WO2006107273A1 - Compounds having progestogenic activity and their use - Google Patents

Abstract

The invention relates to compounds having progestogenic activity and to their use. The invention discloses a compound having the general formula (I) wherein Y, Y', X and X' are independently selected from the group consisting of O, S and N; R1 is selected from the group consisting of optionally substituted C1-8 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C1-6 alkoxy, optionally substituted C1-6 thioalkyl, optionally substituted C4-10 aryl, optionally substituted C6-11 alkylaryl, optionally substituted C1-6 alkylamino, optionally substituted C1-6 alkylcarbonyl, optionally substituted C1-6 alkylsulfonamino, optionally substituted (C1-C6)alkylsulfinyl, optionally substituted C1-6 alkylcarbonylamino, optionally substituted hetero(C5-C10)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; R2, R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen, optionally substituted C1-8 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C1-6 alkoxy, optionally substituted C1-6 thioalkyl, optionally substituted C5-10 aryl, optionally substituted C6-11 alkylaryl, optionally substituted C1-6 alkylamino, optionally substituted C1-6 alkylcarbonyl, optionally substituted C1-6 alkylsulfonamino, optionally substituted (C1-C6)alkylsulfinyl, optionally substituted C1-6 alkylcarbonylamino, optionally substituted hetero(C5-C10)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; ---- indicates an optional double bond.

Description

Compounds having progestogenic activity and their use

Technical Field

The present invention generally relates to compounds having progestogenic activity, to compositions comprising the compounds and to uses thereof.

Background

Progesterone (PR) is a steroid hormone that has an essential role in mammalian reproduction. PR has been implicated in the function of the cardiovascular, immune, bone and central nervous systems. Progestational agents are used for oral contraception, menopausal hormone replacement therapy, and cancer treatments. Progestogens act by binding specifically to the progesterone receptor in responsive cells. The receptor-ligand complex then translocates to the cell nucleus, binds to chromosomal DNA in the promoters of progesterone-responsive genes thereby regulating gene transcription. More recently, non- receptor mediated actions of progesterone have also been described.

The PR receptor is a member of the steroid/nuclear receptor superfamily of transcription factors (Nuclear Receptor Nomenclature Committee, 1999). The 50 or so proteins of this superfamily are involved in diverse physiological functions such as control of embryonic development, cell differentiation, and homeostasis.

There are two natural isoforms of PR, the A- and B-receptors, also referred to herein as PR-A and PR-B, respectively. PR-A and PR-B differ in that PR-B has an additional stretch of 164 amino acids located at the aminoterminus of the receptor. When both receptors are expressed in equimolar quantities, PR-A and PR-B can heterodimerize and transactivate. Like other steroid receptors, the progesterone receptor has four main functional domains, namely the N-terminal transactivation domain, followed by the Hinge, DNA-binding, and Ligand-binding domains. The most conserved domain is the DNA-binding domain. On activation by ligand, the DNA- binding domain binds to progesterone-responsive elements (PRE) which are composed of two palindromic sequences.

The consensus PRE sequence is TGTACAnnnTGTTCT, where n represents any nucleotide. The carboxyl terminal domain is the ligand-binding domain (Bourguet et al 1995, Carson- Jurica etal 1990) which in the presence of progesterone causes nuclear translocation, dimerization and formation of the pre-initiation complex on PRE of progesterone-responsive genes. Compounds with biological activity similar to PR are known as progestogens, progestins or gestagens. The majority of progestogens are steroidal compounds belonging to progesterone and its derivatives, and testosterone and 19- nortestosterones and their derivatives. A smaller number of progestogens that are non-steroidal are also known, including ligands based on sesquiterpenes possessing a ligularenolid skeleton (Kurihara, 1999), tetrahydropyridazines RWJ 26819 (Palmer, 2000),5-Aryl-1 , 2,3, 4- tetrahydrochromeno [3,4-f] quinolin-3-ones (Zhi etal 1999) and the fluorinated phthalides (Lehman et al 2001 ).

Progestins have a wide range of applications such as in oral contraception, hormone replacement therapy, and in cancer from postmenopausal estrogen in women.

Presently, the progestogens available commercially comprise synthetic products such as medroxyprogesterone acetate and progesterol acetate. These synthetic progestogens have been extensively studied by The Women's Health Initiative (WHI), the headquarters of which are located at the Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America. The WHI is a long- term national health study that focuses on strategies for preventing heart disease, breast and colorectal cancer and osteoporosis in postmenopausal women. The WHI studies have found that certain synthetic progestogens result in undesirable side effects. It is suspected that long-term estrogen/progesterone replacement is associated with significant increases in the risk of stroke (41%), heart attacks (29%), cardiovascular disease (22%), thrombosis (200%), breast cancer (26%) and dementia (Rossouw JE, Anderson GL, Prentice RL, et al. (2002). Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. JAMA. 288:321-33.). Women around the globe have decided to discontinue traditional estrogen/progesterone replacement, despite suffering from menopausal symptoms. Studies indicate that it is the progestogen component that to a large extent responsible for these unfavorable effects on breast cancer (Sitruk-Ware R, Plu-Bureau G. Exogenous progestagens and the human breast. Maturitas. 2004 Sep 24;49(1):58-66.) and cardiovascular disease. It is therefore important to develop new progestogens which overcome the above drawbacks.

In addition, considering the growing popularity for drugs based on traditional systems of medicine across the world, herbs and herbal extracts are of prime importance. Thus, phytoprogestogenic compounds would open new avenues of treatment for many people. The use of phytoprogestogens as progestogens are of significant commercial value.

Summary

According to a first aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable amount of a compound having the general formula (I):

and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, in admixture with one or more pharmaceutically acceptable carriers, wherein

Y, Y', X and X' are independently selected from the group consisting of O, S and N;

R₁ is selected from the group consisting of optionally substituted d-β alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted Ci_-6 thioalkyl, optionally substituted C_4-10 aryl, optionally substituted C_6-H alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (Ci-C₆)alkylsulfinyl, optionally substituted Ci_-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₂. R₃. R₄. R₅. R₆ and R₇ are independently selected from the group consisting of hydrogen, optionally substituted Ci_-8 alkyl, optionally substituted C₂^ alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted Ci_-6 thioalkyl, optionally substituted C_5-I0 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted Ci_-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (Ci-C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅- C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

— indicates an optional double bond.

According to a second aspect of the invention, there is provided a compound having the structure (II):

(N)

Y, Y', X and X' are independently selected from the group consisting of O, S and N;

R₁ is selected from the group consisting of optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (d-CβJalkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₂. R3. R4. R5. Re and R₇ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C₁^ alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_4-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C₁^ alkylsulfonamino, optionally substituted (C^CeJalkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅- C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; — indicates an optional double bond. According to a third aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable amount of a compound having the structural formula (II) as defined in the second aspect, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, in admixture with one or more pharmaceutically acceptable carriers.

According to a fourth aspect of the invention, there is provided a compound of structural formula (III):

(III).

According to a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound of structural formula (III) as defined in the fourth aspect, and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, in admixture with one or more pharmaceutically acceptable carriers.

According to a sixth aspect of the invention, there is provided an extract of Ligusticum Chuanxiong comprising a therapeutically effective amount a compound of structural formula (III) as defined in the fourth aspect.

According to a seventh aspect of the invention, there is provided a pharmaceutical composition comprising an extract of Ligusticum Chuanxiong having a therapeutically effective amount a compound of structural formula (III) as defined in the fourth aspect.

According to an eighth aspect of the invention, there is provided one or more compounds selected from the group consisting of compounds having the formula (I), formula (II) and formula (III) as respectively defined in the first, second, and fourth aspects, or an extract as defined in the sixth aspect, for use in medicine.

According to a ninth aspect of the invention, there is provided the use of one or more compounds selected from the group consisting of compounds having the formula (I), formula (II) and formula (III) as respectively defined in the first, second, and fourth aspects, or an extract as defined in the sixth aspect, in the manufacture of a medicament for the treatment of a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries, in a mammal.

According to a tenth aspect of the invention, there is provided a method of treating a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in a mammal, the method comprising the step of administering a therapeutically effective amount of one or more compounds selected from the group consisting of compounds having the structural formula (I), formula (M) and formula (III) as respectively defined in the first, second, and fourth aspects, or an extract as defined in the sixth aspect, to the mammal.

According to an eleventh aspect of the invention, there is provided a kit comprising a pharmaceutical composition as defined in the first aspect, third aspect, fifth aspect or seventh aspect, and instructions for administering the composition to a mammal for the treatment of a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in the mammal.

According to a twelfth aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula (IV):

(IV) wherein

Y, Y', X and X' are independently selected from O, S and N; Ri, R₂. R₃, R₄ and R₆ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C₁^ alkylsulfonamino, optionally substituted (CrCeJalkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅- Cio)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₅ is selected from the group consisting of , optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C₅.₁₀ aryl, optionally substituted C_6-1I alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C₁^ alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (CrC₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; n is 1, 2, 3 or 4; and — indicates an optional double bond.

According to a thirteenth aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound of structural formula (IVA):

wherein

X, X", Y and Y' are independently selected from O, S and N;

Ri. R₂. R₃, R₄ and R₆ are independently selected from the group consisting of hydrogen, optionally substituted Ci_-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C₁^ alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C_rC₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅- C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₅ is selected from the group consisting of , optionally substituted C_1-S alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C₂^ alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted Cs_-10 aryl, optionally substituted C_6-n alkylaryl, optionally substituted C₁^ alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C₁^ alkylsulfonamino, optionally substituted (C₁-C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; n is 1, 2, 3 or 4; and

— indicates an optional double bond.

According to a fourteenth aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound of structural formula (Vl):

(Vl). and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, in admixture with one or more pharmaceutically acceptable carriers.

According to a fifteenth aspect of the invention, there is provided an extract of Ligusticum Chuanxiong comprising a therapeutically effective amount a compound of structural formula (Vl) as defined in the fourteenth aspect.

According to a sixteenth aspect of the invention, there is provided a pharmaceutical composition comprising an extract of Ligusticum Chuanxiong having a therapeutically effective amount of a compound of structural formula (Vl) as defined in the fourteenth aspect.

According to a seventeenth aspect of the invention, there is provided one or more compounds selected from the group consisting of compounds having the structural formula (IV), (V) and (Vl) as respectively defined in the twelfth, thirteenth, and fourteenth aspects, or an extract as defined in the fifteenth aspect, for use in medicine.

