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Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
Full Paper Synthesis and Antimicrobial Activity of Some New Heterocycles Incorporating the Pyrazolopyridine Moiety Sraa Abu-Melha Faculty of Science of Girls, King Khaled University, Abha, Saudi Arabia
2-Cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide (2) was utilized as key intermediate for the synthesis of some new coumarin 3, pyridine 4, pyrrole 5, thiazole 8, pyrido[20 ,30 :3,4]-pyrazolo[5,1-c]triazine 7, and aminopyrazolo 10 compounds. 2-Cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-3-(dimethylamino)acrylamide (11) was synthesized and allowed to react with hydroxylamine, hydrazine, and guanidine to afford regioselectively the isoxazole 13, pyrazole 15, and pyrimidine 17 derivatives, respectively. The reaction of 11 with thiourea and/or with ethyl glycinate in basic medium afforded the regioisomeric pyrimidinethione 18 and 3,5-dioxo-1,4-diazepine-6-carbonitrile 23. All the synthesized products were tested and evaluated as antimicrobial agents. Keywords: Aminopyrazole / Antimicrobial activity / Pyrazolo[3,4-b]pyridine / Pyridine / Pyrimidine Received: May 24, 2013; Revised: August 13, 2013; Accepted: August 15, 2013 DOI 10.1002/ardp.201300195
Introduction 1H-Pyrazolo[3,4-b]pyridines comprise a very interesting class of compounds because of their significant and diverse biological and pharmacological activities, such as antimalarial [1], antiproliferative [2], antimicrobial [3–5], cyclin-dependent kinase-inhibiting [6], cardiovascular [7–9], antiviral [10–12], and antileishmanial activities [13]. Pyrazolefused pyridines and pyrimidines are known to possess a wide range of biological activity. Specifically, pyrazolopyridines exhibit antitubercular and anxiolytic effects [14]. The antibacterial activity caught our attention because antimicrobial resistance developed by important pathogens has increased in the last decade [15]. Besides, emerging and reemerging bacterial infections diseases still cause death and disability worldwide [16]. In view of the above-mentioned findings and as continuation of our effort from our laboratory to identify new candidates that may be of value in designing new potent, selective, and less toxic antimicrobial agents, we report herein the synthesis of some new heterocycles incorporating the pyrazolopyridine moiety starting from 4,6-dimethyl-1H-
Correspondence: Sraa Abu-Melha, Faculty of Science of Girls, King Khaled University, Abha 35516, Saudi Arabia. E-mail: [email protected] Fax: þ966 7 2284691
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
pyrazolo[3,4-b]pyridine-3-amines in order to investigate their antimicrobial activity.
Results and discussion Chemistry The synthetic procedures adopted to obtain the target compounds are depicted in Schemes 1–4. The starting 4,6dimethyl-1H-pyrazolo[3,4-b]pyridine-3-amine 1 was prepared according to the reported method [17, 18]. The increasing importance of N-(cyanoacetyl)aminopyrazolopyridine (2) as a versatile intermediate for the synthesis of several heterocyclic rings with many pharmaceutical applications led to the continuing development of new simple procedures for their synthesis. The most effective and suitable method for the preparation of 2 with high yield and purity is the utilization of 1-cyanoacetyl-3,5-dimethyl-pyrazole as cyanoacetylation reagent [19] (Schemes 1 and 2). Thus, cyclocondensation of compound 2 with salicylaldehyde in boiling ethanol containing a catalytic amount of piperidine afforded the coumarin derivative 3. The reaction of 2 with 1,3-dicarbonyl compound was studied in the aim of formation of pyridine derivatives with potential biological activities [20, 21]. Thus, it reacted with acetylacetone to give the pyridine derivative 4. Structure of the latter product was based on analytical and spectral data. The IR spectrum showed three absorption bands at 3410, 2219, and 1685 cm1
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
CH3
CH3
NH2 N
H3C
H3C
913
NHCOCH2CN
Toluene
CH3 N N COCH2CN
+
N H
N
Novel Pyrazolopyridines
N H3C
N H
N
1
2 Scheme 1. Structures of compounds 1 and 2.
due to NH, CN, and CO groups. Its 1H NMR spectrum revealed the appearance of new three singlets at d 2.38, 2.57, and 5.56 ppm assigned to two methyl protons and the pyridinone H-5. Recently, the reaction of the cyanoacetamide moiety with a-halocarbonyl compounds was reported, which represents a new, simple, and efficient synthetic route for the synthesis of pyrrole derivatives [22]. Therefore, it was interesting to study H3 C
CH3
H N N
CHO
Ar
Ar
O
C
NH
O
N
N
N N H
NH
N2Cl N
H3C
CH3
CH3
N
7
EtOH/ piperidine
3 H3C
N
H N
6 CH3
O
O
Ar
AcOH/
O
OH
H N
H3C
N N NH CN
H N Ar
the reaction of 2 with phenacyl bromide in boiling ethanol using triethylamine as a basic catalyst that furnished the pyrrole derivative 5. The analytical and spectral data are in agreement with the proposed structure. Thus, 1H NMR spectrum of the reaction product showed new two doublet signals at d 4.54 and 5.65 ppm corresponding to the two vicinal pyrrolidine protons H-3 and H-4, respectively, two singlets at d 2.34 and 2.51 ppm corresponding to two CH3
N
N H
O
S Ar
CN
H N
CN PhNCS/ S/ TEA
O
O
Ar
H N
N Ph NH2
O
2
4
S
8
O Ph Ar
Ph
Br
1) PhNCS/ KOH/ DMF 2) MeI
CN
N O
5
Ar
PhHN H N
SCH3 CN
PhHN
NH2NH2.H2O EtOH
O
Ar
N NH
H N
NH2
O
10
9 CH3 Ar = CH3
N
N NH
Scheme 2. Synthesis of compounds 3–10.
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groups, and a multiplet at d 7.30–7.60 ppm corresponding to aromatic protons. The IR spectrum showed the presence of C –– O group stretching at 1690 cm1 and CN group at 2195 cm1. In continuation of the synthesis of bridged-head nitrogen heterocyclic systems, it was found that diazotized heterocyclic amine is an excellent building block for the synthesis of the target compound. Thus, coupling of compound 2 with 4,6dimethyl-1H-pyrazolo[3,4-b]pyridin-3-diazonium chloride in pyridine at 0–5°C afforded the corresponding hydrazono compound 6. When compound 6 is refluxed in acetic acid, it can be cyclized to 7. The formation of 7 may be interpreted through the nucleophilic attack of the ring nitrogen on the cyano group. The IR spectrum of compound 7 showed three absorption bands at 3406, 3288, and 3228 cm1 due to four NH groups besides carbonyl absorption bands at 1672 cm1. Its 1H NMR spectrum revealed three D2O-exchangeable singlets at d 9.12, 9.33, and 10.24 ppm due to four NH protons (see Experimental section). The reaction of compound 2 with both elemental sulfur and phenyl isothiocyanate in warming ethanol containing a catalytic amount of triethylamine gave the thiazole derivative 8. Elemental analysis, IR, 1H NMR, and 13C NMR results are in agreement with the proposed structure. Treatment of compound 2 with phenyl isothiocyanate in DMF, and in the presence of potassium hydroxide, at room temperature, followed by treatment with methyl iodide afforded the novel ketene N,S-acetal 9. The structure of 9 was established on the basis of its elemental analysis and spectral data. Its IR spectrum showed absorption bands at 3350, 3268, and 2190 cm1 due to three NH groups and nitrile functions, respectively. The mass spectrum showed a molecular ion peak at m/z 378 (Mþ), which agrees with its molecular formula C19H18N6OS. Reaction of 9 with hydrazine in refluxing ethanol gave the corresponding pyrazole derivative 10. The chemical structure of 10 was established on the basis of its elemental analysis and spectral data. Its 1H NMR spectrum displayed a broad signal at d 6.92–7.45 ppm related to the aromatic protons, and another three singlets at d 9.97, 10.72, and 11.35 ppm assignable to four NH protons. Treatment of 2 with dimethyformamide-dimethylacetal (DMF-DMA) in refluxing toluene afforded the corresponding enaminonitrile 11. The structure of 11 has been assigned as a reaction product on the basis of analytical and spectral data. The IR spectrum displayed absorption bands at 3230 cm1 due to NH function, and at 2196 cm1 due to conjugated CN function, at 1668 cm1 due to amidic C –– O function. The behavior of enaminonitrile 11 toward some N-nucleophiles to attain polyfunctionally substituted azoles, azines, and related fused systems linked to a thiazole moiety through a carboxamide linkage of potential pharmaceutical interest has been investigated. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
Treatment of 11 with hydroxylamine hydrochloride in refluxing ethanol–dimethylformamide (1:1) mixture in the presence of anhydrous potassium carbonate afforded an orange product that was identified as 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)isoxazole-4-carboxamide 13. The spectral data of the isolated product was in complete agreement with structure 13. The IR spectrum revealed the lack of an absorption band corresponding to a conjugated CN function. The 1H NMR spectrum (DMSO-d6) showed two new broad singlet signals at d 4.56 and 11.44 ppm and sharp singlet signals at d 8.88 ppm characteristic of NH2, NH, and isoxazole-H3 protons, respectively, besides a singlet signal at d 7.71 ppm distinctive for pyridine protons. The mass spectrum showed a molecular ion peak at m/z ¼ 272, corresponding to the molecular formula C12H12N6O2. The 5-aminopyrazole 15 was achieved as a sole product by heating the enaminonitrile 11 with hydrazine hydrate in ethanol. Inspection of 1H NMR spectrum enabled establishing structure 15 for this pyrazole derivative since the pyrazole H-3 appeared as a singlet at d 7.87 ppm. We could not trace in the 1 H NMR spectrum any signals for the tautomeric 3-aminopyrazole as this could reveal pyrazole-H5 as a doublet. The mass spectrum of 15 showed a molecular ion peak (Mþ) at m/z ¼ 271 corresponding to the molecular formula C12H13N7O. Similarly, the enaminonitrile 11 reacted with guanidine in a mixture of ethanol and dimethylformamide (2:1) containing anhydrous potassium carbonate under reflux to yield, in good yield, a product that was identified as 2,4-diamino-N-(4,6dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidine-5-carboxamide 17. The structure of 17 was assigned on the basis of the elemental analysis and spectral data. The IR spectrum showed absorption bands at 3382–3122 cm1, corresponding to two NH2 and two NH functions, and at 1664 cm1 due to amidic C –– O function. The mass spectrum showed a molecular ion peak (Mþ) at m/z ¼ 298, corresponding to the molecular formula C13H14N8O. Formation of compounds 13, 15, and 17 is assumed to take place via a Michael type addition of the amino group of the hydroxylamine, hydrazine, and guanidine to the activated double bond in compound 11 to form the non-isolable intermediates 12, 14, and 16, which readily undergo intramolecular cyclization followed by loss of dimethylamine molecule to form the target compounds (Scheme 3). The site selectivity in cycloaddition of some nitrogen ambident nucleophiles with the enaminonitrile 11 was also studied. Thus, reaction of 11 with thiourea in refluxing ethanol containing a catalytic amount of piperidine afforded a single product 18 (as examined by TLC) for which cycloadduct 18 seemed possible (Scheme 4). However, the pyrimidinethione 18 was assigned for the reaction product on the basis of its elemental analysis and spectral data. The IR spectrum lacked an absorption band due to a nitrile function www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
Ar
H N
H H CN N
NH2OH
CN
Ar
EtOH/ DMF
O
O
2
_ HNMe
OH
Ar
NMe2
NH2NH2 O
NMe2
N O
13
H H CN N
EtOH
Ar O
11
_ HNMe
NH2
NH2
NH
H N
Ar
N O
NMe2
15
HN NH 2 H H CN N NH Ar O NMe2
NH2
EtOH/ DMF
_
Ar
S
H N
O
NH2
EtOH/ Piperidine
Ar
NH2
N
H2N H N
HNMe2
N
O
16
NH2
H N
H2N
2
14
HN
915
O
H N
NH
CN
H N
H2N 2
12
DMF-DMF dry toluene
Ar
Novel Pyrazolopyridines
17
Scheme 3. Synthesis of the isoxazole 13, pyrazole 15, and pyrimidine 17 derivatives.
