Synthesis and Analgesic Activity of η6-(Anisole)- Triscarbonyl-Chromium(0)

The general method for synthesis the η6-(arene)-triscarbonyl-chromium(0) complexes was modified and applied for preparation of η6-(anisole)-triscarbonyl-chromium(0) and the study of its analgesic activity was undertaken. A significant analgesic activity was observed after intraperitoneal injection, in Wistar rats. Two doses (30 and 50 mg/Kg of the body weight) of η6-(anisole)- triscarbonyl-chromium(0) were injected and the analgesic activity was evaluated by the Hot Plate Test method. They showed a significant analgesic effect in comparison with the control group and the group treated with dipyrone standard, but not so high when compared with the group treated with morphine standard. Overall, it was observed that the η6-(anisole)- triscarbonyl-chromium(0) was easily obtained by the modified synthetic method and was effective in increasing the pain threshold.


Introduction
Arene triscarbony chromium complexes have been known since the fifties and have been extensively studied .6. The most common method for their preparation is based on the reaction between hexacarbonyl chromium and arenes . Nevertheless, various problems are encountered. Some organometallic chromium reagent is lost from the reaction medium by sublimation 3, while its low level of reactivity requires the use of high boiling point solvents and/or long reaction times .
Toma and his co-workers observed that yields are low, due to incomplete reaction, when low boiling point solvents are used. However, when high boiling point solvents are used, yields are also reduced due to decomposition4. Many solvents may be used: they include excess of arene, dioxane, acetone, esters, alkanes, amines, ethers, nitriles etc7".
The most used procedure is that of Mahaffy and Pauson with dibutyl ether/THF as solvent, under reflux. Toma and his co-workers modified this method using decalin/butyl acetate4'8, dibutyl ether/butyl acetate , pure decalin , pure butyl acetate, or alkyl formates as solvent5 '7. To the best of our knowledge, neither qe-(anisole)triscarbonyl-chromium(0) nor any other organometallic compound have been screened for analgesic activity.
It is convenient at this point to make some general on analgesic activity. The pain is part of a defensive reaction against dysfunction of the organism or imbalance in its functions, as well as against potentially dangerous stimulus. Analgesia by increase of the pain threshold may be induced by electrical stimulation 12, or, more easily, by drugs.
There are many drus to reduce pain and a few have been used for centuries. They 21 include morphine, dipyrone', aspirin and other salicylates .H owever, we are not aware of any reports on the use of organo-metallic compounds as analgesics. VoL 6, No. 1, 1999 Synthesis and Analgesic Activity of q6-(Anisole)-Triscarbonyl-Chromium(O) The present work reports a modified method for synthesis of q6-(arene)-triscarbonylchromium(0)complexes, represented by the preparation of q e-(anisole)-triscarbonylchromium(0), together with a report on it's analgesic activity.

Materials and Methods Experimental
Synthesis of q6-(Arene)-Triscarbonyl Chromium(0) complexes: Chromium hexacarbonyl, Cr(CO)e (Aldrich) was purified by dissolution in hexane, filtration through an alumina (Brockmann, activity I) column and evaporation of the solvent in a stream of nitrogen.
Representative Preparation: qe-(Anisole)-triscarbonyl chromium(0) Chromium hexacarbonyl (1.1 g, 5 mmol) and anisole (0.7g, 7 mmol) was dissolved in 5 mL of dibutyl ether and mL of butyl acetate or ethyl acetate was added. The mixture was protected from light and deaerated by bubbling with a very slow stream of N and then refluxed using a long Liebig condenser. The sublimed Cr(CO)6 was returned to the flask with a glass rod, and the bubbling of the N continued during all the reflux period, for easily removal of the formed carbon monoxide.
The reflux was continued until no more sublimate appeared at the drop of the condenser (5-6 hours). After this time, the mixture was cooled and the excess of reagent and solvents were removed at 40 60 o under vacuum, and the residue dissolved in acetone, filtered through silica (Merck, 60) in a small column, then eluted with acetone. Acetone was partially removed under vacuum. Hexane was added, and the mixture cooled in freezer during 12 hours. The precipitate qe-(anisole)-triscarbonyl chromium (0)  Male Wistar rats were used, weighing 170g approximately and divided into groups of five animals. The animals were supplied from the Central for Animal Care of University of Franca. The animals were maintained in cages, with water and feeding "ad libitum." Algesimetric Test: Hot-Plate The hot plate method, modified for rats was used6This method utilises an aluminium plate maintained at 51+1C, by means of water from a thermostatic water bath An acrylic box measuring 24 x 18 x 18 cm was placed on the plate to impede the escape of animals.
The act of licking the hind paws was observed as a response, registering the elapsed time from the moment that the animal was placed on the hot plate until the response was obtained 7 (latency time).
To avoid lesions in the animals paws and consequently tissue damage which could affect subsequent readings in the absence of stimuli, the animals were removed from the plate after 30 s exposure. Procedure for Measurement: Before any treatment was given, the latency time was measured in three successive tests, carried out at intervals of 10 minutes. The average of the three measurements was considered as being the "basal latency" value. After intraperitoneal administration, the test latency times were re-measured at time intervals of 10 in 10 minutes, for 40 minutes.
The effects of the various treatments on the latency time were expressed as the Hot Plate Analgesia Index-HPAI (Table I) .

