.
`
`'
`
`0;.
`
`THE PHARMACOLOGICAL ACTION OF SOME
`ANALOGUES OF PHYSOSTIGMINE
`
`JOHN A. AESCHLIMANN AND MARC REINERT
`From tht"Rochs" Chemical and Pharmacological LalJoral;orie., BlUle
`
`Received for publication June I, 1931
`
`Physostigmine (Eserin) is of interest chemically as an example
`of an alkaloid whose pharmacological action depends on the pres(cid:173)
`ence in the molecule of a particular group, in this case the methyl(cid:173)
`carbamic ester group CHaNHCOO. . It has been shown by Sted(cid:173)
`man (1, ·2), Stedman and Stedman (3), White and Stedman (4)
`that other compounds containing this group possess a miotic ac(cid:173)
`tion similar to that of physostigmine. The results of his exten(cid:173)
`sive investigation can be summarized as follows:
`All the compounds which possessed .miotic activity were basi(cid:173)
`cally substituted phenylesters of monoalkylcarbamic acids of the
`general formula RNHCOOC.H4R', where R was a methyl or ethyl
`group and R' a basic substituent such as -N(CH.h or -CHtN-
`(CH,)s, etc.
`.
`The activity was greatest when R was a methyl group,-i.e.
`when the compound was similar to physostigmine in that it
`contained the group CH,NHCOO. No activity was observed
`when R was a phenyl group.
`The miotic activity varied according tb whether Rt was in the
`0, m or p-posiiion and the activity of the hydrochlorides was in
`some cases greater Rnd in others smaller than that of the quater(cid:173)
`nary salts.
`. There is an evident analogy with the case of cocaine, which is a
`benzoic ester of a bicyclic alkamine, whereas phYsostigmine is a
`In the same
`methylcarbamic ester of a tricyclic basic phenol.
`way as many synthetic esters of simpler alkamines possess local
`anesthetic properties similar to those of cocaine, so do the methyl.
`carbamic esters . of the simpler basic phenols synthesized by
`413
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`

`414
`
`JOHN A. AESCHLUlANN AND MARC REiNERT
`
`Stedman pOSBesspharmacological properties similar to those of
`physostigmine. It was therefore of interest to investigate the
`variation of the pharmacological properties in this series on modi(cid:173)
`fying the carbamic acid group on the one hand and the phenolic
`residue on the other. It also seemed probable that a. systematic
`investigation might·result in obtaining a compound suitable for
`therapeutic use which would be free from the inherent disadvant(cid:173)
`ages of physostigmine, particularly the ease with which it de(cid:173)
`composes in solution.
`
`PRELIMINARY WORK
`In the first place several of the compounds whose miotic action
`. had been observed by Stedman were investigated for toxicity,
`miotic action, and action on the rabbit's intestine. It was found
`that many of the quaternary salts of this series were highly toxic
`substances, having the characteristic action on the central ner(cid:173)
`vous system and causing increased salivation like physostigmine.
`The quaternary salts examined were found to have .. a stronger
`action on the intestine than the hydrochlorides of the corresPond(cid:173)
`ing tertiary bases and to be more readily decompOsed. This
`decomposition takes place in 'aqueous or alcoholic solution and
`was also observed by stedman (l,p. 733). It occurs with elim(cid:173)
`ination of alkyl or aryl isocyanate the odor of which is evident
`after some hours standing in the cold or immediately on boiling.
`The carbamic ester group iii transformed into a phenolic hydroxyl
`~oup according to equation A.
`RNHCOOC.H,R' - RNCO + HOC.H,R'
`(A)
`As the presence of a solvent, water or alcohol, is necessary for the
`. decomposition to take place below 100°, it is probable that the
`solvent plays an important part in initiating the decomposition.
`In aqueous solution the isocyanate then reacts with water accord-
`. ing to equation B to form the disubstituted urea.
`2RNCO + HaD -
`(RNH),co + COt
`(B)
`CHaNCO + H.NCtH. - CHaNHCONHC.H.
`(C)
`The change could be particularly welJ followed in the case of
`. phenylcarbamic esters, where insoluble diphenyl urea is produced.
`
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`

`PHARMACOLOGICAL ACTION OF PHYSOSTIGM(NE
`
`415
`
`In the case of the methylisocyanic esters the dimethYl urea
`formed remains in solution but by adding aniline to the solution
`insoluble methylphenyl urea is formed according to equation C.
`In the presence of excess of alkali the end products of thehy(cid:173)
`drolysis are the phenol, amine, and alkali carbonate, but even
`. then the . first stage is an elimination of isocyanate, the odor of
`which can be observed if the solution is made only just alkaline.
`The tentative suggestion had already been made by Stedman
`(l,.p. 733) that the activity of these compounds might be due to
`the action of one of the products of hydrolysis liberated in the
`body. An experiment was therefore made with N-bromaceta-
`
`mide CHa.CON<:r which readily splits offHBr in vitro produc(cid:173)
`ing methylisoeyanate. It was found, however, that it produced
`none of the characteristic symptoms of physostigmine poisonUig.
`When the solution of the carbamic ester is made slightly acid
`(pH on the acid side of 5) the decomposition can be greatly sup(cid:173)
`pressed. It was only after this discovery that we were able to
`obtahi concordant results in the evaluation of the pharmacolog(cid:173)
`icalactivity of many of the compounds mentioned below, which
`were tested in buffered solutions, a method which proved to be
`satisfactory for experimental purposes.
`
`ESTERS OF DISU1JSTITUTED · CARB.A1dIC ACIDS
`In order to obtain compounds which might be less readily de(cid:173)
`composed than the monoalkylcarbamic esters previously pre(cid:173)
`pared, some dialleyl and arylalkylcarbamic esteJ'S were pre(cid:173)
`pared. These contain instead of the group R· NHCOO, the group
`
`:l)NCOO -. It was considered that a decomposition analo(cid:173)
`gous to that of the monosubstituted carbamic esters would be
`far less likely to take place as it would involve the migration of
`an alkyl or aryl radical instead of a hydrogen atom as in. equa(cid:173)
`tion A. H the decomposition took place in an analogous manner'
`a phenolic ether would be produced as in equation D, a course
`which seemed unlikely.
`R )NCOOC.H4R' __ RNCO + R,O.C.H.R'
`R,
`
`(D)
`
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`

