Discovery of new coumarin substituted quinazolines as potential bioactive agents

Article abstract of DOI:10.1007/s11164-015-2140-0

In view of the biological importance of some important pharmacophores such as quinazoline, coumarin, and benzothiazole, we frame these moieties in a single molecular scaffold to be screened in vitro for their antimicrobial activity. The newly synthesized analogs have been examined for their in vitro biological efficacy against two Gram-positive bacteria, three Gram-negative bacteria, two fungi, and for antimycobacterial activity against M. tuberculosis H₃₇Rv. The bioassay results revealed that the majority of final analogs exhibited potential bio efficacies with the remarkable level of MICs comparable to control drugs. The new synthesized compounds were characterized by FT-IR, ¹H NMR, ¹³C NMR, and MS analysis.

Full text of DOI:10.1007/s11164-015-2140-0

Res Chem Intermed
DOI 10.1007/s11164-015-2140-0
Discovery of new coumarin substituted quinazolines
as potential bioactive agents
1
2
Amit B. Patel
•
Rinku M. Raval
Received: 3 May 2015 / Accepted: 11 June 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract In view of the biological importance of some important pharmacophores
such as quinazoline, coumarin, and benzothiazole, we frame these moieties in a
single molecular scaffold to be screened in vitro for their antimicrobial activity. The
newly synthesized analogs have been examined for their in vitro biological efficacy
against two Gram-positive bacteria, three Gram-negative bacteria, two fungi, and
for antimycobacterial activity against M. tuberculosis H Rv. The bioassay results
3
7
revealed that the majority of final analogs exhibited potential bio efficacies with the
remarkable level of MICs comparable to control drugs. The new synthesized
1
13
compounds were characterized by FT-IR, H NMR, C NMR, and MS analysis.
Keywords Quinazoline Á Coumarin Á Benzothiazole Á Antimicrobial Á
Antimycobacterial activity
Introduction
The finding of efficacious new human therapeutic agents is one of humanity’s most
vital tasks. The great expansion in medicinal research in the past has contributed
much to the unparalleled progress of medicine during that period. Improved and
more meaningful biological test procedures have provided better guidance in drug
discovery by pointing out suggestive observations that could be used in the design
Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2140-0)
contains supplementary material, which is available to authorized users.
&
Amit B. Patel
amit.svnit10@gmail.com
1
2
Department of Chemistry, Government College Daman, Daman 396210, UT, India
Department of Chemistry, HVHP Institute of PG Studies and Research, KSV, Kadi 382715,
India
123

A. B. Patel, R. M. Raval
of new therapeutic agents [1]. The elucidation of the structure of many novel
molecular leads in recent years has also made possible more rational study of the
chemical mode of action of such compounds, and their interaction with drugs.
Development of novel classes of drugs with fewer side effects and shorter lengths of
treatment are key in continuing the fight against infectious disease. Hence, the field
of medicinal chemistry has gained extreme importance for several years with better
medical advances [2, 3].
A literature survey revealed that quinazoline, coumarin, and benzothiazole-fused
derivatives are important parts of medicinal chemistry in a program of the drug
discovery process [4–11]. Mosaad et al. [12] synthesized some quinazoline-based
derivatives bearing substitution at the 4th position with ethereal linkage (I), with C–
C bond formation at the same position (II) and ethereal linkage at nitrogen atom
(
III).
X
X
N
HN
NH
H
X
HN
NH
N
O
O
O
NH
O
N
O
O
O
N
O
I
I
I
N
N
O
N
Cl
Cl
Cl
X= O or S
(
I)
(II)
(III)
It was observed that amongst all the three quinazoline derivatives, analog
I) bearing ethereal linkage to the 4th position displayed excellent antimicrobial
(
activities against Gram-positive S. aureus and B. subtilis bacteria. In an attempt to
find new promising pharmacologically active molecules, we report the synthesis and
in vitro biological activity of two series of benzothiazole and coumarin incorporated
quinazolines. The synthesized analogs contain the ethereal linkage between
coumarin and quinazoline rings. The influence of the presence/absence of various
electron withdrawing/donating functional groups on the benzothiazole ring for the
bioassay is elaborated.
Results and discussion
Chemistry
The synthetic route for the target compounds 5a–j is outlined in Scheme 1. The
initial analog 2,4-dichloroquinazoline (3) was synthesized from 2-nitrobenzoic acid
according to the reported literature [13–18]. The structure of compound 3 was
1
23

