Pyrimidine non-nucleoside analogs: A direct synthesis of a novel class of N-substituted amino and N-sulfonamide derivatives of pyrimidines

Article abstract of DOI:10.1080/15257770.2016.1257808

A convenient method for the regioselective synthesis of pyrimidine non-nucleoside analogs was developed. This study reports a novel and efficient method for the synthesis of a new type of N-substituted amino methylsulfanylpyrimidines and the corresponding pyrazolo[3,4-d]pyrimidines. This series of compounds was designed through the reaction of dimethyl N-cyanodithioiminocarbonate with 2-cyano-N′-(thiophen-2-yl-, furan-2-yl- and pyridin-4-ylmethylene)acetohydrazide and N′-(2-cyanoacetyl)arylsulfonohydrazides. The scope and limitation of the method are demonstrated. The antibacterial and antifungal activities of the synthesized compounds were also evaluated.

Full text of DOI:10.1080/15257770.2016.1257808

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Pyrimidine non-nucleoside analogs: A direct
synthesis of a novel class of N-substituted amino
and N-sulfonamide derivatives of pyrimidines
Galal H. Elgemeie, Ali M. Salah, Nermeen S. Abbas, Hoda A. Hussein &
Reham A. Mohamed
To cite this article: Galal H. Elgemeie, Ali M. Salah, Nermeen S. Abbas, Hoda A. Hussein &
Reham A. Mohamed (2017) Pyrimidine non-nucleoside analogs: A direct synthesis of a novel class
of N-substituted amino and N-sulfonamide derivatives of pyrimidines, Nucleosides, Nucleotides and
Nucleic Acids, 36:3, 213-223, DOI: 10.1080/15257770.2016.1257808
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1257808
Published online: 19 Jan 2017.
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Date: 10 February 2017, At: 08:57

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –

http://dx.doi.org/./..
Pyrimidine non-nucleoside analogs: A direct synthesis of a
novel class of N-substituted amino and N-sulfonamide
derivatives of pyrimidines
Galal H. Elgemeie , Ali M. Salah , Nermeen S. Abbasa,b, Hoda A. Husseinc,
a
a
c
and Reham A. Mohamed
a
b
Chemistry Department, Faculty of Science, Helwan University, Helwan, Cairo, Egypt; Chemistry
c
Department, Faculty of Science, Taibah University, Saudi Arabia; Photochemistry Department, National
Research Center, Dokki, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
A convenient method for the regioselective synthesis of pyrimi-
dine non-nucleoside analogs was developed. This study reports
a novel and efficient method for the synthesis of a new type
of N-substituted amino methylsulfanylpyrimidines and the
corresponding pyrazolo[3,4-d]pyrimidines. This series of
compounds was designed through the reaction of dimethyl
Received  February 
Accepted  October 
KEYWORDS
N-substituted amino
methylsulfanylpyrimidines;
dimethyl N-
ꢀ
N-cyanodithioiminocarbonate with 2-cyano-N -(thiophen-2-yl-,
cyanodithioiminocarbonate;
ꢀ
ꢀ
furan-2-yl- and pyridin-4-ylmethylene)acetohydrazide and N -(2-
N -(-cyanoacetyl)arylsul-
cyanoacetyl)arylsulfonohydrazides. The scope and limitation of
the method are demonstrated. The antibacterial and antifungal
activities of the synthesized compounds were also evaluated.
fonohydrazides; antibacterial
and antifungal activities;
ꢀ

-cyano-N -(thiophen--yl-,
furan--yl- and pyridin--
ylmethylene)acetohydrazide;
pyrazolo[,-d]pyrimidines,
purine analogs
Introduction
Pyrimidines and purine bases play key roles in biological systems, since all DNA
bases are pyrimidine and purine derivatives, and many modern pharmaceutical
agents contain pyrimidine and purine fragments. Several examples of biolog-
ically important pyrimidine and purine derivatives are identified, such as the
signal transduction inhibitor imatinib (gleevec) which is highlighted as one of
the most significant new pyrimidine derivatives to have been identified in the
[1]
last few years. Besides their importance in exhibiting optimal cytotoxic effect
against the cancer cell lines, many derivatives are endowed with antiproliferative
effects when evaluated against a panel of tumor cell lines.[ It is worth noting
that sulfonamide derivatives bearing a pyrimidine or purine moieties are attractive
2]
[3]
molecular targets which possess many types of biological activities, includ-
[4]
ing anticancer activity. Consequently, many benzenesulfonamide derivatives,
bearing pyrimidine or purine moieties, has been designed and synthesized as
[5]
antimetabolic agents. We have recently reported different successful approaches
CONTACT Galal H. Elgemeie
versity,  Helwan, Cairo, Egypt.
elgemeie@yahoo.com
Chemistry Department, Faculty of Science, Helwan Uni-
©
 Taylor & Francis Group, LLC

