text:
Isoflavonoid Prodrugs, Compositons Thereof and Therapeutic Methods Involving Same
Phosphate esters of isoflavonoid compounds are prepared for use as prodrugs, medicaments and in formulations, drinks and foodstuffs.
Heaton, Andrew (Abbotsford, AU)
Kelly, Graham Edmund (Northbridge, AU)
NOVOGEN RESEARCH PTY LTD
A61K31/662; A23L1/30; A23L2/52; A61P15/00; C07F9/09; C07F9/655
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CHANDRAKUMAR, NIZAL S
SUGHRUE MION, PLLC (WASHINGTON, DC, US)
1. An isoflavonoid phosphate ester compound of the general formula I: in which R1 R2 and Z are independently M2PO4—, hydrogen, hydroxy, OR9, OC(O)R10, OS(O)R10, CHO, C(O)R10, COOH, CO2R10, CONR3R4, alkyl, haloalkyl, arylalkyl. alkenyl. alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or R2 is as previously defined, and R1 and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from R1 is as previously defined, and R2 and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from W is R1, and A and B taken together with the carbon atoms to which they are attached form a six-membered ring selected from one of wherein R3 is hydrogen, alkyl, aryl, arylalkyl, an amino acid, C(O)R11 where R11, is hydrogen alkyl, aryl, arylalkyl or an amino acid, or CO2R12 where R12 is hydrogen, alkyl, haloalkyl, aryl, heteroaryl or arylalkyl, R4 is hydrogen, alkyl or aryl, or R3 and R4 taken together with the nitrogen to which they are attached comprise pyrrolidinyl or piperidinyl, R5 is M2PO4—, hydrogen, C(O)R11 where R11 is as previously defined, or CO2R12 where R12 is as previously defined, R6, is M2PO4—, hydrogen, hydroxy, alkyl, aryl, amino, thio, NR3R4, COR11 where R11; is as previously defined, CO2R12 where R12 is as previously defined or CONR3R4, R7 is hydrogen, C(O)R11 where R11 is as previously defined, alkyl, haloalkyl, aryl, arylalkyl or Si(R13)3 where each R13 is independently hydrogen, alkyl or aryl, R8 is M2PO4—, hydrogen, hydroxy, alkoxy or alkyl, R9 is alkyl, haloalkyl, aryl, arylalkyl, C(O)R11 where R11 is as previously defined, or Si(R13)3 where R13 is as previously defined, R10, is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an amino acid, alkylamino or dialkylamino, the drawing " " represents either a single bond or a double bond, M is independently hydrogen, a straight or branched alkyl, alkenyl, alkenyl, alkoxyalkyl, alkvlthioalkyl, or aminoalkyl, a substituted or non-substituted cycloalkyl, an aryl, aralkyl, or alkylaryl, and a substituted cycloalkyl where at least one ring contains one or more of a nitrogen, sulfur, oxygen, phoshorous or silicon heteroatom in the at least one rng; T is independently hydrogen, alkyl or aryl, X is 0, NR4 or S, preferably O, and Y is wherein R14, R15 and R16 are independently M2PO4—, hydrogen, hydroxy, OR9, OC(O)R10, OS(O)R10CHO, C(O)R10, COOH, CO2R10, CONR3R4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, and wherein at least one of R1, R2, R5, R6, R8, R14, R15, R16, Z, W or A where present is independently MPO4—, or a pharmaceutically acceptable salt thereof, with the proviso that compounds of the formula: in which R1 is hydrogen or alkyl of 1-3 carbon atoms, R2 and R3 are independently —OPO3H2, hydrogen or a substituent and at least one of R2 and R3 is —OPO3H2, and X and Y are independently hydrogen or F, are specifically excluded.
2. An isoflavonoid phosphate ester according to claim 1 of the general formula II: in which R1, R2, R5, R6, R14, R15, W and Z are as defined in claim 1, and the drawing " " represents either a single bond or a double bond.
3. An isoflavonoid phosphate ester according to claim 2, wherein the drawing " " represents a double bond.
4. An isoflavonoid phosphate ester according to claim 1 of the general formula III: in which R1, R2, R5, R6, R14, R15, W and Z are as defined in ciaim 1.
5. An isoflavonoid phosphate ester according to claim 1 of the general formula IV: in which R1, R2, R5, R6, R14, R15, W and Z are as defined in claim 1.
6. An isoflavonoid compound of claim 1, wherein the phosphate ester moiety is present as a corresponding salt M2PO4—, where M is a pharmaceutically acceptable cation.
7. An isoflavonoid compound of claim 6, wherein the corresponding salt M2PO4— is NaO(HO)P(O)O—.
8. An isoflavonoid compound selected from or a pharmaceutically acceptable salt thereof.
9. A method for the treatment, prophylaxis, amelioration, defence against, and/or prevention of one or more of the therapeutic indications as hereinbefore defined, which comprises administering to a subject a therapeutically effective amount of one or more compounds of formula I as defined in claim 1.
10. (canceled)
11. An agent for the treatment, prophylaxis, amelioration, defence against and/or treatment of one or more of the therapeutic indications as hereinbefore defined which comprises one or more compounds of formula I as defined in claim 1 either alone or in association with one or more carriers or excipients.
12. A method for the treatment, prophylaxis, amelioration, defence against and/or prevention of conditions in a subject associated with abnormal estrogen/androgen balance which method includes the step of administering one or more compounds of formula I as defined in claim 1 either alone or in association with one or more carriers or excipients.
13. A method of claim 12, wherein the subject is a woman.
14. A method of claim 12, wherein the subject is a man.
16. A therapeutic composition which comprises one or more compounds of formula I as defined in claim 1 in association with one or more pharmaceutical carriers and/or excipients.
17. A drink or food-stuff, which contains one or more compounds of formula I as defined in claim 1.
18. A composition comprising one or more compounds of formula I as defined in claim 1, vitamin E, and optionally pharmaceutically, veterinarily or cosmetically acceptable carriers and/or excipients.
This invention relates to compounds, formulations, drinks, foodstuffs, methods and therapeutic uses involving, containing, comprising, including and/or for preparing certain isoflavene prodrugs and analogues thereof. In particular, the invention relates to phosphate esters of isoflavonoids and derivatives, medicaments involving same and therapeutic uses thereof.
Isoflavones and many derivatives thereof possess a very wide range of important biological properties including oestrogenic effects. Isoflavones such as genistein and daidzein have been shown to be involved in the modulation or attenuation of levels of estrogenic steroids in the body. More recently, isoflavenes and in particular dehydroequol have been shown to possess strong chemotherapeutic properties. In some areas of biological activity, there are even some contradictions, for example, some isoflavonoids act as agonists of the estrogen receptor while others act as antagonists of the estrogen receptor. It is believed that there is a strong correlation between lowering levels of biologically active estrogenic steroids in the body with lower incidences of cancer such as breast cancer and many other diseases and conditions.
