Synthesis, structure and antimicrobial evaluation of new derivatives of theophylline sugar hydrazones

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Introduction
Theophylline derivatives in 7-and 8-positions have been investigated in the respect of their bronchospasmolytic 1-4 anticancer 5 and circulatory blood system activity. 6A large amount of work has been performed on the fused systems derived from theophylline, including synthetic procedures and structure determination [7][8][9][10][11][12][13][14][15] but few of the synthesized heterocyclic derivatives were pharmacologically tested to be anti-inflammatory, 16 anti P-388-leukemia 17 and vascular relaxing agents. 18lso, acyclo C-nucleosides represent an important class of compounds characterized by an acyclic sugar moiety attached to the heterocycle through a carbon-carbon bond.
Acyclonucleosides have been recently classified according to the type of glycon rather than the aglycon part.][21][22][23] Sugar hydrazones of theophyllin-7-ylacetic acid were reported by Klosa 24a in 1958 and some of other reported sugar hydrazones have been found to possess antitumor, antibacterial and antiviral activities.24b In view of the biological importance of theophylline derivatives, sugar hydrazones and acyclonucleosides and in continuation of our studies on the synthesis of purines and nucleoside derivatives as potential bioactive compounds, [25][26][27] it was considered worthwhile to study the reaction of 7-ethyl-8-hydrazinotheophylline with sugars.Our goal was the synthesis of [1,2,4]triazolo [4,3-e]theophylline derivatives as new acyclo C-nucleosides via theophylline sugar hydrazones and to explore the biological activities of the products.We report here the synthesis, structure and antimicrobial activity of new theophylline sugar hydrazones.
According to our knowledge, none of these hydrazones 7-11 has been reported hitherto.The structures of obtained hydrazones 7-11 were confirmed by their elemental analyses and spectral (IR, 1 H NMR and FAB-MS) data (see Experimental Section).For example, their 1 H NMR spectra in DMSO-d 6 revealed in each case, a characteristic signal in the region δ = 7.43-7.54assignable to the azamethine N=CH proton.Also, 1 H NMR spectra revealed methyl groups at δ = 3.17The IR spectra of sugar hydrazones showed bands at 3243-3462 cm -1 due to the OH groups, bands at 1629-1699 cm -1 due to the C=O groups.The mass spectra of the hydrazones 9-11 by using FAB technique revealed M + and M+1 + peaks with intensities of 20 Acetylation of the sugar hydrazones 7-11 with acetic anhydride in pyridine at room temperature gave the corresponding poly-O-acetyl derivatives 12-16 (Scheme 1).The IR absorptions of the acetylated sugar hydrazones 12-16 formed showed absorption bands in the carbonyl frequency region at 1745-1752 cm -1 indicating the introduction of O-acetyl groups.Their 1 H NMR spectra exhibited signals in the region of δ = 1.85-2.16confirming the presence of OAc methyl protons.FAB-Mass spectra of 12-16 showed characteristic M+1 + peaks with intensity of 35-52%.
Attempted dehydrogenative cyclization of sugar hydrazones 7-11 or their acetylated derivatives 12-16 with ferric chloride in ethanol, 30,31 copper dichloride in dimethylformamide, 32 bromine in water 33 or in acetic acid, 34 bromine in acetic acid/sodium acetate, 24 bromine in acetic acid, sodium acetate and acetic anhydride 35 or thionyl chloride, 36 as previously used for the cyclization of related sugar hydrazones, did not proceed and no crystalline derivatives of the acyclo C-nucleosides (R= sugar or R`= acetylated sugar) 18 (Scheme 2) could be isolated.The reaction of 7-11 or 12-16 with bromine in acetic acid and sodium acetate did not afford the respective cyclized product 1,2,4-triazolo[4,3-e]theophylline 18, but instead 8-bromo-7ethyltheophylline 17 37 identified by its elemental and spectral analysis (Scheme 2).Further evidence for structure 17 was based on its reaction with hydrazine hydrate giving the starting compound 1.Doering 32 reported that heterocyclization of butanal caffein-8-ylhydrazone 19 was achieved by using copper dichloride to give 5,7,9-trimethyl-3-propyl- [1,2,4]triazolo [4,3-e]purine-6,8dione 20 (Scheme 3) in poor yield (15%).He also suggested that the lower selectivity in the oxidation of 19 indicated that a five-membered ring is less favorable and the difficulties of the heterocyclization of 19 could also be caused by the sterically demanding effect of the methyl group at position 5.This result seems to be in accord with our findings that electrocyclization of sugar hydrazones 7-11 or acetylated sugar hydrazones 12-16 is difficult though this is still under investigation.

Antimicrobial activity
The compounds 9 and 12-16 were tested for their antimicrobial activities using four fungal species, namely Aspergillus fumigatus AF, Penicillium italicum PI, Syncephalastrum racemosum SR and Candida albicans CA, as well as four bacterial species namely Escherichia coli EC, Bacillus subtilis BS, Pseudomonas aeruginosa PA and Staphylococcus aureus SA.The microorganisms were tested against the activity of solutions of concentrations of 5.0, 2.5 and 1.0 mg/ml of each compound and using an inhibition zone diameter in cm (IZD) as criterion for the antimicrobial activity.The fungicide Terbinafin and the bactericide Chloramphenicol were used as references to evaluate the potency of the tested compounds under the same conditions.The results are depicted in Table 1 St. -reference standard; Chloramphenicol (as a standard antifungal); Terbinafin (as a standard antibacterial.The test was done using diffusion agar technique.Well diameter: 0.6 cm…(100 µl of each conc.was tested).Inhibiton values = 0.1-1.5 cm (beyond control = +), 0.6-1.0(beyond control = ++), 1.1-1.5 cm (beyond control = +++), 0 (not detected = -).

