Antibacterial Activity Exerted by some Diazabicyclo-steroid Derivatives Against Staphylococcus Aureus and Streptococcus Pneumoniae

We have synthesize three diazabicyclo-steroid derivatives to evaluate its antibacterial activity. This process involved a series of reactions such as; i) cycloaddition [2 + 2] of 5-hexyn-1ol to OTBS-testosterone (1) or progesterone (2) or OTBS-pregnenolone (3) to form cyclobuteneol-steroid derivatives (4 or 5 or 6); ii) the compounds 4 or 5 or 6 were reacted with ethylenediamine to form steroid-amino conjugates (7 or 8 or 9); iii) alkynylation of 7 or 8 or 9 with 5-hexyn-1-ol to form the steroid-amino-hexynol conjugates (10 or 11 or 12); iv) preparation of the cyclobuta-ynal-steroid derivatives (13 or 14 or 15) by the reaction of 10 or 11 or 12 with DMSO; v) amination of 13 or 14 or 15 with ethylenediamine to form new amino-steroid derivatives 16 or 19 or 21;vi) removal of the tert-butyldimethylsilyl from 16 or 21 with hydrofluoric acid to form hydroxyl-steroids (17 and 22); vii) preparation of 1,4-diazacycloundeca-5,11-dien-steroid derivatives by the reaction 17 or 19 or 22 with Copper(II) chloride. In order to evaluate the possibility of that compounds synthesized may have biological activity; in this study its antibacterial effect on Streptococcus pneumoniae and Staphylococcus aureus bacteria was evaluated. The results indicate that compound 20 exert higher antibacterial activity against Staphylococcus aureus and Streptococcus pneumoniae compared with 18 and 23 via interaction DNa-gyrase. In conclusion, these data indicate that antibacterial activity exerted by the compounds 20 depend of their structure chemical in comparison with the other steroid derivatives involved in this study.


Preparation of 1,4-diazacycloundeca-5,11-dien-steroid derivatives
A solution of 17 or 19 or 22 (0.50 mmol), Copper(II) chloride anhydrous (70 mg, 0.52 mmol), in 5 ml of methanol was stirring for 72 h to room temperature. The reaction mixture was evaporated to dryness under reduced pressure. After, the residue was purified by crystallization from methanol:water (3:1).  -3a,5b-dimethyl-1,2,3,3a4,5,5a,5b,6,7,8,9,11a,12,12a,12b- 2.9. Antimicrobial activity. The evaluation of antimicrobial effect of the different compounds on the bacterial species was made by a previously method described [21]. The bacterial species were incubated on brain/heart Infusion (Streptococcus pneumoniae) and Staphylococcus 110 (Staphylococcus aureus) agars for 24 h at 37 O C. After such time, it was be determined whether growth had taken place or not. In addition, a series of tubes were prepared, the first of which contained 2 ml of culture medium (tripticase soye) at double concentration and the remainder (11tubes), contained the same quantity of medium at single concentrations. From the first tube (double concentration) an aliquot of 2 ml of the studied compound (1 mg/ml) was added and stirred, from this tube an aliquot of 2 ml was taken and added to the following tube (simple concentration) and the process was successively repeated until the last 2 ml of dissolution had been used up. After this process, each tube was inoculated with 0.1 ml of the bacterial suspension, whose concentration corresponded to Mc-Farland scale (9  10 8 cells/ml) and all the tubes were incubated at 37 o C for 24 h. Subsequently, a loop was taken from each of them and inoculated into the appropriate cultures for different bacterial organisms, and were incubated for 24 h at 37 o C. After such time, the minimum inhibitory concentration (MIC) was evaluated to consider the antimicrobial effect of the different compounds. In order to discard the effect of methanol (solvent) on the bacterial species studied, a series of the same number of tubes was prepared in parallel, to which 2 ml of methanol at 60% was added to the first and corresponding successive dilutions were added in the same way as before. In addition a control series was also performed using distilled water to pH 7.0. 2.10. Docking Server. Docking calculations were carried out using Docking Server [22]. The MMFF94 force field [23] was used for energy minimization of ligand molecule using the Docking Server. Gasteiger partial charges were added to the ligand atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on the HERD2 [24] and PARP [25] protein model. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools [26]. Affinity (grid) maps of 20 × 20 × 20-Å grid points and 0.375-Å spacing were generated using the Autogrid program [26]. AutoDock parameter set and distance dependent dielectric functions were used in the calculation of the Van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis and Wets local search method [27]. Initial position, orientation, and torsions of the ligand molecules were set randomly. Each docking experiment was derived from two different runs that were set to terminate after a maximum of 250,000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2Å and quaternion and torsion steps of 5 were applied. 2.11. Statistical analysis. The obtained values are expressed as average ± SE, using each heart (n = 9) as its own control. The data obtained were put under Analysis of Variance (ANOVA) with the Bonferroni correction factor [28] using the SPSS 12.0 program. The differences were considered significant when p was equal or smaller than 0.05.

