Europaisches Patentamt
`
`European Patent Office
`
`Office europeen des brevets
`
`® Publication number:
`
`0193926
`A2
`
`EUROPEAN PATENT APPLICATION
`
`@ Application number: 86102812.4
`
`@ Date of filing: 04.03.86
`
`@)
`
`Int.CI.': C 07 C 125/067, A 61 K 31127
`
`@ Priority: 05.03.85 IL 74497
`
`@ Date of publication of application: 10.09.86
`Bulletin 86/37
`
`® Applicant: YISSUM RESEARCH AND DEVELOPMENT
`COMPANY OF THE HEBREW UNIVERSITY OF
`JERUSALEM, 46, Jabotinsky Street P.O. box 4279,
`Jerusalem 91042 (IL)
`
`@
`
`Inventor: Weinstock Rosin, Marta, Prof., 9 Herzog
`Strasse, Jerusalem (lL)
`Inventor: Chorev, Michael, 135/4 Feinstein Strasse,
`Jerusalem (IL)
`Inventor: Tashma, Zeev, 2 Shahal Strasse, Jerusalem
`(IL)
`
`@l Designated Contracting States: AT BE CH DE FR GB IT
`LILUNLSE
`
`@ Representative: Spott, Gottfried, Dr. et ai, Patentanwlilte
`Spott und Pusch mann Sendlinger-Tor-Platz 11,
`0·8000 Miinchen 2 (DE)
`
`@ Phenyl carbamates.
`
`(@ Phenyl carbamates of the general formula
`
`I
`
`ell) wherein R1 to Rs are as defined in the claims, are useful as
`G) pharmaceuticals .
`
`...
`o
`a. w
`
`ACTORUM AG
`
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`0193926
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`Case 118-6848
`
`PHENYL CARdAMATES
`
`The present invention relates to novel phenyl carbamates wnich
`are useful as pharmaceutical compositions. The invention further
`relates to pharmaceutical compositions having anticholinesterase
`act i vity.
`
`Acetylcholine is a major neurotransmitter which is found in all
`parts of the body. Any reduction in its activity, either as a
`result of neuronal damage, degeneration etc. or as induced by
`drugs or toxins, causes marked changes in the function of the
`organism. Acetylcholine itself h~s an extremely short half life,
`since it is rapidly hydrolysed at its site of action and in
`plasma by specific cholinesterase enzymes. Drugs that inhibit
`acetylcholinesterase, markedly increase and prolong the action of
`acetylcholine, thereby enhancing cholinergic transmission. Three
`such agents are used clinically, i.e., physostigmine, a naturally
`occurring alkaloid, and two synthetic analogues, neostigmine and
`pyridostigmine. Tne latter two agents are strongly ionised at
`physiological prl and therefore are only poorly absorbed from the
`gastro-intestinal tract, and do not penetrate the central nervous
`system to any sljnificant extent. Physostigmine is absorbed after
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`oral administration and readily enters the brain. As a thera(cid:173)
`peutic agent it has several disadvantages. It is chemically
`unstable and must be prepared in solution with an antioxidant,
`and protected from light. It has a relatively short half-life
`(20-40 mins) thereby necessitating frequent administration. The
`1 atter is of parti cul ar importance when the drug is to be admi(cid:173)
`nistered chronically. It has a low therapeutic ratio, a value of
`3-5 being reported in the majority of studies in laboratory ani(cid:173)
`mals, and a small therapeutic window, i.e. small range of dose in
`which it can be given without the accompaniment of side effects.
`Although physostigmine is absorbed from the gastro-intestinal
`tract, this is reported to be irregular and unpredictable, and
`therefore it is usually preferred to administer the drug par(cid:173)
`enterally. This is a serious drawback if it is to be used chroni(cid:173)
`cally on an outpatient basis.
`
`There are a number of clinical and pathological conditions which
`g.re.g.ssociated with cholinergic under-activity which can be
`improved by the administration of an anticholinesterase agent.