`416
`
`JOHN A. AESCHLIMANN AND MARC REINERT
`
`In one compound investigated the nitrogen of the carbamic ester
`group formed part of a heterocyclic ring, Rand R1being in that
`case together represented by the pentamethylene group C.H1o
`so that such a decomposition was completely excluded.
`It was of course possible that owing to the improbability of
`isocyanate liberation, a simple hydrolysis would take place in
`these cases forming the secondary amine and carbon dioxide. On
`heating the neutral aqueous solution of such disubstituted car(cid:173)
`bamic esters no amine could, however be detected or determined
`quantitatively. The ~umption that this class of compounds
`. would be more stable in vitro thus proved to be justified and it
`was found that many of them had a high pharmacological
`activity.
`
`PHARMACOLOGICAL INVESTIGATION
`The results of the investigation ,are given in table 1 and concern
`the following properties: (a) Toxicity (intravenously and orally);
`(b) miotic action; (c) peristaltic action (on the surviving intes(cid:173)
`tine (Magnus) and in some cases in situ (Trendelenburg); (d)
`aQtion on the frOg~s heart.
`.
`The intensity of these effects could be measured quantitatively
`with sufficient accuracy for comparison with the correspondblg
`effe~ts of physostigmine. Using the figures obtained as a basis,
`the compounds could be classified according to their Hphysostig(cid:173)
`mine activity." The various properties were not always present
`in the same degree, 8. few ·substances, for · inatance showing a
`relatively weak miotic action but having a strong action on intes(cid:173)
`tinal peristalsis. The various pharmacological properties are
`therefore treated separately in the discussion.
`
`METHODS
`1. Toxicity. The toxicity was determined in the usual way,
`the minimum dose to cause death of over 80 per cent of the
`animals being. recorded. This naturally gives somewhat higher
`figures for the toxicity than the 1150 per cent-deaths" method
`adopted by White and Stedman (4), but where the same sub(cid:173)
`stances have been examined the toxicities are in the same order
`
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`

`PRARMACOLOGICAL ACTION OF PHYSOSTIGMINE
`
`417
`
`In table 2 the values ob(cid:173)
`as those observed by these authors.
`tained by the "50 per cent-deaths" method are given for com(cid:173)
`parison in the case of two of the compounds and physostigmine,
`the values showing agreement with those of White and Stedman
`for physostigmine.
`In the compounds marked with an asterisk
`the toxicity and other pharmacological effects were measured in
`stabilized solutions of pH about 3.7 . . These were usually pre(cid:173)
`pared by dissolving 1 gram of substance in 95 cc. of decinormal
`glycine-sodium-chloride sOlution + 5 cc. of decinormal hydro(cid:173)
`chloric acid (Sf/frensen buffer solution, Clark (5» a.nd diluting to
`the required strength With Ringer solution. The buffer solution
`itself was non-toxic. Mice were usually used for toxicity
`determinations.
`2. Miotic action. This was observed on the cat, 2 drops of the
`solution to be examined being instilled into one eye and the two
`eyes subsequently compared at interva.ls. To obtain a complete
`comparison of the various compounds it would be necessary to
`take account of the duration as well as the intensity of the maxi(cid:173)
`mum miotic effect produced bya given concentration. The
`duration of the action was only noted in a few cases in which the
`action was strongest.
`3. Action on the small inteatine (raQbit). a. Surviving intestine.
`The action of the substances on the isOlated rabbit intestine sus(cid:173)
`pended in 50 cc. of Da.le's solution kept, like the washing solution,
`at 3", was studied. The apparatus of Guggenheim arid LOilier
`(6), which enables the test to be carried out on two pieces of
`intestine simultaneousiy, was used . . The test pieces were taken
`from various parts of the small intestine and were about 2cm.
`long. Portions of the ileum wereusu~y found to be more sen(cid:173)
`sitivethan those 'from the duodenum. Each substance was
`directly compared with physostigmine so as to eliminate as far
`as possible differences due to varying sensitivity of the test
`object.
`b. Intutina in situ. A few of the substances were a1s9 tested
`by the method of P. Trendelenburg (7) on the intestine in situ
`of a. rabbit kept in deep narcosis by 0.5 cC. per kilogram of
`IIRoehe-Numal," a solution conta.ining 10 per cent of allylisopro-
`
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`