However, the biological activity of isoflavonoids in animals is not conserved across the spectrum of the isoflavonoid family and therefore cannot be predicted, especially where bioavailability is involved. Thus each specific structural variation of the basic isoflavonoid molecule can yield a highly individual biological profile in animals ranging from nil effect through to potent effect. Furthermore, it is thought that some conjugates of biologically active molecules, such as phosphate esters of some biologically active estrogenic steroids, can be largely inactive.
There is a strong need to identify new, improved, better and/or alternative pharmaceutical compositions and agents for the treatment, amelioration and prevention of diseases, conditions and disorders. There is a further need to provide new isoflavonoid compounds and derivatives for the improved formulation, bioavailability and delivery of these compounds. There is also a need for new and different therapies to be available to both physicians and the general public to combat the numerous and various types of diseases and disorders which affect members of the population.
A requirement accordingly exists for the provision of new isoflavonoid compounds and derivatives thereof which are therapeutically beneficial and which show improved, alternative or at least comparable bioactive and bioavailable properties to that of known isoflavonoid compounds.
The present inventors have surprisingly found that phosphate esters of isoflavonoid compounds show good aqueous solubility and bioavailability and exhibit beneficial biological properties. In particular phosphate esters when administered will exhibit a wide range of therapeutic activities including the ability to address oestrogen-levels in the body.
Whilst not wishing to be limited to theory, it is believed that isoflavene prodrugs and derivatives thereof, and in particular isoflavonoid phosphate esters the invention will result in the reduction in the supply of estrogenic steroids, reducing the risk or severity of oestrogen-related diseases and conditions. It is also thought that the isoflavonoid phosphate esters of the invention will provide for the regulation of a range of molecular targets in mammalian cells. Typically, these molecular targets are intimately involved in signal transduction processes that are fundamental to critical cellular processes such as cell growth, differentiation, migration, and death. It can be seen therefore that these surprising biochemical effects have broad and important implications for the health of animals including humans. These and other preferred objects of the invention are described herein.
Thus, according to an aspect of the invention there is provided an isoflavonoid phosphate ester compound of the general formula I:
in which
R1, R2 and Z are independently M2PO4—, hydrogen, hydroxy, OR9, OC(O)R10, OS(O)R10, CHO, C(O)R10, COOH, CO2R10, CONR3R4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or
R2 is as previously defined, and R1 and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from
W is R1, and A and B taken together with the carbon atoms to which they are attached form a six-membered ring selected from
R3 is hydrogen, alkyl, aryl, arylalkyl, an amino acid, C(O)R11 where R11 is hydrogen alkyl, aryl, arylalkyl or an amino acid, or CO2R12 where R12 is hydrogen, alkyl, haloalkyl, aryl, heteroaryl or arylalkyl,
R4 is hydrogen, alkyl or aryl,
or R3 and R4 taken together with the nitrogen to which they are attached comprise pyrrolidinyl or piperidinyl,
R5 is M2PO4—, hydrogen, C(O)R11 where R11 is as previously defined, or CO2R12 where R12 is as previously defined,
R6 is M2PO4—, hydrogen, hydroxy, alkyl, aryl, amino, thio, NR3R4, COR11 where R11 is as previously defined, CO2R12 where R12 is as previously defined or CONR3R4,
R7 is hydrogen, C(O)R11 where R11 is as previously defined, alkyl, haloalkyl, aryl, arylalkyl or Si(R13)3 where each R13 is independently hydrogen, alkyl or aryl,
R8 is M2PO4—, hydrogen, hydroxy, alkoxy or alkyl,
R9 is alkyl, haloalkyl, aryl, arylalkyl, C(O)R11 where R11 is as previously defined, or Si(R13)3 where R13 is as previously defined,
R10 is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an amino acid, alkylamino or dialkylamino,
the drawing " " represents either a single bond or a double bond,
M is independently hydrogen, a straight or branched alkyl, alkenyl, alkynyl, alkoxyalkyl, alkylthioalkyl, or aminoalkyl, a substituted or non-substituted cycloalkyl, an aryl, aralkyl, or alkylaryl, and a substituted cycloalkyl where at least one ring contains one or more of a nitrogen, sulfur, oxygen, phoshorous or silicon heteroatom in the at least one ring;
T is independently hydrogen, alkyl or aryl,
X is O, NR4 or S, preferably O, and
Y is
R14, R15 and R16 are independently M2PO4—, hydrogen, hydroxy, OR9, OC(O)R10, OS(O)R10, CHO, C(O)R10, COOH, CO2R10, CONR3R4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, and
wherein at least one of R1, R2, R5, R6, R8, R14, R15, R16, Z, W or A where present is independently M2PO4—,
In a preferred embodiment, the phosphate ester moiety may be present as the corresponding salt —O—PO(OM)2, where M is hydrogen or a pharmaceutically acceptable counter ion, more preferably Na+, K+, Li+, Mg++ or NH3+, more preferably Na+.
It has surprisingly been found by the inventors that compounds of the general formula I:
R1, R2, W, A, B and Z are as defined above have particular utility and effectiveness in the treatment, prophylaxis, amelioration defence against, and/or prevention of the following diseases and disorders (for convenience hereinafter referred to as the "therapeutic indications"):
(a) all forms of cancer (pre-malignant, benign and malignant) in all tissues of the body. In this regard, the compounds may be used as the sole form of anti-cancer therapy or in combination with other forms of anti-cancer therapy including but not limited to radiotherapy and chemotherapy;
(b) diseases and disorders associated with inflammatory reactions of an abnormal or prolonged nature in any of the body's tissues including but not limited to rheumatoid arthritis, tendonitis, inflammatory bowel disease, ulcerative colitis, Crohn's Disease, sclerosing cholangitis;
(c) papulonodular skin lesions including but not limited to sarcoidosis, angiosarcoma, Kaposi's sarcome, Fabry's Disease
(d) papulosquamous skin lesions including but not limited to psoriasis, Bowen's Disease, and Reiter's Disease;
(e) actinic damage characterized by degenerative changes in the skin including but not limited to solar keratosis, photosensitivity diseases, and wrinkling;
(f) diseases and disorders associated with abnormal angiogenesis affecting any tissue within the body including but not limited to hemangiomas and telangiectasia;
(g) proliferative disorders of bone marrow including but not limited to megaloblastic disease, myelodysplastic syndromes, polycythemia vera, thrombocytosis and myelofibrosis;
(h) autoimmune disease characterized by abnormal immunological responses including but not limited to multiple sclerosis, Type 1 diabetes, systemic lupus erythematosis, and biliary cirrhosis;
(i) neurodegenerative diseases and disorders characterized by degenerative changes in the structure of the neurological system including but not limited to Parkinson's Disease, Alzheimer's Disease, muscular dystrophy, Lou-Gehrig Disease, motorneurone disease;
(j) diseases and disorders associated with degenerative changes within the walls of blood vessels including but not limited to atherosclerosis, atheroma, coronary artery disease, stroke, myocardial infarction, hypertensive vascular disease, malignant hypertension, thromboangiitis obliterans, fibromuscular dysplasia;
(k) diseases and disorders associated with abnormal immunological esponses including but limited to dermatomyositis and scleroderma;
(l) diseases and disorders associated with degenerative changes within the eye including but not limited to cataracts, macular degeneration, retinal atrophy.
