WO2002056832A2 - Methods to treat autoimmune and inflammatory conditions - Google Patents
Methods to treat autoimmune and inflammatory conditions Download PDFInfo
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- WO2002056832A2 WO2002056832A2 PCT/US2002/001361 US0201361W WO02056832A2 WO 2002056832 A2 WO2002056832 A2 WO 2002056832A2 US 0201361 W US0201361 W US 0201361W WO 02056832 A2 WO02056832 A2 WO 02056832A2
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- deoxyuridine
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- 0 *C1NC(*)N(*)C(*)=C1* Chemical compound *C1NC(*)N(*)C(*)=C1* 0.000 description 1
- WRLNIDXFGQFHQQ-UHFFFAOYSA-N CC(C)(C)C(NCCO[O](C)OC)=O Chemical compound CC(C)(C)C(NCCO[O](C)OC)=O WRLNIDXFGQFHQQ-UHFFFAOYSA-N 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
- A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
Definitions
- the present invention is in the field of medicinal chemistry and relates to other areas such as pharmacology and immunology. In particular, it provides methods to treat autoimmune disorders and inflammatory conditions.
- tumor suppressor genes The function of tumor suppressor genes is a major focus of recent attempts to develop innovative therapeutics for the treatment cancer.
- the products of tumor suppressor gene expression are generally characterized as negative regulators of cell proliferation (Knudson, A. G. (1993), Weinberg, R. A. (1995)).
- therapeutic approaches to date include gene therapies to restore inactive or missing tumor suppressor function in cancer cells to re-establish normal cellular function or induce apoptosis (Clayman, G. L. (2000), Knudson, A. G. (1993)).
- Loss of RB/pl6 function can result in similar proinflammatory, proliferative and dedifferentiating effects on cells (Carson, R. A. and Haneji, N. (1999); Shim, J. et al. (2000); Wolff, B. andNaumann, M. (1999); DiCiommo et al. (2000)), and alteration in cell-cell interactions (Plath et al. (2000)).
- Inactivation of tumor suppressor function by somatic mutation or via interaction with virally-encoded proteins is proposed to contribute to the proliferative/inflammatory aspect of athersclerosis, restenosis or other hyperproliferative diseases (Tanaka, K. et al. (1999); Aoki, M. et al.
- MIF macrophage inhibitory factor
- This invention provides methods for treating cells or tissue involved in a pathology selected from the group consisting of an autoimmune disease and an inflammatory condition, by contacting the cells or tissue with an effective amount of a compound selected from the group consisting of a 1,5- substituted pyrimidine derivative or analog and furano-pyrimidone derivative or analog.
- the methods can be practiced in vitro, ex vivo an in vivo, hi one aspect, the cells or tissue are characterized by loss of tumor suppressor function, hi another aspect, the cells overexpress an endogenous intracellular enzyme such as thymidylate synthase.
- the invention When practiced in vivo in a subject, the invention provides a method for treating a subject having an autoimmune disorder or inflammatory condition by delivering to the subject an effective amount of one or more of these compounds.
- Methods for synthesizing the compounds are described herein and in Applicant's prior patent literature, e.g., PCT/US98/16607 and PCT/US99/01332, which describe the compounds as "ECTA" compounds or prodrugs.
- Also provided herein is an assay for selecting agents that inhibit the growth of cells or tissue involved in a pathology selected from the group consisting of an autoimmune disease and an inflammatory condition.
- the methods are useful to treat or ameliorate the symptoms of autoimmune diseases, for example, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, Sj ⁇ gren's syndrome, graft-versus-host disease (GVHD), myasthenia gravis, atherosclerosis, glomerulonephritis, Type 1 diabetes, muscular dystrophy and osteoarthritis.
- autoimmune diseases for example, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, Sj ⁇ gren's syndrome, graft-versus-host disease (GVHD), myasthenia gravis, atherosclerosis, glomerulonephritis, Type 1 diabetes, muscular dystrophy and osteoarthritis.
- GVHD graft-versus-host disease
- myasthenia gravis for example, psoriasis, asthma, ulcerative colitis, scleroderma,
- Figure 2 shows therapeutic effect on paw swelling in animals with collagen- induced arthritis.
- Figure 3 shows histological evaluation of all joints performed by an observer blinded to the treatments received. This figure represents the percentage of joints exhibiting normal, mild or moderate to severe arthritic changes in the joint architecture in different treatment groups. C ⁇ i-square test (2 X 2 correlation) was done to calculate statistical significance of data. P ⁇ 0.05 (*) was considered significant.
- a cell includes a plurality of cells, including mixtures thereof.
- use of "a compound” for treatment or preparation of medicaments as described herein contemplates using one or more compounds of this invention for such treatment or preparation unless the context clearly dictates otherwise.
- compositions and methods include the recited elements, but not excluding others.
- Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
- a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
- Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
- analog is intended to mean a structural derivative of a compound that differs from it by at least one element.
- derivative is intended to mean a compound derived or obtained by another and containing the essential elements of the parent substance.
- a "subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
- an "effective amount” is an amount sufficient to effect beneficial or desired results.
- a therapeutic amount is one that achieves the desired therapeutic effect. This amount may be the same or different from a prophylatically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
- An effective amount can be administered in one or more administrations, applications or dosages.
- autoimmune disorder is any condition in which an organism produces antibodies or immune cells which recognize the organism's own molecules, cells or tissues.
- Non-limiting examples of autoimmune disorders include rheumatoid arthritis, Sj ⁇ gren's syndrome, graft versus host disease, myasthenia gravis, and systemic lupus erythematosus.
- An "inflammatory condition” shall mean those conditions that are characterized by a persistent inflammatory response with pathologic sequelae. This state is characterized by infiltration of mononuclear cells, proliferation of flbroblasts and small blood vessels, increased connective tissue, and tissue destruction.