Discovery of new coumarin substituted quinazolines…
O
OH
Cl
N
O
O
N
COOH
a
N
b
N
c
N
NH2
N
OH
Cl
O
Cl
1
2
3
4
d
R
O
O
N
S
N
N
N
H
5
a–j
3
Scheme 1 Synthetic protocol for analogs 5a–j. Reagents and Conditions a Urea, 180 °C, b POCl ,
DMA, reflux, c 4-hydroxycoumarin, K CO , Acetone, reflux, d 2-aminobenzothiazoles, K CO , Acetone,
2 3 2 3
reflux
1
13
confirmed by H NMR and C NMR (SI Fig S1–S2) spectrometric analysis. The
condensation of 4-hydroxycoumarin with 2,4-dichloroquinzoline (3) in the presence
of potassium carbonate base yields the intermediate analog 4. The reaction of this
intermediate 4 with various 2-aminobenzothiazole derivatives yields desired
1
analogs 5a–j. Structure of compound 5a was confirmed by FT-IR, H NMR, and
1
3
C NMR spectrometric analysis. The spectral details of the analog 5a are discussed
here. In the FT-IR spectrum (SI Fig S3), appearance of the characteristic absorption
-
band at 3214 cm for N–H group confirms the formation of 5a. Moreover, the FT-
1
-
IR spectrum also revealed another two absorption bands at 1691 and 1200 cm for
1
1
the C=O and C–O–C groups. The H NMR spectrum (SI Fig S4) of compound 5a
also confirmed the presence of two singlets at 10.05 and 6.27 ppm for the NH and
coumarin ring proton. Finally, the multiplates were observed between 7.92–7.22 for
quinazoline and coumarin protons. The formation of analog 5a was also supported
1
3
by C NMR spectrum (SI Fig S5), which show peaks at 165.7 and 159.7 ppm due
to the presence of C–O–C linkage. Moreover, the appearance of a peak at
1
further confirmed the assigned structures of 5a by exhibiting the molecular ion
54.5 ppm confirms the presence of the C=O group. The mass spectrum (SI Fig S6)
?
peaks [M?1] at m/z 440.0.
123

A. B. Patel, R. M. Raval
Pharmacology
Antimicrobial activity
Investigation of in vitro antimicrobial activity of the final analogs 5a–j is
summarized in Table 1. The newer analogs were screened against two Gram-
positive and three Gram-negative bacteria. From the bioassay results, it can be
stated that some of the final analogs displayed excellent antibacterial activity against
most of the microorganisms studied. However, the antibacterial property of the each
final analog varies by varying structural features of the benzothiazole ring.
Overall, results suggested that compounds have better inhibitory effects against
Gram-positive bacteria (MICs 6.25–25 lg/mL) than against Gram-negative ones
(
MICs 25–50 lg/mL) in terms of MIC. The hydroxyl group that substituted analog
j (MIC 6.25 lg/mL) was 50 % more potent than Ampicillin (MIC 12.5 lg/mL)
5
against S. aureus, whereas the analog 5h (OCH ) was equipotent to the Ampicillin
3
against the same organism. With regard to the activity against B. cereus, the best
activity was displayed by compounds 5b and 5j (MIC 12.5 lg/mL). On the other
hand, investigation of antibacterial activity of the synthesized compounds against
the three Gram-negative strains revealed that, only a few analogs have been found to
be active against the tested strains. Analog 5h was able to produced good growth
inhibitory activity against E. coli (MIC 25 lg/mL). However, compounds 5d and
5
h (MIC 50 lg/mL) exhibited moderate activity against P. aeruginosa.
Concerning the antifungal activity of the tested compounds 5b, 5d, 5e, 5g, and 5j
showed sensitivity against the mentioned fungal strains, whereas the rest of the
derivatives were reasonably active or insensitive. The methyl group functionalized
derivative 5g displayed exceptional antifungal efficacy 6.25 lg/mL against A. niger.
Moreover, analog 5e was also found to be moderately (MIC 12.5 lg/mL) active
against the same strain. Interestingly, the chloro group substituted analog 5b (MIC
6
.25 lg/mL) displayed the highest inhibition against C. albicans. However, the
synthesized analogs showed significant growth inhibition zones in a range of
7–20 mm, when compared with standards of 33–28 mm against both bacterial as
2
well as fungal species.
Antimycobacterial activity
The investigation of in vitro antimycobacterial screening (Table 2) revealed that all
the new synthesized compounds showed moderate to good inhibition at 3.12–25 lg/
mL against Mycobacterium tuberculosis H Rv.
3
7
The primary screening was conducted at a concentration of 6.25 lg/mL using the
BACTEC MGIT method only for the first selection of active compounds. The
results observed from the BACTEC MGIT method indicated that out of all the
tested compounds, two derivatives 5d and 5j, exhibited the highest inhibition
(
99 %) at a constant concentration level (6.25 lg/mL). However, the results of
secondary biological screening using the Lowenstein-Jensen MIC method revealed
that analog 5d with a fluoro group to the benzothiazole ring showed the highest
inhibition against M. tuberculosis H Rv at 3.12 lg/mL MIC, which is half fold
3
7
1
23