In particular the isoflavene compounds also surprisingly have been found to have a potent effect on the production and function of reproductive hormones such as estrogens and androgens. As a result of this, these compounds may be used in the treatment and prevention of the following disorders and diseases:
(a) conditions in women associated with abnormal estrogen/androgen balance including but not limited to cyclical mastalgia, acne, dysmenorrhoea, uterine fibroids, endometriosis, ovarian cysts, premenstrual syndrome, acute menopause symptoms, osteoporosis, senile dementia, infertility; and
(b) conditions in men associated with abnormal estrogen/androgen balance including but not limited to benign prostatic hypertrophy, infertility, gynecomastia, alopecia hereditaria and various other forms of baldness.
Thus according to another aspect of the present invention there is provided a method for the treatment, prophylaxis, amelioration, defence against, and/or prevention of one or more of the therapeutic indications which comprises administering to a subject a therapeutically effective amount of one or more compounds of formula I as defined above.
According to another aspect of the present invention there is provided the use of compounds of formula I for the manufacture of a medicament for the treatment, amelioration, defence against, prophylaxis and/or prevention of one or more of the therapeutic indications.
According to another aspect of the present invention there is provided the use of one or more compounds of formula I in the treatment, amelioration, defence against, prophylaxis and/or prevention of one or more of the therapeutic indications.
According to another aspect of the present invention there is provided an agent for the treatment, prophylaxis, amelioration, defence against and/or treatment of the therapeutic indications which comprises one or more compounds of formula I either alone or in association with one or more carriers or excipients.
According to another aspect of the present invention there is provided a therapeutic composition which comprises one or more compounds of formula I in association with one or more pharmaceutical carriers and/or excipients.
According to another aspect of the present invention there is provided a drink or food-stuff, which contains one or more compounds of formula I.
According to another aspect of the present invention there is provided a microbial culture or a food-stuff containing one or more microbial strains which microorganisms produce one or more compounds of formula I.
According to another aspect of the present invention there is provided one or more microorganisms which produce one or more compounds of formula I. Preferably the microorganism is a purified culture, which may be admixed and/or administered with one or more other cultures which product compounds of formula I.
Throughout this specification and the claims which follow, unless the text requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
FIG. 1 depicts pharmacokinetic data comparing free and total dehydroequol concentrations in serum from mice injected i.p with bolus dosages of DHE bisphosphate prepared in PBS and dosed at 25 mg/kg.
FIGS. 2a and 2b depict pharmacokinetic data comparing free and total dehydroequol concentrations in serum from mice injected i.p with bolus dosages of dehydroequol prepared in different formulations. DHE bisphosphate formulations were prepared in PBS and dosed at 25 mg/kg. DHE PEG:PBS formulations were prepared in a 1:1 PEG:PBS formulations and dosed at 50 mg/kg. DHE-HPBCD formulations were prepared in 20% HPBCD (HPBCD prepared in PBS) and dosed at 50 mg/kg (total DHE levels not shown).
The term "isoflavonoid" is generally taken to mean ring-fused benzopyran molecules having a pendent phenyl group from the pyran ring based on a 1,2-diphenylpropane system. Thus, the classes of compounds generally referred to as isoflavones, isoflavenes, isoflavans, isoflavanones, isoflavanols and the like are generically referred to herein as isoflavonoids, isoflavonoid compounds, or isoflavone metabolites or derivatives thereof.
Preferred isoflavonoid compounds of invention are the isoflavan-4-ones, isoflavenes, isoflavan-4-ols and isoflavans, which in general are hydrogenated products from the base isoflavones, which compounds may also be optionally substituted.
The term "alkyl" is taken to include straight chain, branched chain and cyclic (in the case of 5 carbons or greater) saturated alkyl groups of 1 to 10 carbon atoms, preferably from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl, cyclopentyl, and the like. The alkyl group is more preferably methyl, ethyl, propyl or isopropyl. The alkyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di-(C1-C4-alkyl)-amino-carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl.
The term "alkenyl" is taken to include straight chain, branched chain and cyclic (in the case of 5 carbons or greater) hydrocarbons of 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, with at lease one double bond such as ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 2-methyl-1-peopenyl, 2-methyl-2-propenyl, and the like. The alkenyl group is more preferably ethenyl, 1-propenyl or 2-propenyl. The alkenyl groups may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di-(C1-C4-alkyl)-amino-carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl.
The term "alkynyl" is taken to include both straight chain and branched chain hydrocarbons of 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, with at least one triple bond such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like. The alkynyl group is more preferably ethynyl, 1-propynyl or 2-propynyl. The alkynyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di-(C1-C4-alkyl)-amino-carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl.
The term "aryl" is taken to include phenyl, biphenyl and naphthyl and may be optionally substituted by one or more C1-C4-alkyl, hydroxy, C1-C4-alkoxy, carbonyl, C1-C4-alkoxycarbonyl, C1-C4-alkylcarbonyloxy or halo.
The term "heteroaryl" is taken to include five-membered and six-membered rings which include at least one oxygen, sulfur or nitrogen in the ring, which rings may be optionally fused to other aryl or heteroaryl rings including but not limited to furyl, pyridyl, pyrimidyl, thienyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isopuinolyl, purinyl, morpholinyl, oxazolyl, thiazolyl, pyrrolyl, xanthinyl, purine, thymine, cytosine, uracil, and isoxazolyl. The heteroaromatic group can be optionally substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di-(C1-C4-alkyl)-amino-carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl. The heteroaromatic can be partially or totally hydrogenated as desired.
The term "halo" is taken to include fluoro, chloro, bromo and iodo, preferably fluoro and chloro, more preferably fluoro. Reference to for example "haloalkyl" will include monohalogenated, dihalogenated and up to perhalogenated alkyl groups. Preferred haloalkyl groups are trifluoromethyl and pentafluoroethyl.
The term "pharmaceutically acceptable salt" refers to an organic or inorganic moiety that carries a charge and that can be administered in association with a pharmaceutical agent, for example, as a counter-cation or counter-anion in a salt. Pharmaceutically acceptable cations, which include the moiety M, are known to those of skilled in the art, and include but are not limited to sodium, potassium, calcium, zinc and quaternary amine. Pharmaceutically acceptable anions are known to those of skill in the art, and include but are not limited to chloride, acetate, citrate, bicarbonate and carbonate.
The term "pharmaceutically acceptable derivative" or "prodrug" refers to a derivative of the active compound that upon administration to the recipient is capable of providing directly or indirectly, the parent compound or metabolite, or that exhibits activity itself.
