WO2009055932A1 - Substituted phenylpropionic acids as stimulators of hematopoiesis and erythropoiesis - Google Patents

Abstract

Compounds, or pharmaceutically acceptable derivatives thereof, of the following general formula (I): n = 0-4, X = O, NH, S, Z = H, C1-C4 (alky I, alkenyl, alkynyl), (II), (III), (IV), Y = OH, C1-C4 (alky!, branched or straight chain), (V), Y = CH3, (VI), A = Oalkyl (branched or straight chain), OH, Oaryl, halogen (F, Cl, Br), CF3, phenyl, B = halogen (F, Cl, Br), CF3, phenyl may be used at least to treat anemia or neutropenia. They may be used as stimulators of at least hematopoiesis or erythropoiesis. They may also be used in combination therapy.

Description

SUBSTITUTED PHENYLPROPIONIC ACIDS AS STIMULATORS OF HEMATOPOIESIS AND ERYTHROPOIESIS

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority benefit of provisional Application No.

60/985,090, filed November 2, 2007.

FIELD OF INVENTION

The present invention relates to the treatment of anemia. This includes the treatment of anemia associated with the use of chemotherapy and radiotherapy as well as the treatment of anemia arising from chronic renal failure or treatment of HIV- infected patients with AZT (zidovudine). The present invention also relates to reducing drug toxicity and enhancing drug efficiency. In particular, the present invention relates to the use of substituted phenylpropionic acids as a stimulator of the production of erythrocyte progenitors, in particular Burst Forming Unit-Erythroid (Erythrocyte) cells or BFU-E cells.

BACKGROUND OF INVENTION Chemotherapy refers to the use of cytotoxic agents such as, but not limited to, cyclophosphamide, doxorubicin, daunorubicin, vinblastine, vincristine, bleomycin, etoposide, topotecan, irinotecan, taxotere, taxol, 5-fluorouracil, methotrexate, gemcitabine, cisplatin, carboplatin, or chlorambucil in order to eradicate cancer cells and tumors. But these agents are non-specific and, particularly at high doses, they are toxic to rapidly dividing, normal cells. This often leads to various side effects in patients undergoing chemotherapy and radiotherapy. Myelosuppression, a severe reduction of blood cell production in bone marrow, is one such side effect. It is characterized by anemia, leukopenia, neutropenia, agranulocytosis, and thrombocytopenia. Severe chronic neutropenia is also characterized by a selective decrease in the number of circulating neutrophils and an enhanced susceptibility to bacterial infections. The essence of treating cancer with chemotherapeutic drugs is to combine a mechanism of cytotoxicity with a mechanism of selectivity for highly proliferating tumor cells over host cells. But it is rare for chemotherapeutic drugs to have such selectivity. The cytotoxicity of chemotherapeutic agents may limit administrable doses, may affect treatment cycles, and may seriously jeopardize the quality of life for the cancer patient.

Although other normal tissues may also be adversely affected, bone marrow is particularly sensitive to proliferation-specific treatments such as chemotherapy or radiotherapy. Acute and chronic bone marrow toxicity is a common side effect of cancer therapies which leads to decreases in blood cell counts and anemia, leukopenia, neutropenia, agranulocytosis, and thrombocytopenia. One cause of such effects is a decrease in the number of replicating hematopoietic cells (e.g., pluripotent stem cells and other progenitor cells) caused by both a lethal effect of cytotoxic agents or radiation on these cells and by differentiation of stem cells provoked by a feedback mechanism induced by the depletion of more mature marrow compartments. The second cause is a reduction in self-renewal capacity of stem cells, which is also related to both direct (mutation) and indirect (aging of stem cell population) effects (Tubiana et al., Radiotherap. Oncol. 29: 1-17, 1993). Thus, cancer treatments often result in a decrease in red blood cells or erythrocytes in the general circulation.

Erythrocytes are non-nucleated, biconcave, disk-like cells which contain hemoglobin and are essential for the transport of oxygen. Hemoglobin is a tetrapeptide which contains four binding sites for oxygen. Anemia refers to that condition which exists when there is a reduction below normal in the number of erythrocytes, the quantity of hemoglobin, or the volume of packed red blood cells in the blood as characterized by a determination of the hematocrit. The hematocrit or "red blood cell volume" is considered to be a particularly reliable indicator of anemia. Typically, in normal adults, average values for red blood cell count (millions/mm³), hemoglobin (g/100 mL), and hematocrit or volume packed red blood cells (mL/100 tnL) for females and males (at sea level) are 4.8 ± 0.6 and 5.4 ± 0.9, 14.0 ± 2.0 and 16.0 ± 2.0 and 42.0 ± 5.0 and 47.0 ± 5.0, as described in Harrison 's Principles of Internal Medicine, 8^th Ed., Appendix-Table A-5, McGraw Hill (1977). In normal humans, erythrocytes are produced by the bone marrow and released in the circulation, where they survive approximately 120 days, They are subsequently removed by the monocyte-phagocyte system.

Anemia is a symptom of various diseases and disorders. Therefore, anemia may be classified in terms of its etiology. For example, aplastic anemia is characterized by absence of regeneration of erythrocytes and is resistant to therapy. In such patients, there is a marked decrease in the population of myeloid, erythroid, and thrombopoietic stem cells, which results in pancytopenia. Hemolytic anemia arises from shortened survival of erythrocytes and the inability of the bone marrow to compensate for their decreased life span. It may be hereditary or may result from chemotherapy, infection, or an autoimmune process. Iron deficiency anemia refers to a form of anemia characterized by low or absent iron stores, low serum iron concentration, low hemoglobin concentration, or decreased hematocrit. Iron deficiency is the most common cause of anemia. Pernicious anemia, which most commonly affects adults, arises from a failure of the gastric mucosa to secrete adequate intrinsic factor, resulting in malabsorption of vitamin B 12. Sickle cell anemia arises from a genetically determined defect in hemoglobin synthesis. It is characterized by the presence of sickle-shaped erythrocytes in the blood. The above are only exemplary of the many different anemias known to medicine. Of particular interest to the inventors is addressing anemia associated with the use of chemotherapy or radiotherapy in the treatment of cancer. According to Bio World Today (July 23, 2002), approximately 1.2 million cancer patients underwent cytotoxic chemotherapy in the United States and about 800,000 or 67% of them became anemic. Additionally, anemia is also associated with end-stage renal disease as is the case for patients who require regular dialysis or kidney transplantation for survival. This falls under the umbrella of chronic renal failure or the clinical situation in which there is a progressive and usually irreversible decline in kidney function.

