GB2184726A - Azoniaspironortropanol esters, processes for the preparation thereof and pharmaceutical compositions containing them - Google Patents

Description

GB 2 184 726 A 1

SPECIFICATION

Azoniaspironortropanol esters, processes for the preparation thereof and pharmaceutical compositions containing them 5 The present invention is concerned with a process for the preparation of azoniaspironortropanol esters, as well as new azoniaspironortropanol esters and pharmaceutical compositions which contain these compounds.

Because of their excellent spasmolyic properties, azoniaspironortropane derivatives are frequently used pharmaceutical ly. These corn pou nds are prepa red from the natu ral ly occu rring tropine but the known 10 processes are laborious and time-consuming and, because of the low yields obtained, are also expensive.

Usual ly, the preparation of azon iaspironortropa ne derivatives takes place according to the following reaction scheme:

is is H 3 c step a H A 20 OH OH OR tropine nortropine 25 step b step W alkylation esterif ication 30 01z Hal e A 35 OH O-C-R' 40 It 0 step c 45 esterif- ication 50 Hal H tep c' -C-R alkylation 55 H O-C-R' 0 of 60 0 In the above scheme, R'signifies the residue of a carboxylic acid and A is an amino protective group.

The oxidative demethylation of tropine to give nortropine (step a) is described by S.P. Findley in J.A.C.S., 65 2 GB 2 184 726 A 2 75,3204/1953. However, this process, which takes place with a supersaturatedtropine solution at WC. and with a reaction time of from 4 to 7 days, cannot be carried out on a technical scale since, under these reaction conditions, the tropine concentration needed for the reaction cannot be kept stable because the tropine precipitates out spontaneously and is thus removed from the further reaction. A homogenisation of the precipitated tropine, for exam pie by an in-line homogeniser, also did not produce any noticeable 5 improvement.

It is also known to carry outthe demethylation by exchange of the methyl radical for an Walkoxycarbonyl radical and subsequent hydrolysis ofthe alkoxycarbamate (see J.C. Kirn, Org. Prep. Proc. Int., 9,1A/1977). In the case of 8-ethoxycarbonyinoi-tropine, the best yield of nortropine to be found in the literature is 16%, referred to tropine (see G. Kraiss and K. Nador, Tetrahedron Letters, 1971, pp. 7-8). Later, itwas even reported 10 that an acidic or alkaline splitting of 8-etho),,ycarbonyinortropine is not possible (see T.A. MonAa, J.1). Matiskella and R.A. Partyka, Tetrahedron Letters, 1974, pp. 1325-1327).

The preparation of azoniaspironortropane derivatives by quaternisation and esterification or bythe reverse reaction sequence is known from Federal Republic of Germany Patent Specification No. 1,194,422 and from Arzneimittelforschung, 17,714-71911967 (steps b and c or steps Wand c'). The hydroxyl group of the is nortropine or of the corresponding azoniaspiro compound is thereby esterified by reaction with the appropriate acid chlorides,the hydroxyl group of hydroxycarboxylic acids and possibly the NH group of the nortropine thereby having to be protected. Adisadvantage of the processes described in these publications is the pooryield, the esterification of the nortropine (step W) and the subsequent reaction with a dihalide (step cl thereby also requiring two further reaction steps. It has long been known to use acid imidazolides as 20 reagents forthe esterification of alcohols (see Chem. Ber., 95,12841297/1962). In particular, Federal Republic of Germany Patent Specification No. 2,003,680 describes the reaction of benzilic acid imidazolidewith alcohois orthioalcohols which contain a tertiary amino group.

It is an object of the present invention to provide a process forthe preparation of azoniaspironortropanol esters which can be carried out on a technical scale and which permits these compounds to be prepared in a 25 simple manner in good yield.

Surprisingly, we have now found that azoniaspironortropanol esters can be prepared in good yield when the demethylation of tropine is carried out in the presence of a Cl-C3- chloroalkane which contains a trichloromethyl radical. The nortropine thus obtained is alkylated with a dihalide in the presence of an amine and the corresponding azoniaspiro compound is esterified by reaction with an acid imidazolide in the 30 presence of a catalyst.

Thus, according to the present invention, there is provided a process for the preparation of azoniaspironortropanol esters of the general formula 35 R A ON G_ ---4 R OH 40 0 R 2 45 IT 0, R wherein R signifies one of thefolloliving radicals: 50 a) an alkylene radical of the general formula:- R3 i -(CM2)n-UH-(U112)n- 55 in which R3 is a hydrogen atom or an alkyi,benzyi,ary] oral koxy radical and n is a whole number offrom 'I to 4, b) an alkenylene radical ofthe general formula:- R4 R5 C=C (CH2)n (CH2).- 3 GB 2 184 726 A 3 in which R4 and R5, which can be the same or different, are hydrogen atoms or alkyl or alkenyl radicals and n is a whole number of from 1 to 4; c) an azaalkylene radical of the general formula:- R6 5 1 -(CH2),,-N-(CH2)n- in which R6 is a hydrogen atom or an alkyl, alkoxycarbonyl or acyl radical and n is a whole number of from 2to 4; 10 d) an oxaalkylene radical of the general formula:

-(CH2)n-O-(CH2)r,- in which n is awhole numberof from 2to4; 15 e) an epoxyalkylene radical of the formula:- -CH2 - CH - CH - CH2- 0 20 f) an o-phenylene radical of the general formula:- X 9 Y 25 -H 2 c CH 2_ 30 g) a peri-naphthylene radical of the general formula- 35 X Y -H 2 c CH 2_ 40 or h) a 2,3-quinoxalinene radical of the general formula:

45 X Y 50 N -H 2 c CH 2_ 55 in which, in formulae f) to h), the symbols X and Y, which can bethe same or different, are hydrogen atomsor alkyl or alkoxy radicals; and wherein R, and R2, which can bethe same or different, are hydrogen or halogen atoms or alkyl, alkoxy, 60 alkoyi, cyclohexyl, phenyl, alkylphenyl, alkoxyphenyl, halophenyl,thienyl orfuryl radicals, the alkyl moieties in the said radicals containing up to 6 carbon atoms and being straight- chained or branched, and A13 isthe anion of a mono-to tribasic mineral acid, by a) demethylation of tropineto give nortropine, b) reaction of nortropine with a dihalideto givethe corresponding azonia compound and 65 4 GB 2 184 726 A 4 c) esterification of the azonia compound, wherein A) the demethylation of tropine of the formula:

H 3 c 5 (11) 10 OH is carried out either by working in a Cl-C3-chloroalkane which contains at least one trichloromethyl radical in 15 the presence of an oxidation agent in basic aqueous solution orthetropine is reacted with a chloroformic acid ester in an inert solvent in the presence of an acid-binding agent to give an 8-alkoxycarbonyinortropine and this is hydrolysed with abase in aqueous solution, B) the nortropine thus obtained of the formula:

20 H 25 (M) OH 30 is reacted at ambienttemperature for 1 or more days in a dipolar aprotic solvent with a compound of the general formula:

A-R-A 35 in which A and R have the above-given meanings, in the presence of a secondary or tertiary amine and C) the compound thus obtained of the general formula:- 40 (IV) 45 oil so in which R a nd 0 h ave the a bove-g iven m ea n i ngs, is esterified in a n a n hyd rou s, d ipo la r, aprotic solvent with a n i m idazo 1 ide of the genera 1 form u la: - OH 55 c - c R 2 (V) 0 R, 60 in wh ich R, a n d R2 have the a bove-g iven mea n i n gs, i n th e presence of a catalyst.

1 n the a bove-def i n ed radica Is, n ca n be the sa me o r d iffe rent, the rad icals n preferably bei ng so selected th at there is a 5- o r 6-mem bered ri ng.

The an ion Ae is preferably a ha 1 ide i on, such as a ch 1 oride, brom ide c r iodide i on, o r a phosp hate, su 1 phate ornitrateion. 65 GB 2 184 726 A 5 Preferred examples of the radical R include the following:

-(CH2)4-; -(CH2)2-CH-(CH2)2-; -CH2-CH=CH-CH2; 1 UN3 5 -CH2-C=CH-CH2_; -(CH2)2-N-(CH2)2- and -(CHA2-0-(CH2)2_.

1 1 UH H or -COCH3 10 H3C-C CH3 15 Within the scope of the present invention,the alkyl radicals, including those present in alkoxy, acyi, alkyiamino and the like radicals, can be straight-chained or branched and contain up to 18 carbon atoms and preferably up to 6 and more preferably up to 4carbon atoms. Specific examples of such radicals include methyl, ethyl, n-propyi, isopropyl, n-butyl, hexyi, lauryl and stearyl radicals. 20 Preferred acyl radicals include the acetyi and benzoyi radicals.

D) When the radical R contains one or more olefinic double bonds in the azonium ring after passing through steps B and/or C, these unsaturated compounds can be hydrogenated in a polar solventwith the help of a noble metal catalystto give the corresponding saturated compounds, compounds of general formula (1) then being obtained in which R is a radical a) as defined above. 25 Step A Th is p rocess step m a kes possi bl e the dem ethyl atio n even on a tech n ica 1 sca le a nd 9 ives nortropi ne i n considerably higher yields in comparison with the prior art. Two process variants can thereby be used, na m ely, oxidative dem ethyl atio n o r th e ca rba mate meth od. 30 Th e adva ntages of th e oxidative m eth od depend u po n the use of a Cl -C3-ch lo roa 1 ka ne co nta i n i ng at 1 east o ne trich lo ro methyl rad ica 1 wh ich is fi n ely dispersed i n th e aq u eo us phase. Exam p[ es of ch lo roa 1 ka nes wh ich ca n be used i ncl u de fo r exa m p 1 e, 1, 1, 1 -trich lo roetha ne, 1, 1, 1 -trich 1 oro propa ne a n d prefera bly ch lorofo rm.

The amount of chloroalkane used is in the range of from 1 - 10% byvolume, preferablyfrom 1 to 5% by vol u me a nd most prefera bly fro m 2 to 4% by vo lu me. 35 Fo r th e dem ethyl atio n, th ere ca n be em p 1 oyed a ny oxidation agent n o rm a] ly used fo r th is pu rpose, potassium ferricyanide being preferred.

The p rocess ca n be ca rried o ut i n a wide tem peratu re ra n ge, for exa m p le of fro m 0 to 1 00'C. but it is preferred to wo rk at a tem peratu re of f ro m 20 to 30'C. When the reactio n is f i n ished, th e produ ct is extracted i n cou nter-cu rrent, p refera b ly with the so Ivent used fo r th e demethylation. 40 The oxidative method results in a considerable saving of time in comparison with the known methods, wh ich ad d itio na i ly i m proves th e eco no my of th e process acco rd i n g to the p resent i nventio n.

However, it is p ref erred to use the ca rba m ate m eth od. Fo r th is pu rpose, tro pi ne is reacted i n an i nert so lvent with a 4 to 6 fol d excess of a ch lo rofo rm!c acid ester a nd genera i ly with ethyl ch 1 orofo rm ate. As so Ivent, th ere is th e reby p refera bly u sed a ch 1 o ri nated hyd roca rbo n, especia 1 ly ch 1 orofo rm. Th e reactio n is 45 ca rried out i n th e p resence of a n acid-bi n d i n g ag ent, p refe ra bly of a n a 1 ka 1 i m eta 1 ca rbo nate o r bica rbo nate.

Wo rki ng is ca rried o ut at a n elevated tem peratu re, prefe rably i n the ra ng e of f ro m 40 to 800C.

