WO2017089301A1

WO2017089301A1 - 3-triazolo-3-deoxy-ingenols

Info

Publication number: WO2017089301A1
Application number: PCT/EP2016/078317
Authority: WO
Inventors: Lars JØRGENSEN; Christian A. KUTTRUFF; Bjarne NØRREMARK
Original assignee: Leo Laboratories Limited
Priority date: 2015-11-27
Filing date: 2016-11-21
Publication date: 2017-06-01

Abstract

The present invention relates to a compound according to formula (I) wherein X represents a bond or -(CR_aR_b)_n-, wherein R_a and R_beach independently represents hydrogen, (C₁-C₄)alkyl or R_a and R_b together with the carbon atom to which they are attached form a (C₃-C₆)cycloalkyl, and n is 1 or 2; wherein Y represents a bond or -O-; wherein R₁ is selected from the group consisting of phenyl, (C₄-C₆)cycloalkyl and (C₄-C₆)alkyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from R₂; wherein R₂ consists of halogen, (C₃-C₆)cycloalkyl, (C₁-C₆)alkyl, halo(₁-C₆)alkyl, (C₁-C₆)alkoxy and halo(C₁-C₆)alkoxy. The invention relates further to intermediates for the preparation of said compounds, to said compounds for use in therapy, to pharmaceutical compositions comprising said compounds, to methods of treating diseases, e.g. dermal diseases, with said compounds, and to the use of said compounds in the manufacture of medicaments.

Description

3-Triazolo-3-deoxy-ingenols

FIELD OF THE INVENTION

This invention relates to novel 3-triazolo-3-deoxy-ingenols and derivatives thereof, to intermediates for the preparation thereof, to said compounds for use in therapy, to pharmaceutical compositions comprising said compounds and to methods of treating diseases with said compounds.

BACKGROUND OF THE INVENTION

Ingenol-3-angelate (PEP005, ingenol mebutate, contained in Picato^®) is a diterpene- ester of the ingenol family which is isolated from various Euphorbia species, particularly from Euphorbia peplus. Picato^® is marketed for the treatment of actinic keratosis and is subject for clinical development for the treatment of non-melanoma skin cancer, squamous cell carcinoma and genital warts.

Ingenol-3-angelate is believed to have a dual mode of action : 1) Induction of cell death by direct cytoxicity or induction of apoptosis and 2) an immunostimulatory effect dominated by neutrophil recruitment and activation (Rosen, R.H., et al., J Am Acad Derm (2012), 66, 486-493; Ersvaer, E., et al., Toxins, (2010), 2, 174-194). Nanomolar concentrations of the agent cause activation and modulation of protein kinase C (PKC) classical and novel isoforms, with particular importance of PKCdelta. Through activation of PKCdelta the agent induces apoptosis in susceptible cells (Hampson, P., et al., Blood, (2005), 106, 1362-1368; Cozzi, SJ.,et al., Cancer Res, (2006), 66, 10083-10091). Rapid cytotoxicity on cancer cells is observed at high micromolar concentrations

(Ogbourne, S. M., et al., Cancer Res (2004), 64, 2833-2839). Through activation of various PKC isoforms the agent also induces pro-inflammatory effects, including release of pro-inflammatory mediators (Challacombe, J. M., et al., J Immunol (2006), 177, 8123- 8132, activation of vascular endothelium (Hampson, P., et al., Cancer Immunol

Immunother, (2008), 57, 1241-1251); chemoattraction of neutrophils through induction of interleukin 8 in keratinocytes and development of specific anti-cancer immune responses by CD8+ cells through adjuvant properties in animal models (Le, T.T., et al., Vacccine, (2009), 27, 3053-3062).

Compounds exerting dual mode of action by induction of cell death by direct cytoxicity or induction of apoptosis, and by an immunostimulatory effect involving neutrophil recruitment and activation, may be useful for treatment of conditions associated with hyperplasia or neoplasia. Compounds inducing cell death by primary and / or secondary necrosis and compounds exhibiting a pro-apoptotic effect may reduce unwanted cell growth and remove unwanted cells, and furthermore, stimulation of the innate immune response and adjuvant effects may augment the biological response against aberrant or transformed cells. WO2012085189 disclose aliphatic ingenol-3-acylates.

WO2012083954 disclose carbocyclic ingenol-3-acylates.

WO2012083953 disclose heterocyclic ingenol-3-acylates and ingenol-3-carbamates.

WO2014001215 disclose 3-O-heteroaryl ingenols.

Liang, X. et. al., Bioorg. Med. Chem. Lett., 23, (2013), 5624-5629 disclose ingenol- mebutate analogues.

Grue-Soerensen, G., et. al., Bioorg. Med. Chem. Lett. 24 (2014) 54-60 disclose ingenol- 3-benzoates.

There is a continuous need to find new ingenol derivatives which induce cell death by cytotoxicity or apoptosis and / or induce an immunostimulatory effect.

SUMMARY OF THE INVENTION

It has surprisingly been found that certain 3-triazolo-3-deoxy-ingenol derivatives exhibit properties which make them useful for treatment of conditions associated with the use of ingenol-3-angelate or useful for conditions which are affected by induction of cell death by cytoxicity or induction of apoptosis and / or by an immunostimulatory effect.

Thus, compounds of the present invention stimulate neutrophil oxidative burst, which is part of the innate immune response.

Some compounds of the present invention stimulate keratinocyte IL-8 release, thus inducing an immunostimulatory effect.

Compounds of the present invention induce rapid necrosis.

Compounds of the present invention exhibit favorable stability properties. Compounds of the present invention may have improved pharmacokinetic and pharmacodynamic properties in comparison with known structurally related compounds.

Accordingly, the present invention relates to a compound according to formula I

I wherein X represents a bond or -(CR_aR_b)_n-, wherein R_a and R_b each independently represents hydrogen, (Ci-C₄)alkyl or R_a and R_b together with the carbon atom to which they are attached form a (C₃-C₆)cycloalkyl, and n is 1 or 2; wherein Y represents a bond or -0-; wherein Ri is selected from the group consisting of phenyl, (C₄-C₆)cycloalkyl and (C₄- C₆)alkyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from R₂; wherein R₂ consists of halogen, (C₃-C₆)cycloalkyl, (d-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci- C₆)alkoxy and halo(Ci-C₆)alkoxy; or pharmaceutically acceptable salts, hydrates or solvates thereof.

In another aspect, the invention relates to a compound according to formula I as defined above for use as a medicament.

In yet another aspect, the invention relates to a compound according to formula I as defined above for use in treatment of physiological disorders or diseases associated with hyperplasia or neoplasia. In yet another aspect, the invention relates to a compound according to formula I as defined above together with a pharmaceutically acceptable vehicle or excipient or pharmaceutically acceptable carrier(s). In yet another aspect, the invention relates to an intermediate for the preparation of a compound according to formula I as defined above, selected from

5,20-Acetonide-3-O-methanesulfonyl-ingenol,

5,20-Acetonide-3-(S)-azido-3-deoxy-ingenol and

3-(S)-azido-3-deoxy-ingenol.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "alkyi" is intended to indicate a radical obtained when one hydrogen atom is removed from a branched or linear hydrocarbon. Said alkyi comprises 1-6, preferably 1- 4, such as 1-3, such as 2-3 or such as 1-2 carbon atoms. The term includes the subclasses normal alkyi (/7-alkyl), secondary and tertiary alkyi, such as methyl, ethyl, n- propyl, isopropyl, /7-butyl, isobutyl, sec-butyl, tert.-butyl, /7-pentyl, isopentyl, neopentyl, /7-hexyl and isohexyl.

The terms "alkyloxy" and "alkoxy" are intended to indicate a radical of the formula -OR', wherein R' is alkyi as indicated herein, wherein the alkyi group is appended to the parent molecular moiety through an oxygen atom, e.g . methoxy (-OCH₃), ethoxy (-OCH₂CH₃), n-propoxy, isopropoxy, butoxy, tert-butoxy, and the like.

The term "cyano" is intended to indicate a -CN group attached to the parent molecular moiety through the carbon atom.

