Synthesis of some new heterocyclic compounds derived from 3-formylchromones and their antimicrobial evaluation

Article abstract of DOI:10.1007/s10593-014-1424-4

The synthesis and antimicrobial evaluation of a series of chromone-linked substituted heterocyclic derivatives is described. The condensation of 3-formylchromone with acetoacetamide under Knoevenagel-Cope reaction conditions was also explored, and the condensation with 4-hydroxy-6-methyl-2H-pyran-2-one constitutes a facile route to pyranopyrone fused systems. Most of the compounds exhibit good antimicrobial properties.

Full text of DOI:10.1007/s10593-014-1424-4

Chemistry of Heterocyclic Compounds, Vol. 49, No. 12, March, 2014 (Russian Original Vol. 49, No. 12, December, 2013)
SYNTHESIS OF SOME NEW HETEROCYCLIC COMPOUNDS
DERIVED FROM 3-FORMYLCHROMONES AND THEIR
ANTIMICROBIAL EVALUATION
2
1
3
4
A. Bari1,2 , S. S. Ali , A. Kadi , I. A. Hashmi , and S. W. Ng
*
The synthesis and antimicrobial evaluation of a series of chromone-linked substituted heterocyclic
derivatives is described. The condensation of 3-formylchromone with acetoacetamide under
Knoevenagel–Cope reaction conditions was also explored, and the condensation with 4-hydroxy-
6-methyl-2H-pyran-2-one constitutes a facile route to pyranopyrone fused systems. Most of the
compounds exhibit good antimicrobial properties.
Keywords: 3-formylchromone, pyranopyrones, antimicrobial activity, enzyme inhibitors, Knoevenagel–
Cope reaction, pharmacophore, urease inhibitory.
3-Formylchromone is considered a versatile synthetic intermediate for the preparation of a variety of
heterocyclic systems due to the presence of an unsaturated system, a conjugated carbonyl group and, more
importantly, an electron deficient center [1]. This center is very reactive and serves as Michael acceptor
resulting in the formation of fused polycyclic heterocycles by reaction with bifunctional nucleophiles.
Moreover, from the biological viewpoint, chromone is an important component of pharmacophores of various
biologically active natural products [2-5]. In addition, the core structure of many flavanoids is based on the
chromone moiety and has been found to possess several therapeutically interesting properties [6-9]. Several
formylchromone derivatives have been examined for their antitumor [10], urease inhibitory [11-12], anti-HIV
[13], and antimicrobial activities [14-15]. It is also known that 3-formylchromone derivatives promote induction
of chloroplast-free mutants in Euglena gracilis [16], have antiproliferative activity [17], and acts as tyrosine
kinase inhibitors [18]. On the other hand, the biological activity of isatin and substituted thiosemicarbazones has
been known for long time, both as anticancer and antiviral compounds [19]. These isatin thiosemicarbazones
undergo cyclization reactions at sulfur and nitrogen afforded oxindoles [20]. Oxindole derivatives have been
used as important intermediates in synthetic approaches to obtain various heterocyclic systems [21];
_______
*To whom correspondence should be addressed, e-mail: abari@ksu.edu.sa.
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box: 2457,
Riyadh 11451, Saudi Arabia.
2Research Center, College of Pharmacy, King Saud University, P.O. Box: 2457, Riyadh 11451, Saudi Arabia;
e-mail: abari@ksu.edu.sa.
3Department of Chemistry, University of Karachi, 75270 Karachi, Pakistan; e-mail: mran_ali_hashmi@yahoo.com.
4Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia; e-mail: weng@yahoo.com.
_________________________________________________________________________________________
Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 12, pp. 1860-1869, December, 2013.
Original article submitted September 24, 2013.
0009-3122/14/4912-1723©2014 Springer Science+Business Media New York
1723

moreover, the methylene group of the thiazolidinone ring could be utilized for further transformations. It was
therefore thought worthwhile to link these oxindoles to the chromone nucleus via condensation. In the last few
decades, much interest has been shown towards systematic studies on condensation reactions of
3-formylchromones with heterocycles and substituted binucleophiles to generate various fused heterocyclic
derivatives possessing different types of biological activities associated with them [22].