According to an eighteenth aspect of the invention, there is provided use of one or more compounds selected from the group consisting of compounds having the structural formula (IV), (V) and (Vl) as respectively defined in the twelfth, thirteenth, and fourteenth aspects, or an extract as defined in the fifteenth aspect, in the manufacture of a medicament for the treatment of a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in a mammal. The condition associated progesterone replacement or progesterone supplementation may be selected from the group consisting of menstrual disorders, amenorrhoea, menorrhagia, polycystic ovarian syndrome, pregnancy complications, endometriosis, contraception, menopause, endometrial hyperplasia and hormonal replacement. According to a nineteenth aspect of the invention, there is provided a method of treating a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in a mammal, the method comprising administering a therapeutically effective amount of one or more compounds selected from the group consisting of compounds having the structural formula (IV), (V) and (Vl) as respectively defined in the twelfth, thirteenth, and fourteenth aspects, or an extract as defined in the fifteenth aspect, to the mammal.

According to a twentieth aspect of the invention, there is provided a kit comprising a pharmaceutical composition as defined in the twelfth, thirteenth or fourteenth aspects, and instructions for administering the composition to a mammal for the treatment of a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in the mammal.

According to a twenty first aspect of the invention, there is provided a combined preparation for use in medicine, the combined preparation comprising compounds of structural formula (I) as defined in the first aspect and compound (IV) as defined in the twelfth aspect.

According to a twenty second aspect of the invention, there is provided a combined preparation for use in a medicament, the combined preparation comprising flavonoid calycosin and at least one compound of structural formula (I) as defined in the first aspect or compound (IV) as defined in the twelfth aspect. Optionally, the flavonoid calycosin and said at least one compound of structural formula (I) as defined in the first aspect or compound (IV) as defined in the twelfth aspect are simultaneously, separately or sequentially administered.

According to a twenty third aspect of the invention, there is provided the use of flavonoid calycosin and at least one compound of structural formula (I) as defined in the first aspect or compound (IV) as defined in the twelfth aspect, in the manufacture of a combined preparation for treating a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in a mammal. The condition associated progesterone replacement or progesterone supplementation may be selected from the group consisting of menstrual disorders, amenorrhoea, menorrhagia, polycystic ovarian syndrome, pregnancy complications, endometriosis, contraception, menopause, endometrial hyperplasia and hormonal replacement.

In one embodiment the flavonoid calycosin and said at least one compound of structural formula (I) as defined in the first aspect or compound (IV) as defined in the twelfth aspect, may be present as separate formulations or as a single formulation. Where in the form of separate formulations, flavonoid calycosin and said at least one compound of structural formula (I) as defined in the first aspect or compound (IV) as defined in the twelfth aspect, may be administered separately, sequentially or simultaneously. According to a twenty fourth aspect of the invention, there is provided a method of producing an extract of Ligusticum Chuanxiong for use in medicine, the method comprising extracting Ligusticum Chuanxiong rhizome in a solvent and for a time to produce a therapeutically effective amount of at least one compound selected from the group having the general formula (III) as defined in the fourth aspect above and the general formula (Vl) as defined in the fourteenth aspect above.

According to a twenty fifth aspect of the invention, there is provided Ligusticum Chuanxiong for use in medicine, produced in a method as defined in the twenty fourth aspect.

According to a twenty sixth aspect of the invention, there is provided use of a reference compound for screening one or more candidate compounds for bioactivity, the reference compound having the general formula (I) as defined in the first aspect or having the general formula (IV) as defined in the twelfth aspect. In one embodiment, the bioactivity is progestogenic activity.

According to a twenty seventh aspect of the invention, there is provided a method of screening candidate compounds for bioactivity, the method comprising the step of: (a) comparing a parameter associated with the candidate compounds to a reference parameter associated with one or more reference compounds having the general formula (I) as defined in the first aspect or having the general formula (IV) as defined in the twelfth aspect. In one embodiment, the bioactivity is progestogenic activity. In one embodiment, the method of the twenty sixth aspect comprises, before step (a), the step of:

(b) separating said one or more candidate compounds using a chromatographic technique.

According to a twenty eighth aspect of the invention, there is provided an assay for use in chromatography, said assay comprising the use of one or more compounds having the general formula (I) as defined in the first aspect or having the general formula (IV) as defined in the twelfth aspect.

Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description. Where chiral carbons are included in chemical structures, unless a particular orientation is depicted, both stereoisomeric forms are intended to be encompassed.

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

As used herein, the term "alkyl group" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 10 carbon atoms, eg, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, the term alkyl includes, but is not limited to, methyl, ethyl, 1- propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1 ,2-dimethylpropyl, 1,1- dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2-dimethylbutyl, 1,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3- ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4- dimethylpentyl, 1 ,2-dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4-dimethylpentyl, 1 ,2,3- trimethylbutyl, 1,1 ,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, and the like. The term "alkenyl group" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms and having at least one double bond, of either E, Z, cis or trans stereochemistry where applicable, anywhere in the alkyl chain. Examples of alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2- propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butentyl, 1 ,3-butadienyl, 1-pentenyl, 2-pententyl, 3-pentenyl, 4-pentenyl, 1 ,3-pentadienyl, 2,4- pentadienyl, 1 ,4-pentadienyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3- hexadienyl, 1 ,4-hexadienyl, 2-methylpentenyl, 1-heptenyl, 2-heptentyl, 3-heptenyl, 1- octenyl, 1-nonenyl, 1-decenyl, and the like.

The term "alkynyl group" as used herein includes within its meaning monovalent ("alkynyl") and divalent ("alkynylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms and having at least one triple bond anywhere in the carbon chain. Examples of alkynyl groups include but are not limited to ethynyl, 1-propynyl, 1-butynyl, 2-butynyl, 1- methyl-2-butynyl, 3-methyl- 1-butynyl, 1-pentynyl, 1-hexynyl, methylpentynyl, 1- heptynyl, 2-heptynyl, 1-octynyl, 2-octynyl, 1 -nonyl, 1-decynyl, and the like.

The term "halogen" or variants such as "halide" or "halo" as used herein refers to fluorine, chlorine, bromine and iodine. The term "alkoxy" as used herein refers to straight chain or branched alkyloxy groups. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocycloxy, heterocycloamino, haloheterocycloalkyl, alkylsulfenyl, alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cyano, cyanate, isocyanate, -C(O)NH(alkyl), and -C(O)N(alkyl)₂. The term "hydroxyl" means -OH.

The term "thiol" means -SH. The term "cyano" means -CN. The term "nitro" means -NO₂. The term "amino" means -NH₂. The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Examples include N, O, S and P.

The term "thioalkyl" refers to an alkyl group, as defined above, having a sulfhydryl or thioether group attached thereto. Examples include -S-alkyl, -S-alkenyl, and -S-alkynyl. The term "aryl" as used herein includes 4-, 5-, 6- and 7-membered single-ring aromatic groups which may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.

The term "alkaryl" refers to an aryl group in which the aromatic ring is substituted with alkyls as defined above. The term "carbonyl" means -C=O.

The term "alkylcarbonyl" refers to an alkyl as defined above bonded to a carbonyl as defined above.

The term "alkylamino" refers to an alkyl as defined above bonded to an amino as defined above. The term "alkylsulfoamino" refers to a -SO₂- group bonded to an alkyl as defined above and an amino as defined above.

The term "alkylsulfinyl" refers to a -SO- group bonded to an alkyl as defined above and an amino as defined above. The term "alkylcarbonylamino" refers to a carbonyl as defined above bonded to an alkyl as defined above and an amino as defined above.

The term "heteroaryl" as used herein refers to an aryl moiety having one or more heteroatoms.

In the context of this invention the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.

In the context of this specification, the term "mammal" includes, without limitation rats, mice, cats, dogs, horses, cattle, cows, pigs, rabbits, non-human primates, and humans.

In the context of this specification, the term "treatment", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. In the context of this specification the terms "therapeutically effective amount" and "diagnostically effective amount", include within their meaning a sufficient but nontoxic amount of a compound or composition of the invention to provide the desired therapeutic or diagnostic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. As used herein, the term "bioactivity" refers to a compound that exerts a therapeutic effect on a mammal following absorption of an effective amount of the compound by the target cells.

The compounds of the invention may find utility as lead compounds in drug development. The term "lead compound" is well known to those skilled in the art and may include the meaning that whilst the compound itself is not used as a drug it is used as a starting-point for the design of other compounds which may have more desirable characteristics, e.g. selectivity in action, potency, stability, solubility, bioavailability etc. The use of a compound of the invention as a lead compound may entail the synthesis of chemical variants of the lead compound, the testing of the variants in one or more assays (which may for example indicate potency, solubility etc.) and the selection of compounds with one or more improved qualities. More specifically, once a lead compound is identified, directed libraries of compounds may be synthesized and tested in one or more assays in order to identify compounds with one or more improved properties (e.g. potency). Generally these directed libraries are combinatorial chemical libraries consisting of compounds with structures related to the lead compound but containing systematic variations including additions, subtractions and substitutions of various structural features. When tested for activity against the target molecule, structural features are identified that either alone or in combination with other features enhance or reduce activity. This information is used to design subsequent directed libraries containing compounds with enhanced activity against the target molecule. After one or several iterations of this process, compounds with substantially increased activity against the target molecule are identified and may be further developed as drugs.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1 % of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Detailed Description Of Disclosed Embodiments

Exemplary, non-limiting embodiments of compounds having progestogenic activity will now be disclosed.

The inventors have surprisingly found that the active compounds as disclosed herein exhibit progestogenic activity. Accordingly, the active compounds disclosed herein, and analogs thereof, can be used as therapeutic agents for administration to mammals, including humans.

The active compounds The compounds of the invention that have surprisingly been found to exhibit progestogenic activity fall into two groups: Compounds of structural formula (I) and compounds of structural formula (IV).

Compounds of structural formula (I)

The compounds of formula (I) are represented by the general formula:

wherein

Y, Y', X and X¹ are independently selected from the group consisting of O, S and N;

R₁ is selected from the group consisting of optionally substituted

alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C₂-₆ alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_4-10 aryl, optionally substituted C_6-H alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C₁^ alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C₁-C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₂. R3. R₄. R₅. R₆ and R₇ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C₂-₆ alkynyl, optionally substituted Ci_₆ alkoxy, optionally substituted C₁^ thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C₁^ alkylsulfonamino, optionally substituted (C_rC₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅- Cio)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

— indicates an optional double bond.

In one embodiment, a preferred compound of compound (I) has the structural formula (II):

(II).

In one embodiment, R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are independently selected from the group consisting of optionally substituted C_1-4 alkyl, optionally substituted C_2-4 alkenyl, optionally substituted C_2-4 alkynyl, optionally substituted C_1-4 alkoxy, optionally substituted C_1-4 thioalkyl, optionally substituted C_M aryl, optionally substituted C_6-8 alkylaryl, optionally substituted C_1-4 alkylamino, optionally substituted C_1-4 alkylcarbonyl, optionally substituted C_1-4 alkylsulfonamino, optionally substituted (C₁- C₄)alkylsulfinyl, optionally substituted C_1-4 alkylcarbonylamino, optionally substituted hetero(C₄-C₈)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; . In one embodiment Ri , R₂, R₃ and R₇ may independently be selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, butenoxy, thiomethyl, thioethyl, thiopropyl, and thiobutyl. In one embodiment Ri , R₂, R₃ and R₇ may independently be selected from the group consisting of propyl, butyl and pentyl.

