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Synthesis of isosafrale To 80 g (0.49 mmol) of safrole was added 100 ml of a 3N solution of potassium hydroxide (KOH) in n-butyl alcohol and the reaction mixture was stirred at room temperature for 3 h. The mixture was poured into a solution of 12 ml of concentrated hydrochloric acid (HCl), and 52 ml of ice water. After neutralization with additional concentrated HCl, the organic layer was extracted with three 35-ml portions of ethyl acetate. The organic extracts were treated with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure, furnishing a crude oily residue. Distillation of this residue, under reduced pressure, furnished 78.4 g (97%) of isosafrole, as a colorless oil. (2) Synthesis of Piperonal A solution of isoafrole (2 g, 12.1 mmol) in acetic acid (10 ml) was saturated with ozone at 0° C., until the formation of a slight blue color. After removal of the excess of ozone by bubbling N2 through for some time, the ozonide was decomposed by stirring in the presence of zinc (Zn) (5 g, 0.076 atg) at 0° C. for 2 h. After filtration, ethyl acetate was added, the reaction mixture was washed with water, and the solvent was evaporated, following which it was dried over anhydrous sodium sulfate, affording 1.42 g (77%) of piperonal, as a white solid, m.p. 36–37° C. (3) Synthesis of Methyl 3,4-Methylenedioxybenzoate To a solution of piperonal (0.30 mmol 0.045 g) in absolute methanol (4 ml) cooled at 0° C., were successively added methanolic solutions (each 3 ml) of iodine (0.100 g, 0.39 mmol) and KOH (0.440 g, 7.85 mmol) at 0° C. After stirring for 1.5 hour at 0° C., small amounts of saturated NaHSO3 solution were added until the disappearance of the brown color. Next, the methanol was almost totally evaporated under reduced pressure. To the residue was added water and the desired methyl 3,4-methylenedioxybenzoate was obtained by filtration, in 90% yield, as a white solid m.p. 53° C. 1NMR (200 MHz) CDCl3/TMS (δ-ppm): 7.63 (dd, H6, Jax-8.2 Hz, Jbx=1.7 Hz); 7.44 (d, H2, Jax=1.6 Hz); 6.82 (d, H5, Jax=8.2 Hz); 6.02 (s, O—CH2—O); 3.87 (s, O—CH3); 13C NMR (50 MHz) CDCl3/TMS (δ-ppm); 166.0 (C═O); 151.4 (C4); 147.5 (C3); 125.1 (C6); 124.0 (C1); 109.3 (C2—AR); 107.7 (C5); 101.6 (O—CH2—O); 51.9 (OCH3); M.S. (70 eV) m/z (relative abundance); 180 (50%); 149 (100%), 121 (20%); 91 (8%), 65 (18%); IR (KBr) cm−1: 1723 (C═O); 1289 (C—O). (4) Synthesis of 3,4-Methylenedioxybenzoylhydrazine To a solution of 2.67 g (14.85 mmol) of methyl 3,4-methylenedioxybenzoate in 10 ml of ethanol, was added 15 ml of 80% hydrazine monohydrate. The reaction mixture was maintained under reflux for 3.5 hours, when thin layer chromatography indicated the end of the reaction. Then, the media was poured on ice, and the resulting precipitate was filtered out, affording the 3,4-methylenedioxybenzoylhydrazine derivative in 70% yield, as a white solid, m.p. 170–171° C. 1H NMR (200 MHz) DMSO/TMS (δ-ppm): 10.74 (s, —CONH—); 7.44 (dd, H6, Jax=8.2 Hz, Jbx=1.6 Hz); 7.36 (s H2) 7.17 (d, H5,J=8.2 Hz); 6.10 (s, O—CH2—O); 4.45 (s, —NH2); 13C NMR (50 MHz) DMSO/TMS (δ-ppm); 165.2 (C═O); 149.6 (C4); 147.3 (C3); 127.2 (C1); 121.9 (C8); 107.9 (C2); 107.1 (C5); 101.6 (O—CH2—O); M.S. (70 eV) m/z (relative abundance); 180 (17%); 149 (100%), 121 (25%); 91 (8%); 65 (19%); IR (KBr) cm−1: 3303.9 NH2); 3220 (NH); 1605 (C═O); 1262 (C—O). (5) Synthesis of 3,4-Methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294) To a solution of 0.150 g (0.83 mmol) of 3,4-methylenedioxybenzoylhydrazine in absolute ethanol (7 ml) containing two drops of 37% hydrochloric acid, was added 0.098 g (0.87 mmol) of thiophene-2-carboxaldehyde. The mixture was stirred at room temperature for 30 minutes, after which extensive precipitation was visualized. Next, the mixture was poured into cold water, and the precipitate formed was filtered out and dried. The 3,4-methylenedioxybenzoyl-2-thienylhydrazone was obtained in 85% yield, after recrystallization in ethanol, as yellow needle crystals, m.p. 204–205° C. 1NMR (200 MHz) DMSO/TMS (δ-ppm): 11.53 (s, —CO—NH—); 8.62 (s, ═CH—); 7.65 (d, H5, J=5.0 Hz); 7.53 (d, H4, J=5.0 Hz); 7.50 (dd, H6, Jax=8.2 Hz, Jax=8.2 Hz, Jbx=0.8 Hz); 7.41 (s, H2); 7.12 (dd, H3′, Jax=4.8 Hz, Jbx=3.9 Hz); 6.12 (s1 O—CH2—O); 13C NMR (50 MHz) DMSO/TMS (-ppm); 162.1 (C═O); 150.2 (C4); 147.4 (C3); 142.6 (═Ch—); 139.2 (C1′); 130.8 (C3′); 128.8 (C2′); 127.9 (C4′); 127.1 (C1); 122.8 (C8); 108.0 (C2); 107.6 (C5); 101.8 (O—CH2—O); IR (KBr) cm−1: 3075 (NH); 2798 (N═CH), 1610 (C═O); 1540 (C═N); 1275 (C—O). Detailed technical methods for performing the described steps can be found in the following references (Barreiro, E. J. and Lima, M. E. F (1992) J. Pharm. Sci. 81, 1219); (Barreiro, E. J., Costa, P. R., Coelho, F. A. S. and Farias, F. M. C. (1985) J. Chem. Res., (M) 2301.); (Yamada, S., Morizono, D., Yamamoto, K. (1992) Tetrahedron Lett., 33, 4329); (Dias, M. L. R., Alvim, J. J. F., Freitas, A. C. C., Barreiro, E. J., Miranda, A. L. P. (1994) Pharm. Acta Helvetiae, 69, 163.).
