Conventional and microwave-assisted syntheses and solid-state structure of new coumarins and angelicins

Article abstract of DOI:10.1080/00397911.2010.502998

New derivatives of coumarin and angelicin, 8-acetyl-7-cyanomethoxy-4- methyl-chromen-2-one (1), 8-acetyl-7-ethoxycarbonylmethoxy-4-methyl-chromen-2- one (2), 8-cyano-4,9-dimethyl-2H-furo[2,3-h]-1-chromen-2-one (3), and 8-ethoxycarbonyl-4,9-dimethyl-(2H-furo[2,3-h]-1-chromen-2-one) (4) were obtained by conventional synthesis and by efficient and high-yielding microwave-assisted synthesis. The solid structures were analyzed using 13C cross polarization/magic angle spinning (CP/MAS) NMR spectroscopy. Compounds 1 and 3 were evaluated for potential anticancer activity in an in vitro screening panel of 60 human tumor cell lines. Selected leukemia, non-small-cell lung, and renal cancer lines showed promising sensitivity to these compounds. Additionally, compound 4 was shown to inhibit growth of Gram-positive strains.

Full text of DOI:10.1080/00397911.2010.502998

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Conventional and Microwave-Assisted
Syntheses and Solid-State Structure of
New Coumarins and Angelicins
Elżbieta Hejchman a , Joanna Trykowska Konc a , Dorota
Maciejewska a & Hanna Kruszewska b
a Department of Organic Chemistry, Faculty of Pharmacy, Medical
University of Warsaw, Warsaw, Poland
b Department of Antibiotics and Microbiology, National Medicines
Institute, Warsaw, Poland
Published online: 14 Jun 2011.
To cite this article: Elżbieta Hejchman , Joanna Trykowska Konc , Dorota Maciejewska &
Hanna Kruszewska (2011) Conventional and Microwave-Assisted Syntheses and Solid-State
Structure of New Coumarins and Angelicins, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 41:16, 2392-2402, DOI:
10.1080/00397911.2010.502998
To link to this article: http://dx.doi.org/10.1080/00397911.2010.502998
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Synthetic Communications1, 41: 2392–2402, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.502998
CONVENTIONAL AND MICROWAVE-ASSISTED
SYNTHESES AND SOLID-STATE STRUCTURE
OF NEW COUMARINS AND ANGELICINS
Elz˙bieta Hejchman,1 Joanna Trykowska Konc,1
Dorota Maciejewska,1 and Hanna Kruszewska2
1Department of Organic Chemistry, Faculty of Pharmacy,
Medical University of Warsaw, Warsaw, Poland
2Department of Antibiotics and Microbiology, National Medicines Institute,
Warsaw, Poland
GRAPHICAL ABSTRACT
Abstract New derivatives of coumarin and angelicin, 8-acetyl-7-cyanomethoxy-4-methyl-
chromen-2-one (1), 8-acetyl-7-ethoxycarbonylmethoxy-4-methyl-chromen-2-one (2),
8-cyano-4,9-dimethyl-2H-furo[2,3-h]-1-chromen-2-one (3), and 8-ethoxycarbonyl-4,9-
dimethyl-(2H-furo[2,3-h]-1-chromen-2-one) (4) were obtained by conventional synthesis
and by efficient and high-yielding microwave-assisted synthesis. The solid structures were
analyzed using 13C cross polarization=magic angle spinning (CP=MAS) NMR spec-
troscopy. Compounds 1 and 3 were evaluated for potential anticancer activity in an
in vitro screening panel of 60 human tumor cell lines. Selected leukemia, non-small-cell
lung, and renal cancer lines showed promising sensitivity to these compounds. Additionally,
compound 4 was shown to inhibit growth of Gram-positive strains.