`These include reduction in cholinergic transmission induced by a
`variety of exogenous substances acting in the peripheral, or
`central nervous system. Peripherally acting agents are gallamine,
`d-tubocurarine and pancuronium, which are used as muscle re(cid:173)
`laxants~ Their action can readily be overcome by an anticholin(cid:173)
`esterase drug. Drugs which interfere with central cholinergic
`transmission are numerous, anticholinergic, atropine-like drugs
`including antiparkinson drugs, tricyclic antidepressants, neuro(cid:173)
`leptics, opiate analgesics, benzodiazepines and some types of
`general anaestnetics. So far the only agent that has proved to be
`of any value in reversing the efFects of the latter group of
`drugs is phYSJstigmine. In all reported cases of drug overdose or
`lack of recov-=fY when the agent was used peri-operatively, physo-
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`stigmine is usually administered parenterally, and administration
`is repeated every'20-30 minutes as required.
`
`Chronic treatment with neuroleptics often results in tardive dys(cid:173)
`kinesias. The widespread use of agents having anticholinesterase
`activity for the treatment of schizophrenia makes this side
`effect an ever increasing possibility. Physostigmine injected
`intravenously produces a significant but short lived improvement
`in a proportion of patients.
`
`A number of pathological and degenerative diseases has also been
`shown to be associated with a reduction or loss of cholinergic
`transmission. This includes myasthenia gravis and Eaton Lambert
`syndrome in which there is an interference with neuromuscular
`transmission.
`
`A selective loss of choline acetyltransferase (the enzyme that
`synthesises acetylcholine) has been found in specific brain
`regions of patients with pre-senile dementia of the Alzheimer
`type. These include the frontal and temporal cortex, hippocampus,
`amygdala, caudate nucleus, substantia innominata. Degeneration of
`cholinergic neurons in some of these areas appears to be asso(cid:173)
`ciated with the aphasia, apraxia, agnosia and loss of short term
`memory that occurs in A1zheimer ' s disease. A similar type of
`dementia is also found in patients with Down's syndrome that
`survive to the age of 40 years and show similar cholinergic
`deficits. There is also a loss of cholinergic transmission in the
`caudate nucleus and putamen of patients with Huntingdon's
`chorea. Physostigmine injections have also been of some benefit
`in this condition. Treatment with a centrally acting anticholin(cid:173)
`esterase should also prove to be beneficial in Friedrich's
`atax i a.
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`There are two major classes of potent inhibitors of the enzyme
`cho 1 inesterase. The first group was mOdelled primari lyon the
`natural alkaloids physostigmine (a carbamate) and an inhibitor of
`cho 1 i nesterase, and d-tubocurarine, an antagoni st of acetyl(cid:173)
`choline. The second group consists of various organophosphorus
`compounds, such as diisopropylfluorophosphonate, paraxon etc. The
`vast majority of the compounds of both these series were designed
`primarily as insecticides. In the first group of carbamate deri(cid:173)
`vatives, almost all of the potent insecticides are monomethyl
`carbamates lacking a charged nitrogen function. This enables the
`molecule to penetrate rapidly the insect cuticle and fatty nerve
`sheath. The dimethyl derivatives are sl ightly less potent but are
`particularly toxic to houseflies and aphids. The monomethyl deri(cid:173)
`vatives tend to be unstable in solution and hydrolyse readily at
`physiological pH. This greatly limits their biological action in
`mammals and makes them less suitable as pharmaceutical or thera(cid:173)
`peutic agents.
`
`The organa-phosphorus group of compounds causes irreversible
`inhibition of cholinesterase and other serine containing enzymes,
`which, together with their high relative toxicity, virtually
`precludes their use in pharmaceutical preparations. The only
`exception is echothiopate, a quaternary ammonium organo(cid:173)
`phosphorus compound, employed in eye drops for the treatment of
`glaucoma.
`
`The synthetic anticholinesterase agents currently employed as
`pharmaceuticals all contain a charged nitrogen func~ion and can
`be broadly classified into 3 groups.
`
`1) Reversible i~hibitorswhich contain a charged nitrogen
`function attached to an aromatic ring, e.g. edrophonium.
`
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`118-6848
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`2) Dimethyl carbamates with an aromatic or heterocyclic ring
`containing a charged nitrogen, neostigmine, pyridostigmine.
