Heterocyclic purine cytokinin derivatives, their use in wound healing and pharmaceutical compositions containing these derivatives

Abstract

The invention relates to heterocyclic purine cytokinin derivatives, their use in wound healing and pharmaceutical preparations containing these derivatives. These compounds stimulate endothelial cell division and migration, angiogenesis and vascularization and exhibit also anti-inflammatory properties.

Description

[0001] Heterocyclic purine cytokinin derivatives, their use in wound healing and pharmaceutical compositions containing these derivatives

[0002] Technical Field

[0003] The invention relates to heterocyclic cytokinin purine derivatives, their use for wound healing and pharmaceutical preparations containing these derivatives.

[0004] Background of the Invention

[0005] Cytokinins are phytohormones identified originally as substances that promote plant cell division in the presence of another phytohormone, auxin. Regarding their chemical structure, plant cytokinins are adenine derivatives substituted at the N6-position with either an isoprenoid or aromatic side chain. Isoprenoid cytokinins include cis- and trans -zeatin (tZ) and their analogues with a saturated side chain (dihydrozeatin) or without hydroxyl group (N6-isopentenyladenine). While isoprenoid cytokinins are present in all plants, aromatic cytokinins with N6-benzyl substituents have only been found in certain taxa. Besides 6-benzylaminopurine (BAP), its hydroxylated derivatives, i.e., topolins, have been described. Kinetin (K), the first identified cytokinin, has a furfuryl side chain. Kinetin was first recognized as the substance responsible for the cytokinin activity of autoclaved herring sperm, which was attributed to thermal DNA damage.

[0006] Because anti-aging activity K was later demonstrated on skin fibroblasts as well as in trans- zeatin (tZ). numerous studies were performed to evaluate the utility of cytokinin bases in skin protection in experiments in vitro and in vivo. For example, cell culture experiments have suggested that tZ could improve skin hydration, as well as prevent detrimental effects of photoaging on those processes (Ji et al. 2010, Int J Mol Med 26:257-263). Treatment with tZ was also able to ameliorate a UV-induced decrease in AQP3 concentrations of protein and membrane water permeability to a large extent. Natural cytokinins (e.g., kinetin and zeatin) and 6,9-disubstituted cytokinin derivatives have been shown to have biological activities related to aging. See, for example, U.S. Patents 5,021,422 (June 4, 1991), 5,371,089 (December 6, 1994), 5,602,139 (February 11, 1997), patent 5,614,407 (published on March 8 and March 25 25.0.2005), all of which are hereby incorporated by reference. Cytokinin bases have also been shown to promote keratinocyte differentiation. Kinetin (40- 200 μM) induced growth arrest and changes in several markers of differentiation in human keratinocytes grown in cell culture (Berge et al. 2006, Ann N Y Acad Sci 1067: 332-336). The effect was enhanced by the presence of Ca2+ions. Other markers of differentiation (trans-glutaminase) were unchanged, suggesting that K-induced differentiation is likely to be mediated by pathways different from those activated by other differentiation-inducing agents. In a follow-up study by Berge et al. (Exp Gerontol 43: 658-662, 2008) reported that K treatment improved the sensitivity of aging keratinocytes to the differentiation effects of Ca2+ions. A positive effect of K on the levels of filaggrin, another marker of keratinocyte differentiation, has been observed in an in vitro reconstructed skin equivalent (Vicanova et al. 2006, Ann N Y Acad Sci 1067: 337-342). In contrast to 2D culture, K promoted keratinocyte growth, as indicated by an increase in the number of Ki67-positive cells. Other dermatological / cosmetic applications of cytokinins relate to the therapy of pigmentation disorders. While K has been reported to reduce hyperpigmentation in dogs (Kimura and Doi 2004, Rejuvenation Res 7: 32-39), BAP has been recognized as a stimulator of melanogenesis (Kim et al. 2009, Arch Dermatol Res 301: 253-258).

[0007] Although heterocyclic purine cytokinins, especially kinetin and zeatin, have significant anti- aging and other properties, it would be desirable to provide additional biologically activities and uses of these natural compounds. It remains desirable to provide additional cytokinin compounds which can be used in a wide variety of therapeutic and cosmetic applications and if such compounds have improved selectivity and efficiency (i.e., less toxic and / or more efficacious) than currently used 6-substituted aminopurines. The present invention provides such wound healing compounds.

[0008] Summary of the Invention

[0009] The object of this invention are heterocyclic purine derivatives of the general formula I and

[0010] II or pharmaceutically acceptable salts thereof with alkali metals, ammonia or amines, in the form of racemates or optically active sugar isomers (β-D, α-D, β-L. α-L), where

[0011] R6 is furfuryl, substituted furfuryl, benzyl, substituted benzyl, alkyl, substituted alkyl, alkenyl and substituted alkenyl group, where substituted furfuryl groups include 3-methoxyfurfuryI, 4-methoxyfurfuryI, and 5- methoxyfurfuryl, substituted benzyl groups include 2 -hydroxybenzyl, 3-hydroxybenzyl, 4- hydroxybenzyl, 2-methoxybenzyI, 3 -methoxybenzyl, 4-methoxybenzyI, 3,4- dihydroxybenzyl, 33.,55--ddiihhyyddrrooxxyybbeennzzyyll,, 2,3-dimethoxybenzyI, 2,4- dimethoxybenzyl, 22.,55--ddiimmeetthhooxxyybbeennzzyyll,, 3,5-dimethoxybenzyI 2,3,4- trimethoxybenzyl, 2-hydroxy-3-methoxybenzyI, 2-hydroxy-5-methoxybenzyI, 2- hydroxy-4-methoxybenzyl, 3-hydroxy-4-methoxybenzyI, 4-hydroxy-3- methoxybenzyl, 4-hydroxy-2-methoxybenzyI, 2,3-dihydroxy-4-methoxybenzyI, 2,4- dihydroxy-3-methoxybenzyl, 2,5-dihydroxy-4-methoxybenzyI, 2,3,5- trimethoxybenzyl, 2,4,6-trimethoxybenzyI, 3,4,5-trimethoxybenzyI, alkyl groups include butyl, 2-methyIbutyI, 3-methyIbutyI and isopropyl, substituted alkyl groups include 4-hydroxy-3-methyIbutyI and 1' -methyl-4-hydroxy- 3-methyIbutyI, alkenyl groups include isopentenyl and 3 -dimethylallyl, substituted alkenyl groups include 3-hydroxymethyl-3-methylallyl, (Z)-(4-hydroxy- 3-methylbut-2-en-1-yl, (E)-(4-hydroxy-3-methylbut-2-en-1-yl, (Z)-(1'-methyl-4- hydroxy-3-methylbut-2-en-1-yl and (Z)-(1' -methyl-4-hydroxy-3-methylbut-2-en-1- yl, and

[0012] R3 and R9 are independently at each occurrence hydrogen and a glucopyranosyl group, for use in wound healing.