`418
`
`JOHN A. AESCHLIMANN AND MARC REINERT
`
`pyl barbituric acid. The injections were made into the vena
`jugularis, into which a cannula which could be closed· by a cock
`was fixed.
`4. Action on the Jrog-heart. The heart action was studied on
`the isolated esculenta heart by the method of Straub.
`5. Actimi. on blood pressure and respiration. The blood pres(cid:173)
`sure tests were carried out on the rabbit narcotized as above.
`The method of P.Trendelenburg (8), which enables the experi(cid:173)
`ment to be carried out for many hours on one animal without
`fear of coagulation, was used .
`. In investigating the action OIl respiration both the effect on the
`volume and on the frequency of respiration was observed. A
`gasometer of 1 liter capacity with two one-way valves regulating
`inspiration and expiration was connected bya cannula to the'
`trachea of the animal.' A spindle on the gasometer malces ,a
`complete revolution when 1 liter of gas passes through the meter
`and carries two radial wires in the form of' a cross. Each time
`250 cc. of gas have passed through the meter one of the wires
`closes a circuit actuating an electromagnet and a stroke is reg(cid:173)
`. istered. In parallel with the meter a Marey's, tambOur was
`arranged to register the respiratory frequency on the sam.~ graph
`as blood-pressure and volume.
`
`DISCUSSION OF RESULTS (TABLE 1)
`TOxiCity.' In discussing the toxicity it is convenient to divide
`the' compounds into three classes, the members of which show
`certain similarities: (a) Salts of weak tertiary aromatic bases
`from which the base is liberated in neutral or slightly alkaline
`solution (i.e., in the intestine) ; (b) salts of strong tertlary bases
`having the nitrogen in the side chain, from which the base is
`liberated only in alkaline solution; (c) quaternary salts, which
`remain in solution in alkali.
`The members of class (a) are in general less toxic than those of
`classes (b) and (c). When given orally salts of class (a) are
`considerably less toxic than when injected intravenously,possibly
`because the free base is only slowly absorbed through the intesti(cid:173)
`nal walls. Salts of class (b) show chemically the closest rela-
`
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`

`PHARMACOLOGICAL ACTION OF PHYSOSTIGMINE
`
`419
`
`tionship to physostigmine and their toxicities are of the same
`order as that of the natural alkaloid. The difference between
`. the .lethal doses by the oral and intravenous route is here less
`pronounced, although several of the compounds are, like phy(cid:173)
`sostigmine, unstable and might be expected to decompose in the
`alimentary tract. The relatively high toxicity of physostigmine
`by the mouth might possibly be due to the inhibiting action it
`exerts on hydrolysis by esterases (Stedman (9» a:s it might con(cid:173)
`ceivably inhibit the action of the enzymes of the alimentary
`tract sufficiently to prevent to some · extent its own hydrolysis.
`Among the compounds of class (c) are some highly toxicsulr
`stances, but throughout this class there is a large .decrease of
`toxicity when the compounds are given orally.
`Irrespective of
`whether we are dealing with compounds which are stable in
`neutral aqueous solution (substances 3, 4, 32, 33, 34, 35, 36,38,
`40~ 42) or SUbstances which readily decompose on boiling their
`solutions (10, 11, 13, 14, 16, 17, is, 20, 21,23,25), the ratio olthe
`oral to the intravenous lethal dose is more than 10 ~nd occasion(cid:173)
`ally over 100 in the different compounds. We do not consider
`tbat this similarity of behavior between the stable and unstable
`substances is due to the fact that the latter were tested in buffered
`solutions which were stable to boiling for a short time, beca~
`the effect of the buffer salts would be overcome in the alimentary
`tract. The solutions were only buffered to prevent decomposi(cid:173)
`tion during testing.
`A large difference between the values obtained for the . lethal
`doses of a compound when administered intravenously or orally
`indicates either that it is only slowly absorbed in the alimentary .
`trac.t so that a lethal concentration is onlyattamed. with high
`doses, or that it is rapidly eliminated either unchanged or after
`decomposition or combination with another substance in the
`. body. Various considerations have led us to abandon the view
`that the cause of the reduced toxicity when given orally is· that
`the substances are all unstable in vivo.
`In the first place· we
`became doubtful of this hypothesis when it was found that in
`vitro stability, as mentioned above, was no criterion of high oral
`toxicity. Further the phenol, substance 4 which cannot undergo
`
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`