`
`3) Bisquaternary structures, e.g. Demacarium, Ambenonium. These
`agents tend to be more selective inhibitors of acetylcholin(cid:173)
`esterase than butyrylcholinesterase, compared with the mono(cid:173)
`quaternary molecules.
`
`The pharmaceutical application of the quaternary anticholin(cid:173)
`esterase agents is limited because of their poor penetration
`through cell membranes. They are therefore used for actions
`outside the central nervous system, and are usually given par(cid:173)
`enterally, since they are not reliably absorbed from the gastro(cid:173)
`intestinal tract. Edrophonium, neostigmine and pyridostigmine and
`the bisquaternary analogues are used in anaesthetic practice for
`the reversal of the action of muscle relaxants. They are also
`used for the treatment of myasthenia gravis, and paralytic ileus.
`
`Physostigmine is the only potent anti-cholinesterase agent which
`has been used clinically to treat conditions in which an ele(cid:173)
`vation of brain acetylcholine activity is desired. These include,
`Alzheimer's disease, tardive dyskinesia, Down's syndrome and
`Huntingdon's chorea. Physostigmine is also used to reverse the
`effects of overdose of anticholinergic agents, anti-Parkinson
`drugs, benzodiazepines and opiate analgesics.
`
`Physostigmine is a natural alkaloid extracted from calabar beans
`and the seeds of the vine Physostigma venenosum and has the
`formula
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`o
`H", U
`N-C-
`CH/
`
`3
`
`There is a need to provide new carbamate derivatives which show
`greater chemical stabil ity than physosti gmine.
`
`Furthermore there is a need to provide new compounds which
`inhibit acetylcholinesterase in the brain for periods exceeding
`3 hours but not more than 12 hours after a single administration.
`
`There is also a need to provide new compounds which will be
`completely and reliably absorbed after oral administration.
`
`There is also a need to provide new compounds which will be
`relatively less toxic than physostigmine. This means that the
`therapeutic ratio, defined as
`
`dose to produce therapeutic effect
`
`dose to produce mortality in 50 % of animals
`
`should be significantly higher than those of physostigmine and
`that the incidence and severity of side effects should be less
`than those of physostigmine at therapeutic doses.
`
`There is also 1 need to provide new compounds which can be given
`orally or par~nterally to treat chronic conditions in which it is
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`desired to raise cholinergic activity in the central nervous
`system. These include, Alzheimer's disease, Down's syndrome,
`Huntingdon's chorea, Friedrich's ataxia.
`
`There is also a need to provide compounds that can be given par(cid:173)
`enterally at the end of operations, and anaesthetic procedures,
`to restore wakefulness, respiration and cardiovascular parameters
`to normal, after the use of anticholinergic, opiates, benzo(cid:173)
`diazepines, neuroleptics and general anaesthetics, thereby
`shortening the stay of patients in the recovery room.
`
`There is also a need to provide compounds that can be given
`together with narcotic analgesics to patients suffering from
`severe pain, e.g. traumatic, post-operative, or due to carcino(cid:173)
`matosis etc. in order to reduce the side effects (respiratory
`depression, somnolence, constipation and urinary retention)
`commonly encountered with narcotics, without impairing their
`analgesic potency.
`
`There is also a need to provide compounds that can be given to
`patients receiving antipsychotic drugs, which have developed
`tardive dyskinesias, in order to diminish or abolish the latter
`syndrome, without exascerbating the psychosis.
`
`According to the present invention it has now been surprisingly
`found that certain novel and known phenyl carbamates also inhibit
`acetylcholinesterase in the mammalian brain after administration
`to provide systemic activity, e.g. oral or parenteral admini(cid:173)
`stration.
`
`Thus according to the present invention there is now provided a
`pharmaceut;Cj1 co:nposition adapted to produce 'anticholinesterase
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`activity in the central nervous system of mammals comprising a
`compound of the general formula I
`
`I
`
`wherein
`Rl
`is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl,
`R2
`is hydrogen, methyl, ethyl or propyl, or
`Rl and R2
`together with the nitrogen to which they are attached
`.form a morpholino or piperidino radical,
`is hydrogen or lower alkyl,
`R3
`R4 and R5 are the same or different and each is a lower alkyl,
`and the dialkylaminoalkyl group is in the meta, ortho or para
`position,
`
`or a pharmacologically acceptable salt thereof and a physiologi(cid:173)
`cally acceptable carrier therefor. Hereinafter these compounds
`are called compounds of the invention.
