Synthesis of 4-Hydroxycoumarin Heteroarylhybrids as Potential Antimicrobial Agents

Full Paper

Synthesis of 4-Hydroxycoumarin Heteroarylhybrids asPotential Antimicrobial Agents

Zeba N. Siddiqui1, Mohammed Musthafa T. N.1, Anis Ahmad2, and Asad U. Khan2

1 Department of Chemistry, Aligarh Muslim University, Aligarh, India2 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India

A new series of 4-hydroxycoumarin derivatives 3a–d was synthesized by the reaction of 3-bromo-4-

hydroxy coumarin 1 with various heteroaldehydes 2a–d in good yields. The synthesized compounds

were characterized on the basis of their elemental and spectral (IR, 1H-NMR and mass spectrometry)

analysis. All target compounds were evaluated for their in-vitro antimicrobial activity against

Streptococcus pyogenes, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella

pneumonia, and Escherichia coli bacterial strains and fungal cultures of Candida albicans, Aspergillus

fumigatus, Trichophyton mentagrophytes, and Penicillium marneffei by disk diffusion assay with slight

modifications. The minimum inhibitory concentration (MIC) was determined for the test

compounds as well as for reference standards. Among the tested compounds, 3a has shown the

most potent antibacterial as well as antifungal activities.

Keywords: Antibacterial activity / Antifungal activity / 3-Bromo-4-hydroxycoumarin / Dicoumarols / Heteroaldehydes

Received: July 26, 2010; Revised: September 4, 2010; Accepted: September 14, 2010

DOI 10.1002/ardp.201000218

Introduction

In recent years, the number of life-threatening infectious dis-

eases caused by multi-drug resistant Gram-positive and Gram-

negative pathogen bacteria have reached an alarming level in

many countries around the world [1, 2]. These enteric bacterial

infections are responsible for morbidity and mortality world-

wide, mostly in developing countries and areas such as the

Indian sub-continent, part of South America, and tropical part

of Africa, etc. [3, 4]. A number of clinical reports in the United

States and worldwide have independently described the

emergence of vancomycin resistance in methicillin-resistant

Staphylococcus aureus (MRSA) isolates and other human

pathogen Gram-negative isolates [5]. Escherichia coli are respon-

sible for the world’s most common and serious infectious

diseases like invasive dysentery and diarrhea [6, 7]. The infec-

tion with these microorganisms may have significant impact

on huge demolition of host tissue and severe diseases [8, 9].

More than 90% of the cases of vaginitis are of candidiasis,

trichomoniasis, and bacterial vaginosis [10]. Thus, the

infections caused by these microorganisms pose a serious

challenge to themedical community and need for an effective

therapy has led to a search for novel antimicrobial agents. For

this reason, the present stratagem for the synthesis of new

compounds is aimed in the direction of developing new

coumarin derivatives to inhibit the growth of Gram-positive,

Gram-negative bacteria, and fungi.

The synthesis of coumarin derivatives has attracted con-

siderable attention of organic andmedicinal chemists as these

compounds are widely used as fragrances, pharmaceuticals,

and agrochemicals [11]. Much research has been focused on

the inhibition of bacterial and fungal growth by naturally

occurring, synthetic coumarin derivatives [12–18]. 3-Acyl- and

3-carbamoyl-4-hydroxycomarins, dicoumarols, and some

other coumarin derivatives like novobiocin and its analogues

have proven as potent antibiotics [19–22]. Furthermore, three

coumarin antibiotics, novobiocin, chlorobiocin, and coumer-

mycin A1 exhibit significant activity on certain tumor cells

[23]. The other biological applications such as anti-inflamma-

tory [24], anticonvulsant [25], antiviral [26], antioxidant [27],

anti-HIV [28], anticarcinogenic [29], and antihistamine [30],

etc., are also cited in the literature. Besides the wide spectrum

biological applications of coumarin and its derivatives, the

chemical literature also embodies their some applications

from the material viewpoint such as cosmetics [11], optical

brightening agents [31], and laser dyes [32].

Correspondence: Zeba N. Siddiqui, Department of Chemistry, AligarhMuslim University, Aligarh-202002, India.E-mail: [email protected]

394 Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In search of more potent antibiotics, herein we report the

one pot, efficient and simple methodology for the synthesis

of coumarin derivatives in good yields by the reaction of

3-bromo-4-hydroxycoumarin with various heteroaldehydes.

