A convenient synthesis and biological evaluation of some novel linear and cyclic α-aminophosphonic acid derivatives containing a quinazolinone ring

Article abstract of DOI:10.1007/s11164-015-2088-0

Synthesis of some novel linear and cyclic α-aminophosphonic acids and their esters bearing a quinazolin-4(3H)-one ring was achieved. The methodology depends on Pudovik reaction conditions in which using 3-(benzylideneamino)-2-phenyl-quinazolin-4(3H)-one (

Full text of DOI:10.1007/s11164-015-2088-0

Res Chem Intermed
DOI 10.1007/s11164-015-2088-0
A convenient synthesis and biological evaluation
of some novel linear and cyclic a-aminophosphonic acid
derivatives containing a quinazolinone ring
Tarik E. Ali1 Somaya M. El-Edfawy1
•
Received: 14 March 2015 / Accepted: 30 April 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Synthesis of some novel linear and cyclic a-aminophosphonic acids and their
esters bearing a quinazolin-4(3H)-one ring was achieved. The methodology depends on
Pudovik reaction conditions in which using 3-(benzylideneamino)-2-phenyl-quinazolin-
4(3H)-one (2) with phosphorous acid, diethyl phosphite and tris(2-chloroethyl) phosphite
gives the corresponding linear a-aminophosphonic acid derivatives 3, 5 and 7, respec-
tively, in good yields. Heterocyclization of the linear compounds to 1,4,2-oxazaphosphi-
nanes as cyclic a-aminophosphonic acid derivatives 4, 6 and 8, respectively, was carried
out. The synthesized compounds were evaluated for their antimicrobial and antioxidant
activities. The cyclic a-aminophosphonic acids and their esters showed better antimicro-
bial and antioxidant activities than the corresponding linear a-aminophosphonic acids and
their esters.
Graphical Abstract Synthesis of some novel linear and cyclic a-aminophosphonic
acids and their esters bearing a quinazolin-4(3H)-one ring as antimicrobial and an-
tioxidant agents from 3-(benzylideneamino)-2-phenyl-quinazolin-4(3H)-one was
achieved.
&
Tarik E. Ali
tarik_elsayed1975@yahoo.com
1
Department of Chemistry, Faculty of Education, Ain Shams University, Roxy, Cairo, Egypt
123

T. E. Ali, S. M. El-Edfawy
OR
O
O
N
O
N
OR
H
N
P
O
N
OR
Ph
P
N
N
H
H
OR
Ph
Ph
Ph
Heterocyclization
Reagents
O
O
O
N
P
N
OR
N
H
Ph
Ph
R=H, CH3CH2, ClCH2CH2
Keywords a-Aminophosphonic acid Á Pudovik reaction Á
1,4,2-Oxazaphosphinanes Á Antimicrobial Á Antioxidant
Introduction
Quinazolin-4(3H)-one is a heterocyclic alkaloid ring system belonging to the
group of N-containing heterocyclic compounds, a group of compounds that has
caused universal concern due to their widely and distinct biopharmaceutical
activities. Researchers have already determined many therapeutic activities of
quinazolin-4(3H)-one derivatives, including antimicrobial [1, 2], antiviral [3],
anti-HIV [4], anticonvulsant [5, 6], anti-inflammatory [7], antihistaminic [8], anti-
tubercular [9], and anticancer activities [10]. On the other hand, a-aminophos-
phonic acids are important isosteres of a-amino acids showing a variety of
biological effects [11]. Apart from this, their potential as herbicides [12],
insecticides [13], fungicides [14], antiviral agents [15], as well as their role for
antibody generation, is well known [16]. A survey of the literature revealed that
compounds containing a-aminophosphonates with a quinazolin-4(3H)-one ring
have not been reported so far. Moreover, 2-alkoxy-2-oxo-1,4,2-oxazaphosphi-
nanes (as cyclic a-aminophosphonate) are rarely reported in literature [17–20] and
they are of interest in regards to their biological activity [21]. Thus, we report
herein a simple method for synthesis of novel linear and cyclic a-amino-
phosphonates containing a quinazolin-4(3H)-one ring in good yields as a part of
our interest in the synthesis of new a-aminophosphonates containing different
bioactive heterocyclic rings [22–26]. The antimicrobial and antioxidant activities
of the synthesized compounds were also evaluated.
123