RESULTS SECTION
There are reports which indicate the preparation of diazahicyclo derivatives as antibacterial agents; nevertheless, expensive reagents and special conditions are required [9][10][11][12][13][14][15][16][17]. Therefore, in this study three diazahicyclo-steroid derivatives were synthetized using several strategies to evaluate the biological activity against Staphylococcus aureus and Streptococcus pneumoniae 3.1. Preparation of three diazecin-steroid-hexahydroazocin derivatives. In this study several straightforward routes are reported for synthesis of three diazahicyclo-steroid derivatives using OTBS-testosterone (1), progesterone (2) and OTBSpregnenolone (3) as chemical tools (Scheme 1). The first stage was achieved by the synthesis of three cyclobutane-steroid derivatives (4 or 5 or 6, Scheme 2); it is important to mention that there are several reports to preparation of cyclobutene rings using some reagents such as Co(PPh 3 ) 2 I 2 /PPh 3 /Zn [29], rodhium [30], nikel [31], ruthenium [32] and others. In this study, the compounds 1, 2 and 3 were reacted with 5-hexyn-1-ol using Cooper II chloride as catalyst to form 4 or 5 or 6. at 208.58 ppm for ketone group. Finally, the presence of 6 was further confirmed from mass spectrum which showed a molecular ion at m/z: 528.39.

Preparation of Cyclobutene-ol-steroid-amino conjugates.
The following stage was achieved by preparation of imino groups involved in the compounds 7 or 8 or 9 (Scheme 2). It is important to mention, that there are several procedures for the synthesis of imino groups which are described in the literature [33,34]. In this study the compounds 7 or 8 or 9 were synthesized (Figure 3) by the reaction of 3 or 5 or 6 with ethylenediamine using boric acid as catalyst, because it is not an expensive reagent and no special conditions are required for use [35]. ii Si Scheme 2. Preparation of cyclobutene-1-ol-steroid-amino conjugates (7 or 8 or 9). Reaction of 1 or 2 or 3 with 5-hexyn-1-ol/Cooper(II) chloride (i) the cyclobuta-3-one-steroid derivatives (4 or 5 or 6). After, 4 or 5 or 6 were reacted with nediamine/boric acid (ii) to form 7 or 8 or 9. In addition, the presence of 9 was further confirmed from mass spectrum which showed a molecular ion at m/z: 570.45.

Alkynylation of amino groups.
There are some studies which shown the reaction of chloro-hexyne derivatives with secondary amines; for example, the preparation of β-Alkynyl-βamino Esters via the Mannich reaction with silyl ketene acetals and alkynyl imines using silver as catalyst [36]. . Other data indicate the preparation of an indole-alkyne derivative by the reaction of 5-chloro-1-pentyne or 6-chloro-1-hexyne with indole-3-acetamide in basic medium [37]. In this investigation the compounds 7, 8 or 9 were reacted with 5-hexyn-2-ol in presence of CopperII chloride to form 10 or 11 or 12 (Scheme 3). The mechanism involves the compounds 7 or 8 via SN 2 mechanism ( Fig.4 and 5). The 1 H NMR spectrum of 10 showed signals at 0.06 and 0.88 ppm for ter-butyldimethylsylane fragment; at 0.68 and 0.98 ppm for methyl groups bound to steroid nucleus; at 0.90, 1.04-1.56, 1.60-1.62, 1.78-2.22, 2.40, 3.53, and 5.60 ppm for steroid nucleus; at 1.58, 1.64, 2.30 and 3.66 ppm for methylene groups bound to both alkyne and hydroxyl groups; at 1.68, 2.26 and 3.52 ppm for methylene groups of arm bound to cyclobutene ring; at 3.20 and 3.56 ppm for methylene groups bound to both amino and imino groups; at 3.80 ppm for both hydroxyl and amino groups; at 5.40 for cyclobutene ring. The 13 C NMR spectra showed chemical shifts at -4.60, 17.80 and 25.72 ppm for terbutyldimethylsylane fragment; at 11.40-15.60 ppm for methyl groups bound to steroid nucleus; at 16