The synthesized compounds were screened for their in-vitro

antibacterial and antifungal activities.

Results and discussion

Chemistry

4-Hydroxycoumarin possesses a nucleophilic character due

to the presence of the carbon–carbon double bond at position

3 and reacts with aldehydes and ketones to give dicoumarol

derivatives [21, 33]. It was therefore expected, that 3-bromo-4-

hydroxycoumarin would also react with aldehydes to give

dicoumarol derivatives. The reaction afforded 3a–c in quan-

titative yields (76–81%) by refluxing 1 and the heteroalde-

hydes 2a–c in the presence of a catalytic amount of pyridine

in methanol (Scheme 1). However, the reaction of 1 with

5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde 2d did

not stop at the dicoumarol product 3e stage rather sub-

sequent cyclization took place to give pyrazolopyrone 3d

in good yield (80%) (Table 1). A probable route for the for-

mation of 3d has been shown in Scheme 2. The structural

assignment of the compounds isolated was done by elemen-

tal and spectral (IR, 1H-NMR, mass) analysis.

The high molecular weight of compound 3b in the electron

spray mass spectrum (Mþ ¼ 494.16) indicated the incorpora-

tion of more than one coumarin moiety in the product. The

infra red (IR) spectrum exhibited a strong absorption band at

1572 cm�1 for the carbon–carbon double bond of the cou-

marin unit whereas carbonyl groups of the chromone and

coumarin moieties appeared at 1636 and 1710 cm�1, respect-

ively. Another absorption band at 3437 cm�1 was assigned to

the OH group of the coumarin unit. The proton nuclear mag-

netic resonance (1H-NMR) spectrum showed a sharp singlet at

Scheme 1. Synthetic route of compounds 3a–d.

Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401 Coumarins as potent antimicrobial agents 395

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.archpharm.com

d 2.42 for the methyl protons of the chromone moiety. The

upfield singlet of themethine proton appeared at d 6.25. The 11

aromatic protons (three protons of the chromonemoiety and 8

protons of the coumarin units) were discernible in the form of

a multiplet at d 7.33–8.01. The diagnostic singlet of the C-2

proton of the chromone moiety was present as a sharp singlet

at d 7.66. A broad singlet (D2O exchangeable) at d 11.20 inte-

grating for more than one proton was assigned to the OH

protons of the coumarin units. Further confirmation for the

structure 3b was provided by the mass spectrum which has

Table 1. Synthesis of compounds 3a–d.

Entry Products Molecular structure Time (h) Yielda (%) M.p. (8C)

1 3a

O O

OH

O

OH

O

OO

3 76 >300

2 3b

O O

OH

O

OH

O

OO

CH33.5 78 >300

3 3cb

O O

OH

O

OH

O

NH

2.5 81 242–245

4 3d

NN

H3C

C6H5

O

O

O

O

OHO

3 80 270–272

a All yields refer to isolated products.b The product was confirmed by elemental analysis, spectral data and compared with [42]

396 Z. N. Siddiqui et al. Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401


given subsequent fragmentation pattern. The spectral studies

of the other compounds followed a similar pattern.

Biological studies

Antibacterial and antifungal activities of the synthesized

compounds were completed by the disk diffusion method

and measured by the Halo Zone Test. The MIC of the synthe-

sized compounds against bacterial and fungal strains was

performed by the macro dilution test and results were

observed visually and spectrophotometrically.

Antibacterial Activity

The newly prepared compounds were screened for their

antibacterial activity against Escherichia coli (ATCC-25922),

methicillin-resistant Staphylococcus aureus (MRSA þVe),

Pseudomonas aeruginosa (ATCC-27853), Streptococcus pyogenes,

and Klebsiella pneumoniae (clinical isolate) bacterial strains

by the disk diffusion method [34, 35]. The results are pre-

sented in Tables 2 and 3.

The investigation of the antibacterial screening data

revealed that all the tested compounds 3a–d showed moder-

ate to good bacterial inhibition. All the compounds showed

good inhibition against S. pyogenes, methicillin-resistant

Staphylococcus aureus (MRSA þVe), P. aeruginosa, K. pneumonia,

and E. coli species. Among the tested compounds, 3a showed a

more potent bactericidal activity against all fungal strains

(MIC 12.5–25 mg/mL). The higher susceptibility of Gram-

positive bacteria is due to absence of a unique outer mem-

brane of peptidoglycon, hence the wall of these bacteria is

permeable to these derivatives. The compounds also have

significant influence on antibacterial profile of Gram-nega-

tive bacteria. The MIC of the compounds was found to be of

12.5–50 mg/mL. The MBC of the compounds was two, three or

four folds higher than the corresponding MIC results.

