EP0577690A1 - Organosulphur compounds useful for the treatment of glaucoma - Google Patents

Abstract

This invention relates to organosulfur compounds, pharmaceutical compositions containing at least one such compound which is active against glaucoma and intraocular hypertension, as well as a method of treatment relating to glaucoma and intra hypertension -ocular.

Description

Organosulphur compounds useful for the treatment of glaucoma

The present invention relates to organosulphur compounds, pharmaceutical preparations containing such compounds and a method for treating glaucoma.

Glaucoma is a very common eye disease affecting millions of people in the later stages of their life. Glaucoma is characterized by abnormally high intraocular pressure and, if untreated, damage to the optic nerves which may cause narrowing of the visual field, and eventually irreversible blindness.

The intraocular pressure is determined by the rates of inflow and outflow, i.e. the dynamics of the aqueous humour. The aqueous humour enters into the posterior chamber of the eye, and then flows through the pupil to the anterior chamber, from where it eventually leaves the eye through the trabecular meshwork.

The aqueous humour supplies nutrients to the lens and cornea, and its proper supply is thus of the utmost importance for maintaining healthy eyes.

Any disturbance of aqueous humour dynamics by either excess inflow, or reduced outflow, results in an increase in the intraocular pressure above the normal value (for adults) of 17 - 20 mm Hg, i.e. the eye becomes hypertensive. A prolonged hypertensive state will result in nerve damage and blindness. Detailed descriptions on glaucoma can be found in "An Outline of Ophthalmology", by R.L. Coakes, and P.J. Holmer Sellars, published by Wright, Bristol (1985), cf. pp. 54/57, and in the series: Current Topics in Eye Research", edited by J.A. Zadunaisky and K. Davson, Academic Press.

All known antiglaucoma drugs on the market lower the intraocular pressure, either by decreasing formation of aqueous humour, or by increasing the outflow, i.e. the elimination of aqueous humour from the eye. Glaucoma drugs are thus all hypotensive agents.

The most common class of antiglaucoma agents are adrenergic antagonists; many of them are 0-blockers (the most widely used of this type is timolol), adrenergic agonists, dopaminergic agents, cholinergic agents (the most widely used of this type is pilocarpine), or several other classes of compounds. For detailed overviews, see for example Annual Reports in Medicinal Chemistry, Vol. 20, chapter 9: "Antiglaucoma Agents", by M.F. Sugrue and R. . Smith (1985, Academic Press), and the text: "The Pharmacological Basis of Therapeutics" by A. Goodman and L. Gilmans.

Thus, one of the characteristics of glaucoma therapy is the fact that an enormous variety of chemical structural types can be used to reduce excessively high intraocular pressure.

None of the currently used drugs is fully satisfactory. There are serious side effects affecting the heart, the kidneys, the lungs and/or the libido. Some of the side effects are, especially in the case of carbonic anhydrase inhibitors, α-adrenergic antagonists and 0-adrenergic antagonists, directly implicated with the different modes of action, while others are not. Furthermore, there are problems of metabolic stability which necessitates several applications of eye drops per day. Great efforts are therefore made to develop new antiglaucoma agents which would be free of the above constraints. Recently, an entirely new chemical structural type of compounds, namely peptides and peptide derivatives, was described as having antiglaucoma activity, i.e. as hypotensive agents. Examples are carboxyalkyl dipeptides (European Patent No. 0088350) and the atrial natriuretic factor, a long peptide of 29 amino acids in length (Fortschritte der Ophthalmologie, Volume 89, pp. 89/91 (1989)).

US Patent Specification No. 4,634,698 describes ophthalmological pharmaceutical compositions comprising carboxyalkyl dipeptides joined through a sulfonamido group to a benzothiadiazinyl sulfonylphenyl moiety and to a method for using said composition in the treatment of glaucoma. The compositions contain as active agent cyclic, proline-type amino acids, which differ substantially from the compounds according to the invention. Thus, the peptide moiety present differs substantially from amino acid like compounds claimed in this invention and also the sulphur atoms obligatorily present are in SO,,-oxidized state and substituted with nitrogen to form sulfonamido groups. The sulfonamido group is also present in the older antiglaucoma drug acetazolamide which is a carbonic anhydrase inhibitor and where the other sulphur atom is bound in a heterocyclic aromatic thiadiazole ring.

Danish Patent Application No. 1315/85, which has lapsed, discloses a process for treatment of glaucoma and/or intraocular hypertension by using ACE inhibitors. The ACE inhibitors mentioned were said to be useful also for lower- ing high blood pressure of different genesis. However, the proposed ACE inhibitors are not of the type proposed in the present invention. Further it is not rendered possible that the compounds have the claimed effect.

Furthermore, hydrolysates of milk proteins were also described as having antiglaucoma activity (WO 86/04217 and EP 210204). The peptide compositions described therein are not well defined chemical compounds as are the compounds of the present invention, rather they are mixtures which resulted from the hydrolysis of milk proteins.

The applicants' previous patent application No. PCT/DK90/00322, filed on December 7, 1990, concerns peptide derivatives of the formula

R₁-A-B-C-D-E-R₂ I

wherein

A is absent or is a non-hydrophobic, uncharged amino acid or a derivative thereof,

B is absent or is an uncharged amino acid or an uncharged N-methylated amino acid,

C is an uncharged amino acid or an uncharged N-methylated amino acid,

D is an uncharged amino acid with a non-hydrophilic "or absent side chain,

E is cysteine or a cysteine homologue, the sulphhydryl group being free or substituted,

R₂ is optionally substituted NH₂, optionally substituted OH,

-0-glycosyl, an L- or D-α-amino acid, or R_ is absent.

These compounds are active with glaucoma and intraocular hypertension. Preferred compounds are H-Asn-Gly-Gly-Val-

Cys(Acm)-NH₂ and H-Asn-Leu-Gly-Val-Cys(Acm)-NH₂. One of the compounds has been tested on human beings and has proved itself suitable against glaucoma and intraocular hypertension by topical application, while no side effect was found on blood pressure or heart rate. The absence of these cardiovascular effects of this compound has also been demonstrated by i.v. administration in rats.

