Heterocyclization of polarized system: synthesis, antioxidant and anti-inflammatory 4-(pyridin-3-yl)-6-(thiophen-2-yl) pyrimidine-2-thiol derivatives

Background Chalcones are intent in the daily diet as a favorable chemotherapeutic compound; on the other hand thiophene moiety is present in a large number of bioactive molecules having diverse biological efficiency. Results Our current goal is the synthesis of (E)-1-(pyridin-3-yl)-3-(thiophen-2-yl) prop-2-en-1-one 3 that’s used as a starting compound to synthesize the novel pyrimidine-2-thiol, pyrazole, pyran derivatives. Chalcones 3 was prepared by condensation of 3-acetylpyridine with thiophene 2-carboxaldehyde which reacted with thiourea to obtain pyrimidinthiol derivative 4. Compound 4 was allowed to react with hydrazine hydrate to afford 2-hydrazinylpyrimidine derivative 5. Compound 5 was used as a key intermediate for a facile synthesis of the targets 6 and 7. In contrast, pyranone 8 was obtained by transformation of compound 5. Using as a precursor for the synthesis of new pyrazolo pyrimidine derivatives 9–10. The major incentive behind the preparation of these compounds was the immense biological activities associated to these heterocyclic derivatives. Conclusions The newly synthesized compounds (1–4) showed potent anti-inflammatory activities both in vitro and in vivo. They also exhibited promising antioxidant vitalities against α, α-diphenyl-β-picrylhydrazyl scavenging activity and lipid peroxidation. In conclusion, compound 1 showed a hopefully anti-inflammatory and antioxidant activities.


Results and discussion
Chemistry Aldol condensation reaction of 3-thiophenecarboxaldehyde 1 with 3-acetylpyridine 2 in ethanolic NaOH solution afforded chalcone 3. The structure of compound 3 was elucidated by its IR, 1 H NMR and 13 C NMR. Its IR spectrum showed a characteristic peak for a conjugated carbonyl group at 1633 cm −1 , and by its 1 H NMR which gave signals at δ 7.53 (d, 1H, J = 12.9 Hz, (CH=C-C=O), and 7.92 (d, 1H, J = 12.9 Hz (CH=C-C=O) and two doublet signals at δ = 7.28 and 7.94 due to thiophenyl-C 4′ H and thiophenyl-C 3′ H and another at 8.11 owing to thiophenyl-C 5′ H whereas, the 13 C NMR spectrum showed a signal at (δ in ppm) 123 caused by ethylene group and 125, 126, 135, 149 and a signal due to C=O groups at 193. [3 + 3] base induced cycloaddition of chalcone 3 with thiourea gave 4-(pyridin-3-yl)-6-(thiophen-2-yl) pyrimidine-2(1H)-thione 4. IR spectra of compounds 4 showed the presence of a C=S band at 1270 cm −1 and an absorption band in the range 3433-3490 cm −1 attributed to the amine (NH). The 1 H NMR spectrum of compound 4 two doublet signals at δ = 7.28 and 7.94 due to thiophenyl-C 4′ H and thiophenyl-C 3′ H and another at 8.11 as a result of thiophenyl-C 5′ H. The spectra displayed a singlet at 8.82 for NH, respectively.
The hydrazinopyrimidine derivative 5 was synthesized by condensation of the thiopyrimidinone 4 with hydrazine hydrate in refluxing alcohol, the structure of compound 5 was confirmed by the IR, 1 H NMR and elemental analysis, where its IR revealed the absorption bands at ν max = 3212 for the NH 2 and 3184 cm −1 for the NH group, 1 H NMR spectrum gave the signals at δ = 8.93-8.95 as a broad singlet for NH 2 , hydrazine NH, respectively (Scheme 1).
Cyclocondensation of chalcone 3 and ethyl cyanoacetate in the presence of sodium ethoxide under the reflux conditions [33] gave pyranone derivative 6. Condensation with hydrazinehydrate [34,35] in refluxing ethanol leads to ring transformation producing corresponding pyridinones 7. The structure of the target 7 was confirmed from its spectral data, where is IR spectra showed absorption bands in the region 2222 and 1688 cm −1 characteristic for C≡N and carbonyl group, respectively (Scheme 2).
The hydrazinopyrimidine derivative 5 was used as a precursor for the synthesis of some heterocyclic compounds. The hydrazinopyrimidine derivative 5 reacted with ethyl acetoacetate in excess manner to afford compound 8. The formation of 8 may be proceeds via the formation of pyrazolone derivative 9 followed by the attack of methylene anion of pyrazolone to ketonic function of ethyl acetoacetate followed by pyran cyclization. IR spectrum of compound 8 revealed the absorption peaks at 1715 cm −1 characteristic of C=O groups respectively, 1 H NMR exhibited the two singlets at δ = 2.25 and 2.32 for 2 CH 3 protons and a singlet at δ = 5.60 ppm for pyranone H. Furthermore, the pyrimidine pyrazolone compound Scheme 1 Synthesis of pyrimidine derivatives 9 was obtained as a result of attack of hydrazinofunction of 5 to ethyl acetoacetate. The pyrazolo pyrimidine 10 was synthesized by heating an alcoholic solution of compound 5 (10.0 mmol.) with acetylacetone (10.0 mmol.) at reflux temperature for 5 h. The IR spectra of 9 and 10 showed the disappearance of the hydrazine group where the 1 H NMR spectrum showed singlet pyrimidine H at δ = 8.95 ppm and two singlets for the two CH 3 protons, respectively (Scheme 3).