Antifungal activity

Antifungal activity was screened out by disk diffusion

method. For assaying antifungal activity Candida albicans,

Aspergillus fumigatus, Penicillium marneffei, and Trichophyton men-

tagrophytes were recultured in DMSO by the agar diffusion

method [36, 37]. The inhibitory results are presented in

Tables 4 and 5.

The antifungal screening data showed good to moderate

activity. All compounds showed good fungicidal activity

Scheme 2. Mechanism for the formation of product 3d.

Table 2. Antibacterial activity of compounds 3a–d.

Compounds Diameter of zone of inhibition (mm)

Gram-positive bacteria Gram-negative bacteria

S. pyogenes MRSAa P. aeruginosa K. pneumoniae E. coli

3a 21.3 � 0.3 18.9 � 0.5 26.1 � 0.2 17.3 � 0.2 22.1 � 0.33b 21.1 � 0.5 20.1 � 0.3 26.3 � 0.5 18.2 � 0.4 21.1 � 0.43c 19.3 � 0.3 18.4 � 0.2 23.2 � 0.4 16.3 � 0.2 20.2 � 0.23d 19.3 � 0.3 18.4 � 0.2 23.2 � 0.4 16.3 � 0.2 20.2 � 0.2Standard 24.0 � 0.3 25.0 � 0.4 32.0 � 0.3 18.0 � 0.2 26.0 � 0.4DMSO – – – – –

Positive control (standard); ciprofloxacin and negative control (DMSO) measured by the Halo Zone Test (mm).a Methicillin-resistant Staphylococcus aureus (MRSA þ Ve).



against C. albicans, A. fumigatus, T. mentagrophytes, and

P. marneffei fungal strains. Among the tested compounds 3a

showed a more potent fungicidal activity against all fungal

strains (MIC 25 mg/mL). The MIC of other compounds was

found to be of 25–100 mg/mL. TheMFC of the compounds was

found to be two, three or four folds higher than the corre-

sponding MIC results.

Conclusion

In summary, we have developed a clean and convenient

method to synthesize new coumarin derivatives in good

yields. The method is advantageous in terms of reduced

reaction time, high yield of products, simple experimental

work-up procedure, etc., thus, adding a useful procedure to

Table 3. MIC and MBC results of 3a–d. Positive control ciprofloxacin.

Compounds Gram-positive bacteria Gram-negative bacteria

S. pyogenes MRSA P. aeruginosa K. pneumoniae E. coli

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

3a 25 50 25 50 25 50 12.5 50 25 503b 25 100 25 100 50 100 25 100 25 503c 50 100 50 >100 50 100 50 >100 50 >1003d 50 100 50 >100 25 100 50 >100 50 >100Standard 12.5 50 12.5 50 12.5 25 12.5 50 12.5 50

MIC (mg/mL) ¼ minimum inhibitory concentration, that is, the lowest concentration of the compound to inhibit the growth ofbacteria completely; MBC (mg/mL) ¼ minimum bactericidal concentration, that is, the lowest concentration of the compound forkilling the bacteria completely.

Table 4. Antifungal activity of 3a–d: Positive control (griseofulvin) and negative control (DMSO) measured by the Halo Zone Test (mm).

Compounds Diameter of zone of inhibition (mm)

CA AF TM PM

3a 19.4 � 0.3 16.5 � 0.2 15.1 � 0.3 13.7 � 0.33b 23.4 � 0.4 19.2 � 0.6 15.2 � 0.4 13.2 � 0.63c 17.9 � 0.5 15.4 � 0.4 15.1 � 0.2 12.9 � 0.53d 18.3 � 0.5 15.8 � 0.4 15.3 � 0.2 13.1 � 0.5Standard 30.0 � 0.2 27.0 � 0.2 24.0 � 0.3 20.0 � 0.5DMSO – – – –

CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes, PM; Penicillium marneffei.

Table 5. MIC and MFC of compounds 3a–d. Positive control griseofulvin.