Additionally, condensations of 3-formylchromones with active methylene and methyl compounds
generated great interest in the earlier research owing to their interesting biological properties [23-24]. This paper
presents the synthetic capability and the exploitation of the above-mentioned types of condensation, achieved
using the Knoevenagel–Cope method, as a very convenient rate-enhancing approach. Several types of
subsequent reactions of the active methylene molecules illustrate the ability of chromone derivatives to serve as
excellent precursors for the synthesis of a wide variety of heterocyclic systems [25].
O
HN
O
S
R1
+
N
N
CHO
R
O
1a,b
O
N
H
2a–c
R1
Ac2O, AcONa
rt, 2 h;
65°C, 20 h
H
N
O
O
N
NH
N
S
R
O
O
3a–f
1 a R = H, b R = Cl; 2 a R1 = H, b R1 = Cl, c R1 = F;
3 a–c R = H, d–f R = Cl; a, d R1 = H, b, e R1 = Cl, c, f R1 = F
Compounds 1a,b were prepared by the literature procedure [26], and the substituted
pseudothiohydantoin derivatives 2a-c were obtained by the condensation of isatin with thiosemicarbazides,
which were cyclized with monochloroacetic acid in glacial AcOH to yield the oxindole products 2a-c as
described in the literature [27]. The reaction of 3-formylchromone 1a,b with substituted oxindoles gave
compounds 3a-f in moderate to good yields when heated at 65°C for 20 h. Further heating of the mixture at
higher temperature resulted in the formation of various side products and lower yields. Compounds 3a-f were
1
characterized by IR, H and 13C NMR spectroscopy, mass spectrometry, and elemental analysis. In the
IR spectrum, absorption bands due to the stretching vibrations of the NH group of indole and the thiazolidine
ring appear in the 3050-3140 cm-1 range. The spectra of compounds 3a-f display also characteristic bands
1
representing C=O groups (intense bands at 1650-1732 cm-1). In the H NMR spectrum of compounds 3a-f the
NH protons of thiazolidinone and isatin moieties appear as two broad singlets at 11.18-11.37 and 12.10-12.28
ppm, while the disappearance of the aldehyde proton signal and appearance of a singlet signal of the methyne
proton at ~7.35 ppm suggest the formation of the desired product. All the compounds 3a-f were isolated as
(E)-isomers, further confirmed by gated-decoupling (GD) measurements in which a strong vicinal resonance
was calculated for vinylic proton. Peaks at 160-166 ppm in the 13C NMR spectra of compounds 3a-f confirm the
presence of three carbonyl groups. Signals of aromatic protons appeared in their respective regions. The
elemental analysis data are also in accord with the proposed structures.
1724

The condensation of formylchromones 1a,b with 4-hydroxy-6-methyl-2H-pyran-2-one in the presence
of 0.5 equiv. of L-proline as catalyst was performed in ethyl acetate at room temperature to get the desired
compounds 4a,b. The same reaction conditions were reported for the condensation of carbohydrate enals with
pyrones and L-proline in the presence of TiCl4 catalysts [28]. However, L-proline provides a cleaner reaction,
consumes less time, and the products are required to be only purified by column chromatography. It was
observed that the reaction proceeds with higher yields at room temperature only, whereas at 0°C or at refluxing
temperature of the solvent using TiCl4 as catalyst gave lower yields and the compounds required rigorous
purification. The reaction is thought to proceed with the in situ formation of the imine salt of L-proline;
moreover, the carbinol amine formed during the reaction, releases a water molecule to equilibrate with the
iminium salt. Regeneration of catalyst and the ring transformation involving 6 π-electrons lead to formation of
the pyranopyrone fused system. The IR spectrum of compounds 4a,b showed bands at 1670 and 1730 cm-1 (for
compound 4a) and at 1680 and 1725 cm-1 (for compound 4b) corresponding to C=O groups. Additionally,
absorption bands at ~3290 cm-1 confirm the presence of the OH group. In the 1H NMR spectrum of compounds
4a,b, two pyran ring protons appeared as two doublets and exhibit characteristic W coupling, having coupling
constants J = 2.1 Hz; this confirms the opening of the pyrone ring and the formation of a new ring. The
13C NMR spectra of the obtained compounds also indicated the presence of two C=O groups at 162.2 and
~189.6 ppm, which supports the postulated structure. The mass spectra are also in accordance with the proposed
structure of compounds 4a,b, indicating the molecular ion peaks [M+H]+ at m/z 285 and 319, as well as base
peaks of fragment [M-C10H4O2]+ at m/z 128 and 162, respectively.
