Synthesis, characterization and biological evaluation of fused thiazolo[3,2-a]pyrimidine derivatives

Article abstract of DOI:10.1039/c4ra02514h

A series of fused thiazolo[3,2-a]pyrimidines (7a-g, 8a-f, 11a-g and 12a,b) have been synthesized in good yields by reaction of fused 3,4-dihydropyrimidin- 2(1H)-thiones (4a-g) with phenacyl bromides (5,6)/3-(2-bromoacetyl)coumarins (9,10) under conventional heating in acetic acid. Analytical and spectral studies as well as single crystal X-ray diffraction data on the representative compound 8e confirmed the structure of all the reaction products. All the synthesized compounds were screened for their antibacterial, antioxidant and DNA cleavage activities. The compound 7e against Escherichia coli, 8a and 8c-e against Pseudomonas aeruginosa have shown prominent antibacterial activity compared to the standard drug Penicillin with MIC 9.375 μg mL^-1, whereas the compounds 11c, 12a and 12b have shown very good antioxidant activity compared to the standard drug Trolox with IC₅₀ values 12.36, 11.12 and 13.88 μM respectively. Compounds 11f and 12b have completely cleaved the DNA even at 50 μg mL^-1 concentration and the remaining compounds have partially cleaved the DNA.

Full text of DOI:10.1039/c4ra02514h

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: J. Banothu, M.
Khanapur, S. Basavoju, R. Bavantula, M. Narra and S. Abbagani, RSC Adv., 2014, DOI:
10.1039/C4RA02514H.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
S
Ar CHO
+
+
H2N NH2
O
2(a-g)
3
1
P(4-VPH)HSO4
Solvent-free, 120 oC
10-20 min
O
O
Br
O
O
O
Ar
N
Ar
Br
Ar
N
R
R
O
N
NH
(5, 6)
(9)
N
S
N
H
S
AcOH, reflux,
3-6 h
AcOH, reflux, 1-3 h
S
4(a-g)
11(a-g)
5/7(a-g):
R = 4-ClC6H4
6/8(a-f): R = 4-BrC6H4
O
O
AcOH, reflux,
2 h
Br
O
O
O
(10)
Ar
N
N
S
12a,b
Ar:
a = C6H5
c = 4-FC6H4
b = 4-OHC6H4
d = 4-OCH3C6H4
e
f
= 4-OH-3-OCH3C6H3
= 3,4-(OCH3)2C6H3
g = 4-ClC6H4
Scheme 1. Synthesis of fused 3,4-dihydropyrimidin-2(1H)-thiones and thiazolo[3,2-
a]pyrimidines.
Figure 1. Biologically potent thiazolo[3,2-a]pyrimidine derivatives.

RSC Advances
Page 2 of 24
View Article Online
DOI: 10.1039/C4RA02514H
Figure 2. ORTEP representation of compound 8e. Thermal ellipsoids are drawn at 50%
probability level.

Page 3 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
Figure 3. DNA cleavage activity of fused thiazolo[3,2-a]pyrimidines (7a-g, 8a-f, 11a-g and
12a,b) at 50 and 100 µg/mL. Where, M-Marker; P-Plasmid pUC18; D-DMSO treated
plasmid; F-FeSO4 (5 mM) treated plasmid; 5-50 µg/mL; 10-100 µg/mL.

RSC Advances
Page 4 of 24
View Article Online
DOI: 10.1039/C4RA02514H
Table 1. Synthesis of fused thiazolo[3,2ꢀa]pyrimidine derivatives.
Producta Ar
Time (h)
Yieldb (%)
82
M.P. (oC)
C6H5
4ꢀOHC6H4
5
4
5
4
3
3
5
5
5
6
5
3
4
1
3
3
2
1
1
2
2
2
319ꢀ320
292ꢀ294
301ꢀ303
260ꢀ262
233ꢀ236
271ꢀ273
317ꢀ318
322ꢀ324
294ꢀ296
298ꢀ300
261ꢀ263
242ꢀ244
280ꢀ282
290ꢀ291
293ꢀ294
277ꢀ279
275ꢀ277
267ꢀ269
261ꢀ263
280ꢀ282
310ꢀ312
264ꢀ266
7a
7b
80
79
82
85
84
82
80
79
80
81
86
84
88
82
80
87
4ꢀFC6H4
7c
4ꢀOCH3C6H4
3,4ꢀ(OCH3)2C6H3
4ꢀOHꢀ3ꢀOCH3C6H3
4ꢀClC6H4
7d
7e
7f
7g
C6H5
8a
4ꢀOHC6H4
8b
4ꢀFC6H4
8c
4ꢀOCH3C6H4
3,4ꢀ(OCH3)2C6H3
4ꢀOHꢀ3ꢀOCH3C6H3
C6H5
4ꢀOHC6H4
4ꢀFC6H4
4ꢀOCH3C6H4
3,4ꢀ(OCH3)2C6H3
4ꢀOHꢀ3ꢀOCH3C6H3
4ꢀClC6H4
C6H5
4ꢀOHC6H4
8d
8e
8f
11a
11b
11c
11d
11e
11f
11g
12a
12b
89
88
83
80
77
a
Reaction conditions: Fused 3,4ꢀdihydropyrimidinꢀ2(1H)ꢀthiones (4aꢀg, 1 mmol), 4ꢀ
chlorophenacyl bromide/4ꢀbromophenacyl bromide/3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone/2ꢀ
(2ꢀbromoacetyl)ꢀ3Hꢀbenzo[f]chromenꢀ3ꢀone (1 mmol), acetic acid (10 mL), reflux.
b Isolated yields.

Page 5 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
Table 2. Antibacterial activity of fused thiazolo[3,2ꢀa]pyrimidine derivatives.
MIC (ꢁg/mL)
Compound
Bs
75
ꢀ
75
ꢀ
ꢀ
ꢀ
75
ꢀ
Sa
Se
75
ꢀ
ꢀ
ꢀ
Ec
75
ꢀ
Pa
Kp
75
ꢀ
ꢀ
ꢀ
18.75
ꢀ
75
150
75
7b
7d
7e
7f
7g
8a
8b
8c
9.375 37.5
ꢀ
ꢀ
18.75 9.375
37.5 37.5
18.75 9.375
18.75 9.375
150
37.5
ꢀ
75
ꢀ
75
ꢀ
ꢀ
ꢀ
150
37.5
75
37.5
18.75
37.5
150
ꢀ
18.75
37.5
9.375
75
ꢀ
37.5
ꢀ
75
75
18.75 18.75
ꢀ
75
75
8d
8e
8f
150
150
150
9.375
75
ꢀ
11a
1.562 1.562
6.25 6.25
3.125 12.5
3.125 6.25
12.5
1.562
6.25
3.125
Penicillin
Streptomycin
Gramꢀpositive bacterial strains:ꢀ Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Se:
Staphylococcus epidermidis;
Gramꢀnegative bacterial strains:ꢀ Ec: Escherichia coli; Pa: Pseudomonas aeruginosa; Kp:
Klebsiella pneumoniae.
“ꢀ” Indicates the concentration >150 ꢁg/mL.

RSC Advances
Page 6 of 24
View Article Online
DOI: 10.1039/C4RA02514H
Table 3. Antioxidant activity of fused thiazolo[3,2ꢀa] pyrimidines.
Compound
IC50 ( ꢁM)
82.58
80.50
25.79
80.91
66.75
41.29
29.67
44.75
46.57
26.01
74.86
63.32
28.76
64.34
18.25
12.36
53.22
47.54
28.45
47.26
11.12
13.88
14.22
10.32
9.84
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
11a
11b
11c
11d
11e
11f
11g
12a
12b
Trolox
Curcumin
Quercetin
Ascorbic acid
3.8

Page 7 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
Graphical abstract
Synthesis, characterization and biological evaluation of fused thiazolo[3,2-a]pyrimidine
derivatives
Janardhan Banothua, Manjulatha Khanapurb, Srinivas Basavojua, Rajitha Bavantulaa*, Muralikrishna Narra,c
Sadanandam Abbaganc*
A series of fused thiazolo[3,2-a]pyrimidines have been synthesized in good yields by reaction
of fused 3,4-dihydropyrimidin-2(1H)-thiones (4a-g) with phenacyl bromides (5,6)/3-(2-
bromoacetyl)coumarins (9,10) under conventional heating in acetic acid. All the synthesized
compounds were characterized by analytical and spectral studies, and screened for their
antibacterial, antioxidant and DNA cleavage activities.

