IT
`
`}
`
`1 1
`
`EXHIBIT
`ACTAVIS, AMNEAL,
`AUROBINDO,
`BRECKENRIDGE,
`
` i"
`
`
`
`(ARHIV ZA KEMIJU)
`
`11/r.LQ-
`
`Z A G R E B
`YUGOSLAVIA
`1956
`
`1j'C'roat. Elhem. Ac7t’c1 :
`
`‘CL"1\'[ENT.«'\ CHEMICAYUGOSLAVICA
`
`CROATICA
`
`"LCHEMICA ACTA
`
`IPR2014-01126- Exhibit 1016 p. 1
`
`

`
`CROATICA CHEM {CA ACTA
`
`Published by The Croatian Chemical Society
`Official journal. of the Croatian Chemical Society
`Official chemical joumml of the University of Zagreb
`and of the Croatian Society of Natural Sciences
`
`COMMITEE OF PUBLICATION:
`Editor: Eoio Teiak
`Assistant Editor: Egon Matijevii: and Velimir Vouk
`(Since September 1, 1956 Petar Ataup
`Secretary: Dionis Sunkn
`(Since Decembre 1, 1958 Dina Keg1evié—BrQ
`Membres: K1-e.§imir Balenovié, Eugen Cerkovnikov, Ivan Filipovié, D1-agutin
`Pavao Mildner, Nikola P1av§ié, Karla Schulz and Karla Weber
`(Since November 15, 1956 Josip Kratohvil, Kazimir Sestanj and Otto We
`Erma:-int office: 19 Maruliéev trg (P. 0. Box 131)
`Zagreb, Croatia, Yugoslavia
`
`CROATICA CI-[EMICA ACTA
`Izdaje Hrvataku kemijskn drufitvo
`Sluébeno glaailo Hrtmtskog kemifskog druitva
`Sluébeno lcemijsko glasilo .5'vem‘51'.h.§ta. 1:. Zngrebu
`i Hrvatskog prirodoslovnog drmétva
`Adresa redakcije '1 aclministracije:
`Zagreb. Maruliéev trg I!) (po§t. pref. 131)
`
`REDAKCIONI ODBOR:
`Glmmi. -uxrednik: E020 Teiak
`Pomaéni. urednicl: Egon Matijevlé i Velimir Vouk
`(Od 1. rujna 1956 Petar Alaupavié)
`Tajnik: Dionis Sunko
`(Od 1.‘p:-osinca 1956 Dina Keglevié-Brovef)
`Clanovi: Krefiimir Balenovié. Eugen Cerkovnikov,
`Ivan Filipovié. Dragutin
`Pavao Mildner, Nikola PIav§'1é, Karla Schulz i Karla Weber
`(Or! 15. studenog 1956 i Josip Kratohvi]. Kazimir Sestanj 1 Otto W'ebEl‘)-
`
`‘
`
`7
`
`_
`
`IPR2014-01126- Exhibit 1016 p. 2
`
`

`
`(cid:3) (cid:55)(cid:75)(cid:76)(cid:86)(cid:3)(cid:80)(cid:68)(cid:87)(cid:72)(cid:85)(cid:76)(cid:68)(cid:79)(cid:3)(cid:80)(cid:68)(cid:92)(cid:3)(cid:69)(cid:72)(cid:3)(cid:83)(cid:85)(cid:82)(cid:87)(cid:72)(cid:70)(cid:87)(cid:72)(cid:71)(cid:3)(cid:69)(cid:92)(cid:3)(cid:38)(cid:82)(cid:83)(cid:92)(cid:85)(cid:76)(cid:74)(cid:75)(cid:87)(cid:3)(cid:79)(cid:68)(cid:90)(cid:3)(cid:11)(cid:55)(cid:76)(cid:87)(cid:79)(cid:72)(cid:3)(cid:20)(cid:26)(cid:3)(cid:56)(cid:17)(cid:54)(cid:17)(cid:3)(cid:38)(cid:82)(cid:71)(cid:72)(cid:12)(cid:3)
`
`IPR2014-01126- Exhibit 1016 p. 3
`
`

`
`6
`
`M. JAEGER, S. ISKRIC AND M. WICKERHAUSER
`
`Of the °0I'tfiEU1‘ati0n Of 0-Inethy1—N-phthaloyl—serine, in this case only the
`of rotation is important. It was thus established that laevorotatory O—meihyP:
`N—phthaIoy1—serine belongs to the I-—series of amino acids.
