Synthesis and antimicrobial evaluation of some new asymmetrically substituted 4-aryl- 2,6-di(coumarinyl) pyridines

Article abstract of DOI:10.4067/S0717-97072016000100009

In the present work the synthesis of various 4-aryl-2-(coumarin-3-yl)-6-(4-methyl-3-phenyl coumarin-6-yl)pyridines (6a-i) and 4-aryl-2-(coumarin-3-yl)-6-(4-methyl-7-methoxy coumarin-8-yl)pyridines (7a-i) have been carried out by reacting 1-[2(H)-1-benzopy

Full text of DOI:10.4067/S0717-97072016000100009

J. Chil. Chem. Soc., 61, Nº 1 (2016)
SYNTHESIS AND ANTIMICROBIAL EVALUATION OF SOME NEW ASYMMETRICALLY SUBSTITUTED
4-ARYL- 2,6-DI(COUMARINYL) PYRIDINES
YOGITA L. CHOVATIYA, KAUSHIK N. KUNDALIYA, RAKESH R. GIRI AND
DINKER I. BRAHMBHATT*
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India
ABSTRACT
In the present work the synthesis of various 4-aryl-2-(coumarin-3-yl)-6-(4-methyl-3-phenyl coumarin-6-yl)pyridines (6a-i) and 4-aryl-2-(coumarin-3-yl)-
6-(4-methyl-7-methoxy coumarin-8-yl)pyridines (7a-i) have been carried out by reacting 1-[2(H)-1-benzopyran-3-yl]-3-aryl-prop-2-en-1-ones (coumarinoyl
chalcones) (3a-f) with appropriate coumarinoyl methyl pyridinium bromide salt 4 and 5 respectively. The target compounds were characterized by the IR,
1H-NMR, 13C-APT and mass spectral analysis. Preliminary examination of target compounds as antimicrobial agents has been carried out using Broth dilution
method.
Keywords: Dicoumarinyl pyridines, Krohnke’s reaction, antimicrobial activity, Broth dilution method
with pyridine moiety by different positions i.e by (3,6’) [A] and (3,8’) [B] as
shown in figure 2 respectively. Additionally, the synthesized compounds were
INTRODUCTION
also evaluated for their antimicrobial potential.
Over past few decades, resistance to antimicrobial agents among bacterial
and fungal pathogens represent major global health problem in terms of
morbidity and mortality1. Infections caused by resistant microorganisms often
fail to respond to the standard treatment, resulting in prolonged illness, higher
health care expenditures, and a greater risk of death. Thus, to conquer such
multidrug resistant pathogens is challenging task in present era. Therefore,
there is crucial need to discover and develop effective antimicrobial agents
with novel mechanisms of action and enhanced activity.
At present, coumarin represents one of the classes of versatile biodynamic
agents, which are plentiful in plant kingdom and present as basic frame in
potent pharmaceutical compounds. The promising biological profile offered
by coumarin and its derivatives attributed to enormous diversity in substitution
patterns on core scaffold. Coumarins play pivotal role in drug discovery
exhibiting various therapeutic actions such as anticoagulant2, antitumor3, anti-
inflammatory4, hypolipidaemic5, vasorelaxant6, CNS depressant7, antioxidant8,
anti-TB9 and antimalarial10 activities.
The interesting biological profiles of the coumarins make them adaptable
targets in organic synthesis. Therefore, a detailed literature survey concerning
the coumarins derivatives was carried out, through which we came across some
3-(2-pyridyl) and 3-(3-pridyl)coumarins which are recognized for their useful
bioactivitiesviz. antifungal11,bactericidal12,fishtoxicity13, mothproofingactivity13
and CNS depressant activity14. Encouraged from the significant biological
essence of pyridine substituted coumarins, a series of new 3-(2-pyridyl)15-20
,
6-(2-pyridyl)21 and 8-(2-pyridyl)coumarins22 and their antimicrobial assessment
were reported from our laboratory. Additionally, we also reported some new
dicoumarinyl substituted pyridines23 in which synthesized coumarins adjoined
pyridine by 3rd position. Thus two cumarin moieties were having symmetrical
linkage with pyridine moiety and the attachment was by (3,3’) positions of
coumarins as shown in figure 1.
