Green synthesis of highly functionalized octahydropyrrolo[3,4-c ]pyrrole derivatives using subcritical water, and their anti(myco)bacterial and antifungal activity

A series of novel 2-(thiazol-2-yl)-octahydropyrrolo[3,4-c ]pyrroles was synthesized by reaction of octahydropyrrolo[3,4-c ]pyrrole N -benzoylthiourea derivatives and α-haloketones in subcritical water at 130 °C in 75-91% yield. Both the thiourea intermediates and the end products were synthesized in subcritical water, which proved a suitable green alternative to acetone by delivering the desired compounds in much shorter reaction times and practically the same yields. The antimicrobial activity of the compounds was determined against five bacterial strains and three fungal strains, and MIC values of 15.62-250  g/mL were observed. Moreover, the compounds exhibited antimycobacterial activity against M. tuberculosis H37Rv with MIC values of 7.81-62.5 µg/mL.


Introduction
2][3][4] Thiazoles are present in a wide range of pharmacologically active compounds exhibiting antibacterial, [5][6][7][8] antimycobacterial, [9][10][11] antifungal, [5][6][7][8] anticancer [12][13][14] and antiviral 15,16 activity.Furthermore, thiazole derivatives are useful as chemosensors 17 and fluorescent sensors. 18he pyrrolidine moiety has been intensively studied in medicinal chemistry 19 due to its presence in the structure of many biologically active compounds such as anisomycin 20 and hygrine. 21Furthermore, pyrrolidine derivatives show various pharmacological activities such as antimicrobial, [22][23][24] antiviral 25 and anticancer activity. 26,27In addition, pyrrolidine-2,5-dione derivatives have also been reported to exhibit a wide range of pharmacological activities such as antimicrobial 28 and anticonvulsant activity. 29Compounds containing pyrrolidine fused to pyrrolidine-2,5-dione represent an important class of bicyclic ring systems in drug research and exhibit various pharmacological activities such as antibacterial 30,31 and anticancer activity. 32ubcritical water is a green alternative to conventional solvents due to being an environmentally friendly, cheap, safe and non-toxic solvent. 33Water, which is heated at a temperature range of 100 °C -374.2 °C and pressurized enough to keep it in the liquid state in this range is defined as subcritical water. 34Subcritical water offers the unique medium for various processes such as oxidation, extraction, 35,36 solubility 37 and organic reaction synthesis 38 through tunable solvent and other physico-chemical properties. 36The dielectric constant that determines the polarity of a solvent can be easily reduced by increasing the temperature in subcritical water. 39Thus, subcritical water acts as a non-polar solvent like ethanol or methanol, providing a favorable medium for organic synthesis.Many reactions such as alkylation, condensation, coupling, decomposition, Diels-Alder, decarboxylation, dehydration, elimination reactions and nano-synthesis can be carried out using subcritical water. 38,40,41n this study, we designed and synthesized octahydropyrrolo [3,4-c]pyrrole N-benzoylthioureas and a series of novel 2-(thiazol-2-yl)-octahydropyrrolo [3,4-c]pyrrole derivatives in subcritical water as green solvent, and investigated their anti(myco)bacterial and antifungal activities.
The synthesis of the octahydropyrrolo [3,4-c]pyrrole N-benzoylthiourea derivatives 3a-b and 2-(thiazol-2yl)-octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j in subcritical water was performed in a reaction apparatus as depicted in Figure 1.The experiments were carried out in a home-made stainless steel reactor with an internal volume of 200 mL equipped with a heater-magnetic stirrer.To synthesize the octahydropyrrolo [3,4c]pyrrole N-benzoylthiourea derivatives 3a-b, the reactor was loaded with octahydropyrrolo [3,4-c]pyrroles 2ab (1 mmol), benzoyl isothiocyanate (2 mmol) and 75 mL of ultra-pure water.After purging the reactor air with nitrogen, the internal pressure of the reactor was fixed at 30 bar supplied by nitrogen.Each experiment was performed at 130 °C for 4 hours under stirring.Finally, the reactor was depressurized and the obtained mixture was extracted using dichloromethane.The crude product was purified by column chromatography (EtOAc:hexane / 1:3) to afford compounds 3a-b in 80-82% yield (Scheme 1, Table 1).For the synthesis of 2-(thiazol-2-yl)-octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j in subcritical water, a similar procedure described above was applied.Briefly, the reactor was loaded with octahydropyrrolo [3,4c]pyrrole N-benzoylthiourea 3a or 3b (1 mmol), an appropriate α-haloketone (1.2 mmol) and 75 mL of ultrapure water.After purging the air in the reactor with nitrogen, the internal pressure of the reactor was fixed at 30 bar supplied by nitrogen.Each experiment was performed at 130 °C for 2 hours under stirring.Finally, the reactor was depressurized and the obtained mixture was extracted using dichloromethane.The crude product was purified by column chromatography (EtOAc:hexane / 1:4) to afford compounds 4a-j in 74-91% yield (Scheme 1, Table 1).
Furthermore, the synthesis of the octahydropyrrolo [3,4-c]pyrrole N-benzoylthiourea derivatives 3a-b and the 2-(thiazol-2-yl)-octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j in subcritical water was compared with their synthesis in conventional organic solvent.The synthesis of compounds 3a-b was performed in acetone at reflux temperature for 30 hours and the products were obtained in 80-82% yield.Similarly, the synthesis of compounds 4a-j was also performed in acetone at reflux temperature for 18 hours and the products were obtained in 67-89% yield.Thus, the synthesis in subcritical water has practically the same yields but has the advantages of a shorter reaction time and of being a green solvent.It can be said that subcritical water is an excellent alternative to conventional organic solvent for the synthesis of both octahydropyrrolo [3,4-  The antimicrobial activity screening of the octahydropyrrolo [3,4-c]pyrrole N-benzoylthiourea derivatives 3a-b and 2-(thiazol-2-yl)-octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j was performed against five standard bacterial strains, one standard mycobacterium strain and three standard fungal strains.The compounds 3a-b and 4a-j exhibited antibacterial activity in the range of 15.62-250 g/mL (Table 2).The compounds 3a-b were less active than the reference compound Ampicillin against S. aureus, B. subtilis and E. coli.However, compound 3a was more active than Ampicillin against the A. hydrophila and A. baumannii strains, and compound 3b showed the same antibacterial activity as Ampicillin against these two strains.The compounds 4a-j exhibited lower antibacterial activity than the reference compound Ampicillin against B. subtilis, A. hydrophila and E. coli.Compound 4b exhibited similar antibacterial activity as Ampicillin against the S. aureus strain, with a MIC value of 31.25 μg/mL.Furthermore, compounds 4d and 4j were more active than Ampicillin against A. baumannii, an opportunistic pathogen with increasing importance in nosocomial infections.The other 2-(thiazol-2-yl)-octahydropyrrolo [3,4-c]pyrrole derivatives 4a-c, 4e-i exhibited similar antibacterial activity with Ampicillin against the A. baumannii strain.