`

`!'fAN_
`
`&TR\."(,.'TUMA&' FORMULA
`
`NUll-
`
`.".
`--
`
`I Methylcarbamic ester
`of phenol
`
`--
`2 Methylcarbamic ester
`of 2-nitrophenol
`
`OCONHCH.
`
`OCONHCH,
`
`CY
`ex NOs
`
`TABLE I .
`
`MCLT1HO
`POINT
`
`LII'l'K"L ..... (MGIIII.,
`Mall. P ••• Oll~)
`
`IDtra ............ y Per 00
`
`MIOTIC ACTION
`(c .. ~)
`
`leoLA ~D ..... tT
`lHTlBTllfa
`
`I8OLA,.aDnnn
`HBART
`
`Si·
`
`>50
`
`>1,000 0.2 per cent, 1 X 10-', no
`no action
`action
`
`56·
`
`33
`
`---
`>50
`
`~ 3
`
`Trlmethylphenyl
`moniumchloride
`
`am-
`
`(CHshN-ct
`I
`C.u.
`
`2Mo
`
`15
`
`OH
`
`1820
`
`25-30
`
`--
`• 3-Oxyphenyl-tri-
`
`methyJammonium-
`iodide
`
`3-Acetoxyphenyl-tri-
`methylammoDium-
`methyJeulfate
`
`--
`5
`
`-
`
`N(CHsc)sJ
`
`OCOCH.
`
`CY
`CY
`
`N (CH.). • SO.CH.
`
`.
`
`m-aoo 2 per eent, 2 X 10-',
`inactive
`definite;
`1 X 10~',
`strODr ac-
`tion
`
`---
`200-250 2 per ce!lt,
`inactive
`
`1 X 10"'"',
`IItroDg ac-
`tioD; 1 X
`10-', defi-
`nite actioD
`
`5 X 10"',
`wealt ac-
`tioD
`
`121·
`
`7.5-10
`
`1,000 1 per eent,
`doubtful
`
`- -
`
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`

`

`6
`
`3.Ethylcarboxydi-
`methylaminophenol-
`hydrochloride
`
`--
`7
`
`3-Ethylcarboxyphenyl.
`trimethylammo-
`niumiodide
`
`--
`8 m-Dimethyl&rnino-
`phenoxyac:etmethyl.
`amid&-dimethyIBul-
`fate
`
`~ -
`
`-
`
`-
`9
`
`--
`10
`
`Bis-(3-Trimethyl.
`phenylammonium)-
`carbonite-di~methyl-
`Bulfate
`
`Bis-(3-dimethylamino·
`
`hydrochloride
`
`phenyl)-carbonate ()
`
`O-CO-o
`
`'D
`'D
`
`N(CH.).HCI
`
`N(CH.hHCI
`
`O-CO-o
`
`()
`
`N(CH.)s
`I
`BOtCH,
`
`N(CH.). .
`I
`BO.CH,
`
`-
`
`---
`
`-
`
`-- -
`
`-
`
`----
`
`- ----- - - -
`
`--- - - -
`
`- -
`
`1310
`
`.
`
`---
`
`153°
`
`OCQOC,H,
`
`()
`
`N(CH.).HCI
`
`OCO<>C,H.
`
`()
`
`N(CH,).· J
`
`OCHtCONHCH,
`
`()
`
`N(CH.) •• SO£lI.
`
`About 1 per <:ent, 2 X 10-',
`500
`inactive
`doubtful
`action
`
`2 X 10--,
`doubtful;
`1 X 10-',
`Blight con-
`traction
`
`1 X 10-', no
`action
`
`25
`
`>500
`
`Ii per cent
`inactive
`
`1210
`
`7.5
`
`---
`1,000 2 per cent
`inactive
`
`2100
`
`2,000- Only IIOluble
`2,500
`inexeeea
`HCl. Too
`irritant
`forteBting
`
`1950 .
`
`12.5
`
`1,000
`
`1 per <:ellt,
`no aetion
`
`1 X 10-',
`definite
`action
`
`1 per cent,
`no definite
`action
`
`-
`
`-
`
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`

`

`HA ...
`
`....
`"171'-
`--
`11- Carbamio ester Qf 3-
`Oxyphenyltrimethyl-
`ammoniummethyl-
`sulfate
`
`--
`12 Methylcarbamic eater
`of 3-oxyphenyldi-
`methylaminehydro-
`chloride
`
`is --
`
`13-
`
`ltfethylcarbamic ester
`of 3-oxyphenyltri-
`methylammonium-
`methylllulfate
`
`1~- Methylcarbamic ellter
`of 3-oxyphenyldi-
`methylethylam-
`moniumbromide
`
`--
`
`15 Methylcarbamic ester
`of m-oxyphenyldi-
`' ethylamine hydro-
`chloride
`
`TABLE I-Cominwd
`
`.. al.nKC
`POINT
`
`LftBAL I>0Il'. (110"".,
`11011 •• "'. 1<011. )
`
`Intnr.venouely ~oe
`
`137·
`
`0.7
`
`500
`
`IIIOTIC ACTION
`(C"'I")
`
`IftOLATItD A".lItT
`Il'tT&IITtN'.
`
`JIIOLAT.I> noo
`HeA.RT
`
`1 per cent,
`inactive '
`
`5 X 10-·,
`strong
`,contrac-
`tion
`
`1 per cent,
`no definite
`action
`
`170·
`
`15
`
`55
`
`5 ~r cent,
`temporary
`
`1 X 10-', ex-
`citation
`
`0.1 per ceni,
`ce88ation
`of beats in
`diastole
`
`157-100·
`
`0.1
`
`2.5
`
`1 per cent,
`temporary
`
`0.5 X 10 .... ,
`definite
`contrac-
`tion
`
`1 per cent,
`ce88ation
`of beats in
`diastole
`
`BTJtUCTO."L POIlM01.A
`
`O-<X> ,NU.
`
`()
`
`N (CH.).-SO.CH.
`
`000 'NHCH.
`
`()
`
`N (CH.), • HCt
`
`000 ·NHCH.
`
`()
`
`N (CH,),-SO,CH.
`
`OCO· NHCH.
`
`(f
`
`N(CH.).· C.H.· Br ,
`
`OCO· NHCH.
`
`()
`
`N(CtH.) •• HCl
`
`IMo
`
`0.15
`
`1~·
`
`5
`
`I't-S
`(rat)
`
`---
`20
`
`1 X 10 .... ,
`active;
`1 X 10-',
`,strongly
`active
`
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`

