Laccase from basidiomycetous fungus-catalyzed synthesis of substituted benzopyranocoumarins via domino reaction

Article abstract of DOI:10.1080/00397911003637484

The present investigation provides a simple and convenient route to organic synthesis of substituted benzopyranocoumarin, which is important because it is a probable HIV protease inhibitor. The reaction of,-unsaturated derivatives of coumarins with catech

Full text of DOI:10.1080/00397911003637484

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Laccase from Basidiomycetous
Fungus–Catalyzed Synthesis of
Substituted Benzopyranocoumarins via
Domino Reaction
Mazaahir Kidwai a , Roona Poddar a , Sarika Diwaniyan b & Ramesh
Chander Kuhad b
a Green Research Laboratory, Department of Chemistry, University of
Delhi, Delhi, India
b Lignocellulose Biotechnology Laboratory, Department of
Microbiology, University of Delhi South Campus, Delhi, India
Version of record first published: 31 Jan 2011
To cite this article: Mazaahir Kidwai, Roona Poddar, Sarika Diwaniyan & Ramesh Chander
Kuhad (2011): Laccase from Basidiomycetous Fungus–Catalyzed Synthesis of Substituted
Benzopyranocoumarins via Domino Reaction, Synthetic Communications: An International Journal for
Rapid Communication of Synthetic Organic Chemistry, 41:5, 695-706
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Synthetic Communications1, 41: 695–706, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911003637484
LACCASE FROM BASIDIOMYCETOUS FUNGUS–
CATALYZED SYNTHESIS OF SUBSTITUTED
BENZOPYRANOCOUMARINS VIA DOMINO REACTION
Mazaahir Kidwai,1 Roona Poddar,1 Sarika Diwaniyan,2 and
Ramesh Chander Kuhad2
1Green Research Laboratory, Department of Chemistry, University of Delhi,
Delhi, India
2Lignocellulose Biotechnology Laboratory, Department of Microbiology,
University of Delhi South Campus, Delhi, India
Abstract The present investigation provides a simple and convenient route to organic
synthesis of substituted benzopyranocoumarin, which is important because it is a probable
HIV protease inhibitor. The reaction of a,b-unsaturated derivatives of coumarins with cat-
echol or 1,4-hydroquinones was catalyzed using laccase in an aqueous medium. Quinones,
generated in situ by the oxidation of the corresponding catechol or 1,4-hydroquinones,
underwent a domino reaction with chalcones to produce benzopyranocoumarins.
Keywords Aqueous medium; benzopyranocoumarin; domino reaction; laccase;
reusability
INTRODUCTION
The rapid spread of acquired immunodeficiency syndrome (AIDS) has
stimulated discovery of therapeutic agents to arrest the replication of the causative
virus, human immunodeficiency virus (HIV). One promising possibility to interrupt
the viral life cycle is the use of virus-encoded protease inhibitors, which are indis-
pensable for viral maturation. The substituted coumarins and analogous compounds
have been identified as active nonpeptide HIV protease inhibitors.[1,2] Benzopyranes
Received November 4, 2009.
Address correspondence to Mazaahir Kidwai, Green Research Laboratory, Department of
Chemistry, University of Delhi, Delhi, India. E-mail: kidwai.chemistry@gmail.com
695

696
M. KIDWAI ET AL.
themselves have also been ascribed biologically important skeletons owing to their
intriguing molecular structure[3] and remarkable pharmacological efficiency. Their
fusion with coumarin results in a superior biological moiety, benzopyranocoumarin.