As used herein, the terms "treatment", "prophylaxis" or "prevention", "amelioration" and the like are to be considered in their broadest context. In particular, the term "treatment" does not necessarily imply that an animal is treated until total recovery. Accordingly, "treatment" includes amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
The invention in particular relates to the compounds of the general formula II and uses thereof:
R1, R2, R5, R6, R14, R15, W and Z are as defined above, the drawing " " represents either a single bond or a double bond, and more preferably where
the drawing " " represents a double bond.
In another aspect, the invention in particular relates to the compounds of the general formula III and uses thereof:
R1, R2, R5, R6, R14, R15, W and Z are as defined above.
In another aspect, the invention in particular relates to the compounds of the general formula IV and uses thereof:
Particularly preferred compounds of the present invention are the isoflavonoid compounds as follows:
Isoflavonoid-O—PO(OM)2
wherein M is independently hydrogen or a counter cation, and
wherein the isoflavene compound or derivative is mono-, di-, or per-phosphorylated and may be derived from the following hydroxyl-containing isoflavanone, isoflavene, isoflavanol and isoflavan compounds and derivatives 1-22 as follows:
R2, R16, W and Z are independently H, OH, Cl, Br, Me or OMe, and
R14 is H, OMe, Me, Cl or Br.
In a most preferred embodiment isoflavonoid compound or derivative is a novel mono-, di- or per-phosphate ester of dihydrodaidzein, dihydrogenestein, tetrahydrodaidzein, dehydroequol or equol, most preferably is a phosphate ester of dehydroequol.
Compounds of the present invention have particular application in the treatment of diseases associated with or resulting from estrogenic effects, androgenic effects, vasodilatory and spasmodic effects, inflammatory effects and oxidative effects.
The amount of one or more compounds of formula I which is required in a therapeutic treatment according to the invention will depend upon a number of factors, which include the specific application, the nature of the particular compound used, the condition being treated, the mode of administration and the condition of the patient. Compounds of formula I may be administered in a manner and amount as is conventionally practised. See, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, 1299 (7th Edition, 1985). The specific dosage utilised will depend upon the condition being treated, the state of the subject, the route of administration and other well known factors as indicated above. In general, a daily dose per patient may be in the range of 0.1 mg to 2 g; typically from 0.5 mg to 1 g; preferably from 50 mg to 200 mg. The length of dosing may range from a single dose given once every day or two, to twice or thrice daily doses given over the course of from a week to many months to many years as required, depending on the severity of the condition to be treated or alleviated. It will be further understood that for any particular subject, specific dosage regimens should be adjust over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
The production of pharmaceutical compositions for the treatment of the therapeutic indications herein described are typically prepared by admixture of the compounds of the invention (for convenience hereafter referred to as the "active compounds") with one or more pharmaceutically or veterinarially acceptable carriers and/or excipients as are well known in the art.
The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier or excipient may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose, for example, a tablet, which may contain from 0.5% to 59% by weight of the active compound, or up to 100% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients.
The formulations of the invention include those suitable for oral, rectal, optical, buccal (for example, sublingual), parenteral (for example, subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.
Formulation suitable for oral administration may be presented in discrete units, such as capsules, sachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture such as to form a unit dosage. For example, a tablet may be prepared by compressing or moulding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound of the free-flowing, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Moulded tablets may be made by moulding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
Formulations suitable for buccal (sublingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
Compositions of the present invention suitable for parenteral administration conveniently comprise sterile aqueous preparations of the active compounds, which preparations are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the compound with water or a glycine buffer and rendering the resulting solution sterile and isotonic with the blood. Injectable formulations according to the invention generally contain from 0.1% to 60% w/v of active compound and are administered at a rate of 0.1 ml/minute/kg.
Formulations suitable for rectal or vaginal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
Formulations or compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combination of two or more thereof. The active compound is generally present at a concentration of from 0.1% to 0.5% w/w, for example, from 0.5% to 2% w/w. Examples of such compositions include cosmetic skin creams.
Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the said active compound.
Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2 M active ingredient.
Formulations suitable for inhalation may be delivered as a spray composition in the form of a solution, suspension or emulsion. The inhalation spray composition may further comprise a pharmaceutically acceptable propellant such as carbon dioxide or nitrous oxide.
The active compounds may be provided in the form of food stuffs, such as being added to, admixed into, coated, combined or otherwise added to a food stuff. The term food stuff is used in its widest possible sense and includes liquid formulations such as drinks including dairy products and other foods, such as health bars, desserts, etc. Food formulations containing compounds of the invention can be readily prepared according to standard practices.
Compounds of the present invention have potent antioxidant activity and thus find wide application in pharmaceutical and veterinary uses, in cosmetics such as skin creams to prevent skin ageing, in sun screens, in foods, health drinks, shampoos, and the like.
It has surprisingly been found that compounds of the formula I interact synergisticly with vitamin E to protect lipids, proteins and other biological molecules from oxidation.
Accordingly a further aspect of this invention provides a composition comprising one or more compounds of formula I, vitamin E, and optionally a pharmaceutically, veterinarily or cosmetically acceptable carriers and/or excipients.
Therapeutic methods, uses and compositions may be for administration to humans or animals, such as companion and domestic animals (such as dogs and cats), birds (such as chickens, turkeys, ducks), livestock animals (such as cattle, sheep, pigs and goats), for use in aquaculture applications and the like.
The isoflavonoid prodrugs and derivatives can also be co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, or antiviral compounds.
The active agent can comprise two or more isoflavones or derivatives thereof in combination or synergistic mixture. The active compounds can also be administered with lipid lowering agents such as probucol and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents such as coumadin; calcium channel blockers such as verapamil, diltiazem, and nifedipine; angiotensin converting enzyme (ACE) inhibitors such as captopril and enalapril, and β-blockers such as propanolol, terbutalol, and labetalol. The compounds can also be administered in combination with nonsteriodal antiinflammatories such as ibuprofen, indomethacin, aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac. The compounds can also be administered with corticosteroids.
The co-administration may be simultaneous or sequential. Simultaneous administration may be effected by the compounds being in the same unit dose, or in individual and discrete unit doses administered at the same or similar time. Sequential administration may be in any order as required and typically will require an ongoing physiological effect of the first or initial active agent to be current when the second or later active agent is administered, especially where a cumulative or synergistic effect is desired.
Isoflavone compounds are suitable starting materials for the synthesis of the isoflavonoid compounds of formula I and these isoflavone starting materials may be prepared by standard methods known to those skilled in the art. Suitable methods may be found in, for example, International Patent Applications WO 98/08503 and WO 00/49009 which are incorporated herein in their entirety by reference. Chemical functional group protection, deprotection, synthons and other techniques known to those skilled in the art may be used where appropriate in the synthesis of the compounds of the present invention. Derivatisation of the hydroxy substituted isoflavones to form the conjugates of the present invention may be performed by any suitable method as known to one skilled in the art.
