One pot synthesis of some new heteroylselenoglycolic acids and their biological activity

Article abstract of DOI:10.1080/10426507.2012.717138

A convenient one pot three-stage synthesis was used for obtaining new heteroylselenoglycolic and di-heteroylselenoglycolic acids by nucleophilic substitution reaction of the starting compounds pyridineselenol, pyridazineselenol, and quinolineselenol with α-chloro- or α,α-dichloroacetic acids for 1-h stirring. The newly synthesized compounds were screened biologically for anti-microbial and anti-oxidant activities. The structure of all new compounds was confirmed by ¹H NMR, ¹³C NMR, Mass, and IR spectroscopy and elemental analyses.

Full text of DOI:10.1080/10426507.2012.717138

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Phosphorus, Sulfur, and Silicon and the
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One Pot Synthesis of Some New
Heteroylselenoglycolic Acids and Their
Biological Activity
Shams H. Abdel-Hafez a b , Laila M. Break a & Amani M. Alsaadi a
a Department of Chemistry, Faculty of Science , Taif University ,
Taif , Kingdom of Saudi Arabia
b Department of Chemistry, Faculty of Science , Assiut University ,
Assiut , Egypt
Accepted author version posted online: 09 Aug 2012.Published
online: 16 Aug 2013.
To cite this article: Shams H. Abdel-Hafez , Laila M. Break & Amani M. Alsaadi (2013) One Pot
Synthesis of Some New Heteroylselenoglycolic Acids and Their Biological Activity, Phosphorus, Sulfur,
and Silicon and the Related Elements, 188:9, 1281-1295, DOI: 10.1080/10426507.2012.717138
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Phosphorus, Sulfur, and Silicon, 188:1281–1295, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.717138
ONE POT SYNTHESIS OF SOME NEW
HETEROYLSELENOGLYCOLIC ACIDS AND THEIR
BIOLOGICAL ACTIVITY
Shams H. Abdel-Hafez,1,2 Laila M. Break,1
and Amani M. Alsaadi1
1Department of Chemistry, Faculty of Science, Taif University, Taif, Kingdom of
Saudi Arabia
2Department of Chemistry, Faculty of Science, Assiut University, Assiut, Egypt
GRAPHICAL ABSTRACT
Abstract A convenient one pot three-stage synthesis was used for obtaining new heteroylse-
lenoglycolic and di-heteroylselenoglycolic acids by nucleophilic substitution reaction of the
starting compounds pyridineselenol, pyridazineselenol, and quinolineselenol with α-chloro-
or α,α-dichloroacetic acids for 1-h stirring. The newly synthesized compounds were screened
biologically for anti-microbial and anti-oxidant activities. The structure of all new compounds
was confirmed by 1H NMR, 13C NMR, Mass, and IR spectroscopy and elemental analyses.
Keywords Selenoglycolic acids; pyridineselenol; pyridazineselenol; quinolineselenol; anti-
oxidant activity; anti-microbial activity
Received 29 May 2012; accepted 9 July 2012.
Address correspondence to Shams H. Abdel-Hafez, Taif University, P.O. Box 888, Alhawiya, Taif, Kingdom
of Saudi Arabia. E-mail: sabdel68@yahoo.co.uk
1281