[0013] Preferred are the heterocyclic purine cytokinin derivatives of general formula I and II selected from the g grroouupp containing: 6-(3-methylbutylamino)purine, 6- isopentenylaminopurine, 6 -(3 , 3 -dimethylallylamino)purine , 6 -(3 -hydroxymethyl-3- methy tally lamino)purine, 6-(Z)-(4-hydroxy-3-methylbut-2-en-1-ylamino)purine, 6-(E)-(4- hydroxy-3-methylbut-2-en-1-ylamino)purine, 6-(Z)-(1' -methyl-4-hydroxy-3-methylbut-2- en-1-ylamino)purine, 6-(E)-(1' -methyl-4-hydroxy-3-methylbut-2-en-1-ylamino)purine, 6- (4-hydroxy-3-methylbutylamino)purine, 6-(1' -methyl-4-hydroxy-3- methylbutylamino)purine, 6-furfurylaminopurine, 6-(3-methoxyfurfurylamino)purine, 6-(4- methoxyfurfurylamino)purine, 6-(5-methoxyfurfurylamino)purine, 6-benzylaminopurine, 6- (2,5-dihydroxy-4-methoxybenzylamino)purine 6-(2-hydroxybenzylamino)purine, 6-(3- hydroxybenzylamino)purine , 6-(4-hydroxybenzylamino)purine, 6-(2- methoxybenzylamino)purine, 6-(3-methoxybenzylamino)purine, 6-(4- methoxybenzylamino)purine, 6-(3,4-dihydroxybenzylamino)purine, 6-(3,5- dihydroxybenzylamino)purine , 6-(3,5-dimethoxybenzylamino)purine, 6-(2,3- dimethoxybenzylamino)purine, 6 -(2 ,4-dimethoxybenzylamino)purine , 6-(2,5- dimethoxybenzylamino)purine, 6-(3 , 5 -dimethoxybenzylamino)purine , 6-(3-hydroxy-4- methoxybenzylamino)purine, 6-(3-hydroxy-5-methoxybenzylamino)purine, 6-(2-hydroxy-3- methoxybenzylamino)purine, 6-(2-hydroxy-5-methoxybenzylamino)purine, 6-(4-hydroxy-3- methoxybenzylamino)purine, 6-(2-hydroxy-4-methoxybenzylamino)purine, 6-(3-hydroxy-4- methoxybenzylamino)purine, 6-(4-hydroxy-2-methoxybenzylamino)purine, 6-(2,3- dihydroxy-4-methoxybenzylamino)purine, 6-(2,4-dihydroxy-3- methoxybenzylamino)purine, 6-(2,3,4-trimethoxybenzylamino)purine, 6-(2,3,5- trimethoxybenzylamino)purine, 6-(3,4,5-trimethoxybenzylamino)purine, 6-(3-methylbutylamino)purine-9-glucopyranosylpurine, 6-isopentenylamino-9- glucopyranosylpurine, 6-(3,3-dimethylallylamino)-9-glucopyranosylpurine, 6-(3- hydroxymethyl-3-methylallyl)amino-9-glucopyranosylpurine, 6-(Z)-(4-hydroxy-3- methylbut-2-en- 1 -ylamino)-9-glucopyranosylpurine, 6-(E)-(4-hydroxy-3-methylbut-2-en- 1 - ylamino)-9-glucopyranosylpurine, 6-(Z)-( 1 ' -methyl-4-hydroxy-3-methylbut-2-en- 1 - ylamino)-9-glucopyranosylpurine, 6-(E)-( 1 ' -methyl-4-hydroxy-3-methylbut-2-en- 1 - ylamino)-9-glucopyranosylpurine, 6-(4-hydroxy-3-methylbutylamino)-9- glucopyr anosylpurine, 6-( 1 '-methyl-4-hydroxy-3-methylbutylamino)-9- glucopyr anosylpurine, 6-furfurylaminopurine, 6-(3-methoxyfurfurylamino)-9- glucopyr anosylpurine , 6-(4-methoxyfurfurylamino)-9-glucopyranosylpurine, 6-(5- methoxyfurfurylamino)-9-glucopyranosylpurine, 6-benzylaminopurine, 6-(2,5-dihydroxy-4- methoxybenzylamino)-9-glucopyranosylpurine 6-(2-hydroxybenzylamino)-9- glucopyr anosylpurine, 6-(3-hydroxybenzylamino)-9-glucopyranosylpurine, 6-(4- hydroxybenzylamino)-9-glucopyranosylpurine, 6-(2-methoxybenzylamino)-9- glucopyr anosylpurine, 6-(3-methoxybenzylamino)-9-glucopyranosylpurine, 6-(4- methoxybenzylamino)-9-glucopyranosylpurine, 6-(3,4-dihydroxybenzylamino)-9- glucopyranosylpurineglucopyranosylpurine, 6-(3,5-dihydroxybenzylamino)-9- glucopyr anosylpurine, 6-(3,5-dimethoxybenzylamino)-9-glucopyranosylpurine, 6-(2,3- dimethoxybenzylamino)-9-glucopyranosylpurine, 6-(2,4-dimethoxybenzylamino)-9- glucopyr anosylpurine, 6-(2,5-dimethoxybenzylamino)-9-glucopyranosylpurine, 6-(3,5- dimethoxybenzylamino)-9-glucopyranosylpurine, 6-(3-hydroxy-4-methoxybenzylamino)-9- glucopyr anosylpurine, 6-(3-hydroxy-5-methoxybenzylamino)-9-glucopyranosylpurine, 6-(2- hydroxy-3-methoxybenzylamino)-9-glucopyranosylpurine, 6-(2-hydroxy-5- methoxybenzylamino)-9-glucopyranosylpurine, 6-(4-hydroxy-3-methoxybenzylamino)-9- glucopyr anosylpurine, 6-(2-hydroxy-4-methoxybenzylamino)-9-glucopyranosylpurine, 6-(3- hydroxy-4-methoxybenzylamino)-9-glucopyranosylpurine, 6-(4-hydroxy-2- methoxybenzylamino) -9-glucopyranosylpurine , 6 -(2 ,3 -dihydroxy-4-methoxybenzylamino) - 9-glucopyranosylpurine, 6-(2,4-dihydroxy-3-methoxybenzylamino)-9- glucopyr anosylpurine, 6-(2,3,4-trimethoxybenzylamino)-9-glucopyranosylpurine, 6-(2,3,5- trimethoxybenzylamino)-9-glucopyranosylpurine, 6-(3,4,5-trimethoxybenzylamino)-9- glucopyr anosylpurine , 6-(3-methylbutylamino)purine-3-glucopyranosylpurine, 6-isopentenylamino-3- glucopyr anosylpurine, 6-(3,3-dimethylallylamino)-3-glucopyranosylpurine, 6-(3- hydroxymethyl-3-methylallyl)amino-3-glucopyranosylpurine, 6-(Z)-(4-hydroxy-3- methylbut-2-en-1-ylamino)-3-glucopyranosylpurine, 6-(E)-(4-hydroxy-3-methylbut-2-en-1- ylamino)-3-glucopyranosylpurine, 6-(Z)-(1' -methyl-4-hydroxy-3-methylbut-2-en-1- ylamino)-3-glucopyranosylpurine, 6-(E)-( 1 ' -methyl-4-hydroxy-3-methylbut-2-en- 1 - ylamino)-3-glucopyranosylpurine, 6-(4-hydroxy-3-methylbutylamino)-3- glucopyr anosylpurine , 6-( 1 '-methyl-4-hydroxy-3-methylbutylamino)-3- glucopyr anosylpurine , 6-furfurylaminopurine, 6-(3-methoxyfurfurylamino)-3- glucopyr anosylpurine , 6-(4-methoxyfurfurylamino)-3-glucopyranosylpurine, 6-(5- methoxyfurfurylamino)-3-glucopyranosylpurine, 6-benzylaminopurine, 6-(2,5-dihydroxy-4- methoxybenzylamino)-3-glucopyranosylpurine 6-(2-hydroxybenzylamino)-3- glucopyranosylpurine, 6-(3-hydroxybenzylamino)-3-glucopyranosylpurine, 6-(4- hydroxybenzylamino)-3-glucopyranosylpurine, 6-(2-methoxybenzylamino)-3- glucopyr anosy Ipurine , 6 -(3 -methoxybenzylamino) -3 -glucopyranosylpurine , 6-(4- methoxybenzylamino)-3-glucopyranosylpurine, 6-(3,4-dihydroxybenzylamino)-3- glucopyr anosy Ipurine, 6-(3,5-dihydroxybenzylamino)-3-glucopyranosylpurine, 6-(3,5- dimethoxybenzylamino)-3-glucopyranosylpurine, 6-(2,3-dimethoxybenzylamino)-3- glucopyranosylpurine, 6-(2,4-dimethoxybenzylamino)-3-glucopyranosylpurine, 6-(2,5- dimethoxybenzylamino)-3-glucopyranosylpurine, 6-(3,5-dimethoxybenzylamino)-3- glucopyranosylpurine, 6-(3-hydroxy-4-methoxybenzylamino)-3-glucopyranosylpurine, 6-(3- hydroxy-5-methoxybenzylamino)-3-glucopyranosylpurine, 6-(2-hydroxy-3- methoxybenzylamino) -3 -glucopyranosylpurine , 6 -(2-hydroxy-5 -methoxybenzylamino) -3 - glucopyr anosy Ipurine, 6-(4-hydroxy-3-methoxybenzylamino)-3-glucopyranosylpurine, 6-(2- hydroxy-4-methoxybenzylamino)-3-glucopyranosylpurine, 6-(3-hydroxy-4- methoxybenzylamino)-3-glucopyranosylpurine, 6-(4-hydroxy-2-methoxybenzylamino)-3- glucopyr anosy Ipurine, 6-(2,3-dihydroxy-4-methoxybenzylamino)-3-glucopyranosylpurine, 6-(2,4-dihydroxy-3-methoxybenzylamino)-3-glucopyranosylpurine, 6-(2,3,4- trimethoxybenzylamino)-3-glucopyranosylpurine, 6-(2,3,5-trimethoxybenzylamino)-3- glucopyr anosy Ipurine, 6-(3,4,5-trimethoxybenzylamino)-3-glucopyranosylpurine, and pharmaceutically acceptable salts thereof with alkali metals, ammonia or amines, in the form of racemates or optically active sugar (β-D, α-D, β-L. α-L) isomers, as well as their acid addition salts for use in wound healing.