- Chronic inflammatory diseases include Crohn's disease, psoriasis, and asthma, are also included within the term "inflammatory condition.”
- Autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus can also result in a chronic inflammatory state.
- to "treat” includes systemic amelioration of the symptoms associated with the pathology and/or a delay in onset of symptons.
- Clinical and sub- clinical evidence of “treatment” will vary with the pathology, the individual and the treatment.
- administration for the treatment of arthritic conditions can result in decreased blood vessel formation in cartilage, specifically joints, resulting in increased mobility and flexibility in these regions.
- administration will reduce dermatological symptoms such as scabbing, flaking and visible blood vessels under the surface of the skin.
- In vitro treatment includes induction of apoptosis, as well as clinical (histological) and sub-clinical (e.g., biochemical and genetic changes associated with a reversal or diminution of the pathological state.) Clinical and sub-clinical evidence of "treatment” will vary with pathology, the individual or subject, the cell or tissue type and the treatment.
- An endogenous intracellular enzyme is one that is expressed by the cell whose regulation or expression can vary, hi one aspect, the enzyme selectively activates a compound whose product inhibits proliferation of the cells or kills them. In one aspect, the enzyme is overexpressed in a diseased cell as compared to a normal healthy cell.
- TS thymidylate synthase
- composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a solid support, a detectable agent or label) or active, such as an adjuvant.
- a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
- the compositions also can include stabilizers and preservatives.
- stabilizers and adjuvants see Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., Mack Publ. Co., Easton, PA (1975).
- Autoimmune diseases and inflammatory conditions are treated by contacting the cells or tissue associated with these pathologies with an effective amount of a compound selected from the group consisting of a 1,5- substituted pyrimidine derivative or analog and furano-pyrimidone derivative or analog.
- the method When practiced in a subject other than a human patient such as a mouse, the method provides an animal model for use in discovering alternative agents and therapies. In a human patient, the method treats an autoimmune disorder or inflammatory condition. Methods for detecting clinical and sub-clinical evidence of effective therapy are known in the art. In each of these methods, an effective amount of a compound selected from the group consisting of a 1,5- substituted pyrimidine derivative or analog and furano-pyrimidone derivative or analog, is delivered or administered to the subject, e.g., mouse or human patient.
- 1,5- substituted pyrimidine derivative or analog is substituted at the 5- position with a group that is extractable from pyrimidine by the endogenous, intracellular enzyme, wherein the substituent at the 1- position is selected from the group consisting of substituted sugar, unsubstituted sugar, substituted thio-sugar, unsubstituted thio-sugar, substituted carbocyclic, unsubstituted carbocyclic, substituted acyclic and unsubstituted acyclic.
- the 1,5- substituted pyrimidine derivative or analog includes, but is not limited to, a 5'-phosphoryl, 5- substituted deoxyuridine derivative or analog or a 5'- phosphoramidate, 5- substituted deoxyuridine derivative or analog. More specifically, the 1,5- substituted pyrimidine derivative or analog includes, but is not limited to, (E)-5-(2- bromovinyl)-2'-deoxy-5'-uridyl phenyl L-alanylphosphoramidate. These compounds and methods to prepare them are provided herein.
- the disease is an autoimmune disease, for example, psoriatic arthritis, atherosclerosis, reactive arthritis, systemic lupus erythematosus, rheumatoid arthritis, Sj ⁇ gren's syndrome, graft- versus-host disease, osteoarthritis, glomerulonephritis, Type 1 diabetes, muscular dystrophy, or myasthenia gravis.
- the disease is an inflammatory condition, for example, psoriasis, asthma, ulcerative colitis, inflammatory bowel disease, scleroderma or Crohn's disease.
- TNF- ⁇ tumor necrosis factor- ⁇
- N-SADDS non-steroidal anti-inflammatory drugs
- the use of operative combinations is contemplated to provide therapeutic combinations that may lower total dosage of each component than may be required when each individual therapeutic method, compound or drug is used alone. A reduction in adverse effects may also be noted.
- the present invention also includes methods involving co-administration of the compounds described herein with one or more additional active agents or methods. Indeed, it is a further aspect of this invention to provide methods for enhancing other therapies and/or pharmaceutical compositions by co-administering a compound of this invention.
- the agents may be administered concurrently or sequentially, hi one embodiment, the compounds described herein are administered prior to the other active agent(s), therapy or therapies.
- the pharmaceutical formulations and modes of administration may be any of those described herein or known to those of skill in the art.
- the compounds of the present invention are also useful in the preparation of medicaments to treat a variety of autoimmune diseases or inflammatory conditions.
- the methods and techniques for preparing medicaments of a compound are known in the art.
- pharmaceutical formulations and routes of delivery- are detailed below.
- any one or more of the compounds described more fully below, including the many specific embodiments can be used by applying standard pharmaceutical manufacturing procedures to prepare medicaments to treat the many disorders described herein.
- Such medicaments can be delivered to the subject by using delivery methods known in the pharmaceutical arts.
- a compound or an agent of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis and the like.
- Methods of delivery include but are not limited to, intra-arterial, intramuscular, intravenous, intranasal, and oral routes, hi a specific embodiment, it may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment; this may be achieved by, for example and not by way of limitation, local infusion during surgery, by injection, or by means of a catheter.
- a tissue sample can be removed from the patient and the cells are assayed for sensitivity to the agent.
- Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the compound as well as whether the compound is used alone or in combination with other agents of therapeutic methods. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
- MLR/MpJ- Ipr/lpr (“MLR-Zpr") (available from Jackson Laboratories, Bal Harbor, Maine ). MLR- Ipr mice develop systemic autoimmune disease.
- Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be readily determined by those of skill in the art. For example, the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, alternatively at about 0.1 mg/kg to about 100 mg/kg, or alternatively at about 0.5 mg/kg to about 50 mg/kg. When the compounds described herein are co-administered with another agent (e.g., as sensitizing agents) or therapy, the effective amount may be less than when the agent is used alone.