It has now surprisingly been found that smaller entities of such a parent compound, containing sulphur, may in themselves be active core structures. As another such class of minimal structures which are significantly different (see the concurrently filed DK patent application No. 0532/91) has been identified, both the mode of action, metabolism and possible side effect profiles may be envisaged to be different. Thus the separation of these new core structures from the parent structure may constitute a significant advantage as a base for design of optimal pharmaceutical preparations targeted specifically towards different forms of glaucoma, treatment profiles and patient groups, while further reducing risk of side effects.

The present invention relates to organosulphur compounds, which lower the intraocular pressure, IOP, in relevant animal models.

The compounds of the invention are of the general formula

R₁-[A]-[[P]-(CH₂)_a-C_χH-]_b-C 0-R₂ I

I I [B] D

I

S-[E]

or an N-C-cyclic or a S-S-bridged form thereof, or a compound transformed into or releasing any of the above basic structures under physiological conditions in humans, wherein

R- is H, straight or branched alkyl or cycloalkyl up to C₂₀, optionally containing double bonds and/or substituted with halogen, nitro, amino, sulpho, phospho or carboxy, or aralkyl or aryl optionally mono- or polysubstituted with halogen, hydroxy, nitro, amino, sulfo, phospho, carboxy or alkyl, or R₁ is glycosyl, nucleosyl or an L- or O-a amino acid or a peptide moiety of 2 to 8 residues connected by bonds of type [P],

a is 0, 1, 2 or 3,

C is a tetrahedral carbon atom (SP3 hybridized) having R or S configuration,

C is a triplanar carbon atom (SP2 hybridized) and D is absent or C is a tetrahedral carbon atom and D is H~,

[A] is absent, a decarboxy amino acid residue or NHR ', wherein R. ' is as defined for R. , and is absent when the compound is an N-C cyclic form,

[P] is absent or is a peptide bond CO-NH,

or isosteres thereof, such as CH₂~NH, CH₂~S, C0-CH₂, retroinverse forms thereof, such as NH-CO,

[B] is absent or C.-C, optionally branched alkyl or cycloalkyl,

S is sulphur

[E] is absent when the compound is in disulfide bridged form, or E is (CR₃R. ) -NHCOR-., wherein R₃ and R. independently are H, CH₃ or halogen, and R-. is H, straight, branched alkyl or cycloalkyl, aralkyl or aryl, all of which are optionally mono- or polysubstituted with halogen, hydroxy, carboxy, sulfo, phospho, amino or nitro, or a decarboxy amino acid or a decarboxy peptide moiety, and c is 1, 2 or 3,

S-CRgR₇Rg, wherein R_g, R„ and R„ are independently H, halogen, straight, branched alkyl or cycloalkyl, aralkyl or aryl, all of which are optionally mono- or polysubstituted as indicated for R₅,

^CR _g ^Rτn^Rιι are independently H, alkyl, aralkyl or aryl, all of which are optionally mono- or polysubstituted as indicated for R..,

or

S-R-₂, wherein R-₂ is C₁-C₁₀ aralkyl or heteroaralkyl,

or

R_₂, wherein R₁₂ is as defined above,

s H, OH, CH₃ or

NR.-R..., wherein R₁₃ and R... are independently H, straight, branched alkyl or cycloalkyl, aralkyl or aryl optionally substituted as defined for R..,

OR.. ₍-, where R- r- is H, straight, branched alkyl or cycloalkyl, aralkyl or aryl, optionally substituted as defined for R--, O-glycosyl, or

an L- or D-α-amino acid, or a peptide moiety of 2 to 8 residues connected by bonds of type [P],

or R₂ is absent, when E is a decarboxy derivative of cysteine or a homologue thereof or the compound is an N-C cyclic form,

b is 1, 2, 3, 4 or 5,

and R₁ , R₂ and R-. together comprise no more than 10 amino acid residues, and

hydrogen atoms may be replaced by fluorine.

Preferred compounds of the invention are of the general formula

R₁-[A]-[[P]-(CH₂)_a-G-]_b-C 0-R₂

I I

[E] D

wherein

G = -CxH is a desamino-Cy ^■*s or -Pen or sidechain

I [B]

I s

homologues of these containing totally 2 - 7 alifatic carbon atoms, S is sulphur

a is 0,

[E] is a sulphur substitution group chosen among

^R %.₁_₃, which is C -C-- alkyl, such as methyl, ethyl, isopropyl, n-butyl, isobutyl, and tert.butyl, n-pentyl, isopentyl, tert.pentyl, 1,2-dimethylpropyl and 2,2- dimethylpropyl or

R.., which is C---C„ homo- or heteroaralkyl, such as phenyl, benzyl, toluyl and pyridyl, or

CH₂NHC0R₁₃ or

CH₂NHC0R₁₄ or

S-R₁₃ or

[E] is absent and the compound is an S-S-dimer, and

R, , [A], [P], Cy, D, Rz and b are as defined in claim 1.

More preferred compounds of the invention are of the general formula I' , wherein

G is desamino-Cys, -Pen or -homocystein or 3-Mercaptopropionic acid, and

S, R-, [A], [P], C , [E], D, R₂, a and b are as defined in claim 2.

Examples of active compounds are H-Cys-(Acm)-Met-NH₂, H-Ile-Gln-Cys(Acm)-NH₂, (H-Ile-Gln)₂~Cystine-(OMe)₂, H-Ile-Gln-Cys(BAM)-NH₂, 3-Mercaptopropionic acid-(Acm)-Tyr-OH

H-(Met)₃-NH₂

[H-Cys(Acm)]₂-Cystine-OMe₂ and H-[Pen(Acm)]₂-NH₂

or pharmaceutically acceptable derivatives thereof.