MIC: The minimal inhibitory concentrations
The compounds 3a-b, 4a-j showed antifungal activity, in the range of 15.62-250 g/mL, but did not exhibit higher antifungal activity than the reference compound Fluconazole, which exhibited antifungal activity against the fungi with MIC values in the range of 3.90-15.62µg/mL (Table 2).It can be said that all of the tested compounds exhibited moderate antifungal activity against the mentioned fungi.Compounds 3a-b, 4a-j were also tested against the Mycobacterium tuberculosis H37Rv strain.Compound 4a performed best with a MIC value of 7.81 µg/mL, but none of the compounds scored better than the reference drug Isoniazid.

Conclusions
The synthesis of octahydropyrrolo [3,4-c]pyrrole N-benzoylthiourea derivatives 3a-b and the 2-(thiazol-2-yl)octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j was successfully performed in subcritical water as a green solvent.Despite the fact that reactive agents such as isothiocyanates and α-haloketones were used, applying subcritical water as a solvent resulted in a significant decrease of reaction times, while maintaining high yields.The synthesized compounds displayed moderate antimicrobial activity against different bacterial and fungal strains and against the Mycobacterium tuberculosis H37Rv strain.

Experimental Section
General.All chemicals used had high-grade commercial products purchased from Merck or Aldrich and all solvents provided by commercial suppliers had reagent grade quality and were used without further purification.A Varian Scimitar Series 1000 FT-IR spectrophotometer, using horizontal ATR, was used.Nuclear magnetic resonance spectra were determined at 400 MHz on a Bruker Ultrashield Plus Biospin GmbH.Chemical shifts are given in parts per million (δ) downfield from TMS as internal standard.Flash column chromatography was performed using silica gel 60 (230-400 mesh).Kieselgel columns were packed with silica gel GF254.Melting points were determined on a Mettler Toledo MP90 device.Mass spectra were recorded by an Agilent 6460 Triple Quad LC/MS/MS mass spectrometer.High resolution mass spectra were recorded by an LC-MS TOF electrospray ionization technique.