A convenient synthesis and biological evaluation of…
Results and discussion
Synthesis of linear a-aminophosphonic acid derivatives and their cyclic
analogues
Owing to their synthetic and biological value, the chemistry of a-aminophosphonic
acids and their esters has received very important attention and still remains of great
interests [27]. In the synthesis of a-aminophosphonic acids, the Pudovik reaction
and the Kabachnik-Fields reaction are powerful and direct methods for construction
of P–C–N bonds [28, 29]. 3-(Benzylideneamino)-2-phenyl-quinazolin-4(3H)-one
(2) was prepared according to the reported method [30, 31] in high yield by
condensation of 3-amino-2-phenyl-quinazolin-4(3H)-one (1) [32, 33] with ben-
zaldehyde using a catalytic amount of glacial acetic acid in absolute ethanol
(Scheme 1).
Addition of phosphorous acid to the imine 2 in toluene containing a catalytic
amount of trifluoroboron etherate (BF3ÁEt2O) under Pudovik reaction conditions
gave {[(4-oxo-2-phenyl-quinazolin-3(4H)-yl)amino] (phenyl)methyl}phosphonic
acid (3) in good yield (Scheme 2). The proposed mechanism suggested that BF3
as a Lewis acid attracted the electron lone pair of the nitrogen atom in the
azomethine bond, which accelerated the nucleophilic phosphorus attack on the
electrophilic carbon atom forming the dipolar intermediate B that produced the
desired product 3 (Scheme 2). The linear a-aminophosphonic acid 3 undergoes
heterocyclization into 1,4,2-oxazaphosphinane as cyclic a-aminophosphonic acid.
Thus, reaction of a-aminophosphonic acid 3 with 1,2-dibromoethane in absolute
ethanol in the presence of anhydrous potassium carbonate afforded 3-(2-hydroxy-2-
oxido-3-phenyl-1,4,2-oxazaphosphinan-4-yl)-2-phenyl-quinazolin-4(3H)-one (4) at
a 63-% yield (Scheme 2).
Consequently, addition of diethyl phosphite to the imine 2 in toluene containing a
catalytic amount of BF3ÁEt2O gave the dipolar intermediate C, which led to the formation
of diethyl {[(4-oxo-2-phenyl-quinazolin-3(4H)-yl)amino](phenyl)methyl}phosphonate
(5) at an 85-% yield (Scheme 3). Heterocyclization of the linear diethyl a-aminophos-
phonate 5 with 2-bromoethanol in ethanolic sodium ethoxide produced the corresponding
1,4,2-oxazaphosphinanyl quinazolinone 6 (Scheme 3).
Similarly, the imine 2 reacted with tris(2-chloroethyl) phosphite under the same
reaction conditions to produce the corresponding a-aminophosphonate 7 in
O
O
N
Ph
NH2
Ph
PhCHO
EtOH
N
N
N
Ph
N
2
1
Scheme 1 Preparation of 3-(benzylideneamino)-2-phenyl-quinazolin-4(3H)-one
123

T. E. Ali, S. M. El-Edfawy
O
N
OH
P
HO
O
..
N
Ph
OH
:
BF3
N
P(OH)3
H-P(OH)2
O
N
..
Ph
Toluene
BF3.Et2O
N
N
Ph
2
Ph
O
A
OH
O
P
H
N
HO
OH
Ph
OH
O
P+
O
82%
Ph
N
N
N
H
H
H
N
N
Ph
Ph
3
B
BrCH2CH2Br
EtOH, K2CO3
63%
O
O
O
N
P
N
OH
Ph
H
N
Ph
4
Scheme 2 Synthesis of the linear a-aminophosphonic acid 3 and its cyclic a-aminophosphonic acid 4
excellent yield (Scheme 4). A possible mechanism for the last reaction could
involve a nucleophilic phosphorus attack on the electrophilic carbon atom of an
azomethine bond to give the dipolar intermediate D, which could be solvated by
water (added during the reaction) to give the transient E [34]. The latter transient
underwent decomposition via removal of a 2-chloroethanol molecule to afford the
final a-aminophosphonate 7 (Scheme 4). Heating of the linear bis(chloroethyl) a-
aminophosphonate 7 in toluene containing a catalytic amount of triethylamine for
12 h achieved the corresponding 1,4,2-oxazaphosphinanyl quinazolinone 8 at a
61-% yield (Scheme 4).
Characterization of linear a-aminophosphonic acid derivatives and their
cyclic analogues
All the compounds were elucidated on the basis of IR, MS, 1H-, 13C- and 31P-NMR
spectra. The elemental analysis data were consistent with the compositions of the
123

