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`REPLACEMENT EXHIBIT 2035
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`REPLACEMENT EXHIBIT Z035REPLACEMENT EXHIBIT Z035
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`AIAUNE,i19'-/17“
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`R
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`A JO U
`i
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`R A .L.
`_ _
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`OF THE‘
`l‘
`A
`_
`- _
`%AM%ER1cA.N:..C;H%EM1cAL SOCIETY
`
`‘
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`.
`
`E
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`W
`
`PAUL D. BARTLETT
`PAUL H..EM1ViE'I"i‘ V
`N. .HowELL FURMAN'
`V HENRY GILMAN
`'
`._
`EDWARD MACK, «IR. 9;
`
`R.
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`.
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`I
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`Nri
`x
`
`~
`
`‘ ARTHUR B.\LA1V‘1LB,<,]’:i3ITOR
`;
`"
`-ASSOCIATE EDITORS‘
`R.‘H.F.MAi~ISKE ‘
`C.‘S. MARVEL
`S. M. M'cELvArN x
`KENNETH S. PITZER‘
`/,CL1'E1=oRD B. PURVES
`JOHN E. R1cc1
`\
`. ‘~\ ‘
`
`’ FREDERICK D. Rossmr
`GEORGE ScA'rcHARDJ
`LEE IRVIN SMITH ‘ L.
`H. B. VICKERY
`‘VINCENT nu VIGNEAUD
`
`;
`
`f it
`~ (5QN.TEil\‘IT'S_i.
`'
`‘GENERAL; PHYSICAL AND ‘I1~'IoRoANIé or/IE1v_u'sTRY‘
`E
`l
`of_ Bonding Existing in Some . Complexes of Ferric Iron
`Stuart Rsyfirinkley. jr.. and S01 Weller: ‘ Generalized Form of
`-
`.
`withSulfonatedPyrocntechnl........................
`'
`the Reaction-Rate Law for Homogeneous Reactions. . . .
`1319 ;
`Webster B. Kay: Vapor Pressure: and Saturated Liquid nnd
`Yierbert C.‘ B_rown and Maddie D. Taylor: Dissociation of
`.2»...
`Vapor Densities of Cyciopenlnne, Methylcyelopentone.
`" 1332 ‘
`‘ Ethylcyelopentnne and Methyleyelohexane. . . . . .
`\ the Addition. Com ound: of Trimethglboron with. the '
`New Optical Method for Observing ‘
`, Gerson Kegeles: A
`R Ethylaminea; Con mutation of the Et ylnmines. . . . . . .
`1525
`obert L. Burwell. Jr.. P. Qllen Smudski and Thomas P.
`Sedimentation Equilibriun:..........................
`L. G. Longsworth: A Moving tnoundnry Method for the
`R May: Ethylene Adsorption isotherms nt --183° . . . . . . .
`obert E. Connick: The interaction offlydrngen Peroxide‘.
`.... . . . . ......................J.....
`.l, , Measurement of Non-electrolyte Transport
`in Mixed
`1' Solvents.Z.. . . .
`'
`Winifred M. Blunt: Activity of Or-
`andilgpochlorohs Acid in Acidic ‘Solutions Containing
`Charles M._Mnson an
`ueuus Solutions at 25° irom
`B Chlon e1on..;........................L......1..'..
`thophosphoric Acid in A
`- F. Daubert R_.nd E. S. Luttun: X-Ray Diiiraction Analyses
`E. m. f. Measurements with
`_
`the Lead Amnl¢am—Lend
`- E. Davis: The Effect of Molecular-,Weight Distribution
`W of Synthetic Unsaturated Monnoid Diglyce;-ides. . .
`. . . ..
`Phosphate Electrode... . .
`.l. Niiray-Szabd and J. Neug bauer: The Crystal Structure
`of Sodium lodate......:....‘...L........‘.......;....