Discovery of new coumarin substituted quinazolines…
123

A. B. Patel, R. M. Raval
Table 2 Results of in vitro antimycobacterial screening of the analogs 5a–j
a
a
Entry
R
BACTEC MGIT method
L. J. MIC method
MIC (lg/mL)
% Inhibition
MIC (lg/mL)
% Inhibition
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
H
[6.25
[6.25
[6.25
6.25
ND
ND
ND
99
250
25
96
98
99
99
96
97
97
96
98
99
Cl
Br
F
12.5
3.12
100
200
250
100
50
NO
2
[6.25
[6.25
[6.25
[6.25
[6.25
6.25
ND
ND
ND
ND
ND
99
CN
g
h
i
CH
3
OCH
OCH
OH
3
2
CH
3
j
6.25
Ethambutol
Pyrazinamide
Rifampicin
Isoniazid
3.12
99
6.25
99
0.25
99
0.20
99
DMSO
–
–
–
–
ND Not determine
a
Each value is the mean of three independent experiments
Bold values indicate highly active compound against respective microorganisms
more active than pyrazinamide. All the remaining derivatives were found to exhibit
moderate to poor activity at MIC ranging from 12.5–50 lg/mL.
A structure-activity relationship study revealed that the presence of strong
electron donating substitution at the benzothiazole ring improves antibacterial
activity. However the presence of electron withdrawing groups is essential for good
antifungal activity. Moreover, the presence of halogenated and hydroxyl derivatives
have shown high antimycobacterial activity.
Conclusion
In conclusion, we report the synthesis of various benzothiazole and coumarin
substituted quinazoline derivatives. The bioassay results reveal that many of the
synthesized final analogs have shown remarkable activity against the Gram-positive
bacteria and fungal strains as compared with the standards. The hydroxyl group
functionalized derivative is found to be half fold more active than ampicillin against
S. aureus, while chloro and methyl derivatives are equipotent to fluconazole against
tested fungal strains. Moreover, the fluoro derivative is half fold more active than
pyrazinamide against M. tuberculosis H Rv. In short, these compounds represent
3
7
new structure scaffolds that could be further optimized for future development of
more potent and selective bioactive agents.
1
23

Discovery of new coumarin substituted quinazolines…
Experimental
General
All reactions were carried out under a nitrogen atmosphere. Air- and moisture-
sensitive solvents and solutions were transferred via syringe or stainless steel cannula.
All chemicals were purchased from Sigma Aldrich, Merck, and Fluka. Solvents used
were of analytical grade. Anhydrous potassium carbonate was stored in a nitrogen-
filled glovebox, ground, and taken out in small quantities and stored in a desiccator.
All reactions were routinely checked by TLC. TLC was performed on aluminum-
backed silica gel plates (silica gel 60 F254 grade, Merck DC) with spots visualized by
UV light. Column chromatography was performed on silica gel LC 60A
(
electronic apparatus VMP–D (Veego Instrument Corporation, Mumbai, India) and are
70–200 micron). Melting points were determined in open capillaries on a Veego
1 13
uncorrected. H NMR and C NMR spectra were recorded on a Varian 400 MHz
model spectrometer using DMSO-d as a solvent and TMS as internal standard with
6
1
13
H resonant frequency of 400 MHz and C resonant frequency of 100 MHz. The
chemical shifts were reported as parts per million (ppm) downfield from TMS
Me Si). The splitting patterns are designated as follows; s, singlet; d, doublet; dd,
(
4
doublet of doublets; t, triplet; m, multiplet. Elemental analyses (C, H, N) were
performed using a Heraeus Carlo Erba 1180 CHN analyzer (Hanau, Germany). The
mass spectra were measured with a Waters Micromass Q-Tof Micro instrument [time
of flight (TOF) mass spectrometer]. Column chromatography was performed on a
1
.5 feet (2.5 cm diameter) glass column using silica gel LC 60A (70–200 microns).
Synthesis of quinazoline-2,4-diol (2)
A flask containing urea (15.0 g, 250 mmol) and 2-aminobenzoic acid (1) (25 mmol)
was heated at 180 °C. After being stirred for 3 h, the reaction mixture was cooled to
1
00 °C and an equal volume of water was added. The obtained suspension was left
to stir for 10 min, after which it was cooled to room temperature. The precipitate
was filtered off and recrystallized from DMF to obtain quinazoline-2,4-diol (2) [14].
Light pink solid, Yield: 81 %, M.P. 298–300 °C (lit. [15] 300 °C).
Synthesis of 2,4-dichloroquinazoline (3)
A mixture of 80 ml POCl , N,N-dimethylaniline (300 mmol) and 2,4-dihydrox-
3
yquinazoline (2) (150 mmol) was refluxed for 2 h. The cooling mixture was poured
into 250 mL ice-water, and the mixture was stirred at room temperature. The
resulting precipitate was filtered off and washed with 50 mL water, and dried to
give 2,4-dichloroquinazoline (3) to be used in the next step without further
purification [16]. Yellow solid, Yield: 87 %, M.P. 118–119 °C (lit. [17]
-
1
1
1
19–120 °C). IR (KBr, cm ): 3064 (Ar–CH), 1610 (CN), 735(C–Cl). H NMR
(
400 MHz, DMSO–d ): d 8.12 (dd, J = 7.2, 1.8 Hz, 1H), 7.97–7.92 (m, 2H, Ar–H),
6
1
3
7
.77–7.73 (m, 1H, Ar–H). C NMR (100 MHz, DMSO–d ): d 162.1, 154.2, 153.9,
6
1
48.0, 132.7, 127.5, 126.2, 123.0.
123