Compounds CA AF TM PM

MFC MIC MFC MIC MFC MIC MFC

3a 25 100 25 50 25 50 25 1003b 25 50 25 100 50 100 50 >1003c 50 100 50 >100 100 100 50 1003d 50 100 50 >100 50 100 50 100Standard 6.25 12.5 12.5 25 6.25 25 12.5 25

CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes, PM; Penicillium marneffei. MIC (mg/mL) ¼ minimuminhibitory concentration, that is, the lowest concentration of the compound to inhibit the growth of fungus completely;MFC (mg/mL) ¼ minimum fungicidal concentration, that is, the lowest concentration of the compound for killing the funguscompletely.



existing methodologies. Further, in-vitro antibacterial and

antifungal evaluation of compounds has proved them as

potent antibacterial and antifungal agents. The importance

of such kind of work lies in the possibility that the new

compounds might be more effective against microbes for

which a thorough study regarding the structure-activity

relationship, toxicity and their biological effects would be

helpful in designing more potent antimicrobial agents.

Experimental

ChemistryMelting points of all synthesized compounds were taken on aRiechert Thermover instrument and are uncorrected. The IRspectra (KBr) were recorded on a Perkin Elmer RXI spectrometer.1H-NMR spectra were recorded on Bruker DRX-300 and BrukerAvance II-400 spectrometer using tetramethyl silane (TMS) as aninternal standard and DMSO-d6 as solvent. Mass spectra wererecorded on Micro mass Quattro II triple quadrupol mass spec-trometer and Jeol-SX-102 (FAB) spectrometer. Elemental analyses(C, H, and N) were conducted using a Carlo Erba analyzer model1108. 3-Bromo-4-hydroxycoumarin [38], 3-formylchromone, 6-methyl-3-formylchromone [39], indole-3-carboxaldehyde [40],and 5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde [41]were prepared by reported procedures. Other chemicals werepurchased from Merck (Mumbai, India) and Sigma-Aldrich(Switzerland) and used without further purification. The purityof all compounds was checked by TLC on glass plates (20 � 5 cm)coated with silica gel (E-Merck G254, 0.5 mm thickness). Theplates were run in chloroform/methanol (3:1) mixture and werevisualized by iodine vapors.

General procedure for the preparation of coumarin

derivatives (3a–d)To a solution of 3-bromo-4-hydroxycoumarin 1 (4.14 mmol) inmethanol (12 mL), containing 0.4 mL pyridine, the heteroalde-hyde 2a–d (2.07 mmol) was added. The reaction mixture wasrefluxed in a heating mantle for 2.5–3.5 h. After completion ofthe reaction (checked by TLC), the reactionmixture was cooled atroom temperature. The solid part separated out was collected byfiltration, dried and recrystallized either from chloroform orfrom benzene to afford 3a–d in pure form.

1-(4-Oxo-4H-1-benzopyran-3-yl)-1,1-bis(4-hydroxy-1-

benzopyran-2-one-3-yl)methane 3aPurified by recrystallization from chloroform. White powder.Yield 76%. M.p.: >3008C; FTIR (KBr) n: 3428 (OH, coumarin),1708 (C––O, coumarin), 1633 (C––O, chromone), 1570 (C––C,coumarin) cm�1; 1H-NMR (300 MHz, DMSO-d6,) d: 6.02 (s, 1H,CH), 7.18–7.79 (m, 11H, Ar-H), 7.88 (d, 1H, C-5, J ¼ 7.8 Hz, chro-mone), 7.98 (s, 1H, C-2, chromone), 11.31 (s, 2H, OH) ppm; ESI-MSm/z: 480.13 (Mþ), 453.12, 451.11, 320.08, 319.08, 317.06, 99.11.Anal. calcd. for C28H16O8: C, 70.01; H, 3.35. Found: C, 70.18; H, 3.42.

1-(6-Methyl-4-oxo-4H-1-benzopyran-3-yl)-1,1-bis(4-

hydroxy-1-benzopyran-2-one-3-yl)methane 3bPurified by recrystallization from chloroform. White powder.Yield 78%. M.p.: >3008C; FTIR (KBr) n: 3437 (OH, coumarin),

1710 (C––O, coumarin), 1636 (C––O, chromone), 1572 (C––C, cou-marin) cm�1; 1H-NMR (400 MHz, DMSO-d6) d: 2.42 (s, 3H, CH3),6.25 (s, 1H, CH), 7.33–8.01 (m, 11H, Ar-H), 7.66 (s, 1H, C-2, chro-mone), 11.20 (s, 2H, OH) ppm; ESI-MS m/z: 494.16 (Mþ), 493.16,318.07, 317.06, 277.15, 99.11. Anal. calcd. for C29H18O8: C, 70.44;H, 3.66. Found: C, 70.56; H, 3.69.