The term "cycloalkyl" is intended to indicate a saturated cycloalkane hydrocarbon radical, including polycyclic radicals such as bicyclic or tricyclic radicals, comprising 3-10 carbon atoms, preferably 3-8 carbon atoms, such as 3-6 carbon atoms, such as 3-5 carbon atoms or such as 3-4 carbon atoms, e.g . cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.

The term "haloalkyl" is intended to indicate an alkyi group as defined herein substituted with one or more halogen atoms as defined herein, e.g. fluoro or chloro, such as difluoromethyl or trifluoromethyl.

The terms "haloalkyloxy" and "haloalkoxy" are intended to indicate an haloalkyl group as defined herein which is appended to the parent molecular moiety through an oxygen atom, such as difluoromethoxy or trifluoromethoxy. The term "halogen" is intended to indicate a substituent from the 7 main group of the periodic ta ble, such as fluoro, chloro and bromo.

The term "heterocycloalkyl" is intended to indicate a cycloalkane radica l as described herein, wherein one or more carbon atoms are replaced by heteroatoms, comprising 1-6 carbon atoms, e.g . 2-5 or 2-4 carbon atoms, further comprising 1-6 heteroatoms, preferably 1, 2, or 3 heteroatoms, selected from 0, N, or S. Representative examples of heterocycloalkyl groups include, but are not limited to azepanyl, azetidinyl, aziridinyl, dioxolanyl, dioxolyl, imidazolidinyl, morpholinyl, oxetanyl, piperazinyl, piperidinyl, pyrrolidinyl, quinoxalinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thietanyl .

The term "4-7 membered heterocycloalkyl" is intended to indicate a "heterocycloalkyl" as described herein comprising 4-7 ring atoms independently selected from C, O, N or S.

The term "hydroca rbon radical" is intended to indicate a radical containing only hydrogen and carbon atoms and it may comprise cyclic moieties in combination with branched or linear moieties. Said hydrocarbon comprises 1-7 carbon atoms, e.g . 3-6, e.g . 1-4, e.g . 1-3, e.g . 1-2 carbon atoms. The term includes alkyl and cycloalkyl as indicated herein .

In some instances, the number of carbon atoms in a hydrocarbon radical is indicated by the prefix "(C_a-C_b)", wherein a is the minimum number and b is the maximum number of ca rbons in the hydrocarbon radical . Thus, for example (Ci-C₄)a lkyl is intended to indicate an alkyl radical comprising from 1 to 4 carbon atoms, and (C₃-C₆)cycloalkyl is intended to indicate a cycloalkyl radical comprising from 3 to 6 carbon ring atoms.

The term "oxo" is intended to indicate an oxygen atom which is connected to the pa rent molecular moiety via a double bond (=0) . When two or more of the above defined terms a re used in combination, such as arylalkyi, heteroarylalkyi, cycloalkylalkyi and the like, it is to be understood that the first mentioned radical is a substituent on the latter mentioned radical, where the point of attachment to the parent molecular moiety is on the latter radical . The g roup C(O) is intended to represent a carbonyl group (C=0)

The term "pharmaceutically acceptable salt" is intended to indicate salts prepared by reacting a compound of formula I, which comprise a basic moiety, with a suitable inorganic or organic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric, phosphoric, formic, acetic, 2,2-dichloroaetic, adipic, ascorbic, L-aspartic, L-glutamic, galactaric, lactic, maleic, L-malic, phthalic, citric, propionic, benzoic, glutaric, gluconic, D-glucuronic, methanesulfonic, salicylic, succinic, malonic, tartaric, benzenesulfonic, ethane-l,2-disulfonic, 2-hydroxy ethanesulfonic acid, toluenesulfonic, sulfamic or fumaric acid. Pharmaceutically acceptable salts of compounds of formula I comprising an acidic moiety may also be prepared by reaction with a suitable base such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, silver hydroxide, ammonia or the like, or suitable non-toxic amines, such as lower alkylamines, hydroxy-lower alkylamines, cycloalkylamines, or benzylamines, or L-arginine or L-lysine. Further examples of pharmaceutical acceptable salts are listed in Berge, S.M. ; J. Pharm. Sci. ; (1977), 66(1), 1-19, which is incorporated herein by reference.

The term "solvate" is intended to indicate a species formed by interaction between a compound, e.g. a compound of formula I, and a solvent, e.g . alcohol, glycerol or water, wherein said species are in a crystalline form. When water is the solvent, said species is referred to as a hydrate.

The term "treatment" as used herein means the management and care of a patient for the purpose of combating a disease, disorder or condition. The term is intended to include the delaying of the progression of the disease, disorder or condition, the amelioration, alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition. The term may also include prevention of the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. Nonetheless, prophylactic (preventive) and therapeutic (curative) treatments are two separate aspects. The phrase "physiological disorders or diseases associated with hyperplasia or neoplasia" in the context of the present invention is intended to cover disorders or diseases such as Cutaneous warts including common warts (Verruca vulgaris), plantar warts (Verruca plantaris) and flat warts (verruca plana); Genital warts (condyloma acuminatum), Pyogenic granuloma, Haemangioma, Scleroderma; Cancers and precancerous lesions such as Actinic keratosis, Squamous cell carcinoma including squamous cell carcinoma in situ (Bowen's disease), invasive squamous cell carcinoma, cutaneous squamous cell carcinoma, mucosal squamous cell carcinoma, head and neck squamous cell carcinoma; Basal cell carcinoma including Superficial basal cell carcinoma and Nodular basal cell ca rcinoma ; Bladder cancer, Lentigo maligna, Cervical dysplasia, Vulva dysplasia and anal dysplasia, Primary melanoma in situ, Head and neck cancer, Cutaneous metastases of any cancer, Kaposi's sa rcoma, Keratoacanthoma, Merkel cell tumor, Prostate cancer, Mycosis fungoides, Intraepithelial neoplasias including anal, cervical, ductal, oral, perianal, prostatic, penile, vaginal and vulvar intraepithelial neoplasia .

Unless otherwise indicated, all exact values provided herein are representative of corresponding approximate values, e.g . exact exemplary values provided with respect to a particular measurement can be considered to also provide a corresponding

approximate measurement, modified by "about" where appropriate.

All references, including publications, patent applications a nd patents, cited herein are hereby incorporated by reference in their entirety a nd to the same extent as if each reference were individually and specifically indicated to be incorporated by reference, rega rdless of any sepa rately provided incorporation of particular documents made elsewhere herein.

Embodiments of the invention

In one or more embodiments the invention provides a compound of general formula I as described above wherein Ri is phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from R₂.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein X represents a bond or -(CR_aR_b)_n-, wherein R_a and R_b each independently represents hydrogen or (Ci-C₄)a lkyl, and n is 1 or 2.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein X represents a bond or -(CR_aRb)_n-, wherein R_a and R_b each independently represents hydrogen or methyl, and n is 1 or 2.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein Ri is phenyl, and wherein said phenyl is optionally substituted with one or more substituents independently selected from methyl, methoxy,

trifluoromethoxy, trifluoromethyl, fluoro and chloro.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein X represents a bond a nd Y represents a bond . In one or more embodiments the invention provides a compound of general formula I as described above, selected from

3-(S) -[4 -(o-Tolyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S) -[4 -(1 -Methyl- l-phenoxy-ethyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S) -(4 -Phenethyltriazol-l-yl)-3-deoxy-ingenol,

3-(S) -(4 -(Phenoxymethyl)triazol-l-yl)-3-deoxy-ingenol,

3-(S) -(4 -Tert-butyltriazol-l-yl)-3-deoxy-ingenol,

3-(S) -[4 -(2-Methoxyphenyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S) -(4 -(3-Fluorophenyl)triazol-l-yl)-3-deoxy-ingenol,

3-(S) -[4 -[3-(Trifluoromethyl)phenyl]triazol-l-yl]-3-deoxy-ingenol,

3-(S) -(4 -Benzyltriazol-l-yl)-3-deoxy-ingenol,

3-(S) -(4 -Cyclohexyltriazol-l-yl)-3-deoxy-ingenol,

3-(S) -[4 -[4-(Trifluoromethoxy)phenyl]triazol-l-yl]-3-deoxy-ingenol,

3-(S) -(4 -Phenyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S) -[4 -[3-(Trifluoromethoxy)phenyl]triazol-l-yl]-3-deoxy-ingenol and

3-(S) -(4 -(4-Chlorophenyl)triazol-l-yl)-3-deoxy-ingenol

or pharmaceutically acceptable salts, hydrates or solvates thereof.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein Ri represents phenyl, butyl, cyclopentyl or cyclohexyl.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein R₂ represents methyl, methoxy, trifluoromethoxy,

trifluoromethyl, fluoro or chloro.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein X represents a bond.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein X represents -CH₂-, -(CH₂)2- or -C(CH₃)₂-.

