US RE33906 E
A compound of formula (I): ##STR1## or a salt or solvate thereof in which R. CO is an acyl group containing a substituted or unsubstituted carbocyclic or heterocyclic aromatic ring, R1 and R2 are independently C1-6 alkyl or together form a C3-6 polymethylene or alkylene group, and p is 1, 2, 3 or 4, is useful for treating pain.
1. A compound of formula I: ##STR26## or a solvate or pharmaceutically acceptable salt thereof in which R has the formula (II):
--(CHR4)n --X--Ar--(R3)m (II)
n is 0, 1 or 2,
m is 0, 1 or 2,
X is a direct bond, or O, S or NR5, in which R5 is hydrogen or C1-6 alkyl,
Ar is phenyl or thienyl,
R3 is halogen, C1-6 alkoxy, cyano, --COOR4, C1-6 alkyl, or amino or, when m is 2, two R3 's form a C3-6 polymethylene group, and R4 is hydrogen or C1-6 alkyl,
R1 and R2 are .[.independently C1-6 alkyl or together form a C3-6 polymethylene or C3-6 alkenylene group.]. .Iadd.each methyl or together with the nitrogen atom to which they are attached form 1-pyrrolidinyl.Iaddend.; and
p is 1, 2, 3 or 4.
2. A compound according to claim 1, in which each of R1 and R2 is methyl.[., ethyl, propyl, butyl, pentyl or hexyl.]..
3. A compound according to claim 1, in which R1 and R2 together .Iadd.with the nitrogen atom to which they are attached .Iaddend.form .[.a propylene, butylene, pentylene or hexylene group.]. .Iadd.1-pyrrolidinyl.Iaddend.. .[.4. The compound according to claim 1, in which R1 and R2 together form a --CH2 --CH═CH--CH2
-- group..]. 5. A compound according to claim 1, in which Ar is phenyl.
A compound according to claim 1 in which p is 2. 7. A compound selected from:
1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methyl piperidine hydrochloride,
1-(3,4-dichlorophenyl)acetyl-2- dimethylaminomethyl piperidine hydrochloride,
(2R)-1-(3,4-dichlorophenylacetyl)-2-(1-pyrrolidinylmethyl)piperidine hydrochloride hydrate,
-(2S)-1-(3,4-dichlorophenylacetyl-2-(1-pyrrolidinylmethyl)piperidine hydrochloride hydrate,
-1-(3,4-dichlorophenylacetyl-2-dimethylaminomethylhexahydroazepine hydrochloride hydrate,
1-(6-tetralylacetyl)-2-(1-pyrrolidinylmethyl) piperidine hydrochloride,
1-(4-chlorophenylacetyl)-2-(1-pyrrolidinylmethyl) piperidine hydrochloride,
1-(2-phenylbutyroil)-2-(1-pyrrolidinylmethyl) piperidine hydrochloride,
hydrochloride. 8. A pharmaceutical composition for treating pain in mammals, comprising an effective amount of a compound according to claim
1, and a pharmaceutically acceptable carrier. 9. A composition according
to claim 8 in unit dosage form. 10. A method of treating pain in mammals which comprises administering an effective non-toxic amount of a compound according to claim 1 to a sufferer.
This invention is concerned with novel azacyclic derivatives, processes for their preparation, and their use in medicine, particularly as analgesics.
Compounds which are K-receptor agonists act as analgesics through interaction with Kappa opioid receptors. The advantage of K-receptor agonists over the classical μ-receptor agonists, such as morphine, lies in their ability of causing analgesia while being devoid of morphine-like behavioural effects and addiction liability.
We have now discovered a novel class of compounds which exhibit K-receptor agonism without the behavioural effects of morphine and morphine analogues, and which are thus of potential therapeutic utility as analgesics.
According to the present invention there is provided a compound, or a solvate or salt thereof, of formula I: ##STR2## in which: R.CO-- is an acyl group containing a substituted or unsubstituted carbocyclic or heterocyclic aromatic ring, R1 and R2 are independently C1-6 alkyl groups or together form a C3-6 polymethylene or alkenylene group, and p is 1,2,3 or 4.