S NH
H N O
18
11 O
OEt
H2N _
b
OEt
O ClH3N
OEt
EtOH/ DMF/ TEA
Ar
H H N
CN a
a
HNMe2
Ar
H N O
O
20
NH
O
NH
NMe2
19
_ HNMe2 _ EtOH
O Ar
NH N O
21
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CN
Scheme 4. Synthesis of pyrimidinethione 18 and 3,5-dioxo-1,4-diazepine-6-carbonitrile 21.
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and revealed absorption bands at 3395–3162, 1670, 1635, and 1279 cm1 characteristic of four NH, two amidic C –– O, and C –– S functions, respectively. The 1H NMR spectrum (DMSO-d6) exhibited a new doublet signal at d 6.50 ppm with coupling constant (J ¼ 6.8 Hz) assignable to pyrimidine-H-6 proton, four broad singlet signals at d 8.98, 11.02, 11.74, and 13.20 ppm, specific for four NH protons, in addition to C3-H of pyridine ring at d 7.71 ppm. The mass spectrum showed a molecular ion peak at m/z ¼ 316, which is in agreement with the molecular formula C13H12N6O2S. It is interesting in this connection that the reaction of 11 with ethyl glycinate hydrochloride in a boiling mixture of ethanol and dimethylformamide containing triethylamine as a catalyst does not afford the pyrrole derivative 20 as could have been expected in analogy to the formation of 18. Actually, the product of this reaction was identified on the basis of its spectral data as 4-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3,5-dioxo-2,3,4,5-tetrahydro-1H-1,4-diazepine-6carbonitrile (21). The IR spectrum has no absorption band characteristic of an ester group and it revealed the presence of CN stretching band at 2205 cm1 and two amidic C –– O stretching bands at 1672 and 1665 cm1. Its mass spectrum showed the molecular ion at m/z ¼ 296. Two peaks at m/z ¼ 151 (37.5%) and 150 (100%, base peak) identify the 1,4diazepine unit. The 1H NMR spectrum of 21 supported its structure, as it revealed the 1,4-diazepine ring protons as two doublet signals at d 3.45 ppm and a singlet signal at d 8.63 ppm assignable to CH2 and 7-H protons, respectively, and a broad singlet signal at 8.91 ppm exchangeable with D2O characteristic of NH proton, besides the other expected signals. The formation of 21 rather than 20 may be attributed to the intermediacy of the non-isolable transamination adduct 19, which underwent cyclization via loss of ethanol and dimethylamine molecules.
Pharmacology Antimicrobial evaluation Sixteen of the newly synthesized targeted compounds were evaluated for their in vitro antibacterial activity against Bacillus subtilis and Bacillus thuringiensis as example of gram-positive bacteria and Escherichia coli and Pseudomonas aeruginosa as examples of gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Fusarium oxysporum and Botrytis fabae fungal strains. Agar-diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Chloramphenicol, cephalothin, and cycloheximide were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the disks in millimeters. The minimum inhibitory concentration (MIN) measurement was determined for compounds that showed significant ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
growth inhibition zones (>14 mm) using twofold serial dilution method [23]. The MIC (mg/mL) and inhibition zone diameters values are recorded in Table 1. The results depicted in Table 1 revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested gram-positive and gram-negative bacterial strains, and also against antifungal strain. In general, most of the tested compounds revealed better activity against the gram-positive rather than the gramnegative bacteria. It would be also noticed that compounds belonging to the pyrimidine and diazepine derivatives exhibited better antibacterial potentials than members of pyridine, pyrazole, and triazole derivatives. Regarding the structure–activity relationship (SAR) of the tested compounds against gram-positive bacteria, the results revealed that compounds 3, 4, 6, 7, 8, 10, 15, 17, 18, and 21 exhibited broad-spectrum antibacterial profile against the tested organisms. Derivatives with electron-withdrawing groups such as CO, Ph, CS, and CN exist in compounds 3, 4, 8, 18, and 21, respectively, recorded with higher activity than compounds 5, 10, 13, 15 and 17. In this view, compounds 2, 18, 21, 6, and 7 were equipotent to chloramphenicol in inhibiting the growth of B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than that of chloramphenicol against B. thuringiensis. Compounds 8, 15, and 17 showed 50% of the activity of chloramphenicol (MIC 6.25 mg/mL) but they were equipotent to cephalothin in inhibiting the growth of B. subtilis and B. thuringiensis (MIC 6.25 mg/mL). Concerning the antibacterial activity of the compounds 2, 3, and 8 revealed weak growth inhibitory against the tested gram-negative bacteria (MIC 50 mg/mL). On the other hand, compounds 18 and 21 showed equipotent activity as chloramphenicol and cephalothin against E. coli and P. aeruginosa. Regarding the activity of pyridine, pyrimidine, pyrazole, and diazepine against antifungal strains, the results revealed that compounds 2, 6, and 18 were 50% lower than cycloheximide in inhibiting the growth of B. fabae and F. oxysporum (MIC 6.25 mg/mL), while the activity of compounds 2 and 21 was 25% lower than cycloheximidine against F. oxysporum (MIC 6.25 mg/mL). The substitution pattern was also crucial. It is worth mentioning that formation of pyrimidine, pyridine, pyrazole, thiazole, and diazepine produced a high antimicrobial activity. On the other hand, conversion of compound 2 to pyrrole and isoxazole 5 and 13 unfortunately produced weak antimicrobial activity. High biological activity can be correlated with low electron density of ring systems. The objective of the present study was to synthesize and investigate the antimicrobial activities of some new functionalized pyrimidines and diazepines with the hope of discovering new structure leads serving as antimicrobial agents. Our www.archpharm.com
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Novel Pyrazolopyridines
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Table 1. Minimal inhibitory concentration (MIC, mg/mL) and inhibition zone (mm) of some newly synthesized compounds. MICa) in mg/mL, and inhibition zone (mm) Bacteria
Fungi
Gram-positive bacteria
Gram-negative bacteria
Compound no.
B. subtilis
B. thuringiensis
E. coli
P. aeruginosa
F. oxysporum
B. fabae
1 2 3 4 5 6 7 8 9 10 11 13 15 17 18 21 Chloramphenicol Cephalothin Cycloheximide
50 (18) 3.125 (43) 25 (27) 25 (27) 50 (18) 3.125 (40) 315.25 (45) 6.25 (39) 25 (33) 50 (32) 12.5 (33) 25 (32) 6.25 (37) 6.25 (38) 3.125 (44) 3.125 (37) 3.125 (44) 6.25 (36) NT
100 (14) 6.25 (37) 100 (15) 100 (15) 100 (14) 6.25 (36) 6.25 (38) 6.25 (39) 50 (20) 6.25 (38) 6.25 (38) 50 (19) 6.25 (37) 6.25 (38) 6.25 (37) 6.25 (37) 3.125 (44) 6.25 (37) NT
50 (20) 50 (19) 50 (19) 100 (15) 100 (15) 100 (15) 25 (25) 50 (20) 100 (14) 100 (15) 50 (19) 100 (14) 100 (15) 25 (27) 6.25 (34) 6.25 (33) 6.25 (37) 6.25 (38) NT
50 (19) 50 (19) 100 (16) 100 (16) 50 (19) 100 (16) 12.5 (33) 50 (20) 100 (15) 50 (19) 50 (20) 6.25 (38) 12.5 (31) 100 (15) 50 (20) 100 (15) 6.25 (38) 6.25 (37) NT
12.5 (33) 12.5 (30) 50 (19) 50 (19) 100 (16) 6.25 (38) 6.25 (30) 100 (15) 100 (16) 100 (15) 50 (19) 100 (16) 25 (27) 100 (16) 6.25 (38) 12.5 (33) NT NT 3.125 (43)
50 (20) 6.25 (32) 100 (14) 100 (16) 50 (18) 50 (20) 25 (27) 50 (19) 100 (16) 100 (15) 100 (15) 25 (26) 100 (15) 100 (16) 50 (19) 12.5 (32) NT NT 3.125 (42)
NT, not tested. a) MIC, minimal inhibitory concentration values with SEM ¼ 0.02.
aim has been verified by the synthesis of four different rings. The obtained results clearly revealed that compounds derived from pyrazolopyridine 2 exhibited better antimicrobial activity than pyrazolopyridine itself. It is also worth mentioning that incorporation of pyrazolopyridine to the pyrimidine and diazepine nucleus at position 3 via a carboxamide linker 2 produced a high antimicrobial activity. Conversion of aminopyrazolopyridine 1 to pyrazolo[3,4-b]pyridine derivative 2 also enhanced the antimicrobial activity. Increasing the nitrogen atoms and the presence of sulfur atom in compounds 18 and 21 gives an excellent and potent value of antimicrobial activity, which indicates that the presence of SH (thiol group) enhances the antimicrobial activities to be equipotent to chloramphenicol (drug reference). The increasing activity of 3, 4, 6, 7, 8, 10, 16, 17, 18, and 21 is due to the presence of heterocyclic ring containing nitrogen atoms such as pyrimidine, pyridine, pyrazole, and diazepine derivatives. In addition, compounds 6 and 7, which have polynuclear heterocyclic system, also showed increasing antimicrobial activity. Moreover, compound 11 showed moderate antimicrobial activity, which may be due to the inductive effect of the two methyl groups attached to the nitrogen atom. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The results for antibacterial activities depicted in Table 1 revealed that compounds 2, 13, and 14 exhibited good activities. On the other hand, most of the prepared compounds, 2 and 10–14, exhibited interesting high antifungal activities against the reference chemotherapeutics. It is worth mentioning that incorporation of antipyrine to the coumarin nucleus at position 3 via a carboxamide linker 2 produced a high antimicrobial activity. Conversion of 5aminopyrazole derivative 9 to pyrazolo[3,4-d]pyrimidine derivative 14 also enhanced the antimicrobial activity. On the other hand, incorporation of the antipyrine nucleus to pyridine at position 1 in 3 unfortunately produced weak antimicrobial activity.
