WO2007053915A2 - Naringenin derivatives with selectivity on ers - Google Patents

Abstract

The present invention relates to naringenin derivatives with selective anti- estrogenic activity and their use in the treatment of diseases or disorders estrogen- deficiency dependent disorders, diseases with excessive estrogen production or conditions benefiting from modulated estrogen production.

Description

Naringenin derivatives with selectivity on ERs

FIELD OF THE INVENTION

The present invention relates to novel compounds that have biological activity with respect to estrogen receptors and to the use of such compounds to treat diseases or disorders related to estrogen activity.

BACKGROUND

17β-Estradiol is a steroidal sex hormone and the main estrogen produced by the gonads. Failure to produce estrogen has profound physiological consequences in males and females, such as decreased bone development or density, increased atherosclerotic deposits, and impaired fertility. However, estrogen also has undesirable effects. Supplementation of estrogen in menopausal women is associated with increased risk for breast and endometrial cancer as well as blood clots.

Until 1995, the stimulation of growth of the uterus by chemicals was considered the hallmark of their estrogenic activity. In 1996 however, a second type of estrogen receptor (ER) was identified, termed ERβ (Kuiper G. G. et al., 1996, Proc. Natl. Acad. Sci. USA 93, 5925-5930). Its genomic structure and chromosomal location are distinct from the ERa, but its molecular structure and tissue distribution overlap partially with ERa (Enmark E. et al. 1997, J. Clin. Endocrinol. Metab. 82, 4258- 4265). It is now generally acknowledged that ERa is the dominant receptor in the adult uterus, while ERβ is expressed at high levels in other estrogen-target tissues such as prostate, salivary glands, testis, ovary, vascular endothelium and smooth muscle, certain neurons in the central and peripheral nervous systems, and the immune system. The initiation of transcription is complex and requires the interaction of many proteins at a target gene promoter. ERa and ERβ belong to the nuclear receptor ligand-activated transcription factors. They are composed of several independent but interacting functional domains: the A/B domain (comprising the activation function (AF) 1), the C or DNA-binding domain, the D domain or hinge region, and the E/F or ligand-binding domain (LBD) with the associated AF2. Following ligand binding, estrogen receptors dimerize and interact with the hormone response element upstream of an estrogen-responsive gene. This triggers the recruitment of transcriptional regulators, with a critical role for the coactivators/corepressors that interact directly with the ER, at one or both activation functions. The activity of AF2 is ligand dependent while that of AF1 is considered to be constitutive, depending on the promoter and the cellular context .

A wide repertoire of structurally distinct compounds bind to the ERs having differing degrees of affinity and potency. Some of these compounds act solely as receptor agonists (e.g., 17β-estradiol, ER's endogenous ligand), while others are classified partial agonists leading to less efficient transactivation compared to 17β-estradiol.

At the down end of this category are compounds that compete efficiently with 17β- estradiol for the ER, have negligible or low estrogenic activity, and thus behave as antiestrogens. ICI-182780 is a full antagonist through disruption of coactivator recruitment to AF2, thereby inhibiting AF1 , and targeting the ER for proteasome- mediated protein breakdown (Jordan, 2003, J. Med Chem. 46, 883-908).

A fourth category of compounds, termed selective ER modulators (SERMs), have the ability to act as both agonists and antagonists depending on the cellular context. Examples include tamoxifen, which inhibits the action of estrogens in the breast, but exerts an estrogenic action in the uterus, and raloxifene, which has no activity in breast or uterus, but seems to be beneficial in bone. It has been shown that this is an ERα-mediated tissue-selective agonism (Pearce and Jordan, 2004, Crit. Rev. Oncol. Hematol. 50, 3-22) that is determined by the presence of specific coactivators and corepressors in the cell, and involves AF1 , as both tamoxifen and raloxifene disrupt AF2 functionality (Koehler et al., 2005, Endocrine Rev. 26, 465- 478). In humans, tamoxifen has proven therapeutic utility in treating and preventing breast cancer, but the compound has been associated with endometrial cancer and blood clots. Newer SERMs have been developed, although no one drug candidate has emerged to fill the needs of women who require the benefits of estrogen replacement and/or treatments for estrogen-dependent cancers (Jordan, V.C., 2003, J. Med. Chem. 46, 1081-1111).

With the discovery of the second estrogen receptor, research has rapidly evolved into the development of a fifth class of estrogens, termed selective ER subtype modulators (Meegan M. J. and Lloyd D. G., 2003, Curr. Med. Chem. 10, 181-210). Their selectivity is based on their differential action on both receptors and, logically, they are believed to have a promising potential in the treatment of diseases that are regulated by any ER subtype. Indeed, the markedly different tissue distribution of ERa and ERβ has opened new areas of research into the (relative) physiological role of both receptors (Pearce and Jordan, 2004, above; Koehler, 2005, above) and into the development of estrogen-receptor subtype agonists or antagonists (Sun J. et al., 1999, Endocrinology 140, 800-804). These endeavours have resulted in the definition of a novel mode of estrogen receptor antagonism, termed "passive antagonism" as elicited by R,f?-5,11-c/s-diethyl-5,6, 11 ,12- tetrahydrochrysene-2,8-diol (THC) (Shiau A. et al., 2002, Nature Struct. Biol. 9, 359-364).

JP09301915 describes 8-alkylnaringenin derivatives as estrogen agonists in general and describes the synthesis of naringenin derivatives with C_1-5 alkyl or with aromatic (benzyl- or phenyl-comprising groups) substitution at position 8 with their estrogen-receptor binding activity.

JP08165238 discloses naringenin derivatives as estrogen agents that can be used for the treatment of diseases related to reduced estrogen levels, such as amenorrhea, anovulatory cycle, menometrorrhagia, uterine hypoplasia, menopausal disorders, osteoporosis, prostate cancer, prostate hypertrophy; it is an alleged advantage of these compounds that side effects commonly observed with estrogen treatments (such as endometrium cancer, breast cancer, etc.) are reduced upon use of these compounds The document however does not provide any indication as to which ER is bound by the compounds and the basis for the alleged effects of these compounds, or the absence of side effects is questionable.

A number of naringenin derivatives isolated from the female flower of hop, including 8-prenyl-naringenin and 8-geranylnaringenin have been described to have estrogen agonistic activity (Schaefer O. et al. 2003, J. Steroid Biochem. MoI. Biol. 84, 359-360; Milligan et al., 2005, J. CHn. Endocrinol. Metab. 85(12):4912- 4915).

SUMMARY OF THE INVENTION

According to a first aspect of the invention, molecules are provided that have been identified as estrogen antagonists and are useful in the treatment or prevention of diseases or symptoms, that are associated with local estrogen activity, such as, but not limited to, estrogen-dependent cancers or male infertility or which require modulation of estrogen production, as well as in the modulation of estrogen- mediated signaling in the hypothalamus., e.g. for induction of ovulation.

According to another aspect of the invention, molecules are provided that have been identified as estrogen antagonists, whereby the antagonistic activity is selective for one of the two estrogen receptors alpha and beta (ERα/ERβ). This property is inter alia advantageous for ensuring a selective antiestrogenic effect, for avoiding side effects associated with the use of pure estrogen antagonists, such as have been described gastrointestinal disturbance, urinary tract infection, vaginitis (Versea et al., 2003; CHn. J. Oncol. Nurs. 7(3), 307-11), but also potentially amenorrhea, menometrorrhagia, uterine hypoplasia, menopausal disorders, and osteoporosis and/or for avoiding the reduced beneficial effects of pure estrogen antagonists on lipids and the skeletal system. According to a particular embodiment of the invention, the compounds are at least partial antagonists, more particularly full antagonists of at least one of the estrogen receptors alpha and beta. According to a further embodiment, the compounds are either partial antaginist, more particularly full antagonists of at least one of the estrogen receptors or are partial agonists of at least one of the estrogen receptors.

The present invention provides novel (synthetic) naringenin derivatives and identifies novel activities of known naringenin derivatives. More specifically, it has been found that naringenin derivatives of the general structural formula I:

I wherein R₂ is a linear or branched C₅ alkyl or a linear or branched alkyl, alkenyl, alkynyl or alkenynyl of six or more carbon atoms, are estrogen antagonists, more particularly, selective ER antagonists.

Thus, according to one aspect, the invention relates to naringenin derivatives as estrogen antagonists, more specifically as selective estrogen antagonists, based on their specific activity on the ERa and ERβ receptors. More specifically, the invention relates to the use of naringenin derivatives according to the general structural formula I:

I or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- Ri₁ Rs, R₉ and R₁₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci_-5 alkyl, Ci_-5 alkoxy, Ci_-5 alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R2 is selected from a C_5-2O alkyl, C_6-2o alkenyl, C_6-2o alkynyl, or C_6-2o alkenynyl wherein each of said alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R₃, R₄, R₅, Re and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

- Xi is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom, in the treatment and/or prevention of disorders which benefit from local estrogen antagonism or modulation of estrogen production. More particularly, the compounds ensure selective ERβ antagonism in combination with either ERa agonism or ERa antagonism.

According to particular embodiments, the invention relates to the use of the 8-aIkyl- naringenin derivatives of the invention in the treatment of estrogen-stimulated cancers, such as breast cancer, in the treatment of osteoporosis, in the treatment of impaired or inadequate hypothalamo-pituitary regulation of gonadal functioning and symptoms associated with increased androgen aromatization like gynaecomastia, prepubertal accelerated growth and bone maturation, to restore bone homeostasis or to restore hair cycle control, or to the use of such a compound to modulate or block estrogen-mediated signalling in the hypothalamus, such as for inducing ovulation.

According to a further aspect, the present invention provides novel synthetic naringenin derivatives according to the general structural formula I:

or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- R₁, R₈, Rg and R₁₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci_-5 alkyl, C-₁-₅ alkoxy, C₁-₅ alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R2 is selected from a branched C₅ alkyl, a C6-20 alkyl, C6-20 alkenyl, C6-20 alkynyl or Cβ-20 alkenynyl, wherein each of said C₆-2₀ alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R3, R₄, R5, Re and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro, or nitroso;

- Xi is selected from O, S; - X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom. wherein the naringenin derivative is not 8-geranylnaringenin;

These novel compounds have been synthesized and are characterized by their antagonistic activity on one or both of the estrogen receptors ERa and ERβ.

A particular embodiment of the present invention relates to novel naringenin derivatives, particularly synthetic naringenin derivatives as described herein directly above, wherein R₂ is selected from the group consisting of 2,2- dimethylpropyl, C_6-2o alkyl, C_6-2O alkenyl, C_6-2o alkynyl or C_6-2o alkenynyl, wherein each of said C_6-2O alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched and wherein the naringenin derivative is not 8-geranylnaringenin.

Yet a further particular embodiment of the present invention relates to naringenin derivatives, particularly synthetic naringenin derivatives, according to the general structural formula II:

or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein R₂ is selected from a branched C₅ alkyl, a C_6-20 alkyl, C_6-2O alkenyl, C_6-20 alkynyl or C_6-20 alkenynyl, wherein each of said alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched; wherein the naringenin derivative is not 8-geranylnaringenin.

Yet a further particular embodiment of the present invention relates to naringenin derivatives, particularly synthetic naringenin derivatives as described directly above, wherein R₂ is selected from 2,2-dimethylpropyl, C_6-2o alkyl, C_6-2o alkenyl, C_{6- 20} alkynyl or C_6-2O alkenynyl, wherein each of said C_6-2o alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched, wherein the naringenin derivative is not 8- geranylnaringenin.

