United States Patent [191
`Rosin et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,948,807
`Aug. 14, 1990
`
`[54] PHENYL CARBAMATES
`
`[75]
`
`Inventors: Marta W. Rosin; Michael Chorev;
`Zeev Tashma, all of Jerusalem, Israel
`
`[73] Assignee: Proterra AG, Zug, Switzerland
`
`[21] Appl. No.: 320,700
`
`[22] Filed:
`
`Mar.8, 1989
`
`Related U.S. Application Data
`[63] Continuation of Ser. No. 185,451, Apr. 25, 1988, aban(cid:173)
`doned, which is a continuation of Ser. No. 835,466,
`~ar. 3, 1986,abandoned.
`
`OTHER PUBLICATIONS
`Stedman, Biochemical Journal, 20 pp. 719-734 (1926).
`Wasserman, Proc. Natl. Acad. Sci., U.S.A., 79 pp.
`4810-4814 (1982).
`Weiden, J. Agr. Food Chern., 13 pp. 200-204 (1965).
`Berry, Biochem. Pharmacal., 20 pp. 3236-3238 (1971).
`Weinstock, Advances in Behavioral Biology, 29 pp.
`539-549 (1986).
`Lange, Haemostasis, 10 pp. 315-347 (1981).
`Meltzer, Entomol. Exp. App., 12 pp. 169-182 (1969).
`Primary Examiner-Michael L. Shippen
`Attorney, Agent, or Firm-Ribis, Graham, Verdon &
`Curtin
`
`Foreign Application Priority Data
`[30]
`~ar. 5, 1985 [IL]
`Israel ........................................ 74497
`
`[57]
`ABSTRACT
`Phenyl carbamates of the general formula
`
`Int. Cl.s ................... C07C 125/067; A61K 31/27
`[51]
`[52] u.s. Cl •.................................... 514/484; 514/330;
`514/487; 514/490; 514/237.5; 544/162;
`546/226; 560/32; 560/115; 560/136
`[58] Field of Search ....................... 560/115, 163, 136;
`514/484, 490, 487
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`1,905,990 4/1933 Aeschlimann ........................ 560/32
`2,208,485 7/1940 Aeschlimann ...................... 560/136
`2,362,508 11/1944 Stevens ............................... 560/136
`2,493,710 1/1950 Aeschlimann ...................... 560/136
`
`FOREIGN PATENT DOCUMENTS
`1037753 7/1956 Fed. Rep. of Germany ...... 560/136
`
`w /Rt
`o-c-N'-
`
`R2
`
`R4
`
`R3
`/
`I
`e-N
`I
`'-.
`CH3
`Rs
`
`wherein Rt toRs are as defmed in the claims, are useful
`as pharmaceuticals.
`
`4 Claims, No Drawings
`
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`

`1
`
`PHENYL CARBAMATES
`
`4,948,807
`
`2
`Chronic treatment with neuroleptics often results in
`tardive dyskinesias; The widespread use of agents hav(cid:173)
`ing anticholinesterase activity for the treatment of
`schizophrenia makes this side effect an ever increasing
`5 possibility. Physostigmine injected intravenously pro(cid:173)
`duces 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 myasthe(cid:173)
`nia gravis and Eaton Lambert syndrome in which there
`is an interference with neuromuscular transmission.
`A selective loss of choline acetyltransferase (the en(cid:173)
`zyme that synthesises acetylcholine) has been found in
`specific brain regions of patients with pre-senile demen(cid:173)
`tia of the Alzheimer type. These include the frontal and
`temporal cortex, hippocampus, amygdala, caudate nu(cid:173)
`cleus, substantia innominata. Degeneration of choliner(cid:173)
`gic neurons in some of these areas appears to be associ(cid:173)
`ated with the aphasia, apraxia, agnosia and loss of short
`term memory that occurs in Alzheimer'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
`anticholinesterase should also prove to be beneficial in
`Friedrich's ataxia.
`There are two major classes of potent inhibitors of
`the enzyme cholinesterase. The first group was mod(cid:173)
`elled primarily on the natural alkaloids physostigmine (a
`carbamate) and an inhibitor of cholinesterase, and d(cid:173)
`tubocurarine, an antagonist of acetylcholine. The sec(cid:173)
`ond group consists of various organophosphorus com-
`pounds, 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 derivatives, 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 slightly less potent
`but are particularly toxic to houseflies and aphids. The
`monomethyl derivatives 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 therapeutic
`agents.