- another agent e.g., as sensitizing agents
- the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders, hi addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
- an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
- the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This maybe achieved, for example, by the intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
- This invention also provides a quick and simple screening assay to enable initial identification of novel compounds and combinations useful to treat or ameliorate symptoms of autoimmune and/or chronic inflammatory conditions.
- the assay requires contacting a first sample comprising suitable cells or tissue ("control sample”) with an effective amount of a compound selected from the group consisting of a deoxyuridine, a substituted deoxyuridine, a substituted deoxyuridine derivative and analogs thereof and contacting a second sample of the suitable cells or tissue ("test sample”) with the agent to be assayed.
- the test agent is contacted with a third sample of cells or tissue comprising normal counterpart cells or tissue to the control and test samples and selecting agents that treat the second sample of cells or tissue but does not adversely effect the third sample.
- a suitable cell or tissue is one involved in pathogenesis of autoimmune or chronic inflammatory conditions.
- the cells are tissue are characterized by the loss of a native tumor suppressor function.
- the assay requires at least two cell types, the first being a suitable control cell. The second cell type is of the same type or tissue as the control cell but differs in that pathogenesis toward disease has begun, hi one aspect, pathogenesis is determined enzymatically by noting enhanced or over expression of an endogenous intracellular enzyme that activates the compound into a toxic entity.
- the compound or agent to be tested can be activated by an endogenous intracellular enzyme that is overexpressed or differentially expressed in a pathological cell as compared to its normal counterpart.
- an enzyme includes, but is not limited to thymidylate synthase.
- a cell genetically modified to differentially express the enzyme or enzymes can be used. Transfection of host cells with polynucleotides encoding the enzyme can be either transient or permanent using procedures well known in the art and described by Chen, L. et al. (1996), Hudziak, R.M. et al. (1988), or Carter, P. et al. (1992), and in the experimental section below.
- the cells can be procaryotic (bacterial such as E. coli) or eucaryotic.
- the cells can be mammalian or non-mammalian cells, e.g., mouse cells, rat cells, human cells, fungi (e.g., yeast) or parasites (e.g., Pneumocystis or Leishmania) which cause disease.
- Suitable vectors for insertion of the cDNA are commercially available from Stratagene, La Jolla, CA and other vendors.
- the amount of expression can be regulated by the number of copies of the expression cassette introduced into the cell or by varying promoter usage.
- the level of expression of enzyme in each transfected cell line can be monitored by immunoblot and enzyme assay in cell lysates, using monoclonal or polyclonal antibody previously raised against the enzyme for immuno-detection. (Chen, L. et al. (1996)).
- Enzymatic assays to detect the amount of expressed enzyme also can be performed as reviewed by Carreras, C.W. and Santi, D.V. (1995), or the method described in the experimental section below.
- more than one species of enzyme can be used to separately transduce separate host cells, so that the effect of the candidate drug with an enzyme can be simultaneously compared to its effect on another enzyme or a corresponding enzyme from another species.
- compositions can be directly added to the cell culture media and the target cell or the culture media is then assayed for the amount of label released from the candidate prodrug if the prodrug contains a detectable label.
- cellular uptake may be enhanced by packaging the prodrug into liposomes using the method described in Lasic, D.D. (1996) or combined with cytofectins as described in Lewis, J.G. et al. (1996).
- the assays are useful to predict whether a subject will be suitably treated by this invention by delivering a compound or composition to a sample containing the cell to be treated and assaying for treatment which will vary with the pathology, hi one aspect, the cell or tissue is obtained from the subject or patient by biopsy.
- Applicants provide kits for determining whether a pathological cell or a patient will be suitably treated by this therapy by providing at least one composition of this invention and instructions for use.
- Kits Applicants also provide kits for determining whether a pathological cell, tissue or patient will be suitably treated by this therapy. Additionally, kits for performance of the assays are provided. These kits contain at least one composition of this invention and instructions for use.
- Therapeutic compounds for use in the methods of this invention are one or more selected from the group consisting a 1,5- substituted pyrimidine derivative or analog and a substituted furano-pyrimidone derivative or analog.
- the 1,5- substituted pyrimidine derivative or analog is substituted at the 5- position with a group that is extractable from pyrimidine by an endogenous, intracellular enzyme.
- the substituent at the 1- position is selected from the group consisting of substituted sugar, unsubstituted sugar, substituted thio-sugar, unsubstituted thio-sugar, substituted carbocyclic, unsubstituted carbocyclic, substituted acyclic and unsubstituted acyclic.
- the 1,5- substituted pyrimidine derivative or analog includes, but is not limited to, a 5'- phosphoryl, 5- substituted deoxyuridine derivative or analog or a 5'- phosphoramidate, 5- substituted deoxyuridine derivative or analog. More specifically, the 1,5- substituted pyrimidine derivative or analog includes, but is not limited to, (E)-5-(2-bromovinyl)-2'- deoxy-5'-uridyl phenyl L-alanylphosphoramidate.
- the compounds are not chemically related to pyrimidines or folates, and can be synthesized based upon known parameters of rational drug design. See Dunn, W.J. et al. (1996).
- R or R 3 may be the same or different and are selected from the group consisting of oxo, OH or NHNH j wherein a is 0 or 1, providing that if a is 0 and R 13 is oxo, then a double bond exits between position 3 and 4 and R 12 is NHNH 2 ; further providing that if a is 0 and R 12 is oxo, then a double bond exists between position 2 and 3 and R 13 is NHNH 2 ; further providing that if a is 1, then R 12 and R 13 are both oxo. While not wishing to be bound by any theory, in one aspect of the above formulae
- R 1 (at the 5-position) is or contains a leaving group which is a chemical entity that has a molecular dimension and electrophilicity compatible with extraction from the pyrimidine ring by an endogenous, intracellular enzyme (e.g., thymidylate synthase).