When R1 and R2 are C1-6 alkyl groups, examples are methyl, ethyl, propyl, butyl, pentyl or hexyl groups, preferably methyl.
When R1 and R2 are polymethylene groups, examples are propylene, butylene, pentylene or hexylene, preferably butylene. As an alkenylene group, R1 -R2 may be typically --CH2 --CH═CH--CH2 --.
The group R preferably has the formula (II)
--(CHR4)n --X--Ar--(R3)m (II)
n is 0, 1 or 2,
m is 1, 1 or 2,
X is a direct bond, or O, S or NR5 in which
R5 is hydrogen or C1-6 alkyl,
Ar is a substituted or unsubstituted carbocyclic or heterocyclic ring,
R3 is halogen, such as chloro or fluoro, C1-6 alkoxy, cyano, --COOR4, C1-6 alkyl, aryl or amino or, when m is 2, two R3 's form a C3-6 polymethylene group, and R4 is hydrogen or C1-6 alkyl.
Examples of R4 are methyl and ethyl, and preferably R4 is hydrogen.
Preferably Ar is phenyl or 2- or 3-thienyl.
Suitable R3 substitutes for Ar are one or more chlorine atoms, or methyl or methoxy groups. When two R3 's are linked they may form a fused cyclopentyl or cyclohexyl ring.
X is typically oxygen or a direct bond, and n is typically 0 or 1.
Examples of R are: ##STR3##
A particularly preferred group of compounds of formula (I) are those in which p=2, i.e. those based on a piperidine ring.
The compounds of formula I or their salts or solvates are preferably in pharmaceutically acceptable or substantially pure form. By pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels.
A substantially pure form will generally contain at least 50% (excluding normal pharmaceutical additives), preferably 75%, more preferably 90% and still more preferably 95% of the compound of formula I or its salt or solvate.
One preferred pharmaceutically acceptable form is the crystalline form, including such form in a pharmaceutical composition. In the case of salts and solvates the additional ionic and solvent moieties must also be non-toxic.
Examples of a pharmaceutically acceptable salt of a compound of formula I include the acid addition salts with the conventional pharmaceutical acids, for example, maleic, hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, mandelic, tartaric, succinic, benzoic, ascorbic and methanesulphonic.
Examples of a pharmaceutically acceptable solvate of a compound of formula I include the hydrate.
The compounds of formula I have at least one asymmetric centre and therefore exist in more than one stereoisomeric form. The invention extends to all such forms and to mixtures thereof, including racemates. The preferred stereoisomeric form is the (S) enantiomer.
The present invention also provide a process for the preparation of a compound of formula I which comprises reacting a compound of formula (III) ##STR4## in which R1 ' and R2 ' and R1 and R2 as defined for formula (I) or a group or atom convertible to R1 and R2, with a compound of formula ##STR5## or an active derivative thereof, in which R' is R as defined for formula (I) or a group convertible to R, to form a compound of formula (Ia) ##STR6## and then performing one or more of the following steps:
(a) where R', R1 ' or R2 ' are other than R, R1 and R2, converting R', R1 ' or R2 - to R, R1 or R2 to obtain a compound of formula (I),
(b) where R', R1 ' and R2 ' are R, R1 and R2, converting one R, R1 or R2 to another R, R1 or R2 to obtain a compound of formula (I),
(c) forming a salt and/or solvate of the obtained compound of formula (I).
Suitable active derivatives of ##STR7## are acid chlorides or acid anhydrides. Another suitable derivative is a mixed anhydride formed between the acid and an alkyl chloroformate.
For example, in standard methods well known to those skilled in the art, the compound of formula (III) may be coupled:
(a) with an acid chloride in the presence of an inorganic or organic base,
(b) with the acid in the presence of dicyclohexyl carbodiimide, N-dimethylaminopropyl-N'-ethyl carbodiimide or carbonyl diimidazole,
(c) with a mixed anhydride generated in situ from the acid and an alkyl (for example ethyl)chloroformate.
It will be appreciated that a compound of formula (Ia) may be converted to a compound of formula (I), or one compound of formula (I) may be converted to another compound of formula (I), by interconversion of suitable substituents. Thus certain compounds of formula (I) and (Ia) are useful intermediates in forming other compounds of the present invention.
