Synthesis of Novel DPPH-Free Radical Scavenger Se-Containing Fused Chalcogenophenes: 2-Alkyl-7-Cyano-4-Imino-3-Phenyl-6-(pyrrolidin-1-yl)-3,4-Dihydroselenopheno[3,2-d]Pyrimidines

Abstract A straightforward and effective strategy for the synthesis of novel 7-cyano-4-imino-3-phenyl-6-(pyrrolidin-1-yl)-2-substituted-3,4-dihydroselenopheno[3,2-d]pyrimidines have been accomplished through a base-promoted cascade addition/cyclization sequence employing phenyl isothiocyanate followed by the S-alkylation of the obtained fused Se-containing heterocyclic framework with various alkyl halides. Free radical scavenging activity of the synthesized selenium-containing structures was studied against DPPH, as well. Antioxidant activities as IC50 values were ranged from 12.19 to 91.08 µM. Unfused 3-amino-2,4-dicyano-5-(pyrrolidin-1-yl)selenophene was much more powerful antioxidant agent than ascorbic acid. Moreover, the promising antioxidant potentials were observed with selenopheno[3,2-d]pyrimidines. Taking into account the best antioxidant activity of S-benzylated derivative amongst pyrimidine-fused heterocycles and significant relationship between antioxidant activity of compounds and many of their biological potencies, using benzyl halides bearing electron donating groups on phenyl ring can probably improve their antioxidant capacities to make them more effective anti-cancer, anti-diabetic, anti-inflammatory and anti-neurodegenerative agents.

The IR spectrum evidence confirms the synthesis of compound 3 through heterocyclization of compound 2 which is based on the disappearance of symmetric and asymmetric NH 2 stretching vibration bands at 3327 and 3427 cm À1 together with the absence of one of the nitriles stretching bands at 2168 cm À1 .The 1 H NMR spectrum of compound 3 showed two multiplet signals around d 2.05-2.11and 3.65-3.79ppm attributed to methylene and azamethylene groups of pyrrolidine, respectively.The presence of two triplet signals at d 6.93 (H meta ) and 7.11 ppm (H para ), and a doublet signal at d 7.69 (H ortho ) ppm corresponding to the aromatic hydrogens as well as a broad singlet signal at d 9.43 ppm due to the NH group clearly confirms the depicted structure.The mass spectrum also showed the molecular ion peak at m/z 400 (M þ ) corresponding to the molecular formula of C 17 H 15 N 5 SSe.
A self-explanatory base-promoted tandem addition/cyclization mechanism employing phenyl isothiocyanate for the synthesis of selenopheno[3,2-d]pyrimidine 3 is proposed in Scheme 2.
Further diversification of the selenopheno [3,2-d]pyrimidine core proceeded through S-alkylation of compound 3 with several alkyl halides in the presence of K 2 CO 3 in boiling ethanol to furnish highly functionalized derivatives 4a-g (Scheme 3).
All physical, chemical and spectral data were in agreement with the newly synthesized compounds 4a-g.For example, the 1 H NMR spectrum of 4f showed two signals at d 1.17 signal at d 7.62 (H ortho ) ppm corresponding to the aromatic hydrogens as well as a D 2 O-exchangable broad singlet at d 9.55 ppm due to the NH group.The 13 C NMR spectrum revealed six signals at d 14.4, 25.9, 33.2, 53.6, 61.4 and 79.1 ppm for the aliphatic carbons and eleven distinct signals for unsaturated carbons at d 99.7, 117.4,122.5, 124.0, 129.0, 139.2, 154.8, 164.5, 166.2, 166.8, and 169.5 ppm in which the latter belongs to the carbon of ester carbonyl group as the most deshielded carbon.In the IR spectrum of 4f, two stretching absorption bands were observed at 2205 and 1733 cm À1 attributed to the nitrile (CN) and carbonyl of ester (CO 2 Et), respectively.Additionally, the observation of the molecular ion peak at m/z 486 in the mass spectrum, along with the complimentary results of the elemental analysis of 4f S-alkylated by ethyl chloroacetate.Analogous results were obtained for the remaining products 4a-e and 4g.
In vitro antioxidant study 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (vitamin C) were supplied from Sigma-Aldrich, and used without further purification.DPPH free radical scavenging was assessed according to the previously published research. 48][51][52] Consequently, the capability of electron or hydrogen radical donation can be a determining factor in predicting these effects.For this purpose, antioxidant activities of all synthesized heterocycles were determined through DPPH free radical scavenging method.Their IC 50 values are reported as the mean of three independent experiments ± standard division in Table 1.
Among the Se-containing heterocycles, the best antioxidant activity was observed with 3amino-2,4-dicyano-5-(pyrrolidin-1-yl)selenophene 2 which was much more effective than ascorbic acid as a positive control.In contrast, selenopheno[3,2-d]pyrimidine 3 had the least activity.The antioxidant effects of S-substituted selenopheno[3,2-d]pyrimidines 4a-g were also between the potency of precursors 2 and 3.It can be probably concluded that -NH 2 , -NHC ¼ S and -SCH 2groups act as hydrogen radical donators in compounds 2, 3 and 4a-g, respectively.The initial radicals formed by the interaction with DPPH radical are shown in Figure 1.According the obtained results, the order of radical stability is 2 0 > 4 0 a-g > 3 0 .As the length of alkyl chain substituted on sulfur element increases from methyl to n-butyl in selenopheno[3,2-d]pyrimidines 4ad, their antioxidant activities predictably increases.In comparison to analogues 4a-d, the activity of selenopheno[3,2-d]pyrimidines 4e-g were improved.This is due to the presence of adjacent radical stabilizing groups including Ph, CO 2 Et and CN.These substituents can stabilize radical centers via resonance delocalization in the following order: Ph > CO 2 Et > CN.
Antiperoxyradical ability of some synthesized selenophene derivatives was studied via differential pulse voltammetry method using DPPH. 53DPPH radical scavenging effects of novel derivatives containing selenophene moiety were assessed by El-Sawy et al.; IC 50 values of effective selenophenes were in the range between 186.75 and 2198.62mg.mlÀ1 . 54Antioxidant activity of systems including the fused rings of selenophene and pyrimidine was evaluated by Kotaiah et al. through DPPH, H 2 O 2 and NO radical scavenging methods. 55Some of them showed acceptable antioxidant properties.

Conclusion
The objective of the present study was to employ an efficient strategy for the synthesis of the novel diversely functionalized selenium-containing fused heterocycles and investigate the antioxidant of them.The reaction of 3-amino-2,4-dicyano-5-(pyrrolidin-1-yl)selenophene 2 with phenyl isothiocyanate was resulted in pyrimidine ring closure through hetero-annulation.In continuation, the treatment of 4-imino-3,4-dihydroselenopheno[3,2-d]pyrimidine 3 with several alkyl halides provided potential biologically active S-alkylated derivatives 4a-g in good yields.Antioxidant potentials of selenophene 2 and its pyrimidine-fused derivatives 3 and 4a-g were evaluated against DPPH radicals.None-fused selenophene 2 was identified as the most effective antioxidant agent.The phenyl ring containing electron donating groups especially hydroxyl in 2benzylthio substituents are expected to improve antioxidant capacities of selenopheno [3,2-d]pyrimidines.The existence of significant relationship between antioxidant activity of compounds and many of their biological effects make them potent candidates to treat cancer, Parkinson's, inflammatory and diabetes diseases.

Table 1 .
Antioxidant activity of Se-containing heterocycles.The initial radicals probably formed by the interaction with DPPH radical.