- An embodiment for the substituent in the R 1 position is one that could undergo an allylic interchange.
- R 4 is a halogen such as I or Br, CN or
- R 1 can be a moiety of the formula:
- R 4 is a toxophore.
- R is or contains a divalent electron conduit moiety.
- R 2 is or contains a mono- or polyunsaturated electron conduit acting to
- R is selected from the group consisting of an unsaturated hydrocarbyl group, an aromatic hydrocarbyl group comprising one or more unsaturated hydrocarbyl groups, and a heteroaromatic group comprising one or more unsaturated hydrocarbyl groups.
- n 0 and R is selected from the group consisting of:
- R 5 is independently the same or different and is selected from the group consisting of a linear or branched alkyl group having from 1 to 10 carbon atoms, a cycloalkyl group having from 3 to 10 carbon atoms, CN and a halogen.
- R 2 is an unsaturated hydrocarbyl group having a structure selected from the group consisting of:
- R 2 is an aromatic hydrocarbyl group having a structure selected from the group consisting of:
- R 2 is a heteroaromatic group having a structure selected from the group consisting of:
- R 3 is a divalent spacer moiety, also referred to as a spacer unit.
- Divalent spacers include, but are not limited to, a moiety having a structure:
- R 5 is the same or different and is independently a linear or branched alkyl group having from 1 to 10 carbon atoms, or a cycloalkyl group having from 3 to 10 carbon atoms.
- R is a divalent spacer moiety having a structure selected from the group consisting of:
- R 2 and R 3 taken together form a structure selected from the group consisting of:
- R 4 (R 4 in Formula D or R 1 in Formulae A, B or C) is or contains a leaving group that is activated or released by an intracellular enzyme
- X a and X b are independently the same or different and are selected from the group consisting of Cl, Br, I, and a potent leaving group and wherein Y a , Yb or Y c are independently the same or different and are hydrogen or F and wherein Z, Z a and Z b are independently the same or different and are selected from the group consisting of O and S; and with respect to Formula C, R 14 is hydrogen or F, providing if R 14 is F, then a is 1 and R , 12 and R 13 are both oxo
- Q is a sugar group, a thio-sugar group, a carbocyclic group or an acyclic carbon group as well as 5'-phosphoryl or phosphoramidate derivatives thereof.
- sugar groups include, but are not limited to, monosaccharide cyclic sugar groups such as those derived from oxetanes (4-membered ring sugars), furanoses (5- membered ring sugars), and pyranoses (6-membered ring sugars).
- furanoses examples include threo-furanosyl (from threose, a four-carbon sugar); erythro-furanosyl (from erythrose, a four-carbon sugar); ribo-furanosyl (from ribose, a five-carbon sugar); ara- furanosyl (also often referred to as arabino-furanosyl; from arabinose, a five-carbon sugar); xylo-furanosyl (from xylose, a five-carbon sugar); and lyxo-furanosyl (from lyxose, a five-carbon sugar).
- threo-furanosyl from threose, a four-carbon sugar
- erythro-furanosyl from erythrose, a four-carbon sugar
- ribo-furanosyl from ribose, a five-carbon sugar
- ara- furanosyl also often
- sugar group derivatives include “deoxy”, “keto”, and “dehydro” derivatives as well as substituted derivatives.
- thio sugar groups include the sulfur analogs of the above sugar groups, in which the ring oxygen has been replaced with a sulfur atom. Similar substitutions can be made to the acyclic carbon group.
- carbocyclic groups include C 4 carbocyclic groups, C 5 carbocyclic groups, and C 6 carbocyclic groups which may further have one or more substituents, such as -OH groups.
- Q is selected from the group consisting of:
- R is attached to Q at the 5' position of Q and is selected from the group consisting of a hydrogen, a hydroxyl, a phosphate group, a phosphodiester group or a phosphoramidate group.
- Any of the members of Formulae F may be in any enantiomeric, diasteriomeric, or stereoisomeric form, including D-form, L-form, ⁇ - anomeric form, and ⁇ -anomeric form.
- Q has the formula:
- R 2 and R 3 are independently the same or different and are independently
- Rg is a hydroxyl protecting group other than acetyl.
- Q has the following structure:
- R 7 is selected from the group consisting of hydrogen, a masked phosphate or a phosphoramidate and derivatives thereof, and wherein R and R 3 are the same or different and are independently hydrogen or -OH.
- R is a phosphoramidate group derived from an amino acid, including, for example, the twenty naturally occurring amino acids, e.g., alanine and tryptophane. Examples of such include, but are not limited to:
- Formula J The group identified herein as Formula J, and methods for its preparation, are described in Abraham et al. (1996).
- Formula K and its method for preparation are described in Freed et al. (1989); Sastry et al. (1992); Farquhar, J. et al. (1994), and Farquhar, J. et al. (1995).
- Formula L and its method for preparation are described in Valette et al. (1996); and Benzaria et al. (1996).
- Formula M and its method of preparation are described in Meier et al. (1997); Meier et al. (1997); and Meier et al. (1997).
- Formula N and its method for preparation are described in Hostetler et al.
- the R 7 forms a cyclic group within Q.
- DMTr 4,4'-dimethoxytrityl
- Boc is t-butyloxycarbonyl
- DCC is 1,3-dicyclohexylcarbodiimide
- 4-DMAP is 4- dimethylaminopyridine
- the compound may be in any enantiomeric, diasteriomeric, or stereoisomeric form, including, D-form, L-form, ⁇ -anomeric form, and ⁇ -anomeric forms.
- the compound may be in a salt form, or in a protected or prodrug form, or a combination thereof, for example, as a salt, an ether, or an ester.
- X d and X e are independently the same or different and are selected from the group consisting of Cl, Br, I, and CN or the nucleoside analogs thereof, hi a more preferred aspect, X is Cl or Br and Xe is hydrogen.
