Preparation of two Cyclobutadiene-steroid derivatives Theoretical Analysis of its Interaction with the μ , d , and k Opiod-receptors

The objective of this investigation was to develop two cyclobutadiene-steroid derivatives (compounds 6 or 7) to evaluate its theoretical interaction on μ, d, and k opioid-receptors. The synthesis of 6 or 7 was carried out using a series of reactions which involves. 1) addition/cyclization: 2) imination, 3) etherification and 4) oxy-functionalization. Chemical structure of all compounds was confirmed using elemental analysis and NMR spectra. In addition, a theoretical analysis on the interaction of compounds 6 or 7 with μ, d, and k opioid-receptors was evaluated using a docking model. The results showed that 6 or 7 may interact with different type of amino acids residues on surface of the μ, d, and k opioid-receptors. Other data, indicated that inhibition constant (Ki) involved in the interaction of compounds 6 or 7 with k-receptor was less compared with the Ki present in the interaction with μ, d, receptors. These data indicated that 1) compounds 6 or 7 show a high affinity by k-receptor; 2) the cyclobutadiene analogs are particularly interesting, because these drugs may constitute a novel therapy for pain


INTROdUCTION
There are several drugs for treatment of pain such as codeine.fentany 1 , buprenorphine, butorphanol and others 1,2 , however some these drugs can cause some adverse effects such addiction 3 .respiratory depressant 4 , sedation, dizziness, nausea, vomiting, constipation 5 .In the search new therapeutic alternatives for treatment of pain, several drugs were developed.In this sense, there is reports that a diethyl-benzamide analog have high affinity by k-receptor 6 .Additionally, a report indicates that an acetamide derivative exert an analgesic effect via k-receptor activation using a rat inodel 7 .Another study showed that a k-receptor agonist (U-50,4H8) was prepared and their biological activity was e valuated in a guinea pig model 8 .Other data describes the synthesis of 2-[(Acylamino)ethyl]-l, 4-benzodiazepines and their interaction with k-receptor using a theoretical docking model 9 .Also, a report showed that arylacetamidc and benzomorphan derivatives can act as agonists of k-receptor using a model based on pharmacophores and coupling 10 .Finally, a report indicated the preparation and interaction of a piperidine analog on k-receptor using a docking model 11 .All these data indicate that some drugs may interact with different types of opioidreceptors; this phenomenon could be due to different functional groups involved in the chemical structure or to diverse protocols used.The objective of this study was synthesizing two cyclobutadiene-steroid analogs to evaluate their theoretical interaction with µ, d, and k opioid-receptors.

Chemical synthesis
The nitro-progesterone was prepared using previously method reported 13 .In addition, all the reagents used in this study were purchased from Sigma-Aldrich Co., Ltd.Infrared spectra (IR) were determined using KBr pellets on a PerkinElmer Lambda 40 spectrometer. 1H and 13 C NMR (nuclear magnetic resonance) spectra were recorded on a Varian VXR300/5 FT NMR spectrometer at 300 and 75.4 MHz (megahertz) in CDCl 3 (deuterated chloroform) using TMS (tetramethylsilane) as an internal standard.EIMS (electron impact mass spectroscopy) spectra were determined using a Finnegan trace gas chromatography Polaris Q-spectrometer.Elementary analysis data were determined from a PerkinElmer Ser.II CHNS/02400 elemental analyser.

Evaluation of physicochemical parameters from compounds 2-7.
Physicochemical factors of compounds 2 to 7 such as lipophilicity degree (LogKow).HBD (hydrogen bond donor groups) and HBA (hydrogen bond acceptor groups), TPSA (topological polar surface area) and number of rotatable bonds (RB) were evaluated using a previously methods reported 13,14 .

Theoretical evaluation
The interaction between compounds 6 or 7 with opioid-receptors was determinate using a Dockingserver 15,16 .In addition, the interaction of compounds 6 or 7 were carried out using a theoretical model for µ (4DKL) 17 , m (4RWA) 18 and k (4DJH) 19 opioid-receptors.

Toxicity analysis.
Theoretical toxicity of compounds 6 or 7 was determined using PASS online software 20 .

Chemical synthesis
The cyclobutadiene-steroid derivatives (compound 6 and 7) were synthesized using some chemical strategies:

Preparation of an imino-steroid derivative
There are some reports that shown the synthesis of several imino analogs; however, the protocols require special conditions 21,22 .The first stage was achieved by reaction of a nitro-progesterone (1) with ethylenediamine Fig. 1 using boric as catalyst to synthesis of an imino-steroid analog (2); it is noteworthy that boric acid did not require special conditions 23 .The 1 H NMR spectra of 2 shows bands at 0.88-0.92ppm for methyl groups bound to steroid nucleus: at 1.80 ppm for methyl bound to imino group: at l.06-1.68,1.84-2.50and 5.14 ppm for steroid moiety; at 3.10-3.82ppm for methylene groups bound to both amino and imino groups: at 4.34 ppm for amino groups.The 13 C NMR spectra showed chemical shifts at 13.22-14.52ppm for methyl groups bound to steroid nucleus; at 16.70 ppm for methyl group bound to imino group: at 21.24-38.53.12 and 63.16-79 22 ppm for steroid moiety: at 41.00-52.84ppm for methylene groups bound to both amino and imino groups; at 156.70-161.60 ppm for imino groups.Finally, the mass spectrum shown a molecular ion at 445.34.

