Synthesis, antibacterial, and antifungal activities of new pyrimidinone derivatives

Article abstract of DOI:10.1515/hc-2015-0066

An efficient synthesis of new pyrrolopyrimidinones 3a-d and isoxazolopyrimidinones 4a-c from the respective aminocyanopyrroles 1a-d and aminocyanoisoxazoles 2a-c is presented. The synthesized compounds were screened for antimicrobial activity against a panel of bacteria and fungi. Compound 4c exhibits remarkable activity against a broad spectrum of Gram-positive and Gram-negative bacteria and pathogenic fungi.

Full text of DOI:10.1515/hc-2015-0066

Heterocycl. Commun. 2015; aop
Preliminary Communication
Oussama Cherif, Fatma Masmoudi, Fatma Allouche, Fakher Chabchoub* and Mohamed Trigui
Synthesis, antibacterial, and antifungal activities
of new pyrimidinone derivatives
DOI 10.1515/hc-2015-0066
Received March 6, 2015; accepted April 12, 2015
group to give an amide and the formylation of the amino
group to give a formamide followed by intramolecular
condensation of these two functionalities, as shown.
All compounds were screened for their antibacterial
Abstract: An efficient synthesis of new pyrrolopyrimi- and antifungal activities against a number of reference
dinones 3a-d and isoxazolopyrimidinones 4a-c from the test organisms, including five Gram-positive, three Gram-
respective aminocyanopyrroles 1a-d and aminocyanoisox- negative, and eight plant pathogenic fungi. The study of
azoles 2a-c is presented. The synthesized compounds antimicrobial activities showed that compounds 1–3 and
were screened for antimicrobial activity against a panel 4a,b exhibit very low activities. A notable exception is
of bacteria and fungi. Compound 4c exhibits remarkable compound 4c, which is active against a broad spectrum of
activity against a broad spectrum of Gram-positive and Gram-positive and Gram-negative bacteria (Table 1). The
Gram-negative bacteria and pathogenic fungi.
same compound 4c shows also high antifungal activity
Table 2).
(
Keywords: antimicrobial activity; isoxazolopyrimidi-
nones; pyrrolopyrimidinones.
Experimental
Melting points were measured on an electrothermal apparatus.
Pyrroles and isoxazoles [1–4] have received intensive
research interests because of their biological activities and Progress of the reactions was monitored by thin-layer chromatogra-
have found a wide range of applications in pharmaceuti- phy (TLC) using aluminum sheets with silica gel 60 F254 from Merck.
Infrared spectra (IR) were recorded on a Perkin-Elmer Paragon FT-IR
cal and agrochemical fields [5–8]. In continuation of our
1
spectrometer using KBr pellets. Unless stated otherwise, H NMR and
previous study on the synthesis and biological activity of
1
3
C NMR spectra were recorded in CDCl on a Bruker spectrometer
3
heterocyclic compounds [9], we now report the prepara-
(1
13
H at 400 MHz, C at 100 MHz). Elemental analyses were performed
tion of a series of pyrrolopyrimidinones 3a-d and isoxa-
on a Carlo Erba 1106 apparatus. Substrates 1a-d and 2a-c were syn-
zolopyrimidinones 4a-c starting with readily available thesized as previously reported [10, 11].
pyrroles 1a-d and isoxazoles 2a-c [10, 11]. Compounds
3
a-d and 4a-c were synthesized by heating the respective
General procedure for the synthesis of compounds 3a-d
and 4a-c
substrates 1a-d and 2a-c under reflux in formic acid in the
presence of a catalytic amount of sulfuric acid. The yields
ranged from 57% to 81% (Scheme 1). Mechanistically, the
reaction may involve the partial hydrolysis of the cyano
3-Amino-4-cyano-substituted pyrrole 1 or 5-amino-4-cyano-substituted
isoxazole 2 (10 mmol) was added portionwise for 2 h to a mildly reflux-
ing mixture of formic acid (20 mL) and concentrated sulfuric acid
(1.2 mL). Af er an additional 30 min, the solution was cooled to 0°C and
poured onto crushed ice. The resulting precipitate was collected by fil-
tration, washed with water, dried, and crystallized from ethanol to give
a pyrrolopyrimidinone 3 or an isoxazolopyrimidinone 4.