`The organo-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 phar(cid:173)
`maceutical preparations. The only exception is echothi(cid:173)
`opate, a quaternary ammonium organophosphorus
`compound, employed in eye drops for the treatment of
`glaucoma.
`The synthetic anticholinesterase agents currently
`employed as pharmaceuticals all contain a charged
`nitrogen function and can be broadly classified into 3
`groups.
`(1) Reversible inhibitors which contain a charged
`nitrogen function attached to an aromatic ring, e.g.
`edrophonium.
`(2) Dimethyl carbamates with an aromatic or hetero(cid:173)
`cyclic ring containing a charged nitrogen, neostig(cid:173)
`mine, pyridostigmine.
`
`30
`
`25
`
`This application is a continuation of application Ser.
`No. 185,451, filed on 04/25/88, entitled Phenyl Carba(cid:173)
`mates which in turn was a continuation of application
`Ser. No. 835,466 filed Mar. 3, 1986, both now aban(cid:173)
`doned.
`The present invention relates to novel phenyl carba(cid:173)
`mates which are useful as pharmaceutical compositions. 10
`The invention further relates to pharmaceutical compo(cid:173)
`sitions having anticholinesterase activity.
`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, degener- 15
`ation etc. or as induced by drugs or toxins, causes
`marked changes in the function of the organism. Ace(cid:173)
`tylcholine itself has an extremely short half life, since it
`is rapidly hydrolysed at its site of action and in plasma
`by specific cholinesterase enzymes. prugs that inhibit 20
`acetylcholinesterase, markedly increase and prolong
`the action of acetylcholine, thereby enhancing choliner(cid:173)
`gic transmission. Three such agents are used clinically,
`i.e., physostigmine, a naturally occurring alkaloid, and
`two synthetic analogues, neostigmine and pyridostig(cid:173)
`mine. The latter two agents are strongly ionised at phys(cid:173)
`iological pH and therefore are only poorly absorbed
`from the gastro-intestinal tract, and do not penetrate the
`central nervous system to any significant extent. Physo-
`stigmine is absorbed after oral administration and
`readily enters the brain. As a therapeutic 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 35
`(20-40 mins) thereby necessitating frequent administra(cid:173)
`tion. The latter is of particular importance when the
`drug is to be administered chronically. It has a low
`therapeutic ratio, a value of 3-5 being reported in the
`majority of studies in laboratory animals, and a small 40
`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(cid:173)
`intestinal tract, this is reported to be iiTegular and un(cid:173)
`predictable, and therefore it is usually preferred to ad- 45
`minister the drug parenterally. This is a serious draw(cid:173)
`back if it is to be used chronically on an outpatient basis.
`There are a number of clinical and pathological con(cid:173)
`ditions which are associated with cholinergic under(cid:173)
`activity which can be improved by the administration of 50
`an anticholinesterase agent. These include reduction in
`cholinergic transmission induced by a variety of exoge(cid:173)
`nous substances acting in the peripheral, or central ner(cid:173)
`vous system. Peripherally acting agents are gallamine,
`d-tubocurarine and pancuronium, which are used as 55
`muscle relaxants. Their action can readily be overcome
`by an anticholinesterase drug. Drugs which interfere
`with central cholinergic transmission are numerous,
`anticholinergic, atropine-like drugs including antipar(cid:173)
`kinson drugs, tricyclic antidepressants, neuroleptics, 60
`opiate analgesics, benzodiazepines and some types of
`general anaesthetics. So far the only agent that has
`proved to be of any value in reversing the effects of the
`latter group of drugs is physostigmine. In all reported
`cases of drug overdose or lack of recovery when the 65
`agent was used peri-operatively, physostigmine is usu(cid:173)
`ally administered parenterally, and administration is
`repeated every 20-30 minutes as required.
`
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`

`4,948,807
`
`3
`(3) Bisquatemary structures, e.g. Demacarium, Am(cid:173)
`benonium. These agents tend to be more selective
`inhibitors of acetylcholinesterase than butyryl(cid:173)
`cholinesterase, compared with the monoquater(cid:173)
`nary molecules.