[0014] Particularly preferred are the heterocyclic purine cytokinin derivatives of general formula I and II selected from the group containing: 6-(3,3-dimethylallylamino)purine, 6- furfurylaminopurine, 6-benzylaminopurine, 6-(3-hydroxybenzylamino)purine, 6-(4- hydroxybenzylamino)purine, 6-(E)-(4-hydroxy-3-methylbut-2-en-1-ylamino)purine, 6-(4- hydroxy-3 -methylbutylamino)purine , 6-furfurylamino-3-β-D-glucopyranosylpurine, 6- benzylaminopurine-3-β-D-glucopyranosylpurine, 6-(3-hydroxybenzylamino)-3-β-D- glucopyranosylpurine , 6-(4-hydroxybenzylamino)-3-β-D-glucopyranosylpurine, 6-(4- hydroxy-3-methylbutylamino)-3-β-D-glucopyranosylpurine, 6-(3,3-dimethylallylamino)-3- β-D-glucopyranosylpurine, 6-(3-hydroxymethyl-3-methylallyl)amino-3-β-D- glucopyr anosy Ipurine , 6-(Z)-(4-hydroxy-3-methylbut-2-en-1-ylamino)-3-β-D- glucopyr anosy Ipurine , 6-(E)-(4-hydroxy-3-methylbut-2-en-1-ylamino)-3-β-D- glucopyr anosy Ipurine, 6-(4-hydroxy-3-methylbutylamino)-3-β-D-glucopyranosylpurine, 6- furfuryIamino-9-β-D-gIucopyranosyIpurine, 6-benzylaminopurine-9-β-D- glucopyr anosy Ipurine , 6-(3-hydroxybenzylamino)-9-β-D-glucopyranosylpurine, 6-(4- hydroxybenzylamino)-9-β-D-glucopyranosylpurine, 6-(4-hydroxy-3-methylbutylamino)-9- β-D-glucopyranosylpurine, 6-(3,3-dimethylallylamino)-9-β-D-glucopyranosylpurine, 6-(3- hydroxymethyI-3-methyIaIIyI)amino-9-β-D-gIucopyranosyIpurine, 6-(Z)-(4-hydroxy-3- methylbut-2-en-1-ylamino)-9-β-D-glucopyranosylpurine, 6-(E)-(4-hydroxy-3-methylbut-2- en-1-ylamino)-9-β-D-glucopyranosylpurine, 6-(4-hydroxy-3-methylbutylamino)-9-β-D- glucopyr anosy Ipurine .

[0015] In a preferred embodiment, the heterocyclic cytokinin derivatives of the general formula I and II are used to promote endothelial cell proliferation and / or migration. Hence, the invention in one or more forms extends though not exclusively, to treating of wounds and the promotion of wound healing.

[0016] A further aspect of the invention is a heterocyclic cytokinin derivative of the general formula I and II for use for treating skin disease conditions.

[0017] Another aspect of the invention is a heterocyclic cytokinin derivative of the general formula I and II for use in stimulating angiogenesis of endothelial cells, for accelerating and promoting angiogenesis, and thus for providing substantially immediate relief of pain and wound healing.

[0018] Examples of endothelial cell types that may be induced to proliferate and / or migrate by heterocyclic cytokinin derivative of the general formula I and II in accordance with the invention include human umbilical vein endothelial cells, human microvascular endothelial cells, and aorta endothelial cells. They can have also different mammalian origin.

[0019] Yet another aspect of the invention is a heterocyclic cytokinin derivative of the general formula I and II for use for wound healing, and / or junction distance modifying agents for the treatment of wounds, including acute, subacute, chronic wounds and wounds with delayed healing.

[0020] The present invention also provides heterocyclic cytokinin derivatives of the general formula I and II for an increase in the rate, extent and / or quality of wound healing through the use of one or more heterocyclic cytokinin derivatives and one or more therapeutic agents, agents useful for wound healing, and / or wound closing modifying agents.

[0021] Yet another aspect of the invention is a heterocyclic cytokinin derivative of the general formula I and II for use in stimulating the migration of fibroblasts, vascular endothelial cells or epithelial cells into a wound, wherein use comprises chemokinesis and stimulating the fibroblasts, vascular endothelial cells, epithelial cells, or a combination thereof.

[0022] In yet another aspect, the invention provides use of heterocyclic cytokinin derivatives of the general formula I and II for promotion or enhancement wound healing, treating, prevention and amelioration of fibrosis or other fibrotic conditions, comprising administering one or more heterocyclic cytokinin agents and one or more therapeutic agents, agents useful for wound healing and / or wound closing modifying agents to a patient in need thereof.

[0023] In a preferred embodiment, the combined use of one or more heterocyclic cytokinin derivatives and one or more therapeutic agents, agents useful for wound healing, and / or wound closing modifying agents has an additive, synergistic or super-additive effect in the promotion of wound treating.

[0024] In a preferred embodiment, the heterocyclic cytokinin derivative of the general formula I and II is used as a drug and / or cosmetics for promoting endothelial cell proliferation and / or migration in a subject in need thereof, comprising treating the subject with at least one cytokinin derivative.

[0025] Another aspect of the invention are the heterocyclic cytokinin derivatives of the general formula I and II for use in skin regeneration, comprising administering to a subject an effective amount of a compound of the invention that increases protein secretion, thereby promoting skin regeneration.

[0026] Tissue sites and wounds that may be treated in accordance with the invention include acute and chronic wounds, burns including burns arising from exposure to ionizing radiation, chemical wounds, surgical wounds, oral wounds, skin and muscle trauma, open skin wounds, diabetic skin sores including diabetic foot ulcers, diabetic naturopathic foot ulcers, ischemic tissue including ischemic naturopathic foot ulcers, venous stasis ulcers, pressure sores, and hypoxic tissue. Examples of ischemic and hypoxic tissues include ischemic heart tissue and hypoxic tissues associated with stroke. Conditions in which the wound healing process may be promoted by the administration of the heterocyclic cytokinin derivatives include also circumstances of delayed wound healing in which healing is impaired or prevented by for example, tissue hypoxia, repeated trauma, or systemic causes such as diabetes and stroke. As used herein, chronic wound may refer to, for example, a wound that is characterized at least in part by one or more of following conditions: 1) a chronic self- perpetuating condition of wound inflammation, 2) a deficient and defective wound extracellular matrix (ECM), 3) poorly responding (senescent) wounded cells especially fibroblasts, limiting ECM production, and 4) failure of re-epithelialization due in part to lack of the necessary components of ECM and lack of scaffold for migration. Chronic wounds include venous ulcers, arterial ulcers, pressure ulcers, vasculitic ulcers, and diabetic ulcers.

[0027] In addition to the definition previously provided, the term wound may also include for example, injuries to the skin and subcutaneous tissue initiated in different ways (e.g., pressure sores from extended bed rest and wounds induced by trauma) and with varying characteristics. Wounds may be classified into one of four grades depending on the depth of the wound: i) Grade I: wounds limited to the epithelium; ii) Grade II: wounds extending into the dermis; iii) Grade III: wounds extending into the subcutaneous tissue; and iv) Grade IV (or full-thickness wounds): wounds where bones are exposed. The term “partial thickness wound” refers to wounds that encompass Grades I-III; examples of partial thickness wounds include burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers. The term “deep wound” is meant to include both Grade III and Grade IV wounds. The compositions and methods of the present invention contemplate treating all wound types, including deep wounds and chronic wounds. The term "chronic” wound refers to a wound that has not healed. Chronic skin wounds include, for example, pressure ulcers, ulcers, abdominal ulcers, diabetic ulcers, venous ulcers, vasculitic ulcers, arterial ulcers, and mixed ulcers. The chronic wound may be an arterial ulcer which comprises ulcerations resulting from complete or partial arterial blockage. The chronic wound may be a venous stasis ulcer which comprises ulcerations resulting from a malfunction of the venous valve and the associated vascular disease. The chronic wound may be a trauma-induced ulcer, a diabetic ulcer, or a vasculitic ulcer.

[0028] In a preferred embodiment, the object of the invention is the pharmaceutical preparation comprising one or more heterocyclic cytokinin derivatives of the general formula I and II or pharmaceutically acceptable salts thereof with alkali metals, ammonia or amines, in the forms of racemates or optically active isomers, or their acid addition salts, and one or more excipients, intended for wound treating and healing.

[0029] A further aspect of the invention is the composition comprising one or more heterocyclic cytokinin derivatives of the general formula I and II or pharmaceutically acceptable salts thereof with alkali metals, ammonia, or amines, in the forms of racemates or optically active isomers, or their acid addition salts, and one or more excipients intended for treating skin disease conditions.

[0030] The composition of the present invention is also useful for treating inflammation-related conditions, such as inflammation, lesions (e.g., accelerating healing thereof), pain and other immunological responses resulting from inflammation (e.g., providing relief thereof) and / or treating inflammation skin diseases.

[0031] The object of the present invention is further use for treating the disease conditions in a mammal, wherein said use comprises applying an amount of a novel heterocyclic cytokinin derivative of the general formula I and II of this invention to the mammalian cells and tissues.

[0032] A further aspect of the invention is the composition comprising one or more heterocyclic cytokinin derivatives of the general formula I and II or pharmaceutically acceptable salts thereof with alkali metals, ammonia or amines, in the forms of racemates or optically active isomers, or their acid addition salts, and one or more excipients intended for wound healing of mammalian epithelial cells.