Experimental Section
General.All evaporations were carried out under reduced pressure at 60 °C.TLC was carried out on aluminum sheet silica gel 60 (Fluka) and detected by short wavelength UV light.All melting points were measured on an electrothermal melting point apparatus and are uncorrected.
The 1 H NMR spectra were recorded in deuterated chloroform (CDCl 3 ) or dimethyl sulfoxide (DMSO-d 6 ) at 300 MHz on a Varian Mercury VXR-300 NMR spectrometer and 13 C NMR spectra were recorded in dimethyl sulfoxide (DMSO-d 6 ) at 75 MHz using a Jeol-300 NMR spectrometer.Chemical shifts were related to that of the solvent.The infrared spectra were recorded in potassium bromide discs on a Pye-Unicam, SP300 and Shimadzu, FT IR 8101 PC infrared spectrophotometers.Biological activity was assessed at the Regional Center for Mycology and Biotechnology at Al-azhar University, Cairo, Egypt.FAB mass spectrometry was carried out at the Institute of Organic Chemistry, Hamburg University, Germany.Elemental analyses were carried out at the Microanalytical Center of Cairo University, Giza, Egypt.Theophylline, sugars, bromine, ethyl iodide and hydrazine hydrate were purchased from Aldrich Chemical Co.Ethanol, methanol, diethyl ether, petroleum ether, chloroform, N,Ndimethylformamide, nitric acid, hydrochloric acid, sodium acetate trihydrate, potassium carbonate, pyridine, glacial acetic acid and acetic anhydride were purchased from EL-Nasr Pharmaceutical and Chemical Co.(ADWIC), Egypt.7-Ethyl-8-hydrazinotheophylline 1, 28 8-chloro-7-ethyltheophylline, 29 8-nitrotheophylline, 38 and 8-chlorotheophylline 38 were prepared according to the in literature reported methods.

General procedure for synthesis of sugar 7-ethyl-8-theophyllinyl-hydrazones 7-11
To a suspension of 7-ethyl-8-hydrazinotheophylline 1 (2.38 g, 10 mmol) in ethanol (30 ml), was added a solution of the appropriate sugar 2-6 (10 mmol) in water (10 ml) and few drops of glacial acetic acid.The mixture was heated at reflux until reaction was judged complete by TLC (2-6 h).The solid product formed upon cooling was filtered off, washed with the minimum amount of ethanol, dried and finally recrystallized from ethanol to afford the respective hydrazones 7-11.The physical constants and the spectral data of the products 7-11 are listed below: The experiments were carried out by pouring a spore suspension of the fungal species (one ml. of sterile water contains approximately 10 8 conidia) or spreading bacterial suspension over a solidified malt agar medium.The layer was allowed to set for 30 min.A solution of the test compounds 9 and 12-16 (5.0, 2.5, 1.0 mg/ml) was placed onto sterile 5 mm filter paper discs and allowed to dry, then the discs were placed on the center of the malt agar plate and incubated at optimum incubation temperature 28 ± 2 ºC.Test organism growth may be affected by the inhibitory action of the test compound.So, a clear zone around the disc appeared as an indication of the inhibition of the test organism growth.The size of clearing zone is proportional to the inhibitory action of the test compound.The fungicide Terbinafin and the bactericide Chloramphenicol were used as standards under the same conditions.Measurements were considered after 72 h for fungi and 24 h for bacteria.The results are shown in Table 1.

Table 1 .
. Compound 9 showed the highest degree of inhibition against AF, PI and SA and weak inhibition against SR.Compounds 9, 12, 13, 15 and 16 displayed a considerable degree of inhibition against SR and EC.While 12 had a considerable degree of inhibition against BS, compounds 13, 15 and 16 had only weak inhibition against BS.Moreover, compounds 9, 12, 15 and 16 have weak inhibition against PI.All compounds, except 9 and 14, exhibited no activity against AF, SA and PA.All compounds except 12 showed no activity against CA.Antimicrobial activity

Reaction of products 7-11(or 12-16) with a mixture of bromine/ acetic acid/ sodium acetate
28 a suspension of 7-11 or 12-16 (2.2 mmol) and anhydrous sodium acetate (6.6 mmol) in acetic acid (15 ml), a solution of bromine (2.2 mmol) in acetic acid (7 ml) was added dropwise and the mixture stirred at room temperature until reaction was judged to be complete by TLC.The solvent was evaporated under vacuum; the oily residue obtained was extracted with chloroform.The precipitate formed was collected, washed with ethanol and recrystallized from ethanol to give orange crystals of product, 8-bromo-7-ethyltheophylline 1737(45% yield), mp 170 o C. C for 2 h, then left to cool and poured into cold water.The solid product was filtered off, washed with cold water, dried and finally recrystallized from ethanol.The product obtained was identical in all respect to 7-ethyl-8-hydrazino-theophylline 1 (0.21 g, 90% yield), mp 254-256 o C [lit.mp 257-8 o C].28Antimicrobial evaluationCultures of four fungi namely Aspergillus fumigatus AF, Penicillium italicum PI, Syncephalastrum racemosum SR and Candida albicans CA were tested as well as four bacteria species namely Escherichia coli EC, Bacillus subtilis BS, Pseudomonas aeruginosa PA and Staphylococcus aureus SA were used to investigate the antimicrobial activity of the compounds 9 and 12-16.The antimicrobial activity was assayed biologically using diffusion plate technique.