Figure 2: Attachment of two coumarin moieties with pyridine by different
positions i.e by (3,6’) [A] and (3,8’) [B].
RESULTS AND DISCUSSION
Chemistry
In the present work, various 4-aryl-2-(coumarin-3-yl)-6- (4-methyl-
3-phenyl
coumarin-6-yl)pyridines 6a-i and 4-aryl-2-(coumarin-3-yl)-6-
(7-methoxy-4-methylcoumarin-8-yl) pyridines 7a-i have been synthesized
by the reacting 1-[2(H)-1-benzopyran-3-yl]-3-aryl-prop-2-en-1-ones 3a-f
with 4-methyl-3-phenyl-6-coumarinoyl methyl pyridinium bromide salt 4
and 7-methoxy-4-methyl-8-coumarinoyl methyl pyridinium bromide salt
5 respectively in the presence of ammonium acetate and acetic acid under
Kröhnke’s reaction condition24 as displayed in scheme 1.
Figure 1: Attachment of two coumarin moieties with pyridine by (3,3’)
linkage.
In glance of our previous study here in, we report the synthesis of
dicoumarinyl substituted pyridine in which the coumarin moieties are attached
2788
e-mail: drdib317@gmail.com

J. Chil. Chem. Soc., 61, Nº 1 (2016)
Compounds
6a, 7a
R
H
R1
H
H
H
H
H
R2
H
H
H
H
H
R3
H
Compounds
6f, 7f
R
OCH3
H
R1
R2
R3
OCH3
H
H
H
6b, 7b
H
CH3
OCH3
H
6g, 7g
Benzo
6c, 7c
H
6h, 7h
H
Benzo
Benzo
CH3
OCH3
6d, 7d
OCH3
OCH3
6i, 7i
H
6e, 7e
CH3
Scheme 1: Synthetic pathway for the preparation of starting precursor 3a-f, target compounds 6a-1 and 7a-i.
The structures of all the synthesized compounds were confirmed on the
respectively. A sharp and intense band observed at 748 cm-1 is due to C-H
out of plane bending vibrations for mono substituted benzene ring. The
1H-NMR spectrum of compound 7a showed a two singlet appeared at 2.69
and 3.90 d integrating for three protons are of methyl group and methoxyl
group respectively. A singlet appeared at 6.21d correspond to proton attached
to C3’’-H. A meta coupled doublet observed at 8.59 d (J= 1.6 Hz) is due to C3-H
proton of pyridine ring. A singlet appeared at 8.76 d and integrating for one
proton is due to proton attached at C’4. The C’4 proton appears in the downfield
region due to the peri effect of nitrogen. The remaining twelve aromatic
protons appeared as multiplet between 7.79-7.06 d. The 13C-APT spectrum of
compound 7a showed signals at 19.61 and 56.47 δ are due to methyl carbon
and methoxyl carbon respectively. The signal appeared at 160.11 and 160.53
δ can be assigned to carbonyl carbon of the d-lactone ring of two coumarin
moieties. The signals for the other aromatic carbons appeared between 108.33
d to 159.18 d. The mass spectrum of compound 7a showed molecular ion peak
at m/z 443.0 along with other fragments corresponding to molecular formula
C31H21NO4. The analytical data for the other derivatives were discussed in
experimental section.
1
basis of H-NMR; 13C-APT; IR; elemental analysis and representative mass
spectral data.