[0033] PHARMACEUTICAL AND COSMETIC PREPARATIONS

[0034] The pharmaceutical preparation comprises from 1 to 95% of the active ingredient, wherein single-use doses preferably comprise from 20 to 90% of the active ingredient and for administration modes which are not single -use preferably comprise from 5 to 20% of the active ingredient. Unit dosage forms are, for example, coated tablets, tablets, ampoules, vials, suppositories, or capsules. Other forms of administration are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions, and the like. Examples are capsules containing from 0.05 g to 1.0 g of the active ingredient.

[0035] Preferably the agents of the invention are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate buffered saline. Suitable diluents and excipients also include, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired substances such as wetting agents or emulsifiers, stabilizing buffering agents may also be present.

[0036] The term “pharmaceutically acceptable carrier” refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. Pharmaceutically acceptable salts can also be present, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

[0037] Suitable carrier materials include any carrier or vehicle commonly used as a base for creams, lotions, gels, emulsions, lotions, or paints for topical administration. Examples include emulsifiers, inert carriers including hydrocarbon bases, emulsifying bases, non-toxic solvents, or water-soluble bases. Particularly suitable examples include pluronics, HPMC, CMC and other cellulose-based ingredients, lanolin, hard paraffin, liquid paraffin, soft yellow paraffin or soft white paraffin, white beeswax, yellow beeswax, cetostearyl alcohol, cetyl alcohol, dimethicones, emulsifying waxes, isopropyl myristate, microcrystalline wax, oleyl alcohol and stearyl alcohol.

[0038] Preferably, the pharmaceutically acceptable carrier or vehicle is a gel, preferably a non-ionic polyoxyethylene-polyoxypropylene copolymer gel, for example, a Pluronic gel, preferably Pluronic F-127 (BASF Corp.). This gel is particularly preferred as it is a liquid at low temperatures but rapidly sets at physiological temperatures, which confines the release of the agent to the site of application or immediately adjacent to that site.

[0039] An adjuvans, such aass casein, gelatine, albumin, glue, ssooddiiuumm alginate, carboxymethylcellulose, methylcellulose, hydroxyethylcellulose or polyvinyl alcohol may also be included in the formulation of the invention. Other suitable formulations include pluronic gel, carboxymethylcellulose (CMC)-based formulations, and hydroxypropylmethylcellulose (HPMC)-based formulations. The composition may be formulated for any desired form of administration, including topical, instillation, parenteral, intramuscular, subcutaneous, or transdermal administration. Other useful formulations include slow or delayed release preparations.

[0040] The pharmaceutical preparations of the present invention are prepared in a known manner, for example by means of conventional mixing, granulating, coating, dissolving or lyophilising processes. Preferably, solutions of active ingredients, and also suspensions or dispersions, especially isotonic aqueous solutions, suspensions or dispersions, are used, which can be prepared before use, for example in the case of lyophilised preparations which comprise the active ingredient alone or together with a carrier, for example mannitol. The pharmaceutical preparations can be sterilised and / or comprise excipients, for example preservatives, stabilisers, humectants and / or emulsifiers, solubilizing agents, salts for regulating the osmotic pressure and / or buffers. They are prepared in a known manner, for example by means of conventional dissolving or lyophilising processes. The solutions or suspensions mentioned can comprise viscosity-increasing substances, such aass sodium carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatine.

[0041] Suspensions in oil comprise, as the oily component, vegetable, synthetic or semi-synthetic oils commonly used for injection purposes. Oils which may be mentioned are, in particular, liquid fatty acid esters which contain, as the acid component, a long-chain fatty acid having 8-22, in particular 12-22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, acid, arachidonic acid and behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, euric acid, brasidic acid and linoleic acid, if appropriate with the addition of antioxidants, for example vitamin E, β-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters does not have more than 6 carbon atoms and is mono- or polyhydric, for example mono-, di- or trihydric alcohol, for example methanol, ethanol, propanol, butanol, or pentanol, and isomers thereof, but in particular glycol and glycerol. Fatty acid esters are therefore, for example: ethyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M 2375“ (polyoxyethylene glycerol trioleate from Gattefosee, Paris), “Labrafil M 1944 CS“ (unsaturated polyglycolated glycerides prepared by an alcoholysis of apricot kernel oil and made up of glycerides and polyethylene glycol esters; from Gattefosee, Paris), “Labrasol“ (saturated polyglycolated glycerides prepared by an alcoholysis of TCM and made up of glycerides and polyethylene glycol esters; from Gattefosee, Paris) and / or “Miglyol 812“ (triglyceride of saturated fatty acids of chain length C8to C12from Huis AG, Germany), and in particular vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and, in particular, groundnut oil.

[0042] Gels or jellies may be produced using a suitable gelling agent including, but not limited to, gelatine, tragacanth, or a cellulose derivative and may include glycerol as a humectant, emollient, and preservative. Ointments are semi-solid preparations that consist of active ingredient incorporated into a fatty, waxy, or synthetic base. Examples of suitable creams include, but are not limited to, water-in-oil and oil-in-water emulsions. Water-in-oil creams may be formulated by using a suitable emulsifying agent with properties similar to those of the fatty alcohols such as cetyl alcohol or cetostearyl alcohol and to emulsifying wax. Oil- in-water creams may be formulated using an emulsifying agent such as cetomacrogol emulsifying wax. Suitable properties include the ability to modify viscosity of the emulsion and both physical and chemical stability over a wide range of pH. Water-soluble or miscible cream base may comprise compression system Vative and may be buffered to maintain acceptable physiological pH.

[0043] The preparation of the injection preparation is carried out in the customary manner under sterile conditions, for example, bottling in ampoules or vials and closing the containers.

[0044] For example, pharmaceutical preparations for oral use can be obtained by combining the active ingredient with one or more solid carriers, optionally by granulating the resulting mixture, and, if required, by processing the mixture or granules to tablets or coated tablets by addition of additional neutral substances.

[0045] Suitable carriers are, in particular, fillers, such as sugars, for example lactose, sucrose, mannitol or sorbitol, cellulose preparations and / or calcium phosphates, preferably tricalcium phosphate, or calcium hydrogen phosphate, furthermore binders such as starches, preferably maize, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and / or, if desired, desintegrators, such as the above mentioned starches, and furthermore carboxymethyl-starch, cross-linked polyvinylpyrrolidone, alginic acid and salts thereof, preferably sodium alginate. Additional neutral substances are flow regulators and lubricants, preferably salicylic acid, talc, stearic acid and salts thereof, such as magnesium stearate and / or calcium stearate, polyethylene glycol or derivatives thereof.

[0046] Coated tablet cores can be coated with suitable coatings which may be resistant to gastric juice, wherein the coatings used are among others concentrated sugar solutions, which may comprise gum arabic, talc, poly vinylpyrrolidine, polyethylene glycol and / or titanium dioxide, furthermore the coating solutions in suitable organic solvents or solvent mixtures or, for the preparation of coatings which are resistant to gastric juice, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be admixed to the tablets or coated tablet, for example for identification or characterisation of different doses of the active ingredient.

[0047] Pharmaceutical preparations, which can be used orally, are also hard capsules of gelatine or soft shell capsules of gelatine and a emollient, such as glycerol or sorbitol. The hard capsules can contain the active ingredient in the form of granules, mixed for example with fillers, such as maize starch, binders or lubricants, such as talc or magnesium stearate, and stabilisers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid substances of neutral nature, such as greasy oil, paraffin oil or liquid polyethylene glycol or fatty acid esters and ethylene or propylene glycol, wherein while it is also possible to add stabilisers and detergents, for example of the polyethylene sorbitan fatty acid ester type.

[0048] Other oral forms of administration are, for example, syrups prepared in a customary manner, which comprise active ingredient, for example, in suspended form and in a concentration of about 5 to 20%, preferably about 10% or in a similar concentration which results in a suitable individual dose, for example, when 5 or 10 ml are measured out. Other forms are, for example, pulverulent or liquid concentrates for preparing shakes, for example in milk. Such concentrates can also be packed in unit dose quantities.

[0049] Pharmaceutical preparations, which can be used rectally, are, for example, suppositories that comprise a combination of the active ingredient with a base. Suitable bases are, for example, naturally occurring or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.

[0050] Preparations which are suitable for parental administration are aqueous solutions of an active ingredient in water-soluble form, for example of water-soluble salt, or aqueous injection suspensions, which comprise viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and / or dextran, and if appropriate stabilisers. The active ingredient can also be present in the form of a lyophilizate, if appropriate together with excipients, and be dissolved before parenteral administration by addition of suitable solvents. Solutions which are used for parental administration can also be used for example also for infusion solutions. Preferred preservatives are preferably antioxidants, such as ascorbic acid, or microbicides, sorbic or benzoic acid.