A. B. Patel, R. M. Raval
Synthesis of 4-((2-chloroquinazolin-4-yl)oxy)-2H-chromen-2-one (4)
Potassium carbonate (10 mmol) was added to a solution of 4-hydroxy coumarin
2 mmol) in anhydrous ethanol (20 mL) and was stirred for 5 min. Then, the
(
intermediate 3 (2 mmol) was added into the reaction mixture. The reaction mixture
was refluxed for 10 h. After cooling to room temperature, the reaction mixture was
treated with 200 mL cold water, and the resulting precipitate was filtered off. The
crude product 4 was recrystallized from DMF [18]. White solid, Yield: 72 %, M.P.
1
92–193 °C.
General synthetic procedure for analogs (5a–j)
A mixture of intermediate 4 (10 mmol), potassium carbonate (12 mmol) and
various 2-aminobenzothiazoles (10 mmol) in acetone solvent (30 mL) was refluxed
for 8 h. The progress of the reaction was monitored by TLC. After cooling to room
temperature, the reaction mixture was treated with 200 mL cold water, and the
resulting precipitate was filtered off. The crude products 5a–j were recrystallized
from DMF [19].
4
-((2-(Benzo[d]thiazol-2-ylamino)quinazolin-4-yl)oxy)-2H-chromen-2-one (5a)
-
1
White solid, Yield: 74 %, M.P. 197–199 °C. IR (KBr, cm ): 3214 (N–H), 2948,
1
2
900 (C–H), 1691 (C=O), 1200 (C–O–C). H NMR (400 MHz, DMSO-d ): d 10.05
6
(
s, 1H, –NH), 8.57 (d, J = 1.7 Hz, 1H), 7.92–7.54 (m, 7H, Ar–H), 7.30–7.22 (m, 4H,
1
3
Ar–H), 6.27 (s, 1H, coumarin). C NMR (100 MHz, DMSO-d ): d 165.7 (1C,
6
Cquinazoline–O–C), 160.0 (1C, Cquinazoline–NH–C), 159.7 (1C, Cbenzothiazole–
NH–C), 154.5 (1C, Ccoumarin–O–C), 153.2 (1C, C=O), 150.6, 150.4, 140.3, 134.3,
1
1
34.0, 131.7, 130.0, 129.6, 129.2, 129.1, 129.0, 127.8, 127.5, 126.4, 121.5, 121.0,
?
11.9, 109.4, 105.4 (19C, Ar–C). MS, m/z 440.0 [M?1] . Anal calcd for
C H N O S: C, 65.74; H, 3.22; N, 12.78; S, 7.31 Found C, 65.98; H, 3.07; N,
2
4 14 4 3
1
2.67; S, 7.19.
4-((2-((6-Chlorobenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5b)
-
1
Light greenish solid, Yield: 71 %, M.P. 232–234 °C. IR (KBr, cm ): 3352 (N–H),
1
008, 2911 (C–H), 1683 (C=O), 1221 (C–O–C), 748 (C–Cl). H NMR (400 MHz,
3
DMSO-d ): d 10.28 (s, 1H, –NH), 8.63 (d, J = 1.7 Hz, 1H), 7.79–7.25 (m, 6H, Ar–
6
1
3
H), 7.24–6.93 (m, 4H, Ar–H), 5.98 (s, 1H, coumarin). C NMR (100 MHz, DMSO-
d6): d 167.3 (1C, Cquinazoline–O–C), 161.7 (1C, Cquinazoline–NH–C), 160.2 (1C,
Cbenzothiazole–NH–C), 158.5 (1C, Ccoumarin–O–C), 155.2 (1C, C=O), 151.1,
1
1
49.8, 142.7, 136.2, 135.8, 132.4, 131.9, 130.2, 129.7, 129.5, 129.2, 128.1, 127.1,
?
27.0, 123.6, 122.4, 115.2, 111.7, 108.0 (19C, Ar–C). MS, m/z 474.2 [M?1] . Anal
calcd for C H ClN O S: C, 60.95; H, 2.77; N, 11.85; S, 6.78 Found C, 60.87; H,
2
4
13
4 3
3
.01; N, 11.89; S, 6.73.
1
23