After su bsta ntia 1 d isti 11 i n g off of the so Ivent, the 8-a 1 koxyca rbo nyl n o rtropi ne th us o btai n ed is hyd ro lysed with a base in aq u eo us so 1 utio n. As base, th ere is prefera biy used potassiu m o r sod iu m hyd roxide, preferably in 16 to 20 fold excess. 50 The nortropine is extracted from the aqueous reaction mixture in the manner described above forthe oxidation demethylation.

According to stepA, nortropine can be obtained in almost quantitative yield, especially according tothe carbamate method.

Step 8 The crude nortropine obtained in step A can be used in step B without fu rther purif ication, in contradistinction to the prior art which requ ires 48 hou rs of continuous extraction a nd crystal] isation f rom diethyl ether. We have, surprising ly, fou nd that the tropine stil 1 present in the crude nortropine as im purity is not quaternised u nder the reaction conditions of step B. 60 Solvents wh ich can be used for quaternising the nortropi ne include, for example, N,N-dimethylformarnide ch loroform or chiorofo rm/acetonitrile. With dihal ides in the presence of secondary or tertiary a mines, after a reaction period of one or more days at am bient temperatu re, the corresponding azoniaspi ro compou nds are obtained in pure form and with h ig h yields. It is preferred to work in an hyd rous so] ution, using nortropine, am ine and dihal ide in a mol e ratio of 1: 2:4. As already mentioned, the product obtained in good yield is of 65 6 GB 2 184 726 A 6 high purity so that a further purification is not necessary.

Secondary am ines which can be used forthis reaction include, for example, dimethylamine,diethylamine, diisopropylamine, dicyclohexylamine and the like. Examples of tertiary amines which can be used include trimethylamine,triethylamine, pyridine, quinoline and the like. The use of diethylamine is preferred.

5 Step C Not onlythe azoniaspiro compounds obtained from step B but also the carboxylic acid imidazolides are generally of low solubility in the anhydrous, dipolar, aprotic solvents usually employed for such a reaction, for example acetone, acetonitrile, dimethylformamide, tetrahydrofuran and the like. If it is endeavoured to overcome the problems therewith involved by increasing the reaction temperature, then the products 10 resulting therefrom are contaminated with a high proportion of by- products. In particular, carboxylicacid imidazolides which have an unprotected hydroxyl group reactwith themselves at an elevated temperature.

Surprisingly have nowfound thatthe reaction of a compound of general formula (R) with a carboxylic acid imidazolide of general formula (V) can be carried out in the presence of an appropriate catalyst even in the above-mentioned anhydrous, dipolar, aproticsolvents by reacting the reaction components in 15 suspension. The advantage of this process is thatthefree hydroxyl groups of the carboxylic acid imidazolides do not haveto be protected and thatthe reaction product precipitates from the above-mentioned solvents and, therefore, can be isolated in a simple manner. The reaction product is not, as wasto have been expected, contaminated by a reactant introduced into the reaction in soliciform.

Furthermore,the reaction takes place undersuch mild conditions that no fragmentation and elimination 20 reactions attributableto the presence of the quaternary ammonium group take place. Consequently, no corresponding by-products can be formed.

4-(Dimethylamino)-pyridine has provedto bethe most advantageous catalystforthis process. This compound can be used in amounts of from 1 to 30 mole % and preferably of from 5to 10 mole %, referredto the benzilic acid imidazolide. 25 As solvents, there can be used the above-mentioned anhydrous, dipolar, aprotic solvents. The reaction is carried out at an elevated temperature and preferably at a temperature of from 60 to WC.

The preparation of the carboxylic acid imidazolides used in this reaction takes place in known mannerby reacting N,N-carbonylclUmidazole with the appropriate carboxylic acids in dry dichloromethane.

30 Step D Since, in the case of the q uaternisation according to step B) with the dihalides su bstituted on the dou ble bond, for example with cis-1,4-dichlorobutene, a much greater speed of reaction is achieved, for exam pie from18daystol hour, it can be advantageous forthe preparation of compounds of general formula (1), in which R has the meaning of a radical such as a), for example a 35 benziloyloxynortropane-8-spiro-l'-pyrrolidinium salt, to choose the route via the corresponding unsaturated compounds with subsequent catalytic hydrogenation either after passing through step B) or steps B) and C).

The hydrogenation of the unsaturated compounds is carried out in a polar solvent, such as water or an alcohol containing up to 4 carbon atoms, preferably methanol, in the presence of a noble metal catalyst, such as platinium dioxide or palladium on active charcoal. 40 In the case of using an unsaturated halide, in carrying outthe quatemisation there is used a considerably smaller excess of dihalide. The mole ratio of nortropine, amine and dihaiide previously stated to be preferably 1:2A in step B) can then be changed to 1:2:2.

The present invention also provides new - azoniaspironortropanol esters of the general formula: 45 R 0+ A 50 OH 0 1 R 55 0 R 1 wherein R, IR,, R2 and AG havethe same meanings as in claim 1, but excluding thefollowing compounds: 60 azoniaspiro-[3u--phenyiglycoloyloxynortropan-8,1'-pyrrolidine] chloride, azoniaspiro-[3u--diphenyigiycoloyloxynortropan-8,1'-pyrrolidine1 chloride, 3et-phenylgiycoloyloxynortropan-8-spiroisoindolinium chloride, 3a-dipheny[glycoloyloxynortropan-8-spiroisoindolinium chloride, 3a-phenyigiycoloyloxynortropan-8-spiro-4'-morpholinium chloride, 65 7 GB 2 184 726 A 7 3a-diphenyiglycoloyloxynortropan-8-spiro-4'-morphol in W m chloride, azoniaspiro-[3oL-cyclohexylphenylgiycoloyinortropan-8,1'-pyrrolidineJ chloride, azoniaspiro-[3a-phenylglycoloyloxynortropan-8,1'-piperidine] chloride and azoniaspiro-[39--diphenylglycoloyfoxynortropan-8,1'-piperidine] chloride.

These compounds possess outstanding spasmolytic properties. 5 In addition, the present invention provides pharmaceutical compositions containing at least one of the compounds according to the present invention, optionally in admixturewith pharmaceutically-acceptable carriers and/or adjuvants.

The following Examples are given forthe purpose of illustrating the present invention:- 10 Example 1

3a-Benziloyloxynortropane-8-spiro- 1'1-pyrrolidinium chloride.

Step A Demethylation of tropine to nortropine 15 In a 300 litre stirrer vessel equipped with a reflux condenser, 1.9 kg. tropine (97%, corresponding to 1.843 kg. of pure compound, equal to 13 mole) are dissolved in 240 litres chloroform and 5.7 kg. sodium hydrogen carbonate powder and 5.3 litres ethyl chloroformate (98%, corresponding to 6.0 kg. and to 55.7 mole) are stirred in. The reaction mixture is heated to the boil and then heated under refluxfor a further2 hours. The progress of the reaction is monitored by means of thin layer chromatography (silica gel 60; 20 dimethylformamide/diethylamine/ethanol/ethyI acetate 5:10:30:6Ov/vlvlv). The reaction mixture isfiltered while hot and the chloroform is distilled off. Asolution of 18 kg. 85% potassium hydroxide in 90 litres wateris added to the residue. The reaction mixture is heated to the boil and then heated under refluxfor a further9 hours. The cooled solution isthen extracted with chloroform using a Karr column. Extraction conditions: the "stationary" phase isthe light phase (aqueous potassium hydroxide solution) which is conveyed at a rateof 25 about 14 litres/hour. The dispersed phase isthe heavy chloroform phasewhich is conveyed at a rate of about 35to 50 litres/hour. Shaking frequency: 200 strokes/minute; temperature 26to 2WC.

In thisway, the nortropine formed is extracted from the aqueous potassium hydroxide solution in almost quantitative yield. Afterstripping off the solvent, the crude product obtained is used in thefollowing step B) withoutfurther purification. There is obtained 1.876 kg. nortropinewith a content of 87% (high pressure 30 liquid chromatography: L-Bondapack C18-column; elution agent: methanol/water 1:9 v/vwith PIC-137). This corresponds to 1.632 kg. of pure tropine and to a yield of 98%.

Step 8 3cL-Hydroxynortropane-8-spiro- 1.pyrrolidinium chloride 35 The composition of the reaction m ixtu re m ust be referred to pu re nortropine and the mole ratio of nortropine:diethylamine: 1,4-dichiorobutane must be exactly 1:2A.

The crude nortropine obtained in step A) (1.186 kg., corresponding to 1. 632 kg. of pure substance andto 12.85 mole) is dissolved in 52 litres N,N-dimethylformarnide and 2.665 litres (1.876 kg.; 25.7 mole) diethylamine and 5.736 litres (6.528 kg.; 5.14 mole) 1,4-dichlorobutane added thereto. The reaction mixture is 40 leftto stand for 18 days at ambient temperature. The crystals which separate out arefiltered off with suction, washed with a little dry acetonitrile and dried at WC. in a vacuum drying cabinet. There are obtained 2.25 kg.

(80%, referred to the amount of tropine used in thefirst step) of pure product; m.p. 250oC Step C 45 3a--Benziloyloxynortropane-8-spiro- 1 -pyrrolidinium chloride (trospium chloride) a) Benzilic acid imidazolide 1.944 kg, (12 mole) N, N-ca rbonyidi imidazole are dissolved in 19.2 litres dry dichloromethane with the exclusion of moisture. 2.736 kg. (12 mole) dry benzilic acid are added thereto, while stirring, at 15 to 2WC. in the course of 6 minutes, whereafterthe reaction mixture is stirred for 1 hour at ambient temperature. The 50 benzilic acid thereby first goes into solution but soon afterwards the benzilic acid imidazoiide begins to separate out in solid form. It is filtered off with suction and washed with 0.8 litres dry dichloromethane. There are obtained 2.4 kg. benzilic acid imidazolide.

b) Preparation of the title compound In a 300 litre stirrer vessel, 1.3 kg. of the compound obtained in step B) are suspended in 230 litres 55 anhydrous acetonitrile and heated to 7WC. A solution of 74.0 g. 4(dimethylamino)-pyridine in 2 litres anhydrous acetonitrile is added thereto. A suspension of 2.086 kg. benzilic acid imidazolide in 9.0 litres anhydrous acetonitrile is then added thereto in three portions at intervals of 30 minutes at 7WC. The reaction mixture is subsequently stirred at 7WC. until there is achieved a total reaction time of 4 hours afterthefirst addition of benzilic acid imidazolide. The reaction mixture is then cooled to 20'C. and further stirred 60 overnight. The suspension formed is filtered off with suction and washed with some acetonitffle. The residue, as well as further product obtained by concentration of the mother liquor (total 2.14 kg.), are recrystallised from isopropanol. There is obtained 1.78 kg. (70%) of pure product; m.p. 258- 263'C.

(decomp.).

8 GB 2 184 726 A FD-MS: m/e = 392 (molecule cation) IR (KBr): y = 3150,1735,1498,1452,747.

Example2

3ot-Benziloyloxynortropane-8-spiro- l-(3-pyrrolidinium) Step 8 3u--Hydroxynortropane-8-spiro- 1'-(3'.p yrrolidinium) chloride 1.05 m]. (10 m Mole) diethylam ine and 1.05 mi. (10 m Mole) cis-1,4-dich lorobut-2-ene a re stirred into a solution of 635 mg. (5 m Mole) nortropine i n 9.5 mi. N,N-dimethylformarn ide. After 1 hou r, the pu re crystalline 10 product is filtered off with suction. The mother 1 iquor is m ixed with ethyl acetate u ntil the commencement of tu rbidity in order to obtain f urther product. The crystals are f iltered off with suction and washed with a little acetone. Yield 984 mg. (91 % of theory); m. p. 204'C.

