GB2389581A - Hydrogenated pyrimidines and their use as inhibitors of deoxyhypusine-synthase or hypusination of eIF-5A - Google Patents

Abstract

Compounds of formula (I), or pharmaceutically acceptable salts thereof, in which the dotted line represents a single bond when R Ü , R Þ and R ý are present and a double bond when R ý and one of R Ü and R Þ are absent. R Ü and R Þ are independently H or an optionally substituted alkyl or aryl group and R ý and R Ú are independently H, carboxyl, acetyl or an acid-labile protecting group. In particular the dotted line is a single bond and each of R Ü , R Þ , R ý and R Ú is H. The compounds may be capable of inhibiting the enzyme deoxyhypusine-synthase and may be used in the treatment or prevention of diseases affected by inhibition of deoxyhypusinesynthase and/or hypusination of the eukaryotic initiation factor 5A (eIF-5A). Specific diseases include viral diseases such as those caused by HIV and diseases associated with abnormal proliferative growth such as carcinomas. <EMI ID=1.1 HE=42 WI=56 LX=803 LY=1419 TI=CF>

Description

Deoxyhypusine-synthase inhibitors, pharmaceuticals comprising said

inhibitors and uses thereof Technical field

The present invention relates to compounds that are capable of inhibiting the enzyme deoxyhypusine-synthase as well as pharmaceutical compositions that comprise one or more of these compounds, methods for preventing or treating diseases that are affected by inhibiting the activity of the enzyme deoxyhypusine-synthase and/or hypusination of the eukaryotic initiation factor 5A (hereinafter also referred to as eIF-5A) and the use of these compounds for the preparation of such a medicament. More particularly, the present invention relates to classes of 1,3Diazacyclohexanes and 1,3-Diazacyclo-1-

hexenes that inhibit the activity of the enzyme deoxyhypusine-synthase and/or hypusination of the eukaryotic initiation factor 5A.

Background Art

Human eukaryotic initiation factor 5A (eIF-5A, previously know as eIF-4D) is a small acidic protein of 154 amino acids with a molecular mass of 16. 7 kDa (Smit-McBride et al., 1989a) eIF-5A is the only cellular protein known to date to contain the unusual amino acid hypusine (Park et al., 1993).

Hypusination occurs at the amino acid position Lys-50 of the human protein. The hypusination of eIF-5A occurs via a biosynthetic pathway involving two major steps. The first step is the reaction of the terminal amino residue of lye-50 of eIF-5A with spermidine which is catalyzed by the cellular enzyme deoxyLypusine-synthase (hereinafter also referred to as OHS").

Thus, during this first step in eIF-5A modification, the aminobutyl group of spermidine is transferred to the terminal

amino group of lye-50 leading to its mono-aminobutylation.

The second step in the biosynthesis of hypusinated eIF-5A is accomplished by hydroxylation of the C-3 moiety of the terminal butylamino group that is catalyzed by the enzyme deoxyhypusyl-hydroxylase (This two step reaction scheme is depicted in Figure 1).

Although hypusine-modified eIF-5A appears to be essential in rapidly dividing cells (e.g. tumorigenic cells) (Park et al., 1994; McCaffrey et al., 1995; Shi et al., 1996; Chen et al., 1996; Caraglia et al., 1999), its precise function remains elusive. Its designation as an Initiation factor" originated from the initial finding that it stimulates the formation of the dipeptide analogue methionyl-puromycin in an in vitro assay that mimics the formation of the first peptide bond in protein synthesis (Park, 1989; Smit-McBride et al., 1989b).

The activity of eIF-5A in this model assay, however, appears to be poor (Kang et al., 1993). More recent in viva data has clearly indicated that eIF-5A is not an initiator of general protein translation. In fact, complete intracellular depletion of eIF-5A in the yeast Saccharomyces cerevisiae does not cause major changes in the overall rate of protein synthesis tKang and Hershey, 1994). Despite this, the precise in vivo function of eIF-5A in eukaryotic cells remains unknown. It is known that the hypusine-containing protein eIF-5A is an essential cellular cofactor of human pathogenic retroviruses (HIV and HTLV).

Investigation of the posttranscriptional regulation of human mmunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) gene expression indicated a novel activity eIF-5A in vivo. HIV1 and HTLV-I require both

the action of viral trans-activator proteins, termed Rev and Rex, respectively, in order to produce progeny viruses (Pollard and Malim, 1998; Hauber, 2001). Rev and Rex constantly shuttle between the nucleus and cytoplasm of host cells and mediate the translocation of unspliced and I incompletely-spliced viral mRNAs across the nuclear envelope.

eIF-5A is an essential factor for this process that binds to Rev or Rex and facilitates efficient interaction of Rev/Rex with the general export receptor CRM1 (Rubl et al., 1993; Katahira et al., 1995; Bevec et al., 1996; Rosorius et al., 1999; Hofmann et al., 2001). Therefore, eIF-5A is part of a specific nucleocytoplasmic RNA transport pathway.

Studies using inhibitors of the hypusine modification of eIF-

5A revealed that the compounds deferiprone and mimosine are able to efficiently interfere with the life cycle of HIV-l by reducing the activity of the enzyme deoxyhypusyl-hydroxylase, I i.e. the second enzyme in the conversion of Lys-50 of eIF-5A to hypusine (Andrus et al., 1998).

WO 91/56553 describes a set of aromatic guanylhydrazones that inhibit DHS and their use against retroviral infection and accompanying diseases.

In addition to the important role that eIF-5A plays in the regulation of HIV-1 and HTLV-l expression, eIF-5A has also been demonstrated to be involved in the regulation of several other biological processes.

Inhibition of hypusine-modification of eIF-5A with DHS inhibitors interferes with TNF-alpha release. Therefore, DHS inhibitors block TNFalpha production by interfering with TNF-alpha mRNA processing (e.g. mRNA transport) and have an anti-inflammatory effect. For example, the multivalent guanylhydrazone CNI-1493 (also known as AXD455) blocks TNF-

alpha release (Cohen et al., 1996) and has been shown to be a DHS inhibitor (see also WO 01/56553 that describes various

pharmaceutically active aromatic guanylhydrazones as inhibitors of DHS).

Inhibition of hypusine-modification of eIF-SA with DHS inhibitors negatively interferes with dendritic cell (hereinafter also referred to as DC") mediated T-cell activation and inhibits DC-mediated immune responses.

Treatment of DC with the DHS inhibitor N-Guanyl-1,7-

diaminoheptane (GC7) results in down regulation of CD83 expression on DC and negatively interferes with DC-mediated T-cell activation (Kruse et al. , 2000). Inhibition of hypusine-modification blocks the postranscriptional processing (e.g. nucleocytoplasmic transport) of CD83-

specific mRNA. Therefore' DHS inhibitors block CD83 expression and, thereby, DC function.

Inhibition of hypusine-modification of eIF-5A with DHS inhibitors has also been shown to have antiproliferative effects and selected compounds have been suggested for the treatment of neoplastic diseases (see Park et al., 1994 and WO 94/15S96).

WO 94/15596 and Jakus et al. disclose that several types of basic compounds structurally related to spermidine act as inhibitors for DHS. Their results indicated that inhibitory compounds associate with the enzyme at the site of spermidine binding and must possess two charged primary amino or guanidino groups, or one of each. The efficiency of inhibition was found to be related to the maximum possible distance between the primary amino groups and is adversely affected by substitutions on the secondary amino group or in the carbon chains of such amines. lN-Guanyl-1,7-

diaminoheptane was found to be the most effective compound in this series with a Kl-value of about 10 nM.

