Synthesis and Biological Evaluation of a Highly Constrained Analogue of Methylthioadenosine ( MTA )

We describe the synthesis and the antibacterial evaluation 2’,N-cyclonucleoside 3 analogue of MTA that is characterized by the presence of an additional linkage between the heterocyclic ring and the sugar moiety.


Introduction
During the last few years, physicians have been faced with little solutions to care bacterial infections and in particular those due to the advent of resistant bacteria such as MRSA (methicillin-resistant Staphylococcus aureus) [1][2][3].
Up to now, antibacterial drug discovery has constantly endeavored to propose to the medical community compounds exhibiting a great selectivity towards pathogens without causing any harm to patients [4].During the recent decades, investigations have bee directed to various microbe specific enzymes that played a key role in the metabolism of pathogens [5][6][7].
Among others, the case of 5'-methylthioadenosine/Sadenosylmethionine nucleosidase is of utmost importance.Indeed, the recovery of 5'-deoxy-5'-methyl-thioadenosine and S-adenosylhomocysteine through the Sadenosylmethionine metabolic pathway is crucial to mammals as well as to various microorganisms for the synthesis of polyamines and the methylation of macromolecules (Figure 1) [8][9][10].
A number of fundamental investigations have disclosed the differences between the metabolic pathways of mammals and those of lower microorganisms such as Escherichia coli, Staphylococcus aureus, Salmonella typhi, Enterococcus faecalis, Mycobacterium tuberculosis, among others.These studies showed that 5'-methyl-thioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is specific for many microbes; whereas a corresponding phosphorylase is active in mammals (Figure 1).Accordingly, the dual substrate enzyme, MTA/ AdoHcy nucleosidase, may be an ideal target for the design of specific antimicrobial drugs [11][12][13].
These nucleoside analogues feature a great variety of chemical structures such as derivatives of acyclic nucleosides, L-nucleosides, carbocyclic nucleosides and many others [16,17].
Herein, we propose the construction of cyclonucleosides that are characterized by the presence of an additional linkage between the heterocyclic ring and the sugar moiety.In this regards, it is noteworthy that cyclonucleosides have been given little attention in view of their application in the medicinal domain [16,17].
Recently, we have defined an efficient access to 2', N 3 -cyclonucleosides derived from adenosine as shown in Figure 3 [18].
After stirring for 24 h, TLC analysis (AcOEt) showed conversion of the starting carbohydrate into a lowerrunning product.The reaction mixture was quenched with a saturated solution of NH 4 Cl (3 mL) and stirred for 30 min, diluted with ether (30 mL) and poured in aq.NaHCO 3 (5 mL).

Antibacterial activity.
As Broth Dilution Method [19] recommended by the Clinical Laboratory Standard Institute, the antibacterial activity of the compound in question was investigated in vitro against eight ATCC strains (American Type Culture Collection): 4  Microorganisms were cultured aerobically in broth Triptych Soy Agar (Difco) at 35.5˚C for 24 h for inoculation, each was adjusted to 0.5 McFarland turbidity (1.5 × 108 CFU/ml) with sterile saline (NaCl 0.85%).The compound tested 3 was dissolved in DMSO for the purchase of two different concentration (1.79 mM and 11.81 mM) and solutions were diluted with Mueller Hinton Broth (Difco) to obtain concentrations 0.023 mM and 1.833 mM in a final volume of 3 mL.The tubes were inoculated with 100 mM of bacterial culture and incubated aerobically at 35.5˚C/24 h.The experiment was done in triplicate.Control experiments were performed using only DMSO.
A subsequent treatment of compound 8 with sodium methylsulfide in DMF at 120˚C led to derivative 9.The spectral data (NMR) confirmed that compound 9 was monotosylated in agreement with the presence of one methyl singlet at δ 2.39 ppm, whereas the methyl singlet at δ 2.00 ppm was due to the presence of the MeS-group at the C-5 position of ribose that is confirmed by viewing the new pattern of the signals ascribed to the methylene at C-5.
In the next step, compound 9 was engaged in the necessary transformations to continue with the introduction of N 6 -benzoyladenine at the anomeric position using the Vorbrügen's method.Thus, removal of the isopropylidene group and in situ acetylation of the resulting diol was accomplished using a mixture of acetic anhydride/ acetic acid containing a catalytic amount of concentrated sulfuric acid.This procedure furnished the diacetate 10 as a 1:1 mixture of α/β diastereoisomers.
Then, the mixture 10 was combined with a previously silylated N 6 -benzoyladenine in the presence a TMS-triflate in acetonitrile under argon atmosphere to provide nucleoside 11.Due to the formation of an intermediate acyloxonium, there is no need to separate the α/β diastereoisomers of mixture 10, the final product being theβ-isomer, exclusively.The spectral data (NMR) of the  Finally, as expected from our previous report, the treatment of precursor 11 in a solution of NH 4 OH/MeOH 1:3 at room temperature gave rise to a cyclization reaction together with adenine debenzoylation leading to compound 3 in 60% yield.The structure of cyclonucleoside 3 was confirmed by the interpretation of its MS and NMR data, including HMBC and NOESY spectra (Table 1).
All compounds have been characterized by IR and NMR spectroscopy.When needed, we utilized 2D NMR spectroscopy.
a The experiment was performed at 500 MHz for 1 H and 100 MHz for 13 C in CDCl 3 and TMS as internal (δ 0.00 ppm).
The antibacterial activity of 2',N 3 -cyclo MTA 3 was further evaluated in vitro against eight bacteria, in which four Gram positive (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 51299 and Micrococcus lentus ATCC 10240) and four Gram negatives (Escherichia coli ATCC 11229, Salmonella typhi ATCC 19430, Pseudomonas aeruginosa ATCC 27853 and Klebisiella pneumonae ATCC 13866).Overall, the MIC ranged between 0.023 mM and 1.833 mM.belonging to the little explored class of adenine 2',N 3cyclonucleosides has been synthesized.Preliminary biological investigations showed the compound to manifest a promising anti-bacterial activity against a number of representative bacterial strains.These results are promising since the constrained nucleoside appeared to be nontoxic in human MRC-5 cells.
Compound 3 showed activity at concentrations above 1.0 mM for all tested bacteria strains, minimal inhibitory concentrations were not determined because the compound precipitated in the culture media above 1.0 mM.

Table 1 . Data of 1 H, 13 C, HMBC and NOESY for compound 3. protected
nucleoside 11 are in agreement with the proposed structure as shown by the presence of the H-1' sugar doublet signal at δ 6.20 ppm and the H-2 singlet signal of adenine at δ 8.20 ppm.