Microwave assisted synthesis of 4-methyl-3-arylpyrano[2,3-f]chromen-2(8H)-one derivatives, evaluation of antiproliferative, and antimicrobial activities

Article abstract of DOI:10.1002/jhet.4103

A series of new 4-methyl-3-arylpyrano[2,3-f]chromen-2(8H)-one derivatives were designed and synthesized through an efficient, an eco-friendly manner under microwave irradiation and conventional heating methods. Structures of final compounds established ba

Full text of DOI:10.1002/jhet.4103

Microwave assisted synthesis of 4-Methyl-3-arylpyrano[2,3-f]chromen-2(8H)-one derivatives,
evaluation of antiproliferative and antimicrobial activities
Ravinder Dharavatha, Madderla Sarasijab, M. Ram Reddya, Nalaparaju Nagarajua, Katta
Ramakrishnaa and D. Ashoka*
aGreen and Medicinal Chemistry Laboratory, Department of Chemistry, Osmania
University, Hyderabad-500007, Telangana, India *E-mail: ashokdou@gmail.com
bDepartment of Chemistry, Satavahana University, Karimnagar-505001, Telangana, India
Abstract
A series of new 4-Methyl-3-arylpyrano[2,3-f]chromen-2(8H)-one derivatives were
designed and synthesized through an efficient, an eco-friendly manner under microwave
irradiation and conventional heating methods. Structures of final compounds established
based on IR, NMR and Mass spectral analysis. The final target compounds were screened
for their in vitro antiproliferative activity by taking cisplatin as a reference, Further,
antibacterial activity by taking strains such as Staphylococcus aurous, Bacillus subtilis,
Klebsiella pneumonia, Escherchia coli. Furthermore, antifungal activity screened with
Aspergillus niger, Aspergillus flavus and Fusarium oxysporum strains. Many compounds
results good activity.
Keywords: 2,4-Dihydroxy acetophenone, 1,1-Carbonyldiimidazole, Microwave
irradiation, Antiproliferative and Antimicrobial activities.
Introduction
One of the life threatening disease like cancer triggered due to rapid development of
abnormal cells is the major problem for several years. A study of public health [1] reveals
that, nearly 10 million new cancer cases were diagnosed every year, which is a real crisis.
Cancer is the second leading cause of deaths[2] worldwide, there is an urgent need to
develop suitable drugs to treat cancer. Infectious diseases caused and spread by
microorganisms are the major reasons for the health of humans and animals.[3] According
to WHO report, in the last few decades, the rate of recurrence of microbial infections has
increased considerably. Diseases caused due to bacterial and fungal infections need to treat
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2
in an effective manner and there is an urgent requirement to develop a new generation of
antimicrobial agents. Simultaneously with the emergence of new anti-microbial resistant
strains, the design and synthesis of resistant antimicrobial drugs are needed.[4]
For the past several years microwave irradiation technique became the most feasible
method and gain much importance in the research field.[5] As microwave irradiation method
provides rapid conversion of reactants into products with greater yields, no side products,
quick reaction times, easy to access and an eco-friendly compared to conventional heating
method was created much interest to chemists in synthesis.[6]
The pyaranochromenones, a class of heterocyclic compounds which shows many
biological, pharmacological applications such as anticancer,[7] antioxidant,[8]
antimicrobial[9] and also they are proved to be promising antiproliferative[10,11] agents.
Some of the compounds containing pyaranochromenones are used as drugs.
An idea of mixing of two or more pharmacophores by molecular hybridization
concept in drug design and development is a powerful tool in the research field. Molecular
hybridization, results, biological efficiency than the existed drugs. Inspiring from the
biological activities of pyaranochromenone, we have approached simple protocol by taking
readily available starting materials to synthesize pyaranochromenone derivatives, further in
evaluating their antiproliferative and antimicrobial activities.
