NO135634B - - Google Patents

Description

This invention relates to an analogous process for the production of new antibacterial hexamethylene bis-biguanides of the general formula I:

where R means hydrogen and R' means a hexyl or pentyl group substituted with one or two methyl groups, or means a cycloalkyl group with more than 6 carbon atoms, a lower-alkyl-cycloalkyl group or a cycloalkyl-lower-alkyl- group, or R and R<1> together with the intervening N atom form an azabicyclo-(3,2,2)-nonane double ring, and pharmaceutically acceptable acid addition salts thereof.

By cycloalkyl here is meant both mono- and polycyclic alkyl groups „ By lower alkyl is meant alkyl groups containing 1-4 carbon atomsc

According to the invention, the new bis-biguanides are produced in the following ways:

a) Hexamethylene bisdicyandiamide with the formula

is reacted with an amine of the general formula RR'NH, where

R and R' have the above meaning. The reaction can take place by melting the substances together at a temperature in the range of 130 - 180°C, or by using a suitable solvent.

b) 1,6-diaminohexane with the formula

H2N-(CH2)6-NH2

is reacted with a dicyandiamide of the general formula

RR'N-C(=NH)-NH-CN,

where R and R' have the meaning stated above, under similar conditions as under section a)„

In general, the amine is used in both alternatives a) and b)

in the form of a salt with an inorganic acid, and hexamethylene-bis-biguanides will then be formed as the corresponding di-salt. The free base can be prepared in the usual way by, for example, reacting the salt with an equivalent amount of base, e.g. NaOH or NH3.

The dicyandiamides used as starting materials are produced by known methods.

The pharmaceutically acceptable acid addition salts of the new hexamethylene bis-biguanides include their salts with one or two equivalents of an appropriate acid. A suitable acid addition salt is e.g. a salt with an inorganic acid, e.g. a hydrochloride, hydrofluoride, nitrate, sulfate or phosphate. Or a salt with an organic acid, e.g. a carboxylic acid, e.g. an acetate, benzoate, tartrate, adipate, lactate, maleate, glutamate, ascorbate, citrate, gluconate, oxalate, succinate, pamoate or salicylate. The salts are valuable, e.g. due to the altered solubility or specific value of the anion. The salts can be prepared from the corresponding base or from salts with other acids according to well-known methods.

Hexamethylene bis-biguanides are known from the past. Thus Norwegian patent 83 394 describes a series of compounds seF where the end groups are made up of substituted phenyl nuclei. One of these compounds, 1,1'-hexamethylene-bis-[5-(4-chlorophenyl)biguanide], has been widely used under the name "chlorhexidine".

The substance has a bactericidal effect, it is active against both gram-positive and gram-negative bacteria, and it has largely replaced disinfectants such as benzalkone and iodine tincture. It is used e.g. in nursing for disinfection of patients' skin before injection or sampling, before operative procedures, puncture or local and spinal anaesthesia" For wound treatment and treatment of traumatic wounds, burns and skin infections, acne Vulgaris etc. For mucous membrane disinfection, e.g. of the urethral opening before catheterization or for washing the urogenital area in connection with childbirth and in flushing fluid for urological use. In eye drops and against eye infections and for nasal treatment. It is also used for disinfection after hand washing, pre-operative hand disinfection, disinfection of utensils of all kinds, anesthetic apparatus, ventilator, storage of sutures and sterile instruments, for liquid for humidification apparatus in ventilators. An important application is the impregnation of bandage material such as e.g. bandages and face masks in surgical departments. Also for technical, non-medical use, impregnation etc. is chlorhexidine valuable.

What has been mentioned above about use in human practice is of course also essentially valid for veterinary practice. In particular, the substance is widely used in the prevention of mastitis.

In dental clinics, chlorhexidine has been used for disinfection of mucous membranes, root canals, disinfection of the work area in endodontic clinics and use in connection with immediate prostheses. Chlorhexidine is also used in caries prophylaxis and against periodontal disorders.

What has recently attracted particular attention is the use of chlorhexidine in the treatment of severe surface lesions (burns etc.) where micro-organisms such as Pseudomonas and other Gram-negative microbes are a problem.

Furthermore, with various fungal infections in the mucous membranes, where Candida albicans in particular is responsible for the more serious disorders. A patient group here consists of people with dentures where the occurrence of fungal infections in the mucous membranes in the oral cavity is not unusual.

Another possible application is the treatment of life-threatening fungal infections in premature infants.

In French patent 1,463,818, a general is indicated

formula that includes an enormous number of compounds, i.a. part of the compounds produced according to the invention.