Erythropoietin (EPO) is a glycoprotein with a molecular weight of 34,000 which is produced in the kidney. EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow (BFU-E cells) and maintains cell viability (inhibition of apoptosis of BFU-E and CFU-E cells). The biological effects of EPO are receptor mediated. Amino acid identity amongst different animals is 92% between human EPO and monkey EPO and 80% between human EPO and mouse EPO. The primary stimulus for the biosynthesis of EPO is tissue hypoxia. As may be seen from the above, however, EPO has significant therapeutic potential for the treatment of certain anemias. For example, EPO can be used to treat anemia arising from a diminished endogenous production of EPO, which may result from a damaged or nonfunctional kidney (e.g., chronic renal failure as discussed above). Alternatively, EPO can be used to treat anemia arising from damaged bone marrow and subsequently diminished proliferation of erythrocyte progenitors (e.g., BFU-E cells), which results from treatment of cancer patients with cytotoxic chemotherapy or radiotherapy (as also discussed above). Various forms of recombinant EPO are available on the market. They differ by their expression system used for their manufacture and by their sites and degree of glycosylation of the protein. Epoetin alpha is expressed in CHO cells and is available under the trade name of Procrit^®, Epogen^® or Eprex^®. Like EPO, Epoetin alpha has three N-linked glycosylation sites at asparagine (Asn) residues; Asn 19, Asn 33 and Asn 78. Epoietin beta is N-glycosylated at three sites but epoetin omega is N- glycosylated at Asn 24, Asn 28, Asn 83 and partially O-glycosylated at serine (Ser 126). Recently, a hyperglycosylated version of EPO has been approved which contains five N-linked glycosylation sites. It is a slow or extended release form of epoetin alpha available under the trade name of Aranesp^®. This protein displays enhanced biological activity compared to the natural form, due to its approximately three-fold longer serum half-life. But the use of these glycosylated proteins is expensive and restricted since they have to be produced by recombinant technology. Such post-therapeutic ameliorative treatments are unnecessary if patients are "chemoprotected" from immune suppression. Therefore, there is a need for novel compositions and methods to reduce the undesirable side effects of myelosuppression induced by chemotherapy and radiotherapy. SUMMARY OF INVENTION

The present invention satisfies the need for treating anemia by providing a novel method for the stimulation of the hematopoietic system in a mammal, including a human patient, in need of such treatment. It also provides a novel method for treating the myelosuppressive effects of chemotherapy and/or radiotherapy and any other situation in which stimulation of the hematopoietic system can be of therapeutic value such as, but not limited to, anemia.

In accordance with this method, a composition containing a therapeutically effective amount of one or more substituted phenylpropionic acids and a pharmaceutically acceptable carrier is administered to a mammal, including a human patient, to significantly reduce the adverse effects of chemotherapy and/or radiotherapy.

Accordingly, compositions containing one or more substituted phenylpropionic acids for the production of chemoprotectives as a single agent or as a combination of two or more agents with and/or without other chemotherapeutic agents or such drugs which induce a state of myelosuppression are provided.

Another object relates to the use of a substituted phenylpropionic acid as a hematopoiesis and/or erythropoiesis stimulating factor.

Furthermore, compositions containing one or more substituted phenylpropionic acids and the use of such compounds for the treatment of myelosuppression and subsequent anemia, and immunosuppression are provided.

It is an object to provide a method effective for providing chemoprotection of a mammal, including a human patient.

Another object is to provide a method effective for increasing the efficacy of chemotherapy and/or radiotherapy in a mammal, including a human patient. Yet another object is to provide a method for using more usual doses or even increasing the dose of chemotherapeutic compositions necessary to achieve a better therapeutic benefit, while avoiding increased side effects.

Still another object is to provide a method effective for reducing or eliminating chemotherapy-induced anemia in a mammal, including a human patient.

Another object is to provide a method for treating anemia arising from chronic renal failure, especially in those human patients with end-stage renal disease.

Yet another object is to provide a method for treating anemia arising from other medical procedures such as orthopedic surgery or the use of other drugs such as AZT.

Finally, another object is to provide a method that minimizes or avoids adverse effects to the recipient. The mammal, including a human patient, may be selected as in need of treatment prior to such treatment.

Further aspects of the invention will be apparent to a person skilled in the art from the following description and claims and generalization therein. For example, a process of making a medicament using one or more substituted phenylpropionic acids to treat any of the aforementioned diseases or conditions.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the effect of compound II on white blood cell count. Figure 2 shows the effect of compound II on red blood cell count.

Figure 3 shows the effect of compound IX on white blood cell count.

Figure 4 shows the effect of compound IX on total bone marrow cell count.

Figure 5 shows the effect of compound I on white cell count.

Figure 6 shows the effect of compound III on spleen white cell count. Figure 7 shows the effect of compound III on white blood cell count.

Figure 8 shows the effect of compound II on neutrophil mobilization. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention includes compounds or therapeutically acceptable derivatives thereof of the following general formula:

n = 0-4

or straight chain)

A = Oalkyl (branched or straight chain), OH, Oaryl, halogen (F, Cl, Br), CF₃, phenyl B = halogen (F, Cl, Br), CF₃, phenyl

It will be appreciated by those skilled in the art that although the invention is described in terms of the formula described above, any minor modification of the formula such as homologation of the propionic acid (e.g., the corresponding substituted phenylbutanoic acid or phenylacetic acid) constitutes a permitted modification. Therefore, such a minor modification will only result in modulation of biological activity, for example, on hematopoiesis or erythropoiesis. Similarly, alteration of the spatial arrangement of the propionic acid moiety and the other (Y-(CH₂)_n-X-) substituent from a para relationship about the core phenyl ring to a meta relationship constitutes a permitted modification.

High-dose chemotherapy and radiotherapy destroy hematopoietic cells in bone marrow. Subsequently, the patient can be severely depleted in erythrocytes, platelets, and neutrophils. Anemia results in fatigue, a lack of energy, and shortness of breath. Thrombocytopenia leads to prolonged clotting time and bleeding disorders. Neutropenia places the patient at increased risk of infection. Myelosuppression is a dose-limiting factor in cancer treatment.

A method of restoring a patient's hematopoietic system is provided. Current methods employed to do the same make use of cytokines or glycoprotein growth factors. For example, erythropoietin can be used to stimulate the proliferation and maturation of responsive bone marrow erythroid cells. Erythropoietin is approved for human use for the treatment of anemia where appropriate: e.g., anemia arising from the inability to produce a sufficient number of erythrocytes. There are limitations, however, which restrict the use of erythropoietin. Indeed, many of these limitations are common to the medical use of recombinant glycoprotein cytokines - availability, toxicity, and efficacy, especially with chronic use. For example, some patients treated with recombinant human erythropoietin develop an immune response to the glycoprotein which results in pure red cell aplasia. When the latter occurs, the antibody developed to the recombinant protein also attacks the patient's equivalent or endogenous protein. Subsequently, the patient develops a worst anemia than before drug treatment.