R1 ' and R2 ' may be alkyl groups and converted to R1 '/R2 ' hydrogen atoms by conventional amine dealkylation. When R1 ' or R2 ' is benzyl or substituted benzyl it may be converted to an R1 or R2 ' hydrogen atom by catalytic hydrogenation or other method of reduction. R1 ' and R2 ' as hydrogen atoms may be converted to R1 and R2 alkyl groups by conventional amine alkylation, or by acylation followed by reduction. R1 ' and R2 ' are preferably R1 and R2 respectively.
The compound ##STR8## is typically of the formula (IIa)
HO--CO--(CH2)n --X--Ar--(R3)m (IIa)
in which R3 ' is R3 as defined by formula (II) or a group or atom convertible to R3, the other variables being as defined for formula (II).
Conversions of substituents R3 ' on the aromatic group Ar to obtain R3 are generally known in the art of aromatic chemistry. R3 ' preferably R3.
The compounds of formula I may be converted into their pharmaceutically acceptable acid addition salts by reaction with the appropriate organic or mineral acids.
Solvates of the compounds of formula I may be formed by crystallization or recrystallization from the appropriate solvent. For example hydrates may be formed by crystallization or recrystallization from aqueous solutions, or solutions in organic solvents containing water.
Also salts or solvates of the compounds of formula I which are not pharmaceutically acceptable may be useful as intermediates in the production of pharmaceutically acceptable salts or solvates. Accordingly such salts or solvates also form part of this invention.
The compounds of formula I exist in more than one stereoisomeric form and the processes of the invention produces mixtures thereof. The individual isomers may be separated one from another by resolution using an optically active acid such as tartaric acid. Alternatively, an asymmetric synthesis would offer a route to the individual form.
The compound of formula (III) may be prepared from a compound of formula (IV) by the reaction scheme shown: ##STR9##
In this scheme, firstly the compound of formula (IV) is nitrogen protected with a conventional protecting group P, such as benzyloxycarbonyl or tert-butyloxycarbonyl, forming the compound of formula (V) which is reacted with the amine R1 'R2 'NH (in which R1 ' and R2 ' are as defined earlier) to obtain N-protected amide (VI). This is conventionally N-deprotected, for example by catalytic debenzylation if P is benzyloxycarbonyl or by acid treatment if P istert-butyloxycarbonyl, and the resulting basic amide (VII) is reduced to the diamine (III) by reaction with lithium aluminium hydride.
Alternatively, the N-protected acid (V) is reduced to a primary alcohol which is esterified, for example with methane sulfonic acid or p-toluenesulfonic acid, and the ester reacted with R1 'R2 'NH. Deprotection of the ring nitrogen gives the diamine (III).
When the starting material of formula (IV) is a racemic mixture, the resulting compounds of formulae (III) and (I) are also racemic. Using a compound of formula (IV) in the R- or S-configuration results in the corresponding optically active products.
The compounds of formula IV are known compounds. When p=1, the compound is R-, S- or R,S-proline. When p=2 it is R-, S-, or R,S-pipecolinic acid (Beilstein 22/IV, 96-97), and when p=3 is it R-, S-, or R,S-hexahydroazepine - 2- carboxylic acid (J. Med. Chem. 14, 501/1971).
Certain of the intermediates described above are novel compounds and, together with the described processes for their preparation, they form a further aspect of this invention.
The activity of the compounds of formula (I) in standard analgesic tests indicates that they are of therapeutic utility in the treatment of pain.
Accordingly the present invention also provides a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use as an active therepeutic substance.
The present invention further provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
The present invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of pain.
Such a medicament, and a composition of this invention, may be prepared by admixture of a compound of the invention with an appropriate carrier. It may contain a diluent, binder, filler, disintegrant, flavouring agent, colouring agent, lubricant or preservative in conventional manner.
These conventional excipients may be employed for example as in the preparation of compositions of known analgesic agents.
Preferably, a pharmaceutical composition of the invention is in unit dosage form and in a form adapted for use in the medical or veterinarial fields. For example, such preparations may be in a pack form accompanied by written or printed instructions for use as an agent in the treatment of pain.