OH
OH
O
Me
O
L-Proline
1a,b
+
O
EtOAc
room temp., 5 h
Me
O
O
R
O
4a,b
a R = H, b R = Cl
Compounds 5a,b were synthesized by reacting compounds 1a,b with acetoacetamide under
Knoevenagel–Cope conditions. Synthesis of compound 5a by a tedious two-step process in the presence of
liquid ammonia was reported previously [29-30]. Under similar conditions, various side products were formed
with low overall yield. We herein report the current reaction in refluxing toluene, which was used in strictly
anhydrous conditions using a Dean–Stark trap. The optimum temperature and conditions for the completion of
this reaction with good yield was determined by a series of reactions of compound 1a with acetoacetamide for
3 h in boiling toluene. The reaction proceeds by the deprotonation of the active methylene group. Piperidinium
acetate formed in situ is used as an active catalyst in this reaction.
H
O
O
O
H2N
OH
O
N
O
O
Acetoacetamide
1a,b
Me
Me
AcOH, piperidine
R
R
PhMe, , 3 h
O
5a,b
a R = H, b R = Cl
The attack at the formyl carbon atom followed by the attack of the NH2 group at the C-1 atom of the
chromone moiety results in the concomitant opening of the ring to give the desired compounds 5a,b in good to
excellent yields. Yellow crystals of compound 5a suitable for X-ray crystallography were obtained by
crystallization from AcOEt–PhMe mixture.
1725

Fig. 1. Crystal structure of compound 5a.
Thermal ellipsoids are shown at the 50% probability level.
The crystal structure of compound 5a is shown in the Figure 1, and the crystal data are listed in Table 1.
1
In the H NMR spectra of compounds 5a,b two protons of the pyridine ring appeared as two doublets with a
characteristic W coupling, having coupling constant J = 2.5 Hz. The NH signal was observed in the downfield
1
region of the H NMR spectrum as a characteristic broad singlet. The 13C NMR spectra of compounds 5a,b
contain signals characteristic for C=O groups. Additionally, in the IR spectrum of compounds 5a,b, strong
absorption bands of C=O, NH, and OH groups are found at 1710-1730, 3050, and 3270 cm-1 (for compound 5a)
and at 1710-1727, 3030, and 3275 cm-1 (for compound 5b), respectively.
TABLE 1. The Main Crystallografic Parameters for Compound 5a
Parameters
Parameters
5a
5a
Empirical formula
Molecular weight
Color, shape
C14H11NO4
257.25
Yellow prism
d
calc, g·cm-3
1.557
0.97
536
µ, cm-1
F(000)
Crystal measurement, mm 0.40×0.30×0.20
Range for , deg
6.7-76.5
T, K
100
Range for indices
-12 < h < 12
-20 < k < 27
-8 < l < 8
Crystal system
Monoclinic
Number of measured
reflections
5543
Space group
Cc
Number of independent
reflections (Rint)
Number of reflections with
2079 (0.017)
2070
Z
4
I > 2σ(I)
a, Å
9.6930(2)
Number of refined
parameters
181
b, Å
c, Å
β, deg
V, Å3
21.8024(4)
6.9173(1)
131.367(2)
1097.10(3)
R1
wR2
GOOF
0.029
0.077
1.05
0.29 / -0.25
Residual electron density,
e·Å-3(dmin/dmax
)
The results of antibacterial activities of compounds 3a-f, 4a,b, and 5a,b against gram-positive bacteria
(S. aureus, S. mutans, B. subtilis) and gram-negative bacteria (E. coli, S. typhi, P. aeruginosa) are summarized
in Table 2. It has been observed that all compounds exhibited interesting antibacterial activities except
compound 3e. Compound 3f showed very high activity against most of the tested bacteria, while compounds
1726

TABLE 2. Antibacterial Activity* of Compounds 3a-f, 4a,b, 5a,b against
Gram-positive and Gram-negative Bacteria
Gram-positive bacteria
Gram-negative bacteria
Compound
S. aureus
S. mutans
B. subtilis
E. coli
S. typhi
P. aeruginosa
—
32
47
40
—
37
46
46
37
41
45
—
22
—
14
—
—
19
24
23
21
—
30
—
21
–
24
24
–
20
33
37
23
—
35
—
22
22
–
–
–
13
15
—
—
19
14
—
—
—
—
—
28
—
—
—
—
—
17
—
—
—
—
11
23
3a
3b
3c
3d
3e
3f
4a
4b
5a
24
–
28
—
—
18
34
5b
Ciprofloxacin
Naldixic Acid
_______
*Values are in millimeter (mm).