FD-MS: mle = 180 (molecule cation) 15 IR (KBr): -V = 3250,1621 cm-1.

H-NMR (90 MHz, D20,8-values referred to TS1=0):

8 = 13-2.7 (8H, H-2, HA H-6, H-7); 3.92 (2H; H-1, H-5); 4.05 (1 H; H-3); 4.14 and 4.31 (each 2H; H-2'and H-5); 5.90 (2H; J-3', H-4% 20 Step C 3ct-Benziloyloxynortropane-8-spiro- l.(3-pyrrolinium) chloride 530 mg. (2.4 mMole) 3oL-hydroxynortropane-8-spiro-l'-(3'-pyrrolinium) chloride are suspended in 353 mi.

anhydrous acetone and stirred in an autoclave for 23 hours at 70'C. with 14 mg. (0.12 mMole) 4-dimethylaminopyridine and 678 mg. (2.4 mMole) benzilic acid imidazolide. Upon cooling to ambient 25 temperature, the product crystallises out of the reaction mixture. It is filtered off with suction and washed with a little acetone. Yield 650 mg. (62% of theory); m.p. 267'C.

FD-MS: mle = 390 (molecule cation) IR (KBr): y = 1722,1595,1490,1445,741 cm-1.

'WNMR (90 MHz, D20,8-values referred to TSP=O): 30 8 = 13-2.8 (8H; H-2, H-4, H-6, H-7); 3.85 (2H; H-1, H-5); 4.09 and 4.37 (each 2H; H-2'and H-5'); 5.24 (1 H; H-3); 5.95 (2H; WX, H-41 7.44 (1 OH; aromatic protons of the benzilic acid).

Step D Conversion of 3a-benziloyloxynortropane-8-spiro- 1 -(3'-pyrrolinium) chloride into 35 3ot-benziloyloxynortropane-8-spiro- l -pyrrolidinium chloride 500 mg. 3a-benziloyloxynortropane-8-spiro-l'-(3'-pyrrolinium) chloride are dissolved in 15 m[. methanol and, afterthe addition of a spatula tip of platinum dioxide, hydrogenated at normal pressure and at a temperature of 25'C. up to the end of the hydrogen take-up. The hydrogenation is carried out in a standard apparatus, such as is illustrated, for example, in Houben-Weyl, Methoden der organischen Chemie, 4th 40 edition, Vol. IW1 c, pub. Georg Thieme Verlag, Stuttgart, New York, 1980, pp. 33-39. Afterfiltering off the catalyst, the filtrate is evaporated to dryness in a vacuum. According to 1 H-N MR spectroscopy, the reaction is quantitative. Crystallisation is carried out as described in Example 1, Step C.

Example3 45

3u--Benziloyloxynortropane-8-spiro-2'1-isoindolinium chloride 1) 3(x-Hydroxynortropane-8-spiro2'-isoindolinium chloride 1.27 g. (10 mMole) nortropine are dissolved in 7 m 1. chloroform and m ixed with 1.46 g. (20 m Mole) diethylami ne and 4 g. (40 m Mole) 1,2-bis-(chloromethyi)-benzene. The clear reaction sol ution is left to stand for 24 hou rs at ambient tem perature in a closed vessel. It is then concentrated to one half and mixed with 50 ethyl acetate i n order to initiate crysta 1 lisation. The crystals are f iltered off with suction and recrystal 1 ised from isopropa noVethyl acetate. Yield 1 g. (38% of theory); m.p. 245 - 247'C.

FID-MS: m/e = 230 (molecule cation) IR (KBr): y = 3168,757,742 cm -1.

1 WNMR (250 MHz, D20, 8-values referred to TSP=O): 55 3 = 2.09 (2H; H-6a, H-7a); 2.40-2.67 (4H; H-2, H-4); 2.59 (2H; H-6b, H- 7b); 4.03 (2H; H-1, H-5); 4.24 (1 H; H-3); 4.82 and 4.99 (4H; H-l'and H-3'); 7.47 (4H; H-4'to H-7').

2) 3ct-Benziloyloxyn ortropan e-8-spiro-2.isoin dolinium chloride 1.33 g. (5 m Mole) 3et-hydroxynortropane-8-spiro-l'-isoindolinium chloride is suspended in 210 m].

anhydrous acetonitrile a nd heated to 78'C. While stirring, there are first introduced 62 mg. (0.5 mMole) 60 4-dimethylaminopyridine and then, within the course of 2.5 hours, portionwise 3.2 g. (11,5 mMole) benzilic.

acid imidazolide. The reaction mixture is further stirred for 5.5 hours at 78'C., then cooled to 22'C. and further stirred overnight. The solution is concentrated to one quarter of its volume and the product causedto crysta 11 ise by th e a d d iti c n of ethyl a cetate. Yiel d 1.3 g. (54% of th eo ry); m. p. 263 - 2 650C.

FD-MS: m/e = 440 (molecule cation). 65 9 GB 2 184 726 A 9 [R (KBr): y = 1740,757,703 em-'. 'WNMR (250 MHz, D20, 8-values referred to TS1=0):

8 = 1.57 (2H; H-6a, H-7a); 2.03 (2H, H-2a, H-4a); 2.07 (2H; H-6b, H-7b); 2.70 (2H, H-2b, H-4b); 3.86 (2H; H-1, H-5); 4.69 amd 4.96 (4H; H-1'- and H-3'); 5.32 (1 H; H-3); 7.40- 7.51 (14H; H4to H-Tand aromatic protons of the benzilic acid). 5 Example 4

3ot-Benziloxyoxynortropane-8-spiro-4'1-morpholinium chloride 1) 3a-Hydroxynortropane-8-spiro-4'-morpholinium chloride 11.8 mi. (113.2 mMole) diethylamine and 26.6 m]. (226.5 mMole) 2,2'-d- chlorodiethyl ether are stirred into a 10 solution of 7.2 g. (56.6 mMole) nortropine and 70 mi. chloroform. The clear reaction solution is leftto stand for3 days at ambient temperature in a closed vessel. The oil-crystal mixture which separates out is homogenised and crystallised through overnight at O'C. The crystals arefiltered off with suction,washed with a little chloroform and dried under a vacuum at40'C. for 2 hours. Additional substance is obtained by evaporating the mother liquor and treating with ethyl acetate. Yield 12.5 g. (95% of theory); m.p. 274- 2760C. 15 (decomp.).

FID-MS: m/e = 198 (molecule cation).

I R (KBr): y = 3320,892 em-'.

N-NIVIR (250 MHz, D20, 8-values referred to TS1=0):

8 = 2.00 (2H; H-6a, H-7a); 2.22-2.62 (6H; H-2, H-4, H-6b, H-7b); 3.50 and 3.65 (4H; H-2'and H-6'); 4.01 and 20 4.08 (4H; H-Tand H-6'); 4.18 (1 H; H03); 4.22 (214; H-1 and H-5).

2) 3u--Benziloxyloxynortropane-8-spiro-4-morpholinium chloride 7.5 9. (32 mMole) 3u--hydroxynortropane-8-spiro-4'-morpholinium chloride are suspended in 650 m].

anhydrous acetonitrile and mixed with 0.587 g. (4.8 mMole) 4(dimethylamino)-pyridine. 26 g. (92.8 mMole) benzilic acid imidazolide are added portionwise at79'C. within the course of 3 hours. The reaction mixture is 25 leftto stand for7 days at ambient temperature and the pure crystalline product isthen filtered offwith suction. The crystals obtainedare dried undervacuum for2 hours at40'C; yield 8.4 g. (60% of theory); m.p.

225'C. (decomp.).

FD-MS: m/e = 408 (molecule cation).

IR (KBr): y = 3410,3183,1731,1492,703 em-'. 30 H-NMR (250 MHz, D20, 8values referred to TS1=0):

8 = 1.51 (2H; H-6a, H-7a); 2.00 (4H; H-2a, H-4a, H-6b, H-7b); 2.63 (2H; H2b, H-4b); 3.38 and 3.64 (4H; H-2' and H-6'); 3.99 and 4.04 (4H; H-3'and H-5'); 4.09 (2H; H-1, H-5); 5.30 (1 H; H-3); 7.46 (1 OH; aromatic protons of the benzilic acid).

35 Example 5

3a-Benziloxyoxynortropane-8-spiro- 1'-pyrrolidino-[3',4'1-bjquinoxalinium bromide 1) 3(x-Hydroxynortropan-8-spiro-l'-pyrrolidino-[3',4'.blquinoxalinium bromide 4.58 g. (43.6 mMole) diethylamine and 13.85 9. (43.6 mMole) 2,3-bis(bromoethyi)-quinoxaline are stirred into a solution of 5.57 g. (43.6 mMole) nortropine and 100 mi. chloroform. The reaction mixture, which has 40 become warm, is cooled to 20'C, the product thereby precipitating out in crystalline form. It is filtered off with suction, washed with chloroform and dried in a vacuum for 22 hours at 550C. Yield 11.1 g. (71 % of theory); m. P. 283'C. (d eco m p.).

FD-MS: mle = 282 (molecule cation).

1 R (KB r): y = 3345,1504,773 em - 1. 45 H-NMR (250 MHz, D20, 8-values referred to TSP=O):

8 = 2.2 1; (2 H; H -6a, H -7 a); 2.53 - 2.89 (6 H; H-2, H-4, H -6 b, H -7 b); 4.29 (2 H; H - 1 a n d H -5); 4.31 (1 H; H -3); 5.21 and 5.41 (each 2H; H-2'a nd H-5'); 7.94 - 8.05 (2H; quinoxal ine o- protons); 8.11 - 8.22 (2H; quinoxal ine m-protons).

2) 3a-Benzilo yloxyn o rtropan e-8-sp iro- 1 p yrro lidin o-[3 'I 4'blquin oxallnium b romide 50 g. (1.39 mMole) 3u--hydroxynortropane-8-spiro-l'-pyrrolidino[3',4'-bl- quinoxalinium bromide are suspended in 130 m]. dried dimethyl sulphoxide and 100 mi. dry acetonitrile. Afterthe addition of 0.26 g. (2.09 mMole) 4-dimethylaminopyridine, the reaction mixture is heated to 78'C. While stirring vigorously, 7.74g.

(27.8 mMole) benzilic acid imidazolide are added in 3 portions at intervals of 30 minutes. The reaction mixture isfurther stirred for 2.5 hours at 78'C and is then cooled to 20'C, and filtered. The filter residue isdiscarded. 55 The filtrate is evaporated to dryness at about 0.2 mbar pressure. The residue is extracted with 500 m]. boiling isopropanol and filtered hot. Thefilter residue is discarded. The filtrate is concentrated to 200 mi. The product crystallises out after standing overnight at ambient temperature. It isfiltered off with suction, washed with cold isopropanol and dried in a vacuum for 2 hours at 55oC. Yield 3.5 g. (44% of theory); m.p. 205'C.

(decomp.). 60 FD-MS: mle = 492 (molecule cation).

]R (KBr): -V = 3375,1730,1504,763 cm-1.

H-NMR (250 MHz, CDC13/CD30D = 3:1 v/v, 8-values referred to TIVIS=O):

GB 2 184 726 A 10 8 = 1.78 (2H; H-6a, H-7a); 2.08 (2H; H-2a, H-4a); 2.20 (2H; H-6b, H-7b); 2.85 (2H; H-2b, H-4b); 4.23 (2H; H-1, H-5); 4.62 (4H; H-2', H-W); 5.35 (1 H; H-3); 7.30-7.48 (1 OH; benzilic acid protons); 7.84-7.97 (2H; quinoxaline o-protons); 8.07-8.22 (2H; quinoxaline m-protons).