`‘
`We also prepared O-ethyl-N—acetylvserine, and subjected it to enzym
`resolution.
`
`'
`
`and [a],_,—I7" respectively.
`
`r:x1=EniMisN'rm..
`
`
`
`All melting points are uncorrected.
`Starting materials
`0-Metilyi-DL-Serine was prepared according to Schiltz and Carter”.
`_
`0—Ethy1-DL—se'ri‘.Ite was prepared according to Wood and du Vigneaudlfi.
`O~Methyl»N-acetyt-nlwserfne was prepared according to Synge”. 0—ELhyt_
`acetyt-DL—serine was also prepared in this manner, for the first time.
`Ar.-ylase I was prepared according to Greenstein, Birnhaum and Levintow
`O-E'thyt-N—acetyt—DL—serine was prepared from n-brainc-fl—etl-ioxypropionic
`(25 g.) after reaction with ammonia. The thus obtained crude mixture of r:-amln _
`ethoxyvpropionic acid and ammonium bromide was treated with excess 5% 5061'
`hydroxide and evaporated to dryness on a water bath. The obtained ainmonia
`mixture of u-arnino—|3—ethoxypropionic acid and sodium. bromide was dissolve
`5% sodium hydroxide [100 m1.), magnesium carbonate (5 g.) was added, and
`mixture treated dropwise with acetic anhydride (18 g.) during an hour. under
`ring. at 5". The reaction mixture was kept at pH 9 with 4N sodium hydr
`during the addition of acetic anhydride. O-Ethyl-N-acety!-DL-serine was '-
`using ethyl acetate, in the usual manner. yield 17.8 g. (80%. based on the a—br'
`ii-ethoxyrupropionic acid used), m. p. 128--133“. Recrystallization from ethyl ac
`petroleum ether gave clusters of colourless needles, m.p. 133_135".
`Amrl. 9.57 mg. sLlbst.: 16.89 mg. C02, 6.42 mg. I-I20
`C:HiaNO.1 (175.18) ca!c'd.: C 4199; H 'T.49°.'u
`iound: C 43.14; H 7.50"}:
`
`Enzymatic resolution of O—methyi-N-ocetyl-Dbserine
`O-Methy1-N-acetyl-DL—serine (20 g.) was dissolved in water (220 ml.) and‘
`S01‘-ltitm ‘b1'01-lght to DH 7.3-7.9 with 2N ammonia. An acylase I solution (33
`corresponding to lg. of amylase 1 powder was added, and the mixture thDrD_ .
`stirred and allowed to stand at 3'?" for 24 hours. The enzymic hydrolysis was
`lowed by the Van Slyke manometric procedure for wcarboxyl nitrogen anal
`Aiter 24 hours the reaction mixture was removed from the thermostat. cooled to
`and treated dropwise with glacial acetic acid to —pH 4.5. Charcoal was added._
`the mixture liltered. The protein-free filtrate was evaporated in vacuo at 40“. A
`of this residue (5 E.) was dissolved in water 001111.] and poured on the top
`column (3.5 ><3B.5 cm.) composed of 20-50 mesh Dowex 50 resin in the acid P
`(the resin was regenerated by two cycles of washing with 5N hy-droch1o1‘i_c
`water, 1N sodium 1'lYd1‘Dxyde. and water, followed by a Iinal 5 N hydrochloflc
`and water Wash). Dilution with water was carried out at the flow rate of 20 1113
`hour. After elution with 800 ml. of effluent, all of the O«methyt—N-acei:.'l—D'3e
`was eluted. yield 1.9 g,
`('?6".’n]. After 1'ec1'ysiallization from ethyl acelaLe—p9t1'°1
`ether.
