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Chinese Chemical Letters 23 (2012) 57–60

www.elsevier.com/locate/cclet

Synthesis and in vitro antimicrobial screening of new

pyrano[4,3-b]pyrane derivatives of 1H-pyrazole
Chetan B. Sangani *, Divyesh C. Mungra, Manish P. Patel, Ranjan G. Patel
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India
Received 22 June 2011
Available online 8 November 2011

Abstract
A new series of pyrano[4,3-b]pyrane 4a–l bearing 1H-pyrazole has been synthesized by one pot base catalyzed cyclocondensa-
tion reaction of 1H-pyrazole-4-carbaldehyde 1a–l, malononitrile 2 and 4-hydroxy-6-methylpyrone 3. All the synthesized
compounds were screened against six bacterial pathogens, namely B. subtilis, C. tetani, S. pneumoniae, S. typhi, V. cholerae,
E. coli and antifungal activity against, two fungal pathogens, A. fumigatus and C. albicans using broth microdilution MIC method.
Some of the compounds are found to be equipotent or more potent than that of commercial drugs, against most of employed strains.
# 2011 Chetan B. Sangani. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Pyranopyrane; MCR; Pyrazole-4-carbaldehyde; Antimicrobial activity

An alarming increment in pathogenic resistance to existing first line standard drugs is a serious problem in
antimicrobial cure and necessitates continuing research into new classes of antimicrobials [1]. Moreover, the
progression of drug-resistant strains has contributed to the inefficiency of the straight antimicrobial therapy. This crop
up an enormous interest in antibacterial research and we strongly believe that there is an urgent call for development of
new drugs with divergent and unique structure and with a probably unusual mechanism of action from that of existing
first line drugs. Consequently, this spot of research is accorded an immense significance and keeps on attracting much
attention of increasing number of medicinal chemists.
Recently, we have been particularly interested in the synthesis of N-arylpyrazole and pyrane incorporating structures for
antimicrobial evaluations [2] on the premise that N-arylpyrazole is chemically useful synthons bearing diverse biological
activities like antimicrobial [3–5], anti-inflammatory (COX-2 inhibitor and ulcerogenic activity) [4], antitubercular [5],
antitumor [6,7], antiangiogenesis [7], anti-parasitic [8], antiviral [9], analgesic and anxiolytic activity [10]. Moreover, the
pyran nucleus is a fertile source of biologically important molecules possessing a wide spectrum of biological and
pharmacological activities, such as antimicrobial [11], antiviral [12], antiproliferative [13], antitumor [14], etc.
Multi component reaction (MCR) approach emerged as the most suitable protocol for the synthesis of
functionalized organic compounds due to the fact that the synthesis can be performed without the isolation of the
intermediates, without discharging any functional groups within short reaction time [15]. This synthetic strategy is
fruitfully employed to achieve new pyrano[4,3-b]pyran derivatives of aryloxypyrazole.

* Corresponding author.
E-mail address: chetansangani1986@yahoo.com (C.B. Sangani).

1001-8417/$ – see front matter # 2011 Chetan B. Sangani. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.09.012
58 C.B. Sangani et al. / Chinese Chemical Letters 23 (2012) 57–60

Thus, in view of biological significance of pyrane, a modification on the 4-position on pyrano[4,3-b]pyrane by 3-

methyl-5-phenoxy-1-phenyl-1H-pyrazole is undertaken to check whether it may bring significant changes in
bioactivities of pyrane derivatives. As a part of our current studies in developing new antimicrobial agents via
combination of two therapeutically active moieties [16], we have synthesized and report herein pyrano[4,3-b]pyrane
derivatives 4a–l via MCR approach.

1. Experimental

All the reagents were obtained commercially and used with further purification. All melting points were taken in
open capillaries in a paraffin bath and are uncorrected. The monitoring of the progress of all reactions and
homogeneity of the synthesized compounds was carried out by thin layer chromatography (TLC). TLC was run using
TLC aluminum sheets silica gel 60 F254 (Merck). Elemental analysis (%C, H, N) was carried out by Perkin-Elmer
2400 series-II elemental analyzer (Perkin-Elmer, USA). The IR spectra were recorded in KBr on a Perkin-Elmer
Spectrum GX FT-IR spectrophotometer (Perkin-Elmer, USA). 1H NMR and 13C NMR spectra were recorded in
DMSO-d6 on a Bruker Avance 400F (MHz) spectrometer (Bruker Scientific Corporation Ltd., Switzerland) using
solvent peak as internal standard at 400 MHz and 100 MHz, respectively. Mass spectra were scanned on a Shimadzu
LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan) (Scheme 1).