But until the unexpected findings disclosed here, the effectiveness of an appropriately substituted phenylpropionic acid (as defined by the general formula above) for the stimulation of production of erythrocytes from erythroid progenitor cells, or erythropoiesis, and hematopoiesis was unknown. Indeed, this discovery was completely unexpected since very little has been reported in the literature with regard to lower molecular weight or smaller molecules than glycoproteins being able to stimulate erythropoiesis. A synthetic dimeric form of an erythropoietin mimetic peptide (EMP) was described by Wrighton et al. (Nature Biotechnol. 15: 1261-1265, 1997). Although considerably smaller than erythropoietin, EMP is a polypeptide which contains twenty amino acids in each monomer. More importantly, EMP is significantly less active than erythropoietin. WO 02/19963 describes synthetic erythropoiesis protein (SEP) as a synthetic stabilized polypeptide with erythropoietin-like biological activity. The reported advantage of SEP is that it is a stabilized, relatively longer, half-life molecule which is made by chemical synthesis and not by relatively more expensive recombinant technology. Stabilization is achieved by the introduction of ethylene glycol units (e.g., PEG) and so this introduces an additional level of complexity into the preparation of SEP. In summary, the prior art teaches that the stimulation of production of erythrocytes requires the use of large polypeptide or protein molecules.

A substituted phenylpropionic acid (as defined by the general formula above) may be used as a hematopoiesis activation or growth factor and, more particularly, as a stimulator of the production of erythrocyte progenitor cells. When used with chemotherapy and/or radiotherapy, an appropriately substituted propionic acid is administered before, during, and/or after the treatment in order to shorten the period of anemia and to accelerate the replenishment of the hematopoietic system. Furthermore, it is possible to use a combination of appropriately substituted phenylpropionic acids at multiple points relative to treatment with chemotherapy and radiotherapy (e.g., prior to treatment, during treatment, subsequent to treatment, or any combination thereof). In severe anemia arising from a diminished production of EPO, an appropriately substituted phenylpropionic acid is used as the therapeutic agent. An appropriately substituted phenylpropionic acid can also be used after bone marrow transplantation in order to stimulate bone marrow stem cells thus shortening the time period for recovery from anemia.

A "therapeutically effective amount" of the compound(s) is used. Such an effective amount may be determined by varying its dose to achieve the desired therapeutic affect(s) such as, for example, treating myelosuppressive effects of chemotherapy and/or radiotherapy, stimulating the production of erythrocyte or other blood cell precursors, reducing the number and/or severity of symptoms of anemia, or any combination thereof. One or more compounds as the active pharmaceutical ingredient(s) can be formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that does not interfere with the physiological effects of the substituted phenylpropionic acid composition, and that is not toxic to a mammal, including a human patient. A pharmaceutical composition may be formulated using an appropriately substituted phenylpropionic acid and pharmaceutically acceptable carriers by methods known to those skilled in the art (see, e.g., The Merck Index, Merck, 14^th Ed., 2006 and Remington: The Science and Practice of Pharmacy, 21^st Ed., Lippincott, 2005). These compositions include, but are not limited to, solids, liquids, oils, emulsions, gels, aerosols, inhalants, capsules, pills, patches, and suppositories.

A method to produce a pharmaceutical composition comprises bringing into contact one or more active ingredients into association with the carrier which constitutes one or more accessory ingredients.

As used herein, the term "chemotherapy" refers to a process of killing proliferating cells using a cytotoxic agent. The phrase "during the chemotherapy" refers to the period in which the effect of the administered cytotoxic agent lasts. On the other hand, the phrase "after the chemotherapy" is meant to cover all situations in which a composition is administered after the administration of a cytotoxic agent regardless of any prior administration of the same, and also regardless of the persistence of the effect of the administered cytotoxic agent.

When applied to chemotherapy, the substituted phenylpropionic acid composition can be administered prior to, during, or subsequent to the chemotherapy (i.e., prior to, during, or subsequent to the administration of a cytotoxic agent).

By "cytotoxic agent" is meant an agent which kills highly proliferating cells: e.g., tumors cells, virally infected cells, or hematopoietic cells. Examples of a cytotoxic agent include, but are not limited to, cyclophosphamide, doxorubicin, daunorubicin, vinblastine, vincristine, bleomycin, etoposide, topotecan, irinotecan, taxotere, taxol, 5- fluorouracil, methotrexate, gemcitabine, cisplatin, carboplatin, or chlorambucil, and an agonist of any of the above compounds. A cytotoxic agent can also be an antiviral agent: e.g., AZT (i.e., 3'-azido-3'-deoxythymidine) or 3TC/lamivudine (i.e., 3- thiacytidine). Such drugs can induce anemia in a mammal, including a human patient. As used herein, the term "chemoprotection" refers to protection provided to a mammal from the toxic effects arising from treatment of the mammal with a chemotherapeutic agent. Most often, the latter is a cytotoxic agent whose therapeutic effect arises from its ability to interfere with or inhibit some aspect of DNA replication, RNA transcription, or subsequent translation of protein. Therefore, a chemoprotective agent refers to any compound administered to a mammal which would protect the mammal, or facilitate the recovery of the animal, from the toxic effects resulting from treatment of the mammal with a chemotherapeutic agent.

Anemia can be diagnosed and its severity can be determined by a person skilled in the art. The term "anemia" may refer to that condition which exists when there is a reduction below normal in the number of erythrocytes, the quantity of hemoglobin, or the volume (hematocrit) of packed red blood cells. Such clinical criteria are subject to variability. Without limitation, anemia may be the result of a reduction in the mass of circulating red blood cell. Efficacy of treatment (including a palliative effect) can be determined by a person skilled in the art.

The pharmaceutical composition may be provided in a form suitable for oral, sublingual, rectal, topical administration or inhalation (nasal spray), intramuscular, intradermal, intraperitoneal, subcutaneous, or intravenous administration for use in the treatment (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery

Systems, 8^th Ed., Lippincott, 2004).

It will be appreciated that the amount of a compound required for use in the treatment will vary with the route of administration, the nature of the condition being treated, the age and condition of the patient, and will ultimately be at the discretion of the attending physician. The desired dose may be conveniently presented in a single dose or as divided doses taken at appropriate intervals, for example as two, three, or more doses per day as necessary to effect or bring about treatment. The term "treatment" or "treating" includes any therapy of existing disease or condition and prophylaxis of the disease or condition (e.g., anemia) in a mammal. This includes (a) preventing the disease or condition from occurring in a patient which may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting or arresting the development of the disease or condition, and (c) relieving the disease or condition by causing its regression or the amelioration of one or more symptoms.

While it is possible that, for use in therapy, an appropriately substituted phenylpropionic acid may be administered as the raw chemical, it may be preferable to formulate that active ingredient as a pharmaceutical composition. A nontoxic composition is formed by the incorporation of any of the normally employed excipients such as, for example but not limited to, mannitol, lactose, trehalose, starch, magnesium stearate, talcum, cellulose, carboxymethyl cellulose, glucose, gelatin, sucrose, glycerol, magnesium carbonate, sodium citrate, sodium acetate, sodium chloride, sodium phosphate, and glycine.

In another embodiment, the pharmaceutical composition is in a form suitable for enteral, mucosal (including sublingual, pulmonary, and rectal), or parenteral (including intramuscular, intradermal, intraperitoneal, subcutaneous, and intravenous) administration. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Methods may include bringing into association the active pharmaceutical ingredient with liquid carriers (e.g., buffered saline, water for injection, etc.) or finely divided solid carriers or both and then, if necessary, shaping the product into the desired form (e.g., gel capsule, tablet, inhalation device, etc.). When desired, the above- described formulations adapted to give sustained release of the active pharmaceutical ingredient may be employed. Sustained release formulations well known to the art include the use of liposomes, biocompatible polymers, a bolus injection, or a continuous infusion.