`The pharmaceutical application of the quaternary
`anticholinesterase agents is limited because of their poor
`penetration through cell membranes. They are there(cid:173)
`fore used for actions outside the central nervous system,
`and are usually given parenterally, since they are not 10
`reliably absorbed from the gastrointestinal tract. Edro(cid:173)
`phonium, neostigmine and pyridostigmine and the his(cid:173)
`quaternary analogues are used in anesthetic practice for
`the reversal of the action of muscle relaxants. They are
`also used for the treatment of myasthenia gravis, and 15
`paralytic ileus.
`Physostigmine is the only potent anti-cholinesterase
`agent which has been used clinically to treat conditions
`in which an elevation of brain acetylcholine activity is
`desired. These include, Alzheimer's disease, tardive 20
`dyskinesia, Down's syndrome and Huntingdon's cho(cid:173)
`rea. 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 25
`calabar beans and the seeds of the vine Physostigma
`venenosum and has the formula
`
`4
`tics 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
`5 suffering from severe pain, e.g. traumatic, post-opera(cid:173)
`tive, or due to carcinomatosis etc. in order to reduce the
`side effects (respiratory depression, somnolence, consti(cid:173)
`pation and urinary retention) commonly encountered
`with narcotics, without impairing their analgesic po(cid:173)
`tency.
`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 exas(cid:173)
`cerbating 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 mam(cid:173)
`malian brain after administration to provide systemic
`activity, e.g. oral or parenteral administration.
`Thus according to the present invention there is now
`provided a pharmaceutical composition adapted to pro(cid:173)
`duce anticholinesterase activity in the central nervous
`system of mammals comprising a compound of the
`general formula I
`
`30
`
`35
`
`w /Rt
`o-c-N"
`R2
`
`~3
`/R.!
`C-N
`I
`"
`CH3
`
`Rs
`
`There is a need to provide new carbamate derivatives
`which show greater chemical stability than physostig(cid:173)
`mine.
`Furthermore there is a need to provide new com- 40
`pounds 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 45
`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, defmed as
`
`dose to produce therapeutic effect
`dose to produce mortality in 50% of animals
`
`should be significantly higher than those of physostig(cid:173)
`mine and that the incidence and severity of side effects 55
`should be less than those of physostigmine at therapeu(cid:173)
`tic doses.
`There is also a need to provide new compounds
`which can be given orally or parenterally to treat
`chronic conditions in which it is desired to raise cholin- 60
`ergic activity in the central nervous system. These in(cid:173)
`clude, Alzheimer's disease, Down's syndrome, Hun(cid:173)
`tingdon's chorea, Friedrich's ataxia.
`There is also a need to provide compounds that can
`be given parenterally at the end of operations, and anes- 65
`thetic procedures, to restore wakefulness, respiration
`and cardiovascular parameters to normal, after the use
`of anticholinergic, opiates, benzodiazepines, neurolep-
`
`wherein
`R1 is hydrogen, lower alkyl, cyclohexyl, allyl or ben(cid:173)
`zyl,
`Rz is hydrogen, methyl, ethyl or propyl, or
`R1 and Rz together with the nitrogen to which they
`are attached form a morpholino or piperidine radi(cid:173)
`cal,
`R3 is hydrogen or lower alkyl,
`R4 and Rs are the same dr different and each is a
`lower alkyl, and the dialkylaminoalkyl group is in
`the meta, ortho or para position,
`so or a pharmacologically acceptable salt thereof and a
`physiologically acceptable carrier therefor. Hereinafter
`these compounds are called compounds of the inven(cid:173)
`tion.
`Especially preferred are pharmaceutical composi(cid:173)
`tions having anticholinesterase activity in the central
`nervous system of mammals, wherein the dialkylamino(cid:173)
`alkyl group is in the meta position, and R4 and Rs are
`both methyl.
`Certain compounds falling within the above formula
`have previously been described i.e. the m disubstituted
`compound in which R1 and R3=H and Rz, 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 R1 and
`Rz are methyl, R3 is H and R4 and Rs are methyl has
`been described as an insecticide. The p and o disubsti-
`tuted derivatives in which R1 and R3=H and Rz, R4and
`Rs=CH3 have been shown to inhibit a preparation of
`
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`

`

`4,948,807
`
`5
`liver cholinesterase. The m disubstituted derivative in
`which Rr =H and R2, R3, ~ 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 5
`phenyl carbamate derivatives of the general formula I'
`
`6
`wherein R3, ~ 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 isocyanate if a compound wherein Rr is
`hydrogen is desired, or with an appropriate carbamoyl
`halogenide, e.g. as described below in processes A and
`B.