An object of the present invention is to provide compounds and pharmaceutical compositions that are capable of

inhibiting the hypusination of the eukaryotic initiation factor 5A.

A further object is to make methods available for preventing or treating diseases that are affected by inhibiting the I activity the eukaryotic initiation factor 5A.

An additional object is to provide a use of compounds that are capable of inhibiting the hypusination of the eukaryotic initiation factor 5A for the preparation of a medicament for preventing or treating diseases that are affected by the eukaryotic initiation factor 5A.

Detailed Description of the Drawings

Figure 1: Schematic representation of the biosynthesis of hypusinated eIF5A.

Best Mode In contrast to the teaching in WO 01/56553 and WO/15596, it was assumed by the inventors that because of the fact that spermidine is required for the eIF-5A activation process, the diaminopropyl group within the substrate must play an essential role within the entire biocatalytical transformation The recognition and effective binding of the diaminopropyl moiety of spermidine by the active site of deoxyhypusinesynthase prior to the aminobutlyation of eIF-5A must be crucial.

It has now been surprisingly and unexpectedly found that molecules bearing an 1,3-diaminopropane moiety within a specific structural arrangement give rise to an alternative DHS-inhibition mechanism with high efficiency. As a consequence, these inhibitors can serve as new drugs for all diseases where an eIF-5A mediated intracellular transport plays a central role.

Thus, one embodiment of the present invention relates to compounds of the general formula (I): /R3 r N R2 R4 wherein the dotted line represents a single bond when R1, R2 and R3 are present and the dotted line represents a double bond when either R1 or R2 are present and R3 is absent, and wherein R1 and R2 can be the same or different and are selected from the group H. straight or branched chain substituted or non-substituted alkyl, substituted or non-

substituted aryl and R3 and R4 can be the same or different and are selected from H. carboxyl, acetyl and an acid-labile protective group, as well as pharmaceutically acceptable salts thereof.

Compounds of the general formula (I) include compounds such as compounds of the general formula (II) and (III): /R3 i \/R1 (II) N/\R2

N \ R1 (III)

N. \ R4 wherein R1, R2, R3 and R4 are defined as above as well as pharmaceutically acceptable salts thereof.

When R1 and/or R2 is a straight chain alkyl, the alkyl group is preferably C1-C6 alkyl such as a methyl, ethyl, propyl, butyl, pentyl or hexyl group.

When R1 and/or R2 is a branched chain alkyl group, the preferred type of branching is secondary, with one branch being C3-C6, such as propyl, butyl, pentyl or hexyl, and the other branch being C1-C4 such as methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, sec-butyl, tert-butyl, methylcyclopropyl and cyclobutyl.

The branched or unbranched chains can contain one or more carbon-carbon double or triple bonds. The alkyl groups chains may contain one or several substituents. Preferred substituents are carboxylic groups, ketone groups, aldehyde groups, hydroxyl groups, ether groups, amino groups, halogens and sulfonic acid groups.

In case of R1 and/or R2 being aryl groups these can be substituted or unsubstituted. Preferred substituents are carboxylic groups, ketone groups, aldehyde groups, hydroxyl groups, ether groups, halogens, amino groups and sulfonic acid groups.

Preferred acid labile protective moieties of R3 and/or R4 are t-butyloxycarbonyl (boc), t-butyldimethylsilyl (tbdms), methoxy-ethoxy-methyl (mem) and tetrahydropyranyl (thp).

In a preferred embodiment of the present invention, the compound is: c: NH The 1,3-Diazanes according to the invention can be synthesized according to modification of the following procedure for the synthesis of hexahydropyrimidine (1,3-

diaza-cyclohexane). O +(H+) H-N N-H

H H H2 NH2 - H2O HXH

A solution of 50 mmol formaldehyde (4.05 ml, 37% aqueous solution) was added slowly to a stirred solution of 50 mmol diaminopropane in 20 ml water. The mixture was stirred for 24h at room temperature and then heated for an additional 2h to cat 70 C. The solution was subsequently cooled to 0 C, saturated with solid NaOH, and then extracted with methylene chloride four times. After drying of the combined CH2Cl2-

extracts with Na2SO4,the solvent was evaporated at 300 mbar.

The purification of the residue takes place by fractionated bulb to bulb distillation in a Kugelrohr" apparatus.

Analytical data for Rl=R2=H: MS (EI, 70ev, direct, cat 100 C): 86 (M+), 85 (M±1), 57 (M+ CH2NH), 42 (C3H6)

1H-NMR (D20) 400,13MHz: = 1.53 q (2H, CH2-CH2-CH2, J =7.3Hz); 2.88 t (4H, J =7.3Hz); 3.65 s (2H, N-CH2-N) Bp.: 80-90 C/45mbar

General procedure for the boc-protection of the 1,3-Diazanes according to the invention.

40 mmol boc-anhydride in 20ml THE was slowly added at room temperature to a solution of 20 mmol 1,3-diazane in 20ml THE.

The mixture was stirred overnight, the solvent evaporated under reduced pressure and the residue fractionated under vacuum. f: 1I for 11 H-N X NH + 2 soc t RXR t -2CO2 The 2-alkyl-3,4, 5,6-tetrahydropyrimidines according to the invention can be synthesized according to following procedure. 0 0

Rat O-Et + NH2 NH2 Rat hi Ntl2 R' N + CaO R 4 H H Mono-N-acetyltrimethylendiamines: 100 mmol (7.4g) 1,3-diaminopropane and 33 mmol of an ethyl-

ester of a carbon acid were sealed in a thick-walled glass tube and heated at 100 C for twenty hours.

The reaction mixture was then transferred to a Kugelrohr"-

apparatus and the low boiling material, consisting of ethanol

and excess 1,3 diaminopropane was removed under diminished pressure. The product was separated by following fractionated bulb to bulb distillation from the bis-acylation product.

Redistillation yielded Mono-N-acetyl-trimethylamine, boiling at 90 C/0. 2mm (R =CH3).

Analytic data for R=CH3: MS (EI, 70ev, direct, ca.70 C): 116 (M+),101 (M+15), 99 (M±

NH3), 73 tM±CH3-CO) b) 2-Alkyl-3,4,5,6-tetrahydropyrimidines 20 mmol mono-N-acetyl-trimethylenamine were sealed with 1 mol finely powdered calcium oxide in a thick walled glass tube and heated for 15h at 250 C. The reaction product was extracted from the solid with absolute ethanol and distilled after removal of the solvent, under diminished pressure.

After redistillation or recrystallization from ethyl acetate/petrolether the pure product was obtained.

Compounds of the invention can form pharmaceutically acceptable salts with organic and inorganic acids. Examples -

of suitable acids for such acid addition salt formation are acetic acid, adipic acid, alpha-toluic acid, ascorbic acid, benzoic acid, camphersulfonic acid, china acid, citric acid, d-o-tolyltartraric acid, ethanesulfonic acid, ethylenesulfonic acid, formic acid, fumaric acid, gluconic acid, hydrobromic acid, hydrochloric acid, hydrogenbenzenesulfonic acid, hydroxyethanesulfonic acid, hydroxymaleic acid, lactic acid, maleic acid, malic acid, malonic acid, mandeLic acid, methanesulfonic acid, naphthylamine sulfonic acid, naphthylsulfonic acid, nitric acid, nitrous acid, o-methylmandelic acid, oxalic acid, -

p-aminobenzoic acid, p-aminosalicylic acid, perchloric acid, -

phenylacetic acid, phosphoric acid, phosphoric acid, = p-hydroxybenzoic acid, picric acid, propionic acid,

f p-toluenesulfonic acid, pyruvic acid, salicylic acid, succinic acid, sulfanilic acid, sulfonic acid, sulfuric acid, tartaric acid, tartronic acid, (o, m, p)-toluic acid, and other mineral or carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.