In one embodiment, R₁ and R₂ are both butyl.

In one embodiment, R₃ and R₄ are both hydrogen.

In one embodiment, R4, R5 and R6 are independently selected from the group consisting of hydrogen, methyl and ethyl. In one embodiment, R4, R5 and R6 are all hydrogen.

In one embodiment of the compound of structural formula (I), X and X' are independently O. In another embodiment, Xi and X^* are both O.

In one embodiment of the compound of structural formula (I), Y and Y' are independently O. In another embodiment, X and X' are both O.

In one embodiment of the compound of structural formula (I), X, X', Y and Y' are all O.

In one embodiment, R₃, R₄, R₅, R₆, and R₇ are independently selected from the group consisting of hydrogen methyl, ethyl, propyl, butyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, butenoxy, thiomethyl, thioethyl, thiopropyl, thiobutyl, Cl and F. In one embodiment, n is 0.

In one embodiment, the bond represented by the symbol " — " indicates a double bond.

In one embodiment of the compound of general formula (I), has the structural formula (HA):

(HA).

In one embodiment of the compound of general formula (I), has the structural formula (HB):

(HB). In one embodiment of the compound of general formula (I), has the structural formula (HC):

(HC).

In one embodiment of the compound of general formula (I), has the structural formula (HD):

(MD). In one embodiment, the compound of general formula (I), has the structural formula (III):

(III).

In one embodiment, the compound of structural formula (I), is represented by the structural formula (MIA):

(MIA). The compound having structure (MIA) is assigned as (3Z, 3Z>(3R, 6S, 7S, 6'S,

3a'S)-3, 8-dihydro-6.6\ 7.3a'-diligustilide. Preferred compounds include compounds of formula (IMA) and analogs thereof. It will be appreciated that where there is a double bond between two carbon atoms, the substituent groups attached to the carbon atoms may be E or Z isomers. Additional exemplary stereo-isomers include: (3E, 3Z')-(3R, 6S, 7S, 6'S, 3a'S)-3, 8-dihydro-6.6\ 7.3a'-diligustilide; (3Z, 3E')-(3R, 6S, 7S, 6'S₁ 3a'S)-3 8-dihydro-6.6', 7.3a'-diligustilide; (3E, 3E')-(3R, 6S, 7S, 6'S, 3a'S)-3, 8-dihydro- 6.6', 7.3a'-diligustilide; (3Z, 3Z>(3R, 6R, 7S, 6¹S, 3a'S)-3, 8-dihydro-6.6', 7.3a'- diligustilide; (3Z, 3Z>(3R, 6R, 7R, 6'S, 3a'S)-3, 8-dihydro-6.6', 7.3a'-diligustilide; and (3Z, 3Z')-(3R, 6S, 7S, 6'S, 3a'R)-3, 8-dihydro-6.6', 7.3a'-diligustilide. Analogs of compound (III) that exhibit progestogenic activity are also encompassed by the present invention. Suitable analogs of compound (III) that have progestogenic activity can be determined by subjecting the analogs compounds of compound (III) to the assay described in the "Biological Studies" section below.

Compounds of structural formula (IV)

The compounds of formula (IV) are represented by the structure:

(IV) wherein

Y, Y', X and X¹ are independently selected from O, S and N; Ri. R₂. R3_> R₄ and R₆ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted Ci_-6 alkylsulfonamino, optionally substituted (CrC^alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅- C_1o)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₅ is selected from the group consisting of , optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C₂^ alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C₁-C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; n is 1, 2, 3 or 4; and — indicates an optional double bond. In one embodiment, R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from the group consisting of optionally substituted C_1-4 alkyl, optionally substituted C_2-4 alkenyl, optionally substituted C_2-4 alkynyl, optionally substituted C_1-4 alkoxy, optionally substituted C_1-4 thioalkyl, optionally substituted C_5-8 aryl, optionally substituted C_6-8 alkylaryl, optionally substituted C_1-4 alkylamino, optionally substituted C₁Wj alkylcarbonyl, optionally substituted C_1-4 alkylsulfonamino, optionally substituted (C₁- C₄)alkylsulfinyl, optionally substituted C₁^ alkylcarbonylamino, optionally substituted hetero(C₄-C₈)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; .

In one embodiment R₁, R₄, and R₅ may be selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, butenoxy, thiomethyl, thioethyl, thiopropyl, and thiobutyl.

In one embodiment R₁ is selected from the group consisting of methyl, ethyl and propyl. In one embodiment, R₁ is both propyl.

In one embodiment R₄ and R₅ are independently hydrogen and/or propyl and hyrdrogen.

In one embodiment, R₂, R3, and R₆ are independently selected from the group consisting of hydrogen, methyl and ethyl. In one embodiment, R₂, R₃, and R₆ are all hydrogen.

In one embodiment of the compound of structural formula (IV), X and X¹ are independently O. In another embodiment, X₁ and X' are both O.

In one embodiment, the bond represented by the symbol "^^ indicates a double bond.

In one embodiment, the compounds of formula (IV) are represented by the structural formula (IVA):

In one embodiment, the compounds of formula (IV) are represented by the structural formula (IVB):

(IVB).

In one embodiment, the compounds of formula (IV) are represented by the structural formula (IVC):

(IVC).

In one embodiment, the compounds of formula (IV) are represented by the structural formula (V):

(V). In one embodiment, the compound has the general formula (Vl):

(Vl). In one embodiment, the compound has the structural formula (VIA):

(VIA). Analogs of compound (Vl) that exhibit progestogenic activity are also encompassed by the present invention. Suitable analogs of compound (Vl) that have progestogenic activity can be determined by subjecting the analogs compounds of compound (Vl) to the assay described in the "Biological Studies" section below.

In one embodiment, there is provided the use of a reference compound for screening one or more candidate compounds for bioactivity, the reference compound having the general formula (I) as defined above or having the general formula (IV) as defined above. The reference compound may be selected from the group consisting of compounds having the structural formula (MA), (NB), (MC), (HD), (III), (IVA), (IVB), (IVC), (V) and (Vl). In one embodiment, the bioactivity is progestogenic activity. The reference parameter may be any characteristic parameter of the candidate compounds that can be used to compare it with the characteristic parameter of the reference compound. Exemplary characteristic parameters include spectrographic data such as NMR peaks, infrared spectra and mass spectra. For example, the peaks of a candidate compound can be analyzed and compared to the peaks of a reference compound. In this way, it is possible to identify those candidate compounds from a compound library that have similar structure to the reference compound and therefore possibly exhibit similar biological activity, such as progestogenic activity. In one embodiment, the reference compound has the structure (IHA):

(IMA).

In another embodiment, the reference compound has the structure (VIA):

(VIA).

In one embodiment, there is provided a method of screening one or more candidate compounds for bioactivity, the method comprising the steps of:

(a) separating said one or more candidate compounds using a chromatographic technique; and (b) screening of the separated compounds by comparing a parameter associated with the separated compounds to a reference parameter associated with one or more reference compounds having the general formula (I) as defined in the first aspect or having the general formula (IV) as defined in the twelfth aspect. In one embodiment, the bioactivity is progestogenic activity. The chromatographic technique may comprise the step of (c) providing said one or more candidate compounds in the bulk of a stationary phase. The separating step may comprise separating said one or more candidate compounds from the bulk of said stationary phase using a mobile phase. The bioactivity may be progestogenic activity.

The separating step may comprise using chromatography to separate the one or more candidate compounds. The chromatography may be High Performance Liquid Chromatography (HPLC). In one embodiment, the parameter associated with the separated compounds is the chromatographic retention time and said reference parameter is the chromatographic retention time of said reference compounds.

In one embodiment, there is provided an assay for use in chromatography, said assay comprising the use of one or more compounds having the general formula (I) as defined above or having the general formula (IV) as defined above. The assay may be used to compare chromatographic data of candidate compounds with chromatographic data of the assay. In this way, the assay can be used to identify candidate compounds from a compound library that exhibit similar biological activity to the compounds having the general formula (I) as defined above or having the general formula (IV) as defined above.

In one embodiment, said assay comprises a compound having the structure (HIA):

(HIA).

In another embodiment, said assay comprises a compound having the structure (VIA):

(VIA).

In one embodiment, there is disclosed the use of one or more compounds having the general formula (I) as defined above or having the general formula (IV) as defined above, as lead compounds.

Derivation of compounds

Compound I is a dimer and could be synthesised by a Deils-Alder adduct reaction of the known compounds Sedanenolide and Liguistilide. Compound (Vl) is riligustilide, a known compound.

Compound (III) is a new compound. The natural derivatives of compound (III) may be derived or isolated from the rhizome Ligusticum Chuanxiong as will be further described below. The compounds disclosed herein may be synthesised utilizing knonwn methodologies disclosed in texts well known to those skilled in the art such as Advanced Organic Chemistry, 3^rd Ed.; John Wiley: New York (1985)) and J. March, Advanced Organic Chemistry - Reactions, Mechanisms and Structure, John Wiley & Sons, New York, 1992.

Synthesis of Compound (III) from Levistolide A

Compound (III) may be synthesised by reduction of Levistolide A in the presence of an organic solvent such as acetone or methanol. A racemic mixture is obtained after reduction of Levistolide A and Compound (III) is isolated using known purification techniques such as HPLC, as described further below.

An exemplary reaction mechanism involves reduction of Levistolide A using NaBm in acetone as follows:

Levistolide A

NaBHU (acetone) Reduction & Purification

(3Z, 3ZM3R, 6S, 7S, 6¹S, 3a'S>3, &Mhydro-&6^t, 7 Ja'-DiligiLstUide

( III ) Levistolide A may be synthesised via a number of reaction schemes. One synthesis scheme involves a reflυxing Z-ligustilide in benzene for 10-40 hours, optionally in the presence of a silver or silver iodide catalyst, to yield Levistolide A via a Diels-Alder reaction. An exemplary reaction scheme may be as follows:

Diels-Alder Ag(syAgl

Z-ligustilide

Levistolide A

Another exemplary reaction scheme for synthesis of Levistolide A involves a retro- Diels-Alder reaction of Tolinolide B to levistolide A. In this reaction, Tokinolide B (a known compound) is heated to 200⁰C for between about 1 hour to about 40hours at a pressure in the range of about 0-20 kBar gauge pressure, in a sealed tube. The overall reaction scheme may be represented as as follows:

Retro-Diels- Alder

Tokinolide B Levistolide A A further reaction scheme for the synthesis of Levistolide A, involves a Diels- Alder reaction of Z-ligustilide and Sedanenolide. An exemplary reaction scheme may be as follows:

Formulations

In accordance with the present invention, when used for the treatment or prevention of microbial infection, compound(s) of the invention may be administered alone. Alternatively, the compounds may be administered as a pharmaceutical, veterinarial, agricultural, or industrial formulation which comprises at least one compound according to the invention. The compound(s) may also be present as suitable salts, including pharmaceutically acceptable salts. In accordance with the present invention, the compounds of the invention may be used in combination with other known treatments or antimicrobial agents, including antifungal treatments, antibiotics, disinfectants, etc. Suitable agents are listed, for example, in the Merck Index, An Encyclopoedia of Chemicals, Drugs and Biologicals, 12^th Ed. ,1996, the entire contents of which are incorporated herein by reference. Combinations of active agents, including compounds of the invention, may be synergistic.

