KR100699968B1 - Analogs of 2-phthalimidinoglutaric acid and their use as inhibitors of angiogenesis - Google Patents

Abstract

The present invention provides novel useful 2-phthaliminoglutaric acid analogs. These analogs include hydroxylated analogs of DL-2-methyl-2-phthaliminoglutaric acid and 2-phthaliminoglutaric acid, wherein R ₃ and R ₄ are independently H or OH. The invention also provides a method of making these analogs. The invention also provides two enantiomers of DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid and (S)-( -)-2-methyl-2-phthaliminoglutaric acid, and a method for separating these individual enantiomers from the racemate. In addition, the present invention provides a method of administering one or more of these compounds to inhibit angiogenesis and to treat angiogenesis-related diseases including cancer and macular degeneration.

2-phthaliminoglutaric acid analogs, angiogenesis inhibitors

Description

ANALOGS OF 2-PHTHALIMIDINOGLUTARIC ACID AND THEIR USE AS INHIBITORS OF ANGIOGENESIS

Cross-reference to related applications

The present invention discloses US Provisional Patent Application 60 / 085,037, filed May 11, 1998, US Provisional Patent Application 60 / 097,384, filed August 21, 1998, and US Provisional Patent Application, filed November 12, 1998. Claim priority on 60 / 108,037.

Field of technology

The present invention relates to 2-methyl glutamic acid derivatives, namely hydroxylated derivatives of 2-methyl-2-phthaliminoglutaric acid and 2-phthaliminodinoglutaric acid. More specifically, the present invention relates to the preparation of 2-phthaliminoglutaric acid analogs and the separation of 2-methyl-2-phthaliminoglutaric acid enantiomers. In addition, the present invention relates to the use of such compounds in treating cancer and treating angiogenesis-related diseases.

Angiogenesis is the production of renal blood vessels into tissues or organs. Under normal physiological conditions, humans and animals cause this angiogenesis only in very specific, limited locations. For example, angiogenesis is commonly observed in wound healing, placental and embryonic development, and corpus luteum, endometrium and placenta formation.

Angiogenesis is controlled through a highly regulated system of angiogenic stimulants and inhibitors. Control of angiogenesis has been found to be altered in certain pathological conditions and in some cases is associated with angiogenesis in which the pathological damage accompanying the disease is not controlled. Both controlled and uncontrolled angiogenesis seem to proceed in a similar way. Endothelial cells and perivascular cells surrounded by the basement membrane form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells lining the lumen of blood vessels extend through the basement membrane. Angiogenic stimulants induce endothelial cells to migrate through the eroded basement membrane. Migrating cells form "sprouts" in the parent vessels where endothelial cells mitosis and proliferate. Endothelial shoots fuse with each other to form capillary loops that create renal vessels.

  Persistent, unregulated angiogenesis occurs during abnormal growth by a number of disease states, tumor metastases and endothelial cells. The various pathological disease states in which unregulated angiogenics are present are grouped together as angiogenic-dependent or angiogenic-related diseases.

One example of a disease mediated by angiogenesis is ocular neovascular disease. The disease is characterized by invasion of the renal vessels into the eye structure, such as the retina or cornea. This is the most common cause of blindness and involves approximately 20 eye diseases. In age-related macular degeneration, a related visual problem is caused by the growth of choroidal capillaries through a defect in the Bruch's membrane following proliferation of ductal tissue under the retinal pigment epithelium. Angiogenic damage is associated with diabetic retinopathy, immature retinopathy, corneal graft rejection, neovascular glaucoma, and posterior capsular fibrosis. Other diseases that involve corneal neovascularization include pandemic keratitis, vitamin A deficiency, contact lens fatigue, atopic keratitis, gastric limbal keratitis, pterygium keratitis dryness, Sjogren's disease, acne injection, phylectenulosis, syphilis, Mycobacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Huff's simplex infections, Huff's zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, terry rims Terrien's marginal degeneration, limbic keratinization, rheumatoid arthritis, systemic lupus, polyarthritis, trauma, Wegner's sarcoidosis, scleritis, Stevens-Johnson's disease, neonatal impetigo, and radiokeratotomy It includes, but is not limited to.

Diseases involving retinal / choroidal neovascularization include diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, resilient fibrous yellow pigmented melanoma, Paget's disease, venous obstruction, arterial obstruction, carotid artery occlusion disease, chronic Uveitis / Volatilitis, Mycobacterial infection, Lyme's disease, Systemic lupus erythematosus, Immature retinopathy, Eales' disease, Behcet's disease, Infection that causes retinitis or choroiditis, Presumptive ocular heath Toplasma, Best's disease, Myopia, Ocular pit, Stargardt's disease, Pars planitis, Chronic retinal detachment, Hyperplasia syndrome, Schistosomiasis Diseases, trauma and post-laser complications, including but not limited to. Other eye-related disorders include, but are not limited to, diseases associated with cutaneous redness (neovascularization of angles) related diseases and abnormal proliferation of ductal or fibrous tissue, including all forms of fertility hyperretinal retinopathy.

Another angiogenesis related disease is rheumatoid arthritis. In the synovial lining of the joint, blood vessels undergo angiogenesis. In addition to forming a renal vascular network, endothelial cells release reactive oxygen species and factors that cause pannus growth and cartilage destruction. Angiogenesis may also be involved in osteoarthritis. Activation of chondrocytes by angiogenic-related factors causes destruction of the joints. In later stages, angiogenic factors promote new bone growth. Therapeutic interventions that interfere with bone destruction can stop the progression of the disease and provide relief for people with arthritis.

Chronic inflammation may also involve pathological angiogenesis. Diseases such as ulcerative colitis and Crohn's disease show histological changes such as the growth of new blood vessels and inflamed tissue. Bartonellaosis, a bacterial infection found in South America, can lead to a chronic stage characterized by the proliferation of vascular endothelial cells. Another pathological role associated with angiogenesis is found in atherosclerosis. Plaques formed in the lumen of blood vessels have been shown to have angiogenic stimulatory activity.

The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971 (Folkman, New Eng. J. Med., 285: 1182-86 (1971)). In the simplest case, this hypothesis states that "once a tumor" take "occurs, the hourly increase in the tumor cell population must proceed by an increase in the renal capillaries concentrated in the tumor. A tumor “take” is interpreted to represent a prevascular state of tumor growth in which a population of tumor cells occupying a few mm ³ volume but not exceeding millions of cells can survive in existing host microtubules. Expansion of tumor volume beyond this condition requires the introduction of renal capillaries. For example, pulmonary micrometastasis in the initial prevascular state in mice cannot be detected except by high magnification microscopy on histological fragments.

Indirect evidence in support of this concept includes the following example.

(1) The growth rate of tumors implanted in a subcutaneous clear chamber in mice is a slow straight line before neovascularization and a fast, nearly exponential function after neovascularization (Algire, et al., J. Nat. Cancer Inst. , 6: 73-85 (1945).

(2) Tumors grown in isolated and perfused organs where blood vessels do not proliferate are limited to 1-2 mm ³ , but when they are transplanted into mice and neovascularized, their volume increases> 100 times faster (Folkman, et al., Annals of Surgery, 164: 491-502 (1966)).

