GB2111489A - N-isopropyl-noratropine-N- oxide - Google Patents

Abstract

The title compound of formula (I> <IMAGE> has marked antibronchospastic activity with extremely low toxicity.

Description

SPECIFICATION New derivative of nor-atropine with anticholinergic activity The present invention relates to a new N-substituted derivative of nor-atropine, processes for its preparation, and pharmaceutical compositions containing it. Particularly, the present invention relates to the compound N-isopropyl-nor-atropine-N-oxide, having formula (I)

its acid addition salts, processes for their preparation and relative pharmaceutical compositions. It is known that some derivatives of isopropyl-nor-atropine are anticholinergic agents endowed with a spasmolytic action at the bronchial smooth musculature level, whereby they are therapeutically used as bronchodilators.

It has now been found that N-isopropyl-nor-atropine-N-oxide, as well as its pharmaceutically acceptable addition salts, exerts a remarkable antibronchospastic activity which together with the extremely low toxicity of the compound confers to the latter a surprisingly high therapeutic index.

The compound having general formula (I) can be prepared, according to the invention, starting from isopropyl-nor-atropine (II) via N-oxidation, according to the following reaction scheme:

N-oxidation of isopropyl-nor-atropine may be carried out with suitable reagents such as those commonly used for the preparation of N-oxides of tertiary amines, such as, for example, hydrogen peroxide, peracetic acid, p-nitro perbenzoic acid, m-chloro perbenzoic acid, metal peroxides or other typical N-oxidizing agents.

The reaction can be carried out in the absence or presence of protic or aprotic solvents, such as H20, alcohols (methyl, ethyl or higher), CH2CI2, CHCl3, acetone, ethyl acetate, acetonitrile, benzene, toluene, dimethylformamide, dimethylsulfoxide and acetic acid.

The reaction temperature can vary from OOC to the solvent's boiling temperature (generally between 0 and 600 C). The isopropyl-nor-atropine (II) used as starting compound is a well known compound, which can be prepared, for instance, according to method described by Schulz et al.

(Arzneim. Forsch. 26, 960 (1976).

The compound of the present invention, as is well shown by the structural formula I, is the biologically active isomer wherein the bond between the nitrogen atom and the isopropyl residue, in position 8, is directed towards the piperidine ring. In order to obtain this, it is necessary to synthetize ex novo the atropine nucleus, as from the above mentioned literature.

The compound (I) can form addition salts with different organic or inorganic salts. Particularly useful salts of this kind, are the acid addition salts with inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric and hydrobromic acids, or with organic acids such as oxalic, maleic, fumaric, malic, tartaric, citric and ascorbic acids.

These acid addition salts can be easily prepared by well known methods, such as, for instance, the addition of the acid (in an amount comprised between an equimolar and an excess amount) to a solution of compound (I) dissolved in a suitable organic solvent, such as methanol, ethanol, isopropanol and acetone.

The present invention is illustrated in greater detail by the following non limitative examples.

EXAMPLE 1 Isopropyl-nor-atropine (31.7 g, 0.1 moles) and 30% H202 (250 ml) were charged in a reaction flask with magnetic stirring. The reaction was left, with stirring, for three days at room temperature and the reaction mixture was then evaporated under reduced pressure. The residue was dissolved in about 800 ml of aceton and acidified to pH 1-0 with 35% HCI. The solid obtained was filtered and washed with acetone. After drying, 33.5 g of a white crystalline product melting at 223-225 C were obtained (90.5% yield). The product was characterised as follows: Spectrum I.R. (KBr) 3400 cm-1 (broad) 2970 cm-1 (P.E. mod. 297) (broad) 2800 cm-l (broad) 1735 cm-1 1430cm-1, 1165 cm-1, 1040cm-1, 745cm-1 SpectrumN.M.R. 1H-NMR (P.E. R-24B) 60 MHz (DMSO-d6) ppm, ) = 7,3 (s, 5H aromatics), 5,0 (t, 1 H, -CH), 4,5-3,5 [m, 8H = 6H (CH+CH2)+2H(OH)], 2,7-1,7 (m,8h, CH2-), 1,3(d, 6H, CH-CH3) Elementary analysis for C19H28CíNO4 (M.W. = 369,86) calcd. % C = 61,69; H = 7,63; N = 3,79 found % C = 61,58; H = 7,64; N = 3,69.