A convenient synthesis and biological evaluation of…
O
H
O
O
N
Ph
..
O
H
O
N
H-P(OEt)2
HO-P(OEt)2
O
N
P
+
N
2
Ph
N
Toluene
BF3.Et2O
N
Ph
Ph
C
85%
O
H
O
O
P
O
O
N
O
H
O
O
N
N
BrCH2CH2OH
P
N
N
Ph
Ph
EtONa-EtOH
58%
H
N
Ph
Ph
5
6
Scheme 3 Synthesis of the linear diethyl a-aminophosphonate 5 and its cyclic ethyl a-aminophosphonic
6
desired products. The gross formula C21H18N3O4P, of acid 3 was confirmed by the
mass spectrum, which exhibited its molecular ion peak at m/z 407 (12 %). The IR
spectrum of acid 3 revealed absorption bands at 3447 (N–H), 3197 (P–OH), 1296
1
(P=O) and 1669 cm-1 (C=O). Its H-NMR spectrum exhibited a singlet at d 4.52
(N–H), a doublet at 4.06 (P–CH) and a broad signal at 2.49 ppm (O–H) [35]. The
13C-NMR spectrum of 3 revealed the characteristic carbon atoms of P–CH and C=O
groups at d 49.0 and 162.2 ppm, respectively (Fig. 1). Moreover, its 31P-NMR
spectrum showed a singlet peak at d 10.5 ppm.
Compounds 5 and 7 showed characteristic IR absorption wave numbers in the
regions around 3447, 1682, 1281 and 1025 cm-1 for NH, C=O, P=O and P–O–C
groups, respectively. Also, the 1H-NMR spectra of these compounds showed
multiplets for aromatic protons in the range of d 7.39–8.24 ppm. Their P–CH
protons resonated as doublets in the region d 4.64 (J = 21 Hz) and 5.62
(J = 19 Hz) ppm, respectively, due to their coupling with phosphorus atoms. In
both compounds, the protons of N–H groups appeared as broad singlets at d 5.35
and 6.20 ppm, respectively. Moreover, protons of the ethoxy group in compound 5
exhibited multiplets at d 0.92–1.10 (CH3) and 3.78–4.10 (CH2) ppm. In compound
7, the methylene protons of OCH2 and CH2Cl resonated as triplets at d 4.20 and
2.11 ppm, respectively. In the 13C-NMR spectra of compounds 5 and 7, the carbon
atoms of the carbonyl groups exhibited signals around d 169.3 ppm. The C-2 of the
quinazolinone ring was recorded around d 153 ppm. In addition, the characteristic
carbon atoms of P–CH groups exhibited doublets in the region d 49.1 and 48.0 ppm
123

T. E. Ali, S. M. El-Edfawy
Cl
Cl
Cl
O
O
H
O
N
Ph
P(OCH2CH2Cl)3
O
N
P
O
N
+
N
Toluene
N
N
2
Ph
Ph
BF3.Et2O
Ph
D
H2O
Cl
Cl
P
Cl
Cl
Cl
Cl
O
H
O
O
H
O
HO
89%
O
N
O
N
P
O
O
H
H
N
N
O
Ph
N
N
H
Ph
Ph
Ph
E
7
Toluene
Et3N
61%
O
O
O
N
P
Cl
N
O
Ph
H
N
Ph
8
Scheme 4 Synthesis of the linear bis(chloroethyl) a-aminophosphonate 7 and its cyclic chloroethyl
a-aminophosphonic 6
Fig. 1 The carbon atom
numbering of compound 3
O
HO
OH
6``
5``
O
P
1``
5
8
4
4``
3
4a
8a
6
7
N
N
H
6` H
2``
3``
5`
4`
2
N
1`
1
2`
3`
123

A convenient synthesis and biological evaluation of…
123

T. E. Ali, S. M. El-Edfawy
with coupling constants of 147.4 and 150 Hz, respectively. Also, the carbon atoms
of ethoxy groups in compound 5 appeared at d 16.0 and 59.7 ppm while the
methylene carbon atoms OCH2 and CH2Cl in compound 7 resonated at d 66.0 and
35.9 ppm, respectively. The 31P-NMR chemical shift of compound 5 appeared at d
22.3 ppm. The mass spectra of these compounds showed their respective molecular
ion peaks at the expected m/z 463 (11 %) and 532 (18 %), respectively.
The IR spectra of compounds 4, 6 and 8 revealed the disappearance of absorption
bands to NH functions and the presence of absorption bands in the ranges of
1668–1682 and 1296–1214 cm-1 assignable to C=O and P=O functions, respec-
1
tively. Their H-NMR spectra showed triplets in the range of d 3.04–2.94 (NCH2)
and 3.94–4.08 (OCH2) ppm for 1,4,2-oxazaphosphinane rings. Also, the protons of
P–CH groups appeared as doublets in range d 4.80–4.88 ppm with phosphorus
coupling constants of 19.8–22.5 Hz. Moreover, the protons of ethoxy groups in both
compounds 6 and 8 appeared at d 1.15 (CH3), 4.20–4.30 (CH2) and 4.10 (OCH2),
2.29 (CH2Cl) ppm, respectively. In addition, the N–H protons of compounds 4, 6
and 8 were absent, which supported the cyclization processes. The signals of P–CH
carbon atoms of compounds 4, 6 and 8 in their 13C-NMR spectra are observed in the
range d 53.0–54.0 ppm (JPC = 146–150 Hz). The carbon atoms of NCH2 and
OCH2 for 1,4,2-oxazaphosphinane rings were resonated in the range d 46.0–47.1
1
and 69.0–71.6 ppm, respectively. Other signals in the H- and 13C-NMR spectra of
compounds 4, 6 and 8 are in agreement with the frameworks of the starting
materials 3, 5 and 7. The 31P-NMR spectra of compounds 4 and 8 exhibited only
one signal for each compound at d 15.6 and 18.9 ppm, respectively. The mass
spectra of the three compounds showed their respective molecular ion peaks at the
expected m/z 433 (6 %), 461 (69 %) and 496 (2 %), respectively.
Biological evaluations
Antimicrobial activity
All the newly synthesized compounds were evaluated in vitro for their antibacterial
activities against Staphylococcus aureus (ATCC 25923) and Bacillus subtilis
(ATCC 6635), as representatives of Gram-positive bacteria and Escherichia coli
(ATCC 25922) and Salmonella typhimurium (ATCC 14028) as examples of Gram-
negative bacteria. They were also examined against Candida albicans (ATCC
10231) and Aspergillus fumigatus as fungi. The agar-diffusion technique was used
for the determination of the preliminary antibacterial and antifungal activities [36,
37]. The minimum inhibitory concentration (MIC, lg mL-1) for the most active
compounds against the same microorganism used in the preliminary screening was
carried out using the tube dilution technique [38]. The obtained results on the
antimicrobial activities of the compounds and control drugs are given in Table 1. In
general, the prepared compounds recorded variable antimicrobial activities towards
the used microorganisms. All compounds exhibited moderate activities against
Gram-positive bacteria, except compounds 7 and 8 that displayed high activities
against S. aureus with MIC values at 250 and 125 lg mL-1, respectively. Most of
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A convenient synthesis and biological evaluation of…
123