RSC Advances
Page 8 of 24
View Article Online
DOI: 10.1039/C4RA02514H
Synthesis, characterization and biological evaluation of fused thiazolo[3,2-
a]pyrimidine derivatives
Janardhan Banothua, Manjulatha Khanapurb, Srinivas Basavojua, Rajitha Bavantulaa*,
Muralikrishna Narra,c Sadanandam Abbaganc*
aDepartment of Chemistry, National Institute of Technology, Warangal-506004, A.P., India.
bDepartment of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad-
500046, A.P., India
cDepartment of Bio-Technology, Kakatiya University, Warangal-506009, A.P., India.
Corresponding Author
Prof. Rajitha Bavantula
Tel.: 0091ꢀ0870ꢀ2459445
Fax: 0091ꢀ0870ꢀ2459547
Eꢀmail: rajitabhargavi@yahoo.com
Abstract
A series of fused thiazolo[3,2ꢀa]pyrimidines (7a-g, 8a-f, 11a-g and 12a,b) have been
synthesized in good yields by reaction of fused 3,4ꢀdihydropyrimidinꢀ2(1H)ꢀthiones (4a-g)
with phenacyl bromides (5,6)/3ꢀ(2ꢀbromoacetyl)coumarins (9,10) under conventional heating
in acetic acid. Analytical and spectral studies as well as single crystal Xꢀray diffraction data
on the representative compound 8e confirmed the structure of all the reaction products. All
the synthesized compounds were screened for their antibacterial, antioxidant and DNA
cleavage activities. The compound 7e against Escherichia coli, 8a and 8c-e against
Pseudomonas aeruginosa have shown prominent antibacterial activity than the standard drug
Penicillin with MIC 9.375 ꢁg/mL, whereas the compounds 11c, 12a and 12b have shown
very good antioxidant activity compared to the standard drug Trolox with IC50 values 12.36,
11.12 and 13.88 ꢂM respectively. Compound 11f and 12b have completely cleaved the DNA
even at 50 ꢁg/mL concentration and remaining compounds have partially cleaved the DNA.
Key words: Antibacterial activity; Antioxidant activity; 3,4ꢀDihydropyrimidinꢀ2(1H)ꢀthiones;
DNA cleavage activity; Thiazolo[3,2ꢀa]pyrimidines.
Introduction
Treatment of bacterial infections still remains an important and challenging therapeutic
problem, due to the emergence of bacteria resistant to current therapeutic agents.1 Reactive

Page 9 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
oxygen species (ROS) are causing damage to nucleic acids, proteins, carbohydrates and lipids
in many types of cells including macrophages,2 eventually leading to many chronic diseases.3
Antioxidants exert their effects by preventing the generation of ROS, which can protect the
formation of free radicals and retard the progress of diseases.3 These are also useful to extend
the shelf life of food and pharmaceuticals during processing, storage and transportation.4
Deoxyribonucleic acid (DNA) is the primary target for most anticancer and antiviral
therapies.5 Therefore, the investigation towards the development of new antibiotics,
antioxidants and molecules that can exhibit DNA binding and cleaving properties have
become a necessary endeavor.
Dihydropyrimidine (DHPM) derivatives are known to possess diverse pharmacological
properties including calcium channel modulators, mitotic kinesin inhibitors, adrenergic
receptor antagonists, antibacterial and antiviral agents.6 Hence, much attention has been
focused on the modification of their core synthon. One such modification was observed when
the reaction of dihydropyrimidinꢀthiones is carried out with 1,2ꢀdielectrophiles such as 2ꢀ
bromo ketones,7 chloroacetyl chloride,8 chloroacetic acid,9 methyl chloroacetate10 and 1,2ꢀ
dichloroethane11 to form ring annulation products, thiazolo[3,2ꢀa]pyrimidine derivatives. The
synthesis of thiazolo[3,2ꢀa]pyrimidines have recently gained considerable interest due to the
broad spectrum of biological activities that include antiꢀinflammatory,9a,12 antihypertensive,13
antifungal,7a antibiofilm, antibacterial,9b antiviral,9c antioxidant,14 anticancer,15 antitumor,16
antiꢀHIV,17 calcium antagonists7b and group 2 metabotropic glutamate receptor antagonists.18
They also serve as inhibitors of CDC25B phosphatase,8 acetylcholinesterase (AChE)
enzymes19 and Bclꢀ2 family proteins.20 The structures of thiazolo[3,2ꢀa]pyrimidine
derivatives and their pharmacological activities are given in Figure 1.6
(Figure 1)
In continuation of our research towards the synthesis of biologically potent heterocyclic
compounds,21 herein we report an efficient method for the synthesis of fused thiazolo[3,2ꢀa]
pyrimidine derivatives and to evaluate their antibacterial, antioxidant and DNA cleavage
activities.
Results and discussion
Fused 3,4ꢀdihydropyrimidinꢀ2(1H)ꢀthiones (4a-g) were prepared in good yields via a
modified Biginelli reaction on 1ꢀtetralone (1), aromatic aldehydes (2a-g) and thiourea (3) in
the presence of poly(4ꢀvinylpyridinium)hydrogen sulfate [P(4ꢀVPH)HSO4] as a catalyst

RSC Advances
Page 10 of 24
View Article Online
DOI: 10.1039/C4RA02514H
under solventꢀfree conditions at 120 oC.22 The title compounds, fused thiazolo[3,2ꢀ
a]pyrimidines (7a-g, 8a-f, 11a-g and 12a,b) were obtained by cyclization of 4a-g with 4ꢀ
chlorophenacyl bromide (5)/4ꢀbromophenacyl bromide (6)/3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromenꢀ
2ꢀone (9)/2ꢀ(2ꢀbromoacetyl)ꢀ 3Hꢀbenzo[f]chromenꢀ3ꢀone (10) under reflux in acetic acid
(Scheme 1, Table 1).
(Scheme 1)
(Table 1)
The formation of title compounds 7a-g, 8a-f, 11a-g and 12a,b from fused 3,4ꢀ
1
dihydropyrimidinꢀ2(1H)ꢀthiones (4a-g) were characterized by IR, H NMR, 13C NMR and
mass spectral data. From the FTIR spectra, the disappearance of absorption bands ranging
from 3144ꢀ3274 cmꢀ1 for thioamide (–N–H) in the starting material and the appearance of a
weak band ranging from 1598ꢀ1611 cmꢀ1 in 7a-g and 8a-f, and 1628ꢀ1636 cmꢀ1 in 11a-g and
12a,b of imine (–C=N–) group was indicative of formation of the products. Compounds 11a-
g and 12a,b were also shown a strong band ranging from 1710ꢀ1740 cmꢀ1 confirm the
1
presence of lactone carbonyl (–O–C=O) group. From the H NMR spectra, the absence of
singlets at 8.98ꢀ9.47 ppm and 9.66ꢀ9.81 ppm for thioamide (–N–H) in the starting material,
and from 13C NMR the disappearance of a signal at 173.2ꢀ174.3 ppm of cyclic thiourea
carbon (–C=S), and the presence of a signal at 160.9ꢀ163.3 ppm of imine carbon (–C=N)
confirmed the formation of the products. In addition, the molecular ion peak from the mass
spectra, elemental analyses data and single crystal Xꢀray diffraction structure of a compound
8e are further evidences for the formation of products.
Single crystal Xꢀray diffraction analysis of compound 8e was carried out on a crystal
obtained from the reaction mixture. The compound was crystallizes in the triclinic space
group Pꢀ1 with one completely protonated 8e, one partially protonated 8e, one Br–, one
partially proton transferred HBr and three molecules of acetic acid in the asymmetric unit.
The ORTEP representation of the molecular structure of 8e is shown in Figure 2. A summary
of the crystallographic data, structure refinement details, the geometrical parameters of
hydrogen bonds and packing diagram is given in the supporting file.
(Figure 2)
All the synthesized compounds were screened for their in vitro antibacterial activity
against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis (Gramꢀ
positive), Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (Gramꢀ
negative) bacterial strains by micro dilution method recommended by CLSI standard

