Long-chain 3-acyl-4-hydroxycoumarins: Structure and antibacterial activity

Article abstract of DOI:10.1002/ardp.200500127

By reacting 4-hydroxycoumarin with long-chain acyl chlorides, a number of new 3-acyl derivatives were prepared and the relationship was studied between their structures and activities against Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, and Propionibacterium acnes ATCC 11827. Antibacterial activity was associated with enolic tautomers of a tricarbonylmethane group bearing a lipophilic side chain (undec-10-enoyl, undec-10-ynoyl, palmitoyl or octadec-9-enoyl). A newly synthesized 2-acyl derivative of 2H-indene-1,3-dione containing the same tricarbonylmethane motif showed a comparable activity.

Full text of DOI:10.1002/ardp.200500127

Arch. Pharm. Chem. Life Sci. 2006, 339, 129–132
G. Cravotto et al.
129
Full Paper
Long-Chain 3-Acyl-4-hydroxycoumarins: Structure and
Antibacterial Activity*
Giancarlo Cravotto1, Silvia Tagliapietra1, Rossella Cappello1, Giovanni Palmisano2,
Massimo Curini3, Marco Boccalini4
1 Dipartimento di Scienza e Tecnologia del Farmaco, Universitꢀ di Torino, Torino, Italy
2 Dipartimento di Scienze Chimiche e Ambientali; Universitꢀ dell'Insubria, Como, Italy
3 Dipartimento di Chimica e Tecnologia del Farmaco, Universitꢀ di Perugia, Perugia, Italy
4 Dipartimento Chimica Organica, Universitꢀ di Firenze, Firenze, Italy
By reacting 4-hydroxycoumarin with long-chain acyl chlorides, a number of new 3-acyl deriv-
atives were prepared and the relationship was studied between their structures and activities
against Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, and Propionibac-
terium acnes ATCC 11827. Antibacterial activity was associated with enolic tautomers of a tricar-
bonylmethane group bearing a lipophilic side chain (undec-10-enoyl, undec-10-ynoyl, palmitoyl
or octadec-9-enoyl). A newly synthesized 2-acyl derivative of 2H-indene-1,3-dione containing the
same tricarbonylmethane motif showed a comparable activity.
Keywords: 4-Hydroxycoumarin / 3-Acyl-4-hydroxycoumarins / 2H-Indene-1,3-dione / Antibacterial /
Received: May 4, 2005; accepted: October 18, 2005
DOI 10.1002/ardp.200500127
their activity on Staphylococcus aureus ATCC 6538, Propio-
nibacterium acnes ATCC 11827, and Staphylococcus epider-
midis ATCC 12228. Results shed light on the relative
importance for antibacterial action of the 4-hydroxycou-
marin nucleus, the tricarbonylmethane motif and the
side-chain length.
Introduction
Many coumarin derivatives are biologically active [2, 3].
Much research has been focused on the inhibition of bac-
terial growth by naturally occurring coumarins (xantho-
toxin, herniarin, umbelliferone, and scopoletin) and on
the antifungal activity of umbelliferone, scopoletin, and
coumarin itself [4–7]. Some coumarin derivatives (novo-
biocin and analogs) have proven very active as antibiotics
[8, 9]. Among synthetic derivatives, several antibacterial
3-acyl- [10–14] and 3-carbamoyl-4-hydroxycoumarins [15,
16] have been described. The simplest one that proved
moderately active, 3-acetyl-4-hydroxycoumarin [12],
exists in its crystalline form, as shown in a X-ray diffrac-
tion study, predominantly as tautomer A (Scheme 1)
with an intramolecular H-bond [17, 18].
