91
`
`7
`
`H' H 1
`92
`mle 183
`Scheme VI-Fragmentation of the mle 183 ion
`
`the spectrum of oxyphenbutazone, analogous peaks were seen at
`mlc 108 (7.6%). (CGHGNO)+, and at mle 107 (11.2%), (CsHsNO)+.
`A characteristic fragmentation of all pyrazolidinediones studied
`was the loss of the elements of phenyl isocyanate either to form the
`radical ion (CGHaNCO)+, m / e 119 [(HOC~HSNCO)+, m / e 135 in
`the case of II] or as a neutral molecule. These pathways have been
`observed in the mass spectrum of aminopyrine (12). Fragmenta-
`tions involving the loss of phenyl isocyanate as a neutral molecule
`are listed in Table 11.
`The ions (X, Table 11) resulting from the loss of the elements of
`phenyl isocyanate as a neutral molecule may then lose CO to give
`
`ions C ~ H ~ N C R I R ~ (Table 111).
`The remaining peaks in the low mass range, below mle 100, are
`common to substituted aromatic systems, and the fragmentations
`observed are illustrated in Figs. 1-6.
`
`REFERENCES
`(1) J. Slingsby and D. A. Zuck, Can. J. Pharm. Sci., 7,
`115( 1972).
`(2) D. V. C. Awang, A. Vincent, and F. Matsui, J. Pharm. Sci.,
`62, 1673(1973).
`
`( 3 ) €3. Unterhalt, Arch. Pharm., 305,334(1972).
`(4) V. G. Pesin, A. M. Khaletskii, and T. Jun-Hsiang, J.
`Gen. Chem. USSR, 28,2841(1958).
`(5) J. Buchi, J . Ammann, R. Lieberherr, and E. Eichenberger,
`Helu. Chin. Acta, 36, 75(1953).
`(6) A. M. Khaletskii, V. G. Pesin, and T. Jun-Hsiang. J.
`Gen. Chem. USSR, 28,2393(1958).
`(7) H. Stenzl, U.S. pat. 2,562,830; through Chem. Abstr., 47,
`1 191 ( 1953).
`(8) J. H. Bowie, G. E. Lewis, and R. G. Cooks, J . Chem. Soc.,
`B, 1967,621.
`(9) J. M. Desmarchelier and R. B. Johns, Org. Mass. Spec-
`trom., 2,697(1969).
`(10) D. P. Maier, G. P. Happ, and T. H. Regan, ibid., 2.
`1289( 1969).
`(11) E. Larsen, I. H. Qureshi, and J. Mdller, ibid., 7,89(1973).
`(12) W. Arnold and H. F. Grutzmacher, 2. Anal. Chem., 247,
`179( 1969).
`
`ACKNOWLEDGMENTS AND ADDRESSES
`Received April 30, 1974, from the Faculty of Pharmacy and
`Pharmaceutical Sciences, University of Alberta, Edmonton, Al-
`berta, Canada, T6G 2H7
`Accepted for publication June 21,1974.
`Presented at the 21st Annual Canadian Conference on Pharma-
`ceutical Research, Ottawa, Canada, 1974.
`Supported in part by Grant MA 3078 from the Medical Re-
`search Council of Canada.
`* Recipient of a Medical Research Council of Canada Summer
`Scholarship.
`To whom inquiries should be directed.
`
`Kinetics of Dehydration of Epitetracycline in Solution
`
`BETTY-ANN HOENER, THEODORE D. SOKOLOSKI x, LESTER A. MITSCHER, and
`LOUIS MALSPEIS
`
`Abstract 0 The dehydration kinetics of epitetracycline in solution
`to form epianhydrotetracycline were studied using UV and visible
`spectrophotometry. The reaction was found to be first order with
`respect to epitetracycline and hydronium-ion concentrations. The
`activation energy for the reaction was 28.3 kcal/mole at pH 2.0 in
`0. I M potassium chloride solutions. Dehydration of epitetracycline
`at pH 2.0 and 70" was found to be slower than that for tetracycline
`under similar solution conditions, although the activation energy
`for both reactants is essentially the same. This result is explicable
`on the basis of conformational differences in the molecules. This
`paper represents a portion of studies of the rates of various degra-
`dation reactions of tetracycline that lead to the toxic material epi-
`anhydrotetracycline.