Keywords Angelicin; coumarin; microwave-assisted synthesis
INTRODUCTION
Coumarin (2H-chromen-2-one) and its derivatives have been targets of our
research because of their diverse biological activities.[1,2] Presently, we are interested
in the synthesis of new, potentially bioactive angular furocoumarins, derivatives of
angelicin [2H-furo(2,3-h)-1-chromen-2-one]. Antifungal activity of angelicin and
its 8-substituted derivatives against Candida albicans, Cryptococcus neoformans,
Received April 21, 2010.
Address correspondence to Dorota Maciejewska, Department of Organic Chemistry, Faculty
of Pharmacy, Medical University of Warsaw, 1 Banacha Str., 02 097 Warsaw, Poland. E-mail:
dmaciejewska@wum.edu.pl
2392

SYNTHESES OF COUMARINS AND ANGELICINS
2393
Saccharomyces cerevisiae, and Aspergillus niger was reported by Sardari et al.[3,4]
Antiproliferative activity of 4-methylangelicin by inhibition of DNA synthesis on
Ehrlich ascites tumor cells was shown.[5,6] The in vitro antiproliferative activities
of some methyl derivatives of 5-methoxyangelicin on HL60 and A431 cell lines of
human cancers were evaluated using 5-methoxypsoralen as the reference compound.
4,9-Dimethyl-5-methoxyangelicin and 3,9-dimethyl-5-methoxyangelicin have better
activity than 5-methoxypsolaren. When the cytotoxicity data were coupled with
the in vivo phototoxicity tests, 4,9-dimethyl-5-methoxyangelicin emerged as a prom-
ising candidate for use in clinical trials because of its high inhibitory effect on the
growth of human cell lines and low skin phototoxicity.[7]
The most traditional and widely used method for the synthesis of angular fur-
ocoumarins is the introduction of the furan ring to the basic coumarin structure.[8]
To prepare derivatives of angelicin with electron-attracting substituents in position
8 of the furan ring, base-catalyzed condensations of acetyl hydroxyheteroarenes with
a-halocarbonyl compounds were applied. 4,9-Dimethylangelicin-8-carboxylic acid
and its ethyl ester were prepared using 8-acetyl-7-hydroxy-4-methyl-chromen-2-one
and diethyl bromomalonate.[9,10] Microwave-induced Claisen rearrangement was
also used for the synthesis of 4,8,9,-substituted angelicins.[11] The microwave-assisted
synthesis has many advantages over traditional methods, which are operational sim-
plicity, good yields, short reaction times, and easy workup procedures. This type of
synthesis was used in our previous studies on coumarins[1,2] as well as in the present
study.
The 13C cross polarization=magic angle spinning (CP=MAS) NMR spectroscopy
in the solid state is used to the investigation of structures of those compounds for which
we could not obtain the crystals suitable for x-ray diffraction (XRD) examinations. Its
application to the studies of biologically active substances is of special interest.[12,13]
Here, we report a new synthetic route for two novel coumarins 1 and 2,
8-cyanoangelicin 3, and previously obtained 8-ethoxycarbonylangelicin 4 (see
Scheme 1) by conventional and microwave-assisted syntheses. Additionally, we
present the application of combined 13C CP=MAS NMR spectroscopy and
Scheme 1. Reagents and conditions used for the preparation of 1, 2, 3, and 4 with atom numbering.

2394
E. HEJCHMAN ET AL.
molecular modeling approach to the structural studies of compounds 1–4. The anti-
cancer and antibacterial activities of compounds 1, 3, and 4 are also discussed.