Results and discussion
Chemistry
We began by reacting under sonochemical conditions
[19] 4-hydroxycoumarin with several long-chain acyl
chlorides (Scheme 1) to prepare a new series of 3-acyl-4-
hydroxycoumarins (Scheme 2). The same reaction, when
carried out on 4-hydroxyquinolin-2-(1H)-one, afforded
the 4-O-acyl derivative (2-oxo-1,2-dihydroquinolin-4-yl-
undec-10-enoate, 2a) exclusively. In microbiological tests
carried out on 2a and on a few previously synthesized 3-
alkyl-4-hydroxycoumarins and 4-alkyloxycoumarins [20]
none of these compounds inhibited bacterial growth to
an appreciable extent. We therefore concluded that anti-
In a search for more active compounds, we prepared a
series of 3-acyl-4-hydroxycoumarins, 1a–d and tested
Correspondence: Giancarlo Cravotto, Dipartimento di Scienza e Tecno-
logia del Farmaco, Universitꢀ di Torino, Via Pietro Giuria 9, 10125 Torino,
Italy.
E-mail: giancarlo.cravotto@unito.it
Fax: +39 011 670-7687
* See Reference [1].
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

130
G. Cravotto et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 129–132
Scheme 1. Tautomers of 3-acyl-4-hydroxycoumarin.
Figure 1. List of compounds synthesized and tested.
MEM ether was removed under acidic conditions with
PTSA in CH2Cl2/MeOH.
Scheme 2. General procedure for the preparation of 3-acyl-4-
hydroxycoumarins.
Antimicrobial activity
As shown in Table 1, most active against Staphylococcus
aureus ATCC 6538 were coumarins 1a, 1b, and the 1H-
inden-1-one derivative 3a; all of them bearing a terminal
unsaturated bond on a C11 side chain. Acute toxicities of
compounds 1a, 1b, and 3a were very low (all mice sur-
vived a single dose of 2 g/kg without any marked symp-
toms of intolerance). The minimum inhibitory concen-
trations (MIC) of all compounds tested fell between 3.6
and 25.6 lg/mL (Table 1). The three most active com-
pounds, 1a, 1b, and 3a, were tested on Propionibacterium
acnes ATCC 11827 and Staphylococcus epidermidis ATCC
12228 (Table 2), confirming that the activities of 1a and
3a were close to each other, while 1b was less active.
bacterial activity required the presence of a tricarbonyl-
methane motif associated with a lipophilic side chain
(C10 or longer). Provided these crucial features were pre-
sumed that replacing the 4-hydroxycoumarin with the
2H-indene-1,3-dione nucleus 3a had no effect on the anti-
microbial activity. We prepared a large number of acyl
derivatives of which only the most active are described
below (1a–d), namely those bearing an undec-10-enoyl-
(1a) [21], undec-10-ynoyl- (1b), palmitoyl- (1c), or (Z)-octa-
dec-9-enoyl- (1d) chain (Fig. 1).
We also prepared 4,7-dihydroxy-8-methyl-3-undec-10-
enoyl-2H-chromen-2-one 4a starting from 2,4-dihydroxy-
3-methylacetophenone [22]. This was selectively pro-
tected (Scheme 3) in position 4 with 2-methoxyethoxy-
methyl chloride (MEM chloride) to yield an ether that
was stable under the harsh conditions (NaH in toluene
under reflux) required for cyclization with diethyl carbo-
nate. After acylation with undec-10-enoyl chloride, the
Table 1. Minimum inhibitory concentration (MIC) (mg/mL) on
Staphylococcus aureus ATCC 6538.
Compound
1a
1b
1c
1d
3a
4a
MIC [mg/mL]
3.6
6.2
25.0
25.6
6. 8 22.6
Scheme 3. Synthesis of 4,7-dihydroxy-8-
methyl-3-undec-10-enoyl-2H-chromen-2-
one 4a.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2006, 339, 129–132
Long-chain 3-Acyl-4-hydroxycoumarins
131
Table 2. Antimicrobial activity (MIC) of compounds 1a, 1b.
General procedure for the preparation of acyl chlorides
In a 50 mL two-necked, round-bottomed flask equipped with a
magnetic stirrer, a dropping funnel and a condenser, long chain
carboxylic acid (45 mmol) and anhydrous CH2Cl2 (0.8 mL/mmol)
were mixed. SOCl2 (1.5 equivalent mol) was added dropwise to
the stirred mixture, which was subsequently heated under
reflux for 10 h. The product was recovered by evaporating the
solvent and the excess of SOCl2 under vacuum.