[0051] Ointments are oil-in-water emulsions, which comprise not more than 70%, but preferably 20 to 50% of water or aqueous phase. The fatty phase consists of, in particular, hydrocarbons, for example vaseline, paraffin oil or hard paraffins, which preferably comprise suitable hydroxy compounds, such as fatty alcohols or esters thereof, for example cetyl alcohol or wool wax alcohols, preferably wool wax, to improve the water-binding capacity. Emulsifiers are corresponding lipophilic compounds, such as sorbitan fatty acid esters (Spans), preferably sorbitan oleate or sorbitan isostearate. Additives to the aqueous phase are, for example, humectants, such as polyalcohols, for example glycerol, propylene glycol, sorbitol and / or polyethylene glycol, or preservatives or odoriferous substances.

[0052] Fatty ointments are anhydrous and comprise, as the base, in particular, hydrocarbons, for example paraffin, vaseline or paraffin oil, and furthermore naturally occurring or semi- synthetic fats, for example hydrogenated coconut-fatty acid triglycerides, or, preferably, hydrogenated oils, for example hydrogenated groundnut or castor oil, and furthermore fatty acid partial esters of glycerol, for example glycerol mono- and / or distearate. They also contain e.g., fatty alcohols, emulsifiers and / or additives mentioned in connection with ointments which increase the uptake of water.

[0053] Creams are oil-in-water emulsions, which comprise more than 50% of water. Oily bases used are, in particular, fatty alcohols, for example lauryl, cetyl or staryl alcohols, fatty acids, for example palmitic or stearic acid, liquid and solid waxes, for example isopropyl myristate, wool wax or beeswax, and / or hydrocarbons, for example vaseline (petrolatum) or paraffin oil. Emulsifiers are surface-active compounds with predominantly hydrophilic properties, such as corresponding non-ionic emulsifiers, for example fatty acid esters of polyalcohols or ethyleneoxy adducts thereof, such as polyglyceric fatty acid esters or polyethylene sorbitan esters (Tween), furthermore polyoxyethylene fatty alcohol ethers or polyoxyethylene fatty acid esters, or corresponding ionic emulsifiers, such as alkali metal salts of fatty alcohol sulphates, preferably sodium lauryl sulphate, sodium cetyl sulphate or sodium stearyl sulphate, which are usually used in the presence of fatty alcohols, for example cetyl stearyl alcohol or stearyl alcohol. Additives to the aqueous phase are, inter alia, agents which prevent the creams from drying out, for example polyalcohols, such as glycerol, sorbitol, propylene glycol and polyethylene glycol, and furthermore preservatives and odoriferous substances.

[0054] Pastes are creams or ointments containing secretion-absorbing powder constituents, such as metal oxides, for example titanium oxide or zinc oxide, and also talc or aluminium silicates, which have the task of binding the moisture or secretion present.

[0055] Foams are administered from pressurised containers, and they are liquid oil-in-water emulsions in aerosol form, wherein as the propellant gases are used halogenated hydrocarbons, such as polyhalogenated alkanes, for example dichlorofluoromethane and dichlorotetrafluoroethane, or, preferably, non-halogenated gaseous hydrocarbons, air, N2O, or carbon dioxide. The oily phases used are the same as for ointments and cremes, and the additives mentioned there are likewise used.

[0056] Tinctures and solutions usually comprise an aqueous-ethanolic base to which, humectants for reducing evaporation, such as polyalcohols, for example glycerol, glycols and / or polyethylene glycol, furthermore re-oiling substances, such as fatty acid esters and lower polyethylene glycols, i.e., lipophilic substances soluble in the aqueous mixture to substitute the fatty substances removed from the skin with the ethanol, and, if necessary, other excipients and additives are admixed.

[0057] This invention further provides veterinary preparations containing at least one active ingredient together with a veterinary carrier. Veterinary carriers are materials for application of a preparation and may be solid, liquid, or gaseous substances, which are inert or acceptable in veterinary medicine and are compatible with the active ingredient. These veterinary preparations can be administered orally, parenterally or by any other desired route.

[0058] The invention also relates to the processes or methods for treatment of the disease states mentioned above. The substances can be administered prophylactically or therapeutically as such or in form of pharmaceutical preparations, preferably in an amount, which is effective against the diseases mentioned, wherein with a warm-blooded animals, for example a human, in need of such treatment, the substance is used, in particular, in form of pharmaceutical preparation. A daily dose of about 0.1 to 5 g, preferably 0.5 g to 2 g, of the substance is administered for a body weight of about 70 kg. Suitable routes for administration include oral, rectal, vascular topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravitreous, intravenous, intradermal, intrathecal, and epidural). The preferred route of administration depends upon the condition of the patient, the toxicity of the compound and the site of infection, among other considerations known to the clinician.

[0059] Brief Description of Drawings

[0060] Fig. 1 shows stimulation of migration of normal human fibroblasts (BJ) in vitro by cytokinins. In scratch assays, trans-zeatin (tZ), trans-zeatin-9-glucoside (tZ9G) and kinetin- 3-glucoside (K3G) at 10 μM stimulate cell migration after 24 h. Untreated cells are indicated as control (Co). Results are compared to control cells stimulated with Vascular Endothelial Growth Factor (VEGF, a positive control) or cells treated with 2- methoxyestradiol (MeO, a negative control).

[0061] Fig. 2 shows an increased migration of normal human fibroblasts (BJ) in vitro after application of cytokinins in various combinations. In scratch assay, both 10 μM trans- zeatin-9-glucoside (tZ9G) and kinetin-3-glucoside (K3G) and in particular their combination strongly stimulate cell migration after 24 h. Untreated cells are indicated as control (Co). Results are compared to control cells stimulated with Vascular Endothelial Growth Factor (VEGF, a positive control) or cells treated with 2-methoxyestradiol (MeO, a negative control).

[0062] Fig. 3 shows stimulation of migration of starved normal human fibroblasts (BJ) in vitro after 24 hours of treatment with kinetin. Cells were starved for 48 h and then kinetin (K) was added to the cells. In the scratch assay, 10 μM K strongly stimulates cells after another 24 h. Results are compared to cells with / without fetal bovine serum (FBS) in two different media (DMEM or M199). Untreated cells are indicated as control (Co). Cells treated with 2- methoxyestradiol (MeO) were used as negative control.

[0063] Fig. 4 shows that kinetin decreases expression of cyclooxygenase-2 (COX-2) in BJ cells stimulated with tumour necrosis factor-a (TNF-α) for 30 min, 1, 6, 12 and 24 h. Decrease of COX-2 may be associated with anti-inflammatory activity and was detected by Western Blot analysis. α-Tubulin indicates equal protein loading. Untreated cells are indicated as a control (Co). Alaptid (A) was used as a positive control (a compound known from prior arts to stimulate wound healing in mammals).

[0064] Fig. 5 shows induction of migration of human umbilical vein endothelial cells (HUVEC) in vitro after application of cytokinins in various combinations. In scratch assay already after 8 hours, combination of two cytokinins, namely 10 μM K (kinetin) and tZ (trans-zeatin) or K9G (kinetin-9-glucoside) and tZ9G (trans-zeatin-9-glucoside), strongly stimulates cell migration. Results are compared to control cells stimulated with Vascular Endothelial Growth Factor (VEGF, a positive control) or cells treated with 2-methoxyestradiol (MeO, a negative control).

[0065] Fig. 6 shows stimulation of vascularization in vitro by cytokinins. Formation of tube-like structures were induced by K, K3G, tZ, tZ9G and their combinations. Cells were seeded on Matrigel® and treated with cytokinins (10 μM or 10 μM + 10 μM). Induction of tubule formation was compared to a positive control (cells with Vascular Endothelial Growth Factor - VEGF) or a negative control (cells with 10 μM 2-methoxyestradiol - MeO). After 16 h, images were taken.

[0066] Fig. 7 shows as cytokinins stimulate adhesion of HUVECs in vitro. VE-cadherin necessary for endothelial cell contacts was upregulated by K and K3G application. Cells were treated with cytokinins (10 μM) for 24 h, fixed and stained using primary monoclonal antibody and secondary conjugated antibody. Induction of expression of VE-cadherin was compared to control cells.

[0067] Fig. 8 shows the vascularisation effects of the heterocyclic cytokinins in Chorioallantoic membrane (CAM) assay. Fertilized chicken eggs were stimulated with VEGF (1 ng) alone or tested cytokinins (45 pg), respectively. All compounds significantly induced vascularization compared to VEGF: Exemplary Embodiments the Invention

[0068] The invention is further illustrated by the following examples, which should not be construed as limiting the scope thereof. All cytokinin derivates according to this invention are common substances, preparation of which is known to a person skilled in the art.