`

`OCO·NHCH.
`
`()
`
`N (C,H.hCH.J
`
`136·
`
`0.1
`
`20
`
`0.5 per cent, 0.5-1.0 X
`10-', defi-
`stron«ly
`active
`niteaction
`
`CH.NHCOO 0
`
`N(CH.).J
`
`165°
`
`2
`
`50
`
`1 X 10-1,
`excitation
`
`---
`
`---
`
`200
`
`0.1
`
`1 per cent,
`no definite
`action
`
`2 X 10-1,
`definite
`contrac-
`tion
`
`IS- Methylcarbamic eater W 1M·
`()
`
`16· Methylcarbamic ester
`of oxyphenylmethyl-
`diethylammonium-
`iodide
`
`--
`17- Methylcarbamic ester
`of p-oxyphenyltri-
`methylammonium-
`iodide
`
`of S-oxyquinoline ·
`methiodide
`
`e
`
`19 Allylcarbamic ester of
`m-oxyphenyldi-
`methylamine hydro-
`chloride
`
`--
`20- Allylcarbamic ellter of
`m-oxyphenyltri-
`methyl ammonium-
`methylsulfate
`
`CH.NHCO . 0
`
`I'"
`CH.J
`
`OCO . ~HCH •. CH= CH,
`
`155·
`
`150
`
`500
`
`2 per cent,
`inactive
`(too
`irri-
`tant)
`
`2 X 10-1,
`stron«
`paralyais ;
`0.5 X 10-',
`definite
`excitation
`
`0.5-Q.25 X
`10-', deft- .
`nite con-
`traction
`
`N(CH.). · .HCl
`
`OCO • NHCH.CH--:-·CH.
`
`()
`
`N(CH.).-SO.CH.
`
`112°
`
`0.75
`
`25
`
`1 per cent,
`definite
`
`1 per cent,
`ceesation
`of beats in
`diastole
`
`0.01 per cent,
`increa.se of
`amplitude;
`0.1 percent,
`cessation
`of beats in
`diastole
`
`1 per cent,
`increase of
`amplitude..
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
`Page 11 of 32
`
`

`

`!fUII-
`
`If.lMa
`
`II""
`--
`21- Ethylcarbamic ester of
`m-oxyphenyltri-
`methylammonium-
`methylsulfate
`
`-.
`TABLE I-Continued
`
`8'r1l ucrua.lL .0'''. ot.A
`
`1I-''''!f0
`PODft
`
`. (Moua ..
`LftIIAL DOe •
`MOil . . . . 1[011.)
`
`Inl .. T ..... uoIT Per ..
`
`---
`
`100
`
`131-
`
`I
`
`M'OTIC AC'I'ION
`(e ..... )
`
`teoLA,2'aD . . . aft
`Inaeft'!(.
`
`""' ..... nohlOG
`• • A ....
`
`-
`
`1 per cent,
`weak
`
`vOCO . NHC,H.
`
`N(CB.h-BO.CB.
`
`--
`22
`
`~ ....
`--
`
`23- Benzylcarbamic ester
`of 3-oxyphenyltri-
`
`methylsufate
`
`.
`
`24,
`
`Phenylcarbamic ester.
`
`OCO-NHC,R,
`
`methylaminehydro-
`chloride
`
`N(CH.).HCl
`
`-
`
`- - - -
`
`Benzylcarbamic ester
`
`OCO - NHcHsCeH,
`
`methylaminehydro-
`chloride
`
`N(CH.)eHCI
`
`000 - NHCHtCeH,
`
`N (CH.l. - SO.CH.
`
`of 3-oxyphenyldi- V
`methylammonium- V
`of 3-oxyphenyldi- V
`
`ISO·
`
`50.0
`
`500
`
`Only eolu-
`blein ex.
`eeII8acid
`
`1090
`
`0.1
`
`33
`
`'1 per cent, no .0.0-{).25 X
`definite
`10-', defi-
`action
`nite &C-
`tion, simi-
`lartophy-
`lIostigmine
`
`0.1 per cent,
`slight in-
`creaee
`of
`amplitude;
`0.01 per
`cent, noac-
`tion
`
`1580
`
`20-30
`
`AbQut
`500
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
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`
`