1.1. General procedure

A mixture of an appropriate 1H-pyrazole-4-carbaldehyde 1a–l (30 mmol), malononitrile 2 (30 mmol) and 4-
hydroxy-6-methylpyrone 3 (30 mmol) in ethanol (20 mL) containing catalytic amount of piperidine was stirred under
reflux for 1–1.5 h. On completion of reaction, monitored by TLC (ethyl acetate:hexane = 1:1), the reaction mixture
was cooled to room temperature, the solid separated was filtered and washed well with ethanol to obtain the pure
compounds 4a–l. Physical, analytical and spectroscopic characterization data of the synthesized compounds 4a and 4k
are given below:

2-Amino-4-[3-methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl]-7-methyl-5-oxo-4,5-dihydropyrano[4,3-b]pyran-3-
carbonitrile (4a): Yield: 80%; mp: 230–231 8C; IR (KBr, n, cm 1): 3400 & 3180 (asym. & sym. str. of –NH2), 2205
(–CBBN str.), 1710 (–C O str.); 1H NMR (400 MHz, DMSO-d6): d 2.00, 2.33 (s, 3H, 2  CH3), 4.28 (s, 1H, H4),
5.68 (s, 1H, H8), 6.57–7.54 (m, 12H, NH2 + Ar–H). 13C NMR (100 MHz, DMSO-d6): d 13.07, 19.52 (CH3), 25.88
(C4), 56.24 (C–CN), 97.95 (4a), 99.17 (C8), 110.82, 114.56, 120.03, 121.63, 123.38, 127.05, 129.63, 130.17,
138.09, 145.35, 148.07, 156.20, 158.36, 158.63, 161.82 (Ar–C), 162.55 (C O); Anal. Calcd. for C26H20N4O4
(452.46): C, 69.02; H, 4.46; N, 12.38%. Found: C, 69.17; H, 4.35; N, 12.22%; MS m/z: 453.52 [M+1]+.

H3C H3C CHO H3C CHO

OH
N O N N O
N DMF Cl DMF N
N
POCl3
K2CO3
R2 4a: R1 = H, R2 = H
R2
R1 R1 R1 4b: R1 = H, R2 = CH3
1a-l R1 4c: R1 = H, R2 = OCH3
4d: R1 = H, R2 = Cl
H 3C CHO 4e: R1 = CH3, R2 = H
N N 4f: R1 = CH3, R2 = CH3
O H3C R2 4g: R1 = CH3, R2 = OCH3
N O
N NC O 4h: R1 = CH3, R2 = Cl
O Ethanol O
+
CN 4i: R1 = Cl, R2 = H
NC Piperidine
H3C OH O 4j: R1 = Cl, R2 = CH3
R2 4k: R1 = Cl, R2 = OCH3
R1 1a-l 2 3 H3C O NH2 4l: R1 = Cl, R2 = Cl
4a-l

Scheme 1. Synthetic pathway for the compounds 1a–l and 4a–l.

 C.B. Sangani et al. / Chinese Chemical Letters 23 (2012) 57–60 59

2-Amino-4-[1-(3-chlorophenyl)-5-(4-methoxyphenoxy)-3-methyl-1H-pyrazol-4-yl]-7-methyl-5-oxo-4,5-dihydro-
pyrano[4,3-b]pyran-3-carbonitrile (4k): Yield: 84%; mp: 240–241 8C; IR (KBr, n, cm 1): 3410 & 3195 (asym. &
sym. str. of –NH2), 2195 (–CBBN str.), 1705 (–C O str.); 1H NMR (400 MHz, DMSO-d6): d 2.03, 2.32 (s, 3H,
2  CH3), 3.66 (s, 3H, OCH3), 4.28 (s, 1H, H4), 5.75 (s, 1H, H8), 6.53–7.61 (m, 10H, NH2 + Ar–H). 13C NMR
(100 MHz, DMSO-d6): d 13.10, 19.41 (CH3), 25.83 (C4), 55.82 (OCH3), 56.01 (C–CN), 98.02 (4a), 99.04 (C8),
111.22, 115.20, 115.42, 119.57, 119.97, 120.92, 126.68, 131.42, 133.98, 139.22, 146.11, 148.86, 149.76, 155.52,
158.38, 158.63, 161.80 (Ar–C), 162.58 (C O); Anal. Calcd. for C27H21ClN4O5 (516.93): C, 62.73; H, 4.09; N,
10.84%. Found: C, 62.95; H, 4.26; N, 10.99%; MS m/z: 517.88 [M+1]+.