A further particular embodiment of the present invention relates to naringenin derivatives according to the general structural formula Il or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein R₂ is selected from n-heptyl, n-nonyl, and π-undecyl; in a particular embodiment, R₂ is 2,2-dimethylpropyl.

BRIEF DESCRIPTION OF THE DRAWINGS

The following description, which is not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which:

Figure 1 : General structural formula I of the present invention with the numbering of the positions of the naringenin skeleton according to one embodiment of the invention.

Figure 2: Synthesis of (±)-8-alkyInaringenins according to one embodiment of the invention. I: (l-i) RLi/RMgBr, ET₂O, -78 ⁰C to rt (l-ii) HSiEt₃ (rt), CF₃COOH, CH₂CI₂, -78 ⁰C to rt; IhAcCI₁ SnCI₄, CH₂CI₂, -10 ⁰C; III: BBr₃, CH₂CI₂, -78 ⁰C to rt; IV: MOMCI, K₂CO₃, acetone, reflux; V: p- MOMO-benzaldehyde, KOH, H₂O-EtOH, O ⁰C to rt; Vl: NaOAc, EtOH, reflux; VII: 3 M HCI, MeOH, reflux

R = (CH₂)₅CH₃ (a); (CH₂)₇CH₃ (b); (CH₂)₉CH₃ (c); C(CH₃)₃ (d); H (e); CH₂CH₃ (f); (CH₂)₃CH₃ (g); CH₂CH(CH₃)₂ (h); CH(CH₃)₂ (i); C₆H₅ Q) Figure 3: Agonist activity of 8-alkylnaringenins. Agonist activity of 8- alkylnaringenins on gene transcription by ERa (A), and by ERβ (B), monitored on an estrogen-responsive ERE-reporter in HuH7 cells, in concentrations ranging from 10^~8 to 10^"5 M. Values represent the mean calculated from four or more separate experiments and are presented as percent response, with the maximal E2-response set at 100%. Error bars represent standard error of the mean. CTRL, control; E2, 17β-estradiol; OHT, 4-hydroxytamoxifen; 8PN, 8- prenylnaringenin; for compound codes, see Table 1.

Figure 4: Antagonist activity of 8-alkylnaringenins. (A) Antagonist activity of 8- alkylnaringenins on gene transcription by ERa and (B) by ERβ, monitored on an estrogen-responsive ERE-reporter in HuH7 cells, in concentrations ranging from 10^'9 to 10^~5 M, in the presence of 10^"9 M E2. Values represent the mean calculated from four or more separate experiments and are presented as percent response, with the response obtained with 10^~9 M E2 set at 100%. Error bars represent standard error of the mean. CTRL, control; E2, 17β-estradiol; OHT, 4- hydroxytamoxifen; for compound codes, see Table 1.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

A "naringenin derivative" as used herein refers to a compound whose general structure corresponds to that of naringenin, i.e. of the general structural formula I:

I

Thus, the term "naringenin derivatives" encompasses both flavanone derivatives, whereby the dotted line is deleted (bond between C2 and C3 is saturated) (as a mixture of stereoisomers or as a pure stereoisomer) and flavone derivatives, whereby the dotted line represents an additional bond (bond between C2 and C3 is unsaturated due to the presence of a double bond).

An "8-alkyInaringenin derivative" as used herein refers to a compound of the general structural formula as described above, wherein the carbon atom at position 8 is substituted by R₂, which is an alkyl, an alkenyl, an alkynyl, alkenynyl an aryl or an arylalkyl. The term 8-alkylnaringenin derivatives includes all stereoisomers of these compounds and is also referred to as (+/-)8-alkylnaringinin derivatives.

As used herein with respect to a substituting radical, and unless otherwise stated, the term "alkyl" relates to a fully saturated hydrocarbon.

As used herein with respect to a substituting radical, and unless otherwise stated, the term "alkenyl" relates to an unsaturated hydrocarbon comprising one or more double bonds.

As used herein with respect to a substituting radical, and unless otherwise stated, the term "alkynyl" relates to an unsaturated hydrocarbon comprising one or more triple bonds.

As used herein with respect to a substituting radical, and unless otherwise stated, the term "alkenynyl" relates to an unsaturated hydrocarbon comprising one or more double bonds and one or more triple bonds. As used herein with respect to a substituting radical, and unless otherwise stated, the term "aryl" relates to carbocyclic aromatic ring systems of 6-20 carbon atoms. Typical aryl groups include, but are not limited to 1 ring, or 2 or 3 rings fused together, radicals derived from benzene, naphthalene, spiro, anthracene, biphenyl, and the like, such as phenyl, naphthyl(1-naphthyl or 2-naphthyl), anthracenyl(1- anthracenyl, 2-anthracenyl, 3-anthracenyl), phenanthrenyl, fluorenyl, indenyl, and the like. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1-(1 ,2,3,4-tetrahydronaphthyl) and 2-(1 , 2,3,4- tetrahydronaphthyl).

"Arylalkyl" as used herein refers to an alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1- yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenyIethan-1-yl and the like. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.

As used herein with respect to the alkyl, alkenyl, alkynyl and alkenynyl radical, C_x or C_x-y indicates the number of carbon atoms present in the alkyl, alkenyl, alkynyl and alkenynyl radical and x and y may be any number between 1 and 20. E.g. C₅ alkyl comprises the group of alkyl radicals comprising 5 carbon atoms in a linear or branched conformation. C_6-2o alkenyl comprises a group of alkenyl radicals comprising 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in a linear or branched conformation.

As used herein a "branched C₅-alkyl" includes 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1 ,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl. As used herein with respect to a substituting radical, and unless otherwise stated, the term "halogen" refers to any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

As used herein with respect to the compounds of the present invention, the term "stereoisomer" refers to all possible different conformational forms which the compounds of the invention may possess, in particular all possible stereochemical^ isomeric forms, such as but not limited to diastereomers, enantiomers or conformers of the basic molecular structure. A stereoisomer of a chemical compound, radical or ion contains the same number of atoms of the same elements but differs in stereochemical conformation.

Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention. As used herein with respect to the compounds of the present invention, the term "tautomer" refers to structurally distinct compounds that are in rapid equilibrium. In most cases, said equilibrium is characterized by a shift of a proton from one atom of the compound to another, e.g. keto-enol tautomerism.

As used herein with respect to the compounds of the present invention, the term "enantiomer" means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e. at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

As used herein with respect to the compounds of the present invention, the term "solvate" includes any combination which may be formed by a naringenin derivative of this invention with a suitable inorganic solvent (e.g. with water to form hydrates) or organic solvent such as, but not limited to alcohols, ketones, esters, ethers, nitriles and the like.

As used herein with respect to the compounds of the present invention, the term "pharmaceutically acceptable salt" refers to any therapeutically active non-toxic addition salt which the naringenin compounds of the present invention are able to form with a salt-forming agent. Such addition salts may conveniently be obtained by treating the naringenin derivatives of the invention with an appropriate salt- forming acid or base. For instance, naringenin derivatives having basic properties may be converted into the corresponding therapeutically active, non-toxic acid addition salt form by treating the free base form with a suitable amount of an appropriate acid following conventional procedures. Alternatively, naringenin derivatives having acidic properties may be converted into the corresponding therapeutically active, non-toxic base addition salt form by treating the free acid form with a suitable amount of an appropriate base following conventional procedures.

"Estrogen-antagonistic activity" as used herein when referring to a compound relates to the fact that the compound is capable of reducing or inhibiting the effect of 17β-estradiol on an estrogen receptor. Thus, antagonism of an ER or ER receptor implies that the compound is capable of preventing the binding of 17β- estradiol to said receptor (optionally by competition with 17β-estradiol for the receptor), whereby the compound itself is not capable of activating the receptor. Methods for determining ERβ- and ERα-antagonism are known to the skilled person and described herein. Full antagonism relates to the full inhibition (75- 100%) of 17β-estradiol to its receptor in the presence of an excess of the antagonist. Partial antagonism refers to the partial (less than 75%) inhibition of the binding of 17β-estradiol to its receptor.

"Estrogen agonistic activity" as used herein relates to the ability of a compound to mimmick the effect of 17β-estradiol on an estrogen receptor. Partial agonistic activity, i.e. only partial activation of the receptor upon binding, can in some circumstances be comparable in effect to partial antagonistic activity, in that the compound, which itself binds to the estrogen receptor (and thus also prevents the binding of 17β-estradiol to its receptor), is only partially capable of activating the receptor. "an absent, positive or negative effect on bone homeostasis" as used herein refers to whether or not the compound is capable of influencing bone degeneration associated with reduced estrogen-levels such as bone decalcification leading to osteoporosis.

The present invention is based on the finding that, generally, introduction of an alkyl group consisting of 5 to 20 carbon atoms at position 8 of naringenin induces an estrogen-antagonistic effect on ERβ, as opposed to the potent estrogen- agonistic activity reported for naringenin derivatives with shorter alkyl chains at the same position. This estrogen-antagonistic effect on ERβ ranges from a weak antagonistic activity (e.g. 8-isopentylnaringenin or 3-methylbutyl) to very pronounced (e.g. 8-2,2-dimethylpropylnaringenin). Moreover, the present invention demonstrates that, within this group of compounds, the estrogen-antagonistic effect on ERβ can be combined with either estrogen-agonistic activity on ERa or estrogen-antagonistic activity on ERa. The anti-estrogen ic effects are more pronounced on ERβ than on ERa, a finding that cannot solely be explained by a difference in binding affinity for the two receptors. In addition, the present invention relates to the surprising finding that introduction of a C₅ alkyl which is double- branched, most particularly a 2,2-dimethylpropyl substituent at position 8 of naringenin results in a potent estrogen-antagonistic activity on ERβ and a potent estrogen-agonistic activity on ERa, an effect that is similar to THC and some 11 β- substituted estradiol derivatives (Zhang J. X. et al., 2004, J. CHn. Endocrinol. Metab 89, 3527-3535; WO 00/31112). The same activity is observed for the known 8-geranyl substituted derivative of naringenin, which was found to have the same ERa agonistic/ ERβ antagonistic properties.

Thus, the present invention demonstrates that naringenin derivatives of the general structural formula I:

wherein R₂ is a linear or branched alkyl of at least five carbon atoms, or an alkenyl, alkynyl or alkenynyl of at least six carbon atoms, are estrogen antagonists, more particularly, selective ER antagonists. Additionally, compounds wherein R2 is an aryl or arylalkyl were found to be ER antagonists, in that they are only partial estrogen receptor agonists.

Estrogen-antagonistic activity is of interest in the treatment of a number of diseases. First, there are diseases that are mediated by sustained estrogen levels such as estrogen-dependent cancers and endometriosis. Secondly, there are conditions that require increased estrogen levels (e.g., anovulation, artificial/increased ovulation for IVF), whereby estrogen antagonists can induce an artificial feedback mechanism to increase local estrogen production.

