Synthesis of 4-hydroxycoumarin heteroarylhybrids as potential antimicrobial agents

Article abstract of DOI:10.1002/ardp.201000218

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

Full text of DOI:10.1002/ardp.201000218

394
Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
Full Paper
Synthesis of 4-Hydroxycoumarin Heteroarylhybrids as
Potential Antimicrobial Agents
Zeba N. Siddiqui1, Mohammed Musthafa T. N.1, Anis Ahmad2, and Asad U. Khan2
1 Department of Chemistry, Aligarh Muslim University, Aligarh, India
2 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
challenge to the medical 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.
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
The synthesis of coumarin derivatives has attracted con-
siderable attention of organic and medicinal 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, Aligarh
Muslim University, Aligarh-202002, India.
E-mail: siddiqui_zeba@yahoo.co.in
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Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
Coumarins as potent antimicrobial agents
395
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.
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-
1
Results and discussion
tal and spectral (IR, H-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 cmꢀ1 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 cmꢀ1, respect-
ively. Another absorption band at 3437 cmꢀ1 was assigned to
the OH group of the coumarin unit. The proton nuclear mag-
netic resonance (1H-NMR) spectrum showed a sharp singlet at
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
Scheme 1. Synthetic route of compounds 3a–d.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
Table 1. Synthesis of compounds 3a–d.
Entry
Products
3a
Molecular structure
Time (h)
Yielda (%)
M.p. (8C)
>300
1
3
76
O
O
OH
OH
O
O
O
O
2
3b
3.5
78
>300
CH3
O
O
OH
OH
O
O
O
O
3
3cb
2.5
81
242–245
H
N
OH
O
OH
O
O
O
4
3d
3
80
270–272
O
O
HO
O
H3C
N
O
N
O
C6H5
a
All yields refer to isolated products.
The product was confirmed by elemental analysis, spectral data and compared with [42]
b
d 2.42 for the methyl protons of the chromone moiety. The
upfield singlet of the methine proton appeared at d 6.25. The 11
aromatic protons (three protons of the chromone moiety 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
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Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
Coumarins as potent antimicrobial agents
397
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.
Scheme 2. Mechanism for the formation of product 3d.
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.
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-
The antifungal screening data showed good to moderate
activity. All compounds showed good fungicidal activity
Table 2. Antibacterial activity of compounds 3a–d.
Compounds
Diameter of zone of inhibition (mm)
Gram-negative bacteria
Gram-positive bacteria
S. pyogenes
MRSAa
P. aeruginosa
K. pneumoniae
E. coli
3a
3b
3c
3d
Standard
DMSO
21.3 ꢁ 0.3
21.1 ꢁ 0.5
19.3 ꢁ 0.3
19.3 ꢁ 0.3
24.0 ꢁ 0.3
–
18.9 ꢁ 0.5
20.1 ꢁ 0.3
18.4 ꢁ 0.2
18.4 ꢁ 0.2
25.0 ꢁ 0.4
–
26.1 ꢁ 0.2
26.3 ꢁ 0.5
23.2 ꢁ 0.4
23.2 ꢁ 0.4
32.0 ꢁ 0.3
–
17.3 ꢁ 0.2
18.2 ꢁ 0.4
16.3 ꢁ 0.2
16.3 ꢁ 0.2
18.0 ꢁ 0.2
–
22.1 ꢁ 0.3
21.1 ꢁ 0.4
20.2 ꢁ 0.2
20.2 ꢁ 0.2
26.0 ꢁ 0.4
–
Positive control (standard); ciprofloxacin and negative control (DMSO) measured by the Halo Zone Test (mm).
a
Methicillin-resistant Staphylococcus aureus (MRSA þ Ve).
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Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
Table 3. MIC and MBC results of 3a–d. Positive control ciprofloxacin.
Compounds
Gram-positive bacteria
S. pyogenes MRSA
Gram-negative bacteria
P. aeruginosa
MIC MBC
K. pneumoniae
E. coli
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
3a
3b
3c
3d
25
25
50
50
50
100
100
100
50
25
25
50
50
50
100
>100
>100
50
25
50
50
25
50
100
100
100
25
12.5
25
50
50
50
100
>100
>100
50
25
25
50
50
50
50
>100
>100
50
Standard
12.5
12.5
12.5
12.5
12.5
MIC (mg/mL) ¼ minimum inhibitory concentration, that is, the lowest concentration of the compound to inhibit the growth of
bacteria completely; MBC (mg/mL) ¼ minimum bactericidal concentration, that is, the lowest concentration of the compound for
killing 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
3b
3c
3d
Standard
DMSO
19.4 ꢁ 0.3
23.4 ꢁ 0.4
17.9 ꢁ 0.5
18.3 ꢁ 0.5
30.0 ꢁ 0.2
–
16.5 ꢁ 0.2
19.2 ꢁ 0.6
15.4 ꢁ 0.4
15.8 ꢁ 0.4
27.0 ꢁ 0.2
–
15.1 ꢁ 0.3
15.2 ꢁ 0.4
15.1 ꢁ 0.2
15.3 ꢁ 0.2
24.0 ꢁ 0.3
–
13.7 ꢁ 0.3
13.2 ꢁ 0.6
12.9 ꢁ 0.5
13.1 ꢁ 0.5
20.0 ꢁ 0.5
–
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
3b
3c
3d
25
25
50
50
100
50
100
100
12.5
25
25
50
50
50
100
>100
>100
25
25
50
100
50
50
100
100
100
25
25
50
50
50
100
>100
100
100
25
Standard
6.25
12.5
6.25
12.5
CA; Candida albicans, AF; Aspergillus fumigatus, TM; Trichophyton mentagrophytes, PM; Penicillium marneffei. MIC (mg/mL) ¼ minimum
inhibitory 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 fungus
completely.
against C. albicans, A. fumigatus, T. mentagrophytes, and Conclusion
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. The MFC of the compounds was
found to be two, three or four folds higher than the corre-
sponding MIC results.
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
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Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
Coumarins as potent antimicrobial agents
399
–
–
–
–
1710 (C O, coumarin), 1636 (C O, chromone), 1572 (C C, cou-
–
–
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.
marin) cmꢀ1
;
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 3c
Purified by recrystallization from chloroform. Orange crystalline