214
G. H. ELGEMEIE ET AL.
Figure . Molecular structure of compound 5a.
for the synthesis of modified nucleoside analogs.[6–13] Derivatives of these ring
systems are interesting as antimetabolites in biochemical reactions.[14–19] In con-
tinuation of our research program and among the strategies addressed to the
synthesis of the pyrimidine and purine nucleoside analogs, the present study
focuses on the novel synthesis of methylsulfanylpyrimidine non-nucleoside analogs
and their corresponding pyrazolo[3,4-d]pyrimidines by the reaction of dimethyl
N-cyanodithioiminocarbonate with substituted hydrazides.
Results and discussion
Chemistry
It has been found that cyanamide reacts with carbon disulfide and methyl iodide in
the presence of alkali to produce the S,S-dimethyl N-cyanodithioiminocarbonate
ꢀ
1
. When compound 1 was treated with 2-cyano-N -(thiophen-2-yl-, furan-2-yl-
and pyridin-4-ylmethylene)acetohydrazide 2a-c at room temperature and in pres-
ence of pulverized potassium hydroxide-1,4-dioxane, the corresponding (1)N-
heterylmethyleneamino-4-methylsulfanylpyrimidines 5 was obtained. The com-
1
13
pounds 5 displayed spectroscopic data (IR, H NMR, C NMR and MS) and
elemental analysis result consistent with the proposed structures. In order to
prove unambiguously the structure of the compound 5a, its crystal structure was
measured. The X-ray analysis confirms the presence of the (1)N-(thiophene-2-
ylmethyleneamino)pyrimidine-6-one form 5a in the solid state (Figs. 1 and 2).
The synthesis of 5 from the reaction of 1 with 2 is assumed to proceed via Michael
addition of active methylene of 2 to the double bond in 1, the formed adducts then
cyclized smoothly via CH SH elimination and additionally to the cyano group.
3
Compounds 5 were treated with hydrazine in refluxing DMF to be converted
into the corresponding pyrazolo[3,4-d] pyrimidin-4(3H)-ones 6. The elemental
analysis and spectral data were compatible with the suggested individual structure
of compounds 6 (Chart 1). In order to investigate the scope of this reaction and
to establish whether the reaction of dimethyl N-cyanodithioiminocarbonate with

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
215
Figure . Packing diagram of 5a, viewed perpendicular to (). Thick dashed bonds represent clas-
sical H-bonds. Atom names correspond to the asymmetric unit.
Chart . Synthesized derivatives 5a-c and 6a-c.

2
16
G. H. ELGEMEIE ET AL.
Chart . Synthesized derivatives 10a,b and 11a,b.
substituted cyanohydrazides could be extended to provide a general approach to
(
1)N-substituted amino methylthiopyrimidines, we studied the reaction of dimethyl
N-cyanodithioiminocarbonate 1 with other functionalized cyanohydrazides. Thus,
it has been found that cyanoacetohydrazide reacts with 4-bromobenzene-1-sulfonyl
chloride and 4-chlorobenzene-1-sulfonyl chloride in ethanol to obtain the corre-
ꢀ
sponding N -(2-cyanoacetyl)arylsulfonohydrazides 7a,b in good yields. Then, the
syntheses of N-(4-methylthio-6-oxopyrimidin-1-yl)arylsulfonamides 10 was car-
ried out by the reaction of 7 with 1 in dioxane in the presence of a catalytic amount
of potassium hydroxide. The structure of 10 was characterized by spectroscopic
1
13
techniques including IR, H NMR and C NMR and elemental analysis. In order
to prove unambiguously the structure of compound 10a, its crystal structure was
measured (Chart 2). The X-ray analysis proves the presence of the pyrimidone
form 10a in the solid state (Figs. 3 and 4). The synthetic route to target compounds
1
0 from 1 and 7 is assumed to proceed via addition of the active methylene group