The ability of a compound to selectively antagonize ER receptors is of interest, as the selective activity of estrogens on these receptors is known to be quite different. Based on experimental data, it has been suggested that signaling through the ERβ receptor retards periosteal bone formation and suppresses gains in bone size and bone strengths, while ERa mediates the positive influence of estrogens on bone formation in endocortical and trabecular bone surfaces (Saxon L. K. and, Turner C. H., 2005, Bone 36, 185-192). The ERα-ERβ selective antagonism moreover allows an estrogen-antagonistic activity without many of the side effects associated with hypo-estrogenic conditions such as bone demineralisation, dry vagina, psychiatric symptoms, anabolic/androgenic effects, etc or makes it possible to maintain the advantageous properties of estrogen activity in some tissues e.g. on the skeletal system and on lipids. Alternatively, it can be of interest to ensure estrogen-agonistic activity (e.g., in the treatment of diseases or conditions such as amenorrhea, anovulatory cycle, uterine hypoplasia, osteoporosis) without the side effects mediated by estrogens in other tissues. Indeed, combination of ERβ-antagonistic activity with ERα-agonistic activity has been demonstrated to provide ancilary benefits to patients treated with tamoxifen for estrogen-dependent cancers, such as reduced cholesterol, reduction in cardiac events, and improved bone density in postmenopausal women. Moreover, in view of the fact that hormone-dependency may vary between patients and at different time points in one patient, the availability of compounds with varying ERα-ERβ-antagonistic activity allows an individualised treatment.

Examples of health conditions where estrogen-agonistic/antagonistic compounds have been demonstrated to be effective in the treatment or prevention of their development are vasomotor and other menopause symptoms (Baracat E. et al., 1999, J. CHn. Endocrinol. Metab 84, 2020-2027), osteoporosis (Albertazzi P. and Purdie D. W., 2001 , Best. Pract. Res. CHn. Rheumatol. 15, 451-468; Gennari L., et al. 2005, Am. J. Epidemiol. 161 , 307-320), male and female infertility (de Ronde W. et al., 2003, CHn. Endocrinol. 58, 529-542; Carr,B. R. 1992, In: Williams Textbook of Endocrinology, pp. 733-798, Edited by Wilson, J. D., and Foster, D. W., Philadelphia, WB Saunders Co; Raman J. D. and Schlegel P. N., 2002, J. Urol. 167, 624-629), estrogen-sensitive cancers (Key T. J. and Pike M. C, 1988, Eur. J. Cancer CHn. Oncol. 24, 29-43; demons M. and Goss P., 2001, N. Engl. J. Med. 344, 276-28; Pearce and Jordan, 2004, above; Hansmann A. et al. Cancer 100, 612-620.), benign prostate hyperplasia (Pearce and Jordan, 2004, above), cardiovascular diseases and lipid metabolism (Gray G. A. et al., 2001 , Trends Pharmacol. Sci. 22, 152-156; Levin E. R., 2002, Trends Endocrinol. Metab. 13, 184-185), hypothalamic-pituitary gonadal axis dysregulation (de Ronde, 2003, above; Carr, 1992, above) symptoms associated with increased androgen aromatization like gynaecomastia, and prepubertal accelerated growth and bone maturation (Perdona S. et al. 2005, Lancet Oncol. 6, 295-300; de Ronde, 2003, above). The treatment of these diseases is thus also envisaged within the context of the present invention. Moreover, selective estrogen agonism/antagonism has also been demonstrated to be useful in ovulation induction (Steiner A. Z. et al., 2005, Hum. Reprod. 20, 1511-1515).

Thus, according to one aspect the invention relates to the use of naringenin derivatives as estrogen antagonists, more specifically as selective estrogen antagonists, based on their specific activity on the ERa and ERβ receptors. More specifically, the invention relates to the use of naringenin derivatives according to the general structural formula I:

I or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- Ri₁ R₈, R₉ and R₁₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci_-5 alkyl, Ci_-5 alkoxy, Ci_-5 alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched; - R₂ is selected from a C_5-2o alkyl, C_6-2o alkenyl, C_6-2O alkynyl or C_6-2o alkenynyl, wherein each of said alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R₃, R₄, R₅, R₆ and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

- X₁ is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom, in the treatment and/or prevention of disorders that benefit from local estrogen antagonism or modulation of estrogen production or in the generation of specific conditions by blocking of estrogen-mediated signaling in the hypothalamus. More particularly, the compounds ensure selective ERβ antagonism in combination with either ERa agonism or either ERa antagonism.

A specific embodiment of the present invention relates to the compounds as described above wherein R₂ is 8-(2,2-dimethylpropyl)naringenin or 8-geranyl- naringenin.

Clinical applications of the naringenin derivatives with ERβ-antagonist activity in combination with ERα-antagonist activity include those diseases caused by an excess/undesired overall estrogen activity or those conditions that are irresponsive to selective estrogen-receptor inhibition (e.g., tamoxifen). Examples are prevention and/or treatment of certain cancers and of symptoms associated with increased androgen aromatization like gynaecomastia and prepubertal accelerated growth and bone maturation.

Clinical applications of the second group of naringenin derivatives with ERβ- antagonistic activity, in combination with ERα-agonistic activity include osteoporosis and bone homeostasis Moreover, they include estrogen deficiency related disorders such as peri-and/or post-menopausal complaints. Accordingly, the invention relates to the use of the naringenin derivatives according to the invention for the manufacture of a medicament in the treatment of estrogen- deficiency related disorders such as osteoporosis and peri-and/or postmenopausal (climacteric) complaints and for the maintenance of bone homeostasis.

The invention further provides compounds for use in the manufacture of a medicament used in HRT (hormone replacement therapy).

The dosage amounts of the present naringenin derivatives will be of the normal order for estradiol derivatives, e.g. of the order of 0.01 to 1000 mg per administration, including, but not limited to dosages of 10, 100, 250, 500 and 750 mg/dosage.

The diseases envisaged to be treated by the selective estrogen antagonists described in the present invention include those diseases that require a local antagonistic effect without a complete hypoestrogenemia.

Examples are the use of such compounds in prevention and/or treatment of breast and other estrogen-stimulated cancers, in improvement of the cellular antioxidant status, in the impaired or inadequate hypothalamo-pituitary regulation of gonadal functioning, in improving bone homeostasis, and hair cycle control. . Further, the invention relates to the use of an 8-prenylnaringenin (hereinafter referred to as 8- PN) derivative according to the invention in the manufacture of a medicament having contraceptive activity. Thus the invention also pertains to the medical indication of contraception, i. e. a method of contraception comprising the administration to a subject, being a woman or a female animal, of a progestogen and an estrogen as is customary in the field, wherein the estrogen is a compound as described hereinbefore (in a suitable pharmaceutical dosage form). The present invention accordingly relates to pharmaceutical compositions comprising one or more of the naringenin derivatives according to the invention mixed with a pharmaceutically acceptable excipients, such as described in the standard reference Gennaro et al., Remmington's Pharmaceutical Sciences, (18th ed., Mack publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture.). The mixture of the naringenin derivatives according to the invention and the pharmaceutically acceptable excipients may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied as an injection preparation in the form of a solution, suspension, emulsion, or as a spray, e.g. nasal spray. For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. The 8-PN derivative of the invention may also be included in an implant, a vaginal ring, a patch, a gel, and any other preparation for sustained release.

Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof used in suitable amounts.

In accordance with the above, the present invention provides methods for the treatment of an estrogen-deficiency dependent disorder, such as those described above, comprising the administration to a patient, being a woman, of a compound as described hereinbefore (in a suitable pharmaceutical dosage form).

A further aspect of the present invention provides novel synthetic naringenin derivatives according to the general structural formula I:

I or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- R-i, R₈, Rg and Ri₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci_-5 alkyl, Ci_-5 alkoxy, Ci_-5 alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R₂ is selected from a branched C₅ alkyl, a C6-20 alkyl, C_6-2o alkenyl, C6-20 alkynyl or C₆-₂₀ alkenynyl, wherein each of said C_6-2o alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R₃, R₄, R₅, R₆ and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

. Xi is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately connecting a hydrogen atom. wherein said naringenin derivative is not 8-geranylnaringenin.

8-Geranylnaringenin is a natural compound which has been isolated from the female flower of hop and has been described as an estrogen agonist (Milligan et al., 2005, J. Clin. Endocrinol. Metab. 85(12):4912-4915). The present invention describes the synthesis of novel synthetic naringenin derivatives and demonstrates that these compounds have estrogen-antagonistic activity, more particularly ERα/ERβ. More particularly, the present invention provides naringenin derivatives according to the general structural formula II:

or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein R₂ is selected from a branched C₅ alkyl, a C_6-2O alkyl, C6_-2o alkenyl, C_6-2O alkynyl or Cβ-₂o alkenynyl, wherein each of said alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched, wherein said naringenin derivative is not 8- geranylnaringenin.

Specific embodiments of the present invention relate to naringenin derivatives according to the general structural formulae I or II, or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein R₂ is selected from n- heptyl; n-nonyl; π-undecyl; Most specifically, the present invention provides a novel naringenin derivative, according to the general structural formulae I or II, or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein R₂ is 2,2-dimethylpropyl.

According to one embodiment, the compounds of the present invention are chemically synthesized using the synthetic pathway illustrated in Figure 2. 2,4,6- Trimethoxybenzaldehyde (1) is used as a starting material, in which the aldehyde serves as a handle for the introduction of desired alkyl groups. Mono-alkylation is effected using organometallics (alkyl lithium, alkylmagnesium bromide) and the resulting secondary benzylic alcohols are efficiently deoxygenated using a method known to the person skilled in the art, preferably upon treatment with triethylsilane and trifluoroacetic acid, whereby C-alkylated 1 ,3,5-trimethoxybenzenes (2a-d) are obtained in high yields from 2,4,6-trimethoxybenzaIdehyde. Subsequent Friedel- Crafts acetylation is effected with acetyl chloride in the presence of tin(IV) chloride yielding 3-alkyl-2,4,6-trimethoxyacetophenones (3a-d). Demethylation of the methoxy substituents is achieved preferably with boron tribromide and furnishes 3- alkyl-2,4,6-trihydroxyacetophenones (4a-d), which are regioselectively protected, preferably b/s-methoxymethylated to give the 3-alkyl-2-hydroxy-4,6- b/s(methoxymethoxy)acetophenones (5a-d). Subsequent Claisen-Schmidt condensation with p-(methoxymethoxy)benzaldehyde affords chalcones (6a-d) having the appropiate functionalities in protected form. Regioselective cyclization, preferably by refluxing with sodium acetate in ethanol, to the corresponding (±)-tris- methoxymethylated flavanones (7a-d) is followed by demethoxymethylation (preferably using hydrogen chloride) and yields the desired (±)-8-alkylnaringenins (8a-d).

Stereoisomers are separated by known chromatographic techniques, preferably high-performance liquid chromatography (HPLC) and, more preferably, reversed- phase HPLC. Diastereoisomers can be readily separated by the person skilled in the art, since retention factors of the compounds differ inherently. As is known to the person skilled in the art, separation of enantiomers requires application of chiral chromatography and examples are described in Kitaoka, M. et al. (1998 Planta Med. 64, 511-515), Milligan, S. et al. (2002, Reproduction 123, 235-242) and in a textbook on Chiral Chromatography by Beesley, T. E. & Scott, R. P. W., Wiley, 1999.