Amongst the compounds 6a-i, the IR spectrum of compound 6a exhibited
the band at 3054 and 2924 cm-1 are due to aliphatic C-H and aromatic C-H
stretching vibrations. The characteristic strong and sharp band observed at
1716 cm-1 for carbonyl stretching of d-lactone ring of coumarin nucleus. The
bands observed at 1611 and 1457 cm-1 are due to aromatic C=C and C=N
stretching vibrations. A sharp and intense band observed at 761 cm-1 is due to
C-H out of plane bending vibrations for mono substituted benzene ring. The
1H-NMR spectrum of compound 6a showed a singlet at 2.47 d integrating for
three protons, which is due to methyl group protons. The signal for C’’ -H
appeared as doublet of doublet in the downfield region centered at 8.35 d 7(J=
8.8 and 1.6 Hz). The C’’5-H appeared as meta coupled doublet at 8.42 d (J= 1.6
Hz). A poorly resolved meta coupled doublet centered at 8.68 d corresponds to
proton attached to C3 of the pyridine ring. The C3-H of pyridine ring appeared
in the downfield region due to peri effect of carbonyl group of δ-lactone. The
signal for C’4 of the coumarin ring appeared in the most downfield region at
8.95 d is due to peri effect of nitrogen N1. The remaining sixteen aromatic
protons appeared as a multiplet between 7.28-7.93d. The 13C-APT spectrum
of compound 6a showed signals at 16.80 δ is due to methyl carbon. The signal
appeared at 160.33 and 160.76 δ are due to carbonyl carbon of the d-lactone
ring of two coumarin moieties. The signals for thirty non equivalent carbons
present in the compound appeared between116.46 d to 156.00d. The mass
spectrum of compound 6a showed molecular ion peak at m/z 533.0 along with
other fragments corresponding to molecular formula C H NO4.
The analytic and spectroscopic data of remaining synthesized compounds
are given in the Supplementary Material to this paper.
Evaluation of Antimicrobial activity
Allthesynthesizedcompoundswerescreenedfortheirinvitroantimicrobial
activity by Broth dilution method25. All the newly synthesized compounds 6a-i
and 7a-i exerted significant inhibitory activity against all the employed strains.
Upon evaluating antimicrobial activity data in Table I, it was observed that
compounds 7b and 7h (MIC= 62.5 μg/ml) exhibited superior activity compare
to Ampicillin (MIC= 250 μg/ml) against gram positive bacteria S. aureus,
while compounds 6e and 7g (MIC= 6.25 μg/ml) were found to be more potent
than Ampicillin (MIC= 250 μg/ml) against gram positive bacteria B. Subtilis.
Against S. aureus, compounds 6d, 6h, 7a, 7e, 7g and 7i (MIC= 100 μg/ml) and
against B. Subtilis compounds 6c, 7a and 7e (MIC= 100 μg/ml) demonstrated
Among the compounds 7a-i, the IR spectrum of36co2m3 pound 7a showed
the band at 3057 and 2924 cm-1 are due to aliphatic C-H and aromatic C-H
stretching vibrations respectively. A strong band at 1724 cm-1 due to carbonyl
stretching of d-lactone ring present in coumarin nucleus. The bands observed
at 1611 and 1457 cm-1 are due to aromatic C=C and C=N stretching vibrations
2789

J. Chil. Chem. Soc., 61, Nº 1 (2016)
more inhibition than Ampicillin (MIC= 250 μg/ml) and comparable activity to
Norfloxacin (MIC= 100 μg/ml). Compounds 6a, 6c, 6e, 6g and 7f (MIC= 125
μg/ml) against S. aureus, while compounds 6a, 6b, 6f, 6g, 6h, 7b, 7d and 7i
(MIC= 125 μg/ml) against B. Subtilis displayed better activity as compare to
Ampicillin (MIC= 250 μg/ml). Moderate inhibition were shown by compounds
6b, 6i, 7c and 7d (MIC= 200 μg/ml) against S. aureus than Ampicillin (MIC=
250 μg/ml). Similarly, compound 6i (MIC= 200 μg/ml) displayed moderate
activity against B.subtilis. Compound 6f (MIC= 250 μg/ml) against S. aureus
and compounds 7c, 7f and 7h (MIC= 250 μg/ml) against B. Subtilis showed
comparable activity to Ampicillin (MIC= 250μg/ml).