An appropriately substituted phenylpropionic acid can also be used in combination with other therapeutically active agents such as growth factors (e.g., granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO), etc.); cytotoxic agents or other anticancer agents (e.g., immune modulating or regulating drugs, therapeutic vaccines, or anti-angiogenesis drugs, etc.); or immune suppressive drugs (including antiinflammatory drugs). The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. The combination referred to above may conveniently be presented for use in the form of a single pharmaceutical formulation and, thus, pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof may be provided.

EXAMPLES

The following examples further illustrate the practice of this invention but are not intended to be limiting thereof.

Preparation of substituted phenylpropionic acid compounds.

General Scheme

protecting group

X, Y, Z, n defined as above

All HPLC chromatograms and mass spectra were recorded on a HP 1 100 LC-MS Agilent instrument using a diode array detector. An analytical Cl 8 column (75 x 4.6 mm, 5 microns) with a gradient of 15-99% acetonitrile- water containing with 0.01% TFA as the elutant in 6 min and a flow of 2 mL/min. Example 1 : Compound I ((±)3-(4-[4-phenoxyphenyl)methoxy]-phenyl)hex-4-ynoic acid) where n = 1 , Z - -C≡C-CH₃, X = O and Y = 3- C₆H₅O- C₆H₅.

A 2-liter flask was charged with 4-hydroxybenzaldehyde (50 g, 409 mmol) and water (400 mL). The temperature of the reaction was kept at 75°C and Meldrum's acid (62 g, 430 mmol) was added as a slurry in water (400 mL). The mixture was stirred for 2 hr then cooled in an ice bath for 2 hr. The product was filtered, rinsed with cold water and dried under vacuum. This gave 5-(4-hydroxybenzylidene)-2,2-dimethyl- [l ,3]dioxane-4,6-dione (95 g, 94%) as a yellow solid. ¹H NMR (400 MHz, DMSO-c/₆) δ 9.75 (br, s, IH); 8.27 (s, IH); 8.24 (d, 2H, J = 10 Hz); 6.98 (d, 2H, J = 10 Hz); 1.76 (s, 6H). MS ESI m/e: 519 (2M+Na). This compound was dissolved in anhydrous tetrahydrofuran (350 mL) and added slowly to a solution of 1 -propylmagnesium bromide in tetrahydrofuran (0.5N, 600 mL). The reaction mixture changed to a yellow suspension that was stirred for 15 min. This was quenched with aqueous ammonium chloride (0.6N, 750 mL) and diluted with hexanes (800 mL). The aqueous layer was then acidified to pH 2 with saturated aqueous potassium hydrogen sulfate and extracted with ethyl acetate (2 x 400 mL). The combined extracts were washed with brine, dried over magnesium sulfate, filtered, and concentrated to give (±)-5-[l-(4-hydroxyphenyl)- but-2-ynyl]-2,2-dimethyl-[l,3]dioxane-4,6-dione (37.0 g, 91%) as a pale yellow solid. ¹H NMR (400 MHz, acetone-tf_tf) δ 8.26 (s, IH); 7.39 (d, 2H, J = 8.5 Hz); 6.76 (d, 2H, J = 8.4 Hz); 4.73 (br, s, IH); 4.46 (d, IH, J = 2.4 Hz); 1.82 (s, 3H); 1.81 (s, 3H); 1.64 (s, 3H). MS ESI m/e: 599 (2M+Na). The phenol derivative (37 g) was suspended in a mixture of diethyl ketone (160 mL) and water (80 mL), then heated to reflux for 48 hr. The aqueous layer was saturated with sodium chloride and separated. The organic layer was dried over magnesium sulfate, filtered, and concentrated to a pale brown oil which was crystallized from hot ethyl acetate: hexanes (1 :2). This gave (±)3-(4- hydroxyphenyl)hex-4-ynoic (20.0 g, 77%) as a white powder. ¹H NMR (400 MHz, DMSO-c/₆) δ 12.2 (s, IH); 9.27 (s, IH); 7.12 (d, 2H, J = 8.5 Hz); 6.67 (d, 2H, J = 8.6 Hz); 3.87 (m, IH); 2.54 (m, 2H); 1.82 (d, 3H, J = 2.4 Hz); MS ESI m/e: 205 (M+H); 227 (M+Na). The acid (23.5 g, 115 mmol) was dissolved in acetone (230 mL) and treated with potassium bicarbonate (1 1.5 g, 1 15 mmol). After 15 min, methyl iodide (5 mL, 80 mmol) was added and the reaction stirred at 40°C overnight. An additional portion of methyl iodide (3 mL, 48 mmol) was added and heating was continued for 24 hr. Insolubles were removed by filtration and rinsed with acetone. The filtrate was concentrated to an oil which was purified on silica gel using 2.5% methanol in dichloromethane as eluent. This gave 3-(4-hydroxyphenyl)hex-4-ynoic acid methyl ester (21.5 g, 85%) as a pale yellow oil. ¹H NMR (400 MHz, acetone-<4) δ 8.2 (br, s, IH); 7.20 (d, 2H, J = 9.5 Hz); 6.77 (d, 2H, J = 9.0 Hz); 3.98 (m, IH); 3.60 (s, 3H); 2.65 (m, 2H); 1.78 (d, 3H, J = 2.5 Hz). MS ESI m/e: 219.1 (M+H); 241 (M+Na). The phenol (0.96 g, 4.4 mmol) and 4-methoxybenzyl chloride (0.72 mL, 5.3 mmol) were dissolved in acetone (9 mL) and treated with cesium carbonate (1.45 g, 4.4 mmol). The reaction mixture was stirred at room temperature overnight. Insolubles were filtered and the solution was evaporated under reduced pressure. This gave 3-[4-(4- methoxybenzyloxy)-phenyl]hex-4-ynoic acid methyl ester (1.67 g, 95%) as a white powder which was used without further purification. To a solution of the ester (1.7 g, 4.25 mmol) in methanol (30 mL) was added 2N potassium hydroxide (aq., 3.2 mL). The reaction was stirred at room temperature overnight. The aqueous solution was adjusted to pH 2 with IN HCl (aq) and extracted with ethyl acetate. The combined organic layers were washed with water, brine and the solvent was removed under reduced pressure. This gave an off-white solid. Recrystallization from ethanol gave pure (±)3-(4-[4-phenoxyphenyl)methoxy]phenyl) hex-4-ynoic acid (1.2 g, 73%) as a white powder. ¹H NMR (400 MHz, D₂O) δ 7.34-7.18 (m, 6H); 6.95 (d, 2H, J = 6.5 Hz); 5.05 (s, 2H); 3.88 (m, IH); 2.47 (d, 2H, J = 8.5 Hz); 2.28 (s, 3H); 1.72 (d, 3H, J = 2.5 Hz). MS ESI m/e: 309.1 (M+H); 331.0 (M+Na).