RESULTS AND DISCUSSION
Chemistry
The O-alkylation reaction of 8-acetyl-4-methyl-7-hydroxycoumarin with the
alkylating agents, chloroacetonitrile, and ethyl chloroacetate under reflux in acetone,
using anhydrous potassium carbonate as a base, was performed, resulting in two
novel O-substituted coumarins, 1 and 2. Heating the 1-methyl-2-pyrrolidone solu-
tions of 8-acetyl-4-methyl-7-hydroxycoumarin with the alkylating agents, chloroace-
tonitrile and ethyl chloroacetate, in the presence of anhydrous potassium carbonate
at the temperature 70–75 ꢀC yielded two products, the novel 8-cyanoangelicin 3 as
well as 8-ethoxycarbonylangelicin 4. Reflux was applied for 11.5 h and for 28.5 h
to obtain the compounds 1 and 2, respectively. Similarly, heating for 3 h and 6.5 h
at 70–75 ꢀC was required to obtain compounds 3 and 4. Because of the prolonged
heating used, the formation of the desired products is accompanied by the decompo-
sition of the starting materials. To speed up these reactions and avoid the undesired
side products, microwave irradiation was used.
The reaction of 8-acetyl-4-methyl-7-hydroxycoumarin with chloroacetonitrile
and ethyl chloroacetate under microwave irradiation afforded the products within
a few minutes. However, the kind of products and their yields were dependent on
the conditions applied. For example, the reflux in acetone resulted in the compounds
1 (43%) and 2 (20%); only traces of the cyclization products 3 and 4 were detected
by thin-layer chromatography (TLC) in spite of the prolonged heating. Using
1-methyl-2-pyrrolidone as a solvent led exclusively to the preparation of the deriva-
tives of angelicin 3 and 4. The major achievement of this procedure is the consider-
able reduction of reaction times: from 3 h 45 min to 3 min for compound 3 and from
6.5 h to 12 min for compound 4. The yields of the products obtained with microwave
heating are greater or comparable with the yields of syntheses carried out in the con-
ventional way. It made the microwave-assisted synthesis our method of choice in this
case. The preparation of O-alkylated coumarins 1 and 2 versus furocoumarins 3 and
4 may be controlled by the choice of the reaction conditions. The formation of fur-
ocoumarins is favored through greater temperature, higher polarity, and basicity of
1-methyl-2-pyrrolidone as well as better solubility of potassium carbonate, resulting
in greater concentration of the base in the reaction medium.
Solid-State Structural Analysis by 13C CP/MAS NMR Spectra
The utilization of combined 13C CP=MAS NMR and computational analysis
to the structures of newly synthesized compounds 1–4 allowed us to propose their
probable stable conformations in the solid state. Exemplary 13C CP=MAS NMR
spectra of 1 and 2 are presented in Fig. 1, and the most probable assignments of reso-
nances in the spectra of 1, 2 and 3, 4 are given in Table 1 (the notation used in the
discussion of the NMR results is given in Scheme 1). Since NMR spectra in the solid
state show local conformational arrangement in opposition to NMR spectra in

SYNTHESES OF COUMARINS AND ANGELICINS
2395
Figure 1. Exemplary 13C CP=MAS NMR spectra of 1, showing simple pattern of resonances, and of 2,
showing double pattern of resonances. Sidebands are marked with an asterisk.
solution, which detect average conformation of molecules, it was necessary to engage
an additional tool for structural analysis: the computation of theoretical shielding
constants for 13C atoms. The density functional theory (DFT) method was used to
examine the geometries and NMR resonances. For the structures corresponding
to local energy minima, we have analyzed the correlation coefficients R2 obtained
for the linear correlations between the experimental chemical shifts d (ppm) and
the theoretical shielding constants r (ppm). We have chosen as the most probable
solid-state structures those for which the greatest values of R2 (0.987–0.996) were
obtained. Examination of the spectra of 1 and 3, 4 reveals the same number of peaks
as the number of chemically distinct carbon sites in these molecules, but in the
spectrum of 2 one can observe a significantly greater number of peaks. Those
multiplicities and intensities of resonances are consistent with an interpretation

2396
E. HEJCHMAN ET AL.
Table 1. Chemical shifts of 13C NMR in solid state d (ppm) for 1, 2 and 3, 4
No.