T. E. Ali, S. M. El-Edfawy
the compounds did not record any remarkable inhibitory effects towards S.
typhimurium. Only compounds 7 and 8 have excellent activity against E. coli with
MIC values at 125 and 62.5 lg mL-1, respectively. All the compounds exhibited
relatively moderate to high inhibitory activities against C. albicans, especially
compound 8 which was effective to a degrre equal to the standard drug.
Furthermore, only compounds 6–8 recorded remarkable inhibitory effects against
A. fumigatus with MIC values at 250–125 lg mL-1. From the above results, it is
clear that connection of a quinazolinone ring to an a-aminophosphonate moiety can
exhibit good antimicrobial effects. Compounds 7 and 8, which have chloroalkyl
groups, recorded highest antimicrobial activity towards all the microorganisms.
These results may help chemists make structural modifications to improve the
antimicrobial activities of these compounds.
Antioxidant activity
The antioxidant activities of linear and cyclic a-aminophosphonic acid and their
esters bearing a quinazolin-4(3H)one ring were evaluated by two in vitro methods in
order to compare the results and to establish some structure–antioxidant activity
relationships. The evaluation was carried out at concentrations ranging from 50 to
400 lmol L-1
.
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity evaluation is a
standard assay in antioxidant activity studies and offers a rapid technique for
screening the radical scavenging activity of specific compounds or extracts [39, 40].
A freshly prepared DPPH solution exhibits a deep purple colour with an absorption
maximum at 517 nm. This purple colour generally disappears when an antioxidant
is present in the medium. Thus, antioxidant molecules can quench DPPH free
radicals (i.e., by providing hydrogen atoms or by electron donation, conceivably)
90
80
70
60
50
40
30
50
100
20
200
300
10
400
0
3
4
5
6
7
8
The Synthesized Compounds
Fig. 2 DPPH radical scavenging activities (%) of the synthesized compounds 3–8 at 50–400 lmol L-1
123

A convenient synthesis and biological evaluation of…
123

T. E. Ali, S. M. El-Edfawy
and convert them to a colorless product (i.e., 2,2-diphenyl-1-picrylhydrazine),
resulting in a decrease in absorbance. Hence, more rapid the absorbance decrease,
the more potent the antioxidant activity of the compound. The percentage activity of
ethanolic solutions of linear and cyclic a-aminophosphonic acids and their esters
were examined and compared (Table 2; Fig. 2). The 50-% inhibitory concentration
IC50 towards DPPH activity of newly synthesized compounds was calculated.
The antioxidant activity of b-carotenoid is based on its radical adducts with free
radicals from linoleic acid. The linoleic acid free radical attacks the highly
unsaturated b-carotene model. The presence of antioxidants can decrease the extent
of b-carotene bleaching by neutralizing the linoleate-free radical and other free
radicals formed in the system [41]. Accordingly, the absorbance decreases rapidly in
the samples without an antioxidant. Whereas in the presence of an antioxidant, they
retain the colour for a longer time. The percentage (%) antioxidant activity of newly
synthesized compounds under study was showed in Table 3 and Fig. 3.
We can conclude from the obtained results that the synthesized compounds
showed promising radical scavenging abilities by the two methods and as compared
with the TBHQ (tertiary butylhydroquinone) as the standard antioxidant. The results
reveal that most of the compounds exhibited poor radical scavenging abilities at
lower concentrations (\50 lmol L-1). However, a gradual increase in the activity
in all cases was observed with an increase in the concentrations of the test
compounds. The cyclic a-aminophosphonic acid and their esters 4, 6 and 8 showed
better antioxidant activities than the corresponding linear a-aminophosphonic acid
and their esters 3, 5 and 7. The a-aminophosphonic acids 3 and 4 showed poor
activities while alkyl a-aminophosphonates 5, 6, 7 and 8 exhibited remarkable
activities at all concentrations, i.e., alkyl groups (electron donating groups) were
much better than acidic OH groups. The presence of chloroalkyl groups in
90
80
70
60
50
40
30
50
20
10
0
100
200
300
400
3
4
5
6
7
8
The Synthesized Compounds
Fig. 3 The percentage (%) antioxidant activity of b-carotenoid is based on its radical adducts with free
radicals from linoleic acid by the synthesized compounds 3–8 at 50–400 lmol L-1
123