The one of the single compounds mentioned and described in the French patent that comes closest to the compounds prepared according to the invention is 1,1<1->hexamethylene-bis-[5-(2-ethylhexyl)biguanide] which in the following for practical reasons referred to as "ethylhexylhexidine".

It is a compound with the above general formula in where

R is hydrogen and R' is 2-ethylhexyl, and it is also known to

have an antibacterial effect..

The hexamethylene bis-biguanides prepared according to

to the invention, differs from chlorhexidine and ethylhexylhexidine in at least one of the following properties: stronger antibacterial effect, or lower toxicity. This makes the compounds valuable in applications e.g. as those described above for chlorhexidine.

The advantages of the new hexamethylene bis-biguanides will be illustrated in the following:

ANTIBACTERIAL EFFECT

Growth inhibition in aqueous solution and subcultivation in liquid medium. Reference: Chlorhexidine=l.

Examination of the effects of the new bisbiguanides on Pseudomonas and Candida albicans has shown that several have a significantly higher effect than chlorhexidine.

At pH6.2, it has thus been found that the effect of compound no. 1 and compound no. 4 against Candida albicans is approx. 25 times greater than that of chlorhexidine. At physiological pH, the following values have been found:

ANTIMYCOTIC EFFECT

Relative growth inhibitory effect on Candida albicans after

2 days.

A similar relationship applies when testing relevant strains of Pseudomonas.

Acne vulgaris is a skin disorder in which Cornebacterium Acne plays a significant role in the development of the disorder.

Treatment has traditionally consisted of local application

of keratolytic compounds, systemic treatment with antibiotics and local treatment with reducing and oxidizing substances with bactericidal effect. Some of the new bisbiguanides produced according to the invention show a very strong effect on even very resistant strains of C.acne.

In a concentration of 0.008%, e.g. connection No. 1

with a working time of 5 min. provide complete killing of lo<7> micro-organisms. Addition of serum in concentrations of 10, 20 and 50% gives a maximum reduction of only 20%.

In these experiments, chlorhexidine showed a much smaller effect on the test microbes. It is to be expected that the new bisbiguanides will be able to become valuable agents in acne therapy not only because of the effect on C.acne, but also because the substances have a keratolytic effect and at the same time have pronounced surfactant properties. The last condition will mean that the substances will more easily penetrate the skin pores where the micro-organisms are located and that, through their emulsifying effect, they can counteract clogging of the follicle openings with sebum plugs (so-called comedones).

A relationship that is generally more difficult to interpret is the binding of the new compounds to proteins. More recent investigations with chlorhexidine have shown that several types of bonds must be taken into account, from weak bond forms such as hydrogen bonds to stronger bonds to carboxyl and sulfate groups in the protein molecule. Some of these lead to a loss of (biological) effect, while other given systems can contribute to an increased effect due to the fact that the contact time to the site of action is increased considerably.

The new bisbiguanides produced according to the invention have

different affinity to protein which, together with differences in effect against microorganisms, can be used in therapy depending on what the purpose of the treatment is.

The difference in affinity to protein can be illustrated by

the following table:

Precipitation of bovine albumin (0.5 mg/ml in TRIS HCl buffer)

with 1.1 mM test solution and varying pH.

As can be seen from the table, compounds 2, 3, 4 and 5 have no tendency or less tendency than the known compounds to precipitate proteins. Also 1 and 8 are better than 9. This is a very valuable characteristic, e.g. in the treatment of skin and mucous membranes and wound treatment where the bis-biguanide could otherwise be inactivated by exuding tissue fluids. In some cases, however, binding to proteins can lead to a desired long-term effect. In contrast, less emphasis will be placed on this characteristic when, for example, disinfection of utensils.

All the compounds produced according to the invention are also clearly distinguished in relation to chlorhexidine by their greater solubility in water and water-ethanol, and in particular by their compatibility with various anions. Thus, even the dihydrochlorides of the new compounds are dissolved in water in the order of percent, while e.g. chlorhexidine dihydrochloride only dissolves 0.06%. They therefore have more than 100 times greater solubility. In the case of pharmaceutical formulations, chlorhexidine's ability to separate a number of organic and inorganic ions is a very big problem. The same applies to inactivation during use by precipitation of sparingly soluble salts.

Liquid preparations, especially pharmaceuticals, often want to be made sterile. This is done in practice either by sterile filtration or by autoclaving. A disadvantage of chlorhexidine is that, during autoclaving, it splits off small amounts of p-chloraniline, which is toxic and can also cause discolouration. The compounds produced according to the invention all have end groups derived from aliphatic or cycloaliphatic amines and thus cannot decompose into harmful aromatic amines.