Page 11 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
protocol23 using Penicillin and Streptomycin as standard drugs. The results are tabulated in
Table 2.
(Table 2)
Evaluation of the antibacterial screening data revealed that, the compound 7e against
Escherichia coli, 8a and 8c-e against Pseudomonas aeruginosa have shown prominent
activity than the standard drug Penicillin (MIC: 12.5 ꢁg/mL) with MIC 9.375 ꢁg/mL (~ 1.33
fold potent than Penicillin, Table 2). The potential activity may be attributed due to the
presence of pharmacologically active moieties like phenyl, 4ꢀfluoro phenyl, 4ꢀmethoxy
phenyl and 3,4ꢀdimethoxy phenyl groups on pyrimidine core and 4ꢀchloro phenyl/4ꢀbromo
phenyl group on thiazole core. Among all the compounds, 8d is active against all the tested
bacterial strains. Remaining compounds derived from 4ꢀchlorophenacyl bromide and 4ꢀ
bromophenacyl bromide has shown weak to moderate activity. Compound 11a (bearing
pyranone moiety) has shown weak activity against Gramꢀpositive bacterial strains whereas
inactive against Gramꢀnegative bacterial strains, rest of the compounds (11b-g and 12a,b)
were inactive against all the tested bacterial strains.
All the synthesized compounds were also screened for free radical scavenging activity in
terms of hydrogen donating or radical scavenging ability by DPPH method.24 Methanol
(95%), DPPH solution and standard compounds (Trolox, Curcumin, Quercetin and Ascorbic
acid) were used as blank, control and reference respectively. The IC50 values of the
compounds under study for antioxidant results are shown in Table 3.
(Table 3)
There is mounting evidence for the radical scavenging capacity of phenol and coumarin
derivatives.25 The free radical scavenging ability of compounds in present study results have
also exhibited good scavenging ability for compounds bearing 4ꢀfluoro phenyl and 2Hꢀ
chromenꢀ2ꢀone (11c, IC50 value 12.36 ꢂM), and phenyl/4ꢀhydroxy phenyl and 3Hꢀ
benzo[f]chromenꢀ3ꢀone (12a, IC50 value 11.12 ꢂM and 12b, IC50 value 13.88 ꢂM). These on
comparison with the standard Trolox with IC50 value of 14.22 ꢂM found to be active by ~
1.15 fold for 11c, ~ 1.27 fold for 12a and ~ 1.02 fold for 12b. Compound possessing 4ꢀ
hydroxy phenyl/4ꢀfluoro phenyl/4ꢀhydroxyꢀ3ꢀmethoxy phenyl/4ꢀchloro phenyl on pyrimidine
core, and 4ꢀchloro phenyl/4ꢀbromo phenyl/2Hꢀchromenꢀ2ꢀone on thiazole core (7c, 7g, 8c, 8f,
11b and 11f) have shown moderate scavenging ability with IC50 values 25.79, 29.67, 26.01,
28.76, 18.25 and 28.45 ꢂM respectively. Remaining compounds have shown weak
scavenging ability/capacity with IC50 values ranging from 41.29ꢀ82.58 ꢂM.

RSC Advances
Page 12 of 24
View Article Online
DOI: 10.1039/C4RA02514H
The DNA cleavage activity of the titled compounds (7a-g, 8a-f, 11a-g and 12a,b) was
determined using agarose gel electrophoresis method.26 DNA cleavage is controlled by
relaxation of supercoiled circular form into nicked circular form and linear form. When
circular plasmid DNA is subjected to electrophoresis, relatively fast migration will be
observed for the intact supercoiled form. If one strand is cleaved, the supercoils will relax to
produce a slower moving open circular form. If both strands are cleaved, a linear form will be
generated and migrates between supercoiled and open circular forms. The pictures of the gels
are presented in Figure 3.
(Figure 3)
The gel after electrophoresis clearly revealed that, the plasmid pUC18 having three
forms of DNA, whereas in case of standard FeSO4 (10 mg/1 mL) complete DNA cleavage
was observed. Compared to standard FeSO4, compound 11f and 12b have completely cleaved
the DNA even at 50 ꢁg/mL, whereas 7c, 7f, 8b and 8e have shown complete cleavage at 100
ꢁg/mL. Remaining compounds have partially cleaved the DNA. From these results we
deduce that, the presence of pyranone moiety on thiazole core, 4ꢀhydroxy phenyl and 4ꢀ
hydroxyꢀ3ꢀmethoxy phenyl groups on pyrimidine core has enhanced the DNA cleavage
activity.
Conclusion
In conclusion, we have synthesized a series of fused thiazolo[3,2ꢀa]pyrimidines under
conventional method in good yields, and evaluated their antibacterial, antioxidant and DNA
cleavage activities. The compound 7e against E.coli, 8a and 8c-e against P.aeruginosa are
shown marked antibacterial activity and the compounds 11c, 12a and 12b are shown very
good antioxidant activity. Similarly, the compound 11f and 12b are shown complete DNA
cleavage even at 50 ꢁg/mL. These results are suggesting that the synthesized compounds can
be better candidates for future investigations.
Experimental
All the reagents and solvents were purchased from Aldrich/Merck and used without
further purifications. Melting points were determined in open capillaries using Stuart SMP30
apparatus (Bibby Scientific Ltd. United Kingdom) and are uncorrected. The progress of the
reactions as well as purity of compounds was monitored by thin layer chromatography with
F254 silicaꢀgel precoated sheets (Merck, Darmstadt, Germany) using hexane/ethyl acetate 8/2
as eluent; UV light and iodine vapours were used for detection. IR spectra were recorded on a
PerkinꢀElmer 100S spectrometer (PerkinꢀElmer Ltd. United Kingdom) utilizing KBr pellets;

Page 13 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
values are expressed in cmꢀ1. 1H NMR and 13C NMR spectra were obtained at 400 MHz and
100 MHz respectively on Bruker spectrometer (Bruker Corporation Ltd., Germany) using
DMSOꢀd6 as solvent and TMS as internal standard, chemical shifts are expressed in parts per
million. Elemental analyses were performed on a CarloꢀErba model EA1108 analytical unit
(Triad Scientific Ltd., New Jersey, USA) and the values are ± 0.4% of theoretical values.
Mass spectra were recorded on a Jeol JMSDꢀ300 spectrometer (Jeol Ltd., Tokyo, Japan).
Synthesis of 4-aryl-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thiones (4a-g)
To a clean and dry round bottom flask were added 1ꢀtetralone (1, 1 mmol), aromatic
aldehyde (2a-g, 1 mmol), thiourea (3, 1.2 mmol) and P(4ꢀVPH)HSO4 (0.015 g). The resulting
o
mixture was heated at 120 C under solventꢀfree conditions for 10ꢀ20 min. After completion
of the reaction (monitored by TLC), water (5 mL) was added and the mixture was stirred at
room temperature for an additional 10 min. The solid separated out was filtered, washed with
water, dried and the crude product was crystallized from ethanol to afford the pure product.
The aqueous layer containing catalyst was recovered under reduced pressure, washed with
dichloromethane, dried and reused for subsequent runs.
4-Phenyl-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione (4a)
o
Yellow solid; Yield: 92%; Mp.: 258ꢀ259 C; IR (KBr) υmax (cmꢀ1): 3159 (NH), 1194 (C=S);
1H NMR (400 MHz, DMSOꢀd6): δ 1.80ꢀ1.88 (m, 1H), 2.15ꢀ2.23 (m, 1H), 2.55ꢀ2.62 (m, 1H),
2.69ꢀ2.77 (m, 1H), 4.95 (s, 1H), 7.17ꢀ7.22 (m, 3H), 7.31ꢀ7.41 (m, 5H), 7.68 (d, J = 7.2 Hz,
1H), 9.08 (s, 1H), 9.76 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 174.2, 142.8, 135.4,
128.6, 127.9, 127.7, 127.5, 126.9, 126.6, 126.3, 121.6, 111.1, 58.4, 27.3, 23.6; MS (ESI) m/z:
293 [M+H]+; Anal. Calcd. for C18H16N2S: C, 73.94; H, 5.52; N, 9.58; Found: C, 73.67; H,
5.71; N, 9.64.
4-(4-Hydroxyphenyl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione (4b)
o
White solid; Yield: 88%; Mp.: 270ꢀ272 C; IR (KBr) υmax (cmꢀ1): 3445 (OH), 3263, 3144
(NH), 1171 (C=S); 1H NMR (400 MHz, DMSOꢀd6): δ 1.79ꢀ1.87 (m, 1H), 2.09ꢀ2.18 (m, 1H),
2.57ꢀ2.63 (m, 1H), 2.67ꢀ2.73 (m, 1H), 4.82 (s, 1H), 6.75 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4
Hz, 2H), 7.15ꢀ7.22 (m, 3H), 7.67 (d, J = 7.2 Hz, 1H), 8.98 (s, 1H), 9.47 (s, 1H), 9.67 (s, 1H);
13C NMR (100 MHz, DMSOꢀd6): δ 173.8, 157.1, 135.4, 133.3, 128.3, 127.8, 127.6, 127.5,
126.4, 126.3, 121.6, 115.3, 111.6, 58.0, 27.3, 23.6; MS (ESI) m/z: 309 [M+H]+; Anal. Calcd.
for C18H16N2OS: C, 70.10; H, 5.23; N, 9.08; Found: C, 70.25; H, 5.17; N, 9.22.
4-(4-Fluorophenyl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione (4c)