(3) In avascular corneas, tumor growth proceeds at a slow linear rate, but after neovascularization, it turns into exponential growth (Gimbrone, Jr., et al., J. Nat. Cancer Inst., 52: 421- 27 (1974).

(4) Tumors suspended in aqueous fluid in the anterior chamber of rabbit eyes are limited to live, avascular size <1 mm ³ . Once transplanted into the iris vascular layer, neovascularization forms and grows rapidly, reaching 16,000 times its original volume within two weeks (Gimbrone, Jr., et al., J. Exp. Med., 136: 261-76). .

(5) When the tumor is transplanted onto the chick embryonic villus, it grows slowly during the avascular phase of> 72 hours but does not exceed the average diameter of 0.93 + 0.29 mm. Rapid tumor spreading occurs within 24 hours of onset of neovascularization, and by day 7 these angiogenic tumors reach an average diameter of 8.0 + 2.5 mm (Knighton, British J. Cancer, 35: 347-56 (1977)).

(6) The vascular circumference of metastasis in rabbit liver shows heterogeneity in the size of metastasis, but shows a relatively constant cut point for the size of where angiogenesis occurs. Tumors are generally avascular up to 1 mm in diameter, but neovascularization is formed above this diameter (Lien, et al., Surgery, 68: 334-40 (1970)).

(7) In transgenic mice that cause carcinoma in the beta cells of the pancreatic islets, the pre-vascular hyperplastic islets are limited to <1 mm in size. At 6-7 weeks, neovascularization forms in 4-10% of the islets, resulting in large angiogenic tumors of at least 1000 times the pre-vascular islet volume (Folkman, et al., Nature, 339: 58-61). (1989)).

(8) Certain antibodies against VEGF (vascular endothelial growth factor) reduce the microtubule density and "significantly or significantly" the growth of three human tumors that depend on VEGF as the only mediator of angiogenesis (in nude mice). Suppress Antibodies do not inhibit the growth of tumor cells in vitro (Kim, et al., Nature, 362: 841-44 (1993)).

(9) Anti-bFGF monoclonal antibodies result in 70% inhibition of the growth of mouse tumors that depend on the secretion of bFGF as the only mediator of angiogenesis. Antibodies do not inhibit the growth of tumor cells in vitro (Hori, et al., Cancer Res., 51: 6180-84 (1991)).

(10) Intraperitoneal injection of bFGF enhances metastasis by stimulating the growth of primary tumors and the growth of intratumoral capillary endothelial cells. Tumor cells themselves lack a receptor for bFGF, and bFGF is not a mitotic material for tumor cells in vitro (Gross, et al., Proc. Am. Assoc. Cancer Res., 31:79 (1990) ).

(11) Certain angiogenesis inhibitors (AGM-1470) inhibit tumor growth and metastasis in vivo, but lack activity in inhibiting tumor cell proliferation in vitro. It inhibits vascular endothelial cell proliferation up to 1/2 at concentrations 4 log lower than inhibiting tumor cell proliferation (Ingber, et al., Nature, 48: 555-57 (1990)). It is also indirect clinical evidence that tumor growth is angiogenesis dependent.

(12) Human retinoblastoma metastasizing to vitreous (developed from the extracted eye and analyzed in vitro) develops into avascular spheroids limited to 1 mm ³ or less despite the fact that it is alive and incorporates ³ H-thymidine. do.

(13) Ovarian cancer metastasizes to the peritoneal membrane in the form of small avascular white seed (1-3 mm ³ ). These implants grow massively after one or more of them are neovascularized.

(14) Breast cancer (Weidner, et al., New Eng. J. Med., 324: 1-8 (1991); Weidner, et al., J Nat. Cancer Inst., 84: 1875-87 (1992)) ; And the intensity of neovascularization in prostate cancer (Weidner, et al., Am. J. Pathol., 143 (2): 401-09 (1993)) is highly correlated with the risk of future metastasis.

(15) Metastases from human skin melanoma are rare before neovascularization. Initiation of neovascularization leads to increased thickness of the wound and increased risk of metastasis (Srivastava, et al., Am. J. Pathol., 133: 419-23 (1988)).

(16) In bladder cancer, the urine level of the angiogenic protein bFGF is a more sensitive indicator of the location and extent of disease than cytology (Nguyen, et al., J. Nat. Cancer Inst., 85: 241-42 (1993) ).

Thus, it is clear that angiogenesis plays a major role in metastasis of cancer. If such angiogenic activity is inhibited or eliminated, no growth occurs even if the tumor is present. In disease states, prevention of angiogenesis can avoid damage caused by invasion of new microvascular systems. Therapies for control of angiogenic progression can lead to the abolition or attenuation of these diseases.

Angiogenesis correlates with many different types of cancer, including solid tumors and hematologic tumors. Angiogenesis-related solid tumors include, but are not limited to, rhabdomyosarcoma, retinoblastoma, Ewing's sarcoma, neuroblastoma, and osteosarcoma. Angiogenesis is associated with blood-based tumors such as leukemia, various acute or chronic tumor diseases of the bone marrow, which involve unlimited proliferation of leukocytes, usually accompanied by anemia, damaged blood clotting, and expansion of lymph nodes, liver and spleen. do. Angiogenesis is thought to be involved in abnormal diseases in the bone marrow that causes tumors such as leukemia.

One of the most frequent angiogenic diseases in childhood is hemangioma. Hemangioma is a tumor consisting of newly formed blood vessels. In most cases, tumors are benign and regress without intervention. In more serious cases, the tumor progresses in the form of large animal infiltrations, leading to clinical complications. Hemangioma, a systemic form of hemangioma, has a high mortality rate. There are hemangiomas that are resistant to treatment that cannot be treated with currently used therapies.

Angiogenesis is also responsible for inherited diseases such as Osler-Weber-Rendu disease, or damage found in hereditary bleeding capillary dilator. It is a hereditary disease characterized by multiple small hemangiomas, blood or lymph vascular tumors. Hemangiomas are found in the skin and mucous membranes and often involve nasal hemorrhage (nose) or gastrointestinal bleeding and sometimes pulmonary or hepatic arteriovenous depression.

Therefore, there is a need for compositions and methods that can inhibit angiogenesis. There is also a need for compositions and methods that can inhibit unwanted growth of blood vessels, particularly in tumors.

Angiogenesis is also involved in normal physiological processes such as regeneration and wound aches. Angiogenesis is an important step in the transplantation of blastocysts after ovulation and fertilization. Prevention of angiogenesis can be used to induce amenorrhea, block ovulation, or prevent transplantation by blastocysts.

In wound soreness, excessive repair or fibrous tissue formation can be a deleterious side effect of the surgical procedure and can be caused or worsened by angiogenesis. Adhesions are a frequent complication of surgery and cause problems such as small intestine obstruction.