`
`Especially preferred are pharmaceutical compositions having anti(cid:173)
`cholinesterase activity in the central nervous system of mammals,
`wherein the dialkylaminoalkyl group is in the meta position, and
`R4 and R5 are both methyl.
`
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`Certain compounds falling within the above formula have pre(cid:173)
`viously been described i.e. the m disubstituted compound in which
`Rl and R3 = Hand R2, R4 and RS = methyl which is known as
`Miotine(R) was claimed to be an insecticide and a myopic agent
`for use in eye drops. The m disubstituted compound in which Rl
`and R2 are methyl, R3 is Hand R4 and RS are methyl has been
`described as an insecticide. The p and 0 disubstituted deri(cid:173)
`vatives in which Rl and R3 = Hand R2, R4 and RS = CH3 have been
`shown to inhibit a preparation of liver cholinesterase. The m
`disubstituted derivative in which Rl = Hand R2, R3, R4 and RS =
`CH3 has also been shown to inhibit liver cholinesterase.
`
`The remaining compounds are believed to be novel and thus the
`present invention also provides novel phenyl carbamate'deriva(cid:173)
`tives of the general formula II
`
`I I
`
`wherein
`Rl
`is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl,
`R2
`is hydrogen, methyl~ ethyl or propyl, or
`Rl and R2
`together with the nitrogen to which they are attached
`form d morpnolino or piperidino radical,
`is hydrogen or lower alkyl,
`
`R3
`
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`R4andR5 are the same or different and each is a lower alkyl,
`and the dialkylaminoalkyl group is in the meta, ortho or para
`position,
`
`and pharmacologically acceptable salts thereof, provided that for
`compounds wherein R4 and RS are both methyl and having the
`dialkylamino group in the meta position, when R2 is methyl and R3
`is hydrogen, Rlis neither hydrogen nor methyl, and when R2 and
`R3 are methyl, Rl is not hydrogen, and for compounds wherein R4
`and RS are both methyl and having the di alkyl amino group in the
`ortho or para position when Rl and R3 are both hydrogen R2 is not
`methyl.
`
`Preferred compounds of the above formula are N-ethyl-3-[l-(di(cid:173)
`methylamino)ethyl]phenyl carbamate, N-propyl-3[1-(dimethylamino)(cid:173)
`ethyl]phenyl carbamate, N-allyl-3-[1-(dimethylamino)ethyl]phenyl
`carbamate, N-ethyl, N-methyl-3[1-( dimethyl amino)ethyl ]phenyl
`carbamate, N,N-diethyl-3[1-(dimethylamino)ethyl]phenyl carbamate,
`N-butyl-3-[1-(dimethylamino)ethyl]phenyl carbamate, N-methyl,
`N-propyl-3[1-(dimethylamino)ethyl]phenyl carbamate and N-ethyl,
`N-methyl-3[1-(dimethylamino)isopropyl]phenyl carbamate.
`
`As indicated, the invention also includes the pharmacologically
`acceptable salts of these compounds such as the acetate, salicy(cid:173)
`late, fumarate, phosphate, sulphate, maleate, succinate, Citrate,
`tartrate, propionate and butyrate sal ts thereof.
`
`The compounds of formula I can be prepared by amidating a
`compound of for~ula II
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`II
`
`wherein R3, R4 and RS are as defined above.
`
`The process can be effected in conventional manner, e.g. by
`reacting the compound of formula II with an appropriate iso(cid:173)
`cyanate if a compound wherein Rl is hydrogen is desired, or with
`an appropriate carbamoyl halogenide, e.g. as described below in
`processes A and B.
`
`PROCESS A:
`
`PROCESS B:
`
`o
`II
`.... R
`NaH n ' Z
`----... U· ~3
`"CH
`1
`C'-N
`~H, 'CH 3
`
`6l0 - C- N'R'
`
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`PROCESS A:
`
`A stirred suspension of a-m-Hydroxyphenylethyldimethylamine or
`a-m-hydroxyphenylisopropyldimethylamine in benzene (0.2 -
`0.3 g/ml) is treated with 2.5 - 3 fold molar excess of the iso(cid:173)
`cyanate. After stirring for 15 -24 hours at ambient temperature
`the reaction mixture is connected to a rotovaporator (20 mm Hg).