EXAMPLE 2 Methanol (200 ml) and isopropyl-nor-atropine (31.7 g, 0.1 moles) were charged in a magnetically stirred reaction flask with an external ice-water bath.

The reaction mixture was stirred till complete dissolution, then cooled to OOC, and keeping at this temperature, peracetic acid (38 g) was slowly charged therein.

When the addition was over, the reaction mixture was stirred overnight allowing the temperature to rise to room temperature; the reaction mixture was then neutralised with sodium bicarbonate, filtered to remove the residue, and evaporated. The residue was treated with acetone/HCI as in Example 1 to give, after washing and drying, 34.3 g of product (yield 92.7%) melting at 222224aC, characterised as in Example 1.

EXAMPLE 3 Methylene chloride (1700 ml) and isopropyl-nor-atropine (15.9 g, 0.05 moles) were charged in a magnetically stirred reaction flask. When the dissolution was over,11.5 g of p-nitroperbenzoic acid were slowly charged at room temperature. During the addition, a slight exothermic reaction (maximum temperature = 35 C), was noticed. After the addition, the reaction mixture was stirred overnight, the solvent was then evaporated under reduced pressure, the residue was treated with acetone/HCI as in Examples 1 and 2, and, in a similar way, 33.7 g (yield 91.1%) of product with m.p.223-225 , with the same characteristics of that Example 1, were obtained.

EXAMPLE 4 The process was completely analogous to that of Example 3, butp-nitroperbenzoic acid was substituted by m-chloroperbenzoic acid (10.8 g), 33,9 g (yield 91.6%) of product melting at 223-225 C with the same characteristics of that of Example 1 were obtained. N-isopropyl-nor atropine-N-oxide, prepared according to the present invention, has been characterised from the toxicological-pharmacological point of view by the following tests: - assessment of the toxicity after single administration (LD50 value); - in vivo action in bronchospasm induced by acetylcholine in the anesthetized guinea-pig; - in vivo action in bronchospasm induced in the anesthetized guinea-pig by the test "acetylcholine aerosol"; - in vitro action on isolated guinea-pig ileum.

In these studies, the activity of compound (I) has been compared with that of ipratropium bromide, a structurally similar compound, known as being anticholinergic.

The results obtained are hereinafter described.

Acute toxicity The toxicity after single administration has been assessed in two different animal species: - male mice, Swiss strain, weighing 20-23 g, by two different administration routes, using in each test 4 or 5 groups with 6-10 animals each; in tests for oral administration the animal fasted, with water ad libitum, for 18 hours before the experiment.

- Male guinea-pigs, Dunkin-Hartley strain, weighing 460-565 g, by the endovenous route, using 3 groups of 6 animals each.

The results of LD50 value, calculated according to Litchfield-Wilcoxon (J. Pharm. Exp. Ther. 96, 99, (1949)), are reported in Table 1, in comparison with those obtained with ipratropium bromide, under the same experimental conditions.

TABLE 1

LD50 Compound Animal species endovenous route oral route Isopropyl nor-atropine N-oxide HCI mouse 195 2.250 (214-178) (2.594-1951) guinea-pig 160 (168-162) ipratropium bromide mouse 13, 2 265 (14,5-12,0) (349- guinea-pig 15,6 (17,2-14, 1) As is evident by the results expressed in the Table, N-isopropyl-nor-atropine-N-oxide is remarkably less toxic than ipratropium bromide.

Action in the bronchospasm by acetylcholine in the guinea pig Male whitish guinea-plgs (Dunkin-Hartley strain), weighing 340-350g, were anesthetized, thermoregulated at 37 C and kept in conditions of constant ventilation. The activity of the compounds under examination was evaluated by measuring the percent inhibition on the bronchospasm induced by endovenous administration of acetylcholine (20-30 g/kg), according to the method described by Konzett-Rössler (Versuchsanordnung zu Untersuchungen an der Bronchialmuskulatur-Arch. Exp.