Results and discussion
1. Chemistry
The protocol for the synthesis of pyaranochromenone derivatives is shown in
Scheme 1. The first step of the synthetic route involves the reaction of 2,4-
dihydroxyacetohenone (1) with propargyl bromide using anhydrous K2CO3 as base and
0
DMF as a solvent at 0 C for 4-5 hrs to obtain selectively 1-(2-hydroxy-4-(prop-2-yn-1-
yloxy)phenyl)ethanone (2). Further, the chromenone (3) is obtained by the intramolecular
0
cyclization of compound (2) by taking N,N-dimethylaniline as solvent[12,13] at 180 C for 6
hrs. Other side the reaction of substituted phenylacetic acid 4 (a-h) by using 1,1-
carbonyldiimidazole (CDI) (5) as a reagent and anhydrous K2CO3 as base and in dry
propanone as solvent at room temperature for 1 hr to obtain 1-(2,3-dihydro-1H-imidazol-1-
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3
yl)-2-phenylethanone in situ 6 (a-h), the second and third step reactions were carried out
under N2 atmospheric conditions. Finally, chromenone (3) treated with 1-(2,3-dihydro-1H-
imidazol-1-yl)-2-phenylethanone in situ 6 (a-h) in the presence of dry propanone solvent
and anhydrous K2CO3 as a base under inert condition for 4.5-5.5 hrs to get target
compounds 7 (a-h).
Scheme- 1
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4
Earlier, the reaction of various phenylacetic acds with DABCO as coupling agent
was reported from literature[14] which could not give encouraging results, further, the
reaction of various phenylacetic acids (1 eq.) and DBU (1 eq.) by using different solvents
like DCM and aromatic hydrocarbon (toluene) reported in the literature[15] could not gave
promising outcome. 1-(2,3-dihydro-1H-imidazol-1-yl)-2-phenylethanone in situ 6 (a-h)
was obtained by the treatment of different phenylacetic acids 4 (a-h) (1 eq.) and 1,1-
carbonyldiimidazole (CDI) (1.2 eq.) using anhydrous K2CO3 as a base and dry propanone
as a solvent.[16] Finally the reaction conditions were successfully optimized with
chromenone 3 (1 eq.), 1-(2,3-dihydro-1H-imidazol-1-yl)-2-phenylethanone 6 (a-h) using
anhydrous K2CO3 as a base and dry propanone as a solvent. Both successive steps were
maintained at nitrogen atmospheric conditions. On the other hand, in microwave
irradiation method, a mixure of compounds 3 and 6 (a-h) with anhydrous K2CO3 as a base
and dry propanone was suspended in Teflon vial irradiated at 180 Watts continued for 7-9
min. with 30 second intervals.
Synthesis of target compounds 7 (a-h) performed in conventional heating as well as
microwave irradiation techniques, however, greater yields obtained through microwave
irradiation (82-88%) method over conventional heating (65-75%) method, the results of the
products depicted in Table 1.
Table 1 Percentage of yields of target compounds 7 (a-h) in conventional heating
and microwave irradiation methods
Conventional method
Time (Hr) (% yield)
MWI method
Product
7a
Time (Min) (% yield)
5
4.5
5
65
68
73
72
75
72
68
66
8
7
7
8
7
7
9
9
85
84
86
85
88
84
82
85
7b
7c
7d
4.5
5.5
5
7e
7f
7g
5
7h
5.5
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5
The final target compound 7 (a) was optimized with different solvents in both
conventional heating and microwave irradiation methods were compiled in Table 2.
Finally, after the optimization, we conclude that best results were achieved when dry
propanone was used as a solvent in both the conventional heating and microwave
irradiation the methods.
Table 2 Optimization of compound 7a with different solvents
Conventional method
MWI method
Entry
Solvent
THF
(% yield)
(% yield)
1
2
3
4
8
6
7
35
20
17
15
15
58
72
40
27
24
20
22
70
85
Acetonitrile
1,4-Dioxane
Ethanol
Methanol
DMF
Propanone
Present work, 1,1 carbonyldiimodazole (CDI) plays a key role to obtain coumarine
skeleton compared to previous methods mentioned in the discussion because it can easily
handle and avoids the usages of different acid chlorides (like thionyl chloride). CDI reagent
is a mild base, fairly soluble in different solvents specially propanone. Progress of reaction
with various phenylacetic acids achieved small duration and reaction mixture can directly
take part in further steps. As CDI offers maximum conversion of products so purification
of crude, simple and convenient due to byproduct imidazole soluble in water to a great
extent. Therefor, we choose CDI as reagents and finally, optimized with propanone as a
solvent.
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6
2 Biological activities
Compounds 7 (a-h) were screened for in vitro antiproliferative activity in C-6,
MCF-6 cell lines, the antibacterial scavenging activity was carried out with Gram-positive
bacteria species like Staphylococcus aureus, Bascillus subtillus and Gram-negative species
Escherchia coli, Klebsilla pneumonia were used as reference. Additionally, antifungal
activity was performed by using species Aspergillus flavus and Fusariumoxy sporum. The
Minimum Inhibition Concentration (MIC) values determined by using the Agar disc
diffusion method. Among the tested compounds, compound 7b, 7e and 7h showed
encouraging results with their standard drugs.