`H on.-the Reduced Viscosity—Concentration Coemcient. .‘
`- L. Fink, M. R.‘_Cines.gF. E. Frey and J’. G. Aston: Solid ‘
`Systems ,
`: William Primalr, R. Keith Oaterheld and Roland Ward: The
`~.2-D1mCIily1bi.ltfl.11C‘2,3-dlfnelilyibhltflfle
`an
`A. 2,2-Di-
`§oluti.on Studies.
`I. Equilibrin in the Dinars;
`Function of Fluxes in the Preparation of infrared Sensi-
`‘tive Phosphor: oi the Alkaline linrth Sulfide: and Selen-
`‘
`ides...............................................
`n1ethy1hutnne—-Cyelopentane. . . . . .". . . .
`; . . . . . . . .. . . . . .
`O
`90116 S. Forbes and Herbert H. Anderson:
`I<‘luoroiso-
`' Max T. Rogers: The Electric Moments of Some Unsaturated
`" tynnntcs of Silicon...,............‘.'................
`“"1 S. Frank and Robert L. Oawalt: The Stability and \
`Aldchydes, Ethcrs, and linlogru Compounds. . . . . . . . . .
`Max T. Rogers: The Magnetic Anisotropy of Coronene,
`Na hthaznrin. and Other Cr sln|!....... .. .. .
`. ..
`........--.......
`"lam D. Harkinsr A General Theory of the Mechanism.
`W‘ Uxllt Absorption of the Complex Ion F:SCN**. . . . . . . .
`Sidney
`. Ron and C. Gardner wnln: Application of the
`W
`R
`of Emulsion Pnlymerization'.. . .1.’
`'
`E. Hunt and Wallace.Davis,‘Jr.: ‘Filters for the Isola-
`Princi
`le of the Concentration _Cell to Kinetic Studies.
`« ll.
`'1‘ e Oxidation of oxalic And by Ceric Sulfate. . . . .
`tion of the 3130 A. Spectral Group of the Mercury Arc.
`A. L
`R. E. Rundle:
`l~Il'ectron Deficient Compounds. . . . . . . . . . . . .
`- Jones and L. B. Yenttnt A Magnetic Study ofthe Type
`..
`..\
`(Conllmud an secandipau of turn)
`
`,’
`
`'
`
`1509
`‘ 1449
`1453\
`
`' 1
`
`50i
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`12r1
`1321
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`1458
`i415_
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`~
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`»
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`‘
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`‘
`
`. 3“PUBLIS1i€§$ ‘.I01~_ITI;ILY' BY THE AM
`ERICAN CI'IEM_ICAL‘ SOCIETY
`.
`st?"
`Copyright 1047. by the American Chemical Society
`'
`,
`I.
`\
`I.
`r
`.
`3 Muller.
`rictontann for moiling ol special
`tr at Easlon. PA. or Second-clas
`Odabrr J. 1917. authorized July (J. 1915‘
`in Section I103. Ad of
`/' Entered at the Past Ofl
`Irate of postage provided for
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`'
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`;
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`’
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`mi hooks sent for review in the
`Iiourxxni of the Anicricmi
`' Manuscripts submitted’ for pubiicatiorfn
`’ditor, Artiulr ii. Lamb.
`cm should be sent to the .
`Chemical Society. and till inquiries rcgurding tli
`12 Oxford Street. Cambridge 38. Massachusetts
`Chemical Lnborniory of Harvard’ Umvcrsity,
`/
`\
`H
`‘
`_
`
`
`
`1266
`
`SAUL W. CHAIKIN
`
`Vol. 69
`
`[CONTRIBUTION FROM THE UNIVERSITY OF CHICAGO TOXICITY LABORATORyl]
`
`Study of the Hydrolysis of Several Physostigmine Analogs
`. By SAUL W. CHAIKIN
`
`Physostigmine (1) and many of its analogs have
`been the subject of much study with regard to
`certain physiological properties which they. exhibit
`o
`II
`CHi
`CH
`H)N-C-e-f)-t-
`
`V'W NJ
`
`CH.CH!