The suitable dosage range for the compounds of the invention depemds on the compound to be employed and on the condition of the patient. It will also depend, inter alia, upon the relation of potency to absorbability and the frequency and route of administration.
The compound or composition of the invention may be formulated for administration by any route, and is preferably in unit dosage form or in a form that a human patient may administer to himself in a single dosage. Advantageously, the composition is suitable for oral, rectal, topical, parenteral, intravenous or intramuscular administration. Preparations may be designed to give slow release of the active ingredient.
Compositions may, for example, be in the form of tablets, capsules, sachets, vials, powders, granules, lozenges, reconstitutable powders, or liquid preparations, for example solutions or suspensions, or suppositories.
The compositions, for example those suitable for oral administration, may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable setting agents such as sodium lauryl sulphate.
Solid compositions may be obtained by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. When the composition is in the form of a tablet, powder, or lozenge, any carrier suitable for formulating solid pharmaceutical compositions may be used, examples being magnesium stearate, starch, glucose, lactose, sucrose, rice flour and chalk. Tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating. The composition may also be in the form of an ingestible capsule, for example of gelatin containing the compound, if desired with a carrier or other excipients.
Compositions for oral administration as liquids may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia, aqueous or non-aqueous vehicles, which include edible oils, for example almond oil, fractionated coconut oil, oily esters, for example esters of glycerine, or propylene glycol, or ethyl alcohol glycerine, water or normal saline; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents.
The compounds of this invention may also be administered by a non-oral route. In accordance with routine pharmaceutical procedure, the compositions may be formulated, for example for rectal administration as a suppository. They may also be formulated for presentation in an injectable form in an aqueous or non-aqueous solution, suspension or emulsion in a pharmaceutically acceptable liquid, e.g. sterile pyrogen-free water of a parenterally acceptable oil or a mixture of liquids. The liquid may contain bacteriostatic agents, anti-oxidants or other preservatives, buffers or solutes to render the solution isotonic with the blood, thickening agents, suspending agents or other pharmaceutically acceptable additives. Such forms will be presented in unit dose form such as ampoules or disposable injection devices or in multi-dose forms such as a bottle from which the appropriate dose may be withdrawn or a solid form or concentrate which can be used to prepare an injectable formulation.
As mentioned earlier, the effective dose of compound depends on the particular compound employed, the condition of the patient and on the frequency and route of administration. A unit dose will generally contain from 20 to 1000 mg and preferably will contain from 30 to 500 mg, in particular 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg. The composition may be administered once or more times a day for example 2, 3 or 4 times daily, and the total daily dose for a 70 kg adult will normally be in the range 100 to 3000 mg. Alternatively the unit dose will contain from 2 to 20 mg of active ingredient and be administered in multiples, if desired, to give the preceding daily dose.
Within the above indicated dosage range, no adverse toxicological effects have been observed with compounds of the invention.
The present invention also provides a method of treating pain in mammals, particularly in humans, which comprises administering an effective amount of a compound of formula (I), or pharmaceutically acceptable salt or solvate thereof, to a sufferer.
Compounds of this invention and their preparation are illustrated in the following Examples.
3 g of crude 2-(1-pyrrolidinyl)methyl Piperidine (0,01783 moles) and 4 g of 3,4-dichlorophenylacetic acid (0.0195 moles) were dissolved in 50 ml of dichloromethane. 5 g of dicyclohexylcarbodiimide (0.0267 moles), dissolved in 30 ml of dichloromethane, was added dropwise to the ice-cooled solution. The reaction mixture was left three days at room temperature, then the precipitated dicyclohexylurea was removed by suction filtration, and the solution evaporated to dryness in vacuo. The remaining oil was extracted three times with 2 N-Hydrochloric acid, the combined acid solutions extracted with ether, and the ether solutions discarded. The acid solution was made alkaline with diluted sodium hydroxide, the oily precipitate was extracted with chloroform, and the organic solution evaporated to dryness in vacuo. The oil residue was dissolved in ethyl ether and the solution brought to acidic pH with HCl saturated ether. The solid precipitate was collected by suction filtration and crystallized from 50 ml of methanol.
Yield 3.7 g
M.P. 246° C.