By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.

For instance, suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention. Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts. S. M. Berge ef a/, describe pharmaceutically acceptable salts in detail in J.

Pharmaceutical Sciences, 1977, 66:1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, asparate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like.

Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration. Depending on the route of administration, the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound. The compound may also be administered parenterally or intraperitoneally. Dispersions of the compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.

In one embodiment of the invention, the compound(s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Suitably, such compositions and preparations may contain at least 1% by weight of active compound. The percentage of the compound(s) of formula (I), (II), (III), (IMA), (IV), (V), (Vl) and/or (VIA) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit. The amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained.

The language "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The compound(s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

In one embodiment, the carrier may be an orally administrable carrier. Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration.

Also included in the scope of this invention are delayed release formulations. Compounds of the invention may also be administered in the form of a "prodrug". A prodrug is an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.

In one embodiment, the compound may be administered by injection. In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the analogue can be incorporated into sustained-release preparations and formulations.

Preferably, the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof. Single or multiple administrations of the pharmaceutical compositions according to the invention may be carried out. One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.

Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using convention course of treatment determination tests. Generally, an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; suitably, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight. More suitably, an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight.

Alternatively, an effective dosage may be up to about 500mg/m². For example, generally, an effective dosage is expected to be in the range of about 25 to about

500mg/m², about 25 to about 350mg/m², about 25 to about 300mg/m², about 25 to about 250mg/m², about 50 to about 250mg/m², and about 75 to about 150mg/m².

Extraction and isolation of compound (HIA) and compound (VIA) from Ligusticum Chuanxiong

Compound (MIA) and compound (VIA) was derived or isolated from the rhizome Ligusticum Chuanxiong (LC) using solvent extraction to derive a crude extract of compound (HIA) and compound (VIA) before isolation using solvent/solvent partitioning with polar and non-polar solvents. The inventors own International PCT Application WO 2004/017983, which describes a method to isolate and obtain a crude extract of LC, is incorporated herein for reference in its entirety.

The amount of compound (IHA) or compound (VIA) or both, in an extract of LC, in weight percent, may be selected from the group consisting of: about 0.1% to about 99%; about 0.5% to about 99%; about 1% to about 99%; about 5% to about 99%; about 10% to about 99%; about 20% to about 99%; about 30% to about 99%; about 40% to about 99%; about 50% to about 99%; about 60% to about 99%; about 70% to about 99%; about 80% to about 99%; about 90% to about 99%; about 0.1% to about 90%; about 0.1% to about 80%; about 0.1% to about 70%; about 0.1% to about 60%; about 0.1% to about 50%; about 0.1% to about 40%; about 0.1% to about 30%; about 0.1 % to about 20%; about 0.1 % to about 10%; and about 0.1 % to about 5%.

In one embodiment there is provided a method of producing an extract of Ligusticum Chuanxiong for use in medicine. The method comprising extracting Ligusticum Chuanxiong rhizome in a solvent and for a time to produce a therapeutically effective amount of compound (HIA) and/or compound (VIA). The method may comprise producing a crude extract of Ligusticum

Chuanxiong (LC) with a polar organic solvent. The solvent may be a low carbon polar organic solvent. The polar organic solvent may have from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. In one embodiment, the low carbon polar organic solvent is selected from the group consisting of alcohols, ethers, aldehydes, ketones and carboxylic acids, and mixtures thereof. The alcohols may be selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and hexanol, and mixtures thereof.

The extracting may be undertaken at a temperature selected from the range group consisting of about 10⁰C to about 60⁰C, 10⁰C to about 40⁰C, about 15⁰C to about 35⁰C, about 20⁰C to about 30⁰C. The extracting may be undertaken at a pressure in the range selected from the group consisting of about 60 kPa to 300 kPa, more preferably from about 95 kPa to 105 kPa. In one embodiment, the exposing occurs at ambient temperature and pressure. The extracting may be undertaken for a period of time selected from the group consisting of: about 1 hour to 120 hours, 1 hour to 80 hours.

The method may further comprise purifying the crude extract to isolate the compound (MIA) and/or compound (VIA). The purifying may comprise one or more of solvent/solvent partitioning using mixtures of polar and non-polar solvents, micro parallel liquid chromatography (MPLC), High Performance Liquid Chromatography (HPLC) and Counter Current Chromatography.

A number of methods to extract and isolate compounds (MIA) and (IVA) will be described below.

Example 1. Method for preparing fractions concentrated for Compound HIA using ethanolic extraction and solvent/solvent partition (first purification step)

The dried roots of LC were ground to a powder and then extracted with 100% ethanol for three days (72 hours) at 78.3°C. The solvent was then filtered and the extract was then subjected to a second extract with 100% ethanol for two days (48 hours) at 78.3°C. Filtered extracts were combined and dried in a rotary evaporator. The dried crude extract yield was 9.9%. These dried extracts contain 0.644 μmol/gm of Compound IMA. Dried extracts were weighed and re-suspended in 100% ethanol with a final concentration of 50mg/ml. To define the conditions under which fractions of concentrated Compound IMA can be obtained, solvent/solvent partition of LC. extracts was performed. Crude LC. extract in ethanol (2L) was mixed with water (2L) and the partitioned with 4L of Dichloromethane (DCM). After solvent-solvent partition, the majority of the bioactivity resided in the DCM and the aqueous fraction had no bioactivity (Table 1 ). The most potent bioactivity was the DCM fraction, where dose- dependent increase in PR activity was observed such that 50 μg/ml of this fraction was equivalent to 40% of 10OnM of progesterone. This method is very useful to obtain fractions enriched for Compound IHA, which can be used as a standard extract, or semi-purified fraction for further separation. Such a DCM fraction contains 1.85.7μmol/gm of Compound IMA as determined by LC/MS/MS.

Table 1 : Bioresponse profiles of fractions from solvent/solvent partition

Progestogenic activity (% of PG 10OnM)

PG 10OnM 100 + 8.47

LC 36.75 + 4.26 (50μg/ml)

Concentrati on of extract

(μg/ml) Crude extract DCM fraction Aqueous fraction

0 0.004 + 0.002

6.25 1.07 + 0.32 3.27 + 0.08 0.001 + 0.0003

12.5 5.84 + 0.45 14.06 + 2.89 0.0014 + 0.009

25 23.21 + 0.23 29.68 + 1.91 0.013 + 0.004

50 31.29 + 0.41 37.1 + 1.83 0.0013 + 0.005

Example 2: Method for preparing fractions concentrated for Compound MIA using MPLC separation and C18 solid matrices (second purification step) and the bioresponse fingerprint of such an extract

As the DCM fraction obtained from solvent/solvent partition exhibited higher concentrations of Compound IHA, it was used for subsequent fractionation. The DCM fraction was subjected to C18 MPLC columns and eluted with solvents of increasing polarity. The fractions were collected and analyzed for bioactivity using the MPLC system and reverse phase C18 powder. 100gm of L.C. (50mg/ml) was applied. Elution of LC. fractions was performed with 2 L (flow rates 50ml/min) each of the following solvents of increasing polarity in the following order: 5%, 30%, 50%, 70%, 80%, 90% and 100% MEOH. Previous studies indicate that fractions from 70 to 90% are bioactive and 8 elutes (each 250ml) were collected form each of these. The fractions were collected separately, dried down, reconstituted in ethanol and their bioactivity measured. Maximum progestogenic activity was in the first 4 fractions eluted with 80% methanol (Table 2). This procedure was highly reproducible and was repeated 10 times to collect sufficient amounts of Fraction 2 for subsequent separation steps. This method is very useful to obtain fractions further enriched for Compound HIA, which can be used as a standard extract, or semi-purified fraction for further separation.

Table 2: Bioresponse fingerprint of fractions from C18 MPLC. Fractions were eluted with increasing concentrations of methanol (5%-100%)

Progestogenic activity (%of PG 10OnM)

PG 10OnM 100 ±12.16

C18 /MPLC fractions (12.5μg/ml)

Vehicle 0.08 ± 0.01

5% 0.99 ±0.15

30% 1.09 ±0.15

50% 0.11 ±0.01

1 0.35 ± 0.05 2 0.12 ±0.02 3 1.31 ±0.19 4 0.94 ±0.14

70% 5 0.46 ± 0.07 6 0.15 ±0.02 7 0.21 ± 0.03 8 0.21 ± 0.03

1 19.73 ±2.88 2 82.43 ±12.5 3 15.28 ±2.23 4 8.11 ±1.18

80% 5 3.5 ±0.51 6 0.82 ±0.12 7 0.67 ± 0.09 8 0.74 ±0.11

1 1.78 ±0.26 2 0.67 ± 0.09 3 0.05 ± 0.001 4 9.8 ±1.43

90% 5 0.08 ± 0.01 6 0.1 ± 0.01 7 0.18 ± 0.03 8 0.06 ± 0.001

1 0.41 ± 0.06 2 0.27 ± 0.04 3 0.05 ± 0.006 4 0.1 ± 0.02

100% 5 0.21 ± 0.03 6 0.27 ± 0.04 7 0.16 ± 0.02 8 0.06 ± 0.03

Example 3: Method for preparing fractions concentrated for Compound MIA using MPLC separation and Diol solid matrices (third purification step) and the bioresponse fingerprint of such an extract

Fraction 2 from one C18 elutes (80% methanol) was dried down and 800 mg of elutes were subjected to second time MPLC separation, and eluted with solvents of increasing polarity. The solvent used (2.4L total over flow rate of 20 ml/min) was in decreasing polarity of 5% hexane: 95% isopropanol to 100% hexane. 120 fractions (20 mis each) were collected, dried and weighed. Fractions 25-28 exhibited the highest progestogenic activity (Table 3b). This method is very useful to obtain fractions further enriched for Compound IMA, which can be used as a standard extract, or semi-purified fraction for further separation.

Table 3a. Progestogenic activity of the fractions collected from the MPLC/Diol Column, where five fractions were combined to be tested.