RSC Advances
Page 14 of 24
View Article Online
DOI: 10.1039/C4RA02514H
o
White solid; Yield: 91%; Mp.: 252ꢀ254 C; IR (KBr) υmax (cmꢀ1): 3179 (NH), 1220 (CꢀF),
1
1190 (C=S); H NMR (400 MHz, DMSOꢀd6): δ 1.79ꢀ1.87 (m, 1H), 2.14ꢀ2.22 (m, 1H), 2.58ꢀ
2.64 (m, 1H), 2.69ꢀ2.73 (m, 1H), 4.99 (s, 1H), 7.17ꢀ7.24 (m, 5H), 7.34ꢀ7.37 (m, 2H), 7.69 (d,
J = 7.2 Hz, 1H), 9.09 (s, 1H), 9.78 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 174.2, 162.9,
139.1, 135.4, 129.0, 128.9, 127.8, 127.5, 126.7, 126.3, 121.7, 115.6, 115.4, 110.9, 57.6, 27.3,
23.5; MS (ESI) m/z: 311 [M+H]+; Anal. Calcd. for C18H15FN2S: C, 69.65; H, 4.87; N, 9.03;
Found: C, 69.52; H, 4.95; N, 9.16.
4-(4-Methoxyphenyl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione (4d)
o
Yellow solid; Yield: 93%; Mp.: 211ꢀ212 C; IR (KBr) υmax (cmꢀ1): 3274, 3196 (NH), 1253
1
(CꢀOꢀC), 1172 (C=S); H NMR (400 MHz, DMSOꢀd6): δ 1.79ꢀ1.87 (m, 1H), 2.12ꢀ2.20 (m,
1H), 2.55ꢀ2.63 (m, 1H), 2.68ꢀ2.74 (m, 1H), 3.74 (s, 3H), 4.88 (s, 1H), 6.93ꢀ6.95 (m, 2H),
7.15ꢀ7.24 (m, 5H), 6.67 (d, J = 7.2 Hz, 1H), 9.00 (s, 1H), 9.69 (s, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 173.9, 158.9, 135.4, 134.9, 128.2, 127.7, 127.6, 127.5, 126.5, 126.3, 121.6,
114.0, 111.4, 57.8, 55.1, 27.3, 23.6; MS (ESI) m/z: 323 [M+H]+; Anal. Calcd. for
C19H18N2OS: C, 70.78; H, 5.63; N, 8.69; Found: C, 70.89; H, 5.54; N, 8.58.
4-(3,4-Dimethoxyphenyl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione (4e)
Yellow solid; Yield: 94%; Mp.: 207ꢀ209 oC; IR (KBr) υmax (cmꢀ1): 3196 (NH), 1258 (CꢀOꢀC),
1
1138 (C=S); H NMR (400 MHz, DMSOꢀd6): δ 1.87ꢀ1.93 (m, 1H), 2.16ꢀ2.21 (m, 1H), 2.61ꢀ
2.65 (m, 1H), 2.69ꢀ2.75 (m, 1H), 3.73 (s, 6H), 4.89 (s, 1H), 6.82ꢀ6.85 (m, 1H), 6.92ꢀ6.97 (m,
2H), 7.16ꢀ7.23 (m, 3H), 7.66 (d, J = 7.2 Hz, 1H), 8.98 (s, 1H), 9.71 (s, 1H); 13C NMR (100
MHz, DMSOꢀd6): δ 174.0, 148.7, 148.5, 135.4, 135.2, 127.8, 127.7, 127.6, 126.6, 126.3,
121.6, 119.0, 111.9, 111.3, 111.0, 58.1, 55.6, 55.5, 27.4, 23.6; MS (ESI) m/z: 353 [M+H]+;
Anal. Calcd. for C20H20N2O2S: C, 68.16; H, 5.72; N, 7.95; Found: C, 68.02; H, 5.88; N, 8.06.
4-(4-Hydroxy-3-methoxyphenyl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione
(4f)
o
Yellow solid; Yield: 90%; Mp.: 230ꢀ232 C; IR (KBr) υmax (cmꢀ1): 3347 (OH), 3177 (NH),
1
1272 (CꢀOꢀC), 1195 (C=S); H NMR (400 MHz, DMSOꢀd6): δ 1.85ꢀ1.93 (m, 1H), 2.12ꢀ2.21
(m, 1H), 2.59ꢀ2.74 (m, 2H), 3.73 (s, 3H), 4.85 (s, 1H), 6.71ꢀ6.89 (m, 3H), 7.71ꢀ7.22 (m, 3H),
7.67 (d, J = 7.2 Hz, 1H), 8.97 (s, 1H), 9.06 (s, 1H), 9.69 (s, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 173.9, 147.5, 146.4, 135.4, 133.8, 127.8, 127.6, 127.5, 126.5, 126.3, 121.6,
119.4, 115.5, 111.5, 58.2, 55.6, 27.4, 23.6; MS (ESI) m/z: 339 [M+H]+; Anal. Calcd. for
C19H18N2O2S: C, 67.43; H, 5.36; N, 8.28; Found: C, 67.31; H, 5.49; N, 8.35.
4-(4-Chlorophenyl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione (4g)

Page 15 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
o
White solid; Yield: 94%; Mp.: 262ꢀ264 C; IR (KBr) υmax (cmꢀ1): 3269, 3194 (NH), 1195
(C=S), 775 (CꢀCl); 1H NMR (400 MHz, DMSOꢀd6): δ 1.79ꢀ1.87 (m, 1H), 2.15ꢀ2.22 (m, 1H),
2.56ꢀ2.64 (m, 1H), 2.69ꢀ2.77 (m, 1H), 4.98 (s, 1H), 7.18ꢀ7.23 (m, 3H), 7.33 (d, J = 8.4 Hz,
13
2H), 7.46 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 7.2 Hz, 1H), 9.10 (s, 1H), 9.81 (s, 1H); C NMR
(100 MHz, DMSOꢀd6): δ 174.3, 141.7, 135.4, 132.4, 128.8, 128.7, 127.8, 127.6, 126.9, 126.3,
121.7, 110.7, 57.6, 27.2, 23.4; MS (ESI) m/z: 327 [M+H]+; Anal. Calcd. for C18H15ClN2S: C,
66.15; H, 4.63; N, 8.57; Found: C, 66.24; H, 4.51; N, 8.69.
Synthesis
of
9-(4-chlorophenyl)-7-aryl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]
quinazolines (7a-g)
4ꢀArylꢀ3,4,5,6ꢀtetrahydrobenzo[h]quinazolineꢀ2(1H)ꢀthione (4a-g, 1 mmol) and 4ꢀchloro
phenacyl bromide (5, 1 mmol) were dissolved in 5 mL of glacial acetic acid and stirred at its
reflux temperature for 3ꢀ5 h. After completion of the reaction (monitored by TLC), the
mixture was kept a room temperature for 4 h, the solid separated out was filtered and
quenched with cold acetic acid. The crude products obtained were purified by
recrystallization from acetic acid.
9-(4-Chlorophenyl)-7-phenyl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazoline (7a)
White solid; IR (KBr) υmax (cmꢀ1): 1604 (C=N), 824 (CꢀCl); 1H NMR (400 MHz, DMSOꢀd6):
δ 1.76 (t, J = 7.6 Hz, 1H), 2.33 (t, J = 8.0 Hz, 1H), 2.57ꢀ2.79 (m, 2H), 6.16 (s, 1H), 6.84 (d, J
= 6.4 Hz, 2H), 7.17ꢀ7.46 (m, 11H), 7.65 (d, J = 6.8 Hz, 1H); MS (ESI) m/z: 427 [M+H]+;
Anal. Calcd. for C26H19ClN2S: C, 73.14; H, 4.49; N, 6.56; Found: C, 73.26; H, 4.59; N, 6.45.
4-(9-(4-Chlorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-7-yl)phenol
(7b)
1
Yellow solid; IR (KBr) υmax (cmꢀ1): 3244 (OH), 1609 (C=N), 836 (CꢀCl); H NMR (400
MHz, DMSOꢀd6): δ 1.75ꢀ1.82 (m, 1H), 2.26ꢀ2.32 (m, 1H), 2.61 (t, J = 8.0 Hz, 1H), 2.78 (t, J
= 7.6 Hz, 1H), 6.04 (s, 1H), 6.52 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 8.4 Hz, 2H), 7.25ꢀ7.40 (m,
6H), 7.50 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 7.2 Hz, 1H), 9.63 (s, 1H); MS (ESI) m/z: 443
[M+H]+; Anal. Calcd. for C26H19ClN2OS: C, 70.50; H, 4.32; N, 6.32; Found: C, 70.37; H,
4.50; N, 6.44.
9-(4-Chlorophenyl)-7-(4-fluorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-
b]quinazoline (7c)
White solid; IR (KBr) υmax (cmꢀ1): 1604 (C=N), 1236 (CꢀF), 837 (CꢀCl); 1H NMR (400 MHz,
DMSOꢀd6): δ 1.70ꢀ1.77 (m, 1H), 2.31 (t, J = 8.4 Hz, 1H), 2.59ꢀ2.64 (m, 1H), 2.75ꢀ2.79 (m,
1H), 6.20 (s, 1H), 6.89ꢀ7.03 (m, 4H), 7.25 (d, J = 8.4 Hz, 3H), 7.32ꢀ7.48 (m, 5H), 7.64 (t, J =