Extensive research efforts have been dedicated to evaluating the new environ-
mentally friendly redox reactions, an important pre-existing field in industrial chem-
istry. In this context, enzyme-catalyzed oxidation reactions are of considerable
current interest.[4–6] Laccase, which belongs to the blue copper oxidase group of
enzymes (benzenediol: oxygen oxidoreductase; EC 1.10.3.2), catalyzes the oxidation
of a broad range of substrates and frequently exhibits high selectivity in aqua-
mediated conditions, which provides a unique solution for a variety of green redox
reactions.[7,8] This blue enzyme for green chemistry finds industrial applications in
various biotechnological processes, including bleaching, detoxification, addition to
washing powders, removal of phenolic browning products from food products, as
well as in treatments of environment pollutants.[9,10] The major advantage attributed
to the use of laccase catalyst is its redox activity, making it ecofriendly and eliminat-
ing water as the sole by-product.[11]
The effective use of biocatalysts is hampered by some peculiar properties of
enzyme proteins, such as nonreusability, high sensitivity to several denaturating
agents, and toxicological effects.[12] Many of these undesirable constraints have been
removed by the use of an aqueous medium. The unique properties of an aqueous
medium, such as high dielectric constant and cohensive energy density, showed an
extraordinary effect on reaction rate. Moreover, its cost-effectiveness, high abun-
dance, non-flammability, and nontoxic nature puts it in the domain of ecofriendly
solvents.[13]
In continuation of our effort to explore chemoenzymatic reactions, it was
thought worthwhile to employ water as a solvent in a biocatalytic reaction.[14] Herein,
we report an efficient and ecofriendly laccase-catalyzed synthesis of benzopyranocou-
marin. The reactions require stoichiometric oxidant in an aqueous medium.
RESULTS AND DISCUSSION
There has been a great deal of interest in the synthesis of benzopyranocoumar-
ins owing to their biological importance and greater therapeutic potentiality as prob-
able HIV protease inhibitors with great therapeutic index.[15] To get more potent
inhibitors, we have successfully attempted a novel and green synthesis of benzopyr-
anocoumarin though fusion of quinones with chalcones (Schemes 1 and 2).
Laccase is a multicopper oxidase that reduces oxygen (source of O2 being a dis-
proponation redox reaction of H2O2 in acidic medium, as handling of H2O2 is much
easier than handling the oxygen gas) to water and simultaneously performs
one-electron oxidation of many aromatic substrates such as phenol and aromatic
amines.[16] Catechol=hydroquinones underwent 1,4-addition reaction with a,b-
unsaturated derivatives of coumarins, leading to nonisolable 6 followed by its oxi-
dation to give 7, which further underwent an intramolecular 1,4-addition reaction
to afford benzopyranocoumarins 3a–h. In other words, the entire domino process
consists of one oxidation and two 1,4-additions[17] (Fig. 1).
Our initial studies were focused on optimization of reaction conditions for the
synthesis of benzopyranocoumarins. The enzymatic synthesis of 3a and 5a from 1a

BENZOPYRANOCOUMARIN SYNTHESIS
697
Scheme 1. Synthesis of benzopyranocoumarina derivatives 3a–h.
were chosen as a model reaction, and the reaction conditions were optimized for
enzymatic synthesis of 3a and 5a from 1a. An experiment was carried out using
1 mmol of 1a and 1.5 mmol of 3a/5a in 15 mL of aqueous medium without the
addition of laccase or H2O2. There was no product formation. The same was also
observed when 0.5 mL of 30% of H2O2 and heat-killed laccase were also added. In
another experiment, when 400 U laccase were added without H2O2, then only traces
of product formation were observed on thin-layer chromatography (TLC). The yield
of 3a/5a was significantly improved with the addition of 0.5 mL of 30% H2O2 along
with 400 U of laccase. An excess of chalcone was required for the reaction; quinones
may undergo competing decomposition, dimerization, or polymerization because of
intrinsic instability. Quinones and a,b-unsaturated derivatives of coumarins were
used in a 1:1.5 ratio to tackle the problem.
The enzymes have been immobilized using several methodologies for their
biocatalytic applicability, affecting their activity, effectiveness of utilization, deacti-
vation, regeneration kinetics, and cost,[18] without considering the toxicity of immo-
bilization reagents and waste disposal problem. In this study, the laccase-catalyzed
synthesis was carried out in an aqueous medium for three consecutive runs and
was monitored by TLC. Table 1 depicts the yield of products benzopyranocoumarin
3a and 5a catalyzed by laccase in three consecutive runs. The yield decreased slightly
after each run.[19] For the model reaction of synthesis of 3a and 5a from 1a, the cata-
lyst could be recovered almost quantitatively and reused several times. In view of the
Scheme 2. R ¼ phenyl, 4-methoxyphenyl, benzo[1,3]dioxol-5-yl, 2-chloroquinolin-3-yl, 2-thienyl, 3-nitro-
phenyl, 4-chlorophenyl, 2H-indol-3-yl.