Pathol. Pharmakol. 195, 71, (1940)). The compounds were administered: (a) as a solution in the jugular vein (b) as a micronized powder directly in the animal's respiratory tree. IDso values (dose producing 50% of maximum inhibition of bronchospasm), determined by the dose-response curve, gave the following::

ID50 nmol/kg Compound endovenous route endotracheal route Isopropyl-nor atropi ne-N-oxi de HCI 9,2 8,9 (7,4-11,5) (7,2-10,9) Ipratropium bromide 2,3 2,8 (2,0-2,5) | (2,5-3,2) Antibronchospastic action in the test "aerosol by acetylcholine" male whitish guinea-pigs (Dunkin-Hartley strain), weighing 300-350 g, were subjected to bronchospasm induced by exposure to an aerosol of acetylcholine.

Thirty minutes before acetylcholine exposure (Ach 1%), the animals were exposed for 5 minutes to an aerosol with the substances under examination. The aerosols (substances under examination or Ach) were delivered with an ultrasound nebulizer De Vilbiss mod. 65 in a 8 1. volume exposure cage.

The E050 values (50% of the effective concentration) on the induction time of a severe bronchospasm, evaluated as animal's dropping on its flank, determined by the dose response curves, gave the following:

Compound EC50 nmol Idl I sopropyl-noratropine- N-oxide HCl | 2,49.105 @pratropium bromide | 4,27.104 For a global evaluation of the antibronchospastic activity of compound (I), on the basis of the data resulting from the described experiments, a calculation of the therapeutic indexes (LD50/ED50), which are expressed in Table II (e.v. = endovenous administration; e.t. = endotracheal administration), was performed.

TABLE 11

Konzett-Rossler Test Therapeutio Indexes e.v. e.t. Test DL50 DL50 DL50 Compounds LD50 ID50 e.v. ID50 e.t. "aerosol by Ach" ID50 ID50 EC50 nmol/kg e.v. nmoles/kg nmoles/kg. EC50nmol/dl e.v. e.t.

Isopropylnor-atropine N-oxide-HCl 4.32.105 9,2 8,9 2,49. 105 4,7. 104 4,9. 104 1,74 Ipratropium Bromide 3.78 + 104 2,3 2,8 4,27. 104 1,6. 104 1,3. 104 0,89 As shown in the Table, isopropyl-nor-atropine-N-oxide HCI shows therapeutic indexes from 2 to 3 times higher than those of ipratropium bromide, therefore proving to have a considerable therapeutic interest.

In vitro " action on guinea-pig isolated ileum The affinity, expressed as pA2, of the compounds under examination for muscarinic receptors of the intestinal smooth musculature, according to the method described by Van Rossum (Cumulative dose-response curves,Arch. Int. Pharmacodyn. Ther. 143,299, (1963)) has been evaluated. Isopropylnor-atropine-N-oxide and ipratropium bromide, although both behaving in this test as pure competitive antagonists of acetylcholine, have however a different affinity for muscarinic receptors of intestinal smooth musculature, as evidenced by pA2 values, respectively of 8.55 + 0.07 and 9.66 + 0.06.

According to these results the compound (I) of the present invention, besides having a marked antibronchospastic activity shows an higher action specificity towards the bronchial smooth musculature.

The compound (I) can be therefore used in therapy for the treatment of bronchoobstructive diseases, in the form of pharmaceutical preparations to be administered by the oral, parenteral or inhalatory route, in combination with excipients commonly used in these formulations. The administration by the inhalatory route, both for bronchial applications (dosed aerosol, solution for inhalation) and for rhinologic applications (spray, rhinologic solutions) turns out to be particularly advantageous, because of the higher action selectivity of the drug.

The following Examples illustrate two formulations, according to the invention, of compound (I).