A number of small peptides which contain some of the basic substructures belonging to the class of compounds defined in the present invention are known, see e.g. EP 0278787, EP 0359399, EP 0179412, WO 88/03535, EP 0399656, Patent Abstract of Japan, 13, 238, US 4968696, CH 0658661, EP 0183245, EP 0161017, Patent Abstract of Japan, 7_, 171, DE 2261926, C.A. 103, 100572b, US 3959519 and US 4024286. None of the compounds disclosed are said to have antiglaucoma effect.

Especially effective compounds for treating intraocular hypertension are

H-Ile-Gln-Cys(BAM)-NH₂,

3-Mercaptopropionic acid-(Acm)-Tyr-OH

H-(Met)₃-NH₂

[H-Cys(Acm)]₂-Cystine-0Me₂ and

H- [Pen(Acm)3₂-NH₂

Further, the invention relates to a pharmaceutical composition containing a compound accβrding to the invention in an amount effective to treat glaucoma or intraocular hypertension and a pharmaceutically acceptable diluent or excipient. Additionally, the invention relates to a method for treating glaucoma or intraocular hypertension, comprising administering to a mammal an effective antiglaucoma or intraocular pressure lowering amount of a compound according to the invention.

The compounds of this invention are preferably used in topically applicable aqueous isotonic and sterile solutions or in sterile solutions or dispersions in an oil as used for the topical treatment of the eye. A typical oil for ocular treatment is sterile castor oil. These topical solutions or dispersions contain 0.01 - 10%, in particular 0.1 - 5%, preferably 0.25 - 1% (percent by weight) of at least one of the organosulphur compounds of this invention. The normal dosage of these solutions is 1 to 5 drops administered to the conjunctival sac of the eye. This dosage is normally administered 2 to 6 times per day. [20 drops of a DAB-9 dropper (Tropfenzahler gemSss "Deutsches Arzneibuch 9") will give about 1 ml] .

In many of the preferred compounds according to the present invention the organosulphur structures are part of peptidic compounds containing amino acids. In this context the term amino acid is to be understood to not only cover the 20 natural amino acids, but also to embrace amino acid replacements and substituents as recognized in the art.

The term alkyl is to be understood to cover all saturated hydrocarbons as exemplified in e.g. IUPAC. As examples are mentioned methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl for C--C. alkyl. In similar way the terms cycloalkyl and aryl are as defined in e.g. IUPAC, and halogen means chlor, brom, iod or fluor.

The term peptide is to be understood to embrace peptide bond replacements and/or peptide mimics, i.e. pseudopeptides, as recognized in the art (see for example: Proceedings of the 20th European Peptide Symposium, edt. G. Jung, E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for topical application as drops, or for oral delivery. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability, or to improve the economy of production, and they are acceptable, provided they do not negatively affect the required biological activity of the peptide as a hypotensive agent suitable for lowering of elevated intraocular pressure and glaucoma.

The actual pharmacological activity effects are envisaged as mediated through binding of the structurally active centre(s) of the molecules to one or more hitherto unestablished and perhaps unknown receptors in the eye. Thus, so far no receptor displacement, in vivo or in vitro, assays performed on the compound HAsnLeuGlyValCys(Acm)NH₂, a potent compound according to PCT DK90/00322, has been able to demonstrate any a- adrenergic agonistic or 0-adrenergic antagonistic effects, cholinergic effects or carbonic anhydrase inhibitory effects.

The pharmacological efficacy, potency and duration of effect may be modulated through additional structural features, such as chain elongation, optical isomerism, the substitution of peptide bond isosters, or substitution with one or more groups, which in case of susceptibility to enzymatic or spontaneous chemical conversion under the pharmacological conditions may also constitute prodrug forms. Different additives and vehicles may also affect pharmacokinetic and therapeutic effects. The modulation may in some cases lead to significant improvement of performance because of enhanced stability, eye penetration, transport to the receptor, or controlled release. An example of the use of amino acid and N-terminal substitutions to enhance stability is given in "Enzyme resistant immunomodulatory peptides" U.S. patent 4,505,583 (1985), Goldstein, G. et al. An example of peptide prodrugs is mentioned in Int. J. of Pharmaceutics 5_^, p. 255 (1989), Bundgaard, H. An example of the use of additives is given in "Evaluation of mucoadhesive polymers in ocular drug delivery. 1. Viscous solutions", Pharmaceuticals Res. £$, p. 1039 (1991), Davies, N.M. et al.

Apart from substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designing bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytic degradation while not impairing activity. An example is given in the paper "Tritiated D-Ala -Peptide T Binding", Smith, C.S. et al, Drug Development Res. _15, pp. 371-379 (1988). Secondly, stability and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in "Conformationally restricted thymopentin-like compounds", U.S. pat. 4,547,489 (1985), Goldstein, G. et al. Finally, the introduction of ketomethylene, methylsulfide or retroinverse bonds to replace peptide bonds, i.e. the interchange of the CO and NH moieties may both greatly enhance stability and potency. An example of the latter type is given in the paper "Biologically active retroinverso analogues of thymopentin", Sisto A. et al in Rivier, J.E. and Marshall, G.R. (eds. ) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), p. 722-773.

A more closely related example of modulation of effect by structural modification not related directly to receptor binding is taken from PCT/DK90/00322 in which the pentapeptide HAsnLeuGlyValCys(Acm)NH₂ was shown both to penetrate the sclera of the eye and to be a potent pressure lowering agent. It further contains two activity centres, one according to the present application and one according to the concurrently filed DK patent application No. 0532/91 together forming the tripeptide moiety

-GlyValCys(Acm)NH₂. However, when the corresponding particular N-α-unprotected tripeptide HGlyValCys(Acm)NH₂ was tested in the stress induced rabbit model for antagonizing effect, this was found to be significantly lower than expected. However, merely acetylating the tripeptide to AcGlyValCys(Acm)NH₂ partially restored activity. Parallel studies, e.g. on HAsnValCys(Acm)NH₂ and HGlyValOBzl, have shown that the lower efficacy is not due to the free amino terminus per se, since these had a good efficacy. Without wanting to be committed to one particular theory, it may be speculated that the overall amphiphilicity of the molecule by acetylation in case of HGlyValCys(Acm)NH₂ is made more favourable, thus improving penetration and transport through the eye and/or to the receptor. Some enhanced enzymatic stability may also be envisaged from the acetylation.