`
`I
`when introduced into the animal body.2-6 The
`phenyl carbamate nucleus has been shown to be
`the active group in the alkaloid. 2
`. Physostigmine
`and certain other phenyl carbamate derivatives
`strongly inhibit choline esterase. The constric(cid:173)
`tion of the pupil of the eye (miosis) of the test
`animal when the compound is instilled into the
`eye has been taken as an index of the physiological
`action and this has been found to depend on varia(cid:173)
`tions in the chemical structure of the carbamate
`used.
`. Physostigmine is not stable in aqueous solution.
`A systematic study of analogs of this compound
`was undertaken by Aeschlimann and Reinert·
`with the object of finding a drug suitable for thera(cid:173)
`peutic use with a greater degree of stability to
`hydrolysis. They found the N,N-dialkyl carba(cid:173)
`mates to be considerably more stable in water
`solution than theN -monoalkyl compounds and to
`have a physiological action of the same order of
`magnitude. The mode of decomposition which
`they proposed for the N-methyl phenyl carba(cid:173)
`mates is the formation, in slightly alkaline solu(cid:173)
`tion, of the phenol and the isocyanate. The odor
`of isocvanate was detected when solutions of
`N-metliyl carbamates were boiled.
`CHaNHCOOCeH4R -+ CHaNCO + HOCeH4R
`With excess alkali
`CH3~CO + 20H- -+ CH3NH2 + C03-
`I t was also reported that hydrolysis was greatly
`suppressed when the PH of the solution was less
`than 5, P. D. Bartlett7 observed
`that the
`methylamine produced on hydrolysis in a solution
`at a hydrogen ion concentration of ca. pH !) ca~
`lyzed further decomposition of an N -methyl car(cid:173)
`bamate.
`Experiments with this class of compounds were
`carried out ill this Laboratory involving tests of
`(I) This work was done under contract with the Medical Division
`of the Chemical Warfare Service.
`(2) E. Stedman, Biochem. J., 10, 719-734 (1926).
`(3) E. Stedman, ibid., IS, 17-24 (1929).
`(4) Aeschlimann and Reinert, J. Pharm. ExP. Therap. ,. U, 413-
`4H (19'31).
`(5) Whit.e and Stedman. ibid., U, 259 (1931).
`(0) Stevens aDd Beutel, TUIS JOU"NAL, II, 308-311 (1941) .
`'. (7) P . n . n"rtlett. ,I al .• unpuhlished data.
`
`toxicity. following subcutaneous injection into
`mice.s It became of some interest to know the
`stability in vitro of the mono- and di-alkyl carba(cid:173)
`mates under conditions of hydrogen ion concen(cid:173)
`tration and temperature similar to those in the
`blood and tissue fluids of animals (pH 7.4 and 38°).
`The compounds studied were the N -methyl
`(M). N,N-dimethyl (DM) and the N,N-penta(cid:173)
`methylene (PM) derivatives of 4-dimethylamino-
`3-isopropylphenyl carbamate methiodide. Since
`o
`0
`II
`
`;;-N ClI.
`
`_<CHI
`
`~ <:
`;;- -N H.
`
`Y-CH(CHah
`
`Y-CH(CHI),
`
`N(CH1)3
`(M)
`
`I
`
`N(CHah
`(DM)
`
`I
`
`o
`/I
`<CH.-CH)c
`O-C-N
`.
`Hi
`( \
`CH.-CHs
`
`Y-CH(CHa)t
`
`N(CH8)a
`I
`(PM)
`the substituents on the benzene ring are the same
`in each case, differences in hydrolytic stability of
`these compounds are due to differences in the car(cid:173)
`bamate residue.
`
`Experimental
`Materlals.-The sample of M used was applied by R. L.
`Shriner. M. E. Synerholm and J. C. Speck, Jr., who
`were the first synthesizers of this compound. The melting
`point was given as 184.5 0 (dec.).