The free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous sodium hydroxide, potassium carbonate, ammonia and sodium bicarbonate. The free base forms differ from their corresponding salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to their corresponding free base forms for purposes of this invention.

The compounds of the present invention cause an inhibition of the hypusination of eIF-5A which can be reversible or irreversible and preferably corresponds to an inhibition of the hypusination of eIF-5A (inhibition of DHS activity) of at least 25%, more preferably at least 50%, still more preferably at least 75% and most preferably at least 90% or greater. A preferred assay for measuring the inhibition of deoxyhypusine synthase activity (hypusination of eIF-5A) is provided in the examples.

Accordingly, the present invention relates to the use of a compound according to the invention for inhibiting deoxyhypusine synthase activity and/or eIF-5A activity in vitro and to assays or kits for directly or indirectly determining DHS activity or eIF-5A hypusination or determining the effects of DHS or eIF-5A in a biological system, said assay or kit comprising a compound according to the invention.

In a further embodiment, the present invention relates to pharmaceutical compositions comprising one or more of the compounds according to the invention and a pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises the compound: NH \NH The preparation of pharmaceutical compositions with an amount of one or more compounds according to the invention and/or their use in the application according to the invention occurs in the customary manner by means of common pharmaceutical technology methods.

For this, the compounds according to the invention are processed together with suitable, pharmaceutically acceptable carriers, e.g. adjuvents, excipients etc., and/or to medicinal forms suitable for the various indications and types of application. Thereby, the medicaments can be produced in such a manner that the respective desired release rate is obtained, for example a quick flooding and/or a sustained or depot effect.

Preparations for parenteral use, to which injections and infusions belong, are included among the systemically employed medicaments for the above mentioned indications.

Preferably, injections are prepared either in the form of vials or also as so-called ready-to-use injection preparations, for example as ready-touse syringes or single

use syringes in addition to perforation bottles for multiple withdrawals. Administration of the injection preparations can occur in the form of subcutaneous (s.c.), intramuscular (i.m.), intravenous (i.v.), internodal (i.n.) or intracutaneous (i.c.) application. The respective suitable injection forms can especially be produced as solutions, crystal suspensions, nanoparticular or colloid-disperse systems, such as for example, hydrosols.

The injectable formulations can also be produced as concentrates which can be adjusted with aqueous isotonic dilution agents to the desired dosage of the compounds of the invention. Furthermore, they can also be produced as powders, such as for example lyophilisates, which are then preferably dissolved or dispersed immediately before application with suitable diluents. The infusions can also be formulated in the form of isotonic solutions, fat emulsions, liposome formulations, microemulsions and liquids based on mixed micells, for example, based on phospholipids As with injection preparations, infusion formulations can also be prepared in the form of concentrates to dilute. The injectable formulations can also be applied in the form of continuous infusions as in stationary as well as in out-

patient therapy, for example in the form of mini-pumps.

Albumin, plasma expanders, surface active compounds, organic solvents, pH influencing compounds, complex forming compounds or polymeric compounds can be added to the parenteral medicinal forms with the aim of decreasing the adsorption of the compounds of the present invention to materials such as injection instruments or packaging materials, for example plastic or glass.

The compounds according to the invention can be bound to nanoparticles in the preparations for parenteral use, for example on finely dispersed particles based on polymeth)acrylates, polyacetates, polyglycolates, polyamino

f acids or polyether urethanes. The parenteral formulations can also be constructively modified as depot preparations, for example on the multiple unit principle, where the compounds of the present invention are incorporated in a most finely distributed and/or dispersed, suspended form or as crystal suspensions, or on the single unit principle, where the compounds according to the invention are enclosed in a medicinal form, for example, a tablet or a seed which is subsequently implanted. Often, these implantation or depot medicaments in single unit and multiple unit medicinal forms consist of so-called biodegradable polymers, such as for example, polyether urethanes of lactic and glycolic acid, polyether urethanes, polyamino acids, polymeth)acrylates or polysaccharides. Sterilized water, pH value influencing substances, such as for example organic and inorganic acids or bases as well as their salts, buffer substances for setting the pH value, agents for isotonicity, such as for example sodium chloride, monosodium carbonate, glucose and fructose, tensides and/or surface active substances and emulsifiers, such as for example, partial fatty acid esters of polyoxyethylene sorbitan (Tween@) or for example fatty acid esters of polyoxethylene (Cremophor@), fatty oils such as for example peanut oil, soybean oil and castor oil, synthetic fatty acid esters, such as for example ethyl oleate, isopropyl myristate and neutral oil (Miglyol) as well as polymer adjuvents such as for example gelatin, dextran, polyvinylpyrrolidone, organic solvent additives which increase solubility, such as for example propylene glycol, ethanol, N,N-dimethylacetamide, propylene glycol or complex forming compounds such as for example citrates and urea, preservatives, such as for example hydroxypropyl benzoate and hydroxymethyl benzoate, benzyl alcohol, anti-oxidants, such as for example sodium sulfite and stabilizers, such as for example EDTA, are suitable as adjuvents and carriers in the production of preparations for parenteral use.

In suspensions, addition of thickening agents to prevent the settling of the compounds of the present invention from tensides and peptizers, to secure the ability of the sediment to be shaken, or complex farmers, such as EDTA, ensues. This can also be achieved with the various polymeric agent complexes, for example with polyethylene glycols, polystyrol, carboxymethylcellulose, Pluronics or polyethylene glycol sorbitan fatty acid esters. The compounds according to the invention can also be incorporated in liquid formulations in the form of inclusion compounds, for example with cyclodextrins. As further adjuvents, dispersion agents are also suitable. For production of lyophilisates, builders are also used, such as for example mannite, dextran, saccharose, human albumin, lactose, PVP or gelatin varieties.

A further systemic application form of importance is peroral administration as tablets, hard or soft gelatin capsules, coated tablets, powders, pellets, microcapsules, oblong compressives, granules, chewable tablets, lozenges, gums or sachets. These solid peroral administration forms can also be prepared as sustained action and/or depot systems. Among these are medicaments with an amount of one or more micronized compounds of the present invention, diffusions and erosion forms based on matrices, for example by using fats, wax-like and/or polymeric compounds, or so-called reservoir systems. As a retarding agent and/or agent for controlled release, film or matrix forming substances, such as for example ethylcellulose, hydroxypropylmethylcellulose, polymeth)acrylate derivatives (for example Eudragit@), hydroxypropylmethylcellulose pUthalate are suitable in organic solutions as well as in the form of aqueous dispersions. In this connection, so-called big-adhesive preparations are also to be named in which the increased retention time in the body is achieved by intensive contact with the mucus membranes of the body. An example of a bio-

adhesive polymer is the group of Carbomers@.