`in. p. 7o~74-2, mi}; —s.7°1o.9u (c, 1.04 in 1 N NaOH).
`Anal. 9.02 mg. sub.-‘it.: 14.85 mg. CO._=, 5.66 mg. }-[50
`Cui'InNO.;
`(161.16) CaIc'd.: C 44.71‘, H 6.88°.I'u
`found: C 44.95: H 'i’.03"fn
`
`After further washing of the column with one liter of water, elution was 133
`with 3.5N hydrochloric acid. The entire O-methyl-L-serine was eluted after ‘W
`or solution had passed through the column. The combined fractions Contam
`
`IPR2014-01126- Exhibit 1016 p. 4
`
`

`
`O-METHYL-N-ACETYL-DL-SERINE.
`
`7
`
`
`
`
`
`y]—L-S€l‘il1.E were neutralized with :1 sodium hydroxide solution (10%) to pH 7,
`rated to dryness in vacuo and converted in the usual manner to O—methyl-L-
`-methyl ester by successive treatment with absolute methanol. dry hydrogen
`,
`_ide_ and a chloroform solution of dry ammonia. O-Methy1—serine methyl ester
`hydrolysed by retluxing with a tenfold quantity of water for 10 hours. and
`rated to dryness. The O-methyl sci-ine thus obtained was optically inactive;
`recrystallization from water-ethanol white leaflets were obtained, with m.}).
`
`tdecomp.)
`(Schiltz and Carter” reported 200—2lfl“', Nobuo Izu.m.iya‘°
`ted 233\234“).
`Aunt. 7.81 mg. subst.: 11.56 mg. C02, 5.28 mg. H20
`C4HnNO,-;
`(119.09) cnlc'd.: C 40.35‘. H '7.59"fn
`found: C 40.42‘, H 'i‘.54"fu
`
`2 g
`
`from Dowex 50
`isolation experiments of O—rnethy1-DL-serine
`'1-, subsequent
`5, 4% ammonia was used instead of 2.5 N hydrochloric acid. After evaporation
`effluent, crude O-methyl-1-—serine was obtained, showing [u]',§ + 150 (c. 1
`in
`C1}. which was converted into 0—methyl—N—phtha1oy1-L-serine without further
`._ication.
`w_o—Methyi-N-pitthuloyt-L-serine was prepared from equimolar quantities of O-
`1-I.—s-erine ([u]n + 16"} and .pl-ithalic anhyciricie. by heating the mixture for half
`guy at 130". The oily O—-methyI-N-phthaloyl-L-serine was purified by precipita-
`Trom dichlorornethane-petroleum ether,
`[u]['," —i8U (c. 5 in methanol).
`Anal. -9.15 mg. subst.: 19.32 mg. C05, 3.84 mg. H20
`C121-IHNO.-. (249.22) calc'd.: C 5'i'.B3;-H 4.45"I'u
`found: C 57.61; H 4.69".v‘o
`
`Th; resolution of 0—ethyl-.N-acen,-i—nL-serthe was carried out in the same man-
`-and O—c1i1yI—N—ncetyl-D-serine was obtained. as white needles, m.p. 15[)—15ri0.
`Mg‘ _17-I (c. 0.9 in 1 N NaOH).
`Anal. 6.01 mg. subst.: 10.82 mg. C02, 4.04 mg. H20
`C7H1aNO,|
`(175.18) ca1c'd.: C 47.99; H 7.49".‘
`found‘. C 43.20; H 'i‘.52".’u
`
`-"Acknowledgments. One of us (M. J.) is indebted to Dr. R. Seiwerth. Head of
`' "B.eseai'ch Institute of nPiiva«, Pharmaceutical and Chemical Works. for facilities
`an-ying out this work.
`, The authors are further indebted to Mrs. Z. Steianac tor the microanalyses.