The compounds of the invention can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E. : "Methoden der organischen Chemie", Volume 15, Band 1 + 2, Synthese von Peptiden, Thieme Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R.B.: "The Peptides", eds. E. Gross, J. Meienhofer., Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. Widmer, F., Johansen, J.T., Carlsberg Res. Commun., Volume 44, pp. 37- 46 (1979), and Kullmann, W. : "Enzymatic Peptide Synthesis", CRC Press Inc., Boca Raton, Florida (1987), and Widmer, F., Johansen, J.T. in "Synthetic Peptides in Biology and Medicine", eds., Alitalo, K. , Partanen, P., Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985)), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy.

The compounds of the invention can be produced by the above listed general synthetic methods, or by an advantageous combination thereof.

The compounds according to the invention can be used for the treatment of glaucoma in pharmaceutical preparations, possibly in combination with pharmaceutical carriers and delivery systems and/or other useful and pharmaceutically acceptable additives.

It was shown in an animal experiment where the intraocular pressure, IOP, in the rabbit eye was experimentally raised above the normal level, that the compounds of the invention were able to achieve a lowering of the intraocular pressure in a similar way as when timolol was applied. Timolol is commonly used to treat glaucoma, but however, being a _*- blocker, it has serious side effects on the heart, lungs and/or sexual functions.

It is anticipated that with the compounds according to the invention, many of these and other side effects can be avoided. Indeed, a particular pentapeptide according to PCT/DK90/00322 containing a characteristic structure of the compounds according to the invention, HAsnLeuGlyValCys(Acm)NH₂, has been especially thoroughly examined for side effects, especially blood pressure and heart rate effects, toxicity and mutagenicity as well as local irritant or anaesthetic effects in a variety of animal and microbial models.

The animal model on which the IOP lowering effect of the antiglaucoma compound(s) was first established, is a clinically relevant model which was developed in the laboratory of one of the inventors who has positively shown in this model the pressure lowering effect of many 5- blockers (such as timolol) and adrenergic agonists, and thus has demonstrated the clinical relevance of the model on known and putative glaucoma drugs.

The main feature of this clinical model is a stress induced elevation of the IOP in the rabbit eye above the initial and normal value. The stress is exerted, i.e. applied, in the form of measuring the pressure (at 12 hour intervals) with the help of a SHIOTZ-Tonometer, which is loaded with 7.5 grams. The pressure first begins to rise after 5 measurements, i.e. after 2 1/2 days, and reaches a maximum after 10 measurements, i.e. after 5 days.

Known antiglaucoma drugs lower the IOP when they are applied after the IOP has clearly been established, in spite of the fact that the trauma, i.e. the measuring of the pressure, continued during the treatment.

If the treatment with the antiglaucoma drugs is started simultaneously with the traumatization, i.e. the exertion of stress by measuring of the pressure at the start of the animal experiment, the active antiglaucoma drugs antagonize the development of an elevated IOP above the initial and normal value, while the inactive compounds will not antagonize, and thus result in an elevated pressure. The relevance of this model has been demonstrated in many experiments with clinically used antiglaucoma drugs. Detailed description of the model is found in: Stainbach, T., Dissertation, Universitats-Augenklinik Hamburg- Eppendorf, 1986: "Adrenergica und neue Peptide bei Augeninnendruck: Beziehung zum Prostaglandin im Kammerwasser von Kaninchen".

The IOP activity of the compounds of the present invention has likewise been demonstrated on this model as shown in the examples. These organosulphur compounds are thus likely candidates for the treatment of glaucoma.

The peptide compositions described in the above doctoral thesis are as mentioned not well defined chemical compounds as are the compounds of the present invention, rather they are mixtures which resulted from the hydrolysis of milk proteins. These peptides and their various activities, among which is antiglaucoma, are described in the European Patent No. 210 204 by one of the present inventors.

The findings of IOP lowering effects in the stress induced rabbit model have been confirmed and further studied by using another elevated eye pressure rabbit model. In this model, the widely applied water load model, elevation of the intraocular pressure is achieved by injecting a large volume of sterile water intraperitoneally into the rabbits. Following onset of eye drop treatment in one eye while the other eye is treated with saline placebo, the IOP of both eyes is then measured at various intervals and the pressure difference between the eyes is taken as an expresion of the pharmaceutical effect. In this model pilocarpine, a well- known pressure lowering cholinergic agent, was shown to have a pressure lowering effect.

The advantage of the compounds of the invention is their defined chemical nature, which allows for proper registration and, if deemed desirable, for logic and systematic structural modification to produce analogues of even better properties than the ones invented and claimed now.

Furthermore, the compounds according to the invention are of low molecular weight (≤ 800), and thus topically applicable, unlike the atrial natriuretic factor described in Fortschritte der Ophthalmologie, Volume 86, p. 89-91 (1989), which has a molecular weight of ^~ 3000, and needs to be administered by injection to achieve an antiglaucoma effect.

Moreover, the atrial natriuretic factor is a cardiovascular hormone and thus not suited to be used for treatment of glaucoma over prolonged periods of time. Finally, both the peptidic protein hydrolysate mixtures (which are not necessarily strictly peptidic in chemical structural terms) and the atrial natriuretic factor are of a size which may give rise to an immune response followed by the production of antibodies. Such a response is unlikely to occur with the low molecular weight compounds according to the invention.