In one or more embodiments the invention provides a compound of general formula I as described above, wherein Y represents a bond. In one or more embodiments the invention provides a compound of general formula I as described above, wherein Y represents -0-. Any combination of two or more embodiments described herein is considered within the scope of the present invention.

The compounds of formula I may be obtained in crystalline form either directly by concentration from an organic solvent or by crystallisation or recrystallisation from an organic solvent or mixture of said solvent and a cosolvent that may be organic or inorganic, such as water. The crystals may be isolated in essentially solvent-free form or as a solvate, such as a hydrate. The invention covers all crystalline forms, such as polymorphs and pseudopolymorphs, and also mixtures thereof.

Compounds of formula I may or may not comprise asymmetrically substituted (chiral) carbon atoms besides the ones present in the ingenol-core which give rise to the existence of isomeric forms, e.g. enantiomers and possibly diastereomers. The present invention relates to all such isomers, either in optically pure form or as mixtures thereof (e.g. racemic mixtures or partially purified optical mixtures). Pure stereoisomeric forms of the compounds and the intermediates of this invention may be obtained by the application of procedures known in the art. The various isomeric forms may be separated by physical separation methods such as selective crystallization and chromatographic techniques, e.g. high pressure liquid chromatography using chiral stationary phases. Enantiomers may be separated from each other by selective crystallization of their diastereomeric salts which may be formed with optically active acids. Optically purified compounds may subsequently be liberated from said purified diastereomeric salts.

Enantiomers may also be resolved by the formation of diastereomeric derivatives.

Alternatively, enantiomers may be separated by chromatographic techniques using chiral stationary phases. Pure stereoisomeric forms may also be derived from the

corresponding pure stereoisomeric forms of the appropriate starting materials, provided that the reaction occur stereoselectively or stereospecifically. Preferably, if a specific stereoisomer is desired, said compound will be synthesized by stereoselective or stereospecific methods of preparation. These methods will advantageously employ chiral pure starting materials.

Furthermore, when a double bond or a fully or partially saturated ring system is present in the molecule geometric isomers may be formed. It is intended that any geometric isomer, as separated, pure or partially purified geometric isomers or mixtures thereof are included within the scope of the invention.

In the compounds of general Formula I, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number found in nature. The present invention is meant to include all suitable isotopic va riations of the compounds of general Formula I. For example, different isotopic forms of hydrogen include ¹H, ²H and ³H and different isotopic forms of carbon include ¹²C, ¹³C and ¹⁴C. Enriching for deuterium (²H) may for example increase in-vivo half-life or reduce dosage regiments, or may provide a compound useful as a standard for characterization of biological samples. Isotopically enriched compounds within general formula I can be prepared by conventional techniques well known to a person skilled in the art or by processes analogous to those described in the general procedures and examples herein using appropriate isotopically enriched reagents and/or intermediates.

In one or more embodiments of the present invention, the compounds of formula l as defined above, optionally in combination with other active compounds, may be useful in therapy and in particular useful for treatment of cutaneous warts, genital warts, actinic keratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC), lentigo maligna, cervical intraepithelial neoplasia, anal intraepithelial neoplasia or vulva intraepithelial neoplasia .

Besides being useful for human treatment, the compounds of the present invention may also be useful for veterinary treatment of animals including mammals such as horses, cattle, sheep, pigs, dogs, and cats.

Pharmaceutical Compositions of the Invention For use in therapy, compounds of the present invention are typically in the form of a pha rmaceutical composition. The invention therefore relates to a pharmaceutical composition comprising a compound of formula I, optionally together with one or more other therapeutically active compound(s), together with a pharmaceutically acceptable excipient, vehicle or carrier(s) . The excipient must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

Conveniently, the active ingredient comprises from 0.0001-99.9% by weight of the formulation.

In the form of a dosage unit, the compound may be administered one or more times a day at appropriate intervals, always depending, however, on the condition of the patient, and in accordance with the prescription made by the medical practitioner. Conveniently, a dosage unit of a formulation contain between 0.001 mg and 1000 mg, preferably between 0.01 mg and 100 mg, such as 0.1-50 mg of a compound of formula I.

A suitable dosage of the compound of the invention will depend, inter alia, on the age and condition of the patient, the severity of the disease to be treated and other factors well known to the practising physician. The compound may be administered either , parenterally, topically, transdermally or interdermally + other routes according to different dosing schedules, e.g. daily, weekly or with monthly intervals. In general a single dose will be in the range from 0.001 to 1 mg/kg body weight. The compound may be administered as a bolus (i.e. the entire daily dosis is administered at once) or in divided doses two or more times a day.

In the context of topical treatment it may be more appropriate to refer to a "usage unit", which denotes a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active material as such or a mixture of it with solid, semisolid or liquid pharmaceutical diluents or carriers.

The term "usage unit" in connection with topical use means a unitary, i.e. a single dose, capable of being administered topically to a patient in an application per square centimetre of the treatment area of from 0.001 microgram to 1 mg and preferably from 0.05 microgram to 0.5 mg of the active ingredient in question.

It is also envisaged that in certain treatment regimes, administration with longer intervals, e.g. every other day, every week, or even with longer intervals may be beneficial.

If the treatment involves administration of another therapeutically active compound it is recommended to consult Goodman & Gilman's The Pharmacological Basis of

Therapeutics, 9^th Ed., J.G. Hardman and L.E. Limbird (Eds.), McGraw-Hill 1995, for useful dosages of said compounds.

The administration of a compound of the present invention with one or more other active compounds may be either concomitantly or sequentially. The formulations include e.g. those in a form suitable for parenteral, transdermal, intradermal or topical administration. The formulations may conveniently be presented in dosage unit form and may be prepared by but not restricted to any of the methods well known in the art of pharmacy, e.g. as disclosed in Remington, The Science and Practice of Pharmacy, 21ed ed., 2005. All methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier, semisolid carrier or a finely divided solid carrier or combinations of these, and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredients, which is preferably isotonic with the blood of the recipient, e.g. isotonic saline, isotonic glucose solution or buffer solution. Furthermore, the formulation may containe cosolvent, solubilising agent and/or complexation agents. The formulation may be conveniently sterilised by for instance filtration through a bacteria retaining filter, addition of sterilising agent to the

formulation, irradiation of the formulation or heating of the formulation. Liposomal formulations as disclosed in e.g. Encyclopedia of Pharmaceutical Technology, vol.9, 1994, are also suitable for parenteral administration.

Alternatively, the compounds of formula I may be presented as a sterile, solid preparation, e.g. a freeze-dried powder, which is readily dissolved in a sterile solvent immediately prior to use. Transdermal formulations may be in the form of a plaster, patch, microneedles, liposomal or nanoparticulate delivery systems or other cutaneous formulations applied to the skin.

Formulations suitable for topical, such as dermal, intradermal or ophthalmic admi- nistration include liquid or semi-solid preparations such as liniments, lotions, gels, applicants, sprays, foams, filmforming sytems, microneedles, micro- or nano-emulsions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops. For topical administration, the compound of formula I may typically be present in an amount of from 0.001 to 20% by weight of the composition, such as 0.01% to about 10 %, but may also be present in an amount of up to about 100% of the composition. In addition to the aforementioned ingredients, the formulations of a compound of formula I may include one or more additional ingredients such as diluents, buffers, flavouring agents, colourant, surface active agents, thickeners, penetration enhancing agents, solubility enhancing agents preservatives, e.g. methyl hydroxy benzoate (in- eluding anti-oxidants), emulsifying agents and the like.

When the active ingredient is administered in the form of salts with pharmaceutically acceptable non-toxic acids or bases, preferred salts are for instance easily water-soluble or slightly soluble in water, in order to obtain a particular and appropriate rate of absorption.