- X f and X g are independently the same or different and are selected from the group consisting of Cl, Br, I, and CN, or the nucleoside analogs thereof.
- X f and X g are the same and are each is Cl or Br.
- X h and Xi are independently the same or different and are selected from the group consisting of Cl, Br, I, and CN, or the nucleoside analogs thereof.
- X h and Xj are independently the same or different and are Cl or Br and in a more preferred embodiment, X h and Xi are both Br.
- R is a lower straight or branched chain alkyl, or the nucleoside analogs thereof.
- R and R are lower straight or branched chain alkyls and R is hydrogen or CH 3; or the nucleoside analogs thereof.
- R , 10 is hydrogen or CH 3 ,or the nucleoside analogs thereof.
- X is selected from the group consisting of CO 2 Et, Cl, and Br; or the nucleoside analogs thereof.
- the above structures are further modified to possess thiophosphodiaziridine instead of phosphodiaziridine groups, using the methods described below.
- the compounds can be combined with a carrier, such as a pharmaceutically acceptable carrier, for use in vitro and in vivo, hi one embodiment, the compound is in a salt form, or in a protected or prodrug form, or a combination thereof, for example, as a salt, an ether, or an ester.
- a carrier such as a pharmaceutically acceptable carrier
- “Pharmaceutically acceptable salt, prodrug or derivative” as used herein, relates to any pharmaceutically acceptable salt, ester, ether, salt of an ester, solvate, such as ethanolate, or other derivative of a compound of the present invention which, upon administration to a recipient, is capable of providing (directly or indirectly in the case of a prodrug) a compound of this invention or an active metabolite or residue thereof.
- Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system).
- Salts of the prodrugs of the present invention may be derived from inorganic or organic acids and bases.
- acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids.
- acids such as oxalic
- bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW 4 + , wherein W is C ⁇ alkyl.
- salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pahnoate, pectinate, .
- salts include anions of the compounds of the present invention compounded with a suitable cation such as Na + , NH4 "1" , and NW 4 + (wherein W is a C M a ky group).
- salts of the compounds of the present invention will be pharmaceutically acceptable.
- salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
- R is selected from (1) straight or branched chain alkyl (for example, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzy
- sulfonate esters such as alkylsulfonyl (for example, methanesulfonyl) or aralkylsulfonyl; (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters.
- the phosphate esters may be further esterified by, for example, a - 2 0 alcohol or reactive derivative thereof, or by a 2,3-di-(C6- 24 )acyl glycerol.
- any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms.
- Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms.
- Any aryl moiety present in such esters advantageously comprises a phenyl group.
- lyxo-furanosyl prodrug derivatives of the present invention include, for example, those with chemically protected hydroxyl groups (e.g., with O-acetyl groups), such as 2'-O- acetyl-lyxo-furanosyl; 3'-O-acetyl-lyxo-furanosyl; 5'-O-acetyl-lyxo-furanosyl; 2',3'-di- O-acetyl-lyxo-furanosyl and 2',3',5'-tri-O-acetyl-lyxo-furanosyl.
- chemically protected hydroxyl groups e.g., with O-acetyl groups
- Ethers of the compounds of the present invention include methyl, ethyl, propyl, butyl, isobutyl, and sec-butyl ethers.
- composition ingredient for In Vivo Administration While it is possible for the composition ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic agents.
- Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
- Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
- the active ingredient may also be presented a bolus, electuary or paste.
- a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. , povidone, gelatin, hydroxypropyhnethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose) and/or surface-active or dispersing agent.
- Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
- the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyhnethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
- Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or fragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
- compositions for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol or oil.
- a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.
- the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, about 0.075 to about 20% w/w, preferably about 0.2 to about 25% w/w and most preferably about 0.5 to about 10% w/w.
- the composition may be employed with either a paraffinic or a water-miscible ointment base.
- the ingredients may be formulated in a cream with an oil-in-water cream base.
- the aqueous phase of the cream base may include, for example, at least about 30%) w/w of a polyhydric alcohol, t.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-l,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
- the topical formulations may desirably include a compound that enhances absorption or penetration of the ingredients through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs.
- the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both an oil and a fat.
- the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
- the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
- Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
- the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
- the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
- Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
- Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the ingredients.
- a suitable carrier especially an aqueous solvent for the ingredients.
- the ingredients are preferably present in such formulation in a concentration of about 0.5 to about 20%>, advantageously about 0.5 to about 10%), particularly about 1.5% w/w.
- Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
- Formulations suitable for vaginal administration may be presented as suppositories, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the ingredients, such carriers as are known in the art to be appropriate.
- Formulations suitable for nasal administration wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
- Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the ingredients.
- Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
- the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable of oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
- compositions of the formula of the present invention may also be presented for the use in the form of veterinary formulations, which may be prepared by methods that are conventional in the art.
- One method requires treatment of 5 -chloromercuri-2' -deoxyuridine with haloalkyl compounds, haloacetates or haloalkenes in the presence of Li 2 PdCl 4 to form, through an organopalladium intermediate, the 5-alkyl, 5-acetyl or 5-alkene derivative, respectively (Wataya, Y. et al. (1979) and Bergstrom, D. E. et al. (1984)).
- C5- modification of pyrimidine nucleosides and nucleotides is the formation of C5-trans- styryl derivatives by treatment of unprotected nucleotide with mercuric acetate followed by addition of styrene or ring-substituted styrenes in the presence of Li 2 PdCl 4 (Bigge, et al. (1980)).
- Pyrimidine deoxyribonucleoside triphosphates can be derivatized with mercury at the 5 position of the pyrimidine ring by treatment with mercuric acetate in acetate buffer at 50° for 3 hours (Dale, et al. (1973)). Such treatment also would be expected to be effective for modification of monophosphates.
- a modified triphosphate can be converted enzymatically to a modified monophosphate, for example, by controlled treatment with alkaline phosphatase followed by purification of monophosphate.