solid. Yield 81%. M.p.: 242–2458C; FTIR (KBr) n: 3398 (OH, cou-
Experimental
–
–
marin), 3065 (NH, indole), 1719 (C O, coumarin), 1572 (C C,
–
;
–
–
–
coumarin), 1290 (C–N, indole) cmꢀ1
1H-NMR (400 MHz, DMSO-
d ) d: 5.04 (s, 1H, CH), 6.46 (s, 1H, –C C–H, indole), 6.81–7.97 (m,
Chemistry
6
Melting points of all synthesized compounds were taken on a
Riechert Thermover instrument and are uncorrected. The IR
spectra (KBr) were recorded on a Perkin Elmer RXI spectrometer.
1H-NMR spectra were recorded on Bruker DRX-300 and Bruker
Avance II-400 spectrometer using tetramethyl silane (TMS) as an
internal standard and DMSO-d6 as solvent. Mass spectra were
recorded 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 model
1108. 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 were
purchased from Merck (Mumbai, India) and Sigma-Aldrich
(Switzerland) and used without further purification. The purity
of all compounds was checked by TLC on glass plates (20 ꢂ 5 cm)
coated with silica gel (E-Merck G254, 0.5 mm thickness). The
plates were run in chloroform/methanol (3:1) mixture and were
visualized by iodine vapors.
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 3d
Purified by recrystallization from benzene. White crystals. Yield
80%. 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) cmꢀ1; 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-MS m/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 evaluation
General procedure for the preparation of coumarin
derivatives (3a–d)
A standard inoculum (1–2 ꢂ 107 cfu/mL 0.5 McFarland stand-
ards) was introduced to the surface of sterile agar plates and a
sterile glass spreader was used for even distribution of the inoc-
ulum. The disks measuring 6 mm in diameter were prepared
from Whatman no. 1 filter paper and sterilized by dry heat
at 1408C for 1 h. The sterile disks previously soaked in a
known concentration of the test compounds were placed in
nutrient agar medium. Solvent and growth controls were kept.
Ciprofloxacin (30 mg) was used as positive control while the disk
poured in DMSO was used as negative control. The plates were
inverted and incubated for 24 h at 378C. The susceptibility was
assessed on the basis of diameter of zone of inhibition against
Gram-positive and Gram-negative strains of bacteria. Inhibition
zones were measured and compared with the controls. The
bacterial zones of inhibition values are given in Table 2.
Minimum inhibitory concentrations (MICs) were determined
by broth dilution technique. The nutrient broth, which
contained logarithmic serially two fold diluted amount of test
compound and controls were inoculated with approximately
5 ꢂ 105 cfu/mL of actively dividing bacteria cells. The cultures
were incubated for 24 h at 378C and the growth was monitored
visually and spectrophotometrically. The lowest concentration
(highest dilution) required to arrest the growth of bacteria was
regarded as the minimum inhibitory concentration (MIC). To
obtain the minimum bactericidal concentration (MBC), 0.1 mL
volume was taken from each tube and spread on agar plates. The
number of cfu was counted after 18–24 h of incubation at 358C.
To a solution of 3-bromo-4-hydroxycoumarin 1 (4.14 mmol) in
methanol (12 mL), containing 0.4 mL pyridine, the heteroalde-
hyde 2a–d (2.07 mmol) was added. The reaction mixture was
refluxed in a heating mantle for 2.5–3.5 h. After completion of
the reaction (checked by TLC), the reaction mixture was cooled at
room temperature. The solid part separated out was collected by
filtration, dried and recrystallized either from chloroform or
from 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 3a
Purified 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) cmꢀ1
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-MS
m/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 3b
Purified by recrystallization from chloroform. White powder.
Yield 78%. M.p.: >3008C; FTIR (KBr) n: 3437 (OH, coumarin),
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Z. N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 394–401
MBC was defined as the lowest drug concentration at which
99.9% of the inoculum were killed. The minimum inhibitory
concentration and minimum bactericidal concentration are
given in Table 3.
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Sabourauds 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 a
suspension of spore of fungal strain for lawn. A loop full of
particular fungal strain was transferred to 3 mL saline to get
a suspension of corresponding species. 20 mL of agar media were
poured into each Petri dish. The excess of suspension was
decanted and the plates were dried by placing in an incubator
at 378C for 1 h. Using an agar punch wells were made and each
well was labeled. A control was also prepared in triplicate and
maintained at 378C for 3–4 days. The fungal activity of each
compound was compared with 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-fold
diluted amount of the test compound and controls was inocu-
lated with approximately 1.6 ꢂ 104–6 ꢂ 104 cfu/mL. The cul-
tures were incubated for 48 h at 358C and the growth was
monitored. The lowest concentration (highest dilution) required
to arrest the growth of fungus was regarded as the minimum
inhibitory concentration (MIC). To obtain the minimum fungi-
cidal concentration (MFC), 0.1 mL volume was taken from each
tube and spread on agar plates. The number of cfu was counted
after 48 h of incubation at 358C. MFC was defined as the lowest
drug concentration at which 99.9% of the inoculums were
killed. The minimum inhibitory concentration and minimum
fungicidal concentration are given in Table 5.
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