RSC Advances
Page 16 of 24
View Article Online
DOI: 10.1039/C4RA02514H
6.8 Hz, 1H); MS (ESI) m/z: 445 [M+H]+; Anal. Calcd. for C26H18ClFN2S: C, 70.18; H, 4.08;
N, 6.30; Found: C, 70.09; H, 4.23; N, 6.44.
9-(4-Chlorophenyl)-7-(4-methoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-
b]quinazoline (7d)
1
Yellow solid; IR (KBr) υmax (cmꢀ1): 1607 (C=N), 1246 (CꢀOꢀC), 822 (CꢀCl); H NMR (400
MHz, DMSOꢀd6): δ 1.75ꢀ1.82 (m, 1H), 2.28ꢀ2.35 (m, 1H), 2.60 (t, J = 8.4 Hz, 1H), 2.75ꢀ2.83
(m, 1H), 3.67 (s, 3H), 6.09 (s, 1H), 6.69ꢀ6.75 (m, 4H), 7.26ꢀ7.40 (m, 6H), 7.49 (d, J = 8.4 Hz,
2H), 7.63 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 161.3, 159.5, 138.5, 135.3,
134.9, 131.3, 130.6, 128.8, 128.5, 128.2, 128.1, 126.7, 123.7, 121.3, 114.0, 112.4, 110.1,
62.1, 55.1, 26.7, 23.2; MS (ESI) m/z: 457 [M+H]+; Anal. Calcd. for C27H21ClN2OS: C, 70.96;
H, 4.63; N, 6.13; Found: C, 70.84; H, 4.72; N, 6.27.
9-(4-Chlorophenyl)-7-(3,4-dimethoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]
quinazoline (7e)
1
Pale yellow solid; IR (KBr) υmax (cmꢀ1): 1609 (C=N), 1245 (CꢀOꢀC), 834 (CꢀCl); H NMR
(400 MHz, DMSOꢀd6): δ 1.77ꢀ1.82 (m, 1H), 2.31 (t, J = 7.6 Hz, 1H), 2.59ꢀ2.79 (m, 2H), 3.47
(s, 3H), 3.67 (s, 3H), 6.13 (s, 1H), 6.56ꢀ6.58 (m, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.25ꢀ7.40 (m,
13
7H), 7.50 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 7.6 Hz, 1H); C NMR (100 MHz, DMSOꢀd6): δ
161.1, 148.9, 147.9, 138.6, 135.3, 134.9, 131.5, 131.1, 128.8, 128.5, 128.1, 126.9, 126.7,
121.3, 119.5, 112.4, 112.1, 110.9, 109.8, 62.4, 55.4, 54.9, 26.7, 23.1; MS (ESI) m/z: 488
[M+H]+; Anal. Calcd. for C28H23ClN2O2S: C, 69.05; H, 4.76; N, 5.75; Found: C, 69.30; H,
4.68; N, 5.81.
4-(9-(4-Chlorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-7-yl)-2-
methoxyphenol (7f)
1
White solid; IR (KBr) υmax (cmꢀ1): 3306 (OH), 1605 (C=N), 1242 (CꢀOꢀC), 821 (CꢀCl); H
NMR (400 MHz, DMSOꢀd6): δ 1.78ꢀ1.84 (m, 1H), 2.26ꢀ2.34 (m, 1H), 2.59ꢀ2.65 (m, 1H),
2.75ꢀ2.81 (m, 1H), 3.47 (s, 3H), 6.09 (s, 1H), 6.42ꢀ6.45 (m, 1H), 6.63 (d, J = 8.4 Hz, 2H),
7.25ꢀ7.40 (m, 6H), 7.51 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1H), 9.20 (s, 1H); MS (ESI)
m/z: 473 [M+H]+; Anal. Calcd. for C27H21ClN2O2S: C, 68.56; H, 4.48; N, 5.92; Found: C,
68.71; H, 4.36; N, 5.98.
7,9-Bis(4-chlorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazoline (7g)
White solid; IR (KBr) υmax (cmꢀ1): 1611 (C=N), 826 (CꢀCl); 1H NMR (400 MHz, DMSOꢀd6):
δ 1.72ꢀ1.79 (m, 1H), 2.31 (t, J = 8.0 Hz, 1H), 2.59ꢀ2.64 (m, 1H), 2.75ꢀ2.79 (m, 1H), 6.19 (s,
1H), 6.89 (d, J = 8.4 Hz, 2H), 7.23ꢀ7.35 (m, 7H), 7.39 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 8.4
Hz, 2H), 7.62 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 161.5, 138.3, 137.5,

Page 17 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
135.3, 135.0, 133.5, 131.4, 128.9, 128.7, 128.5, 128.1, 126.7, 121.4, 111.5, 110.1, 61.8, 26.6,
23.0; MS (ESI) m/z: 462 [M+H]+; Anal. Calcd. for C26H18Cl2N2S: C, 67.68; H, 3.93; N, 6.07;
Found: C, 67.51; H, 4.05; N, 6.19.
Synthesis
of
9-(4-bromophenyl)-7-aryl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-
b]quinazolines (8a-f)
A mixture of 4ꢀarylꢀ3,4,5,6ꢀtetrahydrobenzo[h]quinazolineꢀ2(1H)ꢀthione (4a-f, 1 mmol)
and 4ꢀbromo phenacyl bromide (6, 1 mmol) were taken in 5 mL of glacial acetic acid and
stirred at its reflux temperature for 3ꢀ6 h. After completion of the reaction shown by TLC, the
mixture was kept a room temperature for 4 h, the solid separated out was filtered, washed
with cold acetic acid and recrystallized from acetic acid to afford the pure product.
9-(4-Bromophenyl)-7-phenyl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazoline (8a)
White solid; IR (KBr) υmax (cmꢀ1): 1603 (C=N), 578 (CꢀBr); 1H NMR (400 MHz, DMSOꢀd6):
δ 1.72ꢀ1.80 (m, 1H), 2.28ꢀ2.34 (m, 1H), 2.59 (t, J = 8.0 Hz, 1H), 2.75ꢀ2.81 (m, 1H), 6.15 (s,
1H), 6.84 (d, J = 7.6 Hz, 2H), 7.15ꢀ7.40 (m, 9H), 7.58 (d, J = 8.4 Hz, 3H); MS (ESI) m/z: 472
[M+H]+; Anal. Calcd. for C26H19BrN2S: C, 66.24; H, 4.06; N, 5.94; Found: C, 66.15; H, 4.23;
N, 5.78.
4-(9-(4-Bromophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-7-yl)phenol
(8b)
1
Yellow solid; IR (KBr) υmax (cmꢀ1): 3253 (OH), 1609 (C=N), 631 (CꢀBr); H NMR (400
MHz, DMSOꢀd6): δ 1.76ꢀ1.84 (m, 1H), 2.26ꢀ2.34 (m, 1H), 2.58ꢀ2.65 (m, 1H), 2.76ꢀ2.84 (m,
1H), 6.05 (s, 1H), 6.52 (d, J = 8.4 Hz, 2H), 6.58 (d, J = 8.4 Hz, 2H), 7.20ꢀ7.38 (m, 6H), 7.61ꢀ
7.69 (m, 3H), 9.61 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 161.2, 157.8, 138.6, 135.3,
131.5, 131.4, 128.9, 128.8, 128.2, 127.1, 126.7, 126.6, 123.6, 123.4, 121.3, 115.3, 112.6,
110.0, 62.3, 26.7, 23.2; MS (ESI) m/z: 487 [M]+; Anal. Calcd. for C26H19BrN2OS: C, 64.07;
H, 3.93; N, 5.75; Found: C, 63.95; H, 4.04; N, 5.85.
9-(4-Bromophenyl)-7-(4-fluorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-
b]quinazoline (8c)
White solid; IR (KBr) υmax (cmꢀ1): 1604 (C=N), 1227 (CꢀF), 632 (CꢀBr); 1H NMR (400 MHz,
DMSOꢀd6): δ 1.72ꢀ1.78 (m, 1H), 2.27ꢀ2.34 (m, 1H), 2.59ꢀ2.64 (m, 1H), 2.75ꢀ2.81 (m, 1H),
13
6.19 (s, 1H), 6.89ꢀ7.03 (m, 5H), 7.16ꢀ7.40 (m, 6H), 7.61 (d, J = 8.4 Hz, 2H); C NMR (100
MHz, DMSOꢀd6): δ 163.1, 161.5, 138.4, 135.3, 134.9, 131.5, 131.4, 129.2, 128.9, 128.1,
126.9, 126.7, 123.7, 121.2, 115.6, 115.4, 111.7, 61.8, 26.6, 23.0; MS (ESI) m/z: 490 [M+H]+;
Anal. Calcd. for C26H18BrFN2S: C, 63.81; H, 3.71; N, 5.72; Found: C, 63.90; H, 3.87; N,
5.56.