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
217
Figure . Molecular structure of compound 10a.
of 7 to the double bond of 1 to give intermediate Michael adducts. The latter
loses elements of CH SH to yield the intermediate 8. Cyclization of 8 led to the
3
novel N-(4-methylthio-6-oxopyrimidin-1-yl)arylsulfonamides 10. Compounds 10
is allowed to react with hydrazine in refluxing DMF to give the corresponding
analogs 11. The individual structure of compounds 11 was established on the basis
of elemental analysis and spectral data.
Antibacterial activity
An approach to screen the antimicrobial activity of compounds (5a, 5b, 5c, 6a,
1
0a and 10b) was examined against the bacterial strains Bacillus subtilis, Echerichia
coli, Pseudomonas aeruginosa, Staphylococcus aureus and the fungal strains, Aspe-
rigillusflavus and Candida albicans. Compound 10a has exhibited various degree
Figure . Packing diagram of 10a, viewed perpendicular to (). Thick dashed bonds represent clas-
sical H-bonds. Atom names correspond to the asymmetric unit.

2
18
G. H. ELGEMEIE ET AL.
Table . Antibacterial and antifungal data of the synthesized compounds 5a-c, 6a, 10a,b.
Inhibition zone diameter (mm / mg Sample)
Bacillus Escheric-
Pseudomo- Staphyloco-
Candida
albicans
(Fungus)
subtilis
hia coli nasaeruginosa ccusaureus Aspergillusflavus
Sample
(G+)
(G−)
(G−)
(G+)
(Fungus)
.
Control: DMSO
Standard
.
.
.
.
.
Ampicillin
Antibacterial agent
Amphotericin B
Antifungal agent




—
—
—
.
.
.


.
—
.
.
.
.

—
.
.
.
.

—
.
.
.
.




.
.
.




a
b
c
.
.
.
.
.
.

a


a
b
.
.
.
.
of inhibitory activity on the four pathogenic bacterial strains. It was observed that
this compound has an effect against gram positive and gram negative bacteria. The
bacterial zones of inhibition (mm) values are summarized in Table 1.
Antifungal activity
Compounds 5a and 10a are moderately active compared with the reference drug
(amphotericin) against C. albicans. The fungal zones of inhibition (mm) values are
summarized in Table 1.
Conclusion
In conclusion, searching for novel antimetabolic agents, we have achieved
the synthesis of interesting alkythiopyrimidine non-nucleoside analogs and
their corresponding pyrazolo[3,4-d]pyrimidines by the reaction of dimethyl
ꢀ
N-cyanodithioiminocarbonate with 2-cyano-N -(thiophen-2-yl-, furan-2-yl- and
pyridin-4-ylmethylene)acetohydrazide and N-cyanoacetoarylsulfonylhydrazides.
The compounds obtained seem promising as high potential intermediates for syn-
thesizing other antimetabolic agents and for biological screening.
Experimental
Chemistry
The melting points were determined on a Gallenkamp melting point apparatus and
1
were uncorrected. H NMR spectra were recorded on a Jeol-600 spectrometer oper-
1
13
ating at 600 MHz and 500 MHz for H and C nuclei, respectively, in DMSO-d with
6
Si(CH ) as internal standard at National Research Center, Cairo, Egypt. Shifts were
3
4
given in ppm and the abbreviations were as follows: s (singlet), d (doublet), t (triplet),