Drugs Utilised
Morphine hydrochloride and Sodium Dipyrone were used as standards.
A 10 % hydroalcoholic solution was used to dilute the test compound, physiological solution (NaCI 0,9%) was used to dissolve the standard compounds, and aqueous Tween 80 (10%) for hexacarbonyl chromium and pure anisole samples.
The doses for standards and tested compounds were shown in Table I.

Statistical Analysis
A statistical analysis was made usinj the t-Student and Anova methods for p<0.05.

Results and Discussion
In our preliminary studies we found that the reaction between Cr(CO)6 and anisole in pure butyl acetate gives a low yield of the desired compound due to decomposition. The substitution of butyl acetate by ethyl acetate, however, leads to incomplete reaction. The use of decalin is not convenient for us, because this solvent can not be easily removed under vacuum and flash chromatography would be necessary .
We combined the procedures of Mahaffy and Pauson and of Hudecek and Toma '4''7' and carried out the reaction in a mixture of dibutyl ether with 5% of butyl acetate or of ethyl acetate as coordinating co-solvent, adding the advantages of the Mahaffy procedure (easily isolation) with these of Toma methods esters are more eficient as co-solvents then THF), without the inconvenients of pure decalin. The need for special care with the purity of reagents and solvents'4,'z', was confirmed by us: the yields are very low when impure solvents are used.
We also used a slow bubling of N into the reaction medium, during all the reflux time, for removal of formed CO, because is well known that the reaction between a arene and chromium hexacarbonyl is a equilibrium, and removal of the carbon monoxide is necessary for complete reaction .
While various authors have prepared and studied arene chromium complexes, no tests of biological activity were made.
On the other side, it was demonstrated that transition metal containing moieties( such as chromium tricarbonyl), when coordinated to arenes have electron acceptor properties 6'7 and is also known that the analgesic activity of arenes is increased by the presence of electron acceptor groupsz''4'. For examples, anisole (this work) is a poor analgesic, while phenacetin (4-acetylamino phenetole) is a powerfull analgesic. Aspirin have a electron acceptor group (COOH) and also, the presence of a halogen (CI) atom in analgesics increase their potency ,by increase of their efficiency as prostaglandin biosynthesis inhibitors.
These observations suggested to us that rle-(anisole)-triscarbonyl-chromium(0) may have some degree of analgesic activity.
We carried out analgesic screening of qS-(anisole)-triscarbonyl-chromium(0), using the Hot Plate algesimetric method TM that demonstrate a supra-spinal response . We screened 30 and 50 mg/Kg doses ,injected through the intraperitoneal pathway.
As already mentioned, the analgesic measurements were continued during 40 minutes and expressed as the protection percent This short action time was selected because the toxicology and side effects of administration of q6-(anisole)-triscarbonylchromium(0) were unknown.
We observed that the both 30 and 50 mg/Kg doses showed a significant analgesic effect when compared with these of the control group, and the standards (Table I).

Conclusion
We report a modified method for the synthesis of q-(anisole)-triscarbonylchromium(0) and of some other arene tricarbonyl chromium complexes. Screening shows that this compound exhibits high analgesic effect, the first ever reported for an organometallic complex.