Experimental Melting points were measured with a Gallenkamp apparatus and are uncorrected. IR spectra were recorded for KBr disk on a Mattson 5000 FTIR spectrophotometer. 1H NMR and 13C NMR spectra were measured on a Bruker AC 300 (300 MHz) in CDCl3 or DMSO-d6 as solvent, using TMS as an internal standard, and chemical shifts are expressed as dppm. Mass spectra were determined on Finnigan Incos 500 (70 eV). Elemental analyses were carried out at the Microanalytical Center of Cairo University. All reactions were followed by TLC (silica gel, aluminum sheets 60 F254, Merck). www.archpharm.com
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Synthesis of 2-cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide (2) A solution of aminopyrazolopyridine 1 (0.01 mol) in dry toluene (50 mL) was added to a solution of 1-cyanoacetyl-3,5-dimethylpyrazole (0.01 mol) in the same solvent (50 mL) and the mixture was heated under reflux for 30 min and left to cool at room temperature. The separated solid material was filtered off and recrystallized from ethanol to give 2. Yield (65%); m.p. 210°C; IR (KBr): n (cm1) 3420 (NH), 2220 (CN), 1685 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.55 (s, 3H, CH3), 3.30 (s, 2H, CH2), 7.71 (s, 1H, C3-H pyridine ring), 10.60 (s, 1H, NH), 13.1 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 168.2, 158.5, 154.5, 150.9, 145.9, 122.4, 116.2, 91.5, 25.1, 24.9, 22.2; MS (EI, 70 eV): m/z (%) ¼ 230 (Mþþ1, 3.5), 229 (Mþ, 10), 215 (MþCH3, 15). Anal. calcd. for C11H11N5O (229.24): C, 57.63; H, 4.84; N, 30.55%. Found: C, 57.43; H, 4.70; N, 30.45%.
Synthesis of N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-2-imino-2H-chromene-3-carboxamide (3) To a solution of compound 2 (0.01 mol) in absolute ethanol (20 mL) containing piperidine (0.5 mL), salicylaldehyde (0.01 mol) was added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool. The precipitate that formed was filtered off, washed with ethanol, dried, and recrystallized from ethanol to afford 3. Yield (65%); m.p. 185°C; IR (KBr): n (cm1) 3420 (NH), 3350 (NH), 1680 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.56 (s, 3H, CH3), 7.00 (s, 1H, C4-H coumarin), 7.72 (s, 1H, C3-H pyridine), 6.68–7.36 (m, 4H, Ar–H), 9.15 (s, 1H, NH), 10.21 (s, 1H, NH), 13.20 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 164.1, 158.5, 154.3, 154.0, 150.8, 145.9, 129.4, 128.7, 127.8, 121.3, 120.0, 116.5, 115.8, 91.6, 25.0, 22.1; MS (EI, 70 eV): m/z (%) ¼ 335 (Mþþ2, 3), 334 (Mþþ1, 19.7), 333 (Mþ, 15). Anal. calcd. for C18H15N5O2 (333.34): C, 64.86; H, 4.54; N, 21.01%. Found: C, 64.77; H, 4.45; N, 20.89%.
Synthesis of 1-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3carbonitrile (4) A mixture of compound 2 (0.01 mol) and acetylacetone (0.01 mol) in ethanol (15 mL) containing a few drops of piperidine (three drops) was refluxed for 10 h. The reaction mixture was cooled and the solid obtained was filtered off and recrystallized from ethanol to give 4. Yield (80%); m.p. 230°C; IR (KBr): n (cm1) 3410 (NH), 2219 (CN), 1685 (CO); 1H NMR (DMSO-d6) d (ppm): 1.81 (s, 6H, 2CH3), 2.38 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.56 (s, 1H, C5-H pyridone ring), 7.72 (s, 1H, C3-H pyridine ring), 13.00 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 158.3, 157.7, 154.5, 154.0, 151.0, 146.0, 136.7, 122.4, 115.9, 115.6, 109.0, 90.8, 25.2, 22.0, 19.9, 14.3; MS (EI, 70 eV): m/z (%) ¼ 294 (Mþþ1, 2), 293 (Mþ, 17), 278 (MþCH3, 21). Anal. calcd. for C16H15N5O (293.32): C, 65.52; H, 5.15; N, 23.88%. Found: C, 65.34; H, 5.14; N, 23.80%.
Synthesis of 1-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-2-oxo-5-phenyl-2,3-dihydro-1H-pyrrole-3carbonitrile (5) A mixture of compound 2 (0.01 mol) and phenacyl bromide (0.01 mol) in ethanol (30 mL) containing triethylamine (0.5 mL) was refluxed for 3 h. The formed solid product was filtered off, dried, and recrystallized from ethanol to give compound 5. Yield (45%); m.p. 135°C; IR (KBr): n (cm1) 3410 (NH), 2195 (CN), 1690 (CO); 1H NMR (DMSO-d6) d (ppm): 2.34 (s, 3H, CH3), 2.51 (s, 3H, CH3), 4.54 (d, J ¼ 2.5 Hz, 1H, pyrrole H-3), 5.65 (d, J ¼ 2.5 Hz, 1H, ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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pyrrole H-4), 7.75 (s, 1H, C5-H), 7.30–7.60 (m, 5H, Ar–H), 12.60 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 162.8, 158.0, 154.0, 150.9, 134.0, 128.7, 126.0, 122.5, 121.4, 115.3, 95.5, 91.5, 34.7, 20.51, 22.3; MS (EI, 70 eV): m/z (%) ¼ 331 (Mþþ2, 29), 329 (Mþ, 20). Anal. calcd. for C19H15N5O (293.32): C, 69.29; H, 4.59; N, 21.26%. Found: C, 69.23; H, 4.56; N, 21.24%.
Synthesis of (E)-N 0 -(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-2-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-ylamino)-2-oxoacetohydrazonoyl cyanide (6) To a cold (0–5°C) solution of compound 2 (0.01 mol) in pyridine (25 mL) was added the 4,6-dimethyl-2H-pyrazolo[3,4-b]pyridine-3yl diazonium salt, which was prepared by dissolving sodium nitrite (0.01 mol) in water (2 mL) and adding to a cold solution of 3-amino-4,6-dimethyl-2H-pyrazolo[3,4-b]pyridine (0.01 mol) containing the appropriate amount of hydrochloric acid with continuous stirring portionwise over a period of 30 min. The reaction mixture was kept in an icebox overnight and then diluted with water. The solid that precipitated was filtered off, washed with water, dried, and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 6. Yield (72%); m.p. 200°C; IR (KBr): n (cm1) 3392 (NH), 3350 (br NH), 2195 (CN), 1660 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 6H, 2CH3), 2.55 (s, 6H, 2CH3), 7.10 (s, 1H, NH), 7.72 (s, 2H, C3-H pyridine ring), 13.2 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 403 (Mþþ1, 4), 402 (Mþ, 100). Anal. calcd. for C19H18N10O (402.41): C, 56.71; H, 4.51; N, 34.80%. Found: C, 56.70; H, 4.49; N, 34.71%.
Cyclization of compound 6: Formation of compound 7 A solution of 6 (0.01 mol) in glacial acetic acid (20 mL) was refluxed for 3 h, and then allowed to cool. The precipitate that formed was filtered off, washed with ethanol, and recrystallized from a mixture of ethanol and DMF (1:1) to give compound 7. Yield (80%); m.p. 280°C; IR (KBr): n (cm1) 3406, 3288–3228 (4NH), 1672 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 6H, 2CH3), 2.56 (s, 6H, 2CH3), 7.73 (s, 2H, 2C3-H pyridine ring), 9.12 (s, 1H, NH), 9.33 (s, 1H, NH), 10.24 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 403 (Mþþ1, 70), 402 (Mþ, 4). Anal. calcd. for C19H18N10O (402.41): C, 56.71; H, 4.51; N, 34.81%. Found: C, 56.70; H, 4.48; N, 34.88%.
Synthesis of 4-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-phenyl-2-thioxo-2,3-dihydrothiazole5-carboxamide (8) To a solution of compound 2 (0.01 mol) in ethanol (20 mL) containing triethylamine (0.5 mL), elemental sulfur (0.01 mol) and phenyl isothiocyanate (0.01 mol) were added. The reaction mixture was heated at 60°C for 2 h with continuous stirring and then poured into a beaker containing an ice/water mixture containing few drops of hydrochloric acid. The formed solid product was collected by filtration, dried, and recrystallized from DMF and ethanol (3:1) to give compound 8. Yield (65%); m.p. 222°C; IR (KBr): n (cm1) 3420 (NH2), 3350 (NH), 1693 (CO), 1220 (C –– S); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.50 (sbr, 2H, NH2), 6.50–7.34 (m, 5H, Ar–H), 7.72 (s, 1H, C3-H pyridine ring), 13.10 (s, 2H, 2NH); 13C NMR (DMSO-d6) d (ppm): 188.4, 163.3, 158.8, 158.5, 154.0, 150.9, 145.9, 133.9, 129.1, 126.5, 124.5, 122.4, 90.5, 73.0, 25.1, 22.1; MS (EI, 70 eV): m/z (%) ¼ 397 (Mþþ1, 3), 396 (Mþ, 100). Anal. calcd. for www.archpharm.com
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C18H16N6OS2 (396.49): C, 54.53; H, 4.00; N, 21.20%. Found: C, 54.50; H, 4.00; N, 21.01%.
Synthesis of (E)-2-cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-(methylthio)-3-(phenylamino)acrylamide (9) To a stirred solution of potassium hydroxide (0.01 mol) in dimethylformamide (20 mL) compound 2 (0.01 mol) was added. After stirring for 30 min, phenyl isothiocyanate (0.01 mol) was added to the resulting mixture. Stirring was continued for 6 h, and then methyl iodide (0.01 mol) was added. Stirring was continued for an additional 3 h. Then, the reaction mixture was poured onto ice water. The solid product that formed was filtered off, dried, and recrystallized from ethanol to afford 9. Yield (45%); m.p. 120°C; IR (KBr): n (cm1) 3350, 3268 (3NH), 2190 (CN), 1690 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.25 (s, 3H, S–CH3), 2.57 (s, 3H, CH3), 6.60–7.50 (m, 5H, Ar–H), 7.72 (s, 1H, C3-H pyridine ring), 9.50 (s, 1H, NH), 11.15 (s, 1H, NH); MS (EI, 70 eV): m/z (%) ¼ 380 (Mþþ1, 80), 378 (Mþ, 10). Anal. calcd. for C19H18N6OS (378.45): C, 60.30; H, 4.79; N, 22.21%. Found: C, 60.25; H, 4.71; N, 22.20%.
Synthesis of 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-(phenylamino)-1H-pyrazole4-carboxamide (10) A mixture of compound 9 (0.01 mol) and hydrazine hydrate 98% (0.05 mol) was heated on a steam bath for 1 h and then left to cool. The reaction mixture was triturated with ethanol and the resulting solid was filtered off and recrystallized from ethanol to give compound 10. Yield (80%); m.p. 225°C; IR (KBr): n (cm1) 3400 (NH2), 3240, 3355 (4NH), 1660 (CO); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.42 (sbr, 2H, NH2), 6.92–7.45 (m, 5H, Ar–H), 7.70 (s, 1H, C3-H pyridine ring), 9.97 (s, 1H, NH), 10.72 (s, 1H, NH), 11.35 (s, 2H, 2NH); 13C NMR (DMSO-d6) d (ppm): 164.2, 158.1, 152.0, 151.5, 150.9, 145.0, 143.0, 129.0, 126.0, 122.5, 118.0, 116.0, 91.5, 84.5, 25.10, 22.2; MS (EI, 70 eV): m/z (%) ¼ 364 (Mþþ2, 3), 362 (Mþ, 100), 347 (MþCH3, 30). Anal. calcd. for C18H18N8O (362.39): C, 59.66; H, 5.01; N, 30.92%. Found: C, 59.60; H, 5.00; N, 30.89%.