[0069] Abbreviations and chemical nomenclature

[0070] 3G: 3-p-D-glucopyranosyl;

[0071] 9G: 9-p-D-glucopyranosyl;

[0072] K: kinetin (6-furfurylaminopurine);

[0073] K3G: kinetin-3-glucoside;

[0074] 2iP: 6-isopentenylaminopurine;

[0075] BAP: 6-benzylaminopurine;

[0076] Z: zeatin;

[0077] Z3G: zeatin-3-glucoside; tZ: trans -zeatin; tZ9G: trans-zeatin-9-glucoside; cZ: cis-zeatin; pT: para-topolin (6-(4-hydroxybenzyIamino)purine; pT9G: para-topolin-9-glucoside; mT: meta-topolin (6-(3-hydroxybenzylamino)purine; mT9G: meta-topolin-9-glucoside; oT: ortho -topolin (6-(4-hydroxybenzyIamino)purine;

[0078] MemT: para-methoxytopolin (6-(4-methoxybenzyIamino)purine;

[0079] MeoT: ortho -methoxytopolin (6-(2-methoxybenzyIamino)purine; iP 6-(3,3-dimethyIaIIyIamino)purine;

[0080] 4MeOK: 4-methoxykinetin: 6-(4-methoxyfurfurylamino)purine.

[0081] HUVEC: Human Umbilical Vein Endothelial Cells

[0082] FCS: Fetal Calf Serum

[0083] DMEM: Dulbecco’s Modified Eagle’s Medium

[0084] VEGF: Vascular Endothelial Growth Factor

[0085] SEM, SE: Standard Error of the Mean

[0086] SDS: sodium dodecyl sulphate

[0087] RIP A buffer: Radio Immuno Precipitation Assay buffer

[0088] Tris-HCl: Tris (hydroxymethyl) aminomethane hydrochloride

[0089] EDTA: ethylenediaminetetraacetic acid

[0090] EGTA: ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid NaCl: sodium chloride

[0091] NaF: sodium fluoride

[0092] PMMSF: phenylmethylsulfonyl fluoride

[0093] BSA: Bovine Serum Albumin

[0094] TBS: Tris buffered saline

[0095] PBS: Phosphate buffered saline

[0096] BJ: normal human fibroblasts

[0097] COX-2: cyclooxygenase-2

[0098] TNF-α: tumour necrosis factor-a

[0099] DMSO: dimethyl sulfoxide

[0100] CAM: chick Chorioallantoic Membrane

[0101] TCS: Tumour Cells Survival cAMP: 3',5'-cyclic adenosine monophosphate

[0102] Cells culture

[0103] HUVECs were prepared by digestion of umbilical veins with 0.1 g / 1 collagenase A (Roche, Mannheim, Germany) as described previously and cultured in endothelial cell growth medium (ECGM, Provitro, Berlin, Germany) containing 10% FCS (Biochrom, Berlin, Germany). Experiments were performed using cells at passage 3.

[0104] Statistical analysis

[0105] The number of independently performed experiments and the used statistical tests are stated in the respective figure legend. Representative experiments are shown. Graph data represent means ± SEM. Statistical analysis was performed with the Microsoft Excel. Statistical significance is assumed if p≤0.05.

[0106] EXAMPLE 1

[0107] Migration scratch assay

[0108] Confluent HUVECs were scratched by pipetting tip and immediately treated with either full endothelial cell growth medium (HUVEC) or DMEM (BJ) containing different cytokinins according to the invention or medium without growth factors M199 (serum free, negative control; PAN Biotech, Aidenbach, Germany). After 16 h of incubation, cells were fixed using 4% formaldehyde and stained with Coomasie Blue. Images were captured with a microscope (1X51, Olympus, Tokyo, Japan). Migration was calculated using in-house software and expressed as the ratio between pixels covered by cells and the total number of pixels in the wound area. Cells treated with cytokinins for different time points were compared to the untreated, stimulated (positive control - 10 ng / ml vascular endothelial growth factor, VEGF) or inhibited (negative control - 10 μM 2-methoxyestradiol, MeO) cells. Relative wound area was evaluated for 16 h after the scratch. The data shown are means ± standard error (SE) obtained from at least three independent experiments performed in triplicates.

[0109] Tube formation

[0110] Ibidi 15-well p-slides (ibidi, Munich, Germany) were coated with Matrigel® (Coming, Tewksbury, MA, USA). 1 x 104HUVEC cells were treated with cytokinins and seeded on Martigel. After 16 h of incubation, images were captured using a microscope (1X51, Olympus, Japan). The data shown are means ± SE obtained from three independent experiments performed in triplicates.

[0111] SDS polyacrylamide gel electrophoresis and immunoblotting

[0112] For immunoblotting, harvested cells were lysed in RIP A buffer (20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 2 mM EGTA, 100 mM NaCl, 2 mM NaF, 0.2% Nonidet P-40, 30 mM

[0113] PMMSF, 1 mM of 10 mg / ml of aprotinin and leupeptin). Proteins in lysates were quantified by the Bradford method and then diluted with Laemmli electrophoresis buffer. Proteins were then separated on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes (Bio-Rad Laboratories, CA, USA), and stained with Ponceau S to check equal protein loading. The membranes were blocked with 5% (w / v) BSA and 0.1% Tween-20 in TBS for 2 h and incubated with the specific primary antibodies overnight. After washing in TBS and TBS containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with chemiluminescent detection reagent West Pico Supersignal (Thermo Fisher Scientific, Rockford, USA). To confirm equal protein loading, immunodetection was performed with the anti-α-tubulin monoclonal antibody. The experiments were repeated three times. The protein expressions in treated cells were compared to untreated controls.

[0114] Immunofluorescence labelling methods

[0115] Treated cells in 8-well ibidi p-slides (ibidi, Munich, Germany) were washed with PBS, fixed on the slides with 10% formaldehyde (v / v) for 10 min, labelled with primary antibody (Cell Signaling Technology, Danvers, MA, USA) and fluorescently-conjugated secondary antibody (Thermo Fisher Scientific) and fixed using medium FluorSave (Calbiochem, Merck Millipore, Darmstadt, Germany). The cells were then visualized using a fluorescence microscope (1X51, Olympus, Japan).

[0116] Results Several cytokinins increased human fibroblast and endothelial cell migration in vitro (Fig. 1 - 6). In scratch assay, tZ, tZ9G, K3G, pT9G, Z3G and mT9G at 10 μM stimulated cell migration after 24 h (Fig. 1). In addition, cytokinins in various combinations increased strongly migration of normal human fibroblasts (BJ). Using scratch assay, combination of both 10 μM K3G and tZ9G strongly stimulated cell migration (3 -fold compared to control cells with VEGF) after 24 h (Fig. 2). Moreover, kinetin strongly induced migration of starved normal human fibroblasts (BJ) in vitro after 24 h. Cells were starved for 48 h. Then, kinetin (K) was added to the cells. In scratch assays, 10 μM K strongly stimulates cells after another 24 h (Fig. 3). Results are compared to cells with / without fetal bovine serum (FBS) in two different media (DMEM or Ml 99) to prove the stimulatory potential of cytokinins. In addition, cytokinins in various combinations increased also migration of human umbilical vein endothelial cells (HUVEC) in vitro. In scratch assays, combination of both 10 μM K and tZ or K9G and tZ9G strongly stimulates cell migration already after 8 h (Fig. 5). Results compared to control cells stimulated with VEGF (positive control) again confirmed induction of cell migration.

[0117] Using a tubule formation assay, the ability of cells to form three-dimensional structures was measured. In comparison to control cells with 10 ng / ml of VEGF, tube formation of cytokinin-treated cells was induced significantly (Fig. 6). Strong effect was detected for 10 μM of K, K3G, tZ, tZ9G and their combinations. The networking of cells (junctions), their thickness, number of tubes and therefore the total length of tubes was shown after 16 h of treatment with cytokinins. Cytokinins also increased cell adhesion of HUVEC in vitro. VE- cadherin, endothelial specific adhesion molecule located at junctions between endothelial cells, necessary for endothelial cell contacts was more visible in cells with 10 μM K and K3G after 24 h (Fig. 7). In addition to its adhesive functions, VE-cadherin regulates various cellular processes such as cell proliferation and apoptosis and modulates vascular endothelial growth factor receptor functions. Consequently, VE-cadherin is essential during embryonic angiogenesis (Vestweber, Arterioscler Thromb Vase Biol 2008:223-32).