Many compounds have been used to inhibit angiogenesis. Taylor et al. (Nature, 297: 307 (1982)) used prostamine to inhibit angiogenesis. The toxicity of prostamines limits their practical use as therapeutic agents. Folkman et al. (Science, 221: 719 (1983), and US Pat. Nos. 5,001,116 and 4,994,443) describe the use of heparin and steroids to control angiogenesis. Steroids such as glucocorticoids and tetrahydrocortisol lacking mineralocorticoid activity have been found to be an angiogenic inhibitor.

4 kDa glycoproteins from other factors found endogenously in animals, such as bovine vitreous humor and cartilage inducing factors, have been used to inhibit angiogenesis. Cellular factors such as interferon inhibit angiogenesis. For example, interferon alpha or human interferon beta has been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells. Interferon beta is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells (Sidky, et al., Cancer Res., 47: 5155-61 (1987)). Human recombinant interferon (alpha / A) has been reported to have been used successfully in the treatment of angiogenesis-induced disease, pulmonary angiomatosis (White, et al., New Eng. J. Med., 320: 1197- 1200 (1989).

Other agents used to inhibit angiogenesis include ascorbic acid ethers and related compounds (Japanese Kokai Tokkyo Koho No. 58-13 (1978)). Sulfated polysaccharide DS 4152 also inhibits angiogenesis (Japanese Kokai Tokkyo Koho No. 63-119500). Additional anti-angiogenic compounds include Angiostatin ^R (US Pat. Nos. 5,639,725; 5,792,845; 5,885,795; 5,733,876; 5,776,704; 5,837,682; 5,861,372 and 5,854,221) and Endostatin ^™ (US Pat. No. 5,854,205).

Another compound that has been shown to inhibit angiogenesis is thalidomide (D'Amato, et al., Proc. Natl. Acad. Sci., 90: 4082-85 (1994)). Thalidomide is used in a number of angiogenesis-related diseases such as rheumatoid arthritis (Gutierrez-Rodriguez, Arthritis Rheum., 27 (10): 1118-21 (1984); Gutierrez-Rodriguez, et al., J. Rheumatol , 16 (2): 158-63 (1989)), Bethley's disease (Handley, et al., Br. J. Dermatol., 127 Suppl, 40: 67-8 (1992); Gunzler, Med. Hypotheses, 30 (2): 105-9 (1989)), host rejection for transplantation (Field, et al., Nature, 211 (55): 1308-10 (1996); Heney, et al., Br. J. Haematol. , 78 (1): 23-7 (1991)), mycobacterial diseases (Vicente, et al., Arch. Intern. Med., 153 (4): 534 (1993)), Huff's Simplex and Huff's Joe Ster infection (Naafs, et al., Int. J. Dermatol., 24 (2): 131-4 (1985)), chronic inflammation, ulcerative colitis (Meza, et al., Drug Ther, 23 (11): 74-80, 83 (1993); Powell, et al., Br. J. Dermatol., 113 Suppl 28: 141-4 (1985)), leprosy (Barnes, et al., Infect. Immun., 60 (4) : 1441-46 (1992)) and sleep depressants that have been successfully used to treat Burrows, BMJ, 307: 939-40 (1993). All.

Although thalidomide has minimal side effects in adults, it is a potent teratogenic substance. Thus, pregnant women are concerned when used. Although minimal, there are a number of side effects that limit the desirability of thalidomide as a therapeutic. One of these side effects is drowsiness. In many therapeutic studies, the initial dose of thalidomide must be reduced because the patient is lethargic and unable to function normally. Another side effect of limiting the use of thalidomide is peripheral neuropathy, in which the subject suffers from paralysis and dysfunction of the extremities.

Accordingly, there is a need for improved methods and compositions that can be easily administered and inhibit angiogenesis.

Summary of the Invention

The present invention provides a novel derivative for 2-methyl glutamic acid, which is an analog of 2-phthaliminoglutaric acid. Specifically, the present invention provides a novel compound, 2-methyl-2-phthaliminoglutaric acid and a method for producing the same. The present invention also provides a method for separating individual (R) and (S) enantiomers and (R) and (S) enantiomers of DL-2-methyl-2-phthaliminoglutaric acid.

2-Methyl-2-phthaliminoglutaric acid (2-Me-EM-138) is a derivative of 2-phthaliminoglutaric acid (EM-138) presented by the present invention to inhibit angiogenesis. to be. The present invention also inhibits angiogenesis and protects DL-2-methyl-2-phthaliminoglutaric acid, each individual enantiomer, R-(+)-2-methyl-2-phthaliminoglutar Provided are methods for treating angiogenesis-related diseases using acids and S-(-)-2-methyl-2-phthaliminoglutaric acid.

In addition, the present invention provides hydroxylated derivatives of 2-phthaliminoglutaric acid and methods of making such derivatives. The present invention also provides pharmaceutical compositions containing these derivatives and their use in inhibiting angiogenesis and treating angiogenesis related diseases.

Accordingly, an object of the present invention is to provide a compound DL-2-methyl-2-phthaliminoglutaric acid.

Another object of the present invention is to provide an easy and economical method for producing the compound DL-2-methyl-2-phthaliminoglutaric acid.

It is another object of the present invention to provide a method for separating individual enantiomers of DL-2-methyl-2-phthaliminoglutaric acid.

A further object of the present invention is to provide compounds (R)-(+)-2-methyl-2-phthaliminoglutaric acid and (S)-(-)-2-methyl-2-phthalimininoglutaric acid. There is.

It is another object of the present invention to provide hydroxylated analogs of 2-phthaliminoglutaric acid.

It is another object of the present invention to provide a method for preparing a hydroxylated derivative of 2-phthaliminoglutaric acid.

An object of the present invention is to provide DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid, or (S)-(-)-. There is provided a pharmaceutical composition containing 2-methyl-2-phthaliminoglutaric acid as an active ingredient.

It is an object of the present invention to provide a pharmaceutical composition containing, as active ingredient, a hydroxylated analog of 2-phthaliminoglutaric acid.

It is an object of the present invention to provide a safe and effective method of inhibiting angiogenesis in humans or animals.

It is a further object of the present invention to provide a safe and effective method of inhibiting angiogenesis in humans or animals by administering 2-phthaliminoglutaric acid analogues.

A further object of the present invention is to provide DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid, or (S)-(-). The present invention provides a safe and effective method of inhibiting angiogenesis in humans or animals by administering -2-methyl-2-phthaliminoglutaric acid.

It is a further object of the present invention to provide a safe and effective method of inhibiting angiogenesis in humans or animals by administering hydroxylated analogs of 2-phthaliminoglutaric acid.

It is another object of the present invention to provide a method for the treatment of cancer, more specifically for the treatment of solid and hematological tumors.

Another object of the present invention is to provide a method for treating cancer, more specifically for treating solid and hematologic tumors, by administering analogs of 2-phthaliminoglutaric acid.

Another object of the present invention is to provide DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid, or (S)-(- By administering) -2-methyl-2-phthaliminoglutaric acid, there is provided a method for treating cancer, more specifically for treating solid and hematological tumors.

Another object of the present invention is to provide a method for the treatment of cancer, more specifically for the treatment of solid and hematological tumors, by administering the hydroxylated analogs of 2-phthaliminoglutaric acid.