Example 2: Compound II (3-(4-(3-phenoxybenzylamino)-phenyl)-propionic acid) where n = 1 , Z = H, X = NH and Y = 3-C₆H₅-O-C₆H₅.

To a solution of 3-phenoxybenzaldehyde (3.2 mL, 18.5 mmol) in dichloroethane (60 mL) was added 3-(4-aminophenyl)propionic acid (3.0 g, 18.5 mmol). The mixture was sonicated and transferred in a microwave vial (20 mL). The reaction was irradiated at 100°C for 10 min in the microwave. The solution was transferred to a 500 mL round bottom flask and sodium triacetoxy borohydride (7.8 g, 36.9 mmol) was added in a small portion to the mixture. The reaction was stirred at room temperature for 1 hr. To the resulting slurry was added water (100 mL) and the organic layer was separated. The latter was extracted twice with water (100 mL) and dried over sodium sulfate. Solvent was then removed and the crude product purified by column chromatography on silica gel using hexanes:ethylacetate (1 :1) with trace acetic acid. This gave pure 3-(4-(3-phenoxybenzylamino)-phenyl)propionic acid (5.5 g, 86%) as a low melting point solid. ¹H NMR (400 MHz, CDCl₃) δ 2.40 (t, 2H); 2.63 (t, 2H); 4.21 (s, 2H); 6.09 (bs, IH); 6.44-6.47 (m, 2H); 6.81-6.83 (m, IH); 6.87-6.89 (m, 2H); 6.94-6.97 (m, 2H); 7.07 (bs, IH); 7.11-7.18 (m, 2H); 7.29-7.33 (m, IH); 7.35-7.38 (m, 2H); 12.09 (bs, I H); ¹³C NMR (100 MHz, CD₃OD): δ 175.98, 157.66, 157.49, 146.93, 142.81, 129.64, 129.33, 128.66, 123.06, 122.10, 1 18.60, 1 17.489, 1 16.94, 1 13.29, 36.13, 30.13; MS m/z = 348 (M+H⁺); HPLC: 3.7 min.

Example 3: Compound III (sodium salt of 3-[4-(2-ethylbutoxy)-phenyl]-3- phenylpropionic acid) where n = 1 , Z = C₆H₅, X = O and Y = isopentyl.

min

NaHCO₃, H₂O, MeCN, heat, ultrasounds

In a microwave vial was placed a stirrer, potassium carbonate (2.1 g, 15.1 mmol), 4-hydroxybenzophenone (1.5 g, 7.6 mmol) and 1 -bromo-2-ethylbutane (3.0 g, 18.2 mmol). The mixture was homogenized on the Vortex, and irradiated at 15O⁰C for 10 min in the microwave. The resulting suspension was then homogenized on the Vortex and irradiated again at the same temperature for 10 min. Water was then added and the mixture was extracted three times with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified on a BIOTAGE 4OS column (silica, 1% ethyl acetate/ hexanes) to give 2-ethylbutoxybenzophenone (1.5 g, 72%) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.83-7.74 (m, 4H), 7.56-7.43 (m, 3H), 6.96-6.94 (m, 2H), 3.92 (d, 2H, J = 5.7 Hz), 1.71-1.68 (m, IH), 1.53-1.44 (m, 4H), 0.94 (t, 6H, J = 7.4 Hz); ¹³C NMR (IOO MHz, CD₃OD): δ 195.74, 163.35, 138.60, 132.79, 132.06, 130.04, 129.95, 128.41, 1 14.27, 70.54, 41.02, 23.57, 11.39. A solution of this ketone (1.4 g, 4.9 mmol) in tetrahydrofuran (10 mL) was added to a basic solution of triethylphosphono acetate in tetrahydrofuran (prepared by adding dropwise a solution of the phosphono acetate (1.7 g, 7.4 mmol) under nitrogen in tetrahydrofuran (10 mL) to a suspension of sodium hydride 60% in mineral oil (0.3 g, 7.4 mmol) at room temperature for 15 min. The reaction was then heated at 8O⁰C for 16 hr. Additional amount of tetrahydrofuran (20 ml) was added to the mixture followed by an aqueous solution of lithium hydroxide ( 10 mL, 1.0 g, 24.7 mmol). Methanol (10 mL) was then added and the reaction was stirred at room temperature for 72 hr. The aqueous layer was then acidified to pH 2 with concentrated hydrochloric acid (IN), extracted three times with ethyl acetate and washed with brine. The solvent was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified on a BIOTAGE 4OS column (silica, 20-50% ethyl acetate/ hexanes) to give 3-[4-(2-ethylbutoxy)- phenyl]-3-phenylacrylic acid (0.98 g, 61%) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.39-7.14 (m, 7H), 6.90-6.83 (m, 2H), 6.27and 6.21 (s, IH), 3.89-3.85 (m, 2H), 1.71-1.65 (m, IH), 1.55-1.42 (m, 4H), 0.97-0.91 (m, 6H). MS ESI m/e 325 (MH⁺); HPLC: 5.2 min. The mixture of the cis and trans isomers (0.7 g, 2.2 mmol) were dissolved in ethyl acetate (30 mL) and treated with 10% palladium on charcoal (60.0 mg) at room temperature. The reaction was stirred under hydrogen atmosphere for 16 hr. Insolubles were removed by filtration and the solvent was evaporated to dryness. This gave pure 3-[4-(2-ethylbutoxy)-phenyl]-3-phenylpropionic acid (0.7 g, 93.0%) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.30-7.1 1 (m, 7H), 6.84-6.81 (m, 2H), 4.48 (t, IH, J = 7.9 Hz), 3.81 (d, 2H, J = 5.7 Hz), 3.05 (d, 2H, J = 7.8 Hz), 1.67-1.61 (m, IH), 1.52-1.39 (m, 4H), 0.92 (t, 6H, J = 7.5 Hz); MS ESI m/e 349 (M+Na)⁺; HPLC: 5.0 min. A solution of sodium bicarbonate (172.0 mg, 2.1 mmol) in water (10 mL) was added to the acid (0.67 g, 2.1 mmol). The mixture was than sonicated, heated and stirred at room temperature. Acetonitrile was added to obtain a clear solution. The mixture was filtered and the water was removed by lyophilization. This gave pure sodium salt of 3-[4-(2-ethylbutoxy)-phenyl]-3-phenylpropionic acid (0.7 g, 99.0%) as a white solid. ¹H NMR (400 MHz, D₂O): δ 7.04-7.02 (m, 2H), 6.97-6.94 (m, 2H), 6.85- 6.82 (m, 3H), 6.33-6.31(m, 2H), 4.27-4.23 (m, I H), 3.15-3.14 (m, 2H), 2.80-2.65 (m, 2H), 1.07-0.81 (m, 5H), 0.36-0.32 (m, 6H); ¹³C NMR (100MHz, D₂O): δ 180.5, 157.2, 144.8, 137.1, 128.6, 127.8, 126.2, 1 14.5, 69.8, 47.1, 43.9, 40.4, 22.9, 10.6; MS ESI m/e 309 (M-OH)⁺; HPLC: 5.0 min.