698
M. KIDWAI ET AL.
Figure 1. Proposed mechanism pathway.
environmentally friendly methodologies, recovery and reuse of the catalyst is highly
preferable (Table 1).
To optimize the reaction condition, different solvents were tried. It was
observed that the reaction in an aqueous buffer proceeded successfully using buffer
tablets of NaH2PO4, Na2HPO4, and potassium phthalate with pH 4, the optimum
pH for best laccase activity. While no reaction was observed in phosphate=citrate
buffer medium with pH 5.5, poor yields were obtained in the case of acetic buffer.
The lower percentage yields in other solvent systems were due to a decrease in laccase
activity in nonintermiscible organic and aqueous phases. Moreover, the domino
reaction was shown to exhibit better reactivity and selectivity in an aqueous medium
than in organic solvent (Table 2).
Futher, the reaction temperature was shown to affect on the productivity. The
reaction was studied at different temperatures varying from 5 ꢀC to 60 ꢀC. The best
Table 1. Benzopyranocoumarin derivatives 3a and 5a obtained in
recycled laccase using the optimized conditionsa
Runs
3a yield (%)b
5a yield (%)b
I (fresh)
68
62
57
59
54
51
II
III
aReaction conditions: 1 mmol 2 or 4 and 1.5 mmol of 1a were taken
in 15 mL of buffer solution, and THF was added to dissolve the reac-
tants; 0.5 mL of 30% H2O2 was added to the resultant solution and
stirred well for 4 h.
bYield refers to the isolated and unoptimized yield.

BENZOPYRANOCOUMARIN SYNTHESIS
699
Table 2. Solvent optimization for the synthesis of benzopyranocoumarinaa
Entry
Solvent
3a yield (%)b
5a yield (%)b
1
2
3
4
5
1 M acetete buffer pH 4
Water
THF
24
—
56
—
68
17
—
52
—
59
1 M phosphate=citric buffer pH 5.5
1 M buffer tablet of pH 4 þ THF
aReaction conditions: 1 mmol 2 or 4 and 1.5 mmol of 1a were taken in 15 mL of
solvents; 400 U of laccase and 0.5 mL of 30% H2O2 were added to the resultant sol-
ution and stirred well for 4 h.
bYield refers to the isolated and unoptimized yield.
results were obtained at room temperature (25 ꢀC). This result was attributed to the
increase in rate of decomposition and polymerization of the in situ–generated qui-
nones when higher temperature was employed, while at lower temperature the
insolubility of a,b-unsaturated derivatives of coumarins was observed and finally
retarded the reaction. To combat the insolubility and unstability, the reactions were
carried out at ambient temperature (Fig. 2).
The structures of the synthesized benzopyranocoumarina derivatives 3a were
confirmed on the basis of their spectral data. The infrared (IR) spectrum of products
3a showed two bands, one at 1650–1700 cmꢁ1 for C O stretching and one at
=
1
3350–3300 cmꢁ1 for O-H stretching. The significant H NMR and mass spectrum
further satisfied the proposed product of benzopyranocoumarins. However, IR,
mass, and especially 1H NMR spectra did not confirm the structure of the final pro-
duct of 3a because there is not much difference in proton of H-7 in I structure and
H-12a in structure II. Although the reaction pathway followed in the scheme resulted
in type I product, there are also a chance of getting II (Fig. 2), thus 13C NMR was
found to be informative. 13C NMR confirms the sole product (I) obtained in our pro-
tocol. This can be explained simply. Had C-12a bonded with oxygen on one side, this
would have moved its signal upfield by 70–80 ppm, while the signal of C-6 carbon
atom is downfield because of a double bond (i.e., 145–155 ppm) (structure II). Our
spectra do not consist of such types of signals. Although the spectra show the signal
of C-12a at 166.44 and C-6 signals at 36.15 ppm, that entirely diminishes the possi-
bility of structure II. The purity of compounds was determined by CHN data.
