EHOOPrÁHH^ECRAa XHMH3, 2015, moM 41, № 1, c. 112-120
A CONVENIENT SYNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL THIENO[2,3-c]PYRAZOLE COMPOUNDS AS ANTIMICROBIAL
AND ANTI-INFLAMMATORY AGENTS
© 2015 Adel M. Kamal El-Dean, Remon M. Zaki#, Abdullah Y. Abdulrazzaq
Chemistry Department, Faculty of Science, Assiut University, Assiut 71516, Egypt Received April 3, 2014; in final form June 3, 2014
A new method for synthesizing 4-amino-3-methyl-1-phenyl-1#-5-substituted thieno[2,3-c]pyrazole was reported. The substituted groups at position 5 include carbonitrile, carboxamide, ^-phenyl carboxamide, and benzoyl groups. The newly synthesized compounds and their derivatives were characterized by elemental analysis and spectroscopy (IR, 1H NMR, and mass spectra). Furthermore, some of these synthesized compounds were screened against various pathogenic bacterial and fungal strains. The results demonstrate that most of the synthesized compounds possess a significant antibacterial activity against gram-positive and gram-negative bacteria. In addition, most of these compounds showed a remarkable anti-fungal activity. On the other hand, some of the synthesized compounds possess high anti-inflammatory activity, which was demonstrated using the carrageenan-induced rat paw edema assay.
Keywords: synthesis, reactions, thienopyrazole, thienopyrazolopyrimidine, thienopyrazoloimidazolopyrimidine DOI: 10.7868/S0132342315010054
Pyrazole derivatives display a broad spectrum of biological activities, such as anti-inflammatory [1—5], antimicrobial [5—8], antioxidant , anticancer [9— 11], fungicidal , and antiviral activities [9—11, 13, 14]. Some pyrazole derivatives were reported to possess high affinity and selectivity towards A2b adenos-ine receptor antagonists . Particularly, aryl pyra-zoles are important in medicinal and pesticidal chemistry . Thieno[2,3-c]pyrazoles A are an important class of potent kinase inhibitors .
Literature survey on thienopyrazole revealed that most of papers focus on synthesis of the theinopyra-zole substituted at position 5 similar to structure B. Few reports deal with o-bifunctionalized thienopyrazole similar to structure A [18—33].
In continuation of our previous work in the synthesis of heterocyclic compounds containing pyrazole moiety [34—49] we report a novel facile method of synthesis of thieno[2,3-c]pyrazole substituted at position 4 and 5 similar to structure A.
The difficulty of synthesis of such structures is caused by the fact that most syntheses are based on a starting molecule in which mercapto group is adjacent to cyano group. In the case of pyrazole, the chlorine atom is very difficult to displace with sulfur atom. We
tried to achieve this using thiourea [36, 50—52] as it has been reported in other molecules but all attempts failed.
After several attempts, we displaced chlorine with sulfur in the presence of sodium borohydride and used the product in situ, without isolation, for the next reaction.
RESULTS AND DISCUSSION
5-Methyl-2-phenyl-2,4-dihydropyrazole-3-one (I) treated with Vilsmeier's reagent afforded 5-chloro-3-methyl-1-phenyl-1#-pyrazol-4-carbaldehyde (II). When aldehyde (II) was allowed to react with hydroxyl amine hydrochloride in ethanol in the presence of sodium acetate, the corresponding 5-chloro-3-methyl-1-phenyl-1#-pyrazole-4-carbaldehyde oxime (III)
* Corresponding author (e-mail: firstname.lastname@example.org).
Fig. 1. The substituent positions in thieno[2,3-c]pyrazole.
was obtained. The pyrazole aldehyde oxime (III) was dehydrated using acetic anhydride into the corre-
The attempt to synthesize thieno[2,3-c]pyrazole through converting chloropyrazole carbonitrile (IV) into mercaptopyrazole (V) using thiourea in ethanol, as with other moieties, followed by reacting with a-halogenated compounds, failed. This forced us to search for another method to synthesize thienopyra-zole (VII). The desired result was achieved by the reaction of elemental sulfur with chloropyrazole in the presence of sodium borohydride through reduction of sulfur in ethanol to afford not isolated intermediate sodium salt C, which was used in situ in the next reaction with a-halogenated compound to afford ¿-alkylated mercaptopyrazole carbonitrile (Via—f). Compounds (Via—f) underwent Thorpe-Ziegler cycliza-tion upon heating in ethanolic sodium ethoxide solution to afford thienopyrazole (Vila—f). Conversion of (VIb) to (Vilb) was proven by spectral analysis,
sponding 5-chloro-3-methyl-1-phenyl-1#-pyrazole-4-carbonitrile derivative (IV).