Discovery of new coumarin substituted quinazolines…
4-((2-((6-Bromobenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5c)
-
1
White solid, Yield: 69 %, M.P. 216–218 °C. IR (KBr, cm ): 3283 (N–H), 2931,
1
2
887 (C–H), 1690 (C=O), 1217 (C–O–C). H NMR (400 MHz, DMSO-d ): d 9.97
6
(
s, 1H, –NH), 8.34 (d, J = 1.7 Hz, 1H), 7.67–7.29 (m, 8H, Ar–H), 7.04–6.73 (m, 4H,
1
3
Ar–H), 6.03 (s, 1H, coumarin). C NMR (100 MHz, DMSO-d ): d 165.2 (1C,
6
Cquinazoline–O–C), 161.8 (1C, Cquinazoline–NH–C), 160.1 (1C, Cbenzothiazole–
NH–C), 159.7 (1C, Ccoumarin–O–C), 153.2 (1C, C=O), 152.8, 151.0, 141.4, 134.8,
1
1
34.5, 131.9, 130.4, 130.1, 129.9, 129.6, 129.2, 128.7, 127.1, 126.5, 123.2, 121.9,
?
13.4, 112.6, 107.1 (19C, Ar–C). MS, m/z 518.0 [M?1] . Anal calcd for
C H BrN O S: C, 55.72; H, 2.53; N, 10.83; S, 6.20 Found C, 55.70; H, 2.56; N,
2
4
13
4 3
1
1.05; S, 6.33.
4-((2-((6-Fluorobenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5d)
-
1
White solid, Yield: 73 %, M.P. 254–256 °C. IR (KBr, cm ): 3248 (N–H), 2913,
1
2
901 (C–H), 1687 (C=O), 1228 (C–O–C). H NMR (400 MHz, DMSO-d ): d 10.09
6
(
s, 1H, –NH), 8.51 (d, J = 1.7 Hz, 1H), 7.83–7.41 (m, 8H, Ar–H), 7.17–7.05 (m, 4H,
1
3
Ar–H), 6.13 (s, 1H, coumarin). C NMR (100 MHz, DMSO-d ): d 165.1 (1C,
6
Cquinazoline–O–C), 160.5 (1C, Cquinazoline–NH–C), 159.6 (1C, Cbenzothiazole–
NH–C), 154.1 (1C, Ccoumarin–O–C), 153.8 (1C, C=O), 150.7, 150.1, 141.4, 134.8,
1
1
34.1, 131.6, 131.0, 130.4, 129.7, 129.2, 129.0, 127.6, 127.0, 126.4, 121.8, 121.5,
?
17.5, 109.8, 105.3 (19C, Ar–C). MS, m/z 458.4 [M?1] . Anal calcd for
C H FN O S: C, 63.15; H, 2.87; N, 12.27; S, 7.02 Found C, 63.21; H, 2.69; N,
2
4
13
4 3
1
1.95; S, 6.86.
4
-((2-((6-Nitrobenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-one
5e)
(
-1
Pale yellowish solid, Yield: 64 %, M.P. 203–204 °C. IR (KBr, cm ): 3303 (N–H),
1
2
1
4
960, 2932 (C–H), 1688 (C=O), 1220 (C–O–C). H NMR (400 MHz, DMSO-d ): d
6
0.12 (s, 1H, –NH), 8.56 (d, J = 1.7 Hz, 1H), 7.87–7.56 (m, 8H, Ar–H), 7.21–6.94 (m,
3
H, Ar–H), 6.04 (s, 1H, coumarin). C NMR (100 MHz, DMSO-d ): d 167.7 (1C,
1
6
Cquinazoline–O–C), 163.1 (1C, Cquinazoline–NH–C), 160.4 (1C, Cbenzothiazole–
NH–C), 159.9 (1C, Ccoumarin–O–C), 154.2 (1C, C=O), 152.7, 150.5, 140.8, 135.1,
1
1
5
34.5, 131.1, 130.8, 129.6, 129.1, 128.7, 128.2, 127.0, 126.7, 126.4, 122.1, 121.9, 115.2,
?
11.4, 107.0 (19C, Ar–C). MS, m/z 484.1 [M?1] . Anal calcd for C H N O S: C,
9.62; H, 2.71; N, 14.49; S, 6.63 Found C, 59.78; H, 2.55; N, 14.65; S, 6.47.
2
4 13 5 5
2-((4-((2-Oxo-2H-chromen-4-yl)oxy)quinazolin-2-yl)amino)benzo[d]thiazole-6-
carbonitrile (5f)
-
1
White solid, Yield: 61 %, M.P. 183–184 °C. IR (KBr, cm ): 3249 (N–H), 2925,
1
2
883 (C–H), 2250 (C:N), 1681 (C=O), 1237 (C–O–C). H NMR (400 MHz,
123