1-(Indol-3-yl)-1,1-bis(4-hydroxy-1-benzopyran-2-one-3-yl)

methane 3cPurified by recrystallization from chloroform. Orange crystallinesolid. Yield 81%. M.p.: 242–2458C; FTIR (KBr) n: 3398 (OH, cou-marin), 3065 (NH, indole), 1719 (C––O, coumarin), 1572 (C––C,coumarin), 1290 (C–N, indole) cm�1; 1H-NMR (400 MHz, DMSO-d6) d: 5.04 (s, 1H, CH), 6.46 (s, 1H, –C––C–H, indole), 6.81–7.97 (m,11H, Ar-H), 10.43 (s, 1H, NH), 11.63 (s, 2H, OH) ppm; ESI-MS m/z:451.3 (Mþ), 446.3, 413.5, 377.4, 375.4, 360.6, 358.6, 332.6, 274.6,238.5, 225.5, 134.5, 116.5. Anal. calcd. For C27H17NO6: C,71.83; H,3.79; N, 3.10. Found: C, 71.91; H, 3. 64, N, 3.06.

4-(4-Hydroxy-2-oxo-2H-1-benzopyran-2-one-3-yl)-3-

methyl-1-phenyl pyrazolo[3,4;2,3]-4H-pyrano[3,2-b]-1-

benzopyran-5-one 3dPurified by recrystallization from benzene. White crystals. Yield80%. M.p.: 270–2728C; FTIR (KBr) n: 3480 (OH, coumarin), 1719(C––O, coumarin), 1633 (C––N, pyrazole), 1608 (C––C, coumarin),1368 (C-N, pyrazole) cm�1; 1H-NMR (300 MHz, DMSO-d6) d: 2.50 (s,3H, CH3), 4.67 (s, 1H, CH), 7.09–8.40 (m, 13H, Ar-H), 11.38 (s, 1H,OH) ppm; FAB-MSm/z (%): 490 (Mþ) (25), 329 (50), 331 (60), 346 (5),302 (15), 318 (25), 317 (100), 269 (90), 267 (85), 185 (5), 150 (20),149 (70), 142 (5), 115 (50). Anal. calcd. for C29H18N2O6: C, 71.02; H,3.69; N, 5.71. Found: C, 71.09; H, 3.81; N, 5.63.

Antibacterial evaluationA standard inoculum (1–2 � 107 cfu/mL 0.5 McFarland stand-ards) was introduced to the surface of sterile agar plates and asterile glass spreader was used for even distribution of the inoc-ulum. The disks measuring 6 mm in diameter were preparedfrom Whatman no. 1 filter paper and sterilized by dry heatat 1408C for 1 h. The sterile disks previously soaked in aknown concentration of the test compounds were placed innutrient agar medium. Solvent and growth controls were kept.Ciprofloxacin (30 mg) was used as positive control while the diskpoured in DMSO was used as negative control. The plates wereinverted and incubated for 24 h at 378C. The susceptibility wasassessed on the basis of diameter of zone of inhibition againstGram-positive and Gram-negative strains of bacteria. Inhibitionzones were measured and compared with the controls. Thebacterial zones of inhibition values are given in Table 2.

Minimum inhibitory concentrations (MICs) were determinedby broth dilution technique. The nutrient broth, whichcontained logarithmic serially two fold diluted amount of testcompound and controls were inoculated with approximately5 � 105 cfu/mL of actively dividing bacteria cells. The cultureswere incubated for 24 h at 378C and the growth was monitoredvisually and spectrophotometrically. The lowest concentration(highest dilution) required to arrest the growth of bacteria wasregarded as the minimum inhibitory concentration (MIC). Toobtain the minimum bactericidal concentration (MBC), 0.1 mLvolume was taken from each tube and spread on agar plates. Thenumber of cfu was counted after 18–24 h of incubation at 358C.