C18 H24 Cl2 N2 O.HCl
______________________________________NMR (DMSO) δ = 1.35-1.75 m 6 H 1.75-2.10 m 4 H 2.7-4.0 m 10 H 5.0 m 1 H 7.15-7.55 m 3 H 10.3 broad s 1 H______________________________________
The compounds of these Examples were prepared by the same procedure as Example 1, be reacting together the appropriate amine and acid in the presence of dicyclohexylcarbodiimide in dichloromethane, in the same molar ratio of reactants.
The compounds and starting materials are summarised as follows, with NMR characterising data. Other characteristics of Examples 1 to 17 are summarised Table 1A and 1B.
The compounds in Table 1B are racemates, except in the case of Example 16 which is a diastereoisomer of unassigned configuration separated from the originally obtained diastereoisomeric mixture.
Prepared from 2-dimethylaminomethyl piperidine and 3,4-dichlorophenylacetic acid.
______________________________________NMR (DMSO) δ = 1.35-1.70 m 6 H 2.75 s 6 H 2.9-4.0 m 6 H 5.0 m 1 H 7.15-7.65 m 3 H 10.3 broad s 1 H______________________________________
Prepared from (2R)-2-(1 pyrrolidinyl methyl) piperidine and 3,4-dichlorophenylacetic acid.
______________________________________NMR (DMSO) δ = 1.35-1.75 m 6 H 1.75-2.10 m 4 H 2.7-4.0 m 10 H 5.0 m 1 H 7.15-7.65 m 3 H 10.45 1.broad s 1 H______________________________________
Prepared from (2S)-2-(1-pyrrolidinyl methyl) piperidine and 3,4-dichlorophenylacetic acid.
______________________________________NMR (DMSO) δ = 1.35-1.75 m 6 H 1.75-2.10 m 4 H 2.7-4.0 m 10 H 5.0 m 1 H 7.15-7.65 m 3 H 10.4 1.broad s 1 H______________________________________
Prepared from (2S)-2-dimethylaminomethyl piperidine and 3,4-dichlorophenylacetic acid.
______________________________________NMR (DMSO) δ = 1.35-1.70 m 6 H 2.75 s 6 H 2.9-4.0 m 6 H 5.0 m 1 H 7.15-7.65 m 3 H 10.3 broad s 1 H______________________________________
Prepared from 2-(1-pyrrolidinylmethyl)pyrrolidine and 3,4-dichlorophenylacetic acid.
Prepared from 2-dimethylaminomethyl pyrrolidine and 3,4-dichlorophenylacetic acid.
Prepared from 2-(1-pyrrolidinylmethyl)hexahydroazepine and 3,4-dichlorophenylacetic acid.
Prepared from 2-dimethylaminomethylhexahydroazepine and 3,4-dichlorophenylacetic acid.
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and phenylacetic acid.
______________________________________NMR (CDCl3) δ = 1.3-1.9 broad s 6 H 1.9-2.3 broad s 4 H 2.6-3.1 broad s 3 H 3.3-4.2 m 4 H 3.9 s 2 H 5.2 m 1 H 7.3 s 5 H______________________________________
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 3,4-dimethylphenylacetic acid.
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 5-indaneacetic acid.
______________________________________NMR (CDCl3) δ = 1.1-2.5 m 12 H 2.5-3.1 m 7 H 3.1-4.2 m 7 H 5.2 m 1 H 7.15 m 3 H______________________________________
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 6-tetralylacetic acid.
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 4-chlorophenylacetic acid.
______________________________________NMR (CDCl3) δ = 1.4-1.8 broad s 6 H 1.9-2.4 m 4 H 2.5-3 m 3 H 3.2-4.2 m 5 H 3.9 q 2 H 5.3 m 1 H 7.2 s 4 H______________________________________
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 3,4-dimethoxyphenylacetic acid.