Example 4: Compound IV (sodium salt of 3-[4-(2-ethylbutoxy)-phenyl]-3-(4- fluorophenyl)-propionic acid) where n = 1 , Z = 4-F-C₆H₅, X = O and Y = isopentyl.

The above compound was prepared as in Example 3 except 4- hydroxybenzophenone was replaced by 4-hydroxyphenyl-4-fluorophenylketone. White solid; mp 52°C; ¹H NMR (400 MHz, CD₃OD): δ 7.26-7.20 (m, 2H), 7.14 (d, J = 8.61 Hz, 2H), 6.93 (t, J = 8.80 Hz, 2H), 6.79 (d, J = 6.65 Hz, 2H), 4.48 (t, J = 8.02 Hz, IH), 3.81 (d, J = 5.67 Hz, 2H), 2.81 (d, J = 8.02 Hz, 2H), 1.65-1.58 (m, IH), 1.57-1.35 (m, 4H), 0.92 (t, J = 7.43 Hz, 6H); ¹³C NMR (CD₃OD) δ 179.2, 162.6, 160.2, 157.9,141.8, 137.2, 129.3, 128.6, 1 14.6, 1 14.4, 1 14.1, 69.8, 46.8, 45.0, 41.2, 23.3, 10.32; HPLC: 5.0 min.

Example 5: Compound V (sodium salt of 3-(4-butoxyphenyl)-3-phenylpropionic acid) where n = 1 , Z = C₆H₅, X = O and Y = zero.

The above compound was prepared as in Example 3 except l-bromo-2- ethylbutane was replaced by 1-iodobutane. White solid; ¹H NMR (400 MHz,

D₂O+CD₃OD): δ 7.27-7.07 (m, 7H), 6.67 (d, 2H, J = 8.6 Hz), 4.42 (t, IH, J = 8.0 Hz),

3.67 (t, 2H, J = 6.6 Hz), 2.94-2.83 (m, 2H), 1.50-1.43 (m, 2H), 1.25-1.16 (m, 2H), 0.73 (t, 3H, J = 7.4 Hz); ¹³C NMR (100MHz, D₂CH-CD₃OD): δ 180.33, 156.68, 145.02, 137.42, 128.60, 128.43, 127.59, 126.10, 1 14.51 , 67.89, 44.05, 30.64, 18.67, 13.12; MS ESI m/e 343 (M+Na)⁺; HPLC: 4.4 min.

Example 6: Compound VI (sodium salt of 3-(4-benzyloxyphenyl)-3-phenylpropionic acid) where n = 1 , Z = C₆H₅, X - O and Y = C₆H₅.

The above compound was prepared as in Example 4 except l-bromo-2- ethylbutane was replaced by benzyl bromide. White solid; ¹H NMR (400 MHz, D₂(HCD₃OD): 57.24-7.07 (m, 12H), 6.73 (d, 2H, J = 8.8 Hz), 4.78 (s, 2H), 4.39 (t, IH, J = 8.0 Hz), 2.85-2.81 (m, 2H); ¹³C NMR (100MHz, D₂O+CD₃OD): δ 156.64, 145.33, 138.1 1 , 136.98, 128.85, 128.70, 128.61, 128.20, 127.79, 126.29, 1 15.05, 70.19, 47.57, 44.35; MS ESI m/e 315 (M-OH)⁺; HPLC: 4.3 min.

Example 7: Compound VII (sodium salt of 3-[4-(4-fluorobenzyloxy)-phenyl]-propionic acid) where n = 1 , Z = H, X = O and Y = 4-F-C₆H₅.

In a microwave vial was placed a stirrer, potassium carbonate (0.12 g, 0.83 mmol), 3-(4-hydroxyphenyl)propionic acid methyl ester (0.1 g, 0.56 mmol) and 4- fluorobenzyl bromide (96.0 mg, 0.5 mmol) and acetone (1.5 mL). The mixture was homogenized on the vortex, and irradiated at 15O⁰C for 10 min in the microwave. The resulting suspension was then homogenized on the vortex and irradiated again at the same temperature for 10 min. The mixture was filtered off, washed with acetone and evaporated under reduced pressure. The crude product was purified on a BIOTAGE 12S column (silica, 10% ethyl acetate/ hexanes) to give 3-[4-(4-fluorobenzyloxy)- phenylj-propionic acid methyl ester (0.15 g, 91 %) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.41-7.38 (m, 2H), 7.13-7.04 (m, 4H), 6.89 (d, 2H, J = 8.6 Hz), 4.99 (s, 2H), 3.67 (s, 3H), 2.90 (t, 2H, J = 7.8Hz), 2.60 (t, 2H, J = 7.8Hz); ¹³C NMR (100MHz, CDCl₃): δ 173.65, 163.94, 161.48, 157.35, 133.28, 133.09, 133.06, 129.60, 129.54, 129.51, 1 15.82, 1 15.61 , 115.08, 69.58, 51.85, 36.19, 30.33; MS ESI m/e 289 (MH⁺); HPLC: 4.6 min. To a solution of the ester (0.15 g, 0.5 mmol) in tetrahydrofuran (20 mL) was added an aqueous solution of lithium hydroxide monohydrate (0,84 mL, 2.52 mmol) followed by methanol (0.84 mL). The reaction was stirred at room temperature for 16 hr. The mixture was then acidified with concentrated hydrochloric acid (3M), extracted three times with ethyl acetate and washed with brine. The solvent was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. This gave 3-[4-(4-fluorobenzyloxy)-phenyl]-propionic acid (0.13 g, 94%) as a white solid. ¹H NMR (400 MHz, CDCI₃+CD₃OD): δ 7.38-7.34 (m, 2H), 7.09 (d, 2H, J = 8.8 Hz), 7.02 (t, 2H, J = 8.8H z), 6.84 (d, 2H, J = 8.8 Hz), 4.95 (s, 2H), 2.83 (t, 2H, J = 7.7 Hz), 2.53 (t, 2H, J = 7.8 Hz); ¹³C NMR (100 MHz, CDCI₃+CD₃OD): δ 175.84, 163.86, 161.42, 157.24, 133.40, 133.23, 133.20, 129.53, 129.45, 129.34, 1 15.45, 1 15.24, 1 14.94, 69.42, 36.07, 30.15; MS ESI m/e 319 (M+2Na)⁺; HPLC: 3.8 min. A solution of sodium bicarbonate (0.5 M, 0.94 mL, 0.5 mmol) in water (5 mL) was added to the acid (0.12 g, 0.5 mmol). The mixture was than sonicated, heated and stirred at room temperature. Acetonitrile was added to obtain a clear solution. The mixture was filtered and the water was removed by lyophilization. This gave pure sodium salt of 3-[4-(4- fluorobenzyloxy)-phenyl]propionic acid (0.14 g, 96.0%) as a white solid. ¹H NMR (400 MHz, D₂O): δ 7.28-7.24 (m, 2H), 7.02 (d, 2H, J = 8.6 Hz), 6.95 (t, 2H, J •= 8.9 Hz), 6.77 (d, 2H, J = 8.6 Hz), 4.86 (s, 2H), 2.62 (t, 2H, J = 7.6 Hz), 2.24 (t, 2H, J = 7.8 Hz); ¹³C NMR (100 MHz, D₂O): δ 182.80, 155.99, 135.30, 132.57, 130.38, 130.29, 129.55, 1 15.65, 1 15.43, 1 15.38, 69.90, 39.51 , 31.19; MS ESI m/e 257 (M-OH)⁺; HPLC: 3.9 min. Example 8: Compound VIII (sodium salt of (RS)-3-[4-(4-methoxybenzyloxy)phenyl]- hex-4-ynoic acid) where n=l, Z = -C≡C-CH₃, X = O and Y = 4-MeO-C₆H₅.