Table 3b: Progestogenic activity of the 5 individual fractions with highest progestogenic activity

Example 4: Method for preparing fractions concentrated for Compound HIA using HPLC separation of bioactive fraction 27 (fourth purification step) and the

HPLC and bioresponse fingerprints of such an extract

To further separate the bioactive components of L.C., the MPLC/Diol fraction 27 (in Example 2, Table 3b) was subjected to semi-preparative HPLC separation using Hypersil C18 column (8μm, 20.0x250mm) and JASCO PU-1586 intelligent Prep. Pump and MD-1510 Multiwavelength Detector. The gradient separation condition with a mobile phase comprising of methanol (MeOH) and water at a flow rate of 5 min/ml was:

0 min 60% (MeOH) : 40% (Water) 15 min 70% (MeOH) : 30% (Water) 30 min 80% (MeOH) : 20% (Water) 45 min 85% (MeOH) : 15% (Water) 60 min 85% (MeOH) : 15% (Water) 65 min 90% (MeOH) : 10% (Water)

Under these conditions, Fraction 27 exhibited a typical HPLC chromatogram from which 14 distinct subfractions, with retention times can be recognized (Table 4) by UV detector. These sub fractions (with retention times as noted in Table 4) were dried, weighed and their progestogenic activities determined. Progestogenic activity was greatest in subfraction 10. Our data from this separation indicated that there was one potent bioactive compound in subfraction 10. Thus using this method we can obtain fractions further enriched for Compound HIA, which can be used as a standard extract, or semi-purified fraction for further separation.

Table 4: HPLC and bioresponse profiles of Bioactive fraction 27

Example 5: Method for preparing fractions concentrated for Compound HIA using HPLC separation and bioactive subfraction 10 (fifth purification step) and Bioresponse fingerprints of such an extract

The most active subfraction from Example 4, Table 4 above, subfraction 10 (0.1 mg) was dried down and re-suspended in ethanol and subjected to a further HPLC step using Agilent 1100 Series and a Cadenza CD-C18 (150X2mm) column. Separation was performed in isocratic mode 65% methanol: 35% H₂O. Flow rate 0.2 ml/min and fractions were analyzed with a UV detector at 280nm. Two separate sub-Subfractions were detected at 20.5-22.5 min and 24.0-25.9 min. Sub-Subfraction 1 demonstrated strong progestogenic activity whereas sub-Subfraction 2 was inactive (Table 5). Sub- subfraction 1 and 2 yielded compounds of 80% purity and molecular weight of 382 and 378 respectively by LC/MS/MS. 1 D-NMR showed Sub-subfraction 1 to contain 80% Compound MIA. However there were insufficient amounts for 2D-NMR structural studies. Studies of bioactivity were performed with ng/ml quantities. This technique can be used to prepare sub-subfractions (concentrated for Compound IHA) that are over 35-fold more bioactive than sub-subfraction 2.

Table 5: Progestogenic activity of subfraction 10

Example 6: Method for preparing fractions concentrated for Compound IHA using Counter Current Chromatography and bioactive subfraction 10 (fifth purification step), and the bioresponse fingerprints of such an extract

Fractions concentrated for compound IHA can also be prepared using countercurrent chromatography. 600mg of Fraction 2 from C18 MPLC separation described in example 3 was subjected to solvent/solvent partition using hexane/ethanol/water in the ratio of 6:5:1. Hexane phase (325 ml) was removed and used as the stationary phase in Counter current chromatography. This was run at 1000 rpm/min. Mobile phase (320 ml) was the aqueous phase. Sixty five (65) fractions (each 10 mis) were collected. Highest progestogenic bioactivity resided in the fractions 5-9 (Table 6). Thus using this method we can obtain fractions further enriched for Compound IMA, which can be used as a standard extract, or semi-purified fraction for further separation.

Table 6 Bioresponse profiling of fractions from the counter current chromatography. Activity was tested using 1/1000 of the amount collected in each fraction.

Fractions 31- Bioactivity (% of 65 PG 10OnM)

31 39.51 + 1.13

32 38.37 + 0.64

33 0.13 + 0.05

34 3.54 + 2.5

35 3.88 + 2.74

36 4.11 + 2.9

37 21.35 + 15.1

38 42.71 + 1.77

39 37.06 + 1.33

40 18.85 + 13.32

41 0.58 + 0.02

42 35.81 + 1.33

43 0.83 + 0.05

44-45 20.73 + 14.65

46-47 0.03 + 0.01

48-49 0.35 + 0.07

50-51 1.49 + 1.05

52-53 1.69 + 0.05

54-55 1.31 + 0.92

Example 7: Alternative method for preparing purified fractions of Compound IHA using Counter Current Chromatography and HPLC. and the bioresponse fingerprints of such an extract Fraction 8 from Example 6, table 6 above was subjected to semi-preparative HPLC separation using Hypersil C18 column (8μm, 20.0x250mm) and JASCO PU-1586 intelligent Prep. Pump and MD-1510 Multiwavelength Detector. A gradient mobile phase comprising of Methanol (MeOH) and water was used. The separation conditions (Flow rate 5 min/ml) was : 0 min 60% (Methanol) : 40% (Water)

15 min 70% (Methanol) : 30% (Water)

30 min 80% (Methanol) : 20% (Water)

45 min 85% (Methanol) : 15% (Water)

60 min 85% (Methanol) : 15% (Water) 65 min 90% (Methanol) : 10% (Water)

Under such conditions, 4 peaks were detected using UV at 280nm with maximum bioactivity residing in subfraction 3 (Table 7). Thus using this method we can obtain fractions further enriched for Compound IHA, which can be used as a standard extract, or semi-purified fraction for further separation.

Table 7: Bioresponse profiling of fractions from the counter current chromatography and HPLC

Example 8: HPLC separation and profiling of bioactive subfraction 3

Subfraction 3 was dried down and weighed and 0.5 mg was re-suspended in ethanol and subjected to a further HPLC step using Agilent 1100 Series and a Cadenza CD- C18 (150X2mm) column were used for HPLC analysis, lsocratic mode 65% methanol:35% H2O with a flow rate 0.2 ml/min and fractions were visualized with UV detector at 280nm. Two sub-subfractions were collected, dried and re-suspended in 10μl of ethanol and 2.5μl was used progestogen-driven reporter gene assay. Compound III, (MW 382) with strong progestogenic activity was demonstrated in sub- subfraction I, whereas sub-subfraction 2 (MW 382) was inactive. (Table 8). Table 8 Progestogenic activity of fractions from subfraction 3

Structure of Compounds Example 9: Structure of Novel Compound HIA (MW 382. with strong proqestoqenic activity)

Compound IMA was isolated as colorless powder. Its APCIMS spectrum (positive mode), similar to a known dimeric phthalide Senkyunolide P [1], showed a molecular ion peak at m/z 383 [(M+H)^*], consistent with the molecule formula of C₂₄H₃₀O₄. The MS/MS spectrum of the molecular ion m/z 383 presented only two strong product ions at m/z 193 and m/z 191 with the same intensity. The MS/MS/MS spectrum of product ion m/z 193, similar to Sedanenolide [2], presented ions at m/z 175 (-H₂O), 147 (-H₂O, -CO), 137 (-C₄H₈), 105 (-H₂O, -CO₁ -C₃H₆) and 91 (-H₂O, -CO, -C₄H₈). Similarly, the MS/MS/MS spectrum of product ion m/z 191, similar as Ligustilide [2], presented ions at m/z 173, 145, 135, 117 and 91. This suggests that the Compound I is a phthalic dimer which could be formed by Deils-Alder adduct reaction with Sedanenolide and Liguistilide. Its ¹H-NMR spectra, apparently differing from that of Senkyunolide P, showed very similar profile to another known phthalide dimer 3,8-dihydro-6.6', 7.3a¹- diligustilide [3]. 1D NMR, especially 2D NMR spectra allowed the unambiguous assignment of all the proton and carbon signals from I, indicating it has the same flat structure as 3,8-dihydro-6.6', 7.3a'-diligustilide, with only one chiral carbon showing different configuration in terms of stereochemistry (Table 9). In particular, the signal assignable to H-3 (δ 4.80, 1 H, m) shifted to the lower field in comparison with that (δ 4.54, 1H, m) of 3,8-dihydro-6.6\ 7.3a'-diligustilide. Since the stereochemistry at C-3 of 3, 8-dihydro-6.6', 7.3a'-diligustilide has been determined to be S [3], the configuration of C-3 in I would be therefore deducted as R. The above conclusion is supported by 6, 7, 3', 8'-tetrahydro-8.6, 3.7'-diligustilide [3] and 4, 5-dehydro-3\ 8'-dihydro-8.6',3.7'- diligustilide [3], whose stereochemistry at C-3' were established as R, and the chemical shifts of the two protons were δ4.85 and δ4.83, respectively. On the basis of comparison with known phthalide dimers and 1 D, 2D NMR analysis, the structure of I was finally assigned as (3Z, 3Z')-(3R, 6S, 7S₁ 6¹S, 3a'S)-3, 8-dihydro-6.6\ 7.3a'- diligustilide. Table 9: ¹H-NMR and ¹³C-NMR data of Compound HIA (MW: 382) (CDCL₃, δ in ppm, J in Hz)

Compound I

C/H No δ_c δ _H

1 171.9

3 81.3 4.80 (1H₁ m)

3a 127.6

4 31.1 2.04 (1 H, m), 1.29-1.40 (1 H, m)

5 29.2 1.50-1.60 (1H₁ m), 1.85-1.93 (1 H, m)

6 38.2 2.54 (1H₁ m)

7 41.0 3.21 (1H, m)

7a 167.8

8 26.3 1.10-1.21(1H₁ m), 1.23-1.35 (1H, m)

9 26.2 1.22-1.34 (2H, m)

10 22.4 1.20-1.39 (2H, m)

11 13.9 0.93 (3H, t, J=7.3) r 165.0

3' 150.4

3a' 47.4

4' 31.0 2.05 (1H, m), 1.37-1.51 (1H, m)

5' 25.5 1.31 (1H₁ m), 1.89 (1 H, m)

6' 41.6 2.98 (1H, m)

T 142.0 7.33 (1 H, d, J=6.5)

7a' 134.4

8" 108.8 5.00 (1H, t, J=7.85)

9" 27.4 2.13-2.23 (2H, m)

10' 22.3 1.37-1.51 (2H₁ m)

11" 13.8 0.93 (3H, t, J=7.4)

Example 5: Structure elucidation of Riliαustilide (VIA). MW380 Compound VIA was isolated from the LC. Compound VIA was isolated as fine needle crystals. Compound VIA showed a molecular ion peak at m/z 381 [(M+H)⁺] in its APCIMS spectrum (positive mode), and was determined to be Riligustilide by direct comparison of NMR data (table 3) with literature [3]. Biological Studies

As mentioned above, the compounds of the invention have progestogenic activity. Methods for assessing progestogenic activity are well known in the art and will readily be able to be performed by persons skilled in the art. Assay

Progestogenic activity can, for example, be assessed using the assay described in the inventors' earlier WO2004/017983, the content of which is incorporated herein and which is described below.