A. B. Patel, R. M. Raval
DMSO-d ): d 9.98 (s, 1H, –NH), 8.68 (d, J = 1.7 Hz, 1H), 7.96–7.50 (m, 8H, Ar–H),
6
13
7
.21–7.02 (m, 4H, Ar–H), 6.25 (s, 1H, coumarin). C NMR (100 MHz, DMSO-d6):
d 165.1 (1C, Cquinazoline–O–C), 159.9 (1C, Cquinazoline–NH–C), 159.5 (1C,
Cbenzothiazole–NH–C), 153.8 (1C, Ccoumarin–O–C), 153.2 (1C, C=O), 151.0,
1
1
4
50.4, 140.1, 134.8, 134.2, 131.0, 130.5, 129.1, 129.0, 128.4, 128.1, 127.3, 127.1,
26.5, 121.9, 121.7, 116.0, 113.8, 108.6 (19C, Ar–C), 105.3 (1C, C:N). MS, m/z
?
63.9 [M?1] . Anal calcd for C H N O S: C, 64.79; H, 2.83; N, 15.11; S, 6.92
2
5 13 5 3
Found C, 64.48; H, 2.71; N, 15.23; S, 7.05.
4-((2-((6-Methylbenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5g)
-
1
White solid, Yield: 66 %, M.P. 225–227 °C. IR (KBr, cm ): 3317 (N–H), 2938,
1
2
892 (C–H), 1687 (C=O), 1205 (C–O–C). H NMR (400 MHz, DMSO-d ): d 10.19
6
(
s, 1H, –NH), 8.61 (d, J = 1.7 Hz, 1H), 8.01–7.50 (m, 8H, Ar–H), 7.23–6.80 (m, 4H,
1
3
Ar–H), 5.93 (s, 1H, coumarin), 2.42 (s, 3H, –CH3). C NMR (100 MHz, DMSO-
d6): d 166.2 (1C, Cquinazoline–O–C), 161.8 (1C, Cquinazoline–NH–C), 159.6 (1C,
Cbenzothiazole–NH–C), 157.3 (1C, Ccoumarin–O–C), 153.1 (1C, C=O), 150.5,
1
1
4
50.0, 140.7, 137.2, 133.9, 131.5, 130.2, 129.1, 128.7, 128.1, 127.5, 127.0, 126.1,
25.3, 124.5, 122.6, 115.2, 111.7, 109.1 (19C, Ar–C), 22.4 (1C, CH ). MS, m/z
3
?
53.0 [M?1] . Anal calcd for C H N O S: C, 66.36; H, 3.56; N, 12.38; S, 7.09
2
5 16 4 3
Found C, 66.52; H, 3.60; N, 12.45; S, 7.18.
4-((2-((6-Methoxybenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5h)
-
1
Light brownish solid, Yield: 78 %, M.P. 242–244 °C. IR (KBr, cm ): 3255 (N–H),
1
2
913, 2857 (C–H), 1680 (C=O), 1210 (C–O–C). H NMR (400 MHz, DMSO-d ): d
6
1
0.03 (s, 1H, –NH), 8.51 (d, J = 1.7 Hz, 1H), 7.88–7.64 (m, 8H, Ar–H), 7.25–6.86
1
3
(
m, 4H, Ar–H), 6.21 (s, 1H, coumarin), 4.22 (s, 3H, –OCH ),. C NMR (100 MHz,
3
DMSO-d ): d 165.0 (1C, Cquinazoline–O–C), 160.8 (1C, Cquinazoline–NH–C),
6
1
1
1
59.4 (1C, Cbenzothiazole–NH–C), 154.5 (1C, Ccoumarin–O–C), 153.1 (1C, C=O),
50.5, 150.0, 141.7, 134.5, 134.3, 131.8, 130.4, 129.8, 129.1, 128.5, 127.4, 126.2,
26.0, 125.4, 121.1, 121.0, 114.5, 108.7, 106.2 (19C, Ar–C), 57.8 (1C, OCH ). MS,
3
?
m/z 469.2 [M?1] . Anal calcd for C H N4O4S: C, 64.09; H, 3.44; N, 11.96; S,
2
5 16
6
.84 Found C, 63.88; H, 3.38; N, 11.90; S, 6.99.
4-((2-((6-Ethoxybenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5i)
-1
White solid, Yield: 75 %, M.P. 236–237 °C. IR (KBr, cm ): 3309 (N–H), 2927,
1
2
895 (C–H), 1690 (C=O), 1210 (C–O–C). H NMR (400 MHz, DMSO-d ): d 10.11
6
(
s, 1H, –NH), 8.53 (d, J = 1.7 Hz, 1H), 7.78–7.34 (m, 8H, Ar–H), 7.27–6.89 (m, 4H,
Ar–H), 6.05 (s, 1H, coumarin), 4.01 (q, J = 6.8 Hz, 2H, –OCH CH ), 1.97 (t, J =
2
3
1
3
7
zoline–O–C), 165.7 (1C, Cquinazoline–NH–C), 161.2 (1C, Cbenzothiazole–NH–C),
.2 Hz, 3H, –OCH CH ). C NMR (100 MHz, DMSO-d ): d 168.3 (1C, Cquina-
2
3
6
1
23