Figure 2. Probable structure of benzopyranocoumarina derivatives 3a.

700
M. KIDWAI ET AL.
Table 3. Reaction time and yield for the laccase-catalyzed synthesis of benzopyranocoumarin derivatives
3a–h and 5a–ha
3a–h
Yield (%)b
5a–h
Yield (%)b
S. no.
Entry
R
Time (h)
Time (h)
1
2
3
4
5
6
7
8
a
b
c
Phenyl
68
63
65
55
25
43
52
64
4
45
26
57
24
48
9.5
65
58
4.5
6.5
5
4-Methoxyphenyl
Benzo[1,3]dioxol-5-yl
Thiophen-2-yl
4.5
5.5
3.5
7.5
6
d
e
4.5
8.5
6
3-Nitophenyl
f
4-Chlorophenyl
2H-Indol-3-yl
2-Chloroquinolin-3-yl
g
h
4
4.5
5
4
aReaction conditions: 1 mmol 2 or 4 and 1.5 mmol of 1a were taken in 15 mL of solvents; 400 U of lac-
case and 0.5 mL of 30% H2O2 were added to the resultant solution and stirred well for 4 h.
bYield refers to the isolated and unoptimized yield.
After optimizing the reaction conditions and confirming product, the reaction
was accomplished with diverse a,b-unsaturated derivatives of coumarins. The
choices of aldehyde taken was based on biological activity. For example, in chloro-
benzaldehyde and 2-chloroquinolin-3-yl aldehyde, the chloro group enhances the
biological activity, whereas hetero aromatic aldehydes such as thiophenecarboxal-
dehde, benzo[1,3]dioxol-5-aldehyde, and 2H-indol-3-carboxaldehyde are employed
for superior activity. Nitro- and methoxy groups also possess activity, so these men-
tioned aldehydes were taken to carry out this synthesis.[20–22] The reactivity of the
a,b-unsaturated derivatives of coumarins with the in situ–generated quinone is
shown in Table 3.
CONCLUSION
The laccase–aqueous system was explored for the domino reaction. This syn-
thesis is tunable for catalyst and solvent to integrate the reaction and facilitate the
reduction of waste, with use of more benign solvents. Hence, in addition to the sim-
ple reaction conditions, easy workup procedure and recyclability of catalyst make
our methodology a valid contribution to the existing processes in the field of benzo-
pyranocoumarin synthesis. The other benfit of this protocol is the use of environ-
mentally benign biocatalyst and reaction medium.
EXPERIMENTAL
1H NMR and 13C NMR spectra were recorded on a Burker Top Spin
300-MHz and 75.6-MHz spectrometer with chemical shift values (d) in parts per
million (ppm) downfield from tetramethylsilane (TMS) using dimethylsulfoxide
(DMSO) as solvent. IR spectra of each sample were recorded on a model
Perkin-Elmer Fourier transform FT–IR 1710 spectrometer using KBr. Elemental
analyses were performed using a Heraeus CHN-Rapid Analyzer. Electrospray ioni-
zation (EI) mass spectra were recorded on a turnover frequency (TOF) MS mass

BENZOPYRANOCOUMARIN SYNTHESIS
701
spectrometer. The purity of compounds was checked on silica-gel-coated aluminum
plates (Merck TLC: mass particle size 10–12 mm; particle distribution 5–20 mm; layer
thickness 250 mm; plate height 30 mm). The chalcones were prepared according to the
literature procedure.[23] Guaiacol was purchased from Hi Media Laboratories Pvt.
Ltd. (Mumbai, India). All media components and chemicals used were of analytical
grade.
Microorganism
The Basidiomycetous fungus, RCK-1, isolated from deteriorated sugarcane
bagasse collected from Sugar Mill Industry, Sonipat, Haryana, India, was grown
and maintained on malt extract agar (MEA) containing (gLꢁ1) malt extract
20.0, KH2PO4 0.5, MgSO4 ꢂ 7H2O 0.5, Ca(NO3)2 ꢂ 4H2O 0.5, and agar 20.0 (pH
7.0) at 30 ꢀC.[24,25] Pure cultures were stored at 4 ꢀC and subcultured every
fortnight.
