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Among the synthetic procedures that involve 1,2migration from a carbon atom to a formal six-electron nitrogen atom,1 the Curtius Rearrangement (CR) of carbonyl azides is considered the most convenient and general route,2 especially for the synthesis of fivemembered heteroaryl isocyanates.3 Reactions carried out in anhydrous conditions give isocyanates in very good yields, but in the presence of moisture hydrolytic steps may result in the conversion of carboxylic acids into amines, amides, carbamates or other N-linked heteroarenes.4 The mechanism of the thermal CR process has been the subject of much debate since the pioneering work of Stieglitz.5,6 Other experiments gave results consistent with a concerted route for the thermal reaction.7 The dissociation of five-membered heteroaryl azides exhibits chemical and thermal reactions depend on the 2or 3-azido position on the heteroaryl ring,8 and has been studied as a source of nitrogen heteroaryls.9 Cyclooxygenase enzyme is involved in the process of inflammation. [...]in this study, anti mitotic activity was analyzed by docking to 1jff protein which is alpha-beta tubulin. [...]compound 5 coupled with picolinic acid to obtain title compounds. [...]microtubules have become the main target for synthetic drugs.
ABSTRACT:
The synthesis of piperazine derivatives containing thiophene and oxazole is described. They were prepared from (Z)-3-bromo-N'-phenylthiophene/furan-2-carbohydrazonoyl chloride in a series of steps. All the synthetic compounds are characterized by spectral analyses. They were further analyzed for anti-inflammatory and antimitotic activity and the results are discussed.
KEYWORDS: Synthesis, anti inflammatory activity, antimitotic activity
INTRODUCTION:
Among the synthetic procedures that involve 1,2migration from a carbon atom to a formal six-electron nitrogen atom,1 the Curtius Rearrangement (CR) of carbonyl azides is considered the most convenient and general route,2 especially for the synthesis of fivemembered heteroaryl isocyanates.3
Reactions carried out in anhydrous conditions give isocyanates in very good yields, but in the presence of moisture hydrolytic steps may result in the conversion of carboxylic acids into amines, amides, carbamates or other N-linked heteroarenes.4 The mechanism of the thermal CR process has been the subject of much debate since the pioneering work of Stieglitz.5,6 Other experiments gave results consistent with a concerted route for the thermal reaction.7
The dissociation of five-membered heteroaryl azides exhibits chemical and thermal reactions depend on the 2or 3-azido position on the heteroaryl ring,8 and has been studied as a source of nitrogen heteroaryls.9 Cyclooxygenase enzyme is involved in the process of inflammation. Anti inflammatory activity was evaluated by docking the compound to 5cox. Microtubules are important in cells undergoing mitosis. These contain a and ß-tubulin monomers bound by GTP. Hence in this study, anti mitotic activity was analyzed by docking to 1jff protein which is alpha-beta tubulin.
EXPERIMENTAL SECTION:
Chemical and solvents used were purchased either from Fluka or Merck. All the reagents were of analytical grade. Thin-layer chromatography (TLC) was performed on E. Merck AL silica gel 60 F254 plates and visualized under UV light. IR spectra were recorded as KBr pellet with a perkin-elmer spectrum gx FTIR instrument and only diagnostic and/or intense peaks are reported. JH NMR spectra were recorded in DMSO- d6 with a Varian Mercury plus 400 MHz instrument. Signals due to residual protonated solvent (1 H NMR) served as the internal standard. All the chemical shifts were reported in 5 (ppm) using TMS as an internal standard. The JH NMR chemical shifts and coupling constants were determined assuming first-order behavior. Multiplicity is indicated by one or more of the following: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad); the list of coupling constants (J) corresponds to the order of multiplicity assignment. Mass spectra were recorded with a PE Sciex model API 3000 instrument. All the reactions were carried out under argon atmosphere.
Docking calculations were carried out using Docking Server [10, 11, 12, 13]. The enzyme 5cox (Cycloxygenase) and 1jff(tubulin) was downloaded from RSC PDB and was docked.
tert-butyl4-((3-bromothiophen/furan-2-yl)(2- phenylhydrazono) methyl)-2-methylpiperazine-1- carboxylate (3): Tert-butyl 2-methylpiperazine-1- carboxylate 2 was added to anice cold solution of 3- bromo-N'-phenylfuran/thiophene-2-carbohydrazonoyl chloride 1and triethylamine in anhydrous THF and stirred at rt for 3 h. The progress of the reaction was monitored by TLC [(TLC silica gel plate), EtOAc, using 254 nm UV light to visualize the spot]. Rfvalue of the product is 0.4.After completion of reaction; the reaction mixture was poured in ice water and extracted with EtOAc. The organic layer was dried over anhy.Na2SO4 and concentrated under reduced pressure to get 60 g of crude product. The crude product was absorbed on 120 g of silica gel (100-200 mesh) and loaded over a prepacked column with silica gel [60 mm×80 cm width and height of column; 500g of 100-200 silica gel]. Elution started with 20% ethyl acetate in pet ether and finished with 50% ethyl acetate in pet ether. All pure fractions were collected and concentrated under reduced pressure to afford 31 g of compound-3 (42%).