Preparation of aldehyde-steroid derivative.
The sixth stage was achieved by the synthesis of a aldehyde-steroid derivatives (13 or 14 or 15, Scheme 3); it is noteworthy that there are several reports on the oxidation of primary alcohols to form the corresponding aldehydes. In addition, some reports indicate the preparation of aldehyde derivatives using several reagents such as morpholinium bisulfate [38], calcium hydride [39], 2-(hydroxyalky1)dithianes [40], KN(TMS) 2 [41], chromium(VI) [42], ruthenium [43] and others. However, some these reagents may induce risks of toxicity by generation of several substances involved on the reaction mixtures. Therefore, in this study a method previously reported 37 for oxidation of hydroxyl groups was used for synthesis of 13 or 14 or 15 by the reaction of 10 or 11 or 12 with dimethyl sulfoxide.   All these data indicate that; i) compounds 18, 20 and 22 has different antibacterial potency for Staphylococcus aureus and Streptococcus pneumoniae in comparison with gentamicin (an inhibitor of protein synthesis) [53], and clarithromycin (protein synthesis inhibitor) [54], this phenomenon may be attributed mainly to the different molecular mechanism involved and the characteristic chemical structure of the compounds studied; ii) the compound 20 exerts greater antibacterial activity against Staphylococcus aureus and Streptococcus pneumoniae compared with the compounds 18 and 23; iv) the antibacterial effect of 20 was similar to cycprofloxacin. This phenomenon could depend on the interaction of two diazabicyclo rings involved in the chemical structure of 20 with some cellular structure involved in the microorganisms studied. This hypothesis can be availed by some reports which indicate that antibacterial activity of cycprofloxacin is via interaction with DNA gyrase [54]; v) finally, the different mixtures evaluated in this study do not increase the antibacterial activity compared with the compound 20 against Staphylococcus aureus and Streptococcus pneumoniae. 3.9. Docking evaluation. In order, to evaluate the possibility that the compound 20 could interact with DNA gyrase (PDB ID:2xcr) [55] in this study a molecular docking model (serverdoking) [56] was used [57]. Theoretical results indicate that hydrogeninteraction between compound 20 and DNA gyrase ( Figure 9 and Table 1) involves several amino acid residues such as Leu 704 , Asn 705 , Met 780 , Cys 784 , Met 749 , Leu 762 , Phe 764 , Ser 778 and Met 787 .  Table 1. Aminoacid residues involved between the interaction of diazabicyclo-steroid derivatives (18, 20 and 23) with the DNA-gyrase surface. In addition, other theoretical results showed the decomposed interaction energies (Kcal/mol) between the compound 20 and the amino acid residues from DNA gyrase (table 2). All these data suggest that the interaction of compound 20 with DNA gyrase is conditioned by their physicochemical properties.

CONCLUSIONS
The diazabicyclo-steroid derivative (compound 20) is a particularly interesting drug, because its antibacterial activity exerted against Staphylococcus aureus and Streptococcus pneumoniae involves a molecular mechanism different in comparison with other drugs; this phenomenon may constitute a novel therapy for infectious diseases. Table 2. Descompesed interaction energies (Kcal/mol) involved between the diazabicyclo-steroid derivatives (18, 20 and 23) and DNA-gyrase surface.