"–" indicates either no inhibition or inhibition zone diameter lower than 10 mm.
Values are expressed as mean (n = 3).
4a,b and 5a showed promising results against gram-positive bacteria. On the other hand, it was found that
compounds 3c,d are less active against gram-negative bacteria. Considering the results obtained from
antibacterial tests, it is noteworthy to mention that the tested compounds exhibited more activity towards gram-
positive bacteria than towards gram-negative bacteria.
The results of antifungal activities of all synthesized compounds are evaluated in Table 3. Most of the
studied compounds showed a significant level of activity in comparison with the standard used. Almost all
compounds exhibited medium activity against C. albicans, however, for the species A. niger and A. flavus
compounds 3f, 4a,b and 5a exhibited more potent activity than other compounds.
TABLE 3. Antifungal activity* of compounds 3a-f, 4a,b, and 5a,b against
C. albicans, A. niger, and A. flavus
Compound
C. albicans
A. niger
A. flavus
24
19
—
23
—
20
39
37
27
—
33
21
—
17
—
—
22
31
33
14
—
35
—
17
21
—
—
25
33
35
—
—
30
3a
3b
3c
3d
3e
3f
4a
4b
5a
5b
Amphotericin B
_______
*Values are in millimeter (mm).
"–" indicates either no inhibition or inhibition zone diameter less than 10 mm.
Values are expressed as mean (n = 3).
1727

In conclusion, we have devised an effective and environmentally benign method for the synthesis of
chromone, pyrano[4,3-b]pyran-5(8aH)-one, and pyridin-2(1H)-one derivatives. The notable merits of the
present methods are short reaction times, simple work-up procedure, and moderate to good yield of products.
Many of the obtained products exhibit significant antibacterial and antifungal activity.
EXPERIMENTAL
IR spectra were recorded with a Perkin-Elmer spectrum BX FT-IR spectrometer using KBr pellets.
1H and 13C NMR spectra were recorded on a Bruker instrument (500 and 125 MHz, respectively) in DMSO-d6
(compounds 3a-f) and CDCl3 (compounds 4a,b, 5a,b), TMS was used as internal standard. Mass spectra were
obtained on a JEOL JMS-700 mass spectrometer, ionization method was EI (70 eV). Microanalysis was
performed on a Perkin-Elmer CHNS-2440 analyzer and Thermo Scientific Orion ion selective electrodes.
Melting points were measured with a Thermo Scientific 9100 apparatus and are uncorrected. Thin-layer
chromatography (TLC) was performed with fluorescent silica gel HF254 plates (Merck) and visualized under UV
254 and 265 lights and charring with the EtOH–H2SO4 system. Merck silica gel 60 (230-400 mesh) was used for
column chromatography. Organic solvents used were dried by standard methods. Acetoacetamide, 4-hydroxy-
6-methyl-2H-pyran-2-one, and piperidine were purchased from Aldrich Chemical Co. and were used as
obtained. Chromones 1a,b were prepared by the literature procedure [26], and the substituted pseudothio-
hydantoin derivatives 2a-c were obtained by the method described in [27].
Synthesis of Compounds 3a-f (General Method). A mixture of chromones 1a,b (3.0 mmol),
compounds 2a-c (3.1 mmol), and freshly fused NaOAc (0.25 g, 3.0 mmol) in Ac2O (20 ml) was stirred under N2
atmosphere at room temperature for 2 h, and then at 65°C for 20 h. The obtained residue was purified by
column chromatography on silica gel, eluting with AcOEt–hexane, 2:3.
5-[(4-Oxo-4H-chromen-3-yl)methylene]-2-[(Z)-(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-4-one
(3a). Yield 56%, yellow solid, mp 250-254°C. IR spectrum, ν, cm-1: 1672 (C=O), 1710 (C=O amide), 1732
1
(C=O amide), 3055 (NH), 3110 (NH). H NMR spectrum, δ, ppm (J, Hz): 7.36 (1H, s, =CH–); 7.40 (1H, s,
H Ar); 7.53-7.57 (2H, m, H Ar); 7.65 (1H, t, J = 8.8, H Ar); 7.78 (1H, t, J = 8.7, H Ar); 8.33-8.39 (2H, m,
H Ar); 8.55 (1H, t, J = 8.1, H Ar); 8.68 (1H, s, H-2 chromone); 11.37 (1H, br. s, NH); 12.20 (1H, br. s, NH).