1282
S. H. ABDEL-HAFEZ ET AL.
INTRODUCTION
Organoselenium compounds have continued to attract the attention of investigators
because of a series of their unique properties. There are data showing that organoselenium
compounds are capable of sensitizing processes in living organism.1 Therefore, despite high
toxicity of many selenium compounds,2–4 antitumor,5–7 anticancer,8,9 as well as biologically
active substances exhibiting antiviral10–13 and antimicrobial activities14–17 have been pro-
duced on the basis of organic derivatives of selenium. A literature survey indicates that only
few publications have mentioned the incorporation of a selenium atom into glycolic acid.
Consequently, synthesis and biological screening of selenoglycolic acid may be considered
a virgin research area. Athayde-Filho et al.18 described a convenient one-pot three-stage
synthesis for obtaining three new aroylselenoglycolic acids. In the first stage, powdered
gray selenium reacts with NaBH4 in aqueous solution to form NaHSe. This reacts with
selected aroyl chlorides to form sodium aroylselenides. In the third stage, these react with
α-chloroacetic acid to form the desired aroylselenoglycolic acids. Stimulated by our recent
work on the synthesis of selenium containing sulfa drugs,19 and the synthesis of selenium
containing amino acid analogues,20 we decided to expand our interest to introduce selenium
into heterocyclic molecules followed by α-chloro- or α,α-dichloroacetic acids, hoping to
obtain heteroylselenoglycolic and di-heteroylselenoglycolic acid systems of anticipated
biological and pharmaceutical effects.
RESULTS AND DISCUSSION
Chemistry
The present work was planned to synthesize some new heteroylselenoglycolic
acids and to test their biological activity as anti-bacterial, anti-fungal, and anti-oxidant
agents. Our approach to synthesize target compounds started from 2-chloro-4,6-
dimethylpyridine-3-carbonitrile, 3-chloro-5,6-diphenylpyridazine-4-carbonitrile, and
2-chloro-4,6-dimethylquinoline (1a–c) respectively, which were prepared as described
previously.19, 20
Our work plan consists of the following three steps:
(1) Synthesis of some new heteroylselenoglycolic acids (one pot three-stage synthesis
of 2a–c):
A convenientonepotthree-stagesynthesis was usedforobtainingnewheteroylseleno-
glycolic acids such as (3-cyanopyridin-2-yl)selenoglycolic acid (2a), (4-cyanopyridazin-
3-yl)selenoglycolic acid (2b), and (quinolin-2-yl)selenoglycolic acid (2c) by nucleophilic
substitution reaction of compounds 1a–c with α-chloroacetic acid in the presence of sodium
borohydride as a basic catalyst by 1-h stirring (see Scheme 1).
The first stage:
Reaction of sodium borohydride with powdered gray selenium in methanol or water
as a solvent to produce sodiumhydrogen selenide.
4NaBH4+2Se + 7H2O → 2NaHSe + Na2B4O7+14H2
The second stage:
Reaction of NaHSe with heteroyl chlorides for 1 h with stirring to form sodi-
umheteroyl selenide.

ONE POT SYNTHESIS OF HETEROYLSELENOGLYCOLIC ACIDS
1283
NaBH4/CH3OH/
one hr. stirring
COOH
Cl
COOH
N
Se
2a-c
N
Cl
Cl
COOH
N
Se
NaBH4/CH3OH/
one hr. stirring
COOH
Se
1a-c
Cl
3a-c
N
Cl
Cl
COOH
N
N
NaBH4/CH3OH/
Cl
Se
Se
Se
one hr. stirring
COOH
4a-c
N
CH3
Ph
CH3
N
CN
H3C
CN
Ph
1a-5a,
H3C
; 1b-5b,
; 1c-5c,
N
N
N
Scheme 1 Synthesis of compounds 2a–c and 3a–c.
(1a c)
+
2NaHSe
+
+ H Se
NaCl
2
N
Cl
N
SeNa
The third stage:
Production of heteroylselenoglycolic acids by the reaction of sodiumheteroyl selenide
with α-chloroacetic acid for 1 h.
(2a c)
N
+
+
NaCl
Cl
COOH
SeNa
N
SeCH2 COOH
Trials to prepare compounds 4a–c with trichloroacetic acid failed and this may be
due to steric hindrance effects.
The synthesis of new di-heteroylselenoglycolic acids (one pot three-stage synthesis
of 3a–c) also consists of the following three reaction steps:
4NaBH4 + 2Se + 7H2O → 2NaHSe + Na2B4O7 + 14H2
(1a c)
+
2NaHSe
+
+ H Se
NaCl
2
N
Cl
N
SeNa

1284
S. H. ABDEL-HAFEZ ET AL.
(3a c)
N
Se
C
Cl
N
+
+
NaCl
Cl
COOH
SeNa
N
Se
H
COOH
Unequivocal synthesis of 2a–c and 3a–c through the intermediate com-
pounds 5a–c. To confirm the structure of the new compounds 2a–c and 3a–c, we first
separated compounds 5a–c and then added α,α-dichloroacetic acids in the presence of
anhydrous sodium acetate to yield the identical products 2a–c and 3a–c (see Scheme 2). In
the same manner, the reaction consists of three steps as mentioned above. Trials to prepare
compounds 4a–c with trichloroacetic acid via the intermediate compounds 5a–c failed also.
All the products gave satisfactory mass spectra and elemental analyses. The characteristic
band of the IR spectra is the C O stretch of the carboxyl group at 1730, 1740, and 1720 for
2a, 2b, and 2c respectively. In the 1H NMR spectra of 2a, 2b, and 2c, the hydrogen atoms
of the SeCH2 group have chemical shifts at δ = 3.32, 3.36, and 3.40 ppm respectively. The
carboxy hydrogen atoms exhibit chemical shifts around 14.15 ppm for 2b and 11.53 ppm
for 2c, while the signal of the COOH group disappeared for 2a as a result of the hydrogen
N
Cl
1a-c
COOH
Cl
NaBH4/Se/MeOH/
reflux 2 hrs
COOH
N
Se
anhyd.CH3COONa/EtOH/reflux one hour
2a-c
Cl
N
SeH
5a-c
COOH
Cl
N
Se
Se
COOH
3a-c
anhyd.CH3COONa/EtOH/reflux one hour
N
Cl
Cl
COOH
Cl
N
N
Se
Se
anhyd.CH3COONa/EtOH/reflux one hour
Se
COOH
4a-c
N
CH3
Ph
N
CH3
N
CN
H3C
CN
Ph
1a-5a,
H3C
; 1b-5b,
; 1c-5c,
N
N
Scheme 2 Synthesis of compounds 2a–c and 3a–c through the intermediates 5a–c.