`kinetics in solution,
`Keyphrases Epitetracycline-dehydration
`activation energy, UV and visible spectrophotometry 0 Dehydra-
`tion-epitetracycline
`to epianhydrotetracycline kinetics in solu-
`tion, activation energy, UV and visible spectrophotometry Epi-
`from epitetracycline, dehydration
`anhydrotetracycline-formation
`kinetics in solution, activation energy, UV and visible spectropho-
`in solution
`tometry Kinetics, dehydration-epitetracycline
`
`Studies show that commercially available tetracy-
`cline products contain significant amounts of degra-
`dation products of the antibiotic (1-3). This might be
`expected because tetracyclines can degrade through
`
`at least four different pathways: epimerization, dehy-
`dration, hydrolysis, and oxidation. Since the first two
`reactions are the most commonly encountered, they
`have been of specific interest for study. In solution at
`acid pH, two pathways connect tetracycline and 4-
`epianhydrotetracycline, as shown in Scheme I.
`Epimerization about carbon-4 in tetracycline leads
`to inactive, nontoxic epitetracycline (4). The kinetics
`of this reaction were studied by other workers (4-7).
`Epimerization, which is a reversible first-order pro-
`cess, occurs most rapidly between pH 3 and 5. Dehy-
`dration and aromatization of the C-ring of tetracy-
`cline follow pseudo-first-order kinetics, leading to
`anhydrotetracycline, which is inactive in viuo and
`nontoxic. This reaction occurs in solution at very low
`pH (8) and in the solid state under thermal condi-
`tions (9).
`There are two important steps in the overall degra-
`dation of tetracycline, whose kinetic characteristics
`have not as yet been studied in solution or in the
`solid state. These are the epimerization of anhydrote-
`tracycline and the dehydration of epitetracycline.
`Both reactions lead to the inactive, but toxic, epi-
`
`Vol. 63, No. 12, December 1974 / 1901
`
`Merck Exhibit 2249, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`3.0 c
`
`_ - \
`
`"
`
`200
`
`250
`
`450
`
`500
`
`350
`300
`400
`WAVELENGTH, nrn
`and visible spectra of epitetracycline (-1
`Figure 1-UV
`and epianhydrotetracycline (- - -) at a concentration of 1 X
`M in 0.1 M KC1,pH 2.0, using a 1-cm cell.
`anhydrotetracycline. To supply some of the missing
`kinetic parameters, studies on the effects of pH and
`temperature on the dehydration of epitetracycline
`solutions were conducted.
`Epianhydrotetracycline formation may be conve-
`niently and accurately studied by the appearance of
`an absorption peak at 427 nm or through the loss of
`epitetracycline, as reflected by the decrease in the
`absorption peak at 356 nm (Fig. 1). Analogous to the
`dehydration of tetracycline, epianhydrotetracycline
`formation is the only reaction occurring at pH 2 or
`less (8). In addition to the effects of pH and tempera-
`ture, the influence of citric acid on the rate of dehy-
`dration of epitetracycline is reported, since citric acid
`has been shown to enhance markedly the degradation
`of tetracycline powder (1).
`
`EXPERIMENTAL
`
`Authentic samples of 4-epitetracycline1, 4-epianhydrotetracy-
`cline2, and tetracycline{ were used as obtained. All other chemicals
`were reagent. grade. Solutions were prepared in demineralized,
`double-distilled water, again distilled over ethylenediaminetetraa-
`cetic acid. All reaction solutions were 0.1 M in potassium chloride,
`and hydrochloric acid was added to adjust the pH4. In one experi-
`ment, citric acid a t a concentration of 0.001 M was added to the
`0.1 M KCI solution before adjusting the pH.
`Solutions of 4-epitetracycline were sealed in ampuls for the
`studies carried out at 70.7" and pH 2.00. All other reactions were
`carried out in a sealed reaction vessel (Fig. 2). The 98.5" reaction
`was carried out using a boiling water bath, and those a t 89.8 and
`70.7" were carried out using an oil bath5. For those reactions car-
`ried out in the reaction vessel (Fig. 2), samples of about 4 ml were
`withdrawn oia the syringe and placed in test tubes immersed in ice
`to stop the reaction. The time of the sample was taken as the time
`of placement in the test tube.
`The absorbances of the samples were determined6 after warming
`to room temperature. When ampuls were used, they were removed
`from the constant-temperature bath at appropriate time intervals
`and chilled in an ice bath. The ampuls were then opened, 5 mi was
`withdrawn and diluted to 10 ml with cold 0.1 M KCl-HC1 solution,
`and the absorbance was measured at room temperature.
`The absorbances of all epitetracycline solutions at concentra-
`
`I Ammonium salt, Batch 430, British Pharmacopoeia Commission.
`Hydrochloride salt, Batch 429, British Pharmacopoeia Commission.