Biological activity studies In vitro anti-inflammatory activity
In vitro COX-1 and COX-2 inhibition Compounds (3-6) were calorimetrically evaluated for their anti-inflammatory activities in vitro for COX-1 and COX-2 at 590 nm using ovine COX-1/COX-2 inhibitor screening assay kit [36]. Celecoxib was used as a standard reference drug.
In vitro 5-LOX inhibition A bnova 5 lipoxygenase inhibitor screening assay was used [37]. Meclofenamate sodium was used as a standard reference drug. Results were expressed in Table 1 as IC 50 as means of thee determinations the selectivity index was calculated also as IC 50 (COX-1)/IC 50 (COX-2).

In vivo anti-inflammatory activity
Carrageenan induced rat paw edema in rats: Fifty rats were divided into ten groups (i.e., each group, five rats). The first group (control), received carboxymethyl cellulose. The second group was given diclofenac sodium as a standard anti-inflammatory drug. Groups (3-10) were Scheme 2 Synthesis of pyranone and pyridinones derivatives Scheme 3 Synthesis of isolated and fused pyrimidine derivatives orally given the newly synthesized compounds (3-6) in two dosages (5 and 10 mg/kg). Results were expressed as rat paw edema percent. One hour later after administration of tested doses, carrageenan was injected sub planter in the left hind footpad of each rat as 0.05 ml of 1% solution in sterile distilled water. Plethysmometer was used to measure paw edema volume from 0 to 4 h after carrageenan injection. Paw edema volume was compared with vehicle control group and reduction percent was calculated as the following Where Vt and Vc are the edema volume in the group treated with drug and control, respectively [38]. Results were expressed as mean ± standard deviation (SD). Differences between means were tested for significance using a one-way analysis of variance (ANOVA) followed by Duncan's test (Table 2).

Antioxidant screening
a. DPPH free radical scavenging assay was determined (4). Results were presented in Table 3 as IC 50 (μg/ ml). Ascorbic acid was used as reference standard antioxidant. b. Lipid peroxidation assay (5 and 6) was calculated as IC 50 and recorded in Table 3.
The newly synthesized compounds exhibited a remarkable in vivo and in vitro anti-inflammatory activity. These results are in agreement with those obtained by other researchers [39]. They reported that some novel pyrimidine-pyridine hybrids inhibited cyclooxygenase enzyme and had a significant anti-inflammatory activity comparable to celecoxib as a standard drug. In this concern, other authors [40] reported an investigation of the efficacy of pyridine and pyrimidine analog of acetaminophen as peroxyl radical trapping antioxidants and inhibitors of enzyme catalyzed lipid peroxidation by cyclooxygenase and lipoxygenase. Compounds 3 and 4 exhibited antioxidant activity screening higher scavenging activity towards the DPPH radicals than that of ascorbic acid. Similar results were reported for new pyridine and triazolopyridine derivatives [41][42][43][44][45].

Conclusions
We have reported the synthesis of (E)-1-(pyridin-3-yl)-3-(thiophen-2-yl) prop-2-en-1-one 3 and using to designing a polycyclic heterocyclic compounds containing five and/ or six rings fused. Moreover, we concluded that compounds 3 and 4 showed a significant antioxidant activity regarding cyclooxygenase inhibitory activity, compound 3 presented the highest inhibitory activity in comparison to the standard reference drug [IC 50 as 3.7 and 0.39 µM for COX-1 and COX-2, respectively compared to 5.47 and 0.86 for the standard celecoxib]. Compound 4 also showed a potent inhibitory activity for COX-2 with IC 50 0.44. Compounds 5 and 6 showed inhibitory activity against COX-1 and COX-2 nearly like that of the standard drug. Compound 3 showed the highest inhibitory potential for 5-lipoxygenase with IC 50 (4.71 µM) compared to (6.15 µM) of the standard anti-inflammatory drug meclofenamate sodium.