RSC Advances
Page 18 of 24
View Article Online
DOI: 10.1039/C4RA02514H
9-(4-Bromophenyl)-7-(4-methoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-
b]quinazoline (8d)
1
White solid; IR (KBr) υmax (cmꢀ1): 1606 (C=N), 1245 (CꢀOꢀC), 634 (CꢀBr); H NMR (400
MHz, DMSOꢀd6): δ 1.75ꢀ1.81 (m, 1H), 2.27ꢀ2.35 (m, 1H), 2.56ꢀ2.64 (m, 1H), 2.75ꢀ2.81 (m,
1H), 3.67 (s, 3H), 6.09 (s, 1H), 6.72 (d, J = 8.0 Hz, 4H), 7.19ꢀ7.40 (m, 7H), 7.63 (d, J = 8.4
Hz, 2H); MS (ESI) m/z: 502 [M+H]+; Anal. Calcd. for C27H21BrN2OS: C, 64.67; H, 4.22; N,
5.59; Found: C, 64.52; H, 4.45; N, 5.68.
9-(4-Bromophenyl)-7-(3,4-dimethoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-
b]quinazoline (8e)
1
Pale yellow solid; IR (KBr) υmax (cmꢀ1): 1598 (C=N), 1266 (CꢀOꢀC), 630 (CꢀBr); H NMR
(400 MHz, DMSOꢀd6): δ 1.77ꢀ1.83 (m, 1H), 2.31 (t, J = 8.0 Hz, 1H), 2.57ꢀ2.65 (m, 1H),
2.75ꢀ2.83 (m, 1H), 3.47 (s, 3H), 3.67 (s, 3H), 6.12 (s, 1H), 6.57ꢀ6.59 (m, 1H), 6.81 (d, J = 8.4
Hz, 2H), 7.21ꢀ7.40 (m, 7H), 7.64 (d, J = 8.8 Hz, 2H); MS (ESI) m/z: 532 [M+H]+; Anal.
Calcd. for C28H23BrN2O2S: C, 63.28; H, 4.36; N, 5.27; Found: C, 63.36; H, 4.49; N, 5.06.
4-(9-(4-Bromophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-7-yl)-2-
methoxyphenol (8f)
1
White solid; IR (KBr) υmax (cmꢀ1): 3306 (OH), 1600 (C=N), 1277 (CꢀOꢀC), 634 (CꢀBr); H
NMR (400 MHz, DMSOꢀd6): δ 1.76ꢀ1.84 (m, 1H), 2.25ꢀ2.33 (m, 1H), 2.59ꢀ2.65 (m, 1H),
2.75ꢀ2.81 (m, 1H), 3.48 (s, 3H), 6.07 (s, 1H), 6.44ꢀ6.46 (m, 1H), 6.63 (d, J = 8.0 Hz, 2H),
7.22ꢀ7.40 (m, 6H), 7.60ꢀ7.66 (m, 3H), 9.21 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ
160.9, 146.9, 146.8, 138.6, 135.3, 131.6, 131.4, 129.5, 128.7, 128.1, 127.3, 126.7, 123.6,
123.5, 121.3, 119.7, 116.1, 112.6, 111.5, 109.8, 62.5, 55.1, 26.7, 23.2; MS (ESI) m/z: 518
[M+H]+ Anal. Calcd. for C27H21BrN2O2S: C, 62.67; H, 4.09; N, 5.41; Found: C, 62.40; H,
4.25; N, 5.58.
Synthesis
of
3-(7-aryl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-2H-
chromen-2-one (11a-g)
To a mixture of 4ꢀarylꢀ3,4,5,6ꢀtetrahydrobenzo[h]quinazolineꢀ2(1H)ꢀthione (4a-g, 1
mmol) and 3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone (9, 1 mmol) in 25 mL of round bottom
flask; 5 mL of glacial acetic acid was added and refluxed for 1ꢀ3 h. The progress of the
reaction was monitored by TLC. After completion of the reaction, the solid separated out was
filtered and washed with hot acetic acid to afford the pure product.
3-(7-Phenyl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-2H-chromen-2-one
(11a)

Page 19 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
1
Pale yellow solid; IR (KBr) υmax (cmꢀ1): 1711 (C=O of lactone), 1630 (C=N); H NMR (400
MHz, DMSOꢀd6): δ 1.76 (t, J = 6.4 Hz, 1H), 2.28ꢀ2.36 (m, 1H), 2.57ꢀ2.65 (m, 1H), 2.77ꢀ2.81
(m, 1H), 6.25 (s, 1H), 7.16ꢀ7.26 (m, 6H), 7.34ꢀ7.49 (m, 5H), 7.64ꢀ7.89 (m, 4H); MS (ESI)
m/z: 461 [M+H]+ Anal. Calcd. for C29H20N2O2S: C, 75.63; H, 4.38; N, 6.08; Found: C, 75.47;
H, 4.49; N, 6.15.
3-(7-(4-Hydroxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-2H-
chromen-2-one (11b)
1
Pale yellow solid; IR (KBr) υmax (cmꢀ1): 3430 (OH), 1727 (C=O of lactone), 1633 (C=N); H
NMR (400 MHz, DMSOꢀd6): δ 1.74ꢀ1.79 (m, 1H), 2.26 (t, J = 8.0 Hz, 1H), 2.62 (t, J = 8.0
Hz, 1H), 2.76ꢀ2.80 (m, 1H), 6.10 (s, 1H), 6.52 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H),
7.24ꢀ7.51 (m, 6H), 7.68ꢀ7.74 (m, 3H), 7.92 (s, 1H), 9.61 (s, 1H); 13C NMR (100 MHz,
DMSOꢀd6): δ 161.1, 158.5, 158.0, 153.5, 146.4, 135.4, 133.5, 133.4, 129.4, 129.1, 128.8,
128.5, 128.1, 126.6, 125.0, 121.4, 118.0, 116.2, 115.9, 115.5, 113.4, 112.0, 62.4, 26.7, 23.1;
MS (ESI) m/z: 477 [M+H]+; Anal. Calcd. for C29H20N2O3S: C, 73.09; H, 4.23; N, 5.88;
Found: C, 73.15; H, 4.38; N, 5.60.
3-(7-(4-Fluorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-2H-
chromen-2-one (11c)
White solid; IR (KBr) υmax (cmꢀ1): 1740 (C=O of lactone), 1634 (C=N), 1226 (CꢀF); 1H NMR
(400 MHz, DMSOꢀd6): δ 1.76 (t, J = 6.4 Hz, 1H), 2.30 (t, J = 8.0 Hz, 1H), 2.60ꢀ2.66 (m, 1H),
2.76ꢀ2.83 (m, 1H), 6.27 (s, 1H), 7.02 (t, J = 8.4 Hz, 2H), 7.23ꢀ7.26 (m, 3H), 7.32ꢀ7.50 (m,
5H), 7.63ꢀ7.75 (m, 3H), 7.93 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 163.3, 161.4, 160.9,
158.4, 153.5, 146.9, 135.4, 135.2, 133.6, 133.1, 129.5, 129.4, 128.9, 128.1, 126.6, 125.0,
124.1, 121.5, 117.9, 116.2, 115.9, 115.7, 113.6, 111.3, 61.8, 26.6, 22.9; MS (ESI) m/z: 479
[M+H]+; Anal. Calcd. for C29H19FN2O2S: C, 72.79; H, 4.00; N, 5.85; Found: C, 72.54; H,
4.21; N, 5.72.
3-(7-(4-Methoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-2H-
chromen-2-one (11d)
1
Yellow solid; IR (KBr) υmax (cmꢀ1): 1716 (C=O of lactone), 1608 (C=N), 1248 (CꢀOꢀC); H
NMR (400 MHz, DMSOꢀd6): δ 1.74ꢀ1.79 (m, 1H), 2.28 (t, J = 7.6 Hz, 1H), 2.61 (t, J = 8.0
Hz, 1H), 2.79 (t, J = 7.6 Hz, 1H), 3.58 (s, 3H), 6.18 (s, 1H), 6.69 (d, J = 8.4 Hz, 2H), 7.70 (d,
J = 8.8 Hz, 2H), 7.24ꢀ7.51 (m, 6H), 7.66ꢀ7.87 (m, 4H); MS (ESI) m/z: 491 [M+H]+; Anal.
Calcd. for C30H22N2O3S: C, 73.45; H, 4.52; N, 5.71; Found: C, 73.27; H, 4.72; N, 5.63.
3-(7-(3,4-Dimethoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-
2H-chromen-2-one (11e)