*
Corresponding author: Fakher Chabchoub, Laboratory of Applied
Chemistry Heterocycles, Fats and Polymers, Faculty of Sciences of
Sfax, University of Sfax, Sfax 3018, Tunisia,
e-mail: fakher.chabchoub@yahoo.fr
Oussama Cherif and Fatma Allouche: Laboratory of Applied
Chemistry Heterocycles, Fats and Polymers, Faculty of Sciences of
Sfax, University of Sfax, Sfax 3018, Tunisia
Fatma Masmoudi and Mohamed Trigui: Biopesticides Team (LPIP),
Center of Biotechnology of Sfax, University of Sfax, PO Box 1177,
Sfax 3018, Tunisia
Methylꢀ5,6-dimethyl-4-oxo-4,6-dihydro-3H-pyrrolo[3,4-d]
pyrimidine-7-carboxylate (3a)ꢀYield 81%; yellow solid; mp 231–
-1
1
233°C; IR (cm ): ν 1596, 1692, 3187; H NMR: δ 8.29 (s, 1H, NH), 7.89 (s,
1
3
1H, H-2), 4.01 (s, 3H, CH -7), 2.76 (s, 3H, CH -6), 2.19 (s, 3H, CH -5);
C
3
3
3
NMR: δ 159.3 (C-4), 157.7 (CO Me), 145.2 (C-2), 143.6 (C-5), 133.9 (C-7a),
2
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ꢀꢀꢀꢀꢁꢀO. Cherif et al.: New pyrimidinone derivatives
R2
R2
N
R2
O
N
CN
CONH2
H3C
1
N
NH
-
H2O
NH2
H3C
1
N
H3C
1
NHCHO
R O2C
R O2C
1
a-d
R O2C
HCOOH
H2SO4
3
a-d
Or
R1
O
R1
N
R1
CN
CONH2
NH
-
H2O
N
NH2
N
O
NHCHO
O
O
N
4a-c
2a-c
2
1
3
3
3
3
a: R1 = Me; R = Me
4a: R = Me
1
2
1
b: R = Et; R = Me
4b: R = Et
1
2
1
c: R = Me; R = Et
4c: R = Ph
1
2
d: R = Me; R = Ph
Scheme 1ꢀ
Table 1ꢀAntibacterial activities of compound 4c in vitro.
ꢁꢁ
Bacterial strains
Gram-positive bacteria
ꢁꢁ
Gram-negative bacteria
Bc
ꢁ
Sa
ꢁ
Ef
ꢃꢆ
ꢁ
Ml
ꢁ
Lm
Ec
ꢁ
Se
ꢁ
Kp
Compound ꢂcꢂ
IZa
ꢂ
ꢃꢄ
ꢅ.ꢅꢃ
ꢇꢅ
ꢅ.ꢅꢅ4
–
ꢂ
ꢃꢅ
ꢂ
ꢂ
ꢅ9
ꢂ
ꢃꢇ
ꢂ
ꢃꢃ
ꢂ
ꢃꢇ
ꢂ
ꢃꢈ
b
ꢂ
MIC ꢂ
ꢂ ꢅ.ꢅ4ꢆ
ꢂ ꢅ.ꢅꢄꢈ
ꢂ ꢅ.ꢅꢄꢈ
ꢂ ꢅ.ꢉꢄꢊ
ꢂ ꢅ.ꢅꢄꢈ
ꢂ ꢅ.ꢃꢊꢇ
ꢂ ꢅ.ꢅꢄꢈ
Gentamicinc
DMSO ꢇꢅ%
ꢂ
ꢂ
ꢂ
IZ
ꢂ
ꢂ
ꢇꢅ
ꢂ
ꢇꢅ
ꢂ
ꢃꢄ
ꢂ
ꢇꢅ
ꢂ
ꢇꢈ
ꢂ
ꢇꢈ
ꢂ
ꢇꢇ
MIC ꢂ
ꢂ ꢅ.ꢅꢅ4
ꢂ ꢅ.ꢅꢅ4
ꢂ ꢅ.ꢅꢅ4
ꢂ ꢅ.ꢅꢅꢃ
ꢂ ꢅ.ꢅꢅꢇ
ꢂ ꢅ.ꢅꢅꢇ
ꢂ ꢅ.ꢅꢅꢇ
d
IZ
ꢂ
ꢂ
–
ꢂ
–
ꢂ
–
ꢂ
–
ꢂ
–
ꢂ
–
ꢂ
–
Bacterial strains: Bc, Bacillus cereus ATCC1ꢋ57ꢌ; Sa, Staphylococcus aureus ATCC25ꢌ23; Ef, Enterococcus faecalis ATCC 2ꢌ212; Ml, Micro-
coccus luteus ATCC 1880; Lm, Listeria monocytogenes (FI 2132); Ec, Escherichia Coli ATCC 25ꢌ22; Se, Salmonella enteritidis (food isolate);
Kp, Klebsiella pneumonia CIP 321ꢋ7.
a
Diameter of the inhibition zone (IZ) in mm of a compound, including the diameter of the disc (6 mm).