The isoflavone starting materials may also be obtained in the form of concentrates or extracts from plant sources. Again, those skilled in the art will readily be able to identify suitable plant species, however, for example, plants of particular utility include leguminous plants. More preferably, the isoflavone extract may be obtained from obtained from chickpea, lentils, beans, red clover or subterranean clover species and the like.
The aqueous solubility of isoflavonoids is important for their formulation into pharmaceuticals, foodstuffs and cosmetics, many of which are aqueous-based systems. Low solubility is also frequently an impediment to efficient bioavailability in orally administered products. Low solubility is a particularly serious impediment to formulation of intravenous medications, which are most often delivered in aqueous media. The isoflavonoid phosphate esters of the invention are presented in forms which have increased bioavailability, especially enhanced aqueous solubility relative to the unmodified compounds, while substantially retaining the active properties of such unmodified compounds. The phosphate ester is useful as a pro-drug having a polar (solubilising) leaving group which can be readily hydrolysed under physiological conditions to produce the corresponding isoflavonoid compound.
In preferred embodiments, an alcohol functionality of an isoflavonoid is esterified using a phosphoric acid group yielding a phosphate ester. In general, fluids of the digestive and absorptive gastrointestinal tract, other acids, and various enzymes are capable of hydrolysing the esterified isoflavonoid to the starting isoflavonoid.
The phosphate ester is preferably a (OH)2PO2 group due to the presence of the two polar groups, and that it is a good solubliser and has high biological compatibility. Where the M group for M2PO-4- is not hydrogen, it would generally be expected that the solubility would be less for the compound and would therefore be less favoured. Where M is an alkyl group, for example, the non-polar group is preferably selected to be small.
It is also contemplated to employ metal salt complexes of the esterified isoflavones, especially Li+, Na+, K+, Mg++ and ammonium salts, including NH4+ and low molecular weight mono- or polyalkylammonium counter ions.
The examples that follow are not considered to limit the invention as described.
The isoflavonoid phosphate esters of the invention may be prepared by standard chemical processes known by those skilled in the art from available starting materials and straight forward synthetic methods. In this way, several embodiments of the inventive subject matter can be prepared and characterised. These examples all fall within the group of pro-compounds of formula I having at least one group of the formula M2PO4—. These new phosphate esters are all water soluble and readily hydrolysed in vivo, yet are generally quite stable in aqueous solutions in vitro at normal pH at ambient or body temperature, and are more stable as solids.
Phosphate Esters of Dehydroequol
A solution of dehydroequol (120 mg, 0.5 mmole) and di-tert-butyl phosphoramidite (330 ul, 1.0 mmole) in DMF (1 ml) is stirred under argon while 1H-tetrazole (210 mg in 0.5 ml of DMF; 3.0 mmole) is added dropwise. The solution is cooled to −20° C., then a solution of m-chloroperbenzoic acid (260 mg in 0.5 ml of methylene chloride, 1.5 mmole) is added dropwise. After warming to room temperature, the mixture is diluted threefold with ethyl acetate, then washed with 10% sodium metabisulphite and 10% sodium bicarbonate.
The ethyl acetate solution, containing the butyl esters of the dehydroequol phosphates, is washed with 1M HCl and dried over sodium sulfate. After removal of the solvent in vacuo, the residue is treated with 30% TFA in acetic acid for 90 minutes at room temperature. The solvents are removed in vacuo, and the residue is taken up in ethanol and neutralised with sodium hydroxide to pH 5.5. Removal of the solvent in vacuo affords a mixture of sodium salts of dehydroequol phosphates, 130 mg.
Analysis of the phosphate mixture indicated the presence of the 4′-phosphate, the 7-phosphate and the 4′,7-diphosphate derivatives. Where esterification of the compounds of the invention affords mixtures of phosphate esters, they may be separated into individual components by standard separation techniques including fractional crystallisation, column chromatography and HPLC.
The isoflavonoid phosphate esters prepared by the above methods include:
Likewise, phosphate esters of dihydrodaidzein, tetrahydrodaidzein and equol were synthesised affording the following compounds.
and pharmaceutically acceptable salts thereof
Dehydroequol- 7-phosphate
Dehydroequol with its hydroxy group protected at the pendant phenyl 4′-position undergoes reaction according to Example 1 to afford the corresponding 7-phosphate derivative. Any suitable protective group may be employed including MOM or MEM ethers and benzylic ethers. These groups optionally may be removed after phosphorylation. The protecting groups where used may be incorporated in the synthesis of the isoflavonoid starting materials following any of the methods referred to herein, or may be attached at a later time by taking advantage of synthons, chemical reactivity, polarity, electronic considerations, or steric conditions on or near any of the target hydroxy groups.
By these methods mono- di- and per-phosphorylated derivatives of compounds 1-22 described herein are synthesised. The phosphorus acids and pharmaceutically acceptable salts thereof are thus prepared. Proton or carbon magnetic resonance spectra, IR and/or mass spectra was used to characterise the compounds synthesised.
The bioavailability of the isoflavonoid phosphoric esters of the invention are tested by the in vitro hydrolysis of the dehydroequol phosphates by various enzymes and biological media. Results are determined by measuring the amount of free dehydroequol by HPLC. The sera and media used include human serum, human blood, rat blood, alkaline phosphatase type VII-S (bovine intestinal mucosa) and alkaline phosphatase type XXIV (human placenta).
The bioavailability and conversion rate from the ester depends on a number of factors including the nature of the phosphate ester and substitutions thereon, the media, any enzymes present, the temperature and pH. By controlling these various parameters, it is found that some degree of regulation or control can be obtained by altering the half-life of the ester prodrug to better match the desired bioavailability rate.
The esterified isoflavonoids are found to be readily converted to free isoflavonoids in biological media such as gastrointestinal fluid and blood. Among other things, gastrointestinal fluids often have enzymes and sufficiently high pH to hydrolyse ester bonds, and blood generally contains enzymes such as phosphatases which can hydrolyse phosphate ester bonds.
Pharmacokinetic Experiments
Two separate PK experiments were conducted using dehydroequol (DHE)-bisphosphate formulated in PBS by i.p. and oral modes of delivery. Three animals were to be allocated per timepoint with 5 timepoints (15 min, 30 min, 1 hr, 4 hr and 24 hr) (15 mice per study).
The aim was to determine whether the PK profile was comparable when delivered i.p. vs oral.
Protocol—i.p. Administration
1. Female nude mice were maintained on an isoflavone free diet for at least one week to remove background isoflavone levels in plasma.
2. On day prior to experimentation, 3 mice were assigned per time-point and marked with unique identifiers. Each mouse was weighed to determine the density of DHE bisphosphate required per i.p. injection to achieve a dose of 50 mg/kg for each mouse.
A slight excess of formulated DHE-bisphosphate was prepared and the mass of powder adjusted accordingly. The remaining solution was stored at −20° C. for QA analysis.
3. Each mouse was injected into the lower right or left quadrant of the abdomen, ensuring that the needle was not in a vessel or loop of bowel. Once the DHE-boisphosphate was administered, the mice were placed in a cage until each time point (15 min, 30 min, 1 hr, 4hr, 24 hr).