13C NMR spectrum, δ, ppm: 114.8; 117.0; 118.5; 121.3; 122.1; 123.1; 123.2; 124.7; 128.9; 131.1; 132.4; 149.1;
150.5; 152.1; 153.5; 153.6; 155.7; 155.9; 160.3; 166.1; 166.3. Mass spectrum, m/z (Irel, %): 417 [M+H]+ (100),
403 (77), 389 (44), 259 (21). Found, %: C 60.55; H 2.86; N 13.38; S 7.66. C21H12N4O4S. Calculated, %:
C 60.57; H 2.90; N 13.45; S 7.70.
2-[(Z)-(5-Chloro-2-oxoindolin-3-ylidene)hydrazono]-5-[(4-oxo-4H-chromen-3-yl)methylene]thiazolidin-
4-one (3b). Yield 48%, yellow solid, mp 269-273°C. IR spectrum, ν, cm-1: 1660 (C=O), 1710 (C=O amide),
1728 (C=O amide), 3070 (NH), 3100 (NH). 1H NMR spectrum, δ, ppm (J, Hz): 7.32 (1H, s, =CH–); 7.40 (1H, s,
H Ar); 7.54-7.58 (2H, m, H Ar); 7.65 (1H, t, J = 8.8, H Ar); 7.77 (1H, t, J = 8.8, H Ar); 8.32-8.35 (1H, m,
H Ar); 8.53 (1H, t, J = 8.5, H Ar); 8.66 (1H, s, H-2 chromone); 11.20 (1H, br. s, NH); 12.28 (1H, br. s, NH).
13C NMR spectrum, δ, ppm: 115.1; 117.0; 119.4; 120.8; 123.1; 123.2; 124.1; 124.5; 129.1; 132.2; 134.9; 150.0;
150.7; 153.3; 153.4; 155.5; 158.6; 156.3; 161.2; 165.6; 166.4. Mass spectrum, m/z (Irel, %): 451 [M+H]+ (73), 437
(34), 283 (17), 260 (11). Found, %: C 55.91; H 2.43; Cl 7.83; N 12.40; S 7.09. C21H11ClN4O4S. Calculated, %:
C 55.94; H 2.46; Cl 7.86; N 12.43; S 7.11.
2-[(Z)-(5-Fluoro-2-oxoindolin-3-ylidene)hydrazono]-5-[(4-oxo-4H-chromen-3-yl)methylene]thiazolidin-
4-one (3c). Yield 52%, yellow solid, mp 267-272°C. IR spectrum, ν, cm-1: 1655 (C=O), 1710 (C=O amide),
1732 (C=O amide), 3050 (NH), 3110 (NH). 1H NMR spectrum, δ, ppm (J, Hz): 7.33 (1H, s, =CH–); 7.38 (1H, s,
H Ar); 7.55-7.58 (2H, m, H Ar); 7.66 (1H, t, J = 8.8, H Ar); 7.77 (1H, t, J = 8.2, H Ar); 8.33-8.36 (1H, m,
H Ar); 8.55 (1H, t, J = 8.9, H Ar); 8.65 (1H, s, H-2 chromone); 11.21 (1H, br. s, NH); 12.24 (1H, br. s, NH).
13C NMR spectrum, δ, ppm: 115.2; 116.9; 119.2; 121.2; 123.2; 123.3; 124.0; 124.6; 129.1; 132.2; 135.1; 150.2;
1728

150.8; 153.5; 153.6; 155.5; 156.5; 158.7; 160.7; 165.7; 166.2. Mass spectrum, m/z (Irel, %): 435 [M+H]+ (47),
421 (65), 403 (33), 288 (91). Found, %: C 58.03; H 2.52; F 4.36; N 12.88; S 7.35. C21H11FN4O4S. Calculated,
%: C 58.06; H 2.55; F 4.37; N 12.90; S 7.38.