Organism
MIC [lg/mL]
1a
1b
Propionibacterium acnes ATCC 11827
(1.06103 c.f.u./mL)
0.6
1.2
3.6
0.6
1.8
6.2
Staphylococcus epidermidis ATCC 12228
(1.66103 c.f.u./mL)
General procedure for the acylation
Staphylococcus aureus ATCC 6538
(1.56103 c.f.u./mL)
The reaction was carried out under nitrogen atmosphere in the
above mentioned sonochemical reactor that was thermostated
by four Peltier modules. To the PTFE reaction vessel, 4-hydroxy-
coumarin, 4,7-dihydroxy-8-methylcoumarin or 2H-indene-1,3-
dione, a catalytic amount of piperidine, and anhydrous pyridine
(1 mL/mmol) were added. After the solution was cooled to 08C,
acyl chloride (1.6 equivalent mol) was slowly added through a
septum. The mixture was sonicated (21 kHz, 40 W/cm2) for about
1.5 h under nitrogen at 388C, and monitored by TLC. Work-up:
the reacted mixture was diluted with EtOAc and washed with
5% HCl and NaHCO3, dried over Na2SO4, filtered, and evaporated
to dryness.
Conclusion
We obtained in good yields a number of new 3-acyl deriv-
atives of 4-hydroxycoumarin 1a–d and 4,7-dihydroxy-8-
methylcoumarin 4a that we found to be endowed with a
marked antibacterial activity. This required the presence
of a tricarbonylmethane motif associated with a lipophi-
lic side chain (C10 or longer, best if bearing a terminal
ethylene bond). Provided these crucial features were pre-
served, replacing 4-hydroxycoumarin with 2H-indene-
1,3-dione 3a hardly affected the antibacterial activity.
4-Hydroxy-3-undec-10-ynoyl-2H-chromen-2-one 1b
Compound 1b was obtained as white powder; mp. 1098C. IR
(KBr) m cm– 1 1724, 1618, 1553, 760; 1H-NMR (CDCl3, 300 MHz): d
8.07 (1H, dd, J = 7.9 Hz, 1.5 Hz, H-5), 7.70 (1H, td, J = 7.9 Hz, 1.5 Hz,
H-7), 7.37–7.27 (2H, m, H-8 and H-6), 3.20 (2H, t, J = 7.2 Hz, 29-
CH2), 2.13 (2H, td, J = 6.9 Hz, 2.2 Hz, 99-CH2), 1.95–1.30 (13H, m,
aliph. chain). CIMS: 327 [M+H]+. Rf = 0.45 (hexane/EtOAc (4:1)).
Financial support from Italian MIUR (COFIN 2004; prot.
2004037895) is gratefully acknowledged.
The study was partially supported by Medestea srl and we are
grateful to Dr. Antonio Soleti for assistance in the biological
evaluation.
4-Hydroxy-3-palmitoyl-2H-chromen-2-one 1c [11]
Compound 1c was obtained as white powder; mp. 103–1048C. IR
1
(KBr) m cm– 1 1716, 1606, 1472, 1229, 899; H-NMR (CDCl3, 300
MHz): d 8.08 (1H, dd, J = 7.6 Hz, 1.5 Hz, H-5), 7.69 (1H, td, J = 7.6
Hz, 1.5 Hz, H-7), 7.37–7.26 (2H, m, H-8 and H-6), 3.20 (2H, t, J = 7.3
Hz, 29-CH2), 1.69 (2H, quintet, J = 7.1 Hz, 39-CH2), 1.42–1.16 (24H,
m, aliph. chain), 0.88 (3H, t, J = 6.9 Hz, 169-CH3). CIMS: 401 [M+H]+.
Rf = 0.6 (hexane/EtOAc (4:1)).
Experimental
Chemistry
4-Hydroxy-3-[(9Z)-octadec-9-enoyl]-2H-chromen-2-one
General methods
1d
Anhydrous conditions were achieved (when indicated) by flame-
drying flasks and other equipment. Reactions were monitored
by TLC on Alugram Sil – Macherey-Nagel F254 (0.25 mm) plates
(Macherey-Nagel, Dꢀren, Germany), which were visualized by
UV inspection and stained with a 5% KMnO4 solution or by heat-
ing after a spray of 5% H2SO4 in ethanol. Macherey-Nagel silica
gel was used for column chromatography. A Waters microPora-
sil column 7.8–300 (Waters Milford, MA, USA) was used for semi-
preparative HPLC, a Gilson 133 refractive index refractometer
(Gilson SA, Villiers-le-Bel, France) serving for peak detection.