`

`000- NHC.u.
`
`()
`
`N (CH.), • S04CU.
`
`000 • NHNHC.H.
`
`()
`
`N(CH.),J
`
`OOCNHCH.
`
`0
`
`N-CU1 • CHsN(CsH')2
`I
`CH.
`
`UCt
`
`25-
`
`--
`26
`
`Phenylearbamic eater
`of 3-oltYphenyltri-
`methylammonium-
`methyl sulfate
`
`PheDylhydruinoformie
`ester of 3-oxyphe-
`nyltrimethylammo-
`niumiodide
`
`~ el. --
`
`Z1 Methylearbamio ester
`of 4-oxyphenyldi-
`ethylaminoethyl-
`methylamine hydro-
`chloride
`
`--
`28t Methylcarbamic ·est.er
`of err ,a-hydroxy-
`phenylethyldimethyl-
`amine hydrochloride
`(Miotine)
`
`1~166 1 per cent,
`inactive
`
`156·
`
`2 (buf-
`fered);
`tr7 (un-
`bur-
`fered)
`
`158-
`
`· 0.25
`
`200
`
`1 per .oent,
`no definite
`action
`
`--
`
`0.1 per cent,
`msht de-
`ere . . of
`amplitude
`
`1 X 10-', .
`doubtfUl;
`2 X 10-',
`definite ·
`aotion
`
`0.3 X 10-',
`definite
`actioD;0.6
`X 10-',
`stroDe ac-
`tioD; 1 X
`10-', par-
`alytic ac-
`tion
`
`159·
`
`0.1
`
`25
`
`2 per cent, 2 X 10-', no
`no definite
`definite ac-
`action
`ti<in
`
`---
`
`2.0 O.H).S per
`cent, ·eev-
`eralhoufII;
`1-2 per
`2-l
`cent,
`houfII
`
`0.~.25 X
`10-', defi-
`nite ae·
`tiOD
`
`0.1 per cent,
`lllisht de-
`cre . . o(
`tone; 0.5
`cent,.
`per
`de-
`larr;e
`"cre . . of
`tone
`
`-
`
`169°
`
`1.0
`
`0-000 . NHCR.
`
`CH.CH---'N(CH.hHCI
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
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`
`

`

`"1111-
`SU
`
`JlfAlia
`
`wnlOCl'Oa.A.t. PO_Mm.A
`
`28af Methylcarbamic ester
`of ",.(4-hydroxy-3.
`methoxyphenyl)-
`ethyldimethylamine
`hydrochloride
`
`OCONHCH.
`
`~CH'
`
`TABLE1~ominued
`
`LftII.&L DOIIa (.11011 •• ,
`11011. PD &0".)
`
`lIa.ftMQ
`POln
`
`IDtraYeDOlI8ty Per.,.
`
`---
`1450
`(decom.
`posed)
`
`1-1.5
`
`25
`
`0.5 per cent, 1-2 X 10-·, 0.1 per cent,
`stronp;
`definite in-
`slight de-
`after
`creaee of
`30
`of
`Cfea8e
`minutes
`tone
`tone
`
`MtOTte .AC'ftOK
`(cu)
`
`tBOU"aD 1lA •• rr
`JH1'_TlN&
`
`I&01."''I'.D PROQ
`.mART
`
`--
`29t Methylcarbamic eater
`of _(3-hydroxy-4-
`methoxyphenyl)·
`ethyldimethylamine
`hydrochloride
`
`~
`
`--
`30f Methylcarbamic ester
`of ",.(3-hydroxy-4-
`. methoxyphenyl)-
`ethyltrimethylam-
`moniumiodide
`
`CHaCH . N (CH.h . HCI
`
`OCH.
`VO'NHCH.
`
`CH.CH-N (CH.).HCl
`
`.
`
`OCH.
`().NH~
`
`CH,CH-N(CH,),J
`
`6
`
`1770
`
`5
`
`-
`
`1 X 10-',
`slight ac-
`tion; .1 X
`10-', large
`incr1!&IIe of
`tone
`
`0.1 per cent,
`IIlight de-
`Cfe&lle of
`tone
`
`0.1 per cent,
`no certain
`action
`
`1 per cent,
`maximum
`after
`2
`hours:
`next day
`indefinite;
`0.5 per
`cent, deft-
`niteafter
`2 hours
`0.5 per cent, 2 X In-',
`IItronp;
`lllight in-
`creMe of
`after
`tone; 5 X
`hours;
`10-', mod~
`next day
`indefinite
`erate
`in-
`creaae of
`tone
`_.
`
`2
`
`-
`
`-
`
`-
`
`-
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
`Page 14 of 32
`
`