`The residue obtained is dissolved in dry ether (25 ml) and the
`solution, which is ice cooled, is saturated with dry HCl (g). The
`formed precipitate (the anticipated carbamate) is filtered off,
`washed with dry ether (25 ml) and dried to constant weight in a
`dessicator over KOH pellets under high vacuum (0.1 urn Hg).
`
`PROCESS B:
`
`A solution of ,::t-m-hydroxyphenylethyldimethylamine or a-m-hydroxy(cid:173)
`phenylisopropyldimethylamine in dry acetonitrile (0.1 - 0.5 M) is
`reacted with 50 - 70 % molar excess of the corresponding carba(cid:173)
`moyl chloride in the presence of 200 % molar excess of NaH dis(cid:173)
`persion (50 - 80 % in mineral oil). The reaction mixture is left
`to stir at ambient temperature for 15 - 24 nours. Removal of the
`acetonitrile under reduced pressure (20 mm Hg) is followed by the
`addition of water (10 - 25 m1). The pH of the aqueous solution is
`adj usted to pH = 11 by the add it i on of the appropri ate amount of
`NaOH 0.1 N followed by extraction with ether (3 x 25 ml). The
`combined organic phases are washed with brine (25 ml) dried over
`MgS04 anhydride which is then filtered off. The ice cooled
`etheral filtrate is saturated with a stream of HCl (g) resulting
`in the fonndtion of a heavy precipitate (the anticipated carba(cid:173)
`mate)·which is collected by filtration, washed with dry ether
`(20 ml) and dr1ed to constant weight in a desiccator under high
`vacuum (0.1 mm Hg) over KOH pellets.
`
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`The compounds of the invention e.g. in free form or salt form can
`be utilized by formulating one or more of them in compositions
`such as tablets, capsules or elixirs for oral administration or
`in sterile solutions or suspensions for parenteral admini(cid:173)
`stration. A compound or mixture of compounds of formula (1) or
`physiologically acceptable salt(s) thereof is compounded with a
`physiologically acceptable vehicle, carrier, eXCipient, binder,
`preservative, stabilizer, flavor, etc., in a unit dosage form as
`called for by accepted pharmaceutical practice. The amount of
`active substance in these compositions or preparations is such
`that a suitable dosage is obtained.
`
`Illustrative of the adjuvants which may be incorporated in
`tablets, capsules and the like are the following: a binder such
`as gum tragacanth, acacia, corn starch or gelatin; an excipient
`such as dicalcium phosphate; a disintegrating agent such as corn
`starch, potato starch, alginic acid and the like; a lubricant
`such as mangnesium stearate; a sweetening agent such as sucrose,
`lactose or saccarin; a flavoring agent such as peppermint, oil of
`wintergreen or cherry. When the dosage unit form is a capsule, it
`may contain in addition to materials of the above type a liquid
`carrier such as a fatty oil. Various other mterials may be
`present as coatings or to otherwise modify the physical form of
`the dosage unit. For instance, tablets may be coated with
`shellac, sugar or both. A syrup or elixir may contain the active
`compound, sucrose as a sweetening agent, methyl and propyl
`parabens as preservatives, a dye and a flavoring such as cherry
`or orange flavour.
`
`Sterile compositions for injection can be formulated according to
`conventional pharmaceutical practice by dissolving or suspending
`the active substance in a vehicle such as water for injection.
`
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`Buffers, preservatives, antioxidants and the like can be incorpo(cid:173)
`rated as· required.
`
`Preferred antioxidants for use with the compounds of the present
`invention include sodium metabisulphite and ascorbic acid.