ONE POT SYNTHESIS OF HETEROYLSELENOGLYCOLIC ACIDS
1285
bond formation. The 13C NMR signals appeared at δ 20.58–24.53 (CH3)2, 29.70 (CH2),
108 (C CN), 114 (CN), 122 (H C), 151–152 (C CH3), 155 (C Se), 162 (COOH) for
2a, at δ 56 (CH2), 112 (C CN), 121 (CN), 127.78–128.94 (Ph) 2, 130 (Ph C C), 136
(Ph C N), 161 (C Se), 172 (COOH) for 2b, and at δ 18.6–18.7 (CH3)2, 21.8 (CH2), 120
(CH3 C), 122 (Ph C), 126–147 (Ph), 149 (C Se), 153 (COOH) for 2c. The characteristic
IR band is the C = O stretching of the carboxyl group at 1730, 1742, and 1725 respectively.
In the 1H NMR spectra of 3a, 3b, and 3c, the hydrogen atoms (1H, SeCH) have chemical
shifts at δ 3.34, 4.53, and 3.42 respectively. The hydrogen atoms of the acid group exhibit
chemical shifts around 14.13 ppm for 3b and disappeared in both 3a and 3c as a result of
hydrogen bond formation. The 13C NMR spectra were characterized by peaks average at
δ 20.58–24.53 (CH3)2, especially for 3a and 3a, 29.7 (CH2), 108 (C-CN), 114 (CN), 122
(H-C), 151–152 (C-CH3), 155 (C-Se), and 162 (COOH) for 3a–c.
Biological Activity
Anti-microbial activity. The antimicrobial screening of the synthesized com-
pounds was undertaken using the agar well diffusion assay.21 Table 1 lists the screening
Table 1 Antibacterial activity of 2a–c and 3a–c (inhibition zone in mm)
2a
3a
2b
3b
2c
3c
Bacterial species Sample No.
monopyrd dipyrd monopyrdz dipyrdz monoquino diquino 12∗
Staphylococcus aureus
AUMC No. B-54
Bacillus cereus
AUMC No. B-52
Escherichia coli
AUMC No. B-53
Pseudomonas aeruginosa
AUMC No. B-73
Serratia marcescens
AUMC No. B-55
18
23
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
12
16
0
0
0
0
0
0
27
32
30
24
41
16 p.i.
0
0
0
0
Fungal species Sample No.
2a
28
18
34
32
11
30
3a
0
2b
9
3b
0
2c
14
10
16
16
12
14
3c
9
12∗∗
30
Candida albicans
AUMC No. 418
Geotrichum candidum
AUMC No. 226
Trichophyton rubrum
AUMC No. 1804
Fusarium oxysporum
AUMC No. 5119
Scopulariopsis brevicaulis
AUMC No. 729
8
0
0
0
24
17
10
0
14
12
18
18
0
14
12
0
35
0
22
17
0
26
Aspergillus flavus
AUMC No. 3214
14
13
27
Notes: The amount added in each pore is 50 μL.
12∗ = Choramphenicol as antibacterial standard.
12∗∗ = Clotrimazole as antifungal standard.
p.i. = Partial inhibition.
AUMC = Assiut University Mycological Center.