`

`OCON(CH.),
`
`()
`
`N(CH,).{CHOH,COOH)1
`
`60
`
`Not
`crys-
`°tal_
`lized
`
`OCON(CHih
`
`143·
`
`0.5
`
`12...16
`
`1 per cent,
`alight
`mi08is
`
`0.4 X IO-L
`1 X 10-',
`no definite
`action
`
`0 .&-1 per
`cent, mi-
`osia !leV-
`erai hOUri!
`
`0.4-0.2 X
`10-·,con-
`traction
`
`N(CHI ) •• SO.CHI
`
`OCON (CaH.).
`
`N (CH,), . SO.CH.
`
`OCON (CHaCH=CH.,."
`
`137·
`
`8
`
`---
`71
`
`14 per cent, 1-2 X 10-',
`no definite
`no definite
`action
`action
`
`110·
`
`10
`
`>250
`
`-4 per cent,
`indefinite °
`
`2 X 10-', in-
`definite
`
`1 per cent,
`alight de-
`crease
`of
`amplitude;
`0.1~.01 per
`cent, doubt-
`ful action
`
`(j
`0/
`of 3-oxyphenyltri- (j
`Q--<lCONC,H"
`
`31
`
`Dimethylcarbamic
`ester of 3-oxyphenyl-
`dimethylaminetar-
`trate
`
`--
`32 Dimethylcarbamic
`ester of3-oxyphenyl-
`trimethylammo-
`niummethylsulfate
`
`--
`33 Diethylea:rbamie ester
`of 3-oxyphenyltri-
`methylammonium-
`methylsulfate
`
`~
`
`--
`34 Diallylcarbamic eater
`
`methylammonium-
`iodide
`
`"0
`
`--
`35
`
`Pentamethylenecar-
`bamic eeter of 3-oxy-
`phenyltriinethylam-
`moniummethyhul-
`fate
`
`N(CH,).J
`
`---- ---
`500
`6
`
`159·
`
`2 per cent,
`inactive
`
`- - .
`
`0-
`
`- -
`
`-
`
`N(CH.) • • SOtCH.
`
`2 X 10-',
`eligbt; 1
`X 10-',
`atrong ac-
`tion
`
`-_ . . -- - - -_ .. ~---.-
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
`Page 15 of 32
`
`

`

`CH,
`
`/
`OCON
`
`N (CHa) •• SO.CH.
`
`(CHI)sNCO·O HCI
`
`---
`163"
`
`---
`
`'---
`
`niummethylsul£ate y "'-Cell,
`37 Dimethylcarbamic W 193·
`38 Dimethylcarbamic W 139·
`
`NO ....
`
`H .....
`
`....
`--'-
`36 Methylphenylcarbamic
`ester of 3-oxyphenyl-
`trimethylammo-
`
`--
`
`~
`--
`
`ester of 8-oxyquino-
`Iinehydrochloride
`
`eeter of 8-oxymethyl-
`quinoliniummetbyl-
`sulfate
`
`--
`39
`
`Dimethylcarbamic
`ester of 2-oxybenzyl-
`diethylamine
`
`TABLE I-Continued
`
`ITBUCTU'''AL FOllInlL.l
`
`"a.nK0
`lPOn<r
`
`.. ("O""&,
`L ....... L _
`110 ... ,. •• 1l0II.) ,
`
`IfI&raveDOualy Per""
`---
`75
`
`3.5
`
`1I10Tl0 ACTION
`(cu)
`
`fIIOU'l'D ..... Ilf'l'
`t~1I
`
`JB.OLA'!'aD noo
`HURT
`
`1-2 per cent, 0 .• X 10-·, 0.1 per cent,
`slight mi-
`strong &e-
`no definite
`OBis
`tion
`action
`
`150
`
`400
`
`0.5-1 per
`cent, deli-
`nite
`
`1 X 10-', no
`definite e.c-
`tion
`
`0.5
`
`3J()
`
`0.~.5 per 0.2 X 10-',
`cent, deS-
`de6nite
`nite
`for
`contrac-
`several
`tion
`hour'll
`
`(CH,).NCO • 0
`
`I "'-
`CHI'-
`
`BO,CH.
`
`---
`Not
`crys-
`tal-
`Iized
`
`1.5
`
`5
`
`OCON(CH,h
`
`ex:
`
`CHtN(c,H.),HCl
`
`1 per cent, 1 X 10-', no
`slight
`Action; 2
`X 10-·,
`alight con-
`traction; 2
`X 10-',
`etronccon-
`traction
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
`Page 16 of 32
`
`

`

`40 Dimethylcarbamic
`ester of 2-oxybenzyl-
`methyldiethylam-
`moniumiodide
`
`OCON(CH.h
`
`--
`
`41 Dimethylcarbamic
`
`hydrochloride
`
`~
`'--
`42 Dimethylcarbamic
`
`methiodide
`
`--
`
`of Hannol hydro-
`chloride
`
`ex CHsN(~HlhCHIJ
`ester of Hordenine Q
`ester of Hordenine Q
`, fat Methylcarbamic ester M»N
`
`OOC~(CRah
`
`CH.CHsN(CH.).HCl
`
`OOCN(CH.h
`
`CH,CH,N(CH.hJ
`
`CH.NHCOO
`
`N
`I
`CH,
`
`1 X .1 -I, no
`aer.i( nj 2
`X 10-',
`slight con-
`traction; 1
`X 10-1,
`IItrong
`contrac-
`tion
`
`2 X 10-1, no
`Mtion
`
`2 X 10-', no
`action
`
`1 X 10-', no 0.1 per cent,
`definite &C-
`slackening
`tion; 2 X
`or
`eel!8&-
`tion of pm-
`10-', weak
`action; 4
`sationa
`,
`X 10-'
`weak 'ac-
`tion
`
`,
`
`156"
`
`0.5
`
`75-100
`
`1 per cent, no
`definite
`action
`
`1 per cent,
`trace; 2
`per cent,
`weak mi~
`Ollis
`
`1 per cent,
`indefinite ;
`2 per cent,
`trace
`
`1 per cent,
`weak; 0.5
`per cent,
`indefinite
`miOllis ,
`
`---
`
`206-
`
`---
`75
`
`IS
`
`m·
`
`55
`
`>100
`
`66
`
`1-'
`\.
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
`Page 17 of 32
`
`