In a 100 mL round bottom flask was charged a stirrer, potassium carbonate

(13.8 g, 100 mmol), 4-hydroxybenzaIdehyde (6.13 g, 50.2 mmol) and 1-chloromethyl- 4-methoxybenzene (8.8 mL, 65.3 mmol). The mixture was stirred for 5 h at room temperature and then cooled in an ice bath for few hours. The precipitate was filtered, washed with water and lyophilized. This gave 4-(4-methoxybenzyloxy)benzaldehyde (12.0 g, 98%) as white solid. ¹H NMR (400 MHz, OMSO-d₆): δ 9.84 (s, IH), 7.84 (d, J = 8.8 Hz, 2 H), 7.38 (d, J = 8.8 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 5.12 (s, 2H), 3.73 (s, 3H); MS ESI m/e 243 (M+l). The aldehyde (5.4 g, 22.3 mmol) was dissolved in toluene (17.0 mL) and treated with 2,2-dimethyl-l,3-dioxane- 4,6-dione (3.2 g, 22.3 mmol), concentrated acetic acid (0.7 mL) and piperidine (0.4 mL). The mixture was heated in an oil bath for 3 h at 135⁰C to remove the water. The solution was then cooled in an ice bath for few hours and the precipitate was filtered, washed with cold toluene, and dried under high vacuum. This gave 5-[4-(4- methoxybenzyloxy)-benzylidene]-2,2-dimethyl-[l ,3] dioxane-4,6-dione (5.6 g, 67%) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-^₆): δ 8.28 (s, IH), 8.19 (d, J = 9.0 Hz, 2 H), 7.38 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 5.14 (s, 2H), 3.73 (s, 3H), 1.71 (s, 6H); MS ESI m/e 391 (M+Na). This compound (1.0 g, 2.8 mmol) was dissolved in anhydrous tetrahydrofuran (9.3 mL) under nitrogen atmosphere and was added slowly to a solution of 1 -propynylmagnesium bromide (0.5M, 7.3 mL). The resulting mixture was stirred for 20 min at room temperature and then quenched with a saturated solution of ammonium chloride and water. The aqueous layer was extracted three times with ethyl acetate. The combined extracts were washed with water, dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness. The yellow solid obtained was treated with pyridine (21 ,6 mL) and distilled water (4.3 mL). The resulting solution was placed in a sealed tube and stirred for 36 hr at 100⁰C in an oil bath. The brown solution was then allowed to cool to room temperature and acidified to pH 2 with concentrated hydrochloric acid. Water was then added and the solution was extracted three times with ethyl acetate. The organic layer was washed three times with 10% hydrochloric acid, brine, dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. The resulting residue was recrystallized from 95% ethanol. This gave pure (RS)-3-[4-(4-methoxybenzyloxy)- phenyl]-hex-4-ynoic acid (0.6 g, 67%) as an off-white solid. ¹H NMR (400 MHz, DMSO-flfc): δ 12.22 (s, IH), 7.34 (d, J = 8.6 Hz, 2 H), 7.24 (d, J = 8.8 Hz, 2H), 6.91 (m, 4H), 4.96 (s, 2H), 3.91 (m, IH), 3.73 (s, 3H), 2.57 (m, 2H), 1.75 (d, J = 2.5 Hz, 3H); MS ESI m/e 325 (M+l). The acid (2.2 g, 6.8 mmol) was mixed with sodium bicarbonate (0.6 g, 6.8 mmol) in a 100 mL round bottom flask. Water (17 mL) was added and the mixture was sonicated, heated, and stirred overnight. The resulting solution was then cooled in a dry ice/acetone bath and lyophilized. This gave pure sodium salt of (RS)-3-[4-(4-methoxybenzyloxy)-phenyl]-hex-4-ynoic acid as a white solid (2.3 g, 99%). mp > 209⁰C (decomposition); ¹H NMR (400 MHz, DMSO-dβ): δ 7.33 (d, J = 8.6 Hz, 2 H), 7.21 (d, J - 8.6 Hz, 2H), 6.9 (d, J =8.8 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 4.9 (s, 2H), 3.95 (m, IH), 3.72 (s, 3H), 2.28 (m, 2H), 1.72 (d, J = 2.3 Hz, 3H); ¹³C NMR (101 MHz, DMSCW₆): δ 174.42, 159.59, 157.41, 136.14, 130.07, 129.78, 128.92, 1 15.08, 1 14.48, 83.54, 77.32, 69.54, 55.70, 47.47, 34.31 , 4.05; MS ESl m/e 347 (M+l).

Example 9: Compound IX (sodium salt of (RS)-3-[4-(4-methoxybenzyloxy)- phenyl]butyric acid) where n = 1 , Z = CH₃, X = O and Y = 4-MeO-Ph. The above compound was prepared as in Example 9 except 1 - propynylmagnesium bromide was replaced by methylmagnesium bromide. White solid; mp 190-192⁰C; ¹H NMR (400 MHz, DMSO-c/₆): δ 7.331 (d, J =7.2 Hz, 2 H), 7.105 (d, J -7.6 Hz, 2H), 6.9 (d, J =7.2 Hz, 2H), 6.8 (d, J =7.8 Hz, 2H), 4.9 (s, 2H), 3.7 (s, 3H), 3.0 (m, IH), 2.3 (m, 2H), 1.1 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO-c/₆): δ 174.62, 159.59, 157.26, 139.51, 130.09, 129.83, 128.29, 1 15.21 , 1 14.46, 69.52, 55.67, 44.46, 35.96, 22.89; MS ESI m/e 323 (M+l).

Example 10: In vivo induction of immune cell proliferation or chemoprotection by compound II (3-(4-(3~phenoxy-benzylamino)-phenyl)propionic acid).

Female C57BL/6 mice, 6- to 8-week old, were immunosuppressed by treatment with 200 mg/kg of cyclophosphamide administered intravenously at day 0. To examine the immunoprotective effect of compound III or other compounds, mice were pre- treated orally at day -3, -2 and -1 at day 0 with the compound. Mice were sacrificed at day +5 by cardiac puncture and cervical dislocation. Then, a gross pathological observation of the femurs (as a source of bone marrow cells) was recorded. After sacrifice, tissues were crushed in phosphate buffered saline and cells were counted with a hemacytometer.