General procedure for the synthesis of octahydropyrrolo[3,4-c]pyrrole derivatives (2a,b).
The compounds 2a,b were synthesized using a literature method. 42To a stirred solution of methyl 2-(diphenylmethyleneamino) acetate 1 (1 mmol) in o-xylene (10 mL) was added a solution of N-substituted maleimide (2 mmol) in o-xylene (20 mL) and the reaction mixture was refluxed for 24 h.After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate.The crude mixture was purified by column chromatography (EtOAc:hexane  General procedure for the synthesis of the octahydropyrrolo[3,4-c]pyrrole N-benzoylthiourea derivatives 3a,b in subcritical water.The experiments were carried out in a home-made stainless steel reactor with an internal volume of 200 mL.To synthesize the octahydropyrrolo [3,4-c]pyrrole N-benzoylthiourea derivatives 3a-b, the reactor and magnet were firstly flushed with water and acetone, then thoroughly dried with nitrogen gas.The reactor was loaded with octahydropyrrolo[3,4-c]pyrroles 2a-b (1 mmol), benzoyl isothiocyanate (2 mmol) and 75 mL of ultra-pure water, after that the reactor cover screws were tightly clamped.After purging the air in the reactor with nitrogen, the internal pressure of the reactor was fixed at 30 bar supplied by nitrogen.Each experiment was performed at 130 °C for 4 hours under stirring.Finally, the reactor was cooled, depressurized, and the obtained mixture was extracted using dichloromethane (DCM).The crude product was purified by column chromatography (EtOAc:hexane / 1:3) to afford compounds 3a-b.General procedure for synthesis of the octahydropyrrolo[3,4-c]pyrrole N-benzoylthiourea derivatives (3a,b) in acetone.The octahydropyrrolo [3,4-c]pyrrole N-benzoylthiourea derivatives 3a,b were also synthesized by modification of a literature method. 43,44To a stirred solution of 2a (0.37 g, 1 mmol) or 2b (0.43 g, 1 mmol) in acetone (20 mL) was added a solution of benzoyl isothiocyanate (0.33 g, 2 mmol) in acetone (10 mL) and the mixture was stirred at reflux temperature for 36 h.After completion of the reaction, the solvent was evaporated under reduced pressure and the crude mixture was purified by column chromatography (EtOAc:hexane

General procedure for the synthesis of the 2-(thiazol-2-yl)-octahydropyrrolo[3,4-c]pyrrole derivatives (4a-j) in subcritical water.
The experiments were carried out in steel reactor which was identified in the synthesis of 3a,b and the same operations described above were applied.To synthesize the 2-(thiazol-2-yl)octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j, the required octahydropyrrolo [3,4-c]pyrrole Nbenzoylthiourea derivative 3a or 3b (1 mmol), an appropriate α-haloketone (1.2 mmol) and 75 mL of ultrapure water were loaded to the reactor.After purging the air in the reactor with nitrogen, the internal pressure of the reactor was fixed at 30 bar supplied by nitrogen.Each experiment was performed at 130 °C for 2 hours under stirring.Finally, the reactor was cooled, depressurized, and the obtained mixture was extracted using DCM.The crude product was purified by column chromatography (EtOAc:hexane / 1:4) to afford compounds 4a-j.General procedure for synthesis of the 2-(thiazol-2-yl)-octahydropyrrolo[3,4-c]pyrrole derivatives (4a-j) in acetone.The 2-(thiazol-2-yl)-octahydropyrrolo [3,4-c]pyrrole derivatives 4a-j were synthesized using a literature method. 45To a stirred solution of 3a (0.53 g, 1 mmol) or 3b (0.59 g, 1 mmol) in acetone (20 mL) was added a solution of an appropriate α-haloketone (1.2 mmol) in acetone (10 mL) and the mixture was stirred at reflux temperature for 18 h.After completion of the reaction, the solvent was evaporated under reduced pressure, quenched with saturated aqueous sodium chloride and extracted with DCM.The crude mixture was purified by column chromatography (EtOAc:hexane/1:4) to afford compound 4a-j.Structures of 4a-j were confirmed by NMR and MS techniques.

Figure 1 .
Figure 1.Schematic representation of the reactor equipped with a heater-magnetic stirrer.

Table 1 .
Structure and yield of compounds 3 and 4 a Yield of the reaction in subcritical water.b Yield of the reaction in acetone.

Table 2 .
The MIC values (µg/mL) of the tested compounds against the microbial strains.