Another object of the present invention is ulcerative colitis, Crohn's disease, ulcers, Bedett's syndrome, Stevens-Johnson disease, mycobacterial infection, Huff's simplex infection, Huff's zoster infection, rheumatoid arthritis, osteoarthritis, lupus, Lyme disease To provide a method for treating chronic inflammation, atherosclerosis and hereditary diseases.

Another object of the present invention is to administer 2-phthaliminoglutaric acid analogs to ulcerative colitis, Crohn's disease, ulcers, Beset's syndrome, Stevens-Johnson's disease, Mycobacterial infection, Huff's simplex infection, Huff's zoster A method of treating infection, rheumatoid arthritis, osteoarthritis, lupus, Lyme disease, chronic inflammation, atherosclerosis and hereditary disease.

Another object of the present invention is to provide DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid, or (S)-(- ) -2-methyl-2-phthaliminoglutaric acid was administered to treat ulcerative colitis, Crohn's disease, ulcer, Bedett's syndrome, Stevens-Johnson's disease, mycobacterial infection, Huff's simplex infection, Huff's Zoster infection, A method of treating rheumatoid arthritis, osteoarthritis, lupus, Lyme disease, chronic inflammation, atherosclerosis and hereditary diseases is provided.

Another object of the present invention is to administer hydroxylated analogs of 2-phthaliminoglutaric acid to ulcerative colitis, Crohn's disease, ulcers, Beset's syndrome, Stevens-Johnson disease, mycobacterial infection, Huff's simplex infection. And a method for treating Huff's zoster infection, rheumatoid arthritis, osteoarthritis, lupus, Lyme disease, chronic inflammation, atherosclerosis and hereditary disease.

In addition, the present invention provides a method of controlling wound injuries.

The present invention also provides a method of controlling wound aches by administering a 2-phthaliminoglutaric acid analog.

Further, the present invention provides DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid, or (S)-(-). Provided is a method of controlling wound ache by administering 2-methyl-2-phthaliminoglutaric acid.

In addition, the present invention provides a method of controlling wound ache by administering a hydroxylated analog of 2-phthaliminoglutaric acid.

Another object of the present invention is to induce abortion.

Another object of the present invention is to induce abortion by administering a 2-phthaliminoglutaric acid analog.

Another object of the present invention is to provide DL-2-methyl-2-phthaliminoglutaric acid, (R)-(+)-2-methyl-2-phthaliminoglutaric acid, or (S)-(- ) -2-methyl-2-phthaliminoglutaric acid is administered to induce lactic acid.

Another object of the present invention is to induce abortion by administering a hydroxylated analog of 2-phthaliminoglutaric acid.

1 shows the synthesis of DL-2-methyl-2-phthaliminoglutaric acid and the separation of (R) and (S) enantiomers by chiral HPLC.

2A and 2B show the synthesis of DL-2-methyl-2-phthaliminoglutaric acid dimethyl ester, separation of (R) and (S) enantiomers using ChiroCLEC ^™ -BL, and (R)-(+) Hydrolysis to form 2-methyl-2-phthaliminoglutaric acid and (S)-(-)-2-methyl-2-phthaliminoglutaric acid is shown.

3 shows the synthesis of hydroxylated derivatives of 2-phthaliminoglutaric acid.

4 shows the effect of EM-138 on the inhibition of metastasis in B16-BL6 melanoma cells compared to thalidomide administered intraperitoneally.

5 shows the effect of EM-138 on inhibition of metastasis in B16-BL6 melanoma cells as compared to orally administered thalidomide.

FIG. 6 shows the effect of the number of treatments on the activity of 2-phthaliminoglutaric acid in the B16-BL6 model.

FIG. 7 shows the effect of initial treatment timing on EM-138 activity in B16 / BL6 melanoma cells.

8 shows lung gross pathology of mice treated with 2-phthaliminoglutaric acid (EM-138).

9 shows lung histopathology of mice treated with 2-phthaliminoglutaric acid (EM-138).

10 shows the crystal structure of 2-methyl-2-phthaliminoglutaric acid.

FIG. 11 shows DL-2-methyl-2-phthalimine by HPLC using CH ₃ CN / MeOH / H ₂ O / HOAc (1: 1: 5: 0.1) as eluent at a flow rate of 2 ml / min at 230 nm. The chiral cleavage of the (R) and (S) enantiomers of dinoglutaric acid is shown.

12 depicts the effect of optically-pure enantiomers of 2-methyl-2-phthaliminoglutaric acid on B16-BL6 melanoma metastasis.

13 shows the relative anti-tumor activity of thalidomide, EM-138, and EM-138 analogs. T / C is the ratio of tumor to control. According to this, the lower the T / C ratio, the greater the tumor suppressive activity of the compound.

The present invention provides that compound 2-phthaliminoglutaric acid (EM-138) has angiogenesis inhibitory activity and is useful for treating a variety of diseases including various cancers and macular degeneration. This compound has the following structural formula:

2-phthaliminoglutaric acid (EM-138) is a stable, oral-active analog of thalidomide. Unlike thalidomide, it is relatively resistant to hydrolysis. This is a potent suppressor of metastasis. Although a single dose can inhibit 50% of metastasis and a dose of 0.8 mmol / kg / day has been shown to inhibit metastasis above 90%.

2-Methyl-2-phthaliminoglutaric acid

In the pharmaceutical industry, one enantiomer of a compound is often understood to contain significantly higher levels of advantageous activity compared to other enantiomers. In some cases, it has been found that one of the enantiomers provides a beneficial effect while the other causes toxicity or side effects of the drug.

Because enantiomers readily form racemates, EM-138 cannot be separated into individual enantiomers. Thus, the inventors have synthesized an EM-138 related chiral compound that has similar activity to that of EM-138 and can be separated into individual enantiomers. This compound is 2-methyl-2-phthaliminoglutaric acid (2-Me-EM-138) and has the following structural formula:

This compound can be synthesized in several ways. Preferred synthetic methods are disclosed in 2-methylglutamic acid. 2-methylglutamic acid, phthalic anhydride, and amines such as triethyl amine, diethyl amine, or pyridine are mixed in anhydrous solvents such as anhydrous toluene. The mixture is heated to reflux and the solvent is evaporated. The intermediate 2-methyl-N-phthaloylglutamic acid is crystallized and recovered.

The intermediate is dissolved in an acid such as glacial acetic acid and then zinc dust is added. The mixture is heated under reflux in an inert atmosphere such as nitrogen or argon. 2-methyl-2-phthaliminoglutamic acid is recovered and purified, for example, by recrystallization or eluting on a silica gel column.