2.1 Antiproliferative activities
Total compounds (7a-h) were tested for in vitro antiproliferative activity against C-
6, MCF-7Cell line in MTT[17] and DMEM[18] assay, results depicted in Table 3. From the
IC50 values we conclude that pyaranochromenone derivatives 7 (a–h) exhibited better
activity against the C-6 cell line than MCF- 7 cell line. Compound 7b, 7e and 7h displayed
promising activity than the standard cisplatin. The compounds 7a, 7c and 7g reveals
moderate outcome with standard cisplatin. Further, the remaining compounds showed
comparable activity against the both C-6 and MCF-7 cell lines.
Table 3 IC50 values of target compounds with C-6 and MCF-7 cell lines after 24 incubation
IC50 in µM
Compound
C-6 Cell line
4.638 µM
1.299 µM
4.801µM
4.124 µM
1.638 µM
6.320 µM
3.202 µM
MCF-7 Cell line
2.680 µM
1.270 µM
2.520 µM
3.201µM
7a
7b
7c
7d
7e
7f
1.158 µM
8.124 µM
2.680 µM
7g
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7
7h
1.321µM
1.329 µM
1.210 µM
1.189 µM
Cisplatin
2.2 Antibacterial activities
In vitro antibacterial activity of target compounds performed with Gram-positive
bacteria species like Staphylococcus aureus (MTCC 96), Bascillus subtillus (MTCC 121)
and Gram-negative species Escherchia coli (MTCC 43), Klebsilla pneumonia (MTCC 530)
were used as reference strains and Gatifloxacins was taken as standard drug. For the
evaluation of activity different concentration 10μg/mL and 20μg/mL were used in the Agar
disc diffusion method.[19] The zone of inhibition was measured in mm, Gatifloxacin was
taken as the standard drug and results were listed in Table 4. From the evaluation of
antibacterial activity, compounds 7b, 7e and 7g were highly active than standard
Gatifloxacin drug. Further compounds 7c, 7d and 7a showed moderate results against all
bacterial strains. The screening results conclude that the activities target compounds 7 (a-h)
was influenced by their structures. Finally, 7b, 7e and 7g exhibits better antibacterial
activities against to standard Gatifloxacin drug.
Table 4 Antibacterial activities of compound 7 (a-h) at different concentrations
Zone of inhibition (mm)
Gram-positive bacteria
Gram-negative bacteria
Stapylococus
aeureus
Bacillis
subtilis
Escherichia
coli
Klebsiella
pneumonia
Compound
10
20
10
20
10
20
10
20
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
7a
7b
7c
7d
15
22
18
16
24
32
36
23
16
23
16
15
32
40
34
32
13
16
14
12
16
21
18
16
08
14
09
08
14
20
16
13
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8
7e
23
18
24
15
20
32
25
34
24
30
22
19
22
16
20
42
38
42
28
40
18
15
18
14
15
23
19
25
17
20
13
11
12
06
10
20
17
22
13
18
7f
7g
7h
Gatifloxacin
2.3 Antifungal activities
Final target compounds 7 (a-h) were treated for in-vitro antifungal activity by
taking strains like Aspergillus niger, Aspergillus flavus and Fusarimoxy sporum. The
antifungal activity carried out by agar disc diffusion method[19] with the concentration of 50
µg/mL and results differentiate with Clotrimazole standard drugs. From the results of
antifungal activity, compounds 7b, 7e and 7g were given best activity with standard
Clotrimazole drugs. Finally antifungal activities of target compounds showed promising
results.
Table 5 Antifungal activities of the compound 7 (a-h) at concentration 50µg/mL
Zone of inhibition (mm) at 50µg/mL concentrations
Aspergillus niger Aspergillus flavus Fusariumoxy sporum
Compound
7a
13.6
18.8
12.4
10.6
17.9
15.2
17.6
10.3
17.3
14.2
17.6
14.8
11.8
16.8
14.8
16.8
12.5
16.4
14.8
18.6
15.8
11.6
17.8
16.2
18.6
12.6
18.2
7b
7c
7d
7e
7f
7g
7h
Clotrimazole
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9
3 Experimental
All the chemicals were purchased from Sigma Aldrich, SD-Fine, SRL, Fischer
chemicals and used without further purification. Reactions were performed by using dry
solvents, oven-dried RB Flasks in Nitrogen gas atmosphere. Transfer of solvents into the
RB flask was done with glass syringes. All the final products were synthesized through
conventional heating, microwave irradiation (MWI) by using a dry propanone as a solvent.