Laccase Production
Static cultivation was carried out at 32 ꢀC in 250-ml Erlenmeyer flasks with
50 ml malt extract broth (MEB) containing (gLꢁ1) malt extract 20.0, KH2PO4 0.5,
MgSO4 ꢂ 7H2O 0.5, and Ca(NO3)2 ꢂ 4H2O 0.5 (pH 7.0). The flasks were inoculated
with two fungal discs (8 mm in diameter each) from the periphery of 4-day-old fungal
culture and incubated at 30 ꢀC. After 96 h of growth, 0.5 mL copper sulfate (1 mM)
was added to MEB. The cultures were harvested at regular intervals by filtering
through Whatman No. 1 filter paper after 264 h of growth. The culture filtrate
was centrifuged at 12,000 g for 20 min at 4 ꢀC, and the supernatant obtained was
assayed for laccase activity.
Purification of Laccase
The culture filtrate (900 mL) containing laccase enzyme was precipitated by
addition of ammonium sulfate (80% cutoff) and centrifuged at 10,000 g for 20 min
at 4 ꢀC. The precipitate was resuspended in 50 mM citrate phosphate buffer (pH
5.5) and dialyzed at 4 ꢀC against the same buffer. This partially purified enzyme
(78 mL) was concentrated to 20 ml using a 10-kDa filter membrane (Vivaspin,
Vivascience, Sartorius Group, Stone house, UK) at 4 ꢀC applied to an anion-
exchange DEAE-sepharose (Amersham Pharmacia Biotech, Freiburg, Germany)
column (25 ꢃ 2 cm2), equilibrated with 10 mM citrate phosphate buffer (pH 7.0).
The proteins were eluted by NaCl gradient (0–1 M in equilibrating buffer) at a flow
rate of 0.5 mL minꢁ1 with 1 mL fraction size. Fractions with laccase activity were
pooled, desalted, and assayed for laccase activity.
Enzyme Assay
Guaiacol was used as a substrate for assaying laccase activity.[17] One unit (U)
of laccase was defined as the change in absorbance of 0.01 mlꢁ1 minꢁ1 at 470 nm.

702
M. KIDWAI ET AL.
General Procedure for Synthesis of 9,10-Dihydroxy
Benzopyranocoumarin and 8,11-Dihydroxy Benzopyranocoumarin
Catechol or hydroquinone (1 mmol, 100 mg) and 1.5 mmol of chalcone were
taken in 15 mL of buffer solution, and THF was added to dissolve the reactants.
Then purified laccase (400 U) was added to the resultant solution and stirred well,
0.5 mL of 30% H2O2 was added to oxygenate the reaction mixture. The progress
of the reaction was checked by TLC examination every 30 min. Upon completion
of the reaction, the reaction mixture was extracted with ethyl acetate (3 ꢃ 15). The
organic layer was dried over Na2SO4. The product was purified by using column
chromatography with a mobile phase of 80:20 hexane=ethylacetate and recrystallized
with ethanol. The aqueous phase was reused without any treatment.
Selected Data
8,9-Dihydroxy-7-phenyl-7H-benzopyranocoumarin (3a). HRMS: Mþ
358.2322. Anal. calculated for C22H14O5:C, 73.74, and H, 3.94%. Found:C, 73.85,
and H, 4.02%. IR (t, cmꢁ1, KBr pellet) 3322.44 (OH), 1683.12 (C O). dH
=
(DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 6.69 (dd, 11 J, 4H, Ar), 7.25 (dt, 8 J, 4H,
Ar), 7.61 (d, 9 J, 2H, Ar), 7.89 (d, 5 J, 1H, Ar), 8.90 (s, 2H, OH). dC (DMSO-d6,
75 MHz) 36.38, 103.97, 115.79, 115.87, 118.97, 119.41, 123.55, 124.11, 125.41,
126.17, 126.78, 128.04, 130.84, 131.64, 132.93, 140.84, 145.36, 152.43, 165.02,
166.44, 168.83.