`:' Hydrochloride salt, Lot 48379-171, Lederle Laboratories.
`The pH was determined using a Sargent-Welch pH meter, model DR,
`standardized using pH 4.00 buffer, Lot 69M 388C 6471. Ohio State Reagent
`Laboratory.
`Sargent Thermonitor model ST.
`Cary model lfi spectrophotometer.
`
`1902 / Journal of Pharmaceutical Sciences
`
`RUBBER SEPTUM -9
`
`18 GAUGE NEEDLE
`
`TEFLON TUBING
`
`/
`
`Figure 2-Schematic
`the reaction vessel
`representation of
`used to study the dehydration of epitetracycline and tetra-
`cycline. The flask is immersed in a constant-temperature bath.
`M were read at ambi-
`tions ranging from 8.3 X 10W to 1.82 X
`ent temperatures at 356 and 427 nm, using a 1-cm cell. These ab-
`sorbances measure the content of epitetracycline ( ~ 5 6 = 13,800,
`(427 = 0) and epianhydrotetracycline ( ~ 4 2 7 = 5190, 6356 = 736). The
`absorption spectra for these conipounds are given in Fig. 1. For the
`tetracycline solutions, absorbances at 356 nm measure the content
`of tetracycline and those at 433 nm measure the content of anhy-
`drotetracycline (8).
`
`RESULTS
`Semilogarithmic plots of the difference between absorbance a t a
`given time and absorbance at infinite time (determined after about
`10 half-lives) a t 356 nm and vice versa at 427 nm of the epitetra-
`cycline solutions gave an excellent linear relation. Figure 3 shows
`the results obtained a t pH 2.00 and 70.7' for both wavelengths.
`
`tetracycline
`
`I
`
`epitetracycline
`
`I
`
`anhydrotetracycline
`
`epianhydrotetracycline
`
`Scheme I
`
`Merck Exhibit 2249, Page 2
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`IPR2020-00040
`
`

`

`Table I-Pseudo-First-Order Rate Constants for Dehydration of Epitetracycline or Tetracycline at Various
`Temperatures at pH 2.0
`
`Reactant"
`
`Epitetracycline
`Epitetracycline
`Epitetracycline
`Epitetracycline +
`Epitetracycline
`0.001 M citric acid
`Tetracycline
`
`Temperature
`9 8 . 5 + 0 . 2 "
`8 9 . 8 f 0 . 2 "
`8 0 . 0 =k 0 . 2 O
`70.7 =k 0 . 2 "
`70.7 f 0 . 2 "
`70.7 f 0 . 2 "
`
`Rate Constant. min-I x lo2 f SDb
`at 427< or 433 nmcL
`at 356 nm
`
`5.59,' f 0.09
`3.08 f 0.04
`1 . 1 8 f 0 . 0 2
`0.395, f 0.079
`0.424 f 0.023
`0.553,' f 0.039
`
`5.59, j= 0 . 0 6
`3 . 2 1 + 0 . 0 6
`1 . 2 7 f 0 . 0 3
`0 . 4 2 6 + 0.084
`0.460 f 0.073
`0.599 +C 0.069
`
`' 1 In 0.1 M KCI-HCI, pH 2.00. *Determined by linear regression analynis from the data of a semilogarithmic plot. For epitetracycline. d For tetracycline.
`Average of two delerminalions. The average precision obtained between all pairs of determinations was 3~6.5'12.
`
`Also included in Fig. 3 are the results of an experiment under the
`same conditions utilizing tetracycline.
`The pseudo-first-order rate constants a t various temperatures at
`pH 2.0 are. given in Table I. Also included in Table I is the study
`made in the presence of citric acid. An Arrhenius plot of the epite-
`tracycline dehydration data gave an activation energy, E,, of 28.3
`f 2.4 kcal/mole. The pseudo-first-order rate constants for the
`reaction at 70.7O at three different pH values are presented in
`Table 11. A log (rate constant) uersus pH plot gave a slope of -0.91
`f 0.1.
`
`DISCUSSION
`The dehydration of epitetracycline in solution at low pH to form
`epianhydrotetracycline has been shown to be first order with re-
`spect to epitetracycline. A pH-rate profile also has shown the reac-
`tion to be first order with respect to hydronium ion at below pH
`2.0. The error limits found for the log rate constant-pH relation
`(Table 11) may be due to the limited number of pH values that
`could be used and the change in total ionic strength over the pH
`range used.