For sublingual application, compressives, such as for example nondisintegrating tablets in oblong form of a suitable size with a slow release of the compounds of the present invention, are especially suitable. For purposes of a targeted release of compounds of the present invention in the various sections of the gastrointestinal tract, mixtures of pellets which release at the various places are employable, for example mixtures of gastric fluid soluble and small intestine soluble and/or gastric fluid resistant and large intestine soluble pellets. The same goal of releasing at various sections of the gastrointestinal tract can also be conceived by suitably produced laminated tablets with a core, whereby the coating of the agent is quickly released in gastric fluid and the core of the agent is slowly released in the small intestine milieu. The goal of controlled release at various sections of the gastrointestinal tract can also be attained by multilayer tablets. The pellet mixtures with differentially released agent can be filled into hard gelatin capsules. Anti-stick and lubricant and separating agents, dispersion agents such as flame dispersed silicone dioxide, disintegrants, such as various starch types, PVC, cellulose esters as granulating or retarding agents, such as for example wax-like and/or polymeric compounds on the basis of Eudragit@, cellulose or Cremophor are used as a further adjuvents for the production of compressives, such as for example tablets or hard and soft gelatin capsules as well as coated tablets and granulates.

Anti-oxidants, sweetening agents, such as for example saccharose, xylite or mannite, masking flavors, aromatics, preservatives, colorants, buffer substances, direct tableting agents, such as for example microcrystalline cellulose, starch and starch hydrolysates (for example Celutab@), lactose, polyethylene glycols, polyvinylpyrrolidone and

dicalcium phosphate, lubricants, fillers, such as lactose or starch, binding agents in the form of lactose, starch varieties, such as for example wheat or corn andior rice starch, cellulose derivatives, for example methylcellulose, hydroxypropylcellulose or silica, talcum powder, stearates, such as for example magnesium stearate, aluminum stearate, i calcium stearate, talc, siliconized talc, stearic acid, acetyl alcohol and hydrated fats are used.

In this connection, oral therapeutic systems constructed especially on osmotic principles, such as for example GIT (gastrointestinal therapeutic system) or OROS (oral osmotic system), are also to be mentioned.

Effervescent tablets or tabs, both of which represent! immediately drinkable instant medicinal forms which are quickly dissolved or suspended in water are among the perorally administratable compressives. Among the perorally administratable forms are also solutions, for example drops, juices and suspensions, which can be produced according to the above given method, and can still contain preservatives for increasing stability and optionally aromatics for reasons of easier intake, and colorants for better differentiation as well as antioxidants and/or vitamins and sweeteners such as sugar or artificial sweetening agents. This is also true for inspisated juices which are formulated with water before ingestion. Ion exchange resins in combination with one or more compounds of the present invention are also to be mentioned for the production of liquid ingestable forms.

A special release form consists in the preparation of so-

called floating medicinal forms, for example based on tablets or pellets which develop gas after contact with body fluids and therefore float on the surface of the gastric fluid.

Furthermore, so-called electronically controlled release systems can also be formulated by which release of the

compounds of the present invention can be selectively adjusted to individual needs.

A further group of systemic administration and also optionally topically effective medicinal forms are represented by rectally applicable medicaments. Among these are suppositories and enema formulations. The enema formulations can be prepared based on tablets with aqueous solvents for producing this administration form. Rectal capsules can also be made available based on gelatin or other carriers. Hardened fat, such as for example Witepsol@, Massa Estarinum, Sonatas, coconut fat, glycerolgelatin masses, glycerol-soap-gels and polyethylene glycols are suitable as suppository bases.

For long-term application with a systematic release of the compounds of the present invention up to several weeks, pressed implants are suitable which are preferably formulated on the basis of so-called biodegradable polymers. As a further important group of systemically active medicaments,

transdermal systems are also to be emphasized which distinguish themselves, as with the above-mentioned rectal forms, by circumventing the liver circulation system and/or liver metabolism. These plasters can be especially prepared as transdermal systems which are capable of releasing the compounds of the present invention in a controlled manner over longer or shorter time periods based on different layers and/or mixtures of suitable adjuvents and carriers. Aside from suitable adjuvents and carriers such as solvents and polymeric components r for example based on Eudragit@, membrane infiltration increasing substances and/or permeation promoters, such as for example oleic acid, Azone@, adipinic acid derivatives, ethanol, urea, propylglycol are suitable in the production of transdermal

systems of this type for the purpose of improved and/or accelerated penetration.

As topically, locally or regionally administration medicaments, the following are suitable as special formulations: vaginally or genitally applicable emulsions, creams, foam tablets, depot implants, ovular or transurethral administration installation solutions. For opthalmological application, highly sterile eye ointments, solutions and/or drops or creams and emulsions are suitable.

In the same manner, corresponding otological drops, ointments or creams can be designated for application to the ear For both of the abovementioned applications, the administration of semi-solid formulations, such as for example gels based on Carbopols or other polymer compounds such as for example polyvinylpyrolidone and cellulose derivatives is also possible. For customary application to the skin or also to the mucus membrane, normal emulsions, gels, ointments, creams or mixed phase and/or amphiphilic emulsion systems (oil/water-

water/oil mixed phase) as well as liposomes and transfersomes can be named. Sodium algenate as a gel builder for production of a suitable foundation or celluolose derivatives, such as for example guar or xanthene gum, inorganic gel builders, such as for example aluminum hydroxides or bentonites (so-

called thixotropic gel builder), polyacrylic acid derivatives, such as for example Carbopol@, polyvinylpyrolidone, microcrystalline cellulose or carboxymethylcellulose are suitable as adjuvents and/or carriers. Furthermore, amphiphilic low and high molecular weight compounds as well as phospholipids are suitable. The gels can be present either as hydrogels based on water or as hydrophobic organogels, for example based on mixtures of low and high molecular paraffin hydrocarbons and Vaseline.

Anionic, cationic or neutral tensides can be employed as emulsifiers, for example alkalized soaps, methyl soaps, amine soaps, sulfanated compounds, cationic soaps, high fatty alcohols, partial fatty acid esters of sorbitan and polyoxyethylene sorbitan, for example lanette types, wool wax, lanolin, or other synthetic products for the production of oil/water and/or water/oil emulsions.

Hydrophilic organogels can be formulated, for example, on the basis of high molecular polyethylene glycols. These gel-like forms are washable. Vaseline, natural or synthetic waxes, fatty acids, fatty alcohols, fatty acid esters, for example as mono-, di-, or triglycerides, paraffin oil or vegetable oils, hardened castor oil or coconut oil, pig fat, synthetic fats, for example based on acrylic, caprinic, lauric and stearic acid, such as for example Softisan or triglyceride mixtures such as Miglyol are employed as lipids in the form of fat and/or oil and/or wax-like components for the production of ointments, creams or emulsions.

Osmotically effective acids and bases, such as for example hydrochloric acid, citric acid, sodium hydroxide solution, potassium hydroxide solution, monosodium carbonate, further buffer systems, such as for example citrate, phosphate, Tris-

buffer or triethanolamine are used for adjusting the pH value. Preservatives, for example such as methyl- or propyl benzoate (parabenes) or sorbic acid can be added for increasing stability. Pastes, powders or solutions are to be mentioned as further topically applicable forms. Pastes often contain lipophilic and hydrophilic auxiliary agents with very high amounts of fatty matter as a consistency-giving base.

Powders or topically applicable powders can contain for example starch varieties such as wheat or rice starch, flame dispersed silicon dioxide or silica, which also serve as diluents, for increasing flowability as well as lubricity as well as for preventing agglomerates.

Nose drops or nose sprays serve as nasal application forms.

In this connection, nebulizers or nose creams or ointments can come to use.

Furthermore, nose spray or dry powder formulations as well as controlled dosage aerosols are also suitable for systemic administration of the compounds of the present invention.

These pressure and/or controlled dosage aerosols and dry powder formulations can be inhaled and/or insufflated.