The mechanism, or mechanisms, by way of which the organosulphur compounds according to the invention work, is so far not known in detail and may be of hitherto unknown types or related to some known mechanisms. With the apparent lack of b-blocking effects, α-agonistic effects, cholinergic effects and inhibitory effects on the enzyme carbonic anhydrase other effects on aqueous humour outflow could be working. Some indications of mechanisms of the latter type have been found in in vitro studies. Thus, an in vitro study conducted at an early stage demonstrated that the parent compound HAsnGlyGlyValCys(Acm)NH₂ induced a marked and significant decrease of uptake of glycosamines in cultured bovine trabecular meshwork cells. From this decrease in the synthesis of glucosamineglycanes of importance in the outflow resistance was inferred.

The abbreviations used in this description for amino acids and protecting groups are in agreement with the IUPAC-IUB standard rules for nomenclature. Thus Pen means 0,0- dimethylcysteine.

In addition, and in particular, the following abbreviations are used:

HONSu N-hydroxysuccinimide

DCC Dicyclohexylcarbodiimide

Boc tert.-butyloxycarbonyl

DMF Dimethylformamide

DCU Dicyclohexylurea

TFA Trifluoracetic acid

TEA Triethy1amine

OMe Methylester

Acm Acetamidomethyl

MPR 3-mercaptopropionic acid

BAM Benzamidomethyl

Bzl Benzyl

Bz Benzoyl

The invention is now further explained and documented by way of examples.

Pharmacological examples

Antagonizing of the Intraocular Pressure in stressed Rabbit's Eyes Model

The compound lowers the experimentally increased IOP in the rabbit animal model, or it antagonizes, i.e. prevents the increase in pressure when it is applied simultaneously with the treatment which inflicts the increase in the pressure.

The compound was a freeze-dried powder, and was applied to the rabbit eye as a powder, or as drops, dissolved in 0.9% NaCl aqueous solution. Negative control was 0.9% NaCl solution in water.

Water Load Model Effects in Rabbits' Eyes

The studies utilized a "water load" animal model.

Thirty minutes before drug solution instillation, rabbits were injected intraperitonally with 60 ml/kg of sterile distilled water for injection (30 °C) spiked with an antibiotic mixture (Sigma P9032).

At time zero, 50 ul of a drug solution was instilled to one eye and an equal volume of a saline solution was instilled to the other eye. The IOP in each eye was monitored at the time points indicated. The change in IOP at each time point is computed by subtracting the IOP in the dosed eye from that in the undosed eye.

Plots of this data were made showing the IOP versus time including standard deviation. From these plots were assessed the maximal IOP effect, the time to reach this and the time for returning to a zero or insignificant level of IOP lowering effect. These figures were taken as a measure of potency and duration of effect.

General note for pharmacological examples 1-7

Peptides were tested for the intraocular pressure lowering or antagonising effects in the water load model or the stress induced antagonising model respectively, in groups of four to ten rabbits, as described above. The tests were performed on homogeneous groups of random sexed rabbits, weight 2.5-3.0 kg, but of different breeds in various laboratories several places in the world. Thus, in some cases intergroup variations were found in the absolute starting pressure of the rabbits' eyes.

In the case of the water load model structure, each rabbit served as its own reference control for the duration of the experiment, and in the case of the stress induced antagonistic model, each group of rabbits served as reference control, at the beginning of end of 10 stress units. Usually, the peptides were dissolved in plain isotonic saline, but in two cases in the waterload model, a TRIS-buffer at physiological pH was included.

In both cases a negative saline control group showed no effect on the pressure. The relevant TRIS-buffer control group showed also no effect in the waterload model, while a 2.6% solution of the known miotic glaucoma drug, pilocarpine, gave a similar response to some of the preferred compounds as illustrated in the drawing of Figure 4. The compounds listed in the tables were then classified as active on the following criterion: In the water load model one drop of a 1% solution in one eye resulted in a significant pressure lowering effect corresponding to the control treated eye, which was at maximum at least 1 mm Hg within 1 hour and with a lowering effect duration of at least 90 minutes for the group as average. In the stress induced antagonising model, the pressure increase following 10 stress units for the treated group as average was significant and negative, 0 or less than 2 mm Hg, which compares to normally 8 to 18 mm Hg in untreated controls. The following examples are further explained by means of the drawing in which

fig. 1 shows the change in the intraocular pressure, ΔIOP, in mm Hg as a function of the time in minutes for the compound Peptide No. 119 [H-Cys(Acm)]₂ Cystine0Me₂,

fig. 2 shows the change in the intraocular pressure, ΔIOP, in mm Hg as a function of the time in minutes for the compound Peptide No. 125, H-[Pen(Acm)]₂~NH₂,

fig. 3 shows the change in the intraocular pressure, ΔIOP, in mm Hg as a function of the time in minutes for the compound Peptide No. 1 VI, 1%, HIleGlnCys(Acm)NH₂,

fig. 4 shows the change in the intraocular pressure, ΔIOP, in mm Hg as a function of the time in minutes for the positive reference control, 2,6% pilocarpine, and

fig. 5 shows the mass spectrum for Peptide No. 125, H-[Pen(Acm)]₂-^NH ₂•

Example 1

Antagonizing effect of dipeptides containing the acetamidomethyl (Acm) substitution group on the sulphur atom of cysteine and 3-mercaptopropionic acid (MPR) in C- and N-terminal position, respectively, on the stress induced intraocular pressure in the rabbit's eye, following 10 stress units.

The peptides were applied topically as a 1% solution in 0.9% aqueous NaCl in aliquots of 60 ul three times daily over a period of 5 days. Peptide Antagonizing Effect

H-Val-Cys(Acm)-NH₂ Active MPR(Acm)-Tyr-OH Active

Example 2

Antagonizing effect of di- and tripeptides containing two or more organosulphur atoms, substituted by methyl in methionine or acetamidomethyl (Acm) in cysteine, on the stress induced intraocular pressure in the rabbit's eye, following 10 stress units.

The peptides were applied topically as a 1% solution in 0.9% aqueous NaCl in aliquots of 60 ul three times daily over a period of 5 days.