(a) Isopropyl-nor-atropine-N-oxide HCl, dosed spray The active principle concentration for delivering can vary from 0.04 to 1 mg (preferably from 0.2 and 0.4 mg). The composition of a spray dosed at 0.2 mg/puff, referred to the content of an aerosol bomb for 200 deliveries, is the following: isopropyl-nor-atropine-N-oxide HCI 0.040 g sorbitantrioleate 0.0075 g trichlorofluoromethane 3.5525 g dichlorodifluoromethane 9.4 g For the production of 1000 bombs dosed at 0.2 mg/puff 3552.5 g of trichlorofluoromethane, 40 g of isopropyl-nor-atropine-N-oxide HCI micronized and 7.5 g of sorbitantrioleate are dispersed with a suitable homogenizer. The mixture is homogenized for a time necessary to produce a thin dispersion of the micronized product.If necessary, the weight of suspension is regulated at 3600 g with trichlorofluoromethane. The suspension is cooled at +5"--+100C and distributed in aluminium bombs inserting 3.6 g per bomb. The preparation is completed by pressurizing the active content with 9.4 g of dichlorodifluoromethane. Each puff, dosed with a 50 ul dosage value, weighing 65 mg, contains 0.2 mg of isopropyl-nor-atropine-N-oxide. The product in the described formulation can be provided with a suitable delivery key for endobronchial administrations or for rhinological applications.

(b) Is oprop yl-nor-a tropine-N-oxide HCI, solution for inhalation The concentration of the active principle is comprised between 1 and 5 mg/ml (preferably 1.25 and 2.5 mg/ml). The composition of the solution at a concentration of 2.5 mg/ml, for 100 ml of solution, is the following: isopropyl-nor-atropine-N-oxide HCI 0.25 g monohydrate monobasic sodium phosphate 0.2725 g dihydrate dibasic sodium phosphate 0.0135 g chlorobutanol 0.5 g purified water to 100 ml For the preparation of 10 1 of solution for inhalation, 27.25 g of monohydrate monobasic sodium phosphate, 1.35 g of dihydrate dibasic sodium phosphate and 50 g of chlorbutanol are dissolved in 9500 ml of purified water. Isopropyl-nor-atropine-N-oxide hydrochloride (25 g) is added and dissolved after stirring at room temperature; the volume is then regulated at 10 1. The solution is filtered on a cellulose nitrate membrane having 0.22 porousness and distributed in bottles provided with suitable plugs.

The described formulations can be used for endonasal administration, providing the bottles with suitable mechanically working, microdosing pumps provided with a rhinologic adapter.

The spray dose applied by said device delivers 250 mcg of isopropyl-nor-atropine-N-oxide HCI.

Claims (10)

1. N-substituted derivative of nor-atropine having the formula:

and its addition salts with pharmacologically acceptable acids.

2. The hydrochloride salt of the compound of formula (I).

3. Process for the preparation of compound (I), wherein isopropyl-nor-atropine is subjected to Noxidation with at least one N-oxidizing agent, in the absence or presence of solvents.

4. Process according to claim 3, wherein the N-oxidizing agent is selected from hydrogen peroxide, peracetic acid, p-nitroperbenzoic acid our m-chloro-perbenzoic acid.

5. Pharmaceutical compositions endowed with antibronchospastic activity, containing, as active principle, isopropyl-nor-atropine-N-oxide or one or more pharmacologically acceptable salts thereof, in suitable dosage.

6. Pharmaceutical compositions according to claim 5, intended for administration by the inhalatory route, in the form of spray bombs containing isopropyl-nor-atropine-N-oxide in concentrations of 0.04-1 mg/puff and excipients usually used for the purpose or in the form of a solution for a multidose inhalator device containing from 1 to 5 mg/ml of active principle (I) and suitable excipients.

7. Pharmaceutical compositions according to claim 6, suitable for employment for rhinologic administrations.

8. N-isopropyl-nor-atropine-N-oxide or a pharmaceutically acceptable acid addition salt thereof substantially as herein described.

9. A process for the preparation of N-isopropyl-nor-atropine-N-oxide or a pharmaceutically acceptable acid addition salt thereof substantially as described herein with reference to any one of Examples 1 to 4.

10. Pharmaceutical compositions comprising isopropyl-nor-atropine-N-oxide or one or more pharmacologically acceptable salts thereof substantially as described herein.