Administration forms of this type also certainly have importance for direct, regional application in the lung or bronchi and larynx. Thereby, the dry powder compositions can be formulated for example as invention compound-soft pellets, as an invention compound-pellet mixture with suitable carriers, such as for example lactose and/or glucose. For inhalation or insufflation, common applicators are suitable which are suitable for the treatment of the nose, mouth and/or pharynx. The compounds of the present invention can also be applied by means of an ultrasonic nebulizing device.

As a propellant gas for aerosol spray formulations and/or controlled dosage aerosols, tetrafluoroethane or HFC 134a and /or heptafluoropropane or HFC 227 are suitable, wherein non-

fluorinated hydrocarbons or other propellants which are gaseous at normal pressure and room temperature, such as for example propane, butane or dimethyl ether can be preferred.

Instead of controlled dosage aerosols, propellant-free, manual pump systems can also be used.

The propellant gas aerosols can also suitably contain surface active adjuvents, such as for example isopropyl myristate,

polyoxyethylene sorbitan fatty acid ester, sorbitan trioleate, lecithins or soya lecithin.

The present invention also relates to use of one or more compounds according to the invention for the preparation of a medicament for the prevention and/or treatment of diseases or medical conditions that are responsive to a decrease in hypusine-dependent eIF-SA activity.

Furthermore, the invention relates to a method for the prevention and/or treatment of diseases or medical conditions that are responsive to a decrease in hypusine-dependent eIF 5A activity, wherein an effective amount of a compound! according to the invention or a pharmaceutical composition according to the invention comprising said compound is administered to a subject in need thereof.

Thus, the present invention also preferably relates to use of one or more compounds according to the invention for the preparation of a medicament for the prevention and/or treatment of virally induced infections and diseases, including opportunistic infections.

Furthermore, the invention relates to a method for the prevention and/or treatment of a virally induced infections and diseases, including opportunistic infections, wherein an effective amount of a compound according to the invention or a pharmaceutical composition according to the invention comprising said compound is administered to a subject in need thereof. In a preferred embodiment, the virally induced infections and diseases are caused by a virus selected from a group of retroviruses, hepadnaviruses, herpesviruses, adenoviruses, orthomyxoviruses and flaviviruses.

The group retroviruses include lentiviruses and oncoretroviruses. According to the invention, preferable lentiviruses include HIV, e.g. HIV1 and HIV-2 (Human Immunodeficiency Virus), EIAV (Equine Infectious Anemia Virus), VMV (Visna Maedi Virus), CAEV (Caprine Arthritis- Encephalalitis Virus), BIV! (Bovine Immunodeficiency Virus), FIV (Feline Immunodeficiency Virus) and SIV (Simian Immunodeficiency Virus).

More preferably, the present invention relates to use of one or more compounds according to the invention for the preparation of a medicament for the prevention and/or treatment of HIV induced infections, diseases and/or conditions, such as AIDS.

Opportunistic infections associated with HIV infection are known (see for example WO 01/56553).

The HIV strain can be a T-cell tropic strain, a macrophage-

trophic strain or a drug resistant, for example a protease inhibitor resistant or reverse transcriptase inhibitor resistant, strain of HIV-1 or HIV-2 According to the invention, preferable oncoretroviruses include HTLV, e.g. HTLV-I and HTLV-II (Human T-cell Leukemia Virus) and BLV (Bovine Leukemia Virus).

According to the invention, preferable herpesviruses include human herpesvirus 1 (Herpes Simplex Virus 1, HSV1), human herpesvirus 2 (Herpes Simplex Virus 2, HSV2), human herpesvirus 3 (Varicella Zoster Virus, VZV), human herpesvirus 4 (Epstein-Barr Virus, EBV), human herpesvirus 5 (human Cytomegalovirus, HCMV), human herpesvirus 6 (HHV6), human herpesvirus7 (HHV7) and human herpesvirus 8 (HHV8).

Most preferred are: HSV1, HSV2, VZV, EBV, HCMV, HHV8.

According to the invention, preferable hepadnaviruses include HBV (Hepatitis B Virus), woodchuck hepatitis virus (WHY) and ground squirrel hepatitis virus (GSHV) According to the invention, preferable orthomyxoviruses include influenza viruses.

According to the invention, a preferable flavivirus is hepatitis C virus.

The present invention also relates to use of one or more compounds according to the invention for the preparation of a medicament for the prevention and/or treatment of diseases or medical conditions mediated by TNF-alpha.

Furthermore, the invention relates to a method for the prevention and/or treatment of diseases or medical conditions mediated by TNF-alpha, wherein an effective amount of a compound according to the invention or a pharmaceutical composition according to the invention comprising said compound is administered to a subject in need thereof.

According to the invention, diseases or medical conditions mediated by TNF-alpha include bacterially-induced hemorrhagic fever diseases, hemorrhagic shock syndromes and inflammation.

Moreover, the present invention relates to the use of the compounds of the present invention for the production of a pharmaceutical composition or medicament for regulating an immune response involving the direct or indirect participation of DC (dendritic cells).

Furthermore, the invention relates to a method of treatment or prevention of disorders, diseases and syndromes involving the direct or indirect participation of DC by regulating an immune response, wherein an effective amount of a compound

according to the invention or a pharmaceutical composition according to the invention comprising said compound is administered to a subject.

Thus, the compounds of the present invention can be used to inhibit CD83 protein expression and/or induction of the T cell stimulating mode of DC or induction of so-called "regulatory" T cells and thereby treat or prevent a variety of disorders, diseases and syndromes. "Regulatory" T cells are defined as IL-10-producing non-proliferating CD25+ T cells. For example, the compounds of the present invention can be used to modulate the growth, differentiation and/or i activation of a variety of T cells such as cytotoxic T cells and helper T cells, the differentiation of helper T cells into Thl cells or Th2 cells, the growth, stimulation and/or differentiation of B cells and treat or prevent disorders, diseases and syndromes caused by the failure of the body to regulate these processes in a healthy manner.

In addition, the compounds of the present invention can be used to treat or prevent rejection of tissue and/or organ transplants, particularly xenogenic tissue and/or organ I transplants, that occurs as a result of for example graft vs -host disease or host-vs.-graft disease.

In a further embodiment of the present invention, the compounds of the present invention can be used to treat or prevent undesirable response to foreign antigens and therewith allergies and asthma or similar conditions.

Other disorders, diseases and syndromes that can be treated or prevented by the compounds of the present invention include autoimmune syndromes such as myasthemia gravis, multiple sclerosis and systemic lupus erythematosis, Crohn's

disease, skin diseases such as psoriasis and rheumatoid arthritis. Moreover, the present invention relates to the use of the compounds of the present invention for the production of a pharmaceutical composition or medicament for treating proliferative cell growth associated with malignant or benign disease. Furthermore, the invention relates to a method of treatment proliferative cell growth associated with malignant or benign disease, wherein an effective amount of a compound according to the invention or a pharmaceutical composition according to the invention comprising said compound is administered to a subject. According to the invention, malignant diseases include hematological systemic diseases, carcinomas, sarcomas, myelomas, melanomas, lymphomas and papillomas.

The pharmaceutical composition according to the invention can i also comprise further therapeutic compounds.

For example, when the pharmaceutical composition of the I invention is to be used in the treatment of a virally induced disease or medical condition, the pharmaceutical composition according to the invention can also comprise one or more antiviral agents.

More particularly, when the pharmaceutical composition of the invention is to be used in the treatment of AIDS, the pharmaceutical composition according to the invention can also comprise one or more antiviral agents, for example HIV-1 or HIV-2 protease inhibitors resistant and/or reverse transcriptase inhibitors.

WO 01/56553 provides examples of antiviral agents that are useful in the practice of the present invention and is incorporated by reference.