Example 6 5

3a-Benziloyloxynortropane-8-spiro-2':-(2'-aza-3H-phenolenium) bromide 1) 1.33 mi. (12.7 mMole) dimethylamine and 4 g. (12.7 mMole) 1,8-bis(bromoethyi)-naphthalene are stirred into a solution of 1.62 g. (12.7 mMole) nortropine and 75 mi. N,N- climethylformarnicle. From the reaction mixture, which has become slightly warm, the product crystallises out within 2 hours. It is filtered off with 1() suction, washed with a little N,N-dimethylformarnide and dried in a vacuum at WC. for 2 hours. Yield 3.5 g. 10 (76% of theory); m.p. 330'C. (decomp.).

FD-MS: m/e = 280 (molecule cation):

IR (KBr): y = 3410,1604,1512 cm-1.

H-NMR (250 MHz, CDCI3ICD30D = 3:1 v/v, 8-values referred to TIVIS=O):

8 = 2.02 (2H; H-6a, H-7a); 2.39-2.85 (6H; H-2, H-4, H-6b, H-7b); 3.92 (2H; H-1, H-5); 4.28 (1 H; H-3); 5.01 and 15 5.16 (4H; H-l'and WX); 7.51-7.64 (4H; H-5', H-W, H-7', H-W); 7.93 (2H; H4, H-9,).

2) 3(x-Benziloxyoxynortropane-8-spiro-2'-(2:.aza3H-phenolenium)-bromide 2.95 g. (8.2 mMole) 3u--hydroxynortropane-8-spiro-2'-(2'-aza-3Hphenolenium) bromide are suspended in 1660 mi. dry acetonitrile and 160 m]. dry N,N-dimethyiformamide. Afterthe addition of 152 mg. (1.2 mMole) 4-dimethylaminopyridine, the reaction mixture is heated to 7WC. 4.56 g. (16.4 mMole) benzilic acid 20 imidazolide are added in three portionswith vigorous stirring at intervals of 30 minutes. The reaction mixture isthen stirred for2.5 hours at7WC. and the reaction mixture thereafter evaporated to one half.The precipitated crude product is filtered off with suction at 200C. and suspended in methanol. The material which is insoluble in methanol is filtered off and discarded. The filtrate is concentrated until the crystallisation of the product commences. After crystal lisation overnight at ambienttemperature, the product is filtered off with 25 suction and dried in a vacuum for 2 hours at 550C. Yield 2.1 g. (42% of theory); m.p. 322'C. (decomp.).

FID-MS: m/e 490 (molecule cation).

[R (KBr): y = 3428,3240,1738,1603,1497 cm-1.

H-NMR (250 MHz, CDC13/CD30D = 3:1 v/v, 8-values referred to TMS=O):

8 = 1.75 (2H; H-6a, H-7a); 1.94 (2H; H-2a, H-4a); 2.20 (2H; H-6b, H-7b); 2.80 (2H; H-2b, H-4b); 3.85 (2H; H-1, 30 H-5); 4.93 and 5.19 (each 2H; H-Vand H-3'); 5.45 (1 H, H-3); 7.31-7.46 (1 OH; benzilic acid proton); 7.47-7.67 (4H; H-W, H-W, H-7', H-W); 7.93 (2H; HA', H-9').

Example 7

3u--Benziloyfoxynortropane-8-spiro- 1':-(4'-methyl)-piperidinium chloride 35 1) 3ot-Hydroxynortropane-8-spiro-l':-(4'1-methyl)-piperidinium chloride 7.62 g. (0.06 mole) Nortropine are dissolved in 200 m]. anhydrous N,Ndimethylformarnicle. Afterstirring in 8.76 g. (0.12 mole) diethylamine and 37.18 g. (0.24 mole) 1,5-dichloro-3- methylpentane, the reaction mixture is leftto stand for 18 days at ambient temperature in a closed vessel. The crystals which separate outare filtered off with suction,washed with a little dryacetonitrile and dried in a vacuum drying cabinet at50'C. 40 There are obtained 7.84 g. (53% of theory) of pure product; m.p. 29WC. (decomp.).

FID-MS: m/e = 210 (molecule cation).

IR (KBr): y = 3190 cm-1 H-NMR (250 MHz, D20, y-values in ppm, referred toTSP=O):

8 = 1.01 (m; 3H; CH3); 1.37-2.02 (m; 7H; H-6a, H-7a, WX, HA, H-5'); 2.202.52 (m; 5H; H-2a, H-4a, H-6b, 45 H-7b, OH); 2.60 and 2.67 (2 X t; 2H; H-2b and H-4b); 3.10,120,163 and 3. 74 (4 x m; 4H; H-2'and H-6'); 3.76 and 4.24 (2 X m; 2H; H-1 and H-5); 4.19 (t; 1 H; H-3).

2) 3a-benziloyloxynortropane-8-spiro-l'-(4'-methyi)-piperidinium chloride 7.37 g. (30 mMole) 3et-Hydroxynortropane-8-spiro-l'-(4'-methyi)piperidinium chloride are suspended in 650 mi. anhydrous acetonitrile and heated to 780C, while stirring. Atthis temperatu re, there are firststirred in 50 587 mg. (4.8 mMoie) 4- (dimethylamino)pyridine and then, in the course of 2 hours, 13.35 g. (48 mMole) benzilic acid imidazolide in 4 approximately equal portions. Stirring is continued for 1.5 hours at 7WC. and then the reaction mixture is allowed to cool overnight at ambienttemperature, without stirring. The crystalline product is filtered off with suction and washed with a little acetone. The crude crystallisate is 55 recrystallised from dry isopropanol. The pure crystals are dried in a vacuum for 2 hours at 4WC. Yield 9.56 g. 55 (70% of theory) as a 1: 1 mixed crystallisate with isopropanol; m.p. 256 - 2590C. FID-MS: mle = 420 (molecule cation). IR (KBr)-. y = 1735 cm-1 'H-NMR (250 MHz, D20,8-values in ppm, referred to TSIP=O):

8 = 0.98 (m; 3H; CH3); 1.30-1.65 (m; 4H; H-6a, H-7a, H-3'a, H-5'a); 1.652.03 (m; 7H; H-2a, H-4a, H-6b, H-7b, 60 H-31, H-4', H-5'b); 2.52 and 2,72 (m; 2H; H-2b, H-4b); 3.02,3.19,3,47,3. 72 (t, t; d; 4H; H-2'and H-W); 3.62 and 4.10 (m, m; 2H; H-1 and H-5); 5.30 (t; 1 H; H-3); 7.40-7.40 (m; 1 OH, aromatic protons).

11 GB 2 184 726 A 11 Example 8

3ot-(4,4-Difluorobenziloyloxy)-nortropane-8-spiro- 1'.pyrrolidinium chloride 2.17 g. (0.01 mole) 3u--hydroxynortropane-8-spiro-l-pyrrolidinium chloride are dissolved with 2.02 g. (0.01 mole) sodium heptane-1 -sulphonate, with warming, in 500 m]. anhydrous acetonitrile. Aftercooling to 25to 27C., the sodium chloride precipitate is filtered off with suction with the exclusion of moisture. The solution 5 is mixed with 0.125 g. 4-(dimethylamino)pyridine and transferred to a reaction vessel which is connected to a stirrer vessel in which 4,4'-clifl uorobenzi 1 ic acid imidazolide is prepared. This stirrer vessel is equipped with two dropping funnels. In one dropping funnel, there are placed 2.64 g. (0. 01 mole) 4,4'-difluorobenzilic acid (preparation analogous to the description in Federal Republic of Germany Patent Specification No. 20 34

943), dissolved in 100 mi. anhydrous acetonitrile. In the other dropping funnel there is placed a solution of 10 2.43 g. (0.015 mole) N,N-carbonylciiimiclazole in 150 mi. anhydrous acetonitrile. From each of thetwo solutions, about one quarter of the volume is run in simultaneously, while stirring, into the stirrer vessel,the mixture is then stirred for 15 minutes and the resultant imidazolide solution transferred, with the strict exclusion of moisture, into the reaction vessel in which the solution of 3(x-hydroxynortropane-8-spiro-l'-pyrrolidinium heptane-su 1 phonate is stirred at ambient tem peratu re. This 15 procedure is repeated three times until all the reactants have been combined. The reaction mixture isthen boiled under reflux for 2 hours and subsequently cooled overnightto ambient temperature. The reaction mixture is then evaporated to dryness in a rotary evaporator undervacuum. The residue is purified chromatographically over a silica gel column (silica gel 60,0.063 - 0.200 mm., Merck No. 7734), the mobile phase being 1,2-dich loroethane: acetic acid: methanol: water 57:23:13:7 v/v/v/v). Yield 870 mg. (14% of 20 theory) 3ot-(4,4'-difluorobenziloyloxy)-nortropane-8-spiro-l'pyrrolidinium heptanesulphonate. After passage over a column packed with a strongly basic ion exchanger in the chloride form (Lewatit MP 500), there is obtained the title compound. The crude product is recrystallised from isopropanol, washed with ethyl acetate and dried in a vacuum under vacuum at 400C. to constantweight. Yield 470 mg. as 1: 1 mixed crystallisate with isopropanol; 25 m. p. 242 - 245'C.

F1)-MS.. m/e = 428 (molecule cation) IR (KBr):.y = 1508,1603,1733 cm-1 H-NMR (250 MHz, D20,8-values in ppm, referred toTSP=O):

8 = 1.44-1.67 (m; 2H; H-6a, H-7a); 2.00-2.20 (m; 8H; H-2a, H-4a, H-6b, H7b, WX, HA'); 2.57 and 2.64 (2 x m; 30 2H; H-2b and H-4b); 3.38 and 3.60 (2 X m; 4H; H-2', H-5'); 3.73 (m; 2H; H- 1, H-5); 5.27 (t; 1 H; H-3); 7.19 anc17.42 (2 x m; 8H; aromatic protons).

Example9

3eL-(4,4.Dimethylbenziloyloxy)-nortropane-8-spiro- l-pyrrolidinium chloride 35 The procedure is as in Example 8 but instead of 4,4'-difluorobenzilic acid there is used 4,4'-cli methyl benzilic acid as starting material (preparation analogoustoJ.G. Cannon, Org. Chem., 25,959-962/1960). Yield 1.68g.; m.p. 175'C.

FID-MS: m/e = 420 (molecule cation) 40]R (KBr): y = 1508,1612 (weak), 1718 cm-1 40 WNMR (250 MHz, D20,8-values in ppm, referred to TS1=0):

8 = 1.47-1.51 (m; 2H; H-6a, H-7a); 1.79-2.21 (m; 8H; H-2a, H-4a, H-6b, H7b, WX, HA'); 2.33 (s; 6H; 2 x CH3); 2.48-2.66 (m; 2H; H-2b, H-4b); 3.34 and 3.58 (2 x m; 4H; H-2'and H-5'); 3. 67 (m; 2H; H-1, H-5); 5.23 (t; 1 H; H-3); 7.20-7.31 (m; 8H; aromatic protons).