A significant increase in white blood cell count was observed with oral pre- treatment with compound II in cyclophosphamide treated mice (Fig. 1). Further, some treated animals return to a "baseline level" in terms of the white blood cell count as compared to non-immunosuppressed animals (control).

Furthermore, an increase in red blood cell count was observed with oral pre- treatment with compound II in cyclophosphamide treated mice (Fig. 2). Further exemplification was undertaken to illustrate the effect of compounds on hematopoiesis and erythropoiesis induction or chemoprevention.

A significant increase in white bone marrow cell count was observed with oral pre-treatment with compound IX in cyclophosphamide treated mice (Fig. 3). Some treated animals return to a "baseline level" in terms of the white bone marrow cell count as compared to non-immunosuppressed animals (control). In addition, an increase in total bone marrow cell count was observed with oral pre-treatment with compound IX in cyclophosphamide treated mice (Fig. 4).

Compound I also induces an increase in white blood cell count in cyclophosphamide treated mice (Fig. 5).

A significant increase in spleen white cell count was observed with oral pre- treatment with compound III in cyclophosphamide treated mice (Fig. 6). Furthermore, an increase in white blood cell count was observed with oral pre-treatment with compound III in cyclophosphamide treated mice (Fig. 7).

Example 1 1 : 7« vivo induction of neutrophil mobilization by compound II.

Female C57BL/6 mice, 6- to 8-week old, were treated with 200 mg/kg of compound III administered orally at day 0. To examine the neutrophil mobilization effect of compound III, mice were treated orally at day 0 with the compound. Mice were sacrificed at time 0, 0.5, 1, 2 and 4 hr by cardiac puncture and cervical dislocation. Then, blood was collected. Blood cells were counted on a Coulter counter and stained with Wright/Giemsa staining for a differential cell count analysis.

Figure 8 indicates that compound II induces a significant increase in neutrophil mobilization as seen by an enhancement of the number of neutrophils in blood after 30 and 60 min. The neutrophil count then returns to baseline at 2 hr.

Patents, patent applications and other publications cited herein are incorporated by reference in their entirety.

All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. A claim using the transition "comprising" allows the inclusion of other elements to be within the scope of the claim; the invention is also described by such claims using the transitional phrase "consisting essentially of (i.e., allowing the inclusion of other elements to be within the scope of the claim if they do not materially affect operation of the invention) and the transition "consisting" (i.e., allowing only the elements listed in the claim other than impurities or inconsequential activities which are ordinarily associated with the invention) instead of the "comprising" term. Any of the three transitions can be used to claim the invention.

It should be understood that an element described in this specification should not be construed as a limitation of the claimed invention unless it is explicitly recited in the claims. Thus, the claims are the basis for determining the scope of legal protection granted instead of a limitation from the specification which is read into the claims. In contradistinction, the prior art is explicitly excluded from the invention to the extent of specific embodiments that would anticipate the claimed invention or destroy novelty.

Moreover, no particular relationship between or among limitations of a claim is intended unless such relationship is explicitly recited in the claim (e.g., the arrangement of components in a product claim or order of steps in a method claim is not a limitation of the claim unless explicitly stated to be so). All possible combinations and permutations of the individual elements disclosed herein are considered to be aspects of the invention; similarly, generalizations of the invention's description are considered to be part of the invention.

From the foregoing, it would be apparent to a person of skill in this art that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification.

Claims

WHAT IS CLAIMED IS:

1. A method of at least stimulating hematopoiesis or erythropoiesis in a human patient in need of such treatment, said method comprising administration to said patient of a therapeutically effective amount of at least one compound described by the following general formula:

n = 0-4

branched or straight chain)

A = Oalkyl (branched or straight chain), OH, Oaryl, halogen (F, Cl, Br), CF₃, phenyl B = halogen (F, Cl, Br), CF₃, phenyl

2. The method according to Claim 1, wherein hematopoiesis is stimulated to treat at least neutropenia or anemia or both arising from chemotherapy in said patient.

3. The method according to Claim 1 , wherein hematopoiesis is stimulated to treat at least neutropenia or anemia or both arising from radiotherapy in said patient.

4. The method according to Claim 1 , wherein erythropoiesis is stimulated to treat at least anemia arising from chemotherapy in said patient.

5. The method according to Claim 1, wherein erythropoiesis is stimulated to treat at least anemia arising from radiotherapy in said patient.

6. The method according to Claim 1, wherein erythropoiesis is stimulated to treat at least chronic anemia in said patient.

7. The method according to Claim 1 , wherein erythropoiesis is stimulated to treat at least transient anemia in said patient.

8. The method according to Claim 1, wherein erythropoiesis is stimulated to treat at least anemia arising from chronic kidney disease in said patient.

9. The method according to Claim 1 , wherein erythropoiesis is stimulated to treat at least anemia arising from end-stage renal disease in said patient.

10. The method according to Claim 1, wherein erythropoiesis is stimulated to treat at least drug-induced anemia in said patient.

1 1. The method according to Claim 1 , wherein erythropoiesis is stimulated to treat at least anemia arising from a medical or surgical procedure in said patient.

12. The method according to Claim 1 further comprising simultaneous administration of a therapeutically effective amount of human erythropoietin which is reduced by the administration of a therapeutically effective amount of at least said one or more compounds.

13. The method according to Claim 1 further comprising separate administration of a therapeutically effective amount of human erythropoietin before and/or after administration of a therapeutically effective amount of at least said one or more compounds, but not simultaneous administration.

14. A composition comprising (i) a therapeutically effective amount of at least one or more compounds as defined in Claim 1 and (ii) one or more growth factors.

15. A composition comprising (i) a therapeutically effective amount of at least one or more compounds as defined in Claim 1 and (ii) one or more cytotoxic agents or other anticancer agents.

16. A composition comprising (i) a therapeutically effective amount of at least one or more compounds as defined in Claim 1 and (ii) one or more immune suppressive drugs.

17. A method of at least increasing production of erythrocytes in a mammal comprising administration to said mammal of a therapeutically effective amount of at least one or more compounds as defined in Claim 1.

18. A method of at least increasing production of erythroid progenitor cells in a mammal, said method comprising administration to said mammal of a therapeutically effective amount of at least one or more compounds as defined in Claim 1.

19. A method of at least increasing production of hemoglobin in a mammal, said method comprising administration to said mammal of a therapeutically effective amount of at least one or more compounds as defined in Claim 1.

20. A method of at least increasing hematocrit in a mammal, said method comprising administration to said mammal of a therapeutically effective amount of at least one or more compounds as defined in Claim 1.

21. A method of delaying the progression of renal failure in a human patient with chronic kidney disease, said method comprising administration to said patient of a therapeutically effective amount of at least one or more compounds as defined in Claim 1.

22. A method of inducing neutrophil mobilization in a human patient in need of such treatment, said method comprising administration to said patient of a therapeutically effective amount of at least one or more compounds as defined in Claim 1.