`Unlike the situation with tetracycline powder (I), citric acid at
`this low concentration had no catalytic effect on the dehydration
`of epitetracycline solutions. A most interesting result was that the
`rate of dehydration of epitetracycline was significantly less than
`that of tetracycline under similar conditions (Fig. 3). The rate con-
`stant obtained in this study for tetracycline is in agreement with
`that of Pryves (8). These epimers differ in the configuration of the
`dimethylamino group on carbon-4 in the A-ring of tetracycline. It
`seems likely that the explanation for the considerable difference in
`the rate for the two tetracycline epimers can be found in either the
`difference in electrostatic screening provided by the protonated di-
`methylamino function which is oriented differently in space rela-
`tive to the 66-hydroxy group in the two isomers or through the op-
`eration of the "Barton effect," i.e., conformational transmission
`( 10- 12).
`The transition state for dehydration is favorable in either isomer
`because the elements of water are located antiparallel and trans to
`each other and the 6B-hydroxy group is tertiary, benzylic, and
`axial. Furthermore, the canonical form of the /3-diketo system at
`
`I
`
`1.0
`0.8
`0.6
`
`0.4
`
`0.2
`
`0.1
`
`1.0
`0.8
`0.6
`
`7 ' 0.4
`
`0.2
`
`0.1
`0
`
`120
`
`480
`
`600
`
`240
`360
`MINUTES
`Figure 3-Semilogarithmic
`plot of absorbance change versus
`time for epitetracycline (0 a t 356 nm and 0 a t 427 nm) and
`tetracycline (@ a t 356 n m a n d 0 a t 433 nm) a t 70" and p H 2.0.
`
`C-11, lla, and 12, in which C-11 is olefinic, gives the C-ring a
`naphthaleneoid geometry at the outset (I).
`As in the conformation adopted by chlortetracycline hydrochlo-
`ride in the crystal and supported by solution NMR and circular di-
`chroism studies, the dimethylamino function in tetracycline (11) is
`axial and it is located on the side of the molecule opposite the 60-
`hydroxy group and screened from it by the sigma framework (13,
`14).
`In 4-epitetracycline (III), the equitorial dimethylamino group is
`much closer to the 60-hydroxy group (14) and would be expected
`to inhibit the approach of a hydronium ion more successfully than
`would tetracycline itself. Depending on the extent to which this ef-
`fect would be operative, it would decrease the rate of dehydration
`of 4-epitetracycline relative to tetracycline and thus be in accord
`with the experimental findings.
`The Barton, or conformational transmission, effect has been
`thoroughly substantiated over the years with numerous examples
`(10-12). It has been found in numerous steroids that, in addition
`
`H+
`
`I
`
`I1
`
`I11
`
`6, ,.o,
`H'
`
`. a 0
`
`H'
`
`6, H ,,.o,H,..o
`
`Vol. 63, No. 12, December 1974 1 1903
`
`Merck Exhibit 2249, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`Table 11-Pseudo-First-Order Rate Constants for
`Dehydration of Epitetracycline at Several pH
`Values at 70.7"
`
`Rate Constant, min-' X lo2 f SD"
`at 427 n m
`at 356 nm
`0 . 3 9 5 + 0.O7gc
`1 . 0 3 f 0.01
`3 . 1 9 i 0 . 0 3
`
`0 . 4 2 6 f 0.084c
`1 . 0 4 =t 0 . 0 1
`3 . 1 5 & 0 . 0 1
`
`PHa
`2 .oo
`1 . 5 3
`1 . 0 4
`
`Q
`
`In 0.1 M KC1. *Determined b y linear regression analysis from the data
`of a semilogarithmic plot. C Average of two determinations. The average
`precision obtained for these data sets was +l.!5yc.
`
`to expected electronic effects, conformational distortion in remote
`portions of a molecule, caused by epimeric constituents and the
`like, can be transmitted through the sigma framework of as many
`as four rings and significantly affect the rate and even steric out-
`come of reactions occurring at relatively great distances from the
`site of isomerization (15-25).
`Detailed X-ray, NMR, and circular dichroism studies (13, 14)
`have shown that the A-ring of tetracyclines is somewhat distorted
`in acidic solutions because of the necessity of relieving a transan-
`nular 1,3-diaxial interaction between the 12a- hydroxy and proton-
`ated dimethylamino group at C-4. This apparently provides the
`driving force for epimerization at C-4, since epimerization relieves
`the interaction and releases the strain distortion of ring A. Trans-
`mission of the different strain in these two isomers from the A-ring
`through three to four carbon atoms to the 5a-hydrogen and 6p-
`hydroxy groups, whose geometry relative to one another plays a
`dominant role in the dehydration reaction, could conceivably re-
`sult in rate differences of the magnitude observed in this study
`(Table I).