Peptide Antagonizing Effect

H-Met-Met-Met-NH₂ Active H-Cys(Acm)-Met-NH₂ Active Ac-Cys(Acm)-Met-NH₂ Active

Example 3

Antagonizing effect of different tripeptides containing acetamidomethyl substitution group on the sulphur atom of cysteines in the middle or aminoterminal position, carrying various N-substitutions, on the stress induced intraocular pressure in the rabbit's eye, following 10 stress units. The peptides were applied topically as a 1% solution in 0.9% aqueous NaCl in aliquots of 60 μl three times daily over a period of 5 days.

Peptide Antagonizing Effect

H-Cys(Acm)-Ala-Thr-NH₂ Active H-Cys(Acm)-Val-Tyr-NH₂ Active Ac-Cys(Ac )-Val-Gly-NH₂ Active Bz-Arg-Cys(Acm)-Tyr-NH₂ Active

Example 4

Pressure lowering effect of tripeptides containing the sequence H-Ile-Gln-Cys(R)-NH₂ on which the sulphur atom of cysteine is substituted by R=acetamidomethyl (Acm) or R=benzamidomethyl (BAM) groups on water load induced hypertension in the rabbit's eye by single dose treatment.

50 nl of a 1% solution of the peptides in 0.9% aqueous saline was applied in one eye and 50 ul of 0.9% aqueous saline in the other eye 30 minutes after the interperitonal water loading and IOP was measured in both eyes for 2 hours and the difference calculated.

Peptide Pressure Lowering Effect

H-Ile-Gln-Cys(Acm)-NH₂ Active H-Ile-Gln-Cys(BAM)-NH₂ Active

The time curve for the pressure lowering effect in the waterload model for H-Ile-Gln-Cys(Acm)-NH is given in fig. 3, where the peptide has the designation No. l.VI.

Example 5

Antagonizing effect of a linear tripeptide and a disulfide bridged hexapeptide containing the sequence H-Ile-Gln- Cys(R)-X in which R represents a benzyl substitution or disulfide linkage of sulphur of the cysteines, respectively, and X represents various C-substitutions, on the stress induced IOP in the rabbit's eye, following 10 stress units.

The peptide was applied topically as a 1% solution in 0.9% aqueous NaCl in aliquots of 60 μl three times daily over a period of 5 days.

Peptide Antagonizing Effect

H-Ile-Gln-Cys(Bzl)-NH₂ Active

H-Ile-Gln-Cys-OMe

I (S-S Dimer) Active H-Ile-Gln-Cys-OMe

Example 6

Pressure lowering effect of a tetrapeptide containing the sequence Cys(Acm)-Cystine on water load induced hyper¬ tension in the rabbit's eye by single dose treatment, the tetrapeptide ester (H-Cys(Ac ) )₂~Cystine-OMe₂.

50 ul of a 1% solution of the peptides in 0.9% isotonic saline containing TRIS-buffer pH 7.4 was applied in one eye and 50 ul of 0.9% isotonic saline containing TRIS-buffer 7.4 in the other eye 30 minutes after the intraperitonal water loading and intraocular pressure was measured in both eyes for 2 hours and the difference calculated.

Peptide Pressure Lowering Effect

H-Cys(Ac )-Cys-OMe Active I (S-S Dimer)

H-Cys(Acm)-Cys-OMe

The time curve for the pressure lowering effect in the waterload model for this peptide is given in Fig. 1, where the peptide has the designation No. 119.

Example 7

Pressure lowering effect of a dipeptide containing the sequence Pen(Acm)-Pen(Acm) on water load induced hyper¬ tension in the rabbit's eye by single dose treatment, the dipeptide amide H- ,0-dimethyl(S-acetamidomethyl)- cysteinyl- , -dimethyl(S-acetamidomethyl)-cysteine amide.

50 al of a 1% solution of the peptides in 0.9 % isotonic saline containing TRIS-buffer pH 7.4 was applied in one eye and 50 ul of 0.9% isotonic saline containing TRIS-buffer pH 7.4 in the other eye 30 minutes after the intraperitonal water loading and intraocular pressure was measured in both eyes for 2 hours and the difference calculated. Peptide Pressure Lowering Effect

H-Pen(Acm)-Pen(Acm)-NH₂ Active

The time curve for the pressure lowering effect in the waterload model for this peptide is given in Fig. 2, where the peptide has the designation No. 125.

Synthetic Examples

Example 8

Synthesis of Val-Cys(Acm)-NH₂

Boc-Val-Cys(Acm)-NH₂

77.6 g (405 mmoles) Cys(Acm)NH₂ free base was dissolved in 1350 ml DMF and cooled to 4-5 °C. 85.0 g (270 mmoles) Boc-

Val-ONSu was added and the reaction mixture stirred for one day at 4-5 °C and for one day at room temperature.

Filtration of the crude solution and preparative C_1R RP-

HPLC chromatography using water/ethanol/acetic acid buffer gave fractions containing the pure product, which upon evaporation to dryness gave the dry product as a white powder. Yield 66.4 g (63%).

TFA,H-Val-Cys(Acm)NH₂

47.3 g (121.1 mmoles) Boc-Val-Cys(Acm)NH₂ was dissolved in 120 ml TFA. The product was precipitated after 15 minutes by addition of 450 ml diethylether under stirring. The stirring was kept for two hours to give a more homogeneous product. Filtration and drying under vacuum at 40 °C for one day gave the solid product. Yield 65.0 g (99%). Amino Acid Analysis: Val (1.0) (following acid hydrolysis) Cys identified

Example 9

Cys(BAM)-NH

1.52 g (4.6 mmoles) of S-benzamidomethyl-L- cysteine,methylester,hydrochloride was added to 35 ml of 7 M ammonia solution in dry methanol and stirred for 4 days at room temperature. The solution was then taken to dryness under reduced pressure to give the product as a white powder.