It is obvious that varying naringenin derivatives can be obtained from either appropriately substituted starting materials or from selective substitutions on suitable intermediate reaction products. Thus, a reaction sequence, analogous to that starting from 1 as outlined in Figure 2, can be elaborated starting from 2- hydroxy- or 2-methoxybenzaldehyde carrying substituents at positions 3, 4, 5 and/or 6. For example, 2,3,6-trimethoxybenzaldehyde would lead to I, in which R₁ and Rio = OH; 2,4,5-trimethoxybenzaldehyde would lead to I₁ in which R9 and R-m = OH; 2,3,4,6-tetramethoxybenzaldehyde would lead to I, in which R₁, Rg and R10 = OH.

Similarly, using substituted p-MOMO-benzaldehyde in the formation of protected chalcones (see Figure 2) furnishes I carrying R₃, R₄, Re and R₇ groups. For example, 3,4-dihydroxybenzaldehyde would lead to I, in which R₄ and R₅ = OH; 2,4-dihydroxybenzaldehyde would lead to I, in which R₃ and R₅ = OH; 2,3,4- trihydroxybenzaldehyde would lead to I, in which R₃, R₄ and R₅ = OH.

The phenolic groups can be readily converted to alkoxy (ethers) or alkylcarbonyloxy (esters) groups. Compounds I carrying alkyl groups result from the use of suitably alkylated 2-hydroxy- or 2-methoxybenzaldehyde and p-MOMO- benzaldehyde, respectively, as described above. Rs and R₁0, heteroatom-attached to the flavonoid skeleton, can be introduced by the person skilled in the art using standard halogenation techniques followed by appropriate nucleophilic substitutions. Both positions can be differentiated by virtue of their distinct aliphatic and aromatic character. α-Halogenation of ketones either in acid or basic conditions is straightforward and this reaction should preferably be done on the fully protected flavanone (see Figure 2). As a result, R₈ = Cl is accessible and substitution by cyano or amino is feasible. Introduction of a nitroso group is achieved by diazotation followed by capture of the diazonium salt by nitrosyl chloride. Oxidation of the amine (potassium permanganate) leads to the nitro derivative. A halogen at R₁₀ may result from the presence of a halogen, preferably chlorine, in the starting material or from aromatic halogenation (PCI₅ in chloroform). Substitutions are performed as described above. Similar reactions could be carried out on the suitably substituted aldol-type reaction partner (substituted benzaldehyde, see Figure 2), thereby giving rise to various modifications of the R₃, R₄, Rs₁ RQ and/or R₇ groups.

A Rio alkyl group in compounds I should preferably be present in the starting material, while introduction of a R₈ alkyl group involves a Friedel-Crafts acylation, as is done using acetyl chloride to finally result in formation of the flavanone with R₈ = H. It is clear that reaction of the protected aromatic compound carrying an alkyl group, such as 1-aIkyl-2,4,6-trimethoxybenzene, with any acyl chloride (for example, propionyl chloride) should give rise to I with R₈ = alkyl (for example, methyl).

As to the modification of the heterocyclic ring, substitution of a sulfur for an oxygen is of prime concern. Formation of a cyclic thioether can only be achieved by a thiol- induced intramolecular Michael-type cyclization. This requires the presence of a protected thiol in the substrate, for example methylthio. The same sequence can be elaborated as was done for a phenol. Transformation of a ketone into a thioketone is straightforward and a number of reagents allow this oxygen-to-sulfur conversion to occur. Oyama and Kondo (2004; J. Org. Chem. 69, 5240-5246) reported on reduction (deoxygenation) of the carbonyl in the heterocyclic ring using sodium borohydride. Moreover, they report an efficient method to convert flavanones into flavones by treatment with DDQ (2,3-dichloro-5,6-dicyano-1 ,4- benzoquinone) and phenyl chloride.

EXAMPLES

The following examples are provided for illustration purposes only and should in no way be interpreted as limiting the scope of the present invention. EXAMPLE 1 : Synthesis of the 8-alkylnaringenin compounds

The exact structures of the synthesized compounds have been confirmed using following Nuclear Magnetic Resonance (NMR) techniques: ¹H NMR and ¹³C NMR spectra were obtained with a Varian Mercury 300 spectrometer (¹H NMR: 300 MHz, ¹³C NMR: 75 MHz). All spectra were recorded in DMSO-Cf₆. Chemical shifts (δ) are expressed in parts per million (ppm) relative to the residual solvent peak. All signals assigned to hydroxyl groups were exchangeable with D₂O. Exact mass measurements (High-Resolution Mass Spectrometry, HRMS) were performed on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF 1 , Micromass, Manchester, UK) equipped with a standard electrospray ionization (ESI) interface. Samples were infused in acetonitrile (positive mode: 1% formic acid) at 10 μL/min. Thin layer chromatography (TLC) was carried out on precoated Alugram® SIL G/UV254 silica gel plates (Macherey-Nagel & Co., Dϋren, Germany) and TLC separations were examined under UV light at 254 nm and revealed by a sulfuric acid - anisaldehyde spray.

Purification of the compounds was mainly performed by column chromatography on silica (Ecochrom, ICN Silica 63-200 mesh) from ICN Biomedicals (Eschwege, Germany). Compounds were obtained as amorphous powders or as colorless to light-yellow oils. Technical solvents were purchased from Chemlab (Zedelgem, Belgium), while anhydrous solvents and reagents were obtained from Acros Organics (Geel, Belgium) and Sigma-Aldrich (Bornem, Belgium).

1. Preparation of 2-alkyl-1 ,3,5-trimethoxybenzenes (2a-i)

(i) To a stirred solution of 2,4,6-trimethoxybenzaIdehyde (1) in dry Et₂O (1.5 mL/mmol) was added dropwise at -78°C alkyllithium or alkylmagnesium bromide (1.2 equiv). The cooling bath was removed and, after completion of the reaction (1 - 1.5 h, as monitored by TLC), the reaction mixture was poured on ice and extracted with EtOAc. The organic phase was dried over anhydrous MgSO₄ and the solvent was removed under reduced pressure, (ii) Without purification, the residue was redissolved in dry CH₂CI₂ (1.5 mL/mmol) and HSiEt₃ (2.0 equiv) was added at room temperature together with CF₃COOH (6 equiv) at -78°C. The reaction mixture was allowed to warm up to room temperature (1 h) and stirred until completion of the reaction (30 min - 1 h, as monitored by TLC). After neutralization with saturated aqueous NaHCO₃, the mixture was extracted with Et₂O. The combined organic phases were washed with water, dried over anhydrous MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc) to yield 2a-j.

2. Preparation of 3-alkyl-2 Aβ-trimethoxyacetophenones (3a-i)

To a stirred solution of 2-alkyl-1 ,3,5-trimethoxybenzene (2a-j) in dry CH₂CI₂ (1 mL/mmol) was added dropwise at -10⁰C SnCI₄ (2 equiv) and acetyl chloride (1.5 equiv). The reaction mixture was stirred until completion of the reaction (2 - 3 h, as monitored by TLC), and poured on ice. The separated H₂O-phase was extracted with Et₂O, and both organic phases were washed with saturated aqueous NaHCO₃, dried over anhydrous MgSO₄, and the organic solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc) to yield 3a-j.

3. Preparation of 3-alkyl-2 A 6-trihvdroxyacetophenones (4a-i)

To a solution of 3-alkyl-2,4,6-trimethoxyacetophenone (3a-j) in dry CH₂CI₂ (2 mL/mmol) BBr₃ (4 equiv, 1.0 M in CH₂CI₂) was added dropwise at -78°C. The mixture was allowed to warm up to room temperature and was stirred until completion of the reaction (24 - 48 h, as monitored by TLC). After cooling to O⁰C, the reaction was quenched by pouring on ice. The organic solvent was removed under reduced pressure and the aqueous suspension was repeatedly extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous MgSO₄, and concentrated. The residue was purified by column chromatography (hexane/EtOAc) to provide 4a-j.

4. Preparation of 3-alkyl-2-hvdroxy-4,6-bis(methoxymethoxy)acetophenones (5a-i)

To a stirred mixture of 3-alkyl-2,4,6-trihydroxyacetophenone (4a-j) and anhydrous K₂CO₃ (7 equiv) in dry acetone (3 ml_/mmol 4a-j) was added dropwise MOMCI (2.5 equiv). The mixture was heated at reflux for 1 h, cooled to room temperature, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica column chromatography (hexane/EtOAc), to afford 5a-j.

5. Preparation of 3'-alkyl-2'-hvdroxy-4,4',6'-tris(methoxymethoxy)chalcones (6a-i)

To a stirred mixture of KOH (0.7 g/mmol 5a-j) in H₂O-EtOH (1.5 mL/mmol 5a-j, v/v 2:3) cooled to O⁰C in an ice bath was added dropwise a solution of 3-alkyl- 2-hydroxy-4,6-bis(methoxymethoxy)acetophenone (5a-j) and p-methoxy- methoxybenzaldehyde (1.1 equiv, prepared from p-hydroxybenzaldehyde) in EtOH (2 mL/mmol 5a-j) cooled to 0°C. The reaction mixture was kept in an ice bath for 3 h, then at room temperature for 20 h (until completion of the reaction, as monitored by TLC). The mixture was poured into ice water and extracted with Et₂O. The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and the solvent was evaporated under reduced pressure. The residue was purified by silica column chromatography (hexane/EtOAc), to afford 6a-j.

6. Preparation of (±)-8-alkyl-4',5,7-tris(methoxymethoxy)flavanones (7a-i)

To a solution of 3'-alkyl-2¹-hydroxy-4,4',6'-tris(methoxymethoxy)chalcone (6a-j) in EtOH (5 mL/mmol) were added NaOAc (4 equiv) and H₂O (5 drops). The mixture was heated at reflux for 24 h and, after cooling to room temperature, H₂O was added and the mixture was extracted with Et₂O. The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and the solvent was evaporated under reduced pressure. The residue was purified by silica column chromatography (hexane/EtOAc) to afford 7a-j, while 6a-j was recovered also.

7. Preparation of (+)-8-alkyl-4',5,7-trihvdroxyflavanones (8a-i)

To a stirred solution of (±)-8-aIkyl-4',5,7-tris(methoxymethoxy)flavanone (7a- j) in MeOH (15 mL/mmol) was added dropwise 3 M HCI (5 mL/mmol). The solution was heated at reflux for 1 h (until completion of the reaction, as monitored by TLC), then H₂O was added, and the solution was extracted with Et.₂0. The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and the solvent was evaporated under reduced pressure. The residue was purified by silica column chromatography (hexane/EtOAc), to afford 8a-j.

The following ¹H NMR, ¹³C NMR, and HRMS data support the identity of the synthesised compounds:

2a. ¹H NMR (300 MHz₁ DMSO-Cf₆) £ 0.83 (br t, 3H, J = 7.0 Hz, C(7a)-H), 1.18-1.41 (m, 1OH, C(2a)- H to C(6a)-H), 2.44 (br t, 2H, J = 7.3 Hz, C(Ia)-H), 3.71 (s, 6H, C(I)-OCH₃ and C(3)-OCH₃), 3.72 (s, 3H, C(5)-OCH₃), 6.16 (s, 2H, C(4)-H and C(6)-H); ¹³C NMR (75 MHz, DMSO-e/₆) £ 14.52, 22.66, 22.84, 29.35, 29.75, 29.82, 32.07, 55.60, 56.02, 91.22, 110.98, 158.93, 159.55. HRMS calcd for Ci₆H₂₇O₃ [M + H]⁺ 267.1960; found 267.1944.