5-[(6-Chloro-4-oxo-4H-chromen-3-yl)methylene]-2-[(Z)-(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-
4-one (3d). Yield 52%, yellow solid, mp 263-272°C. IR spectrum, ν, cm-1: 1650 (C=O), 1715 (C=O amide),
1720 (C=O amide), 3090 (NH), 3120 (NH). 1H NMR spectrum, δ, ppm (J, Hz): 7.34 (1H, s, =CH–); 7.41 (1H, s,
H Ar); 7.55-7.59 (2H, m, H Ar); 7.62 (1H, t, J = 8.7, H Ar); 7.78 (1H, t, J = 8.7, H Ar); 8.32-8.36 (1H, m,
H Ar); 8.55 (1H, t, J = 9.0, H Ar); 8.66 (1H, s, H-2 chromone); 11.18 (1H, br. s, NH); 12.21 (1H, br. s, NH).
13C NMR spectrum, δ, ppm: 114.8; 117.0; 118.5; 121.0; 122.1; 123.1; 123.2; 124.7; 128.9; 131.0; 132.2; 149.1;
150.5; 152.1; 153.4; 153.5; 155.6; 155.9; 159.8; 166.1; 166.3. Mass spectrum, m/z (Irel, %): 452 [M+H]+ (41),
437 (51), 416 (33), 389 (21), 261 (77). Found, %: C 55.91; H 2.43; Cl 7.85; N 12.40; S 7.10. C21H11ClN4O4S.
Calculated, %: C 55.94; H 2.46; Cl 7.86; N 12.43; S 7.11.
2-[(Z)-(5-Chloro-2-oxoindolin-3-ylidene)hydrazono]-5-[(6-chloro-4-oxo-4H-chromen-3-yl)methylene]-
thiazolidin-4-one (3e). Yield 55%, yellow solid, mp 282-286°C. IR spectrum, ν, cm-1: 1652 (C=O), 1725 (C=O
1
amide), 1730 (C=O amide), 3060 (NH), 3140 (NH). H NMR spectrum, δ, ppm (J, Hz): 7.35 (1H, s, =CH–);
7.42-7.45 (1H, m, H Ar); 7.54-7.57 (2H, m, H Ar); 7.77 (1H, t, J = 8.8, H Ar); 8.35-8.38 (1H, m, H Ar); 8.54
(1H, t, J = 8.9, H Ar); 8.66 (1H, s, H-2 chromone); 11.20 (1H, br. s, NH); 12.10 (1H, br. s, NH). 13C NMR
spectrum, δ, ppm: 115.3; 117.0; 119.0; 121.2; 122.9; 123.3; 123.9; 124.5; 129.2; 132.2; 135.0; 149.6; 151.1;
152.5; 153.7; 155.6; 155.7; 156.5; 159.9; 166.1; 166.5. Mass spectrum, m/z (Irel, %) 486 [M+H]+ (100), 472
(59), 416 (21), 323 (19), 153 (11). Found, %: C 51.94; H 2.05; Cl 14.58; N 11.51; S 6.59. C21H10Cl2N4O4S.
Calculated, %: C 51.97; H 2.08; Cl 14.61; N 11.54; S 6.61.
5-[(6-Chloro-4-oxo-4H-chromen-3-yl)methylene]-2-[(Z)-(5-fluoro-2-oxoindolin-3-ylidene)hydrazono]-
thiazolidin-4-one (3f). Yield 48%, yellow solid, mp 293-297°C. IR spectrum, ν, cm-1: 1662 (C=O), 1710 (C=O
1
amide), 1730 (C=O amide), 3070 (NH), 3140 (NH). H NMR spectrum, δ, ppm (J, Hz): 7.35 (1H, s, =CH–);
7.42 (1H, s, H Ar); 7.50-7.60 (3H, m, H Ar); 8.36 (1H, t, J = 8.7, H Ar); 8.59-8.62 (1H, m, H Ar); 8.63 (1H, s,
H-2 chromone); 11.21 (1H, br. s, NH); 12.24 (1H, br. s, NH). 13C NMR spectrum, δ, ppm: 115.2; 117.2; 119.1;
120.8; 122.7; 123.2; 124.4; 124.5; 126.2; 129.2; 134.8; 135.2; 150.2; 150.7; 153.2; 153.4; 155.5; 156.5; 160.6;
165.2; 166.5. Mass spectrum, m/z (Irel, %): 469 [M+H]+ (33), 451 (51), 403 (100), 279 (65). Found, %: C 53.76;
H 2.12; Cl 7.54; F 4.02; N 11.91; S 6.82. C21H10ClFN4O4S. Calculated, %: C 53.80; H 2.15; Cl 7.56; F 4.05;
N 11.95; S 6.84.