Preparation of hvdroxy-hexynylamino-4-nitrosteroid derivative
Several studies showed the synthesis of some hexynyl-amino analogs by addition of amine groups to alkyne-derivatives using different reagents such as Cu/DMSO 24 and Ru-Cp 25 .In this investigation, the compound 2 reacted with 1-hexyn-3-ol Fig. 1

Physicochemical parameters
Analyzing some reports, which indicate that some physicochemical factors of several drugs such as hydrogen bond donor groups (HBD) and hydrogen bond acceptor groups (HBA), topological polar surface area (TPSA) and number of rotatable bonds (RB) are used to predict the biological activity of some compounds in different theoretical models [35][36][37] ; therefore, in this study these physicochemical parameters Table 1 were evaluated using the Schrodinger Software38.The results indicate that both HBA, HBD values were < 5, these data indicate that 7 could be well absorbed, such happening with other type of compounds 39 .Another result showed that polarity for 7 was higher compared the compounds 2 to 6; here it is noteworthy some studies indicate that this physicochemical parameter could condition the ability of several drugs to penetrate the bloodbrain barrier affinity and exhibit biological activity on nervous central system 40 .Bond donor groups (HBD) and hydrogen bond acceptor groups (HBA), topological polar surface area (TPSA) and number of rotatable bonds (RB).
On the other hand, also another parameters such as logP and π 41 were used to delineate the structural chemical requirements of compounds 1 to 7,; it is noteworthy that logP can be used to determinate lipophilicity degree of either molecule, therefore, in this study these parameters were calculated.The results shown in the Tables 3 and 4 indicate that aliphatic carbons (-CH 3 , -CH 2 and aromatic carbon) involved in the compound 6 contribute to increase the lipophilicity compared with the compounds 2-5 and 7.All this data suggest that changes in the degree of lipophilicity depend of structural chemical characteristic of compounds studied.

docking evaluation
S o m e s t u d i e s s u g g e s t t h a t s o m e compounds exert their biological activity via opioid receptors; therefore, in this study was evaluated that two cyclobutadiene-steroid derivatives (compound 6 or 7) could interact with opioid receptors such as µ (4DKL) 16 , d (4RWA) 17 and k (4DJH) 18 using a docking model 13 .The results Fig. 3 show the possible interaction of compound 6 with several aminoacid residues involved in the structure of µ opioid receptor such as Gln Also, was evaluated the possibility of that cyclobutadiene-steroid derivative may interact with d opioid-receptor.the results Fig. 4

Thermodynamic parameters
Some studies indicate that thermodynamic parameters are evidences for confirming the interaction drug-protein 42 .In this study a theoretical evaluation ii as carried out on some thermodynamics parameters such as free energy of binding, electrostatic energy, total intermolecular energy, vdW + Hbond + desol energy and inhibition constant.The results showed differences in the intramolecular energy involved in the interaction for compound 6 (Table 5) or 7 (Table 6) with µ, d and k opioidreceptors.In addition.other data shown different inhibition constants involved in the interaction or compounds 6 or 7 (Ki = 11.23;Ki = 0.20 with k-opioid receptor.However, the values of these inhibition constants were lower compared with the Ki involved in the interaction of compounds 6 or 7 with µ and d-opioid receptors; these data are interesting, which implies a higher interaction of 6 or 7 with the k-opioid receptor (which is related to analgesia effect 43 ).This phenomenon opens the possibility that these compounds could be evaluated experimentally in some biological system.In addition, it is important to perform some toxicity studies to evaluate if there are any side effects in the biological activity of the compounds involved in this study.

Theoretical analysis of toxicity
Analyzing the premise above mentioned.in this study also was evaluated the possible toxicity induced by the compounds 6 or 7 using the GUSAR software 44 .The results Table 7 showed that toxicity could be higher via intraperitoneal administration of compounds 6 or 7 compared to the other type of administration routes such as intravenous, oral and subcutaneous.

CONCLUSIONS
In conclusion.all these data indicate that 1 the compounds 6 or 7 show a high affinity by k-receptor.2) the cyclobutadiene-steroid derivatives are particularly interesting, because these drugs may constitute a novel therapy for treatment of pain.

Fig. 3 .
Fig. 3. Contact site of aminoacid residues involved in the surface of µ-opioid receptor (4dKL)and the compound 6 using dockingserver.

Table 6 : Intrramolecular parameters involved between of interaction of the compound 7 and µ,
d and k-pioid receptors