Synthesis and Antioxidant Activity of 7-Thio Derivatives of 6,7-Dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione

New 7-thio derivatives of 6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione have been synthesized by the reaction of 3-cyclohexyl-7-thio-6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione with alkylhalogenides. The synthesized compounds were tested for antioxidant activity on the model of Fe2+-dependent oxidation of adrenaline in vitro. It was found that the antiradical activity of 7-thio derivatives of 6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione significantly depends on the structure of the substituent which is part of the thioether fragment of the base molecule.


Introduction
It is well-known that pyrimidine derivatives are a class of organic compounds which are actively investigated for the presence of pharmacological activity. A large number of highly effective synthetic drugs, based on pyrimidine derivatives, have been widely used.
Pyrimidine derivatives condensed with a five-membered heterocyclic moiety occupy a special place among the large variety of biologically active derivatives of pyrimidine. Wellknown drugs such as allopurinol, sildenafil, and tubercidin represent this type of bioactive pyrimidine.
Although derivatives of cycloalkyl[d]pyrimidine were studied for many types of pharmacological activities, the antioxidant activity of this class of compounds was not investigated. Antioxidants are compounds that inhibit oxidation reactions by neutralizing free radicals which are formed during these reactions. It is known that many diseases are associated with the oxidative damage of biomolecules [12,13]. The use of antioxidants can prevent radical-induced damage and thus allow us to stave off and treat the diseases mentioned above.
In the continuation of our studies for the search of new biologically active compounds among the derivatives of cycloalkyl[d]pyrimidine, the synthesis of new 7-thio derivatives of 6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione has been carried out. For the synthesized compounds, the antioxidant activity was evaluated.
From the 1 H-NMR spectra, it can be seen that there are signals of cyclohexyl and cyclopentyl fragments in the strong field of spectra in the form of multiplets. One of the protons of the cyclopentane fragment which is located near the sulfur atom can be seen as a doublet at 3.99-4.15 ppm with J=5.5-6.7 Hz. The position and the shape of the signal protons of the S-CH 2 fragment essentially depends on the nature of the substituent in this fragment. Thus, in the case of the acetamide substituent, the signal of protons of the S-CH 2 fragment is located at 3.29 ppm as a singlet. In the benzylic substituents, the signal of protons of the S-CH 2 fragment is shifted towards the weak field and located at 3.8 ppm. It should also be noted that the presence of the halogen atom in the ortho-position of the benzyl fragment leads to the appearance of the signal of protons of the S-CH 2 fragment as two doublets with J=12.2-14.0 Hz, that is typical for the AB type spin system. Amide protons are located in the weak field at 11.09-11.44 ppm as a broad singlet.
It was established that compounds 3, 4h, 4a, 4f, and 4e showed high antiradical activity that was even higher than the antiradical activity of the reference substances (ionol, quercetin, ketorolac, diclofenac). Thus, compound 3 that contains the SH group showed the highest antioxidant activity (80%) of all, which is not surprising since thiols are effective interceptors of free radicals [14]. Benzylthioethers that contain halogen atoms in the paraposition and do not contain substituents in the ortho-position also showed high antioxidant activity (4f -64%, 4e -56%). A significant antiradical activity was found for thioderivatives 4h (60%) and 4g (40%) containing ethylacetate and acetamide fragments, respectively. The antiradical activity of compound 4a -a compound containing an orthomethylbenzyl moiety is on par with the activity of ionol. It should also be noted that compound 4b, which contains the metafluorobenzyl moiety, showed but moderate antioxidant activity (33.33%). Despite the fact that the majority of 7-thio derivatives of 6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione showed high antioxidant activity, there are compounds in this series that enhance the oxidation of adrenaline and thus show prooxidant activity. These compounds are orthohalogenated benzylthioethers 4c (−6.67%) and 4d (−60%).
Thus, the antiradical activity of 7-thio derivatives of 6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione, which was evaluated by the model of Fe 2+ -dependent in vitro oxidation of adrenaline, significantly depends on the structure of the substituent attached to the thioether fragment of the base molecule.

3-Cyclohexyl-7-sulfanyl-6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione (3)
The mixture of bromide 2 (0.0064 mol) and thiourea (0.0064 mol) in ethanol (20 mL) was stirred with reflux for 4 h. After the reaction was complete, the crystalline solid was filtered off and added to an aqueous solution of sodium hydroxide (30 mL, 5%). The mixture was heated to boiling. After the reaction mixture was cooled to room temperature and acidified with HCl, the thiol 3 was obtained as a colorless solid. The solid product was filtered off and washed with water. Yield

Antioxidant Activity
The antioxidant activity of the 7-thio derivatives of 6,7-dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione was estimated by the reaction of Fe 2+ -dependent oxidation of adrenaline. The incubation mixture contained 2.5 mL of a carbonate buffer (0.2 M, pH=10.6), 50 µL of a 0.1% solution of adrenaline, and 50 µL of the test compound. The mixture was thoroughly stirred and quickly placed into the SF-46 spectrophotometer. The optical density was measured at 347 nm during 20 min [15]. Into the control probe the carbonate buffer, solution of adrenaline, solution of FeSO 4 , and 50 µL DMSO were added. The mathematical processing of the results was carried out by the methods of variational statistics using the Student's t-test [16].