TOXICITY

Cytotoxic effect on human epithelial cells in vitro after treatment for 5 minutes with Reference: Chlorhexidine=l.

All the investigated compounds produced according to the invention are thus less than or as toxic as chlorhexidine, while ethylhexylhexidine is far more toxic.

ACUTE ORAL TOXICITY IN MICE

The hexamethylene bis-biguanides prepared according to

to the invention all excel in comparison to chlorhexidine and ethylhexylhexidine in at least one of the following properties: stronger antibacterial effect,) stronger antifungal effect,

lower toxicity, better solubility.

As mentioned, the compounds have a number of different areas of application. These areas of application are such by nature that a differentiated spectrum of properties may be desirable. This shall be illustrated in the following:

In some applications, an active substance with an effective effect is desired

on the widest possible spectrum of microorganisms, including fungi.

As can be seen from the tables, here the compounds 1 and 4 will be particularly preferred as they have up to 8 times stronger effect on the indicated microorganisms than ethylhexylhexidine, and from 4 to 16 times stronger effect than chlorhexidine. For most applications it will be important that the disinfectant also has low toxicity, e.g. in the treatment of skin and eye infections etc. In such cases, it is the quotient between therapeutic effect and toxicity that must be given the greatest weight. From the tables it appears that compounds 1, 2, 3 and 4 must all be considered preferred in this respect because they excel over chlorhexidine and ethylhexylhexidine. Their antimicrobial effect is from 1 to 16 times that of chlorhexidine, while their toxicity is from 1/8 to 1 in comparison. In relation to ethylhexylhexidine, the corresponding numbers are 1/8 to 8 and 1/32 to 1/4, respectively. This also applies to a certain extent to 6, 7 and 8. With long-term treatment, the disruption of the normal microflora will have questionable aspects as the risk of infection by pathogenic microorganisms increases, and then e.g. compound 5; be particularly preferred. EXAMPLE 1 7.7 g of 2-amino-6-methylheptane hydrochloride is mixed with 5.5 g of hexamethylene-bis-dicyandiamide in a mortar. The mixture is allowed to react at 155°C for 5 hours, cooled and extracted with boiling water. On cooling, 1,1'-hexamethylene-bis-[5-(1,5-dimethylhexyl)biguanide] dihydrochloride crystallizes out. The product is recrystallized from methanol-ether. EXAMPLE 2 14.5 g of 1,5-dimethylhexyl dicyandiamide is mixed with 6.5 g of hexamethylenediamine dihydrochloride and reacted at 155°C for 5 hours. After processing as in example 1, a compound is obtained which is identical to the product described there. EXAMPLE 3 5.6 g of 1,4-dimethylpentyl dicyandiamide and 2.7 g of hexamethylene diamine dihydrochloride are fused together and stirred at 155°C for 8 hours. The mixture is then dissolved in boiling water and treated with activated charcoal. On cooling, 1,1'-hexamethylenebis-[5-(1,4-dimethylpentyl)biguanide]-dihydrochloride crystallizes. ;Melting point: 22 3°C. EXAMPLE 4 7.6 g of 1,3-dimethylpentyl dicyandiamide is reacted with 3.6 g of hexamethylene diamine hydrochloride for 8 hours at 155°C. Work up as in example 3. 1,1'-4-hexamethylenebis-[5-(1,3-dimethylpentyl)-biguanide]-dihydrochloride is obtained. ;Melting point: 212°C. EXAMPLE 5 10.2 g of 1-methylhexyl dicyandiamide and 5.0. g of hexamethylene-diamine dihydrochloride are mixed and heated to 155 °C for 7 hours, worked up as in example 3. Recrystallized in water/ethanol. 1,1'-Hexamethylenebis-[5-(1-methylhexyl)biguanide]-dihydrochloride is obtained. ;Melting point: 234°C. EXAMPLE 6 4.5 g of (1-adamantyl)dicyandiamide and 1.8 g of hexamethylene diamine dihydrochloride are mixed with 10 ml of nitrobenzene and allowed to react at 160°C for 5 hours with stirring. After cooling, the substance is filtered off and recrystallized in ethanol. One obtains 1,1'-hexa-methylenebis-[5-(1-adamantyl)biguanide]-dihydrochloride. ;Melting point: 268°C. EXAMPLE 7. 3.5 g of 2-aminonorbornane hydrochloride (norbornane-[2,2,1]-bicycloheptane) is mixed with 2.8 g of hexamethylene-bis-dicyandiamide and heated to 155°C for 2 hours .' The substance is crystallized from water, and recrystallized in methanol/ether. 1,1'-Hexamethylenebis-[5-(2-endonorbornyl)biguanide)^dihydrochloride is obtained. ;Melting point: 225°C. ;;;<*> EXAMPLE

7.8 g of cyclohexanemethylamine hydrochloride (hexahydrobenzylamine hydrochloride) and 6.2 g of hexamethylene bis-dicyandiamide are mixed and heated to 155°C for 3 hours. The mixture is extracted with boiling water, decolorized with activated carbon, and crystallizes on cooling.