`Anal. Calcd. for C1,H230 2N2I: 1,33.55. Found: I,
`33.50.
`.
`DMwas first synthesized by Stevens and Beutel8 and
`the sample used in this investigation was submitted by
`H. Gilman. The melting point was reported as 159 0
`(dec.).'
`Anal. Ca1cd. for C15H250,NtI: N. 7.1.'); I, 32.38.
`Found: N, 7.19 and 7.01; 1.32.40.32.13 and 32.41.
`V. A. Englehardt and L. 1. Smith originally prepared
`PM. The melting point was reported as 161 0 (dec.).
`Anal. Calcd. for C18Hu02N2I: C. 50.00; H.6.76.
`Found: C. 50.21; H,7.00.
`
`(8) C. F .' Failey, W . Elder and B. Ginsburg, unpublished data
`University of Chicago Toxicity Laboratory.
`(9) The [ollowing is a note from a letter from H. Gilman giving
`information about the compound DM: "The melting point reported
`by Stevens and Beutel; THIS JOURNAL, ea, 310 (1941) was 170·.
`Subsequently, we learned by private communication that tbe cor(cid:173)
`rected melting point was 165.5°. A meltin&' point determination of
`their sample taken in the same bath and at the same time a. aura W'"
`158° (ours mp.ttiog at 159°). "
`
`
`
`June, l!Hi·
`
`HYDROLYSIS OF SEVERAL PHYSOSTIGMINE ANALOGS
`
`1267
`
`The sample of P (identified later) was synthesized by
`H. Gilman and D. A. Shirley. The melting point was
`given as 205 0 (dec.).
`Anal. Calcd. for Cl,H200~I: I, 39.5. Found: I,
`39.6'and 39.3.
`Procedure.-The course of the hydrolysis was followed
`by measuring the change in absorption of light by solutions
`of carbamate using wave lengths at which the difference in
`absorption by the carbamate and phenol was great. The
`Beckman Quartz Spectrophotometer Model DU was used
`after having been calibrated against solutions of known
`absorption maxima. The carbamate.s were all found to be
`stable in acid solution so that a standard procedure was
`followed involving removing aliquot!; from the buffered
`hydrolyzing solution at intervals and adding them to an
`acid solution to stop the hydrolysis. Spectral absorpti.on
`curves were prepared (Fig. 1) for M, DM, PM and P (a
`pure sample of the phenol which is produced on hydrolysis
`of the carbamates). For purposes of comparison all spec(cid:173)
`trophotometric data have been corrected to a concentration
`of 0.15 mM per liter.
`In all cases the concentrations of
`the solutions prepared were within 5% of this value. The
`curve for P is useful in determining completeness of hy(cid:173)
`drolysis and in ascertaining the effect of hydrolysis prod(cid:173)
`ucts other than phenol on the absorption curve of the com(cid:173)
`pletely hydrolyzed carbamate.
`The hydrolysis rate was measured as follows. A 250-
`m1. volumetric flask containing phosphate buffer (.:\'aH,(cid:173)
`PO. 0.002 1.1l, :-.ia!HPO. 0.01 M) adjusted to pH 7.4 was
`immersed in a water-bath maintained at 38 0 until thermal
`equilibrium was attained. The flask was removed and a
`sample of carbamate weighing about 15 mg. was intro(cid:173)
`duced. The solution was quickly mixed and replaced in
`the bath. At intervals,
`lO-m1. aliquots were with(cid:173)
`drawn and added to 0.17 m1. of 6 N hydrochloric acid
`which so acidified the solution as to stop hydrolysis.
`Absorption of light of 275 mJ.' wave length by the solution
`was then measured. Quartz cuvettes of l.OO-cm. optical
`depth werl! used.
`
`The solutwns used in preparing the curves in Fig. 1
`contained 0.04 10M. of compound in a 250-m1. volumetric
`flask containing phosphate buffer and hydrochloric acid of
`the same concentration as given above. The blank used
`for the readings was a solution of buffer and acid, also of
`th\! above concentrations.