`
`I'
`
`10
`
`PROCESS A
`
`~ /Rt
`
`o-c-N " Rz
`f3
`/14
`C-N
`I
`'-.
`CH3
`Rs
`
`20
`
`30
`
`wherein
`Rr is hydrogen, lower alkyl, cyclohexyl, allyl or ben(cid:173)
`zyl,
`R2 is hydrogen, methyl, ethyl or propyl, or
`Rr and Rz together with the nitrogen to which they
`are attached form a morpholino or piperidino radi- 25
`cal,
`R3 is hydrogen or lower alkyl,
`~ and Rs 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, pro(cid:173)
`vided that for compounds wherein ~ and Rs are both
`methyl and having the dialkylamino group in the meta
`position, when R2 is methyl and R3 is hydrogen, Rr is
`neither hydrogen nor methyl, and when R2 and R3 are 35
`methyl, Rr is not hydrogen, and for compounds
`wherein ~ and Rs are both methyl and having the
`dialkylamino group in the ortho or para position when
`Rr and R3 are both hydrogen R2 is not methyl.
`Preferred compounds of the above formula are N- 40
`ethyl-3-[1-(dimethylamino)ethyl]phenyl carbamate, N(cid:173)
`propyl-3[1-( dimethylamino )ethyl]phenyl
`carbamate,
`N-allyl-3-[1-(dimethylamino)ethyl]phenyl
`carbamate,
`N-ethyl, N-methyl-3[1-(dimethylamino)ethyl]phenyl
`carbamate, N,N-diethyl-3[1-(dimethylamino)ethyl]phe- 45
`nyl carbamate, N-butyl-3-[1-(dimethylamino)ethyl]phe(cid:173)
`nyl
`carbamate, N-methyl, N-propyl-3[1-(dime(cid:173)
`thylamino)ethyl]phenyl carbamate and N-ethyl, N-
`methyl-3[1-( dimethylamino )isopropyl]phenyl
`carba-
`mate.
`As indicated, the invention also includes the pharma(cid:173)
`cologically acceptable salts of these compounds such as
`the acetate, salicylate, fumarate, phosphate, sulphate,
`maleate, succinate, citrate, tartrate, propionate and bu-
`tyrate salts thereof.
`The compounds of formula I can be prepared by
`ari:ridating a compound of formula II
`
`50
`
`55
`
`II
`
`60
`
`@OH
`
`PROCESS B
`
`Rt
`
`CH3 + "N-CO-CI
`/
`Rz
`
`R3
`/
`I
`e-N
`I
`'-.
`CH3
`CH3
`
`NaH >
`
`PROCESS A
`A stirred suspension of a-m-Hydroxyphenylethyl(cid:173)
`dimethylamine or a-m-hydroxyphenylisopropyldime(cid:173)
`thylamine in benzene (0.2-0.3 g/ml) is treated with
`2.5-3 fold molar excess of the isocyanate. 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 mm Hg).
`
`OH
`
`©(f' /~
`
`C-N
`I
`'-.
`CH3
`Rs
`
`PROCESS B
`A solution of a-m-hydroxyphenylethyldimethyla(cid:173)
`mine or a-m-hydroxyphenylisopropyldimethylamine in
`65 dry acetonitrile (0.1-0.5M) is reacted with 50-70%
`molar excess of the corresponding carbamoyl chloride
`in the presence of200% molar excess ofNaH dispersion
`(50-80% in mineral oil). The reaction mixture is left to
`
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`

`4,948,807
`
`7
`stir at ambient temperature for 15-24 hours. Removal of
`the acetonitrile under reduced pressure (20 mm Hg) is
`followed by the addition of water (10-25 ml). The pH of
`the aqueous solution is adjusted to pH= 11 by the addi(cid:173)
`tion of the appropriate amount of NaOH 0.1N followed 5
`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 ftltered off. The ice
`cooled etheral ftltrate is saturated with a stream of HCI
`(g) resulting in the formation of a heavy precipitate (the 10
`anticipated carbamate) which is collected by ftltration,
`washed with dry ether (20 ml) and dried to constant
`weight in a desiccator under high vacuum (0.1 mm Hg)
`over KOH pellets.