Synthesis of (Z)-2-cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-(dimethylamino)acrylamide (11) A mixture of compound 2 (0.015 mol) and dimethylformamide dimethylacetal (2 mL, 0.015 mol) in dry toluene (30 mL) was heated under reflux for 4 h, then left to cool at room temperature. The orange precipitate product was filtered off, washed with petroleum ether, dried well, and recrystallized from toluene to give compound 11. Yield (65%); m.p. 230°C; IR (KBr): n (cm1) 3230 (br NH), 1668 (CO); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.47 (s, 6H, N(CH3)2), 2.55 (s, 3H, CH3), 7.30 (s, 1H, CH–N), 7.71 (s, 1H, C3-H pyridine ring), 11.44 (s, 1H, NH), 13.23 (s, 1H, NH); MS (EI, 70 eV): m/z (%) ¼ 285 (Mþþ1, 30), 284 (Mþ, 100). Anal. calcd. for C14H16N6O (284.32): C, 59.14; H, 5.67; N, 29.56%. Found: C, 59.10; H, 5.61; N, 29.52%.
Synthesis of 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)isoxazole-4-carboxamide (13) A mixture of enaminonitrile 11 (0.01 mol) and hydroxylamine hydrochloride (0.01 mol) in a mixture of ethanol and dimethylß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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formamide (1:1) (30 mL) containing anhydrous potassium carbonate (0.002 mol) was heated under reflux for 6 h, then allowed to cool at room temperature and diluted with ice-cold water (30 mL). The solid product formed was filtered off, washed with water, dried well, and recrystallized from ethanol to afford compound 13. Yield (45%); m.p. 221°C; IR (KBr): n (cm1) 3405 (NH2), 3345– 3234 (2NH), 1666 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.56 (s, 3H, CH3), 4.56 (brs, 2H, NH2), 7.71 (s, 1H, C3-H pyridine ring), 8.88 (s, 1H, H-3 isoxazole), 11.44 (brs, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 273 (Mþþ1, 30), 272 (Mþ, 80). Anal. calcd. for C12H12N6O2 (272.26): C, 52.94; H, 4.44; N, 30.87%. Found: C, 52.90; H, 4.41; N, 30.82%.
Synthesis of 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-1H-pyrazole-4-carboxamide (15) To a solution of enaminonitrile 11 (0.001 mol) in ethanol (20 mL), hydrazine hydrate (80% 0.1 mL, 0.002 mol) was added. The reaction mixture was refluxed for 4 h, and then left overnight at room temperature. The solid product formed was filtered off, washed with ethanol, dried well, and recrystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 15. Yield (76%); m.p. 125°C; IR (KBr): n (cm1) 3420 (NH2), 3330– 3150 (br NH), 1660 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.01 (brs, 2H, NH2), 7.72 (s, 1H, C3-H pyridine ring), 7.87 (s, 1H, H-3 pyrazole), 9.20 (s, 1H, NH), 12.60 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 272 (Mþþ1, 27), 271 (Mþ, 100). Anal. calcd. for C12H13N7O (271.28): C, 53.13; H, 4.83; N, 36.14%. Found: C, 53.10; H, 4.80; N, 36.00%.
Synthesis of 2,4-diamino-N-(4,6-dimethyl-1Hpyrazolo[3,4-b]pyridin-3-yl)pyrimidine-5-carboxamide (17) A mixture of enaminonitrile 11 (0.001 mol) and guanidine nitrate (0.001 mol) in a mixture of ethanol and dimethylformamide (1:1) (30 mL) containing anhydrous potassium carbonate (0.002 mol) was heated under reflux for 8 h, then allowed to cool at room temperature. The reaction mixture was triturated with cold water (50 mL), and few drops of dilute HCl were added (till pH 7). The resultant precipitate was collected by filtration, dried well, and crystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 17. Yield (70%); m.p. 270°C; IR (KBr): n (cm1) 3382–3122 (NH2 and NH), 1664 (CO); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.57 (s, 3H, CH3), 6.53 (s, 2H, NH2), 6.59 (s, 2H, NH2), 7.81 (s, 1H, C3-H pyridine ring), 8.79 (s, 1H, H-6 pyrimidine ring), 11.51 (s, 2H, 2NH), 13.10 (s, 1H, NH); MS (EI, 70 eV): m/z (%) ¼ 299 (Mþþ1, 20), 298 (Mþ, 70). Anal. calcd. for C13H14N8O (298.30): C, 52.34; H, 4.73; N, 37.56%. Found: C, 52.30; H, 4.70; N, 37.51%.
Synthesis of N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5carboxamide (18) A mixture of enaminonitrile 11 (0.001 mol) and thiourea (0.001 mol) in a mixture of ethanol and dimethylformamide (1:1) (20 mL) containing a catalytic amount of piperidine (three drops) was refluxed for 8 h and then left overnight at room temperature. The solid product formed was filtered off, washed with ethanol, dried well, and recrystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 18. www.archpharm.com
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Yield (63%); m.p. 275°C; IR (KBr): n (cm1) 3395–3162 (4NH), 1670 (CO), 1635 (CO), 1279 (C –– S); 1H NMR (DMSO-d6) d (ppm): 2.31 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.50 (d, J ¼ 6.80 Hz, 1H, pyrimidine H-6), 7.71 (s, 1H, C3-H pyridine ring), 8.98 (s, 1H, NH), 11.02 (s, 1H, NH), 11.74 (s, 1H, NH), 13.20 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 175.7, 166.7, 165.5, 158.5, 156.8, 154.0, 150.9, 145.9, 122.4, 106.8, 91.1, 25.1, 22.1; MS (EI, 70 eV): m/z (%) ¼ 317 (Mþþ1, 20), 316 (Mþ, 100). Anal. calcd. for C13H12N6O2S (316.34): C, 49.36; H, 3.82; N, 26.57%. Found: C, 49.31; H, 3.79; N, 26.51%.
Synthesis of 4-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3yl)-3,5-dioxo-2,3,4,5-tetrahydro-1H-1,4-diazepine-6carbonitrile (21) A mixture of enaminonitrile 11 (0.001 mol) and ethyl glycinate hydrochloride (0.001 mol) in a mixture of ethanol and dimethylformamide (1:1) (30 mL) containing a catalytic amount of triethylamine (three drops) was refluxed for 12 h. The solid product formed was filtered off, washed with ethanol, dried well, and recrystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 23. Yield (72%); m.p. 233°C; IR (KBr): n (cm1) 3330 (NH), 2205 (CN), 1672 (CO), 1665 (CO); 1H NMR (DMSO-d6) d (ppm): 2.31 (s, 3H, CH3), 2.55 (s, 3H, CH3), 3.45 (d, J ¼ 8.6 Hz, 2H, diazepin CH2), 7.72 (s, 1H, C3-H pyridine ring), 8.63 (s, 1H, H-7 diazepine ring), 8.91 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 296 (Mþ, 100), 151 (37), 150 (100). Anal. calcd. for C14H12N6O2 (296.28): C, 56.75; H, 4.08; N, 28.36%. Found: C, 56.71; H, 4.00; N, 28.31%.
Antimicrobial evaluation Standard sterilized filter paper disks (5 mm diameter) impregnated with a solution of the tested compound in DMF (1 mg/mL) was placed on an agar plate seeded with the appropriate test organism in triplicates. The utilized test organisms were B. subtilis and B. thuringiensis as examples of gram-positive bacteria and E. coli and P. aeruginosa as examples of gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against F. oxysporum and B. fabae fungal strains. Chloramphenicol, cephalothin, and cycloheximide were used as standard antibacterial and antifungal agents. DMF alone was used as control at the same above-mentioned concentration. The plates were incubated at 37°C for 24 h for bacteria and 48 days for fungi. Compounds that showed significant growth inhibition zones (>14 mm), using the twofold serial dilution technique, were further evaluated for their minimal inhibitory concentrations (MICs).
Minimal inhibitory concentration (MIC) measurement The microdilution susceptibility test in Müller-Hinton broth (Oxoid) and Sabouraud liquid medium (Oxoid) was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, chloramphenicol, cephalothin, and cycloheximide were prepared in DMF at concentration of 1000 mg/mL followed by twofold dilution at concentrations of 500, 250, 3.125 mg/mL. The microorganism suspensions at 106 CFU/mL (colony forming U/mL) concentrations were inoculated to the corresponding wells. Plates were incubated at 36°C for 24–48 h and the minimal inhibitory concentrations (MIC) were determined. Control experiments were also done.
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The author thanks Prof. Dr. Ahmed A. Fadda, Professor of Organic Chemistry, Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt for suggesting the point of research, his continuous guidance, precious advice, real support, and valuable discussion. The author has declared no conflict of interest.
References [1] C. M. S. Menezes, C. M. R. Sant Anna, C. Rangel Rodrigues, E. J. Barreiro, Theochem 2002, 579, 31–39. [2] K. Poreba, A. Opolski, J. Wietrzyk, Acta Pol. Pharm. 2002, 59, 215–222. [3] F. E. Goda, A. A.-M. Abdel-Aziz, O. A. Attef, Bioorg. Med. Chem. 2004, 12, 1845–1851. [4] F. A. Attaby, A. M. Abdel-Fattah, Phosphorus Sulfur Silicon Relat. Elem. 1999, 155, 253. [5] M. A. A. Elneairy, F. A. Attaby, M. S. Elsayed, Phosphorus Sulfur Silicon Relat. Elem. 2000, 167, 161–167. [6] R. N. Misra, H. Y. Xiao, D. B. Rawlins, W. Shan, K. A. Kellar, J. G. Mulheron, J. S. Sack, J. S. Tokarski, S. D. Kimball, K. R. Webster, Bioorg. Med. Chem. Lett. 2003, 13, 2405–2412. [7] J.-P. Stasch, K. Dembowsky, E. Perzborn, E. Stahl, M. Schramm, Br. J. Pharmacol. 2002, 135, 344–349. [8] G. Boerrigter, L. C. Costello-Boerrigter, A. T. Cataliotti, T. Tsuruda, G. J. Harty, H. Lapp, J.-P. Stasch, J. C. Burnett, Circulation 2003, 107, 686–692. [9] D. U. Bawankule, K. Sathishkumar, K. K. Sardar, D. Chanda, A. V. Krishna, V. R. Prakash, S. K. Mishra, J. Pharmacol. Exp. Ther. 2005, 314, 207–214. [10] F. A. Attaby, A. H. H. Elghandour, M. A. Ali, Y. M. Ibrahim, Phosphorus Sulfur Silicon Relat. Elem. 2006, 181, 1087. [11] F. A. Attaby, A. H. H. Elghandour, A. M. Ali, Y. M. Ibrahim, Phosphorus Sulfur Silicon Relat. Elem. 2007, 182, 133–138. [12] A. R. Azevedo, V. F. Ferreira, H. de Mello, L. R. Leao-Ferreira, A. V. Jabor, I. C. P. P. Frugulhetti, H. S. Pereira, N. Moussatche, A. M. R. Bernardino, Heterocycl. Commun. 2002, 8, 427– 432. [13] H. De Mello, A. Echevarria, A. M. Bernardino, M. CantoCavalheiro, L. L. Leon, J. Med. Chem. 2004, 47, 5427– 5433. [14] I. Sekikawa, J. Nishie, S. Tono-Oka, Y. Tanaka, S. Kakimoto, J. Heterocyclic Chem. 1973, 10, 931–936. [15] J. A. Sutcliffe, Bioorg. Med. Chem. Lett. 2003, 13, 4159– 4161. [16] D. M. Moreus, G. K. Folkers, A. S. Fauci, Nature 2004, 430, 242–249. [17] K. Misbahul Ain, B. W. L. Ramas, Quim. Nova 1987, 10 (3), 195–201; Chem. Abstr. 1988, 109, 170356g. [18] M. Hojo, R. Masuda, E. Okada, Synthesis 1990, 347, 347–351. [19] W. Ried, B. Schleimer, Angew. Chem. 1958, 70, 164–169; Chem. Abstr. 1959, 53, 1314f.