`
`While the invention will now be described in connection with
`certai n preferred embodiments in the fo 11 owi ng examples, it wi 11
`be understood that it is not intended to limit the invention to
`these particular embodiments. On the contrary, it is intended to
`cover all alternatives, modifications and equivalents as may be
`included within the scope of the invention as defined by the
`appended claims. Thus, the following examples which include
`preferred embodiments will serve to illustrate the practice of
`this invention, it being understood that the particulars
`descri bed are by way of example and for purposes of illustrative
`discussion of preferred embodiments of the present invention only
`and are presented in the cause of providing what is believed to
`be the most useful and readily understood description of proce(cid:173)
`dures as well as of the principles and conceptual aspects of the
`invention.
`
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`EXAf~PLE 1
`
`0.5 9 (3.03 mmole) of a-m-hydroxyphenylethyldimethylamine are
`dissolved in 15 ml of dry acetonitrile and 0.70 9 (5.2 mmole) of
`diethylcarbamylchloride are added to the mixture with stirring.
`This is followed by NaH 150 mg (50 %) of dispersion. The reaction
`mixture is stirred overnight at 25 - 30 0 C. Removal of acetoni(cid:173)
`trile under reduced pressure is followed by addition of water
`(10 ml) and adjustment of the pH to 11. The product is extracted
`in ether, which is washed by brine, dried over MgS04 and fil(cid:173)
`tered. Upon addition of Hel (g) precipitation occurs immediately,
`the product is filtered off, washed by dry ether and dried in a
`desiccator under high vacuum over KOH pellets.
`
`The carbamate is obtained as a white powder 640 mg (80 %)
`mp. 137 - 138 0 and identified as N,N-diethyl-3-[1-(dimethyl(cid:173)
`amino)ethyl ]phenyl carbamate, having the formul a
`
`EXAMPLE 2
`
`0.75 9 (4.55 mmol) of a-m-hydroxyphenylethyldimethylamine are
`suspended in benzene (3 ml) and 0.898 9 of ethylisocyanate are
`added to the ;ni xture with st; rri ng. After st i rri ng 12 hours at
`room temper1ture the solvent is removed under reduced pressure.
`
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`The residue obtained was dissolved in dry ether. Introduction of
`dry HCl gas into the reaction mixture causes a heavy precipi(cid:173)
`tation. The product is filtered off, washed with ether and dried
`in a desiccator over KOH pellets. The carbamate is obtained as a
`white powder 800 mg (75 %) mp. 177 - 179 0 C and identified as
`N-ethyl-3[1-(dimethylamino)ethyl]phenyl carbamate having the
`formula
`
`O-CO-NH-Et
`
`~CH-N(Me)
`
`'1
`
`,
`CH 1
`
`The compounds of the present invention are useful as pharmaceuti(cid:173)
`cals. In particular they show the following activities in vitro
`and in vivo in the tests specified below.
`
`The values are correct when taken in comparison with the standard
`drug physostigmine.
`
`IN VITRO EXPERIMENTS:
`
`Tests for anticholinesterase activity
`
`A solubilized preparation of acetylcholinesterase was prepared
`from mouse · .. mole brain (minus cerebellum). The brain was homo(cid:173)
`genized with (100 mg/ml) phosphate buffer; pH 8.0, centrifuged,
`the supernatant discarded, and the pellet mixed 'lfith a similar
`volume as above of buffer pH. 8.0 plus 1 % Triton; mixed, centri(cid:173)
`fuged and tn~ ~~~~rnatant which contained most of the solubilized
`enzyme, was j5~d for the subsequent determinations of anti(cid:173)
`Cholinesterase activity.
`
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`The activity of the enzyme (rate of hydrolysis of s~bstrate,
`acetylthiocholine) was measured using at least 4 different con(cid:173)
`centrations of substrate, and at least 3 different concentrations
`of each inhibitor. The enzyme was incubated with inhibitor for
`periods ranging for 2 - 180 mins. at 37 0 C, substrate was then
`added, and its rate of hydrolysis measured by the spectrophoto(cid:173)
`metric method of Ellman et al. (1961).
`
`The molar concentration of each agent that inhibited the activity
`of the enzyme by 50 % (IC50) at the peak time of activity (15 -
`50 min) was calculated from this data and recorded in Table 1
`herei nafter. The compounds in general produce a significant
`iahibition from about 10-5 to about 10-8 molar.