[0118] Furthermore, kinetin decreased expression of cyclooxygenase-2 (COX-2) in BJ cells stimulated with tumor necrosis factor-a ( TNF-α) for 12 h (Fig. 4). Decrease of COX-2 can be joined with anti-inflammatory activity and was detected by Western Blotting analysis

[0119] Table 1: Stimulatory activities of selected heterocyclic cytokinins for HUVEC endothelial cells after 11 h of treatment with 10 or 100 μM of tested compounds

[0120] EXAMPLE 2

[0121] Ex vivo and in vivo vascularisation effects

[0122] The vascularisation effects of the heterocyclic cytokinins were also tested in more complex models ex vivo and in vivo. Vessel formation assay in germinal disc of chicken eggs (CAM assay - Choriallantioc Membrane) was used in this respect. The Chorioallantoic Membrane, also known as the chorioallantois, is a highly vascularized membrane found in the eggs of certain amniotes like birds and reptiles. It is formed by the fusion of the mesodermal layers of two extra membranes - the chorion and the allantois. Chorioallantoic membrane (CAM) assay: Fertilized White Leghorn chicken eggs (Lohmann Tierzucht, Cuxhaven, Germany) were incubated at 37°C for 72 h with constant humidity. Eggs were transferred into dishes, incubated for further 72 h, and stimulated with VEGF (Ing / disc) or VEGF and the indicated compounds (45 pg) using small cellulose discs. The next day, CAMs were photographed using a stereomicroscope (Olympus, Munich, Germany). Cytokinins dose-dependently increased endothelial cell sprouting out of aortic rings (Fig. 8). By applying CAM assays, we found a strong induction of vessel formation by vascular endothelial growth factor (VEGF), which was also stimulated during combined treatment with cytokinins (Fig. 8). As an ultimate in vivo proof of principle, the mouse cornea microdrop assay was used. Intraperitoneally given kinetin strongly induced neovascularization in vivo.

[0123] EXAMPLE 3

[0124] In vitro cytotoxic activity of novel compounds

[0125] Low cytotoxicity of the compounds is the major property determining their cosmetic and therapeutic use. One of the parameters used, as the basis for cytotoxicity assays, is the metabolic activity of viable cells. For example, a microtiter assay, which uses the Calcein AM, is now widely used to quantify cell proliferation and cytotoxicity. For instance, this assay is used in drug screening programs and in chemosensitivity testing. Because Calcein AM cleaves only metabolically active cells, these assays detect viable cells exclusively. The quantity of reduced Calcein AM corresponds to the number of vital cells in the culture.

[0126] Cell lines: human T-lymphoblastic leukaemia CEM; promyelocytic HL-60 and monocytic U937 leukaemia’s; breast carcinoma cell lines MCF-7, BT549, MDA-MB-231; glioblastoma U87MG cells; cervical carcinoma cells HELA; sarcoma cells U2OS and Saos2; hepatocellular carcinoma HepG2; mouse fibroblasts NIH3T3; human fibroblasts BJ; mouse immortalized bone marrow macrophages B2.4 and B10A.4; P388D1 and L1210 leukaemia; B16 and B16F10 melanomas; human osteosarcoma HOS; human myeloid leukaemia K-562; human skin melanoma G-361 were used for routine screening of compounds. The cells were maintained in TTP 80 cm2plastic tissue culture flasks and cultured in cell culture medium (DMEM with 5 g / 1 glucose, 2 mM glutamine, 100 U / ml penicillin, 100 x g / ml streptomycin, 10% fetal calf serum and sodium bicarbonate).

[0127] The cell suspensions that were prepared and diluted according to the particular cell type and the expected target cell density (2,500-30,000 cells per well based on cell growth characteristics) were added by pipette (80 pl) into 96-well microtiter plates. Inoculates were allowed a pre-incubation period of 24 hours at 37°C and 5% CO2for stabilisation. Four-fold dilutions of the intended test concentration were added at time zero in 20 μl aliquots to the microtiter plate wells. Usually, the compound tested was evaluated at six 4-fold dilutions. In routine testing, the highest well concentration was 166.7 μM, but it can be changed depending on the substance. All drug concentrations were examined in duplicate. Incubations of cells with the tested compounds lasted for 72 hours at 37°C, in a 5% CO2atmosphere and 100% humidity. At the end of the incubation period, the cells were assayed by using Calcein AM. Hundred microliters of the stock solution were pipetted into each well and incubated for 1 hour. Fluorescence (FD) was measured with the Labsystem FIA Reader Fluoroscan Ascent (UK). The tumour cell survival (IC50) was calculated using the following calculation: TCS=(FDdrug exposed well / mean FDcontrol wells) x 100%. The GI50value, the drug concentration lethal to 50% of the tumour cells, was calculated from the obtained dose response curves.

[0128] Zero cytotoxicity of the novel compounds is the basic prerequisite for wound healing applications. The cytoxicity of the novel compounds was tested on a panel of cell lines of different histogenetic and species origin (Table 2). We show herein that equal activities were found in all tumour cell lines tested, however, the non-malignant cells, e.g., NIH3T3 fibroblasts and normal human fibroblasts, were resistant to heterocyclic cytokinins induced cytotoxicity. The compounds listed in Tab. 2 can be divided into 2 groups. The first group contains “classical free cytokinin bases” represented by 6-substituted aminopurines (which are known in the prior art). The second group contains the novel heterocyclic cytokinin derivatives substituted at position 3 or 9 by glucose. The results show that the substitution in position 3 or 9 of the purine skeleton generally led to a complete loss in the cytotoxic activity in comparison to the “classical cytokinin” analogues. As demonstrated in Table 2, IC50for BJ fibroblasts (normal human fibroblasts) and NIH3T3 mouse fibroblasts was always higher than 166.7 μM. The novel derivatives show no toxicity to normal and tumour cells in concentrations to 166.7 μM and thus are even more suitable for cosmetic and pharmaceutical applications than natural cytokinins (6-substituted aminopurine derivatives). Table 2: Cytotoxicity of novel compounds for different normal and cancer cell lines

[0129] EXAMPLE 4

[0130] Anti-inflammatory activity

[0131] Rat C6 glioma (ATCC N° CCL107) was cultivated as a monolayer in a serum-free chemically defined medium containing Ham’s FlO-minimal essential medium (1:1 v / v),

[0132] 2 mM L-glutamine, 1% (v / v) of minimal essential medium vitamins (100x), 1% (v / v) of minimal essential medium nonessential amino acids (100x), 100 U / ml penicillin, 100 μg / ml streptomycin and 30 nM sodium selenite. Incubation was performed at 37°C in a humidified atmosphere. The assays were performed in the logarithmic growth phase at a density of

[0133] 2.5x105cells / cm2. Intracellular cAMP synthesis was induced by addition of 5 μM

[0134] (-)isoproterenol. After 30 min of incubation at 37°C the medium was removed, and the cellular amount of cAMP was determined using the cAMP-enzyme immunoassay

[0135] Amersham kit. The I50value was determined from a dose -response curve in duplicate. The effect of the novel heterocyclic purines was measured after simultaneous addition of isoproterenol. The classical cytokinins, known in the prior art, were inactive.

[0136] Table 3: Modulation of the activity of β-adrenergic receptors by substituted purines n. a. means not active

[0137] As P2Y1and A2-like purinergic receptors, negatively and positively coupled to adenylate cyclase, respectively, are present in rat C6 glioma, it remains to be determined whether the modulation of the synthesis of cAMP is due to the inhibition of the activation of β- adrenergic receptors by isoproterenol, or due to the activation of purinergic receptors. The compounds of formula I and II having anti-inflammatory activities can be used for treating inflammation wounding disorders.

[0138] EXAMPLE 5

[0139] Dry Capsules

[0140] 5,000 capsules, each of which contains 0.25 g of one of the compounds mentioned in the preceding Examples as active ingredient, are prepared by following process:

[0141] Composition Active ingredient 1,250 g

[0142] Talc 180 g

[0143] Wheat starch 120 g

[0144] Magnesium stearate 80 g

[0145] Lactose 20 g

[0146] Preparation process: The grounded substances are pressed through a sieve of mesh width 0.6 mm. Dose of 0.33 g of the mixture is transferred to gelatine capsule with the aid of a capsule-filling machine.

[0147] EXAMPLE 6

[0148] Soft Capsules

[0149] 5,000 soft gelatine capsules, each of which contains 0.05 g of one of the compounds mentioned in the preceding Examples as active ingredient, are prepared by following process:

[0150] Composition

[0151] Active ingredient 250 g

[0152] Lauroglykol 2 litres

[0153] Preparation process: The powdered active ingredient is suspended in Lauroglykol® (propylene glycol laurate, Gattefosse S. A., Saint Priest, France) and ground in a wet- pulveriser to a particle size of about 1 to 3 mm. Dose of 0.419 g of the mixture is then transferred to soft gelatine capsules by means of a capsule-filling machine.

[0154] EXAMPLE 7:

[0155] Soft Capsules

[0156] 5,000 soft gelatine capsules, each of which contains 0.05 g of one of the compounds of the formula I mentioned in the preceding Examples as an active ingredient, are prepared by following process: Composition Active ingredient 250 g PEG 400 1 litre

[0157] Tween 80 1 litre

[0158] Preparation process: The powdered active ingredient is suspended in PEG 400 (polyethylene glycol of Mr between 380 and 420, Sigma, Fluka, Aldrich, USA) and Tween® 80 (polyoxyethylene sorbitan monolaurate, Atlas Chem. Ind., Inc., USA, supplied by Sigma, Fluka, Aldrich, USA) and ground in a wet -pulveriser to a particle size of 1 to 3 mm. Dose of 0.43 g of the mixture is then transferred to soft gelatine capsules by means of a capsule- filling machine.