Melting points were obtained on a Bꢀchi SMP-20 apparatus
(Bꢀchi Labortechnik, Flawil, Switzerland) and are uncorrected.
1H-NMR (300 MHz) spectra were recorded on a Bruker 300
Advance spectrometer (Bruker BioSpin GmbH, Rheinstetten,
Germany) at 258C. CIMS were performed on a Finnigan-MAT
TSQ70 (Thermo Electron Corporation, Bremen, Germany) with
isobutane as reactant gas. Unless otherwise noted, commercially
available reagents and solvents were used without further puri-
fication. Some synthetic steps were carried out under high-
intensity ultrasound using a new sonochemical reactor devel-
oped in the authors’ laboratory [22].
Compound 1d was obtained as white powder; mp. 196–1978C.
IR (KBr) m cm– 1 1713, 1607, 1549, 1468, 768; 1H-NMR (CDCl3, 300
MHz): d 8.0 (1H, dd, J = 7.8 Hz, 1.5 Hz, H-5), 7.70 (1H, td, J = 7.8 Hz,
1.5 Hz, H-7), 7.27–7.37 (2H, m, H-8 and H-6), 5.35 (2H, m, H-99 and
H-109), 3.20 (2H, t, J = 7.2 Hz, 29-CH2), 2.01 (4H, m, 89-CH2 and 119-
CH2), 1.73 (2H, quintet, J = 6.7 Hz, 39-CH2), 1.51–1.13 (20H, m,
aliph. chain), 0.88 (3H, t, J = 6.9 Hz, 189-CH3). CIMS: 427 [M+H]+. Rf
= 0.65 (hexane/EtOAc (4:1)).
3-Hydroxy-2-undec-10-enoyl-2H-indene-1,3-dione 3a
Compound 3a was obtained as yellow oil. IR (KBr) m cm– 1 2359,
1
1709, 1655, 1610, 883; H-NMR (CDCl3, 300 MHz): d 7.86–7.82
(2H, m, H-7, H-4), 7.76–7.67 (2H, m, H-5, H-6), 5.87–5.74 (1H, m,
H-109), 5.02–4.91 (2H, m, H-119), 2.97 (2H, t, J = 7.4 Hz, 29-CH2),
2.12 (2H, m, 99-CH2), 1.62 (2H, quintet, 39-CH2), 1.48–1.11 (10H, m,
aliph. chain). CIMS: 313 [M+H]+. Rf = 0.6 (hexane/EtOAc (4:1)).
1-{2-Hydroxy-4-[(2-methoxyethoxy)methoxy]-3-
methylphenyl}ethanone 5
Compound 5 was obtained as white powder; mp. 57–598C. IR
(KBr) m cm– 1 1634, 1499, 1422, 1381, 1333, 1260, 1073, 1101; 1H-
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

132
G. Cravotto et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 129–132
NMR (CDCl3, 300 MHz): d 12.81 (1H, s, H-2), 7.59 (1H, d, J = 9.00
Hz, H-6), 6.72 (1H, d, J = 9.0 Hz, H-5), 5.37 (2H, s, H-19), 3.85 (2H, t, J
= 4.5 Hz, 39-CH2), 3.57 (2H, J = 4.5 Hz, 49-CH2), 3.39 (3H, s, 69-OCH3),
2.59 (3H, s, 299-CH3), 2.13 (3H, s, 3-CH3). CIMS: 255 [M+H]+. Rf = 0.65
(hexane/EtOAc (3:2)).
carbonyl derivative. After 1–4 days of incubation, bacterial
growth was estimated by turbidimetry or by plating on agar
medium.
