Aim:The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates.Materials and Methods:A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA.Results:Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow’s milk (11%), 3 isolates from she-camel’s milk (11%), two isolates from goat’s milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain.Conclusion:These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.

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The synthesis of 2-(6,7-diethoxy-3,4-dihydroisoquinolin-1-yl)acetonitrile (1) has been performed by ring closure of the corresponding amide according to the Bischler-Napieralski method (Scheme 1). Based on spectroscopic data, the tautomeric 2-(tetrahydroisoquinolin-1-ylidene)acetonitrile is the actual compound. The reactions of 1 with a-oxohydrazonoyl halides 4 in the presence of Et 3 N led to 2-(aryldiazenyl)pyrrolo[2,1-a]-isoquinoline derivatives 8 (Scheme 2), whereas with C-(ethoxycarbonyl)hydrazonoyl chlorides 14, 2-(arylhydrazono)pyrrolo[2,1-a]isoquinoline-1-carbonitriles 16 were formed (Scheme 4). The structures of the products were established from their analytical and spectroscopic data and, in the case of 8b, by X-ray crystallography.

Treatment of 6,7-diethoxy-3,4-dihydroisoquinoline (8) and its 1-methyl derivative 12 with hydrazonoyl halides 10 in the presence of Et 3 N in THF under reflux afforded the corresponding 5,6-dihydro-1,2,4-triazolo[3,4-a]isoquinolines 11 and 13, respectively, in high yield (Schemes 2 and 3). The products are formed via regioselective 1,3-dipolar cycloaddition of the intermediate nitrilimines 9 with the isoquinoline CN bond. Reaction of 6,7-diethoxy-3,4-dihydroisoquinoline-1-acetonitrile (4a) with ethyl a-cyanocinnamates 15 in the presence of piperidine in refluxing MeCN yielded benzo[a]quinolizin-4-ones 16 (Scheme 4). Under the same conditions, 12 and arylidene malononitriles 19 reacted to give benzo[a]quinolizin-4-imines 20 (Scheme 5). Instead of 15 and 19, mixtures of an aromatic aldehyde, and ethyl cyanoacetate or malononitrile, respectively, can be used in a one-pot reaction. Introduction. ± Within the class of fused isoquinolines with their cardiovascular [1], anti-inflammatory [2], and antidepressant [3] activities, [1,2,4]triazolo[3,4-a]isoquinolines are of considerable pharmaceutical and agricultural interest [4 ± 7]. Therefore, the synthesis of this ring system is an attractive goal. A convenient approach is the 1,3-dipolar cycloaddition of nitrilimines [8 ± 10], generated by elimination of HX from corresponding hydrazonoyl halides [11 ± 13], to the CN bond of 3,4-dihydroisoquinolines [12] [14]. It has been shown that derivatives of type 1 can be obtained in high yields with R 1 H or alkyl [5] [6] [15] [16]. On the other hand, 3,4-dihydroisoquinolin-1-acetonitriles and hydrazonoyl halides in refluxing THF in the presence of Et 3 N reacted to give pyrrolo[2,1-a]isoquinoline derivatives of type 2 via a cyclocondensation reaction [15] [17]. Spirocyclic adducts of type 3, which could have been formed by 1,3-dipolar cycloaddition with the enamine tautomer of 3,4-dihydroisoquinolin-1-acetamide, have never been observed [6] [15] [17] 3 ).The recently reported results of the reaction of hydrazonoyl halides with 3,4-dihydroisoquinolin-1-acetamide 4 in the presence of Et 3 N [17] are in pronounced contrast to those published earlier [6]. Whereas the reaction of 5 in refluxing THF yielded only the cyclocondensation product 6, the structure of the product obtained from the reaction in CH 2 Cl 2 at room temperature was described as the cycloadduct 1a

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2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (1) condensed with carbaldehydes 2a,b to give the respective thienopyrimidines (3a,b), which reacted with phosphoryl chloride and hydrazine hydrate to afford the respective pyrimidinohydrazines (4a,b). Compound 4a condensed with acetophenone under Vilsmeier conditions to afford the formylated pyrazolopyrimidine 6. Condensation of 4a with active methylenes produced the respective pyrazolopyrimidines (7-11). Besides, 4a condensed with succinic anhydride and with phthalic anhydride, yielding the pyrrolidine-2,5-dione 12 and the isoindoline-1,3-dione 13, respectively. Moreover, 4a reacted with isatin to afford the hydrazono-indolin-2-one 14. Structural elucidations for the new thienopyrimidines were based upon compatible analytical and spectroscopic results. Eleven of the new compounds were tested and found active against influenza A neuraminidase virus (H3N2). Compounds 12 and 13 were the most potent.

The reaction of 3,4-dihydroisoquinolin-1-yl-acetonitrile with DMFDMA afforded the enaminonitrile 5. Compound 5 was reacted with 2-aminobenzimidazole to yield 4-amino-3-(dihydroisoquinolin- 1-yl)-benzo[4,5]imidazo[1,2-a]pyrimidine (11) and with acetonitrile derivatives to afford pyrido[2,1- a]isoquinolines (15a - g).

Treatment of N‐phenyl‐substituted benzenecarbo‐hydrazonoyl chlorides 1a‐d with malononitrile in sodium ethoxide solution gave 5‐amino‐4‐cyanopyrazole derivatives 2‐5. Compounds 2‐5 were converted to formidate derivatives 6‐9 upon treatment with TEOF in acetic anhydride. The reaction of the latter products 6‐9 with hydrazine hydrate gave imino‐amino derivatives 10‐13, which was converted to hydrazino derivatives 14‐17 by refluxing with hydrazine hydrate. Hydrazino as well as imino‐amino derivatives undergo condensation, cyclization, and cycloaddition reactions to give pyrazolo[3,4‐d]pyrimidine 18‐21, pyrazolo[4,3‐e][1,2,4]triazolo‐[3,4‐c]pyrimidine 22‐27, and pyrazolo[3′,4′:4,5]pyrimido[1,6‐b][1,2,4]triazine 42‐44 derivatives. Antimicrobial studies are performed using two Gram‐positive bacteria and two Gram‐negative bacteria. Data indicated that compounds 5, 28D, 29B, and 31D are exploring elevated antibacterial effects against all strains tested. Compound 28D is the most promising antibacterial agent against the delicate bacterial strain Bacillus subtilis and Pseudomonas aeruginosa with high effectiveness (low minimum inhibitory concentration [MIC] value) 40 and 60 μg/mL, respectively.