Discovery of new coumarin substituted quinazolines…
1
1
1
4
55.9 (1C, Ccoumarin–O–C), 155.0 (1C, C=O), 153.8, 152.1, 143.5, 133.8, 133.5,
30.1, 130.0, 129.5, 129.1, 129.0, 128.4, 127.5, 127.1, 126.9, 123.7, 121.5, 117.0,
10.4, 109.6 (19C, Ar–C), 58.9 (1C, –OCH CH ), 15.4 (1C, –OCH CH ). MS, m/z
2
3
2
3
?
82.8 [M?1] . Anal calcd for C H N O S: C, 64.72; H, 3.76; N, 11.61; S, 6.65
2
6 18 4 4
Found C, 64.58; H, 3.49; N, 11.72; S, 6.80.
4-((2-((6-Hydroxybenzo[d]thiazol-2-yl)amino)quinazolin-4-yl)oxy)-2H-chromen-2-
one (5j)
-
1
White solid, Yield: 77 %, M.P. 190–192 °C. IR (KBr, cm ): 3461 (O–H), 3228
1
N–H), 2941, 2905 (C–H), 1684 (C=O), 1235 (C–O–C). H NMR (400 MHz,
(
DMSO-d ): d 10.23 (s, 1H, –NH), 8.44 (d, J = 1.7 Hz, 1H), 7.85–7.37 (m, 8H, Ar–
6
1
H), 7.20–6.84 (m, 4H, Ar–H), 6.09 (s, 1H, coumarin), 5.11 (br s, 1H, OH). C NMR
3
(
100 MHz, DMSO-d ): d 164.6 (1C, Cquinazoline–O–C), 161.1 (1C, Cquinazoline–
6
NH–C), 159.8 (1C, Cbenzothiazole–NH–C), 154.3 (1C, Ccoumarin–O–C), 154.0
1C, C=O), 152.6, 150.3, 147.5, 138.7, 133.4, 132.9, 131.8, 130.3, 129.7, 129.5,
(
1
4
29.2, 128.8, 128.5, 128.0, 124.3, 120.6, 113.2, 111.3, 107.6 (19C, Ar–C). MS, m/z
?
55.1 [M?1] . Anal calcd for C H N O S: C, 63.43; H, 3.11; N, 12.33; S, 7.06
2
4 14 4 4
Found C, 63.59; H, 3.04; N, 12.15; S, 6.89.
Microbiology
Methods for in vitro antimicrobial activity evaluation
The synthesized derivatives 5a–j were examined for their antimicrobial activity
against two Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus),
three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and
Klebsiella pneumoniae) and two fungal species (Aspergillus niger and Candida
albicans) using the paper disc diffusion technique and agar streak dilution method.
The Mueller-Hinton agar media were sterilized (autoclaved at 120 °C for 30 min),
poured at uniform depth of 5 mm, and allowed to solidify. The microbial suspension
5
10 CFU/mL) (0.5 McFarland Nephelometery Standards) was streaked over the
(
surface of media using a sterile cotton swab to ensure even growth of the organisms.
The tested analogs were dissolved in dimethylsulfoxide to give solutions of
3
.12–50 lg/mL. Sterile filter paper discs measuring 6.25 mm in diameter (Whatman
no. 1 filter paper), previously soaked in a known concentration of the respective test
analog in dimethylsulfoxide were placed on the solidified nutrient agar medium that
had been inoculated with the respective microorganism, and the plates were
incubated for 24 h at (37± 1) °C. A control disc impregnated with an equivalent
amount of dimethylsulfoxide without any sample was also used and did not produce
any inhibition. Ampicillin and gentamicina (100 lg/disc) were used as standard
antibacterial drugs while fluconazole (100 lg/disc) was used as an antifungal drug
[
20].
MIC of the analog was determined by the agar streak dilution method. A stock
solution of the synthesized analog (100 lg/mL) in dimethylsulfoxide was prepared
and graded quantities of the test analogs were incorporated in a specified quantity of
123