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 2-phenylbutyric acid. After working-up the reaction mixture as described in Example 1, a mixture of two diastereoisomeric hydrochlorides was obtained. It was dissolved in a minimum amount of water, the solution made alkaline with 5% sodium hydroxide solution, the precipitated oil extracted with methylene chloride, the organic solution dried on sodium sulphate and evaporated to dryness in vacuo. The resulting oily mixture (diastereoisomeric bases) was chromatographed on a silica gel column, eluting first with methylene chloride, amd subsequently with methylene chloride containing increasing amounts of methanol (0.5% to 2.2%). The slow-descending compound, identified as diastereoisomer B (while the fast-moving one was identified as diastereoisomer A) was collected, dissolved in ether, and the solution made acidic with HCL-saturated ether. The precipitated solid was crystallized from acetone/ethanol. M.P. 210°-1° C. The diastereoisomer A was transformed into the hydrochloride in the same way. M.P. 177°-80° C.
Prepared from 2-(1-pyrrolidinylmethyl)piperidine and 4-chlorophenoxyacetic acid.
______________________________________NMR (CDCl3) δ = 1.3-1.9 broad s 6 H 1.9-2.5 m 4 H 2.5-3.1 m 3 H 3.2-4.2 m 5 H 4.95 s 2 H 5.0-5.35 m 1 H 5.9-7.3 m 4 H______________________________________
TABLE IA__________________________________________________________________________SUMMARY OF EXAMPLES ##STR10##EX-AM- Stereo-PLE chemistry Molecular MeltingNO. p R R1 R2 at Carbon 2 Molecular formula Weight Point [α]D__________________________________________________________________________1 2 ##STR11## ##STR12## R,S C18 H24 Cl2 N2 391.77 246 --2 2 " CH3 CH3 R,S C16 H22 Cl2 N2 O.HCl 365.731 207 --3 2 " ##STR13## R C18 H24 Cl2 N2 O.HCl.H2 O 409.783 159-60 +47.6 (c = 2 MeOH)4 2 " " S C18 H24 Cl2 N2 O.HCl.H2 O 409.783 160-1 -49. (c = 2 MeOH)5 2 " CH3 CH3 S C16 H22 Cl2 N2 O.HCl.1.5H2 O 392.755 196-7 -34.5 (c = 0.33 MeOH)6 1 " ##STR14## R,S C17 H22 Cl2 N2 377.741 191 --7 1 " CH3 CH3 R,S C15 H20 Cl2 N2 O.HCl 351.705 175 --8 3 " ##STR15## R,S C19 H26 Cl2 N2 405.793 238 --9 3 " CH3 CH3 R,S C17 H24 Cl2 N2 O.HCl.H2 O 397.773 212 --__________________________________________________________________________
TABLE IB__________________________________________________________________________SUMMARY OF EXAMPLES ##STR16## ##STR17##Example Molecular Molecular MeltingNo. R Salt Formula Weight Point (°C.)__________________________________________________________________________10 ##STR18## HCl C18 H27 N2 OCl 322.869 217-911 ##STR19## HCl C20 H31 N2 OCl 350.921 165-612 ##STR20## HCl C21 H31 N2 OCl 362.931 181-413 ##STR21## HCl C22 H33 N2 OCl 376.957 182-514 ##STR22## HCl C18 H26 N2 OCl 357.318 200-215 ##STR23## HCl C20 H31 N2 O3 Cl 382.92 168-7016 ##STR24## HCl C20 N31 N2 OCl 350.921 210-117 ##STR25## HCl C18 H26 N2 O2 Cl2 373.318 185-7__________________________________________________________________________
The pharmacological activity of the compounds of this invention is illustrated by various in vitro and in vivo models, using the following test procedures, in which the mouse p-phenylquinone writhing test and 60 mouse tail flick test demonstrate analgesic activity.
The results of the tests are given in Tables 2 and 3.
Male Charles River mice, average weight 26 g, are used. Selection is carried out before beginning of experiments: only mice whose reaction time is less than 8 sec are used. They are randomly distributed into groups of 10 and dosed with compounds under test, with positive and negative controls being included.
Compounds under test are administered subcutaneously in isotonic saline in a volume of 20 ml. Kg-1. 30 min later mice are placed again under heat source (Socrel apparatus) and reaction time is recorded.
The analgestic activity of the test compound is expressed as the percent number of mice doubling the initial time within a group. ##EQU1##
Male Charles River mice, average weight 26 g, are randomly distributed into group of 10 and are dosed subcutaneously with compounds under test, dissolved in isotonic saline (20 ml.Kg-1). The experiments are carried out in the presence of positive and negative controls. 20 min after the mince are injected intraperitoneally with p-phenylquinone 0.02%, 10 ml. Kg-1, maintained at 37° C. in a water bath. The mice are placed in a perspex compartmented observation box and observed for a period of 8 min after injection of p-phenylquinone. The dose-response curve is obtained by basing observation on the all or none response.