`The involvement of steric effects in rationalizing the differential
`rates of dehydration of tetracycline and epitetracycline is further
`supported by noting that the activation energy for epitetracycline
`dehydration (28 kcal/mole) is essentially the same as that for the
`dehydration of tetracycline (27 kcal/mole) (8).
`These studies reporting the solution kinetics of the dehydration
`of epitetracycline represent one factor in the overall degradation
`processes available to tetracycline in dilute aqueous solutions and
`in the solid state. Studies are underway to determine the rate of
`epimerization of anhydrotetracycline in solution to complete the
`solution phase picture.
`
`REFERENCES
`
`(1) V. C. Walton, M. R. Howlett, and G. B. Selzer, J . Pharm.
`Sci., 59, 1160(1970).
`(2) W. W. Fike and N. W. Blake, ibid., 61,615(1972).
`
`(3) R. F. Miller, T. D. Sokoloski, L. A. Mitscher, A. C. Bonacci,
`and B. Hoener, ibid., 62,1143(1973).
`(4) J. R. D. McCormick, S. M. Fox, L. L. Smith, B. A. Bitler, J.
`Reichenthal, V. E. Origoni, W. H. Muller, R. Winterbottom, and A.
`P. Doershuk,J. Amer. Chem. SOC., 79,2849(1957).
`(5) E. G. Remers, G. M. Sieger, and A. P. Doerschuk, J .
`Pharm. Sci., 52,752( 1963).
`(6) D. A. Hussar, P. J . Niebergall, E. T. Sugita, and J. T. Dol-
`uisio, J. Pharm. Pharmacol., 20, 239(1968).
`(7) K. D. Schlecht and C. W. Frank, J. Pharm. Sci., 62,
`258(1973).
`(8) H. J. Pryves, M. S. thesis, Columbia University, New York,
`N.Y., 1957.
`(9) E. R. Garrett, J . Pharm. Sci., 51, 1036(1962).
`(10) D. H. R. Barton and A. J. Head, J . Chem. Soc., 1956,932.
`(11) D. H. R. Barton, A. J. Head, and P. J. May, ibid., 1957,935.
`(12) D. H. R. Barton, J. McCapra, P. J. May, and J. Thudium,
`ibid., 1960.1297.
`(13) J. Donohue, J. D. Dunitz, K. N. Trueblood, and M. S. Web-
`ster, J. Amer. Chem. Soc., 85,851(1963).
`(14) L. A. Mitscher, A. C. Bonacci, and T. D. Sokoloski, Antimi-
`crob. Ag. Chemother., 1968,78(1969).
`(15) H. J. Geise, C. Altona, and C. Romers, Tetrahedron, 23,
`439( 1967).
`(16) H. B. Henbest and W. R. Jackson, J. Chem. Soc., 1967,
`2459.
`(17) Ibid., 1967, 2465.
`(18) M. G. McCombe, H. B. Henbest, and W. R. Jackson, J.
`Chem. Soc., 1967,2467.
`(19) R. T. Blickenstaff, K. Atkinson, D. Beraux, E. Foster, Y.
`Kim, and G. C. Wolf, J. Org. Chem., 36, 1271(1971).
`(20) M. B. Rubin, E. C. Blossey, A. P. Brown, and J. E. Vaux, J.
`Chem. Soc., 1970,57.
`(21) V. V. Egorova, A. V. Zakharychev, and S. N. Ananchenko,
`Tetrahedron, 29,301(1973).
`(22) M. J. T. Robinson and W. B. Whalley,
`2123(1963).
`(23) R. Bucourt, Bull. Chim. Sac. Fr., 62, 1983(1963).
`(24) R. Baker and J. Hudec, Chem. Commun., 1967,891.
`(25) Ibid., 1967,479.
`
`ibid., 19,
`
`ACKNOWLEDGMENTS AND ADDRESSES
`Received March 5, 1974, from the College of Pharmacy, Ohio
`State University, Columbus, OH 43210
`Accepted for publication June 28, 1974.
`Presented to the Basic Pharmaceutics Section, APhA Academy
`of Pharmaceutical Sciences, Chicago meeting, August 1974.
`Supported in part by Research Grant l R O l FD-00689-01A1
`from the Food and Drug Administration, U S . Department of
`Health, Education, and Welfare, Rockville, MD 20852
`To whom inquiries should be directed.
`
`1904 /Journal of Pharmaceutical Sciences
`
`Merck Exhibit 2249, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`