3a. ¹H NMR (300 MHz, DMSO-Cf₆) £0.84 (br t, 3H, J = 7.0 Hz, C(7a)-H), 1.18-1.44 (m, 1OH, C(2a)- H to C(6a)-H), 2.36 (s, 3H, C(I)-COCH₃), 2.42 (br t, 2H, J = 7.3 Hz, C(Ia)-H), 3.58, 3.78 and 3.81 (3s, 3H each s, C(2)-OCH_3, C(4)-OCH₃ and C(B)-OCH₃), 6.47 (s, 1 H, C(5)-H); ¹³C NMR (75 MHz, DMSO-Gf₆) £ 14.62, 22.77, 23.46, 29.13, 29.90, 30.07, 31.92, 33.08, 56.55, 56.63, 63.29, 92.98, 116.57, 118.49, 156.17, 156.31 , 160.21 , 201.61. HRMS calcd for C₁₈H₂₉O₄ [M + H]⁺ 309.2066; found 309.2054.

4a. ¹H NMR (300 MHz, DMSO-d₆) £0.82 (br t, 3H, J = 6.6 Hz, C(7a)-H), 1.15-1.40 (m, 1OH, C(2a)- H to C(6a)-H), 2.37 (br t, 2H, J = 7.7 Hz, C(Ia)-H), 2.52 (s, 3H, C(I)-COCH₃), 5.97 (s, 1 H, C(5)-H), 10.15, 10.45 and 13.95 (3s, 1 H each s, C(2)-OH, C(4)-OH and C(B)-OH); ¹³C NMR (75 MHz, DMSOd₆) (514.62, 22.35, 22.79, 29.30, 29.36, 29.78, 32.03, 33.12, 94.59, 104.45, 107.24, 160.72, 163.30, 164.07, 203.10. HRMS calcd for C₁₅H₂₁O₄ [M - H]^" 265.1440; found 265.1430.

5a. ¹H NMR (300 MHz, DMSO-Cf₆) £ 0.82 (br t, 3H, J = 7.0 Hz, C(7a)-H), 1.17-1.45 (m, 10H, C(2a)- H to C(6a)-H), 2.48 (br t, 2H, J = 7.7 Hz, C(Ia)-H), 2.61 (s, 3H, C(I)-COCH₃), 3.38 and 3.43 (2s, 3H each s, C(4)-OCH₃ and C(6)-OCH₃), 5.26 and 5.29 (2s, 2H each s, C(4)-OCH₂ and C(6)-OCH₂), 6.34 (s, 1 H, C(5)-H), 13.81 (s, 1 H, C(2)-OH); ¹³C NMR (75 MHz, DMSO-Cf₆) £ 14.62, 22.49, 22.77, 29.21 , 29.26, 29.73, 31.94, 33.65, 56.71 , 57.18, 92.14, 94.44, 95.21 , 106.79, 111.67, 159.09,

161.35, 163.34, 204.25. HRMS calcd for C₁₉H₂₉O₆ [M - H]^" 353.1964; found 353.1965.

6a. ¹H NMR (300 MHz, DMSO-d₆) £0.84 (br t, 3H, J = 7.0 Hz, C(7a)-H), 1.20-1.44 (m, 1OH, C(2a)- H to C(6a)-H), 2.49 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 3.37, 3.39 and 3.43 (3s, 3H each s, C(4)-OCH₃, C(4')-OCH₃ and C(6')-OCH₃), 5.24, 5.28 and 5.34 (3s, 2H each s, C(4)-OCH₂, C(4')-OCH₂ and C(6')-OCH₂), 6.39 (s, 1 H, C(5')-H), 7.08 (d, 2H, J = 8.8 Hz, C(3)-H and C(5)-H), 7.66 (d, 2H, J = 8.8 Hz, C(2)-H and C(6)-H), 7.70 (d, 1 H, J = 15.8 Hz, C(β)-H), 7.81 (d, 1 H, J = 15.8 Hz, C(α)-H), 13.66 (s, 1 H, C(2')-0H); ¹³C NMR (75 MHz, DMS0-d₆) £ 14.63, 22.67, 22.78, 29.28, 29.77, 31.95, 56.42, 56.74, 57.24, 93.04, 94.39, 94.50, 95.90, 107.74, 112.14, 117.22, 126.04, 129.09, 130.89, 142.98,

158.36, 159.42, 161.17, 163.27, 193.50. HRMS calcd for C₂₈H₃₇O₈ [M - H]^" 501.2488; found 501.2471.

7a. ¹H NMR (300 MHz, DMSO-d₆) £0.79 (br t, 3H, J = 7.0 Hz, C(7a)-H), 1.11-1.46 (m, 1OH, C(2a)- H to C(6a)-H), 2.50 (br t, 2H, J = 6.8 Hz, C(Ia)-H), 2.67 (dd, 1 H, J = 2.9, 16.4 Hz, C(3)-H_A), 2.98 (dd, 1 H, J = 12.6, 16.4 Hz, C(3)-H_B), 3.36, 3.38 and 3.40 (3s, 3H each s, C(4')-OCH₃, C(5)-OCH₃ and C(7)-OCH₃), 5.17, 5.19 and 5.26 (3s, 2H each s, C(4')-OCH₂, C(5)-OCH₂ and C(7)-OCH₂), 5.44 (dd, 1 H, J = 2.9, 12.6 Hz, C(2)-H), 6.48 (s, 1 H, C(6)-H), 7.04 (d, 2H, J = 8.5 Hz, C(3')-H and C(5')- H), 7.41 (d, 2H, J = 8.5 Hz, C(2>H and C(6')-H); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.58, 22.74, 22.90, 29.10, 29.34, 29.53, 31.84, 45.61 , 56.22, 56.70, 56.72, 78.31 , 94.49, 94.54, 95.81 , 96.63, 107.79, 113.04, 116.72, 128.17, 133.18, 157.39, 157.47, 160.57, 161.40, 189.45. HRMS calcd for C₂₈H₃₉O₈ [M + H]⁺ 503.2645; found 503.2633.

8a. ¹H NMR (300 MHz, DMSO-d₆) £ 0.80 (br t, 3H, J = 7.0 Hz, C(7a)-H), 1.11-1.41 (m, 10H, C(2a)- H to C(6a)-H), 2.38 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 2.71 (dd, 1 H, J = 2.9, 16.9 Hz, C(3)-H_A), 3.14 (dd, 1 H, J = 12.5, 16.9 Hz, C(3)-H_B), 5.38 (dd, 1 H, J = 2.9, 12.5 Hz, C(2)-H), 5.95 (s, 1 H, C(6)-H), 6.77 (d, 2H, J = 8.5 Hz, C(3')-H and C(5')-H), 7.28 (d, 2H, J = 8.5 Hz, C(2')-H and C(6')-H), 9.54, 10.63 and 12.09 (3s, 1 H each s, C(4')-OH, C(5)-OH and C(7)-OH); ¹³C NMR (75 MHz, DMS0-d₆) £ 14.62, 22.45, 22.75, 29.15, 29.30, 29.44, 31.89, 42.68, 78.70, 95.94, 102.40, 108.51 , 115.81 , 128.47, 130.02, 158.21 , 160.59, 161.73, 165.30, 197.38. HRMS calcd for C₂₂H₂₅O₅ [M - H]^" 369.1702; found 369.1689. 2b. ¹H NMR (300 MHz, DMSO-Cf₆) 50.83 (br t, 3H, J = 7.0 Hz, C(9a)-H), 1.15-1.39 (m, 14H, C(2a)- H to C(βa)-H), 2.42 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 3.71 (s, 6H, C(I)-OCH₃ and C(3)-OCH₃), 3.72 (s, 3H, C(5)-OCH₃), 6.16 (s, 2H, C(4)-H and C(6)-H); ¹³C NMR (75 MHz, DMSO-Cf₆) 5 14.59, 22.67, 22.78, 29.42, 29.62, 29.68, 29.71 , 29.77, 32.00, 55.72, 56.17, 91.33, 110.90, 158.92, 159.54. HRMS calcd for C₁₈H₃iO₃ [M + H]⁺ 295.2273; found 295.2267.

3b. ¹H NMR (300 MHz, DMSO-d₆) 50.83 (br t, 3H, J = 6.7 Hz, C(9a)-H), 1.16-1.44 (m, 14H, C(2a)- H to C(8a)-H), 2.36 (s, 3H, C(I )-COCH₃), 2.42 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 3.58, 3.78 and 3.81 (3s, 3H each s, C(2)-OCH_3, C(4)-OCH₃ and C(S)-OCH₃), 6.47 (s, 1 H, C(5)-H); ¹³C NMR (75 MHz, DMSO-Cf₆) 5 14.58, 22.77, 23.46, 29.39, 29.49, 29.63, 29.94, 30.06, 31.99, 33.06, 56.51, 56.58, 63.29, 92.90, 116.52, 1 18.45, 156.15, 156.29, 160.19, 201.60. HRMS calcd for C₂₀H₃₃O₄ [M + H]⁺ 337.2379; found 337.2393.

4b. ¹H NMR (300 MHz, DMSO-Cf₆) 50.83 (br t, 3H, J = 7.0 Hz, C(9a)-H), 1.15-1.42 (m, 14H, C(2a)- H to C(βa)-H), 2.36 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 2.52 (s, 3H, C(I)-COCH₃), 5.96 (s, 1 H , C(5)-H),

10.16, 10.45 and 13.95 (3s, 1 H each s, C(2)-OH, C(4)-OH and C(6)-OH); ¹³C NMR (75 MHz, DMSO-Cf₆) 5 14.62, 22.35, 22.77, 29.26, 29.39, 29.69, 29.78, 29.85, 31.97, 33.13, 94.59, 104.45, 107.23, 160.72, 163.30, 164.06, 203.11. HRMS calcd for C₁₇H₂₅O₄ [M - H]^" 293.1753; found 293.1743.

5b. ¹H NMR (300 MHz, DMSO-Cf₆) 50.83 (br t, 3H, J = 7.0 Hz, C(9a)-H), 1.15-1.48 (m, 14H, C(2a)- H to C(8a)-H), 2.48 (br t, 2H, J = 7.7 Hz, C(Ia)-H), 2.61 (s, 3H, C(I)-COCH₃), 3.38 and 3.43 (2s, 3H each s, C(4)-OCH₃ and C(B)-OCH₃), 5.26 and 5.29 (2s, 2H each s, C(4)-OCH₂ and C(B)-OCH₂), 6.34 (S, 1H, C(5)-H), 13.81 (s, 1H, C(2)-OH); ¹³C NMR (75 MHz, DMSO-Cf₆) 514.60, 22.48, 22.76,

29.17, 29.37, 29.59, 29.61 , 29.73, 31.96, 33.65, 56.71 , 57.18, 92.13, 94.44, 95.22, 106.79, 111.66, 159.09, 161.35, 163.34, 204.25. HRMS calcd for C₂₁H₃₃O₆ [M - HJ 381.2277; found 381.2271.