1286

1287

1288
S. H. ABDEL-HAFEZ ET AL.
results of the compounds tested against Gram-negative and Gram-positive bacteria. Also,
Table 1 (continued) lists the screening results of the compounds tested against fungi. Five
bacterial test organisms such as Staphylococcus aureus (B 54), Bacillus cereus (B 52), Es-
cherichia coli (B 53), Serratia marcescens (B 55), and Pseudomonas aeruginosa (B 73),
and six fungi test organisms such as Candida albicans (418), Geotri chum candidum
(226), Trichophyton rubrum (1804), Fusarium oxysporum (5119), Scopulariopsis brevi-
caulis (729), and Aspergillus flavus (3214) were obtained from the Mycological Center
of Assiut University. Chloramphenicol was used as an antibacterial standard, while clotri-
mazole was used as an antifungal standard (Table 2). DMSO was used as a blank, which
exhibited no activity against any of the used organisms. The obtained data revealed that
most of the selected compounds 2a–c and 3a–c showed no activity against all organisms
of bacteria used except for 2a and 2c, which showed remarkably good activity potential
against Staphylococcus aureus and Bacillus cereus with inhibition zone of 18 and 23 mm
for 2a at a concentration of 20 mg/mL, and inhibition zone of 12 and 16 mm for 2c at a con-
centration of 20 mg/mL. The minimum inhibition concentration (MIC) values were found
to be 8 (0.78) and 10 (0.1) mg/mL for 2a, and 8 (3.125) and 12 (3.125) for 2c respectively
(Table 3). All compounds 2a–c and 3a–c showed strong fungicidal effect against all species
of fungi, especially the two compounds 2a and 2c. The MIC values of these compounds
were 11, 12, 12, 10, 8, and 10 mg/mL (0.4%) for 2a and 10, 10, 8, 8, and 10 mg/mL
(6.25%) for 2c. The strong fungicidal and bactericidal effects of these two compounds are
presumably due to the presence of either cyano- or selenoglycolic acid groups in 2a and
the selenoglycolic acid in 2c. In addition, the effect of the pyridine and quinoline nuclei
may play a role. These compounds seem to be good bactericidal and fungicidal candidates
against all species of bacteria and fungi.
Generally, it was noticed that the activities of the tested compounds are less pro-
nounced than those of standard antifungal and antibacterial agents used. Also, organose-
lenium compounds that contain bis-(pyridine, pyridazine, and quinoline) selenylglycolic
acid moieties do not exhibit antimicrobial activity against the tested organisms. It seems
Table 3 Antibacterial activity of chemical compounds tested. Inhibition zone (mm) and MIC (mg/mL) are given
in parentheses
Staphylococcus
aureus
AUMC No.
B-54
Bacillus cereus
AUMC No.
B-52
Sample
2a
2c
8 (0.78)
8 (3.125)
10 (0.15)
10 (0.1)
12 (3.125)
16 (0.15)
12∗ (CHL)
Candida
albicans
AUMC No.
418
Geotrichum
candidum
AUMC No.
226
Fusarium
oxysporum
AUMC No.
5119
Aspergillus
flavus
AUMC No.
3214
Trichophyton Scopulariopsis
rubrum
AUMC No.
1804
brevicaulis
AUMC No.
729
Sample
2a
2c
11 (0.4)
10 (6.25)
26 (0.3)
12 (0.4)
10 (6.25)
22 (0.3)
12 (1.56)
8 (3.125)
18 (0.3)
10 (0.4)
8 (25)
25 (0.3)
8 (0.4)
10 (0.78)
34 (0.3)
10 (6.25)
10 (25)
20 (0.3)
12∗∗ (CLO)
12∗∗ = Clotrimazole (CLO) was used as standard.