45 Example 10

3u--(4,4.Din-butyloxybenziloyloxy)-nortropane-8-spiro-l-pyrrolidinium chloride The procedure is as in Example8 but instead of 4,4'-difluorobenzilic acid there isused 4,4'-di-n-butyloxybenzilic acid as starting material (preparation analogousto J.G. Cannon,J. Org. Chem.25, 959-962/1960). Yield 240 mg. of crystaiswhich deliquesce atambienttemperature. 50 FID-MS: m/e = 536 (molecule cation) [R (KBr): ^1 = 1508,1580 (weak), 1608,1734 cm-1 H-NMR (250 MHz, CDC13, 3-values in ppm, referred toTSP = 0):

8 = 0.96 (t; 6H; 2 X CH3 of n-butyloxy); 1.47 (t; q; 4H; 2 X CH3 of nbutyloxy); 1.53-1.63 (m; 2H; H-6a, H-7a); 1.76 (t, t; 4H; 2 x CH3 of n-butyloxy); 1.80-2.30 (m; 8H; H-2a, H-4a, H- 6b, H-7b, WX, H4); 2.62-2.77 (m; 2H; 55 H-2b, H-4b); 3.65 and 3.99 (2 X m; 4H; H-2'and H-5'); 3.94 (t; 4H; 2 x CH3 of n-butyloxy); 4.16 (m; 2H; H-1, H-5); 5.28 W 1 H; H-3); 6.84 and 7.25 (2 x cl; 8H; aromatic protons).

Example 11

3a-(4-n-butyloxybenziloyloxy)-nortropane-8-spiro- 1'1-pyrrolidinium chloride 60 The procedure is as in Example 8 but instead of 4,4'-difluorobenzilic acid there is used 4-n-butyloxybenzilic acid as starting material (preparation analogous to C.D. Shackiett and H. A. Smith, J.A.C.S., 75 2654-2657/1953). Yield 250 mg.; m.p. 206'C.

FD-MS: m/e = 464 (molecule cation) 65]R (KBr): y = 1512,1609,1742 cm-1 65 12 GB 2 184 726 A 12 H-NMR (250 MHz, CIDC13,8-values in ppm, referred to TIVIS=O):

8 = 0.97 (t; 3H; CH3 of n-butyloxy); 1.50 (t, q; 2H; CH2 of n-butyloxy); 1.56-1.64 (m; 2H; H-6a, H-7a); 1.77 (t,t; 2H; CH2 of n-butyloxy); 1.84-2.45 (m; 8H; H-2a, H-4a, H-6b, H-7b, WX, HA'); 2.65-2.85 (m; 2H; H-2b, H-4b); 3.58 and 3.95 (2 x m; 4H; H-2'and H-5'); 3.95 (t; 2H; CH2 of n-butyloxy); 4.08 (m; 2H; H-1 and H-5); 5.30 (t; 1 H; 6.84 (d) and 7.26-7.37 (m; 9H; aromatic protons). 5 Galenical examples 1. Tablets

40 mg. azoniaspironortropanol ester according to oneof thechemical Examples 10 mg. lactose m g. starch 0.5 mg. magnesium stearate 74.5 mg. microcrystalline cellulose is 15 2. Suppositories mg. azoniaspironortropanol esteraccording to one of the chemical Examples 2 mg. "AerosiV 200 (silicic acid) 2278 mg. Witepsoi (modified triglycerides of saturated plant fatty acids) 20 3. Solution forintravenous injection mg. azoniaspironortropanot esteraccording to one of the chemical Examples 4.6 mg. citric acid monohydrate 14.8 mg. sodium citrate dihydrate ad 2 m].

25 4. Solution forintravenous infusion 500 mg. azoniaspironortropanol ester according to one of the chemical Examples mg. citric acid monohydrate 370 mg. sodium citrate dihydrate ad 50 m].

30 5. Retardform: diffusion pellets Per hard gelatine capsule:

Without With initial initial 35 dose dose sugarspheroids 150 mg. 150 mg.

azoniaspironortropanol ester 40 according to one of the 80 mg. WS 60 mg.

chemical Examples hydroxypropylcellulose (Mucel) 10 mg. 8 mg.

45 acrylic or methacrylic esters:

Endragit RL 2 mg. 2 mg.

Endragit RS 8 mg. 8 mg. 50 polyethyleneglycol (8000) 1 mg. 1 mg.

talc 5 mg. 5 mg.

55 WS 20 mg.

Klucel 2 mg.

6.Retardform: matrix tablet 60 mg. azoniaspironortropanol ester according to one of the chemical Examples mg. lactose mg. ethyl cellulose mg. starch 2 mg. magnesium stearate 65 13 GB 2 184 726 A 13 3 mg. polyethylene glycol (8000) 7. Retardform: two-layertablet with initialdose 1stlayer 2ndlayer 5 retard retard tablet tablet azoniaspironortropanol ester 60 mg. 20 mg.

10 lactose 90 mg. 10M9.

ethyl cellulose 12 mg. - starch 15 mg. 15 mg. 15 magnesium stearate 1.5 mg. 0.3 mg.

polyethylene glycol (8000) 2 mg. - 20 microcrystalline cellulose - 37.2 mg.

8. Dosed aerosol forinhalation Formulation per dosagelspray impulse:

0.1 mg. azoniaspironortropani ester according to one of the chemical Examples 25 0.02 mg. Span 85 (sorbitan mono- and trifatty acid residue based on oleicacid) KI. Frigen 11 (trichlorofluoromethane) Ki. Frigen 12 (dichlorodifluoromethane).

9. Dosedspray for nasal use 30 Formulation per dosagelspray impulse:

2 mg. azoniaspironortropanol ester according to one of the chemical Examples 90 gi. physiological saline 10. Inhalation solution 35 trospium chloride 0.100g.

citric acid monohydrate 0.470 g.

trisodium citrate dihydrate 0.530 g.

sodiumchloride 0.645 g. 40 The solution is prepared by successively dissolving the components in water, followed by sterilising f iltration and placing into 1 ig ht- protected conta iners. The pH val ue of the solution is about 4.2.

11. Dosedaerosol 45 trospium chloride 0.0309.

trichlorofiuoromethane/dichloro- difluoromethane ad 15.0 mi.

so The aerosol is prepared by grinding thetrospium chlorideto a particle size of lessthan 5 Km., suspending it in cooled and liquefied propellant gas and placing into conventional aerosol containers atabout45to 5M.

The valve on the container is so chosen that, per spray impulse, 0.1 mg. trospium chloride is applied.

Examples of compounds which can be used according to the present invention include thefollowing:

1. trospium chloride [(3oL-benzi 1 oyloxyno rtro pan e-8-spi ro-V-pyrro lid in i u m)-ch 1 o ride] 55 m.p. 258 - 26WC. (decomp.); FD-MS: m/e = 392 (molecule cation) IR (KBr): y = 3150,1735,1498,1452,747 em-' 2.3oL-benziloyloxynortropane-8-spiro-l'-(3'-pyrrolinium) chloride m.p. 2670C.; FD-MS: m/e = 390 (molecule cation) 60 IR (KBr): y = 1722,1595,1490,1445,741 em-' 3.3u.-benziloyloxynortropane-8-spiro-2'-isoindolinium chloride m.p. 263 2650C.; FD-MS: m/e = 440 (molecule cation) M (KBr): y = 1740,757,745,703 em-' 14 GB 2 184 726 A 14 4.3a-beniloyloxynortropane-8-spiro-4-morpholinium chloride m.p. 22WC. (decomp.); FD-MS: m/e = 408 (molecule cation IR (KBr): y = 3410,3183,1731,1492,703 cm-1 5.3a-benziloyloxynortropane-8-spiro-l'-pyrrolidino-[3',4'-blquinoxalinium bromide 5 m.p. 20WC. (deco m p.); FD-MS: m/e = 492 (molecule cation) [R (KBr): y = 3375,1730,1504,763 cm-1 6.3oc-benziloyloxynortropane-8-spiro-2'-(2'-aza-3H-phenolenium) bromide m.p. 322'C. (decomp.); 10 FD-MS: mle = 490 (molecule cation) ]R (KBr): y = 3428,3240,1738,1603,1497 cm-1.

Experiments were carried out on isolated rat intestine.

Animalmaterial: is Male and female Wistar rats with a body weight of 150to 250 9. The animals were acclimatised for 1 weekat --L ZC. and ata relative humidity of 50 + 10%. The room illumination was day] ig ht with additional neon tubes with a day/night illumination rhythm of 7.00 to 18.00 hours. The animals were kept in Makrolon cages type 4, each being occupied by 10 rats. The cages had a sawdust bedding. The feed was "ssniff" standard feed (Versuchstierdiaten GmbH, 4770 Soest, Germany) available ad 1 ibitu m and the drinking water, which 20 was tap waterfrom synthetic resin flasks with stainless steel drinking tubes, was available ad libitum.

Substances, dosages:

test substances: compounds of Examples 1 - 6.

solvent: demineralised water 25 concentration: 1.185 X 1 C' g./m[. bath vessel contents (against Carbachol) volume administered: 0.25 mi.

time of action before administration of spasmodic:

3 minutes further substances used: carbamoylcholine(Carbochol) hydrochloride, Merck, Darmstadt (Art No. 500940) 30 sum formula: C6H15CIN202 concentrations: 4x 10-9 g./mi. bath vessel content 2 x 10-" g./mi. bath vessel content 1 X 10-7 g.1m I. bath vessel content 5 x 10-7 g.lmi. bath vessel content 35 2.5 x 10-6 g./m]. bath vessel content 1.25 x 10-5 g.lmi. bath vessel content 6.25 X 10-5 g.lmi. bath vessel content volume administered: 0.25 m].

time of action: 5 minutes 40 Ringer's nutrient solution with the following composition:

sodium chloride = 9.000 g. (E. Merck, Darmstadt) potassium chloride = 0.210 g. (E. Merck, Darmstadt) sodium bicarbonate = 0.500 9. (E. Merck, Darmstadt) 45 glucose monohydrate = 0.500 g. (E. Merck, Darmstadt) calcium chloride monohydrate = 0.318 g. (E. Merck, Darmstadt) Carrying out of the experiments The rats were sacrificed by a neck blow. The abdomen was opened along the median line, an 50 approximately 10 cm. long piece of ileum was removed, immediately transferred to a physiological tempered nutrient solution and then completely and carefully rinsed through twice in toto with the help of a 10 m].

syringe with nutrient solution forthe removal of the intestinal contents. Forthe subsequent experiments, two pieces of intestine of 2 cm. length were separated off and the remaining piece of intestine kept in a refrigerator. Thetwo pieces of intestine were freed in nutrient solution from tissue possibly still attached 55 thereto. Around one end there was applied a sling of silkthread forfixing the piece of intestine in an organ bath, while around the other end was applied a longer thread with a connecting clamp for fixing to a recording layer. The piece of organ was thereafter filled with nutrient solution and suspended in a bath vessel with Carbogen bubbling therethroug hand loaded with 0.5 g. After a resting period of 30 minutes, the experimentcan commence. 60 There was first plotted a dosage action relationship of the spasmodic. The solution to be tested was injected by means of a tuberculin syringe with applied single-use canule into the bath liquid. Depending upon the vol u me to be injected, for the precise maintenance of the bath content there was previously always removed an equal volume of nutrient solution. Concentrations were selected which, in geometric steps of a factor of 5, displayed spasmodic effects of> 10%to 100%, the 100% effect being taken as being the limiting 65 GB 2 184 726 A 15 concentration, exceeding of which brought about nog reater effect. The limiting concentration is taken as reference value and the effects of the lower concentrations were calculated to refer to this 100% value. A complete concentration activity curve was plotted using apiece of intestine.

The period of action of the spasmodic on the organ was 5 m in utes. Thereafter, the content of the bath vessel was changed three times by rinsing and followed by a resting phase (no addition of substance) of 30 5 minutes.

After plotting of the concentration-activity relationship of the spasmodic, the antagonistic strength of action of the substance to be tested was tested. For this purpose, the test substance was injected in a constant concentration into the bath vessel content 3 minutes before application of the spasmodic. The subsequent course of the experiment corresponded to that already described: addition of spasmodic in increasing 10 concentrations, rinsing three times, 30 minute resting phase. Depending upon the effect, the concentrations of the test substance were varied, ten experiments being carried out per concentration.