Analgestic activity is calculated as percentage of animals completely protected from writhing in an 8 min period: ##EQU2##
Radio receptor binding to μ and K sites is performed on fresh guinea pig brain homogenate prepared according to Kosterlitz (1981).
Whole brain without cerebellum is homogenized in 50 mM, Tris-buffer (pH 7.4 at 0° C.) and centrifuged at 49,000×g×10 min.
The pellet is then resuspended in the same buffer, incubated at 37° C. for 45 min. and centrifuged again.
1.9 ml of the final homogenate (1:100 in Tris-pH 7.4, 0° C.) is used for the binding assay.
3 H [D-Ala2, MePhe4, Gly-ol5 ] Enkephalin (3 H-DAGO), an enkephalin analogue that binds selectively to μ receptor, is added to the biological substrate and incubated at 25° C. for 40 min., filtered through Whatman GF-C and washed with ice-cold Tris-buffer.
The filters are then dryed, solubilized in Filtercount and the radioactivity monitored. Non specific binding is determined in the presence of 10-6 M Naloxone.
The binding of tritiated Ethylketocyclazocine to brain homogenate is measured the in presence of 100 nanomolar D-Ala-D-LeuEnkephalin (DADLE) and 100 nanomolar DAGO, added to saturate the δ and μ opioid receptors respectively.
Final homogenate with solutions of the cold ligand and of the labelled ligand is incubated for 40 min. at 25° C., filtered through Whatman GF/C glass filter discs and washed.
The radioactivity bound to the filters is counted by liquid scintillation spectrophotometry.
MR 2266.500 nM is utilized to determine the saturable binding.
For the calculation of the kinetic parameters of the binding of labelled and unlabelled ligands, the equilibrium dissociation constant (KD), the inhibition constant (Ki) and the maximum number of binding sites (B max) are determined from saturation curves and competition experiments (Hill 1910; Scatchard 1949; Cheng and Prusoff 1973; Gillan et al. 1980).
A concentration of radioligand near KD is used in the binding assays evaluating our compounds.
______________________________________Hill, A. V. (1910) J. Physiol. 40, IV-VIII (1910)Scatchard G. (1949) Ann. N.Y. Acad. Sci., 51, 660-674Cheng and Prusoff W. H. (1973) Biochem. Pharmac. 22, 3099-3102Gillan M. G. C., Kosterlitz H. W. Br.J. Pharmac. 70,and Paterson S. Y. (1980) 481-490Kotsterliz H. W., Paterson S. Y. Br.J. Pharmac. 73,and Robson L. E. (1981) 939-949Magnan J., Paterson S. Y., Arch. Pharmacol. 319,Tavani A., and Kosterlits 197-205H. W. (1982)______________________________________
TABLE 2______________________________________PHARMACOLOGICAL DATAIN VIVO TESTS - ED50 mg Kg-1 s.c. RECEPTORExam- Mouse BINDING -ple Mouse p-phenylquinone Ki (nM)Nos. tail flick writhing μ K______________________________________1 0.11 0.20 1860 1.832 0.36 0.65 3730 8.213 17.5 12.4 7110 62.14 0.055 0.12 1559 0.775 1.03 0.44 >1.000 5.018 8.76 -- N.E. 6.51______________________________________
TABLE 3__________________________________________________________________________MOUSE TAIL FLICK OPIATE RECEPTOR BINDINGSUBCUTANEOUS Ki(nm) or % residualExample% Protection ORAL specific binding at 10-6 MNos. 1 mg Kg-1 ED50 mg/Kg-1 ED50 mg/Kg os μ K__________________________________________________________________________10 40 -- -- -- 100%11 60 -- -- -- 67%12 60 -- -- -- 62%13 80 45 3.91 500 5.7214 40 -- -- -- 61%15 40 -- -- -- 100%16 40 -- -- -- --17 80 91 -- -- 9.54__________________________________________________________________________