4. Each moused was killed by cervical dislocation, then the blood collected via the thoracic cavity as per SOP BD-009 using a 20 gauge needle.
5. The blood was allowed to clot then centrifuged at top speed for 3 minutes using a bench-top mini-microfuge at RT.
6. Serum was aspirated into an appropriately labelled eppendorf tube and stored at −20° C. until analysed. Sera from animals dosed with vehicle control and formulated DHE-bisphosphate were stored at −20° C. along with 200 ul aliquots of the vehicle and formulated DHE-bisphosphate for analysis.
Protocol—Oral Administration
1. Female BALB/c mice were maintained on an isoflavone free diet for at least one week to remove background isoflavone levels in plasma.
2. On day prior to experimentation, 3 mice were assigned per time-point and marked with unique identifiers. Each mouse was weighed to determine the density of DHE-bisphosphate required to dose animals at 50 mg/kg.
3. Each mouse was restrained and gavaged an appropriate volume of formulated DHE-bisphosphate to achieve a dose of 50 mg/kg. Once DHE-bisphosphate was administered, the mice were placed in a cage until time point (15 min, 30 min, 1 hr, 4 hr, 24 hr). The control animals were gavaged with 200 μl 1% CMC control. Control animals were culled at 15 min, 30 min, 1 hr, 4 hr, 24 hr timepoints.
4. At the designated time points, each mouse was killed by cervical dislocation, then the blood collected via the thoracic cavity as per SOP BD-009 using a 20 gauge needle.
7. The remaining three animals were gavaged with formulation vehicle at time zero and culled at time 30 min. Serum was stored with the other samples.
When dosed at 25 mg/kg in mice the DHE-bisphosphate molecule was metabolised to the free form of DHE with serum concentrations in blood averaging 98.6 μM 15 mins post i.p. injection. The drug was rapidly excreted at a rate of 62 μM/hr with serum levels lowering to 12 μM 1 hr post administration. Total concentrations of DHE (conjugated +free) reached 120 μM 15 mins post administration and was excreted (120 μM/hr) reaching a serum concentration of 30.85 1 hr post administration (Table 1 and FIG. 1).
Bisphosphate: free vs total
Average (μM) Free:Total
Time (hr) Free Total ratio
0.25 74.39 120.79 1.62
0.50 32.68 63.87 1.95
1 12.00 30.85 2.57
4 0.03 1.36 40.72
24 0.00 0.00 0.00
Comparison of free and total dehydroequol concentrations in sera taken from mice dosed i.p. with DHE-bisphosphate and a DHE-PEG:PBS formulation revealed approximately equal concentrations of the free form of the drug was achieved in serum 15 min post administration however, half the dosage of DHE-bisphosphate (25 mg/kg) was required to achieve this result compared with the DHE-PEG:PBS formulation (50 mg/kg) (74.4 μM vs 62 uM respectively) (Table 1, Table 2 and FIG. 2a). Interestingly, the observed free:total ratios for the DHE-bisphosphate preparation and DHE PEG:PBS formulation were some 5-fold different with more total DHE appearing in the plasma 15 min post-administration from rats dosed with the PEG:PBS formulation when compared to the DHE-bisphosphate preparation (120.8 μM phenoxodiol vs 511.6 μM dehydroequol). Plasma concentrations of free dehydroequol were some 1.8 fold (DHE-bisphosphate) and 2.2 fold (DHE-PEG:PBS) lower than those achieved in mice 15 min post administration of a HPBCD formulation of dehydroequol (50 mg/kg) (FIG. 2b; Table 3).
PEG:PBS free vs total
0.5 14.30 357.09 24.97
1 7.11 387.67 54.55
4 0.32 117.69 366.07
Free DHE HPBCD formulation
Free serum PXD
(μM)
Time (hr) HPBCD
0.25 134.02
Uses of esterified isoflavonoids include any presently known or later discovered uses for isoflavonoids or derivatives thereof including those listed above or described in the literature. The esterified isoflavonoids are found to be indicated in the treatment of osteoporosis and other symptoms of estrogen deficiency in postmenopausal women. Also, the compounds of the present invention are used to prevent osteoporosis and consequent fractures that result from osteoporosis, which are major contributors to morbidity and mortality in the elderly. Still further, the esterified isoflavones are used prophylactically to provide UV protection and in other ways to improve general skin health, to stimulate the immune system, and to reduce undesirable effects of oxidation (i.e., provide antioxidant benefits). Importantly the compounds of the invention are used to treat cancer, including breast, ovarian and prostrate cancers.
The isoflavonoid phosphate esters of the invention quite unexpectedly show some beneficial and/or marked activity in the subjects being treated. This comparison shows the particular utility and effectiveness of conjugated isoflavonoid compounds of the invention, and in particular those conjugates from compounds 1 to 34 described above.
Genistein phosphates are found to have poorer pharmacokinetic properties and profiles compared to the isoflavonoid counterparts described and exemplified above.
Thus, specific embodiments and applications of esterified isoflavonoid compounds have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims.
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 of any two or more of said steps or features.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour.
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English were rich, free, and bold; for them the transmission and the exchange of commodities were easy. The commercial rivalry which set in between the two nations was fatal to the French; when the hour of the final struggle came, the Canadians, though brave, resolute, passionately attached to France, and ready for any sacrifice, were few in number compared with their enemies. Scattered over a vast territory, they possessed but poor pecuniary resources, and could expect from the mother country only irregular assistance, subject to variations of gov ernment and fortune as well as to the chances of maritime warfare and engagements at sea, always perilous for the French ships, which were inferior in build and in number, whatever might be the courage and skill of their commanders. The capture of Louisbourg and of the Island of Cape Breton by the English colonists, in 1745, profoundly disquieted the Canadians. They pressed the government to make an attempt upon Acadia. "The population has remained French," they said; "we are ready to fight for our relatives and friends who have passed under the yoke of the foreigner." The ministry sent the Duke of Anville with a considerable fleet; storms and disease destroyed vessels and crews before it had been possible to attack. A fresh squadron, commanded by the Marquis of La Jonquiere, encountered the English off Cape Finisterre in Spain. Admiral Anson had seventeen ships, M. de La Jonquiere had but six; he, however, fought desperately.