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[20] C. Velazquez, E. E. Knaus, Bioorg. Med. Chem. 2004, 12, 3831– 3840. [21] M. Suzuki, H. Jwasaki, Bioorg. Med. Chem. Lett. 2001, 11, 1285– 1288.
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[22] A. A. Fadda, S. Bondock, A. Tarhoni, Moustsh. Chem. 2008, 139, 153–159. [23] A. H. Shamroukh, M. E. A. Zaki, Arch. Pharm. Chem. Life Sci. 2007, 340, 345–351.
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Full Paper Synthesis and Antimicrobial Activity of Some New Heterocycles Incorporating the Pyrazolopyridine Moiety Sraa Abu-Melha Faculty of Science of Girls, King Khaled University, Abha, Saudi Arabia
2-Cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide (2) was utilized as key intermediate for the synthesis of some new coumarin 3, pyridine 4, pyrrole 5, thiazole 8, pyrido[20 ,30 :3,4]-pyrazolo[5,1-c]triazine 7, and aminopyrazolo 10 compounds. 2-Cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-3-(dimethylamino)acrylamide (11) was synthesized and allowed to react with hydroxylamine, hydrazine, and guanidine to afford regioselectively the isoxazole 13, pyrazole 15, and pyrimidine 17 derivatives, respectively. The reaction of 11 with thiourea and/or with ethyl glycinate in basic medium afforded the regioisomeric pyrimidinethione 18 and 3,5-dioxo-1,4-diazepine-6-carbonitrile 23. All the synthesized products were tested and evaluated as antimicrobial agents. Keywords: Aminopyrazole / Antimicrobial activity / Pyrazolo[3,4-b]pyridine / Pyridine / Pyrimidine Received: May 24, 2013; Revised: August 13, 2013; Accepted: August 15, 2013 DOI 10.1002/ardp.201300195
Introduction 1H-Pyrazolo[3,4-b]pyridines comprise a very interesting class of compounds because of their significant and diverse biological and pharmacological activities, such as antimalarial [1], antiproliferative [2], antimicrobial [3–5], cyclin-dependent kinase-inhibiting [6], cardiovascular [7–9], antiviral [10–12], and antileishmanial activities [13]. Pyrazolefused pyridines and pyrimidines are known to possess a wide range of biological activity. Specifically, pyrazolopyridines exhibit antitubercular and anxiolytic effects [14]. The antibacterial activity caught our attention because antimicrobial resistance developed by important pathogens has increased in the last decade [15]. Besides, emerging and reemerging bacterial infections diseases still cause death and disability worldwide [16]. In view of the above-mentioned findings and as continuation of our effort from our laboratory to identify new candidates that may be of value in designing new potent, selective, and less toxic antimicrobial agents, we report herein the synthesis of some new heterocycles incorporating the pyrazolopyridine moiety starting from 4,6-dimethyl-1H-
Correspondence: Sraa Abu-Melha, Faculty of Science of Girls, King Khaled University, Abha 35516, Saudi Arabia. E-mail: [email protected] Fax: þ966 7 2284691
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
pyrazolo[3,4-b]pyridine-3-amines in order to investigate their antimicrobial activity.
Results and discussion Chemistry The synthetic procedures adopted to obtain the target compounds are depicted in Schemes 1–4. The starting 4,6dimethyl-1H-pyrazolo[3,4-b]pyridine-3-amine 1 was prepared according to the reported method [17, 18]. The increasing importance of N-(cyanoacetyl)aminopyrazolopyridine (2) as a versatile intermediate for the synthesis of several heterocyclic rings with many pharmaceutical applications led to the continuing development of new simple procedures for their synthesis. The most effective and suitable method for the preparation of 2 with high yield and purity is the utilization of 1-cyanoacetyl-3,5-dimethyl-pyrazole as cyanoacetylation reagent [19] (Schemes 1 and 2). Thus, cyclocondensation of compound 2 with salicylaldehyde in boiling ethanol containing a catalytic amount of piperidine afforded the coumarin derivative 3. The reaction of 2 with 1,3-dicarbonyl compound was studied in the aim of formation of pyridine derivatives with potential biological activities [20, 21]. Thus, it reacted with acetylacetone to give the pyridine derivative 4. Structure of the latter product was based on analytical and spectral data. The IR spectrum showed three absorption bands at 3410, 2219, and 1685 cm1
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
CH3
CH3
NH2 N
H3C
H3C
913
NHCOCH2CN
Toluene
CH3 N N COCH2CN
+
N H
N
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N H3C
N H
N
1
2 Scheme 1. Structures of compounds 1 and 2.
due to NH, CN, and CO groups. Its 1H NMR spectrum revealed the appearance of new three singlets at d 2.38, 2.57, and 5.56 ppm assigned to two methyl protons and the pyridinone H-5. Recently, the reaction of the cyanoacetamide moiety with a-halocarbonyl compounds was reported, which represents a new, simple, and efficient synthetic route for the synthesis of pyrrole derivatives [22]. Therefore, it was interesting to study H3 C
CH3
H N N
CHO
Ar
Ar
O
C
NH
O
N
N
N N H
NH
N2Cl N
H3C
CH3
CH3
N
7
EtOH/ piperidine
3 H3C
N
H N
6 CH3
O
O
Ar
AcOH/
O
OH
H N
H3C
N N NH CN
H N Ar
the reaction of 2 with phenacyl bromide in boiling ethanol using triethylamine as a basic catalyst that furnished the pyrrole derivative 5. The analytical and spectral data are in agreement with the proposed structure. Thus, 1H NMR spectrum of the reaction product showed new two doublet signals at d 4.54 and 5.65 ppm corresponding to the two vicinal pyrrolidine protons H-3 and H-4, respectively, two singlets at d 2.34 and 2.51 ppm corresponding to two CH3
N
N H
O
S Ar
CN
H N
CN PhNCS/ S/ TEA
O
O
Ar
H N
N Ph NH2
O
2
4
S
8
O Ph Ar
Ph
Br
1) PhNCS/ KOH/ DMF 2) MeI
CN
N O
5
Ar
PhHN H N
SCH3 CN
PhHN
NH2NH2.H2O EtOH
O
Ar
N NH
H N
NH2
O
10
9 CH3 Ar = CH3
N
N NH
Scheme 2. Synthesis of compounds 3–10.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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groups, and a multiplet at d 7.30–7.60 ppm corresponding to aromatic protons. The IR spectrum showed the presence of C –– O group stretching at 1690 cm1 and CN group at 2195 cm1. In continuation of the synthesis of bridged-head nitrogen heterocyclic systems, it was found that diazotized heterocyclic amine is an excellent building block for the synthesis of the target compound. Thus, coupling of compound 2 with 4,6dimethyl-1H-pyrazolo[3,4-b]pyridin-3-diazonium chloride in pyridine at 0–5°C afforded the corresponding hydrazono compound 6. When compound 6 is refluxed in acetic acid, it can be cyclized to 7. The formation of 7 may be interpreted through the nucleophilic attack of the ring nitrogen on the cyano group. The IR spectrum of compound 7 showed three absorption bands at 3406, 3288, and 3228 cm1 due to four NH groups besides carbonyl absorption bands at 1672 cm1. Its 1H NMR spectrum revealed three D2O-exchangeable singlets at d 9.12, 9.33, and 10.24 ppm due to four NH protons (see Experimental section). The reaction of compound 2 with both elemental sulfur and phenyl isothiocyanate in warming ethanol containing a catalytic amount of triethylamine gave the thiazole derivative 8. Elemental analysis, IR, 1H NMR, and 13C NMR results are in agreement with the proposed structure. Treatment of compound 2 with phenyl isothiocyanate in DMF, and in the presence of potassium hydroxide, at room temperature, followed by treatment with methyl iodide afforded the novel ketene N,S-acetal 9. The structure of 9 was established on the basis of its elemental analysis and spectral data. Its IR spectrum showed absorption bands at 3350, 3268, and 2190 cm1 due to three NH groups and nitrile functions, respectively. The mass spectrum showed a molecular ion peak at m/z 378 (Mþ), which agrees with its molecular formula C19H18N6OS. Reaction of 9 with hydrazine in refluxing ethanol gave the corresponding pyrazole derivative 10. The chemical structure of 10 was established on the basis of its elemental analysis and spectral data. Its 1H NMR spectrum displayed a broad signal at d 6.92–7.45 ppm related to the aromatic protons, and another three singlets at d 9.97, 10.72, and 11.35 ppm assignable to four NH protons. Treatment of 2 with dimethyformamide-dimethylacetal (DMF-DMA) in refluxing toluene afforded the corresponding enaminonitrile 11. The structure of 11 has been assigned as a reaction product on the basis of analytical and spectral data. The IR spectrum displayed absorption bands at 3230 cm1 due to NH function, and at 2196 cm1 due to conjugated CN function, at 1668 cm1 due to amidic C –– O function. The behavior of enaminonitrile 11 toward some N-nucleophiles to attain polyfunctionally substituted azoles, azines, and related fused systems linked to a thiazole moiety through a carboxamide linkage of potential pharmaceutical interest has been investigated. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
Treatment of 11 with hydroxylamine hydrochloride in refluxing ethanol–dimethylformamide (1:1) mixture in the presence of anhydrous potassium carbonate afforded an orange product that was identified as 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)isoxazole-4-carboxamide 13. The spectral data of the isolated product was in complete agreement with structure 13. The IR spectrum revealed the lack of an absorption band corresponding to a conjugated CN function. The 1H NMR spectrum (DMSO-d6) showed two new broad singlet signals at d 4.56 and 11.44 ppm and sharp singlet signals at d 8.88 ppm characteristic of NH2, NH, and isoxazole-H3 protons, respectively, besides a singlet signal at d 7.71 ppm distinctive for pyridine protons. The mass spectrum showed a molecular ion peak at m/z ¼ 272, corresponding to the molecular formula C12H12N6O2. The 5-aminopyrazole 15 was achieved as a sole product by heating the enaminonitrile 11 with hydrazine hydrate in ethanol. Inspection of 1H NMR spectrum enabled establishing structure 15 for this pyrazole derivative since the pyrazole H-3 appeared as a singlet at d 7.87 ppm. We could not trace in the 1 H NMR spectrum any signals for the tautomeric 3-aminopyrazole as this could reveal pyrazole-H5 as a doublet. The mass spectrum of 15 showed a molecular ion peak (Mþ) at m/z ¼ 271 corresponding to the molecular formula C12H13N7O. Similarly, the enaminonitrile 11 reacted with guanidine in a mixture of ethanol and dimethylformamide (2:1) containing anhydrous potassium carbonate under reflux to yield, in good yield, a product that was identified as 2,4-diamino-N-(4,6dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidine-5-carboxamide 17. The structure of 17 was assigned on the basis of the elemental analysis and spectral data. The IR spectrum showed absorption bands at 3382–3122 cm1, corresponding to two NH2 and two NH functions, and at 1664 cm1 due to amidic C –– O function. The mass spectrum showed a molecular ion peak (Mþ) at m/z ¼ 298, corresponding to the molecular formula C13H14N8O. Formation of compounds 13, 15, and 17 is assumed to take place via a Michael type addition of the amino group of the hydroxylamine, hydrazine, and guanidine to the activated double bond in compound 11 to form the non-isolable intermediates 12, 14, and 16, which readily undergo intramolecular cyclization followed by loss of dimethylamine molecule to form the target compounds (Scheme 3). The site selectivity in cycloaddition of some nitrogen ambident nucleophiles with the enaminonitrile 11 was also studied. Thus, reaction of 11 with thiourea in refluxing ethanol containing a catalytic amount of piperidine afforded a single product 18 (as examined by TLC) for which cycloadduct 18 seemed possible (Scheme 4). However, the pyrimidinethione 18 was assigned for the reaction product on the basis of its elemental analysis and spectral data. The IR spectrum lacked an absorption band due to a nitrile function www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
Ar
H N
H H CN N
NH2OH
CN
Ar
EtOH/ DMF
O
O
2
_ HNMe
OH
Ar
NMe2
NH2NH2 O
NMe2
N O
13
H H CN N
EtOH
Ar O
11
_ HNMe
NH2
NH2
NH
H N
Ar
N O
NMe2
15
HN NH 2 H H CN N NH Ar O NMe2
NH2
EtOH/ DMF
_
Ar
S
H N
O
NH2
EtOH/ Piperidine
Ar
NH2
N
H2N H N
HNMe2
N
O
16
NH2
H N
H2N
2
14
HN
915
O
H N
NH
CN
H N
H2N 2
12
DMF-DMF dry toluene
Ar
Novel Pyrazolopyridines
17
Scheme 3. Synthesis of the isoxazole 13, pyrazole 15, and pyrimidine 17 derivatives.