DL-2-methyl-2-phthaliminoglutaric acid activity studies show that this, like EM-138, is a potent inhibitor of angiogenesis. These studies show that the compounds are useful for treating angiogenesis-related diseases. One angiogenesis-related disease group is cancer. Many tumors, including solid tumors and hematologic tumors, require angiogenesis to grow beyond very small sizes. Inhibition of angiogenesis will result in tumor growth inhibition. Examples of certain types of cancer that can be treated with 2-methyl-2-phthaliminoglutaric acid and other derivatives included in the present invention include prostate cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, glioma, hemangioma, kaposi Sarcoma, pancreatic cancer, retinoblastoma, melanoma, bladder cancer, rhabdomyosarcoma, retinoblastoma, Ebbing sarcoma, neuroblastoma, osteosarcoma, leukemia and various acute and chronic neoplastic diseases of the bone marrow. Compound 2-methyl-2-phthaliminoglutaric acid also inhibits metastasis of existing tumors. Examples of metastases that can be inhibited include, but are not limited to, lung metastasis, liver metastasis, and intraperitoneal metastasis.

Another group of angiogenesis-related diseases develops in the eye and around the eye. Examples of ocular diseases that can be treated with 2-methyl-2-phthaliminoglutaric acid and other derivatives included in the present invention include ocular renal vascular disease, macular degeneration with age-related macular degeneration, diabetic retinopathy, immature Retinopathy, corneal graft rejection, neovascular glaucoma, posterior capsular fibrosis, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens fatigue, atopic keratitis, gastric limbal keratitis, pterygium keratitis dryness, terry limbic degeneration, marginal keratitis, radiation cornea Incision, presumptive ocular histoplasmosis, chronic uveitis / supersomalitis, myopia, ocular bovine, planetarity, chronic retinal detachment, hypertensive syndrome, scleritis, trauma and post-laser complications, dermatitis, retinitis or choroiditis Include, but are not limited to, diseases caused by abnormal proliferation of ductal or fibrous tissue, including infectious diseases that cause, and all forms of fertility hyperretinal retinopathy It is not limited.

A further group of angiogenesis-related diseases are those characterized by tissue ulceration or destruction. Many of these diseases also affect the eye. Diseases that can be treated with 2-methyl-2-phthaliminoglutaric acid and other derivatives included in the present invention include Sjogren's disease, ulcerative colitis, Crohn's disease, Bartonella's disease, acne injection, syphilis, sarcoidosis, chemical burns , Bacterial ulcers, fungal ulcers, Bedett's syndrome, Stevens-Johnson's disease, mycobacterial infection, Huff's simplex infection, Huff's Zoster infection, protozoan infection, Murren's ulcer, leprosy, Wegner sarcoidosis, and neonatal impetigo However, it is not limited thereto. Other angiogenesis-related diseases or disorders that can be treated with 2-methyl-2-phthaliminoglutaric acid and other derivatives included herein include rheumatoid arthritis, osteoarthritis, lupus, systemic lupus erythematosus, multiple Arthritis, arterial atresia, venous obstruction, carotid occlusion disease, sickle cell disease, sickle cell disease, Farget's disease, Lyme disease, Best's disease, ale disease, Stargardt's disease, schistosomiasis, lectrenulopathy, lipid Genetic diseases such as degeneration, chronic inflammation, atherosclerosis, Osler-Weber-Lendyr disease, but are not limited thereto. The compounds can be used to control wound sores by inhibiting adhesions and scar formation, block ovulation or prevent blastocysts from inducing amenorrhea and inducing abortion.

(R) and (S) enantiomers of DL-2-methyl-2-phthaliminoglutaric acid

In order to know if one of the enantiomers of DL-2-methyl-2-phthaliminoglutaric acid has greater activity or reduced side effects, the enantiomer must first be separated. Separation can be performed by a number of different methods. In one preferred embodiment of the invention, the enantiomer of DL-2-methyl-2-phthaliminoglutaric acid is partitioned by chiral high pressure liquid chromatography (HPLC) column. The sample is dissolved in a suitable solvent and placed on a column. The sample is eluted with a solvent mixture such as a mixture of CH ₃ CN / MeOH / H ₂ O / HOAc (1: 1: 5: 0.1).

In another preferred embodiment, enantiomers are separated by first forming DL-2-methyl-2-phthaliminoglutaric acid ester. Esters can be produced by known methods. The particular ester formed is not critical. Examples of esters that may be formed include, but are not limited to, methyl esters, ethyl esters, propyl esters, and butyl esters. Dimethyl esters are preferred.

Esters are separated using enantiomer specific hydrolysers such as ChiroCLEC ^™ -BL. ChiroCLEC ^™ -BL hydrolyzes one or two in one ester of enantiomers without affecting other enantiomers. This enantiomer-specific ester hydrolysis allows for subsequent separation of enantiomers using silica gel chromatography. Once separated, the ester is fully hydrolyzed to form the corresponding (R) and (S) acids. Hydrolysis can be performed by any method. One preferred method of hydrolyzing glutaric acid esters is to treat a 1: 1 mixture of glacial acetic acid and concentrated hydrochloric acid.

Polarization analysis shows that the (R) enantiomer of 2-methyl-2-phthaliminoglutaric acid optically rotates in the (+) direction, while the (S) enantiomer rotates optically in the (-) direction. Accordingly, the individual enantiomers of DL-2-methyl-2-phthaliminoglutaric acid are (R)-(+)-2-methyl-2-phthaliminoglutaric acid and (S)-(-)-2. -Methyl-2-phthaliminoglutaric acid.

Hydroxylated derivatives of 2-phthaliminoglutaric acid

We synthesized an EM-138 analogue containing a hydroxyl group in the benzene ring of the phthalimino group. These compounds have the following structural formula:

Wherein R ₃ and R ₄ are each independently H or OH.

These compounds can be synthesized in a number of ways. Preferred synthesis methods start with glutamic acid. Glutamic acid is reacted with a phthalic anhydride or a derivative of phthalic anhydride in a solvent such as pyridine under reflux, and then diacetate is added to the reaction mixture. The resulting intermediate is hydrolyzed in an acidic medium that opens cyclic anhydrides to form glutaric acid derivatives. The glutaric acid intermediate is dissolved in an acid such as glacial acetic acid followed by the addition of zinc dust.

Pharmaceutical Compositions and Methods of Administration

The compounds of the present invention can be administered orally, parenterally, rectal, vaginal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous and the like. The dosage of the active compound will of course depend on the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art. In general, the dosage will range from approximately 100 mg / kg / day to approximately 2000 mg / kg / day.

The compounds of the present invention may be conveniently formulated into pharmaceutical compositions with pharmaceutically acceptable carriers. See Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin (Merck Publ. Co., Easton, Pa.) Describes typical carriers and conventional methods of preparing pharmaceutical compositions that can be used to prepare the compositions of this invention. It may also be administered in conjunction with other active compounds such as organics.

Depending on the intended dosage form, the pharmaceutical composition may be in solid, semisolid, or liquid dosage form. Examples of dosage forms include, but are not limited to, tablets, pills, capsules, suppositories, sachets, granules, powders, creams, lotions, ointments, plasters, liquid solutions, suspensions, and dispersions, emulsions, syrups, and the like. It is not limited. The active ingredient may be encapsulated in liposomes, microparticles, microcapsules or the like.