In the progestogen-driven reporter gene assay, HeLa cells were grown in 24- well microtiter plates and were transiently co-transfected with two plasmids using lipofectamine. The first plasmid consisted of DNA encoding the full-length human progesterone receptor (PR-B), and the second a progesterone reporter gene (PRE2- TATA-LUC) comprising a luciferase reporter gene driven by 2 copies of the progesterone response element from the aminotransferase gene. Cells were exposed to the ligands in RPMI 1640 medium, supplemented with 10% charcoal-stripped fetal calf serum, 2mM L-glutamine, 0.1mM non-essential amino acids and 1mM sodium pyruvate for 42-48 hours at 37°C in a 5% carbon dioxide incubator. Replicate wells exposed to vehicle only (ethanol) and used as negative controls. After the period of incubation, cells were rinsed with PBS buffer, lysis buffer was added, and cell lysates collected for measurement of luciferase activity with luminometer. Progestogenic activity of the ligands was expressed as fold-increase in luciferase activity compared with control cells exposed to vehicle only. All data points were in triplicate. Our assay system represents an effective way to screen for nano-molar quantities of bioactive progestogens. The active compounds may, at 10OnM, have a bioactivity of at least 0.1% or

0.5% or 1% or 5% or 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 100% of that of 10OnM natural hormone progesterone.

There may also be provided a compound whose progestogenic activity may be enhanced by co-administering (whether sequentially, simultaneously or separately) a flavonoid of the calycosin class such as calycosin or genistein.

Example 10: Progestogenic activity of Novel Bioactive Compound IMA (MW 382)

Compound IHA was tested for progestogenic activity with our progestogen- driven reporter gene assays (Table 10). This was compared to a saturating dose of progestogens. Compound IHA has EC₅₀ of 0.035μg/ml (91.6nM) and at this dose, its progestogenic activity was equivalent to that of the natural hormone progesterone (10OnM).

Table 10: Effect of increasing doses of Compound MIA on progestogen-driven reporter > gene assays.

Fold (% of PGIOOnM) ± SE %

PG 10OnM 100 14.9

LC 50μg/ml 41.41 13.6

Compound

HIA (μg/ml) (μM)

0.0 0 0.31 0.23

0.01 0.0261 58.94 11.1

0.1 0.261 114.16 9.0

1 2.617 150.16 19.7

10 26.17 180.68 7.5

20 52.35 123.23 12.4

Example 11: Method to augment the proqestoqenic activity of Novel Bioactive

Compound HIA (MW 382)

By adding the flavonoid calycosin (but not genistein), it was unexpectantly found that it is possible to dose-dependently augment the progestogenic activity of Compound IHA by almost 100%. Thus the resultant bioactivity of calycosin plus Compound IHA was almost doubled that observed with Compound HIA alone. Calycosin on its own does not have any progestogenic activity. This effect was specific to flavonoids of the calycosin type, but was not observed with genistein. This example teaches that the already strong progestogenic activity of Compound IHA can be enhanced by coadministering certain flavonoids of the calycosin class. Resultant bioactivity would be predicted to be much stronger than that achievable by administering the natural hormone alone at maximal doses.

Table 11: Synergistic effect of adding calycosin to Compound IHA on progestogenic activity.

Example 12: Proqestoqenic activity of Compound VIA

Compound VIA was subjected to the same dose response studies as Example 10, which showed that compound VIA (over 95% purity) displayed about 15% of the activity of Progesterone 10OnM at the maximum dose of 150μM. Thus the bioactivity of compound VIA was very much weaker, by several hundred fold, than compound HIA. Table 12: Effect of increasing doses of Compound VIA on progestogen-driven reporter gene assays.

Fold

(% of PGIOOnM) ± SE %

Compound VIA

(μM)

__

0 0.21

12 0.28 0.06

25 0.22 0.053

50 1.45 0.44

100 10.49 0.6

150 15.53 5.15

Comparative Example 13: Structure elucidation of (Levistolide A (VII), MW380)

Compound VII does not form part of the invention and is disclosed herein as a comparative example to the examples of the invention disclosed above.

Compound VII was isolated as a white powder. Compound VII, like VIA, also had a molecular ion peak at m/z 381 [(M+H)⁺] in its APCIMS spectrum (positive mode), and was identified to be Levistolide A by comparing the NMR data (table 13) with those of literature [3]. It was found that Levistolide A is not bioactive in progestogen- driven reporter gene assays. The structure of compound (VII) is as follows:

It can be seen that the structure of compound (VII) is similar to that of compound (IHA), a double bond is shared by C₃ and C₈ while for the structure of compound (IIIA), C₃ is in an R configuration. It has surprisingly and unexpectantly been found by the inventors that compound (III) has progestogenic activity.

Table 6 ¹H-NMR data of Riligustilide (VIA) and Levistolide A (V) (CDCL₃, <5 in ppm, J in Hz)

No VIA

4 2.52-2.63(1 H, m), 2.71-2.80(1 H, m) 2.04-2.10(1H, m), 2.18-2.23(1H, m)

5 2.42-2.50(1 H, m), 2.52-2.63(1 H₁ m) 1.38-1.55(1 H, m), 1.90-1.94(1 H, m)

6 5.93(1 H, dt, J=9.7, 4.1) 2.53-2.56 (1 H, m)

7 6.16(1H, dt, J=9.7, 1.9) 3.25 (1 H, brd, J=8.9)

8 2.94(1H, dt, J=7.8) 5.06 (1 H, t, J=7.9)

9 1.42-1.54 (2H₁ m) 2.29 (2H, m)

10 1.12-1.19 (2H, m) 1.38-1.55 (2H₁ m)

11 0.86 (2H, t, J=7.4) 0.93 (3H, t, J=7.4)

4¹ 2.12-2.25(1H, m), 2.52-2.63 (1H, m) 1.38-1.55 (1 H, m), 2.04 (1 H, m)

5¹ 1.99-2.04 (1H, m), 2.12-2.25(1H, m) 1.28 (1 H, m), 1.87 (1 H, m) 6' 2.52-2.63 (1 H, m) 2.97 (1H, m)

T 3.46(1 H, brd, J=7.4) 7.34 (1 H, d, J=6.5)

8' 5.21(1H, t, J=7.9) 5.01 (1H, t, J=7.7)

9¹ 2.29-2.38 (2H, m) 2.14-2.22 (2H₁ m)

10' 1.42-1.54 (2H₁ m) 1.38-1.55 (2H₁ m)

11' 0.96 (3H, t, J=7.4) 0.92 (3H, t, J=7.4) Comparative Example 14: Structure elucidation of compound VIII (no progestoqenic activity MW 378):

Compound VIII does not form part of the invention and is disclosed herein as a comparative example to the examples of the invention disclosed above.

Compound VIII was isolated as a colorless powder and showed a molecular ion peak at m/z 379 [(M+H)⁺] in its APCIMS spectrum (positive mode). The existence of a benzene ring in the molecule was indicated by the four olefinic signals resonating at δ 7.09 (1 H, d, J=7.4), 7.56 (1 H, t, J=7.9), 7.48 (1H, t, J=7.9) and 7.79 (1H, d, J=7.9) in the low field of the ¹H-NMR spectrum. In addition, two methyl proton signals (<5 0.79, 3H, t, J=7.4; δ 0.55, 3H, t, J=7.4) together with two carboxyl carbon signals (δ 169.5; D 164.2) suggested that Il could be a phthalic dimer formed by 3-butylidenephthalide and Liguistilide through Deils-Alder adduct. 1 D NMR, especially the 2D NMR data confirmed the above hypothesis, and helped assign all the proton and carbon signals unambiguously (Table 7). The most unique NMR character of compound VIII is that the signals (54.56; δ 1.72-1.77, 1.81-1.88; <51.26, 0.97-1.00; 50.55) assigned to H-8\ 9', 10' and 11' shifted to a relatively higher field compared with those (δ 4.62; 51.98, 2.03-2.21 ; δ 1.24-1.38; δ 0.82) of tokinolide B [3]. Only when the butylidene side chain was in the shielding area of benzene ring, could these signals resonate in higher field, and therefore the configuration of C-3 was deduced as R, which is the same as that of tokinolide B. Furthermore, due to the crossing peak between H-8 and H-6' in the NOESY spectrum, both the stereochemistry of C-8 and C-6' can be elucidated as S. Thus, on the basis of the above analysis, the structure of compound VIII was finally determined as (3E, 3Z')-(3S, 8R, 3a'S, 6'R)-4, 5-dehydro-3.3a\ 8.6'-diligustilide, as shown below:

(3E, 3Z')-(3S, 8R, 3a'S, 6'R)-4,5-dehydro-33a, 8.6-digligustilide ( ym)

Compound VIII did not exhibit any progestogenic activity when subjected to the same assay as for the other compounds IHA and VIA described above. Table 7: ¹H-NMR and ¹³C-NMR data of compound VIII (CDCL₃, δ in ppm, J in Hz)

Compound Il

C/H No

. δ_c δ_H

1 169.5

3 88.6

3a 152.5

4 121.0 7.09 (1 H, d, J=7.4)

5 134.8 7.56 (1 H, t, J=7.9)

6 129.5 7.48 (1 H, t, J=7.9)

7 125.2 7.79 (1H, d, J=7.9)

7a 126.2

8 47.2 1.92-1.96 (1 H, m)

9 28.9 1.46-1.51 (1H, m), 1.36-1.38 (1H, m)

10 21.1 1.18-1.22 (1H, m), 0.99-1.02 (1H, m)

11 14.0 0.79 (3H, t, J=7.4)

1' 164.2

3' 148.4

3a' 51.1

4" 27.4 2.52-2.58 (1H, m), 2.14-2.17 (1H₁ m)

5' 17.2 2.12-2.14 (1H, m), 1.36-1.38 (1H, m)

6¹ 37.2 3.16 (1H, br.d)

T 145.3 7.68 (1 H, d, J=6.5)

7a' 133.2

8' 107.2 4.56 (1 H, td, J=6.9, 1.9)

9' 26.7 1.72-1.77 (1H, m), 1.81-1.88 (1H, m)

10' 22.1 1.26 (1H, m), 0.97-1.00 (1 H, m)

11' 13.2 0.55 (3H, t, J=7.4) Industrial Applications

It has surprisingly been found that the compounds disclosed herein exhibit progestogenic activity. Advantageously, the compounds of the present invention can be used in progesterone replacement and progesterone supplementation therapy in humans, and for birth control pills and other animals. The compounds can also be used to treat endometrial hyperplasia, endometrial cancers, menstrual disorders, irregular vaginal bleeding, polycystic ovarian syndrome, endometriosis, fetal and pregnancy support, in recurrent abortions, for assisted reproduction cycles, stroke and brain injuries.