1
2
3
4
C2
C3
160.3
111.6
154.3
114.3
129.9
108.5
156.8
118.0
149.7
199.9
32.9
158.9
110.0
158.6
112.6
153.3
112.6
123.5
109.6
156.3
126.5
—
158.2
113.6
154.8
115.4
124.8
109.6
156.2
141.3
—
C4
154.2
113.4
132.7
C4a
C5
C6
C7
110.0;109.0
156.2
115.6
C8
C8a
C9
149.8
198.3
130.3
114.1
147.7
19.9
124.8
116.8
150.1
19.6
C9a
C9b
C11
C12
C13
C14
C15
32.7;32.2
—
19.3
—
20.1
55.8
65.4;65.3
168.9;168.4
60.3;59.7
14.8;14.4
112.6
10.9
—
160.0
11.4
63.4
116.8
—
—
—
13.9
demanding the presence of a single molecule in the crystallographic asymmetric unit
of compounds 1 and 3, 4 in contrast to compound 2, for which two distinct
molecules are expected. (Double resonances were previously observed for another
O-alkyl-4-methylcoumarin,[2] and XRD measurement revealed two molecules in
the crystallographic asymmetric unit.) The twofold multiplicity of resonances is
particularly apparent in the regions of the spectrum of 2, consisting of peaks due
to the ester and acetyl groups. The small differences between two structures of 2
lie in the orientation of the ester side chain with respect to the fused bicyclic system.
The energies of analyzed conformations are close to each other, and the torsion
angles C8–C7–O–C12 should be very similar for both conformers. The resonances
representing both structures in the spectrum of 2 are located very closely (the values
are within the range of 1 ppm), and we could not deduce structures of both confor-
mers. We decided to show only one conformation with the torsion angle C8–C7–O–
C12 equal to ꢁ165ꢀ, corresponding to the trans ester bond for which the greatest R2
value was obtained (see the hypothetical structure shown on the left-hand side of
Fig. 1). To establish the correct orientations of cyanomethylene in 1 and acetyl sub-
stituents in 1 and 2, we have additionally compared the signals of C6, C8, and C9
atoms in 13C CP=MAS NMR spectra of 1 and 2 with those in solution. This com-
parison reveals a low-frequency shift of C6 resonances in the spectrum of 1 in solid
state, which is in agreement with conformations in which the methylene groups
CH2(12) are directed at C6 atom. An arrangement of methylene group in 1 resembles
those proposed for CH2(12) in 2. Such location of methylene groups force the acetyl
group to deviate from the benzene ring plane. The acetyl group at C8 is principally
perpendicular to the bicyclic plane in 1 and 2 (see the structures in Fig. 1). It is likely
that this orientation is connected with the low-frequency shift of C8 resonance in
solid state. Small high-frequency shift of C2 and C9 atoms in the spectra of 1 and
2 corresponds to the weak hydrogen bonding in which these groups are involved.

SYNTHESES OF COUMARINS AND ANGELICINS
2397
Table 2. Activity of compound 4 against Gram-positive bacterial strains
Strain
MIC (mg=mL)
Micrococcus luteus ATCC 9341
Micrococcus luteus ATCC 10240
Bacillus subtilis ATCC 6633
10
10
7,5
2,5
10
10
10
Bacillus cereus ATCC 11778
Staphylococcus epidermidis ATCC 12228
Staphylococcus aureus ATCC 6538
Staphylococcus aureus ATCC 6538P
Those were not observed for C2 resonances in the spectra of furocoumarins 3 and 4
and could be the evidence that C2¼O groups in these compounds are probably not
engaged in hydrogen bonding. The conformations of 3 and 4, in which the orienta-
tions of all substituents are in plane of the tricyclic fused ring system, are proposed
according to the values of the relevant shielding constants obtained for the
low-energy structures and the greatest values of the correlation coefficient R2.