RSC Advances
Page 20 of 24
View Article Online
DOI: 10.1039/C4RA02514H
1
White solid; IR (KBr) υmax (cmꢀ1): 1734 (C=O of lactone), 1636 (C=N), 1261 (CꢀOꢀC); H
NMR (400 MHz, DMSOꢀd6): δ 1.76ꢀ1.83 (m, 1H), 2.29 (t, J = 8.0 Hz, 1H), 2.61ꢀ2.66 (m,
1H), 2.76ꢀ2.80 (m, 1H), 3.26 (s, 3H), 3.59 (s, 3H), 6.15 (s, 1H), 6.68 (s, 1H), 6.75 (s, 2H),
7.25ꢀ7.50 (m, 6H), 7.66ꢀ7.65 (m, 3H), 7.88 (s, 1H); MS (ESI) m/z: 521 [M+H]+; Anal. Calcd.
for C31H24N2O4S: C, 71.52; H, 4.65; N, 5.38; Found: C, 71.39; H, 4.84; N, 5.47.
3-(7-(4-Hydroxy-3-methoxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-
9-yl)-2H-chromen-2-one (11f)
Pale yellow solid; IR (KBr) υmax (cmꢀ1): 3256 (OH), 1738 (C=O of lactone), 1638 (C=N),
1
1247 (CꢀOꢀC); H NMR (400 MHz, DMSOꢀd6): δ 1.77ꢀ1.81 (m, 1H), 2.27 (t, J = 8.0 Hz,
1H), 2.60ꢀ2.66 (m, 1H), 2.77 (t, J = 7.6 Hz, 1H), 3.23 (s, 3H), 6.09 (s, 1H), 6.61 (d, J = 7.6
Hz, 3H), 7.24ꢀ7.50 (m, 6H), 7.61ꢀ7.74 (m, 3H), 7.93 (s, 1H), 9.18 (s, 1H); 13C NMR (100
MHz, DMSOꢀd6): δ 161.0, 158.5, 153.5, 147.2, 146.4, 135.4, 133.6, 129.7, 129.4, 128.8,
128.1, 126.7, 125.0, 123.7, 121.4, 120.0, 118.0, 116.2, 112.0, 111.8, 62.7, 54.9, 26.7, 23.0;
MS (ESI) m/z: 507 [M+H]+; Anal. Calcd. for C30H22N2O4S: C, 71.13; H, 4.38; N, 5.53;
Found: C, 71.02; H, 4.55; N, 5.41.
3-(7-(4-Chlorophenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-2H-
chromen-2-one (11g)
White solid; IR (KBr) υmax (cmꢀ1): 1740 (C=O of lactone), 1636 (C=N), 840 (CꢀCl); 1H NMR
(400 MHz, DMSOꢀd6): δ 1.73ꢀ1.78 (m, 1H), 2.27ꢀ2.35 (m, 1H), 2.60ꢀ2.66 (m, 1H), 2.76ꢀ2.80
(m, 1H), 6.27 (s, 1H), 7.22ꢀ7.26 (m, 5H), 7.34ꢀ7.51 (m, 5H), 7.64ꢀ7.75 (m, 3H), 7.94 (s, 1H);
13C NMR (100 MHz, DMSOꢀd6): δ 161.5, 158.4, 153.5, 147.0, 137.8, 135.4, 133.9, 133.7,
133.0, 129.4, 129.0, 128.9, 128.1, 126.7, 125.0, 121.5, 117.9, 116.3, 115.7, 113.6, 111.1,
61.8, 26.6, 22.9; MS (ESI) m/z: 495 [M+H]+; Anal. Calcd. for C29H19ClN2O2S: C, 70.37; H,
3.87; N, 5.66; Found: C, 70.50; H, 3.79; N, 5.74.
Synthesis
of
2-(7-aryl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-3H-
benzo[f]chromen-3-one (12a,b)
4ꢀArylꢀ3,4,5,6ꢀtetrahydrobenzo[h]quinazolineꢀ2(1H)ꢀthione (4a,b, 1 mmol) and 2ꢀ(2ꢀ
bromoacetyl)ꢀ3Hꢀbenzo[f]chromenꢀ3ꢀone (10, 1 mmol) were dissolved in 10 mL of glacial
acetic acid and stirred at its reflux temperature for 2 h. After completion of the reaction
(monitored by TLC), the solid separated out was filtered and washed with hot acetic acid to
afford the pure product.
2-(7-Phenyl-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-3H-
benzo[f]chromen-3-one (12a)

Page 21 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
1
Yellow solid; IR (KBr) υmax (cmꢀ1): 1729 (C=O of lactone), 1637 (C=N); H NMR (400
MHz, DMSOꢀd6): δ 1.72ꢀ1.79 (m, 1H), 2.27ꢀ2.34 (m, 1H), 2.61 (t, J = 8.0 Hz, 1H), 2.77ꢀ2.83
(m, 1H), 6.28 (s, 1H), 7.13ꢀ7.41 (m, 8H), 7.52 (s, 1H), 7.64ꢀ7.76 (m, 4H), 8.11 (d, J = 8.0 Hz,
1H), 8.31ꢀ8.34 (m, 2H), 8.72 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 161.4, 158.4, 153.8,
143.0, 138.7, 135.4, 135.1, 133.5, 129.9, 129.0, 128.8, 128.6, 128.1, 127.0, 126.7, 126.4,
122.4, 121.4, 116.4, 114.8, 113.5, 112.4, 111.4, 62.9, 26.6, 23.1; MS (ESI) m/z: 511 [M+H]+;
Anal. Calcd. for C33H22N2O2S: C, 77.62; H, 4.34; N, 5.49; Found: C, 77.74; H, 4.45; N, 5.33.
2-(7-(4-Hydroxyphenyl)-6,7-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9-yl)-3H-
benzo[f]chromen-3-one (12b)
1
Yellow solid; IR (KBr) υmax (cmꢀ1): 3358 (OH), 1710 (C=O of lactone), 1613 (C=N); H
NMR (400 MHz, DMSOꢀd6): δ 1.75ꢀ1.81 (m, 1H), 2.22ꢀ2.30 (m, 1H), 2.60ꢀ2.66 (m, 1H),
2.76ꢀ2.83 (m, 1H), 6.16 (s, 1H), 6.48 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.25ꢀ7.41
(m, 3H), 7.51 (s, 1H), 7.64ꢀ7.70 (m, 4H), 8.12 (d, J = 8.0 Hz, 1H), 8.33ꢀ8.41 (m, 2H), 8.82 (s,
1H), 9.56 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6): δ 161.0, 158.4, 158.0, 153.8, 142.7,
135.4, 135.0, 133.6, 129.9, 129.1, 128.9, 128.8, 128.7, 128.6, 128.1, 126.7, 126.4, 123.9,
122.5, 121.3, 116.4, 115.5, 115.0, 113.4, 112.5, 112.0, 62.6, 26.7, 23.2; MS (ESI) m/z: 527
[M+H]+; Anal. Calcd. for C33H22N2O3S: C, 75.27; H, 4.21; N, 5.32; Found: C, 75.11; H, 4.43;
N, 5.40.
Determination of minimum inhibitory concentration (MIC)
The MIC values for all the synthesized compounds as well as positive control drugs
Penicillin and Streptomycin were determined against the three gramꢀpositive and three gramꢀ
negative bacterial strains by micro dilution method recommended by CLSI Standard Protocol
in liquid medium (Nutrient agar) distributed in 96ꢀwell plates. Serial dilutions of the test
compounds as well as standards were performed at concentrations ranging from 150 to 0.97
ꢁg/mL in a 200 ꢁL culture medium final volume; afterwards each well was seeded with a 50
ꢁL microbial suspension of 0.5 MacFarland density. In each test a microbial culture control
and a sterility control (negative) were performed. The plates were incubated for 24 h at 37 oC.
The lowest concentration which inhibited the visible microbial growth was considered the
MIC.
Diphenylpicrylhydrazyl (DPPH) method
Free radical scavenging activity was done by DPPH method. Briefly, 0.004% w/v of
DPPH radical solution was prepared in methanol and then 900 ꢁL of this solution was mixed