Recrystallises from water. 1,1'-hexamethylene-bis[5-(cyclohexylmethyl)biguanide]dihydrochloride is obtained.

Melting point: 216°C.

EXAMPLE 9

7.8 g of 3-azabicyclo (3,2,2)nonane hydrochloride and 5.8 g of hexamethylene-bis-dicyandiamide are allowed to react at 150° C. for 2 1/2 hours.

Processed as in e.g. 8. Recrystallize in methanol/ether. One gets a connection with formula in where

means 3-azabicyclo(3,2,2)-non-3-yl. Melting point: 216°C

The identity of all compounds produced has been confirmed by infrared spectra.

Fungal infections in the skin and mucous membranes give rise to

a number of serious ailments. A patient group here consists of people with dentures where fungal infection in the mucous membranes and oral cavity is a frequent occurrence. Other serious cases are life-threatening fungal infections in premature babies. These infections are often caused by Candida albicans. From the results presented in the table on page 5, it appears that compounds 1 and 4 have respectively 8 and 16 times better effect against this microorganism than chlorhexidine and the other compounds produced according to the invention, and respectively 2 and 4 times better effect than 2 -the ethylhexyl derivative.' These two compounds simultaneously have lower toxicity and less tendency to inactivate tissue fluids etc. in physiological conditions. They can therefore be said to be particularly preferred in the therapeutic treatment of fungal infections.

Another skin condition where the compounds produced according to the invention can be used is acne vulgaris. In Medd. Norwegian Farm. Selsk-, 38_ (1976) p. 40 very interesting results are shown in experiments with Corynebacterium acnes, the most important microorganism in this disorder. The bactericidal effect is highest for the present compound 1 and the 2-ethylhexyl derivative (known), and these should therefore be the most applicable in acne therapy. If, on the other hand, the substances are tested in a medium with added bl6d, i.e. an environment that more closely corresponds to the relevant physiological conditions, the 2-ethylhexyl derivative turns out to have very little effect. The 1,5-dimethylhexyl derivative (1) also loses much of its effect. In contrast, the 2-norbornyl derivative (5) proves to have a very strong bacteriostatic effect under these conditions. The reason is obviously that the former are inactivated by organic matter, while compound No. 5, which does not haemolyse the blood, preserves its antimicrobial activity. These results show that compound No. 5 is particularly preferred in acne therapy. But these properties also make the compound very interesting in other therapeutic contexts, e.g. to combat and prevent infections in burns and other surface lesions.

For antibacterial purposes, compound no. 3 is also particularly suitable since it has a high antibacterial effect (see the table on page 4), a low toxicity (see the table, at the top of page 8) and little or no tendency to precipitate proteins (see the table on pages 6 and 7).

Claims (5)

1. Analogous method for the preparation of therapeutically active hexamethylene bis-biguanides with the general formula: where R means H, and R' means a hexyl or pentyl group substituted with one or two methyl groups, or means a cycloalkyl group with more than 6 carbon atoms, a lower-alkyl-cycloalkyl group or a cycloalkyl-lower-alkyl -group, or R and R<1 >together with the intervening nitrogen atom form an azabicyclo-(3,2,2)-nonane double ring, and the pharmaceutically acceptable acid addition salts thereof, characterized in that a) Hexamethylene- bis-dicyandiamide is reacted with an amine of the formula RR'NH where R and R<1> have the above meaning, or b) 1,6 diaminohexane is reacted with a dicyandiamide of the general formula RR'N-C(=NH)-NH- CN where R and R' have the meaning given above, since the starting materials can optionally be used in the form of acid addition salts and/or the products formed can optionally be converted into desired acid addition salts. 2. Process according to claim 1, characterized in that starting materials are used where R'=1,5-dimethylhexyl and R=H. 3. Method according to claim 1, characterized in that starting materials are used where R'=1-adamanty1 and R=H. 4. Process according to claim 1, characterized in that starting materials are used where R'=2-norbornyl and R=H. 5. Process according to claim 1, characterized in that starting materials are used where R'=cyclohexylmethyl and R=H.