METHODS OF PREPARATION

The compounds of the present invention can be prepared in a number of ways well known to those skilled in the art of synthesis. The compounds of formula I may for example be prepared using the reactions and techniques outlined below together with methods known in the art of synthetic organic chemistry, or variations thereof as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reactions are carried out in solvents appropriate to the reagents and materials employed and suitable for the transformations being effected. Also, in the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of experiment and work-up procedures, are chosen to be conditions of standard for that reaction, which should be readily recognized by one skilled in the art. Not all compounds falling into a given class may be compatible with some of the reaction conditions required in some of the methods described. Such restrictions to the substituents which are compatible with the reaction conditions will be readily apparent to one skilled in the art and alternative methods can be used. The compounds of the present invention or any intermediate may be purified if required using standard methods well known to a synthetic organist chemist, e.g. methods described in "Purification of Laboratory Chemicals", 6^th ed. 2009, W. Amarego and C. Chai, Butterworth-Heinemann. Starting materials are either known compounds, commercially available, or they may be prepared by routine synthetic methods well known to a person skilled in the art.

Synthetic routes

Compounds of formula I may for example be prepared according to the following non- limiting general methods: cheme 1

The compounds of the general formula e can for example be synthesised according to Scheme 1

As depicted in scheme 1 the 3-azido-3-deoxy-ingenol derivative d may be transformed to 3-triazolo-3-deoxy-ingenol derivatives of the general formula e according to methods for 1,3-dipolar cycloaddition of substituted azides and substituted acetylenes described in the review, but not limited to "Cu-Catalyzed Azide-Alkyne Cycloaddition" by Morten Meldal, and Christian Wenzel Tornoe, Chem. Rev. 2008, 108, 2952-3015 and references cited therein.

Compound e can for example be synthesised by reacting compound d with an Acetylene derivative. The 1,3-dipolar cycloaddition can take place in a suitable solvent such as methanol or DMF, or a mix of solvents such as water/t-butanol. The reaction takes place in the presence of a copper source such as Cu(II)S0₄ or Cu(I)I, sometime the copper source is used in catalytic amount . Sodium ascorbate or tri(carboxyethyl)phosphine (TCEP) is often added as a reduction spices to generate/regenerate Cu(I) in the catalytic cyclus.

The 1,3-dipolar cycloaddition can take place without any additives such as base or a ligand.

The 1,3-dipolar cycloaddition can take place in the presence of a base such as, but not limited to, DIPEA, DBU or Na₂C0₃.

The 1,3-dipolar cycloaddition can take place in the presence of a ligand, an often used ligands for the copper catalysed reaction such as, but not limited to, TBTA (tri(l-benzyl- [ l,2,3]-triazol-4-ylmethyl)amine) or bathophenanthroline.

It may be necessary to use a protecting group in the above schemes. These protecting groups can in general be introduced and removed by standard procedures known to a chemist skilled in the art of organic synthesis (see e.g "Protective Groups in Organic Synthesis", 4^th ed., 2007, Greene T.W. and Wuts P.G.M ., John Wiley & Sons Inc.).

GENERAL PROCEDURES, PREPARATIONS AND EXAMPLES

*H nuclear magnetic resonance (NMR) spectra were recorded at 600 MHz unless otherwise specified. Chemical shift values (δ, in ppm) are quoted relative to internal tetramethylsilane (δ = 0.00) standards. The value of a multiplet, either defined doublet (d), triplet (t), quartet (q)) or not (m) at the approximate midpoint is given unless a range is quoted, (br) indicates a broad peak, whilst (s) indicates a singlet. The organic solvents used were usually anhydrous. Chromatography was performed on Merck silica gel 60 (0.040 - 0-063 mm). The solvent ratios indicated refer to v:v unless otherwise noted. All NMR spectra are recorded in DMSO-d₆ unless another solvent is stated.

The following abbreviations have been used throughout: The following abbreviations have been used throughout:

DCE dichloroethane

DCM dichloromethane

DBU l,8-Diazabicyclo[5.4.0]undec-7-ene

DMF N,N'-Dimethylformamide

DMSO dimethyl sulfoxide

Et ethyl

HATU (l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate)

h hour(s)

HPLC High pressure liquid chromatography

L litre

m milli

Me methyl

MS Mass spectrometry

MTBE Methyl tert- butyl ether

NMR nuclear magnetic resonance

rt room temperature

RT Retention Time

TFA trifluoroacetic acid

THF tetrahydrofuran

v volume Analytical UPLC/MS

Analytical UPLC/MS is performed on a Waters Acquity UPLC-system, Waters LCT Premier XE and SQD-MS.

Column : Waters Acquity HSS T3 1.8μιη, 2.1 x 50mm; Column temperature is 40°C. Solventsystem : A = lOmM Ammonium acetate in water + 0.1% HCOOH and B = acetonitrile + 0.1% HCOOH;

Flow rate = 0.7 mL/min; method (4.8min) :

Gradient: 0.0 99% A 1% B

0.5 94% A 6% B

1.0 94% A 6% B

2.6 5% A 95% B

3.8 5% A 95% B

3.81 99% A 1% B

4.8 99% A 1% B

MS - method : PosNeg_50_1000

UV: 240-400nm

Preparations and Examples

Preparation 1

5,20-Acetonide-3-O-methanesulfonyl-inqenol (Intermediate 1)

Ingenol-5,20-acetonide ( 10000 mg, 25.74 mmol) was dissolved in 200 mL DCM.

Triethylamine was added (3630 mg, 35.9 mmol) and the reaction was stirred under argon. The clear solution was cooled to 0°C. Methanesulfonyl chloride (3833 mg, 33.46 mmol, 2.59 mL) was added slowly. Internal temperature was kept under 5°C.

The reaction was monitored by TLC (eluent heptane/EtOAc 2: 1). After 30 min. TLC showed that all starting material was consumed. The reaction mixture was poured into a separation funnel with 500 ml MTBE and 500ml saturated NaHC0₃ aq. The organic layer was washed with 500ml (water/aq. sat. NaHC0₃ 1 : 1) and then with 500ml brine. The organic layer was dried over sodium sulphate, and concentrated in vacuo.

1H NMR (600 MHz, Chloroform-d) δ 6.10 (q, J = 1.5 Hz, 1H), 5.82 (dq, J = 1.7, 3.4 Hz, 1H), 5.26 (s, 1H), 4.22 (ddt, J = 1.5, 3.0, 14.1 Hz, 1H), 4.14 (ddq, J = 1.6, 4.0, 12.8 Hz, 2H), 3.93 (dp, J = 1.2, 2.1 Hz, 1H), 3.47 (s, 1H), 3.11 (s, 3H), 2.60 - 2.48 (m, 1H), 2.26 (ddd, J = 3.1, 8.5, 15.8 Hz, 1H), 1.85 (d, J = 1.6 Hz, 3H), 1.80 (dt, J = 6.0, 15.9 Hz, 1H), 1.44 (s, 3H), 1.41 (s, 3H), 1.12 (s, 3H), 1.06 (s, 3H), 0.96 (d, J = 7.1 Hz, 3H), 0.93 (dd, J = 8.4, 11.8 Hz, 1H), 0.71 (td, J = 6.2, 8.4 Hz, 1H).

Preparation 2

5,20-Acetonide-3-(S)-azido-3-deoxy-inqenol (Intermediate 2)

Crude intermediate 1 (25.74mmol, 12010 mg) was dissolved in 100 ml DMF. Sodium azide (7000 mg, 108 mmol) was added and the suspension was stirred at 60°C. The reaction was kept for 16 h. at 5°C. The reaction mixture was cooled on an ice bath and water was added slowly. Precipitation was formed. The precipitate was filtered; the solid was washed with water and dried. The solid was dissolved in MTBE and the solution was extracted with brine. The organic phases were dried over sodium sulfate and

concentrated in vacuo. The oil was dissolved in 10 ml of MTBE and 110 ml heptane was added. Crystallization started and the mixture was kept at 5°C for 16h. The product was filtered to afford the title compound , (5.179g, 48% over two steps)

1H NMR (600 MHz, Chloroform-d) δ 5.93 (q, J = 1.6 Hz, 1H), 5.81 (dq, J = 1.5, 3.4 Hz, 1H), 4.23 - 4.11 (m, 4H), 3.94 (t, J = 1.6 Hz, 1H), 3.31 (s, 1H), 2.54 (dtd, J = 3.0, 6.0, 6.8, 12.2 Hz, 1H), 2.26 (ddd, J = 3.1, 8.8, 15.8 Hz, 1H), 1.83 (d, J = 1.6 Hz, 3H), 1.82 - 1.76 (m, 1H), 1.44 (s, 3H), 1.39 (s, 3H), 1.11 (s, 3H), 1.05 (s, 3H), 0.96 (d, J = 7.1 Hz, 3H), 0.91 (dd, J = 8.4, 11.8 Hz, 1H), 0.70 (td, J = 6.3, 8.6 Hz, 1H).