`
`Results and Discussion
`It was found that the solutions of DM and PM
`at :.3~0 and pH 7.4 showed no significant hydroly(cid:173)
`sis during the period of observation of four days.
`~1 was found to be unstable under these con(cid:173)
`ditions. Since the hydrolysis of :\1 is more rapid
`in alkaline solution it is undciubtedly second order,
`but the hydroxyl ion concentration being held
`constant in these experiments, the rate depends
`only on the carbamate concentration. A plot of
`the log of the fraction hydrolyzed against time re(cid:173)
`sults in a straight line (Fig. 2). Calculation of
`reaction constants can therefore be made by
`methods ordinarily applied to first order reactions.
`When complete hydrolysis of M is effected spec(cid:173)
`tral absorption data for the region from 255 to
`;300 m,u. are identical, within experimental error,
`with those of curve P. At shorter wave lengths
`the deviation gradually increases until at 240 m,u.
`it is 5% higher than curve P. This deviation may
`be caused by other products of hydrolysis but
`since it occurs outside the region taken for hy(cid:173)
`drolysis measurements it is of no consequence in
`this connection. The half life for ~1 at 38 0 and
`pH 7.4 is seen from Fig. 2 to be fifty-two minutes
`and the velocity constant 0.0133.
`
`0.7 b.---+-~---+---+--+----!
`
`o
`r'\
`'\.
`20
`
`40
`
`i'..
`\..
`
`"'-}. '" '\
`'\ ."
`\
`
`I
`
`I
`I
`
`IlO
`
`,""
`1'\
`I
`140
`
`20
`
`60
`100
`Time, minutes.
`Fig. 2.-Hydrolysis-timc curve for M at pH 1.4 and :38°.
`
`250
`
`270
`Wave length, mJ.'.
`Fig. l.--Spectral absorption of P, M, DM and PM,
`litl; Na,HPO.,
`concn. of' reagents: KaH,PO •• 0.002
`O.Olllf; Hel, 0.1 M; compound, 0.15 mM; ., P; 0, M;
`0-, DM; O. PM.
`
`290
`
`This study is concerned only with the rate of
`disappearance of the carbamate (or the rate of
`appeara.nce of the phenol) and not with the route
`of mechanism by which such a transformation
`occurs. The work of Bartlett,7 mentioned earlier,
`indicates that the hydrolysis reaction is probably
`
`
`
`1268
`
`C. H. BOATNER, R. T. O'CONNOR, M. C. CURET AND C. S. SAMUELS
`
`Vol. 69
`
`specific amine or. general base catalyzeil10 in which
`case variation of reaction rate with buffer con(cid:173)
`centration (at PH 7.4) may be expected.
`
`Summary
`Hydrolytic stability of three physostigmine
`(10) L. P. Hammett, "Physical Organic Chemistry," 1st ed.,
`McGraw-Hill Book Company, Inc., New York, N. Y., 1940, pp. 344-
`345 and 215-218.
`
`analogs at 38° and pH 7.4 was investigated by
`measur.ement of light absorption of the phenolic
`hydrolysis product at a wave length of 275 m,u.
`The two N,N-dialkyl carbamates were st-able
`under these conditions dur.ing a four. dayobserva(cid:173)
`tion period. The N -methyl carbamate was found
`to be unstable and to have a half life of 52 minutes
`and a velocity constant of 0.0133.
`RECEIVED DECEMBER 12, 1946
`CHICAGO, ILLINOIS
`
`[CONTRIBUTION FROM THE SOUTHERN REGIONAL RESEARCH LABORATORyl J
`The Pigments of Cottonseed.