ONE POT SYNTHESIS OF HETEROYLSELENOGLYCOLIC ACIDS
1289
obvious that quinolineselenyl and pyridineselenylglycolic acids demonstrated the best re-
sults in biological screening.
Screening of In-vitro Antioxidant Activities of Mono- and
Diselenoglycolic Acid Derivatives of 2a–c and 3a–c
Screening of in-vitro antioxidant activities of mono- and diselenoglycolic acid
derivatives of pyridine, pyridazine, and quinoline nuclei was investigated. The antioxi-
dant activities, studied by three methods, viz., Diphenylpicrylhydrazyl (DPPH) assay, β-
carotene/linoleic acid bleaching assay, and ferric reducing power assay, revealed significant
antioxidant activity compared with synthetic antioxidant activity.
Pharmacological Results and Discussion
The potential antioxidant activity of mono- and diselenoglycolic acid derivatives and
the parent selenols of pyridine, pyridazines, and quinoline were evaluated on the basis
of three methods: the scavenging activity of the stable free radical DPPH (EC50 value);
inhibition of the coupled oxidation of linoleic acid and beta-carotene and ferric reducing
antioxidant power. The obtained result was given EC50 values, which is defined as the
half maximum effective concentration. The data are presented in Table 4 and Figure 1.
Generally, each compound with a lower value of EC50 has the higher free radical scaveng-
ing activity. The resulting data showed that the parent pyridineselenol and the mono- and
diselenoglycolic acid derivatives have a good free radical scavenging activity, while quino-
lidineselenol and its mono- and diseleno derivatives have moderate free radical scavenging
activity compared with other tested compounds. Regarding EC50 values of pyridineselenol
and mono- or diseleno derivatives, the DPPH values were 121.01 9.81, 102.39 7.34,
and 95.259
4.60 μg/mL respectively. For EC50 β-carotene, values of 24.51
2.07,
21.13 1.54, and 17.31 1.93 μg/mL were found, and for EC50 ferric, reducing values
of 46.21 3.11, 41.12 2.32, and 35.17 1.77 μg/mL were found. The DPPH values
Table 4 EC50 values (μg/mL) of mono- and diselenoglycolic acids and their parent pyridine-2-selenol,
pyridazine-2-selenol, and quinolineselenol
DPPH assay
EC50
β-carotene assay
Ferric reducing assay
EC50
Sample
EC50
BHT∗
37.14 3.21a
9.64 1.87a
2.54 0.87
3.91 0.73
nt
nt
nt
α-Tocopherol∗
EDTA∗
Pyridine-2-selenol
nt
7.32 1.05
46.21 3.11
41.12 2.32
35.17 1.77
45.40 2.34
39.22 2.11
35.09 3.08
67.20 4.64
51.31 4.12
46.32 2.08 >
121.01 9.81b
102.39 7.34b
95.259 4.60b
256.24 6.87b
235.76 4.67b
226.31 3.14b
145.76 6.78b
140.22 5.91b
138.32 3.72b
24.51 2.07
21.13 1.54
17.31 1.93
56.23 2.43
47.12 2.93
42.23 1.98
41.12 1.93
39.47 1.31
33.45 2.11
Pyridine-monoselenoglycolic acid
Pyridine-diselenoglycolic acid
Pyridazineselenol
Pyridazine-monoselenoglycolic acid
Pyridazine-diselenoglycolic acid
Quinolidineselenol
Quinoline-monoselenoglycolic acid
Quinoline-diselenoglycolic acid
∗Standards; nt = not tested; a, bsignificant compared with standards.

1290
S. H. ABDEL-HAFEZ ET AL.
Figure 1 EC50 values of tested compounds with different antioxidants assays.
for quinolinemono- and -di- derivatives were 145.76 6.78, 140.22 5.91, and 138.32
3.72 μg/mL.
Generally, pyridineselenol and the mono- and diselenoglycolic acid derivatives 5a,
2a, and 3a respectively, have better free radical scavenging activity than quinolineselenol
and the mono- and diselenoglycolic acid derivatives 5c, 2c, and 3c, respectively, which
have a moderate antioxidant activity. The pyridazineselenol and the mono- and diseleno-
glycolic acid 5b, 2b and 3b, respectively have the lowest antioxidant activity in this class
of compounds as compared with other derivatives. The order of decreasing antioxidant
activities was diselenoglycolic acid of pyridine/quinoline > monoselenopyridine/quinoline
> their parent pyridine/quinolineselenols. The data in Fig. 1 show that the efficiency of
the tested compounds as a DPPH free radical scavenger, more effective than the two other
ways β-carotene linoleic acid system and ferric reducing assay, where the seleno compound
reacts with the hydrated free radical (OH.H) to form stable compounds before they destroy
biological macromolecules.22,23 Lipid peroxidation is an oxidative deterioration process of
polyunsaturated fatty acids which is induced by free radicals. In the β-carotene linoleic acid
system assay, effective compounds block the chain reaction of lipid peroxidation mainly
by scavenging the generated intermediate lipid peroxyl radicals.24 In the reducing power
assay, the presence of antioxidants in the tested samples would cause reducing ferricyanide
complex in ferrous form, turning the yellow color of test solution into various shades of
green and blue colors, which depends on the reducing power of antioxidant samples.25
EXPERIMENTAL
Chemistry
Melting points were determined by using the Kofler melting point apparatus, and were
uncorrected. IR (KBr, cm−1) spectra were recorded on a Pye-Unicam SP3–100 instrument.