μg/ml). Against E. Coli, compounds 6d and 6i (MIC= 62.5 μg/ml) depicted
remarkable activity compare to Ampicillin (MIC= 100 μg/ml). Compound 7h
(MIC= 100 μg/ml) against E. Coli and compounds 6c, 6d and 7i (MIC= 100 μg/
ml) against S. Typhi were found to equipotant to Ampicillin (MIC= 100 μg/ml).
Antifungal assessment data of target compounds revealed that Compounds
7e, 7h (MIC= 100 μg/ml) against A. Niger exhibited equal inhibition to
Griseofulvin (MIC= 100 μg/ml) and Nystatin (MIC= 100 μg/ml). Compounds
6d, 7b (MIC= 100 μg/ml) against C. Albicans exerted excellent inhibition
compare to Griseofulvin (MIC= 500 μg/ml) and equal potency to Nystatin
(MIC= 100 μg/ml). Against C. Albicans, compounds 6b and 6g (MIC= 200
μg/ml); compounds
In case of gram negative bacteria, compound 7b (MIC= 50 μg/ml) was
displayed outstanding inhibitory effect against E. Coli compare to Ampicillin
(MIC= 100 μg/ml) and comparable activity to Chloramphenicol (MIC= 50
6h, 7d and 7i (MIC= 250 μg/ml) exhibited significant activity compare to
Griseofulvin (MIC= 500 μg/ml).
Table I. Antibacterial Activity MIC (µg/mL) of the compounds 6a-i and 7a-i.
Gram Positive Bacteria
Gram Negative Bacteria
Fungi
Compounds
S.Aureus
B.Subtilis
MTCC441
E.Coli
S.Typhi
A.Niger
MTCC282
C.Albicans
MTCC227
MTCC96
MTCC443
MTCCC98
6a
125
125
125
100
200
62.5
125
125
125
200
100
125
250
125
100
250
62.5
250
125
250
50
250
200
125
62.5
250
125
250
200
62.5
250
50
125
200
100
100
250
200
125
200
200
250
200
200
250
200
250
200
200
100
100
50
500
250
500
500
>1000
>1000
250
500
1000
500
500
>1000
500
100
500
1000
100
250
-
>1000
200
500
100
500
500
200
250
200
>1000
100
500
250
>1000
500
1000
500
250
-
6b
200
125
100
125
250
125
100
200
100
62.5
200
200
100
125
100
62.5
100
250
50
6c
6d
6e
6f
6g
6h
6i
7a
7b
7c
250
250
200
250
125
100
200
100
50
7d
7e
7f
7g
7h
7i
Ampicillin
Chloramphenicol
Norfloxacin
Griseofulvin
Nystatin
-
-
10
100
-
10
10
-
-
-
-
-
100
100
500
100
-
-
-
-
Structure activity relationship established from the analysis of data
reported in Table.1, lead to some general conclusions that mainly two structural
features have influence on biological potential of synthesized compounds: 1)
Substitution on phenyl ring. 2) Position of attachment of coumarin and pyridine
moieties to each other. Observations indicate that the compounds 6b, 6e, 6h,
7b, 7e and 7h bearing weak electron releasing methyl group (R3=CH3) showed
outstanding activity compare to parent targets. The increased efficiency
attributed to lipophilicity of methyl group. Introduction methoxyl group
(R =OCH3) exerted reduced activity compare to parent analogs. It is interesting
to 3note that compounds 7a-i possess promising antimicrobial activity against
gram positive bacteria B. Subtilis and S. aureus, while compounds 6a-i
demonstrate excellent activity against gram negative bacteria E. Coli and S.