A. B. Patel, R. M. Raval
molten sterile agar, i.e., nutrient agar for evaluation of antibacterial and sabouraud
dextrose agar for antifungal activity, respectively. The medium containing the test
analog was poured into a Petri dish at a depth of 4–5 mm and allowed to solidify
under aseptic conditions. A suspension of the respective microorganism of
5
approximately 10 CFU/mL was prepared and applied to plates with serially
diluted analogs with concentrations in the range of 3.12–50 lg/mL in dimethyl-
sulfoxide and incubated at (37± 1) °C for 24 h (bacteria) or 48 h (fungi). The lowest
concentration of the substance that prevents the development of visible growth is
considered to be the MIC value [21].
Methods for in vitro evaluation of antimycobacterial activity evaluation
BACTEC MGIT method
The mycobacteria growth indicator tubes (MGIT) containing 4 ml of modified
Middle brook 7H9 Broth Base were numbered as per the title compounds to be
tested for antituberculosis efficacy by means of various concentrations prepared.
The suspension was allowed to stand for 20 min and the tubes were centrifuged at
4
7
3
000 rpm for 15 min. After that, 10 –10 CFU/mL of prepared M. tuberculosis
H37RV strain suspension was added in the medium to be incubated and 0.1 mL of
an egg-based medium was also added. The MGIT tubes were then tightly recapped,
mixed well, and incubated into a BACTEC MGIT instrument at 37 °C until
positivity is observed. The readings were measured daily starting from the second
day of incubation. Positive cultures were usually detected within 10 days. For
reading the actual results, the MGIT tubes were removed from the incubator and
placed in the UV light next to a Positive Control tube and an uninoculated tube.
Bright fluorescence detected by the corresponding MGIT tube was noticed in the
form of a bright orange colour in the bottom of the tube and also an orange
reflection on the meniscus [22]. The primary screening was conducted at a
concentration of 6.25 lg/mL against M. tuberculosis H37Rv in the BACTEC MGIT
system. Compounds demonstrating 99 % inhibition in the primary screening were
described as most potent compounds. All the other compounds to be tested were re-
examined for their actual MIC by adopting the conventional L. J. agar dilution
method. The MIC was defined as the lowest concentration inhibiting 99 % of the
inoculum.
Lowenstein and Jensen method
The secondary antimycobacterial screening for test compounds was obtained for M.
Tuberculosis H37Rv, by adopting the L. J. (Lowenstein and Jensen) agar dilution
method [23] for the measurement of MIC, and is defined as the lowest concentration
of drug which inhibits C99 % of bacterial population present at the beginning of the
assay. Stock solutions of 250, 200, 100, 62.5, 50, 25, 12.5, 6.25, 3.12, and 1.56 lg/
mL dilutions of each test compound in DMSO (dimethylsulfoxide) were added in
the liquid L. J. Medium, and then media were sterilized by the inspissation method.
A culture of M. tuberculosis H37Rv growing on L. J. Medium was harvested in
1
23

Discovery of new coumarin substituted quinazolines…
0.85 % saline in bijou bottles. These tubes were then incubated at 37 °C for 24 h
followed by streaking of M. tuberculosis H37Rv (5 9 104 bacilli per tube). These
tubes were then incubated at 37 °C. Growth of bacilli was seen after 12 days,
2
2 days and finally 28 days of incubation. Tubes having the compounds were
compared with control tubes where medium alone was incubated with M.
tuberculosis H37Rv. The concentration at which no development of colonies
occurred or\20 colonies was taken as the MIC concentration of the test compound.
The standard strain M. tuberculosis H37Rv was tested with known drugs Isoniazid,
Rifampicin, Ethambutol, and Pyrazinamide.
Acknowledgments The authors are thankful to Chairman, Kadi Sarva Vishwavidyalaya, Gandhinagar
and Principal Government College Daman for the encouragement, facilities, and financial support. They
are also thankful to SAIF Panjab University and Centre of Excellence, Vapi, India for spectral analyses.
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