S NH
H N O
18
11 O
OEt
H2N _
b
OEt
O ClH3N
OEt
EtOH/ DMF/ TEA
Ar
H H N
CN a
a
HNMe2
Ar
H N O
O
20
NH
O
NH
NMe2
19
_ HNMe2 _ EtOH
O Ar
NH N O
21
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CN
Scheme 4. Synthesis of pyrimidinethione 18 and 3,5-dioxo-1,4-diazepine-6-carbonitrile 21.
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and revealed absorption bands at 3395–3162, 1670, 1635, and 1279 cm1 characteristic of four NH, two amidic C –– O, and C –– S functions, respectively. The 1H NMR spectrum (DMSO-d6) exhibited a new doublet signal at d 6.50 ppm with coupling constant (J ¼ 6.8 Hz) assignable to pyrimidine-H-6 proton, four broad singlet signals at d 8.98, 11.02, 11.74, and 13.20 ppm, specific for four NH protons, in addition to C3-H of pyridine ring at d 7.71 ppm. The mass spectrum showed a molecular ion peak at m/z ¼ 316, which is in agreement with the molecular formula C13H12N6O2S. It is interesting in this connection that the reaction of 11 with ethyl glycinate hydrochloride in a boiling mixture of ethanol and dimethylformamide containing triethylamine as a catalyst does not afford the pyrrole derivative 20 as could have been expected in analogy to the formation of 18. Actually, the product of this reaction was identified on the basis of its spectral data as 4-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3,5-dioxo-2,3,4,5-tetrahydro-1H-1,4-diazepine-6carbonitrile (21). The IR spectrum has no absorption band characteristic of an ester group and it revealed the presence of CN stretching band at 2205 cm1 and two amidic C –– O stretching bands at 1672 and 1665 cm1. Its mass spectrum showed the molecular ion at m/z ¼ 296. Two peaks at m/z ¼ 151 (37.5%) and 150 (100%, base peak) identify the 1,4diazepine unit. The 1H NMR spectrum of 21 supported its structure, as it revealed the 1,4-diazepine ring protons as two doublet signals at d 3.45 ppm and a singlet signal at d 8.63 ppm assignable to CH2 and 7-H protons, respectively, and a broad singlet signal at 8.91 ppm exchangeable with D2O characteristic of NH proton, besides the other expected signals. The formation of 21 rather than 20 may be attributed to the intermediacy of the non-isolable transamination adduct 19, which underwent cyclization via loss of ethanol and dimethylamine molecules.
Pharmacology Antimicrobial evaluation Sixteen of the newly synthesized targeted compounds were evaluated for their in vitro antibacterial activity against Bacillus subtilis and Bacillus thuringiensis as example of gram-positive bacteria and Escherichia coli and Pseudomonas aeruginosa as examples of gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Fusarium oxysporum and Botrytis fabae fungal strains. Agar-diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Chloramphenicol, cephalothin, and cycloheximide were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the disks in millimeters. The minimum inhibitory concentration (MIN) measurement was determined for compounds that showed significant ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2013, 346, 912–921
growth inhibition zones (>14 mm) using twofold serial dilution method [23]. The MIC (mg/mL) and inhibition zone diameters values are recorded in Table 1. The results depicted in Table 1 revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested gram-positive and gram-negative bacterial strains, and also against antifungal strain. In general, most of the tested compounds revealed better activity against the gram-positive rather than the gramnegative bacteria. It would be also noticed that compounds belonging to the pyrimidine and diazepine derivatives exhibited better antibacterial potentials than members of pyridine, pyrazole, and triazole derivatives. Regarding the structure–activity relationship (SAR) of the tested compounds against gram-positive bacteria, the results revealed that compounds 3, 4, 6, 7, 8, 10, 15, 17, 18, and 21 exhibited broad-spectrum antibacterial profile against the tested organisms. Derivatives with electron-withdrawing groups such as CO, Ph, CS, and CN exist in compounds 3, 4, 8, 18, and 21, respectively, recorded with higher activity than compounds 5, 10, 13, 15 and 17. In this view, compounds 2, 18, 21, 6, and 7 were equipotent to chloramphenicol in inhibiting the growth of B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than that of chloramphenicol against B. thuringiensis. Compounds 8, 15, and 17 showed 50% of the activity of chloramphenicol (MIC 6.25 mg/mL) but they were equipotent to cephalothin in inhibiting the growth of B. subtilis and B. thuringiensis (MIC 6.25 mg/mL). Concerning the antibacterial activity of the compounds 2, 3, and 8 revealed weak growth inhibitory against the tested gram-negative bacteria (MIC 50 mg/mL). On the other hand, compounds 18 and 21 showed equipotent activity as chloramphenicol and cephalothin against E. coli and P. aeruginosa. Regarding the activity of pyridine, pyrimidine, pyrazole, and diazepine against antifungal strains, the results revealed that compounds 2, 6, and 18 were 50% lower than cycloheximide in inhibiting the growth of B. fabae and F. oxysporum (MIC 6.25 mg/mL), while the activity of compounds 2 and 21 was 25% lower than cycloheximidine against F. oxysporum (MIC 6.25 mg/mL). The substitution pattern was also crucial. It is worth mentioning that formation of pyrimidine, pyridine, pyrazole, thiazole, and diazepine produced a high antimicrobial activity. On the other hand, conversion of compound 2 to pyrrole and isoxazole 5 and 13 unfortunately produced weak antimicrobial activity. High biological activity can be correlated with low electron density of ring systems. The objective of the present study was to synthesize and investigate the antimicrobial activities of some new functionalized pyrimidines and diazepines with the hope of discovering new structure leads serving as antimicrobial agents. Our www.archpharm.com
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Novel Pyrazolopyridines
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Table 1. Minimal inhibitory concentration (MIC, mg/mL) and inhibition zone (mm) of some newly synthesized compounds. MICa) in mg/mL, and inhibition zone (mm) Bacteria
Fungi
Gram-positive bacteria
Gram-negative bacteria
Compound no.
B. subtilis
B. thuringiensis
E. coli
P. aeruginosa
F. oxysporum
B. fabae
1 2 3 4 5 6 7 8 9 10 11 13 15 17 18 21 Chloramphenicol Cephalothin Cycloheximide
50 (18) 3.125 (43) 25 (27) 25 (27) 50 (18) 3.125 (40) 315.25 (45) 6.25 (39) 25 (33) 50 (32) 12.5 (33) 25 (32) 6.25 (37) 6.25 (38) 3.125 (44) 3.125 (37) 3.125 (44) 6.25 (36) NT
100 (14) 6.25 (37) 100 (15) 100 (15) 100 (14) 6.25 (36) 6.25 (38) 6.25 (39) 50 (20) 6.25 (38) 6.25 (38) 50 (19) 6.25 (37) 6.25 (38) 6.25 (37) 6.25 (37) 3.125 (44) 6.25 (37) NT
50 (20) 50 (19) 50 (19) 100 (15) 100 (15) 100 (15) 25 (25) 50 (20) 100 (14) 100 (15) 50 (19) 100 (14) 100 (15) 25 (27) 6.25 (34) 6.25 (33) 6.25 (37) 6.25 (38) NT
50 (19) 50 (19) 100 (16) 100 (16) 50 (19) 100 (16) 12.5 (33) 50 (20) 100 (15) 50 (19) 50 (20) 6.25 (38) 12.5 (31) 100 (15) 50 (20) 100 (15) 6.25 (38) 6.25 (37) NT
12.5 (33) 12.5 (30) 50 (19) 50 (19) 100 (16) 6.25 (38) 6.25 (30) 100 (15) 100 (16) 100 (15) 50 (19) 100 (16) 25 (27) 100 (16) 6.25 (38) 12.5 (33) NT NT 3.125 (43)
50 (20) 6.25 (32) 100 (14) 100 (16) 50 (18) 50 (20) 25 (27) 50 (19) 100 (16) 100 (15) 100 (15) 25 (26) 100 (15) 100 (16) 50 (19) 12.5 (32) NT NT 3.125 (42)
NT, not tested. a) MIC, minimal inhibitory concentration values with SEM ¼ 0.02.
aim has been verified by the synthesis of four different rings. The obtained results clearly revealed that compounds derived from pyrazolopyridine 2 exhibited better antimicrobial activity than pyrazolopyridine itself. It is also worth mentioning that incorporation of pyrazolopyridine to the pyrimidine and diazepine nucleus at position 3 via a carboxamide linker 2 produced a high antimicrobial activity. Conversion of aminopyrazolopyridine 1 to pyrazolo[3,4-b]pyridine derivative 2 also enhanced the antimicrobial activity. Increasing the nitrogen atoms and the presence of sulfur atom in compounds 18 and 21 gives an excellent and potent value of antimicrobial activity, which indicates that the presence of SH (thiol group) enhances the antimicrobial activities to be equipotent to chloramphenicol (drug reference). The increasing activity of 3, 4, 6, 7, 8, 10, 16, 17, 18, and 21 is due to the presence of heterocyclic ring containing nitrogen atoms such as pyrimidine, pyridine, pyrazole, and diazepine derivatives. In addition, compounds 6 and 7, which have polynuclear heterocyclic system, also showed increasing antimicrobial activity. Moreover, compound 11 showed moderate antimicrobial activity, which may be due to the inductive effect of the two methyl groups attached to the nitrogen atom. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The results for antibacterial activities depicted in Table 1 revealed that compounds 2, 13, and 14 exhibited good activities. On the other hand, most of the prepared compounds, 2 and 10–14, exhibited interesting high antifungal activities against the reference chemotherapeutics. It is worth mentioning that incorporation of antipyrine to the coumarin nucleus at position 3 via a carboxamide linker 2 produced a high antimicrobial activity. Conversion of 5aminopyrazole derivative 9 to pyrazolo[3,4-d]pyrimidine derivative 14 also enhanced the antimicrobial activity. On the other hand, incorporation of the antipyrine nucleus to pyridine at position 1 in 3 unfortunately produced weak antimicrobial activity.