Analysis andapparatus The experimental apparatus consisted of a horizontally fixed, about 66cm. long cylinder-shaped glass 15 surround with inlet and outlet taps in which were melt-sealed two pre- heating spirals which were provided on the outside with inlet pipes and each of which open downwardly into a bath vessel of 25 m[. volume closable below by stopcocks. Dem ineralised water warmed to WC. was circulated by an ultrathermostat of the firm "Colora" through the glass surrounded so that the nutrient solution present in the pre-heating spirals and bath vessels was always uniformly warmed. Incase of need, it was passed from a higher-standing 20 supply vessel via a tube system into the pre-heating spirals. On the bottom of the bath vessel, forthe continuous bubbling through the nutrient solution with Carbogen (95% oxygen and 5% carbon dioxide), there were provided gassing tubes, on the limbs of which, in the lower third thereof, were melt-sealed glass hooks onto which were suspended on one end the previously prepared piece of intestine, whereas the other end was attached with its long thread with a metal recorder lever for M P recorda 1. Finally, the loading was 25 adjusted on the recorder lever and the star recorder of the lever applied to the MP paper on the recorderdrum (diameter 200 mm.) of a kyrnograph. During the experiment, the MP paperwas rolled from the table unrolling device onto the drum. The paper movementwas 2.62 mm. x min'. The recording breadth could be regulated via an MP generatorwith incorporated potentiometer. Fora better current flow, a contact rollerwas additionally applied to the MP paper which was connected with the earthing box of the MP generator. 30 After ending of the experiment, the recordings on the MP paperwere fixed with a special fixing spray.

Ail apparatus necessaryforthe recordings were obtained from the firm Braun, Meisungen, Germany.

Evaluation For each concentration in g./mi. there was obtained the arithmetic average values and their standard devi- 35 ations (x%.t s) of the spasmodic effect.

Results A 50% spasm was obtained with Carbachol alone (blank experiment) at a concentration of 4.3 x 1C3 g./mi.

In the case of the use of the above-mentioned spasmolytically-acting test substances in a concentration of 40 1.18 X 10-8 g./m]., for the initiation of a 50% spasm, carbachol concentrations of the order of 10-6 gJMI.

were needed.

In principle, there are three possibilities for the treatment of diseases dueto asthma and for bronchial diseases: cortisone or corticosteroids, sympathomimetics and parasympatholytics. As is known, corticosteroids involve serious side effectsJor example susceptibility to infections. Sympathomimetics also 45 have considerable symptomatic side effects, for example tachycardia. Parasympatholytics, on the other hand, are characterised by a good measure of success, especially in the case of local administration, bythe absence of oronly small side effects but, on the other hand, thetherapeutic results are not uniform and not certain because of differing response of the symptoms of the disease. In this regard, reference is madetoJ.F.

Keighley, latrogenic asthma associated with adrenergic aerosols, Ann. intern. Med., 65,98511966 and F.E. 50 Speizeretal., Observations on recent increase in mortalityfrom asthma, B. M.J., 1, 33511968.

It is known that some azoniaspironortropane derivatives possess spasmolytic properties (see Federal Republic of Germany Patent Specification No. 1194 422 and Arzneirnittelforschung, 17,714-719/1967.

However, these compounds have hitherto only been used in the urogenital region.

There is a need for new asthma therapeutics and broncholyties with a parasympatholytic character of 55 action but without a systemic accompanying action, i.e. with effect on the circulatory regulation, and with a dependable action.

It is an object of the present invention to improve the treatment of asthmatic diseases and of diseases of the bronchial region.

Thus,the present invention is also concernedwith the use of azoniaspironortropanol esters of thegeneral 60 formula (1) as asthma therapeutics and as broncholytics.

In orderto confirm the effectiveness of the active materials according to the present invention, inhalative provocations were carried out on awake guinea pigswith a cholinergically- effective aerosol. 3 x 10-7 mole kg-1 of active material thereby antagonise an asthmatoid respiratory difficulty broughtabout byan acetyi-p-methylcholine aerosol 15 minutes after intraperitoneal administration. The therapeutic 65 16 GB 2 184 726 A 16 effectiveness of the active materials according to the present invention is markedly stronger than that of equimolar dosages of reference substances, such as atropine and isoproterenol.

Method Animalmaterial animal type: guinea pigs animal strain: Pirbrightwhite origin: LippischeVersuchstierzucht Hagemann GmbH t Co., 4923 External 1, Germany sex: male 10 bodyweight: about 500 - 700 g. acclimatisation time: > 8 days Animal maintenance living space: massive construction, conventional maintenance room temperature: 22 2'C.

atmospheric humidity: 50 - 60% relative humidity room illumination: artificial 12 hour rhythm cages: Macrolon lower part and wire mesh covering with feed and water containers; bedding "ssniff" from "ssniff Versuchstierdibten GmbH, 4770 Soest, Germany 20 feed: Altromin-MS from Aitrogge Spezialfutterwerk, Lage/Lippe, Germany; "ssniff"-MS diet and hay drinking water: tap water ad libitum Trospium chloride = MP 194 = 3a-benziloyloxynortropane-8-spiro-l'- pyrrolidinium chloride dehydrotrospium chloride = WG 71 = 3ot-benzi loyloxyn ortropane-8-spi rol'-(3'-pyrrol in i u m)ch lo ride 25 Substances, dosages andmode ofadministration testsubstance: trospium chloride (MP 194) (M.W. 428) dosage: 3 x 10-7 mole mi-1 kg-1 mode of administration: intraperitoneally testsubstance: dehydrotrospium chloride (WG 71) (M.W. 426) 30 dosage: 3 X 10-7 mole mi-1 kg-1 mode of administration: intraperitoneally referencesubstance: atropine hydrochloride (Serva; MW 325.8) dosage: 3 X 10-7 mole m]-' kg-1 modeof administration: intraperitoneally 35 reference substance: isoproterenol (Fluka; MW 247.72) dosage: 3 x 10-1 mole mi-1 kg-1 mode of administration: intraperitoneally control substance: physiological saline dosage: 1 mi. kg-1 40 mode of administration: intraperitoneally further substances: acetyi-p-methylcholine chloride (Sigma; MW 195.7) concentrations: 0.0316 g. x 100 ml-l double distilledwater 0.0562 g. x 100 mi-1 double distilled water 0.1 g. X 100 mi-1 double distilledwater 45 0.178 9. X 100 mi-1 double distilledwater 0.316 9. X 100 m[-' double distilledwater 0.562 g. X 100 mi-1 double distilledwater mode of administration: 0.5 ml. min-1 by inhalation.

so 50 Grouping distribution to the groups: random animals pergroup: 10 group division: as far as possible, on one day, animals of the experimental and control groups are taken into the experiment. 55 Carrying out of the experiments The guinea pigs intended for an experiment are, after an acclimatisation time of at least 8 days, subjected twice to an aerosol of 0.1 % acetyi-p-methylcholine chloride solution since, as is known from experience, during the firsttwo inhalative provocations, the animals reactwith more distinct respiratory disturbances 60 than in the case of the subsequent provocations (adaption). If, in the case of the two inhalation phases, a non-sensitivity (absence of respiratory disturbances) is observed towards the exposure, these animals are excluded from the actual experiment.

Forthe purpose of aerosol provocation, the guinea pigs are placed individually in an inhalation chamber (see Section 3.6 hereinafter) in which 0.5 mi. of solution per minute are atomised as droplet aerosol by means 65 17 GB 2 184 726 A 17 of a special nozzle (Rhema, Hofheim, Germany). Dependent upon the active material concentration, as well as of a pre-treatment possibly carried out, the aerosol exposure leads to a more or less marked dyspnoea, to attacks of coughing and finally to asphyxia and I oss of consciousness following a tonic-clonic cramp of differeing strength. With the help of a stopwatch, there is recorded the time from the commencement of inhalation to the appearance of the asphyctie state; the animals are i m mediately removed from the inhalation 5 chamber and, as a rule, recover in a very short period of time (recovery of consciousness and normalisation of breathing). If, within 180 seconds, no dyspnoeaoccurs, the inhalation is discontinued.

In order to demonstrate the protective action of the test and reference substances, the animals in the experiment, 15 minutes before the commencement of the inhalation, are pre- treated with these substances according to their body weight (control animals correspondingly with isotonic sodium chloride solution) and 10 subjected to logarithmically graduated concentrations of acetyl-p- methylcholine aerosol. One aerosol concentration is tested per test day; the time between the individual aerosol provocations is at least 1 week.

Analyses and apparatus The inhalation chamber is a Plexig lass container specially made forthis purpose, the lid of which can be 15 closed in an air-tight manner by means of rubber sealing and grip closure means. The internal measurements of the chamber are 285 X 190 X 180 m m., which correspondsto a volume of about 9.751itres. The specia I nozzle (Rhema, Hofheim, Germany, order No. 504104) is fixed to a recess on the I id and ensures a uniform supply of the available cha m ber space with the aerosol. The provocation solution is supplied to the nozzle via an infusion pump (Braun, Meisungen, Germany) (0.5 m]./minute) and there atomised with a 20 superpressure of 180 kPa from an attached pressure gas bottle (artificial air, KW-free). For reasons of safety, the aerosol provocation is carried out under a ventilator.

Evaluation For each of the tested substances (test, reference and control substances) there is taken, in the case of each 25 investigated active material concentration, the percentage proportion of the animals reacting with dyspnoea forthe calculation of the EC50.

The EC50, as well asthe related confidence interval (p >95%), are determined from the probit regression lines of the percentage values (v. supra) provided with weight coefficients after line adaptation bythe "maximum likelihood" method (10 iterations). Furthermore, there is examined the adaption of the linesto 30 the observed data by means of the chiquadrattest. Forthe evaluation, a calculation programme is commercially available (Olivetti).

Results Thetested substances, trospium chlorideaswell as dehydrotrospium chloride, show, after i ntra peritonea 1 35 administration, an outstanding broncholytic effect in the case of cholinergically-induced bronchial cramps on the awake guinea pig. The average effective concentration (EC50) of acetyi-p-methylcholine chloride in the solution to be atomised is, in the case of the control animals, w = 0. 00054 (seethe following Table 1). After pre-treatment with trospium chloride or dehydrotrospium chloride, the corresponding EC50 values are w = 0.00286 and w = 0.00173, respectively (see the following Tables 2 and 3). 40 Atropine and isoproterenol were used as reference substances. For atropine and isoproterenol, there were determined average effective concentrations of the provocation substances of w = 0.00138 and w = 0.00145, respectively (see the following Tables 4 and 5).

Furthermore, the average effective concentrations EC50 of the provocation substances 15 minutes after intraperitoneal administration of MP 194, WG 71, atropine and isoproterenol are illustrated schematically in 45 Figure 1 of the accompanying drawings.