Typhi. We also noticed that among the compounds 6a-i and 7a-i, compounds
bearing 4-methyl-7-methoxycoumarin-8-yl moiety at 6th position of pyridine
ring, i.e. compounds 7a-i were more efficient compare to compounds 6a-i.
EXPERIMENTAL SECTION
All the melting points were determined on μ Thermocal 10 apparatus.
All the IR spectra (KBr disc) were recorded on Shimadzu FT-IR 8400-
S spectrometer. 1H-NMR and 13C-NMR spectra were recorded on Bruker
1
Avance 400 spectrometer operating at 400 MHz for H-NMR and 100 MHz
for 13C-APT. The chemical shift (δ) is reported in ppm using chloroform-d as
a solvent and calibrated standard solvent signal. Mass spectra were recorded
on Shimadzu QP 2010 spectrometer. Elemental analysis was carried out on
Perkin-Elmer 2400 C-H-N-S-O Analyzer Series-II.
1-[2(H)-1-benzopyran-3-yl]-3-aryl-prop-2-en-1-ones
(coumarinoyl
Chalcone) 3a-f23, 4-methyl-3-phenyl-6-coumarinoyl methyl pyridinium
bromide salt 426 and 7-methoxy-4-methyl-8-coumarinoyl methyl pyridinium
bromide salt 527 were prepared according to literature procedure.
General procedure for the synthesis of 4-aryl-2-(coumarin-3-yl)-6-(4-
methyl-3-phenyl coumarin-6-yl)pyridines compounds 6a-i:
2790

J. Chil. Chem. Soc., 61, Nº 1 (2016)
A
solution of 4-methyl-3-phenyl-6-coumarinoyl methyl pyridinium
dilution against all microorganisms. The lowest concentration, which showed
no visible growth (turbidity) after spot subculture was considered as MIC for
each compound.
salt (4) (0.003 mol) in glacial acetic acid (15 mL) was charged in a round
bottom flask. Ammonium acetate (0.03 mol) and an appropriate 1-[2(H)-
1-benzopyran-3-yl]-3-aryl-prop-2-en-1-ones (coumarinoyl Chalcone) 3a-f
(0.003 mol) in glacial acetic acid (15 mL) were add to the solution in the flask
with stirring . The reaction mixture was further stirred for 30 minutes and then
refluxed for 8 hours. It was allowed to cool at room temperature. The reaction
mixture was poured into ice-cold water and extracted with chloroform (3x
30 ml). The organic layer was then washed with water and then dried over
anhydrous sodium sulfate. The removal of chloroform under reduced pressure
gave gummy material which was subjected to column chromatography using
silica gel and chloroform- petroleum ether (60-80) (9:1) as an eluent to give
compounds 6a-i. The compounds were recrystallized from chloroform-hexane.
CONCLUSION
From present study, we summarized that employed synthetic strategy
provide efficient route for the synthesis of asymmetrically substituted 4-aryl-
2,6-di(coumarinyl) pyridines by Krohnke’s protocol in good yield. Moreover
the starting precursors were also easy to prepare from synthesis point of view.
Antimicrobial study on target compounds concluded that the all the compounds
exerted promising activity against gram positive bacteria and gram negative.
The target compounds showed feeble activity against fungal pathogens. The
compounds 6d, 7b, 7g and 7h were most proficient members of the series.
Acknowledgements: The authors YLC, KNK and RRG are thankful to
the Head, Department of Chemistry, Sardar Patel University for providing
research facilities. Financial assistance to the authors from the University
Grants Commission, New Delhi, India, is highly acknowledged.