Microwave reactions were performed in Milestone MultiSYNTH microwave system.
Reaction progress was observed by TLC on silica gel plates 60 F254, the reaction mixture
was taken into crushed ice after gradual stirring, the mixture was filtered and purified with
column chromatography by using 60-120 mesh silica gel (SRL). Determination of melting
points was done with Cintex apparatus. Shimadzu FT-IR-8400S spectrophotometer was
used to record IR spectrum, PMR and CMR recorded with Bruker Avance II 400 and 100
MHz spectrometer (TMS internal standard) by choosing CDCl3 and DMSO-d6 solvents.
Coupling constants (J) introduced in hertz (Hz). Molecular weight determination was
carried out by using Elctron Spray Ionization (ESI) method.
Synthesis procedure
i).
Conventional method: A mixture of 1-(5-Hydroxy-2H-chromen-6-yl)ethanone (3)
(1 mmol), 1-(2,3-dihydro-1H-imidazol-1-yl)-2-phenylethanone 6 (a-h) (2 mmol) and
anhydrous K2CO3 (2 mmol) was refluxed in dry propanone (20 mL) for 4.5-5.5 hr. The
reaction is carried out in N2 atmosphere and the progress of the reaction was monitored by
TLC. After completion of the reaction, the crude was poured into ice cold water and
precipitate obtained was filtered and dried. The compound was purified by column
chromatography with ethyl acetate and n-hexane (1:9 v/v) mixture as an eluent to get pure
target compounds 7 (a-h).
ii).
Microwave Irradiation method: The mixture of 1-(5-Hydroxy-2H-chromen-6-
yl)ethanone (3) (1 mmol) and 1-(2,3-dihydro-1H-imidazol-1-yl)-2-phenylethanone 6 (a-
h) (2 mmol), anhydrous K2CO3 (2 mmol) and dry propanone was suspended in a quartz
tube, inserted into the Teflon vial with screw capped and subjected to microwave
irradiation. The mixture is then irradiated 7-9 min. The Microwave irradiation power set
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10
was to 180 W maximum. After the completion of the reaction, crude was poured into
crushed ice. The precipitate was filtered off, washed with water and dried. The compound
was purified by column chromatography by using ethyl acetate and n-hexane (1:9 v/v)
mixture as an eluent to get pure target compounds 7 (a-h).
4-Methyl-3-phenylpyrano[2,3-f]chromen-2(H)-one (7a).
0
1
Yield: 85%. White solid, M.P: 96-98 C; IR (KBr, cm-1): 1256 (C-O-C), 1714 (C=O), H
NMR spectrum (400 MHz, CDCl3), δ, ppm: 2.25 (s, 3H, CH3); 4.95 (dd, 2H, J = 2.00 Hz, J
= 1.75 Hz); 5.89-5.86 (m, 1H); 6.77-6.75 (m, 1H); 7.05 (d, 1H, J = 8.53 Hz); 7.29 (d, 2H, J
= 9.89 Hz); 7.45-7.43 (m, 4H). 13C NMR spectrum ( 100 MHz, CDCl3), δ, ppm: 30.6, 65.9,
107.5, 113,4, 118.1, 121.0, 121.5, 123.4, 127.8, 129.6, 131.9, 132.4, 138.1, 149.7, 150.6,
154.8, 162.8. MS (ESI Mass): 291 [M+H]+.
3-(4-Fluorophenyl-4-methylpyrano[2,3-f]chromen-2(8H)-one (7b).
0
Yield: 84%, White solid, M.P.: 108-110 C. IR (KBr, cm-1): 1245 (C-O-C), 1712 (C=O),
1H NMR spectrum (400 MHz, CDCl3), δ, ppm: 2.25 (s, 3H, CH3); 4.95 (dd, 2H, J = 2.00
Hz, J = 1.83 Hz); 5.89-5.85 (m, 1H); 6.76 (d, 1H, J = 8.53 Hz); 7.05-7.03 (m, 1H); 7.16-
7.12 (m, 2H); 7.29-7.27 (m, 2H); 7.41 (d, 1H, J = 8.53 Hz). 13C NMR spectrum (100 MHz,
CDCl3, δ, ppm: 29.7, 66.0, 107.3, 113.9, 115.4, 117.7, 118.2, 121.3, 121.8, 123.2, 129.9,
132.0, 141.6, 147.6, 150.9, 155.4, 162.8. MS (ESI Mass): 309 [M+H]+.