References
4-Hydroxy-7-[(2-methoxyethoxy)methoxy]-8-methyl-2H-
chromen-2-one 6
[1] Part XIV in the series: The Chemistry of Coumarin Deriv-
atives. For Part XIII, see: G. Cravotto, G. M. Nano, G. Pal-
misano, S. Tagliapietra, Synthesis 2003, 8, 1286–1291.
To a magnetically stirred suspension of 0.355 g of NaH (8.12
mmol, 2 equivalent/mol) in 15 mL anhydrous toluene 1.03 g
(4.06 mmol, 1 equivalent/mol) of compound 5 were added, fol-
lowed by slowly dropping in 1.23 mL (10.16 mmol, 2.5 equiva-
lent/mol) of diethylcarbonate. The mixture was refluxed for 12 h
and the reaction was monitored by TLC, eluent CHCl3/MeOH
(9:1). Work-up: the reacted mixture was transferred to a separa-
tory funnel and extracted with H2O. When the aqueous phase
was acidified with 2N H2SO4, a voluminous precipitate was
obtained. The solid was collected on a Bꢀchner funnel, washed
with cold H2O, and dried by heating at a temperature no higher
than 608C, yielding 0.76 g of 6 (yield 67%).
[2] R. D. H. Murray, J. Mendez, S. A. Brown, The Natural Cou-
marins, Wiley, Chichester, UK, 1982.
[3] J. R. S. Hoult, M. Paya, Gen. Pharmacol. 1996, 27, 713–722.
[4] L. Jurd, J. Corse, A. D. King, H. Bayne, K. Mihara, Phyto-
chem. 1971, 10, 2971–2974.
[5] L. Jurd, A. D. King, K. Mihara, Phytochem. 1971, 10, 2965–
2970.
[6] M. C. Recio, J. L. Rꢁos, A. Villar, Phytother. Res. 1989, 3,
Compound 6: white powder, mp. 132–1348C. IR (KBr) m cm– 1
1709, 1608, 1555, 1466, 1252, 1196, 972, 762; H-NMR (CDCl3,
300 MHz): d 10.78 (1H, brs, 4-OH), 7.62 (1H, d, J = 8.8 Hz, H-5), 7.09
(1H, d, J = 8.8 Hz, H-6), 5.76 (1H, s, H-3), 5.40 (2H, s, 19-CH2), 3.90
(2H, t, J = 4.4 Hz, 39-CH2), 3.64 (3H, t, J = 4.4 Hz, 49-CH2), 3.42 (3H, s,
69-OCH3), 2.28 (3H, s, 8-CH3). CIMS: 281 [M+H]+. Rf = 0.71(CHCl3/
MeOH (9:1)).
117–125.
1
[7] T. Ojala, S. Remes, P. Haansuu, H. Vuorela, R. Hiltunen,
K. Haahtela, P. Vuorela, J. Ethnopharm. 2000, 73, 299–305.
[8] J. W. Hinman, H. Hoeksema, E. L. Caron, W. G. Jackson, J.
Am. Chem. Soc. 1956, 78, 1072–1074.
[9] P. Laurin, M. Klich, C. Dupis-Hamelin, P. Mauvais, P. Las-
saigne, A. Bonnefoy, B. Musicki, Bioorg. Med. Chem. Lett.
1999, 9, 2079–2084.
4-Hydroxy-7-[(2-methoxyethoxy)methoxy]-8-methyl-3-
[10] B. Musicki, A. M. Periers, P. Laurin, D. Ferroud, Y. Bene-
detti, S. Lachaud, F. Chatreaux, J.-L. Haesslein, A. Iltis, C.
Pierre, Bioorg. Med. Chem. Lett. 2000, 10, 1695–1699.
undec-10-enoyl-2H-chromen-2-one 7
Compound 7 was obtained as white powder; mp. 65–678C. IR
(KBr) m cm– 1 1725, 1613, 1559, 1458, 1248, 1078, 988; H-NMR
1
(CDCl3, 300 MHz): d 7.89 (1H, d, J = 8.9 Hz, H-5), 7.15 (1H, d, J = 8.9
Hz, H-6), 5.85 (1H, m, H-1099), 5.43 (2H, s, 19-CH2), 5.03–4.92 (2H,
m, 1199-CH2), 3.86 (2H, t, J = 4.35 Hz, 39-CH2), 3.57 (2H, t, J = 4.35 Hz,
49-CH2), 3.38 (3H, s, 69-CH3), 3.18 (2H, t, J = 7.3 Hz, 299-CH2), 2.30 (3H,
s, 8-CH3), 2.12 (2H, m, 999-CH2), 1.75 (2H, quintet, J= 7.0 Hz, 399-CH2),
1.57–1.20 (10H, m, aliph. chain). CIMS: 447 [M+H]+. Rf = 0.57 (hex-
ane/EtOAc (3:2)).