4-aryl-2-(coumarin-3-yl)-6-(4-methyl-3-phenyl
coumarin-6-yl)
pyridine (6a): White Solid, Yield 65%,; mp 282˚C; Anal. Calcd. for
C36H23NO4: C, 81.10; H, 4.38; N, 2.59%. Found: C, 81.04; H, 4.34; N, 2.63%;
IR (KBr, nmax cm-1): 3054, 2924, 1716, 1611 and 1457; H NMR (400 MHz,
1
CDCl3): δ 2.47 (3H, s, CH3), 7.28-7.93 (16H, m, Ar-H), 8.35 (1H, dd, J =
8.8 Hz and 1.6 Hz, C’’7-H), 8.42 (1H, poorly resolved doublet, C’’5-H), 8.68
(1H, poorly resolved doublet, C3-H ), 8.95 (1H, s, C’4-H ); 13C APT (100
MHz, CDCl ): δ 16.8 (CH3), 116.5(CH), 117.3(CH), 118.2(CH), 119.5(C),
120.8(C), 1233.7(CH), 124.7(CH), 125.3(C), 127.2(CH), 127.3(CH), 127.8(C),
128.4(CH), 128.5(CH), 128.9(CH), 129.2(CH), 129.3(CH), 130.1(CH),
130.2(CH), 132.3(CH), 134.4(C), 135.7(C), 138.4(C), 142.8(CH), 147.7(C),
150.5(C), 151.7(C), 153.4(C), 154.0(C), 156.0(C), 160.3(CO) and 160.7 (CO).
General procedure for the synthesis of 4-aryl-2-(coumarin-3-yl)-6-(7-
methoxy-4-methyl coumarin-8-yl)pyridines 7a-i:
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4-aryl-2-(coumarin-3-yl)-6-(4-methyl-7-methoxycoumarin-8-yl)
pyridine (7a):
White solid, Yield 60%; mp 280˚C; Anal. Calcd. for C31H21NO : C,78.90;
H, 4.42; N, 3.01%. Found: C, 78.97; H, 4.49; N, 2.97%; IR (KBr, 4nmax cm-1):
1
3057, 2923, 1724, 1611 and 1457; H NMR (400 MHz, CDCl ): δ 2.69 (3H,
s, CH3), 3.90 (3H, s, OCH3), 6.21 (1H, s, C3′′-H), 7.79-7.06 (132H, m, Ar-H),
8.59 (1H, d, J=2 Hz, C -H), 8.76 (1H, s, C′4-H); 13C-APT (100 MHz, CDCl3): δ
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114.31(C), 116.28(CH),3 119.72(C), 121.36(CH), 124.09(CH), 124.50(CH),
126.22(CH), 127.46(CH), 129.04(CH), 129.19(CH), 131.98(CH), 138.30(C),
143.72(CH), 149.29(C), 150.81(C), 150.88(C), 151.48(C), 152.21(C),
153.99(C), 157.10(C), 159.18(C), 160.11(CO), 160.53(CO).
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Biological Assay
All the synthesized compounds were screened for their antimicrobial
activity against two Gram-positive bacteria viz. Bacillus subtilis (MTCC
441) and Staphylococcus aureus (MTCC 96), two Gram-negative bacteria
viz. Escherichia coli (MTCC 443) and Salmonella typhi (MTCC 98) and two
fungi viz. Aspergillus niger (MTCC 282) and Candida albicans (MTCC 227).
All MTCC cultures were collected from Institute of Microbial Technology,
Chandigarh and tested against above mentioned standard drugs. Mueller
Hinton Broth was used as nutrient medium to grow and dilute the compound
suspension for the test bacteria and Sabouraud Dextrose Broth was used for
fungal nutrition. The size of the inoculum for the test strain was adjusted to
108 colony forming unit (CFU) per milliliter by comparing the turbidity. In the
present study, ampicillin and norfloxacin were used as standard antibacterial
drugs, whereas nystatin and griseofulvin were used as standard antifungal drugs.
DMSO was used as a diluent to get the desired concentration of compounds to
test upon standard bacterial strains. Each synthesized compound and standard
drugs were diluted obtaining 2000 μgmL-1 concentration, as a stock solution. In
primary screening 1000, 500 and 250 μgmL-1 concentrations of the synthesized
drugs were taken. The active synthesized compounds found in this primary
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