6b. ¹H NMR (300 MHz, DMSO-d₆) 50.82 (br t, 3H, J = 6.6 Hz, C(9a)-H), 1.12-1.48 (m, 14H, C(2a)- H to C(8a)-H), 2.49 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 3.37, 3.39 and 3.43 (3s, 3H each s, C(4)-OCH₃, C(4')-OCH₃ and C(6')-OCH₃), 5.24, 5.29 and 5.34 (3s, 2H each s, C(4)-OCH₂, C(4')-OCH₂ and C(6')-OCH₂), 6.39 (s, 1 H, C(5')-H), 7.08 (d, 2H, J = 8.8 Hz, C(3)-H and C(5)-H), 7.66 (d, 2H, J = 8.8 Hz, C(2)-H and C(6)-H), 7.71 (d, 1 H, J = 15.8 Hz, C(β)-H), 7.81 (d, 1 H, J = 15.8 Hz, C(α)-H), 13.67 (s, 1 H, C(2')-OH); ¹³C NMR (75 MHz, DMSO-Cf₆) 514.64, 22.78, 29.23, 29.39, 29.58, 29.63, 29.78, 31.98, 56.43, 56.74, 57.25, 92.97, 94.33, 94.44, 95.86, 107.69, 112.06, 117.20, 126.00, 129.05, 130.92, 143.02, 158.36, 159.41 , 161.16, 163.25, 193.50. HRMS calcd for C₃₀H₄₁O₈ [M - H]^" 529.2801 ; found 529.2794. 7b. ¹H NMR (300 MHz, DMSOd₆) £ 0.82 (br t, 3H, J = 7.0 Hz, C(9a)-H), 1.11-1.49 (m, 14H, C(2a)- H to C(βa)-H), 2.49 (br t, 2H, J = 6.8 Hz, C(Ia)-H), 2.67 (dd, 1 H, J = 2.6, 16.4 Hz, C(3)-H_A), 2.98 (dd, 1 H, J = 12.6, 16.4 Hz, C(3)-H_B), 3.36, 3.37 and 3.39 (3s, 3H each s, C(4')-OCH₃, C(5)-OCH₃ and C(7)-OCH₃), 5.17, 5.19 and 5.26 (3s, 2H each s, C(4')-OCH₂, C(5)-OCH₂ and C(7)-OCH₂), 5.44 (dd, 1 H, J = 2.6, 12.6 Hz, C(2)-H), 6.48 (s, 1 H, C(6)-H), 7.04 (d, 2H, J = 8.5 Hz, C(3')-H and C(5')- H), 7.41 (d, 2H, J = 8.5 Hz, C(2')-H and C(6')-H); ¹³C NMR (75 MHz, DMSOd₆) δ 14.64, 22.78, 29.36, 29.44, 29.52, 29.56, 31.95, 45.62, 56.23, 56.71 , 78.28, 94.43, 95.73, 96.53, 107.72, 112.95, 116.69, 128.17, 133.17, 157.35, 157.46, 160.55, 161.39, 189.49. HRMS calcd for C₃₀H₄₃O₈ [M + H]⁺ 531.2958; found 531.2944.

8b. ¹H NMR (300 MHz, DMSOd₆) £0.82 (br t, 3H, J = 7.0 Hz, C(9a)-H), 1.11-1.42 (m, 14H, C(2a)- H to C(8a)-H), 2.37 (br t, 2H, J = 7.3 Hz, C(Ia)-H), 2.70 (dd, 1 H, J = 2.9, 16.9 Hz, C(3)-H_A), 3.15 (dd, 1 H, J = 12.5, 16.9 Hz, C(3)-H_B), 5.38 (dd, 1 H, J = 2.9, 12.5 Hz, C(2)-H), 5.94 (s, 1 H, C(6)-H), 6.77 (d, 2H, J = 8.5 Hz, C(3')-H and C(δ')-H), 7.28 (d, 2H, J = 8.5 Hz, C(2')-H and C(6')-H), 9.55, 10.70 and 12.11 (3s, 1 H each s, C(4')-0H, C(5)-0H and C(7)-0H); ¹³C NMR (75 MHz, DMS0-d₆) £ 14.65, 22.43, 22.79, 29.27, 29.38, 29.44, 29.49, 29.61 , 31.97, 42.71 , 78.70, 95.92, 102.41 , 108.49, 115.80, 128.46, 130.00, 158.21 , 160.60, 161.73, 165.25, 197.41. HRMS calcd for C₂₄H₂₉O₅ [M - H]^" 397.2015; found 397.2018.

2c. ¹H NMR (300 MHz, DMSO-d₆) £0.83 (br t, 3H, J = 7.0 Hz, C(Ha)-H), 1.15-1.42 (m, 18H, C(2a)- H to C(I Oa)-H), 2.42 (br t, 2H, J = 6.7 Hz, C(Ia)-H), 3.69 (s, 6H, C(I)-OCH₃ and C(3)-OCH₃), 3.71 (s, 3H, C(5)-OCH₃), 6.14 (s, 2H, C(4)-H and C(6)-H); ¹³C NMR (75 MHz, DMS0-d₆) £ 14.50, 22.66, 22.83, 29.50, 29.70, 29.80, 32.05, 55.59, 55.98, 91.14, 110.89, 158.87, 159.51. HRMS calcd for C₂₀H₃₅O₃ [M + H]⁺ 323.2586; found 323.2569.

3c. ¹H NMR (300 MHz, DMS0-d₆) £0.81 (br t, 3H, J = 6.7 Hz, C(Ha)-H), 1.22-1.45 (m, 18H, C(2a)- H to C(IOa)-H), 2.34 (s, 3H, C(I )-COCH₃), 2.41 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 3.57, 3.77 and 3.79 (3s, 3H each s, C(2)-OCH_3, C(4)-OCH₃ and C(6)-OCH₃), 6.46 (s, 1 H, C(5)-H); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.53, 22.81 , 23.45, 29.47, 29.55, 29.74, 29.77, 29.80, 30.00, 30.09, 32.03, 32.97, 56.39, 56.49, 63.22, 92.75, 116.47, 118.39, 156.13, 156.26, 160.16, 201.51. HRMS calcd for C₂₂H₃₇O₄ [M + H]⁺ 365.2692; found 365.2687.

4c. ¹H NMR (300 MHz, DMS0-d₆) £0.83 (br t, 3H, J = 7.0 Hz, C(Ha)-H), 1.14-1.44 (m, 18H, C(2a)- H to C(IOa)-H), 2.35 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 2.52 (s, 3H, C(I)-COCH₃), 5.96 (s, 1 H , C(5)- H), 10.18, 10.48 and 13.97 (3s, 1 H each s, C(2)-OH, C(4)-OH and C(6)-OH); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.65, 22.35, 22.80, 29.27, 29.41 , 29.69, 29.71 , 29.75, 29.80, 31.99, 33.18, 94.55, 104.40, 107.19, 160.73, 163.30, 164.06, 203.12. HRMS calcd for C₁₉H₂₉O₄ [M - H]^" 321.2066; found 321.2049.

5c. ¹H NMR (300 MHz, DMSO-d₆) <?0.83 (br t, 3H, J = 7.0 Hz, C(Ha)-H), 1.14-1.46 (m, 18H, C(2a)- H to C(IOa)-H), 2.47 (br t, 2H; J = 7.7 Hz, C(Ia)-H), 2.60 (s, 3H, C(I)-COCH₃), 3.37 and 3.42 (2s, 3H each s, C(4)-OCH₃ and C(6)-OCH₃), 5.26 and 5.29 (2s, 2H each s, C(4)-OCH₂ and C(6)-OCH₂), 6.34 (s, 1 H, C(5)-H), 13.83 (s, 1 H, C(2)-0H); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.64, 22.48, 22.79, 29.18, 29.39, 29.58, 29.65, 29.69, 29.72, 31.97, 33.70, 56.71 , 57.19, 92.07, 94.39, 95.17, 106.73, 111.59, 159.09, 161.34, 163.34, 204.27. HRMS calcd for C₂₃H₃₇O₆ [M - H]^" 409.2590; found 409.2592.

6c. ¹H NMR (300 MHz, DMSO-d₆) £0.81 (br t, 3H, J = 6.6 Hz, C(Ha)-H), 1.14-1.48 (m, 18H, C(2a)- H to C(IOa)-H), 2.50 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 3.36, 3.39 and 3.43 (3s, 3H each s, C(4)-OCH₃, C(4')-OCH₃ and C(6')-OCH₃), 5.24, 5.28 and 5.34 (3s, 2H each s, C(4)-OCH₂, C(4')-OCH₂ and C(6')-OCH₂), 6.39 (s, 1 H, C(5')-H), 7.08 (d, 2H, J = 8.8 Hz, C(3)-H and C(5)-H), 7.66 (d, 2H, J = 8.8 Hz, C(2)-H and C(6)-H), 7.70 (d, 1 H, J = 15.8 Hz, C(β)-H), 7.81 (d, 1 H, J = 15.8 Hz, C(α)-H), 13.70 (S, 1 H, C(2')-OH); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.63, 22.65, 22.80, 29.22, 29.42, 29.62, 29.68, 29.71 , 29.75, 31.99, 56.41 , 56.71 , 57.23, 92.94, 94.33, 94.44, 95.85, 107.65, 112.07, 117.19, 125.98, 129.06, 130.89, 142.98, 158.37, 159.40, 161.16, 163.33, 193.47. HRMS calcd for C₃₂H₄₅O₈ [M - H]^" 557.3114; found 557.3115.

7c. ¹H NMR (300 MHz, DMSO-d₆) £0.82 (br t, 3H, J = 7.0 Hz, C(Ha)-H), 1.11-1.48 (m, 18H, C(2a)- H to C(IOa)-H), 2.48 (br t, 2H, J = 6.8 Hz, C(Ia)-H), 2.66 (dd, 1 H, J = 2.9, 16.4 Hz, C(3)-H_A), 2.97 (dd, 1 H, J = 12.9, 16.4 Hz, C(3)-H_B), 3.36, 3.37 and 3.39 (3s, 3H each s, C(4')-OCH₃, C(5)-OCH₃ and C(7)-OCH₃), 5.17, 5.18 and 5.26 (3s, 2H each s, C(4')-OCH₂, C(5)-OCH₂ and C(7)-OCH₂), 5.44 (dd, 1 H, J = 2.9, 12.9 Hz, C(2)-H), 6.47 (s, 1 H, C(6)-H), 7.03 (d, 2H, J = 8.8 Hz, C(3')-H and C(5')- H), 7.41 (d, 2H, J = 8.8 Hz, C(2')-H and C(6').-H); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.64, 22.79, 22.90, 29.35, 29.41 , 29.46, 29.54, 29.62, 29.70, 29.75, 31.99, 45.63, 56.22, 56.70, 78.28, 94.44, 94.49, 95.75, 96.53, 107.73, 112.96, 116.68, 128.15, 133.17, 157.35, 157.47, 160.55, 161.39, 189.47. HRMS calcd for C₃₂H₄₇O₈ [M + H]⁺ 559.3271 ; found 559.3247.