Biological Activity Assays
Compounds 1 and 3 were accepted for evaluation by the Division of Cancer
Treatment and Diagnosis National Cancer Institute (Bethesda, USA) in the full
panel of 60 different cell lines, representing human leukemia, non-small-cell lung
cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian
cancer, renal cancer, prostate cancer, and breast cancer cell lines. The details are
available at the web site http://dtp.nci.nih.gov. The inhibition of growth was mea-
sured at a single dose of the tested compounds, at the concentration of 10ꢁ5 M.
Compound 1 inhibited the growth of leukemia HL-60(TB) (growth 81.57%),
K-562 (68.99), RPMI-8226 (80.00), non-small-cell lung cancer NCI-H522 (77.26),
and prostate cancer PC-3 (87.23). Compound 3 appeared to be more active. It
inhibited the growth of leukemia CCRF-CEM cell lines (growth 6.77%), and, to
a lesser extent, non-small-cell lung cancer HOP-92 (81.97) and renal cancer A498
(75.10).
The angelicin derivative 4 was screened for activity on 14 microbial strains.
Compound 4 inhibited growth of only Gram-positive strains (Table 2). It inhibited
growth of Bacillus subtilis in the minimal concentration of 2.5 mg=mL (minimal
inhibitory concentration, MIC). The lack of good water solubility of this compound
may prevent it from having better antimicrobial activity.
EXPERIMENTAL
Instruments and Methods
Melting points were determined with a digital melting-point apparatus 9001
and are uncorrected. Microwave oven Plazmatronika (1000 W), equipped with a
single-mode cavity suitable for the microscale synthesis and microwave-choked
outlet connected to external condenser set to 30% power, was used (http://www.

2398
E. HEJCHMAN ET AL.
1
plazmatronika.com.pl). H NMR, 13C NMR, HSQC, and heteronuclear multiple-
bond correlation (HMBC) spectra in solution were recorded at 25 ꢀC with Varian
NMRS-300 or Varian 500 spectrometers. The calculated shielding constants were
used as an aid in an assignment of resonances of 13C atoms. The solid state 13C
CP=MAS NMR spectra were measured using a Bruker Avance DMX 400. The pow-
dered samples were spun at 8 kHz. Contact time of 4 ms, repetition time of 16 s, and
spectral width of 24 kHz were used for the total of 1,000 scans. The notation used in
the detailed description of NMR resonances is given in Scheme 1. Starting geome-
tries of compounds 1–4 were optimized at PM3 semi-empirical level of theory.[14]
Final geometries were obtained using DFT method with B3LYP=6-31ꢂꢂ hybrid func-
tional, and the coupled perturbed Hartree–Fock–gauge including atomic orbitals
(CPHF-GIAO) approach was employed for shielding constants calculations as
implemented in Gaussian 03 package.[15] Infrared (IR) spectra were recorded on a
Fourier transform (FT)–IR Perkin-Elmer instrument. High-resolution mass spectra
(HRMS) were recorded on Quattro LCT (TOF).
Conventional Syntheses
8-Acetyl-7-cyanomethoxy-4-methyl-chromen-2-one
(1). 8-Acetyl-7-
hydroxy-4-methylcoumarin (1.47 mmol, 0.32 g) and chloroacetonitrile (2.7 mmol,
0.17 mL) were dissolved in acetone (15 mL), and anhydrous K2CO3 (2.9 mmol,
0.4 g) was added to this solution. The mixture was refluxed and monitored by
TLC on silica-gel plates (eluent CHCl3–AcOEt 20:1). After completion of the reac-
tion as indicated by TLC, the mixture was poured into the flask with water and ice
(50 mL) and stirred for 30 min. The precipitate was filtered out and dried. Heating:
11 h and 30 min. Rf ¼ 0.10. Yield 0.24 g (64%). The analytical sample was crystallized
from ethanol. Mp 198.5–199 ꢀC; IR (KBr) nmax=cmꢁ1: 3082, 2961, 2935, 2259, 1735,
1
1701, 1591, 1566, 1446, 1493, 1368, 1293, 1102, 839; H NMR (CDCl3, 500 MHz) d
2.44 (d, J ¼ 1.0 Hz, 3H, H-11), 2.63 (s, 3H, H-10), 4.88 (s, 2H, H-12), 6.25 (d,
J ¼ 1.0 Hz, 1H, H-3), 7.03 (d, J ¼ 9.0 Hz, 1H, H-6), 7.66 (d, J ¼ 9.0 Hz, 1H, H-5);
13C NMR (CDCl3, 125 MHz): 18.80 (C-11), 32.55 (C-10), 54.71 (C-12), 109.29
(C-6), 114.11 (C-3), 114.20 (C-13), 116.28 (C-4a), 121.12 (C-8), 126.67 (C-5),
150.97 (C-8a), 151.62 (C-4), 154.80 (C-7), 159.20 (C-2), 198.14 (C-9); TOF MS
ESþ [M þ Na]þ ¼ 280.0539 (C14H11NO4Na).