Experimental Melting points were measured with a Gallenkamp apparatus and are uncorrected. IR spectra were recorded for KBr disk on a Mattson 5000 FTIR spectrophotometer. 1H NMR and 13C NMR spectra were measured on a Bruker AC 300 (300 MHz) in CDCl3 or DMSO-d6 as solvent, using TMS as an internal standard, and chemical shifts are expressed as dppm. Mass spectra were determined on Finnigan Incos 500 (70 eV). Elemental analyses were carried out at the Microanalytical Center of Cairo University. All reactions were followed by TLC (silica gel, aluminum sheets 60 F254, Merck). www.archpharm.com
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Synthesis of 2-cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide (2) A solution of aminopyrazolopyridine 1 (0.01 mol) in dry toluene (50 mL) was added to a solution of 1-cyanoacetyl-3,5-dimethylpyrazole (0.01 mol) in the same solvent (50 mL) and the mixture was heated under reflux for 30 min and left to cool at room temperature. The separated solid material was filtered off and recrystallized from ethanol to give 2. Yield (65%); m.p. 210°C; IR (KBr): n (cm1) 3420 (NH), 2220 (CN), 1685 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.55 (s, 3H, CH3), 3.30 (s, 2H, CH2), 7.71 (s, 1H, C3-H pyridine ring), 10.60 (s, 1H, NH), 13.1 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 168.2, 158.5, 154.5, 150.9, 145.9, 122.4, 116.2, 91.5, 25.1, 24.9, 22.2; MS (EI, 70 eV): m/z (%) ¼ 230 (Mþþ1, 3.5), 229 (Mþ, 10), 215 (MþCH3, 15). Anal. calcd. for C11H11N5O (229.24): C, 57.63; H, 4.84; N, 30.55%. Found: C, 57.43; H, 4.70; N, 30.45%.
Synthesis of N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-2-imino-2H-chromene-3-carboxamide (3) To a solution of compound 2 (0.01 mol) in absolute ethanol (20 mL) containing piperidine (0.5 mL), salicylaldehyde (0.01 mol) was added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool. The precipitate that formed was filtered off, washed with ethanol, dried, and recrystallized from ethanol to afford 3. Yield (65%); m.p. 185°C; IR (KBr): n (cm1) 3420 (NH), 3350 (NH), 1680 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.56 (s, 3H, CH3), 7.00 (s, 1H, C4-H coumarin), 7.72 (s, 1H, C3-H pyridine), 6.68–7.36 (m, 4H, Ar–H), 9.15 (s, 1H, NH), 10.21 (s, 1H, NH), 13.20 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 164.1, 158.5, 154.3, 154.0, 150.8, 145.9, 129.4, 128.7, 127.8, 121.3, 120.0, 116.5, 115.8, 91.6, 25.0, 22.1; MS (EI, 70 eV): m/z (%) ¼ 335 (Mþþ2, 3), 334 (Mþþ1, 19.7), 333 (Mþ, 15). Anal. calcd. for C18H15N5O2 (333.34): C, 64.86; H, 4.54; N, 21.01%. Found: C, 64.77; H, 4.45; N, 20.89%.
Synthesis of 1-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3carbonitrile (4) A mixture of compound 2 (0.01 mol) and acetylacetone (0.01 mol) in ethanol (15 mL) containing a few drops of piperidine (three drops) was refluxed for 10 h. The reaction mixture was cooled and the solid obtained was filtered off and recrystallized from ethanol to give 4. Yield (80%); m.p. 230°C; IR (KBr): n (cm1) 3410 (NH), 2219 (CN), 1685 (CO); 1H NMR (DMSO-d6) d (ppm): 1.81 (s, 6H, 2CH3), 2.38 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.56 (s, 1H, C5-H pyridone ring), 7.72 (s, 1H, C3-H pyridine ring), 13.00 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 158.3, 157.7, 154.5, 154.0, 151.0, 146.0, 136.7, 122.4, 115.9, 115.6, 109.0, 90.8, 25.2, 22.0, 19.9, 14.3; MS (EI, 70 eV): m/z (%) ¼ 294 (Mþþ1, 2), 293 (Mþ, 17), 278 (MþCH3, 21). Anal. calcd. for C16H15N5O (293.32): C, 65.52; H, 5.15; N, 23.88%. Found: C, 65.34; H, 5.14; N, 23.80%.
Synthesis of 1-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-yl)-2-oxo-5-phenyl-2,3-dihydro-1H-pyrrole-3carbonitrile (5) A mixture of compound 2 (0.01 mol) and phenacyl bromide (0.01 mol) in ethanol (30 mL) containing triethylamine (0.5 mL) was refluxed for 3 h. The formed solid product was filtered off, dried, and recrystallized from ethanol to give compound 5. Yield (45%); m.p. 135°C; IR (KBr): n (cm1) 3410 (NH), 2195 (CN), 1690 (CO); 1H NMR (DMSO-d6) d (ppm): 2.34 (s, 3H, CH3), 2.51 (s, 3H, CH3), 4.54 (d, J ¼ 2.5 Hz, 1H, pyrrole H-3), 5.65 (d, J ¼ 2.5 Hz, 1H, ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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pyrrole H-4), 7.75 (s, 1H, C5-H), 7.30–7.60 (m, 5H, Ar–H), 12.60 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 162.8, 158.0, 154.0, 150.9, 134.0, 128.7, 126.0, 122.5, 121.4, 115.3, 95.5, 91.5, 34.7, 20.51, 22.3; MS (EI, 70 eV): m/z (%) ¼ 331 (Mþþ2, 29), 329 (Mþ, 20). Anal. calcd. for C19H15N5O (293.32): C, 69.29; H, 4.59; N, 21.26%. Found: C, 69.23; H, 4.56; N, 21.24%.
Synthesis of (E)-N 0 -(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-2-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin3-ylamino)-2-oxoacetohydrazonoyl cyanide (6) To a cold (0–5°C) solution of compound 2 (0.01 mol) in pyridine (25 mL) was added the 4,6-dimethyl-2H-pyrazolo[3,4-b]pyridine-3yl diazonium salt, which was prepared by dissolving sodium nitrite (0.01 mol) in water (2 mL) and adding to a cold solution of 3-amino-4,6-dimethyl-2H-pyrazolo[3,4-b]pyridine (0.01 mol) containing the appropriate amount of hydrochloric acid with continuous stirring portionwise over a period of 30 min. The reaction mixture was kept in an icebox overnight and then diluted with water. The solid that precipitated was filtered off, washed with water, dried, and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 6. Yield (72%); m.p. 200°C; IR (KBr): n (cm1) 3392 (NH), 3350 (br NH), 2195 (CN), 1660 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 6H, 2CH3), 2.55 (s, 6H, 2CH3), 7.10 (s, 1H, NH), 7.72 (s, 2H, C3-H pyridine ring), 13.2 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 403 (Mþþ1, 4), 402 (Mþ, 100). Anal. calcd. for C19H18N10O (402.41): C, 56.71; H, 4.51; N, 34.80%. Found: C, 56.70; H, 4.49; N, 34.71%.
Cyclization of compound 6: Formation of compound 7 A solution of 6 (0.01 mol) in glacial acetic acid (20 mL) was refluxed for 3 h, and then allowed to cool. The precipitate that formed was filtered off, washed with ethanol, and recrystallized from a mixture of ethanol and DMF (1:1) to give compound 7. Yield (80%); m.p. 280°C; IR (KBr): n (cm1) 3406, 3288–3228 (4NH), 1672 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 6H, 2CH3), 2.56 (s, 6H, 2CH3), 7.73 (s, 2H, 2C3-H pyridine ring), 9.12 (s, 1H, NH), 9.33 (s, 1H, NH), 10.24 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 403 (Mþþ1, 70), 402 (Mþ, 4). Anal. calcd. for C19H18N10O (402.41): C, 56.71; H, 4.51; N, 34.81%. Found: C, 56.70; H, 4.48; N, 34.88%.
Synthesis of 4-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-phenyl-2-thioxo-2,3-dihydrothiazole5-carboxamide (8) To a solution of compound 2 (0.01 mol) in ethanol (20 mL) containing triethylamine (0.5 mL), elemental sulfur (0.01 mol) and phenyl isothiocyanate (0.01 mol) were added. The reaction mixture was heated at 60°C for 2 h with continuous stirring and then poured into a beaker containing an ice/water mixture containing few drops of hydrochloric acid. The formed solid product was collected by filtration, dried, and recrystallized from DMF and ethanol (3:1) to give compound 8. Yield (65%); m.p. 222°C; IR (KBr): n (cm1) 3420 (NH2), 3350 (NH), 1693 (CO), 1220 (C –– S); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.50 (sbr, 2H, NH2), 6.50–7.34 (m, 5H, Ar–H), 7.72 (s, 1H, C3-H pyridine ring), 13.10 (s, 2H, 2NH); 13C NMR (DMSO-d6) d (ppm): 188.4, 163.3, 158.8, 158.5, 154.0, 150.9, 145.9, 133.9, 129.1, 126.5, 124.5, 122.4, 90.5, 73.0, 25.1, 22.1; MS (EI, 70 eV): m/z (%) ¼ 397 (Mþþ1, 3), 396 (Mþ, 100). Anal. calcd. for www.archpharm.com
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C18H16N6OS2 (396.49): C, 54.53; H, 4.00; N, 21.20%. Found: C, 54.50; H, 4.00; N, 21.01%.