Boc-Ile-Gln-OH

8.0 g (24.4 mmoles) Boc-Ile-ONSu was dissolved in 30 ml acetonitrile and poured into a solution of 7.7 g (52.5 mmoles) L-glutamine in a mixture of 100 ml H₂0 and 50 ml acetonitrile which had been adjusted to alkaline pH by addition of 30 ml 2 M aqueous sodium hydroxide. The mixture was stirred for 40 minutes at room temperature to completion of reaction, and pH was lowered to 6 using 10 M aqueous HC1. It was then filtered, taken to dryness, and pH was lowered to 2 using 10 m HC1, after which ethyl acetate was added repeatedly, and the organic phases combined and washed with 10% citric acid, followed by aqueous saturated NaCl solution, dried with MgSO., and taken to dryness under reduced pressure to give the product as a white powder.

Yield 7.85 g (90%) Boc-Ile-Gln-Cys(BAM)-NH₂

1.65 g (4.6 mmoles) Boc-Ile-Gln-OH and 0.56 g (4.8 mmoles) HONSu were dissolved in 7 ml methylene chloride, and after 1 hour at room temperature 0.99 g DCC (4.8 mmoles) was added.

The mixture was filtered and cooled to 0 °C, after which 1.5 g (4.6 mmoles) Cys(BAM)-NH₂ was added as a dry powder.

The reaction was stirred overnight at 4 °C. The reaction mixture was then filtered for DCU, applied to a Waters Preppak 500 C₁₈ column and purified using aqueous ethanol/acetic acid/water buffers. Fractions containing pure product were combined and taken to dryness under reduced pressure to give the product as a white powder.

Yield 0.3 g (12%).

Ile-Gln-Cys(BAM)-NH₂.TFA

To 300 mg Boc-Ile-Gln-Cys(BAM)-NH₂ was added 5 ml TFA. The mixture was stirred for 15 minutes. Following repeated additions of toluene and evaporation to dryness under reduced pressure, Ile-Gln-Cys(BAM)-NH₂ was then dissolved in 10 ml of 50 mM NH.Ac, filtered and applied to a C.,,, reverse phase HPLC column eqvilibrated in H₂0, and partially purified by elution.

Fractions containing purified product were combined and lyophilized. The purification procedure was repeated. The product was eluted with 5% ethanol/water and lyophilized from combined fractions of pure product.

Yield: 65 mg (20%). Example 10

MPR(Acm)TyrQH

148 g (0.835 mole) and 105.7 g (0.919 mole) HONSu were dissolved in 1 liter DMF. The mixture was cooled to 0 °C. A mixture of 175.7 g (0.852 mole) DCC dissolved in 0.7 liter DMF cooled to 0 °C, was added.

The reaction was stirred 2 days at 4 °C and filtered. The mixture was then added slowly (5-6 h) to a solution of 181.6 g (1 mole) L-tyrosine in 400 ml DMF and 1600 ml H₂0 in which pH had been adjusted to 8-9 with concentrated aqueous NaOH.

The reaction was stirred overnight at room temperature, and pH was adjusted to 3 with HC1. The reaction mixture was then filtered, applied to a Waters 20 liter reverse phase C_lβ column and purified using ethanol/water/acetic acid buffers.

Fractions containing pure product were combined and taken to dryness under reduced pressure, repeatedly following addition of dry ethanol to give the product as a dry powder.

Yield: 208.7 g (73%).

Example 11

Synthesis of H-Pen(Acm)-Pen(Acm)NH₂ TFA, H-Pen(Acm)-NH₂

2.4 g Boc-Pen(Acm)-0H and 1.35 g HONSu were dissolved in 50 ml acetonitrile, and following cooling to 0°C, 1.7 g of DCC was added. Following the chemical activation, NH_ gas was bubbled though. The mixture was taken to dryness under reduced pressure, redissolved in DMF and purified by reverse phase HPLC using water/ethanol/acetic acid buffers, and combined fractions of product were taken to dryness by evaporation under reduced pressure to yield 0.8 g of Boc- Pen(Acm)-NH₂. This was then deboced by trifluoracetic acid catalysis to yield TFA, H-Pen(Acm)-NH₂.

AcOH,H-Pen(Acm)-Pen(Acm)-NH₂

1 g of Boc-Pen(Acm)-OH and 0.6 of HONSu were dissolved in 20 ml of DMF and cooled to 0°C, after which 0.7 g of DCC was added. Following the activation, the TFA,H-Pen(Acm)-NH₂ prepared as above was added in 20 ml of DMF containing some TEA to neutralize the TFA. The formed Boc-Pen(Acm)- Pen(Acm)-NH₂ was deboced in 20% TFA in methylene chloride, after which it was taken to dryness under reduced pressure, redissolved and purified by reverse phase HPLC using water/ethanol/acetic acid mixtures. Combined fractions containing pure product was taken to dryness under reduced pressure to yield 0.44 g of AcOH,H-Pen(Acm)-Pen(Acm)-NH₂ as an oil. The mass spectrum of the product is shown in fig. 5. The oil was redissolved in water and lyophilized for later use.

Claims

P A T E N T C L A I M S

1. An organosulphur compound of the general formula:

R₁-CA]-[[P]-(CH₂)_a-C_χH-]_b-C 0-R₂ I

I I

[B] D

I

S-[E]

or an N-C-cyclic or a S-S-bridged form thereof, or a compound transformed into or releasing any of the above basic structures under physiological conditions in humans, wherein

R. is H, straight or branched alkyl or cycloalkyl up to C₂₀, optionally containing double bonds and/or substituted with halogen, nitro, amino, sulfo, phospho or carboxy, or aralkyl or aryl optionally mono- or polysubstituted with halogen, hydroxy, nitro, amino, sulfo, phospho, carboxy or alkyl, or R₁ is glycosyl, nucleosyl or an L- or D-α amino acid or a peptide moiety of 2 to 8 residues connected by bonds of type [P],

a is 0, 1, 2 or 3,

C is a tetrahedral carbon atom (SP3 hybridized) having R or S configuration,

C is a triplanar carbon atom (SP2 hybridized) and D is absent or C is a tetrahedral carbon atom and D is H„,