Preparation 3

3-(S)-azido-3-deoxy-inqenol (Intermediate 3)

Intermediate 2 (1690 mg, 4.087 mmol) dissolved in MeOH (30 mL) was added 4N HCI in water (1.2 mmol, 0.3 mL). The solution was stirred at room temperature for 4 h. To this solution was added a saturated aqueous solution of NaHC0₃ (5 mL) . The obtained mixture was concentrated in vacuo. The residue was taken up in brine and extracted with MTBE. The combined organic phases were dried over sodium sulfate and

concentrated in vacuo. The oil was taken up in 5 ml of acetonitrile to afford a precipitate which was isolated by filtration (1172 mg, 2.982 mmol, 72.96% Yield)

1H NMR (600 MHz, Chloroform-d) δ 6.09 - 6.04 (m, 1H), 5.98 (q, J = 1.6 Hz, 1H), 4.49 - 4.46 (m, 1H), 4.24 - 4.08 (m, 3H), 3.90 - 3.78 (m, 2H), 3.72 (d, J = 1.1 Hz, 1H), 2.39 (qdd, J = 3.2, 4.3, 7.2 Hz, 1H), 2.33 - 2.21 (m, 2H), 1.89 (d, J = 1.6 Hz, 3H), 1.79 (ddd, J = 4.4, 6.4, 15.8 Hz, 1H), 1.10 (s, 3H), 1.06 (s, 3H), 0.98 (d, J = 7.2 Hz, 3H), 0.92 (dd, J = 8.3, 11.9 Hz, 1H), 0.70 (ddd, J = 6.3, 8.3, 9.5 Hz, 1H).

General procedure 1 ; triazole formation

Copper(II) sulfate pentahydrate (0.5 eq., ca. 0.07 mmol), Sodium ascorbic acid (1.5 eq., ca. 0.2mmol) and intermediate 3 (1 eq., ca. 50 mg, 0.13 mmol) was mixed in a 4 ml vial. Methanol (1 mL) and 2-substituted acetylene (2 eq., ca. 0.27 mmol) were added at room temperature. The reaction was typically stirred at 65°C for 0.5-4h. The reaction mixture was taken up in water and extracted with EtOAc. The organic phases were dried over sodium sulfate and concentrated in vacuo. The residue was purified via flash chromatography (silica) : the crude material was taken up in a minimum volume of DCM, and added to the column and eluted with EtOAc/heptane. The eluent was typically 10% EtOAc in heptane -100% EtOAc

Example 1

3-(SM4-(o-Tolvntnazol-l-yl1-3-deoxy-inqenol (compound 1)

The title compound was prepared according to general procedure 1 using

l-ethynyl-2-methyl-benzene as starting material.

1H NMR (300 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.83 (dd, J = 2.0, 7.3 Hz, 1H), 7.39 - 7.19 (m, 3H), 6.08 (d, J = 1.8 Hz, 1H), 5.99 - 5.89 (m, 1H), 5.83 (d, J = 8.3 Hz, 1H), 5.43 (d, J = 1.6 Hz, 1H), 5.25 (s, 1H), 4.79 (t, J = 5.5 Hz, 1H), 4.23 (dd, J = 4.8, 12.1 Hz, 1H), 4.03 - 3.97 (m, 2H), 3.79 (d, J = 8.2 Hz, 1H), 2.64 - 2.52 (m, 1H), 2.46 (s, 3H), 2.06 (dd, J = 9.2, 14.7 Hz, 1H), 1.75 (s, 3H), 1.59 (dt, J = 5.6, 15.6 Hz, 1H), 1.06 - 0.92 (m, 9H), 0.88 - 0.74 (m, 1H), 0.70 - 0.50 (m, 1H).

Example 2

3-(SM4-(l -Methyl- l-phenoxy-ethv0triazol-l-yl^"l-3-deoxy-inqenol (compound 2)

The title compound was prepared according to general procedure 1.

Starting material : l,l-dimethylprop-2-ynoxybenzene.

1H NMR (600 MHz, DMSO-d6) δ 7.72 (s, 1H), 7.16 - 7.07 (m, 2H), 6.98 - 6.91 (m, 1H), 6.68 - 6.55 (m, 2H), 6.07 - 5.99 (m, 1H), 5.92 (dt, J = 1.5, 4.9 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.31 (s, 1H), 5.14 (s, 1H), 4.79 (t, J = 5.6 Hz, 1H), 4.29 - 4.17 (m, 1H), 3.97 (d, J = 5.3 Hz, 2H), 3.72 (d, J = 8.4 Hz, 1H), 2.46 (tq, J = 3.6, 10.4 Hz, 1H), 2.07 (ddd, J = 3.1, 9.1, 15.6 Hz, 1H), 1.71 (s, 3H), 1.70 (s, 3H), 1.67 (d, J = 1.8 Hz, 3H), 1.58 - 1.49 (m, 1H), 1.02 (s, 6H), 0.91 (d, J = 7.2 Hz, 3H), 0.80 (dd, J = 8.3, 11.9 Hz, 1H), 0.59 (td, J = 6.3, 8.7 Hz, 1H).

Example 3

3-(SM4-Phenethyltriazol-l-vO-3-deoxy-inqenol (compound 3)

The title compound was prepared according to general procedure 1.

Starting material : but-3-ynylbenzene.

1H NMR (600 MHz, DMSO-d6) δ 7.49 (s, 1H), 7.26 (t, J = 7.5 Hz, 2H), 7.23 - 7.20 (m, 2H), 7.20 - 7.14 (m, 1H), 6.04 - 5.98 (m, 1H), 5.95 - 5.88 (m, 1H), 5.80 (d, J = 8.4 Hz, 1H), 5.29 (s, 1H), 4.99 (s, 1H), 4.80 (t, J = 5.6 Hz, 1H), 4.24 - 4.18 (m, 1H), 3.97 (d, J = 5.2 Hz, 2H), 3.70 (d, J = 8.3 Hz, 1H), 2.94 (s, 4H), 2.49 - 2.40 (m, 1H), 2.03 (ddd, J = 3.1, 9.0, 15.6 Hz, 1H), 1.64 (d, J = 1.7 Hz, 3H), 1.56 (dq, J = 5.5, 16.5 Hz, 1H), 1.01 (s, 3H), 0.99 (s, 3H), 0.93 (d, J = 7.1 Hz, 3H), 0.80 (dd, J = 8.4, 11.9 Hz, 1H), 0.59 (td, J = 6.3, 8.7 Hz, 1H).

Example 4

3-(SM4-(Phenoxymethv0triazol-l-yl^')-3-deoxy-inqenol (compound 4)

The title compound was prepared according to general procedure 1.

Starting material : prop-2-ynoxybenzene.

1H NMR (600 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.39 - 7.25 (m, 2H), 7.09 - 7.03 (m, 2H), 6.96 (tt, J = 1.2, 7.6 Hz, 1H), 6.06 (s, 1H), 5.93 (dq, J = 1.6, 4.9 Hz, 1H), 5.82 (d, J = 8.5 Hz, 1H), 5.39 (s, 1H), 5.17 (s, 1H), 5.15 (d, J = 1.4 Hz, 2H), 4.80 (s, 1H), 4.28 - 4.15 (m, 1H), 3.98 (s, 2H), 3.74 (d, J = 8.1 Hz, 1H), 2.47 (ddq, J = 3.5, 4.1, 7.2, 11.0 Hz, 1H), 2.03 (ddd, J = 3.1, 9.0, 15.5 Hz, 1H), 1.68 (s, 3H), 1.56 (dt, 1H), 1.01 (s, 3H), 0.99 (s, 3H), 0.94 (d, J = 7.1 Hz, 3H), 0.80 (dd, J = 8.4, 11.9 Hz, 1H), 0.59 (td, J = 6.4, 8.8 Hz, 1H).