3-(4-Chlorophenyl)-4methylpyrano[2,3-f]chromne-2(8H)-one (7c).
0
Yield: 86%, White solid, M.P.: 122-124 C. IR (KBr, cm-1): 1257 (C-O-C), 1712 (C=O).
1H spectrum (400 MHz, DMSO-D6), δ, ppm, 2.20 (s, 3H, CH3); 4.94 (dd, 2H, J = 2.01 Hz,
J = 1.75 Hz); 6.06-6.02 (m, 1H); 6.82 (d, 2H, J = 8.53 Hz); 7.36-7.35 (d, 2H); 7.51 (d, 2H, J
= 8.53 Hz); 7.62 (d, 1H, J = 8.53 Hz). 13C NMR spectrum (100 MHz, CDCl3), δ, ppm:
29.7, 66.1, 110.1, 112.7, 114.7, 117.7, 121.9, 122.3, 123.1, 125.2, 131.6, 132.0, 133.6,
148.4, 148.5, 156.8, 160.7. MS (ESI Mass): 325 [M+H]+.
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11
3-(4-Bromophenyl)-4-methylpyrano[2,3-f]chromen-2(8H)-one (7d).
0
Yield: 85%, White solid, M.P.: 114-116 C. IR (KBr, cm-1): 1245 (C-O-C), 1708 (C=O).
1H NMR spectrum (400 MHz, CDCl3), δ, ppm: 2.25 (s, 3H, CH3); 4.95 (dd, 2H, J = 2.01
Hz, J = 1.83 Hz); 5.89-5.85 (m, 1H); 6.76 (d, 1H, J = 8.78 Hz); 7.03 (d, 1H, J = 10.03 Hz);
13
7.17 (d, 2H, J = 8.53 Hz); 7.41 (d, 1H, J = 8.78 Hz); 7.58 (d, 2H, J = 8.53 Hz). C NMR
spectrum (100 MHz, CDCl3), δ, ppm: 29.7, 66.1, 110.1, 112.7, 114.4, 117.7, 121.9, 122.3,
123.1, 125.1, 131.6, 132.0, 133.6, 148.4, 148.5, 156.8, 160.7. MS (ESI Mass): 369 [M+H]+.
3-(4-Methoxyphenyl)-4-methylpyrano[2,3-f]chromen-2(8H)-one (7e).
0
Yield: 88%, White solid, M.P.: 102-104 C. IR ( KBR, cm-1): 1249 (C-O-C), 1709 (C=O).
1H NMR spectrum ( 400 MHz, CDCl3), δ, ppm: 2.24 (s, 3H, CH3); 3.75 (s, 3H, OCH3);
4.95 (dd, 2H, J = 2.01 Hz, J = 1.68 Hz); 5.89-5.86 (m, 1H); 6.76 (d, 1H, J = 8.53 Hz); 7.03
(d, 1H, J = 8.53 Hz); 7.15 (d, 2H, J = 9.03 Hz); 7.26 (d, 2H, J = 9.03 Hz); 7.41-7.39 (m,
1H). 13C NMR spectrum (100 MHz, CDCl3), δ, ppm: 29.7, 55.4, 65.9, 107.3, 113.3, 113.5,
118.1, 120.8, 121.5, 123.4, 127.2, 130.8, 132.1, 148.6, 150.4, 154.6, 163.1. MS (ESI
Mass): 321 [M+H]+.
4-Methyl-3-(p-tolyl)pyrano[2,3-f]chromen-2(8H)-one (7f).
0
Yield: 84%, White solid, M.P.: 110-112 C. IR (KBr, cm-1): 1245 (C-O-C), 1708 (C=O).