For therapeutic or prophylactic use, the compounds of the present invention are administered to a subject, preferably a mammal, more preferably a human patient, for treatment or prevention in a manner appropriate for the medical indication. A therapeutically effective dosage of a compound of the general formula (I) refers to an amount of the compound that results in an at least partial inhibition of deoxyhypusine synthase. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LDso and EDso. The dosage of the compounds according to the invention lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity. More preferably, the dosage of the compound corresponds to an effective concentration in the range of O.01-10 M. The actual amount of the composition administered will be dependent on the subject being treated, on the subject's weight, the severity of the disease of medical condition, the manner of administration and the judgment of the prescribing physician.

In the following, various aspects of the invention are more closely described via examples. However, it should be understood that the invention should not be considered as being limited to the examples.

Examples

Deoxyhypusine synthase inhibition assay In order to test the ability of compounds to inhibit deoxyhypusine synthase, an assay was developed based on the incorporation of radioactivity from [l4C]-spermidine (Amersham Pharmacia Biotech, UK) into eIF-5A precursor protein. The reaction mixture of 200 pi contained 6 psi [l4C]-spermidine (110 mci/mmol; 50 psi/ml), 1 mM HAD+, 5 fig recombinant eIF-5A precursor protein, 50 g/ml BSA (Sigma Chemical, USA), and various amounts of recombinant deoxyLypusine synthase in 300 mM Glycine-NaOH buffer pH 9.0, 1 mM DTT. This mixtures were incubated in presence of various amounts of DHS- inhibitors for 2 hours at 37 C. The reactions were stopped by addition of 100 pi of 20 mM spermidine in PBS. In order to separate the labeled eIF- 5A protein from excess spermidine, the reaction mixtures were passed through Milipore GSWP02500 filters and filters were washed with 5 ml PBS, air-dried and further processed for scintillation counting Recombinant eIF-5A precursor protein used in these experiments was obtained as follows: Using a premade liver cDNA library (Clontech), the human eIF-

5A c DNA was cloned with polymerase chain reaction (PCR) primers specific for eIF-5A (Smit-McBride et al, 1989a) harboring additional BamHI and EcoRI sites for positional cloning into the bacterial expression vector pGEX-3X (Amersham Pharmacia Biotech) (Bevec et al., 1996). After sequence determination, the pGEX-eIF-5A plasmid was transformed into the E. cold strain BL21 (DE3). An overnight bacterial culture was diluted 1:10 in 500 ml fresh LB-medium (supplemented with 100 pa/ml ampicillin) and grown at 37 C until an OD600nm of 0.5 - 0.6 was reached. Subsequently, IPTG

was added (1 mM) and culturing proceeded for a further 4 h. The cells were pelleted and resuspended in 10 ml PBS containing 1 mM DTT. After sonification (twice for 30 sec at a setting of 10 W) the lysate was centrifuged for 20 min at 14.000 rpm and 4 C in a Sorvall centrifuge (SS34 rotor) and GST-eIF-5A fusion protein was purified from the supernatant by affinity chromatography with glutathione-Sepharose 4B according to the manufacturer's specifications (Amersham

Pharmacia Biotech). The consistency of the fusion protein was tested on a SDS-PAGE gel. Subsequently, the fusion protein was dialyzed overnight against 50 mM Tris-HCl, pH 8.0/100mM NaCL/lmM CaCl. After that, the GSTeIF-5A fusion was cleaved with Factor Xa according to the manufacturer's recommendation (Roche Molecular Biochemicals) and the GST moiety was removed by affinity chromatography with glutathione-Sepharose 4B.

Finally, the remaining eIF-5A protein was dialyzed overnight against PBS.

Recombinant doxyhypusine synthase (DHS) used in these experiments was obtained as follows: Using a HeLa cell cDNA, the human DHS cDNA was cloned with polymerase chain reaction (PCR) primers specific for DHS (Genebank Acc.# L39068) harboring additional BamHI and EcoRI sites for positional cloning into the bacterial expression vector pTrcHisC (Invitrogen). After sequence determination, the pTrcHis-DHS plasmid was transformed into the E. cold strain BL21 (DE3) pLysS. An overnight bacterial culture was diluted 1:10 in 500 ml fresh LB-medium (supplemented with 100 g/ml ampicillin) and grown at 37 C until an OD600nm of 0.5 -

0.6 was reached. Subsequently, IPTG was added (1 mM) and culturing proceeded for a further 4 h. The cells were pelleted and resuspended in 20 ml 20 mM Tris-HCl, pH7.7/500mM NaCl/5mM Imidazol/0.1% Nonidet-P40 supplemented with 50 ug DNAse I (Roche Molecular Biochemicals). After sonification (twice for 30 sec at a setting of 10 W) the lysate was centrifuged for 30 min at 13.000 rpm and 4 C in a Sorvall

centrifuge (SS34 rotor) and the His-tagged DHS fusion protein -

was purified from the supernatant by affinity chromatography with Ni-NTAAgarose according to the manufacturer's specifications (Qiagen). The consistency of the protein was

tested on a SDS-PAGE gel. Subsequently, the protein was dialyzed overnight against PBS.

The controls were reactions in which the DHS protein was omitted or reactions in which the the DHS inhibitor GC7 (1N Guanyl-1,7diaminoheptane; Jakus et al., 1993) was added. i NC = negative control (reaction without DHS) PC = positive control (standard reaction).

The results of the measured number of counts per minute (cpm) are shown in Table 1.

Table 1

NC PC Control for AH4-AHll cpm 2,374 53,687 Control for AH12-AH17 Cpm 2, 374 52,538 Control for AH18-AH23 cpm 3,348_ _ 51,377 An evaluation was then made as whether the two amino groups within 1,3-diaminopropane are essential or can be replaced by other groups. For this purpose the compounds AH4, Ads, AH9, AH10 and AH19 were tested. In these test compounds one or two amino groups are replaced by hydoxy- or methyl groups as well as by hydrogen. The results are shown in Table 2. The number of counts per minute (cpm) resulting from testing compounds AH4, Ads, AH9, AH10 and AHl9 in the above assay at concentrations of 20uM, 40M and 80pM are provided in Table 2.

Table 2

Compound/Formula Conc. of test compound DHS 20M 40 AM 80 AM Inhibitio AH4: H3N:'NH3 x 2 Cl cpm 32,025 21,684 13,537 good AH8: HO POOH

cpm 68,461 72,497 59,064 none AH9: H,,_,N

cpm 65,752 70,951 69,995 none AH10: HO,,_,NH2

cpm 65,421 50,306 60,080 none AH19: C,,_,N

cpm 45,314 49,133 70,266 none As can be readily seen from Table 2, only the parent 1,3-

diaminopropane compound (AH4) is active within this series of test compounds.

In addition, the use of the dihydrochloride of 1,3-

diaminopropane (AH4) in this assay demonstrates the effectiveness of the assay in testing compounds for their activity as DHS-inhibitors.

In a second series of experiments, the parent propano bridge linking the amino termini was exchanged by other bridges. In this respect, either the length of the alkyl bridge (ethano in AHll and AH20 and butano in AH17) or the nature of the propano bridge (hydroy- (AH12), oxo- (AH16) or dimethyl (AH18) substitution at C-2) was varied. The number of counts per minute (cpm) resulting from testing compounds AH11, AH12, AH16, AH17, AH18 and AH20 in the above assay at concentrations of 20M, 40M and 80M are provided in Table 3.