[11] C. Ukita, S. Nojima, M. Matsumoto, J. Am. Chem. Soc. 1950,
72, 5143–5144.
[12] C. Ukita, K. Arakawa, Pharm. Bull. 1953, 1, 255–260.
[13] K. Arakawa, Pharm. Bull. 1953, 1, 331–334.
[14] D. Zavrsnik, F. Basic, F. Becic, E. Becic, S. Jazic, Period. Bio-
logorum 2003, 105, 137–139.
[15] Merck & Co., Inc., GB 856816, 1960, [Chem. Abstr. 1962,
56, 2361].
4,7-Dihydroxy-8-methyl-3-undec-10-enoyl-2H-chromen-
2-one 4a
[16] B. Yang, J. Sutcliffe, C. J. Dutton, (Pfizer Inc., USA) US
Compound 4a was obtained as white powder; mp. 161–1638C.
IR (KBr) m cm– 1 3480, 2922, 1688, 1618, 1568, 1090; 1H-NMR
(CDCl3, 300 MHz): d 9,8 (2H, brs, 26OH), 7.83 (1H, d, J = 8.3 Hz, H-
5), 6.81 (1H, d, J = 8.3 Hz, H-6), 5.80 (1H, m, H-109), 5.02–4.91 (2H,
m, 119-CH2), 3.17 (2H, t, J = 7.3 Hz, 29-CH2), 2.31 (3H, s, 8-CH3), 2.04
(2H, m, 99-CH2), 1.72 (2H, quintet, J = 7.1 Hz, 39-CH2) 1.57–1.27
(10H, m, aliph. chain). CIMS: 359 [M+H]+. Rf = 0.64 (hexane/EtOAc
(3:2)).
5985912, 1999, [Chem. Abstr. 1999, 131, 322536].
[17] V. F. Traven, O. B. Safronova, L. I. Vorob'eva, T. A. Chibi-
sova, I. N. Senchenya, Russ. J. Gen. Chem. 2000, 70, 793–
797.
[18] K. A. Lyssenko, M. Yu. Antipin, Russ. Chem. Bull. Int. Ed.
2001, 3, 418–431.
[19] G. Cravotto, P. Cintas, Chem. Soc. Rev. 2005, 34, 1–17.
[20] G. Cravotto, G. M. Nano, G. Palmisano, S. Tagliapietra,
Antimicrobial activity
Heterocycles 2003, 60, 1351–1358.
Compounds to be tested were dissolved in acetone (10 mg/mL)
and diluted with warm culture medium. Mueller-Hinton Broth
containing 1% Tween 20 was used for tests with Staphylococcus
aureus and Staphylococcus epidermidis; incubations were carried
out aerobically at 36 l 18C. Actinomyces Broth containing 1%
Tween 20 was used for Propionibacterium acnes that was incu-
bated anaerobically at 36 l 18C. Duplicate blanks were incubated
alongside culture aliquots containing scalar dilution of each tri-
[21] G. Cravotto, G. Balliano, S. Tagliapietra, S. Oliaro-Bosso,
G. M. Nano, Chem. Pharm. Bull. 2004, 52, 1171–1174.
[22] P. Laurin, D. Ferroud, M. Klich, C. Dupuis-Hamelin, P.
Mauvais, P. Lassaigne, A. Bonnefoy, B. Musicki, Bioorg.
Med. Chem. Lett. 1999, 9, 2079–2084.
[23] G. Cravotto, G. Omiccioli, L. Stevanato, Ultrason. Sono-
chem. 2005, 12, 213–217.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com