–368.
[18] L. Jing-Zi, S. Bao-An, F. Hui-Tao, P. S. Bhadury, W.-T. Wan, S.
Yang, W. Xu, J. Wu, L.-H. Jin, X. Wei, D.-Y. Hu, S. Zeng, Eur. J.
Med. Chem. 2010, 45, 5108–5112.
[37] K. Smith, G. A. El-Hiti, A. S. Hegazy, Synthesis 2005,
2951–2961.
[38] K. Smith, G. A. El-Hiti, A. S. Hegazy, Synthesis 2010,
1371–1380.
[19] E. D. Naydenova, P. T. Todorov, P. I. Mateeva, R. N. Zamfirova,
N. D. Pavlov, S. B. Todorov, Amino Acids 2010, 39, 1537–1543.
[39] K. Smith, G. A. El-Hiti, A. S. Hegazy, Chem. Commun. 2010, 46,
2790–2792.
[20] J. Zhou, H. T. Fan, B. A. Song, L. H. Jin, P. S. Bhadury, D. Y. Hu,
S. Yang, Phosphorus Sulfur 2011, 186, 81–87.
[40] K. A. Browne, D. D. Deheyn, G. A. El-Hiti, K. Smith, I. Weeks,
J. Am. Chem. Soc. 2011, 133, 14637–14648.
[21] E. K. Fields, J. Am. Chem. Soc. 1952, 74, 1528–1531.
[22] M. Sawamura, Y. Ito, T. Hayashi, Tetrahedron Lett. 1989, 30,
2247–2250.
[41] M. F. Abdel-Megeed, B. E. Badr, M. M. Azaam, G. A. El-Hiti,
Bioorg. Med. Chem. 2012, 20, 2252–2258.
[23] H. Kuntz, S. Laschat, Synthesis 1992, 90–95.
[42] N. Azizi, M. R. Saidi, Tetrahedron 2003, 59, 5329–5332.
[24] T. Yokommatsu, S. Shibuya, Tetrahedron: Asymmetry 1992, 3,
377–380.
[43] P. R. Hsueh, J. C. Chang, L. J. Teng, P. C. Yang, S. W. Ho,
W. C. Hsieh, K. T. Luh, J. Clin. Microbiol. 1997, 35, 1021–1023.
[25] C. Qian, T. Huang, J. Org. Chem. 1998, 63, 4125–4128.
[44] N. J. Leonard, W. V. Ruyle, J. Org. Chem. 1948, 13, 903–910.
[26] B. C. Ranu, A. Hajra, U. Jana, Org. Lett. 1999, 1, 1141–1143.
[45] T. G. Pridham, L. A. Lindenfelser, O. L. Shotwell, F. Stodola,
R. G. Benedict, C. Foley, P. W. Jacks, W. J. Zaumeeyer,
W. H. Perston, J. W. Mitchell, Phytopathology 1956, 46,
568–575.
[27] S. Chandrasekhar, S. J. Prakash, V. Jagadeshwar, Ch.
Narsihmulu, Tetrahedron Lett. 2001, 42, 5561–5563.
[28] Q. M. Wang, Z. G. Li, R. Q. Hung, J. R. Cheng, Heteroatom Chem.
2001, 12, 68–72.
[46] B. N. Meyer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen,
D. E. Nichols, J. L. McLaughlin, Planta Med. 1982, 45,
31–34.
[29] I. El Sayed, S. M. El Kosy, M. A. Hawata, A. A.-A. El Gokha, A.
Tolan, M. M. Abd El-Sattar, J. Am. Sci. 2011, 7, 357–361.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com