Advantageously, embodiments of the compounds are phytoprogestogenic compounds that can be easily extracted from Rhizoma Chuanxiong. Accordingly, embodiments that are derived from Rhizoma Chuanxiong are expected to be highly valued as they can be marketed as an alternative to synthetically derived progestogenic medicines that are currently available. In particular, because these embodiments are extracted from natural products, it is expected that the use of these compounds as progestins will be useful alternatives to many current progestogens such as medroxyprogesterone acetate and progesterol acetate, dienogest, drospirenone, Nestorone (Population Council, New York, NY, USA), nomegestrol acetate and trimegestone (Sitruk-Ware R. New progestogens: a review of their effects in perimenopausal and postmenopausal women. Drugs Aging. 2004;21(13):865-83.)

Additionally, it is expected that embodiments of the disclosed compounds will avoid the undesirable side effects associated with synthetically produced progestogenic medicines, such as increased risk of breast cancer, cardiovascular events, stroke, heart attacks, cardiovascular disease, thrombosis, dementia and excessive androgen activity.

The compounds having progestin activity of the disclosed embodiments have a wide range of applications such as in oral contraception, hormone replacement therapy (in combination with esterogen) and to assist reproductive technology. The compounds having progestogen activity of the disclosed embodiments may also be used to treat inoperable endometrial cancer and may aid in the reducing the risk to endometrial cancer from postmenopausal estrogen in woman. Furthermore, The compounds having progestogen activity of the disclosed embodiments may be used to effectively treat secondary amenorrhea, functional uterine bleeding as well as related menstrual disorders (caused by hormonal deficiency or imbalance). The compounds having progestogen activity of the disclosed embodiments may be used in hormone replacement therapy, normally in conjunction with estrogen to protect the endometrium from hyperplasia and cancer. The compounds having progestogen activity of the disclosed embodiments may be used to treat irregular and abnormal menstrual bleeding, to suppress endometrial growth, and for treating endometrial hyperplasia. The compounds having progestogen activity of the disclosed embodiments may have anti-neoplastic activity against endometrial hyperplasia and cancer. Other uses for The compounds having progestogen activity of the disclosed embodiments may include treatment of premenstrual symptoms such as headaches, depression, water retention and mastodynia. The compounds having progestogen activity of the disclosed embodiments may be used alone or in combination with other steroid hormones for the treatment of endometriosis and may be used in compositions for female birth control pills and hormone replacement therapy.

The compounds having progestogen activity of the disclosed embodiments may also be used to treat luteal phase defects and for luteal support in assisted reproduction cycles for infertile patients. After conception, The compounds having progestogen activity of the disclosed embodiments may be are used for fetal support.

The compounds having progestogen activity of the disclosed embodiments may overcome the disadvantages associated with commercially available synthetic products such as medroxyprogesterone acetate and progesterol acetate. The compounds having progestogen activity of the disclosed embodiments may avoid the undesirable side effects such as excessive androgen activity.

The phytoprogestogenic forms of The compounds having progestogen activity of the disclosed embodiments may be a desirable alternative to the synthetic forms of progestogens that are currently available. It is thought that there may be a large commercial market for these compounds due to the growing popularity for drugs based on traditional systems of medicine across the world, particularly for drugs obtained from herbs and herbal extracts. Accordingly these compounds may open new avenues of treatment for many people. It is also expected that synthetic derived forms of compounds (III) and (Vl) and their analogs will exhibit similar progestogenic activity to forms of compounds (III) and (Vl) that are derived from extracts of LC.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Advantageously, the disclosed compounds can be used in an assay for use in chromatography. The assay comprising the disclosed compounds can be used to identify compounds having bioactivity such as progestogenic activity. Furthermore, the disclosed compounds can be used as lead compounds in reactions for the synthesis of other compounds and, in particular for the synthesis of compounds having progestogenic activity.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

References:

[1] Guillermo Delgado, Rosa G. Reza-Garduno, Ruben A. etc., Secondary Metabolites from the Roots of Ligusticum Porteri (Umbelliferae). X-Ray Structure of Z-6.6¹, 7.3a'- Diligustilide, Heterocycles, Vol. 27, No. 6, 1305-1312, 1988.

[2] X. Zhang, H. Xiao, Q. Xu, X. L, J. Wang and X. Liang, Characterization of phthalides in Liguisticum chuanxiang by Liquid Chromatograpic-Atmospheric Pressure Chemical Ionization-Mass Spectrometry. Journal of Chromatrographic Science, 41 , 428-433, 2003.

[3] Takashi Naito, Takao Katsuhara, kazuke Niitsu, etc, Phthalide dimmers from Ligusticum Chuanxiong Hort. Heterocycles, vol. 32, No.12, 2433-2442, 1991.

Claims

Claims

1. A pharmaceutical composition comprising a pharmaceutically acceptable amount of a compound having the general formula (I):

(D and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, in admixture with one or more pharmaceutically acceptable carriers, wherein

Y, Y', X and X' are independently selected from the group consisting of O, S and N;

R₁ is selected from the group consisting of optionally substituted C_t-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_4-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C^CeJalkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₂. R₃, R₄, R₅, R₆ and R₇ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-1I alkylaryl, optionally substituted C₁^ alkylamino, optionally substituted C₁^ alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C₁- C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; — indicates an optional double bond.

2. A pharmaceutical composition as claimed in claim 1 , wherein the compound of general formula (I) has the structure (II):

(II).

3. A pharmaceutical composition as claimed in claim 1 , wherein R₁, R₂, R₃, R₄, R_5, Re and R₇ are independently selected from the group consisting of optionally substituted C_1-4 alkyl, optionally substituted C_2-4 alkenyl, optionally substituted C_2-4 alkynyl, optionally substituted C_1-4 alkoxy, optionally substituted C_1-4 thioalkyl, optionally substituted C_5-8 aryl, optionally substituted C_6-8 alkylaryl, optionally substituted C_1-4 alkylamino, optionally substituted C_1-4 alkylcarbonyl, optionally substituted C_1-4 alkylsulfonamino, optionally substituted (CrC₄)alkylsulfinyl, optionally substituted C_1-4 alkylcarbonylamino, optionally substituted hetero(C₄-C₈)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol.

4. A pharmaceutical composition as claimed in claim 1 , wherein R₁ , R₂, R₃ and R₇ are independently selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, butenoxy, thiomethyl, thioethyl, thiopropyl, and thiobutyl.

5. A pharmaceutical composition as claimed in claim 1 , wherein R₁ and R₂ are both butyl.

6. A pharmaceutical composition as claimed in claim 1 , wherein R₃ and R₄ are both hydrogen.

7. A pharmaceutical composition as claimed in claim 1 , wherein R₄, R₅ and R₆ are independently selected from the group consisting of hydrogen, methyl and ethyl.

8. A pharmaceutical composition as claimed in claim 1, wherein X, X', Y and Y' are independently oxygen (O).

9. A pharmaceutical composition as claimed in claim 1 , wherein the compound of general formula (I) has the structure (HA):

(HA).

10. A pharmaceutical composition as claimed in claim 1 , wherein the compound of general formula (I) has the structure (HB):

(HB).

11. A pharmaceutical composition as claimed in claim 1 , wherein the compound of general formula (I) has the structure (MC):

(HC).

12. A pharmaceutical composition as claimed in claim 1 , wherein the compound of general formula (I) has the structure (MD):

(HD).

13. A pharmaceutical composition as claimed in claim 1, wherein the compound of general formula (I) has the structure (III):

(III).

14. A pharmaceutical composition as claimed in claim 1, wherein the compound of general formula (I) has the structure (HIA):

(HIA)-

15. A pharmaceutical composition as claimed in claim 1 , wherein the compound is selected from the group consisting of (3Z₁ 3Z'H3R, 6S, 7S, 6'S, 3a'S)-3, 8-dihydro- 6.6", 7.3a'-diligustilide, (3E, 3Z>(3R, 6S, 7S₁ 6'S₁ 3a'S)-3, 8-dihydro-6.6\ 7.3a'- diligustilide; (3Z, 3E')-(3R, 6S₁ 7S₁ 6'S₁ 3a'S)-3 8-dihydro-6.6', 7.3a'-diligustilide; (3E, 3E')-(3R, 6S₁ 7S₁ 6'S₁ Za¹S)-Z, δ-dihydro-6.6¹. 7.3a'-diligustilide; (3Z, 3Z>(3R, 6R₁ 7S₁ 6'S, 3a'S)-3, 8-dihydro-6.6', 7.3a'-diligustilide; (3Z₁ 3Z')-(3R. 6R, 7R, 6'S, 3a'S)-3, δ-dihydro-6.6¹, 7.3a'-diligustilide; (3Z, 3Z')-(3R, 6S, 7S, 6'S, 3a'R)-3, 8-dihydro-6.6\ 7.3a'-diligustilide, and mixtures thereof.

16. A compound represented by the structural formula (M):

wherein

Y, Y', X and X' are independently selected from the group consisting of O, S and N;

R₁ is selected from the group consisting of optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C₁^ thioalkyl, optionally substituted C_4-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C^C^alkylsulfinyl, optionally substituted Ci_-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₂. R3. R4. R5, Re and R7 are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C₁- C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; and

— indicates an optional double bond.

17. A compound as claimed in claim 16, wherein R₁, R₂, R3, R4, R₅. Re and R₇ are independently selected from the group consisting of optionally substituted C_1-4 alkyl, optionally substituted C_2-4 alkenyl, optionally substituted C₂^ alkynyl, optionally substituted C_1-4 alkoxy, optionally substituted C_1-4 thioalkyl, optionally substituted C_5-8 aryl. optionally substituted C_6-8 alkylaryl, optionally substituted C_1-4 alkylamino, optionally substituted Ci_-4 alkylcarbonyl, optionally substituted Ci_-4 alkylsulfonamino, optionally substituted (Ci-C₄)alkylsulfinyl, optionally substituted C_1-4 alkylcarbonylamino, optionally substituted hetero(C₄-C₈)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol.

18. A compound as claimed in claim 16, wherein R₁ , R₂, R₃ and R₇ are independently selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, butenoxy, thiomethyl, thioethyl, thiopropyl, and thiobutyl.

19. A compound as claimed in claim 16, wherein wherein Ri and R₂ are both butyl.

20. A compound as claimed in claim 16, wherein R₃ and R₄ are both hydrogen.

21. A compound as claimed in claim 16, wherein R₄, R₅ and R₆ are independently selected from the group consisting of hydrogen, methyl and ethyl.

22. A compound as claimed in claim 16, wherein X, X', Y and Y' are independently oxygen (O).

23. A compound as claimed in claim 16, wherein the compound has the structure (NC):

(lie).

24. A compound as claimed in claim 16, wherein the compound has the structure (HD):

(MD).

25. A compound as claimed in claim 16, wherein the compound has the structure (IMA):

(MIA).