Table 3

Compound/Formula Conc. of test compound DHS 20M 40 AM 80 AM Inhibitio cpm H3NH3 X 2 cr 32,025 21,604 1d,537 good PXNH2 57,617 62,065 62,740 one AH12: OH

cpm H2N NH2 61,984 62,736 43,290 none AH16: H2N NH2 40,040 42,380 37, 808 none AH17: H2N' - Hi,' - _'NH2 43,143 43,043 39,662 none AH18: \,

cpm HzN NH2 46,492 48,846 43,932 none AH20: NH2

Leg XNH 48,085 48,456 43,533 In all cases of the bridge variation a complete loss of DHS-

inhibitor activity was observed. This demonstrates that the activity requires the presence of a unsubstituted propano moiety. Finally, the influence of substituents within the amino termini as well as the specific arrangement of the 1,3-

diaminopropane moiety within a derivative were evaluated. For this purpose, the DHS-inhibition activity of the alkylated 1,3-diaminopropane derivatives AH14, AH15, AH21 as well as

compound AH23 were investigated. The number of counts per minute (cpm) resulting from testing compounds AX14, AH15, AH21 and AX23 in the above assay at concentrations of HAM, 40M and 80WM are provided in Table 4.

Table 4

Compound/Formula Conc. of test compound DHS 20M 40 AM 80 EM Inhibit.

cpm H3N',_'NH3 X2cr 32,025 21,684 13,537 good AH14- _,N,,,N,, 39,675 62, 944 41,805 none AH15: H

cpm 40,990 53,877 41,180 none AH21: /

cpm N.\ 43, 50g 67,140 46,303 none AH23: AH

pH 9 > 34,090 17,756 15,160 good cpm --4H AH2 3: ASH

pH 3.5 26,591 15,974 10,671 good Cpm --AH _. Only the cyclic molecule 1,3diazacyclohexane (AH23) is active. This clearly demonstrates the significant influence of the precise arrangement of the 1,3diaminopropane moiety within a derivative. A cyclic arrangement within a six-member ring associated with a specific, comparably rigid, orientation of the 1,3-diaminopropane unit is important. It is interesting to note that it is not important whether the

amino groups are protonated or not, since parent 1,3-

diaminopropane and AH4 show comparable activity. This is in contrast to the inhibitors reported in Jakus et al. and WO 94/15596. Moreover, the requirement of the presence of free 1,3-diaminopropane for an effective DHS-inhibition can be neglected since both 1,3-diazacyclohexane dissolved in water, where no cleavage to 1,3-diaminopropane and formaldehyde can take place (pH 9), as well as 1,3-diazacyclohexane dissolved in water at pH 3.5 exhibit comparable activity.

Citations: Andrus,L., Szabo,P, Grady,R.W., Hanauske,A.R., Huima Byron,T., Slowinska,B., Zagulska,S., and Hanauske Abel,.M.

(1998). Antiretroviral effects of deoxyhypusyl hydroxylase inhibitors: a hypusine-dependent host cell mechanism for replication of human immunodeficiency virus type 1 (HIV-1).

Biochem. Pharmacol. 55, 1807-1818.

Bevec,D. et al., Pharmaceutically Active Aromatic Guanylhydrazones, W0 01/56553.

Bevec,D., Jaksche,H., Oft,M., Wohl,T., Himmelspach,M., Pacher,A., Schebesta,M., Koettnitz,K., Dobrovnik,M., Csonga,R., Lottspeich,F., and Hauber,J. (1996). Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A. Science (Wash. DC) 271, 1858-1860.

Caraglia,M., Tagliaferri,P., Budillon,A., and Abbruzzese,A.

(1999). Post-translational modifications of eukaryotic initiation factor5A (eIF-5A) as a new target for anti-cancer therapy. Adv. Exp. Med. Biol. 472, 187-198.

Chen,Z.P., Yan,Y.P., Ding,Q.J., Knapp,S., Potenza,J.A., Schugar,H.J., and Chen,K.Y. (1996). Effects of inhibitors of deoxyhypusine synthase on the differentiation of mouse neuroblastoma and erythroleukemia cells. Cancer Lett. 105, 233-239.;

Cohen,P.S., Nakshatri,H., Dennis,J., Caragine,T., Bianchi,M., Cerami,A., and Tracey,K.J. (1996. CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency. Proc. Natl. Acad. Sci. U. S. A 93, 3967-3971.

Hauber'J. (2001). Nuclear export mediated by the Rev/Rex class of retroviral Trans- activator proteins. Curr. Top.

Microbiol. Immunol 259, 55-76.

Hofmann,W., Reichart,B., Ewald,A., Muller,E., Schmitt,I., Stauber,R.H., Lottspeich,F., Jockusch,B.M., Scheer,U., Hauber,J., and Dabauvalle,M.C. (2001). Cofactor Requirements for Nuclear Export of Rev Response Element (RRE)- and Constitutive Transport Element (CTE)-containing Retroviral RNAs. An unexpected role for actin. J. Cell Biol. 152, 89S-

gl O. J. Jakus, E. C. Wolff, M. Hee Park, J. E. Folk, (1993) Features of the Spermidine-binding Site of Deoxyhypusine Synthase as Derived from Inhibition Studies. J. Biol. Chem. 268, 13151-13159.

J. Jakus, M. Hee Park, E. C. Wolff, Compositions and Methods for inhibiting deoxyhypusine synthase and the growth of cells, WO 94/15596.

Kang,H.A. and Hershey,J.W.B. (1994). Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J. Biol. Chem. 269, 3934-3940.

Kang,H.A., Schwelberger,H.G., and Hershey,J.W.B. (1993).

Effect of initiation factor eIF-5A depletion on cell proliferation and protein synthesis. In Protein Synthesis and Targeting in Yeast. NATO Series H: Cell Biol., A.J.P.Brown, M.F.Tuite, and J.E.G.McCarthy, eds. (Berlin, Germany: Springer Verlag), pp. 123-129.

Katahira,J., Ishizaki,T., Sakai,H., Adachi,A., Yamamoto,K., and Shida,H. (1995). Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited bans

dominantly by a Rex mutant deficient in RNA binding. J. Virol. 69, 31253133.

Kruse,M., Rosorius,O., Kratzer,F., Bevec,D., Kuhnt,C., Steinkasserer,A., Schuler,G., and Hauber,J. (2000).

Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA. J. Exp. Med. 191, 1581-1590.

McCaffrey,T.A., Pomerantz,K.B., Sanborn,T.A., Spoko jny,A.M., Du,B., Park, M.-H., X, Lamberg,A., Kivirikko,K.I., Falcone,D.J., Mehta,S.B., and Hanauske-Abel,H.M. (1995). Specific inhibition of eIF-5A and collagen hydroxylation by a single

agent. Antiproliferative and fibrosuppressive effects on smooth muscle cells from human coronary arteries. J. Clin. Invest. 95, 446-455.

Park,M.H. (1989). The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). J. Biol. Chem. 264, 18531- 18535.

Park,M.H., Wolff,E.C., and Folk,J.E. (1993). Hypusine: its posttranslational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. BioFactors 4, 95-104.

Park,M.H., Wolff,F.C., Lee,Y.B., and Folk,J.E. (1994).

Antiproliferative effects of inhibitors of deoxyLypusine synthase. Inhibition of growth of Chinese hamster ovary cells by guanyl diamines. J. Biol. Chem. 269, 27827-27832.

Pollard,V.W. and Malim,M.H. (1998). The HIV-1 Rev protein.

Annul Rev. Microbiol. 52, 491-532.