`
`IN VIVO EXPERI~ENTS:
`
`a) Assessm~nt of acetylcholinesterase inhibition
`
`The effect of each compound on brain acetylcholinesterase
`in vivo was measured, after subcutaneous or oral admini(cid:173)
`stration to mice. Animals were sacrificed, at different times
`ranging from 0.25 - 8 hours after drug administration. The
`brain was rapidly removed, and the enzyme acetylcholin(cid:173)
`esterase extracted and solubilized with 0.1 % Triton, and its
`ability to hydrolyse acetylthiocholine assessed as described
`above (in vitro experiments), in comparison with the enzyme
`removed from mi ce injected with normal sal i ne. The compounds
`_have in general a potency from about 2 to about 90 %
`that of physiostigmine.
`
`Assessment of acute toxicity
`
`Mice were given one of at least three different doses
`of each compound, orally or subcutaneously, a minimum of
`10 mice allotted to each dose. The number of animals
`which died at
`
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`o 193 926
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`118-6848
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`each dose within 3 hours was determined. From these data, the
`LDSO (dose in mg/kg which was lethal to 50 % of the mice) was
`computed.
`
`This experiment was repeated after the animal s had been pre(cid:173)
`treated with atropine sulphate, which blocks both peripheral
`and central muscarinic receptors. The data from these experi(cid:173)
`ments enabled the assessment of the relative degrees of toxi(cid:173)
`city of the carbamates which result from excessive activation
`of muscarinic receptors, and from respiratory muscle para(cid:173)
`lysis, which is insensitive to this blocking agent.
`
`The incidence and degree of side effects was noted for each
`dose of drug, starting with the lowest that caused any
`significant (> 20 %) inhibition of whole brai.n acetylchol in(cid:173)
`esterase.
`
`c) Antagonism of the somnolent and respiratory depressant
`effects of opiates
`
`Different doses of the carbamate compounds were injected
`intravenously with morphine in rabbits. Respiration rate,
`arterial blood gas tensions and pH were monitored conti(cid:173)
`nuous 1 y before and after drug admi n i str dt i on for 4 -
`5 hours. In another series of experiments the effect of the
`anticholinesterase drugs was assessed on the analgesic effect
`of opiates in rabbits after application of a nociceptive
`stimulus, i.e. electrical stimulation of the sciatic nerve.
`
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`118-6848
`
`o 193 926
`
`All specific examples of formula I'mentioned hereinbefore, e.g.
`on specification page 10 and after especially tables 1 to 3,
`are prepared in analogous manner to example 1, when R1 and R2
`are each other than hydrogen, and example 2, when one of R1
`and R2 is hydrogen. They are thus obtained as hydrochloride
`salts (except where otherwise specified). The specific compounds
`have meta-substitution.
`
`Table 1
`
`In vitro activity on solubilized mouse brain enzyme
`-
`R1
`
`Compound
`(R4=RS=CH3)
`
`R2
`
`R3
`
`1CSO(M)
`
`Physiostigmine H
`(Sal icyl ate)
`
`CH3
`
`H 1.1x10-S
`
`Time of .,>eak
`activity
`(mins)
`
`30
`
`30
`
`120
`
`120
`
`120
`
`120
`
`120
`
`120
`
`120
`
`90
`
`30
`
`30
`
`60
`
`Miotine HCl
`
`RA6 HCl
`
`RA1S HCl
`
`RAI4 HCl
`
`RA13 HCl
`
`RAS HCl
`
`RAI2
`
`RAIO HCl
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`H
`
`CH3 CH3
`
`RA7 HCl
`
`RAS HCl
`
`CH3 C2HS
`C2HS C2HS
`morpholino
`RAU HCl
`RA4 H~~ _____ ~tOPYl
`
`C4H9 n-butyl
`
`cyclohexyl
`
`H 1.3x.10-S
`CH3
`H 4.0x10-7
`C2HS
`C3H7 n-propyl H 1.1x10-7
`H 4.3xI0-7
`C3HS allyl
`C3H7 isopropyl H 1.2x10-S
`H 7.6x10"S
`H 9.3xlO-S
`H 2.7x10-8
`H 1.3x10-6
`H 3.Sx10-5
`H > 2x10 .. 5
`H 1.7x10-6
`-... -- ----
`
`Melting points of compounds (all in hydrochloride fOrl!l except for
`RA12 , which is in free base form as it precipitated from reaction
`mixture before addition of hydrogen chloride), in degreescentigrade:'
`RA6 167-170, RA15 141-143, RA14 147-152, RA13 146-148, RAS 158-162,
`
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`0193 926
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`RA12 75-77, RA10 145, RA7 135-136, RAa 137-13a, RAl1 amorphous,
`RA4 148-149. Compound RAl1 has an Rf value of 0.59 in a system
`~f 95 parts of ethyl acetate and 5 parts of 33 % (w/w) dimethyl(cid:173)
`amine in ethanol.