1H NMR (400 MHz, DMSO-d6):8.5-8.3 (brs, 1H), 7.22- 7.20 (m, 4H), 6.95-6.93 (m, 2H), 3.88-3.87 (m, 2H), 2.8- 3.1 (m, 1H), 2.6 (m, 2H), 1.7 (m, 2H), 1.40 (d, 3H), 1.39 (s, 9H).
(S,Z)-tert-butyl-4-((3-bromofuran-2-yl)(2-phenyl hydrazono)methyl)-2-methylpiperazine-1-carboxylate : 1H NMR (400 MHz, DMSO-d6): 8.4-8.2 (brs, 1H), 7.2- 7.1 (m, 4H), 6.8-6.7 (m, 2H), 3.7-3.6 (m, 2H), 2.7-3.1 (m, 1H), 2.6 (m, 2H), 1.5 (m, 2H), 1.3 (d, 3H), 1.3 (s, 9H).
(2S)tert-Butyl 2-methyl-4-(1-phenyl-1H-thieno/furan [3, 2-c] pyrazol-3-yl) piperidine-1-carboxylate (4):A mixture of compound-2, Copper iodide and potassium carbonate in NMP (10mL) was stirred at 170°C for 1 h. The reaction was monitored by TLC [(TLC silica gel plate), EtOAc using 254 nm UV light to visualize the spot]. Rfvalue of the product is 0.5. After completion of reaction, the reaction mixture was poured in ice water and extracted with EtOAc (3 × 300 mL). The combined organic layer wise washed with water, brine and concentrated under reduced pressure to afford 35 g of crude product. The crude product was absorbed on 70 g of silica gel (100-200 mesh) and loaded over a prepacked column with silica gel [60 mm ×80 cm width and height of column; 300g of 100-200 silica gel]. Elution started with 25% ethyl acetate in pet ether and finished with 50% ethyl acetate in pet ether. All pure fractions were collected and concentrated under reduced pressure to afford 12.2 g of compound-7 (48%).
1H NMR (300 MHz, DMSO-d6):7.82-7.81(d, 1H), 7.71- 7.68 (d, 2H), 7.47-7.42 (t, 2H), 7.19-7.14 (t, 1H), 4.20 (m, 1H), 3.80-3.61 (t, 2H), 3.61-3.57 (d, 1H), 3.22-3.01 (m, 2H), 2.88-2.79 (m, 1H), 1.41-1.38 (s, 9H), 1.24-1.19 (d, 3H).
(2S) tert-Butyl 2-methyl-4-(1-phenyl-1H-thieno/furan [3, 2-c] pyrazol-3-yl) piperidine-1-carboxylate:1H NMR (300 MHz, DMSO-d6): 7.7-7.8b(d, 1H), 7.6-7.5 (d, 2H), 7.4-7.3 (t, 2H), 7.1-7.2 (t, 1H), 4.2 (m, 1H), 3.8- 3.7 (t, 2H), 3.6-3.5 (d, 1H), 3.2-3.0 (m, 2H), 2.8-2.7 (m, 1H), 1.4-1.3 (s, 9H), 1.1-1.0 (d, 3H).
(S)-3-(3-methylpiperazin-1-yl)-1-phenyl-1H-thieno/ furan[3,2-c]pyrazole.HCl (5):4M HCl in 1,4-dioxane was added to a solution of 4in 1,4-dioxane at 0 °C and stirred at rt for 16 h. The reaction was monitored by TLC [(TLC silica gel plate), EtOAc using 254 nm UV light to visualize the spot]. Rfvalue of the product is 0.1. After completion of reaction, the reaction mixture was concentrated under reduced pressure to afford 9.1 g of crude product. The crude product was triturated with diethylether and filtered to afford8.1 g of 6 (98%).
1H NMR (400 MHz, DMSO-d6): 7.88-7.87(d, J = 5.2 Hz, 1H), 7.75-7.73 (d, J = 7.6 2H), 7.58-7.57 (d, J = 5.6 2H), 7.51-7.46 (t, J = 7.6 2H), 7.23-7.20 (t, J = 7.2 1H), 3.9- 3.8 (t, J = 2.4 2H), 3.41-3.35 (m, 2H), 3.35-3.26 (m, 2H), 3.08-3.02 (t, J = 10.8 1H), 1.33-1.32 (d, J = 6 3H).Mass: (m/z = 299.0 [(M-HCl)+H]+).HPLC: 98.43% (215 nm), 98.98% (254 nm).CHIRAL HPLC: 99.90%
(S)-3-(3-methylpiperazin-1-yl)-1-phenyl-1Hfuran[ 3,2-c]pyrazole.HCl: 1H NMR (400 MHz, DMSO-d6): 7.85-7.83 (d, J = 5.2 Hz, 1H), 7.74-7.70 (d, J = 7.6 2H), 7.53-7.57 (d, J = 5.6 2H), 7.50-7.41 (t, J = 7.6 2H), 7.22-7.20 (t, J = 7.2 1H), 3.91-3.82 (t, J = 2.4 2H), 3.41-3.32 (m, 2H), 3.33-3.24 (m, 2H), 3.08-3.01 (t, J = 10.8 1H), 1.31-1.33 (d, J = 6 3H).