Minimum inhibitory concentration in mg/mL.
b
c
Gentamicin was used as a standard antibiotic at 15 μg/mL.
d
Activity not detected.
Table 2ꢀAntifungal activities of compound 4c.
Compounds
ꢁ
ꢁꢁ
Fungal strains
Rnꢁ
Foꢁ
Aaꢁ
Ppꢁ
Fcꢁ
Bcꢁ
Fgꢁ
Af
ꢂc
ꢂ
ꢂ
IZa
ꢂ
ꢃꢃꢂ
ꢅ.ꢉꢄꢊꢂ
ꢃꢅꢂ
ꢅ.ꢆ4ꢆꢂ
–ꢂ
ꢃꢅꢂ
ꢅ.ꢃꢊꢇꢂ
ꢃꢇꢂ
ꢅ.ꢅ4ꢇꢂ
–ꢂ
ꢃꢆꢂ
ꢅ.ꢉꢄꢊꢂ
ꢃꢇꢂ
ꢅ.ꢃꢊꢇꢂ
–ꢂ
ꢃꢇꢂ
ꢅ.ꢆ4ꢆꢂ
ꢃꢄꢂ
ꢅ.ꢆꢃꢇꢂ
–ꢂ
ꢃꢆꢂ
ꢅ.ꢃꢊꢇꢂ
ꢃ4ꢂ
ꢅ.ꢅꢄꢈꢂ
–ꢂ
ꢃꢈꢂ
ꢅ.ꢉꢄꢊꢂ
ꢃꢇꢂ
ꢅ.ꢃꢊꢇꢂ
–ꢂ
ꢃꢅꢂ
ꢅ.ꢆ4ꢆꢂ
ꢃ4ꢂ
ꢅ.ꢆ4ꢆꢂ
–ꢂ
ꢃ9
ꢈ.ꢈ
b
MIC ꢂ
Amphotericinc ꢂ
ꢂ
DMSO ꢇꢅ%
IZ
ꢂ
ꢃꢅ
MIC ꢂ
ꢅ.ꢃꢊꢇ
–
d
ꢂ
IZ
ꢂ
Fungal strains: Rn, Rhizopus nigricans (LPAP26); Fo, Fusarium oxysporum CTM10ꢋ02; Aa, Alternaria alternata CTM 10230; Pp, Pythium
aphanidermatum (LPAP32); Fc, Fusarium culmorum ISPAVE 21W; Bc, Botrytis cinerea (LPAP3ꢋ); Fg, Fusarium graminearum ISPAVE 271;
Af, Aspergillus flavus (food isolate).
a
Diameter of the inhibition zone of a compound, including the diameter of the disc (6 mm).
Minimal inhibitory concentration in mg/mL.
b
c
Amphotericin B was used as antifungal standard at 20 μg/mL.
d
Activity not detected.
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O. Cherif et al.: New pyrimidinone derivativesꢍꢁꢀꢀꢀꢍ3
1
14.1 (C, C-7), 106.6 (C-4a), 31.8 (CH -7), 13.3 (CH -6), 10.6 (CH -5). Anal. (food isolate 824) (Se), and Listeria monocytogenes (food isolate
3
3
3
Calcd for C H N O : C, 54.29; H, 5.01; N, 19. Found: C, 53.80; H, 5.20; 2132) (Lm). The fungi tested were Rhizopus nigricans (LPAP26) (Rn),
1
0
11
3
3
N, 18.33.