`

`. TABLE l-Concluded
`
`NUK-
`
`.....
`--
`«
`
`~
`
`--
`45
`
`"AU..
`
`IrTJtIJC'rUaAL roaJI17t..A.
`
`Physostigmine
`
`CH,
`
`I CH.
`/"-
`.
`CHaNHC00-Q-C
`?H.
`N)m-· -~
`.
`.
`CHi
`I
`CH • .. ·
`
`Physolltigmine methi-
`odide
`
`CuH.10.N., CH.I
`
`MItLTUIQ
`POINT
`
`---
`
`LJ:TIIAL DOe. (MOtJBJI,
`lIa ..... " KOII.)
`
`Intrannoualy Per ..
`
`0.5
`
`3
`
`I
`
`MIone ACT10H
`(CAT)
`
`IBOLAT.D llA,a.lT UOLA'r&DnOQ
`BJu.aT
`IHTAnNI:
`
`0.1...().5 per
`cent, deli-
`niteeev-
`eral hours;
`1-2 per
`cent, 24
`. hours
`
`0.25-0.5 X
`10-1, con-
`traction
`
`0.1 per cent,
`8trong de-
`or
`ereaae
`tone orce8-
`of
`eation
`beats
`
`---
`1880
`
`0.75-1
`
`250-300 0.1 per cent, 0;2 X 10- 1,
`active
`contrac-
`tion
`
`• Tested in bulreJ,'ed lIolution.
`t We are indebted. to Dr. and Mrs. E. Stedman (16) for kindly supplying .the specimens of these live compounds.
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
`Page 18 of 32
`
`

`

`PHARMACOLOGICAL ACTION OF PHYSOSTIGMINE
`
`431
`
`such a hydrolysis, is also 10 times less toxic orally than intrave(cid:173)
`nously .and its carbonic ester (substance 10) is much less toxic
`orally than the phenol which it would form to the extent of over
`90 per cent if hydrolyzed. It would be necessary to assume a
`detoxication of the. phenol in the organism by combination to
`explain these observations.
`It will also be seen from table 1 that physostigmine behaves
`similarly after conversion into its quaternary salt. Although
`the ratio of the oral to the intravenous lethal dose ofphysostig(cid:173)
`mine sulfate is only 6, the corresponding ratio in the case of the
`quaternary salt physostigmine methiodide (su~tance 45) is
`over 100, although there is no great difference in the stability of
`the two salts.
`On careful consideration it was found . that there are many
`indications which point to the fact · that the large difference in
`toxicity by the two routes is a general characteristic of quaternary
`ammonium compounds. Thus curare, which contains 88 active
`. principle the quaternary base curarine, is 70 times less toxic to
`rabbits orally than subcutaneously (K. Sauer (10» and a similar
`relation holds with other animals. . A discussion of the reasons
`for this is given by R. Boehm (11) who points out that it seems
`to be due to · their rapid · elimination, various experimenters
`having . shown that curarine is found almost quantitatively in
`the urine (8. JakabMzy (12». A similar observation was made
`by M. Fuhner (13), in the case of methyl green, the diquaternary
`salt of methyl violet. He found that rabbits showed no re8,Qtion
`to 15 times the intravenous lethal dose if it was given orally. In
`order to have another example of a simple quaternary salt" free
`from ester or hydroxy groups, the toxicity of trimethylphenylam(cid:173)
`monium chloride CJI.N (CH.)aCI, substance 3, was determined on
`mice and found to be 15 mgm. per kilogram intravenously and
`250 mgm. per kilogram orally:
`It therefore seems probable that a much reduced toxicity
`orally compared with intravenously, is a general characteristic
`of quaternary ammonium compounds, probably because they
`are rapidly eliminated from the blood stream.
`In consequence
`it appears justifiable · to conclude that the high value for the
`
`NOVARTIS EXHIBIT 2044
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`IPR2014-00550
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`
`

`

`432
`
`JOHN A. AESCHLIMANN AND MARC REINERT
`
`ratio of the oral to the intravenous lethal dose of the quaternary
`salts of substituted carbamic esters is not solely due to the fact
`that they are hydrolyzed in vivo although hydrolysis probably
`takes place to some extent, particularly in the case of those com(cid:173)
`pounds which are unstable in vitro.
`Miotu; action. Only those BubStances which were carbamic
`esters of phenols containing a basic substituent exhibited any
`miotic action. Relatively slight modmcations of the carbamic
`ester group caUsed the miotic activity to be wea.kened .asin sub(cid:173)
`stance 36, or to disappear, as in substances 2.$, 33 and 34. The
`quaternary salts of the aromatic bases examined were usually
`definitely stronger in their action than the hydrochlorides of· the
`corresponding tertiary bases, but this may be due to the position
`of the basic substituent as observed by Stedman (1, p.732). The
`mono-quaternary salt of physostigmine is almost as active as
`physostigmine, thus forming a contrast to cocaine which loses its
`characteristic properties on conversion to a quaternary salt
`(Ehrlich (14».
`The dimethylc8.fb8.mic esters 31, 32 and 38 were definitely
`more active than the monomethylcarbamic esters of thecorre(cid:173)
`sponding phenol 12, 13 and 18. The dimeth