Synthesis of Compounds 4a,b (General Method). 4-Hydroxy-6-methyl-2H-pyran-2-one (3.2 mmol)
was added to a stirred solution of compounds 1a,b (3.0 mmol) and L-proline (1.5 mmol) in dry EtOAc at room
temperature. The reaction mixture was stirred for a further 5 h; then saturated aq. NaHCO3 was added. The
mixture was extracted with EtOAc (2×100 ml). The organic layer was washed with brine and dried over
Na2SO4. After removal of the solvent in vacuum, the crude product is purified by column chromatography
(EtOAc–PhMe, 1:1).
3-(2-Hydroxybenzoyl)-7-methylpyrano[4,3-b]pyran-5(8aH)-one (4a). Yield 44%, white solid, mp
1
195-198°C. IR spectrum, ν, cm-1: 1670 (C=O lactone), 1730 (C=O), 3295 (OH). H NMR spectrum, δ, ppm
(J, Hz): 1.96 (3H, s, CH3); 5.54 (1H, d, J = 9.0, 8a-CH); 5.98 (1H, d, J = 9.0, H-8); 6.80-6.83 (1H, m, H Ar);
7.48 (1H, t, J = 8.6, H Ar); 7.58-7.63 (2H, m, H Ar); 7.82 (1H, d, J = 2.1, H-4); 8.07 (1H, d, J = 2.1, H-2); 11.22
(1H, br. s, OH). 13C NMR spectrum, δ, ppm: 20.7; 76.2; 98.3; 99.1; 107.1; 118.1; 119.2; 123.3; 123.9; 127.3;
132.4; 142.2; 155.3; 162.2; 165.2; 189.5. Mass spectrum, m/z (Irel, %): 285 [M+H]+ (51), 271 (27), 243 (19),
128 (100). Found, %: C 67.58; H 4.22. C16H12O5. Calculated, %: C 67.60; H 4.25.
3-(5-Chloro-2-hydroxybenzoyl)-7-methylpyrano[4,3-b]pyran-5(8aH)-one (4b). Yield 52%, colorless
1
crystals, mp 210-215°C. IR spectrum, ν, cm-1: 1680 (C=O lactone), 1725 (C=O), 3290 (OH). H NMR
spectrum, δ, ppm (J, Hz): 2.10 (3H, s, CH3); 5.53 (1H, d, J = 9.0, 8a-CH); 5.95 (1H, d, J = 9.0, H-8); 6.82 (1H,
s, H Ar); 7.44 (1H, t, J = 8.8, H Ar); 7.55-7.57 (1H, m, H Ar); 7.80 (1H, d, J = 2.1, H-4); 8.12 (1H, d, J = 2.1,
H-2); 11.19 (1H, br. s, OH). 13C NMR spectrum, δ, ppm: 20.7; 76.6; 98.7; 99.2; 107.1; 118.1; 119.2; 123.3;
1729

123.9; 132.4; 133.3; 142.4; 155.3; 162.2; 165.3; 188.7. Mass spectrum, m/z (Irel, %): 319 [M+H]+ (91), 271 (43), 243
(13), 196 (100). Found, %: C 60.27; H 3.43; Cl 11.10. C16H11ClO5. Calculated, %: C 60.30; H 3.48; Cl 11.12.
Synthesis of Compounds 5a,b (General Method). A mixture of compounds 1a,b (1.00 mmol),
acetoacetamide (1.50 mmol), AcOH (0.49 mmol), and piperidine (0.15 mmol) in PhMe (25 ml) was heated at
reflux using the Dean–Stark trap for 3 h. After removal of the solvent, the residue was purified by column
chromatography (eluent EtOAc–hexane, 3:1).
3-Acetyl-5-(2-hydroxybenzoyl)pyridin-2(1H)-one (5a). Yield 78%, yellow crystals, mp 183-188°C.
1
IR spectrum, ν, cm-1: 1710 (C=O acetyl), 1717 (C=O amide), 1730 (C=O), 3050 (NH), 3270 (OH). H NMR
spectrum, δ, ppm (J, Hz): 2.66 (3H, s, CH3); 6.88-6.91 (1H, m, H Ar); 7.03 (1H, d, J = 2.5, H-4); 7.50-7.55 (2H,
m, H Ar); 8.19 (1H, t, J = 9.0, H Ar); 8.31 (1H, d, J = 2.5, H-6); 11.70 (1H, br. s, OH). 13C NMR spectrum, δ,
ppm: 30.2; 118.1; 118.4; 118.5; 119.5; 119.9; 130.7; 132.5; 136.8; 137.4; 145.1; 162.7; 162.9; 194.2. Mass
spectrum, m/z (Irel, %): 258 [M+H]+ (100), 242 (66), 216 (31), 200 (23). Found, %: C 65.35; H 4.29; N 5.42.