1H NMR Spectrum (400 MHz, CDCl3), δ, ppm: 2.05 (s, 3H, CH3); 2.25 (s, 3H, CH3); 4.95
(dd, 2H, J = 3.24 Hz, J = 1.83 Hz); 5.89-5.86 (m, 1H); 6.76 (d, 1H, J = 8.53 Hz); 7.17 (d,
13
2H, J = 8.53 Hz); 7.41 (d, 2H, J = 8.28 Hz); 7.58 (d, 2H, J = 8.53 Hz). C NMR spectrum
(100 MHz, CDCl3), δ, ppm: 10.2, 29.7, 66.0, 107.3, 114.0, 115.4, 117.7, 118.2, 121.3, 12.8,
123.2, 130.0, 132.1, 141.6, 147.6, 150.8, 155.4, 160.7. MS (ESI Mass): 305 [M+H]+.
3-(3-Methoxyphenyl)-4-methylpyrano[2,3-f]chromen-2(8H)-one (7g).
0
Yield: 82%, White solid, M.P.: 126-128 C. IR (KBr, cm-1): 1248, (C-O-C), 1725 (C=O).
1H NMR spectrum (400 MHz, CDCl3), δ, ppm: 2.20 (s, 3H, CH3); 3.40 (s, 3H, OCH3); 4.90
(s, 2H),; 5.83-5.81 (m, 1H); 6.72-6.70 (m, 1H); 7.01-6.99 (m, 1H); 7.25-7.23 (m, 3H); 740-
7.38 (m, 2H). 13C NMR spectrum (100 MHz, CDCl3), δ, ppm: 10.1, 55.4, 65.9, 107.3,
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12
113.3, 113.5, 118.1, 120.8, 121.5, 123.4, 127.2, 130.8, 132.1, 144.6, 148.6, 150.4, 154.6,
163.1. MS (ESI Mass): 321 [M+H]+.
4-Methyl-3-(m-tolyl)pyrano[2,3-f]chromen-2(8H)-one (7h).
0
Yield: 85%, White solid, M.P.: 120-122 C. IR (KBr, cm-1): 1252 (C-O-C), 1712 (C=O).
1H NMR spectrum, (400 MHz, CDCl3), δ, ppm: 2.25 (s, 3H, CH3); 2.57 (s, 3H, CH3); 4.94
(s, 2H); 5.89-5.86 (m, 1H); 6.77-6.75 (m, 1H); 7.06-7.04 (m, 1H); 7.29-7.26 (m, 3H); 7.45-
7.40 (m, 2H). 13C NMR spectrum (100 MHz, CDCl3), δ, ppm: 10.2, 29.7, 65.9, 107.3,
113.9, 118.1, 120.8, 121.5, 123.4, 127.2, 130.8, 132.1, 141.6, 147.6, 1504., 161.6. Ms (ESI
Mass): 305 [M+H]+.
Antiproliferative activity procedure:
Under sterile conditions, in vitro cytotoxicity assay for synthesized compounds (A-
H) was carried out by employing a standard MTT assay. The MCF 7 (breast
adenocarcinoma cell line) tumor cell line in Dulbecco’s modified eagles medium (DMEM)
(Gibco NY,USA) supplemented with 10% Fetal bovine serum (FBS) (Bio cell, CA, USA)
and 1x antibiotic- anti mycotic solution (contains Penicillin, Streptomycin, Amphotericin-
D). 5× 105 cells/ml was used to test the growth inhibition activity of synthesized
compounds. MCF 7 cells growing exponentially were added to 96 well micro assay, culture
plate at a density of 3×103 cells per well in 200µl culture media and were grown for 24hrs
at 370c in humidified 5% CO2 incubator. The synthesized compounds were A-H, The given
synthesized compounds (A-H) and standard cisplatin were initially soluble in DMSO and
10mM stocks were prepared. The 10mM stocks were diluted to different concentrations of
25µM, 50µM, 100µM, 200µM, 500µM in DMSO. However the final dilution contains less
than 1% DMSO. After 24hrs of seeding cells in 96 well plate, under sterile conditions the
MCF7, C6 cells were subjected to 5µl of giving synthesized compounds and standard
cisplatin of respective concentrations 25µM, 50µM, 100µM and 200µM, 500µM in
duplicates to make the final concentration of 0.625µM, 1.25µM, 2.5µM, 5µM and 10µM
respectively. Control and blank also prepared in the same manner in duplicates, after
treatment the plates were incubated for 24hrs at 370c in humidified 5% CO2 incubator.