`mi?
`to Professor K. Balenovié for his interest in this work.
`RE FERENCES
`
`,-: P. Greenstein. S. M.Bi1'nbaum and M. C. Oty, J. Biol. Chem. 20-1
`(1953) 307.
`EA. Meister, L. Levintow. R. B. Kingsley and J. P. Greenstein.
`‘_ J. Biol. Chem. 192 (1951) 535.
`Li L. Levintow. V. E. Price and J. P. Greenstein, J. Biol. Ciiem. 18-1
`(1950) 55.
`S. M. Birnbaum. L. Leviuiow, R. B. Kingsley and J. P. Greenstein,
`_ J. Biol. Chem.. 194 (1952) 455.
`"C. G. Baker, S. J. Fu. S. M. Birnbauln. H. A. Sober and J. P. Green-
`_ stein, J. Am. Chem. Soc. 1'4 [1952] 4701.
`33. J. S. Frulnn, G. W. Irving Jr. and M. Bergmann. J. Biol. Chem. 133
`(1940) 703.
`" N. F. Albertson, J. Am. Chem. Soc.
`'13 (1951) 452.
`‘N. Stimac and B. Gaépert, Arhiv kem. 26 (1954) 105.
`.. D.F1e§, M. Brajdié and N. Stimac, Amie kem. 2.6 (1954) 183.
`-D. Fleé, B. Balenovié, R. Maruéié “and N. Manger. Arhiv R9111. 2'?
`(1955) 1.
`
`IPR2014-01126- Exhibit 1016 p. 5
`
`

`
`
`
`M. JAEGER, S. ISKRIC AND M. WICKERHAUSER
`
`8 1
`
`1.
`..5°
`13.
`14.
`15.
`16.
`
`in p1:e5S.
`
`!e' and B. Balenovié, J. Am. Chem. Soc,
`wt: wt"?
`Synge, Biochem. J. 33 (1939) 1931.
`a 1
`SF I9 an:
`enovié, N. Eregant, B. Ga§pert, I.Jamb1'e§ié and
`iii, Arhiv Icem. 27 (1955) 207.
`L“
`'31
`L:
`L"
`.
`. Schiltz and H. E. Carter,
`.1’. Biol. Chem. 116 (1936) 793.
`. Woo d and V. du Vigneaud. J. Biol. Chem. 134 (1940) 413.
`J.
`I-‘. Greenstein, S. M. Birnbaum and L. Levintow, Bioche
`34.
`Preparations, Vol. III. J. Wiley, New York 1953, p.
`1'1’. D. Van Slyke, D. D. Dilon and R. T. Mac Fadyen,
`J. Biol. Che
`(1941) 327; D. Van Slyke, R. T. Mac-F_adyen and P. Hamilton, J.
`Chem. 141 (1941) 6'71.
`18. J. P. Gree n stein, Advances in Protein Chemistry,
`Vol. 9. Academic
`New York 1954, p. 121—202.
`19.
`7011.
`No b uu I211 miya. J. Chem. Soc. Japan 71 (1950) 214; cit. from C. A. 45
`
`IZVDD
`
`Enzimatsko cijepanje 0-metil-N-acetil-DL-serina.
`Aminokiseline. XXXII
`
`M. Jneger, S. Isk1'ié i_M. Wickerhauser
`
`Izvede-no je enzimatsko cijepanje 0-:-neti1—N-a.cet§l—DI..—serina prema Green
`L12 djelomiénu racemiz -
`Tako dobiveni O-metil-L-serin daje sa ftalanhidridarn,
`O-metil—N-flaloil-1.-serin.
`KEMIJSKI IN STITUT
`PRIIFLOIJOSLOVNO-N[ATEMA'I‘ICKI FAKULTET
`I
`BIOKEMIJSKA GRUPA
`INSTITUT »R'U!)EH BOSKCVICu
`ZAGREE
`
`Primljeno 2:1. decemblj!
`
`IPR2014-01126- Exhibit 1016 p. 6