ONE POT SYNTHESIS OF HETEROYLSELENOGLYCOLIC ACIDS
1291
1H NMR spectra were obtained on a Varian (400 MHz) EM 390 USA instrument at
King Abdel-Aziz University by using TMS as internal reference.13 C NMR spectra were
recorded on a JNM-LA spectrometer (100 MHz) at King Abdel-Aziz University, Saudi
Arabia. Elemental analyses were obtained on an Elementar Vario EL 1150C analyzer.
Purity of the compounds was checked by thin layer chromatography (TLC) using silica gel
plates. Mass spectra were recorded on a JEOL-JMS-AX 500 at Cairo National Research
Center, Cairo, Egypt and JEOL-JMS 600 at Assiut University, Assiut, Egypt. Purity of the
compounds was checked by TLC.
General procedure (1).
One pot three-stage synthesis of monoheteroylselenoglycolic acids
2a–c. An aqueous solution of NaBH4 (26.5 mmol) was added with magnetic stirring
at room temperature to powder gray selenium (12.7 mmol) suspended in distilled water
or methanol. Within a few minutes, a considerable amount of H2 was liberated and the
selenium was consumed in approximately 5 min. The almost colorless solution of the
resultant NaHSe was used without further treatment. Heteroyl chloride (12.6 mmol) was
added and magnetically stirred for 1 h. A dark red solution was formed, and shortly there-
after α-chloroacetic acid (12.6 mmol) was added in small portions and magnetically stirred
for another 1 h. A black gray solid precipitated, which was poured on crushed ice, then
filtered off, washed with distilled water, dried at room temperature, and recrystallized from
chloroform.
(3-Cyano-4,6-dimethylpyridin-2-yl)selenoglycolic acid (2a)
◦
M.p. 96–97 C (CHCl3), orange crystals, 69% yield. IR: 3410 (OH), 3050 (CHar),
1
2200 (CN), 1730 (CO). H NMR (DMSO-d6): δ 7.43 (s, 1H, CHpyridine), 3.32 (s, 2H,
SeCH2), 2.48 (s, 3H, CH3), 2.47 (s, 3H, CH3). 13C NMR (CDCl3, 75 MHz): δ 20.58–24.53
(CH3)2, 29.70 (CH2), 108(C CN), 114 (CN), 122 (H C), 151–152(C CH3), 155 (C Se),
162 (COOH). MS: m/z (%) 269 [M+1]+. Calcd. for C10H10O2N2Se: C, 44.62; H, 3.74; N,
10.41. Found C, 44.35; H, 3.70; N, 10.33.
(4-Cyano-5,6-diphenylpyridazin-3-yl)selenoglycolic acid (2b)
◦
M.p. 110–112 C (CHCl3), brown crystals, 55% yield. IR: 3405 (OH), 2200 (CN),
1
1740 (CO). H NMR (DMSO-d6): δ 14.15 (s, 1H, COOH), 7.43–7.29 (m, 10H, ArH),
3.36 (s, 2H, SeCH2). 13C NMR (CDCl3, 75 MHz): δ 56 (CH2), 112(C CN), 121 (CN),
127.78–128.94 (Ph) 2, 130 (Ph C C), 136 (Ph C N), 161 (C Se), 172 (COOH). MS:
m/z (%) 394 [M]+. Calcd. for C19H13O2N3Se C, 57.88; H, 3.32; N, 10.66. Found C, 57.65;
H, 3.15; N, 10.23.
(4,6-Dimethylquinolin-2-yl)selenoglycolic acid (2c)
◦
M.p. 115–117 C (CHCl3), beige crystals, 50% yield. IR: 3400 (OH), 1720 (CO).
1H NMR (DMSO-d6): δ 11.53 (s, 1H, COOH), 7.82–7.38 (m, 4H, CHquinoline), 3.40 (s, 2H,
SeCH2), 2.62 (s, 3H, CH3), 2.51 (s, 3H, CH3). 13C NMR (CDCl3, 75 MHz): δ 18.6–18.7
(CH3)2, 21.8 (CH2), 120 (CH3 C Se), 122 (Ph C C Se), 126–147 (Ph), 149 (C Se),
153 (COOH). MS: m/z (%) 294 [M+2]+. Calcd. for C13H13O2NSe: C, 53.07; H, 4.45; N,
4.76. Found C, 52.88; H, 4.19; N, 4.21.
General procedure (2).
One pot three-stages synthesis of di-heteroylselenylglycolic acids (3a–c).
An aqueous solution of NaBH4 (26.5 mmol) was added with magnetic stirring at room tem-
perature to powdered gray selenium (12.7 mmol) suspended in distilled water or methanol.
Within a few minutes, a considerable amount of H2 was liberated, and the selenium was
consumed in approximately 5 min. The almost colorless solution of resultant NaHSe was