A convenient synthesis and biological evaluation of…
compounds 7 and 8 instead of alkyl groups in the same positions in compounds 5
and 6 exhibited significant higher activities. Among the compounds tested,
compound 8 having 1,4,2-oxazaphosphinane and a chloro substituent in the alkyl
groups exhibited promising antioxidative properties with respect to the standard
antioxidant TBHQ.
Conclusion
A convenient synthetic method has been described to synthesize some novel linear
and cyclic a-aminophosphonic acids and their esters bearing a quinazolin-4(3H)-
one ring. The methodology depends on Pudovik reaction conditions in which using
a Schiff base with phosphorylating agents gives linear a-aminophosphonic acid
derivatives. Heterocyclization of the linear a-aminophosphonic acid derivatives to
1,4,2-oxazaphosphinanes as cyclic a-aminophosphonic acid derivatives was also
achieved. The cyclic a-aminophosphonic acid and their esters showed better
antimicrobial and antioxidant activities than the corresponding linear a-aminophos-
phonic acid and their esters. 2-Chloroethoxy-1,4,2-oxazaphosphinane derivative 8
exhibited promising antimicrobial and antioxidant activities.
Experimental
General remarks
Melting points were determined in an open capillary tube on a digital Stuart SMP-3
apparatus. IR spectra were measured on an FT-IR (Nicolet IS10) spectrophotometer
using KBr disks. 1H-NMR spectra were measured on a Gemini-300BB spectrometer
(300 MHz), using DMSO-d6 as a solvent and tetramethylsilane (TMS; d) as an
internal standard. 13C-NMR spectra were measured on a Mercury-300BB
(75 MHz), using DMSO-d6 as a solvent and TMS (d) as an internal standard.
31P-NMR spectra were registered on a Bruker (242 MHz) spectrometer at room
temperature using CdCl3 as a solvent and TMS as an internal standard and 85 %
H3PO4 as an external reference. Mass spectra were recorded on a gas chro-
matograph (Shimadzu GCMS-QP 1000EX) at 70 eV. Elemental microanalyses
were performed using a Perkin-Elmer 2400II at the Chemical War department,
Ministry of Defense. The purity of the synthesized compounds was checked by thin
layer chromatography (TLC) and elemental microanalyses.
Synthesis of a-aminophosphonic acid 3
A mixture of the imine 2 (0.81 g, 2.5 mmol) and phosphorous acid (0.20 g,
2.5 mmol) in toluene (20 mL) in the presence of a catalytic amount of BF3ÁEt2O
(0.05 mL) was heated under reflux for 10 h. After completion of the reaction, the
solution was concentrated. The residue was triturated with diethyl ether. The formed
solid was collected and crystallized from diluted ethanol to give the beige crystals of
123