- Other moieties, organic or nonorganic, with molecular properties similar to mercury but with preferred pharmacological properties could be substituted.
- the above methods would also be applicable to the synthesis of derivatives of 5-substituted pyrimidine nucleosides and nucleotides containing sugars other than ribose or 2'-deoxyribose, for example 2'-3'-dideoxyribose, arabinose, furanose, lyxose, pentose, hexose, heptose, and pyranose.
- An example of a 5-position substituent is the halovinyl group, e.g. (E)-5-(2-bromovinyl)- 2'-deoxyuridylate (Barr, P. J. et al. (1983)).
- 5-bromodeoxyuridine, 5-iododeoxyuridine, and their monophosphate derivatives are available commercially from Glen Research, Sterling, VA (USA), Sigma-Aldrich Corporation, St. Louis, MO (USA), Moravek Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA) and New England Nuclear, Boston, MA (USA).
- Commercially-available 5-bromodeoxyuridine and 5-iododeoxyuridine can be converted to their monophosphates either chemically or enzymatically, through the action of a kinase enzyme using commercial available reagents from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, CA (USA).
- the structures at the 5-position of the 1,5- substituted pyrimidine derivatives or analogs in Formulae A, B and C are referred to as the tethers because they connect a proposed leaving group (toxophore) to the heterocycle.
- the tether also contains a spacer between the toxin and the pyrimidine ring can be unsaturated, e.g., vinyl, allyl, and propargyl units are simple, small, and readily accessible synthetically.
- the vinyl and allyl units have the advantage that they can be prepared in either of two non-interconvertible geometric isomeric forms.
- a yet further approach is based on the structure of TFPe-dUMP and is similar to the vinyl tether approach but has a methylene unit separating the proposed leaving group/toxin and the unsaturated unit and thus contains an allyl or propargyl unit. This is the allyl tether approach.
- the compounds of Formula B are defined by the structure of the uracil base, or modified uracil base present. These classes are compounds where: 1) the base is a furano-pyrimidinone derivative of uracil; 2) the base is 6-fluoro uracil; 3) the base is 4- hydrazone substituted uracil derivative; and 4) the base is uracil.
- the uracil or modified uracil derived base is used to synthesize compounds substituted with toxic leaving groups at the 5 position, attached by an electron conduit tether at this 5 position, and including an appropriate spacer moiety between the electron conduit and the toxic leaving group.
- the compounds can be unphosphorylated, 5' monophosphate, 5' phosphodiester, or 5' protected ("masked") deoxyuridines or comparable derivatives of alternative carbohydrate moieties, as described below.
- Protected 5-substituted deoxyuridine monophosphate derivatives are those in which the phosphate moiety has been blocked through the attachment of suitable chemical protecting groups, hi another embodiment, 5-substituted uracil or uridine derivatives are administered to cells containing nucleoside kinase activity, wherein the 5-substituted uracil/uridine derivative is converted to a 5-substituted uridine monophosphate derivative.
- Uridine derivatives may also be modified to increase their solubility, cell penetration, and/or ability to cross the blood-brain barrier.
- the 3-hydroxy-l-propynyl substituent introduced by the latter reaction can also be accessed by DIBAL-H reduction of a methacrylate group (Cho, Y. M. et al. (1994)), itself arising from the same Heck reaction used in the synthesis of BVdU.
- These palladium-catalyzed reactions can be used to condense very long and elaborately-functionalized propargyl-based tethers to 5-iodo- 2'-deoxyuridines. (Livak, K. J. et al. (1992) and Hobbs, F. W. Jr. (1989)).
- (Z)-AUyl- based tethers are generated by the partial hydrogenation of a propargylic precursor over Undiar catalyst (Robins, M. J. et al. (1983)) whereas the (E)-allyl-based ones are best prepared by Heck coupling of an (E)-tributylstannylated ethylene (Crisp, G. T. (1989)).
- TBDMS-protected propargyl- and (Z)-allytic-tethered nucleosides can serve as convenient precursors to some of the toxophore-equipped targets.
- the known O-tetrahydropyranyl ether derivative is prepared by the literature Heck coupling of an (E)-tributylstannylated ethylene (Crisp, G. T. (1989)).
- furano-pyrimidinones begins with synthesis of a C5 propargylic - alcohol-equipped 2 '-deoxyuridine. Furano-pyrimidmone compounds are then be formed from the O-tetrahydropyranyl ether derivative described above. Synthesis proceeds by reaction of the second carbon of the propargyl bond with the oxygen attached to the C4 position of the pyrimidine ring to yield a fluorescent furano-pyrimidinone which can be readily separated from the reaction mix. Such compounds provide an additional basis for synthesis of compounds through various combinations of specific electron conduits, spacers and toxic leaving groups.
- Furo[2,3-d]pyrimidinone nucleosides (represented by the above generic structure) were prepared by condensing 2',3'-di-O-p-toluoyl or 2',3 l -di-O-acetyl-5-iodo-2'- deoxyuridine with l-(tetrahydropyranyloxy)-2-propyne (Jones, R. G. and Mann, M. J. (1953)) under conditions known to promote the formation of these fluorescent compounds (Robins, M. J. et al.(1983)).
- Proposed toxic R 4 leaving groups can be attached to the furan-2 methyl alcohol using methods similar to those employed to attach toxic leaving groups to the hydroxyl on the C5 propargyl uridine compound, as explained above.
- a variety of alternative toxic leaving groups are envisioned, hi addition, modifications to the length and composition of the R electron conduit component and of the composition of the R spacer element are also envisioned.
- Compounds based on furano-pyrimidinones can also consist of variously modified "Q" moieties.
- Compounds can have a free 5' hydroxyl, a 5' monophosphate, or a 5' phosphoramidate group attached to alternative carbohydrate groups.