Table 1

Control substance: NaCI (W = 0.009) 50 mode of administration: intraperitoneal dosage: 1 mi kg-1 active material: acetyi-p-methylcholine chloride mode of administration: by aerosol inhalation concentration: see below 55 active numberofanimals probit reaction % material analysis calculated concentration tested reacting observed W 60 0.000316 10 2 20.00 22.79 0.000562 10 5 50.00 52.35 0.000750 10 7 70.00 67.81 0.001000 10 9 90.00 80.64 0.001780 10 9 90.00 95.26 65 18 GB 2 184 726 A 18 Testfor linearity: X2 = 1.2629 (FG = 3) linearity can be assumed (a E: 0.05) Result of theprobit analysis Average effective concentration of the active material: 5 EC50: w = 0.000539 confidence interval (P = 0.95): 0.0003390.000855 Table 2

10 testsubstance: trospium chloride mode of administration: intraperitoneal dosage: 3 X 10-7 Moi m[-' kg-1 active material: acetyl-p-methylcholine chloride mode of administration: by aerosol inhalation 15 concentration: see below active number of animals probit reaction % material analysis calculated concentration tested reacting observed 20 W 0.000562 10 0 2.50 5.46 0.001000 10 1 10.00 15.03 0.001780 10 5 50.00 32.00 25 0.003160 10 5 50,00 53.87 0.005620 10 7 70.00 74.66 correction according to Bliss testfor linearity: X2 = 2.0313 (FG = 3) 30 linearity can be assumed (u. 0.05) Resultof theprobitanalysis average effective concentration of the active material EC50: w = 0.00286 35 confidence interval (P = 0.95): 0.00147- 0.00557 Table 3 testsubstance: dehydrotrospium chloride 40 mode of administration: intraperitoneal dosage: 3 x 10-7 Moi mi-1 kg-1 active material: acetyl-p-methylcholine chloride mode of administration: by aerosol inhalation concentration: see below 45 active number of animals probit reaction % material analysis calculated concentration tested reacting observed so W 50 0.001000 10 3 30.00 38.74 0.001780 9 6 66.67 50.60 0.003160 10 6 60.00 62.35 0.005620 10 7 70.00 73.08 55 testfor linearity: X2 = 1.3234 (FG = 2) linearity can be assumed (at 0. 05) Resultoftheprobitanalysis 60 average effective concentration of the active material EC50: w = 0.00173 confidence interval (P = 0.95): 0.00031 - 0.00972 19 GB 2 184 726 A 19 Table 4 reference substance: atropine chloride mode of administration: intraperitoneal dosage: 3 x 10-7 Moi mi-1 kg-1 5 active material: acetyl-p-methylcholine chloride mode of administration: by aerosol inhalation concentration: see below active number of animals probit reaction % 10 material analysis calculated concentration tested reacting observed W 0.000562 10 3 30.00 38.93 15 0.001000 10 6 60.00 45.98 0.001780 9 5 55.56 53.16 0.003160 10 5 50.00 60.20 0.005620 10 7 70.00 66.95 20 testfor linearity: X2 = 1.6245 (FG = 3) linearity can be assumed (ot a 0.05) Result of theprobit analysis average effective concentration of the active material 25 EC50: w = 0.00 138 confidence interval (P = 0.95): 0.00022 - 0.00888 Table 5

30 reference substance: isoproterenol mode of administration: intraperitoneal dosage: 3 x 10-7 Mol mi-1 kg-1 active material: acetyl-p-methylcholine chloride mode of administration: by aerosol inhalation 35 concentration: see below active number of animals probit reaction % material analysis calculated concentration tested reacting observed 40 W 0.001000 9 3 33.33 36.21 0.001780 8 5 62.50 57.88 0.003160 9 7 77.78 77.28 45 0.005620 9 8 88.89 90.30 test fo r 1 i n ea rity: X2 = 0. 1240 (FG = 2) linearity can be assumed (a 2t 0.05) so 50 Result of the probit analysis average effective concentration of the active material EC50: w = 0.00145 confidence interval (P = 0.95): 0.00050- 0.00426 55 Assessment The test substances trospium chloride and dehydrotrospium chloride are, 15 minutes after intraperitoneal administration, comparable with or superiorto the well known pa rasym path olytics atropine and the P-sympathomimetic isoproterenol with regard to their broncholytic effectiveness (see Figure 1 of the accompanying drawings). From the calculations of the concentrations of acetyl-p-methylcholine chloride 60 which, in each case, are on average effective, which is necessary for the inhalation of a dyspnoea, there can be ascertained a greater effectiveness of the test substances, especially of trospium chloride, in comparison with the reference substances.

In orderto testthe receptor specificity of the compounds according to the present invention, the specificity of the chol inerg ic antagon isation must be demonstrated. A model for this is the measurement of the 65 GB 2 184 726 A 20 anticholinergic effectiveness on isolated tracheal spirals of the guinea pig.

The test substance used was 3ot-benziloyloxynortropane-8-spiro-l'pyrrolidiniu m chloride (trospiu m chloride; MP 194), ipratropium bromide being used as comparison substance.

1. Summary 5

MP 194 is competitively antagonistically effective on the isolated tracheal spirals of the guinea pig in comparison with acetyi-p-m ethyl ch ol ine ch loride. Its strength of activity is thereby equivalent to the reference su bsta nce i pratropium bromide but, depending upon the mode of admi nistration and upon the dosage (see Rote Liste, 1985, No. 09015), the usual side effects of ipratropium bromide are considerably reduced or do not occu r at ail. 10 2. Interrogatory There is to be determined the anticholinergic effectiveness of MP 194 in comparison with the reference substance ipratropium bromide on the isolated tracheal spirals of the guinea pig.

15 3. Method 3.1 Animal material 3.1.1. animal species: guinea pig 3.1.2. animal strain: Pirbrightwhite 3.1.3. origin: Hagemann GmbH & Co., 4923 Extertal 1, Germany 20 3.1.4. sex: male 3.1.5. bodyweight: about 500 g.

3.1.6. acclimatisation time: > 8 days 3.2. Animal maintenance 3.2.1. living space: massive construction, conventional maintenance 25 3.2.2. room temperature: 22 -t 2'C.

3.2.3. relative atmospheric humidity: 50 15% 3.2.4. room illumination: artificial dark/light rhythm in 12 hour intervals 3.2.5. animal cages: Makrolon lower part andwire mesh covering with feed and water containers; bedding "ssniff" (Versuchstierdijten GmbH, 4770 Soest, Germany) 30 3.2.6. feed: "ssniff" guinea pig diet 3.2.7. drinking water: tap water ad iibitum 3.3. Substances, dosaging and mode of administration 3.3.1. test substance (test antagonist) trospium chloride (MP 194) (M.W. 428) 35 solvent: tyrode solution bath concentrations: 1 x 10-9 M/m]. bath solution 3.16 x 10-9 M1mL bath solution 1 x 10-8 M/mi. bath solution 1 X 10-7 M/m I. bath solution 40 administration volume: 50 KI.128 mi. bath solution 3.3.2. reference substance (reference antagonist) ipratropium bromide (Atrovent) (M.W. 412.4) solvent: tyrode solution bath concentrations: 1 x 10-9 M/m]. bath solution 45 3.16 x 10-9 M/mi. bath solution 1 x 10-8 M/mi. bath solution 1 X 10-7 M/mi. bath so] ution administration volume: 50 [L1,128 m[. bath solution.

3.3.3. further substances (reference antagonists) 50 3.3.3.1. acetyl-p-methylcholine chloride (Sigma) (M.W. 195.7) solvent: tyrode solution bath concentrations: 1 X 10-7 M/m]. bath solution 1 X 10-6 M/mi. bath solution 1 x 10-5 M/mi. bath solution 55 1 X 10-4 M/mi. bath solution 1 X 10-3 M/m]. bath solution 1 X 10-2 M/mi. bath solution 3.16 x 10-2 M/mi. bath solution administration volume: 501i1J28 mi. bath solution, cumulative 60 3.3.3.2. tyrode solution as nutrient medium 21 GB 2 184 726 A 21 component mMolell. stocksolution mL stock solutionI litre tyrode solution NaCI 139.2 58.00 g/1 (1 M) 139.2 m 1.

KC] 2.7 74.56 g/1 (1 M) 2.7 m 1.

CaC12.2H20 1.8 147.00 g/1 (1 M) 1.8 m I.

M9C12.6H20 0.245 99.62 g/] (0.49 M) 0.49 m].

NaHC03 11.9 21.00 g/] (0.25 M) 47.6 m]. 10 Nal-12P04.1---1200.4 4.00 g/1 (0.03 M) 15.6 mi.

C6H1206 5.5 - 1.0 g.

double distilled water ad 1000 mi.

Calcium chloride is hydroscopic. Therefore, the stock solution must be titrated with the help of a is Chlor-o-Counter (Marius-Chlor-o-Counter, Kipp and Zonen, 6242 Sch6nberg/Taunus, Germany).

in the mixing of the various stock solutions, it is to be noted that calcium precipitates outwith bicarbonate and phosphate when the solutions are mixed together in high concentration. This is avoided byfirst diluting the 1.8 m]. of calcium chloride parent solution with about 100 mi. of double distilled water, the otherstock solutions in a measurement flask already having been substantially made up with double distilled waterand 20 onlythen adding the calcium solution.

3.4. Grouping 3.4.1. division into groups: random 3.4.2. number of preparations: 25 of the test substance group: n = 4 (1 x 10-9M) n = 2 (3.16 x 1 0-91V1) n = 2 (1 x 1 0-"M) n = 4 (1 X 10-7M) of the reference substance 30 group: n = 4 (1 x 10-9M) n = 2 (3.16 X 1 0-91V1) n = 2 (1 x 1 0-"M) n = 4 (1 X 10-7M).

35 3.5. Carrying out of the experiments The guinea pig is stunned by a blow on the neck. Subsequently, the whole of the trachea is roughlyfreed beginning from the larynx up the tracheal bifurcation, removed and transferred to tempered (37'C.) and carbogenised tyrode solution. After surrounding connective tissue has been removed as far as possible,the preparation is cut up spirally by means of fine scissors at an angle of about 450 and separated into two equal 40 sized sections. Afterweighing, both preparations are provided proximally and distallywith a silkthread. One thread servesforfixing the preparation by means of a loopto the bottom of the bath and the otheris connected via a hookwith thetransducer above the bathvessel.

Subsequently, the preparations, corresponding to the calibration, are prestressed with about80 mN and equilibrated from 50- 100 minutes. During the equilibration phase, the nutrient solution in the bath vessels is 45 renewed in 15 minute intervals. As soon as the resting muscle tonus of the preparation has stabilised, there takes placethe cumulative addition of the agonist, wherebythe addition of the next highest concentration firsttakes place when no further increase of contraction is recognisable (plateau). When the maximum contraction height of the preparation is achieved,the cumulative agonist addition is ended andthe preparation is rinsed. After a further equilibration phase (v. supra), the cumulative addition of the agonistis 50 repeated butthis time in the presence of thetest or reference antagonist.