[A] is absent, a decarboxy amino acid residue or NHR₁' wwhheerreeiinn RR. '' iiss aass ddeeffiinneedd ffoorr R.. , and is absent when the compound is an N-C cyclic form, [P] is absent or is a peptide bond CO-NH,

or isosteres thereof, such as CH₂-NH, CH₂-S, C0-CH₂, retroinverse forms thereof, such as NH-CO,

B is absent or C.-Cg optionally branched alkyl or cycloalkyl,

S is sulphur,

E is absent when the compound is in disulfide bridged form, or E is

(CR₃R.) -NHC0R₅, wherein R₃ and R. independently are H, CH₃ or halogen, and R₅ is H, straight, branched alkyl or cycloalkyl, aralkyl or aryl, all of which are optionally mono- or polysubstituted with halogen, hydroxy, carboxy, sulfo, phospho, amino or nitro, or a decarboxy amino acid or a decarboxy peptide moiety, and c is 1, 2 or 3,

S-CRgR₇Rg, wherein R_β, R„ and R„ are independently H, halogen, straight, branched alkyl or cycloalkyl, aralkyl or aryl, all of which are optionally mono- or polysubstituted as indicated for R^,

CR_QR.J-RJ- are independently H, alkyl, aralkyl or aryl, all of which are optionally mono- or polysubstituted as indicated for R,_,

or

S-R-₂, wherein R-₂ is ^C;_I-C,_Q aralkyl or heteroaralkyl,

or R.₂, wherein R.₂ is as defined above,

R₂ is H, OH, CH₃ or

R_₃R₁₄, wherein R-₃ and R.. are independently H, straight, branched alkyl or cycloalkyl, aralkyl or aryl optionally substituted as defined for R..,

0R_lt-, where R₁₅ is H, straight, branched alkyl or cycloalkyl, aralkyl or aryl, optionally substituted as defined for R,_,

O-glycosyl, or

an L- or D-α-amino acid, or a peptide moiety of 2 to 8 residues connected by bonds of type [P],

or R„ is absent, when E is a decarboxy derivative of cysteine or a homologue thereof or the compound is an N-C cyclic form,

b is 1, 2, 3, 4 or 5,

and R» , R~ and R-- together comprise no more than 10 amino acid residues, and

hydrogen atoms may be replaced by fluorine, or a derivative or salt thereof.

2. A compound of claim 1 of the general formula

R ₁-[A]-[[P]-(CH₂)_a-G-]_b-C 0-R2

I I

[E] D wherein

G = -C H is a desamino-Cys or -Pen or sidechain

I

[B]

I s

homologues of these containing totally 2 - 7 alifatic carbon atoms,

S is sulphur,

a is 0,

[E] is a sulphur substitution group chosen among

R-₃, which is C..-C-. alkyl, such as methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, and tert.butyl, n-pentyl, isopentyl, tert.pentyl, 1,2- dimethylpropyl and 2,2-dimethylpropyl, or

R- ., which is C-.-C„ homo- or heteroaralkyl, such as phenyl, benzyl, toluyl and pyridyl, or

CH₂NHC0R₁₃ or

CH₂NHC0R₁₄ or

S-R₁₃ or

S-R₁₄ or

[E] is absent and the compound is an S-S-dimer, and R-i _. [A], [P], C , D, R₂ and b are as defined in claim 1, or a derivative or salt thereof.

3. A compound of claim 2, wherein

G is desamino-Cys, -Pen or -homocystein or 3-Mercaptopropionic acid, and

S, R_, [A], [P], C , [E], D, R₂, a and b are as defined in claim 2, or a derivative or salt thereof.

4. A compound of claim 1, 2 or 3

H-Cys-(Ac )-Met-NH₂,

H-Ile-Gln-Cys(Acm)-NH₂, (H-Ile-Gln)₂-Cystin-(0Me)₂,

H-Ile-Gln-Cys(BAM)-NH₂,

3-Mercaptopropionic acid-(Acm)-Tyr-OH

H-(Met)₃-NH₂,

H-(Pen(Acm))₂-NH₂ or (H-Cys(Acm))₂ Cystine-OMe₂

or a derivative or salt thereof.

5. A compound of any of the claims 1 - 4,

H-Ile-Gln-Cys-X-NH₂

wherein X is BAM (benzamidomethyl) or Acm (acetamidomethyl) or a derivative or salt thereof.

6. A compound of any of the claims 1 - 4,

3-Mercaptopropionic acid-(Acm)-Tyr-OH

or a derivative or salt thereof.

7. A compound of any of the claims 1 - 4,

H-(Met)₃-NH₂

or a derivative or salt thereof.

8. A compound of any of the claims 1 - 4,

H-(Pen(Acm))₂-NH₂

or a derivative or salt thereof.

9. A compound of any of the claims 1 - 4,

(H-Cys(Acm))₂ Cystine-0Me₂

or a derivative or salt thereof.

10. A pharmaceutical composition containing an effective antiglaucoma or intraocular pressure lowering amount of at least one organosulphur compound according to any of the claims 1 - 9 or a pharmaceutical acceptable derivative or salt thereof and a pharmaceutically acceptable diluent or excipient.

11. A pharmaceutical composition according to claim 10, wherein the organosulphur compound is the compound according to any of the claims 5-9 or pharmceutical acceptable derivative or salt thereof.

12. A pharmaceutical composition according to claim 10, wherein the amount of organosulphur compound is 0.01 - 10 percent by weight.

13. A pharmaceutical composition according to claim 10, wherein the amount of organosulphur compound is 0.1 - 5 percent by weight.

14. A pharmaceutical composition according to claim 10, wherein the amount of organosulphur compound is 0.25 - 1 percent by weight.

15. Use of a compound according to any of the claims 1-9 for preparing a pharmaceutical composition useful for treatment of glaucoma and/or intraocular hypertension.

16. A method for treatment of glaucoma or intraocular hypertension, comprising administering to a mammal an effective antiglaucoma or intraocular pressure lowering amount of at least one organosulphur compound, derivative or salt according to any of claims 1 to 9.