8c. ¹H NMR (300 MHz, DMSO-d₆) £0.83 (br t, 3H, J = 7.0 Hz, C(Ha)-H), 1.11-1.42 (m, 18H, C(2a)- H to C(IOa)-H), 2.37 (br t, 2H, J = 7.0 Hz, C(Ia)-H), 2.70 (dd, 1 H, J = 2.9, 17.0 Hz, C(3)-H_A), 3.14 (dd, 1 H, J = 12.6, 17.0 Hz, C(3)-H_B), 5.38 (dd, 1 H, J = 2.9, 12.6 Hz, C(2)-H), 5.94 (s, 1 H, C(6)-H), 6.76 (d, 2H, J = 8.5 Hz, C(3')-H and C(5')-H), 7.28 (d, 2H, J = 8.5 Hz, C(2')-H and C(6')-H), 9.54, 10.66 and 12.10 (3s, 1 H each s, C(4')-OH, C(5)-OH and C(7)-OH); ¹³C NMR (75 MHz, DMSO-d₆) £ 14.63, 22.45, 22.77, 29.27, 29.38, 29.44, 29.47, 29.63, 29.68, 31.97, 42.72, 78.70, 95.95, 102.43, 108.51 , 115.82, 128.44, 130.01 , 158.22, 160.60, 161.74, 165.25, 197.38. HRMS calcd for C₂₆H₃₃O₅ [M - H]^" 425.2328; found 425.2341. 2d. ¹H NMR (300 MHz, DMSOd₆) £ 0.80 (s, 9H, C(3a)-H to C(5a)-H), 2.41 (s, 2H, C(Ia)-H), 3.68 (s, 6H, C(I)-OCH₃ and C(3)-OCH₃), 3.74 (s, 3H, C(5)-OCH₃), 6.18 (s, 2H, C(4)-H and C(6)-H); ¹³C NMR (75 MHz, DMS0-d₆) 5 30.44, 33.56, 35.32, 55.68, 55.86, 91.19, 108.46, 159.67, 159.71. HRMS calcd for C₁₄H₂₃O₃ [M + H]⁺ 239.1647; found 239.1631.

3d. ¹H NMR (300 MHz, DMS0-d₆) 50.82 (s, 9H, C(3a)-H to C(5a)-H), 2.37 (s, 3H, C(I)-COCH₃),

2.41 (S, 2H, C(Ia)-H), 3.52, 3.77 and 3.79 (3s, 3H each s, C(2)-OCH₃, C(4)-OCH₃ and C(6)-OCH₃), 6.47 (s, 1 H, C(5)-H); ¹³C NMR (75 MHz, DMS0-d₆) δ 30.39, 33.10, 33.69, 35.86, 56.15, 56.49, 62.54, 92.51 , 113.60, 118.13, 156.28, 157.47, 160.73, 201.73. HRMS calcd for C₁₆H₂₅O₄ [M + H]⁺ 281.1753; found 281.1742.

4d. ¹H NMR (300 MHz, DMS0-d₆) 50.84 (s, 9H, C(3a)-H to C(5a)-H), 2.35 (s, 2H, C(Ia)-H), 2.53 (s, 3H, C(I)-COCH₃), 5.98 (s, 1 H, C(5)-H), 10.13, 10.47 and 14.09 (3s, 1 H each s, C(2)-OH C(4)- OH and C(6)-OH); ¹³C NMR (75 MHz, DMS0-d₆) 5 30.69, 33.16, 34.00, 35.09, 94.53, 104.34, 105.06, 160.85, 164.22, 165.16, 203.02. HRMS calcd for C₁₃H₁₇O₄ [M - H]^' 237.1127; found 237.1123.

5d. ¹H NMR (300 MHz, DMS0-d₆) 50.87 (s, 9H, C(3a)-H to C(5a)-H), 2.46 (s, 2H, C(Ia)-H), 2.62 (s, 3H, C(I)-COCH₃), 3.38 and 3.44 (2s, 3H each s, C(4)-OCH₃ and C(6)-OCH₃), 5.23 and 5.30 (2s, 2H each s, C(4)-OCH₂ and C(6)-OCH₂), 6.37 (s, 1H, C(5)-H), 13.94 (s, 1 H, C(2)-OH); ¹³C NMR (75 MHz, DMSO-d₆) 530.67, 33.71 , 33.88, 35.20, 56.97, 57.25, 91.89, 94.82, 95.24, 106.59, 109.25, 159.38, 162.54, 164.37, 204.25. HRMS calcd for C₁₇H₂₅O₆ [M - H]^" 325.1651 ; found 325.1636.

6d. ¹H NMR (300 MHz, DMSO-d₆) 50.89 (s, 9H, C(3a)-H to C(5a)-H), 2.49 (s, 2H, C(Ia)-H), 3.37, 3.40 and 3.44 (3s, 3H each s, C(4)-OCH₃, C(4')-OCH₃ and C(6')-OCH₃), 5.24, 5.25 and 5.35 (3s, 2H each s, C(4)-OCH₂, C(4>OCH₂ and C(6>OCH₂), 6.41 (s, 1 H, C(5')-H), 7.08 (d, 2H, J = 8.8 Hz, C(3)-H and C(5)-H), 7.66 (d, 2H, J = 8.8 Hz, C(2)-H and C(6)-H), 7.69 (d, 1 H, J = 15.4 Hz, C(β)-H), 7.80 (d, 1 H, J = 15.4 Hz, C(α)-H), 13.78 (s, 1H, C(2')-OH); ¹³C NMR (75 MHz, DMSO-d₆) 530.67, 33.92, 35.34, 56.43, 56.99, 57.30, 92.72, 94.38, 94.86, 95.89, 107.47, 109.66, 117.23, 126.14, 129.11 , 130.89, 142.88, 158.66, 159.41 , 162.36, 164.31 , 193.51. HRMS calcd for C₂₆H₃₃O₈ [M - H]^" 473.2175; found 473.2188.

7d. ¹H NMR (300 MHz, DMSO-d₆) 50.83 (s, 9H, C(3a)-H to C(5a)-H), 2.45 (d, 1 H, J = 12.9 Hz, C(1a)-H_A), 2.49 (d, 1 H, J = 12.9 Hz, C(Ia)-H₆), 2.60 (dd, 1 H, J = 2.6, 16.4 Hz, C(3)-H_A), 2.97 (dd, 1 H, J = 13.2, 16.4 Hz, C(3)-H_B), 3.37, 3.38 and 3.41 (3s, 3H each s, C(4')-OCH₃, C(5)-OCH₃ and C(7)-OCH₃), 5.19, 5.20 and 5.22 (3s, 2H each s, C(4')-OCH₂, C(5)-OCH₂ and C(7)-OCH₂), 5.38 (dd, 1 H, J = 2.6, 13.2 Hz, C(2)-H), 6.51 (s, 1 H, C(6)-H), 7.05 (d, 2H, J = 8.5 Hz, C(3')-H and C(5')-H),

7.42 (d, 2H, J = 8.5 Hz, C(2')-H and C(6')-H); ¹³C NMR (75 MHz, DMSO-d₆) 530.60, 33.72, 35.69, 46.09, 56.29, 56.75, 56.94, 78.55, 94.49, 94.94, 95.74, 96.35, 107.64, 110.48, 116.73, 128.29, 133.15, 157.36, 157.82, 161.71 , 162.21 , 189.61. HRMS calcd for C₂₆H₃₅O₈ [M + H]⁺ 475.2332; found 475.2329.

8d. ¹H NMR (300 MHz, DMSO-d₆) £ 0.81 (s, 9H, C(3a)-H to C(5a)-H), 2.33 (d, 1 H, J = 12.9 Hz, C(1a)-H_A), 2.36 (d, 1 H, J = 12.9 Hz, C(Ia)-H₆), 2.62 (dd, 1 H, J = 2.9, 17.0 Hz, C(3)-H_A), 3.15 (dd, 1 H, J = 13.2, 17.0 Hz, C(3)-H_B), 5.32 (dd, 1 H, J = 2.9, 13.2 Hz, C(2)-H), 5.96 (s, 1 H, C(6)-H), 6.77 (d, 2H, J = 8.8 Hz, C(3')-H and C(5')-H), 7.28 (d, 2H, J = 8.8 Hz, C(2')-H and C(6')-H), 9.54, 10.66 and 12.21 (3s, 1 H each s, C(5)-OH, C(7)-OH and C(4')-OH); ¹³C NMR (75 MHz, DMS0-d₆) £30.53, 33.84, 35.36, 43.11 , 78.93, 95.94, 102.29, 106.25, 115.82, 128.63, 130.03, 158.19, 161.48, 161.94, 166.29, 197.54. HRMS calcd for C₂oH₂i0₅ [M - H]^" 341.1389; found 341.1397.

EXAMPLE 2: Assays for estrogen receptor modulating activity in vitro

An estrogen is a compound that can bind to one of the estrogen receptors (ERs) and subsequently exert a 17β-estradiol-like physiological response, which is, in most cases (but not limited), the result of binding of the dimerized ligand-containing receptor to estrogen-responsive elements (ERE) in the promotor region of estrogen-responsive genes. After binding, the receptor complex associates with activating or repressing peptides that also interact with the gene transcription machinery. In-vitro estrogenic responses are the growth of breast cancer cells and the expression of certain proteins including the induction of ERE-controlled luciferase- or galactosidase-containing plasmids. An antiestrogen is a compound that antagonizes the effect of 17β-estradiol.

All flavanones were synthesized from the corresponding chalcones via regioselective but not enantioselective cyclization, hence, they are obtained as racemic mixtures. However, recent reports in the literature have indicated for 8- prenylnaringenin that both R- and S-enantiomers show comparable binding affinities and estrogenic activities in vitro and in vivo, on both ER-subtypes (Schaefer et al., 2003, above; Kitaoka et al., 1998). Therefore, in a first instance, all compounds were examined as racemic mixtures using various in-vitro bioassays. Principle of the test

The binding of a compound on the estrogen receptor was tested by incubating a pure preparation of the estrogen receptor with a receptor saturating concentration of radioactively labelled [³H]-17β-estradiol in combination with increasing amounts of the investigated compounds. Subsequently, receptor-bound and free [³H]-17β- estradiol are separated and the bound fraction is measured. Lower values indicate increased displacement from the receptor. The stronger a compound binds to the receptor, the lower the concentration needed to achieve this displacement.

Methods

More specifically, the ER-binding affinities of the naringenin derivatives substituted at position 8 were determined in a competitive radiometric binding assay using purified full-length human ERa and ERβ. In brief, binding of a compound to the estrogen receptor was tested by incubating a pure preparation of the estrogen receptor in buffer with a receptor-saturating concentration of [³H]-17β-estradiol (1 nM) in combination with increasing amounts of the investigated compounds. Subsequently, receptor-bound and free [³H]-17β-estradiol were separated using dextran-coated charcoal and the bound fraction was measured (liquid scintillation).

Results

For compounds 8a to 8d, as well as for a number of reference compounds, the results of the competition reactions are presented in Table 1 and expressed as 50% inhibition values (IC₅₀), which values represent the means of three experiments ± standard deviation. A selectivity ratio > 1 depicts a greater affinity for ERβ compared to ERa.