RSC Advances
Page 22 of 24
View Article Online
DOI: 10.1039/C4RA02514H
with 100 ꢁL of solution containing 10 ꢁg/mL of test compound. The absorbance was
measured at 517 nm after 30 min of incubation.
Agarose gel electrophoresis method
Cleavage products were analyzed by agarose gel electrophoresis method. Test samples
(10 mg/mL) was prepared in DMSO. The samples at two different concentrations (50 and
100 ꢂg) were added to the isolated pUC18 (200 ꢂg/mL) plasmid. The samples were
incubated for 2 h at 37 oC and then 20 ꢂL of DNA sample (mixed with bromophenol blue dye
at 1:1 ratio) was loaded carefully into the electrophoretic chamber wells along with standard
DNA marker containing TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L) and finally
loaded on agarose gel and constant 50 V of electricity was passed for around 30 min. Gel is
removed and stained with 10 ꢂg/mL ethidium bromide for 10ꢀ15 min and the bands were
observed under UV transilluminator and photographed to determine the extent of DNA
cleavage and the results were compared with standard FeSO4.
Acknowledgments
We would like to thank the Director, National Institute of Technology, Warangal for
providing facilities, and wish to thanks Dr. U.S.N. Murthy, Head, Biology division, Indian
Institute of Chemical Technology, Hyderabad, for screening the antibacterial activity of the
synthesized compounds. We thank Dr. Hari Prasad Kokatla, Assistant professor, Department
of Chemistry, NITꢀWarangal for his valuable suggestions. One of the authors JB thanks the
Ministry of Human Resource Development for senior research fellowship.
References and notes
1
2
C. T. Barrett and J. F. Barrett, Curr. Opin. Biotechnol., 2003, 14, 621.
M. Valko, D. Leibfritz, J. Moncol, M. T. Cronin, M. Mazur and J. Telser, Int. J.
Biochem. Cell Biol., 2007, 39, 44.
3
(a) M. D. Stringer, P. G. Gorog, A. Freeman and V. V. Kakkar, Brit. Med. J., 1989,
298, 281; (b) R. J. Perry, P. Watson and J. R. Hodges, Neurophychologia 2000, 38,
252; (c) C. P. Rajneesh, A. Manimaran, K. R. Sasikala and P. Adikappan, Singapore
Med. J., 2008, 49, 640; (d) A. A. Andreadis, S. L. Hazen, S. A. Comhair and S. C.
Erzurum, Free Radic. Biol. Med., 2003, 35, 213; (e) S. B. Abramson, A. R. Amin, R.
M. Clancy and M. Attur, Best Pract. Res. Clin. Rheumatol., 2001, 15, 831; (f) J. M.
McCord, New Eng. J. Med., 1985, 312, 159; (g) N. Sreejayan and M. N. Rao,
Arzneimittelforschung 1996, 46, 169; (h) B. Halliwell and J. M. C. Gutteridge, Free
radicals in biology and medicine, 2nd ed, Oxford University press, USA, 1989, 416ꢀ

Page 23 of 24
RSC Advances
View Article Online
DOI: 10.1039/C4RA02514H
494; (i) T. Finkel and N. J. Holbrook, Nature 2000, 408, 239; (j) A. Bast, G. R. Haenen
and C. J. Doelman, Am. J. Med., (suppl.3C) 1991, 91, 2S; (k) G. B. Bulkley, Surgery
1993, 113, 479.
4.
5.
J. E. Kinsella, E. Frankel, B. German and J. Kanner, Food Technol., 1993, 47, 85.
(a) A. Jarrahpour, D. Khalili, E. De Clercq, C. Salmi and J. M. Brunel, Molecules 2007,
12, 1720; (b) W. J. Zhang, Y. T. Li, Z. Y. Wu, Z. Q. Liu and Z. C. Zheng, J. Chem.
Crystallogr., 2008, 38, 655.
6.
7.
C. O. Kappe, Eur. J. Med. Chem., 2000, 35, 1043.
(a) M. M. Ghorab, S. M. AbdelꢀGawad and M. S. A. ElꢀGaby, Farmaco., 2000, 55,
249; (b) A. Balkan, S. Uma, M. Ertan and W. Wiegrebe, Pharmazie 1992, 47, 687.
S. Kolb, O. Mondesert, M. L. Goddard, D. Jullien, B. O. Villoutreix, B. Ducommun, C.
Garbay and E. Braud, Chem. Med. Chem., 2009, 4, 633.
8.
9.
(a) B. Tozkoparan, M. Ertan, P. Kelicen and R. Demirdamar, Farmaco., 1999, 54, 588;
(b) B. Pan, R. Huang, L. Zheng, C. Chen, S. Han, D. Qu, M. Zhu and P. Wei, Eur. J.
Med. Chem., 2011, 46, 819; (c) S. F. Mohamed, E. M. Flefel, A. EꢀG. E. Amr and D. N.
Abd ElꢀShafy, Eur. J. Med. Chem., 2010, 45, 1494.
10. A. A. AbuꢀHashem, M. M. Youssef and H. A. R. Hussein, J. Chin. Chem. Soc., 2011,
58, 41.
11. D. Bozsing, P. Sohar, G. Gigler and G. Kovacs, Eur. J. Med. Chem., 1996, 31, 663.
12. B. Tozkoparan, M. Ertan, B. Krebs, M. Läge, P. Kelicen and R. Demirdamar, Arch.
Pharm., 1998, 331, 201.
13. E. JeanneauꢀNicolle, M. BenoitꢀGuyod, A. Namil and G. Leclerc, Eur. J. Med. Chem.,
1992, 27, 115.
14. S. Maddila, G. L. V. Damu, E. O. Oseghe, O. A. Abafe, C. V. Rao and P. Lavanya, J.
Korean Chem. Soc., 2012, 56, 334.
15. E. E. Flefel, M. A. Salama, M. ElꢀShahat, M. A. ElꢀHashash and A. F. ElꢀFarargy,
Phosphorus Sulfur 2007, 182, 1739.
16. F. A. AlꢀOmary, G. S. Hassan, S. M. ElꢀMessery and H. I. ElꢀSubbagh, Eur. J. Med.
Chem., 2012, 47, 65.
17. K. Danel, E. B. Pedersen and C. Nielsen, J. Med. Chem., 1998, 41, 191.
18. J. Wichmann, G. Adam, S. Kolczewski, V. Mutel and T. Woltering, Bioorg. Med.
Chem. Lett., 1999, 9, 1573.
19. H. Zhi, L. M. Chen, L. L. Zhang, S. J. Liu, D. C. C. Wan, H. Q. Lin and C. Hu,
Arkivoc 2008, xiii, 266.

RSC Advances
Page 24 of 24
View Article Online
DOI: 10.1039/C4RA02514H
20. B. Zhou, X. Li, Y. Li, Y. Xu, Z. Zhang, M. Zhou, X. Zhang, Z. Liu, J. Zhou, C. Cao, B.
Yu and R, Wang, Chem. Med. Chem., 2011, 6, 904.
21. (a) B. Janardhan, V. Ravibabu, G. Rajitha, B. Rajitha, and P. A. Crooks, Tetrahedron
Lett., 2013, 54, 3862; (b) S. V. Laxmi, Y. T. Reddy, B. S. Kuarm, P. N. Reddy, P. A.
Crooks and B. Rajitha, Bioorg. Med. Chem. Lett., 2011, 21, 4329.
22. B. Janardhan, B. Rajitha and P. A. Crooks, J. Saudi Chem. Soc., 2012,
http://dx.doi.org/10.1016/j.jscs.2012.10.007.
23. Clinical and laboratory standards institute (CLSI) performance standards for
antimicrobial susceptibility testing, 18th informational supplement, 2008, M100ꢀS18,
Wayne, PA (USA).
24. M. Z. Gul, F. Ahmad, A. K. Kondapi, I. A. Qureshi, I. A. Ghazi, BMC Complem.
Altern. Med., 2013, 13, 53.
25. B. Stinefelt, S. S. Leonard, K. P. Blemings, X. Shi and H. Klandorf, Ann. Clin. Lab.
Sci., 2005, 35, 37.
26. T. A. Brown, Essential molecular biology: a practical approach, Vol. I, Oxford
University press, New York, 1990, 51ꢀ52.