C14H11NO4. Calculated, %: C 65.37; H 4.31; N 5.44.
3-Acetyl-5-(5-chloro-2-hydroxybenzoyl)pyridin-2(1H)-one (5b). Yield 68%, colorless crystals, mp
185-190°C. IR spectrum, ν, cm-1: 1710 (C=O acetyl), 1720 (C=O amide), 1727 (C=O), 3030 (NH), 3275 (OH).
1H NMR spectrum, δ, ppm (J, Hz): 2.68 (3H, s, CH3); 6.95-6.98 (1H, m, H Ar); 7.05 (1H, d, J = 2.1, H-4); 7.53-
7.57 (1H, m, H Ar); 8.21 (1H, t, J = 8.8, H Ar); 8.38 (1H, d, J = 2.5, H-6); 11.63 (1H, br. s, OH). 13C NMR
spectrum, δ, ppm: 30.3; 118.2; 118.4; 119.1; 119.4; 126.4; 130.2; 132.1; 137.4; 144.7; 145.9; 162.5; 163.4;
194.1. Mass spectrum, m/z (Irel, %): 292 [M+H]+ (71), 277 (61), 235 (41), 200 (11). Found, %: C 57.62; H 3.43;
Cl 12.12; N 4.76. C14H10ClNO4. Calculated, %: C 57.65; H 3.46; Cl 12.15; N 4.80.
X-ray Crystallographic Study of Compound 5a. Crystals of compound 5a were obtained by
crystallization from EtOAc–PhMe (1:4) by allowing slow solvent evaporation. X-ray structure determination was
performed on an Agilent Technologies SuperNova Dual diffractometer with an Atlas detector. Graphite-
monochromated CuKα radiation (1.54184 Å) was used. The structure was solved by the Patterson heavy atom
method and successive cycles of full-matrix refinement and Fourier difference syntheses. All non-hydrogen atoms
were refined anisotropically by the full-matrix least-squares method. Carbon-bound hydrogen atoms were placed in
calculated positions (C–H 0.95 to 0.98 Å, Uiso(H) 1.2 to 1.5Ueq(C)) and were included in the refinement in the rider
model approximation (Table 1). The hydrogen atoms of hydroxy and amino groups were freely refined. Molecular
graphics were performed from X-SEED software. Crystallographic data for the structure of compound 5 have been
deposited at the Cambridge Crystallographic Data Center (deposit CCDC 918116).
Antimicrobial testing. All synthesized compounds were screened for their in vitro growth inhibitory
activity. For antifungal test, Aspergillus niger MTCC 282 and Apergillus flavus AIIMS and Candida albicans
ATCC 2091 were chosen. Amphotericin B was used for comparison. The bacterial strains used were
Staphylococcus aureus ATCC 6538P, Bacillus subtilis ATCC 6633, and Streptococcus Mutans MTCC 890 (all
gram-positive) and Escherichia coli ATCC 25922, Salmonella typhi MTCC 733, and Pseudomonas aeruginosa
MTCC 741 (all gram-negative).
The plates were incubated at 35°C for 24 and 48 h, respectively, for bacteria and fungi. The inhibition
zones of microbial growth were measured by the paper disc method [31] in which 6-mm paper discs containing
specified amounts of the synthesized compounds (250 µg) were placed on an agar plate inoculated with a
standardized suspension of the microorganisms tested. Ciprofloxacin (15 µg) for gram-positive bacteria,
nalidixic acid (25 µg) for gram-negative bacteria and amphotericin B (25 µg) for fungi were used as standards.
The experiments were repeated thrice, and the diameters of inhibition zones produced by various synthesized
compounds were measured in millimeters.
We thank the Research Center, College of Pharmacy and Deanship of Scientific Research, King Saud
University for supporting this study. S. W. Ng thanks the Ministry of Higher Education of Malaysia (grant No.
UM.C/HIR/MOHE/SC/12) for financial support.
1730

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