Synthesis of (E)-2-cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-(methylthio)-3-(phenylamino)acrylamide (9) To a stirred solution of potassium hydroxide (0.01 mol) in dimethylformamide (20 mL) compound 2 (0.01 mol) was added. After stirring for 30 min, phenyl isothiocyanate (0.01 mol) was added to the resulting mixture. Stirring was continued for 6 h, and then methyl iodide (0.01 mol) was added. Stirring was continued for an additional 3 h. Then, the reaction mixture was poured onto ice water. The solid product that formed was filtered off, dried, and recrystallized from ethanol to afford 9. Yield (45%); m.p. 120°C; IR (KBr): n (cm1) 3350, 3268 (3NH), 2190 (CN), 1690 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.25 (s, 3H, S–CH3), 2.57 (s, 3H, CH3), 6.60–7.50 (m, 5H, Ar–H), 7.72 (s, 1H, C3-H pyridine ring), 9.50 (s, 1H, NH), 11.15 (s, 1H, NH); MS (EI, 70 eV): m/z (%) ¼ 380 (Mþþ1, 80), 378 (Mþ, 10). Anal. calcd. for C19H18N6OS (378.45): C, 60.30; H, 4.79; N, 22.21%. Found: C, 60.25; H, 4.71; N, 22.20%.
Synthesis of 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-(phenylamino)-1H-pyrazole4-carboxamide (10) A mixture of compound 9 (0.01 mol) and hydrazine hydrate 98% (0.05 mol) was heated on a steam bath for 1 h and then left to cool. The reaction mixture was triturated with ethanol and the resulting solid was filtered off and recrystallized from ethanol to give compound 10. Yield (80%); m.p. 225°C; IR (KBr): n (cm1) 3400 (NH2), 3240, 3355 (4NH), 1660 (CO); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.42 (sbr, 2H, NH2), 6.92–7.45 (m, 5H, Ar–H), 7.70 (s, 1H, C3-H pyridine ring), 9.97 (s, 1H, NH), 10.72 (s, 1H, NH), 11.35 (s, 2H, 2NH); 13C NMR (DMSO-d6) d (ppm): 164.2, 158.1, 152.0, 151.5, 150.9, 145.0, 143.0, 129.0, 126.0, 122.5, 118.0, 116.0, 91.5, 84.5, 25.10, 22.2; MS (EI, 70 eV): m/z (%) ¼ 364 (Mþþ2, 3), 362 (Mþ, 100), 347 (MþCH3, 30). Anal. calcd. for C18H18N8O (362.39): C, 59.66; H, 5.01; N, 30.92%. Found: C, 59.60; H, 5.00; N, 30.89%.
Synthesis of (Z)-2-cyano-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-(dimethylamino)acrylamide (11) A mixture of compound 2 (0.015 mol) and dimethylformamide dimethylacetal (2 mL, 0.015 mol) in dry toluene (30 mL) was heated under reflux for 4 h, then left to cool at room temperature. The orange precipitate product was filtered off, washed with petroleum ether, dried well, and recrystallized from toluene to give compound 11. Yield (65%); m.p. 230°C; IR (KBr): n (cm1) 3230 (br NH), 1668 (CO); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.47 (s, 6H, N(CH3)2), 2.55 (s, 3H, CH3), 7.30 (s, 1H, CH–N), 7.71 (s, 1H, C3-H pyridine ring), 11.44 (s, 1H, NH), 13.23 (s, 1H, NH); MS (EI, 70 eV): m/z (%) ¼ 285 (Mþþ1, 30), 284 (Mþ, 100). Anal. calcd. for C14H16N6O (284.32): C, 59.14; H, 5.67; N, 29.56%. Found: C, 59.10; H, 5.61; N, 29.52%.
Synthesis of 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)isoxazole-4-carboxamide (13) A mixture of enaminonitrile 11 (0.01 mol) and hydroxylamine hydrochloride (0.01 mol) in a mixture of ethanol and dimethylß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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formamide (1:1) (30 mL) containing anhydrous potassium carbonate (0.002 mol) was heated under reflux for 6 h, then allowed to cool at room temperature and diluted with ice-cold water (30 mL). The solid product formed was filtered off, washed with water, dried well, and recrystallized from ethanol to afford compound 13. Yield (45%); m.p. 221°C; IR (KBr): n (cm1) 3405 (NH2), 3345– 3234 (2NH), 1666 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.56 (s, 3H, CH3), 4.56 (brs, 2H, NH2), 7.71 (s, 1H, C3-H pyridine ring), 8.88 (s, 1H, H-3 isoxazole), 11.44 (brs, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 273 (Mþþ1, 30), 272 (Mþ, 80). Anal. calcd. for C12H12N6O2 (272.26): C, 52.94; H, 4.44; N, 30.87%. Found: C, 52.90; H, 4.41; N, 30.82%.
Synthesis of 5-amino-N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-1H-pyrazole-4-carboxamide (15) To a solution of enaminonitrile 11 (0.001 mol) in ethanol (20 mL), hydrazine hydrate (80% 0.1 mL, 0.002 mol) was added. The reaction mixture was refluxed for 4 h, and then left overnight at room temperature. The solid product formed was filtered off, washed with ethanol, dried well, and recrystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 15. Yield (76%); m.p. 125°C; IR (KBr): n (cm1) 3420 (NH2), 3330– 3150 (br NH), 1660 (CO); 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.01 (brs, 2H, NH2), 7.72 (s, 1H, C3-H pyridine ring), 7.87 (s, 1H, H-3 pyrazole), 9.20 (s, 1H, NH), 12.60 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 272 (Mþþ1, 27), 271 (Mþ, 100). Anal. calcd. for C12H13N7O (271.28): C, 53.13; H, 4.83; N, 36.14%. Found: C, 53.10; H, 4.80; N, 36.00%.
Synthesis of 2,4-diamino-N-(4,6-dimethyl-1Hpyrazolo[3,4-b]pyridin-3-yl)pyrimidine-5-carboxamide (17) A mixture of enaminonitrile 11 (0.001 mol) and guanidine nitrate (0.001 mol) in a mixture of ethanol and dimethylformamide (1:1) (30 mL) containing anhydrous potassium carbonate (0.002 mol) was heated under reflux for 8 h, then allowed to cool at room temperature. The reaction mixture was triturated with cold water (50 mL), and few drops of dilute HCl were added (till pH 7). The resultant precipitate was collected by filtration, dried well, and crystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 17. Yield (70%); m.p. 270°C; IR (KBr): n (cm1) 3382–3122 (NH2 and NH), 1664 (CO); 1H NMR (DMSO-d6) d (ppm): 2.35 (s, 3H, CH3), 2.57 (s, 3H, CH3), 6.53 (s, 2H, NH2), 6.59 (s, 2H, NH2), 7.81 (s, 1H, C3-H pyridine ring), 8.79 (s, 1H, H-6 pyrimidine ring), 11.51 (s, 2H, 2NH), 13.10 (s, 1H, NH); MS (EI, 70 eV): m/z (%) ¼ 299 (Mþþ1, 20), 298 (Mþ, 70). Anal. calcd. for C13H14N8O (298.30): C, 52.34; H, 4.73; N, 37.56%. Found: C, 52.30; H, 4.70; N, 37.51%.
Synthesis of N-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5carboxamide (18) A mixture of enaminonitrile 11 (0.001 mol) and thiourea (0.001 mol) in a mixture of ethanol and dimethylformamide (1:1) (20 mL) containing a catalytic amount of piperidine (three drops) was refluxed for 8 h and then left overnight at room temperature. The solid product formed was filtered off, washed with ethanol, dried well, and recrystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 18. www.archpharm.com
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Yield (63%); m.p. 275°C; IR (KBr): n (cm1) 3395–3162 (4NH), 1670 (CO), 1635 (CO), 1279 (C –– S); 1H NMR (DMSO-d6) d (ppm): 2.31 (s, 3H, CH3), 2.55 (s, 3H, CH3), 6.50 (d, J ¼ 6.80 Hz, 1H, pyrimidine H-6), 7.71 (s, 1H, C3-H pyridine ring), 8.98 (s, 1H, NH), 11.02 (s, 1H, NH), 11.74 (s, 1H, NH), 13.20 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 175.7, 166.7, 165.5, 158.5, 156.8, 154.0, 150.9, 145.9, 122.4, 106.8, 91.1, 25.1, 22.1; MS (EI, 70 eV): m/z (%) ¼ 317 (Mþþ1, 20), 316 (Mþ, 100). Anal. calcd. for C13H12N6O2S (316.34): C, 49.36; H, 3.82; N, 26.57%. Found: C, 49.31; H, 3.79; N, 26.51%.
Synthesis of 4-(4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3yl)-3,5-dioxo-2,3,4,5-tetrahydro-1H-1,4-diazepine-6carbonitrile (21) A mixture of enaminonitrile 11 (0.001 mol) and ethyl glycinate hydrochloride (0.001 mol) in a mixture of ethanol and dimethylformamide (1:1) (30 mL) containing a catalytic amount of triethylamine (three drops) was refluxed for 12 h. The solid product formed was filtered off, washed with ethanol, dried well, and recrystallized from a mixture of ethanol and dimethylformamide (1:1) to give compound 23. Yield (72%); m.p. 233°C; IR (KBr): n (cm1) 3330 (NH), 2205 (CN), 1672 (CO), 1665 (CO); 1H NMR (DMSO-d6) d (ppm): 2.31 (s, 3H, CH3), 2.55 (s, 3H, CH3), 3.45 (d, J ¼ 8.6 Hz, 2H, diazepin CH2), 7.72 (s, 1H, C3-H pyridine ring), 8.63 (s, 1H, H-7 diazepine ring), 8.91 (s, 2H, 2NH); MS (EI, 70 eV): m/z (%) ¼ 296 (Mþ, 100), 151 (37), 150 (100). Anal. calcd. for C14H12N6O2 (296.28): C, 56.75; H, 4.08; N, 28.36%. Found: C, 56.71; H, 4.00; N, 28.31%.
Antimicrobial evaluation Standard sterilized filter paper disks (5 mm diameter) impregnated with a solution of the tested compound in DMF (1 mg/mL) was placed on an agar plate seeded with the appropriate test organism in triplicates. The utilized test organisms were B. subtilis and B. thuringiensis as examples of gram-positive bacteria and E. coli and P. aeruginosa as examples of gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against F. oxysporum and B. fabae fungal strains. Chloramphenicol, cephalothin, and cycloheximide were used as standard antibacterial and antifungal agents. DMF alone was used as control at the same above-mentioned concentration. The plates were incubated at 37°C for 24 h for bacteria and 48 days for fungi. Compounds that showed significant growth inhibition zones (>14 mm), using the twofold serial dilution technique, were further evaluated for their minimal inhibitory concentrations (MICs).
Minimal inhibitory concentration (MIC) measurement The microdilution susceptibility test in Müller-Hinton broth (Oxoid) and Sabouraud liquid medium (Oxoid) was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, chloramphenicol, cephalothin, and cycloheximide were prepared in DMF at concentration of 1000 mg/mL followed by twofold dilution at concentrations of 500, 250, 3.125 mg/mL. The microorganism suspensions at 106 CFU/mL (colony forming U/mL) concentrations were inoculated to the corresponding wells. Plates were incubated at 36°C for 24–48 h and the minimal inhibitory concentrations (MIC) were determined. Control experiments were also done.
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The author thanks Prof. Dr. Ahmed A. Fadda, Professor of Organic Chemistry, Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt for suggesting the point of research, his continuous guidance, precious advice, real support, and valuable discussion. The author has declared no conflict of interest.
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