2. Results and discussion

The targeted title compounds pyrano[4,3-b]pyranes 4a–l were prepared in moderate to good yield (75–90%) by the
reaction of 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-carbaldehydes, malononitrile and 4-hydroxy-6-methylpyrone
in refluxing ethanol containing piperidine as a basic catalyst. The required 1-aryl-5-chloro-3-methyl-1H-pyrazole-4-
carbaldehydes were prepared according to literature procedure [18] and 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-
carbaldehydes 1a–l by our literature procedure [2a]. The reaction was carried out in aqueous media and under neutral
conditions but failed to proceed even on prolong refluxing. The reaction was also attempted under microwave
irradiation but not succeeded. The reaction proceeded in acetonitrile, methanol, benzene or DMF, in the presence of
morpholine or K2CO3 but required prolong refluxing and resulted only in poor yield. Hence, these conditions were
considered as the most optimized condition for the synthesis of title derivatives.
The plausible mechanism for the formation of the pyrane derivatives 4a–l is outlined in Scheme 2. The reaction
may proceed via an in situ initial formation of the hetarylidene nitrile, containing the electron-poor C C double bond,
from the Knoevenagel condensation between 3-methyl-5-aryloxy-1-arylpyrazole-4-carbaldehyde and malononitrile
by loss of water molecules. Finally, Michael addition of 3 to the initially formed unsaturated nitrile, i.e. nucleophilic
attack of hydroxyl moiety to the cyano olefins afforded cyclized pyrano[4,3-b]pyrane derivatives 4a–l.
1
H NMR (DMSO-d6) spectrum of 4a exhibited a singlet peak around d 4.28, 5.68 stands for H4 and H8 respectively.
Amine and aromatic protons of 4a resonate as multiplets at around d 6.57–7.54. Singlets around d 2.00, 2.33 stands for
methyl protons. 13C NMR of 4a exhibited distinctive signals around d 13.07, 19.52 stands for methyl carbons and d
25.88, 56.24 stands for C4 and C–CN respectively. Aromatic carbons of 4a showed signals around d 110.82–161.82 in
the 13C NMR spectra. Moreover, distinctive signals d 162.55 stands for carbonyl carbon. The IR spectrum of
compound 4a exhibited characteristic absorption bands around 3400–3180 cm 1 and 2205 cm 1 stands for (asym. &
sym. str.) –NH2 and –CN functional groups respectively. The characteristic absorption band of lactone carbon is
observed around 1710 cm 1.
The elemental analysis values and mass spectral data are in good agreement with that of theoretical data. Similarly,
all these compounds were characterized on the basis of spectral studies. All the compounds 4a–l were screened for
their antimicrobial activities using ampicillin, nystatin and griseofulvin as standard drugs by broth microdilution
method as recommended by NCCLS [17]. Reviewing the antimicrobial activity data (Table 1), majority of compounds
are found to be active against Gram-positive bacteria B. subtilis, C. tetani and a fungal pathogen C. albicans. It is worth
mentioning that minor change in the molecular configuration of these compounds profoundly influences the activity.

Pyz Ar Pyz
Pyz CN HO 3 NC
NC
Ar
CHO CN -H2O C
CN
N O
1a-l 2 Hetarylidene nit rile
H
Pyz
Pyz Pyz
H H NC 3 5
NC H NC 4
Ar
Ar Ar
C C H 2N 2 O 6
N O HN O
4a-l

Scheme 2. Plausible mechanistic pathway for the compounds 4a–l.

60 C.B. Sangani et al. / Chinese Chemical Letters 23 (2012) 57–60

Table 1
Antimicrobial activity of compounds 4a–l.
Compound Minimum inhibitory concentration, MIC (mg/mL)
Gram-positive bacteria Gram-negative bacteria Fungal species
B. subtilis C. tetani S. pneumoniae E. coli S. typhi V. cholerae A. fumigatus C. albicans
4a 200 200 250 100 100 250 1000 500
4b 200 200 200 62.5 250 100 500 250
4c 250 500 250 500 500 500 500 250
4d 500 500 100 250 250 250 200 1000
4e 500 250 500 200 250 200 500 250
4f 100 250 500 250 500 250 1000 500
4g 200 250 200 200 250 200 >1000 500
4h 500 200 100 500 500 250 >1000 100
4i 500 250 250 500 500 500 500 250
4j 250 250 250 200 250 200 500 200
4k 250 250 500 62.5 100 100 1000 500
4l 200 500 200 500 200 500 >1000 500
A 250 250 100 100 100 100 – –
B – – – – – – 100 100
C – – – – – – 100 500
A, ampicillin; B, nystatin; C, griseofulvin; ‘–’, ‘not tested’.

Acknowledgment

The authors are grateful to the Department of Chemistry, Sardar Patel University, India for providing laboratory
facilities.

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