`
`Table 2
`
`Anticholinesterase activity of compounds in mouse brain
`compared to that of physostigmine
`
`Compound Relative potency Relative potency % cholinesterase
`to physostigmine
`to physostigmine
`inhibition
`after subcut.
`after oral
`3 hours after
`administration
`(s.c. )
`s.c.
`administration
`administration
`
`--~--
`
`Physo-
`stigmine
`
`Miotine
`
`RA6
`
`RA15
`
`RA14
`
`RAl3
`
`RA5
`
`RA12
`
`RAIO
`
`RA7
`
`RAa
`
`RA4
`
`100
`
`100
`
`11
`
`33
`
`15
`
`2
`
`36
`
`13
`
`81
`
`25
`
`2
`
`13
`
`100
`
`300
`
`19
`
`32
`
`22
`
`5
`
`29
`
`17
`
`92
`
`57
`
`5
`
`29
`
`a
`
`5
`
`35
`
`37
`
`35
`
`30
`
`37
`
`7
`
`41
`
`32
`
`25
`
`----------
`
`'-
`
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`0193 926
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`Tab 1 e 3
`
`Acute toxicity of carbamates in mice
`
`---
`- -
`Degree of*
`LD50
`protection
`~moles/kg
`afforded by
`s.C.
`pretreatment
`with atrQ;:li ne
`-
`
`._--
`Th.erapeut i c LD50 oral
`ratio
`LD50/EDSO
`s.c.
`
`LD50 s.c.
`--
`
`3.3
`
`4.9
`
`11. 9
`
`11.1
`
`11.5
`
`1.6
`
`7.6
`
`5.8
`
`12.7
`
`12.4
`> 10.0
`10.0
`
`4.1
`
`1.2
`
`2.1
`
`4.5
`
`4.4
`
`1.1
`
`S.O
`
`3.6
`
`9.7
`
`1.2
`-
`1.7
`
`3.0
`
`2.4
`
`2.6
`
`4.1
`
`B.O
`
`4.5
`
`5.B
`
`3.B
`
`S.O
`
`10.4
`
`.9
`
`- 4
`
`3.0
`
`4.S
`
`96
`
`31
`
`69
`
`65
`
`19
`
`42
`
`14
`
`46
`> 568
`
`72
`
`Compound
`
`----
`Physostigmine
`
`Miotine
`
`RA6
`
`RA15
`
`RA14
`
`RA13
`
`RA5
`
`RA12
`
`RAlO
`
`RA7
`
`RAB
`
`RA4
`
`*Ratio of LDSO after pretreatment with atropine sulphate 5 mg/kg
`to LDSO of drug alone.
`
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`
`The data in Tables land 2 demonstrate that somewhat larger
`quantities are required of all the drugs of the RA series than of
`physostigmine to inhibit the enzyme acetylcholinesterase.
`However, a comparison of the data in Table 1 with that in
`Table 2, shows that compounds RAS, RA6, RAlS, RAl4, RAlO, RA7 and
`RAg are all relatively more active in vivo compared to physo(cid:173)
`stigmine than one would expect from the in vitro data. This
`greater in vivo potency is particularly marked when the drugs are
`administered orally. This relatively greater in vivo activity may
`be due to:
`
`a) greater chemical stability
`
`b) a slower metabolic degradation orland excretion
`
`c) a higher lipid solubility, enabling a greater proportion of
`the drug to gain access to the enzyme in the central nervous
`system
`
`d) more efficient absorption from gastro-intesti