`III.2 Go ssyfulvin , a Native Cottonseed Pigment
`Related to Gossypo13
`
`By CHARLOTTE H. BOATNER, ROBERT T. O'CONNOR, MAIZIE C. CURET AND CAROLYN S. SAMUELS
`
`ISOLATION OF GOSSYFULVIN FROM COTTONSEED
`Flaked cottonseed
`Diethyl
`ether
`
`I
`
`{-
`Meal
`
`I ...
`Oil, F. F. A., pigments
`(in Et20)
`Aq. NaOH I (Na2S204)
`t
`
`Ka gossypolate, orange pigments, soaps
`(in aq.)
`
`Et20 I HCI
`
`t
`
`Gossypol, orange pigments
`(in Et20)
`I
`HOAc
`I
`
`t
`Soaps
`(in aq.)
`
`i
`...
`Gossypol acetic acid
`(pp.)
`
`An orange colored pigment, gossyfulvin, has
`previously been detected4 in cottonseed. Al(cid:173)
`though gossyfulvin has been shown to differ from
`gossypol in many of its properties it can be readily
`converted into the latter pigment. Since the
`work on gossyfulvin was reported, larger quanti(cid:173)
`ties of the pigment have been prepared by the
`procedur.e outlined in the accompanying diagram
`and it has therefore been possible to obtain fur.ther
`insight into its structur.e and its relationship to
`gossypol.
`Gossyfulvin forms rather large orange-colored
`rhombohedra (Fig. 1), changing at 212° to a more
`deeply colored form which melts with decompo(cid:173)
`sition at 238-239° (cor.). The crystalline form
`and habit of gossyfulvin differentiate it sharply
`from gossypol, which latter pigment, upon re(cid:173)
`crystallization from diethyl ether and petroleum
`naphtha, forms clusters of dog-toothed prisms
`(Fig. 2), m. p. 182.5-183.5°.
`Gossvfulvin reacts with strong mineral acids
`when its chloroform solutions are treated with
`concentrated aqueous solutions of these acids
`yielding gossypol in amounts equal to as much as
`86.8% of the weight of gossyfulvin treated. On
`the basis of the experimentally determined ele(cid:173)
`mentary composition, C34Hs4N20a, the molecular
`weight of gossyfulvin is 598.
`Gossypol obtained after recrystallization from
`diethyl ether and petroleum naphtha at low tem(cid:173)
`peratures and dried without elevation of tem(cid:173)
`perature yields analytical values which agree with
`those calculated for C30H320a.
`Comparison of gossyfulvin with such nitrogen
`derivatives of gossypol as diamino- and dianilino(cid:173)
`gossypol, reveals several significant differences,
`(I) One of the Laboratories of the Bureau of Agricultural and In(cid:173)
`dustrial Chemistry, Agricultural Research Administration, United
`States Department of Agriculture. Article not copyrighted.
`(2) For previous paper of this series see Boatner, Samuels, Hall
`and Curet. THIS JOURNAL, 69, 668-672 (1947).
`.
`(3) Presented before the 109th Meeting of the American Chemical
`Society, AUantic City, New Jersey, April 8 to 12, 1946.
`(4) Boatner, Caravella and Samuels, THIS JOURNAL, 66, 838
`(1944).
`
`r
`...
`Orange pigments
`(in Et20)
`Et20 evaPd.1 Hot acetone
`
`t
`
`Orange C
`(residue)
`
`Gossyfulvin, orange B
`
`{-
`I cooling
`t
`
`t
`
`Gossyfulvin Orange B
`(ppt.)
`(dissolved)
`e. g., the melting point of gossyfulvin lies between
`those of the other nitrogen derivatives. The com(cid:173)
`pounds differ with respect to solubility and sta(cid:173)
`bility. Diaminogossypol dissolved
`in diethyl
`ether or warmed in acetic acid is reported5 to
`evolve ammonia and revert to gossypol. Di(cid:173)
`anilinogossypol, on the other hand, is one of the
`most stable of the compounds formed from gossy(cid:173)
`pol, and is hydrolyzed to gossypol only upon re-
`(5) Miller and Adams, ibid., 1736-1738 (1937).