Table 1. Estrogen-receptor binding affinities of 8-alkylnaringenins, together with those of reference compounds. Compound^* BA on ERa BA on ERβ Selectivity^1"

(i) 17β-Estradiol 1.2 ± 0.2 1.4 ± 0.6 0.9

(i) Hydroxytamoxifen 1.4 ± 0.6 1.3 ± 0.6 1.0

(i) Genistein 1 ,145 ± 368 25 ± 7 45

(i) 8-Prenyl-N 57 ± 10 68 ± 33 0.8

(i) 8-Geranyl-N 180 + 43 78 ± 37 2.6

8-/τ-Heptyl-N (8a) 392 ± 45 69 ± 8 5.7

8-A7-Nonyl-N (8b) 436 ± 105 113 ± 22 3.9

8-n-Undecyl-N (8c) 1 ,017 + 201 421 + 161 2.4

8-(2,2-Dimethylpropyl)-N

216 + 44 141 ± 51 1.5 (8d)

8-n-Pentyl-N (8g) 383 ± 123 97 ± 50 4.0

8-(3-Methylbutyl)-N (8h) 140 ± 31 59 + 4 2.4

^* N: naringenin; BA: binding affinity (mean ± standard deviation, n = 3); ratio BA on ERα/BA on ERβ; (i): known compounds

EXAMPLE 3: ERa- and ERβ-agonist and -antagonist titration curves

Principle of the test

Agonistic and antagonistic activity on both ERs was determined separately using a cell-based assay. Estrogen-receptor null cells were transfected with an estrogen- responsive reporter gene in conjunction with ERa or ERβ. Subsequently, increasing concentrations of the investigated compounds were added to the cells in the absence (agonistic) or the presence (antagonistic) of 17β-estradiol. Methods

Transcriptional activities of the (±)-8-alkyl- and 8-alkenylnaringenins as wel as of other 8-substituted naringenin derivatives, were assayed in human cervical cancer cells (HeIa) and human hepatoma (HuH7) cells transfected with ERa or ERβ and an estrogen-responsive luciferase reporter gene construct, 3xERE-TATA-luciferase reporter and a β-galactosidase reporter. Experiments were performed in triplicate and contained 50ng of ER receptor, 500ng or reporter construct and 20ng of β- Galactosidase per well. Agonistic and antagonistic activities on both ERs were determined separately by increasing concentrations of the compound (1 nM-10 μM) in the absence (agonistic) or presence (antagonistic) of 1 nM 17β-estradiol.

Results

8-PN clearly showed the highest overall potency on both ERa and ERβ. 8a did not display a high degree of agonism on ERa, and was a very weak agonist on ERβ, exerting substantial antagonist character. 8b and 8c elicited no or very little transcriptional activity on both receptors, but they appeared to very effectively antagonize the effect of 17β-estradiol on the ERs.

As expected, naringenins substituted with methyl (8e) and π-propyl (8f) showed full agonist character for ERa and ERβ, with a higher potency for the latter compound. For ERa, 8-n-pentylnaringenin (8g) gave a high response, while its capacity to induce an agonist conformation in ERβ was much lower. Introducing a 3- methylbutyl substituent (8h) gave only partial agonist character on ERβ, while full agonist character on ERa was maintained.

The branched-chain analogs all exhibited full or nearly full agonist character for ERa, but substantial differences in transcriptional activity were noted on ERβ. An equally high response on both receptors was observed for 8-(2- methylpropyl)naringenin (8i). 8d, possessing the short but very bulky 2,2- dimethylpropyl group at C(8), had an uncommon agonist/antagonist profile, acting as a full agonist on ERa and as an antagonist on ERβ. Naringenin substituted with benzyl (8j) showed partial agonist activity on ERβ.

Figure 3A and B demonstrate the agonist and antagonist activity of the naringenin derivatives on gene transcription by ERa and ERβ, respectively, monitored on estrogen-responsive ERE reporter in HuH7 cells, in increasing concentrations.

An overview of the described effects is given in Table 2.

Table 2: Transcriptional activity of 8-aIkylnaringenins detected in HeIa and/or HuH7 cells transfected with either ERa and ERβ, together with an ERE-containing luciferase reporter plasmid.

Compound* ERa ERβ Rating^**

(i) 8-Prenyl-N agonist agonist -

(i) 8-Geranyl-N agonist antagonist 2

8-π-Heptyl-N (8a) Antagonist antagonist 1

8-π-Nonyl-N (8b) antagonist antagonist 1

8-/7-Undecyl-N (8c) antagonist antagonist 1

8-(2,2-Dimethyipropyl)-N (8d) agonist antagonist 2

8-methyl-N (8e) agonist agonist -

8-propyl-N (8f) agonist agonist -

8-pentyl-N (8g) agonist Partial agonist 3

8-3-methyl-butyl-N (8h) agonist Partial agonist 3

8-2-methyl-propyl-N (8i) agonist Agonist -

8-benzyl-N (8j) agonist Partial agonist 3

* N = naringenin ^** Pertains to the ER affinity profile: 1 : antiestrogenic on both ERs; 2: mixed estrogenic ERα/antiestrogenic ERβ; 3: mixed estrogenic ERα/partial agonistic ERβ; (i) known compounds. EXAMPLE 4: Effect of naringenϊn derivatives on bone mineral density and uterus weight in ovariectomized rats

The aim of the study is to examine bone and uterine effects of a 4-week treatment with naringenin derivatives in 3-month-old ovariectomized rats. The derivatives are administered at the dose levels of 0.67 mg/kg, 1 ,77 mg/kg and 18 mg/kg subcutaneously (s.c), once a day, seven times a week. At the end of the study, uterine weight and ex vivo bone mineral density (BMD) of tibia are measured.

Description of study

A total of 30 female 13-14-week-old Sprague-Dawley rats are used. The animals are randomized into 5 groups, with 6 rats/group. Group 1 is sham-operated (SHAM) and groups 2-5 are ovariectomized (OVX).

The mean body weight of the animals is determined before operation. Treatments with the vehicle or test substance are started on the day following operation. The animals in the SHAM group are given a vehicle (e.g. 30% hydroxypropyl- beta - cyclodextrin s.c), and in the OVX groups either the vehicle or the naringenin derivative is given at nominal doses of 0.67 mg/kg/d, 1.77 mg/kg/d or 18 mg/kg/d s.c. Treatments are continued for 4 weeks; the dosing frequency is seven times a week. The animals are sacrificed one day after the last dosing. At the end of the study, left tibiae are excised for ex vivo BMD measurement (peripheral quantitative computed tomography (pQCT)) at proximal tibia and uteri are excised for the determination of absolute and relative weights. Relative uterine weight is calculated as percentage of body weight. The data of body weights, relative uterine weights and BMD are analyzed with oneway analysis of variance.

A post-study histological examination is performed. The formalin-fixed uteri are embedded in paraffin, cut into 5μm transverse sections and are stained with haematoxylin eosin. The sections are then evaluated quantitatively for luminal epithelium cell height.

Claims

1. A naringenin derivative according to the general structural formula I:

I or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- R₁, R₈, Rg and R₁₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci_-5 alkyl, Ci_-5 alkoxy, Ci_-5 alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R₂ is selected from a branched C₅ alkyl, a C_6-2o alkyl, C6-20 alkenyl, C_6-2o alkynyl or C₆-20 alkenynyl, wherein each of said C_6-2o alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R₃, R_4, R₅, Rε and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

- Xi is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom. wherein said naringenin derivative is not 8-geranylnaringenin.

2. A naringenin derivative according to claim 1 wherein:

- R₂ is selected from a branched C5 alkyl, a C6-20 alkyl, C_6-2o alkenyl, C6-20 alkynyl or C_6-2o alkenynyl, wherein each of said C_6-2o alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R_{1 ,} R₅ and R₉ are OH;

- R₃, R₄, R₆, R7, Rs and Ri₀ are H; and

- Xi and X₂ are O.

3. The naringenin derivative according to claim 1 or 2, wherein R₂ is selected from /7-heptyl; n-nonyl; n-undecyl;

4. The naringenin derivative according to claim 1 or 2, wherein R₂ is 2,2- dimethylpropyl.

5. The compounds of any one of claims 1 to 4, for use as a medicament.

6. The use of naringenin derivatives according to the general structural formula I, or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- Ri, R₈, Rg and R₁₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci_-5 alkyl, C_1-5 alkoxy, C_1-5 alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R₂ is selected from a C_5-2O alkyl, C_6-2O alkenyl, C_6-2o alkynyl or C_6-2o alkenynyl, wherein each of said alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched; - R₃, R₄, Rs, R_δ and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

- X₁ is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom, in the manufacture of a medicament for the treatment of estrogen-deficiency dependent disorders, diseases with excessive estrogen production or conditions benefiting from modulated estrogen production.

7. The use according to claim 6, wherein said disease is an estrogen-dependent cancer, endometriose, gynecomastie, or a condition which is the result of impaired or inadequate hypothalamo-pituitary regulation of gonadal functioning.

8. The use according to claim 6, wherein said medicament is an ovulatory agent.

9. The use according to any one of claims 6 to 8, wherein said compound does not negatively affect bone homeostasis.

10. The use according to any one of claims 6 to 9, wherein said compound positively affects bone homeostasis.

11. The use according to any one of claims 6 to 10, wherein:

- R2 is selected from a C_5-2O alkyl, C_6-2O alkenyl, C_6-2O alkynyl or C_6-2O alkenynyl, wherein each of said C_6-2O alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched; R₁, R₅ and R₉ are OH;

- R₃, R₄, Re, R7. Rs and R10 are H; and

12. The use according to claim 11 , wherein said compound is 8-(2,2- dimethylpropyl)naringenin or 8-geranylnaringenin.

13. The use of naringenin derivatives according to the general structural formula I, or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- Ri₁ R₈, Rg and R₁₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, Ci-₅ alkyl, Ci_-5 alkoxy, Ci_-5 alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R₂ is selected from a C_5-2o alkyl, C_6-2o alkenyl, C_6-2o alkynyl or C₆-2o alkenynyl, wherein each of said alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R₃, R₄, R₅, R₆ and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

- Xi is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom, in the preparation of a medicament for the treatment and/or prevention of disorders characterized by the need for ERβ antagonism.

14. The use according to claim 13, wherein said compound is 8-(2,2- dimethylpropyl)naringenin or 8-geranylnaringenin.

15. The use according to claim 13 or 14, wherein said disorder is a disorder of bone homeostasis.

16. The use according to claim 15, wherein said disorder is osteoporosis.

17. A method of treatment or prevention of a disorder which comprises the administration of an naringenin derivatives according to the general structural formula I, or any stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, wherein:

- the dotted line represents optionally a double bond;

- Ri₁ Rs, R9 and Ri₀ are independently selected from OH, H, halogen, cyano, amino, nitro, nitroso, C₁-₅ alkyl, Ci_-5 alkoxy, C₁-₅ alkylcarbonyloxy; wherein each of said alkyl, alkoxy or alkylcarbonyloxy group is linear or branched;

- R₂ is selected from a C_5-2o alkyl, C_6-2o alkenyl, C_6-2o alkynyl or C_6-2o alkenynyl, wherein each of said C₆-₂₀ alkyl, alkenyl, alkynyl or alkenynyl group is linear or branched;

- R₃, R₄, R5, Re and R₇ are independently selected from OH, H, halogen, cyano, amino, nitro or nitroso;

- Xi is selected from O, S;

- X₂ is selected from O, S or the two bonds are each separately formed with a hydrogen atom wherein said naringenin derivative ensures modulated estrogen production or the blocking of estrogen-mediated signalling in the hypothalamus.

18. The method of treatment according to claim 17, wherein said disorder is a disorder of bone homeostasis.

19. The method of treatment according to claim 18, wherein said disorder is osteoporosis.

20. The method of treatment according to claim 17, wherein said disorder is selected from the group consisting of an estrogen-dependent cancer, endometriose, gynecomastie, or a condition which is the result of impaired or inadequate hypothalamo-pituitary regulation of gonadal functioning.