MBC was defined as the lowest drug concentration at which99.9% of the inoculum were killed. The minimum inhibitoryconcentration and minimum bactericidal concentration aregiven in Table 3.

Antifungal evaluationSabourauds agar media were prepared by dissolving peptone(1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 mL)and adjusted to the pH of 5.7. Normal saline was used to make asuspension of spore of fungal strain for lawn. A loop full ofparticular fungal strain was transferred to 3 mL saline to geta suspension of corresponding species. 20 mL of agarmedia werepoured into each Petri dish. The excess of suspension wasdecanted and the plates were dried by placing in an incubatorat 378C for 1 h. Using an agar punch wells were made and eachwell was labeled. A control was also prepared in triplicate andmaintained at 378C for 3–4 days. The fungal activity of eachcompoundwas comparedwith griseofulvin as the standard drug.Inhibition zones were measured and compared with the con-trols. The fungal zones of inhibition values are given in Table 4.The nutrient broth containing a logarithmic serially two-folddiluted amount of the test compound and controls was inocu-lated with approximately 1.6 � 104–6 � 104 cfu/mL. The cul-tures were incubated for 48 h at 358C and the growth wasmonitored. The lowest concentration (highest dilution) requiredto arrest the growth of fungus was regarded as the minimuminhibitory concentration (MIC). To obtain the minimum fungi-cidal concentration (MFC), 0.1 mL volume was taken from eachtube and spread on agar plates. The number of cfu was countedafter 48 h of incubation at 358C. MFC was defined as the lowestdrug concentration at which 99.9% of the inoculums werekilled. The minimum inhibitory concentration and minimumfungicidal concentration are given in Table 5.

Financial assistance in the form of major research project [F.No. 37-15/2009(SR)] from UGC, New Delhi, is gratefully acknowledged. The authors wouldalso like to thank SAIF, CDRI, Lucknow and SAIF, Panjab University,Chandigarh for spectral data.

The authors have declared no conflict of interest

References

[1] I. Berber, C. Cokmus, E. Atalan, Microbiology 2003, 72, 54–59.

[2] L. A. Mitscher, S. P. Pillai, E. J. Gentry, D. M. Shankel,Med. Res.Rev. 1999, 19, 477–496.

[3] F. Qadri, A. M. Svennerholm, A. S. G. Faruque, R. B. Sack, Clin.Microbiol. Rev. 2005, 18, 465–483.

[4] R. A. Devasia, T. F. Jones, J. Ward, L. Stafford, H. Hardin, C.Bopp, M. Beatty, E. Mintz, W. Schaffner, Am. J. Med. 2006, 119,168–176.

[5] V. J. Lee, S. J. Hecker, Med. Res. Rev. 1999, 19, 521–542.

[6] W. Zhang, E. M. Berberoy, J. Freeling, D. He, R. A. Moxley,D. H. Francis, Infect. Immun. 2006, 74, 3107–3114.

[7] H. I. Shaheen, S. B. Khalil, M. R. Rao, R. A. Elyazeed, T. F.Wierzba, L. F. Peruski, Jr, S. Putnam, A. Navarro, B. Z. Morsy,A. Cravioto, J. D. Clemens, A. M. Svennerholm, S. J. Savarino,J. Clin. Microbiol. 2004, 42, 5588–5595.

[8] A. S. Puertoa, J. G. Fernandeza, J. D. L. Castillob, M. Jose,S. Pinoa, G. P. Anguloa, Diagn. Microbiol. Infect. Dis. 2006,54, 135–139.

[9] C. M. Nolan, E. G. Chalhub, G. D. Nash, T. Yamauchi,Antimicrob. Agents Chemother. 1979, 16, 171–175.

[10] P. P. Chong, S. R. Abdul Hadi, Y. L. Lee, C. L. Phan,B. C. Tan, K. P. Ng, H. F. Seow, Infect. Genet. Evol. 2007, 7,449–456.

[11] R. O. Kennedy, R. D. Thornes, Coumarins: Biology, Applicationsand Mode of Action, Wiley & Sons, Chichester 1997.

[12] L. Jurd, J. Corse, A. D. King, H. Bayne, K. Mihara,Phytochemistry 1971, 10, 2971–2974.

[13] L. Jurd, J. Corse, A. D. King, H. Bayne, K. Mihara,Phytochemistry 1971, 10, 2965–2970.