1292
S. H. ABDEL-HAFEZ ET AL.
used without further treatment. The heteroyl chloride (12.6 mmol) was added and magnet-
ically stirred for 1 h. Dark red solution was formed, and shortly thereafter dichloroacetic
acid (12.6 mmol) was added in small portions and magnetically stirred for 1h. Within a few
minutes, a black gray solid precipitated, which was poured on crushed ice, then filtered off,
washed with distilled water, dried at room temperature, and recrystallized from chloroform.
Bis-](3-cyano-4,6-dimethylpyridin-2-yl)selenyl]ethanoic acid (3a)
M.p 105–107 ◦C, deep orange crystals, 35% yield. IR: 3410 (OH), 2200 (CN), 1730
1
(CO). H NMR (DMSO-d6,): δ 7.42 (s, 2H, CHpyridine), 3.34 (s, 1H, SeCH), 2.49–2.46
(s, 12H, CH3), COOH disappeared as a result of H-bond. 13C NMR (CDCl3, 75 MHz): δ
20.28–24.81[(CH3)2)2], 29.70 (CH-COOH), 108–109 (C-CN)2, 114–115 (CN)2, 122–123
(H-C)2, 151–152 (C-CH3)2, 154–155 (C-Se)2, 162 (COOH). MS: m/z (%) 475 [M+1]+.
Calcd. for C18H16O2N4Se2: C, 45.20; H, 3.37; N, 11.71. Found C, 45.00; H, 3.14; N, 11.46.
Bis-[(4-cyano-5,6-diphenylpyridazin-3-yl)selenyl]ethanoic acid (3b)
M.p. 127–129 ◦C, deep brown crystals, 20% yield. IR: 3400 (OH), 2200 (CN), 1742
1
(CO). H NMR (DMSO-d6): δ 14.13 (s, 1H, COOH), 7.54–7.21 (m, 20H, ArH), 3.42 (s,
1H, SeCH). MS: m/z (%) 728 [M–28]+. Calcd. for C36H22O2N6Se2: C, 59.35; H, 3.04; N,
11.54. Found C, 59.30; H, 2.99; N, 11.26.
Bis-[(4,6-dimethylquinolin-2-yl)selenyl]ethanoic acid (3c)
M.p. 150–152 ◦C, yellow crystals, 25% yield. IR: 3410 (OH), 2200 (CN), 1740 (CO).
1H NMR (DMSO-d6): δ 7.84–7.39 (m, 8H, CHquinoline); 3.35 (s, 1H, SeCH), 2.62–2.49 (s,
12H, CH3), COOH disappeared as a result of H-bond. MS: m/z (%) 528 [M]+. Calcd. for
C24H22O2N2Se2: C, 54.56; H, 4.20; N, 5.30. Found C, 54.33; H, 4.10; N, 5.1.
General procedure (3).
Unequivocal synthesis of compounds 2a–c and 3a–c through the in-
termediate compounds 5a–c. Compounds 5a–c were prepared as previously de-
scribed.19,20 All new compounds 2a–c and 3a–c, prepared through procedure (3), were
spectroscopically identical with the compounds obtained via procedures (1) and (2). It is
noticeable that the yields obtained via procedures (1) and (2) are much better compared
with those prepared from procedure (3).
Biological Assay
Antibacterial activity. Five bacterial species, representing both Gram-positive and
Gram-negative strains, were used to test the antibacterial activities of the target compounds
2a–c and 3a–c in vitro in comparison to chloramphenicol as a reference drug using the
standard agar paper disc diffusion method21 against S. aureus (B-54), B. cereus (B-52), E.
coli (B-53), S. marcescens (B-55), and P. aeruginosa (B-73). Cell suspensions of bacterial
strains were prepared from 48-h old cultures grown on potato dextrose agar (PDA) or
Sabouraud agar (SA) media. Cell suspension, 1 mL, was added to Petri dishes of 9 cm in
diameter, and then 15 mL of nutrient agar was poured into plates. Plates were shaken gently
to homogenize the innoculum. Sterile 5-mm filter paper (Whatmann, UK) was saturated
with 10 mg·mL−1 of the test compound, chloramphenicol solutions (200, 100, 50, 25,
15.5, 6 mg·mL−1 concentration) as a reference drug, or DMSO as negative control. Then
impregnated discs were dried for 1 h, and placed in the center of each plate. The seeded
plates were incubated at 35 2 ◦C for 24–48 h. The radii of inhibition zones, in millimeter
of triplicate sets, were measured. The results are given in Tables 1–3.
Antifungal activity. Compounds 2a–c and 3a–c were screened for their antifungal
activity in vitro, in comparison to clotrimazole as a reference drug using the standard agar