8-Acetyl-7-ethoxycarbonylmethoxy-4-methyl-chromen-2-one (2). 8-Acetyl-
7-hydroxy-4-methylcoumarin (1.5 mmol, 0.33 g) and ethyl chloroacetate (1.65 mmol,
0.176 mL) were dissolved in acetone (15 mL), and anhydrous K2CO3 (4.5 mmol,
0.625 g) was added to this solution. The mixture was refluxed and monitored by
TLC on silica-gel plates (eluent CHCl3–AcOEt 20:1). After completion of the reac-
tion as indicated by TLC, the mixture was poured into the flask with water and ice
(50 mL) and stirred for 30 min. The precipitate was filtered out and dried. Heating:
28 h and 30 min. Rf ¼ 0.18. Yield 0.28 g (61%). The product was pure as indicated by
TLC. Mp 105–105.5 ꢀC; IR (KBr) nmax=cmꢁ1: 3093, 2983, 2928, 1757, 1731, 1601,
1
1568, 1491, 1453, 1376, 1208, 1123, 843; H NMR (CDCl3, 300 MHz) d 1.29 (t,
J ¼ 7.2 Hz, 3H, H-15), 2.40 (d, J ¼ 1.2 Hz, 3H, H-11), 2.65 (s, 3H, H-10), 4.26 (q,
J ¼ 7.2 Hz, 2H, H-14), 4.75 (s, 2H, H-12), 6.17 (bq, J ¼ 1.2 Hz, 1H, H-3), 6.76 (d,

SYNTHESES OF COUMARINS AND ANGELICINS
2399
J ¼ 9.0 Hz, 1H, H-6), 7.55 (d, J ¼ 9.0 Hz, 1H, H-5); 13C NMR (CDCl3, 75 MHz)
d 14.32 (C-15), 18.92 (C-11), 32.56 (C-10), 61.91 (C-14), 65.91 (C-12), 108.33
(C-6), 113.37 (C-3), 115.05 (C-4a), 120.43 (C-8), 126.41 (C-5), 150.94 (C-8a),
152.03 (C-4), 156.77 (C-7), 159.90 (C-2), 167.95 (C-13), 199.11 (C-9); TOF MS
ESþ [M þ Na]þ ¼ 327.0827 (C16H16O6Na).
General Procedure for 3 and 4
8-Acetyl-7-hydroxy-4-methylcoumarin (1.5 mmol, 0.325 g) and the alkylating
agent (chloroacetonitrile or ethyl chloroacetate, 1.65 mmol) were dissolved in
1-methyl-2-pyrrolidone (Fluka Chemika) (2 mL), and anhydrous K2CO3 (4.5 mmol,
0.625 g) was added to this solution. The mixture was heated in the oil bath and mon-
itored by TLC on silica-gel plates (eluent CHCl3–AcOEt 20:1). After completion of
the reaction as indicated by TLC, the mixture was poured into the flask with water
and ice (50 mL) and stirred for 30 min. The precipitate was filtered out and dried.