- a method for synthesis of such phosphoramidate compounds is accomplished by reacting a 2-deoxy 3'- hydroxy, 5'-hydroxy unprotected nucleotide with a phosphochloridate in the presence of an HC1 scavenger, hi one aspect, the phosphochloridate comprises a phosphorus substituent which is derived from an amino acid such as alanine.
- the phosphochloridate can be phenyl-L-methoxyalanine phosphorochloridate.
- compounds are synthesized by addition of alternative electron conduits, spacer moieties and toxic leaving groups to either the C6 fluoro-uridine base or the C4 hydrazone modified pyrimidine.
- Methods described above for synthesis of 2-deoxyuridine based compounds can again be employed for the synthesis of such molecules.
- Salient features ⁇ 8.28 (d, 1, H6), 6.10 (pseudo-t, 1, HI*), 5.26 (m, exchanges with D 0, 1, 3'- OH), 5.13 (m, exchanges with D 2 0, 1, 5'-OH), 4.81 (q or dd, 2, propargyl-CH 2 ), 4.24 (m, 1, H3'), 3.57 (m, 2, 5'-CH 2 ), 2.15-2.0 (m, 8, aziridine-CH 2 ).
- Salient features ⁇ 8.29 (d, 1, H6), 6.10 (pseudo-t, 1, HI'), 5.22 (m, exchanges with D 2 0, 1, 3'-OH), 5.10 (m, exchanges with D 2 0, 1, 5'-OH), 4.88 (q or dd, 2, propargyl-CH 2 ), 4.31 (m, 1, H3'), 3.52 (m, 2, 5*-CH 2 ), 2.15-2.0 (m, 8, aziridine-CH 2 ).
- Example 3 3-(2-Deoxy- ⁇ -D-ribofuranosyl)-6-(tetrahydropyran-2-yIoxymethyl)furo[2,3- rf]pyrimidin-2(3H)-one.
- 1H NMR ((CD 3 ) 2 SO) ⁇ 8.80 (s, 1, H4), 6J4 (s, 1, H5), 6.16 (pseudo-t, 1, HI'), 5.27 (d, exchanges with D 2 0, 1, 3'-OH), 5.12 (t, exchanges with D 2 O, 1, 5*-OH), 4J2 (m, 1, THP-H2), 4.56 (q, 2, CH 2 OTHP), 3.92 (m, 1, H4'), 3.64 (m, 2, 5'- CH 2 ), 2.40 (m, 1, H2'a), 2.03 (m, 1, H2'b), 1.68 and 1.50 (m, 8, THP).
- Low-resolution mass spectrum (DCI-NH 3 ) onbis-TMS derivative, m/z
- Example 4 3-(2-Deoxy- ⁇ -D-ribofuranosyl)-6-(hydroxymethyI)furo[2,3- ⁇
- 1H NMR ((CD 3 ) 2 SO) ⁇ 12.0 (bs, 1, OH), 8.24 (s, 1, H4), 6.53 (s, 1, H5), 5.51 (pseudo-t, 1, HI'), 4.42 (m, 2, CH 2 OH).
- Low-resolution mass spectrum DCI-NH 3 ), m/z 167 (B+2H + ), 184 (B+NH 4 + ).
- Salient features ⁇ 8.5 (s, 1, H4), 7.4-7.1 (m, 5, PhO), 6.36 and 6.30 (each s, each 1, H5), 6.23 (m, 1, HI'), 3.67 and 3.65 (each s, each 3, CO 2 Me), 2.69 (m, 1, H2'a), 2.10 (m, 1, H2'b), 1.35 (m, 3, alaninyl- ⁇ -Me).
- Low- resolution mass spectrum DCI-NH 3 ), m/z 525 (MH), 595 (MNH 4 + ).
- Example 7 The 4-nitrophenyl ether derivative of 5-(3-hydroxy-l-propynyl)-2'-deoxyuridine was prepared according to standard ether synthesis as shown below.
- the solution was stirred at -78°C for an additional 2 hours and MeOH (2.5 mL) was added dropwise to destroy any excess DIBAL-H.
- the reaction mixture was cannulated into a mixture of 30%> citric acid solution (50 mL), ice (25 g) and EtOAc (30 mL) over ca. 20 minutes. The phases were separated and the aqueous phase was extracted with EtOAc (2 x 25 mL).
- the title compound can also be prepared from 5-(4-carbomethoxy-l,3- butadienyl)-2'-dexoyuridine (Na, from Example 10) in comparable yield as mentioned above.
- NAIb 5-(4-Bromo-lE,3Z-butadienyl)-2'-dexoyuridine
- E-alanine methyl ester hydrochloride (245.8 g; 1.76 mol) was placed in a 12 liter three-neck round bottom flask (equipped with a mechanical sti ⁇ er and thermometer) followed by 4.0 liters of dichloromethane. The mixture was sti ⁇ ed for 15 minutes at room temperature. Phenyl phosphodichloridate (370.0 g; 1.76 mol) was added to the mixture and stirring was continued for 15 minutes at room temperature. The flask was placed in the bath with dry ice and the stirring was continued for 20 minutes until a uniform suspension was formed.
- Freshly distilled tri-n-butylamine (626.5 g; 3.38 mol) was added dropwise ( ⁇ 90 minutes) with vigorous stirring to the reaction mixture so that the temperature inside the flask was held at ⁇ 0°C. The bath was removed and the stirring was continued for 6 hours at room temperature. The solution was concentrated to ⁇ 2.84 liters by evaporating several portions of the mixture on a rotary evaporator and the mixture was sealed under argon and stored at -20°C. The product was 85% pure by phosphorus ⁇ MR to give an estimated concentration of phenyhnethoxyalaninyl phosphochloridate of -0.5 M.
- the reaction was monitored by reversed phase HPLC and was complete in 3 hours.
- the reaction was quenched by the addition of 100 mL of methanol and the mixture was evaporated to an oil, re-dissolved in 6 liters of dichloromethane and passed through 800 g of silica gel.