3.6Analyses andapparatus 3.6. 1. The perfusion pa rt consists of an L-shaped orga n bath in the hollow space of which (longer limb) ru ns a double g lass spi ral th roug h wh ich the nutrient sol ution is passed into the actual bath vessel (28 m 1. content; 55 shorter 1 i m b), Th is bath vessel is divided i nto two cham bers wh ich are, however, con nected together by two transverse connections. Thus, the supply of the organ with Carbogen (95% oxygen and 5% carbon dioxide) can take place indirectly from the smaller rearmost of the two cham bers, whereby the orga n does not hang directly in the Carbogen inflow wh ich, inter alia, makes possible a more precise recordal of the orga n reactions. The inlet chambers as well as the feeding glass spirals are tempered from the outside to 37'C. by a 60 separate liquid circu lation. This tem pering takes place with the hel p of a "Colora " u ltra-thermostat type K (Col ora Messtech nik G mbH, Dusseldorf, Germa ny) wh ich serves as thermostat and pum p. In order, in case of need, always to have available ready-for-use nutrient solution, above the organ bath is provided a dou ble-wal led storage container in which the nutrient sol ution is a Iso tempered and carbogenised. This is connected via a glass stopcock and a polypropylene tubewith double glass spiral in the interior of the organ bath. 65 22 GB 2 184 726 A 22 3.6.2. The measurement and recording part includes a transducer (StathamUniversa]-Zelle UC-2; Hugo Sachs ElektronicKG, H ugStetten, Germany). By means of a hang ing-in weight, a force of 40 m N is produced on the transducer which passes as electrical signal via a connecting cable to abridge amplifier. The am pi if ication is smoothly so regulated that the force provided corresponds to a constant value on the scale or an analogous value on the millimeter paper of the recorder (dependent upon the amplification; see below). 5 Afterfixing the organ, the preparation is prestressed to the double mark, corresponding to 80 m N. The recorder connected with the amplifier (He] lige, Freiburg/Breisgau, Germany) records all analogue signals on thermosensitive paper with m illimeter divisions. The recorder amplification is thereby so regulated thatthe pulling force of the weight (40 m N) on the transducer corresponds to an indicator stroke of 4cm. (calibration:

force produced by means of the weight -A 4cm. on the analogue protocol). 10 3.7 Evaluation The cumulative addition of the agonist leads on the isolated tracheal spiral to a dosage-dependent contraction force increase which is recorded proportionally on the analogue recorder (see calibration). From these analogue protocois is carried outthe quantitative evaluation of the cumulative dosage action curves according to the method of van Rossum (Cumulative dose-response curves. il. Technique forthe making of sode- response curves in isolated organs and the evaluation of drug parameters, Arch. int. Pharmacodyn., 143,299-330/1963). Forthis purpose, the absolute measurement data (in [mml) is firstconverted on the basis of the maximum effect (EA.. or EAnA, which is taken as being 100%, into percentage values. By means of non-linear regression, from these data there is determined for each individual preparation the ratio of the 20 molar concentrations of the agonists (quotient = x) which are necessary in order preciselyto achieve half of the maximum effect in the presence and absence of the test or reference antagonists of the molar concentration [B] (-log [B] = pAJ. On the basis of the formula pA2 = pAx + log (x- 1) (see H.O. Schild, pA, a new scale for measurement of drug antagonism, Brit.J.Pharmacol., 2, 189/1947; E.J. Ariens and J.M.van Rossum, pDx, pAx and pDx values in the analysis of pharmacodynamics, Arch. int.Pharmacodyn., 110, 25 275-30011957), there is determined the negative decadic logarithm of the molar antagonist concentration (pA2) in the case of which x corresponds to the value of 2, i.e. in the case of the presence of antagonists inthe appropriate molar concentration, the molar agonist concentration must be doubled in orderto achievethe same effect as withoutthe action of the antagonists. The quality of the antagonism (competitive/non-competitive) is tested statistically on the basis of the comparison of the maximum effect in 30 the absence (EA,) and presence (EAn,13) of the test or reference antagonists. (Mestwith paired arrangement).

Finally, the difference of the pA2 t s between testand reference antagonist is examined for significance (t-test of two independent samples).

4. Results 35 MP 194 and ipratropium bromide (Atrovent) show on isolated tracheal spirals from the guinea pig a comparable, dosage-dependent antagonism against the chol inergic agonist acetyi-p-methylchol ine ch loride (see Fig ure 2 of the accom panyi ng drawings). The pA2 -L s determined for M P 194 of 9.26 0.29 does not differ significantly from the pA2 s for ipratropium bromide of 9.31 0. 39(seeTablei).

The quality of the antagonism is competitive not only the case of MP 194 but also in the case of ipratropium 40 bromide, on the basis of the comparison between the maximum effects in the case of the absence and presence of the antagonist in question (heethe following Table 6).

5.Assessment The investigation demonstrates the clear anticholinergic effectiveness of MP 194 on isolated tracheal 45 spirals from the guinea pig and thus supplements the earlier investigations of effectiveness on the awake animal. The better quantification of the results of the in vitro models also permits the conclusion that MP 194 is, with regard to the strength of action, equal to the reference substance ipratropium bromide (seethe pA2 values). Furthermore, on the basis of the investigation, a competitive antagonism of both substances can be assumed (see EAmB/EAm). so Table 6

MP 194 ipratropium bromide 55 pA2.t S 9.26 k 0.29 9.31 0.39 EA B/EAm - S 1.09 L 0.22 1.10-0.24 23 GB 2 184 726 A 23

Claims (18)

1. Process for the preparation of azoniaspironortropanol esters of the general formula:- R N A 5 10 OH 0 R

2 15 0 R wherein R signifies one of the following radicals: 20 a) an alkylene radical of the general formula:R3 1 -(UH2)n-uH-(uH2),- 25 in which R3 is a hydrogen atom or an alkyl, benzy], aryl or alkoxy carbonyl radical and n is a whole numberof from 1 to 4, b) an alkenylene radical of the general formula:

30 R4 R, C=C 1-11 35 -(CH2)n (C H 2)n in which R4 and R5, which can be the same or different, are hydrogen atoms or alkyl or alkenyl radicals and n is a whole nu mber of from 1 to 4, c) an azaalkylene radical of the general formula:- 40 R6 1 -(CH2)n-N-(CH2)n 45 in which R6 is a hydrogen atom or an alkyl, alkoxycarbonyl or acyl radical and n is a whole number of from 2to 4, cl) an oxaalkylene radical of the general formula:

-(CH2),-0-(CH2)n- 50 in which n is a whole number of from 2 to 4, e) an epoxyalkylene radical of theformula:- -CH2 - CH - CH - CH2- 55 0 f) an o-phenylene radical of the general formula:- 24 GB 2 184 726 A 24 X Y 5 -H 2 c CH 2_ g) a peri-naphthylene radical of the general formula. - 10 X Y or H 2 c CH 2_ 15 h) a 2,3-quinoxalinene radical of the genera I formula 20 X Y 25 N N /i -H 2 c CH 2_ 30 in which in formulae f) to h), the symbols X and Y, which can be the same or different, are hydrogen atoms or alkyl or alkoxy radicals; 35 and wherein R, and R2, which can be the same or different, are hydrogen or halogen atoms or a I kyl,alkoxy, alkoyi,cyclohexy], phenyl, a] kylphenyi,alkoxyphenyl, halophenyl,thienyl or fury] radicals, the a lkyl moieties in the said radicals containing up to 6 carbon atoms and being straight- chained or branched, and 0 isthe anion of a mono-to tribasic mineral acid, by a) demethylation of tropine to give nortropine, 40 b) reaction of nortropine with a dihalide to give a corresponding azonia compound, and c) esterification of the azonia compound, wherein A) the demethylation of tropine of the formula:

H c 45 N 50 OH 55 is carried o ut either by wo rki n g i n a Cl -C3-ch lo roa 1 ka ne wh ich co nta i ns at least o ne trich 1 oromethyl rad ica 1 i n the presence of a n oxidation ag ent in basic aq ueo us so] ution or th e tro pi ne is reacted with a ch 1 o rofo rmic acid ester i n a n i n ert solvent i n th e p resence of a n acid-bi ndin g ag ent to g ive a n 8-a 1 koxyca rbo nyl no rtropi ne and this is hyd rolysed with a base i n aq ueo us so] utio n, B) the n o rtropine th us o bta i ned of the fo rm u 1 a: 60 GB 2 184 726 A 25 H N \ 5 OH is reacted at ambient temperature for 'I or more days in a dipolar aprotic solvent with a compound of the general formula:

A-R-A 15 in which A and R havethe above-given meanings, in the presence of a secondary ortertiary amineand C) the compound thus obtained of the general formula:- R A 0- 20 N E ' 1-1 H 25 j OH 30 in which R and A19 have the a bove-g iven mean ings, is esterified in an anhydrous, dipolar, aprotic solvent with an imidazolide of the general formula:

OH 1 35 C C - R2 (V) 11 1 0 R, 40 inwhich R, and R2 have the above-given meanings, in the presence of a catalyst, and D) when the radical R contains one or more olefinic double bonds in the azonium ring after passing through steps B andor C, this unsaturated compound is optionally hydrogenated in a polar solvent with the help of a noble metal catalystto give the corresponding saturated compound of general formula fflin which R is a radical a) as defined hereinbefore. 45 2. Process according to claim 1, wherein in the first variant of step A, the demethyiation is carried outwith potassium ferricyanide, chloroform and sodium hydroxide.

3. Process according to claim 1 or 2, wherein in the first variant of step A, there is used a 1 to 5 fold molar amount of chloroalkane, referred to the tropine.

4. Process according to any of the preceding claims, wherein the reaction temperature in the first.variant 50 of step A is from 20 to 300C.

5. Process according to claim 1, wherein, in the second variant of step A, the reaction is carried out in chloroform in the presence of an alkali metal hydrogen carbonate.

6. Process according to any of the preceding claims, wherein instep B the ratio of nortropine:amine:dihalideisl:2:4. 55

7. Process according to any of the preceding claims, wherein the amine used instep B is diethylamine.

8. Process according to any of the preceding claims, wherein the catalyst used instep C is 4-(N,N-dimethylamino)-pyridine.

9. Process according to any of the preceding claims, wherein the dipolar aprotic solvent used instep C is acetonitrile. 60

10. Process according to any of the preceding claims, wherein the dipolar aprotic solvent used instep B is dimethy[formamide and/or acetonitrile and/or chloroform.

11. Process according to any of the preceding claims, wherein instep D the hydrogenation is carried out in water or in an alcohol containing up to 4 carbon atoms in the presence of platinum dioxide or palladium on activecharcoal. 65 26 GB 2 184 726 A 26

12. Process according to claim 11, wherein the alcohol used is methanol.

13. Process according to claim 1 for the preparation of azoniaspironortropanol esters, substantially as hereinbefore described and exemplified.

14. Azon iaspi ronortropanol esters, whenever prepared by the process according to any of claims 1 to 13.

15. Azoniaspironortropanol esters of the general formula:- - 5 0R +0 Ae N 10 H OH 0 1 15 1 2 R, 20 wherein R, R,, R2 and A8 have the same meanings as in claim 1, but excluding the following compounds:

azoniaspiro-[3a-phenylglycoloyloxynortropan-8,1'-pyrrolidineI chloride, azoniaspiro-[3ct-diphenyigiycoloyloxynortropan-8,1'-pyrrolidine1 chloride, 3ot-phenyigiycoloyloxynortropan-8-spiroisoindolinium chloride, 3(x-diphenyigiycoloyloxynortropan-8-spiroisoindolinium chloride, 25 3a-phenylgiycoloyloxynortropan-8-spiro-4'-morpholinium chloride, 3ot-diphenyiglycoloyloxynortropan-8-spiro-4'-morpholinium chloride, azoniaspiro-[3ot-cyclohexylphenyigiycoloyinortropan-8,1'-pyrrolidineI chloride, azoniaspiro-[3a-phenylgiycoloyloxynortropan-8,1'-piperidine1 chloride and azoniaspiro-[3cL-diphenyigiycoloyloxynortropan-8,1'-piperidinej chloride. 30

16. Azoniaspironortropanol esters according to claim 15 which are hereinbefore specifically exemplified.

17. Pharmaceutical compositions containing at least one compound according to claim 15 or 16 in admixture with conventional pharmaceutical carriers and/or additives.

18. The use of compounds of general formulaffi given in claim 1 as a broncholytics orforthe treatment of asthma. 35 a) demethylation of tropine to give nortropine, b) reaction of nortropine with a dihalide to give a corresponding azonia compound, and c) esterification of the azonia with an aryl imidazolide compound, using specified conditions in each step.

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Published by The Patent Office, 25 Southampton Buildings, London WC2A JAY, from which copies maybe obtained.