[0159] EXAMPLE 8

[0160] Development of ointments

[0161] An ointment formulation was tested during the pilot clinical study in 4 volunteers with psoriatic skin disorders. The names of the formulation ingredients are given according to the terminology of the registering authorities and their quantity is in grams per 100 g.

[0162] Ointment / 100 g trans-zeatin (tZ) 1.0 g butylated hydroxytoluene (Nipanox BHT) 0.2 g butylparaben (Nipabutyl) 0.2 g diethylene glycol monoethyl ether (Transcutol P) 10.0 g glycerol dibehenate (Compritol 888 ATO) 22.0 g propylene glycol laurate (Lauroglycol FCC) 66.6 g

[0163] The volunteers have rated: The possible ointment consistency may be further modified by addition of vaselinum album. It is expected that the transdermal Transcutol P / Lauroglycol FCC system will increase the efficiency of trans-zeatin.

[0164] EXAMPLE 9

[0165] Gel preparation

[0166] An ointment formulation was tested during a pilot clinical study with 4 volunteers with erysipelas which is a skin disease. The ingredients are given in grams per 100 g.

[0167] The gel of this consistence may be additionally modified by addition of silicon dioxide, silica colloidalis anhydrica. It is again expected that the transdermal Transcutol P / Lauroglycol FCC system will increase the efficiency of para-topolin mesylate salt. Silicon dioxide colloidalis anhydrica will probably slow down the penetration of the active ingredient. EXAMPLE 10

[0168] Preparation process of a skin ointment

[0169] The formulation ingredients are given in grams per 200 g:

[0170] Recommended process

[0171] Phase A: 2 grams of para-topolin mesylate salt were dissolved in 20 g of Transcutol P while stirring continuously at a room temperature in a separate glass or stainless-steel container. The dissolution process may be accelerated by heating the solution to a maximal temperature of 40°C.

[0172] Phase B: 0.4 grams of Nipanox BHT and 0.4 g of Nipabutyl were dissolved while stirring continuously in 133.2 g of Lauroglycol FCC at a temperature of approximately 70 °C in another separate glass or stainless-steel container. The clear oily solution is heated to a temperature of approximately 80 °C and 44 g of Compritol 888 ATO are melted in it while stirring continuously. The clear oily solution is cooled down to approximately 60 °C while stirring continuously and cooling down and is mixed with phase A. The resulting whitish ointment is divided into approximately 15 -gram portions and filled into prearranged plastic containers.

[0173] EXAMPLE 11

[0174] Formulation of a preparation for topical application to the skin

[0175] A composition for topical application to the skin contains the following ingredients by weight%:

[0176] Methyl paraben and propyl paraben were dissolved in hot water and subsequently also methylcellulose was dispersed in it. The mixture was chilled to 60°C until the methylcellulose dissolved. The mixture was then heated to 72°C and added to the oil phase which was heated to 70°C while stirring continuously. Para-topolin mesylate salt was added at a temperature of 35°C and the resulting mixture was stirred continuously until dispersed. This composition is applied to the skin on at least a daily basis until the desired skin- ameliorating (anti-aging) effect is reached.

Claims

CLAIMS1. Heterocyclic purine cytokinin derivatives of the general formula I and IIor pharmaceutically acceptable salts thereof with alkali metals, ammonia or amines, in the form of racemates or optically active sugar isomers (β-D, a-D, β-L, α-L), whereR6 is furfuryl, substituted furfuryl, benzyl, substituted benzyl; alkyl, substituted alkyl, alkenyl and substituted alkenyl, where substituted furfuryl groups include 3-methoxyfurfuryl, 4-methoxyfurfuryl, and 5- methoxyfurfuryl, substituted benzyl groups include 2 -hydroxybenzyl, 3-hydroxybenzyl, 4- hydroxybenzyl, 2-methoxybenzyl, 3 -methoxybenzyl, 4-methoxybenzyl, 3,4- dihydroxybenzyl, 3,5-dihydroxybenzyl, 2,3-dimethoxybenzyl, 2,4- dimethoxybenzyl, 2,5-dimethoxybenzyl, 3,5-dimethoxybenzyl 2,3,4- trimethoxybenzyl, 2-hydroxy-3-methoxybenzyl, 2-hydroxy-5-methoxybenzyl, 2- hydroxy-4-methoxybenzyl, 3-hydroxy-4-methoxybenzyl, 4-hydroxy-3- methoxybenzyl, 4-hydroxy-2-methoxybenzyl, 2,3-dihydroxy-4-methoxybenzyl, 2,4- dihydroxy-3-methoxybenzyl, 2,5-dihydroxy-4-methoxybenzyl, 2,3,5- trimethoxybenzyl, 2,4,6-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, alkyl groups include butyl, 2-methylbutyl, 3-methylbutyl and isopropyl, substituted alkyl groups include 4-hydroxy-3-methylbutyl and 1' -methyl-4-hydroxy- 3-methylbutyl, alkenyl groups include isopentenyl and 3 -dimethylallyl, substituted alkenyl groups include 3-hydroxymethyl-3-methylallyl, (Z)-(4-hydroxy- 3-methylbut-2-en-1-yl, (E)-(4-hydroxy-3-methylbut-2-en-1-yl, (Z)-(1'-methyl-4- hydroxy-3-methylbut-2-en-1-yl and (Z)-(1' -methyl-4-hydroxy-3-methylbut-2-en-1- yl,andR3 and R9 are independently at each occurrence hydrogen and a glucopyranosyl group, for use in wound healing.

2. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1, selected from the group containing 6-(3,3-dimethylallylamino)purine, 6- furfurylaminopurine, 6-benzylaminopurine, 6-(3-hydroxybenzylamino)purine, 6-(4- hydroxybenzylamino)purine, 6-(E)-(4-hydroxy-3-methylbut-2-en-1-ylamino)purine, 6-(4- hydroxy-3 -methylbutylamino)purine , 6-furfurylamino-3-β-D-glucopyr anosylpurine, 6- benzylaminopurine-3-β-D-glucopyr anosylpurine, 6-(3-hydroxybenzylamino)-3-β-D- glucopyr anosylpurine , 6-(4-hydroxybenzylamino)-3-β-D-glucopyr anosylpurine, 6-(4- hydroxy-3-methylbutylamino)-3-β-D-glucopyranosylpurine, 6-(3,3-dimethylallylamino)-3-P -D-glucopyranosylpurine , 6-(3-hydroxymethyl-3-methylallyl)amino-3-β-D- glucopyr anosylpurine , 6-(Z)-(4-hydroxy-3-methylbut-2-en-1-ylamino)-3-β-D- glucopyr anosylpurine , 6-(E)-(4-hydroxy-3-methylbut-2-en-1-ylamino)-3-β-D- glucopyr anosylpurine, 6-(4-hydroxy-3-methylbutylamino)-3-β-D-glucopyranosylpurine, 6- furfurylamino-9-β-D-glucopyranosylpurine, 6-benzylamino-9-β-D-glucopyranosylpurine, 6-(3-hydroxybenzylamino)-9-β-D-glucopyranosylpurine, 6-(4-hydroxybenzylamino)-9-β-D- glucopyr anosylpurine, 6-(4-hydroxy-3-methylbutylamino)-9-β-D-glucopyranosylpurine, 6-(3, 3-dimethylallylamino)-9-β-D-glucopyr anosylpurine, 6-(3-hydroxymethyl-3- methylallyl)amino-9-β-D-glucopyranosylpurine, 6-(Z)-(4-hydroxy-3-methylbut-2-en- 1 - ylamino)-9-β-D-glucopyranosylpurine, 6-(E)-(4-hydroxy-3-methylbut-2-en-1-ylamino)-9-β-D-glucopyranosylpurine, and 6-(4-hydroxy-3-methylbutylamino)-9-β-D- glucopyr anosylpurine .

3. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use as a stimulant of migration and angiogenesis of mammalian endothelial cells and fibroblasts.

4. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use as a stimulant of influx of fibroblasts, vascular endothelial cells, or epithelial cells into a wound, wherein the use comprises also intensification of contacts of said fibroblasts, vascular endothelial cells, or epithelial cells.

5. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use as a stimulant of wound healing where said wound is a burn, an ischemic ulcer, a decubitus ulcer, an ulcer resulting from an infectious process or an ulcer resulting from an inflammatory process.

6. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use in treating an injured oral tissue.

7. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use in moisturizing and protecting of dry and / or seborrheic skin.

8. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use in treating inflammation of skin and / or in suppressing immunological responses resulting from inflammation of skin.

9. Heterocyclic cytokinin derivatives of general formula I and II according to claim 1 and 2 for use in treating disease conditions in mammals, where said use comprises application of an amount of a novel heterocyclic derivative according to this invention for the treatment of mammalian cells and tissues.

10. A pharmaceutical preparation characterized in that it comprises one or more heterocyclic cytokinin derivatives of the general formula I and II or pharmaceutically acceptable salts thereof with alkali metals, ammonia, or amines, in the forms of racemates or optically active isomers, or their acid addition salts, according to any of the preceding claims.