SENJU EXHIBIT 2074
`LUPIN v SENJU
`IPR2015-01105
`
`PAGE 1 OF 3
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`TABLE 1. Alkaline Hydrolysis of Benzylpenicillin Anilides
`
`CaH5Cl~lzCONH— cH—— i‘+’\'c(C.”’
`(J:
`CH,
`/“w—H%flMi
`,
`O
`
`R
`
`Melting
`_ l Calculated
`”.
`R
`point
`Empirical
`____fl____ E
`'0 1
`
`(in de ).
`formula
`C
`N
`C
`N
`T‘
`.5
`<: *3. M E
`g
`
`H
`p—CH3
`p-OH
`p~Br
`P-N02
`m-N02
`m-OH
`——NHC5H4 X=OH
`
`9,82 64,50 10,20 4,58
`64,50
`C22H23N3O3S
`129-1303»
`9,43 65,30
`9,91
`5,12
`65,41
`c,,H,,N,o,s
`190-1911:
`9,85 61,90
`9,89 5,50
`60,90
`C3zH33N3O4S
`l28—131C
`8,76 53,80
`8,52 3,91
`169-17013 C2gH23N5O3BIS 53,16
`205—207b
`C22H22N405S
`59,00 12,90 58,20 12,30
`1,02
`159———16lC
`C32H22N4O5S
`58,10 11,87 58,20 12,30 2,50
`1I2—1l5C
`C22H33N3O4S
`00,90
`9,49 61,90
`9,89 4,17
`CuH13N2O4S
`-—
`—~
`——
`——
`——
`
`2,28
`2,39
`2,53
`2,10
`1,83
`1,87
`2,21
`16,50
`
`*Crystallization solvents: a) carbon tetrachloride; b) acetone;
`ethylacetate + petroleum ether.
`
`c)
`
`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 - l0‘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 ,8-lactam ring,when P‘CH3
`
`The reaction constant p has the value——0.l2. The negative sign of p bespeaks a favorable effect of
`electron donor substituents upon the reaction velocity constants, while its low absoluteivalue indicates
`slight sensitivity of the reaction to structural variation in the anilide molecule.
`
`EXPERIMENTAL
`
`Anilide Synthesis. 0.00268 mole of potassium benzylpenicillinate was dissolved in 10 ml water and
`the solution was acidified with 0.5 N HCl to a pH, of ca 2.0. The aqueous phase was saturated with KCl, and
`the liberated benzylpenicillinic acid was extracted several times with 3 ml portions of chloroform. The
`extracts, dried with anhydrous Na2SO4 and cooled to —5°, were mixed with 0.00268 mole of triethylamine
`in chloroform and allowed to stand for 30 min. After addition of 0.00268 mole of ethoxyforrnic 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 of nitroanilines
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`PAGE 2 OF 3
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`73
`
`-1og K
`
`2-75
`
`2.72‘
`
`2'68
`
`2_5o
`
`P‘OH
`j—1—-——
`-—-J
`-——-—l
`Ia I
`0,6
`0,4
`0,2
`~o.2
`0
`-0.4
`Fig. 3. Velocity constants of hydrolysis of
`benzylpenicillin anilides as a function of Ham—
`met's o constants for the respective substituents
`in aniline; p = -0.12.
`
`
`
`COOH is replaced by the arylamide group CONHC6H4R.
`The introduction of electron donor or electron acceptor
`.
`.
`.
`.
`.
`.
`.
`substituents into the benzylpenicillin 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 5—lactam
`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.
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`PAGE 2 OF 3
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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 Nazi-IP04, again with distilled water, and finally dried with anhydrous Na2SO4. 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 benzy1penicil1in—anilide (0.05—0.07 g) was dissolved in a
`50 ml 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 iodometrioally [7]
`for residual, non-hydrolyzed, benzylpenicillin.
`
`P. Doyle, J. H. C. Nayler, and H. R. J. Waddington, J, Chem. Soc., 497 (1963).
`P. Doyle, J. H. C. Nayler, H. Smith, et al., Nature _1_9__1_, 1091 (1961).
`S.
`Khokhlov, E. M, Kleiner, and E. N. Druzhinina, Antibiotics, No. 6, 42 (1958).
`F.
`Paparin and M; V. Solovskii, ibid., No. 11, 1000 (1965).
`D. A. Johson, J. Am. Chem. Soc., lg, 3636 (1953).
`'
`A, Albert and E. P. Serjeant, Ionization Constants of Acids and Bases, London (1962).
`The Analyst, 11. 550 (1949),
`
`
`
`n
`
`LITERATURE CITED
`
`E E A E
`
`o
`
`_<l‘d§U1§:PC.OL\'Jl-*
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`74 PAGE 3 OF 3
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