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
219
and m (multiplet). The mass spectra were run in the Cairo University. The reactions
were monitored by thin-layer chromatography (TLC) using aluminum sheets coated
with silica gel F254 (Merck). Spots were detected by their absorption under a short-
wavelength UV lamp. The reagents and solvents were purchased in commercially
available grade purity.
General procedure for the synthesis of a-c
Dimethyl N-cyanodithioiminocarbonate 1 (0.01 mol) was added to a stirred solu-
tion of the 1-cyanoacetyl-4-aryl methylidenesemicarbazide 3 (0.01 mol) in dry diox-
ane (30 mL) containing potassium hydroxide (0.01 mol) at room temperature. The
mixture was stirred magnetically for 1 h, then the precipitated product was filtered
off and recrystalized from the appropriate solvent to give the desired compound.
2
-Amino-4-(methylthio)-6-oxo-1-(thiophen-2-ylmethyleneamino)-1,6-
dihydropyrimidine-5-carbonitrile (5a). Colorless crystals; (DMF), yield (73.3%),
−
1
1
mp 260°C; IR (KBr, cm ) υ 3436 and 3283 (NH ), 2210 (CN) and 1651 (C=O). H
2
NMR (600 MHz, DMSO-d ): δ 2.49 (s, 3H, SCH ), 7.6 (s, br, 2H, NH ), 7.23–7.97
6
3
2
1
13
(
m, 3H, C H ), 9.1 (s, H, CH). C NMR: (DMSO) δ11.7 (SCH ), 82.0 (C-5), 115.9
4
3
3
ꢀ
ꢀ
ꢀ
ꢀ
(
CN), 127.1 (C-2 ), (C-3 ), 133 (C-4 ), 134.9 (C-1 ), 152.2 (CH=N), 158.0 (C-2),
64.1 (C=O), 171.9 (C-4). MS: m/z = 291. Anal. Calcd. For C H N OS (291.02):
1
11
9
5
2
C, 45.35; H, 3.11; N, 24.04; S, 22.01. Found: C, 45.33; H, 3.11; N, 24.01; S, 22.01.
2
-Amino-1-(furan-2-ylmethyleneamino)-4-(methylthio)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile (5b). Buff solid; (DMF), yield (90.8%), mp
−
1
1
2
68°C; IR (KBr, cm ) υ 3433 and 3279 (NH ), 2210 (CN) and 1656 (C=O). H
2
NMR (600 MHz, DMSO-d ): δ 2.52 (s, 3H, SCH ), 6.7 (s, br, 2H, NH ), 7.3–8.05
6
3
2
13
(
d, t, d, 3H, C H ), 8.5 (s, 1H, CH). C NMR: (DMSO) δ12.8 (SCH ), 82.0 (C-5),
16.1 (CN), 118.0 (C-2 ), (C-3 ), 122.0 (C-4 ), 147.0 (C-1 ), 154.2 (CH=N), 157.0
4
3
3
ꢀ
ꢀ
ꢀ
ꢀ
1
(
(
C-2), 159.1(C=O), 172.5 (C-4). MS: m/z = 275. Anal. Calcd. for C H N O S
1
1
9
5
2
275.29): C, 47.99; H, 3.30; N, 25.44; S, 11.65. Found: C, 47.95; H, 3.30; N, 25.41; S,
11.61.
2
-Amino-4-(methylthio)-6-oxo-1-(pyridin-4-ylmethyleneamino)-1,6-
dihydropyrimidine-5-carbonitrile (5c). White solid; (DMF), yield (50%), mp
−
1
2
58–260°C; IR (KBr, cm ) υ 3734 and 3447 (NH ), 2211 (CN) and 1679 (C=O).
2
1
H NMR (600 MHz, DMSO-d ): δ 2.51 (s, 3H, SCH ), 7.9 (s, br, 2H, NH ), 8.71–
6
3
2
13
8
.76 (m, 4H, C H ), 9.10 (s, 1H, CH). C NMR: (DMSO) δ 12.0 (SCH ), 80.0
5
4
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(C-5), 115.1 (CN), 120.1 (C-2 ), C-6 ), 139.0 (C-3 ), (C-5 ), 150.0 (C-1 ), 156.0
(
CH=N), 157.0 (C-2), 168.0 (C=O), 172.5 (C-4). MS: m/z = 286. Anal. Calcd. for
C H N OS (275.29): C, 50.34; H, 3.52; N, 29.35; S, 11.20. Found: 50.32; H, 3.52;
12
10
6
N, 29.31; S, 11.20.