8,9-Dihydroxy-7-(4-methoxy-enyl)-7H-benzopyranocoumarin
(3b).
HRMS: Mþ 388.0881. Anal. calculated for C23H16O6:C, 71.13, and H, 4.15%.
Found: C, 71.56, and H, 4.28%. IR (t, cmꢁ1, KBr pellet) 3326.24 (OH), 1679.44
=
(C O). dH (DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 3.71 (s, 3H, CH3), 6.48 (dd, 6
J, 4H, Ar), 7.43 (dd, 6 J, 4H, Ar), 8.20 (dd, 2H, Ar), 8.60 (s, 2H, OH). dC (DMSO-d6,
75 MHz) 35.32, 56.63, 102.12, 114.67, 115.76, 119.17, 120.19, 123.55, 124.11,
125.41, 126.17, 126.78, 128.04, 130.84, 131.64, 132.93, 141.08, 145.83, 151.24,
165.46, 167.79, 170.68.
8,9-Dihydroxy-7-(benzo[1,3]dioxol-phenyl)-7H-benzopyranocoumarin
(3c). HRMS: Mþ 402.5658. Anal. calculated for C23H14O7:C, 68.66, and H, 3.51%.
Found:C, 68.35, and H, 3.62%. IR (t, cmꢁ1, KBr pellet) 3312.83 (OH), 1675.84
=
(C O). dH (DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 5.96 (s, 2H, CH2) 6.76 (m,
4H, Ar), 7.66 (dd, 6 J, 4H, Ar), 8.25 (d, 8 J, 1H, Ar), 9.02 (s, 2H, OH). dC
(DMSO-d6, 75 MHz) 34.63, 91.32, 104.35.
8,9-Dihydroxy-7-thiophen-7H-benzopyranocoumarin (3d). HRMS: Mþ
364.5324. Anal. calculated for C20H12O5S:C, 65.93; H, 3.32; S, 8.80%. Found:C,
65.54; H, 3.62; S, 8.59%. IR (t, cmꢁ1, KBr pellet) 3345.18 (OH), 1675.53 (C O).
=
dH (DMSO-d6, 300 MHz) 2.35 (s, 1H, H-7), 6.37–6.85 (m, 3H, Ar), 7.23–7.56 (dd,
8 J, 4H, Ar), 8.02 (d, 11 J, 2H, Ar), 9.02 (s, 2H, OH).
8,9-Dihydroxy-7-(4-nitro-phenyl)-7H-benzopyranocoumarin (3e). HRMS:
Mþ 403.1564 Anal. calculated for C22H13NO7:C, 65.51; H, 3.25; N, 3.47%. Found:C,
65.82; H, 3.38; N, 3.61%. IR (t, cmꢁ1, KBr pellet) 3286.23 (OH), 1694.82 (C O). dH
=

BENZOPYRANOCOUMARIN SYNTHESIS
703
(DMSO-d6, 300 MHz) 2.32 (s, 1H, H-7), 6. 61 (dd, 2H, Ar), 7.12 (m, 4H, Ar), 8.09 (t,
7 J, 2H, Ar), 8.45 (dd, 11 J, 2H, Ar), 8.90 (s, 2H, OH).
8,9-Dihydroxy-7-(4-chloro-phenyl)-7H-benzopyranocoumarin (3f). HRMS:
Mþ 392.2354. Anal. calculated for C22H13ClO5:C, 67.72; H, 3.34; Cl, 9.03%.
Found:C, 67.68; H, 3.56; Cl, 9.21%. IR (t, cmꢁ1, KBr pellet) 3269.89 (OH),
=
1698.84 (C O). dH (DMSO-d6, 300 MHz) 2.63 (s, 1H, H-7), 6.58 (dd, 9 J, 3H,
Ar), 7.35–7.45 (m, 4H, Ar), 8.20 (d, 12 J, 3H, Ar), 9.17 (s, 2H, OH).