"I never saw anybody behave better than the French commander," wrote the captain of the English ship Windsor; "and, to tell the truth, all the officers of that nation showed great courage; not one of them struck until it was absolutely impossible to manoeuvre." The remnants of the French navy, neglected as it had been through the unreflecting economy of Cardinal Fleury, were almost completely destroyed, and England reckoned more than two hundred and fifty ships of war. Neither the successes in the Low Countries and in Germany nor the peace of Aix-la-Chapelle put a serious end to the maritime war; England used her strength to despoil the French forever of the colonies which she envied them. The frontiers of Canada and Acadia had not been clearly defined by the treaties of peace. Distrust and disquiet reigned amongst the French colonists; the ardor of conquest fired the English, who had for a long while coveted the valley of the Ohio and its fertile territories. The covert hostility which often betrayed itself by acts of aggression was destined ere long to lead to open war. An important emigration began amongst the Acadians; they had hitherto claimed the title of neutrals, in spite of the annexation of their territory by England, in order to escape the test oath and to remain faithful to the Catholic faith; the priests and the French agents urged them to do more; more than three thousand Acadians left their fields and their cottages to settle on the French coasts, along the Bay of Fundy. Every effort of the French governors who succeeded one another only too rapidly in Canada was directed towards maintaining the natural or factitious barriers between the two territories. The savages, excited and flattered by both sides, loudly proclaimed their independence and their primitive rights over the country which the Europeans were disputing between themselves. "We have not ceded our lands to anybody," they said; "and we have no mind to obey any king." "Do you know what is the difference between the King of France and the Englishman?" the Iroquois was asked by Marquis Duquesne, the then governor of Canada. "Go and look at the forts which the king had set up, and you will see that the land beneath his walls is still a hunting-ground, he having chosen the spots frequented by you simply to serve your need. The Englishman, on the other hand, is no sooner in possession of land than the game is forced to quit, the woods are felled, the soil is uncovered, and you can scarcely find the wherewithal to shelter yourselves at night."


text:
Q: RESTful Application edit is not reflected on tomcat I followed this tutorial and I was able to successfully get the same result as shown below:
<?xml version="1.0" encoding="UTF-8" standalone="yes"?>
<users>
    <user>
        <id>1</id>
        <name>Mahesh</name>
        <profession>Teacher</profession>
    </user>
</users>

For testing purposes I modified the UserDao.java class with following code:
        User user = new User(1, "Maheshooo", "TeacherDDD");
        User user2 = new User(2, "John", "Brown");
        userList = new ArrayList<User>();
        userList.add(user);
        userList.add(user2);
        saveUserList(userList);

I exported the war file again, then I restarted tomcat server with the new war file but I still get the very same response Mahesh and Teacher like above without 2nd user, I would expect at least the first user to change to Maheshooo and TeacherDDD.
Also in this example I would like to use PUT as well, could you explain how can I add it too?
I am using Win 7, Eclipse Neon, Java 1.8, tomcat 8.5 and jersey 2.25.1. I would be glad if you can give a hand!

A: Because :
     File file = new File("Users.dat");
     if (!file.exists()) {
        User user = new User(1, "Mahesh", "Teacher");
        userList = new ArrayList<User>();
        userList.add(user);
        saveUserList(userList);
     }else{
        FileInputStream fis = new FileInputStream(file);
        ObjectInputStream ois = new ObjectInputStream(fis);
        userList = (List<User>) ois.readObject();
        ois.close();
     }

As par the code file is write at fist time and the it will not go for write it again.
So you will not get affect until you delete Users.dat file.

redpajama_set_name: RedPajamaStackExchange


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It's been a week of TWA and I still could care less about my hair nor what others think. In fact, I wear my hair out to work and though I thought the kids will probably give their comments, it seems they aren't surprised at all. I guess my personality is more than I imagined.
This is by far, the most interesting big chop I've ever done. I have absolutely no interest in this hair at all. It will grow whether I like it or not, so why waste energy on it ?
I've been doing the daily oiling Regimen with my oil mix and wetting the hair twice a day.
I haven't still used any leave in conditioner nor do I have a problem with that.
Actually, it seems my mind knows that my hair will grow so it's tuned off completely.
I don't spend time looking at the growth nor what's even going on with the hair.
For once, all I want to do is finish off items on my to buy list which doesn't have ANY hair product on it. Not that I don't have stuff on my wishlist ; I have a thousand and one but I don't think I care enoughh to get them. What's going on?
I know in a couple of weeks, I'll be back to braiding so I'm nurturing my edges a lot.
There's a program to attend and I might wear a wig for the sake of not dealing with some interesting people. Lol.
My growth partners are also on track and I'll be sharing some wonderful progress with you.


Q: Why couldn't the monster go to sleep?
A: It was afraid there were children under the bed!
Explanation: This joke plays on a common fear of children that there is a monster under the bed- it turns that fear around by having the monster afraid that there are children under the bed.
Listen here for some scary stories.
This entry was posted in Education, ELL, ESL, humor, Joke, jokes, jokes explained, kids jokes, reading, writing and tagged afraid, bed, children, ELL, English, ESL, fear, funny, humor, joke, jokes, kids, kids jokes, monster, reading, sleep, writing. Bookmark the permalink.


Brooklyn, N.Y., May 15, 2013 – MakerBot® made with "Brooklyn pride," is proud to be one of the selected New York City-based designers to be highlighted at the MoMA Design Store Destination: NYC – Made in the USA, that launches May 15, 2013, at the MoMA Design Stores in New York. As part of the MoMA Design Store Destination: NYC – Made in the USA, MakerBot will showcase its MakerBot® Replicator® 2 Desktop 3D Printer (retailing for $2,199), as well as unique and iconic NYC-themed 3D printed novelty items ($7 each), such as the New York Public Library Lions "Patience and Fortitude;" the Statue of Liberty; the Empire State Building; the Chrysler Building Skyline; the Empire State Building Skyline; the Guggenheim Skyline; the Statue of Liberty Skyline; a New York City Subway Car; a New York City Taxi Cab; and iconic New York City street scenes of a subway entrance, a hot dog cart, and a Central Park Bench. There will even be a retro-inspired MakerBot-made MoMA-branded "Mix Tape" MP3 player ($45) and a cool MakerBot-made MoMA 3D printed watch ($68). Destination: NYC – Made in the USA is MoMA Design Store's 11th event in the Destination: Design series. The collection features approximately 200 lifestyle products by 75 New York designers including home accessories, furniture, paper goods, 3D printed items, and jewelry. All products are designed in one of the five boroughs of New York City and manufactured in the United States. Muted colors, graffiti art and pop culture iconography are a few elements of New York's culture that will be celebrated in the product collection. "MakerBot is very proud to have been invited to participate in the MoMA Design Store Destination: NYC – Made in USA program," noted Bre Pettis, CEO of MakerBot. "MakerBots are made with Brooklyn pride and this is a great venue to showcase how 3D printing is helping fuel the Next Industrial Revolution – right here in New York. And the 3D printed MakerBot-made iconic New York scenes are something special to take home." Pettis noted that MakerBot is helping fuel the Next