Conventional non-toxic carriers are agents of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, dextrose, glycerol, magnesium carbonate, triglycerides, oils, solvents, sterile water, and isotonic saline Including but not limited to grades. Solid compositions such as tablets, pills, granules and the like may be coated for convenience. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer and include local anesthetics to relieve pain at the injection site. If desired, the medicament may also contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifiers, pH buffers and the like. Examples of such auxiliary materials include, but are not limited to, sodium acetate, sorbitan monolaurate, triethanolamine, and triethanolamine oleate. The composition of the present invention may comprise excipients such as stabilizers, antioxidants, binders, colorants, flavors, preservatives, and thickening agents.

The invention is further illustrated by the following examples, but is not intended to limit the scope thereof. On the contrary, after reading the specification, it is understood that the skilled person may rely on various aspects, modifications, and equivalents thereof without departing from the spirit of the present invention and / or the scope of the appended claims.

Example 1

Inhibition of metastasis through intraperitoneal administration of EM-138

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. After 3 days, mice are treated intraperitoneally with increasing thalidomide or 2-phthaliminoglutaric acid (EM-138) every other day. 14 days after tumor cell inoculation, the lungs are removed from mice and the surface lung metastases counted. The results of this experiment are shown in FIG. Values shown are averages of 5 mice / group. Bars on the graph represent standard deviations.

Example 2

Inhibition of metastasis through oral administration of EM-138

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. After 3 days, mice are treated orally every other day with increasing amounts of thalidomide or 2-phthaliminoglutaric acid (EM-138). 14 days after tumor cell inoculation, the lungs are removed from mice and the surface lung metastases counted. The results of this experiment are shown in FIG. Values shown are averages of 5 mice / group. Bars on the graph represent standard deviations.

Example 3

Effect of Number of Treatments on EM-138 Activity

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. After 3 days, mice are gavaged with 0.8 mmol / kg of 2-phthaliminoglutaric acid (EM-138). Mice are treated once every other day, five times, or daily for 11 days. 14 days after tumor cell inoculation, lungs are removed from mice and the surface lung metastases counted. The results of this experiment are shown in FIG. Values shown are averages of 5 mice / group. Bars on the graph represent standard deviations.

Example 4

Effect of Early Treatment Timing of EM-138 Activity

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. Mice are treated orally once with 0.8 mmol / kg of 2-phthaliminoglutaric acid (EM-138). Treatment is initiated one day prior to tumor cell introduction or on days 1, 2, 3, 5 or 7 of tumor cell introduction. 14 days after tumor cell inoculation, lungs are removed from mice and the surface lung metastases counted. The results of this experiment are shown in FIG. Values shown are averages of 5 mice / group. Bars on the graph represent standard deviations.

Example 5

Lung Gross Pathology in EM-138 Treated Mice

Mice with pulmonary B16-BL6 metastasis are administered orally with 0.5% carboxymethylcellulose or 0.8 mmol / kg 2-phthaliminoglutaric acid (EM-138). The results of this experiment are shown in FIG. 8 (0.5% carboxymethylcellulose (left panel) and 0.8 mmol / kg EM-138 (right panel)).

Example 6

Lung Tissue Pathology in EM-138 Treated Mice

Mice with melanoma are orally treated with 0.5% carboxymethylcellulose or 0.8 mmol / kg of 2-phthaliminoglutaric acid (EM-138). The lungs of the mice are removed, fixed in formalin and embedded in paraffin. Random sections of representative lungs of each group are stained with hematoxylin and eosin. Shoot the area at 100 magnification. The results are shown in FIG. 9 (0.5% carboxymethylcellulose (left panel) and 0.8 mmol / kg EM-138 (right panel)).

Example 7

General Synthesis Procedure for 2-phthalimino Glutaric Acid Analogues

Equimolar amounts of (dl) -glutamic acid and 3 'and 4' substituted phthalic anhydrides are heated under reflux for 3-4 hours in anhydrous pyridine. After the coupling reaction is complete, the pyridine is evaporated under reduced pressure and the viscous product is mixed with acetic anhydride (3 equiv) and heated to boil for 10 minutes. Evaporation of approximately half of the acetic anhydride volume and cooling the reaction mixture at room temperature yields a white crystalline product which is separated by filtration to replace substituted N-phthaloyl-dl-glutamic anhydride (3a and 3b in FIG. 3) (60- 75% yield) is obtained.

Anhydrides (3a, 3b and 3c in FIG. 3) are hydrolyzed with boiling water, recrystallized and dried under vacuum to yield 98% yield of substituted phthaloyl (dl) glutamic acid (4a, 4b and 4c in FIG. 3). Lose. These products are treated with 5 equivalents of Zn dust in acetic acid to partially reduce to yield 60-80% yield of 2-phthalimino glutaric acid analogs (5a, 5b and 5c in FIG. 3).

Example 8

Preparation of DL-2-methyl-N-phthaloylglutamic acid

A mixture of vacuum dried DL-2-methylglutamic acid (6.8 grams, 40 mmol), phthalic anhydride (5.92 grams, 40 mmol) and 150 ml triethylamine in toluene anhydrous is heated using a Dean-Stark apparatus under reflux. . After 4 hours of heating, 0.7 ml of water are collected. The reaction mixture is heated for an additional 2 hours and then the solvent is evaporated. Dissolving the reaction mixture in 40 ml 1N HCl solution starts to form white crystals. After 18 hours, the crystals were separated from the mother liquor by suction filtration and dried under vacuum to yield 8.1 grams (60%) of white solid product. 1 H-NMR showed the product as DL-2-methyl-N-phthaloylglutamic acid.                 

Example 9

Preparation of DL-2-methyl-2-phthaliminoglutaric acid

1.6 grams (4 mmol) of DL-2-methyl-N-phthaloylglutamic acid prepared in Example 8 is dissolved in 10 ml glacial acetic acid. 1.3 grams (20 mmol) of Zn dust is added to the reaction mixture. The reaction mixture is heated under reflux and a nitrogen atmosphere. After 4 hours of heating, silica gel TLC analysis of the reaction mixture (CHCl ₃ / MeOH 4: 1) shows new product formation with higher Rf values. The reaction mixture is heated for an additional hour, then the hot liquid is filtered off with suction. After evaporating acetic acid under reduced pressure, the viscous product is dissolved in 5 ml water and then the aqueous layer is washed with ethyl ether to remove impurities. Upon standing, white crystals form in the aqueous layer separated overnight by suction filtration. The crystalline material was further purified through a silica gel column and eluted with a CHCl ₃ / MeOH (4: 1) mixture to give a viscous product. The product was recrystallized from water and dried in vacuo to yield 600 mg (60) of a white solid. DL-2-methyl-2-phthalimidoglutaric acid (JHS-171) was confirmed by 1H-NMR. 1H-NMR (DMSO-D6, PPM), 7.7 (3H, m), 7.5 (1H, d), 4.62 (2H, s), 3.4 (2H, wide range), 2.46 (1H, d, t, J = 11.5 , 6.5), 2.3 (1H, t, J = 9.5), 2.1 (2H, m), 1.46 (3H, s). (FIG. 1).