`The compounds of the invention e.g. in free form or 15
`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 suspen(cid:173)
`sions for parenteral administration. A compound or
`mixture of compounds of formula (I) or physiologically 20
`acceptable salt(s) thereof is compounded with a physio(cid:173)
`logically 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 25
`or preparations is such that a suitable dosage is obtained.
`Illustrative of the adjuvants which may be incorpo(cid:173)
`rated 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 30
`disintegrating agent such as corn starch, potato starch,
`alginic acid and the like; a lubricant such as magnesium
`stearate; a sweetening agent such as sucrose, lactose or
`saccharin; a flavoring agent such as peppermint, oil of
`wintergreen or cherry. When the dosage Unit form is a 35
`capsule, it may contain in addition to materials of the
`above type a liquid carrier such as a fatty oil. Various
`other materials may be present as coatings or to other(cid:173)
`wise modify the physical form of the dosage unit. For
`instance, tablets may be coated with shellac, sugar or 40
`both. A syrup or elixir may contain the active com(cid:173)
`pound, sucrose as a sweetening agent, methyl and pro(cid:173)
`pyl parabens as preservatives, a dye and a flavoring
`such as cherry or orange flavour.
`Sterile compositions for injection can be formulated 45
`according to conventional pharmaceutical practice by
`dissolving or suspending the active substance in a vehi(cid:173)
`cle such as water for injection. Buffers, preservatives,
`antioxidants and the like can be incorporated as re-
`quired.
`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 connec(cid:173)
`tion with certain preferred embodiments in the follow- 55
`ing examples, it will be understood that it is not in(cid:173)
`tended to limit the invention to these particular embodi(cid:173)
`ments. On the contrary, it is intended to cover all alter(cid:173)
`natives, modifications and equivalents as may be in(cid:173)
`cluded within the scope of the invention as defined by 60
`the appended claims. Thus, the following examples
`which include preferred embodiments will serve to
`illustrate the practice of this invention, it being under(cid:173)
`stood that the particulars described are by way of exam(cid:173)
`ple and for purposes of illustrative discussion of pre- 65
`ferred 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
`
`50
`
`8
`of procedures as well as of the principles and concep(cid:173)
`tual aspects of the invention.
`
`EXAMPLE 1
`0.5 g (3.03 mmole) of a-m-hydroxyphenylethyldime(cid:173)
`thylamine are dissolved in 15 m1 of dry acetonitrile and
`0.70 g (5.2 mmole) of diethylcarbamoylchloride 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• 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 ftltered. Upon addition of HCl
`(g) precipitation occurs immediately, the product is
`ftltered off, washed by dry ether and dried in a desicca(cid:173)
`tor under high vacuum over KOH pellets.
`The carbamate is obtained as a white powder 640 mg
`(80%) mp. 137•-138• and identified as N,N-diethyl-3-[1-
`(dimethylamino)ethyl]phenyl carbamate, having the
`formula
`
`EXAMPLE2
`0.75 g (4.55 mmol) of a-m-hydroxyphenylethyldime(cid:173)
`thylamine are suspended in benzene (3 ml) and 0.898 g
`of ethylisocyanate are added to the mixture with stir(cid:173)
`ring. After stirring 12 hours at room temperature the
`solvent is removed under reduced pressure.
`The residue obtained was dissolved in dry ether.
`Introduction of dry HCl gas into the reaction mixture
`causes a heavy precipitation. 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• C. and identified as N(cid:173)
`ethyl-3[1-(dimethylamino)ethyl]phenyl carbamate hav(cid:173)
`ing the formula
`
`0-CO-NH-Et
`
`@CH-N(Moh
`
`I
`CH3
`
`The compounds of the present invention are useful as
`pharmaceuticals. In particular they show the following
`activities in vitro and in vivo in the tests specified be(cid:173)
`low.
`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 whole brain (minus cerebellum).
`The brain was homogenized with (100 mg/ml) phos-
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`4,948,807
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`10
`All specific examples of formula I' mentioned herein(cid:173)
`before, e.g. on specification page 10, and after especially
`Tables 1 to 3, are prepared in analogous manner to
`Example 1 when Rt and R2 are each other than hydro(cid:173)
`gen and Example 2 when one of Rt and R2 are hydro(cid:173)
`gen. They are thus obtained as hydrochloride salts (ex(cid:173)
`cept where otherwise specified). The specific com(cid:173)
`pounds have metal substitutions.