T. E. Ali, S. M. El-Edfawy
product 3 in yield (0.83 g, 82 %); mp 236–238 °C. IR (KBr) (mmax, cm-1): 3447
(NH), 3197 (P–OH), 1669 (C=O), 1602 (C=N), 1571, 1559 (C=C), 1296 (P=O), 693
1
(P–O). H-NMR (300 MHz, DMSO-d6): 2.49 (br, 2H, P–OH exchangeable with
D2O), 4.06 (d, 1H, J = 21 Hz, P–CH), 4.52 (s, 1H, NH exchangeable with D2O),
7.49–7.62 (m, 7H, Ar–H), 7.72–7.87 (m, 3H, Ar–H), 8.14–8.20 (m, 4H, Ar–H). 13C-
NMR (75 MHz, DMSO-d6): 49.0 (d, J = 150.4 Hz, P–CH), 120.9 (C-4a), 125.8 (C-
8), 126.5 (C-200,600), 126.6 (C-20,60), 127.4 (C-300,500), 127.5 (C-30,50), 127.7 (C-100),
128.5 (C-400, 6), 128.6 (C-10), 131.3 (C-5), 132.7 (C-40), 134.5 (C-7), 148.7 (C-8a),
152.3 (C-2), 162.2 (C=O). 31P-NMR (242 MHz, CDCl3): 10.5 ppm. MS (EI, m/z):
407 (M?, 12 %). Anal. Calcd for C21H18N3O4P (407.37): C, 61.92; H, 4.45; N,
10.31 %. Found: C, 61.53; H, 4.42; N, 9.99 %.
Synthesis of 3-(2-hydroxy-2-oxido-3-phenyl-1,4,2-oxazaphosphinan-4-yl)-2-
phenyl-quinazolin-4(3H)-one (4)
A mixture of the a-aminophosphonic acid 3 (0.4 g, 1 mmol) and 1,2-dibromoethane
(0.09 mL, 1 mmol) in absolute ethanol (20 mL) in the presence of anhydrous potassium
carbonate (0.5 g), was heated under reflux for 6 h. After cooling, the solution was
poured on cold water. The formed solid was collected and crystallized from benzene to
give pale yellowcrystals of product 4 in yield (0.27 g, 63 %); mp 171–173 °C. IR(KBr)
(mmax, cm-1): 2958 (P–OH), 1668 (C=O), 1602 (C=N), 1570, 1558 (C=C), 1296 (P=O).
1H-NMR (300 MHz, DMSO-d6): 2.50 (s, 1H, P–OH exchangeable with D2O), 3.04 (t,
2H, NCH2), 3.94 (t, 2H, OCH2), 4.80 (d, 1H, J = 19.8 Hz, P–CH), 7.47–7.58 (m, 7H,
Ar–H), 7.71–7.84 (m, 3H, Ar–H), 8.13–8.22 (m, 4H, Ar–H). 13C-NMR (75 MHz,
DMSO-d6): 46.0 (NCH2), 53.0 (d, J = 146 Hz, P–CH), 69.0 (OCH2), 121.0 (C-4a),
126.5(C-8), 126.7(C-200,600), 127.1(C-20,60),127.6(C-300,500),128.5(C-30,50),129.0(C-
100), 129.7 (C-400, 6), 131.0 (C-10), 132.3 (C-5), 132.6 (C-40), 134.5 (C-7), 146.3 (C-8a),
153.0 (C-2), 169.3 (C=O). 31P-NMR (242 MHz, CDCl3): 15.6 ppm. MS (EI, m/z): 433
(M?, 6 %). Anal. Calcd for C23H20N3O4P (433.41): C, 63.74; H, 4.65; N, 9.70 %.
Found: C, 63.42; H, 4.31; N, 9.49 %.
General procedure for the synthesis of a-aminophosphonates 5 and 7
A mixture of the imine 2 (0.81 g, 2.5 mmol) and diethyl phosphite (0.32 mL,
2.5 mmol) or tris(2-chloroethyl) phosphite (0.50 mL, 2.5 mmol) in toluene (20 mL)
in the presence of a catalytic amount of BF3ÁEt2O (0.05 mL) was heated under
reflux for 10 h [added 0.5 mL of distillated water after 2 h in case of tris(2-
chloroethyl) phosphite]. After completion of the reactions, the solutions were
concentrated. The residues were triturated with cold ethanol. The formed solids
were collected and crystallized from absolute ethanol to give white crystals of
products 5 (0.98 g, 85 %) and 7 (1.18 g, 89 %), respectively.
Diethyl {[(4-oxo-2-phenyl-quinazolin-3(4H)-yl)amino](phenyl)methyl}phosphonate (5)
Mp 149–151 °C. IR (KBr) (mmax, cm-1): 3447 (NH), 1682 (C=O), 1608 (C=N),
1587, 1575 (C=C), 1281 (P=O), 1025 (P–O–C). H-NMR (300 MHz, DMSO-d6):
1
123