Fusarium oxysporum CTM10402 (Fo), Alternaria alternata CTM 10230
Aa), Pythium aphanidermatum (LPAP32) (Pp), Fusarium culmorum
(
Ethyl 5,6-dimethyl-4-oxo-4,6-dihydro-3H-pyrrolo[3,4-d]pyrimi- ISPAVE 21W (Fc), Botrytis cinerea (LPAP34) (Bc), Fusarium gramine-
dine-7-carboxylate (3b)ꢀYield 71%; yellow solid; mp 243–245°C; IR arum ISPAVE 271 (Fg), and Aspergillus flavus (food isolate) (Af). The
-
1
1
(
cm ): ν 1597, 1691, 3173; H NMR: δ 8.24 (s, 1H, NH), 8.12 (s, 1H, H-2), bacterial strains were cultivated in Mueller-Hinton (MH) agar (Oxoid
3
4
.43 (q, 2H, J ꢁ=ꢁ 7.5 Hz, CH ), 3.91 (s, 3H, CH -6), 2.61 (s, 3H, CH -5), 1.33 Ltd., UK) at 37°C except for Bacillus species, which were incubated at
2
3
3
3
13
(
t, 3H, J ꢁ=ꢁ 7.5 Hz, CH -7); C NMR: δ 160.1 (C-4), 159.1 (CO Et), 146.2 30°C. The fungi were cultured on potato dextrose agar (PDA) medium
3
2
(
C-2), 144.9 (C-5), 135.3 (C-7a), 115.4 (C-7), 107.0 (C-4a), 59.9 (CH ), 32.9 and incubated at 28°C. Working cultures were prepared by inoculat-
2
(
CH -6), 14.4 (CH -7), 11.0 (CH -5). Anal. Calcd for C H N O : C, 56.16; ing a loopful of each test bacteria in 3 mL of MH broth (Oxoid Ltd.,
3 3 3 11 13 3 3
H, 5.57; N, 17.86. Found: C, 55.66; H, 6.02; N, 17.38.
UK) and were incubated at 37°C for 12 h. For the test, final inoculum
7
concentrations of 10 CFU/mL of bacteria were used. Fungal spore
Methyl 5-ethyl-6-methyl-4-oxo-4,6-dihydro-3H-pyrrolo[3,4-d] suspensions were collected from the surface of such fungal colo-
pyrimidine-7-carboxylate (3c)ꢀYield 73%; yellow solid; mp 247– nies by gently scraping with a loop and suspended in 10 mL potato
-
1
1
2
50°C; IR (cm ): ν 1597, 1680, 3187; H NMR: δ 8.32 (s, 1H, NH), 8.11 dextrose broth. This suspension was mixed vigorously by vortexing
3
(
s, 1H, H-2), 4.09 (s, 3H, CH -7), 4.01 (s, 3H, CH -6), 3.23 (q, 2H, J ꢁ=ꢁ for 15–20 min. The spore suspension stock was diluted to obtain a
3
3
3
13
6
7
.5 Hz, CH ), 1.29 (t, 3H, J ꢁ=ꢁ 7.5 Hz, CH -5); C NMR: δ 160.7 (C-4), concentration of 10 spores/mL (measured by Malassez blade).
2
3
1
58.2 (CO Me), 145.8 (C-2), 141.2 (C-5), 139.5 (C-7a), 111.3 (C-7), 106.0 Antibacterial and antifungal tests were performed by disc diffusion
2
(
C-4a), 51.0 (CH -7), 32.9 (CH -6), 17.5 (CH -5), 13.1 (CH ). Anal. Calcd for method [12] and broth microdilution assay using sterile MH media
3 3 3 2
C H N O : C, 56.16 H, 5.57; N, 17.86 . Found: C, 55.04; H, 5.83; N, 17.29. (Bio-Rad, France) for bacterial strains PDA (Bio-Rad, France) for anti-
1
1
13
3
3
fungal tests. Freshly prepared cell suspension (100 μL) adjusted to
7
6
Methyl 6-methyl-4-oxo-5-phenyl-4,6-dihydro-3H-pyrrolo[3,4-d] 10 CFU/mL for bacteria and 10 spores/mL for fungus were inocu-
pyrimidine-7carboxylate (3d)ꢀYield 65%; yellow solid; mp 232– lated onto the surface of agar plates. Thereaf er, discs with 6 mm in
-1
1
2
(
35°C; IR (cm ): ν 1600, 1693, 3296; H NMR: δ 8.34 (s, 1H, NH), 8.13 diameter were punched in the inoculated agar medium with sterile
s, 1H, H-2), 7.46–7.56 (m, 5H, H-arom), 4.14 (s, 3H, CH -7), 4.01 (s , 3H, Pasteur pipettes, and compounds were added to each disc. Gen-
3
1
3
CH -6); C NMR: δ 161.1 (C-4), 157.7 (CO Me), 145.4 (C-2), 141.4 (C-5), tamicin (10 μg/disc) was used as a positive control for bacteria,
3
2
1
7
27.9–136.9 (C-arom), 115.3 (C-7a), 114.4 (C-7), 107.5 (C-4a), 51.1 (CH3- whereas amphotericin B (20 μg/disc) was used as a positive control
), 35.0 (CH -6). Anal. Calcd for C H N O : C, 63.60; H, 4.63; N, 14.83. for fungal strains. The plate was allowed to stand for 2 h at 4°C to
3
15 13
3
3
Found: C, 62.76; H, 4.64; N, 14.29.