Example 5

3-(SM4-Tert-butyltriazol-l-vO-3-deoxy-inqenol (compound 5)

The title compound was prepared according to general procedure 1.

Starting material : 3,3-dimethylbut-l-yne.

1H NMR (300 MHz, DMSO-d6) δ 7.50 (s, 1H), 6.05 - 5.96 (m, 1H), 5.92 (dd, J = 1.4, 5.0 Hz, 1H), 5.78 (d, J = 8.1 Hz, 1H), 5.32 - 5.21 (m, 1H), 5.00 (s, 1H), 4.78 (t, J = 5.5 Hz, 1H), 4.32 - 4.16 (m, 1H), 3.98 (d, J = 5.5 Hz, 2H), 3.70 (d, J = 8.1 Hz, 1H), 2.68 - 2.52 (m, 1H), 2.04 (ddd, J = 2.9, 8.8, 15.8 Hz, 1H), 1.65 (s, 1H), 1.64 - 1.51 (m, 1H), 1.29 (s, 9H), 1.07 - 0.98 (m, 6H), 0.95 (d, J = 7.0 Hz, 3H), 0.87 - 0.76 (m, 1H), 0.59 (td, J = 6.1, 8.3 Hz, 1H).

Example 6

3-(S^'H4-(2-Methoxyphenyl^')triazol-l-yl^"l-3-deoxy-inqenol (compound 6)

The title compound was prepared according to general procedure 1.

Starting material : l-ethynyl-2-methoxy-benzene.

1H NMR (600 MHz, Chloroform-d) δ 8.23 (dd, J = 1.7, 7.7 Hz, 1H), 7.99 (s, 1H), 7.32

(ddd, J = 1.8, 7.4, 8.3 Hz, 1H), 7.09 (td, J = 1.0, 7.5 Hz, 1H), 6.98 (dd, J = 1.0, 8.4 Hz, 1H), 6.33 - 6.26 (m, 1H), 6.17 - 6.03 (m, 2H), 5.56 (s, 1H), 4.69 (s, 1H), 4.50 (d, J = 12.2 Hz, 1H), 4.30 - 4.24 (m, 1H), 4.19 - 4.13 (m, 1H), 4.11 - 4.06 (m, 1H), 3.95 (d, J = 2.1 Hz, 1H), 3.90 (s, 3H), 2.52 (qt, J = 3.5, 7.2 Hz, 1H), 2.07 (ddd, J = 3.8, 10.6, 17.0 Hz, 1H), 1.78 (s, 3H), 1.62 (ddd, J = 3.9, 6.2, 16.0 Hz, 1H), 1.33 - 1.26 (m, 1H), 1.09 (d, J = 7.3 Hz, 3H), 0.92 (s, 3H), 0.90 - 0.85 (m, 1H), 0.82 (s, 3H), 0.62 (ddd, J = 6.2, 8.2, 10.2 Hz, 1H).

Example 7

3-(S^')-(4-(3-Fluorophenyl^')tnazol-l-yl^')-3-deoxy-inqenol (compound 7)

The title compound was prepared according to general procedure 1.

Starting material : l-ethynyl-3-fluoro-benzene.

1H NMR (300 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.82 - 7.65 (m, 2H), 7.50 (td, J = 6.1, 8.0 Hz, 1H), 7.16 (td, J = 2.7, 8.4 Hz, 1H), 6.08 (s, 1H), 5.92 (d, J = 4.8 Hz, 1H), 5.84 (d, J = 8.3 Hz, 1H), 5.39 (s, 1H), 5.19 (s, 1H), 4.86 - 4.74 (m, 1H), 4.28 - 4.15 (m, 1H), 3.99 (d, J = 5.5 Hz, 2H), 3.75 (d, J = 8.3 Hz, 1H), 2.12 - 1.92 (m, 1H), 1.74 (d, J = 1.3 Hz, 3H), 1.65 - 1.50 (m, 1H), 1.08 - 0.89 (m, 8H), 0.88 - 0.75 (m, 1H), 0.59 (q, J = 8.0 Hz, 1H).

Example 8

3-(S^')-[4-(4-Methoxyphenyl^')triazol-l-yl1-3-deoxy-inqenol (compound 8)

The title compound was prepared according to general procedure 1.

Starting material : l-ethynyl-4-methoxy-benzene.

1H NMR (300 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.80 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.06 (s, 1H), 5.92 (d, J = 4.7 Hz, 1H), 5.81 (d, J = 8.4 Hz, 1H), 5.36 (s, 1H), 5.16 (s, 1H), 4.79 (t, J = 5.5 Hz, 1H), 4.33 - 4.12 (m, 1H), 3.99 (d, J = 5.5 Hz, 2H), 3.79 (s, 3H), 3.75 (d, J = 8.3 Hz, 1H), 2.14 - 1.93 (m, 1H), 1.73 (s, 3H), 1.65 - 1.51 (m, 1H), 1.08 - 0.91 (m, 9H), 0.86 - 0.74 (m, 1H), 0.68 - 0.51 (m, 1H).

Example 9

3-(SM4-r3-(Trifluoromethv0phenvntriazol-l-yl^"l-3-deoxy-inqenol (compound 9)

The title compound was prepared according to general procedure 1.

Starting material : l-ethynyl-3-(trifluoromethyl)benzene.

1H NMR (300 MHz, Chloroform-d) δ 8.00 (s, 1H), 7.94 (d, J = 1.8, 7.3 Hz, 1H), 7.79 (s, 1H), 7.60 - 7.43 (m, 2H), 6.25 (s, 1H), 6.12 (d, J = 4.4 Hz, 1H), 5.79 (d, J = 3.6 Hz, 1H), 5.53 (s, 1H), 4.52 - 4.02 (m, 6H), 2.59 - 2.38 (m, 1H), 2.14 - 1.95 (m, 1H), 1.79 (d, J = 1.5 Hz, 3H), 1.62 (dt, J = 5.6, 16.1 Hz, 1H), 1.06 (d, J = 7.0 Hz, 3H), 0.93 (s, 3H), 0.91 - 0.84 (m, 1H), 0.82 (s, 3H), 0.63 (td, J = 6.3, 8.8 Hz, 1H) .

Example 10

3-(SM4-Benzyltriazol-l-vO-3-deoxy-inqenol (compound 10)

The title compound was prepared according to general procedure 1.

Starting material : prop-2-ynylbenzene.

1H NMR (600 MHz, DMSO-d6) δ 7.57 (s, 1H), 7.33 - 7.24 (m, 4H), 7.23 - 7.17 (m, 1H), 6.02 (s, 1H), 5.93 - 5.89 (m, 1H), 5.77 (d, J = 8.4 Hz, 1H), 5.31 (s, 1H), 5.08 (s, 1H), 4.78 (t, J = 5.6 Hz, 1H), 4.23 - 4.16 (m, 1H), 4.03 - 3.99 (m, 2H), 3.98 - 3.93 (m, 2H), 3.70 (d, J = 8.4 Hz, 1H), 2.50 - 2.43 (m, 1H), 2.03 (ddd, J = 3.2, 9.1, 15.6 Hz,

1H), 1.66 (s, 3H), 1.56 (dt, J = 5.7, 15.6 Hz, 1H), 1.01 (s, 3H), 0.98 (s, 3H), 0.92 (dd, J = 3.3, 7.3 Hz, 3H), 0.79 (dd, J = 8.4, 11.9 Hz, 1H), 0.58 (td, J = 6.4, 8.8 Hz, 1H).

Example 11

3-(S)-(4-Cyclohexyltriazol-l-yl)-3-deoxy-ingenol (compound 11)

The title compound was prepared according to general procedure 1.

Starting material : Cyclohexylacetylene.