8,9-Dihydroxy-7-(2H-indol-3-yl)-7H-benzopyranocoumarin (3g). HRMS:
Mþ 427.1358. Anal. calculated for C26H21NO5:C, 73.06; H, 4.95; N, 3.28%.
Found:C, 73.18; H, 4.82, N, 3.48%. IR (t, cmꢁ1, KBr pellet) 3298.36 (OH),
=
1668.38 (C O). dH (DMSO-d6, 300 MHz) 2.70 (s, 1H, H-7), 6.38–6.85 (dd, 4H,
Ar), 7.01–7.17 (m, 4H, Ar), 7.35–7.45 (dd, 5H, Ar), 8.37 (d, 2H, Ar), 9.02 (d, 2H,
Ar), 930 (s, 2H, OH), 10.02 (s, 1H, NH).
8,9-Dihydroxy-7-(2-chloroquinolin-3-yl)-7H-benzopyranocoumarin (3h).
HRMS: Mþ 473.1558. Anal. calculated for C27H20ClNO5:C, 68.43; H, 4.25; Cl,
7.48; N, 2.96%. Found:C, 68.55; H, 4.36; Cl, 7.61; N, 2.66%. IR (t, cmꢁ1, KBr pellet)
=
3310.94 (OH), 1682.66 (C O). dH (DMSO-d6, 300 MHz) 2.62 (s, 1H, H-7), 6.76 (dd,
10 J, 4H, Ar), 7.05 (d, 2H, Ar), 7.23–7.45 (m,5H, Ar), 7.85–8.31 (dd, 4 h, Ar), 9.20 (d,
2H, Ar), 9.97 (s, 2H, OH).
8,11-Dihydroxy-7-phenyl-7H-benzopyranocoumarin (5a). HRMS: Mþ
358.0846. Anal. calculated for C22H14O5:C, 73.74, and H, 3.94%. Found:C, 73.68,
and H, 4.62%. IR (t, cmꢁ1, KBr pellet) 3326.93 (OH), 1679.84 (C O). dH
=
(DMSO-d6, 300 MHz) 2.56 (s, 1H, H-7), 6.38 (d, 7 J, 3H, Ar), 7.14 (dd, 6 J, 4H,
Ar), 7.56 (m, 4H, Ar), 8.20, 8.95 (s, 2H, OH). dC (DMSO-d6, 75 MHz) 37.23,
105.74, 114.97, 115.78, 119.87, 120.21, 123.22, 123.67, 125.24, 126.45, 127.81,
128.38, 130.34, 131.22, 133.89, 141.24, 145.45, 153.56, 163.50, 166.56, 170.42.
8,11-Dihydroxy-7-(4-methoxy-phenyl)-7H-benzopyranocoumarin (5b).
HRMS: Mþ 388.1532. Anal. calculated for C23H16O6:C, 71.13, and H, 4.15%.
Found:C, 71.56, and H, 4.28%. IR (t, cmꢁ1, KBr pellet) 3326.83 (OH), 1675.62
=
(C O). dH (DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 3.71 (s, 3H, CH3), 6.18 (d, 8
J, 3 H, Ar), 6.89 (t, 4 J, 2H, Ar), 7.34 (m, 3H, Ar), 8.20 (dd, 6 J, 3H, Ar), 8.63 (s,
2H, OH). dC (DMSO-d6, 75 MHz) 35.89, 56.63, 105.79.
8,11-Dihydroxy-7-(benzo[1,3]dioxol-phenyl)-7H-benzopyranocoumarin
(5c). HRMS: Mþ 402.2341. Anal. calculated for C23H14O7:C, 68.66, and H, 3.51%.
Found:C, 68.35, and H, 3.62%. IR (t, cmꢁ1, KBr pellet) 3301.98 (OH), 1676.32
=
(C O). dH (DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 5.96 (s, 2H, CH2), 6.27 (dd, 7
J, 3H, Ar), 6.98 (t, 4 J, 3H, Ar), 8.25 (m, 3H, Ar), 8.93 (s, 2H, OH). dC (DMSO-d6,
75 MHz) 34.35, 90.65, 102.95, 114.97, 115.66, 119.34, 120.32, 123.43, 124.02, 125.14,
126.45, 126.87, 128.12, 130.45, 131.87, 133.78, 141.45, 146.89, 153.23, 166.89, 170.08,
171.56.