Example 10

Lung Metastasis Treatment with 2-methyl-2-phthaliminoglutaric acid

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. Mice were 1.0 ml 2.8% DMSO alone (control), 1.0 ml in DMSO (5.0 mg / ml 2.8% DMSO) 2-phthaliminoglutaric acid (EM-138), or 1.0 ml (5.0 mg / ml 2.8 in DMSO) %) 2-Methyl-2-phthaliminoglutaric acid (2-Me-EM-138) is treated intraperitoneally (ip) four times a day 72 hours after tumor cell injection. At 14 days after tumor cell inoculation, the lungs are removed from the mice and the surface lung metastases are counted. The results of this experiment are shown in the table below.

Processing group Average number of lung metastases (ranges) % Suppression DMSO (n = 5) 210 (164-261) --- EM-138 (n = 5) 85 (51-118) 60% 2-Me-EM-138 84 (1-124) 60% n = number of mice per sample group p <0.01, compared to DMSO-treated control group

Example 11

Separation of 2-methyl-EM-138 Enantiomer by HPLC

Two enantiomers of DL-2-methyl-2-phthaliminoglutaric acid are partitioned by chiral HPLC column. A mixture of CH ₃ CN / MeOH / H ₂ O / HOAc (1: 1: 5: 0.1) with compound DL-2-methyl-2-phthaliminoglutaric acid in methanol placed in Welk-01 (10 mm × 750 mm) Elution with At a dissolution rate of 2 ml / min the retention time of the S (-) isomer is 25.96 minutes and the R (+) isomer is 26 minutes. Absorbance is monitored at 230 nm (FIG. 1).

Example 12

Preparation of DL-2-methyl-2-phthaliminoglutaric acid dimethyl ester

150 mg of DL-2-methyl-2-phthaliminoglutaric acid are dissolved in 10 ml of anhydrous methanol saturated with HCl gas and stirred at 40-50 ° C. for 30 minutes. TLC analysis of the reaction mixture (CHCl ₃ / MeOH 95: 5) shows new product formation with higher Rf values. After evaporation of the solvent, the product is purified on a silica gel column and eluted with a CHCl ₃ / MeOH (4: 1) mixture to give a viscous product (130 mg, 90%). 1 H-NMR showed the product to be DL-2-methyl-2-phthaliminoglutaric acid dimethyl ester (JHS-2-7) (FIG. 2).

Example 13

ChiroCLEC ^TM Separation of (R) and (S) Isomers of 2-Me-EM-138 by -BL Catalyst

To a 130 mg solution of DL-2-methyl-2-phthaliminoglutaric acid dimethyl ester in 2 ml acetone, add 2.5 M, 7.8 ml pH 8.5 phosphate buffer. ChiroCLEC ^™ -BL (0.4 ml, 8 mg) suspension is added. The reaction mixture is heated at 40-50 ° C. for 18 hours. TLC analysis of the reaction mixture (CHCl ₃ / MeOH 95: 5) shows new product formation with higher Rf values than the starting material. After stirring for an additional 2 hours, 4 ml acetone are added and the catalyst is filtered off by suction. After evaporation of the solvent, the two products were separated by silica gel column and eluted with a CHCl ₃ / MeOH (98: 2) mixture to give an initial eluting viscous product, JHS-2-13-P1 (70 mg, 90%), And a late elution product, JHS-2-13-P2 (40 mg, 70%). Polarization analysis of the product JHS-2-13-P1 shows positive rotation and polarization analysis of the product JHS-2-13-P2 shows negative rotation. 1 H-NMR confirmed the product JHS-2-13-P1 as R-(+)-2-methyl-2-phthaliminoglutaric acid dimethyl ester, and JHS-2-13-P2 was identified as S-(- ) -2-methyl-2-phthaliminoglutaric acid monomethyl ester.

Hydrolysis of the products JHS-2-13-P1 and JHS-2-13-P2 by treatment with a 1: 1 mixture of glacial acetic acid and concentrated HCl for 1 hour at 90-100 ° C. resulted in R-(+)-2-methyl 2-phthalimiminoglutaric acid (JHS-2-23-R) and S-(-)-2-methyl-2-phthaliminoglutaric acid (JHS-2-23-S) are obtained ( 2).

Example 14

Pulmonary Metastasis Treatment with R-(+)-2-methyl-2-phthaliminoglutaric acid and S-(-)-2-methyl-2-phthaliminoglutaric acid

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. 1.0 ml 2.8% DMSO alone (control), 1.0 ml (5.0 mg / ml) DL-2-methyl-2-phthaliminoglutaric acid (racemic), 1.0 ml in 2.8% DMSO (5.0 mg / ml) (R)-(+)-2-methyl-2-phthaliminoglutaric acid (JS-2-13-P1), or (S)-(-)-2 in 2.8% DMSO Methyl-2-phthaliminoglutaric acid (JS-2-13-P2) is treated intraperitoneally (ip) four times a day 72 hours after tumor cell injection. 14 days after tumor cell inoculation, the lungs are removed from the mice and the surface lung metastases counted. The experimental results show that the (S) isomer has greater angiogenesis inhibitory activity than the (R) isomer.

Example 15

Lung Metastasis Treatment with 2-phthaliminoglutaric Acid Analogues

B16-BL6 melanoma cells (5 × 10 ⁴ ) are injected intravenously into the tail vein of C57B1 / 6 mice. Mice are treated with one of the following solutions intraperitoneally (ip) four times a day 72 hours after tumor cell injection.

1.0 ml of 2.8% DMSO alone (control)

1.0 ml thalidomide in 2.8% DMSO

1.0 ml of 3'-hydroxy-2-phthaliminoglutaric acid in 2.8% DMSO (Compound 5a)

1.0 ml of 4'-hydroxy-2-phthaliminoglutaric acid in 2.8% DMSO (Compound 5b)

1.0 ml of 2-phthaliminoglutaric acid (EM-138) in 2.8% DMSO (Compound 5c)

1.0 ml of DL-2-methyl-2-phthaliminoglutaric acid (racemate) in 2.8% DMSO (Compound 7)

1.0 ml of (R)-(+)-2-methyl-2-phthaliminoglutaric acid (JS-2-13-P1) in 2.8% DMSO (compound 7-R-(+))

1.0 ml (S)-(-)-2-methyl-2-phthaliminoglutaric acid (JS-2-13-P2) in 2.8% DMSO (compound 7-S-(-))                 

0.4 mmol / kg dose is used for each compound tested. At 14 days after tumor cell inoculation, the lungs are removed from the mice and the surface lung metastases are counted. The results of this experiment are given in the table below.

compound X R ₃ R ₄ T / C ^a Thalidomide - - - 0.9 5a H OH H 0.95 5b H H OH 0.9 5c (EM-138) H H H 0.2 7 CH ₃ H H 0.25 7-R-(+) CH ₃ H H 0.7 7-S-(-) CH ₃ H H 0.15 ^a T / C is defined as the pulmonary metastasis ratio of treated (T) to untreated or control (C) animals

Repeat this experiment with varying doses. The data in the table below represents the average value of three runs of the experiment.

compound X R ₃ R ₄ IC ₅₀ (mmol / kg) Thalidomide - - - 3.2 ^a 5c (EM-138) H H H 0.2 ^a 7 CH ₃ H H 0.2 ^a 7-R-(+) CH ₃ H H Inactive ^b 7-S-(-) CH ₃ H H 0.1 ^a IC ₅₀ = 50% inhibition of surface transition a These data represent the average of three experiments b These enantiomers were tested only once at each dose without showing measurable activity above 0.8 mmol / kg.