1H NMR (600 MHz, DMSO-d6) δ 7.50 (s, 1H), 5.99 (d, J = 1.7 Hz, 1H), 5.90 (dq, J = 1.5, 4.9 Hz, 1H), 5.78 (d, J = 8.3 Hz, 1H), 5.26 (s, 1H), 5.02 (s, 1H), 4.79 (t, J = 5.6 Hz, 1H), 4.22 (d, J = 12.0 Hz, 1H), 3.96 (d, J = 5.4 Hz, 2H), 3.69 (d, J = 8.3 Hz, 1H), 2.64 (dt, J = 5.0, 10.9 Hz, 1H), 2.07 - 1.92 (m, 3H), 1.74 (dd, J = 4.8, 9.0 Hz, 2H), 1.70 - 1.63 (m, 4H), 1.57 (dt, J = 6.0, 15.7 Hz, 1H), 1.43 - 1.31 (m, 4H), 1.24 (s, 2H), 1.00 (d, J = 10.8 Hz, 6H), 0.94 (d, J = 7.0 Hz, 3H), 0.80 (dd, J = 8.4, 11.8 Hz, 1H), 0.59 (td, J = 6.3, 8.5 Hz, 1H).

Example 12

3-(S)-[4-[4-(Trifluoromethoxy)phenyl]triazol-l-yl]-3-deoxy-ingenol (compound 12) The title compound was prepared according to general procedure 1.

Starting material : 4-(trifluoromethoxy)phenylacetylene.

LCMS retention time 2.58 min. M + l = 560

Example 13

3-(S)-(4-Cvclopentyltriazol-l-yl)-3-deoxy-inqenol (compound 13)

The title compound was prepared according to general procedure 1.

Starting material : Cyclopentylacetylene.

LCMS retention time 2.39 min. M + l= 468

Example 14

3-(S)-(4-Phenyl)triazol-l-yl^"l-3-deoxy-inqenol (compound 14)

The title compound was prepared according to general procedure 1.

Starting material : Phenylacetylene

1H NMR (400 MHz, Chloroform-d) δ 7.79 - 7.72 (m, 2H), 7.64 (s, 1H), 7.41 - 7.33 (m, 2H), 7.32 - 7.25 (m, 1H), 6.20 (s, 1H), 6.09 - 6.02 (m, 1H), 5.47 (s, 1H), 4.39 - 4.01 (m, 4H), 2.44 - 2.28 (m, 1H), 2.05 - 1.89 (m, 1H), 1.77 - 1.70 (m, 3H), 1.61 - 1.49 (m, 1H), 0.99 (d, J = 7.1 Hz, 3H), 0.88 (s, 3H), 0.85 - 0.76 (m, 4H), 0.61 - 0.51 (m, 1H).

Example 15

3-(S^')-[4-[3-(Trifluoromethoxy^')phenylltriazol-l-yl1-3-deoxy-inqenol (compound 15) The title compound was prepared according to general procedure 1.

Starting material : 3-(trifluoromethoxy)phenylacetylene

1H NMR (500 MHz, Chloroform-d) δ 7.71 - 7.64 (m, 2H), 7.63 - 7.59 (m, 1H), 7.41 - 7.34 (m, 1H), 7.15 - 7.09 (m, 1H), 6.19 (s, 1H), 6.05 (d, J = 4.7 Hz, 1H), 5.46 (d, J = 1.2 Hz, 1H), 5.40 (s, 1H), 4.30 (d, J = 12.1 Hz, 1H), 4.16 (d, J = 12.2 Hz, 1H), 4.12 - 4.04 (m, 2H), 3.85 (s, 1H), 2.47 - 2.36 (m, 1H), 2.22 - 1.84 (m, 1H), 1.74 (d, J = Hz, 3H), 1.59 - 1.51 (m, 1H), 0.99 (d, J = 7.1 Hz, 3H), 0.88 (s, 3H), 0.84 - 0.80 (i 1H), 0.79 (s, 4H), 0.63 - 0.52 (m, 1H).

Example 16

3-(S)-(4-(4-Chlorophenyl)triazol-l-yl)-3-deoxy-inqenol (compound 16)

The title compound was prepared according to general procedure 1.

Starting material : (4-chlorphenyl)acetylene

1H NMR (500 MHz, Chloroform-d) δ 7.72 - 7.66 (m, 2H), 7.64 (s, 1H), 7.37 - 7.30 (m, 2H), 6.18 (s, 1H), 6.04 (d, J = 4.6 Hz, 1H), 5.45 (s, 1H), 5.20 (s, 1H), 4.28 (d, J = 12.0 Hz, 1H), 4.15 (d, J = 12.1 Hz, 1H), 4.12 - 4.07 (m, 2H), 3.80 (s, 1H), 2.48 - 2.38 (m, 1H), 2.03 - 1.96 (m, 1H), 1.74 (d, J = 1.5 Hz, 3H), 1.62 - 1.55 (m, 1H), 1.53 (s, 1H), 0.99 (d, J = 7.1 Hz, 3H), 0.92 (s, 3H), 0.86 - 0.80 (m, 4H), 0.63 - 0.55 (m, 1H).

Example A

Neutrophil oxidative burst:

PMN's (polymorphonuclear leukocytes) were isolated and purified from fresh buffy coats by sequential sedimentation, density centrifugation and lysis of contaminating erythrocytes. Buffy coats were incubated with 2% methocel for 30-45 min to

differentially sediment red blood cells. The leukocyte-rich supernatant was transferred to lymphoprep tubes to remove mononuclear cells by density centrifugation (400xg, 30 min). The pellet was resuspended and any remaining erythrocytes lysed using 0.2% NaCI for 30 sec before restoring isotonicity by the addition of 1.2% NaCI. This step was repeated until the cell pellet appears relatively free of red blood cells. Cells were resuspended in DPBS (Dulbecco's Phosphate Buffered Saline) (w.o. Ca²⁺, Mg²⁺) and the concentration adjusted to 1.4xl0⁶ cells/ml in HBSS (Hanks Balanced Salt solution) (w Ca²⁺, Mg²⁺) containing 0.1% BSA (Bovine Serum Albumin) and 5mM glucose just prior to assay initiation. Titrated reference and test compounds were pre-mixed with HE

(Hydroethidine) (10μΜ final assay concentration) before addition to 96-well plates containing 2.5xl0⁵ cells. Following 40 min incubation at RT, changes in the respiratory burst was estimated by measuring fluorescence at 579 nm (excitation : 485 nm) using an Envision plate reader.

Test compound titration curves were fitted to a four- para meter sigmoidal curve after normalizing the effect of the test compound to the effect of the positive control (5xl0^~7 M PEP0005). Rel EC₅₀ denotes the concentration of test compound producing an effect that is midway between the fitted top and bottom. Abs EC₅₀ is the concentration of test compound that provokes a response corresponding to 50% of the maximal effect associated with the positive control (5xl0^~7 M PEP0005).

Example B

HeKa cytokine release (IL-8) :

Primary human epidermal keratinocytes, HeKa, were seeded (10.000 cells/well) in 96- well plates the day before the assay. Test compounds were diluted in DMSO (dimethyl sulfoxide) and further diluted in assay medium and pipetted into wells of 96 well-plates containing HeKa cells. The plates were incubated for 6h at 37°C in humidified air with 5% C0₂. Plates were centrifuged briefly to spin down cells at 4°C, the supernatant was removed and analysed by Meso Scale Discovery (MSD) 4-spot cytokine assay (Proinflammatory II Ultra Sensitive kit, MSD, MD, USA). The MSD assay employs a sandwich immunoassay format where capture antibodies are coated in a patterned array on the bottom of the wells of a 4-Spot- Multi-MSD plate. Standard samples were incubated in the MULTI-SPOT plates as well, and the cytokine (IL-8) binds to its corresponding capture antibody spot. The cytokine level was quantitated on a SECTOR™ Imager using a cytokine-specific Detection Antibody labelled with MSD SULFO-TAGTM reagent.

Test compound titration curves were fitted to a four- para meter sigmoidal curve after normalizing the effect of the test compound to the effect of the positive control (1.5xl0^~7 M PEP0005). Rel EC₅₀ denotes the concentration of test compound producing an effect that is midway between the fitted top and bottom. Abs EC₅₀ is the concentration of test compound that provokes a response corresponding to 50% of the maximal effect associated with the positive control (1.5xl0^~7 M PEP0005).