XH NMR revealed the disappearance of the signal at 3.30, characteristic for —CH2— group in compound (VI), and appearance of the signal at 6.90, characteristic for NH2. Also, the IR of (VIIb) showed disappearance of the band characteristic for CN group at 2220 cm-1 in compound (VIb) and appearance of bands characteristic for NH2 group. Mass spectrum of compound (VIb) showed molecular ion peak at 272.14 and showed the base peak at 255.77, which means that the molecular ion lost one molecule of ammonia to give the base peak.
When aminocarboxamide compound (VIIb) was allowed to react with triethyl orthoformate in presence of catalytic amount of acetic acid, 3-methyl-1-pyra-zolothienopyrimidinone (VIII) was obtained.
H 14 ,OH
Me\ 3 4 N
2 N 1
4 N ,
N 2^ s 7 Y 14 10
(VI), (VIIa): Y = CN (VI), (VIIb): Y = CONH2 (VI), (VIIc): Y = CONHPh (VI), (VIId): Y = CONHPhMe-^ (VI), (VIIe): Y = CONHPhOMe-^ (VI), (VIIf): Y = CONHPhCl-^
Regents: i = POCl3/DMF; ii = H2NOH.HCl/AcONa/EtOH; iii = Ac2O; iv = H2NCSNH2/EtOH; v = S8/NaBH4/EtOH; vi = YCH2X/EtOH/AcONa; vii = EtOH/EtONa
Scheme 1. Synthesis of 4-amino-3-methyl-1-phenyl-5-substituted 1#-thieno[2,3-c]pyrazoles (VIIa—f).
On the other hand, the reaction of (VlIb) with ization with sodium carbonate solution afforded the chloroacetyl chloride in dioxane followed by neutral- chloroacetyl amino compound (IX).
NH2 h Cl
Me n Me , 2 Me.
Ph O Ph Ph O
(VIII) (VlIb) (IX)
Reagents: i = HC(OEt)3/AcOH, ii = ClCH2COCl/dioxane
Scheme 2. Reaction of aminothieno[2,3-c]pyrazole carboxamide (VIIb) with triethylorthoformate and chloro-acetyl chloride.
Some of the synthesized compounds were chosen and screened in vitro for their antimicrobial activity against some strains ofbacteria and fUngi. Antifungal and antibacterial activities of tested compounds were evaluated by the reported method using 0.005% (50 ^g/mL) concentration of selected compounds in DMSO as a solvent. The inhibition zone (mm) for the antifungal activity was compared with clotrimazole as a reference. In the case of antibacterial activity, concentration of tested compounds was 2% and the inhibition zone in mm was compared with a series of antibiotics according to the sensitivity of each type of bacteria to the most effective antibiotic for it as a reference.
Some tested compounds showed high antibacterial activity. Aminocarbonitrile compound (Vila) show low antibacterial activity compared with other tested compounds. We found that aminocarboxamide compound (VIIb) showed high antibacterial activity against some strains of gram-negative bacteria, such as Esherichia coli and Klebsiella pneumoniae, with values almost the same as those of the corresponding reference antibiotics (nitrofuratoin and imipenem, respectively). Also, compound (VIIb) showed moderate activity against most of the tested strains of gram-positive bacteria and against Pseudomonas aeruginousa, which is a gram-negative bacterium. The antibacterial activities of 4-amino-^-(4-chlorophenyl)-3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carboxamide compound (VIIe) and 4-amino-^-(4-chlorophenyl)-3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-car-boxamide compound (VIIf) were high compared with other tested compounds, which did not affect any strain. Chloroacetylation of aminocaboxamide (VIIb) afforded chloroacetylamino derivative (IX) showing high antibacterial activity against gram-positive bacteria, such as Staphylococcus aureus and Clostridium difficile. Also, compound (IX) showed moderate activity
against Streptococcus strains, which are gram-positive bacteria, whereas it showed low antibacterial activities against all tested strains of gram-negative bacteria.
On the other hand, compounds (VIIe) and (VIIf) showed high antifungal activity against Candida albicans, Trichophyton rubrum, and Aspergillus flavus, while compound (VIIb) showed the highest antifungal activity against Trichophyton rubrum. However, compounds (VIIa), (VIIb), and (VIIe) showed moderate activity against some species of fungi, while compound (IX) did not affect any of fungi species tested. Compound (VIIa) was inactive with respect to most of the tested species of fungi.
The results of anti-inflammatory activity assessment for some of the synthesized compounds are presented in Figs. 2 and 3.
The inflammatory response is represented as the percentage of the increase in paw swelling upon the anti-inflammatory effect of tested compounds (Fig. 2) and as the percentage of paw edema inhibition (Fig. 3) calculated for each group at each time point using the following ratio:
(Vt - ^control - (Vt - ^treated X 100/(Vt -V0)control,
where Vt is the average volume of paw swelling at a given time point and V0 is the average volume just after carrageenan injection.
From the previous results we found that some of the tested compounds showed high anti-inflammatory activity compared with indomethacin. For example, a significant difference exists between indomethacin and aminocarboxamide (VIIb) with the P value less than 0.05, which means that compound (VIIb) d
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