These data show that EM-138 and 2-methyl-EM-138 have similar anti-tumor activity and both have significantly higher inhibitory activity than thalidomide. These data also show that the activity of 2-methyl-EM-138 is due to the (S) isomer and the (R) isomer has little or no activity. In addition, EM-138 analogs containing hydroxyl groups on the benzene ring of the phthalimino group in the absence of 2-methyl groups seem to show little or no anti-tumor activity at the tested doses.

Of course, the foregoing is directed to preferred embodiments of the invention and many modifications or variations may be made without departing from the spirit and scope of the appended claims.

Claims (23)

delete delete A stereoisomer compound having S-(-)-2-methyl-2-phthaliminoglutaric acid having the formula:

a) reacting 2-methylglutamic acid, phthalic anhydride and amine in anhydrous solvent; b) recovering the 2-methyl-N-phthaloylglutamic acid intermediate; c) dissolving 2-methyl-N-phthaloylglutamic acid intermediate in acid followed by addition of zinc dust; d) the mixture formed in c) is heated under reflux and an inert atmosphere; e) a process for producing 2-methyl-2-phthaliminoglutaric acid, comprising recovering the produced 2-methyl-2-phthaliminoglutaric acid. The method of claim 4 wherein the amine is selected from the group consisting of triethyl amine, diethyl amine, and pyridine. The method of claim 4 wherein the acid is glacial acetic acid. a) placing a DL-2-methyl-2-phthaliminoglutaric acid solution on a chiral HPLC column; b) DL-2-, comprising eluting R-(+)-2-methyl-2-phthaliminoglutaric acid and S-(-)-2-methyl-2-phthaliminoglutaric acid separately Separation method of (S) and (R) enantiomers of methyl-2-phthaliminoglutaric acid. 8. The molar ratio of claim 7 wherein R-(+)-2-methyl-2-phthaliminoglutaric acid and S-(-)-2-methyl-2-phthaliminoglutaric acid is molar ratio 1: 1: 5: 0.1. Eluting separately from the HPLC column using a solvent mixture comprising CH ₃ CN / MeOH / H ₂ O / HOAc. a) forming a diester of DL-2-methyl-2-phthaliminoglutaric acid; b) separating the diester enantiomers using ChiroCLEC ^™ -BL; c) separating the hydrolyzed product on a silica gel column; d) fully hydrolyzing the individual enantiomers to form R-(+)-2-methyl-2-phthaliminoglutaric acid and S-(-)-2-methyl-2-phthaliminoglutaric acid Separation method of (S) and (R) enantiomer of DL-2-methyl-2-phthalimino glutaric acid containing a. delete 10. The process of claim 9, wherein the individual enantiomers are fully hydrolyzed by treating the partially hydrolyzed intermediate formed in step (c) with a 1: 1 mixture of glacial acetic acid and concentrated hydrochloric acid. A pharmaceutical composition for the inhibition of unwanted angiogenesis, comprising a pharmaceutically acceptable carrier and S-(-)-2-methyl-2-phthaliminoglutaric acid. 13. The composition of claim 12 wherein the composition is a tablet, pill, capsule, suppository, sachet, granule, powder, cream, lotion, ointment, plaster, liquid solution, suspension, dispersion, emulsion, syrup, liposome, microparticles, and Pharmaceutical compositions in the form of microcapsules. Administering an angiogenesis inhibitory amount of S-(-)-2-methyl-2-phthaliminoglutaric acid to an animal other than human with unwanted angiogenesis How to suppress angiogenesis. The method of claim 14, wherein the administration is oral, parenteral, rectal, vaginal, topical, transdermal, intravenous, intramuscular, intraperitoneal or subcutaneous administration. The method of claim 14, wherein the effective amount is from 100 mg / kg / day to 2000 mg / kg / day. The method of claim 12, wherein the unwanted angiogenesis is diabetic retinopathy, immature retinopathy, corneal graft rejection, renal vascular glaucoma, posterior fibrosis, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens fatigue, atopic keratitis, stomach Limbic keratitis, pterygium keratitis dryness, Sjogren's syndrome, acne injection, lectrenulosis, syphilis, mycobacterial infection, lipid degeneration, chemical burn, bacterial ulcer, fungal ulcer, Huff's simplex infection, Huff's zoster infection, Protozoan infection, Kaposi's sarcoma, Muren's ulcer, Terrien's marginal degeneration, marginal keratinization, trauma, rheumatoid arthritis, systemic lupus erythematosis, polyarthritis, Wegner's sarcoidosis, scleritis, Stevens-Johnson disease, radiokeratotomy, macular degeneration, Sickle cell anemia, sarcoidosis, respiratory fibrous yellow myeloma, Farget's disease, venous atresia, arterial atresia, carotid artery obstruction, chronic uveitis, chronic vitreous Infections that cause inflammation, Lyme disease, ale disease, Bethle disease, retinitis or choroiditis, presumptive ocular histoplasmosis, best disease, myopia, ocular bovine, stargardt's disease, planetis, chronic retinal detachment Hypertensive syndrome, schistosomiasis, post-laser complications, redness of skin, abnormal proliferation of cognate or fibrous tissue, fertility chorioretinopathy, bartonellosis, hemangioma, Osler-Weber-rendu disease, solid tumor, vascular tumor, acquired immunodeficiency syndrome , Ocular neovascular disease, age-related macular degeneration, osteoarthritis, glioma, disease caused by chronic inflammation, Crohn's disease, ulcerative colitis, rhabdomyosarcoma, retinoblastoma, Eving's sarcoma, neuroblastoma, osteosarcoma, leukemia, psoriasis, A pharmaceutical composition arising from a disease selected from the group consisting of atherosclerosis, auditory neuroma, neurofibroma, trachoma, purulent granulomas, and neonatal impetigo. The pharmaceutical composition of claim 12, which is administered orally, parenterally, rectally, vaginally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously. The pharmaceutical composition according to claim 12, wherein the effective amount is from 100 mg / kg / day to 2000 mg / kg / day. A pharmaceutical composition for the treatment of cancer comprising a therapeutically effective amount of S-(-)-2-methyl-2-phthaliminoglutaric acid. The pharmaceutical composition of claim 20 which is administered orally, parenterally, rectally, vaginally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously. The pharmaceutical composition of claim 20, wherein the effective amount is from 100 mg / kg / day to 2000 mg / kg / day. The method for producing 2-methyl-2-phthaliminoglutaric acid according to claim 4, further comprising the step of recovering and purifying the produced 2-methyl-2-phthaliminoglutaric acid. .

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