ONE POT SYNTHESIS OF HETEROYLSELENOGLYCOLIC ACIDS
1293
paper disc diffusion method21 against six fungi: C. albicans (418), G. candidum (226), T.
rubrum (1804), F. oxysporum (5119), S. brevicaulis (729), and A. flavus (3214). A spore
suspension in sterile distilled water was prepared from 2–3 days old culture of the fungi
growing on potato dextrose agar (PDA) or Sabouraud agar (SA) media. The final spore
concentration was 56104 spores mL−1. About 15 mL of the growth medium was placed into
sterile Petri dishes of 9 cm in diameter and incubated with 1 mL of the spore suspension.
◦
Plates were shaken gently to 28 2 C for 7 days. The radii of the inhibition zones in
millimeter of triplicate sets were measured. The results are shown in Tables 1–3.
Screening of Anti-oxidant Activity
Chemicals. DPPH, methanol, n-hexane, 2-deoxy-2-ribose, β-carotene, and linoleic
acid; BHT; α-tocopherol and EDTA were procured from Sigma (Sigma-Aldrich GmbH,
Sternheim, Germany). Tween 40 and dimethyl sulfoxide (DMSO) were from Merck (Darm-
stadt, Germany). All chemical reagents used were of analytical grade.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. The potential antioxidant activ-
ity of mono- and diseleno derivatives of pyridineselenol, pyridazineselenol, and quinoline-
selenol were measured in terms of hydrogen-donation or radical-scavenging ability, using
the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) as reagent.26 50 μL of each com-
pound (100 μg/mL) was added to 2 mL of 60-μM buffered methanol solution of DPPH
(pH = 5.5). Absorbance measurements were read at 517 nm after 20 min of incubation
time at room temperature. Absorption of a blank sample containing the same amount of
buffered methanol and DPPH solution acted as control, and BHT was used as standard
antioxidant. All determinations were performed in triplicate. The percentage inhibition of
DPPH radical by the samples was calculated according to the following formula:
%Inhibition = [(AB − AA)/AB] × 100,
where AB is the absorption of the blank sample (t = 0 min) and AA is the absorption of the
tested compound or standard substance solution (t = 30 min).
β-Carotene/linoleic acid bleaching assay. The antioxidant activity was deter-
mined by measuring the inhibition of conjugated dienehydroperoxides arising from linoleic
acid oxidation and method running as described by Miraliakbari and Shahidi.27 A stock
solution of β-carotene and linoleic acid was prepared with 0.5 mg of β-carotene in 1 mL
CHCl3, 25 μL of linoleic acid, and 200-mg Tween 40. The solvent was evaporated un-
der vacuum, and 100 mL of aerated distilled water was added to the residue. Each tested
compound dissolved in DMSO, and 350 μL of solution was added to 2.5 mL of the above
◦
mixture in test tubes. The test tubes were incubated in a hot water bath at 50 C for 2 h,
together with two tubes, one containing the antioxidant α-Tocopherol as standard, and the
other containing the same volume of DMSO instead of compounds’ solutions. The test tube
with BHT maintained its yellow color during the incubation period. The absorbencies were
measured at 470 nm on a spectrometer. Antioxidant activities (inhibition percentage, I%)
of the samples were evaluated in terms of the bleaching of β-carotene using the following
equation:
Inhibition% = [A(β − carotene after 2-h assay)/A(initial β-carotene)] × 100,
where A(β-carotene after 2-h assay) is the absorbance of β-carotene after 2-h assay re-
maining in the samples, and A(initial β-carotene) is the absorbance of β-carotene at the

1294
S. H. ABDEL-HAFEZ ET AL.
beginning of the experiments. All tests were carried out in triplicate and inhibition percent-
ages were reported as means SD of triplicates.
Ferric Reducing Antioxidant Power Assay
The reductive potential of the mono- and diseleno derivatives of pyridineselenol, pyri-
dazineselenols, and quinolineselenol and the standard (EDTA) was determined according
to the method of Oyaizu.28 Each tested compound or standard was mixed with phosphate
buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (K3Fe(CN)6: 2.5 mL, 1%).
Then the mixture was incubated at 50 ◦C for 20 min. Thereafter, 2.5 mL of trichloroacetic
acid (10%) was added to the mixture, which was then centrifuged for 10 min at 3000 rpm.
Finally, the upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL)
and FeCl3 (0.5 mL, 0.1% w/v), and the absorbance was measured at 700 nm. Increased
absorbance of the reaction mixture indicated increase of reducing power.
Statistical Analysis
Each of the measurements described was carried out in three replicate experiments,
and the results were recorded as mean standard deviation. The significant difference was
calculated at p ≤ 0.05.
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