The analytical samples were crystallized from ethanol.
8-Cyano-4,9-dimethyl-2H-furo[2,3-h]-1-chromen-2-one (3). Heating: 3 h
and 45 min. Yield 0.123 g (34%). Mp 256–257 ꢀC; IR (KBr) nmax=cmꢁ1: 3090,
2963, 2828, 2225, 1748, 1612, 1481, 1366, 1232, 1074, 852; H NMR (500 MHz,
1
CDCl3) d 2.51 (d, J ¼ 1.5 Hz, 3H, H-11), 2.74 (s, 3H, H-13), 6.32 (bq, J ¼ 1.5 Hz,
1H, H-3), 7.42 (d, J ¼ 8.8 Hz, 1H, H-6), 7.70 (d, J ¼ 8.8 Hz, 1H, H-5); 13C NMR
(CDCl3, 75 MHz) d 10.64 (C-13), 19.48 (C-11), 108.68 (C-6), 111.02 (C-12), 113.65
(C-3), 115.45 (C-4a), 116.00 (C-9a), 124.64 (C-5), 125.99 (C-8), 129.89 (C-9),
149.59 (C-9b), 153.01 (C-4), 157.29 (C-7), 159.66 (C-2); TOF MS ESþ [M þ Na]þ ¼
262.0480 (C14H9NO3Na).
8-Ethoxycarbonyl-4,9-dimethyl-2H-furo[2,3-h]-1-chromen-2-one (4).
Heating: 6.5 h. Yield 0.045 g (10%); mp 191.5–192 ꢀC, lit. 192–192.5 ꢀC[10]; IR
(KBr) nmax=cmꢁ1: 3084, 2986, 2866, 1729, 1609, 1571, 1442, 1471, 1365, 1156,
1
1078, 869; H NMR (CDCl3, 300 MHz) d 1.46 (t, J ¼ 7.0 Hz, 3H, H-15), 2.50 (d,
J ¼ 0.9 Hz, 3H, H-11), 2.91 (s, 3 H, H-13), 4.48 (q, J ¼ 7.0 Hz, 2H, H-14), 6.29 (d,
J ¼ 0.9 Hz, 1H, H-3), 7.46 (d, J ¼ 8.7 Hz, 1H, H-6), 7.64 (d, J ¼ 8.7 Hz, 1H, H-5);
13C NMR (CDCl3, 75 MHz,) d 11.36 (C-13), 14.59 (C-15), 19.72 (C-11), 61.68
(C-14), 109.12 (C-6), 113.27 (C-3), 115.07 (C-4a), 117.98 (C-9a), 123.96 (C-5),
126.02 (C-9), 142.00 (C-8), 150.51 (C-9b), 153.51 (C-4), 156.56 (C-2), 160.19 (C-7),
160.35 (C-12); TOF MS ESþ [M þ Na]þ ¼ 309.0739 (C16H14O5Na).
Microwave-Assisted Synthesis, General Procedure
8-Acetyl-7-hydroxy-4-methylcoumarin, chloroacetonitrile or ethyl chloroace-
tate, and K2CO3 were placed in a microwave flask (the amounts were the same as
used in the traditional procedures), and 3 mL of acetone were added. The mixtures
were refluxed in the monomode microwave oven (300 W) and monitored by TLC on
silica-gel plates (eluent CHCl3–AcOEt 20:1).
Attempts to obtain 8-acetyl-7-cyanomethoxy-4-methyl-chromen-2-one (1) or
8-cyano-4,9–dimethyl-2H-furo[2,3-h]-1-chromen-2-one (3) used 10 cycles. Heating

2400
E. HEJCHMAN ET AL.
time for each cycle was 5 min. Yield of 1, 43%; traces of 3 by TLC. Attempts to obtain
8-acetyl-7-ethoxycarbonylmethoxy-4-methyl-chromen-2-one (2) or 8-ethoxycarbonyl-
4,9-dimethyl-2H-furo[2,3-h]-1-benzopyran-2-one (4) used 16 cycles. Heating time for
each cycle 5 min. Yield of 2, 20%; traces of 4 by TLC.