2
20
G. H. ELGEMEIE ET AL.
General procedure for the synthesis of a-c
A mixture of 5a-c (0.01 mol) and hydrazine hydrate (0.02 mol) was heated in
(30 mL) DMF for 3 h. After cooling of the reaction, the precipitated final product
was filtered off.
3
4
3
,6-Diamino-5-(thiophen-2-ylmethyleneamino)-1H-pyrazolo[3,4-d]pyrimidin-
−
1
(5H)-one(6a). Yellow solid; (EtOH), yield (50%), mp > 300°C; IR (KBr, cm ) υ
1
451(NH ). H NMR (600 MHz, DMSO-d ): δ 5.07 (s, br, 2H, NH ), 5.13 (s, br, 2H,
2
6
2
NH ), 6.87–7.00 (m, 3H, C H ), 8.01 (s, 1H, NH), 9.00 (s, 1H, CH). Anal. Calcd.
2
4
3
for C H N OS (275.29): C, 43.63; H, 3.30; N, 35.62; S, 11.65. Found: C, 43.61; H,
10
9
7
3.30; N, 35.60; S, 11.64.
3
,6-Diamino-5-(furan-2-ylmethyleneamino)-1H-pyrazolo[3,4-d]pyrimidin-
−
1
4
(5H)-one(6b). Buff solid; (EtOH), yield (66.8%), mp > 300°C; IR (KBr, cm )
υ 3451 (NH ). H NMR (600 MHz, DMSO-d ): δ 5.09 (s, br, 2H, NH ), 5.15 (s,
1
2
6
2
2
H, NH ), 7.00–7.70 (m, 3H, C H ), 9.10 (s, 1H, CH),11.46 (s, br, 1H, NH). Anal.
2 4 3
Calcd. for C H N O (259.22): C, 46.33; H, 3.50; N, 37.82. Found: C, 46.31; H,
10
9
7
2
3.50; N, 37.81.
3
,6-Diamino-5-(pyridin-4-ylmethyleneamino)-1H-pyrazolo[3,4-d]pyrimidin-
−
1
4
(5H)-one (6c). Yellow solid; (EtOH), yield (42.36%), mp > 340°C; IR (KBr, cm )
υ 3433 (NH ). H NMR (600 MHz, DMSO-d ): δ 5.10 (s, br, 2H, NH ), 6.21 (s, br,
1
2
6
2
2
H, NH ), 7.00–7.90 (m, 4H, C H ), 8.30 (s, 1H, CH), 10.22 (s, 1H, NH). MS: m/z
270. Anal. Calcd. for C H N O (270.25): C, 48.89; H, 3.73; N, 41.46. Found: C,
11 10 8
2 5 4
=
4
8.87; H, 3.71; N, 41.46.
General procedure for the synthesis of a,b
To a stirred solution of N-cyanoacetoarylsulfonylhydrazides (0.01 mol) in dry
dioxane (30 mL) containing potassium hydroxide was added dimethyl N-
cyanodithioiminocarbonate 1 (0.01 mol). The mixture was stirred magnetically at
room temperature for 30 min, then the precipitated solid was collected by filtration
and recrystallized from DMF.
N-(2-amino-5-cyano-4-(methylthio)-6-oxopyrimidin-1(6H)-yl)-4-
bromobenzenesulfonamide (10a). Yellow crystals; (DMF), yield (40%), mp 210
1
°
8
(
C; H NMR (600 MHz, DMSO-d ): δ 2.51 (s, 3H, SCH ), 7.9 (s, br, 2H, NH ),
6
3
2
13
.71–8.76 (m, 4H, C H ), 9.1 (s, 1H, NH). C NMR: (DMSO) δ 11.5 (SCH ), 81.1
C-5), 115.8 (CN), 122.0 (C-4 ), 127.1 (C-2 ), (C-6 ), 128 (C-3 ), (C-5 ), 148.0 (C-2),
6
4
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
2
1
58.0 (C=O), 168.9 (C-4). Anal. Calcd. for C H BrN O S (416.27): C, 34.62; H,
1
2
10
5
3 2
.42; Br, 19.20; N, 16.82; S, 15.418. Found: C, 34.60; H, 2.42; Br, 19.20; N, 16.82; S,
5.41.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
221
N-(2-amino-5-cyano-4-(methylthio)-6-oxopyrimidin-1(6H)-yl)-4-
1
chlorobenzenesulfonamide (10b). Buff solid; (DMF), yield (20%), mp 220°C; H
NMR (600 MHz, DMSO-d ): δ 2.46 (s, 3H, SCH ), 7.25 (s, br, 2H, NH ), 7.31–7.62
6
3
2
(
m, 4H, C H ), 7.87 (s, br, 1H, NH). Anal. Calcd. for C H Cl N O S (371.82):
6 4 12 10 5 3 2
C, 38.76; H, 2.71; Cl, 9.53; N, 18.84; S, 17.25. Found: C, 38.74; H, 2.71; Cl, 9.52; N,
8.84; S, 17.25.
1