(S)-(2-methyl-4-(1-phenyl-1H-thieno/furan[3,2- c]pyrazol-3-yl)piperazin-1-yl)(pyridin-2- yl)methanone (6):HATU was added to a solution of 5, 2-Picolinic acid and N,N-Diisopropylethylamine in DMF (15 mL) at 0°C and stirred at 0 °C for 1 h. The reaction was monitored by TLC [(TLC silica gel plate), EtOAc using 254 nm UV light to visualize the spot]. Rfvalue of the product is 0.45. After completion of reaction, the reaction mixture was poured in ice water and extracted with EtOAc (3 × 100mL). The combined organic layer was washed with water, brine and concentrated under reduced pressure to afford 1.1 g of crude product. The crude product was absorbed on 5 g of silica gel (100-200 mesh) and loaded over a pre-packed column with silica gel [30 mm ×50 cm width and height of column; 50g of 100-200 silica gel]. Elution started with 25% EtOAc in pet ether and finished with 50% EtOAcin pet ether. All pure fractions were collected and concentrated to afford 0.53 g 6 (55%).
1H NMR (400 MHz, DMSO-d6):8.62 (d, J= 4.4 Hz, 1H), 7.94 (t, J = 7.6 Hz, 1H), 7.84-7.83 (d, J = 4.8 Hz, 1H), 7.73-7.71 (d, J = 8.4 Hz,2H), 7.61-7.45 (m, 5H), 7.20- 7.17 (t, J = 7.6 Hz, 1H), 4.91 (s, 1H), 4.51-4.48 (d, J = 12.4 Hz, 1H), 4.11-3.95 (m, 1H), 3.74-3.52 (m, 3H), 3.15 (bs, 1H), 2.99-2.93 (t, J = 3.6 Hz, 1H), 1.35(s, 3H).Mass: (m/z = 404.0 (M+H)+).HPLC: 99.16% (215 nm), 99.40% (254 nm). CHIRAL HPLC: 99.59%
(S)-(2-methyl-4-(1-phenyl-1H-furan[3,2-c]pyrazol-3- yl)piperazin-1-yl)(pyridin-2-yl)methanone: 1H NMR (400 MHz, DMSO-d6): 8.61 (d, J= 4.4 Hz, 1H), 7.92 (t, J = 7.6 Hz, 1H), 7.82-7.83 (d, J = 4.8 Hz, 1H), 7.71-7.73 (d, J = 8.4 Hz, 2H), 7.63-7.42 (m, 5H), 7.21-7.13 (t, J = 7.6 Hz, 1H), 4.95 (s, 1H), 4.54-4.44 (d, J = 12.4 Hz, 1H), 4.13-3.96 (m, 1H), 3.74-3.53 (m, 3H), 3.16 (bs, 1H), 2.96-2.99 (t, J = 3.6 Hz, 1H), 1.33 (s, 3H).
RESULTS AND DISCUSSION:
3-bromo-N'-phenylfuran/thiophene-2-carbohydrazonoyl chloride 1 reacts with (S)-tert-butyl 2 -methylpiperazine - 1 -carboxylate 2 to offer tert-butyl 4-((3 -bromothiophen/ furan-2-yl)(2-phenylhydrazono)methyl)-2methylpiperazine-1 -carboxylate 3 which undergoes cyclization in presence of Cul to give tert-Butyl 2methyl-4-( 1 -phenyl-1H-thieno/furan[3, 2-c] pyrazol-3yl) piperidine-1 -carboxylate 4. Compound 4 on hydrolysis forms (S)-3-(3 -methylpiperazin-1 -yl) -1phenyl-1H-thieno/furan[3,2-c]pyrazole.HCl 5. Finally compound 5 coupled with picolinic acid to obtain title compounds. The structures of all compounds were confirmed by spectral analyses.
Microtubules play a crucial role in cell division. Hence, microtubules have become the main target for synthetic drugs. The ligand synthesized also binds to ß-tubulin, thereby strengthening against disassembly of microtubules which inhibits mitotis causing apoptosis. Docking of 5cox (cycloxygenase) with the ligand using docking server, showed a free energy of -4.17 kcal/mol making the title compound a probable anti-inflammatory compound. Inhibition constant was 880.29uM which predicts that the ligand is going to inhibit enzyme (Tables 1, 2 and 3). Similarly, docking of ligand with 1jff(alpha-beta tubulin) protein showed a free energy of - 1.09 kcal/mol and an Inhibition constant 158.85uM (Tables 4, 5 and 6). In comparison, the compound was found to be a more potent anti-inflammatory compound than a antimitotic agent.
ACKNOWLEDGEMENTS:
Authors are thankful to Management, Principal and Head, Department of Sciences and Humanities of Sreenidhi Institute of Science and Technology for their encouragement and support for doing the research work.
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