A convenient synthesis and biological evaluation of…
0.92–1.10 (m, 6H, OCH2CH3), 3.78–4.10 (m, 4H, OCH2CH3), 4.64 (d, 1H,
J = 21 Hz, P–CH), 5.35 (brs, 1H, NH), 7.42–7.92 (m, 13H, Ar–H), 8.23 (d, 1H, Ar–
H). 13C-NMR (75 MHz, DMSO-d6): 16.0 (CH3), 49.1 (d, J = 147.4 Hz, P–CH),
59.7 (OCH2), 121.0 (C-4a), 126.5 (C-8), 126.7 (C-200,60), 127.1 (C-20,60), 127.6 (C-
300,500), 128.5 (C-30,50), 129.0 (C-100), 129.7 (C-400, 6), 131.0 (C-10), 132.3 (C-5),
132.5 (C-40), 134.6 (C-7), 146.3 (C-8a), 153.0 (C-2), 169.3 (C=O). 31P-NMR
(242 MHz, CDCl3): 22.3 ppm. MS (EI, m/z): 463 (M?, 11 %). Anal. Calcd for
C25H26N3O4P (463.48): C, 64.79; H, 5.65; N, 9.07 %. Found: C, 64.45; H, 5.33; N,
8.86 %.
Bis(2-chloroethyl){[(4-oxo-2-phenyl-quinazolin-3(4H)-yl)amino](phenyl) methyl}
phosphonate (7)
Mp 143–144 °C. IR (KBr) (mmax, cm-1): 3446 (NH), 1682 (C=O), 1608 (C=N),
1587, 1575 (C=C), 1281 (P=O), 1025 (P–O–C). H-NMR (300 MHz, DMSO-d6):
1
2.11 (t, 2H, ClCH2), 4.20 (t, 2H, OCH2), 5.62 (d, 1H, J = 19 Hz, P–CH), 6.20 (br,
1H, NH), 7.39–7.92 (m, 13H, Ar–H), 8.24 (d, 1H, Ar–H). 13C-NMR (75 MHz,
DMSO-d6): 35.9 (ClCH2), 48.0 (d, J = 150 Hz, P–CH), 66.0 (OCH2), 121.0 (C-4a),
126.3 (C-8), 126.7 (C-200,600), 127.1 (C-20,60), 127.6 (C-300,500), 128.5 (C-30,50), 129.0
(C-100), 129.7 (C-400, 6), 131.0 (C-10), 132.3 (C-5), 132.6 (C-40), 134.5 (C-7), 146.3
(C-8a), 153.0 (C-2), 169.3 (C=O). MS (EI, m/z): 534 (M ? 2, 8 %), 532 (M?,
18 %). Anal. Calcd for C25H24Cl2N3O4P (532.37): C, 56.40; H, 4.54; N, 7.89 %.
Found: C, 56.02; H, 4.32; N, 7.58 %.
Synthesis of 3-(2-ethoxy-2-oxido-3-phenyl-1,4,2-oxazaphosphinan-4-yl)-2-
phenyl-quinazolin-4(3H)-one (6)
A mixture of the diethyl a-aminophosphonate 5 (0.46 g, 1 mmol) and 2-bro-
moethanol (0.07 mL, 1 mmol) in ethanolic sodium ethoxide (0.1 g of sodium metal
in 15 mL of absolute ethanol) was heated under reflux for 18 h. After cooling, the
solution was poured on cold water and neutralized with dilute hydrochloric acid
(5 %). The formed solid was collected and crystallized from ethyl acetate to give
pale yellow crystals of product 6 in yield (0.26 g, 58 %); Mp 158–159 °C. IR (KBr)
(mmax, cm-1): 1682 (C=O), 1594 (C=N), 1560, 1541 (C=C), 1214 (P=O), 1055 (P–
O–C). 1H-NMR (300 MHz, DMSO-d6): 1.15 (t, 3H, OCH2CH3), 2.94 (t, 2H,
NCH2), 4.08 (t, 2H, OCH2), 4.20–4.30 (m, 2H, OCH2CH3), 4.88 (d, 1H,
J = 22.5 Hz, P–CH), 6.99–7.55 (m, 8H, Ar–H), 7.66–7.91 (m, 4H, Ar–H),
8.05–8.16 (m, 2H, Ar–H). 13C-NMR (75 MHz, DMSO-d6): 16.1 (CH3), 47.1
(NCH2), 53.0 (d, J = 147.5 Hz, P–CH), 59.1 (OCH2), 71.6 (OCH2), 121.0 (C-4a),
126.7 (C-8), 126.9 (C-200,600), 127.0 (C-20,60), 127.6 (C-300,500), 128.5 (C-30,50), 128.9
(C-100), 129.7 (C-400,6), 131.0 (C-10), 132.3 (C-5), 132.5 (C-40), 134.5 (C-7), 146.3
(C-8a), 153.0 (C-2), 169.4 (C=O). MS (EI, m/z): 462 (M ? 1, 46 %), 461 (M?,
69 %). Anal. Calcd for C25H24N3O4P (461.46): C, 65.07; H, 5.24; N, 9.11 %.
Found: C, 64.81; H, 4.92; N, 8.95 %.
123

T. E. Ali, S. M. El-Edfawy
Synthesis of 3-[2-(2-chloroethoxy)-2-oxido-3-phenyl-1,4,2-oxazaphosphinan-
4-yl]-2-phenyl-quinazolin-4(3H)-one (8)
A solution of the bis(2-chloroethyl) a-aminophosphonate 7 (0.53 g, 1 mmol) in
toluene (15 mL) containing a catalytic amount of triethylamine (0.1 mL, 1 mmol)
was heated under reflux for 12 h. The precipitated salt was filtered off. The filtrate
was concentrated. The residue was triturated with ethyl acetate. The formed solid
was collected and crystallized from toluene to give yellow crystals of product 8 in
yield (0.30 g, 61 %); mp 168–170 °C. IR (KBr) (mmax, cm-1): 1682 (C=O), 1596
(C=N), 1560, 1541 (C=C), 1214 (P=O), 1055 (P–O–C). 1H-NMR (300 MHz,
DMSO-d6): 2.29 (t, 2H, ClCH2), 2.95 (t, 2H, NCH2), 3.95 (t, 2H, OCH2), 4.10 (t,
2H, OCH2), 4.84 (d, 1H, J = 21 Hz, P–CH), 7.42–7.92 (m, 13H, Ar–H), 8.23 (d,
1H, Ar–H). 13C-NMR (75 MHz, DMSO-d6): 35.9 (ClCH2), 47.1 (NCH2), 54.0 (d,
J = 150 Hz, P–CH), 60.3 (OCH2), 71.4 (OCH2), 120.9 (C-4a), 125.8 (C-8), 126.9
(C-200,600), 127.5 (C-20,60), 127.7 (C-300,500), 128.0 (C-30,50), 128.5 (C-100), 128.9 (C-
400, 6), 131.3 (C-10), 132.7 (C-5), 134.5 (C-40), 136.8 (C-7), 146.1 (C-8a), 152.3 (C-
2), 161.8 (C=O). 31P-NMR (242 MHz, CDCl3): 18.9 ppm. MS (EI, m/z): 496 (M?,
2 %). Anal. Calcd for C25H23ClN3O4P (495.91): C, 60.55; H, 4.67; N, 8.47 %.
Found: C, 60.21; H, 4.43; N, 8.21 %.
Antimicrobial evaluation
All the newly synthesized compounds were evaluated in vitro for their antibacterial
activities against S. aureus (ATCC 25923) and B. subtilis (ATCC 6635) as
representatives of Gram-positive bacteria, and E. coli (ATCC 25922) and S.
typhimurium (ATCC 14028) as examples of Gram-negative bacteria. They were
also examined against Candida albicans (ATCC 10231) and A. fumigatus as fungi.
An agar-diffusion technique was used for the determination of the preliminary
antibacterial and antifungal activities [36, 37]. The test was performed on a medium
of potato dextrose agar (PDA) containing and infusion of 200 g of potatoes, 6 g of
dextrose and 15 g of agar. Uniformly sized filter paper disks (3 disks per compound)
were impregnated with equal volumes (10 lL) from the concentrations of 500 and
1000 lg mL-1 dissolved compounds in dimethylformamide (DMF) and carefully
placed on an inoculated agar surface. After incubation for 36 h at 27 °C in the case
of bacteria and for 48 h at 24 °C in the case of fungi, the antimicrobial activities
were determined by measuring the inhibition zones. Cephalothin, chloramphenicol
and cycloheximide were used as reference drugs (30 lg mL-1) for Gram-positive
bacteria, Gram-negative bacteria and fungi, respectively. The MIC (lg mL-1) for
some selected compounds against some species of microbes was also determined.
The tube dilution technique was applied for the determination of MIC of the tested
compounds against microbes [38]. A dilution series was set up with 250, 125,
62.5…3.25 lg mL-1 of nutrient broth medium to each tube, and 100 mL of
standardized suspension of the test microbes (107 cell mL-1) were added and
incubated at 37 °C for 24 h.
123