8,11-Dihydroxy-7-thiophen-7H-benzopyranocoumarin (5d). HRMS: Mþ
364.0405. Anal. calculated for C20H12O5S:C, 65.93; H, 3.32; S, 8.80%. Found:C,
65.34; H, 3.62; S, 8.96%. IR (t, cmꢁ1, KBr pellet) 3326.93 (OH), 1673.94 (C O).
=

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dH (DMSO-d6, 300 MHz) 2.63 (s, 1H, H-7), 6.12 (dd, 3 J, 3H, Ar), 6.98 (t, 3 J, 2H,
Ar), 8.20 (m, 3H, Ar), 9.05 (s, 2H, OH).
8,11-Dihydroxy-7-(4-nitro-phenyl)-7H-benzopyranocoumarin (5e). HRMS:
Mþ 403.2624. Anal. calculated for C22H13NO7:C, 65.51; H, 3.25; N, 3.47%.
Found:C, 65.34; H, 3.60; N, 3.18%. IR (t, cmꢁ1, KBr pellet) 3326.93 (OH),
=
1679.84 (C O). dH (DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 6.38 (t, 3 J, 2H, Ar),
7.04 (d, 7 J, 3H, Ar), 7.67 (m, 4H, Ar), 8.24 (d, 10 J, 1H, Ar), 8.90 (s, 2H. OH).
8,11-Dihydroxy-7-(4-chloro-phenyl)-7H-benzopyranocoumarin
(5f).
HRMS: Mþ 392.0265. Anal. calculated for C22H13ClO5:C, 67.72; H, 3.34; Cl,
9.03%. Found:C, 67.89; H, 3.18; Cl, 8.85%. IR (t, cmꢁ1, KBr pellet) 3307.63
=
(OH), 1679.84 (C O). dH (DMSO-d6, 300 MHz) 2.51 (s, 1H, H-7), 6.25 (dd, 3 J,
3H, Ar), 7.05 (t, 5 J, 2H, Ar), 7.57 (m, 4H, Ar), 8.58 (t, 6 J, 1H, Ar), 8.78 (s, 2H,
OH).
8,11-Dihydroxy-7-(2H-indol-3-yl)-7H-benzopyranocoumarin (5g). HRMS:
Mþ 427.2131. Anal. calculated for C26H21NO5:C, 73.06; H, 4.95; N, 3.28%.
Found:C, 72.89; H, 4.86; N, 3.11%. IR (t, cmꢁ1, KBr pellet) 3315.37 (OH),
=
1670.55 (C O). dH (DMSO-d6, 300 MHz) 2.75 (s, 1H, H-7), 6.46 (d, 9 J, 2H, Ar),
7.34 (t, 4 J, 3H, Ar), 7.38 (dd, 3 J, 8H, Ar), 8.94 (m, 5H, Ar), 9.20 (s, 2H, OH),
10.13 (s, 1H, NH).
8,11-Dihydroxy-7-(2-chloroquinolin-3-yl)-7H-benzopyranocoumarin (5h).
HRMS: Mþ 473.1558. Anal. calculated for C27H20ClNO5:C, 68.43; H, 4.25; Cl, 7.48;
N, 2.96%. Found:C, 68.33; H, 4.12; Cl, 7.33; N, 2.87%. IR (t, cmꢁ1, KBr pellet)
=
3313.0112 (OH), 1683.546 (C O). dH (DMSO-d6, 300 MHz) 2.5 (s, 1H, H-7), 6.65
(t, 3 J, 4H, Ar), 7.15 (dd, 2 J, 5H, Ar), 7.85 (m, 4H, Ar), 8.68 (m, 4H, Ar), 9.85
(s, 2H, OH).
ACKNOWLEDGMENTS
M. Kidwai and R. Poddar are thankful to the Council of Scientific and Industrial
Research, New Delhi, for providing financial assistance. S. Diwaniyan and R.C. Kuhad
acknowledge financial support from the Department of Biotechnology.
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