`TABLE 1
`In vitro activity on solubilized mouse brain enzyme
`
`Compound
`(R4 = Rs =
`CH3)
`Rt
`Physiostigmine H
`(Salicylate)
`Miotine HCI
`RA6HCI
`RAtsHCI
`RA14HCl
`RA13HCI
`RAsHCl
`RAt2
`RAtoHCl
`RA7HCl
`RA8 HC1
`RAttHCl
`RA4HC1
`
`Rz
`CH3
`
`RJ ICso(M)
`H 1.1 x w- 8
`H u x w- 8
`H
`CH3
`H 4.o x w- 7
`H
`CzHs
`C3H7 n-propyl H 1.1 x w-7
`H
`H 4.3 x w- 1
`H
`C3Hs allyl
`C3H7 isopropy H 1.2 x w- 5
`H
`H 7.6 x w- 8
`C4H9 n-butyl
`H
`H 9.3 x w- 8
`cyclohexyl
`H
`H 2.1 x w- 8
`CH3 CH3
`H 1.3 x w-6
`CH3 CzHs
`H 3.5 x w- 5
`CzHs CzHs
`H >2 x w- 5
`morpholino
`H 1.1 x w- 6
`CH3 propyl
`
`Time
`of peak
`activity
`(mins)
`30
`
`30
`120
`120
`120
`120
`120
`120
`120
`90
`30
`30
`60
`
`9
`phate buffer; pH 8.0, centrifuged, the supernatant dis(cid:173)
`carded, and the pellet mixed with a similar volume as
`above of buffer pH 8.0 plus 1% Triton; mixed, centri(cid:173)
`fuged and the supernatant which contained most of the
`solubilized enzyme, was used for the subsequent deter- 5
`minations of anticholinesterase activity.
`The activity of the enzyme (rate of hydrolysis of
`substrate, acetylthiocholine) was measured using at
`least 4 different concentrations of substrate, and at least
`3 different concentrations of each inhibitor. The en- 10
`zyme was incubated with inhibitor for periods ranging
`for 2-180 mins. at 37• C., substrate was then added, and
`its rate of hydrolysis measured by the spectrophotomet-
`ric method of Ellman et al. (1961).
`The molar concentration of each agent that inhibited 15
`the activity of the enzyme by 50% (IC5o) at the peak
`time of activity (15-60 min) was calculated from this
`data and recorded in Table 1 hereinafter. The com(cid:173)
`pounds in general produce a significant inhibition from
`about 10-5 to about 10-8 molar.
`
`20
`
`IN VIVO EXPERIMENTS
`(a) Assessment of acetylcholinesterase inhibition
`The effect of each compound on brain acetylcholin- 25
`esterase in vivo was measured, after subcutaneous or
`oral administration 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 acetylcholinesterase extracted and solubilized 30
`with 0.1% Triton, and its ability to hydrolyse acetylthi(cid:173)
`ocholine assessed as described above (in vitro experi(cid:173)
`ments), in comparison with the enzyme removed from
`mice injected with normal saline. The compounds have
`in general a potency of from about 2% to about 90% 35
`that of physostigmine.
`
`(b) Assessment of acute toxicity
`Mice were given one of at least three different doses
`of each compound, orally or subcutaneously, a mini- 40
`mum of 10 mice allotted to each dose. The number of
`animals which died at each dose within 3 hours was
`determined. From these data, the LD5o (dose in mg!kg
`which was lethal to 50% of the mice) was computed.
`This experiment was repeated after the animals had 45
`been pretreated with atropine sulphate, which blocks
`both peripheral and central muscarinic receptors. The
`data from these experiments enabled the assessment of
`the relative degrees of toxicity of the carbamates which
`result from excessive activation of muscarinic recep- 50
`tors, and from respiratory muscle paralysis, 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 55
`brain acetylcholinesterase.
`
`Melting points of compounds (all in the hydrochlo(cid:173)
`ride form except for RA12 which is in the free base form
`as it precipitated from the reaction mixture before addi(cid:173)
`tion of hydrogen chloride) are in degrees Centigrade:
`RA6 167-170; RAts 141-143; RAt4 147-152; RAt3
`146-148; RA5 158-162; RA12 75-77; RAw 145; RA1
`135-136;RA8137-138;RA11amorphous;RA4148-149.
`Compound RAu has an RF value of 0.59 in a system
`of 95 parts of ethyl acetate and 5 parts of 33% (w/w)
`dimethylamine in ethanol.