Example C

Necrosis Assay

HeLa cells (ATCC CCL-002) were grown in minimal essential medium (Invitrogen catalog no. 42360) containing 10% fetal bovine serum, lOOIU/ml penicillin and 100μg/ml streptomycin. 4,000-6,000 cells were seeded into 96-well black ViewPlates-plates, clear bottom, (Perkin Elmer) in ΙΟΟμΙ medium and incubated overnight. Compounds were dissolved and pre-diluted in DMSO in 96-well polypropylene plates (Greiner) in a concentration range of 15μΜ to 600μΜ. At the time of the experiment cell plates were placed on heating blocks at 37°C, medium was removed and 40μΙ fresh, pre-warmed medium was added per well. Cells were incubated for 15 min before addition of compounds. In parallel, 3μΙ of compounds were diluted with 197μΙ growth medium on a Tecan freedom-EVO pipetting station using 250μΙ/ε pipetting speed, in order to ensure effective mixing of the highly concentrated compound solutions with the aqueous phase. These pre-dilution plates were then equilibrated on heating blocks at 37°C for lOmin. 80μΙ pre-diluted compound were transferred manually to the corresponding wells containing HeLa cells yielding compound concentrations of ΙΟμΜ to 400μΜ. Control conditions were 1% DMSO in growth medium (100% viability) and 400μΜ ingenol mebutate in growth medium (0% viability). Plates were incubated on the heating blocks at 37°C for 30min. At the end of the incubation 10μΙ PrestoBlue reagent (Invitrogen) were added to each well, plates were sealed with black seal, followed by incubation at 37°C for lOmin with gentle shaking (150rpm). Subsequently, plates were placed at room temperature for 20-30min. Plates were read immediately after on an Envision

Fluorescence reader (Perkin Elmer) with excitation at 535nm and emission at 630nm. Test compound titration curves were fitted to a four- para meter sigmoidal curve after normalizing the effect of the test compound to the effect of the positive control (4 10^"4 M PEP0005/ingenol mebutate). AbsEC₅₀ denotes the concentration of test compound producing 50% effect.

Compounds of the present invention were tested in the neutrophil oxidative burst assay according to the description in example A, in the HeKa cytokine release assay according to the description in example B and in the necrosis assay according to the description in example C.

Example D

Chemical Stability assay at room temperature, buffer pH 7.4

A stock solution was prepared by diluting 50 ul of a ~ 10 mM DMSO solution of the compound with 1.15 ml acetonitrile (Analytical grade). To 0.75 ml stock solution 2.25 ml

Phosphatebuffer (0.067 M) pH 7.4 was added. After filtering (Millipore filter: Millex-LCR

(SLCR013NL)) the solution was placed in an HPLC autosampler (room temperature). The solution was repeatedly injected over a period of 16 hours.

HPLC system :

Stationary Phase: Chromolith Performance RP18(4.6 x 100mm, 2 μιη)

Mobile Phase: A: 25mM Phosphatebuffer B: Acetonitrile Based on the decrease of area of the compound signal (UV detection, suitable wavelength) the recovery of the compound over time was assessed.

Data analysis:

Detection : UV: 235 nm

The absolute area under the curve at t=0 hours equals to 100% recovery.

Calculation of the single recovery values at the measured timepoints:

Recovery [%] at t_x: [Area under the curve (t_x) / Area under the curve (t₀) ] *100

Results are shown in the table below.

Compound no. Neutrophil HeKa cytokine HeLa necrosis Chemical oxidative burst release (IL-8) Abs ECso (μΜ) stability buffer

Abs ECso (nM) Abs ECso (nM) pH 7.4 (%)

1 10 8 78 >95

2 16 36 126 n.a.

3 8 15 140 >95

4 19 22 209 >95

5 27 36 >400 >95

6 9 4 78 >95

7 n.a. 8 n.a. n.a.

8 n.a. 35 n.a. n.a.

9 14 4 49 >95

10 29 17 157 n.a.

11 n.a. 31 n.a. n.a.

12 n.a. 11 n.a. n.a.

13 n.a. 35 n.a. n.a.

14 n.a. 17 n.a. n.a.

15 49 6 n.a. n.a.

16 53 10 n.a. n.a.

Claims

1. A compound according to general formula (I)

I wherein X represents a bond or -(CR_aR )_n-, wherein R_a and R_b each independently represents hydrogen, (Ci-C₄)alkyl or R_a and R_b together with the carbon atom to which they are attached form a (C₃-C₆)cycloalkyl, and n is 1 or 2; wherein Y represents a bond or -0-; wherein Ri is selected from the group consisting of phenyl, (C₄-C₆)cycloalkyl and (C₄- C₆)alkyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from R₂; wherein R₂ consists of halogen, (C₃-C₆)cycloalkyl, (d-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci- C₆)alkoxy and halo(Ci-C₆)alkoxy; or pharmaceutically acceptable salts, hydrates or solvates thereof.

2. The compound according to claim 1 wherein Ri is phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from R₂.

3. The compound according to any one of claims 1-2, wherein X represents a bond or - (CR_aR_b)_n-, wherein R_a and R_b each independently represents hydrogen or (Ci-C₄)alkyl, and n is 1 or 2.

4. The compound according to any one of claims 1-3 wherein X represents a bond or - (CR_aR )_n-, wherein R_a and R each independently represents hydrogen or methyl, and n is 1 or 2.

5. The compound according to any one of claims 1-4 wherein Ri is phenyl, and wherein said phenyl is optionally substituted with one or more substituents independently selected from methyl, methoxy, trifluoromethoxy, trifluoromethyl, fluoro and chloro.

6. The compound according to any one of claims 1-5 wherein X represents a bond and Y represents a bond.

7. A compound according to any one of claims 1-6 selected from

3-(S)-[4-(o-Tolyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S)-[4-(l -Methyl- l-phenoxy-ethyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S)-(4-Phenethyltriazol-l-yl)-3-deoxy-ingenol,

3-(S)-(4-(Phenoxymethyl)triazol-l-yl)-3-deoxy-ingenol,

3-(S)-(4-Tert-butyltriazol-l-yl)-3-deoxy-ingenol,

3-(S)-[4-(2-Methoxyphenyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S)-(4-(3-Fluorophenyl)triazol-l-yl)-3-deoxy-ingenol,

3-(S)-[4-[3-(Trifluoromethyl)phenyl]triazol-l-yl]-3-deoxy-ingenol,

3-(S)-(4-Benzyltriazol-l-yl)-3-deoxy-ingenol,

3-(S)-(4-Cyclohexyltriazol-l-yl)-3-deoxy-ingenol,

3-(S)-[4-[4-(Trifluoromethoxy)phenyl]triazol-l-yl]-3-deoxy-ingenol,

3-(S)-(4-Phenyl)triazol-l-yl]-3-deoxy-ingenol,

3-(S)-[4-[3-(Trifluoromethoxy)phenyl]triazol-l-yl]-3-deoxy-ingenol and

3-(S)-(4-(4-Chlorophenyl)triazol-l-yl)-3-deoxy-ingenol or pharmaceutically acceptable salts, hydrates or solvates thereof.

8. A compound according to any one of claims 1-7 for use as a medicament.

9. A compound according to any one of claims 1-7 for use in treatment of physiological disorders or diseases associated with hyperplasia or neoplasia.

10. A compound according to claim 9 wherein the disorder or disease is selected from cutaneous warts, genital warts, actinic keratosis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC), lentigo maligna, cervical intraepithelial neoplasia, anal intraepithelial neoplasia or vulva intraepithelial neoplasia.

11. A pharmaceutical composition comprising a compound according to any one of claims 1-7 together with a pharmaceutically acceptable vehicle or excipient or pharmaceutically acceptable carrier(s).

12. The pharmaceutical composition according to claim 11 together with one or more other therapeutically active compound(s).

13. A compound selected from

5,20-Acetonide-3-O-methanesulfonyl-ingenol,

5,20-Acetonide-3-(S)-azido-3-deoxy-ingenol and

3-(S)-azido-3-deoxy-ingenol