General Procedure for 3 and 4
8-Acetyl-7-hydroxy-4-methylcoumarin (7.5 mmol, 1.635 g) and the alkylating
agent (chloroacetonitrile or ethyl chloroacetate) (8.25 mmol) were dissolved in
1-methyl-2-pyrrolidone (Fluka Chemika) (10 mL), and anhydrous K2CO3
(22.5 mmol, 3.105 g) was added to this solution. The mixture was heated in the
monomode microwave oven (400 W).
8-Cyano-4,9-dimethyl-2H-furo[2,3-h]-1-chromen-2-one (3). One cycle:
temperature 70–75 ꢀC, time of heating 3 min. After completion of the reaction as
indicated by TLC (silica-gel plates, eluent CHCl3–AcOEt 20:1, Rf ¼ 0.59), ice water
(250 mL) was added to the reaction mixture and stirred magnetically for 30 min. The
precipitate was filtered and dried to give an amorphous light brown solid (1.647 g,
92%).
8-Ethoxycarbonyl-4,9-dimethyl-2H-furo[2,3-h]-1-chromen-2-one (4).
First trial: the mixture was heated in the monomode microwave oven (400 W) for
four cycles: temperature 70–75 ꢀC, time of heating 3 min (each cycle). After com-
pletion of the reaction as indicated by TLC (silica-gel plates, eluent CHCl3–AcOEt
20:1, Rf ¼ 0.47), ice water (50 mL) was added to the reaction mixture and hydro-
chloric acid (1:1) was added dropwise to neutralize the solution. The mixture was
stirred magnetically for 30 min. The precipitate was filtered out and dried to give
an amorphous brown solid. Yield 1.728 g (60%).
Second trial: the mixture was heated in the monomode microwave oven
(400 W) for one cycle: temperature 80–85 ꢀC, time of heating 3 min. After completion
of the reaction as indicated by TLC (silica-gel plates, eluent CHCl3–AcOEt 20:1,
Rf ¼ 0.47), the product was isolated from the reaction mixture as described in the
first trial. Yield 1.155 g (54%).
Antibacterial Evaluation
The following microbial strains with various cell-wall structures were chosen:
bacteria Gram-positive (Staphylococcus aureus ATCC 6538, Staphylococcus aureus
ATCC 6538P, Micrococcus luteus ATCC 9341, Micrococcus luteus ATCC 10240,
Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, and Staphylococcus epi-
dermidis ATCC 12228); bacteria Gram-negative (Escherichia coli ATCC 8739,
Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853, and Pseudo-
monas aeruginosa ATCC 15442), and fungi (yeast strains) (Candida albicans ATCC
10231, Candida parapsilosis ATCC 22019, and Zygosaccharomyces rouxii ATCC
28253). The cylinder-plate method was used in the preliminary antimicrobial activity
tests.[16] One hundred mL of compound were placed into the cylinder in a suspension
of 10% dimethylsulfoxide (DMSO) in a 0.08 M phosphate buffer with pH 7.0. The
cylinders were put on a Muller-Hinton 2 or Sabouraud agar plate inoculated with

SYNTHESES OF COUMARINS AND ANGELICINS
2401
one of the tested strains. The plates with bacterial strains were incubated at 37 ꢀC for
24 h, and plates with yeast strains were incubated at 30 ꢀC for 48 h. MIC was
obtained by mixing with 19 mL of a Mueller-Hinton 2 agar, cooled to 56 ꢀC with
1 mL of appropriate dilution of the compound 4. Then 2 mL of a particular cell sus-
pension of optical density 0.5 unit on the McFarland scale were applied to the sur-
face of the agar. The lowest concentration of compound 4 that totally inhibited
growth of examined strain was evaluated as MIC value.[17]
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