26. An extract of Ligusticum Chuanxiong comprising a therapeutically effective amount a compound having the structural formula (III):

(III).

27. An extract as claimed in claim 26, wherein the compound has the structure (IMA):

28. An extract as claimed in claim 26, produced in a method comprising extracting Ligusticum Chuanxiong rhizome in a solvent.

29. An extract of Ligusticum Chuanxiong as defined in claim 26, for use in medicine.

30. A pharmaceutical composition comprising an extract of Ligusticum Chuanxiong having a therapeutically effective amount a compound of structural formula (III):

(III).

31. A compound for use in medicine, the compound having the general formula (I):

and the pharmaceutically acceptable salts, individual isomers and mixtures of isomers thereof, wherein

Y, Y', X and X^* are independently selected from the group consisting of O, S and N;

R₁ is selected from the group consisting of optionally substituted C₁-_S alkyl, optionally substituted C₂^ alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_4-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C₁^ alkylsulfonamino, optionally substituted (Ci-C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₂. R3. R₄. R₅, Re and R₇ are independently selected from the group consisting of hydrogen, optionally substituted Ci_-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted Ci_-6 alkoxy, optionally substituted Ci_-6 thioalkyl, optionally substituted C₅.₁₀ aryl, optionally substituted C_6-H alkylaryl, optionally substituted C₁^ alkylamino, optionally substituted Ci_-6 alkylcarbonyl, optionally substituted C^ alkylsulfonamino, optionally substituted (C₁- C₆)alkylsulfinyl, optionally substituted Ci_-6 alkylcarbonylamino, optionally substituted hetero(C₅-Cio)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; and

— indicates an optional double bond.

32. The use of one or more compounds of claim 31 , in the manufacture of a medicament for the treatment of a condition associated with progesterone replacement or progesterone supplementation.

33. The use as claimed in claim 32, wherein the condition is selected from the group consisting of menstrual disorders, amenorrhoea, menorrhagia, polycystic ovarian syndrome, pregnancy complications, endometriosis, contraception, menopause, endometrial hyperplasia and hormonal replacement.

34. The use of one or more compounds of claim 31 , in the manufacture of a medicament for the treatment of a condition associated with stroke or a brain injury, in a mammal.

35. A kit comprising a pharmaceutical composition as defined in claim 1 , and instructions for administering the composition to a mammal for the treatment of a condition associated with progesterone replacement or progesterone supplementation in a mammal.

36. A kit comprising a pharmaceutical composition as defined in claim 1, and instructions for administering the composition to a mammal for the treatment of a condition associated with stroke or brain injuries in a mammal.

37. A method of producing an extract of Ligusticum Chuanxiong for use in medicine, the method comprising extracting Ligusticum Chuanxiong rhizome in a solvent and for a time to produce a therapeutically effective amount of a compound having the formula (III):

(III).

38. A method as claimed in claim 37, wherein the compound has the formula (IUA):

(HIA)-

39. A method of treating a condition associated with progesterone replacement or progesterone supplementation in a mammal, the method comprising the step of administering a therapeutically effective amount of one or more compounds as claimed in claim 31, to the mammal.

40. A pharmaceutical composition comprising a pharmaceutically acceptable amount of a compound having the general formula (IV):

(IV) wherein

Y, Y', X and X' are independently selected from O, S and N;

Ri _t R₂. R3. R4 and R₆ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C₅.₁₀ aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C₁- C₆)alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₅ is selected from the group consisting of , optionally substituted C_1-8 alkyl, optionally substituted C₂-e alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C₁^ alkoxy, optionally substituted C_1-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (CrCeJalkylsulfinyl, optionally substituted C₁^ alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; n is 1, 2, 3 or 4; and

— indicates an optional double bond.

41. A pharmaceutical composition as claimed in claim 40, wherein R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from the group consisting of optionally substituted C_1-4 alkyl, optionally substituted C_2-4 alkenyl, optionally substituted C_2-4 alkynyl, optionally substituted C_1-4 alkoxy, optionally substituted C_1-4 thioalkyl, optionally substituted C_5-8 aryl, optionally substituted C_6-S alkylaryl, optionally substituted Ci_-4 alkylamino, optionally substituted Ci_-4 alkylcarbonyl, optionally substituted Ci_-4 alkylsulfonamino, optionally substituted (Ci-C₄)alkylsulfinyl, optionally substituted Ci_-4 alkylcarbonylamino, optionally substituted hetero(C₄-C₈)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol.

42. A pharmaceutical composition as claimed in claim 40, wherein R₁, R₄, and R₅ are selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, ethenyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, butenoxy, thiomethyl, thioethyl, thiopropyl, and thiobutyl.

43. A pharmaceutical composition as claimed in claim 40, wherein Ri is selected from the group consisting of methyl, ethyl and propyl.

44. A pharmaceutical composition as claimed in claim 40, wherein R₄ and R₅ are independently hydrogen and propyl.

45. A pharmaceutical composition as claimed in claim 40, wherein R₂, R₃, and R₆ are independently selected from the group consisting of hydrogen, methyl and ethyl.

46. A pharmaceutical composition as claimed in claim 40, wherein R₂, R₃, and R₆ are all hydrogen.

47. A pharmaceutical composition as claimed in claim 40, wherein X and X' are independently oxygen (O).

48. A pharmaceutical composition as claimed in claim 40, wherein the compound of general formula (IV) has the structure (IVA):

(IVA).

49. A pharmaceutical composition as claimed in claim 40, wherein the compound of general formula (IV) has the structure (IVB):

(IVB).

50. A pharmaceutical composition as claimed in claim 40, wherein the compound of general formula (IV) has the structure (IVC):

(IVC).

51. A pharmaceutical composition as claimed in claim 40, wherein the compound of general formula (IV) has the structure (V):

(V).

52. A pharmaceutical composition as claimed in claim 40, wherein the compound of general formula (IV) has the structure (Vl):

(Vl).

53. A pharmaceutical composition as claimed in claim 40, wherein the compound of general formula (IV) has the structure (VIA):

54. A compound for use in medicine having the general formula (IV):

wherein

Y, Y", X and X' are independently selected from O, S and N; Ri. R₂. R₃, R₄ and R₆ are independently selected from the group consisting of hydrogen, optionally substituted C_1-8 alkyl, optionally substituted C₂^ alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted Ci_-6 thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-H alkylaryl, optionally substituted C₁^ alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (C₁- C₆)alkylsulfinyl, optionally substituted Ci_-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol;

R₅ is selected from the group consisting of , optionally substituted C_1-8 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C₁^ thioalkyl, optionally substituted C_5-10 aryl, optionally substituted C_6-11 alkylaryl, optionally substituted C_1-6 alkylamino, optionally substituted C_1-6 alkylcarbonyl, optionally substituted C_1-6 alkylsulfonamino, optionally substituted (CrC^alkylsulfinyl, optionally substituted C_1-6 alkylcarbonylamino, optionally substituted hetero(C₅-C₁₀)aryl, hydroxyl, halogen, cyano, nitro, amino, formyl, and thiol; n is 1 , 2, 3 or 4; and zzz indicates an optional double bond.

55. The use of one or more compounds of claim 54, in the manufacture of a medicament for the treatment of a condition associated with progesterone replacement or progesterone supplementation.

56. The use as claimed in claim 55, wherein the condition is selected from the group consisting of menstrual disorders, amenorrhoea, menorrhagia, polycystic ovarian syndrome, pregnancy complications, endometriosis, contraception, menopause, endometrial hyperplasia and hormonal replacement.

57. The use of one or more compounds of claim 58, in the manufacture of a medicament for the treatment of a condition associated with stroke or a brain injury, in a mammal.

58. A kit comprising a pharmaceutical composition as defined in claim 40, and instructions for administering the composition to a mammal for the treatment of a condition associated with progesterone replacement or progesterone supplementation in a mammal.

59. A kit comprising a pharmaceutical composition as defined in claim 40, and instructions for administering the composition to a mammal for the treatment of a condition associated with stroke or brain injuries in a mammal.

60. A method of producing an extract of Ligusticum Chuanxiong for use in medicine, the method comprising extracting Ligusticum Chuanxiong rhizome in a solvent and for a time to produce a therapeutically effective amount of a compound having the general formula (Vl):

(Vl).

61. A method as claimed in claim 60, wherein the compound has the structural formula (VIA):

62. A method of treating a condition associated with progesterone replacement or progesterone supplementation in a mammal, the method comprising the step of administering a therapeutically effective amount of one or more compounds as claimed in claim 54, to the mammal.

63. A combined preparation for use in medicine, the combined preparation comprising one or more compounds of claim 31 and one or more compounds of claim 54.

64. A combined preparation for use in medicine, the combined preparation comprising one or more isoflavones and one or more compounds of claim 30 or one or more compounds of claim 54.

65. A combined preparation as defined in claim 64, wherein the isoflavones are selected from the group consisting of calycosin and genistein.

66. A combined preparation as defined in claim 64, wherein said isoflavones and said and one or more compounds of claim 31 or one or more compounds of claim 54, are simultaneously, separately or sequentially administered.

67. A combined preparation as defined in claim 64, in the manufacture of a medicament for treating a condition associated with progesterone replacement, progesterone supplementation, stroke or brain injuries in a mammal.

68. Use of a reference compound for screening one or more candidate compounds for bioactivity, the reference compound having the general formula (I) of claim 1 or having the general formula (IV) of claim 40.

69. Use of a reference compound as claimed in claim 68, wherein the bioactivity is progestogenic activity.

70. Use of a reference compound as claimed in claim 68, wherein the reference compound has the structure (HIA):

(IIIA).

71. Use of a reference compound as claimed in claim 68, wherein the reference compound has the structure (VIA):

72. A method of screening candidate compounds for bioactivity, the method comprising the step of:

(a) comparing a parameter associated with the candidate compounds to a reference parameter associated with one or more reference compounds having the general formula (I) of claim 1 or having the general formula (IV) of claim 40.

73 A method of screening candidate compounds as claimed in claim 72, wherein the method comprises, before step (a), the step of:

(b) separating said one or more candidate compounds using a chromatographic technique.

74 A method as claimed in claim 72, wherein the bioactivity is progestogenic activity.

75 A method of screening as claimed in claim 73, wherein said chromatographic technique is High Performance Liquid Chromatography (HPLC).

76. A method of screening as claimed in claim 73, wherein said parameter associated with the separated candidate compounds is the chromatographic retention time and said reference parameter is the chromatographic retention time of said reference compounds.

77. An assay for use in chromatography, said assay comprising the use of one or more compounds having the general formula (I) of claim 1 or having the general formula (IV) of claim 40.

78. An assay as claimed in claim 77, wherein said compound has the structure (IMA):

79. An assay as claimed in claim 77, wherein said compound has the structure (VIA):

80. Use of compounds having the general formula (I) of claim 1 or having the general formula (IV) of claim 40, as lead compounds.