A convenient synthesis and biological evaluation of…
Antioxidant activity
DPPH radical scavenging activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging effect was carried
out according to the reported method [39, 40]. Compounds of different concen-
trations were prepared in distilled ethanol; 1 mL of each compound solution (50,
100, 200, 300 and 400 lmol L-1) was taken in different test tubes, and 4 mL of
100 lmol L-1 ethanol solution of DPPH was added and shaken vigorously. The
tubes were then incubated in the dark at RT for 20 min. A DPPH blank was
prepared without a compound, and ethanol was used for the baseline correction.
Changes (decrease) in the absorbance at 517 nm were measured using an ultaviolet–
visible spectrophotometer. The radical scavenging activities were expressed as the
inhibition percentage and were calculated using the formula:
%Radical scavenging activity ¼ ðAB À AAÞ=AB Â 100
where AB = absorption of blank and AA = absorption of the tested compound. The
radical scavenging activity of TBHQ was also measured and compared with that of the
different synthesized compounds. The compound concentration providing 50 % inhibi-
tion (IC50) was calculated from the graph ofpercentage againstcompound concentrations.
Antioxidant activity by b-carotene-linoleic acid assay
Each compound at the final concentrations of 50–400 lmol L-1 was incorporated into a
b-carotene-linoleic acid model system independently and the activity was monitored
spectrophotometrically at 470 nm [41]. The substrate suspension was prepared by
addition of b-carotene (4 mg dissolved in 5 mL chloroform) into a covered round-
bottomed flask containing Tween-40 (600 mg) followed by the addition of linoleic acid
(60 mL). The chloroform was removed completely under vacuum using a rotary
evaporator at 40 °C. The resulting solution was diluted with triple-distilled water
(30 mL) and the emulsion was mixed well and diluted with oxygenated water (120 mL).
The aliquot (4 mL) was transferred to different stopper test tubes containing compounds
in distilled ethanol. A controlwasprepared with distilled ethanol(1 mL) and an emulsion
(4 mL). A TBHQ solution as the internal standard of the same concentration was also
analyzed for comparison. Zero adjustment was done using distilled water. As soon as the
emulsion was added to each test tube, the zero time (t = 0) absorbance was measured at
470 nmusing a spectrophotometer and absorbance was measured subsequently for every
30 min, up to 3 h (t = 180). The tubes were placed in a water bath at 50 °C between the
readings. Percentage antioxidant activities of each compound were evaluated in triplicate
in terms of photooxidation of b-carotene using the following formula:
%Antioxidant activity ¼ 100 Â f1 À ðA0 À At=A00 À At0Þg
where A0 = initial absorbance of the sample (t = 0 min), At = absorbance of the
sample after time t (t = 180 min), A00 = initial absorbance of the control
(t = 0 min), and At0 = absorbance of control after time t (t = 180 min).
123

T. E. Ali, S. M. El-Edfawy
Acknowledgments The authors are very grateful to the Department of Microbiology, Faculty of
Agriculture, Al-Azhar University for Girls, Nasr-City, Cairo, Egypt, for performing the antimicrobial
evaluation. Also, evaluation of the antioxidant properties was performed at the Department of Flavour
and Aroma Chemistry, National Research Center (NRC), Egypt.
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