[14] M. C. Rccio, J. L. Rios, A. Villar, Phytother. Res. 1989, 3, 117–125.

[15] T. Ojala, S. Remes, P. Haansuu, H. Vuorela, R. Hiltunen,K. Haahtela, P. Vuorela, J. Ethnopharmacol. 2000, 73, 299–305.

[16] J. N. Modranka, E. Nawrot, J. Graczyk, Eur. J. Med. Chem. 2006,41, 1301–1309.

[17] S. Sardari, Y. Mori, K. Horita, R. G. Micetich, S. Nishibe,M. Daneshtalab, Biorg. Med. Chem. 1999, 7, 1933–1940 .

[18] S. U. Rehman, Z. H. Chohan, F. Gulnaz, C. T. Supuran,J. Enzym. Inhibit. Med. Chem. 2005, 20, 333–340.

[19] B. Musicki, A. M. Periers, P. Laurin, D. Ferroud, Y. Benedetti,S. Lachaud, F. Chatreaux, J. L. Haesslein, A. Iltis, C. Pierre,J. Khider, N. Tessot, M. Airault, J. Demassey, C. Dupuis-Hamelin, P. Lassaigne, A. Bonnefoy, P. Vicat, M. Klich,Bioorg. Med. Chem. Lett. 2000, 10, 1695–1699.

[20] S. Moran, Crop Protect. 2001, 20, 529–533.

[21] N. Hamdi, M. C. Puerta, P. Valerga, Eur. J. Med. Chem. 2008, 43,2541–2548.

[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] M. G. Marcu, T. W. Schulte, L. Neckers, J. Natl. Cancer Inst.2000, 92, 242–248.

[24] C. M. Lin, S. T. Huang, F. W. Lee, H. Sawkuo, M. H. Lin, Bioorg.Med. Chem. 2006, 14, 4402–4409.

[25] M. A. Bhat, N. Siddiqui, S. A. Khan, Indian J. Pharm. Sci. 2006,68, 120–124.

[26] C. Massimo, E. Francesco, M. Federica, M. M. Carla, G. S.Prieto, R. J. Carlos, Aust. J. Chem. 2003, 56, 59–60.

[27] A. K. Tyagi, H. G. Raj, P. Vohra, G. Gupta, R. Kumari,P. Kumar, R. K. Gupta, Eur. J. Med. Chem. 2003, 40, 413–420.

[28] L. Huang, X. Yuon, D. Yu, K. H. Lee, H. C. Chin, Virology 2005,332, 623–628.

[29] C. M. Elinos-Baez, F. Leon, E. Santos, Cell Biol. Int. 2005, 29,703–708.

[30] N. Mohanty, P. C. Rath, M. K. Rout, J. Indian Chem. Soc. 1967,44, 1001–1004.

[31] M. Zahradnik, The production and application of fluorescentbrightening Agents, Wiley & Sons, Chichester 1992.

[32] M. Maeda, Laser dyes, Academic Press, New York 1994.



[33] R. Gasparova, K. Kotlebova, M. Lacova, Nova Biotechnologica2009, 9, 349–354.

[34] R. Cruickshank, J. P. Duguid, B. P. Marmion, R. H. A. Swain,in Medicinal Microbiology, Vol. 2, Churchill Livingstone,London 1975.

[35] A. H. Collins, Microbiological Methods, Butterworth, London1976.

[36] Z. K. Khan, In vitro and in vivo screening techniques for bioactivityscreening and evaluation, Proc. Int. Workshop UNIDO-CDRI,1997, 210–211.

[37] R. S. Varma, Antifungal Agents: Past, Present and Future prospects,National Academy of Chemistry & Biology, Lucknow, India1998.

[38] C. F. Huebner, K. P. Link, J. Am. Chem. Soc. 1945, 67, 99–102.

[39] A.Nohara, T. Umetani, Y. Sanno, Tetrahedron 1974, 30, 3353–3361.

[40] S. S. Panda, P. V. R. Chowdary, Indian J. Pharm. Sci. 2008, 70,208–215.

[41] R. A. Pawar, A. A. Patil, Indian J. Chem. 1994, 33B, 156–158.

[42] M. Kidwai, V. Bansal, P. Mothsra, S. Saxena, R. K. Somvanshi,S. Dey, T. P. Singh, J. Mol. Catal. A: Chem. 2007, 268, 76–81.



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