`TABLE2
`Anticholinesterase activity of compounds in mouse brain compared
`to that of physostigmine
`Relative potency
`Relative
`to physostigmine
`potency to
`physostigmine
`after subcut.
`(s.c.)
`after oral
`administration
`administration
`100
`100
`
`% cholinesterase
`inhibition
`3 hours after
`s.c.
`administration
`0
`
`100
`li
`33
`15
`2
`36
`13
`81
`25
`2
`13
`
`300
`19
`32
`22
`5
`29
`17
`92
`57
`5
`29
`
`5
`35
`37
`35
`
`30
`37
`7
`41
`32
`25
`
`Compound
`Physo(cid:173)
`stigmine
`Miotine
`RA6
`RAts
`RAt4
`RA13
`RAs
`RAt2
`RAto
`RA7
`RA8
`RA4
`
`(c) Antagonism of the somnolent and respiratory
`depressant effects of opiates
`TABLE 3
`Different doses of the carbamate compounds were 60
`Acute toxicit:t of carbamates in mice
`injected intravenously with morphine in rabbits Respi(cid:173)
`Degree of*
`Therapeutic
`protection
`ration rate, arterial blood gas tensions and pH were
`ratio
`afforded by
`LDso
`Com-
`monitored continuously before and after drug adminis-
`LDso oral
`JJ-moles/ pretreatment LDso/EDso
`pound
`tration for 4-5 hours. In another series of experiments
`kg S.C. with atropine
`LD5o s.c.
`S.C.
`the effect of the anticholinesterase drugs was assessed 65 - - - - - - - - - - - - - - - - - - - - -
`3.0
`3.0
`3.3
`4.1
`~~;~~e
`on the analgesic effect of opiates in rabbits after applica-
`tion of a nociceptive stimulus, i.e. electrical stimulation
`Miotine
`of the sciatic nerve.
`RA6
`
`4.5
`96
`
`2.4
`2.6
`
`4.9
`11.9
`
`1.2
`2.1
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`Com-
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`11
`TABLE 3-continued
`Acute toxicit;t of carbamates in mice
`Degree of*
`protection
`Therapeutic
`ratio
`afforded by
`LDso
`J.Lmoles/ pretreatment LDso/EDso
`kg S.C. with attopine
`s.c.
`31
`4.1
`69
`8.0
`4.5
`6S
`19
`5.8
`3.g
`42
`14
`5.0
`10.4
`46
`>S68
`72
`
`I !.I
`RA15
`11.5
`RA14
`1.6
`RA13
`7.6
`RAs
`5.g
`RA12
`12.7
`RAw
`12.4
`RA7
`>10.0
`RAg
`10.0
`4.9
`1.7
`RA4
`•Ratio of LDso after pretreatment with atropine sulphate 5 mg/kg to LDlo of drug
`alone.
`
`LDso oral
`LDso s.c.
`4.5
`4.4
`1.1
`5.0
`3.6
`9.7
`1.2
`
`12
`undesirable side effects such as defaecation, lachryma(cid:173)
`tion, fasciculations or tremor at the doses which inhibit
`the brain enzyme by 50%, while the former 3 side ef(cid:173)
`fects are clearly evident when physostigmine is given at
`5 the appropriate dose (EDso).
`The data in Table 3 show that atropine can afford
`considerably greater protection against the lethality of
`the derivatives RA4, s, 7, to, 13 and 14. This is particu(cid:173)
`larly important in the treatment of drug overdose since
`10 the respiratory muscle paralysis which is not affected by
`atropine and which is the cause of death induced by
`excess drug administration in the presence of atropine
`cannot be satisfactorily reversed by specific antidotes.
`The duration of significant brain enzyme inhibition
`15 ( > 30%) induced by physostigmine (ED so dose) is less
`than 2 hours. Compounds RA4. s, 6, 7, 3, 12. t4, ts all act
`for more than 3 hours at their respective ED so doses and
`RA6 and RA7 still causes significant inhibition (36%)
`after 7 hours. Since none of these drugs caused notice(cid:173)
`able side effects at the EDso doses, an even longer dura(cid:173)
`tion of action may be achieved by giving between 50
`and 100% larger doses. The longer duration of action is
`a distinct advantage, particularly if the drugs are to be
`administered chronically to subjects suffering from
`neurological and behavioural conditions associated
`with