General procedure for the synthesis of a,b
The mixture of compound 10 (0.01 mol) and hydrazine hydrates (0.02 mol) in
(
30 mL) DMF was refluxed for 3 h. After allowing the reaction content to be cooled
to room temperature, then the crude product was filtered off and recrystallized from
the appropriate solvent to afford the desired compound.
4
-Bromo-N-(3,6-diamino-4-oxo-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-
yl)benzenesulfonamide (11a). Buff solid; (DMF), yield (24.5%), mp > 300°C;
IR (KBr) max/cm 3432 (NH). H NMR (600 MHz, DMSO-d ): δ 6.2 (s, br, 2H,
−
1
1
6
NH ), 7.46–7.52 (m, 4H, C H ), 10.19 (s, 1H, NH). Anal. Calcd. for C H BrN O S
2
6
4
11 10
7
3
(
1
400.21): C, 33.01; H, 2.52; Br, 19.97; N, 24.50; S, 8.01. Found: C, 33.01; H, 2.52; Br,
9.95; N, 24.50; S, 8.00.
4
-Chloro-N-(3,6-diamino-4-oxo-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-
yl)benzenesulfonamide (11b). White solid; (DMF), yield (55%), mp > 300°C;
IR (KBr, cm ) υ 3433 (NH). H NMR (600 MHz, DMSO-d ): δ5.20 (s, br, 2H,
−
1
1
6
NH ), 6.30 (s, br, 2H, NH ), 7.46–7.52 (m, 4H, C H ), 8.4 (s, 1H, NH). Anal. Calcd.
2
2
6
4
for C H Cl N O S (355.76): C, 37.14; H, 2.83; Cl, 9.97; N, 27.56; S, 9.01. Found:
11
10
7
3
C, 37.12; H, 2.83; Cl, 9.97; N, 27.55; S, 9.00.
Biological evaluation
In vitro antibacterial screening
The compounds 5a, 5b, 5c, 6a, 10a, 10b were evaluated for their in vitro antibacte-
rial activity, such as Bacillus subtilis, Echerichia coli, Pseudomonas aeruginosa, and
Staphylococcusaureus by a modified Kirby-Bauer disc diffusion.[ The bioassay was
performed using Mueller-Hinton agar (Hi-Media) medium that is rigorously tested
for composition and pH. 100 µL of the tested bacteria were grown in 10 mL of fresh
media until they reached a count of approximately 108 cells/mL.[ 100 µL of bac-
terial suspension was spread onto agar plates corresponding to the broth in which
they were maintained. Standard discs of Ampicillin (antibacterial agent) served as
positive controls for antibacterial activity, filter discs impregnated with 10 µL of sol-
vent (DMSO) were used as a negative control. The zone of inhibition (mm) was
20]
21]
[22]
measured after 24 h incubation at 30°C.

2
22
G. H. ELGEMEIE ET AL.
In vitro antifungal screening
The compounds 5a, 5b, 5c, 6a, 10a, 10b were evaluated for their in vitro antifun-
gal activity such the fungal strains, Asperigillusflavus and C. albicans by a modified
[20]
Kirby-Bauer disc diffusion. 100 µL of the test fungi were grown in 10 mL of fresh
[21]
media until they reached a count of approximately 105 cells/mL. 100 µL of fungal
suspension was spread onto agar plates corresponding to the broth in which they
were maintained. Standard discs of Amphotericin B (antifungal agent) served as
positive controls for antifungal activity, filter discs impregnated with 10 µL of sol-
vent (DMSO) were used as a negative control. Plates inoculated with filamentous
fungi incubated at 30°C for 24–48 hours and, then the diameters of the inhibition
[23]
zones were measured in millimeters.
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