- the major portion of BVdU-PA, referred to herein as NB 1011, was passed through the column during the loading and finally the elution of NB1011 was completed bypassing 5 liters of 5% methanol in dichloromethane. All fractions containing NBIOI 1 were combined and evaporated to an oil, the residue was dissolved in 4 liters of ethyl acetate and the mixture was extracted with water (2 x 2 liters).
- the crude product was purified by two silica gel chromatography using 0-5% MeOH in CH 2 C1 2 and 10% MeOH in CH 2 C1 2 , respectively, as eluent.
- the yield of product (>98% pure) was 64 g.
- Example 16 Using the methods described in Example 15, the phenyl N-methoxy-Z-alanyl phosphoramidates of the following nucleosides were prepared: 1. 5 -(4,4-dibromo- 1 ,3 -butadienyl)-2 ' -deoxyuridine; 2. 5-(2-chlorovinyl)-2'-deoxyuridine;
- the scoring used was: 0, Normal; 1, slight swelling with erythema; 2, pronounced swelling; 3, joint rigidity, hi addition, the extent of paw swelling reflects the degree of edema in affected limbs. Arthritis increased progressively over 10 days as reflected by both clinical score and paw swelling.
- Anti-TNF antibody was used in these experiments was as described by Marinova- Mutafchieva, L. et al. (2000). NB1011 was administered daily by intraperitoneal administration at 2.5 mg total dose per day. Anti-TNF antibody was compared with NBIOI 1 because, at present, antiTNF antibody is the optimal single agent for treatment of collagen induced arthritis (Marino va-Mutafchieva, L. et al. (2000)).
- mice Following immunization with collagen, mice were maintained until a significant clinical score for disease progression was achieved (between 2.5 and 3.5, see Figure 1 and Methods). Mice were then treated with control saline injections, NBIOI 1, or with antiTNF antibody as a positive confrol.
- the results ( Figure 1) show that the NBIOI 1 -treated group exhibited significant disease suppression (p ⁇ 0.05), similar to the anti-TNF control, when compared with the saline-treated control group. There was no significant difference between the NBIOI 1 and anti-TNF groups with regard to clinical score.
- Paw swelling is an alternative measure of CIA disease severity. When paw swelling was used as a criteria for disease suppression, comparable results were observed ( Figure 2).
- both the NBIOI 1 and anti-TNF groups demonstrated significant disease suppression as compared to the saline-treated control group (p ⁇ 0.05). Again, there was no significant difference between the NBIOI 1 and anti-TNF groups, although suppression of swelling may have been less dramatic with NB 1011.
- a further significant outcome of this work is that by comparison with earlier reported work, NBIOI 1 appears to have activity superior to anti-angiogenesis agents, an anti-CD4 immunosuppressive agent, and cannabidiol, a third experimental agent currently being considered for use to treat rheumatoid arthritis, and potentially other autoimmune and inflammatory disorders (Malfait, A. M. et al. (2000); Miotla, J. et al. (2000); Marinova- Mutafchieva, L. et al. (2000)).
- Balzarini J. et al. (1985) Methods Find. Exp. Clin. Pharmacol. 7:19-28 Balzarini, J. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7295-7299
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KR102545956B1 (en) * | 2022-10-19 | 2023-06-22 | 주식회사 카이팜 | Composition for preventing or treating OSCAR-induced diseases comprising brivudine as an active ingredient |
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US7884090B2 (en) * | 1999-03-17 | 2011-02-08 | Ernest L. Bonner, Jr. | Compositions and methods for the treatment of arthritis |
US6765000B2 (en) * | 1999-03-17 | 2004-07-20 | Bonner Jr Ernest L | Treatment for reactive arthritis or bursitis |
US20050137181A1 (en) * | 1999-03-17 | 2005-06-23 | Bonner Ernest L. | Method for treatment of reactive arthritis or bursitis |
WO2001007088A2 (en) * | 1999-07-22 | 2001-02-01 | Newbiotics, Inc. | Methods for treating therapy-resistant tumors |
US6683061B1 (en) * | 1999-07-22 | 2004-01-27 | Newbiotics, Inc. | Enzyme catalyzed therapeutic activation |
US20030212037A1 (en) * | 2000-12-21 | 2003-11-13 | Christopher Boyer | Use of bvdu for inhibiting the growth of hyperproliferative cells |
WO2013158959A1 (en) * | 2012-04-19 | 2013-10-24 | Tarix Pharmaceuticals Ltd. | Compositions and methods for treatment of graft-versus-host disease |
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2002
- 2002-01-18 AU AU2002241911A patent/AU2002241911B9/en not_active Ceased
- 2002-01-18 WO PCT/US2002/001361 patent/WO2002056832A2/en not_active Application Discontinuation
- 2002-01-18 US US10/051,320 patent/US7138388B2/en not_active Expired - Fee Related
- 2002-01-18 EP EP02707508A patent/EP1359921A4/en not_active Withdrawn
- 2002-01-18 CA CA002441350A patent/CA2441350A1/en not_active Abandoned
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2006
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Cited By (1)
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---|---|---|---|---|
KR102545956B1 (en) * | 2022-10-19 | 2023-06-22 | 주식회사 카이팜 | Composition for preventing or treating OSCAR-induced diseases comprising brivudine as an active ingredient |
Also Published As
Publication number | Publication date |
---|---|
EP1359921A2 (en) | 2003-11-12 |
US20020151519A1 (en) | 2002-10-17 |
US20070213307A1 (en) | 2007-09-13 |
US7138388B2 (en) | 2006-11-21 |
WO2002056832A3 (en) | 2003-03-06 |
AU2002241911A1 (en) | 2002-07-30 |
AU2002241911B2 (en) | 2006-12-14 |
CA2441350A1 (en) | 2002-07-25 |
EP1359921A4 (en) | 2006-09-06 |
AU2002241911B9 (en) | 2007-01-25 |
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