permit the diffusion of the compounds followed by incubation at
7°C for 24 h for bacterial strains and 72 h for fungi at 28°C. The anti-
-Methylisoxazolo[5,4-d]pyrimidin-4(5H)-one (4a)ꢀYield 66%; bacterial activity was evaluated by measuring the zones of inhibition
3
3
-
1
1
white solid; mp 232–235°C; IR (cm ): ν 1567, 1707, 3182; H NMR: (clear zone around the disc) against the tested microorganisms. All
13
δ 12.74 (s, 1H, NH), 8.28 (s, 1H, H-6), 2.46 (s, 3H, CH ); C NMR: δ 175.10 tests were repeated three times. Minimum inhibitory concentrations
3
(
C-7a), 157.7 (C-4), 157.2 (C-3), 152.2 (C-6), 101.5 (C-3a), 10.9 (CH ). Anal. (MICs) of compounds were determined in sterile 96-well microplates
3
Calcd for C H N O : C, 47.69; H, 3.33; N, 27.81. Found: C, 46.83; H, 4.16; with a final volume in each microplate well of 200 μL [13]. A twofold
6
5
3
2
N, 2 7.0 5.
serial dilution of the compound was prepared in the microplate wells
over the range 0.01–5.5 mg/mL. To each test well was added 10 μL
6
3
-ethylisoxazolo[5,4-d]pyrimidin-4(5H)-one (4b)ꢀYield 61%; cell suspension to final inoculum concentrations of 10 CFU/mL for
-1
1
5
white solid; mp 185–188°C; IR (cm ): ν 1567, 1702, 3188; H NMR: bacteria and 10 spores/mL for fungi. The plates were then covered
3
δ 12.21 (s, 1H, NH), 8.24 (s, 1H, H-6), 3.03 (q, 2H, J ꢁ=ꢁ 7.7 Hz, CH ), with the sterile plate covers and incubated at 37°C for 24 h for bacte-
2
3
13
1
(
.45 (t, 3H, J ꢁ=ꢁ 7.7 Hz, CH ); C NMR: δ 175.9 (C-7a), 158.9 (C-4); 157.8 rial strains and 72 h for fungi at 28°C. The MIC was defined as the
3
C-3), 154.2 (C-6), 102.7 (C-3a), 15.9 (CH ), 10.6 (CH ). Anal. Calcd for lowest concentration of the compound at which the microorganism
2
3
C H N O : C, 50.91; H, 4.27; N, 25.44. Found: C, 50.95; H, 4.37; N, 25.37.
does not demonstrate visible growth af er incubation. As an indica-
tor of microorganism growth, 25 μL of 0.5 mg/mL p-iodonitrotetrazo-
7
7
3
2
3
-Phenylisoxazolo[5,4-d]pyrimidin-4(5H)-one (4c)ꢀYield 57%; lium chloride, dissolved in sterile water, was added to the wells and
-
1
1
white solid; mp 216–218°C; IR (cm ): ν 1559, 1692, 3190; H NMR: incubated at 37°C for 30 min. The lowest concentration of compound
δ 10.58 (s, 1H, NH), 8.12 (s, 1H , H-6), 7.19–8.24 (m, 5H, H-arom); 13C NMR: showing no growth was taken as its MIC.
δ 170.5 (C-7a), 161.2 (C-4) , 160.2 (C-3), 144.7 (C-6), 123.4–130.7 (C-Carom),
1
15.4 (C-3a). Anal. Calcd for C H N O : C, 61.97; H, 3.31; N, 19.71. Found:
C, 61.32; H, 3.68; N, 18.69.
11 7 3 2
Acknowledgments: The authors acknowledge the Ministry
of Higher Education, Scientific Research, and Technology
in Tunisia for their financial support.
Antimicrobial activity
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