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`SENJU EXHIBIT 2074
`LUPIN v. SENJU
`IPR2015-01097
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`TABLE 1. Alkaline Hydrolysis of Benzylpenicillin Anilides s /-...CH~ CbHsCI4zCONH-- CH--CH "C-...~,, 1~ H p -CHa p -OH p -Br D -NO~ ~n-NO~ m-OH --NHCoH~ X =OH Melting point (in deg)* 129--130a 190--191 b 128--131c 169--170b 205--207b 159--I61c 112--I15c Empirical formula C~2H~3N303S C~H~sN303S C~2H~N,04S C~zH2~NsO3BrS C~H~zN40~S C~H~N405S C22H~NaO4S CI~H lsN ~O~S !Found% C N 64,50 9,82 65,41 9,48 60,90 9,85 53,16 8,76 59,00 12,90 58,10 11,87 60,90 9,49 Calculated % C N 64,50 10,20 65,30 9, 91 61,90 9,89 53,80 8,62 58,20 12,30 58,20 12,30 61,90 9,89 o 4,58 5,12 5,50 3,91 1,02 2,50 4,17 o r o 2,28 2,39 2,53 2,10 1,83 1,87 2,21 16,50 *Crystallization solvents: a) carbon tetrachloride; b) acetone; c) ethylacetate + petroleum ether. -log K 2.76 2.72 2.68 2.64 2.60 m-NOz ,7 p-NOz p-Br p/_o - 0 -0.2 0.2 0.4 0.6 Fig. 3. Velocity constants of hydrolysis of benzylpenicillin anilides as a function of Ham- mat's G constants for the respective substituents in aniline; p -- -0.12. as a pseudounimolecular reaction. The reaction velocity constant was computed, and was also determined graph- ically, as for a first order equation. Velocity constants obtained for hydrolysis of the anilides were found to be about one order of magnitude lower than that of benzylpenicillin (16.5.10-3), but they differed little from each other (see Table 1 and Fig. 1). The substantial difference between the velocities of hydrolysis of benzylpenicillin and of the anilides is due to a sharp decrease in the electron density on the nitrogen atom of the penicillin 3-lactam ring,when COOH is replaced by the arylamide group CONHC6H4R. The introduction of electron donor or electron aceeptor substituents into the benzylpenicfllin anilide molecule leads to no significant change in the velocity constant of hydrolysis. This is caused by a considerable damp- ing of the inductive effect in the intermediate thia- zolidine ring. Even though the position of the sub- stituent was far removed from the reaction site, it was, nonetheless, possible to study its effect upon electron density on the nitrogen atom in the/3-1actam ring. It was found that electron donor substituents increased, and electron acceptor substituents decreased the velocity constant of hydrolysis. A linear relationship was established between the logs of the reaction velocity constants, the pK of the amine (Fig. 2), and Hammet's constant (Fig. 3), thereby confirming ap- plicability of the Hammet equation to the given reactions. The reaction constant p has the value-0.12. The negative sign of p bespeaks a favorable effect of electron donor substituents upon the reaction velocity constants, while its low absolute value indicates slight sensitivity of the reaction to structural variation in the anilide molecule. EXPERIMENTAL Anilide Synthesis. 0.00268 mole of potassium benzylpenieillinate was dissolved in i0 ml wate~ and the solution was acidified with 0.5 N HCI to a PH of ca 2.0. The aqueous phase was saturated with KCI, and the liberated benzylpenicillinic acid was extracted several times with 3 ml portions of chloroform. The extracts, dried with anhydrous Na2SO 4 and cooled to-5 ~ were mixed with 0.00268 mole of triethylamine in chloroform and allowed to stand for 30 rain. After addition of 0.00268 mole of ethoxyformic acid chloro- anhydride in chloroform to the formed triethylamine benzylpenicillinate, the solution was stirred for 30 min at-5 ~ and then treated with 0.00268 mole of the respective amine in chloroform (in the case ofnitroanilines 73
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`and aminophenols acetone was used as the solvent). The reaction went to completion in 3 h at a temperature of 0-5 ~ The reaction product was first washed with distilled water, then with 20 ml of 0.2 M citric acid, 25 ml 0.2 M Na2HPO 4, again with distilled water, and finally dried with anhydrous Na2SO 4. The chloroform was removed under vacuum without the addition of heat, and the residue was crystallized from a suitable to solvent to yield the anilide. Kinetic Studies, A weighed sample of the benzylpenicillin-anilide (0,05-0.07 g) was dissolved in a 50 m! volumetric flask, in 15 ml of dimethylformamide (purified by vacuum distillation), and diluted to the 50 ml mark with a pH 2.0 glycocoll buffer. The resultant solution was kept at 30 ~- 0.2 ~ in a thermostat, and samples were taken from the reaction vessels at given time intervals and analyzed iodometrically [7] for residual, non-hydrolyzed, benzylpenicillin. 1, 2, 3, 4~ 5. 6. 7, LITERATURE CITED E~ P. Doyle, J. H, C, Nayler, and H. R. J, Waddington, J, Chem. Soc., 497 (1963). E. P. Doyle, J. H. C. Nayler, H, Smith, et al., Nature 191, 1091 (1961), A. S. Khokhlov, E. M. Kleiner, and E. N. Druzhinina, Antibiotics, No. 6, 42 (I958). E. F. Paparin and M: V. SolovsMi, ibid, No. 11, 1000 (1965), D. A. Johson, J. Am, Chem. Soc., 75, 3636 (1953). A, Albert and E. P. Serjeant, Ionization Constants of Acids and Bases, London (1962). The Analyst, 74, 550 (1949), 74
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