Industrial Revolution by offering desktop 3D printers that are used for rapid prototyping and personal manufacturing by engineers, architects, industrial designers, artists, educators, and people that just like to make things. MakerBot's 3D Ecosystem makes the design and 3D printing process even easier by offering tools and services that bring the design and 3D printing process closer together, such as MakerBot's website Thingiverse.com, that has more than 80,000 3D designs available for sharing and printing. Thingiverse.com has had more than 17 million downloads of items from its site. MakerBot's 3D Ecosystem is also made up of its MakerBot Replicator 2 Desktop 3D Printer, the MakerBot Store, MakerWare, MakerCare, the MakerBot 3D Photo Booth, Bre Pettis' book "Getting Started with MakerBot," classes and workshops on 3D printing, the soon-to-be-released MakerBot Digitizer Desktop 3D Scanner, and strategic partnerships with like-minded companies, such as Autodesk and its 123D family of design products, OUYA, Nokia, Printcraft, MoMA, and many others. "We are so impressed by the depth of local talent submitted during our Open Call for New York designers last year," says Emmanuel Plat, Director of Merchandising of MoMA's retail division. "The innovative products and unique designs truly reflect urban living and led us to create our largest collection to date." The Destination: NYC product collection will be available in MoMA Design Stores in New York City (11 West 53 Street, 44 West 53 Street, 81 Spring Street) and Tokyo and at MoMAstore.org, MoMAonlinestore.co.kr, and MoMAstore.jp beginning in May 2013 and running through August 2013. Destination: NYC is made possible by the generous support of the International Contemporary Furniture Fair® (ICFF®). MakerBot will also showcase the capabilities of its 3D desktop printers in the MoMA Design Store, and the MoMA Design Store will be an authorized retail outlet where MakerBot Replicator 2 Desktop 3D Printers can be purchased on-site. The two locations offering MakerBot Replicator 2 Desktop 3D Printers will be at the New York City MoMA Design Store locations at 81 Spring Street and 44 West 53rd Street. About MakerBot Founded in 2009, Brooklyn-based MakerBot has grown to be a global leader in desktop 3D printing. There are more than 15,000 MakerBot Desktop 3D Printers in use by engineers, designers, researchers, and people who just like to make things. The MakerBot Replicator Desktop 3D Printer has been named Popular Mechanics "Overall Winner" for best 3D printer and has numerous awards, including being honored as one of Time Magazine's Best Inventions of 2012; "Best Emerging Tech" at the 2012 Consumer Electronics Show; won Popular Mechanics Editor's Choice Award; the Popular Science Product of the Year; and recently was awarded a TechCrunch Crunchies Award for best hardware start-up; a Gold Edison Award for Best Design; and a Fast Company 2012 Innovation by Design Award, as well as being named by Fast Company as "One of the World's Top 10 Most Innovative Companies in Consumer Electronics." The company has been featured on the cover of WIRED, The New York Times, The Wall Street Journal, the Economist, Inc., Worth, The Colbert Report, Fast Company, Engadget, Make: Magazine, Rolling Stone, Time.com, IEEE Spectrum, CNN, Financial Times, NPR, Vogue Italia and many others. Follow MakerBot @MakerBot. About Destination: Design Destination: Design began in 2005 with the following regions explored: Finland, Denmark, Buenos Aires, Berlin, Japan, Seoul, Brazil, Portugal, Istanbul, Mexico and now New York (in that order). Destination: Design reflects MoMA's mission-related commitment to educate the public about innovative items of design, and also fosters other important goals. Products sourced often help local artisans by celebrating traditional craftsmanship and interpreting this work in interesting, new presentations. Sustainability in materials and production techniques are important factors for consideration of all items included in Destination projects. Some of the MoMA Design Store's best selling items are a result of the Destination series including the Kangaroo Desk Organizer (Buenos Aires), Pig Cooking Lid (Japan), Wooden Fishing Play Set and Tea Mug (Seoul), and Chalkboard Vase (Brazil). About MoMA's Retail Division MoMA's Retail Division furthers the Museum's mission of educating the public about modern art and good design. What began in 1939 as a small sales desk in the Museum's lobby now features three store locations in New York and one in Tokyo; e-commerce sites for U.S., Japanese, and Korean markets; a mail order catalog; and a wholesale division. The retail division works with the curatorial departments to select and develop products and publications that reflect MoMA's programs and collections. It offers the public thousands of products, from publications and art reproductions to a varied selection of design objects, jewelry, and personal accessories, and larger design items such as furniture and lighting. In addition, any net revenues generated by the Museum Stores are used solely to advance the Museum's mission, which includes supporting the Museum's programs and exhibitions.


Welcome I hope you enjoy my booth-it is always a work in progress as I am listing new items regularly. You can add me to your favorites so you can get updates when I add new stuff.
I am so NOT the quiet type, in other words..well, you know! I started selling online a few months ago & have really enjoyed meeting & making deals with new friends. All of my items are brand new in box/package, unless I state otherwise. Feel free to stop by my booth any time to make an offer or ask me about any of my items.
Visit me at my other booths for great deals on upscale baby gifts & clothing, handbags & jewelry!
Blazey doesn't have any items in their booth!
From one Kentuckian to another! great customer!! Blessings..
Lisa is such an asset to Bonanzle, always helping us newbies when we get lost she is a wonderful person with fantastic items!
I ENDORSED Blazey/Lisa earlier but want to do so again.
She's A OKAY in all ways around Bonanzle.
and presence around the community.
Make a point of crossing paths with this gal.
You'll be glad you did. She has 3 booths, BTW.
She is absolute SWEETHEART!! This girl is a definite asset to Bonanzle. I wouldn't steer you wrong. She has the neatest stuff. I love just browsing her booth.
Lisa is an asset to the Bonanzle Vommunity.
She takes pride in her booth and offers great product at reasonable prices.
Buy from her if you get the chance. You won't be sorry.
A definite asset to the community! She welcomed me and gave me valuable information that I needed as a newcomer! Thank you Blazey!!
Blazey is a super Bonanzler and wonderful person. Blazey definitely contributes to my wonderful Bonanzle experience. Bonanzle and I are lucky to have Blazey's positive contributions working for our collective success.
Thank you so much Blazey.
Blazey is a fabulous seller and a wonderful friend!
Awesome Person!! Very Friendly!! Great deals!! Fast shipment to Canada !!
This is such a beautiful thoughtful person! She has so many great items in her booth, and a super person to deal with!


"He loved her, and he didn't have a ring," so this romantic Gran did not hesitate. She donated her wedding ring to a waiter. Minutes later, he proposed to the girlfriend in front of the café's visitors, and the woman got her invitation to the upcoming wedding.
Sharon Heinemann from Concord, New Hampshire with two sisters stopped for a meal at Legal Sea Foods in Boston a few weeks ago.
The three sisters were served by Mattheus Gomes, a 'young and handsome' waiter. Mattheus was polite and suave and shared with the ladies he had a beloved girlfriend who worked at the same cafe and the man had long planned an engagement.
Unfortunately, he could not get his wish. Mattheus had no money to buy a ring.
Sharon Heinemann was touched by the story and stepped in. She took her wedding diamond ring off her finger and presented it to Mattheus.
The moment was heartbreaking and moved everybody to tears. Mattheus took the ring and immediately proposed to Maria, his girlfriend, in front of the sisters.
The restaurant thanked ladies by paying for their meals. The newly-engaged thanked them with a wedding invitation.