Rosorius,O., Reichart,B., Kratzer,F., Heger,P., Dabauvalle,M.C., and Hauber,J. (1999). Nuclear pore localization and nucleocytoplasmlc transport of eIF-5A:

evidence for direct interaction with the export receptor CRM1. J. Cell Sci. 112, 2369-2380.

Ruhl,M., Himmelapach,M., Bahr,G.M., Hammerschmid,F., I Jaksche,H., Wolff, B., Aschauer,H., Farrington,G.K., Probst,H., Bevec,D., and Hauber,J. (1993). Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating transactivation. J. Cell Biol. 123, 1309-1320.

Shi,X.P., Yin,K.C., Ahern,A., Davis,J., Stern,A.M., and Waxman,L. (1996). Effects of N1-guanyl-1,7-diaminoheptane: An inhibitor of deoxyhypusine synthase, on the growth of tumorigenic cell lines in culture. Biochim. Biophys. Acta Mol. Cell Res. 1310, 119-126.

Smit-McBride,Z., Dever,T.E., Hershey,J.W.B., and Merrick,W.C.

(1989a). Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein. J. Biol. Chem. 264, 1578-1583.

Smit-McBride,Z., Schnier,J., Kaufman,R.J., and Hershey,J.W.B.

(1989b). Protein synthesis initiation factor eIF-4D. J. Biol.

Chem. 264, 18527-18530.

Claims (1)

1. ó What we claim:

   1. A compound of the general formula (I): /R3 N / ": \/:R2 ()

   N. R4 wherein the dotted line represents a single bond when R1, R2 and R3 are present and the dotted line represents a double bond when either R1 or R2 are present and R3 is absent, and wherein R1 and R2 can be the same or different and are selected from the group consisting of H. straight or branched chain substituted or non-

   substituted alkyl, substituted or non-substituted aryl, and R3 and R4 can be the same or different and are selected from the group consisting of H. carboxyl, acetyl and an acid-labile protective group, as well as pharmaceutically acceptable salts thereof.

   2. The compound according to claim 1, wherein said compound is represented by the general formula (II): R3 N a 1 (II) /\ R2 N R4 wherein R1, R2, R3 and R4 are defined as above as well as pharmaceutically acceptable salts thereof.

   3. The compound according to claim 1, wherein said compound is represented by the general formula (III): N \ R1 (III)

   N Rat wherein R1 and R4 are defined as above as well as pharmaceutically acceptable salts thereof.

   4. The compound according to any of claims 1 to 3, wherein R1 and/or R2 is a straight chain C1-C6 alkyl group.

   5. The compound according to any of claims 1 to 3, wherein R1 and/or R2 is a secondary branch chain with one branch being a C3-C6 alkyl group and the other branch being a C1-C4 alkyl group.

   6. The compound according to any of claims 1 to 5, wherein the branched or unbranched alkyl groups contain one or more carbon-carbon double or triple bonds.

   7. The compound according to any of claims 1 to 6, wherein the alkyl groups contain one or more substituents selected from the group consisting of carboxylic groups, ketone groups, aldehyde groups, hydroxyl groups, ether groups, amino groups, halogens and sulfonic acid groups.

   The compound according to any of claims 1 to 3, wherein R1 and/or R2 is an aryl group.

   3. The compound according to any of claims 1 to 6, wherein the aryl group(s) contain one or more substituents selected from the group consisting of carboxylic groups,

   ( ketone groups, aldehyde groups, hydroxyl groups, ether groups, amino groups, halogens and sulfonic acid groups.

   10. The compound according to any of claims 1 to 9, wherein R3 and/or R4 is H. 11. The compound according to any of claims 1 to 9, wherein R3 and/or R4 is an acid labile protective group selected from the group consisting of t-butyl-oxycarbonyl (boo), t-butyldimethylsilyl (tbdms), methexy-ethexy-methyl (mem) and tetrahydropyranyl (thp).

   12 The compound according to any of claims 1 to 9, wherein R3 and/or R4 is a carboxyl or acetyl group.

   13. A pharmaceutical composition comprising one or more of the compounds according to any of claims 1 to 12 and a pharmaceutically acceptable carrier.

   14. A pharmaceutical composition according to claim 13 comprising further therapeutic compounds.

   15. The use of one or more compounds according to any of claims 1 to 13 for the preparation of a medicament for the prevention and/or treatment of a disease or medical condition that is responsive to a decrease in hypusine-

   dependent eIF-5A activity.

   16. The use according to claim 15, wherein said disease or medical condition is a virally induced infection or disease. 17. The use according to claim 15, wherein said virally induced infection or disease is caused by a virus selected from the group of consisting of retroviruses, hepadnaviruses, herpesviruses, adenoviruses and orthomyxoviruses.

   18. The use according to claim 17, wherein said retrovirus is = selected from the group of lentiviruses consisting of HIV, EIAV, VMV, CAEV, BIV, FIV and SIV or the group of oncoretroviruses consisting of HTLV and BLV. = 19. The use according to any of claims 16 to 18, wherein said virally induced infection or disease is caused by HIV.

   20. The use according to claim 19, wherein said HIV is a strain of HIV-1 or HIV-2 selected from the group consisting of a T-cell tropic strain, a macrophage trophic strain a drug resistant strain, a protease inhibitor resistant strain and a reverse transcriptase inhibitor resistant strain.

   21. The use according to claim 17, wherein said hepadnavirus is HBV.

   22. The use according to claim 17, wherein said herpesviruses is selected from the group consisting of human herpesvirus 1 (Herpes Simplex Virus 1, HSV1), human herpesvirus 2 (Herpes Simplex Virus 2, HSV2), human c herpesvirus 3 (Varicella Zoster Virus, VZV), human -

   herpesvirus 4 (Epstein-Barr Virus, EBV), human herpesvirus 5 (human Cytomegalovirus, HCMV), human -

   herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8).

   23. The use according to claim 15, wherein said disease or medical condition is mediated by TNF-alpha.

   24. The use according to claim 23, wherein said disease or medical condition mediated by TNF-alpha is selected from the group consisting of bacterially-induced hemorrhagic fever diseases, hemorrhagic shock syndromes and -

   inflammation.

   25. The use according to claim 15, wherein said disease or medical condition is an immune response involving the direct or indirect participation of dendritic cells.

   26. The use according to claim 25, wherein said immune response involving the direct or indirect participation of dendritic cells is selected from the group consisting of the growth, differentiation or activation of T cells, the differentiation of helper T cells into Thl cells or Th2 cells, the growth, stimulation or differentiation of B cells.

   27. The use according to claim 2S, wherein said disease or medical condition is rejection of a tissue or organ transplant, a xenogenic tissue or organ transplant, graft-vs.-host disease or host-vs.-graft disease.

   28. The use according to claim 25, wherein said disease or medical condition is selected from the group consisting of allergy, asthma myasthemia gravis, multiple sclerosis and systemic lupus erythematosis, Crohn's disease, skin diseases, psoriasis and rheumatoid arthritis.

   29. The use according to claim 15, wherein said disease or medical condition abnornal proliferative cell growth associated with malignant or benign disease.

   30. The use according to claim 29, wherein said malignant disease is selected from the group consisting of hematological systemic diseases, carcinomas, sarcomas, myelomas, melanomas, lymphomas and papillomas.

   31. The use of a compound according to any of claims 1 to 12 for inhibiting deoxyhypusine synthase activity and/or eIF-5A activity in an in vitro assay.

   ( 32. A kit for directly or indirectly determining deoxyhypusine synthase activity or eI F- 5A hypusination or determining the effects of deoxyhypusine synthase or eIF 5A in an in vitro system, said kit comprising a compound according to any of claims 1 to 12.