After 24hrs, 20µl of MTT dye solution (5mg/ml) was added to each well and incubated for
1hr at 370c in humidified 5% CO2 incubator. MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-di
phenyl tetrazolium bromide) is cleaved by mitochondrial succinate dehydrogenase and
reductase of viable cells, yielding a measurable purple product formazan. This formazan is
directly proportional to the number of viable cells and inversely proportional to the degree
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13
of cytotoxicity. After 1hr the MTT dye solution was aspirated and 100µl of DMSO was
added to soluble the crystals formed. The absorbance was measured at 590nm in ELISA
reader. The graph was plotted between the concentration of synthesized compounds on x
axis (0.625µM, 1.25µM, 2.5µM, 5µM and 10µM) and percentage of cell viability on y-
axis. The data reported percent growth of treated cells, which is presented as percentage
growth of inhibition (GI %).
Antimicrobial Activity procedure
Antibacterial activity:
The synthesized new compounds 7(a-h) were screened for their antibacterial
activity against different types of bacterial strains such as gram-negative bacterial strains of
Klebsiella pneumonia, Escherichia coli, and gram-positive bacterial strains of Bacillus
subtilis, Staphylococcus aeureus at two different concentrations of 10 and 20 µg/mL. The
cultures were diluted with 5 % saline autoclaved, and the final volume was done with
concentration approximately 105–106 CFU/mL. The synthesized compounds were diluted
in DMSO for antibacterial biological assays. For the Agar disc diffusion method, the liquid
form of test compound was soaked on to the disc and then allowed to air dry, such that the
disc gets completely saturated with the test compound. The saturated chemical discs were
introduced onto the upper layer of the medium evenly flooded with the bacteria. The discs
were dipped in different chemical samples, were placed over the evenly spread bacterial
nutrient media, and incubated at 37ºC for 24-48 h for better inhibition of bacteria. The
zones of inhibition were measured after 24-48 h. All the experiments were carried out in
triplicate and the results were expressed as a zone of Inhibition in mm. The zones of
inhibition of synthesized compounds 7(a-h) were compared to the zone of inhibition of
standard antibiotic concentrations of Gatifloxacin (10 and 20 µg/mL).
Antifungal activity procedure:
The antifungal activity of synthesized compounds 7(a-h) was tested against three
pathogenic fungi, namely Aspergillus niger, Aspergilus flavus and Fusariumoxy sporum by
the poison plate technique at a concentration of 50 µg/mL. Three kinds of fungi were
incubated in PDA at 25 ± 1 0C for 5 days to get new mycelium for antifungal assay, then a
mycelium as discs of approximately 0.45 cm diameter cut from the culture medium were
picked up with a sterilized inoculation needle and inoculated in the center of PDA plate.
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14
Test compounds were dissolved in DMSO (10 mL) after that added into the Potato
Dextrose Agar medium (PDA, 90 mL). The final concentration of compounds in the
medium was adjusted to 50 µg/mL. The inoculated plates were incubated at 25 ± 1ºC for 5
days. Acetone was diluted with sterilized distilled water and used as a control, while
Clotrimazole (50 µg/mL) was used as a standard control for each treatment three replicates
of experiments were carried out. The radial growth of the fungal colonies was measured on
the 5th day.
4 Conclusions
The present work constitutes an efficient protocol for the synthesis of compounds 7
(a-h) via conventional heating and microwave irradiation methods. However, microwave
irradiation method results promising yields in shorter reaction time. In vitro
antiproliferative activity done in C-6, MCF-7 cell lines with cisplatin standard. In addition,
Antibacterial activity carried out in Gram-positive, Gram-negative strains and Antifungal
activities carried out with Gatifloxacin and Clotrimazole standard drugs respectively. In
conclusion, in vitro biological evaluations gave evidence that compounds, 7a, 7e and 7h
exhibits excellent activities and compounds 7c, 7d and 7a showed considerable results with
standard reference drugs.
Supporting Information
Spectral analysis data (1H and 13C NMR, Mass Spectrum ESI) of all synthesized
compounds available in supporting information.
Conflicts of interest
No
Acknowledgments
Author Ravinder Dharavath (File No. 09/132(0865)/2017-EMR-I) is grateful to
CSIR, New Delhi for financial assistance in the form of Senior Research. One of the
authers D. Ashok is thankful to UGC, New Delhi for the award of BSR Faculty Fellowship,
Authers MRR, NN and KRK thank to UGC, New Delhi. The authors thank The Head,
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15
Department of Chemistry, Osmania University, Hyderabad for providing laboratory
facilities. We thank CFRD Osmania University, analytical team for providing spectral
analysis facilities.
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